Process for the preparation of heterocyclic shp2 inhibitors

Abstract

The present application belongs to the field of pharmaceutical chemistry, and relates to a preparation method of a heterocyclic SHP2 inhibitor, in particular to a preparation method of (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one or a salt, hydrate, solvate or crystal thereof of formula (I),

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a method for preparing (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidine-4(3H)-one or its salts, hydrates, solvates or crystals. Background Technology

[0002] SHP2 phosphatase is a non-receptor PTP (protein tyrosine phosphatase) encoded by the PTPN11 gene. It comprises two N-terminal SRC (sarcoma gene) homologous domains (SH2), a PTP domain, and a C-terminal tail. X-ray diffraction results indicate that SHP2 interacts with the PTP domain via the N-terminal SH2 domain, blocking ATP entry into the catalytic site; this kinase exists in a self-inhibitory conformation. Some small peptides or proteins that bind to the SH2 domain can activate phosphorylation of this enzyme, promoting cancer development. In cells, SHP2 function is related to downstream receptor tyrosine kinases in the cytoplasm, including RAS-ERK, PI3K-AKT, and JAK-STAT. First, SHP2 binds to RAS and dephosphorylates it, thereby increasing the activity of the effector protein RAF and activating the RAS / ERK / MAPK proliferative signaling pathway. Second, SHP2 participates in the PD-1 / PD-L1 signaling pathway and promotes immune escape. The PD-1 / SHP2 / STAT1 / T-bet signaling axis mediates the immunosuppressive effect of PD-1 on Th1 cells. Therefore, inhibiting PD-1 or SHP2 can restore the immune function of Th1 cells and activate T cells, thereby relieving immunosuppression in the tumor microenvironment.

[0003] SHP2 is associated with the development of a variety of diseases, such as Noonan syndrome, breast cancer, melanoma, gastric cancer, esophageal cancer, lung cancer, colon cancer, head cancer, neuroblastoma, squamous cell carcinoma of the head and neck, anaplastic large cell lymphoma, and glioblastoma.

[0004] Inhibitors targeting the catalytic site of SHP2 generally exhibit poor selectivity and druggability. In recent years, researchers have discovered that inhibiting SHP2 activity through allosteric site modification can improve both activity and selectivity, leading to some progress in drug research. However, the development of superior SHP2 inhibitors remains crucial to obtain drugs with enhanced activity and pharmacokinetic properties for the treatment of SHP2-mediated diseases. Summary of the Invention

[0005] The inventors of this invention have discovered a heterocyclic SHP2 inhibitor, the compound structure of which is shown in Formula (I) below, and its chemical name is (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one (hereinafter referred to as "Compound (I)").

[0006]

[0007] The inventors of this invention have discovered that the compound of formula (I) or its hydrate, solvate or crystals exhibit significant inhibitory activity against SHP2, and are very promising therapeutic agents for SHP2-related diseases.

[0008] As is well known, for human use, due to safety requirements, domestic and international regulatory agencies have very low limits on unproven or undetermined toxicity impurities in active pharmaceutical ingredients (APIs). Impurities in APIs may arise from their own degradation or from the preparation process, including unreacted starting materials, chemical derivatives of impurities contained in the starting materials, and synthetic byproducts. Therefore, it is necessary to study the preparation methods of compounds of formula (I) or their derivatives to obtain methods that are mild in reaction conditions, stable in process, easy to purify, easy to operate, and conducive to large-scale industrial production of compounds of formula (I) or their pharmaceutically acceptable salts, isomers, solvates, or crystals.

[0009] One object of the present invention is to provide a method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, comprising the step of removing an amino protecting group from a compound of formula (II) under the action of an acidic reagent to generate a compound of formula (I), wherein R is an amino protecting group.

[0010]

[0011] In some preferred embodiments, the method for preparing the compound of formula (I) of the present invention or its salt, hydrate, solvate or crystal, wherein R is selected from alkyltrisilylate, aryltrisilylate, alkyl, alkylacyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy and aryloxy, wherein the alkyltrisilylate, aryltrisilylate, alkyl, alkylacyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy and aryloxy are optionally substituted with one or more halogens, hydroxyl, amino, carboxyl, cyano, nitro, alkyl; more preferably, R is selected from C 1-10 Alkyltrisilyl, C 6-10 Aryltrisilyl, C 1-10 Alkyl, C1-10 Alkyl acyl, C 6-10 Aryl acyl, C 1-6 alkylsulfonyl, C 6-10 arylsulfonyl, C 1-6 alkoxycarbonyl, C 6-10 aryloxycarbonyl C 1-6 Alkoxy and C 6-10 aryloxy groups, wherein the alkyltrisyl, aryltrisyl, alkyl, alkylacyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy, and aryloxy groups are optionally substituted with one or more halogens, hydroxyl groups, amino groups, carboxyl groups, cyano groups, nitro groups, C6 groups, C7 groups, C8 groups, C9 ... 1-6 Alkyl substitution; more preferably, R is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, methyl, tert-butyl, allyl, triphenylmethyl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, trifluoroacetyl, benzoyl, methanesulfonyl, ethanesulfonyl, propylsulfonyl, butylsulfonyl, tert-butylsulfonyl, benzomethanesulfonyl, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl, 2-biphenyl-2-propoxycarbonyl, methoxycarbonyl, phenoxycarbonyl, tert-butyloxycarbonyl (t-butyloxy) carbonyl (Boc), methoxy, ethoxy, phenoxy, and trimethylsilylethoxy; more preferably, R is selected from trimethylsilyl, trimethylsilylethoxy, benzyloxycarbonyl, tert-butyloxycarbonyl, 2-biphenyl-2-propoxycarbonyl, methoxycarbonyl, tert-butylsulfonyl, formyl, trifluoroacetyl, triphenylmethyl, and benzyl.

[0012] In some preferred embodiments, the method for preparing the compound according to formula (I) of the present invention further includes the step of removing the amino protecting group from the compound of formula (II) under the action of an acidic reagent to generate a salt of the compound of formula (I), and then reacting the salt of the compound of formula (I) with a basic reagent in an acid-base neutralization reaction to generate the compound of formula (I).

[0013] In some preferred embodiments, the present invention provides a method for preparing the compound of formula (I) of the present invention, wherein the acidic reagent is an inorganic acid or an organic acid; more preferably, the acidic reagent is selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, phosphoric acid, HCl-ethanol and HCl-dioxane; even more preferably, the acidic reagent is selected from HCl-dioxane.

[0014] In some preferred embodiments, the present invention provides a method for preparing the compound of formula (I) of the present invention, wherein the salt of the compound of formula (I) is an inorganic acid salt or an organic acid salt of the compound of formula (I); more preferably, the salt of the compound of formula (I) is selected from the hydrochloride, sulfate, methanesulfonate and phosphate of the compound of formula (I).

[0015] The inventors of this invention further investigated the molar equivalent ratio of the compound shown in formula (II) and the acidic reagent. They found that a low molar equivalent of the acidic reagent resulted in incomplete material conversion, while a high molar equivalent of the acidic reagent did not significantly improve the main reaction. Based on the data, in some preferred embodiments, this invention provides a method for preparing the compound of formula (I), wherein the molar ratio of the compound of formula (II) to the acidic reagent is about 1:1 to about 1:10; more preferably, the molar ratio of the compound of formula (II) to the acidic reagent is about 1:4 to about 1:8; and even more preferably, the molar ratio of the compound of formula (II) to the acidic reagent is about 1:6.

[0016] In some specific embodiments, the method for preparing the compound of formula (I) of the present invention, or its salt, hydrate, solvate, or crystal, further includes the step of reacting the compound of formula (III) with the compound of formula (IV) to generate the compound of formula (II), wherein X is a leaving group and R is an amino protecting group.

[0017]

[0018] In some specific embodiments, the present invention provides a method for preparing a compound of formula (II) of the present invention, wherein the leaving group X is selected from halogen, hydroxyl, alkoxy, acyloxy, aryloxy, heteroaryloxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, acyl and hydroxyl active esters; preferably, X is selected from halogen and hydroxyl active esters; more preferably, X is selected from fluorine, chlorine, bromine, iodine, carboxylic acid ester group, sulfonate group, phosphate ester group and borate ester group. In some specific embodiments, the leaving group X is selected from fluorine, chlorine, bromine, iodine, methanesulfonate group, trifluoromethanesulfonate group, benzenesulfonate group, toluenesulfonate group, p-bromobenzenesulfonate group and p-nitrobenzenesulfonate group.

[0019] In some specific embodiments, the present invention provides a method for preparing the compound of formula (II) of the present invention, wherein the preparation method includes a catalyst, preferably an acidic reagent; more preferably, the catalyst is an organic acid; even more preferably, the catalyst is selected from formic acid, acetic acid, hydrochloric acid, phosphoric acid, sodium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, HCl-ethanol and HCl-dioxane; even more preferably, the catalyst is selected from acetic acid.

[0020] The inventors of this invention discovered that the solvent has a significant impact on the step of reacting compound (III) with compound (IV) to generate compound (II). The reaction solvents were screened, as shown in Table 1. In some preferred embodiments, this invention provides a method for preparing compound (II) of this invention, wherein the reaction solvent is a high-boiling-point solvent. More preferably, the reaction solvent is selected from acetonitrile, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, and dioxane; even more preferably, the reaction solvent is selected from dioxane.

[0021] Table 1

[0022]

[0023] In some specific embodiments, the method for preparing the compound of formula (I) or its salt, hydrate, solvate, or crystal of the present invention further includes the step of reacting the compound of formula (V) or its salt with the compound of formula (VI) in the presence of a basic reagent to generate the compound of formula (IV), wherein X is a leaving group and R is an amino protecting group.

[0024]

[0025] In some preferred embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, wherein the compound of formula (V) or its salt undergoes a condensation or substitution reaction with the compound of formula (VI) in the presence of a basic reagent.

[0026] The inventors of this invention investigated the selection of basic reagents. Extensive experimental data showed that in the step where compound (V) reacts with compound (VI) under the action of a basic reagent to generate compound (IV), the type of base plays a crucial role in the reaction. Data indicates that organic bases are superior to inorganic bases. Some experimental data are shown in Table 2. In some preferred embodiments, this invention provides a method for preparing compound (IV) of this invention, wherein the basic reagent is selected from organic bases, more preferably from DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DBN (1,5-diazabicyclo[4.3.0]non-5-ene), N,N-diisopropylethylamine, triethylamine, 2,4,6-trimethylpyridine, and N-methylmorpholine, and even more preferably from DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).

[0027] Table 2

[0028]

[0029] In some specific embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, which further comprises a catalyst, said catalyst preferably selected from benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0030] The inventors of this invention have discovered that the reaction mechanism between compounds of formula (V) and (VI) is relatively complex. The catalyst not only activates the substrate to catalyze the main reaction but also accelerates the formation of byproducts. The inventors have found that adding the catalyst BOP in batches effectively controls the main reaction and suppresses side reactions. In some specific embodiments, this invention provides a method for preparing compound (IV) of this invention, wherein BOP is added in batches.

[0031] Furthermore, the material ratio plays a crucial role in the reaction process. After considering the feeding method, the inventors of this invention screened the material ratio. They discovered that when the ratio of compound (VI) is too high, it generates impurities with the catalyst; when the ratio is too low, the residue of compound (V) increases. Therefore, in some preferred embodiments, this invention provides a method for preparing compound (IV), wherein the molar ratio of compound (V) to compound (VI) is selected from 1:1. Data shows that in the catalytic system BOP and DBU, when the DBU ratio is too low, there will be a large residue of starting material, while the production of byproducts will also decrease. When the BOP ratio is too low, the material conversion is incomplete. When the BOP ratio is too high, the impact on the main reaction is limited, but the byproducts increase. Therefore, in some preferred embodiments, this invention provides a method for preparing compound (IV), wherein the molar ratio of BOP and DBU is preferably selected from 1.5:5 to 2.0:5.

[0032] In some preferred embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, wherein the molar ratio of the compound of formula (V) to the compound of formula (VI), BOP, and DBU is about 1:0.9:1:5 to 1:1.05:10:5, more preferably about 1:1:1.5:5 to 1:1:6:5, and even more preferably about 1:1:1.5:5 to 1:1:2:5.

[0033] In some preferred embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, wherein the reaction solvent is selected from high-boiling-point solvents, preferably selected from acetonitrile, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dioxane, DMSO and DMF, and more preferably selected from DMF.

[0034] In some specific embodiments, the present invention provides a method for preparing a compound of formula (I) or a salt, hydrate, solvate, or crystal thereof, wherein the method comprises the following steps:

[0035]

[0036] 1) Compound (V-1) reacts with compound (VI-1) under the action of a basic reagent to generate compound (IV-1);

[0037] 2) Compound (III) reacts with compound (IV-1) to form compound (II-1);

[0038] 3) Compound (II-1) reacts with an acidic reagent to form compound (I-1), and compound (I-1) then undergoes an acid-base neutralization reaction with a basic reagent to form compound (I).

[0039] Where x is 1, 2, 3 or 4.

[0040] In some preferred embodiments, the present invention provides a purification method for a compound of formula (I) or its salt, hydrate, solvate or crystal, wherein the method comprises dissolving the compound of formula (I) or its salt, hydrate, solvate or crystal in a solvent and cooling to crystallize; more preferably, the present invention provides a purification method for a compound of formula (I) or its salt, hydrate, solvate or crystal, wherein the method comprises dissolving the compound of formula (I) or its salt, hydrate, solvate or crystal in a solvent and adding a poor solvent to crystallize; wherein the solvent is preferably selected from solvents with high polarity, more preferably selected from N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO); the poor solvent is selected from methane, ethane, propane, butane, pentane, heptane, methanol, ethanol and ethyl acetate.

[0041] The inventors of this invention have discovered that the preparation method of the compound of formula (I) or its salt, hydrate, solvate or crystal provided by this invention has fewer reaction steps, is simpler to operate, is more environmentally friendly, has higher yield and purity, milder reaction conditions, is easier to purify, has stable process, is easy to operate, and can meet the needs of industrial-scale production and application.

[0042] Terminology Explanation

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] The "salt" of this invention can be any salt, particularly a pharmaceutically acceptable salt. In this document, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt formed by the compound described in this invention with the "acid" or "acidic reagent" of this invention. The "acid" or "acidic reagent" of this invention may be selected from hydrochloric acid, hydrobromic acid, phosphoric acid, aminosulfonic acid, nitric acid, p-toluenesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, acetic acid, oxalic acid, phenylacetic acid, propionic acid, malonic acid, trifluoroacetic acid, succinic acid, glycolic acid, stearic acid, ascorbic acid, dihydroxynaphthyl acid, hydroxymaleic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, citric acid, D-gluconic acid, lactic acid, L-malic acid, succinic acid, L-tartaric acid, fumaric acid, α-ketoglutarate, hippuric acid, maleic acid, D-tartaric acid, methanesulfonic acid, or analogs thereof. The pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention containing acidic or basic moieties using conventional chemical methods. Typically, salts of basic compounds can be prepared by stoichiometry or by reacting a free base with a stoichiometric or excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, salts of acidic compounds can be formed by reacting with a suitable inorganic or organic base.

[0045] In this invention, "basic reagent" refers to a compound capable of deprotonating hydroxyl or amino groups. Examples of bases include, but are not limited to, compounds combined with alcohol solvents (C... 1-6 Alkyl) oxide ((C) 1-6 Alkyl)OM), wherein (C) 1-6 Alkyl oxides include, but are not limited to, MeO-, EtO-, n-PrO-, i-PrO-, t-BuO-, i-AmO- (isopentoxy), etc., wherein M is an alkali metal cation, such as Li. + Na + K + Alcohol solvents include (C...) 1-6 Alkyl)OH, such as methanol, ethanol, n-propanol, isopropanol, tert-butanol, isoamyl alcohol, etc. Non-alkoxy bases can also be used, such as sodium hydroxide, potassium hydroxide, sodium hydride, sodium hexamethyldimethoxyamine, lithium hexamethyldimethoxyamine, lithium diisopropylamide, calcium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DBN (1,5-diazabicyclo[4.3.0]non-5-ene), Grignard reagents such as (C 1-6 Alkyl)Mg (halogen), including but not limited to methyl magnesium chloride, methyl magnesium bromide, tert-butyl magnesium chloride, tert-butyl magnesium bromide, etc.

[0046] The term "solvent" refers to the form of the compounds of this invention that form solid or liquid complexes by coordination with solvent molecules. Hydrates are a special form of solvate in which coordination with water occurs. Within the scope of this invention, hydrates are preferred solvates.

[0047] The term "crystallization" refers to the various solid forms formed by the compounds described in this invention, including crystalline and amorphous forms.

[0048] In the compounds of this invention, "hydrogen," "carbon," and "oxygen" include all their isotopes. Isotopes should be understood to include those atoms having the same number of atoms but different mass numbers. For example, isotopes of hydrogen include protium, tritium, and deuterium, and isotopes of carbon include... 13 C and 14 C, oxygen isotopes include 16 O and 18 O etc. Detailed Implementation

[0049] The following representative embodiments are provided to better illustrate the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all materials used in the following embodiments are commercially available.

[0050] Example 1: Preparation of (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0051]

[0052] Step 1: Preparation of (R)-N-((R)-1'-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide

[0053]

[0054] In a 50L glass reactor, N,N-dimethylformamide (DMF, 9.45 kg) was added and stirred at room temperature. Then, (R)-2-methyl-N-((R)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)propane-2-sulfinamide (1.0 kg, 3.24 mol) and 6-amino-5-bromo-3-methylpyrimidine-2,4(1H,3H)-dione (0.71 kg, 3.23 mol) were added and stirred until homogeneous. 1,8-diazabicycloundec-7-ene (DBU, 2.47 kg, 14.6 mol) was slowly added to the reactor. Benzotriazole-1-tris(trimethylamino)-hexafluorophosphate (BOP reagent, 2.16 kg, 4.89 mol) was divided into four portions, each 0.4–0.6 kg, and added to the reaction solution every 10–15 minutes. After the reaction was complete, ethyl acetate (15.0 kg) was added to a 100 L glass reactor at room temperature, and stirring was started. The reaction solution was then added to the stirred ethyl acetate, followed by purified water (50.0 kg), and stirring was continued for 10 min. The organic phase was separated, concentrated under reduced pressure, and dried to obtain 0.93 kg of the title compound, with a yield of 56.2%.

[0055] Step 2: Preparation of (R)-N-((R)-1'-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide

[0056]

[0057] 1,4-Dioxane (19.0 kg) was added to a 50 L glass reactor. Stirring was started, and (R)-N-((R)-1'-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide (0.92 kg, 1.8 mol), sodium 2-(trifluoromethyl)piperidin-3-thiolate (0.51 kg, 2.52 mol), and acetic acid (0.11 kg, 1.8 mol) were added. The temperature was raised and maintained at 60–70 °C. The reaction solution was filtered and concentrated under reduced pressure to obtain 1.07 kg of the crude title compound, with a yield of 97.5%.

[0058] Step 3: Preparation of crude (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0059]

[0060] Weigh 30.0 kg of dichloromethane into a 50 L glass reactor, start stirring, and add (R)-N-((R)-1'-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide (1.07 kg). Cool down, maintaining the reaction solution temperature at 15–25 °C, and add dropwise 2.63 L of hydrogen chloride-dioxane solution. Stir for 2–3 hours. Centrifuge.

[0061] Then, at 20–30°C, slowly add NaOH aqueous solution (0.3 kg sodium hydroxide, 0.6 kg purified water) to adjust the pH of the system to 8–9, and continue stirring for 2–4 hours. Centrifuge the material, wash with purified water, and transfer the collected free alkali solid to a 50 L glass reactor. Add 3.37 kg of purified water / anhydrous ethanol (20:1) solution and slurry. Centrifuge the material, and wash the solid sequentially with purified water and anhydrous ethanol. Dry to obtain 0.51 kg of crude title compound, yield 57.5%.

[0062] Step 4: Preparation of the refined product (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0063]

[0064] Dimethyl sulfoxide (3.37 kg) was added to a 50 L glass reactor, and stirring was started. Then, the crude product obtained in the above reaction (0.51 kg) was added, and the temperature was raised to 75–85 °C with stirring until dissolved. The mixture was kept at this temperature for 0.5–1.0 hours, and anhydrous ethanol (1.58 kg) was added dropwise to precipitate crystals. The heating was then turned off, and the mixture was allowed to cool naturally to about 60 °C. Anhydrous ethanol (2.04 kg) was then added dropwise, and the reaction temperature was controlled at 40–60 °C. After the addition was complete, the mixture was allowed to cool naturally to room temperature (20–30 °C), and stirring was continued for 1–2 hours to precipitate crystals. The mixture was filtered, and the solid was washed with anhydrous ethanol (0.76 kg) and dried to obtain 0.37 kg of solid, with a yield of 72.5%. 1H NMR (400MHz, DMSO-d6) δ8.40 (d, J=4.0Hz, 1H), 7.52-7.50 (m, 1H), 7.43 (d, J= 8.5Hz,1H),7.33(d,J=7.5Hz,1H),7.14(t,J=7.5Hz,1H),6.87(t,J=7.5Hz,1 H),6.77(d,J=8.0Hz,1H),4.12(s,1H),3.63-3.53(m,2H),3.31-3.24(m,5H) ,2.09-2.03(m,1H),1.92-1.89(m,2H),1.86-1.82(m,2H),1.77-1.74(m,1H). ESI-MS m / z: 505.2 [M+H] + .

[0065] Comparative Example 1

[0066] The compound represented by the following formula (compound A) was prepared according to the method disclosed in compound 45 of WO2018 / 172984 (PCT / IB2018 / 051973), and identified by proton NMR and mass spectrometry.

[0067] (Compound A).

[0068] Experiment Example 1: Cell Proliferation Inhibition Experiment

[0069] 1. Experimental Materials

[0070] Test compounds: Compounds of formula (I) of the present invention and compounds prepared in comparative examples, each compound was prepared in 20 mM solution using DMSO. The concentrations of the compounds acting on NCI-H358 cells were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM, respectively.

[0071] Human non-small cell lung cancer cells NCI-H358 were purchased from the American Type Culture Collection (ATCC).

[0072] Reagents: CCK-8 proliferation inhibition assay kit, purchased from Jiangsu Kaiji Biotechnology Co., Ltd., China. Instruments: CKX41 inverted microscope, purchased from Olympus, Japan; multi-functional plate reader, purchased from Molecular Devices, USA; cell incubator, purchased from Thermo Fisher Scientific, USA.

[0073] 2. Experimental Methods

[0074] 2.1 Cell Culture:

[0075] Cell thawing: Remove the NCI-H358 cell cryovials from the liquid nitrogen container and place them in a 37°C water bath. Gently shake to thaw as quickly as possible. After thawing, remove the cryovials, sterilize with alcohol swabs, unscrew the caps, aspirate the cell suspension into centrifuge tubes, add 1 mL of serum-containing complete culture medium, mix well, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add complete culture medium, and repeatedly pipette until the cells are completely dispersed and resuspended. Seed the cells at an appropriate concentration in culture dishes. Incubate at 37°C in a CO2 incubator with 5% CO2 and 95% humidified air.

[0076] Cell passage: When cells reach approximately 80-90% confluence, discard the original culture medium (1640 medium + 10% FBS + 1% penicillin-streptomycin + 1mM sodium pyruvate). Add 1 mL of PBS to wash away any remaining medium, then discard the PBS. Add 1 mL of trypsin digestion solution and digest for 1-2 min. Under a microscope, observe that the pseudopodia of the cells have retracted and become rounded, but the cells have not yet detached in sheets. At this point, discard the trypsin and terminate the digestion with 1-2 mL of complete culture medium. Gently pipette and collect the cell suspension. Centrifuge at 1000 rpm for 5 min. Remove the supernatant, resuspend the cells in complete culture medium, and seed them into culture dishes at the desired density. Incubate in a CO2 incubator at 37°C, 5% CO2, and 95% humidified air. Change the culture medium or passage the cells every 2-3 days depending on cell growth.

[0077] 2.2 Experimental Procedure:

[0078] After passage, NCI-H358 cells were resuspended in fresh culture medium (1640 medium + 3% FBS + 1% penicillin and streptomycin + 1mM sodium pyruvate). Cell counts were then performed at a concentration of 1.5 x 10⁻⁶ cells / mL. 4 Seeds were generated at a density of 100 μL / mL into 96-well cell culture plates, with 100 μL added to each well (equivalent to 1.5 x 10⁻⁶ cells / mL). 3 (cells / well). After 24 hours, 100 μL of fresh medium containing different concentrations (2×) of the drug was added to the original medium. The final concentrations of the compound were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM, with two replicates for each concentration group. After incubation for 168 hours, the medium in the wells was aspirated and dried as much as possible. 100 μL of medium containing CCK-8 was added (CCK-8:medium = 1:10). After incubation for a certain period, the 96-well plate was removed from the incubator and equilibrated at room temperature for 5 minutes. The absorbance (OD value) at 450 nm was measured using a multi-functional plate reader, and the cell proliferation inhibition rate was calculated. The calculation formula is: Inhibition (%) = 100 - (OD)实验孔- OD 空白孔 ) / (OD 溶剂对照孔- OD 空白孔 *100, based on different drug concentrations and their corresponding inhibition rates, IC50 was performed using GraghPad 5.0 software. 50 Curve plotting, data analysis, and final IC calculation. 50 Values. The experimental results are shown in Table 3.

[0079] Table 3

[0080]

[0081] As can be seen from the above experiments, the compound of formula (I) of the present invention exhibits good inhibitory activity against NCI-H358 cells and is very promising as a therapeutic agent for non-small cell lung cancer.

[0082] Experiment Example 2: Pharmacokinetic Experiment

[0083] 1. Experimental Materials

[0084] Compound: The compound of formula (I) of this invention. The drug solvent is Captisol / 50mM sodium acetate, pH 4.6 (10% / 90%, w / v%). Oral administration is prepared as a 0.5 mg / mL clear solution; intravenous administration is prepared as a 0.1 mg / mL clear solution.

[0085] Animals: Male BALB / c mice, SPF grade, purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.; 18-20g. An acclimatization period of 2-3 days was given before the experiment.

[0086] Instruments: AB (Alberts & Arpels) API 4500 triple quadrupole liquid chromatography-mass spectrometry system, equipped with an electrospray ionization source (ESI), LC-30AD dual pump; SIL-30AC autosampler; CTO-30AC column oven; DGU-20A3R degasser; AnalystQSA01.01 chromatography workstation; Milli-Q ultrapure water system (Millipore Inc.); Qilinbeier Vortex-5 shaker; HITACHI CF16R XII benchtop high-speed refrigerated centrifuge.

[0087] 2. Experimental Methods

[0088] (1) Three mice per group. The compound of formula (I) of the present invention was administered by gavage (IG) at a dose of 10 mg / kg and by intravenous administration (IV) at a dose of 1 mg / kg.

[0089] (2) Blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 10 h and 24 h after gavage and intravenous administration and placed in heparinized EP tubes (0.6 mL). After centrifugation at 8000 rpm / min for 5 min, the supernatant plasma was collected, frozen at -20℃ and analyzed by LC-MS / MS.

[0090] (3) Based on the blood drug concentration data obtained in the above steps, a blood drug concentration-time curve was plotted, and the pharmacokinetic parameters were calculated using WinNonlin software. The experimental results are shown in Table 4.

[0091] Table 4

[0092]

[0093] The pharmacokinetics of compound A in the comparative example were determined using the method described in Experimental Example 2. The results showed that the bioavailability (F) of compound A was 51.2%, which was significantly lower than that of the compound of formula (I) of the present invention.

[0094] Experimental results show that the compound of the present invention has a good half-life T. 1 / 2 It has good area under the curve (AUC) and bioavailability (F), indicating good oral absorption and exposure, making it suitable for drug development.

[0095] Although the present invention has been described in detail above, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from its spirit and scope. The scope of the invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

1. A method for preparing a compound of formula (I), comprising the steps of removing an amino protecting group from a compound of formula (II) under the action of an acidic reagent to generate a salt of compound (I), and then reacting the salt of compound (I) with a basic reagent in an acid-base neutralization reaction to generate compound (I), wherein, R is an amino protecting group, which is a tert-butylsulfinyl group. 。 2. The method for preparing the compound of formula (I) according to claim 1, wherein the acidic reagent is an inorganic acid or an organic acid.

3. The method for preparing the compound of formula (I) according to claim 1 or 2, wherein the molar ratio of the compound of formula (II) to the acidic reagent is 1:1 to 1:

10.

4. The method for preparing the compound of formula (I) according to claim 1 or 2, further comprising the step of reacting the compound of formula (III) with the compound of formula (IV) to generate the compound of formula (II), wherein X is a leaving group bromine, and R is an amino protecting group tert-butylsulfinyl. 。 5. The method for preparing the compound of formula (I) according to claim 3, further comprising the step of reacting the compound of formula (III) with the compound of formula (IV) to generate the compound of formula (II), wherein X is a leaving group bromine, and R is an amino protecting group tert-butylsulfinyl. 。 6. The method for preparing the compound of formula (I) according to claim 4, wherein the method comprises the step of reacting the compound of formula (III) with the compound of formula (IV) under a catalyst to generate the compound of formula (II).

7. The method for preparing the compound of formula (I) according to claim 5, wherein the method comprises the step of reacting the compound of formula (III) with the compound of formula (IV) under a catalyst to generate the compound of formula (II).

8. The method for preparing the compound of formula (I) according to claim 6, wherein the catalyst is an acidic reagent.

9. The method for preparing the compound of formula (I) according to claim 7, wherein the catalyst is an acidic reagent.

10. The method for preparing the compound of formula (I) according to claim 4, further comprising the step of reacting the compound of formula (V) or its salt with the compound of formula (VI) in the presence of a basic reagent to generate the compound of formula (IV), wherein X is a leaving group bromine, and R is an amino protecting group tert-butylsulfinyl. 。 11. The method for preparing the compound of formula (I) according to claim 5, further comprising the step of reacting the compound of formula (V) or its salt with the compound of formula (VI) under the action of a basic reagent to generate the compound of formula (IV), wherein X is a leaving group bromine, and R is an amino protecting group tert-butylsulfinyl. 。 12. The method for preparing the compound of formula (I) according to claim 6, further comprising the step of reacting the compound of formula (V) or its salt with the compound of formula (VI) in the presence of a basic reagent to generate the compound of formula (IV), wherein X is a leaving group bromine, and R is an amino protecting group tert-butylsulfinyl. 。 13. The method for preparing the compound of formula (I) according to claim 10, wherein the basic reagent is selected from organic bases.

14. The method for preparing the compound of formula (I) according to claim 11, wherein the basic reagent is selected from organic bases.

15. The method for preparing the compound of formula (I) according to claim 12, wherein the basic reagent is selected from organic bases.

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