WO2025176054A1 - Tricyclic heterocyclic compound, preparation method therefor and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a tricyclic heterocyclic compound, a preparation method therefor and a pharmaceutical use thereof. In particular, the present invention relates to a compound represented by general formula (I), a preparation method therefor, a pharmaceutical composition containing the compound, and a use thereof as a PRMT5 inhibitor. The compound and the pharmaceutical composition containing the compound can be used for treating and/or preventing diseases related to PRMT5 activity, such as non-small cell lung cancer, mesothelioma, neurofibrosarcoma, pancreatic cancer, and solid tumor. The definition of each group in the general formula (I) is the same as that in the description.

Description

Tricyclic heterocyclic compounds and their preparation methods and medical uses Technical Field

The present invention belongs to the field of medical technology, and specifically relates to tricyclic heterocyclic compounds, preparation methods thereof, pharmaceutical compositions containing the same, and uses thereof as PRMT5 inhibitors in the treatment and/or prevention of diseases associated with PRMT5 activity.

Background Art

Epigenetic regulation of gene expression is a crucial biological factor in protein production and cell differentiation, playing a significant role in the pathogenesis of many human diseases. Post-translational modifications (PTMs) are key to proteome diversity. Modifications at one or more sites on a protein can determine its conformation, subcellular localization, interactions with other proteins, stability, and activity. These PTMs are mediated by a variety of enzymes, including phosphorylation, acetylation, ubiquitination, methylation, and hydroxylation. They are also negatively regulated by enzymes such as phosphatases, deubiquitinating enzymes, deacetylases, and demethylases. Arginine methylation, among others, plays a crucial role in cellular processes, including cell signaling, gene transcription, RNA processing, and DNA recombination and repair.

Protein arginine methyltransferases (PRMTs) catalyze the methylation of specific arginine residues by transferring a methyl group from S-adenosylmethionine (SAM) to the guanidine nitrogen of arginine. PRMTs can be divided into three categories based on the specific mode of arginine methylation: type I (PRMT1, 2, 3, 4, 6, and 8) catalyze asymmetric dimethylation, type II (PRMT5 and PRMT9) catalyze symmetric dimethylation, and type III (PRMT7) catalyzes monomethylation. PRMT5 plays an essential role in cell growth and development and homeostatic hematopoiesis, participating in the survival and renewal of stem cells in the neural, muscle, hematopoietic, and reproductive systems. PRMT5 knockout in mice is embryonic lethal, while conditional PRMT5 deletion results in the absence of hematopoietic progenitor cells and lethal bone marrow atrophy. Furthermore, PRMT5-deficient hematopoietic stem and progenitor cells exhibit severe cytokine signaling impairment and upregulation of p53. PRMT5 interacts with the cofactor MEP50, enhancing its binding to SAM and substrates, as well as its methylation activity.

The methylation function of PRMT5 is closely related to tumorigenesis and progression through the regulation of gene expression, mRNA splicing, DNA damage response, cytokine signaling pathways, and tumor immunity. Numerous studies have confirmed that PRMT5 is overexpressed in different types and aggressive cancers, such as glioma, leukemia, B-cell and T-cell lymphoma, metastatic melanoma, breast cancer, prostate cancer, and colon cancer.

PRMT5 exerts important biological functions by epigenetically regulating the expression of target genes or directly methylating key signaling molecules through the methylation of various proteins (including histones and non-histones). PRMT5 methylates the terminal arginine residues of histones to activate or repress the expression of related genes. PRMT5 modification of histones often leads to the silencing of tumor suppressor genes such as p53, ST7, NM23, and Rb, thereby promoting the development and progression of tumors.

Overexpression of PRMT5 in tumor cells can promote tumor progression by inhibiting the expression of histone-dependent oncogenic miRNAs. For example, in B-cell lymphoma, PRMT5 inhibits miR-33b/96, which upregulates cyclin D1 and c-Myc; in lung cancer, PRMT5 inhibits miR-99, which upregulates FGFR3; and in AML, PRMT5 inhibits miR-29b, which upregulates FLT3. PRMT5 methylates H4R3 and H3R8, promoting FGFR3 and eIF4E expression in colorectal cancer and AR expression in prostate cancer. PRMT5-methylated H3R2 participates in transcriptional activation, for example, by recruiting WDR5 and MLL coactivators, leading to trimethylation of H3K4, initiating FOXP1 expression to maintain breast cancer stem cell activity, and activating the transcription of redox-related genes. PRMT5 can also activate or repress the expression of related genes in a histone-independent manner. STRAP recruits PRMT5 in response to DNA damage, methylating p53 to alter its nuclear distribution and the expression of its target genes p21 and PUMA. PRMT5 methylation of p53 promotes lymphomagenesis. PRMT5 can also directly methylate E2F-1 and NF-KB/P65, inducing the expression of their target genes.

PRMT5, as part of the spliceosome, is responsible for pre-mRNA splicing in the spliceosome and can affect mRNA splicing, transport, and degradation through methylation. Loss of Myc or PRMT5 leads to aberrant splicing (exon skipping or intron retention) of genes associated with cell cycle arrest or apoptosis. PRMT5 drives Myc-mediated lymphomagenesis by methylating the splicing factor SRSF1. The splicing factor E2F-1 is also an important substrate of PRMT5. PRMT5-mediated splicing regulates TIP60/KAT5 to promote homologous recombination and genomic integrity.

PRMT5 also plays a crucial role in tumor-driven growth factor signaling pathways. In lung and colon cancer, PRMT5 promotes tumor development by transcriptionally activating FGFR genes. PRMT5 can also directly arginine methylate growth factors such as EGFR, PDGFR, and TFG-β. These signaling pathways are crucial for cancer cell proliferation, differentiation, and survival. Furthermore, PRMT5 regulates the assembly of DDR complexes and the expression of related genes through post-transcriptional modifications.

Loss of tumor suppressor genes is a key driver of tumorigenesis. Loss of tumor suppressor genes often leads to co-deletion of genes adjacent to these tumor suppressor genes. Loss of the tumor suppressor gene CDKN2A at chromosome 9p21 occurs in 15% of human tumors and results in co-deletion of MTAP, a key enzyme in the methionine and adenine salvage pathways. Loss of MTAP leads to elevated levels of MTA, which is structurally similar to the methyl donor SAM, a substrate for the type II methyltransferase PRMT5. Elevated MTA competes with SAM for PRMT5 binding, rendering the methyltransferase hypomorphic and susceptible to further inhibition by PRMT5. Multi-gene panel silencing screens across various tumor cell lines have revealed a strong correlation between MTAP loss and PRMT5 dependency. However, PRMT5 is a known essential gene. Conditional knockout of PRMT5 and siRNA interference studies have shown that PRMT5 inhibition in normal tissues may lead to adverse effects such as pancytopenia, infertility, skeletal muscle atrophy, and cardiac hypertrophy. Therefore, new strategies are needed to exploit this metabolic vulnerability and preferentially target PRMT5 in MTAP-deficient tumors while retaining PRMT5 activity in normal tissues. Targeting PRMT5 with MTA-synergistic small molecule inhibitors can preferentially target the MTA-bound state of PRMT5. In MTAP-deficient tumor cells, the MTA-bound state of PRMT5 is more enriched, providing a therapeutic window superior to that of normal cells.

Summary of the Invention

After intensive research, the inventors designed and synthesized a series of tricyclic heterocyclic compounds and screened them for PRMT5 activity. The results showed that these compounds are potent and selective inhibitors of the PRMT5/MTA complex, with selective inhibitory effects on MTAP-deficient cells, and can be developed as drugs for the treatment and/or prevention of diseases associated with PRMT5 activity.

Therefore, the object of the present invention is to provide a compound represented by general formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

in:

Ring A is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl;

Ring E is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl;

X ¹ and X ² are each independently selected from a bond, CH ₂ , O, S and NH;

X ³ and X ⁴ are each independently selected from CH ₂ , O, S and NH;

each R ¹ is independently selected from hydrogen, halogen, hydroxy, amino, cyano, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and -NR ^a R ^b , wherein said alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more R ^1a ;

each R ² is independently selected from hydrogen, halogen, hydroxy, amino, cyano, alkyl, heteroalkyl, haloalkyl, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently substituted with one or more R ^2a ;

each R ^1a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, oxo, cycloalkyl, -O-cycloalkyl, heterocyclyl, -heterocyclylene-alkyl, -O-heterocyclylene-alkyl, -O-alkyl, heteroalkyl, haloalkyl, -O-haloalkyl, -NH-haloalkyl, hydroxyalkyl, alkyl, -Q-aryl, -Q-heteroaryl, 5-20 membered spiroheterocyclyl, -SF ₅ , HC(═O)-, -L-NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -C(═O)NR ^a R ^b , -CH ₂ NHC(═O)O-alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-alkyl, -CH ₂ -heteroaryl, -CH ₂ NHSO _2- alkyl, -CH ₂ OC(═O)-heterocyclyl, -OC(═O)NR ^a R ^b , —OC(═O)-heterocyclyl, and -alkylene-heterocyclyl, wherein the heterocyclyl or heterocyclylene is optionally substituted with one or more oxo or halogen;

each R ^2a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, heterocyclyl, -O-alkyl, heteroalkyl, haloalkyl, -O-haloalkyl, -NH-haloalkyl, hydroxyalkyl, alkyl, -Q-aryl, -Q-heteroaryl, HC(═O)-, -NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -CH ₂ NHC(═O)O-alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-alkyl, -CH ₂ -heteroaryl, -CH ₂ NHSO _2- alkyl, -CH ₂ OC(═O)-heterocyclyl, -OC(═O)NR ^a R ^b , -OC(═O)-heterocyclyl, and -CH ₂ -heterocyclyl, wherein the heterocyclyl of -CH 2 -heterocyclyl is optionally substituted with one or more oxo groups;

L is selected from a bond, -O-, -NH-, and alkylene, wherein the alkylene is optionally substituted with one or more groups selected from hydroxy, hydroxyalkyl, and heteroaryl;

each Q is independently selected from a bond, -O-, and -NH-;

each R ³ is independently selected from hydrogen, halogen, amino, nitro, cyano, oxo, hydroxy, thiol, carboxyl, ester, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

each R ⁴ is independently selected from hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, heteroalkyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, aminoalkyl, cyanoalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

R ^a and R ^b are each independently selected from hydrogen, halogen, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more groups selected from halogen, amino, nitro, cyano, hydroxy, thiol, carboxyl, ester, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or,

^Ra and ^Rb together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group, which is optionally substituted with one or more groups selected from halogen, amino, nitro, cyano, oxo, hydroxy, thiol, carboxyl, ester, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

m ₁ is 0, 1, 2, or 3;

m ₂ is 0, 1, 2, or 3;

m ₃ is 0, 1, 2 or 3;

s is 0, 1, 2, or 3;

t is 0, 1, or 2.

In a preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein ring A is selected from C _5-7 cycloalkyl, 5 to 7 membered heterocyclyl, phenyl or 5 to 6 membered heteroaryl.

In another preferred embodiment, according to the compound represented by general formula (I) of the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein ring A is phenyl or a 5- to 6-membered heteroaryl group.

In another preferred embodiment, according to the compound represented by general formula (I) of the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, ring A is selected from phenyl, pyridyl, furyl, pyrazolyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl and imidazolyl; more preferably phenyl and pyridyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein:

Ring E is selected from C _5-6 cycloalkyl, 5- to 6-membered heterocyclyl, phenyl and 5- to 6-membered heteroaryl, preferably, ring E is a 5- to 6-membered heterocyclyl or a 5- to 6-membered heteroaryl, more preferably, ring E is selected from cyclopentyl, cyclohexyl, dihydrofuranyl and pyrazolyl;

and/or

Each R ⁴ is independently selected from hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _1-6 cyanoalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, C _6-10 aryl and 5 to 10 _heteroaryl .

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein:

Ring E is selected from C ₅ -C ₆ cycloalkyl, 5- to 6-membered heterocyclyl, phenyl and 5- to 6-membered heteroaryl, preferably cyclopentyl, cyclohexyl, dihydrofuranyl and pyrazolyl;

and/or

Each R ⁴ is independently a C _1-6 alkyl group.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: Selected from

in:

X ¹ is selected from CH ₂ , O, S and NH;

X ² is CH ₂ or a bond;

X ³ is selected from CH ₂ , O and NH;

X ⁴ is CH ₂ ;

t is 0, 1, or 2, preferably 1;

R ¹ , R ² , R ³ , m ₁ , m ₂ and m ₃ are as defined in the general formula (I).

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: Selected from

R ¹ , R ² , R ³ , m ₁ , m ₂ and m ₃ are as defined in the general formula (I).

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: Selected from R ^4a and R ^4b are each independently selected from hydrogen, halogen, amino, hydroxyl, thiol, carboxyl, oxo, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C 1-6 haloalkoxy, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 5 to _{6 membered heterocyclyl, C 6-10 aryl} , 5 to 10 membered heteroaryl, preferably hydrogen or C _1-6 alkyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: for R ^4a is C _1-6 alkyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: for R ^4b is hydrogen or C _1-6 alkyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein:

each R ¹ is independently selected from hydrogen, halogen, hydroxy, amino, cyano, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl, 5 to 10 membered heteroaryl, and -NR ^a R ^b , said C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl, C _{1-6 heteroalkyl, C 1-6 alkoxy} , C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl, and 5 to 10 membered heteroaryl being each independently optionally substituted with one or more R ^1a ;

Each R ^1a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, oxo, C _3-6 cycloalkyl, -OC _3-6 cycloalkyl, 4 to 8 membered heterocyclyl, -4 to 8 membered heterocyclylene-C _1-6 alkyl, -O-4 to 8 membered heterocyclylene-C _1-6 alkyl, -OC _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, -OC _1-6 haloalkyl, -NH-C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 alkyl, -Q-phenyl, -Q-5 to 6 membered heteroaryl, HC(=O)-, -L-NR ^a R ^b , -CH ₂ OC(=O)NR ^a R ^b , -CH ₂ NHC(=O)OC _1-6 alkyl, -CH ₂ NHC(=O)NR ^a R ^b , -CH ₂ NHC(=O)-C _{-C 1-6} alkyl, -CH ₂ -5 to 6 membered heteroaryl, -CH ₂ NHSO ₂ -C _1-6 alkyl, -CH ₂ OC(═O)-4 to 8 membered heterocyclyl, -OC(═O)NR ^a R ^b , -C(═O)NR ^a R ^b , -OC(═O)-4 to 8 membered heterocyclyl, and -C _1-6 alkylene-4 to 8 membered heterocyclyl, wherein the heterocyclyl or heterocyclylene is optionally substituted with one or two oxo groups or halogen;

m ₁ is 0 or 1;

L, Q, ^Ra and ^Rb are as defined in the general formula (I).

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein:

Each R ¹ is independently selected from hydrogen, halogen, cyano, C _2-6 alkynyl, -NR ^a R ^b , C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, C _3-10 membered cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and the above cycloalkyl fused to a 5-7 membered heterocyclic ring, such as hexahydrocyclopentylpyrrolyl), 4- to 10-membered heterocyclic group (e.g., tetrahydropyranyl, dihydropyranyl, tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, etc.), 5- to 10-membered aryl (e.g., phenyl, phenyl fused to a 5-7 membered heterocyclic ring, such as tetrahydroisoquinolinyl) and 5- to 10-membered heteroaryl (e.g., pyrazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, furanyl, thienyl, oxazolyl, isoxazolyl, pyrrolyl, imidazolyl, and the above heteroaryl fused to a 5-7 membered heterocyclic ring, such as tetrahydrothienopyridinyl, tetrahydrothiazolopyridinyl, naphthyridinyl), the alkynyl, C _3-10- membered cycloalkyl, 4- to 10-membered heterocyclyl, phenyl, and 5- to 6-membered heteroaryl are each independently optionally substituted with one or more R ^1a ;

each R ^1a is independently selected from hydrogen, halogen, cyano, C _1-6 alkyl, C _1-6 haloalkyl, -OC _1-6 alkyl, C _1-6 hydroxyalkyl, C _3-6 cycloalkyl, -OC _3-6 cycloalkyl, 4 to 8-membered heterocyclyl, -4 to 8-membered heterocyclylene- _{C 1-6} alkyl, -O-4 to 8-membered heterocyclylene-C _1-6 alkyl, C _1-6 heteroalkyl, -L-NR ^a R ^b , -C(═O)NR ^a R ^b , -C _1-6 alkylene-4 to 8-membered heterocyclyl, and -C _1-6 alkylene-halo 4 to 8-membered heterocyclyl;

m ₁ is 1;

L, ^Ra and ^Rb are as defined in the general formula (I).

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is pyrazolyl, which is optionally further substituted by one or two groups selected from halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _3-6 cycloalkyl, 4 to 8 membered heterocyclyl and C _1-6 heteroalkyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is pyrimidinyl, which is optionally further substituted with one or more C _1-6 alkyl or C _1-6 haloalkyl groups.

In another preferred embodiment, according to the compound of general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is pyridyl, which is optionally further substituted by one or more groups selected from halogen, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 heteroalkyl, C _3-6 cycloalkyl, 4-6 membered heterocyclyl, -4-6 membered heterocyclyl-C _1-6 alkyl, -O-4-6 membered heterocyclyl-C _1-6 alkyl, -NR ^a R ^b , -C(═O)R ^a , -C(═O)NR ^a R ^b , -NHC(═O)R ^a , -L-NR ^a R ^b , -C _1-6 alkylene-4 to 8 membered heterocyclyl and -C _1-6 alkylene-halo 4 to 8 membered heterocyclyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein ^R1 is phenyl, which is optionally further substituted by one or more groups selected from halogen, cyano, _C1-6 alkyl, _C1-6 alkoxy, _C1-6 haloalkyl, _C1-6 heteroalkyl, C3-6 cycloalkyl, _4-6 membered heterocyclyl, -C1-6 ^alkylene - _4-6 membered heterocyclyl, ^-NRaRb , -C(=O) ^Ra , ^-C (=O) ^NRaRb , -NHC(=O) ^Ra and -L- ^{NRaRb .}

In another preferred embodiment, according to the compound represented by general formula (I), general formula (II), general formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein ^R1 is thiazolyl, which is optionally further substituted by one or more substituents selected from cyano, _C1-6 alkyl, _C1-6 haloalkyl, _C3-6 cycloalkyl and -C(=O ^{) NRaRb} .

In another preferred embodiment, according to the compound represented by general formula (I), general formula (II), general formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is oxazolyl, which is optionally further substituted with one or more C _1-6 alkyl or C _1-6 haloalkyl groups.

In another preferred embodiment, according to the compound represented by general formula (I), general formula (II), general formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is pyridin-2(1H)-one, which is optionally further substituted by one or more C _1-6 alkyl or C _1-6 haloalkyl groups.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is C _1-6 haloalkyl.

In another preferred embodiment, according to the compound of general formula (I) of the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, R ¹ is C _3-6 cycloalkyl.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is a 4-10 heterocyclic group, preferably a 5-6 heterocyclic group, which is optionally further substituted by one or more C _1-6 alkyl groups or C _1-6 haloalkyl groups.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ¹ is halogen.

In another preferred embodiment, according to the compound represented by general formula (I), general formula (II), general formula (III) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein ^R1 is _C2-6 alkynyl, which is optionally further substituted by one or more groups selected from _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 heteroalkyl, _C3-6 cycloalkyl, 4-6 membered heterocyclyl and ^-L - ^NRaRb . In another preferred embodiment, the compound represented by the general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R ¹ is selected from hexahydrocyclopentylpyrrolyl, tetrahydroisoquinolyl, tetrahydrothienopyridinyl, tetrahydrothiazolopyridinyl and naphthyridinyl, which is optionally further substituted by one or more groups selected from C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 heteroalkyl, C _3-6 cycloalkyl, 4-6 membered heterocyclyl and -L-NR ^a R ^b .

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: Selected from

in:

R ¹ is selected from hydrogen, halogen, cyano, C _2-6 alkenyl, C _2-6 alkynyl, -NR ^a R ^b , C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl, wherein the C _2-6 alkenyl, C _2-6 alkynyl, 4 to 10 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl are optionally substituted with one or more R ^1a , and R ^1a is as defined in the general formula (I);

R ² , R ³ , m ₂ and m ₃ are as defined above.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein: Selected from

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein each R ² is independently selected from hydrogen, halogen, hydroxyl, amino, cyano, C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to ₈ membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl, wherein the C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl are each independently optionally substituted by one or more R ^2a ;

each R ^2a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, 4 to 8 membered heterocyclyl, -OC _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, -OC _1-6 haloalkyl, -NH-C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 alkyl, -Q-phenyl, -Q-5 to 6 membered heteroaryl, HC(═O)-, -NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -CH ₂ NHC(═O)OC _1-6 alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-C _1-6 alkyl, -CH ₂ -5 to 6 membered heteroaryl, -CH ₂ NHSO ₂ -C _1-6 alkyl, -CH ₂ OC(═O)-4 to 8 membered heterocyclyl, -OC(═O)NR ^a R ^b , -OC(=O)-4 to 8-membered heterocyclyl and -CH ₂ -4 to 8-membered heterocyclyl, wherein the heterocyclyl group of the -CH ₂ -4 to 8-membered heterocyclyl is optionally substituted with one or two oxo groups;

m ₂ is 0, 1 or 2, preferably, m ₂ is 0;

Q, ^Ra and ^Rb are as defined in the general formula (I).

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein R ² is hydrogen.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein _m2 is 0.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein m ₁ is 1.

In another preferred embodiment, the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, wherein each R ³ is independently selected from hydrogen, halogen, amino, nitro, cyano, oxo, hydroxyl, thiol, carboxyl, ester, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _1-6 haloalkoxy, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl; preferably R ³ is hydrogen.

Typical compounds of the present invention include, but are not limited to:

Its tautomers, meso racemates, racemates, enantiomers, diastereomers, or mixtures thereof, or its pharmaceutically acceptable salts.

Another aspect of the present invention provides a method for preparing the compound represented by general formula (I) according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, comprising the following steps:

Compound Ic is subjected to a condensation reaction with compound Id in the presence of a condensation reagent and an alkaline reagent to obtain a compound represented by general formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

Wherein, the condensation reagent is preferably N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, and the alkaline reagent is preferably N-methylimidazole;

wherein Ring A, Ring E, X ¹ , X ² , X ³ , X ⁴ , R ¹ , R ² , R ³ , R ⁴ , m ₁ , m ₂ , m ₃ , t and s are as defined in the general formula (I).

Another aspect of the present invention provides a pharmaceutical composition comprising the compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The present invention further provides use of the compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, or a pharmaceutical composition containing the same, in the preparation of a PRMT5 inhibitor.

The present invention further provides the use of the compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or pharmaceutical composition containing the same in the preparation of a drug for preventing and/or treating diseases related to PRMT5 activity.

The present invention further provides the compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or a pharmaceutical composition containing the same, for use as a medicament.

The present invention further provides the compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or pharmaceutical composition containing the same, which is used as a PRMT5 inhibitor.

The present invention further provides a compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, for use in preventing and/or treating diseases associated with PRMT5 activity.

The present invention further provides a method for inhibiting PRMT5, comprising administering to a subject in need thereof an effective amount of a compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same.

The present invention further provides a method for preventing and/or treating diseases associated with PRMT5 activity, comprising administering to a subject in need thereof a preventive or therapeutically effective amount of a compound according to the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same.

In a preferred embodiment of the present invention, the disease associated with PRMT5 activity according to the present invention may be a solid tumor, such as non-small cell lung cancer, mesothelial tumor, neurofibrosarcoma, pancreatic cancer, etc.

The compounds of the present invention can form pharmaceutically acceptable acid addition salts with acids according to conventional methods in the field of the present invention. The acids include inorganic acids and organic acids, with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like being particularly preferred.

The compounds of the present invention can form pharmaceutically acceptable basic addition salts with bases according to conventional methods in the field of the present invention. The bases include inorganic bases and organic bases. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide, and the like.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral administration, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions and may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide a pleasing and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with nontoxic, pharmaceutically acceptable excipients suitable for tablet preparation. These excipients may include inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as microcrystalline cellulose, croscarmellose sodium, corn starch, or alginic acid; binders such as starch, gelatin, polyvinyl pyrrolidone, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. These tablets may be uncoated or may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained release over a longer period of time. For example, water-soluble taste masking substances such as hydroxypropylmethylcellulose or hydroxypropylcellulose, or time-extending substances such as ethylcellulose, cellulose acetate butyrate may be used.

Oral preparations may also be provided in hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or in soft gelatin capsules wherein the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oily vehicle such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active substance in admixture with excipients suitable for the preparation of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, and gum arabic; dispersing or wetting agents, which may be naturally occurring phospholipids, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain fatty alcohols, such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene oxide sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, for example ethylparaben or n-propylparaben, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oil suspensions can be prepared by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or a mineral oil such as liquid paraffin. Oil suspensions can contain thickeners such as beeswax, hard paraffin or cetyl alcohol. The above-mentioned sweeteners and flavoring agents can be added to provide a palatable preparation. These compositions can be preserved by adding antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

Dispersible powders and granules suitable for preparing aqueous suspensions can be provided with the active ingredient and a dispersant or wetting agent, a suspending agent, or one or more preservatives for mixing by the addition of water. Suitable dispersants or wetting agents and suspending agents are as described above. Other excipients such as sweeteners, flavorings, and coloring agents may also be added. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical composition of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin or a mixture thereof. Suitable emulsifiers can be naturally occurring phospholipids, such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of the partial esters and ethylene oxide, such as polyethylene oxide sorbitol monooleate. Emulsions can also contain sweeteners, flavorings, preservatives, and antioxidants. Syrups and elixirs prepared with sweeteners such as glycerol, propylene glycol, sorbitol, or sucrose can be used. Such preparations can also contain demulcents, preservatives, colorants, and antioxidants.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous solutions. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile injectable formulations may be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in an oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream via local, bolus injection. Alternatively, the solution or microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of the invention. To maintain this constant concentration, a continuous intravenous delivery device may be used.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. Such suspensions may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in a nontoxic, parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile fixed oils may conveniently be used as solvents or suspending media. For this purpose, any blended fixed oil, including synthetic mono- or diglycerides, may be used. Furthermore, fatty acids, such as oleic acid, may also be used to prepare injectable formulations.

The compounds of this invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum and thereby dissolves and releases the drug in the rectum. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

It is well known to those skilled in the art that the dosage of a drug depends on a variety of factors, including but not limited to the following: the activity of the specific compound used, the patient's age, the patient's weight, the patient's health condition, the patient's behavior, the patient's diet, the time of administration, the route of administration, the rate of excretion, the combination of drugs, etc. In addition, the optimal treatment method, such as the mode of treatment, the daily dosage of the general formula compound or the type of pharmaceutically acceptable salt can be verified according to traditional treatment protocols.

The present invention may contain a compound represented by general formula (I), and a pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient, mixed with a pharmaceutically acceptable carrier or excipient to prepare a composition, and prepared into a clinically acceptable dosage form. The derivatives of the present invention can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions. The compounds of the present invention can be used as the sole active ingredient or in combination with other drugs for treating diseases associated with PRMT5 activity. Combination therapy is achieved by administering the individual therapeutic components simultaneously, separately or sequentially.

Definition of terms

Unless otherwise stated, the terms used in the specification and claims have the following meanings.

The carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present invention include their isotopes, that is, the carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present invention are optionally further replaced by one or more corresponding isotopes, wherein carbon isotopes include ¹² C, ¹³ C and ¹⁴ C, hydrogen isotopes include protium (H), deuterium (D, also known as heavy hydrogen), tritium (T, also known as super tritium), oxygen isotopes include ¹⁶ O, ¹⁷ O and ¹⁸ O, sulfur isotopes include ³² S, ³³ S, ³⁴ S and ³⁶ S, nitrogen isotopes include ¹⁴ N and ¹⁵ N, fluorine isotopes include ¹⁹ F, chlorine isotopes include ³⁵ Cl and ³⁷ Cl, and bromine isotopes include ⁷⁹ Br and ⁸¹ Br.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight chain or branched chain group containing 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, an alkyl group containing 1 to 4 carbon atoms or an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment and may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

The term "alkylene" refers to a divalent alkyl group, wherein the alkyl group is as defined above and has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, ₁₈ , 19, or 20) carbon atoms (i.e., _C1-20 alkylene). The alkylene group is preferably an alkylene group having 1 to ₁₂ carbon atoms (i.e., _C1-12 alkylene), more preferably an alkylene group having 1 to 6 carbon atoms (i.e., C1-6 alkylene), and further preferably an alkylene group having 1 to 4 carbon atoms (i.e., _C1-6 alkylene). Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH ₂ —), 1,1-ethylene (—CH(CH ₃ )—), 1,2-ethylene (—CH ₂ CH ₂ )—, 1,1-propylene (—CH(CH ₂ CH ₃ )—), 1,2-propylene (—CH ₂ CH(CH ₃ )—), 1,3-propylene (—CH ₂ CH ₂ CH ₂ —), 1,4-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ —), and the like. The alkylene group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment. The substituents may be selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, preferably an alkenyl group having 2 to 6 (e.g., 2, 3, 4, 5, or 6) carbon atoms, more preferably an alkenyl group having 2 to 4 carbon atoms, such as ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc. The alkenyl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond, preferably an alkynyl group having 2 to 6 carbon atoms, more preferably an alkynyl group having 2 to 4 carbon atoms or an alkynyl group having 3 to 4 carbon atoms, such as ethynyl, propynyl, butynyl, etc. The alkynyl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring comprises 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, even more preferably 5 to 7 carbon atoms, 5 to 6 carbon atoms, or 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

The term " spiroalkyl " refers to a polycyclic group that shares a carbon atom (called spiral atom) between the monocycles of 5 to 20 yuan (for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 yuan), which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of shared spiral atoms between the rings, spiroalkyl is divided into single spiroalkyl, double spiroalkyl or multiple spiroalkyl, preferably single spiroalkyl and double spiroalkyl. More preferably, it is 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 yuan/5 yuan or 5 yuan/6 yuan single spiroalkyl. Non-limiting examples of spiroalkyl include:

The term "fused cycloalkyl" refers to a 5 to 20-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered) all-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic, or polycyclic fused cycloalkyl, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic alkyl. Non-limiting examples of fused cycloalkyls include:

The term "bridged cycloalkyl" refers to a 5 to 20-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-membered) all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged cycloalkyl groups include:

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, tetrahydrobenzofuranyl, tetrahydrobenzoxazolyl, tetrahydrobenzisoxazolyl, cyclopentathienyl, tetrahydrobenzothiazolyl, etc. The cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms, wherein one or more ring atoms is a heteroatom selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 4 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 4 to 10 ring atoms, 4 to 8 ring atoms, 5 to 7 ring atoms, 5 to 6 ring atoms, or 7 to 12 ring atoms, of which 1 to 4 are heteroatoms. The limiting examples of monocyclic heterocyclic radical include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl etc., preferably 1,2,5-oxadiazolyl, pyranyl or morpholinyl.Polycyclic heterocyclic radical includes the heterocyclic radical of spirocycle, condensed ring and bridged ring.

The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group having one atom (called a spiral atom) shared between 5 to 20 monocyclic rings (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 members), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 12 members. Spiroheterocyclyl is divided into monospiro heterocyclyl, dispiro heterocyclyl or polyspiro heterocyclyl according to the number of shared spiral atoms between rings, preferably monospiro heterocyclyl and dispiro heterocyclyl. It is more preferably 4/4 members, 4/5 members, 4/6 members, 5/5 members or 5/6 members monospiro heterocyclyl. Non-limiting examples of spiroheterocyclyl include:

The term "fused heterocyclic radical" refers to a polycyclic heterocyclic group having 5 to 20 members (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 members), wherein each ring in the system shares a pair of adjacent atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 12 members. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic radical, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic radical. Non-limiting examples of fused heterocyclic radicals include:

The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 members) in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 12 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include: wait.

The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylate.

The term "heterocyclylene" refers to a divalent functional group derived from a heterocyclyl group, wherein the heterocyclyl group is as defined above.

The term "aryl" refers to a 6- to 14-membered (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered) all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. More preferably, phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, containing 1 to 3 heteroatoms; more preferably 5 or 6-membered, containing 1 to 2 heteroatoms; preferably, for example, imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, thiazolyl, pyrazolyl or pyrimidinyl, thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylate.

The term "heteroalkyl" refers to a straight or branched chain alkyl group containing 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from O, N, Si and S, wherein the _definition of alkyl is as described above, and wherein N and S may be optionally oxidized and _N may be optionally quaternized, preferably a _C1-6 heteroalkyl group. _Non -limiting examples _of heteroalkyl groups include: _CH3OCH2- , _CH3SCH2- , _{CH3NHCH2- , CH3CH2OCH2- , CH3CH2OCH2CH2- , CH3OCH2CH2- , CH3OCH2OCH2- ,} CH3OCH2OCH2- _{, N} ( _{CH3 )} 2CH2CH2OCH2- _{, and the like} .

The term "alkoxy" refers to -O-(alkyl), wherein the definition of alkyl is as described above. The limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents can be one or more following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "cycloalkoxy" refers to an -O-(cycloalkyl) group, wherein cycloalkyl is as defined above.

The term "heterocycloalkoxy" refers to -O-(heterocyclyl), wherein heterocyclyl is as defined above.

The term "cycloalkylthio" refers to -S-(cycloalkyl) where cycloalkyl is as defined above.

The term "heterocycloalkylthio" refers to an -S-(heterocyclyl) group wherein heterocyclyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "hydroxy" refers to an -OH group.

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

The term "amino" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "oxo" refers to =0.

The term "carboxy" refers to -C(O)OH.

The term "mercapto" refers to -SH.

The term "ester group" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

The term "acyl" refers to a compound containing a -C(O)R group, where R is alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and that the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically/pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

"Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are safe and effective when used in mammals and have the desired biological activity.

"Carrier" refers to a vehicle or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

Synthesis method of the compound of the present invention

In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions.

In some embodiments, the compound represented by formula (I) of the present invention or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt can be prepared by the following scheme.

Step 1: Compound Ia and Compound Ib are subjected to a coupling reaction under heating, in the presence of an alkaline reagent and a catalyst to obtain Compound Ic, wherein the heating condition is preferably 100° C., the alkaline reagent is preferably sodium carbonate, and the catalyst is preferably DPPF palladium dichloride catalyst;

Step 2: In the presence of an alkaline reagent, compound Ic is subjected to a condensation reaction with compound Id to obtain a compound represented by formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the condensation reagent is preferably N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, and the alkaline reagent is preferably N-methylimidazole;

wherein Ring A, Ring E, X ¹ , X ² , X ³ , X ⁴ , R ¹ , R ² , R ³ , R ⁴ , m ₁ , m ₂ , m ₃ , t and s are as defined in the general formula (I).

DETAILED DESCRIPTION

The present invention is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts are reported in units of ^10-6 (ppm). NMR measurements were performed using a Bruker dps300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO- _d6 ), deuterated chloroform ( _CDCl3 ), and deuterated methanol ( _CD3OD ), with tetramethylsilane (TMS) as the internal standard.

LC-MS measurements were performed using an 1100 Series LC/MSD Trap (ESI) mass spectrometer (manufacturer: Agilent).

GC-MS determination was performed using GCMS-QP2010 SE.

Preparative liquid chromatography was performed using an LC3000 and LC6000 high-performance liquid chromatographs (manufacturer: Chuangxin Tongheng). The chromatographic columns were Daisogel C18 10 μm 60A (20 mm × 250 mm).

High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20AD high pressure liquid chromatograph (Agilent TC-C18 250×4.6mm 5μm column) and a Shimadzu LC-2010AHT high pressure liquid chromatograph (Phenomenex C18 250×4.6mm 5μm column).

The thin layer chromatography silica gel plate used was Qingdao Ocean Chemical GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15 mm to 0.2 mm, and the specification used for thin layer chromatography separation and purification products was 0.4 mm to 0.5 mm.

Column chromatography generally uses Qingdao marine silica gel 100-200 mesh and 200-300 mesh silica gel as the carrier.

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, Bailingwei, Yishiming, Shanghai Shuya, Yinuokai, Nanjing Yaoshi, Anaiji Chemical and other companies.

Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

Microwave reactions were performed using a CEM Discover SP microwave reactor.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature, particularly 20°C to 30°C.

The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent systems used in the reactions were: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, and D: acetone. The volume ratio of the solvents was adjusted according to the polarity of the compounds.

The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include: A: dichloromethane and methanol system, B: petroleum ether, ethyl acetate and dichloromethane system, C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

Example 1: Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofurano[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofurano[2,3-b][1,4]oxazin-1-yl)methanone (1)

Step 1: Preparation of tert-butyl (((3,6-dibromo-2,3-dihydrobenzofuran-2-yl)oxy)methyl)carbamate (1a)

At -30°C under a nitrogen atmosphere, N-bromosuccinimide (2.92 g, 11.2 mmol), tert-butyl (2-hydroxyethyl)carbamate (1.75 g, 10.9 mmol), and dichloromethane (50.0 mL) were added to a reaction flask. After stirring for 10 minutes, 6-bromobenzofuran (2.14 g, 10.9 mmol) was added dropwise. The reaction was allowed to react at room temperature for 24 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (mobile phase: PE/EA = 20:1-10:1) to obtain 2.35 g of the title compound as a yellow solid in a yield of 55.6%.

LC-MS: m/z 424.3 [M+H] ⁺ .

Step 2: Preparation of tert-butyl 7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazine-1-carboxylate (1b)

Under a nitrogen atmosphere, compound 1a (2.35 g, 5.56 mmol), silver oxide (6.45 g, 27.8 mmol) and ethyl acetate (50.0 mL) were added to a reaction flask, reacted at 50°C for 3 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (mobile phase: PE/EA = 3:1-1:1) to obtain 1.42 g of the title compound as a yellow liquid, in a yield of 71.1%.

LC-MS: m/z 356.1 [M+H] ⁺ .

Step 3: Preparation of 7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazine (1c)

Under nitrogen atmosphere, compound 1b (200 mg, 0.563 mmol) and hydrochloric acid-dioxane solution (4 M, 2 mL) were added to a reaction flask at 0°C and reacted at 40°C for 3 hours. The filtrate was concentrated under reduced pressure to obtain 140 mg of the title compound as a yellow liquid, with a yield of 97.5%.

LC-MS: m/z 255.1 [M+H] ⁺ .

Step 4: Preparation of (4aS,9bR)-7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazine or (4aR,9bS)-7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazine (1c-2)

38g of compound 1c was subjected to chiral separation (chromatographic column model: CHIRALPAK IG 5cm*25cm, 5μm, mobile phase: _CO2 :MeOH (5% 2mM _NH3 -MeOH), 200mL/min) to obtain 14.36g of light yellow solid compound 1c-1 (first peak) and 15.39g of white solid compound 1c-2 (second peak). The structure of the compound 1c-2 is selected from the group consisting of:

Compound 1c-1:

LC-MS: m/z 255.95 [M+H] ⁺ ;

Compound 1c-2:

LC-MS: m/z 255.95 [M+H] ⁺ .

Step 5: Preparation of 5-bromo-1-methyl-1H-pyrazole-4-carbonitrile (1d)

At room temperature, tert-butyl nitrite (6.33 g, 61.4 mmol), acetonitrile (50 mL), and copper bromide (11.0 g, 49.1 mmol) were added to a reaction flask and reacted at 50°C for 1 hour. 5-Amino-1-methyl-1H-pyrazole-4-carbonitrile was dissolved in acetonitrile (50 mL) and added dropwise to the reaction system. The reaction was allowed to proceed at 45°C for 2 hours. The mixture was filtered, added with water (100 mL), extracted with ethyl acetate (100 mL), washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, and concentrated. The concentrate was dissolved in ethyl acetate (5 mL), and petroleum ether (25-30 mL) was added dropwise. The mixture was filtered to obtain a gray solid compound (5 g, yield: 66%).

Step 6: Preparation of tert-butyl (6-chloro-4-(1,3,6,2-dioxazolidin-2-yl)pyridin-3-yl)carbamate

Under nitrogen atmosphere, tert-butyl (6-chloropyridin-3-yl)carbamate (3.00 g, 13.1 mmol), tetrahydrofuran (7 mL), ethylene glycol dimethyl ether (38 mL), lithium chloride (665 mg, 15.7 mmol), 2,2,6,6-tetramethylpiperidine (6.66 g, 47.2 mmol) and methylmagnesium chloride (3.0 M in THF, 47.2 mmol) were added to a reaction flask and reacted at room temperature for 24 h. A solution of triethyl borate (7.27 g, 49.8 mmol) in tetrahydrofuran (9 mL) was added and the reaction was continued for 30 min. The reaction solution was poured into a mixture of saturated aqueous sodium potassium tartrate solution (30 mL) and 2-methyltetrahydrofuran (30 mL), filtered, and the organic phase of the filtrate was separated and washed with water (100 mL). After drying over anhydrous sodium sulfate, the concentrated product was dissolved in 2-methyltetrahydrofuran (30 mL), and a solution of diethanolamine (1.52 g, 1.44 mmol) in isopropanol (15 mL) was added dropwise. Then, n-hexane (30 ml) was added, filtered, and the filter cake was dried to obtain the title compound as a white solid, 4 g, yield: 89%.

LC-MS: m/z 273.2 [M+H] ⁺ .

Step 7: Preparation of 6-chloro-4-(1,3,6,2-dioxazolidin-2-yl)pyridin-3-amine (1f)

Under nitrogen atmosphere, compound 1e (2 g mg, 5.85 mmol), methanol (10 mL), and hydrochloric acid-dioxane solution (4 M, 5 mL) were added to a reaction flask. The reaction was allowed to proceed at room temperature for 30 min. The filtrate was concentrated under reduced pressure to obtain 2.2 g of the crude title compound as a yellow oil.

LC-MS: m/z 173.2 [M+H] ⁺ .

Step 8: Preparation of methyl 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridine-8-carboxylate (1 g)

Under a nitrogen atmosphere, compound 1f (2.61 g, 10.8 mmol), 1.4-dioxane (32 mL), water (8 mL), potassium phosphate (4.58 g, 21.6 mmol), compound 1d (3.01 g, 16.2 mmol), and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (77 mg, 0.11 mmol) were added to a reaction flask and reacted at 90°C for 2 hours. The mixture was filtered, added with water (50 mL), extracted with ethyl acetate (50 mL), washed with saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 1.8 g of a gray solid. The above gray solid, MeOH (30.0 ml), sodium acetate (3.22 g, 39.2 mmol), Pd(dppf) _Cl₂ (1.44 g, 1.96 mmol), and palladium acetate (440 mg, 1.96 mmol) were added to a reaction vessel at room temperature. The reaction was carried out at 150°C under a CO atmosphere at 1.5 MP pressure for 8 h. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (mobile phase: DCM:MeOH = 10:1) to obtain 1.5 g of the title compound as a brown solid in a yield of 54%.

LC-MS: m/z 258.1 [M+H] ⁺ .

Step 9: Preparation of 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridine-8-carboxylic acid (1h)

Compound 1g (1.5g, 5.84mmol), MeOH (10.0ml), water (15mL), and lithium hydroxide monohydrate (979mg, 23.3mmol) were added to a reaction flask at room temperature and reacted at 50°C for 1h. After cooling to room temperature, water (25mL) was added and the pH was adjusted to 4 with 4M hydrochloric acid. The mixture was filtered under reduced pressure, the filter cake was washed with water, and dried to afford 1.37g of the title compound as a white solid (yield: 96%).

LC-MS: m/z 244.3 [M+H] ⁺ .

Step 10: Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (1i)

Under nitrogen atmosphere, compound 1c-2 (513 mg, 2.00 mmol), compound 1h (535 mg, 2.20 mmol), 10 mL of DMF, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (841 mg, 3.00 mmol), and 1-methylimidazole (493 mg, 6.00 mmol) were added to a reaction flask and reacted at room temperature for 2 h. 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was separated and purified by silica gel column chromatography (mobile phase DCM:MeOH = 10:1) to obtain the title compound as a white solid, 511 mg, yield: 53%.

Step 11: Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (1)

Under nitrogen atmosphere, compound 1i (100 mg, 0.208 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (64.7 g, 0.312 mmol), Pd(dppf) _Cl2 (15 mg, 0.021 mmol), potassium carbonate (86 mg, 0.624 mmol), 1,4-dioxane (4 mL), and water (100 mL) were added to a reaction flask and reacted at 100°C for 12 hours. 10 mL of water was added, and the mixture was extracted with EA (10 mL x 3). The mixture was washed with saturated brine (15 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by preparative liquid chromatography (column model: Daisogei 30 mm*250 mm, C18, 10 μm, 100 Å, mobile phase: acetonitrile/water, gradient: 30%-80%) to give the title compound as a white solid powder, 34 mg, yield: 34%.

LC-MS: m/z 483.2 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d6) δ9.07(d,J＝12.1Hz,1H),8.70–8.54(m,2H),8.16(d,J＝9.2Hz,1H),7.88(d,J＝8.3Hz,1H),7.57(d,J＝7.7Hz,1H),7.36 –7.18(m,2H),7.13(s,1H),6.18(q,J＝7.0Hz,1H),5.99(d,J＝7.1Hz,1H),5.84(d,J＝7.2Hz,1H),4.55(d,J＝2.7Hz,3H),3.86(d,J＝5.4Hz,6H).

Example 7: Preparation of (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-(trifluoromethyl)-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-(trifluoromethyl)-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (7)

Step 1: Preparation of 3-cyano-4-oxotetrahydrofuran (7a)

Sodium hydride (5.55 g, 60%, 138 mmol), ultra-dry tetrahydrofuran (250 ml), and methyl 2-hydroxyacetate (25.0 g, 277 mmol) were added to a reaction flask at room temperature. The temperature was raised to 65°C, and acrylonitrile (17.7 g, 333 mmol) was slowly added. After 2 hours, the temperature was lowered to 25°C and quenched with a 2M aqueous sodium hydroxide solution (250 ml). The mixture was extracted with diethyl ether (500 ml). The aqueous phase was retained, the pH adjusted to 2 with 2M hydrochloric acid, and extracted with dichloromethane (250 mL). The mixture was washed with a saturated aqueous sodium chloride solution (250 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (mobile phase: PE:EA = 90:10) to obtain the title compound as a brown solid, 12.4 g, yield: 40%.

Step 2: Preparation of 4-cyano-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (7b)

Compound 7a (967 mg, 8.79 mmol) was dissolved in ultra-dry dichloromethane (20 ml) at room temperature, cooled to -78°C, and N,N-diisopropylethylamine (2.27 g, 17.6 mmol) and trifluoromethanesulfonic anhydride (3.47 g, 12.3 mmol) were added. After stirring for 30 minutes, the mixture was returned to room temperature and water (250 ml) was added. The mixture was extracted with dichloromethane (25 mL), washed with saturated aqueous sodium chloride solution (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Ether was added to the residue, stirred for 10 minutes, and filtered. The mother liquor was concentrated to give the brown title compound, 1.7 g, yield: 79%.

Step 3: Preparation of methyl 4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridine-8-carboxylate (7c)

Under a nitrogen atmosphere, compound 1f (2.41 g, 10.0 mmol), 1.4-dioxane (30 mL), water (7 mL), potassium phosphate (4.23 g, 20.0 mmol), compound 7b (3.65 g, 15.0 mmol), and bistriphenylphosphine palladium dichloride (70.2 mg, 0.11 mmol) were added to a reaction flask and reacted at 90°C for 4 hours. The mixture was filtered, added with water (50 mL), extracted with ethyl acetate (50 mL), washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 1.21 g of a brown solid. At room temperature, the entire gray solid, MeOH (30.0 ml), sodium acetate (3.22 g, 39.2 mmol), Pd(dppf)Cl ₂ (1.44 g, 1.96 mmol), and palladium acetate (440 mg, 1.96 mmol) were added to a reaction kettle. The reaction was carried out at 150° C. under a CO atmosphere at 1.5 MP pressure for 8 h. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (mobile phase DCM:MeOH=10:1) to obtain 830 mg of the title compound as a brown solid. Yield: 34%

Steps 4 to 6: Preparation of (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-(trifluoromethyl)-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-(trifluoromethyl)-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (7)

The title compound was obtained by the same method as that of compound 1, except that compound 7c was used instead of compound 1g in step 4, and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(trifluoromethyl)-1H-pyrazole was used instead of 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole in step 6.

LC-MS: m/z 525.0 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d6) δ9.01(d,J＝4.4Hz,1H),8.87(d,J＝14.3Hz,1H),8.49(s,1H),7.96(d,J＝29.4Hz,1H),7.62(d,J＝7.7Hz,1H),7.44–7 .34(m,2H),7.11(s,2H),6.23–5.97(m,2H),5.41(t,J＝3.6Hz,2H),5.06(t,J＝3.6Hz,2H),4.23(dt,J＝14.1,7.5Hz,1H),4.03–3.60(m,3H).

The preparation methods of Examples 2-6, 8-12 refer to Examples 1 and 7, and their characterization data are shown in the following table:

Example 13: Preparation of (4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (13)

Step 1: Preparation of methylhydrazine (13a)

At 0°C, tert-butyl 1-methylhydrazine-1-carboxylate (14.6 g, 100 mmol) was added to a reaction flask. Trifluoroacetic acid (70 mL) was added dropwise and stirred for 30 minutes. The mixture was then allowed to return to room temperature and stirred for 16 hours. The mixture was concentrated under reduced pressure to afford 13.9 g of the title compound as a clear oil, which was used without further purification.

LCMS: m/z 47.05 [M+H] ⁺ .

Step 2: Preparation of 5-amino-1,3-dimethyl-1H-pyrazole-4-carbonitrile (13b).

Compound 1a (13.9 g, 100 mmol) and ethanol (100 mL) were added to a reaction flask at room temperature. Triethylamine was added to adjust the pH to 8-9, and 2-(1-ethoxyethylethylene)malononitrile (10.2 g, 75.0 mmol) was added. The mixture was heated to 80°C and stirred under a nitrogen atmosphere for 16 hours. The mixture was cooled to 0°C and allowed to stand for 16 hours. The mixture was filtered, and the filter cake was washed with cold ethanol and dried to obtain 1.09 g of the title compound as a brown solid in a yield of 10.6%.

LCMS: m/z 137.07 [M+H] ⁺ .

Step 3: Preparation of 5-bromo-1,3-dimethyl-1H-pyrazole-4-carbonitrile (13c).

At room temperature, compound 13b (1.09 g, 8.00 mmol), acetonitrile (30 mL), and cuprous bromide (1.72 g, 12.0 mmol) were added to a reaction flask. Tert-butyl nitrite (1.64 g, 16.0 mmol) was added dropwise, and the mixture was stirred under a nitrogen atmosphere for 3 hours. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-30%) to afford 575 mg of the title compound as a green solid in a 36.1% yield.

LCMS: m/z 199.97 [M+H] ⁺ .

Step 4: Preparation of 5-(5-amino-2-chloropyridin-4-yl)-1,3-dimethyl-1H-pyrazole-4-carbonitrile (13d).

At room temperature, compound 1f (1.88 g, 4.10 mmol), tetrahydrofuran (20 mL), compound 13c (950 mg, 4.77 mmol), Pd(PPh ₃ ) ₂ Cl ₂ (288 mg, 0.410 mmol), sodium carbonate (1.74 g, 16.4 mmol), ethanol (5 mL), and water (5 mL) were added to a reaction flask and stirred at 80°C under a nitrogen atmosphere for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: methanol/dichloromethane = 0-5%) to obtain 910 mg of the title compound as a brown solid in an 89.9% yield.

LCMS: m/z 248.06 [M+H] ⁺ .

Step 5: Preparation of 8-chloro-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-4-amine (13e).

Compound 13d (885 mg, 3.58 mmol), N,N-dimethylformamide (10 mL), and potassium carbonate (1.48 g, 10.7 mmol) were added to a reaction flask at room temperature and stirred at 80°C under a nitrogen atmosphere for 16 hours. The reaction mixture was extracted with water and ethyl acetate. The organic phase was concentrated to dryness, and the resulting residue was purified by column chromatography (eluent: methanol/dichloromethane = 0-7%) to afford 544 mg of the title compound as a brown solid, in a yield of 61.5%.

LCMS: m/z 248.06 [M+H] ⁺ .

Step 6: Preparation of methyl 4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridine-8-carboxylate (13f).

Compound 13e (494 mg, 2.00 mmol), methanol (10 mL), Pd(OAc) _₂ (45.0 mg, 0.200 mmol), Pd(dppf) _Cl₂ (147 mg, 0.200 mmol), and sodium acetate (328 mg, 4.00 mmol) were added to a reaction flask at room temperature. The mixture was stirred at 150°C under 1.5 MPa of carbon monoxide for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: methanol/dichloromethane = 0-12%) to afford 380 mg of the title compound as a reddish-brown solid in a yield of 70.1%.

LCMS: m/z 272.11 [M+H] ⁺ .

Step 7: Preparation of 4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridine-8-carboxylic acid (13 g).

Compound 13f (352 mg, 1.31 mmol), methanol (10 mL), lithium hydroxide (166 mg, 3.93 mmol), and water (1 mL) were added to a reaction flask at room temperature and stirred at 50°C for 16 hours. The mixture was concentrated under reduced pressure, diluted with water, and the pH was adjusted to 6-7 with 4.0 M dilute hydrochloric acid. A precipitate formed, which was filtered under reduced pressure and dried to afford 505 mg of the title compound as a brown solid, which was used without further purification.

LCMS: m/z 258.09 [M+H] ⁺ .

Step 8: Preparation of (4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)(7-bromo-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (13h).

At room temperature, compound 13g (478 mg, 0.850 mmol), N,N-dimethylacetamide (5 mL), HATU (486 mg, 1.28 mmol), and N,N-diisopropylethylamine (329 mg, 2.55 mmol) were added to a reaction flask and stirred for 5 minutes. Compound 1c-2 (284 mg, 1.11 mmol) was then added and stirred for 16 hours. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was concentrated and the residue was purified by column chromatography (eluent: methanol/dichloromethane = 0-12%) to afford 307 mg of the title compound as a reddish-brown solid in a yield of 73.0%.

Step 9: Preparation of (4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dimethyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(1-methyl-1H-pyrazol-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (13)

Under nitrogen atmosphere, compound 13h (103 mg, 0.208 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (64.7 g, 0.312 mmol), Pd(dppf) _Cl2 (15 mg, 0.021 mmol), potassium carbonate (86 mg, 0.624 mmol), 1,4-dioxane (4 mL), and water (100 mL) were added to a reaction flask and reacted at 100°C for 12 hours. 10 mL of water was added, and the mixture was extracted with EA (10 mL x 3). The mixture was washed with saturated brine (15 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. After the reaction, the reaction solution was separated by preparative liquid chromatography (chromatographic column model: Daisogei 30 mm*250 mm, C18, 10 μm, 100 Å, mobile phase: acetonitrile/water, gradient: 30%-80%) to obtain 23.0 mg of the title compound as a white solid, in a yield of 22.3%.

LCMS: m/z 497.20 [M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.06(d,J＝12.3Hz,1H),8.61(s,1H),8.15(d,J＝8.1Hz,1 H),7.87(d,J＝6.9Hz,1H),7.45(dd,J＝96Hz,J＝7.8Hz,1H),7.24-7.21(m,2H), 6.18(q,J＝6.8Hz,1H),5.92(dd,J＝52.8Hz,J＝7.0Hz,1H),4.46(s,3H),4.28– 4.21(m,1H),3.95–3.88(m,1H),3.86(s,3H),3.83–3.65(m,2H),2.69(s,3H).

Example 15: Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(3,5-difluoropyridin-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(3,5-difluoropyridin-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (15)

Step 1: ((4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or ( Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (15a)

Under a nitrogen atmosphere, compound 1i (409 mg, 0.85 mmol), dioxane (2 ml), K ₃ PO ₄ (543 mg, 2.4 mmol), and Pd(dppf)Cl ₂ (20 mg, 0.085 mmol) were added to a reaction flask and reacted at 80°C for 1 hour. The mixture was returned to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 10:1) to obtain 398 mg of the title compound as a white solid, in a yield of 88.6%.

LCMS: m/z 529.22 [M+H] ⁺ .

Step 2: Preparation of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-(3,5-difluoropyridin-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-(3,5-difluoropyridin-4-yl)-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (15)

Under nitrogen atmosphere, compound 15a (120 mg, 0.23 mmol), 3,5-difluoro-4-iodopyridine (110 mg, 0.45 mmol), 1,4-dioxane (2 mL), and water (400 μL) were added to a reaction flask. Potassium carbonate (94 mg, 0.68 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (19 mg, 0.02 mmol) were then added, and the mixture was reacted at 80°C for 1.5 h. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by preparative liquid chromatography (Daisogei 30 mm x 250 mm, C18, 10 μm, 100 Å column, mobile phase: acetonitrile/water, gradient: 30%-80%) to give 43 mg of the title compound as a white solid.

LCMS: m/z 516.0 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d6) δ8.84(s,1H),8.48(s,4H),8.28(s,1H),7.65(d,J＝99.9Hz,2H),7.14(dd,J＝7.7,1.5Hz,1 H), 7.03 (s, 1H), 6.27 (d, J = 7.0Hz, 1H), 6.04 (d, J = 54.2Hz, 2H), 4.44 (s, 3H), 3.95 (s, 1H), 3.81 (d, J = 49.9Hz, 3H).

Example 27: Preparation of (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (27)

Step 1: Preparation of (4aS,9bR)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofurano[2,3-b][1,4]oxazine or (4aR,9bS)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofurano[2,3-b][1,4]oxazine (27a).

Compound 1c-2 (306 mg, 1.20 mmol), boron trifluoride-ether complex (6 mL), and N-chlorosuccinimide (193 mg, 1.44 mmol) were added to a reaction flask at room temperature and stirred under a nitrogen atmosphere for 16 hours. Saturated ammonium chloride solution was added to the reaction mixture at 0°C, and the mixture was extracted with ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: methanol/dichloromethane = 0-10%) to afford 279 mg of the title compound as a yellow oil in an 80.4% yield.

LCMS: m/z 289.95 [M+H] ⁺ .

Step 2: Preparation of (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aS,9bR)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone or (4-amino-1,3-dihydrofuro[3,4-c][1,7]naphthyridin-8-yl)((4aR,9bS)-7-bromo-8-chloro-2,3,4a,9b-tetrahydro-1H-benzofuro[2,3-b][1,4]oxazin-1-yl)methanone (27).

At room temperature, compound 7d (88.8 mg, 0.384 mmol), N,N-dimethylacetamide (1 mL), HATU (183 mg, 0.480 mmol), and N,N-diisopropylethylamine (124 mg, 0.960 mmol) were added to a reaction flask and stirred for 5 minutes. Compound 27a (92.5 mg, 0.320 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 16 hours. The reaction solution was concentrated under reduced pressure and separated by preparative liquid chromatography (Daisogei 30 mm x 250 mm, C18, 10 μm, 100 Å column, mobile phase: acetonitrile/water, gradient: 30% - 80%) to afford 5.0 mg of the title compound as a brown solid, in a yield of 2.6%.

LCMS: m/z 503.00 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO) δ8.94(d,J＝20.8Hz,1H),8.06(d,J＝11.9Hz,1H),7.83(s,1H),7.48(d,J＝4.7Hz,1H),7.43(s,1H),6.18(dd,J＝31.2,7.2H z,1H),5.99(dd,J=28.1,7.0Hz,1H),5.44(s,2H),5.08(s,2H),4.33–4.12(m,1H),4.02–3.84(m,2H),3.40–3.21(m,1H),3.15–2.96(m,1H).

The preparation methods of Examples 14, 16-26, and 28 refer to Examples 13, 15, and 27, and their characterization data are shown in the following table:

Intermediate 1: Preparation of 1-methyl-3-cyclopropyl-5-iodo-1H-pyrazole (INT1)

Step 1: Preparation of 3-cyclopropyl-5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (INT1a).

3-Cyclopropyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (960 mg, 5.0 mmol) and anhydrous tetrahydrofuran (10 mL) were added to a reaction flask. LDA was added dropwise in portions at -75°C under a nitrogen atmosphere and allowed to react for 1 h. Elemental iodine (1.5 g, 6.0 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise in portions at -75°C to the reaction mixture. The reaction mixture was poured into 100 mL of water and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0%-10%) to obtain 550 mg of the title compound as a yellow liquid.

Step 2: Preparation of 3-cyclopropyl-5-iodo-1H-pyrazole (INT1b).

Compound INT1a (550 mg, 1.7 mmol), dichloromethane (3 mL), and trifluoroacetic acid (1 mL) were added to a reaction flask at room temperature and reacted under a nitrogen atmosphere for 1 h. The reaction solution was concentrated under reduced pressure, and the resulting crude product was used directly in the next reaction.

LCMS: m/z 234.9 [M+H] ⁺ .

Step 3: Preparation of 1-methyl-3-cyclopropyl-5-iodo-1H-pyrazole (INT1).

Compound INT1b (398 mg, 1.7 mmol), DMF (5 mL), iodomethane (290 mg, 2.0 mmol), and cesium carbonate (1.66 g, 5.1 mmol) were added to a reaction flask and reacted at 80°C under a nitrogen atmosphere for 1 h. The reaction solution was poured into 100 mL of water and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 5%-10%) to obtain 90 mg of the title compound as a white solid.

LCMS: m/z 248.8 [M+H] ⁺ .

Intermediate 2: Preparation of 1-methyl-3-isopropyl-5-iodo-1H-pyrazole (INT2)

The title compound was obtained by the same method as Intermediate 1, except that 3-isopropyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole was used instead of 3-cyclopropyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole in step 1.

LC-MS: m/z 250.8 [M+H] ⁺ .

Intermediate 3: Preparation of 3-bromo-5-fluoro-1-methyl-1H-pyrazole (INT3)

3-Bromo-1-methyl-1H-pyrazole (1 g, 6.2 mmol) and anhydrous tetrahydrofuran (10 mL) were added to a reaction flask and cooled to -65°C under a nitrogen atmosphere. LDA (2 M, 9.3 mL) was then added dropwise in portions and allowed to react for 1 hour. N-Fluoro-N-(phenylsulfonyl)benzenesulfonamide (2.3 g, 7.5 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the reaction mixture. The mixture was allowed to react at -65°C for 2 hours, then allowed to return to room temperature and react overnight. The reaction mixture was poured into 50 mL of water and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 10%-20%) to yield 550 mg of the title compound as yellow crystals.

LCMS: m/z 190.0 [M+H] ⁺ .

Intermediate 4: Preparation of 4-bromo-3-(difluoromethyl)-1-(2-methoxyethyl)-1H-pyrazole (INT4)

Step 1: Preparation of 1-(4-methoxybenzyl)-1H-pyrazole-3-carbaldehyde (INT4a).

1H-pyrazole-3-carboxaldehyde (3.00 g, 31.2 mmol), 1-(chloromethyl)-4-methoxybenzene (4.87 mg, 31.2 mmol), cesium carbonate (15.2 g, 46.8 mmol), and N,N-dimethylformamide (30 mL) were added to a reaction flask at room temperature under a nitrogen atmosphere and allowed to react for 2 hours. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 5/1) to afford 3.50 g of the title compound as a yellow oil in a 51.9% yield.

LC-MS: m/z 216.2 [M+H] ⁺ .

Step 2: Preparation of 3-(difluoromethyl)-1-(4-methoxybenzyl)-1H-pyrazole (INT4b).

Compound INT4a (3.00 g, 13.9 mmol), diethylaminosulfur trifluoride (4.47 g, 27.8 mmol), and dichloromethane (30 mL) were added to a reaction flask at 0°C and reacted under a nitrogen atmosphere for 2 hours at room temperature. The reaction solution was poured into water (100 mL) and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 3/1) to obtain the title compound 2.10 as a yellow oil in a yield of 63.6%.

LC-MS: m/z 239.2 [M+H] ⁺ .

Step 3: Preparation of 3-(difluoromethyl)-1H-pyrazole (INT4c).

Compound INT4b (2.10 g, 8.82 mmol) and trifluoroacetic acid (20 mL) were added to a reaction flask at room temperature and reacted at 70°C under a nitrogen atmosphere for 2 hours. The reaction mixture was poured into water (100 mL) and the pH was adjusted to 7 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 3/1) to obtain 700 mg of the title compound as a yellow oil. Yield: 67.3%.

LC-MS: m/z 119.2 [M+H] ⁺ .

Step 4: Preparation of 4-bromo-3-(difluoromethyl)-1H-pyrazole (INT4d).

Compound INT4c (700 mg, 5.93 mmol), N-bromosuccinimide (1.05 g, 5.93 mmol), and acetonitrile (10 mL) were added to a reaction flask at room temperature under a nitrogen atmosphere and allowed to react for 2 hours. The reaction mixture was poured into water (100 mL) and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 3/1) to afford 530 mg of the title compound as a yellow oil. Yield: 45.6%.

LC-MS: m/z 196.2 [M+H] ⁺ .

Step 5: Preparation of 4-bromo-3-(difluoromethyl)-1-(2-methoxyethyl)-1H-pyrazole (INT4).

Compound INT4d (530 mg, 2.70 mmol), 1-bromo-2-methoxyethane (745 mg, 5.40 mmol), and N,N-dimethylformamide (10 mL) were added to a reaction flask at room temperature under a nitrogen atmosphere and allowed to react for 2 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 5/1) to afford 460 mg of the title compound as a yellow oil in a 67.3% yield.

LC-MS: m/z 254.2 [M+H] ⁺ .

Intermediate 5: Preparation of 5-iodo-1-ethyl-3-(methoxymethyl)-1H-pyrazole (INT5)

Step 1: Preparation of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-acetaldehyde (INT5a).

1H-pyrazole-3-acetaldehyde (5.29 g, 55.0 mmol) and tetrahydrofuran (80 mL) were added to a reaction flask, cooled to below 0°C, and 60% NaH (3.30 g, 82.5 mmol) was added under a nitrogen atmosphere. The mixture was stirred for 0.5 hours, followed by the addition of (2-(chloromethoxy)ethyl)trimethylsilane (9.65 g, 57.8 mmol). The mixture was allowed to return to room temperature under a nitrogen atmosphere and stirred for 2 hours. Water (200 mL) was added to the reaction mixture at 0°C, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-15%) to afford 10.5 g of the title compound as a clear oil in an 84.4% yield.

LCMS: m/z 227.11 [M+H] ⁺ .

Step 2: Preparation of (1-(2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol (INT5b).

Compound INT5a (10.4 g, 46.0 mmol) and ethanol (90 mL) were added to a reaction flask. Sodium borohydride (2.10 g, 55.2 mmol) was added at 0°C and stirred for 16 hours under a nitrogen atmosphere. Saturated ammonium chloride solution (10 mL) was added to the reaction mixture at 0°C to quench the mixture. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-30%) to obtain 9.23 g of the title compound as a clear oil in an 88.0% yield.

LCMS: m/z 229.13 [M+H] ⁺ .

Step 3: Preparation of 3-(methoxymethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (INT5c).

Compound INT5b (9.11 g, 40.0 mmol) and tetrahydrofuran (100 mL) were added to a reaction flask. 60% NaH (4.00 g, 100 mmol) was added at 0°C and stirred for 0.5 hours. Methyl iodide (8.52 g, 60.0 mmol) was then added and the mixture was returned to room temperature under a nitrogen atmosphere and stirred for 3 hours. Water (150 mL) was added dropwise to the reaction solution at 0°C to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain 10.7 g of the title compound as a clear oil, which was used without further purification.

LCMS: m/z 243.15 [M+H] ⁺ .

Step 4: Preparation of 5-iodo-3-(methoxymethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (INT5d).

Compound INT5c (5.10 g, 17.0 mmol) and tetrahydrofuran (70 mL) were added to a reaction flask, cooled to below -70°C, and n-butyllithium (2.5 M, 7.5 mL, 18.7 mmol) was added. The mixture was stirred at this temperature for 1 hour, followed by the addition of a solution of iodine (4.75 g, 18.7 mmol) in tetrahydrofuran (20 mL), and stirred for 2 hours. The reaction mixture was quenched with water at 0°C and concentrated under reduced pressure to remove the tetrahydrofuran. Ethyl acetate was added to dissolve the mixture, and the mixture was washed sequentially with 10% sodium bicarbonate solution and 10% sodium bisulfite solution. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-20%) to obtain 5.60 g of the title compound as a clear oil in an 89.5% yield.

LCMS: m/z 369.04 [M+H] ⁺ .

Step 5: Preparation of 5-iodo-3-(methoxymethyl)-1H-pyrazole (INT5e).

At room temperature, compound INT5d (5.41 g, 14.7 mmol) and dichloromethane (25 mL) were added to a reaction flask, and trifluoroacetic acid (33.6 g, 294 mmol) was added dropwise. The mixture was stirred under a nitrogen atmosphere for 16 hours. The mixture was concentrated under reduced pressure, and ethanol (50 mL) and sodium acetate (6.04 g, 73.5 mmol) were added. The mixture was stirred for 4 hours, filtered, and the filtrate was collected and concentrated. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-40%) to obtain 4.15 g of the title compound as a clear oil, which was used without further purification.

LCMS: m/z 238.96 [M+H] ⁺ .

Step 6: Preparation of 5-iodo-1-ethyl-3-(methoxymethyl)-1H-pyrazole (INT5).

Compound INT5e (595 mg, 2.50 mmol), DMF (7 mL), iodoethane (468 mg, 3.00 mmol), and cesium carbonate (1.23 g, 3.75 mmol) were added to a reaction flask at room temperature and stirred at 100°C under nitrogen for 16 hours. The mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-20%) to obtain 490 mg of the title compound as a clear oil in a yield of 73.6%.

LCMS: m/z 266.99 [M+H] ⁺ .

Intermediate 6: Preparation of 2-bromo-5-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (INT6)

Step 1: Preparation of 2-bromo-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (INT6a).

At 0°C, 4,5,6,7-tetrahydrothieno[3,2-c]pyridine (1 g, 5.69 mmol) and tetrahydrofuran (15 mL) were added to a reaction flask. After stirring for 5 minutes, NBS (1.1 g 6.26 mmol) was slowly added. After stirring at room temperature for 1 hour, the residue was separated and purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 1/3) to obtain 600 mg of the target compound as a yellow oil, yield: 50.1%.

LC-MS: m/z 218 [M+H] ⁺ .

Step 2: Preparation of 2-bromo-5-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (INT6).

Compound INT6a (600 mg, 2.76 mmol), methanol (10 mL), and paraformaldehyde (1 g, 27.6 mmol) were added to a reaction flask at room temperature. After stirring for 40 minutes, sodium cyanoborohydride (664 mg, 8.28 mmol) was slowly added and stirred for 2 hours. The reaction was quenched by adding water (100 mL) and extracted with EA. The combined organic phases were concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 1/2) to obtain 400 mg of the target compound as a yellow oil in a yield of 62.7%.

LC-MS: m/z 232 [M+H] ⁺ .

Intermediate 7: Preparation of 2-(2-methoxyethyl)-1,2,3,3a,4,6a-hexahydrocyclopenta[c]pyrrol-5-yl trifluoromethanesulfonate (INT7)

Step 1: Preparation of tert-butyl 5-(((trifluoromethyl)sulfonyl)oxy)-3,3a,4,6a-tetrahydrocyclopentyl[c]pyrrole-2(1H)-carboxylate (INT7a).

Under a nitrogen atmosphere, tert-butyl 5-oxohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (2.3 g, 10 mmol) and 30 mL of anhydrous tetrahydrofuran were added to a reaction flask. The temperature was cooled to -75°C, and LDA (10 mL, 20.0 mmol) was added dropwise in portions. The mixture was stirred for 1 hour. 1,1,1-Trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (4.3 g, 12.0 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran and added dropwise to the reaction mixture. The mixture was allowed to return to room temperature and react for 16 hours. The reaction mixture was poured into 300 mL of saturated _NaHCO₃ solution and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was used directly in the next reaction.

Step 2: Preparation of 1,2,3,3a,4,6a-hexahydrocyclopenta[c]pyrrol-5-yl trifluoromethanesulfonate (INT7b).

Compound INT7a (714 mg, 2.0 mmol), dichloromethane (3 mL), and a 4 M dioxane hydrochloride solution (3 mL) were added to a reaction flask at room temperature and stirred under a nitrogen atmosphere for 1 h. The reaction solution was concentrated, and the resulting crude product was used directly in the next reaction.

LCMS: m/z 258.1 [M+H] ⁺ .

Step 3: Preparation of 2-(2-methoxyethyl)-1,2,3,3a,4,6a-hexahydrocyclopenta[c]pyrrol-5-yl trifluoromethanesulfonate (INT7).

Compound INT7b (257 mg, 1.0 mmol), 1-bromo-2-methoxyethane (209 mg, 1.5 mmol), anhydrous ethanol (3 mL), and potassium carbonate (414 mg, 3.0 mmol) were added to a reaction flask and reacted at 50°C overnight under a nitrogen atmosphere. The reaction solution was poured into 100 mL of water and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 5%-20%) to obtain 210 mg of the title compound as a brown liquid.

Intermediate 8: Preparation of 3-bromo-5,6,7,8-tetrahydro-1,6-naphthyridine (INT8)

Step 1: Preparation of tert-butyl-3-nitro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (INT8a).

At room temperature, 1-methyl-3,5-dinitropyridin-2(1H)-one (8.01 g, 40.2 mmol), a 7.0 M methanolic ammonia solution (81 mL, 563 mmol), and tert-butyl-4-oxopiperidine-1-carboxylate (8.80 g, 44.3 mmol) were added to a reaction flask and heated to 120°C with stirring for 1 hour. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (eluent: ethyl acetate/dichloromethane = 0-20%) to afford 10.1 g of the title compound as a light yellow oil in an 89.5% yield.

LCMS: m/z 280.12 [M+H] ⁺ .

Step 2: Preparation of tert-butyl-3-amino-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (INT8b).

At room temperature, compound INT8a (8.92 g, 32.0 mmol), DMF (100 mL), and 4,4'-bipyridine (375 mg, 2.40 mmol) were added to a reaction flask. The temperature was cooled below 0°C, and tetrahydroxydiboron (8.62 g, 96.0 mmol) was added portionwise. The mixture was stirred for 5 minutes. The mixture was returned to room temperature, diluted with water, and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to obtain 11.0 g of the title compound as a yellow oil, which was used without further purification.

LCMS: m/z 250.15 [M+H] ⁺ .

Step 3: Preparation of tert-butyl-3-bromo-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (INT8c).

At room temperature, compound INT8b (11.0 g, 28.2 mmol), acetonitrile (140 mL), and copper bromide (9.48 g, 42.3 mmol) were added to a reaction flask. The temperature was cooled to below 0°C, and tert-butyl nitrite (3.52 g, 33.9 mmol) was added dropwise. The mixture was stirred for 1 hour, then returned to room temperature and stirred for 16 hours. The mixture was concentrated under reduced pressure, diluted with water, filtered, and the filtrate was extracted with ethyl acetate. The organic phases were combined and concentrated, and the resulting residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 0-30%) to obtain 8.30 g of the title compound as a yellow oil, which was used without further purification.

LCMS: m/z 313.05 [M+H] ⁺ .

Step 4: Preparation of 3-bromo-5,6,7,8-tetrahydro-1,6-naphthyridine (INT8).

Compound INT8c (2.50 g, 8.00 mmol), ethyl acetate (10 mL), and a solution of hydrogen chloride in 1,4-dioxane (4.0 M, 10 mL, 40.0 mmol) were added to a reaction flask at room temperature and stirred under a nitrogen atmosphere for 16 hours. The mixture was concentrated under reduced pressure, dissolved in dichloromethane, and the pH was adjusted to 9-10 with aqueous potassium carbonate. The aqueous phase was extracted three times with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated to afford 1.65 g of the title compound as a yellow solid in a 97.2% yield.

LCMS: m/z 212.99 [M+H] ⁺ .

Intermediate 9: Preparation of 3-bromo-2-fluoro-5-(piperidin-1-ylmethyl)pyridine (INT9)

Step 1: Preparation of methyl 5-bromo-6-fluoronicotinate (INT9a).

Methyl 5-bromo-6-chloronicotinate (10.0 g, 40.0 mmol), acetonitrile (200 mL), potassium fluoride (7.0 g, 120 mmol), and tetraphenylphosphonium bromide (8.4 g, 20 mmol) were added to a reaction flask and reacted at 80°C under a nitrogen atmosphere for 4 days. The reaction solution was poured into 200 mL of ethyl acetate, filtered, and the filtrate was concentrated. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 30%-50%) to obtain 8.7 g of the title compound as a pale white solid.

LCMS: m/z 233.8 [M+H] ⁺ .

Step 2: Preparation of (5-bromo-6-fluoropyridin-3-yl)methanol (INT9b).

Compound INT9a (8.7 g, 37.5 mmol) and tetrahydrofuran (90 mL) were added to a reaction flask, cooled to 0°C, and lithium aluminum hydride (712 mg, 18.7 mmol) was added portionwise. The resulting mixture was reacted at room temperature under a nitrogen atmosphere for 16 h. 20 mL of water and 20 mL of 15% NaOH solution were added to the reaction solution, stirred for 15 min, and finally an appropriate amount of _MgSO₄ was added. The reaction mixture was filtered, and the filtrate was extracted with EA. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (eluent: methanol/dichloromethane = 3%-10%) to obtain 3.1 g of the title compound as a yellow liquid.

LCMS: m/z 205.9 [M+H] ⁺ .

Step 3: Preparation of 3-bromo-5-(chloromethyl)-2-fluoropyridine (INT9c).

Compound INT9b (410 mg, 2.0 mmol) and dichloromethane (5 mL) were added to a reaction flask at room temperature, followed by the dropwise addition of thionyl chloride (286 mg, 2.4 mmol). The reaction was allowed to proceed for 2 h under a nitrogen atmosphere. The reaction solution was poured into 50 mL of saturated NaHCO₃ _solution and extracted with EA. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether = 10%-20%) to afford 273 mg of the title compound as a colorless liquid.

LCMS: m/z 223.9 [M+H] ⁺ .

Step 4: Preparation of 3-bromo-2-fluoro-5-(piperidin-1-ylmethyl)pyridine (INT9).

Compound INT9c (223 mg, 1.0 mmol), piperidine (128 mg, 1.5 mmol), DMF (2 mL), and cesium carbonate (815 mg, 2.5 mmol) were added to a reaction flask at room temperature and stirred under a nitrogen atmosphere for 2 h. The reaction solution was poured into 50 mL of water and extracted with EA. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (eluent: methanol/dichloromethane = 0%-10%) to obtain 310 mg of the title compound as a colorless liquid.

LCMS: m/z 272.9 [M+H] ⁺ .

The preparation methods of Examples 29-127 refer to Example 15, except that relevant halogenated heteroaryl compounds are used to replace 3,5-difluoro-4-iodopyridine. The characterization data are shown in the following table:

Biological evaluation

Test Example 1: Inhibitory effect of the compounds of the present invention on PRMT5-MTA binding

Reagent preparation:

The biochemical activity of the compounds was evaluated by measuring their inhibitory effects on PRMT5-MTA binding. A PRMT5 (BPS, Cat#51045)-2.6 μM MTA (Sigma, Cat#D5011) solution was prepared in assay buffer. The reference compound MRTX9768 (MCE, Cat#HY-138684) was diluted in DMSO at concentrations of 1000 μM, 8 μM, 64 nM, and 0.51 nM. The test compounds were diluted in DMSO starting at 9.901 μM and then diluted 5-fold over a 10-step concentration gradient. Using an ECHO pipette (Labcyte), 0.1 μL of compound was transferred to a 384-well plate (Corning, 3657). 5 μL of the PRMT5-MTA mixture was added and the plate was centrifuged at 1000 rpm for 30 seconds. The plate was incubated at 25°C for 30 minutes. SAM solution was prepared in assay buffer, 5 μL of SAM solution was added to the assay plate, and the plate was centrifuged at 1000 rpm for 30 seconds. The assay plate was then incubated at 25°C for 90 minutes. ULight-streptavidin detection solution was prepared in 1x LANCE buffer (PE, Cat#CR97-100). After 90 minutes, 10 μL of detection solution (ULight-streptavidin/biotin-labeled peptide (peptide-biotin) 1:6) was added to the assay plate. After 60 minutes, fluorescence data was read at a wavelength of 665 nm (excitation light was 320 or 340 nm) using an Envision Multilabel Analyzer (2104).

Raw data were converted to inhibition percentage using the equation: (1 - (sample - SignalAve_PC) / (SignalAve_VC - SignalAve_PC)) × 100, where SignalAve_PC is the mean fluorescence intensity of the positive control and SignalAve_VC is the mean fluorescence intensity of the negative control. _IC50 values were calculated using a four-parameter curve fit (using the "log(inhibitor) vs. response - variable slope" mode in GraphPad Prism 8).

Table 1 provides the in vitro enzymatic inhibitory activity ( _IC50 ) of the compounds of the present application against PRMT5+2.6μM MTA. In the table, A means _IC50 <100nM; B means _100nM≤IC50 <1μM; and C means _IC50 ≥1μM.

Table 1 Enzymatic activity of the compounds of the present invention on PRMT5 in the presence of MTA

Conclusion: The above data results show that the compounds of the present invention have a significant inhibitory effect on the PRMT5/MTA complex.

Experimental Example 2: Inhibitory effect of the compounds of the present invention on proliferation of wild-type HCT-116 and MTAP knockout HCT-116 cells

The cell activity and selectivity of the compounds were evaluated by detecting the inhibitory effects of the compounds on the proliferation of wild-type and MTAP gene knockout HCT-116 cells.

On day 0, 100 μL of cell suspension containing 500 HCT-116 parental cells (Horizon, HD PAR-034) or HCT-116 MTAP knockout cells (Horizon, HD R02-033) was added to each well of a white 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator.

On day 1, dilute the test compound starting at 10,000 μM. For paclitaxel (Taxol Injection, Shuanglu Pharmaceutical) starting at 1,000 μM, perform a three-fold serial dilution in 100% DMSO, with 9+0 concentrations, in duplicate. Add culture medium to the middle plate, then transfer the serially diluted compound to each well according to the corresponding position. After mixing, transfer 50 μL of the compound to each well of the cell plate. Incubate the cell plate in a 37°C, 5% CO2 incubator for 5 days.

On day 6, cells were digested by adding 75 μL of trypsin-EDTA solution (Invitrogen, 25200056) to each well. The 96-well cell plate was placed in a 37°C, 5% CO2 incubator for 3 minutes. Digestion was terminated by adding 125 μL of culture medium to each well. 135 μL of fresh culture medium was added to a new 96-well plate, and 10 μL of the cell suspension was transferred to a new 96-well plate (1:20 dilution). After adding the same concentration of compound as on day 1, the 96-well cell plate was placed in a 37°C, 5% CO2 incubator for 5 days.

On day 11, Promega CellTiter-Glo assay was performed. The 96-well plate was removed and equilibrated at room temperature for 20 minutes. 40 μL CTG (Promega, G7572) was added to each well and the plate was shaken to mix. The plate was incubated at room temperature for 30 minutes and the fluorescence signal was read using Envision multi-label analyzer.

Raw data were converted to inhibition percentage using the equation: 100 - (sample - SignalAve_PC) / (SignalAve_VC - SignalAve_PC) × 100, where SignalAve_PC is the mean fluorescence intensity of the positive control (blank) and SignalAve_VC is the mean fluorescence intensity of the negative control (0.1% DMSO). _IC50 values were calculated using a four-parameter curve fit ("log(inhibitor) vs. response - variable slope" mode in GraphPad Prism 8).

Table 2 provides the inhibitory activity of the compounds of the present application on the proliferation of HCT-116 parental cells and HCT-116MTAP knockout cells. In the table, a and aa refer to the inhibitory activity of the compounds on the proliferation of HCT-116 parental cells and HCT-116MTAP knockout cells, respectively, with _IC50 <100nM; b and bb refer to _100nM≤IC50 <1000nM; c and cc refer to _IC50 ≥1000nM.

Table 2

Conclusion: The above data show that the compounds of the present invention have significant proliferation inhibitory activity on HCT-116MTAP knockout cells, and the inhibitory activity on HCT-116MTAP knockout cells is higher than that on HCT-116 parental cells.

Experimental Example 3: Pharmacokinetic evaluation of the compound of the present invention in ICR mice

Male 7-8-week-old ICR mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were orally administered with the compounds of the present invention in a solvent containing 0.5% methylcellulose and 0.2% Tween 80 at a dosing volume of 10 mL/kg. Blood was collected from the canthal venous plexus of the mice before and at 0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, and 24.00 hours after dosing. Blood was anticoagulated with sodium heparin and centrifuged at 3500 rpm for 10 minutes at 4°C. Plasma was obtained and stored at -20°C until testing. 10 μL of the plasma sample to be tested was placed in a 96-well plate, and 100 μL of acetonitrile working solution containing 5 ng/mL verapamil hydrochloride (internal standard) (100223-200102, China National Institute for the Control of Pharmaceutical and Biological Products) was added. The mixture was thoroughly mixed by vortexing for 5 minutes and then centrifuged at 4000 rpm for 10 minutes. Pipette 50 μL of supernatant, add 150 μL of acetonitrile and mix well, centrifuge at 4000 rpm for 10 min, take the supernatant, place it in a 96-well sample plate, and analyze the hemorrhagic drug concentration by LC/MS (Waters UPLC I Class/LC 30AD, Waters). Use MassLynx V4.2 SCN977 data processing software to analyze the pharmacokinetic parameters. The main pharmacokinetic parameters of the compounds of the present invention are shown in Table 3 below.

Table 3 Pharmacokinetic parameters of the compounds of the present invention after single oral administration to male ICR mice

Conclusion: The above data show that the compound of the present invention has a higher oral exposure in mice, a higher blood concentration and a longer half-life.

Test Example 4: Inhibitory activity of the compounds of the present invention on hERG

HEK-293 cells (Creacell, A-0320) stably expressing the hERG potassium channel were cultured in DMEM (Gibco, 11995-065) supplemented with 10% fetal bovine serum (Avantor, 76294-180) and 0.8 mg/mL G418 (GPC, AK108) at 37°C and 5% CO2. Prior to patch-clamp analysis, cells were dissociated using TrypLE ^™ Express (Gibco, 12604-013), and 4×10 ³ cells were plated onto coverslips in 24-well plates (final volume: 500 μL). After 18 hours, assays were performed.

The membrane was depolarized to +30 mV for 4.8 seconds and then the voltage was returned to -50 mV for 5.2 seconds to remove inactivation and measure the inactivation tail current. The sampling interval was 15 seconds. The maximum tail current magnitude was used to determine the hERG current amplitude. Blank vector was applied to the cells to establish a baseline. After at least 5 minutes of stable hERG current, the specimen was perfused. The experimental data were acquired by an IPA amplifier (Sutter Instrument) and stored in SutterPatch (with Igor Pro) software.

Drug administration was initiated after the whole-cell hERG currents were stable, and the drug concentration of 10 μM was allowed to act for 5 min (or until the currents stabilized). The assay was repeated three times using at least three cells at each concentration. All electrophysiological experiments were performed at room temperature.

The current after each drug concentration was normalized to the current of the blank control (0.3% DMSO) Then calculate the inhibition rate corresponding to each drug concentration The mean, standard deviation (SD) and standard error (SE) of the inhibition rate at each concentration were calculated. Table 4 provides the inhibition rate of the compounds of the present invention on hERG at a concentration of 10 μM.

Table 4

Conclusion: The above data show that the hERG inhibition rate of the compounds of the present invention is low.

Test Example 5: Liver microsomal metabolic stability of the compounds of the present invention

Experimental Materials:

Liver microsome reaction system:

The reaction system was pre-incubated in a 37°C water bath for 10 minutes. 40 μL of 10 mM NADPH solution was added to the reaction system to start the reaction. The final concentration of NADPH was 1 mM. 40 μL of ultrapure water was used instead of NADPH as a negative control. One replicate was set up for each time point. At 0, 15, 30, 45, and 60 minutes, 50 μL of reaction sample was taken out and quenched by adding 200 μL of acetonitrile containing a final concentration of 200 nM. After the sample was mixed, it was centrifuged at 4000 rpm for 30 minutes. After centrifugation, 100 μL of supernatant was added to 100 μL of pure water, mixed, vortexed, and centrifuged for 5 minutes for HPLC-MS/MS analysis to determine the content of the compound.

LC-MS/MS analysis method:

Mobile phase A: acetonitrile; mobile phase B: 1 mM ammonium acetate aqueous solution (containing 0.1% formic acid);

Chromatographic column: Waters UPLC C18 1.7μm, 2.1*50mm, L1-185.

Table 5 provides the residual rate of the prototype of the compounds of the present invention after incubation in liver microsomes for 60 min.

Table 5

Test Example 6: Pharmacodynamic study of the compound of the present invention

(1) In vivo efficacy study of HCT116 MTAP ^-/- subcutaneous xenograft tumors:

Female Nu/Nu nude mice (6-8 weeks old, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were housed in an SPF animal room at a temperature of 20-25°C, a relative humidity of 40-70%, and a light and dark illumination cycle of 12 hours each. The animals had free access to water and food. The animals were adaptively raised for 5 days before the start of the experiment.

HCT116 MTAP ^-/- cells (from Kyinno Biotechnology) were cultured and expanded in vitro. Cells in the logarithmic growth phase were harvested and resuspended in serum-free McCoy's 5A medium at a cell density of 5 × 10 ⁷ cells/mL. 100 μL of the cell suspension was injected subcutaneously into the right anterior axilla of each mouse. Animals were regularly observed to monitor tumor growth. When tumors reached approximately 150 mm ³ , animals with excessively large, small, or irregularly shaped tumors were removed. Tumor-bearing mice with uniform weight, good condition, and similar tumor volumes were randomly divided into groups of six animals per group. The model group received vehicle (3% DMSO + 0.5% methylcellulose + 0.2% Tween 80). Compounds were suspended in the vehicle and administered orally once daily in the treatment group at a volume of 10 mL/kg. Tumor diameters were measured twice weekly with a vernier caliper, and tumor volumes were calculated. Animal body weights were also recorded.

Tumor volume (TV) was calculated as follows: TV = 1/2 × a × b ² , where a represents the major diameter of the tumor and b represents the minor diameter of the tumor. Tumor growth inhibition (TGI (100%)) was calculated as follows: TGI = [1-(TV _t(T) -TV _initial(T) )/TV _t(C) -TV _initial(C) ] × 100%, where TV _t(T) represents the tumor volume of the dosing group at each measurement, TV _initial(T) represents the tumor volume of the dosing group at the time of dosing, TV _t(C) represents the tumor volume of the vehicle group at each measurement, and TV _initial(C) represents the tumor volume of the vehicle group at the time of dosing. Table 6 provides the growth inhibition rates of HCT116 MTAP ^−/− subcutaneous xenograft tumors by the compounds of the present invention.

Table 6

(2) In vivo efficacy study of NCI-H2228 subcutaneous xenograft tumors:

Female NOG mice (6 weeks old, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were housed in an SPF animal room at a temperature of 20-25°C, a relative humidity of 40-70%, and 12 hours of light and dark lighting. The animals had free access to water and food. The animals were adaptively fed for 5 days before the start of the experiment.

NCI-H2228 cells (sourced from ATCC) were cultured and expanded in vitro. Cells in the logarithmic growth phase were harvested and resuspended in serum-free RPMI-1640 medium at a cell density of 5 × ¹⁰⁷ cells/mL. 100 μL of the cell suspension was injected subcutaneously into the right anterior axilla of each mouse. Animals were regularly observed to monitor tumor growth. When tumors reached approximately 150–200 ^mm3 , animals with excessively large, small, or irregularly shaped tumors were removed. Tumor-bearing mice with uniform weight, good condition, and similar tumor volumes were randomly divided into groups of six animals each. The model group received vehicle (3% DMSO + 0.5% methylcellulose + 0.2% Tween 80). Compounds were suspended in the vehicle and administered once daily by gavage at a volume of 10 mL/kg. Tumor diameters were measured twice weekly with a vernier caliper, and tumor volumes were calculated. Animal body weights were also measured and recorded.

Tumor volume (TV) was calculated as follows: TV = 1/2 × a × b ² . Tumor growth inhibition (TGI (100%)) was calculated as follows: TGI = [1-(TV _t(T) -TV _initial(T) )/TV _t (C) -TV _initial(C) ] × 100%, where TV _t(T) represents the tumor volume of the dosing group at each measurement, TV _initial(T ) represents the tumor volume of the dosing group at the time of dosing, TV _t(C) represents the tumor volume of the vehicle group at each measurement, and TV _initial(C) represents the tumor volume of the vehicle group at the time of dosing. Table 7 provides the growth inhibition rates of the compounds of the present invention on NCI-H2228 subcutaneous xenograft tumors.

Table 7

Claims (14)

A compound represented by general formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,
in: Ring A is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl; Ring E is selected from cycloalkyl, heterocyclyl, aryl and heteroaryl; X ¹ and X ² are each independently selected from a bond, CH ₂ , O, S and NH; X ³ and X ⁴ are each independently selected from CH ₂ , O, S and NH; each R ¹ is independently selected from hydrogen, halogen, hydroxy, amino, cyano, alkyl, alkenyl, alkynyl, heteroalkyl, haloalkyl, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and -NR ^a R ^b , wherein said alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more R ^1a ; each R ² is independently selected from hydrogen, halogen, hydroxy, amino, cyano, alkyl, heteroalkyl, haloalkyl, alkoxy, haloalkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, heteroalkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more R ^2a ; each R ^1a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, oxo, cycloalkyl, -O-cycloalkyl, heterocyclyl, -heterocyclylene-alkyl, -O-heterocyclylene-alkyl, -O-alkyl, heteroalkyl, haloalkyl, -O-haloalkyl, -NH-haloalkyl, hydroxyalkyl, alkyl, -Q-aryl, -Q-heteroaryl, 5-20 membered spiroheterocyclyl, -SF ₅ , HC(═O)-, -L-NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -C(═O)NR ^a R ^b , -CH ₂ NHC(═O)O-alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-alkyl, -CH ₂ -heteroaryl, -CH ₂ NHSO _2- alkyl, -CH ₂ OC(═O)-heterocyclyl, -OC(═O)NR ^a R ^b , —OC(═O)-heterocyclyl, and -alkylene-heterocyclyl, wherein the heterocyclyl or heterocyclylene is optionally substituted with one or more oxo or halogen; each R ^2a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, heterocyclyl, -O-alkyl, heteroalkyl, haloalkyl, -O-haloalkyl, -NH-haloalkyl, hydroxyalkyl, alkyl, -Q-aryl, -Q-heteroaryl, HC(═O)-, -NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -CH ₂ NHC(═O)O-alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-alkyl, -CH ₂ -heteroaryl, -CH ₂ NHSO ₂ -alkyl, -CH ₂ OC(═O)-heterocyclyl, -OC(═O)NR ^a R ^b , -OC(═O)-heterocyclyl, and -CH ₂ -heterocyclyl, wherein the heterocyclyl of -CH ₂ -heterocyclyl is optionally substituted with one or more oxo groups; L is selected from a bond, -O-, -NH-, and alkylene, wherein the alkylene is optionally substituted with one or more groups selected from hydroxy, hydroxyalkyl, and heteroaryl; each Q is independently selected from a bond, -O-, and -NH-; each R ³ is independently selected from hydrogen, halogen, amino, nitro, cyano, oxo, hydroxy, thiol, carboxyl, ester, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; each R ⁴ is independently selected from hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, alkyl, heteroalkyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, aminoalkyl, cyanoalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ^a and R ^b are each independently selected from hydrogen, halogen, hydroxy, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more groups selected from halogen, amino, nitro, cyano, hydroxy, thiol, carboxyl, ester, oxo, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or, ^Ra and ^Rb together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group, which is optionally substituted with one or more groups selected from halogen, amino, nitro, cyano, oxo, hydroxy, thiol, carboxyl, ester, alkyl, alkoxy, haloalkyl, hydroxyalkyl, aminoalkyl, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl; m ₁ is 0, 1, 2, or 3; m ₂ is 0, 1, 2, or 3; m ₃ is 0, 1, 2 or 3; s is 0, 1, 2, or 3; t is 0, 1, or 2. The compound according to claim 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein ring A is selected from C _5-7 cycloalkyl, _5- to 7-membered heterocyclyl, phenyl or 5- to 6-membered heteroaryl. The compound according to claim 1 or 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or a 5- to 6-membered heteroaryl group; preferably phenyl, pyridyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl or imidazolyl, more preferably phenyl or pyridyl. The compound according to any one of claims 1 to 3, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: Selected from in: X ¹ is selected from CH ₂ , O, S and NH; X ² is CH ₂ or a bond; X ³ is selected from CH ₂ , O and NH; X ⁴ is CH ₂ ; t is 0, 1, or 2, preferably 1; R ¹ , R ² , R ³ , m ₁ , m ₂ and m ₃ are as defined in claim 1 ; In particular,
Selected from R ¹ , R ² , R ³ , m ₁ , m ₂ and m ₃ are as defined in claim 1 . The compound according to any one of claims 1 to 4, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: Ring E is selected from C _5-6 cycloalkyl, 5- to 6-membered heterocyclyl, phenyl and 5- to 6-membered heteroaryl, preferably, ring E is a 5- to 6-membered heterocyclyl or a 5- to 6-membered heteroaryl, more preferably, ring E is selected from cyclopentyl, cyclohexyl, dihydrofuranyl and pyrazolyl; and/or Each R ⁴ is independently selected from hydrogen, halogen, amino, nitro, cyano, hydroxyl, thiol, carboxyl, ester, oxo, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _1-6 cyanoalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, C _6-10 aryl and 5 to 10 heteroaryl. The compound according to any one of claims 1 to 5, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: Selected from R ^4a and R ^4b are each independently selected from hydrogen, halogen, amino, hydroxyl, thiol, carboxyl, oxo, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C 1-6 haloalkoxy, C _1-6 hydroxyalkyl, C _1-6 aminoalkyl, C _2-6 alkenyl, _{C 2-6} alkynyl, C _3-6 cycloalkyl, 5 to _{6 membered heterocyclyl, C 6-10 aryl} and 5 to 10 membered heteroaryl, preferably hydrogen or C _1-6 alkyl. The compound according to any one of claims 1 to 6, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: each R ¹ is independently selected from hydrogen, halogen, hydroxy, amino, cyano, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl, 5 to 10 membered heteroaryl, and -NR ^a R ^b , said C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyl, C _{1-6 heteroalkyl, C 1-6 alkoxy} , C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl, and 5 to 10 membered heteroaryl being each independently optionally substituted with one or more R ^1a ; Each R ^1a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, oxo, C _3-6 cycloalkyl, -OC _3-6 cycloalkyl, 4 to 8 membered heterocyclyl, -4 to 8 membered heterocyclylene-C _1-6 alkyl, -O-4 to 8 membered heterocyclylene-C _1-6 alkyl, -OC _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, -OC _1-6 haloalkyl, -NH-C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 alkyl, -Q-phenyl, -Q-5 to 6 membered heteroaryl, HC(=O)-, -L-NR ^a R ^b , -CH ₂ OC(=O)NR ^a R ^b , -CH ₂ NHC(=O)OC _1-6 alkyl, -CH ₂ NHC(=O)NR ^a R ^b , -CH ₂ NHC(=O)-C _{-C 1-6} alkyl, -CH ₂ -5 to 6 membered heteroaryl, -CH ₂ NHSO ₂ -C _1-6 alkyl, -CH ₂ OC(═O)-4 to 8 membered heterocyclyl, -OC(═O)NR ^a R ^b , -C(═O)NR ^a R ^b , -OC(═O)-4 to 8 membered heterocyclyl, and -C _1-6 alkylene-4 to 8 membered heterocyclyl, wherein the heterocyclyl or heterocyclylene is optionally substituted with one or two oxo groups or halogen; m ₁ is 0 or 1; L, Q, ^Ra and ^Rb are as defined in claim 1; Preferably, each R ¹ is independently selected from hydrogen, halogen, cyano, C _2-6 alkynyl, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl and 5 to 10 membered heteroaryl, said C _2-6 alkynyl, C _3-10 cycloalkyl, 4 to 10 membered heterocyclyl, 5 to 10 membered aryl and 5 to 10 membered heteroaryl each independently optionally substituted with one or more R ^1a ; each R ^1a is independently selected from hydrogen, halogen, cyano, C _1-6 alkyl, C _1-6 haloalkyl, -OC _1-6 alkyl, C _1-6 hydroxyalkyl, C _3-6 cycloalkyl, -OC _3-6 cycloalkyl, 4 to 8-membered heterocyclyl, -4 to 8-membered heterocyclylene- _{C 1-6} alkyl, -O-4 to 8-membered heterocyclylene-C _1-6 alkyl, C _1-6 heteroalkyl, -L-NR ^a R ^b , -C(═O)NR ^a R ^b , -C _1-6 alkylene-4 to 8-membered heterocyclyl, and -C _1-6 alkylene-halo 4 to 8-membered heterocyclyl; m ₁ is 1; L, ^Ra and ^Rb are as defined in claim 1. A compound according to any one of claims 1 to 7, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein each R ² is independently selected from hydrogen, halogen, hydroxy, amino, cyano, C _1-6 alkyl, C _1-6 heteroalkyl, C _{1-6 haloalkyl, C 1-6 alkoxy} , C _1-6 haloalkoxy, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl, wherein the C _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 4 to 8 membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl are each independently optionally substituted with one or more R ^2a ; each R ^2a is independently selected from hydrogen, halogen, hydroxy, amino, cyano, 4 to 8 membered heterocyclyl, -OC _1-6 alkyl, C _1-6 heteroalkyl, C _1-6 haloalkyl, -OC _1-6 haloalkyl, -NH-C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _1-6 alkyl, -Q-phenyl, -Q-5 to 6 membered heteroaryl, HC(═O)-, -NR ^a R ^b , -CH ₂ OC(═O)NR ^a R ^b , -CH ₂ NHC(═O)OC _1-6 alkyl, -CH ₂ NHC(═O)NR ^a R ^b , -CH ₂ NHC(═O)-C _1-6 alkyl, -CH ₂ -5 to 6 membered heteroaryl, -CH ₂ NHSO ₂ -C _1-6 alkyl, -CH ₂ OC(═O)-4 to 8 membered heterocyclyl, -OC(═O)NR ^a R ^b , -OC(=O)-4 to 8-membered heterocyclyl and -CH ₂ -4 to 8-membered heterocyclyl, wherein the heterocyclyl group of the -CH ₂ -4 to 8-membered heterocyclyl is optionally substituted with one or two oxo groups; m ₂ is 0, 1 or 2, preferably, m ₂ is 0; Q, ^Ra and ^Rb are as defined in claim 1. The compound according to any one of claims 1 to 8, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein each R ³ is independently selected from hydrogen, halogen, amino, nitro, cyano, oxo, hydroxyl, thiol, carboxyl, ester, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkyl, C _{1-6 hydroxyalkyl, C 1-6} aminoalkyl, C _1-6 haloalkoxy, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 cycloalkyl, 4 to ₈ membered heterocyclyl, phenyl and 5 to 6 membered heteroaryl; preferably R ³ is hydrogen. The compound according to any one of claims 1 to 9, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:















A method for preparing a compound represented by general formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the following steps:
Compound Ic is subjected to a condensation reaction with compound Id in the presence of a condensation reagent and an alkaline reagent to obtain a compound represented by general formula (I) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof. Wherein, the condensation reagent is preferably N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, and the alkaline reagent is preferably N-methylimidazole; wherein Ring A, Ring E, X ¹ , X ² , X ³ , X ⁴ , R ¹ , R ² , R ³ , R ⁴ , m ₁ , m ₂ , m ₃ , s and t are as defined in claim 1 . A pharmaceutical composition comprising the compound according to any one of claims 1 to 10 or its tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Use of a compound according to any one of claims 1 to 10 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 12 in the preparation of a PRMT5 inhibitor. Use of a compound according to any one of claims 1 to 10 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing and/or treating a disease associated with PRMT5 activity, preferably, the disease associated with PRMT5 activity is a solid tumor, such as non-small cell lung cancer, mesothelial tumor, neurofibrosarcoma or pancreatic cancer.