WO2019056374A1 - 酸敏感的硼亚酞菁-吉非替尼配合物及其制备方法和在医药上的应用 - Google Patents

酸敏感的硼亚酞菁-吉非替尼配合物及其制备方法和在医药上的应用 Download PDF

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WO2019056374A1
WO2019056374A1 PCT/CN2017/103185 CN2017103185W WO2019056374A1 WO 2019056374 A1 WO2019056374 A1 WO 2019056374A1 CN 2017103185 W CN2017103185 W CN 2017103185W WO 2019056374 A1 WO2019056374 A1 WO 2019056374A1
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cancer
compound
gefitinib
acid
complex
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PCT/CN2017/103185
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English (en)
French (fr)
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蒋雄杰
黄华静
梁艳红
刘瑶
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深圳市声光动力生物医药科技有限公司
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Priority to PCT/CN2017/103185 priority Critical patent/WO2019056374A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds

Definitions

  • the invention belongs to the field of medicine, and relates to a boron phthalocyanine-gefitinib complex and a preparation method thereof and application thereof in medicine, and the invention discloses the same as a photodynamic therapy-chemotherapy dual therapeutic drug for treating cancer. use.
  • Photodynamic Therapy also known as Photoradiation Therapy (PRT) or Photochemotherapy
  • PDT Photodynamic Therapy
  • PS Photoradiation Therapy
  • the photosensitizer is injected into the body by intravenous injection (for the skin, it can also be applied to the affected area).
  • the tumor tissue is irradiated with light of a specific wavelength, and the photosensitizer enriched in the tumor tissue is excited by light.
  • photodynamic therapy has been successfully applied to lung cancer, gastric cancer, esophageal cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, cholangiocarcinoma, rectal cancer, colon cancer, skin cancer, head and neck cancer, eye tumor, uterine cancer and Treatment of ovarian cancer.
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • Gefitinib is an EGFR tyrosine kinase inhibitor that is a targeted drug for the treatment of tumors. It was first marketed in Japan in 2002 and was approved as a third-line monotherapy for advanced non-small cell lung cancer in the United States and Australia in May 2003. Formally launched in China in 2005, it has been used for clinical treatment of locally advanced or metastatic non-small cell lung cancer.
  • hypoxic microenvironment present in the solid tissue of the tumor results in a lower pH outside the tumor cell at that site (at 6.5 or so), while the normal tissue extracellular pH is about 7.4.
  • the difference in pH between tumor solid tissue and normal tissue provides a new strategy for the design of tumor-targeted drugs.
  • the present invention discloses a series of boron phthalocyanine-gefitinib complexes linked by micro-acid environmentally sensitive bonds outside the tumor tissue.
  • the complex In the normal tissue environment, due to the interaction between the boron phthalocyanine group and the gefitinib group, the complex has low cytotoxicity, but in the extracellular micro-acid environment of the tumor tissue, it can simultaneously release the pole by hydrolysis.
  • High photosensitivity of active boron phthalocyanine fragments and high EGFR tyrosine kinase inhibitory activity of gefitinib derivative fragments can be prepared as a dual-targeted anti-cancer drug for tumor cell extracellular micro-acid environment and EGFR tyrosine kinase dual targeting.
  • the invention discloses a series of micro acid environment sensitive ketal-linked boron phthalocyanine-gefitinib complexes, a preparation method thereof and application in medicine.
  • the present invention relates to a boron phthalocyanine-gefitinib complex represented by the general formula (I), a process for the preparation thereof, and a pharmaceutical composition containing the same, and its use as a photosensitizer, particularly Use in the treatment of cancer.
  • the complex In the normal tissue environment, due to the interaction between the boron phthalocyanine group and the gefitinib group, the complex has low cytotoxicity, but in the extracellular micro-acid environment of the tumor tissue, the ketal is hydrolyzed.
  • the boron phthalocyanine fragment obtained by hydrolysis exhibits extremely high photosensitivity, and the gefitinib derivative obtained by hydrolysis can be used as an epidermal growth factor receptor (EGFR) tyrosinase inhibitor to inhibit tumor growth. They can be prepared as a dual-targeted anti-cancer drug for tumor cell extracellular micro-acid environment and EGFR tyrosine kinase dual targeting.
  • EGFR epidermal growth factor receptor
  • n 1, 2, 3, or 4;
  • n 1, 2, 3, or 4;
  • the compound of formula (I) is structurally stable under normal tissue extracellular pH 7.4. Due to the interaction of the boron phthalocyanine group and the gefitinib group, the complex has lower cytotoxicity.
  • the ketal bond is unstable, and the compound of the formula (I) can be hydrolyzed to obtain the compound of the formula (II) and the formula (III).
  • the structure of the compound of the formula (II) is identical and similar to that of the compounds reported in the literature (Org. Biomol. Chem., 2007, 5, 3987-3992), and these compounds have high uptake rates in tumor cells at very low concentrations. It exhibits very high photosensitivity.
  • Compound (III) is a compound very similar to the structure of gefitinib, which can exhibit the inhibition of epidermal growth factor receptor tyrosine kinase and inhibit tumor growth.
  • Typical compounds of the invention include, but are not limited to:
  • the present invention also provides a process for the preparation of the compound of the formula (I), the reaction equation being as follows, but not limited to the following methods:
  • n 1, 2, 3, or 4;
  • the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform and tetrahydrofuran; the reaction is carried out at a temperature of -5 to 80 ° C;
  • the basic condition is provided by at least one agent selected from the group consisting of pyridine, triethylamine, sodium hydride and 4-N,N-lutidine; the molar ratio of the material a to p-toluenesulfonyl chloride is 1: 0.2 to 2.
  • the solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, ethanol, and methanol; the reaction is carried out at a temperature of 30 to 120 ° C; and the basic condition is selected from the group consisting of pyridine Providing at least one of triethylamine, potassium carbonate, and sodium carbonate; the molar ratio of the intermediate a to the material b is 1:0.3-3.
  • the material b reference method is easily synthesized from gefitinib (Tetrhedron Letters, 46(43), 7381-7384).
  • the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform and tetrahydrofuran; the reaction is carried out at a temperature of -5 to 60 ° C;
  • the basic condition is provided by at least one agent selected from the group consisting of pyridine, triethylamine, sodium hydride and 4-N,N-lutidine; the molar ratio of the intermediate c to the compound of the formula (IV) is 1 : 0.5 to 2.
  • the solvent is selected from the group consisting of toluene and xylene; the reaction is carried out at a temperature of 80 to 160 ° C; and the basic condition is selected from the group consisting of pyridine, triethylamine, sodium hydride and 4-N,N-di A reagent such as methylpyridine is provided; the molar ratio of the chloroboron phthalocyanine to the compound of the formula (V) is 1:0.5-10.
  • the compound can also be purified by methods well known to those skilled in the art, such as by distillation, by silica gel column chromatography or by high performance liquid chromatography (HPLC).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of the formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also relates to the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a photodynamic drug or a photosensitizing drug.
  • the present invention also relates to the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for treating cancer.
  • the cancer described therein is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, cholangiocarcinoma, rectal cancer, colon cancer, skin cancer, head and neck cancer, Eye, uterine and ovarian cancer.
  • the present invention also relates to a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, which is used as a photodynamic drug or a photosensitizing drug.
  • the present invention also relates to a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, which is useful for treating cancer.
  • the cancer described therein is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, breast cancer, bladder cancer, prostate cancer, pancreatic cancer, cholangiocarcinoma, rectal cancer, colon cancer, skin cancer, head and neck cancer, eye tumor, uterine cancer and ovary. cancer.
  • the present invention also relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, and then suitably
  • the light source is illuminated.
  • the suitable light source may be provided by a conventional light source coupled to a suitable filter or by a laser of a particular wavelength, the source having a wavelength in the range of 500 to 900 nm, preferably 550 to 750 nm.
  • the compounds according to the invention may be administered orally, sublingually, parenterally, subcutaneously, Intramuscular administration, intravenous administration, transdermal administration, topical administration, or rectal administration.
  • the active ingredient may be conventionally used.
  • the pharmaceutically acceptable carriers are mixed together and administered to the animal or human in the form of an administration unit.
  • Suitable administration unit forms include oral forms such as tablets, gel capsules, powders, granules and solutions or suspensions for oral administration, sublingual or buccal administration, parenteral, subcutaneous, intramuscular, intravenous, nasal Internal or intraocular administration forms and rectal administration forms.
  • the main active ingredient is mixed with a pharmaceutically acceptable carrier such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • a pharmaceutically acceptable carrier such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like.
  • the tablets may be coated with sucrose or other suitable materials or treated in such a way that they have prolonged or delayed activity and continuously release a predetermined amount of active ingredient.
  • a gel capsule preparation is obtained by mixing the active ingredient with a diluent and by pouring the obtained mixture into a soft or hard capsule.
  • Formulations in the form of syrups or elixirs may contain the active ingredient along with sweetening agents, preservatives, and perfuses, and suitable colorants.
  • the powder or granules which may be dispersed in water may contain the active ingredient in admixture with a dispersing agent, a surfactant, a wetting or suspending agent, and a flavoring or sweetening agent.
  • the pharmaceutical composition contains polyoxyethylene castor oil and its derivatives, dimethyl sulfoxide, ethanol, glycerin, N, N-dimethylformamide, polyethylene glycol 300-3000, cyclodextrin, glucose, Tween One or more of polyethylene glycol monostearate.
  • Suppositories are used for rectal administration, which are prepared using a binder that melts at the rectal temperature, for example, cocoa butter or polyethylene glycol.
  • An aqueous suspension, an isotonic physiological saline solution or a sterile and injectable solution comprising a pharmacologically compatible dispersant and/or wetting agent for parenteral, intranasal or intraocular administration Apply.
  • the pharmaceutical composition contains polyoxyethylene castor oil and its derivatives, dimethyl sulfoxide, ethanol, glycerin, N, N-dimethylformamide, polyethylene glycol 300-3000, cyclodextrin, glucose, Tween One or more of polyethylene glycol monostearate.
  • the active ingredient (possibly together with one or more additive carriers) can also be formulated as a microcapsule.
  • the compounds of the invention can be administered at doses between 0.01 mg/day and 5000 mg/day, in a single dose/day manner or in several doses throughout the day, for example, the same dose twice daily. .
  • the daily dose administered is advantageously between 0.1 mg and 200 mg, even more advantageously between 2.5 mg and 50 mg. It may be desirable to use dosages outside of these ranges, as will be appreciated by those skilled in the art.
  • the pharmaceutical composition may also be formulated for external administration. It can be introduced into the usual form of the application type (i.e., especially lotions, foams, gels, dispersants, sprays), the usual forms having excipients, in particular excipients It is able to penetrate the skin in order to improve the properties and accessibility of the active ingredients.
  • these compositions generally further comprise a physiologically acceptable medium, which usually comprises water or a solvent, for example an alcohol, an ether or an ethylene glycol.
  • the composition may further comprise a surfactant, a preservative, a stabilizer, an emulsifier, a thickener, other active ingredients that produce a complementary effect or a possible synergistic effect, trace elements, fine Oil, flavor, colorant, collagen, chemical or mineral filter.
  • pharmaceutically acceptable is understood to mean that it is used in the preparation of a pharmaceutical composition which is generally safe, non-toxic, biologically or otherwise satisfying needs and said combination Objects can be accepted for use in mammals and humans.
  • a "pharmaceutically acceptable salt” of a compound is understood to mean a salt which is a pharmaceutically acceptable (as defined herein) salt and which possesses the desired pharmacological activity of the parent compound.
  • This salt includes:
  • Acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid , fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, sticky
  • a metal ion such as an alkali metal ion (for example, Na + , K + or Li + ), an alkaline earth metal ion (such as Ca 2+ or Mg 2+ ) or aluminum ion.
  • a salt formed when coordinated with an organic or inorganic base include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like.
  • Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Ts is p-toluenesulfonate.
  • TsCl is p-toluenesulfonyl chloride.
  • the experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the raw material or the manufacturer of the commodity.
  • Reagents without specific source are routine reagents purchased from the market.
  • the material b reference method is easily synthesized from gefitinib (Tetrhedron Letters, 46(43), 7381-7384).
  • Nuclear Magnetic Resonance Instrument Bruker ARX-400 high resolution high resolution nuclear magnetic resonance instrument.
  • Mass Spectrometry QSTAR Elite tandem quadrupole time-of-flight mass spectrometer.
  • MTT test instrument Thermo Scientific Multiskan GO full wavelength microplate reader.
  • PBS buffer phosphate buffer
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR chemical shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • the solvent was determined to be deuterated dimethyl sulfoxide (DMSO-d 6 ), and the internal standard was tetramethylsilane (TMS).
  • TMS tetramethylsilane
  • s is a single peak
  • bs is a broad single peak
  • d is a doublet
  • t is a triplet
  • qdt is a quartet
  • m is a multiple or a large number of peaks
  • dd is a doublet.
  • the thin layer chromatography silica gel plate uses Qingdao GF254 silica gel plate, and the silica gel plate used for thin layer chromatography (TLC) has a specification of 0.15 mm to 0.2 mm, and the thin layer chromatography separation and purification product adopts a specification of 0.4 mm to 0.5 mm.
  • the solution in the reaction means an aqueous solution unless otherwise specified.
  • the temperature of the reaction was room temperature unless otherwise specified.
  • Test Example 2 In vitro anti-tumor cell photosensitivity test
  • Test sample compound 2, compound 4 and compound of formula (III) of the present invention
  • Test cells human lung cancer cell PC9
  • DMEM complete medium Add 500,000 U of penicillin/streptomycin, 56 mL of fetal bovine serum to 500 mL DMEM liquid medium (GIBCO), and mix.
  • MTT solution MTT: 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, purchased from MP Company, USA: 5 mg/mL of powdered MTT The concentration is dissolved in PBS solution, sterilized by filtration, and is now ready for use.
  • the test sample or DMSO was used to prepare a mother liquor with a concentration of 1 mM; 100 ⁇ L of 1 mg/mL mother liquor was taken during the experiment, and 1.15 mL of 0.5% (w/w) polyoxyethylene castor oil pH 7.4 PBS and pH 6.5 PBS buffer were added to prepare. 80 ⁇ g / mL of the drug solution, and diluted with the corresponding PBS buffer into a different concentration of the drug solution, the pH of the drug solution is kept unchanged during the dilution process, the drug solution is allowed to stand at room temperature for 24 hours, the pH value is adjusted to 7.4 After the cell dosing culture. The final concentration of DMSO in each drug and negative control group was ⁇ 1%.
  • Adherent tumor cells in logarithmic growth phase were selected and digested with trypsin, and mixed with DMEM medium containing 10% fetal bovine serum to prepare a suitable concentration of cell suspension, inoculated in 96-well culture plate at 37 ° C, 5% Incubate for 24 hours under CO 2 conditions. Then, 100 ⁇ L of each test drug, solvent and culture solution of different concentrations were added, respectively, and 3 parallel holes per group. After mixing, it was divided into two groups: light and dark. After co-cultivation for 2 hours, the medium was discarded, and the culture medium containing no test sample or medium was added to maintain the culture at 37 ° C and 5% CO 2 . 24 hours.
  • the light source is connected to the insulated water tank by a 200W halogen lamp and a filter larger than 610 nm, and the light dose is 48 J cm -2 .
  • tumor cell growth inhibition rate (%) [(negative control group OD mean - administration group OD mean) / negative control group OD mean value] x 100%.
  • the calculation of the half-inhibitory concentration IC 50 was determined by logit regression.
  • Solid tumors have a slightly acidic environment, such as lung cancer, stomach cancer, esophageal cancer, and milk.
  • Solid tumors such as adenocarcinoma, bladder cancer, prostate cancer, pancreatic cancer, cholangiocarcinoma, rectal cancer, colon cancer, skin cancer, head and neck cancer, eye tumor, uterine cancer and ovarian cancer all have a slightly acidic environment, and the present patent discloses
  • the compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, can be prepared as a photosensitizing drug for the treatment of the above cancer.

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Abstract

本发明涉及微酸环境敏感的缩酮连接的吉非替尼轴向取代硼亚酞菁配合物及其制备方法和在医药上的应用。特别地，本发明涉及通式(I)所示的硼亚酞菁-吉非替尼配合物及其制备方法及含有该配合物的药物组合物，以及其作为光敏剂的用途，特别是在治疗癌症中的用途，其中通式(I)中的各取代基与说明书中的定义相同。在正常组织环境下由于硼亚酞菁基团和吉非替尼基团的相互影响，该配合物有较低的细胞毒性，但在肿瘤组织细胞外微酸环境下，缩酮发生水解反应，水解产物硼亚酞菁碎片展现极高的光敏活性，而水解得到的吉非替尼衍生物可以作为表皮生长因子受体(EGFR)酪氨酸酶抑制剂，抑制肿瘤生长。它们可制备成肿瘤细胞外微酸环境和EGFR酪氨酸激酶双靶向的光动力治疗和化疗双疗效抗癌药物。

Description

酸敏感的硼亚酞菁-吉非替尼配合物及其制备方法和在医药上的应用 技术领域
本发明属于医药领域，涉及硼亚酞菁-吉非替尼配合物及其制备方法及其在医药上的应用，本发明公开了其作为光动力治疗-化疗双疗效药物，用于治疗癌症的用途。
技术背景
光动力治疗(Photodynamic Therapy，简称PDT)，又称光辐射疗法(Photoradiation Therapy，简称PRT)或称光化学疗法(Photochemotherapy)，是一种基于特定化学物质的光化学反应原理的治疗方法。所用的化学物质称为肿瘤化学诊治药物(也称光敏剂，Photosensitizer，简称PS)。PDT疗法过程是通过静脉注射将光敏剂注入体内(对于皮肤也可以将其涂于患处)，经过一定时间后用特定波长的光照射肿瘤组织，富集在肿瘤组织的光敏剂在光的激发下，产生一系列光物理化学反应，产生细胞毒性的活性氧，从而杀死癌细胞破坏肿瘤组织。
1996年被美国FDA批准用于临床，1997年FDA将其列入肿瘤治疗的五类基本方法(手术、放疗、化疗、光动力、生化免疫)之一。和传统的疗法相比，PDT疗法具有创伤很小、毒性低微、选择性好、适用性好、可重复治疗、可姑息治疗、可协同手术提高疗效、可消灭隐性癌病灶、可保护容貌及重要器官功能、治疗时间短等优势。目前光动力疗法已成功应用于肺癌、胃癌、食管癌、乳腺癌、膀胱癌、前列腺癌、胰腺癌、胆管癌、直肠癌、结肠癌、皮肤癌、头颈部癌症、眼肿瘤、子宫癌和卵巢癌的治疗。
近年来以表皮生长因子受体(epidermal growth factor receptor，EGFR)靶点的抗癌药物越来越受到到关注。EGFR是一种膜受体，在多种恶性肿瘤如神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、前列腺癌、肝癌、结肠癌、胃癌等中都有过度表达，激活EGFR会加快肿瘤细胞繁殖，促进肿瘤血管生成，加速肿瘤转移，阻碍肿瘤凋亡。
吉非替尼一种EGFR酪氨酸激酶抑制剂，是一种治疗肿瘤的靶向药物。2002年首次于日本上市，2003年5月在美国及澳大利亚获准作为三线单一治疗药物用于晚期非小细胞肺癌。2005年正式在中国上市，已用于临床治疗局部晚期或转移性非小细胞肺癌。
Figure PCTCN2017103185-appb-000001
肿瘤实体组织部位存在的缺氧微环境导致该部位肿瘤细胞外的pH值较低(在 6.5左右)，而正常组织细胞外pH值为7.4左右。肿瘤实体组织和正常组织之间的pH值差异为肿瘤靶向药物的设计提供了新的策略。
本发明公开了一系列通过肿瘤组织细胞外的微酸环境敏感键连接的硼亚酞菁-吉非替尼配合物。在正常组织环境下由于硼亚酞菁基团和吉非替尼基团的相互影响，该配合物有较低的细胞毒性，但在肿瘤组织细胞外微酸环境下，能通过水解同时释放极高的光敏活性硼亚酞菁碎片分和高EGFR酪氨酸激酶抑制活性吉非替尼衍生物碎片。它们可制备成肿瘤细胞外微酸环境和EGFR酪氨酸激酶双靶向的光动力治疗和化疗双疗效抗癌药物。
发明内容
本发明公开了一系列微酸环境敏感的缩酮连接的硼亚酞菁-吉非替尼配合物及其制备方法和在医药上的应用。特别地，本发明涉及通式(I)所示的硼亚酞菁-吉非替尼配合物及其制备方法及含有该配合物的药物组合物，以及其作为光敏剂的用途，特别是在治疗癌症中的用途。在正常组织环境下由于硼亚酞菁基团和吉非替尼基团的相互影响，该配合物有较低的细胞毒性，但在肿瘤组织细胞外微酸环境下，缩酮发生水解反应，水解得到的硼亚酞菁碎片展现极高的光敏活性，而水解得到的吉非替尼衍生物可以作为表皮生长因子受体(EGFR)酪氨酸酶抑制剂，抑制肿瘤生长。它们可制备成肿瘤细胞外微酸环境和EGFR酪氨酸激酶双靶向的光动力治疗和化疗双疗效抗癌药物。本发明提供的一种通式(I)所示的化合物：
Figure PCTCN2017103185-appb-000002
其中：n＝1，2，3，或4；
或其药学上可接受的盐。
通式(I)所示的化合物在肿瘤组织微酸环境下的水解反应化学式(1)
Figure PCTCN2017103185-appb-000003
其中：n＝1，2，3，或4；
                         --------------------------(1)
在正常组织细胞外pH值为7.4环境下式(I)化合物结构稳定。由于硼亚酞菁基团和吉非替尼基团的相互影响，该配合物有较低的细胞毒性。
但在肿瘤组织细胞外的pH值较低(在6.5左右)环境下，缩酮键不稳定，通式(I)能水解得到式(II)和式(III)化合物。通式(II)化合物的结构与已有文献报道的化合物结构相同和类似(Org.Biomol.Chem.，2007，5，3987-3992)，这些化合物在肿瘤细胞摄取率高，在很低的浓度下展现非常高的光敏活性。而化合物(III)是与吉非替尼结构极为类似的化合物，能展现表皮生长因子受体络氨酸激酶抑制作用，抑制肿瘤生长。
本发明典型的化合物包括，但不限于：
Figure PCTCN2017103185-appb-000004
Figure PCTCN2017103185-appb-000005
或其药学上可接受的盐。
本发明还提供一种制备通式(I)所示的化合物的方法，反应方程式如下，但不仅限于下列方法：
Figure PCTCN2017103185-appb-000006
其中：n＝1，2，3，或4；
第1步，所述有机溶剂选自N，N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、三氯甲烷和四氢呋喃；所述反应在-5～80℃温度下进行；所述碱性条件由选自吡啶、三乙胺、氢化钠和4-N，N-二甲基吡啶中的至少一种试剂提供；所述物料a与对甲基苯磺酸酰氯的摩尔比为1∶0.2～2。
第2步，所述溶剂选自N，N-二甲基甲酰胺、二甲基亚砜、乙醇、甲醇；所述反应在30～120℃温度下进行；所述碱性条件由选自吡啶、三乙胺、碳酸钾、碳酸钠中的至少一种试剂提供；所述中间体a与物料b的摩尔比为1∶0.3～3。物料b参考文献方法以吉非替尼为原料很容易合成得到(Tetrhedron Letters，46(43)，7381-7384)。
第3步，所述有机溶剂选自N，N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、三氯甲烷和四氢呋喃；所述反应在-5～60℃温度下进行；所述碱性条件由选自吡啶、三乙胺、氢化钠和4-N，N-二甲基吡啶中的至少一种试剂提供；所述中间体c与式(IV)化合物的摩尔比为1∶0.5～2。
第4步，所述溶剂选自甲苯、二甲苯；所述反应在80～160℃温度下进行；所述碱性条件由选自吡啶、三乙胺、氢化钠和4-N，N-二甲基吡啶等试剂提供；所述氯硼亚酞菁与通式(V)化合物的摩尔比为1∶0.5～10。
如果有必要，通过本领域技术人员熟知的方法，如通过蒸馏、通过硅胶柱色谱法或者通过高效液相色谱法(HPLC)也可以纯化化合物。
本发明还提供一种药物组合物，其含有治疗有效量的通式(I)所示的化合物或或其药学上可接受的盐，以及药学上可接受的载体、稀释剂或赋形剂。
本发明还涉及通式(I)所示的化合物或其药学上可接受的盐，或包含其的药物组合物在制备光动力药物或光敏药物中的用途。
本发明还涉及通式(I)所示的化合物或其药学上可接受的盐，或包含其的药物组合物在制备治疗癌症的药物中的用途。其中所述的癌症选自其中所述的癌症选自肺癌、胃癌、食管癌、乳腺癌、膀胱癌、前列腺癌、胰腺癌、胆管癌、直肠癌、结肠癌、皮肤癌、头颈部癌症、眼肿瘤、子宫癌和卵巢癌。
本发明还涉及通式(I)所示的化合物或其药学上可接受的盐，或包含其的药物组合物，其用作光动力药物或光敏药物。
本发明还涉及通式(I)所示的化合物或其药学上可接受的盐，或包含其的药物组合物，其用于治疗癌症。其中所述的癌症选自肺癌、胃癌、食管癌、乳腺癌、膀胱癌、前列腺癌、胰腺癌、胆管癌、直肠癌、结肠癌、皮肤癌、头颈部癌症、眼肿瘤、子宫癌和卵巢癌。
本发明还涉及一种治疗癌症的方法，其包括给予所需患者治疗有效量的通式(I)所示的化合物或其药学上可接受的盐，或包含其的药物组合物，然后用适宜的光源照射。所述适宜的光源可以由普通光源连接合适的滤光片来提供或由特定波长的激光来提供，光源的波长范围为500～900nm，优选550～750nm。
根据本发明的化合物可以被口服施用、舌下施用、肠胃外施用、皮下施用、 肌内施用、静脉内施用、经皮施用、局部施用或直肠施用。
在本发明的药用化合物中，对于口服施用、舌下施用、肠胃外施用、皮下施用、肌内施用、静脉内施用、经皮施用、局部施用或直肠施用而言，活性成分可以与常规的药用载体混合在一起，以施用单位的形式施用于动物或人类。适合的施用单位形式包含口服形式如片剂、凝胶胶囊剂、粉剂、颗粒剂和口服的溶液剂或混悬剂，舌下或口腔施用形式，肠胃外、皮下、肌内、静脉内、鼻内或眼内施用形式和直肠施用形式。
当固体组合物被制备成片剂形式时，主要活性成分与药用载体如明胶、淀粉、乳糖、硬脂酸镁、滑石、阿拉伯胶等混合。片剂可以采用蔗糖或其他适合的材料包衣或者以如此的方式处理以至于其具有延长的或延迟的活性并且连续释放预定量的活性成分。
通过将活性成分与稀释剂混合并通过将获得的混合物倾倒入软质或硬质胶囊中来获得凝胶胶囊制剂。
糖浆剂或酊剂形式的制剂可以包含活性成分连同甜味剂、防腐剂以及芳香剂和适当的着色剂。
可分散于水中的粉剂或颗粒剂可以包含活性成分，其与分散剂、表面活性剂、润湿剂或悬浮剂以及与矫味剂或甜味剂混合在一起。其药物组合中含有聚氧乙烯蓖麻油及其衍生物、二甲亚砜、乙醇、甘油、N，N-二甲基甲酰胺、聚乙二醇300-3000、环糊精、葡萄糖、吐温、聚乙二醇单硬脂酸酯中的一种或几种。
栓剂用于直肠施用，其采用在直肠温度下熔化的粘合剂，例如，可可脂或聚乙二醇来制备。
水性混悬剂、等渗的生理盐水溶液剂或无菌的且可注射的溶液剂(其包含药理学上可兼容的分散剂和/或润湿剂)用于肠胃外、鼻内或眼内施用。其药物组合中含有聚氧乙烯蓖麻油及其衍生物、二甲亚砜、乙醇、甘油、N，N-二甲基甲酰胺、聚乙二醇300-3000、环糊精、葡萄糖、吐温、聚乙二醇单硬脂酸酯中的一种或几种。
活性成分(可能与一种或多种添加剂载体一起)也可以被配制成微囊剂。
本发明的化合物能够以介于0.01mg/天和5000mg/天之间的剂量来使用，以单一剂量/天的方式来提供或者以全天内若干剂量的方式来施用，例如，相同剂量每天两次。所施用的日剂量有利地介于0.1mg和200mg之间，甚至更有利地介于2.5mg和50mg之间。使用超出这些范围的剂量可能是需要的，本领域技术人员自身将会意识到这一点。
在本发明的一个特定实施方案中，药物组合物也可以被配制用于外部施用。它可以被引入到该施用类型的常用形式(即，特别是洗剂、泡沫剂、凝胶剂、分散剂、喷雾剂)中，所述常用形式具有赋形剂，所述赋形剂特别地能够穿透皮肤，以便于改善活性成分的性质和可接近性。除了根据本发明的组合物之外，这些组合物通常进一步包含生理上可接受的介质，所述介质通常包含水或溶剂，例如，醇、醚或乙二醇。所述组合物还可以包含表面活性剂、防腐剂、稳定剂、乳化剂、增稠剂、产生互补效果或可能的协同效果的其他活性成分、微量元素、精 油、香料、着色剂、胶原蛋白、化学或矿物过滤剂。
定义
除非有相反陈述，否则下列用在说明书和权利要求书中的术语具有下述含义。
在本发明中，“药学上可接受的”被理解为是指其用于制备药物组合物，所述组合物一般是安全的，无毒的，在生物学或其他方面满足需要并且所述组合物可以被接受用于兽类和人类药物用途。
在本发明中，化合物的“药学上可接受的盐”被理解为指代下列盐，其是药学上可接受的(如本文所定义的)盐并且其具备预期的母体化合物的药理活性。这种盐包括：
(1)与无机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸等形成的酸加成盐，或与有机酸如乙酸、苯磺酸、苯甲酸、樟脑磺酸、柠檬酸、乙磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、乙醇酸、羟萘酸、2-羟基乙磺酸、乳酸、马来酸、苹果酸、扁桃酸、甲磺酸、粘康酸、2-萘磺酸、丙酸、水杨酸、琥珀酸、二苯甲酰基-L-酒石酸、酒石酸、对甲苯磺酸、三甲基乙酸、三氟乙酸等形成的酸加成盐；和
(2)当母体化合物中存在的酸质子被金属离子，例如，碱金属离子(例如，Na+、K+或Li+)，碱土金属离子(如Ca2+或Mg2+)或铝离子代替；或者与有机碱或无机碱配位时形成的盐。可接受的有机碱包括二乙醇胺、乙醇胺、N-甲基葡糖胺、三乙醇胺、氨丁三醇等。可接受的无机碱包括氢氧化铝、氢氧化钙、氢氧化钾、碳酸钠和氢氧化钠。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“Ts”为对甲基苯磺酸基。
“TsCl”为对甲基苯磺酸酰氯。
具体实施方式
通过阅读下列实施例，本领域技术人员将会更好地理解本发明。这些实施例仅用于解释本发明。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。物料b参考文献方法以吉非替尼为原料很容易合成得到(Tetrhedron Letters，46(43)，7381-7384)。
核磁共振仪：Bruker ARX-400型高分辨高分辨核磁共振仪。
质谱：QSTAR Elite串联四级杆飞行时间质谱仪。
MTT检测仪器：Thermo Scientific Multiskan GO全波长酶标仪。
PBS缓冲液：磷酸盐缓冲液。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR化学位 移(δ)以10-6(ppm)的单位给出。测定溶剂为氘代二甲亚砜(DMSO-d6)，内标为四甲基硅烷(TMS)。使用下列缩写：s为单峰，bs为宽单峰，d为二重峰，t为三重峰，qdt为四重峰，m为多重峰或大量峰，dd为双二重峰等。
薄层层析硅胶板使用青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。
实施例中的反应进程的监测采用薄层色谱法(TLC)。
实施例1：化合物1的合成
Figure PCTCN2017103185-appb-000007
第1步
在冰水浴中，把物料a(1.64g，10mmol)，对甲苯磺酸酰氯(1.9g，10mmol)和吡啶(1.6g，20.2mmol)加入二氯甲烷(45mL)，温度升到室温，继续搅拌反应15小时，停止反应，向反应液中加入水(300mL)，搅拌静置，收集有机相，有机相用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为二氯甲烷/甲醇(10∶1)，得到白色油状物为中间体a(1.79g，56％)。MS(ESI)：m/z＝319[M+H]+。
第2步
在冰水浴中，把中间体a(1.59g，5mmol)，物料b(2.16g，5mmol)加入N，N-二甲基甲酰胺(50mL)，向反应液中加入无水碳酸钾(1.38g，10mmol)，反应液加热到90℃，搅拌反应15小时后停止反应。反应停止后减压去除有机溶剂，粗产品加入二氯甲烷100mL搅拌溶解，再加入水200mL搅拌，静置后收集有机相并用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为二氯甲烷/甲醇(15∶1)，得到褐色固体为中间体c(1.82g，62％)。1H NMR(400MHz，DMSO-d6)：δ9.50(s，1H，NH)，8.50(s，1H，Ar-H)，8.08-8.15(m，1H，Ar-H)，7.70-7.83(m，2H，Ar-H)，7.38-7.49(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.10-4.26(m，4H，CH2)，3.55-3.72(m，10H，CH2)，2.44-2.55(m，2H，CH2)，2.34-2.45(m，4H，CH2)，1.94-2.05(m，2H，CH2)，1.41(s，6H，CH3)。MS(ESI)：m/z＝579[M]+。
第3步
在冰水浴中，把化合物c(1.2g，2mmol)，1-1(0.43g，2mmol)加入四氢呋喃(30mL)，缓慢向反应液中加入氢化钠(0.15g，6.3mmol)，反应液加热到40℃继续搅拌反应5小时，缓慢加入5毫升水淬灭反应，减压蒸馏除去有机溶剂，再加入水(100mL)和二氯甲烷(100mL)，搅拌静置，收集有机相。有机相用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为三氯甲烷/甲醇(10∶1)，得到褐色固体为化合物1-2(0.78g，63％)。1H NMR(400MHz，DMSO-d6)：δ9.50(s，1H，NH)，8.50(s，1H，Ar-H)，8.09-8.15(m，1H，Ar-H)，7.71-7.83(m，2H，Ar-H)，7.39-7.50(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.10-4.27(m，4H，CH2)，3.51-3.72(m，14H，CH2)，2.43-2.55(m，2H，CH2)，2.34-2.45(m，4H，CH2)，1.93-2.05(m，2H，CH2)，1.41(s，6H，CH3)。MS(ESI)：m/z＝623[M]+。
第4步
氯硼亚酞菁(0.43g，1mmol)，化合物1-2(0.31g，5mmol)加入到甲苯(25mL)中，向反应溶液中慢慢加入三乙胺(0.5mL)，温度升至120℃反应24小时，减压蒸出溶剂，固体残渣加入水(100mL)和二氯甲烷(100mL)，萃取，收集有机相，用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为二氯甲烷/甲醇(20∶1)，得到红褐色固体1(0.18g，18％)。1H NMR(400MHz，DMSO-d6))：δ9.57-9.66(m，6H，SPc-Hα)，8.50(s，1H，Ar-H)，8.30-8.38(m，6H，SPc-Hβ)，8.08-8.15(m，1H，Ar-H)，7.70-7.83(m，2H，Ar-H)，7.38-7.49(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.10-4.26(m，4H，CH2)，3.55-3.72(m，6H，CH2)，3.27-3.35(m，2H，CH2)，3.02-3.10(m，2H，CH2)，2.34-2.60(m，8H，CH2)，1.94-2.05(m，2H，CH2)，1.63-1.69(m，2H，CH2)，1.31(s，6H，CH3)。MS(ESI)：m/z＝1017[M]+。
实施例2：化合物2的合成
Figure PCTCN2017103185-appb-000008
中间体C按实施例1的方法合成得到。
第1步
在冰水浴中，把化合物c(1.2g，2mmol)，2-1(0.78g，3mmol)加入四氢呋喃(30mL)，缓慢向反应液中加入氢化钠(0.15g，6.3mmol)，反应液加热到40℃继续搅拌反应5小时，缓慢加入5毫升水淬灭反应，减压蒸馏除去有机溶剂，再加入水(100mL)和二氯甲烷(100mL)，搅拌静置，收集有机相。有机相用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为三氯甲烷/甲醇(10∶1)，得到褐色固体为化合物2-2(0.55g，41％)。1H NMR(400MHz，DMSO-d6)：δ9.50(s，1H，NH)，8.50(s，1H，Ar-H)，8.08-8.15(m，1H，Ar-H)，7.70-7.83(m，2H，Ar-H)，7.38-7.50(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.12-4.27(m，4H，CH2)，3.48-3.72(m，18H，CH2)，2.43-2.54(m，2H，CH2)，2.34-2.45(m，4H，CH2)，1.93-2.07(m，2H，CH2)，1.41(s，6H，CH3)。MS(ESI)：m/z＝667[M]+。
第2步
氯亚硼酞菁(0.43g，1mmol)，化合物2-2(0.62g，10mmol)加入到甲苯(30mL)中，向反应溶液中慢慢加入氢化钠(0.15g，6.3mmol)，温度升至120℃反应24小时，减压蒸出溶剂，固体残渣加入100毫升三氯甲烷和100毫升水，萃取，收集有机相，用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为三氯甲烷/甲醇(10∶1)，得到红褐色固体2(0.18g，17％)。1H NMR(400MHz，DMSO-d6))：δ9.56-9.66(m，6H，SPc-Hα)，8.50(s，1H，Ar-H)，8.31-8.38(m，6H，SPc-Hβ)，8.07-8.14(m，1H，Ar-H)，7.71-7.83(m，2H，Ar-H)，7.36-7.49(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.11-4.26(m，4H，CH2)，3.50-3.72(m，10H，CH2)，3.27-3.35(m，2H，CH2)，3.01-3.10(m，2H，CH2)，2.33-2.60(m，8H，CH2)，1.94-2.05(m，2H，CH2)，1.63-1.69(m，2H，CH2)，1.39(s，6H，CH3)。MS(ESI)：m/z＝1061[M]+。
实施例3：化合物3的合成
Figure PCTCN2017103185-appb-000009
中间体C按实施例1的方法合成得到。
第1步
在冰水浴中，把化合物c(1.2g，2mmol)，3-1(1.04g，3mmol)加入四氢呋喃(30mL)，缓慢向反应液中加入氢化钠(0.15g，6.3mmol)，反应液加热到50℃继续搅拌反应5小时，缓慢加入5毫升水淬灭反应，减压蒸馏除去有机溶剂，再加入水(100mL)和二氯甲烷(100mL)，搅拌静置，收集有机相。有机相用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为三氯甲烷/甲醇(10∶1)，得到褐色固体为化合物3-2(0.59g，39％)。1H NMR(400MHz，DMSO-d6)：δ9.50(s，1H，NH)，8.50(s，1H，Ar-H)，8.07-8.15(m，1H，Ar-H)，7.70-7.83(m，2H，Ar-H)，7.38-7.50(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.13-4.27(m，4H，CH2)，3.48-3.72(m，26H，CH2)，2.42-2.54(m，2H，CH2)，2.35-2.43(m，4H，CH2)，1.93-2.05(m，2H，CH2)，1.41(s，6H，CH3)。MS(ESI)：m/z＝755[M]+。
第2步
氯硼亚酞菁(0.43g，1mmol)，化合物3-2(0.38g，5mmol)加入到甲苯(30mL)中，向反应溶液中慢慢加入氢化钠(0.2g，8.3mmol)，反应温度升至120℃反应12小时，减压蒸出溶剂，固体残渣加入100毫升三氯甲烷和100毫升水，萃取，收集有机相，用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为三氯甲烷/甲醇(10∶1)，得到红褐色固体3(0.14g，12％)。1H NMR(400MHz，DMSO-d6))：δ9.56-9.66(m，6H，SPc-Hα)，8.50(s，1H，Ar-H)，8.31-8.38(m，6H，SPc-Hβ)，8.07-8.14(m，1H，Ar-H)，7.71-7.83(m，2H，Ar-H)，7.37-7.49(m，1H，Ar-H)，7.20(s，1H，Ar-H)，4.10-4.26(m，4H，CH2)，3.50-3.73(m，18H，CH2)，3.27-3.32(m，2H，CH2)，3.02-3.10(m，2H，CH2)，2.31-2.60(m，8H，CH2)，1.94-2.05(m，2H，CH2)，1.63-1.69(m，2H，CH2)，1.41(s，6H，CH3)。MS(ESI)：m/z＝1149[M]+。
测试例1：化合物2的水解反应
Figure PCTCN2017103185-appb-000010
取化合物2(110mg，0.1mmol)溶解在50mL四氢呋喃中，再加入50mL pH 6.5磷酸盐缓冲液(PBS溶液)。保持溶液pH值为6.5，室温搅拌反应24小时。减压蒸馏出有机溶剂后加入二氯甲烷(200mL)，震荡静置，收集有机相。有机相用无水硫酸钠干燥，减压蒸馏，粗产品用硅胶层析柱分离纯化，洗脱剂为二氯甲烷/甲醇(20∶1)，得到红固体化合物4(23mg，40％)和褐色固体式(III)化合物(21mg，44％)。
化合物4：1H NMR(400MHz，DMSO-d6)：δ8.81-8.84(m，6H，SPc-Hα)，7.84-7.95(m，6H，SPc-Hβ)，3.67-3.72(m，4H，CH2)，3.42-3.48(m，4H，CH2)，3.25-3.29(m，4H，CH2)，3.01-3.06(m，4H，CH2)，2.54-2.58(m，4H，CH2)，，1.62-1.66(m，4H，CH2)；MS(ESI)：m/z＝567[M+Na]+。
式(III)化合物：1H NMR(400MHz，DMSO-d6)：δ9.50(s，1H，NH)，8.50(s，1H，Ar-H)，8.07-8.14(m，1H，Ar-H)，7.70-7.85(m，2H，Ar-H)，7.37-7.49(m，1H，Ar-H)，7.20(s，1H，Ar-H)，3.96-4.26(m，6H，CH2)，3.55-3.72(m，4H，CH2)，2.94-2.55(m，2H，CH2)，2.34-2.45(m，4H，CH2)，1.94-2.05(m，2H，CH2)。MS(ESI)：m/z＝477[M+H]+。
测试例2：体外抗肿瘤细胞光敏实验
供试品：本发明化合物2，化合物4和式(III)化合物
测试细胞：人肺癌细胞PC9
主要试剂：1)DMEM完全培养液：于500mL DMEM液体培养液(GIBCO公司)中加入青霉素/链霉素10万U，胎牛血清56mL，混匀。2)MTT溶液(MTT：3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐，购于美国MP公司)：将粉状MTT以5mg/mL的浓度溶于PBS溶液，过滤灭菌，现配现用。
实验方法：
将供试品或用DMSO配成浓度为1mM的母液；实验时取100μL1mg/mL的母液，加入1.15mL 0.5％(w/w)聚氧乙烯蓖麻油pH 7.4PBS和pH 6.5PBS缓冲液，配制成80μg/mL药液，并用相对应的PBS缓冲液稀释成不同浓度的药液，稀释过程中保持药液pH值不变，药液配制后在室温静置24小时，调节pH值均为7.4后进行细胞加药培养。各药物和阴性对照组中DMSO的终浓度是≤1％。选用对数生长期的贴壁肿瘤细胞，用胰酶消化后，用含10％胎牛血清的DMEM培养基配成适合浓度的细胞悬液，接种在96孔培养板中在37℃，5％CO2条件下培养24小时。然后分别加入不同浓度的受试药物、溶剂和培养液各100μL，每组3个平行孔。混匀后分为光照和避光两组，均在加药共培养2小时后，弃去培养基，重新加入不含供试品或的培养基置37℃、5％CO2条件下继续培养24小时。24小时后，每孔加入5mg/mL MTT，20μL，37℃、5％CO2条件下孵育4小时后，吸弃上清液，每孔加入200μL DMSO，振荡10分钟，酶标仪检测吸光值，测定波长570nm，激发波长630nm。光源通过200W的卤素灯连接隔热水槽加大于610nm的滤光片提供，光剂量为48J cm-2。
药物对肿瘤细胞生长的抑制率的计算方法：肿瘤细胞生长抑制率(％)＝[(阴性对照组OD均值-给药组OD均值)/阴性对照组OD均值]×100％。半数抑制浓度IC50的计算，采用logit回归法测定。
实验结果：
表1药物在照光时人肺癌细胞PC9的IC50(nM)值
药物 细胞pH 7.4药液培养 细胞pH 6.5药液培养
化合物2 ＞5000 28
化合物4 120 145
式(III)化合物 85 78
实验结果显示，在避光环境下，所有测试的化合物在浓度高达5000nM时，没有显示细胞毒性，但在照光情况化合物4和式(III)化合物在pH 6.5和pH 7.4时对人肺癌细胞PC9的半致死浓度IC50值在78-145nM之间。但化合物2在pH 7.4的药液培养细胞时，浓度高达5000nM，在照光时完全没有光敏活性，但化合物2在pH 6.5的药液进行培养细胞时，展现非常高的光敏活性，IC50值为28nM，这是由于在肿瘤微酸环境下化合物2水解得到高细胞毒性化合物4和(III)化合物。
目前已知，几乎所有的实体肿瘤都存在微酸环境，如肺癌、胃癌、食管癌、乳 腺癌、膀胱癌、前列腺癌、胰腺癌、胆管癌、直肠癌、结肠癌、皮肤癌、头颈部癌症、眼肿瘤、子宫癌和卵巢癌等实体肿瘤均存在微酸环境，本专利公开的化合物或其药学上可接受的盐，或包含其的药物组合物均可以制备成光敏药物治疗上述癌症。
以上所述仅为本发明的实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2017103185-appb-100001
    其中：n＝1,2,3,或4；
    或其药学上可接受的盐。
  2. 根据权利要求1所述的通式(I)所示的化合物，其选自：
    Figure PCTCN2017103185-appb-100002
  3. 一种药物组合物，其含有治疗有效量的根据权利要求1～2中任意一项所述的通式(I)所示的化合物以及药学上可接受的载体。
  4. 根据权利要求1～2中任意一项所述的通式(I)所示的化合物或根据权利要求3所述的药物组合物在制备光动力药物或光敏药物中的用途。
  5. 根据权利要求1～2中任意一项所述的通式(I)所示的化合物或根据权利要求3所述的药物组合物在制备治疗癌症的药物中的用途。
  6. 根据权利要求5所述的用途，其中所述的癌症选自肺癌、胃癌、食管癌、乳腺癌、膀胱癌、前列腺癌、胰腺癌、胆管癌、直肠癌、结肠癌、皮肤癌、头颈部癌症、眼肿瘤、子宫癌和卵巢癌。
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CN103626781A (zh) * 2013-12-12 2014-03-12 福州大学 一种靶向抗癌分子吉非替尼酞菁轭合物及其制备和应用
CN106749478A (zh) * 2016-11-11 2017-05-31 深圳市声光动力生物医药科技有限公司 pH敏感的1,4‑二取代酞菁锌配合物及其制备方法和在医药上的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103626781A (zh) * 2013-12-12 2014-03-12 福州大学 一种靶向抗癌分子吉非替尼酞菁轭合物及其制备和应用
CN106749478A (zh) * 2016-11-11 2017-05-31 深圳市声光动力生物医药科技有限公司 pH敏感的1,4‑二取代酞菁锌配合物及其制备方法和在医药上的应用

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