CN109111439B

CN109111439B - Amide compound, composition containing same and application thereof

Info

Publication number: CN109111439B
Application number: CN201810628061.0A
Authority: CN
Inventors: 王义汉; 赵九洋
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2017-06-26
Filing date: 2018-06-19
Publication date: 2020-09-18
Anticipated expiration: 2038-06-19
Also published as: WO2019001307A1; CN109111439A

Abstract

The invention discloses an amide compound shown as a formula (I), and preparation and application thereof. Specifically, the invention discloses an amide compound shown as a formula (I), or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, a pharmaceutical composition containing the amide compound and an application of the amide compound. The amide compound and the composition containing the same disclosed by the invention have excellent inhibition on protein kinase, have better pharmacokinetic parameter characteristics, and can improve the drug concentration of the compound in an animal body so as to improve the curative effect and safety of the drug.

Description

Amide compound, composition containing same and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an amide compound, a composition containing the amide compound and application of the amide compound.

Background

Protein tyrosine kinases play an important role in cellular regulation and have been observed to be aberrantly expressed or mutated in cancer cells or autoimmune diseases. Protein tyrosine kinases are enzymes that catalyze the transport of phosphate groups from ATP to tyrosines located on protein substrates. Many growth factor receptor proteins function as tyrosine kinases to transmit cellular signals. The interaction between growth factors and their receptors generally controls cell growth, but aberrant signal transduction resulting from mutation or overexpression of any one of the receptors often induces a variety of cancers or autoimmune diseases (e.g., rheumatoid arthritis).

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are molecular targeted drugs against EGFR, which block further transmission of signals into cells, inhibit tumor cell growth and induce apoptosis thereof, mainly by competitively binding with ATP to the binding site of the catalytic domain of EGFR tyrosine kinase located on the cell surface. Currently, EGFR-TKI such as erlotinib and gefitinib are widely used in clinic. Although EGFR inhibitors such as gefitinib and erlotinib have remarkable curative effect on EGFR mutant advanced non-small cell lung cancer (NSCLC), it is subsequently found that EGFR-TKI has primary or secondary resistance when used for treating NSCLC, and that AZD9291 (Osimetinib, Axitinib) can be orally taken for treating EGFR second mutation of lung cancer patients. AZD9291 is an oral, irreversible, third-generation EGFR inhibitor (EGFR-TKI) with improved therapeutic efficacy in NSCLC patients with EGFR-TKI resistance and T790M mutation. However, patients taking AZD9291 still develop symptoms of acquired resistance. The mechanism of acquired resistance mutation of AZD9291 comprises: EGFR C797S mutation, FGFR1 amplification, HER2 amplification, C-Met amplification or MAPK alternative pathway activation, histological transformation (partial transformation to small cell lung cancer), or multiple other gene mutations. (reference: Oxnard et al Nature Medicine,2015,21,560-

AZD9291 forms a covalent bond with cysteine C797 at the ATP binding site, and the C797S mutation affects the binding of the covalent bond, similar to the resistance mechanism of the BTK inhibitor Ibrutinib (Ibrutinib). A heavy article is published in Nature 2.2016, a new generation of targeting drug EAI045 capable of overcoming AZD9291 drug resistance is recorded, and for C797S mutation, EAI045 and erbitux are jointly applied to a mouse model, so that the effective rate is as high as 80%. (reference: M.J.Eck et. Nature,2016,534,129-132)

Therefore, treating advanced NSCLC faces new challenges, and we need to develop new exploration and search for new strategies.

Disclosure of Invention

Aiming at the technical problems, the invention discloses an amide compound, a composition containing the compound and application of the amide compound, wherein the compound has protein kinase inhibitory activity and better pharmacodynamic/pharmacokinetic performance.

In contrast, the invention adopts the following technical scheme:

in one aspect, the present invention relates to an amide-based compound of formula (I), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate, or a solvate thereof:

wherein,

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹and R¹²Each independently selected from hydrogen, deuterium, halogen or trifluoromethyl; with the proviso that the amide-based compound contains at least one deuterium atom.

As a preferred embodiment of the present invention, the compound of formula (I) contains at least one deuterium atom, more preferably two deuterium atoms, more preferably three deuterium atoms, more preferably four deuterium atoms, more preferably five deuterium atoms, more preferably six deuterium atoms, more preferably eight deuterium atoms.

As a preferred embodiment of the present invention, the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

In another embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Preferably, at least one of the deuterium containing groups comprises deuterium, more preferably two deuterium containing groups, more preferably three deuterium containing groups, more preferably four deuterium containing groups, more preferably five deuterium containing groups, more preferably six deuterium containing groups, more preferably seven deuterium containing groups, more preferably eight deuterium containing groups, more preferably nine deuterium containing groups, more preferably ten deuterium containing groups, more preferably eleven deuterium containing groups, and more preferably twelve deuterium containing groups. In particular, the compounds of formula (I) contain at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve deuterium atoms.

As a preferred embodiment of the present invention, R¹And R²Each independently is deuterium or hydrogen.

In another preferred embodiment, R¹Is deuterium.

In another preferred embodiment, R²Is deuterium.

In another preferred embodiment, R¹And R²Is deuterium.

As a preferred embodiment of the present invention, R³And R⁴Each independently is deuterium or hydrogen.

In another preferred embodiment, R³Is deuterium.

In another preferred embodiment, R³And R⁴Is deuterium.

As a preferred embodiment of the present invention, R⁵、R⁶、R⁷And R⁸Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁵、R⁶、R⁷And R⁸Is deuterium.

As a preferred embodiment of the present invention, R⁹、R¹⁰And R¹¹Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁹Is deuterium.

In another preferred embodiment, R¹⁰Is deuterium.

In another preferred embodiment, R¹¹Is deuterium.

In another preferred embodiment, R⁹、R¹⁰And R¹¹Is deuterium.

In another preferred embodiment, R¹²Is deuterium.

In another aspect, the invention also discloses a method for preparing the amide compound shown in the formula (I), which is characterized by comprising substituted or unsubstituted amide compound

As a step of reacting the intermediate with a substituted or unsubstituted 2-aminothiazoline under alkaline conditions.

As a preferred embodiment of the present invention, the base used in the preparation method is selected from at least one of potassium carbonate, sodium hydrogen carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, N-diisopropylethylamine, 4-N, N-dimethylpyridine, or pyridine.

In another aspect, the present invention also discloses a pharmaceutical composition containing a pharmaceutically acceptable excipient and the amide-based compound as described above, or its polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof.

In another aspect, the present invention also discloses a method for preparing the pharmaceutical composition as described above, comprising the following steps: mixing a pharmaceutically acceptable excipient with the amide-based compound as described above, or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, to form a pharmaceutical composition.

In another embodiment, the pharmaceutical composition is an injection, a sachet, a tablet, a pill, a powder or a granule.

In another embodiment, the pharmaceutical composition further comprises an additional therapeutic agent that is an agent for cancer, cardiovascular disease, inflammation, infection, immune disease, cell proliferative disease, viral disease, metabolic disease, or organ transplantation.

In another aspect, the present invention also provides a use of a compound described in the first aspect of the present invention, or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, for the manufacture of a medicament for the treatment and/or prevention of a disease associated with a protein kinase.

In another aspect, the present invention also provides a method for treating and/or preventing a protein kinase-associated disease in a subject, the method comprising administering to the subject an amide-based compound of formula (I), a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate compound thereof, or a pharmaceutical composition thereof.

In another aspect, the present invention also provides an amide-based compound of formula (I), a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, or a pharmaceutical composition thereof, for use in the treatment and/or prevention of a disease associated with a protein kinase.

In another embodiment, the compound or pharmaceutical composition is used for the treatment and/or prevention of the following diseases: cancer, cell proliferative disorders, inflammation, infection, immunological disorders, organ transplantation, viral disorders, cardiovascular disorders or metabolic disorders.

In another embodiment, the cancer includes, but is not limited to: lung cancer, head and neck cancer, breast cancer, prostate cancer, esophageal cancer, rectal cancer, colon cancer, nasopharyngeal cancer, uterine cancer, pancreatic cancer, lymphoma, leukemia, osteosarcoma, melanoma, renal cancer, gastric cancer, liver cancer, bladder cancer, thyroid cancer or carcinoma of large intestine.

In another embodiment, the immune or inflammatory disease includes, but is not limited to: rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gout, asthma, bronchitis, rhinitis, chronic obstructive pulmonary disease, cystic fibrosis.

In another embodiment, the cell proliferative disease is lung cancer, head and neck cancer, breast cancer, prostate cancer, esophageal cancer, rectal cancer, colon cancer, nasopharyngeal cancer, uterine cancer, pancreatic cancer, lymphoma, leukemia, osteosarcoma, melanoma, renal cancer, gastric cancer, liver cancer, bladder cancer, thyroid cancer or colorectal cancer.

In another embodiment, the cancer is non-small cell lung cancer.

In another aspect, the present invention also provides a kit comprising: a first container containing therein a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate of an amide-based compound of formula (I); and optionally, a second container containing an additional therapeutic agent; and optionally, a third container containing pharmaceutically acceptable excipients for diluting or suspending the compound and/or other therapeutic agent.

In another aspect, the invention also discloses the application of the amide compound in preparing a pharmaceutical composition for inhibiting protein kinase. Preferably, it is used for preparing a pharmaceutical composition for inhibiting EGFR kinase.

The compound of formula (I) of the present invention or its polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variant, hydrate or solvate thereof can treat or prevent cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease caused by aberrantly activated B lymphocytes, T lymphocytes or both. Accordingly, the present invention also provides a pharmaceutical composition for treating and/or preventing cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease, comprising the compound of formula (I) of the present invention or its polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variant, hydrate or solvate thereof as an active ingredient.

The invention also includes isotopically-labeled compounds (also referred to as "isotopic variations"), equivalent to those disclosed herein for the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of formula (I) of the present invention, or polymorphs, pharmaceutically acceptable salts, prodrugs, stereoisomers, isotopic variations, hydrates or solvates thereof, containing the aforementioned isotopes or other isotopic atoms are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. In addition, heavier isotopes such as deuterium, i.e.²H, due to its good metabolic stability, may be advantageous in certain therapies, such as increased half-life in vivo or reduced dose, and therefore, may be preferred in certain circumstances. Isotopically-labeled compounds can be prepared by conventional methods by substituting a readily available isotopically-labeled reagent forThe exchange for a non-isotopic reagent can be made using the protocol in the example.

It is to be understood that within the scope of the present invention, each of the above-described technical features, embodiments of the present invention, and each of the technical features specifically described below (e.g., examples) may be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Compared with the prior art, the invention has the beneficial effects that: firstly, the amide compound adopting the technical scheme of the invention has excellent inhibition on protein kinase. Second, the metabolism of the compound in the organism is improved, giving the compound better pharmacokinetic parameters. In this case, the dosage can be varied and a long acting formulation formed, improving the applicability. Thirdly, the medicine concentration of the compound in the animal body is improved, and the medicine curative effect is improved. Fourth, certain metabolites are inhibited, increasing the safety of the compounds.

Defining:

when a range of values is recited, it is intended to include each value and every subrange within the range. E.g. "C₁-C₆Alkyl "includes C₁、C₂、C₃、C₄、C₅、C₆、C₁-C₆、C₁-C₅、C₁-C₄、C₁-C₃、C₁-C₂、C₂-C₆、C₂-C₅、C₂-C₄、C₂-C₃、C₃-C₆、C₃-C₅、C₃-C₄、C₄-C₆、C₄-C₅And C₅-C₆An alkyl group.

It is to be understood that any of the moieties defined below may be substituted with a number of substituents when described herein, and that the corresponding definitions are within their scope as set forth below, including such substituted moieties. Unless otherwise specified, the term "substituted" is as defined below.

"halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "polymorphs" refers to the different arrangements of chemical drug molecules, typically expressed as the presence of the drug material in a solid state. One drug can exist in a plurality of crystal form substances, and different crystal forms of the same drug can be dissolved and absorbed in vivo differently, so that the dissolution and release of the preparation can be influenced.

The term "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail in J.pharmaceutical sciences (1977)66:1-19 by Berge et al. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using methods conventional in the art, e.g., ion exchange methods, are also included. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cypionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, picrate, etc, Stearate, succinate, sulfateTartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Other pharmaceutically acceptable salts include, if appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed with counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

The "subject" to which the drug is administered includes, but is not limited to: a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an older adult)) and/or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and/or a dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

"disease," "disorder," and "condition" are used interchangeably herein.

The term "treating" as used herein includes the effect that occurs when a subject suffers from a particular disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows the progression of the disease, disorder or condition. The term "prevention" as used herein includes an effect that occurs before a subject begins to suffer from a particular disease, disorder or condition.

The compounds of the invention may include one or more asymmetric centers, and thus may exist in a variety of "stereoisomeric" forms, e.g., enantiomeric and/or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from mixtures by methods known to those skilled in the art, including: chiral High Pressure Liquid Chromatography (HPLC) and the formation and crystallization of chiral salts; alternatively, preferred isomers may be prepared by asymmetric synthesis.

One skilled in the art will appreciate that many organic compounds can form complexes with a solvent in which they react or from which they precipitate or crystallize out. These complexes are referred to as "solvates". When the solvent is water, the complex is referred to as a "hydrate". The present invention encompasses all solvates of the compounds of the present invention.

In addition, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo by hydrolysis, for example in the blood, to its active form with a medicinal effect. Pharmaceutically acceptable Prodrugs are described in t.higuchi and v.stella, Prodrugs as Novel Delivery Systems, a.c.s.symposium series, vol.14, Edward b.roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and d.fleisher, s.ramon and h.bara "Improved oral Drug Delivery: the solubility limits over company the use of drivers, Advanced Drug Delivery Reviews (1996)19(2)115-130, each of which is incorporated herein by reference.

A prodrug is any covalently bonded carrier that releases a compound of the invention in vivo when such prodrug is administered to a patient. Prodrugs are typically prepared by modifying functional groups such that the prodrug is cleaved in vivo to yield the parent compound. Prodrugs include, for example, compounds of the present invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, cleaves to form a hydroxy, amino, or sulfhydryl group. Thus, representative examples of prodrugs include, but are not limited to, covalent derivatives of the compounds of the present invention with acetic acid, formic acid, or benzoic acid through a hydroxy, amino, or mercapto functional group therein. In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, and the like may be used. The ester itself may be active and/or may hydrolyze under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable esters include those which readily break down in the human body to release the parent acid or salt thereof.

"pharmaceutically acceptable excipient" for use in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compounds formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Detailed Description

Compound (I)

The invention relates to an amide compound shown as a formula (I), or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvent compound thereof:

wherein,

In a particular embodiment, "R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Each independently selected from hydrogen, deuterium, halogen or trifluoromethyl "includes R¹Selected from hydrogen, deuterium, halogen or trifluoromethyl, R²Selected from hydrogen, deuterium, halogen or trifluoromethyl, R³Selected from hydrogen, deuterium, halogen or trifluoromethyl, and so on, until R²⁰Selected from hydrogen, deuterium, halogen or trifluoromethyl. More specifically, includes R¹Is hydrogen, R¹Is deuterium, R¹Is halogen (F, Cl, Br or I) or R¹Is trifluoromethyl, R²Is hydrogen, R²Is deuterium, R²Is halogen (F, Cl, Br or I) or R²Is trifluoromethyl, R³Is hydrogen, R³Is deuterium, R³Is halogen (F, Cl, Br or I) or R³Trifluoromethyl, and so on, until R²⁰Is hydrogen, R²⁰Is deuterium, R²⁰Is halogen (F, Cl, Br or I) or R²⁰Is a technical scheme of trifluoromethyl.

In a preferred embodiment, the present invention relates to amide compounds of formula (I), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, wherein R is²、R⁹、R¹¹Is hydrogen, R¹、R³-R⁸、R¹⁰、R¹²Each independently selected from hydrogen or deuterium.

In a preferred embodiment, the present invention relates to amide compounds of formula (I), or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof, wherein R is²、R⁹、R¹¹Is hydrogen, R¹Is deuterium, R³-R⁸、R¹⁰、R¹²Each independently selected from hydrogen or deuterium.

In a preferred embodiment, R³And R⁴Are the same.

In a preferred embodiment, R³And R⁴Is deuterium.

In a preferred embodiment, R³Is deuterium.

In a preferred embodiment, R⁵-R⁸Are the same. In a preferred embodiment, R¹⁰Is deuterium.

In a preferred embodiment, the amide compound is of any one of the following structures, or a pharmaceutically acceptable salt thereof, but is not limited to the following structures:

preparation

The following formulation examples illustrate representative pharmaceutical compositions that can be prepared according to the present invention. However, the present invention is not limited to the following pharmaceutical compositions.

Exemplary formulation 1-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press to 0.3-30mg tablets (each tablet containing 0.1-10mg of active compound).

Exemplary formulation 2-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press into 30-90mg tablets (each tablet containing 10-30mg of active compound).

Exemplary formulation 3-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press to form 90-150mg tablets (each tablet containing 30-50mg of active compound).

Exemplary formulation 4-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press into 150-240mg tablets (each containing 50-80mg of active compound).

Exemplary formulation 5-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press to 240-270mg tablets (each containing 80-90mg of active compound).

Exemplary formulation 6-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is shaped in a tablet press into 270-450mg tablets (each containing 90-150mg of active compound).

Exemplary formulation 7-tablet: the compound of the invention in dry powder form may be mixed with the dry gel binder in a weight ratio of about 1: 2. A smaller amount of magnesium stearate was added as a lubricant. The mixture was shaped into 450-900mg tablets (each tablet containing 150-300mg of active compound) in a tablet press.

Exemplary formulation 8-capsule: the compound of the invention in dry powder form may be mixed with a starch diluent in a weight ratio of about 1: 1. The mixture is filled into 250mg capsules (each containing 125mg of active compound).

Exemplary formulation 9-liquid: the compound of the present invention (125mg) may be mixed with sucrose (1.75g) and xanthan gum (4mg), and the resulting mixture may be blended, passed through a No.10 mesh U.S. sieve, and then mixed with a previously prepared aqueous solution of microcrystalline cellulose and sodium carboxymethylcellulose (11:89, 50 mg). Sodium benzoate (10mg), flavouring and colouring agents were diluted with water and added with stirring. Sufficient water may then be added to give a total volume of 5 mL.

Exemplary formulation 10-injection: the compounds of the present invention may be dissolved or suspended in aqueous media, which may be injected in buffered sterile saline, to a concentration of about 5 mg/mL.

Administration of drugs

The pharmaceutical compositions provided by the present invention may be administered by a number of routes including, but not limited to: oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, by implant or other modes of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intracerebrospinal administration, intralesional administration, and intracranial injection or infusion techniques.

Typically, an effective amount of a compound provided herein is administered. The amount of compound actually administered can be determined by a physician, as the case may be, including the condition to be treated, the chosen route of administration, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

When used to prevent a condition according to the invention, a subject at risk of developing the condition is administered a compound provided herein, typically based on physician's advice and under the supervision of a physician, at a dosage level as described above. Subjects at risk of developing a particular disorder, typically include subjects with a family history of the disorder, or those determined to be particularly susceptible to developing the disorder by genetic testing or screening.

The pharmaceutical compositions provided herein may also be administered chronically ("chronic administration"). By long-term administration is meant administration of the compound or pharmaceutical composition thereof over a long period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue for an indefinite period of time, e.g., for the remainder of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a prolonged period of time, e.g., within the therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, the pharmaceutical composition may be administered as a bolus, e.g., in order to rapidly increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the targeted systemic level of the active ingredient, e.g., an intramuscular or subcutaneous bolus dose results in a slow release of the active ingredient, while a bolus delivered directly to the vein (e.g., by IV intravenous drip) can be delivered more rapidly, allowing the concentration of the active ingredient in the blood to rise rapidly to an effective level. In other embodiments, the pharmaceutical composition may be administered as a continuous infusion, e.g., by IV intravenous drip, to provide a steady state concentration of the active ingredient in the body of the subject. Furthermore, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by continuous infusion.

Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More generally, however, the compositions are provided in unit dosage form for convenient administration of the precise dosage. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable for the purpose of producing the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, pre-measured ampoules or syringes of liquid compositions, or in the case of solid compositions, pills, tablets, capsules and the like. In such compositions, the compound is typically a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being various carriers or excipients and processing aids useful in forming the desired form of administration.

For oral dosages, a representative regimen is one to five oral dosages, particularly two to four oral dosages, typically three oral dosages per day. Using these dosing modes, each dose provides about 0.01 to about 20mg/kg of a compound of the invention, with preferred doses each providing about 0.1 to about 10mg/kg, especially about 1 to about 5 mg/kg.

In order to provide a blood level similar to, or lower than, the use of the injected dose, a transdermal dose is generally selected in an amount of from about 0.01 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injection dosage level is in the range of about 0.1 mg/kg/hour to at least 10 mg/kg/hour. To obtain sufficient steady state levels, a preload bolus of about 0.1mg/kg to about 10mg/kg or more may also be administered. For human patients of 40 to 80kg, the maximum total dose cannot exceed about 2 g/day.

Liquid forms suitable for oral administration may include suitable aqueous or nonaqueous carriers, as well as buffers, suspending and dispersing agents, coloring and flavoring agents, and the like. Solid forms may include, for example, any of the following components, or compounds with similar properties: a binder, for example, microcrystalline cellulose, gum tragacanth or gelatin; excipients, for example, starch or lactose, disintegrants, for example, alginic acid, Primogel or corn starch; lubricants, for example, magnesium stearate; glidants, e.g., colloidal silicon dioxide; sweetening agents, for example, sucrose or saccharin; or a flavoring agent, for example, peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based on sterile saline or phosphate buffered saline for injection, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient. When formulated as an ointment, the active ingredient is typically combined with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with a cream base, for example of the oil-in-water type. Such transdermal formulations are well known in the art and typically include other components for enhancing stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope of the present invention.

The compounds of the present invention may also be administered by transdermal means. Thus, transdermal administration can be achieved using a reservoir (reservoir) or porous membrane type, or a patch of various solid matrices.

The above components of the compositions for oral, injectable or topical administration are merely representative. Other materials and processing techniques are described in Remington's Pharmaceutical Sciences,17th edition,1985, Mack Publishing Company, Easton, Pennsylvania, section 8, which is incorporated herein by reference.

The compounds of the present invention may also be administered in sustained release form, or from a sustained release delivery system. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

The invention also relates to pharmaceutically acceptable formulations of the compounds of the invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α -, β -and γ -cyclodextrins consisting of 6,7 and 8 α -1, 4-linked glucose units, respectively, which optionally include one or more substituents on the linked sugar moiety, including but not limited to: methylated, hydroxyalkylated, acylated and sulfoalkyl ether substitution. In some embodiments, the cyclodextrin is sulfoalkyl ether β -cyclodextrin, e.g., sulfobutyl ether β -cyclodextrin, also known as Captisol. See, e.g., U.S.5,376,645. In some embodiments, the formulation includes hexapropyl- β -cyclodextrin (e.g., 10-50% in water).

Examples

Preferred embodiments of the present invention are described in further detail below. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Parts and percentages are parts and percentages by weight unless otherwise indicated.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

The following general preparative routes may be used to synthesize the compounds of the formula (I) of the present invention. The synthetic route is as follows:

example 1

2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl) -N- (thiazol-2-yl-5-d) acetamide (Compound 11) was prepared using the following synthetic route, comprising the steps of:

the method comprises the following steps: synthesis of Compound 3.

4mL of methanesulfonic acid was added to the reaction flask, cooled to 0 deg.C, α -hydroxyhippuric acid (Compound 1, 1g, 5.12mmol) was added, 4-fluoroanisole (646mg, 5.12mmol) was added at 0 deg.C, stirred at room temperature for 1 hour, the reaction was slowly added dropwise to ice water, the white solid was washed out, filtered, the cake was washed with water, and dried under vacuum to give Compound 3(1.3mg, yield 86.6%), LC-MS (APCI): M/z ═ 302(M-1)^-。

Step two: synthesis of Compound 4.

Adding compound 3(1g, 3.3mmol) into a reaction flask, adding 60mL of 6N hydrochloric acid, heating to 100 deg.C, reacting for 36 hours, detecting by TLC (thin layer chromatography) that the raw materials are completely reacted, cooling to room temperature, filtering, concentrating the filtrate to obtain a white solid, and pulping and purifying with ethyl acetate to obtain compound 4(200mg, yield 30.4%), LC-MS (APCI): M/z ═ 198(M-1)^-。

Step three: synthesis of Compound 5.

To a reaction flask, under nitrogen protection, compound 4(200mg, 1mmol), o-phthalaldehyde (134mg, 1mmol) was dissolved in 3mL of acetic acid, heated to 120 ℃ for reaction for 1 hour, cooled to room temperature, and the reaction solution was concentrated to give crude compound 5 (632mg, yield 100%) which was used in the next step without further purification, LC-ms (apci): M/z ═ 314(M-1)^-。

Step four: synthesis of Compound 7.

2-aminothiazole (500mg, 2.9mmol) was added to a reaction flask, Tetrahydrofuran (THF) was added to 20mL of Boc anhydride (1.308g, 6mmol) at room temperature, the reaction was carried out at room temperature for 16 hours, TLC was carried out to detect completion of the reaction of the starting material, the solvent and excess Boc anhydride were removed by concentration, and the product was purified by column chromatography to give Compound 6(859mg, yield 85.9%)

Step five: synthesis of Compound 8.

Adding compound 7(200mg, 1mmol) to a reaction flask, adding 20mL of tetrahydrofuran, cooling to 0 ℃, dropwise adding n-butyllithium (0.52mL, 1.3mmol) at 0 ℃, stirring at 0 ℃ for 1 hour after completion of dropwise addition, quenching the reaction with 4mL of heavy water, extracting the combined organic phases with ethyl acetate, drying over anhydrous sodium sulfate, and purifying by column chromatography to obtain compound 7(200mg, yield 100%), LC-MS (APCI): M/z 399(M +1)⁺。

Step six: synthesis of Compound 9.

Adding the compound 8(200mg, 1mmol) into a reaction bottle, adding 12mL of dichloromethane for dissolving, adding trifluoroacetic acid (570mg, 5mmol) at room temperature for reacting for 3 hours at room temperature, adjusting the pH of the raw material reaction to be alkalescent by using 1M hydrochloric acid through TLC detection, concentrating to remove the solvent, directly using the solvent for the next reaction,¹H NMR(500MHz,MeOD)(/ppm)7.19(s,1H),6.88(d,J＝4.2Hz,0.07H)。

step seven: synthesis of Compound 10.

To a reaction flask were added crude compound 5 (688mg, 2.17mmol) and compound 9(282mg, 2.812mmol), N, N-diisopropylethylamine (DIPEA, 279mg, 2.175mmol), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 1.07g, 2.812mmol), dimethylformamide (DMF, 20mL) was added, the reaction was carried out at 25 ℃ under nitrogen for 3 hours, TLC detected that the starting material was completely reacted, 50mL of water was added to the reaction solution, ethyl acetate was used for extraction 4 times, the organic phases were combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and purified to give compound 9(227mg, yield 26.3%), LC-MS (APCI): M/z 399(M +1)⁺。

Step eight: synthesis of Compound 11.

Add Compound 10(200mg, 0.5 m) to the reaction flaskmol), adding 30mL of dichloromethane, cooling to-10 ℃, and dropwise adding boron tribromide (BBr) into the reaction bottle₃500mg, 2.1mmol), after completion of dropwise addition, warmed to room temperature for 2 hours, TLC detected starting material reacted completely, 30mL of water was added to the reaction flask, extracted 4 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 11 as a white solid (64mg, yield 33.3%), LC-ms (apci): M/z 383(M-1)^-。¹H NMR(500MHz,DMSO)(/ppm)12.59(s,1H),9.94(s,1H),7.72(d,J＝7.5Hz,1H),7.60(t,J＝7.4Hz,1H),7.56(d,J＝7.4Hz,1H),7.50(t,J＝6.3Hz,1H),7.47(d,J＝5.2Hz,1H),7.10(td,J＝8.6,3.1Hz,1H),6.89(dd,J＝8.9,4.8Hz,1H),6.84(dd,J＝9.2,3.0Hz,1H),6.30(s,1H),4.60(d,J＝17.4Hz,1H),3.97(d,J＝17.5Hz,1H)。

Example 2

2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl-3-d) -N- (thiazol-2-yl) acetamide (Compound 21) was prepared using the following synthetic route comprising the steps of:

the method comprises the following steps: synthesis of Compound 12.

Adding compound 4(2.08g, 1045mmol) into a reaction flask, adding 30mL of methanol, cooling to 0 ℃, adding thionyl chloride (1.5mL, 20.9mmol) dropwise, raising the temperature to 75 ℃ after completion of addition, reacting for 3 hours, concentrating after the raw materials are completely reacted to remove methanol and excess thionyl chloride, adding 20mL of methyl tert-butyl ether, and pulping to obtain compound 12(2.15g, yield 98.4%), LC-MS (APCI): M/z 214(M +1)⁺。

Step two: synthesis of Compound 14.

To a reaction flask was added compound 13(2g, 12mmol) and 10mL of thionyl chloride at 0 ℃ to reflux the reaction for 3 hours, and the excess thionyl chloride was concentrated to obtain compound 14(2.74g, yield 100%).

Step three: synthesis of Compound 15.

Triethylamine (TEA, 1.5g, 14.6mmol) was added to the reaction flask, DMF (20mL) was added and the compound was added14(1.04g, 4.88mmol) in DMF (15mL), compound 12 in DMF (15mL), added dropwise to the reaction flask simultaneously at rt for 3 hours, TLC detected complete reaction of starting material and concentrated to give 2.3g of crude compound 15, which was used directly in the next step without further purification, LC-ms (apci) M/z 362(M +1)⁺。

Step four: synthesis of Compound 16.

Adding 2.3g of crude compound 15 into a reaction flask, adding 15mL of thionyl chloride at 0 ℃, raising the temperature to 50 ℃ after dropwise addition, reacting for 5 hours, detecting by TLC that the raw materials are completely reacted, concentrating to remove excessive thionyl chloride, and purifying by column chromatography to obtain white solid compound 16(1.244g, two-step yield 73.16%), LC-MS (APCI): M/z 344(M +1)⁺。

Step five: synthesis of Compound 17.

Adding compound 16(1.244g, 3.568mmol) into a reaction flask, adding zinc powder (2.3g, 35.68mmol), adding acetic acid 10mL, heating to 110 ℃ for reaction for 2 hours, detecting the reaction of the raw materials by TLC, filtering to remove excessive zinc powder, concentrating to remove acetic acid, adding ethyl acetate 40mL, washing with saturated sodium bicarbonate solution, concentrating the organic phase, and purifying by column chromatography to obtain compound 17(315mg, yield 25.5%), LC-MS (APCI): M/z 347(M +1)⁺。

Step six: synthesis of Compound 18.

Adding compound 17(314mg, 0.957mmol), trifluoroacetic acid (TFA, 218mg, 1.914mmol) into a reaction flask, dissolving with 20mL of dichloromethane, cooling to 0 ℃, slowly adding dropwise triethylsilane (333mg, 2.872mmol), reacting at 0 ℃ for 2 hours after completion of dropwise addition, detecting completion of the reaction of the raw materials by TLC, adding 20mL of water into the reaction flask, extracting with dichloromethane three times, combining the organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain compound 18(109mg, yield 34.7%), LC-MS (APCI): M/z ═ 331(M +1)⁺。

Step seven: synthesis of Compound 19.

To a reaction flask was added compound 18(109mg, 0.3313mmol), lithium hydroxide (30mg, 1.7mmol) and water (1mL), tetrahydrofuran (R) ((R))THF, 4mL) at rt for 3 h, TLC checked starting material was completely reacted, adjusted to pH 5-6 with 1M hydrochloric acid and concentrated to give crude 316mg of compound 19, which was used directly in the next reaction without further purification, LC-ms (apci): M/z ═ 362(M-1)^-。

Step eight: synthesis of Compound 20.

Adding crude compound 19 (316mg, 0.33mmol), 2-aminothiophene (43mg, 0.403mmol), DIPEA (55mg, 0.403mmol), HATU (163mg, 0.403mmol) into a reaction flask, adding DMF (10mL), reacting at 25 ℃ for 18 hours under the protection of nitrogen, detecting the completion of the reaction by TLC, adding 50mL of water into the reaction solution, extracting with ethyl acetate for 4 times, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain compound 20(154mg), LC-MS (APCI), wherein M/z is 399(M +1)⁺。

Step nine: synthesis of Compound 21.

Adding compound 20(154mg, 0.387mmol) into a reaction flask, adding 10mL of dichloromethane, cooling to-10 ℃, dropwise adding boron tribromide (1.5mL, 1.16mmol) into the reaction flask, after the dropwise addition is finished, heating to room temperature for reaction for 2 hours, detecting the completion of raw material reaction by TLC, adding 30mL of water into the reaction flask, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain white solid compound 21(92mg, yield 62.2%), LC-MS (APCI): M/z 385(M +1)⁺；¹H NMR(500MHz,DMSO)(/ppm)12.57(s,1H),9.92(s,1H),7.94(s,1H),7.71(d,J＝7.5Hz,1H),7.60(t,J＝7.1Hz,1H),7.56(d,J＝7.5Hz,1H),7.50(d,J＝7.3Hz,1H),7.48–7.42(m,1H),7.09(t,J＝7.0Hz,1H),6.89(dd,J＝8.9,4.7Hz,1H),6.84(d,J＝9.2Hz,1H),6.30(s,1H),4.58(s,0.5H),3.96(s,0.5H)。

Example 3

Preparation of 2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-y-yl-3, 3-d by the following synthetic route₂) -N- (thiazol-2-yl) acetamide (compound 25), comprising the following steps:

the method comprises the following steps: synthesis of Compound 22.

Adding compound 16(1g, 2.91mmol) into a reaction flask, adding zinc powder (10g, 152mmol) 5 times, adding 10mL of deuterated acetic acid, heating to 110 deg.C, reacting for 7 hours, detecting the reaction of the raw materials by TLC, filtering to remove excessive zinc powder, concentrating to remove acetic acid, adding 40mL of ethyl acetate, washing with saturated sodium bicarbonate solution, concentrating the organic phase, and purifying by column chromatography to obtain compound 22(315mg, yield 26.1%), LC-MS (APCI): M/z 332(M +1)⁺。

Step two: synthesis of Compound 23.

To a reaction flask was added compound 22(252mg, 0.76mmol), added lithium hydroxide (159mg, 3.8mmol), added water (1mL), tetrahydrofuran (5mL), reacted at room temperature for 3 hours, TLC detected complete reaction of starting material, adjusted pH to 5-6 with 1M hydrochloric acid, concentrated to give crude 340mg of compound 23, which was used directly in the next reaction without further purification, LC-ms (apci): M/z 316(M-1)^-。

Step three: synthesis of compound 24.

The crude compound 23 (340mg, 0.76mmol), 2-aminothiophene (100mg, 0.968mmol), DIPEA (332mg, 2.57mmol), HATU (374M g, 0.968mmol) were added to a reaction flask, DMF (10mL) was added, the reaction was reacted at 25 ℃ for 5 hours under nitrogen protection, TLC detected that the starting material reacted completely, 40mL of water was added to the reaction solution, extraction was performed 4 times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain compound 24(125mg, two-step yield 41.2%), LC-ms (apci): M/z 400(M +1)⁺。

Step four: synthesis of Compound 25.

Adding compound 24(125mg, 0.312mmol) into a reaction bottle, adding dichloromethane 15mL, cooling to-10 ℃, dropwise adding boron tribromide (1.6mL, 1.6mmol) into the reaction bottle, heating to room temperature after dropwise adding is finished, reacting for 2 hours, detecting that raw materials are completely reacted by TLC, adding water 30mL into the reaction bottle, extracting for 4 times by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid compound 25(53mg, yield 44.1 percent), LC-MS (liquid chromatography-mass spectrometry) ((L) (APCI):m/z＝386(M+1)⁺；¹H NMR(500MHz,DMSO)(/ppm)12.58(s,1H),9.93(s,1H),7.71(d,J＝7.5Hz,1H),7.60(t,J＝7.3Hz,1H),7.55(d,J＝7.4Hz,1H),7.50(d,J＝7.5Hz,1H),7.48–7.45(m,1H),7.25(d,J＝3.5Hz,1H),7.09(td,J＝8.6,3.1Hz,1H),6.89(dd,J＝8.9,4.8Hz,1H),6.84(dd,J＝9.2,3.0Hz,1H),6.30(s,1H)。

Example 4

Preparation of 2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl-4, 5,6,7-d by the following synthetic route₄) -N- (thiazol-2-yl) acetamide (compound 34), comprising the following steps:

the method comprises the following steps: synthesis of Compound 27.

Adding compound 26(1g, 5.88mmol) into a reaction flask, adding 10mL of thionyl chloride at 0 ℃, refluxing for 3 hours, concentrating to remove excess thionyl chloride to obtain compound 27(1.05g, yield-100%)

Step two: synthesis of Compound 28.

Triethylamine (1.56g, 15.48mmol) was added to the reaction flask, DMF (20mL) was added, Compound 12(1.1g, 5.16mmol) was dissolved in DMF (15mL), Compound 27(1.05g, 5.16mmol) was dissolved in DMF (15mL) and added dropwise to the flask at room temperature simultaneously to react at room temperature for 3 hours, TLC detected the starting material was completely reacted and concentrated to give 2.4g of crude Compound 28 which was used directly in the next step, LC-MS (APCI) M/z 366(M +1)⁺。

Step three: synthesis of Compound 29.

Adding 2.4g of crude compound 28 into a reaction flask, adding 15mL of thionyl chloride at 0 ℃, raising the temperature to 50 ℃ after dropwise addition, reacting for 5 hours, detecting by TLC that the raw materials are completely reacted, concentrating to remove excessive thionyl chloride, and purifying by column chromatography to obtain a white solid compound 29(545mg, two-step yield 30.1%), LC-MS (APCI): M/z ═ 348(M +1)⁺。

Step four: synthesis of Compound 30.

Adding chemical combination to a reaction flaskAfter substance 29(545mg, 1.57mmol), zinc powder (1.02g, 15.7mmol) was added, acetic acid (15mL) was added, the reaction was warmed to 110 ℃ for 5 hours, TLC detected that the reaction of the starting material was completed, excess zinc powder was removed by filtration, acetic acid was removed by concentration, ethyl acetate (40 mL) was added, the mixture was washed with a saturated sodium bicarbonate solution, the organic phase was concentrated, and column chromatography was performed to obtain compound 30(165mg, yield 31.7%), LC-ms (apci): M/z 350(M +1)⁺。

Step five: synthesis of Compound 31.

Adding compound 30(165mg, 0.498mmol), trifluoroacetic acid (113mg, 0.997mmol) into a reaction flask, dissolving with 20mL of dichloromethane, cooling to 0 ℃, slowly adding triethylsilane (173mg, 1.495mmol) dropwise, reacting at 0 ℃ for 2 hours after completion of the dropwise addition, detecting by TLC that the raw materials are reacted completely, adding 20mL of water into the reaction flask, extracting with dichloromethane three times, combining the organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain compound 31(97mg, yield 58.7%), LC-MS (APCI): M/z ═ 334(M +1)⁺。

Step six: synthesis of Compound 32.

To a reaction flask was added compound 31(97mg, 0.292mmol), added lithium hydroxide (61mg, 1.46mmol), added water (1mL), tetrahydrofuran (5mL), reacted at room temperature for 3 hours, TLC detected the starting material was completely reacted, adjusted pH to 5-6 with 1M hydrochloric acid, concentrated to give crude 120mg of compound 32, which was used directly in the next reaction without further purification, LC-ms (apci): M/z ═ 318(M-1)^-。

Step seven: synthesis of Compound 33.

Adding compound 32(120mg, 0.292mmol), 2-aminothiophene (30mg, 0.38mmol), DIPEA (100mg, 0.76mmol), HATU (144mg, 0.38mmol) into a reaction flask, adding DMF (5mL), reacting at 25 ℃ for 6 hours under the protection of nitrogen, detecting the completion of the reaction by TLC, adding 50mL of water into the reaction solution, extracting with ethyl acetate for 4 times, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain compound 33(45mg, yield-38.4%), LC-MS (APCI): M/z ═ 402(M +1)⁺。

Step eight: synthesis of compound 34.

Adding compound 33(154mg, 0.387mmol) into a reaction flask, adding 10mL of dichloromethane, cooling to-10 ℃, dropwise adding boron tribromide (1.5mL, 1.16mmol) into the reaction flask, after the dropwise addition is completed, heating to room temperature for reaction for 2 hours, detecting the completion of the raw material reaction by TLC, adding 30mL of water into the reaction flask, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid compound 34(92mg, yield 62.2%), LC-MS (APCI): M/z ═ 388(M +1)⁺；¹H NMR(500MHz,DMSO)(/ppm)12.58(s,1H),9.92(s,1H),7.47(d,J＝3.4Hz,1H),7.25(s,1H),7.09(t,J＝7.0Hz,1H),6.94-6.80(m,2H),6.30(s,1H),4.60(d,J＝17.3Hz,1H),3.97(d,J＝17.4Hz,1H)。

Example 5

The amine 2- (5-fluoro-2-hydroxyphenyl-3-d) -2- (1-oxoisoindolin-2-yl) -N- (thiazol-2-yl) acetamide (compound 42) was prepared using the following synthetic route comprising the steps of:

the method comprises the following steps: synthesis of Compound 35.

P-fluoro anisole (400mg, 3.17mmol) is added into a reaction bottle, 30mL of dichloromethane is added for dissolution, the mixture is cooled to-10 ℃, boron tribromide (1.44g, 15.86mmol) is dropwise added into the reaction, the reaction is carried out at room temperature for 3 hours after the dropwise addition, TLC detection is carried out to ensure that raw materials completely react, 20mL of water is added, dichloromethane is used for extraction for three times, organic phases are combined, anhydrous sodium sulfate is used for drying, and column chromatography purification is carried out to obtain a compound 35(400mg, yield-100%), LC-MS (APCI): M/z ═ 113(M +1)⁺。

Step two: synthesis of Compound 36.

Compound 35(0.4g, 3.57mmol) and sodium hydroxide (71mg, 1.78mmol) were added to a reaction flask, dissolved in 10mL of heavy water, heated to 180 ℃ with microwave for 1 hour, cooled to room temperature, extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give compound 36(400mg, yield-100%), LC-ms (apci): M/z ═ 115(M +1)⁺。

Step three: synthesis of Compound 37.

To a reaction flask was added compound 36(0.4g, 3.57mmol), potassium carbonate (1.323g, 8.9mmol), DMF (15mL) was added, iodomethane (1.25g, 8.9mmol) was added at room temperature, stirred at room temperature for 5 hours, TLC detected that the starting material reacted completely, 30mL of water was added to the reaction, extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give compound 37(400mg, yield 89%), LC-ms (apci): M/z 129(M +1)⁺。

Step four: synthesis of compound 38.

4mL of methanesulfonic acid was added to a reaction flask, the mixture was cooled to 0 ℃ and then the compound 37(426mg, 3.33mmol) was added, α -hydroxyhippuric acid (650mg, 3.33mmol) was added at 0 ℃, the mixture was stirred at room temperature for 1 hour, the reaction mixture was slowly added dropwise to ice water, the white solid was washed off, the mixture was filtered, the cake was washed with water and dried in vacuo to obtain the compound 38(345mg, yield 34.15%), LC-MS (APCI): M/z 303(M-1)^-。

Step five: synthesis of Compound 39.

To a reaction flask was added compound 38(650mg, 2.14mmol), 6N hydrochloric acid (60 mL) was added, the temperature was raised to 100 ℃, reaction was carried out for 48 hours, TLC detected that the starting material reacted completely, cooled to room temperature, filtered, and the filtrate was concentrated to give a white solid, which was slurried with ethyl acetate and purified to give compound 39(345mg, yield 80.7%). LC-MS (APCI) M/z 199(M-1)^-。

Step six: synthesis of Compound 40.

To a reaction flask, under nitrogen protection, compound 39(345mg, 1.725mmol), o-phthalaldehyde (231mg, 1.725mmol) was dissolved in 6mL of acetic acid, warmed to 120 ℃ for reaction for 1 hour, cooled to room temperature, and concentrated to give crude compound 40 (688mg, yield 100%) which was used in the next step without further purification, LC-ms (apci): M/z 315(M-1)^-。

Step seven: synthesis of Compound 41.

To a reaction flask were added crude compound 40 (688mg, 2.17mmol) and 2-aminothiazole (282mg, 2.812mmol), DIPEA (9279mg, 2.175mmol), HATU (1.07g, 2.812 mm)ol) and DMF (20mL) were added, the mixture was reacted at 25 ℃ for 3 hours under nitrogen protection, TLC was used to detect completion of the reaction, 50mL of water was added to the reaction mixture, extraction was performed 4 times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain Compound 41(227mg, yield 26.3%), LC-MS (APCI) M/z 399(M +1)⁺。

Step eight: synthesis of Compound 42.

Adding compound 41(227mg, 0.569mmol) into a reaction bottle, adding 30mL of dichloromethane, cooling to-10 ℃, dropwise adding boron tribromide (713mL, 2.85mmol) into the reaction bottle, after the dropwise addition is completed, raising the temperature to room temperature for reaction for 2 hours, detecting the completion of the raw material reaction by TLC, adding 30mL of water into the reaction bottle, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain white solid compound 42(151mg, yield 68.9), LC-MS (APCI): M/z 383.0(M-1)^-；¹H NMR(500MHz,DMSO)(/ppm)12.58(s,1H),9.92(s,1H),7.72(d,J＝7.6Hz,1H),7.60(t,J＝7.4Hz,1H),7.56(d,J＝7.4Hz,1H),7.50(d,J＝7.6Hz,1H),7.49-7.45(m,1H),7.25(d,J＝3.3Hz,1H),7.09(dd,J＝8.3,3.1Hz,1H),6.84(dd,J＝9.2,3.0Hz,1H),6.30(s,1H),4.60(d,J＝17.4Hz,1H),3.98(d,J＝17.5Hz,1H)。

Example 6

2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl) -N- (thiazol-2-yl) acetamide-2-d (compound 46) is prepared using the following synthetic route comprising the steps of:

the method comprises the following steps: synthesis of Compound 43.

Adding compound 4(800mg, 4mmol) into a reaction flask, adding salicylaldehyde (304mg, 2.32mmol) and 6mL of deuterated acetic acid, heating to 80 ℃ for reaction for 4 hours, detecting by TLC that the raw materials are completely reacted, cooling to room temperature, concentrating to remove deuterated acetic acid, adding 2mL of ethyl acetate, stirring at room temperature for 30 minutes, filtering to obtain a white solid, and drying to obtain compound 43(625mg, yield 78.1%), LC-MS (APCI): M/z ═ 199(M-1)^-。

Step two: synthesis of compound 44.

Under the protection of nitrogen, compound 43(750mg, 3.75mmol), o-phthalaldehyde (502mg, 3.75mmol) were dissolved in 3mL of acetic acid, the temperature was raised to 120 ℃ to react for 1 hour, the reaction mixture was cooled to room temperature, and the reaction mixture was concentrated and purified by column chromatography to obtain compound 44(414mg), LC-MS (APCI) where M/z is 315(M-1)^-。

Step three: synthesis of Compound 45.

Adding compound 44(200mg, 0.63mmol) and 2-aminothiazole (81.9mg, 0.819mmol), DIPEA (80mg, 0.63mmol), HATU (311M g, 0.819mmol) into a reaction flask, adding DMF (15mL), reacting for 4 hours at 25 ℃ under the protection of nitrogen, detecting the completion of the raw material reaction by TLC, adding 30mL of water into the reaction solution, extracting with ethyl acetate for 4 times, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain 316mg of crude compound 45, LC-MS (APCI): M/z is 399(M-1)^-

Step four: synthesis of compound 46.

Adding compound 45(316mg, 0.794mmol) into a reaction flask, adding dichloromethane 15mL, cooling to-10 ℃, dropwise adding boron tribromide (4mL, 4mmol) into the reaction flask, after the dropwise addition is completed, heating to room temperature for reaction for 2 hours, detecting the completion of the raw material reaction by TLC, adding water 30mL into the reaction flask, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, purifying by column chromatography to obtain white solid compound 46(121mg, yield 39.8%), LC-MS (APCI): M/z 383(M-1)^-；¹HNMR(500MHz,DMSO)(/ppm)12.59(s,1H),9.93(s,1H),7.71(d,J＝7.7Hz,1H),7.60(t,J＝7.4Hz,1H),7.56(d,J＝7.5Hz,1H),7.50(d,J＝7.3Hz,1H),7.47(d,J＝3.5Hz,1H),7.26(d,J＝3.3Hz,1H),7.10(t,J＝7.3Hz,1H),6.89(dd,J＝8.9,4.7Hz,1H),6.84(d,J＝9.0Hz,1H),4.60(d,J＝17.5Hz,1H),3.97(d,J＝17.6Hz,1H)。

Example 7

2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl) -N- (thiazol-2-yl-5-d) acetamide-2-d (Compound 48) was prepared using the following synthetic route comprising the steps of:

the method comprises the following steps: synthesis of Compound 47.

Adding compound 44(241mg, 0.76mmol), compound 9(99mg, 0.998mmol), DIPEA (98mg, 0.76mmol), HATU (372M g, 0.998mmol), DMF (10mL) and nitrogen at 25 deg.C for 5 hr, detecting by TLC, adding water 30mL, extracting with ethyl acetate for 4 times, combining organic phases, purifying by column chromatography to obtain compound 47(280mg), LC-MS (APCI), wherein M/z is 400(M +1)⁺。

Step two: synthesis of Compound 48.

Adding compound 47(280mg, 0.701mmol) into a reaction bottle, adding dichloromethane 15mL, cooling to-10 ℃, dropwise adding boron tribromide (3.5mL, 3.5mmol) into the reaction bottle, after the dropwise addition is completed, heating to room temperature for reaction for 2 hours, detecting the completion of the raw material reaction by TLC, adding water 30mL into the reaction bottle, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain a white solid compound 48(38mg, yield 25.2%), LC-MS (APCI): M/z 384(M-1)^-；¹H NMR(500MHz,DMSO)(/ppm)12.59(s,1H),9.93(s,1H),7.71(d,J＝7.5Hz,1H),7.60(t,J＝7.1Hz,1H),7.56(d,J＝7.4Hz,1H),7.50(d,J＝7.3Hz,1H),7.48(d,J＝5.9Hz,1H),7.10(td,J＝8.5,3.0Hz,1H),6.89(dd,J＝8.8,4.7Hz,1H),6.84(d,J＝9.1Hz,1H),4.60(d,J＝17.4Hz,1H),3.99(dd,J＝22.0,12.5Hz,1H)。

Example 8

Preparation of 2- (5-fluoro-2-hydroxyphenyl) -2- (1-oxoisoindolin-2-yl-4, 5,6,7-d by the following synthetic route₄) -N- (thiazol-2-yl-5-d) acetamide (compound 50), comprising the following steps:

the method comprises the following steps: synthesis of Compound 49.

To a reaction flask was added compound 32(212mg, 0.6648mmol), compound 9(56mg, 0.554mmol),DIPEA (194mg, 1.04mmol), HATU (273mg, 0.72mmol), DMF (5mL) was added, the reaction was allowed to react at 25 ℃ for 4 hours under nitrogen, TLC was used to detect completion of the reaction, 50mL of water was added to the reaction mixture, extraction was performed 4 times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 49(108mg, yield 49.1%), LC-MS (APCI): M/z 403(M +1)⁺。

Step two: synthesis of Compound 50.

Adding compound 49(108mg, 0.268mmol) into a reaction bottle, adding 10mL of dichloromethane, cooling to-10 ℃, dropwise adding boron tribromide (0.8mL, 0.8mmol) into the reaction bottle, after the dropwise addition is completed, heating to room temperature for reaction for 2 hours, detecting the completion of the raw material reaction by TLC, adding 30mL of water into the reaction bottle, extracting with dichloromethane for 4 times, combining organic phases, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain a white solid compound 50(63mg, yield 61.1%), LC-MS (APCI): M/z 389(M +1)⁺；¹H NMR(500MHz,DMSO)(/ppm)12.58(s,1H),9.93(s,1H),7.47(s,1H),7.09(td,J＝8.6,3.1Hz,1H),6.89(dd,J＝8.9,4.8Hz,1H),6.84(dd,J＝9.2,3.1Hz,1H),6.30(s,1H),4.60(d,J＝17.4Hz,1H),3.97(d,J＝17.4Hz,1H)。

Example 9

The compounds obtained in the above examples were subjected to biological evaluation to determine their biological activity. In addition, antiproliferative activity in some of these compounds was screened in human A431 skin cancer cells and human NCI-H1975 and HCC827 lung cancer cell lines, and demonstrated activity in the <20nM range. Evaluating the cytotoxicity or growth inhibition of said compound on the tumor cell of interest.

(1) EGFR kinase inhibition

The biological activity of the compounds of examples 1-8 was determined by testing their ability to inhibit a variety of protein kinases of interest. These compounds were found to exhibit potent inhibitory activity against EGFR kinase by testing. The specific method comprises the following steps:

compound preparation: test compounds were dissolved in DMSO to make 20mM stock. Dilutions were made in DMSO at 100-fold final concentration. When adding medicine, the medicine is diluted by buffer solution into 10 times of the dilution solution with final concentration.

EGFR and EGFR [ T790M/L858R ] kinase assays: after buffer preparation, the enzyme was mixed with the pre-diluted compounds at different concentrations for 10 minutes, each concentration being in duplicate wells. The corresponding substrate and ATP were added and the reaction was carried out at room temperature for 20 minutes (negative and positive controls were set). And adding a detection reagent after the reaction is finished, incubating for 30 minutes at room temperature, then performing machine detection, and collecting data. Data analysis and mapping were performed according to Graphpad 5.0 software.

EGFR [ d746-750] kinase assay: after the buffer was prepared, a mixed solution of the enzyme and the antibody was mixed with the compound of different concentrations prepared in advance in duplicate for 10 minutes at each concentration. Kinase trap 199 was added and incubated for 60 minutes at room temperature (where negative and positive controls were set). And after the reaction is finished, the machine is used for detecting, collecting data, and analyzing and drawing.

(2) Cytotoxic effects

The in vitro antiproliferative activity of the compound of the invention on 2 tumor cells cultured in vitro was tested by MTS method. The experimental result shows that the compound has the inhibiting effect on the in vitro proliferation of cancer cells cultured in vitro; wherein the inhibition of in vitro proliferation of lung cancer cells is stronger than the inhibition of in vitro proliferation of skin cancer cells.

Cell line: skin cancer cell a431 (purchased from american standard biological collection (ATCC)); lung cancer cells NCI-H1975 (purchased from american college of standards living things (ATCC)) and HCC827 (purchased from american college of standards living things (ATCC)); all were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin.

Reagents and consumables: RPMI-1640(GIBCO, Cat. No. A10491-01); fetal bovine serum (GIBCO, catalog No. 10099141); 0.25% trypsin-EDTA (GIBCO, cat No. 25200); penicillin-streptomycin; liquid (GIBCO, catalog number 15140- & 122); DMSO (Sigma, cat # D2650); MTS assay kit (Promega, catalog No. G3581), 96-well plate (Corning, catalog No. 3365).

The specific experimental method comprises the following steps:

compound preparation: test compounds were dissolved in DMSO to prepare a 20mM stock solution, which was stored at-20 ℃. Diluted 3-fold with DMSO gradient and 10-fold. When adding medicine, the medicine is diluted 4 times by using cell culture medium.

MTS cell viability assay: cells in the logarithmic growth phase were digested with 0.25% trypsin-EDTA, and 150. mu.l of the compound diluted 4-fold in culture medium was inoculated into a 96-well plate at the optimized density, and 50. mu.l/well (ten concentrations: 100, 33.3, 11.1, 3.70, 1.23, 0.412, 0.137, 0.0457, 0.0152, 0.00508. mu.M were generally selected) was added after 24 hours. Wells to which the same volume of 0.5% DMSO was added served as controls. After the cells were cultured for 72 hours, the MTS measured the cell viability.

The method comprises the following specific operations: adherent cells, medium was discarded and a mixture containing 20. mu.L MTS and 100. mu.l medium was added to each well. The culture was continued for 1 to 4 hours in an incubator and then OD490 was measured using OD650 as a reference. Dose-response curves were generated and IC calculated using GraphPad Prism software₅₀。

The compounds of examples 1 to 8 were subjected to EGFR kinase inhibitory action and cytotoxicity experiments according to the above-mentioned methods, and the results showed that the compounds of the present invention showed potent excellent inhibitory activity against EGFR mutants and no inhibitory activity against EGFR (WT) expressed in normal cells, so that the compounds of the present invention were effective and safe drugs useful for patients with non-small cell lung cancer.

(3) Metabolic stability evaluation

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; mouse liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: an amount of the compound of example was weighed out finely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer solution (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30 min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWherein V-M is equal to 1/protein concentration.

The compounds of the invention and compounds without deuteration were tested simultaneously and compared to evaluate their metabolic stability in human and rat liver microsomes. The half-life and intrinsic hepatic clearance as indicators of metabolic stability are shown in table 1. The non-deuterated compound EAI045 was used as a control in table 1. As shown in table 1, the compounds of the present invention can significantly improve metabolic stability by comparison with EAI045 in human/rat/mouse liver microsome experiments.

TABLE 1 comparative table of metabolic stability indices for the compounds of examples 1-8

(4) Pharmacokinetic experiment of rat

6 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, were divided into 2 groups of 3 per group and compared for pharmacokinetic differences by intravenous or oral administration of a single dose of compound (10 mg/kg oral).

Rats were fed with standard feed and given water. Fasting began 16 hours prior to the experiment. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300 μ L of blood was collected from the orbit into a test tube. In the test tube there was 30. mu.L of 1% heparin salt solution. Before use, the tubes were dried overnight at 60 ℃. After completion of blood collection at the last time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted at least 5 times to ensure mixing and then placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the plasma from the erythrocytes. Pipette 100 μ L of plasma into a clean plastic centrifuge tube, designating the name of the compound and the time point. Plasma was stored at-80 ℃ before analysis. The concentration of the compounds of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

Experiments show that the compound has better pharmacokinetic property in animals, thereby having better pharmacodynamics and treatment effects.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

2. a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. A method of preparing the pharmaceutical composition of claim 2, comprising: mixing a pharmaceutically acceptable excipient with the compound of claim 1 or a pharmaceutically acceptable salt thereof to form a pharmaceutical composition.

4. The pharmaceutical composition of claim 2, further comprising an additional therapeutic agent.

5. The pharmaceutical composition of claim 4, wherein the other therapeutic agent is an agent for treating cancer, cardiovascular disease, inflammation, infection, immunological disease, cell proliferative disease, viral disease, metabolic disease, or organ transplantation.

6. Use of a compound according to claim 1 for the preparation of a medicament for the treatment and/or prevention of a disease associated with a protein kinase.