WO2017152756A1 - cRGD-厄洛替尼缀合物及其制备方法 - Google Patents

cRGD-厄洛替尼缀合物及其制备方法 Download PDF

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WO2017152756A1
WO2017152756A1 PCT/CN2017/074384 CN2017074384W WO2017152756A1 WO 2017152756 A1 WO2017152756 A1 WO 2017152756A1 CN 2017074384 W CN2017074384 W CN 2017074384W WO 2017152756 A1 WO2017152756 A1 WO 2017152756A1
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erlotinib
gly
arg
phe
lys
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PCT/CN2017/074384
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French (fr)
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李书鹏
张海龙
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徐州瑞康生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a targeting compound, in particular to a preparation method of a cRGD-erlotinib/erlotinib conjugate and a conjugate, and belongs to the technical field of tumor targeted delivery drug delivery systems.
  • Targeted drug delivery is site-specific delivery, which provides that the therapeutic agent reaches the disease site without affecting the efficacy of the drug, while reducing the unrelated distribution in normal tissues, thereby increasing local drug concentration and reducing side effects.
  • Erlotinib is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) that prevents ATP binding and further inhibits autophosphorylation and proliferative signaling of tyrosine residues by competitively binding to the catalytic domain of EGFR. Interferes with the growth of cancer cells and is therefore used to treat metastatic non-small cell lung cancer (NSCLC).
  • NSCLC metastatic non-small cell lung cancer
  • erlotinib has many other protein sites of action that promote the interconnection of cancer cell proliferation signals or show pro-apoptotic effects on healthy cells, leading to serious side effects.
  • the present invention is directed to erlotinib having many other protein action sites in the treatment of non-small cell lung cancer, exhibiting a pro-apoptotic effect on healthy cells, thereby causing serious side effects, providing a cancer Cell-targeted cRGD coupled to erlotinib/erlotinib conjugate.
  • the conjugate is formed by direct coupling of erlotinib/erlotinib to an azide-labeled non-natural lysine introduced in a cyclic RGD polypeptide.
  • the introduction of cRGD allows erlotinib/erlotinib to selectively target cancer cells, rather than healthy cells, thereby improving therapeutic efficacy.
  • a method of preparing a compound of formula 1 or 2 comprising the steps of:
  • step 1 includes the following sub-steps:
  • the Fmoc group was removed from the azide-labeled linear polypeptide DfKRG curable resin under conditions of a DMF/DCM/piperidine volume ratio of 1:1:2. Then, after incubation at room temperature for 2 hours under a solution of a mixture of acetic acid/2,2,2-trifluoroethanol (TFE) and DCM in a ratio of 3:1:6, the linear peptide together with the band obtained from the resin 3
  • TFE acetic acid/2,2,2-trifluoroethanol
  • the azide-labeled linear polypeptide H-Asp(OtBu)-D-Phe-Lys(N 3 )-Arg(Pbf)-Gly-OH finally obtained in step 2 was treated with HOBt/HBTU/ under nitrogen protection. DIPEA is used as a condensing agent for liquid phase cyclization. After the reaction solution is concentrated under vacuum, the concentrated solution is further deprotected in a mixed solvent of tetrafluoroethylene, water and dimercaptoethane, and finally an azide-labeled linear polypeptide cyclo(-Arg-Gly-Asp-D- is obtained. Phe-Lys(N 3 )-);
  • step 2 comprises the following substeps: a certain amount of erlotinib or ectinib, cyclo(-Arg-Gly-Asp-D-Phe-Lys(N 3 )-), triethylammonium acetate buffer And ascorbic acid is dissolved in dimethylformamide and then mixed, followed by copper(II)-tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine complex The reaction was continued until the appropriate time; the reaction mixture was concentrated and concentrated using reversed-phase high performance liquid chromatography RP-HPLC to obtain cyclo(-Arg-Gly-Asp-D-Phe-Lys(N 3 )-)-erlotinib conjugate. .
  • reaction of the step 1 is carried out under the protection of an inert gas.
  • reaction of the step 2 is carried out under the protection of an inert gas.
  • the cyclic polypeptide RGD shows a higher affinity and selectivity for binding to the integrin Integrin receptor. It is apparent that the erlotinib conjugate conjugated to the cyclic polypeptide RGD has an integrin receptor that specifically binds to the high abundance expression of cancer cells, thereby promoting the uptake of cancer cells, compared to the erlotinib monomer. Shows better results. At the same time, both erlotinib or ectinib contain an alkyne group, allowing the click chemistry-based alkyne azide cycloaddition to couple another compound or biomolecule.
  • Figure 1 is a structural diagram of the CRDD-erlotinib conjugate in Example 1;
  • Figure 2 is a synthetic route diagram of the CRDD-erlotinib conjugate in Example 1;
  • Figure 3 is a structural diagram of the CRGD-Ektorinib conjugate in Example 2.
  • Figure 4 is a synthetic route diagram of the CRGD-Ekinib conjugate in Example 2.
  • Figure 5 is an ESI high resolution mass spectrometry report of peptide 6 in Example 1;
  • Figure 6 is a HRMS detection report of peptide 6 in Example 1;
  • Figure 7 is an ESI high resolution mass spectrometry report of the final product in Example 1;
  • Figure 8 is a HRMS test report of the final product in Example 1.
  • Figure 9 is an ESI high resolution mass spectrometry report of the final product in Example 2.
  • Figure 10 is a HRMS test report of the final product in Example 2.
  • Figure 11a is a comparison of the sensitivity of cRGD-erlotinib and erlotinib in enhancing PC9 cells produced in Example 1; (inhibition of proliferation of PC9 cells by erlotinib and cRGD-erlotinib)
  • Figure 11b is a graph comparing the sensitivity of cRGD-erlotinib and erlotinib in the enhanced PC9 cells produced in Example 1; (cloning survival assay)
  • Figure 12a is a comparative experiment of cRGD-erlotinib and erlotinib induced apoptosis in PC9 cells produced in Example 1; (flow cytometry analysis of DMSO, erlotinib, and cRGD-erlotinib treatment Effect of PC9 cell apoptosis)
  • Figure 12b is a comparative experiment of cRGD-erlotinib and erlotinib induced apoptosis in PC9 cells produced in Example 1; (PI staining analysis detected DMSO, erlotinib, cRGD-erlotinib treatment on PC9 cells Effect of apoptosis)
  • Figure 12c is a comparative experiment of cRGD-erlotinib and erlotinib induced apoptosis in PC9 cells produced in Example 1. (Western blot analysis of apoptosis-associated proteins, erlotinib and cRGD-erlotinib treatment)
  • the present invention provides a tumor-targeted prodrug for non-small cell lung cancer, which is a cRGD-erlotinib conjugate, and the specific structure is shown in FIG. Among them, cRGD is a cyclic arginine-glycine-aspartate peptide, which can effectively target polypeptides of tumor tissues; erlotinib is a drug for treating non-small cell lung cancer.
  • the present invention provides a method for preparing a tumor-targeted prodrug against non-small cell lung cancer, and the synthetic route thereof is shown in FIG. 2, and specifically includes the following steps:
  • the starting reaction resin (Fmoc-Gly-CTC Resin) obtained in the step 1 is subjected to a Fmoc group from the amino acid in a solution having a DMF/DCM/piperidine volume ratio of 2:1:1 at room temperature. Deprotection reaction. After the reaction for 30 min, the resin was washed twice with DMF.
  • the solid resin is coupled with an Fmoc-protected amino acid or amino acid derivative in a DCM solution for 3 hours, wherein the equivalent weight of the amino acid is 1, and the equivalent of N-hydroxybenzotriazole (HOBt) is 2, O-(benzo
  • the equivalents of triazol-1-yl)-N,N,N',N-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA are 2 and 5, respectively.
  • an amino acid having N-terminal Fmoc protection and side chain protection (in order, Fmoc-arginine (Pbf)-OH, Fmoc-lysine (N 3 )-OH, Fmoc-) Aspartic acid (OtBu)-OH) is attached to the Fmoc-Gly-CTC resin.
  • This coupling reaction was analyzed by the ninhydrin test. It is especially important that lysine introduces azide through the side chain to facilitate the preparation of erlotinib conjugates by click chemical coupling in step 2.
  • the resin was washed twice with 5 ml of DMF and DCM, and dried under vacuum for 12 hours to obtain a linear RGDfK curing resin.
  • the Fmoc group was removed from the linear DfKRG cured resin in a solution having a DMF/DCM/piperidine volume ratio of 1:1:1. Then, the Ffoc-depleted DfKRG-cured resin was subjected to a cleavage reaction in a mixed solution of acetic acid/2,2,2-trifluoroethanol (TFE) and DCM (volume ratio: 3:1:6) at room temperature. After 2 hours, the linear peptide RGDfK with a side chain protecting group was cleaved from the resin 3. After the cleavage reaction, the resin was washed twice with a mixed solution of acetic acid/TFE/DCM. The combined eluates were mixed in 15 volumes of hexane, and the solvent was removed by evaporation using a rotary evaporator to obtain a linear peptide RGDfK 4 with azide labeling and side chain protection.
  • TFE acetic acid/2,2,2-trifluoroethanol
  • DCM volume ratio:
  • the azide label and side chain protected linear polypeptide 4 [H-Asp(OtBu)-D-Phe-Lys(N 3 )-Arg(Pbf)-Gly-OH obtained in step 3 under nitrogen protection conditions Soluble in DMF solution to a concentration of 1.25 ⁇ 10 -3 M. Then, a liquid phase cyclization reaction was carried out using HOBt/HBTU/DIPEA as a condensing agent. The cyclization reaction adds 1 equivalent of HOBt, 2 equivalents of HBTU and 5 equivalents of DIPEA to the DMF solution, and reacts under nitrogen for 24 hours to obtain an azide-labeled and side-chain protected cyclic polypeptide (-arginine).
  • the crude deprotected cyclic peptide 6 was purified by reverse-phase high performance liquid chromatography RP-HPLC using a linear gradient buffer (20-45% of 90% acetonitrile in 0.05% TFA) to give a 98% purity ring on an Agela C18 column.
  • Peptide. Peptide 6 was analyzed by ESI high resolution mass spectrometry. Its retention time is 25 minutes. Its chemical formula is C 27 H 40 N 11 O 7 , and the HRMS calculated by mass spectrometry [M+H] + is 630.31, and the detected value is 629.98.
  • Table 1 shows the sample information of peptide 6:
  • Table 2 shows the results of peptide 6 detection:
  • Table 3 shows the storage conditions of peptide 6:
  • a certain amount of erlotinib was dissolved in 8 ml of DMF (20 mg, 0.051 mmol), and a certain amount of cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-) (26.7 mg, 0.043 mmol) was added. Then, 5.6 ml of triethylammonium acetate buffer (pH 7.0, 2 M) and 1.6 ml of ascorbic acid (5.0 mmol) were added. The solution was mixed at room temperature for 30 minutes, and then 0.8 ml of 10 mM copper(II)-tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine complex was added.
  • the final product was analyzed by ESI high-resolution mass spectrometry with a retention time of 22 minutes, a purity of 100%, a chemical formula of C 49 H 63 N 14 O 11 , and a HRMS calculated by mass spectrometry [M+H] + of 1024.48.
  • the value was 1024.75
  • the HRMS calculated for C 49 H 62 N 14 NaO 11 [M+Na] + was 1046.09
  • the detected value was 1046.50.
  • Table 4 is the sample information of the final product of Example 1:
  • Table 5 shows the test results of the final product of Example 1:
  • Table 6 shows the storage conditions of the final product of Example 1:
  • PC9 cells Human non-small cell lung cancer PC9 cells were grown in DMEM medium containing 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were cultured in a 37-base, 5% CO 2 incubator for 48 hours to reach 80% cell confluence. The medium used to culture the cells was then replaced with different concentrations of the DMSO-containing control, erlotinib and cRGD-erlotinib, respectively, for different times.
  • DMSO-containing control erlotinib and cRGD-erlotinib, respectively, for different times.
  • Ninety-six well plates were used for cell death assays. Six-well plates were used for Annexin V-FITC-PI staining, and 35 mm dishes were used for confocal laser scanning microscopy (CLSM) imaging.
  • CLSM confocal laser scanning microscopy
  • PC9 cells were seeded in 24-well plates (2 plates) (planted in cells/well) and incubated for 72 hours (0 to 2 uM) in increasing concentrations of erlotinib or cRDG-erlotinib medium. After the incubation, 600 post-fertility MTT solutions were added to each well and cultured for 3 hours. Then, 150 ⁇ l of DMSO (SIGMA, D2650) was added to each well and stirred at room temperature for 10 minutes. The absorbance A (OD) at 570 nm was measured on a Bio-Rad microplate reader (Hercules, USA).
  • Three independent Cytotoxicity assays (each drug concentration was repeated four times each) to calculate erlotinib and cRDG-erlotinib IC50, the latter defined as the concentration of drug in inducing 50% of cell death in the cell population.
  • PC9 cells The sensitivity of PC9 cells to drug treatment was analyzed using colony formation assays, which were used to determine cytotoxic agents, such as the effectiveness of chemotherapy.
  • Cells were harvested and seeded in 60-mm culture dishes. After 24 hours, the cells were exposed to 2 uM erlotinib and/or cRDG-erlotinib for 72 hours. The drug was then removed and the cells were allowed to grow for 10 days in fresh growth medium. The cell population was fixed and stained with methanol (25% v/v) containing crystal violet (0.05% w/v) for 30 minutes. Microscopy counts colonies containing at least 30 cells.
  • PC9 cells were exposed to DMSO (solvent control), 20 nM erlotinib or cRDG-erlotinib for 72 hours. Cells were detected with fluorescein isothiocyanate (FITC) conjugated annexin V and propidium iodide (PI) apoptosis assay kit (BD Biosciences, California, USA). Apoptotic cells were then analyzed by flow cytometry.
  • FITC fluorescein isothiocyanate
  • PI propidium iodide
  • PC9 cells were exposed to 30 nM erlotinib and/or cRDG-erlotinib for 48 hours at 37 °C.
  • the medium was changed to an extracellular solution containing 50 ⁇ g/ml propidium iodide (PI) (Invitrogen, Calif.), and after 48 minutes of incubation at 37 ° C, the fluorescence intensity of each well was measured by a microplate reader (Victor 3 ; PerkinElmer, Massachusetts). The number of dead cells is normalized by the total number of cells.
  • PI propidium iodide
  • cRGD-erlotinib conjugate on apoptosis of PC9 cancer cells was further analyzed by immunoblotting by expression of apoptosis-related proteins caspase-3 and PARP.
  • Apoptosis after cell treatment The cells were washed twice with pre-cooled PBS, dissolved in RIPA buffer containing phenylmethylsulfonyl fluoride, and centrifuged at 4 degrees, 12000 acyl fluoride for 20 minutes.
  • the protein was quantified by the BCA method and separated by 6-15% SDS-PAGE and transferred to a nitrocellulose membrane. Incubate with 5% skim milk for 2 hours at 37 °C to block non-specific binding.
  • rabbit anti-caspase-3, rabbit anti-PARP or mouse anti-GAPDH antibody was incubated overnight at 4 degrees with a 1:2000 dilution.
  • the membrane was then washed three times in TBS T and the labeled secondary antibody was incubated for 1 hour at room temperature with a 1:5000 dilution.
  • the protein is shown by chemiluminescence (Millipore).
  • Results are expressed as mean ⁇ standard error and the results are from at least three independent experiments. The results were analyzed using one-way analysis of variance. For clonal analysis, two-way ANOVA was used. After detecting significant differences, Newman Keuls was used to test the difference between the two. Data analysis and statistical comparisons were performed using the GraphPad Prism program (Gradphad, Calif.). P values ⁇ 0.05 were considered statistically significant.
  • cRGD-erlotinib and erlotinib were treated with different concentrations of cRGD-erlotinib and erlotinib (0, 0.2 nm, 2 nm, 20 nm, 200 nm and 2 um), respectively.
  • cRGD-erlotinib and erlotinib showed a dose-response curve for cell viability.
  • the 72-hour survival assay found that the IC50 of erlotinib and cRGD-erlotinib against PC9 cells were approximately 30.35 nM and 5.56 nM, respectively, indicating a significant enhancement of cRGD-erlotinib.
  • Figure 11a and 11b sensitivity of PC9 cells to erlotinib and cRGD-erlotinib.
  • Figure 11a shows the inhibition of proliferation of PC9 cells by erlotinib and cRGD-erlotinib.
  • PC9 cells were treated with erlotinib and cRGD erlotinib at the indicated concentrations for 72 hours. Cell viability was determined by measuring the optical density value (490 nm) by the MTT method.
  • Figure 11b shows that clone survival assays 2 uM erlotinib and cRGD-erlotinib treated PC9 cells for 7 days.
  • Figure 12 Erlotinib and cRGD-erlotinib induce apoptosis in PC9 cells.
  • Figure 12a Flow cytometry analysis of the effects of DMSO, erlotinib and cRGD-erlotinib treatment on PC9 cell apoptosis. After treatment of cells with DMSO, 20 nM erlotinib, 20 nM cRGD-erlotinib for 72 hours, cells were harvested and stained with annexin-V/propidium iodide (PI), followed by flow cytometry analysis.
  • Figure 12b DMSO, erlotinib treatment, PCG cells treated with cRGD-erlotinib for 48 hours, PI staining.
  • the present invention provides a tumor-targeted prodrug for non-small cell lung cancer, which is a cRGD-Ektorinib conjugate, and the specific structure is shown in FIG. Among them, cRGD is a cyclic arginine-glycine-aspartate peptide, which can effectively target polypeptides of tumor tissues; ectinib is a drug for treating non-small cell lung cancer.
  • the present invention provides a method for preparing a tumor-targeted prodrug against non-small cell lung cancer, and the synthetic route thereof is shown in FIG. 4, and specifically includes the following steps:
  • the starting reaction resin (Fmoc-Gly-CTC Resin) obtained in the step 1 is subjected to a Fmoc group from the amino acid in a solution having a DMF/DCM/piperidine volume ratio of 2:1:1 at room temperature. Deprotection reaction. After the reaction for 30 min, the resin was washed twice with DMF.
  • the solid resin is coupled with an Fmoc-protected amino acid or amino acid derivative in a DCM solution for 3 hours, wherein the equivalent weight of the amino acid is 1, and the equivalent of N-hydroxybenzotriazole (HOBt) is 2, O-(benzo
  • the equivalents of triazol-1-yl)-N,N,N',N-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA are 2 and 5, respectively.
  • an amino acid having N-terminal Fmoc protection and side chain protection (in order, Fmoc-arginine (Pbf)-OH, Fmoc-lysine (N 3 )-OH, Fmoc-) Aspartic acid (OtBu)-OH) is attached to the Fmoc-Gly-CTC resin.
  • This coupling reaction was analyzed by the ninhydrin test. It is especially important that lysine introduces azide through the side chain to facilitate the preparation of erlotinib conjugates by click chemical coupling in step 2.
  • the resin was washed twice with 5 ml of DMF and DCM, and dried under vacuum for 12 hours to obtain a linear RGDfK curing resin.
  • the Fmoc group was removed from the linear DfKRG cured resin in a solution having a DMF/DCM/piperidine volume ratio of 1:1:1. Then, the Ffoc-depleted DfKRG-cured resin was subjected to a cleavage reaction in a mixed solution of acetic acid/2,2,2-trifluoroethanol (TFE) and DCM (volume ratio: 3:1:6) at room temperature. After 2 hours, the linear peptide RGDfK with a side chain protecting group was cleaved from the resin 3. After the cleavage reaction, the resin was washed twice with a mixed solution of acetic acid/TFE/DCM. The combined eluates were mixed in 15 volumes of hexane, and the solvent was removed by evaporation using a rotary evaporator to obtain a linear peptide RGDfK 4 with azide labeling and side chain protection.
  • TFE acetic acid/2,2,2-trifluoroethanol
  • DCM volume ratio:
  • the azide label and side chain protected linear polypeptide 4 [H-Asp(OtBu)-D-Phe-Lys(N 3 )-Arg(Pbf)-Gly-OH obtained in step 3 under nitrogen protection conditions Soluble in DMF solution to a concentration of 1.25 ⁇ 10 -3 M. Then, a liquid phase cyclization reaction was carried out using HOBt/HBTU/DIPEA as a condensing agent. The cyclization reaction adds 1 equivalent of HOBt, 2 equivalents of HBTU and 5 equivalents of DIPEA to the DMF solution, and reacts under nitrogen for 24 hours to obtain an azide-labeled and side-chain protected cyclic polypeptide (-arginine).
  • the crude deprotected cyclic peptide 6 was purified by reverse-phase high performance liquid chromatography RP-HPLC using a linear gradient buffer (20-45% of 90% acetonitrile in 0.05% TFA) to give a 98% purity ring on an Agela C18 column. Peptide. Peptide 6 was analyzed by ESI and high resolution mass spectrometry. Its retention time is 25 minutes. C 27 H 40 N 11 O 7 [M + H] + . HRMS calcd 630.31, 629.98 detected value.
  • Ecetinib (40 mg, 0.102 mmol) was dissolved in 15 ml of DMF, and a ring (-Arg-glycine-aspartate-D-phenylalanine-lysine (N 3)-) was added. The solution (42.8 mg, 0.068 mmol) was then added followed by 10.5 mL of triethylammonium acetate buffer (pH 7.0, 0.57 M) and 3.0 mL of ascorbic acid (5.0 mmol). The solution was mixed for 30 minutes at room temperature.
  • Table 7 is the sample information of the final product of Example 2:
  • Table 8 shows the test results of the final product of Example 2:
  • Table 9 shows the storage conditions of the final product of Example 2:

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Abstract

提供了cRGD-厄洛替尼/埃克替尼缀合物及其制备方法，其中，cRGD为环状多肽。厄洛替尼及埃克替尼是表皮生长因子受体(EGFR)的小分子抑制剂，其干扰癌细胞的生长，从而用于治疗转移性非小细胞肺癌(NSCLC)。本发明合成的cRGD-厄洛替尼/埃克替尼缀合物有选择性地将厄洛替尼/埃克替尼递送至非小细胞肺癌细胞，提高了位点递送效率的同时，也减少了癌症患者的副作用。

Description

cRGD-厄洛替尼缀合物及其制备方法 技术领域
本发明涉及一种靶向型化合物，具体来讲是一种cRGD-厄洛替尼/埃罗替尼缀合物及缀合物的制备方法，属于肿瘤靶向递送给药系统技术领域。
背景技术
传统药物的递送通常是被动吸收透过细胞膜。一般情况下，药物会随着全身血液或体液循环而遍布全身大部分组织或器官。对于大多数药物来说，特定区域产生最小有效浓度是需药理学效应所必需的。因此，额外大量的药物量不可避免地对体内细胞功能产生毒性。靶向药物递送是位点特异性递送，该优势为治疗性试剂到达疾病部位，在不影响药物疗效的同时，而减少了正常组织中不相关的分布，从而以增加局部药物浓度和减少副作用。
厄洛替尼是表皮生长因子受体(EGFR)的小分子抑制剂，通过竞争性结合于EGFR的催化结构域，防止ATP结合，进一步抑制酪氨酸残基的自磷酸化和增殖信号传导，干扰癌细胞的生长，从而用于治疗转移性非小细胞肺癌(NSCLC)。然而，厄洛替尼有许多其他的蛋白质作用位点，可促进癌细胞增殖信号的相互联系或显示出对健康细胞的促凋亡作用，导致严重的副作用。
发明内容
本发明针对厄洛替尼具在治疗非小细胞肺癌时有许多其他的蛋白质作用位点，显示出对健康细胞的促凋亡作用，因而导致严重的副作用的不足之处，提供一种具有癌细胞靶向性的cRGD偶联厄洛替尼/埃罗替尼缀合物。该缀合物通过厄洛替尼/埃罗替尼与环状RGD多肽中引入的叠氮标记的非天然性赖氨酸直接偶联而成。作为一种靶向整合素受体的药物定向递送系统，cRGD的引入使得厄洛替尼/埃罗替尼选择性地针对癌细胞，而非健康细胞，从而提高治疗效果。
为实现以上的技术目的，本发明将采取以下的技术方案：
一种选自式I-Ⅱ的化合物及其药学可接受的结构，
Figure PCTCN2017074384-appb-000001
式1，
Figure PCTCN2017074384-appb-000002
式2。
一种制备如结构式1或2化合物的方法，包括以下步骤：
(1)按照多肽固相技术顺序合成叠氮标记的环状多肽cyclic RGDfK-azide【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】；
(2)将步骤1中得到的cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)与厄洛替尼/埃罗替尼通过点击化学共轭得到最终产物。
本发明进一步限定的技术方案为：
进一步的，步骤1包括如下分步骤：
(1)Fmoc固相合成法合成成叠氮标记的线性多肽树脂Fmoc-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-CTC Resin
将2-氯三苯甲基氯树脂、Fmoc-Glycine-OH和无水N，N-二异丙基乙胺溶解于二氯甲烷中搅拌适当时间；加入配好的甲醇和无水N，N-二异丙基乙胺混合溶液继续搅拌；然后，将树脂分别用二甲基甲酰胺(DMF，2次)，DCM(2次)和甲醇(2次)洗涤，真空干燥。最后，按顺序将引入保护基团的氨基酸(按顺序依次为Fmoc-精氨酸(Pbf)-OH，Fmoc-赖氨酸(N3)-OH，Fmoc-天冬氨酸(OtBu)-OH连接Fmoc-Gly-CTC树脂。
(2)合成叠氮标记的线性多肽【H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH】
在DMF/DCM/哌啶体积比为1:1:2的溶液条件下，从叠氮标记的线性多肽DfKRG固化树脂除去Fmoc基团。然后室温条件下，在乙酸/2,2,2-三氟乙醇(TFE)和DCM的混合物比例为3:1:6溶液条件下温育2小时后，线性肽连同从树脂3中获得的带侧链的保护基团在乙酸溶液中裂解。裂解反应后，树脂使用乙酸/TFE/DCM的混合溶液洗涤两次。将合并的洗脱物混合于15倍体积的己烷中，使用旋转蒸发仪进行蒸发除去溶剂以获得具有侧链保护作用的线性肽4(H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH)。
(3)液相环化合成叠氮标记的环状多肽cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)
在氮气保护条件下，将分步骤2中最终得到的叠氮标记的线性多肽H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH以HOBt/HBTU/DIPEA作为缩合剂进行液相环化反应。然后将反应溶液在真空条件下浓缩后，浓缩溶液进一步在四氟乙烯、水及二巯基乙烷混合溶剂中脱保护，最后得到叠氮标记的线性多肽cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)；
进一步的，步骤2包括如下分步骤：一定量的厄洛替尼或者埃克替尼、cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)、乙酸三乙铵缓冲液和抗坏血酸溶于二甲基甲酰胺中混合反应，随后加入铜(II)-三[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺络合物继续反应至适当时间；使用反相高效液相色谱RP-HPLC浓缩纯化反应混合物，获得cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)-厄洛替尼缀合物。
进一步的，分步骤1的反应在惰性气体的保护下进行。
进一步的，步骤2的反应在惰性气体的保护下进行。
本申请申请保护上述两种化合物在制备抗肺癌药物中的用途。
环状多肽RGD对结合整合素Integrin受体显示出更高的亲和性和选择性。显而易见，相比于厄洛替尼单体，环状多肽RGD偶联的厄洛替尼缀合物具有特异性结合癌细胞高丰度表达的整合素Integrin受体，促进癌细胞的摄取，从而表现出更好的疗效。同时，厄洛替尼或者埃克替尼均包含炔基集团，允许基于点击化学的炔叠氮化物环合加成偶联另一种化合物或生物分子。
附图说明
图1是实施例1中cRGD-厄洛替尼缀合物的结构图；
图2是实施例1中cRGD-厄洛替尼缀合物的合成路线图；
图3是实施例2中cRGD-埃克替尼缀合物的结构图；
图4是实施例2中cRGD-埃克替尼缀合物的合成路线图；
图5是实施例1中肽6的ESI高分辨率质谱报告；
图6是实施例1中肽6的HRMS检测报告；
图7是实施例1中最终成品的ESI高分辨率质谱报告；
图8是实施例1中最终成品的HRMS检测报告；
图9是实施例2中最终成品的ESI高分辨率质谱报告；
图10是实施例2中最终成品的HRMS检测报告。
图11a是实施例1制造的cRGD-厄洛替尼与厄洛替尼在增强PC9细胞的敏感性对比实验图；(厄洛替尼和cRGD-厄洛替尼对PC9细胞的增殖抑制作用)
图11b是实施例1制造的cRGD-厄洛替尼与厄洛替尼在增强PC9细胞的敏感性对比实验图；(克隆存活测定)
图12a是实施例1制造的cRGD-厄洛替尼与厄洛替尼诱导PC9细胞凋亡的对比实验；(流式细胞分析检测DMSO，厄洛替尼，和cRGD-厄洛替尼处理对PC9细胞凋亡的影响)
图12b是实施例1制造的cRGD-厄洛替尼与厄洛替尼诱导PC9细胞凋亡的对比实验；(PI染色分析检测DMSO，厄洛替尼,cRGD-厄洛替尼处理对PC9细胞凋亡的影响)
图12c是实施例1制造的cRGD-厄洛替尼与厄洛替尼诱导PC9细胞凋亡的对比实验。(免疫印迹分析细胞凋亡相关蛋白，厄洛替尼和cRGD-厄洛替尼处理)
具体实施方式
下面结合具体实施方式对本发明作进一步阐述，但本发明并不限于以下实施方式。
实施例1
第一方面，本发明提供了一种抗非小细胞肺癌的肿瘤靶向前体药，其为cRGD-厄洛替尼缀合物，具体结构见附图1。其中，cRGD为环状精氨酸-甘氨酸-天冬氨酸肽，能够有效主动靶向肿瘤组织的多肽；厄洛替尼为治疗非小细胞肺癌药物。
第二方面，本发明提供了一种抗非小细胞肺癌的肿瘤靶向前体药的制备方法，其合成路线见附图2，具体包括以下步骤：
(1)制备起始反应树脂(Fmoc-Gly-CTC Resin)
在圆底烧瓶中将1.0g树脂溶于7毫升无水二氯甲烷(DCM)。然后在氮气保护下加入 Fmoc-甘氨酸-OH(891.9毫克，3.0毫摩尔)和无水N，N-二异丙基乙胺(DIPEA)(1.00毫升，5.74毫摩尔)。将该反应溶液在氮气保护下室温搅拌4小时。最后，加入10毫升的MeOH/DIPEA(9∶1)终止混合物反应，并进一步反应20分钟。过滤除去溶液后，分别用5毫升DCM和5毫升二甲基甲酰胺(DMF)各洗涤两次，随后用5毫升甲醇洗涤一次。洗涤后，将树脂在真空下干燥24小时，并储存在4℃备用
(2)固相合成法合成成叠氮标记的线性多肽树脂Fmoc-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-CTC Resin
室温条件下，将分步骤1中得到的起始反应树脂(Fmoc-Gly-CTC Resin)在DMF/DCM/哌啶体积比为2∶1∶1的溶液条件下进行Fmoc基团从氨基酸中的脱保护反应。反应30min后，使用DMF对树脂进行两次洗涤。然后，固体树脂与Fmoc保护的氨基酸或氨基酸衍生物在DCM溶液中偶联反应3小时，其中氨基酸的当量为1，N-羟基苯并三唑(HOBt)的当量为2，O-(苯并三唑-1-基)-N，N，N'，N-四甲基脲六氟磷酸盐(HBTU)和DIPEA的当量分别为2和5。在该偶联反应中，依次将具有N端Fmoc保护且侧链保护的氨基酸(按顺序依次为Fmoc-精氨酸(Pbf)-OH，Fmoc-赖氨酸(N3)-OH，Fmoc-天冬氨酸(OtBu)-OH)连接Fmoc-Gly-CTC树脂。此偶联反应通过茚三酮试验进行分析。尤其重要的是，赖氨酸通过侧链引入叠氮以方便在步骤2通过中的点击化学偶联制备厄洛替尼缀合物。偶联反应结束后，树脂分别使用5毫升DMF和DCM洗涤两次，真空条件下干燥12小时，以获得线性RGDfK固化树脂3.
(3)合成叠氮标记的线性多肽6【H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH)】
在DMF/DCM/哌啶体积比为1∶1∶2的溶液条件下，从线性DfKRG固化树脂除去Fmoc基团。然后室温条件下，脱Fmoc的DfKRG固化树脂在乙酸/2,2,2-三氟乙醇(TFE)和DCM的混合溶液中(体积比为3∶1∶6)进行裂解反应。2小时后，具有侧链保护基团的线性肽RGDfK从树脂3中裂解下来。裂解反应后，树脂使用乙酸/TFE/DCM的混合溶液洗涤两次。将合并的洗脱物混合于15倍体积的己烷中，使用旋转蒸发仪进行蒸发除去溶剂以获得具有叠氮标记和侧链保护作用的线性肽RGDfK 4。
(4)液相环化合成叠氮标记的环状多肽【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】
在氮气保护条件下，将分步骤3中得到的叠氮标记和侧链保护的线性多肽4【H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH】溶于DMF溶液至浓度为1.25×10-3M。然后，以HOBt/HBTU/DIPEA作为缩合剂进行液相环化反应。该环化反应在DMF溶液中加入1当量的HOBt，2当量的HBTU和5当量的DIPEA，在氮气保护下反应24小时，以得到叠氮标记和侧链保护的环状多肽(-精氨酸(Pbf)-甘氨酸-天冬氨酸(OtBu)-D-苯丙氨酸-赖氨酸 (N3)-)5。将反应溶液在真空下浓缩后，在室温条件下，环肽5的残留物用在15mL的去保护溶液(95％TFA，2.5％水和2.5％的乙烷二硫醇)完全脱保护24小时，最后得到叠氮标记和侧链保护的线性多肽6【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】，即环(-精氨酸-甘氨酸-天冬氨酸-D-苯丙氨酸-赖氨酸(N3)-)。粗制的脱保护环肽6通过反相高效液相色谱RP-HPLC纯化，利用线性梯度缓冲液(20-45％的90％乙腈溶于0.05％TFA)在Agela C18柱得到98％纯度的环肽。肽6通过ESI高分辨率质谱分析。其保留时间为25分钟。其化学分子式为C27H40N11O7，质谱[M+H]+的HRMS计算值为630.31，检测值为629.98。
表1为肽6的样品信息：
Figure PCTCN2017074384-appb-000003
表2为肽6的检测结果：
Figure PCTCN2017074384-appb-000004
表3为肽6的存储条件：
Figure PCTCN2017074384-appb-000005
(4)目标产物的制备
一定量厄洛替尼溶于8毫升DMF中(20毫克，0.051毫摩尔)，加入一定量cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)(26.7毫克，0.043毫摩尔)，随后加入5.6毫升乙酸三乙铵缓冲液(pH7.0，2M)和1.6毫升抗坏血酸(5.0毫摩尔)。溶液在室温条件下混合30分钟，随后，加入0.8毫升10mM铜(II)-三[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺络合物(10mM，55％DMSO)。反应的混合物通氩气饱和20分钟后再在氮气保护下继续搅拌24小时。反应得到 的产物经过旋转蒸发仪进行蒸发除去溶剂。最后利用反相高效液相色谱，通过AgelaC18柱线性梯度缓冲液(25-47％的90％乙腈，溶于0.05％TFA)进行分离纯化反应产物，最后获得cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)-厄洛替尼缀合物1共37.5mg(收率为85％)。最终产物通过ESI高分辨率质谱进行分析，其保留时间为22分钟，纯度为100％，化学分子式为C49H63N14O11，质谱[M+H]+的HRMS计算值为1024.48，检测值为1024.75，C49H62N14NaO11[M+Na]+的HRMS计算值为1046.09，检测值为1046.50。
表4为实施例1最终成品的样品信息：
Figure PCTCN2017074384-appb-000006
表5为实施例1最终成品的检测结果：
Figure PCTCN2017074384-appb-000007
表6为实施例1最终成品的存储条件：
存储条件 -20℃
细胞培养和治疗
人非小细胞肺癌PC9细胞生长于含10％胎牛血清、100U/毫升青霉素和100U/毫升链霉素的DMEM培养基中。细胞在37基中、5％CO2温箱中培养48小时达到80％细胞汇合。然后，用于培养细胞的培养基替换为不同浓度的含有DMSO溶剂对照、厄洛替尼和cRGD-厄洛替尼，分别处理不同时间。九十六孔培养板用于细胞死亡测定。六孔培养板用于AnnexinV-FITC-PI染色，和35毫米培养皿用于共聚焦激光扫描显微镜(CLSM)成像。
细胞毒性检测
用MTT法检验体外细胞生存力，以评估药物的敏感性。PC9细胞接种在24孔板(2板 (种在细胞/孔)，并在含递增浓度的厄洛替尼或cRDG-厄洛替尼培养基中孵育72小时(0到2uM)。孵育后，每孔加入600育后的MTT溶液并培养3小时。然后，每孔加入150微升的DMSO(SIGMA，D2650)并在室温下搅拌10分钟。在Bio-Rad公司酶标仪(Hercules，美国)上测量在570nm的吸光度A(OD)。根据A值计算细胞增殖抑制率:细胞增殖抑制率(％)＝(对照孔A值-实验孔A值)/(对照孔A值-空白孔A值)白孔＝(制。进行三次独立的细胞毒性试验(每种药物浓度各自重复四次)，以计算厄洛替尼和cRDG-厄洛替尼IC 50，后者定义为诱导细胞群体中50％的细胞死亡中的药物浓度。
克隆生存分析
PC9细胞对处理药物的敏感度，使用克隆形成实验分析，后者用于确定细胞毒性剂，如化疗有效性进行测定。收集细胞并接种在60对毫米培养皿，24小时后，细胞暴露于2uM厄洛替尼和/或cRDG-厄洛替尼中72小时。然后除去药物，并且使细胞在新鲜的生长培养基增殖10天。细胞群落用含有结晶紫(0.05％重量/体积)的甲醇(25％V/V)固定并染色30分钟。显微镜计计数含有至少30个细胞的集落。
流式细胞仪分析
PC9细胞暴露于DMSO(溶剂对照)，20nM的厄洛替尼或cRDG-厄洛替尼72小时。细胞用异硫氰酸荧光素(FITC)缀合的annexinV和碘化丙啶(PI)凋亡检测试剂盒(BD Biosciences,加利福尼亚，美国)检测。凋亡细胞然后通过流式细胞仪分析。
细胞死亡的定量
为量化厄洛替尼和/或cRDG-厄洛替尼所致的细胞死亡，PC9细胞在37℃下，暴露于30nM厄洛替尼和/或cRDG-厄洛替尼48小时。培养基换为含50微克/毫升碘化丙啶(PI)(Invitrogen公司，加利福尼亚州)的细胞外溶液，在37℃下温育48分钟后，各孔的荧光强度用酶标仪测定(Victor3；PerkinElmer公司，马萨诸塞州)。死亡细胞数按细胞总数标准化。
Western blot分析
cRGD-厄洛替尼缀合物对PC9癌细胞凋亡的影响进一步通过凋亡相关蛋白caspase-3和PARP的表达进行免疫印迹分析。细胞处理后凋亡将细胞用预冷的PBS洗涤两次后，溶解在含有苯甲基磺酰氟的RIPA缓冲液中，在4度，12000酰氟离心20分钟。蛋白用BCA法定量后并通过6-15％SDS-PAGE分离，转移到硝酸纤维素膜上。在37℃，与5％脱脂牛奶孵育2小时以阻断非特异性结合。随后，与兔抗caspase-3，兔抗PARP或小鼠抗GAPDH抗体以1∶2000的稀释液，在4度孵育过夜。然后将膜在TBS T中洗涤三次，和标记的二抗以1∶5000的稀释倍数在室温下孵育1小时。蛋白质通过化学发光法(Millipore)显示。
统计分析
结果表示为平均值±标准误，实验结果来自至少三次的独立实验。使用单因素方差分析分析结果。对于克隆分析，使用双因素方差分析。当检测出显著的差异后，使用纽曼Keuls检验两两差异。数据分析和统计比较采用进行GraphPad Prism程序绘制(Gradphad，加利福尼亚)。P值<0.05为差异有统计学意义。
结果：
cRGD-厄洛替尼增强PC9细胞的敏感性
MTT和克隆存活实验以检测cRGD-厄洛替尼和厄洛替尼对细胞活力的移植作用。不同浓度cRGD-厄洛替尼和厄洛替尼(0，0.2nm，2nm，20nm，200nm和2um的)分别处理PC9细胞。cRGD-厄洛替尼和厄洛替尼显示出对细胞活力的剂量-响应曲线。如图11a，72小时存活测定发现，厄洛替尼和cRGD-厄洛替尼对PC9细胞的IC50分别约为30.35nM和5.56nM，显示cRGD-厄洛替尼的具有显著的增强效果。
如图11a和11b所示：PC9细胞对厄洛替尼和cRGD-厄洛替尼的敏感性。图11a显示，厄洛替尼和cRGD-厄洛替尼对PC9细胞的增殖抑制作用。PC9细胞用指定浓度的厄洛替尼和cRGD厄洛替尼处理72小时。细胞活力以MTT法测定光密度值(490纳米)确定。图11b显示，克隆存活测定2uM厄洛替尼和cRGD-厄洛替尼处理PC9细胞7天。图11b中，1000，10000，100000细胞铺在一式三份的软琼脂培养皿，存活细胞的数目通过各自组DMSO溶剂对照标准化。*P<0.05，单因素方差分析，纽曼-Keuls检验两两差异(n＝3)。
为了进一步研究观察到的细胞敏感性差异，我们进行了克隆存活测定。PC9细胞在2uM的厄洛替尼或cRGD-厄洛替尼处理后，测定其克隆形成。结果表明，相比单独用厄洛替尼处理的细胞，cRGD-厄洛替尼处理后的细胞克隆生存显著减少。
cRGD-厄洛替尼诱导PC9细胞凋亡
然后，我们研究cRGD-厄洛替尼相比厄洛替尼是否增加PC9细胞凋亡。cRGD-厄洛替尼和厄洛替尼对PC9细胞凋亡的作用是通过annexin V和PI双染，并用流式细胞仪检测。如图12a和12b显示，20nM的cRGD-厄洛替尼和厄洛替尼的治疗72小时分别导致了36.56％和79.24％的细胞凋亡，而溶剂对照细胞的细胞凋亡为2.08％(P<0.05)。这些结果表明，cRGD-厄洛替尼能诱导PC9细胞较高的凋亡率。此外，如图12b所示，cRGD-厄洛替尼和厄洛替尼能激活PC9细胞的caspase-3和PARP裂解，表明cRGD-厄洛替尼和厄洛替尼触发caspase蛋白酶级联的细胞凋亡。
图12：厄洛替尼和cRGD-厄洛替尼诱导PC9细胞凋亡。图12a，流式细胞分析检测DMSO，厄洛替尼和cRGD-厄洛替尼处理对PC9细胞凋亡的影响。DMSO，20nM厄洛替尼，20nM cRGD-厄洛替尼处理细胞72小时后，收获细胞并用annexin-V/碘化丙啶(PI)染色，随后通过流式 细胞仪分析。图12b，DMSO，厄洛替尼治疗后，cRGD-厄洛替尼处理PC9细胞48小时后，PI染色。图12c，免疫印迹分析细胞凋亡相关蛋白，厄洛替尼和cRGD-厄洛替尼处理48小时后caspase-3和PARP的激活。GAPDH用作上样对照。*与厄洛替尼组显着差异(P<0.05，n＝3)。
实施例2
第一方面，本发明提供了一种抗非小细胞肺癌的肿瘤靶向前体药，其为cRGD-埃克替尼缀合物，具体结构见附图3。其中，cRGD为环状精氨酸-甘氨酸-天冬氨酸肽，能够有效主动靶向肿瘤组织的多肽；埃克替尼为治疗非小细胞肺癌药物。
第二方面，本发明提供了一种抗非小细胞肺癌的肿瘤靶向前体药的制备方法，其合成路线见附图4，具体包括以下步骤：
(1)制备起始反应树脂(Fmoc-Gly-CTC Resin)
在圆底烧瓶中将1.0g树脂溶于7毫升无水二氯甲烷(DCM)。然后在氮气保护下加入Fmoc-甘氨酸-OH(891.9毫克，3.0毫摩尔)和无水N，N-二异丙基乙胺(DIPEA)(1.00毫升，5.74毫摩尔)。将该反应溶液在氮气保护下室温搅拌4小时。最后，加入10毫升的MeOH/DIPEA(9∶1)终止混合物反应，并进一步反应20分钟。过滤除去溶液后，分别用5毫升DCM和5毫升二甲基甲酰胺(DMF)各洗涤两次，随后用5毫升甲醇洗涤一次。洗涤后，将树脂在真空下干燥24小时，并储存在4℃备用
(2)固相合成法合成成叠氮标记的线性多肽树脂Fmoc-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-CTC Resin
室温条件下，将分步骤1中得到的起始反应树脂(Fmoc-Gly-CTC Resin)在DMF/DCM/哌啶体积比为2∶1∶1的溶液条件下进行Fmoc基团从氨基酸中的脱保护反应。反应30min后，使用DMF对树脂进行两次洗涤。然后，固体树脂与Fmoc保护的氨基酸或氨基酸衍生物在DCM溶液中偶联反应3小时，其中氨基酸的当量为1，N-羟基苯并三唑(HOBt)的当量为2，O-(苯并三唑-1-基)-N，N，N'，N-四甲基脲六氟磷酸盐(HBTU)和DIPEA的当量分别为2和5。在该偶联反应中，依次将具有N端Fmoc保护且侧链保护的氨基酸(按顺序依次为Fmoc-精氨酸(Pbf)-OH，Fmoc-赖氨酸(N3)-OH，Fmoc-天冬氨酸(OtBu)-OH)连接Fmoc-Gly-CTC树脂。此偶联反应通过茚三酮试验进行分析。尤其重要的是，赖氨酸通过侧链引入叠氮以方便在步骤2通过中的点击化学偶联制备厄洛替尼缀合物。偶联反应结束后，树脂分别使用5毫升DMF和DCM洗涤两次，真空条件下干燥12小时，以获得线性RGDfK固化树脂3.
(3)合成叠氮标记的线性多肽6【H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH)】
在DMF/DCM/哌啶体积比为1∶1∶2的溶液条件下，从线性DfKRG固化树脂除去Fmoc基团。然后室温条件下，脱Fmoc的DfKRG固化树脂在乙酸/2,2,2-三氟乙醇(TFE)和DCM的混合溶液中(体积比为3∶1∶6)进行裂解反应。2小时后，具有侧链保护基团的线性肽RGDfK从树脂3中裂解下来。裂解反应后，树脂使用乙酸/TFE/DCM的混合溶液洗涤两次。将合并的洗脱物混合于15倍体积的己烷中，使用旋转蒸发仪进行蒸发除去溶剂以获得具有叠氮标记和侧链保护作用的线性肽RGDfK 4。
(4)液相环化合成叠氮标记的环状多肽【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】
在氮气保护条件下，将分步骤3中得到的叠氮标记和侧链保护的线性多肽4【H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH】溶于DMF溶液至浓度为1.25×10-3M。然后，以HOBt/HBTU/DIPEA作为缩合剂进行液相环化反应。该环化反应在DMF溶液中加入1当量的HOBt，2当量的HBTU和5当量的DIPEA，在氮气保护下反应24小时，以得到叠氮标记和侧链保护的环状多肽(-精氨酸(Pbf)-甘氨酸-天冬氨酸(OtBu)-D-苯丙氨酸-赖氨酸(N3)-)5。将反应溶液在真空下浓缩后，在室温条件下，环肽5的残留物用在15mL的去保护溶液(95％TFA，2.5％水和2.5％的乙烷二硫醇)完全脱保护24小时，最后得到叠氮标记和侧链保护的线性多肽6【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】，即环(-精氨酸-甘氨酸-天冬氨酸-D-苯丙氨酸-赖氨酸(N3)-)。粗制的脱保护环肽6通过反相高效液相色谱RP-HPLC纯化，利用线性梯度缓冲液(20-45％的90％乙腈溶于0.05％TFA)在Agela C18柱得到98％纯度的环肽。肽6通过ESI和高分辨率质谱分析。其保留时间为25分钟。C27H40N11O7[M+H]+的HRMS计算值为630.31，检测值为629.98。
(5)埃克替尼(40毫克，0.102毫摩尔)溶于15ml DMF中，加入环(-Arg-甘氨酸-天冬氨酸-D-苯丙氨酸-赖氨酸(N 3)-)中的溶液(42.8毫克，0.068毫摩尔)然后加入，随后加入10.5毫升三乙基铵醋酸盐缓冲液(pH7.0，0.57M)和3.0毫升抗坏血酸(5.0毫摩尔)。将溶液室温下混合30分钟。然后，加入1.5毫升10毫铜(II)-tris[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺络合物(10毫米，55％DMSO)。将得到的混合物的通氩气20分钟并在氩气下再搅拌24小时。将反应混合物通过RP-HPLC的C18柱线性梯度缓冲液(90％乙腈中的0.05％TFA溶液25-45％)浓缩纯化，以61％的产率获得42.3毫克环(-Arg-甘氨酸-天冬氨酸-D-苯丙氨酸-赖氨酸(N 3)-)-埃克替尼共轭物1。最终结合物1通过ESI高分辨率质谱分析。HPLC TR＝20分钟。纯度：89.35％(HPLC)。HRMS：计算C49H61N14O11：[M+H]1022.09，实测值1022.96。
表7为实施例2最终成品的样品信息：
Figure PCTCN2017074384-appb-000008
Figure PCTCN2017074384-appb-000009
表8为实施例2最终成品的检测结果：
Figure PCTCN2017074384-appb-000010
表9为实施例2最终成品的存储条件：
Figure PCTCN2017074384-appb-000011

Claims (7)

  1. 一种选自式Ⅰ-Ⅱ的化合物及其药学可接受的结构，
    Figure PCTCN2017074384-appb-100001
  2. 一种制备如权利要求1中结构式1或2所述化合物方法，包括以下步骤：
    按照多肽固相技术顺序合成叠氮标记的环状多肽cyclic RGDfK-azide【cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】；
    将步骤1中得到的cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)与厄洛替尼通过点击化学共轭得到最终产物。
  3. 根据权利要求2所述的化合物方法，其特征在于：步骤1包括如下分步骤：
    (1)Fmoc固相合成法合成成叠氮标记的线性多肽树脂【Fmoc-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-CTC Resin】
    将2-氯三苯甲基氯树脂、Fmoc-Glycine-OH和无水N，N-二异丙基乙胺溶解于二氯甲烷中搅拌适当时间；加入配好的甲醇和无水N，N-二异丙基乙胺混合溶液继续搅拌；然后，将树脂分别用二甲基甲酰胺(DMF，2次)，DCM(2次)和甲醇(2次)洗涤，真空干燥；
    最后按顺序引入保护的氨基酸(按顺序依次为Fmoc-精氨酸(Pbf)-OH，Fmoc-赖氨酸(N3)-OH，Fmoc-天冬氨酸(OtBu)-OH连接Fmoc-Gly树脂；
    (2)合成叠氮标记的线性多肽H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH在DMF/DCM/哌啶混合体系中，叠氮标记的线性多肽DfKRG固化树脂除去Fmoc基团；
    然后室温条件下，在乙酸/2,2,2-三氟乙醇(TFE)/DCM的混合溶液中进行裂解反应；
    裂解反应后，树脂使用乙酸/TFE/DCM的混合溶液洗涤两次；
    最后将合并的洗脱物与15倍体积的己烷中混合，使用旋转蒸发仪进行蒸发除去溶剂以获得具有侧链保护作用的线性肽4(H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH)；
    液相环化合成cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)
    在氮气保护条件下，将分步骤2中最终得到的叠氮标记的线性多肽H-Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH以HOBt/HBTU/DIPEA作为缩合剂进行液相环化反应；
    然后将反应溶液在真空条件下浓缩后，浓缩溶液进一步在四氟乙烯、水及二巯基乙烷混合溶剂中脱保护，最后得到叠氮标记的线性多肽cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)。
  4. 根据权利要求2所述的化合物方法，其特征在于：步骤2包括如下分步骤：一定量的厄洛替尼/埃罗替尼、cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)、乙酸三乙铵缓冲液和抗坏血酸溶于二甲基甲酰胺中混合反应，随后加入铜(II)-三[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺络合物继续反应至适当时间；使用反相高效液相色谱RP-HPLC浓缩纯化反应混合物，获得 cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)-厄洛替尼缀合物。
  5. 根据权利要求3所述的化合物方法，其特征在于：分步骤1的反应在惰性气体的保护下进行。
  6. 根据权利要求4所述的化合物方法，其特征在于：步骤2的反应在惰性气体的保护下进行。
  7. 权利要求1中的化合物在制备抗肺癌药物中的用途。
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