WO2017177958A1 - 一种稠环化合物、其制备方法和应用及其中间体化合物 - Google Patents

一种稠环化合物、其制备方法和应用及其中间体化合物 Download PDF

Info

Publication number
WO2017177958A1
WO2017177958A1 PCT/CN2017/080521 CN2017080521W WO2017177958A1 WO 2017177958 A1 WO2017177958 A1 WO 2017177958A1 CN 2017080521 W CN2017080521 W CN 2017080521W WO 2017177958 A1 WO2017177958 A1 WO 2017177958A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
formula
cancer
group
pharmaceutically acceptable
Prior art date
Application number
PCT/CN2017/080521
Other languages
English (en)
French (fr)
Inventor
龙凯
廖立东
王万
Original Assignee
四川赛诺唯新生物技术有限公司
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 四川赛诺唯新生物技术有限公司 filed Critical 四川赛诺唯新生物技术有限公司
Publication of WO2017177958A1 publication Critical patent/WO2017177958A1/zh

Links

Classifications

    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/537—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 spiro-condensed or forming part of bridged ring systems
    • 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
    • A61K31/54—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 sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • 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 invention is in the field of medicinal chemistry and, in particular, relates to a fused ring compound, a process for its preparation and its use as a phosphatidylinositol 3-kinase inhibitor.
  • the invention also relates to intermediate compounds of the compounds.
  • molecularly targeted drugs act on specific molecular targets of tumor cells and inhibit the malignant biological behaviors such as proliferation, invasion and metastasis of tumor cells, thereby producing anti-tumor effects. These molecular targets are specifically expressed or overexpressed in tumor tissues, but are not expressed or expressed in normal tissues, so molecularly targeted drugs are generally capable of selectively killing tumor cells or inhibiting their growth and proliferation, while for normal cells. Less toxic effects.
  • the search for and development of new molecular targeted drugs represents the development direction of precise treatment of tumors.
  • Phosphadylinositol 3-kinase is an important member of the phospholipase kinase family, which specifically catalyzes the phosphorylation of the hydroxylino-inositol 3-hydroxyl group and is a PI3K/Akt/mTOR signaling pathway.
  • the key sites on the key play a key role in cell proliferation, survival and metabolism.
  • PI3K can be divided into three types, I, II and III, depending on its structural characteristics, activation mechanism and substrate selection. Since the most functional research to date is type I PI3K, the so-called PI3K refers to type I PI3K.
  • Type I PI3K is divided into two subclasses, IA and IB, depending on its regulatory subunit and upstream regulatory molecules.
  • the IA subclass PI3K is activated by various receptor tyrosine kinases and Ras, including three subtypes of PI3K ⁇ , PI3K ⁇ and PI3K ⁇ , which are composed of the respective catalytic subunit p110 and the regulatory subunit p85, respectively.
  • the IB subclass PI3K is mainly activated by the G protein-coupled receptor, a subtype of PI3K ⁇ , which consists of the catalytic subunit p110 ⁇ and the regulatory subunit p101 or p84.
  • PI3K ⁇ and PI3K ⁇ are expressed in various organs, while PI3K ⁇ and PI3K ⁇ are mainly distributed in bone marrow cells.
  • the function of the four subtypes of PI3K is also different: PI3K ⁇ is most closely related to tumors. Mutation, amplification and overexpression of PIK3CA, a gene encoding p110 ⁇ , are widely present in many malignant tumors. Therefore, PI3K ⁇ is thought to occur in tumors.
  • PI3K ⁇ is involved in insulin signaling and glucose metabolism; PI3K ⁇ can activate platelets and therefore plays an important role in the development of thrombotic diseases.
  • PI3K ⁇ has been reported in recent years. It plays an important role in PITEN-deficient cancer patients than PI3K ⁇ ; PI3K ⁇ and PI3K ⁇ are closely related to inflammation and immunity.
  • the first reported inhibitor of PI3K synthesis is LY294002, which has a micromolar activity against class I PI3K and an IC 50 value of 0.63 ⁇ M for PI3K ⁇ . It also shows a certain anti-tumor effect in animal experiments, but Due to safety, stability and other reasons, it failed to enter clinical trials.
  • PI3K inhibitors with different structures have been reported, including pyrimidines, thienopyrimidines, and quinazones. , diketenes, imidazoquinolines, imidazopyridines, benzopyridines and other molecular skeleton compounds.
  • GDC-0941 is a pan-PI3K inhibitor developed by Genentech, which has an IC 50 of 3 nM, 33 nM, 3 nM, and 75 nM for p110 ⁇ , p110 ⁇ , p110 ⁇ , and p110 ⁇ , respectively.
  • the clinical phase I trial results of GDC-0941 showed that it has better anti-tumor activity in breast cancer, ovarian cancer and melanoma patients, and is well tolerated.
  • BKM120 is a pan-PI3K inhibitor of 2,4-bismorpholine-substituted pyrimidines developed by Novartis. Its IC 50 for p110 ⁇ , p110 ⁇ , p110 ⁇ , and p110 ⁇ are 52nM, 166nM, 116nM, and 262nM, respectively. The study of metastatic breast cancer is in phase III clinical.
  • IC-87114 is a selective PI3K ⁇ inhibitor with an IC 50 of 0.5 ⁇ M for PI3K ⁇ , which is 58 to 100 times more selective than the inhibitory activity against PI3K ⁇ , PI3K ⁇ , and PI3K ⁇ .
  • CAL-101 (Idelalisib) is also a selective PI3K ⁇ inhibitor with an IC 50 of 2.5 nM for p110 ⁇ , which is 40 to 300 times more selective than the inhibitory activity against p110 ⁇ , p110 ⁇ , and p110 ⁇ .
  • PI3K ⁇ the initial pharmacological studies on CAL-101 focused on its anti-inflammatory and autoimmune diseases.
  • PI3K ⁇ which was mainly expressed in bone marrow cells
  • the pharmacological study of CAL-101 was transferred to the treatment of leukemia and lymphoma.
  • PX866 is a pan-like diketene PI3K inhibitor IC for PI3K ⁇ , PI3K ⁇ , PI3K ⁇ 50 were 6nM, 3nM, 9nM. Currently in Phase I/II clinical trials.
  • BAY80-6946 Bayer is a highly developed active pan-PI3K inhibitor, its PI3K ⁇ , PI3K ⁇ , PI3K ⁇ , PI3K ⁇ the IC 50 were 0.5nM, 3.7nM, 0.7nM, 6.4nM. Currently in phase II clinical trials.
  • PIK75 is an imidazopyridine derivative which is a selective PI3K ⁇ inhibitor with IC 50 of 5.8 nM, 1.3 ⁇ M, 0.51 ⁇ M, and 76 nM for p110 ⁇ , p110 ⁇ , p110 ⁇ , and p110 ⁇ , respectively.
  • CN103788071A claims compounds of the general formula which, in part, exhibit a high inhibitory effect on proliferation in vitro.
  • XL147 is a selective inhibitor of PI3K with an IC for PI3K ⁇ , PI3K ⁇ , PI3K ⁇ , PI3K ⁇ 50 were 39nM, 383nM, 36nM, 23nM.
  • PCT/US2009/045713 discloses a compound of the formula: or a pharmaceutically acceptable salt thereof:
  • 137 compounds including:
  • PI3K small molecule inhibitors As a new class of molecular targeted drugs, PI3K small molecule inhibitors have great potential and broad prospects. Therefore, there is a need for more PI3K inhibitors with novel structures, high biological activity, and good drug-forming properties, for targeted therapy of tumors, and for anti-inflammatory or autoimmune diseases.
  • the present invention provides a novel PI3K selective inhibitor. Specifically, the present invention provides a fused ring compound represented by the following general formula (I): or a pharmaceutically acceptable salt thereof:
  • X represents O, S, NR, SO 2 or NH 2 Cl
  • R is selected from H, C 1-6 alkyl, preferably C 1-4 alkyl
  • R 1 , R 2 , R 3 , R 4 , R 5, R 6, R 7, R 8 are independently selected from the group consisting of H, C 1-6 alkyl, OH or a substituent group having a C 1-6 alkyl group, preferably H, C 1-4 of An alkyl group or a C 1-4 alkyl group having an OH substituent.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 are independently selected from an alkyl group of H, C 1-6 or a C 1-6 alkane having an OH substituent. base.
  • the invention may be selected from the following compounds or pharmaceutically acceptable salts thereof:
  • the present invention also provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.
  • the fused ring compound of the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described above can effectively inhibit the activity of PI3K.
  • the compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as a PI3K inhibitor can be used as an antitumor drug for brain cancer, head and neck cancer, esophageal cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, pancreatic cancer , prostate cancer, colorectal cancer, ovarian cancer, breast cancer, thyroid cancer, skin cancer, leukemia, myelodysplastic syndrome, sarcoma, osteosarcoma or rhabdomyomas.
  • the compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as a PI3K inhibitor can be used as It is used as an anti-inflammatory drug for the treatment of diseases such as chronic obstructive pulmonary disease and asthma.
  • the compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as a PI3K inhibitor can be used as a medicament for treating an autoimmune disease such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus.
  • the compound of the present invention can be produced by the following method.
  • the process comprises the steps of reacting a compound of formula (III') with a compound of formula (IV) to provide a compound of formula (I):
  • the method further comprises the step of reacting a compound of formula (II) with a compound of formula (III) to provide a compound of formula (III'),
  • the invention also provides a process for the preparation of a compound of formula (I), which comprises the step of reacting a compound of formula (IV') with a compound of formula (III) to provide a compound of formula (I):
  • the method further comprises the step of reacting a compound of formula (II) with a compound of formula (IV) to provide a compound of formula (IV'),
  • the desired compound can be obtained in high yield.
  • the present invention provides a compound of the formula (III') or a compound of the formula (IV'),
  • the inventors have extensively explored and experimentally verified that a cyano group is specifically introduced on a fused ring, and the above compound obtained by the present invention or a pharmaceutically acceptable compound thereof is compared with a compound having no cyano group and a compound having a cyano group substituted with another substituent.
  • the accepted salt has an unexpected, non-obvious inhibition activity against PI3K.
  • Step 1 N-(5-(4-Chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
  • 6-Bromo-4-chloroquinoline-3-carbonitrile 140 mg, 0.6 mmol
  • N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2) - dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide 203 mg, 0.618 mmol, 1.03 eq
  • 2N aqueous potassium carbonate 0.9 mL, 1.8 mmol, 3.0 eq
  • the mixture in the ring (6 mL) was degassed and bis(triphenylphosphine)palladium dichloride (21 mg, 0.03 mmol, 0.05 eq) was added.
  • the resulting reaction mixture was degassed and backfilled with argon (three cycles), and then stirred at 100 ° C for 7 hours under an argon atmosphere.
  • the reaction mixture was cooled to room temperature, diluted with water (30 mL) The combined organic layers were washed with EtOAc EtOAc EtOAc. Rate 35%).
  • Step 2 N-(5-(3-Cyano-4-thiomorpholinylquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
  • N-(5-(4-Chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (156 mg, 0.4 mmol) was dissolved in NMP (5 mL) Potassium carbonate (83 mg, 0.6 mmol, 1.5 eq) and thiomorpholine (62 mg, 0.6 mmol, 1.5 eq) were added. The resulting reaction mixture was stirred at 100 ° C for 2 hours.
  • 6-Bromo-4-chloro-quinoline-3-carbonitrile (535 mg, 2.0 mmol) and morpholine (523 mg, 6.0 mmol, 3 eq) were stirred in dioxane (7.5 mL) at 100 °C. After completion of the reaction, the mixture was evaporated to dryness. The combined organic layers were washed with EtOAc EtOAc (EtOAc) Yellow solid (556 mg, yield 87.4%).
  • Step 2 N-(5-(3-Cyano-4-morpholinylquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
  • 6-Bromo-4-morpholinylquinoline-3-carbonitrile (96 mg, 0.3 mmol), N-(2-methoxy-5-(4,4,5,5-tetramethyl) -1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide (118 mg, 0.36 mmol, 1.2 eq) and 2N aqueous potassium carbonate (0.45 mL, 3.0 eq)
  • the mixture in dioxane (4 mL) was degassed then [1,1 '-bis(diphenylphosphino)ferrocene]palladium dichloride (11 mg, 0.015 mmol, 0.05 eq).
  • the resulting reaction mixture was degassed and backfilled with argon (three cycles), and then stirred at 100 ° C for 5 hours under an argon atmosphere.
  • the reaction mixture was cooled to room temperature, diluted with water (30 mL)
  • the combined organic layers were washed with EtOAcqqqqqqqqqHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
  • step 2 is N-(5-(4-chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide and N -Methylpiperazine is reacted at room temperature.
  • step 2) is N-(5-(4-chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide and The reaction was carried out under the same conditions.
  • step 2) is N-(5-(4-chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide and The reaction was carried out under the same conditions.
  • step 2) is N-(5-(4-chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide and The reaction was carried out under the same conditions.
  • step 2) is N-(5-(4-chloro-3-cyanoquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide and The reaction was carried out under the same conditions.
  • Example 10 The remaining compound preparation method of Example 10 was as in Example 1, except that the thiomorpholine was replaced with the starting material in Table 1 below in Step 2.
  • 6-Bromo-4-morpholinylquinoline 105 mg, 0.36 mmol
  • Oxaborolan-2-yl)pyridin-3-yl)methanesulfonamide 141 mg, 0.43 mmol, 1.2 eq
  • 2N aqueous potassium carbonate 0.54 mL, 1.08 mmol, 3.0 eq
  • the mixture in 4 mL) was degassed and then [1,1 '-bis(diphenylphosphino)ferrocene]palladium dichloride (13 mg, 0.018 mmol, 0.05 eq) was added.
  • reaction mixture was degassed and backfilled with argon (three cycles), and then stirred at 100 ° C for 5 hours under an argon atmosphere.
  • the reaction mixture was cooled to room temperature, diluted with water (30 mL) The combined organic layers were washed with brine (30 mL) dry The residue was purified by flash column chromatography eluting elut elut elut elut elut
  • Step 7 N-(5-(3-(Difluoromethyl)-4-morpholinylquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
  • the resulting reaction mixture was degassed and backfilled with argon (three cycles), and then stirred at 100 ° C for 5 hours under an argon atmosphere.
  • the reaction mixture was cooled to room temperature, diluted with water (30 mL) The combined organic layers were washed with EtOAc EtOAc.
  • 6-Bromo-4-chloro-3-iodoquinoline (921 mg, 2.5 mmol), tributyl(1-ethoxyvinyl)tin (948 mg, 2.625 mmol, 1.05 eq) and triphenylarsenic (153 mg)
  • a mixture of 0.5 mmol, 0.2 eq. in DMF (10 mL) was then evaporated, then Pd 2 (dba) 3 (57 mg, 0.0625 mmol, 0.025 eq).
  • the resulting reaction mixture was degassed and backfilled with argon (three cycles), and then stirred at 60 ° C for 6 hours under an argon atmosphere.
  • Step 6 4-(6-Bromo-3-(1,1-difluoroethyl)quinolin-4-yl)morpholine
  • Step 7 N-(5-(3-(1,1-Difluoroethyl)-4-morpholinylquinolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
  • PI103 means: 3-[4-(4-morpholinylpyrido[3',2',4,5]furo[3,2-d]pyrimidin-2-yl)]phenol.
  • Test 1 Inhibitory activity of compounds against kinase PI3K ⁇
  • the compounds in the tube were transferred to one well of a 96-well stock plate, and 30 ⁇ L was transferred to the next well containing 60 ⁇ L of 100% DMSO to dilute the compound in this manner.
  • A1 of a 96-well plate is transferred to the A1 and A2 wells of a 384-well plate.
  • A2 of the 96-well plate was transferred to A3 and A4 of the 384-well plate, and so on.
  • a PI3K ⁇ solution was prepared in 1 ⁇ kinase buffer at a concentration four times the final concentration at the time of the test.
  • test plate was added with 2.5 ⁇ L of kinase solution per well, except for the control well without enzyme (plus 2.5 ⁇ L of 1 ⁇ kinase buffer instead).
  • a substrate solution containing PIP2 substrate and ATP was prepared in 1X Kinase Reaction Buffer, and the concentration of each reagent was twice the final concentration at the time of the test.
  • test plate was started by adding 5 ⁇ L of the substrate solution to each well.
  • test plate was incubated for 1 hour at room temperature.
  • test plate was stopped by adding 10 ⁇ L of Kinase-Glo reagent to each well.
  • Percent inhibition rate 100 - (highest RLU - sample RLU) / (highest RLU - lowest RLU) * 100
  • Highest RLU refers to the RLU value of the enzyme-free control well and "lowest RLU” refers to the RLU value of the DMSO control well.
  • the compounds of the examples all showed inhibitory activity against the kinase PI3K ⁇ , and in particular, the compounds 1 to 6 had high activity: the IC 50 for the kinase PI3K ⁇ was about 10 nM or less.
  • a PI3K gamma solution was prepared in 1X kinase buffer at a concentration four times the final concentration at the time of the test.
  • test plate was added with 2.5 ⁇ L of kinase solution per well, except for the control well without enzyme (plus 2.5 ⁇ L of 1 ⁇ kinase buffer instead).
  • a substrate solution containing PIP2 substrate and ATP was prepared in 1X Kinase Reaction Buffer, and the concentration of each reagent was twice the final concentration at the time of the test.
  • test plate was started by adding 5 ⁇ L of the substrate solution to each well.
  • test plates were incubated for 1 hour at room temperature.
  • Percent inhibition rate 100 - (highest RLU - sample RLU) / (highest RLU - lowest RLU) * 100
  • Highest RLU refers to the RLU value of the enzyme-free control well and "lowest RLU” refers to the RLU value of the DMSO control well.
  • the compounds of Examples 1, 4, and 6 all showed high inhibitory activity against kinase PI3K ⁇ , and their IC 50 was below 10 nM.
  • Test 3 Inhibition of compound phosphorylation of Akt by Serine 473 on MCF-7 cells
  • test compound was formulated into an 8 mM solution in DMSO and then diluted 3 times to 9 concentrations.
  • Torin 1 is used as an internal reference compound; the final concentration of DMSO is 0.5%.
  • This assay is a cell-level activity assay that demonstrates the inhibitory activity of the compound on PI3K on living cells.
  • Compound 6 only added one cyano group in the molecular structure, but its cell activity was significantly higher than the latter, and the IC 50 values differed by more than 4 times. It can be seen that the introduction of the cyano group in the compound 6 has an unexpected and non-obvious effect of increasing cell activity.
  • Test 4 Inhibition of compound phosphorylation of Akt by Serine 473 on MCF-7 cells
  • test method the same as test 3.
  • the activity test at the cell level showed that the inhibitory activities of the compounds 1 to 4 on PI3K in the living cells were significantly better than those in the compound of Comparative Example 1, and the IC 50 values differed by 3 to 5 times.
  • the compounds 1-4 of the present invention all introduce a cyano group at the same substitution position on the fused ring, and these results further indicate that the introduction of the cyano group in the compounds 1-4 has a remarkable effect of enhancing cell activity. .
  • Test 5 CellTiter-Glo (CTG) assay for cell proliferation in U87MG cell lines
  • the inhibitory activity of the compounds on cell proliferation was determined in U87MG cells, and a positive control, a blank control and a vehicle control were set for each cell.
  • the mother liquor was stored frozen at -20 °C.
  • Cells in the logarithmic growth phase were harvested and cell counts were performed using a platelet counter. Cell viability was measured by trypan blue exclusion method to ensure that the viability of each cell line was above 96%.
  • the cell concentration was adjusted by diluting with the culture solution, and 90 ⁇ L of the cell suspension was added to a 96-well plate to bring the cell density to the specified concentration.
  • the cells in the 96-well plates were incubated overnight at 37 ° C, 5% CO 2 , 95% humidity.
  • the test compound was dissolved in a corresponding solvent to form a stock solution and subjected to gradient dilution to obtain a 10-fold solution; a 10-fold solution of the positive drug was also prepared.
  • the cells in the treated 96-well plates were further cultured at 37 ° C, 5% CO 2 , 95% humidity for 96 hours, and subjected to CTG analysis.
  • the CTG reagent was thawed and the cell plates were equilibrated to room temperature for 30 minutes.
  • the cells were lysed by shaking on an orbital shaker for 2 minutes.
  • the cell plates were placed at room temperature for 10 minutes to stabilize the cold light signal.
  • Lum cold light value
  • This experiment is a cell level proliferation inhibition test, which embodies the inhibitory activity of the compound on cell proliferation.
  • the compound of Comparative Example 1 had no cyano group on the fused ring, and the compound of Comparative Example 2 and the compound of Comparative Example 3 replaced the cyano group with other groups at the same substitution position.
  • the results showed that the inhibitory activity of compound 6 on U87MG cells was significantly higher than that of all the compounds in comparison, and the IC 50 values were more than three times. It can be seen that the introduction of the cyano group in the compound 6 has an unexpected and non-obvious effect of increasing cell activity.
  • Test 6 CellTiter-Glo assay for inhibition of proliferation of 5 human tumor cell lines
  • NEAA is a non-essential amino acid
  • This experiment compared the proliferation inhibitory activity of Compound 6 and Comparative Example 1 against different tumor cell lines.
  • Compound 6 only added one cyano group in the molecular structure, but its cell proliferation inhibitory activity was significantly higher than that of the latter.
  • the two were in T47D, MCF7, A2780, NCI-H1975, A549, etc.
  • the IC 50 values on the cell lines differed by about 3 to 6 times. It can be seen that the introduction of the cyano group in the compound 6 has an unexpected and non-obvious effect of increasing cell activity.
  • Test 7 CellTiter-Glo assay for inhibition of proliferation of 5 human tumor cell lines
  • This experiment tested the proliferation inhibitory activity of compound 6 on different tumor cell lines.
  • the results showed that compound 6 was on various tumor cell lines such as BT20, BT474, SK-OV-3, NCI-H460 and NCI-H1650. Both showed excellent proliferation inhibitory activity with IC 50 values below 1 ⁇ M.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明涉及一种稠环化合物、其制备方法和应用及其中间体化合物。该化合物可以用作磷脂酰肌醇3-激酶的抑制剂。

Description

一种稠环化合物、其制备方法和应用及其中间体化合物 技术领域
本发明属于药物化学领域，具体而言，涉及一种稠环化合物以及它们的制备方法和其作为磷脂酰肌醇3-激酶抑制剂的用途。本发明同时涉及该化合物的中间体化合物。
背景技术
恶性肿瘤是一类严重威胁人类健康的重大疾病。临床上应用的大部分小分子抗肿瘤药物或是影响DNA的结构和功能、或是干扰核酸的合成与修复、或是抑制某种管家蛋白(如微管蛋白)的合成与功能，因而具有普遍的细胞毒性，临床应用中存在毒副作用大、易产生耐药性等缺点。
分子靶向药物的出现是抗肿瘤药物发展史上的里程碑式事件。这类药物作用于肿瘤细胞的特定分子靶标，对肿瘤细胞的增殖、侵袭、转移等恶性生物学行为具有抑制作用从而产生抗肿瘤作用。这些分子靶标在肿瘤组织中特异性表达或过高表达，而在正常组织中不表达或常规表达，因此分子靶向药物一般能够选择性地杀伤肿瘤细胞或抑制其生长增殖，而对正常细胞的毒性作用较小。寻找和开发新的分子靶向药物，代表了肿瘤精准治疗的发展方向。
大量研究结果表明，人体内肿瘤的发生和发展与PI3K/Akt/mTOR信号通路的调节和异常活化密切相关。磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase，PI3K)是磷脂激酶家族中的一个重要成员，它能特异性地催化磷脂酰肌醇3位羟基的磷酸化，是PI3K/Akt/mTOR信号通路上的关键位点，对细胞的增殖、生存和代谢等起关键作用。
PI3K依其结构特征、活化机制和底物选择的不同，可分为I、II、III三种类型。由于迄今为止功能研究最多的是I型PI3K，因此通常所说的PI3K就是指I型PI3K。I型PI3K依其调节亚基和上游调节分子的不同又分为IA和IB两个亚类。IA亚类PI3K可被各种受体酪氨酸激酶和Ras激活，包括PI3Kα、PI3Kβ和PI3Kδ三个亚型，分别由各自的催化亚基p110与调节亚基p85组成。IB亚类PI3K主要被G蛋白耦联受体激活，仅PI3Kγ一个亚型，它由催化亚基p110γ和调节亚基p101或p84组成。
以上四种亚型的PI3K在体内的分布有所不同：PI3Kα和PI3Kβ在各种器官均有表达，而PI3Kδ和PI3Kγ主要分布在骨髓细胞中。四种亚型PI3K的功能也有所不同：PI3Kα与肿瘤的关系最为密切，编码p110α的基因PIK3CA的突变、扩增和过表达广泛存在于多种恶性肿瘤中，因此，PI3Kα被认为在肿瘤的发生和发展中起着重要的作用，此外还有研究表明PI3Kα与胰岛素信号传导和葡萄糖代谢有关；PI3Kβ可激活血小板，因此在血栓性疾病的发展中起着重要的作用，此外，PI3Kβ近年来被报道在PTEN缺失的癌症患者中起着比PI3Kα还重要的作用；PI3Kδ和PI3Kγ与炎症和免疫等有密切的关系。
由于PI3K与癌症等疾病的密切关系，以PI3K为靶标的抑制剂的开发受到了国际制药界的高度重视。
第一个报道的PI3K合成抑制剂是LY294002，它对I类PI3K的抑制活性达微摩尔级，对PI3Kα的IC50值达0.63μM，在动物体内实验中也显示了一定的抗肿瘤作用，但由于安全性、稳定性等原因,未能进入临床试验。
Figure PCTCN2017080521-appb-000001
随着PI3K晶体结构的阐明，PI3K抑制剂的药物设计和发现得到了快速推进，目前已有多种具有不同结构的PI3K抑制剂陆续被报道，包括嘧啶类、噻吩并嘧啶类、喹唑酮类、双烯酮类、咪唑并喹啉类、咪唑并吡啶类、苯并吡啶类及其他分子骨架化合物。
嘧啶类、噻吩并嘧啶类及其衍生物：
GDC-0941是由Genentech公司开发的一种泛PI3K抑制剂，其对p110α、p110β、p110δ、p110γ的IC50分别为3nM、33nM、3nM、75nM。GDC-0941的临床I期试验结果表明其在乳腺癌、卵巢癌及黑色素瘤患者中具有较好的抗肿瘤活性，并且耐受性较好。
Figure PCTCN2017080521-appb-000002
BKM120是由Novartis公司开发的一种2,4-双吗啉取代嘧啶类的泛PI3K抑制剂，其对p110α、p110β、p110δ、p110γ的IC50分别为52nM、166nM、116nM、262nM，用于治疗转移性乳腺癌的研究正处于III期临床。
Figure PCTCN2017080521-appb-000003
喹唑酮类及其类似物：
IC-87114是一种选择性PI3Kδ抑制剂，其对PI3Kδ的IC50为0.5μM，较之于对PI3Kα、PI3Kβ、PI3Kγ的抑制活性，选择性高达58到100倍。
Figure PCTCN2017080521-appb-000004
CAL-101(Idelalisib)也是一种选择性PI3Kδ抑制剂，其对p110δ的IC50为2.5nM，较之于对p110α、p110β、p110γ的抑制活性，选择性高达40到300倍。基于PI3Kδ在炎症中的重要作用，最初关于CAL-101的药理研究集中在其抗炎和治疗自身免疫性疾病上。后来基于PI3Kδ主要在骨髓细胞中表达，对CAL-101的药理研究转移到对白血病和淋巴瘤的治疗上。在多个临床试验结果的支持下，美国FDA于2014年7月批准了Idelalisib三个适应症：与利妥昔单抗(Rituxan)联合治疗复发的慢性淋巴细胞白血病、作为单药治疗复发性滤泡B细胞非霍奇金淋巴瘤和复发性小淋巴细胞淋巴瘤。
Figure PCTCN2017080521-appb-000005
双烯酮类及其类似物：
PX866是一种双烯酮类的泛PI3K抑制剂，其对PI3Kα、PI3Kδ、PI3Kγ的IC50分别为6nM、3nM、9nM。目前处于I/II期临床试验中。
Figure PCTCN2017080521-appb-000006
咪唑并喹啉类、咪唑并吡啶类及其类似物：
BAY80-6946是Bayer公司开发的一种高活性的泛PI3K抑制剂，其对PI3Kα、PI3Kβ、PI3Kδ、PI3Kγ的IC50分别为0.5nM、3.7nM、0.7nM、6.4nM。目前处于II期临床试验中。
Figure PCTCN2017080521-appb-000007
PIK75为咪唑并吡啶衍生物，是一种选择性PI3Kα抑制剂，其对p110α、p110β、p110δ、p110γ的IC50分别为5.8nM、1.3μM、0.51μM、76nM。
Figure PCTCN2017080521-appb-000008
苯并吡啶类及其衍生物：
CN103788071A要求保护下述通式化合物，部分化合物显示了较高的体外增殖抑制作用。
Figure PCTCN2017080521-appb-000009
其他类：
XL147是一种选择性PI3K抑制剂，其对PI3Kα、PI3Kβ、PI3Kδ、PI3Kγ的IC50分别为39nM、383nM、36nM、23nM。
Figure PCTCN2017080521-appb-000010
PCT/US2009/045713公开了下述通式化合物或其药学上可接受的盐：
Figure PCTCN2017080521-appb-000011
具体公开了137个化合物，包括：
N-(2-氯-5-(3-甲氧基-6喹啉基)-3-吡啶基)-4-氟苯磺酰胺；
N-(2-氯-5-(4-苯氧基-6-喹啉基)-3-吡啶基)甲磺酰胺；
N-(2-氯-5-(6-喹啉基)-3-吡啶基)-4-氟苯磺酰胺)；
N-(2-甲氧基-5-(4-吗啉基喹啉-6-基)吡啶-3-基)甲磺酰胺等。
PI3K小分子抑制剂作为新一类的分子靶向药物，潜力巨大，前景广阔。因此，需要更多结构新颖、生物活性高、成药性良好的PI3K抑制剂，用于肿瘤的靶向治疗，以及用于抗炎或治疗自身免疫性疾病。
发明内容
本发明提供了一种新型的PI3K选择性抑制剂。具体而言，本发明提供了一种由以下通式(I)表示的稠环化合物或其药学上可接受的盐：
Figure PCTCN2017080521-appb-000012
其中，X表示O、S、NR、SO2或NH2Cl，R选自H、C1-6的烷基，优选C1-4的烷基，R1、R2、R3、R4、R5、R6、R7、R8为独立地选自由H、C1-6的烷基、或具有OH取代基的C1-6的烷基，优选为H、C1-4的烷基或具有OH取代基的C1-4的烷基。R1、R2、R3、R4、R5、R6、R7、R8为独立地选自由H、C1-6的烷基、或具有OH取代基的C1-6的烷基。
具体而言，本发明可以选自以下化合物或其药学上可接受的盐：
Figure PCTCN2017080521-appb-000013
Figure PCTCN2017080521-appb-000014
Figure PCTCN2017080521-appb-000015
Figure PCTCN2017080521-appb-000016
Figure PCTCN2017080521-appb-000017
Figure PCTCN2017080521-appb-000018
Figure PCTCN2017080521-appb-000019
其中，
Figure PCTCN2017080521-appb-000020
指的是
Figure PCTCN2017080521-appb-000021
Figure PCTCN2017080521-appb-000022
指的是
Figure PCTCN2017080521-appb-000023
Figure PCTCN2017080521-appb-000024
指的是
Figure PCTCN2017080521-appb-000025
Figure PCTCN2017080521-appb-000026
指的是
Figure PCTCN2017080521-appb-000027
Figure PCTCN2017080521-appb-000028
指的是
Figure PCTCN2017080521-appb-000029
Figure PCTCN2017080521-appb-000030
指的是
Figure PCTCN2017080521-appb-000031
Figure PCTCN2017080521-appb-000032
指的是
Figure PCTCN2017080521-appb-000033
Figure PCTCN2017080521-appb-000034
指的是
Figure PCTCN2017080521-appb-000035
Figure PCTCN2017080521-appb-000036
指的是
Figure PCTCN2017080521-appb-000037
Figure PCTCN2017080521-appb-000038
指的是
Figure PCTCN2017080521-appb-000039
Figure PCTCN2017080521-appb-000040
指的是
Figure PCTCN2017080521-appb-000041
其他化合物的结构可以参照以上化合物进行解释。
本发明同时提供了包含上述化合物或其药学上可接受的盐的药物组合物。本发明稠环化合物或其药学上可接受的盐或所述的药物组合物可以有效地抑制PI3K的活性。
所述化合物或其药学上可接受的盐，或所述药物组合物作为PI3K抑制剂，可以作为抗肿瘤药物用于脑癌、头颈癌、食管癌、肺癌、肝癌、胃癌、肾癌、胰腺癌、前列腺癌、结直肠癌、卵巢癌、乳腺癌、甲状腺癌、皮肤癌、白血病、骨髓异常增生综合症、肉瘤、骨肉瘤或横纹肌瘤等肿瘤的治疗。
所述化合物或其药学上可接受的盐，或所述药物组合物作为PI3K抑制剂，可以作 为抗炎药物用于慢性阻塞性肺病、哮喘等疾病的治疗。
所述化合物或其药学上可接受的盐，或所述药物组合物作为PI3K抑制剂，可以作为治疗自身免疫性疾病(例如风湿性关节炎、银屑病、系统性红斑狼疮)的药物。
本发明的化合物可以通过以下方法制备。所述方法包括使式(III’)的化合物与式(IV)的化合物反应，得到式(I)的化合物的步骤：
Figure PCTCN2017080521-appb-000042
其中X、R1、R2、R3、R4、R5、R6、R7和R8如前文所定义。
所述方法还包括使式(II)的化合物与式(III)的化合物反应得到式(III’)的化合物的步骤，
Figure PCTCN2017080521-appb-000043
本发明还提供了一种式(I)的化合物的制备方法，所述方法包括使式(IV’)的化合物与式(III)的化合物反应，得到式(I)的化合物的步骤：
Figure PCTCN2017080521-appb-000044
其中X、R1、R2、R3、R4、R5、R6、R7和R8如前文所定义。
所述方法还包括使式(II)的化合物与式(IV)的化合物反应得到式(IV’)的化合物的步骤，
Figure PCTCN2017080521-appb-000045
通过上述方法，能够以高产率得到所需化合物。
另外，本发明还提供了一种式(III’)的化合物或式(IV’)的化合物，
Figure PCTCN2017080521-appb-000046
其中X、R1、R2、R3、R4、R5、R6、R7和R8如前文所定义。式(III’)的化合物或式(IV’)的化合物可以用作合成式(I)的化合物的原料或中间体。
发明人通过大量探索及试验验证，在稠环上特异性的引入氰基，与没有氰基的化合物以及氰基用其他取代基代替的化合物相比，本发明得到的上述化合物或其药学上可接受的盐具有出乎预料的、非显而易见对PI3K的抑制活性。
具体实施方式
以下实施例中使用的原料、试剂均为市购。
实施例1：N-(5-(3-氰基-4-硫代吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物3)
Figure PCTCN2017080521-appb-000047
步骤1：N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺
Figure PCTCN2017080521-appb-000048
将6-溴-4-氯喹啉-3-甲腈(140mg，0.6mmol)，N-(2-甲氧基-5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-3-基)甲磺酰胺(203mg，0.618mmol，1.03eq)和2N碳酸钾水溶液(0.9mL，1.8mmol，3.0eq)于二氧六环(6mL)中的混合物脱气，并加入二(三苯基膦)二氯化钯(21mg，0.03mmol，0.05eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在100℃下氩气气氛中搅拌7小时。将反应混合物冷却至室温，用水(30mL)稀释，乙酸乙酯萃取(30mL×3)。合并有机层并用食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经快速柱色谱(硅胶，二氯甲烷/甲醇＝350:1，v/v)纯化得到黄色固体(82mg，产率35％)。
1H NMR(400MHz,DMSO-d6)δ9.47(s,1H),9.21(s,1H),8.55(d,J＝2.2Hz,1H),8.44(d,J＝1.6Hz,1H),8.39(dd,J＝8.8,1.6Hz,1H),8.29(d,J＝8.8Hz,1H),8.09(d,J＝2.2Hz,1H),4.01(s,3H),3.13(s,3H).
步骤2：N-(5-(3-氰基-4-硫代吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
Figure PCTCN2017080521-appb-000049
将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺(156mg,0.4mmol)溶于NMP(5mL)中，加入碳酸钾(83mg,0.6mmol,1.5eq)和硫代吗啉(62mg,0.6mmol,1.5eq)。将产生的反应混合物在100℃下搅拌2小时。冷却反应混合物，用水(30mL)稀释反应混合物，乙酸乙酯(20mL×3)萃取。合并有机层，依次用水(20mL)和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经快速柱色谱(硅胶，DCM/MeOH＝100:1，v/v)纯化得白色固体(63mg，产率为34.6％)。
1H NMR(400MHz,DMSO-d6)δ9.44(s,1H),8.81(s,1H),8.50(s,1H),8.25–8.15(m,2H),8.13–8.03(m,2H),4.01(s,3H),3.88(br s,4H),3.13(s,3H),2.95(br s,4H).
实施例2：
N-(5-(3-氰基-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物6)
Figure PCTCN2017080521-appb-000050
步骤1：6-溴-4-吗啉基喹啉-3-甲腈
Figure PCTCN2017080521-appb-000051
将6-溴-4-氯-喹啉-3-甲腈(535mg，2.0mmol)和吗啉(523mg，6.0mmol，3eq)于二氧六环(7.5mL)中、在100℃下搅拌。反应结束后真空浓缩，浓缩液用水(20mL)稀释，乙酸乙酯(20mL×3)萃取。合并有机层，依次用水(20mL)和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经快速柱色谱(硅胶，石油醚/乙酸乙酯＝3:1，v/v)纯化得到黄色固体(556mg，产率为87.4％)。
1H NMR(400MHz,DMSO-d6)δ8.81(s,1H),8.20(d,J＝2.0Hz,1H),7.99(dd,J＝8.8,2.0Hz,1H),7.93(d,J＝8.8Hz,1H),3.87(t,J＝4.4Hz,4H),3.68(t,J＝4.4Hz,4H).
步骤2：N-(5-(3-氰基-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
Figure PCTCN2017080521-appb-000052
将6-溴-4-吗啉基喹啉-3-甲腈(96mg，0.3mmol)，N-(2-甲氧基-5-(4,4,5,5-四甲基 -1,3,2-二氧杂环戊硼烷-2-基)吡啶-3-基)甲磺酰胺(118mg，0.36mmol，1.2eq)和2N碳酸钾水溶液(0.45mL，3.0eq)于二氧六环(4mL)中的混合物脱气，然后加入[1,1'-双(二苯基膦基)二茂铁]二氯化钯(11mg，0.015mmol，0.05eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在100℃下氩气气氛中搅拌5小时。将反应混合物冷却至室温，用(30mL)水稀释，用乙酸乙酯(30mL×3)萃取。合并有机层并用食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，浓缩，经快速柱色谱(硅胶，二氯甲烷/甲醇＝200:1，v/v)纯化得到白色固体(104mg，产率为78.9％)。
1H NMR(400MHz,DMSO-d6)δ9.45(s,1H),8.79(s,1H),8.48(d,J＝2.4Hz,1H),8.20(d,J＝1.6Hz,1H),8.18(dd,J＝8.6,2.0Hz,1H),8.08(d,J＝8.6Hz,1H),8.05(d,J＝2.4Hz,1H),4.00(s,3H),3.91(t,J＝4.4Hz,4H),3.73(t,J＝4.4Hz,4H),3.13(s,3H).
实施例3：N-(5-(3-氰基-4-(2,6-二甲基吗啉基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物2)
Figure PCTCN2017080521-appb-000053
制备方法如实施例1，步骤2)中将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺与
Figure PCTCN2017080521-appb-000054
在室温下反应。
1H NMR(400MHz,DMSO)δ9.42(s,1H),8.76(s,1H),8.45(d,J＝2.0Hz,1H),8.26–8.14(m,2H),8.12–8.01(m,2H),4.00(s,3H),3.99–3.90(m,2H),3.85(d,J＝12.4Hz,2H),3.29–3.18(m,2H),3.13(s,3H),1.17(d,J＝6.1Hz,6H).
实施例4：N-(5-(3-氰基-4-(4-甲基哌嗪-1-基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物1)
Figure PCTCN2017080521-appb-000055
制备方法如实施例1，其中步骤2)将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺与N-甲基哌嗪在室温下反应。
1H NMR(400MHz,DMSO-d6)δ9.42(s,1H),8.75(s,1H),8.44(d,J＝2.2Hz,1H),8.19(d,J＝1.6Hz,1H),8.16(dd,J＝8.8,1.6Hz,0H),8.06(d,J＝8.8Hz,1H),8.04(d,J＝2.2Hz,1H),4.00(s,3H),3.72(br s,4H),3.13(s,3H),2.65(br s,4H),2.31(s,3H).
实施例5：(R)-N-(5-(3-氰基-4-(3-甲基吗啉基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰 胺(化合物4)
Figure PCTCN2017080521-appb-000056
制备方法如实施例1，其中步骤2)将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺与
Figure PCTCN2017080521-appb-000057
在同样条件下反应。
1H NMR(400MHz,DMSO-d6)δ9.45(s,1H),8.89(s,1H),8.49(d,J＝2.4Hz,1H),8.33(d,J＝1.6Hz,1H),8.23(dd,J＝8.8,2.0Hz,1H),8.13(d,J＝8.8Hz,1H),8.06(d,J＝2.4Hz,1H),4.18–4.05(m,2H),4.01(s,3H),4.00–3.93(m,1H),3.92–3.82(m,1H),3.82–3.73(m,1H),3.63(dd,J＝10.8,4.0Hz,1H),3.30–3.26(m,1H),3.13(s,3H),1.11(d,J＝6.4Hz,3H).
实施例6：(S)-N-(5-(3-氰基-4-(3-甲基吗啉基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物5)
Figure PCTCN2017080521-appb-000058
制备方法如实施例1，其中步骤2)将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺与
Figure PCTCN2017080521-appb-000059
在同样条件下反应。
1H NMR(400MHz,DMSO-d6)δ9.45(s,1H),8.89(s,1H),8.49(d,J＝2.4Hz,1H),8.32(d,J＝1.6Hz,1H),8.23(dd,J＝8.8,2.0Hz,1H),8.13(d,J＝8.8Hz,1H),8.06(d,J＝2.4Hz,1H),4.18–4.05(m,2H),4.01(s,3H),4.00–3.93(m,1H),3.93–3.72(m,2H),3.63(dd,J＝11.2,4.0Hz,1H),3.30–3.26(m,1H),3.14(s,3H),1.11(d,J＝6.4Hz,3H).
实施例7：N-(5-(3-氰基-4-((2S,6R)-2,6-甲基吗啉基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物56)
Figure PCTCN2017080521-appb-000060
制备方法如实施例1，其中，步骤2)将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶 -3-基)甲烷磺酰胺与
Figure PCTCN2017080521-appb-000061
在同样条件下反应。
1H NMR(500MHz,DMSO-d6)δ9.42(s,1H),8.77(s,1H),8.45(s,1H),8.22–8.15(m,2H),8.11–7.99(m,2H),4.00(s,3H),3.98–3.91(m,2H),3.85(d,J＝12.5Hz,2H),3.23(t,J＝11.5Hz,2H),3.13(s,3H),1.17(d,J＝6.0Hz,6H).
实施例8：N-(5-(3-氰基-4-(1,1-二氧代硫代吗啉基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物67)
Figure PCTCN2017080521-appb-000062
制备方法如实施例1，其中，步骤2)将N-(5-(4-氯-3-氰基喹啉-6-基)-2-甲氧基吡啶-3-基)甲烷磺酰胺与
Figure PCTCN2017080521-appb-000063
在同样条件下反应。
1H NMR(400MHz,DMSO-d6)δ9.44(s,1H),8.88(s,1H),8.59(d,J＝2.0Hz,1H),8.33(d,J＝1.6Hz,1H),8.20(dd,J＝8.8,1.6Hz,1H),8.13(d,J＝8.8Hz,1H),8.08(d,J＝2.0Hz,1H),4.05(br s,4H),4.01(s,3H),3.56(br s,4H),3.13(s,3H).
实施例9：N-(5-(3-氰基-4-(哌嗪-1-基)喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺(化合物68)
Figure PCTCN2017080521-appb-000064
按照实施例1的方法制备中间体4-(3-氰基-6-(6-甲氧基-5-(甲磺酰胺基)吡啶-3-基)喹啉-4-基)哌嗪-1-碳酸叔丁酯，再使用盐酸甲醇溶液脱除Boc基团得到化合物68。
1H NMR(400MHz,DMSO-d6)δ9.63(br s,2H),9.46(s,1H),9.00(s,1H),8.54(d,J＝2.2Hz,1H),8.27(dd,J＝8.8,1.6Hz,1H),8.23(d,J＝1.6Hz,1H),8.17(d,J＝8.8Hz,1H),8.08(d,J＝2.2Hz,1H),4.01(br s,7H),3.46(br s,4H),3.14(s,3H).
实施例10其余化合物制备方法如实施例1，差别在于步骤2中采用下述表1中的原料替换硫代吗啉。
表1
Figure PCTCN2017080521-appb-000065
Figure PCTCN2017080521-appb-000066
Figure PCTCN2017080521-appb-000067
Figure PCTCN2017080521-appb-000068
对比例化合物：
对比例1化合物：N-(2-甲氧基-5-(4-吗啉基喹啉-6-基)吡啶-3-基)甲磺酰胺
Figure PCTCN2017080521-appb-000069
步骤1：4-(6-溴喹啉-4-基)吗啉
将6-溴-4-氯-喹啉(243mg，1.0mmol)和吗啉(523mg，6.0mmol，6eq)于二氧 六环(3mL)中的溶液在110℃下搅拌48小时。在真空中浓缩反应混合物冷却至室温，用水(20mL)稀释，用乙酸乙酯(20mL×3)萃取。用水(20mL)和食盐水(20mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，二氯甲烷/甲醇＝100:1)纯化得到产物为淡黄色油状物(211mg，产率为72％)。
1H NMR(400MHz,DMSO-d6)δ8.75(d,J＝5.0Hz,1H),8.15(d,J＝2.2Hz,1H),7.92(d,J＝8.8Hz,1H),7.84(dd,J＝8.8,2.2Hz,1H),7.07(d,J＝5.0Hz,1H),3.94–3.82(m,4H),3.20–3.09(m,4H).
步骤2：N-(2-甲氧基-5-(4-吗啉基喹啉-6-基)吡啶-3-基)甲磺酰胺
将6-溴-4-吗啉基喹啉(105mg，0.36mmol)，N-(2-甲氧基-5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-3-基)甲磺酰胺(141mg，0.43mmol，1.2eq)和2N碳酸钾水溶液(0.54mL，1.08mmol，3.0eq)于二氧六环(4mL)中的混合物脱气，然后加入[1,1'-双(二苯基膦基)二茂铁]二氯化钯(13mg，0.018mmol，0.05eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在100℃下氩气气氛中搅拌5小时。将反应混合物冷却至室温，用(30mL)水稀释，用乙酸乙酯(30mL×3)萃取。用食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，二氯甲烷/甲醇＝60:1)纯化得到产物为淡黄色固体(114mg，产率为76.4％)。
1H NMR(400MHz,DMSO-d6)δ9.42(s,1H),8.72(d,J＝4.9Hz,1H),8.45(d,J＝2.0Hz,1H),8.16(d,J＝1.2Hz,1H),8.10–7.98(m,3H),7.04(d,J＝5.0Hz,1H),4.00(s,3H),3.95–3.86(m,4H),3.27–3.19(m,4H),3.13(s,3H).
对比例2化合物：N-(5-(3-(二氟甲基)-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
Figure PCTCN2017080521-appb-000070
步骤1：2-(((4-溴苯基)氨基)亚甲基)丙二酸二乙酯
将4-溴苯胺(10.32g，60mmol)和2-乙氧基亚甲基丙二酸二乙酯(12.96g，60mmol，1.0eq)的混合物在150℃下搅拌3小时。将反应混合物冷却至室温，用正己烷(100mL)稀释。抽滤收集产生的白色固体，用正己烷(50mL×2)洗涤，真空中干燥得到产物为白色固体(19.2g，产率为93.9％)，不作进一步纯化直接用于下一步。
步骤2：6-溴-4-羟基喹啉-3-羧酸乙酯
将搅拌中的2-(((4-溴苯基)氨基)亚甲基)丙二酸二乙酯(10g，29.2mmol)和二苯醚(100mL)的混合物加热至回流。在其加热至回流的过程中，将氮气缓慢鼓泡进入反应混合物在，然后在回流过程中在溶剂上方轻轻地吹氮气。将反应混合物回流1小时，然后在氮气流下冷却至室温。用正己烷(100mL)稀释反应混合物，抽滤收集产生的白色固体，用正己烷(50mL×3)洗涤，真空中干燥，得到产物为白色固体(7.8g，产率为90％)，不作进一步纯化直接用于下一步。
步骤3：6-溴-4-氯喹啉-3-羧酸乙酯
将6-溴-4-羟基喹啉-3-羧酸乙酯(10g)于三氯氧磷(100mL)中的混合物在120℃ 下回流3小时。将反应混合物在真空中蒸发除去三氯氧磷。将残留物倒入冰水中，用饱和碳酸氢钠水溶液调节pH值至6。用乙酸乙酯(100mL×3)萃取产生的混合物。用食盐水(100mL×2)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝80:1)纯化得到产物为白色固体(10g，产率为94.1％)。
1H NMR(400MHz,DMSO-d6)δ9.19(s,1H),8.53(d,J＝2.0Hz,1H),8.14(dd,J＝8.9,2.0Hz,1H),8.11(d,J＝8.9Hz,1H),4.44(q,J＝7.1Hz,2H),1.39(t,J＝7.1Hz,3H).
步骤4：6-溴-4-氯喹啉-3-甲醛
在-80℃下氩气气氛中，向6-溴-4-氯喹啉-3-羧酸乙酯(1.573g，5mmol)与无水二氯甲烷(24mL)中的溶液中滴加DIBAL-H溶液(1M甲苯溶液，8mL，8mmol，1.6eq)。将产生的反应混合物在-80℃下搅拌1小时，然后用甲醇淬灭，用水(20mL)稀释，用二氯甲烷(20mL×3)萃取。用水(30mL)和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝15:1)纯化得到产物为白色固体(1.075g，产率为79.5％)。
1H NMR(400MHz,DMSO-d6)δ10.54(s,1H),9.18(s,1H),8.57(d,J＝2.0Hz,1H),8.19(dd,J＝8.8,2.0Hz,1H),8.12(d,J＝8.8Hz,1H).
步骤5：6-溴-4-吗啉基喹啉-3-甲醛
将6-溴-4-氯喹啉-3-甲醛(324mg，1.2mmol)和吗啉(314mg，0.314mL，3.6mmol，3.0eq)于二氧六环(2.5mL)中的混合物在110℃下搅拌2.5小时。用水(30mL)稀释反应混合物，用乙酸乙酯(20mL×3)萃取。用水(30mL)和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝5:1)纯化得到产物为黄色固体(338mg，产率为87.7％)。
1H NMR(400MHz,DMSO-d6)δ10.42(s,1H),8.97(s,1H),8.34(d,J＝2.0Hz,1H),7.97(dd,J＝8.8,2.0Hz,1H),7.93(d,J＝8.8Hz,1H),3.93–3.84(m,4H),3.68–3.57(m,4H).
步骤6：4-(6-溴-3-(二氟甲基)喹啉-4-基)吗啉
在0℃下向6-溴-4-吗啉基喹啉-3-甲醛(321mg，1.0mmol)和乙醇(11.6μL，0.2mmol，0.2eq)于二氯甲烷(5mL)中的溶液中加入二乙胺基三氟化硫(645mg，526μL，4.0mmol，4eq)。将产生的反应混合物在0℃下搅拌0.5小时，在室温下搅拌3小时。将反应混合物倒入冰水中(20mL)，用饱和碳酸氢钠水溶液调节pH至7，用二氯甲烷(20mL×3)萃取。用水(30mL)和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝10:1～4:1)纯化得到产物为黄色固体(296m g，产率为86.3％)。
1H NMR(400MHz,DMSO-d6)δ8.98(s,1H),8.36(d,J＝1.8Hz,1H),8.01(d,J＝9.0Hz,1H),7.97(dd,J＝9.0,1.8Hz,1H),7.53(t,J＝54.4Hz,1H),3.91–3.82(m,4H),3.37–3.24(m,4H).
步骤7：N-(5-(3-(二氟甲基)-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
将4-(6-溴-3-(二氟甲基)喹啉-4-基)吗啉(86mg，0.25mmol)，N-(2-甲氧基-5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-3-基)甲磺酰胺(99mg，0.30mmol，1.4eq)和2N碳酸钾水溶液(0.375mL，0.75mmol，3eq)于二氧六环(4mL)中的混合物脱气，然后加入PdCl2(dppf)(13mg，0.0175mmol，0.07eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在100℃下氩气气氛中搅拌5小时。将反应混合物冷却至室温，用水(30mL)稀释，用乙酸乙酯萃取(30mL×3)。用饱和食盐水(30mL)洗涤 合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，DCM/CH3OH＝100:1)纯化得到产物为黄色固体(95mg，产率为81.8％)。
1H NMR(400MHz,DMSO-d6)δ9.46(s,1H),8.94(s,1H),8.49(d,J＝2.4Hz,1H),8.38(d,J＝1.2Hz,1H),8.17(dd,J＝8.8,1.6Hz,1H),8.14(d,J＝8.8Hz,1H),8.07(d,J＝2.4Hz,1H),7.54(t,J＝54.6Hz,1H),4.01(s,3H),3.93–3.85(m,4H),3.43–3.35(m,4H),3.13(s,3H).
对比例3化合物：N-(5-(3-(1,1-二氟乙基)-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
Figure PCTCN2017080521-appb-000071
步骤1：6-溴-3-碘喹啉-4-酚
将6-溴喹啉-4-酚(2.15g，9.6mmol)和N-碘代丁二酰亚胺(2.6g，11.6mmol，1.2eq)于冰醋酸(25mL)中的混合物在50℃下搅拌1小时。将反应混合物倒入水(100mL)中，抽滤收集产生的黄色固体，用甲醇洗涤，在50℃下烘干，得淡黄色固体(2.7g，产率81％)。该粗产物不作进一步纯化直接用于下一步。
1H NMR(400MHz,DMSO-d6)δ12.37(s,1H),8.57(d,J＝4.6Hz,1H),8.19(d,J＝2.4Hz,1H),7.84(dd,J＝8.8,2.4Hz,1H),7.57(d,J＝8.8Hz,1H).
步骤2：6-溴-4-氯-3-碘喹啉
将6-溴-3-碘喹啉-4-酚(2.769g,7.91mmol)于三氯氧磷(25mL)中的混合物回流2.5小时。在真空中浓缩反应混合物，用水(100mL)稀释残留物。用饱和碳酸氢钠水溶液中和产生的混合物，用乙酸乙酯(100mL×3)萃取。用水(100mL)和食盐水(100mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝25:1)纯化得到产物为白色固体(0.93g，产率32％)。
1H NMR(400MHz,CDCl3)δ9.11(s,1H),8.43(d,J＝2.0Hz,1H),7.95(d,J＝8.8Hz,1H),7.84(dd,J＝8.8,2.0Hz,1H).
步骤3：6-溴-4-氯-3-(1-乙氧基乙烯基)喹啉
将6-溴-4-氯-3-碘喹啉(921mg，2.5mmol)，三丁基(1-乙氧基乙烯基)锡(948mg，2.625mmol，1.05eq)和三苯基砷(153mg，0.5mmol，0.2eq)于DMF(10mL)中的混合物脱气，然后加入Pd2(dba)3(57mg，0.0625mmol，0.025eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在60℃下氩气气氛中搅拌6小时。将反应混合物冷却至室温，用水(100mL)稀释，用乙酸乙酯萃取(50mL×3)。用水(50mL×2)和饱和食盐水(50mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝140:1～100:1)纯化得到产物为黄色固体(461mg，产率为59％)。
1H NMR(400MHz,DMSO-d6)δ8.89(s,1H),8.43(d,J＝1.8Hz,1H),8.06(d,J＝8.8Hz,1H),8.03(dd,J＝8.8,1.8Hz,1H),4.75(d,J＝2.8Hz,1H),4.57(d,J＝2.8Hz,1H),3.98(q,J＝7.0Hz,2H),1.34(t,J＝7.0Hz,3H).
步骤4：1-(6-溴-4-氯喹啉-3-基)乙酮
将6-溴-4-氯-3-(1-乙氧基乙烯基)喹啉(461mg，1.47mmol)和2N HCl水溶液(2.0mL)于THF(10mL)中的混合物在室温下搅拌3小时。将适量硅胶加到反应混合物中，真空中除去挥发物。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝10:1)纯化得到产物为黄色固体(300mg，产率为71.5％)。
1H NMR(400MHz,DMSO-d6)δ9.11(s,1H),8.50(d,J＝1.6Hz,1H),8.13–8.07(m,2H),2.76(s,3H).
步骤5：6-溴-4-氯-3-(1,1-二氟甲基)喹啉
在0℃下向1-(6-溴-4-氯喹啉-3-基)乙酮(350mg，1.23mmol)和乙醇(14.3μL，0.246mmol，0.2eq)于二氯甲烷(7mL)中的溶液中加入二乙胺基三氟化硫(793mg，4.92mmol，4eq)。将产生的反应混合物在0℃下搅拌0.5小时，在室温下搅拌过夜。将反应混合物倒入冰水中(30mL)，用饱和碳酸氢钠水溶液调节pH至7，用二氯甲烷(20mL×3)萃取。用水(30mL)和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝20:1)纯化得到产物为黄色固体(156mg，产率为41.3％)。
1H NMR(400MHz,DMSO-d6)δ9.07(s,1H),8.50(d,J＝2.0Hz,1H),8.14–8.08(m,2H),2.18(t,J＝19.2Hz,3H).
步骤6：4-(6-溴-3-(1,1-二氟乙基)喹啉-4-基)吗啉
将6-溴-4-氯-3-(1,1-二氟甲基)喹啉(137mg，0.45mmol)于吗啉(5mL)中的混合物在100℃下搅拌2小时。用水(30mL)稀释反应混合物，用乙酸乙酯(20mL×3)萃取。用水(30mL)和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，石油醚/乙酸乙酯＝20:1～10:1)纯化得到产物为黄色固体(48mg，产率为30％)。回收原料72mg。
1H NMR(400MHz,DMSO-d6)δ9.01(s,1H),8.43(d,J＝2.0Hz,1H),8.06(d,J＝8.8Hz,1H),7.99(dd,J＝8.8,2.0Hz,1H),3.81(t,J＝4.4Hz,4H),3.26(br s,4H),2.19(t,J＝19.2Hz,3H).
步骤7：N-(5-(3-(1,1-二氟乙基)-4-吗啉基喹啉-6-基)-2-甲氧基吡啶-3-基)甲磺酰胺
将4-(6-溴-3-(1,1-二氟乙基)喹啉-4-基)吗啉(45mg，0.126mmol)，N-(2-甲氧基-5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吡啶-3-基)甲磺酰胺(50mg，0.151mmol，1.2eq)和2N碳酸钾水溶液(0.189mL，0.378mmol，3eq)于二氧六环(4mL)中的混合物脱气，然后加入PdCl2(dppf)(6.5mg，0.0088mmol，0.07eq)。将产生的反应混合物脱气并回充氩气(三个循环)，然后在100℃下氩气气氛中搅拌5小时。将反应混合物冷却至室温，用水(30mL)稀释，用乙酸乙酯萃取(30mL×3)。用饱和食盐水(30mL)洗涤合并的有机层，用无水硫酸钠干燥，过滤，浓缩。残留物经快速柱色谱(硅胶，DCM/CH3OH＝110:1)纯化得到产物为黄色固体(36mg，产率为59.7％)。
1H NMR(400MHz,DMSO-d6)δ9.47(s,1H),8.98(s,1H),8.50(d,J＝2.4Hz,1H),8.44(d,J＝1.3Hz,1H),8.20(d,J＝8.8Hz,1H),8.16(dd,J＝8.8,1.6Hz,1H),8.07(d,J＝2.4Hz,1H),4.01(s,3H),3.84(t,J＝4.0Hz,4H),3.33(br s,4H),3.13(s,3H),2.21(t,J＝19.2Hz,3H).
试验部分
PI103指：3-[4-(4-吗啉基吡啶并[3’,2’,4,5]呋喃并[3,2-d]嘧啶-2-基)]苯酚。
试验1  化合物对激酶PI3Kα的抑制活性
一、试验方法
1.制备1×激酶缓冲液
50mM HEPES，pH 7.5
3mM MgCl2
1mM EGTA
100mM NaCl
0.03％CHAPS
2mM DTT
2.待测化合物DMSO溶液的制备及稀释
1)将化合物用100％DMSO溶解，所配浓度为测试中所需最高抑制浓度的100×。转移100μL该溶液至96孔板的一个孔内。例如，如果所需最高抑制剂浓度是1μM，则这一步需制备100μM的化合物DMSO溶液。
2)对于所有的化合物，将管内的化合物转移至96孔储备板的一个孔内，再转移30μL至含有60μL 100％DMSO的下一个孔内，以此方式对化合物进行稀释。
3)在同一张96孔板上，分别加入100μL 100％DMSO至2个空白孔内作为无化合物对照和无酶对照，这张板记为源板。
3.制备中介板
1)从源板转移4μL化合物至一张新的96孔板，以此作为中介板。
2)中介板每孔内加入96μL 1×激酶缓冲液。
3)在振荡器上将中介板内的化合物混合10min。
4.制备测试板
从中介板的每孔中转移2.5μL的液体至一张384孔板，并设立复孔。例如，96孔板的A1转移到384孔板的A1和A2孔。96孔板的A2转移到384孔板的A3和A4，以此类推。
5.PI3Kα激酶反应
制备4×激酶溶液
1)在1×激酶缓冲液中制备PI3Kα溶液，每种试剂的浓度为测试时最终浓度的4倍。
最终浓度：PI3Kα为1.65nM。
2)测试板每孔加2.5μL激酶溶液，不含酶的对照孔除外(加2.5μL 1×激酶缓冲液代替)。
3)振荡测试板。
制备2×底物溶液
1)在1×激酶反应缓冲液中制备含PIP2底物和ATP的底物溶液，每种试剂的浓度均为测试时最终浓度的2倍。
最终浓度：PIP2为50μM，ATP为25μM。
2)测试板每孔加入5μL底物溶液开始反应。
3)振荡测试板
激酶反应
1)测试板加盖于室温孵育1小时。
6激酶检测
1)将Kinase-Glo试剂平衡至室温。
2)测试板每孔加入10μL的Kinase-Glo试剂终止反应。
3)离心短暂混合，在用读板器测量发光强度之前于振荡器上缓慢振摇15min。
7读板
在Synergy上收集数据
8.曲线拟合
1)从Synergy程序中拷贝RLU(相对光单位)值。
2)将RLU值转换成百分抑制率。
百分抑制率＝100-(最高RLU-样本RLU)/(最高RLU-最低RLU)*100
“最高RLU”指无酶对照孔的RLU值，“最低RLU”指DMSO对照孔的RLU值。
二、结果：
化合物编号 IC50(nM)
化合物1 11
化合物2 4.8
化合物3 4.8
化合物67 5.8
化合物68 54
PI103 10
同样的试验方法测得下述化合物的IC50：
化合物编号 IC50(nM)
化合物4 4.3
化合物5 4.9
化合物6 3.2
PI103 8.4
三、结论：
实施例化合物都显示了对激酶PI3Kα的抑制活性，尤其是化合物1～6具有很高的活性：对激酶PI3Kα的IC50都在10nM左右甚至以下。
试验2  化合物对激酶PI3Kγ的抑制活性
一、试验方法
1.制备1×激酶缓冲液
50mM HEPES，pH 7.5
3mM MgCl2
1mM EGTA
100mM NaCl
0.03％CHAPS
2mM DTT
2.制备4×激酶溶液
1)在1×激酶缓冲液中制备PI3Kγ溶液，每种试剂的浓度为测试时最终浓度的4倍。
终浓度：PI3Kγ为7.6nM。
2)测试板每孔加2.5μL激酶溶液，不含酶的对照孔除外(加2.5μL 1×激酶缓冲液代替)。
3)振荡测试板。
3.制备2×底物溶液
1)在1×激酶反应缓冲液中制备含PIP2底物和ATP的底物溶液，每种试剂的浓度均为测试时最终浓度的2倍。
终浓度：PIP2为50μM，ATP为25μM。
2)测试板每孔加入5μL底物溶液开始反应。
3)振荡测试板
4.激酶反应
测试板加盖于室温孵育1小时。
5.激酶检测
1)将ADP-Glo试剂平衡至室温。
2)从384孔板转移5μL反应混合物至一张新的384孔板。
3)新测试板每孔加入5μL ADP-Glo试剂终止反应。
4)离心短暂混合，于振荡器上缓慢振摇，平衡40min。
5)每孔加入10μL激酶检测试剂，振摇1min，在用读板器测量发光强度之前平衡60min。
6.读板
在Synergy上收集数据。
7.曲线拟合
1)从Synergy程序中拷贝RLU值。
2)将RLU值转换成百分抑制率。
3)数据存于MS Excel，用Graphpad 5.0拟合曲线。
百分抑制率＝100-(最高RLU-样本RLU)/(最高RLU-最低RLU)*100
“最高RLU”指无酶对照孔的RLU值，“最低RLU”指DMSO对照孔的RLU值。
二、结果：
化合物编号 IC50(nM)
化合物1 8.9
化合物4 4.1
化合物6 3.3
PI103 86
三、结论：
实施例化合物1、4、6都显示了很高的对激酶PI3Kγ的抑制活性，其IC50都在10nM以下。
试验3：化合物在MCF-7细胞上对Akt的Serine 473磷酸化的抑制
细胞株
细胞名 供应商 货号 批号 描述
MCF-7 ATCC HTB-22 5105360 人乳腺癌细胞
一、实验方法
第1天  细胞铺板
a.在显微镜下观察细胞株生长状态，在细胞株生长状态良好时开始实验。
b.将细胞培养瓶从培养箱中取出，核对瓶上标记的细胞名称，培养基类型及细胞代数。
c.弃去培养基，用胰酶消化，消化完后，用含血清的培养基中和，吹打细胞，使细胞脱落。
d.用移液管将细胞悬液移入离心管中，1000rpm的转速离心5分钟。
e.吸弃离心管中的细胞上清液。
f.向离心管中加入适当体积的培养基，轻柔吹打使细胞重悬均匀。
g.使用Vi-Cell XR细胞计数仪计数。
h.将细胞悬液调至合适浓度。
i.将细胞悬液加入384孔细胞培养板中，36μL/孔。标记细胞名称，种板密度，日期等详细信息，将培养板放置于CO2培养箱中孵育过夜。
实验条件：
Figure PCTCN2017080521-appb-000072
第2天  加化合物
j.吸弃24μL细胞上清，加24μL新鲜DMEM+10体积％FBS培养基，37℃，5％CO2孵育2h。
k.用DMSO将待测化合物配制成8mM溶液,然后3倍梯度稀释成9个浓度。
l.将梯度稀释的化合物用完全培养基(DMEM+10体积％FBS)配成4×溶液，每孔加12μL化合物到384孔细胞培养板(3701)(化合物的排布和最终浓度如下)，37℃，5％CO2孵育2h。
Figure PCTCN2017080521-appb-000073
注：Torin 1作为内参化合物；DMSO的最终浓度是0.5％。
m.吸弃上清38μL，加10μL 2×裂解缓冲液，室温振30min后-80℃过夜。
第3天AlphaScreen检测
n.室温解冻细胞裂解液，转移10μL裂解液到检测板(Optiplate-384)。
o.加5μL Acceptor beads到检测板，室温孵育2小时。
p.加5μL Donor beads到检测板，室温孵育2小时。
q.用EnSpire Multimode读板器检测，读取结果。
r.在Excel中用XL-fit软件计算化合物抑制的IC50值。
二、结果：
Figure PCTCN2017080521-appb-000074
三、结论：
该实验为细胞水平的活性测试，体现了化合物在活细胞上对PI3K的抑制活性。化合物6相较于对比例1化合物，分子结构中仅增加了一个氰基，但是其细胞活性却显著高于后者，IC50值相差4倍多。可见，化合物6中氰基的引入，具有出乎预料的、非显而易见的提升细胞活性的效果。
试验4：化合物在MCF-7细胞上对Akt的Serine 473磷酸化的抑制
一、试验方法：与试验3相同。
二、结果：
Figure PCTCN2017080521-appb-000075
三、结论：
该细胞水平的活性测试，显示了化合物1～4在活细胞上对PI3K的抑制活性均显著优于对比例1化合物，IC50值相差3到5倍。与对比例1化合物相比，本发明化合物1～4均在稠环上相同取代位置引入一个氰基，这些结果进一步表明，化合物1～4中氰基的引入，具有显著的提升细胞活性的效果。
试验5：CellTiter-Glo(CTG)法测试化合物在U87MG细胞株的细胞增殖实验
一、实验设计
在U87MG细胞中测定化合物对细胞增殖的抑制活性，并每株细胞设定一个阳性对照、一个空白对照和一个溶媒对照。
二、实验材料
细胞系
细胞系 肿瘤类型 生长特性 细胞数量/孔 培养基
U87MG 星形胶质瘤 贴壁 2000 EMEM+10％体积FBS
置于37℃、5％CO2、95％湿度条件下培养。
试剂和耗材
胎牛血清FBS(GBICO)，
Figure PCTCN2017080521-appb-000076
Luminescent Cell Viability Assay(Promega)，96孔透明平底黑壁板(Corning)。
待测药
Figure PCTCN2017080521-appb-000077
母液冻存于-20℃。
阳性药
Figure PCTCN2017080521-appb-000078
三、实验仪器
EnVision多标记微孔板检测仪，PerkinElmer，2104-0010A；
CO2培养箱，Thermo Scientific，Model 3100Series；
生物安全柜，Thermo Scientific，Model 1300Series A2；
倒置显微镜，Olympus，CKX41SF；
电子天平，METTLER-TOLEDO，AL-104；
冰箱，SIEMENS，KK25E76TI。
四、对细胞增殖的抑制活性测定
细胞培养和接种：
收获处于对数生长期的细胞并采用血小板计数器进行细胞计数。用台盼蓝排斥法检测细胞活力，确保各细胞系活力在96％以上。
用培养液稀释调整细胞浓度，添加90μL细胞悬液至的96孔板中使细胞密度达到指定的浓度。
96孔板中的细胞置于37℃、5％CO2、95％湿度条件下培养过夜。
加药：
药物稀释。用相应溶剂溶解被测化合物形成储存液并进行梯度稀释，得到10倍溶液；同样制备阳性药的10倍溶液。
加药。在已接种细胞的96孔板中每孔加入10μL药物溶液，每个细胞浓度设置三个复孔。被测化合物的最高浓度为50μM，9个浓度，3.16倍稀释。
培养。将已加药的96孔板中的细胞置于37℃、5％CO2、95％湿度条件下继续培养96小时，分别进行CTG分析。
终点读板：
融化CTG试剂并平衡细胞板至室温30分钟。
每孔加入等体积的CTG溶液。
在定轨摇床上振动2分钟使细胞裂解。
将细胞板放置于室温10分钟以稳定冷光信号。
用EnVision读取冷光值。
数据处理
使用GraphPad Prism 5.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC50值。
细胞存活率(％)＝(Lum待测药-Lum培养液对照)/(Lum细胞对照-Lum培养液对照)×100％
Lum是指：冷光值。
五、结果：
编号 化合物编号 IC50(μM) IC50(μM)*
1 化合物6 0.322 0.251
2 对比例1化合物 1.445 /
3 对比例2化合物 1.076 /
4 对比例3化合物 1.721 /
*：为另一次以相同方法独立进行的实验结果。
六、结论：
该实验为细胞水平的增殖抑制测试，体现了化合物对细胞增殖的抑制活性。与化合物6相比，对比例1化合物稠环上没有氰基，对比例2化合物和对比例3化合物在相同取代位置用其他基团替代氰基。试验结果表明，化合物6对U87MG细胞的增殖抑制活性显著高于所有对比例化合物，IC50值均相差在3倍以上。可见，化合物6中氰基的引入，具有出乎预料的、非显而易见的提升细胞活性的效果。
试验6  CellTiter-Glo法测试化合物对5株人肿瘤细胞株增殖活性抑制的研究
一、实验方法：与试验5相同。
Figure PCTCN2017080521-appb-000079
NEAA为非必需氨基酸
二、结果：
Figure PCTCN2017080521-appb-000080
三、结论：
该实验比较了化合物6与对比例1化合物对不同肿瘤细胞株的增殖抑制活性。化合物6相较于对比例1化合物，分子结构中仅增加了一个氰基，但是其细胞增殖抑制活 性却显著高于后者，二者在T47D、MCF7、A2780、NCI-H1975、A549等多种细胞株上的IC50值均相差约3～6倍。可见，化合物6中氰基的引入，具有出乎预料的、非显而易见的提升细胞活性的效果。
试验7  CellTiter-Glo法测试化合物对5株人肿瘤细胞株增殖活性抑制的研究
一、实验方法：与试验5相同。
Figure PCTCN2017080521-appb-000081
二、结果：
细胞株 化合物6的IC50(μM)
BT20 0.127
BT474 0.606
SK-OV-3 0.147
NCI-H460 0.346
NCI-H1650 0.475
三、结论：该实验测试了化合物6对不同肿瘤细胞株的增殖抑制活性，结果表明，化合物6在BT20、BT474、SK-OV-3、NCI-H460、NCI-H1650等多种肿瘤细胞株上都表现出了很好的增殖抑制活性，IC50值均在1μM以下。

Claims (18)

  1. 一种由以下通式(I)表示的化合物或其药学上可接受的盐：
    Figure PCTCN2017080521-appb-100001
    其中，X表示O、S、NR、SO2或NH2Cl，R选自H、C1-6的烷基，R1、R2、R3、R4、R5、R6、R7、R8独立地选自H、C1-6的烷基、或具有OH取代基的C1-6的烷基。
  2. 如权利要求1所述的化合物或其药学上可接受的盐，其中，R为C1-4的烷基，R1、R2、R3、R4、R5、R6、R7、R8为独立地选自H、C1-4的烷基、或具有OH取代基的C1-4的烷基。
  3. 如权利要求1所述的化合物或其药学上可接受的盐，其中，R为甲基或乙基，R1、R2、R3、R4、R5、R6、R7、R8为甲基或乙基。
  4. 如权利要求1所述的化合物或其药学上可接受的盐，其中，所述化合物选自以下化合物或其药学上可接受的盐：
    Figure PCTCN2017080521-appb-100002
    Figure PCTCN2017080521-appb-100003
    Figure PCTCN2017080521-appb-100004
    Figure PCTCN2017080521-appb-100005
    Figure PCTCN2017080521-appb-100006
    Figure PCTCN2017080521-appb-100007
    Figure PCTCN2017080521-appb-100008
  5. 一种药物组合物，所述药物组合物包含如权利要求1-4中任一项所述的化合物或其药学上可接受的盐。
  6. 如权利要求1-4中任一项所述的化合物或其药学上可接受的盐、或如权利要求5所述的药物组合物在制备用于抑制磷脂酰肌醇3-激酶的药物中的应用。
  7. 如权利要求6所述的应用，其中所述的药物为抗肿瘤药物。
  8. 如权利要求7所述的应用，其中，所述的肿瘤选自：脑癌、头颈癌、食管癌、肺癌、肝癌、胃癌、肾癌、胰腺癌、前列腺癌、结直肠癌、卵巢癌、乳腺癌、甲状腺癌、皮肤癌、白血病、骨髓异常增生综合症、肉瘤、骨肉瘤或横纹肌瘤。
  9. 如权利要求6所述的应用，其中所述的药物为抗炎药物或治疗自身免疫性疾病的药物。
  10. 如权利要求9所述的应用，其中所述的抗炎药物是用于治疗慢性阻塞性肺病或 哮喘的药物。
  11. 如权利要求9所述的应用，其中自身免疫性疾病选自风湿性关节炎、银屑病或系统性红斑狼疮。
  12. 一种如权利要求1-4任一项所述的式(I)的化合物的制备方法，所述方法包括使式(III’)的化合物与式(IV)的化合物反应，得到式(I)的化合物的步骤：
    Figure PCTCN2017080521-appb-100009
    其中X、R1、R2、R3、R4、R5、R6、R7和R8如权利要求1-4中任一项所定义。
  13. 如权利要求12所述的方法，其中，所述方法包括使式(II)的化合物与式(III)的化合物反应得到式(III’)的化合物的步骤，
    Figure PCTCN2017080521-appb-100010
  14. 一种如权利要求1-4任一项所述的式(I)的化合物的制备方法，所述方法包括使式(IV’)的化合物与式(III)的化合物反应，得到式(I)的化合物的步骤：
    Figure PCTCN2017080521-appb-100011
    其中X、R1、R2、R3、R4、R5、R6、R7和R8如权利要求1-4中任一项所定义。
  15. 如权利要求14所述的方法，其中，所述方法包括使式(II)的化合物与式(IV)的化合物反应得到式(IV’)的化合物的步骤，
    Figure PCTCN2017080521-appb-100012
  16. 一种式(III’)的化合物或式(IV’)的化合物，
    Figure PCTCN2017080521-appb-100013
    其中R1、R2、R3、R4、R5、R6、R7和R8如权利要求1-4中任一项所定义，X表示O、S、NR、SO2或NH2Cl，R为C1-6的烷基。
  17. 如权利要求16所述的式(III’)的化合物或式(IV’)的化合物，其中R为C1-4的烷基。
  18. 如权利要求16所述的式(III’)的化合物或式(IV’)的化合物，其中R为甲基或乙基。
PCT/CN2017/080521 2016-04-15 2017-04-14 一种稠环化合物、其制备方法和应用及其中间体化合物 WO2017177958A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610235304.5A CN105859684B (zh) 2016-04-15 2016-04-15 一种稠环化合物、其制备方法和应用及其中间体化合物
CN201610235304.5 2016-04-15

Publications (1)

Publication Number Publication Date
WO2017177958A1 true WO2017177958A1 (zh) 2017-10-19

Family

ID=56632196

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/080521 WO2017177958A1 (zh) 2016-04-15 2017-04-14 一种稠环化合物、其制备方法和应用及其中间体化合物

Country Status (3)

Country Link
CN (1) CN105859684B (zh)
TW (1) TW201738227A (zh)
WO (1) WO2017177958A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105859684B (zh) * 2016-04-15 2017-03-22 四川赛诺唯新生物技术有限公司 一种稠环化合物、其制备方法和应用及其中间体化合物
CN110642854A (zh) * 2019-11-20 2020-01-03 成都克莱蒙医药科技有限公司 一种稠环化合物的多晶型、其组合物、制备方法及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009155121A2 (en) * 2008-05-30 2009-12-23 Amgen Inc. Inhibitors of pi3 kinase
CN103788071A (zh) * 2012-11-01 2014-05-14 中国人民解放军第二军医大学 N-(5-(喹啉-6-基)吡啶-3-基)苯磺酰胺衍生物、制备方法及治疗用途
CN105859684A (zh) * 2016-04-15 2016-08-17 四川赛诺唯新生物技术有限公司 一种稠环化合物、其制备方法和应用及其中间体化合物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009155121A2 (en) * 2008-05-30 2009-12-23 Amgen Inc. Inhibitors of pi3 kinase
CN103788071A (zh) * 2012-11-01 2014-05-14 中国人民解放军第二军医大学 N-(5-(喹啉-6-基)吡啶-3-基)苯磺酰胺衍生物、制备方法及治疗用途
CN105859684A (zh) * 2016-04-15 2016-08-17 四川赛诺唯新生物技术有限公司 一种稠环化合物、其制备方法和应用及其中间体化合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAN, JINSONG ET AL.: "Structure-Based Optimization Leads to the Discovery of NSC765844, a Highly Potent, Less Toxic and Orally Efficacious Dual PI3K/mTOR Inhibitor", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 122, 23 June 2016 (2016-06-23), pages 684 - 701, XP029705932, DOI: doi:10.1016/j.ejmech.2016.06.030 *

Also Published As

Publication number Publication date
CN105859684A (zh) 2016-08-17
CN105859684B (zh) 2017-03-22
TW201738227A (zh) 2017-11-01

Similar Documents

Publication Publication Date Title
US10780073B2 (en) N4-phenyl-quinazoline-4-amine derivatives and related compounds as ErbB type I receptor tyrosine kinase inhibitors for the treatment of hyperproliferative diseases
AU2018252251B2 (en) Compounds useful as RET inhibitors
CN101809002B (zh) 用于与mtor激酶和/或pi3k相关的疾病中的吗啉代嘧啶衍生物
JP2023159230A (ja) Ｒｅｔキナーゼ阻害剤としての複素環化合物
CN102124000B (zh) 吡啶化合物
JP6035423B2 (ja) 新規な縮合ピリミジン化合物又はその塩
JP6564406B2 (ja) カゼインキナーゼ１デルタ／イプシロン阻害剤としてのイミダゾ−ピリダジン誘導体
CN101801963A (zh) 用于治疗增殖性疾病的三取代的嘧啶衍生物
JP2008505084A (ja) フラノピリミジン
WO2009007390A2 (en) 2-pyraz inylbenz imidazole derivatives as receptor tyrosine kinase inhibitors
CN101155807A (zh) 具有tie2（tek）抑制活性的化合物
WO2007129044A1 (en) Thiazole derivatives and their use as anti-tumour agents
CN101563340A (zh) 用作pi3k和mtor抑制剂用于治疗增殖性疾病的2-苯并咪唑基-6-吗啉代-4-哌啶-4-基嘧啶衍生物
CN109810098B (zh) 含有酞嗪-1(2h)-酮结构的parp-1和pi3k双靶点抑制剂
WO2018145621A1 (zh) 喹啉类化合物、其制备方法及其医药用途
WO2017177958A1 (zh) 一种稠环化合物、其制备方法和应用及其中间体化合物
WO2019100743A1 (zh) 含有苯并呋喃的parp-1和pi3k双靶点抑制剂
US11572359B2 (en) PARP/PI3K double-target inhibit containing pyridopyrimidine structure
CN106866642B (zh) 含芳基酰腙结构的喹唑啉类化合物及其应用
KR102629854B1 (ko) 단백질 키나제 저해제로서 유용한 피리도퀴나졸린 유도체
CN115703760A (zh) 2,4-二取代嘧啶类细胞周期蛋白依赖性激酶酶抑制剂及其制备方法和应用
CN101346371A (zh) 癌症治疗中用作酪氨酸激酶抑制剂的4-(3-氨基吡唑)嘧啶衍生物

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

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

Ref document number: 17781934

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17781934

Country of ref document: EP

Kind code of ref document: A1