WO2016041201A1 - 噻吩并嘧啶类衍生物、其制备方法及其在医药上的应用 - Google Patents

噻吩并嘧啶类衍生物、其制备方法及其在医药上的应用 Download PDF

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WO2016041201A1
WO2016041201A1 PCT/CN2014/086951 CN2014086951W WO2016041201A1 WO 2016041201 A1 WO2016041201 A1 WO 2016041201A1 CN 2014086951 W CN2014086951 W CN 2014086951W WO 2016041201 A1 WO2016041201 A1 WO 2016041201A1
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group
substituted
carbon atoms
unsubstituted
compound
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PCT/CN2014/086951
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English (en)
French (fr)
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安晓霞
别平彦
刘俊
庄戈诗
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上海创诺医药集团有限公司
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Publication of WO2016041201A1 publication Critical patent/WO2016041201A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention relates to the field of medicinal chemistry, and in particular, the present invention provides a thienopyrimidine derivative, a preparation method and application thereof.
  • Tumor is one of the most serious diseases that threaten human health. Its treatment mainly includes radiotherapy, chemotherapy and surgery. In recent years, with the development of cell biology and oncology, the chemical treatment of tumors has undergone tremendous changes. Conventional chemotherapeutic drugs are gradually rejected by killing tumor cells while causing normal cell death by non-specifically blocking cell division, and targeting key node proteins in abnormally activated signaling pathways in tumor cells. It has been found that high-efficiency, low-toxicity and specificity of small molecule inhibitors have become an important direction for the research and development of anti-tumor drugs. Receptor tyrosine kinase (RTK), which is abnormally expressed in tumors, has become a hot spot in anti-tumor drug research because it plays a key role in tumor development, invasion and metastasis, and chemotherapy resistance.
  • RTK Receptor tyrosine kinase
  • Epidermal growth factor receptor also known as HER1 or cerbB1
  • HER1 or cerbB1 is a member of the HER family, the most widely expressed tyrosine kinase in human cancers.
  • the EGFR structure includes three regions: the extracellular region, the transmembrane region, and the intracellular region.
  • the amino terminal of the extracellular domain consists of 622 amino acids with two cysteine-rich segments forming a ligand binding region; the transmembrane region is a single alpha helix; the intracellular region includes the kinase region and has many tyrosines The carboxyl terminal tail of the phosphorylation site.
  • Tyrosine kinase transports the gamma phosphate of ATP to a tyrosine residue.
  • EGFR Upon binding to the ligand, EGFR undergoes homologous or heterodimerization to form a tight junction in the TK region.
  • RTK-mediated tyrosine phosphorylation site phosphorylation at the carboxyl terminal tail creates a binding site for the enzyme and linker proteins (Y992, Y1068, Y1086, Y1148, and Y11730) to initiate intracellular signaling reactions. These signalings form different cellular responses, including proliferation, differentiation, adhesion and angiogenesis, metastasis, and inhibition of apoptosis.
  • EGFR is expressed in non-small cell lung cancer, prostate cancer, breast cancer, colorectal cancer, head and neck cancer, gastric cancer, ovarian cancer, and pancreatic cancer.
  • EGFR activation triggers complex signaling reactions.
  • EGFR proliferates and overexpresses, leading to the loss of control of downstream signaling leading to the formation of various tumors.
  • Mutations in the ATP binding site in EGFR affect the RTK activity of the receptor and interfere with the formation of tumorigenic signals.
  • EGFR is also closely related to tumor progression and poor prognosis.
  • Gefitinib also known as ZD1839 or Iressa
  • Erlotinib is a standard regimen for the treatment of ineffective second- or third-line treatments for advanced NSCLC.
  • the use of multiple targets in cancer therapy is also advantageous, especially for cancer patients who develop resistance to single-target inhibitors.
  • the occurrence and development of tumors is a complex process involving multi-step, multi-stage, in vitro and in vivo interactions involving multiple genes, and most tumors have 4 to 7 independent mutation sites, so multi-target therapy is needed. Ensure the effectiveness and persistence of the anti-tumor effect of the drug.
  • the FDA has approved the launch of several multi-target tyrosine kinase inhibitors, including sorafenib approved in 2005, dasatinib approved in 2006, and obtained in 2007. Batch of sunitinib and lapatinib.
  • the target of lapatinib is epidermal growth factor receptor (EFGR) and human epidermal growth factor receptor 2 (HER-2), which can target these two targets. Produces dual inhibition.
  • EFGR epidermal growth factor receptor
  • HER-2 human epidermal growth factor receptor 2
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl having 1 to 6 carbon atoms, substituted or unsubstituted alkyl acyl group having 1 to 6 carbon atoms, substituted or Unsubstituted cycloalkyl acyl group having 3 to 6 carbon atoms, substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted aryl acyl group having 6 to 10 carbon atoms, a sulfonyl group, a substituted or unsubstituted amide group having 2 to 6 carbon atoms -CONH, a substituted or unsubstituted amino-acyl NHCO having 1 to 6 carbon atoms;
  • R 1 and R 2 together with the nitrogen atom to which they are bonded form a substituted or unsubstituted monocyclic or polycyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms, or a nitrogen atom or an oxygen atom at any position.
  • R 1 , R 2 together with the attached nitrogen atom and the ortho carbon atom of the nitrogen atom constitute a 5-7-membered nitrogen-containing heteroaryl group
  • R 3 is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted 1 to 1 6-carbon atom amide group, substituted or unsubstituted cycloalkyl acyl group having 3 to 6 carbon atoms, substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted 5 An aryl acyl group of 6 carbon atoms, a substituted or unsubstituted alkyl phosphate group having 1 to 6 carbon atoms;
  • R is a substituted aromatic ring or a heteroaromatic ring
  • substituents selected from the group consisting of halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxy, hydroxy, amino, cyano, hydroxy-C1-C4 alkyl, a C2-C10 heterocycloalkyl group, a C2-C10 heterocycloalkyl-oxy group, a carboxyl group, or a C2-C6 carboxylate group;
  • Ar 2 is selected from the group consisting of phenyl, halogen substituted phenyl, C 1 -C 6 alkyl substituted phenyl, biphenyl, halogen substituted biphenyl, naphthyl, pyridyl, thienyl, halogen substituted Thienyl, C 1 -C 3 alkyl substituted thienyl, furyl, halogen substituted furanyl, or C 1 -C 3 alkyl substituted furan;
  • A, B, and D are each independently selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, or none; and at most one of A, B, and D is none;
  • n 0, 1 or 2;
  • substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of halogen, hydroxy, oxo, nitro, C1-C6 haloalkyl, C1 ⁇ .
  • C6 alkyl group C1-C6 alkenyl group, C1-C6 alkynyl group, C3-C12 aryl group, C3-C12 arylalkyl group, C1-C6 alkoxy group, C3-C12 aryl oxygen Amino group, amino group, C1-C6 acylamino group, C1-C6 alkylcarbamoyl group, C3-C12 arylcarbamoyl group, C1-C6 aminoalkyl group, C1-C6 acyl group, carboxyl group, C1-C6 Hydroxyalkyl, C1-C6 alkylsulfonyl, C5-C12 arylsulfonyl, C1-C6 alkylsulfonylamino, C5-C12 arylsulfonylamino, C3-C12 aralkyl a sulfonylamino group, a C1-C6
  • the dotted line is a chemical bond or none.
  • the heterocyclic ring is a saturated ring or an unsaturated ring.
  • any one of R 1 , R 2 , R 3 , R, Ar 2 , A, B, and D is a group corresponding to the specific compound described in Table 1, respectively.
  • A, B, and D are each independently selected from the group consisting of a carbon atom and a nitrogen atom; and one of A, B, and D is a nitrogen atom.
  • only one of A, B, and D is a nitrogen atom.
  • A, B, and D are all carbon atoms
  • Ar 2 is an unsubstituted or halogen substituted phenyl
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, an alkyl group having 1 to 6 carbon atoms or a substituted alkyl group, an alkyl group having 1 to 6 carbon atoms, and 3 to 6 carbon atoms.
  • R 1 and R 2 together with the nitrogen atom to which they are bonded form a substituted or unsubstituted C 3 -C 10 heterocyclic group, wherein the heterocyclic group has 1 to 3 hetero atoms selected from the group consisting of O: , S or N;
  • R 1 and R 2 together with the attached nitrogen atom and the ortho carbon atom of the nitrogen atom constitute a 5- to 7-membered nitrogen-containing heteroaryl group.
  • the compound of formula I is a specific compound shown in Table 1.
  • the compound of formula I is selected from the group consisting of:
  • each group is as defined in the first aspect of the invention.
  • the palladium catalyst is selected from the group consisting of dichlorobis(triphenylphosphine)palladium, tetrakis(triphenylphosphine)palladium, [1,1'-bis(diphenylphosphine). Ferrocene] palladium dichloride, or a combination thereof.
  • the method further comprises the step of reacting a compound of formula (3) with R 3 X to provide a compound of formula I:
  • X is selected from the group consisting of Cl and OTs.
  • a tyrosine kinase inhibitor comprising an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof , tautomers, optical isomers, pharmaceutically acceptable solvates.
  • the tyrosine kinase inhibitor is a dual inhibitor of EGFR/HER2.
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, tautomerous Isomers, optical isomers, pharmaceutically acceptable solvates.
  • the pharmaceutical composition is for treating a disease associated with tyrosine kinase overexpression and/or tyrosine kinase activity, or the pharmaceutical composition is used for treatment and epidermal growth Factor receptor related diseases.
  • the epidermal growth factor receptor-associated disease is selected from the group consisting of abnormal cell proliferation, morphological changes, hyperkinesia, angiogenic diseases, tumor growth, tumor metastasis, or a combination thereof.
  • the disease associated with epidermal growth factor receptor activity is selected from the group consisting of abnormal cell proliferation, morphological changes, hyperkinesia, angiogenic diseases, tumor growth, tumor metastasis, or a combination thereof.
  • the tumor cell is an A431 cell.
  • the tyrosine kinase inhibitor is a multi-target tyrosine kinase inhibitor.
  • the epidermal growth factor receptor is selected from the group consisting of EGFR and/or HER2.
  • the inhibition has an IC50 value of ⁇ 50 nM.
  • the pharmaceutically acceptable salt is a salt of the compound of formula I selected from the group consisting of inorganic acid salts, organic acid salts, alkyl sulfonic acids a salt, an aryl sulfonate, or a combination thereof; preferably, the salt is selected from the group consisting of the hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, Propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethylsulfonate, besylate, p-toluene Acid salt, or a combination thereof;
  • the pharmaceutically acceptable solvate refers to a solvate of a compound of formula I with a solvent selected from the group consisting of water, ethanol, isopropanol, diethyl ether, acetone, or a combination thereof.
  • the present inventors After long-term and intensive research, the present inventors have unexpectedly prepared a class of compounds having the structure shown in formula (I), and the compounds are a class of effective tyrosine kinase inhibitors, and are particularly suitable for use as EGFR. And / or HER2 inhibitors. Based on the above findings, the inventors completed the present invention.
  • the alkyl group includes a linear or branched alkyl group
  • the alkenyl group includes a linear or branched alkenyl group
  • the alkynyl group includes a linear or branched chain.
  • An alkynyl group, said halogen being F, Cl, Br or I, preferably F or Br.
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4.
  • the referenced atoms include all isotopic forms thereof, for example, when referring to "hydrogen atom", it refers to a hydrogen atom, a deuterium atom, a deuterium atom, or a combination thereof.
  • hydrogen atom it refers to a hydrogen atom, a deuterium atom, a deuterium atom, or a combination thereof.
  • the abundance of various isotopic atoms of an element may be a state in which the element naturally exists in nature, or may be a state in which an isotopic is enriched.
  • C1-C4 alkyl refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, Tert-butyl, or a similar group.
  • the number of carbon atoms of the group when the number of carbon atoms of the group is not limited, it means a group having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms.
  • substituted or unsubstituted monocyclic or polycyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms means a nitrogen-containing cyclic group having 3 to 10 carbon atoms, including a monocyclic ring (for example, piperazine, piperazine).
  • the piperazinyl group, piperidinyl group, morpholinyl group and the like, the above polycyclic nitrogen-containing heterocyclic group includes, but is not limited to, azabicyclo[3.2.1]octyl group, or a group shown below:
  • 5-7 membered heteroaryl refers to a heteroaryl group having 5 to 7 carbon atoms or a hetero atom (selected from N, O, S), such as pyrrolyl, pyridyl, furyl, or the like.
  • the heterocyclic ring is a saturated ring or an unsaturated ring.
  • C1-C4 alkoxy refers to a straight or branched alkoxy group having from 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso Butoxy, sec-butoxy, tert-butoxy, or the like.
  • alkyl acyl refers to a group having the structure "-CO-alkyl", such as methyl acyl, ethyl acyl, propyl acyl, isopropyl acyl, butyl acyl, isobutyl acyl, sec-butyl An acyl group, a t-butyl acyl group, or the like.
  • amido group having 2 to 6 carbon atoms means a group having a structure as shown by "C1-C5 alkyl-CONH-".
  • amino-acyl group having 1 to 6 carbon atoms or “aminoacyl group having 1 to 6 carbon atoms” means having, for example, “NH 2 CO-” or “C1-C5 alkyl-NHCO-”
  • the group of the structure is shown, in particular, when the group is “NH 2 CO-", "amino-acyl” is written as “amino-acyl”.
  • oxo refers to two or more hydrogen atoms on the group is substituted with one or more oxygen atoms, e.g., -CH 2 - oxo after being formed -C (O) -Structure.
  • pharmaceutically acceptable solvate refers to a solvate of the corresponding compound with water, ethanol, isopropanol, diethyl ether, acetone.
  • the present invention provides a compound of formula I:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl having 1 to 6 carbon atoms, substituted or unsubstituted alkyl acyl group having 1 to 6 carbon atoms, substituted or Unsubstituted cycloalkyl acyl group having 3 to 6 carbon atoms, substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted aryl acyl group having 6 to 10 carbon atoms, a sulfonyl group, a substituted or unsubstituted amide group having 2 to 6 carbon atoms, a substituted or unsubstituted aminoacyl group having 1 to 6 carbon atoms;
  • R 1 and R 2 together with the nitrogen atom to which they are bonded form a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms, or a nitrogen atom or an oxygen atom at any position.
  • R 1 , R 2 together with the attached nitrogen atom and the ortho carbon atom of the nitrogen atom constitute a 5-7-membered nitrogen-containing heteroaryl group
  • R 3 is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted 1 to 1 6-carbon atom amide group, substituted or unsubstituted cycloalkyl acyl group having 3 to 6 carbon atoms, substituted or unsubstituted alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted 5
  • R is a substituted aromatic ring or a heteroaromatic ring
  • substituents selected from the group consisting of halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxy, hydroxy, amino, cyano, hydroxy-C1-C4 alkyl, a C2-C10 heterocycloalkyl group, a C2-C10 heterocycloalkyl-oxy group, a carboxyl group, or a C2-C6 carboxylate group;
  • Ar 2 is selected from the group consisting of phenyl, halogen substituted phenyl, C 1 -C 6 alkyl substituted phenyl, biphenyl, halogen substituted biphenyl, naphthyl, pyridyl, thienyl, halogen substituted Any one of a thienyl group, a C 1 -C 3 alkyl-substituted thienyl group, a furyl group, a halogen-substituted furyl group, a C 1 -C 3 alkyl-substituted furyl group;
  • A, B, and D are each independently selected from the group consisting of a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, or none; and at most one of A, B, and D is none;
  • n 0, 1 or 2;
  • said one or more hydrogen atoms on the substituent group are substituted with a substituent selected from the group consisting of halogen, Hydroxy, oxo, nitro, C1-C6 haloalkyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C3-C12 aryl, C3-C12 aryl alkane a group, a C1-C6 alkoxy group, a C3-C12 aryloxy group, an amino group, a C1-C6 acylamino group, a C1-C6 alkylcarbamoyl group, a C3-C12 arylcarbamoyl group, C1 ⁇ An aminoalkyl group of C6, an acyl group of C1 to C6, a carboxyl group, a C1-C6 hydroxyalkyl group, a C1-C6 alkylsulfonyl group, a C1
  • the dotted line is a chemical bond or none.
  • A, B, and D are all carbon atoms
  • Ar 2 is a phenyl group
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, an alkyl group having 1 to 6 carbon atoms or a substituted alkyl group, an alkyl group having 1 to 6 carbon atoms, and 3 to 6 carbon atoms.
  • R 1 and R 2 together with the nitrogen atom to which they are bonded form a substituted or unsubstituted C 3 -C 10 heterocyclic group, wherein the heterocyclic group has 1 to 3 hetero atoms selected from the group consisting of O: , S or N;
  • R 1 and R 2 together with the attached nitrogen atom and the ortho carbon atom of the nitrogen atom constitute a 5- to 7-membered nitrogen-containing heteroaryl group (that is, in the case of the compound 3 in Table 1).
  • the compound of formula I is as shown in Table 1.
  • the invention also provides a process for the preparation of a compound of formula I, the process comprising the steps of:
  • a compound of formula (1) i.e., 2-(6-bromo-thiophene[3,2-d]pyrimidin-4-amino)-2-phenyl-ethanol protected with different substituents
  • the compound of formula (1a) (different substituted phenylborate or phenylboronic acid) is subjected to a coupling reaction to give a compound of formula I:
  • the coupling reaction is a Suzuki coupling.
  • the palladium catalyst is selected from the group consisting of dichlorobis(triphenylphosphine)palladium, tetrakis(triphenylphosphine)palladium, [1,1'-bis(diphenylphosphine). Ferrocene] palladium dichloride, or a combination thereof.
  • the process for the preparation of a compound of formula I comprises the step of reacting a compound of formula (3) with R 3 X to give a compound of formula I:
  • X is selected from the group consisting of Cl and OTs.
  • the pharmaceutical forms of the compounds of the invention may include the compounds themselves, as well as other pharmaceutically acceptable modifications, such as optical isomers, cis and trans isomers, or the like, or pharmaceutically acceptable salts or solvates.
  • the pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts such as hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, etc.; organic acid salts such as Acid salt, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, etc.; alkyl sulfonate, such as methyl sulfonate An ethyl sulfonate or the like; an aryl sulfonate such as a benzenesulfonate or a p-toluenesulfonate.
  • inorganic acid salts such as hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, etc.
  • organic acid salts such as Acid salt, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, etc.
  • the pharmaceutically acceptable solvate includes, but is not limited to, a solvate of the compound with water, ethanol, isopropanol, diethyl ether, acetone, and the like.
  • the thienopyrimidine derivative of the present invention has an inhibitory activity against epidermal growth factor receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER2), and therefore, Thienopyrimidine derivatives of the invention or tautomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts thereof, pharmaceutically acceptable Any one or a mixture of the accepted solvates can be used to prepare tyrosine kinase inhibitors, and is particularly useful for the preparation of EGFR and/or HER2 inhibitors.
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • the inhibitor can be applied to the preparation of a medicament for preventing or treating a disease associated with the epidermal growth factor receptor EGFR and/or HER2. Specifically, it can be applied to the preparation of a medicament for preventing or treating abnormal cell proliferation, morphological changes, hyperkinesia, angiogenesis, and tumor metastasis diseases associated with the epidermal growth factor receptor EGFR and/or HER2.
  • the inhibitors are useful in the manufacture of a medicament for the treatment or prevention of tumor growth and metastasis associated with the epidermal growth factor receptor EGFR and/or HER2.
  • the active ingredient of the inhibitor of the present invention is preferably a compound shown in Table 1, or a tautomer, a racemate, an enantiomer, a diastereomer, or a pharmaceutically acceptable compound of the indicated compound. Any one or a mixture of the accepted salts, pharmaceutically acceptable solvates.
  • the thienopyrimidine derivatives provided by the present invention have novel structures, have obvious EGFR inhibitory activities, and some compounds have significant inhibitory activity against VEGFR, and are expected to be developed as tyrosine kinases EGFR or / And VEGFR inhibitors for the prevention or treatment of cell abnormal proliferation, morphological changes, and hyperkinesia associated with epidermal growth factor receptor EGFR and/or angiogenic factor receptor VEGFR and associated with angiogenesis or tumor metastasis Drugs for disease, especially for the preparation of drugs for the treatment or prevention of tumor growth and metastasis associated with the epidermal growth factor receptor EGFR and/or the angiogenic factor receptor VEGFR, for the development of novel drugs with low drug resistance or can alleviate early
  • the inhibitor-resistant tyrosine kinase inhibitor drug provides a new development direction and approach, and has broad application prospects and medicinal value.
  • the 1 H NMR shift ( ⁇ ) is given in parts per million (ppm).
  • the 1 H NMR measurement was performed on a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), internal standard tetramethylsilane (TMS), chemical shift. It is given in units of 10 -6 .
  • the MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQ advantage MAX).
  • ESI FINNIGAN LCQAd
  • the IC 50 value was determined using a NovoStar plate reader (BMG, Germany).
  • the thin layer of silica gel is made of Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate.
  • Silica gel column chromatography was carried out using Yantai Huanghai silica gel 200-300 mesh silica gel as a carrier.
  • the HPLC test was performed 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).
  • the microwave reaction was performed using a CEM Discover-S Model 908860 microwave reactor.
  • reaction was carried out under a nitrogen atmosphere.
  • the argon atmosphere means that the reaction flask is connected to an argon 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 solution in the reaction means an aqueous solution.
  • 6-Bromo-4-chlorothieno[3,2-d]pyrimidine (40 g, 0.16 mol) and 2-methoxy-1-phenyl-ethylamine (29 g, 0.19 mol) were dissolved in N,N- In dimethylformamide (250 ml), N,N-diisopropylethylamine (40 ml) was then added, and the ice bath was removed, and the mixture was warmed to 55 ° C. The reaction was completed until the reaction was completed by TLC, and water was added, ethyl acetate was evaporated. The organic phase was dried over anhydrous sodium sulfate and evaporated. Bromo-thieno[3,2-d]pyrimidin-4-yl)-(2-methoxy-1-phenyl-ethyl)amine (32 g, white solid), yield: 55%.
  • Hydroxylamine hydrochloride (7.2 g, 86 mmol) was dissolved in methanol (30 ml) and water (20 ml), then magnesium oxide (5.1 g, 129 mmol) was added and stirred for 10 minutes, then p-toluenesulfonyl chloride (8 g, 43 mmol) of tetrahydrofuran was added. (300 ml) solution, stir vigorously at room temperature, and react to TLC to monitor the completion of the reaction. After suction filtration with celite, dried over anhydrous magnesium sulfate, and evaporated.
  • N-Hydroxy-4-methyl-benzenesulfonamide (2.8 g, 15 mmol) was dissolved in methanol (12 ml) and water (2 ml), and then potassium carbonate (3.33 g, 24 mmol) and 4-(4-bromo- A solution of phenyl)-but-3-en-2-one (450 mg, 2 mmol) in MeOH (6 mL) was taken to the TLC. The mixture was combined with EtOAc. EtOAc (EtOAc m. (220 mg, yellow solid), yield: 46%.
  • the HTRF kinEASE TK kit (Cat.62TK0PEB, Cisbio) kit of CISBIO was used to test the inhibitory effect of candidate compounds on EGFR enzyme activity.
  • the test method was carried out according to the standard method provided by the manufacturer.
  • the approximate experimental procedure is as follows: In the 10 ⁇ L enzyme reaction system, the enzyme reaction substrate TK-biotin, ATP, EGFR enzyme (Cat. PV3872, Invitrogen) and a certain concentration of compound in 50 mM Hepes/NaOH pH 7.5, 10 mM MgCl 2 were added. , 1 mM EGTA, 0.01% BRIJ-35 enzyme reaction buffer solution was reacted at room temperature for 30 minutes.
  • Each screening concentration of the test compound was determined by a re-pore test, and each experiment was performed with a negative control well without EGFR kinase and a positive control well without compound. After the reaction was completed, 10 ⁇ l of Streptavidin-XL665 and TK antibody europium cryptate (1:100) mixed test solution diluted with 50 mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8 M KF, 20 mM EDTA were added to all wells, and reacted at room temperature. After 1h, use 2104 Fluorescence signals (320 nm stimulation, 665 nm, 615 nm emission) were detected by a Multilabel Reader (Perkinelmer) instrument.
  • the inhibition rate of each well was calculated from the fully active wells and the background signal wells, and the duplicate wells were averaged, and the half-inhibitory activity (IC50) of each test compound was fitted using a professional drawing analysis software GraphPad PRISM 5.0.
  • the HTRF kinEASE TK kit (Cat.62TK0PEB, Cisbio) kit of CISBIO was used to test the inhibitory effect of candidate compounds on HER2 enzyme activity.
  • the test method was carried out according to the standard method provided by the manufacturer.
  • the approximate experimental procedure is as follows: In the 10 ⁇ L enzyme reaction system, the enzyme reaction substrate TK-biotin, ATP, HER2 enzyme (Cat: PV3366, Invitrogen) and a certain concentration of compound in 50 mM Hepes/NaOH pH 7.5, 5 mM MgCl 2 were added. , 1 mM DTT, 50 nM SEB, 1 mM MnCl 2 in an enzyme reaction buffer solution for 30 minutes at room temperature.
  • Each screening concentration of the test compound was determined by a re-pore test, and each experiment was performed with a negative control well without the HER2 enzyme and a positive control well without the compound. After the reaction was completed, 10 ⁇ l of Streptavidin-XL665 and TK antibody europium cryptate (1:100) mixed test solution diluted with 50 mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8 M KF, 20 mM EDTA were added to all wells, and reacted at room temperature. After 1h, use 2104 Fluorescence signals (320 nm stimulation, 665 nm, 615 nm emission) were detected by a Multilabel Reader (Perkinelmer) instrument.
  • the inhibition rate of each well was calculated from the fully active wells and the background signal wells, and the duplicate wells were averaged, and the half-inhibitory activity (IC50) of each test compound was fitted using a professional drawing analysis software GraphPad PRISM 5.0.
  • Fetal bovine serum (Cat#10099-141, GIBCO)
  • test compound was diluted to 500 ⁇ M with medium and diluted 8 times. The cells were added at 25 ⁇ l/well. The final concentration of the compound was diluted from 100 ⁇ M to 0 ⁇ M in 5 fold gradients for a total of 10 concentration points.
  • tumor cell growth inhibition rate % [(A c - A s ) / (A c - A b )] ⁇ 100%
  • a c negative control OA (cell + CCK-8 + DMSO)
  • a b positive control OA (medium + CCK-8 + DMSO)
  • Table 2 Some examples of compounds inhibited tyrosine kinase EGFR, HER2 enzyme activity and A431 cell test results
  • Example 1 Pharmacokinetics of Example 1 (code CDDD-000261) and Example 21 (code CDDD-000257) in rats were administered intravenously or orally.
  • the purpose of this experiment was to administer an effective amount of the compound of Example 21 and the compound of Example 1 to a SD rat test sample by single intravenous (IV) and oral (PO), and blood samples were taken at different time points, and LC/MS/MS was administered.
  • the concentration of the test sample in the rat plasma after the test sample was calculated and the relevant parameters and bioavailability were calculated.
  • Example 21 the compound of Example 21 and the compound of Example 1 were dissolved in water for injection to obtain a solution having a concentration of 5 mg/mL for intravenous and intragastric administration.
  • mice Twenty male SD rats (male weight 130-150 g) of approximately 6-8 weeks old were purchased from Shanghai Sippur-Beikai Experimental Animal Co., Ltd., and 12 animals were used for the test. All groups of animals were fasted overnight (10-14 hours) prior to intravenous and oral administration and fed 4 hours after dosing.
  • Example 21 The compound of Example 21 and the compound of Example 1 were administered by intravenous or oral administration. Drug administration information is shown in the table below.
  • the time points of blood collection in groups 1-8 were: before administration, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h. Each animal received approximately 0.25 mL of blood per heart puncture and K2EDTA was anticoagulated. Blood samples were collected and placed on ice, and plasma was separated by centrifugation (centrifugation conditions: 8000 rpm, 6 minutes, 2-8 ° C). The collected plasma was stored at –80 °C prior to analysis. In addition, there were no excess animals left for experimental studies for the collection of blank blood. The blank plasma after centrifugation was used for bioanalytical method development and biological sample analysis of the test article throughout the study.
  • the mean clearance was 2.60 L/hr/kg.
  • the average half-life (T1/2) was 1.67 hr.
  • the mean value of Cmax after administration was 3218.39 ⁇ g/L, and the average value of Tmax was 0.083 hr.
  • the AUC (0-t) value was 1903.30 hr* ⁇ g/L.
  • the average apparent volume of the final phase distribution was 6.22 L/kg.
  • the average Cmax was 1528.74 ⁇ g/L
  • the average Tmax was 0.50 hr
  • the average AUC (0-t) was 3736.13 hr* ⁇ g/L, half-life (T1). /2)
  • the average is 2.51 hr.
  • the average bioavailability of the compound of Example 21 was 33.01%.
  • the mean clearance was 3.74 L/hr/kg.
  • the average half-life (T1/2) was 5.11 hr.
  • the mean value of Cmax after administration was 616.65 ⁇ g/L, and the average value of Tmax was 0.083 hr.
  • the AUC (0-t) value was 1309.41 hr* ⁇ g/L.
  • the average apparent volume of the final phase distribution was 27.66 L/kg.
  • the average Cmax was 296.20 ⁇ g/L
  • the average Tmax was 5.33 hr
  • the average AUC (0-t) was 3593.95 hr* ⁇ g/L, half-life (T1). /2)
  • the average value is 5.67 hr.
  • the average bioavailability of the compound of Example 1 was 47.71%.
  • CDDD-000257 ie, the compound of Example 21
  • CDDD-000261 ie, the compound of Example 1
  • positive control CDDD-000275 i.e., positive control gefitinib
  • Preparation method all were prepared with 20% PEG400 distilled water.
  • the nude mice were subcutaneously inoculated with human epidermoid carcinoma A431 cells, and after the tumors were grown to 100-200 mm 3 , the animals were randomly grouped (D0).
  • the dosage and administration schedule are shown in Table 1.
  • the tumor volume was measured 2-3 times a week, the rats were weighed, and the data were recorded.
  • the tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width, respectively.
  • T/C(%) (T-T0)/(C-C0) 100 wherein T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

Abstract

本发明提供了一类噻吩并嘧啶类衍生物、其制备方法及其在医药上的应用。具体地，本发明一种如式I所示的化合物，其中各基团的定义如说明书中所述。所述的化合物是一类有效的酪氨酸激酶抑制剂，特别适合用作EGFR和/或HER2抑制剂。

Description

噻吩并嘧啶类衍生物、其制备方法及其在医药上的应用 技术领域
本发明涉及药物化学领域，具体地，本发明提供了一种噻吩并嘧啶类衍生物及其制备方法和应用。
背景技术
肿瘤是威胁人类健康的最严重疾病之一，其治疗主要包括放疗、化疗和手术治疗。近年来随着细胞生物学和肿瘤药理学的发展，肿瘤的化学药物治疗发生了巨大改变。传统的化学治疗药物由于非特异性地阻断细胞***从而在杀死肿瘤细胞的同时也引起正常细胞死亡而逐渐遭到摒弃，同时以肿瘤细胞中异常激活的信号通路中的关键节点蛋白作为靶点，发现高效、低毒、特异性强的小分子抑制剂已成为当今抗肿瘤药物研究开发的重要方向。在肿瘤中异常表达激活的受体酪氨酸激酶(RTK)由于在肿瘤发生发展、侵袭转移、化疗抗性等各个环节均发挥关键作用已成为抗肿瘤药物研究的热点。
表皮生长因子受体(EGFR,epidermal growth factor receptor，又称HER1或cerbB1)是人类癌症中表达最广泛的酪氨酸激酶HER家族成员。EGFR结构包括三个区域：胞外区、跨膜区和胞内区。胞外区的氨基终端由622个氨基酸组成，有形成配体结合区的2个富含半胱氨酸段；跨膜区是个单一的α螺旋；胞内区包括激酶区和有许多酪氨酸磷酸化位点的羧基终端尾部。酪氨酸激酶(RTK)是把ATP的γ磷酸盐转运到酪氨酸残基。在与配体结合后，EGFR发生同源或异源二聚体化而使TK区域形成紧密连接。在羧基终端尾部RTK介导酪氨酸磷酸化位点进行磷酸化，创造了酶和联接子蛋白的结合位点(Y992,Y1068,Y1086,Y1148和Y11730)，从而能开始细胞内信号传导反应。这些信号传导形成不同的细胞反应，包括增殖、分化、粘附和血管形成，转移以及抑制凋亡。
研究表明，EGFR在非小细胞肺癌、***癌、乳癌、大肠癌、头颈癌、胃癌、卵巢癌、和胰腺癌中都有表达，EGFR活化引发复杂信号传导反应。在不同类型的实体瘤中，EGFR有增殖和过度表达，导致下游信号传导失控而引起各种肿瘤的形成。EGFR中ATP结合位点的突变影响受体的RTK活性，干扰致瘤信号的形成，同时，EGFR还与肿瘤的进展和预后差密切相关。
由于EGFR和VEGFR在致瘤中的独特作用，其单克隆抗体和小分子抑制剂已经成为靶向性抗肿瘤药物研发的热点。目前，已经有数个靶向EGFR或VEGFR的抑制剂上市，近20个候选药物处在临床各个研发阶段。其中，吉非替尼和埃罗替尼代表上市较早的靶向EGFR的小分子抑制剂。吉非替尼(Gefitinib，又称ZD1839或Iressa)作为三线单一治疗药物用于晚期非小细胞肺癌(non small cell lung cancer,NSCLC)。埃罗替尼(Erlotinib，又称OSI774或Tarceva)作为标准方案治疗无效的晚期NSCLC的二线或三线治疗药物。
然而，随着这些药物的临床应用，人们发现，并非所有高表达EGFR患者都能对这些药物有效，某些初始对吉非替尼(Gefitinib)有治疗反应的肿瘤在治疗几个月后又出现疾病进展。这些结果表明，目前使用的EGFR抑制剂抗肿瘤药物具有天然或继发性耐药现象，因此，发展新型具有低耐药性或能缓解早期抑制剂耐药性的药物已经成为酪氨酸激酶抑制剂的新发展方向。
另外，多靶点作用在癌症治疗中的应用也是有优势的，尤其是针对单靶点抑制剂产生耐药性的癌症患者。肿瘤的发生发展是由多基因参与的多步骤、多阶段、体内外因素相互作用的复杂过程，且多数肿瘤有4至7个独立的突变位点，因此需要多靶点治疗来 确保药物抗肿瘤作用的有效性和持久性。近年来，FDA先后批准了多个多靶点酪氨酸激酶抑制剂上市，包括2005年获批的索拉非尼(sorafenib)、2006年获批的达沙替尼(dasatinib)、2007年获批的苏尼替尼(sunitinib)和拉帕替尼(lapatinib)。拉帕替尼的作用靶点是表皮生长因子受体(epidermal growth factor receptor,EFGR)和人表皮生长因子受体2(human epidermal growth factor receptor 2,HER-2),可以对这两个靶点产生双重抑制作用。
综上所述，本领域迫切需要开发具有多靶点酪氨酸激酶抑制活性的药物。
发明内容
本发明的目的是提供一类具有多靶点酪氨酸激酶抑制活性的化合物。
本发明的第一方面，提供了一种如式I所示的化合物：
Figure PCTCN2014086951-appb-000001
其中：
R1、R2各自独立地选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基-CONH、取代或未取代的含1～6个碳原子的胺基-酰基NHCO；
或R1、R2与它们相连接的氮原子一起形成碳原子数为3至10个的取代或未取代的单环或者多环含氮杂环基，或任意位置由氮原子、氧原子、硫原子置换的碳原子数为3至10个的取代或未取代的单环或者双环含氮杂环基；
或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基；
R3选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含1～6个碳原子的酰胺基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含5～6个碳原子的芳基酰基、取代或未取代的含1～6个碳原子的烷基磷酸酯基；
R为取代芳环或杂芳环
Figure PCTCN2014086951-appb-000002
上任意氢原子的一个或多个选自下组的取代基：卤素、C1-C4烷基、C1-C4卤代烷基、烷氧基、羟基、氨基、氰基、羟基-C1-C4烷基、C2-C10杂环烷基、C2-C10杂环烷基-氧基、羧基，或C2-C6羧酸酯基；
Ar2选自下组：苯基、卤素取代的苯基、C1-C6烷基取代的苯基、联苯基、卤素取代的联苯基、萘基、吡啶基、噻吩基、卤素取代的噻吩基、C1-C3烷基取代的噻吩基、呋喃基、卤素取代的呋喃基，或C1-C3烷基取代的呋喃基；
A、B、D各自独立地选自下组：碳原子、氮原子、氧原子、硫原子或无；且A、B、D中至多仅有一个为无；
n＝0、1或2；
其中，除非特别说明，所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、羟基、氧代、硝基、C1～C6的卤代烷基、C1～C6的烷基、C1～C6的烯基、C1～C6的炔基、C3～C12的芳基、C3～C12的芳基烷基、C1～C6的烷氧基、C3～C12的芳基氧基、氨基、C1～C6的酰基氨基、C1～C6的烷基氨基甲酰基、C3～C12的芳基氨基甲酰基、C1～C6的氨基烷基、C1～C6的酰基、羧基、C1～C6的羟烷基、C1～C6的烷基磺酰基、C5～C12的芳基磺酰基、C1～C6的烷基磺酰氨基、C5～C12的芳基磺酰氨基、C3～C12的芳烷基磺酰氨基、C1～C6的烷基羰基、C2～6的烯基酰基、C1～C6的酰氧基、C1～C6的羟基-酰基、氰基、脲基、C1～C6的烷基酰基、C1～C6的环烷基酰基、磺酰基、氨基甲酸酯基；
虚线为化学键或无。
在另一优选例中，所述的杂环为饱和环或不饱和环。
在另一优选例中，R1、R2、R3、R、Ar2、A、B、D中任一个分别为表1中所述具体化合物中所对应的基团。
应理解，上述优选基团可相互组合以形成本发明的各种优选化合物。
在另一优选例中，A、B、D各自独立地选自下组：碳原子、氮原子；且A、B、D中有一个为氮原子。
在另一优选例中，A、B、D中仅有一个为氮原子。
在另一优选例中：
A、B、D均为碳原子；
Ar2为未取代或卤素取代的苯基；和/或
R1、R2各自独立地选自下组：氢、含1～6个碳原子的烷基或取代的烷基、含1～6个碳原子的烷基酰基、含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基、取代或未取代的含1～6个碳原子的胺基-酰基；
或R1、R2与它们相连接的氮原子一起形成取代或未取代的C3～C10的杂环基，其中，所述的杂环基具有1～3个选自下组的杂原子：O、S或N；
或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基。
在另一优选例中，所述的式I化合物为表1中所示的具体化合物。
在另一优选例中，所述的式I化合物选自下组：
(S)-(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺；
(S)-{2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]-甲氧乙基}-磷酸二乙酯；
(S)-(2-甲氧基-苯基-乙基)-{6-[4-(5-甲基-异恶唑-3-基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺；
(S)-乙酸-2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]乙酯；
(S)-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮；
(S)-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-丙烯酮；
(S)-2-羟基-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮；
(S)-2-羟基-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄 基}-哌嗪-1-基)-丙烷-1-酮；
(S)-8-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-8-氮杂双环[3.2.1]辛烷-3-醇；
(S)-2-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-1-(4-甲基-哌嗪-1-基)-乙酮；
(S)-(2-甲氧基-1-苯基-乙基)-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺；
(S)-{6-[4-(4-乙基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-(2-甲氧基-1-苯基-乙基)-胺；
(S)-1-(4-乙基-哌嗪-1-基)-2-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-乙酮；
(S)-{6-[4-(4-乙基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺；
(S)-8-(4-{4-[1-(4-氟-苯基)-2-甲氧基-乙胺]-噻吩并[3,2-d]嘧啶-6-基}-苄基)-8-氮杂双环[3.2.1]辛烷-3-醇；
(S)-[1-(4-氟-苯基)-2-甲氧基-乙基]-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺；
(S)-乙酸-2-苯基-2-[6-(4-哌啶-4-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]-乙酯；
(S)-乙酸-2-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-氨基}-2-苯基-乙酯；
(S)-乙酸-2-{6-[6-(4-甲基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙酯；
(S)-乙酸-2-{6-[6-(4-乙基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙酯；
(S)-1-(4-{4-[4-(2-羟基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮。
本发明的第二方面，提供了一种如本发明第一方面所述的式I化合物的制备方法，包括步骤：
在钯催化剂的存在下，用式(1)化合物与式(1a)化合物进行偶联反应，得到式I化合物：
Figure PCTCN2014086951-appb-000003
式中，各基团的定义如本发明第一方面中所述。
在另一优选例中，所述的钯催化剂选自下组：二氯双(三苯基膦)钯、四(三苯基膦)钯、[1,1’-双(二苯基膦)二茂铁]二氯化钯，或其组合。
在另一优选例中，所述方法还包括步骤：用式(3)化合物与R3X反应，得到式I化合物：
Figure PCTCN2014086951-appb-000004
其中，X选自下组：Cl、OTs。
本发明的第三方面，提供了一种酪氨酸激酶抑制剂，所述的抑制剂含有抑制有效量的如本发明第一方面中所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物。
在另一优选例中，所述的酪氨酸激酶抑制剂是EGFR/HER2双重抑制剂。
本发明的第四方面，提供了一种药物组合物，所述的药物组合物含有治疗有效量的如本发明第一方面所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物。
在另一优选例中，所述的药物组合物用于治疗与酪氨酸激酶过表达和/或酪氨酸激酶活性过高相关的疾病，或所述的药物组合物用于治疗与表皮生长因子受体相关的疾病。
在另一优选例中，所述的表皮生长因子受体相关的疾病选自下组：细胞异常增殖、形态变化、运动功能亢进、血管新生疾病、肿瘤生长、肿瘤转移疾病，或其组合。
本发明的第五方面，提供了一种如本发明第一方面所述的式I化合物的用途，用于：
(a)制备酪氨酸激酶抑制剂；
(b)用于体外非治疗性地抑制酪氨酸激酶的活性；
(c)用于体外非治疗性地抑制肿瘤细胞生长；
(d)用于制备治疗表皮生长因子受体活性相关的疾病的药物。
在另一优选例中，所述的表皮生长因子受体活性相关的疾病选自下组：细胞异常增殖、形态变化、运动功能亢进、血管新生疾病、肿瘤生长、肿瘤转移疾病，或其组合。
在另一优选例中，所述的肿瘤细胞为A431细胞。
在另一优选例中，所述的酪氨酸激酶抑制剂为多靶点酪氨酸激酶抑制剂。
在另一优选例中，所述的表皮生长因子受体选自下组：EGFR和/或HER2。
在另一优选例中，所述抑制的IC50值为≤50nM。
在另一优选例中，本发明第三方面或第四方面中，所述药学上可接受的盐是式I化合物的选自下组的盐：无机酸盐、有机酸盐、烷基磺酸盐、芳基磺酸盐，或其组合；较佳地，所述的盐选自下组盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐、甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、甲基磺酸盐、乙基磺酸盐、苯磺酸盐、对甲苯磺酸盐，或其组合；
所述药学上可接受的溶剂合物，是指式I化合物与选自下组的溶剂形成的溶剂合物：水、乙醇、异丙醇、***、丙酮，或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过长期而深入的研究，意外地制备了一类具有如式(I)所示结构的化合物，且所述的化合物是一类有效的酪氨酸激酶抑制剂，特别适合用作EGFR和/或HER2抑制剂。基于上述发现，发明人完成了本发明。
术语
在本发明中，所述的烷基包括直链或支链的烷基，所述的链烯基包括直链或支链的链烯基，所述的链炔基包括直链或支链的链炔基，所述的卤素为F、Cl、Br或I，优选为F或Br。
除非特别说明，在本发明中，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：C1～C4烷基、C3～C7环烷基、C1～C4烷氧基、卤素、羟基、羧基(-COOH)、C1～C4醛基、C2～C4酰基、C2～C4酯基、氨基、苯基；所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C1-C4烷基、氰基、OH、硝基、C3～C7环烷基、C1～C4烷氧基、氨基。
特别地，在本文中，除非特别说明，所提到的原子包括其所有同位素的形式，例如，当提到“氢原子”时，指氢原子、氘原子、氚原子或其组合。在本发明中，某元素各种同位素原子的丰度可以是该元素在自然界中天然存在的状态，也可以是某种同位素富集的状态。
术语“C1～C4烷基”指具有1～4个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
特别地，除非特别说明，在本发明中，当未限定基团的碳原子个数时，指具有1-10个碳原子，优选1-4个碳原子的基团。
术语“碳原子数为3至10个的取代或未取代的单环或者多环含氮杂环基”指具有3～10个碳原子的含氮环基，包括单环(例如哌嗪、哌啶、四氢吡咯、吗啉环等)、二环或多环(包括桥环、螺环)基，其中，上述的单环含氮杂环基例如(但并不限于)：吡咯烷基、哌嗪基、哌啶基、吗啉基等，上述的多环含氮杂环基包括(但并不限于)：氮杂双环[3.2.1]辛烷基，或如下所示的基团：
Figure PCTCN2014086951-appb-000005
或类似基团。
术语“5～7元杂芳基”指具有5～7个碳原子或杂原子(选自N、O、S)的杂芳基，例如吡咯基、吡啶基、呋喃基，或类似基团。
在另一优选例中，所述的杂环为饱和环或不饱和环。
术语“C1～C4烷氧基”指具有1-4个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。
术语“烷基酰基”指具有“-CO-烷基”结构的基团，例如甲基酰基、乙基酰基、丙基酰基、异丙基酰基、丁基酰基、异丁基酰基、仲丁基酰基、叔丁基酰基、或类似基团。
术语“含2～6个碳原子的酰胺基”指具有如“C1-C5烷基-CONH-”所示结构的基团。
术语“含1～6个碳原子的胺基-酰基”或“含1～6个碳原子的胺基酰基”指具有如“NH2CO-”或“C1-C5烷基-NHCO-”所示结构的基团，特别地，当所述的基团为“NH2CO-”时，“胺基-酰基”写作“氨基-酰基”。
如本文所用，术语“氧代”是指基团上的两个或两个以上氢原子被一个或一个以上氧原子所取代，例如，-CH2-被氧代后，形成-C(O)-的结构。
术语“药学上可接受的溶剂合物”指相应的化合物与水、乙醇、异丙醇、***、丙酮的溶剂合物。
式I化合物
本发明提供了一种如式I所示的化合物：
Figure PCTCN2014086951-appb-000006
其中：
R1、R2各自独立地选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基、取代或未取代的含1～6个碳原子的胺基酰基；
或R1、R2与它们相连接的氮原子一起形成碳原子数为3至10个的取代或未取代的单环或者双环的含氮杂环基，或任意位置由氮原子、氧原子、硫原子置换的碳原子数为3至10个的取代或未取代的单环或者双环含氮杂环基；
或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基；
R3选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含1～6个碳原子的酰胺基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含5～6个碳原子的芳基酰基、取代或未取代的含1～6个碳原子的烷基磷酸酯基；较佳地，R3不为H原子；
R为取代芳环或杂芳环
Figure PCTCN2014086951-appb-000007
上任意氢原子的一个或多个选自下组的取代基：卤素、C1-C4烷基、C1-C4卤代烷基、烷氧基、羟基、氨基、氰基、羟基-C1-C4烷基、C2-C10杂环烷基、C2-C10杂环烷基-氧基、羧基，或C2-C6羧酸酯基；
Ar2选自下组：苯基、卤素取代的苯基、C1-C6烷基取代的苯基、联苯基、卤素取代的联苯基、萘基、吡啶基、噻吩基、卤素取代的噻吩基、C1-C3烷基取代的噻吩基、呋喃基、卤素取代的呋喃基、C1-C3烷基取代的呋喃基中的任意一种；
A、B、D各自独立地选自下组：碳原子、氮原子、氧原子、硫原子或无；且A、B、D中至多仅有一个为无；
n＝0、1或2；
其中，所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、 羟基、氧代、硝基、C1～C6的卤代烷基、C1～C6的烷基、C1～C6的烯基、C1～C6的炔基、C3～C12的芳基、C3～C12的芳基烷基、C1～C6的烷氧基、C3～C12的芳基氧基、氨基、C1～C6的酰基氨基、C1～C6的烷基氨基甲酰基、C3～C12的芳基氨基甲酰基、C1～C6的氨基烷基、C1～C6的酰基、羧基、C1～C6的羟烷基、C1～C6的烷基磺酰基、C5～C12的芳基磺酰基、C1～C6的烷基磺酰氨基、C5～C12的芳基磺酰氨基、C3～C12的芳烷基磺酰氨基、C1～C6的烷基羰基、C2～6的烯基酰基、C1～C6的酰氧基、C1～C6的羟基-酰基、氰基、脲基、C1～C6的烷基酰基、C1～C6的环烷基酰基、磺酰基、氨基甲酸酯基；
虚线为化学键或无。
在本发明的优选实施例中，A、B、D均为碳原子；
Ar2为苯基；和/或
R1、R2各自独立地选自下组：氢、含1～6个碳原子的烷基或取代的烷基、含1～6个碳原子的烷基酰基、含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基、取代或未取代的含1～6个碳原子的胺基酰基；
或R1、R2与它们相连接的氮原子一起形成取代或未取代的C3～C10的杂环基，其中，所述的杂环基具有1～3个选自下组的杂原子：O、S或N；
或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基(即形如表1中化合物3的情况)。
更优选地，所述的式I化合物如表1中所示。
表1
Figure PCTCN2014086951-appb-000008
Figure PCTCN2014086951-appb-000009
Figure PCTCN2014086951-appb-000010
式I化合物的制备方法
本发明还提供了一种式I化合物的制备方法，所述方法包括步骤：
在钯催化剂的存在下，用式(1)化合物(即不同取代基保护的2-(6-溴-噻吩[3,2-d]嘧啶-4-氨基)-2-苯基-乙醇)与式(1a)化合物(不同取代的苯硼酸酯或苯硼酸)进行偶联反应，得到式I化合物：
Figure PCTCN2014086951-appb-000011
其中，各基团的定义如上文中所述，特别地，在式(1a)中，二杂氧戊硼烷结构中的“B”代表硼原子。
在另一优选例中，所述的偶联反应是Suzuki偶联。
在另一优选例中，所述的钯催化剂选自下组：二氯双(三苯基膦)钯、四(三苯基膦)钯、[1,1’-双(二苯基膦)二茂铁]二氯化钯，或其组合。
或者所述式I化合物的制备方法包括步骤：用式(3)化合物与R3X反应，得到式I化合物：
Figure PCTCN2014086951-appb-000012
其中，X选自下组：Cl、OTs。
药学上可接受的盐
本发明化合物的药用形式可包括化合物本身，以及药学上可接受的其他变化形式，如光学异构体，顺反异构体等，或者药学上可接受的盐或溶剂合物。
优选地，所述的药学上可接受的盐包括(但并不限于)：无机酸盐，如盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐等；有机酸盐，如甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐等；烷基磺酸盐，如甲基磺酸盐、乙基磺酸盐等；芳基磺酸盐，如苯磺酸盐、对甲苯磺酸盐等。
优选地，所述的药学上可接受的溶剂合物包括(但并不限于)：所述的化合物与水、乙醇、异丙醇、***、丙酮等的溶剂合物。
式I化合物的用途
经研究，本发明所述的噻吩并嘧啶类衍生物具有表皮生长因子受体(EGFR)和/或人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)的抑制活性，因此，本发明所述的噻吩并嘧啶类衍生物或所述衍生物的互变异构体、外消旋体、对映异构体、非对映异构体、药学上可接受的盐、药学上可接受的溶剂合物中的任意一种或几种的混合物，可应用于制备酪氨酸激酶抑制剂，尤其可应用于制备EGFR和/或HER2抑制剂。
同时，所述的抑制剂可应用于制备预防或治疗与表皮生长因子受体EGFR和/或HER2相关疾病的药物。具体说，可应用于制备预防或治疗与表皮生长因子受体EGFR和/或HER2相关的细胞异常增殖、形态变化、运动功能亢进、血管新生及肿瘤转移疾病的药物。
另外，所述的抑制剂可应用于制备治疗或预防与表皮生长因子受体EGFR和/或HER2相关的肿瘤生长和转移的药物。
本专利所述抑制剂的活性成分优选为表1中所示化合物，或所示化合物的互变异构体、外消旋体、对映异构体、非对映异构体、药学上可接受的盐、药学上可接受的溶剂合物中的任意一种或几种的混合物。
本发明的主要优点包括：
与现有技术相比，本发明提供的噻吩并嘧啶类衍生物的结构新颖，具有明显的EGFR抑制活性，而且部分化合物对VEGFR也具有明显抑制活性，可望开发为酪氨酸激酶EGFR或/和VEGFR抑制剂，用于制备预防或治疗与表皮生长因子受体EGFR和/或血管生长因子受体VEGFR相关的细胞异常增殖、形态变化以及运动功能亢进等相关疾病及与血管新生或肿瘤转移相关疾病的药物，尤其可望用于制备治疗或预防与表皮生长因子受体EGFR和/或血管生长因子受体VEGFR相关的肿瘤生长和转移的药物，为发展新型具有低耐药性或能缓解早期抑制剂耐药性的酪氨酸激酶抑制剂药物提供了新的发展方向和途径，具有广阔的应用前景和药用价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
下述实施例中所制得的化合物的结构是通过核磁共振(1HNMR)和质谱(MS)予以确定。
1HNMR位移(δ)以百万分之一(ppm)的单位给出。1HNMR的测定是用BrukerAVANCE-400核磁仪，测定的溶剂为氘代二甲亚砜(DMSO-d6)，氘代氯仿(CDCl3)，内标为四甲基硅烷(TMS)，化学位移是以10-6作为单位给出。
MS的测定是用FINNIGAN LCQAd(ESI)质谱仪(生产商：Therm，型号：Finnigan LCQ advantage MAX)。
IC50值的测定是用NovoStar酶标仪(德国BMG公司)。
薄层硅胶是使用烟台黄海HSGF254或青岛GF254硅胶板。
硅胶柱层析是使用烟台黄海硅胶200～300目硅胶为载体。
HPLC测试是使用安捷伦1200DAD高压液相色谱仪(Sunfire C18150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18150×4.6mm色谱柱)。
微波反应是使用CEM Discover-S 908860型微波反应器。
另外，以下实施例中无特殊说明，反应均在氮气氛下进行。
氩气氛是指反应瓶连接一个约1L容积的氩气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
以下实施例中无特殊说明，反应中的溶液是指水溶液。
实施例1
Figure PCTCN2014086951-appb-000013
第一步：
冰浴下，将60％氢化钠(30g，0.75mol)溶解于四氢呋喃(300ml)中，然后加入溶有L-苯酐氨醇(50g，0.36mol)的四氢呋喃(600ml)溶液，撤去冰浴，室温搅拌2小时，然后在氮气保护下，滴加碘甲烷(24ml)，室温搅拌1小时，再70℃回流2小时。反应直至TLC监测原料反应完全，冷却，体系加入冰盐水中，甲基叔丁基醚萃取，有机相用无水硫酸钠干燥，减压浓缩，得到2-甲氧基-1-苯基-乙胺(51g，无色油状物)，收率：92％。
第二步：
将6-溴-4-氯噻吩并[3,2-d]嘧啶(40g，0.16mol)和2-甲氧基-1-苯基-乙胺(29g，0.19mol)溶解于N,N-二甲基甲酰胺(250ml)中，然后在冰浴下加入N,N-二异丙基乙胺(40ml)，撤去冰浴，升温至55℃反应。反应直至TLC监测原料反应完全，加入水，乙酸乙酯萃取，有机相用无水硫酸钠干燥，减压浓缩，然后加入石油醚重结晶，抽滤，滤饼用正己烷洗涤，得到(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(32g，白色固体)，收率：55％。
第三步：
室温下将化合物(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(18.2g，50mmol)和4-[4-(4,4,5,5-四甲基-[1,3,2]二氧硼戊环-2-基)-苯基]-哌嗪-1-羧酸叔丁酯(22.1g，55mmol)溶解于N,N-二甲基甲酰胺(400ml)中，依次加入四(三苯基膦)钯(1.2g，1mmol)，碳酸钠溶液(1mol/L，80ml)，氮气保护，加热至90℃直至TLC监测原料反应完全，冷却至室温，反应液加入冰水中，抽滤，滤饼用水洗涤，干燥，得到4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-哌嗪-1-羧酸叔丁酯(27g，浅粉色固体)，收率：96％。
第四步：
将4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-哌嗪-1-羧酸叔丁酯(5.6g，10mmol)溶解于二氯甲烷(50ml)中，在冰浴下加入三氟乙酸(15ml，200mmol)，搅拌10分钟，然后撤去冰浴，室温搅拌至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺(5.4g，白色固体)。MS m/z(ESI):460.58[M+1]
1HNMR(400Hz,DMSO-d6):8.38(s,1H),8.33(d,1H),7.82(t,3H),7.46(m,4H),7.31(m,2H),7.22(m,1H),5.67(m,1H),3.80(m,1H),3.62(m,1H),3.52(s,2H),3.32(s,3H),3.17(s,1H),2.41(m,4H),2.40(m,4H).
实施例2
Figure PCTCN2014086951-appb-000014
将2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]乙醇(500mg，1.12mmol)溶解于N,N-二甲基甲酰胺中，加入异丙醇镁(128mg，0.90mmol)，升温至65℃搅拌30分钟，然后降温至50℃，加入对甲苯磺酰甲基膦酸二乙酯(0.43ml，1.68mmol)，搅拌至TLC监测原料反应完全。然后加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得{2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]-甲氧乙基}-磷酸二乙酯(260mg，白色固体)，收率：40％。MS m/z(ESI):596.70[M+1]
1HNMR(400Hz,DMSO-d6):8.36(s,1H),8.18(d,1H),7.80(t,3H),7.43(d,4H),7.31(t,2H),7.22(t,1H),5.45(m,1H),4.98(t,1H),4.02(m,4H),3.75(m,2H),3.50(s,2H),2.77(d,2H),2.59(s,4H),2.40(s,4H),1.23(t,6H).
实施例3
Figure PCTCN2014086951-appb-000015
第一步：
将盐酸羟胺(7.2g，86mmol)溶解于甲醇(30ml)和水(20ml)中，再加入氧化镁(5.1g，129mmol)，搅拌10分钟，然后加入对甲苯磺酰氯(8g，43mmol)的四氢呋喃(300ml)溶液，室温剧烈搅拌，反应至TLC监测原料反应完全。用硅藻土抽滤后，无水硫酸镁干燥，减压浓缩，得到N-羟基-4-甲基-苯磺酰胺(7g，白色固体)，收率：87.5％。
第二步：
将N-羟基-4-甲基-苯磺酰胺(2.8g，15mmol)溶解于甲醇(12ml)和水(2ml)中，依次加入碳酸钾(3.33g，24mmol)和4-(4-溴-苯基)-丁-3-烯-2酮(450mg，2mmol)的甲醇(6ml)溶液，至TLC监测原料反应完全。加水，乙酸乙酯萃取，有机相用无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到3-(4-溴-苯基)-5-甲基-异恶唑(220mg，黄色固体)，收率：46％。
第三步：
将3-(4-溴-苯基)-5-甲基-异恶唑(100mg，0.42mmol)溶解于1,4-二氧六环中，依次加入联硼酸频那醇酯(127mg，0.5mmol)，1,1’-双二苯基膦二茂铁二氯化钯(18mg，0.021mmol)，醋酸钾(42mg，0.42mmol)，氮气置换，升温至95℃搅拌，至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到5-甲基-3-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苯基]异恶唑(98mg，白色固体)，收率：81.6％。
第四步：
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(47mg，0.13mmol)和3(40mg，0.14mmol)溶解于N,N-二甲基甲酰胺中，然后依次加入四(三苯基膦)钯(3mg，0.0026mmol)，1N碳酸钠溶液(1ml)，氮气置换，升温至90℃搅拌，至TLC监测原料反应完全。加水，乙酸乙酯萃取，有机相用无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(2-甲氧基-苯基-乙基)-{6-[4-(5-甲基-异恶唑-3-基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺(50mg，白色固体)，收率：87.7％。MS m/z(ESI):443.62[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.35(d,1H),7.99(s,4H),7.85(s,1H),7.52(m,2H),7.30(m,2H),7.25(m,1H),6.86(s,1H),5.65(m,1H),3.80(m,1H),3.65(m,1H),3.32(s,3H),2.49(s,3H).
实施例4
Figure PCTCN2014086951-appb-000016
第一步：
将4-{4-[4-(2-羟基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-哌嗪-1-羧酸叔丁酯(200mg，0.37mmol)溶解于四氢呋喃中，依次加入醋酸酐(0.17ml，1.83mmol)、三乙胺(0.26ml，1.83mmol)，室温搅拌，至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到4-{4-[4-(2-乙酰氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-哌嗪-1-羧酸叔丁酯(189mg，白色固体)，收率：87％。
第二步：
将4-{4-[4-(2-乙酰氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-哌嗪-1-羧酸叔丁酯(189mg，0.32mmol)溶解于二氯甲烷中，加入三氟乙酸(0.24ml，3.2mmol)，室温搅拌，至TLC监测原料反应完全。向里滴加几滴氨水，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到乙酸-2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]乙酯(120mg，白色固体)，收率：77％。MS m/z(ESI):488.62[M+1]
1HNMR(400Hz,DMSO-d6):8.38(s,1H),8.33(d,1H),7.82(t,3H),7.46(m,4H),7.31(m,2H),7.22(m,1H),5.67(m,1H),3.80(m,1H),3.62(m,1H),3.52(s,2H),2.41(m,4H),2.40(m,4H),2.01(s,3H).
实施例5
Figure PCTCN2014086951-appb-000017
将(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺(150mg，0.33mmol)溶解于甲醇(10ml)中，加入醋酸酐(33mg，0.33mmol)，室温搅拌，至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮(75mg，白色固体)，收率：45.3％。MS m/z(ESI):502.65[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.31(d,1H),7.82(t,3H),7.48(d,4H), 7.32(t,2H),7.24(t,1H),5.67(m,1H),3.80(t,1H),3.56(m,3H),3.45(s,4H),3.36(s,2H),3.31(s,3H),2.41(d,4H),1.98(s,3H).
实施例6
Figure PCTCN2014086951-appb-000018
将(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺(200mg，0.44mmol)溶解于N,N-二甲基甲酰胺(10ml)中，加入丙烯酸(0.04ml，0.52mmol)、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸(166mg，0.52mmol)和N,N-二异丙基乙胺(0.15ml，0.88mmol)，室温反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-丙烯酮(85mg，白色固体)，收率：37.6％。MS m/z(ESI):514.74[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.32(d,1H),7.82(t,3H),7.48(d,4H),7.34(t,2H),7.26(t,1H),6.80(m,1H),6.12(d,1H),5.70(d,2H),3.80(t,1H),3.50-3.66(m,7H),3.32(s,3H),2.40(s,4H).
实施例7
Figure PCTCN2014086951-appb-000019
将(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺(120mg，0.26mmol)溶解于N,N-二甲基甲酰胺(5ml)中，加入乙醇酸(24mg，0.31mmol)、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸(100mg，0.31mmol)和N,N-二异丙基乙胺(0.09ml，0.52mmol)，室温反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到2-羟基-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮(50mg，白色固体)，收率：37.1％。
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.31(d,1H),7.82(t,3H),7.48(d,4H),7.32(t,2H),7.24(t,1H),5.67(m,1H),4.52(t,1H),4.08(d,2H),3.80(t,1H),3.57(m,3H),3.48(s,2H),3.35(s,2H),3.31(s,3H),2.40(s,4H).MS m/z(ESI):518.65[M+1]
实施例8
Figure PCTCN2014086951-appb-000020
将(2-甲氧基-1-苯基-乙基)-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-基]-胺(250mg，0.54mmol)溶解于N,N-二甲基甲酰胺(10ml)中，加入2-羟基丙酸(59mg，0.65mmol)、O-苯并三氮唑-N,N,N’,N’-四甲基脲四氟硼酸(208mg，0.65mmol)和N,N-二异丙基乙胺(0.19ml，1.08mmol)，室温反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到2-羟基-1-(4-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-丙烷-1-酮(85mg，白色固体)，收率：29.6％。MS m/z(ESI):532.65[M+1]
1HNMR(400Hz,DMSO-d6):8.34(s,1H),8.32(d,1H),7.82(t,3H),7.50(d,4H),7.35(t,2H),7.25(t,1H),5.65(m,1H),4.86(d,1H),4.40(t,1H),3.62(m,2H),3.55(s,3H),3.50(d,4H),3.32(s,3H),2.40(s,4H).
实施例9
Figure PCTCN2014086951-appb-000021
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(100mg，0.27mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入8-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-8-氮杂双环[3.2.1]辛烷-3-醇(141mg，0.41mmol)、四(三苯基膦)钯(32mg，0.028mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到8-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-8-氮杂双环[3.2.1]辛烷-3-醇(80mg，黄色固体)，收率：59％。MSm/z(ESI):501.68[M+1]
1HNMR(400Hz,DMSO-d6):10.89(s,1H),8.45(d,2H),7.95(d,5H),7.55(d,2H),7.39(t,2H),7.30(t,1H),5.74(m,1H),5.00(s,1H),4.22(m,2H),3.86(m,3H),3.68(m,3H),3.39(s,3H),3.23(d,1H),2.45(s,3H),2.31(s,2H).
实施例10
Figure PCTCN2014086951-appb-000022
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(350mg，1mmol)溶解于N,N-二甲基甲酰胺(10ml)中，向里依次加入1-(4-甲基-哌嗪-1-基)-2-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苯基]-乙酮(516.4mg，1.5mmol)、四(三苯基膦)钯(120mg，0.1mmol)、1N碳酸钠(1ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完 全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到2-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-1-(4-甲基-哌嗪-1-基)-乙酮(290mg，白色固体)，收率：58％。MS m/z(ESI):502.66[M+1]
1HNMR(400Hz,DMSO-d6):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.67(m,1H),3.80(m,1H),3.78(s,2H),3.62(m,1H),3.49(m,4H),3.31(s,3H),2.25(m,4H),2.16(s,3H).
实施例11
Figure PCTCN2014086951-appb-000023
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(100mg，0.27mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入1-甲基-4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌嗪(174mg，0.55mmol)、四(三苯基膦)钯(31mg，0.027mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(2-甲氧基-1-苯基-乙基)-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺(30mg，白色固体)，收率：23％。MS m/z(ESI):474.65[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.68(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.46(s,3H)
实施例12
Figure PCTCN2014086951-appb-000024
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(200mg，0.55mmol)溶解于N,N-二甲基甲酰胺(10ml)中，向里依次加入1-乙基-4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌嗪(399mg，1.1mmol)、四(三苯基膦)钯(64mg，0.055mmol)、1N碳酸钠(0.9ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到{6-[4-(4-乙基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-(2-甲氧基-1-苯基-乙基)-胺(85mg，白色固体)，收率：31.7％。MS m/z(ESI):487.69[M+1]
1HNMR(400Hz,DMSO-d6):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.70(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.45(m,2H),1.27(t,3H)
实施例13
Figure PCTCN2014086951-appb-000025
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-(2-甲氧基-1-苯基-乙基)胺(100mg，0.27mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入1-(4-乙基-哌嗪-1-基)-2-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苯基]-乙酮(143mg，0.41mmol)、四(三苯基膦)钯(32mg，0.028mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到1-(4-乙基-哌嗪-1-基)-2-{4-[4-(2-甲氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苯基}-乙酮(30mg，白色固体)，收率：21.6％。MS m/z(ESI):516.68[M+1]
1HNMR(400Hz,DMSO-d6):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.70(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.45(m,2H),1.27(t,3H).
实施例14
第一步：
Figure PCTCN2014086951-appb-000026
将1-(4-氟-苯基)-2-甲氧基-乙胺(1g，4.862mmol)溶解于N,N-二甲基甲酰胺(15ml)中，加入6-溴-4-氯噻吩并[3,2-d]嘧啶(809mg，3.24mmol)和三乙胺(1.8ml，12.97mmol)，升温至60℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(6-溴-噻吩并[3,2-d]嘧啶-4-基)-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺(1.08g，黄色固体)，收率：87.1％。
第二步：
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺(100mg，0.26mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入1-乙基-4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌嗪(129mg，0.39mmol)、四(三苯基膦)钯(30mg，0.026mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到{6-[4-(4-乙基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺(65mg，白色固体)，收率：49.2％MS m/z(ESI):506.67[M+1]
1HNMR(400Hz,DMSO-d6):8.37(s,1H),8.27(d,1H),7.77(t,3H),7.50(t,2H),7.47(d,2H),7.15(t,2H),5.62(m,1H),3.78(t,1H),3.58(m,1H),3.49(s,2H),3.30(s,3H),2.37(s,8H),2.31(m,2H),0.92(t,3H).
实施例15
Figure PCTCN2014086951-appb-000027
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺(100mg，0.26mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入8-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-8-氮杂双环[3.2.1]辛烷-3-醇(135mg，0.39mmol)、四(三苯基膦)钯(30mg，0.026mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到8-(4-{4-[1-(4-氟-苯基)-2-甲氧基-乙胺]-噻吩并[3,2-d]嘧啶-6-基}-苄基)-8-氮杂双环[3.2.1]辛烷-3-醇(60mg，白色固体)，收率：44.4％。MS m/z(ESI):519.65[M+1]
1HNMR(400Hz,DMSO-d6):8.37(s,1H),8.28(d,1H),7.76(t,3H),7.48(m,4H),7.15(t,2H),5.64(m,1H),4.30(s,1H),3.78(m,2H),3.58(m,2H),3.31(s,3H),3.04(s,2H),2.08(d,2H),1.90(s,4H),1.59(d,2H).
实施例16
Figure PCTCN2014086951-appb-000028
将(6-溴-噻吩并[3,2-d]嘧啶-4-基)-[1-(4-氟-苯基)-2-甲氧基-乙基]-胺(100mg，0.26mmol)溶解于N,N-二甲基甲酰胺(5ml)中，向里依次加入1-甲基-4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌嗪(124mg，0.39mmol)、四(三苯基膦)钯(30mg，0.026mmol)、1N碳酸钠(0.4ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到[1-(4-氟-苯基)-2-甲氧基-乙基]-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-基}-胺(70mg，白色固体)，收率：54.7％。MS m/z(ESI):492.65[M+1]
1HNMR(400Hz,DMSO-d6):8.36(s,1H),8.28(d,1H),7.77(t,3H),7.48(m,4H),7.13(t,2H),5.62(m,1H),3.75(t,1H),3.58(m,1H),3.48(s,2H),3.29(s,3H),3.37(s,8H),2.15(s,3H).
实施例17
Figure PCTCN2014086951-appb-000029
第一步：
将2-(6-溴-噻吩并[3,2-d]嘧啶-4-氨基)-2-苯基-乙醇(767mg，2.19mmol)溶解于N,N-二甲基甲酰胺(15ml)中，向里依次加入4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌啶-1-甲酸叔丁酯(970mg，2.41mmol)、四(三苯基膦)钯(253mg，0.219mmol)、1N碳酸钠(5ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到4-{4-[4-(2-羟基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌啶-1-甲酸叔丁酯(1.1g，黄色固体)，收率：92％。
第二步：
将4-{4-[4-(2-羟基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌啶-1-甲酸叔丁酯(163mg，0.3mmol)溶解于四氢呋喃(10ml)中，依次加入醋酸酐(153mg，1.5mmol)、三乙胺(152mg，1.5mmol)，升温至30℃搅拌，反应至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到4-{4-[4-(2-乙酰氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌啶-1-甲酸叔丁酯(130mg，黄色固体)，收率：73.9％。
第三步：
将4-{4-[4-(2-乙酰氧基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌啶-1-甲酸叔丁酯(130mg，0.22mmol)溶解于盐酸的乙酸乙酯溶液(2N)，室温搅拌，至TLC监测原料反应完全。减压浓缩，加入水，冰浴下用2N氢氧化钠溶液调PH至12，乙酸乙酯萃取，无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到乙酸-2-苯基-2-[6-(4-哌啶-4-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]-乙酯(20mg，淡黄色固体)，收率：28％。MS m/z(ESI):487.65[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.72(m,1H),4.37(d,2H),3.25(m,2H),2.78(m,4H),1.99(s,3H),1.55～1.30(m,5H).
实施例18
Figure PCTCN2014086951-appb-000030
将2-(6-溴-噻吩并[3,2-d]嘧啶-4-氨基)-2-苯基-乙醇(350mg，1mmol)溶解于N,N-二甲基甲酰胺(10ml)中，向里依次加入1-甲基-4-[4-(4,4,5,5-四甲基-[1,3,2]二杂氧戊硼烷-2-基)-苄基]-哌嗪(348mg，1.1mmol)、四(三苯基膦)钯(58mg，0.05mmol)、1N碳酸钠(2ml)，氮气置换，升温至85℃搅拌，反应至TLC监测原料反应完全。加入水，乙酸乙酯萃取， 无水硫酸钠干燥，减压浓缩，用硅胶柱色谱法纯化所得残留物，得到2-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-氨基}-2-苯基-乙醇(287mg，黄色固体)，收率：62.4％。
将2-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-氨基}-2-苯基-乙醇(143mg，0.3mmol)溶解于四氢呋喃(10ml)中，依次加入醋酸酐(153mg，1.5mmol)、三乙胺(152mg，1.5mmol)，升温至30℃搅拌，反应至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到乙酸-2-{6-[4-(4-甲基-哌嗪-1-甲基)-苯基]-噻吩并[3,2-d]嘧啶-4-氨基}-2-苯基-乙酯(140mg，黄色固体)，收率：89.7％。MS m/z(ESI):502.76[M+1]
1HNMR(400Hz,DMSO-d6):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.68(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),2.78(m,4H),2.55(m,4H),2.46(s,3H),1.99(s,3H).
实施例19
Figure PCTCN2014086951-appb-000031
将2-{6-[6-(4-甲基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙醇(134mg，0.3mmol)溶解于四氢呋喃(10ml)中，依次加入醋酸酐(153mg，1.5mmol)、三乙胺(152mg，1.5mmol)，升温至30℃搅拌，反应至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(S)-乙酸-2-{6-[6-(4-甲基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙酯(131mg，黄色固体)，收率：90％。MS m/z(ESI):489.6[M+1]
1HNMR(400Hz，DMSO-d6)：8.60(d,1H),8.35(s,1H),8.08(d,1H),8.01(dd,1H),7.66(s,1H),7.46(d,2H),7.29(m,3H)，6.96(d,1H),5.44(m,1H),3.78(m,2H),3.62(m,4H)，2.41(m,4H),2.23(s,3H),2.05(s,3H).
实施例20
Figure PCTCN2014086951-appb-000032
将2-{6-[6-(4-乙基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙醇(138mg，0.3mmol)溶解于四氢呋喃(10ml)中，依次加入醋酸酐(153mg，1.5mmol)、三乙胺(152mg，1.5mmol)，升温至30℃搅拌，反应至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(S)-乙酸-2-{6-[6-(4-乙基-哌嗪-1-基)-吡啶-3-基]-噻吩[3,2-d]嘧啶-4-基胺}-2-苯基-乙酯(129mg，黄色固体)，收率：86％。MS m/z(ESI):503.6[M+1]
1HNMR(400Hz，DMSO-d6)：8.60(s,1H),8.34(s,1H),8.09(d,1H),8.01(d,1H), 7.66(s,1H),7.40(d,2H),7.24(m,3H)，6.98(d,1H),5.44(m,1H),3.79(m,2H),3.64(m,4H)，2.47(m,6H),2.07(s,3H),1.07(t,3H).
实施例21
Figure PCTCN2014086951-appb-000033
将2-苯基-2-[6-(4-哌嗪-1-甲基-苯基)-噻吩并[3,2-d]嘧啶-4-氨基]-乙醇(250mg，0.56mmol)溶解于甲醇(10ml)中，加入醋酸酐(57mg，0.56mmol)，室温搅拌，至TLC监测原料反应完全。减压浓缩，用硅胶柱色谱法纯化所得残留物，得到(S)-1-(4-{4-[4-(2-羟基-1-苯基-乙胺)-噻吩并[3,2-d]嘧啶-6-基]-苄基}-哌嗪-1-基)-乙酮(200mg，白色固体)，收率：73.26％。MS m/z(ESI):488.65[M+1]
1HNMR(400Hz，DMSO-d6)：8.37(s,1H),8.20(d,1H),7.82(t,3H),7.45(t,4H),7.32(t,2H),7.22(t,1H),5.44(m,1H),4.99(t,1H),3.74(m,1H),3.56(s,2H),3.45(d,4H),2.33-2.40(m,4H),1.99(s,3H).
生物学实验
1、酪氨酸激酶EGFR酶活抑制IC50评价实验
本实验采用CISBIO公司的HTRF kinEASE TK kit(Cat.62TK0PEB,Cisbio)试剂盒进行候选化合物对EGFR酶活性抑制效果的测试，测试方法按照厂商提供的标准方法进行。大致的实验流程如下：在10μL的酶反应体系中，加入酶反应底物TK-biotin，ATP，EGFR酶(Cat.PV3872,Invitrogen)以及一定浓度的化合物在50mM Hepes/NaOH pH 7.5，10mM MgCl2，1mM EGTA，0.01％BRIJ-35的酶反应缓冲溶液中室温反应30分钟。待测化合物的每个筛选浓度取复孔测试，每次实验均设不加EGFR激酶的阴性对照孔和不加化合物的阳性对照孔。反应完成后，向所有反应孔中加入10μl经50mM Hepes/NaOH pH 7.0，0.1％BSA，0.8M KF，20mM EDTA稀释的Streptavidin-XL665和TK antibody europium cryptate(1:100)混合检测液，室温反应1h后，用2104
Figure PCTCN2014086951-appb-000034
Multilabel Reader(Perkinelmer)仪器检测荧光信号(320nm刺激,665nm，615nm发射)。通过全活性孔和背景信号孔计算出每个孔的抑制率，复孔取平均值，同时用专业的画图分析软件GraphPad PRISM 5.0对每个待测化合物进行半数抑制活性(IC50)的拟合。
2、HER2激酶活性测试方法
本实验采用CISBIO公司的HTRF kinEASE TK kit(Cat.62TK0PEB，Cisbio)试剂盒进行候选化合物对HER2酶活性抑制效果的测试，测试方法按照厂商提供的标准方法进行。大致的实验流程如下：在10μL的酶反应体系中，加入酶反应底物TK-biotin，ATP，HER2酶(Cat:PV3366，Invitrogen)以及一定浓度的化合物在50mM Hepes/NaOH pH 7.5，5mM MgCl2，1mM DTT，50nM SEB，1mM MnCl2的酶反应缓冲溶液中室温反应30分钟。待测化合物的每个筛选浓度取复孔测试，每次实验均设不加HER2酶的阴性对照孔和不加化合物的阳性对照孔。反应完成后，向所有反应孔中加入10μl经50mM Hepes/NaOH pH 7.0，0.1％BSA，0.8M KF，20mM EDTA稀释的Streptavidin-XL665和TK antibody europium cryptate(1:100)混合检测液，室温反应1h后，用2104
Figure PCTCN2014086951-appb-000035
Multilabel Reader(Perkinelmer)仪器检测荧光信号(320nm刺激,665nm，615nm发射)。通过全活性孔和背景信号孔计算出每个孔的抑制率，复孔取平均值，同时用专业的画图分析软件GraphPad PRISM 5.0对每个待测化合物进行半数抑制活性(IC50)的拟合。
3、通过CCK-8检测试剂盒检测化合物对人皮肤鳞状细胞癌细胞株A431的细胞毒性抑制IC50值。
细胞株:
A431人皮肤鳞状细胞癌细胞株(中科院上海细胞库)
试剂和耗材：
Cell Counting Kit-8(Cat#CK04-13，Dojindo)
96孔培养板(Cat#3599，Corning Costar)
胎牛血清(Cat#10099-141，GIBCO)
培养基(Invitrogen)
台式酶标仪SpectraMax M5Microplate Reader(Molecular Devices)
培养基的配制
细胞系 培养基
A431 DMEM+10％FBS
化合物的制备：用DMSO稀释化合物使终浓度为10mM。
细胞培养
a)收集对数生长期细胞，计数，用完全培养基重新悬浮细胞，
b)调整细胞浓度至合适浓度，接种96孔板，每孔接种100μl细胞悬液。
c)细胞在37℃，100％相对湿度，5％CO2培养箱中孵育24小时。
IC50实验
a)收集对数生长期细胞，计数，用完全培养基重新悬浮细胞，调整细胞浓度至合适浓度(依照细胞密度优化试验结果确定)，接种96孔板，每孔加100μl细胞悬液。细胞在37℃，100％相对湿度，5％CO2培养箱中孵育24小时。
b)用培养基将待测化合物稀释至500μM后梯度稀释8次。按25μl/孔加入细胞。化合物作用终浓度从100μM至0μM，5倍梯度稀释，共10个浓度点。
c)细胞置于37℃，100％相对湿度，5％CO2培养箱中孵育72小时。
d)吸弃培养基，加入含10％CCK-8的完全培养基置于37℃培养箱中孵育2-4小时。
e)轻轻震荡后在SpectraMax M5Microplate Reader上测定450nm波长处的吸光度，以650nm处吸光度作为参比，计算抑制率。
数据处理
按下式计算药物对肿瘤细胞生长的抑制率：肿瘤细胞生长抑制率％＝[(Ac-As)/(Ac-Ab)]×100％
As:样品的OA(细胞+CCK-8+待测化合物)
Ac:阴性对照的OA(细胞+CCK-8+DMSO)
Ab:阳性对照的OA(培养基+CCK-8+DMSO)
采用软件Graphpad Prism 5拟合IC50曲线并计算出IC50值。
表2 部分实施例化合物对酪氨酸激酶EGFR、HER2酶活抑制及A431细胞实验结果
Figure PCTCN2014086951-appb-000036
“—”表示活性未测试
上述生物活性结果表明，本发明包括的部分化合物均具有较好的酪氨酸激酶EGFR抑制活性以及HER2抑制活性，而且在细胞水平上实施例1、4、21也显示出明显的抑制A431细胞活性。
单次静脉或口服分别给予实施例1(代号CDDD-000261)和实施例21(代号CDDD-000257)在大鼠体内的药代动力学研究
本实验目的是通过单次静脉(IV)及口服(PO)给予SD大鼠供试品有效量的实施例21化合物和实施例1化合物，于不同时间点采集血样，LC/MS/MS测定给予供试品后大鼠血浆中供试品的浓度并计算相关参数和生物利用度。
供试品的配制
供试品，实施例21化合物和实施例1化合物溶于注射用水中，得到浓度均为5mg/mL的溶液，用于静脉和灌胃给药。
动物分配
从上海西普尔-必凯实验动物有限公司购入大约6-8周龄的20只雄性SD大鼠(雄性体重130-150g)，12只动物用于试验。在静脉和口服给药前，所有组别动物禁食过夜(10-14小时)，给药4小时后给食。
给药
供试品实施例21化合物和实施例1化合物通过静脉或口服单次给药。给药信息见下表
Figure PCTCN2014086951-appb-000037
Figure PCTCN2014086951-appb-000038
Figure PCTCN2014086951-appb-000039
样品采集及处理
第1-8组动物采血时间点为：给药前，5min，15min，30min，1h，2h，4h，6h，8h和24h。每只动物每次经心脏穿刺采约0.25mL血液，K2EDTA抗凝。血液样本采集后置于冰上，离心分离血浆(离心条件：8000转/分钟，6分钟，2-8℃)。收集的血浆分析前存放于–80℃。此外，剩余没有用于实验研究的多余动物用于空白血的采集。离心后的空白血浆用于整个研究中供试品的生物分析方法开发和生物样品分析。
药物代谢动力学分析
根据药物的血药浓度数据，使用药代动力学计算软件WinNonlin5.2非房室模型分别计算供试品的药代动力学参数。任何小于定量下限(LLOQ＝1ng/mL)的数据用0值替换，其平均值和标准差根据替换值进行计算。计算个体动物PK参数时任何小于定量下限的数值将被剔除。
实验结论
雄性SD大鼠血浆中实施例21化合物药代动力学：
动物静脉注射剂量为5mg/kg的实施例21化合物后，总清除率平均值2.60L/hr/kg。半衰期(T1/2)的平均值为1.67hr。给药后Cmax平均值为3218.39μg/L，Tmax平均值为0.083hr。AUC(0-t)值为1903.30hr*μg/L。终期相表观分布容积平均值为6.22L/kg。
动物口服给予剂量为30mg/mL的实施例21化合物后，Cmax平均值为1528.74μg/L，Tmax平均值为0.50hr，AUC(0-t)平均值为3736.13hr*μg/L，半衰期(T1/2)平均值为2.51hr。实施例21化合物平均生物利用度为33.01％。
雄性SD大鼠血浆中实施例1化合物药代动力学：
动物静脉注射剂量为5mg/kg的实施例1化合物后，总清除率平均值3.74L/hr/kg。半衰期(T1/2)的平均值为5.11hr。给药后Cmax平均值为616.65μg/L，Tmax平均值为0.083hr。AUC(0-t)值为1309.41hr*μg/L。终期相表观分布容积平均值为27.66L/kg。
动物口服给予剂量为30mg/mL的实施例1化合物后，Cmax平均值为296.20μg/L，Tmax平均值为5.33hr，AUC(0-t)平均值为3593.95hr*μg/L，半衰期(T1/2)平均值为5.67hr。实施例1化合物平均生物利用度为47.71％。
对人表皮样癌A431裸小鼠皮下移植瘤的疗效比较
药物名称和批号
CDDD-000257(即实施例21化合物)为白色粉末，含量99.7％，批号4010057-78；CDDD-000261(即实施例1化合物)为淡黄色粉末，含量99.1％，批号4010071-08；阳性对照物CDDD-000275(即阳性对照物gefitinib)为白色粉末，含量99％，批号R020-06-130619。
配制方法：均用20％PEG400蒸馏水配制。
实验动物
BALB/cA-nude裸小鼠，6-7周，♀，购自上海西普尔-必凯实验动物有限公司。合格证号：SCXK(沪)2008-0016饲养环境：SPF级。
实验步骤
裸小鼠皮下接种人表皮样癌A431细胞，待肿瘤生长至100-200mm3后，将动物随机分组(D0)。给药剂量和给药方案见表1。每周测2-3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0) 100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
实施例21化合物、实施例1化合物、阳性对照物gefitinib(3、10、30mg/kg,PO,QD 21)均剂量依赖性地抑制高表达EGFR的人表皮样癌A431裸小鼠皮下移植瘤的生长；其中实施例21化合物和实施例1化合物高剂量组分别引起3/6小鼠肿瘤部分消退。荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。总体比较，3个化合物对人表皮样癌A431裸小鼠皮下移植瘤抑瘤活性排序为：实施例21化合物>实施例1化合物>阳性对照物gefitinib。
表1.CDDD-000257、CDDD-000261、CDDD-000275对人表皮样癌A431裸小鼠移植瘤的疗效。
Figure PCTCN2014086951-appb-000040
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种如式I所示的化合物：
    Figure PCTCN2014086951-appb-100001
    其中：
    R1、R2各自独立地选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基、取代或未取代的含1～6个碳原子的胺基-酰基；
    或R1、R2与它们相连接的氮原子一起形成碳原子数为3至10个的取代或未取代的单环或者多环含氮杂环基，或任意位置由氮原子、氧原子、硫原子置换的碳原子数为3至10个的取代或未取代的单环或者双环含氮杂环基；
    或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基；
    R3选自下组：氢、取代或未取代的含1～6个碳原子的烷基、取代或未取代的含1～6个碳原子的烷基酰基、取代或未取代的含1～6个碳原子的酰胺基、取代或未取代的含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含5～6个碳原子的芳基酰基、取代或未取代的含1～6个碳原子的烷基磷酸酯基；
    R为取代芳环或杂芳环
    Figure PCTCN2014086951-appb-100002
    上任意氢原子的一个或多个选自下组的取代基：卤素、C1-C4烷基、C1-C4卤代烷基、烷氧基、羟基、氨基、氰基、羟基-C1-C4烷基、C2-C10杂环烷基、C2-C10杂环烷基-氧基、羧基，或C2-C6羧酸酯基；
    Ar2选自下组：苯基、卤素取代的苯基、C1-C6烷基取代的苯基、联苯基、卤素取代的联苯基、萘基、吡啶基、噻吩基、卤素取代的噻吩基、C1-C3烷基取代的噻吩基、呋喃基、卤素取代的呋喃基，或C1-C3烷基取代的呋喃基；
    A、B、D各自独立地选自下组：碳原子、氮原子、氧原子、硫原子或无；且A、B、D中至多仅有一个为无；
    n＝0、1或2；
    其中，除非特别说明，所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、羟基、氧代、硝基、C1～C6的卤代烷基、C1～C6的烷基、C1～C6的烯基、C1～C6的炔基、C3～C12的芳基、C3～C12的芳基烷基、C1～C6的烷氧基、C3～C12的芳基氧基、氨基、C1～C6的酰基氨基、C1～C6的烷基氨基甲酰基、C3～C12的芳基氨基甲酰基、C1～C6的氨基烷基、C1～C6的酰基、羧基、C1～C6的羟烷基、C1～C6的烷基磺酰基、C5～C12的芳基磺酰基、C1～C6的烷基磺酰氨基、C5～C12的芳基磺酰氨基、C3～C12的芳烷基磺酰氨基、C2～6的烯基酰基、C1～C6的酰氧基、C1～C6的羟基-酰基、氰基、脲基、C1～C6的烷基酰基、C1～C6的环烷基酰基、磺酰基、氨基甲酸酯基；
    虚线为化学键或无。
  2. 根据权利要求1所述的化合物，其特征在于：
    A、B、D均为碳原子；
    Ar2为未取代或卤素取代的苯基；和/或
    R1、R2各自独立地选自下组：氢、含1～6个碳原子的烷基或取代的烷基、含1～6个碳原子的烷基酰基、含3～6个碳原子的环烷基酰基、取代或未取代的含2～6个碳原子的烯基酰基、取代或未取代的含6～10个碳原子的芳基酰基、磺酰基、取代或未取代的含2～6个碳原子的酰胺基、取代或未取代的含1～6个碳原子的胺基-酰基；
    或R1、R2与它们相连接的氮原子一起形成取代或未取代的C3～C10的杂环基，其中，所述的杂环基具有1～3个选自下组的杂原子：O、S或N；
    或R1、R2与相连的氮原子及氮原子的邻位碳原子共同构成5～7元含氮杂芳基。
  3. 如权利要求1所述的式I化合物的制备方法，其特征在于，所述方法包括步骤：
    在钯催化剂的存在下，用式(1)化合物与式(1a)化合物进行偶联反应，得到式I化合物：
    Figure PCTCN2014086951-appb-100003
    式中，各基团的定义如权利要求1中所述。
  4. 如权利要求3所述的制备方法，其特征在于，所述方法包括步骤：用式(3)化合物与R3X反应，得到式I化合物：
    Figure PCTCN2014086951-appb-100004
    其中，X选自下组：Cl、OTs。
  5. 一种酪氨酸激酶抑制剂，其特征在于，所述的抑制剂含有抑制有效量的如权利要求1或2中所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物。
  6. 一种药物组合物，其特征在于，所述的药物组合物含有治疗有效量的如权利要求1或2中所述的式I化合物，或其药学上可接受的盐、互变异构体、光学异构体、药学上可接受的溶剂合物。
  7. 如权利要求6所述的药物组合物，其特征在于，所述的药物组合物用于治疗与酪氨酸激酶过表达和/或酪氨酸激酶活性过高相关的疾病，或所述的药物组合物用于治疗与表皮生长因子受体相关的疾病。
  8. 如权利要求5所述的抑制剂或如权利要求6中所述的药物组合物，其特征在于：
    所述药学上可接受的盐是式I化合物的选自下组的盐：无机酸盐、有机酸盐、烷基磺酸盐、芳基磺酸盐，或其组合；较佳地，所述的盐选自下组盐酸盐、氢溴酸盐、硝酸 盐、硫酸盐、磷酸盐、甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、甲基磺酸盐、乙基磺酸盐、苯磺酸盐、对甲苯磺酸盐，或其组合；
    所述药学上可接受的溶剂合物，是指式I化合物与选自下组的溶剂形成的溶剂合物：水、乙醇、异丙醇、***、丙酮，或其组合。
  9. 如权利要求1或2任一所述的式I化合物的用途，其特征在于，用于：
    (a)制备酪氨酸激酶抑制剂；
    (b)用于体外非治疗性地抑制酪氨酸激酶的活性；
    (c)用于体外非治疗性地抑制肿瘤细胞生长；
    (d)用于制备治疗表皮生长因子受体活性相关的疾病的药物。
  10. 如权利要求9所述的用途，其特征在于，所述的表皮生长因子受体选自下组：EGFR和/或HER2。
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WO2016205590A1 (en) * 2015-06-18 2016-12-22 Cephalon, Inc. Substituted 4-benzyl and 4-benzoyl piperidine derivatives
JP2018524318A (ja) * 2015-06-18 2018-08-30 セファロン、インク． 置換４−ベンジル及び４−ベンゾイルピペリジン誘導体
US10851057B2 (en) 2015-06-18 2020-12-01 89Bio Ltd 1,4-substituted piperidine derivatives
US10919875B2 (en) 2015-06-18 2021-02-16 89Bio Ltd Substituted 4-benzyl and 4-benzoyl piperidine derivatives
JP2021121612A (ja) * 2015-06-18 2021-08-26 エイティナイン バイオ リミテッド 置換４−ベンジル及び４−ベンゾイルピペリジン誘導体
US11702388B2 (en) 2015-06-18 2023-07-18 89Bio Ltd 1,4-substituted piperidine derivatives
JP7334211B2 (ja) 2015-06-18 2023-08-28 エイティナイン バイオ リミテッド 置換４－ベンジル及び４－ベンゾイルピペリジン誘導体
US11878966B2 (en) 2015-06-18 2024-01-23 89Bio Ltd Substituted 4-benzyl and 4-benzoyl piperidine derivates

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