WO2022111541A1 - Pt(Ⅳ)化疗前药及其可控释放用于治疗肿瘤 - Google Patents

Pt(Ⅳ)化疗前药及其可控释放用于治疗肿瘤 Download PDF

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WO2022111541A1
WO2022111541A1 PCT/CN2021/132886 CN2021132886W WO2022111541A1 WO 2022111541 A1 WO2022111541 A1 WO 2022111541A1 CN 2021132886 W CN2021132886 W CN 2021132886W WO 2022111541 A1 WO2022111541 A1 WO 2022111541A1
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complex
radiation
tumor
platinum
cancer
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PCT/CN2021/132886
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English (en)
French (fr)
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刘志博
傅群峰
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北京大学
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Priority to EP21897041.6A priority Critical patent/EP4253392A4/en
Priority to US18/038,629 priority patent/US20240009230A1/en
Priority to CN202180079338.9A priority patent/CN116635396A/zh
Priority to JP2023532203A priority patent/JP2023551676A/ja
Publication of WO2022111541A1 publication Critical patent/WO2022111541A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0086—Platinum compounds
    • C07F15/0093—Platinum compounds without a metal-carbon linkage
    • 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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum 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
    • A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Definitions

  • the invention belongs to the field of medicinal chemistry. Specifically, the present invention relates to a Pt(IV) chemotherapeutic prodrug and its controllable release for treating tumors.
  • Cancer is one of the most serious diseases that threaten human life and health. Surgery, radiotherapy and chemotherapy are the three major treatments for cancer.
  • Radiotherapy is a local treatment method that uses radiation to treat tumors.
  • the efficacy of radiation therapy depends on the radiosensitivity, and the degree of response of different tissues and organs and various tumor tissues after exposure to radiation varies.
  • fibrosarcoma, osteosarcoma, and melanoma are radio-insensitive (resistant) tumors. Radiotherapy is difficult to kill all cancer cells in the tumor, and it has very limited killing effect on cancer cells that are hypoxic.
  • Bivalent platinum drugs have high-efficiency and broad-spectrum anticancer activities, and have become clinically important first-line chemotherapy drugs, and are widely used in lung cancer, bladder cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer and head and neck cancer. Treatment of common malignant tumors such as external tumors.
  • the first-generation platinum-based anticancer drugs are represented by cisplatin
  • the second-generation platinum-based anti-cancer drugs are represented by carboplatin and nedaplatin
  • the third-generation platinum-based anti-cancer drugs are represented by oxaliplatin and leplatin .
  • divalent platinum drugs such as nephrotoxicity, gastrointestinal toxicity, hematological toxicity, nervous system toxicity and ototoxicity limit their application, and tumor resistance also limits their therapeutic effect.
  • divalent platinum drugs such as nephrotoxicity, gastrointestinal toxicity, hematological toxicity, nervous system toxicity and ototoxicity limit their application, and tumor resistance also limits their therapeutic effect.
  • tetravalent platinum drugs people have also carried out research on tetravalent platinum drugs.
  • the tetravalent platinum compound itself has a low ability to kill cancer cells, and can exert anticancer activity by reducing and releasing divalent platinum under physiological conditions.
  • Other unique advantages are brought about by the different coordination structure of tetravalent platinum with divalent platinum.
  • Tetravalent platinum has a d2sp3 six-coordination structure, and its stability is stronger than that of divalent platinum, so the blood stability is higher.
  • the tetravalent platinum complexes possess two additional ligands in the axial direction, providing more options for the design of platinum-based drugs.
  • some tetravalent platinum complexes such as isopropylplatinum or satraplatin have entered clinical research in the last century, no tetravalent platinum drug has been approved for marketing.
  • Pt(IV) complexes can be used as prodrugs, and the prodrugs can be irradiated to release divalent platinum drugs to achieve tumor treatment. If radiotherapy and Pt(IV) complexes are efficiently combined, that is, the controlled release of Pt(IV) complexes by radiation activation, the therapeutic effect can be effectively improved.
  • the present disclosure provides a Pt(IV) complex of formula (I), which is used as a prodrug to be activated by irradiation to treat tumors,
  • L 1 to L 6 are platinum ligands; the complex can release L 5 and L 6 after irradiation to obtain the Pt(II) complex of formula (II),
  • the Pt(II) complex of formula (II) is a cis-configuration Pt(II) complex.
  • the Pt(II) complex of formula (II) is cisplatin, carboplatin (also known as carboplatin), nedaplatin, oxaliplatin, Lobaplatin , also known as lobaplatin), or heptaplatin, Cycloplatin, Miboplatin, Enloplatin, Sebriplatin, Spiroplatin, Zenipaplatin ( Zeniplatin), TRK-710, Aroplatin, bis(isopropylamine)platinum(II) dichloride, or bis(cyclopentylamine)platinum(II) dichloride.
  • the Pt(II) complex of formula (II) is cisplatin, carboplatin (carboplatin), nedaplatin, oxaliplatin, leplatin (lobaplatin), or eplatin .
  • L 5 and L 6 are each independently -OC ( O)-R, wherein R is selected from optionally substituted C 1-20 alkyl, optionally substituted C 1-20 alkyloxy or any Optionally substituted amino, the substituent is selected from C 1-18 alkyl, carboxyl, hydroxyl, halogen, mercapto, amino, di-C 1-3 alkylamino, carbonyl, phenyl, halogenated phenyl, C 1 -6 alkyl substituted phenyl, maleimide, triphenylphosphonium.
  • each R is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, deca Tricarbonyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, carboxymethylene , 2-carboxyethylene, 3-carboxypropylene, 4-carboxybutylene, 5-carboxypentylene, 6-carboxyhexylene, (dimethylamino)methylene, 2-(bis Methylamino) ethylene, 3-(dimethylamino) propylene, 4-(dimethylamino) butylene, 5-(dimethylamino) pentylene, 6-( Dimethylamino) hex
  • the tumor is leukemia, lung cancer, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, Prostate, head and neck, nasopharyngeal, esophageal, testicular, gastric, liver, pancreatic, cervical, endometrial, melanoma, or colorectal cancers.
  • the present disclosure provides a pharmaceutical composition comprising the above-mentioned Pt(IV) complex.
  • the present disclosure also provides the use of the above-mentioned Pt(IV) complex in the preparation of a medicament for activating a tumor by irradiation.
  • the irradiation is from radiotherapy.
  • the present disclosure also provides a method for treating a tumor, comprising: administering the above-mentioned Pt(IV) complex to a subject, and irradiating the subject.
  • the irradiation is from radiotherapy.
  • the radiotherapy is performed 0.5-6 h after the administration of the Pt(IV) complex.
  • the radiation dose is less than 60 Gy.
  • the tumor is leukemia, lung cancer, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, Prostate, head and neck, nasopharyngeal, esophageal, testicular, gastric, liver, pancreatic, cervical, endometrial, melanoma, or colorectal cancers.
  • the present disclosure provides a kit comprising:
  • Figure 1 depicts the broad spectrum of radiation reduction of metal ions.
  • Figure 2 depicts the broad spectrum of radiation reduction metal complexes.
  • Figure 3 depicts radiation-driven Pt(IV) complexes for broad-spectrum and efficient release of FDA-approved Pt(II) drugs.
  • Figure 4 depicts radiation-induced potent controlled release of oxaliplatin in living cells.
  • Figure 5 depicts the radiotherapy-driven oxaliPt(IV)-(OAc) 2 prodrug reductive release of oxaliplatin for chemotherapy of oxaliplatin-sensitive cell line HCT116 tumors to achieve combined chemoradiotherapy.
  • any numerical range recited herein is intended to include all subranges within that range and any combination of the respective endpoints of that range or subrange, such as alkyl (C 1-20 alkyl) including carbon atoms having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
  • alkyl group also includes an alkyl group having a sub-range of carbon atoms of 1-4, 1-6, 1-10, 2-4, or 2-10, etc.
  • the present disclosure employs standard nomenclature and standard laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and coordination chemistry. Unless otherwise specified, the present disclosure adopts traditional methods of mass spectrometry and elemental analysis, and each step and condition may refer to the conventional operation steps and conditions in the art.
  • reagents and starting materials used in the present disclosure are commercially available or can be prepared by conventional chemical synthesis methods.
  • optionally fused to a ring means that it is fused to a ring or not to a ring.
  • optionally substituted refers to being unsubstituted or having at least one non-hydrogen substituent that does not destroy the intended properties possessed by the unsubstituted analog.
  • the number of “substitutions” can be one or more; when there are more than one, it can be 2, 3 or 4. In addition, when the number of the "substitution” is plural, the “substitution” may be the same or different.
  • substitution can be arbitrary unless otherwise specified.
  • axial ligands refers to the two axial ligands in the d2sp3 six-coordination structure of tetravalent platinum, which dissociate from the complex after radiation reduction of the complex.
  • lateral ligands refers to the four lateral ligands in the d2sp3 six - coordination structure of tetravalent platinum, which can still coordinate to the divalent platinum ion after radiation reduction of the complex.
  • neutral ligand or “anionic ligand” refers to a ligand capable of coordinating platinum, which is uncharged or negatively charged in its entirety, but which may locally have a cation such as triphenylphosphonium or ammonium group.
  • treatment may also include prophylaxis.
  • subject or “patient” in this application includes humans and mammals.
  • C1 - C20 alkyl refers to a straight or branched alkane chain containing 1 to 20 carbon atoms.
  • representative examples of C 1 -C 6 alkyl include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), isobutyl (C 4 ), n-pentyl (C 5 ), 3-pentyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butanyl (C 5 ), tert-amyl (C 5 ), n-hexyl (C 6 ) and the like.
  • lower alkyl refers to straight or branched chain alkyl groups having 1 to 4 carbon atoms.
  • Substituted alkyl refers to an alkyl group substituted at any available point of attachment with one or more substituents, preferably 1 to 4 substituents.
  • haloalkyl refers to an alkyl group having one or more halogen substituents including, but not limited to, such as -CH2Br , -CH2I , -CH2Cl , -CH2F , -CHF2, and - groups like CF 3 .
  • alkylene refers to a divalent hydrocarbon group as described above for “alkyl” but having two points of attachment.
  • a methylene group is a -CH2- group and an ethylene group is a -CH2 - CH2- group.
  • alkoxy and alkylthio refer to an alkyl group as described above attached via an oxygen bond (-O-) or sulfur bond (-S-), respectively.
  • substituted alkoxy and substituted alkylthio refer to substituted alkyl groups attached via an oxygen bond or a sulfur bond, respectively.
  • Lower alkoxy is the group OR where R is lower alkyl (an alkyl group containing from 1 to 4 carbon atoms).
  • halogen refers to fluorine, chlorine, iodine or bromine.
  • the radiation sources of the present disclosure may be alpha, beta, gamma rays produced by the decay of radionuclides.
  • X-rays, gamma rays, energetic electrons, protons, heavy ions, and alpha particles from boron neutron capture therapy (BNCT) and other possible exogenous or endogenous radiation from external radiation sources are also suitable for use in the present disclosure.
  • BNCT boron neutron capture therapy
  • the high-energy rays used in radiotherapy have high spatial and temporal resolution, high tissue penetration, and high clinical relevance.
  • the use of high-energy radiation used in radiotherapy to activate prodrug molecules and perform chemical reactions in vivo has the value of basic research and clinical application.
  • High-energy radiation-activated chemical reactions involve the radiolysis of water by radiation to produce large quantities of reactive species, which in turn react with target substrates.
  • the compounds with the highest yield are hydroxyl radicals and hydrated electrons.
  • Organisms are generally in a reducing environment, and a large number of substances such as glutathione and vitamin C will quench hydroxyl radicals and increase the yield of hydrated electrons. Then the use of hydrated electrons for chemical reactions will be a big breakthrough in living chemistry.
  • High-energy rays can be used as external stimuli to reduce the tetravalent platinum complexes to obtain divalent platinum complexes.
  • the prodrugs Due to the high penetrating power of the radiation, as well as the high spatial and temporal resolution of the radiation, the prodrugs can be converted into divalent platinum complexes very efficiently by radiotherapy equipment.
  • X-ray irradiation is used as an external trigger to activate prodrugs. Since the chemical reaction induced by radiation can be controlled in space and time, the area, time and dose of prodrugs converted to their active forms can be precisely controlled.
  • the present disclosure utilizes the property of instantly and efficiently reducing metal complexes by radiation, thereby realizing the release of Pt(II) drugs from Pt(IV) prodrugs, thereby achieving the purpose of controllable release of chemotherapeutic drugs. Therefore, the release of Pt(II) drugs such as oxaliplatin using Pt(IV) prodrugs with less radioreduction toxicity can effectively inhibit various cell lines sensitive to oxaliplatin. In HCT116 tumor-bearing mice, this strategy resulted in almost complete tumor regression. This reduction is mediated by hydrated electrons (e aq ⁇ ) generated by water radiolysis and is suitable for a hypoxic reducing tumor microenvironment. Therefore, the strategy of using radiotherapy to activate prodrugs to release chemotherapeutic drugs has certain potential clinical value.
  • Radiotherapy is required in more than 50% of cancer treatments. Modern radiotherapy techniques can precisely irradiate tumors and deliver high doses of radiation locally.
  • the response of cancer to radiation can be described by radiation sensitivity.
  • Highly radiation-sensitive cancer cells leukemias, most lymphomas, and germ cell tumors
  • Moderately radiation-sensitive cancer cells require higher doses of radiation (60-70 Gy) to kill completely.
  • Some cancers renal cell carcinoma and melanoma
  • Many common moderately radioactive tumors are usually treated with radiation if they are in an early stage. Metastatic cancer is often incurable with radiation therapy because it is impossible to treat the entire body.
  • Radiation therapy itself is painless. Many low-dose palliative treatments (eg, radiation therapy for bone metastases) cause little or no side effects. Higher doses can cause different side effects, including acute side effects during treatment, months or years after treatment (long-term side effects), or after retreatment (cumulative side effects). The nature, severity, and duration of side effects depend on the organ receiving radiation, type of radiation, dose, fractionation, concurrent chemotherapy, and patient. Side effects are dose-dependent; for example, higher doses of radiation to the head and neck may induce cardiovascular complications, thyroid dysfunction, and pituitary axis dysfunction. Modern radiation therapy is designed to minimize side effects and help patients understand and deal with unavoidable side effects.
  • Radiation therapy uses photons or charged particles to damage the DNA of cancer cells. Direct or indirect ionization of the atoms that make up the DNA strand. Indirect ionization ionizes water to form free radicals that then damage DNA. Cells have mechanisms to repair DNA damage, and double-stranded DNA breaks are more difficult to repair and can lead to significant chromosomal abnormalities and gene deletions. Targeting double-strand breaks increases the likelihood that cells will undergo cell death. In the 1950s, experiments conducted by Gray et al. showed that three times higher radiation doses were required to kill hypoxic cells compared to normoxic cells. Due to the limited tolerance of normal tissues to radiation, it is generally not possible to increase the radiation dose to compensate for tumor hypoxia. After radiotherapy, hypoxic tumor cells may persist and then divide, resulting in tumor persistence and the development of a more aggressive tumor phenotype.
  • radiotherapy is limited by the clinically allowable radiation dose (generally less than 60 Gy), and on the other hand, hypoxic tumors are often resistant to radiotherapy, which is not conducive to the DNA damage caused by oxygen-fixed radiation. Therefore, radiotherapy often needs to be combined with chemotherapy drugs to improve the cure rate of tumors.
  • most clinically approved anticancer drugs have a narrow therapeutic window and high systemic toxicity, and often require the introduction of prodrug strategies to further increase the dose and reduce toxicity.
  • the administered dose of prodrugs can exceed 50 times the normal dose, and can overcome the resistance of tumors to chemotherapeutic drugs to a certain extent.
  • prodrug strategies are difficult to achieve clinically.
  • the radiochemical alteration of molecules is the material basis for the study of all radiochemical effects.
  • radiochemical effects There are two main types of radiochemical effects: direct effects in which ionizing radiation causes direct chemical changes in target molecules, and indirect effects in which radiation deposits on environmental molecules cause indirect chemical effects on target molecules.
  • Direct and indirect effects are present at the same time, but indirect effects predominate in living organisms.
  • various active species are mainly produced by the radiolysis of water (Scheme 1a), with the highest yield being hydroxyl radicals ( ⁇ OH) and hydrated electrons (e aq - ).
  • the radiolysis of water is completed within 10-4 seconds, so the radiation-induced reactions tend to occur instantaneously and thus have strong controllability.
  • Protocol 1 Radiation-induced controlled release of metal complexes in tumors.
  • a Radiolysis of water by ionizing radiation.
  • the G value of the hydrated electron is 2.63 (G value refers to the number of molecules formed in the system that absorb 100eV energy).
  • b Radiation-generated hydrated electrons can reduce metal ions and metal complexes.
  • Pt(IV) complexes can be reduced by radiation and release Pt(II) anticancer drugs.
  • the inventors achieved in vivo radiation-induced metal reduction through research, thereby constructing a new in vivo shear chemistry.
  • This strategy was applied to the activation of Pt(IV) prodrugs, making radiotherapy an exogenous stimulus to trigger drug release, thereby completing the release of chemotherapeutic drugs at tumor sites under the guidance of precise radiotherapy.
  • this strategy also helps to solve the radioresistance problem of hypoxic tumors, and can instead improve the drug release efficiency under hypoxic conditions.
  • Direct metal reduction via radiation-induced e aq - can also be extended to other metals or biological complexes (such as metalloproteins), providing an efficient tool for mechanistic dissection of complex biological processes.
  • Pt(IV) complex of formula (I) which is used as a prodrug to be activated by irradiation to treat tumors
  • L 1 to L 6 are platinum ligands; the complex can release L 5 and L 6 after irradiation to obtain the Pt(II) complex of formula (II),
  • the Pt(IV) complexes of formula (I) of the present disclosure release the axial ligands L 5 and L 6 by reduction to give Pt(II) complexes of formula (II).
  • the Pt(IV) complex of the formula (I) is developed on the basis of the Pt(II) complex, and can be regarded as a prodrug of the Pt(II) complex of the formula (II).
  • the lateral ligands L 1 of the Pt(IV) complexes of formula (I) can be determined from the ligands L 1 to L 4 of the Pt(II) complexes of formula (II) to L 4 .
  • the lateral ligands of the Pt(IV) complexes of formula (I) can be in either the cis or trans configuration. In one embodiment, the lateral ligand of the Pt(IV) complex of formula (I) is in the cis configuration.
  • the Pt(II) complex of formula (II) is a cis-configuration Pt(II) complex.
  • the Pt(II) complex of formula (II) may be a divalent platinum ligand known to have anticancer activity.
  • the Pt(II) complex of formula (II) is a bivalent platinum complex that has been marketed or entered the clinic, such as cisplatin, carboplatin, nedaplatin, oxaliplatin, leplatin, or Eplatin, Cycloplatin, Meplatin, Enloplatin, Spaplatin, Spiroplatin, Zenipaplatin, TRK-710, Aroplatin, Bis(isopropylamine)platinum(II), or Bis(cyclopentylamine) Platinum(II) dichloride.
  • the Pt(II) complex of formula (II) is cisplatin, carboplatin, nedaplatin, oxaliplatin, leplatin, or eplatin.
  • L5 and L6 are negative monovalent ligands of Pt(IV), respectively, which can be released from the Pt(IV) complexes of formula (I) upon irradiation.
  • L5 and L6 may be the same or different.
  • L and L are each -OC (O)-R, wherein R is selected from optionally substituted C 1-20 alkyl, optionally substituted C 1-20 alkyloxy, or Optionally substituted amino, the substituent is selected from C 1-18 alkyl, carboxyl, hydroxyl, halogen, mercapto, amino, di-C 1-3 alkylamino, carbonyl, phenyl, halophenyl, C 1-6 alkyl substituted phenyl, maleimide, triphenylphosphonium.
  • Optionally substituted here means that the C 1-20 alkyl or amine group may be substituted or unsubstituted with a substituent.
  • Unsubstituted C 1-20 alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl , tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl.
  • substituents according to the stability of the chemical structure.
  • L and L are each -OC (O)-R, wherein each R is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl base, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, Octadecyl, nonadecyl, eicosyl, carboxymethylene, 2-carboxyethylene, 3-carboxypropylene, 4-carboxybutylene, 5-carboxypentylene, 6-carboxyhexylene, (dimethylamino)methylene, 2-(dimethylamino)ethylene, 3-(dimethylamino)propylene, 4-(dimethylamine) base) butylene, 5-(dimethylamino)pentylene,
  • platinum(IV) complexes are cisplatin-based prodrugs: Compound 1 - Compound 22
  • platinum(IV) complexes are carboplatin-based prodrugs: Compound 23 - Compound 44
  • platinum(IV) complexes are oxaliplatin-based prodrugs: Compound 45 - Compound 66
  • the platinum (IV) complex can be obtained by oxidizing the divalent platinum complex of formula (II) with an oxidizing agent such as hydrogen peroxide to obtain a platinum (IV) dihydroxy complex, and the two hydroxyl groups on the platinum (IV) dihydroxy complex can be acylated by substituted with carboxylate groups by the action of agents such as anhydrides.
  • platinum(IV) complexes can be prepared by the following scheme:
  • the corresponding Pt(II) Drug A (12.6 mmol, 1.0 equiv) was mixed with 12 mL H2O2, diluted with 15 mL H2O and stirred at 50°C for 5 hours. After the Pt(II) drug was completely consumed, the product was collected in a centrifuge tube at room temperature, washed with water, ethanol, and ether, respectively, and the precipitate was lyophilized to obtain a white powder as compound B.
  • Compound C was precipitated with ether and lyophilized to give a white powder.
  • Compound C (1.0 equiv.) and the corresponding carboxylic acid (2.0 equiv.) were dissolved in 5 mL of DMF, added with condensing agent TBTU (2.0 equiv.), heated to 50°C, and reacted overnight in the dark. After evaporating the solvent under reduced pressure, the precipitate was washed with water, and then lyophilized by a freeze dryer to obtain compound D.
  • the tetravalent platinum complexes of the present disclosure are believed to treat tumors primarily by reduction to bivalent platinum in vivo.
  • administration of tetravalent platinum complexes causes the drug to spread to most organs and tissues of the body along with the blood circulation, in each organ and tissue it is reduced to divalent platinum by the cells themselves. different levels.
  • the tumor can be precisely irradiated, delivering high doses of radiation locally, thereby locally increasing the level of reduction of tetravalent platinum to divalent platinum.
  • the tetravalent platinum complex prodrug of the present disclosure can be used to treat leukemia, lung cancer, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder Cancer, thyroid cancer, prostate cancer, head and neck cancer, nasopharyngeal cancer, esophagus cancer, testicular cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, melanoma or colorectal cancer.
  • Another aspect of the present disclosure provides a pharmaceutical composition comprising the above-described Pt(IV) complex and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable or “pharmaceutically acceptable” in this application means: a compound or composition that is chemically and/or toxicologically related to the other ingredients that make up the formulation and/or is prophylactic or therapeutic therewith
  • the disease or disorder is compatible with humans or mammals.
  • excipient in this application refers to an excipient or vehicle used to administer a compound, including but not limited to diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binding agents agents, lubricants, coating materials, etc. Excipients are generally described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • excipients include, but are not limited to, vegetable oils, cyclodextrins, aluminum monostearate, aluminum stearate, carboxymethyl cellulose, sodium carboxymethyl cellulose, crospovidone, glyceryl isostearate, monostearate Glyceryl Stearate, Hydroxyethyl Cellulose, Hydroxymethyl Cellulose, Hydroxy Dioctadecyl Hydroxystearate, Hydroxypropyl Cellulose, Hydroxypropyl Methyl Cellulose, Lactose, Lactose Monohydrate, Hard Magnesium fatty acid, mannitol, microcrystalline cellulose, etc.
  • At least one embodiment of the present disclosure provides a method of making a pharmaceutical composition, the method comprising admixing at least one Pt(IV) complex of the present disclosure with a pharmaceutically acceptable excipient.
  • the Pt(IV) complex of the present disclosure can be formulated into injections and powders for intravenous drip administration after dilution with normal saline or 5% glucose solution.
  • Bivalent platinum drugs are usually administered parenterally and are not suitable for oral administration.
  • the Pt(IV) complexes of the present disclosure can also be formulated into pharmaceutical compositions for oral administration.
  • a pharmaceutical composition for oral administration comprises a suspension of a Pt(IV) complex in at least one pharmaceutically acceptable vegetable oil, animal oil, mineral oil, synthetic oil or semi-synthetic oil.
  • the pharmaceutical composition may be encapsulated in hard gelatin or hydroxypropyl methylcellulose capsules or in soft gelatin capsules, the capsules comprising 50 to 350 mg of the Pt(IV) complex.
  • a pharmaceutical composition for oral administration may comprise the cyclodextrin in the form of an inclusion complex of a Pt(IV) complex by dissolving the Pt(IV) complex in an organic solvent such as acetone, and then allowing Obtained by reaction with a cyclodextrin such as a C1-4 hydroxyalkyl substituted beta or gamma cyclodextrin followed by low pressure sublimation drying to remove the solvent.
  • a cyclodextrin such as a C1-4 hydroxyalkyl substituted beta or gamma cyclodextrin followed by low pressure sublimation drying to remove the solvent.
  • the present disclosure also provides the use of the above-mentioned Pt(IV) complex in the preparation of a medicament for activating a tumor by irradiation.
  • the present disclosure also provides a method of treating a tumor, comprising: administering the above-mentioned Pt(IV) complex to a subject, and irradiating the subject.
  • the radiation is from radiation therapy.
  • Radiation therapy includes: external beam radiation therapy (including conventional external beam radiation therapy; stereotactic radiation; 3-dimensional conformal radiation therapy; intensity-modulated radiation therapy), particle therapy, Auger therapy, contact X-ray, brachytherapy ( particle interventional therapy), radionuclide therapy.
  • external beam radiation therapy including conventional external beam radiation therapy; stereotactic radiation; 3-dimensional conformal radiation therapy; intensity-modulated radiation therapy
  • particle therapy Auger therapy, contact X-ray, brachytherapy ( particle interventional therapy), radionuclide therapy.
  • the equipment that can be used includes: deep X-ray therapy machine, cobalt-60 therapy machine, medical electron linear accelerator, medical proton accelerator, medical heavy ion accelerator, gamma knife, etc.
  • radiotherapy in the present disclosure is different from concurrent radiochemotherapy: concurrent radiotherapy and chemotherapy use low-dose chemotherapy to increase tissue sensitivity to radioactivity; while radiotherapy in the present disclosure promotes the reduction of prodrugs to bivalent platinum drugs at the same time as chemotherapy Play a role.
  • radiation therapy in the present disclosure is also different from sequential chemoradiotherapy: sequential chemoradiotherapy is followed by a chemotherapy group followed by a radiation therapy group or a radiation therapy group followed by a chemotherapy group; whereas radiation therapy in the present disclosure is followed by chemotherapy Radiation therapy is given after a short period of time, eg, 0.5-6 hours.
  • the radiotherapy is performed 0.5-6 h after administration of the Pt(IV) complex.
  • the radiotherapy is performed 0.5, 1 h, 1.5 h, 2 h, 2.5 h, 3 h after administration of the Pt(IV) complex, and the radiotherapy is administered for 1-10 minutes (eg, 1, 2, 3, 4, 5 minute).
  • the radiation therapy equipment is a linear accelerator (such as the Clinac iX of Varian Medical Systems), which produces X-rays with an energy of 6MeV, and the total local irradiation dose of the tumor is 4Gy at a dose rate of 2Gy/min.
  • radiotherapy is performed 2 hours after the administration of the Pt(IV) complex.
  • the radiotherapy time is about 2 minutes, and the interval is 2 days. It needs to be repeated for a total of four weeks.
  • the Pt(IV) complexes of the present disclosure combined with radiotherapy can achieve the treatment of hypoxic tumors that cannot be treated by conventional radiotherapy, such as pancreatic cancer and prostate cancer.
  • the radiotherapy scheme of the present disclosure can be carried out by conventional radiotherapy methods, and can also be carried out with a lower dose than conventional radiotherapy methods, and the side effects of radiotherapy can be reduced when a lower dose is used.
  • the radiation dose is less than 60 Gy.
  • the Pt(IV) complexes of the present disclosure can be used in combination with radiotherapy to treat cancers such as leukemia, lung cancer, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neurosarcoma Blastoma, bladder, thyroid, prostate, head and neck, nasopharyngeal, esophageal, testicular, gastric, liver, pancreatic, cervical, endometrial, melanoma, or colorectal cancers.
  • cancers such as leukemia, lung cancer, malignant lymphoma, breast cancer, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neurosarcoma Blastoma, bladder, thyroid, prostate, head and neck, nasopharynge
  • the present disclosure also provides a kit comprising the above-mentioned Pt(IV) complex or the above-mentioned pharmaceutical composition comprising the Pt(IV) complex, further comprising instructions indicating that the administration is followed by radiotherapy to Treat tumors.
  • the starting materials for the examples are commercially available and/or can be prepared in a variety of ways well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of experiments, and work-up) in the following synthetic methods. Those skilled in the art of organic synthesis will understand that the functional groups present on various parts of the molecule should be compatible with the reagents and reactions presented.
  • Ultra performance liquid chromatography-mass spectrometry was performed on an ACQUITY UPLC H-Class PLUS instrument equipped with a Waters PDA e ⁇ detector and a Waters Acquity QDA mass spectrometer. Absorption spectra were measured by UV-1100 spectrophotometer.
  • the X-ray irradiation was generated by an X-ray generator (RS2000Pro 225, 225kV, 17.7mA; Rad Source Technologies, Inc.
  • the total dose of a single test tube experiment was 0-60 Gy, and the dose rate was 5 Gy/min.
  • the total dose of the cell experiment was 0 to 16 Gy at a dose rate of 1.6 Gy/min.
  • Implanted tumors were irradiated locally using a custom mouse mold. The local irradiation dose was 4 Gy for the tumor at a dose rate of 1 Gy/min, and 5 mm for the rest of the body Thick lead shield.
  • the Pt(IV) complex was prepared by the corresponding Pt(II) drug as described above, and the platinum(IV) dihydroxy complex was obtained after hydrogen peroxide oxidation, and the two hydroxyl groups on the platinum(IV) dihydroxy complex were combined with the corresponding Anhydride reactions prepared compounds 1 to 66.
  • the product was characterized by mass spectrometry.
  • Fe 2+ /Fe 3+ stock solution Dissolve 2.78 mg FeSO 4 ⁇ 7H 2 O and 2.70 mg FeCl 3 ⁇ 6H 2 O in 1 mL of deionized water, respectively, to obtain a 10 mM stock solution, mix 100 ⁇ L of the stock solution in 10 mL Dilute to 100 ⁇ M.
  • Fe 2+ /Fe 3+ probe 54.06 mg of phenanthroline was dissolved in 1 mL of DMSO to give a 300 ⁇ M stock solution, which was used for detection without further dilution. 2.48 mg Fe 3+ probe was dissolved in 1 mL DMSO to give a 10 mM stock solution, 100 ⁇ L was diluted to 100 ⁇ M in 10 mL MeOH/H 2 O (v/v, 1:1).
  • Detection of Fe 2+ /Fe 3+ with phen 100 ⁇ M Fe 2+ /Fe 3+ solution was further attenuated to 80, 60, 40, 20, 0 ⁇ M. Add 3 ⁇ L of the phenol stock solution to 3 mL of the above Fe 2+ /Fe 3+ solution, so the final concentration of phenol is 300 ⁇ M, and the concentration of Fe is hardly affected. UV-Vis absorption was detected at 510 nm.
  • Fe 2+ /Fe 3+ was detected with a Fe 3+ probe.
  • 100 ⁇ M Fe 2+ /Fe 3+ solutions were further attenuated to 80, 60, 40, 20, 0 ⁇ M.
  • 600 ⁇ L Fe 2+ /Fe 3+ solution is mixed with 2400 ⁇ L Fe 3+ probe, so the final concentration of Fe 2+ /Fe 3+ is 20, 16, 10, 8, 4, 0 ⁇ M, and the concentration of Fe 3+ probe is 80 ⁇ M .
  • the reaction system was incubated at 37°C for 20 minutes and detected at 450 nm using a UV-Vis spectrophotometer.
  • Cu 2+ stock solution 2.50 mg CuSO 4 ⁇ 5H 2 O were dissolved in 1 mL of deionized water to obtain 10 mM stock solutions, respectively.
  • Cu 2+ probe Dissolve 34.25 mg sodium diethyldithiocarbamate in 1 mL DMSO to give a 200 mM stock solution.
  • Detection of Cu 2+ with sodium diethyldithiocarbamate 100 ⁇ L of a 10 mM Cu 2+ stock solution was diluted in 10 mL to 100 ⁇ M and further decayed to 80, 60, 40, 20, 0 ⁇ M. Add 3 ⁇ L of sodium diethyldithiocarbamate stock solution to 3 mL of the above Cu 2+ solution, so the final concentration of phen is 200 ⁇ M, and the concentration of Cu 2+ is hardly affected. UV-Vis absorption at 450 nm was detected.
  • Ni 2+ stock solution 2.38 mg of NiCl 2 ⁇ 6H 2 O was dissolved in 1 mL of deionized water to give a 10 mM stock solution, 100 ⁇ L was diluted to 125 ⁇ M in 8 mL.
  • DMG Dimethylacetaldoxime
  • K 2 S 2 O 8 solution 57 mg of K2S2O8 was dissolved in 1.5 mL of deionized water to give a 0.14 mmol solution.
  • Ni 2+ solution was further attenuated to 100, 75, 50, 25, 0 ⁇ M.
  • 50 ⁇ L of K 2 S 2 O 8 solution, 100 ⁇ L of 1M NaOH (aq), and 50 ⁇ L of DMG solution were sequentially added to 800 ⁇ L of Ni 2+ solution, so the final concentrations of Ni 2+ were 100, 80, 60, 40, 20, 0 ⁇ M.
  • the reaction system was incubated at 25°C for 20 minutes and detected at 530 nm with a UV-Vis spectrophotometer.
  • a 10 mM stock solution of Pt(IV) complex 1 was prepared and diluted to 100 [mu]M in a solution of H2O , PBS, 5 mM Tyr, Trp, DMEM and CM (complete medium), FBS. After exposure to X-rays, 200 ⁇ L of ACN was added to each solution, and the supernatant was taken after centrifugation. Repeat 2 more times, and the final concentration is 1/8 of the original concentration. Ligand release was detected by UPLC-MS and quantified by a standard curve for coumarin.
  • the corresponding tetravalent platinum complexes (compounds 1 to 66) were dissolved in DMSO to obtain a tetravalent platinum stock solution (10 mM), which was then diluted to 10 ⁇ M with pure water. After deoxygenation, the solution was subjected to 60 Gy X-ray irradiation (4 Gy/min, 15 min). The crude reaction product was analyzed by UPLC-MS, and the released product was determined by UPLC-MS to be the corresponding divalent platinum drug, and the concentration of the divalent platinum drug was determined by the external standard curve of the platinum drug, and the release efficiency was calculated.
  • the BGC823 cell line was obtained from the National Cell Line Resource Infrastructure (Beijing, China).
  • HCT116, Ls513, HT29, and LoVo were purchased from the American Type Culture Collection (ATCC).
  • HCT116, Ls513, HT29 and BGC823 were grown in RPMI-1640 (Roswell Park Memorial Institute-1640) medium containing 10% FBS and 1% penicillin/streptomycin.
  • LoVo cells were grown in Ham's F-12K (Roswell Park Memorial Institute-1640) medium containing 10% FBS and 1% penicillin/streptomycin. All cell cultures were grown at 37°C and 5% carbon dioxide.
  • HCT116, Ls513, LoVo, HT29 and BGC823 were treated with 200 ⁇ L of RPMI-10% FBS and 1% penicillin/streptomycin at a concentration of 5 ⁇ 10 4 /mL.
  • 1640 or F-12K medium was inoculated in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Cells were then incubated with 10 ⁇ M of oxaliPt(IV)-(OAc) 2 under hypoxia for 24 hours. Cells were then irradiated with 8 Gy X-rays and incubation continued for an additional 3 days.
  • Treatment was initiated when the tumor volume reached 50 mm3 (approximately 6 days) and the treatment regimen is shown in Figure 5 .
  • the mice received radiotherapy, even though the tumor area was irradiated with 4Gy of X-rays.
  • the mouse body weight and tumor size were recorded every two days, and when the mouse tumor size exceeded 1500 mm, the mice were euthanized according to ethics committee guidelines. Recording continued until day 40 after the mice started treatment.
  • Hydrated electrons are one of the strongest reducing agents in water (standard electrode potential, -2.77 V) and one of the main products of water radiolysis ( ⁇ 280 nM/Gy).
  • ⁇ 280 nM/Gy we hypothesized that radiation-driven metal reduction is mediated by e aq - mediated by water radiolysis, as evidenced by the detection of Fe 3+ irradiated in 10 mM methanol, tert-butanol and sodium formate solutions to produce Fe 2+ in a reducing environment ( OH quencher) to facilitate the occurrence of this reaction.
  • NaNO 3 and saturated oxygen solution known as e aq -quencher , greatly reduced the release of Fe 2+ (Fig. 1d).
  • e aq - mediated reduction should be universally applicable to most transition metals, so a series of representative metal ions (100 ⁇ M, aq) were tested under the same conditions (X-ray at 60 Gy) and the reduction yields were determined by the corresponding Absorbance of the complex or other methods to determine. As shown in Figure 1e, radiation-driven reduction reactions are feasible in most cases.
  • the metal reduction yield of radiation is as high as 240 nM/Gy, which is close to the theoretical yield of e aq - .
  • Figure 1 illustrates the broad spectrum of radiation reduction of metal ions.
  • Fe 3+ /Fe 2+ redox reaction was detected by 1,10-phenanthroline (phen).
  • UPLC-MS analysis of Pt(IV) complex 1 after radiation reduction detected the release of the axial ligand. Due to the high potential of Pt(IV) derivatives in clinical applications, the next step will be to test the role of radioreduction in a biological environment, that is, to achieve radiotherapy-driven activation of the original Pt(IV) drug in tumors.
  • the Pt(IV) complexes were dissolved in PBS, 5 mM Tyr, 5 mM Trp, Dulbecco's modified medium (DMEM) and complete medium (CM) and subjected to UPLC-MS analysis after irradiation. As shown in Fig.
  • Figure 2 illustrates the broad spectrum of radiation reduction metal complexes.
  • Transition metal complexes can also be reduced by a medical dose of radiation with a reduction yield higher than the theoretical yield of e aq- .
  • the axial ligand can be released after the Pt(IV) complex (left, 100 ⁇ M, 1% DMSO in water) is reduced by radiation.
  • Pt(IV) complexes (100 ⁇ M) under various biological conditions (Tyr as tyrosine, Trp as tryptophan, 5 mM; DMEM, medium; CM, complete medium; FBS, fetal bovine serum) Radioreduction was performed and the corresponding ligand release was detected by UPLC.
  • the axial ligand release of Pt(IV) complexes can be achieved by hydrolysis or reduction: hydrolysis breaks the ester bond to obtain Pt(IV)-(OH) 2 and axial ligands; while the reduction principle changes the valence state of platinum element , generate the corresponding bivalent platinum drug.
  • hydrolysis breaks the ester bond to obtain Pt(IV)-(OH) 2 and axial ligands; while the reduction principle changes the valence state of platinum element , generate the corresponding bivalent platinum drug.
  • the most common coordination number of Pt(IV) with 5d 6 -valence electron configuration is 6, while Pt(II) with 5d 8 -valence electron configuration tends to form tetragonal complexes.
  • Previous studies on Pt(IV) prodrugs have shown that the reduction of Pt(IV) to Pt(II) leads to a reduction in coordination number and release of ligands.
  • the solution of oxaliPt(IV)-(Suc) 2 (80 mM, D 2 O) was first deoxygenated and then irradiated with 40 kGy of gamma rays ( 60 Co source, 200 Gy/min, 200 min, Figure 3a), and then irradiated with UPLC -MS detected the irradiated solution, and only one new peak was observed, and its retention time (Fig. 3b) and mass spectral signal (Fig. 3c) were consistent with those of the oxaliplatin standard sample.
  • the product was analyzed by nuclear magnetic resonance (NMR).
  • Figure 3 illustrates the broad-spectrum and efficient release of FDA-approved Pt(II) drugs from a radiation-driven Pt(IV) complex.
  • a Schematic illustration of radiation-driven release of Pt(II) drugs from Pt(IV) complexes.
  • b UPLC chromatograms of oxaliPt(IV)-(Suc) 2 , oxaliPt(IV)-(Suc) 2 + radiation, oxaliplatin and oxaliPt(IV)-(OH) 2 , where oxaliplatin and oxaliPt (IV)-(OH) 2 for reference.
  • the main product of radiation-driven release of oxaliPt(IV)-(Suc) 2 has the same retention time as oxaliplatin.
  • the detector wavelength was set to 254 nm.
  • c MS of radiation-driven released product of oxaliPt(IV)-(Suc) 2 indicates that the released product is oxaliplatin.
  • df Nuclear magnetic resonance (NMR) studies on the release of Pt(II) drugs from Pt(IV) complexes.
  • d 195 Pt-NMR spectra of oxaliPt(IV)-(Suc) 2 (1615 ppm, top), radiation irradiation product (-1988 ppm, middle) and external standard (bottom).
  • oxaliPt(IV)-(Suc) 2 has two carboxyl groups as axial ligands with two negative charge and therefore cannot be efficiently enriched in tumors. While the corresponding oxaliPt(IV)-(OAc) 2 has good stability and reactivity, more than 95% of oxaliPt(IV)-(OAc) 2 is still very stable after co-incubating with 20 equivalents of Vc for 24 hours (Fig. 4b). After OxaliPt(IV)-(OAc) 2 (10 ⁇ M PBS solution, deoxygenated) irradiated with 0-60 Gy of X-rays, the released oxaliplatin was found to be positively correlated with a given radiation dose (Fig. 4c).
  • OxaliPt(IV)-(OAc) 2 is 2 to 3 orders of magnitude less toxic than oxaliplatin in oxaliplatin-sensitive cell lines such as HCT116, HT29, LoVo and Ls513 (human colorectal cancer cell lines) IC50 is about sub-micromolar level.
  • CCK-8 assay showed that the cell viability of the group treated with 10 ⁇ M oxaliPt(IV)-(OAc) 2 +8Gy X-ray was significantly lower than that treated with only 10 ⁇ M oxaliPt(IV)-(OAc) 2 or 8Gy X
  • the radiation-treated group (Fig. 4d), demonstrating the feasibility of the strategy of releasing oxaliplatin from oxaliPt(IV)-(OAc) 2 in cells.
  • Figure 4 illustrates that radiation-induced controlled release of oxaliplatin is effective in living cells.
  • a Schematic illustration of radiation-driven release of oxaliplatin, a widely used chemotherapeutic drug, from the prodrug oxaliPt(IV)-(OAc) 2 .
  • b Stability of OxaliPt(IV)-(OAc) 2 prodrug.
  • 10 ⁇ M oxaliPt(IV)-(OAc) 2 was incubated with 20 equivalents of Vc (200 ⁇ M), more than 95% of oxaliPt(IV)-(OAc) 2 remained stable after 24 hours.
  • oxaliPt(IV)-(OAc) 2 released oxaliplatin at clinically relevant doses (0-60 Gy, X-ray), with efficiencies as high as 70%.
  • HCT116, LoVo, Ls513 and HT69 are human colorectal cancer cells that are sensitive to oxaliplatin.
  • mice After injection of 30 ⁇ mol/kg of oxaliPt(IV)-(OAc) 2 into HCT116 tumor-bearing mice via tail vein injection, the mice were sacrificed at selected times. The platinum concentration in blood, tumor, liver and kidney was then measured by ICP-MS. ICP-MS data at multiple time points showed that the prodrug was mainly metabolized by the liver and kidney, with tumor uptake peaking at 1 h after administration ( ⁇ 15 ⁇ M), followed by a gradual decrease and complete clearance 48 h after injection (Fig. 5c). ). One hour after administration, the mice had lower concentrations of the prodrug in the muscles and brain, suggesting that the drug does not have adverse effects on these organs.
  • HCT116 cells were implanted into the right flank of Nu/Nu mice until the average tumor volume reached approximately 50 mm3 .
  • Mice were randomly divided into 7 groups, including control group (PBS only), 3 ⁇ mol/kg oxaliplatin, 30 ⁇ mol/kg oxaliPt(IV)-(OAc) 2 , X-ray, 3 ⁇ mol/kg oxaliplatin + X-ray, 30 ⁇ mol/kg oxaliPt(IV)-(OAc) 2 + X-ray and 3 ⁇ mol/kg transPt(IV)-(OAc) 2 + X-ray group, and the drug was injected on day 0 (Fig.
  • mice in the oxaliPt(IV)-(OAc) 2 +X-ray treatment group did not experience weight loss (Fig. 5h), indicating that our strategy has good biosafety. Therefore, the results of the treatment of HCT116 tumor-bearing mice indicated that this radiation-driven Pt(II) drug release has a high feasibility in vivo.
  • Figure 5 illustrates the radiotherapy-driven oxaliPt(IV)-(OAc) 2 prodrug reductive release of oxaliplatin for chemotherapy of oxaliplatin-sensitive cell line HCT116 tumors to achieve combined chemoradiotherapy.
  • the drug adaptability study in nude mice showed that the maximum tolerated doses of oxaliplatin and oxaliPt(IV)-(OAc) 2 were about 3 ⁇ mol/kg and 30 ⁇ mol/kg, respectively.
  • c Pharmacokinetics of the oxaliPt(IV)-(OAc) 2 prodrug to determine the optimal timing of radiotherapy. Tumor uptake of the OxaliPt(IV)-(OAc) 2 prodrug peaked 1-2 hours after injection and then gradually declined. The remaining prodrugs are rapidly cleared from the blood and excreted through the renal and hepatobiliary systems.
  • This study achieves radiation-induced metal reduction in vivo, thereby constructing a new in vivo shear chemistry.
  • This strategy was applied to the activation of Pt(IV) prodrugs, making radiotherapy an exogenous stimulus to trigger drug release, thereby completing the release of chemotherapeutic drugs at tumor sites under the guidance of precise radiotherapy.
  • this strategy also helps to solve the radioresistance problem of hypoxic tumors, and can instead improve the drug release efficiency under hypoxic conditions.
  • Direct metal reduction via radiation-induced e aq - can also be extended to other metals or biological complexes (such as metalloproteins), providing an efficient tool for mechanistic dissection of complex biological processes.

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Abstract

提供了一种Pt(IV)配合物，其作为前药经辐照激活而释放出Pt(II)配合物以治疗肿瘤。还提供了一种包含所述Pt(IV)配合物的药物组合物，以及Pt(IV)配合物在制备用于经辐照激活治疗肿瘤的药物中的用途。还提供了一种包含所述Pt(IV)配合物和说明书的试剂盒，该说明书指出给药之后进行放疗以治疗肿瘤。

Description

Pt(Ⅳ)化疗前药及其可控释放用于治疗肿瘤 技术领域
本发明属于药物化学领域。具体地，本发明涉及一种Pt(Ⅳ)化疗前药及其可控释放用于治疗肿瘤。
背景技术
癌症是当代最严重的威胁人类生命和健康的疾病之一。手术、放疗和化疗一起并称肿瘤的三大治疗手段。
放疗是是利用放射线治疗肿瘤的一种局部治疗方法。放射治疗的疗效取决于放射敏感性，不同组织器官以及各种肿瘤组织在受到照射后出现变化的反应程度各不相同。例如纤维肉瘤、骨肉瘤、黑色素瘤是放射不敏感(抗拒)的肿瘤。放疗难以杀死肿瘤中所有的癌细胞，而且对乏氧的癌细胞杀伤效果十分有限。
化疗通过使用化学药物杀灭癌细胞达到治疗目的。二价铂类药物具有高效广谱的抗癌活性，已成为临床上重要的一线化疗药物，被广泛用于肺癌、膀胱癌、卵巢癌、宫颈癌、食管癌、胃癌、结直肠癌和头颈部肿瘤等常见恶性肿瘤的治疗。第一代铂类抗癌药物以顺铂为代表，第二代铂类抗癌药物以卡铂、奈达铂为代表，第三代铂类抗癌药物以奥沙利铂和乐铂为代表。二价铂类药物的副作用如肾毒性、肠胃道毒性、血液系统毒性、神经系统毒性和耳毒性限制了其应用，而且肿瘤抗药性也限制了其治疗效果。为了扩展铂类药物，人们也开展了四价铂药物的研究。四价铂化合物自身对癌细胞的杀伤能力较低，可通过在生理条件下还原并释放出二价铂发挥抗癌活性，既保留了传统二价铂类药物的广谱高效抗癌优点，又因四价铂不同于与二价铂的配位结构而带来独特的其它优势。四价铂具有d2sp3六配位结构，稳定性强于二价铂，因此血液稳定性较高。四价铂配合物的轴向拥有两个额外的配体，为铂类药物的设计提供了更多的选择。然而，尽管一些四价铂配合物如异丙铂或赛特铂在上世纪就进入临床研究，但是时至今日仍无四价铂的药物被批准上市。
因此，仍然需要开发一种具有较低毒性和较高疗效的铂药和/或基于铂药的治疗方案。
发明内容
本发明人经过深入的研究和创造性的劳动，发现Pt(Ⅳ)配合物可用作前药，该前药经 辐照释放出二价铂药，实现治疗肿瘤。若将放疗和Pt(Ⅳ)配合物高效结合，即利用辐射激活进行Pt(Ⅳ)配合物的可控释放，则能有效地提高治疗效果。
一方面，本公开提供了一种式(I)的Pt(Ⅳ)配合物，其用作前药经辐照激活治疗肿瘤，
Figure PCTCN2021132886-appb-000001
其中L 1至L 6为铂的配体；该配合物在辐照之后能够释放出L 5和L 6得到式(II)的Pt(II)配合物，
Figure PCTCN2021132886-appb-000002
可选的，式(II)的Pt(II)配合物为顺式构型Pt(II)配合物。例如式(II)的Pt(II)配合物为顺铂(cisplatin)、卡铂(Carboplatin，也称为碳铂)、奈达铂(Nedaplatin)、奥沙利铂(Oxaliplatin)、乐铂(Lobaplatin，也称为洛铂)、或依铂(heptaplatin)、环铂(Cycloplatin)、米铂(Miboplatin)、恩洛铂(Enloplatin)、司铂(Sebriplatin)、螺铂(Spiroplatin)、折尼铂(Zeniplatin)、TRK-710、Aroplatin、双(异丙胺)二氯化铂(II)、或者双(环戊胺)二氯化铂(II)。在一种优选的实施方案中，式(II)的Pt(II)配合物为顺铂、卡铂(碳铂)、奈达铂、奥沙利铂、乐铂(洛铂)、或者依铂。
可选的，L 5和L 6各自独立地为 -O-C(O)-R，其中R选自任选取代的C 1-20烷基、任选取代的C 1-20烷基氧基或者任选取代的胺基，取代基选自C 1-18烷基、羧基、羟基、卤素、巯基、胺基、二C 1-3烷基胺基、羰基、苯基、卤代苯基、C 1-6烷基取代的苯基、马来酰亚胺基、三苯基鏻基。例如，R各自独立地选自甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一碳烷基、十二碳烷基、十三碳烷基、十四碳烷基、十五碳烷基、十六碳烷基、十七碳烷基、十八碳烷基、十九碳烷基、二十碳烷基、羧基亚甲基、2-羧基亚乙基、3-羧基亚丙基、4-羧基亚丁基、5-羧基亚戊基、6-羧基亚己基、(二甲基胺基)亚甲基、2-(二甲基胺基)亚乙基、3-(二甲基胺基)亚丙基、4-(二甲基胺基)亚丁基、5-(二甲基胺基)亚戊基、6-(二甲基胺基)亚己基、5-马来酰亚胺基亚戊基、6-马来酰亚胺基亚己基、7-马来酰亚胺基亚庚基、8-马来酰亚胺基亚辛基、3-(4-碘苯基)亚丙基、3-(3-碘苯基)亚丙基、3-(3,5-二碘苯基)亚丙基、3-(4-溴苯基)亚丙基、3-(3-溴苯基)亚丙基、3-(3,5-二溴苯基)亚丙基、甲胺基、乙胺基、丙胺基、丁胺基、戊胺基、己胺基、庚胺基、辛胺基、壬胺基、癸胺基、十一碳烷胺基、十二碳烷胺基、十三碳烷胺基、十四碳烷胺基、十五碳烷胺基、十六碳烷胺基、十七碳烷胺基、十八碳烷胺基。
可选的，所述肿瘤为白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
又一方面，本公开提供了一种药物组合物，其包含上述Pt(Ⅳ)配合物。
另一方面，本公开还提供了上述Pt(Ⅳ)配合物在制备用于经辐照激活治疗肿瘤的药物中的用途。可选的，所述辐照来自放疗。
又一方面，本公开还提供了一种治疗肿瘤的方法，其包括：向受试者施用上述Pt(Ⅳ)配合物，以及对受试者进行辐照。
可选的，所述辐照来自放疗。
可选的，所述放疗在Pt(Ⅳ)配合物给药的0.5-6h之后进行。
在一种实施方案中，辐射剂量小于60Gy。
可选的，所述肿瘤为白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
又一方面，本公开提供了一种试剂盒，其包含：
上述Pt(Ⅳ)配合物或者上述包含Pt(Ⅳ)配合物的药物组合物，以及
说明书，该说明书指出给药之后进行放疗以治疗肿瘤。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本公开的一些实施例，而非对本发明的限制。
图1描述了辐射还原金属离子的广谱性。
图2描述了辐射还原金属配合物的广谱性。
图3描述了辐射驱动Pt(Ⅳ)配合物广谱高效释放FDA批准的Pt(Ⅱ)药物。
图4描述了辐射诱导能在活细胞中有效地控制释放奥沙利铂。
图5描述了放疗驱动oxaliPt(Ⅳ)-(OAc) 2前药还原释放奥沙利铂用于奥沙利铂敏感细胞系HCT116肿瘤的化疗实现放化疗联合治疗。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例的附图，对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明可在不偏离本发明基本属性的情况下以其它具体形式来实施。应该理解的是，在不冲突的前提下，本发明的任一和所有实施方案都可与任一其它实施方案或多个其它实施方案中的技术特征进行组合以得到另外的实施方案。本发明包括这样的组合得到另外的实施方案。
本公开中提及的所有出版物和专利在此通过引用以它们的全部内容纳入本公开。如通过引用纳入的任何出版物和专利中使用的用途或术语与本公开中使用的用途或术语冲突，以本公开的用途和术语为准。
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。
除非另有规定，本文使用的所有技术术语和科学术语具有要求保护主题所属领域的通常含义。倘若对于某术语存在多个定义，则以本文定义为准。
除了在工作实施例中或另外指出之外，在说明书和权利要求中陈述的表达材料的量、反应条件、持续时间和材料的定量性质等的所有数字应理解为在所有情况中被术语“约”修饰。还应理解的是，本申请列举的任何数字范围意在包括该范围内的所有的子范围和该范围或子范围的各个端点的任何组合，例如具有碳原子数1-20的烷基(C 1-20烷基)包括具有碳原子数1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20的烷基，也包括子范围具有碳原子数1-4、1-6、1-10、2-4、或2-10等的烷基。
本公开的说明书应该被解释为与化学键的法则和原理一致。在一些情况下，可能为了在给定的位置适应取代基而除去氢原子。
本公开中使用的“包括”、“含有”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同，而不排除未记载的要素。本文所用的术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…组成”、或“由…组成”。
应该理解，在本公开中使用的单数形式(如“一种”)可包括复数指代，除非另有规定。
除非另有指明，本公开采用分析化学、有机合成化学和配位化学的标准命名及标准实验室步骤和技术。除非另有说明，本公开采用质谱、元素分析的传统方法，各步骤和条件 可参照本领域常规的操作步骤和条件。
本公开所用试剂和原料是市售可得的或者可通过常规化学合成方法制得的。
本文使用术语“任选”来描述某一情形是指该情形可发生也可不发生。例如，任选地与某环稠合表示其与某环稠合或者不与某环稠合。例如，本文使用的术语“任选取代的”是指为未取代的或者具有至少一个不破坏由未取代的类似物所拥有的目的性能的非氢取代基。
本公开中，如无特殊说明，所述的“取代”的个数可为一个或多个；当为多个时，可为2个、3个或4个。并且，当所述的“取代”的个数为多个时，所述的“取代”可相同或不同。
本公开中，“取代”的位置，如未做特别说明，位置可为任意。
本文使用的术语“轴向配体”是指四价铂的d 2sp 3六配位结构中的两个轴向配体，其在配合物辐照还原之后从配合物中脱离。
本文使用的术语“横向配体”是指四价铂的d 2sp 3六配位结构中的四个横向配体，其在配合物辐照还原之后可以仍然与二价铂离子配位。
本文使用的术语“中性配体”或者“阴离子配体”是指能够与铂配位的配体，该配体整体不带电荷或者带负电荷，然而其局部可以具有阳离子例如三苯基鏻或铵基。
在本申请的上下文中，除非作出相反的具体说明，术语“治疗”也可包括预防。
术语“受试者”或“患者”在本申请中包括人类和哺乳动物。
本文使用的术语“C 1-C 20烷基”是指含有1-20个碳原子的直链或支链烷烃链。例如，C 1-C 6烷基的代表性实例包括但不限于甲基(C 1)、乙基(C 2)、正丙基(C 3)、异丙基(C 3)、正丁基(C 4)、叔丁基(C 4)、仲丁基(C 4)、异丁基(C 4)、正戊基(C 5)、3–戊烷基(C 5)、新戊基(C 5)、3-甲基-2-丁烷基(C 5)、叔戊基(C 5)和正己基(C 6)等。术语“低级烷基”是指具有1至4个碳原子的直链或支链烷基。“经取代的烷基”指在任何可用连接点处经一个或多个取代基优选1至4个取代基取代的烷基。术语“卤代烷基”是指具有一个或多个卤素取代基的烷基，其包括但不限于如-CH 2Br、-CH 2I、-CH 2Cl、-CH 2F、-CHF 2及-CF 3那样的基团。
本文使用的术语“亚烷基”是指如以上就“烷基”所述但具有两个连接点的二价烃基。例如，亚甲基为-CH 2-基团，亚乙基为-CH 2-CH 2-基团。
本文使用的术语“烷氧基”及“烷基硫基”指分别经由氧键(-O-)或硫键(-S-)连接的如上所述的烷基。术语“经取代的烷氧基”及“经取代的烷基硫基”指分别经由氧键或硫键连接的经取代的烷基。“低级烷氧基”为基团OR，其中R为低级烷基(含有1至4个碳原子的烷基)。
本文使用的术语“卤素”是指氟、氯、碘或溴。
本公开的辐射源可以是放射性核素衰变所产生的α、β、γ射线。外部辐射源产生的X 射线，γ射线，高能电子，质子，重离子，以及硼中子俘获治疗(BNCT)产生的α粒子及其他可能的外源或内源辐射也可适用于本公开。
放疗中使用的高能射线具有高时空分辨率、高组织穿透能力，同时具有高度临床相关性。利用放疗使用的高能射线在活体内激活前药分子、进行化学反应具有基础研究价值和临床应用的价值。
高能射线激活的化学反应涉及到射线使水辐解产生大量的活性物质，这些活性物质再与目标底物发生反应。其中水辐解的产物中，产额最高的化合物为羟基自由基以及水合电子。
生物体内一般处于还原性环境，有大量的物质如谷胱甘肽，维生素C等会淬灭羟基自由基，并且提高水合电子的产额。那么利用水合电子进行化学反应将是活体化学中一个大的突破。
高能射线(如X射线及γ射线)可用作外部刺激使四价铂配合物发生还原反应得到二价铂配合物。由于射线的高穿透能力，以及射线的高时空分辨率，前药可以通过放疗设备非常有效地转化为二价铂配合物。例如，X射线辐照作为激活前药的外部触发器，由于在空间和时间上可以控制辐射引发的化学反应，可精确控制前药转化为其活性形式的面积、时间和剂量。
本公开利用辐射即时、高效地还原金属络合物的特性，从而实现Pt(Ⅳ)前药释放Pt(Ⅱ)药物，便能达到化疗药物的可控释放的目的。因此，利用辐射还原毒性较低的Pt(Ⅳ)前药释放出Pt(Ⅱ)药物例如奥沙利铂，便能有效地抑制对奥沙利铂敏感的各种细胞系。在HCT116荷瘤小鼠中，该策略使肿瘤几乎完全消退。这种还原是通过水辐射解产生的水合电子(e aq -)介导的，适用于缺氧的还原性肿瘤微环境。因此，利用放疗激活前药释放化疗药物的策略在临床上具有一定的潜在价值。
肿瘤治疗中超过50％的病例需要接受放疗。现代放疗技术可以精确地照射肿瘤，并局部提供高剂量的辐射。
癌症对辐射的反应可以由辐射敏感性来描述。高度辐射敏感的癌细胞(白血病、大多数淋巴瘤和生殖细胞肿瘤)会被中等剂量的辐射迅速杀死。中等辐射敏感性的癌细胞(大多数上皮癌)需要更高剂量的辐射(60-70Gy)才能完全杀死。有些癌症(肾细胞癌和黑色素瘤)具有显著的抗辐射性，需要比临床中安全的剂量高得多的剂量才能实现根治。许多常见的中度放射反应性肿瘤如果处于早期阶段，通常会接受放疗。转移性癌症通常无法通过放疗治愈，因为不可能治疗整个身体。
放射治疗本身是无痛的。许多低剂量姑息治疗(例如，骨转移瘤的放射治疗)引起的副作用很小或没有副作用。较高剂量会导致不同的副作用，包括在治疗期间产生急性副作用、治疗后数月或数年(长期副作用)或再治疗后(累积副作用)的副作用。副作用的性质、严重程度和持续时间取决于接受放射的器官、放射类型、剂量、分次、同步化疗和患者。副作用是剂量依赖性的；例如，较高剂量的头颈部辐射可能引发心血管并发症、甲状腺功能障碍和垂体轴功能障碍。现代放射治疗旨在将副作用降至最低，并帮助患者了解和处理不可避免的副作用。
放射治疗通过光子或带电粒子破坏癌细胞的DNA。构成DNA链上原子的直接或间接电离。间接电离通过水电离形成自由基然后破坏DNA。细胞具有修复DNA损伤的机制，双链DNA的断裂更难修复，并可能导致显着的染色体异常和基因缺失。靶向双链断裂会增加细胞发生细胞死亡的可能性。在1950年代，Gray等人进行的实验表明，与常氧细胞相比，杀死缺氧细胞需要三倍高的辐射剂量。由于正常组织对辐射的耐受性有限，一般不可能增加辐射剂量来补偿肿瘤缺氧。放疗后，缺氧的肿瘤细胞可能会持续存在然后分裂，导致肿瘤持续存在并发展出更具侵袭性的肿瘤表型。
放疗一方面由于临床允许的辐射剂量有限(一般小于60Gy)，另一方面由于缺氧的肿瘤往往会对放疗产生抗性、不利于氧气固定辐射引起的DNA损伤。因此，放疗往往需要与化疗药物相结合，才能提高肿瘤的治愈率。然而，大多数临床批准的抗癌药物治疗窗口较窄、全身毒性大，往往需要引入前药策略来进一步提高给药剂量、降低毒性。前药的给药剂量可超过正常剂量的50倍，并且能一定程度上克服肿瘤对化疗药物的抵抗。但由于前药激活效率有限，并缺乏肿瘤选择性，前药策略在临床上很难实现。若利用放疗作为一种精确的外源刺激，在肿瘤部位高选择性地激活原药，便能解决以上难题。但目前尚未有活体内辐射诱导的裂解化学在活体内建立起来，过去三十年内，仅有部分工作在试管或细胞水平上实现了该策略。然而，若不在活体内建立辐射激活的化学反应，放疗诱导的化疗药物激活就很难在临床上产生影响。
分子的放射化学改变是研究所有放射化学效应的物质基础。放射化学效应主要有两种：电离辐射引起目标分子的直接化学变化的直接效应，和辐射沉积在环境分子上后引起目标分子的间接化学作用的间接效应。直接和间接效应是同时存在的，但间接效应在活体中占主导地位。因为组织中70～80％是水，因此主要通过水的辐解产生各种活性物质(方案1a)，产额最高的是羟基自由基(·OH)和水合电子(e aq -)。水的辐解在10 -4秒内完成，因此辐射诱导的反应往往在瞬间发生，因此具有较强的可控性。由·OH诱导的裂解化学反应和相 关的荧光探针已成功用于生物成像，但还原性肿瘤微环境对·OH快速的淬灭，阻碍了其在生命体系内的发展。而辐射产生的e aq -的产量，即水辐解的另一个主要产品，在还原性环境中产额增加。因此，我们在此探讨通过精确的放疗局部产生e aq -介导化学剪切反应的可行性(方案1b)，将辐射作为化学工具，以高肿瘤选择性的方式释放目标分子(方案1c)。
Figure PCTCN2021132886-appb-000003
方案1a
Figure PCTCN2021132886-appb-000004
方案1b(M为Fe、Co、Ni、Cu、Ru、Rh、Pd、Ag等)
Figure PCTCN2021132886-appb-000005
方案1c
方案1.辐射诱导的金属复合物在肿瘤中的控制释放。a，电离辐射对水的辐解。水合电子的G值为2.63(G值指系统中吸收100eV能量所形成的分子数)。b，辐射产生的水合电子可以还原金属离子和金属络合物。c，Pt(Ⅳ)配合物可以通过辐射还原，并释放Pt(Ⅱ)抗癌药物。
发明人通过研究实现了活体内的辐射诱导的金属还原，从而构建了新的活体剪切化学。将该策略应用于Pt(Ⅳ)前药的激活，使放疗成为一种触发药物释放的外源刺激，从而在在精确放疗的指导下完成肿瘤部位化疗药物的释放。除此之外，本策略也有助于解决缺氧肿瘤的放疗抗性问题，反而能在乏氧条件下提高药物的释放效率。通过辐射诱导e aq -的直接金属还原还可以拓展到其他金属或生物配合物(如金属蛋白)，为复杂的生物过程的机理剖析提供一个高效的工具。
本公开一方面提供一种式(I)的Pt(Ⅳ)配合物，其作为前药经辐照激活治疗肿瘤，
Figure PCTCN2021132886-appb-000006
其中L 1至L 6为铂的配体；该配合物在辐照之后能够释放出L 5和L 6得到式(II)的Pt(II)配合物，
Figure PCTCN2021132886-appb-000007
本公开的式(I)的Pt(Ⅳ)配合物通过还原释放出轴向配体L 5和L 6得到式(II)的Pt(II)配合物。式(I)的Pt(Ⅳ)配合物是在Pt(II)配合物的基础上研发的，可以看作式(II)的Pt(II)配合物的前药。根据上述式I与式II的对应关系，可以从式(II)的Pt(II)配合物的配体L 1至L 4确定式(I)的Pt(Ⅳ)配合物的横向配体L 1至L 4。
式(I)的Pt(Ⅳ)配合物的横向配体可以为顺式构型或反式构型。在一种实施方案中，式(I)的Pt(Ⅳ)配合物的横向配体为顺式构型。
在一种实施方案中，式(II)的Pt(II)配合物为顺式构型Pt(II)配合物。
式(II)的Pt(II)配合物可以是已知有抗癌活性的二价铂配体。在一种实施方案中，式(II)的Pt(II)配合物是已上市或者进入临床的二价铂配合物，例如顺铂、卡铂、奈达铂、奥沙利铂、乐铂、或依铂、环铂、米铂、恩洛铂、司铂、螺铂、折尼铂、TRK-710、Aroplatin、双(异丙胺)二氯化铂(II)、或者双(环戊胺)二氯化铂(II)。
在一种优选的实施方案中，式(II)的Pt(II)配合物为顺铂、卡铂、奈达铂、奥沙利铂、乐铂、或者依铂。
L 5和L 6分别为Pt(Ⅳ)的负一价配体，其在辐照下能从式(I)的Pt(Ⅳ)配合物中释放出。L 5和L 6可以是相同的或不同的。
在一种实施方案中，L 5和L 6分别为 -O-C(O)-R，其中R选自任选取代的C 1-20烷基、任选取代的C 1-20烷基氧基或者任选取代的胺基，取代基选自C 1-18烷基、羧基、羟基、卤素、巯基、胺基、二C 1-3烷基胺基、羰基、苯基、卤代苯基、C 1-6烷基取代的苯基、马来酰亚胺基、三苯基鏻基。此处任选取代的是指C 1-20烷基或者胺基可以是被取代基取代的或者未取代的。未取代的C 1-20烷基包括甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一碳烷基、十二碳烷基、十三碳烷基、十四碳烷基、十五碳烷基、十六碳烷基、十七碳烷基、十八碳烷基、十九碳烷基、二十碳烷基。本领域技术人员会根据化学结构的稳定性合理地选择取代基。
在一种实施方案中，L 5和L 6分别为 -O-C(O)-R，其中R各自独立地选自甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一碳烷基、十二碳烷基、十三碳烷基、十四碳烷基、十五碳烷基、十六碳烷基、十七碳烷基、十八碳烷基、十九碳烷基、二十碳烷基、羧基亚甲基、2-羧基亚乙基、3-羧基亚丙基、4-羧基亚丁基、5-羧基亚戊基、6-羧基亚己基、(二甲基胺基)亚甲基、2-(二甲基胺基)亚乙基、3-(二甲基胺基)亚丙基、4-(二甲基胺基)亚丁基、5-(二甲基胺基)亚戊基、6-(二甲基胺基)亚己基、5-马来酰亚胺基亚戊基、6-马来酰亚胺基亚己基、7-马来酰亚胺基亚庚基、8-马来酰亚胺基亚辛基、3-(4-碘苯基)亚丙基、3-(3-碘苯基)亚丙基、3-(3,5-二碘苯基)亚丙基、3-(4-溴苯基)亚丙基、3-(3-溴苯基)亚丙基、3-(3,5-二溴苯基)亚丙基、甲胺基、乙胺基、丙胺基、丁胺基、戊胺基、己胺基、庚胺基、辛胺基、壬胺基、癸胺基、十一碳烷胺基、十二碳烷胺基、十三碳烷胺基、十四碳烷胺基、十五碳烷胺基、十六碳烷胺基、十七碳烷胺基、十八碳烷胺基。
例如，铂(Ⅳ)配合物是基于顺铂的前药：化合物1-化合物22
Figure PCTCN2021132886-appb-000008
其中：
Figure PCTCN2021132886-appb-000009
Figure PCTCN2021132886-appb-000010
例如，铂(Ⅳ)配合物是基于卡铂的前药：化合物23-化合物44
Figure PCTCN2021132886-appb-000011
其中：
Figure PCTCN2021132886-appb-000012
Figure PCTCN2021132886-appb-000013
例如，铂(Ⅳ)配合物是基于奥沙利铂的前药：化合物45-化合物66
Figure PCTCN2021132886-appb-000014
其中：
Figure PCTCN2021132886-appb-000015
Figure PCTCN2021132886-appb-000016
铂(Ⅳ)配合物可以通过用氧化剂例如双氧水氧化式(II)的二价铂配合物得到铂(Ⅳ)二羟配合物，铂(Ⅳ)二羟配合物上的两个羟基可以通过酰化剂例如酐的作用下被羧酸盐基团取代。
例如，铂(Ⅳ)配合物可通过以下方案制备：
Figure PCTCN2021132886-appb-000017
将相应的Pt(II)药物A(12.6mmol,1.0当量)与12mL H 2O 2混合，用15mL H 2O稀释并在50℃下搅拌5小时。在Pt(II)药物完全消耗后，用离心管在室温下收集产物，用分别用水、乙醇、乙醚洗涤，将沉淀冻干得到白色粉末为化合物B。
将化合物II(1.0当量)与对应的酸酐(如：丁二酸酐，醋酸酐，N,N-二甲基甘氨酸酐)(1.0当量)混合，用4mL无水DMF溶解后搅拌1小时，得到未纯化的化合物C。
化合物D(氨基甲酸酯键，即R 2代表氨基化合物)：
在上述反应液中加入2mL相应异氰酸酯的无水DMF溶液。反应过夜，在65℃下减压除去溶剂。向油状残留物中加入2mL乙醚，将混合物超声处理1分钟并离心。用4mL DCM和2mL乙醚进一步洗涤固体。将洗过的固体在真空中放置过夜，即可得到化合物D。
化合物D(酯键，即R 2代表烷基化合物)：
利用乙醚将化合物C沉淀，冻干后得到白色粉末。将化合物C(1.0当量)与相应的羧酸(2.0当量)溶解在5mL DMF中，加入缩合剂TBTU(2.0当量)，加热至50℃，暗处反应过夜。减压蒸发溶剂后用水洗沉淀，再用冻干机冻干可得到化合物D。
据信本公开的四价铂配合物主要通过在体内还原成二价铂来治疗肿瘤。尽管四价铂配合物给药(口服、静脉和体腔给药等)会使得药物随着血液循环遍布全身的绝大部分器官和组织，但是在各器官和组织被细胞自身还原成二价铂的水平不同。然而，通过随后的放疗，可精确地照射肿瘤，局部提供高剂量的辐射，从而局部提高四价铂还原成二价铂的水平。
本公开的四价铂配合物前药可用于治疗白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
本公开的另一方面提供药物组合物，其包含上述Pt(Ⅳ)配合物和可药用辅料。
术语“药学上可接受的”或“可药用”在本申请中是指：化合物或组合物在化学上和/或在毒理学上与构成制剂的其它成分和/或与用其预防或治疗疾病或病症的人类或哺乳动物相容。
术语“辅料”在本申请中是指用以将化合物给药的赋形剂或者媒介物，其包括但不限于稀释剂、崩解剂、沉淀抑制剂、表面活性剂、助流剂、粘合剂、润滑剂、包衣材料等。辅料在E.W.Martin的“Remington's Pharmaceutical Sciences”中被一般性描述。辅料的实例包括但不限于植物油、环糊精、单硬脂酸铝、硬脂酸铝、羧甲基纤维素、羧甲基纤维素钠、交聚维酮、异硬脂酸甘油酯、单硬脂酸甘油酯、羟基乙基纤维素、羟基甲基纤维素、羟基硬脂酸羟基二十八酯、羟基丙基纤维素、羟基丙基甲基纤维素、乳糖、乳糖一水合物、硬脂酸镁、甘露醇、微晶纤维素等。
本公开的至少一种实施方案提供制备药物组合物的方法，该方法包括将本公开的至少一种Pt(Ⅳ)配合物与可药用辅料混合。
本公开的Pt(Ⅳ)配合物可以配制成注射剂和粉针剂，使用生理盐水或5％葡萄糖溶液稀释后静脉滴注给药。
二价铂药通常非肠道给药，不适合口服给药。然而本公开的Pt(Ⅳ)配合物还可以配制成口服给药的药物组合物。
例如，口服给药的药物组合物包含Pt(Ⅳ)配合物在至少一种药学可接受的植物油、动 物油、矿物油、合成油或半合成油中的混悬液。在一种实施方案中，药物组合物可以包封在硬明胶或羟丙基甲基纤维素胶囊内或在软明胶胶囊中，胶囊包含50至350mg的Pt(Ⅳ)配合物。
例如，口服给药的药物组合物可包含环糊精与Pt(Ⅳ)配合物的包合络合物的形式，其通过将Pt(Ⅳ)配合物溶解在有机溶剂例如丙酮中，然后使其与环糊精例如C1-4羟烷基取代的β或γ环糊精反应并随后低压升华干燥除去溶剂而获得。
本公开还提供上述Pt(Ⅳ)配合物在制备用于经辐照激活治疗肿瘤的药物中的用途。
在又一方面，本公开还提供一种治疗肿瘤的方法，其包括：向受试者施用上述Pt(Ⅳ)配合物，以及对受试者进行辐照。
在一种实施方案中，所述辐照来自放疗。
放射治疗包括：外照射放射治疗(包括常规外照射放射治疗；立体定向辐射；3维适形放射治疗；调强放射治疗)、粒子疗法、俄歇疗法、接触式X射线、近距离放射治疗(粒子介入治疗)、放射性核素治疗。
可以使用的设备包括：深部X线治疗机、钴-60治疗机、医用电子直线加速器、医用质子加速器、医用重离子加速器、伽玛刀等。
请注意本公开的放疗与同步放化疗不同：同步放化疗是用小剂量化疗增加组织对放射性的敏感度；而本公开的放疗是在化疗的同时，辐照促进前药还原成二价铂药发挥作用。请注意本公开的放疗也不同于序贯放化疗：序贯放化疗是先进行一组化疗然后进行一组放疗或者先进行一组放疗然后进行一组化疗；而本公开的放疗是在化疗之后短时间内例如在0.5-6小时之后进行放疗。
在一种实施方案中，所述放疗在给药Pt(Ⅳ)配合物之后0.5-6h进行。
例如，所述放疗在给药Pt(Ⅳ)配合物之后0.5、1h、1.5h、2h、2.5h、3h进行，放疗进行辐照1-10分钟(例如，1、2、3、4、5分钟)。
例如，放疗设备为直线加速器(例如瓦里安医疗系统的Clinac iX)，产生能量为6MeV的X射线，肿瘤的局部照射总剂量为4Gy，剂量率为2Gy/min，治疗方案，每周进行两次治疗(药物+放疗为一次治疗)，Pt(IV)配合物给药之后2小时进行放疗，放疗时间为约2分钟，间隔时间为2天，一共需要重复四周，
Figure PCTCN2021132886-appb-000018
本公开的Pt(IV)配合物联合放疗可以对常规放疗无法治疗的乏氧肿瘤例如胰腺癌和前列腺癌实现治疗。
本公开的放疗方案可以采用常规的放疗方法进行，也可以采用比常规的放疗方法更低的剂量进行，当采用较低剂量进行时可以降低放疗的副作用。
在一种实施方案中，辐射剂量小于60Gy。
本公开的Pt(Ⅳ)配合物可联合放疗用于治疗癌症，例如白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
在又一方面，本公开还提供一种试剂盒，其包含上述Pt(Ⅳ)配合物或者上述包含Pt(Ⅳ)配合物的药物组合物，还包含说明书，该说明书指出给药之后进行放疗以治疗肿瘤。
实施例
实施例的起始材料是市售可得的和/或可以以有机合成领域技术人员熟知的多种方法进行制备。有机合成领域的技术人员会在下述合成方法的中适当地选择反应条件(包括溶剂、反应气氛、反应温度、实验的持续时间和后处理)。有机合成领域的技术人员会理解，存在于分子各部分上的官能团应当与所提出的试剂和反应相容。
试剂和仪器
所有化学试剂均购自能源化工(中国)、百灵威(中国)、伊诺凯(中国)和国药集团(中国)，均按原样使用，无需进一步提纯。溶剂在使用前用Na或CaH 2除水后蒸馏出来。细胞计数试剂盒-8(CCK-8)购自碧云天生物技术研究所。整个过程中使用的超纯水(18.2MΩ/cm)来自Milli-Q参考系统(Millipore)。核磁共振(NMR)光谱由布鲁克AVANCE 400MHz光谱仪记录。超高效液相色谱-质谱法(UPLC-MS)是在配备Waters PDA eλ检测器和Waters Acquity QDA质谱仪的ACQUITY UPLC H-Class PLUS仪器上进行。吸收光谱由UV-1100分光光度计测量。X射线照射是由X射线发生器产生(RS2000Pro 225，225kV，17.7mA；Rad Source Technologies,Inc.单次试管实验的总剂量为0～60Gy，剂量率为5Gy/min。细胞实验的总剂量为0～16Gy，剂量率为1.6Gy/min。使用一个定制的小鼠模具对植入的肿瘤进行局部照射。肿瘤的局部照射剂量为4Gy，剂量率为1Gy/min，身体的其他部位用5毫米厚的铅屏蔽。γ射线辐照由 60Co源提供。
1.Pt(Ⅳ)配合物的合成与表征
Pt(Ⅳ)配合物如前所述通过相应的Pt(II)药物制备，双氧水氧化后得到铂(Ⅳ)二羟配合物，铂(Ⅳ)二羟配合物上的两个羟基通过与相应的酸酐反应制备化合物1至化合物66。产物通过质谱得到表征。
Figure PCTCN2021132886-appb-000019
Figure PCTCN2021132886-appb-000020
Figure PCTCN2021132886-appb-000021
2.分析方法
2.1铁离子和络合物的检测
Fe 2+/Fe 3+储备液：将2.78mg FeSO 4·7H 2O和2.70mg FeCl 3·6H 2O分别溶于1mL去离子水，得到10mM的储备液，将100μL的储备液在10mL中稀释到100μM。
Fe 2+/Fe 3+探针：将54.06mg菲罗啉溶于1mL DMSO中，得到300μM的储备溶液，该溶液无需进一步稀释就可用于检测。将2.48mg Fe 3+探针溶解在1mL DMSO中，得到10mM的储备溶液，100μL在10mL MeOH/H 2O(v/v，1：1)中稀释到100μM。
[Fe(phen) 3] 2+溶液。将15μL苯酚原液加入15mL 100μM的Fe 2+溶液中，得到[Fe(phen) 3] 2+复合物。
用phen检测Fe 2+/Fe 3+：将100μM的Fe 2+/Fe 3+溶液进一步衰减到80、60、40、20、0μM。在3mL上述Fe 2+/Fe 3+溶液中加入3μL苯酚储备液，因此苯酚的最终浓度为300μM，Fe的浓度几乎不受影响。在510nm下检测UV-Vis吸收。
用Fe 3+探针检测Fe 2+/Fe 3+。100μM的Fe 2+/Fe 3+溶液被进一步衰减到80、60、40、20、0μM。600μL Fe 2+/Fe 3+溶液和2400μL Fe 3+探针混合，因此Fe 2+/Fe 3+最终浓度为20、16、10、8、4、0μM，Fe 3+探针的浓度为80μM。反应体系在37℃下孵育20分钟，用紫外可见分光光度计在450nm下检测。
Figure PCTCN2021132886-appb-000022
2.2铜离子和络合物的检测
Cu 2+储备液：将2.50mg CuSO 4·5H 2O溶解在1mL去离子水中，分别得到10mM的储备液。
Cu 2+探针：将34.25mg二乙基二硫代氨基甲酸钠溶于1mL DMSO中，得到200mM的储备溶液。
用二乙基二硫代氨基甲酸钠检测Cu 2+：将100μL的10mM Cu 2+原液在10mL中稀释到100μM，再进一步衰减到80、60、40、20、0μM。在3mL上述Cu 2+溶液中加入3μL二乙基二硫代氨基甲酸钠原液，因此phen的最终浓度为200μM，Cu 2+的浓度几乎不受影响。检测450nm下的紫外可见吸收。
2.3镍离子和络合物的检测
Ni 2+储备溶液：将2.38mg NiCl 2·6H 2O溶于1mL去离子水，得到10mM的储备溶液，100μL在8mL中稀释到125μM。
二甲基乙醛肟(DMG)溶液：将89mg DMG溶于1.5mL 10M NaOH(aq)，得到0.51M的溶液。
K 2S 2O 8溶液。将57mg K 2S 2O 8溶于1.5mL去离子水，得到0.14mmol溶液。
Ni 2+的检测：125μM的Ni 2+溶液被进一步衰减为100、75、50、25、0μM。将50μL K 2S 2O 8溶液、100μL 1M NaOH(aq)、50μL DMG溶液依次加入到800μL Ni 2+溶液中，因此Ni 2+的最终浓度为100、80、60、40、20、0μM。反应体系在25℃下培养20分钟，用紫外可见分光光度计在530nm下检测。
2.4无探针的离子/配合物的检测
准备金属离子或络合物与相应化合物的10mM储备溶液，依次稀释到100、80、60、40、20、0μM。用以下方法检测离子或络合物
1.ICP-AES.
离子/配合物 化合物
Co 2+ CoCl 2·6H 2O
Pd 2+ Pd(OAc) 2
Ag + Ag 2SO 4
2.紫外可见光谱检测:
离子/配合物 化合物 检测波长/nm
Rh 3+ RhCl 3·3H 2O 280
Ru 3+ RuCl 3 395
Fe(CN) 6 3- K 3Fe(CN) 6 420
Co(NH 3) 6 3+ Co(NH 3) 6Cl 3 210
RuCl 6 2- K 2RuCl 6 320
Ru(NH 3) 6 3+ Ru(NH 3) 6Cl 3 275
PdCl 4 2- K 2PdCl 4 320
PtCl 6 2- K 2PtCl 6 270
AuCl 4 - KAuCl 4 250
IrCl 6 2- (NH 4) 2IrCl 6 495
3.UPLC-MS
配合物 化合物
Co(ⅡI)VB 12 VB 12
Pt(Ⅳ) Pt(Ⅳ)配合物1
2.5检测不同溶液Pt(Ⅳ)配合物1的配体释放情况
准备Pt(Ⅳ)配合物1的10mM储备溶液，将其在H 2O、PBS、5mM Tyr、Trp、DMEM和CM(完全培养基)、FBS的溶液中稀释至100μM。接受X射线照射后，在每个溶液中加入200μL ACN，离心后取上清液。再重复2次，终浓度为原浓度的1/8。用UPLC-MS检测配体释放，通过香豆素的标准曲线进行定量。
2.6检测Pt(Ⅳ)-(Suc) 2的对应铂药释放情况
利用 195Pt NMR进行测定(图3，def)
将Pt(Ⅳ)-(Suc) 2溶解在1mL氘水(80mmol)中，用NaOH调节pH＝7。脱氧后，将该溶液暴露于40kGyγ射线辐照下( 60Co源，200Gy/min，200min)。反应结束后，加入DMSO 以重新溶解沉淀物(对于oxaliPt(Ⅳ)-(Suc) 2)，或者直接以反应后的澄清溶液(对于cisPt(Ⅳ)-(Suc) 2对于carboPt(Ⅳ)-(Suc) 2)进行 195Pt NMR测定。
利用UPLC-MS进行产物确定(图3，bc)
将oxaliPt(Ⅳ)-(Suc) 2溶于1mL去离子水(1mM)，用NaHCO 3调节pH＝7。脱氧后，将溶液暴露在1kGyγ射线照射下( 60Co源，100Gy/min，10min)。用UPLC-MS分析反应粗品，通过UPLC-MS确定释放的产物为奥沙利铂。
2.7利用UPLC-MS进行释放效率测定
将对应的四价铂配合物(化合物1～66)溶于DMSO中得到四价铂储备液(10mM)，然后用纯水将其稀释至10μM。脱氧后，使溶液接受60Gy X射线照射(4Gy/min，15min)。用UPLC-MS分析反应粗品，通过UPLC-MS确定释放的产物为对应二价铂药，并通过铂药的外标曲线进行二价铂药浓度的确定，并计算释放效率。
3.生物学方法
3.1细胞培养
BGC823细胞系来自国家细胞系资源基础设施(北京，中国)。HCT116，Ls513，HT29，及LoVo购自美国典型培养物保藏中心(ATCC)。HCT116,Ls513,HT29和BGC823生长在含有10％的FBS和1％的青霉素/链霉素的RPMI-1640(Roswell Park Memorial Institute-1640)培养基中。LoVo细胞生长在含有10％的FBS和1％的青霉素/链霉素的Ham's F-12K(Roswell Park Memorial Institute-1640)培养基中。所有的细胞培养物都在37℃和5％的二氧化碳条件下进行培养。
3.2细胞存活率测定
用CCK-8检测法评估细胞活力。每个试验重复三次。
为了检测oxaliPt(Ⅳ)-(OAc) 2的细胞毒性，将HCT116,Ls513,LoVo,HT29和BGC823以5×10 4/mL浓度的200μL含10％FBS和1％青霉素/链霉素的RPMI-1640或F-12K培养基接种于96孔板中，并在37℃的5％CO 2培养箱培养24小时。然后将细胞与10μM的oxaliPt(Ⅳ)-(OAc) 2在缺氧状态下培养24小时。然后用8Gy X射线照射细胞，再继续孵育3天。孵育结束后，向细胞中加入含有终浓度为0.5mg/mL CCK-8的空白培养基。将96 孔板在37℃、5％CO 2下继续培养2小时，在450nm处测量吸光度。将处理过的细胞的吸光度与对照组的吸光度进行比较，其中未进行处理的对照组存活率被设定为100％。
3.3肿瘤模型
所有的动物实验都是按照北京大学伦理委员会批准的准则进行的。
6周大的雌性Nu/Nu小鼠从维通利华实验室(中国北京)订购，在特定的无病原体，充足水和食物条件下生长。将含有2×10 6个HCT116细胞的100μL PBS注射入小鼠右肩皮下，构建肿瘤异种移植物模型。肿瘤体积等于1/2长*宽 2。
当肿瘤体积达到50mm 3时(约6天)开始进行治疗，治疗方案显示于图5中。根据oxaliPt(Ⅳ)-(OAc) 2的药代动力学，注射前药1小时后，小鼠接受放疗，即使肿瘤区域接受X射线4Gy的照射。每隔两天记录一次小鼠体重与肿瘤大小，当小鼠肿瘤大小超过1500mm 3时，根据伦理委员会指导，对小鼠实施安乐死。持续记录至小鼠开始治疗后第40天。
放疗剂量辐射对金属还原效率的影响
工业中通常使用10-500kGy的辐射处理废水，用于沉淀污染水中的有毒的重金属离子。因此，本研究首先尝试利用10-60Gy(临床放疗剂量)的X射线辐照FeCl 3(100μM，aq)溶液(图1a)。1,10-菲罗啉是检测Fe 3+/Fe 2+氧化还原反应的经典探针，Fe(phen) 3 3+的水溶液是无色的，而Fe(phen) 3 2+是橘红色的。在辐照后立即向FeCl 3溶液中加入1,10-菲罗啉(30mM，DMSO)(1,10-菲罗啉的最终浓度为300μM)，溶液立即变成橘红色(图1b)，说明生成了Fe 2+。通过Fe(phen) 3 2+的标准曲线确定，Fe 2+的产量与辐射吸收剂量呈线性关系(图1c)。此外，对Fe 3+进行定量检测，发现Fe 3+的消耗几乎与Fe 2+的生成相等(图1c)，因此，Fe 2+是Fe 3+辐射还原的主要产物。
水合电子是水中最强的还原剂之一(标准电极电位，-2.77V)，也是水辐射分解的主要产物之一(～280nM/Gy)。我们假设，辐射驱动的金属还原是由水辐射分解的e aq -介导的，通过检测Fe 3+在10mM甲醇、叔丁醇和甲酸钠溶液中辐照产生Fe 2+的结果证明，还原性环境(·OH的淬灭剂)有利于该反应的发生。而NaNO 3和饱和氧溶液，即已知的e aq -淬灭剂，则大大减少了Fe 2+的释放量(图1d)。e aq -介导的还原应该普遍适用于大多数过渡金属，因此在相同的是按条件下(60Gy的X射线)测试一系列有代表性的金属离子(100μM，aq)，还原产量由相应的复合物的吸光度或其他方法确定。如图1e所示，辐射驱动的还原反应在大多数情况下是可行的。辐射的金属还原产率要高达240nM/Gy，接近e aq -的理论产率。
图1示例说明了辐射还原金属离子的广谱性。利用1,10-菲罗啉(phen)检测Fe 3+/Fe 2+氧化还原反应。Fe 3+溶液经X射线照射(0～60Gy)后立即加入phen，定量地与Fe(Ⅱ)形成橙红色的复合物(λ max＝510nm)，证明了辐射驱动Fe 3+(100μM，aq)向Fe 2+的还原。辐射驱动还原的滴定染色法的(a)示意图和(b)照片。c，Fe 3+的消失几乎等于[Fe(phen) 3] 2+的生成，与吸收的辐射剂量呈线性关系。其G值为200nM/Gy，接近e aq -的理论G值(280nM/Gy)。d，用羟基自由基淬灭剂处理时，辐射驱动的Fe 3+/Fe 2+的还原产额增加，用e aq -淬灭剂处理时则减少。e，辐射驱动的金属离子还原一般适用于过渡金属。
随后，本研究进一步探究辐射还原金属络合物的可行性。金属络合物的还原电势可能会发生改变，导致辐射还原的反应性降低。因此，我们制备了100μM金属配合物，在脱氧后照射0～60Gy的X射线，令人欣喜的是，金属配合物也都取得了较好的结果。根据各个金属配合物的标准曲线可知，辐射驱动的金属还原产率超过200nM/Gy，对于某些金属来说可以高达350nM/Gy(图2a)，超过了e aq -的理论产率。这因为高原子序数的金属原子能沉积更多的X射线能量，从而放大了电离辐射剂量。因此，辐射还原金属的反应具有广泛、高效和高选择性的特性，有希望发展成为活体内剪切化学的工具。
此外，对Pt(Ⅳ)配合物1的辐射还原后的UPLC-MS分析，检测到了轴向配体的释放。由于Pt(Ⅳ)衍生物在临床上有较高的应用潜力，下一步将测试辐射还原在生物环境中的作用，即在肿瘤中实现放疗驱动的Pt(Ⅳ)原药激活。为了测试生物相容性，将Pt(Ⅳ)复合物溶于PBS、5mM Tyr、5mM Trp、’杜氏改良培养基(DMEM)和完全培养基(CM)，辐照后进行UPLC-MS分析。如图2b所示，在上述溶液中均实现了Pt(Ⅳ)复合物1轴向配体的释放。即使在完全的胎牛血清(FBS)中，反应产率与在水中相差不大，因此辐射还原Pt(Ⅳ)复合物1释放轴向配体的策略，在生物体内复杂的条件下也有较高的可行性。
图2示例说明了辐射还原金属配合物的广谱性。a，过渡金属复合物也可以被医学剂量的辐射还原，还原产量高于e aq -的理论产量。同时，在Pt(Ⅳ)复合物(左图，100μM，1％DMSO水溶液)被辐射还原后能释放出轴向配体。b，在各种生物条件下(Tyr为酪氨酸，Trp为色氨酸，5mM；DMEM，培养基；CM，完全培养基；FBS，胎牛血清)对Pt(Ⅳ)复合物(100μM)进行辐射还原，用UPLC检测相应的配体释放。
这种辐射驱动的释放机制有两种可能性。Pt(Ⅳ)复合物的轴向配体释放可以通过水解或还原实现：水解打破酯键，得到Pt(Ⅳ)-(OH) 2和轴向配体；而还原则改变了铂元素的价态，生成相应的二价铂药物。根据配体场理论，具有5d 6价电子构型的Pt(Ⅳ)最常见的配位数是6，而5d 8价电子构型的Pt(Ⅱ)倾向于形成四边形配合物。已有的关于Pt(Ⅳ)前药的研究表 明，Pt(Ⅳ)还原为Pt(Ⅱ)会导致配位数的减少和配体的释放。
为了探究反应过程，首先使oxaliPt(Ⅳ)-(Suc) 2(80mM，D 2O)的溶液脱氧后接受40kGy的γ射线( 60Co源，200Gy/min，200min，图3a)照射，用UPLC-MS检测辐照后的溶液，只观察到一个新的峰，其保留时间(图3b)、质谱信号(图3c)均与奥沙利铂标准样品一致。用核磁共振(NMR)分析产物。 195Pt-NMR显示，Pt(Ⅳ)复合物1615ppm的峰在辐照后几乎消失(图3d上)，而在-1988ppm处出现了一个新的单峰(图3d中)，位于Pt(Ⅱ)复合物的化学位移范围内，且与奥沙利铂的化学位移吻合(图3d下)。上述实验均证明了轴向配体的释放是通过Pt(Ⅳ)的辐射还原而不是水解导致的。
为了探究本策略的普适性，我们进一步对另两款全球通用的铂类药物——卡铂和顺铂进行相同的研究。核磁共振表征发现，cisPt(Ⅳ)-(Suc) 2和carboPt(Ⅳ)-(Suc) 2在D 2O中用γ射线辐射后会释放出相应的Pt(Ⅱ)药物(图3e,f)。鉴于铂类药物在化疗中的广泛应用，本工作提出的辐射驱动Pt(Ⅳ)前药还原后控制释放出Pt(Ⅱ)的策略，在实现放疗驱动的精准化疗十分有希望。
图3示例说明了辐射驱动Pt(Ⅳ)配合物广谱高效释放FDA批准的Pt(Ⅱ)药物。a，辐射驱动的从Pt(Ⅳ)配合物中释放Pt(Ⅱ)药物的示意图。b，oxaliPt(Ⅳ)-(Suc) 2、oxaliPt(Ⅳ)-(Suc) 2+辐射、奥沙利铂和oxaliPt(Ⅳ)-(OH) 2的UPLC色谱图，其中奥沙利铂和oxaliPt(Ⅳ)-(OH) 2作为参考。oxaliPt(Ⅳ)-(Suc) 2的辐射驱动释放的主要产物与奥沙利铂具有相同的保留时间。检测器波长设置为254nm。c，oxaliPt(Ⅳ)-(Suc) 2的辐射驱动所释放的产物的MS表明释放的产物是奥沙利铂。d-f，核磁共振(NMR)研究从Pt(Ⅳ)配合物中的释放Pt(Ⅱ)药物。d，oxaliPt(Ⅳ)-(Suc) 2(1615ppm，上)、辐射照射产物(-1988ppm，中)和外标(下)的 195Pt-NMR光谱。e，cisPt(Ⅳ)-(Suc) 2(1082ppm，上)、辐射照射产物(-2150ppm，中)和外标(下)的 195Pt-NMR光谱。f，carboPt(Ⅳ)-(Suc) 2(1883ppm，上)、辐射照射产物(1707ppm，中)和外标(下)的 195Pt-NMR光谱。 195Pt-NMR谱表明，经FDA批准的Pt(Ⅱ)药物的辐射驱动的释放是有效的，普遍适用于Pt(Ⅳ)配合物。
开发一款成功的前药的关键在于平衡对稳定性和生理条件下反应性的需求。化疗药物中的金属配合物，其致命的缺点在于其有限的生物稳定性。事实上，对于迄今为止报道的大多数Pt(Ⅳ)前药，在细胞内生物还原性条件下便能释放出活性的Pt(Ⅱ)抗癌药物。因此，Pt(Ⅳ)前药的生物稳定性是实现本策略的重要前提。基于已有的研究，四羧基Pt(Ⅳ)具有明显的稳定性优势。因此，我们进一步设计了oxaliPt(Ⅳ)-(OAc) 2(图4a),因为oxaliPt(Ⅳ)-(Suc) 2有两个羧基作为轴向配体，在体内生理环境下带有两个负电荷，因此不能在肿瘤中有效地 富集。而相应的oxaliPt(Ⅳ)-(OAc) 2则具有良好的稳定性和反应性，在与20当量的Vc共孵育24小时后，超过95％的oxaliPt(Ⅳ)-(OAc) 2仍十分稳定(图4b)。OxaliPt(Ⅳ)-(OAc) 2(10μM PBS溶液，脱氧)照射0-60Gy的X射线后，研究发现释放的奥沙利铂与给定的辐射剂量呈正相关(图4c)。OxaliPt(Ⅳ)-(OAc) 2对奥沙利铂敏感的细胞系，如HCT116、HT29、LoVo和Ls513(人类结直肠癌细胞系)的毒性比奥沙利铂低2至3个数量级，其IC50约为亚微摩尔级别。
为了检验辐射驱动产生的奥沙利铂，是否在细胞环境中释放并发挥其抗癌功能，我们用几种细胞系进行了oxaliPt(Ⅳ)-(OAc) 2+X射线的细胞活力测定。本实验以完全培养基作为对照，分别用8Gy的X射线、10μM oxaliPt(Ⅳ)-(OAc) 2和10μM oxaliPt(Ⅳ)-(OAc) 2+8Gy X射线处理细胞。培养96小时后，CCK-8检测显示，经10μM oxaliPt(Ⅳ)-(OAc) 2+8Gy X射线处理的组的细胞活力明显低于仅经过10μM oxaliPt(Ⅳ)-(OAc) 2或8Gy X射线处理的组(图4d)，表明从oxaliPt(Ⅳ)-(OAc) 2释放的奥沙利铂的策略在细胞中的可行性。
图4示例说明了奥沙利铂的辐射诱导控制释放在活细胞中有效。a，放疗驱动的奥沙利铂从前药oxaliPt(Ⅳ)-(OAc) 2释放的示意图，奥沙利铂是一种广泛使用的化疗药物。b,OxaliPt(Ⅳ)-(OAc) 2前药的稳定性。将10μM oxaliPt(Ⅳ)-(OAc) 2与20当量Vc(200μM)一起孵育，24小时后超过95％的oxaliPt(Ⅳ)-(OAc) 2保持稳定状态。c,10μM oxaliPt(Ⅳ)-(OAc) 2在临床相关剂量(0～60Gy，X射线)下释放的奥沙利铂，效率其中高达70％。d，奥沙利铂体外控释的细胞活力测定(前药：[oxaliPt(Ⅳ)-(OAc) 2]＝10μM(h)；X射线，8Gy；n＝6)。HCT116、LoVo、Ls513和HT69是人结直肠癌细胞，对奥沙利铂敏感。
为了找到最佳的给药剂量，我们评估了在健康小鼠上不同剂量下奥沙利铂和前药对小鼠健康的影响。与治疗时的给药方式相同，小鼠每隔两天给药一次，然后记录体重曲线。可以看出，注射3μmol/kg的奥沙利铂或30μmol/kg的oxaliPt(Ⅳ)-(OAc) 2并不会使小鼠体重降低，但是超过10μmol/kg的奥沙利铂以及100μmol/kg的oxaliPt(Ⅳ)-(OAc) 2展现出明显的副作用，导致小鼠出现失重的现象(图5a)。当奥沙利铂的给药剂量达到30μmol/kg时，小鼠体重下降严重，并且在第8天出现死亡，直至第16天全部死亡(图5b)。从小鼠的长时间存活曲线和体重曲线可以得出，3μmol/kg的奥沙利铂以及30μmol/kg oxaliPt(Ⅳ)-(OAc) 2无明显副作用，可作为合适的给药剂量。为达到最佳的放化疗效果，我们用ICP-MS研究了HCT116荷瘤小鼠的oxaliPt(Ⅳ)-(OAc) 2的药代动力学，以探究最佳放疗时间。通过尾静脉注射给HCT116荷瘤小鼠注射30μmol/kg的oxaliPt(Ⅳ)-(OAc) 2后，在选定的时间将小鼠牺牲。然后利用ICP-MS分别检测血液、肿瘤、肝脏和肾脏中的铂药浓 度。多个时间点的ICP-MS数据显示，该前药主要通过肝脏和肾脏代谢，肿瘤摄取量在给药1小时后达到峰值(约15μM)，随后逐渐减少，注射后48小时完全清除(图5c)。给药1小时后，小鼠的肌肉和大脑中前药浓度较低，说明该药物不会对这些器官产生副作用。
随后进一步评估了辐射介导的奥沙利铂在小鼠体内的释放及相应疗效。将HCT116细胞植入Nu/Nu小鼠的右腹，直到肿瘤平均体积达到约50mm 3。将小鼠随机分为7组，包括对照组(仅PBS)、用3μmol/kg奥沙利铂、30μmol/kg oxaliPt(Ⅳ)-(OAc) 2、X射线、3μmol/kg奥沙利铂+X射线、30μmol/kg oxaliPt(Ⅳ)-(OAc) 2+X射线和3μmol/kg transPt(Ⅳ)-(OAc) 2+X射线组，并在第0天注射药物(图5d)。对于治疗组，在注射后1小时给予4Gy的X射线，在第10-12天重复治疗循环。到开始治疗后18天，对照组的肿瘤大小达到1500mm 3；而单独注射30μmol/kg的oxaliPt(Ⅳ)-(OAc) 2对肿瘤生长并没有明显的影响，30μmol/kg oxaliPt(Ⅳ)-(OAc) 2+X射线组肿瘤被明显抑制(图5e,f)，并提高了小鼠的生存时间(图5g)，这说明该治疗效应是由辐射介导的奥沙利铂释放导致的。同时，oxaliPt(Ⅳ)-(OAc) 2+X射线治疗组的小鼠也没有出现体重下降(图5h)，表明我们的策略具有较好的生物安全性。因此，对HCT116荷瘤小鼠的治疗结果表明，本辐射驱动的Pt(Ⅱ)药物释放在活体内具有较高的可行性。
图5示例说明了放疗驱动oxaliPt(Ⅳ)-(OAc) 2前药还原释放奥沙利铂用于奥沙利铂敏感细胞系HCT116肿瘤的化疗实现放化疗联合治疗。静脉注射不同剂量的oxaliPt(Ⅳ)-(OAc) 2和奥沙利铂后小鼠的体重变化曲线(a)和存活曲线(b)。裸鼠的药物适应性研究表明，奥沙利铂和oxaliPt(Ⅳ)-(OAc) 2的最大耐受剂量分别约为3μmol/kg和30μmol/kg。c,oxaliPt(Ⅳ)-(OAc) 2前药的药代动力学，以确定放疗的最佳时机。OxaliPt(Ⅳ)-(OAc) 2前药的肿瘤吸收在注射后1-2小时达到顶点，然后逐渐下降。其余前药迅速从血液中清除，并通过肾和肝胆系统排出体外。d，治疗方案。e-h，放疗驱动的奥沙利铂释放用于肿瘤治疗(每组小鼠n＝6)。e，单个小鼠的肿瘤体积。f，肿瘤的平均体积。前药通过静脉注射，共注射4次，剂量为30μmol/kg。静脉注射后1小时注射后，照射组肿瘤部位进行4Gy X线照射。每两天测量一次每组的肿瘤体积，持续40天。g，小鼠的生存曲线。根据北京大学动物伦理委员会的指导，当肿瘤体积达到1500mm 3时处死小鼠。h，小鼠体重曲线。没有观察到明显的副作用，突出了这种新型治疗策略的生物学安全性。
本研究实现了活体内的辐射诱导的金属还原，从而构建了新的活体剪切化学。将该策略应用于Pt(Ⅳ)前药的激活，使放疗成为一种触发药物释放的外源刺激，从而在在精确放疗的指导下完成肿瘤部位化疗药物的释放。除此之外，本策略也有助于解决缺氧肿瘤的放 疗抗性问题，反而能在乏氧条件下提高药物的释放效率。通过辐射诱导e aq -的直接金属还原还可以拓展到其他金属或生物配合物(如金属蛋白)，为复杂的生物过程的机理剖析提供一个高效的工具。
根据以上描述，本领域技术人员可以容易地确定本发明的基本特征，并且在不脱离本发明的精神和范围的情况下，可以对本发明进行各种改变和修改以使其适应各种用途和条件。因此，其他实施例也在所附权利要求的范围内。
本申请要求于2020年11月25日递交的中国专利申请第202011337782.X号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。

Claims (16)

  1. 一种式(I)的Pt(Ⅳ)配合物，其用作经辐照激活治疗肿瘤的前药，
    Figure PCTCN2021132886-appb-100001
    其中L 1至L 6为铂的配体；该配合物在辐照之后能够释放出L 5和L 6得到式(II)的Pt(II)配合物，
    Figure PCTCN2021132886-appb-100002
  2. 权利要求1的Pt(Ⅳ)配合物，其中式(II)的Pt(II)配合物为顺式构型Pt(II)配合物。
  3. 权利要求1的Pt(Ⅳ)配合物，其中式(II)的Pt(II)配合物为顺铂、卡铂、奈达铂、奥沙利铂、乐铂、或依铂、环铂、米铂、恩洛铂、司铂、螺铂、折尼铂、TRK-710、Aroplatin、双(异丙胺)二氯化铂(II)、或者双(环戊胺)二氯化铂(II)。
  4. 权利要求3的Pt(Ⅳ)配合物，其中式(II)的Pt(II)配合物为顺铂、卡铂、奈达铂、奥沙利铂、乐铂、或者依铂。
  5. 权利要求1-4任一项的Pt(Ⅳ)配合物，其中L 5和L 6分别为 -O-C(O)-R，其中R选自任选取代的C 1-20烷基、任选取代的C 1-20烷基氧基或者任选取代的胺基，取代基选自C 1-18烷基、羧基、羟基、卤素、巯基、胺基、二C 1-3烷基胺基、羰基、苯基、卤代苯基、C 1-6烷基取代的苯基、马来酰亚胺基、三苯基鏻基。
  6. 权利要求5的Pt(Ⅳ)配合物，其中R各自独立地选自甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一碳烷基、十二碳烷基、十三碳烷基、十四碳烷基、十五碳烷基、十六碳烷基、十七碳烷基、十八碳烷基、十九碳烷基、二十碳烷基、羧基亚甲基、2-羧基亚乙基、3-羧基亚丙基、4-羧基亚丁基、5-羧基亚戊基、6-羧基亚己基、(二甲基胺基)亚甲基、2-(二甲基胺基)亚乙基、3-(二甲基胺基)亚丙基、4-(二甲基胺基)亚丁基、5-(二甲基胺基)亚戊基、6-(二甲基胺基)亚己基、5-马来酰亚胺基亚戊基、6-马来酰亚胺基亚己基、7-马来酰亚胺基亚庚基、8-马来酰亚胺基亚辛基、3-(4-碘苯基)亚丙基、3-(3-碘苯基)亚丙基、3-(3,5-二碘苯基)亚丙基、3-(4-溴苯基)亚丙基、3-(3-溴苯基)亚丙基、3-(3,5-二溴苯基)亚丙基、甲胺基、乙胺基、丙胺基、丁胺基、戊胺基、己胺基、庚胺基、 辛胺基、壬胺基、癸胺基、十一碳烷胺基、十二碳烷胺基、十三碳烷胺基、十四碳烷胺基、十五碳烷胺基、十六碳烷胺基、十七碳烷胺基、十八碳烷胺基。
  7. 权利要求1-6任一项的Pt(Ⅳ)配合物，其中所述肿瘤为白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
  8. 一种药物组合物，其包含权利要求1-7任一项的Pt(Ⅳ)配合物。
  9. 权利要求1-7任一项的Pt(Ⅳ)配合物在制备用于经辐照激活治疗肿瘤的药物中的用途。
  10. 权利要求9的用途，其中所述辐照来自放疗。
  11. 一种治疗肿瘤的方法，其包括：
    向受试者施用权利要求1-7任一项的Pt(Ⅳ)配合物，以及
    对受试者进行辐照。
  12. 权利要求11的方法，其中所述辐照来自放疗。
  13. 权利要求12的方法，所述放疗在给药的0.5-3h之后进行。
  14. 权利要求12或13的方法，其中辐射剂量小于60Gy。
  15. 权利要求11-14任一项的方法，其中所述肿瘤为白血病、肺癌、恶性淋巴瘤、乳腺癌、卵巢癌、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、母细胞瘤、神经母细胞瘤、膀胱癌、甲状腺癌、前列腺癌、头颈部肿瘤、鼻咽癌、食道癌、睾丸癌、胃癌、肝癌、胰腺癌、宫颈癌、子宫内膜癌、黑色素瘤或结直肠癌。
  16. 一种试剂盒，其包含：
    权利要求1-7中任一项的Pt(Ⅳ)配合物或者权利要求8的药物组合物，以及
    说明书，该说明书指出给药之后进行放疗以治疗肿瘤。
PCT/CN2021/132886 2020-11-25 2021-11-24 Pt(Ⅳ)化疗前药及其可控释放用于治疗肿瘤 WO2022111541A1 (zh)

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