A hypoxia-responsive prodrug and its preparation method and application

Abstract

The present invention discloses a hypoxia-responsive prodrug and its preparation method and application. The hypoxia-responsive prodrug includes a compound of formula I, a pharmaceutically acceptable salt thereof, and a solvate thereof. The present invention uses an azo bond as a functional linker and a hypoxia-responsive group to temporarily block the fluorescence and biological activity of a photosensitizer. Under hypoxic conditions, the prodrug can selectively break and release the original photosensitizer and chemotherapy drug, thereby achieving "off-on" fluorescence and biological activity in response to hypoxia stress, thereby improving selectivity for tumor cells. The "off-on" fluorescence properties of the prodrug are expected to be used for tumor diagnosis and efficacy monitoring, and are expected to achieve integrated diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine 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 cell proliferation and differentiation under the influence of genetic and environmental factors. Currently, cancer is one of the most serious health issues affecting people's well-being worldwide, killing millions of people each year. The main characteristic of cancer is the unlimited proliferation and division of cancer cells, forming tumors and invading surrounding tissues and organs. Early diagnosis and treatment have greatly improved cancer survival and cure rates. However, current cancer treatment still faces significant challenges, including poor treatment selectivity and side effects, which often affect patients' quality of life and treatment effectiveness.

[0003] Hypoxia is a key physiological characteristic of many different solid tumors and a key driver of malignancy. In recent years, it has been recognized as an important target for cancer therapy. The sensitization of hypoxic cells in tumors and the improvement of the hypoxic tumor microenvironment have always been hot topics 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 target hypoxia to release therapeutic drugs and improve the hypoxic microenvironment, such as using hypoxia-activated prodrugs for tumor-targeted therapy. After the prodrug is enriched in the hypoxic area, it releases the active prodrug under the stress of the hypoxic microenvironment, thereby selectively killing adjacent hypoxic tumor cells. Therefore, the design and synthesis of hypoxia-responsive anti-tumor prodrugs, and then the selective release of the prodrug in the hypoxic tumor microenvironment, is expected to improve the bioavailability and selectivity of the drug, reduce the potential toxic side effects of the prodrug, and improve the anti-tumor efficacy.

[0004] Photodynamic therapy (PDT) is a method of selectively irradiating photosensitizers (PS) that accumulate 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 PDT is currently clinically approved in numerous countries worldwide, primarily for the treatment of neoplastic diseases. However, the photosensitizers currently in clinical use are primarily macrocyclic molecules based on tetrapyrrole rings. These complex molecular structures are challenging to synthesize, and the "always on" nature of traditional photosensitizers makes them less selective, inevitably damaging normal cells while killing tumor cells. Summary of the Invention

[0005] The present invention aims to address at least one of the aforementioned technical problems in the prior art. To this end, the present invention provides a hypoxia-responsive prodrug, its preparation method, and its use. The antitumor activity and fluorescence properties of this tumor hypoxia-responsive prodrug are essentially hidden and quenched under normoxic conditions, but can selectively release a photosensitizer and chemical anticancer drug under hypoxic stress, potentially reducing potential adverse side effects of the original drug and improving therapeutic efficacy. Furthermore, under hypoxic stress, the fluorescence properties of the photosensitizer are restored, potentially enabling early diagnosis of hypoxic tumors and therapeutic efficacy monitoring.

[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 coupling it with N,N-dialkylaniline; then chlorinating it to obtain an intermediate;

[0015] S2: The intermediate is reacted with 2-methylquinolin-8-amine through N-alkylation reaction; 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 is carried out using POCl3; the reaction temperature is 20-100°C.

[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 50-80° C.; and the reaction time is 10-20 h.

[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 a pharmaceutically acceptable salt or solvate thereof.

[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 as described herein, wherein the target cell typically undergoes 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 600 nm to 900 nm (e.g., about 600 nm to 700 nm), wherein the malignant tumor cell or the infectious bacterial cell typically causes a hypoxic microenvironment, and selectively releasing the photosensitizer compound 1 by the compound of Formula I activated by light and in the hypoxic microenvironment. and nitrogen mustard chemotherapy drugs, compound 1 generates cytotoxic free radicals / radical ions or singlet oxygen 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 cells are tumor cells or cancer cells. In some embodiments, the target cells are bacterial cells, which can be Gram-positive bacteria or Gram-negative bacteria, such as Escherichia coli or Staphylococcus aureus. In some embodiments, the target cells are in the body of a subject, such as a human or animal subject. Therefore, the methods described in the present invention effectively provide a treatment 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 a tumor diagnostic agent and / or therapeutic agent.

[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 can 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 medicaments 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 E.W. 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. Preparations administered by inhalation (e.g., aerosols) or for oral, rectal, or vaginal administration are also contemplated.

[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 aid passage through the host's skin. For example, a transdermal device is in the form of a bandage comprising a backing member, a reservoir containing the compound and an optional carrier, an optional rate-controlling barrier for delivering the compound to the host's skin at a controlled and predetermined rate over an extended period of time, and a device for securing the device to the skin. Matrix transdermal formulations can also be used.

[0034] For oral administration, the pharmaceutical composition or medicament can 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 oil, 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 desired, (d) disintegrants, for example, starch (for example, potato starch or sodium starch glycolate), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, for example, sodium lauryl sulfate and / or (f) absorbents, colorants, flavorings 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 optionally contain buffer salts, flavorings, coloring agents, and / or sweeteners. If desired, the preparation for oral administration can be suitably formulated to give a 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 for regulating osmotic pressure and / or buffers. Alternatively, the active ingredient can be in powder form and is composed of a suitable medium, such as sterilized pyrogen-free water, before use. In addition, they can also contain other substances of therapeutic value. The composition is prepared according to conventional mixing, granulation or coating methods, and contains about 0.1% to 75% (mass fraction) of active ingredient, preferably about 1% to 50% (mass fraction) of active ingredient.

[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 pressurized aerosols, the dosage unit can 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 can 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 formulation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. For example, the compound of formula I can be formulated with a suitable polymeric material or hydrophobic material (e.g., in an acceptable oil as an emulsion) or an ion exchange resin, or as a slightly soluble derivative, e.g., as a slightly soluble salt.

[0040] The pharmaceutical compositions or medicaments of the present invention comprise (i) an effective amount of a compound of Formula I as described herein that acts as an effective photosensitizer, and (ii) another therapeutic agent. When used with a compound of the present invention, such therapeutic agent 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 can be administered together in the same pharmaceutical formulation.

[0041] dose

[0042] The pharmaceutical composition or drug can be administered to a subject in 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 elicit an effective therapeutic response in the subject.

[0043] The dosage of the active agent administered depends on the subject's weight, age, individual condition, surface area or volume of the area to be treated, and the form of administration. The size of the dosage will also be determined by the presence, nature, and extent of any side effects associated with the administration of a particular compound in a particular subject. For example, each compound in the compounds of Formula I described herein can have a unique dosage. A unit dose for oral administration to a mammal of about 50kg to 70kg can contain about 5mg to 500mg of the compound. Typically, the dosage of the compounds of Formula I of the present invention is a dosage sufficient to achieve the desired effect. The optimal dosage regimen can be calculated by the measured values ​​of the reagent accumulated in the subject's body. Typically, the dosage can be administered once or multiple times daily, weekly, or monthly. One skilled in the art can easily determine the optimal dosage, administration methodology, and repetition frequency.

[0044] In order to achieve the desired therapeutic effect, the compound of formula I can be administered for many days with a daily dose that is effective for the treatment of. Therefore, the effective administration of the treatment of the compound requires periodic (for example, every day) administration, and continues for three days to two weeks or longer cycles to treat the associated 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 dose is the preferred approach to achieve a therapeutically effective dose, even if the compound is not administered every day, as long as repeated administration is frequently performed to maintain a therapeutically effective concentration of the compound in the object, then therapeutic beneficial effects can be achieved. For example, reagents can be administered every other day or every three days, or if a higher dosage range is adopted and tolerated by the object, reagents can be administered once a week.

[0045] Optimal dosages, toxicity, and therapeutic effects of compounds may vary depending on the relative potency of 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 all deaths) 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 Certain 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 in order 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 lies within the range that includes the ED 50 The dose 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 initially estimated from cell culture assays. The dose can be formulated in animal models to achieve the IC values ​​determined in cell culture. 50 The range of circulating plasma concentrations of the compound that achieves half-maximal inhibition of symptoms can be calculated. 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 can be 0.1-0.5 mg / eye. The compound can be administered orally at 5 mg-1000 mg, or by intravenous infusion at 10 mg / mL-500 mg / mL. In an alternative, the compound can be administered by intravenous injection or infusion at 50 mg / mL-500 mg / mL (over 120 minutes); 1 mg / kg-500 mg / kg (over 60 minutes); or 1 mg / kg-100 mg / kg (push) five times a week. The compound of Formula I can 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. Other therapeutic compounds can be administered simultaneously with the main active ingredient (for example, the compound of formula I of the present invention), or even administered together with the main active ingredient in the same composition. Other therapeutic compounds can also be administered individually, in a separate composition, or in a dosage form different from the main active ingredient. The main ingredient of some dosages, for example the compound of formula I as described herein, can be administered simultaneously with other therapeutic compounds, while other dosages 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 generally performed by those skilled in the art.

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

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

[0052] (2) The "Always ON" property of traditional photosensitizers makes them less selective, and while killing tumor cells, they will inevitably damage normal cells. The present invention uses an azo bond as a functional linker and hypoxia-responsive group to temporarily block the fluorescence and biological activity of the photosensitizer. Under hypoxia, the original photosensitizer and chemotherapy drugs can be selectively released, achieving "OFF-ON" fluorescence and biological activity in response to hypoxia stress, thereby improving the selectivity for tumor cells. 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 and does not require very strict experimental conditions and operation requirements, which is conducive to scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 For intermediate 3 1 H NM (400 MHz, CDCl3);

[0055] Figure 2 For intermediate 3 13 C NMR (101 MHz,CDCl3);

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

[0057] Figure 4 For intermediate 4 13 C NMR (101 MHz, CDCl3);

[0058] Figure 5 For intermediate 5 1 H NMR (400 MHz, CDCl3);

[0059] Figure 6 For intermediate 5 13C NMR (101 MHz, CDCl3);

[0060] Figure 7 For compound 7 1 H NMR (500 MHz, CDCl3);

[0061] Figure 8 For compound 7 13 C NMR (126 MHz, CDCl3);

[0062] Figure 9 For intermediate 3a 1 H NMR (400 MHz, CDCl3);

[0063] Figure 10 For intermediate 3a 13 C NMR (101 MHz, CDCl3);

[0064] Figure 11 For intermediate 4a 1 H NMR (400 MHz, CDCl3);

[0065] Figure 12 For intermediate 4a 13 C NMR (101 MHz, CDCl3);

[0066] Figure 13 For intermediate 5a 1 H NMR (400 MHz, CDCl3);

[0067] Figure 14 For intermediate 5a 13 C NMR (101 MHz, CDCl3);

[0068] Figure 15 For compound 8 1 H NMR (400 MHz, CDCl3);

[0069] Figure 16 For compound 8 13 C NMR (101 MHz, CDCl3);

[0070] Figure 17 For compound 1 1 H NMR (600 MHz, CDCl3);

[0071] Figure 18A, B, and C are the UV absorption spectra of compounds 1, 7, and 8 in different solvents, respectively; D is the fluorescence spectra of compounds 1, 7, and 8 in H2O:MeCN (1:1);

[0072] Figure 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] Figure 20 A is the fluorescence value of compound 7 at 680 nm (excitation wavelength λex = 625 nm) in the presence of different ions, active substances and bioreductants; B is the fluorescence spectrum of compound 7 incubated with liver microsomes and NADPH for different lengths of time

[0074] Figure 21 HPLC profiles of compounds 7 and 8 after incubation with different concentrations of Na2S2O4;

[0075] Figure 22 The fluorescence spectra of compounds 7 and 8 after incubation with different concentrations of Na2S2O4;

[0076] Figure 23 The test results of the active oxygen generation performance of the compounds; A is the singlet oxygen probe DPBF detection of active oxygen generation in different groups; B is the image of compounds 1, 7, 8, 7 + Na2S2O4 and 8 + Na2S2O4 in acetonitrile aqueous solution (1:1) under visible light;

[0077] Figure 24 Figure A shows the production of reactive oxygen species in different groups detected by the singlet oxygen probe SOSG (fluorescence value at 530 nm); Figure B shows the production of superoxide anions in different groups detected by the superoxide anion fluorescent probe DHR123 (fluorescence value at 530 nm);

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

[0079] Figure 26Figures 7 and 8 show the cytotoxicity test results of compounds 7 and 8; Figure A shows the cytotoxicity test results of compound 7 on different tumor cells (A549, MCF-7, and Hela cells) and normal Hek 293T cells under normoxic and hypoxic conditions; Figure B shows the cytotoxicity test results of compound 8 at different concentrations incubated with A549 cells under normoxic and hypoxic conditions for different incubation times; Figure C shows the cytotoxicity test results of compound 7 incubated with A549 cells under hypoxic conditions for 6 hours and then irradiated with different light for different times; Figure D shows the cytotoxicity test results of compound 8 on A549 lung cancer cells under normoxic and hypoxic conditions;

[0080] Figure 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] Figure 28 The results of compound 7 producing reactive oxygen species in cells are shown in the figure. Scale bar: 50 μm.

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

[0083] Figure 30 The experimental results of compound 7 inducing LAMP1 protein expression in cells, scale: 5 μm;

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

[0085] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0086] Example 1

[0087] In this example, compound 7 (compound of formula I) was prepared by the following process:

[0088]

[0089] S1: Preparation of intermediate 3:

[0090] A 100 mL round-bottom flask was charged with compound 2 (500.0 mg, 4.06 mmol) and a 10 mol / L aqueous HCl solution (7 mL). The mixture was stirred in an ice-water bath until compound 2 was completely dissolved. Subsequently, 1.5 mL of sodium nitrite solution (560.0 mg, 8.13 mmol, NaNO₂) was slowly added dropwise to the reaction mixture, and the mixture was stirred at 0°C for 2 h. Saturated aqueous sodium acetate was then added to the reaction mixture to adjust the pH to approximately 2. After the reaction was continued for 0.5 h, urea (365.0 mg, 6.09 mmol) and a solution of N,N-dihydroxyethylaniline in acetic acid (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 completion of the reaction, as monitored by TLC, saturated aqueous sodium bicarbonate (NaHCO₃) was added to the mixture until the pH was approximately 7, and the crude product was extracted with ethyl acetate (EA: 25 mL × 3). The combined organic layers were washed with saturated sodium chloride solution (NaCl: 30 mL × 2) and dried over anhydrous sodium sulfate (Na2SO4), 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) δ 7.82 (d, J = 9.2 Hz, 2H), 7.79 (d, J =8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 9.2 Hz, 2H), 4.68 (s, 2H), 3.81 (t, J = 5.9 Hz, 4H), 3.68 (t, J = 5.9 Hz, 4H) ppm. 13 C NMR (101 MHz, DMSO- d 6) δ 151.87, 151.30, 144.53, 142.75, 127.58, 125.22, 122.07, 111.77, 63.04, 58.61,53.72, 39.82 ppm. LC-MS: m / z 316.32 [M+H] + . Figure 1 For intermediate 3 1 H NMR spectrum, Figure 2 For intermediate 3 13C 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 (POCl₃, 5 mL) was gradually added dropwise. The mixture was then stirred and heated to 85°C under reflux for 5 h. After completion of the reaction, monitored by TLC, the round-bottom flask was placed in an ice-water bath and saturated sodium carbonate (Na₂CO₃) solution was slowly poured into the flask to neutralize the solution to a pH of approximately 7. The crude product was then extracted with ethyl acetate (EA: 25 mL × 3). The combined organic layers were washed with saturated sodium chloride solution (NaCl: 30 mL × 2) and dried over anhydrous sodium sulfate (Na₂SO₄). The crude product was purified by column chromatography to yield compound 4 (470.0 mg, 40%) as an orange solid. 1 H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 4.65 (s, 2H), 3.84 (t, J = 6.8 Hz, 4H), 3.70 (t, J = 6.7 Hz, 4H) ppm. 13 C NMR (101 MHz, CDCl3) δ 152.82, 148.78, 144.60, 139.00, 129.45, 125.51, 122.80, 111.70, 53.48, 46.03,40.38 ppm. 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] A 100 mL round-bottom flask was charged with compound 4 (360.0 mg, 973.43 μmol), 2-methylquinolin-8-amine (140 mg, 884.93 μmol), potassium carbonate (K2CO3, 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, after which 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). The combined organic phases were washed with saturated ammonium chloride solution (NH4Cl: 30 mL × 2) and sodium chloride solution (NaCl: 30 mL × 2), respectively, and dried over anhydrous sodium sulfate (Na2SO4). After removal of the solvent, the residue was purified by column chromatography to yield the orange compound 5 (295.0 mg, 68%). 1 H NMR (400 MHz, CDCl3) δ 7.94-7.82 (m, 5H), 7.53 (d, J = 8.3 Hz, 2H), 7.27-7.19 (m, 2H), 7.01 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 9.1 Hz, 2H), 6.59 (d, J = 7.6Hz, 1H), 4.59 (s, 2H), 3.71 (t, J = 6.9 Hz, 4H), 3.59 (t, J = 6.8 Hz, 4H), 2.69(s, 3H) ppm. 13 C NMR (101 MHz,CDCl3) δ 155.86, 152.31, 148.47, 144.73, 144.00,141.72, 136.24, 127.97, 126.79, 126.70, 125.31, 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 (Formula I)

[0097] To a 50 mL double-necked round-bottom flask were added compound 5 (270.0 mg, 548 μmol), compound 6 (227.0 mg, 822 μmol), and a mixture of methanol and ethyl acetate (6 mL, MeOH:EA = 4:1). Silver carbonate (AgCO3, 302.0 mg, 1.10 mmol) was added slowly in portions. The reaction mixture was refluxed at 85°C for 4 h. The reaction mixture was then cooled to room temperature and filtered through Celite. The Celite filter was washed with a mixture of methanol and dichloromethane until the filtrate was colorless. The concentrated organic layer was purified by column chromatography to afford a dark green solid. The solid was redissolved in 5 mL of dichloromethane and acidified with 0.25 mL of methanolic hydrochloric acid (HCl: 1 mol / L in MeOH). The mixture was then concentrated to afford compound 7 (168.0 mg, 45%) as a dark green solid. 1 H NMR (500 MHz, Methanol- d 4) δ 9.21 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 9.6 Hz,1H), 7.87-7.77 (m, 5H), 7.59 (d, J = 8.3 Hz, 2H), 7.48 (dd, J = 9.6, 2.5 Hz, 1H),7.40 (s, 1H), 7.33 (d, J = 2.5 Hz, 1H), 6.87 (d, J = 9.2 Hz, 2H), 5.02 (s, 2H), 3.88 (t, J = 6.8 Hz, 4H), 3.73 (dt, J = 18.0, 6.8 Hz, 8H), 2.83 (s, 3H), 1.33 (t, J = 7.1 Hz, 6H) ppm. 13 C NMR (101 MHz, 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, 11.72 ppm. HRMS (ESI): calcd for C 37 H 39 Cl2N7SCl ([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) δ 7.77(dd, J = 14.8, 8.6 Hz, 4H), 7.46 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 4.58 (s, 2H), 3.05 (s, 6H) ppm. 13 C NMR (101 MHz, DMSO- d 6) δ 152.89, 151.84,144.71, 143.05, 127.55, 112.03, 63.04, 39.95 ppm. LC-MS: m / z 256.69 [M+H] + . Figure 9 For intermediate 3a1 H NMR spectrum, Figure 10 For intermediate 3a 13 C NMR spectrum proved that the compound was successfully prepared.

[0103] S2: Preparation of compound 4a

[0104] The preparation of compound 4a refers to the synthetic steps of compound 4. 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J =9.0 Hz, 2H), 7.83(d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 9.1Hz, 2H), 4.64 (s, 2H), 3.0952(s, 6H) ppm. 13 C NMR (101 MHz, CDCl3) δ 153.00,152.60, 143.62, 138.36, 129.32,125.20, 122.52, 111.58, 46.02, 40.36 ppm. LC-MS: m / z 274.37 [M+H]+. Figure 11 For intermediate 4a 1 H NMR spectrum, Figure 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 synthetic steps of compound 5. 1 H NMR (400 MHz, CDCl3) δ 7.94 (d, J =8.4 Hz, 1H), 7.84 (dd, J = 19.4, 8.7 Hz, 4H), 7.53 (d, J = 8.3 Hz, 2H), 7.24 (dt, J = 7.4, 3.7 Hz, 2H), 7.01 (d, J = 7.9 Hz, 1H), 6.74 (d, J = 9.1 Hz, 2H), 6.60 (d,J = 7.5 Hz, 1H), 4.62 (s, 2H), 3.06 (s, 6H), 2.70 (s, 3H) ppm. 13 C NMR (101 MHz,CDCl3) δ 155.78, 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, 25.21 ppm. LC-MS: m / z 396.66. Figure 13 For intermediate 5a 1 H NMR spectrum, Figure 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) δ 8.98(d, J = 8.3 Hz, 1H), 7.91 (d, J = 9.5 Hz, 1H), 7.71 (dd, J = 23.8, 8.3 Hz, 5H),7.53 (d, J = 7.8 Hz, 2H), 7.37 (d, J = 9.0 Hz, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 6.69 (d, J = 8.8 Hz, 2H), 4.89 (s, 2H), 3.72-3.64 (m, 4H), 3.02 (s, 6H), 2.74(s, 3H), 1.32 (t, J = 6.9 Hz, 6H) ppm. 13 C NMR (101 MHz, 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, 124.63, 122.26, 118.22, 111.06, 104.94, 101.84, 47.60, 45.72, 38.94,23.56, 11.79 ppm. HRMS (ESI): calcd for C 35 H 37 N7SCl ([M-Cl] + ): 586.2747, found586.2788. Figure 15 Compound 8 1 H NMR spectrum, Figure 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 by the following process:

[0111]

[0112] A 50 mL two-necked round-bottom flask was charged with compound 6 (140.0 mg, 0.5 mmol), 2-methylquinolin-8-amine (40.4 mg, 0.25 mmol), and a mixture of methanol and ethyl acetate (6 mL, MeOH:EA = 4:1) and refluxed at 85°C. Silver carbonate (Ag2CO3, 140.0 mg, 0.5 mmol) was then added portionwise. After reflux for 2 h, the reaction mixture was cooled to room temperature, filtered through celite, and washed with a mixture of methanol and dichloromethane (DCM) until the filtrate was colorless. The organic layer was concentrated to yield a blue solid, which was redissolved in 5 mL of dichloromethane and acidified with 0.25 mL of methanolic hydrochloric acid (HCl: 1 mol / L in methanol). The mixture was concentrated to yield compound 1 (31.1 mg, 32%) as a dark blue solid. 1 H NMR (600 MHz,CDCl3) δ 8.98 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 9.3 Hz, 1H), 7.83 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.16 (dd, J= 9.4, 2.6 Hz, 1H), 6.82 (d, J = 2.6 Hz, 1H), 3.62(q, J = 7.2 Hz, 4H), 2.74 (s, 3H), 1.36 (t, J = 7.0 Hz, 6H) m / z 391.19. Figure 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. The compounds were diluted to 10 μM using MeCN, DCM, DMSO, EG, EtOH, and H2O:MeCN (v / v=1:1) solvents and then detected by UV-visible spectrophotometry ( Figure 18 A, B, C).

[0117] The experimental results are as follows Figure 18 As shown, compounds 1, 7 and 8 basically have strong spectral absorption peaks at 660 nm in different solvents. However, in H2O:CH3CN solution, the absorption intensity of the compounds becomes weaker and the absorption peak becomes broader, especially for prodrug compound 7 and control compound 8. This may be due to the influence of strong hydrogen bonding in H2O:MeCN solution.

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

[0119] Fluorescence emission spectrum detection: The mother solutions of compounds 1, 7 and 8 were diluted to 10 μM with H2O:MeCN (1:1), and their fluorescence emission spectra were detected by fluorescence spectrophotometer. Ex =625 nm, λ Em =650~900 nm.

[0120] The experimental results are as follows Figure 18 As shown in D: Compound 1 has the highest peak at 680 nm 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 of the compound (λ Ex =625 nm, λ Em =650~900 nm)( Figure 19 A, B, C).

[0123] B. pH stability: The compound was dissolved in deionized water at pH 3, 4, 5, 6, 7, 8, 9, and 10 to a final concentration of 10 μM. The fluorescence spectra were measured using a fluorescence spectrophotometer (λ Ex =625 nm, λ Em =650-900 nm)( Figure 19 Middle D).

[0124] The experimental results are as follows Figure 19 As shown, the fluorescence of compound 1 gradually decreases under continuous illumination, indicating that the compound will gradually undergo photobleaching with prolonged illumination. However, the fluorescence of compounds 7 and 8 remains essentially unchanged at different illumination times, indicating that the modified prodrug 7 and the control compound 8 are more photostable and do not undergo photobleaching. In addition, the fluorescence of compounds 1, 7, and 8 remains essentially unchanged under different pH conditions, indicating that the compounds have excellent pH stability and do not undergo acidic or alkaline decomposition.

[0125] Test Example 2

[0126] This test example studies the in vitro release performance of the compound. 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 10 mM): Ca 2+ 、Cu 2+ , K + Mg 2+ 、Na + 、Zn 2+ .

[0130] 2) Active oxygen / active sulfur / active nitrogen and other active substances group (active substance concentration is 100 μM): ClO - 、H2O2、HS - 、NO2 - .

[0131] 3) Amino acid and bioreductant group (concentration of 1 mM): GSH, VC, Ala, Glu, Arg, Pro, glucose, Na2S2O4.

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

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

[0134] Experimental method: The DMSO stock solution (10 mM) of the prodrug compound 7 was diluted with H2O:MeCN (1:1) to a final concentration of 10 μM. Liver microsomal enzymes (final concentration of 0.5 mg / mL) and NADPH (final concentration of 500 μM) were added and incubated at 37°C with shaking for different times. The drug release performance of compound 7 under the action of liver microsomal enzymes was then measured using a fluorescence spectrophotometer. (As shown in Figure 2). Figure 20 Middle B)

[0135] The experimental results are as follows Figure 20 Prodrug compound 7 showed little response to various metal ions, reactive oxygen species, reactive sulfur species, reactive nitrogen species, amino acids, and bioreductants. However, under the action of the azoreductase chemical mimetic Na2S2O4, it was able to release strong fluorescence in response to stress, with the fluorescence value reaching as high as 648, achieving a fluorescence transition from "OFF" to "ON". This indicates that prodrug compound 7 can effectively release the original drug under reducing conditions and has good hypoxia response specificity. In addition, further verification of prodrug compound 7 under the action of azoreductase liver microsomes showed that the fluorescence intensity gradually increased with increasing incubation time. After 24 hours of incubation, the fluorescence value increased from 58 at 0 hours 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) Concentration response characteristics of prodrug compounds 7 and 8 to the azoreductase chemical mimetic Na2S2O4

[0137] Method 1: The mother solution of compound 7 or 8 was diluted with deionized water to an aqueous solution with a final concentration of 25 μM. Freshly prepared Na2S2O4 solution (mother solution concentration was 1 M) was then added to the solution to prepare mixtures of different concentration ratios. The mixtures were then incubated at 37°C for 1 h and detected by HPLC. HPLC analysis conditions: High performance liquid chromatograph: Shimadzu LC-20A, column: Athena (C18-WP 100A, 4.6 × 250 mm, 5 μM), detector: spd20a, detection band: 254 nm, flow rate: 1 mL / min. Mobile phase composition (phase A: acetonitrile containing 0.1% trifluoroacetic acid; phase B: water containing 0.1% trifluoroacetic acid); gradient elution conditions were 40-100% acetonitrile (containing 0.1% trifluoroacetic acid), and the elution time was 30 min. (e.g. Figure 21 )

[0138] The experimental results are as follows Figure 21 As shown in the figure, after the prodrug compound is co-incubated with Na2S2O4, a new peak will be generated in the mixed solution, and its retention time is consistent with the retention time of the original drug peak 1, indicating that the prodrug compound 7 or 8 will release the original drug after co-incubation with Na2S2O4, and with the increase of Na2S2O4 concentration, the release of the original drug will gradually increase within the same incubation time, indicating that the release of prodrug compounds 7 and 8 and the azoreductase chemical mimetic Na2S2O4 show a good concentration dependence.

[0139] Method 2: Prepare a stock solution of compound 7 or 8 with deionized water to a final concentration of 25 μM. Add a freshly prepared aqueous solution of Na2S2O4 (stock solution concentration is 1 M) to each solution to increase the final concentration of Na2S2O4 in the mixture from 0 to 2 mM. Incubate at 37°C for 1 h. Detect the fluorescence spectrum of the mixture after co-incubation using a fluorescence spectrophotometer. Ex =625 nm, λ Em =650-900 nm. (e.g. Figure 22 )

[0140] The experimental results are shown in Figure 22 As shown in the figure, with the increase of Na2S2O4 concentration in the reaction system, 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 showed that the release of the original drug increased with the increase of Na2S2O4 concentration, which was 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. 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 water solution; single-drug compound 7 treatment group: 10 μM compound 7 + 40 μM DPBF in acetonitrile water solution; single-drug compound 8 treatment group: 10 μM compound 8 + 40 μM DPBF in acetonitrile water solution; compound 7 / 8 and Na2S2O4 mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na2S2O4 + 40 μM DPBF in acetonitrile water solution; and 10 μM compound 8 + 800 μM Na2S2O4 + 40 μM DPBF acetonitrile aqueous solution. The above solutions were respectively irradiated with 660 nm laser (84 mw / cm 2 ) After irradiation for 0 s, 5 s, 10 s, 20 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s, the absorbance of the compound at 410 nm was measured. (For example Figure 23 Middle A)

[0144] The experimental results are as follows Figure 23 Figure A shows that the original drug compound 1 can effectively generate singlet oxygen under light, while the simple prodrug compounds 7 and 8 basically do not generate singlet oxygen under light. However, when co-incubated with Na2S2O4, they can effectively generate reactive oxygen species under light, and as the light exposure time increases, the generation of reactive oxygen species gradually increases, indicating that the singlet oxygen generation performance of the prodrug compounds 7 and 8 is also effectively quenched, and can be effectively activated by the reducing agent Na2S2O4 to regenerate singlet oxygen. Figure 23 B in the figure is the color of the acetonitrile aqueous solution (water:acetonitrile, 1:1) of compounds 1, 7, and 8, as well as the color of the acetonitrile aqueous solution of 7 and 8 after incubation with Na2S2O4, further indicating that compounds 7 and 8 were reduced to generate compound 1 after incubation with Na2S2O4.

[0145] (2) 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 Na2S2O4 mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na2S2O4 + 3 μM SOSG in acetonitrile aqueous solution; and 10 μM compound 8 + 800 μM Na2S2O4 + 10 μM SOSG in acetonitrile aqueous solution. Then, the cells were illuminated with a 660 nm laser (84 mw / cm 2 ) were irradiated for 0, 30, 60, 120, 180, 300 and 480 s, and the fluorescence emission spectrum of the compound was detected by fluorescence spectrophotometer. Ex =488 nm, λ Em =530 nm. (e.g. Figure 24 Middle A)

[0146] The experimental results are as follows Figure 24 As shown in Figure A, the original drug compound 1 can effectively generate singlet oxygen under light (fluorescence value 893.8), while the prodrug compound 7 (fluorescence value 34.11) and the control compound 8 (fluorescence value 25.18) can basically not generate singlet oxygen. However, when co-incubated with Na2S2O4 respectively, they can also effectively generate reactive oxygen species under light (fluorescence value 646.10 for compound 7 and fluorescence value 609.9 for compound 8), which is basically consistent with the results detected by DPBF and UV spectrophotometer, further proving that the prodrug compound 7 and the control compound 8 have good hypoxia response properties and can effectively release reactive oxygen species under light and hypoxia stress response.

[0147] (3) The superoxide anion fluorescent probe DHR123 (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 (all final concentrations below) 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 Na2S2O4 mixed solution treatment group (concentration ratio 1:80, pre-incubated at 37°C for 1 h): 10 μM compound 7 + 800 μM Na2S2O4 + 5 μM DHR123 in acetonitrile aqueous solution; and 10 μM compound 8 + 800 μM Na2S2O4 + 5 μM DHR123 in acetonitrile aqueous solution. Then, a 660 nm laser (84 mw / cm 2 ) were irradiated for 0, 10, 30, 60, 120 and 180 s, and the fluorescence emission spectrum of the compound was detected by fluorescence spectrophotometer. Ex =488 nm, λ Em =530 nm. (e.g. Figure 24 Middle B)

[0148] The experimental results are as follows Figure 24 As shown in Figure B, the original drug compound 1 can effectively generate superoxide anion radicals under light, producing highly 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 radicals), and Figure 23 China A and Figure 24 A in Figure 1 indicates that compound 1 has a Type II mechanism of action (generating singlet oxygen). This combination of Type I and Type II mechanisms of action indicates that compound 1 possesses excellent photodynamic properties and is an excellent photosensitizer. Similarly, the superoxide anion radical production ability of the prodrug compound 7 (fluorescence value 427.2) and the control compound 8 (fluorescence value 223.8) can be effectively and temporarily quenched. However, upon co-incubation with the chemical reducing agent Na2S2O4 (fluorescence value 4344 for compound 7 and 3635 for compound 8), the original drug compound 1 can be effectively released, further generating superoxide anion radicals under light.

[0149] Test Example 4

[0150] This test case studies the cytotoxicity of the compound. The specific process is as follows:

[0151] First, establish the hypoxic cell model. Experimental method: select A549 cells in the logarithmic growth phase and 4 Cells / well were seeded in a 6-well plate and cultured at 37°C under normoxia (21% O2) for 2 h. After the cells attached, the cell culture plates were placed in normoxia (21% O2) and hypoxia (1% O2) incubators for 12 h or 24 h, respectively. After removing the culture medium, wash with PBS three times, add 80 μL of RIPA (Radio Immunoprecipitation Assay) lysis buffer containing protease inhibitors to each well, lyse on ice for 15 min, use a cell scraper to thoroughly mix the cells and lysis buffer, collect the supernatant, and determine the protein concentration by BCA method. Equal amounts of protein samples (20 μg) were subjected to SDS-PAGE and transferred to a carbonate cellulose membrane (NC membrane), blocked with 5% skim milk for 2 h, and then incubated with HIF-1α primary antibody dilution at 4 °C for 8 h, and then incubated with HRP-conjugated secondary antibody dilution at room temperature for 2 h. Immunoreactive bands were observed using ECL immunoblotting detection reagent. (As shown in Figure 2) Figure 25 , ** indicates p < 0.05)

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

[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 a concentration gradient of 5×10 3 Cells were seeded at a density of 100 μg / well in a 96-well plate and cultured in normoxia for 2 h. After normal cell attachment, cells were placed in a normoxia or hypoxia incubator for 24 h. The old culture medium was removed, and then culture medium containing prodrug compound 7 (final working concentrations of 0 μM, 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM) was added. After a further 4 h of culture, the drug-containing culture medium was removed and fresh culture medium was added. The cells were then irradiated with a 660 nm laser for 3 min (84 mw / cm 2 ), placed in normoxic and hypoxic incubators for another 20 h, then added 10 μL of MTT (Sigma) to each well of the culture plate. The plates were incubated in an incubator for 4 h, followed by 100 μL of DMSO per well. The plates were shaken for 10 min, and the absorbance at 570 nm was measured using a microplate reader. Calculate cell viability: Cell viability = 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 test of compound 8 in A549 cells was performed as the prodrug control group. The experimental method was as described above.

[0154] (2) Hypoxia incubation time cytotoxicity experiment: A549 cells were selected as representatives. 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. The cells were then placed in normoxia and hypoxia incubators for incubation 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 being cultured overnight in normoxia, the medium containing the drug was added. The plate with the drug was then placed in an anoxic incubator and incubated for 6 h. The medium containing the drug was removed and new medium was added. The cells were irradiated with a 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 Figure 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 ( Figure 26 A in the middle), 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. Figure 26 Middle B) and light exposure time ( Figure 26 The increase of C) in the above table indicates that the prodrug prepared by the present invention has good hypoxia and light control properties. Figure 26 Figure D is the cytotoxicity test result of compound 8 in A549 cells. Compared with compound 7 linked to the chemotherapy drug nitrogen mustard, the cell killing ability is smaller, indicating that the prodrug compound 7 linked to the photosensitizer and chemotherapy drug has a stronger killing effect.

[0157] Test Example 5

[0158] This test case 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 3×10 4 Cells were seeded in confocal microscopy. After the cells adhered, they were placed in a normoxic or hypoxic incubator overnight. After the culture medium was removed, drug-containing culture medium (final concentration of 2 μM) was added and the cells were cultured in a normoxic or hypoxic incubator for 4 h. After the culture medium was removed, the cells were washed three times with PBS and the release of the prodrug compound in the cells was observed using a laser confocal microscope (λ Ex =633 nm, λ Em = 647-759 nm). ( Figure 27 Middle A)

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

[0162] A549 cells in the logarithmic growth phase were taken and 3×10 4 Cells were seeded in 6-well plates. After the cells adhered, they were cultured overnight in a hypoxic incubator for 12 hours. Compound 7 was then added at concentrations of 0, 0.0625, 0.125, 0.25, 0.5, and 1 μM, and the hypoxic incubation continued for 4 hours. The drug-containing medium was removed, and the cells were washed three times with PBS. The cells were digested with EDTA, the cell pellet was collected, and the cells were resuspended in 500 μL of PBS and analyzed by flow cytometry. Figure 27 Middle B)

[0163] (2) Release of prodrugs under different oxygen concentrations

[0164] A549 cells in the logarithmic growth phase were selected and 3×10 4 Cells were seeded in 6-well plates. The plates were placed in a normoxic incubator to adhere for 2 hours. The plates were then incubated overnight in incubators with 1%, 10%, and 20% oxygen concentrations. Drug-containing culture medium (final concentration 2 μM) was added and hypoxic culture was continued for 4 hours at different oxygen concentrations. The drug-containing culture medium was removed, and the cells were washed three times with PBS. The cells were digested with EDTA, the cell pellet was collected, and the cells were resuspended in 500 μL of PBS. Flow cytometry was used for analysis. Figure 27 Middle C)

[0165] The experimental results are as follows Figure 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 ( Figure 27 As the compound concentration increases, more original drug compound 1 is released and the fluorescence becomes stronger ( Figure 27 The lower the oxygen concentration, the stronger the fluorescence, indicating that compound 7 is more easily converted into prodrug compound 1 under lower hypoxia conditions ( Figure 27 Middle C).

[0166] Test Example 6

[0167] This test example studies the generation of reactive oxygen species in cells under light exposure. The specific process is as follows:

[0168] A549 cells in the logarithmic growth phase were selected and 3×10 4 Cells / dish were seeded in a confocal dish and cultured in a normoxic incubator for 2 h before being transferred to a hypoxic workstation for overnight culture. Culture medium containing 2 μM compound 7 was added and cultured in the hypoxic workstation for another 4 h. The drug-containing culture medium was removed and fresh culture medium was added. The procedure was performed according to the instructions of the reactive oxygen species 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 reactive oxygen species in the cells was detected using a confocal microscope (λ Ex =488 nm, λ Em = 493 - 630nm).

[0169] The experimental results are as follows Figure 28 As shown, hypoxic A549 cells were incubated with compound 7 for a period of time, and then exposed to light, the reactive oxygen species (ROS) probe DCFH-DA was added. Almost no green fluorescence was observed in the blank control (MERGED) and the no-light treatment (Bright) groups. However, with increasing light exposure time, the intracellular green fluorescence gradually increased. This indicates that compound 7 can generate ROS under hypoxic light conditions.

[0170] Test Example 7

[0171] This experiment was conducted to conduct a subcellular organelle colocalization experiment. The specific process was as follows:

[0172] 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 lysosomes and mitochondria of the cells, respectively, and the cell nucleus was stained with a nuclear dye (Hoechst33342). Finally, the localization and distribution of compound 7 in the cells (λ Ex1 =488 nm, λ Em1 = 493 - 630 nm; λEx2 =633 nm, λ Em2 = 647 - 759 nm).

[0173] The experimental results are as follows Figure 29 As shown in the figure, 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.

[0174] Example 4

[0175] This example detects the expression of autophagy proteins induced by prodrugs in cells. The specific process is as follows:

[0176] A549 cells in the logarithmic growth phase were selected and 5×10 4 Cells / dish were seeded in 20 mm culture dishes and allowed to adhere to the culture medium for 2 h under normoxia. The cells were then placed in a hypoxic workstation for overnight culture. The culture medium was removed and fresh culture medium containing different concentrations of compound 7 was added. The cells were then incubated in hypoxia for 4 h. After removing the drug-containing culture medium, fresh culture medium was added. Except for the blank group, each dish was irradiated with a 660 nm laser for 3 min (84 mw / cm 2 ). Wash the cells three times with PBS buffer solution, add 1 mL of 4% paraformaldehyde to each dish and let it stand for 30 minutes to fix the cells. After removing the paraformaldehyde, wash the cells three times with PBS and incubate with 0.1% Triton X-100 solution for 10 minutes. Remove the excess Triton X-100, wash three times with PBS, add LAMP1 primary antibody (1:1000) in PBST buffer and incubate at 4°C overnight. 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 2 hours. Recover the fluorescent secondary antibody, wash three times with PBS, incubate with the nuclear dye DAPI (20 μg / mL) in the dark, and finally seal the slices with anti-fluorescence decay mounting medium. Laser confocal microscopy was used to detect the expression of autophagy protein LAMP1 in cells treated with compound 7 (λ Ex1 =488 nm, λ Em1 = 493 - 630 nm; λ Ex2 =633nm,λ Em2 = 647 - 759 nm; λ Ex3 =405 nm, λ Em3 = 410 - 581 nm).

[0177] The experimental results are as follows Figure 30As 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.

[0178] Example 5

[0179] This example conducts a cell apoptosis experiment, and the specific process is as follows:

[0180] A549 cells in the logarithmic growth phase were selected and 3×10 5 Cells were seeded into 6-well plates, cultured in normoxia for 2 h, and then placed in normoxia and hypoxia incubators overnight. The old culture medium was removed, and culture medium containing different concentrations of compound 7 was added. The cells were then cultured in a hypoxia workstation and a normoxia incubator for 4 h. After removing the drug-containing culture medium and adding new culture medium, the cells were irradiated with a 660 nm laser for 3 min (84 mw / cm 2 ), and then placed in hypoxic and normoxic incubators for 4 hours, respectively. After digesting the cells with EDTA-free trypsin, the cells and supernatant were collected into a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After washing three times with PBS buffer, follow the procedures of the cell apoptosis detection kit: resuspend the cells in 100 μL of binding buffer, add 10 μL of Annexin V-FITC / PI kit staining buffer diluent and 5 μL of Annexin V-FITC, incubate on ice in the dark for 15 minutes, and then add 400 μL of binding buffer and mix thoroughly. The samples were analyzed by flow cytometry for the cell apoptosis induced by compound 7 under normoxic and hypoxic conditions.

[0181] The experimental results are as follows Figure 31 (*** indicates p < 0.001) As shown, the apoptosis rate of the control group was 2.71%, while the apoptosis rate after intervention with 0.25 μM 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 0.5 μM prodrug compound 7, the apoptosis rate under normoxic illumination treatment was 12.88%, while the apoptosis rate under hypoxic illumination treatment was 41.63%, indicating that under the same treatment conditions, compound 7 is more likely to induce cell apoptosis under hypoxia than under normoxia, and the effect is positively correlated with its concentration. Therefore, prodrug compound 7 can also kill tumor cells by inducing cell apoptosis.

[0182] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof: 。 2. Compounds of formula II, or pharmaceutically acceptable salts thereof: 。 3. The method for preparing the compound of formula (I) according to claim 1, wherein: The following steps are involved: S1: diazotizing p-aminobenzyl alcohol and coupling it with N,N-dihydroxyethylaniline; After chlorination reaction, an intermediate is obtained; S2: After the intermediate is reacted with 2-methylquinolin-8-amine through N-alkylation reaction; It is prepared by cyclization under the action of silver carbonate.

4. The method for preparing the compound of formula II according to claim 2, wherein: The following steps are involved: S1: diazotizing p-aminobenzyl alcohol and coupling it with N,N-dimethylaniline; After chlorination reaction, an intermediate is obtained; S2: After the intermediate is reacted with 2-methylquinolin-8-amine through N-alkylation reaction; It is prepared by cyclization under the action of silver carbonate.

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

6. A pharmaceutical composition comprising the compound of formula I according to claim 1 and / or a pharmaceutically acceptable salt thereof.

7. Use of the compound of formula I according to claim 1, a pharmaceutically acceptable salt thereof and / or the pharmaceutical composition according to claim 6 in the preparation of a drug for inhibiting cell proliferation.

8. Use of the compound of formula I according to claim 1, a pharmaceutically acceptable salt thereof and / or the pharmaceutical composition according to claim 6 in the preparation of a tumor diagnostic agent and / or therapeutic agent.

9. The use according to claim 8, characterized in that: The tumor includes at least one of lung cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, lymphoma, penile cancer, prostate cancer, skin cancer, gastrointestinal stromal tumor, head tumor, and neck tumor.

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