CN115304613A - Preparation method of heterocyclic SHP2 inhibitor - Google Patents

Abstract

The invention belongs to the field of medicinal chemistry, relates to a preparation method of a heterocyclic SHP2 inhibitor, and particularly relates to (R) -6-amino-2- (3-amino-3H-spiro [ benzofuran-2,4' -piperidine) in a formula (I)]A process for the preparation of (E) -1' -yl) -3-methyl-5- ((2- (trifluoromethyl) pyridin-3-yl) sulfanyl) pyrimidin-4 (3H) -one or a salt, hydrate, solvate or crystal thereof,

Description

Preparation method of heterocyclic SHP2 inhibitor

technical field

The invention belongs to the field of medicinal chemistry, in particular to (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methanol A method for producing yl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one or a salt, hydrate, solvate or crystal thereof.

Background technique

SHP2 phosphatase is a non-receptor PTP (protein tyrosine phosphatase) encoded by PTPN11 gene. It includes two N-terminal SRC (sarcoma gene) homology domains (SH2), a PTP domain and a C-terminal tail. The results of X-ray diffraction showed that SHP2 interacted with the PTP domain through the N-terminal SH2 and blocked ATP from entering the catalytic site. The kinase was in a self-inhibited configuration. Some small peptides or proteins that can bind to the SH2 domain can activate the phosphorylation of this enzyme and promote the development of cancer. In cells, the function of SHP2 is related to the downstream receptor tyrosine kinases in the cytoplasm, including RAS-ERK, PI3K-AKT, and JAK-STAT. First, SHP2 can bind to RAS and dephosphorylate RAS, thereby increasing the effect of the effector protein RAF and activating the RAS/ERK/MAPK signaling pathway to promote proliferation. 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, inhibition of PD-1 or SHP2 can restore the immune function of Th1 and the activation of T cells, and relieve the immune suppression in the tumor microenvironment.

SHP2 is associated with the occurrence of various 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.

Inhibitors targeting its catalytic site are generally poor in selectivity and druggability. In recent years, researchers have found that inhibiting the activity of SHP2 through the allosteric site can improve the activity and selectivity, and some progress has been made in drug research. However, there is still a need to develop better SHP2 inhibitors in order to obtain drugs with superior activity and better pharmacokinetic properties for the treatment of SHP2-mediated related diseases.

Contents of the invention

The inventors of the present invention have discovered a heterocyclic SHP2 inhibitor, the compound structure of which is shown in the following formula (I), 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)pyrimidine- 4(3H)-ketone (hereinafter referred to as "compound of formula (I)"):

The inventors of the present invention have found that the compound of formula (I) or its hydrate, solvate or crystal has significant inhibitory activity on SHP2, and is very promising as a therapeutic agent for SHP2-related diseases.

It is well known that for human medicinal products, domestic and international regulatory agencies have very low limits for unidentified or undetermined toxic impurities in APIs due to safety concerns. Impurities in APIs may be produced due to their own degradation, or may originate from the preparation process, for example, including unreacted starting materials, chemical derivatives of impurities contained in starting materials, synthetic by-products, etc. Therefore, it is necessary to study the preparation method of the compound of formula (I) or its derivatives in order to obtain a compound of formula (I) or its pharmaceutically acceptable preparation with mild reaction conditions, stable process, easy purification, and easy operation, which is conducive to large-scale industrial production. Accepted salts, isomers, solvates or methods of crystallization.

One object of the present invention is to provide a preparation method of the compound shown in formula (I) or its salt, hydrate, solvate or crystal, comprising making the compound of formula (II) remove the amino protecting group under the action of an acidic reagent to generate formula (1) the step of compound, wherein, R is an amino protecting group,

In some preferred embodiments, the present invention provides the preparation method of the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention, wherein R is selected from alkyl trisilyl, aryl trisilane group, alkyl, alkylacyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy and aryloxy, the alkyltrisilyl , aryltrisilyl, alkyl, alkylacyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy and aryloxy are optionally One or more halogen, hydroxyl, amino, carboxyl, cyano, nitro, alkyl substitution; more preferably, R is selected from C _1-10 alkyl trisilyl, C _6-10 aryl trisilyl, C _1-10 alkyl, C _1-10 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, the alkyl trisilyl, aryl trisilyl, alkyl, alkyl acyl, aryl acyl, Alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy and aryloxy are optionally replaced by one or more of halogen, hydroxy, amino, carboxyl, cyano, nitro, C _1-6 alkyl substitution; more preferably, R is selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenyl Silyl, methyl, tert-butyl, allyl, trityl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl , trifluoroacetyl, benzoyl, methanesulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, tert-butylsulfonyl, phenylmethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl , 2-biphenyl-2-propoxycarbonyl, Wat methoxycarbonyl, phenoxycarbonyl, t-butyloxycarbonyl (t-butyloxycarbonyl, Boc), methoxy, ethoxy, phenoxy and trimethyl Silylethoxy; More preferably, R is selected from trimethylsilyl, trimethylsilylethoxy, benzyloxycarbonyl, tert-butoxycarbonyl, 2-biphenyl-2-propoxycarbonyl, Watt methoxycarbonyl, tert-butylsulfonyl, formyl, trifluoroacetyl, trityl and benzyl.

In some preferred embodiments, according to the preparation method of the compound represented by the formula (I) of the present invention, it further comprises making the compound of the formula (II) remove the amino protecting group under the action of an acidic reagent to generate the compound of the formula (I) Salt, a step in which the salt of the compound of formula (I) reacts with an alkaline reagent to generate the compound of formula (I) through an acid-base neutralization reaction.

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; more preferably, the acidic reagent is selected from HCl-dioxane.

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 or organic acid salt of the compound of formula (I); more Further preferably, the salt of the compound of formula (I) is selected from hydrochloride, sulfate, methanesulfonate and phosphate of the compound of formula (I).

The inventor of the present invention further investigated the molar equivalent ratio of the compound shown in formula (II) and the acidic reagent, and the inventor of the present invention found that the molar equivalent of the acidic reagent was low, and the material conversion was incomplete; the molar equivalent of the acidic reagent On the high side, there is no significant improvement on the main reaction. Comprehensive data, in some preferred embodiments, the present invention provides the preparation method of the compound of formula (I) of the present invention, wherein the molar ratio of compound of formula (II) and acidic reagent is about 1:1 to about 1:10; More More preferably, the molar ratio of the compound of formula (II) to the acidic reagent is about 1:4 to about 1:8; even more preferably, the molar ratio of the compound of formula (II) to the acidic reagent is about 1:6.

In some specific embodiments, the preparation method of the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention further comprises reacting the compound of formula (III) with the compound of formula (IV) to form The step of the compound of formula (II), wherein X is a leaving group, R is an amino protecting group,

In some specific embodiments, the present invention provides a method for preparing the compound of formula (II) of the present invention, wherein the leaving group X is selected from halogen, hydroxyl, alkoxy, acyloxy, aryloxy, hetero Active esters of aryloxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, acyl and hydroxy; preferably Preferably, X is selected from active esters of halogen and hydroxyl; further preferably, X is selected from fluorine, chlorine, bromine, iodine, carboxylate, sulfonate, phosphate and borate. In some specific embodiments, the leaving group X is selected from fluorine, chlorine, bromine, iodine, mesylate, triflate, benzenesulfonate, tosylate , brosylate group and p-nitrobenzenesulfonate group.

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, the catalyst is an acidic reagent; more preferably, the catalyst is Organic acid; 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; still more preferably, the The catalyst is selected from acetic acid.

The inventors of the present invention found that in the step of reacting the compound of formula (III) with the compound of formula (IV) to generate the compound of formula (II), the solvent had a great influence on the reaction, and the reaction solvent was screened, see Table 1. In some preferred embodiments, the present invention provides the preparation method of the compound of formula (II) of the present 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; more preferably, the reaction solvent is selected from dioxane.

Table 1

In some specific embodiments, the preparation method of the compound of formula (I) or its salt, hydrate, solvate or crystal of the present invention further comprises making the compound of formula (V) or its salt and the compound of formula (VI) Under the action of basic reagent, reaction occurs to generate the step of compound of formula (IV), wherein X is a leaving group, R is an amino protecting group,

In some preferred embodiments, the present invention provides the preparation method of the compound of formula (IV) of the present invention, wherein, the compound of formula (V) or its salt and the compound of formula (VI) are condensed or substituted under the action of basic reagent reaction.

The inventors of the present invention have investigated the selection of alkaline reagents, and a large number of experimental data show that in the step of reacting the compound of formula (V) with the compound of formula (VI) under the action of alkaline reagents to generate the compound of formula (IV), The type of base plays a key role in the reaction. The data show that organic bases are better than inorganic bases. Some experimental data are shown in Table 2. In some preferred embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, wherein the basic reagent is selected from organic bases, more preferably selected 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-collidine and N-methylmorpholine, more preferably DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).

Table 2

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, preferably selected from the group consisting of benzotriazol-1-yloxytris(dimethylamino) ) Phosphonium hexafluorophosphate (BOP).

The inventors of the present invention have found that the reaction mechanism between the compound of formula (V) and the compound of formula (VI) is relatively complicated, wherein the catalyst can not only activate the substrate to catalyze the main reaction, but also accelerate the formation of by-products. The inventor of the present invention It is found that when the catalyst BOP is added in batches, the main reaction can be better controlled and the side reaction can be suppressed. In some specific embodiments, the present invention provides a method for preparing the compound of formula (IV) of the present invention, wherein BOP is added in batches.

In addition, the ratio of materials plays a key role in the reaction process. After combining the feeding method, the inventors of the present invention screened the ratio of materials. The inventors of the present invention found that when the ratio of the compound of formula (VI) is too high, it will Impurities are produced with the catalyst, the proportioning ratio is too low, and the residue of the compound of formula (V) increases. Therefore, in some preferred embodiments, the present invention provides a preparation method of 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) is selected from 1:1. According to the data, the catalyst system BOP and DBU, when the ratio of DBU is low, there will be a large residue of starting materials, and the generation of by-products will also be reduced. When the ratio of BOP is low, the conversion of materials is incomplete. On the high side, the influence on the main reaction is limited, but the by-products increase. Therefore, in some preferred embodiments, the present invention provides a process for the preparation of the compound of formula (IV) of the present invention, wherein the molar ratio of BOP and DBU is preferably selected from 1.5:5 to 2.0:5.

In some preferred embodiments, the present invention provides the preparation method of compound of formula (IV) of the present invention, wherein the mol ratio of compound of formula (V) and compound of formula (VI), BOP, 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, more preferably about 1:1:1.5:5 to 1:1:2:5 .

In some preferred embodiments, the present invention provides the preparation method of the compound of formula (IV) of the present invention, the reaction solvent is selected from high boiling point solvents, preferably selected from acetonitrile, ethyl acetate, tetrahydrofuran, N,N-dimethylformaldehyde Amide, dioxane, DMSO and DMF are further preferably selected from DMF.

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:

1) The compound of formula (V-1) reacts with the compound of formula (VI-1) under the action of an alkaline reagent to generate the compound of formula (IV-1);

2) The compound of formula (III) reacts with the compound of formula (IV-1) to generate the compound of formula (II-1);

3) The compound of formula (II-1) reacts under the action of an acidic reagent to generate a compound of formula (I-1), and the compound of formula (I-1) reacts with an alkaline reagent to generate a compound of formula (I);

Wherein, x is 1, 2, 3 or 4.

In some preferred embodiments, the present invention provides a purification method of the compound of formula (I) or its salt, hydrate, solvate or crystallization, wherein said method comprises the compound of formula (I) or its salt, hydration Substances, solvates or crystals are dissolved in a solvent, and the temperature is reduced to crystallize; further preferably, the present invention provides a refining method for the compound of formula (I) or its salt, hydrate, solvate or crystals, wherein the method Including dissolving the compound of formula (I) or its salt, hydrate, solvate or crystal in a solvent, adding a poor solvent to crystallize; wherein the solvent is preferably selected from a more polar solvent, more preferably selected from N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO); the poor solvent is selected from methane, ethane, propane, butane, pentane, heptane, methanol, ethanol and ethyl acetate.

The inventors of the present invention have found that the preparation method of the compound of formula (I) provided by the present invention or its salt, hydrate, solvate or crystal has fewer steps in the reaction route, is easy to operate, is more environmentally friendly, has higher yield and Purity, mild reaction conditions, easy purification, stable process, easy operation, and can meet industrial scale production and application.

Glossary

Unless defined otherwise, 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 belongs.

The "salt" of the present invention may be any salt, especially a pharmaceutically acceptable salt. Herein, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt formed by the compound of the present invention and the "acid" or "acidic agent" of the present invention, and the "acid" or "acidic agent" of the present invention The "reagent" may be selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfamic acid, nitric acid, p-toluenesulfonic acid, benzenesulfonic acid, p-sulfanilic acid, sulfuric acid, acetic acid, oxalic acid, phenylacetic acid, propionic acid, Malonic acid, trifluoroacetic acid, succinic acid, glycolic acid, stearic acid, ascorbic acid, pamoic acid, hydroxymaleic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, trans Butenedioic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, citric acid, D-gluconic acid, lactic acid, L-malic acid, succinic acid, L-tartaric acid, fumaric acid, α-ketoglutaric acid , hippuric acid, maleic acid, D-tartaric acid, methanesulfonic acid or their analogs. "Pharmaceutically acceptable salts" of compounds of the present invention can be synthesized by conventional chemical methods from compounds of the present invention that contain acidic or basic moieties. Usually, salts of basic compounds can be prepared by example exchange chromatography, or the free Bases are prepared by reacting stoichiometric or excess amounts of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, salts of acidic compounds may be formed by reaction with a suitable inorganic or organic base.

The "basic reagent" in the present invention refers to a compound capable of deprotonating a hydroxyl group or an amino group. Examples of bases include, but are not limited to, (C _1-6 alkyl)oxides ((C _1-6 alkyl)OM) in combination with alcohol solvents, where (C _1-6 alkyl)oxides include, but are not limited to MeO-, EtO-, n-PrO-, i-PrO-, t-BuO-, i-AmO-(isoamyloxy), etc., and wherein M is an alkali metal cation, such as Li ⁺ , Na ⁺ , K ⁺ Wait. Alcoholic solvents include (C _1-6 alkyl)OH such as, for example, methanol, ethanol, n-propanol, isopropanol, tert-butanol, isoamyl alcohol, and the like. Non-alkoxy bases such as sodium hydroxide, potassium hydroxide, sodium hydride, sodium hexamethyldisilazide, lithium hexamethyldisilazide, lithium diisopropylamide, calcium hydride can also be used , sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, DBU (1,8-diazabicyclo [5.4.0] undec-7-ene), DBN (1,5-di Azabicyclo[4.3.0]non-5-ene), Grignard reagents such as (C _1-6 alkyl)Mg(halogen), which include but not limited to methylmagnesium chloride, methylmagnesium bromide, tert-butyl Magnesium chloride, tert-butylmagnesium bromide, etc.

The term "solvate" refers to a form of a compound of the present invention that forms a solid or liquid complex by coordination with solvent molecules. Hydrates are a special form of solvates in which coordination with water occurs. Within the scope of the present invention, solvates are preferably hydrates.

The term "crystal" refers to various solid forms formed by the compounds of the present invention, including crystalline forms and amorphous forms.

"Hydrogen", "carbon", and "oxygen" in the compounds of the present invention include all isotopes thereof. Isotopes are understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, tritium and deuterium, isotopes of carbon include ¹³ C and ¹⁴ C, and isotopes of oxygen include ¹⁶ O and ¹⁸ O, etc.

Detailed ways

The following representative examples are for better illustrating the present invention, but not for limiting the protection scope of the present invention. Materials used in the following examples are commercially available unless otherwise specified.

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

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

Add N,N-dimethylformamide (DMF, 9.45kg) into a 50L glass reactor, start stirring at room temperature, and then add (R)-2-methyl-N-((R)-3H-spiro[ Benzofuran-2,4'-piperidin]-3-yl)propane-2-sulfinamide (1.0kg, 3.24mol) and 6-amino-5-bromo-3-methylpyrimidine-2,4( 1H,3H)-diketone (0.71kg, 3.23mol), stir well. 1,8-diazabicycloundec-7-ene (DBU, 2.47kg, 14.6mol) was slowly added to the reaction kettle, and benzotriazole-1-tris(trimethylamino)-hexafluorophosphoric acid The ester (BOP reagent, 2.16kg, 4.89mol) was divided into 4 parts, each 0.4-0.6kg, and added to the reaction solution every 10-15min. 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, then the above reaction solution was put into the stirred ethyl acetate, purified water (50.0 kg) was added, and stirred for 10 min. The organic phase was separated, concentrated under reduced pressure, and dried to obtain 0.93kg of the title compound, yield 56.2%

Step 2: (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 preparation of

Take 1,4-dioxane (19.0kg) and add it to a 50L glass reactor, start stirring, add (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 (0.92kg, 1.8mol), 2-(trifluoromethyl)piperidine-3-thiolate sodium (0.51kg, 2.52mol) and acetic acid (0.11kg, 1.8mol), warming up, maintaining the reaction solution The reaction temperature is 60-70°C. The reaction solution was suction filtered and concentrated under reduced pressure to obtain 1.07 kg of the crude product of the title compound, with a yield of 97.5%.

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

Weigh dichloromethane (30.0kg) into a 50L glass reactor, start stirring, 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'-piperidine ]-3-yl)-2-methylpropane-2-sulfinamide (1.07kg), lower the temperature, keep the temperature of the reaction solution at 15-25°C, add hydrogen chloride-dioxane solution (2.63L) dropwise, and stir for 2 -3 hours. centrifugal.

Then slowly add NaOH aqueous solution (sodium hydroxide 0.3kg, purified water 0.6kg) at 20-30°C to adjust the pH of the system to 8-9, and continue stirring for 2-4 hours. The material was centrifuged, rinsed with purified water, and the collected free base solid was transferred to a 50L glass reactor, and purified water/absolute ethanol (20:1) solution (3.37kg) was added to make a slurry. The material was centrifuged, and the solid was washed successively with purified water and absolute ethanol. After drying, 0.51 kg of the crude product of the title compound was obtained, with a yield of 57.5%.

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

Add dimethyl sulfoxide (3.37kg) into a 50L glass reactor, start stirring, then add the crude product (0.51kg) obtained in the above reaction, heat up to 75-85°C, stir to dissolve, and keep warm for 0.5-1.0 hours. Add absolute ethanol (1.58kg) dropwise to precipitate crystals, turn off the heating, and continue to add absolute ethanol (2.04kg) dropwise when the temperature is naturally cooled to about 60°C, and control the reaction temperature at 40-60°C. After the addition, it was naturally lowered to room temperature at 20-30°C and continued to stir and crystallize for 1-2 hours. After suction filtration, the solid was washed with absolute ethanol (0.76 kg) and dried to obtain 0.37 kg of solid with a yield of 72.5%. ¹ H NMR (400MHz, DMSO-d ₆ )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, 1H), 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] ⁺ .

Comparative example 1

The compound (compound A) represented by the following formula was prepared according to the method disclosed in compound 45 in WO2018/172984 (PCT/IB2018/051973), and was identified by hydrogen spectrum and mass spectrum,

(化合物A)。

(Compound A).

Experimental Example 1: Cell Proliferation Inhibition Experiment

1. Experimental materials

Test compound: the compound of formula (I) of the present invention and the compound prepared in the comparative example, each compound was prepared in 20 mM with 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, 0.00038 μM;

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

Reagents: CCK-8 Proliferation Inhibition Detection Kit, purchased from China Jiangsu Kaiji Biotechnology Co., Ltd. Instrument: CKX41 Inverted Microscope, purchased from Olympus Company, Japan; Multi-functional plate reader, purchased from Molecular Devices Company, USA; The incubator was purchased from Thermo Company, USA.

2. Experimental method

2.1 Cell culture:

Cell recovery: Take out the NCI-H358 cell cryopreservation tube from the liquid nitrogen tank and place it in a 37°C water bath, shake it gently to thaw as soon as possible. After thawing, take out the cryopreservation tube, unscrew the cap after disinfection with alcohol cotton ball, suck out the cell solution and pour it into the centrifuge tube, add 1mL of complete medium containing serum, mix well and place in a centrifuge at 1000rpm for 5min. Then discard the supernatant, add complete medium and pipette repeatedly until the cells are completely dispersed and resuspended. Inoculate in a petri dish at an appropriate concentration. Place them in a CO ₂ incubator with 5% CO ₂ and 95% humid air at 37°C.

Cell passage: Cells grow to about 80-90% confluence, discard the original culture medium (1640 medium + 10% FBS + 1% penicillin + 1mM sodium pyruvate), add 1mL of PBS to wash the remaining medium After cleaning, aspirate and discard, add 1mL trypsin digestion solution, digest for 1-2min, observe under the microscope that the pseudopods of the cells retract and become round, but the cells have not fallen off in pieces, at this time, aspirate the trypsin and stop digestion with 1-2mL complete medium. Gently pipette and collect the cell suspension, centrifuge at 1000rpm for 5min. Remove the supernatant, resuspend the cells with complete medium, inoculate them in culture dishes according to the required density, and culture them in a CO ₂ incubator at 37°C, 5% CO ₂ , and 95% humid air. -Change the culture medium every 3 days or carry out subculture.

2.2 Experimental steps:

After subculture, NCI-H358 cells were resuspended with fresh medium (1640 medium + 3% FBS + 1% penicillin + 1mM sodium pyruvate), and after cell counting, inoculated to 96 cells at a density of ^1.5X10 cells/mL. In a well cell culture plate, add 100 μL to each well (that is, 1.5×10 ³ cells/well). After 24 hours, on the basis of the original old medium, 100 μL of fresh medium containing different concentrations (2×) of drugs was added. The final concentrations of the compounds 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, 0.00038 μM, and two replicate wells were set for each concentration group. After continuing to culture in the incubator for 168 hours, discard the culture medium in the wells, blot as dry as possible, and add 100 μL of the medium to which CCK-8 has been added (CCK-8: medium = 1:10). After continuing to culture in the incubator for a certain period of time, take the 96-well plate out of the incubator, place it at room temperature for 5 minutes, place it in a multi-function plate reader, detect the absorbance (OD value) at 450nm, and calculate the cell proliferation Inhibition rate. The calculation formula is Inhibition (%)=100-(OD _{experimental well-} OD _{blank well} )/(OD _{solvent control well-} OD _{blank well} )*100, according to different drug concentrations and their corresponding inhibition rates, using GraghPad 5.0 software The IC ₅₀ curve was drawn, and the data was analyzed to obtain the final IC ₅₀ value. The experimental results are shown in Table 3.

table 3

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

Experimental example 2 pharmacokinetic experiment

1. Experimental materials

Compound: a compound of formula (I) of the present invention. The drug vehicle is Captisol/50mM sodium acetate, pH4.6 (10%/90%, w/v%). Oral drug is formulated as 0.5mg/mL, clear solution; intravenous drug is formulated as 0.1mg/mL, clear solution.

Animals: male BALB/c mice, SPF grade, purchased from Shanghai Xipro-Bicai Laboratory Animal Co., Ltd.; 18-20g. An adaptation period of 2 to 3 days was given before the experiment.

Instrument: API4500 type triple quadrupole liquid mass spectrometer of American AB Company, equipped with electrospray ion source (ESI), LC-30AD double pump; SIL-30AC automatic sampler; CTO-30AC column thermostat; DGU- 20A3R degasser; AnalystQSA01.01 chromatographic workstation; Milli-Q ultrapure water device (Millipore Inc); Qilinbeier Vortex-5 oscillator; HITACHICF16RXⅡ desktop high-speed refrigerated centrifuge.

2. Experimental method

(1) 3 mice in each group. Intragastric (I.G.) administration of 10 mg/kg of the compound of formula (I) of the present invention, intravenous (IV) administration of 1 mg/kg of the compound of formula (I) of the present invention;

(2) Blood was collected from the orbital venous plexus at 5min, 15min, 30min, 1h, 2h, 6h, 10h, and 24h after gavage and intravenous administration, and was centrifuged at 8000rpm/min for 5min to take the upper layer Plasma, stored at -20°C, analyzed by LC-MS/MS;

(3) Draw a blood drug concentration-time curve according to the blood drug concentration data obtained in the above steps, and use WinNonlin software to calculate the pharmacokinetic parameters. The experimental results are shown in Table 4.

Table 4

The pharmacokinetics of Compound A of Comparative Example was measured using the method of Experimental Example 2. The experimental results show that the bioavailability (F) of compound A is 51.2%, which is obviously lower than that of the compound of formula (I) of the present invention.

Experimental results show that the compound of the present invention has good half-life T _1/2 , area under the curve AUC, bioavailability F, good oral absorption and exposure, and is suitable for pharmaceutical preparation.

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

Claims (10)

1. A method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof, comprising the step of deprotecting a compound of formula (II) in the presence of an acidic reagent to produce a compound of formula (I), wherein R is an amino-protecting group,

2. a process for the preparation of a compound of formula (I), or a salt, hydrate, solvate or crystal thereof, according to claim 1, wherein R is selected from the group consisting of alkyltrisilyl, aryltrisilyl, alkyl, alkanoyl, arylacyl, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy, and aryloxy, which are optionally substituted with one or more halogen, hydroxy, amino, carboxy, cyano, nitro, alkyl.

3. A process for producing a compound of formula (I), or a salt, hydrate, solvate or crystal thereof according to claim 1 or 2, which comprises the step of subjecting a compound of formula (II) to amino-protecting group removal with an acidic reagent to produce a salt of the compound of formula (I), and subjecting the salt of the compound of formula (I) to acid-base neutralization with a basic reagent to produce the compound of formula (I).

4. A process for the preparation of a compound of formula (I), or a salt, hydrate, solvate or crystal thereof, as claimed in any one of claims 1 to 3, wherein the acidic agent is an inorganic or organic acid.

5. A process for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 1 to 4 wherein the molar ratio of compound of formula (II) to acidic agent is from about 1:1 to about 1.

6. A process for the preparation of a compound of formula (I), or a salt, hydrate, solvate or crystal thereof according to any one of claims 1 to 5, further comprising the step of reacting a compound of formula (III) with a compound of formula (IV) to produce a compound of formula (II) wherein X is a leaving group and R is an amino protecting group,

7. the method for preparing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 6, wherein the leaving group X is selected from the group consisting of halogen, hydroxy, alkoxy, acyloxy, aryloxy, heteroaryloxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, optionally substituted arylsulfonyloxy, acyl, and an active ester of hydroxy.

8. A process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 6 or 7, which comprises the step of reacting a compound of formula (III) with a compound of formula (IV) in the presence of a catalyst to produce a compound of formula (II), preferably the catalyst is an acidic reagent.

9. The process for producing a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to any one of claims 6 to 8, which further comprises the step of reacting a compound of formula (V) or a salt thereof with a compound of formula (VI) in the presence of a basic reagent to produce a compound of formula (IV) wherein X is a leaving group and R is an amino-protecting group,

10. a process for the preparation of a compound of formula (I) or a salt, hydrate, solvate or crystal thereof according to claim 9 wherein the basic agent is selected from organic bases.