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WO2025112584A1 - 一种含硒元素类化合物及其用途 - Google Patents

一种含硒元素类化合物及其用途 Download PDF

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Publication number
WO2025112584A1
WO2025112584A1 PCT/CN2024/107560 CN2024107560W WO2025112584A1 WO 2025112584 A1 WO2025112584 A1 WO 2025112584A1 CN 2024107560 W CN2024107560 W CN 2024107560W WO 2025112584 A1 WO2025112584 A1 WO 2025112584A1
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alkyl
cycloalkyl
heterocyclyl
cyano
alkoxy
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French (fr)
Inventor
姜正羽
尤启东
杨爽
于泽洲
王馨仪
闫蕾昕
陈学涛
郭小可
徐晓莉
王磊
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China Pharmaceutical University
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the invention belongs to the field of medical technology, and specifically relates to a selenium-containing compound and a use thereof.
  • selenium is rarely used in the field of medicinal chemistry and drug design.
  • researchers in the field of medicinal chemistry have gradually increased their interest in the application of selenium in drug design, development and synthesis.
  • Selenium has unique oxidation-reduction properties and nucleophilicity due to its larger atomic structure and loose outer valence electron properties.
  • selenium-containing molecules usually show higher biological reactivity than sulfur-containing molecules, are easy to form Se-S adducts, and significantly enhance the selective absorption effect of cancer cells. Therefore, selenium-containing molecules are often used as antioxidants, free radical scavengers or pro-oxidants to participate in inflammation suppression therapy or anti-tumor therapy.
  • Selenium atoms are also used as bioisosteres of oxygen atoms in drug molecule design and natural product modification. Many compounds have thus acquired unique drug activity. The introduction or replacement of selenium atoms has also become a new perspective for the strategy of improving the properties of compounds.
  • GLP-1 is a fast-metabolizing endogenous incretin hormone, produced from intestinal tissue and central nervous tissue, and acts on GLP-1R.
  • Intestinal GLP-1 is widely distributed in tissues throughout the body along the circulatory system, and mainly plays a role in regulating blood sugar content and metabolism; central GLP-1 plays a neurotransmitter function and inhibits appetite and food intake.
  • GLP-1R agonists Due to its important blood sugar and metabolic regulation functions, GLP-1R agonists have become important drugs for the treatment and control of metabolic diseases such as diabetes.
  • the GLP-1R agonists currently approved for marketing are all peptidomimetic compounds that simulate GLP-1 peptides, such as exenatide, liraglutide, dulaglutide, semaglutide and other GLP-1 analogs.
  • GLP-1R agonists including iteratively optimized GLP-1 peptide analogs, orally available peptide agonists, small molecule agonists, allosteric agonists, etc.
  • Danuglipron (PF-06882961) is a small molecule direct GLP-1R agonist of the benzimidazole-6-carboxylic acid class developed by Pfizer. It has picomolar-level GLP-1R-cAMP signaling pathway agonist activity (EC 50 ) and complete agonist effect (E max ), and is a partial agonist of GLP-1R. Structural biology information shows that Danuglipron compounds occupy part of the GLP-1 peptide action site, inducing and simulating the conformation of key ligand-receptor interactions with structural water networks. Danuglipron shows primate species-specific agonist effects, and oral administration shows acceptable oral absorption and in vivo metabolism.
  • Glucose tolerance tests show that Danuglipron can effectively improve the glucose tolerance level of experimental subjects, promote insulin release, and effectively control the blood sugar level of experimental subjects.
  • the Phase II clinical trial of Danuglipron for blood sugar control indications has been completed, and Phase III clinical trials are underway.
  • the first purpose of the present invention is to provide a selenium-containing compound; the second purpose of the present invention is to provide uses of the selenium-containing compound, including uses for preparing GLP-1 receptor agonists and for preparing drugs for diseases that are directly or indirectly treated or alleviated by stimulating the GLP-1 receptor.
  • the present invention develops a small molecule GLP-1R agonist containing selenium and effective orally based on the new structure of Danuglipron, which is used to prepare drugs for metabolic diseases that can regulate GLP-1R related signals.
  • this type of small molecule agonist is effective orally; at the same time, it improves the pharmacokinetic properties and cardiac toxicity of Danuglipron.
  • the present invention provides a compound of general formula I or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof:
  • Ring A is selected from C 3-8 cycloalkyl, C 2-9 heterocyclyl, C 5-12 spirobicyclyl, C 5-12 spiroheterobicyclyl, C 5-12 fused bicyclyl, C 5-12 fused heterobicyclyl, C 5-12 bridged cyclyl or C 5-12 bridged heterocyclyl;
  • L is selected from -C(O)-, -O-, -S-, -CR 4 R 5 or -NR 6 -;
  • R 4 and R 5 are each independently selected from hydrogen, deuterium, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, -NR 5a R 5b , 6-10 membered aryl, 5-8 membered heteroaryl, 3-8 membered saturated or partially saturated cycloalkyl, 3-8 membered saturated or partially saturated heterocyclyl; the C1-C6 alkyl or C1-C6 alkoxy is optionally substituted by one or more of the following substituents: halogen, oxo, cyano, hydroxyl, C3-C6 saturated or partially saturated cycloalkyl; the aryl, heteroaryl, saturated or partially saturated cycloalkyl, saturated or partially saturated heterocyclyl is optionally substituted by one or more of the following substituents: halogen, oxo, cyano or -NR 5a R 5b ;
  • R 5a , R 5b are each independently selected from hydrogen or C1-C6 alkyl
  • R 6 is selected from hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, heterocyclyl, -S(O) 2 R 6a or -S(O) 2 N(R 6a )(NR 6b ); the cycloalkyl or heterocyclyl is optionally substituted by C1-C6 alkyl, -F or cyano;
  • R 6a , R 6b are each independently selected from -H, C1-C6 alkyl or C3-C10 cycloalkyl;
  • R1 is selected from C1-C6 alkyl substituted with 3-6 membered heterocyclyl, C1-C6 alkyl substituted with C3-C6 cycloalkyl, C1-C6 alkyl substituted with 5-8 membered heteroaryl, C1-C6 alkyl substituted with C1-C6 alkoxy, 3-6 membered heterocyclyl, C3-C6 cycloalkyl or 5-8 membered heteroaryl; the 3-6 membered heterocyclyl, C3-C6 cycloalkyl, 5-8 membered heteroaryl or C1-C6 alkoxy is optionally substituted with one of the following: substituted by one or more substituents: H, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, cyano, cyano-substituted C1-C6 alkyl, hydroxy,
  • R2 is each independently selected from a hydrogen atom, deuterium, halogen, cyano, hydroxyl, oxo, C1-C6 alkyl, C1-C6 alkoxy, amino, 6-10 membered aryl, 5-8 membered heteroaryl, 3-8 membered saturated or partially saturated cycloalkyl, 3-8 membered saturated or partially saturated heterocyclic group;
  • R3 is each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, cyano, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • X 1 , X 2 , X 3 and X 4 are the same or different and are each independently selected from -CR 7 or an N atom;
  • R 7 is selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclic, aryl or heteroaryl;
  • G is selected from G1 or G2; the structural formulas of G1 and G2 are as follows:
  • Ring B and Ring C are each independently selected from a 6-12 membered aryl group or a 6-12 membered heteroaryl group;
  • R 8 and R 11 are the same or different and are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl are optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, cyano, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R9 , R10 , R12 , R13 are the same or different and are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • EE is selected from -COOH or a carboxyl surrogate; the carboxyl surrogate is selected from:
  • n 0, 1, 2 or 3;
  • n 0, 1, 2, 3 or 4;
  • o 0, 1, 2, 3, 4, or 5;
  • p 0, 1, 2, 3, 4 or 5.
  • the present invention also provides a compound of general formula II or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof:
  • Ring A is selected from C 3-8 cycloalkyl, C 2-9 heterocyclyl, C 5-12 spirobicyclyl, C 5-12 spiroheterobicyclyl, C 5-12 fused bicyclyl, C 5-12 fused heterobicyclyl, C 5-12 bridged cyclyl or C 5-12 bridged heterocyclyl;
  • L is selected from -C(O)-, -O-, -S-, -CR 4 R 5 or -NR 6 -;
  • R 4 and R 5 are each independently selected from hydrogen, deuterium, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, -NR 5a R 5b , 6-10 membered aryl, 5-8 membered heteroaryl, 3-8 membered saturated or partially saturated cycloalkyl, 3-8 membered saturated or partially saturated heterocyclyl; the C1-C6 alkyl or C1-C6 alkoxy is optionally substituted by one or more of the following substituents: halogen, oxo, cyano, hydroxyl, C3-C6 saturated or partially saturated cycloalkyl; the aryl, heteroaryl, saturated or partially saturated cycloalkyl, saturated or partially saturated heterocyclyl is optionally substituted by one or more of the following substituents: halogen, oxo, cyano or -NR 5a R 5b ;
  • R 5a , R 5b are each independently selected from hydrogen or C1-C6 alkyl
  • R 6 is selected from hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, heterocyclyl, -S(O) 2 R 6a or -S(O) 2 N(R 6a )(NR 6b ); the cycloalkyl or heterocyclyl is optionally substituted by C1-C6 alkyl, -F or cyano;
  • R 6a , R 6b are each independently selected from -H, C1-C6 alkyl or C3-C10 cycloalkyl;
  • R1 is selected from C1-C6 alkyl substituted with 3-6 membered heterocyclyl, C1-C6 alkyl substituted with C3-C6 cycloalkyl, C1-C6 alkyl substituted with 5-8 membered heteroaryl, C1-C6 alkyl substituted with C1-C6 alkoxy, 3-6 membered heterocyclyl, C3-C6 cycloalkyl or 5-8 membered heteroaryl; the 3-6 membered heterocyclyl, C3-C6 cycloalkyl, 5-8 membered heteroaryl or C1-C6 alkoxy is optionally substituted with one of the following: substituted by one or more substituents: H, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, cyano, cyano-substituted C1-C6 alkyl, hydroxy,
  • R2 is each independently selected from a hydrogen atom, deuterium, halogen, cyano, hydroxyl, oxo, C1-C6 alkyl, C1-C6 alkoxy, amino, 6-10 membered aryl, 5-8 membered heteroaryl, 3-8 membered saturated or partially saturated cycloalkyl, 3-8 membered saturated or partially saturated heterocyclic group;
  • R3 is each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, cyano, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • X 1 , X 2 , X 3 and X 4 are the same or different and are each independently selected from -CR 7 or an N atom;
  • R 7 is selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclic, aryl or heteroaryl;
  • G is selected from G1 or G2; the structural formulas of G1 and G2 are as follows:
  • Ring B and Ring C are each independently selected from a 6-12 membered aryl group or a 6-12 membered heteroaryl group;
  • R 8 and R 11 are the same or different and are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl are optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, cyano, amino, nitro, cyano, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R9 , R10 , R12 , R13 are the same or different and are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 14 is each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more of the following substituents: halogen, alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, cyano, amino, nitro, hydroxy, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • EE is selected from -COOH or a carboxyl surrogate; the carboxyl surrogate is selected from:
  • n 0, 1, 2 or 3;
  • n 0, 1, 2, 3 or 4;
  • o 0, 1, 2, 3, 4, or 5;
  • p 0, 1, 2, 3, 4 or 5;
  • q 0, 1, 2, or 3.
  • Ring A is selected from C 3-8 cycloalkyl or C 2-9 heterocyclyl
  • L is selected from -CR 4 R 5 ;
  • R1 is selected from C1-C6 alkyl substituted by 3-6 membered heterocyclyl, C1-C6 alkyl substituted by C3-C6 cycloalkyl, C1-C6 alkyl substituted by 5-8 membered heteroaryl, C1-C6 alkyl substituted by C1-C6 alkoxy;
  • EE is selected from -COOH or carboxyl surrogate
  • R 4 and R 5 are as defined in Formula I or Formula II.
  • Ring A is selected from C 2-9 heterocyclic groups
  • Y and Z are each independently selected from -Se- or -Se(O)-;
  • EE is selected from -COOH
  • the compounds of the general formula I or II of the present invention are preferably the compounds shown in the following Table 1:
  • the above-mentioned compounds of general formula I or general formula II of the present invention may also exist in the form of their salts, hydrates, solvates, which are converted into compounds of general formula I or general formula II in vivo.
  • the compounds of the present invention are converted into the form of pharmaceutically acceptable salts, and they are used in the form of salts.
  • Certain compounds of the present invention may exist in multiple crystalline forms.
  • the present invention also provides a pharmaceutical composition, which comprises a compound of Formula I or Formula II or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polycrystal thereof and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir.
  • the pharmaceutical composition of the present invention can be administered alone or in combination with other drugs.
  • Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions and solutions.
  • Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, adhesives, colorants, fillers, emulsifiers, etc.
  • Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art.
  • Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.
  • the actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the dosage level selected depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.
  • the present invention also provides the use of the compound of formula I or formula II or its pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph in the preparation of GLP-1 receptor agonist.
  • the present invention also provides the use of a compound of Formula I or Formula II or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate or polymorph thereof in the preparation of a drug for preventing and/or treating a disease associated with stimulating the GLP-1 receptor.
  • the drug directly or indirectly treats or alleviates a disease by stimulating the GLP-1 receptor.
  • the diseases associated with stimulating the GLP-1 receptor include diabetes, diabetic complications, metabolic syndrome, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, Parkinson's disease, Alzheimer's disease, hypertension, Hyperlipidemia, atherosclerosis, cardiovascular risk, coronary heart disease or stroke.
  • the present invention provides a class of selenium-containing compounds, which have a complete GLP-1R-cAMP signaling pathway agonist effect (E max ), and the compound GLP-1R-cAMP half-effective concentration (EC 50 ) reaches the nanomolar level, and the EC 50 of some compounds reaches the picomolar level, which is an excellent GLP-1 receptor agonist.
  • the representative compound 1 has good drug properties. Therefore, the selenium-containing compounds provided by the present invention have the prospect of being developed as drugs for preventing and/or treating diseases that are directly or indirectly treated or alleviated by stimulating the GLP-1 receptor.
  • FIG1 is a standard curve of the rHLM test of compound 1.
  • FIG. 2 is a standard curve of the rHLM test of PF-06882961.
  • Figure 3 is the c-t curve of the PK test of PF-06882961.
  • Figure 4 is the c-t curve of the PK test of the low-dose group of compound 1.
  • Figure 5 is the c-t curve of the PK test of the high-dose group of compound 1.
  • the following is a detailed description of the essential contents of the present invention in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.
  • the experimental methods without specific conditions in the embodiments of the present invention are usually conventional conditions, or the conditions recommended by the raw material or commodity manufacturer; the reagents without source are usually conventional reagents available through commercial channels.
  • NMR The 1 H-NMR spectra of the compounds of the present invention were measured using a Bruker nuclear magnetic resonance spectrometer (300 MHz) and a Bruker nuclear magnetic resonance spectrometer (500 MHz). The 13 C-NMR spectra were measured using a Bruker nuclear magnetic resonance spectrometer (300 MHz). Chemical shifts ( ⁇ ) are expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used.
  • MS The mass spectra of the compounds of the present invention were measured using a Shimadzu LC/MS-8050 triple quadrupole mass spectrometer, using ESI as the ionization method, and the molecular weight was accurate to one decimal place.
  • HRMS The high-resolution mass spectra of the compounds of the present invention are measured by LC/MS mass spectrometers of Shimadzu and Agilent, the test method is Q-TOF, and the molecular weight is accurate to four decimal places.
  • Compound 1 was prepared using starting material 1-1 according to the following route.
  • Step 1 Preparation of 1-(tert-butyl)-4-methyl-4-(6-bromopyridin-2-yl)piperidine-1,4-dicarboxylic acid (Compound 1-3).
  • Step 2 Preparation of 4-(6-bromopyridin-2-yl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (Compound 1-4).
  • Step 3 Preparation of tert-butyl 4-(6-bromopyridin-2-yl)piperidine-1-carboxylate (Compound 1-5).
  • the intermediate 1-4 was dissolved in 45 mL 1,2-dichloroethane (DCE), heated to 95°C for 4 h. After the reaction was completed by TLC, the product 1-5 was dried under reduced pressure to obtain a pale yellow-white solid (1.52 g, 86.4%).
  • DCE 1,2-dichloroethane
  • Step 4 Preparation of tert-butyl 4-(6-selenohydroxypyridin-2-yl)piperidine-1-carboxylate (Compound 1-6).
  • Step 5 Preparation of tert-butyl 4-(6-((4-cyano-2-fluorobenzyl)selenyl)pyridin-2-yl)piperidine-1-carboxylate (Compound 1-8).
  • Step 6 Preparation of 3-fluoro-4-((6-(piperidin-4-yl)pyridin-2-yl)selenyl)methyl)benzonitrile (Compound 1-9) p-toluenesulfonate.
  • Step 7 Preparation of (S)-methyl 2-(4-(6-(4-cyano-2-fluorobenzyl)seleno)pyridin-2-yl)piperidin-1-methyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate (Compound 1-11).
  • Compound 7 was synthesized using 2-methoxy-4-(trifluoromethyl)benzyl bromide and intermediate 1-6 as starting materials according to the synthetic route of compound 1.
  • Compound 7 was a white solid, 45 mg in total, with a yield of 60.5%.
  • Compound 8 was prepared using starting materials 1-1 and 8-2 according to the following route.
  • Step 1 Preparation of tert-butyl 4-(6-bromopyridin-2-yl)piperazine-1-carboxylate (Compound 8-2).
  • Step 2 Preparation of tert-butyl 4-(6-selenohydroxypyridin-2-yl)piperazine-1-carboxylate (Compound 8-3).
  • Step 3 Preparation of tert-butyl 4-(6-(4-cyano-2-fluorobenzyl)selenyl)pyridin-2-yl)piperazine-1-carboxylate (Compound 8-4).
  • Compound 10 was prepared using starting materials 1-9 according to the following route.
  • Step 1 Preparation of 3-fluoro-4-((6-(piperidin-4-yl)pyridin-2-yl)selensulfonyl)methyl)benzonitrile (Compound 10-1).
  • Compound 11 was prepared using starting material 11-1 according to the following route.
  • Step 1 Preparation of 3-fluoro-4-hydrogenselenidebenzonitrile (Compound 11-2).
  • Step 2 Preparation of tert-butyl 6-(hydroxymethyl)-3',6'-dihydro-(2,4'-bipyridine)-1'(2'H)-carboxylate (Compound 11-5).
  • 6-Chloro-2-hydroxymethylpyridine (compound 11-3, 231 mg, 1.62 mmol) was dissolved in 20 mL of dioxane and 2 mL of H 2 O, and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (compound 11-4, 999 mg, 3.23 mmol), Pd(dppf)Cl 2 (118 mg, 0.16 mmol) and Cs 2 CO 3 (1153 mg, 3.55 mmol) were added. The mixture was protected by N 2 and heated to 90°C and stirred for 2 h. TLC showed that the reaction was complete. The reaction solution was extracted with an equal volume of water and EA.
  • Step 3 Preparation of tert-butyl 4-(6-(hydroxymethyl)pyridin-2-yl)piperidine-1-carboxylate (Compound 11-6).
  • Step 4 Preparation of tert-butyl 4-(6-(bromomethyl)pyridin-2-yl)piperidine-1-carboxylate (Compound 11-7).
  • Step 5 Preparation of tert-butyl 4-(6-(4-cyano-2-fluorophenyl)selenomethyl)pyridin-2-yl)piperidine-1-carboxylate (Compound 11-8).
  • Compound 12 was prepared using compound 1 as a raw material according to the following route.
  • the intermediate 12-2 (58 mg, 0.08 mmol) was dissolved in 5 mL of methanol, and a methanol solution of TFA (6.2 ⁇ L, 0.08 mmol) was added dropwise, and stirred at room temperature for 2 h.
  • Compound 13 was prepared using compound 1 as a raw material according to the following route.
  • Compound 14 was prepared using starting material 14-1 according to the following route.
  • Step 2 Preparation of (S)-tert-butyl((4-nitro-3-((oxetanylamino-2-ylmethyl)amino)phenyl)sulfonyl)carbamate (Compound 14-3).
  • Step 3 Preparation of (S)-tert-butyl((4-amino-3-((oxetanylamino-2-ylmethyl)amino)phenyl)sulfonyl)carbamate (Compound 14-4).
  • Step 4 Preparation of (S)-tert-butyl((2-(chloromethyl)-1-(oxetanin-2-ylmethyl)-1H-benzo[d]imidazol-6-yl)sulfonyl)carbamate (Compound 14-5).
  • Step 5 Preparation of (S)-tert-butyl ((2-((4-(6-((4-cyano-2-fluorobenzyl)selenyl)pyridin-2-yl)piperidin-1-yl)methyl)-1-(oxetanylamino-2-ylmethyl)-1H-benzo[d]imidazol-6-yl)sulfonyl)carbamate (Compound 14-6).
  • Step 6 Preparation of (S)-2-((4-(6-((4-cyano-2-fluorobenzyl)selenyl)pyridin-2-yl)piperidin-1-yl)methyl)-1-(oxetanin-2-ylmethyl)-1H-benzo[d]imidazole-6-sulfonamide (Compound 14-7).
  • Compound 16 was synthesized using 1-(bromomethyl)-2-fluoro-4-(trifluoromethyl)benzene and intermediate 1-6 as starting materials according to the synthetic route of compound 1.
  • Compound 16 was a white solid, 28.4 mg in total, with a yield of 59.9%.
  • Compound 17 was synthesized using 4-(bromomethyl)-3-methoxybenzonitrile and intermediate 1-6 as starting materials according to the synthetic route of compound 1.
  • Compound 17 was a white solid, 42.3 mg in total, with a yield of 59.8%.
  • Compound 18 was prepared using starting material 18-1 according to the following route.
  • Step 1 Preparation of methyl 3-((2-methoxyethyl)amino)-4-nitrobenzoate (18-3).
  • Step 2 Preparation of methyl 4-amino-3-((2-methoxyethyl)amino)benzoate (18-4).
  • Step 3 Preparation of methyl 2-(chloromethyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-6-carboxylate (18-5).
  • Step 4 Preparation of methyl 2-((4-(6-((4-cyano-2-fluorobenzyl)seleno)pyridin-2-yl)piperidin-1-yl)methyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-6-carboxylate (18-6).
  • Step 5 Preparation of 2-((4-(6-((4-cyano-2-fluorobenzyl)seleno)pyridin-2-yl)piperidin-1-yl)methyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-6-carboxylic acid (Compound 18).
  • Compound 20 was synthesized using (1-ethyl-1H-imidazol-5-yl)methylamine and compound 18-1 as starting materials according to the synthetic route of compound 18.
  • Compound 20 was a white solid, with a total of 64.92 mg and a yield of 49.3%.
  • Compound 22 was prepared using starting material 22-1 according to the following route.
  • Step 1 Preparation of tert-butyl (6-bromopyridin-2-yl)carbamate (22-3).
  • Step 2 Preparation of tert-butyl (6-bromopyridin-2-yl)(2-oxoethyl)carbamate (22-4).
  • Step 3 Preparation of tert-butyl 2-(2-((6-bromopyridin-2-yl)(tert-butoxycarbonyl)amino)ethyl)hydrazine-1-carboxylate (22-6).
  • the intermediate 22-4 (9.75 g, 30.94 mmol) was dissolved in 150.00 mL DCE, and compound 22-5 (8.23 g, 61.87 mmol) was added, and stirred at room temperature for 1 h.
  • NaBH 3 CN (5.84 g, 92.81 mmol)
  • NaBH(OAc) 3 (19.78 g, 92.81 mmol) were added, and stirred at room temperature for about 3 h.
  • the reaction was quenched with a saturated aqueous solution of NaHCO 3 , extracted with DCM, and the organic phases were combined. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • Step 4 Preparation of 6-bromo-N-(2-hydrazineethyl)pyridin-2-amine (22-7).
  • the intermediate 22-6 (6.86 g, 15.76 mmol) was dissolved in 120 mL of DCM, and 60 mL of a 4 M solution of hydrogen chloride in dioxane was added. The mixture was stirred at room temperature for about 5 h, and a large amount of white solid precipitated. After the reaction was completed by TLC, the solvent was directly evaporated under reduced pressure, and the hydrochloride obtained was directly used for the next step without purification.
  • LC-MS: [M+H] + 231.1.
  • Step 5 Preparation of 6-bromo-N-(2-hydrazineethyl)pyridin-2-amine (22-8).
  • Step 6 Preparation of 4-(6-bromopyridin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (22-9).
  • Step 7 Preparation of tert-butyl 4-(6-bromopyridin-2-yl)-5,6-dihydro-1,2,4-triazine-1(4H)-carboxylate (22-10).
  • Step 8 Preparation of (S)-2-((4-(6-((4-cyano-2-fluorobenzyl)seleno)pyridin-2-yl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl)methyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylic acid (Compound 22).
  • Compound 23 was synthesized from methyl 3,5-difluoro-4-nitrobenzoate and (S)-oxetane-2-methylamine according to the synthetic route of compound 18.
  • Compound 23 was a white solid, 52.25 mg in total, with a yield of 60.8%.
  • Compound 24 was synthesized from intermediate 22-10, intermediate 1-7 and methyl 3,5-difluoro-4-nitrobenzoate according to the synthetic route of compound 22.
  • Compound 24 was a white solid, 39.82 mg in total, with a yield of 57.9%.
  • Compound 25 was synthesized from intermediate 22-10, 2-methoxy-4-(trifluoromethyl)benzyl bromide and methyl 3,5-difluoro-4-nitrobenzoate according to the synthetic route of compound 22.
  • Compound 25 was a white solid, 45.07 mg in total, with a yield of 60.4%.
  • Compound 26 was synthesized from intermediate 22-10, 4-(bromomethyl)-3-methoxybenzonitrile and methyl 3,5-difluoro-4-nitrobenzoate according to the synthetic route of compound 22.
  • Compound 26 was a white solid, 43.73 mg in total, with a yield of 67.4%.
  • Compound 27 was synthesized from intermediate 22-10, 1-(bromomethyl)-2-fluoro-4-(trifluoromethyl)benzene and methyl 3,5-difluoro-4-nitrobenzoate according to the synthetic route of compound 22.
  • Compound 27 was a white solid, 37.46 mg in total, with a yield of 59.8%.
  • Compound 28 was synthesized from intermediate 22-10, 1-(bromomethyl)-4-chloro-2-fluorobenzene and methyl 3,5-difluoro-4-nitrobenzoate according to the synthetic route of compound 22.
  • Compound 28 was a white solid, 39.03 mg in total, with a yield of 56.6%.
  • the present invention uses a cAMP signal agonist effect detection method to characterize the agonist activity of a compound on GLP-1R.
  • HEK cell line that stably expresses GLP-1R (constructed by Beijing Aisi Yipu Biotechnology Co., Ltd., expressing the human GLP-1R gene), and the number of cells used for testing was 1000/well.
  • the cell lines were cultured in DMEM+100 ⁇ g/mL Hygromycin B medium containing 10% fetal bovine serum, the culture temperature was 37°C, and the carbon dioxide concentration was 5%.
  • the old culture medium was removed and washed once with PBS, and then 1mL TrypLE TM Express solution (Beijing Aisi Yipu Biotechnology Co., Ltd.) was added and incubated at 37°C for about 2min.
  • the test was performed using the LANCE Ultra cAMP kit.
  • the kit includes: Stimulation Buffer, Eu-cAMP, Detection buffer, and ULight-anti-cAMP.
  • Step 1 All reagents in the kit should be equilibrated to room temperature before use. Prepare 1 ⁇ Stimulation Buffer according to the kit instructions.
  • Step 2 The compound to be tested was diluted with DMSO in a gradient manner, and then diluted to 10 ⁇ with 1 ⁇ Stimulation Buffer.
  • the actual starting test concentration was set to 5000nM, and 10 groups were diluted in a 5-fold gradient, where each test group contained 2 sets of duplicate test wells.
  • Each test group was set with a DMSO concentration of 0.1% as the negative control group, and a GLP-1 (7-37) concentration of 100nM (provided by Beijing Aisiyipu Biotechnology Co., Ltd.) as the positive control group.
  • Step 3 Stably transfected cells were cultured to 80% confluence; cells were collected by trypsin digestion and counted, and then inoculated into 384-well plates at 9 ⁇ L/well.
  • Step 4 Take 1 ⁇ L of the 10 ⁇ compound diluted in step 2 and add it to the corresponding experimental well. After centrifugation, incubate at 37°C for 30 min.
  • Step 5 Dilute Eu-cAMP to the working concentration with Detection buffer and add 5 ⁇ L/well to the corresponding experimental wells.
  • Step 6 Dilute ULight-anti-cAMP to the working concentration with detection buffer, and then add 5 ⁇ L/well to the corresponding experimental wells; after centrifugation, incubate at room temperature for 1 hour.
  • Step 7 After incubation, use Biotek multifunctional microplate reader to detect the readings at 665nm and 620nm. Calculate the ratio of the readings under the corresponding wavelength conditions R 665/620 , and calculate the average ratio of the corresponding test group (R Group ) and the average ratio of the positive reference The average ratio of negative reference
  • the agonist rate A was calculated according to the formula, and the agonist effect of the compound at the 5000nM level was determined, and the compounds with agonist effect (A 5000nM >50.00%) were selected.
  • the A of the compound test concentration with an agonist effect greater than 80% was selected and plotted against the corresponding concentration, and the curve fitting and half agonist activity concentration (EC 50 ) were calculated using the nonlinear regression method of GraphPad Prism software.
  • the calculation result shall be rounded to 4 significant figures.
  • test results show that the compounds of the present invention can activate the downstream signal transduction of GLP-1R by stimulating the cAMP signaling pathway, and have a complete GLP-1R agonist effect and a low half-agonist concentration.
  • the present invention uses LC-MS/MS method to detect the rat liver microsome stability of compound 1, compound 17 and compound 26 and PF-06882961.
  • Chromatographic column GL Science ODS-3, 3 ⁇ m, 2.1mm*50mm;
  • Mobile phase binary high pressure gradient elution, 0-2min, pump A: methanol, 20%; pump B: water (containing 0.1% formic acid), 80%; 2-5min: pump A: methanol, 80%; pump B: water (containing 0.1% formic acid), 20%; 5-7min, pump A: methanol, 20%; pump B: water (containing 0.1% formic acid), 80%;
  • Detection wavelength 254nm; detection time: 7min; injection volume: 5 ⁇ L;
  • Test system 120 ⁇ L PBS, 20 ⁇ L 5mg/mL inactivated liver microsomes, 10 ⁇ L 100mM MgCl 2 solution, 10 ⁇ L compound, mix well, incubate at 37°C for 5 min, add 40 ⁇ L NADPH solution. A total of 200 ⁇ L, in the test system, rHLM was diluted 10 times, MgCl 2 was diluted 20 times, compound was diluted 20 times, and NADPH was diluted 5 times.
  • the final concentration of the compound detection is 80 times the concentration of the original solution, precipitate protein, centrifuge at 12000 rpm, 4 ° C for 5 minutes, and take 20 ⁇ L of the supernatant.
  • the final concentration of the test falls between 1000 ng/mL-0.1 ng/mL, LC-MS/MS injection 5 ⁇ L detection, using the internal standard method, the concentration ratio (C) versus the area ratio (A) to draw a standard curve.
  • the standard curve formulas for compound 1 and PF-06882961 were prepared using the above test method.
  • the fitting results showed that the peak area response of compound 1 and PF-06882961 in the range of 0.1 ng/mL-1000 ng/mL was linearly related to the concentration, and the fitting regression degree was good.
  • the standard curve of compound 1 is shown in FIG1
  • the standard curve of PF-06882961 is shown in FIG2 .
  • the test method described in step (2) was used for detection in three parallel groups.
  • the concentration of the test sample was 4 ⁇ g/mL.
  • the timing was started after the addition of NADPH solution to initiate the reaction.
  • Samples were taken at 0, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min.
  • the residual concentration of the compound at each time point was calculated, and the compound retention percentage (HLMRemaining, %) was calculated with the compound concentration at 0 min as 100%.
  • the average value of the natural logarithm of the retention percentage was linearly fitted with the incubation time to obtain the elimination rate constant k (min -1 ) and SEM.
  • the clearance-related parameters were calculated according to the following formula:
  • test results showed that the stability of compound 1, compound 17 and compound 26 in rat liver microsomes was better than that of the positive molecule PF-06882961 in rat liver microsomes.
  • the present invention uses the LC-MS/MS method to calculate the resident concentrations (c) of compound 1 and PF-06882961 in the plasma of adult rats at different time points (t) under different dosing conditions, draws the c-t curves of corresponding conditions/corresponding compounds, and uses the Winnolin7.0 software non-modal model to fit and analyze the c-t parameters.
  • Rats SD strain, male, 6-8 weeks old, weight 180 g-220 g;
  • intravenous injection (1 mg/kg or 10 mg/kg); oral administration (5 mg/kg or 50 mg/kg);
  • the solvent formula is: 10% DMSO + 40% PEG-400 + 50% saline (containing 20% w/v hydroxypropyl- ⁇ -cyclodextrin), the solution concentration is 1 mg/mL or 5 mg/mL;
  • Chromatographic column GL Science ODS-3, 3 ⁇ m, 2.1mm*50mm;
  • Mobile phase binary high pressure gradient elution, 0-2min, pump A: methanol, 20%; pump B: water (containing 0.1% formic acid), 80%; 2-5min: pump A: methanol, 80%; pump B: water (containing 0.1% formic acid), 20%; 5-7min, pump A: methanol, 20%; pump B: water (containing 0.1% formic acid), 80%;
  • Detection wavelength 254nm; Detection time: 7min; Injection volume: 10 ⁇ L or 1 ⁇ L;
  • Rats were weighed and the dosing volume was calculated.
  • Blood collection time points were set at 1min, 5min, 10min, 30min, 45min, 60min, 120min, 240min, 360min, 480min, 540min, and 1440min, for a total of 12 time points, and 4 parallel experimental groups were set up for each dosing group. The timing started at the time point when the dosing was completed, and 100 ⁇ L to 200 ⁇ L of blood was collected from the orbital venous plexus of the rat at the set blood collection time point, and the plasma was separated according to the general plasma separation method (4500rpm, R.T., 5min), and then 20 ⁇ L of sample plasma was drawn.
  • the general plasma separation method 4500rpm, R.T., 5min
  • CT curves were plotted with compound concentration versus time, and the pharmacokinetic parameters were calculated using Winnolin7.0 software non-compartmental statistical moment method.
  • subscript iv represents intravenous injection, and po represents oral administration by gavage.
  • C max and T max are both measured values, and the AUC last value is calculated by the trapezoidal area method.
  • the area under the curve of 0-+ ⁇ time AUC int_obs AUC last + C last / HL_Lambda_z, where C last represents the last identifiable effective minimum concentration, and HL_Lambda_z represents the elimination phase half-life.
  • Oral bioavailability F (AUC int_obsp.o. / D po )/(AUC int_obsi.v. / D iv )*100%, where AUC represents the area under the curve, and D represents the dosage.
  • the c-t curve of PF-06882961 is shown in Figure 3
  • the c-t curve of the low-dose group of compound 1 is shown in Figure 4
  • the c-t curve of the high-dose group of compound 1 is shown in Figure 5.
  • the PK parameters obtained from the test are shown in Table 7.
  • *PF-06882961 is administered orally in the form of its Tris salt, which is synthesized by the synthesis method disclosed in patent WO2018109607A1;
  • the data results show that the intravenous half-life of the low-dose group of compound 1 is short, the oral half-life is long, the drug clearance rate is fast under intravenous administration, and the plasma drug inventory is lower than that of the positive drug; under oral administration, the drug absorption and metabolic distribution are in balance, and the plasma drug inventory is significantly improved compared with the intravenous condition, showing the characteristics of tissue drug accumulation and sustained release, resulting in an oral bioavailability that is better than the positive drug PF-06882961, which is 20.84%.
  • the half-life of the intravenous and oral conditions of the high-dose group is moderate, at 1.37h and 2.65h respectively; the in vivo absorption degree and average AUC value are higher than those of the positive drug, and the oral high-dose group has a higher bioavailability than PF-06882961.
  • the present invention uses the LC-MS/MS method to calculate the resident concentration (c) of compound 17 in the plasma of adult rats at different time points (t) under different administration conditions, draws the c-t curve of the corresponding conditions/corresponding compounds, and uses the Winnolin7.0 software non-mode model to fit and analyze the c-t parameters.
  • Rats SD strain, male, 6-8 weeks old, weight 180 g-220 g;
  • the pharmacokinetic properties of compound 17 were evaluated under the conditions of intravenous injection of 5 mg/kg and oral gavage of 30 mg/kg.
  • the experimental results showed that the intravenous and oral half-lives of compound 17 were moderate, the average plasma drug exposure, apparent distribution volume and clearance were relatively maintained, and the oral bioavailability reached 28.91%.
  • the above experimental results evaluated the in vitro drug properties of compound 17.

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Abstract

一类含硒元素类化合物及其用途。该类化合物具备完全的GLP-1R-cAMP信号通路激动效应(E max)，且化合物GLP-1R-cAMP半数效应浓度(EC 50)均达到纳摩尔级，部分化合物的EC 50达皮摩尔级，是优异的GLP-1受体激动剂。同时，该类化合物的成药性质良好。因此，上述含硒元素类化合物能够用于制备GLP-1受体激动剂，用于制备预防和/或治疗与激动GLP-1受体相关疾病的药物。

Description

一种含硒元素类化合物及其用途 技术领域
本发明属于医药技术领域，具体涉及一种含硒元素类化合物及其用途。
背景技术
硒元素作为第四周期-第六主族元素，较少地被应用于药物化学领域和药物设计领域，但近些年药化领域的研究者们逐渐提升了硒元素在药物设计、开发及合成阶段中的应用兴趣。硒元素因其较大的原子结构和松散的外层价电子性质，显示出独特的氧化-还原性质和亲核性。在生物体内含硒分子通常显示出比含硫分子更高的生物反应活性，易形成Se-S加合物，并显著提升癌细胞的选择性吸收效应，因此含硒分子常作为抗氧化剂、自由基清除剂或促氧化剂等参与炎症抑制治疗或抗肿瘤治疗。硒原子也作为氧原子的生物电子等排体被应用于药物分子设计和天然产物改造之中，很多化合物也由此获得独特的药物活性，硒原子引入或替换也成为化合物性质改良策略的一类新视角选择。
GLP-1是一类快代谢型内源性肠促胰岛素激素，自肠组织与中枢神经组织中产生，作用于GLP-1R。肠源性GLP-1随着循环系统广泛地分布于全身组织，主要起到血糖含量及代谢调节功能；中枢性GLP-1发挥神经递质功能，起到食欲和食物摄取抑制功能。因其重要的血糖及代谢调控功能，GLP-1R激动剂成为糖尿病等代谢性疾病治疗、控制的重要药物。当前获批上市的GLP-1R激动剂均为模拟GLP-1肽的拟肽化合物，如艾塞那肽、利拉鲁肽、度拉糖肽、司美格鲁肽等GLP-1类似物。当前人们仍在探索新类型与新剂型的GLP-1R激动剂，包括迭代性质优化的GLP-1肽类似物，可口服的肽类激动剂，小分子激动剂，变构激动剂等。
Danuglipron(PF-06882961)是由辉瑞公司开发的苯并咪唑-6-羧酸类小分子直接GLP-1R激动剂，具备皮摩尔级的GLP-1R-cAMP信号通路激动活性(EC50)和完全的激动效应(Emax)，是GLP-1R的偏向激动剂。结构生物学信息显示Danuglipron化合物占据部分GLP-1肽作用位点，以结构水网络诱导与模拟关键配体-受体相互作用的构象。Danuglipron显示灵长类物种特异性的激动作用，口服给药表现出可接受的口服吸收以及体内代谢状况，葡萄糖耐量实验显示Danuglipron能有效提升实验对象的葡萄糖耐受水平，促进胰岛素的释放，可有效控制实验对象的血糖水平。现对Danuglipron的血糖控制适应症的Ⅱ期临床试验已经结束，正开展Ⅲ期的临床试验。
GLP-1R小分子激动剂目前报道较少，但随着GLP-1R与配体结合前全长结构的揭示，小分子GLP-1R激动剂的结构类型将多样化。Danuglipron分子的报道为这类结构优化提供了模板，但其药代动力学性质以及毒性方面尚不理想。
发明内容
发明目的：本发明第一目的在于提供一种含硒元素类化合物；本发明第二目的在于提供所述含硒元素类化合物的用途，包括用于制备GLP-1受体激动剂和用于制备通过激动GLP-1受体而直接或间接得到治疗或缓解的疾病的药物的用途。
本发明开发了基于Danuglipron的新结构的含硒元素可口服有效的小分子GLP-1R激动剂，用于制备与GLP-1R相关信号可调节的代谢性疾病的药物。与肽类药物相比，该类小分子激动剂可口服有效；同时改善Danuglipron的药代动力学性质以及心脏毒性。
技术方案：本发明的目的通过下述技术方案实现：
本发明提供了一种通式I的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体：
其中，
环A选自C3-8环烷基、C2-9杂环基、C5-12螺双环基、C5-12螺杂双环基、C5-12稠合双环基、C5-12稠合杂双环基、C5-12桥环基或C5-12桥杂环基；
L选自-C(O)-、-O-、-S-、-CR4R5或-NR6-；
R4、R5各自独立地选自氢、氘、卤素、氰基、C1-C6烷基、C1-C6烷氧基、-NR5aR5b、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；所述C1-C6烷基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基、羟基、C3-C6饱和或部分饱和的环烷基；所述芳基、杂芳基、饱和或部分饱和的环烷基、饱和或部分饱和的杂环基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基或-NR5aR5b；
R5a、R5b各自独立地选自氢或C1-C6烷基；
R6选自氢、C1-C6烷基、C3-C10环烷基、杂环基、-S(O)2R6a或-S(O)2N(R6a)(NR6b)；所述环烷基或杂环基任选地被C1-C6烷基、-F或氰基取代；
R6a、R6b各自独立地选自-H、C1-C6烷基或C3-C10环烷基；
R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基、3-6元杂环基、C3-C6环烷基或5-8元杂芳基；所述3-6元杂环基、C3-C6环烷基、5-8元杂芳基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：H、卤素、C1-C6烷基、C3-C6环烷基、C1-C6烷氧基、C1-C6烷氨基、氰基、氰基取代的C1-C6烷基、羟基、羟基取代的C1-C6烷基、卤代C1-C6烷基、卤代C3-C6环烷基、卤代C1-C6烷氧基、卤代C1-C6烷氨基、酰基、酰胺基、氨酰基、磺酰基或氨基；
R2各自独立地选自氢原子、氘、卤素、氰基、羟基、氧代、C1-C6烷基、C1-C6烷氧基、氨基、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；
R3各自独立地选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
X1、X2、X3和X4相同或不同，且各自独立地选自-CR7或N原子；
R7选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
G选自G1或G2；G1、G2结构式如下：
环B、环C各自独立地选自6-12元芳基或6-12元杂芳基；
Y、Z各自独立地选自-CH2-、-S-、-SO-、-SO2-、-(O＝S＝NH)-、-Se-或-Se(O)-；
R8、R11相同或不同，各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
R9、R10、R12、R13相同或不同，且各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
EE选自-COOH或羧基替代物；所述羧基替代物选自：
m为0、1、2或3；
n为0、1、2、3或4；
o为0、1、2、3、4或5；
p为0、1、2、3、4或5。
本发明还提供了一种通式II的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体：
其中，
环A选自C3-8环烷基、C2-9杂环基、C5-12螺双环基、C5-12螺杂双环基、C5-12稠合双环基、C5-12稠合杂双环基、C5-12桥环基或C5-12桥杂环基；
L选自-C(O)-、-O-、-S-、-CR4R5或-NR6-；
R4、R5各自独立地选自氢、氘、卤素、氰基、C1-C6烷基、C1-C6烷氧基、-NR5aR5b、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；所述C1-C6烷基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基、羟基、C3-C6饱和或部分饱和的环烷基；所述芳基、杂芳基、饱和或部分饱和的环烷基、饱和或部分饱和的杂环基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基或-NR5aR5b；
R5a、R5b各自独立地选自氢或C1-C6烷基；
R6选自氢、C1-C6烷基、C3-C10环烷基、杂环基、-S(O)2R6a或-S(O)2N(R6a)(NR6b)；所述环烷基或杂环基任选地被C1-C6烷基、-F或氰基取代；
R6a、R6b各自独立地选自-H、C1-C6烷基或C3-C10环烷基；
R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基、3-6元杂环基、C3-C6环烷基或5-8元杂芳基；所述3-6元杂环基、C3-C6环烷基、5-8元杂芳基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：H、卤素、C1-C6烷基、C3-C6环烷基、C1-C6烷氧基、C1-C6烷氨基、氰基、氰基取代的C1-C6烷基、羟基、羟基取代的C1-C6烷基、卤代C1-C6烷基、卤代C3-C6环烷基、卤代C1-C6烷氧基、卤代C1-C6烷氨基、酰基、酰胺基、氨酰基、磺酰基或氨基；
R2各自独立地选自氢原子、氘、卤素、氰基、羟基、氧代、C1-C6烷基、C1-C6烷氧基、氨基、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；
R3各自独立地选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
X1、X2、X3和X4相同或不同，且各自独立地选自-CR7或N原子；
R7选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
G选自G1或G2；G1、G2结构式如下：
环B、环C各自独立地选自6-12元芳基或6-12元杂芳基；
Y、Z各自独立地选自-CH2-、-S-、-SO-、-SO2-、-(O＝S＝NH)-、-Se-或-Se(O)-；
R8、R11相同或不同，各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
R9、R10、R12、R13相同或不同，且各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
R14各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
EE选自-COOH或羧基替代物；所述羧基替代物选自：
m为0、1、2或3；
n为0、1、2、3或4；
o为0、1、2、3、4或5；
p为0、1、2、3、4或5；
q为0、1、2或3。
在某些优选的实施方式中，
环A选自C3-8环烷基或C2-9杂环基；
L选自-CR4R5；
R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基；
EE选自-COOH或羧基替代物；
R4、R5如通式I或通式II中定义。
在某些优选的实施方式中，
环A选自C2-9杂环基；
Y、Z各自独立地选自-Se-或-Se(O)-；
EE选自-COOH、
本发明通式I或通式II的化合物优选以下表1所示化合物：
表1优选化合物

本发明涉及的上述通式I或通式II化合物还可以以其盐、水合物、溶剂合物的形式存在，它们在体内转化为通式I或通式II化合物。例如，在本发明的范围内，按照本领域已知的工艺，将本发明化合物转化为药学上可接受的盐的形式，并且以盐形式使用它们。
本发明的某些化合物可以以多晶形式存在。
本发明通式I或通式II化合物的所有的互变异构形式均包括在本发明的范围之内。本发明的化合物可以存在特定的几何或立体异构体形式。烷基等取代基中可存在另外的不对称碳原子，所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
本发明还提供了一种药物组合物，所述药物组合物包括通式I或通式II的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体以及药学上可接受的载体或赋形剂。
本发明的药物组合物可以采用各种已知的方式施用，例如口服、胃肠外施用、通过吸入喷雾施用或经由植入的贮库施用。本发明的药物组合物可单独给药也可与其他药物联合用药。口服组合物可以是任何口服可接受的剂型，包含但不限于片剂、胶囊剂、乳剂以及混悬剂、分散物和溶液。常用的药学上可接受的载体或赋形剂包括稳定剂、稀释剂、表面活性剂、润滑剂、抗氧化剂、粘合剂、着色剂、填充剂、乳化剂等。
无菌可注射组合物可按照本领域已知的技术使用适合的分散剂或润湿剂和助悬剂来配制。可以使用的药学上可接受的载体和溶剂包括水、甘露醇、氯化钠溶液等。
可以改变本发明的药物组合物中活性成分的实际剂量水平以获得对特定患者、组合物和施用方式而言可以有效实现所需治疗响应、对患者无毒的活性成分的量。所选择的的剂量水平取决于多种因素，包括所用的具体的本发明的化合物或其盐的活性、施用途径、施用时间、所用的具体组合物的排泄速率、治疗的持续时间、与所用的具体组合物组合使用的其它药物、化合物和/或材料、所治疗的患者的年龄、性别、体重、一般健康状况和既往病史以及医学领域中公知的类似因素。
本发明还提供了通式I或通式II的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体在制备GLP-1受体激动剂中的用途。
本发明还提供了通式I或通式II的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体在制备预防和/或治疗与激动GLP-1受体相关疾病的药物中的用途。所述药物通过激动GLP-1受体而直接或间接治疗或缓解疾病。所述与激动GLP-1受体相关疾病包括糖尿病、糖尿病并发症、代谢综合征、肥胖症、非酒精性脂肪性肝病、非酒精性脂肪性肝炎、帕金森病、阿尔兹海默症、高血压症、 高血脂症、动脉粥样硬化、心血管风险、冠心病或脑卒中。
有益效果：
本发明提供了一类含硒元素类化合物，该类化合物具备完全的GLP-1R-cAMP信号通路激动效应(Emax)，且化合物GLP-1R-cAMP半数效应浓度(EC50)均达到纳摩尔级，部分化合物的EC50达皮摩尔级，是优异的GLP-1受体激动剂。同时，代表化合物1的成药性质良好。因此，本发明提供的含硒元素类化合物具备开发为预防和/或治疗通过激动GLP-1受体而直接或间接得到治疗或缓解的疾病的药物的前景。
附图说明
图1为化合物1的rHLM测试标准曲线。
图2为PF-06882961的rHLM测试标准曲线。
图3为PF-06882961的PK测试c-t曲线。
图4为化合物1低剂量组的PK测试c-t曲线。
图5为化合物1高剂量组的PK测试c-t曲线。
具体实施方式
下面结合实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。本发明实施例中未注明具体条件的实验方法，通常为常规条件，或按照原料或商品制造厂商所建议的条件；未注明来源的试剂，通常为通过商业途径可购得的常规试剂。
化合物的鉴定、表征及纯度测定方法：
1、NMR：本发明的化合物1H-NMR谱是用Bruker公司核磁共振波谱仪(300MHz)与Bruker公司核磁共振波谱仪(500MHz)测定而得，13C-NMR谱是用Bruker公司核磁共振波谱仪(300MHz)测定而得。化学位移(δ)用ppm表示。使用四甲基硅烷内标准(0.00ppm)。1H-NMR的表示方法：s＝单峰，d＝双重峰，t＝三重峰，m＝多重峰，br＝变宽的，dd＝双重峰的双重峰，dt＝三重峰的双重峰。若提供偶合常数时，其单位为Hz。
2、MS：本发明的化合物质谱是用Shimadzu公司的LC/MS-8050三重四极杆质谱仪测定而得，离子化方式为ESI，分子量精确至小数点后一位数字。
3、HRMS：本发明的化合物高分辨质谱是用Shimadzu公司与Agilent公司的LC/MS质谱仪测定而得，测试方法为Q-TOF，分子量精确至小数点后四位数字。
4、纯度测定：本发明化合物的纯度测定是用Shimadzu公司的LC-20AT高效液相色谱仪测定而得，测试条件：AgilentXDS-C18色谱柱(4.6mm*250mm)，流动相为甲醇：水(含0.1％甲酸)＝70：30，测试化合物样品为浓度约为1mg/mL的色谱甲醇溶液，进样量10μL，测定波长254nm，检测时间10min。纯度使用保留时间(tR)与面积比(％)表征，精确至小数点后两位数字。
实施例1 化合物1的制备
(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物1)
按照如下路线利用起始原料1-1制备化合物1。
步骤1：1-(叔丁基)-4-甲基-4-(6-溴吡啶-2-基)哌啶-1,4-二羧酸(化合物1-3)的制备。
将原料2,6-二溴吡啶(化合物1-1,1.6g，6.7mmol)和N-Boc-哌啶-4-羧酸甲酯(化合物1-2,1.75g，8.1mmol)溶于3mL无水THF，N2保护下降温至-78℃。缓慢注入17mLLiHMDS的THF溶液(17mmol)，反应30min后，升温至室温搅拌2.5h。TLC检测反应结束后，向反应液中滴加稀HCl溶液，pH调节至中性。反应液用EA萃取，饱和氯化钠溶液洗涤两次，浓缩得到无色油状液体，即化合物1-3(2.06g,77.1％)。1H NMR(300MHz,DMSO-d6)δ7.78(t,J＝7.8Hz,1H),7.59(dd,J＝7.9,0.7Hz,1H),7.53-7.49(m,1H),3.70(d,J＝4.4Hz,1H),3.65(s,4H),3.08(s,2H),2.35-2.23(m,2H),1.97(ddd,J＝13.9,7.2,3.2Hz,2H),1.41(s,9H)。LC-MS：[M+H]+＝398.1/400.1(Br同位素)。
步骤2：4-(6-溴吡啶-2-基)-1-(叔丁氧羰基)哌啶-4-羧酸(化合物1-4)的制备。
将中间体1-3(2.06g，5.16mmol)溶于20mL甲醇，逐滴加入NaOH水溶液(2M，26mL)，60℃加热搅拌2h，TLC检测反应完成。稀盐酸酸化至pH小于6，EA萃取，浓缩得到白色晶体，不经纯化直接下一步。
步骤3：4-(6-溴吡啶-2-基)哌啶-1-羧酸叔丁酯(化合物1-5)的制备。
将中间体1-4溶于45mL1,2-二氯乙烷(DCE)，升温至95℃加热4h。TLC检测反应结束后，减压干燥即得产物1-5，为淡黄白色固体(1.52g,86.4％)。1H NMR(300MHz,DMSO-d6)δ7.70(t,J＝7.7Hz,1H),7.49(d,J＝7.8Hz,1H),7.37(d,J＝7.6Hz,1H),4.07(d,J＝13.1Hz,2H),2.86(tt,J＝11.9,2.8Hz,3H),1.83(d,J＝12.8Hz,2H),1.55(td,J＝12.5,4.2Hz,2H),1.43(s,9H)。LC-MS：[M+H]+＝340.1/341.9。
步骤4：4-(6-硒羟基吡啶-2-基)哌啶-1-甲酸叔丁酯(化合物1-6)的制备。
将NaOH(22mg,0.53mmol)与单质硒粉(42mg,0.53mmol)溶于3mL无水DMF，N2保护，注入水合肼(80％,18μL,0.29mmol)，加热至40℃搅拌2h。将中间体1-5(100mg,0.29mmol)的无水DMF溶液加入到反应体系，升温至160℃搅拌4h，TLC显示反应完全。将反应液过滤，用等体积的水和EA萃取，浓缩后经柱层析(洗脱体系为石油醚：乙酸乙酯＝30：1，v/v)纯化得黄色固体，即化合物1-6(55mg,55.2％)。1H NMR(300MHz,DMSO-d6)δ7.71–7.55(m,2H),7.24–7.09(m,1H),4.11–3.98(m,2H),2.90–2.72(m,3H),1.84–1.73(m,2H),1.53(td,J＝12.5,4.2Hz,2H),1.42(s,9H)。LC-MS：[M+H]+＝343.1。
步骤5：4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲酸叔丁酯(化合物1-8)的制备。
将中间体1-6(411mg,1.20mmol)混悬于10mLPEG-400，N2保护。加入NaBH4(136mg,3.60mmol)，室温搅拌0.5h。加入4-氰基-2-氟溴苄(化合物1-7,257mg,1.20mmol)，搅拌15min，体系中析出白色不溶物。TLC显示反应完全，反应液用等体积的水和EA萃取，经柱层析(洗脱体系为石油醚：乙酸乙酯＝50：1，v/v)纯化得无色油状液体，即化合物1-8(420mg,73.7％)。
1H NMR(300MHz,Chloroform-d)δ7.66(t,J＝7.7Hz,1H),7.59–7.54(m,1H),7.49(q,J＝1.6Hz,1H),7.48–7.43(m,1H),7.28(d,J＝8.1Hz,1H),7.07(d,J＝7.6Hz,1H),4.73–4.58(m,2H),4.52–4.27(m,2H),2.99(tt,J＝12.0,4.1Hz,3H),2.06(d,J＝12.9Hz,2H),1.98–1.88(m,2H),1.63(s,9H)。LC-MS：[M+H]+＝476.2。
步骤6：3-氟-4-((6-(哌啶-4-基)吡啶-2-基)硒基)甲基)苯甲腈(化合物1-9)对甲苯磺酸盐的制备。
将中间体1-8(420mg,0.88mmol)溶于10mLEA，加入p-TSA·H2O(502mg,2.64mmol)，60℃加热 搅拌，TLC监测反应进程。反应完毕后蒸干溶剂得到化合物1-9粗品，不经纯化直接投入下一步。
步骤7：(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸甲酯(化合物1-11)的制备。
将中间体1-9粗品溶于10mLMeCN，加入原料(S)-2-(氯甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸甲酯(化合物1-10,235mg,0.80mmol)和K2CO3(552mg，4.00mmol)，升温至50℃搅拌。TLC监测反应完全。经柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得浅白色油状物，即化合物1-11(247mg,48.8％)。1H NMR(300MHz,Chloroform-d)δ8.09(d,J＝1.6Hz,1H),7.92–7.87(m,1H),7.70(d,J＝7.7Hz,1H),7.43(t,J＝7.6Hz,1H),7.32(t,J＝7.7Hz,1H),7.25(s,2H),7.04(d,J＝7.8Hz,1H),6.86(d,J＝7.7Hz,1H),5.14(d,J＝6.0Hz,1H),4.69–4.49(m,3H),4.44(d,J＝1.4Hz,2H),4.32(dt,J＝9.7,6.2Hz,1H),3.92(d,J＝11.6Hz,2H),3.88(d,J＝2.7Hz,3H),2.97(s,2H),2.78–2.59(m,2H),2.45–2.35(m,1H),2.24(s,2H),1.83(d,J＝25.3Hz,4H)。LC-MS：[M+H]+＝634.2。
步骤8：化合物1的制备。
将中间体1-11(247mg,0.39mmol)溶于15mLMeCN，加入TBD(114mg,0.82mmol)的水溶液，室温搅拌过夜。TLC检测反应完毕，使用CA水溶液调pH至5-6。DCM萃取，浓缩经柱层析(洗脱体系为二氯甲烷：甲醇＝30：1，v/v)纯化得到暗白色固体化合物1(152mg,62.9％)。
1H NMR(300MHz,DMSO-d6)δ12.80(s,1H),8.28(s,1H),7.81(d,J＝9.2Hz,2H),7.72–7.49(m,4H),7.29(d,J＝7.9Hz,1H),7.08(d,J＝7.5Hz,1H),5.11(s,1H),4.72(dd,J＝37.1,11.4Hz,2H),4.52(s,2H),4.40(q,J＝8.9,8.4Hz,2H),3.90(dd,J＝54.5,13.8Hz,2H),3.12–2.82(m,2H),2.79–2.55(m,2H),2.44(s,1H),2.31–2.09(m,2H),1.90–1.68(m,4H)。HRMS(ESI):计算值C31H30FN5O3Se，[M+H]+620.1506，实测值620.1556。HPLC：tR＝2.89min，纯度99.56％。
实施例2 化合物2的制备
(S)-2-(4-(6-苄基硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物2)
利用原料溴化苄和中间体1-6为起始原料，按照化合物1的合成路径合成化合物2。化合物2为白色固体，共50.4mg，收率62.5％。1H NMR(300MHz,DMSO-d6)δ8.27(d,J＝1.6Hz,1H),7.80(dd,J＝8.5,1.6Hz,2H),7.76–7.69(m,2H),7.64(d,J＝8.5Hz,1H),7.60–7.50(m,3H),7.28(d,J＝7.7Hz,1H),7.07(d,J＝7.5Hz,1H),5.18–5.03(m,1H),4.86–4.60(m,2H),4.53(s,2H),4.36(dt,J＝14.7,6.3Hz,2H),4.04–3.69(m,2H),3.28(d,J＝6.0Hz,1H),3.04(d,J＝10.9Hz,1H),2.88(d,J＝11.1Hz,1H),2.65(td,J＝17.0,14.4,6.4Hz,2H),2.21(dd,J＝22.5,11.2Hz,2H),1.86(td,J＝13.0,12.5,6.7Hz,4H)。HRMS(ESI):计算值C30H32N4O3Se,[M+H]+577.1712,实测值577.1708。HPLC：tR＝2.45min，纯度97.02％。
实施例3 化合物3的制备
(S)-2-(4-(6-(4-氰基苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物3)
利用原料4-氰基溴苄和中间体1-6为起始原料，按照化合物1的合成路径合成化合物3。化合物3为白色固体，共46.7mg，收率42.2％。1H NMR(300MHz,DMSO-d6)δ12.83(s,1H),8.30(d,J＝1.6Hz,1H),7.88–7.71(m,4H),7.67(d,J＝8.4Hz,1H),7.63–7.52(m,3H),7.31(d,J＝7.8Hz,1H),7.10(d,J＝7.6Hz,1H),5.18–5.04(m,1H),4.89–4.63(m,2H),4.56(s,2H),4.41(tq,J＝12.3,5.6Hz,2H),4.06–3.76(m,2H),3.29(d,J＝6.0Hz,1H),3.07(d,J＝11.1Hz,1H),2.91(d,J＝11.0Hz,1H),2.80–2.60(m,2H),2.24(dd,J＝22.4,11.1Hz,2H),1.88(dd,J＝15.5,9.4Hz,4H)。HRMS(ESI):计算值C31H31N5O3Se,[M+H]+602.1665,实 测值602.1651。HPLC：tR＝3.15min，纯度98.92％。
实施例4 化合物4的制备
(S)-2-(4-(6-(4-氯苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物4)
利用原料4-氯溴苄和中间体1-6为起始原料，按照化合物1的合成路径合成化合物4。化合物4为白色固体，共43.7mg，收率47.1％。1H NMR(300MHz,DMSO-d6)δ12.74(s,1H),8.27(s,1H),7.80(d,J＝8.5Hz,1H),7.65(d,J＝8.6Hz,1H),7.52(t,J＝7.7Hz,1H),7.40(d,J＝8.1Hz,2H),7.28(dd,J＝15.0,7.9Hz,3H),7.06(d,J＝7.6Hz,1H),5.09(d,J＝7.3Hz,1H),4.86–4.58(m,2H),4.54–4.29(m,4H),4.09–3.72(m,2H),3.27(t,J＝6.0Hz,1H),3.11–2.83(m,2H),2.67(s,2H),2.23(d,J＝22.0Hz,2H),1.84(s,4H)。HRMS(ESI):计算值C30H31ClN4O3Se,[M+H]+611.1323,实测值611.1317。HPLC：tR＝5.39min,纯度96.75％。
实施例5 化合物5的制备
(S)-2-(4-(6-(2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物5)
利用原料2-氟溴苄和中间体1-6为起始原料，按照化合物1的合成路径合成化合物5。化合物5为白色固体，共42.3mg，收率38.3％。1H NMR(300MHz,DMSO-d6)δ8.27(s,1H),7.85–7.75(m,1H),7.65(d,J＝8.5Hz,1H),7.53(t,J＝7.7Hz,1H),7.45(t,J＝7.5Hz,1H),7.25(dd,J＝10.6,7.0Hz,2H),7.20–7.01(m,3H),5.10(d,J＝7.7Hz,1H),4.88–4.57(m,2H),4.48(s,2H),4.43–4.31(m,2H),4.09–3.71(m,2H),2.96(dd,J＝49.8,11.0Hz,2H),2.80–2.59(m,2H),2.44(d,J＝8.8Hz,1H),2.35–2.11(m,2H),1.85(dt,J＝13.5,6.5Hz,4H)。HRMS(ESI):计算值C30H31FN4O3Se,[M+H]+595.1618,实测值595.1617.HPLC：tR＝6.51min,纯度94.38％。
实施例6 化合物6的制备
(S)-2-(4-(6-(4-氯-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物6)
利用原料4-氯-2-氟溴苄和中间体1-6为起始原料，按照化合物1的合成路径合成化合物6。化合物6为白色固体，共62.5mg，收率75.9％。1H NMR(300MHz,DMSO-d6)δ12.74(s,1H),8.27(s,1H),7.80(d,J＝8.5Hz,1H),7.65(d,J＝8.6Hz,1H),7.52(t,J＝7.7Hz,1H),7.40(d,J＝8.1Hz,2H),7.28(dd,J＝15.0,7.9Hz,3H),7.06(d,J＝7.6Hz,1H),5.09(d,J＝7.3Hz,1H),4.86–4.58(m,2H),4.54–4.29(m,4H),4.09–3.72(m,2H),3.27(t,J＝6.0Hz,1H),3.11–2.83(m,2H),2.67(s,2H),2.23(d,J＝22.0Hz,2H),1.84(s,4H)。HRMS(ESI):计算值C30H30ClFN4O3Se,[M+H]+629.1256,实测值629.1218。HPLC：tR＝5.79min,纯度97.66％。
实施例7 化合物7的制备
(S)-2-(4-(6-((2-甲氧基-4-(三氟甲基)苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物7)
利用原料2-甲氧基-4-(三氟甲基)苄基溴和中间体1-6为起始原料，按照化合物1的合成路径合成化合物7。化合物7为白色固体，共45mg，收率60.5％。1H NMR(300MHz,DMSO-d6)δ8.26(s,1H),7.85–7.76(m,1H),7.64(d,J＝8.5Hz,1H),7.52(dt,J＝7.8,3.8Hz,2H),7.29–7.15(m,3H),7.06(d,J＝7.6Hz,1H),5.10(d,J＝7.6Hz,1H),4.80(dd,J＝15.0,7.2Hz,1H),4.71–4.60(m,1H),4.48–4.31(m,4H),3.98(d,J＝13.5Hz,1H),3.88(s,3H),3.79(d,J＝13.4Hz,1H),3.05(d,J＝11.0Hz,1H),2.90(d,J＝11.1Hz,1H),2.71(s,2H),2.44(d,J＝8.4Hz,1H),2.48–2.14(m,2H),1.85(s,4H)。HRMS(ESI):计算值C32H33F3N4O4Se,[M+H]+675.1619,实测值675.1657。HPLC：tR＝7.22min,纯度96.63％。
实施例8 化合物8的制备
(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌嗪-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物8)
按照如下路线利用原料1-1和8-2制备化合物8。
步骤1：4-(6-溴吡啶-2-基)哌嗪-1-羧酸叔丁酯(化合物8-2)的制备。
将原料2,6-二溴吡啶(化合物1-1,346mg,1.46mmol)溶于10mLDMF中，加入原料1-Boc-哌嗪(化合物8-1,353mg,1.89mmol)和K2CO3(405mg,2.93mmol)，80℃加热搅拌4h。TLC显示反应完全，经柱层析(洗脱体系为石油醚：乙酸乙酯＝20：1，v/v)纯化得白色固体8-2(331mg,66.1％)。1H NMR(300MHz,Chloroform-d)δ7.33(dd,J＝8.4,7.4Hz,1H),6.81(d,J＝7.5Hz,1H),6.56(d,J＝8.3Hz,1H),3.56(s,8H),1.51(s,9H)。LC-MS：[M+H]+＝342.1。
步骤2：4-(6-硒羟基吡啶-2-基)哌嗪-1-羧酸叔丁酯(化合物8-3)的制备。
将NaOH(180mg,4.50mmol)与单质硒粉(356mg,4.50mmol)溶于6mL无水DMF，N2保护，注入水合肼(80％,150μL,3.0mmol)，加热至40℃搅拌2h。将中间体8-2(514mg,1.50mmol)溶于5mL无水DMF后加入到反应体系，升温至160℃搅拌4h，TLC显示反应完全。将反应液过滤，用等体积的水和EA萃取，浓缩经柱层析(洗脱体系为石油醚：乙酸乙酯＝15：1，v/v)纯化得黄色固体8-3(331mg,64.3％)。1H NMR(300MHz,Chloroform-d)δ7.45(t,J＝7.4Hz,1H),7.12(dd,J＝7.5,1.5Hz,1H),6.87(dd,J＝7.5,1.5Hz,1H),6.19(s,1H),3.62(s,8H),1.46(s,9H)。LC-MS：[M+H]+＝344.0。
步骤3：4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌嗪-1-羧酸叔丁酯(化合物8-4)的制备。
将中间体8-3(169mg,0.49mmol)混悬于10mLPEG-400，N2保护。加入NaBH4(56mg,0.49mmol)，室温搅拌0.5h。加入4-氰基-2-氟溴苄(化合物1-7,105mg,0.49mmol)，搅拌15min，体系中析出白色不溶物。TLC显示反应完全，反应液用等体积的水和EA萃取，经柱层析(洗脱体系为石油醚：乙酸乙酯＝30：1，v/v)纯化得无色油状液体，即化合物8-4(128mg,73.7％)。1H NMR(300MHz,Chloroform-d)δ7.54(t,J＝7.7Hz,1H),7.43–7.34(m,3H),6.72(d,J＝7.4Hz,1H),6.47(d,J＝8.4Hz,1H),4.51(d,J＝1.4Hz,2H),3.62(s,8H),1.55(s,9H)。LC-MS：[M+H]+＝477.1。
步骤4：化合物8的制备。
依照化合物1的合成方法，以中间体8-4为原料制备化合物8。化合物8为淡黄色固体，共64.5mg，收率43.7％。1H NMR(300MHz,DMSO-d6)δ8.28(d,J＝1.6Hz,1H),7.86–7.77(m,2H),7.69–7.55(m,3H),7.35(t,J＝7.9Hz,1H),6.69(d,J＝7.4Hz,1H),6.59(d,J＝8.4Hz,1H),5.11(d,J＝7.3Hz,1H),4.81(dd,J＝ 15.4,7.2Hz,1H),4.71–4.62(m,1H),4.55–4.33(m,4H),4.05–3.77(m,2H),3.52(d,J＝5.7Hz,4H),2.70(d,J＝9.2Hz,1H),2.57(d,J＝5.2Hz,4H),2.42(d,J＝9.0Hz,1H)。HRMS(ESI):计算值C30H29FN6O3Se,[M+H]+621.1523,实测值621.1523。HPLC：tR＝5.00min,纯度97.48％。
实施例9 化合物9的制备
(S)-2-(4-(6-(4-氯-2-氟苄基)硒基)吡啶-2-基)哌嗪-1-甲基)-1-(氧杂环丁烷-2-甲基)-1H-苯并[d]咪唑-6-羧酸(化合物9)
利用原料4-氯-2-氟溴苄和中间体8-4，按照化合物8的合成路径合成化合物9。化合物9为黄色固体，共79.4mg，收率62.5％。1H NMR(300MHz,DMSO-d6)δ9.07(s,1H),8.22(s,1H),7.82(d,J＝8.4Hz,1H),7.57(d,J＝8.4Hz,1H),7.47–7.30(m,3H),7.20(dd,J＝8.3,2.2Hz,1H),6.68(d,J＝7.4Hz,1H),6.58(d,J＝8.4Hz,1H),5.10(dt,J＝9.4,4.7Hz,1H),4.72(ddd,J＝40.5,15.3,5.1Hz,2H),4.49(q,J＝7.3Hz,1H),4.38(d,J＝9.1Hz,3H),4.04–3.94(m,1H),3.81(d,J＝13.4Hz,1H),3.27(t,J＝5.9Hz,4H),3.20(t,J＝5.8Hz,4H),2.77–2.66(m,1H),2.44(t,J＝9.2Hz,1H)。HRMS(ESI):计算值C29H29ClFN5O3Se,[M+H]+630.1108,实测值630.1180。HPLC：tR＝4.99min,纯度94.42％。
实施例10 化合物10的制备
2-(4-(6-(4-氰基-2-氟苄基)亚硒砜基)吡啶-2-基)哌啶-1-甲基)-1-((S)-氧杂环丁烷-2-基)甲基)-1H-苯并[d]咪唑-6-羧酸(化合物10)
按照如下路线利用原料1-9制备化合物10。
步骤1：3-氟-4-((6-(哌啶-4-基)吡啶-2-基)亚硒砜基)甲基)苯甲腈(化合物10-1)的制备。
将中间体1-9粗品(108mg,0.29mmol)溶于5mLDCM和5mLMeOH，冰浴降温至0℃。加入NCS(46.5mg,0.35mmol)，搅拌0.5h。向体系中加入5mLDCM，加入10％的NaOH水溶液(2.9mL,10mL/mmol)，搅拌5min。TLC显示反应完全。使用等体积的水和DCM萃取，浓缩经柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得无色油状液体10-1(81mg,71.6％)。
1H NMR(300MHz,Chloroform-d)δ7.72(t,J＝7.7Hz,1H),7.39(ddd,J＝17.5,7.8,1.3Hz,2H),7.27–7.14(m,3H),4.35–4.12(m,2H),3.33–3.21(m,2H),2.84(m,J＝22.2,14.8,11.9,3.2Hz,3H),1.99–1.71(m,4H)，LC-MS：[M+H]+＝392.0。
步骤2：化合物10的制备。
依照化合物1的合成方法，以中间体10-1为原料制备化合物10。化合物10为白色固体，共18.0mg，收率40.5％。1H NMR(300MHz,DMSO-d6)δ9.07(s,1H),8.22(s,1H),7.82(d,J＝8.4Hz,1H),7.57(d,J＝8.4Hz,1H),7.47–7.30(m,3H),7.20(dd,J＝8.3,2.2Hz,1H),6.68(d,J＝7.4Hz,1H),6.58(d,J＝8.4Hz,1H),5.10(dt,J＝9.4,4.7Hz,1H),4.72(ddd,J＝40.5,15.3,5.1Hz,2H),4.49(q,J＝7.3Hz,1H),4.38(d,J＝9.1Hz,3H),4.04–3.94(m,1H),3.81(d,J＝13.4Hz,1H),3.27(t,J＝5.9Hz,4H),3.20(t,J＝5.8Hz,4H),2.77–2.66(m,1H),2.44(t,J＝9.2Hz,1H)。HRMS(ESI):计算值C31H30ClFN5O4Se,[M+H]+630.1520,实测值630.1579。HPLC：tR＝4.99min,纯度94.42％。
实施例11 化合物11的制备
(S)-2-(4-(6-(4-氰基-2-氟苯基)硒基甲基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪 唑-6-羧酸(化合物11)
按照如下路线利用原料11-1制备化合物11。
步骤1：3-氟-4-氢亚硒苯甲腈(化合物11-2)的制备。
将NaOH(243mg,6.07mmol)与单质硒粉(480mg,6.07mmol)溶于5mL无水DMF，N2保护，注入水合肼(80％,128μL,2.02mmol)，加热至40℃搅拌2h。将3-氟-4-碘苯甲腈(化合物11-1,500mg,2.02mmol)溶于5mL无水DMF溶液加入到反应体系，升温至160℃搅拌4h，TLC显示反应完全。将反应液过滤，用等体积的水和EA萃取，浓缩经柱层析(洗脱体系为石油醚：乙酸乙酯＝20：1，v/v)纯化得黄色固体，即化合物11-2(410mg,50.9％)。1H NMR(300MHz,Chloroform-d)δ7.71(dd,J＝8.1,6.8Hz,1H),7.45–7.30(m,2H)。LC-MS：[M+H]+＝201.9。
步骤2：6-(羟甲基)-3',6'-二氢-(2,4'-联吡啶)-1'(2'H)-羧酸叔丁酯(化合物11-5)的制备。
将6-氯-2-羟甲基吡啶(化合物11-3,231mg,1.62mmol)溶于20mL二氧六环和2mLH2O，加入N-Boc-1,2,5,6-四氢吡啶-4-硼酸频哪醇酯(化合物11-4,999mg,3.23mmol)、Pd(dppf)Cl2(118mg,0.16mmol)和Cs2CO3(1153mg,3.55mmol)，N2保护，升温至90℃搅拌2h，TLC显示反应完全。反应液用等体积的水和EA萃取。浓缩经柱层析(洗脱体系为石油醚：乙酸乙酯＝50：1，v/v)纯化得无色油状液体，即化合物11-5(369mg,78.7％)。1H NMR(300MHz,Chloroform-d)δ7.67(t,J＝7.7Hz,1H),7.11(d,J＝7.7Hz,2H),6.56(s,1H),4.78(s,2H),4.31(s,2H),2.91(dd,J＝12.2,4.3Hz,2H),1.99(s,2H),1.79(dd,J＝12.6,4.4Hz,2H),1.54(s,9H)。LC-MS：[M+H]+＝291.3。
步骤3：4-(6-(羟甲基)吡啶-2-基)哌啶-1-甲酸叔丁酯(化合物11-6)的制备。
将中间体11-5(369mg,1.27mmol)溶于10mLMeOH，加入等质量的Pd/C(10％)，H2置换，搅拌2h，LC-MS/MS显示反应结束，过滤，旋干溶剂，得到无色油状液体，即化合物11-6(369mg,99.2％)。1HNMR(300MHz,Chloroform-d)δ7.67(t,J＝7.7Hz,1H),7.11(d,J＝7.7Hz,2H),4.78(s,2H),4.31(s,2H),2.91(m,4H),1.99(s,2H),1.79(d,J＝12.6Hz,2H),1.54(s,9H)。LC-MS：[M+H]+＝293.3。
步骤4：4-(6-(溴甲基)吡啶-2-基)哌啶-1-甲酸叔丁酯(化合物11-7)的制备。
将中间体11-6(369mg,1.27mmol)溶于10mLDCM，加入CBr4(420mg,1.27mmol)和PPh3(499mg,1.90mmol)，室温搅拌3h，TLC显示反应完全。经柱层析(洗脱体系为石油醚：乙酸乙酯＝20：1，v/v)纯化得黄白色固体，即化合物11-7(234mg,52.2％)。1H NMR(300MHz,Chloroform-d)δ7.64(t,J＝7.7Hz,1H),7.31–7.27(m,1H),7.07(d,J＝7.8Hz,1H),4.53(s,2H),4.25(d,J＝13.1Hz,2H),2.86(d,J＝12.6Hz,3H),1.92(d,J＝13.1Hz,2H),1.76–1.63(m,2H),1.48(d,J＝1.1Hz,9H)。LC-MS：[M+H]+＝355.1。
步骤5：4-(6-(4-氰基-2-氟苯基)硒基甲基)吡啶-2-基)哌啶-1-甲酸叔丁酯(化合物11-8)的制备。
将中间体11-2(66mg,0.33mmol)和NaBH4(37.4mg,0.99mmol)溶于2mL无水乙醇，N2保护， 滴加中间体11-7(234mg,0.66mmol)的乙醇溶液(3mL)，室温搅拌过夜。TLC显示反应完全，反应液用等体积的水和EA萃取，经柱层析(洗脱体系为石油醚：乙酸乙酯＝30：1，v/v)纯化得无色油状液体，即化合物11-8(90mg,73.7％)。1H NMR(300MHz,Chloroform-d)δ7.72(dt,J＝8.1,5.7Hz,1H),7.56(td,J＝10.4,9.1,5.3Hz,1H),7.37–7.22(m,2H),7.15(d,J＝7.6Hz,1H),7.01(d,J＝7.7Hz,1H),4.29(d,J＝21.9Hz,4H),2.82(d,J＝12.6Hz,3H),1.86(d,J＝13.0Hz,2H),1.69(dt,J＝12.4,6.7Hz,2H),1.49(s,9H)。LC-MS：[M+H]+＝476.1。
步骤6：化合物11的制备。
依照化合物1的合成方法，以中间体11-8为原料制备化合物10。化合物10为暗白色固体，共35mg，收率67.4％。1H NMR(300MHz,DMSO-d6)δ13.12–12.50(m,1H),8.28(d,J＝1.6Hz,1H),7.98–7.87(m,1H),7.86–7.74(m,2H),7.72–7.57(m,3H),7.30(d,J＝7.7Hz,1H),7.13(d,J＝7.7Hz,1H),5.11(dt,J＝9.3,4.7Hz,1H),4.83(dd,J＝15.2,7.1Hz,1H),4.68(dd,J＝15.1,2.8Hz,1H),4.56–4.44(m,3H),4.40(dt,J＝9.0,5.9Hz,1H),3.97(d,J＝13.5Hz,1H),3.79(d,J＝13.4Hz,1H),3.01(d,J＝11.0Hz,1H),2.87(d,J＝11.1Hz,1H),2.80–2.68(m,1H),2.60(d,J＝11.1Hz,1H),2.45(dd,J＝9.9,7.4Hz,1H),2.30–2.10(m,2H),1.70(dd,J＝24.9,11.4Hz,4H)。HRMS(ESI):计算值C31H30FN5O3Se,[M+H]+620.1571,实测值620.1531。HPLC：tR＝3.26min,纯度98.20％。
实施例12 化合物12的制备
(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-酰基羟胺(化合物12)
按照如下路线利用化合物1为原料制备化合物12。
步骤1：中间体12-2的制备
将化合物1(70mg，0.11mmol)溶于5mL无水DMF，加入EDCI(65mg，0.34mmol)、HOBt(45.8mg，0.34mmol)、DIPEA(59μL，0.34mmol)以及NH2OTHP(化合物12-1,26.5mg，0.22mmol)，室温搅拌3h。TLC检测反应完毕，反应液用等体积的水和EA萃取，合并有机相并浓缩得粗品中间体12-2(58mg，48.8％)。LC-MS：[M+H]+＝719.2。
步骤2：化合物12的制备
将中间体12-2(58mg，0.08mmol)溶于5mL甲醇，滴加TFA(6.2μL，0.08mmol)的甲醇溶液，室温搅拌2h。TLC检测反应完毕，经经柱层析(洗脱体系为二氯甲烷：甲醇＝20：1，v/v)纯化得到化合物12，为白色固体(36mg,70.3％)。1H NMR(300MHz,DMSO-d6)δ11.17(s,1H),8.99(s,1H),8.08(s,1H),7.81(d,J＝9.8Hz,1H),7.65(d,J＝8.2Hz,1H),7.61(s,3H),7.53(d,J＝7.7Hz,1H),7.29(d,J＝7.6Hz,1H),7.08(d,J＝7.4Hz,1H),5.13(s,1H),4.76(t,J＝11.0Hz,1H),4.62(d,J＝14.9Hz,1H),4.52(s,2H),4.41(q,J＝6.9Hz,2H),3.97(d,J＝13.4Hz,1H),3.78(d,J＝13.5Hz,1H),3.03(d,J＝11.0Hz,1H),2.89(d,J＝11.0Hz,1H),2.76–2.64(m,3H),2.21(dd,J＝22.6,11.5Hz,2H),1.82(d,J＝11.9Hz,4H).HRMS(ESI):计算值C31H31FN6O3Se,[M+H]+635.1607,实测值635.1650。HPLC：tR＝2.51min,纯度99.60％。
实施例13 化合物13的制备
(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-酰胺甲基磺酰胺(化合物13)
按照如下路线利用化合物1为原料制备化合物13。
步骤1：化合物13的制备。
将化合物1(40mg，0.06mmol)溶于5mL无水DCM，冰浴下加入2-氯-1-甲基吡啶碘化物(33mg，0.13mmol)、DMAP(11.8mg，0.10mmol)和Et3N(27μL，0.19mmol),搅拌0.5h后加入甲基磺酰胺(化合物13-1,6.8mg，0.07mmol)，室温搅拌10h。TLC检测反应完毕，反应液用等体积的水和DCM萃取，合并有机相，浓缩经柱层析(洗脱体系为二氯甲烷：甲醇＝15：1，v/v)纯化得到化合物13，共27mg，收率60.0％。1H NMR(300MHz,DMSO-d6)δ8.35(d,J＝1.6Hz,1H),7.83(t,J＝8.8Hz,2H),7.73(d,J＝8.5Hz,1H),7.70–7.52(m,3H),7.32(d,J＝7.8Hz,1H),7.11(d,J＝7.6Hz,1H),5.12(d,J＝7.1Hz,1H),4.80(dd,J＝15.3,7.0Hz,1H),4.65(d,J＝13.6Hz,1H),4.50(d,J＝21.6Hz,2H),4.41(dd,J＝9.2,6.2Hz,3H),2.86(s,1H),2.79–2.64(m,2H),2.41(s,1H),2.27–2.12(m,2H),1.98(s,3H),1.94(s,2H),1.47(s,1H),1.34(d,J＝4.7Hz,3H)。HRMS(ESI):计算值C32H33FN6O4SSe,[M+H]+697.1433,实测值697.1486。HPLC：tR＝2.90min,纯度93.43％。
实施例14 化合物14的制备
(S)-2-(4-(6-(4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-甲基磺酰脲(化合物14)
按照如下路线利用原料14-1制备化合物14。
步骤1：((3-氟-4-硝基苯基)磺酰)氨基甲酸叔丁酯(化合物14-2)的制备
将氨基甲酸叔丁酯(10.74g,91.7mmol)溶于100mL无水乙腈，N2保护下降温至-78℃。将化合物3-氟-4-硝基苯磺酰氯(化合物14-1,18.26g,76.4mmol)溶于150mL无水乙腈后快速加入。反应半小时以后，升温至室温搅拌1.5h。TLC检测反应结束后，向反应液中滴加稀HCl溶液，调节pH到中性。反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物经过柱层析(洗脱体系为石油醚：乙酸乙酯＝3：1，v/v)纯化得到中间体14-2(10.47g,42.8％)。
1H NMR(300MHz,DMSO-d6)δ8.19(dd,J＝8.8,7.3Hz,1H),7.78–7.65(m,2H),1.22(s,9H)。LC-MS：[M+H]+＝321.0。
步骤2：(S)-((4-硝基-3-((氧杂环丁胺-2-基甲基)氨基)苯基)磺酰)氨基甲酸叔丁酯(化合物14-3)的制备。
将中间体14-2(10.47g,32.71mmol)溶于50mL乙腈，加入DIPEA(6.83ml,39.25mmol)和(S)-氧杂环丁烷-2-基甲胺(3.42g,39.25mmol)，升温至60℃反应4h。TLC检测反应结束后，加入饱和氯化铵 溶液淬灭。反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，得到中间体14-3(9.18g,72.5％)。1H NMR(300MHz,DMSO-d6)δ8.41(t,J＝5.7Hz,1H),8.19(d,J＝8.9Hz,1H),7.49(d,J＝1.8Hz,1H),7.01(dd,J＝9.0,1.8Hz,1H),5.00(t,J＝5.9Hz,1H),4.62–4.39(m,2H),3.72–3.57(m,3H),2.76–2.52(m,2H),1.26(d,J＝9.3Hz,9H)。LC-MS：[M+H]+＝388.1。
步骤3：(S)-((4-氨基-3-((氧杂环丁胺-2-基甲基)氨基)苯基)磺酰)氨基甲酸叔丁酯(化合物14-4)的制备。
将中间体14-3(9.18g,23.72mmol)溶于40mL甲醇，加入10％Pd/C(918mg)，N2保护下升温至50℃反应2h。TLC检测反应结束后，硅藻土过滤反应液并低压浓缩除去溶剂，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝100：1，v/v)纯化得到中间体14-4(5.1g,60.2％)。
1H NMR(300MHz,DMSO-d6)δ11.01(s,1H),6.99(dd,J＝8.2,2.1Hz,1H),6.86(d,J＝2.1Hz,1H),6.58(d,J＝8.2Hz,1H),5.60(s,2H),4.95(dt,J＝14.1,6.0Hz,2H),4.61–4.41(m,2H),3.31(ddt,J＝18.9,13.2,6.9Hz,2H),2.67(dtd,J＝10.9,8.1,6.2Hz,1H),2.50–2.35(m,1H),1.30(s,9H)。LC-MS：[M+H]+＝358.1。
步骤4：(S)-((2-(氯甲基)-1-(氧杂环丁胺-2-基甲基)-1H-苯并[d]咪唑-6-基)磺酰)氨基甲酸叔丁酯(化合物14-5)的制备。
将中间体14-4(5.1g,14.24mmol)溶于30mL乙腈，加入对甲苯磺酸水合物(2.4g,1.42mmol)和2-氯-1,1,1-三甲氧基乙烷(2.63g,17.09mmol)，升温至60℃反应4h。TLC检测反应结束后，反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝200：1，v/v)纯化得到中间体14-5(2.4g,40.6％)。
1H NMR(300MHz,DMSO-d6)δ11.64(s,1H),8.30(d,J＝1.8Hz,1H),7.88(d,J＝8.6Hz,1H),7.74(dd,J＝8.6,1.8Hz,1H),5.21–5.09(m,2H),5.05(td,J＝7.2,2.7Hz,1H),4.79(dd,J＝15.5,6.9Hz,1H),4.67(dd,J＝15.5,2.9Hz,1H),4.47(td,J＝8.1,7.7,5.8Hz,1H),4.32(dt,J＝9.1,5.9Hz,1H),2.79–2.61(m,1H),2.46–2.29(m,1H),1.27(s,9H)。LC-MS：[M+H]+＝416.1。
步骤5：(S)-((2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(氧杂环丁胺-2-基甲基)-1H-苯并[d]咪唑-6-基)磺酰)氨基甲酸叔丁酯(化合物14-6)的制备。
将中间体1-9(4.18g,5.82mmol)溶于15mL乙腈，加入K2CO3(3.98g,28.85mmol)室温搅拌0.5h，再加入化合物14-5(2.4g,5.77mmol)升温至50℃反应2h。TLC检测反应结束后，加入饱和氯化铵溶液淬灭。反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝50：1，v/v)纯化得到中间体14-6(3g,68.9％)。
1H NMR(300MHz,DMSO-d6)δ11.58(s,1H),8.23(d,J＝1.7Hz,1H),7.90–7.77(m,2H),7.76–7.49(m,4H),7.30(d,J＝7.8Hz,1H),7.08(d,J＝7.6Hz,1H),5.78(s,1H),5.12(t,J＝8.1Hz,1H),4.82(dd,J＝15.3,7.1Hz,1H),4.69(d,J＝15.0Hz,1H),4.53(s,2H),4.47–4.30(m,2H),4.03(d,J＝13.6Hz,1H),3.87(d,J＝13.6Hz,1H),3.07(d,J＝11.0Hz,1H),2.93(d,J＝11.1Hz,1H),2.69(s,2H),2.49–2.34(m,1H),2.27(dd,J＝20.3,10.4Hz,2H),1.80(dd,J＝24.2,11.6Hz,3H),1.27(s,9H)。LC-MS：[M+H]+＝755.2。
步骤6：(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(氧杂环丁胺-2-基甲基)-1H-苯并[d]咪唑-6-磺酰胺(化合物14-7)的制备。
将中间体14-6(3g,3.98mmol)溶于20mL二氯甲烷，逐滴加入CF3COOH(4.54g,39.8mmol)，室温搅拌2h。TLC检测反应结束后，加入饱和碳酸氢钠溶液淬灭。反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，得到中间体14-7(2.18g,83.8％)。
1H NMR(300MHz,DMSO-d6)δ8.13(d,J＝1.7Hz,1H),7.84–7.50(m,6H),7.29(t,J＝3.8Hz,3H),7.08(d,J＝7.6Hz,1H),5.76(s,1H),5.11(d,J＝6.8Hz,1H),4.79(dd,J＝15.2,7.1Hz,1H),4.70–4.60(m,1H),4.52(s,2H),4.45–4.34(m,2H),3.98(d,J＝13.5Hz,1H),3.80(d,J＝13.5Hz,1H),3.03(d,J＝11.2Hz,1H),2.88(d,J＝11.2Hz,1H),2.68(s,2H),2.43(q,J＝8.3Hz,1H),2.23(dt,J＝21.4,11.5Hz,2H),1.88–1.73(m,3H)。LC-MS：[M+H]+＝655.1。
步骤7：化合物14的制备。
将中间体14-7(500mg,0.76mmol)溶于5mL乙腈，加入N-甲基氨基甲酸苯酯(127mg,0.84mmol)和DBU(139mg,0.92mmol)，室温搅拌2h。TLC检测反应结束后，加入饱和氯化铵溶液淬灭。反应液用等体积的乙酸乙酯和水分液萃取3次，合并有机层并低压浓缩除去溶剂，残留物经过柱层析(洗脱体系为二氯甲烷：甲醇＝20：1，v/v)得到化合物14(397mg,73.1％)。1H NMR(300MHz,DMSO-d6)δ10.69(s,1H), 8.25(d,J＝1.7Hz,1H),7.87–7.76(m,2H),7.72(dd,J＝8.6,1.7Hz,1H),7.63(d,J＝5.9Hz,2H),7.55(t,J＝7.7Hz,1H),7.30(d,J＝7.8Hz,1H),7.09(d,J＝7.6Hz,1H),6.41(d,J＝4.8Hz,1H),5.77(s,1H),5.14–5.06(m,1H),4.81(dd,J＝15.2,7.0Hz,1H),4.74–4.63(m,1H),4.53(s,2H),4.47–4.30(m,2H),3.09(s,1H),2.94(s,1H),2.76–2.59(m,2H),2.48(s,1H),2.34–2.13(m,1H),1.85(s,4H),1.49(s,1H),1.34(s,1H),1.24(d,J＝4.3Hz,3H)。HRMS(ESI):计算值C32H34FN7O5SSe,[M+H]+712.1542,实测值712.1582。HPLC：tR＝3.60min,纯度94.67％。
实施例15 化合物15的制备
(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-N-(甲基氨基甲酰基)-1-(氧杂环丁-2-基甲基)-1H-苯并[d]咪唑-6-环丙基磺酰脲(化合物15)
以环丙基氨基甲酸苯酯和中间体14-7为原料，按照化合物14的合成路径合成化合物15。化合物15为白色固体，共62mg，收率66.2％。1H NMR(300MHz,DMSO-d6)δ10.71(s,1H),8.39–8.29(m,1H),7.98–7.74(m,3H),7.72–7.56(m,3H),7.36(d,J＝7.9Hz,1H),7.13(d,J＝7.5Hz,1H),7.02–6.96(m,1H),5.77(s,1H),5.04(d,J＝7.0Hz,1H),4.89(s,2H),4.75(d,J＝15.2Hz,1H),4.59–4.43(m,2H),4.36(d,J＝8.6Hz,1H),3.83(s,2H),3.04(s,1H),2.74(s,1H),2.37(d,J＝7.4Hz,2H),2.34–2.11(m,3H),1.55–1.45(m,1H),1.37–1.22(m,1H),1.24(s,5H),0.85(m,1H)。HRMS(ESI):计算值C34H36FN7O4SSe,[M+H]+738.1771,实测值738.1735。HPLCtR＝4.21min，纯度99.25％。
实施例16 化合物16的制备
(S)-2-((4-(6-((2-氟-4-(三氟甲基)苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(氧杂环丁-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物16)
利用原料1-(溴甲基)-2-氟-4-(三氟甲基)苯和中间体1-6为起始原料，按照化合物1的合成路径合成化合物16。化合物16为白色固体，共28.4mg，收率59.9％。1H NMR(300MHz,DMSO-d6)δ8.28(s,1H),7.82(d,J＝8.4Hz,1H),7.78–7.54(m,2H),7.51(dd,J＝11.8,7.9Hz,4H),7.32(dd,J＝17.3,7.8Hz,1H),7.09(dd,J＝7.7,5.0Hz,1H),5.11(dd,J＝7.2,2.8Hz,1H),4.81(dd,J＝15.2,7.1Hz,1H),4.66(dd,J＝15.1,3.0Hz,1H),4.54(d,J＝3.5Hz,3H),4.49–4.30(m,1H),4.11–3.93(m,1H),3.81(d,J＝13.4Hz,1H),3.44–3.34(m,1H),3.33–3.14(m,1H),2.94–2.83(m,1H),2.79–2.59(m,1H),2.49–2.36(m,1H),2.35–2.14(m,1H),2.09–1.96(m,2H),1.87(dd,J＝11.2,5.0Hz,2H),1.85–1.66(m,1H)。HRMS(ESI):计算值C31H30F4N4O3Se,[M+H]+663.1492,实测值663.1467。HPLCtR＝4.17min，纯度95.77％。
实施例17 化合物17的制备
(S)-2-((4-(6-((4-氰基-2-甲氧基苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物17)
利用原料4-(溴甲基)-3-甲氧基苯腈和中间体1-6为起始原料，按照化合物1的合成路径合成化合物17。化合物17为白色固体，共42.3mg，收率59.8％。1H NMR(300MHz,DMSO-d6)δ12.79(s,1H),8.27(s,1H),7.81(d,J＝8.4Hz,1H),7.65(d,J＝8.4Hz,1H),7.48(dd,J＝14.3,6.7Hz,2H),7.28(dd,J＝19.4,7.8Hz,2H),7.05(d,J＝7.6Hz,1H),5.11(h,J＝6.1Hz,1H),4.80(dd,J＝15.2,7.1Hz,1H),4.65(dd,J＝15.2,2.9Hz,1H),4.43(s,2H),4.48–4.37(m,1H),4.35(dd,J＝10.4,4.9Hz,1H),3.98(d,J＝13.5Hz,1H),3.84(d,J＝10.4Hz, 4H),3.05(d,J＝11.1Hz,1H),2.91(d,J＝11.2Hz,1H),2.79–2.59(m,2H),2.43(s,1H),2.35–2.15(m,2H),1.81(dd,J＝23.9,11.7Hz,4H)。HRMS(ESI):计算值C32H33N5O4Se,[M+H]+632.1771,实测值632.1757。HPLCtR＝7.88min，纯度94.36％。
实施例18 化合物18的制备
2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(2-甲氧基乙基)-1H-苯并[d]咪唑-6-羧酸(化合物18)
按照如下路线利用原料18-1制备化合物18。
步骤1：3-((2-甲氧基乙基)氨基)-4-硝基苯甲酸甲酯(18-3)的制备。
将化合物18-1(1.90g,9.54mmol)溶于20mLACN，加入化合物18-2(1.00mL,11.45mmol)和DIPEA(3.32mL,19.08mmol)，加热至60℃搅拌8h。TLC检测反应结束后，用水和EA萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝70:1，v/v)纯化得到黄色固体18-3(1.72g，收率70.26％)。1H NMR(300MHz,DMSO-d6)δ8.18(dd,J＝9.1,4.1Hz,2H),7.57(d,J＝1.7Hz,1H),7.15(dd,J＝8.9,1.8Hz,1H),3.89(s,3H),3.66–3.50(m,4H),3.34(s,3H)。LC-MS：[M+H]+＝255.1。
步骤2：4-氨基-3-((2-甲氧基乙基)氨基)苯甲酸甲酯(18-4)的制备。
将中间体18-3(1.72g,6.69mmol)溶于20mLTHF，加入10％Pd/C(170mg)，在氢气保护下常温搅拌2h，TLC检测反应结束后，用硅藻土过滤反应液，DCM洗涤，滤液减压浓缩。经柱层析(洗脱体系为PE:EA＝10:1，v/v)纯化得到无色油状液体18-4(1.32g，收率86.73％)。
1H NMR(300MHz,DMSO-d6)δ7.17(dd,J＝8.1,1.9Hz,1H),6.99(d,J＝1.9Hz,1H),6.56(d,J＝8.1Hz,1H),5.46(s,2H),4.65(t,J＝5.5Hz,1H),3.73(s,3H),3.56(t,J＝5.6Hz,2H),3.30(s,3H),3.22(q,J＝5.6Hz,2H)。LC-MS：[M+H]+＝225.1。
步骤3：2-(氯甲基)-1-(2-甲氧基乙基)-1H-苯并[d]咪唑-6-甲酸甲酯(18-5)的制备。
将中间体18-4(1.32g,5.80mmol)溶于20mLACN，加入p-TSA·H2O(220.02mg,1.16mmol)和2-氯-1,1,1-三甲氧基乙烷(1.56mL,11.60mmol)，加热至60℃搅拌4h。TLC检测反应结束后，用水和EA萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝20:1，v/v)纯化得到无色油状液体(989.27mg，收率59.97％)。
1H NMR(300MHz,DMSO-d6)δ8.29(dd,J＝1.7,0.7Hz,1H),7.87(dd,J＝8.5,1.6Hz,1H),7.73(dd,J＝8.5,0.6Hz,1H),5.10(s,2H),4.61(t,J＝5.1Hz,2H),3.90(s,3H),3.70(t,J＝5.1Hz,2H),3.21(s,3H)。LC-MS：[M+H]+＝283.1。
步骤4：2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(2-甲氧基乙基)-1H-苯并[d]咪唑-6-羧酸甲酯(18-6)的制备。
以中间体18-5和中间体1-6为原料，以化合物1的合成路线得到白色固体18-6(307mg，收率66.8％)。
步骤5：2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(2-甲氧基乙基)-1H-苯并[d]咪唑-6-羧酸(化合物18)的制备。
以中间体18-6为原料，按照化合物1的合成路径合成化合物18。化合物18为白色固体，共265.28mg，收率66.1％。1H NMR(300MHz,DMSO-d6)δ12.81(s,1H),8.19(s,1H),7.81(d,J＝8.7Hz,2H),7.68–7.58(m,3H),7.52(d,J＝9.9Hz,1H),7.28(d,J＝7.9Hz,1H),7.07(d,J＝7.4Hz,1H),4.62(s,2H),4.50(s,2H),3.87(s,2H),3.75(s,2H),3.52(s,1H),2.95(d,J＝10.6Hz,2H),2.71(s,1H),2.24(s,2H),1.82(s,5H)。HRMS(ESI):计算值C30H30FN5O3Se,[M+H]+608.1571,实测值608.1551；HPLCtR＝5.85min，纯度97.32％。
实施例19 化合物19的制备
2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(四氢呋喃-3-基)-1H-苯并[d]咪唑-6-羧酸(化合物19)
以3-氨基四氢呋喃和化合物18-1为起始原料，按照化合物18的合成路径合成化合物19。化合物19为白色固体，共193.39mg，收率72.5％。1H NMR(300MHz,DMSO-d6)δ12.79(s,1H),8.34(s,1H),7.81(d,J＝9.1Hz,2H),7.71–7.48(m,4H),7.28(d,J＝7.8Hz,1H),7.07(d,J＝7.5Hz,1H),5.67(s,1H),4.52(s,2H),4.28–4.17(m,1H),4.11(dd,J＝10.5,3.0Hz,1H),4.02–3.89(m,2H),3.84(d,J＝13.4Hz,1H),3.64(q,J＝8.7Hz,1H),2.95(d,J＝10.9Hz,1H),2.84(d,J＝10.9Hz,1H),2.71(s,1H),2.20(q,J＝11.9Hz,3H),1.82(s,3H),1.74(d,J＝11.7Hz,2H)。HRMS(ESI):计算值C31H30FN5O3Se,[M+H]+620.1571,实测值620.1551。HPLCtR＝4.16min，纯度97.96％。
实施例20 化合物20的制备
2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-((1-乙基-1H-咪唑-5-基)甲基)-1H-苯并[d]咪唑-6-羧酸(化合物20)
以(1-乙基-1H-咪唑-5-基)甲胺和化合物18-1为起始原料，按照化合物18的合成路径合成化合物20。化合物20为白色固体，共64.92mg，收率49.3％。1H NMR(300MHz,DMSO-d6)δ12.70(s,1H),8.07(s,1H),7.86–7.76(m,3H),7.72–7.48(m,4H),7.28(d,J＝7.8Hz,1H),7.02(d,J＝7.6Hz,1H),6.44(s,1H),4.50(s,2H),4.07–3.92(m,2H),3.84(s,2H),3.27(t,J＝6.0Hz,3H),3.24–3.13(m,3H),2.92(d,J＝10.9Hz,2H),2.18(m,2H),1.88(dd,J＝12.0,6.0Hz,4H)。HRMS(ESI):计算值C31H30FN5O3Se,[M+H]+658.1839,实测值658.1834。HPLCtR＝7.92min,纯度94.66％。
实施例21 化合物21的制备
2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-1-(噻唑-5-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物21)
以噻唑-5-甲胺和化合物18-1为起始原料，按照化合物18的合成路径合成化合物21。化合物21为白色固体，共58.72mg，收率55.6％。1H NMR(300MHz,DMSO-d6)δ8.86(s,1H),8.10(s,1H),7.96(s,1H),7.80–7.67(m,2H),7.60(d,J＝8.4Hz,1H),7.52(s,1H),7.47(t,J＝7.7Hz,2H),7.21(d,J＝7.8Hz,1H),6.98(d,J＝7.6Hz,1H),5.90(s,2H),4.44(s,2H),3.83(s,2H),2.94(d,J＝10.9Hz,2H),2.63(m,1H),2.18(m,2H),1.73(s,3H),1.73–1.59(m,1H)。HRMS(ESI):计算值C31H27FN6O2SSe,[M+H]+646.1138,实测值646.1132。HPLCtR＝4.81min,纯度97.65％。
实施例22 化合物22的制备
(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-1-(氧杂环丁烷-2-基 甲基)-1H-苯并[d]咪唑-6-羧酸(化合物22)
按照如下路线利用原料22-1制备化合物22。
步骤1：(6-溴吡啶-2-基)氨基甲酸叔丁酯(22-3)的制备。
将化合物22-1(10.00g,36.76mmol)溶于150mLDMF，加入Cs2CO3(35.91g,110.29mmol)，室温搅拌0.5h，加入化合物22-2(9.50mL,110.29mmol)，加热至70℃搅拌3h。TLC检测反应结束后，用水和EA萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝200:1，v/v)纯化得到无色油状液体22-3(10.51g，收率91.25％)。
1H NMR(300MHz,DMSO-d6)δ7.77–7.63(m,2H),7.36(dd,J＝6.3,2.0Hz,1H),5.98–5.79(m,1H),5.17–5.03(m,2H),4.43(dt,J＝5.4,1.6Hz,2H),1.46(s,9H)。LC-MS：[M+H]+＝313.1。
步骤2：(6-溴吡啶-2-基)(2-氧代乙基)氨基甲酸叔丁酯(22-4)的制备。
将中间体22-3(10.51g,33.52mmol)溶于200mLTHF:H2O＝1:1(v/v)的混合溶液，加入锇酸钾二水合物(105.05mg,0.33mmol)和NaIO4(36.25g,167.62mmol)，室温搅拌1h，反应析出大量白色固体。TLC检测反应结束后，倒入饱和硫代硫酸钠水溶液，搅拌10min后用EA萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝100:1，v/v)纯化得到无色油状液体22-4(9.75g，收率92.65％)。1H NMR(300MHz,DMSO-d6)δ9.63(s,1H),7.82(d,J＝8.2Hz,1H),7.74(t,J＝7.9Hz,1H),7.37(d,J＝7.4Hz,1H),4.68(s,2H),1.45(s,9H)。LC-MS：[M+H]+＝315.1。
步骤3：2-(2-((6-溴吡啶-2-基)(叔丁氧羰基)氨基)乙基)肼-1-甲酸叔丁酯(22-6)的制备。
将中间体22-4(9.75g,30.94mmol)溶于150.00mLDCE，加入化合物22-5(8.23g,61.87mmol)，室温搅拌1h，TLC检测反应结束后，加入NaBH3CN(5.84g,92.81mmol,)和NaBH(OAc)3(19.78g,92.81mmol)，室温搅拌约3h后，用饱和NaHCO3水溶液淬灭反应后，用DCM萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝10:1，v/v)纯化得到无色油状液体(6.86g，收率50.79％)。1H NMR(300MHz,DMSO-d6)δ8.13(s,1H),7.76–7.66(m,1H),7.62(dd,J＝8.2,0.9Hz,1H),7.37(dd,J＝7.5,0.9Hz,1H),4.58(s,1H),3.83(t,J＝7.2Hz,2H),2.87(t,J＝7.2Hz,2H),1.46(s,9H),1.38(s,9H)。LC-MS：[M+H]+＝431.1。
步骤4：6-溴-N-(2-肼乙基)吡啶-2-胺(22-7)的制备。
将中间体22-6(6.86g,15.76mmol)溶于120mLDCM中，加入4M浓度的氯化氢的二氧六环溶液60mL，室温搅拌约5h，有大量白色固体析出，TLC检测反应结束后，直接减压蒸干溶剂，得到的盐酸盐无需纯化直接进行下一步反应。LC-MS：[M+H]+＝231.1。
步骤5：6-溴-N-(2-肼乙基)吡啶-2-胺(22-8)的制备。
将上一步的产物盐酸盐溶于80mL冰醋酸，加入40mL原甲酸三甲酯，N2保护，加热至100℃搅拌12h。TLC检测反应结束后，蒸干溶剂，加入饱和NaHCO3水溶液，用EA萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝2:1，v/v)纯化得到白色固体22-8(4.18g，收率91.85％)。1H NMR(300MHz,DMSO-d6)δ8.49(s,1H),7.87(d,J＝13.9Hz,1H),7.72(m,1H),7.27(m,2H),3.94–3.80(m,2H),3.85(s,2H)。LC-MS：[M+H]+＝268.9。
步骤6：4-(6-溴吡啶-2-基)-1,4,5,6-四氢-1,2,4-三嗪(22-9)的制备。
将中间体22-8(4.18g,15.29mmol)溶于100mLDCM:MeOH＝1:1(v/v)的混合溶液，在冰浴(0℃) 条件下，滴加4M的盐酸水溶液15.28mL，室温搅拌过夜。TLC检测反应结束后，加入大量饱和NaHCO3水溶液，用DCM萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝1:2，v/v)纯化得到黄色固体22-9(1.62g，收率43.67％)。1H NMR(300MHz,DMSO-d6)δ7.75(s,1H),7.62(t,J＝8.0Hz,1H),7.08(dd,J＝16.4,7.9Hz,2H),6.35(s,1H),3.73(t,J＝5.0Hz,2H),3.12(t,J＝5.1Hz,2H)。LC-MS：[M+H]+＝241.0。
步骤7：4-(6-溴吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-羧酸叔丁酯(22-10)的制备。
将中间体22-9(1.62g,6.66mmol)溶于30mLDCM，加入DMAP(81.42mg,0.66mmol)、TEA(1.15mL,8.00mmol)和Boc2O(3.18mL,13.32mmol)，室温搅拌过夜。TLC检测反应结束后，用水和DCM萃取，合并有机相，有机层用饱和食盐水洗涤，用无水硫酸钠干燥，减压浓缩。经柱层析(洗脱体系为PE:EA＝4:1，v/v)纯化得到白色固体22-10(780.03mg，收率34.47％)。
1H NMR(300MHz,DMSO-d6)δ7.80(s,1H),7.70(t,J＝7.9Hz,1H),7.22(dd,J＝8.0,5.1Hz,2H),3.82(tt,J＝4.6,2.7Hz,4H),1.46(s,9H)。LC-MS：[M+H]+＝341.0。
步骤8：(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物22)的制备。
以中间体22-10为原料，按照化合物1的合成路径合成化合物22。化合物22为白色固体，共45.84mg，收率75.2％。1H NMR(300MHz,DMSO-d6)δ12.77(s,1H),8.28(s,1H),7.95(s,1H),7.84–7.77(m,2H),7.68–7.51(m,4H),7.02(d,J＝7.5Hz,1H),6.84(d,J＝8.3Hz,1H),5.10–5.01(m,1H),4.83(dd,J＝15.5,7.1Hz,1H),4.70(d,J＝14.1Hz,1H),4.56–4.30(m,6H),3.85(s,2H),3.14(d,J＝5.2Hz,2H),2.69(dd,J＝16.5,4.7Hz,1H),2.39(t,J＝9.4Hz,1H)。HRMS(ESI):计算值C29H26FN7O3Se,[M+H]+620.1319,实测值620.1325。HPLCtR＝4.50min,纯度98.18％。
实施例23 化合物23的制备
(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)哌啶-1-基)甲基)-4-氟-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物23)
以3,5-二氟-4-硝基苯甲酸甲酯和(S)-氧杂环丁烷-2-甲胺为原料，按照化合物18的合成路径合成化合物23。化合物23为白色固体，共52.25mg，收率60.8％。1H NMR(300MHz,DMSO-d6)δ13.07(s,1H),8.15(s,1H),7.81(d,J＝9.9Hz,1H),7.67–7.46(m,4H),7.29(d,J＝7.8Hz,1H),7.08(d,J＝7.6Hz,1H),5.15–5.06(m,1H),4.84(d,J＝16.0Hz,1H),4.68(d,J＝14.8Hz,1H),4.52(s,2H),4.39(q,J＝8.2Hz,3H),3.99(d,J＝13.8Hz,1H),3.81(d,J＝13.8Hz,1H),3.03(d,J＝10.1Hz,1H),2.88(d,J＝11.1Hz,1H),2.69(s,3H),2.26(s,1H),1.82–1.77(m,4H)。HRMS(ESI):计算值C31H29F2N5O3Se,[M+H]+638.1476,实测值628.1463。HPLC tR＝4.36min,纯度98.19％。
实施例24 化合物24的制备
(S)-2-((4-(6-((4-氰基-2-氟苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-4-氟-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物24)
以中间体22-10、中间体1-7和3,5-二氟-4-硝基苯甲酸甲酯为原料，按照化合物22的合成路径合成化合物24。化合物24为白色固体，共39.82mg，收率57.9％。1H NMR(300MHz,DMSO-d6)δ13.11(s,1H),8.17(s,1H),7.96(s,1H),7.81(d,J＝10.0Hz,1H),7.63–7.47(m,4H),7.03(d,J＝7.6Hz,1H),6.84(d,J＝8.0Hz,1H),5.07(d,J＝8.2Hz,1H),4.88(d,J＝15.1Hz,1H),4.73(d,J＝15.2Hz,1H),4.47(t,J＝10.8Hz,5H),4.37(s,1H),3.86(s,2H),3.17(s,2H),2.69(s,1H),2.39(s,1H)。HRMS(ESI):计算值C29H25F2N7O3Se, [M+H]+638.1225,实测值638.1236。HPLCtR＝5.84min,纯度96.71％。
实施例25 化合物25的制备
(S)-4-氟-2-((4-(6-((2-甲氧基-4-(三氟甲基)苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物25)
以中间体22-10、2-甲氧基-4-(三氟甲基)苄基溴和3,5-二氟-4-硝基苯甲酸甲酯为原料，按照化合物22的合成路径合成化合物25。化合物25为白色固体，共45.07mg，收率60.4％。
1H NMR(300MHz,DMSO-d6)δ13.11(s,1H),8.18(s,1H),7.98(s,1H),7.58–7.44(m,3H),7.27–7.16(m,2H),7.00(d,J＝7.6Hz,1H),6.84(dd,J＝14.7,8.4Hz,1H),5.07(d,J＝6.9Hz,1H),4.88(dd,J＝15.5,7.0Hz,1H),4.80–4.68(m,1H),4.59–4.28(m,6H),3.88(s,5H),3.37(s,1H),3.17(d,J＝6.5Hz,1H),2.67(d,J＝8.6Hz,1H),2.41(dd,J＝16.7,8.0Hz,1H)。HRMS(ESI):计算值C30H28F4N6O4Se,[M+H]+693.1346,实测值693.1344。HPLCtR＝4.81min,纯度98.68％。
实施例26 化合物26的制备
(S)-2-((4-(6-((4-氰基-2-甲氧基苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-4-氟-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物26)
以中间体22-10、4-(溴甲基)-3-甲氧基苯腈和3,5-二氟-4-硝基苯甲酸甲酯为原料，按照化合物22的合成路径合成化合物26。化合物26为白色固体，共43.73mg，收率67.4％。1H NMR(300MHz,DMSO-d6)δ13.12(s,1H),8.17(s,1H),7.97(s,1H),7.52(dd,J＝9.6,6.4Hz,2H),7.44(d,J＝6.1Hz,2H),7.31(d,J＝7.8Hz,1H),7.00(d,J＝7.5Hz,1H),6.81(d,J＝8.3Hz,1H),5.07(d,J＝8.6Hz,1H),4.88(dd,J＝15.5,6.9Hz,1H),4.74(d,J＝15.1Hz,1H),4.50(dd,J＝22.7,13.9Hz,3H),4.39(s,3H),3.87(d,J＝7.8Hz,5H),3.36(s,1H),2.69(s,1H),2.51(s,2H),2.41(d,J＝10.0Hz,1H)。HRMS(ESI):计算值C30H28FN7O4Se,[M+H]+650.1425,实测值650.1432。HPLCtR＝4.13min,纯度99.00％。
实施例27 化合物27的制备
(S)-4-氟-2-((4-(6-((2-氟-4-(三氟甲基)苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物27)
以中间体22-10、1-(溴甲基)-2-氟-4-(三氟甲基)苯和3,5-二氟-4-硝基苯甲酸甲酯为原料，按照化合物22的合成路径合成化合物27。化合物27为白色固体，共37.46mg，收率59.8％。
1H NMR(300MHz,DMSO-d6)δ13.14(s,1H),δ8.16(s,1H),7.97(s,1H),7.63(d,J＝9.0Hz,2H),7.56(d,J＝8.6Hz,1H),7.50(dd,J＝7.4,2.8Hz,2H),7.03(d,J＝7.5Hz,1H),6.84(d,J＝8.3Hz,1H),5.06(d,J＝7.6Hz,1H),4.86(dd,J＝15.4,7.0Hz,1H),4.73(d,J＝14.9Hz,1H),4.58–4.35(m,5H),4.39–4.28(m,1H),3.86(t,J＝4.9Hz,2H),3.16(q,J＝5.7Hz,2H),2.67(d,J＝8.8Hz,1H),2.39(t,J＝8.6Hz,1H)。HRMS(ESI):计算值C29H25F5N6O3Se,[M+H]+681.1146,实测值681.1154。HPLCtR＝4.51min,纯度98.96％。
实施例28 化合物28的制备
(S)-2-((4-(6-((4-氯-2-氟苄基)硒基)吡啶-2-基)-5,6-二氢-1,2,4-三嗪-1(4H)-基)甲基)-4-氟-1-(氧杂环丁烷-2-基甲基)-1H-苯并[d]咪唑-6-羧酸(化合物28)
以中间体22-10、1-(溴甲基)-4-氯-2-氟苯和3,5-二氟-4-硝基苯甲酸甲酯为原料，按照化合物22的合成路径合成化合物28。化合物28为白色固体，共39.03mg，收率56.6％。
1H NMR(300MHz,DMSO-d6)δ13.11(s,1H),8.17(s,1H),7.97(s,1H),7.55(d,J＝6.8Hz,2H),7.52–7.33(m,2H),7.19(d,J＝8.3Hz,1H),7.02(d,J＝7.6Hz,1H),6.83(d,J＝8.3Hz,1H),5.11–5.02(m,1H),4.87(dd,J＝15.2,7.0Hz,1H),4.73(d,J＝15.0Hz,1H),4.58–4.30(m,2H),4.37(s,4H),3.86(s,2H),3.16(d,J＝6.2Hz,2H),2.70(d,J＝11.6Hz,1H),2.39(t,J＝8.6Hz,1H)。HRMS(ESI):计算值C28H25ClF2N6O3Se,[M+H]+647.0883,实测值647.0883。HPLCtR＝4.83min,纯度97.94％。
实施例29 GLP-1R-cAMP激动效果评价
本发明使用cAMP信号激动效应检测方法表征化合物对GLP-1R的激动活性。
(1)实验试剂及仪器装置(见表2、表3)
表2实验试剂与耗材
表3实验仪器装置
(2)实验细胞
本发明中，我们采用了稳定表达GLP-1R的HEK细胞系(北京爱思益普生物科技股份有限公司构建，表达人的GLP-1R基因)，测试用细胞个数为1000个/孔。细胞系均在含有10％胎牛血清的DMEM+100μg/mL Hygromycin B培养基中培养，培养温度为37℃，二氧化碳浓度为5％。除去旧培养基并用PBS洗一次，然后加入1mL TrypLETMExpress溶液(北京爱思益普生物科技股份有限公司)，37℃孵育2min左右。当细胞从皿底脱离，加入约2mL 37℃预热的完全培养基DMEM。将细胞悬液用吸管轻轻吹打使聚集的细胞分离。将细胞悬液转移至无菌的离心管中，1000rpm离心5min收集细胞，用于实验或传代培养。为维持细胞的生理活性，实验细胞融合度控制在80％左右。细胞传代、复苏及冻存均按常规方法进行。所有的操作都遵循北京爱思益普生物科技股份有限公司细胞培养的标准操作流程。
(3)实验方法
测试使用LANCE Ultra cAMP kit试剂盒。
试剂盒中包含：Stimulation Buffer、Eu-cAMP、Detection buffer、ULight-anti-cAMP。
步骤1：试剂盒中所有试剂，使用前平衡至室温。按照试剂盒说明书配制1×Stimulation Buffer待用。
步骤2：将待测化合物用DMSO进行梯度稀释，然后用1×Stimulation Buffer稀释化合物至10×。设定实际起始测试浓度为5000nM，以5倍梯度共稀释10组，其中每测试组包含2组复测试孔。每测试组设置浓度为0.1％的DMSO为阴性对照组，设置浓度为100nM的GLP-1(7-37)(北京爱思益普生物科技股份有限公司提供)为阳性对照组。
步骤3：稳转系细胞培养至80％融合度；胰酶消化处理收集细胞，经计数后接种9μL/孔于384孔板中。
步骤4：取1μL步骤2中稀释好的10×化合物加入至相应实验孔中，离心后置于37℃孵育30min。
步骤5：将Eu-cAMP用Detectionbuffer稀释至工作浓度，取5μL/孔加入相应实验孔中。
步骤6：将ULight-anti-cAMP用Detection buffer稀释至工作浓度，然后取5μL/孔加入相应实验孔中；离心后放置于室温孵育1h。
步骤7：孵育完成后，利用Biotek多功能酶标仪检测665nm和620nm读值。计算对应波长条件读值的比值R665/620，计算对应测试组的平均比值(RGroup)、阳性参照平均比值和阴性参照平均比值按照公式计算激动率A，测定化合物在5000nM水平的激动效应，选定有激动效应的化合物(A5000nM>50.00％)。选择激动效应大于80％的进行化合物测试浓度的A与对应浓度作图，用GraphPad Prism软件非线性回归方法进行曲线拟合及半数激动活性计浓度(EC50)计算。
(4)计算方法
计算结果保留4位有效数字。
(5)实验结果(见表4、表5)
对照药品Danuglipron(PF-06882961)：参照专利WO2018109607A1合成。
表4 5000nM化合物cAMP激动效应
表5化合物对cAMP信号的激动EC50
测试结果显示，本发明的化合物可通过激动cAMP信号通路激活GLP-1R的下游信号传导，具备完全的GLP-1R激动效应和低的半数激动浓度。
实施例30 大鼠肝微粒体稳定性测试
本发明使用LC-MS/MS方法检测化合物1、化合物17和化合物26与PF-06882961的大鼠肝微粒体稳定性。
(1)LC-MS/MS条件
LC-MS/MS型号：Shimadzu LCMS-8050；
色谱柱：GL Science ODS-3，3μm，2.1mm*50mm；
流动相：二元高压梯度洗脱，0-2min，泵A：甲醇，20％；泵B：水(含0.1％甲酸)，80％；2-5min：泵A：甲醇，80％；泵B：水(含0.1％甲酸)，20％；5-7min，泵A：甲醇，20％；泵B：水(含0.1％甲酸)，80％；
检测波长：254nm；检测时间：7min；进样体积：5μL；
计算方法：克拉霉素-内标法；
内标浓度：10ng/mL；
(2)标准曲线的建立
配制100mM的MgCl2的PBS溶液，5mM的NADPH的PBS溶液，5mg/mL的大鼠肝微粒体(rHLM，购买自上海权洋生物科技有公司)的PBS稀释测试液(4℃保存)，5mg/mL的灭活大鼠肝微粒体(rHLM，灭活条件为60℃加热1h)的PBS稀释测试液(4℃保存)，1mM的化合物MeOH母液。将化合物母液使 用PBS稀释为80μM的测试液，使用PBS以二倍梯度稀释十组。
测试体系：120μL PBS，20μL 5mg/mL灭活肝微粒体，10μL 100mM的MgCl2溶液，10μL化合物，混匀，37℃孵育5min，加40μL NADPH溶液。共200μL，测试体系中rHLM稀释10倍，MgCl2稀释20倍，化合物稀释20倍，NADPH稀释5倍。
取样10μL，加入30μL 12ng/mL的克拉霉素的乙腈内标稀释，涡旋混匀，此时化合物检测终浓度为原液稀释80倍的浓度，沉淀蛋白，12000rpm，4℃离心5min，取上清液20μL。测试终浓度落于1000ng/mL-0.1ng/mL之间，LC-MS/MS进样5μL检测，以内标法，将浓度比(C)对面积比(A)绘制标准曲线。
使用上述测试方法制作了化合物1和PF-06882961的标准曲线公式，拟合结果显示化合物1和PF-06882961在0.1ng/mL-1000ng/mL范围内峰面积响应与浓度呈线性关系，拟合回归程度好。
化合物1的标准曲线如图1所示，PF-06882961的标准曲线如图2所示。
(3)测试方法
按步骤(2)中描述测试方法检测，平行三组。测试用样品浓度为4μg/mL，在加入NADPH溶液启动反应后开始计时，在0，5，10，15，20，30，45，60，90，120min时间点取样处理。计算各时间点化合物的剩余浓度，并以0min时间点化合物浓度为100％计算化合物保留百分比(HLMRemaining，％)。将保留百分比的自然对数的平均值与孵育时间作直线拟合，获得消除速率常数k(min-1)与SEM。按如下公式计算清除相关参数：
半衰期t1/2(min)＝0.693/|k|；内在清除率CLint(mL/min/kg)＝(0.693/t1/2)*(1/CrHLM)*比例因子(大鼠：1792)；
使用化合物的rHLM条件下降解的t1/2和CLint表征化合物在大鼠肝微粒体中的稳定性。结果见表6：
表6化合物的rHLM稳定性
测试结果显示：化合物1、化合物17和化合物26在大鼠肝微粒体中稳定性均优于阳性分子PF-06882961的大鼠肝微粒体稳定性。
实施例31 药物代谢动力学测试
本发明使用LC-MS/MS方法，计算在不同给药方式条件下不同时间点(t)化合物1与PF-06882961在成年大鼠血浆内的驻留浓度(c)，绘制对应条件/对应化合物的c-t曲线，使用Winnolin7.0软件非方式模型对c-t参数拟合分析。
(1)前期准备
大鼠：SD品系，雄性，6～8周龄，体重180g～220g；
给药方式：静脉注射给药(1mg/kg或10mg/kg)；灌胃口服给药(5mg/kg或50mg/kg)；
配制溶剂配方：10％DMSO+40％PEG-400+50％生理盐水(含20％w/v羟丙基-β-环糊精)，溶液浓度为1mg/mL或5mg/mL；
(2)LC-MS/MS条件
LC-MS/MS型号：Shimadzu LCMS-8050；
色谱柱：GL Science ODS-3，3μm，2.1mm*50mm；
流动相：二元高压梯度洗脱，0-2min，泵A：甲醇，20％；泵B：水(含0.1％甲酸)，80％；2-5min：泵A：甲醇，80％；泵B：水(含0.1％甲酸)，20％；5-7min，泵A：甲醇，20％；泵B：水(含0.1％甲酸)，80％；
检测波长：254nm；检测时间：7min；进样体积：10μL或1μL；
计算方法：克拉霉素-内标法；
内标浓度：10ng/mL；
(3)标准曲线建立
使用色谱乙腈配制受试化合物的10000ng/mL、1000ng/mL、500ng/mL、200ng/mL、100ng/mL、50ng/mL、10ng/mL、1ng/mL、0.1ng/mL标准浓度测试样品，取20μL测试样品，使用40μL20ng/ml的乙腈溶液和20μL空白大鼠血浆稀释，按已描述的LC-MS方法分析，每次进样10μL，计算对应离子峰面积。使用内标法，以浓度比(C)对面积比(A)绘制对应化合物的标准曲线。
(4)取样及处理计算
大鼠称重，计算给药体积。设立采血时间点1min，5min，10min，30min，45min，60min，120min，240min，360min，480min，540min，1440min，共计12时间点，每一给药组设置4个平行实验组。于给药完毕时间点开始计时，在设定采血时间点自大鼠眼眶静脉丛处取血100μL～200μL，按通用血浆分离法(4500rpm,R.T.,5min)分离血浆，随后吸取20μL样品血浆。向样品血浆内加入40μL20ng/mL的克拉霉素乙腈溶液和20μL的乙腈，涡旋混匀，沉淀蛋白。以12000rpm，4℃条件离心5min，吸取上清液40μL，按上述LC-MS方法分析，每样品进样10μL，计算对应离子峰面积，带入内标法标准曲线计算化合物浓度。
各以化合物浓度对时间作C-T曲线，使用Winnolin7.0软件非房室模型统计矩法计算药代动力学参数。式中下标i.v.代表静脉注射，p.o.代表灌胃口服。其中Cmax和Tmax均为实测值，AUClast值为梯形面积法计算所得，0-+∞时间的曲线下面积AUCint_obs＝AUClast+Clast/HL_Lambda_z，式中Clast代表最后可识别有效最低浓度，HL_Lambda_z代表消除相半衰期。口服生物利用度F＝(AUCint_obsp.o./Dp.o.)/(AUCint_obsi.v./Di.v.)*100％，式中AUC代表曲线下面积，D代表给药剂量。
(5)测试参数
PF-06882961的c-t曲线如图3所示，化合物1低剂量组的c-t曲线如图4所示，化合物1高剂量组的c-t曲线如图5所示。测试所得PK参数如表7所示。
表7化合物的药物代谢动力学参数
*PF-06882961的口服给药形式为其Tris盐，通过专利WO2018109607A1内公开的合成方法合成所得；
**口服组Vz_obs及CLobs数据均显示表观参数。
数据结果显示：化合物1的低剂量组的静注半衰期短，口服半衰期长，静注给药条件下药物清除速率快，血浆药物存量相对阳性药更低；口服给药条件下药物吸收与代谢分布处于平衡，血浆药物存量相对静注条件明显提升，显示出组织药物蓄积与缓释特征，致使口服生物利用度优于阳性药PF-06882961，为20.84％。高剂量组的静注及口服条件的半衰期适中，分别为1.37h和2.65h；体内吸收程度及平均AUC值均高于阳性药，且口服高剂量组的生物利用度要高于PF-06882961。
上述实验结果评价了化合物1的体外成药性质。
实施例32 药物代谢动力学测试
本发明使用LC-MS/MS方法，计算在不同给药方式条件下不同时间点(t)化合物17在成年大鼠血浆内的驻留浓度(c)，绘制对应条件/对应化合物的c-t曲线，使用Winnolin7.0软件非方式模型对c-t参数拟合分析。
(1)前期准备
大鼠：SD品系，雄性，6～8周龄，体重180g～220g；
给药方式：静脉注射给药(5mg/kg)；灌胃口服给药(30mg/kg)；
配制溶剂配方：10％DMSO+40％PEG-400+50％生理盐水(含20％w/v羟丙基-β-环糊精)，溶液浓度为5mg/mL；
(2)LC-MS/MS条件
与实施例31相同。
(3)标准曲线建立
与实施例31相同。
(4)取样及处理计算
与实施例31相同。
(5)测试参数
测试所得PK参数如表8所示。
表8化合物的药物代谢动力学参数
以静脉注射给药5mg/kg和口服灌胃给药30mg/kg的条件评价了化合物17的药代动力学性质。实验结果显示：化合物17的静注及口服半衰期适中，平均血浆药物暴露量、表观分布容积以及清除率等相对保持，口服生物利用度达到了28.91％。上述实验结果评价了化合物17的体外成药性质。
至此我们获得了GLP-1R激动活性良好且具备可接受的口服给药效果的GLP-1R小分子激动剂，扩展了硒元素在药物设计与合成领域的应用。但以化合物1、化合物17为代表的硒连接链化合物系列的结构设计改造仍相对保守，在生物利用度等成药性质方面以及心脏毒性方面仍有继续提升的潜力。
如上所述，尽管参照特定的优选实施例已经表示和表述了本发明，但其不得解释为对本发明自身的限制。在不脱离所附权利要求定义的本发明的精神和范围前提下，可对其在形式上和细节上作出各种变化。

Claims (10)

  1. 一种通式I的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体：
    其中，
    环A选自C3-8环烷基、C2-9杂环基、C5-12螺双环基、C5-12螺杂双环基、C5-12稠合双环基、C5-12稠合杂双环基、C5-12桥环基或C5-12桥杂环基；
    L选自-C(O)-、-O-、-S-、-CR4R5或-NR6-；
    R4、R5各自独立地选自氢、氘、卤素、氰基、C1-C6烷基、C1-C6烷氧基、-NR5aR5b、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；所述C1-C6烷基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基、羟基、C3-C6饱和或部分饱和的环烷基；所述芳基、杂芳基、饱和或部分饱和的环烷基、饱和或部分饱和的杂环基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基或-NR5aR5b；
    R5a、R5b各自独立地选自氢或C1-C6烷基；
    R6选自氢、C1-C6烷基、C3-C10环烷基、杂环基、-S(O)2R6a或-S(O)2N(R6a)(NR6b)；所述环烷基或杂环基任选地被C1-C6烷基、-F或氰基取代；
    R6a、R6b各自独立地选自-H、C1-C6烷基或C3-C10环烷基；
    R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基、3-6元杂环基、C3-C6环烷基或5-8元杂芳基；所述3-6元杂环基、C3-C6环烷基、5-8元杂芳基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：H、卤素、C1-C6烷基、C3-C6环烷基、C1-C6烷氧基、C1-C6烷氨基、氰基、氰基取代的C1-C6烷基、羟基、羟基取代的C1-C6烷基、卤代C1-C6烷基、卤代C3-C6环烷基、卤代C1-C6烷氧基、卤代C1-C6烷氨基、酰基、酰胺基、氨酰基、磺酰基或氨基；
    R2各自独立地选自氢原子、氘、卤素、氰基、羟基、氧代、C1-C6烷基、C1-C6烷氧基、氨基、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；
    R3各自独立地选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
    X1、X2、X3和X4相同或不同，且各自独立地选自-CR7或N原子；
    R7选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
    G选自G1或G2；G1、G2结构式如下：
    环B、环C各自独立地选自6-12元芳基或6-12元杂芳基；
    Y、Z各自独立地选自-CH2-、-S-、-SO-、-SO2-、-(O＝S＝NH)-、-Se-或-Se(O)-；
    R8、R11相同或不同，各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
    R9、R10、R12、R13相同或不同，且各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、 烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
    EE选自-COOH或羧基替代物；所述羧基替代物选自：
    m为0、1、2或3；
    n为0、1、2、3或4；
    o为0、1、2、3、4或5；
    p为0、1、2、3、4或5。
  2. 根据权利要求1所述的化合物，其特征在于，
    环A选自C3-8环烷基或C2-9杂环基；
    L选自-CR4R5；
    R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基；
    EE选自-COOH或羧基替代物。
  3. 根据权利要求2所述的化合物，其特征在于，
    环A选自C2-9杂环基；
    Y、Z各自独立地选自-Se-或-Se(O)-；
    EE选自-COOH、
  4. 根据权利要求1所述的化合物，其特征在于选自：

  5. 一种药物组合物，其特征在于：包括权利要求1～4任一项所述的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体以及药学上可接受的载体或赋形剂。
  6. 根据权利要求1～4任一所述的化合物在制备GLP-1受体激动剂中的用途，在制备预防和/或治疗与激动GLP-1受体相关疾病的药物中的用途。
  7. 根据权利要求6所述的用途，其特征在于，所述与激动GLP-1受体相关疾病包括糖尿病、糖尿病并发症、代谢综合征、肥胖症、非酒精性脂肪性肝病、非酒精性脂肪性肝炎、帕金森病、阿尔兹海默症、高血压症、高血脂症、动脉粥样硬化、心血管风险、冠心病或脑卒中。
  8. 一种通式II的化合物或其药学上可接受的盐、对映异构体、立体异构体、水合物、溶剂合物或多晶体：
    其中，
    环A选自C3-8环烷基、C2-9杂环基、C5-12螺双环基、C5-12螺杂双环基、C5-12稠合双环基、C5-12稠合杂双环基、C5-12桥环基或C5-12桥杂环基；
    L选自-C(O)-、-O-、-S-、-CR4R5或-NR6-；
    R4、R5各自独立地选自氢、氘、卤素、氰基、C1-C6烷基、C1-C6烷氧基、-NR5aR5b、6-10元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；所述C1-C6烷基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基、羟基、C3-C6饱和或部分饱和的环烷基；所述芳基、杂芳基、饱和或部分饱和的环烷基、饱和或部分饱和的杂环基任选地被以下一个或多个取代基所取代：卤素、氧代、氰基或-NR5aR5b；
    R5a、R5b各自独立地选自氢或C1-C6烷基；
    R6选自氢、C1-C6烷基、C3-C10环烷基、杂环基、-S(O)2R6a或-S(O)2N(R6a)(NR6b)；所述环烷基或杂环基任选地被C1-C6烷基、-F或氰基取代；
    R6a、R6b各自独立地选自-H、C1-C6烷基或C3-C10环烷基；
    R1选自3-6元杂环基取代的C1-C6烷基、C3-C6环烷基取代的C1-C6烷基、5-8元杂芳基取代的C1-C6烷基、C1-C6烷氧基取代的C1-C6烷基、3-6元杂环基、C3-C6环烷基或5-8元杂芳基；所述3-6元杂环基、C3-C6环烷基、5-8元杂芳基或C1-C6烷氧基任选地被以下一个或多个取代基所取代：H、卤素、C1-C6烷基、C3-C6环烷基、C1-C6烷氧基、C1-C6烷氨基、氰基、氰基取代的C1-C6烷基、羟基、羟基取代的C1-C6烷基、卤代C1-C6烷基、卤代C3-C6环烷基、卤代C1-C6烷氧基、卤代C1-C6烷氨基、酰基、酰胺基、氨酰基、磺酰基或氨基；
    R2各自独立地选自氢原子、氘、卤素、氰基、羟基、氧代、C1-C6烷基、C1-C6烷氧基、氨基、6-10 元芳基、5-8元杂芳基、3-8元饱和或部分饱和的环烷基、3-8元饱和或部分饱和的杂环基；
    R3各自独立地选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
    X1、X2、X3和X4相同或不同，且各自独立地选自-CR7或N原子；
    R7选自氢原子、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
    G选自G1或G2；G1、G2结构式如下：
    环B、环C各自独立地选自6-12元芳基或6-12元杂芳基；
    Y、Z各自独立地选自-CH2-、-S-、-SO-、-SO2-、-(O＝S＝NH)-、-Se-或-Se(O)-；
    R8、R11相同或不同，各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；所述烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、氰基、氨基、硝基、氰基、羟烷基、环烷基、杂环基、芳基或杂芳基；
    R9、R10、R12、R13相同或不同，且各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
    R14各自独立地选自氢、卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基，所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选地被以下一个或多个取代基所取代：卤素、烷基、烯基、炔基、烷氧基、卤代烷基、卤代烷氧基、羟烷基、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基或杂芳基；
    EE选自-COOH或羧基替代物；所述羧基替代物选自：
    m为0、1、2或3；
    n为0、1、2、3或4；
    o为0、1、2、3、4或5；
    p为0、1、2、3、4或5；
    q为0、1、2或3。
  9. 根据权利要求8所述的化合物，其特征在于选自：

  10. 根据权利要求8或9所述的化合物在制备GLP-1受体激动剂中的用途，在制备预防和/或治疗与激动GLP-1受体相关疾病的药物中的用途。
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