WO2021022984A1 - 一种偶氮杯芳烃药用辅料及其用途 - Google Patents

一种偶氮杯芳烃药用辅料及其用途 Download PDF

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WO2021022984A1
WO2021022984A1 PCT/CN2020/102054 CN2020102054W WO2021022984A1 WO 2021022984 A1 WO2021022984 A1 WO 2021022984A1 CN 2020102054 W CN2020102054 W CN 2020102054W WO 2021022984 A1 WO2021022984 A1 WO 2021022984A1
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cac4a
azocalixarene
drugs
drug
treatment
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French (fr)
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郭东升
刘阳
王宏磊
黄帆
张天行
张展展
岳宇昕
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唐山天泉科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
    • C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
    • C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
    • C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This application relates to the field of pharmaceuticals, in particular to a pharmaceutical composition comprising an azocalixarene supramolecular compound as a targeted pharmaceutical adjuvant for hypoxia response, and a new use of the azocalixarene supramolecular compound in pharmacy.
  • Hypoxia is one of the important pathological and physiological characteristics of many diseases, such as cancer (Nature Reviews Cancer, 2011, 11:393-410) and myocardial infarction (N.Engl.J.Med., 2017,377:1240-1249) , Stroke (Age and Aging, 2002, 31: 10-12), Atherosclerosis (Curr. Opin. Lipidol. 2009, 20: 409-14), Rheumatoid arthritis (J. Biomed Sci., 2016, 23:62), inflammatory bowel disease (J. Inflamm Res. 2014, 7: 113-120), chronic hypoxic lung disease (Lancet Respir Med. 2016, 4: 225), chronic kidney disease (Int. J. Mol. Sci. 2017, 18: 950) and so on.
  • hypoxia is also an effective target for these diseases.
  • a common response strategy is to covalently link the drug with hypoxia-responsive groups such as nitro, quinone, and azo. In the hypoxic environment, the response group is reduced, causing the release of the drug.
  • hypoxia-responsive groups such as nitro, quinone, and azo.
  • the response group is reduced, causing the release of the drug.
  • covalent strategies face problems such as complex molecular design, time-consuming and high cost synthesis and separation, and changes in activity and toxicity caused by covalent linkage.
  • Azocalixarene compounds are an important class of functional calixarene derivatives, which have been widely used in metal ion complexation (see CN108404854A; Lilin Lu et al., Analytica Chimica Acta 535(2005):183-187; Tae Hyun Kim et al. Human, Bull. Korean Chem. Soc. 2013, Vol. 34, No. 11: 3377-3380), organic small molecule binding (J. Cameron Tyson et al., Journal of Inclusion Phenomena and Molecular Recognition in Chemistry 29 (1997): 109-118), ion selective electrodes (Jianquan Lu et al., Journal of Electroanalytical Chemistry, 528 (2002), 33-38), dyes (Shobhana K.
  • this azocalixarene body with accurate structure, fixed molecular weight, stable batch synthesis, easy derivatization and unique cavity bonding properties as a drug-carrying molecular container platform that responds to hypoxia. Targeted drug release in hypoxic areas of the body.
  • An object of the present invention is to provide a hypoxia-responsive targeted drug-loaded molecular container platform, thereby improving the effectiveness, targeting and safety of active drug molecule delivery.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one pharmaceutically active substance and an azocalixarene compound of formula (I):
  • n 4 or 5;
  • R 1 is selected from H, C 1-6 alkyl and -(CH 2 CH 2 O) m CH 3 , wherein m is an integer selected from 1-3;
  • n is 4 preferably.
  • R 1 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopropyl, neopentyl, tert-pentyl , Pent-3-yl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 3,3-di Methyl butyl and so on.
  • R 2 is selected from -COOM and -SO 3 M, wherein M is independently selected from H and Na; more preferably, R 2 is selected from -COOH, -SO 3 H and -SO 3 Na.
  • the pharmaceutically active substance used for targeted administration using the drug-loaded molecular container platform of the present invention is selected from drugs for the treatment of one or more of the following diseases: cancer, myocardial infarction, stroke, atherosclerosis Sclerosis, rheumatoid arthritis, inflammatory bowel disease, chronic hypoxic lung disease, and chronic kidney disease.
  • the drug for treating cancer is selected from: adriamycin, paclitaxel, camptothecin, hydroxycamptothecin, irinotecan, topotecan, oxaliplatin, belotecan, tilatinib and methoxy Estradiol.
  • the drugs used to treat myocardial infarction are selected from: aspirin and clopidogrel.
  • the drugs used for the treatment of stroke are selected from: aspirin, heparin, alteplase, nimodipine, ropizole, tirazat and elirodil.
  • the drugs for treating atherosclerosis are selected from: lovastatin and ezetimibe.
  • the drug for treating rheumatoid arthritis is selected from: methotrexate, tofacitinib, baritinib, etanercept, abatacept, leflunomide, hydroxychloroquine and sulfasalazine.
  • the drugs for the treatment of inflammatory bowel disease are selected from: Mesalazine, Azathioprine, Mercaptopurine, Methotrexate, Infliximab, Adalimumab, Certuzumab, Natalizumab and Thali degree.
  • the drug for the treatment of chronic hypoxic lung disease is selected from: tadalafil, lioxigua and treprostinil.
  • the drugs used to treat chronic kidney disease are selected from: rosastat and vidastat.
  • preferred pharmaceutical active substances include (hydrochloride) doxorubicin, oxaliplatin, paclitaxel, camptothecin, hydroxycamptothecin, irinotecan, (hydrochloride) topotecan, clopidogrel, irirotide, hydroxychloroquine One or more of lovastatin and tadalafil.
  • the invention also relates to the use of the azocalixarene compound of formula (I) in the preparation of hypoxia-responsive targeted drug adjuvants.
  • Azocalixarene compounds are a class of targeted drug excipients that respond to hypoxia, which can improve the solubility and stability of drug molecules and reduce their impact on normal cells.
  • Hypoxia is a typical feature of cancer, myocardial infarction, stroke, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, chronic hypoxic lung disease, chronic kidney disease and other diseases. In order to survive under hypoxic conditions, hypoxic cells often obtain energy through glycolysis, which becomes an effective way to resist hypoxia.
  • Figure 1 shows a schematic diagram of the hypoxia response of the DOX/CAC4A host-guest inclusion complex.
  • Figure 6a shows the UV absorption curve of CAC4A before and after adding SDT
  • Figure 6b shows the fluorescence curve of DOX/CAC4A before and after adding SDT.
  • Figure 7 shows the UV absorption spectrum of CAC4A over time under hypoxic conditions.
  • Figure 8 is a graph showing the effect of different concentrations of CAC4A on cell viability in an MTT cytotoxicity experiment.
  • Figure 9 is a graph showing the effects of different concentrations of DOX and DOX/CAC4A on cell viability in the MTT cytotoxicity experiment.
  • Figures 10a-c show graphs showing the changes of AST, ALP and ALT in the blood of mice in each group after administration of PBS, CAC4A, DOX, and DOX/CAC4A.
  • Figure 11 shows the fluorescence imaging photos of each organ in the SiPcN 2 and SiPcN 2 /CAC4A mice.
  • Figure 12 shows a graph of the statistical analysis of the fluorescence intensity of the tumor in the SiPcN 2 and SiPcN 2 /CAC4A groups of mice.
  • Figure 13a shows the changes in tumor volume of mice in each group after PBS, CAC4A, DOX, and DOX/CAC4A administration
  • Figure 13b shows the changes in body weight of mice in each group after PBS, CAC4A, DOX, and DOX/CAC4A administration.
  • Figure 14a shows the tumor photos of mice in the PBS, CAC4A, DOX, and DOX/CAC4A administration groups
  • Figure 14b shows the tumor weights of the mice in the PBS, CAC4A, DOX, and DOX/CAC4A administration groups.
  • Figure 15 shows confocal micrographs of TUNLE and Ki67 staining of mouse tumors in each group.
  • Figure 16 shows confocal micrographs of H&E staining of mouse tumor tissues in each group.
  • the present invention adopts conventional methods such as mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the technical scope of the art.
  • mass spectrometry NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology
  • nomenclature and laboratory operations and techniques related to the analytical chemistry, synthetic organic chemistry, and medicine and medicinal chemistry described herein are known to those skilled in the art.
  • the aforementioned techniques and steps can be implemented by conventional methods well known in the art and described in various general documents and more specific documents, which are cited and discussed in this specification.
  • alkyl refers to an aliphatic hydrocarbon group, which can be a branched or straight chain alkyl group. According to the structure, the alkyl group may be a monovalent group or a divalent group (ie, an alkylene group). In the present invention, the alkyl group is preferably an alkyl group having 1 to 8 carbon atoms, more preferably a "lower alkyl group” having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. Typical alkyl groups include but are not limited to methyl, ethyl, propyl, butyl, pentyl, hexyl and the like.
  • alkyl includes all possible configurations and conformations of the alkyl group.
  • the "propyl” mentioned herein includes n-propyl and isopropyl
  • butyl includes n-butyl.
  • Pentyl includes n-pentyl, isopropyl, neopentyl, tert-pentyl, and pent-3-yl.
  • alkoxy refers to -O-alkyl, where alkyl is as defined herein. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy and the like.
  • alkoxyalkyl means that an alkyl group as defined herein is substituted with an alkoxy group as defined herein.
  • aromatic group refers to a planar ring having a delocalized ⁇ electron system and containing 4n+2 ⁇ electrons, where n is an integer.
  • the aromatic ring can be composed of five, six, seven, eight, nine, or more than nine atoms.
  • Aromatic groups may be optionally substituted.
  • aryl includes carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaryl”) groups (e.g., pyridine).
  • the term includes monocyclic or fused-ring polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups.
  • aryl means that each atom of the aromatic ring is a carbon atom.
  • the aryl ring can be composed of five, six, seven, eight, nine, or more than nine atoms.
  • Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, phenanthryl, anthracenyl, fluorenyl, and indenyl.
  • the aryl group may be a monovalent group or a divalent group (ie, an arylene group).
  • hydroxyl refers to the -OH group.
  • Carboxy refers to the chemical moiety of formula -COOH.
  • “Ester group” refers to a chemical moiety having the formula -COOR, where R is selected from alkyl, cycloalkyl, aryl and the like.
  • amino refers to the -NH 2 group.
  • alkylamino refers to an amino substituent further substituted by one or two alkyl groups, specifically referring to the group -NRR', wherein R and R'are each independently selected from hydrogen or lower alkyl, with the condition of- NRR' is not -NH 2 .
  • Alkylamino includes groups of compounds in which the nitrogen of -NH 2 is attached to at least one alkyl group. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, and the like.
  • Dialkyl amino includes groups wherein the nitrogen -NH 2 group is connected to at least two additional alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino, diethylamino, and the like.
  • alkylaminoalkyl means that an alkyl group as defined herein is substituted with an alkylamino group as defined herein.
  • hydroxyalkyl or "hydroxyalkyl” refers to an alkyl substituent further substituted with one or more hydroxy groups.
  • sulfo group or "sulfonic acid group” means a functional group -SO 3 H in formula.
  • quaternary ammonium group refers to -N + RR'R", where R, R', and R" are each independently selected from alkyl groups having 1-8 carbon atoms.
  • optional means that one or more events described later may or may not occur, and include both events that occur and events that do not occur.
  • the term “optionally substituted” or “substituted” means that the mentioned group may be substituted by one or more additional groups, which are each and independently selected from alkyl, cycloalkyl , Aryl, heteroaryl, heterocyclyl, hydroxyl, alkoxy, cyano, halogen, amido, nitro, haloalkyl, amino, methanesulfonyl, alkylcarbonyl, alkoxycarbonyl, heteroaryl Alkyl, heterocycloalkylalkyl, aminoacyl, amino protecting group, etc.
  • the amino protecting group is preferably selected from the group consisting of pivaloyl, tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, p-methoxybenzyl, allyloxycarbonyl, and trifluoroacetyl.
  • the present invention provides a hypoxia-responsive targeted drug-loading molecular container platform, which is an azocalixarene compound of formula (I):
  • n 4 or 5;
  • R 1 is selected from H, C 1-6 alkyl and -(CH 2 CH 2 O) m CH 3 , wherein m is an integer selected from 1-3;
  • n is 4 particularly preferably.
  • R 1 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopropyl, neopentyl, tert-pentyl , Pent-3-yl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 3,3-di Methyl butyl and so on.
  • R 2 is selected from -COOM and -SO 3 M, wherein M is independently selected from H and Na; more preferably, R 2 is selected from -COOH, -SO 3 H and -SO 3 Na.
  • the azocalixarene compound of the present invention as a hypoxia-responsive targeted drug-loaded molecular container platform is selected from the following compounds:
  • the azocalixarene compound of the present invention can form a host-guest clathrate through hydrogen bonding, electrostatic, hydrophobic and other non-covalent interactions with the pharmaceutically active substance, and the bonding constant is 10 4 or more, more preferably 10 5 or more, even more preferably 10 6 or more, particularly preferably 10 7 or more.
  • the azo bond of the azocalixarene compound of the present invention is reduced and broken under the action of the over-expressed azo reductase, so that the active drug substance is released by the prototype, realizing the drug
  • the targeting property reduces the dosage and improves safety.
  • the azocalixarene compound of the present invention can form a stable host-guest non-covalent binding structure with various small molecule pharmaceutical active substances, and target the release of the pharmaceutical active substances in the hypoxic region of the body, it can be used as hypoxia Responsive targeted drug-loaded molecular container platform or pharmaceutical excipients are applied in the pharmaceutical field.
  • the application also provides a pharmaceutical composition, which includes at least one pharmaceutically active substance and an azocalixarene compound of formula (I).
  • the pharmaceutical composition of the present invention may also include other pharmaceutically acceptable carriers or excipients, and other therapeutic agents.
  • the pharmaceutically active substance used for targeted administration using the drug-loaded molecular container platform of the present invention is selected from drugs for the treatment of one or more of the following diseases: cancer, myocardial infarction, stroke, atherosclerosis Sclerosis, rheumatoid arthritis, inflammatory bowel disease, chronic hypoxic lung disease, and chronic kidney disease.
  • the preferred cancer treatment drugs are selected from: doxorubicin, paclitaxel, camptothecin, hydroxycamptothecin, irinotecan, topotecan, oxaliplatin, belotecan, tilatinib and A Oxyestradiol.
  • the preferred drugs for treating myocardial infarction are selected from: aspirin and clopidogrel.
  • the preferred drugs for the treatment of stroke are selected from: aspirin, heparin, alteplase, nimodipine, ropizole, tirazate and elirodil.
  • the preferred drugs for treating atherosclerosis are selected from: lovastatin and ezetimibe.
  • Preferred drugs for treating rheumatoid arthritis are selected from: methotrexate, tofacitinib, baritinib, etanercept, abatacept, leflunomide, hydroxychloroquine and sulfasalazine.
  • the preferred drugs for the treatment of inflammatory bowel disease are selected from: mesalazine, azathioprine, mercaptopurine, methotrexate, infliximab, adalimumab, certuzumab, natalizumab And Sali.
  • Preferred drugs for the treatment of chronic hypoxic lung disease are selected from the group consisting of tadalafil, riosigua and treprostinil.
  • the preferred medicine for treating chronic kidney disease is selected from: rosastat and vidastat.
  • more preferred pharmaceutically active substances include (hydrochloride) doxorubicin, oxaliplatin, paclitaxel, camptothecin, hydroxycamptothecin, irinotecan, (hydrochloride) topotecan, clopidogrel, irirotide , Hydroxychloroquine, lovastatin and tadalafil one or more.
  • the drug containing the pharmaceutical excipient and the pharmaceutically active substance of the present invention can be administered to the patient by at least one of injection, oral administration, inhalation, rectal and transdermal administration.
  • the amount of a given drug depends on many factors, such as the specific dosing regimen, the type of disease or condition and its severity, and the subject in need of treatment Or the uniqueness of the host (e.g. body weight).
  • the dosage can be routinely determined by methods known in the art.
  • the administered dose is typically in the range of 0.02-5000 mg/day, for example, about 1-1500 mg/day.
  • the required dose can conveniently be expressed as one dose, or simultaneous (or within a short period of time) or divided doses at appropriate intervals, such as two, three, four or more divided doses per day.
  • the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the doctor's diagnosis.
  • the compound of formula (I) can be synthesized using standard synthesis techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein.
  • the solvent, temperature and other reaction conditions given herein can be changed according to the skill in the art.
  • the following synthesis methods can also be used.
  • the reactions can be used sequentially to provide the compounds described herein; or they can be used to synthesize fragments that are subsequently added by the methods described herein and/or methods known in the art.
  • a method similar to that described below can be used to synthesize the compound by using appropriate optional starting materials.
  • the starting materials used to synthesize the compounds described herein can be synthesized or can be obtained from commercial sources.
  • the compounds described herein and other related compounds with different substituents can be synthesized using techniques and raw materials known to those skilled in the art.
  • the general methods for preparing the compounds disclosed herein can be derived from reactions known in the art, and the reactions can be modified by reagents and conditions deemed appropriate by those skilled in the art to introduce various moieties in the molecules provided herein.
  • reaction product can be separated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and other methods. These products can be characterized using conventional methods, including physical constants and spectral data.
  • the general synthetic route of the azocalixarene compound of the present invention is as follows.
  • Example 1 Determination of the bonding constant between azocalixarene compound and drug molecule
  • Test method fluorescence titration method.
  • Test tool The quartz cuvette is used as the sample cell for the test, the test light path is 10mm, the instrument model is Varian Cary Eclipse, and the model is Cary Single-cuvette Peltier cuvette temperature control device.
  • Rhodamine B was purchased from Shanghai Macleans Biochemical Technology Co., Ltd.
  • Doxorubicin hydrochloride was purchased from Ailan (Shanghai) Chemical Technology Co., Ltd.
  • Paclitaxel was purchased from Dalian Meilun Biotechnology Co., Ltd.
  • Camptothecin was purchased in Dalian Meilun Biotechnology Co., Ltd.
  • Irinotecan was purchased and placed in Shanghai Yuanye Biotechnology Co., Ltd.
  • Oxaliplatin was purchased in Shanghai Yuanye Biotechnology Co., Ltd.
  • Clopidogrel was purchased from Dalian Meilun Biotechnology Co., Ltd.
  • Hydroxychloroquine was purchased from Tianjin Medema Biotechnology Co., Ltd.
  • Lovastatin was purchased from Shanghai Myrell Chemical Technology Co., Ltd.
  • Tadalafil was purchased in Beijing Warwick Chemical Co., Ltd.
  • Topotecan hydrochloride was purchased from Dalian Meilun Biotechnology Co., Ltd.
  • Hydroxycamptothecin was purchased from Dalian Meilun Biotechnology Co., Ltd.
  • the synthetic route of SiPcN 2 dye is as follows:
  • PBS phosphate buffer solution
  • the azocalixarene compound of the present invention has a strong binding strength to the active drug molecule, and the bonding constant reaches 10 4 or more, preferably 2 ⁇ 10 4 or more, more preferably 10 5 or more, and even more preferably 10 6 or more, particularly preferably 10 7 or more. Therefore, the azocalixarene compound of the present invention can form a stable host-guest non-covalent bond with the active drug molecule.
  • Example 2 The same procedure as in Example 1 was followed to determine the bonding constant of DOX/CAC4A at 37°C.
  • Example 3 Determination of the bonding constant of the dye SiPcN 2 , Rhodamine B and CAC4A
  • Test method fluorescence titration method.
  • Test tool The quartz cuvette is used as the sample cell for the test, the test light path is 10mm, the instrument model is Varian Cary Eclipse, and the model is Cary Single-cuvette Peltier cuvette temperature control device.
  • the bonding constant of the dye SiPcN 2 and CAC4A was measured at room temperature (25°C).
  • First, prepare CAC4A and SiPcN 2 mother liquors, and dissolve them in phosphate buffer solution (PBS, 10mM, pH 7.4) respectively, and the preparation concentration is 100 ⁇ M.
  • PBS phosphate buffer solution
  • the preparation concentration is 100 ⁇ M.
  • the bonding constant between Rhodamine B and CAC4A is (4.9 ⁇ 0.2) ⁇ 10 6 (25°C).
  • Test method fluorescence titration method.
  • Test tool The quartz cuvette is used as the sample cell for the test, the test light path is 10mm, the instrument model is Varian Cary Eclipse, and the model is Cary Single-cuvette Peltier cuvette temperature control device.
  • the Job curve is shown in Figure 5, and the ordinate represents the difference in fluorescence intensity between DOX and DOX/CAC4A at the same DOX concentration.
  • the experimental results show that DOX and CAC4A form a 1:1 inclusion compound.
  • Test method ultraviolet-visible spectroscopy, fluorescence spectroscopy.
  • Test tool Shimadzu UV-3600 ultraviolet-visible spectrophotometer, equipped with a temperature control module (model: PTC-348WI), the test sample is Shimadzu's own quartz cuvette, and the optical path is 10mm.
  • the fluorescent instrument model is Varian Cary Eclipse, equipped with a cuvette temperature control device model Cary Single-cuvette Peltier. The quartz cuvette is used as the sample cell for the test, and the test light path is 10mm.
  • the dotted line represents the fluorescence curve of DOX/CAC4A (10 ⁇ M/10 ⁇ M), and the solid line represents the fluorescence curve after adding SDT. After SDT was added, the fluorescence of DOX recovered, indicating that DOX was released from the CAC4A cavity.
  • Test method ultraviolet-visible spectroscopy.
  • Test tool Shimadzu UV-3600 ultraviolet-visible spectrophotometer, equipped with a temperature control module (model: PTC-348WI), the test sample is Shimadzu's own quartz cuvette, and the optical path is 10mm.
  • NADPH Reduced coenzyme II
  • DT-lipoamide dehydrogenase (DT-diaphorase) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.
  • Test method MTT method for cytotoxicity test.
  • Murine 4T1 breast cancer cells were purchased from Tianjin Yibo Hengtai Biotechnology Co., Ltd.
  • Fetal bovine serum (FBS) and DMEM medium were purchased from Thermo Fisher Scientific in the United States.
  • Thiazole Blue (MTT, 98.6% purity) was purchased from Beijing Soleibao Technology Co., Ltd.
  • Penicillin and streptomycin were purchased from Tianjin Baisaisi Biotechnology Co., Ltd.
  • DMEM fetal calf serum
  • penicillin streptomycin a maltylcholine
  • the cancer cells were incubated in a 37°C, 5% CO 2 cell incubator. Before each experiment, the cells were pre-cultured until they reached confluence. Collect 4T1 logarithmic phase cells, adjust the concentration of cell suspension, add 100 ⁇ L to each well, plate to adjust the density of the cells to be tested to 1000-10000 per well, and fill the edge holes with sterile PBS.
  • cell survival rate (OD 492 (samples)-OD 492 (blank))/(OD 492 (control)-OD 492 (blank)) ⁇ 100%.
  • Example 8 Toxicity test of DOX/CAC4A and DOX under normoxia and hypoxia conditions
  • the concentration represents the concentration of DOX
  • the concentration ratio of DOX to CAC4A in the DOX/CAC4A group is 1:1. It can be seen from the cytotoxicity in Figure 9 that the cytotoxicity of the inclusion compound DOX/CAC4A is reduced under normal oxygen conditions. Under hypoxic conditions, the cytotoxicity of the inclusion compound DOX/CAC4A is greater than that under normoxia, indicating that the inclusion compound DOX/CAC4A has the characteristics of hypoxia release.
  • mice Select female Balb/c, 6-8 week old mice (purchased from Beijing Weitong Lihua Company), and randomly divide them into four groups: PBS, CAC4A, DOX, and DOX/CAC4A, each with 4 mice, each mouse It weighs about 20g.
  • the drug was administered by tail vein injection. The dose was 1.16 mg/kg DOX, and each dose was 200 ⁇ L.
  • the drug concentrations of each group were CAC4A (200 ⁇ M), DOX (200 ⁇ M), and DOX/CAC4A (200/200 ⁇ M).
  • the safety experiment was determined according to the kit provided by the Nanjing Jiancheng Institute of Biological Engineering.
  • BUN Blood urea nitrogen
  • Lactate dehydrogenase lactate dehydrogenase
  • the indexes of DOX group were higher than those of other groups, indicating that free DOX has certain damage to liver and kidney, and CAC4A and CAC4A/DOX are safer.
  • the AST, ALP, and ALT indicators show that the CAC4A/DOX group is significantly different from the DOX group, which proves that the CAC4A/DOX group has better safety than the DOX group.
  • mice Select female Balb/c, 6-8 weeks old mice (purchased from Beijing Weitong Lihua Company), and randomly divide them into two groups, SiPcN 2 and SiPcN 2 /CAC4A, 3 mice in each group, and the drug is injected into the tail vein.
  • the dose was calculated as 1.5 mg/kg SiPcN 2 , and the drug concentration of each group was SiPcN 2 (200 ⁇ M), SiPcN 2 /CAC4A (200/200 ⁇ M).
  • Mice were dissected at 12h, 48h, 72h, and various organs were taken and used for fluorescence imaging with excitation wavelength of 605nm and emission wavelength of 700nm.
  • the liver fluorescence intensity of the SiPcN 2 /CAC4A group was significantly decreased, and the tumor fluorescence intensity was significantly increased, while the fluorescence of the free SiPcN 2 tumor site gradually decreased, proving that SiPcN 2 /CAC4A has the characteristic of specifically targeting tumor sites to release drugs.
  • mice 1x10 6 4T1 cells were injected in situ into the mammary glands of female Balb/C mice aged 6-8 weeks, and waited until the tumor size grew to 50mm 3 .
  • the mice were randomly divided into four groups: PBS, CAC4A, DOX, and DOX/CAC4A, with 5 mice in each group.
  • Each group of drugs was injected into the tail vein, and the long and short axis diameters of the mice were measured before each injection. It was administered every other day, 5 times for 9 days.
  • Each dose was 200 ⁇ L, and the drug concentration of each group was CAC4A (200 ⁇ M), DOX (200 ⁇ M), DOX/CAC4A (200/200 ⁇ M).
  • the tumor volume and body weight of mice were measured from the first day of administration.
  • the CAC4A/DOX group has a lower tumor growth rate and short-term inhibition after stopping the administration, which proves that CAC4A/DOX has a good tumor suppressing effect.
  • Monitoring the weight of the mice showed that there was no significant toxicity in the four groups.
  • TUNLE staining steps freeze the mouse tumors and stain them with PBS for 15 minutes and follow the experimental manual provided by Roach.
  • Ki67 staining steps take out the frozen section from -80°C, return to room temperature, treat with 0.1% Triton X-100 for 15 minutes, wash off Triton with PBS, block with 5% BSA (Sigma) for 1 hour, add primary antibody (Ki67 primary antibody) , Rat source, sigma), protected from light overnight at 4°C. After 12 hours, the primary antibody was washed off with PBS, and then fluorescent-labeled secondary antibody (goat anti-rat, Alexa Fluro488, Sigma) was added. One hour later, the slides were mounted and photographed with a confocal microscope for analysis.
  • Ki67 labeled cell proliferation-related antigens.
  • the degree of cell proliferation in the DOX/CAC4A group was lower than that in the control group.
  • mice tumor cells were fixed in 4% paraformaldehyde (sigma) for 24 hours, and then sent to Tianjin Yishengyuan Biotechnology Co., Ltd. for paraffin section and H&E staining experiments. Subsequent microscopic analysis.
  • the DOX/CAC4A group observed pyknosis of the nuclei, separation of nuclei and cytoplasm, and outflow of nuclei from the 40 ⁇ microscope.
  • the degree of cell necrosis in the DOX/CAC4A group was significantly higher than that of the other three groups, which proved that the DOX/CAC4A group has a good tumor killing effect.
  • the invention provides an azocalixarene supramolecular compound as a targeted drug adjuvant for hypoxia response.
  • the compounds of the present invention can be combined with appropriate active drug molecules to prepare corresponding pharmaceutical compositions, which are suitable for industrial applications.

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Abstract

一种包括式（I）的偶氮杯芳烃超分子化合物作为乏氧响应的靶向药用辅料的药物组合物、以及偶氮杯芳烃超分子化合物在制药中的用途。

Description

一种偶氮杯芳烃药用辅料及其用途 技术领域
本申请涉及制药领域，特别是涉及一种包括偶氮杯芳烃超分子化合物作为乏氧响应的靶向药用辅料的药物组合物、以及偶氮杯芳烃超分子化合物在制药中的新用途。
背景技术
乏氧是许多疾病的重要病理、生理特征之一，例如癌症(Nature Reviews Cancer,2011,11:393-410)、心肌梗塞(N.Engl.J.Med.,2017,377：1240-1249)、中风(Age and Aging,2002,31:10-12)、动脉粥样硬化(Curr.Opin.Lipidol.2009,20:409-14)、类风湿性关节炎(J.Biomed Sci.,2016,23:62)、炎症性肠病(J.Inflamm Res.2014,7:113-120)、慢性缺氧性肺病(Lancet Respir Med.2016,4:225)、慢性肾病(Int.J.Mol.Sci.2017,18:950)等。同时，乏氧也是这些疾病的有效靶点。常见的响应策略是将药物与乏氧响应基团——如硝基、醌、偶氮等共价相连。在乏氧环境下，响应基团发生还原后，引起药物的释放。然而，共价策略面临着分子设计复杂、合成分离耗时且成本高、以及共价连接带来活性变化和毒性等问题。
鉴于此，在制药领域中存在开发新的主客体非共价策略用于乏氧治疗的需求。主客体相互作用是组装过程中重要的驱动力之一，其多样的键合选择性，使得其在医药、生物化工、材料科学等领域存在潜在的应用前景。杯芳烃及其衍生物能够与有机分子或金属离子形成配合物，因此被认为是极具发展潜力的第三代超分子主体分子，在主客体化学领域得到广泛的研究。杯芳烃是一类由苯酚单元通过亚甲基在酚羟基的邻位连接而形成的环状低聚物。现有技术中已经开发出采用两亲性磺化杯芳烃(参见CN103550156A；CN104174026A)应用于药物递送的技术。
偶氮杯芳烃化合物是一类重要的功能性杯芳烃衍生物，已经广泛应用于金属离子络合(参见CN108404854A；Lilin Lu等人，Analytica Chimica Acta 535(2005):183-187；Tae Hyun Kim等人，Bull.Korean Chem.Soc.2013,Vol.34,No.11:3377-3380)、有机小分子结合(J. Cameron Tyson等人，Journal of Inclusion Phenomena and Molecular Recognition in Chemistry 29(1997):109-118)、离子选择电极(Jianquan Lu等人，Journal of Electroanalytical Chemistry,528(2002),33-38)、染料(Shobhana K.Menon等人，J.Incl.Phenom.Macrocycl.Chem.(2010)67:73-79；Serkan
Figure PCTCN2020102054-appb-000001
等人，J.Incl.Phenom.Macrocycl.Chem.(2012)77:259-267)等领域。有论文称该类化合物也有被应用于包络药物活性分子而用于药物递送的前景。然而，据报道该类化合物仅仅被用于包络抗菌类药物或抗精神病类药物并起到药物缓释作用，没有在体内的特定靶点定向释放药物的功能。
本发明人经过研究，利用这种结构准确、分子量固定、批合成稳定、易于衍生化且具有独特空腔键合性质的偶氮杯芳烃主体，作为乏氧响应的载药分子容器平台，可实现体内乏氧区域的靶向药物释放。
发明内容
本发明的一个目的是提供一种乏氧响应的靶向载药分子容器平台，从而提高活性药物分子递送的有效性、靶向性和安全性。
更具体地，本发明提供一种药物组合物，包括至少一种药物活性物质和式(I)的偶氮杯芳烃化合物：
Figure PCTCN2020102054-appb-000002
其中，
n为4或5；
R 1选自H、C 1-6烷基和-(CH 2CH 2O) mCH 3，其中m为选自1-3的整数；
R 2选自-COOM、-SO 3M、-N(CH 3) 3X、和-C(＝O)NH(CH 2CH 2)SO 3M，其中M独立地选自H、Na和K，X是独立地选自Cl、Br和I的抗衡离子。
在优选的实施方式中，优选n为4。
优选地，R 1选自H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异丙基、新戊基、叔戊基、戊-3-基、正己基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、3,3-二甲基丁基等。
另外优选地，R 2选自-COOM和-SO 3M，其中M独立地选自H和Na；更优选R 2选自-COOH、-SO 3H和-SO 3Na。
在优选的实施方式中，用于使用本发明的载药分子容器平台进行靶向给药的药物活性物质选自治疗以下一种或多种疾病的药物：癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
优选地，治疗癌症的药物选自：阿霉素、紫杉醇、喜树碱、羟基喜树碱、伊立替康、拓扑替康、奥沙利铂、贝洛替康、替拉替尼和甲氧雌二醇。
治疗心肌梗塞的药物选自：阿司匹林和氯吡格雷。
治疗中风的药物选自：阿司匹林、肝素、阿替普酶、尼莫地平、罗吡唑、替拉扎特和依利罗地。
治疗动脉粥样硬化的药物选自：洛伐他汀和依泽替米贝。
治疗类风湿性关节炎的药物选自：甲氨蝶呤、托法替尼、巴瑞替尼、依那西普、阿巴西普、来氟米特、羟基氯喹和柳氮磺胺吡啶。
治疗炎症性肠病的药物选自：美沙拉秦、硫唑嘌呤、巯嘌呤、甲氨蝶呤、英夫利昔单抗、阿达木单抗、赛妥珠单抗、那他珠单抗和沙利度。
治疗慢性缺氧性肺病的药物选自：他达拉非、利奥西呱和曲前列环素。
治疗慢性肾病的药物选自：罗沙司他和维达司他。
其中优选药物活性物质包括(盐酸)阿霉素、奥沙利铂、紫杉醇、喜树碱、羟基喜树碱、伊立替康、(盐酸)拓扑替康、氯吡格雷、依利罗地、羟基氯喹、洛伐他汀和他达拉非等中的一种或多种。
本发明还涉及式(I)的偶氮杯芳烃化合物在制备乏氧响应的靶向药物辅料中的用途。偶氮杯芳烃化合物是一类乏氧响应的靶向药物辅料， 可以改善药物分子的溶解性和稳定性，降低其对正常细胞的影响。乏氧是癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病、慢性肾病等疾病的典型特征。为了在乏氧条件下生存，乏氧细胞往往通过糖酵解获得能量，成为抵抗缺氧的有效途径。然而，这种途径会导致乳酸水平和还原酶的升高，例如细胞色素b5还原酶、NADPH硝基还原酶和DT-硫辛酰胺脱氢酶等。当药物分子被包结在本发明的偶氮杯芳烃化合物主体空腔内时，在正常组织，药物分子不被释放；在乏氧环境中，遇到过度表达的还原酶(偶氮还原酶)，杯芳烃偶氮键断裂，药物分子脱离主体空腔，在乏氧区域可控地释放，实现在乏氧区域的靶向治疗，使偶氮杯芳烃化合物成为一类具有刺激响应的靶向药物辅料。
附图说明
图1显示DOX/CAC4A主客体包合物乏氧响应的示意图。
图2a显示25℃下，PBS缓冲溶液中(10mM，pH＝7.4)，阿霉素(DOX)与CAC4A-SiPcN2(0.4/0.5μM)的荧光滴定曲线(λ ex＝610nm)；图2b显示DOX与CAC4A的键合常数拟合曲线，由主客体1:1竞争键合模型进行拟合(λ em＝690nm)。
图3a显示37℃下，PBS缓冲溶液中(10mM，pH＝7.4)，阿霉素(DOX)与CAC4A-SiPcN2(0.4/0.5μM)的荧光滴定曲线(λ ex＝610nm)；图3b显示DOX与CAC4A的键合常数拟合曲线，由主客体1:1竞争键合模型进行拟合(λ em＝690nm)。
图4a显示25℃下，PBS缓冲溶液中(10mM，pH＝7.4)，SiPcN 2与CAC4A的荧光滴定曲线(λ ex＝610nm)；图4b显示SiPcN 2与CAC4A的键合常数拟合曲线，由主客体1:1竞争键合模型进行拟合(λ em＝690nm)。
图5显示DOX和CAC4A的Job曲线，λ ex＝540nm，λ em＝594nm，[DOX]+[CAC4A]＝2.0μM，T＝37℃。
图6a显示加入SDT前后，CAC4A的紫外吸收曲线；图6b显示加入SDT前后，DOX/CAC4A的荧光曲线。
图7显示乏氧条件下，CAC4A的紫外吸收随时间变化的谱图。
图8显示MTT法细胞毒性实验中，不同浓度的CAC4A对于细胞存活率的影响的图。
图9显示MTT法细胞毒性实验中，不同浓度的DOX、DOX/CAC4A对于细胞存活率的影响的图。
图10a-c显示PBS、CAC4A、DOX、和DOX/CAC4A给药后各组小鼠血液中AST、ALP和ALT变化的图。
图11显示SiPcN 2和SiPcN 2/CAC4A两组小鼠中各器官的荧光成像照片。
图12显示SiPcN 2和SiPcN 2/CAC4A两组小鼠中肿瘤部位荧光强度统计分析的图。
图13a显示PBS、CAC4A、DOX、和DOX/CAC4A给药后各组小鼠的瘤子体积变化；图13b显示PBS、CAC4A、DOX、和DOX/CAC4A给药后各组小鼠的体重变化。
图14a显示PBS、CAC4A、DOX、和DOX/CAC4A给药组小鼠的瘤子照片；图14b显示PBS、CAC4A、DOX、和DOX/CAC4A给药组小鼠的瘤子重量。
图15显示各组小鼠肿瘤的TUNLE和Ki67染色的共聚焦显微镜照片。
图16显示各组小鼠肿瘤组织的H&E染色的共聚焦显微镜照片。
具体实施方式
术语
除非另外定义，所有本文使用的科技术语都具有与要求保护的主题所属领域的技术人员一般理解相同的含义。
除非另有说明，本发明采用本领域技术范围内的质谱、NMR、HPLC、蛋白质化学、生物化学、重组DNA技术和药理学等常规方法。除非提供具体的定义，否则与本文描述的分析化学、合成有机化学、以及医学和药物化学等化学上相关的命名和实验室操作和技术，是本领域技术人员已知的。一般而言，前述技术和步骤可以通过本领域众所周知的和在各种一般文献和更具体文献中描述的常规方法来实施，这些文献在本说明书中被引用和讨论。
术语“烷基”是指脂肪族烃基团，可以是支链或直链的烷基。根据结构，烷基可以是单价基团或双价基团(即亚烷基)。在本发明中，烷基优选是具有1-8个碳原子的烷基，更优选具有1-6个碳原子的“低级烷基”，甚至更优选具有1-4个碳原子的烷基。典型的烷基包括但不限于甲基、乙基、丙基、丁基、戊基、己基等。应理解，本文提到的“烷基”包括可能存在的所有构型和构象的该烷基，例如本文提到的“丙基”包括正丙基和异丙基，“丁基”包括正丁基、异丁基和叔丁基，“戊基”包括正戊基、异丙基、新戊基、叔戊基、和戊-3-基等。
术语“烷氧基”是指-O-烷基，其中烷基如本文中定义。典型的烷氧基包括但不限于甲氧基、乙氧基、丙氧基、丁氧基、戊氧基、己氧基等。
术语“烷氧基烷基”是指本文定义的烷基被本文定义的烷氧基取代。
术语“芳香基”是指平面环具有离域的π电子系统并且含有4n+2个π电子，其中n是整数。芳香基环可以由五、六、七、八、九或多于九个原子构成。芳香基可以是任选取代的。术语“芳香基”包括碳环芳基(例如苯基)和杂环芳基(或“杂芳基”或“杂芳香基”)基团(例如吡啶)。该术语包括单环或稠环多环(即共用相邻的碳原子对的环)基团。
本文使用的术语“芳基”是指芳香基环中每一个构成环的原子都是碳原子。芳基环可以由五、六、七、八、九或多于九个原子构成。芳基可以是任选取代的。芳基的实例包括但不限于苯基、萘基、菲基、蒽基、芴基和茚基。根据结构，芳基可以是单价基团或双价基团(即亚芳基)。
术语“羟基”是指-OH基团。
术语“羧基”是指式-COOH的化学部分。“酯基”是指具有式-COOR的化学部分，其中R选自烷基、环烷基、芳基等。
术语“氨基”是指-NH 2基团。
术语“烷基氨基”是指进一步被一个或两个烷基取代的氨基取代基，具体是指基团-NRR’，其中R和R’各自独立地选自氢或低级烷基，条件是-NRR’不是-NH 2。“烷基氨基”包括其中-NH 2的氮连接至少一个 烷基基团的化合物的基团。烷基氨基基团的例子包括但不限于，甲基氨基、乙基氨基等。“二烷基氨基”包括其中-NH 2的氮连接至少两个其它烷基基团的基团。二烷基氨基基团的例子包括但不限于，二甲基氨基、二乙基氨基等。
术语“烷基氨基烷基”是指本文定义的烷基被本文定义的烷基氨基取代。
术语“羟烷基”或“羟基烷基”是指进一步被一个或多个羟基取代的烷基取代基。
术语“磺基”或“磺酸基”是指式-SO 3H的官能团。术语“砜基”或“磺酰基”是指磺酸失去羟基后的官能团，具体是指-S(＝O) 2-基团。
术语“季铵基”是指-N +RR’R”，其中R、R’和R”各自独立地选自具有1-8个碳原子的烷基。
属于“偶氮基”或“重氮基”是指两个氮原子相互连接组成的二价原子团，具体是指-N＝N-基团。
术语“任选”指后面描述的一个或多个事件可以发生或可以不发生，并且包括发生的事件和不发生的事件两者。术语“任选取代的”或“取代的”是指所提及的基团可以被一个或多个额外的基团取代，所述额外的基团各自并且独立地选自烷基、环烷基、芳基、杂芳基、杂环基、羟基、烷氧基、氰基、卤素、酰胺基、硝基、卤代烷基、氨基、甲磺酰基、烷基羰基、烷氧基羰基、杂芳基烷基、杂环烷基烷基、氨酰基、氨基保护基等。其中，氨基保护基优选选自新戊酰基、叔丁氧羰基、苄氧羰基、9-芴甲氧羰基、苄基、对甲氧苄基、烯丙氧羰基、和三氟乙酰基等。
本发明的偶氮杯芳烃化合物及其载药用途
本发明提供一种乏氧响应的靶向载药分子容器平台，其为式(I)的偶氮杯芳烃化合物：
Figure PCTCN2020102054-appb-000003
其中，
n为4或5；
R 1选自H、C 1-6烷基和-(CH 2CH 2O) mCH 3，其中m为选自1-3的整数；
R 2选自-COOM、-SO 3M、-N(CH 3) 3X和-C(＝O)NH(CH 2CH 2)SO 3M，其中M独立地选自H、Na和K，X是独立地选自Cl、Br和I的抗衡离子。
在优选的实施方式中，特别优选n为4。
优选地，R 1选自H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异丙基、新戊基、叔戊基、戊-3-基、正己基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、3,3-二甲基丁基等。
另外优选地，R 2选自-COOM和-SO 3M，其中M独立地选自H和Na；更优选R 2选自-COOH、-SO 3H和-SO 3Na。
特别优选地，本发明的作为乏氧响应的靶向载药分子容器平台的偶氮杯芳烃化合物选自以下化合物：
Figure PCTCN2020102054-appb-000004
Figure PCTCN2020102054-appb-000005
对于各个变量，上述基团的任意组合也在本文考虑之中。可以理解的是：本文所提供的化合物上的取代基和取代模式可以由本领域技术人员进行选择，以便提供化学上稳定的且可以使用本领域已知的技术以及本文阐述的技术合成的化合物。
本发明的偶氮杯芳烃化合物能够与药物活性物质通过氢键、静电、疏水等非共价相互作用形成主客体包合物，键合常数为10 4以上，更优选10 5以上，再更优选10 6以上，特别优选10 7以上。如图1所示，在体内乏氧区域，本发明的偶氮杯芳烃化合物的偶氮键在过表达的偶氮还原酶作用下被还原断裂，使得药物活性物质被原型释放出来，实现了药物的靶向性，减少了给药量，提高了安全性。
由于本发明的偶氮杯芳烃化合物能够与各种小分子药物活性物质形成稳定的主客体非共价结合结构，并在体内乏氧区域将药物活性物 质靶向释放出来，因此可以用作乏氧响应的靶向载药分子容器平台或药物辅料而在制药领域中应用。
本发明的药物组合物
本申请还提供药物组合物，其包括至少一种药物活性物质和式(I)的偶氮杯芳烃化合物。任选地，本发明的药物组合物还可以包括其它药学可接受的载体或赋形剂、以及其它治疗剂。
在优选的实施方式中，用于使用本发明的载药分子容器平台进行靶向给药的药物活性物质选自治疗以下一种或多种疾病的药物：癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
其中，优选的治疗癌症的药物选自：阿霉素、紫杉醇、喜树碱、羟基喜树碱、伊立替康、拓扑替康、奥沙利铂、贝洛替康、替拉替尼和甲氧雌二醇。
优选的治疗心肌梗塞的药物选自：阿司匹林和氯吡格雷。
优选的治疗中风的药物选自：阿司匹林、肝素、阿替普酶、尼莫地平、罗吡唑、替拉扎特和依利罗地。
优选的治疗动脉粥样硬化的药物选自：洛伐他汀和依泽替米贝。
优选的治疗类风湿性关节炎的药物选自：甲氨蝶呤、托法替尼、巴瑞替尼、依那西普、阿巴西普、来氟米特、羟基氯喹和柳氮磺胺吡啶。
优选的治疗炎症性肠病的药物选自：美沙拉秦、硫唑嘌呤、巯嘌呤、甲氨蝶呤、英夫利昔单抗、阿达木单抗、赛妥珠单抗、那他珠单抗和沙利度。
优选的治疗慢性缺氧性肺病的药物选自：他达拉非、利奥西呱和曲前列环素。
优选的治疗慢性肾病的药物选自：罗沙司他和维达司他。
其中，更优选的药物活性物质包括(盐酸)阿霉素、奥沙利铂、紫杉醇、喜树碱、羟基喜树碱、伊立替康、(盐酸)拓扑替康、氯吡格雷、依利罗地、羟基氯喹、洛伐他汀和他达拉非等中的一种或多种。
在治疗过程中，可以根据情况单独或与一种或多种其它的治疗剂 组合使用。可以通过注射、口服、吸入、直肠和经皮施用中的至少一种将包含本发明的药物辅料和药物活性物质的药物施用给患者。
在本发明的实施方式中，在根据本发明对患者进行治疗时，给定药物的量取决于诸多因素，如具体的给药方案、疾病或病症类型及其严重性、需要治疗的受治疗者或宿主的独特性(例如体重)。但是，根据特定的周围情况，包括例如已采用的具体药物、给药途径、治疗的病症、以及治疗的受治疗者或宿主，施用剂量可由本领域已知的方法常规决定。通常，就成人治疗使用的剂量而言，施用剂量典型地在0.02-5000mg/天，例如约1-1500mg/天的范围。该所需剂量可以方便地被表现为一剂、或同时给药的(或在短时间内)或在适当的间隔的分剂量，例如每天二、三、四剂或更多分剂。本领域技术人员可以理解的是，尽管给出了上述剂量范围，但具体的有效量可根据患者的情况并结合医师诊断而适当调节。
偶氮杯芳烃化合物的制备
使用本领域技术人员已知的标准合成技术或使用本领域已知的方法与本文描述的方法组合，可以合成式(I)的化合物。另外，本文给出的溶剂、温度和其它反应条件可以根据本领域技术而改变。作为进一步的指导，也可以利用以下的合成方法。
所述反应可以按顺序使用，以提供本文描述的化合物；或它们可以用于合成片段，所述片段通过本文描述的方法和/或本领域已知的方法随后加入。
可以使用与下述类似的方法，通过使用适当的可选择的起始原料，合成化合物。用于合成本文描述的化合物的起始原料可以被合成或可以从商业来源获得。本文描述的化合物和其它相关具有不同取代基的化合物可以使用本领域技术人员已知的技术和原料合成。制备本文公开的化合物的一般方法可以来自本领域已知的反应，并且该反应可以通过由本领域技术人员所认为适当的试剂和条件修改，以引入本文提供的分子中的各种部分。
如果需要，反应产物可以使用常规技术分离和纯化，包括但不限于过滤、蒸馏、结晶、色谱等方法。这些产物可以使用常规方法表征， 包括物理常数和图谱数据。
本发明的偶氮杯芳烃化合物的通用合成路线如下。
Figure PCTCN2020102054-appb-000006
将2.5mmol对氨基苯甲酸(或对氨基苯磺酸等)固体与500μL浓盐酸分别加入3.75mL水中，将所得溶液放置到冰盐浴中，冷却到2℃。将2.7g(40mmol)NaNO 2溶解于2.5mL水中，将溶液逐滴滴加到对氨基苯甲酸(或对氨基苯磺酸等)和盐酸的混合液中。反应放热，为保证重氮盐稳定，反应过程中控制温度于5℃以下，搅拌30分钟，可见溶液变为淡黄色，得到溶液1。将1mmol CnA(表示杯[n]芳烃，n为4-8，典型地为杯[4]芳烃或杯[5]芳烃；杯[4]芳烃的合成参照文献Org.Synth.1990,68,238和J.Am.Chem.Soc.1982,104,2652；杯[5]芳烃的合成参照文献J.Org.Chem.1998,63,489)与4.08g(30mmol)三水合乙酸钠溶于26mL MeOH-DMF(体积比5:8)中，置于冰水浴中冷却至5℃以下，得到溶液2。将溶液1缓慢逐滴滴加到溶液2中，滴加过程中控制温度不超过5℃。滴加完成后，在5℃下搅拌15分钟，转移至室温环境下，继续搅拌2.5小时，静置待悬浊液沉降完全后向其中加入37.5mL稀盐酸(0.25％)，搅拌并于60℃加热30分钟。过滤后用蒸馏水和MeOH洗涤数次，得到红色固体即为粗产物。将粗产物溶于25mL的热NaHCO 3(0.5M)溶液中，过滤除去滤渣。冷却后向滤液中加入3mL浓盐酸，并于60℃下加热搅拌30分钟，过滤，滤饼蒸馏水洗涤数次后，放入真空干燥箱中干燥过夜，得到纯化产物。产物的 1H NRM和MS数据如下。
CAC4A： 1H NMR(400MHz,DMSO-d 6)δ8.02(d,8H,J＝8.6Hz,Ar-H),7.81(d,8H,J＝8.6Hz,ArH),7.80(s,8H,calix-H),4.37 and 3.72(s,8H,Ar-CH 2-Ar)；MS(MALDI-TOF):calcd.for C 56H 39N 8O 12 -[M-H] -1015.269,found 1015.195。
CAC5A： 1H NMR(400MHz,DMSO-d 6)δ8.05(d,10H,J＝8.0Hz,Ar-H),7.81(s,10H,calix-H),7.80(d,10H,J＝8.0Hz,ArH),3.94(s,10H, Ar-CH 2-Ar)；MS(MALDI-TOF):calcd.for C 70H 50N 10O 15Na +[M+Na] +1293.335,found 1293.335。
SAC4A： 1H NMR(400MHz,DMSO-d 6)δ7.78(s,8H,calix-H),7.66-7.72(m,16H,Ar-H),4.39and 3.66(s,8H,Ar-CH 2-Ar)；MS(MALDI-TOF):calcd.for C 52H 36N 8O 16S 4Na 5 +[M+Na] +1271.062,found 1271.062。
SAC5A： 1H NMR(400MHz,DMSO-d 6)δ7.79(s,10H,calix-H),7.70-7.75(m,20H,Ar-H),3.94(s,10H,Ar-CH 2-Ar)。
QAC4A、QAC5A、TCAC4A和TCAC5A等化合物参照文献Chemistry Letters,1989,vol.18,issue 6,pp 931-934中所述的方法和类似方法合成。
测试实施例
实施例1：偶氮杯芳烃化合物与药物分子键合常数的测定
测试方法：荧光滴定法。
测试工具：测试选用石英比色皿为样品池，测试光路10mm，仪器型号为Varian Cary Eclipse，配有型号为Cary Single-cuvette Peltier的比色皿控温装置。
试剂：罗丹明B购置于上海麦克林生化科技有限公司。
盐酸阿霉素(DOX)购置于艾览(上海)化工科技有限公司。
紫杉醇购置于大连美仑生物技术有限公司。
喜树碱购置于大连美仑生物技术有限公司。
伊立替康购置于上海源叶生物科技有限公司。
奥沙利铂购置于上海源叶生物科技有限公司。
氯吡格雷购置于大连美仑生物技术有限公司。
依利罗地购置于北京华威锐科化工有限公司。
羟基氯喹购置于天津美德玛生物科技有限公司。
洛伐他汀购置于上海迈瑞尔化学技术有限公司。
他达拉非购置于北京华威锐科化工有限公司。
拓扑替康盐酸盐购置于大连美仑生物技术有限公司。
羟基喜树碱购置于大连美仑生物技术有限公司。
SiPcN 2染料的合成路线如下：
Figure PCTCN2020102054-appb-000007
将N,N-二甲基乙醇胺(0.089g，2.87mmol)、二氯硅(IV)酞菁(0.22g，0.36mmol)和吡啶(0.5ml)溶于干燥甲苯(15ml)中，回流加热4小时。冷却后，旋去溶剂。用水(500ml)和正己烷(250ml)洗涤残余物，然后溶解于CH 2Cl 2(100ml)中并经无水Na 2SO 4干燥。旋去CH 2Cl 2，得到蓝色固体产物1(0.18g，70％)。 1H NMR(CDCl 3)，δ9.62-9.64(m,8H,Pc-H α),8.32-8.34(m,8H,Pc-H β),0.59(s,12H,CH 3),-0.89(t,J＝6.5Hz,4H,CH 2),-1.96(t,J＝6.5Hz,4H,OCH 2)。 13C NMR(CDCl 3)δ149,136,131,124,58,54,44。
将产物1(0.136g，0.19mmol)与碘甲烷(0.29g，2.04mmol)溶于CHCl 3(10ml)中，室温下搅拌过夜。将溶剂蒸发至干燥，用正己烷处理残余物得到沉淀。将沉淀溶解在水中后，通过过滤去除不溶性固体。滤液经冷冻干燥，得到产物SiPcN 2(0.154g，81％)。 1H NMR(CDCl 3)，δ9.70-9.68(m,8H,Pc-H α),8.47-8.49(m,8H,Pc-H β),1.17(s,12H,CH 3),0.83(t,J＝4.4Hz,4H,CH 2),-1.64(virtual t,J＝4.4Hz,4H,OCH 2)。 13C NMR(DMSO-d 6)δ149,135,132,124,63,51,49。MS(MALDI-TOF):calcd.for C 42H 42N 10O 2Si 2+[M-2I] 2+373.163,found 373.163。
CAC4A和光致发光分子的荧光滴定实验均在室温(25℃)进行。首先配制CAC4A、SiPcN 2、罗丹明B的母液，将其分别溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)中，配置浓度均为100μM。测试时先将CAC4A-SiPcN 2(0.4/0.5μM)或者CAC4A-罗丹明B(0.3/0.3μM)荧光传感对配置于荧光池内，PBS定容到体积2.5mL。将活性药物分子溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)，配置浓度100μM， 并向其加入荧光传感对，使荧光传感对浓度与荧光池内一致，PBS定容至1mL。随后将活性药物分子溶液以预定体积加入荧光池内，记录荧光强度变化。荧光滴定数据由主客体1:1竞争键合模型进行拟合，测定主客体包结的键合常数Ka。结果参见图2a和图2b。
各种活性药物分子与CAC4A的键合常数测试结果如下表1所示。
表1.活性药物分子与CAC4A的键合常数
Figure PCTCN2020102054-appb-000008
Figure PCTCN2020102054-appb-000009
Figure PCTCN2020102054-appb-000010
主客体的键合常数越大，说明CAC4A与药物的包结能力越强，形成的包合物越稳定，药物越不容易泄露。如实验结果所示，本发明的偶氮杯芳烃化合物与活性药物分子具有较强的结合强度，键合常数达到10 4以上，优选2×10 4以上，更优选10 5以上，再更优选10 6以上，特别优选10 7以上。因此，本发明的偶氮杯芳烃化合物能够与活性药物分子形成稳定的主客体非共价结合。
实施例2：(阿霉素)DOX/CAC4A键合常数的测定
按照与实施例1相同的步骤测定DOX/CAC4A在37℃的键合常数。将DOX(0～3.44μM)与CAC4A/SiPcN 2(0.4/0.5μM)在37℃，PBS缓冲液(pH 7.4，10mM)中进行荧光滴定，激发波长610nm。
测试结果如图3a和3b所示。DOX/CAC4A在37℃的键合常数经测定为(8.13±0.81)×10 7M -1。
实施例3：染料SiPcN 2、罗丹明B与CAC4A键合常数的测定
测试方法：荧光滴定法。
测试工具：测试选用石英比色皿为样品池，测试光路10mm，仪器型号为Varian Cary Eclipse，配有型号为Cary Single-cuvette Peltier的比色皿控温装置。
染料SiPcN 2与CAC4A键合常数测定在室温(25℃)进行。首先配制CAC4A、SiPcN 2母液，将其分别溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)中，配制浓度均为100μM。测试时先将染料SiPcN 2母液加入于荧光池内，使其浓度为0.5μM，用PBS定容到体积为2.5mL，。取CAC4A母液溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)，配置浓度30μM，并向其加入SiPcN 2，使SiPcN 2浓度与荧光池内一致，定容至1mL。随后将CAC4A溶液以预定体积加入荧光池内，记录荧光强度变化。测试结果如图4a和4b所示。SiPcN 2/CAC4A的键合常数经测定为(5.4±0.4)×10 7M -1(25℃)。
依同样的方法，测得罗丹明B与CAC4A键合常数为(4.9±0.2)×10 6(25℃)。
实施例4：CAC4A/DOX键合比的确定
测试方法：荧光滴定法。
测试工具：测试选用石英比色皿为样品池，测试光路10mm，仪器型号为Varian Cary Eclipse，配有型号为Cary Single-cuvette Peltier的比色皿控温装置。
实验步骤：首先配制CAC4A、DOX的母液，将其分别溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)中，配制浓度均为100μM。随后分别按下表2和3配制1-18样品，PBS定容至2.5mL。测试各样品荧光强度，λ ex＝540nm。
表2：
μM 1 2 3 4 5 6 7 8 9
DOX 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
CAC4A 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2
表3：
μM 10 11 12 13 14 15 16 17 18
DOX 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Job曲线如图5所示，纵坐标表示相同DOX浓度下DOX与DOX/CAC4A的荧光强度差。实验结果表明DOX与CAC4A形成1:1的包合物。
实施例5：连二亚硫酸钠(SDT)还原实验
测试方法：紫外-可见分光光谱法、荧光分光光谱法。
测试工具：日本岛津UV-3600紫外-可见分光光度计，配有控温模块(型号：PTC-348WI)，测试样品选用岛津自带石英比色皿，光程10mm。荧光仪器型号为Varian Cary Eclipse，配有型号为Cary Single-cuvette Peltier的比色皿控温装置。测试选用石英比色皿为样品池，测试光路10mm。
实验步骤：首先配制CAC4A、DOX的母液，将其分别溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)中，配制浓度均为100μM。取CAC4A母液稀释到10μM CAC4A，体积2.5mL，测试420nm处紫外吸收随时间变化，6分钟时加入1.0mM SDT，测试结果如图6a所示。图6a是加入SDT前后，CAC4A紫外吸收曲线。从图6a中看出，加入SDT后，CAC4A中偶氮键对应的紫外吸收随时间逐渐下降，说明CAC4A能被SDT还原，具有乏氧响应性。
取CAC4A、DOX母液，配制DOX/CAC4A(10μM/10μM)的溶液，PBS定容至2.5mL，测试荧光光谱。随后加入1.0mM SDT，再次测试荧光光谱，见图6b。
图6b中，虚线代表DOX/CAC4A(10μM/10μM)荧光曲线，实线代表加入SDT后的荧光曲线。SDT加入后，DOX的荧光恢复，说明DOX被从CAC4A空腔释放出来。
实施例6：乏氧响应验证-酶还原反应实验
测试方法：紫外-可见分光光谱法。
测试工具：日本岛津UV-3600紫外-可见分光光度计，配有控温模块(型号：PTC-348WI)，测试样品选用岛津自带石英比色皿，光程10mm。
试剂及其来源：
还原型辅酶II(NADPH)购置于北京伊诺凯科技有限公司。
DT-硫辛酰胺脱氢酶(DT-diaphorase)购置于西格玛奥德里奇(上海)贸易有限公司。
实验步骤：首先配制CAC4A、DT-硫辛酰胺脱氢酶、NADPH的母液，将其溶于磷酸盐缓冲溶液(PBS，10mM，pH＝7.4)中，配制浓度均为100μM。分别取CAC4A、DT-硫辛酰胺脱氢酶、NADPH母液，配制体积2.5mL含3μM CAC4A、1.0μM DT-硫辛酰胺脱氢酶、50μM NADPH的试样。然后将试样转移到荧光池内，向其鼓吹氮气，除氧气，营造乏氧条件，测试不同时刻下的紫外吸收，测试结果如图7所示。CAC4A中偶氮键对应的紫外吸收随时间逐渐下降，说明CAC4A能被DT-硫辛酰胺脱氢酶和NADPH还原，具有乏氧响应性。
实施例7：CAC4A的毒性实验
测试方法：MTT法进行细胞毒性实验。
试剂及其来源：
鼠源4T1乳腺癌细胞购置于天津益博恒泰生物科技有限公司。
胎牛血清(FBS)和DMEM培养基购置于美国Thermo Fisher Scientific公司。
噻唑蓝(MTT，纯度98.6％)购置于北京索莱宝科技有限公司。
青霉素链霉素购置于天津百赛斯生物科技有限公司。
实验步骤：
1、培养基使用DMEM，加有10％FBS和1％青霉素链霉素。将癌细胞置于37℃、5％CO 2细胞培养箱中孵育，每次在实验之前，将细胞预培养直至达到汇合。收集4T1对数期细胞，调整细胞悬液浓度，每孔加入100μL，铺板使待测细胞调密度至1000-10000每孔，边缘孔用 无菌PBS填充。
2、在5％CO 2，37℃下孵育，至细胞单层铺满孔底(96孔平底板)，加入浓度梯度的CAC4A(1μM、2μM、4μM、8μM、16μM、32μM)。
3、常氧条件：将96孔板置于5％CO 2，37℃培养箱中培养24小时。乏氧条件：将96孔板置于乏氧小室，通入5％CO 2，94％N 2，1％O 2混合气体，随后关闭进气口和出气口，将其置于37℃培养箱中培养24小时。
4、小心吸去孔内培养液，每孔加入20uL MTT溶液(5mg/ml，即0.5％MTT)，继续培养4小时。
5、终止培养，小心吸去孔内培养液，每孔加入150μl二甲基亚砜，置摇床上低速振荡10分钟，使结晶物充分溶解。在酶联免疫检测仪OD 490nm处测量各孔的吸光值。细胞存活率可以通过下面的公式计算得到：细胞存活率＝(OD 492(samples)-OD 492(blank))/(OD 492(control)-OD 492(blank))×100％。
从图8的MTT法细胞毒性结果看出，CAC4A载体在常氧/乏氧条件下均无细胞毒性。
实施例8：DOX/CAC4A和DOX在常氧和乏氧条件下的毒性实验
按照与实施例7相同的方式进行实验，结果参见图9。
图9中，浓度表示DOX的浓度，DOX/CAC4A组中DOX与CAC4A的浓度比为1:1。从图9的细胞毒性看出，常氧条件下，包合物DOX/CAC4A细胞毒性降低。乏氧条件下，包合物DOX/CAC4A细胞毒性比常氧条件的下细胞毒性大，说明包合物DOX/CAC4A具有乏氧释放的特性。
实施例9：动物安全性实验
选取雌性Balb/c、6-8周小鼠(购买于北京维通利华公司)，随机分为PBS、CAC4A、DOX、和DOX/CAC4A四组，每组4只小鼠，每只小鼠重约20g。采取尾静脉注射给药，给药剂量按1.16mg/kg DOX计，每次给药200μL，各组药物浓度分别是CAC4A(200μM)，DOX(200μM)，DOX/CAC4A(200/200μM)。安全性实验根据南京建成 生物工程研究所提供的试剂盒测定。
血生化测试结果参见下表4。各缩写字母代表含义如下：
Aspartate transaminase(AST)谷草转氨酶
Alkaline phosphatase(ALP)碱性磷酸(酯)酶
Blood urea nitrogen(BUN)血尿素氮
Creatinine(CRE)肌酐
Lactate dehydrogenase(LDH)乳酸脱氢酶
Alanine aminotransferase ALT谷丙转氨酶
表4.血生化测试结果。
Figure PCTCN2020102054-appb-000011
DOX组各项指标高于其它组，说明游离DOX对肝脏肾脏有一定损伤，而CAC4A及CAC4A/DOX的安全性较高。
根据图10的差异显著性分析，AST、ALP、ALT指标说明CAC4A/DOX组与DOX组存在一定差异显著性，证明CAC4A/DOX组与DOX组相比具有更好的安全性。
实施例10：器官离体成像
选取雌性Balb/c，6-8周小鼠(购买于北京维通利华公司)，随机分为SiPcN 2和SiPcN 2/CAC4A两组，每组3只小鼠，尾静脉注射药物，给药剂量按1.5mg/kg SiPcN 2计，各组药物浓度分别是SiPcN 2(200μM)，SiPcN 2/CAC4A(200/200μM)。于12h、48h、72h解剖小鼠，取各脏器，荧光成像，激发波长605nm，发射波长700nm。
如图11所示，SiPcN 2/CAC4A组肝脏荧光强度明显下降，肿瘤荧光强度显著增强，而游离SiPcN 2肿瘤部位荧光逐渐减弱，证明SiPcN 2/CAC4A具有特异性靶向肿瘤部位释放药物的特性。
对小鼠肿瘤部位的平均荧光强度进行统计分析，结果如图12所示： 72小时后，SiPcN 2/CAC4A组肿瘤组织荧光强度是游离SiPcN 2组荧光强度的2倍，证明SiPcN 2/CAC4A可在肿瘤部位特异性响应肿瘤乏氧微环境释放SiPcN 2。
实施例11：动物抑瘤实验
将1x10 6个4T1细胞原位注射到6-8周的雌性Balb/C小鼠乳腺中，等到瘤子体积大小长到50mm 3。肿瘤大小通过游标卡尺测量，并使用以下公式计算肿瘤体积：V＝W 2×L/2，其中W和L分别是肿瘤的最短和最长直径。将小鼠随机分为PBS、CAC4A、DOX、和DOX/CAC4A四组，每组5只小鼠。尾静脉注射各组药物，每次注射前测量小鼠长短轴直径。隔天给药，给药5次共9天。每次给药200μL，各组药物浓度分别是CAC4A(200μM)，DOX(200μM)，DOX/CAC4A(200/200μM)。从给药第一天开始测量小鼠瘤子体积及小鼠体重。
如图13a和13b所示，与对照组相比，CAC4A/DOX组肿瘤生长速度降低，停止给药后有短期抑制作用，证明CAC4A/DOX具有良好的抑瘤效果。监测小鼠体重可知，四组均无显著毒性。
药后第15天，将小鼠杀死并取出肿瘤用于拍照和瘤子重量检测，结果参见图14a和14b。根据差异显著性分析，DOX/CAC4A组肿瘤重量与对照组相比具有显著差异性(p＜0.0001)，证明DOX/CAC4A具有良好的抑瘤效果。
实施例12：TUNLE和Ki67染色
TUNLE染色步骤：将小鼠肿瘤进行冰冻切片处理，PBS湿润15min后遵循Roach公司提供的实验手册进行染色。
Ki67染色步骤：将冰冻切片从-80℃取出，恢复至室温，0.1％Triton X-100处理15分钟后，PBS洗掉Triton，5％BSA(Sigma)封闭1小时后加入一抗(Ki67一抗，大鼠来源，sigma)，4℃避光过夜。12小时后PBS洗掉一抗，然后加入荧光标记的二抗(山羊抗大鼠，alexa Fluro488，Sigma)。1小时后封片共聚焦显微镜拍照分析。
如图15所示，在Tunle染色照片中，蓝色为细胞核，红色是细胞凋亡的标志(标记断裂DNA)。DOX/CAC4A组细胞凋亡程度明显高 于其它三组。
Ki67染色照片中，Ki67标记细胞增殖相关抗原。DOX/CAC4A组细胞增殖程度低于对照组。
两者均证明DOX/CAC4A具有良好的肿瘤杀伤效果。
实施例13：肿瘤组织H&E染色切片
将小鼠肿瘤细胞置于4％多聚甲醛(sigma)中固定24小时后，送天津易生源生物技术有限公司进行石蜡切片及H&E染色实验。随后进行显微镜分析。
如图16所示，由40×显微镜观察到DOX/CAC4A组细胞核固缩，核质分离，细胞核外流。DOX/CAC4A组细胞坏死程度明显高于其他三组，证明DOX/CAC4A组具有良好的杀伤肿瘤的效果。
工业应用性
本发明提供一种偶氮杯芳烃超分子化合物作为乏氧响应的靶向药物辅料。本发明的化合物可以与适当的活性药物分子一起制成相应的药物组合物，适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (12)

  1. 一种药物组合物，包括至少一种药物活性物质和式(I)的偶氮杯芳烃化合物：
    Figure PCTCN2020102054-appb-100001
    其中，
    n为4或5；
    R 1选自H、C 1-6烷基和-(CH 2CH 2O) mCH 3，其中m为选自1-3的整数；
    R 2选自-COOM、-SO 3M、-N(CH 3) 3X、和-C(＝O)NH(CH 2CH 2)SO 3M，其中M独立地选自H、Na和K，X是独立地选自Cl、Br和I的抗衡离子，
    所述至少一种药物活性物质选自治疗以下一种或多种疾病的药物：癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
  2. 如权利要求1所述的药物组合物，其中所述至少一种药物活性物质为治疗癌症的药物。
  3. 如权利要求1所述的药物组合物，其中R 1选自H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异丙基、新戊基、叔戊基、戊-3-基、正己基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基和3,3-二甲基丁基。
  4. 如权利要求1所述的药物组合物，其中R 2选自-COOM和-SO 3M，其中M独立地选自H和Na。
  5. 如权利要求1所述的药物组合物，其中所述式(I)的偶氮杯芳烃化合物选自：
    Figure PCTCN2020102054-appb-100002
  6. 如权利要求1-5中任一项所述的药物组合物，其中所述药物活性物质选自以下一种或多种：选自阿霉素、紫杉醇、喜树碱、羟基喜树碱、伊立替康、拓扑替康、奥沙利铂、贝洛替康、替拉替尼和甲氧雌二醇的治疗癌症的药物；选自阿司匹林和氯吡格雷的治疗心肌梗塞的药物；选自阿司匹林、肝素、阿替普酶、尼莫地平、罗吡唑、替拉 扎特和依利罗地的治疗中风的药物；选自洛伐他汀和依泽替米贝的治疗动脉粥样硬化的药物；选自甲氨蝶呤、托法替尼、巴瑞替尼、依那西普、阿巴西普、来氟米特、羟基氯喹和柳氮磺胺吡啶的治疗类风湿性关节炎的药物；选自美沙拉秦、硫唑嘌呤、巯嘌呤、甲氨蝶呤、英夫利昔单抗、阿达木单抗、赛妥珠单抗、那他珠单抗和沙利度的治疗炎症性肠病的药物；选自他达拉非、利奥西呱和曲前列环素的治疗慢性缺氧性肺病的药物；选自罗沙司他和维达司他的治疗慢性肾病的药物。
  7. 如权利要求6所述的药物组合物，其中所述药物活性物质包括选自(盐酸)阿霉素、奥沙利铂、紫杉醇、喜树碱、羟基喜树碱、伊立替康、(盐酸)拓扑替康、氯吡格雷、依利罗地、羟基氯喹、洛伐他汀和他达拉非中的一种或多种。
  8. 式(I)的偶氮杯芳烃化合物用于乏氧响应的靶向药物辅料的用途，
    Figure PCTCN2020102054-appb-100003
    其中，
    n为4或5；
    R 1选自H、C 1-6烷基和-(CH 2CH 2O) mCH 3，其中m为选自1-3的整数；
    R 2选自-COOM、-SO 3M、-N(CH 3) 3X、和-C(＝O)NH(CH 2CH 2)SO 3M，其中M独立地选自H、Na和K，X是独立地选自Cl、Br和I的抗衡离子。
  9. 如权利要求8所述的偶氮杯芳烃化合物的用途，其中R 1选自H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、 异丙基、新戊基、叔戊基、戊-3-基、正己基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、和3,3-二甲基丁基。
  10. 如权利要求8所述的偶氮杯芳烃化合物的用途，其中R 2选自-COOM和-SO 3M，其中M独立地选自H和Na。
  11. 如权利要求8所述的偶氮杯芳烃化合物的用途，其中所述式(I)的偶氮杯芳烃化合物选自：
    Figure PCTCN2020102054-appb-100004
  12. 如权利要求8-11中任一项所述的偶氮杯芳烃化合物的用途，其中所述药物是选自治疗以下一种或多种疾病的药物：癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
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