WO2018086609A1 - 3,4-二吡啶基吡唑类衍生物、其制备方法及其在医药上的应用 - Google Patents

3,4-二吡啶基吡唑类衍生物、其制备方法及其在医药上的应用 Download PDF

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WO2018086609A1
WO2018086609A1 PCT/CN2017/110686 CN2017110686W WO2018086609A1 WO 2018086609 A1 WO2018086609 A1 WO 2018086609A1 CN 2017110686 W CN2017110686 W CN 2017110686W WO 2018086609 A1 WO2018086609 A1 WO 2018086609A1
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group
compound
cancer
formula
mixture
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PCT/CN2017/110686
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English (en)
French (fr)
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杨方龙
张羚
沈光远
贺峰
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CA3043383A priority Critical patent/CA3043383A1/en
Priority to JP2019523621A priority patent/JP2020500177A/ja
Priority to KR1020197015521A priority patent/KR20190077048A/ko
Priority to BR112019008516A priority patent/BR112019008516A2/pt
Priority to US16/349,165 priority patent/US10899741B2/en
Priority to EP17868813.1A priority patent/EP3539957A4/en
Priority to AU2017357333A priority patent/AU2017357333A1/en
Priority to MX2019004822A priority patent/MX2019004822A/es
Priority to CN201780016854.0A priority patent/CN108779100B/zh
Publication of WO2018086609A1 publication Critical patent/WO2018086609A1/zh

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    • 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
    • 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
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the invention belongs to the field of medicine and relates to a novel 3,4-dipyridylpyrazole derivative, a preparation method thereof and a pharmaceutical composition containing the same and as a therapeutic agent, in particular as an inhibitor of TGF- ⁇ Uses and use in the manufacture of a medicament for the treatment, prevention or reduction of cancer mediated by TGF- ⁇ overexpression.
  • TGF- ⁇ Transforming growth factor
  • BMPs Bone morphogenetic proteins
  • GDFs Differentiation factors
  • MIS Müllerian-inhibiting substances
  • TGF- ⁇ has three subtypes of TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3, which are involved in the regulation of cell proliferation and differentiation, wound healing, extracellular matrix production and immunosuppression. See, eg, Massague, J. Ann. Rev, Cell. Biol. 6: 594-641 (1990); Roberts, ABPeptide Growth Factor and Their receptors, 95: 419-472 Berlin: Springer-Verlag (1990); Roberts, AB and Sporn MBGrowth Factor 8: 1-9 (1993); and Alexandrow, MG, Moses, HL Cancer Res. 55: 1452-1457 (1995). Three subtypes of TGF- ⁇ are present in most cells along with their receptors.
  • Each TGF- ⁇ isoform is synthesized as a precursor protein that is cleaved intracellularly into a C-terminal region (latency related peptide, LAP) and an N-terminal portion, termed mature or active TGF. - ⁇ .
  • LAP latency related peptide
  • LAP is typically linked to mature TGF- ⁇ in a non-covalent manner.
  • the LAP-TGF- ⁇ complex does not bind to the TGF- ⁇ receptor and is not biologically active.
  • TGF-[beta] is typically released (and is active) from the complex by a variety of mechanisms including, for example, interaction with thrombospondin-1 or plasmin.
  • TGF- ⁇ 1 transduces signals through two highly conserved single transmembrane serine/threonine kinases, namely type I (ALK5) and type II TGF- ⁇ receptors.
  • type II receptor hyperphosphorylates the serine/threonine residue in the ALK5GS region, causing activation of ALK5 by creating a binding site for the Smad protein.
  • Activated ALK5 in turn phosphorylates the Smad2 and Smad3 proteins at the C-terminal SSXS-motif, causing them to dissociate from the receptor and form a heteromeric complex with Smad4.
  • the Smad complex is readily located in the nucleus and assembles with specific DNA-binding cofactors and co-regulators, ultimately activating the transcription of extracellular matrix components and matrix-degrading protease inhibitors.
  • TGF- ⁇ signaling pathway The extreme activity of the TGF- ⁇ signaling pathway is responsible for many human diseases such as excessive deposition of extracellular matrices, abnormally high levels of inflammatory responses, fibrotic disorders, and progressive cancer.
  • tumor cells and intratumoral stromal cells In the advanced stages of various cancers, tumor cells and intratumoral stromal cells generally overexpress TGF- ⁇ . This causes stimulation of angiogenesis and cell movement, inhibition of the immune system, and increased interaction of tumor cells with extracellular matrices (e.g., Hojo, M. et al, Nature 397: 530-534 (1999)). Therefore, tumor cells become more aggressive and metastasize to distant organs. For example, Maehara, Y. et al., J. Clin. Oncol. 17: 607-614 (1999); Picon, A. et al., Cancer Epidemiol. Biomarkers Prev. 7: 497-504 (1998)).
  • TGF- ⁇ TGF- ⁇
  • Thy-1 rat model of proliferative glomerulonephritis rabbit anti-GBM glomerulonephritis and bureau
  • a 5/6 nephrectomized rat model of focal segmental glomerulosclerosis has recently been reviewed (eg, Bitzer, M. et al., Kidney Blood Press. Res. 21:1-12 (1998)).
  • Neutralizing antibodies to TGF- ⁇ improve glomerular histology in the Thy-1 nephritis model (e.g., Border, W. A. et al, Nature 346: 371-374 (1990)).
  • TGF- ⁇ 1 and its receptor are overexpressed in damaged vascular and fibroproliferative vascular injury, causing excessive production of extracellular matrix (eg, Saltis, J. et al., Clin. Exp. Pharmacol. Physiol. 23: 193). -200 (1996); McCaffrey, TA et al, J. Clin. Invest. 96: 2667-2675 (1995)).
  • TGF- ⁇ 2 levels are elevated in most eyes with aqueous glaucoma with juvenile glaucoma and almost half in eyes with primary open angle glaucoma (POAG) (eg, Picht, G. et al., Graefes Arch. Clin) .Exp. Ophthalmol. 239: 199-207 (2001)). Both TGF- ⁇ 1 and TGF- ⁇ 2 subtypes have been reported to increase the extracellular matrix production of cultured human Tenon's capsule fibroblasts from patients with pseudo-exfoliative glaucoma and POAG (eg, Kottler, UB et al., Exp. Eye Res. 80: 121-134 (2005)).
  • POAG primary open angle glaucoma
  • Modulators (e.g., antagonists) of the disclosed TGF-[beta] family member receptors include WO2004111046, WO2012000595, WO2012002680, WO2013009140, WO2016106266.
  • the present invention provides a novel structure of a TGF- ⁇ receptor kinase inhibitor, and finds that a compound of such a structure has good selective activity against ALK5 and toxicity test results show that the compound of such structure has good safety. And showed excellent TGF- ⁇ receptor inhibition and tumor inhibition effect.
  • the object of the present invention is to provide a compound of the formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer thereof, or a mixture form, or a pharmaceutically acceptable salt thereof,
  • Ring A is a cycloalkyl or heterocyclic group
  • R 1 is the same or different and is each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, -S(O) m R 5 , -C(O)OR 5 , cycloalkyl and heterocyclic;
  • R 2 is the same or different and is each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, and an aromatic group.
  • Base heteroaryl, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 ;
  • R 3 are the same or different and are each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, an alkoxy group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, an amino group, a cyano group, and a nitro group;
  • R 4 is the same or different and is each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group. base;
  • R 5 is selected from the group consisting of a hydrogen atom, an alkyl group, an amino group, a halogenated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • R 6 and R 7 are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a halogenated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;
  • n 0, 1 or 2;
  • s 0, 1, 2, 3, 4 or 5;
  • p 0, 1, 2 or 3;
  • q 0, 1, 2, 3 or 4;
  • n 0, 1, or 2.
  • the compound of the formula (I) is a compound of the formula (II):
  • G 1 is CH 2 , NR 8 or O;
  • R 8 is selected from -S(O) m R 5 , -C(O)OR 5 , a hydrogen atom or an alkyl group;
  • R 2 , R 4 and R 5 are as defined in the general formula (I);
  • s 0, 1 or 2;
  • n 0, 1 or 2;
  • r 0, 1, 2 or 3.
  • the compounds of the present invention include all conformational isomers thereof, such as cis and trans isomers; and all optical isomers and stereoisomers thereof, and mixtures thereof.
  • the compounds of the invention have asymmetric centers and therefore different enantiomers and diastereomers.
  • the invention relates to the use of the compounds of the invention, and to all pharmaceutical compositions and methods of treatment which may be employed and contained therein.
  • the invention relates to the use of all such isomers and mixtures thereof.
  • Typical compounds of the invention include, but are not limited to:
  • a tautomer a meso form, a racemate, an enantiomer, a diastereomer, or a mixture thereof or a pharmaceutically acceptable salt thereof.
  • the present invention also relates to a compound of the formula (I-A) which is an intermediate for the synthesis of a compound of the formula (I):
  • X is a halogen, preferably bromine
  • Rings A, R 1 , R 4 , n and q are as defined in the general formula (I).
  • the compound represented by the formula (I-A) includes, but is not limited to:
  • Another aspect of the invention relates to a process for the preparation of a compound of formula (I), which process comprises:
  • the compound of the formula (I-A) and the compound of the formula (I-B) are subjected to a Suzuki reaction in the presence of a catalyst under basic conditions to give a compound of the formula (I).
  • X is a halogen, preferably bromine
  • Ring A, R 1 to R 4 , n, s, p and q are as defined in the formula (I).
  • Another aspect of the invention relates to a process for the preparation of a compound of formula (II), the process comprising:
  • the compound of the formula (II-A) and the compound of the formula (I-B) are subjected to a Suzuki reaction under basic conditions in the presence of a catalyst to obtain a compound of the formula (II).
  • X is a halogen, preferably bromine
  • G 1 , R 2 , R 4 , r, s and q are as defined in the formula (I).
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I) or a tautomer, a mesogen, a racemate thereof, Enantiomers, diastereomers, or mixtures thereof or pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers Body, diluent or excipient.
  • the present invention also relates to a process for the preparation of the above composition, which comprises the compound of the formula (I) or a tautomer thereof, a mesogen, a racemate, an enantiomer, a non- The enantiomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is admixed with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for the preparation of a medicament for inhibiting a TGF- ⁇ and/or activin (especially human TGF- ⁇ and/or activin) signaling pathway.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for the preparation of a medicament for the treatment, prevention or reduction of metastasis of tumor cells, particularly human tumor cells.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for the preparation of a medicament for the treatment, prevention or reduction of cancer mediated by TGF- ⁇ overexpression, in particular by inhibiting the human TGF- ⁇ signaling pathway Use in a medicament for preventing or reducing cancer mediated by TGF- ⁇ overexpression.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or Use of a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for the preparation of a medicament for the treatment, prevention or alleviation (especially human) selected from the group consisting of vascular injury, glomerulonephritis, diabetic nephropathy , lupus nephritis, hypertension-induced nephropathy, renal interstitial fibrosis, renal fibrosis caused by drug exposure complications, HIV-associated nephropathy, graft nephropathy, liver fibrosis of various causes, return Hepatic dysfunction due to infection, alcohol-induced hepatitis, cystic fibrosis, interstitial lung disease, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease,
  • the invention further relates to a method of treating, preventing or reducing human tumor cell metastasis comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer thereof, a mesogen thereof, A racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
  • the invention further relates to a method of treating, preventing or reducing cancer mediated by TGF- ⁇ overexpression, in particular by treating, preventing or reducing cancer mediated by TGF- ⁇ overexpression by inhibiting the TGF- ⁇ signaling pathway
  • a method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer A composition, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
  • the invention further relates to a method of treating, preventing or ameliorating (particularly human) a disease selected from the group consisting of vascular injury, glomerulonephritis, diabetic nephropathy, lupus nephritis, hypertension-induced nephropathy, kidney Interstitial fibrosis, renal fibrosis caused by drug exposure complications, HIV-associated nephropathy, graft nephropathy, liver fibrosis of various causes, liver dysfunction attributable to infection, alcohol-induced hepatitis, Cystic fibrosis, interstitial lung disease, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung disease caused by infectious or toxic factors, post-infarction cardiac fibrosis, congestion Heart failure, dilated cardiomyopathy, myocarditis, intimal thickening, vascular stenosis, vascular remodeling caused by hypertension, pulmonary hypertension, coronary restenosis
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or Its pharmaceutical composition, its action drug.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which acts as a TGF-beta receptor kinase inhibitor.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for use in the treatment, prevention or reduction of metastasis of tumor cells, particularly human tumor cells.
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for use in the treatment, prevention or reduction of cancer mediated by TGF- ⁇ overexpression, in particular by inhibiting the TGF- ⁇ signaling pathway for treatment, prevention or reduction Cancer mediated by overexpression of TGF- ⁇ .
  • the invention further relates to a compound of the formula (I) or a tautomer, a mesophil, a racemate, an enantiomer, a diastereomer, or a mixture thereof Or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which is used for the treatment, prevention or alleviation (especially human) of a disease selected from the group consisting of vascular injury, glomerulonephritis, diabetic nephropathy, lupus Nephritis, hypertension-induced nephropathy, renal interstitial fibrosis, renal fibrosis caused by drug exposure complications, HIV-associated nephropathy, graft nephropathy, liver fibrosis of various causes, attributable to Infected liver dysfunction, alcohol-induced hepatitis, cystic fibrosis, interstitial lung disease, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, by infectious or toxic
  • the active ingredient-containing pharmaceutical composition may be in a form suitable for oral administration, such as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Tincture.
  • Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives, To provide a pleasing and tasty pharmaceutical preparation. Tablets contain the active ingredient and non-toxic pharmaceutically acceptable excipients suitable for the preparation of a tablet for admixture.
  • the aqueous suspension contains the active substance and excipients suitable for the preparation of the aqueous suspension for mixing.
  • excipients are suspending agents.
  • Oil suspensions can be formulated by suspending the active ingredient in vegetable oil.
  • the oil suspension may contain a thickening agent.
  • the above sweeteners and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of an antioxidant.
  • the dispersible powders and granules suitable for the preparation of aqueous suspensions can be provided by the addition of water to provide the active ingredient and dispersing or wetting agents, suspending agents or one or more preservatives. Suitable dispersing or wetting agents and suspending agents can be used to illustrate the above examples. Other excipients such as sweetening, flavoring, and coloring agents may also be added. These compositions are preserved by the addition of an antioxidant.
  • compositions of the invention may also be in the form of an oil-in-water emulsion.
  • the oil phase can be a vegetable oil.
  • Suitable emulsifiers can be naturally occurring phospholipids.
  • the emulsions may also contain sweeteners, flavoring agents, preservatives, and antioxidants. Such formulations may also contain a demulcent, a preservative, a colorant, and an antioxidant.
  • compositions of the invention may be in the form of a sterile injectable aqueous solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase.
  • compositions of the invention may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable non-toxic diluent or solvent.
  • a sterile fixed oil may conveniently be employed as a solvent or suspension medium.
  • the compounds of the invention may be administered in the form of a suppository for rectal administration.
  • These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug.
  • the dosage of the drug to be administered depends on a variety of factors including, but not limited to, the following factors: the activity of the particular compound used, the age of the patient, the weight of the patient, the health of the patient, and the patient's behavior.
  • the dosage, the patient's diet, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal treatment mode such as the mode of treatment, the daily dosage of the compound of formula (I) or a pharmaceutically acceptable salt The type can be verified according to traditional treatment options.
  • alkyl refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group of 1 to 20 carbon atoms, preferably an alkyl group having 1 to 12 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-methylpentyl,
  • lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently selected from alkyl, alkenyl, alkynyl, Alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy
  • One or more substituents of cycloalkylthio, heterocycloalkylthio, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 Replaced.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms.
  • the carbon atoms most preferably containing from 3 to 6 carbon atoms, for example 3, 4, 5 or 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • the group, cyclooctyl group and the like are preferably a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group; and the polycyclic cycloalkyl group includes a spiro ring, a fused ring and a bridged ring cycloalkyl group.
  • spirocycloalkyl refers to a polycyclic group that shares a carbon atom (referred to as a spiro atom) between 5 to 20 members of a single ring, which may contain one or more double bonds, but none of the rings have a fully conjugated ⁇ electronic system. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • the spirocycloalkyl group is classified into a monospirocycloalkyl group, a bispirocycloalkyl group or a polyspirocycloalkyl group, preferably a monospirocycloalkyl group and a bispirocycloalkyl group, depending on the number of common spiro atoms between the rings.
  • spirocycloalkyl groups include:
  • fused cycloalkyl refers to 5 to 20 members, and each ring in the system shares an all-carbon polycyclic group of an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or Multiple double bonds, but none of the rings have a fully conjugated ⁇ -electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • fused cycloalkyl groups include:
  • bridged cycloalkyl refers to an all-carbon polycyclic group of 5 to 20 members, any two rings sharing two carbon atoms which are not directly bonded, which may contain one or more double bonds, but none of the rings have complete Conjugate ⁇ -electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic ring, a tricyclic ring or a tetracyclic ring, and more preferably a bicyclic ring or a tricyclic ring.
  • bridged cycloalkyl groups include:
  • the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring to which the parent structure is attached is a cycloalkyl group, non-limiting examples include indanyl, tetrahydronaphthalene Base, benzocycloheptyl and the like.
  • the cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, Halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio Substituting one or more substituents of -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 .
  • heterocyclyl refers to a saturated/or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S (O). a hetero atom of m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • the heterocyclic group contains from 3 to 10 ring atoms, most preferably heterocyclic groups Contains 3 to 6 ring atoms, for example 3, 4, 5 or 6 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazine
  • the group or the like is preferably an oxetanyl group and a tetrahydrofuranyl group; and the polycyclic heterocyclic group includes a heterocyclic group of a spiro ring, a condensed ring and a bridged ring.
  • spiroheterocyclyl refers to a polycyclic heterocyclic group in which one atom (called a spiro atom) is shared between 5 to 20 members of a single ring, wherein one or more ring atoms are selected from nitrogen, oxygen or S (O). ) m (where m is an integer 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • the spiroheterocyclyl group is classified into a monospiroheterocyclic group, a dispiroheterocyclic group or a polyspirocyclic group according to the number of shared spiro atoms between the ring and the ring, and is preferably a monospirocycloalkyl group and a bispirocycloalkyl group. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6-membered monospiroheterocyclic group.
  • Non-limiting examples of spiroheterocyclyl groups include:
  • fused heterocyclyl refers to 5 to 20 members, and each ring in the system shares an adjacent pair of atomic polycyclic heterocyclic groups with other rings in the system, and one or more rings may contain one or more Double bond, but none of the rings have a fully conjugated ⁇ -electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the remaining rings
  • the atom is carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members.
  • fused heterocyclic groups include:
  • bridge heterocyclyl refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms which are not directly bonded, which may contain one or more double bonds, but none of the rings have a total A ⁇ -electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), the remaining ring atoms being carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. 7 to 10 yuan.
  • bridge heterocyclic groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclic group, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy.
  • alkylthio alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Substituted by one or more substituents of alkylthio, heterocycloalkylthio, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 .
  • aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (ie, a ring that shares a pair of adjacent carbon atoms) groups which are polycyclic rings having a conjugated ⁇ -electron system (ie, The ring group adjacent to a carbon atom is preferably 6 to 10 members such as a phenyl group and a naphthyl group.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio Substituted by one or more substituents of a heterocycloalkylthio group, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 .
  • the substituent is preferably one or more of the following groups, which are independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy,
  • heteroaryl refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, pyridyl Azole Base, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio group, alkylamino group, halogen, thiol, hydroxyl group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkyl sulfide Substituted by one or more substituents in the group, heterocycloalkylthio, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 .
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio group, alkylamino group, halogen, thiol, hydroxy, amino, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Substituted by one or more substituents of alkylthio, heterocycloalkylthio, -OR 5 , -C(O)R 5 , -S(O) m R 5 and -C(O)NR 6 R 7 .
  • hydroxyalkyl refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted by one or more halogens, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • halogen means fluoro, chloro, bromo or iodo.
  • amino means -NH 2.
  • cyano refers to -CN.
  • nitro refers to -NO 2 .
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions and can be determined by those skilled in the art without undue effort (by experiment or Theory) may or may not be replaced. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention which is safe and effective for use in a mammal and which possesses the desired biological activity.
  • Figure 1 is the effect of Compound 1 on the volume of transplanted tumor of B16-F1 tumor-bearing C57 mice.
  • Figure 2 is the effect of Compound 1 on the body weight of C57 mice.
  • a method for preparing a medicinal salt comprising the steps of:
  • the compound of the formula (I-1) and the compound of the formula (I-2) are subjected to a Suzuki reaction under basic conditions in the presence of a catalyst to obtain a compound of the formula (I-3);
  • the compound of the formula (I-6) is reacted with a halogenating reagent to obtain a compound of the formula (I-A);
  • the compound of the formula (I-A) and the compound of the formula (I-B) are subjected to a Suzuki reaction under basic conditions in the presence of a catalyst to obtain a compound of the formula (I);
  • the reagents providing basic conditions include organic bases including, but not limited to, triethylamine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, and inorganic bases. , bis-trimethylsilylamino lithium, potassium acetate, sodium t-butoxide or potassium t-butoxide, said inorganic bases including but not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, carbonic acid Antimony, sodium hydroxide and lithium hydroxide;
  • Catalysts include, but are not limited to, palladium on carbon, Raney nickel, tetra-triphenylphosphine palladium, palladium dichloride, palladium acetate, [1,1'-bis(diphenylphosphino)ferrocene] dichloride Palladium, 1,1'-bis(dibenzylphosphine) dichlorodipentadium iron palladium or tris(dibenzylideneacetone) dipalladium, preferably [1,1'-bis(diphenylphosphino)dilenole Iron] palladium dichloride or tetrakistriphenylphosphine palladium;
  • Halogen reagents include, but are not limited to, liquid bromine, hydrogen bromide, N-bromosuccinimide (NBS), PBr 3 , POBr 3 , pyridinium bromide hydrobromide (PHP), tetrabromocycloketone (TBCO), diethyl bromomalonate, tetrabutylammonium bromide, N-chlorosuccinimide, PCl 3 and POCl 3 ;
  • the above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-two.
  • a solvent including, but not limited to, acetic acid, methanol, ethanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-two.
  • Oxy hexacyclohexane water, N,N-dimethylformamide and mixtures thereof;
  • G 2 and G 3 are each independently selected from
  • X is a halogen, preferably bromine
  • Ring A, R 1 to R 4 , n, s, p and q are as defined in the formula (I).
  • a method for preparing a medicinal salt comprising the steps of:
  • the reagents providing basic conditions include organic bases including, but not limited to, triethylamine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, and inorganic bases. , bis-trimethylsilylamino lithium, potassium acetate, sodium t-butoxide or potassium t-butoxide, said inorganic bases including but not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, carbonic acid Antimony, sodium hydroxide and lithium hydroxide;
  • Catalysts include, but are not limited to, palladium on carbon, Raney nickel, tetra-triphenylphosphine palladium, palladium dichloride, palladium acetate, [1,1'-bis(diphenylphosphino)ferrocene] dichloride Palladium, 1,1'-bis(dibenzylphosphine) dichlorodipentadium iron palladium or tris(dibenzylideneacetone) dipalladium, preferably [1,1'-bis(diphenylphosphino)dilenole Iron] palladium dichloride or tetrakistriphenylphosphine palladium;
  • the above reaction is preferably carried out in a solvent including, but not limited to, acetic acid, methanol, ethanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-two.
  • a solvent including, but not limited to, acetic acid, methanol, ethanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4-two.
  • Oxy hexacyclohexane water, N,N-dimethylformamide and mixtures thereof;
  • G 2 is selected from
  • X is a halogen, preferably bromine
  • G 1 , R 2 , R 4 , r, s and q are as defined in the formula (I).
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • NMR was measured using a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), internal standard was four.
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methyl silane
  • the measurement of the MS was carried out using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • ESI FINNIGAN LCQAd
  • the HPLC was measured using an Agilent 1200 DAD high pressure liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 x 4.6 mm column).
  • Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate.
  • the specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm.
  • the specification for thin layer chromatography separation and purification is 0.4mm. ⁇ 0.5mm.
  • the CombiFlash Rapid Preparer uses the Combiflash Rf200 (TELEDYNE ISCO).
  • the known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Companies such as chemicals.
  • the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L volume.
  • the pressurized hydrogenation reaction was carried out using a Parr Model 3916EKX hydrogenation apparatus and a clear blue QL-500 type hydrogen generator or a HC2-SS type hydrogenation apparatus.
  • the hydrogenation reaction is usually evacuated, charged with hydrogen, and operated three times.
  • the microwave reaction used a CEM Discover-S Model 908860 microwave reactor.
  • the solution means an aqueous solution.
  • reaction temperature is room temperature and is 20 ° C to 30 ° C.
  • TLC thin layer chromatography
  • the developing agent used for the reaction the column chromatography eluent system used for the purification of the compound
  • the thin layer chromatography developing solvent system including: A: Methylene chloride/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: acetone, E: dichloromethane/acetone system, F: ethyl acetate/dichloromethane system , G: ethyl acetate / dichloromethane / n-hexane, H: ethyl acetate / dichloromethane / acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound, a small amount of triethylamine and acetic acid may also be added. Adjust with alkaline or acidic reagents.
  • Boric acid 1b (5066.1 mg, 25.33 mmol), sodium carbonate (5369.1 mg, 50.66 mmol) and tetrakistriphenylphosphine palladium (2926.7 mg, 2.53 mmol) were stirred under a argon atmosphere for 16 hours at 100 °C.
  • the reaction was monitored by LC-MS to EtOAc (EtOAc).
  • EtOAc EtOAc
  • Test Example 1 Determination of the inhibitory effect of the compound of the present invention on TGF ⁇ RI kinase activity
  • the inhibitory effect of the compound of the present invention on the activity of TGF ⁇ RI kinase ALK5 was determined by the following experimental method:
  • Enzyme activity assay using TGF ⁇ RI kinase assay kit (V4093, Promega), 2 ⁇ l of lysis buffer (40 mM Tris pH 7.5, 20 mM MgCl 2 , 0.1 mg/ml BSA) was added to 384-well plates (4514, Corning).
  • the enzyme solution (the final concentration of the enzyme in the reaction system is 2 ng / ⁇ L), 1 ⁇ l of a 3-fold gradient dilution of the compound dissolved in 5% DMSO, 2 ⁇ l of a mixture of ATP and TGF ⁇ RI substrate polypeptide (ATP final concentration of 50 ⁇ M, substrate final The concentration was 0.2 ⁇ g/ ⁇ L), and after reacting at 27 ° C for 2.5 hours, 5 ⁇ l of the ADP-Glo solution in the kit was added to each well, and the mixture was allowed to stand at 27 ° C for 40 minutes, and 10 ⁇ l of the kinase detection reagent was further added to each well, and the mixture was allowed to stand at 27 ° C for 30 minutes. Chemiluminescent signal values were measured using a Victor 3 (PerkinElmer) multi-plate reader. IC 50 values of inhibitory activity of the compound is calculated using Graphpad prism software corresponding to each concentration of compound according to the signal value.
  • the compounds of the examples of the present invention have a significant inhibitory effect on the activity of TGF ⁇ RI kinase ALK5.
  • Test Example 2 Determination of the inhibitory effect of the compound of the present invention on VEGFR2 kinase activity
  • VEGFR2 kinase activity in vitro was tested by the following method.
  • Enzyme activity detection Kinase Assay Kit-Tyrosine 1Peptide (PV3190, Invitrogen) kit 5 ⁇ l of reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.05% BRIJ-35) was sequentially added to a 384-well plate (4513, Corning).
  • the compounds of the present invention have a weak inhibitory effect on VEGFR2 kinase activity, indicating that the compounds of the present invention have a selective inhibitory effect on TGF ⁇ RI kinase.
  • Test Example 3 Determination of the inhibitory effect of the compound of the present invention on p38 ⁇ kinase activity
  • Enzyme activity assay using p38 ⁇ kinase assay kit (V9591, Promega), 2 ⁇ l of lysis buffer (40 mM Tris pH 7.5, 20 mM MgCl 2 , 0.1 mg/ml BSA) was added to 384-well plates (4514, Corning).
  • Enzyme solution (final concentration of enzyme in the reaction system is 0.5 ng/ ⁇ L), 1 ⁇ l of a 3-fold gradient dilution of the compound in 5% DMSO, 2 ⁇ l of a mixture of ATP and p38 substrate polypeptides (ATP final concentration of 50 ⁇ M, substrate final The concentration was 0.2 ⁇ g/ ⁇ L), and after reacting at 27 ° C for 2.5 hours, 5 ⁇ l of the ADP-Glo solution in the kit was added to each well, and the mixture was allowed to stand at 27 ° C for 40 minutes, and 10 ⁇ l of the kinase detection reagent was further added to each well, and the mixture was allowed to stand at 27 ° C for 30 minutes. Chemiluminescent signal values were measured using a Victor 3 (PerkinElmer) multi-plate reader. IC 50 values of inhibitory activity of the compound is calculated using Graphpad prism software corresponding to each concentration of compound according to the signal value.
  • the compounds of the present invention have a weak inhibitory effect on p38 ⁇ kinase activity, indicating that the compound of the present invention has a selective inhibitory effect on TGF ⁇ RI kinase.
  • Test Example 4 Determination of the inhibitory effect of the compound of the present invention on RIPK2 kinase activity
  • Enzyme activity assay using RIPK2 Kinase Assay Kit (V4084, Promega), 2 ⁇ l of a reaction buffer (40 mM Tris pH 7.5, 20 mM MgCl 2 , 0.1 mg/ml BSA) was sequentially added to a 384-well plate (4514, Corning).
  • RIPK2 enzyme solution final concentration of enzyme in the reaction system is 0.5 ng/ ⁇ L
  • 1 ⁇ l of a 3-fold gradient dilution of Compound 1 solution in 5% DMSO 2 ⁇ l of a mixture of ATP and MBP substrate polypeptides (ATP final concentration of 50 ⁇ M, The final concentration of the substrate was 0.2 ⁇ g/ ⁇ L.
  • the compounds of the present invention have a weak inhibitory effect on RIPK2, indicating that the compounds of the present invention have a selective inhibitory effect on TGF ⁇ RI kinase.
  • Test Example 4 Inhibition of proliferation of NIH3T3 cells by the compounds of the present invention
  • the compounds of the present invention have significant inhibitory activity on the proliferation of NIH3T3 cells.
  • Test Example 5 Determination of the inhibitory activity of the compound of the present invention on the Smad signaling pathway of TGF ⁇ RI
  • HepG2 TCHu 72, Cell Culture Bank of the Chinese Academy of Sciences
  • EPM medium 42360-099, Gibco
  • the cells were incubated overnight at 37 ° C, 5% CO 2 .
  • the cells will continue to culture at 37 ° C, 5% CO 2 . hour.
  • EMEM medium containing 0.5% FBS 90 ⁇ l was replaced per well and starved for 6 hours.
  • the compound was configured as a 20 mM stock solution, diluted to a 400x concentration with a 100% DMSO gradient, and diluted 40-fold with EMEM containing 0.5% FBS.
  • ONE-Glo TM Luciferase Assay (E6110 , Promega), at room temperature in the dark for 10 minutes, using Victor3.0 (PerkinElmer) chemiluminescent signal read value.
  • Compound IC 50 value calculated using Graphpad Prism software corresponding to each concentration of compound according to the signal value.
  • the compounds of the present invention have significant inhibitory activities against the Smad signaling pathway of TGF ⁇ RI.
  • Rats were used as test animals, and the concentration of the drug in plasma at different times after administration of the compound of Example 1 and the compound of Example 2 by intragastric administration was determined by LC/MS/MS method.
  • the pharmacokinetic behavior of the compounds of the invention in rats was investigated and their pharmacokinetic characteristics were evaluated.
  • a certain amount of the drug was weighed, and 5% by volume of DMSO, 5% by volume of Tween 80, and 90% by volume of physiological saline were weighed to prepare a clear transparent solution of 0.2 mg/mL.
  • the SD rats were intragastrically administered overnight after fasting, and the dose was 2.0 mg/kg, and the administration volume was 10.0 mL/kg.
  • Example 1 and the compound of Example 2 were administered orally by the rats, and 0.2 mL of blood was collected from the eyelids before and after the administration, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after administration, and heparinized.
  • the tubes were separated by centrifugation at 4 ° C, 3500 rpm for 10 minutes in a test tube, stored at -20 ° C, and fed 2 hours after administration.
  • the content of the test compound in the plasma of rats after different doses of the drug was measured: 25 ⁇ L of rat plasma at each time after administration, 30 ⁇ L of camptothecin (100 ng/mL), 225 ⁇ L of acetonitrile, vortex The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (3600 rpm), and plasma samples were taken for 5.0 ⁇ L of the supernatant for LC/MS/MS analysis.
  • the pharmacokinetic parameters of the compounds of the invention are as follows:
  • the compound of the present invention has good pharmacological absorption and has obvious pharmacokinetic advantages.
  • TGF ⁇ inhibitor was administered to SD rats by repeated administration for 7 days toxicity test.
  • the TGF ⁇ inhibitor Compound 1 was evaluated for toxicity to SD rats by repeated administration for 7 days.
  • Feeding conditions SPF grade, animals were housed in plastic transparent rat cages, 2-3 per cage. Room temperature 20-26 ° C, relative humidity 40 ⁇ 70%. Control lighting for 12 hours / dark for 12 hours. Free and unlimited intake of pellet feed for rats. Drink high-temperature sterilized water freely through the drinking water bottle.
  • the rats in the 7-day toxicity test were given doses of 30 and 100 mg/kg (qd) for 7 days.
  • Route of administration intragastric administration, administration volume: 10 ml/kg, frequency of administration and administration period: daily administration in the morning, continuous administration for 7 days.
  • Test Example 8 Therapeutic effect of TGF ⁇ compound on B16-F1 transplanted tumor C57 mice
  • Example 1 The effect of the compound of Example 1 on the growth of murine melanoma cell B16-F1 xenografts on C57 mice was evaluated.
  • Compound 1 was formulated into a 5 mg/ml aqueous solution with 0.5% CMC-Na + 0.25% (v/v) Polysorbate 80 at a concentration of 50 mg/kg and an oral gavage volume of 10 ml/kg.
  • mice Female
  • mice purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd. (Shanghai, China, certificate SCXK (Shanghai) 2008-0016), purchased at 16-20 g.
  • the experiment was started after 3 days of adaptive feeding. Feeding conditions: SPF grade.
  • Animal feeding method 12/12 hours light/dark cycle adjustment, temperature 23 ⁇ 1°C, humidity 40-50%, animals were given standard sterilized rat feed, free to eat and drink.
  • Blank group 10 0.5% CMC (i.g/bid)
  • Compound 1 group 10 50 mg/kg of Compound 1 (i.g/bid)
  • bid is administered twice a day.
  • the adaptively fed C57 mice were fasted overnight in a metabolic cage, and were weighed into the following groups after randomization: blank control group and compound 1 group, 10 rats in each group.
  • the right flank of C57 mice was prepared for skin one day in advance, and B16-F1 cells (1 ⁇ 10 4 each) were inoculated subcutaneously, and administration was started on the third day after inoculation.
  • Each mouse was intragastrically administered (ig, 10 ml/kg) twice a day, and the blank group was administered with the corresponding solvent. Tumor volume was measured twice a week, body weight was weighed and data was recorded.
  • V 1/2 ⁇ L length ⁇ L short 2
  • Tumor inhibition rate (%) (C-T) / C (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • C and T are the blank control group (Blank) at the end of the experiment and the average tumor volume of the experimental group.
  • Compound 1 was administered on the fourth day after tumor cell transplantation, twice daily, and the tumor volume was significantly small on the 19th day.
  • the tumor inhibition rate was calculated to be 71.12%, and the administration had no effect on the body weight of the mice.

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Abstract

本发明涉及3,4-二吡啶基吡唑类衍生物、其制备方法及其在医药上的应用。具体而言，本发明涉及一种通式(I)所示的新的3,4-二吡啶基吡唑类衍生物、其制备方法及含有该衍生物的药物组合物及其作为治疗剂，特别是作为TGF-β抑制剂的用途和在制备治疗、预防或减少由TGF-β过度表达介导的癌症的药物中的用途，其中通式(I)的各取代基与说明书中的定义相同。

Description

3,4-二吡啶基吡唑类衍生物、其制备方法及其在医药上的应用 技术领域
本发明属于医药领域，涉及一种新的3,4-二吡啶基吡唑类衍生物、其制备方法及含有该衍生物的药物组合物及其作为治疗剂特别是作为TGF-β的抑制剂的用途和在制备治疗、预防或减少由TGF-β过度表达介导的癌症的药物中的用途。
背景技术
转化生长因子TGF-β(Transforming Growth Factorβ)是二聚多肽生长因子超家族中的一员，其包括例如活化素、抑制素、骨形成蛋白(Bone morphogenetic proteins，BMPs)、生长和分化因子(Growth differentiation factors，GDFs)和繆勒氏管抑制物(Müllerian-inhibiting substance，MIS)。
TGF-β有TGF-β1、TGF-β2和TGF-β3三种亚型，它们参与细胞增殖与分化、伤口愈合、细胞外基质产生和免疫抑制的调控。参见如Massague,J.Ann.Rev,Cell.Biol.6:594-641(1990)；Roberts,A.B.Peptide Growth Factor and Their receptors,95:419-472Berlin:Springer-Verlag(1990)；Roberts,A.B.和Sporn M.B.Growth Factor 8:1-9(1993)；以及Alexandrow,M.G.,Moses,H.L.Cancer Res.55:1452-1457(1995)。TGF-β的三种亚型与其受体一起存在于大多数的细胞中。每种TGF-β亚型被合成作为前体蛋白，该前体蛋白在细胞内裂解成C-端区域(潜伏相关肽latency associated peptide，LAP)和N-端部分，被称为成熟或活性TGF-β。从细胞中分泌前，LAP一般与成熟TGF-β以非共价的方式连接。LAP-TGF-β复合体不能与TGF-β受体结合且不具有生物学活性。TGF-β通常通过多种机制包括例如，与血小板反应蛋白-1或纤溶酶相互作用，从复合体中释放出来(并具有活性)。TGF-β1通过两种高保守性单跨膜丝氨酸/苏氨酸激酶转导信号，即I型(ALK5)和II型TGF-β受体。一旦配体诱发低聚化，II型受体过度磷酸化ALK5GS区中的丝氨酸/苏氨酸残基，通过创建Smad蛋白的结合位点引起ALK5的活化。活化的ALK5继而磷酸化C-末端SSXS-基序处的Smad2和Smad3蛋白，从而导致它们从受体中离解，并且与Smad4生成异质复合体(heteromeric complex)。Smad复合体易位于核，与特异性DNA-结合性辅因子和辅调控剂装配，最终活化细胞外基质组分和基质-降解性蛋白酶抑制剂的转录。
TGF-β信号通路的极度活跃是许多人类疾病(如细胞外基质的过量沉积、炎症应答的异常高水平、纤维变性病症以及进行性癌)的原因。在各种癌症的晚期，肿瘤细胞和肿瘤内基质细胞一般过度表达TGF-β。这引起血管生成与细胞运动的刺激、免疫系统的抑制和肿瘤细胞与细胞外基质的相互作用增加(例如,Hojo,M.等人,Nature 397:530-534(1999))。所以，肿瘤细胞变得更有侵袭性，转移至远侧器官例如,Maehara,Y.等人,J.Clin.Oncol.17:607-614(1999)；Picon,A.等人,Cancer  Epidemiol.Biomarkers Prev.7:497-504(1998))。
大量实验性动物研究证明了TGF-β的肾小球表达与纤维化之间的关联，包括增殖性肾小球性肾炎的Thy-1大鼠模型、兔抗-GBM肾小球性肾炎和局灶性节段性肾小球硬化的5/6肾切除大鼠模型，最近已有评述(例如，Bitzer,M.等，Kidney Blood Press.Res.21:1-12(1998))。TGF-β的中和抗体改进Thy-1肾炎模型中的肾小球组织学(如,Border,W.A.等人,Nature 346:371-374(1990))。
TGF-β1及其受体在受损血管和纤维增殖性血管损伤中被过度表达，引起细胞外基质的过度产生(例如，Saltis,J.等人,Clin.Exp.Pharmacol.Physiol.23:193-200(1996)；McCaffrey,T.A.等人，J.Clin.Invest.96:2667-2675(1995))。
TGF-β2水平在大多数患有少年青光眼的眼房水肿瘤眼中和几乎一半患有原发性开放角度青光眼(POAG)的眼中都有增加(例如，Picht,G.等人,Graefes Arch.Clin.Exp.Ophthalmol.239:199-207(2001))。TGF-β1和TGF-β2亚型据报道都增加培养的来自假性剥脱性青光眼和POAG患者的人特农氏囊成纤维细胞的细胞外基质产生(例如，Kottler,U.B.等人,Exp.Eye Res.80:121-134(2005))。
因此希望开发出对TGF-β家族成员的抑制剂来预防和/或治疗包括这种信号通路的疾病。公开的TGF-β家族成员受体的调节剂(如拮抗剂)专利申请包括WO2004111046、WO2012000595、WO2012002680、WO2013009140、WO2016106266。
为了达到更好的治疗效果的目的，更好的满足市场需求，发明人希望能开发出新一代的高效低毒的TGF-β受体激酶抑制剂。本发明将提供一种新型结构的TGF-β受体激酶抑制剂，并发现此类结构的化合物对ALK5具有良好的选择性活性和毒性试验结果显示此类结构的化合物具有较好的安全性，并表现出优异的TGF-β受体抑制作用和抑瘤效果。
发明内容
本发明的目的在于提供一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐，
Figure PCTCN2017110686-appb-000001
其中：
环A为环烷基或杂环基；
R1相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、-S(O)mR5、-C(O)OR5、环烷基和杂环基；
R2相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7；
R3相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氨基、氰基和硝基；
R4相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
R5选自氢原子、烷基、氨基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
R6和R7各自独立地选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
n为0、1或2；
s为0、1、2、3、4或5；
p为0、1、2或3；
q为0、1、2、3或4；且
m为0、1或2。
在本发明的一个优选的实施方案中，所述的通式(I)所示的化合物，其为通式(II)所示的化合物：
Figure PCTCN2017110686-appb-000002
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
G1为CH2、NR8或O；
R8选自-S(O)mR5、-C(O)OR5、氢原子或烷基；
R2、R4和R5如通式(I)中所定义；
s为0、1或2；
m为0、1或2；且
r为0、1、2或3。
在本发明的一个优选的实施方案中，所述的通式(I)所示的化合物，其中R2选自氰基、-C(O)R5或-S(O)mR5，R5为羟基或烷基，优选R2为氰基或甲磺酰基，且s为1。
在本发明的一个优选的实施方案中，所述的通式(I)所示的化合物，其R4为烷基。
本发明的化合物包括其所有构象异构体，例如顺式和反式异构体；以及其所有旋光异构体和立体异构体及其混合物。本发明的化合物具有不对称中心，因此存在不同的对映异构体与非对映异构体。本发明涉及本发明化合物的用途，和可以采用和含有它们的所有药物组合物和治疗方法。本发明涉及所有这类异构体及其混合物的用途。
本发明的典型化合物包括但不限于：
Figure PCTCN2017110686-appb-000003
Figure PCTCN2017110686-appb-000004
Figure PCTCN2017110686-appb-000005
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐。
本发明还涉及一种通式(I-A)所示的化合物，其为合成通式(I)化合物的中间体：
Figure PCTCN2017110686-appb-000006
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
其中：
X为卤素，优选为溴；
环A、R1、R4、n和q如通式(I)中所定义。
通式(I-A)所示的化合物包括，但不限于：
Figure PCTCN2017110686-appb-000007
Figure PCTCN2017110686-appb-000008
本发明的另一方面涉及一种制备通式(I)所示的化合物的方法，该方法包括：
Figure PCTCN2017110686-appb-000009
通式(I-A)的化合物和通式(I-B)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(I)的化合物，
其中：
G2为
Figure PCTCN2017110686-appb-000010
X为卤素，优选为溴；
环A、R1～R4、n、s、p和q如通式(I)中所定义。
本发明的另一方面涉及一种制备通式(II)所示的化合物的方法，该方法包括：
Figure PCTCN2017110686-appb-000011
通式(II-A)的化合物和通式(I-B)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(II)的化合物，
其中：
G2为
Figure PCTCN2017110686-appb-000012
X为卤素，优选为溴；
G1、R2、R4、r、s和q如通式(I)中所定义。
本发明的另一方面涉及一种药物组合物，所述药物组合物含有治疗有效剂量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或可药用的盐，以及一种或多种药学上可接受的载 体、稀释剂或赋形剂。本发明还涉及一种制备上述组合物的方法，其包括将通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐与一种或多种药学上可接受的载体、稀释剂或赋形剂相混合。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，在制备用于抑制TGF-β和/或活化素(特别是人类TGF-β和/或活化素)信号传导途径的药物中的用途。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，在制备用于治疗、预防或减少肿瘤细胞(特别是人类肿瘤细胞)转移的药物中的用途。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物在制备用于治疗、预防或减少由TGF-β过度表达介导的癌症的药物中的用途，特别是通过抑制人类TGF-β信号传导途径来治疗、预防或减少由TGF-β过度表达介导的癌症的药物中的用途。
本发明进一步涉及通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物在制备用于治疗、预防或减轻(特别是人类的)选自下述疾病的药物中的用途：血管损伤、肾小球性肾炎、糖尿病性肾病、狼疮性肾炎、高血压-诱发的肾病、肾间质性纤维化、由药物暴露并发症所致肾纤维化、与HIV有关的肾病、移植物肾病、各种病因的肝纤维化、可归因于感染的肝功能障碍、酒精诱发的肝炎、囊性纤维化病、间质性肺病、急性肺损伤、成人呼吸窘迫综合征、特发性肺纤维化、慢性阻塞性肺病、由感染性或毒性因子引起的肺病、梗塞后心纤维化、充血性心力衰竭、扩张型心肌病、心肌炎、内膜增厚、血管狭窄、高血压引起的血管重构、肺动脉高压、冠状动脉再狭窄、周围动脉再狭窄、颈动脉再狭窄、支架诱导的再狭窄、动脉粥样硬化、眼部瘢痕形成、角膜瘢痕形成、增生性玻璃体视网膜病变、青光眼、眼内压高、发生在由创伤或手术伤口所致伤口愈合期间的过度性或肥厚性真皮瘢痕或瘢痕疙瘩形成、腹膜与皮下粘连、硬皮病、纤维硬化、进行性系统性硬化病、皮肌炎、多肌炎、关节炎、骨质疏松、溃疡、神经系统功能减低、男性勃起功能障碍、佩罗尼氏病、杜普伊特伦氏挛缩、阿尔茨海默氏病、雷诺氏综合征、辐射-诱发的纤维化、血栓形成、肿瘤转移性生长、多发性骨髓瘤、 黑素瘤、神经胶质瘤、胶质母细胞瘤、白血病、肉瘤、平滑肌瘤、间皮瘤、乳腺癌、宫颈癌、肺癌、胃癌、直肠癌、结肠癌、胰腺癌、脑癌、皮肤癌、口腔癌、前列腺癌、骨癌、肾癌、卵巢癌、膀胱癌和肝癌。
本发明进一步涉及一种治疗、预防或减少人类肿瘤细胞转移的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物。
本发明进一步涉及一种治疗、预防或减少由TGF-β过度表达介导的癌症的方法，特别是通过抑制TGF-β信号传导途径来治疗、预防或减少由TGF-β过度表达介导的癌症的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物。
本发明进一步涉及一种治疗、预防或减轻(特别是人类的)选自下述疾病的方法：血管损伤、肾小球性肾炎、糖尿病性肾病、狼疮性肾炎、高血压-诱发的肾病、肾间质性纤维化、由药物暴露并发症所致肾纤维化、与HIV有关的肾病、移植物肾病、各种病因的肝纤维化、可归因于感染的肝功能障碍、酒精诱发的肝炎、囊性纤维化病、间质性肺病、急性肺损伤、成人呼吸窘迫综合征、特发性肺纤维化、慢性阻塞性肺病、由感染性或毒性因子引起的肺病、梗塞后心纤维化、充血性心力衰竭、扩张型心肌病、心肌炎、内膜增厚、血管狭窄、高血压引起的血管重构、肺动脉高压、冠状动脉再狭窄、周围动脉再狭窄、颈动脉再狭窄、支架诱导的再狭窄、动脉粥样硬化、眼部瘢痕形成、角膜瘢痕形成、增生性玻璃体视网膜病变、青光眼、眼内压高、发生在由创伤或手术伤口所致伤口愈合期间的过度性或肥厚性真皮瘢痕或瘢痕疙瘩形成、腹膜与皮下粘连、硬皮病、纤维硬化、进行性系统性硬化病、皮肌炎、多肌炎、关节炎、骨质疏松、溃疡、神经系统功能减低、男性勃起功能障碍、佩罗尼氏病、杜普伊特伦氏挛缩、阿尔茨海默氏病、雷诺氏综合征、辐射-诱发的纤维化、血栓形成、肿瘤转移性生长、多发性骨髓瘤、黑素瘤、神经胶质瘤、胶质母细胞瘤、白血病、肉瘤、平滑肌瘤、间皮瘤、乳腺癌、宫颈癌、肺癌、胃癌、直肠癌、结肠癌、胰腺癌、脑癌、皮肤癌、口腔癌、前列腺癌、骨癌、肾癌、卵巢癌、膀胱癌和肝癌，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含 其的药物组合物，其作用药物。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，其作用TGF-β受体激酶抑制剂。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，其用于治疗、预防或减少肿瘤细胞(特别是人类肿瘤细胞)转移。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，其用于治疗、预防或减少由TGF-β过度表达介导的癌症，特别是通过抑制TGF-β信号传导途径来治疗、预防或减少由TGF-β过度表达介导的癌症。
本发明进一步涉及一种通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用盐或包含其的药物组合物，其用于治疗、预防或减轻(特别是人类的)选自下述的疾病：血管损伤、肾小球性肾炎、糖尿病性肾病、狼疮性肾炎、高血压-诱发的肾病、肾间质性纤维化、由药物暴露并发症所致肾纤维化、与HIV有关的肾病、移植物肾病、各种病因的肝纤维化、可归因于感染的肝功能障碍、酒精诱发的肝炎、囊性纤维化病、间质性肺病、急性肺损伤、成人呼吸窘迫综合征、特发性肺纤维化、慢性阻塞性肺病、由感染性或毒性因子引起的肺病、梗塞后心纤维化、充血性心力衰竭、扩张型心肌病、心肌炎、内膜增厚、血管狭窄、高血压引起的血管重构、肺动脉高压、冠状动脉再狭窄、周围动脉再狭窄、颈动脉再狭窄、支架诱导的再狭窄、动脉粥样硬化、眼部瘢痕形成、角膜瘢痕形成、增生性玻璃体视网膜病变、青光眼、眼内压高、发生在由创伤或手术伤口所致伤口愈合期间的过度性或肥厚性真皮瘢痕或瘢痕疙瘩形成、腹膜与皮下粘连、硬皮病、纤维硬化、进行性系统性硬化病、皮肌炎、多肌炎、关节炎、骨质疏松、溃疡、神经系统功能减低、男性勃起功能障碍、佩罗尼氏病、杜普伊特伦氏挛缩、阿尔茨海默氏病、雷诺氏综合征、辐射-诱发的纤维化、血栓形成、肿瘤转移性生长、多发性骨髓瘤、黑素瘤、神经胶质瘤、胶质母细胞瘤、白血病、肉瘤、平滑肌瘤、间皮瘤、乳腺癌、宫颈癌、肺癌、胃癌、直肠癌、结肠癌、胰腺癌、脑癌、皮肤癌、口腔癌、前列腺癌、骨癌、肾癌、卵巢癌、膀胱癌和肝癌。
含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂。可按照本领域任何已知制备药用组合物的方法制备口服组合物，此类组合物可含有一种或多种选自以下的成分：甜味剂、矫味剂、着色剂和防腐剂，以提供悦目和可口的药用制剂。片剂含有活性成分和用于混合的适宜制备片剂的无毒的可药用的赋形剂。
水悬浮液含有活性物质和用于混合的适宜制备水悬浮液的赋形剂。此类赋形剂是悬浮剂。
油混悬液可通过使活性成分悬浮于植物油中配制而成。油悬浮液可含有增稠剂。可加入上述的甜味剂和矫味剂，以提供可口的制剂。可通过加入抗氧化剂保存这些组合物。
通过加入水可使适用于制备水混悬的可分散粉末和颗粒提供活性成分和用于混合的分散剂或湿润剂、悬浮剂或一种或多种防腐剂。适宜的分散剂或湿润剂和悬浮剂可说明上述的例子。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂保存这些组合物。
本发明的药物组合物也可以是水包油乳剂的形式。油相可以是植物油。适宜的乳化剂可以是天然产生的磷脂。乳剂也可以含有甜味剂、矫味剂、防腐剂和抗氧剂。此类制剂也可含有缓和剂、防腐剂、着色剂和抗氧剂。
本发明的药物组合物可以是无菌注射水溶液形式。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。
本发明的药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在肠胃外可接受的无毒稀释剂或溶剂中制备的无菌注射溶液或混悬液此外，可方便地用无菌固定油作为溶剂或悬浮介质。
可按用于直肠给药的栓剂形式给予本发明化合物。可通过将药物与在普通温度下为固体但在直肠中为液体，因而在直肠中会溶化而释放药物的适宜的无刺激性赋形剂混合来制备这些药物组合物。
如本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用具体化合物的活性、患者的年龄、患者的体重、患者的健康状况、患者的行被、患者的饮食、给药时间、给药方式、排泄的速率、药物的组合等；另外，最佳的治疗方式如治疗的模式、通式化合物(I)的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。
发明的详细说明
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基 团，优选含有1至12个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至6个碳原子，例如3、4、5或6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选为环丙基、环丁基、环戊基和环己基；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“螺环烷基”指5至20元的单环之间共用一个碳原子(称螺原子)的多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基或多螺环烷基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺环烷基。螺环烷基的非限制性实例包括：
Figure PCTCN2017110686-appb-000013
术语“稠环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。稠环烷基的非限制性实例包括：
Figure PCTCN2017110686-appb-000014
术语“桥环烷基”指5至20元，任意两个环共用两个不直接连接的碳原子的全碳多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更有选为双环或三环。桥环烷基的非限制性实例包括：
Figure PCTCN2017110686-appb-000015
所述环烷基环可以稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性实例包括茚满基、四氢萘基、苯并环庚烷基等。环烷基可以是任选取代的或非取代的，当被取代时，取代基优选为独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“杂环基”指饱和/或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子，例如1、2、3或4个是杂原子；更优选杂环基包含3至10个环原子，最优选为杂环基包含3至6个环原子，例如3、4、5或6个环原子。单环杂环基的非限制性实例包括氧杂环丁基、四氢呋喃基、四氢吡喃基、吡咯基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等，优选为氧杂环丁基和四氢呋喃基；多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性实例包括：
Figure PCTCN2017110686-appb-000016
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性实例包括：
Figure PCTCN2017110686-appb-000017
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性实例包括：
Figure PCTCN2017110686-appb-000018
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2017110686-appb-000019
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“芳基”指6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，其为具有共轭的π电子体系的多环(即其带有相邻对碳原子的环)基团，优选为6至10元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2017110686-appb-000020
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、吡唑 基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2017110686-appb-000021
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、氨基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7中的一个或多个取代基所取代。
术语“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH2。
术语“氰基”指-CN。
术语“硝基”指-NO2。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或 理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“可药用盐”是指本发明化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。
m和R5～R7如通式(I)中所定义。
附图说明：
图1为化合物1对B16-F1荷瘤C57小鼠移植瘤体积的影响。
图2为化合物1对C57小鼠体重的影响。
具体实施方式
本发明化合物的合成方法
为了完成本发明的目的，本发明采用如下技术方案：
方案一
本发明通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2017110686-appb-000022
Figure PCTCN2017110686-appb-000023
第一步，通式(I-1)的化合物和通式(I-2)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(I-3)的化合物；
第二步，通式(I-3)的化合物和联硼酸频那醇酯在碱性条件下，在催化剂存在下反应，得到通式(I-B)的化合物；
第三步，通式(I-4)的化合物和通式(I-5)的化合物在碱性条件下反应，得到通式(I-6)的化合物；
第四步，通式(I-6)的化合物和卤代试剂反应，得到通式(I-A)的化合物；
第五步，通式(I-A)的化合物和通式(I-B)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(I)的化合物；
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、双三甲基硅基胺基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、醋酸钾、碳酸铯、氢氧化钠和氢氧化锂；
催化剂包括但不限于钯/碳、雷尼镍、四-三苯基膦钯、二氯化钯、醋酸钯、[1,1'-双(二苯基膦基)二茂铁]二氯化钯、1,1’-双(二苄基磷)二氯二戊铁钯或三(二亚苄基丙酮)二钯，优选为[1,1'-双(二苯基膦基)二茂铁]二氯化钯或四-三苯基膦钯；
卤代试剂包括但不限于液溴、溴化氢、N-溴代丁二酰亚胺(NBS)、PBr3、POBr3、过溴化吡啶氢溴酸盐(PHP)、四溴环酮(TBCO)、溴代丙二酸二乙酯、四丁基溴化铵，N-氯代琥珀酰亚胺，PCl3和POCl3；
上述反应优选在溶剂中进行，所用溶剂包括但不限于：醋酸、甲醇、乙醇、甲苯、四氢呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亚砜、1,4-二氧六环、水、N,N-二甲基甲酰胺及其混合物；
其中：
G2和G3各自独立的选自
Figure PCTCN2017110686-appb-000024
X为卤素，优选为溴；
环A、R1～R4、n、s、p和q如通式(I)中所定义。
方案二
本发明通式(II)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2017110686-appb-000025
通式(II-A)的化合物和通式(I-B)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(II)的化合物；
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、双三甲基硅基胺基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、醋酸钾、碳酸铯、氢氧化钠和氢氧化锂；
催化剂包括但不限于钯/碳、雷尼镍、四-三苯基膦钯、二氯化钯、醋酸钯、[1,1'-双(二苯基膦基)二茂铁]二氯化钯、1,1’-双(二苄基磷)二氯二戊铁钯或三(二亚苄基丙酮)二钯，优选为[1,1'-双(二苯基膦基)二茂铁]二氯化钯或四-三苯基膦钯；
上述反应优选在溶剂中进行，所用溶剂包括但不限于：醋酸、甲醇、乙醇、甲苯、四氢呋喃、二氯甲烷、石油醚、乙酸乙酯、正己烷、二甲基亚砜、1,4-二氧六环、水、N,N-二甲基甲酰胺及其混合物；
其中：
G2选自
Figure PCTCN2017110686-appb-000026
X为卤素，优选为溴；
G1、R2、R4、r、s和q如通式(I)中所定义。
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商:Thermo,型号：Finnigan LCQ advantage MAX)。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
手性HPLC分析测定使用LC-10A vp(Shimadzu)或者SFC-analytical(Berger Instruments Inc.)；
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
手性制备柱层析使用Prep Star SD-1(Varian Instruments Inc.)或SFC-multigram(Berger Instruments Inc.)。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
激酶平均抑制率及IC50值的测定用NovoStar酶标仪(德国BMG公司)。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，C：石油醚/乙酸乙酯体系，D：丙酮，E：二氯甲烷/丙酮体系，F：乙酸乙酯/二氯甲烷体系，G：乙酸乙酯/二氯甲烷/正己烷，H：乙酸乙酯/二氯甲烷/丙酮，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
实施例1
4-(1-环丁基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000027
第一步
4-溴-2-(4-(甲磺酰基)苯基)吡啶1c
将2,4-二溴吡啶1a(6g，25.33mmol)溶于126mL甲苯、乙醇和水(V:V:V＝4:2:1)混合溶剂中，加入(4-(甲磺酰基)苯基)硼酸1b(5066.1mg，25.33mmol)，碳酸钠(5369.1mg，50.66mmol)和四三苯基膦钯(2926.7mg，2.53mmol)，氩气保护下，100℃下搅拌反应16小时。LC-MS监测反应至原料消失，停止反应，冷却反应，加入乙酸乙酯和水，用乙酸乙酯萃取(80mL×2)，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物1c(7.09g，白色固体)，产率：89.79％。
第二步
2-(4-(甲磺酰基)苯基)-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)吡啶1d
将1c(210mg，0.67mmol)，联硼酸频那醇酯(205mg，0.81mmol)，醋酸钾(131.5mg，1.34mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(49mg，0.067mmol)溶于10mL 1,4-二氧六环中，升温至90℃搅拌反应3小时。反应液减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物1d(130mg，黑色固体)，产率：50％。
第三步
2-(1-环丁基-1H-吡唑-3-基)-6-甲基吡啶1g
将2-甲基-6-(1H-吡唑-3-基)吡啶1e(2.01g，12.6mmol，采用公知的方法“Bioorganic and Medicinal Chemistry,2015,23(6),1260-1275”制备而得)溶于100mLN,N-二甲基甲酰胺中，再加入甲苯-4-磺酸环丁酯1f(4.29g，18.9mmol，采用专利申请“WO2009093029”公开的方法制备而得)和碳酸铯(8.23g，25.3mmol)，加热至60℃搅拌反应16小时。冷却至室温，旋干N,N-二甲基甲酰胺，加入水，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓 缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题化合物1g(1.81g，无色油状物)，产率：67.3％。
第四步
2-(4-溴-1-环丁基-1H-吡唑-3-基)-6-甲基吡啶1h
将1g(91mg，0.43mmol)溶于10mL二氯甲烷中，加入N-溴代琥珀酰亚胺(76mg，0.43mmol)，室温搅拌过夜。反应结束后，减压浓缩反应液，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题化合物1h(110mg，黄色油状物)，产率：88％。
第五步
4-(1-环丁基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶1
将1h(540mg，1.85mmol)，1d(563.36mg，2.03mmol)，1,1'-双(二苯基膦基)二茂铁(102.46mg，0.185mmol)，碳酸钾(510.89mg，3.7mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(135.24mg，0.185mmol)溶于12mL 1,4-二氧六环和水(V:V＝10:1)混合溶剂中，升温至80℃搅拌反应12小时。反应液减压浓缩，所得残余物中加入水，乙酸乙酯萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，用高效液相色谱法纯化所得残余物，得到标题化合物1(400mg，黄白色固体)，产率：48.7％。
MS m/z(ESI):445.5[M+1]
1H NMR(400MHz,CDCl3)δ8.66(d,1H),8.11(dd,4H),7.98(s,1H),7.86(s,1H),7.69(t,1H),7.56(d,1H),7.41(s,1H),7.20(d,1H),4.96-4.92(m,1H),3.13(s,3H),2.72-2.60(m,4H),2.55(s,3H),2.01-1.94(m,2H)。
实施例2
4-(1-环丙基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000028
第一步
2-(1-环丙基-1H-吡唑-3-基)-6-甲基吡啶2b
将1e(700mg，4.40mmol)，环丙基硼酸2a(756mg，8.80mmol)，一水合乙酸铜(1.76g，8.80mmol)，碳酸钠(933mg，8.80mmol)和2,2-联吡啶(1.37g，8.80mmol)溶于25mL 1,2-二氯乙烷中，升温至50℃搅拌反应16小时。反应结束后，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题化合物2b(440mg，黄色粘稠物)，产率：50.2％。
第二步
2-(4-溴-1-环丙基-1H-吡唑-3-基)-6-甲基吡啶2c
将2b(330mg，1.66mmol)溶于8mL二氯甲烷中，加入N-溴代琥珀酰亚胺(295mg，1.66mmol)，室温搅拌1小时。反应结束后，反应液中加入水，二氯甲烷萃取(10mL×3)，合并有机相，减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物2c(335mg，黄色粘稠物)，产率：72.6％。
第三步
4-(1-环丙基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶2
将2c(40mg，0.144mmol)，1d(103.5mg，0.288mmol)，磷酸钾(183.4mg，0.864mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(10.5mg，0.0144mmol)溶于2mL 1,4-二氧六环中，升温至90℃搅拌反应16小时。反应液减压浓缩，所得残余物中加入水，乙酸乙酯萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，用高效液相色谱法纯化所得残余物，得到标题化合物2(10mg，黄白色固体)，产率：16％。
MS m/z(ESI):431.5[M+1]
1H NMR(400MHz,CD3OD)δ8.55(dd,1H),8.32(s,1H),8.12-8.05(m,4H),7.91(dd,1H),7.82(t,1H),7.53(d,1H),7.37-7.34(m,2H),3.86-3.81(m,1H),3.18(s,3H),2.48(s,3H),1.30-1.26(m,2H),1.18-1.13(m,2H)。
实施例3
4-(1-环戊基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000029
Figure PCTCN2017110686-appb-000030
第一步
(2-(4-(甲磺酰基)苯基)吡啶-4-基)硼酸3a
将1c(1.6g，5.12mmol)，联硼酸频那醇酯(1.56g，6.15mmol)，醋酸钾(1g，10.24mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(375mg，0.512mmol)溶于50mL 1,4-二氧六环中，氩气保护下，90℃搅拌反应16小时。反应液减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物3a(1.41g，棕色固体)，产率：100％。
MS m/z(ESI):278.4[M+1]
第二步
2-(1-环戊基-1H-吡唑-3-基)-6-甲基吡啶3c
将1e(700mg，4.40mmol)溶于10mL N,N-二甲基甲酰胺中，加入甲苯-4-磺酸环戊酯3b(1.056g，4.40mmol，采用专利申请“WO2009062990”公开的方法制备而得)和碳酸铯(2.87g，8.80mmol)，60℃搅拌反应2小时。冷却至室温，加入50mL水，乙酸乙酯萃取(50mL×3)，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，用CombiFlash快速制备仪以洗脱剂体系B纯化所得残余物，得到标题化合物3c(600mg，淡黄色油状物)，产率：60％。
第三步
2-(4-溴-1-环戊基-1H-吡唑-3-基)-6-甲基吡啶3d
将3c(600mg，2.64mmol)溶于15mL二氯甲烷中，加入N-溴代琥珀酰亚胺(470mg，2.64mmol)，室温搅拌1小时。反应结束后，反应液减压浓缩，用CombiFlash快速制备仪以洗脱剂体系B纯化所得残余物，得到标题化合物3d(760mg，淡黄色油状物)，产率：94.0％。
MS m/z(ESI):306.3[M+1]
第四步
4-(1-环戊基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶3
将3d(61mg，0.2mmol)，3a(67mg，0.24mmol)，1,1'-双(二苯基膦基)二茂铁(11mg，0.02mmol)，碳酸钾(55mg，0.4mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(15mg，0.02mmol)溶于5.5mL 1,4-二氧六环和水(V:V＝10:1)混合溶剂中，氩气保护下，升温至80℃搅拌反应6小时。反应液减压浓缩，用高效液相色谱法纯 化所得残余物，得到标题化合物3(20mg，白色固体)，产率：21.9％。
MS m/z(ESI):459.4[M+1]
1H NMR(400MHz,CDCl3)δ8.68(d,1H),δ8.18(d,2H),δ8.08(d,2H),7.84(s,1H),7.70(t,1H),7.60-7.56(m,1H),7.35-7.30(m,2H),7.21(d,1H),4.89-4.78(m,1H),3.14(d,3H),2.54(d,3H),2.36-2.29(m,2H),2.22-2.15(m,2H),2.01-1.92(m,2H),1.87-1.78(m,2H)。
实施例4
(S)-4-(3-(6-甲基吡啶-2-基)-1-(四氢呋喃-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000031
第一步
(S)-2-甲基-6-(1-(四氢呋喃-3-基)-1H-吡唑-3-基)吡啶4b
将1e(328mg，2.06mmol)溶于10mL N,N-二甲基甲酰胺中，加入(R)-甲苯-4-磺酸四氢呋喃-3-基酯4a(500mg，2.06mmol，采用专利申请“WO2016021192”公开的方法制备而得和碳酸铯(1.3g，4.12mmol)，60℃搅拌反应2小时。反应液减压浓缩，残余物中加入水，乙酸乙酯萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品标题化合物4b(370mg，无色透明油状物)，产品不经纯化直接进行下一步反应。
第二步
(S)-2-(4-溴-1-(四氢呋喃-3-基)-1H-吡唑-3-基)-6-甲基吡啶4c
将粗品4b(800mg，3.47mmol)溶于18mL二氯甲烷中，加入N-溴代琥珀酰亚胺(741.76mg，4.16mmol)，室温搅拌1小时。反应结束后，反应液中加入水，二氯甲烷萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物4c(123mg，黄白色固 体)，产率：11.39％。
第三步
(S)-4-(3-(6-甲基吡啶-2-基)-1-(四氢呋喃-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶4
将4c(123mg，0.4mmol)溶于5.5mL 1,4-二氧六环和水(V:V＝10:1)混合溶剂中，加入1d(166mg，0.6mmol)，1,1'-双(二苯基膦基)二茂铁(22mg，0.04mmol)，碳酸钾(110mg，0.8mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(29mg，0.04mmol)，氩气保护下，升温至80℃搅拌反应16小时。反应液减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物4(20mg，黄色固体)，产率：10.86％。
MS m/z(ESI):461.5[M+1]
1H NMR(400MHz,CD3OD)δ8.54(d,1H),8.30(s,1H),8.06(q,4H),7.92(s,1H),7.79(t,1H),7.55(d,1H),7.37-7.31(m,2H),5.17(m,1H),4.18(q,2H),4.12(m,1H),3.98(t,1H),3.13(s,3H),2.57-2.50(m,2H),2.49(s,3H)。
实施例5
(R)-4-(3-(6-甲基吡啶-2-基)-1-(四氢呋喃-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000032
第一步
(R)-2-甲基-6-(1-(四氢呋喃-3-基)-1H-吡唑-3-基)吡啶5b
将1e(637mg，4.0mmol)溶于20mL N,N-二甲基甲酰胺中，加入(S)-甲苯-4-磺酸四氢呋喃-3-基酯5a(1.45g，6.0mmol，采用专利申请“WO2014049133”公开的方法制备而得和碳酸铯(2.61g，8.0mmol)，60℃搅拌反应16小时。反应液减压浓缩，用CombiFlash快速制备仪以洗脱剂体系B纯化所得残余物，得到标题化合物 5b(610mg，无色油状物)，产率：66.5％。
第二步
(R)-2-(4-溴-1-(四氢呋喃-3-基)-1H-吡唑-3-基)-6-甲基吡啶5c
将5b(600mg，2.62mmol)溶于30mL二氯甲烷中，加入N-溴代琥珀酰亚胺(466mg，2.62mmol)，室温搅拌16小时。反应结束后，减压浓缩，用CombiFlash快速制备仪以洗脱剂体系B纯化所得残余物，得到标题化合物5c(795mg，黄色油状物)，产率：98.6％。
第三步
(R)-4-(3-(6-甲基吡啶-2-基)-1-(四氢呋喃-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶5
将5c(100mg，0.32mmol)溶于15mL 1,4-二氧六环和水(V:V＝10:1)混合溶剂中，加入1d(135mg，0.49mmol)，1,1'-双(二苯基膦基)二茂铁(18mg，0.032mmol)，碳酸钾(88mg，0.64mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(23mg，0.032mmol)，氩气保护下，升温至80℃搅拌反应16小时。反应结束后，反应液减压浓缩，用CombiFlash快速制备仪以洗脱剂体系A纯化所得残余物，得到标题化合物5(58mg，浅褐色固体)，产率：38.9％。
MS m/z(ESI):461.5[M+1]
1H NMR(400MHz,CDCl3)δ8.66-8.65(d,1H),8.16-8.14(d,2H),8.06-8.04(d,2H),7.92(s,1H),7.88(s,1H),7.68-7.64(t,1H),7.51-7.49(d,1H),7.36-7.34(dd,1H),7.20-7.18(d,1H),5.19(m,1H),4.19-4.24(m,2H),4.14-4.10(m,1H),4.03-4.01(m,1H),3.13(s,3H),2.66-2.57(m,1H),2.55(s,3H),2.51-2.42(m,1H)。
实施例6
4-(3-(6-甲基吡啶-2-基)-1-(氧杂环丁-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶
Figure PCTCN2017110686-appb-000033
Figure PCTCN2017110686-appb-000034
第一步
2-甲基-6-(1-(氧杂环丁-3-基)-1H-吡唑-3-基)吡啶6b
将1e(749mg，4.7mmol)溶于15mL N,N-二甲基甲酰胺中，加入甲苯-4-磺酸氧杂环丁-3-基酯6a(1.61g，7.05mmol，采用专利申请“WO2013056070”公开的方法制备而得)和碳酸铯(3.06g，9.4mmol)，60℃搅拌反应3小时。反应液减压浓缩，用CombiFlash快速制备仪以洗脱剂体系A纯化所得残余物，得到标题化合物6b(420mg，淡黄色油状物)，产率：41.6％。
第二步
2-(4-溴-1-(氧杂环丁-3-基)-1H-吡唑-3-基)-6-甲基吡啶6c
将6b(420mg，1.95mmol)溶于30mL二氯甲烷中，加入N-溴代琥珀酰亚胺(347mg，1.95mmol)，室温搅拌16小时。反应结束后，减压浓缩，用CombiFlash快速制备仪以洗脱剂体系A纯化所得残余物，得到标题化合物6c(479mg，淡黄色油状物)，产率：83.4％。
第三步
4-(3-(6-甲基吡啶-2-基)-1-(氧杂环丁-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶6
将6c(50mg，0.17mmol)溶于5.5mL 1,4-二氧六环和水(V:V＝10:1)混合溶剂中，加入1d(91.6mg，0.25mmol)，1,1'-双(二苯基膦基)二茂铁(9.42mg，0.02mmol)，碳酸钾(46.99mg，0.34mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(12.44mg，0.02mmol)，氩气保护下，升温至80℃搅拌反应18小时。LC-MS监测反应至原料消失，停止反应。冷却反应，浓缩大部分溶剂，加入10mL水，用乙酸乙酯萃取(10mL×3)，合并有机相，用饱和氯化钠溶液洗涤(10mL×2)，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题化合物6(41mg，黄色固体)，产率：52.43％。
MS m/z(ESI):447.4[M+1]
1H NMR(400MHz,CDCl3)δ8.65(d,1H),8.07(dd,4H),7.99(s,1H),7.91(s,1H),7.64(t,1H),7.54(d,1H),7.34(d,1H),7.18(d,1H),5.64-5.58(m,1H),5.17-5.13(m,4H),3.10(s,3H),2.51(s,3H)。
实施例7
4-(4-(1-环丙基-3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)吡啶-2-基)苯甲腈7
Figure PCTCN2017110686-appb-000035
采用实施例2合成路线，将第三步原料化合物1d替换为4-(4-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)吡啶-2-基)苯甲腈(采用专利申请“KR20160025776”公开的方法制备而得)，制得标题化合物7(10mg)。
MS m/z(ESI):378.4[M+1]
1H NMR(400MHz,CD3OD)δ8.67-8.66(d,1H),8.48(s,1H),8.25-8.21(m,1H),8.13-8.11(m,3H),7.91-7.89(d,2H),7.75-7.72(m,2H),7.56-7.54(d,1H),3.98-3.92(m,1H),2.77(s,3H),1.35-1.28(m,2H),1.22-1.18(m,2H)。
实施例8
(R)-4-(3-(6-甲基吡啶-2-基)-1-(1-(甲磺酰基)吡咯烷-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶8
Figure PCTCN2017110686-appb-000036
第一至三步
(R)-3-(3-(6-甲基吡啶-2-基)-4-(2-(4-(甲磺酰基)苯基)吡啶-4-基)-1H-吡唑-1-基)吡咯烷-1-甲酸叔丁酯8d
采用实施例3合成路线，将第二步原料化合物3b替换为(S)-3-(对甲基苯基磺酰基氧基)吡咯烷-1-甲酸叔丁酯8a(采用专利申请“WO2016034134”公开的方法制备而得)，制得标题化合物8d(190mg)。
第四步
(R)-4-(3-(6-甲基吡啶-2-基)-1-(吡咯烷-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶盐酸盐8e
将化合物8d(190mg，0.3395mmol)溶于10mL乙酸乙酯中，加入4M氯化氢的1,4-二氧六环溶液(0.42mL，1.69mmol)，搅拌反应4小时。反应液减压浓缩，得粗品标题化合物8e(200mg)，产物不经纯化直接下一步反应。
第五步
(R)-4-(3-(6-甲基吡啶-2-基)-1-(1-(甲磺酰基)吡咯烷-3-基)-1H-吡唑-4-基)-2-(4-(甲磺酰基)苯基)吡啶8
将粗品化合物8e(100mg，0.2176mmol)溶于3mL吡啶，滴加入甲烷磺酰氯(74.8mg，0.6528mmol)，搅拌反应16小时。反应液减压浓缩，加入20mL饱和硫酸酮溶液，用乙酸乙酯萃取(10mL×3)，合并有机相，得标题化合物8(20mg)，产率：17.1％。
MS m/z(ESI):538.5[M+1]
1H NMR(400MHz,CD3OD)δ8.55-8.54(d,1H),8.35(s,1H),8.12-8.10(m,2H),8.06-8.04(d,2H),7.93(s,1H),7.82-7.78(m,1H),7.56-7.54(d,1H),7.39-7.37(dd,1H),7.33-7.31(d,1H),5.22-5.15(m,1H),3.93-3.87(m,2H),3.76-3.69(m,1H),3.62-3.56(m,1H),3.17(s,3H),2.94(s,3H),2.62-2.57(m,2H),2.45(s,3H)。
测试例：
生物学评价
测试例1、本发明化合物对TGFβRI激酶活性的抑制作用的测定
体外TGFβRI激酶活性的抑制作用通过以下的方法进行测试。
本发明化合物对TGFβRI激酶ALK5活性的抑制作用采用如下实验方法测定：
酶活性检测使用TGFβRI激酶检测试剂盒(V4093，Promega)，在384孔板(4514，Corning)中依次加入2μl用反应缓冲液(40mM Tris pH7.5，20mM MgCl2，0.1mg/ml BSA)配制的酶溶液(反应体系中酶终浓度为2ng/μL)，1μl溶于5％DMSO的3倍梯度稀释的化合物，2μl ATP和TGFβRI底物多肽的混合溶液(ATP终浓度为50μM，底物终浓度为0.2μg/μL)，27℃反应2.5小时后，每孔加入5μl试剂盒中的ADP-Glo溶液，27℃放置40分钟，每孔再加入10μl激酶检测试剂，27℃放置30分钟。使用Victor 3(PerkinElmer)多功能酶标仪检测化学发光信号值。用Graphpad prism软件根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC50值。
本发明化合物的生物活性通过以上的试验进行测定，测得的IC50值见下表1。
表1本发明化合物对TGFβRI激酶ALK5活性抑制的IC50
实施例编号 IC50(nM)
1 208
2 270
3 241
8 370
结论：本发明实施例化合物对TGFβRI激酶ALK5活性均有明显地抑制作用。
测试例2、本发明化合物对VEGFR2激酶活性的的抑制作用的测定
体外VEGFR2激酶活性的的抑制作用通过以下的方法进行测试。
以下所述实验方法用来测定本发明化合物对VEGFR2激酶活性的抑制作用：
酶活性检测使用
Figure PCTCN2017110686-appb-000037
Kinase Assay Kit-Tyrosine 1Peptide(PV3190，Invitrogen)试剂盒，在384孔板(4513，Corning)中依次加入5μl用反应缓冲液(50mM HEPES pH7.5，10mM MgCl2，1mM EGTA，0.05％BRIJ-35)配制的重组人VEGFR2酶(PV3660，Invitrogen)和VEGFR2底物多肽(反应体系中酶终浓度为0.14ng/μL,底物终浓度为2μM)，2.5μl溶于5％DMSO的2倍梯度稀释的化合物，2.5μL ATP溶液(ATP终浓度为50μM)，25℃反应2小时后，每孔加入5μL检测试剂，25℃放置1小时后，用NOVOstar(BMG)多功能酶标仪检测发射波长445nm和520nm的荧光信号值。用Graphpad prism软件根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC50值。
本发明化合物的生物活性通过以上的试验进行测定，测得的IC50值见下表2。
表2本发明化合物对VEGFR2激酶活性的抑制作用的IC50
实施例编号 IC50(μM)
1 2.5
2 4.2
结论：本发明实施例化合物对VEGFR2激酶活性抑制作用较弱，说明本发明实施例化合物对TGFβRI激酶具有选择性抑制作用。
测试例3、本发明化合物对p38α激酶活性的抑制作用的测定
体外p38α激酶活性的抑制通过以下的方法进行测试。
以下所述实验方法用来测定本发明化合物对p38α激酶活性的抑制作用：
酶活性检测使用p38α激酶检测试剂盒(V9591，Promega)，在384孔板(4514，Corning)中依次加入2μl用反应缓冲液(40mM Tris pH7.5，20mM MgCl2，0.1mg/mlBSA)配制的酶溶液(反应体系中酶终浓度为0.5ng/μL)，1μl溶于5％DMSO的3倍梯度稀释的化合物，2μl ATP和p38底物多肽的混合溶液(ATP终浓度为50μM，底物终浓度为0.2μg/μL)，27℃反应2.5小时后，每孔加入5μl试剂盒中的ADP-Glo溶液，27℃放置40分钟，每孔再加入10μl激酶检测试剂，27℃放置30分钟。使用Victor 3(PerkinElmer)多功能酶标仪检测化学发光信号值。用Graphpad  prism软件根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC50值。
本发明化合物的生物活性通过以上的试验进行测定，测得的IC50值见下表3。
表3本发明化合物对p38α激酶活性的抑制作用的IC50
实施例编号 IC50(μM)
1 2.2
2 1.0
结论：本发明实施例化合物对p38α激酶活性抑制作用较弱，说明本发明实施例化合物对TGFβRI激酶具有选择性抑制作用。
测试例4、本发明化合物对RIPK2激酶活性的抑制作用的测定
体外RIPK2激酶活性的抑制通过以下的方法进行测试。
以下所述实验方法用来测定本发明化合物对RIPK2激酶活性的抑制作用：
酶活性检测使用RIPK2激酶检测试剂盒(V4084，Promega)，在384孔板(4514，Corning)中依次加入2μl用反应缓冲液(40mM Tris pH7.5，20mM MgCl2，0.1mg/mlBSA)配制的RIPK2酶溶液(反应体系中酶终浓度为0.5ng/μL)，1μl溶于5％DMSO的3倍梯度稀释的化合物1溶液，2μl ATP和MBP底物多肽的混合溶液(ATP终浓度为50μM，底物终浓度为0.2μg/μL)，27℃反应2.5小时后，每孔加入5μl试剂盒中的ADP-Glo溶液，27℃放置40分钟，每孔再加入10μl激酶检测试剂，27℃放置30分钟。使用Victor 3(PerkinElmer)多功能酶标仪检测化学发光信号值。用Graphpad prism软件根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC50值。
本发明化合物的生物活性通过以上的试验进行测定，测得的IC50值见下表4。
表4本发明化合物对RIPK2激酶活性的抑制作用的IC50
实施例编号 IC50(μM)
1 1.2
结论：本发明实施例化合物对RIPK2抑制作用弱，说明本发明实施例化合物对TGFβRI激酶具有选择性抑制作用。
测试例4、本发明化合物对NIH3T3细胞增殖的抑制测定
下面的体外试验是用来测定本发明化合物对NIH3T3细胞增殖的抑制活性。
以下所述实验方法用来测定本发明化合物对NIH3T3细胞增殖的抑制作用：
在96孔透明底白板(3903，Corning)中用含10％FBS的DMEM培养基(SH30243.01，GE)每孔接种100μL NIH3T3细胞(GNM6，中国科学院典型培养物保藏委员会细胞库)，接种密度为2000细胞/孔，细胞在37℃，5％CO2条件下培养过夜。过夜培养后，每孔更换为90μL含0.5％FBS的DMEM培养基，然后加入10μL用含0.5％FBS的DMEM培养基3倍梯度稀释的化合物，放置37℃， 5％CO2细胞培养箱中培养72小时。最后每孔加入50μL CellTiter-Glo(G7573，Promega)，室温孵育10分钟后使用Victor3酶标仪(PerkinElmer)读取化学发光信号值。用Graphpad Prism软件根据化合物各浓度与相应的信号值计算化合物的IC50值。
本发明化合物生物活性由上述分析所得，计算所得的IC50值如下表4：
表4本发明化合物对NIH3T3细胞增殖的抑制的IC50
实施例编号 IC50(nM) 最大抑制率(％)
1 88 105％
2 194 107％
3 366 105％
8 490 114％
结论：本发明化合物对NIH3T3细胞增殖有明显的抑制活性。
测试例5、本发明化合物对TGFβRI的Smad信号通路的抑制活性的测定
下面的体外试验是用来测定本发明化合物对TGFβRI的Smad信号通路的抑制活性。
以下所述实验方法用来测定本发明化合物对TGFβRI的Smad信号通路的抑制活性：
在96孔板中用含10％FBS的EMEM培养液(42360-099，Gibco)每孔接种100μl HepG2(TCHu 72，中国科学院典型培养物保藏委员会细胞库)细胞，接种密度为2.5x104细胞/孔，细胞在37℃，5％CO2条件下培养过夜。更换含10％FBS的EMEM新鲜培养液，每孔转染0.1μg 3TP-lux质粒(11767，普如汀生物技术(北京)有限公司)，细胞继续在37℃，5％CO2条件下培养24小时。每孔更换90μl含0.5％FBS的EMEM培养液，饥饿6小时。将化合物配置成20mM的储存液，用100％DMSO梯度稀释成400×的浓度，再用含0.5％FBS的EMEM稀释40倍。取出细胞培养板，每孔分别加入10μl稀释后的化合物或对照(0.25％DMSO)，轻轻振荡混匀，放置37℃，5％CO2培养箱中培养18小时，最后每孔加入100μl检测试剂ONE-GloTM Luciferase Assay(E6110，Promega)，室温避光放置10分钟，采用Victor3.0(PerkinElmer)读取化学发光信号值。用Graphpad Prism软件根据化合物各浓度与相应的信号值计算化合物的IC50值。
本发明化合物生物活性由上述分析所得，计算所得的IC50值如下表5：
表5本发明化合物对TGFβRI的Smad信号通路抑制的IC50
实施例编号 IC50(nM) 最大抑制率(％)
1 77 101％
2 174 100％
3 57 101％
7 168 98％
8 218 98％
结论：本发明化合物均对TGFβRI的Smad信号通路具有明显的抑制活性。
药代动力学评价
测试例6、本发明化合物的药代动力学测试
1.摘要
以大鼠为受试动物，应用LC/MS/MS法测定了大鼠灌胃给予实施例1化合物和实施例2化合物后不同时刻血浆中的药物浓度。研究本发明化合物在大鼠体内的药代动力学行为，评价其药动学特征。
2.试验方案
2.1试验药品
实施例1化合物和实施例2化合物。
2.2试验动物
健康成年SD大鼠8只，雌雄各半，平均分成2组，每组4只，购自上海西普尔-必凯实验动物有限公司，动物生产许可证号：SCXK(沪)2008-0016。
2.3药物配制
称取一定量药物，加5％体积的DMSO、5％体积的吐温80和90％体积的生理盐水配制成0.2mg/mL的澄清透明溶液。
2.4给药
SD大鼠禁食过夜后灌胃给药，给药剂量均为2.0mg/kg，给药体积均为10.0mL/kg。
3.操作
大鼠灌胃给药实施例1化合物和实施例2化合物，于给药前及给药后0.5，1.0，2.0，4.0，6.0，8.0，11.0，24.0小时由眼眶采血0.2mL，置于肝素化试管中，4℃、3500转/分钟离心10分钟分离血浆，于-20℃保存，给药后2小时进食。
测定不同浓度的药物灌胃给药后大鼠血浆中的待测化合物含量：取给药后各时刻的大鼠血浆25μL，加入内标溶液喜树碱30μL(100ng/mL)，乙腈225μL，涡旋混合5分钟，离心10分钟(3600转/分钟)，血浆样品取上清液5.0μL进行LC/MS/MS分析。
4.药代动力学参数结果
本发明化合物的药代动力学参数如下：
Figure PCTCN2017110686-appb-000038
Figure PCTCN2017110686-appb-000039
结论：本发明化合物的药代吸收良好，具有明显的药代动力学优势。
毒性试验
测试例7、TGFβ抑制剂对SD大鼠灌胃重复给药7天毒性实验
1.实验目的
评估TGFβ抑制剂化合物1灌胃重复给药7天对SD大鼠毒性。
2.实验药品
化合物1，纯度100％，白色固体
含盐分子量444.5，游离碱分子量444.5(以游离碱计)
储存条件：室温保持
3.实验设计和实验方法
3.1.实验动物和饲养条件
SD大鼠，购入26只，180～200g左右，6-7周龄，雌雄各半，订购于上海杰思捷实验动物有限责任公司，SPF级。
饲养条件：SPF级，动物饲养于塑料透明大鼠饲养笼中，每笼2-3只。室温20-26℃，相对湿度40～70％。控制光照12小时/黑暗12小时交替。自由不限量摄取鼠用颗粒饲料。经饮水瓶自由饮用高温灭菌水。
3.2.动物分组：
随机分为溶媒对照组、化合物1-30mg/kg和化合物1-100mg/kg及相应毒代组，共为5组，溶媒对照组、化合物1-30mg/kg和化合物1-100mg/kg为6只/组，毒代组为4只/组，雌雄各半。
Figure PCTCN2017110686-appb-000040
3.3.实验方法：
本次大鼠7天毒性试验供试品剂量分别为：30和100mg/kg(qd)，连续给药7天。给药途径：灌胃，给药容量：10ml/kg，给药频率及给药期：每日上午给药，连续给药7天。
3.4.数据表达和统计学处理
各项指标检查结果经计算机Excel录入和SPSS软件包录入与统计处理。
临床症状、摄食量及解剖肉眼大体观察结果不进行统计学计算处理，只列出方案中观察项目的观察数据。体重、脏器重量和临检等指标使用SPSS进行统计。
4.结果
给药期间化合物1各剂量组动物临床观察未见明显异常。与溶媒对照组相比，各剂量组动物体重及体重增长率未见明显差异(p>0.05)，摄食量均未见明显变化。
化合物1各剂量组血液生化指标未见毒理学改变。与溶媒对照组相比，各组动物凝血功能和尿液各项指标未见明显异常。
末次给药后动物麻醉安乐死尸检结果显示，化合物1各剂量组动物大体病理解剖均未见明显异常。
与溶媒对照组相比，各给药组脏器系数变化无明显异常。心脏组织病理学检查各给药组升主动脉壁各层未见明显病变，冠状动脉壁未见病变，房室瓣膜无异常。心脏病理学检查未见明显异常。
5.结论：在本次实验条件下，化合物1具有较好的安全性。
药效试验
测试例8、TGFβ化合物对B16-F1移植瘤C57小鼠疗效实验
1.实验目的
评价实施例1化合物抑制鼠黑色素瘤细胞B16-F1移植瘤在C57小鼠上生长的作用。
2.实验药品
化合物1用0.5％CMC-Na+0.25％(v/v)Polysorbate 80配制成5mg/ml的水溶液，给药浓度为50mg/kg，口服灌胃体积为10ml/kg。
3.实验方法和实验材料
3.1.实验动物和饲养条件
实验用C57BL/6J鼠，雌性，购自上海西普尔-必凯实验动物有限公司(中国上海，合格证SCXK(沪)2008-0016)，购入时16-20g。动物购进后，进行3天适应性饲养后开始实验。饲养条件：SPF级。动物饲养方式：给予12/12小时光/暗周期调节，温度23±1℃，湿度40～50％，动物均给予标准灭菌鼠饲料，自由进食饮水。
3.2.动物分组：
C57BL/6J鼠适应性饲养后，分组如下：
分组 n 给药方式
空白组 10 0.5％CMC(i.g/bid)
化合物1组 10 50mg/kg化合物1(i.g/bid)
注：bid为一天给药2次。
3.3.实验方法：
将适应性喂养的C57小鼠置于代谢笼中禁食过夜，称完体重后随机分成以下几组：空白对照组和化合物1组，每组10只。C57小鼠右肋部提前一天备皮，皮下接种B16-F1细胞(1×104每只)，接种后第3天，开始给药。每只小鼠一天两次灌胃(i.g.，10ml/kg)化合物1，空白组灌胃相应的溶剂。每周测定2次瘤体积，称量体重并记录数据。
3.4.数据统计
使用Excel统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
抑瘤率(％)＝(C-T)/C(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。C、T分别为实验结束时的空白对照组(Blank)及实验组的平均肿瘤体积。
4.结果
化合物1对B16-F1裸小鼠移植瘤的疗效数据见表4.1和图1。
化合物1对C57小鼠体重的影响见图2。
表4.1化合物1对B16-F1裸小鼠移植瘤的疗效
Figure PCTCN2017110686-appb-000041
*p<0.05
5.结论
化合物1在肿瘤细胞移植后第四天开始给药,每天两次,至第19天肿瘤体积明显小
于空白对照组。经计算抑瘤率为71.12％，且给药对小鼠体重没有影响。

Claims (14)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2017110686-appb-100001
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    环A为环烷基或杂环基；
    R1相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、-S(O)mR5、-C(O)OR5、环烷基和杂环基；
    R2相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基、杂芳基、-OR5、-C(O)R5、-S(O)mR5和-C(O)NR6R7；
    R3相同或不同，且各自独立地选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氨基、氰基和硝基；
    R4相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
    R5选自氢原子、烷基、氨基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
    R6和R7各自独立地选自氢原子、烷基、卤代烷基、环烷基、杂环基、芳基和杂芳基；
    n为0、1或2；
    s为0、1、2、3、4或5；
    p为0、1、2或3；
    q为0、1、2、3或4；且
    m为0、1或2。
  2. 根据权利要求1所述的通式(I)所示的化合物，其为通式(II)所示的化合物：
    Figure PCTCN2017110686-appb-100002
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    G1为CH2、NR8或O；
    R8选自-S(O)mR5、-C(O)OR5、氢原子或烷基；
    R2、R4和R5如权利要求1中所定义；
    s为0、1或2；
    m为0、1或2；且
    r为0、1、2或3。
  3. 根据权利要求1或2所述的通式(I)所示的化合物，其中R2选自氰基、-C(O)R5或-S(O)mR5，R5为羟基或烷基，优选R2为氰基或甲磺酰基，且s为1。
  4. 根据权利要求1～3中任一项所述的通式(I)所示的化合物，其中R4为烷基。
  5. 根据权利要求1～4中任一项所述的化合物，其选自：
    Figure PCTCN2017110686-appb-100003
    Figure PCTCN2017110686-appb-100004
  6. 一种通式(I-A)所示的化合物：
    Figure PCTCN2017110686-appb-100005
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式或其可药用的盐，
    其中：
    X为卤素，优选为溴；
    环A、R1、R4、n和q如权利要求1中所定义。
  7. 根据权利要求4所述的通式(I-A)所示的化合物，其选自：
    Figure PCTCN2017110686-appb-100006
    Figure PCTCN2017110686-appb-100007
  8. 一种制备根据权利要求1所述的通式(I)所示的化合物的方法，该方法包括：
    Figure PCTCN2017110686-appb-100008
    通式(I-A)的化合物和通式(I-B)的化合物在碱性条件下，在催化剂存在下经Suzuki反应，得到通式(I)的化合物，
    其中：
    G2为
    Figure PCTCN2017110686-appb-100009
    X为卤素，优选为溴；
    环A、R1～R4、n、s、p和q如权利要求1中所定义。
  9. 一种药物组合物，所述药物组合物含有治疗有效量的根据权利要求1～5中任一项所述的通式(I)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  10. 根据权利要求1～5中任一项所述的的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求9所述的药物组合物，其作用药物。
  11. 根据权利要求1～5中任一项所述的的化合物或其互变异构体、内消旋体、 外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求9所述的药物组合物，其作用TGF-β受体激酶抑制剂。
  12. 根据权利要求1～5中任一项所述的的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求9所述的药物组合物，在制备用于治疗、预防或减少肿瘤细胞转移的药物中的用途。
  13. 根据权利要求1～5中任一项所述的化合物或其互变异构体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求9所述的药物组合物在制备用于治疗、预防或减少由TGF-β过度表达介导的癌症的药物中的用途。
  14. 根据权利要求1～5中任一项所述的化合物或其互变异构体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐或根据权利要求9所述的药物组合物在制备用于治疗、预防或减轻选自下述疾病的药物中的用途：血管损伤、肾小球性肾炎、糖尿病性肾病、狼疮性肾炎、高血压-诱发的肾病、肾间质性纤维化、由药物暴露并发症所致肾纤维化、与HIV有关的肾病、移植物肾病、各种病因的肝纤维化、可归因于感染的肝功能障碍、酒精诱发的肝炎、囊性纤维化病、间质性肺病、急性肺损伤、成人呼吸窘迫综合征、特发性肺纤维化、慢性阻塞性肺病、由感染性或毒性因子引起的肺病、梗塞后心纤维化、充血性心力衰竭、扩张型心肌病、心肌炎、内膜增厚、血管狭窄、高血压引起的血管重构、肺动脉高压、冠状动脉再狭窄、周围动脉再狭窄、颈动脉再狭窄、支架诱导的再狭窄、动脉粥样硬化、眼部瘢痕形成、角膜瘢痕形成、增生性玻璃体视网膜病变、青光眼、眼内压高、发生在由创伤或手术伤口所致伤口愈合期间的过度性或肥厚性真皮瘢痕或瘢痕疙瘩形成、腹膜与皮下粘连、硬皮病、纤维硬化、进行性系统性硬化病、皮肌炎、多肌炎、关节炎、骨质疏松、溃疡、神经系统功能减低、男性勃起功能障碍、佩罗尼氏病、杜普伊特伦氏挛缩、阿尔茨海默氏病、雷诺氏综合征、辐射-诱发的纤维化、血栓、肿瘤转移性生长、多发性骨髓瘤、黑素瘤、神经胶质瘤、胶质母细胞瘤、白血病、肉瘤、平滑肌瘤、间皮瘤、乳腺癌、宫颈癌、肺癌、胃癌、直肠癌、结肠癌、胰腺癌、脑癌、皮肤癌、口腔癌、前列腺癌、骨癌、肾癌、卵巢癌、膀胱癌和肝癌。
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