CN116802182A

CN116802182A - Pyrazolopyridine derivatives and uses thereof

Info

Publication number: CN116802182A
Application number: CN202280013553.3A
Authority: CN
Inventors: S·博纳齐; A·塞尔尼延科; J·S·科布; N·A·戴尔斯; J·杜赫斯特; M·J·赫西; R·贾殷; J·R·克里根; H·A·马利克; J·R·曼宁; G·奥布赖恩; A·W·帕特森; N·M-F·汤姆森; P·Y·汀
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2021-03-15
Filing date: 2022-03-14
Publication date: 2023-09-22

Abstract

The present disclosure relates to compounds and pharmaceutical compositions having formula (I) and their use in reducing widely spaced zinc finger motif (WIZ) expression levels, or inducing fetal hemoglobin (HbF) expression, and in treating hereditary blood diseases (e.g., hemoglobinopathies, e.g., β -hemoglobinopathies), e.g., sickle cell disease and β -thalassemia.

Description

Pyrazolopyridine derivatives and uses thereof

Priority claim

The present application claims priority from U.S. provisional application number 63/161139 filed on day 3 and 15 of 2021 and U.S. provisional application number 63/164130 filed on day 3 and 22 of 2021, the disclosures of which are incorporated herein by reference in their entireties.

Technical Field

The present disclosure relates to pyrazolopyridine derivative compounds and pharmaceutical compositions and their use in reducing widely spaced zinc finger motif (WIZ) protein expression levels and/or inducing fetal hemoglobin (HbF) protein expression levels, and in treating hereditary blood diseases (hemoglobinopathies, e.g., β -hemoglobinopathies), e.g., sickle cell disease and β -thalassemia.

Background

Sickle Cell Disease (SCD) is a serious group of hereditary blood diseases that cause red blood cells to twist into sickle shape. These cells can cause blockage of blood flow, resulting in severe pain, organ damage, and premature death. Beta thalassemia is a group of hereditary blood diseases caused by a decrease or deficiency in beta globulin synthesis, thereby causing anemia.

Fetal hemoglobin (HbF) induction is known to ameliorate symptoms in patients with SCD and beta-thalassemia and has been clinically validated genetically (single nucleotide polymorphisms in the globulin control locus and BCL 11A) and pharmacologically (hydroxyurea) (Vinjamur, D.S. et al (2018), the British Journal of Haematology [ J.UK hematopathy ],180 (5), 630-643). Hydroxyurea is the current standard of care for SCD and is believed to provide benefits via induction of HbF, but is genotoxic, causing dose-limiting neutropenia, and a response rate of less than 40%. Other mechanisms of clinical and preclinical targeting include inhibition of HDAC1/2 (Shearstone et al, 2016, PLoS One [ public science library. Complex ],11 (4), e 0153767), LSD1 (river et al, 2018,Experimental Hematology [ experimental hematology ],67,60-64), DNMT1, PDE9a (McArthur et al, 2019, haemoalogic [ hematology ] doi: 10.3324/haemato.2018.213462), HRI kinase (Grevat et al, 2018, science [ science ],361 (6399), 285-290) and G9a/GLP (Krvega et al, 2015, blood [ blood ],126 (5), 665-672; renneville et al, blood [ blood ],126 (16), 1930-1939). In addition, the immunomodulators pomalidomide and lenalidomide induce HbF in human primary erythroid cells ex vivo (Moutouh-de Parseval, L.A. et al (2008), the Journal of Clinical Investigation [ journal of clinical investigation ],118 (1), 248-258) and in vivo (Meiler, S.E. et al (2011), blood [ Blood ],118 (4), 1109-1112). WIZ is widely expressed and plays a role in targeting G9a/GLP histone methyltransferases to genomic loci to regulate chromatin structure and transcription (Bian, chen et al (2015), eLife 2015; 4:e05606).

Disclosure of Invention

The present disclosure relates to therapeutic agents effective in reducing the level of WIZ protein expression and/or inducing fetal hemoglobin (HbF) expression. The present disclosure further relates to pyrazolopyridine compounds, pharmaceutically acceptable salts thereof, compositions thereof, and their use in the treatment of conditions and purposes detailed above that are effective in reducing the level of WIZ protein expression and/or inducing fetal hemoglobin (HbF) expression.

In a first aspect, the present disclosure provides a compound having formula (I "), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

R ^x selected from hydrogen, C ₁ -C ₆ Alkyl, halo (e.g. F, cl), C ₁ -C ₆ Alkoxy and C ₃ -C ₈ Cycloalkyl;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

each R ² Independently selected from C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl, halo and oxo, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution; or 2R on non-adjacent carbon atoms ² Forms a bridged ring with the non-adjacent carbon atoms to which they are attached;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ³ selected from hydrogen, C ₁ -C ₈ Alkyl, CN ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ 、C ₁ -C ₆ Haloalkyl, -C (=o) -O- (R) ⁵ )、-C(＝O)-(R ⁶ )、C ₃ -C ₁₀ Cycloalkyl and 4-to 10-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O and S, wherein the C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl groups are each independently R from 0 to 3 occurrences ^3a Substitution, and wherein said C ₃ -C ₁₀ Cycloalkyl and 4-to 10-membered heterocyclyl are each independently substituted with 0-3 occurrences of R ^3b Substitution;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atom to which it is attached, form a 5-or 6-membered heterocyclic group comprising 0-1 additional heteroatoms selected from N, O and S, wherein the 5-or 6-membered heterocyclic group is substituted with 0-2 occurrences of oxo;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 10-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, 5-to 10-membered heteroaryl containing 1-4 heteroatoms selected independently from N, O and S, C ₆ -C ₁₀ Aryl, C ₁ -C ₆ Alkoxy, hydroxy and-C (=o) -NR ⁷ R ⁸ Wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 10-membered heteroaryl and C ₆ -C ₁₀ R in which the aryl radical is present from 0 to 4 times ^3b Substitution;

each R ^3b Independently selected from C ₁ -C ₆ Alkoxy, halo, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Alkyl, -CN, -SO ₂ NR ⁷ R ⁸ 、-SO ₂ R ⁴ And hydroxyl;

R ⁴ selected from C ₃ -C ₈ Cycloalkyl, C ₁ -C ₆ Alkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, C ₆ -C ₁₀ Aryl and-NR ^4b R ^4c Wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^4a Substitution;

R ^4a selected from C ₃ -C ₈ Cycloalkyl, C ₆ -C ₁₀ Aryl and C ₁ -C ₆ An alkoxy group;

R ^4b selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^4c selected from hydrogen, C ₁ -C ₆ Alkyl and C ₃ -C ₈ Cycloalkyl;

R ⁵ selected from C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl and C ₆ -C ₁₀ An aryl group;

R ⁶ selected from C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl, C ₆ -C ₁₀ Aryl, 4-to 10-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O and S, and-NR ^4b R ^4c Wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^6a Substitution, said C ₃ -C ₈ Cycloalkyl is substituted with 0-1 occurrences of R ^6b Substituted and the 4-to 10-membered heterocyclic group is C-substituted with 0-1 occurrences ₁ -C ₆ Alkyl substitution;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ^6b selected from halogenated, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy and C ₁ -C ₆ An alkyl group;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

R ⁷ And R is ⁸ Together with the nitrogen atom to which they are attached, form a 5-or 6-membered heterocyclyl containing 0 to 1 additional heteroatoms selected from N, O and S;

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

In a further aspect, the present disclosure provides a compound having formula (I'), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

Is a single bond or a double bond;

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ³ selected from hydrogen, C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ 、C ₁ -C ₆ Haloalkyl, -C (=o) -O- (R) ⁵ ) and-C (=o) - (R ⁶ ) Wherein said C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl is independently substituted with 0-3 occurrences of R ^3a Substitution;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atom to which it is attached, form a 5-or 6-membered heterocyclyl comprising 0-1 additional heteroatoms selected from N, O and S;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, a 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, a cycloalkyl containing 1-4 heteroatoms selected independently from5-to 10-membered heteroaryl of heteroatoms of N, O and S, C ₆ -C ₁₀ Aryl, C ₁ -C ₆ Alkoxy, hydroxy and-C (=o) -NR ⁷ R ⁸ Wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 10-membered heteroaryl and C ₆ -C ₁₀ R in which the aryl radical is present from 0 to 4 times ^3b Substitution;

R ⁴ selected from C ₃ -C ₈ Cycloalkyl, C ₁ -C ₆ Alkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O and S, and C ₆ -C ₁₀ Aryl, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^4a Substitution;

R ⁶ selected from C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl and C ₆ -C ₁₀ Aryl, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^6a Substitution, and the C ₃ -C ₈ Cycloalkyl is substituted with 0-1 occurrences of R ^6b Substitution;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

In a further aspect, the present disclosure provides a compound having formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

x is selected from CH, CF and N;

R' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, 5-to 10-membered heteroaryl containing 1-4 heteroatoms selected independently from N, O and S, C ₆ -C ₁₀ Aryl, C ₁ -C ₆ Alkoxy, hydroxy and-C (=o) -NR ⁷ R ⁸ Wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 10-membered heteroaryl and C ₆ -C ₁₀ R in which the aryl radical is present from 0 to 4 times ^3b Substitution;

R ⁶ selected from C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl and C ₆ -C ₁₀ Aryl, wherein said C ₁ -C ₆ Alkyl quiltR of 0-1 occurrence ^6a Substitution, and the C ₃ -C ₈ Cycloalkyl is substituted with 0-1 occurrences of R ^6b Substitution;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

In a further aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier or excipient.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use as a medicament.

In a further aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of treating or preventing a disorder affected by reduced or modulated levels of WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of inhibiting expression of a WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of degrading a WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein, comprising administering to the subject a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of inducing or promoting fetal hemoglobin in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of reactivating fetal hemoglobin production or expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of increasing fetal hemoglobin expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of treating a hemoglobinopathy, e.g., β -hemoglobinopathy, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of treating sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a method of treating β -thalassemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder selected from sickle cell disease and β -thalassemia.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease or disorder affected by reduced levels of WIZ protein.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease or disorder affected by inhibition or reduction of WIZ protein expression.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease or disorder affected by WIZ protein degradation.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inducing or promoting fetal hemoglobin.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in reactivating fetal hemoglobin production or expression.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in increasing fetal hemoglobin expression.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating hemoglobinopathy.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating sickle cell disease.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating β -thalassemia.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by increased expression of fetal hemoglobin.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by inhibition, reduction, or elimination of WIZ protein activity or WIZ protein expression.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by induction or promotion of fetal hemoglobin.

In a further aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by reactivating fetal hemoglobin production or expression.

Various other aspects of the disclosure are described herein and in the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification and claims, the singular form also includes the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. The references cited herein are not to be considered prior art to the claimed disclosure. In case of conflict, the present specification, including definitions, will control. In addition, these materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the compounds, compositions, and methods disclosed herein will be apparent from the following detailed description and claims.

Drawings

FIG. 1A depicts a volcanic plot of differentially expressed genes from WIZ KO cells compared to a scrambled gRNA control. Each dot represents a gene. The HBG1/2 gene is differentially up-regulated from WIZ_6 and WIZ_18 gRNA targeting WIZ KO.

FIG. 1B depicts a bar graph showing the frequency of HbF+ cells due to shRNA-mediated loss of WIZ in human mobilized peripheral blood CD34+ derived erythroid cells.

Fig. 1C depicts a bar graph showing the frequency of hbf+ cells due to CRISPR/Cas9 mediated deletion of WIZ in human mobilized peripheral blood cd34+ derived erythroid cells.

Detailed Description

The compounds disclosed herein are effective in reducing WIZ protein expression levels, or inducing fetal hemoglobin (HbF) expression. Without wishing to be bound by any theory, it is believed that the disclosed compounds may treat hematological disorders, such as hereditary hematological disorders, such as sickle cell disease and β -thalassemia, by inducing fetal hemoglobin HbF expression.

Definition of the definition

The term "compounds of the present disclosure (compounds of the present disclosure, compounds of the disclosure or compound of the disclosure)" refers to compounds, exemplary compounds, salts (particularly pharmaceutically acceptable salts), hydrates, solvates, prodrugs thereof, and all stereoisomers (including diastereomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties, having the formula (I "), (I '), (I), (Ia"), (Ia '), (Ib), (Ic "), (Ic '), (Ic), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), and (Ie).

Groups (groups) defined belowp, radial) or moiety, the number of carbon atoms is usually indicated before the group, e.g. C ₁ -C ₈ Alkyl means an alkyl group having 1 to 8 carbon atoms. In general, for a group comprising two or more subunits, the last mentioned group is a group attachment point, e.g. "alkylaryl" means a monovalent group of the formula alkyl-aryl-, and "arylalkyl" means a monovalent group of the formula aryl-alkyl-.

Furthermore, the use of terms representing monovalent groups wherein divalent groups are appropriate should be understood to mean the corresponding divalent groups and vice versa. Unless otherwise indicated, the conventional definition of the term control and the conventional valency of the stabilizing atom are assumed, and are embodied in all formulae and groups. The articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "and/or" means "and" or "unless indicated otherwise.

The term "substituted" means that a particular group or moiety bears one or more suitable substituents, where the substituents may be attached to the particular group or moiety at one or more positions. For example, an aryl substituted with a cycloalkyl group may indicate that the cycloalkyl group is attached to one atom of the aryl group through a bond or is fused to the aryl group and shares two or more common atoms.

As used herein, the term "C ₁ -C ₈ Alkyl "refers to a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, which is free of unsaturation, has one to eight carbon atoms, and is attached to the remainder of the molecule by a single bond. The term "C ₁ -C ₃ Alkyl "," C ₁ -C ₄ Alkyl "," C ₁ -C ₆ Alkyl "should be construed accordingly. C (C) ₁ -C ₈ Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl (iso-butyl or i-butyl)), 1-dimethylethylPhenyl (tert-butyl), n-pentyl, 3-pentyl, n-hexyl, n-heptyl, 4-heptyl, n-octyl, 2-isopropyl-3-methylbutyl.

As used herein, the term "C _1- C ₆ Alkoxy "means having the formula-OR _a Wherein R is a group of _a Is C as defined above in general _1- C ₆ An alkyl group. C (C) ₁ -C ₆ Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentoxy, and hexoxy.

As used herein, the term "C ₁ -C ₆ Haloalkyl "means C as defined above substituted with one or more halo groups as defined herein ₁ -C ₆ An alkyl group. C (C) ₁ -C ₆ Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl 1, 1-difluoroethyl, 2-trifluoroethyl, 2-fluoropropyl, 1-trifluoropropyl 2, 2-difluoropropyl, 3-difluoropropyl and 1-fluoromethyl-2-fluoroethyl, 1, 3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl, 1, 2-tetrafluoropropyl and 1, 4-trifluorobutan-2-yl.

As used herein, the term "C ₁ -C ₆ Haloalkoxy "means C as defined herein substituted with one or more halo groups ₁ -C ₆ An alkoxy group. C (C) ₁ -C ₆ Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, fluoromethoxy, trichloromethoxy, 1-difluoroethoxy, 2-trifluoroethoxy, 1-fluoromethyl-2-fluoroethoxy, pentafluoroethoxy, 2-fluoropropoxy, 3-difluoropropoxy and 3-dibromopropoxy. Preferably C ₁ -C ₆ One or more of the halo groups of the haloalkoxy group is fluorine. Preferably C ₁ -C ₆ Haloalkoxy is selected from the group consisting of trifluoromethoxy, difluoromethoxy, fluoromethoxy, 1-difluoroethoxy, 2-trifluoroethoxy, 1-fluoromethyl-2-fluoroethoxy and pentafluoroethoxy.

The term "halogen" or "halo" means fluorine, chlorine, bromine or iodine.

As used herein, the term "cycloalkyl" means a monocyclic or polycyclic saturated or partially unsaturated carbocycle containing 3 to 18 carbon atoms, wherein there is no common delocalized pi electron (aromaticity) between the ring carbons. The term "C ₃ -C ₁₀ Cycloalkyl "," C ₃ -C ₈ Cycloalkyl "," C ₄ -C ₁₀ Cycloalkyl radicals "and" C ₄ -C ₇ Cycloalkyl "is to be construed accordingly. The term polycyclic encompasses bridged (e.g., norbornane), fused (e.g., decalin) and spiro cycloalkyl groups. Preferably cycloalkyl radicals, e.g. C ₃ -C ₁₀ Cycloalkyl is a monocyclic, bridged or spiro hydrocarbon group of 3 to 10 carbon atoms.

Examples of cycloalkyl groups include, but are not limited to, cyclopropenyl, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl (norboryl), norbornenyl (norboryl), spiro [3.3] heptanyl (e.g., spiro [3.3] heptan-6-yl), bicyclo [2.2.2] octanyl, adamantyl, and derivatives thereof. Preferably, the cycloalkyl group is saturated.

C ₃ -C ₁₀ Preferred examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro [3.3]]Heptyl (e.g. spiro [3.3] ]Heptane-6-yl), bicyclo [1.1.1]Amyl, bicyclo [2.1.1]Hexyl, bicyclo [2.1.1]Heptyl, bicyclo [2.2.2]Octyl and adamantyl.

"heterocyclyl" means a saturated or partially saturated monocyclic or polycyclic ring containing carbon and at least one heteroatom selected from oxygen, nitrogen and sulfur (O, N and S), and wherein there is no shared delocalized pi electron (aromaticity) between the ring carbons or heteroatoms. The terms "4-to 10-membered heterocyclyl", "4-to 6-membered heterocyclyl" and "5-or 6-membered heterocyclyl" should be construed accordingly. The heterocyclyl ring structure may be substituted with one or more substituents. The substituents themselves may be optionally substituted. The heterocyclic group may be bonded via a carbon atom or heteroatom. The term polycyclic encompasses bridged, fused and spiro heterocyclyl groups.

Examples of heterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, oxazolinyl, isoxazolinyl, oxazolidinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxanyl, piperidinyl, morpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, aza Radical, oxa->Radical, diaza->Group, tropanyl, oxazolidonyl, 1, 4-dioxanyl, dihydrofuranyl, 1, 3-dioxolanyl, imidazolidinyl, dihydroisoxazolinyl, pyrrolinyl, pyrazolinyl, oxazal>Radicals, dithiolane radicals, homotropane radicals, dihydropyrane radicals (e.g. 3, 6-dihydro-2H-pyranyl radical), oxaspiro heptane radicals (e.g. 2-oxaspiro [3.3]]Heptan-6-yl), diazabicyclo [3.2.1]Octan-3-yl, 2-azaspiro [3.3]]Heptyl (e.g. 2-azaspiro [3.3]]Heptane-6-yl) and the like.

Preferred examples of heterocyclyl groups include, but are not limited to, oxetanyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, dihydroisoxazolinyl, tetrahydropyranyl, morpholinyl, dihydropyranyl (e.g., 3, 6-dihydro-2H-pyranyl), 2-azaspiro [3.3] heptanyl (e.g., 2-azaspiro [3.3] heptan-6-yl), and oxaspiro heptanyl (e.g., 2-oxaspiro [3.3] heptan-6-yl).

As used herein, the term "aryl" as used herein means a monocyclic, bicyclic, or polycyclic carbocyclic aromatic ring. Examples of aryl groups include, but are not limited to, phenyl, naphthyl (e.g., naphthalen-1-yl, naphthalen-2-yl), anthracenyl (e.g., anthracen-1-yl, anthracene- 9-yl), phenanthryl (e.g., phenanthren-1-yl, phenanthren-9-yl), and the like. Aryl is also intended to include monocyclic, bicyclic, or polycyclic carbocyclic aromatic rings substituted with carbocyclic aromatic rings. Representative examples are biphenyl (e.g., biphenyl-2-yl, biphenyl-3-yl, biphenyl-4-yl), phenylnaphthalenyl (e.g., 1-phenylnaphthalen-2-yl, 2-phenylnaphthalen-1-yl), and the like. Aryl is also intended to include partially saturated bicyclic or polycyclic carbocycles having at least one unsaturated moiety (e.g., a benzo moiety). Representative examples are indanyl (e.g., indan-1-yl, indan-5-yl), indenyl (e.g., inden-1-yl, inden-5-yl), 1,2,3, 4-tetrahydronaphthalen-1-yl, 1,2,3, 4-tetrahydronaphthalen-2-yl, 1,2,3, 4-tetrahydronaphthalen-6-yl), 1, 2-dihydronaphthalen-1-yl, 1, 2-dihydronaphthalen-4-yl, 1, 2-dihydronaphthalen-6-yl, fluorenyl (e.g., fluoren-1-yl, fluoren-4-yl, fluoren-9-yl), and the like. Aryl is also intended to include partially saturated bicyclic or polycyclic carbocyclic aromatic rings containing one or two bridges. Representative examples are benzonorbornyl (e.g., benzonorbornan-3-yl, benzonorbornan-6-yl), 1, 4-methano-1, 2,3, 4-tetrahydronaphthalenyl (e.g., 1, 4-methano-1, 2,3, 4-tetrahydronaphthalen-2-yl, 1, 4-methano-1, 2,3, 4-tetrahydronaphthalen-10-yl), and the like. The term "C ₆ -C ₁₀ Aryl "should be construed accordingly.

Preferred examples of aryl groups include, but are not limited to, indenyl (e.g., inden-1-yl, inden-5-yl), phenyl (C) ₆ H ₅ ) Naphthyl (C) ₁₀ H ₇ ) (e.g., naphthalen-1-yl, naphthalen-2-yl), indanyl (e.g., indan-1-yl, indan-5-yl), and tetrahydronaphthalenyl (e.g., 1,2,3, 4-tetrahydronaphthalenyl).

Preferably C ₆ -C ₁₀ Aryl refers to a monocyclic or bicyclic carbocyclic aromatic ring.

C ₆ -C ₁₀ Preferred examples of aryl groups include, but are not limited to, phenyl and naphthyl. In one embodiment, C ₆ -C ₁₀ Aryl is phenyl.

As used herein, the term "heteroaryl" as used herein is intended to include monocyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are pyrrolyl, furanyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isothiazideOxazolyl, isoxazolyl, triazolyl (e.g., 1,2, 4-triazolyl), oxadiazolyl (e.g., 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl), thiadiazolyl (e.g., 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3, 4-thiadiazolyl), tetrazolyl, pyranyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2, 3-triazinyl, 1,2, 4-triazinyl, 1,3, 5-triazinyl, thiadiazinyl, aza A radical, an azedocaine radical (azecinyl), and the like.

Heteroaryl is also intended to include bicyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are indolyl, isoindolyl, benzofuranyl, benzothienyl, indazolyl, benzopyranyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazinyl, benzotriazolyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, cinnolinyl, quinolinyl, isoquinolinyl, quinazolinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolopyridinyl, furopyridinyl, thiophenopyridinyl, imidazopyridinyl, imidazopyrimidinyl, pyrazolopyridinyl, pyrazolopyrimidinyl, pyrazolotriazinyl, triazolopyridinyl, triazolopyrimidinyl, imidazothiazolyl, triazolopyridinyl, triazolopyrimidinyl, and the like.

Heteroaryl is also intended to include polycyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are carbazolyl, phenoxazinyl, phenazinyl, acridinyl, phenothiazinyl, carbolinyl, phenanthroline, and the like.

Heteroaryl is also intended to include partially saturated monocyclic, bicyclic or polycyclic heterocyclic groups containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are imidazolinyl, indolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzopyranyl, dihydropyridoxazinyl, dihydrobenzodioxinyl (e.g., 2, 3-dihydrobenzo [ b ] [1,4] dioxinyl), benzodioxolyl (e.g., benzo [ d ] [1,3] dioxole), dihydrobenzooxazinyl (e.g., 3, 4-dihydro-2H-benzo [ b ] [1,4] oxazin), tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydroimidazo [4,5-c ] pyridinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, and the like.

The heteroaryl ring structure may be substituted with one or more substituents. The substituents themselves may be optionally substituted. Heteroaryl rings may be bonded via a carbon atom or heteroatom.

The term "5-to 10-membered heteroaryl" should be construed accordingly.

Examples of 5-10 membered heteroaryl groups include, but are not limited to, indolyl, imidazopyridinyl, isoquinolyl, benzoxazolonyl, pyridinyl, pyrimidinyl, pyridonyl, benzotriazolyl, pyridazinyl, pyrazolotriazinyl, indazolyl, benzimidazolyl, quinolinyl, triazolyl (e.g., 1,2, 4-triazolyl), pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, pyrrolyl, oxadiazolyl (e.g., 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl), imidazolyl, pyrrolopyridinyl, tetrahydroindazolyl, quinoxalinyl, thiadiazolyl (e.g., 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl), pyrazinyl, oxazolopyridinyl, pyrazolopyrimidinyl, benzoxazolyl, indolinyl, isoxazolyl [ e.g., 1,3, 4-dihydro [ 2, 3-dihydro ] benzo [ 2, 4] oxa ] benzo [ 2, 3-dihydro ] oxa [ b ] oxa ] benzo [ 2, 4] oxazinyl, and [ H ] [ 2, 4-dihydro ] [ 2, 4-oxazinyl ] benzo [ b ] [ 2,3, 4] oxazinyl.

As used herein, the term "oxo" refers to the group = O.

"cyano" or "-CN" means a substituent having a nitrogen atom and a carbon atom attached through a triple bond, such as C.ident.N.

The term "C" as used herein ₂ -C ₆ Alkenyl "means a branched or straight chain hydrocarbon group having 2 to 6 carbon atoms and at least one double bond. Representative examples are vinyl(ethenyl or vinyl), propenyl (e.g., prop-1-enyl, prop-2-enyl), 2-methylpropan-1-enyl, 2-methylpropan-2-enyl, 1- (dimethyl) prop-2-enyl, butadienyl (e.g., but-1, 3-dienyl), butenyl (e.g., but-1-en-1-yl, but-2-en-1-yl), 2-methylbutan-1-enyl, pentenyl (e.g., pent-1-enyl, pent-2-enyl), hexenyl (e.g., hex-1-enyl, hex-2-enyl, hex-3-enyl), 2-methylpent-3-enyl, and the like.

The term "bridged ring" as used herein means that two non-adjacent carbon atoms (linked to form C) in the heterocycloalkyl moiety of formula (I) ₁ -C ₃ Alkylene linker), wherein one of the carbon atoms of the linker is optionally replaced with a heteroatom selected from nitrogen, oxygen and sulfur. In a preferred embodiment, the alkylene linker contains only carbon atoms.

As used herein, the term "C ₁ -C ₃ Alkylene "refers to a straight hydrocarbon chain divalent group consisting of only carbon and hydrogen atoms, with no unsaturation present in the group, having one to three carbon atoms.

As used herein, the term "optionally substituted" includes unsubstituted or substituted.

As used herein,indicating attachment points to other parts of the molecule.

As used herein, the term nitrogen Protecting Group (PG) in a compound having formula (X) or any intermediate in any of schemes 1 to 5 and sub-formulae thereof refers to a group that should protect the relevant functional group from undesired secondary reactions such as acylation, etherification, esterification, oxidation, solvolysis and the like. It can be removed under deprotection conditions. Depending on the protecting group used, the skilled person will know how to remove the protecting group to obtain the free amine NH by referring to known methods ₂ A group. These include reference to textbooks and literature methods of organic chemistry, e.g., J.F.W.McOmie, "Protective Groups in Organic Chemistry [ protecting groups in organic chemistry ]]", plenum Press [ Plenum Press ]]London and New YorkAbout 1973; T.W.Greene and P.G.M.Wuts, "Greene's Protective Groups in Organic Synthesis [ protecting groups in Green organic Synthesis ] ]"fourth edition, wiley [ Wiley Verlag]New york 2007; in "The Peptides]"; vol.3 (editions: E.Gross and J.Meienhofer), academic Press [ Academic Press ]]London and new york 1981; kocienski, "Protecting Groups [ protecting group ]]", third edition, georg Thieme Verlag [ Georg's Siemens Press ]]Stuttgart and New York 2005; in "Methoden der organischen Chemie" (Methods of Organic Chemistry) [ organic chemistry methods ]]Houben Weyl, 4 th edition, volume 15/I, georg Thieme Verlag [ George, mei Press ]]Stuttgart 1974.

Preferred nitrogen protecting groups generally include: c (C) ₁ -C ₆ Alkyl (e.g., tert-butyl), preferably C ₁ -C ₄ Alkyl, more preferably C ₁ -C ₂ Alkyl, most preferably C ₁ Alkyl, which is mono-, di-or tri-substituted with: trialkylsilyl-C ₁ -C ₇ Alkoxy (e.g. trimethylsilylethoxy), aryl (preferably phenyl) or a heterocyclic group (e.g. benzyl, cumyl, benzhydryl, pyrrolidinyl, trityl, pyrrolidinylmethyl, 1-methyl-1, 1-dimethylbenzyl, (phenyl) methylbenzene), wherein the aryl ring or heterocyclic group is unsubstituted or substituted with one or more (e.g. two or three) residues, e.g. selected from the group consisting of: c (C) ₁ -C ₇ Alkyl, hydroxy, C ₁ -C ₇ Alkoxy (e.g., p-methoxybenzyl (PMB)), C ₂ -C ₈ Alkanoyl-oxy, halogen, nitro, cyano, and CF ₃ aryl-C ₁ -C ₂ Alkoxycarbonyl (preferably phenyl-C) ₁ -C ₂ Alkoxycarbonyl groups (e.g. benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), pivaloyloxymethyl (POM)), C ₁ -C ₁₀ -alkenyloxycarbonyl, C ₁ -C ₆ Alkylcarbonyl (e.g. acetyl or pivaloyl), C ₆ -C ₁₀ -an arylcarbonyl group; c (C) ₁ -C ₆ Alkoxycarbonyl groups such as t-butoxycarbonyl (Boc), methylcarbonyl, trichloroethoxycarbonyl (Troc), pivaloyl [ ]Piv), allyloxycarbonyl), C ₆ -C ₁₀ -aryl C ₁ -C ₆ Alkoxycarbonyl (e.g. 9-fluorenylmethoxycarbonyl (Fmoc)), allyl or cinnamyl, sulfonyl or sulfenacyl, succinimidyl, silyl groups (e.g. triarylsilyl, trialkylsilyl, triethylsilyl (TES), trimethylsilylethoxymethyl (SEM), trimethylsilyl (TMS), triisopropylsilyl or tert-butyldimethylsilyl).

Preferred nitrogen Protecting Groups (PGs) according to the present disclosure may be selected from the group comprising: tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), p-methoxybenzyl (PMB), 2, 4-Dimethoxybenzyl (DMB), methyloxycarbonyl, trimethylsilylethoxymethyl (SEM) and benzyl. The nitrogen Protecting Group (PG) is preferably an acid labile protecting group such as t-butoxycarbonyl (Boc), 2, 4-Dimethoxybenzyl (DMB).

In some embodiments, the compounds of the present disclosure are selective for other proteins.

As used herein, the term "therapeutic agent" in connection with a method of reducing the level of expression of a WIZ protein and/or inducing expression of fetal hemoglobin (HbF) refers to a substance that results in a detectable reduction in expression of the WIZ gene or WIZ protein or a reduction in the level of activity of the WIZ protein as compared to the level in the absence of such a substance.

As used herein, "modulator" or "degradant" means, for example, a compound of the present disclosure that is effective to modulate, reduce, or decrease the level of a specific protein (e.g., WIZ) or degrade a specific protein (e.g., WIZ). The amount of degraded specific protein (e.g., WIZ) can be measured by comparing the amount of specific protein (e.g., WIZ) remaining after treatment with a compound of the disclosure to the initial amount or level of specific protein (e.g., WIZ) present measured prior to treatment with a compound of the disclosure.

As used herein, "selective modulator," "selective degradation agent," or "selective compound" means, for example, a compound of the present disclosure that is effective in modulatingReducing, or reducing the level of or degrading the specific protein (e.g., WIZ) to a greater extent than any other protein. For example, a "selective modulator," "selective degradation agent," or "selective compound" can be identified by comparing the ability of a compound to modulate, reduce, or decrease the level of a specific protein (e.g., WIZ) or degrade a specific protein to the ability of the compound to modulate, reduce, or decrease the level of or degrade other proteins. In some embodiments, the EC of a compound may be measured by ₅₀ Or IC (integrated circuit) ₅₀ And (5) identifying selectivity. Degradation may be achieved by mediating an E3 ligase (e.g., an E3-ligase complex comprising the protein Cereblon).

In one embodiment, the degraded specific protein is a WIZ protein. In one embodiment, at least about 30% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 40% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 50% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 60% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 70% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 75% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 80% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 85% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 90% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 95% of the WIZ is degraded compared to the initial level. In one embodiment, more than 95% of the WIZ is degraded compared to the initial level. In one embodiment, at least about 99% of the WIZ is degraded compared to the initial level.

In one embodiment, the wip is degraded in an amount from about 30% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 40% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 50% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 60% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 70% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 80% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 90% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 95% to about 99% compared to the initial level. In one embodiment, the wip is degraded in an amount from about 90% to about 95% compared to the initial level.

As used herein, the term "induce fetal hemoglobin", "fetal hemoglobin induction", or "increase fetal hemoglobin expression" refers to increasing the percentage of HbF in the blood of a subject. In one embodiment, the amount of total HbF in the blood of the subject is increased. In one embodiment, the amount of total hemoglobin in the blood of the subject is increased. In one embodiment, the amount of HbF is increased by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or more than 100%, for example at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or more than 10-fold, as compared to the absence of any of the compounds disclosed herein.

In one embodiment, total hemoglobin in blood (e.g., blood of a subject) is increased by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or more than 100%, e.g., at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or more than 10-fold, as compared to any of the absence of a compound disclosed herein.

The term "therapeutically effective amount" of a compound of the present disclosure refers to an amount of a compound of the present disclosure that will elicit a biological or medical response (e.g., a decrease or inhibition of enzymatic or protein activity, or an improvement in symptoms, alleviation of a condition, slowing or delaying the progression of a disease, or preventing a disease, etc.) in a subject. In one embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present disclosure that, when administered to a subject, is effective to: (1) At least partially alleviating, preventing and/or ameliorating a condition or disorder or disease that is (i) mediated by wip, or (ii) associated with wip activity, or (iii) characterized by wip activity (normal or abnormal); (2) reducing or inhibiting WIZ activity; or (3) reduce or inhibit expression of WIZ. In another embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present disclosure that is effective to at least partially reduce or inhibit the activity of WIZ when administered to a cell, or tissue, or non-cellular biological material, or medium; or at least partially reduce or inhibit expression of WIZ.

By "HbF dependent disease or disorder" is meant any disease or disorder that is directly or indirectly affected by modulation of HbF protein levels.

As used herein, the term "subject" refers to primates (e.g., humans (male or female)), dogs, rabbits, guinea pigs, rats, and mice. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

As used herein, the term "inhibit" refers to a reduction or inhibition of a given condition, symptom or disorder, or disease, or a significant decrease in a baseline activity of a biological activity or process.

As used herein, the term "treating (treat, treating or treatment)" of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the progression of the disease or at least one clinical symptom thereof); or reducing or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those that may not be distinguishable for the patient.

As used herein, the term "prevention" of any disease or disorder refers to the prophylactic treatment of a disease or disorder; or delay the onset or progression of a disease or disorder.

As used herein, a subject is "in need of" such treatment if such subject would benefit biologically, medically, or in quality of life from the treatment.

As used herein, the terms "a" and "an" and "the" and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Various enumerated embodiments of the present disclosure are described herein. It should be appreciated that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the present disclosure.

Examples are given

Example 1 a compound having the formula (I "), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ³ selected from hydrogen, C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ 、C ₁ -C ₆ Haloalkyl, -C (=o) -O- (R) ⁵ )、-C(＝O)-(R ⁶ )、C ₃ -C ₁₀ Cycloalkyl and 4-to 10-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O and S, wherein the C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl groups are each independently R from 0 to 3 occurrences ^3a Substitution, and wherein said C ₃ -C ₁₀ Cycloalkyl and 4-to 10-membered heterocyclyl are each independently substituted with 0-3 occurrences of R ^3b Substitution;

or alternatively

each R ^3b Independently and separatelySelected from C ₁ -C ₆ Alkoxy, halo, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Alkyl, -CN, -SO ₂ NR ⁷ R ⁸ 、-SO ₂ R ⁴ And hydroxyl;

R ^4b selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^4c selected from hydrogen, C ₁ -C ₆ Alkyl and C ₃ -C ₈ Cycloalkyl;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

Example 2 a compound having formula (I'), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

Example 3 a compound having formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

X is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

each R ² Independently selected from C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl, halo and oxo, whereinThe C is ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution; or 2R on non-adjacent carbon atoms ² Forms a bridged ring with the non-adjacent carbon atoms to which they are attached;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

Example 4 the compound of any one of examples 1 to 3, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₃ Alkyl group ；

R ¹ Selected from hydrogen and C ₁ -C ₃ An alkyl group;

each R ² Independently selected from unsubstituted C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl and halo; or 2R on non-adjacent carbon atoms ² Forms a bridged ring with the non-adjacent carbon atoms to which they are attached;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atom to which it is attached, form a 5-or 6-membered heterocyclyl containing 0 to 1 additional heteroatoms selected from N and O;

R ⁴ selected from C ₃ -C ₈ Cycloalkyl, C ₁ -C ₆ Alkyl group, bagA 4-to 6-membered heterocyclic group containing 1-2 heteroatoms independently selected from N, O and S, and C ₆ -C ₁₀ Aryl, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^4a Substitution;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ^6b selected from chlorine, fluorine, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy and C ₁ -C ₆ An alkyl group;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2 or 3;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

Example 5 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is selected from hydrogen and methyl;

R ¹ selected from hydrogen and methyl;

each R ² Independently selected from unsubstituted C ₁ -C ₆ Alkyl and halo; or 2R on non-adjacent carbon atoms ² Together with the non-adjacent carbon atoms to which they are attached form C ₁ -C ₃ An alkylene bridge ring;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atom to which it is attached, form a 5-or 6-membered heterocyclyl containing 0-1 additional O heteroatoms;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, 5-to 10-membered heteroaryl containing 1-4 heteroatoms selected independently from N, O and S, and phenyl, wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 10-membered heteroaryl and phenyl are each substituted with 0-4 occurrences of R ^3b Substitution;

R ⁵ selected from C ₁ -C ₆ Alkyl, C ₃ -C ₆ Cycloalkyl and C ₆ -C ₁₀ An aryl group;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2 or 3;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

Example 6 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

each R ² Independently selected from unsubstituted C ₁ -C ₆ Alkyl and fluoro; or 2R on non-adjacent carbon atoms ² Together with the non-adjacent carbon atoms to which they are attached form C ₁ -C ₃ An alkylene bridge ring;

R ³ selected from C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ And C ₁ -C ₆ Haloalkyl, wherein said C ₁ -C ₈ R in which the alkyl radical is present from 0 to 2 times ^3a Substitution, and the C ₁ -C ₆ R in which the haloalkyl is present 0 to 1 times ^3a Substitution;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N and O, 5-to 6-membered heteroaryl containing 1-3 heteroatoms selected independently from N, O and S, and phenyl, wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 6-membered heteroaryl and phenyl are each substituted with 0-4 occurrences of R ^3b Substitution;

each R ^3b Independently selected from halo, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Alkyl and hydroxy;

n is 0, 1, 2 or 3;

m is 1 or 2; and is also provided with

p is 0 or 1.

Example 7 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

each R ² Independently selected from unsubstituted C ₁ -C ₆ An alkyl group; or 2R on non-adjacent carbon atoms ² Together with the non-adjacent carbon atoms to which they are attached form C ₁ -C ₃ An alkylene bridge ring;

R ³ selected from C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ And unsubstituted C ₁ -C ₆ Haloalkyl, wherein said C ₁ -C ₈ R in which the alkyl radical is present from 0 to 2 times ^3a Substitution;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N and O, 5-to 6-membered heteroaryl containing 1-3 heteroatoms selected independently from N, O and S, and phenyl, wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 6-membered heteroaryl and phenyl are each substituted with 0-3 occurrences of R ^3b Substitution;

R ⁴ selected from C ₃ -C ₈ Cycloalkyl, C ₁ -C ₆ Alkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms independently selected from N, O and S, and C ₆ -C ₁₀ Aryl, wherein said C ₁ -C ₆ R in which the alkyl radical is present 1 time ^4a Substitution;

n is 0, 1 or 2;

m is 1 or 2; and is also provided with

p is 1.

Example 8 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

each R ² Independently selected from unsubstituted C ₁ -C ₃ An alkyl group;

R ³ selected from C ₁ -C ₆ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ And unsubstituted C ₁ -C ₆ Haloalkyl, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 2 times ^3a Substitution;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1O heteroatom, 6-membered heteroaryl containing 1-2N heteroatoms, and phenyl, wherein the C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 6-membered heteroaryl and phenyl are substituted with 0-2 occurrences of R ^3b Substitution;

each R ^3b Independently selected from chlorine, fluorine, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy and C ₁ -C ₆ An alkyl group;

R ⁴ selected from C ₃ -C ₈ Cycloalkyl, C ₁ -C ₆ Alkyl, 4-to 6-membered heterocyclyl containing 1O heteroatom, and phenyl, wherein the C ₁ -C ₆ R in which the alkyl radical is present 1 time ^4a Substitution;

R ^4a selected from C ₃ -C ₈ Cycloalkyl and phenyl;

n is 0, 1 or 2;

m is 1 or 2; and is also provided with

p is 1.

Example 9 a compound as described in example 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ia "):

example 10 the compound of any one of examples 1 and 2, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ia'):

example 11 a compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ia):

example 12. The compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Selected from C ₁ -C ₆ Alkyl and-CH ₂ -R ^3a 。

Example 13 the compound of any one of examples 1 and 9, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ib "):

example 14 the compound of any one of examples 1, 2, 9 and 10, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ib'):

example 15 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ib):

example 16 the compound of any one of examples 1 and 9, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ic "), wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

R ^2b selected from hydrogen, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl and halo, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

R ^2c selected from hydrogen and C ₁ -C ₆ Alkyl, wherein the C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

or R is ^2b And R is ^2c Attached to themTogether form an oxo group;

R ^2d and R is ^2e Each independently selected from hydrogen, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl, halo and oxo, wherein said C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

R ^2f is hydrogen;

or R is ^2b And R is ^2e Or R is ^2b And R is ^2f Together with the carbon atoms to which they are attached, form a bridged ring;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy; and is also provided with

R ³ Is defined according to any one of the preceding embodiments.

Embodiment 17 the compound of any one of embodiments 1, 2, 9, and 10, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ic'), wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

or R is ^2b And R is ^2c Together with the carbon atoms to which they are attached, form an oxo group;

R ^2f is hydrogen;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy; and is also provided with

R ³ Is defined according to any one of the preceding embodiments.

Embodiment 18 the compound of any one of embodiments 1 to 12, 16, and 17, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ic), wherein:

x is selected from CH, CF and N;

R ^2f is hydrogen;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy; and is also provided with

R ³ Is defined according to any one of the preceding embodiments.

Embodiment 19 the compound of any one of embodiments 16 to 18 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2b selected from hydrogen, C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl and halo, wherein said C ₁ -C ₃ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

R ^2c selected from hydrogen and C ₁ -C ₃ Alkyl, wherein the C ₁ -C ₃ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

R ^2d and R is ^2e Each independently selected from hydrogen, C ₁ -C ₃ Alkyl, C ₁ -C ₃ Haloalkyl, halo and oxo, wherein said C ₁ -C ₃ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution;

R ^2f is hydrogen;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ³ selected from C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ 、C ₁ -C ₆ Haloalkyl, -C (=o) -O- (R) ⁵ ) and-C (=o) - (R ⁶ ) Wherein said C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl is independently substituted with 0-3 occurrences of R ^3a Substitution;

R ⁵ selected from C ₁ -C ₆ Alkyl and C ₆ -C ₁₀ An aryl group;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

R ⁷ And R is ⁸ Together with the nitrogen atom to which they are attached, form a 5-or 6-membered heterocyclyl containing 0 to 1 additional heteroatoms selected from N, O and S; and is also provided with

m is 1 or 2.

Embodiment 20 the compound of any one of embodiments 16 to 19, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2b 、R ^2c 、R ^2d and R is ^2e Each independently selected from hydrogen and unsubstituted C ₁ -C ₃ An alkyl group;

R ^2f is hydrogen;

or R is ^2b And R is ^2e Or R is ^2b And R is ^2f Together with the carbon atom to which they are attached form C ₁ -C ₃ An alkylene bridge ring;

R ³ selected from C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ And C ₁ -C ₆ Haloalkyl, wherein said C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl is independently substituted with 0-3 occurrences of R ^3a Substitution;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

Or alternatively

m is 1 or 2.

Embodiment 21 the compound of any one of embodiments 16 to 20 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2f is hydrogen;

each R ^3b Independently selected from C ₁ -C ₆ Alkoxy, halo, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Alkyl and hydroxy;

R ^4a selected from C ₃ -C ₈ Cycloalkyl, C ₆ -C ₁₀ Aryl and C ₁ -C ₆ An alkoxy group; and is also provided with

m is 1.

Embodiment 22 the compound of any one of embodiments 1, 9 and 16, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Id "), wherein:

R ^2b 、R ^2c and R is ^2e Is defined according to any one of embodiments 16 to 21.

Example 23 the compound of any one of examples 1, 2, 9, 10, 16, 17, and 22, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Id'), wherein:

Embodiment 24 the compound of any one of embodiments 1 to 12 and 16 to 23, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Id), wherein:

Embodiment 25 the compound of any one of embodiments 1 to 12 and 16 to 24, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Id-1), wherein:

R ^2b Selected from hydrogen and C ₁ -C ₄ An alkyl group; and is also provided with

X and R ³ Defined in any one of the preceding embodiments.

Example 26 the compound of example 25, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

x is CH or N;

R ^2b selected from hydrogen and C ₁ -C ₄ An alkyl group;

R ³ selected from C ₁ -C ₈ Alkyl, -SO ₂ R ⁴ and-C (=o) - (R ⁶ ) Wherein said C ₁ -C ₈ R in which the alkyl radicals are independently present from 0 to 3 times ^3a Substitution;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, C ₁ -C ₆ Alkoxy and hydroxy, wherein said C ₃ -C ₁₀ Cycloalkyl and R wherein the 4-to 6-membered heterocyclic group is present 0-2 times ^3b Substitution;

each R ^3b Independently selected from C ₁ -C ₆ Alkoxy, halo, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₁ -C ₆ Alkyl, -CN, -SO ₂ R ⁴ And hydroxyl groups.

Example 27 the compound of any one of examples 25 and 26, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Id-2) or (Id-3):

embodiment 28 the compound of any one of embodiments 1, 9, 13, 16 and 22, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ie "), wherein:

Example 29 the compound of any one of examples 1, 2, 9, 10, 13, 14, 16, 17, 22, 23, and 28, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ie'), wherein:

Embodiment 30 the compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ie), wherein:

Embodiment 31 the compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X is CH.

Embodiment 32. The compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X is N.

Embodiment 33. The compound of any one of embodiments 1 to 15, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein n is selected from 0 and 1, and m is selected from 1 and 2.

Embodiment 34. As in any one of embodiments 1 to 15The compound or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ² Is unsubstituted C ₁ -C ₆ Alkyl, such as methyl, and n is 1.

Embodiment 35 the compound of any one of embodiments 1 to 20 and 33, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein m is 1.

Example 36 the compound of any one of examples 1 to 12, 16 to 27 and 31 to 35, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Is C ₁ -C ₆ Alkyl, wherein the C ₁ -C ₆ R in which the alkyl radical is present 1 time ^3a And (3) substitution.

Example 37 the compound of any one of examples 1 to 12, 16 to 27 and 31 to 36, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, 2-propyl, butyl, isobutyl, 2-butyl, 3-methyl-2-butyl, isopentyl, 3-pentyl, neopentyl, 2, 4-dimethylpentanyl and-CH ₂ -(CH ₂ ) _0-1 -R ^3a 。

Example 38 the compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^3a Is C ₃ -C ₁₀ Cycloalkyl, wherein said C ₃ -C ₁₀ R in which cycloalkyl is present 0 to 4 times ^3b Substitution, wherein each R ^3b Independently selected from C ₁ -C ₆ Alkoxy, chloro, fluoro, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy and C ₁ -C ₆ An alkyl group.

Example 39A compound according to any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or stereoisomer thereofIsomers, or tautomers, wherein R ^3a Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl,

Example 40. The compound of any one of the preceding examples, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^3a Is C ₃ -C ₇ Cycloalkyl, wherein said C ₃ -C ₇ Cycloalkyl groups are substituted with 0-2 occurrences of fluorine.

Embodiment 41 the compound of any one of embodiments 16 to 40 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b And R is ^2e Each independently selected from hydrogen and unsubstituted C ₁ -C ₃ An alkyl group; and R is ^2c Is hydrogen.

Embodiment 42 the compound of any one of embodiments 16 to 41 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b And R is ^2e Each independently selected from hydrogen and methyl; and R is ^2c Is hydrogen.

Embodiment 43 the compound of any one of embodiments 16 to 42 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b Is unsubstituted C ₁ -C ₃ Alkyl (e.g., methyl); r is R ^2c Is hydrogen; and R is ^2e Selected from hydrogen and unsubstituted C ₁ -C ₃ An alkyl group.

Example 44 the compound of any one of examples 16 to 43, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b Is methyl and R ^2c 、R ^2d 、R ^2e And R is ^2f Are all hydrogen.

Example 45 the compound of any one of examples 1-15 and 31-44, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ² Is unsubstituted C ₁ -C ₃ Alkyl, and n is 1.

The compound of any one of examples 1, 2, 4 to 10, 12 to 14, 16, 17, 19 to 23, 28, 29 and 31 to 45,is a double bond.

Example 47 the compound according to any one of the preceding examples, whereinIs a single bond.

Example 48 a compound as described in example 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, selected from the group consisting of:

/>

embodiment 49 the compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an acid addition salt.

Embodiment 50. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier or excipient.

Embodiment 51 the compound of any one of embodiments 1 to 49 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof for use as a medicament.

Embodiment 52. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt thereof.

Example 53 a method of treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Embodiment 54 a method of treating a disease or disorder affected by modulation of WIZ protein levels, comprising administering to a patient in need thereof a compound of any one of embodiments 1-49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 55. A method of inhibiting WIZ protein expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 56 a method of degrading WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of examples 1-49, or a pharmaceutically acceptable salt thereof.

Embodiment 57. A method of inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein, comprising administering to the subject a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 58 a method of inducing or promoting fetal hemoglobin in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 59. A method of reactivating fetal hemoglobin production or expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of examples 1-49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 60 a method of increasing fetal hemoglobin expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 61. A method of treating a hemoglobinopathy, e.g., β -hemoglobinopathy, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 62. A method of treating sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of examples 1-49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Embodiment 63. A method of treating β -thalassemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Example 64 a method for reducing WIZ protein level in a subject, the method comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Embodiment 65 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease or disorder in a subject in need thereof.

Embodiment 66. The compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment of a disease or disorder selected from sickle cell disease and β -thalassemia.

Embodiment 67 the compound of any one of embodiments 1 to 49 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof for use in treating or preventing a disorder affected by inhibition of WIZ protein levels in a subject in need thereof.

Embodiment 68 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof.

Embodiment 69 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment or prevention of a disease or disorder affected by WIZ protein degradation.

Embodiment 70 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting, reducing, or eliminating WIZ protein activity or WIZ protein expression in a subject in need thereof.

Embodiment 71 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inducing or promoting fetal hemoglobin in a subject in need thereof.

Embodiment 72 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in reactivating fetal hemoglobin production or expression in a subject in need thereof.

Embodiment 73 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in increasing fetal hemoglobin expression in a subject in need thereof.

Embodiment 74 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a hemoglobinopathy in a subject in need thereof.

Embodiment 75 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating sickle cell disease in a subject in need thereof.

Embodiment 76 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating β -thalassemia in a subject in need thereof.

Embodiment 77 the compound of any one of embodiments 1 to 49 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof for use in the treatment of a disease or disorder affected by increased expression of hemoglobin in a fetus.

Embodiment 78 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder that is affected by inhibition, reduction, or elimination of WIZ protein activity or WIZ protein expression.

Embodiment 79 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by induction or promotion of hemoglobin in a fetus.

Embodiment 80 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by reactivation of fetal hemoglobin production or expression.

Embodiment 81 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting WIZ protein expression in a subject in need thereof.

Embodiment 82 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in degrading WIZ protein in a subject in need thereof.

Embodiment 83 the use of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder that is affected by reduced levels of WIZ protein, inhibition of WIZ protein expression, or degradation of WIZ protein.

Embodiment 84 the use of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder that is affected by induction or promotion of fetal hemoglobin.

Embodiment 85 the use of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder affected by reactivation of fetal hemoglobin production or expression.

The use of a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder that is affected by increased fetal hemoglobin expression.

Embodiment 87 the use of a compound of any one of embodiments 83 to 86, wherein the disease or disorder is selected from sickle cell disease and β -thalassemia.

Embodiment 88 the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder that is affected by reduced levels of WIZ protein, inhibition of WIZ protein expression, or degradation of WIZ protein.

Embodiment 89 the use of the compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the treatment of a disease or disorder affected by induction of fetal hemoglobin, reactivation of fetal hemoglobin production or expression, or increase of fetal hemoglobin expression.

Embodiment 90 the use of embodiment 88 or 89 wherein the disease or disorder is selected from sickle cell disease and β -thalassemia.

Embodiment 91 a pharmaceutical combination comprising a compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more additional therapeutic agents.

Depending on the choice of starting materials and procedure, the compounds may be present in the form of possible isomers or as mixtures thereof (e.g. as pure optical isomers or as isomer mixtures, such as racemates and diastereomeric mixtures), depending on the number of asymmetric centers. The present disclosure is intended to include all such possible isomers, including racemic mixtures, enantiomerically enriched mixtures, diastereomeric mixtures, and optically pure forms. Optically active (R) -and (S) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a di-or tri-substituted cycloalkyl, the one or more cycloalkyl substituents may have a cis or trans configuration. The present disclosure includes cis and trans configurations of substituted cycloalkyl groups, and mixtures thereof. All tautomeric forms are also included. In particular, when the heteroaryl ring containing N as the ring atom is 2-pyridone, then for example tautomers are included in which the carbonyl group is described as hydroxy (e.g. 2-hydroxypyridine).

Pharmaceutically acceptable salts

As used herein, the term "salt" refers to an acid addition salt or a base addition salt of a compound of the present disclosure. "salt" includes in particular "pharmaceutically acceptable salt". The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure, and are typically not biologically or otherwise undesirable. Compounds of the present disclosure are capable of forming acid and/or base salts due to the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, formic acid, trifluoroacetic acid and the like.

Pharmaceutically acceptable base addition salts may be formed with inorganic and organic bases. Inorganic bases from which salts may be derived include, for example, ammonium salts and metals from columns I to XII of the periodic Table of the elements. In certain embodiments, the salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

Organic bases from which salts may be derived include, for example, primary, secondary and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, choline salts, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

In another aspect, the present disclosure provides a compound in the form: acetate, ascorbate, adipate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, decanoate, chloride/hydrochloride, chlorouronium (chlorohydrozincate), citrate, ethanedisulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodite/iodide, isethionate, lactate, lactobionic aldehyde, dodecyl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, mucinate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, triphenylacetate (triphenate), trifluoroacetate, or a form of naphthacene.

In another aspect, the present disclosure provides a compound in the form: sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, copper, isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine or tromethamine salt forms.

Preferably, the pharmaceutically acceptable salts of the compounds of formulae (I), (Ia), (Ib), (Ic), (Id) and (Ie) are acid addition salts.

Isotopically-labelled compounds

Any formulae given herein are also intended to represent unlabeled as well as isotopically labeled forms of the compounds. Isotopically-labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Co-ordination that may be incorporated into compounds of the present disclosureExamples of the potential element include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine and iodine, for example, respectively ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁸ O、 ¹⁵ N、 ¹⁸ F、 ¹⁷ O、 ¹⁸ O、 ³⁵ S、 ³⁶ Cl、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I. The present disclosure includes various isotopically-labeled compounds as defined herein, for example, wherein a radioisotope (e.g. ³ H and ¹⁴ c) Or to incorporate non-radioactive isotopes therein (e.g ² H and ¹³ c) Those of (3). Such isotopically-labeled compounds are useful in metabolic studies (with ¹⁴ C) Reaction kinetics studies (using, for example ² H or ³ H) Detection or imaging techniques (e.g., positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays), or for radiation therapy of a patient. In particular the number of the elements to be processed, ¹⁸ the F compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds having the formula (I "), (Ia '), (Ia), (Ib"), (Ib '), (Ib), (Ic "), (Ic '), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), and (Ie) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and general schemes (e.g., general schemes 1 to 5), using an appropriate isotopically-labeled reagent in place of the unlabeled previously used reagent.

In one embodiment of any aspect of the present disclosure, the hydrogen in the compound having formula (I), formula (I') or formula (I ") is present in its normal isotopic abundance. In another embodiment, the hydrogen is enriched in the form of the isotope deuterium (D), and in one particular embodiment of the present disclosure, one or more hydrogens of the Dihydropyrimidine (DHU) or uracil moiety in the compound having formula (I) or (I') are enriched in the form of D, e.g.

Deuterated dihydropyrimidine and uracil moieties can be prepared as described in Hill, R.K. et al, journal of Labelled Compounds and Radiopharmaceuticals [ labeled compounds and journal of radiopharmaceuticals ], volume XXII, phase 2, pages 143-148.

In addition, the use of heavier isotopes, particularly deuterium (i.e., ² h or D) may provide certain therapeutic advantages derived from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements or improved therapeutic index). It is to be understood that deuterium herein is considered to be a substituent of a compound having formulas (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie). The concentration of such heavier isotopes, in particular deuterium, may be defined by an isotopic enrichment factor. The term "isotopically enriched factor" as used herein means a ratio between the isotopic abundance and the natural abundance of a specified isotope. If substituents in compounds of the present disclosure indicate deuterium, such compounds have an isotopic enrichment factor for each named deuterium atom of at least 3500 (52.5% deuterium incorporation on each indicated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

Pharmaceutically acceptable solvates according to the present disclosure include those in which the crystallization solvent may be isotopically substituted, e.g., D ₂ O/d ₆ Acetone, d ₆ -DMSO。

The compounds of the present disclosure, i.e., compounds having the formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), and (Ie), containing a group capable of acting as a donor or acceptor of hydrogen bonds may be capable of forming co-crystals with a suitable co-crystal former. These co-crystals can be prepared from compounds having the formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie) by known co-crystal formation methods. Such methods include milling, heating, co-sublimating, co-melting, or contacting in solution compounds having the formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie) with a co-crystal former under crystallization conditions and isolating the co-crystal formed thereby. Suitable eutectic formers include those described in WO 2004/078163.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed.

Any asymmetric center (e.g., carbon, etc.) of one or more compounds of the present disclosure can exist in racemic or enantiomerically enriched form, such as the (R) -, (S) -or (R, S) -configuration. In certain embodiments, for example, as a mixture of enantiomers, each asymmetric center exists in at least 10% enantiomeric excess, at least 20% enantiomeric excess, at least 30% enantiomeric excess, at least 40% enantiomeric excess, at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess. In certain embodiments, for example in enantiomerically enriched form, each asymmetric center exists in at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess. Thus, the compounds of the present disclosure can exist in racemic mixtures or enantiomerically enriched forms or enantiomers, or as mixtures of diastereomers.

In the formula of the application, at C-sp ³ Terminology of the aboveIndicating absolute stereochemistry, (R) or (S). In the formula of the application, at C-sp ³ The term->Indicating absolute stereochemistry, (R) or (S). In the formula of the application, at C-sp ³ The term->Represents a covalent bond in which the stereochemistry of the bond is not defined. This means that, at C-sp ³ The term->Including the (S) or (R) configuration of the respective chiral center. In addition, mixtures may also be present. Thus, the present disclosure encompasses mixtures of stereoisomers, such as mixtures of enantiomers (e.g., racemates), and/or mixtures of diastereomers.

For the avoidance of doubt, for any R group (e.g. R in formula (I) ² ) When depicting structures of compounds having undefined stereochemistry, e.g. by bondBy this is meant that the asymmetric center has the (R) -or (S) -configuration, or is present as a mixture thereof and so stated.

Thus, as used herein, a compound of the present disclosure may be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers, or mixtures thereof, for example, in the form of substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers, racemates, or mixtures thereof.

Any resulting stereoisomer mixture may be separated into pure or substantially pure geometric or optical isomers, diastereomers, racemates based on the physicochemical differences of the components, for example by chromatography and/or fractional crystallization.

Any resulting racemate of a compound or intermediate of the present disclosure may be resolved into the optical isomers (enantiomers) by known methods, for example, by separating the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound. In particular, compounds of the present disclosure may thus be resolved into their optical enantiomers using basic moieties, for example by fractional crystallization with optically active acids, such as tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O, O' -p-toluoyltartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. The racemic compounds or racemic intermediates of the present disclosure can also be resolved by chiral chromatography (e.g., high Pressure Liquid Chromatography (HPLC) using chiral adsorbents).

In addition, the compounds of the present disclosure (including salts thereof) may also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present disclosure may form solvates, either inherently or by design, with pharmaceutically acceptable solvents (including water); accordingly, the present disclosure is intended to include both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of a compound of the present disclosure (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules (e.g., water, ethanol, etc.) are those commonly used in the pharmaceutical arts, which are known to be harmless to the recipient. The term "hydrate" refers to a complex in which the solvent molecule is water. One skilled in the art can use tools (e.g., NMR) to identify the presence of solvates.

The compounds of the present disclosure (including salts, hydrates, and solvates thereof) may form polymorphs either inherently or by design.

Preparation method

The compounds of the present disclosure can be prepared in a number of ways well known to those skilled in the art of organic synthesis. For example, compounds of the present disclosure may be synthesized using methods described below, as well as synthetic methods known in the art of synthetic organic chemistry or variations thereof as understood by those skilled in the art.

In general, compounds having formula (I), formula (I) and formula (I') can be prepared according to the schemes provided below.

General scheme 1

The starting materials for the above reaction schemes are commercially available or may be prepared according to methods known to those skilled in the art or by the methods disclosed herein. Generally, compounds of the present disclosure are prepared according to reaction scheme 1 above as follows:

in a polar solvent (e.g., N-Dimethylformamide (DMF)), a suitable ligand (e.g., dppf), and a base (e.g., potassium carbonate (K) ₂ CO ₃ ) Cross-coupling reactions in the presence of, for example, palladium (Pd) -catalyzed coupling of I-1 with a borane-based coupling partner having the formula I-2A (e.g., prepared by hydroboration of a suitable olefin with 9-BBN), can provide cross-coupled product I-3 (where X is CH) in step 1. Removal of the protecting group (e.g., boc) under acidic conditions at room temperature can provide the free amine I-4A, where z=2, 4-Dimethoxybenzyl (DMB). Alternatively, removal of the protecting group under acidic conditions and with heat may provide I-4B (step 2). I-4A and I-4B may then be converted to I-5A and I-5B, respectively, via reductive amination with the appropriate aldehyde (step 3-I) in the presence of a borohydride reagent, such as sodium borohydride acetate. Can be used for Alternatively, in an amine or basic carbonate and a polar solvent (e.g. Diisopropylethylamine (DIPEA) or potassium carbonate (K) ₂ CO ₃ ) And Dimethylformamide (DMF)) by alkylation with an appropriate alkyl halide, mesylate, tosylate or triflate (step 3-ii). Alternatively, the amide coupling reaction is carried out by reacting the compound with an appropriate carboxylic acid, an activator (e.g., HATU) and a base (e.g., DIPEA) (step 3-iii), at which time R ³ Forming an amide with the nitrogen to which it is attached. Alternatively, by acylation or sulfonylation with an appropriate acid chloride or sulfonyl chloride and a base (e.g., DIPEA or TEA) (step 3-iv), wherein R ³ Forming an amide or sulfonamide with the nitrogen to which it is attached. Removal of the protecting group of I-5A under acidic conditions and with heat can provide I-5B (step 4).

General scheme 2

The starting materials for the above reaction schemes are commercially available or may be prepared according to methods known to those skilled in the art or by the methods disclosed herein. Generally, compounds of the present disclosure are prepared according to reaction scheme 2 above as follows:

in an organic solvent (e.g., toluene) and water, a phosphine ligand (e.g., ruPhos or Xphos) and a base (e.g., cesium carbonate (Cs) ₂ CO ₃ ) Cross-coupling reactions in the presence of, for example, palladium (Pd) catalyzed coupling of I-1 with a trifluoroborate (potassium salt) coupling partner having the formula II-2B, wherein X is N, can provide cross-coupled products I-3 in step 1. The compound I-3 prepared in this manner can be converted to a compound having the formula I-5B by the method of steps 2-4 of general scheme 1.

General scheme 3

The starting materials for the above reaction schemes are commercially available or may be prepared according to methods known to those skilled in the art or by the methods disclosed herein. Generally, compounds of the present disclosure are prepared according to reaction scheme 3 above as follows:

cross-coupling reactions, such as nickel (Ni) -catalyzed coupling of I-1 with an alkyl bromide coupling partner having the formula III-2C, in the presence of a polar solvent (e.g., DMA), a salt (e.g., sodium iodide (NaI)), zinc (Zn), and a ligand (e.g., pyridine-2, 6-bis (formamidine) dihydrochloride) can provide cross-coupled product I-3 in step 1, where X is CH or CF. The compound I-3 prepared in this manner can be converted to a compound having the formula I-5B by the method of steps 2-4 of general scheme 1.

General scheme 4

In general scheme 4, a compound having formula I-5A or I-5B is subjected to oxidizing conditions (e.g., mnO) in a suitable solvent (e.g., toluene) (e.g., at room temperature) or in the presence of N, O-bis (trimethylsilyl) trifluoroacetamide ₂ ) To produce a compound of formula IV-5C (i.e., formula (I ') when z=h), followed by an optional deprotection step when the Z group represents a nitrogen protecting group, to give a compound of formula IV-5D (i.e., formula (I'))).

General scheme 5

In general scheme 5, the compound having formula I-5A is subjected to a claisen condensation followed by a selenization/oxidation/elimination sequence to give the compound having formula V-5E. The compound having formula V-5E is subjected to hydrolysis followed by copper catalyzed decarboxylation to give the compound having formula IV-5C. Subsequent deprotection, e.g., under acidic conditions and heating, provides compounds having formulas IV-5D (i.e., formulas (I') or (I ")).

For schemes 1 to 5, X, R ² 、R ³ N, m and p are as defined herein, particularly as defined in any one of embodiments 1 to 49.

In a further embodiment, there is provided a compound having formula I-1, which is

1- (5-bromopyrazolo [1,5-a ] pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione.

In a further embodiment, there is provided a compound having formula (X-1) or a salt thereof,

wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

z is selected from hydrogen and 2, 4-Dimethoxybenzyl (DMB);

R ^N selected from hydrogen and nitrogen protecting groups PG (e.g., t-butyloxycarbonyl (Boc));

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

In a further embodiment, there is provided a compound having formula (X) or a salt thereof,

wherein:

x is selected from CH, CF and N;

z is selected from hydrogen and 2, 4-Dimethoxybenzyl (DMB);

R ^N selected from hydrogen and nitrogen protecting groups PG (e.g. tert-butyloxycarbonyl (Boc))

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

In one embodiment of formula (X-1) or (X), PG is an acid labile protecting group.

In one embodiment of formula (X-1) or (X), PG is a Boc protecting group (t-butyloxycarbonyl).

In a further embodiment of formula (X-1) or (X), there is provided a compound or salt thereof selected from:

tert-butyl (2 s,4 r) -4- ((3- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

(S) -5-methyl-1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -2-methyl-4- ((3- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperazine-1-carboxylate;

tert-butyl (2 s,4 r) -2-methyl-4- ((3- (5-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperidine-1-carboxylate;

5-methyl-1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

5-fluoro-1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

Tert-butyl (2 s,4 r) -4- ((3- (5-fluoro-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

(S) -5-fluoro-1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -4- ((3- (5-fluoro-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate;

5-chloro-1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (2 s,4 r) -4- ((3- (5-chloro-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

5-methoxy-1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (2 s,4 r) -4- ((3- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

(S) -5-methoxy-1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -4- ((3- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate;

(S) -5-cyclopropyl-1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -4- ((3- (5-cyclopropyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate;

5-cyclopropyl-1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (2 s,4 r) -4- ((3- (5-cyclopropyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

(S) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -4- ((3- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate;

tert-butyl (2 s,4 r) -4- ((3- (3, 4-dimethylbenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate;

3- (3, 4-dimethylbenzyl) -1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

Tert-butyl 4- ((3- (3, 4-dimethylbenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperazine-1-carboxylate;

3- (3, 4-dimethylbenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

1- (5- (piperazin-1-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl 4- ((3- (3, 4-dimethylbenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperidine-1-carboxylate;

3- (3, 4-dimethylbenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione;

tert-butyl (S) -4- ((3- (3, 4-dimethylbenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate;

(S) -3- (3, 4-dimethylbenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione; and

(S) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione.

In one embodiment of formula (X-1) or (X), the salt is selected from the group consisting of HCl salt and TFA salt.

In a further aspect, the present disclosure provides a process for preparing a compound having formula (I), (I'), (I ") or a subformula thereof in free form or in pharmaceutically acceptable salt form, the process comprising the steps of:

1) The aryl bromide of formula (I-1) is coupled with a borane-based coupling partner of formula I-2A or II-2B under cross-coupling conditions to give a compound of formula (I-3) as defined herein.

The borane-based coupling partner of step 1 may optionally be prepared by hydroboration of a precursor olefin, for example with 9-BBN.

1) The aryl bromide of formula (I-1) is coupled with an alkyl bromide of formula (III-2C) under cross-coupling conditions to give a compound of formula (I-3) as defined herein.

The cross-coupling reaction conditions used in any of the preceding method steps or hereinbelow involve the reaction of a phosphine ligand (e.g., pd (OAc) ₂ And RuPhos or Xphos), and a base (e.g., cesium carbonate (Cs) ₂ CO ₃ ) In the presence of a suitable solvent (e.g., toluene, water, or mixtures thereof), a Pd catalyst is used.

Cross-coupling reaction conditions (e.g. in Sp ² -Sp ³ In the case of coupling) may alternatively include the use of Ni (II) complexes (e.g., niCl) ₂ (DME)), a ligand (e.g., pyridine-2, 6-bis (formamidine) dihydrochloride), an additive (e.g., naI), a metal transfer agent (e.g., zn or Mn), a suitable solvent (e.g., DMA), and heating at room temperature to 150 ℃ (e.g., 70 ℃) for a period of, for example, 12 hours.

In one embodiment of any of the method aspects above, there is provided the further step of:

2) Deprotection of a compound of formula (I) -3 to give a compound of formula (I) -4A or (I) -4B as defined herein;

3-a) reacting a compound having formula (I) -4A or (I) -4B under reductive amination conditions to give a compound having formula (I) -5A or (I) -5B as defined herein; or alternatively

3-B) reacting a compound having formula (I) -4A or (I) -4B under alkylation conditions to give a compound having formula (I) -5A or (I) -5B as defined herein; or alternatively

3-c) reacting a compound having formula (I) -4A or (I) -4B under amide coupling conditions to give a compound having formula (I) -5A or (I) -5B as defined herein; or alternatively

3-d) reacting a compound having formula (I) -4A or (I) -4B under acylation or sulfonylation conditions to give a compound having formula (I) -5A or (I) -5B as defined herein; and

4) Deprotection of a compound of formula (I) -5A gives a compound of formula (I) as defined herein.

The reductive amination conditions used in any of the foregoing process steps or hereinbelow involve the use of the corresponding aldehyde, a suitable hydride reagent (e.g., naBH (OAc) ₃ ) The reaction is carried out at room temperature (r.t.), a suitable solvent (e.g. DMF).

Alkylation reaction conditions for any of the foregoing process steps or following involve the reaction of a catalyst in a suitable base (e.g., DIPEA) or basic carbonate (e.g., K ₂ CO ₃ ) The reaction is carried out at a suitable temperature (e.g. room temperature to 100 ℃, e.g. 80 ℃) using the corresponding alkyl halide, mesylate, tosylate or triflate in the presence of a polar solvent (e.g. DMF), optionally under microwaves.

The amide coupling reaction conditions used in any of the preceding process steps or below involve conducting the reaction at a suitable temperature (e.g., room temperature) for a suitable time, e.g., 12 hours, using the corresponding carboxylic acid, activator (e.g., HATU), suitable base (e.g., DIPEA or NMM), suitable solvent (e.g., DMF).

The acylation or sulfonylation reaction conditions used in any of the preceding process steps or hereinafter include the use of the corresponding acid chloride or sulfonyl chloride in the presence of a suitable solvent (e.g., DCM) and a base (e.g., DIPEA or TEA), the reaction being carried out at a suitable temperature (e.g., room temperature).

In a further embodiment, a process for preparing a compound having formula (I') or (I ") is provided, the process comprising the steps of:

1) Under cross-coupling reaction conditions, a reaction product having the formulaAryl bromides of (wherein hal is halo, preferably I) and +.>Coupling to give a compound of formula (I) as defined hereinWherein>Is a double bond or a single bond, R ¹ 、R’、X、R ² 、n、m、p、R ^x Is as defined herein, for example, according to any one of enumerated examples 1 to 49, PG is a nitrogen protecting group (e.g., boc) as defined herein, and R ^N Selected from hydrogen and nitrogen protecting groups PG (e.g., t-butoxycarbonyl (Boc)). In one embodiment, R ¹ And R' are both hydrogen.

The cross-coupling conditions used in the foregoing process may involve the use of copper as a catalyst, such as ullmann reaction conditions. For example, the reaction conditions may use copper (I) iodide as a catalyst, a ligand (e.g., N- (2-cyanophenyl) picolinamide), a base (e.g., K) ₃ PO ₄ ) Suitable solvents (e.g., DMSO), heating at room temperature to 130 ℃ (e.g., 70 ℃ to 120 ℃, e.g., 110 ℃). The reaction may be heated for 72 hours.

In a further embodiment, a process according to any one of general schemes 1 to 5 for preparing a compound having formula (I), (I'), (I ") or a sub-formula thereof in free form or in pharmaceutically acceptable salt form is provided.

The compounds of formula (I) -1, (X-1) and (X) as defined herein may be used to prepare compounds of the present disclosure, for example compounds of formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie). Accordingly, in one aspect, the present disclosure relates to compounds having formula (I) -1, (X-1) or (X) or salts thereof. In another aspect, the present disclosure relates to the use of a compound having formula (I) -1, (X-1) or (X) or a salt thereof in the manufacture of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), and (Ie). The present disclosure further includes any variation of the methods of the present disclosure, wherein the intermediate product obtainable at any stage thereof is used as starting material and the remaining steps are performed, or wherein the starting material is formed in situ under the reaction conditions, or wherein the reaction components are used in the form of their salts or optically pure substances.

Pharmaceutical composition

In another aspect, the present disclosure provides pharmaceutical compositions comprising one or more compounds described herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutical composition" refers to a compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in a form suitable for oral or parenteral administration, and at least one pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that can be used to prepare or use a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delaying agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof, as known to those skilled in the art (see, e.g., remington The Science and Practice of Pharmacy [ leimington: pharmaceutical science and practice ], 22 nd edition, pharmaceutical Press [ pharmaceutical publishing company ],2013, pages 1049 to 1070).

In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. For purposes of this disclosure, solvates and hydrates are generally considered to be compositions unless specified otherwise. Preferably, the pharmaceutically acceptable carrier is sterile. The pharmaceutical compositions may be formulated for particular routes of administration, such as oral, parenteral, rectal, and the like. In addition, the pharmaceutical compositions of the present disclosure can be made in solid form (including but not limited to capsules, tablets, pills, granules, powders, or suppositories), or in liquid form (including but not limited to solutions, suspensions, or emulsions). The pharmaceutical compositions may be subjected to conventional pharmaceutical procedures, such as sterilization, and/or may be formulated to contain conventional inert diluents, lubricants or buffers, and adjuvants (e.g., preservatives, stabilizers, wetting agents, emulsifying agents, buffers, and the like).

Typically, the pharmaceutical composition is a tablet or gelatin capsule comprising the active ingredient and one or more of the following:

a) Diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine;

b) Lubricants, for example silica, talc, stearic acid, magnesium or calcium salts thereof and/or polyethylene glycol;

c) Binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone;

d) Disintegrants, for example starch, agar, alginic acid or a sodium salt thereof, or effervescent mixtures; and

e) Adsorbents, colorants, flavors, and sweeteners.

In one embodiment, the pharmaceutical composition is a capsule comprising only the active ingredient.

The tablets may be film coated or enteric coated according to methods known in the art.

Compositions suitable for oral administration include an effective amount of a compound of the present disclosure in the form of a tablet, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, or syrup or elixir, solution or solid dispersion. Compositions intended for oral use are prepared according to any method known in the art for manufacturing pharmaceutical compositions, and in order to provide pharmaceutically elegant and palatable preparations, such compositions may contain one or more agents selected from the group consisting of: sweeteners, flavoring agents, coloring agents, and preservatives. Tablets may contain the active ingredient in admixture with non-toxic, pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Such excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Formulations for oral use may be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Some injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, the composition may contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1% -75%, or contain about 1% -50% of the active ingredient.

Compositions suitable for transdermal applications include an effective amount of a compound of the present disclosure and a suitable carrier. Suitable carriers for transdermal delivery include absorbable pharmacologically acceptable solvents that aid in passage through the skin of the host. For example, transdermal devices are in the form of bandages comprising a backing, a reservoir containing a compound and optionally a carrier, a rate controlling barrier to deliver the compound to the skin of a host, optionally over an extended period of time, at a controlled and predetermined rate, and means for securing the device to the skin.

Compositions suitable for topical application (e.g., to the skin and eyes) include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such a topical delivery system would be particularly suitable for dermal applications, for example for the treatment of skin cancer, for example for prophylactic use in sunscreens, lotions, sprays and the like. It is therefore particularly suitable for topical use, including cosmetics, formulations well known in the art. Such systems may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein, topical application may also involve inhalation or intranasal application. They may be conveniently delivered in the form of a dry powder (alone as a mixture, e.g. dry blend with lactose, or mixed component particles, e.g. mixed component particles with phospholipids) from a dry powder inhaler or in the form of an aerosol spray from a pressurized container, pump, spray, nebuliser or nebulizer, with or without the use of a suitable propellant.

The compounds of formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie) in free form or in pharmaceutically acceptable salt form exhibit valuable pharmacological properties, such as WIZ regulatory properties or WIZ degradation properties or HbF induction properties, for example as shown in vitro assays provided in the examples, and thus are indicated to be useful in therapy or as research chemicals, for example as tool compounds.

Additional properties of the disclosed compounds include good potency, favorable safety, and favorable pharmacokinetic properties in the bioassays described herein.

Diseases and disorders

In one embodiment of the present disclosure, compounds of the present disclosure, or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, or tautomers thereof, are provided that are effective for reducing WIZ protein expression levels and/or inducing fetal hemoglobin (HbF) expression.

The compounds of the present disclosure may be used to treat one or more of the diseases or disorders described below. In one embodiment, the disease or disorder is affected by a decrease in the expression level of WIZ protein and/or induction of the expression level of fetal hemoglobin protein. In another embodiment, the disease or disorder is a hemoglobinopathy, such as beta hemoglobinopathy, including Sickle Cell Disease (SCD) and beta-thalassemia.

Application method

All the previous and following examples relating to methods of reducing the level of WIZ protein expression and/or inducing fetal hemoglobin (HbF) expression are equally applicable:

a compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of reducing the level of WIZ protein expression and/or inducing fetal hemoglobin (HbF) expression;

A compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment of the above-described diseases or disorders according to the present disclosure;

use of a compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the treatment of a disease or disorder described above according to the present disclosure; and

a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder described above according to the present disclosure.

In view of their activity as WIZ modulators or degradants, compounds of formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') and (Ie) in free or pharmaceutically acceptable salt form may be used to treat conditions that may be treated by, for example, modulating the expression level of WIZ protein, reducing the expression level of WIZ protein or inducing fetal hemoglobin (HbF) in a hematologic disorder (e.g., a hereditary hematologic disorder such as sickle cell disease or β -thalassemia). In one aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of inhibiting WIZ protein expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of degrading a WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein, the method comprising administering to the subject a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of inducing or promoting fetal hemoglobin in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of reactivating fetal hemoglobin production or expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of increasing fetal hemoglobin expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of treating a hemoglobinopathy, e.g., β -hemoglobinopathy, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of treating sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the present disclosure provides a method of treating β -thalassemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In one embodiment, heavy or moderate β -thalassemia is the result of homozygous null mutations or complex heterozygous mutations that result in β -globulin deficiency and phenotypic complications of β -thalassemia, whether transfusion dependent or not.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), or a pharmaceutically acceptable salt, hydrate, solvate, hydrate, or hydrate thereof, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate thereof, solvates, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of inhibiting WIZ protein expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, and/or a pharmaceutically acceptable salt thereof Stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of degrading WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia "), (Ib '), (Ib"), (Ib), (Ic "), (Ic), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, inter alia, of formula (I "), (Ia"), (Ib '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie') Stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein, the method comprising administering to the subject a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, and/or a pharmaceutically acceptable salt thereof Stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of inducing or promoting fetal hemoglobin in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, hydrate, or hydrate thereof, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of reactivating fetal hemoglobin production or expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate thereof, solvates, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of increasing fetal hemoglobin expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib"), (Ic '), (Ic), (Id "), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, or hydrate thereof, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating a hemoglobinopathy, e.g., β -hemoglobinopathy, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic'), (Ic), (Id"), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie') or (Ie), or a pharmaceutically acceptable salt thereof, hydrates, solvates, prodrugs, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id-1), (Id-2), (Id-3), (Ie"), (Ie ') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, and/or a pharmaceutically acceptable salt thereof, stereoisomers, or tautomers.

In another aspect, the present disclosure provides a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating β -thalassemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie') or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, hydrate, or hydrate thereof, prodrugs, stereoisomers, or tautomers.

Dosage of

For a subject of about 50-70kg, the pharmaceutical composition or combination of the present disclosure may be in a unit dose of about 1-1000mg of one or more active ingredients, or about 1-500mg or about 1-250mg or about 1-150mg or about 0.5-100mg, or about 1-50mg of active ingredient. The therapeutically effective dose of a compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or the severity thereof.

The above dose characteristics can be demonstrated in vitro and in vivo tests using advantageous mammals (e.g., mice, rats, dogs, monkeys) or isolated organs, tissues and preparations thereof. The compounds of the present disclosure can be used in vitro in the form of solutions (e.g., aqueous solutions), and in vivo, e.g., enterally, parenterally (advantageously, intravenously) in suspension or in aqueous solution. The in vitro dosage can be about 10 ^-3 Molar sum 10 ^-9 In the range between the molar concentrations. The in vivo therapeutically effective amount may range between about 0.1-500mg/kg, or between about 1-100mg/kg, depending on the route of administration.

The activity of a compound according to the present disclosure can be assessed by in vitro methods described in the examples.

Combination therapy

In another aspect, the present disclosure provides a pharmaceutical combination comprising a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more additional therapeutic agents for simultaneous, separate or sequential use in therapy. In one embodiment, the additional therapeutic agent is a bone marrow inhibitor, such as hydroxyurea.

Combination therapies include administering to a subject an additional combination of a compound with other bioactive ingredients (e.g., without limitation, a second, different anti-neoplastic agent or therapeutic agent targeting HbF or another cancer target) and non-drug therapies (e.g., without limitation, surgery or radiation therapy). For example, the compounds of the present application may be used in combination with other pharmaceutically active compounds (preferably, compounds capable of enhancing the effect of the compounds of the present application).

The compounds of the present disclosure may be administered simultaneously with, or before or after, one or more other therapeutic agents. The compounds of the present disclosure may be administered alone, by the same or different routes of administration, or together with other agents in the same pharmaceutical composition. A therapeutic agent is, for example, a chemical compound, peptide, antibody fragment, or nucleic acid that has therapeutic activity or enhances therapeutic activity when administered to a patient in combination with a compound of the disclosure. Thus, in one embodiment, the present disclosure provides a combination comprising a therapeutically effective amount of a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more additional therapeutically active agents.

In one embodiment, the present disclosure provides a product comprising a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), and at least one other therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is treatment of a disease or disorder modulated by WIZ. Products provided as a combined preparation include compositions comprising a compound having the formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie) together with one or more other therapeutic agents in the same pharmaceutical composition, or having the formula (I "), (I '), (I), (Ia"), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic '), (Ic), (Id '), (Id-1), (Id-2), (Id-3), (Ie "), (Ie '), or (Ie) and one or more other therapeutic agents in separate forms, for example in kit form.

In one embodiment, the present disclosure provides a pharmaceutical composition comprising a compound having formula (I "), (I '), (I), (Ia"), (Ia'), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic'), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie'), or (Ie), and one or more additional therapeutic agents. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier as described above.

In one embodiment, the present disclosure provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound having formula (I "), (I '), (I), (Ia"), (Ia '), (Ia), (Ib "), (Ib '), (Ib), (Ic"), (Ic), (Id "), (Id '), (Id-1), (Id-2), (Id-3), (Ie"), (Ie '), or (Ie). In one embodiment, the kit comprises means for separately retaining the compositions, such as a container, a separate bottle, or a separate foil pouch. Examples of such kits are blister packs, as are typically used for packaging tablets, capsules and the like.

Kits of the present disclosure can be used to administer different dosage forms (e.g., oral and parenteral), to administer separate compositions at different dosage intervals, or to titrate separate compositions relative to one another. To facilitate compliance, the kits of the present disclosure typically include instructions for administration.

In combination therapies of the present disclosure, the compounds of the present disclosure and the other therapeutic agents may be manufactured and/or formulated by the same or different manufacturers. Furthermore, the compounds of the present disclosure and other therapeutic agents may be put together in combination with combination therapies in the following cases: (i) Prior to release of the combination product to the physician (e.g., in the case of a kit comprising a compound of the present disclosure and another therapeutic agent); (ii) Shortly before administration, by the physician himself (or under the direction of the physician); (iii) In the patient himself, for example during sequential administration of the compound of the disclosure and another therapeutic agent.

Preparation of the Compounds

It is to be understood that in the following description, combinations of substituents and/or variables of the depicted formulae are permitted only when such combinations result in stable compounds.

It will also be appreciated by those skilled in the art that in the methods described below, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, phenol, amino, and carboxylic acid. Suitable protecting groups for hydroxyl or phenol include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, substituted benzyl, methyl, and the like. Suitable protecting groups for amino, amidino and guanidino groups include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters.

Protecting groups may be added or removed according to standard techniques, which are well known to those skilled in the art and as described herein. The use of protecting groups is described in detail in J.F.W.McOmie, "Protective Groups in Organic Chemistry [ protecting groups in organic chemistry ]", plenum Press [ Plenum Press ], london and New York 1973; T.W.Greene and P.G.M.Wuts, "Greene's Protective Groups in Organic Synthesis [ protecting group in Green organic Synthesis ]", fourth edition, wiley [ Wiley Verlag ], new York 2007; kocienski, "Protecting Groups [ protecting group ]", third edition, georg Thieme Verlag [ gerglatiramer publication ], stuttgart and new york 2005; and in "Methoden der organischen Chemie" (Methods of Organic Chemistry) [ organic chemistry methods ], houben Weyl, 4 th edition, volume 15/I, georg Thieme Verlag [ George, mei-Verlag ], stuttgart 1974.

The protecting group may also be a polymeric resin, such as Wang's (Wang) resin or 2-chlorotrityl-chloride resin.

The following reaction schemes illustrate methods for preparing compounds of the present disclosure. It will be appreciated that one skilled in the art will be able to manufacture these compounds by similar methods or by methods known to those skilled in the art. In general, the starting components and reagents may be obtained from sources such as Sigma Aldrich, lank synthesis limited (Lancaster Synthesis, inc.), maybridge, matrix science (Matrix Scientific), TCI, fluorochemistry (Fluorochem USA), strem, other commercial suppliers, or may be synthesized according to sources known to those skilled in the art, or prepared according to the present disclosure.

Analytical methods, materials and apparatus

Reagents and solvents as received from commercial suppliers were used unless otherwise indicated. Proton Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker Avance spectrometer or a Varian Oxford 400MHz spectrometer, unless otherwise indicated. Spectra are given in ppm (δ) and coupling constants J are reported in hertz. Tetramethylsilane (TMS) was used as an internal standard. Chemical shifts are reported in ppm relative to dimethyl sulfoxide (δ2.50), methanol (δ3.31), chloroform (δ7.26) or other solvents shown in NMR spectroscopic data. A small amount of the dried sample (2-5 mg) was dissolved in the appropriate deuterated solvent (1 mL). Chemical names were generated using ChemBioDraw Ultra v from cambridge soft.

Mass spectra (ESI-MS) were collected using a Waters system (acquisition UPLC and Micromass ZQ mass spectrometer) or Agilent-1260 informativity (6120 quadrupole); unless otherwise indicated, all masses reported are m/z of the protonated parent ion. The sample is dissolved in a suitable solvent (such as MeCN, DMSO, or MeOH) and injected directly into the column using an automated sample processor. Analysis was performed on a Waters Acquity UPLC system (column: waters Acquity UPLC BEH C18.7 μm,2.1x 30mm; flow rate: 1mL/min;55 ℃ (column temperature; solvent a: 0.05% formic acid in water, solvent B: 0.04% formic acid in MeOH; gradient: from 0 to 0.10min,95% solvent a; from 0.10 to 0.50min,95% solvent a to 20% solvent a; from 0.50 to 0.60min,20% solvent a to 5% solvent a; from 0.6 to 0.8min, hold at 5% solvent a; from 0.80 to 0.90min,5% solvent a to 95% solvent a; and hold at 95% solvent a) from 0.90 to 1.15 min).

Abbreviations:

ACN acetonitrile

AcOH acetic acid

AIBN azo-bis-isobutyronitrile

aq. Water-based

B ₂ pin ₂ Bis (pinacolato) diboron

9-BBN 9-boron bicyclo [3.3.1] nonane

Boc ₂ Di-tert-butyl O dicarbonate

Bn benzyl

BnBr benzyl bromide

br broad peak

d double peak

dd double peak

ddd double peak

ddq double quadruple peak

ddt double triplet

dq double quartet

dt double triplet

dtbbpy 4,4 '-di-tert-butyl-2, 2' -bipyridyl

dtd double triplet

Cs ₂ CO ₃ Cesium carbonate

DCE 1, 2-dichloroethane

DCM dichloromethane

DHP dihydropyran

DIBAL-H diisobutylaluminum hydride

DIPEA (DIEA) diisopropylethylamine

DIPEA N, N-diisopropylethylamine

DMA N, N-dimethylacetamide

DMAP 4-dimethylaminopyridine

DMB 2, 4-dimethoxy benzyl

DME 1, 2-dimethoxyethane

DMF N, N-dimethylformamide

DMP dess-Martin periodate or 1, 1-tris (acetoxy) -1, 1-dihydro-1, 2-phenyliodi-3- (1H) -one

DMSO dimethyl sulfoxide

EC ₅₀ Half maximum effective concentration

ELSD evaporative light scattering detector

EtOH ethanol

Et ₂ O-diethyl ether

Et ₃ N-triethylamine

EtOAc ethyl acetate

HATU 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

HCl hydrogen chloride

heptad peak of hept

HPLC high performance liquid chromatography

h or hr hours

HRMS high resolution mass spectrometry

g

g/min

IC ₅₀ Half maximal inhibitory concentration

IPA (iPrOH) isopropyl alcohol

Ir[(dF(CF ₃ )ppy) ₂ dtbbpy]PF ₆ [4,4' -bis (1, 1-dimethylethyl) -2,2' -bipyridine-N1, N1 ] ']Bis [3, 5-difluoro-2- [5- (trifluoromethyl) -2-pyridinyl-N ]phenyl-C]Iridium (III) hexafluorophosphate

K ₂ CO ₃ Potassium carbonate

KI potassium iodide

KOAc potassium acetate

K ₃ PO ₄ Tripotassium phosphate

LCMS liquid chromatography mass spectrometry

LDA lithium diisopropylamide

m multiple peaks

MeCN acetonitrile

MeOH methanol

mg

MHz megahertz (MHz)

min

mL of

mmol millimoles

M mole

MS mass spectrometry

NaH sodium hydride

NaHCO ₃ Sodium bicarbonate

NaBH(OAc) ₃ Sodium triacetoxyborohydride

Na ₂ SO ₄ Sodium sulfate

NBS N-bromosuccinimide

NMM N-methylmorpholine

NMP N-methyl-2-pyrrolidone

NMR nuclear magnetic resonance

on overnight

Pd/C palladium carbon

PdCl ₂ (dppf). DCM complexed [1,1' -bis (diphenylphosphino) ferrocene with dichloromethane]Palladium dichloride (II)

Pd(PPh ₃ ) ₄ Tetrakis (triphenylphosphine) palladium (0)

PMB p-methoxybenzyl

q quartet

qd quadruple double peak

quint five-element peak

quintd five-fold double peak

rbf round bottom flask

RockPhos G3 Pd [ (2-di-tert-butylphosphino-3-methoxy-6-methyl-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) -2- (2-aminobiphenyl) ] methanesulfonic acid palladium (II)

rt or r.t. room temperature

Rt retention time

RuPhos dicyclohexyl (2 ',6' -diisopropyloxy- [1,1' -biphenyl ] -2-yl) phosphanes unimodal

SEM 2- (trimethylsilyl) ethoxymethyl

SnBu ₃ Tributyltin

t triplet

td triplet double peak

tdd triple dual doublet

TBAI tetrabutylammonium iodide

TEA(NEt ₃ ) Triethylamine

TFA trifluoroacetic acid

TfOH triflic acid

THF tetrahydrofuran

THP tetrahydropyran

TMP 2, 6-tetramethylpiperidine

Ts tosyl group

triplet tt triplet

ttd triple triplet-doublet

TLC thin layer chromatography

UPLC ultra-high performance liquid chromatography

XPhos Pd G2 chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (II)

Microwaved mu W or uW

Preparation of intermediates

Preparation of 3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione.

Step 1.Tert-butyl (3- ((2, 4-dimethoxybenzyl) amino) -3-oxopropyl) carbamate.

To a solution of 3- ((tert-butoxycarbonyl) amino) propionic acid (200 g,1.06mol,1.00 eq.) in DCM (1200 mL) was added CDI (189 g,1.16mol,1.10 eq.). The reaction mixture was stirred at 20℃for 2h. The reaction mixture was slowly added to a solution of (2, 4-dimethoxyphenyl) methylamine (212 g,1.27mol,191mL,1.20 eq.) and DMAP (12.9 g,106mmol,0.10 eq.) in DCM (1000 mL). The solution was stirred at 20℃for 12h. The reaction mixture was slowly poured into water (2L) and stirred at room temperature for 10min. The organic phase was separated and the aqueous phase was extracted with DCM (800 ml x 2). The combined organic phases were washed with brine (1L x 2) and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 50:1 to 2:1 petroleum ether: ethyl acetate) to give a white solidT-butyl (3- ((2, 4-dimethoxybenzyl) amino) -3-oxopropyl) carbamate (210 g, 6271 mmol,59% yield). ¹ H NMR(400MHz,DMSO-d6)δ8.06(t,J＝5.6Hz,1H),7.08-7.01(m,1H),6.72(br t,J＝5.3Hz,1H),6.55-6.51(m,1H),6.46(dd,J＝2.4,8.4Hz,1H),4.13(d,J＝5.6Hz,2H),3.77(s,3H),3.73(s,3H),3.13(q,J＝7.0Hz,2H),2.28(t,J＝7.3Hz,2H),1.37(s,9H)。

And 2, step 2.3-amino-N- (2, 4-dimethoxybenzyl) propanamide hydrochloride

To a solution of tert-butyl (3- ((2, 4-dimethoxybenzyl) amino) -3-oxopropyl) carbamate (210 g, 6271 mmol,1.00 eq.) in DCM (1000 mL) was added HCl/dioxane (4 m,1000mL,6.45 eq.) and the mixture was stirred at 20 ℃ for 5h. The reaction mixture was concentrated to give 3-amino-N- (2, 4-dimethoxybenzyl) propanamide hydrochloride (180 g, crude, HCl salt) as a white solid, which was used in the next step without further purification. ¹ H NMR(400MHz,DMSO-d6)δ8.38(br t,J＝5.6Hz,1H),8.07(br s,3H),7.08(d,J＝8.3Hz,1H),6.53(d,J＝2.4Hz,1H),6.46(dd,J＝2.4,8.3Hz,1H),4.15(d,J＝5.6Hz,2H),3.75(d,J＝15.6Hz,6H),2.96(sxt,J＝6.3Hz,2H),2.59-2.52(m,2H)。

And 3, step 3.3- (2, 4-Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To a mixture of 3-amino-N- (2, 4-dimethoxybenzyl) propionamide hydrochloride (180 g, 650 mmol,1.00 eq.) and DIPEA (212 g,1.64mol, 284 mL,2.50 eq.) in DCE (1200 mL) was added CDI (127 g,786mmol,1.20 eq.) at 0deg.C. The mixture was stirred at 0 ℃ for 0.5h, then heated to 100 ℃ and stirred for 12h. The reaction mixture was slowly poured into water (1000 mL) and stirred at 20 ℃ for 20min. The organic phase was separated and the aqueous phase was extracted with DCM (500 ml x 2). The combined organic phases were washed with brine (500 ml x 2), dried over Na ₂ SO ₄ Dried, filtered and concentrated. The residue was purified by silica gel chromatography (45:1 to 0:1 petroleum ether: ethyl acetate) to give 3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione (120 g,454mmol,69% yield) as a white solid. ¹ H NMR(400MHz,CDCl ₃ )δ6.99(d,J＝8.3Hz,1H),6.48-6.37(m,2H),5.79(br s,1H),4.93(s,2H),3.80(d,J＝15.3Hz,6H),3.41(dt,J＝2.6,6.8Hz,2H),2.76(t,J＝6.8Hz,2H)。

Preparation of potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate.

Step 1.((3R) -4- (tert-Butoxycarbonyl) -3-methylpiperazin-1-ium-1-yl) methyl) trifluoroborate

To a solution of potassium (bromomethyl) trifluoroborate (2.00 g,9.96 mmol) in THF (10 mL) was added tert-butyl (R) -2-methylpiperazine-1-carboxylate (2.09 g,15.7 mmol). The reaction mixture was stirred at 80℃for 16h. The reaction mixture was filtered and the filter cake was washed with THF (2 x10 mL) and the filter cake was collected and dried to give (((3R) -4- (tert-butoxycarbonyl) -3-methylpiperazin-1-ium-1-yl) methyl) trifluoroborate as a white solid (4.3 g, crude). The crude product was used in the next step without any further purification. ¹ H NMR(400MHz,DMSO-d6)δ8.45-8.44(m,1H),4.31-2.92(m,1H),3.87-3.82(m,1H),3.67-3.54(m,1H),3.27-3.04(m,2H),2.99-2.77(m,2H),1.99(br s,2H),1.83-1.70(m,1H),1.50-1.37(m,9H),1.21(br d,J＝7.2Hz,3H)。

And 2, step 2.Potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate.

To a solution of (((3R) -4- (tert-butoxycarbonyl) -3-methylpiperazin-1-ium-1-yl) methyl) trifluoroborate (4.3 g, crude) in acetone (20 mL) was added K ₂ CO ₃ (2.10 g,15.2 mmol) and the reaction mixture was stirred at 25℃for 16h. The reaction mixture was filtered and the filter cake was washed with acetone (2 x 10 ml) and the filtrate was concentrated to give potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate (1.1 g, crude) as a white solid. The crude material was used in the next step without any further purification. ¹ H NMR(400MHz,DMSO-d6)δ4.09(br s,1H),3.79-3.60(m,1H),3.51-3.21(m,1H),2.98(br s,3H),1.71-1.46(m,2H),1.39(s,9H),1.15(d,J＝7.2Hz,3H)。

Additional borates prepared by the above process:

the borates in the following table are prepared by the method of potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate except as indicated using the appropriate commercially available piperazine in step 1.

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Preparation of (R) -1- (cyclohexylmethyl) -2- (methoxymethyl) piperazine hydrochloride.

Step 1.Tert-butyl (R) -4- (cyclohexylmethyl) -3- (hydroxymethyl) piperazine-1-carboxylic acid ester

To a stirred solution of tert-butyl (R) -3- (hydroxymethyl) piperazine-1-carboxylate (6.0 g,27.7 mmol) and cyclohexane-formaldehyde (4.6 g,41.6 mmol) in DCM (70 mL) was added Et ₃ N (11.7 mL,83.2 mmol). The reaction mixture was stirred at room temperature for 30min, then sodium triacetoxyborohydride (11.7 g,55.5 mmol) was added in portions at 0 ℃. The reaction mixture was allowed to stir at room temperature for 16h. The reaction was diluted with DCM and water, and the organic layer was taken up over Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -20% EtOAc in hexanes) to give tert-butyl (R) -4- (cyclohexylmethyl) -3- (hydroxymethyl) piperazine-1-carboxylate (4.2 g,13.4mmol,48% yield). LCMS [ M+H-tBu ]] ⁺ ：257.2。

And 2, step 2.Tert-butyl group(R) -4- (cyclohexylmethyl) -3- (methoxymethyl) piperazine-1-carboxylic acid ester

To a stirred solution of tert-butyl (R) -4- (cyclohexylmethyl) -3- (hydroxymethyl) piperazine-1-carboxylate (0.70 g,2.2 mmol) in DMF (10 mL) cooled to 0deg.C under an inert atmosphere was added NaH (0.13 g,3.36 mmol). The reaction mixture was stirred at 0deg.C for 30min, then MeI (0.47 g,3.36 mmol) was added at 0deg.C. The reaction was diluted with EtOAc and water, and the organic layer was dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -20% EtOAc in hexanes) to give tert-butyl (R) -4- (cyclohexylmethyl) -3- (methoxymethyl) piperazine-1-carboxylate (0.45 g,1.37mmol, 61%). LCMS [ M+H] ⁺ ：327.1

And 3, step 3.(R) -1- (cyclohexylmethyl) -2- (methoxymethyl) piperazine hydrochloride

To a stirred solution of tert-butyl (R) -4- (cyclohexylmethyl) -3- (methoxymethyl) piperazine-1-carboxylate (0.45 g,1.37 mmol) in DCM (7.0 mL) cooled to 0 ℃ was added HCl solution (4.0M in dioxane, 4.0 mL). The reaction mixture was stirred at room temperature for 3h and then concentrated. The crude compound was washed with diethyl ether to give (R) -1- (cyclohexylmethyl) -2- (methoxymethyl) piperazine hydrochloride (0.40 g, crude). LCMS [ M+H ] ⁺ ：227.1。

Preparation of (R) -1-isobutyl-2- (methoxymethyl) piperazine hydrochloride.

Prepared by the method of (R) -1- (cyclohexylmethyl) -2- (methoxymethyl) piperazine hydrochloride using isobutyraldehyde in step 1. LCMS [ M+H] ⁺ ：187.1。

Preparation of (R) -1- (cyclohexylmethyl) -2- (difluoromethyl) piperazine hydrochloride.

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To a stirred solution of tert-butyl (R) -3- (hydroxymethyl) piperazine-1-carboxylate (6.0 g,27.75 mmol) and cyclohexane-carbaldehyde (4.6 g,41.62 mmol) in DCM (70 mL) was added Et ₃ N (11.69 mL,83.25 mmol). The reaction mixture was stirred at room temperature for 30min. Sodium triacetoxyborohydride (11.7 g,55.50 mmol) was then added slowly at 0 ℃. The reaction mixture was stirred at room temperature for 16h. After completion, the reaction was diluted with DCM and water, and the organic layer was taken up in Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -20% EtOAc in hexanes) to give tert-butyl (R) -4- (cyclohexylmethyl) -3- (hydroxymethyl) piperazine-1-carboxylate (4.2 g,13.44mmol,48% yield). LCMS [ M+H-tBu ]] ⁺ ：257.2。

And 2, step 2.Tert-butyl (R) -4- (cyclohexylmethyl) -3-formylpiperazine-1-carboxylic acid ester

To a stirred solution of oxalyl chloride (2.04 mL,24.0 mmol) in DCM (25 mL) at-78deg.C under an inert atmosphere was added DMSO (3.41 mL,48.0 mmol) dropwise. The reaction mixture was stirred at-78 ℃ for 15min, then a solution of tert-butyl (R) -4- (cyclohexylmethyl) -3- (hydroxymethyl) piperazine-1-carboxylate (2.5 g,8.0 mmol) in DCM (5.0 mL) was added dropwise at-78 ℃. The reaction mixture was stirred at-78 ℃ for 1h and Et was slowly added ₃ N (11.24 mL,80.01 mmol). The reaction mixture was stirred at-78 ℃ for 1h and allowed to warm to room temperature. The reaction was diluted with DCM and water, and the organic layer was taken up over Na ₂ SO ₄ Dried, filtered and concentrated to give crude tert-butyl (R) -4- (cyclohexylmethyl) -3-formylpiperazine-1-carboxylate (2.7 g, crude). LCMS [ M+H] ⁺ ：311.1。

And 3, step 3.Tert-butyl (R) -4- (cyclohexylmethyl) -3- (difluoromethyl) piperazine-1-carboxylate

DAST (2.29 mL,17.4 mmol) was added to a stirred solution of tert-butyl (R) -4- (cyclohexylmethyl) -3-formylpiperazine-1-carboxylate (2.7 g,8.69 mmol) in DCM (30 mL) at 0deg.C under an inert atmosphere. The reaction mixture was stirred at 0 ℃ for 2h. After completion, the reaction was saturatedNaHCO ₃ The aqueous solution was quenched and diluted with DCM. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -15% etoac in hexanes) to give tert-butyl (R) -4- (cyclohexylmethyl) -3- (difluoromethyl) piperazine-1-carboxylate (0.41 g,1.23mmol,14% yield). LCMS [ M+H ] ⁺ ：333.5。

And 4, step 4.(R) -1- (cyclohexylmethyl) -2- (difluoromethyl) piperazine hydrochloride

To a stirred solution of tert-butyl (R) -4- (cyclohexylmethyl) -3- (difluoromethyl) piperazine-1-carboxylate (0.41 g,1.2 mmol) in DCM (7.0 mL) at 0 ℃ was added a solution of HCl in dioxane (4.0 m,4.0 mL). The reaction mixture was stirred at room temperature for 3h. After completion, the mixture was concentrated, and the crude compound was washed with diethyl ether to give (R) -1- (cyclohexylmethyl) -2- (difluoromethyl) piperazine hydrochloride (0.33 g,1.2mmol,100% yield). LCMS [ M+H] ⁺ ：232.9。

Preparation of 1- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine hydrochloride.

Step 1.Tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropionyl) piperazine-1-carboxylate

To a stirred solution of 3, 3-trifluoro-2, 2-dimethylpropionic acid (1.0 g,6.4 mmol) in DMF (15 mL) was added DIPEA (3.35 mL,19.2 mmol) under inert atmosphere followed by HATU (3.65 g,9.60 mmol). The reaction mixture was stirred at room temperature for 15min. After 15min, t-butylpiperazine-1-carboxylic acid ester (1.43 g,7.68 mmol) was added and the mixture was stirred at room temperature for 16h. The reaction was poured into cold water and the precipitate was filtered and dried under vacuum to give crude tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropionyl) piperazine-1-carboxylate (1.0 g,3.08, 48% yield). LCMS [ M+H-tBu ] ] ⁺ ：269.1。

And 2, step 2.Tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine-1-carboxylate

In the inert stateTo a stirred solution of tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropionyl) piperazine-1-carboxylate (1.0 g,3.08 mmol) in THF (15 mL) at 0 ℃ under an atmosphere was added BH ₃ DMS (15.4 mL,30.8mmol,1M in THF). The reaction mixture was then stirred at 50℃for 16h. The reaction was quenched with MeOH and concentrated. The residue was dissolved in DCM and sequentially washed with aqueous 2M NaHCO ₃ The solution and brine were washed. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -12% EtOAc in hexanes) to give tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine-1-carboxylate (0.5 g,1.61mmol,52% yield). LCMS [ M+H-tBu ]] ⁺ ：254.9。

And 3, step 3.1- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine hydrochloride

To a stirred solution of tert-butyl 4- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine-1-carboxylate (500 mg,1.61 mmol) in DCM (7 mL) was added 4M HCl in dioxane (3 mL) and the mixture was stirred at room temperature for 2h. The mixture was concentrated, and the residue was washed with diethyl ether to give 1- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazine hydrochloride (500 mg, crude). LCMS [ M+H ] ⁺ ：211.2。

Preparation of (3-iodopyrazolo [1,5-a ] pyridin-5-yl) methyl 4-methylbenzenesulfonate.

Step 1.3-iodopyrazolo [1,5-a ]]Pyridine-5-carboxaldehydes

NIS (1.4 g,5.92 mmol) was added to pyrazolo [1,5-a ] at 0deg.C]Pyridine-5-carbaldehyde (79mg, 5.41 mmol) in DMF (10 mL). The mixture was then stirred at room temperature for 8h. After completion, the reaction was quenched with water, and the precipitated solid was collected by filtration and dried under vacuum to give 3-iodopyrazolo [1,5-a]Pyridine-5-carbaldehyde (1.2 g,4.4mmol,81% yield). LCMS [ M+H] ⁺ ：273.0。

And 2, step 2.(3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methanol

At 0 ℃, 3-iodopyrazolo [1,5-a]To a stirred solution of pyridine-5-carbaldehyde (1.2 g,4.40 mmol) in MeOH: THF (2:1) (10 mL) was added NaBH ₄ (250 mg,6.60 mmol). The reaction mixture was stirred for 1h and then concentrated. The residue was diluted with water and extracted with DCM. The organic layer was washed with brine, dried over Na ₂ SO ₄ Drying, filtering and concentrating to obtain (3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methanol (800 mg, crude). LCMS [ M+H] ⁺ ：274.7。

And 3, step 3.(3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl 4-methylbenzenesulfonate

To (3-iodopyrazolo [1, 5-a)]To a stirred solution of pyridin-5-yl) methanol (700 mg,2.55 mmol) in DCM (10 mL) at 0deg.C was added TEA (0.8 mL,3.66 mmol). The mixture was stirred for 10min, then p-toluenesulfonyl chloride (610 mg,3.06 mmol) and DMAP (38 mg,0.25 mmol) were added. The reaction was stirred at room temperature for 1h. The mixture was then diluted with DCM and water, and the organic layer was taken up in Na ₂ SO ₄ Drying, filtering and concentrating to obtain (3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl 4-methylbenzenesulfonate (1.0 g, crude). The crude material was used without further purification.

Preparation of trans-3-methoxycyclobutane-1-carbaldehyde

To a solution of trans-3-methoxycyclobutylmethanol [ see WO 2021/124172,2021, A1] (0.30 g,2.6mmol,1.0 eq.) in DCM (15 mL) at 0deg.C was added DMP (1.2 g,2.8mmol,1.1 eq.). The reaction mixture was stirred at 0 ℃ for 2h. The reaction mixture was then concentrated. The crude material was purified by neutral alumina chromatography (eluting with 15% etoac in hexanes) to give trans-3-methoxycyclobutane-1-carbaldehyde as a colorless oil (0.21 g,1.8mmol,71% yield).

Preparation of cis-3-methoxycyclobutane-1-carbaldehyde.

To cis-3-methoxycyclobutylmethanol [ see WO 2021/124172,2021, A1]]To a solution of (0.10 g,0.86mmol,1.0 eq.) in DCM (7 mL) at 0deg.C was added DMP (0.40 g,0.94mmol,1.1 eq.). The reaction mixture was stirred at 0 ℃ for 2h. The reaction mixture was then concentrated. The crude material was taken up in Et ₂ O (10 mL) was diluted and filtered through celite, followed by Et ₂ And (3) washing. The filtrate was concentrated to give crude cis-3-methoxycyclobutane-1-carbaldehyde (0.12 g,1.1 mmol). The crude material was used in the next step without any further purification.

Preparation of (R) -3, 3-difluorocyclopentane-1-carbaldehyde.

To a solution of (R) - (3, 3-difluorocyclopentyl) methanol (0.230 g,1.69mmol,1.0 eq.) in DCM (5 mL) at 0deg.C was added DMP (0.788 g,1.86mmol,1.1 eq.). The reaction mixture was stirred at room temperature for 2h. The reaction mixture was then concentrated. The crude material was diluted with EtOAc (10 mL) and filtered through celite. The filtrate was washed with water, over MgSO ₄ Dried, filtered and concentrated. The crude material was purified by neutral alumina chromatography (eluting with 20% etoac in hexanes) to give (R) -3, 3-difluorocyclopentane-1-carbaldehyde as a yellow oil (0.13 g,0.97mmol,57% yield).

Preparation of (S) -3, 3-difluorocyclopentane-1-carbaldehyde.

To a solution of (S) - (3, 3-difluorocyclopentyl) methanol (0.170 g,1.24mmol,1.0 eq.) in DCM (10 mL) at 0deg.C was added DMP (0.258 g,1.37mmol,1.1 eq.). The reaction mixture was stirred at room temperature for 2h. The reaction mixture was then concentrated. The crude material was purified by neutral alumina chromatography (eluting with 20% EtOAc in hexanes) to give (S) -3, 3-difluorocyclopentane-1-carbaldehyde (0.15 g) as a colorless oil.

Preparation of (1 r,3R, 4S) -3, 4-difluorocyclopentane-1-carbaldehyde.

To ((1 r,3R, 4S) -3, 4-difluorocyclopentyl) methanol [ see EP 2275114,2011, A1 ]]To a solution of (0.170 g,1.24mmol,1.0 eq.) in DCM (10 mL) at 0deg.C was added DMP (1.58 g,3.74mmol,3.0 eq.). The reaction mixture was stirred at room temperature for 2h. The reaction mixture was then taken up in Et ₂ Dilute O and filter through neutral alumina before Et ₂ And (3) washing. The filtrate containing crude (1 r,3r,4 s) -3, 4-difluorocyclopentane-1-carbaldehyde was used in the next step without any further purification.

Preparation of 2-oxaspiro [3.3] heptane-6-carbaldehyde.

Balano (2-oxaspiro [3.3]]To a solution of heptane-6-yl) methanol (0.25 g,1.95mmol,1.0 eq.) in DCM (10 mL) at 0deg.C was added DMP (1.65 g,3.90mmol,2 eq.). The reaction mixture was stirred at room temperature for 1h. The reaction mixture was then filtered through celite and the filtrate was treated with NaHCO ₃ And (5) diluting the solution. The mixture was extracted with DCM and the DCM layer was washed with water, brine, over Na ₂ SO ₄ Dried and concentrated to give crude 2-oxaspiro [3.3] as a colorless oil]Heptane-6-carbaldehyde (0.1 g). The crude material was used without further purification.

The borates in the following table are prepared by the process of potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate using the appropriate commercially available piperazine in step 1.

Preparation of potassium (S) - ((1, 1-hexahydro-5H-isothiazolo [2,3-a ] pyrazin-5-yl) methyl) trifluoroborate.

Step 1.Tert-butyl (R) -4- (methylsulfonyl) -3- (((methylsulfonyl) oxy) methyl) piperazine-1-carboxylic acid Esters of

To tert-butyl (R) -3- (hydroxymethyl) piperazine-1-carboxylate (4.00 g,18.5 mmol) and Et ₃ To a solution of N (5.15 mL,37.0 mmol) in DCM (10 mL) at-20deg.C was added methanesulfonyl chloride (3.1 g,27.7 mmol). The reaction mixture was stirred for 10min and then saturated NaHCO ₃ Diluting the aqueous solution. The mixture was extracted with DCM and the organic layer was dried over MgSO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 10% MeOH in DCM) provided tert-butyl (R) -4- (methylsulfonyl) -3- (((methylsulfonyl) oxy) methyl) piperazine-1-carboxylate (3.0 g,8.1mmol,44% yield) as a viscous solid. The crude product was used in the next step without any further purification.

And 2, step 2.Tert-butyl (S) -hexahydro-5H-isothiazolo [2,3-a ]]Pyrazine-5-carboxylic acid ester 1, 1-dioxide

To a solution of tert-butyl (R) -4- (methylsulfonyl) -3- (((methylsulfonyl) oxy) methyl) piperazine-1-carboxylate (3.0 g,8.1 mmol) in THF (10 mL) at-78deg.C was added LHMDS solution (1.0M in THF, 24.3mL,24.3 mmol). The reaction mixture was stirred at-78 ℃ for 3h and then saturated NaHCO ₃ Diluting the aqueous solution. The mixture was extracted with DCM and the organic layer was dried over MgSO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 50% EtOAc in hexanes) afforded tert-butyl (S) -hexahydro-5H-isothiazolo [2,3-a as a white solid]Pyrazine-5-formate 1, 1-dioxide (2.5 g). ¹ H NMR (400 MHz, methanol-d 4) delta ppm 4.35-4.12 (m, 2H) 3.39-3.37 (m, 1H) 3.28-3.10 (m, 3H) 2.80-2.40 (m, 3H) 2.39-2.36 (m, 1H) 2.03-1.94 (m, 1H) 1.59 (s, 9H).

Step 3:(S) -hexahydro-2H-isothiazolo [2,3-a ]]Pyrazine 1, 1-dioxideHydrochloride salt

HCl solution (4.0M in dioxane, 3 mL) was added to a solution of tert-butyl (S) -hexahydro-5H-isothiazolo [2,3-a ] pyrazine-5-carboxylate 1, 1-dioxide (2.5 g,9.0 mmol) and the mixture stirred at room temperature for 2H. The reaction was then concentrated to give the crude (S) -hexahydro-2H-isothiazolo [2,3-a ] pyrazine 1, 1-dioxide hydrochloride salt, which was used without further purification.

And 4, step 4.(S) - ((1, 1-hexahydro-5H-isothiazolo [2, 3-a)]Pyrazin-5-yl) methyl) trifluoroboric acid Potassium

To a solution of (S) -hexahydro-2H-isothiazolo [2,3-a ] pyrazine 1, 1-dioxide hydrochloride salt (1.7 g,8.0 mmol) in THF (20 mL) was added NaH (0.463 g,19.3 mmol), (bromomethyl) potassium trifluoroborate (1.9 g,9.6 mmol) and tetrabutylammonium iodide (0.178 g, 0.480 mmol). The reaction mixture was stirred at room temperature for 16h. The reaction mixture was then concentrated to give potassium (S) - ((1, 1-hexahydro-5H-isothiazolo [2,3-a ] pyrazin-5-yl) methyl) trifluoroborate (3 g, crude) as a white solid.

Preparation of tert-butyl (2S) -4- (bromomethyl) -4-fluoro-2-methylpiperidine-1-carboxylate.

To tert-butyl (S) -2-methyl-4-methylenepiperidine-1-carboxylate [ see example 71 ]]To a solution of (400 mg,1.89 mmol) in DCM (10 mL) at 0deg.C was added triethylamine trihydrofluoride (0.77 mL,4.73 mmol). The reaction mixture was stirred at 0deg.C for 30min, then NBS (500 mg,2.83 mmol) was added. The mixture was stirred at room temperature for 2h. The reaction mixture was then treated with saturated NaHCO ₃ The aqueous solution was basified and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel chromatography (eluting with 10% EtOAc/hexanes) to give tert-butyl (2S) -4- (bromomethyl) -4-fluoro-2-methylpiperidine-1-carboxylate (0.35 g,60% yield) as a mixture of diastereomers, which was used without further purification.

Preparation of (cis) -4- (bromomethyl) -1-isobutyl-2- (trifluoromethyl) piperidine.

Step 1.(cis) -methyl-1-isobutyl-2- (trifluoromethyl) piperidine-4-carboxylic acid ester

Isobutyraldehyde (0.767 g,10.7 mmol) and triethylamine (3.07 mL,21.3 mmol) were added to (cis) -methyl 2- (trifluoromethyl) piperidine-4-carboxylate [ see WO 2021/158948,2021, A1 ]](1.5 g,7.1 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 30min, then sodium triacetoxyborohydride (4.51 g,21.3 mmol) was added. The reaction mixture was stirred at room temperature for 4h and then saturated NaHCO ₃ The aqueous solution was quenched and extracted three times with DCM. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated to give crude (cis) -methyl 1-isobutyl-2- (trifluoromethyl) piperidine-4-carboxylate (1.0 g) which was used without further purification.

And 2, step 2.(cis) - (1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methanol

LAH solution (2M in THF, 1.02mL,2.05 mmol) was added to a solution of (cis) -methyl 1-isobutyl-2- (trifluoromethyl) piperidine-4-carboxylate (0.5 g,1.87 mmol) in THF (5 mL) at 0deg.C. The mixture was stirred at 0 ℃ for 2h and then quenched with EtOAc. The mixture was washed with saturated aqueous ammonium chloride solution, and the organic layer was washed with Na ₂ SO ₄ Dried, filtered and concentrated to give crude (cis) - (1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methanol (150 mg) which was used without further purification.

And 3, step 3.(cis) -4- (bromomethyl) -1-isobutyl-2- (trifluoromethyl) piperidine

Triphenylphosphine (427 mg,1.62 mmol) and carbon tetrabromide (537 mg,1.62 mmol) were added in portions to a solution of (cis) - (1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methanol (130 mg,0.54 mmol) in DCM (4 mL) at 0deg.C. The reaction mixture was stirred at room temperature for 2h. After completion of the reaction, the mixture was diluted with DCM and washed with water And (5) washing. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 0-50% EtOAc in hexanes) to give (cis) -4- (bromomethyl) -1-isobutyl-2- (trifluoromethyl) piperidine (60 mg,0.20mmol,37% yield).

Preparation of example Compounds

Example 1.preparation of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 1)

Step 1:1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -diones

3- (2, 4-Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (400 mg,1.51 mmol), 5-bromo-3-iodopyrazolo [1,5-a ] in a microwave vial]A mixture of pyridine (499 mg,1.54 mmol), cesium carbonate (986 mg,3.03 mmol) and CuI (57.7 mg,0.303 mmol) in 1, 4-dioxane (12 mL) was purged with nitrogen. (. + -.) -trans-1, 2-diaminocyclohexane (0.036 mL,0.30 mmol) was added and the mixture was again purged with nitrogen. The vials were capped and heated in a heated block at 80 ℃ overnight. The reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 0-100% EtOAc in heptane) afforded 1- (5-bromopyrazolo [1, 5-a) as a pale yellow foamy solid ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -d. LCMS [ M+H] ⁺ ：459.2。 ¹ H NMR(500MHz,CDCl ₃ )δ8.23(dd,J＝7.3,0.8Hz,1H),7.90(s,1H),7.49(dd,J＝2.1,0.8Hz,1H),7.14(d,J＝8.9Hz,1H),6.86(dd,J＝7.3,2.1Hz,1H),6.49-6.39(m,2H),5.03(s,2H),3.83(s,3H),3.80(m,5H),2.96(t,J＝6.6Hz,2H)。

Step 2:tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) Pyrazolo [1,5-a]Pyridine-5-Methyl) piperidine-1-carboxylic acid ester

a) A microwave vial containing tert-butyl 4-methylenepiperidine-1-carboxylate (500 mg,2.53 mmol) was purged with nitrogen for 15min before a solution of 9-BBN (0.5M in THF, 5.07mL,2.53 mmol) was added. The vials were capped and the mixture was heated at 80 ℃ for 3.5h and then cooled to room temperature.

b) The reaction mixture from part a was added via syringe to a mixture containing 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1048 mg, 2.281mmol), K ₂ CO ₃ (438 mg,3.17 mmol) and PdCl ₂ (dppf).CH ₂ Cl ₂ The adduct (54 mg,0.066 mmol) was in a microwave vial of a mixture of DMF (14 mL) and water (1.4 mL). The vials were capped and the reaction mixture was heated at 60 ℃ overnight. The reaction mixture was then cooled to room temperature and diluted with ethyl acetate and washed sequentially with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated. Silica gel column chromatography (eluting with 0-100% EtOAc in heptane) provided tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as a pale yellow foamy solid ]Pyridin-5-yl) methyl) piperidine-1-carboxylic acid ester. LCMS [ M+H] ⁺ ：578.4。

Step 3:3- (2, 4-Dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride

HCl solution (4.0M in dioxane, 15ml,60 mmol) was added to tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl piperidine-1-carboxylate (1270 mg,2.154 mmol) and the mixture was stirred at room temperature for 2h. The reaction was then concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride. LCMS [ M+H] ⁺ ：478.4。

Step 4:1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H,3H) -diketones

To 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (100 mg,0.195 mmol) in DMF (3 mL) was added potassium carbonate (81 mg,0.58 mmol) and (bromomethyl) cyclohexane (0.081 mL,0.58 mmol). The mixture was heated at 80 ℃ for 4h and then cooled to room temperature. The mixture was diluted with ethyl acetate and washed sequentially with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：574.4。

Step 5:1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydro Pyrimidine-2, 4 (1H, 3H) -diones

TFA (2 mL,26 mmol) was added to crude 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (30 mg,0.0525 mmol) and the mixture was heated at 80℃overnight. The mixture was cooled to room temperature, concentrated, and the residue was dissolved in toluene and concentrated again. The residue was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% formic acid. The product-containing fractions were combined, frozen and lyophilized to give 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]A formate salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：424.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.44(s,1H),8.58(d,J＝7.1Hz,1H),8.16(s,1H),8.00(s,1H),7.37(d,J＝1.8Hz,1H),6.79(dd,J＝7.2,1.9Hz,1H),3.77(t,J＝6.7Hz,2H),3.59-3.26(m,2H),3.19(d,J＝12.2Hz,3H),2.79(t,J＝6.7Hz,2H),2.60(d,J＝6.8Hz,2H),2.56(s,1H),1.82-1.58(m,9H),1.41(q,J＝12.4Hz,2H),1.30-1.09(m,3H),0.96-0.82(m,2H)。

The compounds in the following table were prepared by the method of example 1 using the appropriate commercially available halides, mesylate, tosylate or triflate in step 4.

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Example 63.1- (5- ((1- (piperidin-4-ylmethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 63)Is prepared from

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By the method of example 1, t-butyl is used in step 4The base 4- (bromomethyl) piperidine-1-carboxylic acid ester was prepared instead of (bromomethyl) cyclohexane. LCMS [ M+H] ⁺ ：425.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.45(d,J＝6.6Hz,1H),8.61(dd,J＝7.2,3.9Hz,1H),8.40(d,J＝11.9Hz,1H),8.02(d,J＝3.2Hz,1H),7.47-7.35(m,1H),6.80(dd,J＝7.2,1.9Hz,1H),3.78(td,J 6.7,4.0Hz,2H),3.51(d,J＝11.8Hz,2H),3.30(d,J＝12.8Hz,2H),2.98(t,J＝6.1Hz,2H),2.94-2.82(m,4H),2.79(t,J＝6.7Hz,2H),2.62(d,J＝6.6Hz,2H),2.09(dt,J＝7.7,3.8Hz,1H),1.84(dq,J＝29.1,16.0,14.5Hz,5H),1.50(q,J＝13.1Hz,2H),1.41-1.28(m,2H)。

Examples 64 and 65.1- (5- ((1- (((1 r,4 r) -4-methoxycyclohexyl) methyl) piperidin-4-yl) methyl) pyri-dine Azolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 64) and 1- (5- ((1- (((1 s,4 s) -4- Methoxy cyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) s Diketone (example 65)Is prepared from

Prepared by the method of example 1 using a commercially available mixture of cis and trans 1- (bromomethyl) -4-methoxycyclohexane instead of (bromomethyl) cyclohexane in step 4. These stereoisomers were purified by reverse phase HPLC (eluting with ACN/water/0.1% TFA) after step 5.

Example 64.1- (5- ((1- (((1 r,4 r) -4-methoxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

The minor isomer eluted first. LCMS [ M+H] ⁺ ：454.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.45(d,J＝4.6Hz,1H),8.60(d,J＝7.1Hz,1H),8.01(s,1H),7.47-7.27(m,1H),6.80(dd,J＝7.1,1.9Hz,1H),3.78(td,J＝6.7,3.0Hz,2H),3.47(d,J＝12.1Hz,2H),3.24(d,J＝5.2Hz,4H),3.05(ddt,J＝16.9,10.7,5.3Hz,1H),2.93-2.83(m,3H),2.79(t,J＝6.7Hz,2H),2.62(d,J＝6.6Hz,2H),2.00(d,J＝12.3Hz,2H),1.92-1.63(m,6H),1.49(q,J＝13.1Hz,2H),1.19-1.07(m,2H),0.98(q,J＝12.9Hz,2H)。

Example 65.1- (5- ((1- (((1 s,4 s) -4-methoxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

The second eluting is the major isomer. LCMS [ M+H] ⁺ ：454.3。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.45 (d, j=7.1 hz, 1H), 8.02 (s, 1H), 7.49-7.25 (m, 1H), 6.85 (dd, j=7.1, 1.8hz, 1H), 3.90 (t, j=6.8 hz, 2H), 3.61-3.44 (m, 3H), 3.35 (s, 3H), 2.92 (dt, j=13.6, 6.9hz, 6H), 2.71 (d, j=7.1 hz, 2H), 2.09-1.82 (m, 6H), 1.70-1.46 (m, 6H), 1.44-1.31 (m, 2H).

Example 66.1- (5- (((1R, 5S) -8- (cyclohexylmethyl) -8-azabicyclo [ 3.2.1)]Octane-3-yl) methyl ester Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

By the method of example 1, steps 2-5, tert-butyl (1R, 5S) -3-methylene-8-azabicyclo [3.2.1 was used in step 2]Octane-8-formate is used for replacing tert-butyl 4-methylene piperidine-1-formate. LCMS [ M+H] ⁺ ：450.4。 ¹ H NMR(400MHz,DMSO-d6)δ10.43(s,1H),8.53-8.52(m,1H),8.29-8.20(m,1H),7.97(d,J＝2.4Hz,1H),7.42-7.29(m,1H),6.77(d,J＝7.2Hz,1H),3.75-3.60(m,2H),3.23(br s,2H),2.84-2.70(m,3H),2.24(br s,2H),2.11-1.82(m,4H),1.81-1.56(m,6H),1.56-1.37(m,4H),1.33-1.09(m,4H),0.91-0.78(m,2H)。

Example 67.1- (5- (((1R, 5S) -8-isobutyl-8-azabicyclo [ 3.2.1)]Octane-3-yl) methyl) pyrazole And [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

By the method of example 1, steps 2-5, tert-butyl (1R, 5S) -3-methylene-8-azabicyclo [3.2.1 was used in step 2]Octane-8-formate substituted tert-butyl 4-methylenepiperidine-1-methylAcid esters were prepared and 1-iodo-2-methylpropane was used instead of (bromomethyl) cyclohexane in step 4. LCMS [ M+H ] ⁺ ：410.3。 ¹ H NMR(400MHz,CDCl ₃ )ppm 11.33(s,1H),8.40(m,J＝6.8Hz,1H),7.94(s,1H),7.75(s,1H),6.67(m,J＝7.3,11.7Hz,1H),3.93-3.84(m,4H),3.19(s,1H),2.97-2.89(m,3H),2.78-2.71(m,4H),2.42-2.25(m,2H),2.15(s,1H),1.92(m,J＝8.6Hz,2H),1.68(s,3H),1.14(m J＝6.4Hz,6H)。

Example 68.1- (5- (((1R, 5S) -8- (pyridin-3-ylmethyl) -8-azabicyclo [ 3.2.1)]Octane-3-yl Methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

By the method of example 1, steps 2-5, tert-butyl (1R, 5S) -3-methylene-8-azabicyclo [3.2.1 was used in step 2]Octane-8-carboxylate instead of tert-butyl 4-methylenepiperidine-1-carboxylate and 3- (bromomethyl) pyridine instead of (bromomethyl) cyclohexane was used in step 4. LCMS [ M+H] ⁺ ：445.3。 ¹ H NMR 1H NMR (400 MHz, methanol-d 4) ppm=8.54 (s, 1H), 8.46-8.34 (m, 2H), 8.00-7.96 (m, 1H), 7.89 (m, j=7.8 hz, 1H), 7.44-7.30 (m, 2H), 6.81 (d, j=2.1, 7.1hz, 1H), 3.88 (dt, j=4.6, 6.7hz, 2H), 3.60 (d, j=4.4 hz, 2H), 3.18 (s, 2H), 2.89 (m, 3H), 2.58 (d, j=7.2 hz, 1H), 2.23-2.17 (m, 1H), 2.10-2.03 (m, 2H), 1.94-1.81 (m, 1H), 1.68-1.60 (m, 1H), 1.49 (d, j=4.4 hz, 2H), 3.18 (s, 2H), 2.23-2.17 (m, 1H), 2.10-2.03 (m, 2H).

EXAMPLE 69 preparation of 1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.3- (2, 4-Dimethoxybenzyl) -1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]To a stirred solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (100 mg,0.209 mmol) and 2-chloro-4-methoxypyrimidine (30.2 mg,0.209 mmol) in MeCN (1 mL) was added DIPEA (54 mL,0.418 mmol). The mixture was stirred at 120℃for 2h. After completion, the reaction was cooled to room temperature and diluted with EtOAc and water. The organic layer was purified by Na ₂ SO ₄ Drying and concentrating. The crude compound was purified by silica gel chromatography (eluting with 10% MeOH in DCM) to give 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (90 mg,0.153mmol,90% yield). LCMS [ M+H] ⁺ ：586.3。

And 2, step 2.1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine- 3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]To a stirred solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (90 mg,0.145 mmol) in TFA (1 mL) was added TfOH (0.1 mL) and the reaction mixture was stirred at 70℃for 2h. After completion, the reaction was concentrated. The crude compound was purified by PREP HPLC using: mobile phase: a = 0.1% HCOOH in water, B = acetonitrile, column: JUPITER Phenomenex (250 mm. Times.21.2 mm), 5.0 μm, flow rate: 20mL/min. The collected fractions were concentrated under reduced pressure to give 1- (5- ((1- (4-methoxypyrimidin-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) as an off-white solid ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (25 mg,0.053mmol,27% yield). LCMS [ M+H] ⁺ ：436.2；HPLC：Rt＝4.794min。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.43 (d, j=7.1 hz, 1H), 8.00 (s, 1H), 7.96 (d, j=6.4 hz, 1H), 7.37 (s, 1H), 6.85 (dd, j=7.1, 1.8hz, 1H), 6.21 (d, j=6.5 hz, 1H), 4.56 (d, j=9.6 hz, 2H), 3.89 (t, j=6.8 hz, 2H), 3.05 (t, j=12.9 hz, 2H), 2.89 (t, j=6.8 hz, 2H), 2.68 (d, j=7.3 hz, 2H), 2.10-1.97 (m, 1H), 1.83 (d, j=13.2 hz, 2H), 1.33 (qd, j=12.3, 3.3hz, 3H), no solvent exchange was observed due to NH.

EXAMPLE 70 preparation of 1- (5- ((1- (3-methylbutan-2-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1- (3-methylbutan-2-yl) -4-methylenepiperidine

To a solution of 4-methylenepiperidine hydrochloride (2.0 g,15 mmol) in DCM (20 mL) was added TEA (6.25 mL,44.9 mmol), tiCl4 (0.8 mL,7.48 mmol) and 3-methylbutan-2-one (1.4 g,16.5 mmol). The mixture was stirred at room temperature for 12h, then NaBH was added ₃ CN (2.8 g,45 mmol). The reaction was stirred at room temperature for 4h. After completion, the mixture was diluted with DCM and washed with water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give 1- (3-methylbutan-2-yl) -4-methylenepiperidine (0.4 g, crude). ¹ H NMR(300MHz,CDCl ₃ )δ4.60(s,2H),2.61-2.53(m,2H),2.36-2.31(m,2H),2.25-2.09(m,4H),1.65-1.59(m,1H),1.11-1.06(m,3H),0.96-0.92(m,3H),0.89-0.85(m,3H)。

And 2, step 2.1- (5- ((1- (3-methylbutan-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 70) Prepared from 1- (3-methylbutan-2-yl) -4-methylenepiperidine using the procedure of example 1, steps 2 and 5, using 1- (3-methylbutan-2-yl) -4-methylenepiperidine instead of tert-butyl 4-methylenepiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：398.3。 ¹ H NMR(300MHz,CD ₃ OD) δ8.42 (d, j=7.2 hz, 1H), 8.00 (s, 1H), 7.35 (s, 1H), 6.82 (dd, j=7.2 hz,1.2hz, 1H), 3.87 (t, j=6.4 hz, 2H), 3.58-3.47 (m, 2H), 3.13-2.96 (m, 3H), 2.87 (t, j=6.8 hz, 2H), 2.69 (d, j=6.4 hz, 2H), 2.24-2.20 (m, 1H), 1.98-1.94 (m, 3H), 1.59-1.55 (m, 2H), 1.27 (d, j=6.8 hz, 3H), 1.04 (d, j=6.8 hz, 3H), 0.98 (d, j=6.8 hz, 3H), no NH was observed due to the replacement of the proton solvent.

EXAMPLE 71 preparation of 1- (5- (((2S, 4S) -1- (cyclohexylmethyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (S) -2-methyl-4-methylenepiperidine-1-carboxylic acid ester。

To dry t-BuOK (1.58 g,14.1 mmol) in THF (20 mL) was added methyltriphenylphosphonium bromide (5.02 g,14.07 mmol) at 0deg.C, and the mixture was stirred at room temperature for 2h. The mixture was cooled to 0 ℃ and a solution of tert-butyl (S) -2-methyl-4-oxopiperidine-1-carboxylate (2 g,9.38 mmol) in THF (5 mL) was slowly added. The reaction mixture was stirred at room temperature for 14h. The reaction mixture was treated with saturated NH ₄ Aqueous Cl (50 mL) was quenched and extracted with EtOAc (2X). The combined organic layers were concentrated to give the crude product. The crude product was purified by flash chromatography on silica gel (eluting with 0-10% EtOAc/petroleum ether) to give tert-butyl (S) -2-methyl-4-methylenepiperidine-1-carboxylate (1.7 g,8.1mmol,86% yield) as a yellow oil. ¹ H NMR(400MHz,CDCl ₃ )δ4.85(d,J＝1.6Hz,1H),4.74(d,J＝1.6Hz,1H),4.51-4.48(m,1H),4.04-4.01(m,1H),2.89-2.82(m,1H),2.42-237(m,1H),2.17-2.13(m,2H),2.03-2.00(m,1H),1.47(s,9H),1.07(d,J＝6.8Hz,3H)。

And 2, step 2.1- (5- (((2S, 4S) -1- (cyclohexylmethyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5 ] a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H,3H) Diketone (example 71)) Prepared from tert-butyl (S) -2-methyl-4-methylenepiperidine-1-carboxylate using the method of example 1, steps 2-5, wherein tert-butyl (S) -2-methyl-4-methylenepiperidine-1-carboxylate was used instead of tert-butyl 4-methylenepiperidine-1-carboxylate. The final product contains a small amount of trans isomer, which is removed by SFC purification: column: chiralpak IG-3.50X4.6mm I.D.,3 μm; mobile phase: phase A is CO ₂ Phase B is IPA (0.05% DEA); gradient elution: in CO ₂ 40% IPA (0.05% DEA); flow rate: 3mL/min; a detector: a PDA; column temperature: 35 ℃; back pressure: 100 bar. The product was peak 1 with a retention time of 3.1min. LCMS [ M+H] ⁺ ：438.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.40 (d, j=7.2 hz, 1H), 7.98 (s, 1H),7.33(s,1H),6.81-6.79(m,1H),3.89-3.86(m,2H),3.04(br d,J＝12.0Hz,1H),2.89-2.87(m,2H),2.66-2.56(m,3H),2.20-1.84(m,4H),1.75-1.44(m,8H),1.37-1.11(m,5H),1.10-1.03(m,3H),0.99-0.81(m,2H)。

The compounds in the following table were prepared by the method of example 71 using the appropriate commercially available halides in the alkylation step.

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The compounds in the following table were prepared using the method of example 71, using tert-butyl (R) -2-methyl-4-oxopiperidine-1-carboxylate instead of tert-butyl (S) -2-methyl-4-oxopiperidine-1-carboxylate and using the appropriate commercially available halide in the alkylation step.

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Example 79.1- (5- ((1-isobutyl-2, 2-dimethylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine-3- Radical) dihydropyrimidine-2, 4 (1H, 3H) -dioneIs prepared from

Prepared using the method of example 71, using tert-butyl 2, 2-dimethyl-4-oxoPiperidine-1-carboxylic acid ester replaces tert-butyl (S) -2-methyl-4-oxopiperidine-1-carboxylic acid ester. LCMS [ M+H] ⁺ ：412.6。 ¹ H NMR(400MHz,CD ₃ OD)δ8.44(d,J＝7.2Hz,1H),8.39(s,1H),8.02(s,1H),7.36(s,1H),6.85-6.83(m,1H),3.90(t,J＝7.2Hz,2H),3.53-3.50(m,1H),3.22-3.13(m,2H),2.89(t,J＝6.8Hz,2H),2.67-2.65(m,3H),2.21(brs,1H),2.03-1.82(m,3H),1.65-1.52(m,2H),1.43(s,3H),1.36(s,3H),1.10-1.06(m,6H)。

Example 80.preparation of 1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridine- 3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

DIPEA (0.042 mL,0.24 mmol) and cyclohexylmethanesulfonyl chloride (14 mg,0.073 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] at room temperature ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (25 mg,0.049 mmol) in DCM (1.5 mL). The mixture was stirred at room temperature for 1h, then diluted with DCM and washed sequentially with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (31 mg,0.049 mmol). LCMS [ M+H] ⁺ ：638.4。

Step 2:1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridine- 3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 80)From 1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 1 step 5 using 1- (5- ((1- ((cyclohexylmethyl) sulfonyl) piperidin-4-yl) methyl) pyri-dineAzolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H ] ⁺ ：488.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.44(s,1H),8.57(d,J＝7.1Hz,1H),8.00(s,1H),7.38(d,J＝1.7Hz,1H),6.80(dd,J＝7.2,1.9Hz,1H),3.77(t,J＝6.7Hz,2H),3.56(d,J＝12.2Hz,2H),2.85(d,J＝6.2Hz,2H),2.79(t,J＝6.7Hz,2H),2.70(td,J＝12.1,2.4Hz,2H),2.60(d,J＝6.9Hz,2H),1.92-1.78(m,3H),1.79-1.64(m,5H),1.59(dd,J＝10.3,6.4Hz,1H),1.31-1.19(m,4H),1.18-1.00(m,3H)。

The compounds in the following table were prepared by the method of example 80 using the appropriate commercially available sulfonyl chloride or chloroformate in step 1.

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Example 97 preparation of tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperidine-1-carboxylate (example 97)

Step 1.1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

TFA (1.5 mL,19 mmol) was added to 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (95 mg,0.21 mmol) and the mixture was heated at 80℃overnight. The mixture was then cooled to room temperature, concentrated, and the residue was dissolved in toluene and concentrated again to give crude 1- (5-bromopyrazolo [1, 5-a) as TFA salt]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：309.1。

Step 1.Tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridine-5- Methyl) piperidine-1-carboxylic acid ester

From 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 2, wherein 1- (5-bromopyrazolo [1,5-a ] is used ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione instead of 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. Purification by reverse phase HPLC using ACN/water/0.1% TFA. LCMS [ M+H] ⁺ ：428.3。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.30 (d, j=7.1 hz, 1H), 7.88 (s, 1H), 7.25 (s, 1H), 6.72 (d, j=7.2 hz, 1H), 3.95 (d, j=13.3 hz, 2H), 3.78 (t, j=6.7 hz, 2H), 2.79 (t, j=6.8 hz, 2H), 2.61 (d, j=15.8 hz, 2H), 2.53 (d, j=7.2 hz, 2H), 1.74 (s, 1H), 1.56 (d, j=13.3 hz, 2H), 1.34 (s, 9H), 1.13-0.98 (m, 2H).

Example 98.preparation of 1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1, 5-a]pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

HATU (28 mg,0.073 mmol) and isobutyric acid (6.2 μl,0.097 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (25 mg,0.049 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 5min, then DIPEA (0.034 ml,0.19 mmol) was added. The mixture was stirred at room temperature for 1h, then diluted with ethyl acetate and washed sequentially with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (27 mg,0.049 mmol). LCMS [ M+H] ⁺ ：548.3。

Step 2:1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine Pyridine-2, 4 (1H, 3H) -diones(example 98)

From 3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, wherein 3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyrylpiperidin-4-yl) methyl) pyrazolo [1,5-a is used]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：488.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.43(s,1H),8.56(d,J＝7.1Hz,1H),7.99(s,1H),7.37(d,J＝1.7Hz,1H),6.80(dd,J＝7.1,1.9Hz,1H),4.39(d,J＝13.1Hz,1H),3.93(d,J＝13.6Hz,1H),3.77(t,J＝6.7Hz,2H),2.97(t,J＝12.8Hz,1H),2.86(h,J＝6.7Hz,1H),2.79(t,J＝6.7Hz,2H),2.58(d,J＝7.2Hz,2H),2.47(d,J＝12.9Hz,1H),1.86(ddd,J＝11.2,7.5,3.8Hz,1H),1.73-1.56(m,2H),1.22-1.01(m,2H),0.99(dd,J＝12.3,6.7Hz,6H)。

The compounds in the following table are prepared by the method of example 98, using the appropriate commercially available carboxylic acid in step 1.

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Example 104.1- (5- ((1-acetylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine Pyridine-2, 4 (1H, 3H) -dionesIs prepared from

Step 1.1- (5- ((1-acetylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4- Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

DIPEA (0.049 mL,0.28 mmol) and acetic anhydride (0.016 mL,0.17 mmol) were added to a solution of 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (27 mg,0.057 mmol) in DCM (1.5 mL) at room temperature. The mixture was stirred at room temperature for 30min, then partitioned between DCM and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated to give crude 1- (5- ((1-acetylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ m+h ]:520.4.

Step 2:1- (5- ((1-acetylpiperidin-4-yl) methyl) pyri-dineAzolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidines 2,4 (1H, 3H) -diones

From 1- (5- ((1-acetylpiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, using 1- (5- ((1-acetylpiperidin-4-yl) methyl) pyrazolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H]：370.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.41(s,1H),8.55(d,J＝7.2Hz,1H),7.98(s,1H),7.35(d,J＝1.6Hz,1H),6.78(dd,J＝7.1,1.8Hz,1H),4.34(d,J＝13.1Hz,1H),3.77(q,J＝6.3Hz,3H),3.03-2.90(m,1H),2.78(t,J＝6.7Hz,2H),2.57(d,J＝7.2Hz,2H),2.49-2.41(m,1H),1.97(s,3H),1.83(ddd,J＝10.9,7.4,3.6Hz,1H),1.61(t,J＝12.6Hz,2H),1.08(dqd,J＝47.6,12.4,4.2Hz,2H)。

Example 105.preparation of 1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.3- (2, 4-Dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

2, 2-Dimethylethyleneoxide (63 mg,0.87 mmol) and DIPEA (0.15 mL,0.87 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (150 mg,0.29 mmol) in solution in THF (2 mL) and MeOH (2 mL). The mixture was heated at 70 ℃ overnight, then cooled to room temperature and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：550.3。

And 2, step 2.3- (2, 4-Dimethoxybenzyl) -1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) Methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

Trimethyloxyonium tetrafluoroborate (40 mg,0.27 mmol) and DIPEA (0.072 mL,0.41 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (40 mg,0.068 mmol) in DCM (2 mL). The mixture was stirred at room temperature overnight, diluted with DCM and then sequentially saturated NaHCO ₃ Aqueous solution and brine wash. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：564.3。

Step 3:1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine- 3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

From 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, wherein 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-methoxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a is used]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：414.4。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.46 (d, j=7.1 hz, 1H), 8.03 (s, 1H), 7.39 (s, 1H), 6.86 (d, j=7.2 hz, 1H), 3.90 (q, j=6.0 hz, 4H), 3.45 (s, 3H), 3.30-3.12 (m, 4H), 2.90 (t, j=6.8 hz, 2H), 2.78 (d, j=7.3 hz, 2H), 2.04 (d, j=9.8 hz, 1H), 1.96-1.77 (m, 4H), 1.46 (s, 6H).

Example 106.1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo[1,5-a]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

From 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (from step 2 of example 105) was prepared by the method of example 1, step 5, using 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：400.4。 ¹ H NMR(400MHz,DMSO-d6)δ10.42(s,1H),8.58(dd,J＝7.2,2.8Hz,1H),8.00(s,1H),7.36(d,J＝3.2Hz,1H),6.78(dd,J＝7.2,1.8Hz,1H),4.00-3.62(m,2H),3.57(d,J＝12.8Hz,2H),3.30-3.10(m,3H),3.07-2.89(m,3H),2.79(t,J＝6.6Hz,2H),2.63(dd,J＝28.7,7.0Hz,1H),1.91(d,J＝53.7Hz,1H),1.68(dt,J＝42.6,14.6Hz,4H),1.23(d,J＝5.3Hz,6H)。

Example 107.preparation of 1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.3- (2, 4-Dimethoxybenzyl) -1- (5- ((1- ((1-hydroxycyclohexyl) methyl) piperidin-4-yl) methyl Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]To a stirred solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (250 mg, 0.323 mmol) in acetonitrile (5.0 mL) was added lithium perchlorate (110 mg,1.046 mmol). The reaction was stirred for 10min, followed by the addition of 1-oxaspiro [2.5 ]]Octane (293 mg,2.61 mmol). The mixture is mixedStirred for 4h at 90 ℃. After cooling to room temperature, the reaction was diluted with EtOAc and water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- ((1-hydroxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (200 mg, crude). LCMS [ M+H ] ⁺ ：590.3。

And 2, step 2.3- (2, 4-Dimethoxybenzyl) -1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- ((1-hydroxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]To a stirred solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (80 mg,0.135 mmol) in DCM (10 mL) at 0deg.C was added DAST (43 mg,0.271 mmol). The reaction was stirred at 0℃for 1h. The reaction was then diluted with DCM and water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (50 mg, crude). LCMS [ M+H] ⁺ ：592.2。

Step 3:1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 107)

From 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (10 mg,0.016 mmol) was prepared using the method of example 1, step 5, using 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. The crude compound was purified by chiral HPLC: column: CHIRALPAK IG,250mm X20mm X5 μm; mobile phase: hexane (A), 0.1% DEA (B) in MeOH: etOH (1:1); flow rate: 15mL; isocratic: 75 25 (A) and (B)). The collected fractions were concentrated to give 1- (5- ((1- ((1-fluorocyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] as an off-white solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (3 mg,0.006mmol,33% yield). LCMS [ M+H] ⁺ ：442.3。HPLC Rt＝4.95min。 ¹ H NMR (300 MHz, methanol-d 4) delta 8.40 (d, j=7.1 hz, 1H), 7.98 (s, 1H), 7.34 (s, 1H), 6.81 (d, j=7.2 hz, 1H), 3.88 (t, j=6.6 hz, 2H), 2.98-2.84 (m, 4H), 2.61 (d, j=6.5 hz, 2H), 2.42 (d, j=23.5 hz, 2H), 2.07 (t, j=11.2 hz, 2H), 1.82 (s, 2H), 1.69-1.47 (m, 10H), 1.43-1.24 (m, 3H). No NH protons were observed due to solvent exchange.

Example 108 preparation of 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

TFA (25 mL,5.28 mmol) was added to tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) ]Pyridin-5-yl) methyl) piperidine-1-carboxylic acid ester (3.05 g,5.28 mmol). The mixture was heated in a sealed vial at 85 ℃ overnight. The mixture was then cooled to room temperature and concentrated, and dried azeotropically with toluene to afford the crude product (2.33 g,5.28 mmol). A portion (about 20 mg) of the crude material was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA. The fractions containing the product were combined, frozen and lyophilized to give 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]TFA salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 108) (8 mg,0.018 mmol). LCMS [ M+H] ⁺ ：328.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.44(s,1H),8.59(d,J＝7.1Hz,1H),8.52(d,J＝11.4Hz,1H),8.23(d,J＝11.4Hz,1H),8.01(s,1H),7.39(d,J＝1.8Hz,1H),6.80(dd,J＝7.2,1.9Hz,1H),3.77(t,J＝6.7Hz,2H),3.26(d,J＝12.6Hz,2H),2.90-2.76(m,4H),2.61(d,J＝7.0Hz,2H),1.89(ddh,J＝14.7,7.3,3.6Hz,1H),1.80-1.72(m,2H),1.35(tdd,J＝14.3,12.0,4.0Hz,2H)。

Example 109 preparation of 1- (5- ((1-isobutylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Isobutyraldehyde (1.90 g,26.4 mmol) and triethylamine (1.10 mL,7.92 mmol) were added to 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (example 108) (2.33 g,5.28 mmol) in DCM (30 mL) and MeOH (2 mL). The reaction mixture was stirred at room temperature for 30min, then sodium triacetoxyborohydride (5.59 g,26.4 mmol) was added. The reaction mixture was stirred at room temperature overnight and then saturated NaHCO ₃ The aqueous solution was quenched and extracted three times with DCM. The combined organic extracts were washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 0-100% EtOAc/EtOH (3:1), heptane and 0.1% TEA) afforded a light brown solid which was triturated with diethyl ether to give 1- (5- ((1-isobutylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] as an off-white solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. (1055 mg,2.738mmol,52% yield). LCMS [ M+H] ⁺ ：384.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.42(s,1H),8.53(d,J＝7.1Hz,1H),7.97(d,J＝1.4Hz,1H),7.34(s,1H),6.83-6.63(m,1H),3.76(t,J＝6.6Hz,2H),2.78(t,J＝6.8Hz,4H),2.55(d,J＝6.5Hz,2H),1.97(d,J＝7.4Hz,2H),1.75(dt,J＝19.4,9.2Hz,3H),1.56(d,J＝11.5Hz,3H),1.31-1.09(m,2H),0.83(d,J＝6.5Hz,6H)。

The compounds in the following table were prepared by the method of example 109 using the appropriate commercially available aldehyde.

Example 112.1 preparation of 1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

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Step 1.1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Piirae-type pyridine Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (200 mg, 0.319 mmol) and 2-cyclohexyl-2, 2-difluoroacetaldehyde (1.36 g,8.38 mmol) [ see Org. Lett. [ quick report on organic chemistry ] ]2009,11,943-946]NaBH (OAc) was added to a solution in DCE (2 mL) ₃ (133 mg, 0.6278 mmol) and then the mixture was stirred for 16h. The reaction mixture was diluted with water and then extracted with ethyl acetate. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give the crude product. The crude product was purified by preparative HPLC (column: waters Xridge C18 150X25 mmX10 μm; mobile phase: [ water (0.225% FA) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:24% -54%,10 min), the eluate was concentrated to remove MeCN and lyophilized to give 1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) as a yellow solid]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (100 mg,0.16mmol,38% yield). LCMS [ M+H] ⁺ ：624.6。

Step 2:1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 1112)

From 1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, using 1- (5- ((1- (2-cyclohexyl-2, 2-difluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclo)Hexyl methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：474.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.41(s,1H),8.53(d,J＝6.8Hz,1H),7.97(s,1H),7.34(s,1H),6.77-6.75(m,1H),3.76-3.73(m,2H),2.85-2.82(m,2H),2.78-2.75(m,2H),2.70-2.59(m,2H),2.55-2.52(m,2H),2.08-2.07(m,2H),1.99-1.85(m,1H),1.85-1.67(m,4H),1.64-1.61(m,1H),1.55-1.52(m,3H),1.27-1.05(m,7H)。

The compounds in the following table were prepared by the method of example 112 using the appropriate commercially available aldehyde and TEA or DIPEA (2 equivalents) in step 1.

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Example 118.preparation of 1- (5- ((1- ((3-methyloxetan-3-yl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

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From 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 108) was prepared using the method of example 1, step 4, wherein 3- (bromomethyl) -3-methyl oxetane was used instead of (bromomethyl) cyclohexane. LCMS [ M+H] ⁺ ：412.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.42(s,1H),8.70-8.39(m,1H),8.00(d,J＝1.9Hz,1H),7.37(s,1H),6.78(dd,J＝7.2,2.0Hz,1H),4.45(d,J＝6.2Hz,2H),4.24-4.20(m,2H),3.82-3.67(m,3H),3.60-3.31(m,3H),3.22(d,J＝12.2Hz,2H),3.15-2.88(m,2H),2.78(t,J＝6.8Hz,2H),2.60(d,J＝6.4Hz,1H),1.78(d,J＝14.6Hz,3H),1.70-1.38(m,4H)。

Example 119.1- (5- ((1- (oxetan-3-ylmethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

From 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 108) was prepared using the method of example 1, step 4, wherein 3- (bromomethyl) oxetane was used instead of (bromomethyl) cyclohexane. LCMS [ M+H ] ⁺ ：398.4。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.45 (d, j=7.2 hz, 1H), 8.03 (d, j=1.8 hz, 1H), 7.38 (s, 1H), 6.84 (d, j=7.2 hz, 1H), 4.84 (d, j=7.1 hz, 2H), 4.50 (t, j=6.0 hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.77-3.67 (m, 1H), 3.67-3.42 (m, 4H), 3.29-3.23 (m, 1H), 3.04-2.88 (m, 4H), 2.77-2.65 (m, 2H), 2.12-1.92 (m, 3H), 1.53 (t, j=14.3 hz, 2H).

Example 120.preparation of 1- (5- ((1- (2- (1H-imidazol-4-yl) ethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 108) was prepared using the method of example 1, step 4, using 4- (2-chloroethyl) -1H-imidazole instead of (bromomethyl) cyclohexane and adding KI (1.5 eq). LCMS [ M+H] ⁺ ：422.4。 ¹ H NMR(500MHz,DMSO-d6)δ10.43(s,1H),9.73-9.28(m,1H),9.02(s,1H),8.59(d,J＝7.1Hz,1H),8.00(s,1H),7.53(s,1H),7.37(s,1H),6.79(dd,J＝7.1,1.9Hz,1H),3.76(t,J＝6.7Hz,2H),3.56-3.43(m,2H),3.39-3.29(m,2H),3.10(t,J＝7.9Hz,2H),3.02-2.87(m,2H),2.78(t,J＝6.7Hz,2H),2.65-2.56(m,2H),1.96-1.77(m,3H),1.52-1.38(m,2H)。

Example 121.1 preparation of 1- (5- ((1- (3-hydroxy-2- (hydroxymethyl) propyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From the following components1- (5- ((1- (oxetan-3-ylmethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine-3- Radical) dihydropyrimidine-2, 4 (1H, 3H) -dioneExample 119), using the procedure of example 1, step 5, using1-(5- ((1- (oxetan-3-ylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesInstead of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：416.3。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.45 (d, j=7.2 hz, 1H), 8.03 (d, j=1.6 hz, 1H), 7.40 (d, j=11.5 hz, 1H), 6.86 (dd, j=9.2, 7.1hz, 1H), 5.51 (d, j=1.5 hz, 1H), 3.91 (t, j=6.6 hz, 2H), 3.77-3.67 (m, 4H), 3.58 (dd, j=10.7, 7.6hz, 2H), 3.43-3.36 (m, 1H), 3.25 (d, j=6.8 hz, 2H), 3.02-2.88 (m, 4H), 2.72 (d, j=6.8 hz, 2H), 2.32 (s, 1H), 2.02 (t, j=18.2H), 1.55 (j=13.2 hz, 2H).

Example 122.preparation of 1- (5- ((1- (2-methoxybenzyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

DIPEA (0.053 mL,0.31 mmol) and 1- (chloromethyl) -2-methoxybenzene (11 mg,0.073 mmol) were added to 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 108) (20 mg,0.061 mmol) in DCM. The mixture was stirred at room temperature for 30min. 1- (chloromethyl) -2-methoxybenzene (11 mg,0.073 mmol) and DIPEA (0.053 mL,0.31 mmol) were added again, and the mixture was stirred at room temperature for 2h. The reaction was then diluted with DCM and sequentially with water and brine And (5) washing. The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA. The product-containing fractions were combined, frozen and lyophilized to give 1- (5- ((1- (2-methoxybenzyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]TFA salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (2.3 mg,0.0039mmol,6% yield). LCMS [ M+H] ⁺ ：448.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.45(d,J＝14.6Hz,1H),8.60(dd,J＝12.9,7.2Hz,1H),8.01(d,J＝8.5Hz,1H),7.54-7.42(m,2H),7.42-7.33(m,1H),7.14(d,J＝8.3Hz,1H),7.05(q,J＝7.8Hz,1H),6.78(dd,J＝7.2,1.9Hz,1H),4.22(d,J＝4.9Hz,2H),3.84(s,3H),3.77(t,J＝6.7Hz,2H),3.36(d,J＝12.1Hz,2H),2.95(d,J＝11.6Hz,2H),2.78(t,J＝6.8Hz,2H),2.59(d,J＝6.7Hz,2H),1.95 -1.70(m,3H),1.44(q,J＝12.2,11.0Hz,2H)。

Example 123.preparation of 1- (5- ((1- (cyclohexylmethyl) -4-fluoropiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) Pyrazolo [1,5-a]Pyridin-5-yl) methyl) -4-fluoropiperidine-1-carboxylic acid ester

To the dried vial was added tert-butyl 4- (bromomethyl) -4-fluoropiperidine-1-carboxylate (0.308 g,1.04 mmol), 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.367 g,0.8 mmol), niCl ₂ (DME) (8.8 mg,0.040 mmol), pyridine-2, 6-bis (formamidine) dihydrochloride (9.4 mg,0.040 mmol), naI (0.030 g,0.20 mmol) and Zn (0.105 g,1.60 mmol). The vial was sealed with a septum cap, evacuated and refilled 3 times with nitrogen. DMA (2.7 mL) and TFA (6 μl,0.08 mmol) were added and the reaction was stirred at room temperature for 2min. The vial was carefully evacuated and refilled 3 times with nitrogen to remove any H ₂ . The reaction was then heated at 70 ℃ overnight to form a brown reaction mixture. The reaction was cooled to room temperature, diluted with EtOAc and filtered through a plug of silica gel eluting with EtOAc. The eluate was concentrated and the residue was purified by silica gel column chromatography (eluting with 0-100% EtOAc in heptane) to give tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoropiperidine-1-carboxylate (0.47 g,0.80mmol,98% yield, purity 70%). LCMS [ M+H] ⁺ :596.4. the product was used without further purification.

Step 2:1- (5- ((1- (cyclohexylmethyl) -4-fluoropiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Radical) dihydropyrimidine-2, 4 (1H, 3H) -dione

From tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoropiperidine-1-carboxylate was prepared using the method of example 109, using cyclohexane-formaldehyde instead of isobutyraldehyde. LCMS [ M+H] ⁺ ：442.2。 ¹ H NMR(400MHz,DMSO-d6)δ10.44(s,1H),8.62(dd,J＝7.1,0.9Hz,1H),8.04(d,J＝1.9Hz,1H),7.43(d,J＝30.9Hz,1H),6.92-6.71(m,1H),3.81-3.74(m,2H),3.43(d,J＝12.2Hz,2H),3.28-2.86(m,6H),2.82-2.73(m,2H),2.14-1.84(m,4H),1.82-1.53(m,6H),1.18(dt,J＝30.0,12.3Hz,3H),1.01-0.84(m,2H)。

Example 124.preparation of 1- (5- ((4-fluoro-1-isobutylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 123, wherein isobutyraldehyde was used instead of cyclohexane formaldehyde. LCMS [ M+H ] ⁺ ：402.4。 ¹ H NMR(400MHz,DMSO-d6)δ10.44(s,1H),8.62(d,J＝7.0Hz,1H),8.04(d,J＝1.9Hz,1H),7.47(s,1H),6.82(d,J＝7.3Hz,1H),3.78(t,J＝6.8Hz,2H),3.44(d,J＝12.5Hz,2H),3.15-2.89(m,6H),2.83-2.73(m,2H),2.17-1.87(m,5H),0.93(dd,J＝6.7,2.0Hz,6H)。

Example 125.1 preparation of 1- (5- ((1- (cyclobutylmethyl) -4-fluoropiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 123, wherein cyclobutane-formaldehyde was used instead of cyclohexane-formaldehyde. LCMS [ M+H] ⁺ ：402.4。 ¹ H NMR(400MHz,DMSO-d6)δ10.44(s,1H),8.61(dd,J＝7.2,1.0Hz,1H),8.04(s,1H),7.46(d,J＝1.6Hz,1H),6.93-6.73(m,1H),3.78(t,J＝6.7Hz,2H),3.34(d,J＝12.3Hz,2H),3.23-2.93(m,6H),2.79(t,J＝6.8Hz,2H),2.67(p,J＝7.3,6.9Hz,1H),2.15-1.92(m,5H),1.91-1.71(m,5H)。

Example 126.preparation of 1- (5- ((1-benzyl-4-fluoropiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoropiperidine-1-carboxylate was prepared using the method of example 1, steps 3 to 5, using benzyl bromide instead of (bromomethyl) cyclohexane. LCMS [ M+H] ⁺ ：436.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.44(s,1H),8.61(d,J＝7.1Hz,1H),8.03(s,1H),7.46(d,J＝16.6Hz,6H),6.80(d,J＝7.3Hz,1H),4.34(d,J＝5.1Hz,2H),3.77(t,J＝6.6Hz,2H),3.21-3.01(m,6H),2.77(t,J＝6.7Hz,2H),2.64(s,1H),2.07-1.80(m,3H)。

Example 127.preparation of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1-amino-4- ((tert-butoxycarbonyl) amino) -3-methylpyridin-1-ium 2, 4-dinitrophenol salt

Tert-butyl (3-methylpyridin-4-yl) carbamate (2.447 g,10.0 mmol) and O- (2, 4-dinitrophenyl) hydroxylamine (2.19 g,11.0 mmol) were added to the reaction flask. 2-MeTHF (20 mL) was added and the reaction was heated at 40℃for 1h and then stirred at room temperature overnight. O- (2, 4-dinitrophenyl) hydroxylamine (600 mg,3.00mmol,0.3 eq.) was added and the reaction stirred for a further 2h at 40 ℃. The reaction was diluted with isopropanol and concentrated to give a yellow solid which was suspended in cold IPA, filtered and dried to give 1-amino-4- ((tert-butoxycarbonyl) amino) -3-methylpyridin-1-ium 2, 4-dinitrophenol salt (5.5 g, crude) which was used without further purification. LCMS [ M ] ] ⁺ ：224.1。

Step 2. Ethyl radical5- ((tert-Butoxycarbonyl) amino) -4-methylpyrazolo [1,5-a ]]Pyridine-3-carboxylic acid ester

Potassium carbonate (5.60 g,40.5 mmol) was added to a mixture of 1-amino-4- ((tert-butoxycarbonyl) amino) -3-methylpyridin-1-ium 2, 4-dinitrophenol salt (5.5 g,13.5 mmol) in DMF (13.5 mL) at 0deg.C. After 5min, ethyl propiolate (1.50 ml,14.8 mmol) was added and the reaction was stirred at 0 ℃ and allowed to warm to room temperature overnight. Both regioisomers were presented by LCMS. The reaction was concentrated and the residue was suspended in water, filtered, and the solid was purified by silica gel chromatography to give ethyl 5- ((tert-butoxycarbonyl) amino) -4-methylpyrazolo [1,5-a as a single regioisomer]Pyridine-3-carboxylic acid ester (849 mg,2.66mmol,20% yield). LCMS [ M+H] ⁺ ：320。 ¹ H NMR(500MHz,CDCl ₃ )δ8.37(d,J＝1.8Hz,1H),8.34(d,J＝7.6Hz,1H),7.80(d,J＝7.6Hz,1H),6.58(s,1H),4.37-4.27(m,2H),2.77(d,J＝1.8Hz,3H),1.55(d,J＝1.7Hz,9H),1.39(td,J＝7.1,1.8Hz,3H)。

And 3, step 3.Ethyl 5-amino-4-methylpyrazolo [1,5-a ]]Pyridine-3-carboxylic acid ester

TFA (3.3 mL) was added to ethyl 5- ((tert-butoxycarbonyl) amino) -4-methylpyrazolo [1,5-a ] at room temperature]Pyridine-3-carboxylic acid ester (850 mg,2.66 mmol) in DCM (10 mL). The reaction was stirred at room temperature for 1h and then concentrated. The residue was azeotropically dried with toluene to give a pale yellow colorEthyl 5-amino-4-methylpyrazolo [1,5-a ] as a coloured solid ]Pyridine-3-carboxylic acid ester (850 mg,2.66 mmol). LCMS [ M+H] ⁺ ：220。

And 4, step 4.Ethyl 5-iodo-4-methylpyrazolo [1,5-a ]]Pyridine-3-carboxylic acid ester

A solution of sodium nitrite (37.9 mg,0.550 mmol) in water (0.50 mL) was added dropwise to ethyl 5-amino-4-methylpyrazolo [1,5-a ] at 0deg.C]Pyridine-3-carboxylic acid ester (167 mg,0.5 mmol) in a suspension of MeCN (0.83 mL) and aqueous 6M HCl (2.5 mL). The reaction turned bright yellow and was stirred at 0 ℃ for 1h. A solution of potassium iodide (166 mg,1.00 mmol) in water (0.50 mL) was added dropwise to the vigorously stirred reaction; the reaction turned dark brown and bubbled, and a precipitate formed. After 15min, the reaction was diluted with water, filtered, and the solid was washed with water. The solid was then dissolved in EtOH/DCM and concentrated. The solid was suspended in cold methanol, filtered and dried to give ethyl 5-iodo-4-methylpyrazolo [1,5-a ] as a pale yellow solid]Pyridine-3-carboxylic acid ester (141 mg,0.427mmol,85% yield). LCMS [ M+H] ⁺ ：331。

Step 5 ethyl radical5- ((1- (tert-Butoxycarbonyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ]]Piirae-type pyridine Pyridine-3-carboxylic acid ester

From ethyl 5-iodo-4-methylpyrazolo [1,5-a ]]Pyridine-3-carboxylic acid ester prepared by the method of example 1 step 2, using ethyl 5-iodo-4-methylpyrazolo [1,5-a ] ]Pyridine-3-carboxylic acid ester substituted for 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H-tBu ]] ⁺ ：346.1。

And 6, step 6.5- ((1- (tert-Butoxycarbonyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ]]Pyridine-3- Formic acid

Sodium hydroxide (360 mg,9.00 mmol) was added to ethyl 5- ((1- (tert-butoxycarbonyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ]]Pyridine-3-carboxylic acid ester (723 mg,1.80 mmol) in EtOH (7.2 mL) and water (1.8 mL). The reaction was heated at 60 ℃ for 3h, then cooled to room temperature and concentrated. The residue was dissolved in water, filtered, and then aqueous 6M HCl was added dropwise until the product precipitated. Decanting the supernatant and concentratingThe solid was washed with water, dried and purified by silica gel chromatography (eluting with 0-50% AcOH in heptane (1% AcOH in 3:1 etoac/EtOH)) to give 5- ((1- (tert-butoxycarbonyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ] as an off-white solid]Pyridine-3-carboxylic acid (636 mg,1.703mmol,95% yield). LCMS [ M+H-tBu ]] ⁺ ：318。

And 7, step 7.Tert-butyl 4- ((3- ((ethoxycarbonyl) amino) -4-methylpyrazolo [1, 5-a)]Pyridin-5-yl) methyl esters Base) piperidine-1-carboxylic acid ester

DIPEA (675 μl,3.86 mmol) was added to 5- ((1- (tert-butoxycarbonyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1,5-a ] at room temperature ]Pyridine-3-carboxylic acid (481 mg,1.29 mmol) in dioxane (4.3 mL). The bright yellow mixture was stirred at room temperature for 3h, then EtOH (1.5 mL,25.8 mmol) was added and the reaction was heated at 100deg.C for 15min. The reaction was cooled to room temperature, diluted with water and brine and extracted with EtOAc. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. The residue was purified by silica gel chromatography (eluting with 0-60% EtOAc/heptane) to give tert-butyl 4- ((3- ((ethoxycarbonyl) amino) -4-methylpyrazolo [1, 5-a) as a colorless oil]Pyridin-5-yl) methyl) piperidine-1-carboxylic acid ester (479 mg,1.150mmol,89% yield). LCMS [ M+H-tBu ]] ⁺ ：361。

And 8, step 8.Tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) -4-methylpyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-5-yl) methyl) piperidine-1-carboxylic acid ester

Acrylamide (14.2 mg,0.200 mmol) and tert-butyl 4- ((3- ((ethoxycarbonyl) amino) -4-methylpyrazolo [1, 5-a)]Pyridin-5-yl) methyl piperidine-1-carboxylate (41.7 mg,0.1 mmol) was added to the vial followed by tBuOH (0.5 mL) and potassium tert-butoxide (110. Mu.l, 0.110 mmol) (1.0M in THF) -the reaction turned pale yellow. The mixture was heated at 60 ℃ overnight. Saturated NaHCO for reaction ₃ Aqueous solution and water quenching. The mixture was extracted with EtOAC and taken up in Na ₂ SO ₄ Dried, filtered and concentrated to give crude tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) -4-methylpyrazolo [1, 5-a)]Pyridin-5-yl) methyl piperidine-1-carboxylic acid ester (39 mg,0.088mmol,88% yield). LCMS [ M+H-Boc] ⁺ ：342。

Step 9.1- (4-methyl-5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

TFA (215 μl) was added to tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) -4-methylpyrazolo [1, 5-a) at room temperature]Pyridin-5-yl) methyl piperidine-1-carboxylate (38 mg,0.086 mmol) in DCM (645 μl). The reaction was stirred at room temperature for 30min and then concentrated to give crude 1- (4-methyl-5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：342。

Step 10.1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) -4-methylpyrazolo [1, 5-a)]Pyridine- 3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

From 1- (4-methyl-5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione is prepared by the method of example 109, wherein cyclohexane formaldehyde is used instead of isobutyraldehyde. LCMS [ M+H ] ⁺ ：438.5。 ¹ H NMR(500MHz,DMSO-d6)δ10.45(s,1H),8.60(s,1H),8.44(d,J＝7.1Hz,1H),7.98(s,1H),6.73(d,J＝7.1Hz,1H),3.79(dt,J＝13.3,7.1Hz,1H),3.65(dt,J＝12.6,6.4Hz,2H),2.88-2.80(m,3H),2.76(t,J＝6.8Hz,2H),2.68-2.59(m,2H),2.35(s,3H),1.86-1.58(m,10H),1.53(d,J＝12.6Hz,2H),1.33-1.07(m,4H),0.92(d,J＝12.8Hz,2H)。

The compounds in the following table were prepared by the method of example 1 using tert-butyl 3-methylenepyrrolidine-1-carboxylate in step 2 and the appropriate commercially available halide in step 4.

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EXAMPLE 133.1 preparation of 1- (5- (azetidin-3-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 1, steps 2 and 5, using tert-butyl 3-methyleneazetidine-1-carboxylate instead of tert-butyl 4-methylenepiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：300.0。 ¹ H NMR(300MHz,CD ₃ OD)δ8.53(s,1H),8.44(d,J＝7.2Hz,1H),8.01(s,1H),7.37(s,1H),6.82-6.79(m,1H),4.09(t,J＝8.7Hz,2H),3.92-3.86(m,4H),3.34(m,1H),3.03(d,J＝8.1Hz,2H),2.88(t,J＝7.2Hz,2H)。

Example 134.1 preparation of 1- (5- ((1-isobutylazetidin-3-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 1- (5- (azetidin-3-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 133), prepared using the method of example 109, using 1- (5- (azetidin-3-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione instead of 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate. LCMS [ M+H] ⁺ ：356.3。 ¹ H NMR(300MHz,CD ₃ OD) δ8.55 (s, 1H), 8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.37 (s, 1H), 6.81-6.79 (m, 1H), 3.96-3.87 (m, 4H), 3.63-3.61 (m, 2H), 3.13-3.09 (m, 1H), 3.00 (d, j=8.0 hz, 2H), 2.89 (d, j=7.2 hz, 2H), 2.79 (d, j=6.8 hz, 2H), 1.84-1.81 (m, 1H), 0.96 (d, j=6.8 hz, 6H), no NH protons are observed due to the exchange of solvents.

Example 135.1 preparation of 1- (5- ((1- (cyclohexylmethyl) azetidin-3-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 1- (5- (azetidin-3-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 133), prepared using the method of example 109, using 1- (5- (azetidin-3-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione instead of 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate and cyclohexane-formaldehyde replace isobutyraldehyde. LCMS [ M+H] ⁺ ：396.1。 ¹ H NMR(300MHz,CD ₃ OD)δ8.54(s,1H),8.42(d,J＝7.2Hz,1H),8.00(s,1H),7.35(s,1H),6.80-6.77(m,1H),3.95-3.86(m,4H),3.62-3.56(m,2H),3.09-3.07(m,1H),2.99-2.97(m,2H),2.88(d,J＝6.6Hz,2H),2.80-2.78(m,2H),1.73-1.70(m,5H),1.51(s,1H),1.30-0.95(m,5H)。

Example 136.1 preparation of 1- (5- ((1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(4-methylenepiperidin-1-yl) (tetrahydro-2H-pyran-4-yl) methanones

To a stirred solution of tetrahydro-2H-pyran-4-carboxylic acid (4.0 g,27.7 mmol) in THF (80 mL) at 0deg.C was added HATU (15.81 g,41.60 mmol), DIPEA (14.2 mL,83.2 mmol). The mixture was stirred for 10min, followed by the addition of a solution of 4-methylenepiperidine (4.41 g,33.3 mmol) in THF (20 mL). The reaction mixture was then stirred at room temperature for 12h. The reaction was then quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 50% EtOAc in hexanes) to give (4-methylenepiperidine-1-Yl) (tetrahydro-2H-pyran-4-yl) methanone (3.8 g,18.1mmol,59% yield). LCMS [ M+H] ⁺ ：210.0。

And 2, step 2.4-methylene-1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidine

ZrCl was added to a stirred solution of (4-methylenepiperidin-1-yl) (tetrahydro-2H-pyran-4-yl) methanone (1.0 g,4.7 mmol) in THF (12 mL) at-20deg.C ₄ (1.09 g,4.7 mmol) and the mixture was stirred for 30min. Adding MeMgBr. Et ₂ 0 (9.4 mL,28.2mmol, 3.0M) and the mixture was stirred at-20deg.C for 10min and then at room temperature for 2h. After completion, the reaction was quenched with water (10 mL) and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 50% etoac in hexanes) to give 4-methylene-1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidine (250 mg,1.12mmol,24% yield). LCMS [ M+H] ⁺ ：224.0。

Step 3:1- (5- ((1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesPrepared from 4-methylene-1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidine using the procedure of example 1, steps 2 and 5, using 4-methylene-1- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidine instead of tert-butyl 4-methylenepiperidine-1-carboxylate. LCMS [ M+H ] ⁺ ：454.2。 ¹ H NMR(400MHz,DMSO-d6):δ10.44(s,1H),8.59(d,J＝7.2Hz,1H),8.00(s,1H),7.83(s,1H),7.35(s,1H),6.78(d,J＝6.8Hz,1H),3.91-3.88(m,2H),3.74(t,J＝6.8Hz,2H),3.28(s,2H),2.96-2.93(m,2H),2.78(t,J＝6.4Hz,2H),2.61-2.59(m,2H),2.42(brs,1H),2.02-1.80(m,5H),1.58-1.48(m,4H),1.35-1.28(m,2H),1.20(s,6H)。

Example 137.1- (5- ((1- (2-methyl-1- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperidin-4-yl) methyl) Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the procedure of example 136, wherein 2- (tetrahydro-2H-pyran-4-yl) acetic acid was used instead of tetrahydro-2H-pyran-4-carboxylic acid. LCMS [ M+H] ⁺ ：468.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.44(s,1H),8.60(d,J＝7.1Hz,1H),8.28(t,J＝8.3Hz,1H),8.00(s,1H),7.36(s,1H),6.78(d,J＝7.1Hz,1H),3.84-3.72(m,4H),3.47(s,1H),3.29(t,J＝11.4Hz,2H),2.90(q,J＝11.7Hz,2H),2.78(t,J＝6.7Hz,2H),2.61(d,J＝6.4Hz,2H),1.92(s,2H),1.85(d,J＝14.6Hz,2H),1.68-1.52(m,5H),1.46(t,J＝12.9Hz,2H),1.38-1.13(m,3H),1.30(s,6H)。

Example 138.1- (5- ((1- (2-cyclobutylpropan-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the method of example 136, wherein cyclobutanecarboxylic acid was used instead of tetrahydro-2H-pyran-4-carboxylic acid. LCMS [ M+H] ⁺ ：424.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.36 (s, 1H), 7.08 (s, 1H), 6.83 (dd, j=7.1, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.58-3.48 (m, 2H), 2.97-2.86 (m, 3H), 2.82-2.74 (m, 1H), 2.68 (d, j=6.7 hz, 1H), 2.64 (d, j=5.9 hz, 2H), 2.09-1.90 (m, 6H), 1.50-1.26 (m, 10H).

Example 139.1- (5- ((1- (2, 4-dimethylpentan-2-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the method of example 136, wherein 3-methylbutanoic acid was used in place of tetrahydro-2H-pyran-4-carboxylic acid. LCMS [ M+H] ⁺ ：426.3。 ¹ H NMR (300 MHz, methanol-d 4) δ8.53 (s, 1H), 8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.38 (s, 1H), 6.84 (d, j=7.3 hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.71-3.48 (m, 3H), 2.97 (t, j=12.2 hz, 2H), 2.89 (t, j=6.6 hz, 3H), 2.69 (d, j=6.3 hz, 2H), 2.00 (d, j=13.2 hz, 3H), 1.75 (hept, j=6.3 hz, 1H), 1.63 (d, j=5.4 hz, 2H), 1.61-1.49 (m, 3H), 1.41 (s, 6H), 1.03 (d, j=6.6 hz, 6H). No NH protons were observed due to solvent exchange.

Example 140.1- (5- ((1- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the method of example 136, using 4, 4-difluorocyclohexane-1-carboxylic acid instead of tetrahydro-2H-pyran-4-carboxylic acid. LCMS [ M+H] ⁺ ：488.4。 ¹ H NMR(400MHz,CD ₃ OD) [ delta ] 8.43 (d, j=7.2 hz, 1H), 8.00 (s, 1H), 7.35 (s, 1H), 6.82 (dd, j=7.6 hz,2.0hz, 1H), 3.87 (t, j=6.8 hz, 2H), 3.59 (d, j=12.4 hz, 2H), 3.06-2.98 (m, 2H), 2.87 (t, j=6.8 hz, 2H), 2.70 (d, j=6.8 hz, 2H), 2.08-2.10 (m, 2H), 2.01-1.75 (m, 8H), 1.64-1.46 (m, 4H), 1.33 (m, 6H). No NH protons were observed due to solvent exchange.

Example 141.1 preparation of 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (S) -4- (methoxymethylene) -2-methylpiperidine-1-carboxylate

KOTBu solution (63.78 mL,63.78mmol,1M in THF) was added dropwise to a stirred suspension of (methoxymethyl) triphenylphosphonium chloride (21.86 g,63.78 mmol) in THF (70 mL) at 0deg.C. The red solution was stirred at room temperature for 30min and then cooled again to 0 ℃. A solution of tert-butyl (S) -2-methyl-4-oxopiperidine-1-carboxylate (8.0 g,37.5 mmol) in THF (30 mL) was added and the reaction mixture stirred at room temperature for 16h. The reaction mixture was treated with saturated NH ₄ Quench with aqueous Cl solution and useExtraction with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 15% -20% EtOAc in hexanes) to give tert-butyl (S) -4- (methoxymethylene) -2-methylpiperidine-1-carboxylate (7.5 g,31mmol,82% yield) as a mixture of E: Z isomers. LCMS [ M+H-tBu ]] ⁺ ：186。 ¹ H NMR(300MHz,CDCl ₃ )δ5.96(s,1H),5.78(s,1H),4.46-4.39(m,2H),4.00-3.89(m,2H),3.56(s,3H),3.53(s,3H),2.86-2.74(m,2H),2.62-2.47(m,2H),2.31-2.24(m,1H),2.02-1.94(m,4H),1.90-1.81(m,3H),1.46(s,18H)1.04(d,J＝6.6Hz,3H)。

And 2, step 2.Tert-butyl (2S) -4-formyl-2-methylpiperidine-1-carboxylate

HCl solution (2.0M in water, 75 mL) was added to a solution of tert-butyl (S) -4- (methoxymethylene) -2-methylpiperidine-1-carboxylate (7.5 g,31 mmol) in MeCN (220 mL) at room temperature. The reaction was heated to 40 ℃ and stirred for 40min. The reaction was then cooled to room temperature and purified by addition of solid NaHCO ₃ Quenching. Brine was added and the reaction was extracted 3 times with EtOAc. The combined organic layers were taken up over Na ₂ SO ₄ Dried, filtered and concentrated to give crude tert-butyl (2S) -4-formyl-2-methylpiperidine-1-carboxylate (7 g,31 mmol) as an about 5:1 mixture of cis: trans isomers. The crude material was used for the next reaction without further purification. LCMS [ M+H-tBu ]] ⁺ ：172。

And 3, step 3.Tert-butyl (2S, 4R) -4- (hydroxymethyl) -2-methylpiperidine-1-carboxylate

NaOMe (335 mg,6.15 mmol) was added to a stirred solution of crude tert-butyl (2S) -4-formyl-2-methylpiperidine-1-carboxylate (7 g,31 mmol) in MeOH (70 mL) at 0deg.C. The reaction mixture was kept at 4 ℃ for 24h. After 24h, the reaction mixture was placed in an ice bath and NaBH was added at 0deg.C ₄ (4.65 g,123 mmol) and then the reaction was stirred at room temperature for 10min. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 20% EtOAc in hexanes) to giveTert-butyl (2S, 4R) -4- (hydroxymethyl) -2-methylpiperidine-1-carboxylate (5.4 g,76% yield) was obtained. LCMS [ M+H-tBu ]] ⁺ ：174。 ¹ H NMR(400MHz,CDCl ₃ )δ4.45-4.41(m,1H),3.99-3.94(m,1H),3.38-3.35(m,2H),2.90(brs,1H),1.87-1.84(m,1H),1.75-1.72(m,1H),1.65-1.62(m,1H),1.46(s,9H),1.32-1.25(m,2H),1.17(d,J＝6.4Hz,3H),1.05-1.01(s,1H)。

And 4, step 4.Tert-butyl (2S, 4R) -4- (bromomethyl) -2-methylpiperidine-1-carboxylate

Triphenylphosphine dibromide (1.2 g,28.5 mmol) was added to a solution of imidazole (2.10 g,30.6 mmol) and tert-butyl (2 s,4 r) -4- (hydroxymethyl) -2-methylpiperidine-1-carboxylate (5.4 g,23.5 mmol) in DCM (50 mL) at 0 ℃. The reaction mixture was stirred at room temperature for 16h. After the reaction was complete, the mixture was diluted with DCM and washed with water. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 10% EtOAc in hexanes) to give tert-butyl (2 s,4 r) -4- (bromomethyl) -2-methylpiperidine-1-carboxylate (3.5 g,12.2mmol,50% yield). LCMS [ M+H-tBu ] ] ⁺ ：236。 ¹ H NMR(300MHz,CDCl ₃ )δ4.45(brs,1H),4.01(brs,1H),3.30-3.20(m,2H),2.88-2.80(m,1H),2.04-1.94(m,1H),1.85-1.81(m,1H),1.70-1.65(m,1H),1.29-1.03(m,15H)。

And 5, step 5.Tert-butyl (2S, 4R) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1) (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylic acid ester

The Zn powder was activated by taking commercial material and vigorously stirring in aqueous 1M HCl solution for 10 min. The material was then filtered and the large pieces were broken up with a spatula. The solid was washed with distilled water, then EtOH, et ₂ And (3) washing. The solid was then heated under vacuum at 50 ℃ overnight.

To a dried 2-necked flask was added tert-butyl (2S, 4R) -4- (bromomethyl) -2-methylpiperidine-1-carboxylate (2.98 g,10.2 mmol), 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (3.90 g,8.5 mmol), niCl ₂ (DME)(0.093g0.425 mmol), pyridine-2, 6-bis (formamidine) dihydrochloride (0.100 g,0.425 mmol), zn powder (1.11 g,17.0 mmol) and sodium iodide (0.319 g,2.125 mmol). The flask was sealed with a septum, evacuated and refilled 3 times with argon. DMA (34.0 mL, degassed by bubbling argon for a few minutes) was added and the reaction was stirred at 70 ℃ overnight. The reaction was cooled to room temperature and poured into water, and the grey precipitate formed was collected by filtration. The solid was diluted with EtOH (200 mL) and filtered through celite (washed with EtOH) to remove Zn solids. The filtrate was concentrated and the crude material was purified by silica gel chromatography (with 10% -100% (3:1 etoac/EtOH w/0.1% Et ₃ N)/heptane elution) to give tert-butyl (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as an off-white solid]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate (4.30 g,6.69mmol,79% yield). LCMS [ M+H-Boc] ⁺ ：492.2。

And 6, step 6.3- (2, 4-dimethoxybenzyl) -1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazole And [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride

HCl solution (4.0M in dioxane, 40 mL) was added to tert-butyl (2S, 4R) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate (5.1 g,8.61 mmol) and the mixture was stirred at room temperature for 3h and then concentrated. The crude compound was triturated with diethyl ether to give 3- (2, 4-dimethoxybenzyl) -1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (4.8 g, crude). LCMS [ M+H] ⁺ ：492.3。

And 7, step 7.1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

TEA (3.3 mL,23.7 mmol) and 4, 4-difluorocyclohexane-1-carbaldehyde (1.4 g,9.48 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) diA solution of hydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (2.5 g,4.74 mmol) in DCM solution (25 mL) was stirred for 1.5h. The reaction was cooled to 0deg.C and sodium triacetoxyborohydride (2.00 g,9.48 mmol) was added. The reaction mixture was stirred at room temperature for 16h. After completion, the reaction was diluted with DCM and water, and the organic layer was taken up in Na ₂ SO ₄ Dried, filtered and concentrated to give crude 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1.7 g, crude). LCMS [ M+H] ⁺ ：624.5。

And 8, step 8.1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

TFA (3 mL) was added to crude 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. The reaction mixture was stirred at 90 ℃ for 16h and then concentrated. The crude compound was purified by reverse phase HPLC using: mobile phase: a=0.1% hcooh in water, b=acetonitrile; column: x SELECT (250 mm. Times.21.2 mm), 5.0 μm; flow rate: 20mL/min. The collected fractions were concentrated under reduced pressure to obtain the product in the form of formate. The product was dissolved in 20% MeOH in DCM with saturated NaHCO ₃ The aqueous solution was washed and concentrated to give 1- (5- (((2 s,4 r) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) as an off-white solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (554 mg,1.05mmol,48% yield). HPLC:98.40% [ Rt= 4.893min]。LCMS[M+H] ⁺ ：474.5。 ¹ H NMR(500MHz,DMSO-d6)δ10.42(s,1H),8.54(d,J＝7.1Hz,1H),7.98(s,1H),7.34(s,1H),6.78(dd,J＝7.3,1.9Hz,1H),3.76(t,J＝6.7Hz,2H),2.91(s,1H),2.79(t,J＝6.7Hz,2H),2.53(d,J＝9.4Hz,2H),2.45-2.34(m,2H),2.27-2.12(m,2H),2.04-1.92(m,2H),1.92-1.85(m,1H),1.85-1.67(m,4H),1.60-1.47(m,2H),1.47-1.35(m,2H),1.24-1.14(m,1H),1.13-1.00(m,2H),0.88(d,J＝6.5Hz,3H)。

The compounds in the following table were prepared by the method of example 141, using the appropriate commercially available aldehyde in step 7.

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Example 151.1- (5- ((1- (((1 r,4 r) -4-hydroxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the procedure of example 141, steps 7-8, using trans-4- (benzyloxy) cyclohexane-1-carbaldehyde [ see WO 2020/232470,2020, A1 ]]Instead of 4, 4-difluorocyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：440.1。 ¹ H NMR(400MHz,CD ₃ OD) delta 8.42 (s, 1H), 8.00 (s, 1H), 7.36 (s, 1H), 6.98 (d J =5.6 hz, 1H), 3.87 (t, j=6.8 hz, 2H), 3.56-3.48 (m, 2H), 2.93-2.85 (m, 6H), 2.68-2.65 (m, 2H), 1.97-1.80 (m, 7H), 1.58-1.55 (m, 2H), 1.29.1.27 (m, 4H), 1.11-1.08 (m, 2H), no NH and OH protons are observed due to the exchange of solvents.

Example 152.preparation of 1- (5- (((2S, 4R) -1- ((3, 3-difluorocyclobutyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (see example 141, step 6) was prepared using the method of example 1, steps 4 to 5, wherein 3- (bromomethyl) -1, 1-difluorocyclobutane was used instead of (bromomethyl) cyclohexane. LCMS [ M+H] ⁺ ：446.3。 ¹ H NMR(400MHz,CD ₃ OD) δ8.51 (s, 1H), 8.43 (d, j=7.1 hz, 1H), 8.00 (s, 1H), 7.36 (s, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.48 (s, 1H), 3.08 (d, j=41.8 hz, 3H), 2.89 (t, j=6.8 hz, 2H), 2.76 (d, j=7.6 hz, 2H), 2.68 (d, j=7.2 hz, 2H), 2.43 (d, j=29.2 hz, 3H), 2.18 (d, j=10.5 hz, 1H), 1.90-1.62 (m, 3H), 1.51 (s, 1H), 1.37-1.17 (m, 4H). No NH protons were observed due to solvent exchange.

The compounds in the following table were prepared from tert-butyl (R) -2-methyl-4-oxopiperidine-1-carboxylate using the procedure of example 141, using the appropriate commercially available aldehyde in step 7.

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Example 156.preparation of 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine)Pyridin-1 (2H) -yl Pyrazolo [1,5-a]Pyridin-5-yl) methyl) piperazine-1-carboxylic acid ester

To 1- (5-bromopyrazolo [1, 5-a) at room temperature]To a suspension of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (45 mg,0.098 mmol) in toluene (2 mL) and water (0.2 mL) was added Cs ₂ CO ₃ (128 mg, 0.390 mmol), { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Potassium methyl } (trifluoro) borate (60.0 mg,0.196 mmol) and RuPhos (9.14 mg, 0.020mmol) followed by Pd (OAc) ₂ (2.2 mg, 9.8. Mu. Mol). The mixture was stirred at 90 ℃ for 3h, then cooled to room temperature and partitioned between EtOAc and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated to give crude tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylic acid ester (56 mg,0.098 mmol). LCMS [ M+H] ⁺ :579.4. the crude material was used without further purification.

Step 2:3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride

HCl solution (4.0M in dioxane, 2mL,8 mmol) was added to tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) ]Pyridin-5-yl) methyl) piperazine-1-carboxylate (55 mg,0.095 mmol) and the mixture was stirred at room temperature for 2h. The reaction was then concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (46 mg,0.095 mmol) was used without further purification. LCMS [ M+H] ⁺ ：479.4。

Step 3:1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Potassium carbonate was added to a solution of pyridin-3-yl dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (22 mg,0.043 mmol) in DMF (1 mL)30mg,0.21 mmol) and (bromomethyl) cyclohexane (0.012 mL,0.085 mmol). The mixture was heated at 80 ℃ for 4h and then cooled to room temperature. The mixture was diluted with ethyl acetate and washed sequentially with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (25 mg,0.043 mmol) was used without further purification. LCMS [ M+H ] ⁺ ：575.4。

Step 4:1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydro Pyrimidine-2, 4 (1H, 3H) -diones

TFA (1.5 mL,19 mmol) was added to crude 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (24 mg,0.042 mmol) and the mixture was heated at 80℃overnight. The mixture was cooled to room temperature, concentrated, and the residue was dissolved in toluene and concentrated again. The residue was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA. The product-containing fractions were combined, frozen and lyophilized to provide 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]TFA salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (5.5 mg,10umol,24% yield). LCMS [ M+H] ⁺ ：425.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.48(s,1H),8.66(d,J＝7.2Hz,1H),8.06(s,1H),7.53(s,1H),6.91(d,J＝6.9Hz,1H),4.59(s,6H),3.80(t,J＝6.7Hz,2H),3.72(s,1H),3.47(s,1H),3.01(s,4H),2.80(t,J＝6.7Hz,2H),1.69(td,J＝29.6,13.7Hz,6H),1.21(dq,J＝36.0,12.2Hz,3H),0.95(q,J＝11.9Hz,2H)。

The compounds in the following table were prepared by the method of example 156 using the appropriate commercially available halides, mesylate or triflate in step 4.

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Examples 179 and 180.1- (5- ((4- (((1 r,4 r) -4-methoxycyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 179) and preparation of 1- (5- ((4- (((1 s,4 s) -4-methoxycyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example (180)

Using the procedure of example 156, a commercially available mixture of cis and trans 1- (bromomethyl) -4-methoxycyclohexane was used in place of (bromomethyl) cyclohexane. After the last step, the stereoisomers were purified by reverse phase HPLC (eluting with ACN/water/0.1% TFA).

Example 179.1- (5- ((4- (((1 r,4 r) -4-methoxycyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

The minor isomer eluted first. LCMS [ M+H] ⁺ ：455.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.48(s,1H),8.66(d,J＝7.2Hz,1H),8.07(s,1H),7.54(s,1H),6.91(dd,J＝7.3,1.8Hz,1H),4.59(s,4H),3.80(t,J＝6.7Hz,4H),3.42(d,J＝45.9Hz,1H),3.24(s,3H),3.06(ddd,J＝14.6,10.7,4.2Hz,6H),2.80(t,J＝6.7Hz,2H),2.01(d,J＝12.1Hz,2H),1.86-1.54(m,3H),1.20-0.77(m,4H)。

Example 180.1- (5- ((4- (((1 s,4 s) -4-methoxycyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

The second eluting is the major isomer. LCMS [ M+H] ⁺ ：455.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.48(s,1H),8.66(d,J＝7.2Hz,1H),8.07(s,1H),7.54(s,1H),6.91(dd,J＝7.1,1.8Hz,1H),4.60(s,4H),3.80(t,J＝6.7Hz,4H),3.47(s,1H),3.38(d,J＝4.9Hz,1H),3.21(s,3H),3.02(s,5H),2.80(t,J＝6.7Hz,2H),1.91-1.62(m,3H),1.56-1.36(m,4H),1.32-1.18(m,2H)。

Example 181.1 preparation of 5- ((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(4-Benzylpiperazin-1-yl) (4, 4-difluorocyclohexyl) methanone

HATU (8.26 g,21.9 mmol) and DIPEA (9.53 mL,54.75 mmol) were added to a solution of 4, 4-difluorocyclohexane-1-carboxylic acid (3.0 g,18.25 mmol) in THF (10 mL) at 0 ℃. The mixture was stirred for 10min, then 1-benzylpiperazine (3.2 g,18.3 mmol) was added and the reaction was stirred at room temperature for 16h. The reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 50% EtOAc in hexanes) to give (4-benzylpiperazin-1-yl) (4, 4-difluorocyclohexyl) methanone (1.5 g,4.65mmol,25% yield). LCMS [ M+H] ⁺ ：323.5。

And 2, step 2.1-benzyl-4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine

ZrCl was added to a stirred solution of (4-benzylpiperazin-1-yl) (4, 4-difluorocyclohexyl) methanone (1.5 g,4.65 mmol) in THF (10 mL) at-20deg.C ₄ (1.84 g,4.65 mmol) and the mixture was stirred for 30min. Adding MeMgBr. Et ₂ 0 (9.4 mL,28.2mmol, 3.0M) and the mixture was stirred at-20deg.C for 10min and then at room temperature for 16h. After completion, the reaction was quenched with water (10 mL) and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 10% -15% EtOAc in hexanes) to give 1-benzyl-4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine (500 mg,1.49mmol,31% yield).

And 3, step 3.1- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine

Pd/C (100 mg) was added to a stirred solution of 1-benzyl-4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine (500 mg,1.49 mmol) in EtOAc (15 mL) at room temperature under an inert atmosphere. The flask was evacuated and refilled with hydrogen from the balloon and stirred at room temperature for 36h. The reaction was then purged with argon and filtered through celite. The filtrate was concentrated to give crude 1- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine (400 mg, crude). The material was used without further purification.

And 4, step 4.((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) potassium trifluoroborate

To a stirred solution of 1- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazine (400 mg,1.62 mmol) in THF (10 mL) at room temperature was added K ₂ CO ₃ (4478 mg,3.25 mmol) and potassium (bromomethyl) trifluoroborate (326 mg,1.62 mmol). The reaction was stirred at 80 ℃ for 12h, then cooled to room temperature and concentrated,to obtain potassium ((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) trifluoroborate (1.5 g, crude). The material was used without further purification.

Step 5:1- (5- ((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) pyrazolo [1, 5-a]pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

Prepared from ((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) trifluoroborate by the method of example 156, steps 1 and 4, using ((4- (2- (4, 4-difluorocyclohexyl) propan-2-yl) piperazin-1-yl) methyl) trifluoroborate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl }]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：489.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.51 (d, j=6.9 hz, 1H), 8.06 (s, 1H), 7.54 (d, j=14.9 hz, 1H), 7.00 (dd, j=7.4, 1.8hz, 1H), 3.91 (t, j=6.7 hz, 2H), 3.48 (s, 3H), 3.26-3.10 (m, 2H), 2.89 (t, j=6.7 hz, 2H), 2.67 (s, 3H), 2.16 (d, j=23.5 hz, 2H), 1.82 (d, j=14.0 hz, 4H), 1.55-1.18 (m, 7H). Due to peak broadening, 4 protons were not counted. No NH protons were observed due to solvent exchange.

Example 182.1 preparation of 1- (5- ((4- (2- (tetrahydro-2H-pyran-4-yl) propan-2-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of 181, wherein tetrahydro-2H-pyran-4-carboxylic acid is used instead of 4, 4-difluorocyclohexane-1-carboxylic acid. LCMS [ M+H] ⁺ ：474.8。 ¹ H NMR(400MHz,DMSO-d6)δ10.46(s,1H),8.74,(s,1H),8.62(s,1H),8.13(s,1H),8.03(s,1H),7.48(s,1H),6.90(s,1H),3.89(d,J＝11.0Hz,2H),3.78(t,J＝6.5Hz,2H),3.63(s,1H),3.32(s,9H),2.78(t,J＝6.7Hz,2H),1.85(d,J＝110.7Hz,2H),1.52(d,J＝12.5Hz,2H),1.43-1.07(m,6H),0.86(s,3H)。

Example 183.1- (5- ((4- (2-hydroxy-2-methylpropyl) piperazin-1-yl) methyl) pyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the method of example 106 using 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione replaces 3- (2, 4-dimethoxybenzyl) -1- (5- ((1- (2-hydroxy-2-methylpropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：401.4。 ¹ H NMR(400MHz,DMSO-d6)δ10.46(s,1H),8.66(d,J＝7.1Hz,1H),8.07(d,J＝1.7Hz,1H),7.56(s,1H),6.92(dd,J＝7.3,1.9Hz,1H),4.70(s,4H),3.96-3.67(m,4H),3.29(s,2H),2.97(d,J＝36.7Hz,5H),2.79(t,J＝6.8Hz,2H),1.22(d,J＝1.8Hz,6H)。

EXAMPLE 184.1 preparation of 1- (5- ((4- (1- (trifluoromethyl) cyclopropane-1-carbonyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

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Prepared using the method of example 98 using 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride instead of 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride and 1- (trifluoromethyl) cyclopropane-1-carboxylic acid replace isobutyric acid. LCMS [ M+H] ⁺ ：465.2。 ¹ H NMR(400MHz,DMSO-d6)δ10.51(d,J＝2.2Hz,1H),8.76(d,J＝7.1Hz,1H),8.14(d,J＝1.9Hz,1H),7.72(s,1H),6.99(dd,J＝7.2,2.0Hz,1H),4.34(s,2H),3.82(td,J＝6.4,1.8Hz,2H),3.64(s,4H),3.19(s,4H),2.79(t,J＝6.7Hz,2H),1.47-0.84(m,4H)。

EXAMPLE 185.1 preparation of 1- (5- ((4- (isopropylsulfonyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 80 using 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride instead of 3- (2, 4-dimethoxybenzyl) -1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride and propane-2-sulfonyl chloride was used instead of cyclohexylmethanesulfonyl chloride. LCMS [ M+H] ⁺ ：435.4。 ¹ H NMR (500 mhz, dmso-d 6) delta 10.45 (s, 1H), 8.67 (d, j=6.7 hz, 1H), 8.06 (s, 1H), 7.62 (s, 1H), 6.90 (d, j=7.4 hz, 1H), 4.27 (bs, 4H), 3.74 (t, j=6.6 hz, 2H), 3.23-2.83 (m, 4H), 2.72 (t, j=6.7 hz, 2H), 1.16 (d, j=6.8 hz, 6H) (missing protons are masked by water peaks).

Example 186.1 preparation of- (5- ((4-Isobutylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diketones

TFA (4 ml,52 mmol) was added to tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylic acid ester (200 mg,0.346 mmol). The mixture was heated in a sealed vial at 85 ℃ overnight. The mixture was then cooled to room temperature and concentrated, and azeotropically dried with toluene to provide crude 1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate, which was used without further purification. LCMS [ M+H] ⁺ ：329.2。

Step 2:1- (5- ((4-isobutylpiperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidines 2,4 (1H, 3H) -diones

Isobutyraldehyde (10 mg,0.14 mmol) and triethylamine (0.014 mL,0.10 mmol) were added to 1- (5)- (piperazin-1-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (30 mg,0.068 mmol) in DCM (2 mL) and MeOH (2 mL). The reaction mixture was stirred at room temperature for 10min, then sodium triacetoxyborohydride (43 mg,0.20 mmol) was added. The reaction mixture was stirred at room temperature overnight then quenched with saturated aqueous NaHCO3 and extracted three times with DCM. The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA. The product-containing fractions were combined, frozen and lyophilized to give 1- (5- ((4-isobutylpiperazin-1-yl) methyl) pyrazolo [1, 5-a) ]TFA salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (12 mg,0.023mmol,33% yield). LCMS [ M+H] ⁺ ：385.3。 ¹ H NMR(500MHz,DMSO-d6)δ10.46(s,1H),8.56(d,J＝7.1Hz,1H),8.05(s,1H),7.50(s,1H),6.89(d,J＝7.1Hz,1H),3.79(t,J＝6.7Hz,2H),3.72-3.55(m,4H),3.41(s,4H),3.00(d,J＝39.0Hz,4H),2.79(t,J＝6.7Hz,2H),2.11-1.98(m,1H),0.94(d,J＝6.6Hz,6H)。

The compounds in the following table were prepared by the method of example 186 using the appropriate commercially available aldehyde in step 2.

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EXAMPLE 192 preparation of (S) -1- (5- ((4- (cyclohexylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1 (2H)) Group) pyriAzolo [1,5-a ]]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylic acid ester

To 1- (5-bromopyrazolo [1, 5-a) at room temperature]To a suspension of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1.2 g,2.61 mmol) in toluene (20 mL) and water (2 mL) was added Cs ₂ CO ₃ (2.55 g,7.84 mmol), potassium tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate (3.35 g,10.45 mmol) [ see ChemMedChem [ chemical and pharmaceutical chemistry ]],2016,11,2640-2648]And RuPhos (242 mg,0.52 mmol), followed by Pd (OAc) ₂ (59 mg,0.26 mmol). The mixture was stirred at 100 ℃ overnight, then cooled to room temperature and partitioned between EtOAc and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated. Silica gel column chromatography [ elution with 0-100% EtOAc in heptane/EtOH (3:1) ] ]Tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) is provided as an off-white foamy solid]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate (1.12 g,2.61mmol,71% yield). LCMS [ M+H] ⁺ ：593.4。

And 2, step 2.(S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a]pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate

TFA (4 mL,2 mmol) was added to tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate (1.2 g,2.0 mmol) in DCM (12 mL) and the mixture stirred at room temperature for 2h. The reaction was then concentrated and azeotropically dried with toluene to give crude (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (1.2 g,1.4 mmol) was used without further purification. LCMS [ M+H] ⁺ ：493.2。

And 3, step 3.(S) -1- (5- ((4- (cyclohexylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Piirae-type pyridine Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (100 mg,0.20 mmol) in DCE (2 mL) was added cyclohexane-formaldehyde (21 mg,0.20 mmol), naBH (OAc) ₃ (120 mg,0.60 mmol), 4A MS (100 mg) and DIPEA (113 mg,0.15mL,0.95 mmol). The reaction was stirred at room temperature for 2h. The suspension was filtered through a pad of celite and the filtrate was washed with saturated NaHCO ₃ The aqueous solution was diluted and extracted with DCM. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give (S) -1- (5- ((4- (cyclohexylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (200 mg,0.34mmol,72% purity). The crude product was used in the next step without any further purification. LCMS [ M+H] ⁺ ：589.2。

Step 4:(S) -1- (5- ((4- (cyclohexylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a] Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 192)Prepared using the method of example 156, step 4, using (S) -1- (5- ((4- (cyclohexylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：439.1。 ¹ H NMR(400MHz,DMSO-d6)δ10.43(s,1H),8.57-8.55(m,1H),8.00(s,1H),7.43(s,1H),6.88-6.86(m,1H),3.76(s,2H),3.45-3.44(m,2H),2.80-2.72(m,3H),2.57(br s,1H),2.47-2.40(m,1H),2.35-2.26(m,1H),2.23-2.08(m,2H),2.03-1.77(m,3H),1.63(br s,4H),1.42-1.39(m,1H),1.30-1.02(m,4H),0.92-0.91(m,3H),0.87-0.70(m,2H)。

The compounds in the following table were prepared from 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione hydrochloride by the method of example 192, step 3-4, using the appropriate commercially available aldehyde in step 3.

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The compounds in the following table were prepared by the method of example 192, using the appropriate commercially available aldehyde in step 3.

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Example 201.preparation of 1- (5- (((S) -4- (((1 r, 4S) -4-hydroxycyclohexyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, steps 3-4, wherein trans-4- (benzyloxy) cyclohexane-1-carbaldehyde (see WO 2020/232470,2020, a1, incorporated herein by reference) was used instead of cyclohexane-carbaldehyde. LCMS [ M+H] ⁺ ：455.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.44 (d, j=7.1 hz, 1H), 8.00 (d, j=11.5 hz, 1H), 7.38 (s, 1H), 6.83 (d, j=6.2 hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.63-3.44 (m, 3H), 2.96-2.85 (m, 6H), 2.73-2.64 (m, 2H), 2.08-1.72 (m, 7H), 1.58 (q, j=12.1, 11.1hz, 2H), 1.37-1.22 (m, 4H), 1.11 (q, j=12.5 hz, 2H), no NH and OH protons are observed due to solvent exchange.

The compounds in the following table were prepared from (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate by the method of example 156, step 3-4, using the appropriate commercially available halide, mesylate or triflate in step 3.

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Example 203 (alternative Synthesis): (S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 203) preparation

Step 1.(S) -3- (2, 4-Dimethoxybenzyl) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (2.2 g,3.6 mmol) in DCM (20 mL) was added Et ₃ N (0.51 mL,3.6 mmol) followed by isobutyraldehyde (1.31 g,18.1 mmol). The mixture was stirred at room temperature for 30min, then NaBH (OAc) was added ₃ (3.84 g,18.1 mmol). The reaction was stirred at room temperature overnight. Saturated NaHCO for reaction ₃ Quench with aqueous solution and extract with DCMTaking. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 10% -100% EtOAc in hexanes (25% etoh) provided (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a) as a white solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1.25 g,2.28mmol,63% yield). LCMS [ M+H] ⁺ ：549.3。

And 2, step 2.(S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 203)

TFA (10 mL) was added to (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1.25 g,2.28 mmol). The reaction mixture was stirred at 90 ℃ for 16h and then concentrated. The crude material was dissolved in DCM and saturated NaHCO ₃ Washing with aqueous solution. The layers were separated and the aqueous layer was extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 10% -100% EtOAc in hexanes (25% etoh) provided (S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (800 mg,1.97mmol,86% yield). LCMS [ M+H ] ⁺ ：399.4。 ¹ H NMR (500 MHz, methanol-d 4) delta 8.46 (dd, j=7.1, 0.9hz, 1H), 8.02 (s, 1H), 7.51 (dd, j=1.9, 1.0hz, 1H), 7.01 (dd, j=7.2, 1.8hz, 1H), 3.91 (t, j=6.7 hz, 2H), 3.63-3.49 (m, 2H), 2.91 (t, j=6.8 hz, 3H), 2.73 (dd, j=28.7, 10.7hz, 2H), 2.39 (dd, j=71.2, 21.3hz, 4H), 2.02 (d, j=45.3 hz, 2H), 1.81 (s, 1H), 1.04 (d, j=6.2 hz, 3H), 0.93 (dd, j=7.7, 6.6hz, 6H).

Examples 210 and 211.1 preparation of 1- (5- (((S) -4- (((1 r, 4S) -4-methoxycyclohexyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 210) and 1- (5- (((S) -4- (((1S, 4R) -4-methoxycyclohexyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 211)

Prepared using the procedure of example 202, using a commercially available mixture of cis and trans 1- (bromomethyl) -4-methoxycyclohexane instead of trans-4- (benzyloxy) cyclohexane-1-carbaldehyde. After the last step, the stereoisomers were purified by reverse phase HPLC (eluting with ACN/water/0.1% TFA).

Example 210.1- (5- (((S) -4- (((1 r, 4S) -4-methoxycyclohexyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

The minor isomer eluted first. LCMS [ M+H] ⁺ ：469.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.45(s,1H),8.64(d,J＝7.2Hz,1H),8.05(s,1H),7.52(s,1H),6.90(dd,J＝7.2,1.8Hz,1H),4.99(s,4H),3.79(t,J＝6.7Hz,2H),3.72(s,2H),3.23(s,4H),3.13-2.94(m,4H),2.79(t,J＝6.7Hz,3H),2.37(d,J＝30.0Hz,1H),2.00(s,2H),1.92-1.57(m,2H),1.26(s,3H),1.07(dt,J＝37.0,13.5Hz,4H)。

Example 211.1- (5- (((S) -4- (((1S, 4R) -4-methoxycyclohexyl) methyl) -3-methylpiperazin-1-yl) Methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

The second eluting is the major isomer. LCMS [ M+H] ⁺ ：469.3。 ¹ H NMR 400MHz,DMSO-d6)δ10.44(s,1H),8.63(d,J＝7.2Hz,1H),8.04(s,1H),7.50(s,1H),6.90(dd,J＝7.2,1.8Hz,1H),3.78(t,J＝6.7Hz,2H),3.67(s,5H),3.23(s,3H),3.20(s,3H),3.17-2.84(m,4H),2.79(t,J＝6.7Hz,2H),2.46-2.21(m,1H),1.92-1.69(m,3H),1.55(s,1H),1.51-1.29(m,4H),1.25(s,3H)。

EXAMPLE 212 preparation of (S) -1- (5- ((4- (cyclopentylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Preparation using the procedure of example 186Wherein tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) is used]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate instead of tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylate and cyclopentane-formaldehyde was used instead of isobutyraldehyde. LCMS [ M+H] ⁺ ：425.3。 ¹ H NMR(400MHz,DMSO-d6)δ10.44(s,1H),8.63(d,J＝7.2Hz,1H),8.04(s,1H),7.51(s,1H),6.90(dd,J＝7.2,1.9Hz,1H),4.39(s,3H),3.78(t,J＝6.7Hz,2H),3.61(d,J＝44.7Hz,3H),3.30(d,J＝20.9Hz,2H),3.00(d,J＝14.0Hz,3H),2.79(t,J＝6.7Hz,2H),2.18(s,1H),1.79(d,J＝20.7Hz,2H),1.58(ddd,J＝38.4,7.7,4.0Hz,4H),1.42-1.10(m,5H)。

EXAMPLE 213 preparation of (S) -1- (5- ((4- (cyclobutylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 186 using tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) ]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate instead of tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylate and uses cyclobutane-formaldehyde instead of isobutyraldehyde. LCMS [ M+H] ⁺ ：411.5。 ¹ H NMR(500MHz,DMSO-d6)δ10.44(s,1H),8.58(d,J＝7.1Hz,1H),8.02(s,1H),7.45(s,1H),6.89(dd,J＝7.1,1.8Hz,1H),3.78(t,J＝6.7Hz,2H),3.47(s,2H),2.79(t,J＝6.7Hz,2H),2.76-2.57(m,3H),2.56(s,2H),2.14(d,J＝47.7Hz,3H),1.99(s,3H),1.92-1.73(m,3H),1.65(s,2H),1.03(d,J＝50.1Hz,3H)。

The compounds in the following table were prepared using the method of example 192, using potassium (R) - ((4- (tert-butoxycarbonyl) -3-methylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and using the appropriate commercially available aldehyde in place of cyclohexane formaldehyde.

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The compounds in the following table were prepared using the method of example 192, using potassium (R) - ((4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) methyl) trifluoroborate [ see j.med. Chem. [ journal of pharmaceutical chemistry ]2012,55,7796-7816] instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and using the appropriate commercially available aldehyde instead of cyclohexane formaldehyde.

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The compounds in the following table were prepared using the method of example 192, using potassium (S) - ((4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) methyl) trifluoroborate [ see j.med. Chem. [ journal of pharmaceutical chemistry ]2012,55,7796-7816] instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and using the appropriate commercially available aldehyde instead of cyclohexane formaldehyde.

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EXAMPLE 226 preparation of (S) -1- (5- ((4- ((3, 3-difluorocyclobutyl) methyl) -2-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 156, using potassium (S) - ((4- (tert-butoxycarbonyl) -2-methylpiperazin-1-yl) methyl) trifluoroborate [ see j.med. Chem. [ journal of pharmaceutical chemistry ]]2012,55,7796-7816]Instead of potassium ((4- (tert-butoxycarbonyl) piperazin-1-yl) methyl) trifluoroborate, and instead of (bromomethyl) cyclohexane, (3, 3-difluorocyclobutyl) methyl 4-methylbenzenesulfonate was used. LCMS [ M+H] ⁺ ：447.0。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.45 (d, j=7.1 hz, 1H), 8.37 (s, 1H), 8.01 (s, 1H), 7.51 (s, 1H), 6.98 (dd, j=7.1, 1.8hz, 1H), 4.19 (d, j=13.7 hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.34-3.27 (m, 1H), 3.07-2.94 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.86-2.63 (m, 6H), 2.53 (t, j=11.2 hz, 1H), 2.49-2.24 (m, 5H), 1.23 (d, j=6.2 hz, 3H), no NH protons are observed due to the exchange solvent.

EXAMPLE 227 preparation of (S) -1- (5- ((3-ethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using potassium (S) - ((4- (tert-butoxycarbonyl) -3-ethylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and using tetrahydro-2H-pyran-4-carbaldehyde Instead of cyclohexane-formaldehyde. LCMS [ M+H] ⁺ ：455.5。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.48 (d, j=7.2 hz, 1H), 8.23 (s, 1H), 8.04 (s, 1H), 7.52 (s, 1H), 7.00 (dd, j=7.3, 1.9hz, 1H), 3.96 (dd, j=11.5, 4.2hz, 2H), 3.90 (t, j=6.8 hz, 2H), 3.86-3.50 (m, 4H), 3.50-3.39 (m, 2H), 3.28-2.51 (m, 9H), 1.96 (d, j=78.4 hz, 3H), 1.75 (d, j=13.3 hz, 2H), 1.66 (d, j=13.2 hz, 1H), 1.49-1.31 (m, 2H), 0.96 (t, j=7.4 hz, 3H), no protons are observed due to NH.

EXAMPLE 228 preparation of (S) -1- (5- ((3-ethyl-4-isobutylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using (S) - ((4- (tert-butoxycarbonyl) -3-ethylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt and isobutyraldehyde in place of cyclohexane formaldehyde. LCMS [ M+H] ⁺ ：413.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.48 (d, j=7.1 hz, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.51 (s, 1H), 7.00 (d, j=7.2 hz, 1H), 3.90 (t, j=6.6 hz, 2H), 3.74 (d, j=13.7 hz, 1H), 3.63 (d, j=13.7 hz, 1H), 3.48 (s, 1H), 3.23-3.01 (m, 3H), 2.89 (t, j=7.0 hz, 5H), 2.75-2.50 (m, 2H), 2.12-1.99 (m, 1H), 1.95-1.69 (m, 2H), 1.05 (t, j=6.1 hz, 6H), 0.96 (t, j=7.4 hz, 3H), no protons were observed due to the exchange of the solvent.

EXAMPLE 229 preparation of (S) -1- (5- ((4-isobutyl-3-isopropylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192 using (S) - ((4- (tert-butoxycarbonyl) -3-isopropylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt and allowingThe cyclohexane formaldehyde is replaced by isobutyraldehyde. LCMS [ M+H] ⁺ ：427.5。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.48 (d, j=7.4 hz, 1H), 8.07 (s, 1H), 8.04 (s, 1H), 7.53 (s, 1H), 7.00 (dd, j=6.9, 1.8hz, 1H), 3.90 (t, j=6.8 hz, 2H), 3.86-3.53 (m, 3H), 3.41-2.95 (m, 4H), 2.89 (t, j=6.8 hz, 2H), 2.55 (d, j=44.4 hz, 4H), 2.12 (s, 1H), 1.11-1.01 (m, 9H), 1.00 (s, 3H), no NH protons are observed due to the exchange of solvents.

EXAMPLE 230 preparation of (S) -1- (5- ((3-isopropyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using (S) - ((4- (tert-butoxycarbonyl) -3-isopropylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt and tetrahydro-2H-pyran-4-carbaldehyde in place of cyclohexane formaldehyde. LCMS [ M+H ] ⁺ ：469.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.49 (d, j=7.2 hz, 1H), 8.30 (s, 1H), 8.04 (s, 1H), 7.55 (s, 1H), 7.00 (dd, j=7.2, 1.8hz, 1H), 3.95 (dd, j=11.5, 4.2hz, 2H), 3.90 (t, j=6.8 hz, 2H), 3.85 (d, j=13.7 hz, 1H), 3.72 (d, j=13.6 hz, 1H), 3.53-3.38 (m, 3H), 3.18-2.92 (m, 5H), 2.89 (t, j=6.8 hz, 2H), 2.59 (t, j=10.8 hz, 2H), 2.50 (dd, j=12.6, 10.0hz, 1H), 2.41-2.28 (m, 1.7hz, 1H), 3.53-3.38 (m, 3.18-2.92 (m, 5H), 3.9-2.92 (m, 5H), 2.9 (t, j=6.8 hz, 2H), 2.50 (j=1.9, 2H), 2.41-2.28 (d, 1H), 1.7hz, 1H), 1.7 (d, 1H), 1.7hz (1H), 1.7 (d, 1H), 1.3.3.3 (1H), 1H (1H).

Example 231.preparation of 1- (5- ((4- (cyclohexylmethyl) -3, 3-dimethylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192 using ((4- (tert-butoxycarbonyl) -3, 3-dimethyl)Piperazine-1-yl) methyl trifluoroborate replaces the potassium salt of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate. LCMS [ M+H] ⁺ ：453.3。 ¹ H NMR (400 MHz, methanol-d 4) δ8.52 (s, 1H), 8.46 (d, j=7.2 hz, 1H), 8.02 (s, 1H), 7.49 (s, 1H), 7.00 (dd, j=7.4, 1.7hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.61 (s, 2H), 2.89 (t, j=6.8 hz, 2H), 2.99-2.32 (m, 5H), 1.92-1.59 (m, 6H), 1.42-1.15 (m, 10H), 1.04 (q, j=11.4 hz, 2H), no NH protons are observed due to the exchange of solvents.

Example 232.1 preparation of 1- (5- ((3, 3-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using ((4- (tert-butoxycarbonyl) -3, 3-dimethylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt and tetrahydro-2H-pyran-4-carbaldehyde in place of cyclohexane formaldehyde. LCMS [ M+H] ⁺ ：455.1。 ¹ H NMR (300 MHz, methanol-d 4) delta 8.45 (d, j=7.2 hz, 1H), 8.27 (s, 1H), 8.01 (s, 1H), 7.47 (s, 1H), 6.98 (d, j=7.3 hz, 1H), 3.94 (d, j=12.8 hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.71-3.31 (m, 9H), 3.05 (s, 5H), 2.87 (t, j=6.7 hz, 2H), 2.08-1.58 (m, 3H), 1.32 (d, j=24.2 hz, 7H). No NH protons were observed due to solvent exchange.

Example 233.1 preparation of 1- (5- ((4-isobutyl-3, 3-dimethylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using ((4- (tert-butoxycarbonyl) -3, 3-dimethylpiperazin-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt, and isobutyraldehyde And (3) cyclohexane formaldehyde. LCMS [ M+H] ⁺ ：413.3。 ¹ H NMR (300 MHz, methanol-d 4) delta 8.47 (d, j=7.1 hz, 1H), 8.03 (s, 1H), 7.50 (s, 1H), 7.00 (d, j=7.1 hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.81-3.36 (m, 2H), 3.11 (d, j=39.0 hz, 2H), 2.89 (t, j=6.8 hz, 2H), 2.83-2.42 (m, 3H), 2.01 (ddd, j=25.6, 12.6,5.4hz, 2H), 1.59-1.20 (m, 8H), 1.08 (d, j=6.5 hz, 6H).

Example 234.1 preparation of 1- (5- (((1R, 4R) -5- (cyclohexylmethyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, using (((1R, 4R) -5- (tert-butoxycarbonyl) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl) trifluoroborate instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate. LCMS [ M+H] ⁺ ：437.2。

Example 235.1 preparation of 1- (5- (((1R, 4R) -5- (pyridin-3-ylmethyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, using (((1R, 4R) -5- (tert-butoxycarbonyl) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl) potassium trifluoroborate instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and nicotinaldehyde was used instead of cyclohexane-formaldehyde. LCMS [ M+H ] ⁺ ：432.2。 ¹ H NMR(400MHz,CD ₃ OD) delta 8.80 (brs, 1H), 8.60 (d, j=7.2 hz, 1H), 8.42 (m, 1H), 8.13 (s, 1H), 7.90 (brs, 1H), 7.77 (s, 1H), 7.07 (d, j=7.2 hz, 1H), 4.49-4.44 (m, 1H), 4.32-4.20 (m, 3H), 4.08-3.87 (m, 5H), 3.62-3.61 (m, 3H), 3.04-3.01 (m, 1H), 2.90 (t, j=6.8 hz, 2H), 2.31 (s, 2H), no NH protons are observed due to the exchange of solvents.

Example 236.1 preparation of 1- (5- (((1S, 4S) -5- (cyclohexylmethyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192 using (((1S, 4S) -5- (tert-butoxycarbonyl) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl) trifluoroborate instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate. LCMS [ M+H] ⁺ ：437.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.46 (d, j=7.2 hz, 1H), 8.03 (s, 1H), 7.52 (s, 1H), 7.02 (d, j=7.4 hz, 1H), 4.23 (s, 1H), 3.97-3.86 (m, 2H), 3.83-3.54 (m, 2H), 3.28 (s, 1H), 3.19-2.95 (m, 3H), 2.95-2.84 (m, 2H), 2.84-2.61 (m, 1H), 2.23 (d, j=11.7 hz, 1H), 2.13-2.04 (m, 1H), 1.91-1.69 (m, 6H), 1.59 (s, 1H), 1.42-1.20 (m, 4H), 1.15-0.95 (m, 2H), no NH was observed due to the exchange of the solvent.

Example 237.1 preparation of 1- (5- (((1S, 4S) -5- (pyridin-3-ylmethyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192 using (((1S, 4S) -5- (tert-butoxycarbonyl) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl) potassium trifluoroborate instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylate and nicotinaldehyde was used instead of cyclohexane-formaldehyde. LCMS [ M+H] ⁺ ：432.1。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.83 (s, 1H), 8.75 (d, j=5.6 hz, 1H), 8.59 (d, j=7.3 hz, 1H), 8.52 (d, j=8.1 hz, 1H), 8.12 (s, 1H), 7.96 (t, j=6.9 hz, 1H), 7.78 (s, 1H), 7.07 (dd, j=7.2, 1.9hz, 1H), 4.49 (d, j=13.3 hz, 1H), 4.33 (d, j=13.2 hz, 1H), 4.28 (s, 1H), 4.20 (d, j=14.7 hz, 1H), 4.05 (d, j=14.7 hz, 1H), 3.93 (t, j=6.8 hz, 2H), 3.84 (s, 1H), 3.62 (d, j=11.3 hz, 1H), 3.33 (d, 1H), 3.38.8 hz, 1H)dd, j=12.0, 2.7hz, 1H), 2.89 (t, j=6.8 hz, 2H), 2.31 (s, 2H), NH protons are not observed due to solvent exchange.

EXAMPLE 238.1 preparation of 1- (5- (((1S, 4S) -5- (isopropylsulfonyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1- (5- (((1S, 4S) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl pyrazolo [1,5 ] a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 1- (5- (((1S, 4S) -2, 5-diazabicyclo [ 2.2.1) ]Heptane-2-yl) methyl pyrazolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (175 mg,0.32 mmol) [ prepared in example 236]TfOH (0.2 mL) was added to a stirred solution in TFA (5 mL) and the reaction mixture was stirred at 70 ℃ for 2h. The reaction was concentrated to give the crude compound. The crude compound was dissolved in 10% MeOH in DCM and basified with Amberlyst-a21 (free base) resin, then filtered. Concentrating the filtrate to obtain 1- (5- (((1S, 4S) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (150 mg, crude). LCMS [ M+H] ⁺ ：341.3。

And 2, step 2.1- (5- (((1S, 4S) -5- (isopropylsulfonyl) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2- Group) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 1- (5- (((1S, 4S) -2, 5-diazabicyclo [ 2.2.1) at-20deg.C]Heptane-2-yl) methyl pyrazolo [1,5-a ]]To a stirred solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (130 mg,0.38 mmol) in DCM (5 mL) was added Et ₃ N (0.076 mL,0.76 mmol) and propane-2-sulfonyl chloride (0.035 mL,0.45 mmol). The reaction mixture was stirred at 0 ℃ for 2h, then quenched with methanol and concentrated to give the crude compound. The crude compound was purified by PREP HPLC using: mobile phase: a = 0.1% TFA in water, B = acetonitrile; column: ATLA (automatic Tesla) NTIS (250 mm×21.2 mm), 5.0 μ; flow rate: 20mL/min. The collected fractions were concentrated under reduced pressure to give 1- (5- (((1S, 4S) -5- (isopropylsulfonyl) -2, 5-diazabicyclo [ 2.2.1) as an off-white solid]Heptane-2-yl) methyl pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (12 mg,0.022mmol,5.8% yield). LCMS [ M+H] ⁺ ：447.2。HPLC：Rt＝5.406min。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.63 (dd, j=7.2, 3.1hz, 1H), 8.15 (d, j=1.7 hz, 1H), 7.83 (d, j=2.8 hz, 1H), 7.07 (dd, j=7.4, 2.1hz, 1H), 4.59 (dd, j=13.5, 7.5hz, 3H), 4.44 (d, j=12.3 hz, 1H), 3.95 (t, j=6.8 hz, 2H), 3.72 (s, 2H), 3.50 (d, j=9.1 hz, 1H), 2.91 (t, j=6.8 hz, 2H), 2.47 (d, j=12.0 hz, 1H), 2.23 (d, j=13.0 hz, 1H), 1.49 (d, j=2.2 hz, 2H), 1.35 (d, j=6.8 hz, 2H), 3.72 (s, 2H), 3.50 (d, j=9.8 hz, 1H), 2H).

Example 239.1 preparation of 1- (5- (((1R, 4R) -5- (isopropylsulfonyl) -2, 5-diazabicyclo [2.2.1] heptan-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 238 using 1- (5- (((1R, 4R) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl pyrazolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-Dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione [ prepared in example 234 ]Instead of 1- (5- (((1S, 4S) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-yl) methyl pyrazolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：446.8。

Example 240.preparation of 1- (5- ((4- (3-methylbutan-2-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 240)

Prepared using the method of example 156, steps 1 and 4, using trifluoro ((4- (3-methylbutane)-2-yl) piperazin-1-yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：399.0。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.47 (d, j=7.1 hz, 1H), 8.39 (s, 1H), 8.03 (s, 1H), 7.52 (s, 1H), 6.99 (d, j=7.2 hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.70 (s, 2H), 3.31 (m, 4H), 3.07 (d, j=6.5 hz, 1H), 2.89 (t, j=6.8 hz, 2H), 2.81 (bs, 4H), 2.30-2.16 (m, 1H), 1.25 (d, j=6.7 hz, 3H), 1.04 (d, j=6.7 hz, 3H), 0.97 (d, j=6.7 hz, 3H).

Example 241.1 preparation of 1- (5- ((4- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using potassium trifluoro ((4- (3, 3-trifluoro-2, 2-dimethylpropyl) piperazin-1-yl) methyl) borate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl ] ]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：453.2。 ¹ H NMR (400 MHz, methanol-d 4) δ8.61 (d, j=7.0 hz, 1H), 8.13 (s, 1H), 7.75 (s, 1H), 7.00 (dd, j=7.3, 1.9hz, 1H), 4.35 (s, 2H), 3.93 (t, j=6.8 hz, 2H), 3.48 (d, j=12.2 hz, 1H), 3.08 (t, j=11.8 hz, 1H), 2.98-2.67 (m, 8H), 2.55 (d, j=10.8 hz, 2H), 1.14 (d, j=2.1 hz, 6H). No NH protons were observed due to solvent exchange.

Example 242.preparation of 1- (5- ((4- (cyclohexylmethyl) -3-oxopiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using ((4- (cyclohexylmethyl) -3-oxopiperazin-1-yl) methyl) trifluoroborate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：439.2。 ¹ H NMR (400 MHz, methanol-d 4) δ8.50 (d, J=7.1 Hz, 1H), 8.04 (s, 1H), 7.57 (s, 1H)7.00 (dd, j=7.1, 1.8hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.81 (s, 2H), 3.43 (t, j=5.5 hz, 2H), 3.38-3.31 (m, 2H), 3.25 (d, j=7.2 hz, 2H), 2.89 (t, j=6.8 hz, 4H), 1.80-1.59 (m, 6H), 1.24 (q, j=9.3, 6.0hz, 3H), 0.97 (q, j=11.9 hz, 2H), NH protons are not observed due to solvent exchange.

Example 243.preparation of 1- (5- ((4- (2-cyclohexylethyl) -3-oxopiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using ((4- (2-cyclohexylethyl) -3-oxopiperazin-1-yl) methyl) trifluoroborate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：452.9。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.52 (d, j=7.2 hz, 1H), 8.06 (s, 1H), 7.62-7.58 (m, 1H), 7.00 (dd, j=7.1, 1.9hz, 1H), 4.01-3.78 (m, 3H), 3.51-3.38 (m, 5H), 3.12-2.96 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 1.83-1.60 (m, 5H), 1.46 (dt, j=9.7, 6.8hz, 2H), 1.37-1.15 (m, 5H), 0.96 (qd, j=14.1, 12.9,4.4hz, 2H), no NH protons are observed due to solvent exchange.

EXAMPLE 244 preparation of (R) -1- (5- ((4- (cyclohexylmethyl) -3- (methoxymethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using potassium (R) - ((4- (cyclohexylmethyl) -3- (methoxymethyl) piperazin-1-yl) methyl) trifluoroborate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：469.4。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.46 (s, 1H), 8.45 (s, 1H), 8.02 (s, 1H), 7.49 (s, 1H), 3.89 (t, J=6.8 Hz, 2H), 3.70-3.53 (m, 4H), 3.36 (s, 4H), 3.19 (s, 1H), 3.03 (dd, J=12.0, 8.2Hz, 1H), 2.97-2.82 (m, 5H), 2.67-2.47 (m, 3H), 1.90 (d, J=13.1 Hz, 1) H) 1.82-1.63 (m, 5H), 1.41-1.18 (m, 4H), 1.09-0.92 (m, 2H), no NH protons are observed due to solvent exchange.

EXAMPLE 245 preparation of (R) -1- (5- ((4-isobutyl-3- (methoxymethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using potassium (R) -trifluoro- (4-isobutyl-3- (methoxymethyl) piperazin-1-yl) methyl) borate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：429.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.46 (d, j=7.1 hz, 1H), 8.42 (1H, s) 8.02 (s, 1H), 7.50 (s, 1H), 6.98 (d, j=7.1 hz, 1H), 3.89 (t, j=6.7 hz, 2H), 3.73-3.56 (m, 4H), 3.45 (d, j=13.1 hz, 1H), 3.37 (s, 3H), 3.31 (s, 1H), 3.12-2.96 (m, 2H), 2.94-2.84 (m, 4H), 2.72 (dd, j=13.0, 5.5hz, 1H), 2.67-2.55 (m, 2H), 2.05 (H, j=6.8 hz, 2H), 1.03 (d, j=6.8 hz, 3H), 1.01 (d, 6.8hz, 3H), and no proton exchange due to the proton exchange was observed.

EXAMPLE 246 preparation of (R) -1- (5- ((4- (cyclohexylmethyl) -3- (difluoromethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using potassium (R) - ((4- (cyclohexylmethyl) -3- (difluoromethyl) piperazin-1-yl) methyl) trifluoroborate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：475.4。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.56 (d, j=7.2 hz, 1H), 8.09 (s, 1H), 7.66 (s, 1H), 7.01 (dd, j=7.4, 2.0hz, 1H), 6.35 (t, j=54.3 hz, 1H), 4.12 (s, 1H), 3.92 (t, j=6.8 hz, 2H), 3.53 (t, j=17.9 hz, 1H), 3.10 (d, j=49.1 hz, 4H), 2.89 (t, j=6.8 hz, 3H), 2.59 (s, 1H), 2.12 (dd, j=35.5, 24.8hz, 1H), 1.98-1.45 (m, 7H), 1.26 (dq, j= 21.1,12.4,11).8hz, 4H), 1.08-0.83 (m, 2H), no NH protons are observed due to solvent exchange.

EXAMPLE 247 preparation of (R) -1- (5- ((3- (difluoromethyl) -4-isobutylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using potassium (R) - ((3- (difluoromethyl) -4-isobutylpiperazin-1-yl) methyl) trifluoroborate in place of { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：434.7。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.58 (d, j=7.2 hz, 1H), 8.11 (s, 1H), 7.70 (s, 1H), 7.01 (dd, j=7.1, 1.9hz, 1H), 6.34 (t, j=54.1 hz, 1H), 4.23 (d, j=24.8 hz, 2H), 3.92 (t, j=6.7 hz, 2H), 3.38 (s, 2H), 3.12 (m, 4H), 2.90 (t, j=6.7 hz, 4H), 2.52 (s, 1H), 1.90 (s, 1H), 1.04-0.84 (m, 6H) were not observed due to the exchange of solvents.

Example 248.preparation of 1- (5- ((1, 4-diazepan-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, steps 1 and 4, using ((4- (tert-butoxycarbonyl) -1, 4-diazepan-1-yl) methyl) trifluoroboric acid potassium salt instead of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt. LCMS [ M+H] ⁺ ：343.1。 ¹ HNMR (400 MHz, methanol-d 4) δ8.56 (t, j=6.8 hz, 1H), 8.10 (d, j=4.4 hz, 1H), 7.71 (d, j=21.5 hz, 1H), 7.14-6.99 (m, 1H), 4.27 (d, j=44.9 hz, 2H), 3.92 (t, j=6.7 hz, 2H), 3.68 (t, j=4.7 hz, 2H), 3.60-3.50 (m, 3H), 3.45-3.37 (m, 2H), 3.26 (s, 1H), 2.89 (t, j=6.8 hz, 2H), 2.29-2.16 (m, 2H), no NH protons are observed due to the replacement of solvents.

Example 249.1 preparation of 1- (5- ((4- (cyclohexylmethyl) -1, 4-diazepan-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192 using ((4- (tert-butoxycarbonyl) -1, 4-diazepan-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-borane) methyl) piperazine-1-carboxylic acid potassium salt. LCMS [ M+H] ⁺ ：439.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.52 (d, j=7.2 hz, 1H), 8.07 (s, 1H), 7.61 (s, 1H), 7.03 (dd, j=7.3, 2.0hz, 1H), 4.02 (s, 2H), 3.91 (t, j=6.8 hz, 2H), 3.51 (s, 4H), 3.23 (s, 1H), 3.14-3.03 (m, 5H), 2.90 (t, j=6.8 hz, 2H), 2.25-2.10 (m, 2H), 1.87-1.66 (m, 6H), 1.44-1.19 (m, 4H), 1.07 (t, j=11.7 hz, 2H), no NH protons are observed due to the exchange of solvents.

Example 250.preparation of 1- (5- ((4-isobutyl-1, 4-diazepan-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, using ((4- (tert-butoxycarbonyl) -1, 4-diazepan-1-yl) methyl) trifluoroborate in place of tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-borane) methyl) piperazine-1-carboxylic acid potassium salt and isobutyraldehyde in place of cyclohexane formaldehyde. LCMS [ M+H] ⁺ ：399.3。 ¹ H NMR(400MHz,CD ₃ OD) δ8.44 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.49 (s, 1H), 7.02 (d, j=6.6 hz, 1H), 3.88 (t, j=7.2 hz, 2H), 3.73 (s, 2H), 3.14-3.07 (m, 4H), 2.90-2.69 (m, 8H), 1.97 (m, 3H), 0.97 (d, j=6.6 hz, 6H), NH protons are not observed due to the exchange of solvents.

Example 251.1 preparation of 1- (5- (((1R, 5S) -8- (3-fluorobenzyl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (1R, 5S) -3- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1) (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid ester

In a glove box, at room temperature, 1- (5-bromopyrazolo [1, 5-a) was introduced into]To a suspension of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (600 mg,1.30 mmol) in t-amyl-OH (6 mL) was added Cs ₂ CO ₃ (2.6 mL,1.5M aqueous solution), ((1R, 5S) -8- (tert-butoxycarbonyl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) methyl potassium trifluoroborate (518 mg,1.56 mmol) and Ad ₂ n-BuP-Pd-G ₃ (44 mg,0.06 mmol). The reaction mixture was stirred under an inert atmosphere at 90 ℃ for 16h. The reaction mixture was then filtered and concentrated. The crude product was purified by silica gel chromatography (eluting with ethyl acetate in petroleum ether) to give tert-butyl (1 r,5 s) -3- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as a pale yellow solid]Pyridin-5-yl) methyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid ester. LCMS [ M+H] ⁺ ：605.2。

Step 2:1- (5- (((1R, 5S) -8- (3-fluorobenzyl) -3, 8-diazabicyclo [ 3.2.1)]Octane-3-yl) methyl ester Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

Prepared using the procedure of example 156, step 2-4, using tert-butyl (1R, 5S) -3- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid ester substituted tert-butyl 4- ((3- (3- (2, 4-dimethoxy benzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) ]Pyridin-5-yl) methyl) piperazine-1-carboxylate and 1- (bromomethyl) -3-fluorobenzene was used instead of (bromomethyl) cyclohexane. LCMS [ M+H] ⁺ ：463.1。 ¹ H NMR(400MHz,DMSO-d6)δ＝10.43(br s,1H),8.57(d,J＝7.2Hz,1H),7.99(s,1H),7.41(s,1H),7.38-7.28(m,1H),7.20-7.17(m,2H),7.07-6.98(m,1H),6.91-6.88(m,1H),3.76(t,J＝6.8Hz,2H),3.49-3.47(m,4H),3.04(br s,2H),2.76(t,J＝6.8Hz,2H),2.54-2.52(m,2H),2.32-2.25(m,2H),1.94-1.83(m,2H),1.81-1.71(m,2H)。

Example 252.preparation of 1- (5- (((1R, 5S) -8- (cyclohexylmethyl) -3, 8-diazabicyclo [3.2.1] oct-3-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, step 2-4, using tert-butyl (1 r,5 s) -3- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid ester substituted tert-butyl 4- ((3- (3- (2, 4-dimethoxy benzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylic acid ester. LCMS [ M+H] ⁺ ：451.1。 ¹ H NMR(400MHz,DMSO-d6)δ10.43(br s,1H),8.56(d,J＝7.1Hz,1H),7.99(s,1H),7.40(s,1H),6.89-6.87(m,1H),3.75(t,J＝6.8Hz,2H),3.45-3.42(m,2H),3.01(br s,2H),2.76(t,J＝6.8Hz,2H),2.48(br s,2H),2.24-2.21(m,2H),2.06(d,J＝7.2Hz,2H),1.87-1.53(m,9H),1.09(br s,4H),0.91-0.74(m,2H)。

Example 253.1- (5- (((1R, 5S) -8- (pyridin-3-ylmethyl) -3, 8-diazabicyclo [ 3.2.1)]Octane- 3-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Prepared using the procedure of example 192, step 2-4, using tert-butyl (1 r,5 s) -3- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid ester substituted tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxy benzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) ]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate and reactingNicotinaldehyde is used to replace cyclohexane formaldehyde. NaBH used in THF/EtOH ₃ CN、ZnCl ₂ And DIPEA. LCMS [ M+H] ⁺ ：446.1。 ¹ H NMR(400MHz,CDCl ₃ )δ8.58(d,J＝2.0Hz,1H),8.50-8.48(m,1H),8.35(d,J＝7.2Hz,1H),7.92(s,1H),7.79-7.67(m,2H),7.27-7.24(m,2H),6.91-6.90(m,1H),3.88(t,J＝6.7Hz,2H),3.51(d,J＝16.0Hz,4H),3.10(br s,2H),2.91(t,J＝6.8Hz,2H),2.57-2.55(m,2H),2.39-2.37(m,2H),1.99-1.84(m,4H)。

EXAMPLE 254 preparation of 1- (5- ((4-isobutyl-2-oxopiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.4-Isobutylpiperazin-2-one

To a stirred solution of piperazin-2-one (500 mg,4.99 mmol) in DCM (20 mL) was added TEA (2.0 mL,14.97 mmol) and isopropyl aldehyde (720 mg,9.98 mmol) at room temperature. The mixture was stirred for 30min, then NaBH (OAc) was added ₃ (2.1 g,9.98 mmol). The reaction was stirred at room temperature for 4h, then diluted with DCM and water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give 4-isobutylpiperazin-2-one (500 mg, crude). LCMS [ M+H] ⁺ ：157.0。

And 2, step 2.1- ((3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-isobutylpiperazin-2-one。

To a stirred solution of 4-isobutylpiperazin-2-one (184 mg,1.16 mmol) in THF (5 mL) at 0deg.C was added NaH (88.0 mg,2.23 mmol). The mixture was stirred for 30min and allowed to warm to room temperature. Addition of (3-iodopyrazolo [1,5-a ]]A solution of pyridin-5-yl) methyl 4-methylbenzenesulfonate (500 mg,1.16 mmol) in THF (5 mL) and the reaction was stirred at room temperature for 1h. The reaction was diluted with EtOAc and water and the organic layer was taken up in Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 60% EtOAc in hexanes) to give 1- ((3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-isobutylpiperidineOxazin-2-one (130 mg). LCMS [ M+H] ⁺ ：413.0。

Step 3:1- (5- ((4-isobutyl-2-oxopiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) di Hydropyrimidine-2, 4 (1H, 3H) -dionePrepared using the procedure of example 1, steps 1 and 5, using 1- ((3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-isobutylpiperazin-2-one instead of 5-bromo-3-iodopyrazolo [1,5-a]Pyridine. LCMS [ M+H] ⁺ ：399.1。 ¹ H NMR(400MHz,DMSO-d6):δ10.45(s,1H),8.60(d,J＝7.6Hz,1H),8.16(brs,2H),8.03(s,1H),7.43(s,1H),6.73(d,J＝7.2Hz,1H),4.54(s,2H),3.77(t,J＝6.4Hz,2H),3.23-3.21(m,2H),3.05(s,2H),2.77(t,J＝6.8Hz,2H),2.60-2.50(m,2H),2.11-2.09(m,2H),1.78-1.72(m,1H),0.85(d,J＝6.4

Hz,6H)。

Example 255.1 preparation of- (5- ((4- (cyclohexylmethyl) -2-oxopiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 254, wherein cyclohexane-formaldehyde was used instead of iso-propanal. LCMS [ M+H] ⁺ ：439.2。 ¹ H NMR(400MHz,DMSO-d6)δ10.45(s,1H),8.60(d,J＝7.3Hz,1H),8.33(s,1H),8.03(s,1H),7.43(s,1H),6.73(dd,J＝7.3,1.9Hz,1H),4.54(s,2H),3.77(t,J＝6.7Hz,2H),3.21(t,J＝5.4Hz,2H),3.04(s,2H),2.77(t,J＝6.7Hz,2H),2.58(t,J＝5.5Hz,2H),2.53-2.50(m,1H),2.14(d,J＝7.3Hz,2H),1.76-1.56(m,5H),1.47(ddt,J＝10.3,6.2,3.3Hz,1H),1.29-1.07(m,3H),0.82(q,J＝11.5Hz,2H)。

Example 256.1 preparation of- (5- (((3S, 5R) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (3S, 5R)-3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine-1-carboxylic acid Esters of

To a stirred solution of tert-butyl (3 s,5 r) -3, 5-dimethylpiperazine-1-carboxylate (200 mg,0.933 mmol) in DCM (5 mL) at 0 ℃ was added tetrahydro-2H-pyran-4-carbaldehyde (1599 mg,1.39 mmol) and TEA (0.39 mL,2.79 mmol). The mixture was stirred for 30min, then NaBH (OAc) was added ₃ (3995 mg,1.86 mmol) and the mixture was stirred at room temperature for 2h. The reaction was diluted with DCM and water and the organic layer was taken up over Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% MeOH in DCM) to give tert-butyl (3 s,5 r) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine-1-carboxylate (200 mg,0.64mmol,69% yield). LCMS [ M+H] ⁺ ：313.2。

And 2, step 2.(2S, 6R) -2, 6-dimethyl-1- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine hydrochloride

To a stirred solution of tert-butyl (3 s,5 r) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine-1-carboxylate (200 mg,0.64 mmol) in DCM (2 mL) was added 4M HCl in dioxane (2 mL) and the mixture stirred at room temperature for 2H. The reaction was then concentrated to give crude (2 s,6 r) -2, 6-dimethyl-1- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine hydrochloride (200 mg, crude), which was used without further purification. LCMS [ M+H] ⁺ ：213.2。

And 3, step 3.5- (((3S, 5R) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) Phenyl) -3-iodopyrazolo [1,5-a]Pyridine compound

To (3-iodopyrazolo [1, 5-a)]To a stirred solution of pyridin-5-yl) methyl 4-methylbenzenesulfonate (200 mg,0.467 mmol) in DMF (4.0 mL) was added Cs ₂ CO ₃ (458 mg,1.401 mmol). The mixture was stirred at 0deg.C for 30min, then a solution of (2S, 6R) -2, 6-dimethyl-1- ((tetrahydro-2H-pyran-4-yl) methyl) piperazine hydrochloride (99.1 mg,0.467 mmol) in DMF (1 mL) was added. The mixture was stirred at room temperature for 1h. The reaction was diluted with EtOAc and water, and the organic layer was dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (used in hexane30% EtOAc elution) to give 5- (((3 s,5 r) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) -3-iodopyrazolo [1, 5-a)]Pyridine (80 mg,0.17mmol,40% yield). LCMS [ M+H] ⁺ ：469.0。

Step 4:1- (5- (((3 s,5 r) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) Methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesPrepared using the procedure of example 1, steps 1 and 5, using 5- (((3 s,5 r) -3, 5-dimethyl-4- ((tetrahydro-2H-pyran-4-yl) methyl) piperazin-1-yl) methyl) -3-iodopyrazolo [1,5-a]Pyridine substituted 5-bromo-3-iodopyrazolo [1,5-a ]]Pyridine. LCMS [ M+H] ⁺ ：455.4。 ¹ H NMR(400MHz,CD ₃ OD):δ8.46(d,J＝7.2Hz,1H),8.02(s,1H),7.49(s,1H),6.99-6.97(m,1H),3.97-3.87(m,4H),3.63(s,2H),3.42(t,J＝11.6Hz,4H),3.05-2.98(m,4H),2.88(t,J＝6.4Hz,2H),2.30-2.25(m,2H),1.94-1.93(m,2H),1.79-1.75(m,2H),1.42-1.28(m,7H)。

Example 257.1- (5- (((3S, 5R) -4-isobutyl-3, 5-dimethylpiperazin-1-yl) methyl) pyrazolo [1,5 ] a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones Is prepared from

Prepared using the method of example 256, wherein isobutyraldehyde was used instead of tetrahydro-2H-pyran-4-carbaldehyde. LCMS [ M+H] ⁺ ：413.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.46 (d, j=7.1 hz, 1H), 8.03 (s, 1H), 7.50 (s, 1H), 6.99 (dd, j=7.1, 1.8hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.60 (s, 2H), 3.30-3.13 (m, 2H), 3.00-2.77 (m, 6H), 2.21 (t, j=10.7 hz, 2H), 2.00-1.85 (m, 1H), 1.26 (s, 2H), 1.25 (s, 2H), 1.05 (s, 2H), 1.04 (s, 2H). No NH protons were observed due to solvent exchange.

Example 258.preparation of 1- (5- (((3S, 5S) -4- (cyclohexylmethyl) -3, 5-dimethylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 256, wherein tert-butyl (3 s,5 s) -3, 5-dimethylpiperazine-1-carboxylate is used instead of tert-butyl (3 s,5 r) -3, 5-dimethylpiperazine-1-carboxylate and cyclohexane-formaldehyde is used instead of tetrahydro-2H-pyran-4-carbaldehyde. LCMS [ M+H] ⁺ ：453.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.51 (d, j=7.3 hz, 1H), 8.06 (s, 1H), 7.56 (s, 1H), 7.01 (d, j=7.7 hz, 1H), 3.95-3.64 (m, 6H), 3.20-2.49 (m, 8H), 1.91-1.65 (m, 6H), 1.59-1.17 (m, 9H), 1.08 (q, j=12.0 hz, 2H). No NH protons were observed due to solvent exchange.

Example 259.preparation of 1- (5- (((3S, 5S) -4-isobutyl-3, 5-dimethylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 259)

Prepared using the method of example 256, wherein tert-butyl (3 s,5 s) -3, 5-dimethylpiperazine-1-carboxylate is used instead of tert-butyl (3 s,5 r) -3, 5-dimethylpiperazine-1-carboxylate and isobutyraldehyde is used instead of tetrahydro-2H-pyran-4-carbaldehyde. LCMS [ M+H] ⁺ ：413.2。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.47 (d, j=7.3 hz, 1H), 8.07 (s, 1H), 8.03 (s, 1H), 7.49 (s, 1H), 7.00 (d, j=7.1 hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.82 (s, 1H), 3.76-3.51 (m, 3H), 3.09 (dd, j=13.4, 9.7hz, 1H), 3.04-2.93 (m, 2H), 2.93-2.75 (m, 3H), 2.62 (d, j=12.5 hz, 1H), 2.46 (t, j=11.2 hz, 1H), 2.16-2.01 (m, 1H), 1.50-1.33 (m, 7H), 1.06 (t, j=6.9 hz, 7H). No NH protons were observed due to solvent exchange.

EXAMPLE 260 preparation of (S) -1- (5- ((hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 260)

Prepared using the procedure of example 156 steps 1 and 4,wherein (S) -trifluoro ((hexahydropyrrolo [1, 2-a) is used]Substituted potassium pyrazin-2 (1H) -yl) methyl borate for { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：369.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.46 (d, J=7.2 Hz, 1H), 8.03 (s, 1H), 7.52 (d, J=0.4 Hz, 1H), 7.03-7.01 (m, 1H), 3.93-3.89 (m, 2H), 3.74-3.57 (m, 2H), 3.12-2.99 (m, 3H), 2.95-2.82 (m, 3H), 2.42-2.29 (m, 2H), 2.25-2.19 (m, 2H), 2.05-1.95 (m, 1H), 1.91-1.78 (m, 3H), 1.50-1.36 (m, 1H).

EXAMPLE 261 preparation of (R) -1- (5- ((hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using (R) -trifluoro- (hexahydropyrrolo [1, 2-a)]Substituted potassium pyrazin-2 (1H) -yl) methyl borate for { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：369.1。 ¹ H NMR (400 MHz, methanol-d 4) δ=8.47-8.41 (m, 1H), 8.01 (s, 1H), 7.50 (d, j=0.8 hz, 1H), 7.06-6.92 (m, 1H), 3.95-3.86 (m, 2H), 3.71-3.60 (m, 2H), 3.10-2.97 (m, 3H), 2.93-2.83 (m, 3H), 2.39-2.29 (m, 2H), 2.21 (d, j=8.8 hz, 2H), 2.01-1.96 (m, 1H), 1.86-1.74 (m, 3H), 1.47-1.33 (m, 1H).

EXAMPLE 262 preparation of (S) -1- (5- ((octahydro-2H-pyrido [1,2-a ] pyrazin-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 156, steps 1 and 4, using (S) -trifluoro ((octahydro-2H-pyrido [1, 2-a)]Pyrazin-2-yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：383.1。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.48 (d, j=7.2 hz, 1H), 8.04 (s, 1H), 7.54 (s, 1H), 6.99 (dd, j=7.2, 1.9hz, 1H), 4.88 (br.s, 2H),3.90(t,J＝6.8Hz,2H),3.76(s,2H),3.58-3.37(m,2H),3.22-3.01(m,3H),2.89(t,J＝6.8Hz,2H),2.87-2.74(m,2H),2.67-2.52(m,2H),2.52-2.36(m,2H)。

EXAMPLE 263 preparation of (R) -1- (5- ((hexahydropyrazino [2,1-c ] [1,4] oxazin-8 (1H) -yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using (R) -trifluoro ((hexahydropyrazino [2, 1-c)][1,4]Oxazin-8 (1H) -yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：385.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.44 (d, j=7.1 hz, 1H), 8.25 (s, 1H), 8.00 (s, 1H), 7.50 (s, 1H), 6.97 (dd, j=7.1, 1.8hz, 1H), 3.91-3.82 (m, 3H), 3.72-3.58 (m, 4H), 3.24 (d, j=11.1 hz, 1H), 2.98-2.71 (m, 6H), 2.61-2.38 (m, 4H), 1.99 (t, j=10.9 hz, 1H). No NH protons were observed due to solvent exchange.

EXAMPLE 264 preparation of (S) -1- (5- ((hexahydropyrazino [2,1-c ] [1,4] oxazin-8 (1H) -yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 156, steps 1 and 4, using (S) -trifluoro ((hexahydropyrazino [2, 1-c)][1,4]Oxazin-8 (1H) -yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：385.2。 ¹ H NMR(400MHz,CD ₃ OD)δ8.45(d,J＝7.2Hz,1H),8.24(brs,1H),8.01(s,1H),7.50(s,1H),6.98(dd,J＝7.2Hz,1.2Hz,1H),3.89-3.84(m,3H),3.72-3.61(m,4H),3.27-3.24(m,1H),2.97-2.78(m,6H),2.62-2.44(m,4H),2.03-1.98(m,1H)。

Example 265.1 preparation of- (5- (((2S, 4R) -1- (((1 r, 4S) -4-methoxycyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein trans-4-methoxycyclohexane-1-carbaldehyde was used in place of 4, 4-difluorocyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：468.1。 ¹ H NMR(400MHz,CD ₃ OD)δ8.55(s,1H),8.43(d,J＝7.2Hz,1H),8.01(s,1H),7.36(s,1H),6.83(dd,J＝7.1,1.9Hz,1H),3.89(t,J＝6.7Hz,2H),3.62(s,1H),3.35(s,3H),3.16(ddd,J＝15.1,7.5,4.4Hz,3H),2.89(t,J＝6.7Hz,4H),2.68(d,J＝7.3Hz,1H),2.25-2.07(m,3H),1.96-1.56(m,7H),1.38-1.04(m,8H)。

Example 266.1 preparation of 1- (5- ((1- (((1 r,4 r) -4-ethoxycyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, step 3-4, using trans-4-ethoxycyclohexane-1-carbaldehyde [ see U.S. Pat. No. 2016/122318,2016, A1 ]]Instead of cyclohexane-formaldehyde. LCMS [ M+H] ⁺ ：468.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.45(d,J＝4.5Hz,1H),8.60(dt,J＝7.1,1.4Hz,1H),8.02(d,J＝1.8Hz,1H),7.46-7.28(m,1H),6.80(dd,J＝7.2,1.9Hz,1H),3.78(td,J＝6.7,2.8Hz,2H),3.45(p,J＝6.6Hz,4H),3.26-3.08(m,2H),2.92-2.83(m,3H),2.79(td,J＝6.7,2.0Hz,2H),2.61(d,J＝6.6Hz,2H),1.98(d,J＝12.1Hz,2H),1.92-1.65(m,6H),1.49(q,J＝13.1Hz,2H),1.11(s,2H),1.09(t,J＝7.0Hz,3H),0.99(dd,J＝14.4,11.3Hz,2H)。

Example 267.1 preparation of- (5- (1- (1-isobutylpiperidin-4-yl) ethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.5- (trimethylstannyl) pyrazolo [1,5-a]Pyridine compound

To 5-bromopyrazolo [1,5-a ]]To a stirred solution of pyridine (3 g,15.2 mmol) in dioxane (40 mL) was added Pd (PPh) ₃ ) ₄ (877 mg,0.76 mmol), hexamethyl-ditin (4.97 g,15,2 mmol), and the reaction was stirred at 90℃for 4h. After completion, the reaction mixture was filtered through celite and concentrated to give 5- (trimethylstannyl) pyrazolo [1,5-a]Pyridine (3.2 g, crude). The material was used without further purification. LCMS [ M+H] ⁺ ：282.9。

And 2, step 2.Benzyl 4- (pyrazolo [1, 5-a)]Pyridine-5-carbonyl) piperidine-1-carboxylic acid ester

To 5- (trimethylstannyl) pyrazolo [1,5-a]To a stirred solution of pyridine (3.0 g,10.7 mmol) in THF (30 mL) was added benzyl 4- (chlorocarbonyl) piperidine-1-carboxylate (3.0 g,10.7 mmol) and the solution was degassed by bubbling nitrogen through the solution for 10 min. Allylpalladium (II) chloride dimer (390 mg,1.07 mmol) and molecular sieve (500 mg) were added, and the reaction was stirred at 60℃for 4h. After completion, the reaction mixture was concentrated. The crude compound was purified by silica gel chromatography (eluting with 50% EtOAc in hexanes) to give benzyl 4- (pyrazolo [1, 5-a)]Pyridine-5-carbonyl) piperidine-1-carboxylic acid ester (1.8 g,4.95mmol,46% yield). LCMS [ M+H] ⁺ ：364.0。

And 3, step 3.Benzyl 4- (1- (pyrazolo [1, 5-a)]Pyridin-5-yl) vinyl) piperidine-1-carboxylic acid ester

To a stirred solution of methyltriphenylphosphonium bromide (1.47 g,4.12 mmol) in THF (10 mL) at 0deg.C was slowly added 1M KOTBu (4.1 mL,4.12 mmol) appearing yellow. The reaction mixture was stirred at room temperature for 30min, and then benzyl 4- (pyrazolo [1, 5-a) was added dropwise at 0 ℃]Pyridine-5-carbonyl) piperidine-1-carboxylic acid ester (0.50 g,1.37 mmol) in THF (2 mL). The reaction mixture was allowed to stir at room temperature for 3h. After completion, the reaction was saturated with NH ₄ The aqueous Cl solution was quenched, and the mixture was diluted with ethyl acetate and washed with water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (30% EtO in hexanesAc elution) to give benzyl 4- (1- (pyrazolo [1, 5-a)]Pyridin-5-yl) vinyl) piperidine-1-carboxylic acid ester (0.21 g,0.58mmol,42% yield). ¹ H NMR(400MHz,CD ₃ OD)δ8.41(d,J＝7.2Hz,1H),7.93(d,J＝2.1Hz,1H),7.45(s,1H),7.36-7.30(m,4H),6.76(dd,J＝7.2Hz,2.1Hz,1H),6.49(d,J＝2.1Hz,1H),5.34(s,1H),5.13-5.11(m,3H),4.28(s,2H),2.89-2.82(m,2H),2.63-2.56(m,2H),1.85-1.81(m,2H),1.43-1.40(m,2H)。

And 4, step 4.5- (1- (piperidin-4-yl) ethyl) pyrazolo [1,5-a]Pyridine compound

Pd/C (0.10 g) was added to a stirred solution of benzyl 4- (1- (pyrazolo [1,5-a ] pyridin-5-yl) vinyl) piperidine-1-carboxylate (0.20 g,0.55 mmol) in EtOH (10 mL) under nitrogen. The flask was evacuated and refilled with hydrogen from the balloon and stirred at room temperature for 16h. After completion, the reaction mixture was filtered through celite and washed with EtOH. The filtrate was concentrated to give 5- (1- (piperidin-4-yl) ethyl) pyrazolo [1,5-a ] pyridine (0.18 g, crude). The compound was used in the next step without further purification.

And 5, step 5.Tert-butyl 4- (1- (pyrazolo [1, 5-a)]Pyridin-5-yl) ethyl) piperidine-1-carboxylic acid ester

To 5- (1- (piperidin-4-yl) ethyl) pyrazolo [1,5-a]Et is added to a stirred solution of pyridine (0.18 g,0.78 mmol) in DCM (7 mL) at 0deg.C ₃ N (0.33 mL,2.4 mmol) followed by di-tert-butyl dicarbonate (0.26 g,1.17 mmol) and DMAP (9.59 mg,0.078 mmol). The reaction mixture was stirred at room temperature for 16h. After completion, the reaction was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude compound was purified by silica gel chromatography (eluting with 10% -15% EtOAc in hexanes) to give tert-butyl 4- (1- (pyrazolo [1,5-a ])]Pyridin-5-yl) ethyl) piperidine-1-carboxylate (110 mg,0.33mmol,42% yield). ¹ H NMR(300MHz,CD ₃ OD)δ8.39(d,J＝7.5Hz,1H),7.91(s,1H),6.56(dd,J＝7.2Hz,2.1Hz,1H),6.40(d,J＝1.2Hz,1H),2.65-2.45(m,3H),1.84-1.80(m,1H),1.61-1.49(m,1H),1.43-1.38(m,10H),1.28-1.02(m,6H)。

And 6, step 6.Tert-butyl 4- (1- (3-iodopyrazine)Azolo [1,5-a ]]Pyridin-5-yl) ethyl) piperidine-1-carboxylic acid ester

To tert-butyl 4- (1- (pyrazolo [1, 5-a) under an inert atmosphere]To a stirred solution of pyridin-5-yl) ethyl piperidine-1-carboxylate (0.10 g,0.30 mmol) in DMF (5 mL) at 0deg.C was added NIS (68.2 mg,0.30 mmol). The reaction mixture was stirred at room temperature for 3h. After completion, the reaction was quenched with water and the yellow precipitate formed was collected by filtration. The solid was washed with water and dried under vacuum to give tert-butyl 4- (1- (3-iodopyrazolo [1,5-a ])]Pyridin-5-yl) ethyl) piperidine-1-carboxylate (120 mg,0.26mmol,86% yield). LCMS [ M+H] ⁺ ：456.0。

Step 7: tert-butyl 4- (1- (3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1 (2H)) Radical) pyrazolo [1,5-a]Pyridin-5-yl) ethyl) piperidine-1-carboxylic acid esterPrepared using the procedure of example 1, step 1, using tert-butyl 4- (1- (3-iodopyrazolo [1,5-a ])]Pyridin-5-yl) ethyl) piperidine-1-carboxylic acid ester instead of 5-bromo-3-iodopyrazolo [1,5-a]Pyridine. LCMS [ M+H] ⁺ ：592.2。

Step 8:1- (5- (1- (1-isobutylpiperidin-4-yl) ethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine Pyridine-2, 4 (1H, 3H) -dionesPrepared using the procedure of example 141, step 6-8, using tert-butyl 4- (1- (3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) ethyl) piperidine-1-carboxylate instead of tert-butyl (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate and isobutyraldehyde was used instead of 4, 4-difluorocyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：398.3。 ¹ H NMR (400 MHz, methanol-d 4) delta 8.51 (s, 1H), 8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.36 (s, 1H), 6.84 (dd, j=7.4, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.74-3.61 (m, 1H), 3.23-3.14 (m, 2H), 2.95 (s, 1H), 2.89 (t, j=6.7 hz, 2H), 2.69 (d, j=7.2 hz, 2H), 2.21 (s, 1H), 2.06 (d, j=19.5 hz, 2H), 1.81 (dd, j=37.0, 15.2hz, 7H), 1.59 (s, 1H), 1.35 (t, j=11.2 hz, 5H).

Example 268.1 preparation of 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Methanesulfonic acid (2.0 mL,31 mmol) was added to 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) in DCM (8 mL)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (1.24 g,1.99 mmol). The resulting pale red solution was stirred at 40 ℃ overnight. The reaction mixture was quenched with 50% aqueous sodium bicarbonate and extracted three times with 4:1 dichloromethane:trifluoroethanol. The combined organic phases were taken up in Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by silica gel chromatography (eluting with 15% -100%3:1etoac: etoh in heptane, 0.1% TEA as modifier) to give an impure product. The material was further purified by C18 reverse phase chromatography (eluting with 25% -75% acetonitrile in water, 0.1% NH ₄ OH as modifier) and the fractions containing the product were pooled and poured into phosphate buffer at pH 7. The aqueous phase was extracted three times with 4:1 dichloromethane/trifluoroethanol. The combined organic phases were concentrated in vacuo, diluted with 2:1 acetonitrile: water (3 mL) and lyophilized to give 1- (5- (((2 s,4 r) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) as a white solid ]Pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione (3.5 mg,0.0072mmol,0.36% yield). LCMS [ M+H] ⁺ ：472.3。 ¹ H NMR(400MHz,DMSO-d ₆ )δ11.48(s,1H),8.61(d,J＝7.1Hz,1H),8.12(s,1H),7.70(d,J＝7.8Hz,1H),7.33(s,1H),6.85(dd,J＝7.2,1.8Hz,1H),5.69(d,J＝7.8Hz,1H),2.96-2.83(m,1H),2.57-2.50(m,2H),2.46-2.35(m,2H),2.17(qd,J＝12.5,7.2Hz,2H),2.02-1.90(m,2H),1.90-1.84(m,1H),1.84-1.62(m,4H),1.59-1.44(m,2H),1.44-1.33(m,2H),1.22-1.12(m,1H),1.05(q,J＝13.7,12.7Hz,2H),0.86(d,J＝6.6Hz,3H)。

Example 269.1 preparation of- (5- (azepan-4-ylmethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 1, steps 2 and 5, wherein tert-butyl 4-methyleneazepane-1-carboxylate is used in step 2 [ see WO 2021/158829,2021, A1 ]]Instead of tert-butyl 4-methylenepiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：341.8。 ¹ H NMR(400MHz,CD ₃ OD) delta 8.43 (s, 1H), 8.41 (s, 1H), 8.00 (d, j=2.2 hz, 1H), 7.36 (s, 1H), 6.84 (dd, j=7.1, 2.0hz, 1H), 3.88 (td, j=6.8, 2.1hz, 2H), 3.28-3.21 (m, 2H), 3.20-3.04 (m, 2H), 2.89 (td, j=6.8, 1.8hz, 2H), 2.75-2.61 (m, 2H), 2.09-1.89 (m, 4H), 1.86-1.73 (m, 1H), 1.62 (ddt, j=18.5, 13.0,6.5hz, 1H), 1.46-1.29 (m, 1H). NH was missing due to solvent exchange.

Example 270 preparation of tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) azepane-1-carboxylate

TEA (0.061 mL,0.44 mmol) and di-tert-butyl dicarbonate (0.015 mL,0.32 mmol) were added to 1- (5- (azepan-4-ylmethyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 269) (100 mg,0.21 mmol) in DCM (5 mL). The mixture was stirred at room temperature for 4h, then partitioned between DCM and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was dissolved in DMSO, filtered through a1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% formic acid. The fractions containing the product were combined, frozen and lyophilized to give tert-butyl 4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as an off-white solid ]Pyridin-5-yl) methyl) azepane-1-carboxylate. LCMS [ M+H-tBu ]] ⁺ ：385.9。 ¹ H NMR(400MHz,CD ₃ OD)δ8.40(d,J＝7.1Hz,1H),7.98(d,J＝2.7Hz,1H),7.34(s,1H),6.82(dd,J＝7.1,1.9Hz,1H),3.88(t,J＝6.7Hz,2H),3.55(dtJ=16.6, 5.4hz, 1H), 3.37 (dd, j=15.6, 8.8hz, 2H), 3.25-3.12 (m, 1H), 2.89 (t, j=6.8 hz, 2H), 2.64 (d, j=7.1 hz, 2H), 1.90-1.73 (m, 4H), 1.56 (s, 1H), 1.45 (d, j=5.7 hz, 9H), 1.40-1.19 (m, 2H). NH was missing due to solvent exchange.

Example 271 preparation of 1- (5- ((1-methylazepan-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 1- (5- (azepan-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 269), prepared using the method of example 109, wherein paraformaldehyde is used in place of isobutyraldehyde. LCMS [ M+H] ⁺ ：355.9。 ¹ H NMR(400MHz,CD ₃ OD)δ8.46-8.35(m,2H),8.00(s,1H),7.35(s,1H),6.83(dd,J＝7.0,1.9Hz,1H),3.89(t,J＝6.8Hz,2H),3.22(d,J＝12.2Hz,3H),2.94-2.83(m,5H),2.74-2.60(m,2H),2.09(ddt,J＝10.3,7.2,3.6Hz,1H),2.01-1.80(m,4H),1.77-1.65(m,1H),1.48-1.28(m,2H)。

Example 272.preparation of 1- (5- ((1- (cyclohexylmethyl) azepan-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

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From 1- (5- (azepan-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 269), prepared using the method of example 109, wherein cyclohexane formaldehyde is used instead of isobutyraldehyde. LCMS [ M+H] ⁺ ：438.2。 ¹ H NMR(400MHz,CD ₃ OD)δ8.42(d,J＝7.3Hz,1H),8.38(s,1H),8.01(s,1H),7.35(s,1H),6.83(dd,J＝7.1,1.9Hz,1H),3.89(t,J＝6.7Hz,2H),3.41(s,2H),3.22(s,2H),2.98(d,J＝6.7Hz,2H),2.89(t,J＝6.8Hz,2H),2.68(qd,J＝13.5,7.1Hz,2H),2.11-1.61(m,12H),1.45-1.16(m,4H),1.04(q,J＝12.1Hz,2H)。

Example 273 preparation of 1- (5- (((2S, 4R) -2-methyl-1- (2, 3-tetrafluoropropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydro Pyrimidine-2, 4 (1H, 3H) -diones

TFA (2 mL) was added to 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (340 mg,0.57 mmol). The reaction mixture was stirred at 90 ℃ for 16h and then concentrated. The residue was taken up in DCM, stirred with basic resin, filtered and concentrated again. The crude material was triturated sequentially with pentane and diethyl ether to give crude 1- (5- (((2 s,4 r) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) as a yellow semi-solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (360 mg). LCMS [ M+H] ⁺ ：341.9。

Step 2:1- (5- (((2 s,4 r) -2-methyl-1- (2, 3-tetrafluoropropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione

To 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (150 mg,0.43 mmol) in DCM (5 mL) was added triethylamine (131 mg,1.29 mmol) and 2, 3-tetrafluoropropyl triflate (137 mg,0.51 mmol). The reaction mixture was allowed to stir at room temperature for 16h. The reaction mixture was diluted with water (20 mL) and extracted with DCM (2×30 mL). The organic layer was washed with brine solution (10 mL), dried over sodium sulfate and concentrated. The crude material was purified by reverse phase HPLC using ACN/water/0.1% formic acid. The fractions containing the product were combined, frozen and lyophilized to give 1- (5- (((2 s,4 r) -2-methyl-1- (2, 3-tetrafluoropropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] as a white solid ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：456.1。 ¹ H NMR(300MHz,CD ₃ OD)δ8.40(d,J＝7.1Hz,1H),8.23(s,1H),7.98(s,1H),7.33(s,1H),6.81(dd,J＝7.1,1.9Hz,1H),6.39-5.96(m,1H),3.88(t,J＝6.8Hz,2H),3.04(d,J＝14.0Hz,2H),2.89(t,J＝6.8Hz,3H),2.81-2.69(m,1H),2.62(dd,J＝17.2,5.7Hz,3H),2.00(d,J＝23.6Hz,1H),1.65-1.47(m,3H),1.35-1.27(m,1H),1.02(d,J＝6.7Hz,3H)。

Example 274.1 preparation of- (5- (((2S, 4R) -1- (2, 2-difluoroethyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared by the method of example 273, wherein 2, 2-difluoroethyl triflate is used in place of 2, 3-tetrafluoropropyl triflate in step 2. LCMS [ M+H] ⁺ ：406.2。 ¹ H NMR (400 mhz, meod) delta 8.41 (dd, j=7.2, 0.9hz, 1H), 8.17 (s, 1H), 7.99 (s, 1H), 7.34 (dd, j=1.9, 1.0hz, 1H), 6.82 (dd, j=7.2, 1.9hz, 1H), 5.98 (tt, j=55.7, 4.2hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.21 (d, j=6.5 hz, 1H), 3.05-2.92 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.81 (dd, j=8.0, 3.4hz, 2H), 2.64 (d, j=7.3 hz, 2H), 2.10-1.99 (m, 1H), 1.69 (dd, j=6.8 hz, 2H), 3.21 (d, j=6.5 hz, 1H), 3.05-2.92 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.81 (d, 3.7.7 hz, 1H). No NH protons were observed due to solvent exchange.

Example 275.1 preparation of- (5- (((2S, 4R) -2-methyl-1- (2, 2-trifluoroethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared by the method of example 273, wherein 2, 2-trifluoroethyl triflate is used in place of 2, 3-tetrafluoropropyl triflate in step 2. LCMS [ M+H ] ⁺ ：424.0。 ¹ H NMR(400MHz,MeOD)δ8.40(dd,J＝7.2,0.9Hz,1H),7.98(s,1H),7.34(dd,J＝1.9,0.9Hz,1H),6.81(dd,J＝7.2,1.8Hz,1H),3.88(t,J＝6.8Hz,2H),3.17-2.95(m,3H),2.89(t,J＝6.8Hz,2H),2.81-2.65(m,2H),2.61(d,J=7.3 hz, 2H), 1.97 (H, j=6.1 hz, 1H), 1.66-1.49 (m, 3H), 1.34 (dtd, j=12.9, 10.7,4.4hz, 1H), 1.02 (d, j=6.6 hz, 3H). No NH protons were observed due to solvent exchange.

Example 276.1 preparation of- (5- (((2S, 4R) -2-methyl-1- (3, 3-trifluoropropyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared by the method of example 273, wherein 3, 3-trifluoropropyl triflate is used in place of 2, 3-tetrafluoropropyl triflate in step 2. LCMS [ M+H] ⁺ ：438.0。 ¹ H NMR (400 mhz, meod) delta 8.42 (d, j=7.2 hz, 1H), 7.99 (s, 1H), 7.35 (s, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 3.88 (t, j=6.8 hz, 2H), 2.98 (s, 2H), 2.89 (t, j=6.8 hz, 2H), 2.66 (d, j=7.3 hz, 2H), 2.53 (s, 2H), 2.15-2.00 (m, 2H), 1.84-1.56 (m, 4H), 1.51-1.39 (m, 2H), 1.16 (d, j=6.7 hz, 3H). No NH protons were observed due to solvent exchange.

EXAMPLE 277 preparation of 1- (5- (((2S, 4R) -2-methyl-1- (oxetan-2-ylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione is prepared by the method of example 109, in which oxetan-2-carbaldehyde is used instead of isobutyraldehyde. LCMS [ M+H ] ⁺ ：412.0。 ¹ H NMR(400MHz,MeOD)δ8.44(s,1H),8.42(s,1H),8.01(s,1H),7.36(s,1H),6.86-6.79(m,1H),5.39-5.21(m,1H),5.20-4.99(m,1H),4.76-4.52(m,3H),4.43-4.26(m,2H),3.89(t,J＝6.8Hz,2H),3.63(s,1H),3.23-3.00(m,2H),2.89(t,J＝6.8Hz,2H),2.82-2.48(m,4H),2.03(d,J＝6.0Hz,1H),1.89-1.57(m,2H),1.40-1.25(m,3H)。

EXAMPLE 278 preparation of 1- (5- (((2S, 4R) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:1- (5- (((2S, 4R) -1- (3- ((tert-butyldiphenylsilyl) oxy) -2, 2-difluoropropyl) o-propyl) 2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -diones

To 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (0.17 g,0.32 mmol) and K ₂ CO ₃ (0.133 g,0.96 mmol) to a mixture of dioxane (5 mL) was added 3- ((tert-butyldiphenylsilyl) oxy) -2, 2-difluoropropyl trifluoromethanesulfonate (0.156 g,0.32 mmol). The reaction mixture was stirred at 90℃for 16h. The reaction mixture was cooled to room temperature and was then quenched with water and saturated NaHCO ₃ Diluting the aqueous solution. The mixture was extracted with DCM and the organic layer was extracted with Na ₂ SO ₄ Dried, filtered and concentrated to give crude 1- (5- (((2S, 4R) -1- (3- ((tert-butyldiphenylsilyl) oxy) -2, 2-difluoropropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.24 g) was used without further purification. LCMS [ M+H ] ⁺ ：824.6。

Step 2:1- (5- (((2 s,4 r) -1- (2, 2-difluoro-3-hydroxypropyl) -2-methylpiperidin-4-yl) methyl) pyri-dine Azolo [1,5-a ]]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To 1- (5- (((2S, 4R) -1- (3- ((tert-butyldiphenylsilyl) oxy) -2, 2-difluoropropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]To a solution of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.24 g,0.29 mmol) in THF (3 mL) was added TBAF (1.0M in THF, 2 mL) and the reaction mixture was stirred at room temperature for 2h. The reaction is carried outThe mixture was diluted with water and extracted with DCM. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude 1- (5- (((2 s,4 r) -1- (2, 2-difluoro-3-hydroxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) as a brown solid]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.11 g,0.15 mmol). LCMS [ M+H] ⁺ ：586.1。

Step 3:1- (5- (((2S, 4R) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To 1- (5- (((2 s,4 r) -1- (2, 2-difluoro-3-hydroxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] at 0 ℃C ]To a stirred solution of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (80 mg,0.136 mmol) in DMF (2 mL) was added sodium hydride (10 mg,0.41 mmol). After 5min, methyl iodide (39 mg,0.27 mmol) was added and the reaction mixture was stirred at room temperature for 10min. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 30% EtOAc in hexanes) afforded 1- (5- (((2 s,4 r) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (80 mg). LCMS [ M+H] ⁺ ：600.2。

Step 4:1- (5- (((2S, 4R) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 278)From 1- (5- (((2 s,4 r) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 1 step 5 using 1- (5- (((2S, 4R) -1- (2, 2-difluoro-3-methoxypropyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxyBenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：450.0。 ¹ H NMR(400MHz,MeOD)δ8.42-8.36(m,1H),8.24(s,1H),7.98(s,1H),7.33(s,1H),6.81(dd,J＝7.3,1.9Hz,1H),3.88(t,J＝6.8Hz,2H),3.65(q,J＝12.9Hz,2H),3.41(s,3H),2.95(d,J＝15.0Hz,1H),2.89(t,J＝6.8Hz,2H),2.85-2.68(m,3H),2.60(d,J＝7.2Hz,2H),2.02(q,J＝7.7Hz,2H),1.61(d,J＝12.3Hz,2H),1.54(dd,J＝7.7,4.0Hz,2H),1.04(d,J＝6.7Hz,3H)。

Example 279 preparation of 1- (5- (((2S, 4R) -2-methyl-1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (180 mg,0.41 mmol) in THF (10 mL) was added oxetan-3-one (85 mg,1.2 mmol), dibutyltin dichloride (62 mg,0.20 mmol) and triethylamine (0.2 mL,1.2 mmol). The mixture was stirred at 80 ℃ for 1h then cooled to 0 ℃ and phenylsilane (45 mg,0.41 mmol) was added. The reaction was stirred in a capped vial at 80 ℃ for 4h. The reaction was cooled to room temperature, diluted with DCM and washed sequentially with water and brine. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. The crude material was purified by reverse phase HPLC using ACN/water/0.1% formic acid. The product-containing fractions were combined, frozen and lyophilized to give 1- (5- (((2 s,4 r) -2-methyl-1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) as a white solid ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：398.2。 ¹ H NMR (400 mhz, meod) delta 8.44 (dd, j=7.2, 0.9hz, 1H), 8.01 (s, 1H), 7.36 (d, j=1.8 hz, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 4.84-4.74 (m, 3H), 4.52 (s, 1H), 3.89 (t, j=6.8 hz, 2H), 3.81 (s, 1H), 3.24-3.02 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.67 (d, j=9.1 hz, 2H), 2.25 (d, j=19.4 hz, 1H), 2.00-1.20 (m, 8H). No NH protons were observed due to solvent exchange.

Example 280.preparation of 1- (5- (((2S, 4R) -1-cyclobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 279, wherein cyclobutanone was used instead of oxetan-3-one. LCMS [ M+H] ⁺ ：396.0。 ¹ H NMR (400 mhz, meod) delta 8.44 (s, 1H), 8.01 (s, 1H), 7.36 (s, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.70 (s, 1H), 3.14 (s, 1H), 2.89 (t, j=6.8 hz, 2H), 2.68 (d, j=7.0 hz, 2H), 2.31 (s, 2H), 2.25-2.12 (m, 3H), 1.95-1.80 (m, 4H), 1.30 (s, 7H). No NH protons were observed due to solvent exchange.

Example 281.1 preparation of- (5- ((1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 279 using 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：384.1。 ¹ H NMR(400MHz,MeOD)δ8.42(dd,J＝7.1,0.9Hz,1H),8.25(s,1H),7.99(s,1H),7.39-7.31(m,1H),6.82(dd,J＝7.2,1.9Hz,1H),4.75(t,J＝7.1Hz,2H),4.67(t,J＝6.6Hz,2H),3.88(t,J＝6.8Hz,3H),3.09(d,J＝11.7Hz,2H),2.89(t,J＝6.8Hz,2H),2.68(d,J＝6.8Hz,2H),2.30(t,J＝12.1Hz,2H),1.88-1.78(m,3H),1.45(td,J＝14.3,7.4Hz,2H)。

Example 282.1 preparation of- (5- ((1-Cyclobutylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 279 using 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione and cyclobutanone is used instead of oxetan-3-one. LCMS [ M+H] ⁺ ：382.0。 ¹ H NMR (400 mhz, meod) δ8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.37 (s, 1H), 6.83 (dd, j=7.2, 1.8hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.59 (s, 1H), 3.41 (d, j=12.2 hz, 2H), 3.35 (s, 2H), 2.89 (t, j=6.8 hz, 2H), 2.78-2.65 (m, 3H), 2.36-2.26 (m, 2H), 2.17 (d, j=13.8 hz, 2H), 2.04-1.79 (m, 4H), 1.49 (d, j=13.3 hz, 2H). No NH protons were observed due to solvent exchange.

Example 283.preparation of 1- (5- (((2S, 4R) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -1- (ethylsulfonyl) -2-methylpiperidine) and its preparation 4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

Triethylamine (0.066 mL,0.47 mmol) and ethanesulfonyl chloride (37 mg,0.28 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (50 mg,0.095 mmol) in DCM (2 mL) at 0deg.C. The mixture was stirred at room temperature for 2h, then diluted with DCM and washed with brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 3- (2, 4-dimethoxybenzyl) -1- (5- (((2 s,4 r) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：584.4。

Step 2:1- (5- (((2S, 4R) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl)) Pyrazolo [1,5 ] a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 283)From 3- (2, 4-dimethoxybenzyl) -1- (5- (((2 s,4 r) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 1 step 5 using 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：434.1。 ¹ H NMR(500MHz,DMSO)δ10.44(s,1H),8.57(dd,J＝7.1,0.9Hz,1H),7.99(s,1H),7.37(dd,J＝1.9,0.9Hz,1H),6.81(dd,J＝7.2,1.9Hz,1H),4.12-4.00(m,1H),3.77(t,J＝6.7Hz,2H),3.61-3.45(m,1H),3.15-2.92(m,3H),2.79(t,J＝6.7Hz,2H),2.54(d,J＝7.4Hz,2H),2.03(dd,J＝7.6,3.9Hz,1H),1.65-1.49(m,2H),1.37(td,J＝12.9,5.4Hz,1H),1.25-1.15(m,6H),1.11(td,J＝12.6,4.6Hz,1H)。

The compounds in the following table were prepared by the method of example 283 using the appropriate commercially available sulfonyl chloride in step 1.

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Example 287.1 preparation of 1- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1:1- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5 ] a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidinePyridine-2, 4 (1H, 3H) -diones

HATU (43 mg,0.11 mmol) and cyclopropanecarboxylic acid (6.5 mg,0.076 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (40 mg,0.076 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 5min, followed by the addition of DIPEA (0.040 mL,0.23 mmol). The mixture was stirred at room temperature for 12h, then diluted with ethyl acetate and washed sequentially with brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give crude 1- (5- (((2 s,4 r) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：560.4。

Step 2:1- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5 ] a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesExample 287

From 1- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, using 1- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：410.2。 ¹ H NMR(500MHz,DMSO-d6)δ10.43(s,1H),8.56(d,J＝7.1Hz,1H),7.99(s,1H),7.36(s,1H),6.81(dd,J＝7.2,1.8Hz,1H),4.85-4.48(m,1H),4.19(dd,J＝89.0,13.8Hz,1H),3.77(t,J＝6.7Hz,2H),3.22-3.04(m,1H),2.79(t,J＝6.7Hz,2H),2.67-2.54(m,2H),2.09(d,J＝11.9Hz,1H),1.93(ddt,J＝16.7,13.1,5.8Hz,1H),1.71-1.47(m,2H),1.43-0.92(m,5H),0.82-0.58(m,4H)。

The compounds in the following table are prepared by the method of example 287 using the appropriate commercially available carboxylic acid in step 1.

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Example 291.1 preparation of 1- (5- (((2S, 4R) -2-methyl-1- (pyrrolidin-1-ylsulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Triethylamine (89 mg,0.87 mmol) and pyrrolidine-1-sulfonyl chloride (60 mg,0.35 mmol) were added to 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (100 mg,0.29 mmol) in DCM (4 mL). The mixture was stirred at room temperature for 16h, then saturated NaHCO ₃ Diluting the aqueous solution. The mixture was extracted with DCM and the organic layer was washed with brine. The organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by reverse phase HPLC using ACN/water/0.1% formic acid. The product-containing fractions were combined, frozen and lyophilized to give 1- (5- (((2 s,4 r) -2-methyl-1- (pyrrolidin-1-ylsulfonyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a) as a white solid]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：475.2。 ¹ HNMR (400 mhz, meod) δ8.41 (dd, j=7.2, 0.9hz, 1H), 7.98 (s, 1H), 7.37-7.32 (m, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 4.10 (t, j=6.4 hz, 1H), 3.88 (t, j=6.7 hz, 2H), 3.52 (dt, j=13.2, 3.6hz, 1H), 3.24-3.18 (m, 4H), 3.05 (td, j=13.2, 2.7hz, 1H), 2.89 (t, j=6.8 hz, 2H), 2.60 (d, j=7.1 hz, 2H), 2.07 (ddd, j=11.9, 7.7,4.0hz, 1H), 1.94-1.87 (m, 4H), 1.24-3.18 (m, 4H), 3.05 (td, j=13.2, 2.7hz, 1H), 2.89 (t, j=6.8 hz, 2H). No NH protons were observed due to solvent exchange.

The compounds in the following table were prepared by the method of example 291 using the appropriate commercially available sulfamoyl chloride, carbamoyl chloride or isocyanate.

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Example 297 preparation of 1- (5- ((1- (pyrrolidin-1-ylsulfonyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

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Prepared using the method of example 291, using 1- (5- (piperidin-4-ylmethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：461.3。 ¹ H NMR (400 mhz, meod) delta 8.40 (dd, j=7.2, 0.9hz, 1H), 7.98 (s, 1H), 7.36 (dd, j=1.9, 1.0hz, 1H), 6.82 (dd, j=7.2, 1.9hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.66 (d, j=12.1 hz, 2H), 3.29-3.24 (m, 3H), 2.89 (t, j=6.8 hz, 2H), 2.77 (td, j=12.3, 2.4hz, 2H), 2.65 (d, j=7.0 hz, 2H), 1.93-1.87 (m, 4H), 1.74 (d, j=13.9 hz, 3H), 1.40-1.27 (m, 3H). No NH protons were observed due to solvent exchange.

Example 298 preparation of N-cyclopentyl-4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) piperidine-1-sulfonamide

Use example 291, wherein cyclopentylsulfamoyl chloride is used instead of pyrrolidine-1-sulfonyl chloride and 1- (5- (piperidin-4-ylmethyl) pyrazolo [1,5-a ] is used ]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：475.3。 ¹ H NMR (400 mhz, meod) delta 8.41 (dd, j=7.1, 0.9hz, 1H), 7.99 (s, 1H), 7.39-7.31 (m, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 3.88 (t, j=6.7 hz, 2H), 3.67-3.56 (m, 3H), 2.89 (t, j=6.7 hz, 2H), 2.71-2.61 (m, 3H), 1.90 (q, j=5.5 hz, 2H), 1.78-1.66 (m, 4H), 1.61-1.46 (m, 5H), 1.32 (dd, j=18.1, 6.0hz, 4H). No NH protons were observed due to solvent exchange.

EXAMPLE 299 preparation of (2S, 4R) -4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide

Step 1:(2S, 4R) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1 (2H)) Radical) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide

Triethylamine (74 mg,0.73 mmol) and triphosgene (213 mg,0.73 mmol) were added to 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (120 mg,0.24 mmol) in DCM (5 mL). The mixture was stirred at room temperature for 10min, then N-methylethylamine (22 mg,0.36 mmol) was added. The reaction was stirred at room temperature for 4h, then diluted with water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product was purified by flash chromatography on silica gel (eluting with 6.5% MeOH/DCM) to give (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as an off-white solid ]Pyridin-5-yl) methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide (0.10 g,0.17mmol,71% yield). LCMS [ M+H] ⁺ ：576.9。

Step 2:(2S, 4R) -4- ((3- (2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridine-5- Methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide (example 299)From (2S, 4R) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide prepared by the method of example 1 step 5 using (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -N-ethyl-N, 2-dimethylpiperidine-1-carboxamide instead of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：427.2。 ¹ H NMR (400 mhz, meod) delta 8.41 (dd, j=7.2, 0.9hz, 1H), 7.98 (s, 1H), 7.34 (d, j=1.6 hz, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 4.03 (t, j=6.5 hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.42 (d, j=13.8 hz, 1H), 3.24-3.10 (m, 3H), 3.03 (td, j=13.2, 2.7hz, 1H), 2.89 (t, j=6.8 hz, 2H), 2.79 (s, 3H), 2.59 (d, j=7.2 hz, 2H), 2.11 (s, 1H), 1.62 (dd, j=29.5, 13.2 hz), 1.39-3.10 (m, 3H), 3.03 (td, 3H), 2.7.7 hz, 1H), 2.89 (t, 1H). No NH protons were observed due to solvent exchange.

Example 300.preparation of 1- (5- ((1- (((1 s,3 s) -3-methoxycyclobutyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 112, wherein cis-3-methoxycyclobutane-1-carbaldehyde was used in place of 2-cyclohexyl-2, 2-difluoroacetaldehyde in step 1. LCMS [ M+H] ⁺ ：426.2。 ¹ H NMR (400 mhz, cd3 od) delta 8.42 (d, j=7.2 hz, 1H), 8.00 (s, 1H), 7.36 (s, 1H), 6.82 (dd, j=7.4, 1.8hz, 1H), 3.88 (t, j=6.8 hz, 2H), 3.79 (p, j=7.3 hz, 1H), 3.22 (d, j=4.2 hz, 3H), 2.89 (t, j=6.8 hz, 4H), 2.67 (d, j=6.7 hz, 4H), 2.56-2.44 (m, 2H), 2.24-2.03 (m, 2H), 1.85 (d, j=14.0 hz, 3H), 1.63 (dt, j=11.7, 9.2hz, 2H), 1.46 (d, j=13.0 hz, 2H), 1.30 (s, 1H). Due to moreBy changing the solvent, no NH protons were observed.

Example 301.1 preparation of 1- (5- ((1- (((1 r,3R, 4S) -3, 4-difluorocyclopentyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 112, wherein (1 r,3r,4 s) -3, 4-difluorocyclopentane-1-carbaldehyde was used in place of 2-cyclohexyl-2, 2-difluoroacetaldehyde in step 1. LCMS [ M+H] ⁺ ：446.3。 ¹ HNMR (400 mhz, meod) δ8.43 (dd, j=7.2, 0.9hz, 1H), 8.01 (s, 1H), 7.36 (dd, j=1.9, 0.9hz, 1H), 6.83 (dd, j=7.2, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.50-3.40 (m, 2H), 3.06 (d, j=7.2 hz, 2H), 2.89 (t, j=6.8 hz, 2H), 2.85-2.75 (m, 2H), 2.70 (d, j=6.8 hz, 2H), 2.43 (dq, j=15.2, 7.9hz, 1H), 2.37-2.19 (m, 2H), 1.91 (d, j=14.4 hz, 3H), 1.75 (ddq, j=6.8 hz, 2H), 2.85-2.75 (m, 2H), 2.70 (d, j=6.8 hz, 1H), 2.43 (d, 1.7.9 hz, 1H). No NH protons were observed due to solvent exchange.

Example 302 preparation of 1- (5- (((2S, 4R) -1- (((1 r, 3S) -3-methoxycyclobutyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein trans-3-methoxycyclobutane-1-carbaldehyde was used instead of 4, 4-difluorocyclohexane-1-carbaldehyde in step 7. LCMS [ M+H] ⁺ ：440.3。 ¹ H NMR (400 mhz, cd3 od) delta 8.43 (d, j=7.3 hz, 1H), 8.01 (d, j=1.8 hz, 1H), 7.37 (d, j=7.3 hz, 1H), 6.87-6.78 (m, 1H), 4.05-3.65 (m, 4H), 3.63-3.36 (m, 1H), 3.26-3.07 (m, 6H), 2.89 (t, j=6.7 hz, 2H), 2.80-2.47 (m, 4H), 2.19 (d, j=8.3 hz, 3H), 1.95-1.65 (m, 4H), 1.38 (ddd, j=34.9, 6.8,4.2hz, 4H). NH was missing due to solvent exchange.

EXAMPLE 303 preparation of 1- (5- (((2S, 4R) -1- (((1 s, 3R) -3-methoxycyclobutyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein cis-3-methoxycyclobutane-1-carbaldehyde was used instead of 4, 4-difluorocyclohexane-1-carbaldehyde in step 7. LCMS [ M+H] ⁺ ：440.4。 ¹ H NMR (400 mhz, cd3 od) delta 8.43 (dd, j=7.4, 1.8hz, 1H), 8.01 (d, j=1.6 hz, 1H), 7.36 (s, 1H), 6.83 (dd, j=7.1, 1.9hz, 1H), 3.89 (td, j=6.8, 3.4hz, 2H), 3.75-3.67 (m, 1H), 3.26-3.21 (m, 4H), 3.19-3.06 (m, 3H), 2.89 (t, j=6.8 hz, 2H), 2.66 (d, j=7.1 hz, 2H), 2.60-2.47 (m, 2H), 2.30-2.12 (m, 3H), 1.87 (q, j=14.hz, 2H), 1.70 (dddt, j=17.3, 12.0,9, 3.0 hz, 2H), 2.89 (t, 2.8 hz, 2H), 2.30-2.30 (d, j=7.1 hz, 1.7H). No NH protons were observed due to solvent exchange.

EXAMPLE 304 preparation of 1- (5- (((2S, 4R) -1- (((1 r,3R, 4S) -3, 4-difluorocyclopentyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein (1 r,3r,4 s) -3, 4-difluorocyclopentane-1-carbaldehyde was used instead of 4, 4-difluorocyclohexane-1-carbaldehyde in step 7. LCMS [ M+H] ⁺ ：460.3。 ¹ HNMR(400MHz,MeOD)δ8.49(s,1H),8.43(dd,J＝7.2,0.9Hz,1H),8.01(s,1H),7.36(dd,J＝1.9,0.9Hz,1H),6.83(dd,J＝7.2,1.9Hz,1H),5.14-4.93(m,1H),3.89(t,J＝6.8Hz,2H),3.64(s,1H),3.22-3.00(m,4H),2.89(t,J＝6.8Hz,2H),2.69(d,J＝7.2Hz,1H),2.46-2.15(m,4H),1.93-1.64(m,5H),1.58(s,1H),1.32(q,J＝6.3Hz,5H)。

EXAMPLE 305.preparation of 1- (5- (((2S, 4R) -2-methyl-1- (((1 r, 4S) -4- (trifluoromethyl) cyclohexyl) methyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein (1 r,4 r) -4- (trifluoromethyl) cyclohexane-1-carbaldehyde was used instead of 4, 4-difluorocyclohexane-1-carbaldehyde in step 7. LCMS [ M+H] ⁺ ：506.4。 ¹ HNMR (300 mhz, cd3 od) delta 8.44 (d, j=7.1 hz, 1H), 8.02 (s, 1H), 7.37 (s, 1H), 6.84 (d, j=6.9 hz, 1H), 3.89 (t, j=6.7 hz, 2H), 3.74 (s, 1H), 2.98 (s, 2H), 2.89 (t, j=6.7 hz, 2H), 2.70 (d, j=7.1 hz, 2H), 2.25-2.13 (m, 2H), 2.05-1.75 (m, 8H), 1.49-1.26 (m, 8H), 1.14 (d, j=12.7 hz, 2H). No NH protons were observed due to solvent exchange.

Example 306.preparation of 1- (5- (((2S, 4R) -1- (((R) -3, 3-difluorocyclopentyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein (R) -3, 3-difluorocyclopentane-1-carbaldehyde was used in place of 4, 4-difluorocyclohexane-1-carbaldehyde in step 7. LCMS [ M+H] ⁺ ：460.3。 ¹ H NMR (400 mhz, meod) delta 8.44 (d, j=7.1 hz, 1H), 8.01 (s, 1H), 7.36 (d, j=2.3 hz, 1H), 6.84 (ddd, j=7.1, 4.9,1.9hz, 1H), 3.89 (td, j=6.8, 1.9hz, 2H), 3.82 (s, 1H), 3.69-3.40 (m, 1H), 3.23-3.06 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.72 (dd, j=42.6, 7.3hz, 2H), 2.63-2.35 (m, 2H), 2.34-2.03 (m, 4H), 2.01-1.50 (m, 6H), 1.40 (dd, j=41.6, 6.8hz, 4H). No NH protons were observed due to solvent exchange.

Example 307.1 preparation of- (5- (((2S, 4R) -1- (((S) -3, 3-difluorocyclopentyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 141, wherein (S) -3,3-Difluoro-cyclopentane-1-carbaldehyde replaces 4, 4-difluoro-cyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：460.3。 ¹ H NMR (400 mhz, meod) delta 8.43 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.36 (d, j=2.1 hz, 1H), 6.88-6.80 (m, 1H), 3.89 (td, j=6.8, 1.8hz, 2H), 3.82 (s, 1H), 3.23-3.05 (m, 2H), 2.89 (t, j=6.8 hz, 2H), 2.67 (d, j=7.0 hz, 2H), 2.62-2.37 (m, 2H), 2.31-2.05 (m, 4H), 1.98-1.52 (m, 6H), 1.47-1.22 (m, 5H). No NH protons were observed due to solvent exchange.

Examples 308 and 309.1 preparation of- (5- (((2S) -1- ((4, 4-difluorocyclohexyl) methyl) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1) (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylic acid ester

To the dried vial was added tert-butyl (2S) -4- (bromomethyl) -4-fluoro-2-methylpiperidine-1-carboxylate (0.270 g,0.87 mmol), 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.250 g,0.544 mmol), niCl ₂ (DME) (5 mg,0.027 mmol), pyridine-2, 6-bis (formamidine) dihydrochloride (6 mg,0.027 mmol), naI (0.020g, 0.14 mmol) and Zn (0.070 g,1.1 mmol). The vial was sealed with a septum cap, evacuated and refilled 3 times with nitrogen. DMA (3 mL) was added and the reaction was stirred at 70℃for 17h. The reaction was cooled to room temperature, diluted with EtOAc and filtered through a plug of silica gel eluting with EtOAc. The eluate was concentrated and the residue was purified by silica gel column chromatography (eluting with 50% -70% EtOAc in heptane) to give tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) as a mixture of diastereomers ]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate. Diastereoisomers were separated by chiral HPLC purification: column: lux cell-4X 21.2mm I.D.,5 μm; mobile phase: phase A is n-hexanePhase B was 1:1EtOH: meOH (0.1% HCOOH); isocratic elution: 50% (A) 50% (B); flow rate: 15mL/min; peak 1 (145 mg), LCMS [ M+H-Boc] ⁺ :510.3, hplc rt=17.04 min; peak 2 (245 mg), LCMS [ M+H-Boc] ⁺ ：510.3，HPLC rt＝17.64min。

Step 2:1- (5- (((2S) -1- ((4, 4-difluorocyclohexyl) methyl) -4-fluoro-2-methylpiperidin-4-yl) methyl) Pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 308)

From tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Prepared by the method of pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 1) using example 141 step 6-8, using tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) in step 6]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 1) instead of tert-butyl (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：492.1。 ¹ H NMR(400MHz,MeOD)δ8.44(dd,J＝7.2,0.9Hz,1H),8.33(s,1H),8.03(s,1H),7.42(s,1H),6.87(d,J＝7.2Hz,1H),3.90(t,J＝6.8Hz,2H),3.18-3.06(m,2H),2.98(s,2H),2.89(t,J＝6.8Hz,2H),2.81(s,1H),2.19-1.72(m,12H),1.45-1.25(m,6H)。

1- (5- (((2S) -1- ((4, 4-difluorocyclohexyl) methyl) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 309)

From tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Prepared by the method of pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 2) using example 141 step 6-8, using tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a) in step 6]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 2) instead of tert-butyl (2 s,4 r) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetraHydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：492.1。 ¹ H NMR(400MHz,MeOD)δ8.44(d,J＝7.2Hz,1H),8.33(s,1H),8.03(s,1H),7.43(s,1H),6.87(d,J＝7.2Hz,1H),3.90(t,J＝6.7Hz,2H),3.15-2.96(m,4H),2.89(t,J＝6.8Hz,2H),2.69(s,1H),2.13-1.72(m,10H),1.29(t,J＝5.0Hz,8H)。

Example 310.preparation of 1- (5- (((2S) -4-fluoro-2-methyl-1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1- (5- (((2S) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl Dihydropyrimidine-2, 4 (1H, 3H) -diones

TFA (2 mL) was added to tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 1 from example 308, step 1) (350 mg, 0.514 mmol). The mixture was heated in a sealed vial at 90 ℃ for 24h. The mixture was then cooled to room temperature and concentrated, and azeotropically dried with toluene to provide crude 1- (5- (((2S) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H] ⁺ ：360.0。

Step 2:1- (5- (((2S) -4-fluoro-2-methyl-1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazole And [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 310)From 1- (5- (((2S) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 279 using 1- (5- (((2S) -4-fluoro-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride. LCMS[M+H] ⁺ ：416.0。 ¹ H NMR (400 mhz, meod) delta 8.41 (dd, j=7.2, 0.9hz, 1H), 8.00 (s, 1H), 7.40 (d, j=1.7 hz, 1H), 6.90-6.79 (m, 1H), 4.71-4.60 (m, 4H), 3.96 (q, j=7.0 hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.06 (d, j=24.6 hz, 2H), 2.89 (t, j=6.8 hz, 2H), 2.79 (ddt, j=17.7, 12.0,5.9hz, 2H), 2.37 (dt, j=11.9, 5.7hz, 1H), 1.95-1.79 (m, 3H), 1.72 (td, j=13.1, 6.6hz, 1H), 0.98 (dd, j=6.7, 1.7hz, 3H). No NH protons were observed due to solvent exchange.

Example 311.1 preparation of 1- (5- (((2S) -4-fluoro-2-methyl-1- (oxetan-3-yl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 310 using tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 2 from example 308 step 1) replaces tert-butyl (2S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -4-fluoro-2-methylpiperidine-1-carboxylate (chiral peak 1 from example 308, step 1). LCMS [ M+H] ⁺ ：416.0。 ¹ H NMR (400 mhz, meod) delta 8.41 (dd, j=7.2, 0.9hz, 1H), 8.00 (s, 1H), 7.42 (s, 1H), 6.87 (d, j=7.2 hz, 1H), 4.71-4.54 (m, 4H), 3.89 (t, j=6.8 hz, 2H), 2.99 (d, j=22.4 hz, 2H), 2.89 (t, j=6.8 hz, 2H), 2.58 (d, j=11.9 hz, 1H), 2.40 (s, 1H), 2.20-2.10 (m, 1H), 1.85-1.65 (m, 3H), 1.64-1.37 (m, 2H), 0.87 (d, j=6.4 hz, 3H). No NH protons were observed due to solvent exchange.

EXAMPLE 312 preparation of (cis) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(cis) -3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidine) -4-) Group) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesTo the dried vial was added (cis) -4- (bromomethyl) -1-isobutyl-2- (trifluoromethyl) piperidine (14 mg,0.049 mmol), 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (17.5 mg,0.038 mmol), niCl ₂ (DME) (1 mg,0.001 mmol), pyridine-2, 6-bis (formamidine) dihydrochloride (1 mg,0.001 mmol), naI (1 mg, 0.09 mmol) and Zn (4 mg,0.076 mmol). The vial was sealed with a septum cap, evacuated and refilled 3 times with nitrogen. DMA (2 mL) was added and the reaction was stirred at 100deg.C for 17h. The reaction was cooled to room temperature, diluted with EtOAc and filtered through a plug of silica gel eluting with EtOAc. The eluate is concentrated to give crude (cis) -3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione, which was used without further purification. LCMS [ M+H ] ⁺ ：602.2。

Step 2:(cis) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methyl) pyrazolo [1, 5-a)] Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 312)

From (cis) -3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared using the method of example 1, step 5, wherein (cis) -3- (2, 4-dimethoxybenzyl) -1- (5- ((1-isobutyl-2- (trifluoromethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a is used]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((1- (cyclohexylmethyl) piperidin-4-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：452.4。 ¹ H NMR(400MHz,MeOD)δ8.40(dd,J＝7.2,0.9Hz,1H),7.98(s,1H),7.37(dd,J＝1.9,1.0Hz,1H),6.84(dd,J＝7.2,1.9Hz,1H),3.87(t,J＝6.8Hz,2H),3.27(d,J＝8.1Hz,1H),3.24-3.12(m,1H),2.89(t,J＝6.8Hz,2H),2.72(dt,J＝9.1,6.4Hz,2H),2.46(d,J＝7.4Hz,2H),2.25(dq,J＝11.6,5.6Hz,1H),2.10-2.00(m,2H),1.73(dddd,J＝13.3,10.5,7.9,3.2Hz,4H),0.91(d,J＝6.5Hz,3H),0.86(d,J＝6.7Hz,3H)。

EXAMPLE 313 preparation of (R) -1- (5- ((octahydro-2H-pyrido [1,2-a ] pyrazin-2-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using (R) -trifluoro ((octahydro-2H-pyrido [1, 2-a)]Pyrazin-2-yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：383.0。 ¹ H NMR (400 mhz, meod) δ8.49 (d, j=7.1 hz, 1H), 8.04 (s, 1H), 7.55 (s, 1H), 6.99 (dd, j=7.2, 1.8hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.79 (s, 2H), 3.51-3.41 (m, 2H), 3.28-3.12 (m, 3H), 3.04 (td, j=12.9, 3.2hz, 1H), 2.89 (t, j=6.8 hz, 2H), 2.63 (s, 1H), 2.37 (s, 1H), 1.94 (dd, j=30.0, 13.6hz, 3H), 1.77 (q, j=13.4 hz, 1H), 1.69-1.43 (m, 2H), 1.37-1.27 (m, 1H). No NH protons were observed due to solvent exchange.

EXAMPLE 314 preparation of (R) -1- (5- ((4-oxohexahydropyrazino [2,1-c ] [1,4] oxazin-8 (1H) -yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using (R) -trifluoro ((4-oxo-hexahydropyrazino [2, 1-c)][1,4]Oxazin-8 (1H) -yl) methyl potassium borate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：399.0。 ¹ H NMR (400 mhz, meod) delta 8.45 (dd, j=7.2, 0.9hz, 1H), 8.01 (s, 1H), 7.51 (dd, j=1.9, 1.0hz, 1H), 7.01 (dd, j=7.2, 1.8hz, 1H), 4.49 (ddd, j=13.3, 3.3,1.8hz, 1H), 4.10 (d, j=1.7 hz, 2H), 3.98 (dd, j=12.0, 4.6hz, 1H), 3.90 (t, j=6.8 hz, 2H), 3.74-3.51 (m, 4H), 2.98-2.82 (m, 5H), 2.14 (td, j=11.6, 3.2hz, 1H), 2.06-1.94 (m, 1H). No NH protons were observed due to solvent exchange.

EXAMPLE 315 preparation of (S) -1- (5- ((1, 1-hexahydro-5H-isothiazolo [2,3-a ] pyrazin-5-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 156, steps 1 and 4, using (S) - ((1, 1-hexahydro-5H-isothiazolo [2, 3-a)]Pyrazin-5-yl) methyl) potassium trifluoroborate instead of { [4- (tert-butoxycarbonyl) -1-piperazinyl ]Methyl } (trifluoro) potassium borate. LCMS [ M+H] ⁺ ：419.0。 ¹ H NMR (400 mhz, meod) delta 8.45 (d, j=7.1 hz, 1H), 8.01 (s, 1H), 7.50 (s, 1H), 7.00 (dd, j=7.2, 1.8hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.65 (q, j=13.7 hz, 2H), 3.26-3.10 (m, 3H), 3.04 (d, j=11.3 hz, 1H), 2.96 (d, j=11.5 hz, 1H), 2.92-2.86 (m, 3H), 2.40-2.14 (m, 2H), 2.09-1.90 (m, 3H). No NH protons were observed due to solvent exchange.

Example 316.1 preparation of 1- (5- (((S) -4- (((1 r, 3S) -3-methoxycyclobutyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein trans-3-methoxycyclobutane-1-carbaldehyde was used instead of cyclohexane-carbaldehyde in step 3. LCMS [ M+H] ⁺ ：441.1。 ¹ H NMR (400 mhz, cd3 od) delta 8.45 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.48 (s, 1H), 6.97 (dd, j=7.1, 1.9hz, 1H), 4.03-3.94 (m, 1H), 3.88 (t, j=6.8 hz, 2H), 3.69-3.56 (m, 2H), 3.40 (d, j=12.6 hz, 3H), 3.22 (d, j=4.3 hz, 3H), 3.10 (d, j=11.6 hz, 2H), 3.02 (s, 1H), 2.87 (t, j=6.7 hz, 2H), 2.72 (d, j=8.3 hz, 2H), 2.52 (s, 2H), 2.16 (ddt, j=36.3, 12.7,7.4hz, 1.34 (d, 6hz, 4H). No NH protons were observed due to solvent exchange.

EXAMPLE 317 preparation of 1- (5- (((S) -4- (((1S, 3R) -3-methoxycyclobutyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein cis-3-methoxycyclobutane-1-carbaldehyde was used in place of cyclohexane-carbaldehyde in step 3. LCMS [ M+H] ⁺ ：441.3。 ¹ H NMR (400 mhz, cd3 od) delta 8.46 (d, j=7.2 hz, 1H), 8.02 (s, 1H), 7.50 (s, 1H), 6.99 (dd, j=7.1, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.81 (p, j=7.2 hz, 1H), 3.70-3.56 (m, 2H), 3.22 (s, 6H), 3.06-2.84 (m, 6H), 2.60-2.46 (m, 3H), 2.36 (s, 1H), 2.22 (qd, j=9.1, 6.6hz, 1H), 1.75-1.60 (m, 2H), 1.30 (d, j=6.3 hz, 3H). No NH protons were observed due to solvent exchange.

Example 318.1 preparation of- (5- (((S) -4- (((1 r,3R, 4S) -3, 4-difluorocyclopentyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein (1 r,3R, 4S) -3, 4-difluorocyclopentane-1-carbaldehyde was used in place of cyclohexane-carbaldehyde in step 3. LCMS [ M+H] ⁺ ：461.4。 ¹ H NMR (400 mhz, meod) delta 8.46 (dd, j=7.2, 0.9hz, 1H), 8.02 (s, 1H), 7.50 (d, j=1.8 hz, 1H), 6.99 (dd, j=7.2, 1.8hz, 1H), 5.08-4.92 (m, 1H), 4.81 (s, 1H), 3.89 (t, j=6.8 hz, 2H), 3.68-3.54 (m, 2H), 3.20-3.11 (m, 1H), 2.99 (t, j=11.1 hz, 1H), 2.93-2.75 (m, 5H), 2.53 (d, j=75.2 hz, 3H), 2.39-2.06 (m, 4H), 1.83-1.56 (m, 2H), 1.17 (d, j=6.1 hz, 3H). No NH protons were observed due to solvent exchange.

Example 319.1 preparation of 1- (5- (((S) -4- (((R) -3, 3-difluorocyclopentyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein (R) -3, 3-difluorocyclopentane-1-carbaldehyde was used in place of cyclohexane-carbaldehyde in step 3. LCMS [ M+H] ⁺ ：461.2。 ¹ H NMR(300MHz,CD ₃ OD) δ8.45 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.50 (s, 1H), 7.05-6.94 (m, 1H), 3.89 (t, j=6.7 hz, 2H), 3.56 (d, j=3.6 hz, 2H), 2.89 (t, j=6.7 hz, 3H), 2.74 (t, j=12.3 hz, 3H), 2.61-1.91 (m, 11H), 1.38-1.25 (m, 2H), 1.06 (d, j=6.1 hz, 2H). No NH protons were observed due to solvent exchange.

Example 320.preparation of 1- (5- (((S) -4- (((S) -3, 3-difluorocyclopentyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein (S) -3, 3-difluorocyclopentane-1-carbaldehyde was used in place of cyclohexane-carbaldehyde in step 3. LCMS [ M+H] ⁺ ：461.1。 ¹ H NMR (400 mhz, meod) delta 8.44 (d, j=7.1 hz, 1H), 8.01 (s, 1H), 7.49 (s, 1H), 6.99 (dd, j=7.2, 1.8hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.54 (d, j=6.3 hz, 1H), 2.89 (t, j=6.8 hz, 3H), 2.79-2.63 (m, 3H), 2.50-1.90 (m, 12H), 1.32 (d, j=16.3 hz, 1H), 1.04 (dd, j=6.1, 4.1hz, 3H). No NH protons were observed due to solvent exchange.

EXAMPLE 321 preparation of (S) -1- (5- ((4- (cyclopropylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 192, wherein cyclopropanecarbaldehyde was used in place of cyclohexane carbaldehyde in step 3. LCMS [ M+H] ⁺ ：397.4。 ¹ H NMR(500MHz,DMSO)δ10.47(s,1H),8.64(d,J＝7.2Hz,1H),8.05(s,1H),7.52(s,1H),6.91(dd,J＝7.2,1.8Hz,1H),3.79(t,J＝6.7Hz,2H),3.68(s,4H),3.36(s,1H),3.25-3.12(m,2H),3.04(d,J＝14.6Hz,3H),2.80(t,J＝6.7Hz,2H),2.31(d,J＝11.9Hz,1H),1.25(d,J＝6.4Hz,3H),1.05(s,1H),0.65(s,2H),0.39(d,J＝29.5Hz,2H)。

EXAMPLE 322 preparation of (S) -1- (5- ((3-methyl-4- ((1-methylcyclobutyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 192, wherein 1-methylcyclobutane-1-carbaldehyde was used in place of cyclohexane carbaldehyde in step 3. LCMS [ M+H] ⁺ ：425.2。 ¹ H NMR(500MHz,DMSO)δ10.47(s,1H),8.64(d,J＝7.2Hz,1H),8.05(s,1H),7.51(s,1H),6.91(d,J＝7.2Hz,1H),3.79(t,J＝6.7Hz,6H),2.96(s,6H),2.80(t,J＝6.7Hz,3H),2.10(d,J＝13.4Hz,1H),2.01(d,J＝9.1Hz,1H),1.88(s,1H),1.84-1.71(m,2H),1.67(s,1H),1.27(s,6H)。

EXAMPLE 323 preparation of 1- (5- (((S) -3-methyl-4- (((1 r, 4S) -4- (trifluoromethyl) cyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(S) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine Pyridine-2, 4 (1H, 3H) -diones

TFA (5 mL) was added to tert-butyl (S) -4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylpiperazine-1-carboxylate (0.24 g,0.40 mmol). The mixture was heated in a sealed vial at 70 ℃ for 2h. The mixture was then cooled to room temperature and concentrated, and azeotropically dried with toluene to afford the crude product (0.3 g), which was used without further purification. LCMS [ M+H ] ⁺ ：343.9。

And 2, step 2.1- (5- (((S) -3-methyl-4- (((1 r, 4S) -4- (trifluoromethyl) cyclohexyl) methyl) piperazine-1- Radical) methyl radical) Pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -diones

(1 r,4 r) -4- (trifluoromethyl) cyclohexane-1-carbaldehyde (0.126 g,0.70 mmol) and triethylamine (0.14 mL,1.05 mmol) were added to (S) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione (0.12 g,0.35 mmol) in DCM (4 mL). The reaction mixture was stirred at room temperature for 90min, then sodium triacetoxyborohydride (0.148 g,0.70 mmol) was added. The reaction mixture was stirred at room temperature for 3h then quenched with saturated aqueous NaHCO3 and extracted three times with DCM. The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% formic acid. The product-containing fractions were combined, frozen and lyophilized to provide 1- (5- (((S) -3-methyl-4- (((1 r, 4S) -4- (trifluoromethyl) cyclohexyl) methyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ]]A formate salt of pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H ] ⁺ ：507.3。 ¹ H NMR (400 mhz, cd3 od) delta 8.46 (d, j=7.3 hz, 1H), 8.03 (s, 1H), 7.51 (s, 1H), 6.99 (dd, j=7.1, 1.9hz, 1H), 3.90 (t, j=6.7 hz, 2H), 3.70-3.59 (m, 2H), 3.38 (s, 1H), 3.21 (q, j=7.4 hz, 2H), 3.04-2.95 (m, 1H), 2.89 (t, j=6.7 hz, 3H), 2.60 (s, 2H), 2.37 (s, 1H), 2.15 (dd, j=23.1, 8.1hz, 1H), 1.98 (d, j=12.3 hz, 2H), 1.86 (d, j=13.1 hz, 1H), 1.72 (s, 1H), 1.43-1.23 (m, 1H), 2.7.7 hz, 3H), 2.60 (s, 2H). No NH protons were observed due to solvent exchange.

Example 324.1 preparation of- (5- (((3S) -3-methyl-4- (oxetan-2-ylmethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 323, wherein oxetane-2-carbaldehyde was used in place of (1 r,4 r) -4- (trifluoromethyl) cyclohexane-1-carbaldehyde in step 2. LCMS [ M+H] ⁺ ：413.0。 ¹ H NMR(400MHz,MeOD)δ8.47(dd,J＝7.2,0.9Hz,1H),8.03(s,1H),7.50(s, 1H), 6.98 (dd, j=7.2, 1.9hz, 1H), 5.18 (q, j=9.2 hz, 1H), 4.76-4.56 (m, 2H), 3.90 (t, j=6.8 hz, 2H), 3.81-3.61 (m, 3H), 3.57-3.37 (m, 3H), 3.24-3.10 (m, 2H), 3.08-2.78 (m, 4H), 2.66-2.29 (m, 3H), 1.41-1.26 (m, 3H). No NH protons were observed due to solvent exchange.

EXAMPLE 325 preparation of (S) -1- (5- ((4- ((2-oxaspiro [3.3] heptan-6-yl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 323, wherein 2-oxaspiro [3.3] was used in step 2]Heptane-6-carbaldehyde replaces (1 r,4 r) -4- (trifluoromethyl) cyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：453.2。 ¹ H NMR(300MHz,cd3od)δ8.47(s,1H),8.45(s,1H),8.03(s,1H),7.49(s,1H),6.98(dd,J＝7.2,1.9Hz,1H),4.75(s,2H),4.57(s,2H),3.89(t,J＝6.7Hz,2H),3.67-3.56(m,2H),3.21(d,J＝24.5Hz,2H),2.89(t,J＝6.5Hz,6H),2.56-2.41(m,4H),2.38-2.21(m,1H),2.04(d,J＝3.8Hz,2H),1.76(td,J＝10.7,5.4Hz,1H),1.27(d,J＝6.5Hz,3H)。

EXAMPLE 326 preparation of (S) -1- (5- ((4- ((6, 6-difluorospiro [3.3] heptan-2-yl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 323, wherein 6, 6-difluorospiro [3.3] was used in step 2]Heptane-2-carbaldehyde replaces (1 r,4 r) -4- (trifluoromethyl) cyclohexane-1-carbaldehyde. LCMS [ M+H] ⁺ ：487.3。 ¹ H NMR (400 mhz, meod) delta 8.45 (dd, j=7.2, 1.0hz, 1H), 8.02 (d, j=1.5 hz, 1H), 7.49 (d, j=1.9 hz, 1H), 6.98 (dt, j=7.3, 1.9hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.68 (dd, j=5.5, 4.0hz, 1H), 3.63-3.52 (m, 2H), 3.22-3.11 (m, 2H), 2.93-2.75 (m, 6H), 2.75-2.55 (m, 3H), 2.55-2.37 (m, 3H), 2.35-1.93 (m, 5H), 1.33-1.17 (m, 3H). Due to the solvent exchange, no observation was madeNH protons.

EXAMPLE 327 preparation of (S) -1- (5- ((4- (2, 2-difluoroethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate is prepared by the method of example 156, steps 3-4, using 2, 2-difluoroethyl trifluoromesylate in step 3. LCMS [ M+H ] ⁺ ：407.0。 ¹ H NMR (400 mhz, meod) delta 8.47 (dd, j=7.2, 0.9hz, 1H), 8.02 (s, 1H), 7.53 (dd, j=1.9, 1.0hz, 1H), 7.00 (dd, j=7.2, 1.8hz, 1H), 5.93 (tt, j=56.0, 4.2hz, 1H), 3.90 (t, j=6.8 hz, 2H), 3.72-3.62 (m, 2H), 3.15-2.95 (m, 2H), 2.89 (t, j=6.7 hz, 2H), 2.82 (ddd, j=9.4, 6.8,3.7hz, 2H), 2.76-2.58 (m, 3H), 2.44 (j=10.9, 2.8hz, 1H), 2.15 (dd, j=12.8, 8.95 (m, 2H), 3.15 (d, 1H). No NH protons were observed due to solvent exchange.

EXAMPLE 328 preparation of (S) -1- (5- ((4- (2, 2-difluoro-3-methoxypropyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared by the method of example 278, wherein (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a is used in step 1]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride instead of 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride. LCMS [ M+H] ⁺ ：451.1。 ¹ H NMR (300 MHz, methanol-d 4) delta ppm 8.46 (d, J=7.3 Hz, 1H), 8.01 (s, 1H), 7.51 (s, 1H), 6.99 (br d, J=6.9 Hz, 1H), 3.89 (t, J=6.8 Hz, 2H), 3.73-3.53 (m, 3H) 3.39 (s, 3H), 3.18-2.99 (m, 2H), 2.88 (t, J=6.76H) z, 2H), 2.38-2.80 (m, 7H), 2.09-2.24 (m, 1H), 1.05 (d, j=6.3 hz, 3H). No NH protons were observed due to solvent exchange.

Example 329 preparation of (S) -1- (5- ((4-cyclobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(S) -1- (5- ((4-cyclobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Phenyl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]To a solution of pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione hydrochloride (50 mg,0.094 mmol) in THF (2 mL) was added cyclobutanone (20 mg,0.28 mmol), dibutyltin dichloride (86 mg,0.28 mmol) and triethylamine (48 mg,0.47 mmol). The mixture was stirred at room temperature for 1h, then phenylsilane (20 mg,0.19 mmol) was added. The reaction was stirred in a capped vial at 80 ℃ for 12h. The reaction was cooled to room temperature, diluted with DCM and washed sequentially with water and brine. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude (S) -1- (5- ((4-cyclobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. The crude product was used in the next step without any further purification. LCMS [ M+H ] ⁺ ：547.6。

Step 2:(S) -1- (5- ((4-cyclobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Base) dihydropyrimidine-2, 4 (1H, 3H) -dione (example 329) Prepared using the method of example 156, step 4, using (S) -1- (5- ((4-cyclobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione in place of 1- (5- ((4- (cyclohexylmethyl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：397.2。 ¹ H NMR(500MHz,DMSO)δ10.47(s,1H),8.64(d,J＝7.1Hz,1H),8.05(s,1H),7.50(s,1H),6.90(d,J＝7.0Hz,1H),3.79(t,J＝6.7Hz,6H),3.12(d,J＝33.9Hz,4H),3.03-2.85(m,2H),2.79(t,J＝6.7Hz,2H),2.17(d,J＝8.0Hz,4H),1.83-1.63(m,2H),1.26(dd,J＝31.8,6.6Hz,3H)。

The compounds in the following table were prepared by the method of example 329, using the appropriate commercially available ketone in step 1.

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EXAMPLE 336 preparation of (S) -1- (5- ((3-methyl-4- (2-methyl-2-azaspiro [3.3] heptan-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.Tert-butyl (S) -6- (4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1) (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) methyl) -2-methylpiperazin-1-yl) -2-azaspiro [3.3]Heptane-2-carbonitrile Acid estersPrepared using the method of example 329 step 1, using tert-butyl 6-oxo-2-azaspiro [3.3] ]Heptane-2-carboxylic acid ester replaces cyclobutanone. LCMS [ M+H] ⁺ ：688.3。

And 2, step 2.(S) -3- (2, 4-Dimethoxybenzyl) -1- (5- ((3-methyl-4- (2-azaspiro [3.3 ])]Heptane-6- Yl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate

TFA (4 mL) was added to a solution of tert-butyl (S) -6- (4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazin-1-yl) -2-azaspiro [3.3] heptane-2-carboxylate (300 mg,0.43 mmol) in DCM (2 mL) at room temperature. The reaction was stirred at room temperature for 2h and then concentrated. The residue was azeotropically dried with toluene to give crude (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methyl-4- (2-azaspiro [3.3] heptan-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate, which was used without further purification.

And 3, step 3.(S) -3- (2, 4-Dimethoxybenzyl) -1- (5- ((3-methyl-4- (2-methyl-2-azaspiro [3.3 ])] Heptane-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesWas prepared using the procedure of example 329, step 1, using paraformaldehyde instead of cyclobutanone and omitting triethylamine. LCMS [ M+H ] ⁺ ：602.3。

And 4, step 4.(S) -1- (5- ((3-methyl-4- (2-methyl-2-azaspiro [3.3 ])]Heptane-6-yl) piperazin-1-yl) methyl ester Radical) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesFrom (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methyl-4- (2-methyl-2-azaspiro [ 3.3)]Heptane-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 156, step 4. LCMS [ M+H] ⁺ ：452.2。 ¹ H NMR (400 mhz, meod) delta 8.44 (d, j=7.2 hz, 1H), 8.01 (s, 1H), 7.48 (s, 1H), 6.98 (dd, j=7.1, 1.8hz, 1H), 4.16 (s, 2H), 4.03 (s, 2H), 3.89 (t, j=6.8 hz, 2H), 3.16-3.09 (m, 1H), 2.93-2.79 (m, 5H), 2.70 (s, 2H), 2.60-2.17 (m, 7H), 2.00 (s, 1H), 1.68 (s, 1H), 1.48-1.37 (m, 2H), 1.09 (d, j=6.4 hz, 3H). No NH protons were observed due to solvent exchange.

EXAMPLE 337 preparation of (S) -1- (5- ((3-methyl-4- (2- (methylsulfonyl) -2-azaspiro [3.3] heptan-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.(S) -1- (5- ((3-methyl-4- (2-azaspiro [3.3 ])]Heptane-6-yl) piperazin-1-yl) methyl) pyrazole And [1,5-a ]]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate

TFA (5 mL) was added to tert-butyl (S) -6- (4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a ] pyridin-5-yl) methyl) -2-methylpiperazin-1-yl) -2-azaspiro [3.3] heptane-2-carboxylate (152 mg,0.22 mmol). The reaction was stirred at 90 ℃ for 16h and then concentrated. The residue was azeotropically dried with toluene to give crude (S) -1- (5- ((3-methyl-4- (2-azaspiro [3.3] heptan-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate, which was used without further purification.

Step 2:(S) -1- (5- ((3-methyl-4- (2- (methylsulfonyl) -2-azaspiro [ 3.3)]Heptane-6-yl) piperazine Oxazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesFrom (S) -1- (5- ((3-methyl-4- (2-azaspiro [3.3 ])]Heptane-6-yl) piperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate is prepared by the method of example 283 step 1, wherein methanesulfonyl chloride is used in place of ethanesulfonyl chloride. LCMS [ M+H] ⁺ ：516.3。 ¹ H NMR(400MHz,DMSO)δ10.42(s,1H),8.60-8.49(m,1H),8.13(s,1H),8.00(s,1H),7.43(s,1H),6.86(dd,J＝7.2,1.8Hz,1H),3.88(s,2H),3.80-3.71(m,4H),3.45(q,J＝13.7Hz,1H),2.93(s,4H),2.77(t,J＝6.7Hz,2H),2.60(s,1H),2.41(s,1H),2.37-2.28(m,3H),2.28-1.92(m,6H),0.94(d,J＝6.3Hz,3H)。

Example 338.1 preparation of 1- (5- ((4-cyclohexylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 329, wherein 3- (2, 4-dimethoxybenzyl) -1- (5- (piperazin-1-ylmethyl) pyrazolo [1, 5-a) is used in step 1]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dionesInstead of (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione and cyclohexanone is used instead of cyclobutanone. LCMS [ M+H] ⁺ ：411.2。 ¹ H NMR(300MHz,CD ₃ OD) δ8.46 (d, j=7.1 hz, 1H), 8.03 (s, 1H), 7.50 (s, 1H), 6.99 (dd, j=7.2, 1.8hz, 1H), 3.89 (t, j=6.8 hz, 2H), 3.67 (s, 2H), 3.11 (s, 2H), 2.89 (t, j=6.8 hz, 8H), 2.12 (d, j=9.2 hz, 2H), 1.94 (d, j=10.7 hz, 2H), 1.72 (d, j=12.7 hz, 1H), 1.53-1.15 (m, 6H). No NH protons were observed due to solvent exchange.

EXAMPLE 339 preparation of (S) -1- (5- ((4- (ethylsulfonyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

From (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione prepared by the method of example 283. LCMS [ M+H] ⁺ ：435.1。 ¹ H NMR(500MHz,DMSO)δ10.53(s,1H),8.75(d,J＝7.2Hz,1H),8.14(s,1H),7.71(s,1H),7.01(d,J＝7.2Hz,1H),4.27(d,J＝76.7Hz,5H),3.90-3.77(m,2H),3.71(s,1H),3.35(s,2H),3.17(tq,J＝14.3,7.1Hz,3H),2.80(dd,J＝7.4,6.1Hz,2H),1.33(d,J＝7.0Hz,3H),1.21(t,J＝7.3Hz,3H)。

EXAMPLE 340 preparation of (S) -1- (5- ((4- (cyclopropanecarbonyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared by the method of example 287, using (S) -3- (2, 4-dimethoxybenzyl) -1- (5- ((3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] in step 1]Pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione instead of 3- (2, 4-dimethoxybenzyl) -1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine compound-3-yl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：411.1。 ¹ H NMR(500MHz,DMSO)δ10.54(s,1H),8.77(d,J＝7.2Hz,1H),8.15(s,1H),7.73(s,1H),7.10-6.93(m,1H),4.80(s,1H),4.37(s,2H),3.84(ddd,J＝10.1,8.3,5.0Hz,4H),3.37(s,2H),3.01(s,1H),2.80(t,J＝6.7Hz,2H),1.97(t,J＝6.4Hz,1H),1.35(s,1H),1.19(d,J＝10.9Hz,2H),0.75(s,5H)。

EXAMPLE 341 preparation of (S) -1- (5- ((4-isobutyryl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 340, wherein isobutyric acid was used in place of cyclopropanecarboxylic acid in step 1. LCMS [ M+H] ⁺ ：413.2。 ¹ H NMR(500MHz,DMSO)δ10.54(s,1H),8.77(d,J＝7.2Hz,1H),8.15(s,1H),7.73(s,1H),7.02(d,J＝7.3Hz,1H),4.82(s,2H),4.06(s,2H),3.92-3.73(m,3H),3.39(d,J＝41.9Hz,2H),2.83(dt,J＝28.3,6.7Hz,3H),2.56(s,1H),1.34(d,J＝6.9Hz,1H),1.18(d,J＝7.1Hz,2H),0.99(dd,J＝24.2,6.9Hz,7H)。

EXAMPLE 342 preparation of (S) -1- (5- ((4- (cyclohexanecarbonyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 340, wherein cyclohexane carboxylic acid was used instead of cyclopropanecarboxylic acid in step 1. LCMS [ M+H] ⁺ ：453.1。 ¹ H NMR(500MHz,DMSO)δ10.54(s,1H),8.77(d,J＝7.2Hz,1H),8.15(s,1H),7.72(s,1H),7.02(d,J＝7.2Hz,1H),4.81(s,1H),4.41(d,J＝69.3Hz,3H),3.82(dd,J＝7.0,5.0Hz,4H),3.40(dd,J＝35.2,21.7Hz,3H),2.94(d,J＝30.7Hz,2H),2.80(dd,J＝7.3,6.1Hz,2H),1.71(d,J＝13.2Hz,2H),1.64(d,J＝14.5Hz,2H),1.31(td,J＝24.7,14.1Hz,5H),1.17(d,J＝7.3Hz,3H)。

EXAMPLE 343 preparation of (S) -1- (5- ((4- (cyclopentanecarbonyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 340, wherein cyclopentanecarboxylic acid was used instead of cyclopropanecarboxylic acid in step 1. LCMS [ M+H] ⁺ ：439.1。 ¹ H NMR(500MHz,DMSO)δ10.54(s,1H),8.77(s,1H),8.15(s,1H),7.72(s,1H),7.02(s,1H),4.81(s,1H),4.37(s,4H),3.87-3.58(m,2H),3.39(d,J＝14.3Hz,3H),3.09-2.86(m,2H),2.80(t,J＝6.7Hz,2H),1.75(d,J＝21.2Hz,3H),1.56(d,J＝33.7Hz,5H),1.33(d,J＝6.9Hz,1H),1.18(d,J＝6.9Hz,2H)。

EXAMPLE 344 preparation of (S) -1- (5- ((4- (cyclobutanecarbonyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 340, wherein cyclobutanecarboxylic acid was used instead of cyclopropanecarboxylic acid in step 1. LCMS [ M+H] ⁺ ：425.1。 ¹ H NMR(500MHz,DMSO)δ10.53(s,1H),8.76(s,1H),8.14(s,1H),7.71(s,1H),7.00(s,1H),4.58(d,J＝188.4Hz,5H),3.90-3.72(m,3H),3.50-3.17(m,3H),2.97(s,1H),2.80(t,J＝6.7Hz,2H),2.27-2.01(m,4H),1.91(p,J＝8.7Hz,1H),1.76(d,J＝9.3Hz,1H),1.29(d,J＝6.9Hz,1H),1.18(d,J＝7.1Hz,2H)。

EXAMPLE 345.preparation of 1- (5- (1- (4-isobutylpiperazin-1-yl) ethyl) pyrazolo [1,5-a ] pyridin-3-yl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1.1- (5-Acetylpyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine The reaction of pyridine-2, 4 (1H,3H) -diketones

Tributyl (1-ethoxyvinyl) stannane (757 mg,2.09 mmol) and Pd (PPh) were added at room temperature ₃ ) ₂ Cl ₂ (122 mg,0.174 mmol) was added to 1- (5-bromopyrazolo [1, 5-a)]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (800 mg,1.74 mmol) in DMF (8 mL). The mixture was stirred at 90 ℃ for 6h, then cooled to room temperature and acidified with 1N aqueous HCl. The mixture was partitioned between EtOAc and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by flash chromatography on silica gel (eluting with 45% EtOAc/hexanes) to give 1- (5-acetylpyrazolo [1, 5-a) as an orange solid ]Pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：423.2。

And 2, step 2.Tert-butyl 4- (1- (3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidine-1 (2H)) Radical) pyrazolo [1,5-a]Pyridin-5-yl) ethyl) piperazine-1-carboxylic acid ester

To 1- (5-acetylpyrazolo [1, 5-a)]To a solution of pyridin-3-yl) -3- (2, 4-dimethoxybenzyl) dihydropyrimidine-2, 4 (1H, 3H) -dione (170 mg,0.402 mmol) and tert-butylpiperazine-1-carboxylate (89 mg,0.48 mmol) in THF (5 mL) was added dibutyltin dichloride (244 mg,0.804 mmol) and triethylamine (0.17 mL,1.2 mmol). The mixture was stirred at 80℃for 2h, then phenylsilane (87 mg,0.80 mmol) was added. The reaction was stirred in a capped vial at 80 ℃ for 12h. The reaction was cooled to room temperature, diluted with EtOAc and washed with water. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated to give crude tert-butyl 4- (1- (3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) ethyl) piperazine-1-carboxylic acid ester. The crude product was used in the next step without any further purification. LCMS [ M+H] ⁺ ：593.0。

And 3, step 3.1- (5- (1- (4-isobutylpiperazin-1-yl) ethyl) pyrazolo [1, 5-a)]Pyridin-3-yl) dihydropyrimidine Pyridine-2, 4 (1H, 3H) -dionesPrepared using the procedure of example 192, step 2-4, wherein tert-butyl 4- (1- (3- (2, 4-dimethoxybenzyl) was used in step 2) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1,5-a]Pyridin-5-yl) ethyl piperazine-1-carboxylate instead of tert-butyl 4- ((3- (3- (2, 4-dimethoxybenzyl) -2, 4-dioxotetrahydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) piperazine-1-carboxylate, and isobutyraldehyde was used in place of cyclohexane-formaldehyde in step 3. LCMS [ M+H] ⁺ ：399.2。 ¹ H NMR (400 mhz, meod) delta 8.47 (d, j=7.3 hz, 1H), 8.03 (s, 1H), 7.51-7.47 (m, 1H), 7.02 (dd, j=7.3, 1.9hz, 1H), 3.90 (t, j=6.8 hz, 2H), 3.67 (q, j=6.6 hz, 1H), 3.25-2.55 (m, 12H), 2.08 (dq, j=15.4, 7.6hz, 1H), 1.45 (d, j=6.7 hz, 3H), 1.02 (d, j=6.6 hz, 6H). No NH protons were observed due to solvent exchange.

Example 346.1- (5- (((2S, 4R) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Piirae-type pyridine Pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dionesIs prepared from

Step 1.Tert-butyl (2S, 4R) -2-methyl-4- (pyrazolo [1, 5-a)]Pyridin-5-ylmethyl) piperidine-1-carboxylic acid Esters of

Addition of 5-bromopyrazolo [1,5-a ] to a dried vial]Pyridine (100 mg, 507. Mu. Mol), nickel chloride, dimethoxyethane adduct (5.6 mg, 25. Mu. Mol), pyridine-2, 6-bis (formamidine) -hydrochloride (6.0 mg, 25. Mu. Mol), active zinc (83 mg,1.3 mmol), tert-butyl (2S, 4R) -4- (bromomethyl) -2-methylpiperidine-1-carboxylate (178 mg, 609. Mu. Mol) and sodium iodide (19 mg, 127. Mu. Mol). Seal the reaction septum cap and use N via needle ₂ And (5) purging. DMA (2 mL) was added and the reaction was heated at 70℃overnight. The reaction was cooled to room temperature, diluted with EtOAc and filtered through a plug of silica gel eluting with EtOAc. The eluate was concentrated and the residue was purified by silica gel column chromatography (eluting with 0-100% EtOAc in heptane) to give tert-butyl (2 s,4 r) -2-methyl-4- (pyrazolo [1, 5-a) as a viscous solid]Pyridin-5-ylmethyl) piperidine-1-carboxylic acid ester. LCMS [ M+H] ⁺ ：330.3。

Step 2:tert-butyl (2S, 4R) -4- ((3-iodopyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylPiperidine- 1-Carboxylic acid ester

To tert-butyl (2S, 4R) -2-methyl-4- (pyrazolo [1, 5-a)]To a solution of pyridin-5-ylmethyl) piperidine-1-carboxylic acid ester (220 mg, 668. Mu. Mol) in MeCN (5 mL) at 0deg.C was added NIS (180 mg, 801. Mu. Mol). The reaction was then stirred at room temperature for 1h. The reaction was carried out by adding Na ₂ S ₂ O ₃ The aqueous solution was quenched and extracted with EtOAc. The organic layer was washed sequentially with water and brine, over Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (eluting with 0-100% etoac in heptane) provided tert-butyl (2 s,4 r) -4- ((3-iodopyrazolo [1, 5-a) as a clear viscous solid]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：456.1。

Step 3:tert-butyl (2S, 4R) -4- ((3- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylic acid ester

Addition of tert-butyl (2S, 4R) -4- ((3-iodopyrazolo [1, 5-a) to a dried vial]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate (80 mg, 176. Mu. Mol) in DMSO (0.5 mL), pyrimidine-2, 4 (1H, 3H) -dione (uracil) (26 mg, 228. Mu. Mol), potassium phosphate (78 mg, 369. Mu. Mol), N- (2-cyanophenyl) picolinamide (16 mg, 70. Mu. Mol) and copper (I) iodide (6.7 mg, 35. Mu. Mol). Seal the vial with septum cap and use N via needle ₂ And (5) purging. The reaction was heated at 110℃for 72h. The reaction was carried out with 1M KHSO ₄ The aqueous solution was quenched and extracted with EtOAc. The organic layer was purified by Na ₂ SO ₄ Dried, filtered and concentrated. Silica gel column chromatography (EtOAc/heptane) provided tert-butyl (2S, 4R) -4- ((3- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate. LCMS [ M+H] ⁺ ：440.2。

Step 4:1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) azoxystrobin Pyridine-2, 4 (1H, 3H) -dione trifluoroacetate

To tert-butyl (2S, 4R) -4- ((3- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) pyrazolo [1, 5-a)]Solution of pyridin-5-yl) methyl) -2-methylpiperidine-1-carboxylate (35 mg, 80. Mu. Mol) in DCM (2 mL) TFA (2 mL) was added. The reaction was stirred at room temperature for 45min. The reaction was concentrated and the crude material was azeotropically dried with toluene to give crude 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione trifluoroacetate, which was used without further purification. LCMS [ M+H] ⁺ ：340.2。

Step 5:1- (5- (((2S, 4R) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridine- 3-yl) pyrimidine-2, 4 (1H, 3H) -dione (example 346)

To 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]To a solution of pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (36 mg, 79. Mu. Mol) in DCM (2 mL) and MeOH (500. Mu.L) was added isobutyraldehyde (14. Mu.L, 159. Mu. Mol) and triethylamine (10. Mu.L, 71. Mu. Mol). The reaction was stirred at room temperature for 10min, then sodium triacetoxyborohydride (84 mg, 397. Mu. Mol) was added. The reaction was stirred at room temperature overnight. The reaction was quenched with a drop of TFA and then concentrated. The crude material was dissolved in DMSO, filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA to give 1- (5- (((2 s,4 r) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H ] ⁺ ：396.2. ¹ H NMR(500MHz,DMSO)δ11.51(s,1H),8.67(t,J＝26.8Hz,1H),8.14(s,1H),7.71(d,J＝7.8Hz,1H),7.36(d,J＝14.1Hz,1H),6.87(s,1H),5.71(s,1H),3.59(d,J＝89.5Hz,1H),3.23-2.69(m,2H),2.59(d,J＝47.7Hz,1H),2.36(s,1H),2.23-1.81(m,2H),1.78-1.14(m,7H),0.89(d,J＝69.0Hz,8H)。

Example 347.1 preparation of 1- (5- (((2S, 4R) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methylpyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-methylpyrimidine-2, 4 (1 h,3 h) -dione is used in place of pyrimidine-2, 4 (1 h,3 h) -dione in step 3. LCMS [ M+H] ⁺ ：410.2。 ¹ HNMR(500MHz,DMSO)δ11.53(d,J＝6.5Hz,1H),8.73-8.54(m,1H),8.15(d,J＝1.4Hz,1H),7.62(dd,J＝8.8,1.4Hz,1H),7.42-7.23(m,1H),6.89(dd,J＝7.2,1.9Hz,1H),3.69(s,1H),3.24-2.90(m,3H),2.89-2.69(m,2H),2.60(h,J＝6.6Hz,1H),2.23-1.88(m,2H),1.83(d,J＝1.2Hz,3H),1.77-1.61(m,3H),1.61-1.43(m,1H),1.34(d,J＝6.6Hz,1H),1.22(d,J＝6.8Hz,2H),1.03-0.88(m,6H)。

Example 348.1 preparation of- (5- (((2S, 4R) -1- (cyclopropylmethyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methylpyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-methylpyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3, and cyclopropanecarbaldehyde is used instead of isobutyraldehyde in step 5. LCMS [ M+H] ⁺ ：408.2。 ¹ H NMR(500MHz,DMSO)δ11.53(d,J＝5.4Hz,1H),8.69(dt,J＝7.2,1.4Hz,1H),8.16(d,J＝1.4Hz,1H),7.63(dd,J＝9.4,1.4Hz,1H),7.37(dd,J＝14.6,1.8Hz,1H),6.90(ddd,J＝7.2,3.6,1.8Hz,1H),3.65(s,1H),3.44(s,1H),3.37-2.93(m,3H),2.75(dd,J＝14.9,7.2Hz,1H),2.69-2.56(m,1H),2.26-2.03(m,1H),1.83(d,J＝1.2Hz,3H),1.80-1.53(m,3H),1.51-1.37(m,1H),1.32(d,J＝6.6Hz,1H),1.23(d,J＝6.9Hz,2H),1.05(dt,J＝27.2,7.1Hz,1H),0.73-0.53(m,2H),0.49-0.22(m,2H)。

Example 349.5 preparation of fluoro-1- (5- (((2S, 4R) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

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Prepared using the method of example 346, wherein 5-fluoropyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3. LCMS [ M+H] ⁺ ：414.4。 ¹ HNMR(500MHz,DMSO)δ12.04(dd,J＝8.4,5.2Hz,1H),8.69(dd,J＝7.3,2.7Hz,1H),8.29-8.06(m,2H),7.51-7.37(m,1H),6.97-6.82(m,1H),3.38(d,J＝11.0Hz,1H),2.97(dt,J＝12.7,6.1Hz,3H),2.90-2.70(m,2H),2.61(tt,J＝13.0,7.0Hz,1H),2.29-1.91(m,2H),1.68(d,J＝4.5Hz,3H),1.62-1.44(m,1H),1.35(d,J＝6.6Hz,1H),1.23(d,J＝6.8Hz,2H),1.08-0.83(m,6H)。

Example 350.preparation of 1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-fluoropyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-fluoropyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3 and 4, 4-difluorocyclohexane-1-carbaldehyde is used instead of isobutyraldehyde in step 5. LCMS [ M+H ] ⁺ ：490.4。 ¹ H NMR(500MHz,DMSO)δ8.92(s,1H),8.68(dd,J＝7.2,3.2Hz,1H),8.22(dd,J＝10.6,6.5Hz,1H),8.14(d,J＝2.0Hz,1H),7.43(d,J＝17.7Hz,1H),6.89(d,J＝7.2Hz,1H),3.40-2.98(m,4H),2.90(t,J＝6.2Hz,1H),2.76-2.53(m,2H),2.23-1.98(m,3H),1.96-1.42(m,9H),1.28(dd,J＝54.9,6.8Hz,5H)。

Example 351.1 preparation of- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methylpyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-methylpyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3, and 4, 4-difluorocyclohexane-1-carbaldehyde is used instead of isobutyraldehyde in step 5. LCMS [ M+H] ⁺ ：486.4。 ¹ H NMR(500MHz,DMSO)δ8.89(s,1H),8.67(dd,J＝7.2,3.2Hz,1H),8.14(d,J＝2.0Hz,1H),7.61(dd,J＝9.8,1.5Hz,1H),7.34(d,J＝16.0Hz,1H),6.87(dd,J＝7.2,1.9Hz,1H),3.73(s,1H),3.53(s,1H),3.28-2.97(m,2H),2.89(t,J＝6.1Hz,1H),2.75-2.53(m,2H),2.10(d,J＝80.9Hz,3H),1.93-1.42(m,12H),1.27(dd,J＝53.7,6.8Hz,5H)。

Example 352.5 preparation of chloro-1- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

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Prepared using the method of example 346, wherein 5-chloropyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3 and 4, 4-difluorocyclohexane-1-carbaldehyde is used instead of isobutyraldehyde in step 5. LCMS [ M+H] ⁺ ：506.4。 ¹ H NMR(500MHz,DMSO)δ8.86(s,1H),8.68(dd,J＝7.2,3.0Hz,1H),8.24(d,J＝9.9Hz,1H),8.16(d,J＝2.0Hz,1H),7.43(d,J＝19.0Hz,1H),6.91-6.85(m,1H),3.41-2.99(m,4H),2.90(t,J＝6.2Hz,1H),2.75-2.54(m,2H),2.24-1.95(m,3H),1.95-1.41(m,9H),1.27(dd,J＝54.4,6.8Hz,5H)。

Example 353.1 preparation of- (5- (((2S, 4R) -1- ((4, 4-difluorocyclohexyl) methyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-methoxypyrimidine-2, 4 (1 h,3 h) -dione is used in place of pyrimidine-2, 4 (1 h,3 h) -dione in step 3, and 4, 4-difluorocyclohexane-1-carbaldehyde is used in place of isobutyraldehyde in step 5. LCMS [ M+H ] ⁺ ：502.2。 ¹ H NMR(500MHz,DMSO)δ11.67(s,1H),8.63(d,J＝7.1Hz,1H),8.15(s,1H),7.36(d,J＝4.2Hz,2H),6.97-6.76(m,1H),3.63(s,3H),2.91(s,1H),2.44-2.32(m,3H),2.19(s,2H),2.05-1.68(m,8H),1.47(d,J＝52.8Hz,4H),1.22(d,J＝25.8Hz,1H),1.08(d,J＝12.7Hz,2H),0.88(s,3H)。

Example 354.5 preparation of cyclopropyl-1- (5- (((2S, 4R) -1-isobutyl-2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 346, wherein 5-cyclopropyl pyrimidine-2, 4 (1 h,3 h) -dione was used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 3. LCMS [ M+H] ⁺ ：436.2。 ¹ HNMR(500MHz,DMSO)δ11.52(d,J＝7.0Hz,1H),8.68(dd,J＝7.2,2.7Hz,1H),8.15(d,J＝1.5Hz,1H),7.44-7.24(m,2H),6.88(d,J＝7.1Hz,1H),3.70(s,2H),3.24-2.90(m,2H),2.90-2.70(m,2H),2.69-2.56(m,3H),2.30-1.92(m,1H),1.85-1.42(m,4H),1.34(d,J＝6.5Hz,1H),1.22(d,J＝6.8Hz,2H),1.09-0.87(m,6H),0.71(dt,J＝8.8,2.9Hz,2H),0.68-0.57(m,2H)。

Example 355.1 preparation of- (5- (((2S, 4R) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

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HATU (38 mg,0.099 mmol) and cyclopropanecarboxylic acid (8.5 mg,0.099 mmol) were added to 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at room temperature]Pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (30 mg,0.066 mmol) in DMF (1.5 mL). The mixture was stirred at room temperature for 5min, then DIPEA (0.035 ml,0.19 mmol) was added. The mixture was stirred at room temperature overnight, then filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA to give 1- (5- (((2 s,4 r) -1- (cyclopropanecarbonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) pyrimidine-2, 4 (1 h,3 h) -dione. LCMS [ M+H] ⁺ ：408.2。 ¹ H NMR(500MHz,DMSO)δ11.52(d,J＝2.3Hz,1H),8.66(dd,J＝7.2,0.9Hz,1H),8.15(s,1H),7.73(d,J＝7.8Hz,1H),7.37(d,J＝1.6Hz,1H),6.90(dd,J＝7.2,1.9Hz,1H),5.72(dd,J＝7.8,2.3Hz,1H),4.68(d,J＝66.7Hz,1H),4.19(dd,J＝89.6,13.7Hz,1H),3.12(t,J＝13.1Hz,1H),2.86-2.56(m,2H),2.19-1.82(m,2H),1.74-1.45(m,2H),1.43-1.22(m,1H),1.21-0.90(m,4H),0.80-0.46(m,4H)。

EXAMPLE 356 preparation of- (5- (((2S, 4R) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Triethylamine (0.046 mL,0.33 mmol) and ethanesulfonyl chloride (0.019 mL,0.19 mmol) were added to 1- (5- (((2S, 4R) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a) at 0deg.C]Pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione trifluoroacetate (30 mg,0.066 mmol) in DCM (2 mL). The mixture was stirred at room temperature overnight, then filtered through a 1 micron filter and purified by reverse phase HPLC using ACN/water/0.1% TFA to give 1- (5- (((2 s,4 r) -1- (ethylsulfonyl) -2-methylpiperidin-4-yl) methyl) pyrazolo [1, 5-a)]Pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione (8 mg, 14. Mu. Mol,21% yield). LCMS [ M+H] ⁺ ：432.5. ¹ H NMR(500MHz,DMSO)δ11.52(d,J＝2.3Hz,1H),8.66(d,J＝7.1Hz,1H),8.15(s,1H),7.73(dd,J＝7.8,1.2Hz,1H),7.37(s,1H),6.89(dd,J＝7.2,1.8Hz,1H),5.72(dd,J＝7.8,2.3Hz,1H),4.05(t,J＝6.3Hz,1H),3.49(s,1H),3.14-2.86(m,3H),2.59-2.54(m,2H),2.03(s,1H),1.55(dd,J＝32.4,13.3Hz,2H),1.37(td,J＝12.8,5.3Hz,1H),1.18(t,J＝7.3Hz,6H),1.14-1.07(m,1H)。

EXAMPLE 357 preparation of (S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Step 1:tert-butyl (S) -2-methyl-4- (pyrazolo [1, 5-a)]Pyridin-5-ylmethyl) piperazine-1-carboxylic acid ester

At room temperature, to 5-bromopyrazolo [1,5-a ]]To a suspension of pyridine (500 mg,2.54 mmol) in toluene (10 mL) and water (1 mL) was added Cs ₂ CO ₃ (2.48 g,7.61 mmol), tert-butyl (S) -2-methyl-4- ((trifluoro-l 4-boranyl) methyl) piperazine-1-carboxylic acid potassium salt (2.44 g,7.61 m)mol) and RuPhos (237 mg,0.508 mmol), followed by Pd (OAc) ₂ (57 mg, 0.25. Mu. Mol). The mixture was stirred at 100 ℃ overnight, then cooled to room temperature and partitioned between EtOAc and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated. Silica gel column chromatography (EtOAc/EtOH/heptane) provided tert-butyl (S) -2-methyl-4- (pyrazolo [1, 5-a)]Pyridin-5-ylmethyl) piperazine-1-carboxylic acid ester (730 mg,1.9mmol,75% yield). LCMS [ M+H] ⁺ ：331.4。

And 2, step 2.(S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a]Pyridine-3- Base) pyrimidine-2, 4 (1H, 3H) -dionesPrepared by the method of example 346, step 2-5, wherein tert-butyl (S) -2-methyl-4- (pyrazolo [1, 5-a) is used in step 2]Pyridin-5-ylmethyl) piperazine-1-carboxylic acid ester instead of tert-butyl (2 s,4 r) -2-methyl-4- (pyrazolo [1, 5-a)]Pyridin-5-ylmethyl) piperidine-1-carboxylic acid ester. LCMS [ M+H] ⁺ ：397.2。 ¹ H NMR (500 mhz, dmso) δ11.54 (s, 1H), 8.80-8.57 (m, 1H), 8.19 (s, 1H), 7.74 (d, j=7.8 hz, 1H), 7.48 (d, j=22.7 hz, 1H), 6.98 (d, j=7.1 hz, 1H), 5.73 (d, j=8.2 hz, 1H), 3.64 (d, j=14.1 hz, 1H), 3.52 (t, j=15.9 hz, 1H), 3.21-2.99 (m, 1H), 2.92 (d, j=13.0 hz, 2H), 2.80-2.64 (m, 1H), 2.49-2.28 (m, 2H), 2.29-1.58 (m, 2H), 1.29 (d, j=6.4 hz, 2H), 0.98 (dd, j=15.9 hz, 1H), 3.21-2.99 (m, 1H), 2.92 (d, 1.80 hz, 5H). The two missing protons are due to overlap with the solvent.

EXAMPLE 358 preparation of (S) -5-fluoro-1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 357, wherein 5-fluoropyrimidine-2, 4 (1 h,3 h) -dione was used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 2. LCMS [ M+H] ⁺ ：415.2。 ¹ HNMR(500MHz,DMSO)δ12.05(d,J＝5.3Hz,1H),8.74(d,J＝7.2Hz,1H),8.23(d,J＝6.4Hz,1H),8.19(s,1H),7.59(s,1H),6.99(dd,J＝7.1,1.8Hz,1H),3.51(s,4H),3.21-2.72(m,7H),2.21-1.75(m,1H),1.27(s,3H),0.97(t,J＝6.7Hz,6H)。

EXAMPLE 359 preparation of (S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methylpyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 357, wherein 5-methylpyrimidine-2, 4 (1 h,3 h) -dione was used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 2. LCMS [ M+H] ⁺ ：411.2。 ¹ HNMR(500MHz,DMSO)δ11.52(s,1H),8.71(dd,J＝7.1,0.9Hz,1H),8.17(s,1H),7.61(q,J＝1.2Hz,1H),7.50(s,1H),6.97(dd,J＝7.2,1.8Hz,1H),3.72(s,5H),3.05(s,4H),2.75(d,J＝59.3Hz,2H),2.01(s,1H),1.81(d,J＝1.3Hz,3H),1.26(d,J＝6.4Hz,3H),0.94(t,J＝6.7Hz,6H)。

EXAMPLE 360 preparation of (S) -1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 357, wherein 5-methoxypyrimidine-2, 4 (1 h,3 h) -dione was used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 2. LCMS [ M+H] ⁺ ：427.2。 ¹ HNMR(500MHz,DMSO)δ11.71(s,1H),8.73(d,J＝7.2Hz,1H),8.21(s,1H),7.53(s,1H),7.37(s,1H),6.98(d,J＝7.2Hz,1H),3.64(s,8H),2.93(d,J＝47.3Hz,5H),2.38(s,1H),2.03(s,1H),1.49-1.14(m,3H),0.96(t,J＝6.3Hz,6H)。

EXAMPLE 361 preparation of (S) -5-cyclopropyl-1- (5- ((4-isobutyl-3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Prepared using the procedure of example 357, wherein 5-cyclopropyl pyrimidine-2, 4 (1 h, 3) was used in step 2H) -diketones instead of pyrimidine-2, 4 (1 h,3 h) -diketones. LCMS [ M+H ] ⁺ ：437.2。 ¹ HNMR(500MHz,DMSO)δ11.53(s,1H),8.72(d,J＝7.1Hz,1H),8.19(s,1H),7.49(d,J＝14.4Hz,1H),7.35(d,J＝0.9Hz,1H),6.97(dd,J＝7.2,1.8Hz,1H),3.52(s,5H),2.94(d,J＝49.6Hz,5H),2.38(s,1H),2.03(s,1H),1.65(ddd,J＝11.0,8.6,5.3Hz,1H),1.39-1.16(m,3H),0.96(t,J＝6.6Hz,6H),0.81-0.67(m,2H),0.65-0.47(m,2H)。

Example 362 preparation of (S) -1- (5- ((4- (cyclopropylmethyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) -5-methylpyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 357, wherein 5-methylpyrimidine-2, 4 (1 h,3 h) -dione is used instead of pyrimidine-2, 4 (1 h,3 h) -dione in step 2, and cyclopropanecarbaldehyde is used instead of isobutyraldehyde. LCMS [ M+H] ⁺ ：409.2。 ¹ H NMR(500MHz,DMSO)δ11.54(s,1H),8.73(d,J＝7.3Hz,1H),8.19(s,1H),7.64(d,J＝1.4Hz,1H),7.50(s,1H),6.98(dd,J＝7.2,1.8Hz,1H),3.67(d,J＝13.9Hz,2H),3.35(s,2H),3.17(d,J＝11.8Hz,2H),3.01(t,J＝13.6Hz,3H),2.25(t,J＝11.8Hz,1H),1.83(d,J＝1.3Hz,3H),1.32(d,J＝6.6Hz,1H),1.24(d,J＝6.4Hz,3H),1.05(s,1H),0.65(d,J＝13.3Hz,2H),0.50-0.15(m,2H)。

Example 363 preparation of (S) -1- (5- ((4, 4-difluorocyclohexyl) methyl) -3-methylpiperazin-1-yl) methyl) pyrazolo [1,5-a ] pyridin-3-yl) pyrimidine-2, 4 (1H, 3H) -dione

Prepared using the method of example 357, wherein 4, 4-difluorocyclohexane-1-carbaldehyde was used in place of isobutyraldehyde in step 2. LCMS [ M+H] ⁺ ：473.2。 ¹ H NMR(500MHz,DMSO)δ11.56(s,1H),8.74(d,J＝7.2Hz,1H),8.21(s,1H),7.74(d,J＝7.7Hz,1H),7.52(s,1H),7.14-6.62(m,1H),5.82-5.52(m,1H),4.19(s,2H),3.68(s,3H),3.29(s,2H),2.99(s,3H),2.36(d,J＝19.6Hz,1H),2.04(s,2H),1.96-1.59(m,5H),1.26(s,5H)。

Biological data

Abbreviations (abbreviations)

BSA bovine serum albumin

Cas9 CRISPR associated protein 9

CRISPR clustered regularly interspaced short palindromic repeats

crRNA CRISPR RNA

Improved eagle medium of DMEM dulbeck

DMSO dimethyl sulfoxide

DTT dithiothreitol

EDTA ethylenediamine tetraacetic acid

eGFP enhanced green fluorescent protein

FACS fluorescence activated cell sorting

FBS fetal bovine serum

FITC fluorescein

Flt3L Fms-related tyrosine kinase 3 ligands, flt3L

HbF fetal hemoglobin

HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid)

IMDM Iskov modified Dulbecco's Medium (Iscove's modified Dulbecco's medium)

KCl potassium chloride

Peripheral blood mobilized by mPB

PBS phosphate buffered saline

recombinant human erythropoietin of rhEPO

rhIL-3 recombinant human interleukin-3

rhIL-6 recombinant human interleukin-6

rhSCF recombinant human stem cell factor

rhTPO recombinant human thrombopoietin

RNP ribonucleoprotein

short hairpin RNA of shRNA

trans-activating crRNA by tracrRNA

WIZ protein containing widely spaced zinc fingers

Materials and methods

Example 364: quantification of WIZ protein levels in HiBit tag fusion protein assays

The HiBit system from Promega, promega was used to develop high throughput and quantitative assays to measure changes in WIZ protein levels in response to compounds. The HiBit tag is derived from a split nanoluciferase and has the following protein sequence: VSGWRLFKKIS (SEQ ID NO: 1). Complementary fragments of nano-luciferase (known as LgBit from plagmager) were added to the HiBit tag to form an active nano-luciferase whose activity can be precisely measured. In this way, the level of the HiBit tagged fusion protein can be quantified in the cell lysate.

Construction of lentiviral vectors (Invitrogen based) ^TM The pLenti6.2/V5 DEST backbone), which places the HiBit tag upstream of WIZ and expresses the fusion protein from the HSVTK promoter.

To ensure moderate and consistent expression of the HiBit-WIZ fusion protein in all cells in the population, stable cell lines were constructed from cells with single copy constructs. Using a DNA fragment from Invitrogen ^TM ViraPower of (A) ^TM The kit prepares the lentivirus packaged with the construct. At low multiplicity of infection, 293T cells (catalog number: CRL-3216) from the ATCC were infected with the virus and selected in the medium by 5. Mu.g/mL blasticidin for 2 weeks.

The level of HiBit-WIZ-tagged fusion protein in the compound-treated cell lines was measured as follows:

on day 1, cells were diluted to 1.0x10 in normal growth medium ⁶ Individual cells/ml. mu.L of the cell suspension was inoculated into each well of a solid white 384-well plate. The plates were incubated at 37℃with 5% CO ₂ Incubate overnight in a humidified tissue incubator.

On day 2, serial dilutions of the compound were prepared in 384 well plates. Compound plates were provided in columns 1, 2, 23, 24DMSO, compound plates were provided with 10-point dilution series at columns 3-12 and columns 13-22. 10mM stock solutions of compounds were placed in column 3 or column 13 and serial 1:5 dilutions were performed until there was a 10 point dilution series for each compound. By passing through Sound transfer (Labcyte corporation) 50nL of diluted compound was transferred into plated cells. The highest concentration of the compound was 25. Mu.M. The plates were incubated at 37℃with 5% CO ₂ Incubate overnight (about 18 hours) in a humidified tissue incubator.

On day 3, the plates were removed from the incubator and allowed to equilibrate for 60 minutes at room temperature. As described in the manufacturer's protocol, hiBit substrate was added (Nano-HiBit Lytic detection System, promega Corp catalog number: n3050). Plates were incubated for 30 min at room temperature and +.>Reader->The light emission is read. Use->The software package analyzes and visualizes the data.

WIZ degradation Activity of Compounds (Table 1)

Table 1 shows the wip degradation activity of the compounds of the present disclosure in a wip HiBit assay in 293T cells. WIZ Amax reflects DMSO normalization and the percentage curve fit of WIZ-HiBit was kept at 25uM. The calculation is performed by: DMSO controls were normalized to 100%, parametric curve fitting (10 points, 5-fold) was performed on dose response data, followed by calculation of the response at 25uM using the fit equation (nd=undetermined).

Table 1:

/>

example 365: small molecule HbF induction assay

Cryopreserved primary human CD34 was obtained from Allcells, LLC ⁺ Hematopoietic stem cells and progenitor cells. Isolation of CD34 from peripheral blood of healthy donors after mobilization by administration of granulocyte colony-stimulating factor ⁺ And (3) cells. Cells were differentiated ex vivo towards erythroid lineages using a 2-stage culture method. In the first stage, the cells were incubated at 37℃with 5% CO ₂ The following is supplemented with rhSCF (50 ng/mL,company), rhIL-6 (50 ng/mL,/-A)>Company), rhIL-3 (50 ng/mL,/-A)>Company) and rhFlt3L (50 ng/mL,)>Company) and 1X antibiotic-antifungal agent (life technologies (Life Technologies), sameimers technology (Thermo Fisher Scientific)) ^TM Serum-free expansion medium (SFEM) (stem cell technology company (STEMCELL Technologies inc.)) for 6 days. During the second stage, the cells were incubated at 37℃with 5% CO ₂ The cells were cultured in erythroid differentiation medium at 5,000 cells/mL for 7 days in the presence of the compound. The erythroid differentiation medium included IMDM (Life technologies Co.) supplemented with insulin (10. Mu.g/mL, sigma Aldrich Co.), heparin (2U/mL, sigma Aldrich Co.), holohexan (330. Mu.g/mL, sigma Aldrich Co.), human serum AB (5%, sigma Aldrich Co.), hydrocortisone (1. Mu.M, stem cell technologies Co.), rhSCF (100 ng/mL) >Company), rhIL-3 (5 ng/mL,/-A)>Company), rhEPO (3U/mL,/->Company) and 1X antibiotic-antifungal agents. All compounds were dissolved and diluted into dimethyl sulfoxide (DMSO) and then added to the medium to a final concentration of 0.3% DMSO, thus testing in a 7 point, 1:3 dilution series starting at 30 uM.

Staining and flow cytometry

To make aliveForce analysis, washing and resuspending the samples in Phosphate Buffered Saline (PBS) and using LIVE/DEAD ^TM The purple dead cell staining kit (Life technologies Co., L34963) can be fixed for 20 minutes. Cells were then washed again with PBS and resuspended in PBS supplemented with 2% Fetal Bovine Serum (FBS) and 2mM EDTA in preparation for cell surface marker analysis. Cells were labeled with allophycocyanin-conjugated CD235a (1:100, BD biosciences, 551336) antibody and Brilliant Violet-conjugated CD71 (1:100, BD biosciences, 563767) antibody for 20 minutes. For analysis of cytoplasmic fetal hemoglobin (HbF), a fixed buffer is used according to the manufacturer's protocol420801 And permeabilization washing buffer (>421002 Cells were fixed and permeabilized. During the permeabilization step, the cells were treated with phycoerythrin-conjugated or FITC-conjugated HbF-specific antibodies (1:10-1:25, invitrogen ^TM MHFH 04-4) staining for 30 minutes. In FACSCanto ^TM II flow cytometer or LSRFortessa ^TM The stained cells were washed with phosphate buffered saline before analysis on (BD biosciences). Using FlowJo ^TM The software (BD biosciences) performs data analysis.

HbF-inducing Activity of Compounds (Table 2)

mPB cd34+ cells were expanded for 6 days and then erythroid differentiated in the presence of the compound for 7 days. Cells were fixed, stained and analyzed by flow cytometry. Table 2 shows HbF-inducing activity of the compounds. HbF Amax = the highest percentage of HbF positive stained cells (% hbf+ cells) in the fitted dose response curve. Baseline% hbf+ cells of DMSO-treated cells were approximately 30% -40%.

Table 2:

example 366: cell culture for shRNA and CRISPR assays

HEK293T cells were maintained in DMEM high glucose complete medium containing sodium pyruvate, non-essential amino acids, 10% FBS, 2mM L-glutamine, 100U/mL pen/strep, 25mM HEPES. All reagents used to culture HEK293T cells were obtained from Invitrogen unless otherwise indicated ^TM Obtained.

Mobilized peripheral blood (mPB) CD34+ cells (Australian Seer) were maintained at StemSpan supplemented with 50ng/mL each of rhTPO, rhIL-6, rhFLT3L, rhSCF prior to shRNA transduction or WIZ-targeted Ribonucleoprotein (RNP) electroporation ^TM Serum-free expansion Medium (SFEM) (Stem cell technology Co.) for 2-3 days. All cytokines were derived fromObtained by the company. The cell culture was maintained at 37℃and 5% CO ₂ In a humidified tissue incubator.

Generation of WIZ-targeted shRNA lentiviral clones

5' -phosphorylated sense and antisense complementary single stranded DNA oligonucleotides for respective shrnas of WIZ were synthesized by integrated DNA technologies (Integrated DNA Technologies, inc., IDT). Each DNA oligonucleotide was designed with a PmeI/AscI restriction overhang at the 5 '-end and 3' -end, respectively, for subsequent compatible ligation into the lentiviral vector backbone. Equimolar complementary oligonucleotides were incubated in NEB buffer 2 (New England) by heating at 98℃for 5 min on a heating block followed by cooling to room temperature on a benchCompany). The annealed double-stranded DNA oligonucleotides were ligated into the pHage lentiviral backbone digested with PmeI/AscI using a T4 DNA ligase kit (new england biosystems). The ligation reaction was transformed into chemically competent Stbl3 cells (Invitrogen) according to the manufacturer's protocol ^TM ). Positive clones were verified using sequencing primers (5'-ctacattttacatgatagg-3'; SEQ ID NO: 2) and plasmids were passed through Alta organisms Purification by technical company (Alta Biotech LLC).

According to the manufacturer's instructions (Invitrogen) ^TM ) Lentiviral particles of the corresponding shRNA constructs were generated by co-transfecting HEK293T cells with envelope plasmids expressing pCMV-dr8.91 and pCMV-VSV-G using Lipofectamine 3000 reagent in the form of 150mm tissue culture dishes. Lentiviral supernatants were harvested 48 hours after co-transfection, filtered through a 0.45 μm filter (Millipore) and concentrated using Amicon Ultra 15 (Millipore) with an Ultracel-100 membrane. After serial dilution and infection of HEK293T cells, the infection unit of each lentiviral particle was determined by flow cytometry using eGFP expression as a transduction marker.

The shRNA sequence is as follows:

shWIZ_#1 5’-AGCCCACAATGCCACGGAAAT-3’(SEQ ID NO:3)；

shWIZ_#2 5’-GCAACATCTACACCCTCAAAT-3’(SEQ ID NO:4)；

shWIZ_#4 5’-TGACCGAGTGGTACGTCAATG-3’(SEQ ID NO:5)；

shWIZ_#5 5’-AGCGGCAGAACATCAACAAAT-3’(SEQ ID NO:6)。

lentiviral shRNA transduction and FACS of mPB cd34+ cells

mPB cd34+ transduction was performed on retronectin coated non-tissue culture treated 96-well flat bottom plates (Corning, inc.). Briefly, the plate was used with 100. Mu.L(1. Mu.g/mL) (Takarabio, inc.) was coated, sealed and incubated overnight at 4 ℃. Then remove->Plates were incubated with BSA (bovine serum albumin) (1%) in PBS for 30 min at room temperature. Subsequently, BSA (bovine serum albumin) was aspirated and replaced with 100. Mu.L of lentiviral concentrate and centrifuged at 2000Xg for 2 hours at room temperature. Next, the remaining supernatant was gently aspirated, and mPB cd34+ cells were prepared for transduction. One thousand cells were inoculated into 150. Mu.L of rhTPO, rh supplemented with 50ng/mL each StemSpan of IL-6, rhFLT3L and rhSCF ^TM In serum-free amplification medium (SFEM) to initiate transduction. Cells were cultured for 72 hours and then transduction efficiency was assessed using eGFP expression as a marker.

In FACSAria ^TM eGFP positive cells were sorted on III (BD biosciences). Briefly, the transduced mPB cd34+ cell population was washed and resuspended with FACS buffer containing 1x Hank buffered saline solution, EDTA (1 mM) and FBS (2%). The sorted eGFP positive cells were used in erythroid differentiation assays.

Targeted CRISPR knockout of WIZ

Alt-R CRISPR-Cas9 crRNA and tracrRNA (5'-AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU-3'; SEQ ID NO: 7) were purchased from integrated DNA technologies Inc. The equimolar crrnas were annealed with WIZ-targeted crrnas in Tris buffer (10 mm, ph 7.5) (table 3) by heating at 95 ℃ for 5 min using a Polymerase Chain Reaction (PCR) machine (burle company (Bio-Rad)) followed by cooling to room temperature on a bench. Subsequently, by annealing the tracrRNA crRNA with 6ug of Cas9 at 37℃in the presence of HEPES (100 mM), KCl (50 mM), mgCl ₂ (2.5 mM), glycerol (0.03%), DTT (1 mM) and Tris pH 7.5 (2 mM) in 1 Xbuffer for 5 minutes to form Ribonucleoprotein (RNP) complexes.

According to the manufacturer's proposal, in 4D-Nucleofector ^TM Electroporation of RNP complex was performed on a Lonsha company (Lonza)). Briefly, 50,000 mPB cd34+ cells resuspended in primary cell P3 buffer containing supplement (dasha corporation) were premixed with 5 μl RNP complex per well in a nuclear cuvette and incubated for 5 minutes at room temperature. Subsequently, the mixture was subjected to electroporation using the CM-137 procedure. Cells were cultured for 72 hours after RNP electroporation, and erythroid differentiation was then started. The crRNA sequences are shown in table 3 below.

Table 3.

Erythroid differentiation of shRNA-transduced or RNP-electroporated mPbCD34+ cells

Erythroid differentiation was initiated by seeding 8,000 RNP electroporated or FACS sorted egfp+mpbc34+ cells per well in 96 well tissue culture plates. Basal differentiation medium consisted of IMDM (Dulbecco's medium modified by Iskov), human AB serum (5%), transferrin (330. Mu.g/mL), insulin (10. Mu.g/mL) and heparin (2 IU/mL). Differentiation medium was supplemented with rhSCF (100 ng/mL), rhIL-3 (10 ng/mL), rhEPO (2.5U/mL) and hydrocortisone (1. Mu.M). After 4 days of differentiation, cells were allowed to divide in fresh medium (1:4) to maintain optimal growth density. Cells were further cultured for 3 days and used to assess fetal hemoglobin (HbF) expression.

HbF gene expression by RNA-seq analysis

In mpbccd34+ HSCs, wip was subjected to two independent targeted CRISPR/Cas9 Knockouts (KO) using wip_6 and wip_18 gRNA or non-targeted scrambled gRNA negative controls. Cells from KO and negative controls were then cultured for 7 days for erythroid differentiation and used for total RNA isolation (Zymo Research, catalog No. R1053). The amount of isolated RNA was determined prior to sequencing using the Agilent RNA 6000 Pico kit (Agilent, catalog number 5067-1513).

RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA sample preparation protocol and sequenced using the Illumina Novaseq6000 platform (Illumina). The samples were sequenced to have a length of 2x76 base pairs. For each sample, the sequenced fragment was mapped to annotated transcripts in the human genome hg38 provided by the ENSEMBL database using salcon version 0.8.2 (Patro et al 2017; doi: 10.1038/nmet.4197). The expression level of each gene was obtained by adding the counts of transcript level counts using txamport (Soneson et al 2015; doi:10.12688/f1000 research.7563.1). DESeq2 was used to normalize library size and transcript length differences and to test for differential expression between samples treated with WIZ-targeted gRNA and samples treated with a scrambled gRNA control (Love et al 2014; doi:10.1186/s 13059-014-0550-8). Data was visualized using ggplot2 (Wickham H (2016): ggplot2: elegant Graphics for Data Analysis [ ggplot2: exquisite chart for data analysis ]. Springer-Verlag New York [ Scheplerger Press, N.Y. ]. ISBN 978-3-319-24277-4; https:// ggplot2.Tidyverse. Org).

HbF intracellular staining

One hundred thousand cells were aliquoted into U-bottom 96-well plates and stained with diluted LIVE/DEAD in the dark for 20min with a purple vital dye according to manufacturer's recommendations (Invitrogen). Cells were washed with FACS staining buffer followed by staining with anti-CD 71-BV711 (BD biosciences) and anti-CD 235a-APC (BD biosciences) for 20min in the dark. After washing two rounds with three volumes of 1X PBS, cells were fixed with 1X BD Cytofix/Cytoperm (BD biosciences) and permeabilized for 30 minutes at room temperature in the dark. Subsequently, the cells were washed twice with three volumes of 1 XPRAM/wash buffer (BD biosciences). anti-HbF-FITC (thermo scientific) was diluted in 1 Xperm/wash buffer (1:25), added to permeabilized cells and incubated at room temperature in the dark for 30 min. Next, the cells were washed twice with three volumes of 1x perm/wash buffer and analyzed by flow cytometry using LSR Fortessa (BD biosciences). Data was analyzed using FlowJo software.

Results

WIZ KO up-regulates HBG1/2 expression upon erythroid differentiation

Targeting KO to WIZ using two independent grnas (wiz_6 and wiz_18) showed up-regulation of fetal hemoglobin gene (HBG 1/2) as shown in fig. 1A.

WIZ deletion induces fetal hemoglobin in mPB CD34 ⁺ Expression in cells of the derived erythroid lineage

To verify if WIZ is a negative regulator of HbF expression, shRNA and CRISPR-Cas9 mediated knockdown and knockdown functional genetics methods were employed. Will mPB CD34 ⁺ Cells were treated with shRNA or CRISPR-Cas9 reagent and erythroid differentiated for 7 days, and then flow cytometry analysis was performed. Targeted knockdown of WIZ transcripts resulted in 78% -91% HbF ⁺ Cells, in contrast, negative control scrambled shRNA resulted in 40% HbF ⁺ And (3) cells. Error bars represent standard error of two biological replicates, each biological replicateThere are three technical iterations (fig. 1B). CRISPR/Cas9 mediated targeted deletion of WIZ results in 62% -88% HbF ⁺ Cells, in contrast, randomly oriented crrnas result in 39% HbF ⁺ And (3) cells. Error bars represent the standard error of a biological sample with four technical replicates (fig. 1C). In summary, the results indicate that the absence of WIZ induces HbF in human primary erythroid cells. Thus, the zinc finger transcription factor widely spaced zinc finger motif (WIZ) was identified as a new target for HbF induction. These data provide genetic evidence that WIZ is a regulator of fetal hemoglobin expression and is a new target for the treatment of sickle cell disease and beta-thalassemia.

Having thus described several aspects of several embodiments, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the disclosure. Accordingly, the foregoing description and drawings are by way of example only.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Sequence listing

<110> North Co., ltd (NOVARTIS AG)

<120> pyrazolopyridine derivative and use thereof

<130> PAT059039-WO-PCT

<140> 63/164,130

<141> 2021-03-22

<160> 14

<170> patent In version 3.5

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Claims

1. A compound having the formula (I "), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein:

Is a single bond or a double bond;

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atoms to which they are attached form a further heteroatom comprising 0 to 1 of a member selected from N, O and SA 5-or 6-membered heterocyclic group of (2), wherein the 5-or 6-membered heterocyclic group is substituted with 0-2 occurrences of oxo group;

R ^4b selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^4c selected from hydrogen, C ₁ -C ₆ Alkyl and C ₃ -C ₈ Cycloalkyl;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

2. A compound having the formula (I "), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^x Selected from hydrogen, C ₁ -C ₆ Alkyl and halo (e.g., F, cl).

3. The compound of any one of claims 1 and 2, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (I'), wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N, O and S, 5-to 10-membered heteroaryl containing 1-4 heteroatoms selected independently from N, O and S, C ₆ -C ₁₀ Aryl, C ₁ -C ₆ Alkoxy, hydroxy and-C (=o) -NR ⁷ R ⁸ Wherein said C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterogeniesCyclic groups, 5-to 10-membered heteroaryl groups and C ₆ -C ₁₀ R in which the aryl radical is present from 0 to 4 times ^3b Substitution;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (I), wherein:

x is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

or alternatively

R ³ To which is attached a nitrogen atom and R ² Together with the carbon atoms to which they are attached form a chain comprising 0 to 1A 5-or 6-membered heterocyclyl of a further heteroatom selected from N, O and S;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

5. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH, CF and N;

r' is selected from hydrogen and C ₁ -C ₃ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₃ An alkyl group;

or alternatively

R ³ And to thisAttached nitrogen atom and R ² Together with the carbon atom to which it is attached, form a 5-or 6-membered heterocyclyl containing 0 to 1 additional heteroatoms selected from N and O;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2 or 3;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

6. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is selected from hydrogen and methyl;

R ¹ selected from hydrogen and methyl;

or alternatively

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

n is 0, 1, 2 or 3;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.

7. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

each R ^3a Independently selected from C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl containing 1-2 heteroatoms selected independently from N and O, 5-to 6-membered heterocychc containing 1-3 heteroatoms selected independently from N, O and SAryl, and phenyl, wherein the C ₃ -C ₁₀ Cycloalkyl, 4-to 6-membered heterocyclyl, 5-to 6-membered heteroaryl and phenyl are each substituted with 0-4 occurrences of R ^3b Substitution;

n is 0, 1, 2 or 3;

m is 1 or 2; and is also provided with

p is 0 or 1.

8. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

n is 0, 1 or 2;

m is 1 or 2; and is also provided with

p is 1.

9. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

r' is hydrogen;

R ¹ is hydrogen;

R ^4a selected from C ₃ -C ₈ Cycloalkyl and phenyl;

n is 0, 1 or 2;

m is 1 or 2; and is also provided with

p is 1.

10. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Selected from C ₁ -C ₆ Alkyl and-CH ₂ -R ^3a 。

11. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ia):

12. the compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ib):

13. the compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Ic), wherein:

x is selected from CH, CF and N;

R ^2f is hydrogen;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ³ is defined according to any one of the preceding claims; and is also provided with

m is 1 or 2.

14. The compound of claim 13, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2f is hydrogen;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

R ⁵ selected from C ₁ -C ₆ Alkyl and C ₆ -C ₁₀ An aryl group;

R ^6a selected from C ₆ -C ₁₀ Aryl and C ₃ -C ₈ Cycloalkyl;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

or alternatively

m is 1 or 2.

15. The compound of claim 13 or 14, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2f is hydrogen;

R ³ selected from C ₁ -C ₈ Alkyl, C ₂ -C ₆ Alkenyl, -SO ₂ R ⁴ 、C ₁ -C ₆ Haloalkyl, wherein said C ₁ -C ₈ Alkyl and C ₁ -C ₆ Haloalkyl is independently substituted with 0-3 occurrences of R ^3a Substitution;

R ⁷ selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ⁸ selected from hydrogen and C ₁ -C ₆ An alkyl group;

Or alternatively

m is 1 or 2.

16. The compound of any one of claims 13 to 15, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

X is selected from CH and N;

R ^2f is hydrogen;

m is 1.

17. The compound of any one of claim 1 to 11 and 13 to 16, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having formula (Id),

wherein, the liquid crystal display device comprises a liquid crystal display device,

x is selected from N and CH;

R ^2b 、R ^2c and R is ^2e Is defined according to any one of claims 11 to 14, e.g. R ^2b Is C ₁ -C ₃ Alkyl, and R ^2c And R is ^2e Are all hydrogen; and is also provided with

R ³ Is defined according to any one of the preceding claims.

18. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, having the formula (Ie),

x is selected from N and CH;

R ³ Is defined according to any one of the preceding claims.

19. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein X is CH.

20. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable thereofSalts, hydrates, solvates, prodrugs, stereoisomers, or tautomers, wherein R ² Is unsubstituted C ₁ -C ₆ Alkyl radicals, e.g. C ₁ -C ₄ Alkyl, and n is 1.

21. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein m is 1.

22. The compound of any one of claims 1 to 11, 13 to 17, and 19 to 21, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Is C ₁ -C ₆ Alkyl, wherein the C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^3a And (3) substitution.

23. The compound of any one of claims 1 to 11, 13 to 17, and 19 to 22, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ³ Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, 2-propyl, butyl, isobutyl, 2-butyl, 3-methyl-2-butyl, isopentyl, 3-pentyl, neopentyl, 2, 4-dimethylpentanyl and-CH ₂ -(CH ₂ ) _0-1 -R ^3a 。

24. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^3a Is C ₃ -C ₁₀ Cycloalkyl, wherein said C ₃ -C ₁₀ R in which cycloalkyl is present 0 to 4 times ^3b Substitution, wherein each R ^3b Independently selected from C ₁ -C ₆ Alkoxy, chloro, fluoro, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy and C ₁ -C ₆ An alkyl group.

25. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^3a Is C substituted by 0-2 occurrences of fluorine ₃ -C ₇ Cycloalkyl groups.

26. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^3a Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl,

27. The compound of any one of claims 13 to 26, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b And R is ^2e Each independently selected from hydrogen and unsubstituted C ₁ -C ₃ An alkyl group; and R is ^2c Is hydrogen.

28. The compound of any one of claims 13 to 27, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b And R is ^2e Each independently selected from hydrogen and methyl; and R is ^2c Is hydrogen.

29. The compound of any one of claims 13 to 28, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein

R ^2b Is unsubstituted C ₁ -C ₃ Alkyl (e.g., methyl);

R ^2c is hydrogen; and is also provided with

R ^2e Selected from hydrogen and unsubstituted C ₁ -C ₃ An alkyl group.

30. The compound of any one of claims 13 to 29, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, wherein R ^2b Is methyl and R ^2c 、R ^2d 、R ^2e And R is ^2f Are all hydrogen.

31. A compound according to any one of claims 1 to 3 whereinIs a double bond.

32. A compound as claimed in any one of the preceding claims whereinIs a single bond.

33. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, selected from the group consisting of:

/>

34. The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an acid addition salt.

35. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier or excipient.

36. A compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use as a medicament.

37. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

38. A method of treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

39. A method of treating a disease or disorder affected by modulation of WIZ protein levels, the method comprising administering to a patient in need thereof a compound of any one of claims 1-34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

40. A method of inhibiting WIZ protein expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

41. A method of degrading WIZ protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

42. A method of inhibiting, reducing, or eliminating the activity of a WIZ protein or expression of a WIZ protein, comprising administering to the subject a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

43. A method of inducing or promoting fetal hemoglobin in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

44. A method of reactivating fetal hemoglobin production or expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

45. A method of increasing fetal hemoglobin expression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

46. A method of treating a hemoglobinopathy, e.g., β -hemoglobinopathy, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

47. A method of treating sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

48. A method of treating β -thalassemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

49. A method for reducing WIZ protein levels in a subject, the method comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

50. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease or disorder in a subject in need thereof.

51. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment of a disease or disorder selected from sickle cell disease and β -thalassemia.

52. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disorder affected by inhibition of WIZ protein levels in a subject in need thereof.

53. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disorder affected by reduced levels of WIZ protein in a subject in need thereof.

54. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment or prevention of a disease or disorder affected by WIZ protein degradation.

55. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting, reducing, or eliminating WIZ protein activity or WIZ protein expression in a subject in need thereof.

56. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inducing or promoting fetal hemoglobin in a subject in need thereof.

57. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in reactivating fetal hemoglobin production or expression in a subject in need thereof.

58. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in increasing fetal hemoglobin expression in a subject in need thereof.

59. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a hemoglobinopathy in a subject in need thereof.

60. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating sickle cell disease in a subject in need thereof.

61. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating β -thalassemia in a subject in need thereof.

62. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by increased expression of hemoglobin in a child.

63. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by inhibition, reduction, or elimination of WIZ protein activity or WIZ protein expression.

64. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by or facilitated by fetal hemoglobin.

65. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating a disease or disorder affected by reactivation of fetal hemoglobin production or expression.

66. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in inhibiting WIZ protein expression in a subject in need thereof.

67. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in degrading WIZ protein in a subject in need thereof.

68. Use of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder affected by reduced levels of WIZ protein, inhibition of WIZ protein expression, or degradation of WIZ protein.

69. Use of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder that is affected by induction or promotion of fetal hemoglobin.

70. Use of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder affected by reactivation of fetal hemoglobin production or expression.

71. Use of a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for treating a disease or disorder affected by increased fetal hemoglobin expression.

72. The use of a compound according to any one of claims 68-71, wherein the disease or disorder is selected from sickle cell disease and β -thalassemia.

73. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment of a disease or disorder affected by reduced levels of WIZ protein, inhibition of WIZ protein expression, or degradation of WIZ protein.

74. The compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the treatment of a disease or disorder affected by induction of fetal hemoglobin, reactivation of fetal hemoglobin production or expression, or increase of fetal hemoglobin expression.

75. The use of claim 73 or 74, wherein the disease or disorder is selected from sickle cell disease and β -thalassemia.

76. A pharmaceutical combination comprising a compound of any one of claims 1 to 34, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and one or more additional therapeutic agents.

77. A compound having the formula (X-1) or a salt thereof,

wherein:

is a single bond or a double bond;

x is selected from CH, CF and N;

z is selected from hydrogen and 2, 4-Dimethoxybenzyl (DMB);

R ^N selected from hydrogen and nitrogen protecting groups PG, such as t-butyloxycarbonyl (Boc);

r' is selected from hydrogen and C ₁ -C ₆ An alkyl group;

R ¹ selected from hydrogen and C ₁ -C ₆ An alkyl group;

each R ² Independently selected from C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl, halo and use thereofOxo, wherein the C ₁ -C ₆ R in which the alkyl radical is present from 0 to 1 times ^2a Substitution; or 2R on non-adjacent carbon atoms ² Forms a bridged ring with the non-adjacent carbon atoms to which they are attached;

R ^2a selected from C ₁ -C ₆ Alkoxy and hydroxy;

n is 0, 1, 2, 3 or 4;

m is 0, 1 or 2; and is also provided with

p is 0 or 1.