WO2019001307A1

WO2019001307A1 - Amide compound, composition containing same, and use thereof

Info

Publication number: WO2019001307A1
Application number: PCT/CN2018/091829
Authority: WO
Inventors: 王义汉; 赵九洋
Original assignee: 深圳市塔吉瑞生物医药有限公司
Priority date: 2017-06-26
Filing date: 2018-06-19
Publication date: 2019-01-03
Also published as: CN109111439B; CN109111439A

Abstract

An amide compound of formula (I), a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate, or a solvate thereof, a pharmaceutical composition containing the compound, and a pharmaceutical use thereof. The compound and the pharmaceutical composition have excellent protein kinase inhibitory activity and good pharmacokinetic properties, can increase drug concentrations of the compound in animals, and improve the efficacy and safety of the drug.

Description

Amide compound and composition containing the same and use thereof

Technical field

The invention belongs to the technical field of medicine, and in particular relates to an amide compound and a composition comprising the same and use thereof.

Background technique

Protein tyrosine kinases play an important role in cell regulation, and abnormal expression or mutations have been observed in cancer cells or autoimmune diseases. Protein tyrosine kinases are enzymes that catalyze the transport of phosphate groups from ATP to tyrosine located on a protein substrate. Many growth factor receptor proteins function as tyrosine kinases to transmit cellular signals. The interaction between growth factors and their receptors usually controls cell growth, but abnormal signaling caused by mutation or overexpression of any of the receptors often induces a variety of cancers or autoimmune diseases (eg rheumatoid joints) inflammation).

EGFR tyrosine kinase inhibitor (EGFR-TKI) is a molecularly targeted drug targeting EGFR, which binds to the EGFR tyrosine kinase catalytic domain binding site on the cell surface by competitively binding to ATP, blocking signaling into cells. Further delivery inhibits tumor cell growth and induces apoptosis. Currently, EGFR-TKI such as erlotinib and gefitinib have been widely used in clinical practice. Although EGFR inhibitors such as gefitinib and erlotinib have achieved remarkable results in EGFR-mutant advanced non-small cell lung cancer (NSCLC), it has been found that existing EGFR-TKIs may be primary in the treatment of NSCLC. Sexually or secondaryly, the second mutation in EGFR in patients with lung cancer can be treated with oral AZD9291 (Osimertinib, Oxytinib). AZD9291 is an oral, irreversible, third-generation EGFR inhibitor (EGFR-TKI) that has a better therapeutic effect in patients with NSCLC who are already resistant to EGFR-TKI and have T790M mutations. However, patients taking AZD9291 will still develop symptoms of acquired resistance. The mechanisms of AZD9291 acquired resistance mutations include: EGFR C797S mutation, FGFR1 amplification, HER2 amplification, c-Met amplification or MAPK alternative pathway activation, histological transformation (partial conversion to small cell lung cancer), or other genes mutation. (Reference: Oxnard et al. Nature Medicine, 2015, 21, 560-562)

AZD9291 forms a covalent bond with cysteine C797 at the ATP binding site, and the C797S mutation affects the binding of covalent bonds, similar to the resistance mechanism of the BTK inhibitor Ibrutinib. In February 2016, "Nature" published a heavy article describing a new generation of targeted drug EAI045 that can overcome AZD9291 resistance, and combined EAI045 with Erbitux for mouse models for C797S mutation. Among them, the efficiency is as high as 80%. (Reference: M.J. Eck et al. Nature, 2016, 534, 129-132)

Therefore, the treatment of advanced NSCLC faces new challenges, and we need to carry out new explorations and find new countermeasures.

Summary of the invention

In view of the above technical problems, the present invention discloses an amide compound, a composition comprising the same, and a use thereof, which have protein kinase inhibitory activity and have better pharmacodynamic/pharmacokinetic properties.

In this regard, the present invention adopts the following technical solutions:

In one aspect, the invention relates to an amide compound of formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent compound thereof:

among them,

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl. With the proviso that the above amide compound contains at least one ruthenium atom.

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

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

Specifically, in the present invention, the ytterbium isotope in each of the deuterated positions of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² The content is at least 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more preferably More than 40%, more preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70%, more preferably More than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 99%.

In another specific embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² of the compound of formula (I) , at least one of which contains bismuth, more preferably two 氘, more preferably three 氘, more preferably four 氘, more preferably five 氘, more preferably six 氘, more preferably Seven contain sputum, more preferably eight sputum, more preferably nine sputum, more preferably ten sputum, more preferably eleven sputum, more preferably twelve sputum. Specifically, the compound of formula (I) contains at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve deuterium atoms. .

As a preferred embodiment of the invention, R ¹ and R ² are each independently hydrazine or hydrogen.

In another preferred embodiment, R ¹ is deuterium.

In another preferred embodiment, R ² is deuterium.

In another preferred embodiment, R ¹ and R ² are deuterium.

As a preferred embodiment of the invention, R ³ and R ⁴ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ³ is deuterium.

In another preferred embodiment, R ³ and R ⁴ are deuterium.

As a preferred embodiment of the invention, R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁵ , R ⁶ , R ⁷ and R ⁸ are deuterium.

As a preferred embodiment of the invention, R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ⁹ is deuterium.

In another preferred embodiment, R ¹⁰ is deuterium.

In another preferred embodiment, R ¹¹ is hydrazine.

In another preferred embodiment, R ⁹ , R ¹⁰ and R ¹¹ are deuterium.

In another preferred embodiment, R ¹² is deuterium.

In another aspect, the present invention also discloses a process for the preparation of an amide compound of the formula (I), characterized in that it comprises a substituted or unsubstituted

The step of reacting as an intermediate with a substituted or unsubstituted 2-aminothiazoline under basic conditions.

As a preferred embodiment of the present invention, the base used in the production method is selected from the group consisting of potassium carbonate, sodium carbonate, sodium hydrogencarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, N, N-di At least one of isopropylethylamine, 4-N,N-lutidine or pyridine.

In another aspect, the present invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an amide compound as described above, or a polymorphic form thereof, a pharmaceutically acceptable salt, or a prodrug , stereoisomers, isotopic variations, hydrates or solvates.

In another aspect, the present invention also discloses a process for the preparation of a pharmaceutical composition as described above, comprising the steps of: pharmaceutically acceptable excipients with an amide compound as described above, or a polymorph thereof The pharmaceutically acceptable salts, prodrugs, stereoisomers, isotopic variations, hydrates or solvates are combined 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, which is cancer, cardiovascular disease, inflammation, infection, immune disease, cell proliferative disease, viral disease. , a metabolic disease, or a drug for organ transplantation.

In another aspect, the invention provides a compound of the first aspect of the invention, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent thereof Use of the compound for the preparation of a medicament for the treatment and/or prevention of a disease associated with a protein kinase.

In another aspect, the invention provides a method of treating and/or preventing a protein kinase-associated disease in a subject, the method comprising administering to the subject an amide of formula (I) A compound having a polymorphic form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound, or a pharmaceutical composition thereof.

In another aspect, the present invention provides a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent compound of the amide compound of formula (I), or A pharmaceutical composition for treating and/or preventing a disease associated with a protein kinase.

In another embodiment, the compound or pharmaceutical composition is for treating and/or preventing a disease: cancer, cell proliferative disease, inflammation, infection, immune disease, organ transplantation, viral disease, cardiovascular disease Or metabolic disease.

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, blood cancer , osteosarcoma, melanoma, kidney cancer, stomach cancer, liver cancer, bladder cancer, thyroid cancer or colorectal cancer.

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

In another embodiment, the cell proliferative disorder refers to lung cancer, head and neck cancer, breast cancer, prostate cancer, esophageal cancer, rectal cancer, colon cancer, nasopharyngeal cancer, uterine cancer, pancreatic cancer, lymphoma, blood cancer. , osteosarcoma, melanoma, kidney cancer, stomach 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 provides a kit comprising: a first container comprising an amide compound of the formula (I), a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, An isotope variant, hydrate or solvent compound; and, optionally, a second container containing other therapeutic agent; and, optionally, a third container containing for diluting or suspending the compound and/or other therapeutic agent A pharmaceutically acceptable excipient.

In another aspect, the invention also discloses the use of an amide compound as described above for the preparation of a pharmaceutical composition for inhibiting protein kinases. Preferably, it is used to prepare a pharmaceutical composition that inhibits EGFR kinase.

The compound of formula (I) of the present invention, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotope variant, hydrate or solvate thereof, can treat or prevent abnormally activated B lymphocytes A cancer, a tumor, an inflammatory disease, an autoimmune disease, or an immune-mediated disease caused by cells, T lymphocytes, or both. Accordingly, the present invention also provides a pharmaceutical composition for treating and/or preventing cancer, a tumor, an inflammatory disease, an autoimmune disease or an immune-mediated disease, comprising the compound of the formula (I) of the present invention or Polymorphic forms, pharmaceutically acceptable salts, prodrugs, stereoisomers, isotopic variations, hydrates or solvates are included as active ingredients.

The invention also includes isotopically labeled compounds (also referred to as "isotopic variants"), equivalent to the original compounds disclosed herein. Examples of isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, respectively. , ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. A compound of the formula (I) of the present invention containing the above isotope or other isotopic atom, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof It is within the scope of the invention. Certain isotopically-labeled compounds of the present invention, such as the radioisotopes of ³ H and ¹⁴ C, are also among them, useful in tissue distribution experiments of drugs and substrates.氚, ie ³ H and carbon-14, ie ¹⁴ C, are easier to prepare and detect and are preferred in isotopes. In addition, heavier isotopic substitutions such as guanidine, or ² H, are preferred in certain therapies due to their good metabolic stability, such as increased half-life or reduced dosage in vivo, and therefore may be preferred in certain circumstances. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.

It should be understood that within the scope of the present invention, the above various technical features, embodiments, and technical features specifically described in the following (such as the embodiments) may be combined with each other to constitute a new or preferred technique. Program. Due to space limitations, we will not repeat them here.

Compared with the prior art, the beneficial effects of the present invention are as follows: First, the amide compound using the technical scheme of the present invention has excellent inhibitory effect on protein kinase. Second, the metabolism of the compound in the organism is improved, giving the compound better pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability. Third, the drug concentration of the compound in the animal is increased, and the drug efficacy is improved. Fourth, certain metabolites are inhibited and the safety of the compounds is increased.

definition:

When a range of values is recited, each value and sub-range within the range are intended to be included. For example, "C ₁ -C ₆ alkyl" includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₁ -C ₆ , C ₁ -C ₅ , C ₁ -C ₄ , C ₁ - C ₃ , C ₁ -C ₂ , C ₂ -C ₆ , C ₂ -C ₅ , C ₂ -C ₄ , C ₂ -C ₃ , C ₃ -C ₆ , C ₃ -C ₅ , C ₃ -C ₄ C ₄ -C ₆ , C ₄ -C ₅ and C ₅ -C ₆ alkyl.

It will be understood that any of the moieties defined below may be substituted by a number of substituents, as described herein, and the corresponding definitions are within the scope of their inclusion, including such substitutions. Unless otherwise stated, the term "substituted" is as defined below.

"Halogen" means fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "polymorph" refers to a different arrangement of chemical drug molecules, generally expressed as the presence of a pharmaceutical material in a solid state. A drug may exist in a plurality of crystalline forms, and different crystal forms of the same drug may have different dissolution and absorption in the body, thereby affecting the dissolution and release of the formulation.

The term "pharmaceutically acceptable salt" means that, within the scope of sound medical judgment, it is suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergies, etc., and with reasonable benefits/dangers. Those salts that are proportionate. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., pharmaceutically acceptable salts as described in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and inorganic and organic 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 salts with organic acids such as acetic acid, oxalic acid, Maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also included are salts formed using conventional methods in the art, for example, ion exchange methods. Other pharmaceutically acceptable salts include: adipic acid salts, alginate, ascorbate, aspartate, besylate, benzoate, disulfate, borate, butyrate, camphor Acid salt, camphor sulfonate, citrate, cyclopentanoate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerol Phosphate, 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 Salt, pectin ester, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, Thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from suitable bases include the alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. If appropriate, other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed with counterions, counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, Nitrate, lower alkyl sulfonate and aryl sulfonate.

"Subjects" for administration include, but are not limited to, humans (ie, males or females of any age group, eg, pediatric subjects (eg, infants, children, adolescents) or adult subjects (eg, young Adults, middle-aged adults or older adults) and/or non-human animals, for example, mammals, for example, primates (eg, cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep , goats, rodents, cats and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "person," "patient," and "subject" are used interchangeably herein.

"Disease," "disorder," and "disorder" are used interchangeably herein.

The term "treating" as used herein includes the effect that occurs when a subject has a particular disease, disorder or condition that 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 the effect that occurs before a subject begins to have 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 "stereoisomer" forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be in the form of individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, A racemic mixture and a mixture rich in one or more stereoisomers are included. The isomers can be separated from the mixture by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of a chiral salt; or preferred isomers can be passed Prepared by asymmetric synthesis.

Those skilled in the art will appreciate that many organic compounds can form complexes with solvents in which they react or precipitate or crystallize from the solvent. 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 invention.

In addition, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound which is converted in vivo to an active form thereof having a medical effect by, for example, hydrolysis in blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, ACSSymposium 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. Barbra "Improved oral drug delivery: Solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, each This article is incorporated herein by reference.

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

"Pharmaceutically acceptable excipient" as used in the present invention refers to a non-toxic carrier, adjuvant or vehicle which does not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin) ), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated plant fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, Sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based material, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-embedded Segment polymer, polyethylene glycol and lanolin.

Detailed ways

Compound

The present invention relates to an amide compound of the formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotope variant, hydrate or solvent compound thereof:

among them,

In a particular embodiment, "R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each independently selected from the group consisting of hydrogen and deuterium. , halogen or trifluoromethyl" includes R ¹ selected from hydrogen, deuterium, halogen or trifluoromethyl, R ^{2 is} selected from hydrogen, deuterium, halogen or trifluoromethyl, and R ^{3 is} selected from hydrogen, deuterium, halogen or tri fluoromethyl, and so on until ²⁰ is selected from hydrogen, deuterium, halogen or trifluoromethyl aspect R. More specifically, R ¹ is hydrogen, R ¹ is deuterium, R ¹ is halogen (F, Cl, Br or I) or R ¹ is trifluoromethyl, R ² is hydrogen, R ² is deuterium, and R ² is Halogen (F, Cl, Br or I) or R ² is trifluoromethyl, R ³ is hydrogen, R ³ is deuterium, R ³ is halogen (F, Cl, Br or I) or R ³ is trifluoromethyl And so on, until R ²⁰ is hydrogen, R ²⁰ is deuterium, R ²⁰ is halogen (F, Cl, Br or I) or R ²⁰ is a trifluoromethyl group.

In a preferred embodiment, the invention relates to an amide compound of formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent compound thereof, Wherein R ² , R ⁹ and R ¹¹ are hydrogen, and R ¹ , R ³ -R ⁸ , R ¹⁰ and R ¹² are each independently selected from hydrogen or hydrazine.

In a preferred embodiment, the invention relates to an amide compound of formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent compound thereof, Wherein R ² , R ⁹ and R ¹¹ are hydrogen, R ¹ is hydrazine, and R ^{3 to} R ⁸ , R ¹⁰ and R ¹² are each independently selected from hydrogen or hydrazine.

In a preferred embodiment, R ³ and R ⁴ are the same.

In a preferred embodiment, R ³ and R ⁴ are deuterium.

In a preferred embodiment, R ³ is deuterium.

In a preferred embodiment, R ^{5 -} R ⁸ are the same. In a preferred embodiment, R ¹⁰ is deuterium.

In a preferred embodiment, the amide compound is any 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 in accordance with the present invention. However, the invention is not limited to the following pharmaceutical compositions.

Exemplary Formulation 1 - Tablet: The compound of the present invention in dry powder form can be mixed with the dried 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 into 0.3-30 mg tablets (each tablet contains 0.1-10 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 2 - Tablet: The compound of the present invention in dry powder form can be mixed with the dried gel binder in a weight ratio of about 1:2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is formed into a 30-90 mg tablet (each tablet contains 10-30 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 3 - Tablet: The compound of the present invention in dry powder form can be mixed with the dried 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 into 90-150 mg tablets (30-50 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 4-Tablet: The compound of the invention in dry powder form can be combined with the dried gel binder in a weight ratio of about 1:2. A smaller amount of magnesium stearate was added as a lubricant. The mixture is formed into a 150-240 mg tablet (each tablet contains 50-80 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 5 - Tablet: The compound of the invention in dry powder form can be combined with the dried 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 into 240-270 mg tablets (each tablet contains 80-90 mg of active compound per tablet) in a tablet press.

Exemplary Formulation 6-Tablet: The compound of the invention in dry powder form can be combined with the dried 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 into a 270-450 mg tablet (each tablet contains 90-150 mg of active compound) in a tablet press.

Exemplary Formulation 7-Tablet: The compound of the invention in dry powder form can be combined with the dried 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 into 450-900 mg tablets (each tablet contains 150-300 mg of active compound per tablet) in a tablet press.

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

Exemplary Formulation 9-Liquid: The compound of the present invention (125 mg) can be mixed with sucrose (1.75 g) and xanthan gum (4 mg), and the resulting mixture can be blended, passed through a No. 10 mesh U.S. sieve, and then It was mixed with an aqueous solution of microcrystalline cellulose and sodium carboxymethylcellulose (11:89, 50 mg) prepared in advance. Sodium benzoate (10 mg), flavor and color are diluted with water and added with stirring. Then, sufficient water can be added to give a total volume of 5 mL.

Exemplary Formulation 10 - Injection: The compound of the invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of about 5 mg/mL.

Administration

The pharmaceutical composition provided by the present invention can be administered by a variety of routes including, but not limited to, oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration. Drug, administration by implant or other means of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intra-articular administration, intra-arterial administration, intrasynovial administration, intrasternal administration. , intracerebroventricular administration, intralesional administration, and intracranial injection or infusion techniques.

Generally, an effective amount of a compound provided herein is administered. Depending on the circumstances, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc., the amount of compound actually administered can be determined by the physician. .

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

The pharmaceutical compositions provided herein ("long-term administration") can also be administered chronically. Long-term administration refers to administration of a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or can be continuously administered indefinitely, For example, the rest of the subject. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a prolonged period of time, for example, within a therapeutic window.

The pharmaceutical composition of the present invention can be further delivered using various methods of administration. For example, in some embodiments, a pharmaceutical composition can be administered by bolus injection, for example, in order to rapidly increase the concentration of the compound in the blood to an effective level. The bolus dose will depend on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous bolus dose will allow the active component to be slowly released, while a bolus delivered directly to the vein (eg, via IV IV drip) can be more Delivered rapidly so that the concentration of the active ingredient in the blood is rapidly increased to an effective level. In other embodiments, the pharmaceutical composition can be administered in a continuous infusion form, for example, by IV intravenous drip to provide a steady state concentration of the active ingredient in the subject's body. Moreover, in other embodiments, a bolus dose of the pharmaceutical composition can be administered first, followed by continued infusion.

Oral compositions can be in the form of a bulk liquid solution or suspension or bulk powder. More generally, however, the composition is provided in unit dosage form for ease of precise dosing. 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 ingredient suitable to produce the desired therapeutic effect with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, pre-measured ampoules or syringes of the liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being useful for forming the desired form of administration. A carrier or excipient and a processing aid.

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

In order to provide a blood level similar to the use of an injectable dose, or a lower blood level than the injectable 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 20% by weight, preferably about 0.1. To about 10% by weight, and more preferably from about 0.5 to about 15% by weight.

The injection dose level ranges from about 1 mg/kg/hr to at least 10 mg/kg/hr from about 1 to about 120 hours, especially 24 to 96 hours. In order to obtain a sufficient level of steady state, a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more can also be administered. For a human patient of 40 to 80 kg, the maximum total dose cannot exceed about 2 g/day.

Liquid forms suitable for oral administration may include suitable aqueous or nonaqueous vehicles as well as buffers, suspending and dispersing agents, coloring agents, flavoring agents, and the like. The solid form may include, for example, any of the following components, or a compound having similar properties: a binder, for example, microcrystalline cellulose, tragacanth or gelatin; an excipient such as starch or lactose, a disintegrant, For example, alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silica; a sweetener such as sucrose or saccharin; or a flavoring agent such as mint, water Methyl salicylate or orange flavoring.

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

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

The compounds of the invention may also be administered by transdermal means. Thus, transdermal administration can be accomplished using a reservoir or a porous membrane type, or a patch of a plurality of solid matrices.

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

The compounds of the 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 further 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 alpha-, beta- and gamma-cyclodextrins consisting of 6, 7 and 8 alpha-1,4-linked glucose units, respectively, optionally including one on the attached sugar moiety. Or a plurality of substituents including, but not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substituted. In some embodiments, the cyclodextrin is a sulfoalkyl ether beta-cyclodextrin, eg, sulfobutylether beta-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645. In some embodiments, the formulation comprises hexapropyl-β-cyclodextrin (eg, 10-50% in water).

Example

Preferred embodiments of the invention are further described in detail below. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

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

Example 1

Preparation of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl)-N-(thiazol-2-yl-5-d)B by the following synthetic route The amide (Compound 11) comprises the following steps:

Step 1: Synthesis of Compound 3.

4 mL of methanesulfonic acid was added to the reaction flask, cooled to 0 ° C, α-hydroxy hippuric acid (Compound 1, 1 g, 5.12 mmol) was added, and 4-fluoroanisole (646 mg, 5.12 mmol) was added at 0 ° C, room temperature. After stirring for 1 hour, the reaction mixture was slowly added dropwise to ice water, washed with a white solid, filtered, and the filter cake was washed with water and dried in vacuo to give compound 3 (1.3 mg, yield 86.6%), LC-MS (APCI) ): m/z=302(M-1) ^- .

Step 2: Synthesis of Compound 4.

Compound 3 (1 g, 3.3 mmol) was added to the reaction flask, 60 mL of 6N hydrochloric acid was added, the temperature was raised to 100 ° C, and the reaction was carried out for 36 hours. The reaction of the starting material was completely detected by TLC (thin layer chromatography), cooled to room temperature, filtered, and the filtrate was concentrated to give white. The solid was slurried with ethyl acetate and purified compound 4 (200mg, yield 30.4%), LC-MS ( APCI): m / z = 198 (m-1) -.

Step 3: Synthesis of Compound 5.

Under a nitrogen atmosphere, the compound 4 (200 mg, 1 mmol) and o-phthalaldehyde (134 mg, 1 mmol) were dissolved in 3 mL of acetic acid under nitrogen atmosphere, and the mixture was heated to 120 ° C for 1 hour, cooled to room temperature, and concentrated to give a crude compound. (632 mg, yield 100%) was used directly in the next step without further purification, LC-MS (APCI): m / z = 314 (m-1) -.

Step 4: Synthesis of Compound 7.

2-Aminothiazole (500 mg, 2.9 mmol) was added to the reaction flask, and 20 mL of tetrahydrofuran (THF) was added thereto. Boc anhydride (1.308 g, 6 mmol) was added at room temperature, and the reaction was carried out for 16 hours at room temperature. The reaction of the starting material was completely confirmed by TLC. Boc anhydride, purified by column chromatography to give compound 6 (859 mg, yield 85.9%)

Step 5: Synthesis of Compound 8.

Compound 7 (200 mg, 1 mmol) was added to the reaction flask, 20 mL of tetrahydrofuran was added, and the mixture was cooled to 0 ° C. n-butyllithium (0.52 mL, 1.3 mmol) was added dropwise at 0 ° C, and the mixture was stirred at 0 ° C for 1 hour after completion of the dropwise addition. The reaction was quenched with 4 mL of EtOAc (EtOAc) (EtOAc). z=399(M+1) ⁺ .

Step 6: Synthesis of Compound 9.

Compound 8 (200 mg, 1 mmol) was added to the reaction flask, and 12 mL of dichloromethane was added to dissolve. Trifluoroacetic acid (570 mg, 5 mmol) was added at room temperature, and the mixture was reacted at room temperature for 3 hours. The reaction of the starting material by TLC was adjusted to pH with 1 M hydrochloric acid. weakly basic, the solvent is concentrated out directly in the next ^{reaction, 1 H NMR (500MHz, MeOD} ) (δ / ppm) 7.19 (s, 1H), 6.88 (d, J = 4.2Hz, 0.07H).

Step 7: Synthesis of Compound 10.

The crude product of compound 5 (688 mg, 2.17 mmol) and compound 9 (282 mg, 2.812 mmol), N,N-diisopropylethylamine (DIPEA, 279 mg, 2.175 mmol), 2-(7-even) were added to the reaction flask. Nitrobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.07 g, 2.812 mmol), added with dimethylformamide (DMF, 20 mL), nitrogen The reaction was carried out at 25 ° C for 3 hours, and the reaction of the starting material was completed by TLC. 50 mL of water was added to the reaction mixture, and the mixture was extracted 4 times with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. after purification to give compound 9 (227mg, yield 26.3%), LC-MS ( APCI): m / z = 399 (m + 1) +.

Step 8: Synthesis of Compound 11.

Compound 10 (200 mg, 0.5 mmol) was added to the reaction flask, 30 mL of dichloromethane was added, and the mixture was cooled to -10 ° C, and boron tribromide (BBr ₃ , 500 mg, 2.1 mmol) was added dropwise to the reaction flask. The reaction was carried out to room temperature for 2 hours, and the reaction of the starting material was completed by TLC. 30 mL of water was added to the reaction flask, and the mixture was extracted 4 times with dichloromethane. The organic phase was combined and dried over anhydrous sodium sulfate. 11 (64mg, yield 33.3%), LC-MS ( APCI): m / z = 383 (m-1) -. ¹ H NMR (500 MHz, DMSO) (δ / ppm) 12.59 (s, 1H), 9.94 (s, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H) , 7.56 (d, J = 7.4 Hz, 1H), 7.50 (t, J = 6.3 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.10 (td, J = 8.6, 3.1 Hz, 1H) , 6.89 (dd, J=8.9, 4.8 Hz, 1H), 6.84 (dd, J=9.2, 3.0 Hz, 1H), 6.30 (s, 1H), 4.60 (d, J = 17.4 Hz, 1H), 3.97 ( d, J = 17.5 Hz, 1H).

Example 2

Preparation of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl-3-d)-N-(thiazol-2-yl)B by the following synthetic route The amide (Compound 21) comprises the following steps:

Step 1: Synthesis of Compound 12.

Compound 4 (2.08 g, 1045 mmol) was added to the reaction flask, 30 mL of methanol was added thereto, and the mixture was cooled to 0 ° C. Thionyl chloride (1.5 mL, 20.9 mmol) was added dropwise, and the mixture was added to 75 ° C for 3 hours. After completion of the reaction, the methanol and excess thionyl chloride were removed by concentration, and 20 mL of methyl t-butyl ether was added to give a compound 12 (2.15 g, yield: 98.4%), LC-MS (APCI): m/z = 214 (M) +1) ⁺ .

Step 2: Synthesis of Compound 14.

Compound 13 (2 g, 12 mmol) was added to the reaction flask, and 10 mL of thionyl chloride was added thereto at 0 ° C, and the reaction was refluxed for 3 hours, and the excess chlorosulfoxide was concentrated to give compound 14 (2.74 g, yield 100%).

Step 3: Synthesis of Compound 15.

Triethylamine (TEA, 1.5 g, 14.6 mmol) was added to a reaction flask, DMF (20 mL) was added, and Compound 14 (1.04 g, 4.88 mmol) was dissolved in DMF (15 mL) and Compound 12 was dissolved in DMF (15 mL) In the reaction flask, it was added dropwise to the reaction flask at room temperature, and reacted at room temperature for 3 hours. The reaction of the starting material was completely confirmed by TLC. After concentration, 2.3 g of the crude compound 15 was obtained, which was directly used for the next reaction, without further purification, LC-MS (APCI) ): m/z=362(M+1) ⁺ .

Step 4: Synthesis of Compound 16.

2.3 g of crude compound 15 was added to the reaction flask, and 15 mL of thionyl chloride was added at 0 ° C. After the completion of the dropwise addition, the reaction was carried out to 50 ° C for 5 hours, and the reaction of the starting material was completely confirmed by TLC, and the excess thionyl chloride was concentrated. purified by column chromatography to give a white 16 (1.244g, 73.16% yield for two steps) as a solid compound, LC-MS (APCI): m / z = 344 (m + 1) +.

Step 5: Synthesis of Compound 17.

Compound 16 (1.244 g, 3.568 mmol) was added to the reaction flask, and zinc powder (2.3 g, 35.68 mmol) was added to 10 mL of acetic acid, and the mixture was heated to 110 ° C for 2 hours. The reaction of the starting material was completely detected by TLC, and excess zinc powder was removed by filtration and concentrated. The acetic acid was removed, 40 mL of ethyl acetate was added, and the mixture was washed with saturated aqueous sodium hydrogen carbonate, and the organic phase was concentrated and purified by column chromatography to give compound 17 (315 mg, yield: 25.5%), LC-MS (APCI): m/z = 347 (M+1) ⁺ .

Step 6: Synthesis of Compound 18.

Compound 17 (314 mg, 0.957 mmol), trifluoroacetic acid (TFA, 218 mg, 1.914 mmol) was dissolved in 20 mL dichloromethane, cooled to 0 ° C, and triethylsilane (333 mg, 2.872 mmol) was slowly added dropwise. After the completion of the dropwise addition, the reaction was carried out at 0 ° C for 2 hours, and the reaction of the starting material was completed by TLC. Water (20 mL) was added to the reaction flask, and the mixture was extracted three times with dichloromethane. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. after purification by column chromatography to give compound 18 (109mg, yield 34.7%), LC-MS ( APCI): m / z = 331 (m + 1) +.

Step 7: Synthesis of Compound 19.

Compound 18 (109 mg, 0.3313 mmol) was added to the reaction flask, lithium hydroxide (30 mg, 1.7 mmol) was added, water (1 mL), tetrahydrofuran (THF, 4 mL) was added, and the reaction was carried out for 3 hours at room temperature. adjusted with 1M HCl to pH 5-6, and concentrated to give 316mg crude compound 19 was used directly in the next reaction without further purification, LC-MS (APCI): m / z = 362 (m-1) -.

Step 8: Synthesis of Compound 20.

The crude product of compound 19 (316 mg, 0.33 mmol), 2-aminothiophene (43 mg, 0.403 mmol), DIPEA (55 mg, 0.403 mmol), HATU (163 mg, 0.403 mmol), DMF (10 mL), nitrogen The reaction was carried out under the protection of 25 ° C for 18 hours, and the reaction of the starting material was completed by TLC. 50 mL of water was added to the reaction mixture, and the mixture was extracted 4 times with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. after analysis to give compound 20 (154mg), LC-MS (APCI): m / z = 399 (m + 1) +.

Step 9: Synthesis of Compound 21.

Compound 20 (154 mg, 0.387 mmol) was added to the reaction flask, and 10 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (1.5 mL, 1.16 mmol) was added dropwise to the reaction flask. The mixture was reacted to room temperature for 2 hours, and the reaction was completed by TLC. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography. , yield 62.2%), LC-MS (APCI): m/z = 385 (M+1) ⁺ ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.57 (s, 1H), 9.92 (s, 1H), 7.94 (s, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.1 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.48 - 7.42 (m, 1H), 7.09 (t, J = 7.0 Hz, 1H), 6.89 (dd, J = 8.9, 4.7 Hz, 1H), 6.84 (d, J = 9.2) Hz, 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-oxoisoindoline-2-yyl-3,3-d ₂ )-N-(thiazole- ₂ ) using the following synthetic route -yl)acetamide (compound 25), comprising the following steps:

Step 1: Synthesis of Compound 22.

Compound 16 (1 g, 2.91 mmol) was added to the reaction flask, and zinc powder (10 g, 152 mmol) was added in 5 portions, and 10 mL of deuterated acetic acid was added thereto, and the mixture was heated to 110 ° C for 7 hours, and the reaction of the starting material was completely detected by TLC, and excess was filtered off. Zinc powder, concentrated to remove acetic acid, 40 mL of ethyl acetate was added, washed with saturated sodium bicarbonate solution, and the organic phase was concentrated and purified by column chromatography to give compound 22 (315 mg, yield 26.1%), LC-MS (APCI): m /z=332(M+1) ⁺ .

Step 2: Synthesis of Compound 23.

Add compound 22 (252 mg, 0.76 mmol) to a reaction flask, add lithium hydroxide (159 mg, 3.8 mmol), add water (1 mL), tetrahydrofuran (5 mL), and react for 3 hours at room temperature. hydrochloric acid to adjust to pH 5-6, and concentrated to give the crude compound 340mg 23, was used directly in the next reaction without further purification, LC-MS (APCI): m / z = 316 (m-1) -.

Step 3: Synthesis of Compound 24.

The crude product of compound 23 (340 mg, 0.76 mmol), 2-aminothiophene (100 mg, 0.968 mmol), DIPEA (332 mg, 2.57 mmol), HATU (374 g, 0.968 mmol), DMF (10 mL), The reaction was carried out at 25 ° C for 5 hours under a nitrogen atmosphere, and the reaction of the starting material was completed by TLC. 40 mL of water was added to the reaction mixture, and the mixture was extracted 4 times with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. chromatography to give compound 24 (125mg, yield for two steps 41.2%), LC-MS ( APCI): m / z = 400 (m + 1) +.

Step 4: Synthesis of Compound 25.

Compound 24 (125 mg, 0.312 mmol) was added to the reaction flask, and 15 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (1.6 mL, 1.6 mmol) was added dropwise to the reaction flask. The mixture was reacted to room temperature for 2 hours, and the reaction of the starting material was completed by TLC. Water (30 mL) was added to the reaction flask, and the mixture was extracted with methylene chloride for 4 times. The organic phase was combined and dried over anhydrous sodium sulfate. 53 mg, yield 44.1%), LC-MS (APCI): m/z = 386 (M+1) ⁺ ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.58 (s, 1H), 9.93 (s , 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H) ), 7.48 - 7.45 (m, 1H), 7.25 (d, J = 3.5 Hz, 1H), 7.09 (td, J = 8.6, 3.1 Hz, 1H), 6.89 (dd, J = 8.9, 4.8 Hz, 1H) , 6.84 (dd, J = 9.2, 3.0 Hz, 1H), 6.30 (s, 1H).

Example 4

The following synthetic route was used to prepare 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl-4,5,6,7-d ₄ )-N-( Thiazol-2-yl)acetamide (Compound 34), comprising the following steps:

Step 1: Synthesis of Compound 27.

Compound 26 (1 g, 5.88 mmol) was added to the reaction flask, 10 mL of thionyl chloride was added at 0 ° C, and the reaction was refluxed for 3 hours. The excess thionyl chloride was removed to give compound 27 (1.05 g, yield -100%).

Step 2: Synthesis of Compound 28.

To the reaction flask was added triethylamine (1.56 g, 15.48 mmol), DMF (20 mL), Compound 12 (1.1 g, 5.16 mmol) dissolved in DMF (15 mL) Soluble in DMF (15mL), add dropwise to the reaction flask at room temperature, react at room temperature for 3 hours, complete the reaction of the starting material by TLC, concentrate to obtain 2.4g of crude compound 28, directly used in the next step, LC-MS (APCI ): m/z=366(M+1) ⁺ .

Step 3: Synthesis of Compound 29.

2.4 g of crude compound 28 was added to the reaction flask, and 15 mL of thionyl chloride was added at 0 ° C. After the completion of the dropwise addition, the reaction was carried out to 50 ° C for 5 hours. The reaction of the starting material was completely confirmed by TLC, and the excess thionyl chloride was concentrated. , a white solid was purified by column chromatography to give compound 29 (545mg, yield for two steps 30.1%), LC-MS ( APCI): m / z = 348 (m + 1) +.

Step 4: Synthesis of Compound 30.

Compound 29 (545 mg, 1.57 mmol) was added to the reaction flask, zinc powder (1.02 g, 15.7 mmol) was added, 15 mL of acetic acid was added, and the mixture was heated to 110 ° C for 5 hours. The reaction of the starting material was completely detected by TLC, and excess zinc powder was removed by filtration. The acetic acid was concentrated to remove acetic acid, and ethyl acetate (40 mL) was added and washed with saturated sodium hydrogen carbonate solution, and the organic phase was concentrated and purified by column chromatography to give compound 30 (165 mg, yield 31.7%), LC-MS (APCI): m/z = 350 (M+1) ⁺ .

Step 5: Synthesis of Compound 31.

Compound 30 (165 mg, 0.498 mmol), trifluoroacetic acid (113 mg, 0.997 mmol) was dissolved in 20 mL of dichloromethane, cooled to 0 ° C, and triethylsilane (173 mg, 1.495 mmol) was slowly added dropwise. After the completion of the dropwise addition, the reaction was carried out at 0 ° C for 2 hours, and the reaction of the starting material was completed by TLC. 20 mL of water was added to the reaction flask, and the mixture was extracted three times with dichloromethane. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. chromatography to give compound 31 (97mg, yield 58.7%), LC-MS ( APCI): m / z = 334 (m + 1) +.

Step 6: Synthesis of Compound 32.

Compound 31 (97 mg, 0.292 mmol) was added to the reaction flask, lithium hydroxide (61 mg, 1.46 mmol) was added, water (1 mL), tetrahydrofuran (5 mL) was added, and the reaction was carried out for 3 hours at room temperature. hydrochloric acid to adjust to pH 5-6, and concentrated to give 120mg crude compound 32 was used directly in the next reaction without further purification, LC-MS (APCI): m / z = 318 (m-1) -.

Step 7: Synthesis of Compound 33.

Compound 32 (120 mg, 0.292 mmol), 2-aminothiophene (30 mg, 0.38 mmol), DIPEA (100 mg, 0.76 mmol), HATU (144 mg, 0.38 mmol), DMF (5 mL) The reaction was carried out at 25 ° C for 6 hours, and the reaction of the starting material was completed by TLC. 50 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. to give the compound 33 (45mg, yield ~ 38.4%), LC-MS (APCI): m / z = 402 (m + 1) +.

Step 8: Synthesis of Compound 34.

Compound 33 (154 mg, 0.387 mmol) was added to the reaction flask, and 10 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (1.5 mL, 1.16 mmol) was added dropwise to the reaction flask. The mixture was reacted to room temperature for 2 hours, and the reaction was completed by TLC. Water (30 mL) was added to the reaction flask, and the mixture was extracted with methylene chloride for 4 times. The combined organic phases were dried over anhydrous sodium sulfate and purified by column chromatography. , yield 62.2%), LC-MS (APCI): m/z = 388 (M+1) ⁺ ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.58 (s, 1H), 9.92 (s, 1H), 7.47 (d, J = 3.4 Hz, 1H), 7.25 (s, 1H), 7.09 (t, J = 7.0 Hz, 1H), 6.94 - 6.80 (m, 2H), 6.30 (s, 1H), 4.60 (d, J = 17.3 Hz, 1H), 3.97 (d, J = 17.4 Hz, 1H).

Example 5

Preparation of the amine 2-(5-fluoro-2-hydroxyphenyl-3-d)-2-(1-oxoisoindoline-2-yl)-N-(thiazol-2-yl) by the following synthetic route Acetamide (Compound 42), including the following steps:

Step 1: Synthesis of Compound 35.

To the reaction flask, p-fluoroanisole (400 mg, 3.17 mmol) was dissolved in 30 mL of dichloromethane, cooled to -10 ° C, and boron tribromide (1.44 g, 15.86 mmol) was added dropwise to the reaction. The reaction was carried out for 3 hours, and the reaction of the starting material was completed by TLC. 20 mL of water was added, and the mixture was extracted three times with dichloromethane. The organic phase was combined, dried over anhydrous sodium sulfate and purified by column chromatography to give compound 35 (400 mg, yield -100%), LC - MS (APCI): m/z = 113 (M + 1) ⁺ .

Step 2: Synthesis of Compound 36.

Compound 35 (0.4 g, 3.57 mmol) and sodium hydroxide (71 mg, 1.78 mmol) were added to the reaction flask, dissolved in 10 mL of heavy water, heated in a microwave to 180 ° C for 1 hour, cooled to room temperature and extracted three times with dichloromethane. The combined organic phase was dried over anhydrous sodium sulfate, and concentrated to give compound 36 (400mg, yield ~ 100%), LC-MS (APCI): m / z = 115 (m + 1) +.

Step 3: Synthesis of Compound 37.

Compound 36 (0.4 g, 3.57 mmol), potassium carbonate (1.323 g, 8.9 mmol) was added to the reaction mixture, and DMF (15 mL) was added, and methylene chloride (1.25 g, 8.9 mmol) was added at room temperature, and stirred at room temperature for 5 hours, TLC The reaction of the starting material was completed, 30 mL of water was added to the reaction, and the mixture was extracted three times with ethyl acetate. The organic phase was combined, dried over anhydrous sodium sulfate and concentrated to give compound 37 (400 mg, yield 89%), LC-MS (APCI) :m/z=129(M+1) ⁺ .

Step 4: Synthesis of Compound 38.

4 mL of methanesulfonic acid was added to the reaction flask, and the mixture was cooled to 0 ° C. Compound 37 (426 mg, 3.33 mmol) was added, and α-hydroxy hippuric acid (650 mg, 3.33 mmol) was added at 0 ° C, and the mixture was stirred at room temperature for 1 hour. The mixture was slowly added dropwise to ice water, washed with a white solid, filtered, and the filter cake was washed with water and dried in vacuo to give compound 38 (345 mg, yield 34.15%), LC-MS (APCI): m/z = 303 ( M-1) ^- .

Step 5: Synthesis of Compound 39.

Compound 38 (650 mg, 2.14 mmol) was added to the reaction flask, 60 mL of 6N hydrochloric acid was added, the temperature was raised to 100 ° C, and the reaction was carried out for 48 hours. The reaction of the starting material was completed by TLC, cooled to room temperature, filtered, and the filtrate was concentrated to give a white solid. Ester ester purification gave compound 39 (345 mg, yield 80.7%). LC-MS (APCI): m / z = 199 (M-1) -.

Step 6: Synthesis of Compound 40.

Under a nitrogen atmosphere, the compound 39 (345 mg, 1.725 mmol), o-phthalaldehyde (231 mg, 1.725 mmol) was dissolved in 6 mL of acetic acid, and the mixture was heated to 120 ° C for 1 hour, cooled to room temperature, and concentrated to give compound 40. the crude product (688 mg, yield 100%) was used directly in the next step without further purification, LC-MS (APCI): m / z = 315 (m-1) -.

Step 7: Synthesis of Compound 41.

The crude product of compound 40 (688 mg, 2.17 mmol) and 2-aminothiazole (282 mg, 2.812 mmol), DIPEA (9279 mg, 2.175 mmol), HATU (1.07 g, 2.812 mmol) was added to the reaction flask, and DMF (20 mL) was added. The reaction was carried out at 25 ° C for 3 hours under a nitrogen atmosphere, and the reaction of the starting material was completed by TLC. 50 mL of water was added to the reaction mixture, and the mixture was extracted 4 times with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. chromatography to give compound 41 (227mg, yield 26.3%), LC-MS ( APCI): m / z = 399 (m + 1) +.

Step 8: Synthesis of Compound 42.

Compound 41 (227 mg, 0.569 mmol) was added to the reaction flask, 30 mL of dichloromethane was added, and the mixture was cooled to -10 ° C, and boron tribromide (713 mL, 2.85 mmol) was added dropwise to the reaction flask. The mixture was reacted at room temperature for 2 hours, and the reaction of the starting material was completed by TLC. Water (30 mL) was added to the reaction flask, and the mixture was extracted with methylene chloride. , yield 68.9), LC-MS (APCI): m/z = 383.0 (M-1) ^- ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.58 (s, 1H), 9.92 (s, 1H) ), 7.72 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.49-7.45 (m, 1H), 7.25 (d, J = 3.3 Hz, 1H), 7.09 (dd, J = 8.3, 3.1 Hz, 1H), 6.84 (dd, J = 9.2, 3.0 Hz, 1H), 6.30 (s, 1H), 4.60 (d, J = 17.4 Hz, 1H), 3.98 (d, J = 17.5 Hz, 1H).

Example 6

Preparation of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl)-N-(thiazol-2-yl)acetamide-2- using the following synthetic route d (Compound 46), comprising the following steps:

Step 1: Synthesis of Compound 43.

Compound 4 (800 mg, 4 mmol) was added to the reaction flask, and salicylaldehyde (304 mg, 2.32 mmol) and 6 mL of deuterated acetic acid were added, and the mixture was heated to 80 ° C for 4 hours. The reaction of the starting material was completely confirmed by TLC, cooled to room temperature, and concentrated to remove deuterated. acetic acid, was added 2mL of ethyl acetate, stirred for 30 minutes at room temperature, the white solid was filtered, dried to give compound 43 (625mg, yield 78.1%), LC-MS ( APCI): m / z = 199 (m-1) - .

Step 2: Synthesis of Compound 44.

Under a nitrogen atmosphere, compound 43 (750 mg, 3.75 mmol), o-phthalaldehyde (502 mg, 3.75 mmol), dissolved in 3 mL of acetic acid, heated to 120 ° C for 1 hour, cooled to room temperature, and concentrated. Analysis give compound 44 (414mg), LC-MS (APCI): m / z = 315 (m-1) -.

Step 3: Synthesis of Compound 45.

Compound 44 (200 mg, 0.63 mmol) and 2-aminothiazole (81.9 mg, 0.819 mmol), DIPEA (80 mg, 0.63 mmol), HATU (311 g, 0.819 mmol), DMF (15 mL) The reaction was carried out under the protection of 25 ° C for 4 hours, and the reaction of the starting material was completed by TLC. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. After purification and purification, 316 mg of compound 45 was obtained, LC-MS (APCI): m/z = 399 (M-1) ^-

Step 4: Synthesis of Compound 46.

Compound 45 (316 mg, 0.794 mmol) was added to the reaction flask, and 15 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (4 mL, 4 mmol) was added dropwise to the reaction flask. The reaction was carried out for 2 hours, and the reaction of the starting material was completed by TLC. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with methylene chloride for 4 times. The organic phase was combined and dried over anhydrous sodium sulfate. Yield 39.8%), LC-MS (APCI): m/z = 383 (M-1) ^- ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.59 (s, 1H), 9.93 (s, 1H) ), 7.71 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.47 (d, J = 3.5 Hz, 1H), 7.26 (d, J = 3.3 Hz, 1H), 7.10 (t, J = 7.3 Hz, 1H), 6.89 (dd, J = 8.9, 4.7 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H), 4.60 (d, J = 17.5 Hz, 1H), 3.97 (d, J = 17.6 Hz, 1H).

Example 7

Preparation of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl)-N-(thiazol-2-yl-5-d)B by the following synthetic route Amide-2-d (Compound 48), comprising the following steps:

Step 1: Synthesis of Compound 47.

Compound 44 (241 mg, 0.76 mmol), compound 9 (99 mg, 0.998 mmol), DIPEA (98 mg, 0.76 mmol), HATU (372 g, 0.998 mmol), DMF (10 mL) After reacting for 5 hours at °C, the reaction of the starting material was completed by TLC, 30 mL of water was added, and the mixture was extracted 4 times with ethyl acetate. The organic phase was combined and purified by column chromatography to give compound 47 (280 mg), LC-MS (APCI): m/z=400 (M+1) ⁺ .

Step 2: Synthesis of Compound 48.

Compound 47 (280 mg, 0.701 mmol) was added to the reaction flask, and 15 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (3.5 mL, 3.5 mmol) was added dropwise to the reaction flask. The mixture was reacted to room temperature for 2 hours, and the reaction of the starting material was completed by TLC. 30 mL of water was added to the reaction flask, and the mixture was extracted 4 times with dichloromethane. The organic phase was combined and dried over anhydrous sodium sulfate. 38 mg, yield 25.2%), LC-MS (APCI): m/z = 384 (M-1) ^- ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.59 (s, 1H), 9.93 (s , 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.60 (t, J = 7.1 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H) ), 7.48 (d, J = 5.9 Hz, 1H), 7.10 (td, J = 8.5, 3.0 Hz, 1H), 6.89 (dd, J = 8.8, 4.7 Hz, 1H), 6.84 (d, J = 9.1 Hz) , 1H), 4.60 (d, J = 17.4 Hz, 1H), 3.99 (dd, J = 22.0, 12.5 Hz, 1H).

Example 8

The following synthetic route was used to prepare 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxoisoindol-2-yl-4,5,6,7-d ₄ )-N-( Thiazol-2-yl-5-d)acetamide (Compound 50), comprising the following steps:

Step 1: Synthesis of Compound 49.

Compound 32 (212 mg, 0.6648 mmol), compound 9 (56 mg, 0.554 mmol), DIPEA (194 mg, 1.04 mmol), HATU (273 mg, 0.72 mmol), DMF (5 mL) The reaction was carried out for 4 hours, and the reaction of the starting material was completed by TLC. 50 mL of water was added to the reaction mixture, and the mixture was combined with ethyl acetate. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. compound 49 (108mg, yield 49.1%), LC-MS ( APCI): m / z = 403 (m + 1) +.

Step 2: Synthesis of Compound 50.

Compound 49 (108 mg, 0.268 mmol) was added to the reaction flask, and 10 mL of dichloromethane was added thereto, and the mixture was cooled to -10 ° C, and boron tribromide (0.8 mL, 0.8 mmol) was added dropwise to the reaction flask. The mixture was reacted to room temperature for 2 hours, and the reaction of the starting material was completed by TLC. 30 mL of water was added to the reaction flask, and the mixture was extracted with dichloromethane for 4 times. The combined organic phases were dried over anhydrous sodium sulfate. , yield 61.1%), LC-MS (APCI): m/z = 389 (M+1) ⁺ ; ¹ H NMR (500 MHz, DMSO) (δ/ppm) 12.58 (s, 1H), 9.93 (s, 1H), 7.47 (s, 1H), 7.09 (td, J = 8.6, 3.1 Hz, 1H), 6.89 (dd, J = 8.9, 4.8 Hz, 1H), 6.84 (dd, J = 9.2, 3.1 Hz, 1H) ), 6.30 (s, 1H), 4.60 (d, J = 17.4 Hz, 1H), 3.97 (d, J = 17.4 Hz, 1H).

Example 9

The compounds obtained in the above examples were subjected to biological evaluation to determine their biological activities. In addition, anti-proliferative 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 the activity was demonstrated to be in the range of <20 nM. The cytotoxic or growth inhibitory effects of the compounds on the tumor cells of interest were evaluated.

(1) EGFR kinase inhibition

The ability of the compounds of Examples 1-8 to inhibit a variety of protein kinases of interest was tested to determine their biological activity. These compounds were found to exhibit potent inhibitory activity against EGFR kinase by tests. The specific method is:

Compound Formulation: Test compounds were dissolved in DMSO to make a 20 mM stock solution. The solution was diluted in DMSO to a final concentration of 100 times the dilution. Dilute to 10 times the final concentration of the dilution solution with the buffer.

EGFR and EGFR [T790M/L858R] kinase assay: After buffer preparation, the enzyme was mixed with different concentrations of pre-diluted compounds for 10 minutes, each double well. The corresponding substrate and ATP were added and reacted at room temperature for 20 minutes (in which a negative positive control was set). After the reaction is completed, the detection reagent is added, and after incubation at room temperature for 30 minutes, the machine is detected and data is collected. Data analysis and mapping according to Graphpad 5.0 software.

EGFR [d746-750] Kinase Assay: After the buffer was prepared, the mixed solution of the enzyme and the antibody was mixed with the different concentrations of the compound prepared by pre-dilution for 10 minutes, and the concentration was doubled. Kinase tracer 199 was added and incubated for 60 minutes at room temperature (where a negative positive control was set). After the reaction is completed, the machine is tested, data is collected, and analysis and mapping are performed.

(2) Cytotoxicity

The anti-proliferative activity of the compound of the present invention against two tumor cells cultured in vitro was examined by the MTS method. The experimental results show that the compound of the present invention has an inhibitory effect on the in vitro proliferation of cancer cells cultured in vitro; wherein the inhibition of proliferation of lung cancer cells in vitro is stronger than that of skin cancer cells in vitro.

Cell lines: skin cancer A431 (purchased from the American Standard Collection of Biological Products (ATCC)); lung cancer cells NCI-H1975 (purchased from the American Standard Collection of Biological Products (ATCC)) and HCC827 (purchased from the US Standard Collection of Biological Products) (ATCC)); both were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

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

Specific experimental methods:

Compound preparation: The test compound was dissolved in DMSO to prepare a 20 mM mother liquor and stored at -20 °C. Dilute 3 times with a gradient of DMSO and the like, and dilute 10 times. The drug medium was diluted 4 times with the drug.

MTS cell viability assay: 0.25% trypsin-EDTA digested logarithmic growth phase cells, inoculated with 150 μl in 96-well plates at an optimized density, and added to the medium 4 times after dilution for 4 hours, 50 μl/well (generally 10 Concentrations: 100, 33.3, 11.1, 3.70, 1.23, 0.412, 0.137, 0.0457, 0.0152, 0.00508 μM). A well of the same volume of 0.5% DMSO was added as a control. After the cells were cultured for 72 hours, MTS was assayed for cell viability.

Specific procedure: adherent cells, discard the medium, and add a mixture containing 20 μL of MTS and 100 μl of medium to each well. OD490 was detected after being placed in an incubator for 1-4 hours, with the OD650 value as a reference. Using GraphPad Prism software to make dose-response curves and calculate IC _50.

The compounds of Examples 1-8 were subjected to EGFR kinase inhibition and cytotoxicity experiments according to the above methods, and the results showed that the compounds of the present invention exhibited potent and excellent inhibitory activities against EGFR mutants and no expression of EGFR (WT) expressed in normal cells. The inhibitory activity, so the compound of the present invention is an effective safe drug for patients with non-small cell lung cancer.

(3) Metabolic stability evaluation

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

Preparation of the stock solution: A certain amount of the powder of the example compound was accurately weighed and dissolved in 5 mM with DMSO.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH of the solution was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

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

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL 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 were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of each reaction solution was taken at 10, 30, and 90 min, respectively, and added to a stop plate, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

t _1/2 (min); CL _int (μL/min/mg).

The metabolic stability of human and rat liver microsomes was evaluated by simultaneously testing the compounds of the present invention and their compounds without deuteration. The half-life and liver intrinsic clearance as indicators of metabolic stability are shown in Table 1. The undeuterated compound EAI045 was used as a control in Table 1. As shown in Table 1, in the human/rat/mouse liver microsome experiment, the compound of the present invention can significantly improve metabolic stability by comparison with EAI045.

Table 1 Comparison table of indicators of metabolic stability of the compounds of Examples 1-8

(4) Rat pharmacokinetic experiments

Six male Sprague-Dawley rats, aged 7-8 weeks, weighing approximately 210 g, were divided into two groups of 3 animals each, and their pharmacokinetic differences were compared by intravenous or oral single dose of compound (10 mg/kg orally). .

Rats were fed a standard diet and given water. Fasting began 16 hours before the test. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

Rats were briefly anesthetized after inhalation of ether, and 300 μL of blood samples were collected from the eyelids in test tubes. There was 30 μL of 1% heparin salt solution in the test tube. The tubes were dried overnight at 60 ° C before use. After the blood sample collection was completed at the last time point, the rats were anesthetized with ether and sacrificed.

Immediately after the blood sample is collected, gently invert the tube at least 5 times to ensure adequate mixing and place on ice. Blood samples were centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate plasma from red blood cells. Pipette 100 μL of plasma into a clean plastic centrifuge tube, indicating the name and time of the compound. Plasma was stored at -80 °C prior to analysis. The concentration of the compound 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 have shown that the compounds of the present invention have better pharmacokinetic properties in animals and therefore have better pharmacodynamics and management effects.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

.

Claims

An amide compound of the formula (I), or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof:

among them,

R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl. ;

The additional condition is that the above amide compound contains at least one ruthenium atom.
The amide compound of the formula (I) according to claim 1, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof, wherein R 1 and R 2 are each independently hydrazine or hydrogen.
The amide compound of the formula (I) according to claim 1, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof, wherein R 3 and R 4 are each independently hydrazine or hydrogen.
The amide compound of the formula (I) according to claim 1, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof, wherein R 5 , R 6 , R 7 and R 8 are each independently hydrazine or hydrogen.
The amide compound of the formula (I) according to claim 1, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvate thereof, wherein R 9. R 10 , R 11 and R 12 are each independently hydrazine or hydrogen.
The amide compound of the formula (I) according to any one of claims 1 to 5, or a polymorph, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variation, hydrate or solvent thereof The amide compound of the formula (I) may be selected from any of the following structures:
A process for the preparation of the amide compound of claim 1 comprising substituted or unsubstituted

The step of reacting as an intermediate with a substituted or unsubstituted 2-aminothiazoline under basic conditions.
The method according to claim 7, wherein the base used is selected from the group consisting of potassium carbonate, sodium carbonate, sodium hydrogencarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, N, N. At least one of diisopropylethylamine, 4-N,N-dimethylpyridine or pyridine.
A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an amide compound of the formula (I) according to any one of claims 1 to 6, or a polymorphic form thereof, a pharmaceutically acceptable salt thereof , prodrugs, stereoisomers, isotopic variations, hydrates or solvates.
A process for the preparation of a pharmaceutical composition according to claim 9, comprising: a pharmaceutically acceptable excipient and the amide compound of the formula (I) according to any one of claims 1 to 6, or Polymorphic forms, pharmaceutically acceptable salts, prodrugs, stereoisomers, isotopic variations, hydrates or solvate compounds are combined to form a pharmaceutical composition.
The pharmaceutical composition according to claim 10, which further comprises other therapeutic agents, which are cancer, cardiovascular disease, inflammation, infection, immune disease, cell proliferative disease, viral disease, metabolic disease, Or a drug for organ transplantation.
Use of an amide compound of the formula (I) according to any one of claims 1 to 6 for the preparation of a medicament for the treatment and/or prevention of a disease associated with a protein kinase.