WO2018121610A1 - 针对Smoothened突变株的刺猬通路抑制剂 - Google Patents

针对Smoothened突变株的刺猬通路抑制剂 Download PDF

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WO2018121610A1
WO2018121610A1 PCT/CN2017/119014 CN2017119014W WO2018121610A1 WO 2018121610 A1 WO2018121610 A1 WO 2018121610A1 CN 2017119014 W CN2017119014 W CN 2017119014W WO 2018121610 A1 WO2018121610 A1 WO 2018121610A1
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group
formula
compound
pharmaceutically acceptable
solvate
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PCT/CN2017/119014
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French (fr)
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宋淳
张成城
黄牛
张承智
赵昕
张衡
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山东大学
济南成城生物技术有限公司
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Priority to CN201780080339.9A priority Critical patent/CN110099900B/zh
Publication of WO2018121610A1 publication Critical patent/WO2018121610A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/28—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the present disclosure provides a hedgehog pathway inhibitor against a Smoothened mutant strain, and the use thereof for treating various diseases caused by abnormal Hh pathway.
  • Hedgehog (Hh) pathway plays an important role in mammalian embryonic development and maintenance of mature stem cells and tissue damage repair. Abnormalities in the Hh pathway can lead to a variety of diseases, such as decreased Hh pathway activity during embryonic development, which can cause congenital defects such as no-brain deformity of the forebrain and one-eye, and overexpression of the Hh pathway is associated with the occurrence of various cancers, including Basal cell carcinoma, medulloblastoma, and the like. Therefore, Hh pathway modulators play an important role in the treatment of various diseases.
  • the main members of the Hh pathway include: three Hh proteins (including Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and Indian Hedgehog (IHh)), 12 transmembrane protein Ptch (Patched), 7 transmembrane protein Smo (Smoothened), and Zinc refers to the transcription factor Gli.
  • Hh proteins including Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and Indian Hedgehog (IHh)
  • 12 transmembrane protein Ptch Patched
  • 7 transmembrane protein Smo Smoothened
  • Zinc refers to the transcription factor Gli.
  • Each member can serve as a target for Hh pathway modulators, and each member has been reported to have a corresponding inhibitor, such as Ptch antibody 5E1, Hh inhibitor RU-SKI 43, BRD6851, GK03795, BAS 13382637, SMO inhibitor cyclopamine GDC-0449 (Vismodegib), NVP-LDE225 (Sonidegib) and Gli inhibitor GANTs, HPIs, and the like.
  • GDC-0449 Vismodegib
  • NVP-LDE225 Nonidegib
  • Gli inhibitor GANTs HPIs, and the like.
  • the present disclosure relates to a compound of Formula I, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate thereof, or a compound obtained by isotopic substitution of any atom of the compound of Formula I,
  • a and B are each independently an aromatic ring, an aromatic heterocyclic ring, an aromatic fused ring, an aromatic fused heterocyclic ring, a carbocyclic ring or a heterocyclic ring;
  • X is absent or methylene, NR 6 CO, CONR 6 , COO, OOC, SONR 6 , NR 6 SO, SO 2 NR 6 , NR 6 SO 2 , NHCONH, NHC(NH)NH, wherein R 6 is halogen , alkyl, substituted alkyl, alkoxyalkyl, carbonyl, heterocyclic, alkylaminoalkyl, aryl, substituted aryl, alkoxy, carbonyl, alkylamino, arylamino, alkylamino Formyl, alkylaminosulfonyl, sulfonyl;
  • R 1 is hydrogen, halogen, hydroxy, amino, cyano, nitro, carboxyl, aldehyde, carbamoyl, aminosulfonyl, sulfonylamino, trifluoromethyl, alkyl, alkoxy, trifluoromethoxy Base, alkylamino group, alkylsulfone group, cycloalkyl group, heterocycloalkyl group, hydroxyalkyl group, aminoalkyl group, alkoxyalkyl group, alkylaminoalkyl group;
  • R 2 , R 3 and R 4 are each independently hydrogen, halogen, hydroxy, amino, cyano, nitro, carboxyl, aldehyde, aminoacyl, aminosulfonyl, sulfonylamino, trifluoromethyl;
  • R 5 is hydrogen, halogen, hydroxy, amino, cyano, nitro, carboxyl, aldehyde, carbamoyl, aminosulfonyl, sulfonylamino, trifluoromethyl, alkyl, alkoxy, trifluoromethoxy Base, alkylamino group, alkyl sulfone group, cycloalkyl group, heterocycloalkyl group, aryl group, substituted aryl group, heteroaryl group, substituted heteroaryl group, hydroxyalkyl group, aminoalkyl group, alkoxyalkyl group, alkane Aminoalkyl group;
  • n 0-3;
  • n 0-3.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the above formula I, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate thereof, or any atom of a compound of formula I via its isotope
  • a pharmaceutically acceptable carrier or excipient comprising at least one compound of the above formula I, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate thereof, or any atom of a compound of formula I via its isotope
  • the present disclosure also relates to a compound of the formula I, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate thereof, or a compound obtained by isotopic substitution of any of the compounds of the formula I, for use in the treatment of a disease or Use in a condition or a medicament for alleviating the severity of the disease or condition, or in the preparation of a medicament as a hedgehog pathway inhibitor.
  • the disease or condition is a tumor or a cancer, such as non-small cell lung cancer, basal cell carcinoma (BCC), medulloblastoma, melanoma, medulloblastic carcinoma, pancreatic cancer, prostate cancer, colon cancer, Breast, colon or stomach cancer.
  • the present disclosure also relates to a method of treating or lessening the severity of a disease or condition, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula I, a pharmaceutically acceptable salt thereof, and a difference thereof A compound obtained by subjecting a compound, a solvate, or any of the compounds of the formula I to an isotope substitution thereof, wherein the diseased tumor or cancer, such as non-small cell lung cancer, basal cell carcinoma (BCC), medulloblastoma, melanin Tumor, medulloblastic cell carcinoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, colon cancer or gastric cancer.
  • the diseased tumor or cancer such as non-small cell lung cancer, basal cell carcinoma (BCC), medulloblastoma, melanin Tumor, medulloblastic cell carcinoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, colon cancer or gastric cancer.
  • the present disclosure also relates to at least one compound of the above formula I, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate thereof, or a compound obtained by isotopic substitution of any atom of the compound of formula I, said compound, a pharmaceutically acceptable salt thereof, an isomer thereof, a solvate, or a compound obtained by isotopic substitution of any of the compounds of the formula I, for use in treating a disease or condition or reducing the severity of the disease or condition, or Inhibition of the hedgehog pathway.
  • the diseased tumor or cancer such as non-small cell lung cancer, basal cell carcinoma (BCC), medulloblastoma, melanoma, medulloblastic cell carcinoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, Colon or stomach cancer.
  • BCC basal cell carcinoma
  • medulloblastoma medulloblastoma
  • melanoma medulloblastic cell carcinoma
  • pancreatic cancer prostate cancer
  • colon cancer colon cancer
  • breast cancer Colon or stomach cancer.
  • formula I R 6 is selected from alkyl, substituted alkyl, alkoxyalkyl, alkylcarbonyl, heterocycloalkyl, dialkylaminoalkyl, aryl, substituted aryl, alkylamino Formyl, alkylaminosulfonyl, sulfonyl;
  • a in Formula I is selected from the group consisting of, but not limited to, the group:
  • B of Formula I is selected from the group consisting of, but not limited to, the group:
  • the compound of Formula I has the structure of Formula Ia:
  • R 1 is selected from alkyl, carbocyclic or heterocyclic rings, each of which is optionally hydroxy, halo, cyano, amino, carbonyl, acyl, alkyl, haloalkyl, trifluoromethyl, nitro, alkoxy, Carboxyl, aldehyde, methylamino, carboxamide, alkylsulfonyl, alkylsulfinyl, alkylcarbamoyl, alkanoylamino, alkylsulfamoyl, alkylsulfonamide, alkylsulfinyl, Alkoxy, alkylcarbamoyl, alkanoylamino, alkylaminoacyl, alkylsulfonyl, carbocyclic or heterocyclic substituent, optionally amino, halo, hydroxy, carbonyl, haloalkyl, alkyl, alkane Substituted by an oxy or acyl substituted
  • the compound of Formula I has the structure of Formula Ib:
  • R 2 , R 3 , R 4 are each taken from halogen, hydroxy, alkyl, acyl or alkoxy, each of which is optionally hydroxy, halo, amino, nitro, alkyl, acyl, alkylsulfonyl or alkane Oxygen substitution;
  • X is taken from
  • the compound of Formula I has the structure shown in Formula Ic:
  • R 5 in each case is independently hydroxy, halo, amino, carboxy, decyl, decyl, carbonyl, nitro, cyano, acyl, alkyl, haloalkyl, sulfonyl, sulfinyl, alkoxy
  • R 6 is, in each case, independently selected from substituted alkyl groups (eg methyl, trifluoromethyl, dimethylaminomethyl, piperidinylmethyl, morpholinomethyl); halogen (eg chlorine); Alkoxy (such as methoxy); carbonyl (such as morpholinocarbonyl, acetyl); heterocycle (such as morpholine, N-methylpiperazin-4-yl, N-acetylpiperazin-4-yl) , 1H-1,2,4-triazole); alkylamino (such as isobutylamino, benzylamino, hydroxyethylamino, methoxyethylamino, morpholinoethylamino, morpholinopropyl Amino, imidazoliumethylamino); arylamino (such as phenylamino); alkylcarbamoyl (such as dimethylcarbamoyl, isobutylaminocarbon
  • n 0-3.
  • the compound of Formula I has the structure shown in Formula Id:
  • R 10 is selected from the group consisting of hydrogen, halogen, -C(O)R 14 ;
  • R 11 is selected from hydrogen, -C(O)OCH 3 ;
  • R 12 is selected from hydrogen or hydrazine, halogen; and
  • R 13 is selected from hydrogen, halogen, methyl;
  • R 14 is selected from:
  • the compound of Formula I has the structure shown in Formula Ie:
  • R 12 is selected from hydrogen or hydrazine, halogen; and R 13 is selected from the group consisting of hydrogen, halogen, and methyl.
  • the compound of Formula I has the structure shown in Formula If:
  • R 12 is selected from hydrogen or hydrazine, halogen; and R 13 is selected from hydrogen and halogen.
  • the compound of Formula I has the structure shown in Formula Ig:
  • R 14 is selected from:
  • the compound of Formula I has the structure shown in Formula Ig:
  • R 12 is hydrogen or deuterium
  • R 13 is hydrogen or chlorine
  • R 16 is R 15 is a methyl group, an amino group
  • the compound of Formula I has the structure shown in Formula Ih:
  • R 12 is hydrogen or deuterium
  • R 13 is hydrogen or halogen chloride
  • R 16 is R 15 is a methyl group, an amino group
  • halogen means fluoro, chloro, bromo or iodo.
  • Preferred halogen groups are fluorine, chlorine or bromine.
  • a more preferred halogen group is chlorine.
  • the pharmaceutically acceptable salts include acid addition salts and base addition salts.
  • a pharmaceutically acceptable acid addition salt refers to a salt which retains the biological effectiveness and properties of the free base and which is not biologically or otherwise undesirable, formed with inorganic acids and organic acids, such as hydrochloric acid, hydrobromine Acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, etc.
  • the organic acid may be selected from the group consisting of a fatty acid, a cyclic fatty acid, an aromatic acid, an aromatic fatty acid, a heterocyclic acid, a carboxylic acid, and a sulfonic acid, such as formic acid, acetic acid, propionic acid, glycolic acid.
  • gluconic acid lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, Benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid.
  • Pharmaceutically acceptable base addition salts include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
  • Salts derived from pharmaceutically acceptable inorganic organic bases include primary, secondary and tertiary ammonium, substituted ammonium including naturally substituted ammonium, refilled and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine , triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, beet a salt of a base, ethylenediamine, glucosamine, theobromine, hydrazine, piperazine, piperidine, N-, and piperidine, a polyamine resin or the like.
  • Particularly preferred non-toxic organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • the compound of Formula I described in the present disclosure is selected from the group consisting of
  • a pharmaceutical composition comprises a compound of formula I and a conventional pharmaceutical carrier or excipient.
  • the pharmaceutical composition can be administered by, for example, oral or parenteral routes.
  • the pharmaceutical compositions of the present disclosure can be prepared into various dosage forms including, but not limited to, tablets, capsules, solutions, suspensions, granules or injections, etc., by conventional methods in the art, for example, by oral or parenteral administration.
  • the dosage and method of use of the compounds described in the present disclosure depend on a number of factors, including the age, weight, sex, natural health, nutritional status of the patient, the strength of the activity of the compound, the time of administration, the rate of metabolism, and the severity of the condition. The degree and subjective judgment of the doctor.
  • a preferred use dose is between 0.01 and 100 mg/kg body weight per day.
  • the compound of formula E is prepared by substituting Suzuki for the substitution of pyridine A and boronate B, and then reducing the nitro group and reacting with the corresponding benzoyl chloride to obtain the product.
  • the compound of formula J is also first coupled via Suzuki, and after hydrolysis of the ester group, it is reacted with the corresponding aniline to give the target compound.
  • the compound of formula N is first subjected to a condensation reaction to obtain intermediate M, which is then condensed with the corresponding amine to give the desired product.
  • 6-bromonicotinic acid methyl ester D (1.00 g, 4.63 mmol), 2-nitrophenylboronic acid E (1.16 g, 6.94 mmol), tetratriphenylphosphine palladium (0.16 g, 0.14 mmol) and sodium carbonate (0.74) g, 6.94 mmol) was dissolved in methanol / dioxane (1:3) solution (40 mL) and warmed to 80 ° C under argon atmosphere overnight. The reaction mixture was cooled and concentrated under reduced pressure. EtOAc (EtOAc)EtOAc. A pale yellow solid F (0.98 g, 81.99%).
  • the synthesized compound also has:
  • Test compounds were dissolved in DMSO and diluted to different gradients for use.
  • TM3Hh12 cells pTA-8xGli-Luc containing Hh-responsive reporter gene construct
  • F12Ham's/DMEM medium containing 5% horse serum, 2.5% fetal bovine serum and 15 mM HEPES buffer solution (Ph 7.3) with trypsin
  • F12Ham's/DMEM (1:1) medium containing 5% horse serum and 15 mM HEPES pH 7.3
  • the cells were added to the assay plate containing the compound and incubated at 37 ° C for 30 minutes in a 5% CO 2 atmosphere.
  • C3H10T1/2 cells were transfected with wild-type or D473H mutant SMO and Gli fluorescently labeled plasmids.
  • the transfection process of DNA and GeneJuice was as follows: 3 ⁇ L of Opti-MEM and 0.15 ⁇ L of GeneJuice transfection reagent were added to an empty 96-well plate and incubated for 5 minutes at room temperature. Add 20 ng of 8x-Gli1 fluorescent label, 1 ng of p-RL-TK and 10 ng of pcDNA-Smo-WT, pcDNA-Smo-D473H or empty pcDNA3.1 control, carefully mix, incubate for 15 minutes at room temperature and add to the cells. .
  • Smo-containing cell membranes were prepared using CHO-K1 cells transfected with mouse or human Smo cDNA, and the test compounds were formulated into a series of gradients and placed in an empty assay plate for later use.
  • the cell membrane containing Smo was added to the assay plate, and 1.5 nM of [3H]Hh-Ag 1.5 was prepared by using a buffer solution (50 mM Tris-HCl, 10 mM EDTA, and 5 mM magnesium chloride, pH 7.2), and the assay plate was placed at 37. Incubate for 48 hours at °C.
  • the 96-well Unifilter GF/B filter plate was pre-incubated with 0.5% polyethyleneimine and 1% fetal bovine serum for 60 minutes and washed three times with 2% HPCD distilled water.
  • the culture solution in the assay plate was transferred to the filter plate using a Unifilter-96 pipette.
  • the loaded filter plate was washed three times with 2% HPCD buffer, and the unbound [3H]Hh-Ag1.5 was washed away. Dry at 60 ° C for 30 minutes and cool.
  • the bottom end was sealed, 50 ⁇ L of Microscint-O was added to each well, the tip was sealed, and incubated for 100 minutes-overnight.
  • the binding amount of [3H]Hh-Ag1.5 was obtained using a TopCount (Perkin-Elmer) scintillation counter. The number of saturated bindings was analyzed using Graphpad Prizm software.
  • the cell membrane was diluted to 0.01-0.02 mg/ml with a buffer solution containing 50 mM HEPES and 3 mM MgCl2, pH 7.2. 200 ⁇ l of the cell membrane suspension was added to 100 ⁇ l of a buffer solution containing fluorescent ligands, various concentrations of inhibitors and protease inhibitors, and 0.02% fetal calf serum was added to reduce non-specific binding. Binding experiments were performed on 96-well plates. The 96-well plates were incubated for 5 hours at 23 ° C to bring the inhibitors to equilibrium, and the free labeled ligands were quickly vacuum filtered off with an infiltrated glass fiber filter (0.3% polyethyleneimine).
  • the filtrate was washed twice with 0.3 ml of cold phosphate buffered saline containing 0.01% Triton X-100.
  • the fluorescence activity of the filtrate was quantified using a TopCount liquid scintillation counter.
  • 2X106 HEK-293 cells were seeded into 10 cm plates, and cells were transfected with 3 ⁇ g of SMO-expressed GeneJuice plasmid. After 40 hours, the cells were transferred to a 1 mmol/L EDTA solution, treated with 4% paraformaldehyde in PBS for 10 minutes and washed three times with PBSA. One 96-well plate was added, and 2 ⁇ 105 cells were added to each well. 50 mM unlabeled GDC-0449 or blank control was added, and then 5 nM of [3H]-GDC-0449 was added, and incubated at 37 ° C for one hour in PBS. The cells were transferred to a filter tray, washed 6 times and dried. Radioactivity was measured using a Microscint-20 scintillation fluid and a Topcount reader. The results are presented in raw data or removed from the background and presented as SMO-WT.
  • Fluorescence binding test with BODIPY FL or 558/568 labeled cyclopamine was carried out as follows: CHO cells expressing human or mouse SMO were added to a 384-well plate, treated with 4% paraformaldehyde for 15 minutes at room temperature, and added with 0.5% after washing. Fetal bovine serum in PBS buffer, then fluorescently labeled BODIPY-cyclopamine (20 nM) and test compound were added and incubated for 4 hours at 37 °C. The cells were washed with PBS and stained with Hoechst 33258. Ultra imager analysis.
  • Endogenous mouse Gli and Ptch1 or human Gli and Ptch1 gene expression was expressed by rat NIH 3T3 or human HEPM cells.
  • NIH 3T3 and HEPM cells were cultured in 6-well plates cultured in EMEM medium containing 0.5% FBS, and cultured with recombinant ShhN (200 ng/ml), Smo inhibitor or DMSO control. All RNA was obtained with TRIzol reagent, followed by extraction with chloroform and purification using a RNeasy mini-column column. Reverse transcription was performed using a high efficiency cDNA reverse transcriptase kit. Quantified by TaqMan gene expression analysis, the internal standard was VIC/MGB Probe. The TaqMan Gene Expression Master Mix kit was used for RT-PCR and the workstation was ABI Prism 7900HT Fast RT-PCR.
  • TM3-SV40 cells were obtained by transfecting TM3 cells with 1 ⁇ g of the selective pGL3-SV40-Luc plasmid in the presence of neomycin, and operating with Fugene reagent according to the instructions. Cells were screened selectively with G418 for two weeks and the remaining cells were collected for later use.
  • 293T-STF-Luc cells were obtained by transfecting 293T cells with the SuperTopflash-Luc vector responsive to Wnt in the presence of neomycin. Screening was selectively performed with G418 for two weeks and the remaining cells were collected for later use.
  • a 384-well plate containing 30 ⁇ l of DMEM + 5% FBS per well was added, and 10,000 cells were added per well. After 12 hours, the diluted inhibitor solution was added. 20 ⁇ l of Wnt medium was added and incubated for 48 hours, and the fluorescence activity was measured using a BrightGlo fluorescent kit.
  • 293T-NFexcellentB-Luc cells were obtained by transfecting 293T cells with a Luc vector responsive to NF MéB in the presence of neomycin. Screening was selectively performed with G418 for two weeks and the remaining cells were collected for later use.
  • a 384-well plate containing 40 ⁇ l of DMEM + 5% FBS per well was added to each well and 10,000 cells were added per well. After 12 hours, the diluted inhibitor solution was added, and after 30 minutes, 10 ⁇ l of the culture medium containing recombinant TNF ⁇ was added. Fluorescence activity was measured using a BrightGlo Fluorescence Kit after 24 hours of incubation.
  • Ptch +/- p53-/- transgenic mice were divided into multiple fragments and passaged in nude mice.
  • Ptch+/-p53-/-MB cells were isolated from transplanted mice and used in a non-adhesive 10 cm Petri dish with Neurobasal medium containing 2% B-27, 1% N2 additive and 1% penicillin/streptomycin. Culture, 1 x 107 cells per well. Three days after cell culture, the wild type or D477G mutant lentiviral supernatant was infected. The Smo expression level was tested by RT-PCR, and the proliferation assay was performed using the Click-iT EdU Cell Proliferation Assay Kit according to the instructions.
  • CGNP cerebellar granule neuron progenitor
  • CGNP was isolated from 4 day old C57BL/6. Cultured in a 384-well plate supplemented with poly-D-lysine with Neurobasal medium containing 2% B-27 additive, 1 mM sodium pyruvate, 2 mM L-glutamine and 1% penicillin/streptomycin. The cell concentration was 105 and then recombinant ShhN (200 ng/ml) and inhibitor were added.
  • the Click-iT EdU Cell Proliferation Assay Kit performs cell proliferation experiments as described above.
  • HEPM cells are derived from human embryonic mesenchymal tissue and obtained from ATCC. HEPM cells were cultured in a minimum basic medium (MEM) + Earle's Salts + L-glutamine + 10% fetal bovine serum, 1 x sodium pyruvate and 1 x MEM non-essential amino acids at 35 ° C with 5% CO 2 . The cells were dispensed twice a week with 0.05% trypsin EDTA solution and then placed in a 75 cm Erlenmeyer flask. Cells were added to 96-well plates in an amount of 50,000 per well for Gli1 RT-PCR analysis. Next, 50 nM of agonist and different concentrations of inhibitor were added. At 48 hours, cells were collected for RT-PCR.
  • MEM minimum basic medium
  • the medium was removed, 50 ⁇ l of different concentrations of the inhibitor was added, followed by 50 ⁇ l of the agonist, and incubated for 48 hours.
  • 50 ⁇ l of the agonist Hh Ag1.5 (1:1000) diluted with the medium was added, and the concentration of the agonist per well was 50 nM.
  • the inhibitor was obtained by diluting 10 mM DMSO solution 1:3 and then diluting 1000 times with the culture solution.
  • Quantitative expression of mRNA was performed by RT-PCR using Taqman reagent on an Applied Biosystems 7900HT Fast RT-PCR or 7900HT Sequence Detection System.
  • the instrument setting procedure is as follows: cancel ROX, the first stage is 50 ° C for 2 min, the second stage is 95 ° C for 10 min, the third stage is 95 ° C for 15 sec, and the last 60 ° C for 1 min is cycled 40 times.
  • Five solutions are required during the reaction: 2x Master mix, primers for Gli and betaactin, and the desired cDNA and water for the reaction. Note that the 2xMaster mix needs to contain the following ingredients: Amplitaq Gold DNA Polymerase, Amperase UNG, dNTP's, with UTP, negative control ROX.
  • Real-time PCR data removes betaactin effects.
  • IC50 values were calculated using XLfit4using Fit Designer Dose Response Model 202.
  • 1.0 ⁇ 106 or 5.0 ⁇ 106 Ptch+/-Hic-/- mouse MB cells obtained from tumor samples were subcutaneously inoculated into the right abdomen of nude mice or nude rats.
  • the drug was started 7 days after transplantation, and was randomly grouped according to the tumor size before administration.
  • the average tumor size of each group was 250 mm3.
  • Tumor volume and body weight of each group of mice were recorded 2-3 times per week for later analysis.
  • the dose of the drug was changed with the body weight at any time, and the control between the different treatment groups was calculated by the ANOVA (Tukey) rank sum test method.
  • E3LZ10.7 cells were injected subcutaneously into male CD1 nude mice, and tumors were removed after 2-4 weeks and cut into small pieces of about 1 mm3. 6-8 weeks old male CD1 nude mice were anesthetized with anesthetic gas, and opened under the rib of 1 cm on the left side of the abdomen until the spleen and pancreatic appendages were visible, and a small opening was placed in the pancreas to implant the prepared tumor and suture.
  • L3.6pl group 106 cells were suspended in 50 ⁇ L of PBS/Matrigel (1:1) mixture and directly injected into the mouse pancreas. After 21 days, the volume of the tumor was measured with ultrasound. The tumor size was divided into four groups: control, cyclopamine (25 mg/kg feed supplemented, once every two days), gemcitabine (100 mg/kg, once every four days) and combination group, continuous administration 30 days.
  • the L3.6pl group only needed to give cyclopamine for 15 days, 50 mg/kg/d.
  • mice After 30 days, the mice were sacrificed and the tumor spread of the kidney, liver, spleen, small intestine, peritoneum and lung was examined and preserved with formalin. Tumor samples were rapidly frozen for RNA analysis. In addition to direct observation, each mouse should be randomly selected for kidney, liver, spleen, small intestine, peritoneum and lung tissue for microscopic examination to observe the presence or absence of micrometastases. Obtain local lymph node samples, make sections and check for lesions. The L3.6pl group only needs to observe the lesions of the liver and peritoneum.
  • the compound was dissolved in a solution of 5% NMP + 10% PEG400 + 35% of 10% ETPGS + 50% D5W, and male C57BL mice (2 per time point) were injected intravenously (1 mg/kg, 0.2 mg). /mL) or gavage (10mg/kg, 1mg/ml). Blood samples were taken at a fixed time after 24 hours of dosing (2 per time point). Plasma was separated by centrifugation and the concentration of the compound in the plasma was measured by HPLC/MS/MS. Related plasma pharmacokinetic constants such as clearance, volume of distribution and half-life are calculated as non-compartmental models.
  • Sprague-Dawley rat tail vein injection (1mg/kg, 1mg/mL compound solution prepared by dissolving 1.5eq HCl, 20% captisol in pH 8 buffer solution, 2 times each time) or gavage (10mg/kg, 0.5)
  • the sodium carboxymethylcellulose suspension was administered in three doses each time. After 24 hours, blood samples were taken at fixed times and centrifuged to obtain plasma. The concentration of plasma compounds was determined by HPLC/MS/MS and the relevant plasma pharmacokinetic constants such as clearance, volume and half-life were calculated as non-compartmental models.
  • the compound was dissolved in 20% captisol and formulated into a 0.4 mg/ml solution, and the fasted male Beagle dogs (3) were given a single dose (0.1 mg/kg) or orally (0.3 mg/kg). After 24 hours, blood samples were taken at fixed times and centrifuged to obtain plasma. The concentration of plasma compounds was determined by HPLC/MS/MS and the relevant plasma pharmacokinetic constants such as clearance, volume and half-life were calculated as non-compartmental models.
  • the A549 cell suspension in the logarithmic growth phase was diluted to 4 ⁇ 10 4 - 5 ⁇ 10 4 / mL, and seeded in a 96-well plate (100 ⁇ L / well). After incubating for 8 h at 37 ° C, 5% CO 2 , 100 ⁇ L of different concentrations of compound dilution (diluted in medium) were added to each well, and three replicate wells were set for each concentration. After incubation for 48 h, 10 ⁇ L of 0.5% MTT staining solution was added to each well, and incubation was continued for 4 h.

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Abstract

提供了通式I 所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I 化合物中的任意原子经其同位素替换后得到的化合物，以及该化合物的用途。

Description

针对Smoothened突变株的刺猬通路抑制剂 技术领域
本公开提供了一种针对Smoothened突变株的刺猬通路抑制剂，以及其用于治疗Hh通路异常引起的各种疾病的用途。
背景技术
刺猬(Hedgehog，Hh)通路在哺乳动物胚胎发育及成熟个体干细胞的维持及组织损伤修复的过程中起着重要的作用。Hh通路的异常会导致多种疾病的发生，如胚胎发育过程中Hh通路活性降低会引起前脑无裂畸形、独眼等先天性缺陷，而Hh通路的过度表达与多种癌症的发生有关，包括基底细胞癌、髓母细胞瘤等。因此，Hh通路调节剂在各种疾病的治疗过程中起着重要的作用。
Hh通路主要成员包括：三个Hh蛋白(包括Sonic Hedgehog(SHh)，Desert Hedgehog(DHh)和Indian Hedgehog(IHh))，12跨膜蛋白Ptch(Patched)，7跨膜蛋白Smo(Smoothened)，和锌指转录因子Gli。每个成员都可以作为Hh通路调节剂的靶点，据报道每个成员都有相应的抑制剂，如Ptch抗体5E1，Hh抑制剂RU-SKI 43、BRD6851、GK03795、BAS 13382637，SMO抑制剂cyclopamine、GDC-0449(Vismodegib)、NVP-LDE225(Sonidegib)和Gli抑制剂GANTs、HPIs等。随着GDC-0449(Vismodegib)和NVP-LDE225(Sonidegib)的成功上市，证明了Hh通路抑制剂作为抗肿瘤抑制剂的可行性，Hh通路抑制剂日益受到人们的重视。
发明内容
本公开涉及通式I所示化合物、其药学上可接受的盐、其异构体、溶剂化物，或式I化合物中的任意原子经其同位素替换后得到的化合物，
Figure PCTCN2017119014-appb-000001
其中A、B各自独立地为芳香环、芳香杂环、芳香稠合环、芳香稠合杂环、碳环或杂环；
X不存在或是亚甲基、NR 6CO、CONR 6、COO、OOC、SONR 6、NR 6SO、SO 2NR 6、NR 6SO 2、NHCONH、NHC(NH)NH，其中R 6为卤素、烷基、取代的烷基、烷氧基烷基、羰基、杂环、烷基氨基烷基、芳基、取代芳基、烷氧基、羰基、烷基氨基、芳基氨基、烷基氨基甲酰基、烷基氨基磺酰基、磺酰基；
R 1为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基甲酰基、氨基磺酰基，磺酰胺基、三氟甲基、烷基、烷氧基、三氟甲氧基、烷氨基、烷基砜基、环烷基、杂环烷基、羟基烷基、氨基烷基、烷氧基烷基、烷氨基烷基；
R 2、R 3、R 4各自独立的为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基酰基，氨基磺酰基、磺酰胺基、三氟甲基；
R 5为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基甲酰基、氨基磺酰基，磺酰胺基、三氟甲基、烷基、烷氧基、三氟甲氧基、烷氨基、烷基砜基、环烷基、杂环烷基、芳基、取代芳基，杂芳基、取代杂芳基、羟基烷基、氨基烷基、烷氧基烷基、烷氨基烷基；
m是0-3；
n是0-3。
本公开还涉及药物组合物，所述药物组合物包含至少一种上述通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，以及药学上可接受的载体或赋形剂。
本公开还涉及所述通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物在制备用于治疗疾病或病症或减轻所述疾病或病症严重性的药物中的用途，或者在制备作为刺猬通路抑制剂的药物中的用途。优选地，其中所述疾病或病症为肿瘤或癌症，例如非小细胞肺癌、基底细胞癌(BCC)、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
本公开还涉及治疗疾病或病症或减轻所述疾病或病症严重性的方法，所述方法包括给予需要这种治疗的患者治疗有效量的通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其中所述疾病肿瘤或癌症，例如非小细胞肺癌、基底细胞癌(BCC)、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
本公开还涉及至少一种上述通式I化合物、其药学上可接受的盐、其异构体、溶 剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，所述化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物用于治疗疾病或病症或减轻所述疾病或病症严重性，或者用于抑制刺猬通路。优选地，其中所述疾病肿瘤或癌症，例如非小细胞肺癌、基底细胞癌(BCC)、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
在某些实施方案中，通式I中R 6选自烷基、取代的烷基、烷氧基烷基、羰基、杂环、烷基氨基烷基、芳基、取代芳基、烷基氨基甲酰基、烷基氨基磺酰基、磺酰基；
在某些实施方案中，通式I中A任选自下述基团但不限于下述基团：
Figure PCTCN2017119014-appb-000002
在某些实施方案中，通式I中B任选自下述基团但不限于下述基团：
Figure PCTCN2017119014-appb-000003
在某些实施方案中，通式I化合物具有如式Ia所示结构：
Figure PCTCN2017119014-appb-000004
其中R 1选自烷基、碳环或杂环，其各自任选被羟基、卤素、氰基、氨基、羰基、酰基、烷基、卤代烷基、三氟甲基、硝基、烷氧基、羧基、醛基、甲氨基、甲酰胺基、烷基磺酰基、烷基亚磺酰基、烷基氨基甲酰基、烷酰基氨基、烷基氨磺酰基、烷基磺 酰胺、烷基亚磺酰基、烷氧基、烷基氨基甲酰基、烷酰基氨基、烷基氨基酰基、烷基磺酰基、碳环或杂环取代基、任选被氨基、卤素、羟基、羰基、卤代烷基、烷基、烷氧基或者酰基取代的碳环或杂环取代；A选自：
Figure PCTCN2017119014-appb-000005
在某些实施方案中，通式I化合物具有如式Ib所示结构：
Figure PCTCN2017119014-appb-000006
其中R 2、R 3、R 4分别取自卤素、羟基、烷基、酰基或烷氧基，其各自任选被羟基、卤素、氨基、硝基、烷基、酰基、烷基磺酰基或烷氧基取代；X取自
Figure PCTCN2017119014-appb-000007
在某些实施方案中，通式I化合物具有如式Ic所示结构：
Figure PCTCN2017119014-appb-000008
其中，B为
Figure PCTCN2017119014-appb-000009
R 5在每种情况下独立的为羟基、卤素、氨基、羧基、脒基、胍基、羰基、硝基、氰基、酰基、烷基、卤代烷基、磺酰基、亚磺酰基、烷氧基、烷硫基、氨基甲酰基、 酰胺基、氨磺酰基、磺酰胺、碳环或杂环；其中所述氨基、脒基、烷基、酰基、磺酰基、亚磺酰基、烷氧基、烷硫基、氨基甲酰基、酰胺基、氨磺酰基、磺酰胺、碳环或杂环取代基任选被羟基、羧基、氨基、羰基、卤素、卤代烷、烷基、烷氧基、酰基、磺酰基、亚磺酰基取代，
R 6在每种情况下独立的选自取代的烷基(如甲基、三氟甲基、二甲氨基甲基、哌啶基甲基、吗啉代甲基)；卤素(如氯)；烷氧基(如甲氧基)；羰基(如吗啉代羰基、乙酰基)；杂环(如吗啉、N-甲基哌嗪-4-基、N-乙酰基哌嗪-4-基、1H-1,2,4-三唑)；烷基氨基(如异丁基氨基，苄基氨基、羟乙基氨基、甲氧乙基氨基、吗啉代乙基氨基、吗啉代丙基氨基、咪唑乙基氨基)；芳基氨基(如苯基氨基)；烷基氨基甲酰基(如二甲基氨基甲酰基、异丁基氨基羰基)；烷基氨基磺酰基(如丙基氨基磺酰基、异丁基氨基磺酰基、二甲氨基磺酰基、二甲氨基乙酰基、羟乙基氨基磺酰基、甲氧乙基磺酰基)；磺酰基(如甲基磺酰基、以及磺酰基、氨基磺酰基、二甲氨基丙基磺酰基、N-甲基哌嗪-4-基磺酰基、吗啉代-4-基磺酰基、三氟甲基磺酰基)，
n是0-3。
在某些实施方案中，通式I化合物具有如式Id所示结构：
Figure PCTCN2017119014-appb-000010
其中：R 10选自氢、卤素、-C(O)R 14；R 11选自氢、-C(O)OCH 3；R 12选自氢或氘、卤素；R 13选自氢、卤素、甲基；
R 14选自：
Figure PCTCN2017119014-appb-000011
Figure PCTCN2017119014-appb-000012
在某些实施方案中，通式I化合物具有如式Ie所示结构：
Figure PCTCN2017119014-appb-000013
其中：R 12选自氢或氘、卤素；R 13选自氢、卤素、甲基。
在某些实施方案中，通式I化合物具有如式If所示结构：
Figure PCTCN2017119014-appb-000014
其中：R 12选自氢或氘、卤素；R 13选自氢、卤素。
在某些实施方案中，通式I化合物具有如式Ig所示结构：
Figure PCTCN2017119014-appb-000015
其中R 14选自：
Figure PCTCN2017119014-appb-000016
Figure PCTCN2017119014-appb-000017
在某些实施方案中，通式I化合物具有如式Ig所示结构：
Figure PCTCN2017119014-appb-000018
其中：R 12为氢或氘；R 13为氢或氯；R 16为
Figure PCTCN2017119014-appb-000019
R 15为甲基、氨基、
Figure PCTCN2017119014-appb-000020
在某些实施方案中，通式I化合物具有如式Ih所示结构：
Figure PCTCN2017119014-appb-000021
其中：R 12为氢或氘；R 13为氢或卤素氯；R 16为
Figure PCTCN2017119014-appb-000022
R 15为甲基、氨基、
Figure PCTCN2017119014-appb-000023
本公开中，所用的术语“卤素”意指氟，氯，溴或碘。优选的卤素基团为氟、氯或溴。更优选的卤素基团为氯。
本公开中使用的化合物名称与化学结构式不一致时，以化学结构式为准。
本公开中，所述药学上可接受的盐包括酸加成盐和碱加成盐。药学上可接受的酸加成盐是指保留游离碱的生物有效性和性质并且不是生物学或其它方面不期望的、与无机酸和有机酸形成的盐，所述无机酸例如盐酸、氢溴酸、硫酸、硝酸、碳酸、磷酸等，所述的有机酸可以选自脂肪酸、环脂肪酸、芳香酸、芳香脂肪酸、杂环酸、羧酸和磺酸，例如甲酸、乙酸、丙酸、乙醇酸、葡萄糖酸、乳酸、丙酮酸、草酸、苹果酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、天冬氨酸、抗坏血酸、谷氨酸、邻氨基苯甲酸、苯甲酸、肉桂酸、扁桃酸、扑酸、苯乙酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸。
药学上可接受的碱加成盐包括衍生自无机碱的盐如钠、钾、锂、铵、钙、镁、铁、锌、铜、锰、铝盐等。特别优选的是铵、钾、钠、钙和镁盐。衍生自药学可接受的无机有机碱的盐包括伯胺、仲铵和叔铵、包括天然取代铵在内的取代铵、换装安和碱性 离子交换树脂如异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、2-二乙胺基乙醇、三甲胺、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-以及哌啶、多胺树脂等的盐。特别优选的无毒有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环已胺、胆碱和咖啡因。
在某些实施方案中，本公开所述的通式I化合物选自：
Figure PCTCN2017119014-appb-000024
Figure PCTCN2017119014-appb-000025
Figure PCTCN2017119014-appb-000026
Figure PCTCN2017119014-appb-000027
Figure PCTCN2017119014-appb-000028
Figure PCTCN2017119014-appb-000029
Figure PCTCN2017119014-appb-000030
Figure PCTCN2017119014-appb-000031
Figure PCTCN2017119014-appb-000032
Figure PCTCN2017119014-appb-000033
Figure PCTCN2017119014-appb-000034
Figure PCTCN2017119014-appb-000035
根据本公开，药物组合物包含通式I化合物与常规药用载体或赋形剂。该药物组合物可通过例如口服或非肠道等途径给药。本公开的药物组合物可按本领域常规方法 制备成各种剂型，包括但不限于片剂、胶囊、溶液、悬浮液、颗粒剂或注射剂等，经例如口服或非肠道等途径给药。
另外需要指出，本公开所述的化合物使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。优选的使用剂量介于0.01－100mg/kg体重/天。
合成路线
化合物的合成采用本领域已知的合成方法，除非特殊说明所使用的试剂均选用商业化的原料及中间体。通式I化合物可以采用以下合成路线合成：
路线一
Figure PCTCN2017119014-appb-000036
制备式E的化合物以取代吡啶A和硼酸酯B先经Suzuki偶联，还原硝基后与相应的苯甲酰氯反应即可得到产物。
路线二
Figure PCTCN2017119014-appb-000037
式J的化合物同样先经Suzuki偶联，水解酯基之后与相应的苯胺反应，得到目标化合物。
路线三
Figure PCTCN2017119014-appb-000038
式N化合物先经缩合反应，得到中间体M，后与相应的胺缩合成环得到目标产物。
具体实施方式
下面结合实施例进一步说明本公开，应该理解的是，下面实施例仅仅是用于说明本公开，不是对本公开的限制，本公开的范围与核心内容依据权利要求书加以确定。下面实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
制备2-氯-4-甲砜基-N-(3-(吡啶-2-基)-苯基)-苯甲酰胺(化合物1)
Figure PCTCN2017119014-appb-000039
将4-甲砜基-2-氯苯甲酸A(0.71g，3.0mmol)溶于30ml无水CH 2Cl 2，室温下滴加1ml SOCl 2，并滴加6滴N,N-二甲基甲酰胺(DMF)，加热至回流，反应6h。反应完成后旋蒸除去多余的SOCl 2和CH 2Cl 2。产物用30ml无水四氢呋喃(THF)溶解，室温下滴加间氨基苯乙酮B(0.40g，3mmol)的THF溶液(3ml)，加0.70ml Et 3N，室温反应4h。反应物浓缩，加水(30ml)后用乙酸乙酯萃取(15ml×3)，合并有机相后用无水硫酸钠干燥，硅胶柱色谱分离得中间体C(1.02g，96.2％)。 1H NMR (400MHz,DMSO-d 6)δ10.90(s,1H),8.31(t,J＝1.8Hz,1H),8.14(d,J＝1.6Hz,1H),8.02(dd,J＝8.0,1.7Hz,1H),7.96–7.89(m,2H),7.76(dt,J＝7.8,1.2Hz,1H),7.54(t,J＝7.9Hz,1H),3.36(s,3H),2.59(s,3H).HRMS m/z 352.0439。
将对甲苯磺酸(32mg，0.17mmol)，三氟甲磺酸铜(10mg，0.028mmol)，1,3-丙二胺(105mg，1.42mmol)和4-甲砜基-2-氯-N-(3-乙酰基苯基)苯甲酰胺C(100mg，0.28mmol)溶于10mlN,N-二甲基乙酰胺中，氧气环境下升温至110℃反应24h。反应液浓缩，加入饱和碳酸氢钠后用CH 2Cl 2萃取(15ml×3)，饱和食盐水洗涤，无水硫酸钠干燥，硅胶柱色谱分离得到化合物1(61mg，55.47％)。 1H NMR(600MHz,Chloroform-d)δ:8.64(s,1H),8.44(s,1H),8.21(s,1H),7.96(s,1H),7.92(d,J＝7.9Hz,1H),7.84(d,J＝7.9Hz,1H),7.81–7.67(m,4H),7.52(t,J＝7.8Hz,1H),7.25(s,1H),3.08(s,3H).HRMS m/z 387.0605。
依据实施例1所述的方法制备以下化合物：
Figure PCTCN2017119014-appb-000040
Figure PCTCN2017119014-appb-000041
实施例2制备化合物7
Figure PCTCN2017119014-appb-000042
将6-溴烟酸甲酯D(1.00g，4.63mmol)，2-硝基苯硼酸E(1.16g，6.94mmol)，四三苯基磷钯(0.16g，0.14mmol)和碳酸钠(0.74g，6.94mmol)溶于甲醇/二氧六环(1:3)溶液(40mL)中，氩气保护下升温至80℃反应过夜。反应液冷却后减压浓缩，加乙酸乙酯100mL溶解，依次用饱和碳酸氢钠、蒸馏水、饱和食盐水洗，无水硫酸钠干燥，乙酸乙酯重结晶。得浅黄色固体F(0.98g，81.99％)。 1H NMR(400MHz,Chloroform-d)δ9.32(dd,J＝2.2,0.9Hz,1H),8.94(t,J＝2.0Hz,1H),8.45(ddd,J＝7.8,1.7,1.1Hz,1H),8.43(dd,J＝8.2,2.1Hz,1H),8.33(ddd,J＝8.2,2.3,1.1Hz,1H),7.91(dd,J＝8.3,0.9Hz,1H),7.70(t,J＝8.0Hz,1H),4.00(s,3H,CH 3).
将中间体F(0.60g，2.32mmol)溶于30mL无水乙醇中，加入无水氯化亚锡(2.20g，11.62mmol)后升温至70℃反应3h。反应液冷却浓缩后用乙酸乙酯(50mL)溶解， 饱和碳酸氢钠调节pH>7，硅藻土过滤后有机层依次水洗、饱和氯化钠洗、无水硫酸钠干燥，硅胶柱色谱分离的白色固体G(0.43g，81.08％)。 1H NMR(400MHz,Chloroform-d)δ9.25(dd,J＝2.3,0.9Hz,1H),8.32(dd,J＝8.3,2.2Hz,1H),7.77(dd,J＝8.3,0.9Hz,1H),7.45(t,J＝2.0Hz,1H),7.38(ddd,J＝7.7,1.7,1.0Hz,1H),7.27(t,J＝7.9Hz,1H),6.79(ddd,J＝7.9,2.4,1.0Hz,1H),3.97(s,3H,CH 3),3.70(s,2H,NH 2).
将2-氯-4-甲砜基苯甲酸(0.36g，1.44mmol)溶于二氯甲烷(20mL)中，加氯化亚砜(1.05mL)和DMF几滴后升温至50℃回流反应3h。反应液冷却后减压蒸干，用四氢呋喃(20mL)溶解后，缓慢滴加到中间体G(0.30g，1.31mmol)和三乙胺(275μL)的四氢呋喃(30mL)溶液中，反应室温搅拌过夜。反应液减压浓缩，加乙酸乙酯溶解，饱和碳酸氢钠洗、水洗、饱和氯化钠洗，无水硫酸钠干燥，硅胶柱色谱分离得白色固体H(0.36g,61.77％)。 1H NMR(400MHz,DMSO-d 6)δ10.87(s,1H),9.18(dd,J＝2.2,0.8Hz,1H),8.60(t,J＝1.9Hz,1H),8.39(dd,J＝8.3,2.3Hz,1H),8.14(d,J＝1.7Hz,1H),8.10(dd,J＝8.4,0.9Hz,1H),8.02(dd,J＝8.0,1.8Hz,1H),7.92(d,J＝8.0Hz,1H),7.92(dd,J＝8.0,1.6Hz,1H),7.81(ddd,J＝8.1,2.2,1.0Hz,1H),7.55(t,J＝7.9Hz,1H),3.92(s,3H),3.36(s,3H).
将中间体H(100mg，0.22mmol)溶于甲醇/水(4:1，5mL)中，加入氢氧化锂(16mg，0.67mmol)后搅拌4h。反应液减压浓缩，加水(15mL)后用乙酸乙酯(20mL)萃取，水层用1N盐酸调节pH<7，收集固体得白色固体I(64mg，66.80％)。 1H NMR(600MHz,DMSO-d 6)δ13.43(s,1H),10.89(s,1H),9.17(d,J＝2.2Hz,1H),8.61(t,J＝1.9Hz,1H),8.37(dd,J＝8.3,2.2Hz,1H),8.15(d,J＝1.6Hz,1H),8.08(d,J＝8.4Hz,1H),8.03(dd,J＝7.9,1.7Hz,1H),7.93(t,J＝8.0Hz,2H),7.81(dd,J＝8.1,1.0Hz,2H),7.55(t,J＝7.9Hz,1H),3.37(s,3H).
将中间体I(100mg，0.23mmol)、HATU(109mg，0.28mmol)和三乙胺(81μL，0.46mmol)溶于DMF(3mL)中，搅拌10分钟后加入异丙基胺(25μL，0.28mmol)，室温搅拌6h。反应液倒入50mL水中，搅拌10分钟后收集固体并干燥，固体用三氯甲烷和甲醇重结晶得化合物7(47.3mg，43.18％)。 1H NMR(600MHz,DMSO-d 6)δ10.87(s,1H),9.09(d,J＝1.5Hz,1H),8.56(s,1H),8.47(d,J＝7.7Hz,1H),8.30(dd,J＝8.4,2.3Hz,1H),8.15(d,J＝1.6Hz,1H),8.03(dd,J＝8.1,3.4Hz,2H),7.93(d,J＝7.9Hz,1H),7.90(d,J＝7.8Hz,1H),7.84–7.80(m,1H),7.53(t,J＝7.9Hz,1H),4.14(h,J＝6.8Hz,1H),3.37(s,3H),1.21(d,J＝6.6Hz,6H).
依据实施例2所述的方法制备以下化合物：
Figure PCTCN2017119014-appb-000043
Figure PCTCN2017119014-appb-000044
Figure PCTCN2017119014-appb-000045
按上述合成方法，所合成的化合物还有：
Figure PCTCN2017119014-appb-000046
Figure PCTCN2017119014-appb-000047
Figure PCTCN2017119014-appb-000048
Figure PCTCN2017119014-appb-000049
Figure PCTCN2017119014-appb-000050
Figure PCTCN2017119014-appb-000051
实施例3活性测定
3.1活性测定方法
1.TM3-Gli-Luc报告基因检测试验(TM3-Gli-Luc reporter gene assay(Gli shift))
测试化合物用DMSO溶解，稀释成不同梯度的溶液备用。TM3Hh12细胞(含有Hh-响应报告基因构建的pTA-8xGli-Luc)用含有5％马血清，2.5％胎牛血清和15mM的HEPES缓冲溶液(Ph 7.3)的F12Ham’s/DMEM培养液培养，用胰蛋白酶处理后移至含5％马血清和15mM HEPES(pH 7.3)的F12Ham’s/DMEM(1:1)培养液中。细胞加入到含化合物的分析板中，置于37℃，5％的CO2环境下孵育30分钟。加入1nM或者25nM的Hh Ag1.5，相同环境下孵育48小时。孵育完成后，加入Bright-Glo(Promega E2650)或者MTS试剂(Promega G258B)，测试荧光度或492nM下的吸收度。IC50值(即曲线的拐点值)由Gli驱动的荧光素酶发光或者MTS分析法的吸收信号对测试化合物的log10值做曲线得到，曲线用R统计软件得到。
2.Gli reporter gene assay with a Smo D473H mutant cell line.
C3H10T1/2细胞转染野生型或D473H突变的SMO和Gli荧光标记的质粒。DNA和GeneJuice的转染过程如下：向空96孔板内加入3μL的Opti-MEM和0.15μL的GeneJuice转染试剂，室温下孵育5分钟。加入20ng的8x-Gli1荧光标记，1ng的p-RL-TK和10ng的pcDNA-Smo-WT，pcDNA-Smo-D473H或空pcDNA3.1对照，小心混匀，室温孵育15分钟后加入到细胞中。小心摇动板子确保分布均匀，37℃含5％CO 2条件下孵育。转染6小时后培养液换100μL的完全生长液。转染24小时后加入不同浓度的抑制剂(0.0001-10μM)或DMSO对照，培养24小时。加入抑制剂24小时后测定荧光活性，计算相对于空白的抑制率，求出IC50值。
3.靶点结合实验
(1)Smoothened膜滤结合试验(Smoothened membrane filter binding assays)
用转染了小鼠或者人Smo cDNA的CHO-K1细胞制得含Smo的细胞膜，测试化合物配成系列梯度放于空分析板内备用。将含有Smo的细胞膜加入到分析板内，加入用缓冲溶液(50mM Tris-HCl,10mM EDTA,and 5mM magnesium chloride,pH 7.2)制得1.5nM的[3H]Hh-Ag 1.5，分析板置于37℃下孵育48小时。96孔Unifilter GF/B滤板先用0.5％的聚乙烯亚胺和1％的胎牛血清预先孵育60分钟，用2％HPCD蒸馏水溶液洗三次。将分析板内的培养液用Unifilter-96移液枪移至滤板内，已上样的滤板 用2％的HPCD缓冲液洗三次，洗去未结合的[3H]Hh-Ag1.5，60℃干燥30分钟，冷却。封住底端，每个孔加50μL的Microscint-O，封住顶端，孵育100分钟-过夜。[3H]Hh-Ag1.5的结合量用TopCount(Perkin-Elmer)闪烁计数仪得到。饱和结合数用Graphpad Prizm软件分析得到。
(2) 3H-cyclopamine
细胞膜用含有50mM HEPES和3mM MgCl2，pH为7.2的缓冲溶液稀释至0.01-0.02mg/ml。先向100μl含有荧光配体、不同浓度的抑制剂和蛋白酶抑制剂的缓冲溶液内加200μl的细胞膜悬浮液，再加0.02％的胎牛血清以减少非特异性结合。结合试验在96孔板上进行。96孔板在23℃下孵育5小时使抑制剂达到结合平衡，用浸润好的玻璃纤维滤膜(0.3％的聚乙烯亚胺)将游离的标记配体快速真空滤去。滤出物用0.3ml含有0.01％Triton X-100的冷的磷酸缓冲盐冲洗2次。滤出物的荧光活性用TopCount液体闪烁计数器定量。
(3) 3H-GDC-0449
2X106个HEK-293细胞接种到10cm的板子里，用3μg SMO表达的GeneJuice质粒转染细胞。40小时后，细胞转移至1mmol/L EDTA溶液中，用含4％多聚甲醛的PBS溶液处理10分钟后用PBSA洗三次。取96孔板一个，每孔加细胞2X105个，加50mM的未标记GDC-0449或者空白对照，然后加入5nM的[3H]-GDC-0449，PBS中37℃孵育一小时。细胞转移到滤盘中，洗6次并干燥。用Microscint-20闪烁液和Topcount读数仪测量放射性。结果以原始数据呈现或者去除背景后以SMO-WT呈现。
(4)BODIPY-cyclopamine
荧光结合试验用BODIPY FL or
Figure PCTCN2017119014-appb-000052
558/568标记的环巴胺进行，方法大体如下：将表达了人或者小鼠SMO的CHO细胞加入到384孔板内，用4％的多聚甲醛室温处理15分钟，洗涤后加入含0.5％胎牛血清的PBS缓冲溶液，然后加入荧光标记的BODIPY-环巴胺(20nM)和测试化合物，37℃孵育4小时。细胞用PBS冲洗，Hoechst 33258染色，用
Figure PCTCN2017119014-appb-000053
Ultra成像仪分析。
4.TaqMan基因表达试验(TaqMan Gene Expression Assay)
内源性的小鼠Gli和Ptch1或人Gli和Ptch1基因表达由大鼠NIH 3T3或者人HEPM细胞表达得到。NIH 3T3和HEPM细胞含0.5％FBS的EMEM培养液培养的6-孔板培养，培养时用加入重组ShhN(200ng/ml)、Smo抑制剂或者DMSO对照。全部的RNA用TRIzol试剂取得，接着用氯仿萃取，然后用RNeasy小型离心柱纯化。 用高效cDNA逆转录酶试剂盒进行逆转录。用TaqMan基因表达分析法定量，内标为VIC/MGB Probe。RT-PCR用TaqMan Gene Expression Master Mix试剂盒，工作站为ABI Prism 7900HT快速RT-PCR。
5.通路选择性试验(Control reporter assays)
TM3-SV40细胞的构建用1μg的选择性pGL3-SV40-Luc质粒在新霉素存在下转染TM3细胞得到，用Fugene试剂按说明书操作即可。细胞用G418选择性筛选两周，收集剩下的细胞备用。
293T-STF-Luc细胞用对Wnt响应的SuperTopflash-Luc载体，在新霉素存在的情况下转染293T细胞得到。用G418选择性筛选两周，收集剩下的细胞备用。为了检测抑制剂对Wnt的响应，取384孔板一个每孔含30μl DMEM+5％FBS的培养液，每孔加10000细胞。12小时后，加入稀释好的抑制剂溶液。加20μl Wnt培养液，孵育48小时，用BrightGlo荧光试剂盒测定荧光活性。
293T-NFкB-Luc细胞用对NFкB响应的Luc载体在新霉素的存在下转染293T细胞得到。用G418选择性筛选两周，收集剩下的细胞备用。为了检测抑制剂对NFкB的响应，取384孔板一个每孔含40μl DMEM+5％FBS的培养液，每孔加10000细胞。12小时后，加入稀释好的抑制剂溶液，30分钟后加10μl含重组TNFα的培养液。孵育24小时后用BrightGlo荧光试剂盒测定荧光活性。
6.化合物体外细胞增殖抑制试验
(1)MB增殖试验(Medulloblastoma Proliferation Assay)
从Ptch+/-p53-/-转基因小鼠取到的肿瘤分成多个片段利用裸鼠进行传代。Ptch+/-p53-/-MB细胞从移植的老鼠身上分离出来，在非粘着性的10cm Petri盘中用含2％B-27,1％N2添加剂和1％青霉素/链霉素的Neurobasal培养液培养，每孔1x107个细胞。细胞培养三天后，用野生型的或D477G突变的慢病毒上清液感染。用RT-PCR测试Smo表达水平，增殖实验用Click-iT EdU细胞增殖分析试剂盒按照说明书进行操作即可。简单操作如下：细胞先用5mM的EdU冲洗12小时，用4％的低聚甲醛固定，用含有0.5％Triton X-100的PBS缓冲溶液渗透，冲洗，然后加入荧光基团(500nM Alexa Fluor 488azide，1mM Cu2SO4和5mM的维生素C)一起孵育。孵育完以后用PBS冲洗，Hoechst 33258染色，ImageXpress Ultra imaging system分析。
(2)CGNP细胞增殖试验(CGNP Proliferation Assay)(CGNP,小脑颗粒神经元祖细胞)
CGNP是从4天大的C57BL/6上分离得到。在固定了poly-D-lysine的384孔板上用含2％B-27添加剂，1mM的丙酮酸钠，2mM L-谷氨酰胺和1％青霉素/链霉素添加剂的Neurobasal培养液培养，每孔细胞浓度105，然后加入重组ShhN(200ng/ml)和抑制剂。Click-iT EdU细胞增殖分析试剂盒进行细胞增值实验，方法同上。
(3)Gli mRNA抑制试验(Gli mRNA inhibition in the HEPM assay)
HEPM细胞是从人胚胎腭间质组织衍生而来，从ATCC获得的。HEPM细胞用最小基础培养液(MEM)+Earle’s Salts+L-谷氨酰胺+10％胎牛血清，1x丙酮酸钠和1x的MEM非必须氨基酸情况下，在35℃含5％CO2条件下培养。细胞用0.05％胰蛋白酶EDTA溶液每周分装两次，然后放置于75cm的锥形瓶中。将细胞以每孔50000的数量加入到96孔板中，用于Gli1RT-PCR分析。接下来加入50nM的激动剂和不同浓度的抑制剂。48小时候收集细胞，用于RT-PCR。
细胞在35℃ 5％的条件下培养后，除去培养基，加入50μl不同浓度的抑制剂，接着加50μl激动剂，孵育48小时。加入的50μl用培养基稀释的激动剂Hh Ag1.5(1:1000),使每个孔的激动剂的浓度为50nM。抑制剂则是以1:3稀释10mM的DMSO溶液，然后用培养液稀释1000倍得到的。
用带有移液枪收集并纯化96孔板上的细胞RNA。取新的96孔板，预先加入4μL的逆转录酶，然后加入上述的RNA溶液16μL。得到的混合液转移到ABI96孔板上，用PCR仪在25℃孵育5分钟，42℃孵育30分钟，85℃孵育5分钟。反应完成以后，每个孔加20μL的不含DNA酶/RNA酶的水使每个孔的液体为40μL。将样品储存在-20℃下备用。
mRNA的定量表达用RT-PCR法，使用Taqman试剂在Applied Biosystems7900HT快速RT-PCR仪或者7900HT序列检测系统上进行。仪器设置流程如下：取消ROX,第一阶段50℃ 2min,第二阶段95℃ 10min,第三阶段95℃ 15sec,最后60℃ 1min循环40次。反应过程中需要5种溶液：2xMaster mix，Gli和betaactin的引物，反应所需的cDNA和水。注意2xMaster mix需要包含以下成分：Amplitaq Gold DNA聚合酶,Amperase UNG,dNTP’s,with UTP,阴性对照ROX。
实时PCR的数据去除betaactin影响。要想得到IC50值，需要先扣除DMSO的背景值，计算相对于没加抑制剂只含激动剂的抑制率。IC50值用XLfit4using Fit Designer Dose Response Model 202计算。
7.化合物动物体内抑制肿瘤活性的动物模型的建立
(1)皮下MB移植试验(Subcutaneous Ptch+/-Hic-/-medulloblastoma allograft models)
取1.0×106或者5.0×106个从肿瘤样本中取得的Ptch+/-Hic-/-小鼠MB细胞，分别皮下接种到裸鼠或者裸大鼠右侧腹部。移植后7天开始喂药，喂药前根据肿瘤大小随机分组，以每个组的平均肿瘤大小为250mm3为宜。每组老鼠的肿瘤体积和体重需要每周记录2-3次，用于以后分析。喂药剂量随着体重随时更改，不同治疗组之间的对照用ANOVA(Tukey)rank sum test法计算。
(2)orthotopic pancreatic cancer xenografts
E3LZ10.7细胞皮下注射入雄性CD1裸鼠体内，2-4周后摘取肿瘤，切成约1mm3的小块。6-8周的雄性CD1裸鼠用麻醉性气体麻醉，腹部左侧1cm的肋下打开，直到能看见脾脏和胰腺附属物，在胰腺上开一个小口植入准备好的肿瘤并缝合。
L3.6pl组中，106个细胞悬浮于50μL的PBS/Matrigel(1:1)混合液中，直接注入小鼠胰腺内。21天以后，用超声测量肿瘤的体积。以肿瘤大小为标准，平均分为四组：对照，环巴胺(25mg/kg饲料添加喂养，两天一次)，吉西他滨(100mg/kg，每四天注射一次)和联合用药组，持续给药30天。L3.6pl组只需要给环巴胺15天，50mg/kg/d。
30天以后，处死小鼠，检查肾脏，肝脏，脾脏，小肠，腹膜和肺的肿瘤扩散情况，并用福尔马林浸泡保存。肿瘤样本快速冷冻用于RNA分析。除直接观察以外，每只小鼠应随机选取肾脏，肝脏，脾脏，小肠，腹膜和肺组织用于镜检，观察有无微转移(micrometastases)。获取局部淋巴结样本，做成切片并检查是否发生病变。L3.6pl组只需要观察肝脏和腹膜的病变情况。
8.化合物动物体内药物代谢试验
(1)小鼠体内单剂量生物利用度和药代动力学试验
化合物溶于由5％NMP+10％PEG400+35％的10％ETPGS+50％D5W配成的溶液中，雄性C57BL小鼠(2只每个时间点)尾静脉注射(1mg/kg,0.2mg/mL)或者灌胃(10mg/kg,1mg/ml)。给药24小时后固定时间心脏穿刺取血样(2只每个时间点)。离心分离血浆，用HPLC/MS/MS测量血浆中化合物的浓度。相关血浆药代动力学常数如清除率，分布体积和半衰期按非房室模型计算。
(2)大鼠体内单剂量生物利用度和药代动力学试验
Sprague-Dawley鼠尾静脉注射(1mg/kg，由1.5eq HCl，20％captisol溶于pH 8 缓冲溶液配成的1mg/mL的化合物溶液,每次2只)或者灌胃(10mg/kg，0.5％羧甲基纤维素钠悬浮液，每次3只)的方法给药。24小时后按照固定的时间取血样，离心得血浆。血浆内化合物的浓度用HPLC/MS/MS测得，相关血浆药代动力学常数如清除率，分布体积和半衰期按非房室模型计算。
(3)狗体内单剂量生物利用度和药代动力学试验
化合物溶于20％captisol中，配成0.4mg/ml的溶液，禁食的雄性比格犬(3只)单剂量注射(0.1mg/kg)或者口服(0.3mg/kg)。24小时后按照固定的时间取血样，离心得血浆。血浆内化合物的浓度用HPLC/MS/MS测得，相关血浆药代动力学常数如清除率，分布体积和半衰期按非房室模型计算。
9.对A549非小细胞肺癌细胞系的抑制活性试验
将对数生长期的A549细胞悬浮液，稀释为4×10 4-5×10 4个/mL，接种于96孔板中(100μL/孔)。于37℃，5％CO 2条件下孵育8h后，每孔加入100μL不同浓度的化合物稀释液(培养基稀释)，每个浓度设三个复孔。孵育48h后，每孔加入10μL0.5％的MTT染色液,继续孵育4h，后离心弃去上清液，在每孔中加入150μL DMSO，于37℃摇床孵育5-10min。用酶标仪测定570nM处的OD值，并计算化合物的抑制率和IC 50值。
3.2实验结果
根据上述3.1节所述的活性测定方法，测定了本公开所述化合物对SMO的结合活性以及对A549非小细胞肺癌细胞系的抑制活性，部分化合物的活性测试结果如表1所示：
表1
Figure PCTCN2017119014-appb-000054
Figure PCTCN2017119014-appb-000055
备注：表1中A代表IC 50<100nM；B代表100nM<IC 50<500nM；C代表IC 50>500nM。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解：根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (18)

  1. 通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，
    Figure PCTCN2017119014-appb-100001
    其中：
    A、B各自独立地为芳香环、芳香杂环、芳香稠合环、芳香稠合杂环、碳环或杂环；
    X不存在或是亚甲基、NR 6CO、CONR 6、COO、OOC、SONR 6、NR 6SO、SO 2NR 6、NR 6SO 2、NHCONH、NHC(NH)NH，其中R 6为卤素、烷基、取代的烷基、烷氧基烷基、羰基、杂环、烷基氨基烷基、芳基、取代芳基、烷氧基、羰基、烷基氨基、芳基氨基、烷基氨基甲酰基、烷基氨基磺酰基、磺酰基；
    R 1为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基甲酰基、氨基磺酰基，磺酰胺基、三氟甲基、烷基、烷氧基、三氟甲氧基、烷氨基、烷基砜基、环烷基、杂环烷基、羟基烷基、氨基烷基、烷氧基烷基、烷氨基烷基；
    R 2、R 3、R 4各自独立的为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基酰基，氨基磺酰基、磺酰胺基、三氟甲基；
    R 5为氢、卤素、羟基、氨基、氰基、硝基、羧基、醛基、氨基甲酰基、氨基磺酰基，磺酰胺基、三氟甲基、烷基、烷氧基、三氟甲氧基、烷氨基、烷基砜基、环烷基、杂环烷基、芳基、取代芳基，杂芳基、取代杂芳基、羟基烷基、氨基烷基、烷氧基烷基、烷氨基烷基；
    m是0-3；
    n是0-3。
  2. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其中，A任选自：
    Figure PCTCN2017119014-appb-100002
  3. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其中，B选自下述基团：
    Figure PCTCN2017119014-appb-100003
  4. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有式Ia所示的结构：
    Figure PCTCN2017119014-appb-100004
    其中：
    R 1选自烷基、碳环或杂环，R 1任选被羟基、卤素、氰基、氨基、羰基、酰基、烷基、卤代烷基、三氟甲基、硝基、烷氧基、羧基、醛基、甲氨基、甲酰胺基、烷基磺酰基、烷基亚磺酰基、烷基氨基甲酰基、烷酰基氨基、烷基氨磺酰基、烷基磺酰胺、烷基亚磺酰基、烷氧基、烷基氨基甲酰基、烷酰基氨基、烷基氨基酰基、烷基磺酰基、碳环或杂环取代基、任选被氨基、卤素、羟基、羰基、卤代烷基、烷基、烷氧基或者酰基取代的碳环或杂环取代；
    A选自：
    Figure PCTCN2017119014-appb-100005
  5. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有式Ib所示的结构：
    Figure PCTCN2017119014-appb-100006
    其中R 2、R 3、R 4各自独立地选自卤素、羟基、烷基、酰基或烷氧基，所述烷基、酰基或烷氧基任选被羟基、卤素、氨基、硝基、烷基、酰基、烷基磺酰基或烷氧基取代；
    X选自：
  6. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有式Ic所示的结构：
    Figure PCTCN2017119014-appb-100008
    其中：
    B选自
    Figure PCTCN2017119014-appb-100009
    R 5在每种情况下独立的为羟基、卤素、氨基、羧基、脒基、胍基、羰基、硝基、氰基、酰基、烷基、卤代烷基、磺酰基、亚磺酰基、烷氧基、烷硫基、氨基甲酰基、酰胺基、氨磺酰基、磺酰胺、碳环或杂环；其中所述氨基、脒基、烷基、酰基、磺酰基、亚磺酰基、烷氧基、烷硫基、氨基甲酰基、酰胺基、氨磺酰基、磺酰胺、碳环或杂环取代基任选被羟基、羧基、氨基、羰基、卤素、卤代烷、烷基、烷氧基、酰基、磺酰基或亚磺酰基取代；
    n是0-3。
  7. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其中，R 6在每种情况下独立的选自取代的烷基(如甲基、三氟甲基、二甲氨基甲基、哌啶基甲基、吗啉代甲基)；卤素(如氯)；烷氧基(如甲氧基)；羰基(如吗啉代羰基、乙酰基)；杂环(如吗啉、N-甲基哌嗪-4-基、N-乙酰基哌嗪-4-基、1H-1,2,4-三唑)；烷基氨基(如异丁基氨基，苄基氨基、羟乙基氨基、甲氧乙基氨基、吗啉代乙基氨基、吗啉代丙基氨基、咪唑乙基氨基)；芳基氨基(如苯基氨基)；烷基氨基甲酰基(如二甲基氨基甲酰基、异丁基氨基羰基)；烷基氨基磺酰基(如丙基氨基磺酰基、异丁基氨基磺酰基、二甲氨基磺酰基、二甲氨基乙酰基、羟乙基氨基磺酰基、甲氧乙基磺酰基)；磺酰基(如甲基磺酰基、以及磺酰基、氨基磺酰基、二甲氨基丙基磺酰基、N-甲基哌嗪-4-基磺酰基、吗啉代-4-基磺酰基、三氟甲基磺酰基)。
  8. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有式Id所示的结构：
    Figure PCTCN2017119014-appb-100010
    其中：R 10选自氢、卤素、-C(O)R 14；R 11选自氢、-C(O)OCH 3；R 12选自氢、氘、卤素；R 13选自氢、卤素、甲基；
    R 14选自：
    Figure PCTCN2017119014-appb-100011
    Figure PCTCN2017119014-appb-100012
  9. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有如式Ie所示结构：
    Figure PCTCN2017119014-appb-100013
    其中：R 12选自氢、氘、卤素；R 13选自氢、卤素、甲基。
  10. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有如式If所示结构：
    Figure PCTCN2017119014-appb-100014
    其中：R 12选自氢、氘、卤素；R 13选自氢、卤素。
  11. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有如式Ig所示结构：
    Figure PCTCN2017119014-appb-100015
  12. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有如式Ig所示结构：
    Figure PCTCN2017119014-appb-100016
  13. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶 剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其具有如式Ih所示结构：
    Figure PCTCN2017119014-appb-100017
    其中：R 12为氢或氘；R 13为氢或卤素氯；R 16为
    Figure PCTCN2017119014-appb-100018
    R 15为甲基、氨基、
    Figure PCTCN2017119014-appb-100019
  14. 权利要求1所述的通式I所示的化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其选自：
    Figure PCTCN2017119014-appb-100020
    Figure PCTCN2017119014-appb-100021
    Figure PCTCN2017119014-appb-100022
    Figure PCTCN2017119014-appb-100023
    Figure PCTCN2017119014-appb-100024
    Figure PCTCN2017119014-appb-100025
    Figure PCTCN2017119014-appb-100026
    Figure PCTCN2017119014-appb-100027
    Figure PCTCN2017119014-appb-100028
    Figure PCTCN2017119014-appb-100029
    Figure PCTCN2017119014-appb-100030
    Figure PCTCN2017119014-appb-100031
  15. 药物组合物，其包含至少一种权利要求1至14任一项所述的通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，以及任选的药学上可接受的载体或赋形剂。
  16. 权利要求1至14任一项所述的通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物在制备用于治疗疾病或病症或减轻所述疾病或病症严重性的药物中的用途，或者在制备作为刺猬通路抑制剂的药物中的用途，
    优选地，其中所述疾病或病症为肿瘤或癌症，例如非小细胞肺癌、基底细胞癌、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
  17. 一种治疗疾病或病症或减轻所述疾病或病症严重性的方法，所述方法包括给予需要这种治疗的患者治疗有效量的权利要求1至14任一项所述的通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，其中所述疾病肿瘤或癌症，例如非小细胞肺癌、基底细胞癌、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
  18. 权利要求1至14任一项所述的通式I化合物、其药学上可接受的盐、其异构体、溶剂化物、或式I化合物中的任意原子经其同位素替换后得到的化合物，用于治疗疾病或病症或减轻所述疾病或病症严重性，或者用于抑制刺猬通路，
    优选地，其中所述疾病肿瘤或癌症，例如非小细胞肺癌、基底细胞癌、髓母细胞瘤、黑色素瘤、成神经管细胞癌、胰腺癌、前列腺癌、结肠癌、乳腺癌、结肠癌或胃癌。
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