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WO2025218365A1 - 芳基烷基胺类化合物及其制备方法和用途 - Google Patents

芳基烷基胺类化合物及其制备方法和用途

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Publication number
WO2025218365A1
WO2025218365A1 PCT/CN2025/079912 CN2025079912W WO2025218365A1 WO 2025218365 A1 WO2025218365 A1 WO 2025218365A1 CN 2025079912 W CN2025079912 W CN 2025079912W WO 2025218365 A1 WO2025218365 A1 WO 2025218365A1
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Prior art keywords
compound
group
mmol
pharmaceutically acceptable
alkoxy
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English (en)
French (fr)
Inventor
闫弘毅
张恺
牛长群
孙晓彦
董宗跃
付香杰
刘育苗
张浩楠
史云瑞
张�杰
孙汇蕾
贾琦
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Renhe Yikang Group Co Ltd
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Renhe Yikang Group Co Ltd
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Publication of WO2025218365A1 publication Critical patent/WO2025218365A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/14—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • 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
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
    • A61P5/20—Drugs for disorders of the endocrine system of the parathyroid hormones for decreasing, blocking or antagonising the activity of PTH
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D405/02—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 two hetero rings
    • C07D405/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D405/02—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 two hetero rings
    • C07D405/10—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 two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • the present invention belongs to the field of medical technology, and specifically relates to an arylalkylamine compound, a preparation method thereof, and the use of the arylalkylamine compound for allosterically stimulating calcium-sensing receptors (CaSR).
  • CaSR calcium-sensing receptors
  • SHPT secondary hyperparathyroidism
  • the present invention aims to provide an arylalkylamine compound with enhanced activity and safety.
  • the compound of the present invention exhibits excellent CaSR agonist activity, high bioavailability, high in vivo metabolic stability, and enhanced safety.
  • the present invention is achieved through the following technical solutions.
  • the present invention provides a compound having a structure represented by the following formula (I) or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof:
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from a hydrogen atom; a C 2-6 carboxylic acid group; a C 1-7 alkoxy group (which may be a cycloalkoxy group or a chain alkoxy group), which may be substituted by a substituent selected from the following: halogen, cyano, nitro, oxygen, a C 1-6 carboxylic acid group, an amino group, a C 3-6 cycloalkyl group, an aryl C 1-6 alkyloxy group (e.g., benzyloxy), a hydroxyl group, a C 1-6 alkoxy group, a halogenated C 1-6 alkyl group and a halogenated C 1-6 alkoxy group; halogen; a C 1-6 alkyl group; a halogenated C 1-6 alkyl group;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are not H at the same time; 2) when the substituent is a C 2-6 carboxylic acid group, the substitution sites are R 2 -R 4 , and at least two of R 1 -R 5 are not H;
  • R 1 and R 5 are selected from H, halogen (eg, F, Cl), C 1-6 alkyl (eg, methyl, ethyl), and C 1-6 alkoxy (eg, methoxy, ethoxy).
  • halogen eg, F, Cl
  • C 1-6 alkyl eg, methyl, ethyl
  • C 1-6 alkoxy eg, methoxy, ethoxy
  • At least one of R 2 -R 4 is an unsubstituted or substituted C 2-6 carboxylic acid group, preferably an acetyl group, and at least two of R 1 -R 5 are not H. That is, at least the para or meta position is an acetyl group (ie, -CH 2 COOH), and at least one of the other positions is substituted.
  • At least one of R 2 -R 4 is an unsubstituted or substituted acetyl group, and at least two of R 1 -R 5 are not H. That is, at least the para or meta position is an acetyl group (ie, -CH 2 COOH), and at least one of the other positions is substituted.
  • R 1 , R 2 , R 4 , and R 5 are each independently selected from H, halogen (e.g., F, Cl), C 1-6 alkyl (e.g., methyl, ethyl), C 1-6 alkoxy (e.g., methoxy, ethoxy, etc.), which is substituted by a substituent selected from the following: hydroxyl, C 1-6 alkoxy, and R 1 , R 2 , R 4 , and R 5 are not H at the same time; R 3 is a C 2-6 carboxylic acid group.
  • halogen e.g., F, Cl
  • C 1-6 alkyl e.g., methyl, ethyl
  • C 1-6 alkoxy e.g., methoxy, ethoxy, etc.
  • R 3 is a C 2-6 carboxylic acid group.
  • R 1 , R 2 , R 4 , and R 5 are each independently selected from H, halogen (e.g., F, Cl), C 1-6 alkyl (e.g., methyl, ethyl), C 1-6 alkoxy (e.g., methoxy, ethoxy, etc.), which are substituted by substituents selected from the following: hydroxyl, C 1-6 alkoxy, and R 1 , R 2 , R 4 , and R 5 are not H at the same time; R 3 is acetoxy.
  • halogen e.g., F, Cl
  • C 1-6 alkyl e.g., methyl, ethyl
  • C 1-6 alkoxy e.g., methoxy, ethoxy, etc.
  • the compound of formula (I) is selected from:
  • the compound of formula (I) of the present invention may be in free form or in the form of a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable inorganic acid salts include hydrochlorides, sulfates, phosphates, or hydrobromides.
  • Pharmaceutically acceptable organic acid salts include acetic acid, fumaric acid, oxalic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or maleic acid.
  • a metal ion salt such as an alkali metal salt or alkaline earth metal salt such as a sodium salt, potassium salt, or calcium salt.
  • the present invention provides a method for preparing the compound of formula (I), comprising reacting a compound of formula A with a compound of formula (II) to form a compound of formula (I):
  • X represents a leaving group selected from halogen, hydroxyl, lower alkylsulfonyloxy (eg, C 1-6 alkylsulfonyloxy), sulfonyloxy, etc., preferably Br, Cl, trifluoromethanesulfonyloxy.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof and a pharmaceutically acceptable carrier or excipient.
  • the compound of formula (I) of the present invention or its pharmaceutically acceptable salt when used as a medical active ingredient, can be used together with an inert carrier suitable for each method of administration, and can be formulated into conventional pharmaceutical preparations, such as tablets, granules, capsules, powders, solutions, suspensions, emulsions, injections, etc.
  • the inert carrier When used as solid preparations, the inert carrier includes a binder (gum arabic, gelatin, sorbitol, polyvinyl pyrrolidone, etc.), an excipient (lactose, galactose, corn starch, sorbitol, etc.), a lubricant (magnesium stearate, talc, polyethylene glycol, etc.), a disintegrant (potato starch, etc.), etc.
  • a binder glycose, galactose, corn starch, sorbitol, etc.
  • a lubricant magnesium stearate, talc, polyethylene glycol, etc.
  • a disintegrant potato starch, etc.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof for medical use of the present invention can be administered orally, by intravenous injection, intramuscular injection, subcutaneous injection, transdermal absorption, etc.
  • the dosage and administration volume can be determined according to factors such as the characteristics of the drug, the administration route, the age, weight or disease state of the patient, etc.
  • the present invention provides the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or tautomer, or a pharmaceutical composition comprising the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or tautomer and a pharmaceutically acceptable carrier or excipient. It produces multiple pharmacological effects by activating CaSR and is used to prevent and treat diseases related to CaSR, such as inhibiting the production of PTH and regulating the levels of calcium and phosphorus in the blood by activating CaSR.
  • the present invention includes all stereoisomers of the compound. Unless specific stereo configuration is specified, the present invention is intended to include all such isomeric forms of these compounds.
  • the asymmetric centers in the compounds of the present invention can all have (R) configuration or (S) configuration independently of each other.
  • the bond connected to the chiral carbon is represented as a straight line in the structural formula of the present invention, it should be understood that the (R) and (S) configurations of the chiral carbon and two enantiomers and mixtures thereof are all included in the formula.
  • it means enantiomer (R) or (S).
  • C1-6 carboxyl groups and C2-6 carboxyl groups are lower alkyl groups that further contain a carboxyl group.
  • C1-6 carboxyl groups can be formate (e.g., -COOH), acetate, propionate, butyrate, valerate, or hexanoate.
  • C2-6 carboxyl groups can be acetate, propionate, butyrate, valerate, or hexanoate.
  • the compound of formula (I) of the present invention allosterically activates the CaSR on parathyroid cells, inhibiting the synthesis and secretion of parathyroid hormone, thereby reducing parathyroid hormone levels in the blood and stabilizing blood calcium and phosphorus concentrations. It can be used to prevent or treat hyperparathyroidism, exhibiting excellent CaSR allosteric agonism and PTH-lowering effects in vivo. CaSR allosteric activation experiments, a SD rat adenine model, and a SD rat 5/6 nephrectomy model confirmed that the compound has CaSR allosteric activation and PTH-lowering effects in model rats.
  • the present invention's formula (I) compound or its pharmaceutically acceptable salt as an active ingredient not only has excellent activity, but also has certain in vivo stability.
  • the present invention carried out rat liver microsome incubation experiments to verify that the compound does have metabolic stability.
  • the present invention's formula (I) compound or its pharmaceutically acceptable salt as an active ingredient not only has excellent CaSR activation effect, but also has high safety. Compared with the prior art, the present application not only has excellent activity and good safety, but also has high metabolic stability in vivo and high relative bioavailability in vivo.
  • the known starting materials disclosed herein can be synthesized using methods known in the art, or can be purchased from chemical companies such as Anergy Chemical, J&K Technologies, Maclean, Aladdin, and Bede Pharmaceuticals.
  • solution refers to an aqueous solution
  • room temperature refers to 20°C to 30°C
  • inert gas refers to argon or nitrogen
  • the developing solvents used for thin layer chromatography (TLC) monitoring of the reaction and column chromatography purification are dichloromethane/methanol system, n-hexane/ethyl acetate system, petroleum ether/ethyl acetate system, petroleum ether/ethyl acetate/methanol system, etc.
  • the proportions of the system components are adjusted according to the polarity of the compound, and can also be adjusted by adding a small amount of alkaline or acidic reagents such as triethylamine and acetic acid.
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • HPLC high-performance liquid chromatography
  • the synthetic route is as follows:
  • Step 2 (3S)-2-methylpropan-2-yl 3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydropyrrole-1-carboxylate (A-3)
  • compound A-2 (45.00g, 0.12mol), (1R)-1-(naphthalen-1-yl)ethan-1-amine (17.60g, 0.10mol), potassium phosphate (21.23g, 0.10mol), and 150mL of acetonitrile were added. The temperature was raised to 75°C and stirred for 24h. The mixture was cooled to room temperature, filtered, and the filter cake was rinsed with 60mL of acetonitrile. The mixture was concentrated under reduced pressure until no solvent was removed. 90mL of ethyl acetate and 90mL of saturated brine were added, stirred for 0.5h, and allowed to stand until distinct layers separated.
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 2-fluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetate (1-2)
  • Step 3 ⁇ 2-Fluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetic acid (1)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 3-fluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetate (2-2)
  • Step 3 ⁇ 2-Fluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetic acid (2)
  • the synthetic route is as follows:
  • Step 3 ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-3-methylphenyl ⁇ acetic acid (3)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-3-methylphenyl ⁇ acetate (4-2)
  • Step 3 ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-methylphenyl ⁇ acetic acid (4)
  • the synthetic route is as follows:
  • Step 1 Ethyl ⁇ 2,6-difluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetate (5-1)
  • Step 2 ⁇ 2,6-difluoro-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetic acid (5)
  • the synthetic route is as follows:
  • Step 6 Ethyl ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-3-methoxyphenyl ⁇ acetate (6-6)
  • Step 7 ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-3-methoxyphenyl ⁇ acetic acid (6)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-methoxyphenyl ⁇ acetate (7-2)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 2-ethoxy-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetate (8-2)
  • Step 3 ⁇ 2-ethoxy-4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]phenyl ⁇ acetic acid (8)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-(propan-2-yloxy)phenyl ⁇ acetate (9-2)
  • Step 3 ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-(propan-2-yloxy)phenyl ⁇ acetic acid (9)
  • the synthetic route is as follows:
  • Step 2 Ethyl ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-(propyloxy)phenyl ⁇ acetate (10-2)
  • Step 3 ⁇ 4-[(3S)-3- ⁇ [(1R)-1-(naphthalen-1-yl)ethyl]amino ⁇ tetrahydro-1H-pyrrol-1-yl]-2-(propyloxy)phenyl ⁇ acetic acid (10)
  • the CaSR belongs to the C family of G protein-coupled receptors (GPCRs).
  • GPCRs G protein-coupled receptors
  • Preparation before plating 12 hours before plating cells, coat 384-well cell culture plates with 0.1 mg/mL poly-lysine.
  • Cell Plating Resuspend the digested CaSR-CHO cells in complete medium. Count the cells using a cell counter and adjust the cell density to 5 ⁇ 105 cells/mL. Inoculate 25 ⁇ L of the cell suspension per well. After plating, incubate the cells in a 37°C, 5% CO2 incubator for 18 hours.
  • a hyperparathyroidism model was established in SD male rats by removing 5/6 of the kidneys and inducing a special diet.
  • Parathyroid hormone (PTH) in the blood was used as the detection indicator.
  • the ELISA enzyme-linked immunosorbent assay was used to detect the changes in PTH concentration in the rat plasma after administration of the compound to evaluate the in vivo activity of the compound.
  • Model Construction 7- to 8-week-old SPF male Sprague-Dawley rats were used. They were fed a normal diet for 1 week, followed by 2/3 left kidney removal and recovery with a normal diet for 1 week, followed by right kidney removal and recovery with a normal diet for 1 week, and then fed a special diet (0.5% calcium and 1.2% total phosphorus) for 2 weeks.
  • Dosing and Activity Testing A single dose of the compound was administered orally. Blood samples were collected before and after administration and processed using the above method to obtain serum. PTH levels were then measured by ELISA. This method confirmed that the compound had the effect of reducing PTH levels in the blood of model rats.
  • the samples were pretreated with rat liver microsomes, testosterone (positive control), tolbutamide (internal standard), the test compound, and PBS (pH 7.4) at 37°C for 0, 5, 10, 20, 30, and 60 minutes, quenched with methanol, and analyzed by LC-MS.
  • the data were processed (plotted with the peak area of the test compound/the peak area of the internal standard as the ordinate and time as the abscissa), and t 1/2 was calculated.
  • Table 3 The test results for some compounds of the present invention are shown in Table 3.
  • liver microsome stability test is affected by the activity of liver microsomes.
  • the longest incubation time for in vitro tests is 60 min.
  • a t 1/2 > 60 min indicates that the compound has high metabolic stability in vivo.
  • Blood collection points Blood was collected from the tail vein before administration and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration, and 100 ⁇ L of blood sample was collected at each blood collection point.
  • Evocalcet stock solution (0.2 mg/mL): Accurately weigh 10 mg of the compound into a 50 mL volumetric flask, dissolve it in methanol, and dilute to the mark. Shake well to obtain the compound. (Preparation of compound stock solution is the same as for Evocalcet)
  • Preparation of standard solutions Accurately measure the stock solutions of Evocalcet, compound 2, and compound 3, dilute them with 50% methanol, and prepare standard solutions with concentrations of 80, 200, 1000, 5000, 10000, 20000, and 40000 ng/mL.
  • clenbuterol hydrochloride solution Preparation of internal standard clenbuterol hydrochloride solution: Accurately weigh 10 mg of clenbuterol hydrochloride into a 10 mL volumetric flask, dilute to volume with methanol, and shake well to obtain a 1.0 mg/mL clenbuterol hydrochloride stock solution. Accurately measure an appropriate amount of the stock solution and dilute with methanol to a 2000 ng/mL internal standard working solution.
  • Standard curve sample treatment Take 90 ⁇ L of blank matrix, add 10 ⁇ L of each standard series solution and 50 ⁇ L of internal standard solution, vortex mix for 30 seconds, add 350 ⁇ L of methanol precipitation protein solution, vortex mix, centrifuge the sample at 12000 rpm in a centrifuge for 10 minutes, take 200 ⁇ L of supernatant, add 600 ⁇ L of water and mix, and take the supernatant for sampling and analysis.
  • Blood sample processing Take 50 ⁇ L of plasma sample, add 25 ⁇ L of internal standard solution, vortex mix for 30 seconds, add 175 ⁇ L of methanol protein precipitation solution, vortex mix, centrifuge the sample at 12000 rpm for 10 minutes, take 100 ⁇ L of supernatant, add 300 ⁇ L of water and mix, and take the supernatant for analysis.

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Abstract

本发明涉及芳基烷基胺类化合物及其制备方法和用途，本发明的芳基烷基类化合物具有下式(I)的结构：其中R1、R2、R3、R4、R5如说明书所定义。本发明的化合物具有优异CaSR激动活性，生物利用度高，体内代谢稳定性高，且安全性高。

Description

芳基烷基胺类化合物及其制备方法和用途 技术领域
本发明属于医药技术领域，具体地说，涉及一种芳基烷基胺类化合物及其制备方法和变构激动钙敏感受体(Cacium-sensing receptor,CaSR)的用途。
背景技术
慢性肾病会引起继发性甲状旁腺功能亢进症(SHPT)。目前，国内外治疗SHPT的方法包括许多。但是，存在成本高、治疗窗口窄、不彻底、易复发和不耐受等缺点。拟钙剂类药物直接作用于CaSR，能够有效控制PTH的合成和分泌，更具有临床优势。
田边三菱制药株式会社开发出拟钙剂类药物Evocalcet(WO2005115975)，并于2018年3月首次在日本获批上市，据Evocalcet的审查报告书报道，其不良反应主要包括低钙血症、呕吐、恶心、寒战、QT延长、情绪不良、心律失常、血压下降及痉挛等(オルケディア錠1mg、同錠2mg審議結果報告書.2018年3月)。
因此，开发具有更高活性和安全性的拟钙剂类药物，特别是芳基烷基胺类化合物，用于治疗患者的SHPT，具有极其重要的临床价值和社会价值。
发明内容
为了解决现有技术的缺陷，本发明旨在提供一种具有更高活性和安全性的芳基烷基胺类化合物。本发明的化合物具有优异的CaSR激动活性，生物利用度高，体内代谢稳定性高，且安全性高等特点。本发明是通过以下技术方案来实现的。
一方面，本发明提供具有如下式(I)所示结构的化合物或其药学上可接受的盐、立体异构体或互变异构体：
其中，R1、R2、R3、R4、R5各自独立地选自氢原子；C2-6羧酸基；C1-7烷氧基(可以是环烷氧基或者链烷氧基)，其可以被选自以下的取代基取代：卤素、氰基、硝基、氧、C1-6羧酸基、胺基、C3-6环烷基、芳基C1-6烷基氧基(例如苄基氧基)、羟基、C1-6烷氧基、卤代C1-6烷基和卤代C1-6烷氧基；卤素；C1-6烷基；卤代C1-6烷基；
条件是：1)R1、R2、R3、R4、R5不同时为H；2)当取代基为C2-6羧酸基时，取代位点为R2-R4，且R1-R5中至少2个不是H；
优选地，R1、R5选自H、卤素(例如F、Cl)、C1-6的烷基(例如甲基、乙基)、C1-6的烷氧基(例如甲氧基、乙氧基)。
优选地，R2-R4中至少一个为未取代或取代C2-6羧酸基，优选为乙酸基，且R1-R5中至少2个不是H。即至少在对位或间位为乙酸基(即-CH2COOH)，其他至少有一个位点被取代。
优选地，R2-R4中至少一个为未取代或取代乙酸基，且R1-R5中至少2个不是H。即至少在对位或间位为乙酸基(即-CH2COOH)，其他至少有一个位点被取代。
优选地，R1、R2、R4、R5各自独立地选自H、卤素(例如F、Cl)、C1-6的烷基(例如甲基、乙基)、C1-6的烷氧基(例如甲氧基、乙氧基等)，其被选自以下的取代基取代：羟基、C1-6烷氧基，且R1、R2、R4、R5不同时为H；R3为C2-6羧酸基。
优选地，R1、R2、R4、R5各自独立地选自H、卤素(例如F、Cl)、C1-6的烷基(例如甲基、乙基)、C1-6的烷氧基(例如甲氧基、乙氧基等)，其被选自以下的取代基取代：羟基、C1-6烷氧基，且R1、R2、R4、R5不同时为H；R3为乙酸基。
在一个具体实施方案中，式(I)的化合物选自：
本发明的通式(I)化合物可以是游离形式或药学上可接受的盐形式。药物上可接受的无机酸盐，如盐酸盐、硫酸盐、磷酸盐或氢溴酸盐等。药物上可接受的有机酸盐，如醋酸、富马酸、草酸、柠檬酸、甲磺酸、苯磺酸、对甲苯磺酸或马来酸等。此外，当化合物具有羧基等酸性基团时，可以成金属离子的盐，如钠盐、钾盐、钙盐等碱金属盐或碱土金属盐。
另一方面，本发明提供前述式(I)化合物的制备方法，该制备方法包括使式A的化合物与式(II)的化合物反应，形成式(I)化合物的步骤：
其中，X代表离去基团，选自卤素、羟基、低级烷基磺酰氧基(例如C1-6烷基磺酰氧基)、磺酰基氧基等，优选Br、Cl、三氟甲磺酰氧基。
再一方面，本发明提供包含上述化合物或其药学上可接受的盐、立体异构体或互变异构体以及药学上可接受的载体或赋形剂的药物组合物。
具体来说，本发明的式(I)化合物或其药物上可接受的盐，作为医用的有效成分时，可与适合每种给药方法的惰性载体一起使用，并可配制成常规药物制剂，如片剂、颗粒、胶囊、粉末、溶液、悬浮液、乳液、注射液等。当作为固体制剂使用时，惰性载体包括粘合剂(阿拉伯胶、明胶、山梨醇、聚乙烯吡咯烷酮等)，赋形剂(乳糖、半乳糖、玉米淀粉、山梨糖醇等)，润滑剂(硬脂酸镁、滑石粉、聚乙二醇等)，崩解剂(马铃薯淀粉等)等。当作为注射液使用时，可用注射用蒸馏水、生理盐水、葡萄糖水溶液等进行配制。
本发明用于医疗用途的式(I)化合物或其药物上可接受的盐，给药途径可以为口服、静脉注射、肌内注射、皮下注射、透皮吸收等方式给药，给药剂量与给药体积可以根据药物的特性、给药途径、患者的年龄、体重或疾病状态等因素来确定。
本发明提供上述化合物或其药学上可接受的盐、立体异构体或互变异构体或包含上述化合物或其药学上可接受的盐、立体异构体或互变异构体以及药学上可接受的载体或赋形剂的药物组合物。其通过激活CaSR产生多种药理作用，用于预防和治疗与CaSR相关的疾病，如通过激活CaSR，抑制PTH的产生，调控血液中钙磷水平，用于治疗原发性甲状旁腺功能亢进，继发性甲状旁腺功能亢进，三发性甲状旁腺功能亢进，慢性肾功能衰竭(伴随透析或不伴随透析)，慢性肾脏疾病(伴随透析或不伴随透析)，甲状旁腺腺瘤，甲状旁腺增生，甲状旁腺癌，血管钙化和瓣膜钙化，异常钙稳态(如高钙血症)，异常磷稳态(如低磷血症)，由甲状旁腺功能亢进引起、慢性肾脏疾病或甲状旁腺癌而引起的骨相关疾病或并发症，肾移植后的骨质流失，囊性纤维性骨炎，无力型骨病，肾性骨病，由甲状旁腺功能亢进或慢性肾脏疾病引起的心血管并发症，Ca2+异常高的某些恶性肿瘤。
除非另有说明，说明书和权利要求书中使用的下列术语具有以下给出的含义。
本发明包括所述化合物的所有立体异构体型式。除非指明具体的立体构型，否则本发明意图包含这些化合物的所有此类异构形式。本发明化合物中的不对称中心均可以彼此独立地具有(R)构型或(S)构型。当与手性碳连接的键在本发明的结构式中表示为直线时，应理解手性碳的(R)和(S)构型以及两种对映异构体及其混合物都包含在该式中。当描绘特定构型时，意指对映异构体(R)或(S)。类似地，当记载化合物名称而没有手性碳的手性命名时，应理解，手性碳的(R)和(S)构型以及单个对映异构体及其混合物都被该名称包括。在获得这些立体异构体或混合物的实施例中可以鉴定特定立体异构体或其混合物的制备，但这决不限制所有立体异构体及其混合物包括在本公开的范围内。
术语“羧基”是有机化学中的基本官能团，由一个碳原子、两个氧原子和一个氢原子组成，化学式为–COOH。C1-6羧酸基、C2-6羧酸基是进一步含羧基的低级烷基。C1-6羧酸基可以是甲酸基(例如-COOH)、乙酸基、丙酸基、丁酸基、戊酸基、己酸基。C2-6羧基可以是乙酸基、丙酸基、丁酸基、戊酸基、己酸基。
本发明的式(I)化合物或其药学上可接受的盐作为有效成分，通过变构激活甲状旁腺细胞上的CaSR，抑制甲状旁腺激素的合成和分泌，从而降低血液中甲状旁腺激素水平，进而稳定维持血钙和血磷浓度，可以用于预防或治疗甲状旁腺功能亢进症，具有优异的CaSR变构激动效应和降低体内PTH作用。通过CaSR变构激活效应实验、SD大鼠腺嘌呤模型或SD大鼠切除5/6肾模型，确认化合物具有CaSR变构激活作用和降低模型大鼠体内PTH作用。
本发明的式(I)化合物或其药学上可接受的盐作为有效成分，不仅具有优异的活性，而且还具有一定的体内稳定性。本发明进行了大鼠肝微粒体孵育实验验证了所述化合物确实具有代谢稳定性。本发明的式(I)化合物或其药学上可接受的盐作为有效成分，不仅具有优异的CaSR激活效果，而且具有较高安全性。与现有技术相比较，本申请不仅活性优异，安全性好，而且在体内代谢稳定性高，体内相对生物利用度高。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自安耐吉化学、百灵威科技、麦克林、阿拉丁、毕得医药等化学品公司。
实施例中无特殊说明，溶液是指水溶液，室温是指20℃～30℃，惰性气体是指氩气或氮气，薄层色谱板(TLC)监测反应和柱层析纯化所用的展开溶剂为二氯甲烷/甲醇体系、正己烷/乙酸乙酯体系、石油醚/乙酸乙酯体系、石油醚/乙酸乙酯/甲醇体系等，体系组分比例根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
实施例中无特殊说明，结构确认用的质谱(Mass spectrum,MS)、核磁共振谱(Nuclear Magnetic Resonance，NMR)和高效液相色谱(High performance liquid chromatography,HPLC)，仪器设备型号分别为Agilent InfinityLab LC/MSD iQ G6160A,Bruker AVANCE 400或Bruke AVANCE NEO 600和Shimadzu LC-2010AHT。NMR的测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，氘代甲醇(CD3OD)或氘代重水(D2O)，内标为四甲基硅烷(TMS)。
实施例1：(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢吡咯二盐酸盐(A)的合成
合成路线如下：
步骤1：2-硝基苯磺酸-(3R)-1-{[(2-甲基丙-2-基)氧基]羰基}四氢吡咯-3-基酯(A-2)
500mL三口瓶，加入化合物A-1(25.00g,0.13mol),三乙胺(17.57g,0.17mol),三甲胺盐酸盐(2.55g,0.03mol)和二氯甲烷125mL搅拌；冷却至0℃～10℃，滴加2-硝基苯磺酰氯(32.55g,0.15mol)的二氯甲烷125mL溶液；反应完毕，加入150mL纯化水，用6N盐酸调pH至2.0～4.0；静置至明显分层，分相，保留有机相，用50mL二氯甲烷萃取水相一次，合并有机相，无水硫酸钠干燥，过滤；减压浓缩滤液至恒重，得化合物A-2(47.40g,y＝95％)。LCMS(ESI)m/z:373.1[M+H]+.
步骤2：(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢吡咯-1-甲酸-2-甲基丙-2-基酯(A-3)
100mL三口瓶，加入化合物A-2(45.00g,0.12mol),(1R)-1-(萘-1-基)乙-1-胺(17.60g,0.10mol),磷酸钾(21.23g,0.10mol)和乙腈150mL，升温至75℃，搅拌反应24h；降至室温，抽滤，60mL乙腈淋洗滤饼，减压浓缩至无溶剂蒸出；加入90mL乙酸乙酯和90mL饱和食盐水搅拌0.5h，静置至明显分层，分相，保留有机相，45mL乙酸乙酯萃取水相一次，合并有机相，无水硫酸钠干燥，减压浓缩滤液至恒重，得化合物A-3(32.62g，y＝96％)。LCMS(ESI)m/z:341.2[M+H]+.
步骤3：(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢吡咯二盐酸盐(A)
500mL三口瓶，加入化合物A-3(30.00g,0.09mol)和乙酸乙酯150mL搅拌，冷却至0℃～10℃，滴加浓盐酸22.5mL，滴毕，保温搅拌0.5h，升温至55℃反应2.0h；冷却至室温，过滤，50mL乙酸乙酯淋洗滤饼，45℃鼓风干燥至恒重，得化合物A(19.87g,y＝92％)。LCMS(ESI)m/z:241.2[M+H]+.1H NMR(400MHz,MeOH-d4):δ8.29(d,J＝8.8Hz,1H),8.04-7.97(m,2H),7.94(d,J＝7.2Hz,1H),7.72-7.58(m,3H),5.60-5.51(m,1H),3.98-3.87(m,1H),3.65-3.54(m,1H),3.51-3.38(m,2H),3.29-3.20(m,1H),2.55-2.44(m,1H),2.43-2.32(m,1H),1.89(d,J＝6.8Hz,3H).
实施例2：{2-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(1)的合成
合成路线如下：
步骤1：(3-溴-2-氟苯基)乙酸乙酯(1-1)
250mL单口瓶，加入化合物B(2.00g,8.58mmol)和60mL乙醇搅拌，冷却至0℃，滴加浓硫酸1mL，升温回流反应3.0h；冷却至室温，减压浓缩至无溶剂蒸出，加入50mL乙酸乙酯和50mL纯化水搅拌，滴加饱和碳酸钠溶液调pH至8.0，静置至明显分层，分相，保留有机相，用50mL乙酸乙酯萃取水相一次，合并有机相，无水硫酸钠干燥，过滤；减压浓缩滤液至恒重，得化合物1-1(2.00g,y＝89％)。LCMS(ESI)m/z:261.0[M+H]+.
步骤2：{2-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸乙酯(1-2)
250mL单口瓶，加入游离的化合物A(1.00g,4.16mmol),化合物1-1(1.31g,4.99mmol),Pd(OAc)2(50mg,0.20mmol),X-Phos(100mg,0.20mmol),Cs2CO3(4.10g,12.48mmol)和50mL甲苯，置换三次氮气，升温至100℃搅拌反应12.0h，冷却至室温，加入30mL饱和食盐水搅拌，静置至明显分层，分相，保留有机相，20mL乙酸乙酯萃取水相一次，合并有机相，无水硫酸钠干燥，过滤，减压浓缩滤液至无溶剂蒸出，残留物柱层析(200-300目硅胶，石油醚/乙酸乙酯：80/20→50/50)纯化，减压浓缩，得化合物1-2(0.70g,y＝40％)。LCMS(ESI)m/z:421.2[M+H]+.
步骤3：{2-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(1)
100mL单口瓶，加入1-2(0.60g,1.43mmol),氢氧化钠(572mg,14.3mmol),纯化水10mL和乙醇10mL，60℃搅拌反应3.0h，冷却至室温，用1N盐酸调pH至固体明显析出，过滤，10mL纯化水洗涤滤饼，45℃鼓风干燥至恒重，得化合物1(0.30g,y＝53％)。LCMS(ESI)m/z:393.3[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.34(d,J＝8.4Hz,1H),7.96(d,J＝7.9Hz,1H),7.87(d,J＝8.6Hz,2H),7.56(dd,J＝14.2,5.9Hz,3H),7.05(t,J＝8.6Hz,1H),6.24(d,J＝8.9Hz,2H),4.96(s,1H),3.43(s,3H),3.36(q,J＝7.7Hz,2H),3.13(q,J＝7.8Hz,2H),2.07(m,2H),1.52(s,3H).
实施例3：{3-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(2)的合成
合成路线如下：
步骤1：(4-溴-3-氟苯基)乙酸乙酯(2-1)
250mL单口瓶，加入化合物C(2.00g,8.58mmol)和60mL乙醇搅拌，冷却至0℃，滴加浓硫酸1mL，合成与后处理操作参考化合物1-1，得化合物2-1(1.80g,y＝81％)。LCMS(ESI)m/z:261.0[M+H]+.
步骤2：{3-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸乙酯(2-2)
250mL单口瓶，加入游离的化合物A(1.00g,4.16mmol),化合物2-1(1.31g,4.99mmol),Pd(OAc)2(50mg,0.20mmol),X-Phos(100mg,0.20mmol),Cs2CO3(4.10g,12.48mmol)和50mL甲苯，合成与后处理操作参考化合物1-2，得化合物2-2(0.89g,y＝51％)。LCMS(ESI)m/z:421.2[M+H]+.
步骤3：{2-氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(2)
100mL单口瓶，加入2-2(0.80g,1.90mmol),氢氧化钠(760mg,19.0mmol),纯化水10mL和乙醇10mL，合成与后处理操作参考化合物1，得化合物2(0.39g,y＝52％)。LCMS(ESI)m/z:393.3[M+H]+.1H NMR(400MHz,DMSO-d6)δ8.33-8.26(m,1H),7.93(dd,J＝7.0,2.3Hz,1H),7.79(d,J＝8.1Hz,1H),7.72(d,J＝7.2Hz,1H),7.51(dtd,J＝10.2,7.2,4.8Hz,3H),6.91(dd,J＝14.9,2.0Hz,1H),6.85(dd,J＝8.2,2.0Hz,1H),6.56(t,J＝9.0Hz,1H),4.72(q,J＝6.6Hz,1H),3.42-3.28(m,4H),3.19(p,J＝6.9,5.9Hz,2H),3.09(ddd,J＝8.9,5.7,2.7Hz,1H),1.96(dq,J＝12.3,6.1Hz,1H),1.83(dq,J＝13.6,7.2Hz,1H),1.40(d,J＝6.5Hz,3H).
实施例4：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲基苯基}乙酸(3)的合成
合成路线如下：
步骤1：(4-溴-3-甲基苯基)乙酸乙酯(3-1)
250mL单口瓶，加入化合物D(1.00g,4.37mmol)和60mL乙醇搅拌，冷却至0℃，滴加浓硫酸1mL，合成与后处理操作参考化合物1-1，得化合物3-1(0.87g,y＝77％)。LCMS(ESI)m/z:257.0[M+H]+.
步骤2：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲基苯基}乙酸乙酯(3-2)
250mL单口瓶，加入游离的化合物A(1.00g,4.16mmol),化合物3-1(1.28g,4.99mmol),Pd(OAc)2(50mg,0.20mmol),X-Phos(100mg,0.20mmol),Cs2CO3(4.10g,12.48mmol)和50mL甲苯，合成与后处理操作参考化合物1-2，得化合物3-2(0.73g,y＝42％)。LCMS(ESI)m/z:417.2[M+H]+.
步骤3：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲基苯基}乙酸(3)
100mL单口瓶，加入3-2(0.70g,1.68mmol),氢氧化钠(0.34g,8.40mmol),纯化水10mL和乙醇10mL，合成与后处理操作参考化合物1，得化合物3(0.44g,y＝67％)。LCMS(ESI)m/z:389.2[M+H]+.1H NMR(400MHz,DMSO-d6)δ8.32(d,J＝8.0Hz,1H),7.93(dd,J＝7.3,2.1Hz,1H),7.80(d,J＝8.1Hz,1H),7.73(d,J＝7.1Hz,1H),7.51(qd,J＝8.1,7.3,2.3Hz,3H),6.97-6.86(m,2H),6.71-6.64(m,1H),4.71(q,J＝6.5Hz,1H),3.38(s,2H),3.18(s,1H),3.13-3.06(m,2H),3.02(m,1H),2.92(dd,J＝9.2,6.3Hz,1H),2.11(s,3H),1.99(dq,J＝12.6,6.5Hz,1H),1.86-1.73(m,1H),1.41(d,J＝6.5Hz,3H).
实施例5：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-甲基苯基}乙酸(4)的合成：
合成路线如下：
步骤1：(4-溴-2-甲基苯基)乙酸乙酯(4-1)
250mL单口瓶，加入化合物E(1.00g,4.37mmol)和60mL乙醇搅拌，冷却至0℃，滴加浓硫酸1mL，合成与后处理操作参考化合物1-1，得化合物4-1(0.93g,y＝83％)。LCMS(ESI)m/z:257.0[M+H]+.
步骤2：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲基苯基}乙酸乙酯(4-2)
250mL单口瓶，加入游离的化合物A(1.00g,4.16mmol),化合物4-1(1.28g,4.99mmol),Pd(OAc)2(50mg,0.20mmol),X-Phos(100mg,0.20mmol),Cs2CO3(4.10g,12.48mmol)和50mL甲苯，合成与后处理操作参考化合物1-2，得化合物4-2(0.73g,y＝42％)。LCMS(ESI)m/z:417.2[M+H]+.
步骤3：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-甲基苯基}乙酸(4)
100mL单口瓶，加入4-2(0.50g,1.20mmol),氢氧化钠(0.24g,6.00mmol),纯化水5mL和乙醇5mL，合成与后处理操作参考化合物1，得化合物4(0.32g,y＝69％)。LCMS(ESI)m/z:389.2[M+H]+.1H NMR(400MHz,DMSO-d6)δ8.30(d,J＝8.0Hz,1H),7.92(dd,J＝7.3,2.1Hz,1H),7.81(d,J＝8.1Hz,1H),7.73(d,J＝7.1Hz,1H),7.53(qd,J＝8.1,7.3,2.3Hz,3H),6.97-6.86(m,2H),6.71-6.64(m,1H),4.71(q,J＝6.5Hz,1H),3.38(s,2H),3.18(s,1H),3.13-3.06(m,2H),3.02(m,1H),2.91(dd,J＝9.2,6.3Hz,1H),2.10(s,3H),1.99(dq,J＝12.6,6.5Hz,1H),1.86-1.72(m,1H),1.43(d,J＝6.5Hz,3H).
实施例6：{2,6-二氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(5)的合成
合成路线如下：
步骤1：{2,6-二氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸乙酯(5-1)
250mL单口瓶，加入游离的化合物A(861mg,3.58mmol),化合物F(1.00g,3.58mmol),Pd(OAc)2(80mg,0.36mmol),X-Phos(171mg,0.36mmol),Cs2CO3(3.50g,10.74mmol)和50mL甲苯，合成与后处理操作参考化合物1-2，得化合物5-1(1.44g,y＝74％)。LCMS(ESI)m/z:439.2[M+H]+.
步骤2：{2,6-二氟-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(5)
100mL单口瓶，加入5-1(1.44g,3.28mmol),氢氧化钠(0.66g,16.43mmol),纯化水5mL和乙醇5mL，合成与后处理操作参考化合物1，得化合物5(1.25g,y＝93％)。LCMS(ESI)m/z:411.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.30(s,1H),7.94(s,1H),7.85-7.78(m,1H),7.73(dd,J＝10.9,6.0Hz,1H),7.57-7.47(m,J＝6.5,5.5Hz,3H),6.08(dq,J＝10.2,5.2Hz,2H),4.75(dd,J＝11.7,6.0Hz,1H),3.42(q,J＝5.5,5.0Hz,2H),3.37-3.23(m,3H),3.11(s,1H),2.99(s,1H),2.05-1.98(m,1H),1.94-1.88(m,1H),1.42(p,J＝5.7Hz,3H).
实施例7：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲氧基苯基}乙酸(6)的合成
合成路线如下：
步骤1：4-溴-3-甲氧基苯甲酸甲酯(6-1)
100mL单口瓶，加入游离的化合物G(10.00g,0.046mol),碘甲烷(19.62g,0.14mol),碳酸钾(25.47g,0.18mol)和50mL DMF搅拌反应过夜，加入500mL饱和食盐水和100mL乙酸乙酯搅拌，静置至明显分层，分相，保留有机相，50mL乙酸乙酯萃取水相两次，合并有机相，无水硫酸钠干燥，过滤，减压浓缩滤液至无溶剂蒸出，得化合物6-1(10.50g,y＝93％)。LCMS(ESI)m/z:245.0[M+H]+.
步骤2：(4-溴-3-甲氧基苯基)甲醇(6-2)
500mL三口瓶，加入化合物6-1(10.50g,23.8mmol)和100mL无水四氢呋喃搅拌，冷却至0℃，分批加入四氢铝锂(1.95g,51.4mmol)，保温反应4.0h，缓慢加入1N盐酸150mL搅拌，静置至明显分层，分相，保留有机相，50mL乙酸乙酯萃取水相两次，合并有机相，无水硫酸钠干燥，过滤，减压浓缩滤液至无溶剂蒸出，残留物柱层析纯化(200-300目硅胶，石油醚/乙酸乙酯:95/5→80/20)，减压浓缩，得化合物6-2(5.80g,y＝62％)。LCMS(ESI)m/z:217.0[M+H]+.
步骤3：1-溴-4-(溴甲基)-2-甲氧基苯(6-3)
500mL三口瓶，加入化合物6-2(5.00g,23.00mmol),三苯基膦(7.30g,27.60mmol),四溴化碳(8.40g,25.30mmol)和100mL二氯甲烷搅拌过夜，减压浓缩滤液至无溶剂蒸出，残留物柱层析纯化(200-300目硅胶，石油醚/乙酸乙酯:95/5→85/15)，减压浓缩，得化合物6-3(5.80g,y＝90％)。LCMS(ESI)m/z:279.0[M+H]+.
步骤4：(4-溴-3-甲氧基苯基)乙腈(6-4)
500mL三口瓶，加入化合物6-3(5.00g,17.9mmol),TBAF(5.60g,21.4mmol)和50mL乙腈搅拌，冷却至0℃，分批加入TMSCN(2.13g,21.4mmol),搅拌过夜，减压浓缩滤液至无溶剂蒸出，残留物柱层析纯化(200-300目硅胶，石油醚/乙酸乙酯:95/5→85/15)，减压浓缩，得化合物6-4(3.60g,y＝89％)。LCMS(ESI)m/z:226.0[M+H]+.
步骤5：(4-溴-3-甲氧基苯基)乙酸乙酯(6-5)
500mL三口瓶，加入化合物6-4(3.00g,13.27mmol)和50mL乙醇搅拌，冷却至0℃，滴加浓硫酸7.5mL,合成与后处理操作参考化合物1-1，得化合物6-5(3.20g,y＝88％)。LCMS(ESI)m/z:273.0[M+H]+.
步骤6：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲氧基苯基}乙酸乙酯(6-6)
100mL单口瓶，加入游离的化合物A(1.00g,4.61mmol),化合物6-5(1.10g,4.99mmol),Pd(OAc)2(19mg,0.08mmol),X-Phos(40mg,0.08mmol),Cs2CO3(4.00g,12.50mmol)和20mL甲苯，合成与后处理操作参考化合物1-2，得化合物6-6(1.20g,y＝67％)。LCMS(ESI)m/z:433.2[M+H]+.
步骤7：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-3-甲氧基苯基}乙酸(6)
100mL单口瓶，加入6-6(1.00g,2.31mmol),氢氧化钠(0.28g,6.93mmol),纯化水5mL和乙醇5mL，合成与后处理操作参考化合物1，得化合物6(0.31g,y＝33％)。LCMS(ESI)m/z:405.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.32(d,J＝8.2Hz,1H),7.92(dd,J＝7.7,1.8Hz,1H),7.79(d,J＝8.2Hz,1H),7.72(dd,J＝7.3,1.2Hz,1H),7.55-7.46(m,3H),6.73(d,J＝2.0Hz,1H),6.65(dd,J＝8.1,1.9Hz,1H),6.48(d,J＝8.1Hz,1H),4.71(s,1H),3.64(s,3H),3.40(s,2H),3.22(dt,J＝7.9,6.0Hz,2H),3.14(dt,J＝9.4,6.8Hz,2H),3.01(t,J＝7.9Hz,1H),1.93(dt,J＝13.2,6.6Hz,1H),1.77(dd,J＝12.5,6.7Hz,1H),1.40(d,J＝6.6Hz,3H).
实施例8：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-甲氧基苯基}乙酸(7)的合成
合成路线如下：
步骤1：4-溴-2-甲氧基苯乙酸乙酯(7-1)
500mL三口瓶，加入化合物H(10.00g,40.80mmol)和80mL乙醇搅拌，冷却至0℃，滴加浓硫酸1.5mL,合成与后处理操作参考化合物1-1，得化合物7-1(10.50g,y＝94％)。LCMS(ESI)m/z:245.0[M+H]+.
步骤2：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-甲氧基苯基}乙酸乙酯(7-2)
100mL单口瓶，加入游离的化合物A(1.47g,6.11mmol),化合物7-1(2.00g,7.32mmol),Pd(OAc)2(70mg,0.31mmol),X-Phos(150mg,0.31mmol),Cs2CO3(5.96g,12.50mmol)和20mL甲苯，合成与后处理操作参考化合物1-2，得化合物7-2(1.60g,y＝61％)。LCMS(ESI)m/z:433.2[M+H]+.
步骤3：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-甲氧基苯基}乙酸乙酯(7)
100mL单口瓶，加入7-2(1.60g,3.70mmol),氢氧化钠(0.59g,14.75mmol),纯化水5mL和乙醇5mL，合成与后处理操作参考化合物1，得化合物7(0.50g,y＝33％)。LCMS(ESI)m/z:405.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.33(d,J＝8.3Hz,1H),7.93(d,J＝7.8Hz,1H),7.81(d,J＝8.0Hz,1H),7.75(d,J＝7.2Hz,1H),7.51(q,J＝7.9,6.8Hz,3H),6.88(d,J＝8.2Hz,1H),5.98(d,J＝2.3Hz,1H),5.96(dd,J＝8.4,2.2Hz,1H),4.76(s,1H),3.70(s,3H),3.32(s,4H),3.12(dt,J＝9.2,7.3Hz,1H),2.99(s,1H),2.04(s,1H),1.91(s,1H),1.42(s,3H).
实施例9：{2-乙氧基-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(8)的合成
合成路线如下：
步骤1：4-溴-2-乙氧基苯甲酸乙酯(8-1)
250mL三口瓶，加入化合物I(2.00g,7.72mmol),碳酸钾(3.20g,23.00mmol),溴乙烷(0.93g,8.40mmol)和50mL乙腈，60℃搅拌反应24.0h；冷却至室温，减压浓缩至无溶剂蒸出，加入50mL乙酸乙酯和50mL纯化水搅拌，静置至明显分层，分相，保留有机相，用50mL乙酸乙酯萃取水相一次，合并有机相，无水硫酸钠干燥，过滤；减压浓缩滤液至恒重，得化合物8-1(2.02g,y＝91％)。LCMS(ESI)m/z:287.0[M+H]+.
步骤2：{2-乙氧基-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸乙酯(8-2)
100mL单口瓶，加入游离的化合物A(1.00g,4.16mmol),化合物8-1(2.00g,7.32mmol),Pd(OAc)2(70mg,0.31mmol),X-Phos(150mg,0.31mmol),Cs2CO3(4.51g,13.90mmol)和20mL甲苯，合成与后处理操作参考化合物1-2，得化合物8-2(1.60g,y＝86％)。LCMS(ESI)m/z:447.3[M+H]+.
步骤3：{2-乙氧基-4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]苯基}乙酸(8)
100mL单口瓶，加入8-2(1.60g,3.53mmol),氢氧化钠(0.60g,15.00mmol),纯化水10mL和乙醇10mL，合成与后处理操作参考化合物1，得化合物8(0.52g,y＝35％)。LCMS(ESI)m/z:419.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ11.70(s,1H),10.23(s,1H),9.78(s,1H),8.39(d,J＝8.5Hz,1H),8.12(s,1H),8.02(t,J＝8.9Hz,2H),7.64(d,J＝7.5Hz,2H),7.60(t,J＝7.5Hz,1H),6.93(d,J＝8.0Hz,1H),6.02(d,J＝9.1Hz,2H),5.42(s,1H),3.96(q,J＝7.0Hz,2H),3.82(s,1H),3.40(t,J＝7.1Hz,3H),3.15(q,J＝8.1Hz,1H),2.34(s,1H),2.26(s,1H),1.76(s,3H),1.29(t,J＝7.0Hz,3H).
实施例10：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙-2-基氧基)苯基}乙酸(9)的合成
合成路线如下：
步骤1：[4-溴-2-(丙-2-基氧基)苯基]乙酸乙酯(9-1)
250mL三口瓶，加入化合物I(2.00g,7.72mmol),碳酸钾(3.20g,23.15mmol),溴代异丙烷(1.04g,8.49mmol)和50mL乙腈，合成与后处理操作参考化合物8-1，得化合物9-1(1.95g,y＝84％)。LCMS(ESI)m/z:301.0[M+H]+.
步骤2：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙-2-基氧基)苯基}乙酸乙酯(9-2)
100mL单口瓶，加入游离的化合物A(0.80g,3.32mmol),化合物9-1(1.20g,3.32mmol),Pd(OAc)2(40mg,0.16mmol),X-Phos(80mg,0.16mmol),Cs2CO3(3.25g,9.96mmol)和20mL甲苯，合成与后处理操作参考化合物1-2，得化合物9-2(0.80g,y＝52％)。LCMS(ESI)m/z:461.3[M+H]+.
步骤3：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙-2-基氧基)苯基}乙酸(9)
100mL单口瓶，加入9-2(0.80g,1.70mmol),氢氧化钠(0.20g,5.20mmol),纯化水10mL和乙醇10mL，合成与后处理操作参考化合物1，得化合物9(0.50g,y＝68％)。LCMS(ESI)m/z:433.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.32(d,J＝8.0Hz,1H),7.92(dd,J＝7.2,2.3Hz,1H),7.79(d,J＝8.1Hz,1H),7.74(d,J＝7.2Hz,1H),7.51(qd,J＝7.2,5.1Hz,3H),6.87(d,J＝8.7Hz,1H),5.94(dq,J＝4.4,2.3Hz,2H),4.73(d,J＝6.9Hz,1H),4.50-4.42(m,1H),3.29(d,J＝4.1Hz,3H),3.24(t,J＝6.3Hz,1H),3.20(dd,J＝9.0,6.6Hz,1H),3.09(dt,J＝9.1,7.2Hz,1H),2.92(dd,J＝9.1,5.5Hz,1H),2.02(dd,J＝12.2,6.3Hz,1H),1.87(s,1H),1.40(d,J＝6.6Hz,3H),1.21(d,J＝6.0Hz,6H).
实施例11：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙基氧基)苯基}乙酸(10)的合成
合成路线如下：
步骤1：[4-溴-2-(丙基氧基)苯基]乙酸乙酯(10-1)
250mL三口瓶，加入化合物I(2.00g,7.72mmol),碳酸钾(3.20g,23.15mmol),溴代正丙烷(1.04g,8.49mmol)和50mL乙腈，合成与后处理操作参考化合物8-1，得化合物10-1(1.57g,y＝68％)。LCMS(ESI)m/z:301.0[M+H]+.
步骤2：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙基氧基)苯基}乙酸乙酯(10-2)
100mL单口瓶，加入游离的化合物A(1.04g,4.31mmol),化合物10-1(1.57g,5.20mmol),Pd(OAc)2(50mg,0.22mmol),X-Phos(100mg,0.22mmol),Cs2CO3(4.25g,13.04mmol)和20mL甲苯，合成与后处理操作参考化合物1-2，得化合物10-2(0.45g,y＝23％)。LCMS(ESI)m/z:461.3[M+H]+.
步骤3：{4-[(3S)-3-{[(1R)-1-(萘-1-基)乙基]氨基}四氢-1H-吡咯-1-基]-2-(丙基氧基)苯基}乙酸(10)
100mL单口瓶，加入10-2(0.45g,0.98mmol),氢氧化钠(0.12g,3.00mmol),纯化水10mL和乙醇10mL，合成与后处理操作参考化合物1，得化合物10(0.38g,y＝90％)。LCMS(ESI)m/z:433.2[M+H]+.1H NMR(600MHz,DMSO-d6)δ8.33(d,J＝8.3Hz,1H),7.94(d,J＝7.8Hz,1H),7.82(s,1H),7.76(s,1H),7.52(dt,J＝15.4,8.8Hz,3H),6.88(d,J＝8.3Hz,1H),5.95(s,2H),4.78(s,1H),3.84(t,J＝6.3Hz,2H),3.32(s,4H),3.27(s,1H),3.11(q,J＝7.8Hz,1H),2.99(s,1H),2.06(s,1H),1.94(s,1H),1.68(h,J＝6.7Hz,2H),1.44(s,3H),0.97(t,J＝7.4Hz,3H).
实验例1：CaSR变构激活效应体外实验
CaSR属于G蛋白偶联受体(GPCR)C家族一员。当细胞外Ca2+或化合物激活细胞上的CaSR时，细胞内Ca2+浓度升高。因此，应用稳转hCaSR-CHO的细胞系，并以细胞内Ca2+浓度作为检测指标，通过检测细胞内Ca2+浓度变化，评价化合物对CaSR的变构激活效应。
一、实验步骤：
1、铺板前准备：细胞铺板前12h，用0.1mg/mL多聚赖氨酸包被384孔细胞培养板。
2、细胞铺板：将消化后的CaSR-CHO细胞用完全培养基重悬，细胞计数器计数并将细胞密度调整为5×105cells/mL的密度，每孔接种细胞悬液体积为25μL。细胞接种后，放置于37℃，5％ CO2培养箱继续培养18h。
3、配制化合物测试液：将待测化合物倍比稀释至所需浓度(10μM、2μM、400nM、80nM、16nM、3.2nM和0.64nM)，并加入384孔加样板，备用。此处，由于待检测的细胞培养板中已有Fluo-4 NW检测试剂25μL，仪器设置给药体系为20μL，因此，此时加样板中的化合物浓度是实际检测浓度的2.25倍，即10μM化合物上样孔的化合物浓度为22.5μM，以此类推。
4、检测试剂准备：根据试剂盒Fluo-4 NW Calcium Assay Kits(Thermo,F36206)说明书的要求，在Component B中加入1mL assay buffer，制备成250mM的丙磺舒溶液；然后取100μL丙磺舒溶液加入10mL assay buffer中，充分混匀后加入Component A中，制备成1X dye loading solution备用。
5、加入荧光染料：弃去完全培养基，使用multidrop自动分液器将25μL 1X dye loading solution加到384孔板中，避光放置37℃孵育30min，转移至室温再放置30min，转入Fliper Tetra(Molecular Devices)仪器。
6、读取原始实验数据：加入测试化合物溶液20μL，设置激发波长470-495nm范围，检测515-575nm范围的发射光，仪器质控板读板确定无误后开始检测，连续检测时间长度660秒，读取结束后导出数值。
7、数据处理：加药浓度后的最大荧光值(MAX取40sec-80sec均值)减去最低荧光值(MIN取14sec-21sec均值)，得出荧光强度变化数值，以此表示细胞内钙信号变化情况。使用GraphPad Prism 8.0，以加药浓度为横坐标，以荧光强度变化为纵坐标作图，计算出个化合物的EC50值。
二、实验结果：
本发明部分化合物的体外药效结果，见表1。
表1.化合物的细胞药效实验数据
实验例2：抑制造模大鼠血液中PTH浓度的体内实验
通过切除5/6肾+特殊饲料诱导方式，构建SD雄性大鼠甲状旁腺功能亢进模型，以血液中甲状旁腺激素(PTH)作为检测指标，应用ELISA酶联免疫法检测给予化合物后大鼠血浆中PTH浓度的变化，用于评价化合物的体内活性。
一、实验步骤：
1、模型构建:应用SPF级7～8周龄雄性SD大鼠。正常饲料适应性饲养1周；切除2/3左肾并用正常饲料恢复饲养1周；切除右肾并用正常饲料恢复饲养1周；特殊饲料(钙含量0.5％和总磷含量1.2％)喂养2周。
2、检测与分组：尾静脉采集约300μL血液至促凝管，室温放置30分钟，12000rpm离心3分钟，得血清样品，并用MicroVue Intact PTH EIA-96Test(Quidel,catalog No.60-2500)检测血清中全段PTH(1-84)浓度；根据大鼠PTH浓度水平进行分组，每组大鼠PTH浓度均值基本相同；
3、给药与活性测试：单次灌胃给予一定剂量的化合物，并采集给药前后的血样，并用上述方法处理得血清，ELISA检测PTH含量。根据该方法，证实化合物具有降低模型大鼠血液PTH水平的作用。
二、实验结果：
本发明部分化合物降低模型大鼠血液PTH的效果，见表2。
表2.化合物的体内药效实验数据
注：与0h PTH浓度相比，给药后1h降低PTH的效率。
实验例3：大鼠肝微粒体稳定性试验
以一定浓度的大鼠肝微粒体，睾酮(阳性对照)，甲苯磺丁脲(内标)，待测化合物和PBS(pH 7.4)，在37℃孵育，分别于0、5、10、20、30和60分钟，甲醇淬灭，预处理样品，并用LC-MS方法检测，数据处理(以待测化合物峰面积/内标峰面积为纵坐标和时间为横坐标作图)，计算得出t1/2。本发明部分化合物的测试结果如表3所示。
表3.化合物的大鼠肝微粒稳定性实验数据

注：肝微粒体稳定性试验受肝微粒体活性影响，体外试验最长孵育时间为60min，t1/2＞60min预估化合物在体内代谢稳定性高。
实验例4：SD大鼠药代动力学实验
一、实验步骤：
1)动物信息：SD正常大鼠，7～8周龄，雄性，3～5只/组
2)给药信息：给药前禁食12h以上，单次灌胃给药。
3)采血点：给药前及给药后0.25、0.5、1、2、4、6、8和24h尾静脉采血，每个采血点收集100μL血样。
4)样品处理及保管：血样采集后转移至1.5mL EP管中，每管50μL，每个样本2管，2h内完成离心，全血样品在离心前冰浴保存，离心条件为：4000r/min，2-8℃，10min。吸取上清后若不立即检测，于-80℃以下保存。
5)检测
a)色谱柱：Infinitylab poroshell 120SB C18 2.1*100mm，2.7μm；柱温：30℃；流速：0.3ml/min；运行时间：9.5min；后运行时间：2min；进样体积：2μL；流动相：甲醇-0.1％甲酸。按下表进行梯度洗脱：
b)质谱条件：

c)储备液、工作溶液样品的配制：
所有配制的储备液都保存在-80℃冰箱中，工作液放置4℃冰箱。Evocalcet贮备液(0.2mg/mL)：精密称量本品10mg至50ml量瓶中，用甲醇溶解并定容至刻度，摇匀，即得。(化合物贮备液配制同Evocalcet)
标准溶液的配制：精密量取Evocalcet、化合物2和化合物3的贮备液，用50％甲醇稀释，配制浓度为80、200、1000、5000、10000、20000和40000ng/mL的标准溶液。
内标盐酸克伦特罗溶液的配制：精密称量盐酸克伦特罗10mg至10ml量瓶中，用甲醇溶液并定容至刻度，摇匀，即得盐酸克伦特罗贮备液(1.0mg/mL)。精密量取贮备液适量，用甲醇稀释成2000ng/mL的内标工作液。
d)样品处理过程：
标准曲线样品处理：取空白基质90μL，分别加入标准系列溶液各10uL，内标溶液50μL，涡旋混匀30S，加350μL甲醇沉淀蛋白溶液，涡旋混合，样品于离心机中以12000rpm，离心10min，取上清液200μL加入600μL水混匀，取上清液进样分析。
血样处理：取血浆样品50μL，加内标溶液25μL，涡旋混匀30s，加175μL甲醇沉淀蛋白溶液，涡旋混合，样品于离心机中以12000rpm，离心10min，取上清液100μL加入300μL水混匀，取上清液进样分析。
二、实验结果
结果如表4所示。
表4

Claims (9)

  1. 具有如式(I)所示结构的化合物或其药学上可接受的盐、立体异构体或互变异构体：
    其中，R1、R2、R3、R4、R5各自独立地选自氢原子；C2-6羧酸基；C1-7烷氧基，其被选自以下的取代基取代：卤素、氰基、硝基、氧、C1-6羧酸基、胺基、C3-6环烷基、芳基C1-6烷基氧基、羟基、C1-6烷氧基、卤代C1-6烷基和卤代C1-6烷氧基；卤素；C1-6烷基；卤代C1-6烷基；
    条件是：1)R1、R2、R3、R4、R5不同时为H；2)当取代基为C2-6羧酸基，取代位点为R2-R4，且R1-R5中至少2个不是H。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐、立体异构体或互变异构体，其中，R1、R5选自H、卤素、C1-6的烷基、C1-6的烷氧基。
  3. 根据权利要求1或2所述的化合物或其药学上可接受的盐、立体异构体或互变异构体，其中，R2-R4中至少一个为未取代或取代C2-6羧酸基，优选为乙酸基，且R1-R5中至少2个不是H；优选地，R2-R4中至少一个为未取代或取代乙酸基，且R1-R5中至少2个不是H。
  4. 根据权利要求1至3中任一项所述的化合物或其药学上可接受的盐、立体异构体或互变异构体，其中，R1、R2、R4、R5各自独立地选自H、卤素、C1-6的烷基、C1-6的烷氧基，其被选自以下的取代基取代：羟基、C1-6烷氧基，且R1、R2、R4、R5不同时为H；R3为C2-6羧酸基；
    优选地，R1、R2、R4、R5各自独立地选自H、卤素、C1-6的烷基、C1-6的烷氧基，其被选自以下的取代基取代：羟基、C1-6烷氧基，且R1、R2、R4、R5不同时为H；R3为乙酸基。
  5. 根据权利要求1至4中任一项所述的化合物或其药学上可接受的盐、立体异构体或互变异构体，其中，式(I)的化合物选自
  6. 一种权利要求1至5中任一项所述的式(I)化合物的制备方法，该制备方法包括使式A的化合物与式(II)的化合物反应，形成式(I)化合物的步骤：
    其中，X代表离去基团，选自卤素、羟基、低级烷基磺酰氧基(例如C1-6烷基磺酰氧基)、磺酰基氧基等，优选Br、Cl、三氟甲磺酰氧基。
  7. 一种包含权利要求1至5中任一项所述的化合物或其药学上可接受的盐、立体异构体或互变异构体以及药学上可接受的载体或赋形剂的药物组合物。
  8. 权利要求1至5中任一项所述的化合物或其药学上可接受的盐、立体异构体或互变异构体或权利要求6所述的药物组合物在用于制备预防和/或治疗与CaSR相关的疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其中，所述疾病选自原发性甲状旁腺功能亢进，继发性甲状旁腺功能亢进，三发性甲状旁腺功能亢进，慢性肾功能衰竭，慢性肾脏疾病，甲状旁腺腺瘤，甲状旁腺增生，甲状旁腺癌，血管钙化和瓣膜钙化，异常钙稳态，异常磷稳态，由甲状旁腺功能亢进引起、慢性肾脏疾病或甲状旁腺癌而引起的骨相关疾病或并发症，肾移植后的骨质流失，囊性纤维性骨炎，无力型骨病，肾性骨病，由甲状旁腺功能亢进或慢性肾脏疾病引起的心血管并发症，Ca2+异常高的某些恶性肿瘤。
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CN113121398B (zh) * 2019-12-31 2023-06-20 上海拓界生物医药科技有限公司 芳基烷基胺化合物、其制备方法及其在医药上的应用

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