WO2021164741A1 - Phenyl bisamide compound - Google Patents

Abstract

Disclosed in the present invention is a phenyl bisamide compound. Specifically, disclosed is a compound as represented by Formula (P) or a pharmaceutically acceptable salt thereof.

Description

Phenyl bisamide compounds

The present invention claims the following priority:

CN202010105349.7, application date: February 20, 2020.

Technical field

The present invention relates to a class of phenyl bisamide compounds, in particular to a compound represented by formula (P) or a pharmaceutically acceptable salt thereof.

Background technique

The RET gene is a proto-oncogene that rearranges during the transfection process, hence its name. This gene encodes a cell membrane receptor tyrosine kinase. When the growth factor binds to the extracellular region of RET, it will Trigger a series of chain chemical reactions within the cell, according to the signal received by the receptor, cause the cell to divide and mature, and then play a corresponding function on the development of the organ and the homeostasis of the tissue. RET protein plays an important role in the development of several nerves (including the intestine and autonomic nervous system).

RET plays a key role in regulating the development of sympathetic, parasympathetic, motor and sensory nerves in the enteric nervous system (ENS). The RET gene knockout mice lack enteric neurons and have other neurological abnormalities, which means that a functional RET kinase protein product is required during growth. Hirschsprung patients with loss-of-function RET mutations and lack of normal colonic nerve distribution can cause colonic obstruction. Therefore, inhibiting RET in the enteric nervous system is a new treatment strategy for normalizing nerve function and alleviating the symptoms of irritable bowel syndrome (IBS) patients. GSK3179106 is a RET inhibitor of the pyridone class and is currently in phase I clinical study.

The present invention aims to develop a class of quinoline derivatives as RET inhibitors for the treatment of tumors or intestinal diseases related to abnormal activation of RET.

Summary of the invention

The compound represented by formula (P) or a pharmaceutically acceptable salt thereof,

in,

R ₁ and R ₂ are each independently selected from H, F, Cl, Br, I, OH, NH 2, CN, C 1-3 alkyl group and a C _1-3 alkoxy group, a C _{1- 3} alkyl and a C _1-3 alkoxy group optionally substituted with 1, 2 or 3 R _a;

Each R _{3 is} independently selected from H, F, Cl, Br and I;

R _{4 is} selected from H, F, Cl, Br and I;

R _{5 is} selected from H, F, Cl, Br, I and C _1-3 alkyl, the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R _{6 is} selected from H, F, Cl, Br, I and C _1-3 alkyl, the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _c ;

Each R _a is independently selected from H, F, Cl, Br and I;

Each R _{b is} independently selected from H, F, Cl, Br and I;

Each R _{c is} independently selected from H, F, Cl, Br, I and C _1-3 alkylamino group, said C _1-3 alkylamino group is optionally substituted by 1, 2 or 3 R;

Each R is independently selected from H, F, Cl, Br and I;

m is selected from 0, 1, 2 and 3.

In some aspects of the present invention, the R ₁ and R ₂ are each independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) _2. OCH ₃ , OCH ₂ CH ₃ and OCH(CH ₃ ) ₂ , the CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ , OCH ₃ , OCH ₂ CH ₃ and OCH(CH ₃ ) _{2 are} optional 1, 2 or 3 substituents R _a, the other variables are as defined in the present invention.

In some aspects of the present invention, the R ₁ and R ₂ are each independently selected from OCH ₃ , and other variables are as defined in the present invention.

In some aspects of the present invention, the R _{5 is} selected from H, F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ and CH(CH ₃ ) ₂ , the CH ₃ , CH ₂ CH ₃ and CH (CH ₃ ) _{2 is} optionally substituted with 1, 2 or 3 R _b , and other variables are as defined in the present invention.

In some aspects of the present invention, the R _{5 is} selected from CF ₃ , and other variables are as defined in the present invention.

In some aspects of the present invention, the R _{6 is} selected from H, F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ and CH(CH ₃ ) ₂ , the CH ₃ , CH ₂ CH ₃ and CH (CH ₃ ) _{2 is} optionally substituted with 1, 2 or 3 R _c , and other variables are as defined in the present invention.

In some aspects of the present invention, the R _{6 is} selected from

Other variables are as defined in the present invention.

In some aspects of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from,

Wherein, R ₁ , R ₂ and R _{6 are} as defined in the present invention.

There are also some schemes of the present invention that come from any combination of the above-mentioned variables.

The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment of RET disease.

Technical effect

The compound of the present invention has a good inhibitory effect on wild-type and V804M mutant RET kinase. It has good one-phase metabolic stability and good drug interaction safety. It is not absorbed into the blood after oral administration, avoiding the toxic side effects of drugs.

Definition and description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears in this article, it is meant to refer to its corresponding commodity or its active ingredient.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound 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 salt or similar salts. When the compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the compound 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 including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include 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, which can be converted into any base or acid addition salt.

The pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both.

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 Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.

Unless otherwise specified, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.

Unless otherwise specified, the term "cis-trans isomer" or "geometric isomer" is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.

Unless otherwise specified, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror-image relationship.

Unless otherwise specified, "(+)" means right-handed, "(-)" means left-handed, and "(±)" means racemic.

Unless otherwise specified, use wedge-shaped solid line keys

And wedge-shaped dashed key

Represents the absolute configuration of a three-dimensional center, with a straight solid line key

And straight dashed key

Indicates the relative configuration of the three-dimensional center, using wavy lines

Represents a wedge-shaped solid line key

Or wedge-shaped dashed key

Or use wavy lines

Represents a straight solid line key

And straight dashed key

Unless otherwise specified, when there is a double bond structure in the compound, such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond, and each atom on the double bond is connected to two different substituents (including a nitrogen atom) In the double bond, a lone pair of electrons on the nitrogen atom is regarded as a substituent connected), if the atom on the double bond in the compound and its substituent are separated by a wavy line

Linked means the (Z) isomer, (E) isomer or a mixture of two isomers of the compound. For example, the following formula (A) means that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2) The following formula (B) means that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of two of formula (B-1) and formula (B-2) A mixture of isomers exists. The following formula (C) represents that the compound exists as a single isomer of formula (C-1) or formula (C-2) or as two isomers of formula (C-1) and formula (C-2) Exist as a mixture.

Unless otherwise specified, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached. For example, proton tautomer (also called prototropic tautomer) includes interconversion through proton migration, such as keto-enol isomerization and imine-ene Amine isomerization. Valence isomers (valence tautomer) include some recombination of bonding electrons to carry out mutual transformations. A specific example of keto-enol tautomerization is the tautomerism between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise specified, the term "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in enantiomers" refers to one of the isomers or pairs of The content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.

Unless otherwise specified, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .

The optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).

The compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), or C-14 ( ¹⁴ C). For another example, deuterium can be substituted for hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention. "Optional" or "optionally" means that the event or condition described later may but not necessarily occur, and the description includes a situation in which the event or condition occurs and a situation in which the event or condition does not occur.

The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent. The substituent may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the compound after substitution Is stable. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.

When any variable (such as R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group can optionally be substituted with up to two Rs, and R has independent options in each case. In addition, combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.

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

When one of the variables is selected from a single bond, it means that the two connected groups are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent is absent. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom. For example, a pyridyl group can pass through any one of the pyridine ring as a substituent. The carbon atom is attached to the substituted group.

When the listed linking group does not indicate its linking direction, its linking direction is arbitrary, for example,

The middle linking group L is -MW-, at this time -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right

It can also be formed by connecting ring A and ring B in the opposite direction to the reading order from left to right

Combinations of the linking groups, substituents, and/or variants thereof are only permitted if such combinations result in stable compounds.

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 connection method of the chemical bond is not positioned, and there is a H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will correspondingly decrease with the number of chemical bonds connected to become the corresponding valence number的组。 The group. The chemical bond between the site and other groups can be a straight solid bond

Straight dashed key

Or wavy line

Express. For example _{, the straight solid bond in -OCH 3} means that it is connected to other groups through the oxygen atom in the group;

The straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;

The wavy line in indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms;

Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These four connection methods, even though the H atom is drawn on -N-, but

Still include

The group in this connection mode, only when one chemical bond is connected, the H at this position will decrease by one and become the corresponding monovalent piperidinyl group.

As otherwise specified, the term "C _1-3 alkyl" is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) . Examples of C _1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, the term "C _1-3 alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule through an oxygen atom. The C _1-3 alkoxy group includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy groups and the like. Examples of C _1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

Unless otherwise specified, the term "C _1-3 alkylamino" refers to those alkyl groups containing 1 to 3 carbon atoms attached to the rest of the molecule through an amino group. The C _1-3 alkylamino group includes C _1-2 , C ₃ and C ₂ alkylamino groups and the like. Examples of C _1-3 alkylamino groups include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH ₂ CH ₃ , -NHCH ₂ CH ₂ CH ₃ ,- NHCH ₂ (CH ₃ ) ₂ and so on.

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, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.

Compounds are based on conventional naming principles in the field or use

The software is named, and the commercially available compounds use the supplier catalog name.

Detailed ways

The present invention will be described in detail through the following examples, but it is not meant to impose any disadvantageous restriction on the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Will be obvious.

Example 2

Step 1: Preparation of compound 2

Compound 1-5 (0.15 g, 462.49 μmol) and compound 2-1 (140.04 mg, 508.74 μmol) were added to pyridine (3.0 mL), and tri-n-propyl cyclic phosphoric anhydride 50% ethyl acetate solution (1.47 g, 2.31 mmol, 1.38 mL), react at 20°C for 1 hour. The reaction solution was added to 10mL saturated ammonium chloride, extracted with ethyl acetate (10mL×2), the organic phases were combined, washed with 10mL saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by HPLC (column: Phenomenex Luna C18 150×30mm×5μm; fluidity: [water (0.05% HCl)-ACN]; ACN%: 15%-35%, 12min) to obtain compound 2. ¹ H NMR (400MHz, d ₄ -MeOH) δ = 9.57 (s, 2H), 8.81 (s, 1H), 8.40 (s, 1H), 8.27-8.20 (m, 2H), 8.13-8.07 (m, 1H) ), 7.94(s, 1H), 7.78(s, 1H), 7.56(s, 1H), 4.53-4.50(m, 1H), 4.17(s, 3H), 4.12(s, 3H), 3.98(s, 2H), 3.80 (t, J=5.6 Hz, 2H), 3.42 (t, J=5.6 Hz, 2H), 3.01 (s, 6H); MS m/z=582.3 [M+H] ⁺ .

Experimental example 1: Evaluation of in vitro inhibitory activity of human wild-type and V804M mutant RET kinase

^{The 33} P isotope-labeled kinase activity test of Reaction Biology Corp was used to _{determine the IC 50} value to evaluate the inhibitory ability of the test compound against human wild-type and V804M mutant RET.

Buffer conditions: 20mM hydroxyethylpiperazine B sulfuric acid (Hepes) (pH 7.5), 10mM MgC l2, 1mM EGTA (EGTA), 0.02% polyoxyethylene dodecyl ether ( Brij35), 0.02mg/ml BSA, 0.1mM Na ₃ VO ₄ , 2mM dithiothreitol (DTT), 1% DMSO.

Compound treatment: The test compound is dissolved in 100% DMSO and serially diluted with DMSO by Integra Viaflo Assist to a specific concentration.

Test procedure: Dissolve the substrate in the newly prepared buffer, add the tested kinase to it and mix gently. Using acoustic technology (Echo 550), the DMSO solution with the test compound dissolved in the above mixed reaction solution was added and incubated at room temperature for 20 minutes. The compound concentration in the reaction solution was 3μM, 1μM, 0.333μM, 0.111μM, 0.0370μM, 0.0123μM, 4.12nM, 1.37nM, 0.457nM, 0.152nM. After 15 minutes of incubation, ³³ P-ATP (activity 0.01μCi/μl, km concentration) was added to start the reaction. After the reaction was carried out at room temperature for 120 minutes, the radioactivity was detected by the filter binding method. The kinase activity data is expressed by comparing the kinase activity of the test compound with that of the blank group (only containing DMSO). The IC ₅₀ value is obtained by curve fitting with Prism4 software (GraphPad). The experimental results are shown in Table 1.

Table 1

Compound number	RET(WT)IC 50 (nM)	RET(V804M)IC 50 (nM)
Compound 2	0.3	1.0

Conclusion: In vitro enzyme activity test results show that the test compound has a good inhibitory effect on wild-type and V804M mutant RET kinase.

Experimental example 2 Microsome stability study (Microsome stability)

Purpose:

Test the one-phase metabolic stability of the compounds of the present invention in CD-1 mice and human liver microsomes.

experimental method:

Add 10 μL of the working solution of the test product and 80 μL of the microsomal working solution (0.625 mg/mL liver microsomal protein) to the reaction plate and NCF60 plate respectively, add only the microsomal working solution to the Blank60 plate, and then place the above incubation plate on Pre-incubate in a 37°C water bath for about 10 minutes.

After the pre-incubation, except for the NCF60 plate and T0 plate, add 10 μL of NADPH regeneration system working solution to each sample well to start the reaction, and add 10 μL of potassium phosphate buffer to each well of the NCF60 plate. The final concentration of the test substance or reference substance is 1 μM, and the concentration of liver microsomes is 0.5 mg/mL.

After incubating for an appropriate time, 300 μL of stop solution (acetonitrile solution containing 200 ng/mL tolbutamide) was added to each sample well to stop the reaction. Add the stop solution to the T0 plate sample first and then add the NADPH regeneration system working solution.

All sample plates were shaken well and centrifuged at 4000 rpm for 20 minutes, and then 100 μL of supernatant per well was diluted into 300 μL of pure water for LC-MS/MS analysis.

data processing:

The in vitro elimination rate constant ke of the test substance and the reference substance is obtained by converting the ratio of the peak area of the sample to the internal standard in the following formula into the remaining percentage:

CL _int(mic) = 0.693/half-life/mg microsome protein per mL (microsome concentration during incubation)

CL _int(liver) = CL _int(mic) × mg microsomal protein/g liver weight × liver weight to weight ratio

mg microsomal protein/g liver weight: the value is 45 for both animal and human species

Liver weight to weight ratio: The parameters in mice and humans are 88 and 20g/kg, respectively

The experimental results are shown in Table 2.

Table 2

Note: MMS stands for microsomal metabolic stability, H stands for human, and M stands for mouse.

Experimental conclusion: The compound has good one-phase metabolic stability.

Experimental example 3 Cytochrome enzyme inhibition study (CYP inhibition)

Purpose:

A mixed probe substrate of CYP isoenzymes was used to evaluate the inhibitory properties of the compounds of the present invention on human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4).

experimental method:

The test product will be prepared with DMSO into a 10mM stock solution. Add 178 μL of human liver microsomes and substrate mixture solution to the inhibitor-free control (NIC) and test sample wells of the reaction plate, and place on ice. Take 2μL of the blank solvent and the working solution of the test product from the dilution plate and add them to the reaction plate (final concentration is 0.05-50μM). Preheat the reaction plate in a 37±0.2°C water bath for 10 minutes. Use a liquid processing workstation to take 20 μL of the cofactor solution and add it to the reaction plate to start the reaction.

After 10 minutes, 400 μL of stop solution was added to the reaction plate to stop the reaction, and the reaction plate was placed on ice for 5 minutes. Shake the plate for 10 minutes to make the solution uniform, centrifuge at 4000 rpm for 20 minutes, then take out the supernatant and add ultrapure water in an appropriate ratio. Liquid chromatography tandem mass spectrometry (LC/MS/MS) was used to detect the peak area of the substrate and product. The samples are stored at 2-8°C before testing.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the ratio of the metabolite produced by the probe substrate to the peak area of the internal standard. The retention time of analytes and internal standards, chromatogram acquisition and chromatogram integration are processed by the software Analyst (AB Sciex, Framingham, Massachusetts, USA).

data processing:

Use SigmaPlot (V.11) to make a non-linear regression analysis of the average percentage activity versus concentration of the test product. _{The IC 50} value is calculated by a three-parameter or four-parameter inverse logarithmic equation. When the CYP percentage activity is greater than 50% under the highest concentration (50μM) of the test substance, the IC ₅₀ value is marked as ">50μM".

Three-parameter inverse logarithmic equation:

Four-parameter inverse logarithmic equation:

max: Maximum enzyme activity.

min: minimum enzyme activity.

x: The concentration of the test product or positive control inhibitor.

y: Enzyme activity at the corresponding concentration, hillslope: slope.

IC ₅₀ : half inhibitory concentration.

When the minimum enzyme activity is within ±10%, a four-parameter recursive logarithmic equation is used, otherwise a three-parameter equation is used.

The experimental results are shown in Table 3.

table 3

Experimental conclusion: The compound has little inhibitory effect on human liver microsomal cytochrome P450 isoenzymes, and has high drug interaction safety.

Experimental Example 4 Study on pharmacokinetics in mice

Purpose:

This experiment aims to study the pharmacokinetics of the compound of the present invention in the plasma of male CD-1 mice after intravenous injection and oral administration.

experimental method:

The animals were randomly divided into two groups with 2 males in each group. The compound is formulated into the designated preparation, the intravenous injection preparation is prepared with 5% DMSO/95% (6% hydroxypropyl-β-cyclodextrin) to obtain a clear solution, and the oral preparation is prepared with 0.1% Tween80/0.5% HPMC aqueous solution to obtain a uniform mixture. Suspension.

Animals collect whole blood samples from the saphenous vein at 5, 15, 30 minutes, 1, 2, 4, 8, 12, and 24 hours after administration. The whole blood sample was added to a centrifuge tube containing anticoagulant, centrifuged at 3200g for 10 min at 4°C, the supernatant plasma was quickly frozen on dry ice, and then stored in a refrigerator at -70±10°C until LC-MS/MS analysis was performed.

data processing:

WinNonlin ^TM Version 6.3.0 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process the plasma drug concentration data of the compound in a non-compartmental model. The peak concentration (C _max ), peak time (T _max ) and quantifiable end time can be obtained directly from the plasma concentration-time diagram.

The following pharmacokinetic parameters were calculated using the log-linear trapezoidal method: plasma clearance (CL), volume of distribution (Vd), elimination phase half-life (T _1/2 ), and the average residence time of the drug in the body from 0 o'clock to the end time. (MRT _0-last ), the average residence time of the drug in the body from 0 o’clock to infinite time (MRT _0-inf ), 0 o’clock to the end time point-area under the plasma concentration curve (AUC _0-last ), 0 o’clock to infinity Area under the time-plasma concentration curve (AUC _0-inf ) and bioavailability (F).

The experimental results are shown in Table 4.

Table 4

Note: ND stands for not detected.

Experimental conclusion: The compound is not absorbed into the blood after oral administration, avoiding the toxic side effects of the drug.

Claims (11)

1. The compound represented by formula (P) or a pharmaceutically acceptable salt thereof,

   

   in,

   R ₁ and R ₂ are each independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, C _1-3 alkyl and C _1-3 alkoxy, the C _1-3 alkyl and a C _1-3 alkoxy group optionally substituted with 1, 2 or 3 R _a;

   Each R _{3 is} independently selected from H, F, Cl, Br and I;

   R _{4 is} selected from H, F, Cl, Br and I;

   R _{5 is} selected from H, F, Cl, Br, I and C _1-3 alkyl, the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

   R _{6 is} selected from H, F, Cl, Br, I and C _1-3 alkyl, the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _c ;

   Each R _a is independently selected from H, F, Cl, Br and I;

   Each R _{b is} independently selected from H, F, Cl, Br and I;

   Each R _{c is} independently selected from H, F, Cl, Br, I and C _1-3 alkylamino group, said C _1-3 alkylamino group is optionally substituted by 1, 2 or 3 R;

   Each R is independently selected from H, F, Cl, Br and I;

   m is selected from 0, 1, 2 and 3.
2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ₁ and R ₂ are each independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ , OCH ₃ , OCH ₂ CH ₃ and OCH(CH ₃ ) ₂ , the CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ , OCH ₃ , OCH ₂ CH ₃ and OCH (CH _{3) 2} optionally substituted with 1, 2 or 3 R _a.
3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ₁ and R ₂ are each independently selected from OCH ₃ .
4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R _{5 is} selected from H, F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ and CH(CH ₃ ) ₂ , the CH _3. CH ₂ CH ₃ and CH(CH ₃ ) _{2 are} optionally substituted with 1, 2 or 3 R _b .
5. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R _{5 is} selected from CF ₃ .
6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R _{6 is} selected from H, F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ and CH(CH ₃ ) ₂ , the CH _3. CH ₂ CH ₃ and CH(CH ₃ ) _{2 are} optionally substituted with 1, 2 or 3 R _c .
7. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R _{6 is} selected from

   
8. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the compound is selected from,

   

   Wherein, R ₁ , R ₂ and R _{6 are} as defined in any one of claims 1-7.
9. The compound represented by the following formula or a pharmaceutically acceptable salt thereof,

   
10. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1-9 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.
11. The use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 10 in the preparation of a medicine for the treatment of RET-related diseases.