WO2018219281A1 - 一类4‐嘧啶二胺类小分子有机化合物及其衍生物及其应用 - Google Patents

一类4‐嘧啶二胺类小分子有机化合物及其衍生物及其应用 Download PDF

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WO2018219281A1
WO2018219281A1 PCT/CN2018/088932 CN2018088932W WO2018219281A1 WO 2018219281 A1 WO2018219281 A1 WO 2018219281A1 CN 2018088932 W CN2018088932 W CN 2018088932W WO 2018219281 A1 WO2018219281 A1 WO 2018219281A1
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pyrimidinediamine
fluoro
dimethylpiperazinyl
phenyl
methoxy
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PCT/CN2018/088932
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French (fr)
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陈益华
刘明耀
郭伟凯
邢雅婧
黄懂霞
易正芳
杜冰
张乾森
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华东师范大学
上海邦耀生物科技有限公司
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Definitions

  • the present invention relates to a class of 4 -pyrimidinediamine small molecule organic compounds and derivatives thereof and uses thereof.
  • the compound or a pharmaceutical composition containing the same can be used for the preparation of an antitumor drug for treating various malignant tumors, including tumor metastasis and the like, and can be used as a B cell lymphokine factor 6 (BCL6) inhibitor for prevention.
  • BCL6 mediated diseases such as autoimmune diseases, allergies and inflammation).
  • Cancer is a general term for malignant cells that are not controlled by organisms and can proliferate indefinitely. In the current society, cancer has become the second most common cause of death in humans other than cardiovascular disease. According to the World Health Organization (WHO)'s latest Global Cancer Report 2014, the number of cancer patients worldwide is growing rapidly, from 14 million in 2012 to 24 million in 2035. The report also shows that half of the world's new cancer patients in 2012 came from Asia, with China accounting for the majority, and China has become a high-risk area for cancer.
  • WHO World Health Organization
  • Surgical treatment is still the most effective means of treating cancer so far.
  • surgical treatment is only suitable for early cancer patients.
  • radiation therapy kills normal cells while killing tumor cells.
  • Chemotherapy can be divided into traditional chemotherapy drugs and targeted drug therapy.
  • Traditional chemotherapy drugs often have great side effects because they are not selective.
  • Targeted drug therapy often aims to treat cancer by specifically targeting the oncogenic sites of tumor cells and specifically interfering with certain signaling pathways of tumor cells. Targeted therapy can specifically kill tumor cells. Therefore, compared with traditional chemotherapy, the toxic side effects of targeted therapy are smaller and have attracted widespread attention.
  • BCL6 Proto-oncogene B cell lymph factor 6
  • BCL6 is a transcriptional repressor, and BCL6 is involved in the formation of germinal centers in vivo.
  • BCL6 is a member of the BTB/POZ/Zinc Finger Nuclease family and consists of 706 amino acids.
  • BCL6 can be divided into three parts according to its structure and function: the N-terminal BTB/POZ domain (amino acid residues 1-130), and the central region predicted to have little or no fixed structure (amino acid residues 129-517) , six zinc finger domains at the C-terminus (amino acid residues 518-681).
  • the N-terminal BTB/POZ domain can interact with three known helper repressors: SMRT, N-CoR and BCOR in a mutually exclusive manner, thereby exerting transcriptional inhibition.
  • the gene inhibited by BCL6 is involved in cell activation, differentiation, apoptosis, cell cycle arrest, etc.
  • BCL6 can inhibit the expression of PRDM1, cyclinD2, p53 and other genes, which are plasma cell differentiation, cell cycle regulation, and apoptosis. , DNA repair and important genes that maintain genomic stability. Therefore, if the normal downregulation of BCL6 is blocked, the cells are prone to differentiation, prolonged proliferation, long-term survival, and genetic instability, eventually leading to malignant transformation. Its function is to prevent cell differentiation and death, and promote cell development and proliferation.
  • BCL6 has a very important relationship with the occurrence and development of various tumors.
  • Non-Hodgkin's lymphoma is a high-grade malignant lymphoma that ranks 6th among common tumors and is divided into three basic types based on cell origin: B cells, T cells, and NK/T cell NHL. Most of the clinical NHL is B-cell type, accounting for 70%-85% of the total. Among them, Diffuse Large B-Cell Lymphoma (DLBCL) is the most common B-cell type NHL, accounting for about 40% of all NHL. %. Depending on the gene expression profile, DLBCL can be divided into a germinal center B cell-like (GCB-like) type, an activated B cell-like (ABC-like) type, and a primary mediastinal B-cell-like (PMBL) type.
  • GCB-like germinal center B cell-like
  • ABSC-like activated B cell-like
  • PMBL primary mediastinal B-cell-like
  • BCL6 in GCB-DLBCL is as high as 86%, and the expression in ABC-DLBCL is 34%.
  • the resistance of DLBCL may be related to the high expression of BCL6.
  • R-CHOP can selectively kill BCL6 negative.
  • DLBCL has little effect on BCL6-positive DLBCL.
  • BCL6 is also highly expressed in follicular lymphomas (FLs) and the like.
  • FLs follicular lymphomas
  • BCL6 is a very important target for the treatment of DLBCL and FLs.
  • the researchers found that BCL6 also plays an important role in autoimmune diseases.
  • BCL6 is an important regulator of germinal centers (GC), and BCL6 is closely related to germinal center abnormalities.
  • GC-like structures are present in salivary glands, meninges, and synovium of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and type 1 diabetes. , a variety of tissue lesions such as the thyroid, skin, pancreas and kidney (Aloisi F et al. / Nat Rev Immunol. 2006 Mar; 6 (3): 205-17).
  • Myasthenia gravis (MG) is a T cell-dependent antibody-mediated organ-specific autoimmune disease. To date, there are no specific immunosuppressive agents that selectively target MG.
  • BCL6 can be used not only as a target for the treatment of MG, but also as a therapeutic target for other antibody-mediated autoimmune diseases, such as systemic. Lupus erythematosus, rheumatoid arthritis, etc. (N. Xin et al. / Molecular and Cellular Neuroscience 58 (2014) 85-94).
  • BCL6 peptide inhibitor can reactivate BCL6 target gene by competitively binding to the N-terminal BTB/POZ domain of BCL6, and can kill BCL6 in vitro and in vivo.
  • Highly expressed DLBCL but BPI has problems such as low oral bioavailability and high cost, thus limiting its further clinical application.
  • FX1 was modified on the basis of 79-6, and its activity was greatly improved compared with 79-6, and the IC 50 was about 36 ⁇ M (The Journal of Clinical Investigation, June 3, 2016.).
  • FX1 still has a major disadvantage, and FX1 still has poor inhibitory activity against tumor cell proliferation, and this disadvantage has limited their further clinical development.
  • the invention adopts diaminopyrimidine as a basic structural skeleton, and is structurally transformed into a novel type of 4 -pyrimidinediamine small molecular organic compound.
  • the compounds of the invention are capable of binding to the NBT-terminal BTB/POZ domain of BCL6 in a competitive manner, thereby reactivation of target genes downstream of BCL6.
  • the compound of the present invention can strongly inhibit the proliferation of DLBCL and promote the apoptosis of DLBCL, and its antiproliferative activity reaches 1 ⁇ M or less.
  • the compounds of the present invention have greatly improved activity and completely different structures, and can be used as a lead compound of BCL6 and a potential clinical candidate drug.
  • the present invention provides a novel class of 4-pyrimidinediamino small molecule organic compounds and related analogs which are novel as BCL6 inhibitors and antitumor drugs, including useful salts thereof, esters and the like.
  • the 4-pyrimidinediamino small molecule organic compound or related analog or pharmaceutically acceptable salt provided by the present invention has the structure as shown in (I):
  • R 1 and R 2 are selected from one or more of the following groups: hydrogen (wherein R 1 and R 2 are not hydrogen at the same time), halogen, C 1 -C 3 alkoxy, C 1 -C 3 alkylamine Base, hydroxyl, nitro, amino, carboxyl, Wherein R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile base.
  • R 3 is selected from one of the following groups: a C 1 -C 5 chain alcohol amino group, a C 1 -C 5 fatty chain amino group, a C 3 -C 6 cyclic amino group, a tetrahydropyrrolyl group, a piperazinyl group, C 1 -C 3 alkylpiperazinyl, piperidinyl, C 1 -C 3 alkylpiperidinyl, morpholinyl, C 1 -C 3 alkylmorpholinyl, thiomorpholinyl, C 1 - C 3 alkylthiomorpholinyl.
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 1 and R 2 are selected from one or more of the group consisting of hydrogen (wherein R 1 and R 2 are not simultaneously hydrogen), halogen, C 1 -C 3 alkoxy, C 1 -C 3 alkylamine , hydroxyl, nitro, amino, carboxyl, Wherein R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile base.
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 5 and R 6 are each selected from one of the following groups: hydrogen, C 1 -C 3 alkyl.
  • X is O or N
  • R 1 is selected from one or more of the group consisting of hydrogen (wherein R 1 and R 2 are not simultaneously hydrogen), halogen, C 1 -C 3 alkoxy, C 1 -C 3 alkylamino, hydroxy, Nitro, amino, carboxyl,
  • R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile base.
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 5 and R 6 are each selected from one of the following groups: hydrogen, C 1 -C 3 alkyl.
  • R 7 is a C 1 -C 3 alkyl group.
  • X is O or N
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 5 and R 6 are each selected from one of the following groups: hydrogen, C 1 -C 3 alkyl.
  • R 7 is a C 1 -C 3 alkyl group.
  • R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile.
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 5 and R 6 are each selected from one of the following groups: hydrogen, C 1 -C 3 alkyl.
  • R 7 is a C 1 -C 3 alkyl group.
  • R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile.
  • R 4 is selected from one of the following groups: hydrogen, halogen, nitrile, trifluoromethyl, C 1 -C 3 alkoxy, hydroxy, C 1 -C 3 alkyl.
  • R 7 is a C 1 -C 3 alkyl group.
  • R 8 is selected from one or more of the group consisting of hydrogen, halogen, hydroxy, carboxy, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, ester, nitro, amino, nitrile.
  • the present invention also provides the 4-pyrimidinediamine small molecule organic compound or a related analog thereof or a pharmaceutically acceptable salt thereof, the disubstituted pyrimidinediamine small molecule organic compound forming an acid addition salt with an acid
  • the acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid. , succinic acid.
  • the 4-pyrimidinediamine small molecule organic compound or a related analog or pharmaceutically acceptable salt thereof in the present invention wherein the 4 -pyrimidinediamine small molecule organic compound can be combined with a radioactive group and a fluorescent group
  • the group or biotin combines to form a label.
  • the 4-pyrimidineamine small molecule organic compound or related analog or pharmaceutically acceptable salt thereof provided by the present invention comprises:
  • the present invention also provides a pharmaceutical composition wherein the pharmaceutical composition contains the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions are formulated as injectable fluids, aerosols, creams, gels, pills, capsules, syrups, transdermal patches or excipients.
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition as a BCL6 inhibitor.
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for inhibiting the interaction of a BCL6 protein with a polypeptide SMRT.
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for inhibiting the formation of a germinal center.
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for the preparation of a medicament for preventing and/or treating BCL6 mediated diseases;
  • the diseases include, but are not limited to, autoimmune diseases, allergies or inflammation; wherein the autoimmune diseases include lupus erythematosus, multiple sclerosis, arthritis, dermatomyositis, chronic thyroiditis, juvenile diabetes, Abnormal anemia, atrophic gastritis, non-specific ulcerative colitis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune hemolytic anemia, and special Leukopenia.
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for preparing an antitumor drug; the 4-pyrimidinediamine of the present invention Small molecule organic compounds, hydrates or pharmaceutically acceptable salts or pharmaceutical compositions can inhibit proliferation, growth, migration and infiltration of tumor cells.
  • the tumor cells include diffuse large B-cell lymphoma (eg, human lymphoma cell line SUDHL4), follicular lymphoma, Burkitt's lymphoma, non-small cell lung cancer cells, breast cancer cells, B-cell acute lymphocytes Cell leukemia, chronic myelogenous leukemia, esophageal cancer cells, ovarian cancer cells, head and neck squamous cell carcinoma cells, colon cancer cells, prostate cancer cells, liver cancer cells.
  • diffuse large B-cell lymphoma eg, human lymphoma cell line SUDHL4
  • follicular lymphoma e.g, follicular lymphoma
  • Burkitt's lymphoma non-small cell lung cancer cells
  • breast cancer cells breast cancer cells
  • B-cell acute lymphocytes Cell leukemia chronic myelogenous leukemia
  • esophageal cancer cells ovarian cancer cells
  • head and neck squamous cell carcinoma cells colon cancer cells
  • prostate cancer cells
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for preparing a medicament for preventing and/or treating a malignant tumor;
  • the malignant tumor includes diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, non-small cell lung cancer, breast cancer, B-cell acute lymphoblastic leukemia, chronic myeloid leukemia, esophageal cancer, Ovarian cancer, head and neck squamous cell carcinoma, colon cancer, prostate cancer, liver cancer.
  • the present invention provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for up-regulating the expression of BCL6 downstream genes CXCR4, CD69, Caspase8 and DUSP5; preferably
  • the 4 -pyrimidinediamine small molecule organic compound is N 2 -((3S,5R)-3,5-dimethylpiperazinyl)-N 4 -(4-(6-bromo-2-pyridyl) Amido)phenyl)-5-fluoro- 2,4-pyrimidinediamine (WH-007), N 2 -((3S,5R) ⁇ 3,5-dimethylpiperazinyl)-N 4 ⁇ ( (3-Methoxy-4-yl(3-bromobenzamide))phenyl)-5-fluoro- 2,4-pyrimidinediamine (WH-009), N 2 ⁇ ((3S,5R) ⁇ 3,5-Dimethylpiperaziny
  • the present invention also provides that the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition up-regulates BCL6 downstream gene CXCR4, CD69 in BCL6-positive cell line and BCL6-negative cell.
  • the application in the expression of CASPASE8 and DUSP5 preferably, the BCL6-positive cell strain is SUDHL4, and the BCL6-negative cell strain is TOLEDO; preferably, the 4-pyrimidinediamine-based small molecule organic compound is the 4 ⁇
  • a small molecule organic compound of pyrimidinediamine is N 2 -((3S,5R)-3,5-dimethylpiperazinyl)-N 4 -(4-(6-bromo-2-pyridylamino)phenyl -5-fluoro- 2,4-pyrimidinediamine (WH-007), N 2 -((3S,5R) ⁇ 3,5-dimethylpiperazinyl)-N 4 ⁇ ((
  • the present invention also provides the use of the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition for inhibiting the expression of LPS-stimulated TNF ⁇ .
  • the 4-pyrimidinediamine small molecule organic compound or related analog or pharmaceutically acceptable salt or pharmaceutical composition may be used alone or in combination with other drugs.
  • the beneficial effects of the present invention are that the 4-pyrimidinediamine small molecule organic compound or related analog or the pharmaceutically acceptable salt or the pharmaceutical composition of the formula (I) to (VI) provided by the present invention can be remarkably inhibited.
  • the interaction of BCL6 fragment protein with the polypeptide SMRT inhibits the proliferation of diffuse large B-cell lymphoma (human lymphoma cell line SUDHL4), inhibits the formation of germinal centers, inhibits the expression of LPS-stimulated TNF ⁇ , and can be used to prevent and/or treat itself.
  • Immune diseases, allergies or inflammation inhibit the proliferation, growth, migration and infiltration of malignant tumors, and have broad application prospects.
  • Fig. 1 is a view showing the effect of detecting the binding effect of a part of the compound of the present invention and BCL6 protein. Among them, the abscissa represents time (seconds), and the ordinate represents reflectance units.
  • Figure 2 is a graph showing the effect of the compound of the present invention on up-regulation of the downstream gene of BCL6.
  • Figures A, B, C and D are the up-regulation effects of the compounds of the present invention on the genes CD69, CXCR4, CASSASE8 and DUSP5, respectively.
  • Fig. 3A is a schematic diagram showing the expression level of BCL6 protein in human diffuse large B cell lines SUDHL4 and TOLEDO.
  • Figure 3B is a graph showing the up-regulation effects of the compounds WH-007 and WH-023 of the present invention on the downstream genes B69 and CDX4 in the cell lines SUDHL4 and TOLEDO.
  • Figure 4 is a graph showing the effect of the compound WH-007 of the present invention on inhibiting the formation of germinal centers in mice.
  • Figure A is a statistical diagram of the weight of the mouse spleen (wherein the ordinate indicates the weight of the spleen (g))
  • B is a flow chart of the formation of the germinal center of the compound WH-007 of the present invention
  • C is a compound of the present invention.
  • WH-007 inhibited the statistical map of germinal center formation in mice at a gavage dose of 50 mg/kg/d.
  • Figure 5 is a graph showing the effect of the compound WH-023 of the present invention on inhibiting the formation of germinal centers in mice.
  • Figure A is a statistical diagram of the weight of the spleen of the mouse (wherein the ordinate indicates the weight of the spleen (g)
  • B is a flow chart of the formation of the germinal center of the compound WH-023 of the present invention
  • C is a compound of the present invention WH -023 A graph that inhibits the formation of germinal centers in mice at a gavage dose of 50 mg/kg/d (the upper part of the figure is the ratio of germinal center B cells.
  • Figure 6 is a graph showing the inhibition of LPS-stimulated TNFa expression by a compound of the invention.
  • Fig. 7 shows a significant therapeutic effect of the compound of the present invention on arthritis; wherein Figure A is a typical effect diagram of the compound of the present invention for treating arthritis, and Figure B is a statistical score chart of the therapeutic effect of arthritis.
  • FIG. 8 is a schematic diagram of a compound having a terminal piperazine-based functional group and a morpholine-functional group in the present invention, which can form a stable hydrogen bond with the H14 residue of the BTB region of the BCL6 protein; wherein A is a graph showing the hydrogen bond formation of the piperazine compound, Figure B is an effect diagram showing the formation of hydrogen bonds by morpholine compounds.
  • Example 1 ⁇ 06 Compound N 2 ⁇ ((3S,5R) ⁇ 3,5-Dimethylpiperazinyl)-N 4 ⁇ ((3 ⁇ methoxy-4-4 ⁇ (6 ⁇ bromo-2-phenyl) Preparation of Amido))phenyl)-5-fluoro-2,4-pyrimidinediamine (WH ⁇ 006)
  • 6-Bromo-2-picolinic acid (120.0 mg, 0.6 mmol) was dissolved in 1.5 mL of DMF, and EDC ⁇ HCl (118 mg) and HOBt (112 mg) were added at room temperature for 10 min and then added to 2-((3S,5R) -3,5-Dimethylpiperazinyl)-5-fluoro-N-(3-methoxy-4-ylaminophenyl)pyrimidine-4-amine (173.2 mg, 0.5 mmol), stirred overnight. After work-up: extraction with dichloromethane and water three times, the organic phases were combined and dried over anhydrous sodium sulfate.
  • 3-Bromo-2-pyridine benzoic acid 114.7 mg, 0.57 mmol was dissolved in 1.5 mL of DMF, and EDC ⁇ HCl (118 mg) and HOBt (112 mg) were added at room temperature for 10 min, then added 2-((3S,5R) -3,5-Dimethylpiperazyl)-5-fluoro-N-(4-aminophenyl)pyrimidine-4-amine (151.0 mg, 0.47 mmol), stirred overnight, extracted with dichloromethane and water three times The organic phase was combined, dried over anhydrous sodium sulfate and dichloromethane was evaporated.
  • 5-Phenyl-2-thiophenecarboxylic acid (118.5 mg, 0.58 mmol) was dissolved in 1.5 mL of DMF, and EDC ⁇ HCl (118 mg) and HOBt (112 mg) were added at room temperature for 10 min and then added to 2 - (3S, 5R)-3,5-Dimethylpiperazinyl)-5-fluoro-N-(3-methoxy-4-ylaminophenyl)pyrimidine-4-amine (100.0 mg, 0.29 mmol) was stirred overnight. Work-up: extraction with dichloromethane and water three times. The organic phases were combined and dried over anhydrous sodium sulfate.
  • Example 1-36 N 2 ⁇ ((3S,5R) ⁇ 3,5-dimethylpiperazinyl)-N 4 ⁇ ((3 ⁇ (5 ⁇ bromo-2-phenylthioethyl)) ⁇ 4 ⁇ Preparation of methoxyphenyl)-5-fluoro- 2,4-pyrimidinediamine (WH-036)
  • Example 2 Homogeneous Time-Resolved Fluorescence (HTRF) assay for the inhibition of the interaction of a compound of the invention with a BCL6 fragment protein and its co-suppressor SMRT
  • HTRF Homogeneous Time-Resolved Fluorescence
  • HTRF Homogeneous Time-Resolved Fluorescence
  • FRET fluorescence resonance energy transfer
  • TRF time-resolved fluorescence
  • the energy donor selected for the present invention is GST-Tb and the receptor is 6His-XL665 (Cisbio).
  • the proteins expressed and purified by the present invention also carry GST and His tags, respectively, BCL6-GST and 6His-SMRT.
  • the transcriptional repressor activity domain of BCL6 is a domain that binds to the transcriptional co-repressor SMRT (polypeptide SMRT).
  • the BTB/POZ domain sequence of the N-terminus of the BCL6 protein required by the present invention is ADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKT-VLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASE-GST.
  • the sequence of the SMRT polypeptide is 6HISGL-VATVKEAGRSIHEIPREEL.
  • the HTRF experiment requires that the fluorescence ratio of the two proteins bound at a certain concentration has a better S/N ratio than the fluorescence ratio when not bound.
  • the selected 6His-SMRT concentration of the present invention is 200 nM, BCL6.
  • the concentration of -GST is 6.25 nM.
  • the experiment was a 20 ⁇ L system. First, 4 ⁇ L of 5 ⁇ BCL6-GST and 4 ⁇ L of 5 ⁇ 6 His-SMRT were added to each well of a 384-well plate for 30 min at room temperature, and two replicate wells were set for each group. Then 2 ⁇ L of 10 ⁇ different concentrations of compound were added, and finally 5 ⁇ L of GST-Tb and 6His-XL665 were added, incubated overnight at room temperature, and the next day was read on a Cytation 5 cell imaging microplate reader. The experimental results were processed with GraphPad Prism and the experiments were repeated 3 times independently.
  • the results of the experiment are shown in Table 2.
  • the compound of the present invention inhibits the interaction between the BCL6 fragment protein and the polypeptide SMRT, wherein all the compounds of the present invention (WH-001 to WH-053) can effectively inhibit the interaction between the fragment protein and the polypeptide SMRT.
  • the half-inhibitory concentration (IC 50 ) is below 20 ⁇ M, such as compounds WH-001-WH005, WH017, WH018, WH-026, WH-028, WH-032, WH-035, WH-037 to WH-046, WH
  • the IC 50 of -051 to WH-053 is between 10-20 ⁇ M; the compounds WH-010 to WH016, WH-019 to WH-022, WH-036, WH-047 and WH-050 interact with BCL6 fragment protein and SMRT
  • the half-inhibitory concentration (IC 50 ) is between 5 and 10 ⁇ M; the compounds WH-006 to WH-009, WH-023 to WH-025, WH-027, WH-030, WH-031 and WH-033 for the BCL6 fragment
  • the half-inhibitory concentration (IC 50 ) of the interaction of the protein with SMRT is below 5 ⁇ M.
  • Table 2 compounds inhibit BCL6 protein and its fragments of IC 50 values cosuppression SMRT interaction factor of the present invention
  • Example 3 Compounds of the invention inhibit proliferation of diffuse large B-cell lymphoma
  • the human lymphoma cell SUDHL4 (human diffuse large B-cell lymphoma cell SUDHL4) used in the present invention is from the laboratory of Professor Zheng Wei of the Shanghai Key Laboratory of Control Biology. The cells were cultured in a 37 ° C incubator (95% humidity, 5% CO 2 concentration), wherein the SUDHL4 medium was RPMI-1640 (Gibco) containing 20% fetal calf serum (Gemini).
  • Cell proliferation was measured by the CCK8 method.
  • the experimental results are shown in Table 3.
  • the half effective inhibitory concentration (IC 50 ) of the compounds of the present invention WH-001 to WH053 inhibiting the proliferation of human diffuse large B-cell lymphoma cells SUDHL4 are all below 10 ⁇ M, indicating that the compound of the present invention is against lymphoma.
  • Cell proliferation has a significant inhibitory effect.
  • compounds WH-023, WH-024, WH-027, WH-029, WH-030, WH-031, WH-033, WH-034 and WH-035 have half-inhibitory concentration IC 50 for cell SUDHL4 proliferation. Below 1 ⁇ M, it shows a strong anti-tumor cell proliferation effect.
  • Example 4 Affinity detection of the compound of the present invention and BCL6 protein
  • This experiment detects the affinity between a compound of the invention and a BCL6 fragment protein containing a BTB/POZ domain by using a Biacore Biomacromolecular Interaction Instrument.
  • the invention selects four compounds WH-007, WH-009, WH-023 and WH-025 which have the effects of HTRF test and inhibits the proliferation of tumor cells, and tests the binding effect of BCL6 protein.
  • BCL6 Fragment Protein was immobilized on the surface of the chip by amino coupling.
  • the carboxyl group on the surface of the Fc2 channel of the CM5 chip was activated by the NHS and EDC mixed solution.
  • the BCL6 fragment protein was diluted into a sodium acetate buffer of pH 4.0 and coupled via the Fc2 channel.
  • the carboxyl group which was not bound to the BCL6 fragment protein after activation was blocked with ethanolamine.
  • Affinity test The compounds of the present invention, WH-007, WH-009, WH-023 and WH-025, were serially diluted into PBS buffer to prepare test samples of various concentrations of 0.01-10 ⁇ M.
  • the Fc1 channel which does not bind to the BCL6 fragment protein was used as a reference, and the Fc2 was used as a test channel.
  • the compound test samples were respectively flowed through the above two channels, and the response values of the binding of the BCL6 fragment proteins were tested for the same compound to obtain the concentration.
  • the dependence curve, and based on the 1:1 binding model fits the affinity between the tested compound and the protein.
  • Example 5 The compound of the present invention up-regulates the expression of a downstream gene of BCL6
  • Real-time quantitative PCR is a method for real-time detection of changes in the amount of amplified products in each PCR amplification reaction using changes in fluorescence signals, and quantitative analysis of the starting template by analysis of Ct values and standard curves.
  • the present invention employs qRT-PCR to examine the regulation of the expression of a downstream gene of BCL6 by a compound of the invention.
  • BCL6 is a transcriptional repressor, and BCL6 function is inhibited and its downstream gene expression is up-regulated.
  • the relevant detection of the compound of the present invention is shown in Fig. 2 and Fig. 3; wherein, Fig.
  • FIG. 2 is an example of the compounds of the present invention on the BCL6 positive cell line SUDHL4 by taking WH-007, WH-009, WH-023 and WH-025 as examples.
  • FIG. 3 is an example of the results of detecting the concentration of the compounds CD69 and CXCR4 in the BCL6-positive cell line SUDHL4 and the BCL6-negative cell line TOLEDO by using WH-007 and WH-023 as examples.
  • Reverse transcription mRNA is reversed to cDNA.
  • the reaction system was 20 ⁇ L, wherein ddH 2 O was 15 ⁇ L, 5 ⁇ Mix buffer was 4 ⁇ L, and RNA was 1 ⁇ l (1000 ng).
  • the reaction conditions are: 37 ° C reaction for 30 min, 85 ° C reaction for 5 s, 16 ° C;
  • the primer sequences of BCL6 downstream gene CD69 are CTGGTCACCCATGGAAGTG and CATGCTGCTGACCTCTGTGT, the primer sequences of CXCR4 are AGGCCCTAGCTTTCTTCCAC and CTGCTCACAGAGGTGAGTGC, the primer sequences of Caspase8 are CTGGGAGAAGGAAAGTTGGA and CAAGGCTGCTGCTTCTCTCT, and the primer sequences of DUSP5 are: ATGGATCCCTGTGGAAGACA and TCACAGTGGACCAGGACCTT.
  • the primer sequences of the internal reference gene actin are GTACGCCAACACAGTGCTG and CGTCATACTCCTGCTTGCTG.
  • the reaction conditions were: one cycle of pre-denaturation (95 ° C, 5 min), and 40 cycles of PCR reaction (95 ° C, 30 s, 60 ° C, 30 s, 72 ° C, 30 s).
  • FIG. 2 Some experimental results are shown in Fig. 2 and Fig. 3.
  • compounds WH-007, WH-009, WH-023 and WH-025 can up-regulate the expression of BCL6 downstream genes CXCR4, CD69, CASSASE8 and DUSP5, among which WH
  • the effect of -023 was the most significant, and its effect on the up-regulation of the gene CASSASE8 was about 10 times, the effect of up-regulating the expression of the genes CXCR4 and DUSP5 was several tens of times, and the up-regulation effect on the gene CD69 was more than 2000 times.
  • Figure 3A is a graph showing the expression of BCL6 protein in cell lines SUDHL4 and TOLEDO.
  • Example 6 Inhibition of the formation of germinal centers in mice by the compounds of the invention in vivo
  • the germinal center response is a process of a T cell-dependent antigenic immune response in which activated B cells undergo clonal proliferation, functional maturation to plasma cells that differentiate to produce high affinity antibodies, and differentiate into memory B cells.
  • germinal center (GC) B cells are characterized by high expression of cell surface proteins GL7 and FAS.
  • BCL6 the most important regulator of germinal center formation, is a transcriptional repressor necessary for the development of germinal center B cells. In the absence of BCL6, the formation of germinal centers is inhibited.
  • WH-007 and WH-023 were taken as examples to test the effect of the compound of the present invention on inhibiting BCL6 function and thereby affecting the germinal center.
  • mice were intragastrically administered with 50 mg/kg/d (low concentration) and 100 mg/kg/d (high concentration) of the compound of the present invention, and the compound was dissolved in sodium carboxymethylcellulose (CMC-Na), blank control The group was intragastrically given an equal volume of solvent CMC-Na;
  • mice were sacrificed on the 12th day of immunization, the spleens of the mice were taken and the weight of the spleen was weighed;
  • the mouse spleen was placed in a 70 ⁇ m cell strainer and ground with a syringe core.
  • the cell suspension was collected by washing with MACS buffer (PBS + 2% FBS);
  • FIG. 4A and 5A are statistical graphs of mouse spleen weight, indicating that the compounds of the present invention WH-007 and WH-023 have no effect on mouse spleen.
  • the compounds of the invention WH-007 (Fig. 4B) and WH-023 (Fig. 5B) showed significant inhibition of the formation of germinal centers in mice, GCB cell ( The proportion of GL7+FAS+) has dropped significantly.
  • Fig. 4C and Fig. 5C are graphs showing the percentage of germinal center B cells at an administration dose of 50 mg/kg/d.
  • Example 7 The compounds of the invention inhibit the expression of LPS-stimulated TNF ⁇ .
  • Mouse RAW 264.7 cells (10000 cells/well) were separately added to a 96-well culture plate, and then LPS (100 ng/ml) was added for treatment for 6 hours, and then the compound of the present invention WH-001 to WH-053 was added at a concentration of 100 nM to continue treatment. After the supernatant was collected, the cytokine content in the supernatant was detected using an ELISA kit of BD TNF- ⁇ .
  • the compound of the present invention has the ability to inhibit the expression of LPS-activated TNF ⁇ at a lower concentration, particularly the compounds WH-007, WH-009, WH-015, WH-023, WH-025, WH. -030, WH-036, WH-037 and WH-053 all have the ability to strongly inhibit the expression of TNF ⁇ activated by LPS, and have a good anti-inflammatory effect.
  • Example 8 The compound of the present invention has a significant therapeutic effect on arthritis
  • Collagen emulsification In the experiment, the model was prepared by two immunization methods, and both were completely emulsified with chicken type II collagen using a Freund's adjuvant, and the volume ratio was 1:1.
  • mice On day 0, the emulsified collagen was injected subcutaneously at the base of the tail of the mouse, 100 ul each; at the same time, the compounds of the present invention, WH-007, WH-009, WH-015, at a concentration of 50 mg/kg/2d. , WH-023, WH-025, WH-030, WH-036, WH-037, and WH-053 were administered to mice by gavage. On the 21st day, a second immunization was performed, also in the tail of the mouse. Inject emulsified collagen next to one immunization point, each 100 ul.
  • mice were observed every 2 days from the 22nd day, and the standard four-point method was used for the score and the thickness of the sole of the foot, and the experimental results were recorded and analyzed.
  • No abnormality 1 Have a toe redness 2 Have two or more toes red and swollen 3 The entire sole of the foot is swollen 4 The entire sole of the foot is red and swollen and the joints are stiff.
  • FIG. 7 is a typical therapeutic effect diagram. From the figure, it can be found that the compounds of the present invention are administered WH-007, WH-009, WH-015, WH-023. After treatment with WH-025, WH-030, WH-036, WH-037 and WH-053 for a period of time, the symptoms of arthritis in mice were greatly alleviated, as evidenced by the score in Figure 7B, illustrating the present invention. The compounds have the potential to significantly treat arthritis.
  • the compound of the present invention has been evaluated for therapeutic effects in other autoimmune diseases, and as a result, it has been found that the compound of the present invention is in an autoimmune disease such as lupus erythematosus, multiple sclerosis, arthritis, dermatomyositis, chronic thyroiditis, Juvenile diabetes, aplastic anemia, atrophic gastritis, non-specific ulcerative colitis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune hemolysis Both anemia and idiopathic leukopenia have obvious therapeutic effects.
  • an autoimmune disease such as lupus erythematosus, multiple sclerosis, arthritis, dermatomyositis, chronic thyroiditis, Juvenile diabetes, aplastic anemia, atrophic gastritis, non-specific ulcerative colitis, autoimmune glomerulonephritis,
  • Example 9 The compound of the present invention containing a terminal piperazine and a morpholine functional group plays a key role in binding to the BCL6 protein.
  • the compounds of the present invention containing terminal piperazines and morpholine functional groups are bound in the BCL-6 BTB domain for docking.
  • the BCL-6 BTB domain structure is derived from the RCSB protein database (PDB number 1R2B).
  • PDB number 1R2B RCSB protein database
  • the BCL-6 BTB region is crystallized in the form of a dimer, while a small peptide of SMRT protein (residue number from S1424 to I1425) is also present in the crystal.
  • the SWISS ⁇ MODEL repository was used to complement the BCL ⁇ 6 BTB structure, and then the Gromocs program was used to hydrogenate and minimize the energy of the protein using the amber force field.
  • the resulting protein serves as a molecular docking protein structure file.
  • the terminal piperazine and morpholine compound molecules used in the present invention were optimized using the quantum chemical calculation software Gaussion 03.
  • the optimization application of the density functional B3LYP method is optimized at the 6-31G(d) basis set level.
  • the rotatable bond and twist angle of the optimized drug molecule were determined according to AutoDock 4.0, and then subjected to charge treatment to obtain a small molecular structure file for molecular docking.
  • the invention uses the molecular docking software AutoDock4.0 to obtain the binding information of the terminal piperazine and morpholine molecules and the BCL-6 BTB domain.
  • the protein and small molecule structure files and parameter files required for molecular docking are automatically generated by the program.
  • the free energy function and the Lamarckian genetic algorithm are chosen for the calculation.
  • the binding region of the SMRT small peptide in the BCL-6 BTB domain in the crystal structure is used as a binding pocket, and the protein side chain and the small molecule are kept flexible for docking, and each molecule performs 500 independent docking operations. The combination of the highest scores was selected for analysis.
  • the piperazine-based functional group plays a key role in the binding of the compound of the present invention WH-006 to the BTB region of the BCL-6 protein: the compound of the present invention containing a terminal piperazine-based functional group can be located at the BCL- 6
  • the 14-position histidine (H14) in the BTB region forms a stable hydrogen bond, which is a polar hydrogen on the piperazine-based functional group as a hydrogen donor and a nitrogen atom on the H14 side chain imidazole ring without hydrogen. Hydrogen bonds are formed to stabilize the binding of the compound molecules to the BCL-6 molecule.
  • the presence of this hydrogen bond plays a key role in the specific binding of the compound to the protein.
  • Other terminal piperazine-containing functional compounds such as WH-007 to WH-053 in the present invention have similar binding modes.
  • the terminal morpholine-containing functional group plays a key role in the binding of the compound of the present invention WH-005 to the BTB region of the BCL-6 protein: the compound of the present invention containing a terminal morpholine functional group can be combined with BCL.
  • the H14 residue in the BTB region of the -6 protein forms a stable hydrogen bond.
  • the specific mode of action is that the morpholine functional group oxygen atom acts as a hydrogen acceptor and a polar hydrogen on the nitrogen atom of the H14 side chain imidazole ring.
  • the stabilized morpholine molecule binds to the BCL-6 molecule. The presence of this hydrogen bond plays a key role in the specific binding of the compound to the protein.
  • the compound WH-002 was prepared by substituting ethylamine for isobutylamine according to the method for preparing compound WH-001.
  • 1 H NMR 500 MHz, DMSO
  • the compound WH-003 was prepared by substituting ethylamine for ethanolamine and preparing the compound WH-001.
  • 1 H NMR 500 MHz, DMSO
  • the compound WH-004 was prepared by substituting ethylamine for propanolamine according to the procedure for preparing compound WH-001.
  • 1 H NMR 500 MHz, DMSO
  • the compound WH-008 was prepared by replacing 6-bromo-2-picolinic acid with 3-bromobenzoic acid according to the procedure for preparing compound WH-007.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-009 was prepared by replacing 6-bromo-2-picolinic acid with 3-bromobenzoic acid according to the procedure for preparing compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-010 was prepared by the method of the preparation of the compound WH-006.
  • NMR 500 MHz, DMSO
  • the compound WH-011 was prepared by replacing 4-nitroaniline with 3-fluoro-4-nitroaniline, replacing 6-bromo-2-picolinic acid with 3-bromobenzoic acid, and preparing compound WH-007.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 9.59 s, 1H
  • 8.08 s, 1H
  • Example 1-16 N 2 -((3S,5R)-3,5-dimethylpiperazinyl)-N 4 -((3-methoxy-4-yl (3-bromobenzamide)) Preparation of phenyl)-6-methyl- 2,4-pyrimidinediamine (WH-016)
  • the compound WH-018 was prepared by the method of preparing the compound WH-006 by substituting 6-bromo-2-pyridinecarboxylic acid to thiophene-2-carboxylic acid.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 9.51 s, 1H
  • 9.36 s, 1H
  • 7.98 s, 1H
  • 7.50 (d, J 8.6 Hz, 2H)
  • 3.83 s, 3H
  • the compound WH-019 was prepared by substituting 6-bromo-2-picolinic acid to 5-bromothiophene-2-carboxylic acid according to the procedure for preparing compound WH-006.
  • 1 H NMR 500 MHz, DMSO
  • the compound WH-022 was prepared by substituting 6-bromo-2-pyridinic acid for p-chloroforman-2-carboxylic acid and preparing the compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 9.43 s, 1H
  • 9.30 s, 1H
  • 8.09 s, 1H
  • 7.68-7.58 m, 2H
  • 6.75 s, 1H
  • the compound WH-024 was prepared by substituting phenylboronic acid to 2-methoxyphenylboronic acid according to the method for preparing compound WH-023.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-026 was prepared by substituting phenylboronic acid to 3-pyridineboronic acid by the method of preparing compound WH-023.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-028 was prepared by substituting 6-bromo-2-picolinic acid for p-5-bromofuran-2-carboxylic acid to give the compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-029 was prepared by substituting 6-bromo-2-picolinic acid into p--3-bromofuran-2-carboxylic acid to prepare the compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 9.44 s, 1H
  • 9.10 s, 1H
  • 8.01 s, 1H
  • 7.62 s, 1H
  • 7.33 7.8 Hz, 1H
  • the compound WH-030 was prepared by substituting phenylboronic acid into 3-furan boronic acid according to the procedure for preparing compound WH-023.
  • the compound WH-031 was prepared by the method of preparing the compound WH-006 by substituting 6-bromo-2-picolinic acid for p-benzothiophene-2-carboxylic acid.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-032 was prepared by the method of preparing the compound WH-006 by substituting 6-bromo-2-pyridinic acid into p-benzofuran-2-carboxylic acid.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-033 was prepared by the method of preparing the compound WH-006 by substituting 6-bromo-2-picolinic acid into p-oxo-2-carboxylic acid.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-034 was prepared by substituting 6-bromo-2-pyridinic acid into p-chloroindole-2-carboxylic acid to prepare the compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • Example 1-35 N 2 ⁇ ((3S,5R) ⁇ 3,5-dimethylpiperazinyl)-N 4 ⁇ ((3 ⁇ methoxy-4-4 ⁇ (5 ⁇ fluoro ⁇ 2 ⁇ ) Preparation of Amido))phenyl)-5-fluoro-2,4-pyrimidinediamine (WH-035)
  • the compound WH-035 was prepared by substituting 6-bromo-2-pyridinic acid into p-fluoroindol-2-carboxylic acid to prepare the compound WH-006.
  • 1 H NMR 500MHz, DMSO
  • the compound WH-037 was prepared by the procedure of the compound WH-036 by substituting 5-bromo-2-thiophene bromide bromide to 5-nitro-2-furanyl bromide.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 8.84 s, 1H
  • 7.10-7.09 m, 1H
  • the compound WH-038 was prepared by substituting 5-bromo-2-thiophene bromide bromide to 5-nitro-2-thiophene bromide bromide according to the procedure for preparing compound WH-036.
  • 1 H NMR 500 MHz, DMSO
  • 8.88 (s, 1H) 8.88 (s, 1H)
  • the compound WH-039 was prepared by the method of preparing the compound WH-006 by substituting 3-methoxy-4-nitroaniline for 3-nitro-4-methoxybenzene.
  • 1 H NMR 500MHz, DMSO
  • ⁇ 10.35 s, 1H
  • 9.35 s, 1H
  • 9.14 s, 1H
  • 8.92 s, 1H
  • 8.07 (d, J 8.3 Hz, 1H)
  • 8.04 s, 1H
  • Example 1-44 N 2 ⁇ ((3S,5R) ⁇ 3,5-Dimethylpiperazinyl)-N 4 ⁇ ((3 ⁇ (3 ⁇ bromo-2-phenylphenamido)) ⁇ 4 ⁇ Preparation of methoxyphenyl)-5-fluoro- 2,4-pyrimidinediamine (WH-044)

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Abstract

本发明公开了一类4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其结构如式(I)‐(VI)所示。本发明还公开了所述4‐嘧啶二胺类小分子有机化合物或其药物组合物在制备预防和/或治疗各种恶性肿瘤、自身免疫性疾病、炎症等药物中的用途，作为B细胞淋巴因子6(BCL6)的抑制剂在制备预防和/或治疗BCL6介导的疾病药物中的用途。

Description

一类4‐嘧啶二胺类小分子有机化合物及其衍生物及其应用 技术领域
本发明涉及一类4‐嘧啶二胺类小分子有机化合物及其衍生物及其用途。所述化合物或含有该类化合物的药物组合物能够用于制备抗肿瘤药物，用于治疗各种恶性肿瘤，包括肿瘤转移等相关疾病，作为B细胞淋巴因子6(BCL6)抑制剂能够用于预防和/或治疗BCL6介导的疾病(如自身免疫性疾病、过敏和炎症)。
背景技术
癌症是一种不受有机体控制的能够无限增殖的恶性肿瘤细胞的统称。当前社会，癌症已经成为除了心血管疾病之外人类的第二大致死病因。根据世界卫生组织(WHO)最新发布的《全球癌症报告2014》称，目前全球癌症患者数目正成快速增长态势，将会由2012年的1400万人，增加到2035年的2400万人。报告还显示，全球2012年新增癌症患者中有一半来自亚洲，其中中国占据了大部分，中国俨然成为了癌症的高发区。
现有的治疗癌症的常规疗法包括：手术治疗、放射治疗、化疗等。到目前为止手术治疗仍然是治疗癌症的最有效的手段，然而手术治疗只适用于早期的癌症患者，对于复发的和转移性的恶性肿瘤，手术治疗则不能达到治疗的目的。放射治疗是使用辐射线杀死肿瘤细胞，然而放疗在杀死肿瘤细胞的同时也会杀死正常细胞。化疗可以分为传统的化疗药物治疗和靶向药物治疗，其中传统的化疗药物因为没有选择性，而常常具有很大的副作用。靶向药物治疗往往是通过特异性的靶向肿瘤细胞的致癌位点，特异性的干预肿瘤细胞的某些信号通路，从而达到治疗癌症的目的，靶向治疗因为能够特异性的杀死肿瘤细胞，因此与传统的化疗相比，靶向治疗的毒副作用更小，广泛受到人们的关注。
原癌基因B细胞淋巴因子6(B-cell lymphoma 6,BCL6)是一种转录抑制因子，在生物体内BCL6与生发中心的形成有十分重要的关系。BCL6是BTB/POZ/锌指核酶家族中的一员，由706个氨基酸组成。根据结构和功能可将BCL6分为三个部分：N端的BTB/POZ结构域(1-130号氨基酸残基)、预测具有很少或没有固定结构的中心区域(129-517号氨基酸残基)、C端的六个锌指结构域(518-681号氨基酸残基)。其中N端的BTB/POZ结构域可以以相互排斥的方式与三种已知的辅助抑制子：SMRT、N-CoR和BCOR相互作用，从而发挥转录抑制作用。 被BCL6抑制的基因涉及细胞的活化、分化、凋亡、细胞周期阻滞等方面，如：BCL6可以抑制PRDM1、cyclinD2、p53等基因的表达，这些基因是浆细胞分化、细胞周期调控、凋亡、DNA修复和维持基因组稳定性的重要基因。因此，BCL6的正常下调如果被阻断，细胞易发生分化阻滞、持续增殖、长期生存、基因不稳定，最终导致恶变。它的功能是阻止细胞分化和淍亡，促进细胞的发育增殖。BCL6与多种肿瘤的发生、发展有十分重要的关系。
非霍奇金淋巴瘤(Non-Hodgkin's lymphoma,NHL)是一类高发的恶性淋巴瘤，在常见肿瘤中排名第6位，依据细胞来源将其分为三种基本类型：B细胞、T细胞和NK/T细胞NHL。临床大多数NHL为B细胞型，占总数70％～85％，其中弥漫性大B细胞淋巴瘤(Diffuse Large B-Cell Lymphoma,DLBCL)是最常见的B细胞型NHL，约占全部NHL的40％。根据基因表达谱的不同，可将DLBCL分为生发中心B细胞样(GCB-like)型、活化B细胞样(ABC-like)型、原发性纵隔B细胞样(PMBL)型。目前，治疗DLBCL的一线疗法是R-CHOP(美罗华+环磷酰胺+阿霉素+长春新碱+强的松)疗法，经过化疗后有>50％的患者可以被治愈。但仍然有1/3以上患者经治疗后复发或难治疗。因此开发一种新的治疗DLBCL耐药的药物十分重要(NATURE REVIEWS|CLINICAL ONCOLOGY VOLUME 11|JANUARY 2014)。
据文献报道，BCL6在GCB-DLBCL的表达量高达86％，在ABC-DLBCL的表达量为34％，DLBCL的耐药可能与BCL6的高表达有关，R-CHOP能够选择性的杀死BCL6阴性的DLBCL，而对BCL6阳性的DLBCL几乎没有作用。不仅如此，BCL6在滤泡性淋巴瘤(follicular lymphomas,FLs)等中也高表达。BCL6是治疗DLBCL和FLs的十分重要的靶点。除此之外，研究者发现，BCL6在自身免疫性疾病方面也发挥着十分重要的作用。BCL6是生发中心(GC)的重要调控因子，BCL6与生发中心异常密切相关。已有报道证实，生发中心样(GC-like)结构存在于自身免疫性疾病如系统性红斑狼疮(SLE)、类风湿性关节炎(RA)以及1型糖尿病等患者的唾液腺、脑膜、滑膜、甲状腺、皮肤、胰腺及肾脏等多种组织病灶中(Aloisi F et al./Nat Rev Immunol.2006 Mar；6(3):205-17)。人重症肌无力症(Myasthenia gravis，MG)是一种T细胞依赖性抗体介导的器官特异性自身免疫性疾病。到目前为止还没有选择性靶向MG的特异性免疫抑制剂，使用皮质类固醇 和其它非特异性免疫抑制药物，会带来与全球非特异性抑制免疫应答相关的潜在副作用和风险，包括感染和恶性肿瘤，因此开发MG特异性的免疫抑制剂具有十分重要的意义。有文章报道，在实验性自身免疫性重症肌无力(Experimental autoimmune myasthenia gravis，EAMG)的动物模型中BCL6和IL-21的表达明显提高，用siRNA干扰沉默掉BCL6的表达后，能够明显缓解EAMG的严重程度，提示，BCL6可以作为MG的治疗靶标，不仅如此，文章中还提到，BCL6不仅可以作为治疗MG的靶标，同时可以作为其它抗体介导的自身免疫性疾病的治疗靶标，如系统性红斑狼疮、类风湿性关节炎等(N.Xin et al./Molecular and Cellular Neuroscience 58(2014)85–94)。
BCL6多肽类的抑制剂(BCL6 peptide inhibitor,BPI)能够通过与共抑制因子SMRT等竞争性的结合到BCL6的N端BTB/POZ结构域从而重新激活BCL6的靶基因，并且能在体内外杀死BCL6高表达的DLBCL，但是BPI具有口服生物利用率低，花费大等问题，因此限制了它在临床上的进一步应用。79-6是第一个被文献报道的BCL6小分子抑制剂，但它与BCL6的亲和力很差(IC50＝212μM)(Cancer Cell 17,400–411,April 13,2010)。FX1是在79-6的基础上改造而得，它的活性与79-6相比有很大提高，IC 50约为36μM(The Journal of Clinical Investigation,June 3,2016.)。但是，FX1仍然存在较大劣势，FX1对肿瘤细胞增殖抑制活性仍然较差，由于这个劣势限制了它们在临床上的进一步研发。
发明内容
本发明以二氨基嘧啶为基本的结构骨架，经过结构改造成一类新型的4‐嘧啶二胺类小分子有机化合物。在分子水平上，本发明化合物能够通过竞争性的方式结合到BCL6的N端的BTB/POZ结构域，从而使BCL6下游的靶基因重新激活。在细胞水平上，本发明化合物能够强烈的抑制DLBCL的增殖，促进DLBCL的凋亡，其抗增殖活性达到1μM以下。本发明化合物与已报到的BCL6小分子抑制剂79-6和FX1比较，其活性得到极大提高，结构完全不同，可作为BCL6的先导化合物和潜在的临床候选药物。
本发明提供了一类结构新颖的可作为BCL6抑制剂和抗肿瘤药物的4‐嘧啶二氨基类小分子有机化合物及其相关类似物，包括其可用盐、以及酯等。
本发明提供的4‐嘧啶二氨基类小分子有机化合物或相关类似物或药学上可 接受的盐，其结构如(I)所示：
Figure PCTCN2018088932-appb-000001
其中：
R 1和R 2选自下列基团中的一个或多个：氢(其中，R 1和R 2不同时为氢)、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
Figure PCTCN2018088932-appb-000002
Figure PCTCN2018088932-appb-000003
其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000004
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
R 3选自下列基团中的一个：C 1‐C 5的链状醇氨基、C 1‐C 5的脂肪链状氨基、C 3‐C 6环氨基、四氢吡咯基、哌嗪基、C 1‐C 3烷基哌嗪基、哌啶基、C 1‐C 3烷基哌啶基、吗啉基、C 1‐C 3烷基吗啉基、硫代吗啉基、C 1‐C 3烷基硫代吗啉基。
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
本发明所述式(I)中，当R 3为哌嗪基或者2,6‐二烷基哌嗪时，所述4‐嘧啶二氨基类小分子有机化合物结构如下式(II)所示：
Figure PCTCN2018088932-appb-000005
其中：
R 1和R 2选自下列基团中的一个或多个：氢(其中R 1和R 2不同时为氢)、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
Figure PCTCN2018088932-appb-000006
其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000007
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基。
本发明所述式(I)中，当R 3为哌嗪或者2,6‐二烷基哌嗪，R 2为C 1‐C 3烷氧基或C 1‐C 3烷胺基时，所述4‐嘧啶二氨基类小分子有机化合物结构如下式(III)所示：
Figure PCTCN2018088932-appb-000008
其中：
X为O或者N；
R 1选自下列基团中的一个或多个：氢(其中R 1和R 2不同时为氢)、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
Figure PCTCN2018088932-appb-000009
其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000010
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基。
R 7为C 1‐C 3烷基。
本发明中所述结构式(III)中，当R 1为
Figure PCTCN2018088932-appb-000011
时，所述4‐嘧啶二氨基类小分子有机化合物结构如下式(IV)所示：
Figure PCTCN2018088932-appb-000012
其中：
X为O或者N；
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基。
R 7为C 1‐C 3烷基。
R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000013
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
本发明所述式(IV)中，当X为氧原子时，其结构如式(V)所示：
Figure PCTCN2018088932-appb-000014
其中：
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基。
R 7为C 1‐C 3烷基。
R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000015
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
本发明中所述结构式(V)中，当X为氧原子时，R 5和R 6为顺势甲基时，其结构如下式(VI)所示：
Figure PCTCN2018088932-appb-000016
其中：
R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
R 7为C 1‐C 3烷基。
R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基。
Figure PCTCN2018088932-appb-000017
选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
本发明还提供了所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，所述双取代嘧啶二胺类小分子有机化合物与酸形成酸加成盐；其中，所述酸包括但不限于是：盐酸、氢溴酸、硫酸、磷酸、乙酸、酒石酸、水杨酸、柠檬酸、甲磺酸、对甲苯磺酸、乳酸、丙酮酸、马来酸、琥珀酸。
本发明中所述4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其中，所述4‐嘧啶二胺类小分子有机化合物可以与放射性基团、荧光基团或者生物素结合形成标记物。
本发明提供的4‐嘧啶胺类小分子有机化合物或其相关类似物或药学上可接 受的盐，包括：
N 2‐乙氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐异丁氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐乙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐丙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐吗啉基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(6‐溴‐2‐吡啶酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(6‐溴‐2‐吡啶酰胺基)苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(对氯苯酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐氟‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲氧基‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氯‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲基‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐氯‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐甲基‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐三 氟甲基‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(4‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐甲氧基)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐氯)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐吡啶基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐噻吩基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐呋喃基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氟‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐呋喃乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氨基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(4‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(3‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐呋喃酰胺基))‐4‐甲氧基苯基)‐5‐ 氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氯‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐苯并噻吩酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐恶唑酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐嘧啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(5‐溴‐3‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(4‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(6‐溴‐4‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
本发明还提供了一种药物组合物，其中药物组合物含有所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐，以及药学上可接受的载体。所述药物组合物被配制成可注射流体、气雾剂、乳膏、凝胶剂、丸剂、胶囊剂、糖浆剂、透皮贴剂或赋形剂。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在作为BCL6抑制剂中的应用。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在抑制BCL6蛋白与多肽SMRT的相互作用中的应用。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在抑制生发中心的形成中的应用。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学 上可接受的盐或药物组合物在制备预防和/或治疗BCL6介导的疾病的药物中的应用；其中，所述疾病包括但不限于自身免疫性疾病、过敏或炎症；其中，所述自身免疫性疾病包括红斑狼疮、多发性硬化症、关节炎、皮肌炎、慢性甲状炎，青少年型糖尿病、恶习性贫血、萎缩性胃炎、非特异性溃疡性结肠炎、自身免疫性肾小球肾炎、肺肾出血性综合症、特发性血小板减少性紫癜、重症肌无力、自身免疫性溶血性贫血和特发性白细胞减少症。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在制备抗肿瘤药物中的应用；本发明所述4‐嘧啶二胺类小分子有机化合物、水合物或药学上可接受的盐或药物组合物可以抑制肿瘤细胞的增殖、生长、迁移和浸润。其中，所述肿瘤细胞包括弥漫性大B细胞淋巴瘤(如，人淋巴瘤细胞SUDHL4)、滤泡性淋巴瘤、伯基特淋巴瘤、非小细胞肺癌细胞、乳腺癌细胞、B细胞急性淋巴细胞白血病、慢性骨髓性白血病、食管癌细胞，卵巢癌细胞，头颈部鳞状细胞癌细胞、结肠癌细胞、前列腺癌细胞、肝癌细胞。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在制备预防和/或治疗恶性肿瘤的药物中的应用；所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物用于抑制恶性肿瘤的增殖、生长、浸润、转移、复发。其中，所述恶性肿瘤包括弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、伯基特淋巴瘤、非小细胞肺癌、乳腺癌、B细胞急性淋巴细胞白血病、慢性骨髓性白血病、食管癌，卵巢癌，头颈部鳞状细胞癌、结肠癌、前列腺癌、肝癌。
本发明提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在上调BCL6下游基因CXCR4，CD69，Caspase8和DUSP5表达中的应用；优选地，所述4‐嘧啶二胺类小分子有机化合物为N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(6‐溴‐2‐吡啶酰胺基)苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-007)，N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-009)，N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-023)和N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐氯)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶 二胺(WH-025)。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在BCL6阳性细胞株和BCL6阴性细胞中BCL6上调BCL6下游基因CXCR4，CD69，CASPASE8和DUSP5表达中的应用；优选地，所述BCL6阳性细胞株为SUDHL4，所述BCL6阴性细胞株为TOLEDO；优选地，所述4‐嘧啶二胺类小分子有机化合物为所述4‐嘧啶二胺类小分子有机化合物为N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(6‐溴‐2‐吡啶酰胺基)苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-007)，N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-009)，N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-023)和N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐氯)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-025)。
本发明还提供了所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物在抑制LPS刺激的TNFα的表达中的应用。
本发明中，所述4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物可以单独使用或与其它药物联合使用。
本发明的有益效果在于，本发明提供的式(I)～(VI)所示的4‐嘧啶二胺类小分子有机化合物或相关类似物或药学上可接受的盐或药物组合物能够显著抑制BCL6片段蛋白与多肽SMRT的相互作用，抑制弥漫大B细胞淋巴瘤的增殖(人淋巴瘤细胞SUDHL4)，抑制生发中心的形成，抑制LPS刺激的TNFα的表达，能够用于预防和/或治疗自身免疫性疾病、过敏或炎症，抑制恶性肿瘤的增殖、生长、迁移和浸润，具有广泛的应用前景。
附图说明
图1为举例说明本发明部分化合物与BCL6蛋白结合效果检测的效果图。其中，横坐标表示时间(秒)，纵坐标表示反射率单位。
图2为本发明化合物对BCL6下游基因上调的效果图。其中图A、B、C和D分别为本发明化合物对基因CD69、CXCR4、CASPASE8和DUSP5的上调效果图。
图3A为人弥漫大B细胞株SUDHL4和TOLEDO中BCL6蛋白表达水平的 示意图。图3B为本发明化合物WH-007和WH-023在细胞株SUDHL4和TOLEDO中对BCL6下游基因CD69和CXCR4的上调效果图。
图4为本发明化合物WH-007抑制小鼠生发中心形成的效果图。其中A图为小鼠脾脏重量的统计图(其中，纵坐标表示脾脏重量(g))，B图为本发明化合物WH-007抑制小鼠生发中心形成的流式图，C图为本发明化合物WH-007在50mg/kg/d的灌胃剂量下抑制小鼠生发中心形成的统计图。
图5为本发明化合物WH-023抑制小鼠生发中心形成的效果图。其中A图为小鼠脾脏重量的统计图(其中，纵坐标表示脾脏重量(g)，B图为本发明化合物WH-023抑制小鼠生发中心形成的流式图，C图为本发明化合物WH-023在50mg/kg/d的灌胃剂量下抑制小鼠生发中心形成的统计图(图中上方为生发中心B细胞比例。
图6为本发明化合物抑制LPS刺激的TNFα的表达。
图7为本发明化合物对关节炎具有显著的治疗效果；其中图A为本发明化合物治疗关节炎的典型效果图，图B为关节炎治疗效果统计评分图。
其中，图中，*表示p<0.05，**表示p<0.01，***表示p<0.001。
图8为本发明含有末端哌嗪类官能团和吗啉类官能团的化合物可与BCL6蛋白的BTB区域H14残基形成稳定的氢键作用；其中A图为哌嗪类化合物形成氢键的效果图，B图为吗啉类化合物形成氢键的效果图。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。
1H-NMR用Bruker 500MHz型仪测定；所有溶剂在使用前均经过重新蒸馏，所使用的无水溶剂均是按标准方法干燥处理获得；除说明外，所有反应均是在氮气保护下进行并用TLC跟踪，后处理时均经饱和食盐水洗和无水硫酸钠干燥过程；产品的纯化除说明外均使用硅胶(200-300目)的柱色谱法；所使用的硅胶，包括200-300目和GF254为青岛海洋化工厂或烟台缘博硅胶公司生产。
实施例1：4‐嘧啶二胺类小分子有机化合物的制备
实施例1‐01、N 2‐乙氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐001)的制备
取3‐氯‐4‐甲氧基苯胺(1.567g,10.0mmol)于无水乙醇(30mL)中，加入5‐氟‐2,4‐二氯嘧啶(1.837g,11.0mmol)和5.2mL的DIEA，升温至40℃搅拌过夜。后处理：减压出去大部分的有机溶剂，用二氯甲烷和水萃取三次，将有机相合并后用无水硫酸钠干燥，减压除去二氯甲烷，通过柱层析得到中间体2‐氯‐5‐氟‐N‐(3‐氯‐4‐甲氧基苯基)嘧啶‐4‐胺(2.707g,94％)。
将2‐氯‐5‐氟‐N‐(3‐氯‐4‐甲氧基苯基)嘧啶‐4‐胺(144.06mg,0.5mmol)于5mL的正丁醇中，加入2mL的乙胺和0.26mL的DIEA，升温至120℃搅拌过夜。后处理：减压出去大部分的有机溶剂，用二氯甲烷和水萃取三次，将有机相合并后用无水硫酸钠干燥，减压除去二氯甲烷，通过柱层析得到产物WH‐001(115mg)。 1H NMR(500MHz,DMSO)δ9.10(s,1H),8.06(s,1H),7.91(d,J＝3.9Hz,1H),7.69–7.68(m,1H),7.09(d,J＝9.0Hz,1H),6.75(s,1H),3.83(s,3H),3.26–3.19(m,2H),1.11(t,J＝7.1Hz,3H).
实施例1‐06、化合物N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(6‐溴‐2‐吡啶酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐006)的制备
取3‐甲氧基‐4‐硝基苯胺(841.0mg,5.0mmol)于无水DMF(5mL)中，加入5‐氟‐2,4‐二氯嘧啶(1.25g,7.5mmol)和碳酸铯(4.89g,15.0mmol)常温下反应过夜。后处理：用二氯甲烷和水萃取三次，将有机相合并后用无水硫酸钠干燥，减压除去二氯甲烷，通过柱层析得到中间体2‐氯‐5‐氟‐N‐(3‐甲氧基‐4‐硝基苯基)嘧啶‐4‐胺(944mg,63％)。
将2‐氯‐5‐氟‐N‐(3‐甲氧基‐4‐硝基苯基)嘧啶‐4‐胺(944.0mg,3.16mmol)溶于15mL的正丁醇中，在体系中加入顺式‐2,6‐二甲基哌嗪(1.08g,9.48mmol)和DIEA(1.63mL,9.48mmol)加热至120℃回流过夜。后处理：减压除去大部分溶剂，常规处理后柱层析得到中间体2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(3‐甲氧基‐4‐硝基苯基)嘧啶‐4‐胺(786mg,66％)。
将2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(3‐甲氧基‐4‐硝基苯基)嘧啶‐4‐胺 (786.0mg,2.09mmol)溶于50mL无水甲醇中密封，在氮气保护下加入35mg钯/炭，将体系全部置换成氢气，反应过夜。后处理：减压除去溶剂，得到粗产物2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(3‐甲氧基‐4‐氨基苯基)嘧啶‐4‐胺直接用于下一步反应。
将6‐溴‐2‐吡啶甲酸(120.0mg,0.6mmol)，溶于1.5mL的DMF中，加入EDC·HCl(118mg)、HOBt(112mg)常温下搅拌10min后加入2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(3‐甲氧基‐4‐氨基苯基)嘧啶‐4‐胺(173.2mg,0.5mmol)，搅拌过夜。后处理：用二氯甲烷和水萃取三次，将有机相合并后加入无水硫酸钠干燥，减压除去二氯甲烷，柱层析得到WH‐006(192mg)。 1H NMR(500MHz,DMSO)δ10.06(s,1H),9.44(s,1H),8.29(d,J＝8.8Hz,1H),8.18(d,J＝7.5Hz,1H),8.09(d,J＝3.5Hz,1H),8.04(t,J＝7.7Hz,1H),7.96(d,J＝7.9Hz,1H),7.68(d,J＝2.1Hz,1H),7.35–7.34(m,1H),4.58(d,J＝13.4Hz,2H),3.95(s,3H),3.22–3.21(m,2H),2.86–2.81(m,2H),1.26(d,J＝6.4Hz,6H).
实施例1-07、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(6‐溴‐2‐吡啶酰胺基)苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐007)的制备
取4‐硝基苯胺(276.26mg,2.0mmol)于无水DMF(2mL)中，加入5‐氟‐2,4‐二氯嘧啶(400.7mg,2.4mmol)和碳酸铯(2.0g,6.0mmol)在常温下反应过夜。后处理：用二氯甲烷和水萃取三次，将有机相合并后加入无水硫酸钠干燥，减压除去二氯甲烷，柱层析得到中间体2‐氯‐5‐氟‐N‐(4‐硝基苯基)嘧啶‐4‐胺(227mg,42.3％)。
将2‐氯‐5‐氟‐N‐(4‐硝基苯基)嘧啶‐4‐胺(227mg,0.85mmol)溶于5mL正丁醇中，加入顺式‐2,6‐二甲基哌嗪(289mg,2.54mmol)和DIEA(0.44mL,2.54mmol)加热至120℃回流过夜。后处理：减压除去大部分溶剂，常规处理后，柱层析得到中间体2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐硝基苯基)嘧啶‐4‐胺(173mg,59％)。
将2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐硝基苯基)嘧啶‐4‐胺(173mg,0.5mmol)于20mL无水甲醇中之后密封，在氮气保护下加入20mg钯/炭，将体系全部置换成氢气反应过夜。后处理：减压除去溶剂，得到粗产物2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐氨基苯基)嘧啶‐4‐胺直接用于下一步反应。
将3‐溴‐2‐吡啶苯甲酸(114.7mg,0.57mmol)溶于1.5mL的DMF中，加入EDC·HCl(118mg)、HOBt(112mg)常温下搅拌10min后加入2-((3S,5R)-3,5- 二甲基哌基)‐5‐氟‐N‐(4‐氨基苯基)嘧啶‐4‐胺(151.0mg,0.47mmol)，搅拌过夜，用二氯甲烷和水萃取三次，将有机相合并后加入无水硫酸钠干燥，减压除去二氯甲烷，柱层析得到产物WH‐007(119mg)。1H NMR(500MHz,DMSO)δ10.37(s,1H),9.27(s,1H),8.14(d,J＝7.4Hz,1H),8.01(d,J＝7.7Hz,1H),7.99(d,J＝2.5Hz,1H),7.92(d,J＝7.9Hz,1H),7.82(d,J＝8.8Hz,2H),7.73(d,J＝8.9Hz,2H),4.35(d,J＝12.5Hz,2H),2.68–2.64(m,2H),2.33–2.38(m,2H),1.00(d,J＝6.1Hz,6H).
实施例1‐23、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐023)的制备
取一个干燥的圆底烧瓶将其密封后用氮气保护，加入5‐溴噻吩‐2‐甲酸甲酯(221.07mg,1.0mmol)，Pd(PPh 3) 4(35mg)于2.5mL的DME和5mL Na 2CO 3(2.0mol/L)，体系在氮气保护下常温搅拌10min。在混合物中加入苯硼酸(243.8mg,2.0mmol)和5mL的甲醇，将其密封，在氮气保护下加热至85℃，反应过夜。后处理：减压把溶剂蒸干，用二氯甲烷和水萃取三次，合并水相，将水相调至酸性，抽虑，烘干得到中间体5‐苯基‐2‐噻吩甲酸(178mg)。
将5‐苯基‐2‐噻吩甲酸(118.5mg,0.58mmol)，溶于1.5mL的DMF中，加入EDC·HCl(118mg)、HOBt(112mg)常温下搅拌10min后加入2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(3‐甲氧基‐4‐氨基苯基)嘧啶‐4‐胺(100.0mg,0.29mmol)，搅拌过夜。后处理：用二氯甲烷和水萃取三次，将有机相合并后加入无水硫酸钠干燥，减压除去二氯甲烷，柱层析得到产物WH‐023(74mg)。 1H NMR(500MHz,DMSO)δ9.57(s,1H),9.39(s,1H),8.08(d,J＝2.9Hz,1H),8.00(d,J＝2.4Hz,1H),7.75(d,J＝7.5Hz,2H),7.67(s,1H),7.60(d,J＝3.5Hz,1H),7.53(d,J＝8.5Hz,1H),7.47(t,J＝7.4Hz,2H),7.39(t,J＝7.3Hz,1H),7.30(d,J＝8.6Hz,1H),4.53(d,J＝12.8Hz,2H),3.85(s,3H),3.09–3.08(m,2H),2.74–2.64(m,2H),1.18(d,J＝5.5Hz,6H).
实施例1‐36、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐036)的制备
取5‐溴噻吩‐2‐甲醛(421.0mg,2.2mmol)于50mL干燥的圆底烧瓶中，加入10mL的无水乙醇，在冰浴下加入硼氢化钠(417.0mg,11.01mmol)，之后常温搅拌10min。减压把溶剂蒸干，用二氯甲烷和水萃取三次，合并有机相，加入无水硫 酸钠干燥，减压除去有机溶剂，得到5‐溴噻吩‐2‐甲醇(400mg)。
取5‐溴噻吩‐2‐甲醇(400.0mg,2.07mmol)于100mL的圆底烧瓶中，加入20mL的无水二氯甲烷，在冰浴下滴加三溴化磷(0.59mL,6.22mmol)，滴加完后在常温下搅拌过夜。后处理：减压把溶剂蒸干，用二氯甲烷和水萃取三次，合并有机相，加入无水硫酸钠干燥，减压除去二氯甲烷，得到5‐溴噻吩‐2‐溴甲基(514mg)。
取4‐甲氧基‐3‐硝基苯胺(841.0mg,5.0mmol)于无水DMF(5mL)中，加入5‐氟‐2,4‐二氯嘧啶(1.25g,7.5mmol)和碳酸铯(4.89g,15mmol)常温下反应过夜。用二氯甲烷和水萃取三次，将有机相合并后加入无水硫酸钠干燥，减压除去二氯甲烷，柱层析得到中间体2‐氯‐5‐氟‐N‐(4‐甲氧基‐3‐硝基苯基)嘧啶‐4‐胺(944mg,63％)。
将2‐氯‐5‐氟‐N‐(4‐甲氧基‐3‐硝基苯基)嘧啶‐4‐胺(944.0mg,3.16mmol)溶于15mL正丁醇中，加入顺式‐2,6‐二甲基哌嗪(1.08g,9.48mmol)和DIEA(1.63mL,9.48mmol)加热至120℃回流过夜。后处理：减压除去大部分溶剂，常规处理后柱层析，得到中间体2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐甲氧基‐3‐硝基苯基)嘧啶‐4‐胺(786mg,66％)。
将2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐甲氧基‐3‐硝基苯基)嘧啶‐4‐胺(786.0mg,2.09mmol)溶于50mL无水甲醇中将其密封，体系在氮气保护下加入35mg钯/炭，将体系全部置换成氢气搅拌过夜。后处理：减压除去溶剂，得到粗产物2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐甲氧基‐3‐氨基苯基)嘧啶‐4‐胺直接用于下一步反应。
取5‐溴噻吩‐2‐溴甲基(255.96mg,1.0mmol)于50mL的圆底烧瓶中，加入10mL的无水乙醇，之后加入2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐5‐氟‐N‐(4‐甲氧基‐3‐氨基苯基)嘧啶‐4‐胺(346.4mg,1.0mmol)，常温下搅拌过夜。后处理：减压把溶剂蒸干，用二氯甲烷和水萃取三次，合并有机相，加入无水硫酸钠干燥，减压除去有机溶剂，柱层析得到产物WH‐036(204mg)。 1H NMR(500MHz,DMSO)δ9.23(s,1H),8.81(t,J＝6.0Hz,1H),8.48(d,J＝2.8Hz,1H),7.97(d,J＝3.7Hz,1H),7.67–7.66(m,1H),7.12(d,J＝9.0Hz,1H),7.05(d,J＝3.7Hz,1H),6.85(d,J＝3.7Hz,1H),4.57(d,J＝5.9Hz,2H),4.40–4.39(m,2H),3.88(s,3H),2.68–2.64(m,2H),2.29–2.24(m,2H),1.01(d,J＝6.2Hz,6H).
实施例1‐1到1‐53化合物WH‐001到WH‐053的制备方法如表1所示
Figure PCTCN2018088932-appb-000018
Figure PCTCN2018088932-appb-000019
Figure PCTCN2018088932-appb-000020
Figure PCTCN2018088932-appb-000021
Figure PCTCN2018088932-appb-000022
Figure PCTCN2018088932-appb-000023
Figure PCTCN2018088932-appb-000024
Figure PCTCN2018088932-appb-000025
实施例2：均相时间分辨荧光技术(HTRF)检测本发明化合物抑制BCL6片段蛋白与其共抑制因子SMRT的相互作用
技术方法：
均相时间分辨荧光(HTRF)是结合了荧光共振能量转移(FRET)和时间分辨荧光(TRF)两种技术的一种用来检测纯液相体系中待测物的方法。在这一体系中，本发明选定的能量供体是GST-Tb，受体是6His-XL665(Cisbio)。相应的，本发明表达纯化出的蛋白也分别带有GST和His标签，分别为BCL6-GST和6His-SMRT。BCL6的转录抑制活性结构域BTB domain是和转录共抑制因子SMRT(多肽SMRT)结合的结构域。本发明所需BCL6蛋白N端的BTB/POZ结构域序列为ADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKT-VLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASE-GST。SMRT多肽的序列为6HISGL-VATVKEAGRSIHEIPREEL。HTRF实验需要两个蛋白在某一浓度下结合后的荧光比值相对于不结合时的荧光比值有一个较好的性噪比，根据研究发现，本发明选定的6His-SMRT浓度为200nM，BCL6-GST的浓度为6.25nM。实验为20μL的体系，首先于384孔板中每孔加入4μL的5×BCL6-GST和4μL的5×6His-SMRT室温孵育30min，各组设2个复孔。之后加入2μL的10×不同浓度化合物，最后加入GST-Tb和6His-XL665各5μL，室温孵育过夜，第二天于Cytation 5细胞成像微孔板检测仪上读值。实验结果用GraphPad Prism处理，实验独立重复3次。
实验结果如表2所示，本发明化合物能抑制BCL6片段蛋白与多肽SMRT的相互作用，其中，所有本发明化合物(WH‐001至WH‐053)均能有效抑制片段蛋白与多肽SMRT的相互作用，半数抑制浓度(IC 50)均在20μM以下，如化合物WH‐001～WH005，WH017、WH018、WH-026，WH-028，WH-032，WH-035，WH-037至WH-046，WH-051至WH-053的IC 50在10‐20μM之间；化合物WH-010至WH016、WH-019至WH-022，WH-036，WH-047和WH-050对BCL6片段蛋白与SMRT相互作用的半数抑制浓度(IC 50)在5‐10μM之间；化合物WH-006至 WH-009，WH-023至WH-025，WH-027，WH-030，WH-031和WH-033对BCL6片段蛋白与SMRT相互作用的半数抑制浓度(IC 50)在5μM以下。
表2为本发明化合物抑制BCL6片段蛋白与其共抑制因子SMRT的相互作用的IC 50值
Figure PCTCN2018088932-appb-000026
实施例3：本发明化合物抑制弥漫大B细胞淋巴瘤的增殖
技术方法：
1.细胞的培养
本发明中所用人淋巴瘤细胞SUDHL4(人弥漫大B细胞淋巴瘤细胞SUDHL4)来自上海市调控生物学重点实验室郑彪教授实验室。细胞培养于37℃ 恒温培养箱(湿度95％，CO 2浓度5％)中，其中SUDHL4培养基为含20％胎牛血清(Gemini)的RPMI-1640(Gibco)。
2.细胞增殖实验
用CCK8法测定细胞增殖。人淋巴瘤细胞株SUDHL4以2×10 4个/孔的密度接种于96孔板(90μL)，24h后加入10μL不同浓度化合物，各组设3个复孔。继续培养48h后，避光加入10μL的CCK8于37℃孵育1-4小时，用酶标仪检测450nm处的吸光度值。实验独立重复3次。细胞存活率(％)＝加药物OD值/对照组OD值×100％。
实验结果如表3所示，本发明化合物WH‐001至WH053对人弥漫大B细胞淋巴瘤细胞SUDHL4增殖的抑制的半数有效抑制浓度(IC 50)都在10μM以下，说明本发明化合物对淋巴瘤细胞增殖具有明显的抑制效果。另外，化合物WH-023，WH-024，WH-027，WH-029，WH-030，WH-031，WH-033，WH-034和WH-035对细胞SUDHL4增殖的半数抑制浓度IC 50均在1μM以下，显示出强烈的抗肿瘤细胞增殖效果。
在相似的抑制实验还发现，本发明化合物WH‐001至WH053对滤泡性淋巴瘤、伯基特淋巴瘤、非小细胞肺癌、乳腺癌、B细胞急性淋巴细胞白血病、慢性骨髓性白血病、食管癌，卵巢癌，头颈部鳞状细胞癌、结肠癌、前列腺癌和肝癌细胞的增殖也有相似的抑制效果。
表3.本发明化合物在无毒剂量下对BCL6高表达肿瘤细胞增殖的抑制效果
Figure PCTCN2018088932-appb-000027
Figure PCTCN2018088932-appb-000028
实施例4：本发明化合物与BCL6蛋白亲和力检测
该实验通过采用Biacore生物大分子相互作用仪检测本发明化合物与含有BTB/POZ结构域的BCL6片段蛋白之间的亲和力。本发明挑选了HTRF测试效果和抑制肿瘤细胞增殖效果均好的四个化合物WH-007，WH-009，WH-023和WH-025进行与BCL6蛋白结合效果的检测实验。
技术方法：
1.BCL6片段蛋白的固定：首先，利用氨基偶联法将BCL6片段蛋白固定到芯片表面。通过NHS与EDC现混溶液活化CM5芯片Fc2通道表面的羧基。然后，将BCL6片段蛋白稀释到pH4.0的醋酸钠缓冲液中通过Fc2通道进行偶联。最后，利用乙醇胺将活化后未结合BCL6片段蛋白的羧基进行封闭。
2.亲和力测试：将本发明化合物WH-007，WH-009，WH-023和WH-025系列稀释到PBS缓冲液中，制备成0.01-10μM的多个浓度的测试样品。以未结合BCL6片段蛋白的Fc1通道作为参比，以Fc2作为测试通道，将化合物测试样品分别流经上述两个通道，针对同一化合物测试多个不同浓度与BCL6片段蛋白结合的响应值，获得浓度依赖性曲线，并基于1:1结合模型拟合出所测化合物与蛋白之间的亲和力。
实验结果如图1所示，随着本发明化合物WH-007，WH-009，WH-023和WH-025的浓度上升，其与BCL6片段蛋白的响应值也随之提高，两者的结合具有浓度依赖性，说明本发明化合物和BCL6片段蛋白能特异性的结合。根据不同 浓度化合物与蛋白的响应值曲线作图，结果发现本发明化合物的平衡解离常数K D处于0.4-5μM之间，K D值越小，代表两者之间亲和力越强。化合物WH-007的K D值为1.61μM，WH-009的K D值为3.05μM，WH-023的K D值为0.47μM，WH-025的K D值为3.6μM，表明本发明化合物WH-007，WH-009，WH-023和WH-025能与BCL6蛋白强烈结合。
实施例5：本发明化合物上调BCL6下游基因的表达
实时荧光定量PCR技术(qRT-PCR)是利用荧光信号的变化实时检测PCR扩增反应中每一个循环扩增产物量的变化，通过Ct值和标准曲线的分析对起始模板进行定量分析。本发明采用qRT-PCR来检查本发明化合物对BCL6下游基因表达的调节情况。BCL6为转录抑制因子，BCL6功能受到抑制其下游基因表达则会上调。对本发明化合物进行的相关检测如图2和图3所示；其中，图2是以WH-007，WH-009，WH-023和WH-025为例说明本发明化合物在BCL6阳性细胞株SUDHL4上的检测结果；图3是以WH-007和WH-023为例说明本发明化合物在BCL6阳性细胞株SUDHL4和BCL6阴性细胞株TOLEDO中设置浓度梯度对基因CD69和CXCR4的检测结果。
技术方法：
1.RNA的提取：
(1)在6cm皿中接入1.5mL细胞悬液和1.5mL用培养基稀释的双倍浓度的药物(终浓度5μM)，细胞经药物处理24h后离心收集细胞，使用预冷PBS洗2遍；
(2)加入1mL Trizol试剂，用枪吹打裂解细胞，此时样品可放置于-80℃保存数星期；
(3)将裂解后的细胞12000rpm离心5分钟，取上清至新的1.5mL无RNAse的EP管，加入200μL三氯甲烷(氯仿)，轻颠数次，静置5分钟后12000rpm4℃离心15min；
(4)将上清吸入干净无RNAse的EP管中，注意不要吸到中间液相层，加入500μL的异丙醇，轻颠数次，放置10分钟沉淀RNA；
(5)将样品12000rpm 4℃离心10min，尽量吸除上清，加入1mL 75％乙醇(使用DEPC水配制)洗涤沉淀两次，室温晾干，之后加适量DEPC水溶解RNA 沉淀；
(6)紫外可见分光光度计测定RNA浓度和纯度。
2、qRT-PCR：
(1)反转录：mRNA逆转为cDNA。反应体系为20μL，其中ddH 2O为15μL，5×Mix buffer为4μL，RNA为1Μl(1000ng)。反应条件为：37℃反应30min，85℃反应5s，16℃保存；
(2)qRT-PCR反应：根据Takara SYBR试剂盒进行。反应体系(25mL)如下：ddH 2O为10μL，2×SYBR Mix buffer为12.5μL，上下游引物(10μM)各1μL，模板cDNA为0.5μL。BCL6下游基因CD69的引物序列为CTGGTCACCCATGGAAGTG和CATGCTGCTGACCTCTGTGT，CXCR4的引物序列为AGGCCCTAGCTTTCTTCCAC和CTGCTCACAGAGGTGAGTGC，Caspase8的引物序列为CTGGGAGAAGGAAAGTTGGA和CAAGGCTGCTGCTTCTCTCT，DUSP5的引物序列为:ATGGATCCCTGTGGAAGACA和TCACAGTGGACCAGGACCTT。内参基因actin的引物序列为GTACGCCAACACAGTGCTG和CGTCATACTCCTGCTTGCTG。反应条件为：预变性(95℃、5min)1个循环，PCR反应(95℃、30s，60℃、30s，72℃、30s)40个循环。
部分实验结果如图2和图3所示，图2中，化合物WH-007，WH-009，WH-023和WH-025均能上调BCL6下游基因CXCR4，CD69，CASPASE8和DUSP5的表达，其中WH-023的效果最为显著，其对基因CASPASE8表达上调的效果达10倍左右，对基因CXCR4和DUSP5表达上调的效果达几十倍，对基因CD69的上调效果达2000多倍。图3A为细胞株SUDHL4和TOLEDO中BCL6蛋白的表达图。图3B中，化合物WH-007和WH-023在BCL6阳性细胞株SUDHL4中对CD69和CXCR4上调的效果明显优于阴性细胞株TOLEDO，尤其是化合物WH-023，其在SUDHL4细胞中对基因CD69上调效果达2000多倍，而在TOLEDO中上调基因CD69只有10倍左右。
实施例6：本发明化合物体内抑制小鼠生发中心的形成
生发中心反应是一种T细胞依赖性抗原免疫应答的过程，在这个过程中活化的B细胞经历克隆增殖、功能成熟到分化产生高亲和力抗体的浆细胞以及分化 成记忆B细胞。在大多数的抗体应答抗原蛋白的反应中，生发中心(GC)B细胞以细胞表面蛋白GL7、FAS高表达为特征。生发中心形成的最主要调控因子BCL6，是生发中心B细胞发育中所必须的转录抑制因子。在BCL6缺失的情况下，生发中心的形成被抑制。本实验采用小鼠体内生发中心形成模型，以WH-007和WH-023为例来检测本发明化合物抑制BCL6功能进而对生发中心形成影响的效果。
技术方法：
1.动物免疫：采用8-10周的C57/BL6，腹腔注射100μg 4-羟基-3-硝基苯基乙酰基(NP)欧联的鸡γ球蛋白(CGG)，免疫两天后开始灌胃给药；
2.每天灌胃给药小鼠50mg/kg/d(低浓度)和100mg/kg/d(高浓度)的本发明化合物，化合物溶于羧甲基纤维素钠(CMC-Na)，空白对照组灌胃给等体积的溶剂CMC-Na；
3.给药10天，即免疫12天处死小鼠，取小鼠脾脏并称量脾脏重量；
4.将小鼠脾脏置于70μm的cell strainer，用注射器内芯研磨。用MACS buffer(PBS+2％FBS)冲洗收集细胞悬液；
5.1500rpm离心5min，去上清；
6.加入红细胞裂解液ACK buffer，每个脾脏2mL，处理2min；
7.加入4倍体积的MACS buffer终止裂解，用70μm的cell strainer过滤去除杂质，1500rpm离心5min，去上清；
8.MACS buffer重悬细胞，并进行细胞计数；
9.将106个细胞重悬到100μL的MACS buffer里，分别加入1μL的APC-B220，PE-FAS和FITC-GL7抗体于4℃孵育40min。
10.加入MACS buffer 1500rpm离心5min洗涤细胞，用500μL的MACS buffer重悬细胞并转移至流式管，上机检测。
实验结果如图4和图5所示，本发明化合物对小鼠生发中心的形成有抑制作用。图4A和图5A是小鼠脾脏重量的统计图，表明本发明化合物WH-007和WH-023对小鼠脾脏没有影响。在较低的给药浓度下(50mg/kg/d)，本发明化合物WH-007(图4B)和WH-023(图5B)对小鼠生发中心的形成就有显著的抑制，GCB cell(GL7+FAS+)的比例就有明显下降。图4C和图5C是在50mg/kg/d的给药剂 量下，生发中心B细胞的百分比统计图。
实施例7：本发明化合物抑制LPS刺激的TNFα的表达。
小鼠RAW 264.7细胞(10000个/孔)分别加入96孔培养板中，然后加入LPS(100ng/ml)处理6小时，再加入浓度为100nM的本发明化合物WH-001至WH-053继续处理12个小时，收集上清后用BD公司TNF-α的ELISA试剂盒对上清中的细胞因子含量进行检测。结果如图6所示，本发明化合物在较低浓度下都有抑制LPS激活的TNFα表达的能力，特别是化合物WH-007、WH-009、WH-015、WH-023、WH-025、WH-030、WH-036、WH-037和WH-053都有强烈抑制LPS激活的TNFα表达的能力，具有较好的抗炎效果。
实施例8：本发明化合物对关节炎具有显著的治疗效果
胶原诱导关节炎模型Collagen‐Induced Arthritis in Mice
材料
1.8‐10周、雌性C57BL/6小鼠
2.100％冰醋酸(Merck,cat.no.1.00063.1000)
3.鸡II型胶原(Chondrex Inc.；cat.no.20011)。使用0.1M冰醋酸溶解胶原至浓度为2mg/ml。
4.弗氏完全佐剂CFA，5mg/ml(Chondrex Inc.；cat.no.7023)
方法
1.胶原乳化：实验中模型制备采用两次免疫的方法，两次均使用完全弗氏佐剂与鸡II型胶原乳化，体积比为1：1。
2.免疫小鼠：第0天，在小鼠尾根部皮下注射已经乳化好的胶原，每只100ul；同时以50mg/kg/2d浓度的本发明化合物WH-007、WH-009、WH-015、WH-023、WH-025、WH-030、WH-036、WH-037和WH-053对小鼠进行灌胃给药，第21天，进行第二次免疫，同样在小鼠尾根部第一次免疫点旁边注射乳化好的胶原，每只100ul。
3.小鼠观察评分：从第22天起每个2天观察小鼠，采用标准的四分法进行评分及脚掌厚度的测量，记录并分析实验结果。
附：临床评分标准四分法
分数 评分标准
0 无异常
1 有一个脚趾红肿
2 有两个或两个以上脚趾红肿
3 整个脚掌红肿
4 整个脚掌红肿且关节出现僵直
实验结果表明，本发明化合物具有显著治疗关节炎的能力，图7是一个典型的治疗效果图，从图中可以发现，给予本发明化合物WH-007、WH-009、WH-015、WH-023、WH-025、WH-030、WH-036、WH-037和WH-053一段时间治疗后，小鼠关节炎症状得到了极大的减轻，图7B评分上也证实了这一点，说明本发明化合物具有显著治疗关节炎的潜力。同时，本发明化合物还在其它自身免疫性疾病进行了治疗效果的评价，结果发现，本发明化合物在自身免疫性疾病如红斑狼疮、多发性硬化症、关节炎、皮肌炎、慢性甲状炎，青少年型糖尿病、恶习性贫血、萎缩性胃炎、非特异性溃疡性结肠炎、自身免疫性肾小球肾炎、肺肾出血性综合症、特发性血小板减少性紫癜、重症肌无力、自身免疫性溶血性贫血和特发性白细胞减少症等上都有明显的治疗效果。
实施例9：本发明化合物中含有末端哌嗪类和吗啉类官能团的化合物与BCL6蛋白的结合中发挥了关键作用
为验证末端哌嗪类和吗啉类官能团是否在本发明化合物与BCL6蛋白的结合中发挥了关键作用，本发明进行了如下对接实验：
技术方法：将本发明中含末端哌嗪类和吗啉类官能团的化合物在BCL‐6 BTB结构域中结合进行对接。BCL‐6 BTB结构域结构来自于RCSB蛋白质数据库(PDB编号为1R2B)。在该结构中，BCL‐6 BTB区域是以二聚体形式结晶，同时在晶体中还存在SMRT蛋白的一段小肽(残基号从S1424到I1425)。分子对接前，采用SWISS‐MODEL资源库对BCL‐6 BTB结构补全残基，然后采用Grmoacs程序并用amber力场对蛋白质进行加氢和能量最小化。得到的蛋白质作为分子对接的蛋白质结构文件。
进行对接前，本发明中用到的末端哌嗪类和吗啉类化合物分子运用量子化学计算软件Gaussion 03进行结构构型优化。优化应用密度泛函B3LYP方法在6‐31G(d)基组水平上进行计算优化。优化后的药物分子的可旋转键及扭转角根据 AutoDock4.0进行确定，然后进行加电荷处理，得到用于分子对接的小分子结构文件。
本发明采用分子对接软件AutoDock4.0获取末端哌嗪和吗啉类分子与BCL‐6 BTB结构域结合信息。分子对接所需的蛋白和小分子结构文件和参数文件由程序自动生成。计算时选择自由能函数和Lamarckian遗传算法。分子对接过程中，以晶体结构中SMRT小肽在BCL‐6 BTB结构域中的结合区域作为结合口袋，蛋白质侧链和小分子都保持柔性进行对接，每个分子进行500次独立的对接操作，选取打分最高的结合构象进行分析。
从图8A对接结果中可以发现，哌嗪类官能团在本发明化合物WH‐006与BCL‐6 蛋白的BTB区域结合中发挥了关键作用：含末端哌嗪类官能团的本发明化合物能与位于BCL‐6 BTB区域14位组氨酸(H14)形成一个稳定的氢键，具体作用方式为哌嗪类官能团氮原子上的极性氢作为氢供体能与H14侧链咪唑环上不带氢的氮原子形成氢键，以此来稳定化合物分子与BCL‐6分子结合。这个氢键的存在对于化合物与蛋白特异性结合发挥了关键作用。本发明中其它含末端哌嗪类官能团的化合物如WH‐007至WH‐053等都有相似的结合模式。
从图8B对接结果中可以发现，含末端吗啉类官能团在本发明化合物WH‐005与BCL‐6蛋白的BTB区域结合中发挥了关键作用：含末端吗啉类官能团的本发明化合物能与BCL‐6蛋白的BTB区域H14残基形成一个稳定的氢键作用，具体作用方式为吗啉类官能团氧原子作为氢受体与H14侧链咪唑环含氮原子上的极性氢作用，以此来稳定吗啉类化合物分子与BCL‐6分子结合。这个氢键的存在对于化合物与蛋白特异性结合发挥了关键作用。
这两部分的结果表明末端哌嗪类和吗啉类官能团的存在对本发明化合物与BCL6蛋白的特异性结合有着非常重要的作用。
以下实施例提供了本发明001‐053部分化合物的制备方法及产物检测结果。
实施例1‐02、N 2‐异丁氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐002)的制备
将乙胺置换成异丁胺，按制备化合物WH‐001的方法制备化合物WH‐002。 1H NMR(500MHz,DMSO)δ9.10(s,1H),8.08(s,1H),7.89(d,J＝3.9Hz,1H),7.66–7.64–7.63(m,1H),7.09(d,J＝9.0Hz,1H),6.86(s,1H),3.83(s,3H),3.03– 3.00(m,2H),1.88–1.80(m,1H),0.88(d,J＝6.7Hz,6H).
实施例1‐03、N 2‐乙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐003)的制备
将乙胺置换成乙醇胺，按制备化合物WH‐001的方法制备化合物WH‐003。 1H NMR(500MHz,DMSO)δ9.11(s,1H),7.98(s,1H),7.91(d,J＝3.9Hz,1H),7.72–7.71(m,1H),7.09(d,J＝9.0Hz,1H),6.61(t,J＝5.7Hz,1H),4.63(t,J＝5.4Hz,1H),3.83(s,3H),3.53–3.49(m,2H),3.29–3.26(m,2H).
实施例1‐04、N 2‐丙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐004)的制备
将乙胺置换成丙醇胺，按制备化合物WH‐001的方法制备化合物WH‐004。 1H NMR(500MHz,DMSO)δ9.10(s,1H),8.03(s,1H),7.90(d,J＝3.9Hz,1H),7.72–7.70–7.69(m,1H),7.09(d,J＝9.0Hz,1H),6.73(s,1H),4.43(t,J＝5.1Hz,1H),3.83(s,3H),3.47(m,2H),3.25–3.24(m,2H),1.71–1.63(m,2H).
实施例1‐05、N 2‐吗啉基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐005)的制备
将乙胺置换成吗啉，按制备化合物WH‐001的方法制备化合物WH‐005。 1H NMR(500MHz,DMSO)δ9.30(s,1H),8.03(d,J＝3.7Hz,1H),7.87(d,J＝2.6Hz,1H),7.67–7.62(m,1H),7.13(d,J＝9.0Hz,1H),3.83(s,3H),3.67–3.61(m,4H),3.60–3.54(m,4H).
实施例1‐08、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐008)的制备
将6‐溴‐2‐吡啶甲酸置换成3‐溴苯甲酸，按制备化合物WH‐007的方法制备化合物WH‐008。 1H NMR(500MHz,DMSO)δ10.35(s,1H),9.38(s,1H),8.16(s,1H),8.04(d,J＝3.5Hz,1H),7.97(d,J＝7.8Hz,1H),7.80(d,J＝8.8Hz,1H),7.74(d,J＝9.0Hz,2H),7.69(d,J＝9.1Hz,2H),7.51(t,J＝7.9Hz,1H),4.49(d,J＝13.0Hz,2H),3.12–3.00(m,2H),2.71–2.64(m,2H),1.19(d,J＝6.5Hz,6H).
实施例1‐09、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐009)的制备
将6‐溴‐2‐吡啶甲酸置换成3‐溴苯甲酸，按制备化合物WH‐006的方法制备化 合物WH‐009。 1H NMR(500MHz,DMSO)δ9.65(s,1H),9.44(s,1H),8.12(d,J＝20.7Hz,2H),7.96(d,J＝3.6Hz,1H),7.79(d,J＝5.5Hz,1H),7.62(s,1H),7.55(s,1H),7.49(s,1H),7.31(d,J＝6.3Hz,1H),4.60(d,J＝12.9Hz,2H),3.83(s,3H),3.30–3.20(m,2H),2.99–2.83(m,2H),1.29(d,J＝6.2Hz,6H).
实施例1‐10、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(对氯苯酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐010)的制备
将6-溴-2-吡啶甲酸置换成对氯苯甲酸，按制备化合物WH-006的方法制备化合物WH-010。1H NMR(500MHz,DMSO)δ9.56(s,1H),9.44(s,1H),9.00(d,J＝24.2Hz,1H),8.09(d,J＝5.4Hz,1H),7.98(d,J＝5.4Hz,2H),7.60–7.59(m,3H),7.31(s,1H),4.60(d,J＝6.4Hz,2H),3.83(s,3H),3.30–3.15(m,2H),2.97–2.80(m,2H),1.28(d,J＝0.6Hz,6H).
实施例1-11、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐氟‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH-011)的制备
将4‐硝基苯胺置换成3‐氟‐4硝基苯胺，将6‐溴‐2‐吡啶甲酸置换成3‐溴苯甲酸，按制备化合物WH‐007的方法制备化合物WH‐011。 1H NMR(500MHz,DMSO)δ9.59(s,1H),8.46(d,J＝2.3Hz,1H),8.39(d,J＝3.9Hz,1H),8.08(s,1H),7.92(d,J＝7.5Hz,1H),7.88(d,J＝8.6Hz,1H),7.82(d,J＝7.7Hz,1H),7.67(d,J＝8.7Hz,1H),7.60(s,1H),7.53(t,J＝7.8Hz,1H),3.03–3.02(m,2H),3.00(d,J＝11.3Hz,2H),2.31–2.30(m,2H),1.02(d,J＝6.1Hz,6H).
实施例1‐12、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲氧基‐2,4‐嘧啶二胺(WH‐012)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成5‐甲氧基‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐012。 1H NMR(500MHz,DMSO)δ9.63(s,1H),8.81(s,1H),8.14(s,1H),7.96(d,J＝7.2Hz,1H),7.84(s,1H),7.80(d,J＝7.4Hz,1H),7.67(s,1H),7.54–7.48(m,2H),7.40(d,J＝8.4Hz,1H),4.60(d,J＝13.3Hz,2H),3.86(s,3H),3.83(s,3H),3.27–3.26(m,2H),2.86–2.77(m,2H),1.27(d,J＝5.6Hz,6H).
实施例1‐13、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氯‐2,4‐嘧啶二胺(WH‐013)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成5‐氯‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐013。 1H NMR(500MHz,DMSO)δ9.62(s,1H),9.54(s,1H),8.14(s,1H),7.97(d,J＝10.9Hz,2H),7.79(d,J＝4.4Hz,1H),7.61(s,1H),7.55–7.44(m,2H),7.09(d,J＝5.7Hz,1H),4.73(d,J＝11.3Hz,2H),3.84(s,3H),3.29–3.22(m,2H),2.97–2.83(m,2H),1.30(d,J＝5.5Hz,6H).
实施例1‐14、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲基‐2,4‐嘧啶二胺(WH‐014)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成5‐甲基‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐014。 1H NMR(500MHz,DMSO)δ9.60(s,1H),9.27(s,1H),8.14(s,1H),7.96(d,J＝7.4Hz,1H),7.79(d,J＝7.5Hz,2H),7.69(s,1H),7.54(d,J＝8.4Hz,1H),7.27(dd,J＝20.4,7.8Hz,1H),5.97(s,1H),4.64(d,J＝12.4Hz,2H),3.83(s,3H),3.17(s,2H),2.98(s,2H),2.17(s,3H),1.13(d,J＝6.1Hz,6H).
实施例1‐15、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐氯‐2,4‐嘧啶二胺(WH‐015)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成6‐氯‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐015。 1H NMR(500MHz,DMSO)δ9.60(s,1H),9.33(s,1H),8.14(s,1H),7.96(d,J＝7.8Hz,1H),7.93(d,J＝5.6Hz,1H),7.78(d,J＝8.0Hz,1H),7.74(s,1H),7.49(d,J＝8.1Hz,2H),7.01(dd,J＝8.6,2.0Hz,1H),6.05(d,J＝5.7Hz,1H),4.54(d,J＝12.1Hz,2H),3.84(s,3H),3.17(d,J＝3.9Hz,2H),2.76(s,2H),1.03(d,J＝6.0Hz,6H).
实施例1‐16、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐甲基‐2,4‐嘧啶二胺(WH‐016)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成6‐甲基‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐016。 1H NMR(500MHz,DMSO)δ9.61(s,1H),8.27(s,1H),8.14(s,1H),7.96(d,J＝7.7Hz,1H),7.84(s,1H),7.79(d,J＝7.7Hz,1H),7.57(s,1H),7.53(d,J＝8.5Hz,1H),7.50(d,J＝7.9Hz,1H),7.29(dd,J＝8.6,1.9Hz,1H),4.55(d,J＝12.2Hz,2H),3.82(s,3H),3.17(s,2H),3.02(s,2H),2.08(s,3H),1.15(d,J＝6.0Hz,6H).
实施例1‐17、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐三氟甲基‐2,4‐嘧啶二胺(WH‐017)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴苯甲酸，将5‐氟‐2,4‐二氯嘧啶置换成6‐三氟甲基‐2,4‐二氯嘧啶，按制备化合物WH‐006的方法制备化合物WH‐017。 1H NMR(500MHz,DMSO)δ9.89(s,1H),9.65(s,1H),8.14(s,1H),7.96(d,J＝7.0Hz,1H),7.79(d,J＝7.8Hz,1H),7.67(s,1H),7.57(d,J＝8.6Hz,1H),7.50(d,J＝7.9Hz,1H),7.05(dd,J＝8.6,2.0Hz,1H),6.44(s,1H),4.59(d,J＝11.6Hz,2H),3.85(s,3H),3.00(s,2H),2.64(s,2H),1.24(s,1H),1.13(d,J＝6.0Hz,6H).
实施例1‐18、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐018)的制备
将6-溴-2-吡啶甲酸置换成噻吩-2-甲酸，按制备化合物WH-006的方法制备化合物WH-018。 1H NMR(500MHz,DMSO)δ9.51(s,1H),9.36(s,1H),8.06(d,J＝2.8Hz,1H),7.98(s,1H),7.83(d,J＝4.6Hz,1H),7.80(d,J＝8.3Hz,1H),7.68(s,1H),7.50(d,J＝8.6Hz,2H),4.50(d,J＝12.4Hz,2H),3.83(s,3H),3.03–2.94(m,2H),2.64–2.56(m,2H),1.11(d,J＝5.4Hz,6H).
实施例1‐19、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐019)的制备
将6‐溴‐2‐吡啶甲酸置换成5‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐019。 1H NMR(500MHz,DMSO)δ9.67(s,1H),9.45(s,1H),8.92(d,J＝2.2Hz,1H),8.10(d,J＝2.2Hz,1H),7.85(d,J＝4.5Hz,1H),7.60(s,1H),7.47(d,J＝7.3Hz,1H),7.34(s,1H),4.60(d,J＝13.4Hz,2H),3.83(s,3H),3.20–3.16(m,2H),2.92–2.85(m,2H),1.27(d,J＝3.0Hz,6H).
实施例1‐20、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(4‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐020)的制备
将6‐溴‐2‐吡啶甲酸置换成4‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐020。 1H NMR(500MHz,DMSO)δ9.68(s,1H),9.29(s,1H),8.07(s,1H),8.02(d,J＝3.6Hz,1H),7.98(d,J＝1.3Hz,1H),7.76(d,J＝2.2Hz,1H),7.45(d,J＝8.6Hz,1H),7.25(dd,J＝8.6,2.2Hz,1H),4.38(d,J＝10.0Hz,2H),3.84(s,3H),2.71–2.64(m,2H),2.33–2.26(m,2H),0.99(d,J＝6.2Hz,6H).
实施例1‐21、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐021)的制备
将6‐溴‐2‐吡啶甲酸置换成3‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐021。 1H NMR(500MHz,DMSO)δ9.52(s,1H),9.46(s,1H),8.83(s,1H),8.10(d,J＝3.6Hz,1H),7.96(d,J＝5.2Hz,1H),7.64(d,J＝1.9Hz,1H),7.34(dd,J＝8.8,1.9Hz,1H),7.31(d,J＝5.2Hz,1H),4.61(d,J＝14.5Hz,2H),3.93(s,3H),3.29–3.23(m,2H),2.93–2.83(m,2H),1.28(d,J＝6.4Hz,6H).
实施例1‐22、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐022)的制备
将6‐溴‐2‐吡啶甲酸置换成对5‐氯呋喃‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐022。 1H NMR(500MHz,DMSO)δ9.43(s,1H),9.30(s,1H),8.09(s,1H),7.68–7.58(m,2H),7.38(d,J＝2.0Hz,1H),7.30(d,J＝8.1Hz,1H),6.75(s,1H),4.60(d,J＝11.2Hz,2H),3.85(s,3H),3.28–3.18(m,2H),2.89–2.77(m,2H),1.25(d,J＝1.2Hz,6H).
实施例1‐24、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐甲氧基)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐024)的制备
将苯硼酸置换成2‐甲氧基苯硼酸，按制备化合物WH‐023的方法制备化合物WH‐024。 1H NMR(500MHz,DMSO)δ9.49(s,1H),9.45(s,1H),8.10(s,1H),7.96(s,1H),7.83(d,J＝5.6Hz,1H),7.67(s,1H),7.62(s,1H),7.56(d,J＝7.3Hz,1H),7.38(s,1H),7.32(d,J＝6.2Hz,1H),7.19(d,J＝6.8Hz,1H),7.07(d,J＝5.5Hz,1H),4.61(d,J＝11.4Hz,2H),3.95(s,3H),3.85(s,3H),3.29–3.19(m,2H),2.96–2.81(m,2H),1.29(d,J＝1.2Hz,6H).
实施例1‐25、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐氯)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐025)的制备
将苯硼酸置换成2‐氯苯硼酸，按制备化合物WH‐023的方法制备化合物WH‐025。 1H NMR(500MHz,DMSO)δ9.66(s,1H),9.48(s,1H),8.11(d,J＝3.5Hz,1H),8.03(d,J＝3.5Hz,1H),7.71(dd,J＝7.0,2.3Hz,1H),7.63(dd,J＝6.9,1.9Hz,2H),7.52(dd,J＝7.9,6.4Hz,2H),7.48–7.44(m,2H),7.33(dd,J＝8.6,1.9Hz,1H),4.61(d,J＝13.0Hz,2H),3.85(s,3H),3.30–3.29(m,2H),2.91–2.89(m,2H),1.29(d,J＝6.3Hz,6H).
实施例1‐26、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐吡啶基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐026)的制备
将苯硼酸置换成3‐吡啶硼酸，按制备化合物WH‐023的方法制备化合物WH‐026。 1H NMR(500MHz,DMSO)δ9.65(s,1H),9.42(s,1H),8.99(s,1H),8.56(s,1H),8.21–7.94(m,3H),7.73(d,J＝4.3Hz,1H),7.63(d,J＝3.4Hz,1H),7.51(d,J＝2.3Hz,2H),7.32(s,1H),4.59(d,J＝5.5Hz,2H),3.85(s,3H),3.27–3.17(m,2H),2.92–2.75(m,2H),1.25(d,J＝3.6Hz,6H).
实施例1‐27、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐噻吩基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐027)的制备
将苯硼酸置换成3‐噻吩硼酸，按制备化合物WH‐023的方法制备化合物WH‐027。 1H NMR(500MHz,DMSO)δ9.53(s,1H),9.36(s,1H),8.06(d,J＝3.5Hz,1H),7.96(d,J＝3.8Hz,1H),7.91(dd,J＝2.8,1.2Hz,1H),7.69(dd,J＝4.9,2.9Hz,2H),7.54–7.50(m,2H),7.48(d,J＝3.9Hz,1H),7.29(dd,J＝8.6,1.9Hz,1H),4.50(d,J＝12.0Hz,2H),3.85(s,3H),3.08–2.87(m,2H),2.60–2.59(m,2H),1.13(d,J＝6.0Hz,6H).
实施例1‐28、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐028)的制备
将6‐溴‐2‐吡啶甲酸置换成对5‐溴呋喃‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐028。 1H NMR(500MHz,DMSO)δ9.40(s,1H),9.30(s,1H),8.08(d,J＝1.9Hz,1H),7.64(d,J＝5.9Hz,1H),7.62(s,1H),7.34(s,1H),7.29(d,J＝8.5Hz,1H),6.83(d,J＝3.3Hz,1H),4.54(d,J＝13.0Hz,2H),3.85(s,3H),3.14–3.12(m,2H),2.73–2.66(m,2H),1.19(d,J＝4.8Hz,6H).
实施例1‐29、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐029)的制备
将6‐溴‐2‐吡啶甲酸置换成对3‐溴呋喃‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐029。 1H NMR(500MHz,DMSO)δ9.44(s,1H),9.10(s,1H),8.10(d,J＝1.1Hz,1H),8.01(s,1H),7.95(d,J＝8.0Hz,1H),7.62(s,1H),7.33(d,J＝7.8Hz,1H),6.95(s,1H),4.60(d,J＝12.8Hz,2H),3.89(s,3H),3.30–3.24(m,2H),2.94–2.83(m,2H),1.28(d,J＝3.2Hz,6H).
实施例1‐30、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐呋喃基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐030)的制备
将苯硼酸置换成3‐呋喃硼酸，按制备化合物WH‐023的方法制备化合物WH‐030。 1H NMR(500MHz,DMSO)δ9.53(s,1H),9.44(s,1H),8.23(s,1H),8.10(d,J＝3.0Hz,1H),7.95(s,1H),7.80(s,1H),7.62(s,1H),7.54(d,J＝8.5Hz,1H),7.37(d,J＝3.5Hz,1H),7.32(d,J＝8.6Hz,1H),6.92(s,1H),4.61(d,J＝12.6Hz,2H),3.85(s,3H),3.31–3.24(m,2H),2.90–2.89(m,2H),1.28(d,J＝5.4Hz,6H).
实施例1‐31、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐031)的制备
将6‐溴‐2‐吡啶甲酸置换成对苯并噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐031。 1H NMR(500MHz,DMSO)δ9.80(s,1H),9.47(s,1H),8.35(s,1H),8.11(d,J＝3.5Hz,1H),8.05(d,J＝7.5Hz,1H),7.99(d,J＝7.0Hz,1H),7.64(d,J＝2.0Hz,1H),7.60(d,J＝8.6Hz,1H),7.52–7.40(m,2H),7.34(dd,J＝8.6,2.0Hz,1H),4.61(d,J＝12.4Hz,2H),3.86(s,3H),3.30–3.26(m,2H),2.96–2.84(m,2H),1.29(d,J＝6.4Hz,6H).
实施例1‐32、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐032)的制备
将6‐溴‐2‐吡啶甲酸置换成对苯并呋喃‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐032。 1H NMR(500MHz,DMSO)δ9.48(s,1H),9.47(s,1H),8.10(d,J＝3.0Hz,1H),7.83(t,J＝8.3Hz,2H),7.74(d,J＝7.2Hz,2H),7.66(s,1H),7.52(t,J＝7.6Hz,1H),7.38(t,J＝7.5Hz,1H),7.34(d,J＝8.6Hz,1H),4.61(d,J＝12.7Hz,2H),3.90(s,3H),3.28–3.27(m,2H),2.91–2.89(m,2H),1.29(d,J＝6.1Hz,6H).
实施例1‐33、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐033)的制备
将6‐溴‐2‐吡啶甲酸置换成对吲哚‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐033。 1H NMR(500MHz,DMSO)δ11.74(s,1H),9.42(d,J＝3.8Hz,2H),8.08(d,J＝0.7Hz,1H),7.67(s,2H),7.46(d,J＝7.1Hz,1H),7.37–7.28(m,2H),7.25–7.19(m,1H),7.11–7.02(m,1H),4.56(d,J＝10.9Hz,2H),3.87(s,3H),3.20–3.10(m,2H),2.84–2.69(m,2H),1.21(d,J＝6.4Hz,6H).
实施例1‐34、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐034)的制备
将6‐溴‐2‐吡啶甲酸置换成对5‐氯吲哚‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐034。 1H NMR(500MHz,DMSO)δ11.95(s,1H),9.53(s,1H),9.37(s,1H),8.06(s,1H),7.78(d,J＝8.1Hz,1H),7.71(d,J＝16.4Hz,2H),7.62(d,J＝7.2Hz,1H),7.50(d,J＝6.8Hz,1H),7.47(d,J＝8.4Hz,1H),7.36–7.32(m,1H),4.50(d,J＝11.5Hz,2H),3.85(s,3H),3.32–3.30(m,2H),3.05–2.95(m,2H),1.12(d,J＝5.6Hz,6H).
实施例1‐35、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氟‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐035)的制备
将6‐溴‐2‐吡啶甲酸置换成对5‐氟吲哚‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐035。 1H NMR(500MHz,DMSO)δ11.85(s,1H),9.49(s,1H),9.39(s,1H),8.07(s,1H),7.68(s,1H),7.64(s,1H),7.44(d,J＝6.0Hz,2H),7.31(d,J＝10.5Hz,2H),7.08(s,1H),4.53(d,J＝10.3Hz,2H),3.85(s,3H),3.12–2.99(m,2H),2.69–2.59(m,2H),1.15(d,J＝6.0Hz,6H).
实施例1‐37、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐呋喃乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐037)的制备
将5‐溴‐2‐噻吩溴乙基溴置换为5‐硝基‐2‐呋喃溴乙基溴，按制备化合物WH‐036的方法制备化合物WH‐037。 1H NMR(500MHz,DMSO)δ8.84(s,1H),7.91(d,J＝3.8Hz,1H),7.63(d,J＝3.7Hz,1H),7.10–7.09(m,1H),6.92(d,J＝2.4Hz,1H),6.80(d,J＝8.7Hz,1H),6.64(d,J＝3.8Hz,1H),4.41(d,J＝6.2Hz,2H),4.35–4.34(m,2H),3.79(s,3H),2.67–2.64(m,2H),2.29–2.25(m,2H),0.99(d,J＝6.2Hz,6H).
实施例1‐38、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐038)的制备
将5‐溴‐2‐噻吩溴乙基溴置换为5‐硝基‐2‐噻吩溴乙基溴，按制备化合物WH‐036的方法制备化合物WH‐038。 1H NMR(500MHz,DMSO)δ8.88(s,1H),8.02(d,J＝4.2Hz,1H),7.91(d,J＝3.7Hz,1H),7.14(d,J＝4.2Hz,1H),7.08–7.07(m,1H),6.85(d,J＝2.3Hz,1H),6.82(d,J＝8.7Hz,1H),4.53(d,J＝6.2Hz,2H),4.37(d,J＝12.0Hz,2H),3.81(s,3H),2.79–2.69(m,2H),2.38–2.28(m,2H),1.02 (d,J＝5.9Hz,6H).
实施例1‐39、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐039)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，按制备化合物WH‐006的方法制备化合物WH‐039。 1H NMR(500MHz,DMSO)δ10.35(s,1H),9.35(s,1H),9.14(s,1H),8.92(s,1H),8.40(d,J＝8.4Hz,1H),8.07(d,J＝8.3Hz,1H),8.04(s,1H),7.24(d,J＝8.8Hz,1H),7.12(d,J＝8.9Hz,1H),4.68(d,J＝13.4Hz,2H),3.93(s,3H),3.27–3.26(m,2H),2.81–2.76(m,2H),1.20(d,J＝5.2Hz,6H).
实施例1‐40、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐040)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐硝基噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐040。 1H NMR(500MHz,DMSO)δ10.13(s,1H),9.19(s,1H),8.29(d,J＝1.8Hz,1H),8.20(d,J＝4.3Hz,1H),8.07(d,J＝3.8Hz,1H),7.96(d,J＝3.1Hz,1H),7.44–7.43(m,1H),7.10(d,J＝8.9Hz,1H),4.34(d,J＝11.8Hz,2H),3.83(s,3H),2.64–2.62(m,2H),2.24–2.20(m,2H),0.91(d,J＝6.0Hz,6H).
实施例1‐41、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氨基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐041)的制备
取化合物N2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N4‐((3‐(5‐硝基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺100mg溶于10ml的甲醇中密封，在氮气保护下加入钯/炭35mg密封，将体系完全置换成氢气，反应过夜，简单处理后，柱层析得到WH‐041。 1H NMR(500MHz,DMSO)δ9.29(s,1H),8.87(s,1H),8.63(s,1H),8.10(d,J＝3.5Hz,1H),7.63(d,J＝4.0Hz,1H),7.37–7.34(m,1H),7.14(d,J＝8.9Hz,1H),6.58(s,2H),6.01(d,J＝4.0Hz,1H),4.64(d,J＝11.4Hz,2H),3.96(s,3H),3.17–2.97(m,2H),2.62–2.48(m,2H),1.22(d,J＝4.0Hz,6H).
实施例1‐42、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐042)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐042。 1H NMR(500MHz,DMSO)δ9.66(s,1H),9.30(s,1H),8.33(d,J＝2.4Hz,1H),8.02 (d,J＝3.6Hz,1H),7.88(d,J＝4.0Hz,1H),7.36(d,J＝4.0Hz,1H),7.31–7.30(m,1H),7.08(d,J＝9.0Hz,1H),4.59(d,J＝13.3Hz,2H),3.83(s,3H),3.17–3.16(m,2H),2.74–2.69(m,2H),1.19(d,J＝6.2Hz,7H).
实施例1‐43、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(4‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐043)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对4‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐043。 1H NMR(500MHz,DMSO)δ9.70(s,1H),9.29(s,1H),8.34(d,J＝2.5Hz,1H),8.11(s,1H),8.02(d,J＝3.4Hz,2H),7.34–7.33(m,1H),7.09(d,J＝9.0Hz,1H),4.57(d,J＝12.2Hz,2H),3.84(s,3H),3.18–3.17(m,2H),2.67–2.61(m,2H),1.15(d,J＝0.7Hz,6H).
实施例1‐44、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(3‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐044)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对3‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐044。 1H NMR(500MHz,DMSO)δ9.63(s,1H),9.32(s,1H),8.97(d,J＝2.3Hz,1H),8.04(d,J＝3.6Hz,1H),8.00(d,J＝5.2Hz,1H),7.34(d,J＝5.3Hz,1H),7.23–7.22(m,1H),7.11(d,J＝8.9Hz,1H),4.68(d,J＝12.2Hz,2H),3.93(s,3H),3.31–3.20(m,2H),2.77–2.69(m,2H),1.20(d,J＝2.5Hz,6H).
实施例1‐45、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐呋喃酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐045)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐溴呋喃‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐045。 1H NMR(500MHz,DMSO)δ9.27(s,1H),9.21(s,1H),8.50(d,J＝2.6Hz,1H),7.98(d,J＝3.7Hz,1H),7.34(d,J＝3.6Hz,1H),7.07(d,J＝9.0Hz,1H),6.84(d,J＝3.6Hz,1H),4.43(d,J＝11.9Hz,2H),3.85(s,3H),2.81–2.80(m,2H),2.38–2.34(m,2H),1.00(d,J＝5.9Hz,6H).
实施例1‐46、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氯‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐046)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐氯噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐046。 1H NMR(500MHz,DMSO)δ9.69(s,1H),9.33(s,1H),8.32(d,J＝2.6Hz,1H),8.03(d,J＝3.6Hz,1H),7.94(d,J＝4.1Hz,1H),7.31–7.30(m,1H),7.27(d,J＝4.1Hz,1H),7.09(d,J＝9.0Hz,1H),4.62(m,2H),3.83(s,3H),2.84–2.76(m,2H),2.09–2.08(m,2H),1.23(d,J＝6.1Hz,6H).
实施例1‐47、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐苯并噻吩酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐047)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对苯并噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐047。 1H NMR(500MHz,DMSO)δ9.75(s,1H),9.29(s,1H),8.47(d,J＝1.8Hz,1H),8.36(s,1H),8.05(d,J＝7.6Hz,1H),8.03–7.97(m,2H),7.51–7.47(m,2H),7.34–7.33(m,1H),7.11(d,J＝8.9Hz,1H),4.57(d,J＝13.0Hz,2H),3.87(s,3H),3.06–3.05(m,2H),2.59–2.58(m,2H),1.12(d,J＝6.4Hz,6H).
实施例1‐48、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐噁唑酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐048)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对噁唑甲酸，按制备化合物WH‐006的方法制备化合物WH‐048。1H NMR(500MHz,DMSO)δ10.00(s,1H),9.52(s,1H),8.94(s,1H),8.79(s,1H),8.67(s,1H),8.02(s,1H),7.26(d,J＝7.0Hz,1H),7.09(d,J＝8.0Hz,1H),4.60(d,J＝12.0Hz,2H),3.90(s,3H),3.23–3.08(m,2H),2.98–2.87(m,2H),1.28(d,J＝6.2Hz,6H).
实施例1‐49、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐嘧啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐049)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐嘧啶甲酸，按制备化合物WH‐006的方法制备化合物WH‐049。 1H NMR(500MHz,DMSO)δ10.12(s,1H),9.52(s,1H),9.37(s,2H),9.27(s,1H),8.42(s,1H),8.03(d,J＝3.6Hz,1H),7.38–7.36(m,1H),7.10(d,J＝9.0Hz,1H),4.57(d,J＝13.5Hz,2H),3.83(s,3H),3.17–3.16(m,2H),2.96–2.89(m,2H),1.24(d,J＝6.1Hz,6H).
实施例1‐50、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐050)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对吡啶‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐050。 1H NMR(500MHz,DMSO)δ10.54(s,1H),9.54(d,J＝9.6Hz,1H),9.16(d,J＝2.5Hz,1H),8.99(d,J＝8.5Hz,1H),8.77(d,J＝4.5Hz,1H),8.13–8.12(m,1H),7.74–7.71(m,1H),7.23–7.22(m,1H),7.13(d,J＝8.9Hz,1H),4.69(d,J＝12.6Hz,2H),3.95(s,3H),3.33–3.30(m,2H),2.94–2.85(m,2H),1.22(d,J＝5.2Hz,6H).
实施例1‐51、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐3‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐051)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对5‐溴噻吩‐2‐甲酸，按制备化合物WH‐006的方法制备化合物WH‐051。 1H NMR(500MHz,DMSO)δ9.93(s,1H),9.33(s,1H),9.07(s,1H),8.93(s,1H),8.54(s,1H),8.43(s,1H),8.03(s,1H),7.34(d,J＝8.9,1H),7.10(d,J＝9.0Hz,1H),4.60(d,J＝15.7Hz,2H),3.84(s,3H),3.17–3.16(m,2H),2.74–2.68(m,2H),1.16(d,J＝6.2Hz,6H).
实施例1‐52、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(4‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐052)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对4‐溴‐2‐吡啶甲酸，按制备化合物WH‐006的方法制备化合物WH‐052。 1H NMR(500MHz,DMSO)δ10.42(s,1H),9.36(s,1H),9.17(d,J＝2.5Hz,1H),8.67(d,J＝5.2Hz,1H),8.28(d,J＝1.6Hz,1H),8.08–8.01(m,2H),7.24(d,J＝8.8,1H),7.13(d,J＝8.9Hz,1H),4.69(d,J＝13.6Hz,2H),3.94(s,3H),3.29–3.28(d,J＝5.1Hz,2H),2.83–2.78(m,2H),1.21(d,J＝5.8Hz,6H).
实施例1‐53、N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(6‐溴‐4‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺(WH‐053)的制备
将3‐甲氧基4‐硝基苯胺置换为3‐硝基‐4甲氧基苯，将6‐溴‐2‐吡啶甲酸置换成对6‐溴‐4‐吡啶甲酸，按制备化合物WH‐006的方法制备化合物WH‐053。 1H NMR(500MHz,DMSO)δ9.99(s,1H),9.36(s,1H),8.61(d,J＝4.8Hz,1H),8.39(s,1H),8.10(s,1H),8.04(d,J＝3.3Hz,1H),7.89(d,J＝4.4Hz,1H),7.35(d,J＝6.9Hz, 1H),7.11(d,J＝8.9Hz,1H),4.63(d,J＝13.3Hz,2H),3.84(s,3H),3.26–3.25(m,2H),2.83–2.77(m,2H),1.20(d,J＝5.7Hz,6H)。
Figure PCTCN2018088932-appb-000029
Figure PCTCN2018088932-appb-000030
Figure PCTCN2018088932-appb-000031
Figure PCTCN2018088932-appb-000032

Claims (15)

  1. 一类4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，其结构如下式(I)所示：
    Figure PCTCN2018088932-appb-100001
    其中：
    R 1和R 2选自下列基团中的一个或多个：氢、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
    Figure PCTCN2018088932-appb-100002
    且R 1和R 2不同时为氢；
    其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100003
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃；
    R 3选自下列基团中的一个：C 1‐C 5的链状醇氨基、C 1‐C 5的脂肪链状氨基、C 3‐C 6环氨基、四氢吡咯基、哌嗪基、C 1‐C 3烷基哌嗪基、哌啶基、C 1‐C 3烷基哌啶基、吗啉基、C 1‐C 3烷基吗啉基、硫代吗啉基、C 1‐C 3烷基硫代吗啉基；
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基。
  2. 根据权利要求1所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，当R 3为哌嗪基或者2,6‐二烷基哌嗪时，其结构如下式(II)所示：
    Figure PCTCN2018088932-appb-100004
    其中：
    R 1和R 2选自下列基团中的一个或多个：氢、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
    Figure PCTCN2018088932-appb-100005
    且R 1和R 2不同时为氢；
    其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100006
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃；
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基；
    R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基；
  3. 根据权利要求2所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，当R 2为C 1‐C 3烷氧基或C 1‐C 3烷胺基时，其结构如下式(III)所示：
    Figure PCTCN2018088932-appb-100007
    X为O或者N；
    R 1选自下列基团中的一个或多个：氢、卤素、C 1‐C 3烷氧基、C 1‐C 3烷胺基、羟基、硝基、氨基、羧基、
    Figure PCTCN2018088932-appb-100008
    且R 1和R 2不同时为氢；其中，R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100009
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃；
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基；
    R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基；
    R 7为C 1‐C 3烷基。
  4. 根据权利要求3所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，当R 1为
    Figure PCTCN2018088932-appb-100010
    时，其结构如下式(IV)所 示：
    Figure PCTCN2018088932-appb-100011
    其中：
    X为O或者N；
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基；
    R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基；
    R 7为C 1‐C 3烷基；
    R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100012
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
  5. 根据权利要求4所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，当X为氧原子时，其结构如下式(V)所示：
    Figure PCTCN2018088932-appb-100013
    其中：
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基；
    R 5和R 6分别选自下列基团中的一个：氢、C 1‐C 3烷基；
    R 7为C 1‐C 3烷基；
    R 8选自下列基团中的一个或多个：氢、卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3 烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100014
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
  6. 根据权利要求5所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，当X为氧原子时，R 5和R 6为顺势甲基时，其结构如下式(VI)所示：
    Figure PCTCN2018088932-appb-100015
    其中：
    R 4选自下列基团中的一个：氢、卤素、腈基、三氟甲基、C 1‐C 3烷氧基、羟基、C 1‐C 3烷基；
    R 7为C 1‐C 3烷基；
    R 8选自下列基团中的一个或多个：卤素、羟基、羧基、C 1‐C 3烷氧基、C 1‐C 3烷基、酯基、硝基、氨基、腈基；
    Figure PCTCN2018088932-appb-100016
    选自下列芳香基或杂环芳香基中的任意一个或者两个串联而成：苯环、吡啶、咪唑、呋喃、噻吩、吡唑、苯并噻吩、吲哚、喹啉、苯并呋喃，苯基噻吩，苯基呋喃。
  7. 4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，其特征在于，选自：
    N 2‐乙氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐异丁氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐乙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐丙醇氨基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐吗啉基‐N 4‐(3‐氯‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(6‐溴‐2‐吡啶酰胺基))苯基)‐5‐氟 ‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(6‐溴‐2‐吡啶酰胺基)苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐(4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲氧基‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氯‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(对氯苯酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐氟‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐5‐甲基‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐氯‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐甲基‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴苯甲酰胺基))苯基)‐6‐三氟甲基‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(4‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐呋喃酰胺基))苯基)‐5‐氟 ‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐甲氧基)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(2‐氯)苯基‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐吡啶基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐噻吩基)‐2‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(3‐溴‐2‐呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐(3‐呋喃基)‐噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并噻吩酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(苯并呋喃酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氯‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐甲氧基‐4‐(5‐氟‐2‐吲哚酰胺基))苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐呋喃乙基))‐4‐甲氧基苯基)‐5‐氟 ‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩乙基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐硝基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氨基‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(4‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(3‐溴‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐溴‐2‐呋喃酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐氯‐2‐噻吩酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐苯并噻吩酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐恶唑酰胺基)‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(5‐嘧啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4‐((3‐(2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(5‐溴‐3‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(4‐溴‐2‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐ 嘧啶二胺
    N 2‐((3S,5R)‐3,5‐二甲基哌嗪基)‐N 4((3‐(6‐溴‐4‐吡啶酰胺基))‐4‐甲氧基苯基)‐5‐氟‐2,4‐嘧啶二胺。
  8. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐，以及药学上可接受的载体。
  9. 如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在制备BCL6抑制剂中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在制备抑制BCL6蛋白与多肽SMRT的相互作用，或上调BCL6下游基因CXCR4，CD69，Caspase8和DUSP5的表达药物中的应用。
  11. 如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在制备抑制生发中心的形成的药物中的应用。
  12. 如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在在制备预防和/或治疗BCL6介导的疾病药物中的应用；其中，所述疾病包括自身免疫性疾病、过敏或炎症；其中，所述自身免疫性疾病选自红斑狼疮、多发性硬化症、关节炎、皮肌炎、慢性甲状炎，青少年型糖尿病、恶习性贫血、萎缩性胃炎、非特异性溃疡性结肠炎、自身免疫性肾小球肾炎、肺肾出血性综合症、特发性血小板减少性紫癜、重症肌无力、自身免疫性溶血性贫血和特发性白细胞减少症。
  13. 如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在制备预防和/或治疗肿瘤的药物中的应用。
  14. 如权利要求11所述的应用，其特征在于，所述肿瘤包括恶性肿瘤，所述4‐嘧啶二胺类小分子有机化合物或其相关类似物或药学上可接受的盐在制备抑制肿瘤细胞的增殖、生长、迁移和浸润、复发药物中的应用；其中，所述肿瘤或恶性肿瘤选自弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、伯基特淋巴瘤、非小细胞肺癌、乳腺癌、B细胞急性淋巴细胞白血病、慢性骨髓性白血病、食管癌，卵巢癌，头颈部鳞状细胞癌、结肠癌、前列腺癌、肝癌。
  15. 如权利要求1‐7之任一项所述的4‐嘧啶二胺类小分子有机化合物或其相关类 似物或药学上可接受的盐在制备抑制LPS刺激的TNFα的表达的抑制剂中的应用。
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