Tricyclic compounds and their applications

Abstract

The present invention discloses a series of tricyclic compounds, and specifically discloses a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

Description

[0001] The present invention claims the following priority:

[0002] CN202010144417.0, application date: March 4, 2020;

[0003] CN202010464135.9, application date: May 27, 2020. Technical Field

[0004] The present invention relates to a class of tricyclic compounds, and in particular to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. Background Art

[0005] Sphingosine-1-phosphate (S1P) is a cell membrane-derived lysophospholipid signaling molecule that exerts physiological functions mainly by stimulating some members of the G protein-coupled receptor family, mainly the sphingosine-1-phosphate receptors (S1PRs) family. Currently, five different S1PR subtypes have been identified in mammals, including sphingosine-1-phosphate receptor 1 (S1PR1 or EDG1), sphingosine-1-phosphate receptor 2 (S1PR2 or EDG5), sphingosine-1-phosphate receptor 3 (S1PR3 or EDG3), sphingosine-1-phosphate receptor 4 (S1PR4 or EDG6), and sphingosine-1-phosphate receptor 5 (S1PR5 or EDG8). S1PR1-3 are widely expressed in various tissues, S1PR4 is mainly expressed in the lymphatic system and blood system, and S1PR5 is mainly expressed in the central nervous system. Lymphocytes sense S1P concentration gradients through S1PR1, thereby regulating lymphocytes from secondary lymphoid organs into lymphatic and blood circulation. S1PR1 agonists can trigger the endocytosis of S1PR1 on the surface of lymphocytes, making lymphocytes unable to sense S1P concentration gradients, preventing lymphocytes from migrating to lymphatic and blood circulation, triggering lymphocyte homing, reducing the number of lymphocytes in the peripheral circulatory system, preventing lymphocytes from reaching the location of inflammatory lesions or transplants, reducing excessive inflammation, and having an immune regulatory effect.

[0006] Autoimmune diseases are a general term for a class of diseases caused by the body's immune response to its own antigens, which causes the immune system to mistakenly attack its own tissues. Currently, there are more than 80 types of diseases that have been precisely defined, and excessive inflammatory response is a common feature. S1PR1 agonists can effectively reduce excessive inflammation and can be used to treat or prevent autoimmune diseases, including multiple sclerosis, inflammatory bowel disease (divided into Crohn's disease and ulcerative colitis), systemic lupus erythematosus and psoriasis.

[0007] At present, the in vivo efficacy studies of S1PR1 agonists are used to treat or prevent autoimmune diseases. Novartis' first-generation non-selective S1PRs agonist Fingolimod was approved by the FDA for relapsing multiple sclerosis (RMS) in September 2010, and Novartis' second-generation selective S1PR1 and S1PR5 agonist Siponimod was also approved by the FDA for relapsing multiple sclerosis (RMS) in March 2019. The discovery and application of new S1PR agonists has broad prospects. Summary of the invention

[0008] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0009]

[0010] in,

[0011] E is selected from O;

[0012] Ring A is selected from oxazolyl, 1,2,4-oxadiazolyl, thiazolyl, 1,3,4-thiadiazole, 1,2,4-thiadiazolyl, pyrimidinyl and pyrazinyl;

[0013] R1 is selected from C 1-3 Alkyl and cyclobutyl, the C 1-3 The alkyl and cyclobutyl groups are optionally substituted with 1, 2 or 3 R a replace;

[0014] R2 is selected from F, Cl, Br, CN and CHF2;

[0015] R3 is selected from C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with 1, 2 or 3 R b replace;

[0016] R a Each is independently selected from F, Cl, Br, OH, NH2 and COOH.

[0017] R b are independently selected from F, Cl and Br.

[0018] In some embodiments of the present invention, the ring A is selected from Other variables are as defined herein.

[0019] In some embodiments of the present invention, the above R a are independently selected from OH and COOH, and other variables are as defined in the present invention.

[0020] In some embodiments of the present invention, the above R1 is selected from CH3, CH2CH3, CH2CH2CH3 and The CH3, CH2CH3 and Optional 1, 2 or 3 R a The other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, the above R1 is selected from CH2CH2OH, CH2COOH, CH2CH2COOH and Other variables are as defined herein.

[0022] In some embodiments of the present invention, the above R2 is selected from Br and CN, and other variables are as defined in the present invention.

[0023] In some embodiments of the present invention, the above R3 is selected from C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R b The other variables are as defined in the present invention.

[0024] In some embodiments of the present invention, the above R3 is selected from CH(CH3)2, and other variables are as defined in the present invention.

[0025] In some embodiments of the present invention, the above structural unit Selected from Other variables are as defined herein.

[0026] In some embodiments of the present invention, the above compound or a pharmaceutically acceptable salt thereof is selected from:

[0027]

[0028] in,

[0029] R1 is as defined in any one of claims 1, 4 or 5;

[0030] R2 is as defined in claim 1 or 6;

[0031] R3 is as defined in any one of claims 1, 7 or 8;

[0032] E as defined in claim 1;

[0033] T1 is selected from O and S.

[0034] Some other solutions of the present invention are obtained by any combination of the above variables.

[0035] The present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0036]

[0037]

[0038] The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases related to S1PR1.

[0039] Technical Effects

[0040] The compounds of the present invention have significant and even unexpected S1PR1 agonistic activity and good bioavailability.

[0041] Related definitions

[0042] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0043] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0044] The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

[0045] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0046] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0047] 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). For another example, deuterium can be used to replace hydrogen to form a deuterated drug. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

[0048] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0049] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include a variant of deuterium and hydrogen, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituents may be arbitrary on the basis of chemical achievable.

[0050] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0051] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0052] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0053] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease with the number of connected chemical bonds to become a group with the corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group; It means that any connectable site on the piperidine group can be connected to other groups through one chemical bond, including at least These four connection methods, even if the H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

[0054] Unless otherwise specified, the term “C 1-6 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. 1-6 Alkyl groups include C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4, C6 and C5 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, and the like.

[0055] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0056] Unless otherwise specified, C n-n+m or C n -C n+m Any specific case including n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , and C 12 , also includes any range from n to n+m, such as C 1-12 Including C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 Etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any range from n to n+m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.

[0057] The compounds of the present invention can be prepared by a variety of synthetic methods well 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 well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0058] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0059] The solvent used in the present invention is commercially available.

[0060] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names. DETAILED DESCRIPTION

[0061] The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed therein. It will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0062] Example 1

[0063]

[0064] Synthesis route:

[0065]

[0066] first step

[0067] Compound 1a (1.00 g, 4.31 mmol) and 2-iodopropane (1.47 g, 8.62 mmol) were dissolved in toluene (8 mL), and then silver carbonate (3.57 g, 12.9 mmol) was added, and the reaction solution was stirred at 50° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was diluted with water (100 mL), extracted with ethyl acetate (100 mL×3), and the combined organic phase was washed with saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1b.

[0068] MS-ESI calculated value [M+H] + 274 and 276, measured values ​​274 and 276.

[0069] 1 H NMR (400MHz, CDCl3) δ=8.72 (d, J=2.1Hz, 1H), 8.38 (d, J=2.1Hz, 1H), 5.48-5.37 (m, 1H), 3.92 (s, 3H), 1.42 (d, J=6.2Hz, 6H).

[0070] Step 2

[0071] Compound 1b (1.00 g, 3.65 mmol) was dissolved in anhydrous N, N-dimethylformamide (15 mL), zinc cyanide (857 mg, 7.30 mmol), tris(dibenzylideneacetone)dipalladium (334 mg, 365 μmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropylbiphenyl (348 mg, 730 μmol) were added, and the reaction solution was stirred at 90° C. for 12 hours under nitrogen protection. The reaction solution was concentrated, the residue was diluted with water (50 mL), extracted with ethyl acetate (30 mL×3), the combined organic phases were washed with saturated brine (40 mL×1), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated and purified by silica gel column chromatography (10:1, petroleum ether / ethyl acetate, Rf=0.4) to obtain compound 1c.

[0072] MS-ESI calculated values ​​[M+H- i Pr] + 179, measured value 179.

[0073] Step 3

[0074] Compound 1c (100 mg, 454 μmol) was dissolved in tetrahydrofuran (1 mL) and methanol (0.5 mL), and a solution of lithium hydroxide monohydrate (57.2 mg, 1.36 mmol) in water (0.5 mL) was added dropwise to the reaction solution, and the reaction solution was stirred at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure to remove the organic solvent, and the residue was adjusted to pH 6 with 1N hydrochloric acid aqueous solution, then diluted with water (50 mL), extracted with ethyl acetate (50 mL×3), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by thin layer silica gel chromatography (10:1, dichloromethane / methanol, Rf=0.21) to obtain compound 1d.

[0075] 1H NMR (400MHz, CDCl3) δ = 9.18-8.74 (m, 1H), 8.70-8.23 (m, 1H), 5.60-5.38 (m, 1H), 1.43 (d, J = 6.1Hz, 6H).

[0076] Step 4

[0077] Compound 1f (56.0 g, 308 mmol) was dissolved in N, N-dimethylformamide (200 mL), potassium tert-butoxide (34.6 g, 308 mmol) was added, the reaction solution was stirred at 25°C for 2 hours, and then compound 1e (50.0 g, 237 mmol) was slowly added, and the reaction solution was stirred at 25°C for 12 hours. Water (800 mL) was added to the reaction solution, and it was extracted with ethyl acetate (400 mL×3), the organic phases were combined, and the organic phases were washed with water (500 mL×1) and saturated brine (500 mL×1), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated and purified by column chromatography (10:1, petroleum ether / ethyl acetate, Rf=0.6, 0.7) to obtain compound 1g.

[0078] MS-ESI calculated value [M+H] + 267 and 269, measured values ​​267 and 269.

[0079] 1 H NMR (400MHz, CDCl3) δ = 7.57-7.48 (m, 1H), 7.39-7.29 (m, 1H), 7.24-7.10 (m, 1H) ), 6.55-6.27(m, 1H), 3.81-3.70(m, 3H), 3.63-3.37(m, 2H), 3.33-3.00(m, 2H).

[0080] Step 5

[0081] Compound 1g (100 g, 374 mmol) was dissolved in dimethyl sulfoxide (400 mL), cesium carbonate (97.6 g, 299 mmol) was added, and nitromethane (68.6 g, 1.12 mol) was slowly added dropwise, and the reaction solution was stirred at 70°C for 16 hours. Water (1600 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (800 mL×3), and the organic phases were combined and washed with water (1000 mL×1) and saturated brine (1000 mL×1), respectively, dried over anhydrous sodium sulfate, and concentrated to obtain compound 1h.

[0082] 1H NMR (400MHz, CDCl3) δ=7.41 (dd, J=7.2, 1.6Hz, 1H), 7.12-7.04 (m, 2H), 4.89 (d, J=11.6Hz, 1H), 4.81 (d, J=1 1.6Hz, 1H), 3.86 (s, 3H), 3.04-2.96 (m, 3H), 2.78 (d, J=16.4Hz, 1H), 2.44-2.35 (m, 1H), 2.26-2.16 (m, 1H).

[0083] Step 6

[0084] Compound 1h (100 g, 305 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (100 mL), and ammonium chloride (48.90 g, 914 mmol) and iron powder (51.1 g, 914 mmol) were added, and the reaction solution was stirred at 80°C for 15 hours. The reaction solution was filtered through diatomaceous earth, the filtrate was diluted with water (1000 mL), extracted with ethyl acetate (500 mL×3), the combined organic phases were washed with saturated brine (500 mL×1), dried over anhydrous sodium sulfate, concentrated, and the crude product was added to a mixed solution of ethyl acetate / n-heptane (1:6, 1180 mL), stirred at 25°C for 3 days, filtered, and the filter cake was dried in vacuo to obtain compound 1i.

[0085] MS-ESI calculated value [M+H] + 266 and 268, measured values ​​266 and 268.

[0086] 1 H NMR (400MHz, CDCl3) δ=7.52-7.32(m, 1H), 7.25-7.06(m, 2H), 7.06-6.87(m, 1H ), 3.63-3.32(m, 2H), 3.08-2.83(m, 2H), 2.69-2.41(m, 2H), 2.36-2.10(m, 2H).

[0087] Step 7

[0088] Compound 1i (182 g, 589 mmol) and compound 1j (422 g, 1.77 mol) were dissolved in N, N-dimethylformamide (360 mL), cesium carbonate (575 g, 1.77 mol) and sodium iodide (44.1 g, 294 mmol) were added, and the reaction solution was stirred at 80°C for 16 hours. The reaction solution was filtered, the filter cake was washed with ethyl acetate (400 mL×4), the filtrate was diluted with water (4000 mL), the organic phase was collected by separation, and the aqueous phase was extracted with ethyl acetate (2000 mL×2). The combined organic phases were washed with saturated brine (2000 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1k.

[0089] MS-ESI calculated value [M+H] + 424 and 426, measured values ​​424 and 426.

[0090] Step 8

[0091] Compound 1k (470 g, 587 mmol) was dissolved in anhydrous N,N-dimethylformamide (1200 mL), and zinc cyanide (103 g, 880 mmol), tris(dibenzylideneacetone)dipalladium (21.5 mg, 23.5 mmol), and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (22.4 mg, 47.0 mmol) were added to the reaction solution in sequence. Under nitrogen protection, the reaction solution was stirred at 90°C for 16 hours. The reaction solution was concentrated under reduced pressure, and water (6000 mL) was added to the residue, extracted with ethyl acetate (2000 mL×3), the organic phases were combined, washed with saturated brine (2000 mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was filtered through neutral alumina (800 g), and the filter cake was washed with a mixed solvent of petroleum ether / ethyl acetate / dichloromethane (3 / 1 / 1, 15 L). The filtrate was concentrated under reduced pressure, the crude product was diluted with n-heptane (1.0 L), stirred at 25°C for 16 hours, filtered, and the filter cake was diluted with n-heptane (500 mL), stirred at 25°C for 16 hours, filtered, and the filter cake was washed with n-heptane (150 mL×1). The filter cake was dried under vacuum to obtain compound 11.

[0092] MS-ESI calculated value [M+H] + 371, measured value 371.

[0093] Step 9

[0094] Compound 11 (120 g, 324 mmol) was dissolved in ethanol (600 mL), and diisopropylethylamine (83.7 g, 647 mmol) and hydroxylamine hydrochloride (45.0 g, 647 mmol) were added in sequence. The reaction was stirred at 40°C for 16 hours. Water (700 mL) was added for dilution, and the mixture was extracted with ethyl acetate (400 mL × 3). The combined organic phases were washed with saturated brine (400 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was added to a mixed solution of n-heptane / isopropanol (5 / 1, 560 mL), stirred at 15°C for 65 hours, filtered, and the filter cake was washed with a mixed solution of n-heptane / isopropanol (5:1, 100 mL × 2). The filter cake was dried under vacuum to obtain compound 1m.

[0095] 1 H NMR (400MHz, CDCl3) δ = 7.45-7.37 (m, 1H), 7.31-7.25 (m, 2H), 4.84 (s, 2H), 3.81 (t, J = 5.3Hz, 2H), 3.67 (d, J = 9.6Hz, 1H), 3.61 (d, J = 9, 6Hz, 1H), 3 .48 (t, J=5.3Hz, 2H), 3, 13 (t, J=7.1Hz, 2H), 2.69 (d, J=16.8Hz, 1H), 2.5 9(d, J=16.8Hz, 1H), 2.33-2.08(m, 2H), 0.89(s, 9H), 0.10-0.04(m, 6H).

[0096] Step 10

[0097] Compound 1d (51.1 mg, 248 μmol) was dissolved in N, N-dimethylformamide (2 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (57.0 mg, 297 μmol) and 1-hydroxybenzotriazole monohydrate (40.2 mg, 297 μmol) were added to the solution, and the reaction solution was stirred at 25°C for 15 minutes. Compound 1m (100 mg, 248 μmol) was then added, and the mixture was stirred at 25°C for 1 hour and at 80°C for 16 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL×3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: Phenomenex Luna C18 150*25mm*10μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 43%-73%, 10 minutes} to obtain compound 1.

[0098] MS-ESI calculated value [M+H] + 460, measured value 460.

[0099] 1 H NMR (400MHz, CDCl3) δ=9.16 (d, J=2.3Hz, 1H), 8.65 (d, J=2.3Hz, 1H), 8.11-8.08 (m, 1H), 7.46-7.41 (m, 2H), 5.62-5.51 (m, 1H), 3.89-3.82 (m, 2H), 3 .69-3.61(m, 2H), 3.57-3.47(m, 2H), 3.43-3.32(m, 2H), 2.79(d, J=16.9H z, 1H), 2.69 (d, J=16.9Hz, 1H), 2.41-2.23 (m, 2H), 1.48 (d, J=6.2Hz, 6H).

[0100] Example 3

[0101]

[0102] Synthesis route:

[0103]

[0104] first step

[0105] Compound 3a (200 mg, 862 μmol) and isopropyl iodide (176 mg, 1.03 mmol) were dissolved in anhydrous toluene (3 mL), and silver carbonate (238 mg, 862 μmol) was added to the reaction solution, and the reaction solution was stirred at 50° C. for 16 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (20 mL), extracted with ethyl acetate (20 mL×1), and the combined organic phase was washed with saturated brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 3b.

[0106] MS-ESI calculated values ​​[M+H- i Pr] + 232 and 234, measured values ​​232 and 234.

[0107] Step 2

[0108] Compound 3b (260 mg, 949 μmol) was dissolved in anhydrous tetrahydrofuran (3 mL), anhydrous methanol (3 mL) and water (1 mL), and lithium hydroxide monohydrate (79.6 mg, 1.90 mmol) was added to the reaction solution, and the reaction solution was stirred at 25°C for 12 hours. The reaction solution was concentrated under reduced pressure, and 1N hydrochloric acid was added to the residue to adjust the pH to about 3. The reaction solution became turbid, and the filter cake was collected by filtration. The filter cake was dried under reduced pressure to obtain a crude compound 3c. MS-ESI calculated value [M+H- i Pr] +218 and 220, measured values ​​218 and 220.

[0109] Step 3

[0110] Compound 3c (90.2 mg, 347 μmol) was dissolved in anhydrous N, N-dimethylformamide (5 mL), 1-hydroxybenzotriazole (56.3 mg, 416 μmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (79.8 mg, 416 μmol) were added to the reaction solution, and the reaction solution was stirred at 25°C for 1 hour. 1m (140 mg, 347 μmol) was added to the reaction solution, and the reaction solution was stirred at 25°C for 1 hour and at 80°C for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography {hydrochloric acid adjustment, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 66%-86%, 7 minutes} to obtain compound 3.

[0111] MS-ESI calculated value [M+H] + 513 and 515, measured values ​​513 and 515.

[0112] 1H NMR (400MHz, CDCl3) δ = 8.93 (d, J = 2.1Hz, 1H), 8.60-8.58 (m, 1H), 8.13-8.08 (m, 1H), 7.45-7.42 (m, 2H), 5.52-5.45 (m, 1H), 3.87 (t, J = 5.0Hz, 2H), 3 .69-3.62(m, 2H), 3.59-3.50(m, 2H), 3.43-3.37(m, 2H), 2.81(d, J=16.9H z, 1H), 2.70 (d, J=16.9Hz, 1H), 2.39-2.27 (m, 2H), 1.47 (d, J=6.2Hz, 6H).

[0113] Example 4

[0114]

[0115] Synthesis route:

[0116]

[0117] first step

[0118] Compound 4a (5 g, 25.1 mmol) and isopropyl iodide (5.13 g, 30.2 mmol) were dissolved in anhydrous toluene (40 mL), and silver carbonate (3.46 g, 12.6 mmol) was added to the reaction solution, and the reaction solution was stirred at 50° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was diluted with water (100 mL), extracted with ethyl acetate (100 mL×1), and the combined organic phase was washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 4b.

[0119] MS-ESI calculated values ​​[M+H- i Pr] + 199 and 201, measured values ​​199 and 201.

[0120] 1H NMR (400MHz, CDCl3) δ=8.29 (d, J=2.5Hz, 1H), 7.86 (d, J=2.5Hz, 1H), 5.32-5.25 (m, 1H), 1.32 (d, J=6.2Hz, 6H).

[0121] Step 2

[0122] Compound 4b (200 mg, 830 μmol), bis-naphthalene borate (253 mg, 996 μmol) and potassium acetate (163 mg, 1.66 mmol) were dissolved in anhydrous dioxane (3 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (60.7 mg, 83.0 μmol) was added to the reaction solution, and the reaction solution was reacted at 120° C. for 3 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (20 mL), extracted with ethyl acetate (20 mL×1), the combined organic phase was washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.7) to obtain compound 4c.

[0123] MS-ESI calculated values ​​[M+H- i Pr] + 247, measured value 247.

[0124] 1H NMR (400MHz, CDCl3) δ=8.64 (d, J=1.8Hz, 1H), 8.20 (d, J=1.8Hz, 1H), 5.50-5.42 (m, 1H), 1.39 (d, J=6.1Hz, 6H), 1.25 (s, 12H).

[0125] Step 3

[0126] Compound 1k (9 g, 21.2 mmol), bis-naphthalene borate (8.08 g, 31.8 mmol) and potassium acetate (4.16 g, 42.4 mmol) were dissolved in anhydrous dioxane (100 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (1.55 g, 2.12 mmol) was added to the reaction solution, and the reaction solution was stirred at 80°C for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (200 mL), extracted with ethyl acetate (150 mL×2), the combined organic phase was washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated by column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.13) and purified to obtain compound 4d.

[0127] MS-ESI calculated value [M+H] + 472, measured value 472.

[0128] Step 4

[0129] Compound 4d (200 mg, 424 μmol), compound 4e (84.6 mg, 424 μmol) and potassium phosphate (180 mg, 848 μmol) were dissolved in anhydrous dioxane (5 mL) and water (1 mL). 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (31.0 mg, 42.4 μmol) was added to the reaction solution under nitrogen atmosphere, and the reaction solution was stirred at 100° C. for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (30 mL), extracted with ethyl acetate (30 mL×1), the combined organic phase was washed with saturated brine (30 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 4f.

[0130] MS-ESI calculated value [M+H] + 508 and 510, measured values ​​508 and 510.

[0131] Step 5

[0132] Compound 4f (220 mg, 433 μmol), compound 4c (125 mg, 433 μmol) and potassium phosphate (184 mg, 865 μmol) were dissolved in anhydrous dioxane (5 mL) and water (1 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (31.7 mg, 43.3 μmol) was added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (30 mL), extracted with ethyl acetate (30 mL×1), the combined organic phase was washed with saturated brine (30 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (2:1, petroleum ether / ethyl acetate, Rf=0.2) to obtain compound 4g.

[0133] MS-ESI calculated value [M+H] + 590, measured value 590.

[0134] Step 6

[0135] Compound 4g (113 mg, 192 μmol) was dissolved in ethyl acetate (1 mL), and hydrochloric acid / ethyl acetate (4 M, 1 mL) was added to the mixture, and the reaction solution was stirred at 20°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 56%-76%, 7 minutes) to obtain compound 4.

[0136] MS-ESI calculated value [M+H] + 476, measured value 476.

[0137] 1H NMR (400MHz, CDCl3) δ = 9.33 (d, J = 2.3Hz, 1H), 8.82 (d, J = 2.3Hz, 1H), 8.05-8.02 (m, 1H), 7.49-7.43 (m, 2H), 5.57-5.50 (m, 1H), 3.90-3.85 (m, 2H), 3. 68-3.65 (m, 2H), 3.61-3.48 (m, 2H), 3.37 (t, J=7.2Hz, 2H), 2.80 (d, J=16.8 Hz, 1H), 2.71 (d, J=16.8Hz, 1H), 2.46-2.32 (m, 2H), 1.47 (d, J=6.2Hz, 6H).

[0138] Example 5

[0139]

[0140] Synthesis route:

[0141]

[0142] first step

[0143] Compound 1d (500 mg, 2.42 mmol) was dissolved in anhydrous dichloromethane (5 mL), N,N-dimethylformamide (17.7 mg, 242 μmol) and oxalyl chloride (923 mg, 7.27 mmol) were added to the reaction solution, and the reaction solution was stirred at 25° C. for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude compound 5a.

[0144] Step 2

[0145] Compound 5a (520 mg, 2.31 mmol) and 2-aminoethanol (212 mg, 3.47 mmol) were dissolved in anhydrous dichloromethane (5 mL), and N,N-diisopropylethylamine (598 mg, 4.63 mmol) was added to the reaction solution, and the reaction solution was stirred at 25°C for 2 hours. The reaction solution was diluted with water (50 mL), extracted with dichloromethane (30 mL×2), and the organic phase was washed with water (50 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 5b.

[0146] MS-ESI calculated value [M+H] + 250, measured value 250.

[0147] Step 3

[0148] Compound 5b (580 mg, 2.33 mmol) was dissolved in anhydrous dichloromethane (10 mL), thionyl chloride (830 mg, 6.98 mmol) was added to the reaction solution, and the reaction solution was stirred at 25° C. for 12 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (3:1, petroleum ether / ethyl acetate, Rf=0.3) to obtain compound 5c.

[0149] MS-ESI calculated value [M+H] + 268, measured value 268.

[0150] 1H NMR (400MHz, CDCl3) δ = 8.68 (d, J = 2.4Hz, 1H), 8.24 (d, J = 2.4Hz, 1H), 6.52-6.42 (m, 1 H), 5.45-5.38 (m, 1H), 3.77-3.72 (m, 2H), 3.70-3.65 (m, 2H), 1.35 (d, J=6.2Hz, 6H).

[0151] Step 4

[0152] Compound 5c (300 mg, 1.12 mmol) was dissolved in anhydrous dichloromethane (3 mL), triethylamine (340 mg, 3.36 mmol) was added to the reaction solution, and the reaction solution was stirred at 80°C for 12 hours. The reaction solution was concentrated under reduced pressure, diluted with water (30 mL), extracted with dichloromethane (30 mL×2), the organic phase was washed with water (30 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 5d.

[0153] MS-ESI calculated value [M+H] + 232, measured value 232.

[0154] Step 5

[0155] Compound 5d (220 mg, 851 μmol) was dissolved in carbon tetrachloride (5 mL), N-bromosuccinimide (508 mg, 2.85 mmol) and azobisisobutyronitrile (7.81 mg, 47.6 μmol) were added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (3:1, petroleum ether / ethyl acetate, Rf=0.31) to obtain compound 5e.

[0156] MS-ESI calculated value [M+H] + 308 and 310, measured values ​​308 and 310.

[0157] Step 6

[0158] Compound 5e (90.2 mg, 293 μmol), compound 4d (138 mg, 293 μmol) and potassium phosphate (124 mg, 585 μmol) were dissolved in anhydrous dioxane (3 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (21.4 mg, 29.3 μmol) was added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (20 mL), extracted with ethyl acetate (20 mL×1), the combined organic phase was washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated by thin layer chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.13) and purified to obtain compound 5f.

[0159] MS-ESI calculated value [M+H] + 573, measured value 573.

[0160] Step 7

[0161] Compound 5f (100 mg, 175 μmol) was dissolved in ethyl acetate (1 mL), hydrochloric acid / ethyl acetate (4 M, 1.2 mL) was added to the mixture, and the reaction solution was stirred at 20° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and the crude product was purified by HPLC {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 45%-65%, 7 minutes} to obtain compound 5.

[0162] MS-ESI calculated value [M+H] + 459, measured value 459.

[0163] 1 H NMR (400MHz, CDCl3) δ=9.05 (d, J=2.4Hz, 1H), 8.52 (d, J=2.3Hz, 1H), 7.68 (d, J =7.6Hz, 1H), 7.41-7.36 (m, 2H), 7.29 (d, J = 7.7Hz, 1H), 5.55-5.48 (m, 1H), 3.8 9-3.84 (m, 2H), 3.68-3.61 (m, 2H), 3.58-3.51 (m, 2H), 3.18 (t, J=7.1Hz, 2H), 2 .82-2.76 (m, 1H), 2.71-2.65 (m, 2H), 2.41-2.29 (m, 2H), 1.46 (d, J=6.2Hz, 6H).

[0164] Example 6

[0165]

[0166] Synthesis route:

[0167]

[0168] first step

[0169] Compound 4c (200 mg, 694 μmol), compound 6a (198 mg, 694 μmol) and potassium phosphate (295 mg, 1.39 mmol) were dissolved in anhydrous dioxane (4 mL) and water (1 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (51.0 mg, 69.7 μmol) was added to the reaction solution, and the reaction solution was stirred at 80°C for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (30 mL), extracted with ethyl acetate (30 mL×2), the combined organic phase was washed with saturated brine (30 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 6b.

[0170] MS-ESI calculated value [M+H] + 319 and 321, measured values ​​319 and 321.

[0171] Step 2

[0172] Compound 6b (102 mg, 318 μmol), compound 4d (150 mg, 318 μmol) and potassium phosphate (135 mg, 636 μmol) were dissolved in anhydrous dioxane (3 mL) and water (1 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (23.3 mg, 31.8 μmol) was added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure, the crude product was diluted with water (30 mL), extracted with ethyl acetate (30 mL×1), the combined organic phase was washed with saturated brine (30 mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.25) to obtain compound 6c.

[0173] MS ESI calculated value [M+H] + 584, measured value 584.

[0174] Step 3

[0175] Compound 6c (86 mg, 147 μmol) was dissolved in ethyl acetate (1 mL), hydrochloric acid / ethyl acetate (4 M, 1 mL) was added to the mixture, and the reaction solution was stirred at 20° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 50%-70%, 7 minutes} to obtain compound 6.

[0176] MS-ESI calculated value [M+H] + 470, measured value 470.

[0177] 1H NMR (400MHz, CDCl3) δ=9.42 (d, J=2.1Hz, 1H), 8.95 (d, J=2.1Hz, 1H), 8.88 (s, 2H ), 7.45-7.37(m, 2H), 7.32(d, J=7.1Hz, 1H), 5.58-5.51(m, 1H), 3.91-3.83(m, 2H ), 3.74-3.64(m, 2H), 3.60-3.49(m, 2H), 3.09-3.02(m, 2H), 2.86-2.78(m, 1H), 2.74-2.67 (m, 1H), 2.66-2.63 (m, 1H), 2.34-2.22 (m, 2H), 1.46 (d, J=6.1Hz, 6H).

[0178] Example 7

[0179]

[0180] Synthesis route:

[0181]

[0182] first step

[0183] Compound 4c (300 mg, 1.04 mmol), compound 7a (171 mg, 1.04 mmol) and potassium phosphate (442 mg, 2.08 mmol) were dissolved in anhydrous dioxane (5 mL) and water (1 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (76.2 mg, 104 μmol) was added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL×2), and the combined organic phase was washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.53) to obtain compound 7b.

[0184] MS-ESI calculated value [M+H] + 246, measured value 246.

[0185] Step 2

[0186] Compound 7b (92 mg, 375 μmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), N-bromosuccinimide (134 mg, 750 μmol) was added to the reaction solution at 0°C, and the reaction solution was stirred at 60°C for 3 hours. The reaction solution was diluted with water (30 mL), extracted with ethyl acetate (30 mL×2), the combined organic phase was washed with saturated brine (30 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.64) to obtain compound 7c.

[0187] MS-ESI calculated value [M+H] + 324 and 326, measured values ​​324 and 326.

[0188] Step 3

[0189] Compound 7c (62.0 mg, 191 μmol), compound 4d (90.2 mg, 191 μmol) and potassium phosphate (81.2 mg, 382 μmol) were dissolved in anhydrous dioxane (3 mL) and water (1 mL). Under nitrogen protection, 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (14.0 mg, 19.1 μmol) was added to the reaction solution, and the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, the crude product was diluted with water (15 mL), extracted with ethyl acetate (15 mL×2), the combined organic phase was washed with saturated brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by thin layer chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.40) to obtain compound 7d.

[0190] MS-ESI calculated value [M+H] + 589, measured value 589.

[0191] Step 4

[0192] Compound 7d (102 mg, 173 μmol) was dissolved in ethyl acetate (1 mL), hydrochloric acid / ethyl acetate (4 M, 1 mL) was added to the mixture, and the reaction solution was stirred at 20° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and the crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 52%-72%, 7 minutes} to obtain compound 7.

[0193] MS-ESI calculated value [M+H] + 475, measured value 475.

[0194] 1H NMR (400MHz, CDCl3) δ = 8.90 (s, 1H), 8.46 (s, 1H), 7.95 (s, 1H), 7.45 (d, J = 6.8Hz, 1H), 7.38-7.28 (m, 2H), 5.54-5.46 (m, 1H), 3.86 (br s, 2H), 3.73-3.62 (m, 2H), 3.60-3.48 (m, 2H), 3.20-3.10 (m, 2H), 2.84-2 .75 (m, 1H), 2.73-2.63 (m, 1H), 2.38-2.23 (m, 2H), 1.45 (d, J=6.1Hz, 6H).

[0195] Example 8

[0196]

[0197] Synthesis route:

[0198]

[0199] first step

[0200] Compound 1i (500 mg, 1.88 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). Potassium tert-butoxide (316 mg, 2.82 mmol) was added to the mixture at 0°C. The reaction solution was stirred at 0°C for 0.5 hours. Compound 8a (941 mg, 5.64 mmol) was added to the mixture at 0°C. The reaction solution was stirred at 20°C for 2 hours. 30 mL of water was added to the reaction solution for dilution, and the mixture was extracted with ethyl acetate (30 mL*3). The organic phases were combined, washed with 30 mL of water and 30 mL of saturated brine in sequence, and dried over anhydrous sodium sulfate. The crude product was separated by column chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.39) to obtain compound 8b.

[0201] MS-ESI calculated value [M+H] + 352 and 354, measured values ​​352 and 354.

[0202] 1H NMR (400MHz, CDCl3) δ=7.31 (d, J=7.8Hz, 1H), 7.19 (d, J=7.5Hz, 1H), 7.07-7. 01 (m, 1H), 4.14 (q, J = 7.2Hz, 2H), 4.06-4.03 (m, 2H), 3.53 (d, J = 9.4Hz, 1H), 3 .47 (d, J=9.4Hz, 1H), 2.90 (t, J=7.2Hz, 2H), 2.64 (d, J=16.9Hz, 1H), 2.54 (d, J=16.9Hz, 1H), 2.31-2.22 (m, 1H), 2.18-2.09 (m, 1H), 1.22 (t, J=7.2Hz, 3H).

[0203] Step 2

[0204] Compound 8b (467 mg, 1.33 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and zinc cyanide (234 mg, 1.99 mmol), tripalladium benzalkonium (60.7 mg, 66.3 μmol), and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (63.2 mg, 133 μmol) were added to the mixture. Under nitrogen protection, the reaction solution was stirred at 90° C. for 12 hours. The reaction solution was concentrated, and the crude product was separated and purified by column chromatography (5:1-3:1, petroleum ether / ethyl acetate, Rf=0.24) to obtain compound 8c.

[0205] MS-ESI calculated value [M+H] + 299, measured value 299.

[0206] Step 3

[0207] Hydroxylamine hydrochloride (143 mg, 2.07 mmol) and triethylamine (209 mg, 2.07 mmol) were dissolved in anhydrous ethanol (5 mL), and compound 8c (206 mg, 690 μmol) was added to the mixture under nitrogen protection. The reaction solution was stirred at 80° C. for 4 hours under nitrogen protection. The reaction solution was concentrated to remove the solvent, and the crude product was purified by high performance liquid chromatography {neutral conditions, column model: Waters Xbridge C18 150*50mm*10 μm; mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; B%: 8%-38%, 11.5 minutes} to obtain compound 8d.

[0208] MS-ESI calculated value [M+H] + 332, measured value 332.

[0209] Step 4

[0210] Compound 1d (79.6 mg, 386 μmol) was dissolved in anhydrous N, N-dimethylformamide (10 mL), 1-hydroxybenzotriazole (62.6 mg, 463 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88.8 mg, 463 μmol) were added to the mixture, and the reaction solution was stirred at 25°C for 1 hour, compound 8d (128 mg, 386 μmol) was added to the reaction solution, and the reaction solution was stirred at 25°C for 2 hours, and the reaction solution was further stirred at 80°C for 8 hours. 30 mL of water was added to the reaction solution, and it was extracted with dichloromethane (30 mL*2), the organic phases were combined, the organic phases were washed with 20 mL of water and 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound 8e.

[0211] MS-ESI calculated value [M+H] + 502, measured value 502.

[0212] Step 5

[0213] Compound 8e (188 mg, 375 μmol) was dissolved in anhydrous tetrahydrofuran (8 mL) and anhydrous methanol (4 mL), and a solution of lithium hydroxide monohydrate (62.9 mg, 1.50 mmol) in water (2 mL) was added to the mixture, and the reaction solution was stirred at 25° C. for 12 hours. The reaction solution was concentrated to remove the solvent, and the crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_PhenomenexLuna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; B%: 20%-40%, 8 minutes} to obtain compound 8f.

[0214] MS-ESI calculated value [M+H] + 432, measured value 432.

[0215] 1 H NMR (400MHz, DMSO-d6) δ = 13.48 (br s, 1H), 12.85 (br s, 1H), 8.80 (d, J = 2.6Hz, 1H), 8.71 (d, J = 2.7Hz, 1H), 8.00-7.96 (m, 1H), 7.63 (d, J = 7.3Hz, 1H), 7.49-7.43 (m, 1H), 4.08-3.97 (m , 2H), 3.56 (s, 2H), 3.27 (t, J=7.2Hz, 2H), 2.68 (d, J=16.5Hz, 1H), 2.54 (d, J=16.5Hz, 1H), 2.38-2.28 (m, 1H), 2.23-2.13 (m, 1H).

[0216] Step 6

[0217] Compound 8f (38.4 mg, 88.9 μmol) and 2-iodopropane (45.4 mg, 267 μmol) were dissolved in anhydrous toluene (3 mL), silver carbonate (24.5 mg, 88.9 μmol) was added to the mixture, and the reaction solution was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the crude product was purified by thin layer chromatography (2:1, petroleum ether / ethyl acetate, Rf=0.15) to obtain compound 8g.

[0218] MS-ESI calculated value [M+H] + 516, measured value 516.

[0219] Step 7

[0220] Compound 8g (22.0 mg, 42.7 μmol) was dissolved in anhydrous tetrahydrofuran (2 mL), and a solution of sodium hydroxide (6.83 mg, 170 μmol) in water (0.5 mL) was added to the mixture, and the reaction solution was stirred at 20°C for 12 hours. The reaction solution was concentrated, and the crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 52%-72%, 7 minutes} to obtain compound 8.

[0221] MS-ESI calculated value [M+H] + 474, measured value 474.

[0222] 1 H NMR (400MHz, CDCl3) δ=9.15 (d, J=2.3Hz, 1H), 8.64 (d, J=2.3Hz, 1H), 8.09 (d, J=7.5 Hz, 1H), 7.51 (d, J=7.6Hz, 1H), 7.43 (t, J=7.6Hz, 1H), 5.62-5.51 (m, 1H), 4.30-4.15 (m, 2H), 3.71 (d, J = 9.4Hz, 1H), 3.63 (d, J = 9.4Hz, 1H), 3.38 (t, J = 6.9Hz, 2H), 2.82 ( d, J=17.0Hz, 1H), 2.72 (d, J=17.0Hz, 1H), 2.48-2.23 (m, 2H), 1.48 (d, J=6.2Hz, 6H).

[0223] Example 9

[0224]

[0225] Synthesis route:

[0226]

[0227] first step

[0228] Compound 1i (5.00 g, 18.8 mmol) was dissolved in anhydrous N, N-dimethylformamide (50 mL), zinc cyanide (3.31 g, 28.2 mmol), tris(dibenzylideneacetone)dipalladium (516 mg, 564 μmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropylbiphenyl (537 mg, 1.13 mmol) were added to the mixture, and the reaction solution was stirred at 90°C for 12 hours under nitrogen protection, 100 mL of water was added to the reaction solution for dilution, and it was extracted with ethyl acetate (60 mL*3), the organic phases were combined, the organic phases were washed with 50 mL of water and 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated. 50 mL of ethanol was added to the crude product, the reaction solution was stirred at 20°C for 12 hours, filtered, and the filter cake was dried under reduced pressure to obtain compound 9a.

[0229] MS-ESI calculated value [M+H] + 213, measured value 213.

[0230] 1 H NMR (400MHz, CDCl3) δ=7.53 (d, J=7.8Hz, 1H), 7.50 (d, J=7.8Hz, 1H), 7.34 (t, J=7.6Hz, 1H), 5.97 (br s, 1H), 3.55-3.47 (m, 2H), 3.16 (t, J=7.2Hz, 2H), 2.65-2.51 (m, 2H), 2.42-2.25 (m, 2H).

[0231] Step 2

[0232] Compound 9a (100 mg, 471 μmol) was dissolved in anhydrous ethanol (2 mL). Hydroxylamine hydrochloride (98.2 mg, 1.41 mmol) and triethylamine (143 mg, 1.41 mmol) were added to the mixture under nitrogen. The reaction solution was stirred at 80°C for 4 hours under nitrogen protection. The reaction solution was concentrated, 5 mL of ethyl acetate was added to the crude product, the reaction solution was stirred at 20°C for 12 hours, filtered, and the filter cake was dried under reduced pressure to obtain compound 9b.

[0233] MS-ESI calculated value [M+H] + 246, measured value 246.

[0234] Step 3

[0235] Compound 1d (123 mg, 594 μmol) was dissolved in anhydrous N, N-dimethylformamide (10 mL), 1-hydroxybenzotriazole (107 mg, 793 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (152 mg, 793 μmol) were added to the mixture, the reaction solution was stirred at 25°C for 1 hour, compound 9b (162 mg, 660 μmol) was added to the reaction solution, the reaction solution was stirred at 25°C for 2 hours, and the reaction solution was further stirred at 80°C for 8 hours. 20 mL of water was added to the reaction solution, and it was extracted with ethyl acetate (30 mL*2), the organic phases were combined, the organic phases were washed with 20 mL of water and 20 mL of saturated brine, and dried over anhydrous sodium sulfate, and the crude product was purified by thin layer chromatography (0:1, petroleum ether / ethyl acetate, Rf=0.42) to obtain compound 9c.

[0236] MS-ESI calculated value [M+H] + 416, measured value 416.

[0237] Step 4

[0238] Compound 9c (100 mg, 241 μmol) was dissolved in anhydrous tetrahydrofuran (10 mL). Potassium tert-butoxide (35.1 mg, 313 μmol) was added to the mixture at 0°C, and the reaction solution was stirred at 0°C for 0.5 hours. 3-bromopropanol (100 mg, 722 μmol) was added to the mixture at 0°C, and the reaction solution was stirred at 20°C for 2 hours. 30 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL*3). The organic phases were combined, washed with 30 mL of water and 30 mL of saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by thin layer chromatography (0:1, petroleum ether / ethyl acetate, Rf=0.46) to obtain compound 9d.

[0239] MS-ESI calculated value [M+H] + 474, measured value 474.

[0240] Step 5

[0241] Compound 9d (31.0 mg, 65.6 μmol) was dissolved in anhydrous dichloromethane (2 mL), and Dess-Martin periodinane (46.1 mg, 109 μmol) was added to the mixture. The reaction solution was stirred at 20°C for 2 hours, and a saturated 10% sodium bicarbonate solution (10 mL) was added to the reaction solution. The mixture was extracted with dichloromethane (10 mL*2), and the organic phases were combined, washed with 10 mL of water and 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound 9e.

[0242] MS-ESI calculated value [M+H] +472, measured value 472.

[0243] Step 6

[0244] Compound 9e (35.0 mg, 74.2 μmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and tert-butanol (1 mL). To the mixture were added 2-methyl-2-butene (52.0 mg, 742 μmol), disodium hydrogen phosphate (105 mg, 742 μmol) and sodium chlorite (10.0 mg, 111 μmol) in water (1 mL). The reaction solution was stirred at 20°C for 1 hour. Water (10 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL*2). The organic phases were combined, washed with 10 mL of water and 10 mL of saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex Luna C18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 50%-70%, 7 minutes} was purified to obtain compound 9.

[0245] MS-ESI calculated value [M+H] + 488, measured value 488.

[0246] 1 H NMR (400MHz, CDCl3) δ=9.14 (d, J=2.3Hz, 1H), 8.63 (d, J=2.3Hz, 1H), 8.07 (d, J=6.2Hz, 1H), 7.46-7.34 (m, 2H), 5.6 3-5.51 (m, 1H), 3.74-3.56 (m, 4H), 3.42-3.30 (m, 2H), 2.80-2.60 (m, 4H), 2.40-2.18 (m, 2H), 1.48 (d, J=6.1Hz, 6H).

[0247] Example 10

[0248]

[0249] Synthesis route:

[0250]

[0251] first step

[0252] Compound 1i (2.00 g, 7.52 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and lithium aluminum hydride (570 mg, 15.0 mmol) was slowly added to the reaction solution in batches, and the reaction solution was stirred at 70 ° C for 3 hours. The reaction solution was quenched by adding saturated ammonium chloride solution (50 mL), filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was diluted with water (100 mL), extracted with ethyl acetate (100 mL × 2), and the combined organic phase was washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude compound 10a.

[0253] MS-ESI calculated value [M+H] + 252 and 254, measured values ​​252 and 254.

[0254] Step 2

[0255] Compound 10a (1.22 g, 4.84 mmol) was dissolved in anhydrous dichloromethane (20 mL), di-tert-butyl dicarbonate (1.27 g, 5.81 mmol) and triethylamine (1.18 g, 11.6 mmol) were added to the mixture, and the reaction solution was stirred at 25° C. for 2 hours. The reaction solution was concentrated, and the crude product was separated and purified by silica gel column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.7) to obtain compound 10b.

[0256] MS-ESI calculated values ​​[M+H- t Bu] + 296 and 298, measured values ​​296 and 298.

[0257] Step 3

[0258] Compound 10b (1.44 g, 4.09 mmol), zinc cyanide (720 mg, 6.13 mmol) and 2-dicyclohexylphospho-2,4,6-triisopropylbiphenyl (156 mg, 327 μmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL). Tris(dibenzylideneacetone)dipalladium (150 mg, 164 μmol) was added to the mixture under nitrogen protection, and the reaction solution was stirred at 90°C for 12 hours. The reaction solution was diluted with water (100 mL), extracted with ethyl acetate (100 mL×2), and the combined organic phase was washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (5:1, petroleum ether / ethyl acetate, Rf=0.25) to obtain compound 10c.

[0259] MS-ESI calculated values ​​[M+H- t Bu] + 243, measured value 243.

[0260] Step 4

[0261] Compound 10c (720 mg, 2.41 mmol) was dissolved in anhydrous ethanol (10 mL), hydroxylamine hydrochloride (502 mg, 7.22 mmol) and triethylamine (731 mg, 7.22 mmol) were added to the mixture, and the reaction solution was stirred at 80° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and water (25 mL) was added to the residue for dilution. The reaction solution became turbid, and was filtered. The filter cake was vacuum dried to obtain a crude compound 10d.

[0262] MS-ESI calculated value [M+H] + 332, measured value 332.

[0263] Step 5

[0264] Compound 1d (790 mg, 3.83 mmol) was dissolved in anhydrous N, N-dimethylformamide (10 mL), 1-hydroxybenzotriazole (621 mg, 4.60 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (882 mg, 4.60 mmol) were added to the mixture, the reaction solution was stirred at 25°C for 1 hour, compound 10d (1.27 g, 3.83 mmol) was added to the reaction solution, the reaction solution was stirred at 25°C for 2 hours, and the reaction solution was stirred at 80°C for 8 hours. 80 mL of water was added to the reaction solution, extracted with ethyl acetate (60 mL*3), the organic phases were combined, the organic phases were washed with saturated brine (60 mL*3), dried over anhydrous sodium sulfate, and concentrated to obtain compound 10e.

[0265] MS-ESI calculated values ​​[M+H- t Bu] + 446, measured value 446.

[0266] Step 6

[0267] Compound 10e (1.93 mg, 2.74 mmol) was dissolved in ethyl acetate (30 mL), and ethyl acetate hydrochloride (4 M, 6.85 mL) was added to the mixture. The reaction solution was stirred at 25°C for 1 hour, filtered, and the filter cake was collected. Ethyl acetate (50 mL) was added to the filter cake, and stirred at 20°C for 12 hours. The filter cake was filtered and dried under reduced pressure to obtain the hydrochloride salt of compound 10f.

[0268] MS-ESI calculated value [M+H] + 402, measured value 402.

[0269] Step 7

[0270] The hydrochloride of compound 10f (50.0 mg, 114 μmol) and compound 10g (43.8 mg, 343 μmol) were dissolved in 1,2-dichloromethane (4 mL), glacial acetic acid (3.43 mg, 57.9 μmol) was added to the mixture, and the reaction solution was stirred at 20°C for 6 hours, sodium acetate borohydride (72.6 mg, 343 μmol) was added to the reaction solution, and the reaction solution was stirred at 20°C for 12 hours. The mixture was concentrated under reduced pressure, 10% aqueous sodium bicarbonate (10 mL) was added to the crude product, and the mixture was extracted with ethyl acetate (10 mL*2), the organic phases were combined, washed with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 10h.

[0271] MS-ESI calculated value [M+H] + 514, measured value 514.

[0272] Step 8

[0273] Compound 10h (140 mg, 273 μmol) was dissolved in anhydrous tetrahydrofuran (12 mL), and a solution of sodium hydroxide (43.6 mg, 1.09 mmol) in water (3 mL) was added to the mixture, and the reaction solution was stirred at 20°C for 12 hours. A 1M aqueous hydrochloric acid solution was used to adjust the pH value to about 5, and the mixture was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: 3_Phenomenex LunaC18 75*30mm*3μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 34%-54%, 7 minutes} to obtain compound 10.

[0274] MS-ESI calculated value [M+H] + 500, measured value is 500.

[0275] 1 H NMR (400MHz, MeOH-d4) δ=9.19 (d, J=2.4Hz, 1H), 8.80 (d, J=2.4Hz, 1H), 8.12 (d, J=7.8Hz, 1H), 7.61 (d, J=7.3Hz, 1H), 7.53-7.45 (m, 1H), 5 .68-5.53 (m, 1H), 4.07-3.67 (m, 3H), 3.55-3.36 (m, 4H), 3.11-2.95 (m, 1H), 2.78-2.67 (m, 2H), 2.58-2.23 (m, 6H), 1.47 (d, J=6.2Hz, 6H).

[0276] Embodiment 11

[0277]

[0278] Synthesis route:

[0279]

[0280] first step

[0281] Compound 4d (200 mg, 424 μmol), compound 11a (103 mg, 424 μmol), potassium phosphate (180 mg, 848 μmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (31.0 mg, 42.4 μmol) were dissolved in dioxane (3 mL) and water (1 mL). Under nitrogen protection, the reaction solution was stirred at 80° C. for 12 hours. The reaction solution was decompressed, 15 mL of water was added to the residue, and the mixture was extracted with ethyl acetate (10 mL×2). The organic phases were combined, washed with saturated brine (10 mL), and dried over anhydrous sodium sulfate. The crude product was separated and purified by thin layer chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.17) to obtain compound 11b.

[0282] MS-ESI calculated value [M+H] + 508 and 510, measured values ​​508 and 510.

[0283] Step 2

[0284] Compound 11b (40.0 mg, 78.6 μmol), compound 4c (22.6 mg, 78.6 μmol), potassium phosphate (33.4 mg, 157 μmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (5.76 mg, 7.87 μmol) were dissolved in dioxane (3 mL) and water (1 mL). Under nitrogen protection, the reaction solution was stirred at 80°C for 12 hours. The reaction solution was concentrated under reduced pressure, and water (15 mL) was added to the residue. The mixture was extracted with ethyl acetate (10 mL×2). The organic phases were combined, washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and concentrated to obtain compound 11c.

[0285] MS-ESI calculated value [M+H] + 590, measured value 590.

[0286] Step 3

[0287] Compound 11c (56.0 mg, 94.9 μmol) was dissolved in ethyl acetate (1 mL), and a hydrochloric acid ethyl acetate (4 M, 1 mL) solution was added to the mixture. The reaction solution was stirred at 25°C for 1 hour, and the reaction solution was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography {hydrochloric acid conditions, column model: Phenomenex luna C18 150*25mm*10μm; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 38%-68%, 10 minutes) to obtain compound 11.

[0288] MS-ESI calculated value [M+H] + 476, measured value 476.

[0289] 1 H NMR (400MHz, CDCl3) δ = 8.93 (d, J = 2.4Hz, 1H), 8.52 (d, J = 2.4Hz, 1H), 7.86-7.76 (m, 1H), 7.48-7.35 (m, 2H), 5.58-5.47 (m, 1H), 3.91-3.78 ( m, 2H), 3.72-3.45 (m, 4H), 3.40 (t, J=7.2Hz, 2H), 2.79 (d, J=16.8Hz, 1H), 2.69 (d, J=16.8Hz, 1H), 2.43-2.24 (m, 2H), 1.46 (d, J=6.4Hz, 6H).

[0290] Test Example 1: In vitro evaluation of the agonist activity of the compounds of the present invention on S1PR1

[0291] Experimental purpose: To detect the agonist activity of compounds on S1PR1

[0292] 1. Cell Treatment

[0293] 1. Thaw the PathHunter cell line according to standard procedures;

[0294] 2. Inoculate the cells in 20 μl of a 384-well microplate and incubate at 37°C for an appropriate time.

[0295] 2. Agonists

[0296] 1. For agonist assays, cells are incubated with the sample to be tested to induce a response;

[0297] 2. Dilute the stock solution to be tested 5-fold into the buffer solution;

[0298] 3.5 μl of the 5-fold dilution was added to the cells and incubated at 37°C for 90-180 minutes. The vehicle concentration was 1%.

[0299] 3. Signal Detection

[0300] 1. Add 12.5 μl or 15 μl of 50% volume ratio PathHunter detection reagent once, and then incubate at room temperature for 1 hour to generate detection signal;

[0301] 2. Read the microplate using a PerkinElmer EnvisionTM instrument to detect chemiluminescent signals.

[0302] 4. Data Analysis

[0303] 1. Use CBIS data analysis suite (ChemInnovation, CA) to analyze compound activity;

[0304] 2. Calculation formula: % activity = 100% x (average test sample RLU - average solvent RLU) / (average maximum control ligand - average solvent RLU)

[0305] The experimental results are shown in Table 1:

[0306] Table 1 S1PR1 agonist activity test results

[0307] Test product S1PR1 agonist activity, Emax Compound 1 0.134nM, 99.3% Compound 3 4.46nM, 62.8% Compound 4 20.9nM, 67.6% Compound 5 24.3nM, 63.5% Compound 6 7.1nM, 78.5% Compound 7 284nM, 88.1% Compound 8 1.25nM, 110% Compound 9 0.613nM, 104% Compound 10 0.474nM, 92.0% Compound 11 6.12nM, 104%

[0308] Conclusion: The compounds of the present invention all have significant and even unexpected S1PR1 agonist activity.

[0309] Test Example 2: Pharmacokinetic Evaluation of Compounds in Rats

[0310] Experimental purpose: To test the pharmacokinetics of the compound in SD rats

[0311] Experimental Materials:

[0312] Sprague Dawley rats (male, 200-300 g, 7-9 weeks old, Shanghai Slake)

[0313] Experimental operation:

[0314] The pharmacokinetic characteristics of rodents after intravenous and oral administration of the compound were tested according to the standard protocol. In the experiment, the candidate compound was prepared into a clear solution and given a single intravenous injection and oral administration to rats. The intravenous solvent was a 5:95 DMSO and 10% hydroxypropyl β-cyclodextrin aqueous solution, and the oral solvent was a 0.5% w / v methylcellulose and 0.2% w / v Tween 80 aqueous solution. Whole blood samples were collected within 24 hours, centrifuged at 3000g for 15 minutes, and the supernatant was separated to obtain plasma samples. Four volumes of acetonitrile solution containing internal standard were added to precipitate proteins, and the supernatant was centrifuged and added with an equal volume of water. The supernatant was centrifuged again and the supernatant was injected. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis method, and pharmacokinetic parameters such as peak concentration, clearance, tissue distribution, half-life, area under the drug-time curve, bioavailability, etc. were calculated.

[0315] Experimental results:

[0316] Table 2 Pharmacokinetic test results

[0317]

[0318] Conclusion: The compounds of the present invention show good bioavailability, higher area under the concentration-time curve and lower clearance in the pharmacokinetics of SD rats.

[0319] Test Example 3: Pharmacokinetic Evaluation of Compounds in Mice

[0320] Experimental purpose: To test the pharmacokinetics of compounds in CD-1 mice

[0321] Experimental Materials:

[0322] CD-1 mice (male, 20-40 g, 6-10 weeks old, Shanghai Bikai)

[0323] Experimental operation:

[0324] The pharmacokinetic characteristics of the compounds after intravenous and oral administration in rodents were tested by standard protocols. In the experiment, the candidate compounds were prepared into clear solutions and given a single intravenous injection and oral administration to two rats. The intravenous solvent was 5:95 DMSO and 10% hydroxypropyl β-cyclodextrin aqueous solution, and the oral solvent was 0.5% w / v methylcellulose and 0.2% w / v Tween 80 aqueous solution. Whole blood samples were collected within 24 hours, centrifuged at 3200g for 10 minutes, and the supernatant was separated to obtain plasma samples. Four volumes of acetonitrile solution containing internal standard were added to precipitate proteins, and the supernatant was centrifuged and added with an equal volume of water. The supernatant was centrifuged again and the supernatant was injected. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis method, and pharmacokinetic parameters such as peak concentration, tissue distribution, clearance, half-life, area under the drug-time curve, bioavailability, etc. were calculated.

[0325] Experimental results:

[0326] Table 3 Pharmacokinetic test results

[0327]

[0328] Conclusion: The compounds of the present invention show good bioavailability, higher area under the concentration-time curve and lower clearance in the pharmacokinetics of CD-1 mice.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, E is selected from O; Ring A is 1,2,4-oxadiazolyl; R1 is selected from C 1-3 Alkyl and cyclobutyl, the C 1-3 The alkyl and cyclobutyl groups are optionally substituted with 1, 2 or 3 R a replace; R2 is selected from F, Cl, Br, CN and CHF2; R3 is selected from C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with 1, 2 or 3 R b replace; R a Each independently selected from F, Cl, Br, OH, NH2 and COOH; R b are independently selected from F, Cl and Br.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: Ring A is 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R a are independently selected from OH and COOH.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from CH3, CH2CH3, CH2CH2CH3 and The CH3, CH2CH3 and Optional 1, 2 or 3 R a replace.

5. The compound according to any one of claims 1, 3 or 4 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from CH2CH2OH, CH2COOH, CH2CH2COOH and 6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R2 is selected from Br and CN.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: R3 is selected from C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R b replace.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein: R3 is selected from CH(CH3)2.

9. The compound according to claim 1 or 8 or a pharmaceutically acceptable salt thereof, wherein: Structural unit Selected from 10. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is selected from: in, R1 is as defined in claim 1; R2 as defined in claim 1; R3 as defined in claim 1; E as defined in claim 1.

11. A compound represented by the following formula or a pharmaceutically acceptable salt thereof:

12. Use of the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases associated with S1PR1.

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