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WO2024235013A1 - 一种含吲唑基的羟肟酸衍生物及其应用 - Google Patents

一种含吲唑基的羟肟酸衍生物及其应用 Download PDF

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Publication number
WO2024235013A1
WO2024235013A1 PCT/CN2024/090847 CN2024090847W WO2024235013A1 WO 2024235013 A1 WO2024235013 A1 WO 2024235013A1 CN 2024090847 W CN2024090847 W CN 2024090847W WO 2024235013 A1 WO2024235013 A1 WO 2024235013A1
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alkyl
substituted
compound
halogenated
pharmaceutically acceptable
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PCT/CN2024/090847
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English (en)
French (fr)
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冯子侠
曹正洪
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南昌双天使生物科技开发有限公司
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Publication of WO2024235013A1 publication Critical patent/WO2024235013A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of biomedicine, and in particular to an indazole-containing hydroxamic acid derivative and application thereof.
  • Tyrosine kinases are a class of proteins with tyrosine kinase activity. They can catalyze the transfer of phosphate groups on ATP to tyrosine residues of many important proteins, causing phosphorylation and thus activating downstream signal transduction pathways. Protein tyrosine kinases occupy a very important position in the intracellular signal transduction pathways, regulating a series of physiological and biochemical processes such as cell growth, differentiation, and death. Dysregulation of protein tyrosine kinase function can cause a series of diseases in the body, including tumors. The occurrence, development, and metastasis of many tumors and the formation of tumor angiogenesis are closely related to the abnormal expression of tyrosine kinases.
  • tyrosine kinase receptors are abnormally expressed in solid tumor cells, among which vascular endothelial cell growth factor receptor (VEGFR) is highly expressed in many tumor cells and tumor vascular endothelial cells, and platelet-derived growth factor receptor (PDGFR) is abnormally expressed in fibroblasts in the tumor stroma.
  • the autocrine loop formed by its ligand and receptor is directly involved in the occurrence and development of tumor cells.
  • vascular endothelial growth factor receptor (VEGFR) exists in melanoma
  • platelet-derived growth factor receptor (PDGFR) exists in glioma
  • stem cell growth factor receptor (KIT) exists in small cell lung cancer, etc.
  • similar loops also exist in meningiomas, neuroendocrine tumors, ovarian cancer, prostate cancer and pancreatic cancer. This loop is closely related to the occurrence and development of tumors.
  • Tumor angiogenesis is an important process of tumor cell infiltration, migration and proliferation.
  • the vascular endothelial growth factor receptor family (VEGFR) and the platelet-derived growth factor receptor family (PDGFR) have a direct relationship with the occurrence and development of tumors and the formation of tumor angiogenesis.
  • vascular endothelial growth factor (VEGF) plays an important role in the proliferation, migration and vascular construction of endothelial cells. Its expression level and the degree of vascularization of tumor tissue show a significant positive correlation.
  • VEGF mainly exerts its biological effects by acting on the high-affinity receptors VEGFR-1 and KDR on endothelial cells to phosphorylate their tyrosine kinases, and the two have different signal transduction pathways.
  • KDR plays a key role in tumor growth, metastasis and tumor angiogenesis.
  • Platelet-derived growth factor (PDGF) and its receptor (PDGFR) are involved in the pathogenesis of various tumors and play an important role in angiogenesis.
  • PDGFR platelet-derived growth factor
  • PDGF expresses its cellular biological effects through its receptor (PDGFR).
  • PDGFR maintains the integrity of the vascular wall by regulating the proliferation and migration of pericytes and vascular smooth muscle cells in the vascular wall and promotes the formation of new blood vessels in tumors. In addition, it promotes tumor growth by changing the microenvironment within the tumor.
  • tyrosine kinase Since the abnormal expression of tyrosine kinase is closely related to the occurrence, development and metastasis of tumors and the generation of tumor angiogenesis, the development of drugs targeting tyrosine kinase has become a hot spot in international anti-tumor drug research. In particular, targeting neovascularization to inhibit tumor angiogenesis, block the nutrient supply and migration pathways of tumors, and prevent tumor growth and metastasis has become a new strategy for treating tumors.
  • the abnormal expression of KDR or PDGFR receptors plays a key role in the process of tumor angiogenesis, and they have become the most ideal targets for anti-tumor drug treatment.
  • Histone deacetylases are a class of intracellular metalloproteinases that play an important role in the structural modification of chromosomes and the regulation of gene expression.
  • HDACs Histone deacetylases
  • overexpression of HDACs leads to increased binding of histones to DNA, causing abnormal conformational changes in chromosomes.
  • the expression of cell cycle inhibitors is inhibited, the stability and DNA binding ability of the tumor suppressor p53 decrease, and the expression of hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) increases.
  • HIF-1 hypoxia inducible factor-1
  • VEGF vascular endothelial growth factor
  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • HDAC1mRNA and protein levels are highly expressed in gastric cancer, prostate cancer, colon cancer and liver cancer tissues, and are related to the TNM stage and lymph node metastasis of the tumor.
  • Hydroxamic acid small molecule HDACi is a type of HDACi that has received much attention in recent years, showing good antitumor activity both in vivo and in vitro. HDAC has developed into a relatively mature drug as an anticancer target.
  • One of the purposes of the present invention is to provide a novel indazole-containing hydroxamic acid derivative that can simultaneously exert a good inhibitory effect on multiple targets (especially histone deacetylase 1, histone deacetylase 6, and two tyrosine kinases (vascular endothelial growth factor receptor and platelet-derived growth factor receptor)).
  • Another object of the present invention is to provide a pharmaceutical composition comprising the novel indazole-containing hydroxamic acid derivative.
  • Another object of the present invention is to provide the novel indazole-containing hydroxamic acid derivatives or the pharmaceutical composition containing the novel indazole-containing hydroxamic acid derivatives for use in the preparation of drugs for preventing and/or treating diseases mediated by tyrosine kinase and/or histone deacetylase.
  • Ra and Rb are independently selected from hydrogen, C1-20 alkyl, 3-10 membered heterocycloalkyl, 5-12 membered aromatic group, and 5-12 membered heteroaryl group; wherein the C1-20 alkyl, 3-10 membered heterocycloalkyl, 5-12 membered aromatic group, and 5-12 membered heteroaryl group are each independently unsubstituted or substituted by one, two, three or more substituents selected from the following substituents: fluorine, chlorine, bromine, iodine, nitro, cyano, mercapto, hydroxyl, amino, amino substituted with C1-6 alkyl, C1-10 alkyl, halogenated C1-6 alkyl, nitro-substituted C1-6 alkyl, cyano-substituted C1-6 alkyl, mercapto-substituted C1-6 alkyl, amino substituted with C1-6 alkyl, hydroxyl-substituted C1-6 alkyl, C1-6 alk
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from unsubstituted C 1-20 alkyl groups, or independently selected from C 1-20 alkyl groups substituted by one or more substituents selected from fluorine, chlorine, bromine, iodine, nitro, cyano, mercapto, hydroxyl, and amino;
  • Each R c is the same or different and is independently selected from hydrogen, C 1-20 alkyl, C 1-20 alkoxy, fluorine, chlorine, bromine, iodine, nitro, cyano, mercapto, hydroxyl, amino, amino substituted with C 1-6 alkyl, halogenated C 1-6 alkyl, nitro substituted with C 1-6 alkyl, cyano substituted with C 1-6 alkyl, mercapto substituted with C 1-6 alkyl, amino substituted with C 1-6 alkyl;
  • R d and Re are independently selected from hydrogen, C 1-20 alkyl, C 1-6 alkyl substituted by nitro, C 1-6 alkyl substituted by cyano, C 1-6 alkyl substituted by mercapto, and C 1-6 alkyl substituted by amino;
  • Rg has a chain structure, the number of atoms constituting the main chain of the chain structure is 3-15 and the number of heteroatoms is no more than 2, and the heteroatoms are selected from oxygen, sulfur, and nitrogen;
  • x is 1, 2, 3, or 4.
  • the C 1-20 alkyl group may be a C 1-10 alkyl group or a C 10-20 alkyl group; including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, hexyl, heptyl, and the like.
  • the number of heteroatoms in the atoms constituting the main chain of the chain structure is 1 or 2, and one of the heteroatoms is adjacent to the indazole ring.
  • the benzene rings are connected.
  • the chain structure is a saturated straight chain or a saturated branched chain.
  • the compound has a structure shown in general formula (II):
  • Ra , Rb , Rc , Rd , Re and x are respectively the same as Ra , Rb , Rc , Rd, Re and x in the general formula (I);
  • a and B are independently CH2 , O, S or -N( R6 )-;
  • R6 is hydrogen or C1-6 alkyl;
  • m and n are independently selected from integers of 0-10, and 3 ⁇ m+n ⁇ 10.
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • B is CH 2 :
  • A is CH 2 ; (2) A is selected from O, S or —N(R 6 )—, and m is 0; (3) n is 0.
  • A is selected from CH 2 , O, S or —NH—
  • B is selected from CH 2 , O, —N(CH 3 )—, —N(CH 2 CH 3 )— or —N(CH 2 CH 2 CH 3 )—.
  • A is O
  • B is CH 2
  • m+n is 3, 4 or 5.
  • Ra and Rb are not hydrogen at the same time.
  • Ra and Rb are selected from unsubstituted or substituted pyrimidinyl, pyrazolyl, pyridyl, pyrazinyl, imidazolyl, pyridazinyl, thiazolyl, phenyl; and the other is selected from hydrogen, or unsubstituted or substituted C1-6 alkyl.
  • the compound has a structure shown in formula (III):
  • R c , R d , Re , x, A, B, m, n are the same as R c , R d , Re , x, A, B, m, n in the general formula (II) respectively;
  • R b is selected from hydrogen, or C 1-6 alkyl which is unsubstituted or substituted by 1 to 3 halogens;
  • R 7 is selected from fluorine, chlorine, bromine, iodine, hydroxyl, mercapto, cyano, amino, methylamino, ethylamino, nitro, C 1-6 alkyl, halogenated C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy, hydroxymethyl, mercaptomethyl, C( ⁇ O)CH 3 , C( ⁇ O)CH 2 CH 3 , C( ⁇ O)NHCH 3 , C( ⁇ O)NHCH 2 CH 3 , NHC( ⁇ O)CH 3 , N
  • the compound has a structure shown in formula (IV):
  • R b , R c , R d , Re , x, A, B, m, n are respectively the same as R b , R c , R d , Re , x, A, B, m, n in the general formula (III);
  • R 8 and R 9 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, halogenated methyl, halogenated ethyl, halogenated n-propyl, halogenated isopropyl, halogenated n-butyl, halogenated isobutyl, methoxy, ethoxy, halogenated methoxy, halogenated ethoxy.
  • the halogenated group may be substituted with one or more halogens selected from fluorine, chlorine, bromine and iodine, for example, it may be substituted with 1 fluorine, 2 fluorines, 1 chlorine and 1 fluorine, 2 bromines and 1 fluorine, etc.
  • R b , R d , and Re are independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, or isobutyl.
  • each R c is the same or different and is independently selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, or ethoxy.
  • R b , R c , R d , and Re are all hydrogen.
  • Rg is selected from:
  • the compound has a structure shown in formula (V):
  • R 8 and R 9 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, halogenated methyl, halogenated ethyl, halogenated n-propyl, halogenated isopropyl, halogenated n-butyl, halogenated isobutyl, methoxy, ethoxy, halogenated methoxy, halogenated ethoxy.
  • the structural formula of the compound is as shown in any one of formulas (ST-01) to (ST-16):
  • a pharmaceutical composition which comprises the above-mentioned compound, its pharmaceutically acceptable salt, hydrate or prodrug, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also contain a diluent or an excipient.
  • the pharmaceutical composition is a composition for preventing and/or treating malignant tumors, including but not limited to renal cancer, liver cancer, colon cancer, gastrointestinal stromal tumors, lung cancer, breast cancer, pancreatic cancer, glioma, lymphoma, fibrosarcoma, ovarian cancer, prostate cancer, leukemia, and lymphoma.
  • malignant tumors including but not limited to renal cancer, liver cancer, colon cancer, gastrointestinal stromal tumors, lung cancer, breast cancer, pancreatic cancer, glioma, lymphoma, fibrosarcoma, ovarian cancer, prostate cancer, leukemia, and lymphoma.
  • Another technical solution provided by the present invention is: use of the above-mentioned compound, its pharmaceutically acceptable salt, hydrate or prodrug, or the above-mentioned pharmaceutical composition in the preparation of drugs for preventing and/or treating diseases mediated by tyrosine kinase and/or histone deacetylase.
  • the disease mediated by tyrosine kinase and/or histone deacetylase is a malignant tumor, which includes but is not limited to renal cancer, liver cancer, colon cancer, gastrointestinal stromal tumor, lung cancer, breast cancer, pancreatic cancer, glioma, lymphoma, fibrosarcoma, ovarian cancer, prostate cancer, leukemia, and lymphoma.
  • the compound includes not only a single compound form, but also a mixture of multiple compounds whose structures meet the requirements of general formula (I), as well as different isomeric forms of the same compound such as racemates, enantiomers, diastereomers, etc.
  • the pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydrobromide, phosphate, sulfate, acetate, trifluoroacetate, maleate, methanesulfonate, benzenesulfonate, benzoate, toluenesulfonate, succinate, fumarate, tartrate, gallate, citrate and the like.
  • the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof is usually administered via the oral route in the form of a pharmaceutical preparation comprising an active ingredient or a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt, in a pharmaceutically acceptable dosage form.
  • a pharmaceutical preparation comprising an active ingredient or a pharmaceutically acceptable salt or solvate thereof, or a solvate of such a salt, in a pharmaceutically acceptable dosage form.
  • the composition can be administered at different doses.
  • the pharmaceutical formulations of the compounds represented by the general formula (I) described above can be prepared for oral administration, specifically in the form of tablets or capsules, and particularly involve techniques aimed at providing colon-targeted drug release.
  • compositions of the compounds represented by the general formula (I) described above may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • compositions suitable for oral administration may include one or more physiologically compatible carriers and/or excipients, and may be in the form of solid or liquid. Tablets and capsules may be prepared with adhesives, fillers, lubricants and/or surfactants (such as sodium lauryl sulfate). Liquid compositions may contain conventional additives, such as suspending agents, emulsifiers and/or preservatives. Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.
  • Oral formulations are preferred, particularly tablets or capsules, which can be formulated by methods known to those skilled in the art to provide a dose of the active compound in the range of 0.001 mg to 10000 mg, for example, a dose of the active compound in the range of 0.01 mg to 5000 mg, or a dose of the active compound in the range of 0.1 mg to 1000 mg, etc.
  • the prodrug of the compound represented by general formula (I) refers to a prodrug that can be converted into at least one compound of general formula (I) or its salt by metabolism or chemical reaction (such as enzymatic hydrolysis or hydrolysis) in the subject after being administered by an appropriate method.
  • Another technical solution provided by the present invention is an intermediate for preparing the above-mentioned compound, its pharmaceutically acceptable salt, hydrate or prodrug, wherein the intermediate has a structure shown in the general formula (VI):
  • Ra , Rb , Rc , Rd , Re , Rg , and x are respectively the same as Ra , Rb , Rc , Rd , Re , Rg , and x in the aforementioned general formula; and R is a C1-6 alkoxy group.
  • R may be methoxy, ethoxy, propoxy, or tert-butoxy.
  • Another technical solution provided by the present invention is a method for preparing the above-mentioned compound, its pharmaceutically acceptable salt, hydrate or prodrug, which comprises the step of reacting the above-mentioned intermediate with hydroxylamine.
  • the hydroxylamine is obtained by adding a hydroxylamine inorganic acid salt, and the hydroxylamine inorganic acid salt includes but is not limited to hydroxylamine hydrochloride and/or hydroxylamine phosphate.
  • the reaction of the intermediate and hydroxylamine is carried out under alkaline conditions in an alcohol solvent.
  • the alkaline conditions are formed by adding an alkaline substance, and the alkaline substance includes but is not limited to sodium hydroxide, potassium hydroxide, and the like.
  • the alcohol solvent includes but is not limited to methanol and the like.
  • the present invention has the following advantages compared with the prior art:
  • the compounds of the present invention can combine multiple important targets into one, especially can simultaneously inhibit histone deacetylase VI (HDAC6, It has a strong inhibitory effect on the two tyrosine kinases (vascular endothelial growth factor receptor, Vascular endothelial cell growth factor2, VEGFR2) and platelet-derived growth factor receptor (platelet-derived growth factor- ⁇ , PDGFR- ⁇ ), which can better avoid the problems caused by different properties and metabolism when multiple drugs are used in combination, give full play to the synergistic effect to increase biological activity, and has a good application prospect.
  • HDAC6 histone deacetylase VI
  • C 1-20 alkyl refers to a straight or branched saturated hydrocarbon group of 1 to 20 carbon atoms ("C 1-C 20 -alkyl").
  • the alkyl group contains 1 to 10 carbon atoms, or contains 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5 or 6 carbon atoms.
  • Some non-limiting examples include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl and 2,2-dimethylpropyl.
  • alkoxy refers to an alkyl group attached to a parent molecular moiety via an oxygen atom.
  • the alkoxy group contains 1 to 6 carbon atoms and can be expressed as a C 1-6 alkoxy group.
  • Some non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and tert-butoxy.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
  • halogen refers to fluorine (F), chlorine (Cl) or bromine (Br).
  • amino-substituted alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by an amino group.
  • amino-substituted C 1-6 alkyl refers to an alkyl group in which at least one hydrogen atom of the C 1-6 alkyl group is replaced by an amino group.
  • aminoalkyl refers to an alkyl group in which 1, 2 or 3 hydrogen atoms of the alkyl group are replaced by an amino group.
  • Non-limiting examples of aminoalkyl groups may be aminomethyl and 1-aminoethyl, etc.
  • amino-substituted alkoxy refers to an alkoxy group in which at least one hydrogen atom of the alkoxy group is replaced by an amino group.
  • an amino-substituted C 1-6 alkoxy group refers to a C 1-6 alkoxy group in which at least one hydrogen atom is replaced by an amino group.
  • aminoalkoxy refers to an alkoxy group in which 1, 2 or 3 hydrogen atoms of the alkoxy group are replaced by an amino group.
  • Non-limiting examples of aminoalkoxy groups are aminomethoxy and 1-aminoethoxy, etc.
  • nitro-substituted alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by a nitro group.
  • a nitro-substituted C 1-6 alkyl group refers to a C 1-6 alkyl group in which at least one hydrogen atom is replaced by a nitro group.
  • nitro-substituted alkoxy refers to an alkoxy group in which at least one hydrogen atom of the alkoxy group is replaced by a nitro group.
  • a nitro-substituted C 1-6 alkoxy group refers to a C 1-6 alkoxy group in which at least one hydrogen atom is replaced by a nitro group.
  • cyano-substituted alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by a cyano group.
  • a cyano-substituted C 1-6 alkyl group refers to a C 1-6 alkyl group in which at least one hydrogen atom is replaced by a cyano group.
  • cyano-substituted alkoxy refers to an alkoxy group in which at least one hydrogen atom of the alkoxy group is replaced by a cyano group.
  • a cyano-substituted C 1-6 alkoxy group means that at least one hydrogen atom of the C 1-6 alkoxy group is replaced by a cyano group.
  • thiol-substituted alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by a thiol group.
  • a thiol-substituted C 1-6 alkyl group refers to a C 1-6 alkyl group in which at least one hydrogen atom is replaced by a thiol group.
  • mercapto-substituted alkoxy refers to an alkoxy group wherein at least one hydrogen atom of the alkoxy group is substituted with a mercapto group.
  • a mercapto-substituted C 1-6 alkoxy group means that at least one hydrogen atom of the C 1-6 alkoxy group is replaced by a mercapto group.
  • hydroxy substituted alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by a hydroxy group.
  • a hydroxy substituted C 1-6 alkyl group refers to a C 1-6 alkyl group in which at least one hydrogen atom is replaced by a hydroxy group.
  • hydroxy substituted alkoxy refers to an alkoxy group in which at least one hydrogen atom of the alkoxy group is replaced by a hydroxy group.
  • a hydroxy substituted C 1-6 alkoxy group means that at least one hydrogen atom of the C 1-6 alkoxy group is replaced by a hydroxy group.
  • halogenated alkyl refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by a halogen, such as fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
  • a halogenated C 1-6 alkyl group means that at least one hydrogen atom of the C 1-6 alkyl group is replaced by a halogen.
  • halogenated alkoxy refers to an alkoxy group in which at least one hydrogen atom of the alkoxy group is replaced by a halogen, such as fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
  • a halogenated C 1-6 alkoxy group means that at least one hydrogen atom of the C 1-6 alkoxy group is replaced by a halogen.
  • heterocycloalkyl refers to a saturated monocyclic, bicyclic, or multicyclic ring with more rings condensed.
  • heterocycloalkyl refers to a monocyclic, bicyclic, or multicyclic ring with more rings condensed of 3 to 10 ring atoms, wherein 1, 2, 3 or 4 of the ring atoms are heteroatoms selected from N, O and S, and the remaining ring atoms are carbon.
  • heterocycloalkyl examples include azetidin-3-yl, azetidin-2-yl, oxetan-3-yl, oxetan-2-yl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, pyrrolidinyl (e.g., pyrrolidin-2-yl), morpholino, morpholin-2-yl, morpholin-3-yl, pyrrolidinyl (e.g., pyrrolidin-3-yl), piperazinyl (e.g., piperazin-1-yl), 3-azabicyclo[3.1.0]hexan-6-yl, or 2,5-diazabicyclo[2.2.1]heptan-2-yl.
  • pyrrolidinyl e.g., pyrrolidin-2-yl
  • morpholino morpholin-2-yl
  • morpholin-3-yl morpholin
  • aromatic group refers to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated ⁇ electron system, which is an aromatic cyclic group.
  • aromatic group is preceded by a restriction on the number of ring atoms, such as a 5-12-membered aromatic group, it means that the aromatic group has 5-12 ring atoms.
  • Representative examples of aromatic groups include, but are not limited to, phenyl, naphthyl, or similar groups.
  • heteroaryl refers to an aromatic heterocyclic group having one to multiple (preferably 1, 2, 3, 4 or 5) heteroatoms, which may be a monocyclic (monocyclic) or polycyclic (bicyclic, tricyclic or polycyclic) group fused together or covalently linked, each heterocyclic group containing heteroatoms may have one or more (e.g., 1, 2, 3, 4) heteroatoms independently selected from the following group: oxygen, sulfur and nitrogen.
  • a number limit before the heteroaryl it refers to the number of ring atoms of the heteroaryl, for example, a 5-12 membered heteroaryl refers to a heteroaryl having 5-12 ring atoms.
  • heteroaryl include, but are not limited to: pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, tetrazolyl, 1H-pyrazolo[3,4-d]pyrimidin-6-yl, or similar groups.
  • hydroxyl refers to an -OH group.
  • amino refers to the —NH 2 group.
  • cyano refers to a -CN (nitrile) group.
  • nitro refers to the group -NO2 .
  • mercapto refers to the group -SH.
  • pharmaceutically acceptable salt refers to those salts that retain the biological effects and properties of free bases or free acids, which are formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., especially hydrochloric acid) and organic acids (such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, lactic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc.).
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., especially hydrochlor
  • salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, etc.
  • Salts derived from organic bases include, but are not limited to, the following Salts of: primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, and the like).
  • hydrate refers to an association formed between a solvent molecule being water and a compound of the present invention via one, two, three or more water molecules.
  • the term "treating" includes: (1) inhibiting a state, disorder or condition of at least one clinical or subclinical symptom of a disease (e.g., in the case of maintenance therapy, preventing, alleviating or delaying the development of a disease or its recurrence); and/or (2) relieving a condition (i.e., causing a state, disorder or condition of a disease or at least one clinical or subclinical symptom to subside).
  • the benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician.
  • a drug is administered to a patient to treat a disease, the result may not always be an effective treatment.
  • one or more refers to the range from one substituent to the highest number of possible substituents, that is, replacement of one hydrogen by a substituent until all hydrogens are replaced by substituents, for example, "one or more” can mean one, two, three, four, five or six, etc.
  • composition and “pharmaceutical formulation” (or “formulation”) are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active drug ingredient and a pharmaceutically acceptable excipient, which is administered to a mammal, e.g., a human in need thereof.
  • excipient refers to any pharmaceutically acceptable ingredient in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant used to formulate the drug product.
  • compound 1 5 g, 65.71 mmol was dissolved in 200 ml of anhydrous tetrahydrofuran, and sodium cyanide (2.89 g, 72.28 mmol) was added. After stirring at 0°C for 30 minutes, a solution of tert-butyldiphenylsilyl chloride (18.1 g, 65.71 mmol) dissolved in 50 ml of tetrahydrofuran was added. The reaction mixture was placed at room temperature and stirred overnight. The reaction mixture was then quenched with aqueous ammonium chloride at 0°C and extracted with ethyl acetate.
  • compound 6 (1.0 g, 3.80 mmol) was dissolved in 20 ml of anhydrous tetrahydrofuran, and compound 5 (853 mg, 4.18 mmol), triphenylphosphine (1.5 g, 5.7 mmol) and diethyl azodicarboxylate (1.54 g, 7.6 mmol) were added.
  • the reaction mixture was stirred at room temperature for 2 hours.
  • the reaction mixture was then concentrated in vacuo and purified by silica gel column chromatography (petroleum ether-ethyl acetate, 4:1) to obtain 1.0 g of yellow liquid compound 7 in a yield of 58.5%.
  • compound 1 (6.0 g, 22.81 mmol) was dissolved in 60 ml of anhydrous tetrahydrofuran, and compound 2 (5.6 g, 25.09 mmol), triphenylphosphine (8.97 g, 34.21 mmol) and diethyl azodicarboxylate (9.22 g, 45.62 mmol) were added.
  • the reaction mixture was placed at room temperature and stirred for 2 hours.
  • the reaction mixture was then concentrated in vacuo and purified by silica gel column chromatography (petroleum ether-ethyl acetate, 4:1) to obtain 3.5 g of yellow liquid compound 3, with a yield of 36.5%.
  • Example 5 Evaluation of the inhibitory effects of compound ST-01, compound ST-04 hydrochloride, compound ST-05, and compound ST-06 on HDAC1, HDAC6, VEGFR2, and PDGFR- ⁇ activities, respectively
  • the purpose of the experiment is to detect the inhibitory ability of small molecule compounds on kinase activity through a kinase-based drug screening system.
  • the experimental principle of the HTRF method is: kinase phosphorylates substrate; Eu-labeled antibody binds to the phosphorylated site of the substrate; streptavidin-XL665 binds to the substrate biotin; when Eu and XL665 are close, Eu as a donor receives excitation from the light source (320nm), emits emission light (615nm) and resonates energy transfer to the adjacent receptor XL665, and the receptor emits emission light (665nm) after being excited; the specific signal is proportional to the phosphorylated substrate.
  • the inhibitor When the inhibitor is added, the phosphorylation level is inhibited, and the emission light of 665nm cannot be detected, and only the emission light of 615nm can be detected. In this way, the inhibitory level of the compound on the kinase activity is evaluated.
  • the experimental principle of the ADP-Glo method is: the kinase and its substrate undergo enzymatic reactions, consuming ATP to produce ADP, and the amount of the product is detected using the ADP-Glo reagent and luminescence method to reflect the activity of the kinase.
  • VEGFR2 and PDGFR- ⁇ Substrate Formula 1 VEGFR2 and PDGFR- ⁇ Substrate Formula 1
  • VEGFR2 and PDGFR- ⁇ detection steps are VEGFR2 and PDGFR- ⁇ detection steps:
  • the reading value of the negative control (1% DMSO well) was set as 0% inhibition rate, and the reading value of the positive control (positive drug well) was set as 100% inhibition rate, and the inhibition rate of each test solution was calculated.
  • IC50 half maximal inhibitory concentration
  • Min is the positive control Ratio value
  • Max is the negative control DMSO Ratio value
  • SD is standard error and AVE is the mean value of Ratio.
  • the (CTG) method was used to measure the in vitro efficacy of a test compound and a positive control drug on 20 cell lines.
  • the IC 50 of one test compound and one positive compound was determined in 20 cell lines.
  • the drug treatment time was 3 days, 9 concentrations were used, and 3 replicates were performed for each concentration.
  • Cell line Note: All cells were cultured at 37°C and 5% CO2 .
  • NEAA (GIBCO, item number: 11140050)
  • Penicillin-Streptomycin (Pen-Strep) (Gibco, Cat. No.: 15070063)
  • V sample is the reading of the drug-treated group
  • V vehicle control is the average value of the solvent control group.
  • GraphPad Prism 9 software was used to draw a S-shaped dose-survival curve using a nonlinear regression model and calculate the IC 50 value. The data also include the maximum inhibition rate.
  • the compound of the present invention can simultaneously inhibit histone deacetylase VI (HDAC6, histone deacetylases 6), histone deacetylase 1 (HDAC1) and two tyrosine kinases (vascular endothelial cell growth factor receptor, Vascular endothelial cell growth factor2, VEGFR2) and platelet-derived growth factor receptor (platelet-derived growth factor- ⁇ , PDGFR- ⁇ ), indicating that the compound of the present invention combines multiple targets in one, can be used as a multi-target inhibitor, and has the pharmacological effects of sunitinib and vorinostat.
  • HDAC6 histone deacetylase 6
  • HDAC1 histone deacetylase 1
  • two tyrosine kinases vascular endothelial cell growth factor receptor, Vascular endothelial cell growth factor2, VEGFR2
  • platelet-derived growth factor receptor platelet-derived growth factor- ⁇ , PDGFR- ⁇
  • the compound of the present invention can be used to treat diseases mediated by tyrosine kinases and histone deacetylases, such as malignant tumors.
  • Treatable malignant tumors include, but are not limited to, solid tumors such as renal cancer, liver cancer, colon cancer, gastrointestinal stromal tumors, lung cancer, breast cancer, pancreatic cancer, glioma, lymphoma, fibrosarcoma, ovarian cancer, prostate cancer, and malignant tumors of the blood system such as leukemia and lymphoma.
  • This type of compound combines multiple targets in one, which can not only give full play to the synergistic effect to increase biological activity, but also avoid the problems caused by the different properties and metabolism when multiple drugs are used in combination. It has good practicality and good prospects.
  • the compound ST-04 hydrochloride showed strong activity in 20 tumor cell lines, most of which exceeded the positive control drug, and the rest were also equivalent to the positive control drug, showing good prospects for application in the treatment of malignant tumors.

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Abstract

本发明公开了一种具有通式(Ⅰ)所示的化合物，其可药用盐、水合物或前药，及其在制备预防和/或治疗由酪氨酸激酶和/或组蛋白去乙酰化酶介导的疾病的药物中的应用，该化合物能够针对多种靶点(尤其是组蛋白去乙酰化酶1、组蛋白去乙酰化酶6、两种酪氨酸激酶(血管内皮细胞生长因子受体及血小板衍生生长因子受体))同时起到较好抑制作用，有望用于恶性肿瘤的治疗中。

Description

一种含吲唑基的羟肟酸衍生物及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种含吲唑基的羟肟酸衍生物及其应用。
背景技术
酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质，它们能催化ATP上的磷酸基转移到许多重要蛋白质的酪氨酸残基上，使其发生磷酸化，从而激活下游的信号传导通路。蛋白酪氨酸激酶在细胞内的信号传导通路中占据了十分重要的地位，调节着细胞体内生长、分化、死亡等一系列生理生化过程。蛋白酪氨酸激酶功能的失调则会引发生物体内一系列的疾病，其中包括肿瘤等疾病。许多肿瘤的发生发展及转移以及肿瘤新生血管的生成都与酪氨酸激酶的异常表达有着极其密切的联系。特别是某些酪氨酸激酶受体在实体瘤的细胞有异常表达，其中血管内皮细胞生长因子受体(vascular endothelial cell growth factor,VEGFR)在许多肿瘤细胞及肿瘤血管内皮细胞中均呈高表达，血小板衍生生长因子受体(platelet-derived growth factor，PDGFR)在肿瘤基质内成纤维细胞中异常表达。其配体与受体形成的自身分泌环路直接参与肿瘤细胞的发生及发展，例如，血管内皮细胞生长因子受体(VEGFR)存在于黑色素瘤；血小板衍生生长因子受体(PDGFR)存在于胶质瘤；干细胞生长因子受体(KIT)存在于小细胞肺癌等。另外，相似的环路也在脑膜瘤，神经内分泌肿瘤，卵巢癌，前列腺癌和胰腺癌中存在。这一环路与肿瘤的发生发展有着极其密切的关系。
此外，实体肿瘤的发生、发展和转移均依赖于肿瘤的新生血管生成，它为肿瘤的生长提供了必需的营养和氧气。肿瘤血管生成(tumor angiogenesis)是肿瘤细胞的浸润、迁移、增殖的重要过程。其中血管内皮细胞生长因子受体家族(VEGFR)和血小板衍生生长因子受体家族(PDGFR)与肿瘤的发生发展及肿瘤新生血管的生成有着直接的关系。血管内皮生长因子(VEGF)作为已知最强的血管渗透剂和内皮细胞特异的有丝分裂源，在内皮细胞的增殖，迁移和血管构建中起着重要的作用。它的表达水平和肿瘤组织的血管化程度呈现明显的正相关。VEGF主要是通过作用于内皮细胞上高亲和力的受体VEGFR-1和KDR使之酪氨酸激酶发生磷酸化而发挥其生物学作用，两者具有不同信号转导途径。其中，KDR在肿瘤的生长、转移以及肿瘤新生血管形成中起着关键的作用。血小板衍生生长因子(PDGF)和其受体(PDGFR)涉及多种肿瘤的发病机制并在血管生成中起着重要的作用。血小板衍生生长因子(PDGF)经由其受体(PDGFR)而表现其细胞生物效应。PDGFR通过调节血管壁的周细胞及血管平滑肌细胞增殖、迁移来维持血管壁的完整性，促进肿瘤新生血管的形成。并且，通过改变肿瘤内的微环境促进肿瘤生长。
由于酪氨酸激酶的异常表达与肿瘤的发生发展及转移以及肿瘤新生血管的生成有着极其密切的联系，因此，以酪氨酸激酶为靶点的药物研发已成为国际上抗肿瘤药物研究的热点。尤其是以新生血管为靶点来抑制肿瘤血管形成、阻断肿瘤的营养供应和迁移途径，防止肿瘤生长及转移已成为目前治疗肿瘤的新策略。而KDR或PDGFR受体异常表达在肿瘤的新生血管形成过程中起着关键作用，它们已成为最为理想的抗肿瘤药物治疗的靶点。而且，美国FDA批准的两个主要抑制KDR和PDGFR受体酪氨酸激酶的抗肿瘤药物 索拉非尼(Sorafenib)和舒尼替尼(SU11248，Sunitinib)在临床上已充分证实了疗效高且副作用少的抗肿瘤治疗效果。
组蛋白去乙酰化酶(histone deacetylases,HDACs)是一类细胞内的金属蛋白酶，对染色体的结构修饰和基因表达调控发挥着重要的作用。在癌细胞中，HDACs的过度表达导致组蛋白与DNA结合力增加使染色体发生异常变构。与此同时，细胞周期抑制因子的表达被抑制，肿瘤抑制因子p53的稳定性和与DNA结合的能力下降，而缺氧诱导因子(Hypoxia induciblefactor-1,HIF-1)和血管内皮生长因子(Vascular endothelial growth factor,VEGF)表达量升高。在哺乳动物细胞中，乙酰化和去乙酰化的平衡在基因转录和不同细胞蛋白功能中起到关键的作用。组蛋白乙酰化状态由组蛋白乙酰转移酶(Histone Acetyltransferases,HATs)和组蛋白去乙酰化酶(Histone Deacetylases,HDACs)调节。HATs和HDACs之间的动态平衡控制着染色质的结构和基因的表达，它们的功能紊乱是肿瘤发生发展的重要分子机制之一。HDACs属于去乙酰化酶超家族。HDACs在染色质重塑、基因阻遏、调节细胞周期和分化中起到重要作用。肿瘤细胞中组蛋白去乙酰化酶功能异常可导致许多基因转录抑制，抑制抑癌基因表达。有资料报道在胃癌、前列腺癌、结肠癌及肝癌组织中HDAC1mRNA水平和蛋白水平均高表达，并和肿瘤的TNM分期及淋巴结转移相关。研究发现，抑制HDACs的活性能有效的抑制癌细胞增殖，诱导细胞周期停滞和促进细胞凋亡，因此，HDACs成为抗癌药物设计的新靶点，开发HDACs抑制剂(HDACi)被视为肿瘤治疗一个有效的策略。异羟肟酸类小分子HDACi是近年来备受重视的一类HDACi，在体内和体外均显示出良好的抗肿瘤活性。HDAC作为抗癌靶点成药已发展较为成熟。2006年，美国药企默沙东推出了治疗皮肤T细胞淋巴瘤的伏立诺他这是首个上市的HDAC抑制剂。此后，数款HDAC抑制剂相继获批上市，分别是治疗T细胞淋巴瘤的罗米地辛(2009年上市，原研厂家美国新基公司)、用于治疗复发性或耐药性外周T细胞淋巴瘤的贝利司他(2014年上市，原研厂家Spectrum生物医药公司)、治疗多发性骨髓瘤的帕比司他(2015年上市，原研厂家瑞士诺华公司)等。2015年，深圳微芯依托中国本土市场，成功推出了西达本胺，这是中国市场上首个治疗淋巴瘤的HDAC抑制剂。越来越多的HDACi陆续进入临床用于治疗和辅助治疗结肠癌、肺癌等实体瘤以及白血病、淋巴癌等血液系统的恶性肿瘤。
发明内容
本发明的目的之一是提供一种能够针对多种靶点(尤其是组蛋白去乙酰化酶1、组蛋白去乙酰化酶6、两种酪氨酸激酶(血管内皮细胞生长因子受体及血小板衍生生长因子受体))同时起到较好抑制作用的新型的含吲唑基的羟肟酸衍生物。
本发明的另一个目的是提供一种包含上述新型的含吲唑基的羟肟酸衍生物的药物组合物。
本发明的再一个目的是提供上述新型的含吲唑基的羟肟酸衍生物或上述包含上述新型的含吲唑基的羟肟酸衍生物的药物组合物在制备预防和/或治疗由酪氨酸激酶和/或组蛋白去乙酰化酶介导的疾病的药物 中的应用。
为达到上述目的，本发明采用的一种技术方案是：
一种具有通式(Ⅰ)所示的化合物，其可药用盐、水合物或前药，
其中：Ra、Rb独立地选自氢、C1-20烷基、3-10元杂环烷基、5-12元芳香基、5-12元杂芳基；其中，所述的C1-20烷基、3-10元杂环烷基、5-12元芳香基、5-12元杂芳基各自独立地未被取代或被选自如下取代基中的一个、两个、三个或更多个所取代：氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基、C1-6烷基取代的氨基、C1-10烷基、卤代的C1-6烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基、羟基取代的C1-6烷基、C1-6烷氧基、卤代的C1-6烷氧基、硝基取代的C1-6烷氧基、氰基取代的C1-6烷氧基、巯基取代的C1-6烷氧基、氨基取代的C1-6烷氧基、羟基取代的C1-6烷氧基、C2-6烯基、C2-6炔基、-C(＝O)R1、-C(＝O)NHR2、-C(＝O)OR3、-NHC(＝O)R4、-OC(＝O)R5；
R1、R2、R3、R4、R5独立地选自未被取代的C1-20烷基，或独立地选自被氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基中的一个或多个取代基所取代的C1-20烷基；
各个Rc相同或者不同，且各自独立地选自氢、C1-20烷基、C1-20烷氧基、氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基、C1-6烷基取代的氨基、卤代的C1-6烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基；
Rd、Re独立地选自氢、C1-20烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基；
Rg具有链状结构，构成所述链状结构的主链的原子数为3-15个且其中杂原子个数不超过2个，所述杂原子选自氧、硫、氮；
x为1、2、3或4。
在一些实施方式中，C1-20烷基可以是C1-10烷基或C10-20烷基；非限制性地包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、己基、庚基等等。
根据本发明的一些优选方面，构成所述链状结构的主链的原子中杂原子的个数为1个或2个，且其中一个杂原子与吲唑环中的苯环连接。
根据本发明的一些优选方面，所述链状结构为饱和直链或饱和支链。
根据本发明的一些优选且具体的方面，所述的化合物具有通式(Ⅱ)所示的结构：
其中：Ra、Rb、Rc、Rd、Re、x分别与通式(Ⅰ)中的Ra、Rb、Rc、Rd、Re、x相同，A、B独立地为CH2、O、S或-N(R6)-，R6为氢或C1-6烷基，m、n独立地选自0-10的整数，且3≤m+n≤10。
在一些实施方式中，m为0、1、2、3、4、5、6、7、8、9、10。在一些实施方式中，n为0、1、2、3、4、5、6、7、8、9、10。
根据本发明的一些优选方面，当存在如下任一情形时，B为CH2：
(1)A为CH2；(2)A选自O、S或-N(R6)-，且m为0；(3)n为0。
根据本发明的一些优选方面，A选自CH2、O、S或-NH-，B选自CH2、O、-N(CH3)-、-N(CH2CH3)-或-N(CH2CH2CH3)-。
在本发明的一些实施方式中，A为O，B为CH2，m+n为3、4或5。
根据本发明的一些优选方面，Ra、Rb不同时为氢。
进一步地，Ra、Rb中，一个选自未被取代的或被取代基取代的嘧啶基、吡唑基、吡啶基、吡嗪基、咪唑基、哒嗪基、噻唑基、苯基；另一个选自氢，或者选自未被取代的或被1-3个卤素取代的C1-6烷基。其中，取代所采用的取代基包括：氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基、C1-6烷基取代的氨基、C1-10烷基、卤代的C1-6烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基、羟基取代的C1-6烷基、C1-6烷氧基、卤代的C1-6烷氧基、硝基取代的C1-6烷氧基、氰基取代的C1-6烷氧基、巯基取代的C1-6烷氧基、氨基取代的C1-6烷氧基、羟基取代的C1-6烷氧基、C2-6烯基、C2-6炔基、-C(＝O)R1、-C(＝O)NHR2、-C(＝O)OR3、-NHC(＝O)R4、-OC(＝O)R5；R1、R2、R3、R4、R5独立地选自未被取代的C1-20烷基，或独立地选自被氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基中的一个或多个取代基所取代的C1-20烷基。
在一些实施方式中，所述的化合物具有通式(Ⅲ)所示的结构：
其中：Rc、Rd、Re、x、A、B、m、n分别与通式(ⅠI)中的Rc、Rd、Re、x、A、B、m、n相同；Rb选自氢，或者选自未被取代的或被1-3个卤素取代的C1-6烷基；R7选自氟、氯、溴、碘、羟基、巯基、氰基、氨基、甲氨基、乙氨基、硝基、C1-6烷基、卤代的C1-6烷基、C1-6烷氧基、卤代的C1-6烷氧基、羟基甲基、巯基甲基、C(＝O)CH3、C(＝O)CH2CH3、C(＝O)NHCH3、C(＝O)NHCH2CH3、NHC(＝O)CH3、NHC(＝O)CH2CH3、NHCH3、N(CH3)2、NHCH2CH3，t为0、1、2、3、4或5，D、E独立地选自CH或N。
在一些实施方式中，当t为4时，D、E中至少一个为CH，当t为5时，D、E均为CH。
在一些实施方式中，所述的化合物具有通式(Ⅳ)所示的结构：
其中：Rb、Rc、Rd、Re、x、A、B、m、n分别与通式(ⅠII)中的Rb、Rc、Rd、Re、x、A、B、m、n相同，R8、R9独立地选自氢、氟、氯、溴、碘、甲基、乙基、正丙基、异丙基、正丁基、异丁基、卤代的甲基、卤代的乙基、卤代的正丙基、卤代的异丙基、卤代的正丁基、卤代的异丁基、甲氧基、乙氧基、卤代的甲氧基、卤代的乙氧基。
本发明中，卤代的可以是选自氟、氯、溴、碘中的一个或多个卤素进行取代，例如可以是1个氟、2个氟、1个氯1个氟、2个溴1个氟等等进行取代。
在一些实施方式中，Rb、Rd、Re独立地选自氢、甲基、乙基、正丙基、异丙基、正丁基或异丁基。
在一些实施方式中，各个Rc相同或者不同，且各自独立地选自氢、氟、氯、溴、甲基、乙基、正丙基、异丙基、正丁基、异丁基、甲氧基或乙氧基。
在一些实施方式中，Rb、Rc、Rd、Re均为氢。
在一些实施方式中，Rg选自：
在一些实施方式中，所述的化合物具有通式(Ⅴ)所示的结构：
其中：p为2、3、4、5、6、7、8、9或10，R8、R9独立地选自氢、氟、氯、溴、碘、甲基、乙基、正丙基、异丙基、正丁基、异丁基、卤代的甲基、卤代的乙基、卤代的正丙基、卤代的异丙基、卤代的正丁基、卤代的异丁基、甲氧基、乙氧基、卤代的甲氧基、卤代的乙氧基。
在本发明的一些实施方式中，所述化合物的结构式如式(ST-01)至(ST-16)中任一项所示：


本发明提供的又一技术方案：一种药物组合物，该药物组合物包含上述所述的化合物，其可药用盐、水合物或前药，以及药学上可接受的载体。
进一步地，该药物组合物还可以包含稀释剂或赋形剂。
进一步地，所述药物组合物为用于预防和/或治疗恶性肿瘤的组合物，所述恶性肿瘤包括但不限于肾癌、肝癌、结肠癌、胃肠道间质瘤、肺癌、乳腺癌、胰腺癌、神经胶质瘤、淋巴癌、纤维肉瘤、卵巢癌、前列腺癌、白血病、淋巴癌。
本发明提供的又一技术方案：上述所述的化合物，其可药用盐、水合物或前药，或者上述所述的药物组合物在制备预防和/或治疗由酪氨酸激酶和/或组蛋白去乙酰化酶介导的疾病的药物中的应用。
进一步地，所述由酪氨酸激酶和/或组蛋白去乙酰化酶介导的疾病为恶性肿瘤，所述恶性肿瘤包括但不限于肾癌、肝癌、结肠癌、胃肠道间质瘤、肺癌、乳腺癌、胰腺癌、神经胶质瘤、淋巴癌、纤维肉瘤、卵巢癌、前列腺癌、白血病、淋巴癌。
根据本发明，所述的化合物，其不仅包括单一的某种化合物形式，还包括多种结构满足通式(Ⅰ)要求的化合物的混合物形式，以及同一化合物的不同异构体形式例如外消旋体、对映异构体、非对映异构体等。
所述的可药用盐包括但不限于盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、醋酸盐、三氟醋酸盐、马来酸盐、甲磺酸盐、苯磺酸盐、苯酸盐、甲基苯磺酸盐、琥珀酸盐、延胡索酸盐、富马酸盐、酒石酸盐、没食子酸盐、柠檬酸盐等。
具有通式(Ⅰ)所示的化合物或其药学上可接受的盐通常会经由口服途径以如下药物制剂形式施用，这些药物制剂包含活性成分或其药学上可接受的盐或溶剂化物、或这样的盐的溶剂化物，呈药学上可接受的剂型。取决于待治疗的障碍和患者，组合物可以按不同剂量施用。
以上所述的具有通式(Ⅰ)所示的化合物的药物配制品可被制备用于口服施用，具体地呈片剂或胶囊形式，并且尤其涉及目的在于提供靶向结肠的药物释放的技术。
以上所述的具有通式(Ⅰ)所示的化合物的药物配制品可以方便地以单位剂型施用，并且可以通过制药领域中熟知的任何方法制备。
适于口服施用的药物配制品可以包含一种或多种生理上可相容的载体和/或赋形剂，并且可以是呈固体或液体的形式。片剂和胶囊可以用粘合剂、填充剂、润滑剂和/或表面活性剂(如十二烷基硫酸钠)制备。液体组合物可以含有常规添加剂，如悬浮剂、乳化剂和/或防腐剂。液体组合物可以包封在例如明胶中以提供单位剂型。
口服配制品是优选的，特别是片剂或胶囊，其可以通过本领域技术人员已知的方法配制，以提供0.001mg至10000mg范围内的活性化合物的剂量，例如可以提供以0.01mg至5000mg范围内的活性化合物的剂量，也可以提供0.1mg至1000mg范围内的活性化合物的剂量等。
本发明中，通式(Ⅰ)所示的化合物的前药是指当采用适当的方法施用后，可在受试者体内进行代谢或化学反应(例如酶解或水解)而转变成通式(Ⅰ)的至少一种化合物或其盐的前体药物。
本发明提供的又一技术方案：一种中间体，用于制备上述所述的化合物，其可药用盐、水合物或前药，所述中间体具有通式(Ⅵ)所示结构：
其中：Ra、Rb、Rc、Rd、Re、Rg、x分别与前述通式中的Ra、Rb、Rc、Rd、Re、Rg、x相同，R为C1-6烷氧基。
在一些实施方式中，R可以为甲氧基、乙氧基、丙氧基或叔丁氧基。
本发明提供的又一技术方案：一种制备上述所述的化合物，其可药用盐、水合物或前药的方法，该方法包括使上述所述的中间体与羟胺进行反应的步骤。
在一些实施方式中，所述羟胺通过添加羟胺无机酸盐转化制得，所述羟胺无机酸盐包括但不限于羟胺盐酸盐和/或羟胺磷酸盐。
在一些实施方式中，使上述所述的中间体与羟胺的反应在碱性条件下、在醇溶剂中进行。在一些实施方式中，所述碱性条件通过添加碱性物质形成，所述碱性物质包括但不限于氢氧化钠、氢氧化钾等。
在一些实施方式中，所述醇溶剂包括但不限于甲醇等。
由于上述技术方案运用，本发明与现有技术相比具有下列优点：
本发明化合物可以将多种重要的靶点集于一身，尤其是可以同时对组蛋白去乙酰化酶VI(HDAC6， histone deacetylases 6)，组蛋白去乙酰化酶1(HDAC1)和两种酪氨酸激酶(血管内皮细胞生长因子受体，Vascular endothelial cell growth factor2，VEGFR2)及血小板衍生生长因子受体(platelet-derived growth factor-β，PDGFR-β)具有很强的抑制作用，可以较好地避免多种药物联合用药时可能存在的因为不同性质和代谢所引起的问题，能充分发挥协同作用增加生物活性，应用前景较好。
具体实施方式
除非另有定义，否则本文所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员理解的相同含义。尽管与本文所述方法或材料相似或等同的方法和材料都可用于本发明的实施或测试，但适合的方法和材料描述于下文中。
术语“C1-20烷基”是指1至20个碳原子(“C1-C20-烷基”)的直链或支链饱和烃基团。在一些实施例中，烷基基团含有1至10个碳原子，或者含有1至6个碳原子，例如1、2、3、4、5或6个碳原子。一些非限制性实例包括甲基、乙基、丙基、2-丙基(异丙基)、正丁基、异丁基、仲丁基、叔丁基和2,2-二甲基丙基。
术语“烷氧基”是指经由氧原子附接到母体分子部分上的烷基基团。在一些实施例中，烷氧基基团含有1至6个碳原子，可以表述为C1-6烷氧基。一些非限制性实例包括甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基和叔丁氧基。
术语“卤素”是指氟(F)、氯(Cl)、溴(Br)或碘(I)。优选地，术语“卤素”是指氟(F)、氯(Cl)或溴(Br)。
术语“氨基取代的烷基”是指其中烷基基团的至少一个氢原子被氨基基团代替的烷基基团。例如氨基取代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被氨基基团代替。优选地，“氨基烷基”是指其中烷基基团的1、2或3个氢原子被氨基基团代替的烷基基团。氨基烷基的非限制性实例可以是氨基甲基和1-氨基乙基等。
术语“氨基取代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被氨基基团取代。例如氨基取代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被氨基基团代替。优选地，“氨基烷氧基”是指一种烷氧基基团，其中该烷氧基基团的1、2或3个氢原子被氨基基团取代。氨基烷氧基的非限制性的实例是氨基甲氧基和1-氨基乙氧基等。
术语“硝基取代的烷基”是指一种烷基基团，其中该烷基基团的至少一个氢原子被硝基基团取代。例如硝基取代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被硝基基团代替。
术语“硝基取代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被硝基基团取代。例如硝基取代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被硝基基团代替。
术语“氰基取代的烷基”是指一种烷基基团，其中该烷基基团的至少一个氢原子被氰基基团取代。例如氰基取代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被氰基基团代替。
术语“氰基取代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被氰基基团取代。例如氰基取代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被氰基基团代替。
术语“巯基取代的烷基”是指一种烷基基团，其中该烷基基团的至少一个氢原子被巯基基团取代。例如巯基取代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被巯基基团代替。
术语“巯基取代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被巯基基团取代。 例如巯基取代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被巯基基团代替。
术语“羟基取代的烷基”是指一种烷基基团，其中该烷基基团的至少一个氢原子被羟基基团取代。例如羟基取代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被羟基基团代替。
术语“羟基取代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被羟基基团取代。例如羟基取代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被羟基基团代替。
术语“卤代的烷基”是指一种烷基基团，其中该烷基基团的至少一个氢原子被卤素例如氟(F)、氯(Cl)、溴(Br)或碘(I)取代。例如卤代的C1-6烷基，则是指C1-6烷基基团的至少一个氢原子被卤素代替。
术语“卤代的烷氧基”是指一种烷氧基基团，其中该烷氧基基团的至少一个氢原子被卤素例如氟(F)、氯(Cl)、溴(Br)或碘(I)取代。例如卤代的C1-6烷氧基，则是指C1-6烷氧基基团的至少一个氢原子被卤素代替。
术语“杂环烷基”是指饱和的单环、双环，或更多环稠合的多环，例如“3-10元杂环烷基”则是指3至10个环原子的单环、双环，或更多环稠合的多环，其中所述环原子中的1、2、3或4个为选自N、O和S的杂原子，其余环原子为碳。杂环烷基的一些非限制性实例包括氮杂环丁烷-3-基、氮杂环丁烷-2-基、氧杂环丁烷-3-基、氧杂环丁烷-2-基、1-哌啶基、2-哌啶基、3-哌啶基、4-哌啶基、吡咯烷基(例如，吡咯烷-2-基)、吗啉代、吗啉-2-基、吗啉-3-基、吡咯烷基(例如，吡咯烷-3-基)、哌嗪基(例如，哌嗪-1-基)、3-氮杂双环[3.1.0]己烷-6-基或2,5-二氮杂双环[2.2.1]庚烷-2-基。
术语“芳香基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，是一种芳香环状基团，当芳香基前面具有环原子数限定，如5-12元芳香基，则指所述的芳香基具有5-12个环原子。芳香基代表性示例包括但不限于：苯基、萘基，或类似基团。
术语“杂芳基”指具有一个到多个(优选为1、2、3、4或5个)杂原子的芳族杂环基团，其可以是单环(单环的)或者稠合在一起或共价地连接的多环(二环的、三环的或多环的)基团，每个含有杂原子的杂环基团上可以具有一个或多个(如1、2、3、4个)各自独立选自下组的杂原子：氧、硫和氮。当杂芳基前有元数限定时，指的是杂芳基的环原子个数，例如5-12元杂芳基指的是具有5-12个环原子的杂芳基。杂芳基代表性的例子包括但不限于：吡咯基、吡唑基、咪唑基、噁唑基、噻唑基、呋喃基、吡啶基、吡嗪基、嘧啶基、哒嗪基、三氮唑基、四氮唑基、1H-吡唑并[3,4-d]嘧啶-6-基，或类似基团。
术语“羟基”是指–OH基团。
术语“氨基”是指–NH2基团。
术语“氰基”是指–CN(腈)基团。
术语“硝基”是指基团-NO2。
术语“巯基”是指基团-SH。
术语“可药用盐”是指保留游离碱或游离酸的生物效果和性质的那些盐，这些盐用无机酸(诸如盐酸、氢溴酸、硫酸、硝酸、磷酸等，特别是盐酸)和有机酸(诸如乙酸、三氟乙酸、丙酸、乙醇酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、乳酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸、N-乙酰半胱氨酸等)形成。另外，这些盐可通过向游离酸中添加无机碱或有机碱来制备。衍生自无机碱的盐包括但不限于钠、钾、锂、铵、钙、镁盐等。衍生自有机碱的盐包括但不限于以下 各项的盐：伯胺、仲胺和叔胺、取代胺(包括天然存在的取代胺)、环胺和碱性离子交换树脂(诸如异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、赖氨酸、精氨酸、N-乙基哌啶、哌啶、聚亚胺树脂等)。
术语“水合物”是指溶剂分子是水，且通过一个、两个、三个或者更多个水分子与本发明化合物所形成的缔合物。
如本文所用的术语“治疗”包括：(1)抑制疾病的至少一种临床或亚临床症状的状态、病症或病情(例如，在维持治疗的情况下，阻止、减轻或延迟疾病的发展或其复发)；和/或(2)缓解病情(即，使疾病的状态、病症或病情或者至少一种临床或亚临床症状消退)。对待治疗患者的益处在统计学上是显著的或者至少是患者或医生可察觉的。然而，应当理解，当向患者施用药物以治疗疾病时，结果可能并不总是有效的治疗。
当指示取代基的数量时，术语“一个或多个(one or more)”是指从一个取代基到最高数量的可能的取代基的范围，即通过取代基取代一个氢直至所有氢被取代基取代，例如“一个或多个”可以是指一个、两个、三个、四个、五个或六个等。
术语“药物组合物”和“药物制剂(pharmaceutical formulation)”(或“制剂”)可互换使用，且表示包含治疗有效量的活性药物成分与药学上可接受的赋形剂的混合物或溶液，其施用于哺乳动物，例如，此需要的人。
术语“赋形剂”、“药用载体”和“治疗惰性赋形剂”可互换使用，且表示药物组合物中没有治疗活性且对被施用的个体无毒的任何药学上可接受的成分，诸如用于调制药物产品的崩解剂、粘合剂、填充剂、溶剂、缓冲剂、张力剂、稳定剂、抗氧化剂、表面活性剂、载体、稀释剂或润滑剂。
以下结合具体实施例对上述方案做进一步说明；应理解，这些实施例是用于说明本发明的基本原理、主要特征和优点，而本发明不受以下实施例的范围限制；实施例中采用的实施条件可以根据具体要求做进一步调整，未注明的实施条件通常为常规实验中的条件。
下述实施例中未作特殊说明，所有原料均来自于商购或通过本领域的常规方法制备而得。
实施例1化合物ST-01的合成
实验过程：
1)化合物2的制备
在-78℃下，将化合物1(11.6克，46.03毫摩尔)溶解于100毫升无水四氢呋喃中，再加入二异丙基氨基锂(30毫升，59.83毫摩尔，2摩尔每升的四氢呋喃溶液)。-78℃搅拌1小时后，将二氧化碳鼓入反应并继续搅拌1小时。然后用6M盐酸将反应混合物调节pH值至2，用乙酸乙酯萃取，用盐水洗涤，有机相用无水硫酸钠干燥并真空浓缩，得到14.8g黄色固体粗化合物2，其直接用于下一步反应。1H NMR(400MHz,DMSO-d6)δ13.92(s,1H),7.62(dd,J＝8.6,1.5Hz,1H),7.05(t,J＝8.8Hz,1H),3.85(s,3H).
2)化合物3的制备
将化合物2(16.6克，56.07毫摩尔)和二甲基甲酰胺(0.3毫升)加入50毫升氯化亚砜中，80℃加热3小时。反应混合物真空浓缩，得到粗产物2-氟-6-碘-3-甲氧基苯甲酰氯。上述粗产物在0℃下加入50毫升无水四氢呋喃和50毫升氨水，继续0℃搅拌0.5小时，将反应混合物真空浓缩，得到10.0克棕色固体粗化合物3，直接用于下一步反应。1H NMR(400MHz,DMSO-d6)δ7.99(s,1H),7.73(s,1H),7.57(dd,J＝8.8,1.4Hz,1H),6.99(t,J＝8.8Hz,1H),3.84(s,3H).
3)化合物4的制备
将化合物3(10.7克，36.27毫摩尔)和氯化亚砜(22.0克，181.33毫摩尔)加入60毫升二甲基甲酰胺中，加热至115℃反应过夜。将反应混合物倒入冷却水中并用乙酸乙酯萃取。有机层用饱和碳酸氢钠水溶液和盐水洗涤，用无水硫酸钠干燥并浓缩。剩余物用硅胶柱层析纯化(石油醚-乙酸乙酯，10:1)，得到4.4g红色固体化合物4，收率43.8％。1H NMR(400MHz,DMSO-d6)δ7.80(dd,J＝8.8,1.3Hz,1H),7.36(t,J＝8.8Hz,1H),3.89(s,3H).
4)化合物5的制备
在-78℃下将化合物4(1.0克，3.61毫摩尔)溶解于10毫升二氯甲烷中，加入三溴化硼22毫升。将反应混合物置于室温并搅拌过夜。然后将反应混合物倒入冷水中并用二甲基甲酰胺萃取。用盐水洗涤有机层，用无水硫酸钠干燥并浓缩。剩余物用硅胶柱层析纯化(石油醚-乙酸乙酯，7:1)，得到850mg棕色固体化合物5，收率89.5％。1H NMR(400MHz,DMSO-d6)δ10.96(s,1H),7.62(dd,J＝8.8,1.2Hz,1H),7.09(t,J＝8.8Hz,1H).
5)化合物7的制备
将化合物5(1.0克，3.80毫摩尔)溶解于10毫升乙腈中，加入化合物6(1.0克，4.56毫摩尔)和碳 酸钾(1.6克，11.41毫摩尔)。将反应混合物在60℃下加热6小时，然后真空浓缩。剩余物用硅胶柱层析纯化(石油醚-乙酸乙酯，5:1)，得到1.5克黄色固体化合物7，收率97.4％。1H NMR(400MHz,DMSO-d6)δ7.76(dd,J＝8.8,1.3Hz,1H),7.36(t,J＝8.8Hz,1H),4.09(t,J＝6.4Hz,2H),3.58(s,3H),2.30(t,J＝7.2Hz,2H),1.76–1.67(m,2H),1.58–1.49(m,2H),1.44–1.28(m,4H).LC-MS:(ESI)m/z[M+H]+＝406.0
6)化合物8的制备
将化合物7(1.0克，2.47毫摩尔)和水合肼(927毫克，18.51毫摩尔)加入10毫升正丁醇中，110℃加热1小时。然后将反应混合物真空浓缩并用硅胶柱层析纯化(二氯甲烷-甲醇，30:1)，得到508毫克黄色固体化合物8，收率49.3％。1H NMR(400MHz,DMSO-d6)δ11.91(s,1H),7.19(d,J＝7.6Hz,1H),6.50(d,J＝7.6Hz,1H),4.97(s,2H),4.06(t,J＝6.4Hz,2H),3.58(s,3H),2.31(t,J＝7.2Hz,2H),1.80–1.70(m,2H),1.55(dt,J＝15.2,7.2Hz,2H),1.46(dd,J＝15.2,7.2Hz,2H),1.38–1.29(m,2H).LC-MS:(ESI)m/z[M+H]+＝418.0
7)化合物10的制备
将化合物8(1.1克，2.64毫摩尔)溶解于12毫升二甲醚和4毫升水的混合溶液中，加入化合物9(1.2克，5.27毫摩尔)、四(三苯基膦)钯(305毫克，0.26毫摩尔)和碳酸钠(838毫克，7.91毫摩尔)。将反应混合物通氮气并加热至80℃反应过夜。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，15:1)，得到868毫克棕色固体化合物10，收率86.1％。1H NMR(400MHz,DMSO-d6)δ11.70(s,1H),7.07(d,J＝8.4Hz,2H),6.70(d,J＝7.6Hz,1H),6.65(d,J＝8.4Hz,2H),6.57(d,J＝7.6Hz,1H),5.19(s,2H),4.31(s,2H),4.09(t,J＝6.4Hz,2H),3.58(s,3H),2.33(t,J＝7.2Hz,2H),1.82–1.73(m,2H),1.57(dt,J＝14.8,7.2Hz,2H),1.49(dd,J＝15.2,7.6Hz,2H),1.36(dd,J＝14.8,8.0Hz,2H).LC-MS:(ESI)m/z[M+H]+＝383.2
8)化合物12的制备
在0℃下将化合物10(860毫克，2.25毫摩尔)溶解于8毫升二甲基甲酰胺中，加入化合物11(299毫克，2.25毫摩尔)。将反应混合物置于室温搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯 化(二氯甲烷-甲醇，20:1)纯化，得到680毫克黄色固体化合物12，收率58.7％。1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),8.75(s,1H),8.63(s,1H),7.55(d,J＝8.4Hz,2H),7.34(d,J＝8.4Hz,2H),7.32(s,1H),7.25(d,J＝8.4Hz,1H),7.16(t,J＝7.6Hz,1H),6.80(d,J＝7.2Hz,1H),6.76(d,J＝7.6Hz,1H),6.67(d,J＝7.6Hz,1H),4.30(s,2H),4.12(t,J＝6.4Hz,2H),3.58(s,3H),2.33(t,J＝7.6Hz,2H),2.29(s,3H),1.84–1.74(m,2H),1.59(dd,J＝15.0,7.6Hz,2H),1.54–1.46(m,2H),1.40–1.31(m,2H).LC-MS:(ESI)m/z[M+H]+＝516.2
9)化合物ST-01的制备
将4.2克盐酸羟胺和2.4克氢氧化钠加入25毫升甲醇中，0℃搅拌半小时。过滤反应混合物以获得滤液。将化合物12(1.5克，2.91毫摩尔)和氢氧化钠(465毫克，11.64毫摩尔)加入15毫升上述滤液中，室温搅拌2小时。然后用1摩尔每升的盐酸将反应混合物调节pH值至7。过滤反应混合物以获得固体，用甲醇和水洗涤固体，得到1.0克白色固体化合物ST-01，收率66.5％。1H NMR(400MHz,DMSO-d6)δ11.83(s,1H),10.38(s,1H),8.80–8.62(m,3H),7.57(d,J＝8.0Hz,2H),7.39–7.31(m,3H),7.27(d,J＝8.0Hz,1H),7.17(t,J＝7.6Hz,1H),6.80(d,J＝7.2Hz,1H),6.75(d,J＝7.6Hz,1H),6.68(d,J＝7.6Hz,1H),4.32(s,2H),4.11(t,J＝6.0Hz,2H),2.29(s,3H),1.99(t,J＝7.2Hz,2H),1.83–1.75(m,2H),1.58–1.47(m,4H),1.34(d,J＝6.8Hz,2H).13C NMR(101MHz,DMSO-d6)δ169.7,153.0,148.9,143.9,140.1,139.3,138.5,134.2,133.2,129.7,129.1,127.9,123.1,120.0,119.2,118.5,115.9,112.3,106.4,68.2,32.7,29.1,28.9,25.6,25.6,21.7.LC-MS:(ESI)m/z[M+H]+＝517.2
实施例2化合物ST-04的合成
实验过程：
1)采用实施例1中的方法制备化合物10
2)化合物12的制备
在0℃下将化合物10(1.0克，2.61毫摩尔)溶解于10毫升二甲基甲酰胺中，然后加入化合物11(394毫克，2.61毫摩尔)。将反应混合物置于室温搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，20:1)纯化，得到560毫克白色固体化合物12，收率40.1％。1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),9.17(s,1H),8.52(s,1H),8.01(d,J＝7.7Hz,1H),7.56(d,J＝8.6Hz,2H),7.36 (d,J＝8.5Hz,2H),7.11(dd,J＝11.3,8.3Hz,1H),6.83–6.78(m,1H),6.76(d,J＝7.8Hz,1H),6.67(d,J＝7.6Hz,1H),4.29(s,2H),4.12(t,J＝6.4Hz,2H),3.58(s,3H),2.33(t,J＝7.4Hz,2H),2.28(s,3H),1.85–1.75(m,2H),1.59(dd,J＝15.1,7.5Hz,2H),1.50(dd,J＝15.6,7.5Hz,2H),1.41–1.32(m,2H).LC-MS:(ESI)m/z[M+H]+＝534.2
3)化合物ST-04和ST-04盐酸盐的制备
将4.2克盐酸羟胺和2.4克氢氧化钠加入25毫升甲醇中，0℃搅拌半小时。过滤反应混合物以获得滤液。将化合物12(760毫克，1.42毫摩尔)和氢氧化钠(227毫克，5.68毫摩尔)加入10毫升上述滤液中，室温搅拌2小时。然后用1摩尔每升的盐酸将反应混合物调节pH值至7。过滤反应混合物以获得固体。用甲醇和水洗涤固体，得到400毫克白色固体化合物ST-04，收率52.5％。然后将990毫克化合物ST-04加入水中，80℃加热半小时。将反应混合物冷却至室温并过滤以获得固体。将上述固体加入30毫升2摩尔每升的盐酸中，室温搅拌1小时，冷冻干燥，得到760毫克化合物ST-04盐酸盐(含一分子盐酸)。
化合物ST-04：1H NMR(400MHz,DMSO-d6)δ11.81(s,1H),10.34(s,1H),9.21(s,1H),8.65(s,1H),8.54(d,J＝2.1Hz,1H),8.01(d,J＝6.4Hz,1H),7.56(d,J＝8.5Hz,2H),7.36(d,J＝8.4Hz,2H),7.11(dd,J＝11.3,8.4Hz,1H),6.83–6.78(m,1H),6.76(d,J＝7.8Hz,1H),6.68(d,J＝7.7Hz,1H),4.29(s,2H),4.12(t,J＝6.3Hz,2H),2.28(s,3H),1.97(t,J＝7.3Hz,2H),1.84–1.73(m,2H),1.59–1.45(m,4H),1.34(dd,J＝14.6,7.6Hz,2H).
化合物ST-04盐酸盐：
1H NMR(400MHz,DMSO-d6)δ13.26(br,1H),10.42(br,1H),9.79(s,1H),8.78(s,1H),7.98(dd,J＝7.8,1.7Hz,1H),7.61(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),7.09(dd,J＝11.2,8.3Hz,1H),6.99(d,J＝7.9Hz,1H),6.93(d,J＝7.8Hz,1H),6.85–6.75(m,1H),4.18(t,J＝6.2Hz,2H),2.27(s,3H),1.98(t,J＝7.3Hz,2H),1.86–1.73(m,2H),1.60–1.44(m,4H),1.36–1.29(m,2H).19F NMR(400MHz,DMSO-d6)δ-134.30.13C NMR(101MHz,DMSO-d6)δ169.6,152.8,152.0,149.7,144.1,139.7,134.7,133.9,133.9,132.1,129.7,127.6,127.5,123.2,122.9,121.6,118.6,115.1,114.9,112.7,108.5,68.6,32.7,28.9,28.8,25.6,25.6,21.2.LC-MS:(ESI)m/z[M+H]+＝535.2。
实施例3化合物ST-05的合成
实验过程：
1)化合物2的制备
在0℃下，将化合物1(5克，65.71毫摩尔)溶解于200毫升无水四氢呋喃中，加入氰化钠(2.89克，72.28毫摩尔)。0℃搅拌30分钟后，加入溶解于50毫升四氢呋喃中的叔丁基二苯基氯硅烷(18.1克，65.71毫摩尔)溶液。将反应混合物置于室温并搅拌过夜。然后在0℃下用氯化铵水溶液猝灭反应混合物，并用乙酸乙酯萃取。有机层用盐水洗涤，用硫酸钠干燥，真空浓缩。剩余物通过硅胶柱层析纯化(石油醚-乙酸乙酯，10:1)，得到13.6克无色油状化合物2，收率65.8％。1H NMR(400MHz,DMSO-d6)δ7.63(dd,J＝7.4,1.6Hz,4H),7.50–7.38(m,6H),3.74(t,J＝6.4Hz,2H),3.53(t,J＝6.3Hz,2H),1.70(p,J＝6.3Hz,2H),1.00(s,9H).
2)化合物4的制备
将化合物2(13.6克，43.24毫摩尔)溶解于170毫升叔丁醇中，加入化合物3(8.31克，64.86毫摩尔)和碳酸铯(14.09克，43.24毫摩尔)。室温搅拌6小时后，将反应混合物倒入水中并用乙酸乙酯萃取。有机层用盐水洗涤，用硫酸钠干燥，真空浓缩。剩余物通过硅胶柱层析纯化(石油醚-乙酸乙酯，10:1)，得到11.5克无色油状化合物4，收率60％。1H NMR(400MHz,DMSO-d6)δ7.63–7.60(m,4H),7.49–7.38(m,6H),3.70(t,J＝6.3Hz,2H),3.53(t,J＝6.2Hz,2H),3.48(t,J＝6.2Hz,2H),2.38(t,J＝6.2Hz,2H),1.74(p,J＝6.2Hz,2H),1.36(s,9H),0.99(s,9H).
3)化合物5的制备
将化合物4(0.5克，1.13毫摩尔)溶解于5毫升无水四氢呋喃中，加入四丁基氟化铵(2.30毫升，2.26毫摩尔，1摩尔每升的四氢呋喃溶液)。室温搅拌2小时，将反应混合物倒入水中并用乙酸乙酯萃取。有机层用盐水洗涤，用硫酸钠干燥，真空浓缩。剩余物通过硅胶柱层析纯化(石油醚-乙酸乙酯，3:1)，得到180毫克淡黄色油状化合物5，收率78％。1H NMR(400MHz,DMSO-d6)δ4.34(t,J＝5.1Hz,1H),3.53(t,J＝6.2Hz,2H),3.41(t,J＝6.8Hz,4H),2.39(t,J＝6.2Hz,2H),1.68–1.56(m,2H),1.40(s,9H).
4)化合物7的制备
在0℃下，将化合物6(1.0克，3.80毫摩尔)溶解于20毫升L无水四氢呋喃中，加入化合物5(853毫克，4.18毫摩尔)、三苯基膦(1.5克，5.7毫摩尔)和偶氮二甲酸二乙酯(1.54克，7.6毫摩尔)。将反应混合物置于室温搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(石油醚-乙酸乙酯，4:1)，得到1.0克黄色液体化合物7，收率58.5％。1H NMR(400MHz,DMSO-d6)δ7.57(dd,J＝8.8,1.7Hz,1H),6.96(t,J＝8.6Hz,1H),4.13(t,J＝6.3Hz,2H),3.67(t,J＝6.3Hz,2H),3.61(t,J＝5.9Hz,2H),2.47(dd,J＝7.5,5.0Hz,2H),2.06(p,J＝6.1Hz,2H),1.43(s,9H).LC-MS:(ESI)m/z[M+Na]+＝472.0
5)化合物8的制备
将化合物7(180毫克，0.4毫摩尔)和水合肼(140毫克，2.8毫摩尔)加入6毫升正丁醇中，混悬液加热至110℃恒温2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，30∶1)，得到100毫克黄色固体化合物8，收率54.1％。1H NMR(400MHz,CDCl3)δ7.28(d,J＝8.0Hz,1H),6.42(d,J＝8.0Hz,1H),4.18(t,J＝6.2Hz,2H),3.69(t,J＝6.3Hz,2H),3.63(t,J＝6.0Hz,2H),2.49(t,J＝6.3Hz,2H),2.08(p,J＝6.1Hz,2H),1.44(s,9H).LC-MS:(ESI)m/z[M+H]+＝462.2
6)化合物10的制备
将化合物8(1.4克，3.03毫摩尔)溶解于12毫升二甲醚和4毫升水的混合溶液中，加入化合物9(996毫克，4.54毫摩尔)、四(三苯基膦)钯(350毫克，0.3毫摩尔)和碳酸钠(963毫克，9.09毫摩尔)。将反应混合物加热至80℃并通氮气反应过夜。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，20:1)，得到1.2克棕色固体化合物10，收率92.7％。1H NMR(400MHz,DMSO-d6)δ11.71(s,1H),7.07(d,J＝8.4Hz,2H),6.69(d,J＝7.7Hz,1H),6.65(d,J＝8.4Hz,2H),6.57(d,J＝7.7Hz,1H),5.19(s,2H),4.31(s,2H),4.14(t,J＝6.2Hz,2H),3.61(dt,J＝9.2,6.2Hz,4H),2.43(t,J＝6.1Hz,2H),1.99(p,J＝6.1Hz,2H),1.37(s,9H).LC-MS:(ESI)m/z[M+H]+＝427.2
7)化合物12的制备
在0℃下，将化合物10(1.0克，2.34毫摩尔)溶解于10毫升二甲基甲酰胺中，加入化合物11(354毫克，2.34毫摩尔)。将反应混合物置于室温搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，20:1)，得到760毫克白色固体化合物12，收率56.1％。1H NMR(400MHz,DMSO-d6)δ11.84(s,1H),9.17(s,1H),8.52(d,J＝2.5Hz,1H),8.01(dd,J＝7.9,1.9Hz,1H),7.56(d,J＝8.6Hz,2H),7.36(d,J＝8.6Hz,2H),7.11(dd,J＝11.4,8.3Hz,1H),6.84–6.78(m,1H),6.75(d,J＝7.8Hz,1H),6.67(d,J＝7.7Hz,1H),4.30(s,2H),4.17(t,J＝6.2Hz,2H),3.62(dt,J＝12.2,6.2Hz,4H),2.44(t,J＝6.1Hz,2H),2.28(s,3H),2.01(p,J＝6.1Hz,2H),1.37(s,9H).4.31(s,2H),4.14(t,J＝6.2Hz,2H),3.61(dt,J＝9.2,6.2Hz,4H),2.43(t,J＝6.1Hz,2H),1.99(p,J＝6.1Hz,2H),1.37(s,9H).LC-MS:(ESI)m/z[M+H]+＝578.4
8)化合物ST-05的制备
将8.4克盐酸羟胺和4.8克氢氧化钠加入50毫升甲醇中，混悬液在0℃下搅拌半小时。过滤反应混合物以获得滤液。将化合物12(2.54克，4.40毫摩尔)和氢氧化钠(704毫克，17.6毫摩尔)加入30毫升上述滤液中，60℃搅拌2小时。然后用1摩尔每升的盐酸将反应混合物调节pH值至7。过滤反应混合物以获得固体。用甲醇和水洗涤固体，得到724毫克白色固体化合物ST-05，收率31％。1H NMR(400MHz,DMSO-d6)δ11.86(s,1H),10.43(s,1H),9.20(s,1H),8.79(s,1H),8.54(d,J＝2.0Hz,1H),8.03(d,J＝6.4Hz,1H),7.57(d,J＝8.4Hz,2H),7.37(d,J＝8.4Hz,2H),7.12(dd,J＝11.2,8.4Hz,1H),6.79(dd,J＝16.4,6.4Hz,2H),6.69(d,J＝7.6Hz,1H),4.33(s,2H),4.18(t,J＝6.0Hz,2H),3.62(t,J＝5.2Hz,4H),2.28(s,3H),2.22(t,J＝6.0Hz,2H),2.06–1.96(m,2H).LC-MS:(ESI)m/z[M+H]+＝537.2
实施例4化合物ST-06的合成
实验过程：
1)化合物3的制备
在0℃下，将化合物1(6.0克，22.81毫摩尔)溶解于60毫升无水四氢呋喃中，再加入化合物2(5.6克，25.09毫摩尔)、三苯基膦(8.97克g，34.21毫摩尔)和偶氮二甲酸二乙酯(9.22克，45.62毫摩尔)。将反应混合物置于室温并搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(石油醚-乙酸乙酯，4:1)，得到3.5克黄色液体化合物3，收率36.5％。1H NMR(400MHz,DMSO-d6)δ7.78(d,J＝8.8Hz,1H),7.41(t,J＝8.9Hz,1H),4.25(s,2H),3.54(s,2H),2.84(d,J＝9.5Hz,3H),1.34(d,J＝25.7Hz,9H).LC-MS:(ESI)m/z[M-55]+＝365.0
2)化合物4的制备
将化合物3(100毫克，0.21毫摩尔)加入4毫升4摩尔每升的溶于二恶烷的盐酸溶液中，混悬液室温搅拌半小时。然后将反应混合物真空浓缩，得到75毫克黄色固体粗化合物4，收率98.4％。LC-MS:(ESI)m/z[M+H]+＝321.0
3)化合物6的制备
将化合物4(300毫克，0.94毫摩尔)溶解于5毫升甲醇中，再加入化合物5(149毫克，0.94毫摩尔)、三乙酰氧基硼氢化钠(398毫克，1.88毫摩尔)和乙酸(5.83毫克，0.094毫摩尔)。在室温下搅拌半小时后，将反应混合物倒入冷水中并用二氯甲烷萃取。用盐水洗涤有机层，用无水硫酸钠干燥并浓缩。剩余物通过硅胶柱层析纯化(二氯甲烷-甲醇，20:1)，得到340毫克黄色液体化合物6，收率78.5％。1H NMR(400MHz,DMSO-d6)δ7.77(dd,J＝8.8,1.3Hz,1H),7.38(t,J＝8.9Hz,1H),4.17(t,J＝5.6Hz,2H),2.70(t,J＝5.6Hz,2H),2.36(t,J＝7.0Hz,2H),2.21(s,3H),2.17(t,J＝7.3Hz,2H),1.59(p,J＝7.2Hz,2H),1.37(s,9H).LC-MS:(ESI)m/z[M+H]+＝463.2
4)化合物7的制备
将化合物6(340毫克，0.74毫摩尔)和水合肼(556毫克，11.1毫摩尔)加入8毫升正丁醇中，混悬液在110℃下加热2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(石油醚-乙酸乙酯，1∶1)，得到280毫克黄色固体化合物7，收率80.3％。LC-MS:(ESI)m/z[M+H]+＝475.1
5)化合物9的制备
将化合物7(500毫克，1.05毫摩尔)溶解于12毫升二甲醚和4毫升水的混合溶液中，再加入化合物8(276毫克，1.26毫摩尔)、四(三苯基膦)钯(121毫克，1.05毫摩尔)和碳酸钠(223毫克，2.1毫摩尔)。将反应混合物加热至80℃并通氮气反应过夜。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，20:1)，得到300毫克黄色固体化合物9，收率64.8％。1H NMR(400MHz,DMSO-d6)δ11.65(br,1H),7.07(d,J＝8.3Hz,2H),6.74(d,J＝7.7Hz,1H),6.65(d,J＝8.3Hz,2H),6.58(d,J＝7.7Hz,1H),5.18(s,2H),4.31(s,2H),4.19(t,J＝5.7Hz,2H),2.80(s,2H),2.43(s,2H),2.28(s,3H),2.22(t,J＝7.2Hz,2H),1.70–1.60(m,2H),1.38(s,9H).LC-MS:(ESI)m/z[M+Na]+＝462.2
6)化合物11的制备
在0℃下将化合物9(1.8克，4.1毫摩尔)溶解于10毫升二甲基甲酰胺中，再加入化合物10(620毫克，4.1毫摩尔)。将反应混合物置于室温并搅拌2小时。然后将反应混合物真空浓缩并通过硅胶柱层析纯化(二氯甲烷-甲醇，15:1)，得到1.2克黄色固体化合物11，收率49.6％。LC-MS:(ESI)m/z[M+H]+＝591.3
7)化合物ST-06的制备
将4.2克盐酸羟胺和2.4克氢氧化钠加入25毫升甲醇中，0℃搅拌半小时。过滤反应混合物以获得滤液。将化合物11(1.2克，2.19毫摩尔)和氢氧化钠(1.75克，4.38毫摩尔)加入10毫升上述滤液中，加热至60℃恒温搅拌2小时。然后用1摩尔每升的盐酸将反应混合物调节pH值至7。过滤反应混合物以获得固体，通过反相高效液相色谱法纯化(0.1％乙酸-乙腈和水)。得到590毫克白色固体化合物ST-06，收率49.1％。1H NMR(400MHz,DMSO-d6)δ10.48(br,1H),9.34(s,1H),8.63(s,1H),8.24(s,1H),8.00(dd,J＝7.8,1.4Hz,1H),7.58(d,J＝8.4Hz,2H),7.37(d,J＝8.4Hz,2H),7.11(dd,J＝11.2,8.4Hz,1H),6.81(d,J＝ 7.7Hz,2H),6.70(d,J＝7.6Hz,1H),4.36–4.24(m,2H),3.03(s,2H),2.64(t,J＝7.0Hz,2H),2.45(s,3H),2.28(s,3H),2.06(t,J＝6.9Hz,2H),1.87–1.66(m,2H).13C NMR(101MHz,DMSO-d6)δ169.5,164.4,152.7,152.0,149.6,149.0,143.4,139.1,134.2,134.0,133.3,129.8,128.3,127.6,127.5,123.2,123.2,121.5,112.0,118.4,115.1,114.9,112.3,106.7,65.6,56.8,55.5,42.1,30.4,22.4,21.2.19F NMR(376MHz,DMSO)δ-134.69.LC-MS:(ESI)m/z[M+H]+＝550.2
实施例5化合物ST-01、化合物ST-04盐酸盐、化合物ST-05、化合物ST-06分别对HDAC1、HDAC6、VEGFR2和PDGFR-β活性抑制的评价
5.1、目的背景
实验目的是通过基于激酶的药物筛选体系来检测小分子化合物分别对于激酶活性的抑制能力。HTRF方法实验原理为：激酶磷酸化底物；Eu标记的抗体结合底物磷酸化的位点；链霉亲和素-XL665结合底物生物素；当Eu和XL665接近时，Eu作为供体收到光源(320nm)的激发后，发出发射光(615nm)并将能量共振转移到临近的受体XL665上，受体收到激发后发出发射光(665nm)；特定的信号与磷酸化底物成正比，当添加抑制剂后，磷酸化水平被抑制，不能检测到665nm的发射光，仅能检测到615nm的发射光。以此评价化合物对激酶活性的抑制水平。ADP-Glo方法实验原理为：激酶与其底物进行酶学反应，消耗ATP产生ADP，利用ADP-Glo试剂和发光的方法检测产物的量用以反映激酶的活性。
5.2、试验方法
5.2.1、酶反应试剂配方
5.2.1.1、酶反应配方
酶反应配方
5.2.1.2、底物配方
HDAC底物配方
VEGFR2和PDGFR-β底物配方
5.2.2、试剂、耗材
5.2.3、仪器
5.2.4、试验步骤
HDAC检测步骤：
1)用DMSO将待测化合物从10μM起稀释10个浓度，取6μL的化合物加入54μL的DMSO中，进行3倍稀释。
2)使用Echo转移50nL待测物到384反应板中,每个化合物两个复孔，1000rpm，离心1分钟，DMSO终浓度均为1％。
3)添加2.5μL HDAC到384反应板中，1000rpm离心1分钟,25℃孵育10分钟。
4)添加2.5μL底物到384反应板中，1000rpm离心1分钟，37℃孵育30分钟。
5)添加5μL显影剂混合物到384反应板中，1000rpm离心1分钟，25℃孵育10分钟。
6)用酶标仪读读取FI信号(em:340；ex:450nm)。
VEGFR2和PDGFR-β检测步骤：
1)用DMSO将待测化合物从10μM起稀释10个浓度，取6μL的化合物加入54μL的DMSO中，进行3倍稀释。
2)使用Echo转移25nL待测物到384反应板中,每个化合物两个复孔，1000rpm，离心1分钟，DMSO终浓度均为1％。
3)添加2.5μLkinasesolution到384反应板中，1000rpm离心1分钟。25℃孵育10分钟。
4)添加2.5μLATP&SubMixture到384反应板中，1000rpm离心1分钟，25℃孵育50分钟。
5)添加5μLSa-XL 665/TK-antibody-Cryptate到384反应板中，1000rpm离心1分钟，25℃孵育1小时。
6)用BMG高通量药筛多功能酶标仪读HTRF信号(Ratio 665/620nm)。
5.2.3、数据处理方法
将阴性对照(1％ DMSO孔)的读值设为0％抑制率，阳性对照(阳性药孔)的读值设为100％抑制率，计算各测试溶液的抑制率。
：10μM Reference
： 1％ DMSO
利用以下非线性拟合公式来得到化合物的IC50(半数抑制浓度)：
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)×HillSlope))
X：化合物浓度Log值
Y：化合物抑制率(％inhibition)
Z’因子计算方程：
Z’＝1-3(SDmin+SDmax)/(AVEmax-AVEmin)
其中：
Min为阳性对照Ratio值，Max为阴性对照DMSO Ratio值。
SD为标准误差，AVE为Ratio平均值。
5.2.4、实验结果

阳性对照品：Vorinostat，伏立诺他：
阳性对照品：Sunitinib，舒尼替尼：
实施例6化合物ST-04盐酸盐在20株细胞系中的体外药效检测
1.研究目的
用(CTG)法测量1个待测化合物和1个阳性对照药物对20个细胞系的体外药效检测。
2.实验设计
在20个细胞系中测定1个待测化合物和1个阳性化合物的IC50，药物处理时间为3天，9个浓度，每个浓度3个复孔。
3.材料
Cell line:


注：所有细胞均在37℃、5％CO2条件下培养。
4.试剂和耗材
96孔板(Corning,货号:3610)
MEM(Invitrogen,货号:11095080)
RPMI 1640(Invitrogen,货号:C22400500BT)
FBS(ExCell Bio,货号:FND500)
CellTiter-Glo(CTG)(Promega，货号：G7573)
NEAA(GIBCO，货号：11140050)
Penicillin-Streptomycin(Pen-Strep)(Gibco，货号：15070063)
5.测试样品
待测药：
6.仪器
EnVision 2104多标记微孔板检测仪(Envision 2104Multilabel Reader):PerkinElmer(仪器编号：TAREA0011)；
二氧化碳培养箱：SANYO Electric Co.,Ltd.(仪器编号：02100400059)；
生物安全柜，苏净安泰，BFC-1300IIA2(仪器编号：TBBSC0140)；
倒显微镜：重庆光电XDS-1B(仪器编号：TAMIC0200)；
活细胞计数仪：Vi-Cell XR,Beckman Coulter(仪器编号：TACEL0030)；
电子天平：Mettlertoledo AL104(仪器编号：TBBAL0560)；
7.方法
7.1.待测化合物对细胞的2D抑制作用
细胞培养
1)用培养基复苏细胞，维持细胞生长至生长对数期，用胰酶进行消化，离心机1000rpm 5分钟收集细胞。离心完毕后去上清，加入适量培养基重悬细胞。
第一天：细胞铺板
2)取上述细胞悬液用细胞计数仪进行细胞计数；
3)根据所需测试数量进行细胞铺板，在96孔板中每孔加入90μL细胞悬液，设置T0板；
4)在37℃、5％CO2、95％湿度条件下进行细胞培养；
第二天：T0板读值
5)每孔加入10μL含有溶媒的培养基后进行CTG分析；
6)融化CTG试剂并平衡细胞板至室温30分钟；
7)每孔加入50μL CTG溶液；
8)在定轨摇床上振动2分钟使细胞裂解；
9)把细胞板放置于室温10分钟以稳定冷光信号；
10)用EnVision读取冷光值；
第二天：加入待测药
11)用溶媒溶解被测化合物形成储存液并进行梯度稀释，然后用培养基再稀释20倍，得到10倍溶液；
用培养基制备阳性药的10倍溶液；
12)每孔加入10μL 10X药物溶液(每个细胞浓度设置三个复孔)。被测化合物最高浓度为20μM,9个浓度，3倍稀释；
13)在37℃、5％CO2条件下进行细胞培养；
第五天：试验板读值(药物处理72小时)
14)融化CTG试剂并平衡细胞板至室温30分钟；
15)每孔加入100μL CTG溶液；
16)在定轨摇床上振动2分钟使细胞裂解；
17)把细胞板放置于室温10分钟以稳定冷光信号；
18)用EnVision读取冷光值；
8.数据分析
细胞存活率用公式：(Vsample-VMedium control)/(Vvehicle control-VMedium control)×100％计算。其中Vsample为药物处理组的读数，Vvehicle control为溶剂对照组的平均值。应用GraphPad Prism 9软件,使用非线性回归模型绘制S型剂量-存活率曲线并计算IC50值。数据同时还包括最大抑制率。
9.实验结果
9.1.待测化合物对细胞的IC50和最大抑制率抑制

上述实验证明了化合物的结构准确，相比于对照样品，本发明化合物能够同时对组蛋白去乙酰化酶VI(HDAC6，histone deacetylases 6)，组蛋白去乙酰化酶1(HDAC1)和两种酪氨酸激酶(血管内皮细胞生长因子受体，Vascular endothelial cell growth factor2，VEGFR2)及血小板衍生生长因子受体(platelet-derived growth factor-β，PDGFR-β)具有明显的抑制作用，表明本发明化合物集多种靶点于一身，可作为多靶点抑制剂，同时具有舒尼替尼和伏立诺他的药理作用。因此，本发明化合物可以应用于治疗由酪氨酸激酶和组蛋白去乙酰化酶介导的疾病，如恶性肿瘤等。可治疗的恶性肿瘤包括但不限于肾癌、肝癌、结肠癌、胃肠道间质瘤、肺癌、乳腺癌、胰腺癌、神经胶质瘤、淋巴癌，纤维肉瘤、卵巢癌，前列腺癌等实体瘤和白血病、淋巴癌等血液系统的恶性肿瘤等。
该类化合物集多重靶点于一身，既能充分发挥协同作用增加生物活性，又能避免多种药物联合使用时不同性质和代谢所引起的问题，实用性好，前景良好。
此外，化合物ST-04盐酸盐在20株细胞系中的体外药效检测中，对二十个肿瘤细胞系的体外药效检测都显示出很强的活性，大部分超过了阳性对照药物，其余也都是和阳性对照药物相当。显示了在治疗恶心肿瘤应用方面良好的前景。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。

Claims (21)

  1. 具有通式(Ⅰ)所示的化合物，其可药用盐、水合物或前药，
    其中：
    Ra、Rb独立地选自氢、C1-20烷基、3-10元杂环烷基、5-12元芳香基、5-12元杂芳基；其中，所述的C1-20烷基、3-10元杂环烷基、5-12元芳香基、5-12元杂芳基各自独立地未被取代或被选自如下取代基中的一个、两个、三个或更多个所取代：氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基、C1-6烷基取代的氨基、C1-10烷基、卤代的C1-6烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基、羟基取代的C1-6烷基、C1-6烷氧基、卤代的C1-6烷氧基、硝基取代的C1-6烷氧基、氰基取代的C1-6烷氧基、巯基取代的C1-6烷氧基、氨基取代的C1-6烷氧基、羟基取代的C1-6烷氧基、C2-6烯基、C2-6炔基、-C(＝O)R1、-C(＝O)NHR2、-C(＝O)OR3、-NHC(＝O)R4、-OC(＝O)R5；
    R1、R2、R3、R4、R5独立地选自未被取代的C1-20烷基，或独立地选自被氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基中的一个或多个取代基所取代的C1-20烷基；
    各个Rc相同或者不同，且各自独立地选自氢、C1-20烷基、C1-20烷氧基、氟、氯、溴、碘、硝基、氰基、巯基、羟基、氨基、C1-6烷基取代的氨基、卤代的C1-6烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基；
    Rd、Re独立地选自氢、C1-20烷基、硝基取代的C1-6烷基、氰基取代的C1-6烷基、巯基取代的C1-6烷基、氨基取代的C1-6烷基；
    Rg具有链状结构，构成所述链状结构的主链的原子数为3-15个且其中杂原子个数不超过2个，所述杂原子选自氧、硫、氮；
    x为1、2、3或4。
  2. 根据权利要求1所述的化合物，其可药用盐、水合物或前药，其特征在于，构成所述链状结构的主链的原子中杂原子的个数为1个或2个，且其中一个杂原子与吲唑环中的苯环连接。
  3. 根据权利要求1所述的化合物，其可药用盐、水合物或前药，其特征在于，所述链状结构为饱和直链或饱和支链。
  4. 根据权利要求1所述的化合物，其可药用盐、水合物或前药，其特征在于，所述的化合物具有通式(Ⅱ)所示的结构：
    其中：
    Ra、Rb、Rc、Rd、Re、x分别与通式(Ⅰ)中的Ra、Rb、Rc、Rd、Re、x相同；
    A、B独立地为CH2、O、S或-N(R6)-，R6为氢或C1-6烷基，m、n独立地选自0、1、2、3、4、5、6、7、8、9、10，且3≤m+n≤10；
    当存在如下任一情形时，B为CH2：
    (1)A为CH2；(2)A选自O、S或-N(R6)-，且m为0；(3)n为0。
  5. 根据权利要求4所述的化合物，其可药用盐、水合物或前药，其特征在于，A选自CH2、O、S或-NH-，B选自CH2、O、-N(CH3)-、-N(CH2CH3)-或-N(CH2CH2CH3)-。
  6. 根据权利要求4所述的化合物，其可药用盐、水合物或前药，其特征在于，A为O，B为CH2，m+n为3、4或5。
  7. 根据权利要求1至6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，Ra、Rb不同时为氢。
  8. 根据权利要求7所述的化合物，其可药用盐、水合物或前药，其特征在于，Ra、Rb中，一个选自未被取代的或被取代基取代的嘧啶基、吡唑基、吡啶基、吡嗪基、咪唑基、哒嗪基、噻唑基、苯基；另一个选自氢，或者选自未被取代的或被1-3个卤素取代的C1-6烷基。
  9. 根据权利要求4至6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，所述的化合物具有通式(Ⅲ)所示的结构：
    其中：
    Rc、Rd、Re、x、A、B、m、n分别与通式(ⅠI)中的Rc、Rd、Re、x、A、B、m、n相同；
    Rb选自氢，或者选自未被取代的或被1-3个卤素取代的C1-6烷基；
    R7选自氟、氯、溴、碘、羟基、巯基、氰基、氨基、甲氨基、乙氨基、硝基、C1-6烷基、卤代的C1-6烷基、C1-6烷氧基、卤代的C1-6烷氧基、羟基甲基、巯基甲基、C(＝O)CH3、C(＝O)CH2CH3、C(＝O)NHCH3、C(＝O)NHCH2CH3、NHC(＝O)CH3、NHC(＝O)CH2CH3、NHCH3、N(CH3)2、NHCH2CH3，t为0、1、2、3、4或5，D、E独立地选自CH或N。
  10. 根据权利要求9所述的化合物，其可药用盐、水合物或前药，其特征在于，当t为4时，D、E中至少一个为CH，当t为5时，D、E均为CH。
  11. 根据权利要求9所述的化合物，其可药用盐、水合物或前药，其特征在于，所述的化合物具有通式(Ⅳ)所示的结构：
    其中：Rb、Rc、Rd、Re、x、A、B、m、n分别与通式(ⅠII)中的Rb、Rc、Rd、Re、x、A、B、m、n相同；
    R8、R9独立地选自氢、氟、氯、溴、碘、甲基、乙基、正丙基、异丙基、正丁基、异丁基、卤代的甲基、卤代的乙基、卤代的正丙基、卤代的异丙基、卤代的正丁基、卤代的异丁基、甲氧基、乙氧基、卤代的甲氧基、卤代的乙氧基。
  12. 根据权利要求1-6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，Rb、Rd、Re独立地选自氢、甲基、乙基、正丙基、异丙基、正丁基或异丁基。
  13. 根据权利要求1-6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，各个Rc相同或者不同，且各自独立地选自氢、氟、氯、溴、甲基、乙基、正丙基、异丙基、正丁基、异丁基、甲氧基或乙氧基。
  14. 根据权利要求1-6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，Rb、Rc、Rd、Re同时为氢。
  15. 根据权利要求1-6中任一项权利要求所述的化合物，其可药用盐、水合物或前药，其特征在于，Rg选自：

  16. 根据权利要求1所述的化合物，其可药用盐、水合物或前药，其特征在于，所述的化合物具有通式(Ⅴ)所示的结构：
    其中：p为2、3、4、5、6、7、8、9或10；R8、R9独立地选自氢、氟、氯、溴、碘、甲基、乙基、正丙基、异丙基、正丁基、异丁基、卤代的甲基、卤代的乙基、卤代的正丙基、卤代的异丙基、卤代的正丁基、卤代的异丁基、甲氧基、乙氧基、卤代的甲氧基、卤代的乙氧基。
  17. 根据权利要求1所述的化合物，其可药用盐、水合物或前药，其特征在于，所述化合物的结构式如式(ST-01)至(ST-16)中任一项所示：


  18. 一种药物组合物，其特征在于，该药物组合物包含权利要求1-17中任一项权利要求所述的化合物，其可药用盐、水合物或前药，以及药学上可接受的载体。
  19. 根据权利要求18所述的药物组合物，其特征在于，所述药物组合物为用于预防和/或治疗恶性肿瘤的组合物，所述恶性肿瘤包括肾癌、肝癌、结肠癌、胃肠道间质瘤、肺癌、乳腺癌、胰腺癌、神经胶质瘤、淋巴癌、纤维肉瘤、卵巢癌、前列腺癌、白血病、淋巴癌。
  20. 权利要求1-17中任一项权利要求所述的化合物，其可药用盐、水合物或前药，或者权利要求18所述的药物组合物在制备预防和/或治疗由酪氨酸激酶和/或组蛋白去乙酰化酶介导的疾病的药物中的应用。
  21. 根据权利要求20所述的应用，其特征在于，所述酪氨酸激酶包括血管内皮细胞生长因子受体和血小板衍生生长因子受体，所述组蛋白去乙酰化酶包括组蛋白去乙酰化酶1和组蛋白去乙酰化酶6。
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