New heterocyclic covalent inhibitor of menin, preparation method therefor and pharmaceutical use thereof

Abstract

Provided in the present invention are a compound of formula (I), a pharmaceutically acceptable salt thereof, a preparation method therefor and the use thereof. The compound can be used as a medicament in the treatment of diseases and disorders, including cancers and other diseases mediated by menin-MLL interactions.

Description

A novel heterocyclic menin covalent inhibitor, its preparation method and medical use Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a novel heterocyclic menin covalent inhibitor, a preparation method thereof, and medical uses. Background Art

[0002] Mixed lineage leukemia-rearranged (MLL-r) is an acute leukemia with a poor prognosis caused by a spontaneous translocation of the MLL1 gene. The resulting MLL-r fusion protein binds with high affinity to the nuclear protein Menin. The Menin-MLL interaction drives a specific transcriptional program leading to leukemogenesis.

[0003] Currently, there are no approved drugs or therapies for acute myeloid leukemia (AML) that target MLL-r or NPM1 mutations. The presence of MLL fusion proteins is a hallmark of poor prognosis in leukemia. Patients with MLL-r leukemia respond poorly to currently available treatments, with a five-year overall survival rate of approximately 35%. Translocations in the MLL gene are associated with a 13-34% decrease in five-year disease-free survival in children, while the average five-year survival rate for adult patients with MLL-r AML is only 5-10%. The five-year overall survival rate for NPM1-mutant AML is approximately 50%. Furthermore, AML has a high relapse rate, with approximately 50% of patients relapsing within one to two years, and most relapsed AML eventually progresses to relapsed / refractory AML. Currently, there are no effective treatment options for relapsed / refractory AML. AML exhibits significant molecular heterogeneity, and patients with different mutations can have vastly different prognoses. The focus of AML drug research and development is on more precisely targeting different mutations and developing targeted drugs for these mutations.

[0004] Menin-MLL interaction inhibitors are epigenetic inhibitors that block the interaction between Menin and the MLL fusion protein (MLL-r) in leukemia cells. Two compounds are currently in clinical development: SNDX-5613, which is undergoing a Phase 1 / 2 study for patients with relapsed / refractory acute leukemia (AML) harboring MLL rearrangements or NPM1 mutations, and KO-539, which is undergoing a Phase 1 study for patients with relapsed / refractory AML harboring MLL rearrangements or NPM1 mutations.

[0005] Furthermore, according to the U.S. Centers for Disease Control and Prevention, 537 million adults worldwide have diabetes. In the United States alone, 37.3 million adults, or 11.3% of the total population, have diabetes. Medications on the market primarily mitigate rising blood sugar levels, but no cure for diabetes has yet been developed.

[0006] In 2007, researchers at Stanford University (*Menin Controls Growth of Pancreatic β-Cells in Pregnant Mice and Promotes Gestational Diabetes. Science, (2007), 801-806, 318) demonstrated preclinically that during pregnancy in mice, prolactin downregulates Menin, which leads to the proliferation of maternal pancreatic β-cells, increased insulin production, and the maintenance of normal glucose levels to prevent gestational diabetes.

[0007] The small molecule menin inhibitor BMF-219 is reportedly a potential new diabetes drug that may address the root cause of diabetes – the loss of beta cell mass and function. Through exploration, the present invention has developed a class of menin inhibitors that can lower blood glucose levels in models.

[0008] In summary, Menin-MLL interaction inhibitors have great application prospects as drug development, and there is a great clinical demand for the development of new, highly effective and low-toxic Menin-MLL interaction inhibitors.

[0009] Summary of the Invention

[0010] The present application provides a compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof:

[0011] in:

[0012] A is an optionally substituted or unsubstituted 8-15 membered heterocycle, wherein the substituent is selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, -OH, cyano or C1-C4 haloalkyl;

[0013] C is an optionally substituted or unsubstituted 5-15 membered aromatic ring, an optionally substituted or unsubstituted 5-15 membered aliphatic ring, an optionally substituted or unsubstituted 5-15 membered aliphatic heterocycle, or an optionally substituted or unsubstituted 5-15 membered aromatic heterocycle;

[0014] R1 is independently selected from 4-9 membered heterocyclyl, wherein the 4-9 membered heterocyclyl may be optionally substituted with one or more -OH, halogen, cyano, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl or C1-C4 haloalkyl;

[0015] X and Y are each independently selected from -NH- or -C(=O)-, and X and Y are not the same;

[0016] Z1 and Z2 are each independently selected from -CH2-, -NRz- or a chemical bond;

[0017] D1, D2, B1 and B2 are each independently selected from N or -CH-, and when Z is -NRz, D can only be -CH;

[0018] Rz is selected from H or C1-C4 alkyl;

[0019] G is in for Among them, R g1 、R g2 and R g3 Each is independently absent, H, C1-C4 alkyl, C1-C4 cycloalkyl, C1-C4 alkoxy, C1-C4 aminoalkyl, C1-C4 hydroxyalkyl, halogen, hydroxy, CN or CF3;

[0020] R2, R3, Rm and Rn are each independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 haloheteroalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C 10 Carbocyclyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 6-12 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, CN, SF5, -C(=O)OR 30 、-C(=O)R 30 、-C(=O)NR 31 R 32 、-NR 33 C(=O)R 34 、-NR 31 R 32 、-OC(=O)R 30 、-OC(=O)NR 31 R 32 、-NR 33 C(=O)NR 31 R 32 、-NR 33 C(=O)OR 30 、-S(=O)NR 31 R 32 、-S(=O)2NR 31 R 32 、-S(=O)R 35 、-S(=O)(=NR 37 )R 35 、-S(=O)2R 35 、-NR 33 S(=O)2R 35 、-S(=O)2NR31 R 32 、-OR 37 、-SR 37 、-C(=NR 38 )NR 31 R 32 、-C(=NR 38 )R 32 、-NR 33 C(=NR 38 )NR 31 R 32 、-N=NR 38 ;

[0021] R 30 、R 37 、R 39 and R 40 Each is independently selected from hydrogen, halogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 haloalkyl, C1-C8 haloheteroalkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, 4-10 membered heterocyclyl, C6-C 12 Aryl, 5-12 membered heteroaryl, C1-C8 alkylene-C 6- C 12 Aryl and C1-C8 alkylene-(5-10 membered heteroaryl);

[0022] R 31 、R 32 、R 33 and R 34 Each is independently selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 haloalkyl, C1-C8 haloheteroalkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, 4-10 membered heterocyclyl, C6-C 12 Aryl, 5-12 membered heteroaryl, C1-C8 alkylene-C6-C 12 Aryl and C1-C8 alkylene-(5-10 membered heteroaryl);

[0023] R 35 Selected from C1-C8 alkyl, C 1- C8 alkoxy, C 3- C8 cycloalkyl, 4-10 membered heterocyclic group, C 6- C 12 Aryl, 5-10 membered heteroaryl, C 1- C8 alkylene-C 6- C 12 Aryl and C 1- C8 alkylene-(5-10 membered heteroaryl);

[0024] R 36is selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C1-C6 haloheteroalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C 10 Carbocyclyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 6-12 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, CN, SF5;

[0025] R 38 Selected from hydrogen, hydroxy, cyano, nitro, -S(=O)R 35 and -S(=O)2R 35 ;

[0026] m is 0, 1, 2, 3, 4, 5 or 6;

[0027] n is 0, 1, or 2;

[0028] p is 0, 1, 2, 3, 4, 5, or 6;

[0029] G is selected from:

[0030] In some embodiments, the C is phenyl or pyridyl.

[0031] In some specific embodiments, the compound of formula (I) described herein is selected from:

[0032] In another aspect, the present invention also relates to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating diseases associated with the menin-MLL protein.

[0033] Specifically, the diseases associated with menin-MLL protein refer to mixed lineage leukemia (MLL), MLL-related leukemia, MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, rearranged mixed lineage leukemia (MLL-r), leukemia associated with MLL rearrangement or MLL gene rearrangement, acute leukemia, chronic leukemia, lymphoblastic leukemia, lymphocytic leukemia, myeloid leukemia, myeloid leukemia, childhood leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, Myeloid leukemia (AML), acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), therapy-related leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), myeloproliferative neoplasia (MPN), plasma cell neoplasm, multiple myeloma, myelodysplasia, cutaneous T-cell lymphoma, lymphoid neoplasm, hairy cell leukemia, leukemic meningitis, multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma (malignant lymphoma).

[0034] On the other hand, the present application also relates to a process for synthesizing the compound of formula (I),

[0035] Method 1:

[0036] The steps include:

[0037] Among them, A, B1, B2, Z1, Z2, D1, D2, G, R1, R2, R3, Rm, Rn, R g1 、R g2 、R g3 , p, m and n are as defined above;

[0038] P is a protecting group selected from: Cbz, Boc, Fmoc, Alloc or Teoc;

[0039] Hal is a halogen selected from: -I, -Br or -Cl;

[0040] F is chlorine or hydroxyl;

[0041] 1) The starting material M-1 and the boronic acid intermediate M-2 are subjected to a Suzuki coupling reaction to generate the intermediate compound IA-1;

[0042] 2) Intermediate IA-1 reacts with intermediate M-3 in the presence of a condensation reagent to produce intermediate compound IA-2;

[0043] 3) Removal of the protecting group of intermediate IA-2 under appropriate conditions to generate intermediate compound IA-3;

[0044] 4) Intermediate IA-3 is reacted with commercially available intermediate M-4 through acylation to obtain the compound of formula (IA).

[0045] Method 2:

[0046] The steps include:

[0047] Among them, A, B1, B2, Z1, Z2, D1, D2, G, R1, R2, R3, Rm, Rn, R g1 、R g2 、R g3 , p, m and n are as defined above;

[0048] P is a protecting group selected from: Cbz, Boc, Fmoc, Alloc or Teoc;

[0049] Hal is a halogen selected from: -Br ​​or -Cl;

[0050] F is chlorine or hydroxyl;

[0051] 1) The starting material M-3 and the boronic acid intermediate M-2 are reacted via Suzuki coupling reaction to produce the intermediate compound IIA-1;

[0052] 2) Intermediate IIA-1 reacts with intermediate M-3 in the presence of a condensation reagent to produce intermediate compound IIA-2;

[0053] 3) Intermediate IIA-2 is reacted with an alkaline reagent to generate intermediate compound IIA-3;

[0054] 4) Removal of the protecting group of intermediate IA-3 under appropriate conditions to generate intermediate compound IA-4;

[0055] 5) Intermediate IA-4 is reacted with commercially available intermediate M-4 through acylation to obtain the compound of formula (IIA).

[0056] The present invention has discovered a new class of menin-MLL interaction inhibitors with a structure as shown in the compound of formula (I), which have good cell inhibitory activity and good pharmacokinetic properties, and are a new generation of high-efficiency and low-toxic menin-MLL interaction inhibitors.

[0057] As another embodiment of the present invention, the compound described herein, its isomer or pharmaceutically acceptable salt thereof is used in the preparation of a drug for preventing or treating diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the effects of the test substance of the present application on fasting blood glucose in STZ type 1 rats (Mean ± SD);

[0059] FIG2 shows the changes in blood biochemical indicators after administration of the test substance of the present application to STZ-induced diabetic rats. DETAILED DESCRIPTION

[0060] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] The structures of the compounds were determined by mass spectrometry (MS) or nuclear magnetic resonance (NMR) 1 HNMR) to determine.

[0062] Nuclear magnetic resonance (NMR) 1 HNMR) shifts (δ) are given in parts per million (ppm); nuclear magnetic resonance ( 1 HNMR) was measured using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvent was deuterated dimethyl sulfoxide (DMSO), the internal standard was tetramethylsilane (TMS), and the chemical shift was 10 -6 The units are given in ppm.

[0063] Mass spectrometry (MS) was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQ advantage MAX).

[0064] Thin layer silica gel uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate.

[0065] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0066] In the present invention, "nitrogen protection" means, for example, connecting the reaction bottle to a 1 L nitrogen balloon.

[0067] Unless otherwise specified in the present invention, the solution mentioned in the reaction of the present invention is an aqueous solution.

[0068] The term "room temperature" in the present invention refers to a temperature between 10°C and 25°C.

[0069] Intermediate M1 4-(Chloromethyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (M1)

[0070] Synthesis route:

[0071] Step 1: 4-(6-iodo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)morpholine (M1-1)

[0072] 4-Chloro-6-iodo-7H-pyrrolo[2,3-D]pyrimidine (3 g, 10.7 mmol) was dissolved in isopropanol (200 ml), and morpholine (1.4 g, 16.1 mmol) and DIPEA (2.77 g, 21.5 mmol) were added and reacted at 90°C for 5 h. The reaction was completed after LC / MS detection. After cooling, the mixture was filtered and the solid was washed with petroleum ether / ethyl acetate = 5 / 1 (500 ml) and dried to obtain 3.55 g of a crude yellow solid, which was used directly in the next step.

[0073] ESI-MS m / z:331[M+H] + .

[0074] Step 2: 4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)aniline (M1-2)

[0075] The above-mentioned intermediate M1-1 (3.5 g, 10.6 mmol), 4-aminophenylboronic acid pinacol ester (3.48 g, 15.9 mmol), potassium carbonate (2.93 g, 21.2 mmol), and Pd(dppf)Cl2 (0.77 g, 1.06 mmol) were dissolved in 1,4-dioxane (50 ml) and reacted at 100°C for 12 h. LC / MS confirmed the completion of the reaction. After cooling, the mixture was filtered, and the filter cake was washed with 100 ml of ethyl acetate. The filtrate was concentrated and purified by column chromatography (D / M = 20 / 1) to obtain 2.61 g of the product (yield: 83.6%).

[0076] ESI-MS m / z:296.1[M+H] + .

[0077] 1 H NMR (400MHz, DMSO-d6) δ11.90(s,1H),8.12(s,1H),7.68–7.48(m,2H),6.83(d,J=2.2H z,1H),6.68–6.43(m,2H),5.30(s,2H),3.88–3.80(m,4H),3.74(dd,J=5.6,3.8Hz,4H).

[0078] Step 3: 4-(Hydroxymethyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (M1-3)

[0079] The above intermediate (M1-2) (2.5 g, 8.46 mmol), 4-(hydroxymethyl)pyridine-2-carboxylic acid (1.56 g, 10.16 mmol), DIEA (3.27 g, 25.38 mmol), and HATU (4.83 g, 12.69 mmol) were added to DMF (100 mL). The reaction mixture was stirred at room temperature overnight. LCMS monitored the reaction completion. The reaction mixture was extracted three times with water and EA, and the organic phase was concentrated. Column chromatography afforded 2.71 g of the desired product (M1-3) as an off-white solid (yield: 74.55%).

[0080] ESI-MS m / z:430.2[M+H] + .

[0081] Step 4: 4-(Chloromethyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (M1)

[0082] The above intermediate (M1-3) (2.5 g, 5.82 mmol) was added to DMF (10 mL). The reaction mixture was cooled to 0°C and SOCl2 (2 mL) was added dropwise. The reaction mixture was warmed to room temperature and stirred overnight. After completion of the reaction as monitored by LCMS, the mixture was added to 50 mL of water, stirred thoroughly, and filtered. The solid was dried to obtain 2.26 g of the desired product (M1) as a gray solid (yield: 86.61%).

[0083] ESI-MS m / z:448.2[M+H] + .

[0084] Intermediate M4 4-(4-(3-methoxyazetidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)aniline

[0085] Synthesis route:

[0086] Referring to the preparation method of M1, 4-(4-(3-methoxyazetidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)aniline (M4) was prepared and purified. ESI-MS: m / z = 296.1 [M+H] + ;

[0087] Intermediate M9 (S)-1-(6-(4-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-ol

[0088] Synthesis route:

[0089] Referring to the preparation method of M1, (S)-1-(6-(4-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-ol (M9) was prepared and purified. ESI-MS: m / z = 296.1 [M+H] + ;

[0090] Intermediate M10

[0091] Synthesis of tert-butyl 2-((2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)-2,6-diazaspiro[3.5]nonane-6-carboxylate

[0092] Step 1: Synthesis of tert-butyl (4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamate (M10-1) 4-Chloro-6-iodo-7H-pyrrolo[2,3-d]pyrimidine (2.0 g, 7.17 mmol) was added to a reaction flask, followed by tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate (2.0 g, 6.45 mmol), potassium carbonate (5.0 g, 35.84 mmol), 1,4-dioxane (100 mL), and water (20 mL). The mixture was degassed with nitrogen five times, and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (519 mg, 0.72 mmol) was added to the reaction flask. The mixture was refluxed at 100°C for 12 h. The solution turned black. The mixture was cooled to room temperature, and extracted three times with water and ethyl acetate. The organic phase was filtered through celite, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a dark gray residue M10-1 (1.7 g, yield: 78%). ESI-MS m / z: 345.1 [M+H]+.

[0093] Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)aniline (M10-2)

[0094] M10-1 (800 mg, 2.32 mmol) was dissolved in 10 mL of ACN and cooled on an ice bath for 15 min. Then, 3 mL of TMSCl was added, followed by dropwise addition of 1 mL of tetrabutylammonium fluoride. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction as monitored by LC-MS, the reaction was filtered and the filter cake dried to afford M10-2 (500 mg, 88.3% yield) as a yellow solid. ESI-MS m / z: 245.1 [M+H]+.

[0095] Step 3: Synthesis of methyl 4-(chloromethyl)picolinate (M10-3)

[0096] Methyl 4-(hydroxymethyl)picolinate (3.0 g, 17.96 mmol) was added to a reaction flask, dissolved in 50 mL of DCM, and then added 0.5 mL of DMF. SOCl₂ (6.4 g, 53.89 mmol) was slowly added dropwise to the reaction solution at 0°C. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After completion of the reaction, the mixture was purified by column chromatography using a 20:1 ratio of DCM:MeOH. M10-3 (3.0 g, 90% yield) was obtained as a pale yellow solid. ESI-MS m / z: 186.2 [M+H]⁺ was used.

[0097] Step 4: Synthesis of tert-butyl 2-((2-(methoxycarbonyl)pyridin-4-yl)methyl)-2,6-diazaspiro[3.5]nonane-6-carboxylate (M10-4)

[0098] The above-mentioned 1-3 (1.0 g, 5.4 mmol) was added to a reaction flask, followed by tert-butyl 2,6-diazaspiro[3.5]nonane-6-carboxylate (1.2 g, 5.4 mmol), potassium carbonate (2.2 g, 16.21 mmol), and potassium iodide (0.45 g, 2.7 mmol). The mixture was dissolved in 10 mL of DMF and heated under reflux in an 80°C oil bath for 4 h. After completion of the reaction as monitored by LC-MS, the mixture was cooled to room temperature and filtered directly. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using a 15:1 ratio of DCM:MeOH. M10-4 (1.2 g, 60% yield) was obtained as an off-white solid. ESI-MS m / z: 376.2 [M+H]+.

[0099] Step 5: Synthesis of 4-((6-(tert-Butyloxycarbonyl)-2,6-diazaspiro[3.5]non-2-yl)methyl)picolinic acid (M10-5)

[0100] The above 1-4 (1200 mg, 3.2 mmol) and NaOH (192 mg, 4.8 mmol) were added to a reaction flask, dissolved in 5 mL of THF, and 1 mL of water was added. The mixture was heated under reflux at 50°C in an oil bath for 2 h. After the reaction was complete as monitored by LC-MS, the mixture was cooled to room temperature and concentrated under reduced pressure to remove THF. An appropriate amount of water was added, and dilute hydrochloric acid was added dropwise under ice bath to adjust the pH to 5-6. The mixture was then rapidly lyophilized. A white solid was obtained after lyophilization. The white solid was washed with an appropriate amount of methanol, filtered, and the filtrate was concentrated under reduced pressure to obtain an off-white solid M10-5 (1050 mg, yield: 90.9%). ESI-MS m / z: 362.2 [M+H]+.

[0101] Step 6: Synthesis of tert-butyl 2-((2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)-2,6-diazaspiro[3.5]nonane-6-carboxylate (M10)

[0102] The above 1-2 (500 mg, 2.05 mmol) and 1-5 (1100 mg, 3.07 mmol) were dissolved in 10 mL of DMF, 10 drops of DIPEA were added dropwise, and after stirring at room temperature for 10 min, HATU (1500 mg, 3.95 mmol) was added and the reaction was continued at room temperature for 4 h. After the reaction was complete as monitored by LC-MS, water was added until the product precipitated, filtered, and the filter cake was dried to obtain a yellow solid M10 (800 mg, yield: 66.7%). ESI-MS m / z: 588.2 [M+H] +. Intermediate M11 5-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)pyridin-2-amine (M11)

[0103] Synthesis route:

[0104] The above intermediate (M1-1) (11 g, 33.3 mmol), 2-aminopyridine-5-boronic acid pinacol ester (8.03 g, 36.7 mmol), and KOAc (19.2 g, 200 mmol) were added to a solution of dioxane (200.0 mL) and H₂O (20 mL). The mixture was stirred at 25°C for 0.5 h. Pd(dppf)Cl₂ (2.44 g, 3.33 mmol) was then added under nitrogen. The mixture was stirred at 100°C for 12 h. TLC (dichloromethane / methanol = 10 / 1, Rf = 0.47) indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove the dioxane. The residue was diluted with H₂O (150.0 mL) and then extracted with EtOAc (300.0 mL x 5). The combined organic layers were washed with brine (300.0 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was triturated with MeOH (60.0 mL) at 25° C. for 2 hours to afford 4.68 g of brown solid intermediate M11 (yield: 47.5%).

[0105] ESI-MS m / z:297.1[M+H] + .

[0106] Example 1 4-((3aS,7aR)-1-acryloyl-6H-pyrrolo[2,3-c]pyridin-6-yl)methyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (1)

[0107] Step 1: Tert-butyl (3aS, 7aR)-6-(2-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate (1-1)

[0108] The above intermediate (M1) (120 mg, 0.27 mmol) and potassium carbonate (111 mg, 0.80 mmol) were added to DMF (2 ml) and stirred for five minutes. Tert-butyl (3aS, 7aR)-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate (66.7 mg, 0.3 mmol) and potassium iodide (3.3 mg, 0.02 mmol) were added and reacted at 80°C under nitrogen for 16 hours. The reaction was complete as determined by LC / MS. Water (10 ml) was added to quench the reaction and the mixture was extracted three times with ethyl acetate (10 ml). The organic phases were combined and dried to give 200 g of a light yellow solid (1-1), which was used directly in the next reaction.

[0109] ESI-MS m / z:638.3[M+H] + .

[0110] Step 2: 4-((3aR,7aR)-octahydro-6H-pyrrolo[2,3-c]pyridin-6-yl)methyl)-N-(4-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (1-2)

[0111] The above intermediate (1-1) (200 mg, 0.27 mmol) was dissolved in acetonitrile (5 ml), and trimethylsilyl chloride (2 ml) was added. The reaction was carried out at 50°C for 16 h. The reaction was detected by LC / MS. The reaction was quenched with 5 ml of water and freeze-dried to obtain 260 mg of a crude brown solid product (1-2), which was used directly in the next step.

[0112] ESI-MS m / z:538.3[M+H] + .

[0113] Step 3: 4-((3aS,7aR)-1-acryloyl-6H-pyrrolo[2,3-c]pyridin-6-yl)methyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (1)

[0114] The above intermediate (1-2) (260 mg, 0.27 mmol) was added to 10 ml of THF. NaH (21.6 mg, 0.54 mmol) was added under ice-cooling. The mixture was stirred at room temperature for 30 minutes. After cooling under ice-cooling, acryloyl chloride (27 mg, 0.3 mmol) was added dropwise with stirring. The reaction was maintained at room temperature for 3 hours. LC-MS confirmed the completion of the reaction. The mixture was quenched with water under ice-cooling, solubilized with DMF, purified and isolated by high pressure preparative purification, and lyophilized to obtain 15 mg of the product as a light yellow solid (yield: 9.5%). ESI-MS m / z: 592.3 [M+H] + .

[0115] 1 H NMR (400MHz, DMSO-d6) δ12.20(s,1H),10.71(s,1H),8.64(t,J=5.6Hz,1H),8.19(s,1H),8.06(d,J=9.7H z,1H),8.03–7.96(m,2H),7.91(d,J=8.7Hz,2H),7.57–7.48(m,1H),7.17(d,J=2.1Hz,1H),6.77(td,J=1 7.2,10.4Hz,1H),6.11(ddd,J=34.4,16.7,2.6Hz,1H),5.58(ddd,J=77.6,10.4,2.5Hz,1H),4.27–4.13( m,1H),4.05–3.69(m,10H),3.50–3.36(m,2H),2.99–2.62(m,2H),2.32–2.14(m,2H),1.94–1.33(m,5H).

[0116] Example 2 4-(6-acryloyl-2,6-diazaspiro[3.5]non-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (2)

[0117] Referring to the preparation method of Example 1, the intermediate M was reacted with tert-butyl 2,6-diazaspiro[3.5]nonane-6-carboxylate, and prepared and purified by the steps described in Example 1 to obtain 4-(6-acryloyl-2,6-diazaspiro[3.5]nonan-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (2).

[0118] ESI-MS: m / z = 593.3 [M+H] + .

[0119] 1H NMR (400MHz, DMSO-d6) δ12.20(d,J=2.2Hz,1H),10.73(s,1H),8.67(d,J=4.9Hz,1H),8.18(s,1H ),8.07(s,1H),8.03–7.97(m,2H),7.95–7.88(m,2H),7.56(d,J=5.0Hz,1H),7.16(d,J=2.1Hz,1H ),6.95-6.75(m,1H),6.10(t,J=16.7Hz,1H),5.70(dd,J=41.4,10.4Hz,1H),3.95–3.63(m,12H) ,3.45(t,J=5.5Hz,2H),3.06(s,2H),2.88(s,2H),1.76(t,J=6.1Hz,2H),1.41(d,J=15.2Hz,2H).

[0120] Example 3 4-(7-Acryloyl-2,7-diazaspiro[4.4]non-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (3)

[0121] Referring to the preparation method of Example 1, the intermediate M1 was reacted with tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate, and the mixture was prepared and purified by the steps described in Example 1 to obtain 4-(7-acryloyl-2,7-diazaspiro[4.4]nonan-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (3).

[0122] ESI-MS: m / z = 593.3 [M+H] + .

[0123] 1H NMR(400MHz,DMSO-d6)δ12.20(s,1H),10.74(s,1H),8.71–8.65(m,1H),8.19(s,1H),8.15–8.11( m,1H),8.04–7.97(m,2H),7.95–7.88(m,2H),7.62(dd,J=5.0,1.6Hz,1H),7.17(d,J=1.7Hz,1H), 6.60-6.52(m,1H),6.11(ddd,J=16.8,5.6,2.5Hz,1H),5.65(dt,J=10.4,2.2Hz,1H),3.89(t,J=4 .8Hz,4H),3.76(dd,J=6.3,3.2Hz,6H),3.63-3.29(m,4H),2.74–2.40(m,4H),2.03–1.70(m,4H).

[0124] Example 4 4-(1-acryloylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)pyridineamide (4)

[0125] Referring to the preparation method of Example 1, the intermediate M1 was reacted with tert-butyl hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate, and the mixture was prepared and purified by the steps described in Example 1 to obtain 4-(1-acryloylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)pyridineamide (4).

[0126] ESI-MS: m / z = 579.3 [M+H] + .

[0127] 1H NMR (400MHz, DMSO-d6) δ12.20(s,1H),10.73(s,1H),8.68(dd,J=5.0,2.8Hz,1H),8.18(s,1H) ,8.10(s,1H),7.99(d,J=8.5Hz,2H),7.96–7.89(m,2H),7.59(d,J=5.6Hz,1H),7.16(s,1H),6. 70–6.51(m,1H),6.12(ddd,J=16.9,8.6,2.4Hz,1H),5.65(ddd,J=10.4,6.5,2.4Hz,1H),3.95– 3.70(m,10H),3.69–3.59(m,2H),2.88–2.54(m,4H),2.28(t,J=6.3Hz,1H),2.10–1.69(m,3H).

[0128] Example 5 4-((2-acryloyl-5-oxa-2,8-diazaspiro[3.5]non-8-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (5)

[0129] Referring to the preparation method of Example 1, the intermediate M1 was reacted with tert-butyl 5-oxo-2,8-diazaspiro[3.5]nonane-2-carboxylate, and the mixture was prepared and purified by the steps described in Example 1 to obtain 4-(2-acryloyl-5-oxa-2,8-diazaspiro[3.5]non-8-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (5).

[0130] ESI-MS: m / z = 595.3 [M+H] + .

[0131] 1H NMR(400MHz,DMSO-d6)δ12.20(s,1H),10.75(s,1H),8.75–8.67(m,1H),8.18(s,1H),8.16(d, J=1.3Hz,1H),8.04–7.97(m,2H),7.95–7.89(m,2H),7.64(dd,J=4.9,1.6Hz,1H),7.16(s,1H), 6.32(dd,J=17.0,10.3Hz,1H),6.09(dd,J=17.0,2.2Hz,1H),5.66(dd,J=10.3,2.2Hz,1H),4.1 0(q,J=9.3Hz,2H),3.89(t,J=4.8Hz,4H),3.83–3.61(m,10H),2.69–2.53(m,2H),2.43(s,2H).

[0132] Example 6 4-((3aR,7aS)-1-acryloyl-6H-pyrrolo[2,3-c]pyridin-6-yl)methyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (6)

[0133] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl (3aR, 7aS)-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate, and the mixture was prepared and purified by the steps described in Example 1 to obtain 4-((3aR, 7aS)-1-acryloyl-6H-pyrrolo[2,3-c]pyridin-6-yl)methyl)-N-(4-(4-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (6).

[0134] ESI-MS m / z:592.3[M+H] + .

[0135] 1H NMR (400MHz, DMSO-d6) δ12.20(s,1H),10.71(s,1H),8.64(t,J=5.6Hz,1H),8.19(s,1H),8.06(d,J=9.7H z,1H),8.03–7.96(m,2H),7.91(d,J=8.7Hz,2H),7.57–7.48(m,1H),7.17(d,J=2.1Hz,1H),6.77(td,J=1 7.2,10.4Hz,1H),6.11(ddd,J=34.4,16.7,2.6Hz,1H),5.58(ddd,J=77.6,10.4,2.5Hz,1H),4.27–4.13( m,1H),4.05–3.69(m,10H),3.50–3.36(m,2H),2.99–2.62(m,2H),2.32–2.14(m,2H),1.94–1.33(m,5H).

[0136] Example 7 4-(((5-acryloyl-5-azaspiro[2.5]oct-8-yl)amino)methyl)-N-(4-(4-morpholinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (7)

[0137] Step 1: Synthesis of tert-butyl 8-(((2-(methoxycarbonyl)pyridin-4-yl)methyl)amino)-5-azaspiro[2.5]octane-5-carboxylate (7-1)

[0138] 8-Amino-5-azaspiro[2.5]octane-5-carboxylic acid tert-butyl ester (500 mg, 2.2 mmol) was added to a 25 mL reaction bottle and dissolved in 10 mL of acetonitrile. Methyl 4-(chloromethyl)picolinate (688.2 mg, 3.7 mmol) and K2CO3 (456.1 mg, 3.3 mmol) were added in sequence. The temperature was raised to 80°C for reaction for 8 h. The mixture was cooled to room temperature, filtered, rotary evaporated, and purified by column chromatography to obtain 7-1 (423.1 mg, yield: 51.3%) as a white solid.

[0139] ESI-MS m / z:376.2[M+H] + .

[0140] Step 2: Synthesis of 4-(((5-(tert-Butyloxycarbonyl)-5-azaspiro[2.5]octan-8-yl)amino)methyl)picolinic acid (7-2)

[0141] The above-mentioned tert-butyl 8-(((2-(methoxycarbonyl)pyridin-4-yl)methyl)amino)-5-azaspiro[2.5]octane-5-carboxylate (423.1 mg, 1.1 mmol) was dissolved in 5 mL of methanol, cooled in an ice bath, and 2 mL of water and LiOH (81.2 mg, 3.3 mmol) were added. The reaction was completed in an ice-water bath for 2 h, 5 mL of water was added, and dilute hydrochloric acid was added to adjust the pH to 6. The crude product 7-2 (475 mg) was directly used in the next step.

[0142] ESI-MS m / z:362.2[M+H] + .

[0143] Step 3: Synthesis of tert-butyl 8-(((2-((4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)amino)-5-azaspiro[2.5]octane-5-carboxylate (7-3)

[0144] The above 4-(((5-(tert-butyloxycarbonyl)-5-azaspiro[2.5]octan-8-yl)amino)methyl)picolinic acid (475.3 mg, 1.1 mmol) was dissolved in 5 mL of N,N-dimethylformamide and cooled in an ice bath. 4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)aniline (332.8 mg, 1.1 mmol), 2-(7-azabenzotriazole)-N,N,N', N'-tetramethyluronium hexafluorophosphate (627.1 mg, 1.65 mmol), diisopropylethylamine (425.7 mg, 3.3 mmol), moved to room temperature and reacted for 5 h until the reaction was complete, added 15 mL of water, extracted with EA (10 mL×3), dried over Na2SO4, filtered, concentrated, and purified by column chromatography (dichloromethane / methanol=10 / 1) to obtain a white solid 7-3 (301.9 mg, yield: 43.1%).

[0145] ESI-MS m / z:638.3[M+H] + .

[0146] Step 4: Synthesis of benzyl (S)-2-((2,3-dioxo-3-(4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)methyl)pyrrolidine-1-carboxylate (7-4)

[0147] The above tert-butyl 8-(((2-((4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)amino)-5-azaspiro[2.5]octane-5-carboxylate (301.9 mg, 0.47 mmol) was placed in a 25 mL single-necked bottle, 5 mL of trimethylsilyl chloride was added, and the reaction was carried out at room temperature for 4 h until the reaction was complete. Most of the trimethylsilyl chloride was removed by rotary evaporation, 15 mL of water was added, and the mixture was lyophilized to obtain crude product 7-4 (317.3 mg). ESI-MS m / z: 538.3 [M+H] + .

[0148] Step 5: Synthesis of 4-(((5-acryloyl-5-azaspiro[2.5]octan-8-yl)amino)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (7)

[0149] The above-mentioned benzyl (S)-2-((2,3-dioxo-3-(4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)propoxy)methyl)pyrrolidine-1-carboxylate (317.3 mg, 0.59 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran, cooled in an ice bath, protected by nitrogen, and sodium hydride (28.3 mg, 1.18 mmol) was added. The reaction was maintained at this temperature for 0.5 h, and acryloyl chloride (58.7 mg, 0.65 mmol) was added. The reaction was moved to room temperature for 1 h. After the reaction was complete, dilute hydrochloric acid was placed in an ice-water bath to cool. At the same time, the reaction solution was cooled and then added dropwise with glacial dilute hydrochloric acid. DMF was added to dissolve it, filtered, and purified by high-pressure preparative column to obtain a white solid 7 (58.4 mg, yield: 16.7%).

[0150] ESI-MS m / z:593.3[M+H] + .

[0151] 1H NMR (400MHz, DMSO-d6) δ12.20(s,1H),10.73(s,1H),8.66(d,J=4.9Hz,1H),8.20(d,J=8.5Hz,2H),8.03–7.97(m,2H),7.91( d,J=8.8Hz,2H),7.69–7.63(m,1H),7.16(d,J=1.9Hz,1H),6.79(ddd,J=38.2,16.7,10.5Hz,1H),6.08(dt,J=16.7,2.4Hz,1H ),5.63(ddd,J=13.1,10.3,2.6Hz,1H),3.97–3.83(m,7H),3.76(t,J=4.8Hz,4H),3.70–3.63(m,1H),3.50–3.40(m,1H),3.1 2(d,J=13.6Hz,1H),2.20(s,1H),1.80–1.53(m,2H),0.49(d,J=12.9Hz,1H),0.42(dt,J=9.4,4.7Hz,1H),0.38–0.24(m,2H).

[0152] Example 8 4-(8-Acryloyl-5-oxa-2,8-diazaspiro[3.5]non-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (8)

[0153] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl 5-oxo-2,8-diazaspiro[3.5]nonane-8-carboxylate, and compound 8 was prepared and purified by the steps described in Example 1 to obtain compound 8.

[0154] ESI-MS m / z:595.3[M+H] + .

[0155] 1H NMR (400MHz, DMSO-d6) δ12.2 (s, 1H), 10.73 (s, 1H), 8.68 (d, J = 4.9Hz, 1H), 8.18 (s, 1H), 8. 08(s,1H),8.00(d,J=8.9Hz,2H),7.96–7.88(m,2H),7.58(dd,J=5.0,1.6Hz,1H),7.16(d,J =2.0Hz,1H),6.94–6.70(m,1H),6.16(d,J=16.7Hz,1H),5.75(dd,J=30.5,10.5Hz,1H),3.8 9(t,J=4.8Hz,4H),3.83(s,2H),3.79–3.69(m,6H),3.54(s,4H),3.30(s,2H),2.96(s,2H).

[0156] Example 9 Synthesis of 4-(2-acryloyl-2,6-diazaspiro[3.5]non-6-yl)methyl)-N-(4-(4-morpholinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (9)

[0157] Referring to the preparation method of Example 1, intermediate M1 was reacted with 9-2, and compound 9 was prepared and purified by the steps described in Example 1 to obtain compound 9.

[0158] ESI-MS m / z:593.2[M+H] + .

[0159] 1 H NMR (400MHz, DMSO-d6) δ12.20(d,J=2.1Hz,1H),10.74(s,1H),8.69(d,J=4.9Hz,1H) ,8.16(d,J=17.2Hz,2H),8.05–7.87(m,4H),7.62(dd,J=5.0,1.6Hz,1H),7.17(d,J=2 .1Hz,1H),6.30(dd,J=17.0,10.3Hz,1H),6.07(dd,J=17.0,2.3Hz,1H),5.63(dd,J=1 0.3, 2.3Hz, 1H), 3.99–3.53 (m, 14H), 2.40 (d, J = 37.0Hz, 4H), 1.57 (d, J = 25.2Hz, 4H).

[0160] Example 10 4-(8-Acrylamide-2-oxa-6-azaspiro[3.4]octan-6-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (10)

[0161] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl (2-oxa-6-azaspiro[3.4]octan-8-yl)carbamate, and compound 10 was prepared and purified by the steps described in Example 1 to obtain compound 10.

[0162] ESI-MS m / z:595.3[M+H] +

[0163] 1 H NMR (400MHz, DMSO-d6) δ12.30(s,1H),10.75(s,1H),8.70-8.69(d,J=4Hz,1H),8.29-8.27(d,J=8Hz,1 H),8.18(s,1H),8.12(s,1H),8.05-7.88(m,4H),7.65-7.59(m,1H),7.16(s,1H),6.30-6.28(m,1H),6 .18-6.14(m,1H),5.66-5.63(dd,J=8,4Hz,1H),4.61-4.60(d,J=4Hz,1H),4.54-4.48(m,2H),4.42-4. 40(d,J=8Hz,1H),4.30-4.28(d,J=8Hz,1H),3.90-3.88(m,4H),3.78-3.74(m,6H),3.05-2.83(m,4H).

[0164] Example 11 4-(6-Acryloyl-2,6-diazaspiro[3.4]octan-2-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (11)

[0165] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl 2,6-diazaspiro[3.4]octane-6-carboxylate, and compound 11 was prepared and purified by the steps described in Example 1.

[0166] ESI-MS m / z:579.3[M+H] + .

[0167] 1H NMR (400MHz, DMSO-d6) δ12.20(d,J=2.1Hz,1H),10.74(s,1H),8.68(d,J=5.0Hz,1H),8.19(s,1H),8. 09(d,J=2.1Hz,1H),8.00(d,J=8.8Hz,2H),7.92(d,J=8.8Hz,2H),7.59(dt,J=4.8,2.2Hz,1H),7.16( d,J=2.1Hz,1H),6.56(ddd,J=16.6,10.3,6.0Hz,1H),6.12(ddd,J=16.8,2.5,1.3Hz,1H),5.66(dt,J =10.3,2.9Hz,1H),3.97–3.64(m,12H),3.60–3.49(m,2H),3.26(s,4H),2.07(dt,J=42.9,7.0Hz,2H).

[0168] Example 12 Synthesis of 4-((8-acrylamido-5-azaspiro[2.5]octan-5-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (12)

[0169] Referring to the preparation method of Example 1, intermediate M1 was reacted with 12-2, and compound 12 was prepared and purified by the steps described in Example 1.

[0170] ESI-MS m / z:593.3[M+H] + .

[0171] 1H NMR (400MHz, DMSO-d6) δ12.20(d,J=2.2Hz,1H),10.73(s,1H),8.67(dd,J=4.9,0.8Hz,1H),8.18(s,1H),8.15(t,J=1.2Hz,1H),8. 03–7.97(m,2H),7.95–7.89(m,2H),7.81(d,J=8.4Hz,1H),7.60(dd,J=5.0,1.6Hz,1H),7.16(d,J=2.1Hz,1H),6.29(dd,J=17.0,10 .2Hz,1H),6.07(dd,J=17.0,2.3Hz,1H),5.57(dd,J=10.2,2.3Hz,1H),3.94–3.85(m,4H),3.76(t,J=4.9Hz,5H),3.64(d,J=6.6Hz, 2H),2.73(s,1H),2.42(d,J=9.4Hz,2H),2.30–2.18(m,2H),1.81–1.64(m,2H),0.60–0.51(m,1H),0.34–0.27(m,1H),0.21(s,2H).

[0172] Example 13 N-(4-(4-(((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-acryloyl-2,6-diazaspiro[3.5]nonan-2-yl)methyl)picolinamide (13)

[0173] Step 1: tert-Butyl 2-((2-((4-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)carbamoyl)pyridin-4-yl)methyl)-2,6-diazaspiro[3.5]nonane-6-carboxylate (13-3)

[0174] Intermediate (M10) (160 mg, 0.28 mmol) and intermediate (13-2) (69 mg, 0.51 mmol) were dissolved in 5 mL of acetonitrile, and N,N-diisopropylethylamine (330 mg, 2.56 mmol) was added. The mixture was allowed to react at 80°C for 16 h. LCMS confirmed the reaction was complete. The product was concentrated to yield 200 mg of crude product, which was then purified using a spherical reverse-phase medium-pressure column (C18, 40 g) in a 30% gradient of 0.5‰ trifluoroacetic acid / acetonitrile to afford intermediate (13-3). After lyophilization, 100 mg of a yellow solid was obtained in a 56.4% yield.

[0175] Step 2: 4-((2,6-diazaspiro[3.5]nonan-2-yl)methyl)-N-(4-(4-(((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (13-4)

[0176] The above intermediate (13-3) (100 mg, 0.15 mmol) was dissolved in trifluoroacetic acid (3 ml) and reacted at room temperature for 1 h. The reaction was completed after LC / MS detection. The mixture was concentrated to obtain 100 mg of crude intermediate (13-4) as a yellow oil.

[0177] Step 3: N-(4-(4-(((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-acryloyl-2,6-diazaspiro[3.5]nonan-2-yl)methyl)picolinamide (13)

[0178] Intermediate (13-4) (100 mg, crude) and N,N-diisopropylethylamine (198.5 mg, 1.53 mmol) were dissolved in 3 ml of THF and stirred at 0°C for 10 min. Acryloyl chloride (20.8 mg, 0.23 mmol) was added, and the mixture was stirred at 0°C for 1 h. LC-MS confirmed the completion of the reaction. Reverse medium-pressure preparative purification was performed using 0.05 mM ammonium bicarbonate / acetonitrile, followed by a 5‰ trifluoroacetic acid / acetonitrile gradient to obtain the final product 13. After lyophilization, 2.2 mg of a yellow solid was obtained, with a yield of 1.3% and a purity of 96.36%.

[0179] ESI-MS m / z:605.3[M+H] +

[0180] 1H NMR (400MHz, DMSO-d6) δ12.12(s,1H),10.72(s,1H),8.67-8.66(d,J=4.92Hz,1H),8.12(s,1H),8.06(s,1H),7.9 9-7.97(m,2H),7.90-7.86(m,2H),7.56(s,1H),7.03(s,1H),6.96-6.69(m,1H),6.20-6.00(t,J=16.64Hz,1H),5. 66-5.63(d,J=10.44Hz,1H),5.23(s,1H),4.75(s,1H),3.94-3.82(m,2H),3.81-3.61(m,6H),3.49-3.43(m,2H),3 .09-2.99(m,2H),2.91-2.86(m,2H),2.00-1.95(m,1H),1.94-1.87(m,1H),1.83-1.70(m,2H),1.52-1.37(m,2H).

[0181] Example 14 N-(4-(4-(((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-acryloyl-2,6-diazaspiro[3.5]nonan-2-yl)methyl)picolinamide (14)

[0182] Referring to the preparation method of Example 13, intermediate M10 was reacted with 14-2, and compound 14 was prepared and purified by the steps described in Example 13.

[0183] ESI-MS m / z:605.2[M+H] +

[0184] 1H NMR (400MHz, DMSO-d6) δ12.12(s,1H),10.72(s,1H),8.67-8.66(d,J=4.92Hz,1H),8.12(s,1H),8.06(s,1H),7 .99-7.97(m,2H),7.90-7.86(m,2H),7.56(d,1H),7.03(s,1H),6.96-6.69(m,1H),6.16-6.05(t,J=16.64Hz,1H ),5.66-5.63(dd,J=41.72,10.43Hz,1H),5.23(s,1H),4.75(s,1H),4.05-3.56(m,8H),3.50-3.40(m,2H),3.1 2-2.96(m,2H),2.92-2.78(m,2H),2.06-1.96(m,1H),1.94-1.85(m,1H),1.82-1.70(m,2H),1.52-1.35(m,2H).

[0185] Example 15 Synthesis of 4-(2-acryloyl-2,6-diazaspiro[3.4]octan-6-yl)methyl)-N-(4-(4-morpholinyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (15)

[0186] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate, and compound 15 was prepared and purified by the steps described in Example 1 to obtain compound 15.

[0187] ESI-MS m / z:579.3[M+H] + .

[0188] 1H NMR (400MHz, DMSO--d6) δ12.20(d,J=2.1Hz,1H),10.74(s,1H),8.68(d,J=5.0Hz,1H),8.18(s,1H),8.12(d,J =1.9Hz,1H),8.00(d,J=8.9Hz,2H),7.94–7.88(m,2H),7.61(dd,J=5.0,1.7Hz,1H),7.16(d,J=2.2Hz,1H),6. 29(dd,J=17.0,10.3Hz,1H),6.08(dd,J=17.0,2.3Hz,1H),5.64(dd,J=10.3,2.3Hz,1H),4.15(s,2H),3.88(d d,J=9.9,5.0Hz,6H),3.76(t,J=2.7Hz,6H),2.79–2.69(m,2H),2.59(q,J=6.6Hz,2H),2.07(t,J=7.1Hz,2H).

[0189] Example 16 Synthesis of N-(4-(4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-acryloyl-2,6-diazaspiro[1.5]nonan-2-yl)methyl)picolinamide (16)

[0190] Referring to the preparation method of Example 13, intermediate M10 was reacted with 16-1 and prepared and purified by the steps described in Example 13 to obtain compound 16.

[0191] ESI-MS m / z:633.3[M+H] + .

[0192] Example 17 Synthesis of 4-(((3aS,7aS)-1-acryloyloctahydro-4H-pyrrolo[3,2-b]pyridin-4-yl)methyl)-N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)picolinamide (17)

[0193] Referring to the preparation method of Example 1, intermediate M1 was reacted with (3aS,7aS)-octahydro-1H-pyrrolo[3,2-b]pyridine-1-carboxylic acid tert-butyl ester, and prepared and purified by the steps described in Example 1 to obtain compound 17.

[0194] ESI-MS m / z:593.3[M+H] + .

[0195] 1H NMR (400MHz, DMSO-d6) δ12.25–12.16(m,1H),10.74(s,1H),8.68(d,J=4.9Hz,1H),8.19–8.13(m,2H),8.00(d,J=8.8Hz,2H),7.94–7. 88(m,2H),7.66–7.61(m,1H),7.16(d,J=1.9Hz,1H),6.59(ddd,J=48.4,16.7,10.3Hz,1H),6.13(ddd,J=16.8,4.1,2.4Hz,1H),5.64(d dd,J=10.0,7.3,2.5Hz,1H),4.04(dq,J=20.4,7.1,6.6Hz,1H),3.90(dd,J=10.3,5.6Hz,5H),3.81(s,1H),3.75(dd,J=10.4,5.7Hz,5 H),3.51(ddq,J=23.9,12.6,6.5Hz,2H),2.44(d,J=18.1Hz,2H),2.14(dt,J=35.3,10.7Hz,1H),2.00–1.71(m,2H),1.60–1.21(m,3H).

[0196] Example 18 Synthesis of N-(4-(4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-acetyl-2,6-diazaspiro[1.5]nonan-2-yl)methyl)picolinamide (18)

[0197] Referring to the preparation method of Example 13, intermediate M10 was reacted with 2-oxa-6-azaspiro[3.3]heptane hemioxalate, and compound 18 was prepared and purified by the steps described in Example 13 to obtain compound 18.

[0198] ESI-MS m / z:605.2[M+H]+

[0199] Example 19 Synthesis of N-(4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)-4-((6-propyl-2,6-diazaspiro[3.5]nonan-2-yl)methyl)picolinamide (19)

[0200] Referring to the preparation method of Example 1, intermediate M1 was reacted with tert-butyl 2,6-diazaspiro[3.5]nonane-6-carboxylate, and compound 19 was prepared and purified by the steps described in Example 1 to obtain compound 19.

[0201] ESI-MS m / z:591.3[M+H]+ .

[0202] 1 H NMR (400MHz, DMSO-d6) δ12.20(s,1H),10.73(d,J=2.7Hz,1H),8.67(d,J=5.0Hz,1H),8.18(s,1H),8.11–7.87(m,5H),7.56(d,J=4.9Hz,1H),7.16(d, J=2.2Hz,1H),3.95–3.63(m,12H),3.61(t,J=5.6Hz,1H),3.42(t,J=5.6Hz ,1H),3.19–3.00(m,3H),2.88(s,2H),1.76(d,J=6.0Hz,2H),1.24(s,2H).

[0203] Example 20 4-(7-Acryloyl-2,7-diazaspiro[4.4]nonan-2-yl)methyl)-N-(5-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)pyridin-2-yl)pyridineamide (20)

[0204] Step 1: Methyl 4-(chloromethyl)picolinate (20-1)

[0205] Methyl 4-(hydroxymethyl)picolinate (350 mg, 2.1 mmol) was dissolved in anhydrous DCM. 2.4 g of thionyl chloride was added dropwise at room temperature. The mixture was allowed to react at room temperature for 16 h. TLC analysis (PE / EA = 2 / 1) indicated the reaction was complete. The mixture was then concentrated to dryness to afford 450 mg of a yellow oil (yield: 100%).

[0206] ESI-MS m / z:186[M+H] + .

[0207] Step 2: tert-Butyl 7-((2-(methoxycarbonyl)pyridin-4-yl)methyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (20-2)

[0208] The above intermediate 20-1 (450 mg, 2.1 mmol) was dissolved in anhydrous DMF, and tert-butyl 2,7-diazoaspirin[4.4]nonane-2-carboxylate (570 mg, 2.52 mmol), potassium carbonate (870 mg, 6.3 mmol), and potassium iodide (35 mg, 0.21 mmol) were added at room temperature and reacted overnight at room temperature. The reaction was detected by LC-MS. The product was purified and separated by column chromatography (DCM / MeOH = 20 / 1) to obtain 520 mg of the product (yield: 66.07%).

[0209] ESI-MS m / z:376.2[M+H] + .

[0210] Step 3: 4-(7-(tert-Butyloxycarbonyl)-2,7-diazaspiro[4.4]nonan-2-yl)methylpyridinic acid (20-3)

[0211] The above intermediate 20-2 (520 mg, 1.39 mmol) was dissolved in THF, and an equal volume of water was added. Lithium hydroxide (100 mg, 4.16 mmol) was added at room temperature. The reaction was allowed to react at room temperature for 6 h. The reaction progress was monitored by TLC. After completion of the reaction, the THF was removed by rotary evaporation, and the water was lyophilized to obtain 622 mg of the crude product as a gray solid (yield: 100%). ESI-MS m / z: 362.2 [M+H] + .

[0212] Step 4: Tert-butyl 7-(2-((5-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)pyridin-2-yl)carbamoyl)pyridin-4-yl)methyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (20-4)

[0213] The above intermediate 20-3 (620 mg, 1.39 mmol), M1 (494 mg, 1.67 mmol), HATU (635 mg, 1.67 mmol), and pyridine (330 mg, 4.17 mmol) were dissolved in DMF and reacted at room temperature for 16 h. The reaction was completed after LC-MS detection. The product was diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was recovered by rotary evaporation to obtain 1.1 g (yield: 100%) of a crude yellow oil.

[0214] ESI-MS m / z:640.3[M+H] + .

[0215] Step 5: 4-(2,7-diazaspiro[4.4]nonan-2-yl)methyl)-N-(5-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)pyridin-2-yl)picolinamide (20-5)

[0216] The above intermediate 20-4 (1.1 g, 1.39 mmol) was dissolved in acetonitrile (10 ml), and TMS-Cl (3 ml) was added. The reaction was allowed to react at room temperature for 2 h. The reaction was detected by LC-MS. The product was filtered, and the filter cake was washed with acetonitrile and dried to obtain 430 mg of the product as a gray solid (yield: 57.33%).

[0217] ESI-MS m / z:540.3[M+H] + .

[0218] Step 6: 4-(7-Acryloyl-2,7-diazaspiro[4.4]nonyl-2-methyl)-N-(5-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)pyridin-2-yl)pyridineamide (20)

[0219] Intermediate 20-5 (150 mg, 0.28 mmol) was added to dry NMP (3 ml). Sodium hydride (22 mg, 0.56 mmol) was added at room temperature and stirred for 30 minutes. Acryloyl chloride (25 mg, 0.28 mmol) was then added and allowed to react for 2 hours. LC-MS confirmed the reaction was complete. The sodium hydride was quenched with ice water in an ice bath and purified by high-pressure preparative liquid chromatography. After lyophilization, 6.5 mg of an off-white product was obtained (yield: 3.95%).

[0220] ESI-MS m / z:594.3[M+H] + .

[0221] 1 H NMR (400MHz, DMSO-d6) δ12.37(s,1H),10.52(s,1H),8.99–8.93(m,1H),8.70(d,J=4.9Hz,1H),8.40(dd,J=8.7,2 .4Hz,1H),8.33(dd,J=8.7,1.9Hz,1H),8.21(s,1H),8.17(s,1H),7.67(dd,J=5.0,1.7Hz,1H),7.32(d,J=1.7Hz, 1H),6.56(ddd,J=16.9,10.3,5.2Hz,1H),6.11(ddd,J=16.8,5.0,2.4Hz,1H),5.68–5.61(m,1H),3.91(t,J=4.8H z,4H),3.77(dd,J=9.4,4.4Hz,6H),3.62–3.35(m,4H),2.73–2.53(m,2H),2.48-2.44(m,2H),2.00–1.72(m,4H).

[0222] Test Example 1 Cell experiment (MV4-11 cell proliferation inhibition experiment)

[0223] Cell viability was determined by CCK-8 reagent

[0224] Day 1: Cell plating

[0225] 1) Collect the cell suspension in the culture flask into a 15 mL centrifuge tube and centrifuge the cell suspension at 1000 rpm for 4 minutes.

[0226] 2) Discard the supernatant from the centrifuge tube and add an appropriate amount of fresh complete culture medium (IMDM + 10% FBS + 1% P / S) to resuspend the cells. Take 20 μL of the cell suspension and add 20 μL of trypan blue. Count the cells using a CounterStar.

[0227] 3) Calculate the required volume of cell suspension and complete medium based on the viable cell density and the number of cells required for plating. The plating density of MV4-11 cells is 1*10 4 per well, 100 μL per well.

[0228] 4) Use an electric dispenser to pipette 100 μL of the cell suspension into a 96-well plate. Add 200 μL of complete culture medium to the blank control wells. Add an appropriate volume of PBS to the surrounding wells to prevent evaporation. Place the 96-well plate in an incubator and incubate overnight.

[0229] Day 2: Compound Preparation

[0230] 1) Take out the compound dissolved in DMSO and perform a serial dilution (96-well PCR plate dilution, from left to right: column A, column B, etc., to column I).

[0231] Column A has an initial concentration of 1 mM;

[0232] B: dilute 2 μL of A to 8 μL of DMSO to obtain 200 μM;

[0233] C: Dilute 2 μL of B into 8 μL of DMSO to obtain 40 μM;

[0234] D. Dilute 2 μL of C to 8 μL of DMSO to obtain 8 μM.

[0235] E was diluted from D, 2 μL to 8 μL DMSO to obtain 1.6 μM;

[0236] F took E and diluted 2 μL into 8 μL DMSO to obtain 0.32 μM;

[0237] G took F and diluted 2 μL into 8 μL DMSO to obtain 0.064 μM;

[0238] H took G and diluted 2 μL into 8 μL DMSO to obtain 0.0128 μM;

[0239] I took H and diluted 2 μL into 8 μL DMSO to obtain 0.00256 μM;

[0240] Centrifuge at 1000 rpm for 1 min.

[0241] 2) 4× compound preparation: (dilution in 96-well cell culture plate)

[0242] Use a 10 μL manual dispenser to dispense 2.4 μL of cells from columns A through I into 197.6 μL of complete medium to obtain final concentrations of 12,000, 2,400, 480, 96, 19.2, 3.84, 0.768, 0.1536, and 0.03072 nM, respectively. Add 2.4 μL of DMSO to 197.6 μL of complete medium as a control. Shake on a shaker at 500 rpm for 5 min.

[0243] Compound treatment:

[0244] 1) Using a manual dispenser set to 50 μL, pipette the prepared 4X compound solution into the corresponding wells, with a total incubation volume of 200 μL per well. The final concentrations of the compounds in the 96-well plate are 3 μM, 0.6 μM, 120 nM, 24 nM, 4.8 nM, 0.96 nM, 0.192 nM, 0.0384 nM, and 0.00768 nM. Shake on a shaker at 500 rpm for 5 minutes.

[0245] 2) Place the prepared 96-well plate in a CO2 incubator and culture for 72 hours.

[0246] Day 5: Result test:

[0247] 1) Using a dispenser, set the volume to 20 μL and add 20 μL / well of CCK-8 detection solution.

[0248] 2) Place the 96-well cell culture plate in a CO2 incubator and incubate for 4 hours.

[0249] 3) Use BMG microplate reader to read the absorbance at 450 nm and calculate the inhibition rate. GraphPad calculates IC 50 value.

[0250] Table 1 below provides the data on the inhibition of MV4-11 cell proliferation by some test compounds (IC 50: nM). “n / a” means data not available;

[0251] Table 1 Inhibitory effects of representative compounds on MV4-11 cell proliferation

[0252] The test results show that compounds 1-4, 6-9, 11, 13-15, 17, 19, and 20 of the present invention have a good inhibitory effect on MV4-11 cells. Test Example 2: Pharmacokinetic study of oral administration in rats

[0253] 1. Test principle

[0254] SD rats were used as test animals, and the LC-MS / MS method was used to determine the plasma concentration of the compound of the present application at different times after oral administration to the rats, to obtain the pharmacokinetic parameters of the compound of the present application in rats, and to study its pharmacokinetic characteristics.

[0255] 2. Test materials

[0256] 2.1 Compounds:

[0257] Positive drug: BMF-219, provided by Chengdu Yuandong Biopharmaceutical Co., Ltd., batch number D0060-11-001-20230608;

[0258] Test drugs: Compound 2; Compound 3; Compound 4; Compound 6; Compound 7;

[0259] 2.2 Test instruments:

[0260] Shimadzu LC-30A AB API4500 tandem mass spectrometer, vacuum blood collection tubes, blood collection needles, filter paper, syringes, etc.

[0261] 2.3 Experimental Animals

[0262] Female SD rats weighing 180-220 g were used in each group. The animals were housed in an animal room after purchase and allowed to adapt for at least 3 days before being used in the experiment after passing quarantine.

[0263] 3. Test methods

[0264] 3.1. Grouping: The rats were randomly divided into groups according to Table 1. After grouping, there was no statistical difference in body weight between the groups.

[0265] 3.2. Solvent: po: 10% DMSO+10% Solutol+80% (10% HP-β-CD)

[0266] Table 2 Trial groups and dosing regimens

[0267] 3.2. Blood sample collection and measurement:

[0268] According to Table 2, the rats in each group were gavaged with the corresponding test drugs. Before administration and 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours after administration, a fixed volume of blood was collected from the jugular vein and placed in an EDTA-K2 anticoagulant tube. The blood was centrifuged at 4500 rpm for 10 minutes, and the plasma was separated and placed in a centrifuge tube and frozen at -80°C.

[0269] 3.3 Analysis Method

[0270] The plasma at each time point stored at -80°C was taken out, acetonitrile was added, vortexed at 1500 rpm for 2 min, and centrifuged for 15 min (3500 r / min). A fixed volume of the supernatant was taken for LC-MS / MS analysis.

[0271] 4. Calculation of pharmacokinetic parameters:

[0272] The pharmacokinetic behavior of the test compounds was fitted with a non-compartmental model, and the main pharmacokinetic parameters (T 1 / 2 、T max 、C max , AUC last wait).

[0273] 5. Test results:

[0274] Table 3 Pharmacokinetic parameters of the compounds in the examples

[0275] It can be seen from the test results in Table 3 that, at the same dose, the plasma exposure and maximum blood drug concentration of Compound 2, Compound 3, Compound 4, Compound 6, and Compound 7 of the present application are superior to those of BMF-219 after oral administration, indicating that the compounds obtained in the examples of the present application have significantly improved pharmacokinetic properties compared with BMF-219.

[0276] Experimental Example 3: Blood glucose changes in type 1 diabetic rats induced by streptozotocin (STZ)

[0277] 1. Compounds and Reagents

[0278] Test drug: Compound 3 was prepared by the synthesis method of Example 3;

[0279] Positive drug: BMF-219 / pioglitazone, provided by MCE;

[0280] DMSO: purchased from MP Biomedicals;

[0281] Solutol HS15: purchased from Sigma;

[0282] HP-β-CD: purchased from Aladdin;

[0283] Citrate buffer: purchased from Ruji Biotechnology

[0284] 2. Test materials and instruments

[0285] Blood glucose meter (Roche, USA), blood glucose test strips (Roche, USA);

[0286] 3. Experimental Animals

[0287] STZ type I-SD rats, male, were purchased from Zhanyuan Biotechnology (Chengdu) Co., Ltd.

[0288] 4. Test methods and results

[0289] 4.1 Grouping and Dosing

[0290] The model rats were randomly divided into 4 groups according to the pre-measured blood glucose levels, namely, vehicle group, pioglitazone group, BMF-219 group, and compound 3 group, with 8 rats in each group. The first day of administration was marked as D1, and the corresponding test substances were administered according to the following groups:

[0291] Table 4 Note: Solvent (v / v / v): 10% DMSO + 10% Solutol HS15 + 80% (10% HP-β-CD), where 10% HP-β-CD was prepared with citric acid aqueous solution (pH = 3, 50 mM); the solvent for pioglitazone was 0.25% CMC + 1% Tween 80; the solvent for BMF-219 and compound 3 was 10% DMSO + 10% Solutol HS15 + 80% (10% HP-β-CD)

[0292] 4.2 Detection indicators

[0293] On D1 and D8, fasting blood glucose levels were measured (the first drop of blood was discarded and the second drop of blood was tested). After the measurement, the subjects resumed eating.

[0294] The serum biochemistry of each group of animals was measured on D1 and D17.

[0295] 4.3 Test results

[0296] 1) Effects of the test substances on fasting blood glucose in STZ-induced diabetic rats

[0297] In the STZ-induced type 1 diabetes model, after 7 days of treatment, compound 3 significantly reduced fasting blood glucose levels (fasting blood glucose value: 11.46±8.19, P=0.0018) compared with the vehicle group (fasting blood glucose value: 23.71±5.71), as shown in Figure 1.

[0298] 2) Changes in blood biochemical parameters after administration of the test substance to STZ-induced diabetic rats

[0299] As shown in Figure 2, compared with the vehicle control group, the levels of ALT (alkaline phosphatase), AST (aspartate aminotransferase) and r-GT (r-glutamyl transpeptidase) in all drug-treated groups increased to a certain extent. Among them, the activity of liver enzymes of BMF-219 increased the most, and the increase in liver function indicators in the compound 3 group was weaker than that of BMF-219. Total protein (TP) and albumin (ALB) decreased, and urea nitrogen (BUN) increased. There was no significant change in serum creatinine (Scr). The P and Ca levels in the compound 3 group were relatively stable, and the CK level changed little. The CK level in the BMF-219 group increased significantly at D17. This suggests that compound 3 may have a certain degree of renal protective effect. The effects of BMF-219 and compound 3 on TG and TC were not significant. After treatment with compound 3, blood sugar decreased at D17, and its hypoglycemic effect was better than that of BMF-219.

[0300] Experimental Example 4 STZ type II diabetes rat model experimental plan

[0301] 1 Experimental Materials

[0302] 1.1 Model Animals

[0303] High-fat diet (HFD)-fed streptozotocin (STZ)-induced type 2 diabetes (type 2 DM) in Wistar rats

[0304] 1.2 Drugs and reagents

[0305] Pioglitazone, test compound.

[0306] 1.3. Instruments and Equipment

[0307] Blood glucose meter, blood glucose test strips; insulin test kit; biochemical analyzer

[0308] 2 Animal grouping and drug administration

[0309] Wistar rats were randomly divided into a blank control group (n=8, male) and a diabetic model group (n=40, male). The diabetic model rats were randomly divided into a model group, a pioglitazone group, a BMF-219 group, a low-dose compound 3 group, and a high-dose compound 3 group according to the pre-measured blood glucose values, with 8 rats in each group, for a total of 6 groups. After grouping, the rats were weighed and dosed, with the first day of administration designated as D1. The corresponding test substances were administered according to the following groups: the blank control group and the model group were administered with the solvent 10% DMSO + 10% Solutol HS15 + 80% (10% HP-β-CD). Note: Solvent (v / v / v): 10% DMSO + 10% Solutol HS15 + 80% (10% HP-β-CD), where 10% HP-β-CD was prepared with citric acid aqueous solution (pH = 3, 50 mM); the solvent for pioglitazone was 0.25% CMC + 1% Tween 80; the solvent for BMF-219 and compound 3 was 10% DMSO + 10% Solutol HS15 + 80% (10% HP-β-CD)

[0310] 3 Observation items and tests

[0311] 3.1 Clinical Observation: During the trial (after the animals were grouped), the diet and drinking water conditions of all animals, their mental state, changes in their weight, fur color, and changes in their urine and feces were observed.

[0312] 3.2 Fasting blood glucose: Non-fasting blood glucose was measured on the 1st, 8th, 14th and 17th day of the experiment. The effects of different doses of the positive control drug pioglitazone and compound 3 on the blood glucose concentration of type 2 diabetic rats were compared.

[0313] 3.3 OGTT test: On days 15 and 29, 2 g / kg glucose solution (10 mL / kg) was administered by oral gavage. Blood glucose concentration was measured by tail vein blood sampling at the following time points: 0 (before glucose loading), 15, 30, 60, 90, and 120 minutes.

[0314] 3.4 Insulin level determination: Enzyme-linked immunosorbent assay (ELISA) was used to detect the non-fasting insulin levels in rat serum at 1 day, 8 days, 14 days and 17 days.

[0315] 3.5 Blood biochemical test: Use a biochemical analyzer to detect the serum biochemical indicators of rats on the 1st and 17th days.

Claims

1. A compound of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof: in: A is an optionally substituted or unsubstituted 8-15 membered heterocycle, wherein the substituent is selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, -OH, cyano or C1-C4 haloalkyl; C is an optionally substituted or unsubstituted 5-15-membered aromatic ring, an optionally substituted or unsubstituted 5-15-membered aliphatic ring, an optionally substituted or unsubstituted 5-15-membered aliphatic heterocyclic ring, or an optionally substituted or unsubstituted 5-15-membered aromatic heterocyclic ring; R1 is independently selected from 4-9 membered heterocyclyl, wherein the 4-9 membered heterocyclyl may be optionally substituted with one or more -OH, halogen, cyano, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl or C1-C4 haloalkyl; X and Y are each independently selected from -NH- or -C(=O)-, and X and Y are not the same; Z1 and Z2 are each independently selected from -CH2-, -NRz- or a chemical bond; D1, D2, B1 and B2 are each independently selected from N or -CH-, and when Z is -NRz, D can only be -CH; Rz is selected from H or C1-C4 alkyl; G is in for Among them, R g1 , R g2 and R g3 Each is independently absent, H, C1-C4 alkyl, C1-C4 cycloalkyl, C1-C4 alkoxy, C1-C4 aminoalkyl, C1-C4 hydroxyalkyl, halogen, hydroxy, CN or CF3; R2, R3, Rm and Rn are each independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 haloheteroalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C 10 Carbocyclic group, substituted or unsubstituted 3-10 membered heterocyclic group, substituted or unsubstituted 6-12 membered aryl group, substituted or unsubstituted 5-10 membered heteroaryl group, CN, SF5, -C(=O)OR 30 、-C(=O)R 30 、-C(=O)NR 31 R 32 、-NR 33 C(=O)R 34 、-NR 31 R 32 、-OC(=O)R 30 、-OC(=O)NR 31 R 32 、-NR 33 C(=O)NR 31 R 32 、-NR 33 C(=O)OR 30 、-S(=O)NR 31 R 32 、-S(=O)2NR 31 R 32 、-S(=O)R 35 、-S(=O)(=NR 37 )R 35 、-S(=O)2R 35 、-NR 33 S(=O)2R 35 、-S(=O)2NR 31 R 32 、-OR 37 、-SR 37 、-C(=NR 38 )NR 31 R 32 、-C(=NR 38 )R 32 、-NR 33 C(=NR 38 )NR 31 R 32 、-N=NR 38 ; R 30 , R 37 , R 39 and R 40 are each independently selected from hydrogen, halogen, C 1- C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 haloalkyl, C1-C8 haloheteroalkyl, C1-C8 heteroalkyl, C 3- C8 cycloalkyl, 4-10 membered heterocyclic group, C6-C 12 Aryl, 5-12 membered heteroaryl, C1-C8 alkylene-C6-C 12 Aryl and C1-C8 alkylene-(5-10 membered heteroaryl); R 31 , R 32 , R 33 and R 34 Each is independently selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 haloalkyl, C1-C8 haloheteroalkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, 4-10 membered heterocyclyl, C6-C 12 Aryl, 5-12 membered heteroaryl, C1-C8 alkylene-C6-C 12 Aryl and C1-C8 alkylene-(5-10 membered heteroaryl); R 35 Selected from C1-C8 alkyl, C1-C8 alkoxy, C3-C8 cycloalkyl, 4-10 membered heterocyclic group, C6-C 12 Aryl, 5-10 membered heteroaryl, C1-C8 alkylene-C6-C 12 Aryl and C1-C8 alkylene-(5-10 membered heteroaryl); R 36 is selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 haloalkyl, substituted or unsubstituted C1-C6 haloheteroalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C 10 Carbocyclyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 6-12 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, CN, SF5; R 38 is selected from hydrogen, hydroxy, cyano, nitro, -S(=O)R 35 and -S(=O)2R 35 ; m is 0, 1, 2, 3, 4, 5 or 6; n is 0, 1, or 2; p is 0, 1, 2, 3, 4, 5 or 6; G is selected from:

2. The compound of formula (I) according to claim 1, its isomer or a pharmaceutically acceptable salt thereof, characterized in that: The C is phenyl or pyridyl.

3. The compound of formula (I) according to claim 1, its isomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from:

4. A method for preparing a compound of formula (I) as claimed in any one of claims 1 to 3, characterized in that: The steps include: Among them, A, B1, B2, Z1, Z2, D1, D2, G, R1, R2, R3, Rm, Rn, R g1 , R g2 , R g3 , p, m and n are as defined in any one of claims 1 to 3; P is a protecting group selected from: Cbz, Boc, Fmoc, Alloc or Teoc; Hal is a halogen selected from: -I, -Br or -Cl; F is chlorine or hydroxyl; 1) The starting material M-1 and the boronic acid intermediate M-2 are subjected to Suzuki coupling reaction to generate the intermediate compound IA-1; 2) intermediate IA-1 reacts with intermediate M-3 in the presence of a condensation reagent to generate intermediate compound IA-2; 3) Removing the protecting group of intermediate IA-2 under appropriate conditions to generate intermediate compound IA-3; 4) Intermediate IA-3 is reacted with commercially available intermediate M-4 through acylation to obtain the compound of formula (IA).

5. A method for preparing a compound of formula (I) as claimed in any one of claims 1 to 3, characterized in that: The steps include: Among them, A, B1, B2, Z1, Z2, D1, D2, G, R1, R2, R3, Rm, Rn, R g1 , R g2 , R g3 , p, m and n are as defined in any one of claims 1 to 3; P is a protecting group selected from: Cbz, Boc, Fmoc, Alloc or Teoc; Hal is a halogen selected from: -Br ​​or -Cl; F is chlorine or hydroxyl; 1) The starting material M-3 and the boronic acid intermediate M-2 are subjected to Suzuki coupling reaction to generate the intermediate compound IIA-1; 2) intermediate IIA-1 reacts with intermediate M-3 in the presence of a condensation reagent to generate intermediate compound IIA-2; 3) Intermediate IIA-2 is reacted in the presence of an alkaline reagent to generate intermediate compound IIA-3; 4) Removing the protecting group of intermediate IA-3 under appropriate conditions to generate intermediate compound IA-4; 5) Intermediate IA-4 is reacted with commercially available intermediate M-4 through acylation to obtain a compound of formula (IIA).

6. Use of the compound according to any one of claims 1 to 3, its isomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating a disease associated with the Menin-MLL protein.

7. The use according to claim 6, characterized in that The diseases associated with Menin-MLL protein include mixed lineage leukemia (MLL), MLL-related leukemia, MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, rearranged mixed lineage leukemia (MLL-r), leukemia associated with MLL rearrangement or MLL gene rearrangement, acute leukemia, chronic leukemia, lymphoblastic leukemia, lymphocytic leukemia, myeloid leukemia, myeloid leukemia, childhood leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), therapy-related leukemia, myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), myeloproliferative neoplasia (MPN), plasma cell neoplasm, multiple myeloma, myelodysplasia, cutaneous T-cell lymphoma, lymphoid neoplasm, hairy cell leukemia, leukemic meningitis, multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma (malignant lymphoma).

8. Use of the compound according to any one of claims 1 to 3, its isomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating diabetes.

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