Hypoxia response prodrug as well as preparation method and application thereof

Abstract

The invention discloses a hypoxia response prodrug and a preparation method and application thereof.The hypoxia response prodrug comprises a compound shown in the formula I, pharmaceutically acceptable salt of the compound and solvate: # imgabs0 #. According to the hypoxia response prodrug and the preparation method and application thereof, azo bonds serve as a functional connecting group and a hypoxia response group, and then fluorescence and biological activity of a photosensitizer are temporarily closed; the photosensitizer and the chemotherapeutic drug can be selectively broken and released under the hypoxia condition, OFF-ON (off-on) fluorescence and biological activity of hypoxia stress response can be achieved, the selectivity to tumor cells is further improved, the fluorescence characteristic of the prodrug OFF-ON is expected to be used for tumor diagnosis and curative effect monitoring, and integration of diagnosis and treatment is expected to be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a hypoxia responsive prodrug and a preparation method and application thereof. Background Art

[0002] Cancer (malignant tumor) is a disease caused by abnormal proliferation and differentiation of cells under the influence of genetic and environmental factors. At present, cancer is one of the most serious health problems affecting people's life happiness index worldwide, and millions of people die from cancer every year. The main characteristics of cancer are the unlimited proliferation and division of cancer cells, the formation of tumors and invasion of surrounding tissues and organs. Through early diagnosis and treatment, the survival rate and cure rate of cancer have been greatly improved. However, there are still great challenges in current cancer treatment, one of which is the poor selectivity of treatment and side effects, which often affect the quality of life and treatment effect of patients.

[0003] Hypoxia is an important physiological feature of many different solid tumors and a key driver of malignant tumors. In recent years, it has been recognized as an important target for cancer treatment. The sensitization of hypoxic cells in tumors and the improvement of hypoxic tumor microenvironment have always been hot issues in tumor research, and hypoxia is also one of the most interesting therapeutic targets in current cancer research. One of the main strategies for treating hypoxic tumors is to release therapeutic drugs in response to hypoxia and improve the hypoxic microenvironment, such as using hypoxia-activated prodrugs for tumor targeted therapy. When the prodrug is enriched in the hypoxic area, it will release the active original drug under the stress of the hypoxic microenvironment, thereby selectively killing adjacent hypoxic tumor cells. Therefore, designing and synthesizing hypoxia-responsive anti-tumor prodrugs, and then selectively releasing the original drug in the hypoxic tumor microenvironment, is expected to improve the bioavailability and selectivity of the drug, reduce the potential toxic side effects of the original drug, and improve the anti-tumor efficiency.

[0004] Photodynamic therapy (PDT) is a method of selectively irradiating photosensitizers (PS) that are enriched in diseased tissues (such as tumors) using a light source of a specific wavelength, thereby generating cytotoxic reactive oxygen species (ROS, such as singlet oxygen 1 O 2 ) to selectively kill tumor cells. PDT has been clinically approved in many countries around the world and is mainly used to treat tumor diseases. However, the photosensitizers currently used in clinical practice are basically macrocyclic molecules based on tetrapyrrole ring structures, which have complex molecular structures and are difficult to synthesize. In addition, the "Always ON" characteristic of traditional photosensitizers makes the photosensitizers less selective, and while killing tumor cells, they will inevitably damage normal cells. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a hypoxia-responsive prodrug and its preparation method and application, the anti-tumor activity and fluorescence performance of the tumor hypoxia stress-responsive prodrug are basically hidden and quenched under normoxic conditions, and can selectively release photosensitizers and chemical anticancer drugs under hypoxic stress, which is expected to reduce the potential adverse side effects of the original drug and improve the therapeutic efficacy, and under hypoxic stress, the fluorescence performance of the photosensitizer will be restored, which is expected to be used for early diagnosis of hypoxic tumors and efficacy monitoring in treatment.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a compound of formula I, a pharmaceutically acceptable salt, or a solvate thereof:

[0008]

[0009] In the present invention, the compound of formula I can selectively release photosensitizers and nitrogen mustard chemotherapy drugs in the hypoxic microenvironment of tumors, and can serve as a hypoxia-responsive prodrug; it has high selectivity for tumor cells and can selectively kill tumor cells under hypoxic conditions; it can kill tumor cells by inducing cell apoptosis and autophagy.

[0010] The second aspect of the present invention provides a compound of formula II, a pharmaceutically acceptable salt, and a solvate thereof:

[0011]

[0012] In the present invention, the compound of formula II is a control compound of formula I.

[0013] The third aspect of the present invention provides a method for preparing a compound of formula I or a compound of formula II, comprising the following steps:

[0014] S1: diazotizing p-aminobenzyl alcohol and subjecting it to coupling reaction with N,N-dialkylaniline; and subjecting it to chlorination reaction to obtain an intermediate;

[0015] S2: The intermediate is subjected to N-alkylation reaction with 2-methylquinolin-8-amine; and then cyclized with 2-thiosulfate-4-N,N-diethyl-p-phenylenediamine under the action of silver carbonate to obtain the product.

[0016] In some embodiments of the present invention, in S1, the reaction temperature of the coupling reaction is -10 to 5°C; and the reaction time is 5 to 15 hours.

[0017] In some embodiments of the present invention, in S1, the chlorination reaction uses POCl 3 Carry out; the reaction temperature is 20~100℃.

[0018] In some embodiments of the present invention, in S2, the N-alkylation reaction is carried out under the action of potassium carbonate and potassium iodide; the reaction temperature is 50 to 80° C.; and the reaction time is 10 to 20 hours.

[0019] In some embodiments of the present invention, in S2, the cyclization reaction temperature is 70-100° C. and the reaction time is 2-6 h.

[0020] In some embodiments of the present invention, in S1, when the N,N-dialkylaniline is N,N-dihydroxyethylaniline, the final product of S2 is a compound of formula I.

[0021] In some embodiments of the present invention, in S1, when the N,N-dialkylaniline is N,N-dimethylaniline, the final product of S2 is a compound of formula II.

[0022] The fourth aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I and / or its pharmaceutically acceptable salt or solvate.

[0023] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0024] The fifth aspect of the present invention provides a method for inhibiting cell proliferation. The method comprises the following steps: (a) contacting a target cell with an effective amount of a compound of formula I described herein, the target cell usually undergoing inappropriate proliferation, such as a malignant tumor cell or an infectious bacterial cell; and (b) exposing the compound together with the target cell in its vicinity to light having an appropriate wavelength of about 600nm to 900nm (e.g., about 600nm to 700nm), the malignant tumor cell or the infectious bacterial cell usually leading to a hypoxic microenvironment, selectively releasing the photosensitizer compound 1 by the compound of formula I activated by light and the hypoxic microenvironment. and nitrogen mustard chemotherapy drugs, the cytotoxic free radicals / radical ions or singlet oxygen generated by compound 1 under light; nitrogen mustard chemotherapy drugs can also kill target cells. Under the combined action of the two, the compound of formula I causes the death of target cells. In some embodiments, the target cell is a tumor cell or a cancer cell. In some embodiments, the target cell is a bacterial cell, which can be a gram-positive bacterium or a gram-negative bacterium, such as Escherichia coli or Staphylococcus aureus. In some embodiments, the target cell is in the body of a subject, such as a human or animal subject. Therefore, the method described in the present invention effectively provides a method for treating a proliferative disease of a subject, including various types of cancer or infections involving microbial pathogens such as bacteria and viruses. Tumors / cancers that can be treated in this manner include lung cancer, pancreatic cancer, breast cancer, colorectal cancer, colon cancer, esophageal cancer, oral cancer, lymphoma, penile cancer, prostate cancer, skin cancer, gynecological cancer, gastrointestinal stromal tumors, head tumors, neck tumors, eye tumors, etc. Bacterial infections that can be treated in this manner include infections caused by gram-positive and gram-negative bacteria (e.g., Escherichia coli or Staphylococcus aureus). In some embodiments, the methods described herein effectively provide methods for treating ophthalmic diseases (AMD), arthritis, atherosclerosis, and restenosis. Compounds can be delivered to subjects using a variety of routes of administration, including oral ingestion, topical application, and injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, and intratumoral injection).

[0025] The sixth aspect of the present invention provides the use of the compound of formula I and / or the pharmaceutical composition in the preparation of tumor diagnostic agents and / or therapeutic agents.

[0026] In some embodiments of the present invention, the tumor comprises at least one of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, colon cancer, esophageal cancer, oral cancer, lymphoma, penile cancer, prostate cancer, skin cancer, gynecological cancer, gastrointestinal stromal tumor, head tumor, neck tumor, and eye tumor.

[0027] In the present invention, the pharmaceutically acceptable salt includes but is not limited to at least one of inorganic acid salts, organic acid salts, alkyl sulfonates and aryl sulfonates; preferably, the inorganic acid salt includes but is not limited to at least one of hydrochloride, hydrobromide, nitrate, sulfate and phosphate; preferably, the organic acid salt includes but is not limited to at least one of formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate and citrate; preferably, the alkyl sulfonate includes but is not limited to at least one of methyl sulfonate and ethyl sulfonate; the aryl sulfonate includes but is not limited to at least one of benzene sulfonate and p-toluene sulfonate.

[0028] The carriers of the pharmaceutical composition include, but are not limited to, liposomes, nanoparticles, vesicles, microbubbles, microspheres, nanobubbles, micelles, emulsions, gels, liquid crystals, biomedical materials, etc. The composition may be composed of general delivery media or excipients, including but not limited to ethanol, polyethylene glycol, dimethyl sulfoxide, Tween, glycerol, castor oil, buffers, etc.

[0029] The pharmaceutical compositions or drugs used in the present invention can be formulated using one or more physiologically acceptable carriers or excipients by standard techniques. Suitable pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences" by EW Martin. The compounds of formula I of the present invention and / or their physiologically acceptable salts and solvates can be formulated for administration by appropriate routes, including administration via inhalation, topical, nasal, oral, enteral or rectal administration.

[0030] Typical formulations for topical administration include creams, ointments, sprays, lotions and patches. However, the pharmaceutical composition can be formulated for any type of administration using a syringe or other device, for example, intradermal, subcutaneous, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection. Also contemplated are formulations for administration by inhalation (e.g., aerosols) or for oral, rectal or vaginal administration.

[0031] Route of administration

[0032] Suitable formulations for topical application, for example to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well known in the art. These formulations may contain solubilizers, stabilizers, isotonicity enhancers, buffers and / or preservatives.

[0033] Suitable formulations for transdermal administration include an effective amount of a compound of formula I of the present invention and a carrier. Preferred carriers include absorbable pharmacologically acceptable solvents that assist in passing through the host's skin. For example, a transdermal device is in the form of a bandage that includes a backing member, a reservoir containing the compound and an optional carrier, an optional rate-controlling barrier that delivers the compound to the host's skin at a controlled and predetermined rate over an extended period of time, and a device that secures the device to the skin. Matrix transdermal formulations can also be used.

[0034] For oral administration, the pharmaceutical composition or medicament may take the form of tablets or capsules prepared, for example, by conventional means with pharmaceutically acceptable excipients. Preferred are tablets and gelatin capsules comprising the active ingredient, i.e., a compound of formula I, together with (a) a diluent or filler, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and / or calcium hydrogen phosphate, calcium sulfate; (b) a lubricant, for example, silicon dioxide, talc, stearic acid, its magnesium or calcium salts, metal stearates, colloidal silicon dioxide, hydrogenated vegetable oils, corn starch, sodium benzoate, acetic acid sodium and / or polyethylene glycol; for tablets, together with (c) binders, for example, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and / or hydroxypropyl methylcellulose; if necessary, (d) disintegrants, for example, starch (for example, potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, for example, sodium lauryl sulfate and / or (f) absorbents, colorants, flavoring agents and sweeteners.

[0035] Tablets can be film-coated or enteric-coated according to methods known in the art. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or they can be presented as dry products composed of water or other suitable media before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents, such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats; emulsifiers, such as lecithin or gum arabic; non-aqueous media, such as almond oil, oily esters, ethanol or fractionated vegetable oils; and preservatives, such as methylparaben or propylparaben or sorbic acid. The preparation can also contain buffer salts, flavoring agents, colorants and / or sweeteners as appropriate. If necessary, the preparation for oral administration can be appropriately formulated to give controlled release of the compound of formula I of the present invention.

[0036] The compound of formula I of the present invention can be formulated for parenteral administration by injection, for example, by push injection or continuous infusion. Injectable preparations can be present in unit dosage forms, for example, in ampoules or in multi-dose containers, and preservatives are added. The injectable composition is preferably an isotonic aqueous solution or suspension, and the suppository is preferably prepared by a fat emulsion or suspension. The composition can be sterilized and / or contain adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts and / or buffers for regulating osmotic pressure. Alternatively, the active ingredient can be in powder form, composed of a suitable medium, such as sterilized pyrogen-free water, before use. In addition, they can also contain other therapeutically valuable substances. The composition is prepared according to conventional mixing, granulation or coating methods, respectively, and contains about 0.1% to 75% (mass fraction) of active ingredients, preferably about 1% to 50% (mass fraction) of active ingredients.

[0037] For administration by inhalation, the active ingredient, e.g., a compound of formula I of the present invention, can be conveniently delivered in the form of an aerosol spray using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0038] The compounds of formula I may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0039] In addition, the compound of formula I can be formulated as a reservoir preparation. Such long-acting preparations can be administered by implantation (such as subcutaneous or intramuscular) or intramuscular injection. For example, the compound of formula I can be formulated with a suitable polymeric material or hydrophobic material (for example, as an emulsion in an acceptable oil) or an ion exchange resin, or as a slightly soluble derivative, for example, as a slightly soluble salt.

[0040] The pharmaceutical composition or medicament of the present invention comprises (i) an effective amount of a compound of formula I described herein that is used as an effective photosensitizer, and (ii) another therapeutic agent. When used with the compounds of the present invention, such therapeutic agents can be used alone, sequentially, or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and a compound of formula I of the present invention). Administration can be by the same or different routes of administration, or administered together in the same pharmaceutical preparation.

[0041] dose

[0042] The pharmaceutical composition or drug can be administered to a subject at a therapeutically effective dose to prevent, treat or manage a proliferative disease as described herein. The pharmaceutical composition or drug is administered to a subject in an amount sufficient to induce an effective therapeutic response in the subject.

[0043] The dosage of the active agent administered depends on the weight, age, individual condition, surface area or volume of the area to be treated and the form of administration of the subject. The size of the dosage will also be determined by the presence, nature and degree of any side effects associated with the administration of a specific compound in a specific subject. For example, each compound in the compound of formula I described herein can have a unique dosage. A unit dose of about 50kg to 70kg of a mammal administered orally can contain about 5mg to 500mg of the compound. Typically, the dosage of the compound of formula I of the present invention is a dosage sufficient to achieve the desired effect. The best dosage regimen can be calculated by the measured value of the reagent accumulated in the body of the subject. Typically, the dosage can be given once or more every day, every week or every month. Those skilled in the art can easily determine the optimal dose, administration methodology and repetition frequency.

[0044] In order to achieve the desired therapeutic effect, the compound of formula I can be administered for multiple days with a therapeutically effective daily dose. Therefore, the effective administration of the compound for treatment requires periodic (e.g., every day) administration, and continues for three days to two weeks or longer cycles to treat the relevant disease states or diseases described herein of the object. Typically, the compound of formula I will be administered for at least three consecutive days, typically at least five consecutive days, more typically at least ten consecutive days, and sometimes for 20 consecutive days, 30 days or more days. Although continuous daily doses are preferred approaches to achieve therapeutically effective doses, even if the compound is not administered every day, as long as frequent repeated administration is sufficient to maintain the therapeutically effective concentration of the compound in the object, then therapeutic beneficial effects can be achieved. For example, the agent can be administered every other day or every three days, or if a higher dosage range is adopted and tolerated by the object, the agent is administered once a week.

[0045] Optimal doses, toxicity, and therapeutic effects of a compound may vary depending on the relative potency of the individual compounds and may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose that causes 50% of the population to die) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 Particular compounds that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the affected tissue site to minimize potential damage to normal cells and thereby reduce side effects.

[0046] For example, data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds preferably ranges from about 10 to about 20 mg / kg to about 10 mg / kg. 50 The dosage may vary within this range depending on the dosage form and route of administration employed. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve an IC value including that determined in cell culture. 50 (the concentration at which the compound achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. For example, levels in plasma can be measured by high performance liquid chromatography (HPLC). Generally, for a typical subject, the dose equivalent of the compound is about 1 ng / kg to 100 mg / kg.

[0047] Exemplary dosages of the compounds of formula I of the present invention. In the case of intravitreal administration (e.g., 5 mg / kg-30 mg / kg), an exemplary dosage of the compound of formula I may be 0.1-0.5 mg / eye. The compound may be administered orally at 5 mg-1000 mg, or by intravenous infusion at 10 mg / mL-500 mg / mL. In an alternative, the compound may be administered by intravenous injection or infusion at 50 mg / mL-500 mg / mL (within 120 minutes); 1 mg / kg-500 mg / kg (within 60 minutes); or 1 mg / kg-100 mg / kg (push) five times a week. The compound of formula I may be administered subcutaneously at 10 mg-500 mg; 0.1 mg / kg-500 mg / kg intravenously twice a day, or about 50 mg once a week, or 25 mg twice a week.

[0048] The pharmaceutical composition of the present invention can be administered alone or in combination with at least one other therapeutic compound. Exemplary favorable therapeutic compounds include systemic anti-inflammatory agents and local anti-inflammatory drugs, analgesics, antihistamines, anesthetic compounds, antibiotics, etc. Additional therapeutic compounds can be administered simultaneously with the main active ingredient (e.g., the compound of formula I of the present invention), or even administered together with the main active ingredient in the same composition. Additional therapeutic compounds can also be administered individually, in a separate composition, or in a dosage form different from the main active ingredient. The main ingredients of some doses, such as the compound of formula I described herein, can be administered simultaneously with additional therapeutic compounds, while other doses are administered separately according to the specific symptoms and characteristics of the individual.

[0049] The dosage of the pharmaceutical composition of the present invention can be adjusted throughout the course of treatment according to the severity of symptoms, frequency of relapses and physiological response to the treatment regimen. Such adjustments of treatment regimens are usually performed by those skilled in the art.

[0050] The beneficial effects of the present invention are:

[0051] (1) In a hypoxic microenvironment, the compound of formula I of the present invention can simultaneously release the photosensitizer compound 1 having an anti-tumor effect and the nitrogen mustard chemotherapy drug, thereby enhancing the anti-tumor effect.

[0052] (2) The "Always ON" property of traditional photosensitizers makes the photosensitizers less selective, and will inevitably damage normal cells while killing tumor cells. The present invention uses azo bonds as functional linkers and hypoxia response groups to temporarily block the fluorescence and biological activity of the photosensitizer, and can selectively release the original photosensitizer and chemotherapeutic drugs under hypoxia, so as to achieve "OFF-ON" fluorescence and biological activity in response to hypoxia stress, thereby improving the selectivity for tumor cells, and the "OFF-ON" fluorescence property of the prodrug is expected to be used for tumor diagnosis and efficacy monitoring, and is expected to achieve integrated diagnosis and treatment.

[0053] (3) The synthesis and preparation process of the compound of formula I of the present invention is simple, does not require very strict experimental conditions and operation, and is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 For intermediate 3 1 H NM(400MHz,CDCl 3 );

[0055] Figure 2 For intermediate 3 13 C NMR (101 MHz, CDCl 3 );

[0056] Figure 3 For intermediate 4 1 H NMR (400 MHz, CDCl 3 );

[0057] Figure 4 For intermediate 4 13 C NMR (101 MHz, CDCl 3 );

[0058] Figure 5 For intermediate 5 1 H NMR (400 MHz, CDCl 3 );

[0059] Figure 6 For intermediate 5 13 C NMR (101 MHz, CDCl 3 );

[0060] Figure 7 For compound 7 1 H NMR (500 MHz, CDCl 3 );

[0061] Figure 8 For compound 7 13 C NMR (126 MHz, CDCl 3 );

[0062] Fig. 9 For intermediate 3a 1 H NMR (400 MHz, CDCl 3 );

[0063] Fig.10 For intermediate 3a 13 C NMR (101 MHz, CDCl 3 );

[0064] Fig.11 For intermediate 4a 1 H NMR (400 MHz, CDCl 3 );

[0065] Fig.12 For intermediate 4a 13 C NMR (101 MHz, CDCl 3 );

[0066] Fig.13 For intermediate 5a 1 H NMR (400 MHz, CDCl 3 );

[0067] Fig.14 For intermediate 5a 13 C NMR (101 MHz, CDCl 3 );

[0068] Fig.15 For compound 8 1 H NMR (400 MHz, CDCl 3 );

[0069] Fig.16 For compound 8 13 C NMR (101 MHz, CDCl 3 );

[0070] Fig.17 For compound 1 1 H NMR (600 MHz, CDCl 3 );

[0071] Fig.18 A, B, and C are the UV absorption spectra of compounds 1, 7, and 8 in different solvents, respectively; D is the UV absorption spectra of compounds 1, 7, and 8 in H 2 Fluorescence spectrum of O:MeCN (1:1);

[0072] Fig.19 A, B, and C are the fluorescence spectra of compounds 1, 7, and 8 at different illumination times, respectively; D is the fluorescence spectra in solutions with different pH values; the excitation wavelength λex = 625 nm;

[0073] Fig. 20 A is the fluorescence value of compound 7 at 680nm (excitation wavelength λex = 625nm) in the presence of different ions, active substances and bioreductants; B is the fluorescence spectra of compound 7 incubated with liver microsomes and NADPH for different lengths of time

[0074] Fig.21 Compounds 7 and 8 with different concentrations of Na 2 S 2 O 4 HPLC profile after incubation;

[0075] Fig. 22 Compounds 7 and 8 with different concentrations of Na 2 S 2 O 4 Fluorescence spectra after incubation;

[0076] Fig.23 The test results of the active oxygen generation performance of the compounds; A is the singlet oxygen probe DPBF to detect the active oxygen generation in different groups; B is the active oxygen generation of compounds 1, 7, 8, 7+Na under visible light 2 S 2 O 4 and 8+Na 2 S 2 O 4 Pictures in acetonitrile-water solution (1:1);

[0077] Fig.24 A is the detection of reactive oxygen species production in different groups by singlet oxygen probe SOSG (fluorescence value at 530 nm); B is the detection of superoxide anion production in different groups by superoxide anion fluorescent probe DHR123 (fluorescence value at 530 nm);

[0078] Fig.25The expression of HIF-1α protein in A549 cells in normoxic and hypoxic incubators (left) and statistical results (right);

[0079] Fig.26 Cytotoxicity test results of compounds 7 and 8; wherein A is a cytotoxicity test result of compound 7 on different tumor cells (A549, MCF-7 and Hela cells) and normal cells Hek 293T cells under normoxic and hypoxic conditions; B is a cytotoxicity test result of compound 8 at different concentrations incubated with A549 cells under normoxic and hypoxic conditions for different times; C is a cytotoxicity test result of compound 7 incubated with A549 cells under hypoxic conditions for 6 hours and then illuminated for different times; D is a cytotoxicity test result of compound 8 on A549 lung cancer cells under normoxic and hypoxic conditions;

[0080] Fig. 27 The release results of the compounds under different oxygen concentrations; A is the confocal experimental results of compounds 7 and 8 (2 μM) under normoxic and hypoxic conditions, respectively, scale: 50 μm; B is the flow cytometry results of different concentrations of compound 7 under hypoxic conditions; C is the cell flow cytometry results of compound 7 under different oxygen concentrations;

[0081] Fig.28 The results of compound 7 producing reactive oxygen species in cells, scale bar: 50 μm;

[0082] Fig.29 The intracellular subcellular organelle localization result diagram of compound 7 (scale: 50 μm) and the corresponding statistical results;

[0083] Fig.30 The experimental results of compound 7 inducing intracellular LAMP1 protein expression are shown in Figure 1. Scale bar: 5 μm;

[0084] Fig.31 This is the experimental result of compound 7 inducing cell apoptosis. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0086] Example 1

[0087] In this example, compound 7 (compound of formula I) was prepared, and the specific process was as follows:

[0088]

[0089] S1: Preparation of intermediate 3:

[0090] Compound 2 (500.0 mg, 4.06 mmol) and HCl (10 mol / L, 7 mL) aqueous solution were added to a 100 mL round-bottom flask and stirred in an ice-water bath until compound 2 was completely dissolved. Then, 1.5 mL of sodium nitrite solution (NaNO 2 560.0 mg, 8.13 mmol) and stirred at 0°C for 2 h. Then, a saturated aqueous sodium acetate solution was added to the reaction mixture to adjust the pH to about 2. After continuing the reaction for 0.5 h, urea (365.0 mg, 6.09 mmol) and an acetic acid solution of N,N-dihydroxyethylaniline (1.10 g, 6.09 mmol) were added, and the reaction mixture was stirred for 6.5 h to obtain an orange-red solution. After the reaction was completed by TLC monitoring, saturated sodium bicarbonate (NaHCO 3 ) aqueous solution to pH about 7, and extract the crude product with ethyl acetate (EA: 25 mL×3). The combined organic layer was washed with saturated sodium chloride solution (NaCl: 30 mL×2) and washed with anhydrous sodium sulfate (Na 2 SO 4 ) and then the crude product was purified by column chromatography to obtain orange solid compound 3 (1.1 g, 86%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.82(d,J=9.2Hz,2H),7.79(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),6.89(d ,J=9.2Hz,2H),4.68(s,2H),3.81(t,J=5.9Hz,4H),3.68(t,J=5.9Hz,4H)ppm. 13 C NMR (101 MHz, DMSO-d 6 )δ151.87,151.30,144.53,142.75,127.58,125.22,122.07,111.77,63.04,58.61,53.72,39.82ppm.LC-MS:m / z 316.32[M+H] + . Figure 1 For intermediate 3 1 H NMR spectrum, Figure 2 For intermediate 3 13 C NMR spectrum proved that the compound was successfully prepared.

[0091] S2: Preparation of intermediate 4

[0092] Compound 3 (1 g, 3.17 mmol) was added to a 100 mL round-bottom flask and placed in an ice-water bath. After the reaction system cooled to 0 °C, phosphorus oxychloride (POCl 3 , 5 mL), then stirred and heated to 85 ° C reflux for 5 h. After the reaction was completed by TLC monitoring, the round-bottom flask was placed in an ice-water bath and saturated sodium carbonate (Na 2 CO 3 ) solution, and neutralized the solution to a pH of about 7. The crude product was then extracted with ethyl acetate (EA: 25 mL×3). The combined organic layers were washed with a saturated sodium chloride solution (NaCl: 30 mL×2) and washed with anhydrous sodium sulfate (Na 2 SO 4 The crude product was purified by column chromatography to obtain orange solid compound 4 (470.0 mg, 40%). 1 H NMR (400 MHz, CDCl 3 )δ7.91(d,J=8.5Hz,2H),7.85(d,J=8.3Hz,2H),7.50(d,J=8.2Hz,2H),6.78(d ,J=8.4Hz,2H),4.65(s,2H),3.84(t,J=6.8Hz,4H),3.70(t,J=6.7Hz,4H)ppm. 13 C NMR (101 MHz, CDCl 3 )δ152.82,148.78,144.60,139.00,129.45,125.51,122.80,111.70,53.48,46.03,40.38ppm.LC-MS:m / z 370.47[M+H] + .

[0093] Figure 3 For intermediate 4 1 H NMR spectrum, Figure 4 For intermediate 4 13 C NMR spectrum proved that the compound was successfully prepared.

[0094] S3: Preparation of Intermediate 5

[0095] Compound 4 (360.0 mg, 973.43 μmol), 2-methylquinolin-8-amine (140 mg, 884.93 μmol), potassium carbonate (K 2 CO 3, 366.9 mg, 2650 μmmol), potassium iodide (KI, 15.0 mg, 88.49 μmol) and N,N-dimethylformamide (DMF, 4 mL). The reaction mixture was reacted at 65 ° C for 16 h, and then the mixture was cooled to room temperature and 20 mL of water was added. The reaction mixture was extracted with ethyl acetate (EA: 25 mL × 3), and the combined organic phases were respectively washed with saturated ammonium chloride solution (NH 4 Cl: 30 mL × 2) and sodium chloride solution (NaCl: 30 mL × 2), and washed with anhydrous sodium sulfate (Na 2 SO 4 After removing the solvent, the residue was purified by column chromatography to give orange compound 5 (295.0 mg, 68%). 1 H NMR (400 MHz, CDCl 3 )δ7.94-7.82(m,5H),7.53(d,J=8.3Hz,2H),7.27-7.19(m,2H),7.01(d,J=8.1Hz,1H),6.68(d,J=9.1Hz ,2H),6.59(d,J=7.6Hz,1H),4.59(s,2H),3.71(t,J=6.9Hz,4H),3.59(t,J=6.8Hz,4H),2.69(s,3H)ppm. 13 C NMR (101 MHz, CDCl 3 )δ155.86,152.31,148.47,144.73,144.00,141.72,136.24,127.97,126.79,126.70,1 25.31,122.74,122.31,114.33,111.68,105.50,53.47,47.57,40.39,25.28.LC-MS:m / z 492.43[M+H] + . Figure 5 For intermediate 5 1 H NMR spectrum, Figure 6 For intermediate 5 13 C NMR spectrum proved that the compound was successfully prepared.

[0096] S4: Preparation of Compound 7 (Compound of Formula I)

[0097] Compound 5 (270.0 mg, 548 μmol), compound 6 (227.0 mg, 822 μmol) and a mixed solvent of methanol and ethyl acetate (6 mL, MeOH:EA=4:1) were added to a 50 mL double-necked round-bottom flask. Silver carbonate (Ag 2 CO 3, 302.0 mg, 1.10 mmol). The system was refluxed at 85°C for 4 h, then the reaction solution was cooled to room temperature and filtered with diatomaceous earth, and the diatomaceous earth filter layer was washed with a mixed solvent of methanol and dichloromethane until the filtrate was colorless. The concentrated organic layer was purified by column chromatography to obtain a dark green solid compound, which was redissolved with 5 mL of dichloromethane and acidified with 0.25 mL of a methanolic hydrochloric acid solution (HCl: 1 mol / L, dissolved in MeOH) and then concentrated to obtain a dark green solid compound 7 (168.0 mg, 45%). 1 H NMR (500MHz, Methanol-d 4 )δ9.21(d,J=8.5Hz,1H),8.08(d,J=9.6Hz,1H),7.87-7.77(m,5H),7.59(d,J=8.3Hz,2H),7.48(dd,J=9.6,2.5Hz,1H),7.40(s,1H),7.33(d,J =2.5Hz,1H),6.87(d,J=9.2Hz,2H),5.02(s,2H),3.88(t,J=6.8Hz,4H),3.73(dt,J=18.0,6.8Hz,8H),2.83(s,3H),1.33(t,J=7.1Hz,6H)ppm. 13 C NMR (101MHz, Methanol-d 4 )δ160.71,152.51,151.78,143.95,139.12,138.74,138.32,137.46,133.60,132.85,132.38,127.96,126.81,124. 91,122.46,118.31,113.06,111.48,105.04,53.73,52.75,47.60,45.71,40.10,23.58,11.72ppm.HRMS(ESI):calcd for C 37 H 39 Cl 2 N 7 SCl([M-Cl] + ):682.2281,found 682.2353. Figure 7 For compound 7 1 H NMR spectrum,. Figure 8 For compound 7 13 C NMR spectrum proved that the compound was successfully prepared.

[0098] Example 2

[0099] In this example, compound 8 was prepared by the following process:

[0100]

[0101] S1: Preparation of intermediate 3a

[0102] The preparation of compound 3a followed the synthetic steps of compound 3. 1 H NMR (400 MHz, DMSO-d 6 )δ7.77(dd,J=14.8,8.6Hz,4H),7.46(d,J=8.2Hz,2H),6.83(d,J=9.0Hz,2H),4.58(s,2H),3.05(s,6H)ppm. 13 C NMR (101 MHz, DMSO-d 6 )δ152.89,151.84,144.71,143.05,127.55,112.03,63.04,39.95ppm.LC-MS:m / z 256.69[M+H] + . Fig. 9 For intermediate 3a 1 H NMR spectrum, Fig.10 For intermediate 3a 13 The CNMR spectrum proved that the compound was successfully prepared.

[0103] S2: Preparation of compound 4a

[0104] The preparation of compound 4a followed the synthetic steps of compound 4. 1 H NMR (400 MHz, CDCl 3 )δ7.89(d,J=9.0Hz,2H),7.83(d,J=8.3Hz,2H),7.49(d,J=8.3Hz,2H),6.77(d,J=9.1Hz,2H),4.64(s,2H),3.0952(s,6H)ppm. 13 C NMR (101 MHz, CDCl 3 )δ153.00,152.60,143.62,138.36,129.32,125.20,122.52,111.58,46.02,40.36ppm.LC-MS: m / z274.37[M+H]+. Fig.11 For intermediate 4a 1 HNMR spectrum, Fig.12 For intermediate 4a 13 C NMR spectrum proved that the compound was successfully prepared.

[0105] S3: Preparation of intermediate 5a

[0106] The preparation of compound 5a refers to the synthesis steps of compound 5. 1 H NMR (400 MHz, CDCl 3 )δ7.94(d,J=8.4Hz,1H),7.84(dd,J=19.4,8.7Hz,4H),7.53(d,J=8.3Hz,2H),7.24(dt,J=7.4,3.7Hz,2H),7. 01(d,J=7.9Hz,1H),6.74(d,J=9.1Hz,2H),6.60(d,J=7.5Hz,1H),4.62(s,2H),3.06(s,6H),2.70(s,3H)ppm. 13 C NMR (101 MHz, CDCl 3 )δ155.78,152.52,152.39,144.02,143.74,141.01,137.60,136.17,127.86,126.69,126 .61,124.92,122.46,122.18,114.18,111.54,105.42,47.58,40.33,25.21ppm.LC-MS:m / z 396.66. Fig.13 For intermediate 5a 1 H NMR spectrum, Fig.14 For intermediate 4a 13 C NMR spectrum proved that the compound was successfully prepared.

[0107] S4: Preparation of Compound 8

[0108] The preparation of compound 8 followed the synthetic steps of compound 7. 1 H NMR (400 MHz, Methanol-d 4 )δ8.98(d,J=8.3Hz,1H),7.91(d,J=9.5Hz,1H),7.71(dd,J=23.8,8.3Hz,5H),7.53(d,J=7.8Hz,2H),7.37(d,J=9.0Hz,1H),7.25 (s,1H),7.18(s,1H),6.69(d,J=8.8Hz,2H),4.89(s,2H),3.72-3.64(m,4H),3.02(s,6H),2.74(s,3H),1.32(t,J=6.9Hz,6H)ppm. 13 C NMR (101MHz, Methanol-d 4)δ160.51,152.55,151.21,142.96,138.92,137.66,137.39,132.71,132.19,128.06,126.04,124.6 3,122.26,118.22,111.06,104.94,101.84,47.60,45.72,38.94,23.56,11.79ppm.HRMS(ESI):calcd for C 35 H 37 N 7 SCl([M-Cl] + ):586.2747,found586.2788. Fig.15 Compound 8 1 H NMR spectrum, Fig.16 For intermediate 4a 13 C NMR spectrum proved that the compound was successfully prepared.

[0109] Example 3

[0110] In this example, compound 1 was prepared, and the specific process was as follows:

[0111]

[0112] Compound 6 (140.0 mg, 0.5 mmol), 2-methylquinolin-8-amine (40.4 mg, 0.25 mmol) and a mixed solvent of methanol and ethyl acetate (6 mL, MeOH:EA=4:1) were added to a 50 mL two-necked round-bottom flask and refluxed at 85°C. Then, silver carbonate (Ag 2 CO 3 , 140.0 mg, 0.5 mmol), after reflux reaction for 2 h, the reaction mixture was cooled to room temperature, filtered through diatomaceous earth, and washed with a mixed solvent of methanol and dichloromethane (DCM) until the filtrate was colorless. The organic layer was concentrated to obtain a blue solid, which was redissolved with 5 mL of dichloromethane and acidified with 0.25 mL of hydrochloric acid methanol solution (HCl: 1 mol / L, dissolved in methanol). The mixture was concentrated to obtain dark blue solid compound 1 (31.1 mg, 32%). 1 H NMR (600 MHz, CDCl 3)δ8.98(d,J=8.3Hz,1H),7.90(d,J=9.3Hz,1H),7.83(s,1H),7.58(d,J=8.4Hz,1H),7.16(dd,J=9. 4,2.6Hz,1H),6.82(d,J=2.6Hz,1H),3.62(q,J=7.2Hz,4H),2.74(s,3H),1.36(t,J=7.0Hz,6H)m / z 391.19. Fig.17 Compound 1 1 The H NMR spectrum proved that the compound was successfully prepared.

[0113] Test Example 1

[0114] This test example studies the photophysical properties of the compound. The specific process is as follows:

[0115] (1) The UV-visible absorption spectra of compounds 1, 7 and 8 were measured using a UV-visible spectrophotometer.

[0116] Experimental method: Compounds 1, 7 and 8 were dissolved in DMSO to prepare a 10 mM test stock solution. MeCN, DCM, DMSO, EG, EtOH and H 2 The compound was diluted to 10 μM in a solvent of O:MeCN (v / v=1:1) and then detected by UV-visible spectrophotometer ( Fig.18 A, B, C).

[0117] The experimental results are as follows Fig.18 As shown in Figure 2, compounds 1, 7, and 8 all have strong spectral absorption peaks at 660 nm in different solvents, but in H 2 O:CH 3 In CN solution, the absorption intensity of the compounds became weaker and the absorption peaks became broader, especially for prodrug compound 7 and control compound 8, which may be due to the 2 The influence of strong hydrogen bonding in O:MeCN solution.

[0118] (2) Fluorescence emission spectra of compounds 1, 7 and 8 were measured using a fluorescence spectrophotometer

[0119] Fluorescence emission spectroscopy detection: The mother solutions of compounds 1, 7 and 8 were eluted with H 2 O:MeCN (1:1) was diluted to 10 μM, and its fluorescence emission spectrum was detected by fluorescence spectrophotometer. Ex =625nm,λ Em =650~900nm.

[0120] The experimental results are as follows Fig.18As shown in D: Compound 1 has the highest peak at 680nm with a fluorescence value of 275, while compounds 7 and 8 are only 30, indicating that the azo bond in the structure of compounds 7 and 8 can effectively quench the fluorescence of the photosensitizer and achieve fluorescence "OFF".

[0121] (3) Study on the photostability and pH stability of compounds 1, 7 and 8

[0122] A. Photostability test: The stock solutions of compounds 1, 7 and 8 were diluted with PBS to a concentration of 10 μM. The samples were irradiated with a 660 nm laser for 0, 0.5, 1, 2, 3, 4, 6, 8 and 10 min (84 mw / cm 2 ) and then the fluorescence spectrum (λ Ex =625nm,λ Em =650~900nm)( Fig.19 A, B, C).

[0123] B. pH acid-base stability: The compounds were dissolved in deionized water at pH 3, 4, 5, 6, 7, 8, 9, and 10 to prepare a solution with a final concentration of 10 μM. The changes in the fluorescence spectra (λ Ex =625nm,λ Em =650-900nm)( Fig.19 D).

[0124] The experimental results are as follows Fig.19 As shown, the fluorescence of compound 1 gradually decreases under continuous illumination, indicating that the compound will gradually undergo photobleaching as the illumination time increases, while the fluorescence of compounds 7 and 8 at different illumination times is basically unchanged, indicating that the prodrug 7 obtained after modification and the control compound 8 have better light stability and will not undergo photobleaching. In addition, under different pH conditions, the fluorescence of compounds 1, 7, and 8 also basically does not change, indicating that the compounds have good pH stability and will not undergo acid and alkaline decomposition.

[0125] Test Example 2

[0126] This test example studies the in vitro release performance of the compound, and the specific process is as follows:

[0127] (1) In vitro specific release experiment of prodrug compound 7:

[0128] The prodrug compound 7 was dissolved in aqueous solutions containing the following substances to prepare solutions with a final concentration of 10 μM.

[0129] 1) Metal ion group (ion concentration is 10mM): Ca 2+ , Cu 2+ , K + Mg 2+ 、Na+ 、Zn 2+ .

[0130] 2) Active oxygen / active sulfur / active nitrogen and other active substances (active substance concentration is 100μM): ClO - , H 2 O 2 , HS - 、NO 2 - .

[0131] 3) Amino acids and bioreductants group (concentration of 1 mM): GSH, VC, Ala, Glu, Arg, Pro, glucose, Na 2 S 2 O 4 .

[0132] The prepared sample solution was incubated at 37°C for 1 h, and the changes in its fluorescence spectrum were detected by a fluorescence spectrophotometer. Ex =625nm,λ Em =650-900nm. (e.g. Fig. 20 A)

[0133] (2) Study on the drug release performance of prodrug compound 7 under the action of azobioreductase liver microsomes

[0134] Experimental method: The DMSO stock solution (10 mM) of prodrug compound 7 was treated with H 2 The O:MeCN (1:1) system was diluted to a final concentration of 10 μM, and liver microsomal enzymes (final concentration of 0.5 mg / mL) and NADPH (final concentration of 500 μM) were added, and incubated at 37°C for different periods of time with shaking. The drug release performance of compound 7 under the action of liver microsomal enzymes was then detected by a fluorescence spectrophotometer. (e.g. Fig. 20 B)

[0135] The experimental results are as follows Fig. 20 The prodrug compound 7 had no response to various metal ions, reactive oxygen, reactive sulfur, reactive nitrogen, amino acids and biological reductants, but had no response to the azoreductase chemical mimetic Na 2 S 2 O 4However, under the action of azobioreductase liver microsomes, it can release strong fluorescence in response to stress, with the highest fluorescence value reaching 648, realizing the transition of fluorescence from "OFF" to "ON", indicating that prodrug compound 7 can effectively release the original drug under reducing conditions and has good hypoxia response specificity. In addition, it was further verified that under the action of azobioreductase liver microsomes, the fluorescence intensity of prodrug compound 7 gradually increased with the increase of incubation time. After 24h of incubation, the fluorescence value increased from 58 at 0h to 228, indicating that prodrug compound 7 can release the original drug under the action of bioreductase liver microsomes, and shows a certain time dependence, further verifying the hypoxia stress response performance of prodrug compound 7.

[0136] (2) Prodrug compounds 7 and 8 for the azoreductase chemical mimetic Na 2 S 2 O 4 Concentration response characteristics

[0137] Method 1: The mother solution of compound 7 or 8 was diluted with deionized water to a final concentration of 25 μM. Then, freshly prepared Na 2 S 2 O 4 The solution (the mother solution concentration is 1M) was prepared into mixtures of different concentration ratios, and then incubated at 37°C for 1h and detected by HPLC. HPLC analysis conditions: High performance liquid chromatograph: Shimadzu LC-20A, chromatographic column: Athena (C18-WP 100A, 4.6×250mm, 5μM), detector: spd20a, detection band: 254nm, flow rate: 1mL / min. Mobile phase composition (Phase A: acetonitrile, containing 0.1% trifluoroacetic acid; Phase B: water, containing 0.1% trifluoroacetic acid); gradient elution conditions are 40-100% acetonitrile (containing 0.1% trifluoroacetic acid), and the elution time is 30min. (such as Fig.21 )

[0138] The experimental results are as follows Fig.21 As shown, the current drug compound and Na 2 S 2 O 4 After co-incubation, a new peak will be generated in the mixed solution, and its retention time is consistent with that of the original drug peak 1, indicating that the prodrug compound 7 or 8 is 2 S 2 O 4 After co-incubation, the original drug is released and along with Na 2 S 2 O 4 With the increase of the concentration, the release of the original drug gradually increased within the same incubation time, indicating that the release of prodrug compounds 7 and 8 and the azoreductase chemical mimetic Na 2 S 2 O 4It showed good concentration dependence.

[0139] Method 2: The mother solution of compound 7 or 8 was prepared into an aqueous solution with a final concentration of 25 μM using deionized water, and freshly prepared Na 2 S 2 O 4 aqueous solution (mother liquor concentration is 1M), so that Na 2 S 2 O 4 The final concentration of was increased from 0 to 2 mM. Then they were incubated at 37°C for 1 h. The fluorescence spectrum of the mixed solution after co-incubation was detected using a fluorescence spectrophotometer. Ex =625nm,λ Em =650-900nm. (e.g. Fig. 22 )

[0140] The experimental results are shown in Fig. 22 As shown, as Na 2 S 2 O 4 With the increase of Na concentration, the fluorescence intensity of the reaction solution also gradually increased. The fluorescence value of compound 7 reached 580, and the fluorescence value of compound 8 reached 560, which further indicated that the release of the original drug increased with the increase of Na 2 S 2 O 4 The results of method 1 are basically consistent with the conclusion of method 1.

[0141] Test Example 3

[0142] This test example studies the active oxygen generation performance of the compound, and the specific process is as follows:

[0143] (1) The singlet oxygen probe DPBF (1,3-diphenylisobenzofuran) was used to detect the active oxygen generation performance of the compounds by UV-visible spectrophotometry. The detection groups were as follows: DPBF group: 40 μM DPBF (the following are all final concentrations) in acetonitrile / water solution (1:1); single drug compound 1 treatment group: 10 μM compound 1 + 40 μM DPBF in acetonitrile solution; single drug compound 7 treatment group: 10 μM compound 7 + 40 μM DPBF in acetonitrile solution; single drug compound 8 treatment group: 10 μM compound 8 + 40 μM DPBF in acetonitrile solution; compound 7 / 8 and Na 2 S 2 O 4 Mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na 2 S 2 O 4+ 40 μM DPBF in acetonitrile; and 10 μM compound 8 + 800 μM Na 2 S 2 O 4 +40μM DPBF in acetonitrile solution. 2 ) After irradiation for 0s, 5s, 10s, 20s, 30s, 60s, 90s, 120s, 150s, and 180s, the absorbance of the compound at 410nm was then detected. Fig.23 A)

[0144] The experimental results are as follows Fig.23 A shows that the original drug compound 1 can effectively generate singlet oxygen under light irradiation, while the simple prodrug compounds 7 and 8 basically do not generate singlet oxygen under light irradiation, but when combined with Na 2 S 2 O 4 After co-incubation, reactive oxygen species can be effectively generated under light, and the generation of reactive oxygen species gradually increases with the extension of light exposure time, indicating that the singlet oxygen generation performance of prodrug compounds 7 and 8 is also effectively quenched and can be reduced by the reducing agent Na 2 S 2 O 4 Effectively activated, regenerating singlet oxygen. Fig.23 B is the color of acetonitrile aqueous solution (water: acetonitrile, 1:1) of compounds 1, 7, and 8, and the color of 7 and 8 with Na 2 S 2 O 4 The color of the acetonitrile aqueous solution after incubation further indicated that compounds 7 and 8 2 S 2 O 4 After incubation, the compound 1 is reduced.

[0145] (2) The singlet oxygen probe SOSG (Singlet Oxygen Sensor Green) was used to detect the active oxygen generation performance of the compounds by fluorescence spectrophotometry. The detection groups were as follows: SOSG group: 3 μM SOSG (the following are all final concentrations) in acetonitrile aqueous solution (1:1); compound 1 treatment group: 10 μM compound 1 + 3 μM SOSG in acetonitrile aqueous solution; single drug group compound 7 and compound 8 group: 10 μM compound 7 or 8 + 3 μM SOSG in acetonitrile aqueous solution; compound 7, 8 and Na 2 S 2 O 4 Mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na 2 S 2 O 4+ 3 μM SOSG in acetonitrile; and 10 μM compound 8 + 800 μM Na 2 S 2 O 4 +10μM SOSG in acetonitrile solution. Then use 660nm laser (84mw / cm 2 ) for 0, 30, 60, 120, 180, 300 and 480 s, and then the fluorescence emission spectrum of the compound was detected by fluorescence spectrophotometer, λ Ex =488nm,λ Em =530nm. (e.g. Fig.24 A)

[0146] The experimental results are as follows Fig.24 As shown in A, the original drug compound 1 can effectively generate singlet oxygen (fluorescence value 893.8) under light, while the prodrug compound 7 (fluorescence value 34.11) and the control compound 8 (fluorescence value 25.18) can hardly generate singlet oxygen. 2 S 2 O 4 After co-incubation, reactive oxygen species can also be effectively generated under light (fluorescence value of compound 7 is 646.10, and fluorescence value of compound 8 is 609.9), which is basically consistent with the results detected by DPBF and UV spectrophotometer, further proving that prodrug compound 7 and control compound 8 have good hypoxia response properties and can effectively release reactive oxygen species under light and hypoxia stress response.

[0147] (3) Superoxide anion fluorescent probe DHR123 (Dihydrorhodamine 123, dihydrorhodamine 123) was used to detect the production of reactive oxygen species by fluorescence spectrophotometry: the detection groups were as follows: DHR123 group: 5 μM DHR123 (the following are all final concentrations) in acetonitrile aqueous solution (1:1); compound 1 treatment group: 10 μM compound 1 + 5 μM DHR123 in acetonitrile aqueous solution; single drug group: compound 7, 8 group: 10 μM compound 7, 8 + 5 μM DHR123 in acetonitrile aqueous solution; compound 7 / 8 and Na 2 S 2 O 4 Mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na 2 S 2 O 4 + 5 μM DHR123 in acetonitrile; and 10 μM compound 8 + 800 μM Na 2 S 2 O 4 +5μM DHR123 in acetonitrile solution. Then use 660nm laser (84mw / cm2 ) for 0, 10, 30, 60, 120 and 180 s, and then the fluorescence emission spectrum of the compound was detected by a fluorescence spectrophotometer. Ex =488nm,λ Em =530nm. (e.g. Fig.24 B)

[0148] The experimental results are as follows Fig.24 As shown in B, the original drug compound 1 can effectively generate superoxide anion free radicals under light, producing strong fluorescent rhodamine 123 (fluorescence value 6250), and the fluorescence becomes stronger as the light exposure time increases, indicating that more superoxide anions are generated, proving that the original drug compound 1 has a Type I PDT mechanism (generating superoxide anion free radicals), and Fig.23 A and Fig.24 A shows that compound 1 has a Type II mechanism of action (generating singlet oxygen). This mechanism of action, which combines Type I and Type II, indicates that compound 1 has excellent photodynamic properties and is an excellent photosensitizer. Similarly, the superoxide anion radical generation ability of prodrug compound 7 (fluorescence value 427.2) and control compound 8 (fluorescence value 223.8) can also be effectively temporarily quenched, and when combined with the chemical reducing agent Na 2 S 2 O 4 After co-incubation (fluorescence value of compound 7 is 4344, and fluorescence value of compound 8 is 3635), the original drug compound 1 can be effectively released, and then superoxide anion radicals are generated under light.

[0149] Test Example 4

[0150] This test example studies the cytotoxicity of the compound, and the specific process is as follows:

[0151] First, a hypoxic cell model was established. The experimental method was to select A549 cells in the logarithmic growth phase and 4 Cells were seeded / well in 6-well plates and cultured in normoxia (21% O 2 ) at 37°C for 2 h. After the cells adhered to the wall, the cell culture plates were placed in normoxia (21% O 2 ) and hypoxia (1% O 2)Incubator for 12h or 24h. After removing the culture medium, wash three times with PBS, add 80μL of RIPA (Radio Immunoprecipitation Assay) lysis buffer containing protease inhibitors to each well, lyse on ice for 15min, mix the cells and lysis buffer thoroughly with a cell scraper, collect the supernatant, and determine the protein concentration by BCA method. An equal amount of protein sample (20μg) was subjected to SDS-PAGE and transferred to a carbonate cellulose membrane (NC membrane), blocked with 5% skim milk for 2h, and then incubated with HIF-1α primary antibody dilution at 4℃ for 8h, and then incubated with HRP-coupled secondary antibody dilution at room temperature for 2h. Use ECL immunoblotting detection reagent to observe immune reaction bands. (such as Fig.25 , ** indicates p<0.05)

[0152] The experimental results are as follows Fig.25 As shown, the establishment of the hypoxia model was verified by detecting the expression of HIF-1α (hypoxia inducible factor). When the cells were cultured in the hypoxia workstation, the expression of HIF-1α was significantly higher than that of the cells cultured in normoxic conditions, indicating that the hypoxia cell model was successfully constructed.

[0153] (1) Concentration gradient cytotoxicity experiment: Non-small cell lung cancer cells A549, breast cancer cells MCF-7, mouse breast cancer cells 4T1, human cervical cancer cells HeLa, and human embryonic kidney cells HEK293T were cultured at 5×10 3 / well were inoculated in a 96-well plate, cultured in normoxia for 2 hours, and then placed in a normoxia or hypoxia incubator for 24 hours after the cells adhered normally. The old culture medium was removed, and then the culture medium containing the prodrug compound 7 (final working concentrations were 0 μM, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM) was added. After continuing to culture for 4 hours, the drug-containing culture medium was removed, and new culture medium was added. The cells were irradiated with a 660 nm laser for 3 minutes (84 mw / cm 2 ), and then placed in the normoxic and hypoxic incubators for 20 hours, 10 μL of MTT (Sigma) was added to each well of the culture plate, and the plate was placed in the incubator for 4 hours, and then 100 μL of DMSO was added to each well, and the plate was shaken for 10 minutes, and then the absorbance at a wavelength of 570 nm was detected by an ELISA reader. Calculation of cell survival rate: Cell survival rate = OD 实验组 / OD 对照组 The experiment was repeated three times, and the data were processed using Graphad Prism 9.0. The cytotoxicity experiment of compound 8 in A549 cells was used as the prodrug control group. The experimental method was as described above.

[0154] (2) Hypoxic incubation time cytotoxicity experiment: A549 cells were selected as a representative. After being cultured overnight in normoxia, medium containing drugs was added and cultured for 6 h. The medium containing drugs was removed and new medium was added. Then, the cells were placed in normoxic and hypoxic incubators for 4 h, 6 h, and 12 h, respectively. The cells were irradiated with a 660 nm laser for 3 min (84 mw / cm 2 ), placed in a normoxic incubator for 20 h, and then added with MTT for detection.

[0155] (3) Illumination time cytotoxicity experiment: A549 cells were selected as a representative. After overnight normoxic culture, medium containing drugs was added, and then the plate with drugs was placed in a hypoxic incubator for 6 h. The medium containing drugs was removed, and new medium was added. The cells were irradiated with 660 nm laser for 1, 3, and 5 min (84 mw / cm 2 ), and then placed in a normoxic incubator for 20 h, after which MTT was added for detection.

[0156] The experimental results are as follows Fig.26 As shown, the prodrug provided by the present invention exhibits good hypoxia selectivity in a variety of tumor cells including lung cancer A549 cells, breast cancer MCF-7 cells, breast cancer 4T1 cells and cervical cancer HeLa cells, and has little toxicity to normal embryonic kidney cells HEK293T regardless of hypoxia or normoxia ( Fig.26 A), indicating that the hypoxic stress-responsive prodrug developed by the present invention has good tumor selectivity and safety. In addition, the cytotoxicity of the prodrug compound will increase with the hypoxic incubation time (e.g. Fig.26 B) and light exposure time ( Fig.26 C) increases, indicating that the prodrug prepared by the present invention has good hypoxia and light control properties. Fig.26 D is the result of the cytotoxicity test of compound 8 in A549 cells. Compared with compound 7 linked to the chemotherapy drug nitrogen mustard, the cell killing ability of compound 8 is smaller, indicating that the prodrug compound 7 linked to the photosensitizer and the chemotherapy drug has a stronger killing effect.

[0157] Test Example 5

[0158] This test example studies the drug release of the compound in cells. The specific process is as follows:

[0159] (1) Detection of prodrug release in cells using laser confocal microscopy

[0160] A549 cells in the logarithmic growth phase were selected and cultured at 3×10 4Cells / dish were seeded in confocal dishes. After the cells adhered to the wall, they were placed in normoxic or hypoxic incubators overnight. After the culture medium was removed, drug-containing culture medium (final concentration was 2 μM) was added and placed in normoxic or hypoxic incubators for 4 h. After the culture medium was removed, the cells were washed with PBS three times and the release of prodrug compounds in cells was observed using a laser confocal microscope (λ Ex =633nm,λ Em =647-759nm). Fig. 27 A)

[0161] (2) Detecting the release of prodrug in cells using flow cytometry:

[0162] A549 cells in the logarithmic growth phase were taken and 3×10 4 Cells / dish were seeded in 6-well plates, and after the cells adhered, they were placed in an anaerobic incubator for overnight culture for 12 hours. Then, 0, 0.0625, 0.125, 0.25, 0.5, and 1 μM of compound 7 were added in sequence, and the anaerobic culture was continued for 4 hours. The drug-containing medium was removed, and the cells were washed 3 times with PBS. The cells were digested with EDTA, the cell pellets were collected, and the cells were resuspended in 500 μL PBS and detected by flow cytometry.

[0163] ( Fig. 27 B)

[0164] (2) Release of prodrugs under different oxygen concentrations

[0165] A549 cells in the logarithmic growth phase were selected and cultured at 3×10 4 Cells / dish were seeded in 6-well plates, and the 6-well plates were placed in a normoxic incubator for 2 hours, and then placed in an incubator with 1%, 10%, and 20% oxygen concentrations for overnight culture. Drug-containing culture medium (final concentration 2μM) was added, and hypoxic culture was continued for 4 hours under different oxygen concentrations. The drug-containing culture medium was removed, and the cells were washed 3 times with PBS. The cells were digested with EDTA, the cell pellets were collected, and the cells were resuspended in 500μL PBS. The cells were detected using a cell flow cytometer. ( Fig. 27 C)

[0166] The experimental results are as follows Fig. 27 As shown: Compared with normoxic cells, prodrugs 7 and 8 have stronger fluorescence in hypoxic cells, indicating that under hypoxic conditions, the highly fluorescent original drugs can be effectively released ( Fig. 27 A). As the compound concentration increases, more original drug compound 1 is released and the fluorescence becomes stronger ( Fig. 27 B). The lower the oxygen concentration, the stronger the fluorescence, indicating that the lower the degree of hypoxia, the easier it is for compound 7 to be converted into prodrug compound 1 ( Fig. 27 C).

[0167] Test Example 6

[0168] This test example studies the generation of reactive oxygen species in cells under light exposure of the compound. The specific process is as follows:

[0169] A549 cells in the logarithmic growth phase were selected and cultured at 3×10 4 Cells / dish were planted in a confocal dish, cultured in a normoxic incubator for 2 hours, and then transferred to a hypoxic workstation for overnight culture. Culture medium containing 2 μM compound 7 was added, and culture was continued in the hypoxic workstation for 4 hours. The drug-containing culture medium was removed, and new culture medium was added, and the operation was performed according to the instructions of the reactive oxygen detection kit. Dichlorodihydrofluorescein-acetoacetate (DCFH-DA) staining solution was added. The cells were irradiated with a 660 nm laser for 0, 30, 60, and 120 s (84 mw / cm 2 ), and then placed in a hypoxic workstation for 20 min. The drug-containing culture medium was removed, washed three times with PBS, and then PBS was added. The release of active oxygen in the cells by compound 7 was detected using a confocal microscope (λ Ex =488nm,λ Em =493-630nm).

[0170] The experimental results are as follows Fig.28 As shown in the figure, A549 cells treated with hypoxia were incubated with compound 7 for a period of time, and the reactive oxygen detection probe DCFH-DA was added after illumination. Almost no green fluorescence was observed in the blank control group (MERGED) and the non-illumination treatment group (Bright), but as the illumination time increased, the green fluorescence in the cells gradually increased. This shows that compound 7 can produce reactive oxygen under hypoxic illumination conditions.

[0171] Test Example 7

[0172] This test example conducts a subcellular organelle co-localization experiment, and the specific process is as follows:

[0173] A549 cells in the logarithmic growth phase were selected and 5×10 4 Cells / dish were plated in 20 mm culture dishes, cultured in normoxia for 2 hours to adhere to the wall, and then placed in a hypoxic workstation for overnight culture. The culture medium was removed, and a culture medium containing 2 μM compound 7 was added, and incubated in hypoxia for 4 hours. After washing three times with PBS buffer, the lysosome probe (Lyso-tracker) or mitochondrial probe (Mito-tracker) was used to selectively stain the cell lysosomes and mitochondria, respectively, and the cell nucleus was stained with a nuclear dye (Hoechst33342). Finally, a laser confocal microscope was used to detect the localization and distribution of compound 7 in the cells (λ Ex1 =488nm,λ Em1 =493-630nm; Ex2 =633nm,λ Em2 =647-759nm).

[0174] The experimental results are as follows Fig.29 As shown, the fluorescence generated by the original drug of compound 7 released after cell hypoxic culture and the fluorescence generated by the lysosomal probe can overlap well, but overlaps less with the fluorescence generated by the mitochondrial probe, indicating that compound 7 is mainly enriched in lysosomes.

[0175] Example 4

[0176] This example detects the expression of autophagy proteins induced by prodrugs in cells, and the specific process is as follows:

[0177] A549 cells in the logarithmic growth phase were selected and 5×10 4 Cells / dish were inoculated in a 20 mm culture dish and adhered to the wall for 2 h under normoxia. Then, the dish was placed in a hypoxic workstation for overnight culture. After the culture medium was removed, new culture medium containing different concentrations of compound 7 was added and incubated for 4 h under hypoxia. After the drug-containing culture medium was removed, new culture medium was added. Except for the blank group, each dish was irradiated with 660 nm laser for 3 min (84 mw / cm 2 ). Wash the cells three times with PBS buffer solution, add 1mL4% paraformaldehyde to each dish and let it stand for 30min to fix the cells. After removing the paraformaldehyde, wash the cells three times with PBS, add 0.1% Triton X-100 solution and incubate for 10min. Remove excess Triton X-100, wash three times with PBS, add LAMP1 primary antibody (1:1000) in PBST buffer and incubate overnight at 4°C. Recover the primary antibody, wash three times with PBS, and then add the primary antibody's green fluorescent secondary antibody (1:5000) in PBST buffer and incubate at room temperature in the dark for 2h. Recover the fluorescent secondary antibody, wash three times with PBS, add the nuclear dye DAPI (20μg / mL) incubation in a dark environment, and finally seal the slices with anti-fluorescence attenuation sealing agent. Laser confocal microscopy was used to detect the expression of intracellular autophagy protein LAMP1 after treatment with compound 7 (λ Ex1 =488nm,λ Em1 =493-630nm; Ex2 =633nm,λ Em2 =647-759nm; λ Ex3 =405nm,λ Em3 =410-581nm).

[0178] The experimental results are as follows Fig.30 As shown, under hypoxic culture conditions, compound 7 can induce the expression of autophagy protein LAMP1 after illumination, and as the concentration of compound 7 increases, the green fluorescence after illumination gradually increases, indicating that the expression of LAMP1 protein gradually increases. Therefore, compound 7 can also kill cancer cells by inducing cell autophagy.

[0179] Example 5

[0180] This example conducts a cell apoptosis experiment, and the specific process is as follows:

[0181] A549 cells in the logarithmic growth phase were selected and cultured at 3×10 5 Cells / well were inoculated in 6-well plates, and then cultured in normoxic and hypoxic incubators for overnight culture. The old culture medium was removed, and culture medium containing different concentrations of compound 7 was added. The cells were then cultured in hypoxic workstations and normoxic incubators for 4 hours. After removing the drug-containing culture medium and adding new culture medium, the cells were irradiated with 660nm laser for 3min (84mw / cm 2 ), and then placed in hypoxic and normoxic incubators for 4 hours respectively. After digesting the cells with EDTA-free trypsin, collect the cells and supernatant into a 1.5mL centrifuge tube and centrifuge at 1000rpm for 5 minutes. After washing three times with PBS buffer, follow the operation of the cell apoptosis detection kit: resuspend the cells with 100μL binding solution, add 10μL Annexin V-FITC / PI kit staining buffer diluent and 5μL Annexin V-FITC, incubate on ice in the dark for 15 minutes, and then add 400μL binding solution and mix evenly. The samples were detected and analyzed by flow cytometry for the apoptosis caused by compound 7 under normoxic and hypoxic conditions.

[0182] The experimental results are as follows Fig.31 (where *** indicates p<0.001) As shown, the apoptosis rate of the control group was 2.71%, while the apoptosis rate after intervention with 0.25μM prodrug compound 7 under normoxic culture conditions and illumination was 7.96%, and the apoptosis rate after illumination under hypoxic culture was 17.91%; in addition, after intervention with 0.5μM prodrug compound 7, the apoptosis rate of normoxic illumination treatment was 12.88%, while the apoptosis rate of hypoxic illumination treatment was 41.63%, indicating that compound 7 is more likely to induce apoptosis in hypoxia than in normoxic conditions under the same treatment conditions, and is positively correlated with its concentration. Therefore, prodrug compound 7 can also kill tumor cells by inducing apoptosis.

[0183] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Compounds of formula I, and pharmaceutically acceptable salts and solvates thereof:

2. Compounds of formula II, and pharmaceutically acceptable salts and solvates thereof:

3. The method for preparing the compound of formula I or formula II according to claim 1, characterized in that: The following steps are involved: S1: diazotizing p-aminobenzyl alcohol and subjecting it to coupling reaction with N,N-dialkylaniline; and subjecting it to chlorination reaction to obtain an intermediate; S2: The intermediate is subjected to N-alkylation reaction with 2-methylquinolin-8-amine; and then cyclized with 2-thiosulfate-4-N,N-diethyl-p-phenylenediamine under the action of silver carbonate to obtain the product.

4. The preparation method according to claim 3, characterized in that: In S2, the reaction temperature of the N-alkylation reaction is 50-80°C and the reaction time is 10-20 hours; the reaction temperature of the cyclization reaction is 70-100°C and the reaction time is 2-6 hours.

5. The preparation method according to claim 3, characterized in that: In S1, when the N,N-dialkylaniline is N,N-dihydroxyethylaniline, the final product of S2 is a compound of formula I.

6. The preparation method according to claim 3, characterized in that: In S1, when the N,N-dialkylaniline is N,N-dimethylaniline, the final product of S2 is a compound of formula II.

7. A pharmaceutical composition comprising the compound of formula I according to claim 1 and / or its pharmaceutically acceptable salt or solvate.

8. Use of the compound of formula I according to claim 1 and / or the pharmaceutical composition according to claim 5 in the preparation of a drug for inhibiting cell proliferation.

9. Use of the compound of formula I according to claim 1 and / or the pharmaceutical composition according to claim 5 in the preparation of a tumor diagnostic agent and / or therapeutic agent.

10. The use according to claim 9, characterized in that: The tumor includes at least one of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, colon cancer, esophageal cancer, oral cancer, lymphoma, penile cancer, prostate cancer, skin cancer, gynecological cancer, gastrointestinal stromal tumor, head tumor, neck tumor, and eye tumor.

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