Pyrimidine amino compounds as JAK inhibitors and PHD inhibitors

Abstract

The present invention discloses pyrimidine amino compounds as JAK inhibitors and PHD inhibitors. Specifically, the present invention discloses a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, which can be used to treat inflammatory bowel disease.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to pyrimidine amino compounds serving as JAK inhibitors and PHD inhibitors. Background Art

[0002] The JAK family of cellular protein tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an important role in cytokine signal transduction. When cytokines bind to their receptors, the cytokines activate the JAKs, which then phosphorylate the cytokine receptors, thereby creating docking sites for signal transduction molecules, notably members of the signal transducer and activator of transcription (STAT) family that ultimately lead to gene expression. Many cytokines are known to activate the JAK family. These cytokines include interferon IFN family (IFN-α, IFN-β, IFN-ω, limitin, IFN-γ, IL-10, IL-19, IL-20, IL-22), gp130 family (IL-6, IL-11, OSM, LIF, CNTF, NNT-1 / BSF-3, G-CSF, CT-1, leptin, IL-12, IL-23), γC family (IL-2, IL-7, TSLP, IL-9, IL-15, IL-21, IL-4, IL-13), IL-3 family (IL-3, IL-5, GM-CSF), single chain family (EPO, GH, PRL, TPO), receptor tyrosine kinase (EGF, PDGF, CSF-1, HGF), and G-protein coupled receptor (AT1). JAK1 is expressed at different levels in all tissues. Animal studies have shown that JAK1 is essential for the development, function and homeostasis of the immune system. Modulating immune activity by inhibiting JAK1 kinase activity has been shown to be useful in treating a variety of immune disorders.

[0003] Inflammatory bowel disease (IBD) is a chronic disease in which inflammation and ulcers are caused in the intestinal mucosa due to excessive immune response. IBD includes, for example, ulcerative colitis and Crohn's disease. It is known that in the pathological conditions of IBD, the expression of genes related to the barrier function of the gastrointestinal epithelium is induced by hypoxia-inducible factor 1α (HIF-1α). HIF-1α is one of the subtypes of hypoxia-inducible factor α (HIF-α). HIF-α becomes stable in a hypoxic environment (hypoxia), and then it responds to hypoxia and activates the transcription of several genes. In contrast, the proline residues of HIF-α are hydroxylated by prolyl hydroxylases (PHDs) in an oxygen-rich environment (normoxia), and then the HIF-α is degraded by the proteasome pathway. It is known that PHD has 3 subtypes, namely PHD1, PHD2 and PHD3. Summary of the invention

[0004] The invention provides a compound with a novel structure and capable of simultaneously inhibiting JAK1 and PHD2.

[0005] In one aspect of the present invention, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0006]

[0007] Ring A is selected from phenyl, pyridyl or pyrazolyl, wherein the phenyl, pyridyl or pyrazolyl is optionally substituted by 1 halogen;

[0008] L is selected from -(CH2) n1 -NH-(CH2) n2 -、-(CH2) n1 -O-(CH2) n2 -N(CH2CH2NBoc)-or-(CH2) n1 -O-(CH2) n2 -NH-, -(CH2) n1 -NH-(CH2) n2 -、-(CH2) n1 -O-(CH2) n2 -N(CH2CH2NBoc)-, or -(CH2) n1 -O-(CH2) n2 -NH- is optionally substituted with 1 methyl group;

[0009] R 1 is selected from H or halogen;

[0010] n1 and n2 are each independently 0, 1 or 2, and n1 and n2 are not 0 at the same time.

[0011] According to an embodiment of the present invention, ring A is selected from The remaining variables are as defined in the present invention.

[0012] According to an embodiment of the present invention, L is selected from -CH2NH-, -CH2CH2OCH2CH2N(CH2CH2NBoc)-, -CH2N(CH3)-, -CH(CH3)NH- or -CH2CH2OCH2CH2NH-, and the remaining variables are as defined in the present invention.

[0013] According to an embodiment of the present invention, R 1 is selected from H or F, and the remaining variables are as defined in the present invention.

[0014] According to an embodiment of the present invention, the structure of the compound represented by formula (I) is shown as one of the following:

[0015]

[0016] According to an embodiment of the present invention, the structure of the compound represented by formula (I) is as follows:

[0017]

[0018] According to an embodiment of the present invention, the structure of the compound represented by formula (I) is as follows:

[0019]

[0020] According to an embodiment of the present invention, the structure of the compound represented by formula (I) is shown as one of the following:

[0021]

[0022] In another aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises the aforementioned compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0023] In another aspect of the present invention, the present invention provides the use of the aforementioned compound or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of a drug for treating diseases mediated by PHD2 and JAK1.

[0024] In some embodiments of the present invention, the PHD2 and JAK1 mediated disease is inflammatory bowel disease.

[0025] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or with organic acids, such as nitric acid, phosphoric acid, carbonic acid, etc.; organic acids such as propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions; or coordination compounds formed with organic bases, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. In addition to the form of salts, compounds provided by the present invention also exist in prodrug forms. Prodrugs of compounds described herein easily undergo chemical changes under physiological conditions to be converted into compounds of the present invention. In addition, prodrugs can be converted to compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0026] As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

[0027] The compounds of the invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0028] The term "optionally substituted" means that the group may be substituted or unsubstituted, and unless otherwise specified, the type and number of the substituents may be any on the basis of what is chemically feasible.

[0029] The compound shown in formula (I) of the present invention can be prepared using a synthetic method known in the art or using a method known in the art in combination with the method described in the present invention. The solvent, temperature and other reaction conditions given in the present invention are exemplary and can be changed according to methods well known in the art. The embodiment compounds described in the present invention can be synthesized according to the method described in the embodiment using appropriate starting materials according to their specific structure, or can be synthesized using a similar method described in the embodiment. The starting materials used to synthesize the embodiment compounds of the present invention can be prepared by known synthetic methods or similar methods described in the literature or obtained from commercial sources. The compound can be further separated by methods well known in the art, such as crystallization, chromatography, etc. to obtain its stereoisomers as needed, and the separation conditions are easily obtained by those skilled in the art through conventional means or limited experiments. As a further illustration, the compound of formula (I) of the present invention can be synthesized using the following method, wherein the solvent, temperature and other reaction conditions in each step can be the same or similar to those described in the following embodiments, or reaction conditions known in the art can be used. DETAILED DESCRIPTION

[0030] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0031] The present invention is described in detail below by way of examples, but it is not intended to limit the present invention in any adverse way. The present invention has been described in detail herein, and its specific embodiments are also disclosed. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0032] The following abbreviations are used throughout this disclosure.

[0033] THF represents tetrahydrofuran, MeOH represents methanol, Pd2(dba)3 represents tris(dibenzylideneacetone)dipalladium, Cs2CO3 represents cesium carbonate, Na2SO4 represents sodium sulfate, DMA represents dimethylacetamide, DMF represents N,N-dimethylformamide, Pd(Oac)2 represents palladium acetate, Boc represents tert-butyloxycarbonyl, Ts represents p-toluenesulfonyl, DCM represents dichloromethane, DMSO represents dimethyl sulfoxide, DIPEA represents N,N-diisopropylethylamine, EtOAc represents ethyl acetate, TFA represents trifluoroacetic acid, HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, Cbz represents benzyloxycarbonyl, and Xantphos represents 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.

[0034] Example 1

[0035]

[0036] Step 1:

[0037] Compound 1-1 (1 g, 3.079 mmol) was dissolved in NMP (2 mL), 4-aminobenzylcarbamic acid benzyl ester (0.87 g, 3.387 mmol), Pd2(dba)3 (0.28 g, 0.308 mmol) and Cs2CO3 (2.01 g, 6.157 mmol), Brettphos (0.49 g, 0.616 mmol) were added, and the mixture was reacted at 100 ° C for 2 hours under a nitrogen atmosphere. The reaction was quenched with water (50 mL), extracted with ethyl acetate (30 mL × 3), and dried over Na2SO4. After filtration, it was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH / DCM=1:9) to obtain compound 1-2.

[0038] Step 2:

[0039] Compound 1-2 (1 g, 1.836 mmol) was dissolved in MeOH (10 mL), 10% Pd / C (0.20 g, 1.836 mmol) was added, and the mixture was reacted for 2 hours under a hydrogen atmosphere. After filtration and concentration, the crude product 1-3 was directly used in the next step. LCMS: 411.2 [M+H] + .

[0040] Step 3:

[0041] Compound 1-3 (500 mg, 1.218 mmol) was dissolved in DMSO (3 mL), 2-chloro-N-((6-cyanopyridin-3-yl)methyl)-5-hydroxy-1,7-naphthyridine-6-carboxamide (206.90 mg, 0.609 mmol) and DIPEA (0.503 mL, 3.045 mmol) were added, and the mixture was reacted at 100 ° C for 18 hours. The reaction was quenched with water (50 mL) and extracted with EtOAc (30 mL×3). The organic phase was washed with saturated brine (30 mL×3), dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (0-8% MeOH / DCM) to obtain compound 1-4. LCMS: 714.4 [M+H] + .

[0042] Step 4:

[0043] Compound 1-4 (200 mg, 0.280 mmol) was added to EtOAc / HCl (6 mL) and reacted at room temperature for 1 hour. The mixture was filtered, washed with EtOAc (10 mL×3), and dried under reduced pressure to obtain compound 1-5. LCMS: 614.3 [M+H] + .

[0044] Step 5:

[0045] Compound 1-5 (60 mg, 0.098 mmol) was dissolved in DMF (3 mL), and DIPEA (58.31 mg, 0.490 mmol), T3P (50% EtOAc solution) (155.82 mg, 0.490 mmol) and (1S)-2,2-difluorocyclopropane-1-carboxylic acid (11.93 mg, 0.098 mmol) were added, and the mixture was reacted at room temperature for 1 hour. The reaction was diluted with EtOAc (50 mL) and water (30 mL). The organic phase was washed with saturated brine (30 mL). The mixture was concentrated under reduced pressure, and the crude product was separated and purified by preparative liquid phase to obtain compound 1. LCMS: 718.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ13.28(s,1H),9.74(t,J=6.2Hz,1H),9.08(s,1H),8.76( s,1H),8.39(s,1H),8.18-8.05(m,2H),8.03-7.93(m,3H),7.71-7.61(m,2H),7. 29(d,J=8.4Hz,2H),7.07(d,J=9.2Hz,1H),6.22(dd,J=10.7,6.1Hz,1H),4.75-4 .51(m,6H),4.15(s,2H),3.24-2.85(m,3H),1.99-1.63(dd,J=96.0,47.5Hz,6H).

[0046] Example 2

[0047]

[0048] Step 1:

[0049] Compound 2-1 (3 g, 9.453 mmol) was dissolved in DMF (30 mL), 4-nitropyrazole (1.56 g, 13.764 mmol) and Cs2CO3 (6.90 g, 21.176 mmol) were added, and the mixture was reacted at 80°C for 1 hour. The reaction was quenched with water (100 mL) and extracted with EtOAc (100 mL×3). The organic phase was washed with saturated brine (40 mL×3), dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (30% EtOAC / petroleum ether) to obtain compound 2-2. LCMS: 323 [M+Na] + .

[0050] Step 2:

[0051] Compound 2-2 (2.9 g, 9.657 mmol) was added to HCl / EtOAc (10 mL) and reacted at room temperature for 1 hour. The mixture was filtered, washed with EtOAc (3 mL), and dried to obtain compound 2-3. LCMS: 201.2 [M+H] + .

[0052] Step 3:

[0053] Compound 2-3 (750 mg, 3.746 mmol) was dissolved in DMF (10 mL), tert-butyl (2-bromoethyl) carbamate (839.54 mg, 3.746 mmol), KI (434.57 mg, 3.746 mmol) and NaHCO3 (944.17 mg, 11.239 mmol) were added, and the mixture was reacted at 50°C for 5 hours. The reaction was quenched with water (50 mL) and extracted with EtOAc (40 mL×3). The organic phase was washed with saturated brine (40 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain compound 2-4. LCMS: 344.2 [M+H] + .

[0054] Step 4:

[0055] Compound 2-4 (850 mg, 2.475 mmol) was dissolved in THF (8 mL), H2O (8 mL), NaHCO3 (311.94 mg, 3.713 mmol) and CbzCl (0.348 mL, 2.475 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction was quenched with EtOAc (100 mL) and water (40 mL). The organic phase was washed with saturated brine (30 mL) and dried to obtain compound 2-5.

[0056] Step 5:

[0057] Compound 2-5 (500 mg, 1.047 mmol) was added to MeOH (12 mL), and then NaBH4 (158.44 mg, 4.188 mmol) and NiCl2 (135.07 mg, 1.047 mmol) were added, and the mixture was reacted at room temperature for 1 hour. The reaction was quenched with EtOAc (100 mL) and water (40 mL). The organic phase was washed with saturated brine, dried and concentrated. The crude product was purified by column chromatography (0-10% MeOH / DCM) to obtain compound 2-6. LCMS: 448.2 [M+H] + .

[0058] Step 6:

[0059] Compound 2-6 (200 mg, 0.447 mmol) and 2-6' (176.30 mg, 0.536 mmol) were added to MeCN (6 mL), TsOH (25.47 mg, 0.134 mmol) was added, and the mixture was reacted at 50°C for 6 hours. After the reaction was quenched, it was washed with saturated brine and concentrated under reduced pressure. The crude product was purified by column chromatography (0-5% MeOH / DCM) to obtain compound 2-7. LCMS: 740.4 [M+H] + .

[0060] Step 7:

[0061] 10% Pd / C (30 mg) was added to a solution of compound 2-7 (300 mg, 0.405 mmol) in MeOH (8 mL), and the mixture was reacted for 3 hours under a hydrogen atmosphere. The mixture was filtered and concentrated to obtain compound 2-8. LCMS: 606.4 [M+H] + .

[0062] Step 8:

[0063] Compound 2-8 (140 mg, 0.231 mmol) was dissolved in DMSO (0.5 mL), 2-chloro-N-((6-cyanopyridin-3-yl)methyl)-5-hydroxy-1,7-naphthyridine-6-carboxamide (78.53 mg, 0.231 mmol) and DIPEA (0.229 mL, 1.387 mmol) were added, and the reaction was carried out at 100 ° C for 16 hours. The reaction was quenched with water (50 mL) and extracted with EtOAc (30 mL×3). The organic phase was washed with saturated brine (40 mL), dried over Na2SO4, concentrated under reduced pressure, and the crude product was separated and purified by preparative liquid phase to obtain compound 2. LCMS: 909.4 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ13.29(s,1H),9.71(s,1H),8.84-8.80(m,2H),8.36(s,1H), 8.15(d,J=8.8Hz,1H),8.00(s,2H),7.90(d,J=5.5Hz,1H),7.75(s,1H),7.45(s,1H) ,7.25(s,1H),6.91(s,1H),6.08(s,1H),4.71-4.54(m,4H),4.19-4.15(m,3H),3.77 -3.62(m,8H),3.20-3.17(m,3H),3.01-2.79(m,2H),1.98-1.63(m,6H),1.30(s,9H).

[0064] Example 3

[0065]

[0066] Step 1:

[0067] Compound 3-1 (1.00 g, 7.34 mmol) and 2-chloro-N-((6-cyanopyridin-3-yl)methyl)-5-hydroxy-1,7-naphthyridine-6-carboxamide (2.49 g, 7.34 mmol) were dissolved in THF (10 mL) and DMF (5 mL), TEA (1.53 mL, 11.0 mmol) was added, and the mixture was reacted at 70 ° C for 12 hours. The reaction was quenched with water (20 mL), extracted with EtOAc (20 mL×3), and the organic phase was dried over Na2SO4, filtered and concentrated to obtain compound 3-2. LCMS: 440.2 [M+H] + .

[0068] Step 2:

[0069] Compound 3-2 (200 mg, 0.455 mmol) and 1-1 (147 mg, 0.455 mmol) were dissolved in DMA (2 mL), Pd(OAc)2 (10.2 mg, 0.0460 mmol), Xantphos (52.6 mg, 0.091 mmol) and Cs2CO3 (296.5 mg, 0.910 mmol) were added, and the mixture was stirred at 130 ° C for 2 hours under nitrogen atmosphere. The reaction was quenched with water (10 mL), extracted with EtOAc (10 mL×3), and the organic phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by preparative TLC to obtain compound 3-3. LCMS: 728.5 [M+H] + .

[0070] Step 3:

[0071] Compound 3-3 (100 mg, 0.137 mmol) was dissolved in DCM (1.5 mL), TFA (0.5 mL, 0.137 mmol) was added, and the mixture was reacted at room temperature for 2 hours. After concentration, compound 3-4 was obtained. LCMS: 628.5 [M+H] + .

[0072] Step 4:

[0073] Compound 3-4 (80.0 mg, 0.127 mmol) and (1S)-2,2-difluorocyclopropane-1-carboxylic acid (15.5 mg, 0.127 mmol) were dissolved in DCM (1 mL), HATU (58.1 mg, 0.153 mmol) and DIPEA (0.042 mL, 0.255 mmol) were added, and the mixture was reacted at room temperature for 2 hours. After concentration to obtain a crude product, it was separated and purified by preparative liquid phase to obtain compound 3. LCMS: 732.5 [M+H] + . 1HNMR(400MHz,DMSO)δ13.35(s,1H)9.75(t,J=5.94Hz,1H)9.07(s,1H)8.76(s,1H)8.44(s,1H)8.26 (d,J=9.38Hz,1H)7.93-8.01(m,3H)7.66(dd,J=8.44,4.19Hz,2H)7.35(d,J=9.38Hz,1H)7.18(d,J= 8.38Hz,2H)6.21(dd,J=10.82,6.19Hz,1H)4.90(s,2H)4.63(d,J=6.13Hz,2H)3.97-4.25(m,2H)3.2 2(s,3H)3.08-3.20(m,2H)2.82-3.04(m,2H)1.92-2.06(m,2H)1.68-1.89(m,4H)1.57-1.67(m,1H).

[0074] Example 4

[0075]

[0076] Prepared in a similar manner to Example 1. LCMS: 733.5 [M+H] + . 1 HNMR(400MHz,MeOD)δ12.12(brs,1H),9.81-9.68(m,1H),8.78-8.74(m,2H),8.67(s, 1H),8.39(s,2H),8.25(d,J=2.0Hz,1H),7.99(s,2H),7.59(d,J=3.5Hz,1H),7.33(br d,J=8.6Hz,1H),7.01(d,J=3.6Hz,1H),4.62(br d,J=6.3Hz,2H),3.80(br d,J=8.3Hz,2H),3.72(br s,2H),3.63(br d,J=8.3Hz,2H),3.55-3.48(m,3H),3.22(s,3H),2.83(br t,J=6.1Hz,2H).

[0077] Example 5

[0078]

[0079] Prepared in a similar manner to Example 1. LCMS: 719.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ13.28(s,1H),9.75(s,1H),9.28(s,1H),8.82-8.80(m,2H),8.37(s,1H),8.20-8.18(m,2H),8.06-8.04(m,4H),7.33 (d,J=8.6Hz,1H),7.14(d,J=9.5.12Hz,1H),6.33-6.17(m,1H),4.94-4.48(m,7H),4.12-4.11(s,2H),3.20-2.85(m,3H),2.08-1.36(m,8H).

[0080] Example 6

[0081]

[0082] Prepared in a similar manner to Example 1. LCMS: 733.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.33 (s, 1H), 9.75 (s, 1H), 8.76-8.78 (d, J = 9.7Hz, 2H), 8.42 (s, 1H),8.25-8.27(d,J=9.4Hz,1H),8.10-8.13(m,1H),7.98-8.0(d,J=6.2Hz,3H),7.36-7. 39(d,J=9.4Hz,1H),7.19-7.21(d,J=8.5Hz,1H),6.24-6.28(m,1H),4.96(s,2H),4.62-4 .70(m,4H),4.12-4.10(m,2H),3.33-3.31(m,3H),2.90-3.26(m,3H),1.58-2.05(m,6H).

[0083] Example 7

[0084]

[0085] Prepared in a similar manner to Example 1. LCMS: 784.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ13.26(s,1H),9.75-9.74(m,1H),9.33(s,1H),8.76(s,1H),8.37(s,1H),8.14-7.93(m,5H),7.75(s,1H),7.45(d,J=5. 4Hz,1H),7.02-7.01(m,1H),4.70-4.55(m,5H),4.20-4.18(m,4H),3.83 -3.82(m,2H),3.62-3.61(m,4H),3.15-3.13(m,2H),2.08-1.61(m,6H).

[0086] Example 8

[0087]

[0088] Prepared in a similar manner to Example 1. LCMS: 736.2 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ13.05(s,1H),9.77(s,1H),9.32(s,1H),8.76(s, 1H),8.39(s,1H),8.22(s,1H),8.15(d,J=8.9Hz,1H),8.01-8.00(m,3H),7 .76-7.74(m,1H),7.40-7.38(m,2H),7.06(d,J=9.3Hz,1H),6.28(dd,J=1 0.9,5.9Hz,1H),4.64-4.62(m,6H),2.95-2.93(m,2H),1.99-1.65(m,9H).

[0089] Biological test cases

[0090] JAK

[0091] With Echo 655 (Beckman, 655SYSTEM) to transfer 40nL of compound to a 384 reaction plate (manufacturer: Greiner, catalog number: 784075). Prepare 2× kinase solution with 1× kinase reaction buffer (50mM Hepes, 10mM MgCl2, 0.01% Brij-35, 1mM EGTA, 2mM DTT), and transfer 2μL of JAK1 (25nM, manufacturer: Carna, catalog number: 08-144) solution to the 384 reaction plate. Centrifuge at 1000rpm for 1 minute and incubate at 25℃ for 10 minutes. Prepare a 2× mixture of substrate (IRS1: 0.05mg / mL) and ATP (20μM, manufacturer: Promega, catalog number: V915B) with kinase reaction buffer, start the reaction after adding 2μL of the mixture of substrate and ATP to the reaction plate, and centrifuge at 1000rpm for 1 minute. Incubate at 25°C for 60 minutes. Transfer 4 μL of ADP-Glo ​​to the 384 assay plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 40 minutes. Transfer 8 μL of the assay solution to the 384 assay plate, centrifuge at 1000 rpm for 1 minute, incubate at 25°C for 40 minutes, and read the luminescence signal of each well in the ADP-Glo ​​mode on an ELISA reader (manufacturer: BMG, model: PHERAstar FSX). Set the reading of the negative control to 0% inhibition and the reading of the positive control to 100% inhibition, calculate the inhibition rate of each test solution, and then perform data analysis using GraphPad 8 software, and use the nonlinear fitting formula to obtain the IC of the compound. 50 (half inhibitory concentration), the results are shown in Table 1 below.

[0092] PHD2

[0093] Use DMSO to make gradient dilutions of the test compound. Use ECHO to add 40nL / well of the compound to the 384-well reaction plate. Seal the experimental plate and centrifuge at 1000rpm for 1 minute. Add 4μL / well 2× PHD2 enzyme working solution to the 384-well reaction plate with the above compounds. Seal the experimental plate, centrifuge at 1000rpm for 1 minute, and incubate at 25℃ for 30 minutes. Prepare 2× substrate working solution (CODD peptide and α-ketoglutarate) and add 4μL / well 2× substrate working solution to the 384-well reaction plate with the above compounds. Seal the experimental plate, centrifuge at 1000rpm for 1 minute, and incubate at 25℃ for 30 minutes. Add 4μL / well 4× prepared stop working solution to the 384-well reaction plate to stop the reaction. Seal the experimental plate and centrifuge at 1000rpm for 1 minute. Add 4 μL / well 4× detection working solution (Alphascreen streptavidin donor beads, AlphaScreen A protein acceptor beads and hydroxy-HIF-1α (Pro564) (D43B5) Rabbit mAb) into a 384 assay plate. The assay plate was sealed, centrifuged at 1000 rpm for 1 minute, and incubated at 25°C for 30 minutes. The fluorescence signal was detected on an Envision 2104 Reader. The results are shown in Table 1 below.

[0094] Table 1

[0095]

[0096] Although the specific embodiments of the present invention have been described in detail, according to all the teachings disclosed, those skilled in the art can make various modifications and substitutions to the details of the technical solution of the present invention, and these changes are within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, Ring A is selected from phenyl, pyridyl or pyrazolyl, wherein the phenyl, pyridyl or pyrazolyl is optionally substituted by 1 halogen; L is selected from -(CH2) n1 -NH-(CH2) n2 -、-(CH2) n1 -O-(CH2) n2 -N(CH2CH2NBoc)-or-(CH2) n1 -O-(CH2) n2 -NH-, -(CH2) n1 -NH-(CH2) n2 -、-(CH2) n1 -O-(CH2) n2 -N(CH2CH2NBoc)-or-(CH2) n1 -O-(CH2) n2 -NH- is optionally substituted with 1 methyl group; R 1 is selected from H or halogen; n1 and n2 are each independently 0, 1 or 2, and n1 and n2 are not 0 at the same time.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L is selected from -CH2NH-, -CH2CH2OCH2CH2N(CH2CH2NBoc)-, -CH2N(CH3)-, -CH(CH3)NH- or -CH2CH2OCH2CH2NH-.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The structure of the compound represented by formula (I) is shown as one of the following:

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The structure of the compound represented by formula (I) is shown below:

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The structure of the compound represented by formula (I) is shown below:

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The structure of the compound represented by formula (I) is shown as one of the following:

8. A pharmaceutical composition, wherein The pharmaceutical composition comprises the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating diseases mediated by PHD2 and JAK1.

10. The use according to claim 9, wherein: The PHD2 and JAK1 mediated disease is inflammatory bowel disease.

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