Pyruvate kinase activators for the treatment of blood disorders

Abstract

Described herein are compounds that activate pyruvate kinase, pharmaceutical compositions, and methods of using the same. The compounds are represented by Formula (I), wherein R 1 , R 2 , R a , R b , R j , R k , and Q are as defined herein.

Description

[0001] This application is a divisional application of Chinese invention patent application (application date: August 15, 2018; application number: 201880053315.9 (international application number: PCT / US2018 / 000128); invention title: pyruvate kinase activator for treating blood disorders).

[0002] Cross-references to related applications

[0003] This application claims the benefit of priority to U.S. Provisional Patent Applications Nos. 62 / 673,526 and 62 / 673,533, both filed May 18, 2018. This application also claims the benefit of priority to International Patent Application No. PCT / CN2017 / 097496, filed August 15, 2017. Each of the aforementioned priority applications is incorporated herein by reference in its entirety. Background Technology

[0004] Pyruvate kinase deficiency (PKD) is a red blood cell disorder caused by a deficiency of pyruvate kinase R (PKR) enzyme due to a recessive mutation in the PKLR gene (Wijk et al., Human Mutation, 2008, 30(3) 446-453). PKR activators may be beneficial in the treatment of PKD, thalassemia (e.g., β-thalassemia), hereditary ellipocytosis, abetalipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria, anemia (e.g., congenital anemia (e.g., enzyme disease), hemolytic anemia (e.g., hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, chronic hemolytic anemia caused by phosphoglycerate kinase deficiency, anemia of chronic disease, nonspheric hemolytic anemia or hereditary spherocytosis). Summary of the Invention

[0005] This article describes compounds of formulas (I), (II), (III), (IV), and (V) (collectively referred to herein as "formulas (I)-(V)") that activate wild-type and / or mutant pyruvate kinase R (PKR) enzymes (such as those described herein).

[0006] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Where R 1 R 2 R a R b R j Rk And Q as defined in this article.

[0009] In one embodiment, the compound or a pharmaceutically acceptable salt thereof is selected from Table 1 or... Figure 1 Compounds in [the compound].

[0010] Pharmaceutical compositions comprising compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers are also provided.

[0011] This disclosure also provides a method of treating anemia in a subject, comprising administering to the subject an effective amount of (1) the compound described herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutically acceptable composition comprising the compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the anemia is erythropoietic anemia, such as type I, II, III, or IV congenital erythropoietic anemia.

[0012] This disclosure also provides a method of treating sickle cell disease in a subject, comprising administering to the subject an effective amount of (1) the compound disclosed herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutical composition comprising the compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0013] This disclosure also provides a method for treating a subject with hemolytic anemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinase deficiency, Blood Cells Mol Dis, 2011; 46(3): 206), comprising administering to the subject an effective amount of (1) the compound disclosed herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutical composition comprising the compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the hemolytic anemia is hereditary and / or congenital, acquired, or anemia as part of a multisystem disease. In some embodiments, the hemolytic anemia is congenital. In some embodiments, the hemolytic anemia is hereditary (e.g., nonspherocytic hemolytic anemia or hereditary spherocytosis).

[0014] This disclosure also provides a method for treating a subject with thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipocytosis, abeta-lipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme disease)), sickle cell disease, or anemia of chronic disease, comprising administering to the subject an effective amount of (1) the compound disclosed herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutical composition comprising the compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In one embodiment, acquired hemolytic anemia includes congenital anemia. In some embodiments, the provided method is for treating thalassemia. In some embodiments, the thalassemia is β-thalassemia.

[0015] This disclosure also provides a method for treating a subject with pyruvate kinase deficiency (PKD), the method comprising administering to the subject an effective amount of (1) a compound disclosed herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the PKD is PKR deficiency. In some embodiments, the PKR deficiency is associated with a pyruvate kinase R mutation.

[0016] The compounds and pharmaceutical compositions described herein are PKR activators with lower activity compared to the wild type, and therefore can be used in the methods of this disclosure. In some embodiments, the PKR is wild-type. In some embodiments, the PKR is a mutant. Mutations in such PKR can affect enzyme activity (catalytic efficiency), regulatory properties (regulated by fructose diphosphate (FBP) / ATP), and / or the enzyme's thermostability. Examples of such mutations are described in Valentini et al., JBC 2002. Some examples of mutants activated by the compounds described herein include G332S, G364D, T384M, R479H, R479K, R486W, R532W, K410E, R510Q, and R490W. Without being limited by theory, in some embodiments, the compounds described herein affect the activity of PKR mutants by activating FBP-insensitive PKR mutants, restoring the thermostability of mutants with reduced stability, or restoring the catalytic efficiency of damaged mutants. The activation activity of the compounds of the present invention for PKR mutants can be tested according to the methods described in the examples. In some implementations, the compounds described herein are also activators of wild-type PKR.

[0017] In one embodiment, this disclosure provides a method for activating a PKR in erythrocytes in a subject with this need, comprising administering to the subject an effective amount of (1) a compound disclosed herein or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the PKR is wild-type. In some embodiments, the PKR is a mutant.

[0018] In one embodiment, the mutant PKR is selected from G332S, G364D, T384M, K410E, R479H, R479K, R486W, R532W, R510Q, and R490W. In some embodiments, the mutant PKR is selected from A468V, A495V, I90N, T408I, Q421K, and R498H. In some embodiments, the mutant PKR is R532W, K410E, or R510Q. Attached Figure Description

[0019] Figure 1 This is a list of exemplary compound structures of the present invention.

[0020] Figure 2 The synthesis of the exemplary intermediates used in Examples 1-10 is shown. Invention Details

[0021] The construction details and arrangement of components set forth in the following description or shown in the accompanying drawings are not intended to be limiting. The embodiments can be practiced or implemented in various ways. The phrases and terms used herein are for descriptive purposes and should not be considered limiting.

[0022] definition

[0023] The compounds described herein may contain one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. The isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH Tables of Resolving Agents and Optical Resolutions page 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0024] The compounds described herein may also exist in multiple tautomer forms, in which case all tautomer forms of the compounds described herein are obviously included, even if they may only be represented as a single tautomer form (e.g., alkylation of a ring system may result in alkylation at multiple sites; all such reaction products are obviously included). All such isomers of such compounds are obviously included. If the tautomer of a compound is aromatic, then the compound is aromatic. Similarly, if the tautomer of a substituent is a heteroaryl, then the substituent is a heteroaryl.

[0025] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 10 carbon atoms ("C10"). 1-10 Alkyl group). C 1-6Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentyl, amyl, neopentyl, 3-methyl-2-butyl, tert-pentyl), and hexyl (C6) (e.g., n-hexyl). Unless otherwise stated, each alkyl group is independently unsubstituted (“unsubstituted alkyl”) or substituted by one or more substituents (e.g., halogens such as F) (“substituted alkyl”). In some embodiments, the alkyl group is an unsubstituted -C 1-10 Alkyl group. In some embodiments, the alkyl group is a substituted -C group. 1-10 alkyl.

[0026] The term "haloalkyl" refers to a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a halogen (e.g., fluorine, chlorine, bromine, or iodine), and includes alkyl groups in which all hydrogen atoms are replaced by a halogen (e.g., perfluoroalkyl). In some embodiments, the haloalkyl group has 1 to 8 carbon atoms ("C"). 1-8 (Halogenated alkyl group).

[0027] The term "alkoxy (alkoxyl)" refers to -O-alkyl. For example, -O-alkyl having 1-6 carbon atoms.

[0028] The term "aryloxy group" refers to -O-aryl. In some embodiments, the aryloxy group is a phenoxy group.

[0029] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having 2-10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). -C 2-4 Examples of alkenyl groups include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), heptenyl (C7), octenyl (C8), octetrinyl (C8), etc. Unless otherwise stated, each alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted by one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is an unsubstituted -C 2-10 Alkenyl group. In some embodiments, the alkenyl group is a substituted -C group. 2-10 Alkenyl. In alkenyl groups, the C=C double bond can be an (E)- or (Z)- double bond.

[0030] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group ("C-H") having 2-10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds). 2-10 The alkynyl group ("alkynyl group"). Examples of alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), heptyynyl (C7), octyynyl (C8), etc. Unless otherwise stated, each alkynyl group is independently unsubstituted ("unsubstituted alkynyl group") or substituted by one or more substituents ("substituted alkynyl group"). In some embodiments, the alkynyl group is an unsubstituted -C 2-10 Alkyne group. In some embodiments, the alkynyl group is a substituted -C group. 2-10 Alkyne group.

[0031] The term "carbocyclic" or "carbocyclic" refers to a non-aromatic ring system having 3-14 ring carbon atoms (C). 3-14 A carbocyclic group is a non-aromatic monocyclic, bicyclic, tricyclic, or polycyclic hydrocarbon ring system with 0 heteroatoms. The carbocyclic group includes fully saturated ring systems (e.g., cycloalkyl) and partially saturated ring systems. In some embodiments, the carbocyclic group has 3 to 10 ring carbon atoms (“C”). 3-10 (Carbon cyclo group).

[0032] As used herein, the term "cycloalkyl" includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 14 carbon atoms, comprising a specified number of rings and carbon atoms (e.g., C3-C4). 14 Single ring, C4-C 14 Second Ring Road, C5-C 14 Three Rings or C6-C 14 (Polycyclic cycloalkyl). In some embodiments, "cycloalkyl" is a monocyclic cycloalkyl. Examples of monocyclic cycloalkyl include cyclopentyl (C5), cyclohexyl (C5), cyclopropyl (C3), cyclobutyl (C4), cycloheptyl (C7), and cyclooctyl (C8). In some embodiments, "cycloalkyl" is a dicyclic cycloalkyl. Examples of dicyclic cycloalkyl include bicyclo[1.1.0]butane (C4), bicyclo[1.1.1]pentane (C5), spiro[2.2]pentane (C5), bicyclo[2.1.0]pentane (C5), bicyclo[2.1.1]hexane (C6), and bicyclo[3.3.3]undecane (C8). 11 ), decahydronaphthalene (C 10 ), bicyclo[4.3.2]undecane (C 11 ), spiro[5.5]undecane (C 11 ) and bicyclic [4.3.3]dodecane (C 12 In some embodiments, "cycloalkyl" is a tricyclic cycloalkyl. Examples of tricyclic cycloalkyl include adamantyl (C46- ...12 Unless otherwise stated, each cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted by one or more substituents (“substituted cycloalkyl”). In some embodiments, the cycloalkyl group is an unsubstituted C14. 3-14 Cycloalkyl. In some embodiments, the cycloalkyl group is a substituted C-type alkyl group. 3-14 Cycloalkyl.

[0033] The term "heterocyclic group" or "heterocyclic" refers to a group having a 3- to 14-membered non-aromatic ring system with a ring carbon atom and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3- to 14-membered heterocyclic group"). In heterocyclic groups containing one or more nitrogen atoms, the bonding point may be a carbon or nitrogen atom, provided the valence state allows. Heterocyclic groups may be monocyclic ("monocyclic heterocyclic group") or polycyclic (e.g., fused ring, bridged ring, or spirocyclic systems, such as bicyclic systems ("bicyclic heterocyclic group") or tricyclic systems ("tricyclic heterocyclic group")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclic polycyclic systems may contain one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems in which the heterocyclic ring as defined above is fused with one or more carbocyclic groups, wherein the connection point is on the carbocyclic ring or on the heterocyclic ring; or ring systems in which the heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the connection point is on the heterocyclic ring, in which case the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Unless otherwise stated, each heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted by one or more substituents ("substituted heterocyclic group"). In some embodiments, the heterocyclic group is an unsubstituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a substituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a 5-10 membered non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heterocyclic group"). Exemplary heterocyclic groups include azircyclopropane, oxacyclopropane, thiohexacyclopropane, azircyclobutane, oxacyclobutane, thiohexacyclobutane, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiopheneyl, dihydrothiopheneyl, pyrrolyl, dihydropyrrolyl, pyrrolyl-2,5-dine, dioxopentyl, oxathiopheneyl, dithiopheneyl, triazolinyl, oxadiazolinyl, thiadiazolinyl, piperidinyl, tetrahydropyranyl, dihydropyridinyl, thiohexyl, piperazine, morpholinyl, dithiohexyl, dioxacyclohexyl, triazinealkyl, and azircycloheptane. Alkyl, oxetane heptyl, thioheptane, azirane octyl, oxetane octyl, thioheptane octyl, dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydro-1,8-naphthidyl, decahydropyrrolo[3,2-b]pyrrole, dihydroindolyl, phthalimide, naphthalimide, chromenyl, 1H-benzo[e][1,4]diaza The compounds include 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolithyl, 5,6-dihydro-4H-furano[3,2-b]pyrrolithyl, 6,7-dihydro-5H-furano[3,2-b]pyrrolithyl, 5,7-dihydro-4H-thieno[2,3-c]pyrrolithyl, 2,3-dihydro-1H-pyrroli[2,3-b]pyridyl, 2,3-dihydrofurano[2,3-b]pyridyl, 4,5,6,7-tetrahydro-1H-pyrroli[2,3-b]pyridyl, 4,5,6,7-tetrahydrofurano[3,2-c]pyridyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridyl, and 1,2,3,4-tetrahydro-1,6-naphthidyl.

[0034] The term "aryl" refers to a group ("C") having a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared π electrons arranged in a ring) that provides 6-14 ring carbon atoms and 0 heteroatoms in an aromatic ring system. 6-14 Aryl group (“C6 aryl”). In some embodiments, the aryl group has 6 ring carbon atoms (“C6 aryl”; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms (“C6 aryl”). 10 Aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“C”). 14 "Aryl"; for example, anthracene. "Aryl" also includes ring systems in which the aryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the linking group or linking point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Unless otherwise stated, each aryl group is independently unsubstituted ("unsubstituted aryl") or substituted by one or more substituents ("substituted aryl"). In some embodiments, the aryl group is an unsubstituted C 6-14 Aryl group. In some embodiments, the aryl group is a substituted C group. 6-14 Aryl.

[0035] The term "heteroaryl" refers to a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6 shared π electrons arranged in a ring) having a cyclic carbon atom and 1-4 cyclic heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In some embodiments, the heteroaryl may be a 5- or 6-membered monocyclic heteroaryl containing 1-4 heteroatoms. In some embodiments, the heteroaryl may be an 8- or 12-membered bicyclic heteroaryl having 1-6 heteroatoms. "5- or 6-membered monocyclic heteroaryl" or "5- or 6-membered monocyclic heteroaryl" refers to a 5- or 6-membered monocyclic and non-fused 4n+2 aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary monocyclic 5- or 6-membered heteroaryl groups include pyrrole, furanyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and tetraazinyl. As used herein, if the tautomer of a group is a heteroaryl, then the group is heteroaryl. The term “tautomer” or “tautomerism” refers to two or more interconvertible compounds / substituents resulting from at least one formal migration of a hydrogen atom and at least one change in valence state (e.g., from single to double, triple to single, or vice versa). The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. Tautomerization (i.e., the reaction that provides tautomer pairs) can be catalyzed by acids or bases. Exemplary tautomerizations include ketone to enol, amide to imine, lactam to lactimide, enamine to imine, and enamine to (different enamines) tautomerization.

[0036] The term "saturated" refers to a portion that does not contain double or triple bonds; that is, the portion contains only single bonds.

[0037] The term "optionally substituted" refers to either substituted or unsubstituted. Generally, the term "substituted" indicates that at least one hydrogen atom present on a group is replaced by an allowable substituent, such as a substituent that, upon substitution, yields a stable compound, for example, a compound that does not spontaneously undergo transformation by reactions such as remodeling, cyclization, elimination, or others. Unless otherwise stated, a "substituted" group has a substituent at one or more substituted positions on the group (e.g., C10, C20, C30, C40, C50, C60, C60, C7 ... 1-6 Alkyl, halogen, nitro, cyano, hydroxyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, C 1-6 Acyl group, C 3-6 cycloalkyl, C 6-10Aryl, monocyclic or bicyclic heteroaryl, and monocyclic or bicyclic heterocyclic groups, when substituted at more than one position in a specified structure, may have the same or different substituents at each position. The term "substituted" is contemplated to include substitution with all permissible organic compound substituents, and includes any substituents described herein that result in the formation of a stable compound. The invention contemplates any and all such combinations to obtain a stable compound. For the purposes of this invention, a heteroatom (such as nitrogen) may have a hydrogen substituent and / or satisfy the valence state of that heteroatom and result in the formation of a stable moiety by any suitable substituent as described herein. This invention is not intended to be limited in any way to the exemplary substituents described herein.

[0038] "Substitutable cyclic carbon atom" refers to a carbon atom on an aryl / heteroaryl / carbocyclic / heterocyclic ring, wherein the carbon atom has at least one hydrogen atom that is replaced by an allowable substituent as defined above. "Substitutable cyclic nitrogen atom" refers to a nitrogen atom on a heteroaryl-heterocyclic ring, wherein the nitrogen atom has at least one hydrogen atom that is replaced by an allowable substituent.

[0039] Unless otherwise stated, a “substituted” group has substituents at one or more substituted positions on the group, where the substituents at each position are the same or different when more than one position is substituted in a specified structure. The term “substituted” is contemplated to include substitution by all permissible substituents of an organic compound, and includes any substituents described herein that result in the formation of a stable compound. This invention contemplates any and all such combinations to obtain a stable compound. For the purposes of this invention, heteroatoms (such as nitrogen) may have hydrogen substituents and / or satisfy the valence state of that heteroatom and result in the formation of a stable moiety by any suitable substituents described herein. This invention is not intended to be limited in any way to the exemplary substituents described herein.

[0040] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0041] The term "acyl" refers to a group having the following general formula: -C(=O)R X1 -C(=O)OR X1 -C(=O)-OC(=O)R X1 -C(=O)SR X1 -C(=O)N(R) X1 )2、-C(=S)R X1 -C(=S)N(R) X1 )2 and -C(=S)S(R X1 -C (=NR) X1 )R X1 -C(=NR) X1 OR X1 -C(=NR) X1)SR X1 and -C(=NR) X1 )N(R X1 )2, where, when the price state allows, R X1 It is hydrogen; halogen; substituted or unsubstituted hydroxyl group; substituted or unsubstituted thiol group; substituted or unsubstituted amino group; substituted or unsubstituted acyl group; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain C 1-10 Alkyl; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain C 2-10 Alkenyl; substituted or unsubstituted C 2-10 Alkyne group; substituted or unsubstituted C 6-12 Aryl groups, substituted or unsubstituted heteroaryl groups. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas.

[0042] In some embodiments, the substituents present on the nitrogen atom, oxygen atom, or sulfur atom are nitrogen protecting groups, oxygen protecting groups, or sulfur protecting groups, respectively. Nitrogen, oxygen, and sulfur protecting groups are well known in the art and include those described in detail below: Protecting Groups in Organic Synthesis, TWGreene and PGMWuts, 3 rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference. For example, nitrogen-protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate (Fmoc), tert-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 2-(trimethylsilyl)ethoxy]methyl (SEM), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), phenthiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, etc. Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), tetrahydropyranyl (THP), mesylate, benzylsulfonic acid, and toluenesulfonic acid (Ts).

[0043] The term “leaving group” is given in its general sense in the field of synthetic organic chemistry, referring to an atom or group that can be replaced by a nucleophilic group. Examples of suitable leaving groups include, but are not limited to, halogens (such as F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkylsulfonyloxy, arylsulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), sulfonates such as tosylate (-OTs), mesylate (-OMs), p-bromobenzenesulfonyloxy (-OBs), -OS(=O)2(CF2)3CF3 (perfluorobutanesulfonate, -ONf), or trifluoromethanesulfonate (-OTf).

[0044] The term "pharmaceutically acceptable salt" refers to those salts that, within reasonable medical judgment, are suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., in J. Pharmaceutical Sciences, 1977, 66, 1-19, describe pharmaceutically acceptable salts in detail, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic or organic acids, or formed using other methods known in the art such as ion exchange, where the inorganic acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, and the organic acids include acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptate, glycerophosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1-4 Alkyl)4 -Salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. When appropriate, other pharmaceutically acceptable salts include those using non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonates, and aryl sulfonates.

[0045] The terms “composition” and “formulation” are used interchangeably.

[0046] The term "subject" to which the drug is intended is defined as a human (i.e., male or female of any age group, such as a minor (e.g., an infant, child, or adolescent) or an adult (e.g., a young adult, middle-aged adult, or elderly person)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog) or a bird (e.g., a commercially relevant bird, such as a chicken, duck, goose, or turkey)). In some embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal can be male or female at any developmental stage. The non-human animal can be a transgenic or genetically engineered animal. In some embodiments, the subject is a patient. The term "patient" refers to a human subject who requires treatment for a disease. In some embodiments, the term "patient" is a human adult over 18 years of age who requires treatment for a disease. In some embodiments, the term "patient" is a human child under 18 years of age who requires treatment for a disease. In some implementations, the patient is not receiving regular intravenous infusions (e.g., no more than 4 infusion events within a 12-month period). In some implementations, the patient is receiving regular intravenous infusions (e.g., at least 4 infusion events within a 12-month period). In some implementations, the subject has undergone splenectomy. In some implementations, the subject has undergone splenectomy and is not receiving regular intravenous infusions.

[0047] The terms “administration,” “giving,” or “drug administration” refer to the implantation, absorption, ingestion, injection, inhalation, or other introduction of the compound or a combination thereof described herein into or onto a subject.

[0048] The term "treatment" refers to reversing, alleviating, delaying the onset of the disease described herein, or inhibiting the progression of the disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have been developed or observed (i.e., therapeutic treatment). In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to susceptible subjects before the onset of symptoms (i.e., preventative treatment) (e.g., taking into account a history of symptoms and / or based on exposure to a pathogen). Treatment may also continue after symptoms have been resolved to, for example, delay or prevent recurrence. In some embodiments, treatment includes delaying the onset of at least one symptom of the condition for a certain period of time.

[0049] The terms “symptom,” “disease,” and “disorder” are used interchangeably.

[0050] The term "effective amount" of the compounds described herein refers to an amount sufficient to elicit the desired biological response. The effective amount of the compounds described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition to be treated, the route of administration, and the age and health of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount will produce a hemoglobin response in the subject with an increase in Hb concentration ≥1.5 g / dL relative to baseline. The subject's baseline Hb concentration is the average of all available Hb concentrations prior to treatment with the compound. In some embodiments, the effective amount will produce a hemoglobin response in the subject with an increase in Hb concentration ≥1.0 g / dL relative to baseline. In some embodiments, the effective amount will produce a hemoglobin response in the subject with an increase in Hb concentration ≥2.0 g / dL relative to baseline. In some embodiments, the effective amount is the amount of the compound described herein in a single dose. In some embodiments, the effective amount is a combined amount of the compound described herein in multiple doses. In some embodiments, the effective amount is a therapeutically effective amount.

[0051] The "therapeuticly effective amount" of a compound described herein is an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. Therapeuticly effective amount of a compound represents the amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides therapeutic benefit in the treatment of a condition. The term "therapeuticly effective amount" may include amounts that improve overall treatment, reduce or avoid symptoms, signs, or causes of the condition, and / or enhance the therapeutic efficacy of another therapeutic agent. In some embodiments, the therapeutically effective amount is an amount sufficient to induce measurable activation of wild-type or mutant PKR. In some implementations, the therapeutically effective amount is an amount sufficient to regulate the levels of 2,3-bisphosphoglycerate and / or ATP in the blood where such regulation is desired, or for the treatment of pyruvate kinase deficiency (PKD), hemolytic anemia (e.g., chronic hemolytic anemia, hereditary nonspherocytic anemia), sickle cell disease, thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipocytosis, abeta-lipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme disease)), anemia of chronic disease, or for the treatment of diseases or conditions associated with elevated 2,3-bisphosphoglycerate levels (e.g., liver disease). In some implementations, the therapeutically effective amount is an amount sufficient to induce measurable activation of wild-type or mutant PKR and is used to regulate 2,3-bisphosphoglycerate levels in the blood where this is desired or to treat pyruvate kinase deficiency (PKD), hemolytic anemia (e.g., chronic hemolytic anemia, hereditary nonspherocytic anemia), sickle cell disease, thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipocytosis, abeta-lipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme disease)), anemia of chronic disease, or to treat diseases or conditions associated with elevated 2,3-bisphosphoglycerate levels (e.g., liver disease). In one aspect, the therapeutically effective amount is the amount required to produce a hemoglobin response in the subject such that the Hb concentration increases by ≥1.0 g / dL relative to baseline (e.g., ≥1.5 g / dL or ≥2.0 g / dL). The subject's baseline Hb concentration is the average of all available Hb concentrations over at least 2 weeks (e.g., 3, 4, 5, or 6 weeks) prior to treatment with the compound described herein. In some aspects, the therapeutically effective amount is the amount required to reduce the patient's infusion burden. In one aspect, the therapeutically effective amount is a compound provided at a dose of 0.01-100 mg / kg body weight / day, such as, for example, 0.1-100 mg / kg body weight / day. In some embodiments, the therapeutically effective amount will reduce the patient's infusion burden.

[0052] As used herein, reduced infusion burden means a reduction of at least 20% in the number of RBC units infused over at least 5 weeks of treatment. In some embodiments, reduced infusion burden is a reduction of ≥33% in the number of RBC units infused over at least 5 weeks of treatment. In some embodiments, reduced infusion burden is observed over at least 10 weeks (e.g., at least 20 weeks or at least 24 weeks).

[0053] As used herein, sickle cell disease (SCD), hemoglobin SS disease, and sickle cell anemia are used interchangeably. Sickle cell disease (SCD) describes a group of inherited red blood cell disorders. In some embodiments, a subject with SCD has an abnormal hemoglobin in their red blood cells, referred to as hemoglobin S or sickle hemoglobin. In some embodiments, people with SCD have at least one abnormal gene that causes the body to produce hemoglobin S. In some embodiments, people with SCD have two hemoglobin S genes, hemoglobin SS.

[0054] Thalassemia is a genetic blood disorder in which the body produces an abnormal form of hemoglobin. In some embodiments, the abnormal form of hemoglobin leads to a deficiency of alpha or beta globulin. In some embodiments, the condition causes the destruction of large numbers of red blood cells, which causes anemia. In some embodiments, thalassemia is alpha thalassemia. In some embodiments, thalassemia is beta thalassemia.

[0055] As used herein, the term "activator" means an agent that (measurably) increases the activity of wild-type pyruvate kinase R (wt PKR) or causes an increase in the activity of wild-type pyruvate kinase R (wt PKR) to a level above the basal activity level of wt PKR, or an agent that (measurably) increases the activity of mutant pyruvate kinase R (mPKR) or causes an increase in the activity of mutant pyruvate kinase R (mPKR) to a level above the basal activity level of mutant PKR, for example, to 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the wild-type PKR activity.

[0056] As used herein, the term "hematocrit" or PRBC refers to red blood cells obtained from whole blood units by centrifugation and removal of most of the plasma. In some embodiments, the PRBC unit has a hematocrit of at least about 95%. In some embodiments, the PRBC unit has a hematocrit of at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%.

[0057] As used herein, the term “ex vivo” refers to a method that occurs outside a living organism. For example, cells (e.g., red blood cells), tissues, or blood (containing at least red blood cells, plasma, and hemoglobin) may be extracted from a organism to be brought into contact with one or more compounds provided herein or their pharmaceutically acceptable salts or pharmaceutical compositions thereof, optionally under artificially controlled conditions (e.g., temperature).

[0058] As used herein, the term "in vitro" refers to a method that occurs outside of an organism and is contained within an artificial environment. For example, cells (e.g., red blood cells), tissues, or blood (containing at least red blood cells, plasma, and hemoglobin) may be extracted from an organism to be in contact with one or more compounds provided herein or their pharmaceutically acceptable salts or pharmaceutical compositions thereof in a closed artificial environment (e.g., a culture system), such as in test tubes, in culture media, in flasks, in microtiter plates, in Petri dishes, etc.

[0059] compound

[0060] This document describes compounds and compositions that activate wild-type PKR and / or mutant PKR, such as those described herein. In one embodiment, compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof, are provided.

[0061] In a first embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided:

[0062]

[0063] in:

[0064] R 1 It is hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, -OR o1 -C(=O)R c1 Or nitrogen protecting group; wherein:

[0065] R o1 It is a hydrogen, optionally substituted alkyl, or oxygen protecting group;

[0066] R c1 It is an substituted alkyl group or -N(R) cn )2, where each R cn Independently hydrogen, -C 1-6 Alkyl or nitrogen protecting groups;

[0067] R 2Q and Q are each independently optional substituted 5- or 6-membered monocyclic heteroaryl groups;

[0068] R a and R b Each of these can be independently hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, or -OR. o3 -N(R) n1 )2、-C(=O)N(R n1 )2 or -C(=O)R c2 Or alternatively, R a and R b They can combine with the carbon atoms to which they are attached to form optionally substituted cycloalkyl or optionally substituted heterocyclic groups; wherein:

[0069] Each R n1 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0070] R o3 It is hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group; and

[0071] R c2 It is an optionally substituted -C1-C6 alkyl group; and

[0072] R j and R k Each is independently hydrogen, halogen, -CN, -OR o7 -N(R) n5 )2、-N(R n5 )C(=O)R c5 -C(=O)N(R) n5 )2、-C(=O)R c5 -C(=O)OR o7 -SR js -S(=O)2R js -S(=O)R js Or optionally substituted -C1-C6 alkyl; or optionally R j and R k They can form C=O, optionally substituted C1-C6 monocyclic cycloalkyl rings, or optionally substituted C3-C6 monocyclic heterocyclic rings together with the carbon atoms they are attached to; wherein:

[0073] Each R n5 Independently hydrogen, optionally substituted -C1-C6 alkyl, -OR o8 Or nitrogen-protecting group, wherein R o8 It is hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0074] Each R o7Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0075] Each R c5 Independently, it is an optionally substituted -C1-C6 alkyl group; and

[0076] Each R js Independently, it is an optionally substituted -C1-C6 alkyl group, or an optionally substituted C... 6-12 Aryl, optionally substituted heteroaryl, or sulfur-protected groups.

[0077] In a second embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented by R is replaced at each substituted ring carbon atom. p Optional substitution and R at each substituted cyclic nitrogen atom n6 Optional substitution; where:

[0078] Each R p Independently, it is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heteroaryl, -OR o6 -SR s2 -N(R) n3 )2、-C(=O)N(R n3 )2、-N(R n3 )C(=O)R c4 -C(=O)R c4 -C(=O)OR o6 -OC(=O)R c4 -S(=O)R s2 -S(=O)2R s2 -S(=O)OR o6 -OS(=O)R c4 -S(=O)2OR o6 -OS(=O)2R c4 -S(=O)N(R) n3 )2、-S(=O)2N(R n3 )2、-N(R n3 )S(=O)R s2 -N(R) n3 )S(=O)2R s2 -N(R) n3 )C(=O)OR o6 -OC(=O)N(R) n3 )2、-N(R n3)C(=O)N(R n3 )2、-N(R n3 )S(=O)N(R n3 )2、-N(R n3 )S(=O)2N(R n3 )2、-N(R n3 )S(=O)OR o6 -N(R) n3 )S(=O)2OR o6 -OS(=O)N(R) n3 )2 or -OS(=O)2N(R n3 )2; or alternatively, two R atoms can be attached to the same or adjacent carbon atoms. p They can combine with the carbon atoms to which they are attached to form optionally substituted cycloalkyl or heterocycloalkyl groups; wherein:

[0079] Each R n3 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0080] Each R o6 Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting groups; and

[0081] Each R c4 It is an optionally substituted -C1-C6 alkyl group;

[0082] Each R s2 Independently, it is an optionally substituted -C1-C6 alkyl or sulfur protecting group; and

[0083] R n6 It is hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0084] The remaining variables are as defined in the first implementation scheme.

[0085] In a third embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0086]

[0087]

[0088] in:

[0089] Each R nc and R nd Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0090] p can be 0, 1, 2, 3, or 4, depending on the valence state.

[0091] The remaining variables are as defined in the first or second implementation scheme.

[0092] In a fourth embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0093]

[0094] The remaining variables are defined as in the first, second, or third implementation scheme.

[0095] In a fifth embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0096]

[0097] The remaining variables are as defined in the first, second, third, or fourth implementation scheme.

[0098] In a sixth embodiment of the invention, a compound of formula (II) or a pharmaceutically acceptable salt thereof is provided:

[0099]

[0100] Where q is 0, 1, 2, or 3; and the remaining variables are as defined in the first, second, third, fourth, or fifth embodiments. In some embodiments, a compound or a pharmaceutically acceptable salt thereof is provided, which is one of the following formulas:

[0101]

[0102] In a seventh embodiment of the invention, a compound of formula (III) or a pharmaceutically acceptable salt thereof is provided:

[0103]

[0104] Where q is 0, 1, 2 or 3; and the remaining variables are as defined in the first, second, third, fourth, fifth or sixth implementation schemes.

[0105] In an eighth embodiment of the invention, a compound of formula (IV) or a pharmaceutically acceptable salt thereof is provided:

[0106]

[0107] Where q is 0, 1, 2 or 3; and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth or seventh implementation schemes.

[0108] In a ninth embodiment of the invention, a compound of formula (V) or a pharmaceutically acceptable salt thereof is provided:

[0109]

[0110] Where q is 0, 1, 2 or 3; and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh or eighth implementation schemes.

[0111] In a tenth embodiment of the invention, a compound of formula (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R n6 Is it hydrogen or -C? 1-4 Alkyl; wherein the remaining variables are as defined in the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiments.

[0112] In the eleventh embodiment of the invention, compounds of formula (II), (III), (IV) or (V) or pharmaceutically acceptable salts thereof are provided, wherein each R p Independently hydrogen, halogen, or optionally substituted C 1-4 Alkyl, -CN, -NO2, -N3, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2、-C(=O)R c3 or -C(=O)OR o4 The remaining variables are as defined in the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth implementation schemes.

[0113] In a twelfth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0114]

[0115] in:

[0116] Each R n Independently, it is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heteroaryl, -OR o4 -SR s1-N(R) n2 )2、-C(=O)N(R n2 )2、-N(R n2 )C(=O)R c3 -C(=O)R c3 -C(=O)OR o4 -OC(=O)R c3 -S(=O)R s1 -S(=O)2R s1 -S(=O)OR o4 -OS(=O)R c3 -S(=O)2OR o4 -OS(=O)2R c3 -S(=O)N(R) n2 )2、-S(=O)2N(R n2 )2,-N(R n2 )S(=O)R s1 -N(R) n2 )S(=O)2R s1 -N(R) n2 )C(=O)OR o4 -OC(=O)N(R) n2 )2、-N(R n2 )C(=O)N(R n2 )2、-N(R n2 )S(=O)N(R n2 )2、-N(R n2 )S(=O)2N(R n2 )2、-N(R n2 )S(=O)OR o4 -N(R) n2 )S(=O)2OR o4 -OS(=O)N(R) n2 )2 or -OS(=O)2N(R n2 )2; or two R atoms connected to the same or adjacent carbon atoms n Together with the carbon atoms to which they are attached, they form optionally substituted cycloalkyl or heterocycloalkyl groups; wherein:

[0117] Each R n2 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0118] Each R o4 Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0119] Each R c3 Independently, it is an optionally substituted -C1-C6 alkyl group;

[0120] Each R s1 Independently, it is an optional substituted -C1-C6 alkyl or sulfur protecting group;

[0121] When the valence state allows, n is 0, 1, 2, or 3; and

[0122] R na R nb and R nd Each of them is independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen protecting group.

[0123] The remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh implementation schemes.

[0124] In a thirteenth embodiment of the invention, compounds of formula (I), (II), (III), (IV) or (V) or pharmaceutically acceptable salts thereof are provided, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0125]

[0126] The remaining variables include the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth implementation schemes.

[0127] In a fourteenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0128]

[0129] The remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth implementation schemes.

[0130] In a fifteenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein Q is The remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiments. In further embodiments, compounds of formulas (I), (II), (III), (IV), or (V) or pharmaceutically acceptable salts thereof are provided, wherein Q is... The remaining variables are defined as in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth implementation schemes.

[0131] In a sixteenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein Q is The remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiments. In further embodiments, compounds of formulas (I), (II), (III), (IV), or (V) or pharmaceutically acceptable salts thereof are provided, wherein Q is... The remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiments. In some embodiments, R 2 Q is the same. In some implementations, R... 2 It is different from Q.

[0132] In a seventeenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R na Is it hydrogen or -C? 1-4 Alkyl; wherein the remaining variables are as defined in embodiments 12, 13, 14 or 15.

[0133] In a seventeenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R nb Is it hydrogen or -C? 1-4 Alkyl; wherein the remaining variables are as defined in embodiments 12, 13, 14 or 15.

[0134] In a seventeenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R nd Is it hydrogen or -C? 1-4 Alkyl; wherein the remaining variables are as defined in embodiments 12, 13, 14 or 15.

[0135] In an eighteenth embodiment of the invention, compounds of formula (I), (II), (III), (IV) or (V) or pharmaceutically acceptable salts thereof are provided, wherein each R n Independently hydrogen, halogen, or optionally substituted C 1-4Alkyl, -CN, -NO2, -N3, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2、-C(=O)R c3 or -C(=O)OR o4 The remaining variables are as defined in the twelfth, thirteenth, fourteenth, fifteenth, sixteenth, or seventeenth implementation schemes.

[0136] In a nineteenth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R 1 It is hydrogen or -C1-C4 alkyl; wherein the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiments.

[0137] In a twentieth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R 1 It is methyl; wherein the other variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or eighteenth embodiments.

[0138] In a twenty-first embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R j and R k Each is independently hydrogen, halogen, -OR o7 or -C1-C4 alkyl; or Optional R j and R k Combined, they form =O, where the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth implementation schemes.

[0139] In a twenty-second embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R j and R kEach of them is hydrogen; the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth or twenty-first implementation schemes.

[0140] In a twenty-third embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein R a and R b Each is hydrogen; the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first or twenty-second implementation schemes.

[0141] In the twenty-fourth embodiment of the invention, a compound or a pharmaceutically acceptable salt thereof of formula (I), (II), (III), (IV) or (V) is provided, wherein q is 0 or 1; wherein the remaining variables are as defined in the fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second or twenty-third embodiments.

[0142] In the twenty-fifth embodiment of the invention, a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof is provided, wherein n is 0 or 1; wherein the remaining variables are as defined in the twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third or twenty-fourth embodiments.

[0143] In one embodiment, the compound or a pharmaceutically acceptable salt thereof is selected from Example 1 and Table 1 and Figure 1 Compounds of formula (I)-(V) in the text.

[0144] The compounds described herein can be used as activators of PKR mutants with lower activity compared to the wild type, and therefore can be used in the methods of this invention. Such mutations in PKR can affect the enzyme's activity (catalytic efficiency), regulatory properties (mediated by fructose diphosphate (FBP) / ATP), and / or thermal stability. Examples of such mutations are described in Valentini et al., JBC 2002. Some examples of mutants activated by the compounds described herein include G332S, G364D, T384M, R479H, R479K, R486W, R532W, K410E, R510Q, and R490W. Without being limited by theory, the compounds described herein affect the activity of PKR mutants by activating FBP-insensitive PKR mutants, restoring the thermal stability of mutants with reduced stability, or restoring the catalytic efficiency of damaged mutants. The activation activity of the compounds of this invention against PKR mutants can be detected according to the methods described in Examples 11-17. The compounds described herein can also be used as activators of wild-type PKR.

[0145] In one embodiment, to increase the lifespan of red blood cells, the compounds, compositions, or pharmaceutical compositions described herein can be added directly in vitro to whole blood or hematocrit red blood cells or administered directly to a patient (e.g., via intraperitoneal injection, intravenous injection, intramuscular injection, oral administration, inhalation (aerosol delivery), transdermal delivery, sublingual delivery, and other delivery routes). Without being theoretically limited, the compounds described herein increase the lifespan of RBCs, thus counteracting the aging of stored blood by affecting the levels of 2,3-DPG and / or ATP in the blood. A decrease in 2,3-DPG concentration induces a leftward shift of the oxyhemoglobin dissociation curve and shifts the allosteric equilibrium towards the R or oxidized state, thus therapeutically inhibiting intracellular polymerization caused by sickle cell formation by increasing oxygen affinity due to 2,3-DPG depletion, thereby stabilizing more soluble oxyhemoglobin. Therefore, in one embodiment, the compounds and pharmaceutical compositions described herein can be used as anti-sickle cell formation agents. In another embodiment, to modulate 2,3-diphosphoglycerate, the compounds, compositions, or pharmaceutical compositions described herein may be added directly in vitro to whole blood or hematocrit cells or administered directly to a patient (e.g., via intraperitoneal injection, intravenous injection, intramuscular injection, oral administration, inhalation (aerosol delivery), transdermal delivery, sublingual delivery, and other delivery routes). In another embodiment, the compounds, compositions, or pharmaceutical compositions described herein may increase ATP levels and help protect cells from reactive oxygen species (Mol Cell. 2012 Oct26; 48(2): 158-167).

[0146] In Table 1, the compounds described herein may have AC of wild-type PKR, PKR K410E, or PKR 510Q. 50 "A" refers to AC. 50Less than 0.300 μM; "B" refers to AC 50 The range is from 0.301 μM to 0.800 μM; and "C" refers to AC. 50 Greater than 0.800 μM. For some compounds, the AC of wild-type PKR... 50 It can also be measured in cell-based ATP assays. "AA" refers to AC. 50 Less than or equal to 1 μM, "BB" refers to AC 50 Greater than 1 μM.

[0147] Table 1. Activation of wild-type and mutant PKR by compounds from the examples

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] The asterisk (*) indicates that the value is based on the average of multiple test results.

[0166] The compounds described herein can be prepared using a variety of synthetic techniques illustrated in the examples. Synthetic chemical transformations and group protection methods (protection and deprotection) that can be used to synthesize the compounds described herein are known in the art and include, for example, those described in: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Watts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); 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) and subsequent editions thereof.

[0167] Certain activator compounds that can be used as wild-type and / or mutant PKR activators are those that have demonstrated specificity and activation levels of PKR enzymes (wild-type and / or mutant enzymes) in the absence of FBP greater than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% in the presence of FBP.

[0168] Synthesis of the compounds of the present invention

[0169] Option 1

[0170]

[0171] In some embodiments, the compound of formula (I) can be prepared using the method described in Scheme 1. The thiazolyl aldehyde of formula S1 reacts with ethyl azide under nucleophilic addition conditions (e.g., a base) in a suitable solvent (e.g., ethanol) to give the intermediate of formula S2. The hydroxyl group of formula S2 can be converted to a leaving group and undergo elimination to give formula S3. Cyclization of the amino group and subsequent functionalization provide a bicyclic compound of formula S5, which undergoes nucleophilic substitution with sodium methanethiol followed by oxidation to give formula S7. Formula S7 is further cyclized in the presence of hydrazine, followed by oxidation with LG in the presence of a base. 1 -CH2-Q 1Nucleophilic substitution provides an intermediate of formula S9. The sulfur group in formula S9 can be oxidized to a sulfinyl or sulfonyl group to provide formula S10 or S11, which are substrates for further nucleophilic substitution to produce general formula S12. As used herein, X 1 It is a leaving group as defined herein. In some embodiments, X 1 It includes halogens, alkylsulfonyloxy groups, arylsulfonyloxy groups, diazo compounds, alkyl diazenes, aryl diazenes, alkyl triazenes, aryl triazenes, nitro groups, alkyl nitrate esters, aryl nitrate esters, alkyl phosphate esters, aryl phosphate esters, alkyl carbonyloxy groups, aryl carbonyloxy groups, alkoxy carbonyloxy groups, aryloxy carbonyloxy groups, ammonia, alkylamines, arylamines, hydroxyl groups, alkoxy groups, and aryloxy groups; LG 1 It is a leaving group as defined in this article; Q 1 It is an optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl (e.g., optionally substituted 5- or 6-membered monocyclic heteroaryl); and Nu 1 It is a nucleophilic group as defined herein (e.g., an optionally substituted 5- or 6-membered monocyclic heteroarylene alkylene). The Nu of compounds of formula S12 1 It can be further converted into other functional groups using standard chemical transformations. R 1 As defined in the first embodiment. In some embodiments, Q 1 It is an optional substituted heteroaryl group.

[0172] Option 2

[0173]

[0174] In some embodiments, compounds of formula (I) can be prepared using the method shown in Scheme 2. Similar to Scheme 1, formula S21 can be prepared from a thiazolyl aldehyde of formula S13. Halogenation of formula S21 yields formula S22, which can undergo an organic coupling reaction with an alkyl metal, alkenyl metal, alkynyl metal, aryl metal, heteroaryl metal, heterocyclic metal, or cycloalkyl metal to give compounds of formula S23. As used herein, X 3 It is halogen; R 1 As defined in the first embodiment of the present invention; LG 2 It is a leaving group as defined in this article; Q 2 It is an optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl (e.g., an optionally substituted 5- or 6-membered monocyclic heteroaryl); M 1 It is a metal (e.g., Li, Na, K, Mg, Zn, Sn, B, Pd, Si, Cu, etc.), X 4 It is a halogenated or alkyl sulfonate or aryl sulfonate; R r1It can be an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, the organocoupling reaction is a Negishi reaction; X 3 It is I; and M 1 It is Zn.

[0175] The compounds of formulas S22 and S23 are in X 3 and / or R r1 Useful intermediates that introduce more functional groups at the position (Scheme 3). In some embodiments, the compound of formula 23-i can be further oxidized to form formula S24. Nucleophilic addition of S24 with a suitable nucleophile yields the compound of formula S25. In another embodiment, the compound of formula S22 can be coupled with a vinyl metal to introduce a vinyl group onto the thiazole ring. Oxidation of the vinyl followed by nucleophilic addition provides the compound of formula S28. As used herein, Nu 2 It is a nucleophilic reagent. In some implementations, R r1 It is an optionally substituted 5- or 6-membered monocyclic heteroarylendene, Q 2 It is an optional substituted 5- or 6-membered monocyclic heteroaryl group.

[0176] Option 3

[0177]

[0178] As used in this article, R r2 It is an optionally substituted alkyl, optionally substituted alkenyl, optionally substituted ynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, or optionally substituted heteroaryl; and Q 2 As defined in Scheme 2. In some implementations, R r2 and Q 2 Each is independently a substituted 5- or 6-membered monocyclic heteroaryl group.

[0179] As used herein, a nucleophile is a chemical substance that contributes an electron pair to an electrophile to form a reaction-related chemical bond. All molecules or ions having a free electron pair or at least one π bond can be used as nucleophiles. Exemplary nucleophiles include at least one group having a nucleophilic functional group, such as an α-carbon (e.g., a carbon adjacent to a carbonyl, sulfonyl, sulfinyl, aryl, or heteroaryl group), a thiol, a hydroxyl group, a primary amine group, a secondary amine group, a halide, a cyanide, an azide, an alcoxide, an organometallic compound, or an inorganic base.

[0180] In some embodiments, the compounds described herein can be prepared using the method shown in Scheme 4. Nucleophilic substitution of formulas S30 and S33 with secondary cyclic amines yields formulas S31 and S34, respectively. Organic coupling reactions of compound S35 (e.g., Suzuki coupling, Stille coupling, etc.) yield compounds of formulas S36(i)-(ii).

[0181] Option 4

[0182]

[0183] As used in this article, This indicates a 5- or 6-membered monocyclic heteroaryl ring A containing nitrogen as a ring atom. As used herein, This indicates a 5- or 6-membered monocyclic heteroaryl ring B with the connection point on a carbon ring atom.

[0184] R 1 As defined in the first implementation scheme. X 4 It is a halogen or -OTf. M 4 It is an organometallic metal, which, when the valence state allows, has suitable ligands (organic or inorganic) as needed. An example is M... 4 Including but not limited to organic Li, Sn, B (e.g., boric acid and borate esters), Zn, Mg, Si, Pd and Cu.

[0185] Treatment

[0186] In one embodiment, a method of treating a disease, condition, or disorder as described herein (e.g., treatment) is provided, comprising administering a compound described herein, a pharmaceutically acceptable salt of the compound, or a pharmaceutical composition comprising the compound (e.g., as described in the examples and Table 1 and Table 2). Figure 1 Compounds of formula (I)-(V) in the text.

[0187] The compounds and compositions described herein may be administered to cultured cells (e.g., in vitro or ex vivo) or to subjects (e.g., in vivo) to treat and / or diagnose a variety of conditions, including those described below.

[0188] In one embodiment of the invention, a method is provided to increase the lifespan of red blood cells (RBCs) in need, comprising mixing blood with an effective amount of (1) the compounds described herein (e.g., in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0189] In a further embodiment, the compound or pharmaceutical composition is added directly to whole blood or hematocrit cells (e.g., in vitro). In another embodiment, the compound or pharmaceutical composition is administered to a subject in need of it.

[0190] In one embodiment of the invention, a method is provided for adjusting 2,3-diphosphoglycerate levels in blood where such adjustment is desired, comprising mixing blood with an effective amount of (1) the compound described herein (e.g., in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0191] In one embodiment of the invention, a method for treating sickle cell disease is provided, comprising administering to a subject in need an effective amount of (1) the compound described herein (e.g., as shown in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0192] As used herein, sickle cell disease (SCD), hemoglobin SS disease, and sickle cell anemia are used interchangeably. Sickle cell disease (SCD) describes a group of inherited red blood cell disorders. In some embodiments, subjects with SCD have an abnormal hemoglobin in their red blood cells called hemoglobin S or sickle hemoglobin. In some embodiments, subjects with SCD have at least one abnormal gene that causes the body to produce hemoglobin S. In some embodiments, subjects with SCD have two hemoglobin S genes, hemoglobin SS.

[0193] In one embodiment of the invention, a method for treating a subject with pyruvate kinase deficiency (PKD) is provided, comprising administering to the subject an effective amount of (1) the compound described herein (e.g., as shown in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0194] As described herein, PKD is a PKR deficiency. In some embodiments, this PKR deficiency is associated with a PKR mutant. In some embodiments, PKD refers to the presence of at least two mutant alleles in the PKLR gene. In some embodiments, at least one of the at least two mutant alleles in the PKLR gene is a missense mutation. In some embodiments, the Hb concentration of a patient with PKD is less than or equal to 10.0 g / dL. In some embodiments, the patient does not receive regular infusions (e.g., no more than 4 infusion events in 12 months). In some embodiments, the patient receives regular infusions (e.g., at least 4 infusion events in 12 months). In some embodiments, the patient receives regular infusions and has at least 6 infusion events in 12 months. In some embodiments, a patient receiving regular infusions has a hemoglobin (Hb) ≤12.0 g / dL (if male) or ≤11.0 g / dL (if female). In some embodiments, the patient has undergone splenectomy.

[0195] In one implementation, the PKR mutant is selected from: A31V, A36G, G37Q, R40W, R40Q, L73P, S80P, P82H, R86P, I90N, T93I, G95R, M107T, G111R, A115P, S120F, H121Q, S130P, S130Y, V134D, R135D, A137T, G143S, I153T, A154T, L155P, G159V, R163C, R163L, T164N, G165V, L167M, G169G, E172Q, W201R, I219T, A221Y, D221N, G222A, I224T, G23 2C, N253D, G263R, G263W, E266K, V269F, L272V, L272P, G275R, G275R, E277K , V280G, D281N, F287V, F287L, V288L, D293N, D293V, A295I, A295V, I310N, I 314T, E315K, N316K, V320L, V320M, S330R, D331N, D331G, D331E, G332S, V33 5M, A336S, R337W, R337P, R337Q, D339N, D339Q, G341A, G341D, I342F, K348N, A352D, I357T, G358R, G358E, R359C, R359H, C360Y, N361D, G364D, K365M, V3 68F, T371I, L374P, S376I, T384M, R385W, R385K, E387G, D390N, A392T, N393 D. N393S, N393K, A394S, A394D, A394V, V395L, D397V, G398A, M403I, G406R, E407K, E407G, T408P, T408A, T408I, K410E, G411S, G411A, Q421K, A423A, A42 3A, R426W, R426Q, E427A, E427N, A431T, R449C, I457V, G458D, A459V, V460M , A468V, A468G, A470D, T477A, ​​R479C, R479H, S485F, R486W, R486L, R488Q, R 490W, I494T, A495T, A495V, R498C, R498H, A503V, R504L, Q505E, V506I, R51 0Q, G511R, G511E, R518S, R531C, R532W, R532Q, E538D, G540R, D550V, V552M,G557A, R559G, R559P, N566K, M568V, R569Q, R569L, Q58X, E174X, W201X, E241X, R270X, E440X, R486X, Q501X, L508X, R510X, E538X, and R559X. These mutants are described in Canu et al., Blood Cells, Molecules and Diseases 2016, 57, pp. 100-109. In one embodiment, the mutant PKR is selected from G332S, G364D, T384M, K410E, R479H, R479K, R486W, R532W, R510Q, and R490W. In some embodiments, the mutant PKR is selected from A468V, A495V, I90N, T408I, Q421K, and R498H. In some embodiments, the mutant PKR is R532W, K410E, or R510Q.

[0196] In one embodiment of the invention, a method for treating anemia in a subject is provided, comprising administering to the subject an effective amount of (1) the compound described herein (e.g., as described in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formulas (I)-(V) herein, or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers. In some embodiments, the anemia is an erythropoietic anemia, such as congenital erythropoietic anemia type I, II, III, or IV. In some embodiments, the anemia is hemolytic anemia. In some embodiments, the hemolytic anemia is a congenital and / or hereditary form of hemolytic anemia, such as PKD, sickle cell disease, thalassemia (e.g., α or β type), hereditary spherocytosis, hereditary elliptic polycythemia, paroxysmal nocturnal hemoglobinuria, or aβ-lipoproteinemia (Bassen-Kornzweig syndrome). In some embodiments, the hemolytic anemia is acquired hemolytic anemia, such as autoimmune hemolytic anemia or drug-induced hemolytic anemia. In some implementations, hemolytic anemia is anemia that is part of a multisystem disease, such as congenital erythrocytic purpura anemia, Fanconi anemia, and Diamond-Blackfan anemia.

[0197] As used herein, the term “anemia” refers to a deficiency of red blood cells (RBCs) and / or hemoglobin. As used herein, anemia includes all types of clinical anemia, such as (but not limited to): microcytic anemia, iron deficiency anemia, hemoglobinopathies, heme synthesis defects, globulin synthesis defects, sideroblastic defects, normocytic anemia, anemia of chronic disease, aplastic anemia, hemolytic anemia, macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia of preterm infants, Fanconi anemia, hereditary spherocytosis, sickle cell disease, warm antibody-type autoimmune hemolytic anemia, cold agglutinin hemolytic anemia, osteosclerosis, thalassemia, and myelodysplastic syndromes.

[0198] In some embodiments, anemia can be diagnosed based on a complete blood count. In some embodiments, anemia can be diagnosed based on measurements of one or more hemolytic markers (e.g., RBC count, hemoglobin, reticulocytes, cleavage cells, lactate dehydrogenase (LDH), haptoglobin, bilirubin, and ferritin) and / or hemosiderinuria mean corpuscular volume (MCV) and / or red blood cell distribution width (RDW). In the context of this invention, anemia is present if an individual's hemoglobin (Hb) level is less than ideal, for example, an Hb concentration less than 14 g / dL, more preferably less than 13 g / dL, more preferably less than 12 g / dL, more preferably less than 11 g / dL, or most preferably less than 10 g / dL.

[0199] In some embodiments, this document provides a method for increasing the amount of hemoglobin in a subject with a need by administering an effective amount of a compound as described herein, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable composition thereof. In some embodiments, the provided method increases the hemoglobin concentration of the subject. In some embodiments, the provided method increases the Hb concentration to an ideal level, for example, above 10 g / dL, more preferably above 11 g / dL, more preferably above 12 g / dL, more preferably above 13 g / dL, or most preferably above 14 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 0.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 1.0 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 1.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 2.0 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 2.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 3.0 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 3.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 4.0 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 4.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 5.0 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 5.5 g / dL. In some embodiments, the provided method increases the Hb concentration by at least about 6.0 g / dL.

[0200] In one embodiment of the invention, a method for treating hemolytic anemia is provided, comprising administering to a subject an effective amount of (1) the compound described herein (e.g., as shown in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.

[0201] In further embodiments, hemolytic anemia is hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, or anemia as part of a multisystem disease. In some embodiments, the hemolytic anemia is congenital. In some embodiments, the hemolytic anemia is hereditary (e.g., nonspherocytic hemolytic anemia or hereditary spherocytosis).

[0202] In one embodiment of the invention, a method for treating the following diseases is provided: thalassemia; hereditary spherocytosis; hereditary ellipocytosis; aβ-lipoproteinemia or Bassen-Kornzweig syndrome; paroxysmal nocturnal hemoglobinuria; acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme disease)); sickle cell disease; or chronic anemia, comprising administering to a subject an effective amount of (1) the compound described herein (e.g., in the examples and Table 1 and Figure 1 (1) Compounds of formula (I)-(V) herein, or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers. In one embodiment, the acquired hemolytic anemia includes congenital anemia. In some embodiments, the provided method is used to treat thalassemia. In some embodiments, the thalassemia is β-thalassemia.

[0203] As used herein, thalassemia is a hereditary blood disorder in which the body produces an abnormal form of hemoglobin. In some embodiments, the disease causes the destruction of a large number of red blood cells, which leads to anemia. In some embodiments, thalassemia is alpha-thalassemia. In some embodiments, thalassemia is beta-thalassemia.

[0204] In one embodiment of the invention, a method for activating mutant PKR in erythrocytes is provided, comprising administering to a subject in need an effective amount of (1) the compound described herein (e.g., as shown in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formulas (I)-(V) herein, or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers. In one embodiment, the method is an in vitro method. In another embodiment, the method is an in vitro method. In some embodiments, the blood or red blood cells are derived from or obtained from a subject suffering from or susceptible to a disease or condition selected from: pyruvate kinase deficiency (PKD), thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipocytosis, abetalipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria, anemia (e.g., dyserythropoetic anemia), hemolytic anemia, and anemia of chronic disease. In some embodiments, the hemolytic anemia is hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, or anemia as part of a multisystem disease.

[0205] In one embodiment of the invention, a method for activating wild-type PKR in erythrocytes is provided, comprising administering to a subject in need an effective amount of (1) the compound described herein (e.g., in the examples and Table 1 and Table 2). Figure 1 (1) Compounds of formulas (I)-(V) herein, or pharmaceutically acceptable salts thereof; (2) Pharmaceutically acceptable compositions comprising the compounds described herein, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers. In one embodiment, the method is an in vitro method. In another embodiment, the method is an in vitro method. In some embodiments, the blood or red blood cells are derived from or obtained from a subject suffering from or susceptible to a disease or condition selected from the group consisting of: pyruvate kinase deficiency (PKD), thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipocytosis, abeta-lipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria, anemia (e.g., ischemic anemia), hemolytic anemia, and anemia of chronic disease. In some embodiments, the hemolytic anemia is hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, or anemia as part of a multisystem disease.

[0206] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier for the preparation of a medicament for increasing the lifespan of red blood cells (RBCs) in need of such medicament.

[0207] In a further embodiment, the compound or pharmaceutical composition is formulated for direct in vitro addition to whole blood or hematocrit cells. In another embodiment, the compound or pharmaceutical composition is formulated for administration to a subject in need.

[0208] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (i) compounds of formula (I)-(V) or pharmaceutically acceptable salts thereof; (ii) the use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for regulating the level of 2,3-diphosphoglycerate in the blood where such regulation is desired.

[0209] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1(1) The use of compounds of formula (I)-(V) herein, or pharmaceutically acceptable salts thereof; (2) The use of a pharmaceutically acceptable composition comprising the compounds described herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, in the preparation of a medicament for treating anemia. In some embodiments, the anemia is an erythropoietic anemia, such as congenital erythropoietic anemia type I, II, III, or IV. In some embodiments, the anemia is hemolytic anemia. In some embodiments, the hemolytic anemia is a congenital and / or hereditary form of hemolytic anemia, such as PKD, sickle cell disease, thalassemia (e.g., α or β type), hereditary spherocytosis, hereditary elliptic polycythemia, paroxysmal nocturnal hemoglobinuria, or abeta-lipoproteinemia (Bassen-Kornzweig syndrome). In some embodiments, the hemolytic anemia is acquired hemolytic anemia, such as autoimmune hemolytic anemia or drug-induced hemolytic anemia. In some implementations, the hemolytic anemia is anemia that is part of a multisystem disease, such as congenital erythrocytic purpura anemia, Fanconi anemia, or Diamond-Blackfan anemia.

[0210] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for the treatment of hemolytic anemia.

[0211] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for the treatment of sickle cell disease.

[0212] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use in the preparation of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the treatment of a subject with pyruvate kinase deficiency (PKD).

[0213] As described in this article, PKD is a PKR deficiency. In some implementations, this PKR deficiency is associated with PKR mutants.

[0214] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) The use of a pharmaceutically acceptable composition comprising the compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for the treatment of: thalassemia; hereditary spherocytosis; hereditary elliptic polycythemia; abeta-lipoproteinemia or Bassen-Kornzweig syndrome; paroxysmal nocturnal hemoglobinuria; acquired hemolytic anemia; or anemia of chronic disease.

[0215] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for activating mutant PKR in erythrocytes.

[0216] In one embodiment of the invention, (1) the compounds described herein (e.g., in the examples and Table 1 and) are provided. Figure 1 (1) Compounds of formula (I)-(V) herein) or pharmaceutically acceptable salts thereof; (2) Use of a pharmaceutically acceptable composition comprising the compounds described herein or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for activating wild-type PKR in erythrocytes.

[0217] In one embodiment of the invention, a method for activating pyruvate kinase R (PKR) is provided, comprising reacting PKR with an effective amount of (1) the compound described herein (e.g., in the examples and Table 1 and Table 2). Figure 1(1) A compound of formula (I)-(V) or a pharmaceutically acceptable salt thereof; or (2) a pharmaceutically acceptable composition comprising the compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In one embodiment, the PKR is a wild-type PKR. In another embodiment, the PKR is a mutant PKR. In some embodiments, the PKR is expressed in erythrocytes. In one embodiment, the method is an in vitro method. In another embodiment, the method is an in vitro method. In some embodiments, the blood or erythrocytes are derived from or obtained from a subject suffering from or susceptible to a disease or condition selected from: pyruvate kinase deficiency (PKD), thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria, anemia (e.g., dyserythropoetic anemia), hemolytic anemia, and anemia of chronic disease. In some implementations, the hemolytic anemia is hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, or anemia as part of a multisystem disease.

[0218] Since the compounds and compositions described herein act through the same biological pathways and have similar modes of action as those described in WO2012 / 151451, the compounds and compositions described herein can activate PKR mutants as described in WO2012 / 151451.

[0219] Composition and route of administration

[0220] The compositions described herein comprise compounds described herein (e.g., compounds described herein) and other therapeutic agents (if present) in amounts that are effective in modulating diseases or disease symptoms (including those described herein).

[0221] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compound provided thereto, and that does not impair its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver an effective amount of the compound.

[0222] Pharmaceutically acceptable carriers, adjuvants, and media that may be used in the pharmaceutical compositions provided herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrins or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins or other solubilizing derivatives, can also be advantageously used to enhance the delivery of compounds of the formulas described herein.

[0223] The pharmaceutical compositions provided herein can be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir, preferably orally or by injection. The pharmaceutical compositions provided herein may contain any conventional, non-toxic, pharmaceutically acceptable carrier, adjuvant, or medium.

[0224] The pharmaceutical compositions described herein can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions, aqueous suspensions, dispersants, and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions and / or emulsions, the active ingredient may be suspended or dissolved in the oil phase in combination with emulsifiers and / or suspending agents.

[0225] When the compositions provided herein comprise a combination of a compound of the formula described herein with one or more other therapeutic or preventative agents, both the compound and the other agents shall be administered at a dose level of about 1 to 100%, more preferably about 5 to 95%, of the dose normally administered in a single treatment regimen. The other agents may be administered separately from the compounds provided herein as part of a multi-dose regimen. Alternatively, those agents may be mixed with the compounds provided herein in a single composition as part of a single dosage form.

[0226] The compounds described herein may be administered, for example, by injection, intravenous, intraarterial, subdermal, intraperitoneal, intramuscular, or subcutaneous routes; or orally, buccally, nasally, transmucosally, topically, via ocular formulations, or by inhalation, at doses ranging from about 0.5 to about 100 mg / kg body weight, optionally every 4–120 hours or, depending on the specific drug requirements, at doses from 1 mg to 1000 mg per dose. The amount of active ingredient that may be combined with a carrier substance to prepare a single dosage form will vary depending on the subject being treated and the specific route of administration. Typical formulations will contain about 5% to about 95% (w / w) of the active compound. Alternatively, such formulations may contain about 20% to about 80% of the active compound.

[0227] experiment

[0228] Abbreviation list:

[0229]

[0230]

[0231] General test

[0232] In the following examples, the chemical reagents were purchased from commercial sources (such as Alfa, Acros, Sigma Aldrich, TCI, and Shanghai Chemical Reagent Co., Ltd.) and used without further purification. Rapid chromatography was performed on an Ez Purifier III column containing 200-300 mesh silica gel particles. Analytical and preparative thin-layer chromatography plates (TLC) were HSGF 254 (0.15-0.2 mm thick, Shanghai Anbang Company, China). Nuclear magnetic resonance (NMR) spectra were recorded using a Brucker AMX-300 or AMX-400 NMR (Brucker, Switzerland). Chemical shifts are reported in parts per million (ppm, δ) eterO (ESI) from a Waters LCT TOF mass spectrometer (Waters, USA). HPLC chromatograms were recorded on an Agilent 1200 liquid chromatograph (Agilent, USA, column: Ultimate 4.6m x 50mm, 5M, mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile). Microwave reactions were performed on an Initiator 2.5 microwave synthesizer (Biotage, Sweden).

[0233] The HPLC conditions used in the experiments described in this article are as follows:

[0234] Method 1:

[0235] Instrument: Shimadzu LC-2010AHT

[0236] Column: YMC-Triart C18, 50×4.6mm, 5μm

[0237] Mobile phase: Solvent A: H2O / CH3OH / TFA = 90 / 10 / 0.1

[0238] Solvent B: H₂O / CH₃OH / TFA = 90 / 10 / 0.1

[0239] Flow rate: 2.5 mL / min; Column temperature: 35℃; Wavelength: 220 nm / 254 nm

[0240] Method 2:

[0241] Instrument: Shimadzu LC-2010AHT

[0242] Column: YMC-Triart C18, 50×4.6mm, 5μm

[0243] Mobile phase: Solvent A: H2O / CH3OH / TFA = 90 / 10 / 0.1

[0244] Solvent B: H₂O / CH₃OH / TFA = 90 / 10 / 0.1

[0245] Flow rate: 2.5 mL / min; Column temperature: 35℃; Wavelength: 220 nm / 254 nm

[0246] The preparative HPLC conditions used in the experiments described in this article are as follows:

[0247] Instrument: Waters 2545B / 2767

[0248] Column: YMC-Triart C18, 50×4.6mm, 5μm

[0249] Mobile phase: Solvent A: H2O (0.1% FA)

[0250] Solvent B: CH3OH or CH3CN

[0251] Flow rate: 20 mL / min; Column temperature: 35℃; Wavelength: 220 nm / 254 nm

[0252] Example 1. Synthesis of 6-(3-methoxybenzyl)-2,4-dimethyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one.

[0253]

[0254] Step A. (Z)-2-Azide-3-(2-methylthiazolyl-5-yl)ethyl acrylate. Add dropwise a solution of 2-methylthiazolyl-5-carboxaldehyde (500 mg, 3.93 mmol) and anhydrous EtOH (3 mL) of ethyl 2-azidoacetate (1.53 g, 11.79 mmol) to a solution of NaOEt (803 mg, 11.79 mmol) in 10 mL of EtOH at about -10 °C to about -5 °C. Stir the reaction mixture for about 1 hour while maintaining the temperature below 0 °C, then warm to room temperature and stir for another 2 hours. The resulting mixture is poured into a saturated aqueous solution of NH4Cl (50 mL) at 0 °C and extracted with EtOAc. The combined organic layers are washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure to give the desired product (500 mg), which is used directly in the next step without any purification. LCMS: m / z 239 (M+H) + .

[0255] Step B. Ethyl 2-methyl-4H-pyrrolo[2,3-d]thiazolyl-5-carboxylate (Z)-2-azido-3-(2-methylthiazolyl-5-yl)acrylate (500 mg, 2.1 mmol) was stirred at 140 °C for 2 hours, then cooled to room temperature and subsequently purified directly by silica gel column chromatography (eluent: pentane / EtOAc = 6 / 1) to give the desired product (220 mg, 49.8% yield). LCMS: m / z 211 (M+H) + .

[0256] Step C. Ethyl 2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate. NaH (36.5 mg, 1.52 mmol) was added to a DMF (3 mL) solution of ethyl 2-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (160 mg, 0.76 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 0.5 h, followed by the addition of CH3I (47 μL, 0.76 mmol). The resulting mixture was stirred at room temperature for 0.5 h, then poured into a saturated aqueous solution of NH4Cl at 0 °C and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: pentane / ethyl acetate = 6 / 1) to give the desired product (124 mg, 72.6% yield). LCMS: m / z 225 (M+H) + .

[0257] Step D. Ethyl 6-formyl-2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate. At 0 °C, POCl3 (122.5 μL, 1.338 mmol) was added to a mixture of ethyl 2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (100 mg, 0.446 mmol) in DMF (1 mL). The reaction mixture was stirred at 100 °C for 2 hours, followed by extraction with EtOAc after being poured into a saturated aqueous solution of NaHCO3 at 0 °C. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: pentane / ethyl acetate = 5 / 1) to give the desired product (57 mg, 50.7% yield). LCMS: m / z 253 (M+H) + .

[0258] Step E. 2,4-Dimethyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. N₂H₄·H₂O (53.7 μL, 1.130 mmol) was added to a mixture of ethyl 6-formyl-2,4-dimethyl-4H-pyrrolo[2,3-d]thiazo-5-carboxylate (57 mg, 0.226 mmol) in 2-ethoxyethanol (2 mL). The reaction mixture was stirred at 100 °C for 1 h, then poured into H₂O and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: pentane / ethyl acetate = 5 / 1) to give the desired product (49 mg, 98.4% yield). LCMS: m / z 221 (M+H) + .

[0259] Step F. 6-(3-methoxybenzyl)-2,4-dimethyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. At 0 °C, t-BuOK (50.8 mg, 0.454 mmol) was added to a mixture of 2,4-dimethyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one (49 mg, 0.223 mmol) in DMF (1 mL). The reaction mixture was stirred at room temperature for 0.5 h, followed by the addition of 1-(chloromethyl)-3-methoxybenzene (34.9 mg, 0.223 mmol). The resulting mixture was stirred at room temperature for 1 h, then poured into a saturated aqueous solution of NH4Cl at 0 °C and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: pentane / ethyl acetate = 3 / 1) to obtain the desired product. LCMS: m / z 341 (M+H)+ . 1 H NMR (400MHz, DMSO-d6) δ 8.56 (s, 1H), 7.23 (t, 1H), 6.92 -6.72 (m, 3H), 5.32 (s, 2H), 4.26 (s, 3H), 3.72 (s, 3H), 2.85 (s, 3H).

[0260] Example 1 provides an exemplary synthesis of compound E1-3. It should be understood that different compounds can be synthesized using suitable heteroaryl-CH2-halides.

[0261] Example 2. Preparation of compound E2-vii using scheme 2

[0262] Option E2

[0263]

[0264] Where X is a leaving group (e.g., Cl, Br or I, OMs or OTs); M is an organometallic complex (e.g., organoboron complexes, such as boric acid or pinacolboron complexes, organotin complexes, such as -Sn(Bu) t )3; organozinc complexes, such as α-Zn (halogen); Q and R 2 Each is independently a optionally substituted 5- or 6-membered heteroaryl group. Similar to the synthesis of compounds of formula E1-v in Example 1, compound E2-iv can be synthesized from thiazoaldehyde E2-i with several modifications (e.g., compound E2-ii reacts with MsCl followed by elimination to give compound E2-iii; the tricyclic system can be formed using a catalytic amount of (cat.) AcOH in 2-methoxyethanol). Substitution and iodination of compound E2-iv provide compound E2-vi. Coupling of compound E2-vi with an organometallic compound in the presence of a catalyst yields compound E2-vii.

[0265] Example 2A. Synthesis of 4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo-[2,3-d]pyridazin-5-one

[0266]

[0267] Step A. Ethyl 2-azido-3-hydroxy-3-(thiazol-5-yl)propionate. Sodium (12.2 g, 0.531 mol) was slowly added at room temperature to a stirred solution of dry EtOH (300 mL). The reaction mixture was then cooled to -20 °C, followed by dropwise addition of anhydrous EtOH (100 mL) solution of ethyl 2-azido-3-ethyl acetate (68.5 g, 0.531 mol) and thiazol-5-carboxaldehyde (20.0 g, 0.177 mol) while maintaining the temperature between -20 °C and -15 °C. After the addition, the reaction mixture was stirred at -20 °C for 1 hour, and then poured into a saturated aqueous solution of NH4Cl (1 L). The resulting mixture was saturated with NaCl and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using eluent: PE / EtOAc = 6 / 1 to 5 / 1 to 1 / 1) to give the desired product (34 g) as a grayish-white substance. LCMS: m / z = 243 (M+H) + .

[0268] Step B. (Z)-2-Azide-3-(thiazol-5-yl)ethyl acrylate. At -35°C, MsCl (146 g, 1.28 mol) was added to a stirred mixture of ethyl 2-azido-3-hydroxy-3-(thiazol-5-yl)propionate (103 g, 0.426 mol) in dry DCM (1.5 L), followed by dropwise addition of TEA (301 g, 2.98 mol), while maintaining the temperature between -35°C and -30°C. After the addition, the reaction mixture was stirred at -30°C for 15 min, then poured into a saturated aqueous solution of NH4Cl (1.5 L). The resulting mixture was saturated with NaCl and extracted twice with DCM. The combined organic layers were washed successively with aqueous HCl (1 M) and brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using eluent: PE / EtOAc = 5 / 1) to give the desired product (82.0 g, 86.3% yield). LCMS: m / z = 225 (M+H) + .

[0269] Step CE for the synthesis of 4H-pyrrolo[2,3-d]thiazole-5-carboxylate, 4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate and 6-formyl-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate is similar to the method in Example 1.

[0270] Step F. (E)-6-(hydrazinemethyl)-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate. At room temperature, ethyl 6-formyl-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (4.8 g, 20 mmol) was added to a stirred mixture of N₂H₄·H₂O (2.0 g, 98%, 40 mmol) in 2-methoxyethanol (50 mL), followed by 20 drops of AcOH. The reaction mixture was stirred at room temperature for about 30 minutes until the mixture became clear. The resulting mixture was poured into water (100 mL) while stirring and extracted with DCM. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the desired product, which was used for the next step without further purification. LCMS: m / z = 253 (M+H) + .

[0271] Step G. 4-Methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. At room temperature, 20 drops of AcOH were added to a stirred suspension of (E)-6-(hydrazinemethyl)-4-methyl-4H-pyrrolo[2,3-d]thiazo-5-carboxylate (4.8 g, 0.19 mol) in 50 mL of 2-methoxyethanol. The reaction suspension was stirred at 105 °C for 3 hours, followed by filtration. The filter cake was washed with water and dried under high vacuum to obtain the first desired product. The filtrate was diluted with water and extracted twice with DCM. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain the second desired product. The combined two desired products (2.5 g) were used directly in the next step without further purification. LCMS: m / z = 207 (M+H) + .1H NMR (400MHz, DMSO) δ12.68(s,1H), 9.35(s,1H), 8.55(s,1H), 4.30(s,3H)

[0272] Example 3. Synthesis of compounds E3-vii and E3-viii

[0273] Solution E3

[0274]

[0275] Where Hal is a halogen (e.g., Cl, Br, or I); LG is a leaving group (e.g., Cl, Br, I, OMs, or OTs); Q is an optionally substituted 5- or 6-membered heteroaryl; and Q' is a further functionalized 5- or 6-membered heteroaryl. Aromatic substitution of compound E3-i with sodium methanethiol yields compound E3-ii, which can be converted to compound E3-v using the synthesis of compounds E1-iii to E1-vi. Oxidation of compound E3-v with mCPBA yields compounds E3-vi and E3-vii, respectively.

[0276] Example 3A. Synthesis of 4-methyl-2-(meththio)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0277]

[0278] Step A: Ethyl 4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate. NaSMe (240.0 mg, 3.5 mmol) was added to a mixture of ethyl 2-bromo-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (500.0 mg, 1.73 mmol) in EtOH (10.0 mL). The reaction mixture was stirred at 25 °C for 3 hours, then quenched with ice water and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give the desired product (460 mg), which was used directly in the next step without any purification. LC-MS: m / z 257 (M+H) + .

[0279] Step B: Ethyl 6-formyl-4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate. POCl3 (550.0 mg, 3.6 mmol) was added to a DCE (10 mL) solution of ethyl 4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (460.0 mg, 1.8 mmol) and N-methyl-N-benzamide (490 mg, 3.6 mmol). The resulting mixture was stirred at 130 °C for 3 hours, then quenched with ice water and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 8 / 1) to give the desired product (320.0 mg, 63% yield). LC-MS: m / z 285 (M+H) + .

[0280] Step C: 4-Methyl-2-(methylthio)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. N₂H₄·H₂O (2 mL, 98% wt) was added to a solution of ethyl 6-formyl-4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazo-5-carboxylate (300.0 mg, 1.06 mmol) in EtOH (5.0 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 60 °C and maintained overnight, followed by cooling. The solid was collected by filtration and dried under high vacuum to give the desired product (180.0 mg, 67% yield). LC-MS: m / z 253 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ12.61(s,1H), 8.48(s,1H), 4.22(s,3H), 2.81(s,3H).

[0281] Example 3B. Synthesis of 6-((2-aminopyridin-4-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0282]

[0283] Step A. 2-Bromo-4-(Bromomethyl)pyridine. A mixture of 2-bromo-4-methylpyridine (1 g, 5.81 mmol), NBS (1.1 g, 6.39 mmol), and a catalytic amount of AIBN (100 mg) in CCl4 (10 mL) was stirred overnight at 80 °C. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 200 / 1) to give the desired product E3-4 (500 mg, 34.28% yield).

[0284] Step B. 6-((2-bromopyridin-4-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. A mixture of 4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (100 mg, 0.40 mmol) and K2CO3 (164 mg, 1.19 mmol) in DMF (8 mL) was stirred at 60 °C for 2 hours, followed by the addition of a DMF (2 mL) solution of 2-bromo-4-(bromomethyl)pyridine (199 mg, 0.80 mmol) and a catalytic amount of TBAB (13 mg). The mixture was stirred at 60 °C overnight, then quenched with water (20 mL) and extracted with EtOAc. The combined organic layers were washed with saturated NH4Cl aqueous solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 10 / 1) to give the desired product (150 mg, 89.62% yield). LCMS: m / z 423 (M+H) + .

[0285] Step C. (4-((4-methyl-2-(methylthio)-5-oxo-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate. 6-((2-bromopyridin-4-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (100m A mixture of 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) g (0.24 mmol), tert-butyl carbamate (83 mg, 0.71 mmol), K3PO4 (201 mg, 0.95 mmol), Pd2(dba)3 (18 mg, 0.02 mmol), and Xantphos (4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (11 mg, 0.02 mmol) in dioxane (10 mL) was stirred overnight at 100 °C under nitrogen. The resulting mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: PE / EtOAc = 3 / 1) to give the desired product (100 mg, 92.10% yield). LCMS: m / z 459 (M+H) + .

[0286] Step D. (4-((4-methyl-2-(methylsulfinyl)-5-oxo-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate. At 0 °C, 3-chloroperoxybenzoic acid (38 mg, 0.22 mmol) was added to a mixture of (100 mg, 0.22 mmol) of tert-butyl carbamate in DCM (5 mL). The reaction mixture was stirred at 0 °C for 1 hour, then quenched with water and extracted with DCM. The combined organic layers were washed with a saturated aqueous solution of NaHCO3, dried over anhydrous Na2SO4, and concentrated under reduced pressure to give the desired product (100 mg, 96.63% yield), which was used directly in the next step without any purification. LCMS: m / z 475 (M+H) + .

[0287] Step E. 6-((2-aminopyridin-4-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. A mixture of (100 mg, 0.21 mmol) of tert-butyl carbamate in TFA (1 mL) and DCM (3 mL) was stirred at room temperature for 1 hour, followed by concentration under reduced pressure. The residue was purified by preparative HPLC to give the desired product (20 mg, 25.35% yield). LCMS: m / z 375 (M+H) + . 1 HNMR (400MHz, DMSO-d6) δ8.67(s,1H), 7.73(d,1H), 6.30(d,1H), 6.12(s,1H), 5.89(s,2H), 5.24 -5.03(m,2H), 4.29(s,3H), 3.03(s,3H).

[0288] The following compounds were prepared using suitable starting materials and the method described above. Standard protection and deprotection procedures may be used when necessary.

[0289]

[0290] Example 3C. Synthesis of 6-((2-aminothiazolyl-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0291]

[0292] Steps AB. Similar to Example 3B, 2-bromo-5-(bromomethyl)thiazole and 6-((2-bromothiazol-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one were synthesized.

[0293] Step C. 6-((2-((2,4-dimethoxybenzyl)amino)thiazolyl-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. A mixture of 6-((2-bromothiazolyl)methyl)-4-methyl-2-(methylthio)-4H-thiazolyl[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (130 mg, 0.30 mmol) and DIPEA (0.1 mL) in NMP (0.1 mL) and (2,4-dimethoxyphenyl)methylamine (0.1 mL) was stirred at 150 °C for 4 hours. The reaction mixture was then quenched with water (10 mL) and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with PE / EtOAc = 5 / 1) to give the desired product (60 mg, 38.4% yield). LC-MS: m / z 515 (M+H) + .

[0294] Step D. 6-((2-((2,4-dimethoxybenzyl)amino)thiazolyl-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Potassium persulfate (oxone) (61 mg, 0.10 mmol) was added to a mixture of 6-((2-((2,4-dimethoxybenzyl)amino)thiazolyl-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg, 0.10 mmol) in THF (3 mL) at 0 °C. The mixture was stirred at 0 °C for 1 hour, then quenched with saturated Na₂S₂O₃ aqueous solution (5 mL) and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give the desired product (30 mg, 50.1% yield), which was used directly in the next step without further purification. LC-MS: m / z 531 (M+H) + .

[0295] Step E. Similar to Example 3B, synthesize 6-((2-aminothiazol-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS: m / z 381 (M+H) + . 1 HNMR (400MHz, DMSO-d6) δ8.70(s,1H), 6.97(s,1H), 6.87(s,2H), 5.28(s,2H), 4.29(s,3H), 3.11(s,3H).

[0296] The following compounds were prepared using suitable starting materials and the methods described above. Standard protection and deprotection procedures may be used when necessary.

[0297]

[0298]

[0299] Example 4. Synthesis of 6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0300] Example 4A. Synthesis of 3-((benzenesulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

[0301]

[0302] Step A. Methyl 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate was added to a THF (1 L) solution of methyl 1H-pyrazole-3-carboxylate (90 g, 0.72 mol) under nitrogen atmosphere at 0 °C with stirring. The resulting mixture was slowly warmed to room temperature and stirred for 1 hour. The reaction mixture was then cooled to 0 °C, and SEMCl (151.5 mL, 0.842 mol) was added dropwise. Stirring was continued for another 2 hours, followed by quenching with saturated NH4Cl and extraction with ethyl acetate (3x). The combined organic layers were washed with brine and dried over Na2SO4. The solvent was removed under vacuum to provide 210 g of crude product, which was used unpurified for the next step.

[0303] Step B. Crude methyl 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methanol was added to a suspension of LAH (16.9 g, 0.44 mol) in THF (760 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was slowly warmed to room temperature and stirred for 1 hour. The reaction mixture was cooled back to 0 °C, and H2O (15.6 mL), 10% NaOH (15.6 mL), and H2O (15.6 mL) were added sequentially. The resulting mixture was filtered through a diatomaceous earth filter and washed with MTBE (4x). The combined organic fractions were dried over Na2SO4. The solvent was removed under reduced pressure to provide 69.4 g of crude product, which was used unpurified for the next step. LC-MS: m / z 229 (M+H) + .

[0304] Step C. 3-(iodomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole. At 0 °C under a nitrogen atmosphere, TEA (55.42 mL, 0.393 mol) was added to a stirred solution of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methanol (61.5 g, theoretically 0.262 mol) in THF (310 mL), followed by MsCl (24 mL, 0.314 mol). The reaction was warmed to room temperature and stirred for 1 hour, followed by the introduction of NaI (196.5 g, 1.31 mol, in 310 mL DMF). The resulting mixture was stirred for another 1 hour, quenched with ice water, and extracted with MTBE (3x). The combined organic layers were washed with saturated Na₂S₂O₃ and brine, dried over Na₂SO₄, and concentrated to provide 77.5 g of crude product for the next step. LC-MS: m / z 339 (M+H) + .

[0305] Step D. 3-((benzenesulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole. Sodium benzenesulfinate (53.5 g, 0.32 mol) was added to a solution of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methanol (77.5 g, theoretically 0.229 mol) in DMF (600 mL) under stirring at 0 °C and a nitrogen atmosphere, and stirred at 0 °C for 1 hour. After warming to room temperature, the reaction mixture was quenched with ice water and saturated Na₂S₂O₃ and extracted with ethyl acetate (3x). The combined organic layers were washed successively with saturated NaHCO₃ and brine, and dried over Na₂SO₄. The solvent was removed under vacuum, and the residue was purified by rapid chromatography (silica gel, 20%–70% ethyl acetate in petroleum ether solution) to provide 56.7 g of a pale yellow oil. LCMS: [M+H] + 353.1H NMR (400MHz, DMSO) δ7.85-7.77(m,4H), 7.62(dd,2H), 6.19(d,1H), 5.35(d,2H), 4.70(d,2H), 3.44-3.38(m,2H), 0.88-0.77(m,2H), -0.01(s,9H).

[0306] Example 4B. Synthesis of 6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0307]

[0308] Step A. 4-Methyl-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LiHMDS (7.5 mL, 7.5 mmol) was added dropwise to a dry THF (30 mL) solution of 3-((benzenesulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (1.8 g, 5.1 mmol) at -40 °C. The mixture was stirred at room temperature for 30 min, followed by the addition of a suspension of 4-methyl-2-(methylsulfinyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (580 mg, 2.7 mmol) in dry THF (30 mL) at room temperature. The mixture was stirred again at room temperature for 1 hour, then poured into ice-cooled saturated NH4Cl aqueous solution (20 mL) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with water (60 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, 0–2.5% methanol in dichloromethane solution) to give the desired product (800 mg, 66%). LC-MS (ESI) result: 557 (M+H). + . 1H NMR (400MHz, DMSO) δ12.78(s,1H), 8.65(s,1H), 8.03(d,1H), 7.84-7.78(m,3H), 7.67-7.59(m,2H) ), 6.94(s,1H), 6.72(d,1H), 5.48(d,2H), 4.29(s,3H), 3.56(dd,2H), 0.88(dd,2H), 0.00(s,9H).

[0309] Step B. 4-Methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Under N2 and in an ice bath, SmI2 (0.1M / THF, 45mL) was added dropwise to a mixture of 4-methyl-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (0.8g, 1.41mmol) in THF (5mL) and MeOH (10mL). After stirring for 10 min, the reaction was quenched with saturated NH4Cl aqueous solution (50 mL) and extracted with EOAc (50 mL x 3). The combined organic layers were washed with water (60 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, 0–3% methanol in dichloromethane solution) to give the desired product (310 mg, 51.0%). LC-MS result: 417 (M+H) + . 1 H NMR (400MHz, CDCl3) δ 8.31 (s, 1H), 7.60 (d, 1H), 6.39 (d, 1H), 5.49 (s, 2H), 4.58 (s, 2H), 4.43 (s, 3H), 3.62 (t, 2H), 0.95 (t, 2H), 0.0 (s, 9H).

[0310] Step C. 3-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-6(5H)-yl)methyl)-1H-pyrazol-1-carboxylic acid tert-butyl ester. Argon was introduced into a mixture of 4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (30 mg, 0.07 mmol) and K₂CO₃ (25 mg, 0.18 mmol) in DMF (5.0 mL), and the mixture was stirred at 50 °C for 1 hour. Then, tert-butyl 3-(bromomethyl)-1H-pyrazol-1-carboxylic acid (37 mg, 0.14 mmol) was added. After stirring overnight at 50 °C under argon, the reaction mixture was cooled to room temperature, quenched with saturated aqueous NH₄Cl solution (20 mL), and extracted with EAOAC (30 mL x 3). The combined organic layers were washed with water (40 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by preparative TLC (DCM:MeOH = 15:1) to obtain the desired product (15 mg, 35.0%). LC-MS (ESI) result: 597 (M+H). + .

[0311] Step D. 6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Add TFA (1 mL) to a DCM (5 mL) solution of 3-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)-1H-pyrazol-1-carboxylic acid tert-butyl ester (15 mg, 0.025 mmol). The reaction mixture was stirred overnight at room temperature, then concentrated and purified by preparative TLC (DCM:MeOH = 15:1) to give the desired product (5 mg, 50%). LC-MS result: 367 (M+H). + . 1 H NMR (400MHz, DMSO) δ12.80 (s, 1H), 12.65 (s, 1H), 8.50 (s, 1H), 7.71 (brs, 1H), 7 .60(brs,1H), 6.26(d,1H), 6.11(d,1H), 5.32(s,2H), 4.49(s,2H), 4.27(s,3H).

[0312] Example 5. Synthesis of 4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0313]

[0314] At room temperature, 3-chloro-peroxybenzoic acid (0.77 g, 3.8 mmol) was added to a suspension of 4-methyl-2-(methylthio)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one (1.01 g, 4.0 mmol) in DCM (20 mL) under stirring. The mixture was stirred at room temperature for 2 hours. The mixture was then filtered, washed with EtOAc, and ground with MeOH. The solid was dried under vacuum to give 4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one (600 mg). LCMS: m / z 269 (M+H) + .1H NMR (400MHz, DMSO) δ12.78(s,1H), 8.64(s,1H), 4.28(s,3H), 3.11(s,3H).

[0315] Example 6. Synthesis of 4-methyl-2-(methanesulfonyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0316]

[0317] At 20°C, m-CPBA (61.5 g, 3 equivalents) was added in three portions to a three-necked flask containing a solution of 30 g, 0.119 mol, 1.0 equivalent of 4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one (30 g, 0.119 mol, 1.0 equivalent) in DCM (600 mL). The mixture was stirred overnight at 30°C, and LC-MS showed 100% consumption of the starting material, forming 20% ​​E4-2 and 80% E6-1. The mixture was cooled to room temperature, and another portion of m-CPBA (1.0 equivalent) was added. The reaction mixture was stirred at 30°C for 2 hours, and LC-MS showed E4-2 (LCMS: m / z 269(M+H)). +<8%. The mixture was cooled to room temperature and filtered. The filter cake was suspended in MeOH (500 mL) and stirred at room temperature for 1 hour. The solid was collected by filtration, washed with ethyl acetate, and dried under vacuum to give 28 g of a mixture of 5% E4-2 and 95% E6-1. This mixture (28 g) was suspended in DMSO (600 mL) and heated to 120-130 °C to form a clear solution. It was then cooled to room temperature, and the solid precipitated. The mixture was filtered and dried to provide 23 g of pure E6-1, LCMS: m / z 285 (M+H). + . 1H NMR (400MHz, DMSO) δ12.87(s,1H), 8.69(s,1H), 4.32(s,3H), 3.56(s,3H).

[0318] Example 7. Synthesis of compounds E7-v and E7-viii

[0319] Solution E7

[0320]

[0321] Nucleophilic aromatic substitution between compound E7-i and compounds E4-2 and / or E6-1 yielded intermediate E7-ii. Reduction of the benzenesulfonyl group of compound E7-ii gave intermediate E7-iii. Standard alkylation of compound E7-ii in the presence of a base (e.g., K2CO3, K3PO4, t-BuOK, or Cs2CO3) using E7-iv and compound E7-iii yielded compound E7-vi, where X... a These are leaving groups, such as Cl, Br, I, OMs, OTs; Ar1 ​​and Ar2 are each independently optional substituted 5- or 6-membered monocyclic heteroaryl groups. Compound E7-vi can also be synthesized from compound E7-iii by reacting E7-v with Mitsunobu in toluene using cyanomethylenetributylphosphine.

[0322] Example 7A. Synthesis of 4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0323]

[0324] Step A. 4-Methyl-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one was added to a solution of 3-((benzenesulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (26 g, 78.86 mmol, 2.1 equivalents) in anhydrous THF (700 mL) at -40 °C. LiHMDS (81.2 mL, 81.2 mmol, 1 M THF solution, 2.31 equivalents) was added to the solution. The reaction was then warmed to room temperature and stirred for 1 hour. Subsequently, 10 g (35.17 mmol, 1 equivalent) of 4-methyl-2-(methanesulfonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one was added to the mixture at room temperature. The reaction was stirred at room temperature for another 0.5 h. The reaction mixture was poured into a saturated ammonium chloride solution (500 mL) at 0 °C and extracted twice with ethyl acetate. The combined organic layers were washed with water and brine, dried, and concentrated to give a yellow residue. The residue was purified by silica gel chromatography (dichloromethane:methanol = 100:1 to 30:1) to give 13 g of 4-methyl-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS ESIM / Z = 557(M+H) + . 1H NMR (400MHz, DMSO) δ12.81(s,1H), 8.65(s,1H), 8.03(d,1H), 7.84-7.78(m,3H), 7.66(t,2H), 6.94(s,1H), 6.72(d,1H), 5.50(d,2H), 4.29(s,3H), 3.56(dd,2H), 0.91(dd,2H), 0.02(s,9H).

[0325] Step B. 4-Methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. At room temperature, SmI2 (503 mL, 50.29 mmol, 0.1 M THF solution, 3.5 equivalents) was added to a solution of 4-methyl-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (8 g, 14.37 mmol, 1 equivalent) in 500 mL of THF and 500 mL of MeOH. The reaction was stirred for 0.5 hours and then concentrated. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate: methanol = 75:25:0 to 20:20:1) to give 4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (4.3 g). LC-MS ESIM / Z = 417 (M+H) + . 1 H NMR (400MHz, CDCl3) δ8.31 (s, 1H), 7.60 (d, 1H), 7.28 (s, 1H), 6.39 (d, 1H), 5 .49(s,2H), 4.58(s,2H), 4.43(s,3H), 3.62(t,2H), 0.95(t,2H), 0.0(s,9H).

[0326] Example 7B. Synthesis of 6-((1H-imidazol-2-yl)methyl)-2-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0327]

[0328] Step A. 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-carboxaldehyde: Under N2, the NaH sample was washed with hexane (2 x 10 mL). Dry DMF (20 mL) was added to a flask, and 1H-imidazol-2-carboxaldehyde (500 mg, 5.2 mmol) was added aliquots. After stirring at room temperature for 1.5 h, SEMCl (864 mg, 5.2 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give crude 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-carboxaldehyde (800 mg). LCMS: 227 (M+H) + .

[0329] Step B. (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methanol: At 0 °C, NaBH4 (1.34 g, 35 mmol) was added to a mixture of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-carboxaldehyde (1.6 g, 7 mmol) in THF (20 mL) under stirring. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was poured into an aqueous solution of NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give crude (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methanol (1.3 g).

[0330] Step C. 2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole: At room temperature, NCS (466 mg, 3.5 mmol) and PPh3 (920 mg, 3.5 mmol) were added to a mixture of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-yl)methanol (400 mg, 1.75 mmol) in DCM (20 mL) under stirring. The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water and extracted with DCM. The mixture was washed with water, and the organic layer was concentrated under reduced pressure. The residue was purified by preparative TLC (PE:EtOAc = 1:1) to give 2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole. LCMS: 247 (M+H) +At 60°C and under N2, K2CO3 (66 mg, 0.48 mmol) was added to a mixture of 4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg, 0.12 mmol) in dry DMF (5 mL) under stirring. After 20 minutes, compound E7-2 (60 mg, 0.24 mmol) was added to dry DMF (2 mL) at 60°C and under N2. The mixture was stirred at 60°C and N2 for 1.5 hours. The reaction mixture was cooled to room temperature and adjusted to pH 5–6 with 0.5N hydrochloric acid aqueous solution. The mixture was then extracted with EtOAc and washed with water and brine. The organic layer was dried over Na₂SO₄, concentrated under reduced pressure, and purified by preparative TLC (PE:EtOAc = 1:1.5) to obtain 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methyl)-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (25 mg). LCMS: 627 (M+H) + A mixture of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methyl)-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (25 mg, 0.04 mmol) in DCM / TFA (2 mL / 2 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated. The residue was purified by preparative HPLC to give the desired product (1.3 mg). LCMS: 367 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.51(s,1H), 7.66(d,1H), 6.9(s,2H), 6.27(d,1H), 5.34(s,2H), 4.51(s,2H), 4.27(s,3H).

[0331] Following the methods of scheme E7 and Examples 7A-7B, the following compounds were synthesized using suitable starting materials. Standard protection and deprotection methods were used when necessary.

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] Example 8. Synthesis of compounds E8-v, E8-vi and E8-viii

[0360] Solution E8

[0361]

[0362] Compound E8-i can be converted to intermediate E8-ii via alkylation reactions similar to those in Examples E7-iii to E7-vi, or via a Mitsunobu reaction (see route (i) in scheme E8). E8-ii is then oxidized with mCPBA or potassium persulfate to yield compounds E8-v and / or E8-vi. Alternatively, compounds E8-i can be converted to E8-v and E8-vi via prior oxidation followed by alkylation or a Mitsunobu reaction (see route (ii) in scheme E8). Where X a It is a leaving group (e.g., Cl, Br, I, OMs, OTs); compounds E8-v and / or E8-vi can be converted to intermediate E8-vii by nucleophilic aromatic substitution reaction with compounds E8-xi using bases such as LiHMDS or t-BuOK. Compound E8-viii can be synthesized from compound E8-vii using SmI2 or Zn in AcOH or TES in AlCl3. As used herein, Ar1 and Ar2 are each independently an optionally substituted 5- or 6-membered heteroaryl group.

[0363] Example 8A. Synthesis of 2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and 6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazol-3-carbonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0364]

[0365] Step AN-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]dodec-1(8),2(6),4,11-tetraen-10-yl}methyl)pyridin-2-yl]carbamate tert-butyl ester under N2 at 70°C, 4-methyl-2-(methanesulfonyl)-4H-thiazo[5',4' A mixture of [4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (7.5 g, 26.4 mmol) and K3PO4 (8.3 g, 39.3 mmol) in anhydrous MeCN (300 mL) was stirred for 1 hour, followed by the addition of a MeCN (30 mL) solution of N-[(tert-butoxy)carbonyl]-N-[6-(bromomethyl)pyridin-2-yl]carbamate tert-butyl ester (11.2 g, 29.0 mmol). After stirring at 70 °C under N2 for 2.5 hours, the reaction mixture was quenched with saturated NH4Cl and extracted with EA (300 mL x 3). The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, and the organic phase was concentrated. The crude product was purified by rapid chromatography (silica gel, petroleum ether solution of 0–50% ethyl acetate) to give N-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]dodec-1(8),2(6),4,11-tetraen-10-yl}methyl)pyridin-2-yl]carbamate (5.5 g). LC-MS (ESI) result: 591.1 (M+H) + .

[0366] Step B. (6-((4-methyl-5-oxo-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate. At -40°C under argon, LiHMDS (50 mL, 1 M THF solution) was added to a mixture of 3-((benzenesulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (11.9 g, 33.8 mmol) in anhydrous THF (200 mL) under stirring. After 10 min, the mixture was heated to 10 °C and stirred for 1 hour, followed by the addition of N-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]dodec-1(8),2(6),4,11-tetraen-10-yl}methyl)pyridin-2-yl]carbamate (9.1 g, 15.4 mmol, in 35 mL THF). The reaction was stirred at 10 °C for another 30 min. The reaction mixture was poured into an aqueous solution of NH4Cl and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by rapid chromatography (silica gel, petroleum ether solution of 0–50% ethyl acetate) to give 6.6 g of (6-((4-methyl-5-oxo-2-((benzenesulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate. LC-MS (ESI) result: 763.2 (M+H) + .

[0367] Step C. (6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate) Ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate tert-butyl ester (6.0 g, 7.86 mmol) in EtOH / AcOH (35 mL / 50 mL) solution was heated to 50 °C and vigorously stirred for 40 min in the presence of Zn (2.55 g, 117.9 mmol). Additional zinc (2.55 g, twice, was added every 40 min, with the reaction monitored by TLC / LC-MS to avoid byproducts and over-reduction products). The solution was filtered, and the filter cake was washed with DCM. The filtrate was partially evaporated, neutralized with saturated NaHCO3 solution, dried over MgSO4, and the solvent was removed under vacuum. The crude product was purified by rapid chromatography (silica gel, DCM:MeOH = 40:1) to give tert-butyl 6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate (3.1 g). LC-MS (ESI) result: 623.3 (M+H). + .

[0368] Step D. 2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one was added to a mixture of tert-butyl carbamate (3.0 g, 4.8 mmol) in ethanol (30 mL) and HCl (30 mL, 4 M dioxane solution). The reaction mixture was stirred at 80 °C for 40 min. The reaction mixture was cooled to room temperature, filtered, and the solid was collected, suspended in water, and neutralized with an aqueous solution of NaHCO3 at 10°C. Filtering yielded the desired compound 2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (1.5 g). LC-MS (ESI) result: 393.2 (M+H) + . 1 HNMR (400MHz, DMSO-d6) δ12.78(s,1H), 8.53(s,1H), 7.72(s,1H), 7.25(dd,1H), 6.33 -6.24(m,2H), 6.08(d,1H), 5.90(s,2H), 5.19(s,2H), 4.49(s,2H), 4.26(s,3H).

[0369] Step E. Synthesis of (6-((4-methyl-5-oxo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-6(5H)-yl)methyl)pyridin-2-yl)tert-butyl carbamate to (6-((4-methyl-5-oxo-2-((1-(( K₂CO₃ (88 mg, 0.64 mmol) was added to a DMF (2 mL) solution of 2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate (100 mg, 0.16 mmol). The mixture was stirred at 70 °C for 8 hours. The mixture was poured into water, filtered, and the precipitate was collected and purified by preparative TLC (2% MeOH in DCM solution) to give tert-butyl 6-((4-methyl-5-oxo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate (20 mg). LC-MS (ESI): m / z 637 (M+H) + .

[0370] Step F. Synthesis of 6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. HCl (1 mL, 4 mol / L in dioxane) was added to a solution of tert-butyl carbamate (20 mg, 0.03 mmol) in EtOH (1 mL). The mixture was stirred at 80 °C for 1 hour and then cooled. The precipitate was collected by filtration, neutralized with saturated NaHCO3, washed with water, and dried to give 5 mg of 6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 407 (M+H) + . 1H NMR (400MHz, DMSO-d6) δ: 8.75(s,1H), 7.96(s,1H), 7.50(s,1H), 7.31-7.2 2(m,1H), 6.31(d,1H), 6.14(d,1H), 5.91(s,2H), 5.23(s,2H), 4.38(s,3H).

[0371] Example 8B. Synthesis of 6-((1H-pyrazol-3-yl)methyl)-2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0372]

[0373] Step A. (6-(dimethylamino)pyridin-2-yl)methanol. Pd(OAc)₂ (78 mg, 0.35 mmol), Xantphos (4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene)) (170 mg, 0.29 mmol), and t-BuONa (385 mg, 4.01 mmol) were added to a solution of (6-chloropyridin-2-yl)methanol (500 mg, 2.67 mmol) in THF solution of dimethylamine (35 mL). The reaction mixture was stirred at 100 °C for 18 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, 0–35% ethyl acetate in petroleum ether) to give (6-(dimethylamino)pyridin-2-yl)methanol (180 mg). LCMS: 153 (M+H) + .

[0374] Step B. 6-(chloromethyl)-N,N-dimethylpyridin-2-amine. At 0°C, SOCl2 (665 mg, 5.6 mmol) was added to a mixture of (6-(dimethylamino)pyridin-2-yl)methanol (170 mg, 1.1 mmol) in DCM (10 mL) under stirring. The reaction mixture was stirred at room temperature for 1 hour. The pH of the reaction mixture was adjusted to 7–8 with aqueous NaHCO3 solution. The mixture was then extracted with DCM and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give 6-(chloromethyl)-N,N-dimethylpyridin-2-amine (70 mg). LCMS: 171 (M+H) + .

[0375] Step 1: CN,N-Dimethyl-6-((benzenesulfonyl)methyl)pyridine-2-amine. At room temperature, 1.44 g (8.82 mmol) of PhSO₂Na was added to a mixture of 6-(chloromethyl)-N,N-dimethylpyridine-2-amine (500 mg, 2.94 mmol) in DMSO (10 mL) under stirring. The mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into water and extracted with DCM. The mixture was washed with water and the organic layer was concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, 0–20% ethyl acetate in petroleum ether) to give N,N-dimethyl-6-((benzenesulfonyl)methyl)pyridine-2-amine (380 mg). LCMS: 277 (M+H) + .

[0376] Step D.2-((6-(dimethylamino)pyridin-2-yl)(benzenesulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Under N2 and at 60°C, N,N-dimethyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (180 mg, 0.36 mmol) (whose synthesis is similar to compound E8-1 in Example 8A) was added to a mixture of 4-methyl-6-((benzenesulfonyl)methyl)pyridin-2-amine (120 mg, 0.44 mmol) and t-BuOK (122 mg, 1.1 mmol) in dry THF (10 mL) under stirring. The mixture was stirred under N2 at 60°C for 2 hours. The mixture was then poured into water, extracted with EtOAc, and washed with water and brine. The organic layer was dried over Na₂SO₄, concentrated under reduced pressure, and purified by preparative TLC (PE:EtOAc = 1:1.5) to obtain 2-((6-(dimethylamino)pyridin-2-yl)(benzenesulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg). LCMS: 691 (M+H) + .

[0377] Step E.2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. At room temperature and under N2, SmI2 (5 mL, 0.1 M THF solution) at -40 °C was added to a mixture of 2-((6-(dimethylamino)pyridin-2-yl)(benzenesulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg, 0.07 mmol) in THF (5 mL) and MeOH (5 mL). The reaction mixture was stirred at -40 °C for 10 min, followed by quenching with water. The subsequent mixture was extracted twice with EtOAc. The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EtOAc = 1 / 1.5) to obtain the desired product (10 mg). LCMS: m / z 551 (M+H) + .

[0378] Step F. 6-((1H-pyrazol-3-yl)methyl)-2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. A mixture of 2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (10 mg, 0.018 mmol) in DCM / TFA (2 mL / 2 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated. The residue was purified by preparative HPLC to give the desired product (1.4 mg). LCMS:421(M+H) + . 1 H NMR (400MHz, DMSO) δ8.51(s,1H), 7.55(s,1H), 7.48(dd,1H), 6.64(d,1H), 6. 54(d,1H), 6.11(d,1H), 5.32(s,2H), 4.44(s,2H), 4.26(s,3H), 3.05(s,6H).

[0379] The following compounds were synthesized using suitable starting materials according to scheme E8 and example 8C. Standard protection and deprotection may be used when necessary.

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397] Example 9. Synthesis of compounds E9-vi and E9-vii

[0398] Solution E9

[0399]

[0400] Compound E9-iv can be synthesized via two methods (i) and (ii) of Scheme 9. For method (i), compound E9-ii can be synthesized from compound E9-i via an alkylation reaction as shown in Example 7 or Example 8. As used herein, X aIt is a leaving group. The formylation reaction of compound E9-ii with LiHMDS and DMF provides intermediate E9-iii. E9-iii reacts with a reducing agent (e.g., NaBH4) to provide compound E9-iv. Alternatively, in method (ii), halogenation of compound E9-i yields compound E9-ix. Compound E9-ix undergoes a Stille reaction, ozone decomposition, and reduction to modify compound E9-x. Compound E9-x can be alkylated with E9-viii to provide compound E9-iv. In scheme 9(iii), compound E9-iv undergoes halogenation to give intermediate E9-v(X). b It is a halogen, such as Cl or Br). E9-v is provided by catalytic coupling with a metal (e.g., Pd or Cu) of organotin, boron, zinc or magnesium. As used herein, M is an organometallic complex (e.g., an organoboron complex, such as boric acid or pinacolboron complex; an organotin complex, such as -SN(Bu t )3; organozinc complexes, such as -Zn (halogen); compound E9-v can also react with some nucleophiles (such as nitrogen in the heterocycle) to give product E9-vii. As used herein, Ar1 and Ar2 are each independently an optionally substituted 5- or 6-membered heteroaryl group, X a It is a leaving group (e.g., Br, I, OMs, or OTs); and X b It is halogen.

[0401] Example 9A. Synthesis of 6-((1H-indazol-4-yl)methyl)-4-methyl-2-(thiazol-4-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0402]

[0403] Step A. Synthesis of 4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde. At -78°C, LiHMDS (1M, 11.14mL, 2.0 equivalents) was added to a mixture of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (2.6 g, 5.57 mmol, 1 equivalent) in dry THF (30 mL). The mixture was stirred at -78°C for 2 hours. DMF (2.04 g, 27.86 mmol, 2.14 mL, 5.0 equivalents) was then added dropwise to the mixture. The mixture was stirred at -78 °C for 2 hours. TLC (PE:EA = 2:1, UV = 254 nm) showed the formation of a new major spot. The mixture was poured into cold saturated NH4Cl (20 mL). The mixture was then warmed to room temperature. The mixture was extracted with EtOAc (40 mL x 3). The organic layer was washed with water (20 mL x 3) and concentrated under vacuum to give the desired product (2.6 g, crude). LCMS: m / z 495.2 [M+H] +

[0404] Step B. Synthesis of 2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. NaBH4 (152.97 mg, 4.04 mmol, 2 equivalents) was added to a mixture of crude 4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde (1.0 g, 2.02 mmol, 1 equivalent) in THF (10 mL) and MeOH (10 mL). The mixture was stirred at 30°C for 14 hours. TLC (DCM:MeOH = 10:1, UV = 254 nm) showed complete consumption of the starting material and the formation of a new major spot. The reaction was quenched by the addition of water (20 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL). The organic phase was concentrated under vacuum. The residue was analyzed by rapid silica gel chromatography (…). 40g Purification was performed using a silica gel rapid column chromatography with 0–5% MeOH / DCM eluent at 30 mL / min. The eluent was then concentrated under vacuum to obtain the desired product (382 mg). LCMS: m / z 497.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δppm 8.61(s,1H), 8.25(s,1H), 7.66(d,1H), 7.38(t,1H), 7.05(d,1H), 6.36(t,1H), 5.74(s ,2H), 5.68(s,2H), 4.89(d,2H), 4.26(s,3H), 3.50(t,2H), 0.78(t,2H), -0.12(s,9H).

[0405] Step C. Synthesize 2-(chloromethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. 4-Methylbenzenesulfonyl chloride (75.0 mg, 393.40 μmol, 1.30 equivalent) was added to a mixture of 2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-5(6H)-one (150.0 mg, 302.02 μmol, 1 equivalent) and Et3N (61.12 mg, 604.04 μmol, 84.08 μL, 2.0 equivalent) in DCM (5 mL). The mixture was stirred at 30 °C for 5 hours. TLC (PE:EA = 4:1, UV = 254 nm) showed complete consumption of the starting materials. Water (10 mL) and DCM (20 mL) were added to the mixture. The organic layer was concentrated under vacuum to obtain a yellow gel-like substance (0.1 g). The residue was subjected to rapid silica gel chromatography (HPLC). 4g Purification was performed using a silica gel rapid column chromatography with a petroleum ether gradient solution of 0–20% ethyl acetate as eluent at 30 mL / min. The desired fractions were concentrated under vacuum to obtain the desired product (40.0 mg, 76.88 μmol). LCMS: m / z 515.1 [M+H] + . 1¹H NMR (400MHz, chloroform-d) δppm 8.27 (d, 1H), 8.26 (s, 1H), 7.52 (d, 1H), 7.39 (dd, 1H), 7.25 (s, 1H), 5.76 (s, 2H), 5.72 (s, 2H), 4.96 (s, 2H), 4.40 (s, 3H), 3.50–3.57 (m, 2H), 0.84–0.90 (m, 2H), -0.09 to -0.06 (m, 9H).

[0406] Step D. Synthesis of 4-methyl-2-(thiazolyl-4-ylmethyl)-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. Pd(PPh3)4 (402 mg, 2.91 mmol) was added to a toluene (4 mL) solution of 2-(chloromethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg, 0.97 mmol) and 4-(tributylsilyl)thiazole (114 mg, 2.91 mmol). The mixture was then heated in a MW reactor at 120 °C for 30 min under N2. The solution was poured into water, extracted with EtOAc, and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure. The residue was purified by rapid chromatography (silica gel, petroleum ether solution of 0–50% ethyl acetate) to give 4-methyl-2-(thiazol-4-ylmethyl)-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (30 mg). LCMS: 564 (M+H) + . 、

[0407] Step E. Synthesis of 6-((1H-indazol-4-yl)methyl)-4-methyl-2-(thiazol-4-ylmethyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. At room temperature and under N2, TFA (3 mL) was added to a mixture of compound E9-4 (30 mg, 0.05 mmol) in DCM (3 mL). The reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (C18, 0–90% acetonitrile in H2O solution containing 0.1% formic acid) to give the desired product (3.9 mg). LCMS: 434 (M+H) + . 1H NMR (400MHz, DMSO) δ13.12(s,1H), 9.12(d,1H), 8.56(s,1H), 8.14(s,1H), 7.71(d,1H), 7.45(d,1H), 7.35 -7.24(m,1H), 6.96(d,1H), 5.65(s,2H), 4.70(s,2H), 4.27(s,3H).

[0408] Example 9B. Synthesis of 4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0409]

[0410] Step A. Synthesis of 2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one at -78 °C: LiHMDS (4.3 mL, 1 M THF solution) was added to a mixture of 4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (640 mg, 2.15 mmol) in THF (10 mL). After 30 min, dry DMF (0.84 mL, 10.8 mmol) was added to the mixture. After the starting material was completely consumed, NaBH4 (164 mg, 4.3 mmol) was added to a mixture of EtOH (4 mL), and the mixture was stirred for 5 min. The mixture was then poured into saturated NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by rapid chromatography (silica gel, 0–5% MeOH in DCM solution) to give 2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (220 mg). LC-MS (ESI): m / z 328 (M+H) + .

[0411] Step B. Synthesis of 2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one Et3N (0.43 mL, 3.1 mmol) and MsCl (0.12 mL, 1.5 mmol) were added to a mixture of 2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (100 mg, 0.31 mmol) in DCM (5 mL). The reaction was stirred at room temperature for 6 hours. The mixture was then washed with saturated NH4Cl (aqueous solution), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by rapid chromatography (silica gel, PE solution of 0–50% EtOAc) to give 65 mg of 2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 346 (M+H) + .

[0412] Step C. Synthesis of 4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one: Under nitrogen atmosphere, Pd(PPh3)4 (17 mg, 0.014 mmol) was added to a mixture of 2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (50 mg, 0.14 mmol) and 2-(tributyltinyl)pyridine (0.14 mL, 0.43 mmol) in toluene (3 mL). The reaction mixture was stirred overnight at 100 °C. The mixture was then cooled and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: 10% MeOH in DCM solution) to give 2 mg of 4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 389 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ 8.56 (m, 3H), 8.48 (dd, 1H), 7.81 (td, 1H), 7.71 (d, 1H), 7.50 (d, 1H), 7.37 -7.30(m,2H), 5.38(s,2H), 4.67(s,2H), 4.25(s,3H).

[0413] Example 9C. Synthesis of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and 6-((1H-pyrazol-3-yl)methyl)-2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0414]

[0415] Step A. Synthesis of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxaldehyde. At -40 °C, 2-(chloromethoxy)ethyl-trimethylsilane (26.03 g, 156.11 mmol, 27.63 mL, 1.5 equivalent) was added dropwise to a suspension of 1H-pyrazole-3-carboxaldehyde (10.0 g, 104.07 mmol, 1 equivalent) and DIPEA (33.63 g, 260.18 mmol, 45.32 mL, 2.5 equivalent) in DCM (500 mL). The reaction mixture was then warmed to room temperature and stirred for 16 hours. TLC (petroleum ether:EtOAc = 5:1) showed complete consumption of the starting materials and the formation of two new spots. The reaction mixture was concentrated under vacuum. The residue was combined with two other batches (10.0 g each) and purified by Combiflash (a petroleum ether solution of 100% petroleum ether to 40% EtOAc) to give 60.0 g of the desired product. (Note: a mixture of two positional isomers in a ratio of approximately 5 / 4). 1 H NMR (400MHz, chloroform-d) δppm 10.06(s,1H), 10.00(s,1H), 7.68(d,1H), 7.67(d,1H), 7.03(d,1H), 6.92(d,1H), 5. 87(s,2H), 5.56(s,2H), 3.61-3.67(m,4H), 0.91-1.01(m,4H), -0.09-0.05(m,18H).

[0416] Step B. Synthesis of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methanol. NaBH4 (7.52 g, 198.81 mmol, 1.50 equivalent) was added fractionally to a solution of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxaldehyde (30 g, 132.54 mmol, 1 equivalent, a mixture of two positional isomers in a ratio of approximately 5 / 4) in 200 mL THF / 100 mL MeOH at 0 °C. The reaction mixture was stirred at 0 °C to room temperature for 18 hours. TLC (petroleum ether: EtOAc = 2:1) showed complete consumption of the starting material and the formation of two new spots. The solvent was concentrated under vacuum. The residue was purified by combiflash (100% petroleum ether to 100% EtOAc) to give 25 g of the desired product. (Note: This is a mixture of two positional isomers in a ratio of approximately 3 / 2). 1 H NMR (400MHz, chloroform-d) δppm7.55(d,1H), 7.47(brs,1H), 6.36(d,1H), 6.34(d,1H), 5.57(s,2H) , 5.42(s,2H), 4.74-4.76(m,4H), 3.55-3.60(m,4H), 0.85-0.96(m,4H), 0.00-0.06(m,18H).

[0417] Step C. Synthesis of 3-(bromomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole. At 0 °C, CBr4 (46.76 g, 141.00 mmol, 1.4 equivalent) was added to a solution of (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-yl)methanol (23 g, 100.72 mmol, 1 equivalent, a mixture of the two positional isomers in a ratio of approximately 3 / 2) and PPh3 (36.98 g, 141.00 mmol, 1.4 equivalent) in 200 mL of DCM. The reaction mixture was stirred at 0 °C for 3 hours. TLC (petroleum ether:EtOAc = 5:1) showed complete consumption of the starting material and the formation of a new spot. The reaction mixture was concentrated under vacuum. The residue was combined with another batch (2.0 g) and purified by Combiflash (a petroleum ether solution of 100% petroleum ether to 50% EtOAc) to give 22.0 g (75.53 mmol) of the desired product. 1 ¹H NMR (400MHz, chloroform-d) δppm 7.51 (d, 1H), 6.39 (d, 1H), 5.38 (s, 2H), 4.50 (s, 2H), 3.52–3.57 (m, 2H), 0.86–0.96 (m, 2H), -0.03–0.02 (m, 9H).

[0418] Step D. Synthesis of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. 4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (1.0 g, 4.85 mmol, 1 A suspension of 3-(bromomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (2.12 g, 7.27 mmol, 1.5 equivalents), K3PO4 (2.57 g, 12.12 mmol, 2.5 equivalents), and NaI (218.05 mg, 1.45 mmol, 0.3 equivalents) in DMF (15 mL) was stirred at 60 °C under N2 for 18 hours. TLC (petroleum ether: EtOAc = 1:1) showed complete consumption of the starting materials and the formation of a new spot. The reaction mixture was combined with three other batches (1.0 g each) and poured into ice water (250 mL). The mixture was extracted with EtOAc (150 mL x 3). The combined organic layers were washed with water (120 mL x 2) and brine (120 mL) and dried over Na2SO4. The solvent was concentrated under vacuum. The crude product was purified by Combiflash (a petroleum ether solution of 100% petroleum ether to 80% EtOA) to give the desired product (3.6 g). 1 ¹H NMR (400MHz, chloroform-d) δppm 8.91 (s, 1H), 8.27 (s, 1H), 7.49 (d, 1H), 6.36 (d, 1H), 5.51 (s, 2H), 5.40 (m, 2H), 4.45 (s, 3H), 3.52–3.58 (m, 2H), 0.85–0.90 (m, 2H), -0.05 (s, 9H).

[0419] Step E. Synthesis of 4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde. Under argon atmosphere and at -78°C, LiHMDS (1.0 M, 8.18 mL, 2 equivalents) was slowly added to a THF (30 mL) solution of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (1.7 g, 4.09 mmol, 1 equivalent), and the reaction mixture was stirred at -70°C for 1 hour. A solution of DMF (1.49 g, 20.45 mmol, 1.57 mL, 5 equivalents) in THF (3 mL) was then added dropwise to the mixture. The resulting mixture was stirred at -70 °C for 1 hour. TLC (petroleum ether: EtOAc = 1:1) showed the formation of a new spot. The reaction mixture was added dropwise to an aqueous solution of NH4Cl (50 mL) at 0 °C, followed by extraction with EtOAc (30 mL x 3). The combined organic layers were washed with brine (40 mL) and dried over Na2SO4. The solvent was removed under vacuum to give the desired crude product (1.8 g), which was used for the next step without further purification.

[0420] Step F. Synthesize 2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. At 0 °C, NaBH4 (245.08 mg, 6.48 mmol, 2 equivalents) was added to a solution of 4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-2-carboxaldehyde (1.8 g, 3.24 mmol, 1 equivalent) in THF (20 mL) / MeOH (10 mL), and the reaction mixture was stirred at room temperature for 18 hours. TLC (petroleum ether:EtOAc = 1:2) showed complete consumption of the starting material and the formation of a new spot. The reaction mixture was concentrated under vacuum, and the residue was purified by Combiflash (from 100% DCM to a DCM solution of 5% MeOH). The desired product (1.1 g) was obtained. 1H NMR (400MHz, DMSO-d6) δppm8.63(s,1H), 7.86(d,1H), 6.44(t,1H), 6.27(d,1H), 5.40-5. 42(m,4H), 4.98(d,2H), 4.34(s,3H), 3.55-3.61(m,2H), 0.86-0.91(m,2H), 0.00(s,9H).

[0421] Step G. Synthesis of (4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-yl)methylmethanesulfonate. At 0 °C, MsCl (269.32 mg, 2.35 mmol, 181.97 μL, 1.5 equivalent) was added dropwise to a solution of 2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-5(6H)-one (700 mg, 1.57 mmol, 1 equivalent) and Et3N (317.21 mg, 3.13 mmol, 436.33 μL, 2.0 equivalent) in DCM (15 mL). The reaction mixture was stirred at room temperature for 1 hour. TLC (petroleum ether:EtOAc = 1:1) showed complete consumption of the starting material and the formation of a new spot. The reaction mixture was diluted with EtOAc (80 mL), washed with water (30 mL x 4) and brine (40 mL), and dried over Na2SO4. The solvent was removed under vacuum to give the crude product (700 mg). LCMS: (m / z) 525.5 (M+H).

[0422] Step H. Synthesize 2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and 2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. At 60 °C and under N2, a mixture of (4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-yl)methylmethanesulfonate (150 mg, 285.88 μmol, 1 equivalent), 1H-1,2,4-triazole (197.45 mg, 2.86 mmol, 10 equivalents) and CsF (86.85 mg, 571.77 μmol, 21.08 μL, 2 equivalents) in MeCN (8 mL) was stirred for 18 hours. LCMS showed complete consumption of the starting material and the formation of two new peaks. The reaction mixture was concentrated under vacuum, and the residue was purified by Combiflash (a DCM solution of 100% DCM to 8% MeOH). The products 2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (55 mg) and 2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (30 mg) were obtained.

[0423] Step I. Synthesize 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. H₂O (1.99 mg, 110.52 μmol, 1 equivalent) was added to a suspension of 2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (55 mg, 110.52 μmol, 1 equivalent) and HCl / dioxane (4 M, 1 mL, 36.19 equivalents) in DCM (3 mL). The reaction mixture was stirred at room temperature for 18 hours. LCMS showed complete consumption of the starting material and 84% of the desired product was found. The reaction mixture was concentrated under vacuum, and the residue was purified by preparative HPLC to obtain the desired product (24.1 mg, 65.60 μmol, 59.35% yield). Column: Ultimate C18 150*25 mm*5 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 13%-43%, 11.2 min. LCMS: m / z 367.9 (M+H). +1 HNMR (400MHz, methanol-d4) δppm 8.72 (s, 1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.52 (brs, 1H), 6.26 (d, 1H), 5.92 (s, 2H), 5.43 (s, 2H), 4.30 (s, 3H).

[0424] Step J. Synthesis of 6-((1H-pyrazol-3-yl)methyl)-2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. H₂O (50.00 mg, 2.78 mmol, 0.05 mL, 46.04 equivalences) was added to a suspension of 2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-5(6H)-one (30 mg, 60.28 μmol, 1 equivalent) and HCl / dioxane (4 M, 1 mL, 66.35 equivalents) in DCM (3 mL). The reaction mixture was stirred at room temperature for 18 hours. LCMS showed complete consumption of the starting material and identification of the desired product. The reaction mixture was concentrated under vacuum, and the residue was purified by preparative HPLC to give the desired product (2.1 mg, 5.72 μmol). Column: Ultimate C18 150*25mm*5um; Mobile phase: [Water (0.225% FA)-ACN]; B%: 13%-43%, 11.2min. LCMS: m / z 368.0 (M+H) + . 1 ¹H NMR (400MHz, methanol-d⁴) δppm 8.74 (s, 2H), 8.38 (s, 1H), 7.52 (d, 1H), 6.26 (d, 1H), 5.84 (s, 2H), 5.44 (s, 2H), 4.31 (s, 3H).

[0425] Following the methods of scheme E9 and Examples 9A-9B, the following compounds were synthesized using suitable starting materials. Standard protection and deprotection were performed where necessary.

[0426]

[0427]

[0428]

[0429] Example 9D Synthesis of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0430]

[0431] Step A. Synthesis of 2-chloro-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LiHMDS (30.5 mL) was added dropwise to a mixture of 3 g (14.5 mmol) of 4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one in 80 mL of dry THF at -65 °C. After stirring for 1 hour, a solution of hexachloroethane (1.8 mL, 16 mmol) in 20 mL of dry THF was added. The reaction mixture was heated to -20 °C over 3 hours. The mixture was then quenched with saturated NH4Cl and stirred at room temperature for 20 min. The precipitate was collected by filtration and washed with EtOAc to give 3.5 g of 2-chloro-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 241 (M+H) + .

[0432] Step B. Synthesis of 4-methyl-2-vinyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Under nitrogen atmosphere, Pd(PPh3)4 (0.36 g, 0.31 mmol) was added to a mixture of 2-chloro-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (1.5 g, 6.2 mmol) and tributyl(vinyl)stanane (5.5 mL, 18.7 mmol) in DMF (30 mL). The reaction mixture was stirred at 100 °C for 2 hours. The mixture was then cooled and diluted with EtOAc, washed with water and brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by rapid chromatography (silica gel, 0–10% MeOH in DCM solution) to give 1.4 g of 4-methyl-2-vinyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 233 (M+H) + .

[0433] Step C. Synthesis of 4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde. A mixture of 500 mg (2.15 mmol) of 4-methyl-2-vinyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one in DCM / MeCN (500 mL, 1:1 volume) was purged with O3 for 20 min at -60 °C. The reaction was then quenched with dimethylsolfane and concentrated to give 500 mg of 4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde. LC-MS (ESI): m / z 235 (M+H) + .

[0434] Step D. Synthesis of 2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. At 0 °C, NaBH4 (81 mg, 2.13 mmol) was added to a mixture of 4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carboxaldehyde (500 mg, 2.13 mmol) in EtOH (3 mL). The reaction was stirred at room temperature for 5 min. The mixture was then poured into saturated NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by rapid chromatography (silica gel, 0–8% MeOH in DCM solution) to give 120 mg of 2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 237 (M+H) + .

[0435] Step E. Synthesis of 6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. K₂CO₃ (351 mg, 2.54 mmol) was added to a mixture of 2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (200 mg, 0.85 mmol) in DMF (8 mL). After stirring at 60 °C for 30 min, a DMF solution of 2-chloro-4-(chloromethyl)pyrimidine (276 mg, 1.7 mmol) was added (2 mL). The reaction mixture was stirred for another 4 hours, poured into saturated NH₄Cl, and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by rapid chromatography (silica gel, 0–8% MeOH in DCM solution) to give 160 mg of 6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 363 (M+H) + .

[0436] Step F. Synthesis of 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Bis(2,4-dimethoxybenzyl)amine (280 mg, 0.88 mmol) and AcOH (1 drop) were added to a mixture of 6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (160 mg, 0.44 mmol) in MeCN (5 mL). The reaction mixture was stirred overnight at 80 °C. The reaction mixture was evaporated, and the residue was purified by preparative TLC (eluent: 5% MeOH in DCM solution) to give 75 mg of 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 644 (M+H) + .

[0437] Step G. Synthesis of 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. At 0 °C, Et3N (0.16 mL, 1.16 mmol) and MsCl (0.05 mL, 0.58 mmol) were added to a 5 mL solution of 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (75 mg, 0.12 mmol) in DCM. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM, washed with saturated NH4Cl and brine, dried over anhydrous Na2SO4, and concentrated to give 70 mg of crude 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 662 (M+H) + .

[0438] Step H. Synthesis of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-5(6H)-one: A mixture of 4H-1,2,4-triazole (39 mg, 0.57 mmol) and K2CO3 (78 mg, 0.57 mmol) in DMF (3 mL) was stirred at 60 °C for 30 min. Add 75 mg (0.11 mmol) of 6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and stir for another 30 min. Pour the suspension into saturated NH4Cl and extract with EtOAc. Wash the organic layer with brine, dry over anhydrous Na2SO4 and concentrate. The residue was purified by rapid chromatography (silica gel, 0–10% DCM in MeOH solution) to give 60 mg of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 695 (M+H) + .

[0439] Step G. Synthesis of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one: HCl (0.5 mL, 4 M dioxane solution) was added to a mixture of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (30 mg, 0.043 mmol) in EtOH (2 mL). The reaction mixture was stirred overnight at 80 °C. The mixture was then cooled, poured into saturated NaHCO3, and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by preparative HPLC to give 8 mg of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. LC-MS (ESI): m / z 395 (M+H) + . 1 HNMR (400MHz, DMSO-d6) δ8.80(s,1H), 8.62(s,1H), 8.12-8.10(m,2H), 6.60(s,2H), 6.19(d,1H), 6.01(s,2H), 5.19(s,2H), 4.26(s,3H).

[0440]

[0441] Example 10. Synthesis of compound E10-ii

[0442] Solution E10

[0443]

[0444] The reaction of E10-i with LiHMDS and an aldehyde yields compound E10-ii, which can be separated by chiral HPLC or SFC to give two enantiomers. As used herein, Ar1 and Ar2 are each independently an optionally substituted 5- or 6-membered monocyclic heteroaryl group.

[0445] Example 10A. Synthesis of (R)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and (S)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

[0446]

[0447] Step A. Synthesize 2-(hydroxy(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. Under argon atmosphere, LiHMDS (1.0 M, 2.41 mL, 2 equivalents) was slowly added to a THF (10 mL) solution of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (0.5 g, 1.20 mmol, 1 equivalent) and the reaction mixture was stirred at -70 °C for 1 h. Subsequently, a THF (1 mL) solution of 1-(2-trimethylsilylethoxymethyl)pyrazol-3-carboxaldehyde (816.97 mg, 3.61 mmol, 3 equivalents) was added to the reaction mixture. The resulting mixture was stirred at -70 °C for 1 h. TLC (petroleum ether:EtOAc = 1:1) showed the formation of two new spots. The reaction mixture was quenched at -70°C with 5 mL of aqueous NH₄Cl solution, followed by warming to room temperature. The mixture was diluted with 10 mL of water and extracted with EtOAc (8 mL x 3). The combined organic layers were washed with 10 mL of brine and dried over Na₂SO₄. The solvent was concentrated under vacuum. The residue was purified by combiflash (from 100% petroleum ether to 100% EtOAc) to give a crude product (130 mg) of a light brown gel, which was used for the next step without further purification. LCMS: m / z 643.2 [M+H] +

[0448] Step B. Synthesize 6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. TFA (4.62 g, 40.52 mmol, 3 mL, 200.38 equivalents) was added to a solution of 2-(hydroxy(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-5(6H)-one (0.13 g, 202.20 μmol, 1 equivalent) in DCM (8 mL), followed by the addition of H2O (500.00 mg, 27.75 mmol, 0.5 mL, 137.26 equivalents). The reaction mixture was then stirred at room temperature for 18 h, followed by heating to 40 °C and maintaining the temperature for 18 h. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated under vacuum, and the residue was purified by preparative HPLC to obtain the desired product (20.5 mg). LCMS: m / z 382.9 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δppm 12.72 (brs, 1H), 12.61 (brs, 1H), 8.52 (s, 1H), 7.56-7.62 (m, 2H), 6.82 (b rs, 1H), 6.18 (d, 1H), 5.99-6.09 (m, 2H), 5.24-5.32 (m, 2H), 4.19 (s, 3H).

[0449] Step C. Synthesize (R)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one and (S)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one. The compound 6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one was separated by SFC. SFC conditions: column was DAICELCHIRALCEL OJ-H (250mm*30mm, 5um); mobile phase: A: 55% CO2; B: 45% [0.1% NH3H2O ​​in EtOH solution] / min. SFC isolated 2-[(R)-hydroxy(1H-pyrazol-3-yl)methyl]-4-methyl-6-(1H-pyrazol-3-ylmethyl)thiazo[3,4]pyrrolo[1,3-d]pyridazin-5-one and 2-[(S)-hydroxy(1H-pyrazol-3-yl)methyl]-4-methyl-6-(1H-pyrazol-3-ylmethyl)thiazo[3,4]pyrrolo[1,3-d]pyridazin-5-one. One isomer (4.4 mg): LCMS: m / z 383 [M+H] + . 1 ¹H NMR (400MHz, methanol-d⁴) δppm 8.39 (s, 1H), 7.61 (brs, 1H), 7.55 (brs, 1H), 6.34 (brs, 1H), 6.25 (brs, 1H), 6.18 (brs, 1H), 5.44 (s, 2H), 4.29 (s, 3H). Another isomer (4.1 mg): LCMS: m / z 383 (M+H) + . 1 H NMR (400MHz, methanol-d4) δppm 8.40 (s, 1H), 7.59 (brs, 1H), 7.54 (brs, 1H), 6.34 (brs, 1H), 6.26 (brs, 1H), 6.19 (brs, 1H), 5.44 (s, 2H), 4.29 (s, 3H).

[0450] Example 10B: Synthesis of 6-((1H-pyrazol-3-yl)methyl)-2-(difluoro(1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0451]

[0452] Step A. 3-(6-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazol-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazol-1-sulfonamide. MnO2 (72.85 mg, 838.00 μmol) was added to a mixture of 3-((2-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazole-3-yl)(hydroxy)methyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (50 mg, 83.80 μmol, similar to E10-1 preparation) in DCM (1.5 mL), and the mixture was stirred at 15 °C for 1.5 h. The reaction mixture was filtered and concentrated under reduced pressure to give crude product (60 mg, crude). LCMS: m / z 595.1 (M+H) + . 1 H NMR (400MHz, CDCl3) δ8.34(s,1H), 8.12(d,1H), 7.90(d,1H), 7.41(d,1H), 6.38(d,1H), 5.54(s,2H), 4.49(s,3H), 3.10(s,6H)2.93(s,6H).

[0453] Step B. 3-((2-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazol-3-yl)difluoromethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazol-1-sulfonamide. BAST (1.34 g, 6.05 mmol, 1.33 mL) was added to a DCE (4 mL) solution of 3-(6-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazole-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (240 mg, 403.60 μmol), and the mixture was stirred at 50 °C for 12 h. The reaction mixture was diluted with dichloromethane (20 mL), washed with saturated NaHCO3 (10 mL * 2), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a crude product (300 mg, crude product), which was used for the next step without further purification. LCMS: m / z 617.1 (M+H)+ .

[0454] Step C. 6-((1H-pyrazol-3-yl)methyl)-2-(difluoro(1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. TFA (2.07 g, 18.15 mmol, 1.34 mL) was added to a mixture of 3-((2-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazol-3-yl)difluoromethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazol-1-sulfonamide (240 mg, 163.47 μmol) in DCM (2 mL). The mixture was heated to 50 °C and maintained for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Agela ASB 150*25mm*5um; mobile phase: [water (0.05% HCl)-ACN]; B%: 30%-60%, 8 min) to obtain the desired product (3.9 mg, 5.45% yield, 92% purity), which was a white solid. LCMS: m / z 403.1 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.67(s,1H), 7.95(s,1H), 7.61(s,1H), 6.72(s,1H), 6.16(s,1H), 5.36(s,2H), 4.28(s,3H).

[0455] Example 10C: Synthesis of 2-(1-(1H-pyrazol-3-yl)ethyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0456]

[0457] Step A: 3-((2-(1-(1-(N,N-dimethylaminosulfonyl)-1H-pyrazol-3-yl)-1-hydroxyethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazol-1-sulfonamide. To a solution of 3-(6-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazole-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (160 mg, 269.07 μmol) in THF (3 mL), CH3MgBr (3 M, 179.38 μL) was added, and the reaction mixture was stirred at 0 °C for 3 hours. The reaction mixture was poured into saturated NH4Cl (10 mL) at 0 °C and extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was analyzed by rapid silica gel chromatography (…). 12g Purification was performed using a silica gel fast column with an eluent gradient of 0–90% ethyl acetate / petroleum ether at 40 mL / min to obtain the desired product (50 mg, 81.87 μmol). LCMS: m / z 611.1 (M+H) + .

[0458] Step B: 2-(1-(1H-pyrazol-3-yl)ethyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one. At 0 °C, Et3SiH (19.04 mg, 163.75 μmol, 26.15 μL) was added to a mixture of 3-((2-(1-(1-(N,N-dimethylaminosulfonyl)-1H-pyrazole-3-yl)-1-hydroxyethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (50 mg, 81.87 μmol) in DCE (0.5 mL), followed by the addition of TFA (1.54 g, 13.51 mmol, 1 mL, 164.96 equivalents). The mixture was stirred at 0 °C for 1 hour. The mixture was then warmed to 50 °C and maintained for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Agela ASB 150*25mm*5um; mobile phase: [water (0.05% HCl)-ACN]; B%: 25%-55%, 7 min) to obtain 7.0 mg of the desired product. LCMS: m / z 381.2 (M+H) + . 1 H NMR (400MHz, CD3OD) δ 8.46 (s, 1H), 8.15 (d, 2H), 6.78 (d, 1H), 6.74 (d, 1H), 5.60 (s, 2H), 5.00 (q, 1H), 4.33 (s, 3H), 1.94 (d, 3H).

[0459] Example 10D: Synthesis of 6-((1H-pyrazol-3-yl)methyl)-4-methyl-2-(1H-pyrazol-3-carbonyl)-4,6-dihydro-5H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-5-one

[0460]

[0461] Step A. 4-Methyl-2-(1H-pyrazole-3-carbonyl)-6-(1H-pyrazole-3-ylmethyl)thiazo[3,4]pyrrolo[1,3-d]pyridazin-5-one. TFA (2.31 g, 20.26 mmol, 1.5 mL) was added to a solution of 3-(6-((1-(N,N-dimethylaminosulfonyl)-1H-pyrazole-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazo[5',4':4,5]pyrrolo[2,3-d]pyridazin-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide (50 mg, 84.08 μmol) in DCE (1.5 mL). The reaction mixture was heated to 50 °C and maintained for 12 hours. The reaction mixture was concentrated under vacuum. The residue was purified by preparative HPLC to yield 6.0 mg of the desired product. LCMS: m / z 381.1 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ8.72(s,1H), 7.96(d,1H), 7.64(d,1H), 7.49(d,1H), 6.19(d,1H), 5.37(s,2H), 4.38(s,3H).

[0462] Example 11. Detection of PKR mutants

[0463] method :

[0464] Dilute the PKR or PKR mutant enzyme solution to the reaction mixture containing 1x buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) and PEP (concentration depends on the enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT, and 0.03% BSA; the final detection concentration is shown after a 1.11-fold dilution.

[0465] First, add 2 μL of the test compound to the well, followed by 180 μL of the reaction mixture.

[0466] Collect the reaction mixture containing the test compound, excluding ADP, and store the plate at room temperature for 60 minutes.

[0467] 20 μL of ADP was added at room temperature to initiate the reaction, and the reaction progress was measured by the change in absorbance at a wavelength of 340 nm at room temperature.

[0468] Preparation of test compounds:

[0469] Prepare a 100x concentration stock solution (10 mM) of the test compound in 100% DMSO.

[0470] For the 11 points, 1 to 3-fold dilutions were performed (i.e., adding 50 μl of the first concentration to 100 μl of 100% DMSO yielded 3.33 mM, adding 50 μl of the same concentration to 100 μl of DMSO yielded 1.11 mM, and so on).

[0471] • Dilute 1 to 100 times in the detection solution (2 μl in 200 μl) to obtain an initial concentration of 100 μM. For 11 sites, reduce by 3 times.

[0472] Detection buffer : 100mM KCl, 50mM Tris 7.5, 5mM MgCl2, 1mM DTT, 0.03% BSA

[0473] reaction mixture PKR mutant enzyme: 40-400 ng / well; ADP: 0.2-1.65 mM; PEP: 0.1-0.5 mM; NADH: 180 μM; LDH: 0.5 units (Sigma#59023); DTT: 1 mM; BSA: 0.03%.

[0474] Example 12. Detection of single-point activation percentage of wild-type PKR

[0475] The compound described herein was diluted with DMSO and tested at a concentration of 1 μM. The enzyme was diluted in an enzyme solution containing 1x buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) and PEP (concentration depending on the enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT, and 0.03% BSA; the final detection concentration after a 1.11-fold dilution is shown.

[0476] First, add 2 μL of the compound solution to each well, followed by 180 μL of the enzyme solution. Collect the assay analytes except for ADP and store the plate at room temperature for 60 minutes. Add 20 μL of ADP to initiate the assay, and evaluate the assay output using OD340. This assay was performed at room temperature.

[0477] Final concentrations: PKR wild type (100 ng / well), Tris pH 7.5 (50 mM), KCl (100 mM), MgCl2 (5 mM), ADP (0.48 mM), PEP (0.15 mM), NADH (180 μM), LDH (0.5 units, Sigma 59023), DTT (1 mM) and BSA (0.03%).

[0478] Example 13. PKR R510Q Single-Point Activation Percentage Detection

[0479] The compound described herein was diluted with DMSO and detected at a concentration of 1 μM. The enzyme was diluted in an enzyme solution containing 1x buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) and PEP (concentration depending on the enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT, and 0.03% BSA; the final detection concentration after a 1.11-fold dilution is shown.

[0480] First, add 2 μL of the compound solution to each well, followed by 180 μL of the enzyme solution. Collect the assay analytes except for ADP and store the plate at room temperature for 60 minutes. Add 20 μL of ADP to initiate the assay, and evaluate the assay output using OD340. This assay was performed at room temperature.

[0481] Final concentrations: PKR R510Q (40 ng / well), Tris pH 7.5 (50 mM), KCl (100 mM), MgCl2 (5 mM), ADP (0.2 mM), PEP (0.11 mM), NADH (180 μM), LDH (0.5 units, Sigma 59023), DTT (1 mM), and BSA (0.03%).

[0482] Example 14. PKR R532W Single-Point Activation Percentage Detection

[0483] The compound described herein was diluted with DMSO and tested at a concentration of 1 μM. The enzyme was diluted with an enzyme solution containing 1x buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) and PEP (concentration depending on the enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT, and 0.03% BSA; the final detection concentration after a 1.11-fold dilution is shown.

[0484] First, add 2 μL of the compound solution to each well, followed by 180 μL of the enzyme solution. Collect the assay analytes except for ADP and store the plate at room temperature for 60 minutes. Add 20 μL of ADP to initiate the assay, and evaluate the assay output using OD340. This assay was performed at room temperature.

[0485] Final concentrations: PKR R532W (100 ng / well), Tris pH 7.5 (50 mM), KCl (100 mM), MgCl2 (5 mM), ADP (0.36 mM), PEP (0.1 mM), NADH (180 μM), LDH (0.5 units, Sigma 59023), DTT (1 mM), and BSA (0.03%).

[0486] Example 15. PKR T384M Single-Point Activation Percentage Detection

[0487] The compound described herein was diluted with DMSO and tested at a concentration of 1 μM. The enzyme was diluted in an enzyme solution containing 1x buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) and PEP (concentration depending on the enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT, and 0.03% BSA; the final detection concentration after a 1.11-fold dilution is shown.

[0488] First, add 2 μL of the compound solution to each well, followed by 180 μL of the enzyme solution. Collect the assay analytes except for ADP and store the plate at room temperature for 60 minutes. Add 20 μL of ADP to initiate the assay, and evaluate the assay output using OD340. This assay was performed at room temperature.

[0489] Final concentrations: soluble PKR T384M (300 ng / well), Tris pH 7.5 (50 mM), KCl (100 mM), MgCl2 (5 mM), ADP (0.08 mM), PEP (0.23 mM), NADH (180 μM), LDH (0.5 units, Sigma 59023), DTT (1 mM), and BSA (0.03%).

[0490] Example 16. Red blood cell (RBC) purification

[0491] Collect fresh blood from healthy volunteers into a K2EDTA tube. Precipitate the whole blood by rotating at 500g for 10 minutes. Cut the infusion bag port of the Purecell leukocyte removal neofilter (Fisher NC0267633) one (1) inch above the filter. Connect a 10 ml syringe barrel to the remaining cut tube connected to the neofilter. Remove the plasma layer from the whole blood pellet and resuspend the pellet in 2x volumes of phosphate-buffered saline (PBS). Transfer 9 ml of the resuspended blood pellet to a prepared 10 ml syringe connected to the neofilter. Allow the whole blood to flow through the filter under gravity until all fluid has passed through the upper tube into the filter disc. Add a plunger to the syringe, remove the syringe from the clamp, and invert the filter, then inject air into the syringe filter system. Using a new 5 ml syringe, remove the filtered RBCs from the bag through the syringe port and transfer the purified RBCs to a 5 ml snap-cap tube that has been incubated on ice. Incubate the 5ml snap-capped tube at 15℃ with 500g for 10 minutes, aspirate the supernatant, and then... (The sentence is incomplete and requires more context to translate accurately). 9Cells / mL were resuspended in AGAM (1x PBS, 1% glucose, 170 mg / L adenine, 5.25 g / L mannitol).

[0492] Example 17. Cell-based ATP detection

[0493] For cell-based ATP assays, a 10 mM stock solution of the compound described herein was prepared in 100% DMSO. Serial dilutions (1:4) were performed in 96-well V-bottom storage plates, followed by a 1:100 addition to 96-well V-bottom plates containing AGAM. RBCs were diluted in AGAM medium to a density of 1 x 10⁻⁶. 7 Cells / mL, followed by 90 μL / well added to a black transparent-bottomed assay plate (final compound concentration: 0.1% DMSO). The assay plate was sealed with aluminum foil and incubated overnight at 37°C in a humidified chamber. ATP levels were read using a Cell-Titer-Glo (Promega) assay.

[0494] Therefore, several aspects of several embodiments have been described, and it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the invention. Therefore, the foregoing description and figures are merely illustrative.

[0495] This application relates to the following implementation scheme:

[0496] 1. Compounds represented by the following structural formulas or their pharmaceutically acceptable salts:

[0497]

[0498] in:

[0499] R 1 It is hydrogen, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, -OR o1 -C(=O)R c1 Or nitrogen protecting group; wherein:

[0500] R o1 It is a hydrogen, optionally substituted alkyl, or oxygen protecting group;

[0501] R c1 It is an substituted alkyl group or -N(R) cn )2, where each R cn Independently hydrogen, -C 1-6 Alkyl or nitrogen protecting groups;

[0502] R2 Q and Q are each independently optional substituted 5- or 6-membered monocyclic heteroaryl groups;

[0503] R a and R b Each of these can be independently hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, or -OR. o3 -N(R) n1 )2、-C(=O)N(R n1 )2 or -C(=O)R c2 Or alternatively, R a and R b They can combine with the carbon atoms to which they are attached to form optionally substituted cycloalkyl or optionally substituted heterocyclic groups; wherein:

[0504] Each R n1 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0505] R o3 It is hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group; and

[0506] R c2 It is an optionally substituted -C1-C6 alkyl group; and

[0507] R j and R k Each is independently hydrogen, halogen, -CN, -OR o7 -N(R) n5 )2、-N(R n5 )C(=O)R c5 -C(=O)N(R) n5 )2、-C(=O)R c5 -C(=O)OR o7 -SR js -S(=O)2R js -S(=O)R js Or optionally substituted -C1-C6 alkyl; or optionally R j and R k They can form C=O, optionally substituted C1-C6 monocyclic cycloalkyl rings, or optionally substituted C3-C6 monocyclic heterocyclic rings together with the carbon atoms they are attached to; wherein:

[0508] Each R n5 Independently hydrogen, optionally substituted -C1-C6 alkyl, -OR o8 Or nitrogen-protecting group, wherein R o8 It is hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0509] Each Ro7 Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0510] Each R c5 Independently, it is an optionally substituted -C1-C6 alkyl group; and

[0511] Each R js Independently, it is an optionally substituted -C1-C6 alkyl group, or an optionally substituted C... 6-12 Aryl, optionally substituted heteroaryl, or sulfur-protected groups.

[0512] 2. The compound or a pharmaceutically acceptable salt thereof according to embodiment 1, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is optionally replaced by R at each substituted ring carbon atom. p Substitution, and optionally R at each substituted cyclic nitrogen atom. n6 Replace; of which:

[0513] Each R p Independently, it is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heteroaryl, -OR o6 -SR s2 -N(R) n3 )2、-C(=O)N(R n3 )2、-N(R n3 )C(=O)R c4 -C(=O)R c4 -C(=O)OR o6 -OC(=O)R c4 -S(=O)R s2 -S(=O)2R s2 -S(=O)OR o6 -OS(=O)R c4 -S(=O)2OR o6 -OS(=O)2R c4 -S(=O)N(R) n3 )2、-S(=O)2N(R n3 )2、-N(R n3 )S(=O)R s2 -N(R) n3 )S(=O)2R s2 -N(R) n3 )C(=O)OR o6 -OC(=O)N(R) n3 )2、-N(R n3)C(=O)N(R n3 )2、-N(R n3 )S(=O)N(R n3 )2、-N(R n3 )S(=O)2N(R n3 )2、-N(R n3 )S(=O)OR o6 -N(R) n3 )S(=O)2OR o6 -OS(=O)N(R) n3 )2 or -OS(=O)2N(R n3 )2; or alternatively, two R atoms can be attached to the same or adjacent carbon atoms. p They can combine with the carbon atoms to which they are attached to form optionally substituted cycloalkyl or heterocycloalkyl groups; wherein:

[0514] Each R n3 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0515] Each R o6 Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting groups; and

[0516] Each R c4 It is an optionally substituted -C1-C6 alkyl group;

[0517] Each R s2 Independently, it is an optionally substituted -C1-C6 alkyl or sulfur protecting group; and

[0518] R n6 It is hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group.

[0519] 3. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 and 2, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0520]

[0521]

[0522] in:

[0523] Each R nc and R nd Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen protecting group; and

[0524] When the valence state allows, p can be 0, 1, 2, 3, or 4.

[0525] 4. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-3, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0526]

[0527] 5. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-4, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following:

[0528]

[0529] 6. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein the compound is represented by the following structural formula:

[0530]

[0531] Where q is 0, 1, 2 or 3.

[0532] 7. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein the compound is represented by the following structural formula:

[0533]

[0534] Where q is 0, 1, 2 or 3.

[0535] 8. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein the compound is represented by the following structural formula:

[0536]

[0537] Where q is 0, 1, 2 or 3.

[0538] 9. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-5, wherein the compound is represented by the following structural formula:

[0539]

[0540] Where q is 0, 1, 2 or 3.

[0541] 10. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 2-9, wherein R n6 Is it hydrogen or -C? 1-4 alkyl.

[0542] 11. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 2-7, wherein each R p Independently hydrogen, halogen, or optionally substituted C 1-4 Alkyl, -CN, -NO2, -N3, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2、-C(=O)R c3 or -C(=O)OR o4 .

[0543] 12. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-11, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0544]

[0545] in:

[0546] Each R n Independently, it is hydrogen, halogen, -CN, -NO2, -N3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic, optionally substituted heteroaryl, -OR o4 -SR s1 -N(R) n2 )2、-C(=O)N(R n2 )2、-N(R n2 )C(=O)R c3 -C(=O)R c3 -C(=O)OR o4 -OC(=O)R c3 -S(=O)R s1 -S(=O)2R s1 -S(=O)OR o4 -OS(=O)R c3 -S(=O)2OR o4 -OS(=O)2R c3 -S(=O)N(R) n2 )2、-S(=O)2N(R n2 )2、-N(R n2 )S(=O)R s1 -N(R) n2 )S(=O)2R s1 -N(R) n2 )C(=O)OR o4 -OC(=O)N(R) n2 )2、-N(R n2)C(=O)N(R n2 )2、-N(R n2 )S(=O)N(R n2 )2、-N(R n2 )S(=O)2N(R n2 )2、-N(R n2 )S(=O)OR o4 -N(R) n2 )S(=O)2OR o4 -OS(=O)N(R) n2 )2 or -OS(=O)2N(R n2 )2; or two R atoms connected to the same or adjacent carbon atoms n Together with the carbon atoms to which they are attached, they form optionally substituted cycloalkyl or heterocycloalkyl groups; wherein:

[0547] Each R n2 Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen-protecting group;

[0548] Each R o4 Independently hydrogen, optionally substituted -C1-C6 alkyl or oxygen protecting group;

[0549] Each R c3 Independently, it is an optionally substituted -C1-C6 alkyl group;

[0550] Each R s1 Independently, it is an optional substituted -C1-C6 alkyl or sulfur protecting group;

[0551] When the valence state allows, n is 0, 1, 2, or 3; and

[0552] R na R nb and R nd Each is independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen protecting group.

[0553] 13. The compound according to any one of embodiments 1-12, or a pharmaceutically acceptable salt thereof, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0554]

[0555] 14. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-13, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following:

[0556]

[0557] 15. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-14, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is

[0558] 16. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-14, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is

[0559] 17. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-15, wherein R na Is it hydrogen or -C? 1-4 alkyl.

[0560] 18. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-17, wherein each R n Independently hydrogen, halogen, or optionally substituted C 1-4 Alkyl, -CN, -NO2, -N3, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2、-C(=O)R c3 or -C(=O)OR o4 .

[0561] 19. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-18, wherein R 1 It is hydrogen or -C1-C4 alkyl.

[0562] 20. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-19, wherein R 1 It is a methyl group.

[0563] 21. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-20, wherein R j and R k Each is independently hydrogen, halogen, -OR o7 or -C1-C4 alkyl, or R j and R k They combine to form =O.

[0564] 22. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-21, wherein R j and R k Each is hydrogen.

[0565] 23. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein R a and Rb Each is hydrogen.

[0566] 24. The compound according to any one of embodiments 5-23, wherein q is 0 or 1.

[0567] 25. The compound according to any one of embodiments 12-24, wherein n is 0 or 1.

[0568] 26. A pharmaceutical composition comprising an effective amount of a compound as described in any one of embodiments 1-25 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0569] 27. A method for increasing the lifespan of red blood cells (RBCs), comprising contacting the red blood cells with an effective amount of (1) a compound according to any one of embodiments 1-25 or a pharmaceutically acceptable salt thereof; or (2) a pharmaceutical composition according to embodiment 26.

[0570] 28. The method according to embodiment 27, wherein the compound or pharmaceutical composition is added directly in vitro to whole blood containing red blood cells or to hematocrit cells containing red blood cells.

[0571] 29. The method according to embodiment 28, wherein the compound or pharmaceutical composition is administered to a subject containing red blood cells.

[0572] 30. A method for regulating the level of 2,3-diphosphoglycerate in the blood, comprising contacting the blood with an effective amount of (1) the compound according to any one of embodiments 1-25 or a pharmaceutically acceptable salt thereof; or (2) the pharmaceutical composition according to embodiment 26.

[0573] 31. A method for treating anemia in a subject, comprising administering to the subject an effective amount of (1) a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) a pharmaceutical composition according to embodiment 26.

[0574] 32. The method according to implementation scheme 31, wherein the anemia is erythropoiesis disorder anemia.

[0575] 33. A method for treating hemolytic anemia in a subject, comprising administering to the subject an effective amount of (1) a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) a pharmaceutical composition according to embodiment 26.

[0576] 34. The method according to embodiment 33, wherein the hemolytic anemia is hereditary and / or congenital hemolytic anemia, acquired hemolytic anemia, or anemia as part of a multisystem disease.

[0577] 35. A method of treating sickle cell disease in a subject, comprising administering to the subject an effective amount of (1) a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) a pharmaceutical composition according to embodiment 26.

[0578] 36. A method of treating a subject with pyruvate kinase deficiency (PKD), comprising administering to the subject an effective amount of (1) a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) a pharmaceutical composition according to embodiment 26.

[0579] 37. A method of treating a subject for thalassemia, hereditary spherocytosis, hereditary ellipocytosis, abeta-lipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia or anemia of chronic disease, comprising administering to the subject an effective amount of (1) the compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) the pharmaceutical composition according to embodiment 26.

[0580] 38. A method for treating a subject with thalassemia, comprising administering to the subject an effective amount of (1) a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) a pharmaceutical composition according to embodiment 26.

[0581] 39. The method according to embodiment 38, wherein the thalassemia is β-thalassemia.

[0582] 40. A method for activating mutant pyruvate kinase R (PKR) in erythrocytes in a subject with such need, comprising administering to the subject an effective amount of (1) the compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) the pharmaceutical composition according to embodiment 26.

[0583] 41. A method for activating wild-type pyruvate kinase R (PKR) in erythrocytes in a subject with such need, comprising administering to the subject an effective amount of (1) the compound according to any one of embodiments 1-25 or a pharmaceutically acceptable salt thereof; or (2) the pharmaceutical composition according to embodiment 26.

[0584] 42. A method for increasing hemoglobin levels in subjects in need, comprising administering to the subject an effective amount of (1) the compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-25; or (2) the pharmaceutical composition according to embodiment 26.

Claims

1. Compounds represented by the following structural formulas or their pharmaceutically acceptable salts: (I) in: R 1 It is a -C1-C6 alkyl group; R 2 yes ； Q is: ； When the valence state allows, p is 0, 1, 2, 3, or 4; Each R p Independently hydrogen, halogen, optionally substituted C1-C4 alkyl, -CN, -NO2, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2 or -C(=O)OR o4 ; Each R nc and R nd Independently hydrogen, optionally substituted -C1-C6 alkyl or nitrogen protecting group; and When the valence state allows, n is 0, 1, 2, or 3; R n Independently hydrogen, halogen, or optionally substituted C 1- C4 alkyl, -CN, -OR o4 -N(R) n2 )2、-C(=O)N(R n2 )2 or -C(=O)OR o4 ; Each R n2 Independently hydrogen or optionally substituted -C1-C6 alkyl; Each R o4 Independently hydrogen or optionally substituted -C1-C6 alkyl; R na It is hydrogen or optionally substituted -C1-C6 alkyl; R a and R b Each is hydrogen R j and R k Each is independently hydrogen, halogen, -OR o7 or -C1-C6 alkyl; or alternatively R j and R k They can combine with the carbon atoms they are attached to to form C=O; R o7 It is hydrogen or -C1-C6 alkyl; Unless otherwise stated, the optional substitution of C 1- C4 or C1-C6 alkyl groups are optionally substituted with one or more halogens or hydroxyl groups.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 2 The 5- or 6-membered monocyclic heteroaryl group represented is selected from one of the following: 。 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: (II) Where q is 0, 1, 2 or 3.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: (III) Where q is 0, 1, 2 or 3, and R n6 It is hydrogen or C1-C4 alkyl.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: (IV) Where q is 0, 1, 2 or 3, and R n6 It is hydrogen or C1-C4 alkyl.

6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: (V) Where q is 0, 1, 2 or 3.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the 5- or 6-membered monocyclic heteroaryl group represented by Q is selected from the following: 。 8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Q represents a 5- or 6-membered monocyclic heteroaryl group. or 。 9. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R na Is it hydrogen or -C? 1- C4 alkyl.

10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is a methyl group.

11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R j and R k Each is hydrogen.

12. The compound according to any one of claims 1-11, wherein q is 0 or 1.

13. The compound according to any one of claims 1-11, wherein n is 0 or 1.

14. A pharmaceutical composition comprising an effective amount of the compound according to any one of claims 1-11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

15. The compound according to claim 1, wherein the compound is selected from the group consisting of: 。 16. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 17. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 18. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 19. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 20. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 21. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 22. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 23. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 24. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 25. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 26. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 27. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 28. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 29. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 30. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 31. The compound of claim 15 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 32. A pharmaceutical composition comprising an effective amount of any one of claims 1-11 or 15-31 of the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

33. Use of a composition comprising any one of the compounds according to claims 1-11 or 15-31 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of pyruvate kinase deficiency (PKD).

34. Use of a composition comprising any one of the compounds of claims 1-11 or 15-31 in the preparation of a medicament for treating sickle cell disease.

35. Use of a composition comprising any one of the compounds of claims 1-11 or 15-31 in the preparation of a medicament for treating hemolytic anemia.

36. Use of a composition comprising any one of the compounds of claims 1-11 or 15-31 in the preparation of a medicament for the treatment of thalassemia.

37. The use according to claim 36, wherein the thalassemia is α-thalassemia or β-thalassemia.

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