WO2022057838A1 - 异色满类化合物 - Google Patents

异色满类化合物 Download PDF

Info

Publication number
WO2022057838A1
WO2022057838A1 PCT/CN2021/118597 CN2021118597W WO2022057838A1 WO 2022057838 A1 WO2022057838 A1 WO 2022057838A1 CN 2021118597 W CN2021118597 W CN 2021118597W WO 2022057838 A1 WO2022057838 A1 WO 2022057838A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
present
compounds
group
pharmaceutically acceptable
Prior art date
Application number
PCT/CN2021/118597
Other languages
English (en)
French (fr)
Inventor
蔡哲
孙飞
丁照中
陈曙辉
Original Assignee
南京明德新药研发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京明德新药研发有限公司 filed Critical 南京明德新药研发有限公司
Priority to US18/044,791 priority Critical patent/US20230348503A1/en
Priority to EP21868660.8A priority patent/EP4215523A4/en
Priority to CN202180064048.7A priority patent/CN116249698A/zh
Publication of WO2022057838A1 publication Critical patent/WO2022057838A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/76—Benzo[c]pyrans
    • 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
    • C07D405/12—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 chain containing hetero atoms as chain links

Definitions

  • the present invention relates to isochroman compounds with novel structures, in particular to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the field of therapy applications in .
  • Cancer tumor is a major disease that seriously threatens human life and health.
  • Current treatments include surgery, chemotherapy, and targeted therapy.
  • Chemotherapy is a treatment method that uses chemical drugs to kill tumor cells and inhibit the growth of tumor cells. It is a systemic treatment method. Due to the heterogeneity of malignancies, chemotherapy remains an important method for the treatment of tumors. However, it is this systemic treatment that causes chemotherapy to have significant side effects. There is a huge unmet clinical need for developing targeted chemotherapeutics.
  • Aldehyde-keto reductase is a member of the aldo-keto reductase family and is mainly involved in hormone synthesis and toxin removal.
  • AKR1C3 can be overexpressed by factors such as smoking, alcohol, hepatitis B or C infection.
  • AKR1C3 is overexpressed in a variety of refractory cancers, such as liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate adenocarcinoma, acute lymphoblastic leukemia, especially liver cancer, with a high expression rate of more than 60%.
  • Taiwan Haoding Company reported a compound OBI-3424 targeting the AKR1C3 enzyme.
  • OBI-3424 is a selective prodrug that releases a potent DNA alkylating agent in tumor cells with high expression of AKR1C3 enzyme, selectively kills tumor cells with high expression of AKR1C3, and enables chemical drugs to have obvious targeting effects.
  • HCC hepatocellular carcinoma
  • CRPC castrated prostate cancer
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • T is N or CH
  • R 1 and R 2 are each independently H, F, Cl, Br, I or C 1-3 alkyl, wherein said C 1-3 alkyl is optionally substituted with 1, 2 or 3 R a ;
  • each Ra is independently F, Cl, Br, I, -CN, -OH, or -NH2 ;
  • R 3 and R 4 are each independently H, F, Cl, Br, I, CN, C 1-3 alkyl, C 1-3 alkoxy, wherein the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R e ;
  • R b and R c are each independently H, -CH 3 , -CH 2 CH 3 , -(CH 2 ) 2 CH 3 or -CH(CH 3 ) 2 ;
  • R d is -CH 3 , -CH 2 CH 3 , -(CH 2 ) 2 CH 3 or -CH(CH 3 ) 2 ;
  • Each R e is independently F, Cl, Br, I, -CN, -OH or -NH2 .
  • the above-mentioned compound has the structure represented by formula (I-1) or formula (I-2):
  • R 1 , R 2 , R 3 and R 4 are as defined in the present invention.
  • the above-mentioned compound has the structure represented by formula (I-3) or (I-4):
  • the carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer form; R 1 , R 2 , R 3 and R 4 as defined in the present invention.
  • R 1 and R 2 are each independently H, F, Cl, Br, I or -CH 3 , wherein the -CH 3 is optionally represented by 1, 2 or 3 R a Substitution, Ra and other variables are as defined herein.
  • R 1 is H, F, Cl, Br, I or -CH 3 , and other variables are as defined in the present invention.
  • R2 is H, and other variables are as defined herein.
  • R 1 is H, F, Cl, Br, I or -CH 3 ;
  • R 2 is H, and other variables are as defined in the present invention.
  • R 3 and R 4 are each independently H, F, Cl, Br, I, CN, -CH 3 , -OCH 3 , wherein the -CH3 is optionally substituted with 1, 2 or 3 R e , R e and other variables are as defined in the present invention.
  • R 3 and R 4 are each independently H, F, -CH 3 , -CHF 2 , -OCH 3 , Other variables are as defined in the present invention.
  • R 3 and R 4 are each independently H, F, -CH 3 , -CHF 2 , -OCH 3 , and other variables are as defined in the present invention.
  • R3 and R4 above are each independently H.
  • the present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof,
  • the present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof,
  • the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a drug targeting AKR1C3 enzyme.
  • the present invention also provides the application of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver cancer.
  • the present invention provides a compound targeting AKR1C3 with novel structure.
  • the compounds of the present invention have excellent anti-proliferative activity on tumor cells with high expression of AKR1C3, and weak activity on tumor cells with low AKR1C3 expression, showing excellent selectivity.
  • the compounds of the present invention exhibited significant antitumor efficacy.
  • salts refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
  • the compounds provided herein also exist in prodrug forms.
  • Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the present invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.
  • tautomer or “tautomeric form” refers to isomers of different functional groups that are in dynamic equilibrium and are rapidly interconverted at room temperature.
  • a chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution).
  • proton tautomers also called prototropic tautomers
  • prototropic tautomers include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence tautomers include interconversions by recombination of some bonding electrons.
  • keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in one enantiomer” refer to one of the isomers or pairs
  • the enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • isomeric excess or “enantiomeric excess” refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .
  • Optically active (R)- and (S)-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art
  • the diastereoisomers were resolved and the pure enantiomers recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • deuterated drugs can be formed by replacing hydrogen with deuterium, and the bonds formed by deuterium and carbon are stronger than those formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • oxygen it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically achievable basis.
  • any variable eg, R
  • its definition in each case is independent.
  • the group may optionally be substituted with up to two Rs, with independent options for R in each case.
  • combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituents When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate through which atom it is attached to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be through any one of the pyridine ring The carbon atom is attached to the substituted group.
  • the direction of attachment is arbitrary, for example,
  • the linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right. It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.
  • Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • any one or more sites in the group can be linked to other groups by chemical bonds.
  • the chemical bond connecting the site to other groups can be represented by straight solid line bonds straight dotted key or wavy lines Express.
  • a straight solid bond in -OCH 3 indicates that it is connected to other groups through the oxygen atom in this group;
  • the straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;
  • the wavy lines in the phenyl group indicate connections to other groups through the 1 and 2 carbon atoms in the phenyl group.
  • the number of atoms in a ring is generally defined as the number of ring members, eg, "5-7 membered ring” refers to a “ring” of 5-7 atoms arranged around it.
  • C 1-3 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (eg methyl), divalent (eg methylene) or multivalent (eg methine) .
  • Examples of C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • C1-3alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms attached to the remainder of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy and the like.
  • Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • Cn-n+m or Cn - Cn+m includes any particular instance of n to n+ m carbons, eg C1-12 includes C1 , C2 , C3, C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also including any one range from n to n+m, eg C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 , etc.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, a nucleophilic substitution reaction).
  • a substitution reaction eg, a nucleophilic substitution reaction
  • representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-
  • hydroxy protecting group refers to a protecting group suitable for preventing hydroxyl side reactions.
  • Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.
  • alkyl groups such as methyl, ethyl and tert-butyl
  • acyl groups such as alkanoyl (eg acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenyl
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the present invention adopts the following abbreviations:
  • Pd/C Pd/C catalyst palladium content 10w% DCM Dichloromethane THF tetrahydrofuran EtOAc Ethyl acetate TBME tert-butyl methyl ether Boc tert-Butoxycarbonyl, an amine protecting group Cbz Benzyloxycarbonyl, an amine protecting group DMF N,N-Dimethylformamide TFA Trifluoroacetate PE Petroleum ether DMSO dimethyl sulfoxide EtOH Ethanol MeOH methanol AcOH Acetic acid DIPEA Diisopropylethylamine SiO2 100-200 mesh silica powder for column chromatography psi Pound force per square inch, pressure unit p-HPLC Preparative high performance liquid chromatography for compound purification
  • the solvents used in the present invention are commercially available and do not require further purification.
  • the reaction is generally carried out in an anhydrous solvent under inert nitrogen.
  • Proton NMR data were recorded on a Bruker Avance III 400 (400 MHz) spectrometer and chemical shifts are expressed in ppm at the upfield of tetramethylsilane.
  • LC/MS or Shimadzu MS contains a DAD: SPD-M20A (LC) and Shimadzu Micromass 2020 detector.
  • the mass spectrometer is equipped with an electrospray ionization source (ESI) operating in positive or negative mode.
  • ESI electrospray ionization source
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuK ⁇ radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • SXRD single crystal X-ray diffraction method
  • the cultured single crystal is collected by Bruker D8 venture diffractometer
  • the light source is CuK ⁇ radiation
  • the scanning mode is: After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • High performance liquid chromatography was performed on a Shimadzu LC20AB system equipped with a Shimadzu SIL-20A autosampler and Shimadzu DAD: SPD-M20A detector, using an Xtimate C18 (3 ⁇ m packing, size 2.1 ⁇ 300 mm) chromatographic column.
  • 0-60AB_6 min method Apply a linear gradient starting with 100% A (A is 0.0675% TFA in water) and ending with 60% B (B is 0.0625% TFA in MeCN), the whole process is 4.2 minutes, then eluted with 60% B for 1 minute. The column was re-equilibrated for 0.8 minutes to 100:0 for a total run time of 6 minutes.
  • 10-80AB_6 min method apply a linear gradient starting with 90% A (A is 0.0675% TFA in water) and ending with 80% B (B is 0.0625% TFA in acetonitrile), the whole process is 4.2 minutes followed by 1 minute elution with 80% B.
  • the column was re-equilibrated for 0.8 minutes to 90:10 for a total run time of 6 minutes.
  • the column temperature was 50°C and the flow rate was 0.8 mL/min.
  • the diode array detector scans at a wavelength of 200-400 nm.
  • TLC Thin-layer chromatography
  • Preparative chromatography was performed on a Gilson-281 Prep LC 322 system with a Gilson UV/VIS-156 detector using Agella Venusil ASB Prep C18 (5 ⁇ m packing, size 150 ⁇ 21.2mm), Phenomenex Gemini C18 (5 ⁇ m packing, size 150 ⁇ 30mm), Boston Symmetrix C18 (5 ⁇ m packing, size 150 ⁇ 30mm) or Phenomenex Synergi C18 (4 ⁇ m packing, size 150 ⁇ 30mm).
  • Compounds were eluted with a low gradient of acetonitrile/water (10 mM ammonium bicarbonate in water) at a flow rate of approximately 25 mL/min for a total run time of 8-15 minutes.
  • Figure 1 shows the tumor volume growth curve of each group during the administration period.
  • Figure 2 is the relative body weight growth curve of each group of animals during the administration period.
  • Figure 3 is a tumor growth signal-time curve.
  • Figure 4 is a schematic diagram of the tumor weight at the end point of the experiment.
  • Figure 5 is an animal body weight-time curve.
  • Figure 6 is an animal body weight change-time curve.
  • Step A Compound 1-1 (5 g, 44.21 mmol) was dissolved in DMF (50 mL), potassium carbonate (18.33 g, 132.64 mmol) and compound 1-2 (9 g, 48.63 mmol) were added. The reaction solution was stirred at 50°C for 12 hours. The reaction solution was concentrated under reduced pressure, water (50 ml) was added to the residue, and the pH was adjusted to 1 with dilute hydrochloric acid (1 mol/L). After filtration, the filter cake was vacuum dried to obtain compound 1-3.
  • Step C Compound 1-5 (2.5 g, 6.37 mmol) was added to toluene (50 mL), cooled to minus 60 degrees Celsius, and diisobutylaluminum hydride (1 mol/L, 12.11 mL) was added. The reaction solution was stirred at minus 60 degrees Celsius for 2 hours. The reaction solution was quenched by adding water (1 mL) at minus 60 degrees Celsius, then adding sodium tartrate aqueous solution (4.5 g dissolved in 100 mL), the mixture was stirred for 1.5 hours, and then extracted with EtOAc (100 mL ⁇ 2), and the organic phases were combined. Washed with brine (50 mL ⁇ 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude compound 1-6 was obtained.
  • Step D Compound 1-6 (2.4 g, 1.06 mmol), triethylsilylhydrogen (2.12 g, 18.26 mmol), was added to DCM (50 mL), TFA (2.08 g, 18.26 mmol). The mixture was slowly raised to 25°C and stirred for 2 hours. The reaction solution was added with DCM (100 mL), washed with sodium bicarbonate (50 mL ⁇ 1), the combined organic phase was washed with brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • Step F Compound 1-8 (190 mg, 620.42 ⁇ mol) was dissolved in THF (8 mL), lithium hexamethyldisilazide (1 mol/L, 930.63 ⁇ L) was added at minus 60°C, and mixed The liquid was stirred at minus 60 degrees Celsius for 15 minutes under nitrogen protection. Phosphorus oxychloride (190.26 mg, 1.24 mmol) was added at minus 60 degrees Celsius, and the mixture was stirred at minus 60 degrees Celsius under nitrogen protection for 15 minutes. Additional 2-bromoethylamine hydrobromide (1.02 g, 4.96 mmol) and diisopropylethylamine (641.46 mg, 4.96 mmol) were added.
  • reaction solution was stirred at 0°C for 1 hour under nitrogen protection.
  • the reaction solution was added with water (10 mL) and extracted with EtOAc (50 mL ⁇ 3).
  • the combined organic phases were washed with brine (10 mL ⁇ 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • Step G Compound 1-9 (360 mg, 601.85 ⁇ mol) was dissolved in THF (18 mL) and silver oxide (4.18 g, 18.06 mmol) was added. The mixture was stirred at 63°C for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to p-HPLC (separating column: Welch Ultimate XB-SiOH (specification: 250 mm ⁇ 50 mm, particle size: 10 ⁇ m); mobile phase: [n-hexane-isopropanol]; elution gradient: isopropanol 20%- 60%, 15 min) to give compound 1.
  • Synthesis of compound 2 refers to the synthesis method of steps A to G of compound 1.
  • the starting material 1-1 can be replaced with 2-fluorophenol.
  • RPMI-1640 medium penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • CellTiter-Glo Cell Viability Chemiluminescence Detection Reagent
  • the NCI-H460 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo Multilabel Analyzer (PerkinElmer).
  • NCI-H460 cells (lung cancer) were seeded in 96-well plates, 80 ⁇ L of cell suspension per well, which contained 4000 NCI-H460 cells. Cell plates were incubated overnight in a carbon dioxide incubator. The compounds to be tested were diluted 5-fold to the ninth concentration, that is, from 2 mM to 5.2 nM, and a double-well experiment was set up. Add 78 ⁇ L of medium to the middle plate, and then transfer 2 ⁇ L of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 ⁇ L of each well to the cell plate. Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.026 nM.
  • the cell plate was cultured in a carbon dioxide incubator for 2 hours, then the drug-containing medium was removed, the cell plate was rinsed once with fresh medium, and 100 ⁇ L of fresh medium without drug was added to each well for 70 hours. Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • the IC 50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on NCI-H460 cell proliferation.
  • the compounds of the present invention have excellent anti-proliferative activity against NCI-H460 which highly expresses AKR1C3.
  • DMEM medium penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • CellTiter-Glo Cell Viability Chemiluminescence Detection Reagent
  • the HepG2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multilabel Analyzer (PerkinElmer).
  • HepG2 cells liver cancer
  • white 384-well plates 25 ⁇ M cell suspension per well, which contained 1000 HepG2 cells.
  • Cell plates were incubated overnight in a carbon dioxide incubator.
  • the compound to be tested was diluted 3-fold to the ninth concentration, that is, from 200 ⁇ M to 30 nM, and a double-well experiment was set up.
  • the concentration of compounds transferred to the cell plate ranged from 1 ⁇ M to 0.15 nM.
  • the cell plates were placed in a carbon dioxide incubator for 5 days.
  • Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • the IC 50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).
  • Table 2 provides the inhibitory activity of the compounds of the present invention on HepG2 cell proliferation.
  • the compounds of the present invention have excellent anti-proliferative activity against HepG2 which highly expresses AKR1C3.
  • EMEM medium penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • CellTiter-Glo Cell Viability Chemiluminescence Detection Reagent
  • the Hep3B cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multilabel Analyzer (PerkinElmer).
  • Hep3B cells liver cancer
  • a white 96-well plate 80 ⁇ L of cell suspension per well, which contained 3000 Hep3B cells.
  • Cell plates were incubated overnight in a carbon dioxide incubator.
  • the compounds to be tested were diluted 5-fold to the ninth concentration, that is, from 2 mM to 5.12 nM, and a double-well experiment was set up.
  • Add 78 ⁇ L of medium to the middle plate and then transfer 2 ⁇ L of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 ⁇ L of each well to the cell plate.
  • Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.0256 nM.
  • the cell plates were placed in a carbon dioxide incubator for 3 days.
  • Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • the IC 50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).
  • Table 3 provides the inhibitory activity of the compounds of the present invention on Hep3B cell proliferation.
  • the compound of the present invention has no anti-proliferative activity against Hep3B which expresses low AKR1C3, and shows high selectivity.
  • mice Female NU/NU nude mice (number: 90; age: 6-8 weeks), human hepatoma cells HepG2, MEM culture medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, PBS , Matrigel, etc.
  • FBS fetal bovine serum
  • trypsin trypsin
  • cyan-chain double antibody PBS , Matrigel, etc.
  • Routine cell culture was carried out in 5% CO 2 , 37°C, and MEM medium containing 10% fetal bovine serum; 0.25% trypsinization was used for passage; according to cell growth, passage 2 to 3 times a week, with a passage ratio of 1:1: 3 to 1:6.
  • Hep G2 cells in logarithmic growth phase were collected, counted and resuspended in 50% serum-free MEM medium and 50% Matrigel, and the cell concentration was adjusted to 2.5 ⁇ 10 7 cells/mL; After dispersing evenly, put it into a 50mL centrifuge tube, and place the centrifuge tube in an ice box; draw the cell suspension with a 1mL syringe and inject it subcutaneously into the axilla of the front right limb of nude mice, inoculate 200 ⁇ L per animal (5 ⁇ 106 cells/mouse) , established the Hep G2 nude mouse xenograft model.
  • the animal status and tumor growth were observed regularly, and the tumor diameter was measured using an electronic vernier caliper.
  • the data were directly input into an Excel spreadsheet to calculate the tumor volume.
  • the tumor volume reached 100-300 mm 3
  • the day of the grouping was taken as the first day of the experiment (D1).
  • the tumor diameter was measured twice a week, the tumor volume was calculated, and the body weight of the animals was weighed and recorded.
  • the animal grouping and dosing schedule are shown in Table 4. Dosing started on the day of the grouping, and the experiment was terminated after 3 weeks (or the tumor volume in the solvent control group reached more than 2000 mm3 , whichever came first), and the dosing volume was 10 mL ⁇ kg -1 .
  • Group 1 as a solvent control group, was given DMSO & 30% HP- ⁇ -CD (10:90, v:v) by intravenous injection, once a week for 3 consecutive weeks; Groups 2 and 3 were given compounds 1A and 1B by intravenous injection , and the doses were all 1 mg ⁇ kg -1 .
  • TGI 100% ⁇ [1-(TV t(T) -TV initial(T) )/(TV t(C) -TV initial(C) )] where, TV t(T) represents the tumor volume measured each time in the treatment group; TV initial(T) represents the tumor volume in the treatment group during group administration; TV t(C) represents the tumor volume measured each time in the solvent control group; TV initial( C) represents the tumor volume of the solvent control group when administered in groups.
  • BW t represents the animal body weight measured each time during the administration period
  • BW initial represents the animal body weight during group administration.
  • mice Female Balb/C nude mice, 6-8 weeks old, body weight 18-22 g, fetal bovine serum (PBS), medium EMEM (Cat. No. 30-2003), phosphate buffered saline, double antibody (Cat. No. 15240-062), Matrigel, trypsin.
  • PBS fetal bovine serum
  • EMEM fetal bovine serum
  • EMEM fetal bovine serum
  • phosphate buffered saline Cat. No. 15240-062
  • Matrigel Matrigel
  • trypsin trypsin.
  • the experimental indicator is whether tumor growth can be delayed or whether the tumor can be cured.
  • the bioluminescence signal and animal body weight of the animals were detected once a week until the end of the observation period.
  • the bioluminescence signal value can be used to calculate T/C (where T is the dosed group and C is the mean bioluminescence intensity value of the blank control group at the set time).
  • Tumor inhibition rate TGI calculation formula: TGI(%) [1-(T i -T 0 )/( V i -V 0 )] ⁇ 100, where Ti is the average intensity of bioluminescence in the treatment group at the set time; T 0 is the mean intensity of bioluminescence at the start of dosing. Vi is the average intensity of bioluminescence in the blank control group at the set time; V 0 is the average intensity of bioluminescence at the starting point of administration.
  • This experiment evaluated the efficacy of compound 1B in HepG2 liver cancer orthotopic xenograft tumor model. After 21 days of administration, Compound 1B significantly inhibited tumor growth at a dose of 1 mg/kg, with p ⁇ 0.05 compared with the vehicle control group. When the dose of compound 1B was increased to 3 mg/kg, the tumor-inhibiting effect was significantly enhanced.
  • the p value was obtained by using one-way ANOVA to analyze the relative value of tumor volume (RBL). There was a significant difference in the F value between each group (p ⁇ 0.001), and the Games-Howell test was used.
  • c.p value was obtained by one-way ANOVA and vehicle treatment group to analyze tumor weight, F value was significantly different (p ⁇ 0.001), and it was analyzed by Games-Howell method.
  • the compound of the present invention has the effect of significantly inhibiting tumor growth, and the body weight of the animals in the administration group does not decrease significantly, showing good safety.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种式(I)所示的异色满类化合物或其药学上可接受的盐，其作为醛酮还原酶(AKR1C3)抑制剂，用于治疗肝癌。

Description

异色满类化合物
本申请主张如下优先权：
CN202010991814.1，申请日2020年09月18日。
技术领域
本发明涉及结构新颖的异色满类化合物，具体涉及式(I)所示化合物或其药学上可接受的盐，以及式(I)所示化合物或其药学上可接受的盐在治疗相关领域中的应用。
背景技术
恶性肿瘤是一种严重威胁人类生命健康的重大疾病。目前的治疗手段主要有手术治疗、化学治疗、靶向治疗等。化学治疗是利用化学药物杀死肿瘤细胞、抑制肿瘤细胞生长的一种治疗方式，是一种全身性治疗手段。由于恶性肿瘤的异质性，化学疗法仍然是治疗肿瘤的重要方法。然而正是这种全身性治疗，导致化学疗法具有很大的副作用。开发具有靶向作用的化疗药物存在巨大的未满足的临床需求。
醛酮还原酶(AKR1C3)是醛酮还原酶家族成员，主要参与激素合成和毒素的清除。AKR1C3可被吸烟、酒精、乙型肝炎或丙型肝炎感染等因素诱发过度表达。AKR1C3在多种难治性癌症中过度表达，如肝癌、肺癌、胃癌、食道癌、结直肠癌、前例腺癌，急性淋巴性白血病，尤其是肝癌，其高表比例在60％以上。
目前在临床上有开发AKR1C3抑制剂药物，但没有取得很好的进展。台湾浩鼎公司报道了一种靶向AKR1C3酶化合物OBI-3424。OBI-3424是一个选择性前药，在高表达AKR1C3酶的肿瘤细胞中释放出强效DNA烷基化剂，选择性杀伤高表达AKR1C3的肿瘤细胞，使化学药物具有明显的靶向作用。
Figure PCTCN2021118597-appb-000001
目前这个靶点的研究还处在早期阶段，只有OBI-3424进入临床一期，适应症主要是肝细胞癌(HCC)和去势性前列腺癌(CRPC)，其有效性和安全性还在验证中。所以这一领域还需要更多的探索和研究。
发明内容
本发明提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2021118597-appb-000002
其中，T为N或CH；
R 1和R 2各自独立地为H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R a所取代；
各R a独立地为F、Cl、Br、I、-CN、-OH或-NH 2；
R 3和R 4各自独立地为H、F、Cl、Br、I、CN、C 1-3烷基、C 1-3烷氧基、
Figure PCTCN2021118597-appb-000003
其中所述C 1-3烷基任选被1、2或3个R e所取代；
R b和R c各自独立地为H、-CH 3、-CH 2CH 3、-(CH 2) 2CH 3或-CH(CH 3) 2；
R d为-CH 3、-CH 2CH 3、-(CH 2) 2CH 3或-CH(CH 3) 2；
各R e独立地为F、Cl、Br、I、-CN、-OH或-NH 2。
在本发明的一些方案中，上述化合物具有式(I-1)或式(I-2)所示结构：
Figure PCTCN2021118597-appb-000004
其中，R 1、R 2、R 3和R 4如本发明所定义。
在本发明的一些方案中，上述化合物具有式(I-3)或(I-4)所示结构：
Figure PCTCN2021118597-appb-000005
其中，带“*”的碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；R 1、R 2、R 3和R 4如本发明所定义。
在本发明的一些方案中，上述R 1和R 2各自独立地为H、F、Cl、Br、I或-CH 3，其中所述-CH 3任选被1、2或3个R a所取代，R a及其他变量如本发明所定义。
在本发明的一些方案中，上述R 1为H、F、Cl、Br、I或-CH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2为H，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1为H、F、Cl、Br、I或-CH 3；R 2为H，其他变量如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H、F、Cl、Br、I、CN、-CH 3、-OCH 3、
Figure PCTCN2021118597-appb-000006
Figure PCTCN2021118597-appb-000007
其中所述-CH 3任选被1、2或3个R e所取代，R e及其他变量如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H、F、-CH 3、-CHF 2、-OCH 3、
Figure PCTCN2021118597-appb-000008
Figure PCTCN2021118597-appb-000009
其他变量如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H、F、-CH 3、-CHF 2、-OCH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 3和R 4各自独立地为H。
本发明还有一些方案是由上述变量任意组合而来。
本发明还提供了下式化合物或其药学上可接受的盐，
Figure PCTCN2021118597-appb-000010
本发明还提供了下式化合物或其药学上可接受的盐，
Figure PCTCN2021118597-appb-000011
本发明还提供了上述化合物或其药学上可接受的盐在制备靶向AKR1C3酶的药物中的应用。
本发明还提供了上述化合物或其药学上可接受的盐在制备治疗肝癌的药物中的应用。
技术效果
本发明提供了一种结构新颖的靶向AKR1C3的化合物。本发明化合物对高表达AKR1C3的肿瘤细胞具有优异的抗增殖活性，对低表达AKR1C3的肿瘤细胞活性很弱，展示出优异的选择性。在肝癌皮下模型和肝癌原位模型中，本发明的化合物都展示出显著的抗肿瘤药效。
定义
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通 过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含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。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。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。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2021118597-appb-000012
和楔形虚线键
Figure PCTCN2021118597-appb-000013
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021118597-appb-000014
和直形虚线键
Figure PCTCN2021118597-appb-000015
表示立体中心的相对构型，用波浪线
Figure PCTCN2021118597-appb-000016
表示楔形实线键
Figure PCTCN2021118597-appb-000017
或楔形虚线键
Figure PCTCN2021118597-appb-000018
或用波浪线
Figure PCTCN2021118597-appb-000019
表示直形实线键
Figure PCTCN2021118597-appb-000020
和直形虚线键
Figure PCTCN2021118597-appb-000021
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其 任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2021118597-appb-000022
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2021118597-appb-000023
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2021118597-appb-000024
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2021118597-appb-000025
直形虚线键
Figure PCTCN2021118597-appb-000026
或波浪线
Figure PCTCN2021118597-appb-000027
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2021118597-appb-000028
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2021118597-appb-000029
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1-12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲核取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指 适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明采用下述缩略词：
Pd/C Pd/C催化剂，钯含量10w％
DCM 二氯甲烷
THF 四氢呋喃
EtOAc 乙酸乙酯
TBME 叔丁基甲醚
Boc 叔丁氧羰基，是一种胺保护基团
Cbz 苄氧羰基，是一种胺保护基团
DMF N,N-二甲基甲酰胺
TFA 三氟乙酸
PE 石油醚
DMSO 二甲亚砜
EtOH 乙醇
MeOH 甲醇
AcOH 乙酸
DIPEA 二异丙基乙基胺
SiO 2 100-200目硅胶粉，用于柱层析
psi 磅力/平方英寸，压力单位
p-HPLC 制备高效液相色谱，用于化合物的纯化
本发明化合物依据本领域常规命名原则或者使用
Figure PCTCN2021118597-appb-000030
软件命名，市售化合物采用供应商目录名称。
本发明所使用的溶剂可经市售获得且不需要进一步纯化。反应一般是在惰性氮气下、无水溶剂中进行的。质子核磁共振数据记录在Bruker Avance III 400(400MHz)分光仪上，化学位移以四甲基硅烷高场处的(ppm)表示。LC/MS或Shimadzu MS包含一个DAD：SPD-M20A(LC)和Shimadzu Micromass 2020检测器。质谱仪配备有一个正或负模式下操作的电喷雾离子源(ESI)。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2021118597-appb-000031
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
用配有Shimadzu SIL-20A自动进样器和日本岛津DAD：SPD-M20A探测器的岛津LC20AB系统进行高效液相色谱分析，采用Xtimate C18(3μm填料，规格为2.1×300mm)色谱柱。0-60AB_6分钟的方法：应用线性梯度，以100％A(A为0.0675％TFA的水溶液)开始洗脱，并以60％B(B为0.0625％TFA的MeCN溶液)结束洗脱，整个过程为4.2分钟，然后以60％B洗脱1分钟。将色谱柱再平衡0.8分钟达到100：0，总运行时间为6分钟。10-80AB_6分钟的方法：应用线性梯度，以90％A(A为0.0675％TFA的水溶液)开始洗脱，并以80％B(B为0.0625％TFA的乙腈溶液)结束洗脱，整个过程为4.2分钟，然后以80％B洗脱1分钟。将色谱柱再平衡0.8分钟达到90：10，总运行时间为6分钟。柱温为50℃，流速为0.8mL/min。二极管阵列检测器扫描波长为200-400nm。
在Sanpont-group的硅胶GF254上进行薄层色谱分析(TLC)，常用紫外光灯照射检出斑点，在某些情况下也采用其他方法检视斑点，在这些情况下，用碘(10g硅胶中加入约1g碘并彻底混合而成)、香草醛(溶解大约1g香草醛于100mL 10％H 2SO 4中制得)、茚三酮(从Aldrich购得)或特殊显色剂(彻底混合25g(NH 4)6Mo 7O 24·4H 2O、5g(NH 4)2Ce(IV)(NO 3)6、450mL H 2O和50mL浓H 2SO 4而制得)展开薄层板，检视化合物。采用Still，W.C.；Kahn，M.；and Mitra，M.Journal of Organic Chemistry，1978，43，2923-2925.中所公开技术的类似方法，在Silicycle的40-63μm(230-400目)硅胶上进行快速柱色谱。快速柱色谱或薄层色谱的常用溶剂是二氯甲烷/甲醇、EtOAc/甲醇和石油醚/EtOAc的混合物。
在使用Gilson UV/VIS-156检测器的Gilson-281 Prep LC 322系统上进行制备色谱分析，所采用的色谱柱为Agella Venusil ASB Prep C18(5μm填料，规格为150×21.2mm)、Phenomenex Gemini C18(5μm填料，规格为150×30mm)、Boston Symmetrix C18(5μm填料，规格为150×30mm)或Phenomenex Synergi C18(4μm填料，规格为150×30mm)。在流速约为25mL/min时，用低梯度的乙腈/水(水中含有10mM碳酸氢铵)洗脱化合物，总运行时间为8-15分钟。
附图说明
图1为给药期间各组肿瘤体积增长曲线。
图2为给药期间各组动物相对体重增长曲线。
图3为肿瘤生长信号-时间曲线。
图4为实验终点肿瘤重量示意图。
图5为动物体重-时间曲线。
图6为动物体重变化-时间曲线。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
Figure PCTCN2021118597-appb-000032
步骤A：将化合物1-1(5克，44.21毫摩尔)溶解在DMF(50毫升)中，加入碳酸钾(18.33克，132.64毫摩尔)和化合物1-2(9克，48.63毫摩尔)。反应液在50摄氏度下搅拌12小时。反应液减压浓缩，残余物加水(50毫升)，用稀盐酸(1摩尔/升)将pH调到1。过滤，滤饼真空干燥，得到化合物1-3。 1H NMR(DMSO-d 6,400MHz)δ13.88-13.37(m,1H),8.55(d,J＝2.0Hz,1H),8.21-8.16(m,2H),7.97(ddd,J＝1.6,8.1,9.9Hz,1H),7.50(dd,J＝5.0,7.8Hz,1H),7.29(d,J＝8.8Hz,1H)。
步骤B：将化合物1-3(6克,21.57毫摩尔)溶解在二氧六环(150毫升)中，加入二氯双(4-甲基异丙基苯基)钌(II)(1.32克，2.16毫摩尔)，碳酸胍(1.94克，10.78毫摩尔)，化合物1-4(3.91克,29.55毫摩尔)和AcOH(1.30克，21.57毫摩尔)。氮气保护下，反应液在105摄氏度下搅拌16小时。反应液减压浓缩。粗产品经柱层析纯化(SiO 2，PE：EtOAc＝1:0-5:1)得到化合物1-5。 1H NMR(DMSO-d 6,400MHz)δ8.64(s,1H),8.14(td,J＝1.5,4.8Hz,1H),7.92(ddd,J＝1.7,8.1,10.0Hz,1H),7.80(s,1H),7.60(s,1H),7.52-7.42(m,4H),7.39-7.32(m,2H),5.42(d,J＝1.2Hz,2H)。
步骤C：将化合物1-5(2.5克，6.37毫摩尔)加入到甲苯(50毫升)中，冷却到零下60摄氏度，加入二异丁基氢化铝(1摩尔/升，12.11毫升)。反应液在零下60摄氏度下搅拌2小时。反应液在零下60摄氏度下加入水(1毫升)淬灭，再加入酒石酸钠水溶液(4.5克溶于100毫升)，混合液搅拌1.5小时，然后用EtOAc萃取(100毫升×2)，合并有机相用盐水洗涤(50毫升×1)，无水硫酸钠干燥，减压浓缩。得到粗品化合物1-6。
步骤D：将化合物1-6(2.4克，1.06毫摩尔)，三乙基硅氢(2.12克，18.26毫摩尔)，加入到DCM (50毫升)中，在0摄氏度下加入TFA(2.08克，18.26毫摩尔)。混合液缓慢升到25摄氏度搅拌2小时。反应液加入DCM(100毫升)，用碳酸氢钠(50毫升×1)洗涤，合并的有机相用食盐水(20毫升)洗涤，有机相用无水硫酸钠干燥，过滤后减压浓缩。粗产品经柱层析纯化(SiO 2，PE/DCM/EtOAc＝20:1:1-5:1:1)得到化合物1-7。 1H NMR(DMSO-d 6,400MHz)δ8.07(s,1H),8.04(s,1H),7.99(br d,J＝4.9Hz,1H),7.57(ddd,J＝1.4,8.2,10.1Hz,1H),7.52(s,1H),7.47-7.40(m,2H),7.38-7.30(m,4H),4.84(s,2H),4.75(s,2H)。
步骤E：将化合物1-7(400毫克，1.06毫摩尔)溶解在DCM(50毫升)中，冷却到零下70摄氏度，通入臭氧约15分钟，多余的臭氧用氮气流吹走。在零下20摄氏度加入硼氢化钠(131.09毫克，3.47毫摩尔)和甲醇(5毫升)的混合液，反应液在0到20摄氏度下搅拌1小时。在20摄氏度下向反应液加入水(10毫升)淬灭，减压浓缩。粗产品经柱层析纯化(SiO 2，PE：EtOAc＝100:0-1:1)得到化合物1-8。 1H NMR(DMSO-d 6,400MHz)δ8.10(td,J＝1.5,4.8Hz,1H),7.94(s,1H),7.77(ddd,J＝1.5,8.1,10.1Hz,1H),7.44(ddd,J＝0.6,4.8,7.9Hz,1H),7.25(s,1H),5.76(d,J＝6.2Hz,1H),4.83-4.67(m,2H),4.55(q,J＝6.1Hz,1H),3.94(dd,J＝4.9,11.4Hz,1H),3.58(dd,J＝6.8,11.4Hz,1H)。
步骤F：将化合物1-8(190毫克，620.42微摩尔)溶解在THF(8毫升)中，零下60摄氏度下加入六甲基二硅基氨基锂(1摩尔/升，930.63微升)，混合液在氮气保护下，零下60摄氏度搅拌15分钟。在零下60摄氏度加入氧氯化磷(190.26毫克，1.24毫摩尔)，混合液在氮气保护下零下60摄氏度搅拌15分钟。再加入2-溴乙胺氢溴酸盐(1.02克，4.96毫摩尔)和二异丙基乙胺(641.46毫克，4.96毫摩尔)。反应液在氮气保护下于0摄氏度搅拌1小时。反应液加水(10毫升)，EtOAc(50毫升×3)萃取，合并有机相用盐水(10毫升×1)洗涤，无水硫酸钠干燥，过滤并减压浓缩。粗产品经柱层析纯化(SiO 2，PE：EtOAc＝100:0-0:1)得到化合物1-9。
步骤G：将化合物1-9(360毫克，601.85微摩尔)溶解在THF(18毫升)中，加入氧化银(4.18克，18.06毫摩尔)。混合液在63摄氏度搅拌12小时。反应液过滤，滤液减压浓缩。粗产品经p-HPLC(分离柱:Welch Ultimate XB-SiOH(规格：250mm×50mm，粒径：10μm)；流动相:[正己烷-异丙醇]；洗脱梯度：异丙醇20％-60％，15分钟)得到化合物1。 1H NMR(CDCl 3,400MHz)δ8.00(td,J＝1.6,4.8Hz,1H),7.66(s,1H),7.48(ddd,J＝1.7,7.8,9.6Hz,1H),7.20-7.16(m,2H),5.42-5.29(m,1H),4.87-4.59(m,2H),4.00(d,J＝4.4Hz,2H),2.13-1.96(m,8H)。步骤H：
Figure PCTCN2021118597-appb-000033
化合物1经手性拆分(分离柱:DAICEL CHIRALPAK AD(规格：250mm×30mm，粒径：10μm)；流动相:[中性-异丙醇]；洗脱梯度：异丙醇35％-35％)得到化合物1A(保留时间＝1.671分钟)和化合物1B(保留时间＝1.870分钟)。
化合物1A： 1H NMR(CDCl 3,400MHz)δ8.00(td,J＝1.6,4.8Hz,1H),7.66(s,1H),7.48(ddd,J＝1.7,7.9,9.6Hz,1H),7.21-7.15(m,2H),5.35(dt,J＝8.76,4.25Hz,1H),4.87-4.57(m,2H),4.00(d,J＝4.4Hz,2H), 2.14-1.96(m,8H)。ee值(对映体过量)：100％。
化合物1B： 1H NMR(CDCl 3,400MHz)δ8.00(td,J＝1.5,4.8Hz,1H),7.66(s,1H),7.48(ddd,J＝1.7,7.9,9.6Hz,1H),7.19-7.16(m,2H),5.35(dt,J＝8.63,4.32Hz,1H),4.84-4.63(m,2H),4.00(d,J＝4.4Hz,2H),2.13-1.95(m,8H)。ee值(对映体过量)：98％。
实施例2
Figure PCTCN2021118597-appb-000034
化合物2的合成参考化合物1步骤A到G的合成方法。在化合物2的合成路线中，将起始原料1-1替换成2-氟苯酚即可。
化合物2： 1H NMR(400MHz,CDCl 3)δ7.62(s,1H),7.18-7.10(m,4H),7.08(s,1H),5.31(td,J＝4.2,8.7Hz,1H),4.82-4.73(m,1H),4.68-4.58(m,1H),3.99(d,J＝4.4Hz,2H),2.08-1.92(m,8H)。
生物活性
实验例1：本发明化合物对NCI-H460细胞系的抗增殖活性
实验材料：
RPMI-1640培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。NCI-H460细胞系购自南京科佰生物科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：
将NCI-H460细胞(肺癌)种于96孔板中，80μL细胞悬液每孔，其中包含4000个NCI-H460细胞。细胞板置于二氧化碳培养箱中过夜培养。将待测化合物用排枪进行5倍稀释至第9个浓度，即从2mM稀释至5.2nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养2个小时，之后将含药物的培养基去掉，用新鲜的培养基润洗细胞板一次，每孔再加入100μL不含药物的新鲜培养基继续培养70小时。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入25μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。向细胞板中加入每孔25μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表1提供了 本发明的化合物对NCI-H460细胞增殖的抑制活性。
表1本发明化合物对NCI-H460细胞系的抗增殖活性数据
化合物编号 IC 50(nM)
化合物1 3.8
化合物1A 10.1
化合物1B 16.6
化合物2 21.6
结论：本发明化合物对高表达AKR1C3的NCI-H460具有优异的抗增殖活性。
实验例2：本发明化合物对HepG2细胞系的抗增殖活性
实验材料：
DMEM培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。HepG2细胞系购自中国科学院细胞库。Nivo多标记分析仪(PerkinElmer)。
实验方法：
将HepG2细胞(肝癌)种于白色384孔板中，25μM细胞悬液每孔，其中包含1000个HepG2细胞。细胞板置于二氧化碳培养箱中过夜培养。将待测化合物用排枪进行3倍稀释至第9个浓度，即从200μM稀释至30nM，设置双复孔实验。向中间板中加入99μM培养基，再按照对应位置，转移1μM每孔的梯度稀释化合物至中间板，混匀后转移25μM每孔到细胞板中。转移到细胞板中的化合物浓度范围是1μM至0.15nM。细胞板置于二氧化碳培养箱中培养5天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入20μM细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。向细胞板中加入每孔20μM的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表2提供了本发明的化合物对HepG2细胞增殖的抑制活性。
表2本发明化合物对HepG2细胞系的抗增殖活性数据
化合物编号 IC 50(nM)
化合物1A 9.3
化合物1B 7.4
结论：本发明化合物对高表达AKR1C3的HepG2具有优异的抗增殖活性。
实验例3：本发明化合物对Hep3B细胞系的抗增殖活性
实验材料：
EMEM培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。Hep3B细胞系购自中国科学院细胞库。Nivo多标记分析仪(PerkinElmer)。
实验方法：
将Hep3B细胞(肝癌)种于白色96孔板中，80μL细胞悬液每孔，其中包含3000个Hep3B细胞。细胞板置于二氧化碳培养箱中过夜培养。将待测化合物用排枪进行5倍稀释至第9个浓度，即从2mM稀释至5.12nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.0256nM。细胞板置于二氧化碳培养箱中培养3天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入25μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。向细胞板中加入每孔25μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表3提供了本发明的化合物对Hep3B细胞增殖的抑制活性。
表3本发明化合物对Hep3B细胞系的抗增殖活性数据
化合物编号 IC 50(nM)
化合物1A ＞10,000
化合物1B ＞10,000
结论：本发明化合物对低表达AKR1C3的Hep3B无抗增殖活性，显示出很高的选择性。
实验例4：本发明化合物对人肝癌Hep G2裸鼠移植瘤模型的体内药效学研究
实验目的：
本试验使用人肝癌Hep G2裸鼠移植瘤模型肿瘤体内生长的抑制作用。
实验材料：
雌性NU/NU裸小鼠(只数：90只；周龄：6～8周龄)，人肝癌细胞HepG2，MEM培养液，胎牛血清(FBS)，胰蛋白酶，青-链双抗，PBS，基质胶等。
实验方法和步骤：
1.细胞培养
在5％CO 2、37℃以及含10％胎牛血清MEM培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代2到3次，传代比例为1:3到1:6。
2.动物模型制备
收取对数生长期Hep G2细胞，细胞计数后重悬于含50％无血清MEM培养基和50％基质胶中，调整细胞浓度至2.5×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中；用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种200μL(5×106细胞/只)，建立Hep G2裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物48只，根据肿瘤体积采用随机区组法分为8组(n＝6)，同时尽量保证每组平均体重保持一致。以分组当天为实验第一天(D1)，实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
肿瘤体积(TV)计算公式如下：TV(mm 3)＝l×w 2/2
其中，l表示肿瘤长径(mm)；w表示肿瘤短径(mm)。
3.动物分组及给药：
动物分组及给药方案见表4，于分组当天开始给药，3周后结束实验(或溶剂对照组肿瘤体积达到2000mm 3以上，以先到指标为准)，给药体积均为10mL·kg -1。第1组作为溶剂对照组，静脉注射给予DMSO&30％HP-β-CD(10:90，v:v)，每周1次，连续3周；第2、3组均静脉注射给予化合物1A和1B，给药剂量都为1mg·kg -1。
表4.Hep G2裸鼠移植瘤模型药效实验给药方案
Figure PCTCN2021118597-appb-000035
4.实验指标：
肿瘤生长抑制率(GI)的计算公式为：TGI＝100％×[1－(TV t(T)－TV initial(T))/(TV t(C)－TV initial(C))]其中，TV t(T)表示治疗组每次测量的肿瘤体积；TV initial(T)表示分组给药时治疗组的肿瘤体积；TV t(C)表示溶剂对照组每次测量的肿瘤体积；TV initial(C)表示分组给药时溶剂对照组的肿瘤体积。相对动物体重的计算公式为：相对动物体重＝BW t/BW initial*100
其中，BW t表示给药期间每次测量的动物体重；BW initial表示分组给药时的动物体重。
5.化合物对HepG2肝癌裸鼠皮下移植肿瘤生长的抑制作用：
本实验评价了化合物1A和1B在HepG2肝癌移植瘤模型模型中的药效。给药21天时，化合物1B在1mg/kg给药剂量下均有显著的抑制肿瘤生长作用，于溶媒对照组相比均为p<0.05。肿瘤体积和抑制率见表5，肿瘤生长曲线详见图1。
表5给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2021118597-appb-000036
6.体重变化情况：
此模型中，所有治疗组动物体重均没有较大的波动，整体动物体重均值下降未超过10％，详见图2。结论：本发明化合物展示出良好的的抗肿瘤药效。
实验例5：本发明化合物对人肝癌Hep G2原位异种移植瘤模型的体内药效学研究
实验目的：
本试验使用HepG2原位异种移植肿瘤裸小鼠模型评价化合物的抗肿瘤作用。
实验材料：
雌性Balb/C裸小鼠，周龄为6-8周龄，体重为18～22克，胎牛血清(PBS)，培养基EMEM(货号30-2003)，磷酸盐缓冲液，双抗(货号15240-062)，Matrigel基质胶，胰酶。
实验方法与步骤
1.细胞培养准备：HepG2-luc细胞体外单层培养，培养条件为EMEM培养基中加10％热灭活胎牛血清，于37℃含5％CO 2培养箱中培养。一周两次用胰酶-EDTA进行消化处理传代。当细胞饱和度为80％-90％时，细胞用胰酶-EDTA消化，计数，重悬于PBS和基质胶中(PBS：基质胶＝1:1)，密度为166.67×10 6个细胞/mL。
2.肿瘤细胞接种分组：经肌肉注射60mg/kg舒泰50+1.5mg/kg甲苯噻嗪将动物麻醉，待动物至深麻时，将动物进行适当固定，用75％酒精棉球清洁腹部皮肤，用手术剪开约10mm的创口，将0.03mL(PBS：Matrigel＝1:1)的HepG2-luc细胞原位接种于每只小鼠的肝左大叶上，然后将肌肉层创口用可吸收肠线缝合，皮肤创伤口用缝合器缝合。将手术完成的动物放置在保温毯上保温至苏醒。为减少动物的疼痛，将在术后连续3天给予2mg/kg的美洛昔康(皮下注射给药，每天一次)。随机择取15只动物检测信号生长情况，当信号开始上升时，根据生物荧光信号值随机分组，开始给药治疗，详细治疗方案见表6。
表6实验动物分组及给药方案
Figure PCTCN2021118597-appb-000037
3.实验指标
实验指标是肿瘤生长能否被延迟或肿瘤能否被治愈。肿瘤接种后每周检测1次动物的生物发光信号和动物体重，持续至观察期结束。生物发光信号数值可被用来计算T/C(其中T为给药组，C为空白对照组在设定时间的生物发光平均强度值)。肿瘤抑制率TGI计算公式：TGI(％)＝[1-(T i-T 0)/(V i-V 0)]×100，其中T i为治疗组在设定时间的生物发光平均强度；T 0为给药起始点的生物发光平均强度。V i为空白对照组在设定时间的生物发光平均强度；V 0为给药起始点生物发光平均强度。
4.化合物对HepG2肝癌裸鼠皮下移植肿瘤生长的抑制作用
本实验评价了化合物1B在HepG2肝癌原位异种移植瘤模型中的药效。给药21天时，化合物1B在1mg/kg给药剂量下均有显著的抑制肿瘤生长作用，于溶媒对照组相比均为p<0.05。增加化合物1B的给药剂量至3mg/kg，抑瘤效果显著增强。
实验结果：见图3和表7、8、9。实验终点瘤重见图4和表10。
表7本发明化合物对HepG2原位异种移植瘤模型的抑瘤效果
Figure PCTCN2021118597-appb-000038
注：a.平均值±SEM，n＝6。
表8 HepG2异种移植瘤模型各组间相对肿瘤信号生长值(RBL)比较的p值
组别 组1 组2 组3 组4
1 N/A 0.038 0.029 0.024
2 0.029 0.921 N/A 0.682
3 0.024 0.384 0.682 N/A
注：p值运用one-way ANOVA进行分析肿瘤体积相对值(RBL)所得，各组间F值有显著差异(p<0.001)，运用Games-Howell发进行检验。
表9各组不同时间点的瘤组织生物发光信号值
Figure PCTCN2021118597-appb-000039
注:a.平均值±SEM，n＝6
表10各组肿瘤重量
Figure PCTCN2021118597-appb-000040
注：a.平均值±SEM，n＝6。
b.肿瘤生长抑制由T/C weight＝TW treatment/TW 溶媒计算。
c.p值运用one-way ANOVA与溶媒治疗组进行分析肿瘤重量所得，F值有显著性差异(p<0.001)，应用Games-Howell法进行分析。
5.体重变化情况
此模型中，所有治疗组动物体重均没有较大的波动，整体动物体重均值下降未超过5％，详见图5和6。结论：本发明化合物具有显著地抑制肿瘤生长的作用，且给药组动物体重没有明显降低，显示出良好的安全性。

Claims (11)

  1. 式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2021118597-appb-100001
    其中，T为N或CH；
    R 1和R 2各自独立地为H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R a所取代；各R a独立地为F、Cl、Br、I、-CN、-OH或-NH 2；
    R 3和R 4各自独立地为H、F、Cl、Br、I、CN、C 1-3烷基、C 1-3烷氧基、
    Figure PCTCN2021118597-appb-100002
    其中所述C 1-3烷基任选被1、2或3个R e所取代；
    R b和R c各自独立地为H、-CH 3、-CH 2CH 3、-(CH 2) 2CH 3或-CH(CH 3) 2；
    R d为-CH 3、-CH 2CH 3、-(CH 2) 2CH 3或-CH(CH 3) 2；
    各R e独立地为F、Cl、Br、I、-CN、-OH或-NH 2。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其中所述化合物具有式(I-1)或式(I-2)所示结构：
    Figure PCTCN2021118597-appb-100003
    其中，R 1、R 2、R 3和R 4如权利要求1所定义。
  3. 根据权利要求2所述的化合物或其药学上可接受的盐，其中所述化合物具有式(I-3)或(I-4)所示结构：
    Figure PCTCN2021118597-appb-100004
    其中，带“*”的碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；R 1、R 2、 R 3和R 4如权利要求2所定义。
  4. 根据权利要求1～3任一项所述的化合物或其药学上可接受的盐，其中R 1和R 2各自独立地为H、F、Cl、Br、I或-CH 3，其中所述-CH 3任选被1、2或3个R a所取代。
  5. 根据权利要求4所述的化合物或其药学上可接受的盐，其中R 1为H、F、Cl、Br、I或-CH 3；R 2为H。
  6. 根据权利要求1～3或5任一项所述的化合物或其药学上可接受的盐，其中R 3和R 4各自独立地为H、F、Cl、Br、I、CN、-CH 3、-OCH 3、
    Figure PCTCN2021118597-appb-100005
    其中所述-CH 3任选被1、2或3个R e所取代。
  7. 根据权利要求6所述的化合物或其药学上可接受的盐，其中R 3和R 4各自独立地为H、F、-CH 3、-CHF 2、-OCH 3、
    Figure PCTCN2021118597-appb-100006
  8. 根据权利要求7所述的化合物或其药学上可接受的盐，其中R 3和R 4各自独立地为H。
  9. 下式化合物或其药学上可接受的盐，
    Figure PCTCN2021118597-appb-100007
  10. 下式化合物或其药学上可接受的盐，
    Figure PCTCN2021118597-appb-100008
  11. 根据权利要求1～10任一项所述的化合物或其药学上可接受的盐在制备治疗肝癌的药物中的应用。
PCT/CN2021/118597 2020-09-18 2021-09-15 异色满类化合物 WO2022057838A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US18/044,791 US20230348503A1 (en) 2020-09-18 2021-09-15 Isochroman compound
EP21868660.8A EP4215523A4 (en) 2020-09-18 2021-09-15 ISOCHROMANE COMPOUND
CN202180064048.7A CN116249698A (zh) 2020-09-18 2021-09-15 异色满类化合物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010991814.1 2020-09-18
CN202010991814 2020-09-18

Publications (1)

Publication Number Publication Date
WO2022057838A1 true WO2022057838A1 (zh) 2022-03-24

Family

ID=80776441

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/118597 WO2022057838A1 (zh) 2020-09-18 2021-09-15 异色满类化合物

Country Status (4)

Country Link
US (1) US20230348503A1 (zh)
EP (1) EP4215523A4 (zh)
CN (1) CN116249698A (zh)
WO (1) WO2022057838A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023169462A1 (zh) * 2022-03-10 2023-09-14 深圳扬厉医药技术有限公司 异色满类化合物的晶型
WO2023174319A1 (zh) * 2022-03-15 2023-09-21 深圳艾欣达伟医药科技有限公司 治疗brca突变癌症患者的方法
WO2024078568A1 (zh) * 2022-10-12 2024-04-18 深圳艾欣达伟医药科技有限公司 Akr1c3激活抗癌前药化合物与镇痛药物联用治疗伴有疼痛的癌症患者

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107530556A (zh) * 2015-03-10 2018-01-02 深圳艾衡昊医药科技有限公司 Dna烷化剂
CN108290911A (zh) * 2015-11-16 2018-07-17 深圳艾衡昊医药科技有限公司 (r)-及(s)-1-(3-(3-n,n-二甲基胺基羰基)苯氧基-4-硝苯基)-1-乙基-n,n’-双(伸乙基)胺基磷酸酯、组合物及其使用及制备方法
WO2021068952A1 (zh) * 2019-10-12 2021-04-15 南京明德新药研发有限公司 靶向醛酮还原酶1c3的苯并二氢吡喃类化合物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MY198613A (en) * 2015-04-02 2023-09-11 Ascenta Pharmaceuticals Ltd Nitrobenzyl derivatives of anti-cancer agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107530556A (zh) * 2015-03-10 2018-01-02 深圳艾衡昊医药科技有限公司 Dna烷化剂
CN108290911A (zh) * 2015-11-16 2018-07-17 深圳艾衡昊医药科技有限公司 (r)-及(s)-1-(3-(3-n,n-二甲基胺基羰基)苯氧基-4-硝苯基)-1-乙基-n,n’-双(伸乙基)胺基磷酸酯、组合物及其使用及制备方法
WO2021068952A1 (zh) * 2019-10-12 2021-04-15 南京明德新药研发有限公司 靶向醛酮还原酶1c3的苯并二氢吡喃类化合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP4215523A4
STILL, W.C.KAHN, M.MITRA, M., JOURNAL OF ORGANIC CHEMISTRY, vol. 43, 1978, pages 2923 - 2925

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023169462A1 (zh) * 2022-03-10 2023-09-14 深圳扬厉医药技术有限公司 异色满类化合物的晶型
WO2023174319A1 (zh) * 2022-03-15 2023-09-21 深圳艾欣达伟医药科技有限公司 治疗brca突变癌症患者的方法
WO2024078568A1 (zh) * 2022-10-12 2024-04-18 深圳艾欣达伟医药科技有限公司 Akr1c3激活抗癌前药化合物与镇痛药物联用治疗伴有疼痛的癌症患者

Also Published As

Publication number Publication date
US20230348503A1 (en) 2023-11-02
EP4215523A4 (en) 2024-03-06
CN116249698A (zh) 2023-06-09
EP4215523A1 (en) 2023-07-26

Similar Documents

Publication Publication Date Title
WO2022057838A1 (zh) 异色满类化合物
WO2021068952A1 (zh) 靶向醛酮还原酶1c3的苯并二氢吡喃类化合物
CN115335372B (zh) 八氢吡嗪并二氮杂萘啶二酮类化生物
EP2253625A1 (en) Pyridazinones, the preparation and the use thereof
JPH09502457A (ja) 置換アザインドリリデン化合物及びその製造方法
CN113993860B (zh) 作为kras g12c突变蛋白抑制剂的七元杂环类衍生物
TWI765451B (zh) 作為erk抑制劑的螺環類化合物及其應用
TWI758999B (zh) 作為erk抑制劑的噻唑并內醯胺類化合物及其應用
JP7260718B2 (ja) ジアザインドール誘導体及びそのＣｈｋ１阻害剤としての使用
CN113825755B (zh) 作为irak4抑制剂的咪唑并吡啶类化合物
WO2018084321A1 (ja) Ｅｇｆｒ阻害及び腫瘍治療に有用な新規化合物
CN115819418B (zh) Plk1激酶抑制剂及其制备方法和应用
JP2023526490A (ja) フルオロピロロピリジン系化合物及びその使用
WO2020239107A1 (zh) 作为Cdc7抑制剂的四并环类化合物
CN114008046A (zh) 作为cdk9抑制剂的氮杂吲哚连吡唑类化合物
KR20220127900A (ko) Jak 키나아제 관련 질병의 치료를 위한 약물의 제조에 있어서 jak 억제제의 용도
WO2021218912A1 (zh) 含苯基并内磺酰胺的化合物
CN114072401B (zh) 作为irak4和btk多靶点抑制剂的噁唑类化合物
CN115551842B (zh) 联苯类化合物
JP2021527116A (ja) 選択的ａ２ａ受容体アンタゴニスト
WO2023143169A1 (zh) 含吲哚的大环类化合物及其应用
WO2022121900A1 (zh) 吡咯并吡啶类化合物及其应用
WO2022179469A1 (zh) 噻吩并嘧啶二酮类化合物及其应用
WO2022100614A1 (zh) 苯并脲环衍生物及其制备方法和应用
TW202233617A (zh) 吡啶並嘧啶酮類化合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21868660

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021868660

Country of ref document: EP

Effective date: 20230418