WO2021129678A1 - 具有辐射响应基团的缀合物及其用途 - Google Patents

具有辐射响应基团的缀合物及其用途 Download PDF

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WO2021129678A1
WO2021129678A1 PCT/CN2020/138682 CN2020138682W WO2021129678A1 WO 2021129678 A1 WO2021129678 A1 WO 2021129678A1 CN 2020138682 W CN2020138682 W CN 2020138682W WO 2021129678 A1 WO2021129678 A1 WO 2021129678A1
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group
radiation
conjugate
aryl
heteroaryl
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French (fr)
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刘志博
傅群峰
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北京大学
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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure relates to the medical and imaging fields, in particular to a conjugate containing a radiation-responsive group and a therapeutic agent or an imaging agent and its use.
  • prodrugs through physiological stimulation or external stimulation has a very promising prospect in the treatment of diseases and has attracted widespread attention. Because the prodrug itself is low-toxic or non-toxic, but through certain stimuli, such as light, heat, pH changes, and enzymatic reactions, the chemical structure of the prodrug can be changed to restore it to an active structure.
  • these stimulation methods have their limitations, especially prodrugs with photosensitizing activity.
  • the photosensitive prodrug restores the drug activity through the photochemical removal reaction under ultraviolet-visible light.
  • the penetration ability of ultraviolet-visible light is limited (a few millimeters) and cannot penetrate the tissue well, so it cannot effectively activate the prodrug molecules in the deep tissues. The purpose of treatment. Therefore, there is still a need to develop a prodrug that can be activated in a non-enzymatic manner in the body, such as deep in tissues.
  • Hydroxyl radicals can be derived from mammals (including humans).
  • the hydroxide radicals may be generated by irradiating the aqueous solution or by a chemical reaction such as the decomposition of hydrogen peroxide.
  • it is necessary to develop a probe with radiation response performance in order to improve the imaging performance of body tissues and organs in an environment where hydrogen and oxygen free radicals are generated by irradiation, it is necessary to develop a probe with radiation response performance.
  • One aspect of the present disclosure provides a conjugate of general formula (I),
  • P is a radiation-responsive group containing aryl or heteroaryl
  • L is absent or is a divalent linking group
  • D is an imaging agent or a therapeutic agent.
  • the radiation-responsive group P in the conjugate undergoes a chemical reaction, so that the radiation-responsive group P or the radiation-responsive group P and the linker L (if present) together fall off from the conjugate and release ⁇ Active Molecule D.
  • the radiation-responsive group is a group capable of chemically reacting with the hydroxyl radical generated by radiation to be detached from the conjugate, or the radiation-responsive group is capable of reacting with The hydroxyl radical generated by radiation is a group that undergoes a chemical reaction and is released from the conjugate together with the linking group L.
  • the aryl or heteroaryl group is, for example, selected from the following group: phenyl, naphthyl, anthryl, phenanthryl, biphenyl, pyridyl, pyridazinyl , Pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, Azolyl, iso Azole, Diazolyl, Triazolyl, furazanyl, thiazolyl, isothiazolyl, indolyl, benzothiazolyl, benzodioxolyl, benzo Azolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimid
  • the aryl or heteroaryl group is selected from phenyl, pyrrolyl, furyl, thienyl, pyrazolyl, and imidazolyl, and the aryl group or Heteroaryl groups are optionally substituted with one or more amino, amino, -NHCOCH 3 , phenyl, methoxy, ethoxy, methyl, ethyl, hydroxyl, alkenyl, F, Cl, Br, I, -N 3 or methyl.
  • the aryl group is an optionally substituted resorcinol group.
  • D is a fluorescent imaging agent or a therapeutic agent for treating cancer.
  • Another aspect of the present disclosure also provides the use of the above-mentioned conjugate in a method of treatment or imaging.
  • the above-mentioned conjugate is used for qualitative or quantitative detection of hydroxyl radicals.
  • Figure 1a shows the release of Fmoc-lys from 10Gy of compound 13 in different solutions
  • Figure 1b shows the UV absorption peaks of compound 14 after 1000 Gy of ⁇ radiation and after incubation at 37°C;
  • Figure 2a shows the fluorescence emission spectrum of the conjugate Fqf-OH under a radiation dose gradient
  • Figure 2b shows the relationship between the fluorescence intensity of the conjugate Fqf-OH and the radiation dose
  • Figure 2c shows the fluorescence response of the conjugate Fqf-OH to different ROS/RNS
  • Figure 3 shows the confocal imaging study of the conjugate Fqf-OH releasing fluorescent molecules in vitro after gamma radiation
  • Figure 4 shows the release of fluorescent molecules in vivo of the conjugate Fqf-OH.
  • Figure 5 shows that compound 27 as a prodrug molecule releases drug molecules after gamma radiation.
  • the active molecule D in the conjugate may remove a hydrogen atom and directly connect to the radiation responsive group or connect to the radiation responsive group through the linker L.
  • imaging used in the present disclosure is not limited to development imaging, but also includes detection based on luminescence intensity, for example, qualitative or quantitative detection of hydroxyl radicals based on the intensity of fluorescence excited by near infrared.
  • the radiation source of the present disclosure may be ⁇ , ⁇ , and ⁇ rays produced by the decay of radionuclides.
  • X-rays, gamma rays, high-energy electrons, protons, heavy ions generated by external radiation sources, alpha particles generated by boron neutron capture therapy (BNCT) and other possible exogenous or endogenous radiation are also applicable to the present disclosure.
  • High-energy rays can be used as external stimuli to chemically react with prodrug molecules or imaging agent precursors to release drug molecules or imaging agents. Due to the high penetration ability of radiation and the high temporal and spatial resolution of radiation, prodrug molecules or imaging agent precursors can be activated very effectively by radiotherapy equipment. For example, X-ray irradiation is used as an external trigger to activate prodrugs. Since the chemical reaction caused by radiation can be controlled in space and time, the area, time and dose of such prodrugs can be precisely controlled to convert to their active form.
  • the biggest problem with the use of high-energy rays as an activation method is that the chemical reactions triggered by high-energy rays usually lack selectivity.
  • the protective group of the prodrug molecule or the precursor of the imaging agent it is possible to remove the drug molecule or the protective group at the same time.
  • the structure of the imaging agent is broken. Therefore, the urgent problem to be solved is to find a group with high reactivity, high reaction rate, and high selectivity to irradiation as a protective group for drug molecules or imaging agents.
  • conjugate refers to a compound obtained by coupling active molecules such as drugs and imaging agents with a radiation-responsive group through a linker or not through a linker.
  • the compound is irradiated such as X-rays or After gamma rays, the radiation responsive group and the linking group (if present) are separated from the active molecule to release the active molecule.
  • the conjugate contains a radiation-responsive group, it does not mean that the conjugate must be used in a radiation environment. For example, in the oxidation treatment of wastewater by the Fenton reagent method, the free radicals generated by hydrogen peroxide may also cause radiation.
  • the responsive group and the linking group (if present) are separated from the active molecule to release the active molecule.
  • C 1 -C 6 alkyl refers to a straight or branched alkane chain containing 1 to 6 carbon atoms.
  • Representative examples of C1-C6 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 ), etc.
  • lower alkyl refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms.
  • substituted alkyl refers to an alkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.
  • haloalkyl refers to an alkyl group with one or more halogen substituents, which includes, but is not limited to, -CH 2 Br, -CH 2 I, -CH 2 Cl, -CH 2 F, -CHF 2 and- Groups like CF 3.
  • alkylene refers to a divalent hydrocarbon group as described above for “alkyl” but having two points of attachment.
  • methylene is a -CH 2 -group
  • ethylene is a -CH 2 -CH 2 -group.
  • alkoxy and alkylthio refer to an alkyl group as described above connected via an oxygen bond (-O-) or a sulfur bond (-S-), respectively.
  • substituted alkoxy and substituted alkylthio refer to a substituted alkyl group connected via an oxygen bond or a sulfur bond, respectively.
  • Lower alkoxy is the group OR, where R is a lower alkyl (alkyl containing 1 to 4 carbon atoms).
  • halogen refers to fluorine, chlorine, iodine or bromine.
  • radiation responsive group refers to a group that can undergo a chemical change after irradiating, for example, X-rays or ⁇ -rays, thereby detaching from a molecule.
  • the conjugate contains a radiation-responsive group, it does not mean that the conjugate must be used in a radiation environment.
  • the responsive group and the linking group are separated from the active molecule to release the active molecule.
  • the radiation responsive group P of the present disclosure contains an aryl group or a heteroaryl group.
  • the radiation-responsive group P is an optionally substituted aryl or heteroaryl, that is, the aryl or heteroaryl may be unsubstituted or substituted.
  • the hydroxyl radicals generated by irradiation cause the radiation-responsive group P to undergo a chemical reaction, so that the radiation-responsive group P or the radiation-responsive group P and the linking group L (if present) together from the conjugate It falls off and releases the active molecule D.
  • aryl refers to a monovalent aromatic hydrocarbon group having 6-20 carbon atoms (C 6 -C 20 ) obtained by removing one hydrogen atom from a single carbon atom in the aromatic nucleus of an aromatic hydrocarbon molecule.
  • Typical aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, biphenyl and the like.
  • heteroaryl refers to a monovalent aromatic group as a 5- or 6-membered ring and a fused ring system of 5-20 atoms (in which at least one ring is aromatic), which contains one or more Heteroatoms independently selected from nitrogen, oxygen and sulfur, if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent.
  • Exemplary monocyclic heteroaryl groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl , Tetrazolyl, Azolyl, iso Azole, Diazolyl, Triazolyl, furazanyl, thiazolyl, isothiazolyl, etc.
  • Exemplary bicyclic heteroaryl groups include indolyl, benzothiazolyl, benzodioxolyl, benzo Azolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indazinyl, benzofuranyl, chromonyl, coumarone Primyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridyl, dihydroisoindolyl, tetrahydroquinolinyl and the like.
  • Exemplary tricyclic heteroaryl groups include carbazolyl, dibenzofuranyl, dibenzothienyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl ( phenanthridinyl), xanthene, etc.
  • the aryl or heteroaryl group has no substituent other than the linking group L attached.
  • the aryl or heteroaryl group is substituted by one or more substituents, and the substituents include hydroxy, alkoxy (e.g., methoxy, ethoxy, propoxy, Butoxy), amino, methylamino, dimethylamino. It is believed that these substituents have electron donating properties and can increase the electron cloud density of the aryl or heteroaryl group, which is conducive to the chemical reaction of the radiation-responsive group under irradiation to fall off from the conjugate.
  • substituents include hydroxy, alkoxy (e.g., methoxy, ethoxy, propoxy, Butoxy), amino, methylamino, dimethylamino. It is believed that these substituents have electron donating properties and can increase the electron cloud density of the aryl or heteroaryl group, which is conducive to the chemical reaction of the radiation-responsive group under irradiation to fall off from the conjugate.
  • the aryl or heteroaryl group is substituted by one or more substituents, and the substituents include halogen, such as fluorine, chlorine, bromine, and iodine, and more preferably include iodine.
  • the aryl or heteroaryl group is substituted by one or more substituents, and the substituents include -N 3 .
  • the radiation responsive group can be selected from:
  • X is the connection site. It can be seen from the position of X above that the radiation-responsive group P can be directly connected to L or D through an aryl or heteroaryl group, or through a divalent group on the aryl or heteroaryl group such as methylene and L Or D is connected.
  • the divalent linking group L does not exist in the conjugates of the present disclosure, that is, the conjugate of general formula (I) actually has a P—D structure.
  • the radiation-responsive group P falls off from the conjugate after irradiation, releasing the active molecule D.
  • the hydroxyl radicals generated by the irradiated aqueous solution cause the radiation-responsive group P to undergo a chemical reaction, so that the radiation-responsive group P falls off from the conjugate and releases the active molecule D.
  • a divalent linking group L in the conjugate of the present disclosure.
  • the divalent linking group L couples the radiation-responsive group P and the active molecule D together, and the divalent linking group L is irradiated. Together with the radiation-responsive group P, the active molecule D is released from the conjugate.
  • the hydroxyl radicals generated by the irradiated aqueous solution cause the radiation-responsive group P to undergo a chemical reaction, so that the radiation-responsive group P and the divalent linking group L to which it is connected fall off from the conjugate and release the active molecule D.
  • the term "active molecule” as used herein refers to a molecule with activity released from the conjugate, which is derived from D in the PLD of the conjugate.
  • the active molecule is a therapeutic agent or an imaging agent.
  • the therapeutic agent or imaging agent does not have an electron-rich aromatic ring or heteroaromatic ring.
  • the therapeutic agent or imaging agent has an aromatic ring or a heteroaromatic ring, it has a strong electron withdrawing group such as a quaternary ammonium ion, -CN, -COOH, -CHO, and -SO on the aromatic or heteroaromatic ring. 3 H, -CF 3, etc., so that the therapeutic agent or imaging agent is relatively stable under irradiation.
  • the radiation reactivity of the radiation-responsive group P is significantly higher than that of the linker L and the active molecule D.
  • the radiation-responsive group P and the active molecule D have both an aromatic ring or a heteroaromatic ring, the radiation reactivity of P is significantly higher than that of D.
  • the reason may be one or more of the following: the aromatic ring or heteroaromatic ring of P
  • the electron cloud density of the aromatic ring itself is higher than that of the aromatic ring or heteroaromatic ring of D; the existence of large substituents on the ring of D causes steric hindrance; the large groups around the ring of D cause steric hindrance;
  • the ring of P has one or more strong electron donating groups; the ring of D has weak electron donating groups or has one or more electron withdrawing groups.
  • the therapeutic agent in the conjugate P-L-D may be any therapeutic agent that can be coupled with P-L or P, as long as it can be separated from P-L or P under the action of radiation and release a molecule with therapeutic activity.
  • the therapeutically active molecule released is generally the same structure as the therapeutic agent D in the conjugate P-L-D.
  • the released therapeutically active molecule has a slightly different structure from the therapeutic agent D in the conjugate PLD, that is, the therapeutic agent molecule undergoes a chemical reaction under the action of radiation but does not significantly affect its therapeutic activity .
  • Suitable molecules that can be used as therapeutic agents include, but are not limited to, polypeptides, oligopeptides, peptidomimetics, amino acids, enzyme inhibitors, hormones, toxins, antibiotics, anti-inflammatory substances, and the like.
  • the active molecule is a therapeutic agent for the treatment of cancer.
  • the active molecule is a therapeutic agent for the treatment of cancer
  • the combined treatment of radiotherapy and chemotherapy can be realized by this means of activating the prodrug of radiotherapy, that is, when the cancer is treated by radiation, the radiation also activates the release of chemical antitumor drugs. Realize chemotherapy.
  • the present disclosure uses drugs containing primary or secondary amines as therapeutic agents.
  • the present disclosure uses anticancer drugs containing primary or secondary amines as therapeutic agents.
  • Drugs containing primary or secondary amines include, for example, ibrutinib, acatinib, zebutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin Chlorine, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazide, Yunnan mold Hormones, gemcitabine, cytarabine, dolastatin, dacarbazine, 5-fluorouracil and their derivatives.
  • Drugs containing primary or secondary amines also include amino derivatives of drugs that do not naturally contain amino groups.
  • a drug that does not originally contain an amino group can be chemically modified to have an amino group, and then coupled to a radiation-responsive group or a radiation-responsive group and a linking group by the primary or secondary amine coupling method described in the present disclosure.
  • the therapeutic agent is monomethyl auristatin E.
  • the present disclosure uses hydroxyl-containing drugs as therapeutic agents.
  • the present disclosure uses hydroxyl-containing anticancer drugs as therapeutic agents.
  • Hydroxy-containing therapeutic agents include, for example, paclitaxel, docetaxel, gemcitabine, cytarabine and the like.
  • the hydroxy-containing therapeutic agent also includes hydroxy derivatives of drugs that do not naturally contain a hydroxy group. In other words, a drug that does not originally contain a hydroxyl group can be chemically modified to have a hydroxyl group, and then coupled to a radiation-responsive group or a radiation-responsive group and a linking group by the hydroxyl coupling method described in the present disclosure.
  • the present disclosure uses sulfhydryl-containing drugs as therapeutic agents.
  • the present disclosure uses sulfhydryl-containing anticancer drugs as therapeutic agents.
  • the sulfhydryl-containing therapeutic agent includes, for example, 6-mercaptopurine and the like.
  • Sulfhydryl-containing therapeutic agents also include sulfhydryl derivatives of drugs that do not naturally contain sulfhydryl groups.
  • a drug that does not originally contain a sulfhydryl group can be chemically modified to have a sulfhydryl group, and then the sulfhydryl group coupling method described in the present disclosure can be coupled to the radiation-responsive group or coupled to the radiation-responsive group and the linking group.
  • the imaging agent in the conjugate P-L-D may be any imaging agent that can be coupled to P-L or P, as long as it can be separated from P-L or P under the action of radiation and release a molecule capable of imaging.
  • the released molecules capable of imaging are generally the same structure as the imaging agent in the conjugate P-L-D.
  • the released imageable molecules have a slightly different structure from the imaging agent in the conjugate P-L-D, that is, the imaging agent molecules undergo a chemical reaction under the action of radiation but do not significantly affect its imaging performance.
  • Imaging agents that can be used in the present disclosure include, but are not limited to, dyes, PET imaging agents (such as 18 F-FDG), radiolabeled agents, and the like.
  • dyes include rhodamine dyes, cyanine dyes, and fluorescein dyes.
  • rhodamine dyes include, but are not limited to, 5-carboxytetramethylrhodamine (5-TAMRA), rhodamine B, rhodamine 6G, TRITC, Texas Red, rhodamine 123, sulforhodamine 101 Wait.
  • fluorescein dyes include, but are not limited to, fluorescein, fluorescein maleimide, 5-aminofluorescein, 6-aminofluorescein, fluorescein isocyanate (FITC), NHS-fluorescein, and the like.
  • radiolabeled reagents can be used as imaging agents of the present teachings.
  • the rhodamine dye or fluorescein dye may be isotopically labeled.
  • isotopes suitable for inclusion in the conjugate include hydrogen (e.g. 2 H and 3 H), carbon (e.g. 11 C, 13 C and 14 C), chlorine (e.g. 36 Cl), fluorine (e.g.
  • Isotopes of iodine e.g., 123 I and 125 I
  • nitrogen e.g., 13 N and 15 N
  • oxygen e.g., 15 O, 17 O, and 18 O
  • phosphorus e.g., 32 P
  • sulfur e.g., 35 S
  • Isotope-labeled conjugates can be prepared by conventional techniques known to those skilled in the art, using appropriate isotope-labeled reagents instead of unlabeled reagents.
  • the above-mentioned imaging agent has a hydroxyl group, a sulfhydryl group or an amino group, it can be coupled to the radiation responsive group or the radiation responsive group and the linking group by the hydroxyl, sulfhydryl or amino coupling method described in the present disclosure. It is also possible to chemically modify the imaging agent that does not originally contain a hydroxyl, sulfhydryl or amino group to have a hydroxyl, sulfhydryl or amino group, and then couple with a radiation-responsive group or respond to radiation by the hydroxyl, sulfhydryl or amino coupling method described in the present disclosure. The group and the linking group are coupled.
  • the conjugate P-L-D in which D is an imaging agent is used as an imaging agent under an irradiation environment.
  • the conjugate PLD in which D is an imaging agent is not used in an irradiated environment, but is used in an environment capable of generating hydroxide radicals, for example, it can be used in the oxidation treatment of wastewater by the Fenton reagent method.
  • Fluorescence detection agent to detect the concentration of hydroxyl radicals in the aqueous solution.
  • the conjugate P-L-D reacts with the hydroxide radicals in the solution to release the imaging agent D, the fluorescence intensity of the imaging agent is used to detect the fluorescence intensity, and the concentration of the hydroxide radicals in the solution is calculated.
  • P and D are coupled through the linker L, or there is no linker L but P and D are directly coupled, that is, the conjugate is actually P-D.
  • the compounds of the present disclosure can be prepared according to various methods known to those skilled in the art of organic synthesis. In the process of synthesis, it may be necessary to use a protecting group to modify the group to reduce (especially eliminate) undesirable side reactions at the protected site.
  • Suitable hydroxyl protecting groups include trimethylsilyl ether (TMS), triethylsilyl ether (TES), tert-butyldimethylsilyl ether (TBDMS), tert-butyldiphenylsilyl ether (TBDPS) ), triisopropyl silyl ether group (TIPS) and so on.
  • Suitable sulfhydryl protecting groups include trimethoxybenzyl (Tmob), benzhydryl, trityl, tert-butyl, fluorenylmethyl (Fm), 2,4-dinitro-phenethyl (Dnpe) , 9-fluorenylmethyleneoxycarbonyl (Fmoc) and so on.
  • Suitable amino protecting groups include trityl (Trt), 2,4-dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB), phthaloyl (Pht), p-toluenesulfonyl Acyl (Tos), trifluoroacetyl (Tfa), o-(p)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl ( Teoc), methyl (or eth)oxycarbonyl, trifluoroacetyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc), etc.
  • a person skilled in the art can easily determine whether such protection is required.
  • protecting groups and their uses see Greene and Wuts (Protective Group
  • the conjugate P-L-D contains a urea group, which can be reacted with triphosgene by a primary or secondary amine, and then reacted with another molecule of amine to synthesize urea.
  • Commonly used solvents are: dichloromethane, tetrahydrofuran and so on.
  • Commonly used bases include triethylamine, diisopropylethylamine and the like.
  • the conjugate PLD contains a ureido group, which can be combined with a chloroformate such as p-nitrochloroformate under basic conditions (for example, in the presence of diisopropylethylamine) with a primary or secondary amine. Phenyl ester is reacted to obtain the corresponding alkoxycarbonamide, which is then reacted with another molecule of amine to synthesize urea. If a secondary amine is used to react with the chloroformate, it is usually carried out in DMAP-CH 3 CN.
  • a chloroformate such as p-nitrochloroformate under basic conditions (for example, in the presence of diisopropylethylamine)
  • Phenyl ester is reacted to obtain the corresponding alkoxycarbonamide, which is then reacted with another molecule of amine to synthesize urea.
  • a secondary amine is used to react with the chloroformate, it is usually carried out in DMAP-CH
  • the conjugate P-L-D contains a thiourea group, which can be obtained by reacting a thio reagent (such as Lawsson's reagent) with the urea prepared as above.
  • a thio reagent such as Lawsson's reagent
  • the conjugate P-L-D contains a thiourea group, which can be synthesized by reacting a primary or secondary amine with thiophosgene and then reacting with another molecule of amine to synthesize thiourea.
  • Type (wherein X, Y are each independently O, S or NR', R'is methyl or hydrogen) conjugates can be through R-OH, R-SH or R-NHR'(R' is methyl Or hydrogen) under alkaline conditions (for example, in the presence of diisopropylethylamine) with a chloroformate such as p-nitrophenyl chloroformate, and then with another molecule of R 1 -OH, R 1 -SH or R 1- NHR'(R' is methyl or hydrogen) prepared by reaction.
  • a chloroformate such as p-nitrophenyl chloroformate
  • the compound is formulated into an aqueous solution, and then subjected to different doses of radiation, and then the active molecules released in the solution are quantitatively analyzed by HPLC-MS, and the amount of released active molecules is plotted against the radiation dose, and the unit dose generation is calculated Release rate.
  • the starting materials of the examples are commercially available and/or can be prepared in a variety of methods well known to those skilled in the art of organic synthesis. Those skilled in the field of organic synthesis will appropriately select the reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of the experiment, and post-treatment) in the following synthesis methods. Those skilled in the field of organic synthesis will understand that the functional groups present on each part of the molecule should be compatible with the proposed reagents and reactions.
  • NMR was recorded using Bruker AVANCE 400MHz spectrometer.
  • the high-resolution mass spectrometer is measured by Bruker Fourier Transform Ion Cyclotron resonance mass spectrometer.
  • the liquid chromatography-mass spectrometry used is Waters e2695 instrument, equipped with Waters 2995 PDA and Waters Acquity QDA mass spectrometer.
  • 1.D is an exemplary synthesis of Fmoc-lysine conjugate P-L-D
  • the crude product was purified by silica gel chromatography, using hexane/ethyl acetate (4:1), ethyl acetate (100%), dichloromethane/methanol (25:1), ethyl acetate/methanol (10:1) in sequence. ) Elution to obtain the conjugate PLD.
  • Dissolve the compound in physiological saline at room temperature prepare a 10 ⁇ M aqueous solution, and then use 60Co as a ⁇ -ray source (or a radiotherapy instrument as an X-ray source) for irradiation (both 4Gy/min). After irradiation for different periods of time (time It is 2.5min, 5min, 10min, 15min, 20min, 25min), and the Fmoc-Lys produced in the solution is quantitatively analyzed by HPLC-MS. Then plot the amount of Fmoc-lys released against the radiation dose to find the release rate.
  • Reagents and conditions (a) K 2 CO 3 , CH 3 I, DMF, rt, 12h; (b) 2N NaOH, MeOH, rt, 10h; (c) Tf 2 O, pyridine, CH 2 Cl 2 , 0°C To room temperature; (d) Pd 2 (dba) 3 ⁇ CHCl 3 , xantphos, Cs 2 CO 3 , benzophenone imine, toluene, 105° C., 12 h; (e) 1N HCl, THF, rt, 30 min; ( f) Triphosgene, TEA, THF, rt, 19h; (g) iPrNEt 2 ,3,5-bis(tert-butyldimethylsilyloxy)phenyl]methanol, CH 2 Cl 2 ,rt, 1h; (h) TBAF, THF, rt, 10h.
  • Step a under N 2 atmosphere, slowly add K 2 CO 3 (9.88 g, 71.50 mmol) to a solution of fluorescein (10 g, 28.60 mmol) in N,N-dimethylformamide (90 mL), and react The mixture was stirred at room temperature for 1 h. Then, methyl iodide (12.18 g, 85.80 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was completed, ice water was added to the reaction mixture and stirred at 0°C for 30 minutes. The yellow solid was filtered and washed with water to completely wash off the K 2 CO 3 . The solid was dried to obtain the desired compound (10.30 g) in a quantitative yield.
  • step c pyridine (0.91 g, 12 mmol) was added to a solution of compound 31 (1.00 g, 2.89 mmol) in anhydrous dichloromethane (12 mL), and the reaction mixture was stirred at room temperature for 20 min.
  • the reaction mixture was cooled to 0°C, and trifluoromethanesulfonic anhydride (1.63 g, 5.79 mmol) was slowly added to the reaction mixture within 30 minutes.
  • the reaction mixture was warmed to room temperature and stirred for 3 hours.
  • the reaction was quenched with water and extracted with dichloromethane.
  • the organic layer was washed with 1N HCl solution followed by water and brine.
  • the combined organic layer was dried over anhydrous sodium sulfate, and the volatiles were removed under reduced pressure.
  • the crude residue was purified by flash chromatography to obtain compound 32 (0.98 g) in a yield of 71%.
  • steps d and e all glassware is dried in an oven before the reaction.
  • Pd 2 (dba) 3 ⁇ CHCl 3 (0.21 g, 0.24 mmol)
  • xantphos (0.18 g, 0.31 mmol).
  • the reaction mixture was purged with nitrogen, and then cesium carbonate (0.93 g, 2.86 mmol) and benzophenone imine (0.44 g, 2.45 mmol) were added thereto.
  • the reaction mixture was heated at 105°C for 12 hours under a nitrogen atmosphere.
  • the reaction mixture was filtered through a short pad of celite, and then it was washed with dichloromethane. The filtrate was concentrated under reduced pressure, and the crude residue was used in the next reaction without further purification.
  • the crude compound was dissolved in tetrahydrofuran (30 mL), and then 3 mL 1N HCl was added. The reaction mixture was stirred at room temperature for 30 minutes, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the volatiles were removed under reduced pressure.
  • Step f before the reaction, all glassware is dried in an oven.
  • compound 34 (0.83 g, 2.40 mmol) was dissolved in 16 mL of anhydrous tetrahydrofuran.
  • Triphosgene (0.713 g, 2.403 mmol) was dissolved in 16 mL of anhydrous tetrahydrofuran and then injected into the reactor. Then add distilled 2.92 mL triethylamine (21.63 mmol). White smoke (gaseous phosgene) is formed immediately.
  • the reactor was sealed, and the solution was vigorously stirred at room temperature for 19 hours. The mixture was concentrated under reduced pressure.
  • the crude residue was quickly filtered, and the solid was washed with anhydrous tetrahydrofuran.
  • the solvent was removed under reduced pressure to obtain compound 6 as a brown oil (quantitative), which was used without further purification.
  • Step g under an ice water bath, add triethylamine (0.037g, 0.29mmol) in dichloromethane (2mL) solution to compound 35 (0.05g, 0.14mmol) in dichloromethane (4mL) solution, and then add 3 ,5-Bis(tert-butyldimethylsiloxy)phenyl]methanol (0.063g, 0.17mmol).
  • the reaction mixture was stirred at room temperature for 1 h.
  • the solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography to obtain compound 36 (0.075 g) in a yield of 72%.
  • step h at 0°C, a solution of 1.0M tetrabutylammonium fluoride (TBAF) (230 ⁇ L, 0.23 mmol) was added dropwise to a solution of 36 (75 mg, 0.1 mmol). The mixture was stirred at room temperature for 10 hours. The solution was poured into water and extracted three times with chloroform. The combined organic phases were washed with brine, dried over anhydrous MgSO 4, filtered through a celite pad, and concentrated by rotary evaporator. The residue was purified by silica gel flash column chromatography to obtain 37 (35.8 mg, 0.07 mmol, 70%) as an orange solid.
  • TBAF tetrabutylammonium fluoride
  • the inventors introduced a highly reactive resorcinol structure into fluorescent molecules. Under the action of the hydroxyl radical generated by the irradiation, the fluorescent precursor molecule Fqf-OH undergoes a removal reaction, thereby releasing the fluorescent molecule.
  • the total dose of the solution was irradiated with a gradient of 10Gy, and the received doses were 0Gy, 10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 60Gy, 70Gy.
  • the fluorescence intensity (measured at 518nm, excitation wavelength is 477nm) gradually increases with increasing dose (Figure 2a) and has a good linear relationship (Figure 2b).
  • Fqf-OH has high selectivity to hydroxyl radicals
  • Fqf-OH was contacted with different RNS and ROS, and it was found that only under the action of hydroxyl radicals, fluorescent molecules can be released, and its reactivity is 30% of other ROS and RNS. Times more than ( Figure 2c).
  • Fqf-OH is stable against various metal ions such as sodium, potassium, calcium, iron, zinc ions and amino acids.
  • Fqf-OH is stable in the cell, and the addition of high-concentration substances in the cell cannot restore the fluorescence activity.
  • Compound 27 was dissolved in physiological saline at room temperature to prepare a 10 ⁇ M aqueous solution. Then use 60 Co as a ⁇ -ray source (or a radiotherapy instrument as an X-ray source) for irradiation (both are 4Gy/min), and after irradiation for different times (times are 2.5min, 5min, 10min, 15min, 20min, 25min), pass The concentration of MMAE generated in the solution was quantitatively analyzed by HPLC-MS (see the upper right corner of Figure 5, showing the change in the mass spectrum signal peak of the drug molecule after irradiation), and then the amount of released MMAE was plotted against the radiation dose. The drug molecule concentration has a good linear relationship with the dose, and the release rate is about 40 nM/Gy (see the lower left corner of Figure 5).
  • the unirradiated 10 ⁇ M aqueous solution of compound 27 (prodrug molecule) was diluted to 10nM, and then incubated with the cells. It was found that the unirradiated aqueous solution of compound 27 had almost no cytotoxicity, but it was irradiated with 60 Co gamma rays for 10 Gy. The later cytotoxicity recovered to be comparable to the drug molecule MMAE. From the graph of cell survival vs. log concentration (bottom right corner of Figure 5), it can be seen that the cytotoxicity of the irradiated solution is significantly improved compared with the unirradiated prodrug molecule.
  • the embodiments of the present disclosure illustrate the feasibility of the strategy of releasing active molecules by irradiation in principle through fluorescent precursor molecules. Furthermore, the embodiments of the present disclosure use MMAE drug molecules as a model to illustrate the feasibility of the radiation release strategy. The examples of the present disclosure also illustrate that the concentration of the active molecule released from the conjugate has a good linear relationship with the dose.
  • the present invention specifically relates to the following items:
  • P is a radiation-responsive group containing aryl or heteroaryl
  • L is absent or is a divalent linking group
  • D is an imaging agent or a therapeutic agent.
  • the radiation-responsive group is a group capable of chemically reacting with hydroxyl radicals generated by radiation to fall off from the conjugate, or the radiation-responsive group It is a group that can chemically react with the hydroxyl radicals generated by radiation to fall off from the conjugate together with the linking group L.
  • aryl or heteroaryl is selected from the group consisting of phenyl, naphthyl, anthryl, phenanthryl, biphenyl, pyridyl, pyridazinyl, pyrimidine Group, pyrazinyl, triazinyl, tetrazinyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, Azolyl, iso Azole, Diazolyl, Triazolyl, furazanyl, thiazolyl, isothiazolyl, indolyl, benzothiazolyl, benzodioxolyl, benzo Azolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, in
  • R 1 and R 2 are independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 3 -C 10 cycloalkyl, C 6 -C 20 Aryl or heteroaryl having 5-20 ring atoms, the alkyl, alkenyl, cycloalkyl, aryl and heteroaryl groups are optionally substituted by one or more halogen, hydroxyl, mercapto, -CN , Nitro, -N 3 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 3 -C 10 cycloalkyl, C 6 -C 20 aryl or heteroaryl substitution with 5-20 ring atoms.
  • aryl or heteroaryl is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, pyrazolyl and imidazolyl, the aryl or heteroaryl group Optionally substituted with one or more amino, amino, -NHCOCH 3 , phenyl, methoxy, ethoxy, methyl, ethyl, hydroxyl, alkenyl, F, Cl, Br, I, -N 3 Or methyl.
  • the therapeutic agent is selected from one or more of the following: monomethyl auristatin E, monomethyl auristatin F, ibrutinib, Aka Tinib, Zebutinib, Doxorubicin, Mitomycin-C, Mitomycin-A, Daunorubicin, Aminopterin, Actinomycin, Bleomycin, 9-Amino Camptotheca Alkali, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazide, Yunnanmycin, gemcitabine, cytarabine, dolastatin, dacarbazine , 5-Fluorouracil; Paclitaxel, Docetaxel, Gemcitabine, Cytarabine; 6-Mercaptopurine.
  • the imaging agent is selected from rhodamine dyes, cyanine dyes, fluorescein dyes, PET imaging agents, and radiolabeled agents.
  • a treatment or imaging method comprising oral or infusion of the conjugate of any one of items 1-16 to a patient, and then the patient receives precise irradiation.
  • a method for qualitatively or quantitatively detecting hydroxyl radicals including adding Fqf-OH to the solution to be tested, and measuring the fluorescence intensity of the solution.

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Abstract

一种具有辐射响应基团的缀合物及其用途，该缀合物具有通式P—L—D，其中P为含有芳基或杂芳基的辐射响应基团；L不存在或者为二价连接基；D为成像剂或治疗剂。在辐照下，所述缀合物中的辐射响应基团P发生化学反应，从而辐射响应基团P或者辐射响应基团P和连接基L(若存在)一起从缀合物中脱落，释放出活性分子D。

Description

具有辐射响应基团的缀合物及其用途
本申请要求于2019年12月24日递交的中国专利申请第201911351250.9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。
技术领域
本公开涉及医疗和成像领域，具体涉及一种含有辐射响应基团与治疗剂或成像剂的缀合物及其用途。
背景技术
通过生理刺激或者外部刺激激活前药在疾病治疗中具有非常远大的前景，引起了广泛的关注。因为前药本身是低毒性或无毒的，但通过某些刺激，如光、热、pH改变以及酶促反应，可以改变前药的化学结构，使其恢复到具有活性的结构。但是这些刺激方法都有其局限性，尤其是具有光敏活性的前药。光敏前药通过紫外可见光下的光化学脱除反应恢复药物活性，然而紫外可见光的穿透能力有限(数毫米)，无法很好穿透组织，故不能有效的激活组织深处的前药分子，达到治疗的目的。因此，仍然需要开发一种能够通过非酶促方式在体内例如组织深处激活的前药。
另一方面，在具有氢氧自由基的特殊环境下，可能需要开发一种能够灵敏响应氢氧自由基的探针。氢氧自由基可以是来自哺乳动物(包括人)体内的。或者，氢氧自由基可以是辐照水溶液产生的也可以是化学反应例如过氧化氢分解产生的。在某些情形下，为了提高在辐照产生氢氧自由基的环境下对身体组织器官的成像性能，需要开发一种具有辐射响应性能的探针。在另一些情形下，为了检测溶液中氢氧自由基例如体内或Fenton试剂法氧化处理废水时的氢氧自由基浓度，也可能需要开发一种能够灵敏响应自由基的探针。
发明内容
本公开的一个方面提供一种通式(I)的缀合物，
P——L——D   (I)
其中P为含有芳基或杂芳基的辐射响应基团；L不存在或者为二价连接基；D为成像剂或治疗剂。在辐照下，所述缀合物中的辐射响应基团P发生化学反应，从而辐射响应基团P或者辐射响应基团P和连接基L(若存在)一起从缀合物中脱落，释放出活性分子D。
在本公开的一种实施方式中，所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而从缀合物中脱落的基团，或者所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而与连接基L一起从缀合物中脱落的基团。
在本公开至少一实施例提供的缀合物中，所述芳基或杂芳基例如选自下组：苯基、萘基、蒽基、菲基、联苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基、呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、三唑基、四唑基、
Figure PCTCN2020138682-appb-000001
唑基、异
Figure PCTCN2020138682-appb-000002
唑基、
Figure PCTCN2020138682-appb-000003
二唑基、
Figure PCTCN2020138682-appb-000004
三唑基、呋咱基、噻唑基、异噻唑基、吲哚基、苯并噻唑基、苯并二氧杂环戊烯基、苯并
Figure PCTCN2020138682-appb-000005
唑基、苯并噻吩基、喹啉基、四氢异喹啉基、异喹啉基、苯并咪唑基、苯并吡喃基、吲嗪基、苯并呋喃基、色酮基、香豆素基、苯并吡喃基、噌啉基、喹喔啉基、吲唑基、吡咯并吡啶基、呋喃并吡啶基、二氢异吲哚基、四氢喹啉基、咔唑基、二苯并呋喃基、二苯并噻吩基、苯并吲哚基、菲咯啉基、吖啶基、菲啶基和呫吨基；其中所述芳基或杂芳基任选取代有一个或者多个选自下组的取代基：卤素、-R 1、-NR 1R 2、-CN、-NO 2、-N 3、-OR 1、-SR 1、-NHCOR 1、-O-COR 1、-CH＝CR 1R 2、-C(＝O)-R 1、-C(＝O)-OR 1、-C(＝O)-Cl、-C(＝O)-NH 2、-C(＝O)-NH-R 1和-C(＝O)-NR 1R 2；其中R 1和R 2独立地选自H、C 1-C 6烷基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基，所述烷基、烯基、环烷基、芳基和杂芳基任选地被一个或多个卤素、羟基、巯基、-CN、硝基、-N 3、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基取代。
例如，在本公开至少一实施例提供的缀合物中，所述芳基或杂芳基选自苯基、吡咯基、呋喃基、噻吩基、吡唑基和咪唑基，所述芳基或杂芳基任选 取代有一个或多个氨基、胺基、-NHCOCH 3、苯基、甲氧基、乙氧基、甲基、乙基、羟基、烯基、F、Cl、Br、I、-N 3或者甲基。
在本公开优选的一种实施方式中，所述芳基或杂芳基上没有直接与其相连的氧代、-C(＝O)-、-C(＝S)-、-CN、-SO 3H、-CF 3。
在本公开优选的一种实施方式中，所述芳基为任选取代的间苯二酚基。
在本公开至少一实施例提供的缀合物中，L例如选自-SS-、线性的C 1-C 6-亚烷基-、支链的C 3-C 6-亚烷基-、-R 3-X-C(＝O)-Y-、-R 3-X-C(＝S)-Y-、-R 3-X-、-X-C(＝O)-Y-、-X-C(＝S)-Y-或者-CH 2-X-C(＝Z)-Y-CH 2CH 2-E-C(＝J)-F-，其中R 3选自C 1-C 3亚烷基或者-CH 2-C 2烯基-；X、Y、E和F独立地选自O、NH或S；Z和J独立地选自O或S；L可任选取代有羟基、巯基、NH 2、F、Cl、Br、I、-N 3、甲基。
在本公开优选的一种实施方式中，L为-CH 2-X-(C＝O)-Y-、-CH 2-X-(C＝S)-Y-、-CH 2-X-或者-X-(C＝O)-Y-，其中X和Y独立地选自O、NH、S。在本公开更优选的一种实施方式中，L为-CH 2-O-(C＝O)-、-CH 2-O-(C＝O)-NH-或者-O-(C＝O)-NH-。
在本公开至少一实施例提供的缀合物中，D为荧光成像剂或者治疗癌症的治疗剂。
本公开的另一方面还提供上述缀合物在治疗或成像的方法中的用途。
在本公开的一种实施方式中，上述缀合物用于定性或定量检测氢氧自由基。
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1a示出了化合物13在不同溶液中10Gy的γ辐射释放Fmoc-lys的情况；
图1b示出了化合物14在1000Gy的γ辐射之后以及在37℃孵育之后的UV吸收峰；
图2a示出了缀合物Fqf-OH在辐照剂量梯度下的荧光发射谱；
图2b示出了缀合物Fqf-OH的荧光强度与辐照剂量的关系；
图2c示出了缀合物Fqf-OH对于不同ROS/RNS的荧光响应；
图3示出了共聚焦成像研究缀合物Fqf-OH在γ辐射之后体外释放荧光分子的情况；
图4示出了缀合物Fqf-OH的活体内荧光分子释放；和
图5示出了化合物27作为前药分子在γ辐射之后释放出药物分子的情况。
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。
除非另外定义，本公开使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
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。例如活性分子D在缀合物中可能除去一个氢原子与辐射响应基团直接相连或者通过连接基L与辐射响应基团相连。
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，而不排除其他要素。
本公开中使用的“成像”并不局限于显影成像，还包括根据发光强度进行检测，例如根据近红外激发的荧光的强度进行定性或定量检测氢氧自由基。
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。
高能射线(如X射线及γ射线)可用作外部刺激使前药分子或者成像剂前体发生化学反应，释放出药物分子或成像剂。由于射线的高穿透能力，以及射线的高时空分辨率，前药分子或者成像剂前体可以通过放疗设备非常有效的激活。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，可精确控制此类前药转化为其活性形式的面积、时间和剂量。
但是，利用高能射线作为激活手段目前存在的最大问题是高能射线引发的化学反应通常缺乏选择性，在脱除前药分子或者成像剂前体中的保护基团时，同时有可能将药物分子或者成像剂的结构破坏。所以迫切需要解决的问题是：找到一个对辐照具有高反应活性，高反应速率，高选择性的基团作为药物分子或者成像剂的保护基团。
本文使用的术语“缀合物(conjugate)”是指通过连接基或者不通过连接基将药物、成像剂等活性分子与辐射响应基团偶联得到的化合物，该化合物在辐照例如X射线或γ射线之后辐射响应基团和连接基(若存在)与活性分子分离从而释放出活性分子。请注意，虽然缀合物包含辐射响应基团，但是并不是说该缀合物一定要在辐射环境下使用，例如在Fenton试剂法氧化处理废水中，过氧化氢产生的自由基也可能使辐射响应基团和连接基(若存在)与活性分子分离从而释放出活性分子。
本文使用的术语“C 1-C 6烷基”是指含有1-6个碳原子的直链或支链烷烃链。C1-C6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活性的非氢取代基。
辐射响应基团P
本文使用的术语“辐射响应基团”是指在辐照例如X射线或γ射线之后能够发生化学变化，从而从分子中脱落的基团。请注意，虽然缀合物包含辐射响应基团，但是并不是说该缀合物一定要在辐射环境下使用，例如在Fenton试剂法氧化处理废水中，过氧化氢产生的自由基也可能使辐射响应基团和连接基(若存在)与活性分子分离从而释放出活性分子。
本公开的辐射响应基团P含有芳基或杂芳基。在一种实施方式中，辐射响应基团P为任选取代的芳基或杂芳基，即芳基或杂芳基可以是未取代也可以是取代的。
在一种实施方式中，辐照产生的氢氧自由基使得辐射响应基团P发生化学反应，从而辐射响应基团P或者辐射响应基团P和连接基L(若存在)一起从缀合物中脱落，释放出活性分子D。
术语"芳基"表示通过由芳烃分子的芳核中的单个碳原子除去一个氢原子而得到的具有6-20个碳原子(C 6-C 20)的一价芳族烃基。典型的芳基包括但不限于苯基、萘基、蒽基、菲基、联苯基等。
术语“杂芳基”是指作为5-或6-元环的单价芳族基团及包括5-20个原子的稠环系统(其中至少一个环是芳族的)，其含有一个或多个独立选自氮、氧和硫的杂原子，如果该环含有多个氧原子，则这些氧原子不是直接相邻的。
示范性的单环杂芳基包括吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、 四嗪基、呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、三唑基、四唑基、
Figure PCTCN2020138682-appb-000006
唑基、异
Figure PCTCN2020138682-appb-000007
唑基、
Figure PCTCN2020138682-appb-000008
二唑基、
Figure PCTCN2020138682-appb-000009
三唑基、呋咱基、噻唑基、异噻唑基等。
示范性的二环杂芳基包括吲哚基、苯并噻唑基、苯并二氧杂环戊烯基、苯并
Figure PCTCN2020138682-appb-000010
唑基、苯并噻吩基、喹啉基、四氢异喹啉基、异喹啉基、苯并咪唑基、苯并吡喃基、吲嗪基、苯并呋喃基、色酮基、香豆素基、苯并吡喃基、噌啉基、喹喔啉基、吲唑基、吡咯并吡啶基、呋喃并吡啶基、二氢异吲哚基、四氢喹啉基等。
示范性的三环杂芳基包括咔唑基、二苯并呋喃基、二苯并噻吩基、苯并吲哚基、菲咯啉基(phenanthrolinyl)、吖啶基(acridinyl)、菲啶基(phenanthridinyl)、呫吨等。
在一种实施方式中，所述芳基或杂芳基除了连接有连接基L之外没有取代基。
在另一种实施方式中，所述芳基或杂芳基被一个或多个取代基取代，所述取代基包括但不限于卤素、-R 1、-NR 1R 2、-CN、-NO 2、-N 3、-OR 1、-SR 1、-NHCOR 1、-O-COR 1、-CH＝CR 1R 2、-C(＝O)-R 1、-C(＝O)-OR 1、-C(＝O)-Cl、-C(＝O)-NH 2、-C(＝O)-NH-R 1和-C(＝O)-NR 1R 2，其中R 1和R 2独立地选自H、C 1-C 6烷基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基或者具有5-20个环原子的杂芳基，针对所述取代基描述的烷基、烯基、环烷基、芳基和杂芳基任选地被一个或多个卤素、羟基、巯基、-NH 2、-CN、-NO 2、-N 3、-NHCOH、-O-C(＝O)H、-C(＝O)H、-C(＝O)-OH、-C(＝O)-Cl、-C(＝O)-NH 2、-C(＝O)-NH-CH 3、-C(＝O)-CH 3、-C(＝O)-OCH 3、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基取代。
在一种优选的实施方式中，所述芳基或杂芳基被一个或多个取代基取代，所述取代基包括羟基、烷氧基(例如甲氧基、乙氧基、丙氧基、丁氧基)、氨基、甲胺基、二甲胺基。据信这些取代基具有给电子性，能提高芳基或杂芳基的电子云密度，这有利于辐射响应基团在辐照下发生化学反应从而从缀合物中脱落。
在一种优选实施方式中，所述芳基或杂芳基被一个或多个取代基取代， 所述取代基包括卤素，例如氟、氯、溴、碘，更优选包括碘。
在一种优选实施方式中，所述芳基或杂芳基被一个或多个取代基取代，所述取代基包括-N 3。
在一种优选的实施方式中，所述芳基或杂芳基上没有直接与其相连的氧代(即＝O)、-C(＝O)-、-C(＝S)-、-CN、-SO 3H、-CF 3。据信这些基团有强的吸电子性，会降低芳基或杂芳基的电子云密度，不利于辐射响应基团在辐照下发生化学反应。
例如，辐射响应基团可以选自：
Figure PCTCN2020138682-appb-000011
其中X为连接位点。从上述X的位置可以看出，辐射响应基团P可以通过芳基或杂芳基直接与L或者D相连，也可以通过芳基或杂芳基上的二价基团例如亚甲基与L或者D相连。
二价连接基L
在一种实施方式中，本公开的缀合物中不存在二价连接基L，即，通式(I)的缀合物实际上为P——D的结构。此情形下，辐射响应基团P在辐照后从缀合物中脱落，释放出活性分子D。例如，辐照水溶液产生的氢氧自由基使得辐射响应基团P发生化学反应，从而辐射响应基团P从缀合物中脱落，释放出活性分子D。
在一种实施方式中，本公开的缀合物中存在二价连接基L，二价连接基L将辐射响应基团P与活性分子D偶联在一起，在辐照后二价连接基L同辐射响应基团P一起从缀合物中脱落，释放出活性分子D。例如，辐照水溶液产生的氢氧自由基使得辐射响应基团P发生化学反应，从而辐射响应基团P与其连接的二价连接基L一起从缀合物中脱落，释放出活性分子D。
在一种实施方式中，连接基L选自-SS-、线性的C 1-C 6-亚烷基-、支链的C 3-C 6-亚烷基-、-R 3-X-C(＝O)-Y-、-R 3-X-C(＝S)-Y-、-R 3-X-、-X-C(＝O)-Y-、-X-C(＝S)-Y-或者-CH 2-X-C(＝Z)-Y-CH 2CH 2-E-C(＝J)-F-，其中R 3选自C 1-C 3亚烷基或者-CH 2-C 2烯基-；X、Y、E和F独立地选自O、NH或S；Z和J独立地选自O或S；连接基L可任选取代有羟基、巯基、NH 2、F、Cl、Br、I、-N 3、甲基。
在一种优选的实施方式中，L为-CH 2-X-(C＝O)-Y-、-CH 2-X-(C＝S)-Y-、-CH 2-X-或者-X-(C＝O)-Y-，其中X和Y独立地选自O、NH、S。在一种更优选的实施方式中，L为-CH 2-O-(C＝O)-、-CH 2-O-(C＝O)-NH-或者-O-(C＝O)-NH-。
活性分子D
本文使用的术语“活性分子”是指从缀合物中释放出来具有活性的分子，其衍生自缀合物P-L-D中的D。一种实施方式中，所述活性分子为治疗剂或成像剂。一种优选的实施方式中，所述治疗剂或成像剂不具有富电子云的芳环或杂芳环。例如，所述治疗剂或成像剂尽管具有芳环或杂芳环，但是在芳环或杂芳环上具有强的吸电子基团诸如季铵离子、-CN、-COOH、-CHO、-SO 3H、-CF 3等，从而所述治疗剂或成像剂在辐照下相对稳定。
需要说明的是，在所述缀合物P-L-D中，辐射响应基团P的辐射反应活性(即在辐照下发生化学反应的活泼性)明显高于连接基L和活性分子D。例 如，辐射响应基团P和活性分子D同时具有芳环或杂芳环时，P的辐射反应活性明显高于D，其原因可能是以下中的一种或多种：P的芳环或杂芳环的电子云密度本身高于D的芳环或杂芳环的电子云密度；D的环上存在大的取代基造成空间位阻；D的环周围有大的基团造成空间位阻；P的环上具有一个或多个强给电子基团；D的环上具有弱的给电子基团或者具有一个或多个吸电子基团。
治疗剂
在缀合物P-L-D中的治疗剂可以是能够与P-L或P偶联的任意治疗剂，只要其在辐射作用下能够与P-L或P分离并释放出有治疗活性的分子即可。在本公开中，释放出的有治疗活性的分子通常与在缀合物P-L-D中的治疗剂D的结构相同。在一些实施方式中，释放出的有治疗活性的分子与在缀合物P-L-D中的治疗剂D的结构略有不同，即治疗剂分子在辐射作用下发生了化学反应但是不显著影响其治疗活性。
可用作治疗剂的合适分子包括但不限于多肽、寡肽、拟肽、氨基酸、酶抑制剂、激素、毒素、抗生素、抗炎物质等。
在一种优选的实施方式中，所述活性分子为治疗癌症的治疗剂。
当所述活性分子为治疗癌症的治疗剂时，可以通过这种放疗激活前药的手段实现放疗和化疗的联合治疗，即，在放射线治疗癌症的同时，辐照还激活释放出化学抗肿瘤药实现化疗。
在一种实施方式中，本公开使用含伯胺或仲胺的药物作为治疗剂。在一种优选实施方式中，本公开使用含伯胺或仲胺的抗癌药作为治疗剂。含伯胺或仲胺的药物例如包括依鲁替尼、阿卡替尼、泽布替尼、阿霉素、丝裂霉素-C、丝裂霉素-A、柔红霉素、氨基蝶呤、放线菌素、博来霉素、9-氨基喜树碱、N8-乙酰基亚精胺、1-(2-氯乙基)-1,2-二甲烷磺酰基酰肼、云南霉素、吉西他滨、阿糖胞苷、多拉司他汀、达卡巴嗪、5-氟尿嘧啶以及它们的衍生物。含伯胺或仲胺的药物还包括天然不包含氨基的药物的氨基衍生物。换言之，可以通过化学修饰使本来不包含氨基的药物具有氨基，进而通过本公开所描述的伯胺或仲胺偶联方法与辐射响应基团偶联或者与辐射响应基团和连接基 偶联。
在一种优选的实施方式中，所述治疗剂为一甲基澳瑞他汀E。
在一种实施方式中，本公开使用含羟基的药物作为治疗剂。在一种优选实施方式中，本公开使用含羟基的抗癌药作为治疗剂。含羟基的治疗剂例如包括紫杉醇、多西他赛(Docetaxel)、吉西他滨、阿糖胞苷等。含羟基的治疗剂还包括天然不包含羟基的药物的羟基衍生物。换言之，可以通过化学修饰使本来不包含羟基的药物具有羟基，进而通过本公开所描述的羟基偶联方法与辐射响应基团偶联或者与辐射响应基团和连接基偶联。
在一种实施方式中，本公开使用含巯基的药物作为治疗剂。在一种优选实施方式中，本公开使用含巯基的抗癌药作为治疗剂。含巯基的治疗剂例如包括6-巯基嘌呤等。含巯基的治疗剂还包括天然不包含巯基的药物的巯基衍生物。换言之，可以通过化学修饰使本来不包含巯基的药物具有巯基，进而通过本公开所描述的巯基偶联方法与辐射响应基团偶联或者与辐射响应基团和连接基偶联。
成像剂
在缀合物P-L-D中的成像剂可以是能够与P-L或P偶联的任意成像剂，只要其在辐射作用下能够与P-L或P分离并释放出能够显像的分子即可。在本公开中，释放出的能够显像的分子通常与在缀合物P-L-D中的成像剂的结构相同。在一些实施方式中，释放出的能够显像的分子与在缀合物P-L-D中的成像剂的结构略有不同，即成像剂分子在辐射作用下发生了化学反应但是不显著影响其成像性能。
可用作本公开的成像剂包括但不限于染料、PET成像剂(例如 18F-FDG)、放射性标记的试剂等。
染料例如罗丹明染料、花青染料、荧光素染料等。罗丹明染料的代表性实例包括但不限于5-羧基四甲基罗丹明(5-TAMRA)、罗丹明B、罗丹明6G、TRITC、德克萨斯红、罗丹明123、磺基罗丹明101等。荧光素染料的实例包括但不限于荧光素、荧光素马来酰亚胺、5-氨基荧光素、6-氨基荧光素、异氰酸荧光素(FITC)、NHS-荧光素等。
在一些实施方式中，放射性标记的试剂可用作本教导的成像剂。在一些实施方式中，罗丹明染料或荧光素染料可为同位素标记的。适于包含在缀合物中的同位素的实例包括氢(例如 2H和 3H)、碳(例如 11C、 13C和 14C)、氯(例如 36Cl)、氟(例如 18F)、碘(例如 123I和 125I)、氮(例如 13N和 15N)、氧(例如 15O、 17O和 18O)、磷(例如 32P)和硫(例如 35S)的同位素。
同位素标记的缀合物可通过本领域的技术人员已知的常规技术，使用适当的同位素标记的试剂代替未标记的试剂来制备。
如果上述成像剂具有羟基、巯基或氨基，则可通过本公开所描述的羟基、巯基或氨基偶联方法与辐射响应基团偶联或者与辐射响应基团和连接基偶联。也可以通过化学修饰使本来不包含羟基、巯基或氨基的成像剂具有羟基、巯基或氨基，然后通过本公开所描述的羟基、巯基或氨基偶联方法与辐射响应基团偶联或者与辐射响应基团和连接基偶联。
在一些实施方式中，其中D为成像剂的缀合物P-L-D在辐照环境下用作成像剂。
在一些实施方式中，其中D为成像剂的缀合物P-L-D并不在辐照环境下使用，而是在能够产生氢氧自由基的环境下使用，例如在Fenton试剂法氧化处理废水时可用作荧光检测剂以检测水溶液中的氢氧自由基的浓度。在此情形中，缀合物P-L-D与溶液中的氢氧自由基反应释放出成像剂D，利用成像剂的荧光特性检测荧光强度，进而计算出溶液中的氢氧自由基的浓度。
偶联反应
在缀合物P-L-D中，通过连接基L使得P和D偶联，或者没有连接基L而是P和D直接偶联，即缀合物实际上是P—D。
本公开化合物可按有机合成领域技术人员已知的多种方法来制备。在合成的过程中，可能需要使用保护基来修饰基团以减轻(特别是排除)在经保护的位点发生不希望的副反应。
当活性分子D同时含有羟基、巯基和氨基或者含有多个羟基、巯基或氨基时，可以先对其它羟基、巯基或氨基进行保护，然后与辐射响应基团P或者与辐射响应基团P和连接基L进行偶联。适当的羟基保护基包括三甲基硅 醚基(TMS)、三乙基硅醚基(TES)、叔丁基二甲基硅醚基(TBDMS)、叔丁基二苯基硅醚基(TBDPS)、三异丙基硅醚基(TIPS)等。适当的巯基保护基包括三甲氧基苄基(Tmob)、二苯甲基、三苯甲基、叔丁基、芴甲基(Fm)、2,4-二硝基-苯乙基(Dnpe)、9-芴基亚甲基氧基羰基(Fmoc)等。适当的氨基保护基包括三苯甲基(Trt)、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、邻苯二甲酰基(Pht)、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、邻(对)硝基苯磺酰基(Ns)、特戊酰基、苯甲酰基、烯丙氧羰基(Alloc)、三甲基硅乙氧羰基(Teoc)、甲(或乙)氧羰基、三氟乙酰基、叔丁氧羰基(BOC)、苄氧羰基(CBz)和9-芴基亚甲基氧基羰基(Fmoc)等。本领域技术人员可容易地确定是否需要所述保护。对于保护基和它们用途的一般描述，参见Greene和Wuts(Protective Groups In Organic Synthesis，Wiley-Interscience，3rd Edition，1999)。
在一种实施方式中，缀合物P-L-D含有脲基，其可通过伯胺或仲胺与三光气反应，然后与另一分子胺反应合成脲。常用的溶剂有：二氯甲烷、四氢呋喃等。常用的碱例如三乙胺、二异丙基乙胺等。
Figure PCTCN2020138682-appb-000012
在一种实施方式中，缀合物P-L-D含有脲基，其可通过伯胺或仲胺在碱性条件下(例如二异丙基乙胺存在下)与氯甲酸酯例如氯甲酸对硝基苯酯反应得到相应的烷氧基碳酰胺，然后再与另一分子胺反应合成脲。如果使用仲胺与氯甲酸酯反应，通常在DMAP-CH 3CN中进行。
在一种实施方式中，缀合物P-L-D含有硫脲基，其可通过硫代试剂(如Lawsson试剂)与如上所制备的脲反应得到硫脲。
在一种实施方式中，缀合物P-L-D含有硫脲基，其可通过伯胺或仲胺与硫光气反应，然后与另一分子胺反应合成硫脲。
对于
Figure PCTCN2020138682-appb-000013
类型(其中X、Y各自独立地为O、S或NR’， R’为甲基或氢)的缀合物，可通过R-OH、R-SH或R-NHR’(R’为甲基或氢)在碱性条件下(例如二异丙基乙胺存在下)与氯甲酸酯例如氯甲酸对硝基苯酯反应，然后与另一分子R 1-OH、R 1-SH或R 1-NHR’(R’为甲基或氢)反应制得。以下以
Figure PCTCN2020138682-appb-000014
为例说明此类缀合物的制备。
Figure PCTCN2020138682-appb-000015
将相应的醇ROH(0.6mmol，1.0当量)溶于二氯甲烷(4mL，无水)中，并冷却至0℃。加入DIPEA(232mg，1.8mmol，3.0当量)，然后加入化合物14(503mg，1.5mmol，2.5当量)。在1小时内将反应混合物温热至室温，此时将其用水淬灭并用二氯甲烷(3×20mL)萃取。合并有机层，经无水Na 2SO 4干燥，并在减压下旋干。粗产物通过硅胶色谱法用己烷/乙酸乙酯(9:1)纯化。
Figure PCTCN2020138682-appb-000016
在圆底烧瓶中，将R 1OH(1mmol，1.0当量)和DIPEA(2mmol，2.0当量)溶解在30mL THF中，并搅拌2分钟。加入化合物15(1mmol，1.0当量)，并将混合物在室温搅拌1h。完全反应完成后，将反应溶液用乙酸乙酯萃取，并先后用水和饱和NaCl溶液洗涤两次。合并有机层，经无水Na 2SO 4干燥，并在减压下旋干。粗产物通过硅胶色谱法纯化。
活性分子释放实验：
将化合物配制成水溶液，再接受不同剂量的辐照，然后通过HPLC-MS对溶液中释放出的活性分子进行定量分析，以释放出的活性分子的量对辐射剂量作图，求出单位剂量产生释放率。
Figure PCTCN2020138682-appb-000017
在化合物13的水溶液中添加10mM的H 2O 2、t-BuOH或者DMSO，来 改变在辐照下氢氧自由基的产额。以10μM化合物13的磷酸钾缓冲液作为对照组，在添加了10mM H 2O 2的溶液中，氢氧自由基的产额大大提高，在此条件下，释放的Fmoc-lys的量增大。与之相对的是添加了10mM t-BuOH及10mM DMSO这两种可以淬灭氢氧自由基的物质，释放的Fmoc-lys的量明显减少，参见图1a。图1a示出了化合物13(10μM，0.02M磷酸钾缓冲液,pH＝7.4,0.1％DMF)在4种溶液(对照组；含10mM H 2O 2；含10mM t-BuOH；含10mM DMSO)中10Gy的γ辐射之后释放出Fmoc-lys的情形。
不受任何特定理论的束缚，申请人认为高能射线使水发生辐解产生大量的氢氧自由基，然后氢氧自由基与芳环发生亲电加成生成酚，再通过1,4或者1,6-消除反应释放出活性成分Fmoc-lys(如以下反应式所示)。
Figure PCTCN2020138682-appb-000018
对高浓度的化合物14(1mM，在纯水中)进行100Gy/min的γ射线照射( 60Co)辐照10min后接受了总量为1000Gy的剂量，发明人发现出现了一个新的紫外吸收峰(参见图1b)，其分子量表明是在化合物14加成上了羟基。在37℃保存1h后，新生成的UV峰几乎完全消失，而赖氨酸的质谱峰大大增强。说明该反应是由于氢氧自由基与高电子云密度的芳环发生加成反应，再发生1,4或1,6消除所产生的。此外，在化合物12的辐照研究中发现，通过质谱的单离子通道模式，发明人发现了有酚羟基加成的脱除产物出现。
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。NMR使用Bruker AVANCE 400MHz光谱仪记录。高分辨质谱使用Bruker Fourier Transform Ion Cyclotron共振质谱仪进行测量。所使用的液相色谱-质谱联用是Waters e2695仪，装备有Waters 2995 PDA和Waters Acquity QDA质谱仪。
1.D为Fmoc-赖氨酸的缀合物P-L-D的示例性合成
步骤一：中间体化合物15的合成
Figure PCTCN2020138682-appb-000019
将相应的醇R-OH(0.6mmol，1.0当量)溶于无水二氯甲烷(4mL)中，并冷却至0℃。加入N,N-二异丙基乙基胺(232mg，1.8mmol，3.0当量)，然后加入对硝基苯基氯甲酸酯(503mg，1.5mmol，2.5当量)。在1小时内将反应混合物温热至室温，此时将其用水淬灭并用二氯甲烷(3×20mL)萃取。合并有机层，经无水Na 2SO 4干燥，并在减压下旋干。粗产物通过硅胶色谱法用己烷/乙酸乙酯(9∶1)纯化得到化合物15。
步骤二：缀合物P-L-D的合成
Figure PCTCN2020138682-appb-000020
在圆底烧瓶中，将Fmoc-赖氨酸(1mmol，1.0当量)和N,N-二异丙基乙基胺(2mmol，2.0当量)溶解在15mL THF和15mL H 2O中，并搅拌2分钟。加入化合物15(1mmol，1.0当量)，并将混合物在室温搅拌1h。反应完成后，将反应溶液用乙酸乙酯萃取，并先后用水和饱和NaCl溶液洗涤两次。合并有机层，经无水Na 2SO 4干燥，并在减压下旋干。粗产物通过硅胶色谱法纯化，依次用己烷/乙酸乙酯(4∶1)，乙酸乙酯(100％)，二氯甲烷/甲醇(25∶1)，乙酸乙酯/甲醇(10∶1)洗脱，得到缀合物P-L-D。
通过选择不同的ROH，按照上述方法合成了表1的化合物。示例性化合物的NMR和MS数据：
Figure PCTCN2020138682-appb-000021
1H NMR(400MHz,DMSO-d 6)δ12.58(s,1H),7.90(d,J＝7.5Hz,2H),7.73(d,J＝7.5Hz,2H),7.62(d,J＝8.1Hz,1H),7.42(t,J＝7.5Hz,2H),7.33(t,J＝7.5,2H),7.07(t,J＝5.7Hz,1H),4.25–4.17(m,1H),3.96(q,J＝7.1Hz,2H),3.90(m,1H),2.95(q,J＝6.5Hz,2H),1.75–1.50(m,2H),1.43–1.27(m,4H),1.14(t,J＝7.0Hz,3H)。
13C NMR(101MHz,DMSO-d 6)δ174.47,156.70,156.61,144.31,144.27,141.18,141.17,128.10,127.53,125.76,125.74,120.58,66.05,59.88,54.28,47.12,30.90,29.49,23.40,15.16。
HRMS(ESI)：计算值C 24H 29N 2O 6([M+H] +):441.2020，实测值:441.2019。
Figure PCTCN2020138682-appb-000022
1H NMR(400MHz,DMSO-d 6)δ12.60(s,1H),7.90(d,J＝7.5Hz,2H),7.73(d,J＝7.5Hz,2H),7.62(d,J＝7.9Hz,1H),7.41(q,J＝7.7Hz,2H),7.40–7.23(m,8H),5.00(s,2H),4.32–4.18(m,3H),3.90(m,1H),2.98(q,J＝6.5Hz,2H)1.75–1.50(m,2H),1.43–1.27(m,4H)。
13C NMR(101MHz,DMSO-d 6)δ174.49,156.62,156.54,144.30,144.26,141.17,137.72,128.81,128.20,128.10,127.53,125.75,120.58,66.04,65.58,54.29,47.11,40.51,39.85,30.90,29.45,23.41。
HRMS(ESI)：计算值C 29H 31N 2O 6([M+H] +):503.2177，实测值:503.2166。
Figure PCTCN2020138682-appb-000023
1H NMR(400MHz,DMSO-d 6)δ12.58(s,1H),7.89(d,J＝7.5Hz,2H),7.73(d,J＝7.5Hz,2H),7.63(d,J＝8.0Hz,1H),7.42(t,J＝7.5Hz,2H),7.33(t, J＝7.5Hz,2H),7.29(d,J＝5.7Hz,1H),6.50(d,J＝2.3Hz,2H),6.42(t,J＝2.3Hz,1H),4.94(s,2H),4.29(d,J＝7.9Hz,2H),4.25–4.21(m,1H),3.91(m,1H),3.72(s,6H),3.00(q,J＝6.5Hz,2H),1.75–1.56(m,2H),1.39(m,4H)。
13C NMR(101MHz,DMSO-d 6)δ174.45,160.91,156.63,156.48,144.30,141.17,140.10,128.10,127.53,125.76,120.57,105.74,99.81,66.05,65.40,55.62,54.24,47.12,40.51,30.86,29.44,23.41。
HRMS(ESI)：计算值C 31H 35N 2O 8([M+H] +):563.2388，实测值:563.2388。
Figure PCTCN2020138682-appb-000024
1H NMR(400MHz,DMSO-d 6)δ12.59(s,1H),9.26(s,2H),7.90(d,J＝7.5Hz,2H),7.73(d,J＝7.4Hz,2H),7.65(d,J＝7.9Hz,1H),7.41(t,J＝7.4Hz,2H),7.33(t,J＝7.4,2H),7.25(t,J＝5.7Hz,1H),6.15(d,J＝2.2Hz,2H),6.08(t,J＝2.3Hz,1H),4.82(s,2H),4.35–4.18(m,3H),3.91(m,1H),2.98(q,J＝6.5Hz,2H),1.77–1.54(m,2H),1.38(m,4H)。
13C NMR(101MHz,DMSO-d 6)δ174.47,158.81,156.64,156.56,144.30,141.16,139.69,128.11,127.54,125.77,120.58,105.94,102.18,66.04,65.54,54.25,47.11,39.83,30.85,29.48,23.43。
HRMS(ESI)：计算值C 29H 31N 2O 8([M+H] +):535.2075，实测值:535.20734。
2.D为Fmoc-赖氨酸的缀合物P-L-D的辐照释放活性分子实验
在室温下将化合物溶解在生理盐水中，配制成10μM的水溶液，然后使用60Co作为γ射线源(或者放疗仪器作为X射线源)进行辐照(均为4Gy/min)，照射不同时间后(时间是2.5min，5min，10min，15min，20min，25min)，通过HPLC-MS定量分析溶液中产生的Fmoc-Lys。然后以释放Fmoc-lys的量对辐射剂量作图，求出释放率。
表1：缀合物P-L-D在4Gy/min的X射线或γ射线下释放率
Figure PCTCN2020138682-appb-000025
Figure PCTCN2020138682-appb-000026
3.Fqf-OH的制备
Figure PCTCN2020138682-appb-000027
Fqf-OH的合成方案
试剂和条件：(a)K 2CO 3,CH 3I,DMF,rt,12h；(b)2N NaOH,MeOH,rt,10h；(c)Tf 2O，吡啶，CH 2Cl 2,0℃至室温；(d)Pd 2(dba) 3·CHCl 3，xantphos,Cs 2CO 3,二苯甲酮亚胺，甲苯，105℃,12h；(e)1N HCl,THF,rt,30min；(f)三光气,TEA,THF,rt,19h；(g)iPrNEt 2,3,5-双(叔丁基二甲基甲硅烷氧基)苯基]甲醇,CH 2Cl 2,rt,1h；(h)TBAF,THF,rt,10h。
Figure PCTCN2020138682-appb-000028
步骤a，在N 2气氛下，向荧光素(10g，28.60mmol)的N,N-二甲基甲酰胺(90mL)溶液中缓慢加入K 2CO 3(9.88g，71.50mmol)，并将反应混合物在室温搅拌1h。然后，将碘甲烷(12.18g，85.80mmol)加入到反应混合物中，并将混合物在室温搅拌12h。通过TLC监控反应，反应完成后，向反应混合物中加入冰水，并在0℃下搅拌30分钟。过滤黄色固体，并用水洗涤以完全洗去K 2CO 3。干燥固体，以定量收率得到所需化合物(10.30g)。
1H NMR(400MHz,DMSO-d 6)δ8.21(dd,J＝8.0,1.1Hz,1H),7.87(td,J＝7.5,1.4Hz,1H),7.78(td,J＝7.5,1.2Hz,1H),7.50(dd,J＝7.5,1.1Hz,1H),7.23(d,J＝2.7Hz,1H),6.89(dd,J＝8.7,2.3Hz,1H),6.84(d,J＝9.1Hz,1H), 6.80(d,J＝9.6Hz,1H),6.39(dd,J＝9.6,1.8Hz,1H),6.24(d,J＝1.8Hz,1H),3.91(s,3H),3.58(s,3H)。
13C NMR(100MHz,DMSO-d 6)δ184.38,165.72,164.42,158.89,154.10,150.58,134.43,133.73,131.24,130.89,130.58,130.03,129.91,129.38,117.17,114.82,114.10,105.12,101.11,56.82,52.84。
HRMS(ESI +):m/z 计算值C 22H 17O 5[M+H] +:361.1076，实测值：361.1072。
Figure PCTCN2020138682-appb-000029
步骤b，向化合物30(10.30g，28.60mmol)的甲醇(150mL)溶液中缓慢加入2N NaOH(50mL)的水溶液。将反应混合物在室温搅拌10小时。减压蒸发甲醇，水层用乙醚洗涤两次。然后将水层用6N HCl酸化。过滤固体，用水洗涤以完全除去HCl以达到pH＝7，并干燥以定量收率得到所需化合物31(9.45g)。
1H NMR(400MHz,DMSO-d 6)δ10.16(s,1H),8.00(d,J＝7.3Hz,1H),7.79(td,J＝7.5,1.2Hz,1H),7.72(td,J＝7.5,0.9Hz,1H),7.27(d,J＝7.8Hz,1H),6.93(d,J＝2.7Hz,1H),6.74-6.67(m,2H),6.64(d,J＝8.7Hz,1H),6.57(d,J＝1.4Hz,2H),3.81(s,3H)。
13C NMR(100MHz,DMSO-d 6)δ169.20,161.56,160.11,153.00,152.41,152.30,152.22,136.17,130.67,129.63,129.48,126.58,125.20,124.54,113.34,112.42,111.53,109.98,102.73,101.31,83.31,65.44,56.18。
HRMS(ESI +):m/z C 21H 15O 5计算值[M+H] +:347.0920，实测值：347.0916。
Figure PCTCN2020138682-appb-000030
步骤c，向化合物31(1.00g，2.89mmol)的无水二氯甲烷(12mL)溶液中添加吡啶(0.91g，12mmol)，并将反应混合物在室温搅拌20min。将反应混合物 冷却至0℃，并在30分钟内将三氟甲磺酸酐(1.63g，5.79mmol)缓慢加入到反应混合物中。将反应混合物温热至室温并搅拌3小时。用水淬灭反应，并用二氯甲烷萃取。有机层先后用1N HCl溶液和水和盐水洗涤。合并的有机层经无水硫酸钠干燥，并在减压下除去挥发物。通过快速色谱法纯化粗残余物，以71％的产率得到化合物32(0.98g)。
1H NMR(400MHz,氯仿-d)δ8.05(d,J＝7.3Hz,1H),7.71(td,J＝7.5,1.2Hz,1H),7.66(td,J＝7.4,1.2Hz,1H),7.25(d,J＝2.4Hz,1H),7.18(d,J＝7.5,1H),7.00–6.87(m,2H),6.80(d,J＝2.5Hz,1H),6.76–6.63(m,2H),3.85(s,3H)。
13C NMR(100MHz,氯仿-d)δ168.96,161.67,152.63,152.06,151.89,149.98,135.38,130.18,130.09,129.02,126.30,125.34,123.88,120.27,119.82,116.71,112.47,110.54,110.49,100.93,81.64,55.67。
HRMS(ESI +):m/z C 22H 14F 3O 7S计算值[M+H] +:479.0412，实测值：479.0410。
Figure PCTCN2020138682-appb-000031
步骤d和e，反应之前，将所有玻璃器皿在烘箱中干燥。向化合物32(0.98g，2.04mmol)在甲苯(20mL)中的溶液中添加Pd 2(dba) 3·CHCl 3(0.21g，0.24mmol)和xantphos(0.18g，0.31mmol)。用氮气吹扫反应混合物，然后向其中加入碳酸铯(0.93g，2.86mmol)和二苯甲酮亚胺(0.44g，2.45mmol)。将反应混合物在氮气气氛下在105℃下加热12小时。通过TLC确认反应完成后，将反应混合物通过短硅藻土垫过滤，随后将其用二氯甲烷洗涤。滤液在减压下浓缩，粗残余物无需进一步纯化即可用于下一步反应。将粗化合物溶解在四氢呋喃(30mL)中，然后加入3mL 1N HCl。将反应混合物在室温搅拌30分 钟，并用乙酸乙酯萃取。有机层经无水硫酸钠干燥，并在减压下除去挥发物。通过快速柱色谱法用洗脱溶剂(PE∶EA＝7∶3)纯化粗残余物，以82％的收率得到化合物34(0.58g)。
1H NMR(400MHz,DMSO-d 6)δ7.98(d,J＝7.8Hz,1H),7.79(td,J＝7.5,0.9Hz,1H),7.71(td,J＝7.5,0.9Hz,1H),7.25(d,J＝7.3Hz,1H),6.91(d,J＝2.3Hz,1H),6.68(dd,J＝8.9,2.5Hz,1H),6.60(d,J＝8.7Hz,1H),6.45(d,J＝2.3Hz,1H),6.38(d,J＝8.2Hz,1H),6.34(dd,J＝8.5,2.1Hz,1H),5.66(s,2H),3.81(s,3H))。
13C NMR(100MHz,DMSO-d 6)δ169.29,161.43,153.03,152.66,152.50,151.88,136.00,130.49,129.37,129.02,126.92,125.04,124.52,112.06,111.87,111.79,105.82,101.27,99.60,84.20,56.15。
HRMS(ESI +):m/z C 21H 16NO 4计算值[M+H] +:346.1079，实测值：346.1071。
Figure PCTCN2020138682-appb-000032
步骤f，反应之前，将所有玻璃器皿在烘箱中干燥。在反应器中，将化合物34(0.83g，2.40mmol)溶解在16mL无水四氢呋喃中。将三光气(0.713g，2.403mmol)溶解在16mL无水四氢呋喃中，然后注入反应器中。然后加入蒸馏的2.92mL三乙胺(21.63mmol)。立即形成白色烟雾(气态光气)。密封反应器，并将溶液在室温下剧烈搅拌19小时。混合物减压浓缩。将粗残余物快速过滤，并将固体用无水四氢呋喃洗涤。减压除去溶剂，得到化合物6，为棕色油(定量)，其无需进一步纯化即可使用。
Figure PCTCN2020138682-appb-000033
步骤g，在冰水浴下，向化合物35(0.05g，0.14mmol)的二氯甲烷(4mL)溶液中添加三乙胺(0.037g，0.29mmol)的二氯甲烷(2mL)溶液，然后加入3,5- 双(叔丁基二甲基甲硅烷氧基)苯基]甲醇(0.063g，0.17mmol)。将反应混合物在室温搅拌1h。减压蒸发溶剂，并将残余物通过快速柱色谱纯化，以72％的收率得到化合物36(0.075g)。
1H NMR(400MHz,氯仿-d)δ8.02(dt,J＝7.3,1.1Hz,1H),7.66(td,J＝7.5,1.2Hz,1H),7.61(td,J＝7.5,1.2Hz,1H),7.55(s,1H),7.25(d,J＝2.4Hz,1H),7.18(d,J＝7.5,1H),7.00–6.87(m,2H),6.80(d,J＝2.5Hz,1H),6.76–6.63(m,2H),6.49(d,J＝2.2Hz,2H),6.30(t,J＝2.2Hz,1H),5.08(s,2H),3.84(s,3H),0.97(s,18H),0.22(s,12H)；
13C NMR(100MHz,氯仿-d)δ161.38,156.73,152.45,151.98,139.86,137.55,135.03,129.72,129.02,128.80,125.05,123.92,113.17,111.92,111.78,111.02,100.84,77.23,67.04,55.60,25.68,18.22,-4.36。
HRMS(ESI +):m/z C 41H 50NO 8Si 2计算值[M+H] +:740.3069，实测值：740.3072。
Figure PCTCN2020138682-appb-000034
步骤h，在0℃下，将1.0M氟化四丁基铵(TBAF)(230μL，0.23mmol)的溶液滴加到36(75mg，0.1mmol)的溶液中。将混合物在室温搅拌10小时。将该溶液倒入水中，并用氯仿萃取三次。合并的有机相用盐水洗涤，用无水MgSO 4干燥，通过硅藻土垫过滤，并用旋转蒸发仪浓缩。将残余物通过硅胶快速柱色谱纯化，得到橙色固体状的37(35.8mg，0.07mmol，70％)。
1H NMR(400MHz,乙腈-d 3)δ8.09(s,1H),7.66(td,J＝7.5,1.2Hz,1H),7.61(td,J＝7.5,1.2Hz,1H),7.55(s,1H),7.25(d,J＝2.4Hz,1H),7.18(d,J＝7.5,1H),7.00–6.87(m,2H),6.80(d,J＝2.5Hz,1H),6.76–6.63(m,2H),6.49(d,J＝2.2Hz,2H),6.30(t,J＝2.2Hz,1H),5.08(s,2H),3.83(s,3H)。
13C NMR(100MHz,乙腈-d 3)δ169.55,162.23,158.71,153.88,152.90,152.18,141.76,139.72,135.87,130.56,129.65,129.23,127.00,125.27,124.43,114.87,113.92,112.28,111.73,106.65,106.00,102.44,101.33, 66.68,55.98。
HRMS(ESI +):m/z C 29H 22NO 8计算值[M+H] +:512.1340，实测值：512.1339。
4.Fqf-OH的试管内荧光分子释放
为了证明在辐照条件下小分子释放策略的广谱可行性，以及为了便于检测，发明人将具有高反应活性的间苯二酚结构引入到荧光分子中。在辐照产生的羟基自由基的作用下，荧光前体分子Fqf-OH发生脱除反应，从而释放荧光分子。
Figure PCTCN2020138682-appb-000035
将Fqf-OH溶于0.02M的磷酸缓冲液(pH＝7.4)中，配成10μM的溶液(0.1％的DMF作为助溶剂)，再接受1Gy/min的γ射线照射( 60Co)。通过控制照射时间，使溶液受辐照的总剂量以10Gy为梯度，所受剂量分别为0Gy、10Gy、20Gy、30Gy、40Gy、50Gy、60Gy、70Gy。荧光强度(在518nm测量，激发波长为477nm)随剂量增强而逐渐增大(图2a)且具有良好的线性关系(图2b)。为了验证Fqf-OH对羟基自由基具有高选择性，将Fqf-OH与不同的RNS及ROS接触，发现只有在羟基自由基的作用下可以释放荧光分子，其反应能力是其他ROS及RNS的30倍以上(图2c)。Fqf-OH针对各种金属离子例如钠、钾、钙、铁、锌离子和氨基酸是稳定的。而且Fqf-OH在细胞内是稳定的，添加细胞内高浓度存在的物质，并不能使荧光活性恢复。
5.Fqf-OH的细胞内荧光分子释放实验
验证了Fqf-OH对辐照下产生羟基自由基的高选择性及高反应性后，将其引入细胞实验中，验证Fqf-OH在细胞内的释放。在引入细胞实验之前，检测它的毒性，在10μM的Fqf-OH下，细胞存活率大于95％。
HeLa细胞在共聚焦皿中培养24h后，移除培养基，用HBSS洗3次，再 以10μM Fqf-OH的HBSS溶液(pH＝7.4,0.1％DMF)孵育30min后接受 60Co源γ射线1Gy/min照射1min及5Gy/min照射1、2、4min后进行激发波长488nm的共聚焦成像(图3的上部图)，释放的荧光分子的量随剂量的增大而增加。在扣除细胞自荧光背景后，1Gy的剂量下，荧光强度增强了2.6倍，5Gy，10Gy，20Gy下分别增长6.6、12、18倍(图3的下部柱状图示出了具有Fqf-OH的三种细胞共聚焦成像得到的荧光强度)。在4T1细胞以及MC38上可以得到同样的实验结果(图3)。
上述细胞实验说明了前体分子可以在绝大多数细胞中通过γ射线释放荧光分子。
6.活体内荧光分子释放
为了验证荧光前体分子在小鼠肿瘤内的释放，我们借助了医院的放疗仪器。由于放疗仪器产生的是X射线，我们先证明在X射线下与γ射线荧光前体分子在相同剂量下有着相同的释放效果。在4T1荷瘤小鼠上局部注射20μL 200μM(1％DMF作为助溶剂)荧光前体Fqf-OH的PBS溶液，半小时后让小鼠接受4Gy的照射。为了模拟肿瘤治疗实验，我们重复此过程3次(图4)。将小鼠处死后，剖出肿瘤，切片做荧光成像，发现注射荧光前体分子的实验组比注射生理盐水的对照组有非常明显的荧光信号增强(图4)，在扣除组织自荧光背景之后，荧光强度在处理之后增加了2.5倍。该实验说明了荧光前体分子在活体内可以通过X射线的辐照释放荧光分子。
7.D为一甲基澳瑞他汀E的缀合物P-L-D的合成
以下以化合物27为例说明缀合物P-L-D的合成，其中P为间苯二酚基，L为-CH 2-O-C(＝O)-，D为一甲基澳瑞他汀E(Monomethyl auristatin E，简称MMAE)。
Figure PCTCN2020138682-appb-000036
中间体化合物24
将化合物23(5mmol)溶于二氯甲烷(50mL)中，再加入N,N-二异丙基乙基胺(15mmol,3equiv)，在室温下剧烈搅拌十分钟。在冰水浴下加入叔丁基二甲基甲硅烷基氯(10.5mmol,2.1equiv)，30分钟后，使其恢复到室温，再反应2小时。将反应液用水稀释，再使用乙酸乙酯萃取三次。将有机相合并，依次用pH＝3的盐酸溶液，饱和碳酸氢钠溶液，水，饱和食盐水洗涤。旋干后，使用石油醚/乙酸乙酯体系(10:1)作为洗脱剂，通过硅胶柱纯化得到化合物24，产率为95％。
中间体化合物25
将化合物24(4.5mmol)溶于超干的四氢呋喃(50mL)中，在冰水浴下向其分批加入LiAlH4(4.5mmol,1equiv)，30分钟后，使其恢复到室温，再反应2小时。在冰水浴下向反应液中缓慢滴加水淬灭反应。垫硅藻土过滤，滤液用水稀释，再使用乙酸乙酯萃取三次。将有机相合并，依次用pH＝3的盐酸溶液，饱和碳酸氢钠溶液，水，饱和食盐水洗涤。旋干后，使用石油醚/乙酸乙酯体系(4:1)作为洗脱剂，通过硅胶柱纯化得到化合物25，产率为98％。
中间体化合物26
将化合物25(4mmol)溶于DCM(120mL)中，加入N,N-二异丙基乙基胺(6mmol,1.5equiv)后，搅拌10分钟。再加入对硝基苯基氯甲酸酯(4.8mmol,1.2equiv)。2小时后。将反应液用水稀释，再使用乙酸乙酯萃取三次。将有机相合并，依次用pH＝3的盐酸溶液，饱和碳酸氢钠水溶液，水，饱和食盐水洗涤。旋干后，使用石油醚/乙酸乙酯体系(4:1)作为洗脱剂，通过硅胶柱纯化得到化合物26，产率为81％。
化合物27
将化合物26(0.6mmol)溶于DCM(20mL)中，加入1-羟基苯并三唑(0.7mmol,1.2equiv)与N,N-二异丙基乙基胺(1.2mmol,2equiv)后，搅拌10分钟。再加入MMAE(0.6mmol,1.0equiv)。反应过夜。将反应液用水稀释，再使用乙酸乙酯萃取三次。将有机相合并，依次用pH＝3的盐酸溶液，饱和碳酸氢钠水溶液，水，饱和食盐水洗涤。旋干后，再将反应粗产品溶于THF(20mL)中,再加入四丁基氟化铵的四氢呋喃溶液1.4mL(1.4mmol,1.2equiv)，反应2小时，旋干，使用二氯甲烷/甲醇体系(20:1)作为洗脱剂，通过硅胶柱纯化得到化合物27，产率为58％。高分辨质谱：C 47H 74N 5O 11[M+H] +计算值884.5379,测定值884.5365。
8.化合物27的辐照释放活性分子实验
Figure PCTCN2020138682-appb-000037
在室温下将化合物27溶解在生理盐水中，配制成10μM的水溶液。然后使用 60Co作为γ射线源(或者放疗仪器作为X射线源)进行辐照(均为4Gy/min)，照射不同时间后(时间是2.5min，5min，10min，15min，20min，25min)，通过HPLC-MS定量分析溶液中产生的MMAE的浓度(参见图5的右上角，示出了辐照之后的药物分子质谱信号峰的变化)，然后以释放MMAE的量对辐射剂量作图。药物分子浓度与剂量有良好的线性关系，释放率约为40nM/Gy(参见图5的左下角)。
将未辐照处理的10μM的化合物27(前药分子)水溶液稀释至10nM，然后与细胞共孵育，发现未辐照处理的化合物27水溶液几乎无细胞毒性，但是在通过 60Co的γ射线照射10Gy后的细胞毒性恢复至与药物分子MMAE相当。从细胞存活与浓度对数的关系图(图5的右下角)可看出：辐照后的溶液与未经辐照的前药分子相比，溶液的细胞毒性有显著提升。
本公开的实施例通过荧光前体分子在原理上说明了辐照释放活性分子策略的可行性。进一步，本公开的实施例以MMAE药物分子作为模型说明了辐照释放药物策略的可行性。本公开的实施例还说明从缀合物中释放出的活性分子的浓度与剂量有良好的线性关系。
以上所述仅是本发明的示范性实施方式，而非用于限制本发明的保护范围，本发明的保护范围由所附的权利要求确定。
本发明具体涉及以下各项：
1、一种通式(I)的缀合物，
P——L——D   (I)
其中P为含有芳基或杂芳基的辐射响应基团；L不存在或者为二价连接基；D为成像剂或治疗剂。
2、根据项1所述的缀合物，其中所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而从缀合物中脱落的基团，或者所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而与连接基L一起从缀合物中脱落的基团。
3、根据项1所述的缀合物，其中所述芳基或杂芳基选自下组：苯基、萘 基、蒽基、菲基、联苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基、呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、三唑基、四唑基、
Figure PCTCN2020138682-appb-000038
唑基、异
Figure PCTCN2020138682-appb-000039
唑基、
Figure PCTCN2020138682-appb-000040
二唑基、
Figure PCTCN2020138682-appb-000041
三唑基、呋咱基、噻唑基、异噻唑基、吲哚基、苯并噻唑基、苯并二氧杂环戊烯基、苯并
Figure PCTCN2020138682-appb-000042
唑基、苯并噻吩基、喹啉基、四氢异喹啉基、异喹啉基、苯并咪唑基、苯并吡喃基、吲嗪基、苯并呋喃基、色酮基、香豆素基、苯并吡喃基、噌啉基、喹喔啉基、吲唑基、吡咯并吡啶基、呋喃并吡啶基、二氢异吲哚基、四氢喹啉基、咔唑基、二苯并呋喃基、二苯并噻吩基、苯并吲哚基、菲咯啉基、吖啶基、菲啶基和呫吨基；
其中所述芳基或杂芳基任选取代有一个或者多个选自下组的取代基：卤素、-R 1、-NR 1R 2、-CN、-NO 2、-N 3、-OR 1、-SR 1、-NHCOR 1、-O-COR 1、-CH＝CR 1R 2、-C(＝O)-R 1、-C(＝O)-OR 1、-C(＝O)-Cl、-C(＝O)-NH 2、-C(＝O)-NH-R 1和-C(＝O)-NR 1R 2；
其中R 1和R 2独立地选自H、C 1-C 6烷基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基，所述烷基、烯基、环烷基、芳基和杂芳基任选地被一个或多个卤素、羟基、巯基、-CN、硝基、-N 3、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基取代。
4.根据项3所述的缀合物，其中所述芳基或杂芳基选自苯基、吡咯基、呋喃基、噻吩基、吡唑基和咪唑基，所述芳基或杂芳基任选取代有一个或多个氨基、胺基、-NHCOCH 3、苯基、甲氧基、乙氧基、甲基、乙基、羟基、烯基、F、Cl、Br、I、-N 3或者甲基。
5.根据项3所述的缀合物，其中所述芳基或杂芳基上没有直接与其相连的氧代、-C(＝O)-、-C(＝S)-、-CN、-SO 3H、-CF 3。
6.根据项5所述的缀合物，其中所述芳基为任选取代的间苯二酚基。
7、根据项1-6中任一项所述的缀合物，其中，
L选自-SS-、线性的C 1-C 6-亚烷基-、支链的C 3-C 6-亚烷基-、-R 3-X-C(＝O)-Y-、-R 3-X-C(＝S)-Y-、-R 3-X-、-X-C(＝O)-Y-、-X-C(＝S)-Y-或者-CH 2-X-C(＝Z)-Y-CH 2CH 2-E-C(＝J)-F-，其中R 3选自C 1-C 3亚烷基或者-CH 2-C 2烯基-；X、Y、E和F独立地选自O、NH或S；Z和J独立地选自O或S； L可任选取代有羟基、巯基、NH 2、F、Cl、Br、I、-N 3、甲基。
8、根据项7所述的缀合物，其中，L为-CH 2-X-(C＝O)-Y-、-CH 2-X-(C＝S)-Y-、-CH 2-X-或者-X-(C＝O)-Y-，其中X和Y独立地选自O、NH、S。
9、根据项8所述的缀合物，其中，L为-CH 2-O-(C＝O)-、-CH 2-O-(C＝O)-NH-或者-O-(C＝O)-NH-。
10.根据项1所述的缀合物，其中D为荧光成像剂或者治疗癌症的治疗剂。
11.根据项10所述的缀合物，其中所述荧光成像剂或者治疗癌症的治疗剂中不存在芳基或杂芳基。
12.根据项10所述的缀合物，其中所述荧光成像剂或者治疗癌症的治疗剂中存在芳基或杂芳基，并且芳基或杂芳基上存在直接与其相连的氧代、-C(＝O)-、-C(＝S)-、-CN、-SO 3H或-CF 3基团。
13.根据项1所述的缀合物，其中所述治疗剂选自以下的一种或多种：一甲基澳瑞他汀E、一甲基澳瑞他汀F、依鲁替尼、阿卡替尼、泽布替尼、阿霉素、丝裂霉素-C、丝裂霉素-A、柔红霉素、氨基蝶呤、放线菌素、博来霉素、9-氨基喜树碱、N8-乙酰基亚精胺、1-(2-氯乙基)-1,2-二甲烷磺酰基酰肼、云南霉素、吉西他滨、阿糖胞苷、多拉司他汀、达卡巴嗪、5-氟尿嘧啶；紫杉醇、多西他赛(Docetaxel)、吉西他滨、阿糖胞苷；6-巯基嘌呤。
14.根据项13所述的缀合物，其中所述治疗剂为一甲基澳瑞他汀E、一甲基澳瑞他汀F、或者5-氟尿嘧啶。
15.根据项1所述的缀合物，其中所述成像剂选自罗丹明染料、花青染料、荧光素染料、PET成像剂、放射性标记的试剂。
16.根据项15所述的缀合物，所述缀合物为Fqf-OH。
17.一种治疗或者成像方法，包括将项1-16中任一项的缀合物口服或者注入患者，然后患者接受精准的辐照。
18.根据项17的方法，其中所述患者是患有癌症的人或哺乳动物。
19.项1-16中任一项的缀合物在定性或定量检测氢氧自由基中的用途。
20.一种在定性或定量检测氢氧自由基的方法：包括将Fqf-OH加入待测溶液中，测量溶液的荧光强度。

Claims (10)

  1. 一种通式(I)的缀合物，
    P——L——D  (I)
    其中P为含有芳基或杂芳基的辐射响应基团；L不存在或者为二价连接基；D为成像剂或治疗剂。
  2. 根据权利要求1所述的缀合物，其中所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而从缀合物中脱落的基团，或者所述辐射响应基团是能够与辐射产生的氢氧自由基发生化学反应从而与连接基L一起从缀合物中脱落的基团。
  3. 根据权利要求1所述的缀合物，其中所述芳基或杂芳基选自下组：苯基、萘基、蒽基、菲基、联苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基、呋喃基、噻吩基、吡咯基、吡唑基、咪唑基、三唑基、四唑基、
    Figure PCTCN2020138682-appb-100001
    唑基、异
    Figure PCTCN2020138682-appb-100002
    唑基、
    Figure PCTCN2020138682-appb-100003
    二唑基、
    Figure PCTCN2020138682-appb-100004
    三唑基、呋咱基、噻唑基、异噻唑基、吲哚基、苯并噻唑基、苯并二氧杂环戊烯基、苯并
    Figure PCTCN2020138682-appb-100005
    唑基、苯并噻吩基、喹啉基、四氢异喹啉基、异喹啉基、苯并咪唑基、苯并吡喃基、吲嗪基、苯并呋喃基、色酮基、香豆素基、苯并吡喃基、噌啉基、喹喔啉基、吲唑基、吡咯并吡啶基、呋喃并吡啶基、二氢异吲哚基、四氢喹啉基、咔唑基、二苯并呋喃基、二苯并噻吩基、苯并吲哚基、菲咯啉基、吖啶基、菲啶基和呫吨基；其中所述芳基或杂芳基任选取代有一个或者多个选自下组的取代基：卤素、-R 1、-NR 1R 2、-CN、-NO 2、-N 3、-OR 1、-SR 1、-NHCOR 1、-O-COR 1、-CH＝CR 1R 2、-C(＝O)-R 1、-C(＝O)-OR 1、-C(＝O)-Cl、-C(＝O)-NH 2、-C(＝O)-NH-R 1和-C(＝O)-NR 1R 2；其中R 1和R 2独立地选自H、C 1-C 6烷基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基，所述烷基、烯基、环烷基、芳基和杂芳基任选地被一个或多个卤素、羟基、巯基、-CN、硝基、-N 3、C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6卤代烷基、C 2-C 6烯基、C 3-C 10环烷基、C 6-C 20芳基、或者具有5-20个环原子的杂芳基取代。
  4. 根据权利要求3所述的缀合物，其中所述芳基或杂芳基选自苯基、吡咯基、呋喃基、噻吩基、吡唑基和咪唑基，所述芳基或杂芳基任选取代有一个或多个氨基、胺基、-NHCOCH 3、苯基、甲氧基、乙氧基、甲基、乙基、羟基、烯基、F、Cl、Br、I、-N 3或者甲基。
  5. 根据权利要求3所述的缀合物，其中所述芳基或杂芳基上没有直接与其相连的氧代、-C(＝O)-、-C(＝S)-、-CN、-SO 3H、-CF 3。
  6. 根据权利要求5所述的缀合物，其中所述芳基为任选取代的间苯二酚基。
  7. 根据权利要求1-6中任一项所述的缀合物，其中，
    L选自-SS-、线性的C 1-C 6-亚烷基-、支链的C 3-C 6-亚烷基-、-R 3-X-C(＝O)-Y-、-R 3-X-C(＝S)-Y-、-R 3-X-、-X-C(＝O)-Y-、-X-C(＝S)-Y-或者-CH 2-X-C(＝Z)-Y-CH 2CH 2-E-C(＝J)-F-，其中R 3选自C 1-C 3亚烷基或者-CH 2-C 2烯基-；X、Y、E和F独立地选自O、NH或S；Z和J独立地选自O或S；L可任选取代有羟基、巯基、NH 2、F、Cl、Br、I、-N 3、甲基。
  8. 根据权利要求7所述的缀合物，其中，L为-CH 2-X-(C＝O)-Y-、-CH 2-X-(C＝S)-Y-、-CH 2-X-或者-X-(C＝O)-Y-，其中X和Y独立地选自O、NH、S。
  9. 根据权利要求8所述的缀合物，其中，L为-CH 2-O-(C＝O)-、-CH 2-O-(C＝O)-NH-或者-O-(C＝O)-NH-。
  10. 根据权利要求1所述的缀合物，其中D为荧光成像剂或者治疗癌症的治疗剂。
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