A compound for combined photothermal-immunotherapy of tumors and its application

Abstract

The purpose of the invention is to solve the above-mentioned problems existing in the prior art (mainly for the adverse reactions of using photothermal therapy alone and immunotherapy alone), by constructing a supramolecular photothermal agent, and proposing a compound for photothermal and immune combined therapy and its pharmaceutical composition. This type of compound can self-assemble into a micro-nano structure in water, and through the "passive targeting" of the EPR effect, the photothermal agent is enriched in the tumor tissue. When the photothermal agent emits light under 808nm laser irradiation, it releases heat to achieve integrated diagnosis and treatment, with the advantages of high photothermal conversion efficiency, excellent photothermal stability and easy degradation and high safety. At the same time, because the immune checkpoint inhibitor NLG919 used in this project in the tumor can inhibit IDO-1 activity, reverse the immunosuppressive microenvironment, activate the anti-tumor cascade immune effect, and induce cancer cell immunogenic cell death (ICD), and through NLG919-mediated IDO1 inhibition, it effectively delays the degradation of tryptophan to the immunosuppressant kynurenine, thereby promoting the decrease in the level of regulatory T cells and the increase in the level of effector T cells, achieving the therapeutic effect of immune killing.

Description

Technical Field

[0001] The present invention belongs to the field of chemical pharmaceuticals, and relates to a class of supramolecular photothermal agents with "passive" targeting effects and immune checkpoint inhibitors that are bonded through stimulus-responsive disulfide bonds. They can be supramolecularly assembled into nanoparticles in water, and can respond to the tumor microenvironment and release immune checkpoint inhibitors, thereby achieving combined photothermal and immunotherapy. Background Art

[0002] Cancer, a common medical problem faced by all mankind, is characterized by rapid growth, strong invasiveness, and the ability to spread from the primary site to other parts of the body. In addition to traditional cancer treatments such as surgery, radiotherapy, and chemotherapy, researchers are constantly exploring and innovating.

[0003] With the increasing incidence of cancer, posing a significant threat to human health and life, researchers are constantly exploring new treatments, with laser photothermal therapy gradually gaining attention. Photothermal therapy (PTT) uses a beam of near-infrared light to irradiate tumor tissue. The fluorescent photothermal agent emits heat simultaneously, which kills cancer cells and achieves a therapeutic effect. It has attracted widespread attention for its high specificity for tumors, minimal invasiveness to surrounding normal tissues, and spatial and temporal selectivity.

[0004] However, recent studies have revealed that photothermal therapy alone still has some issues, the most prominent of which is the production of heat shock proteins. Sustained heat causes cancer cells to produce heat shock proteins to resist sudden environmental changes, while sustained high temperatures can also adversely affect normal tissues. Furthermore, tumor-driven immunosuppression causes the entire tumor microenvironment to transmit immunosuppressive signals, paralyzing the immune system and leading to a high likelihood of tumor recurrence and metastasis.

[0005] Immunotherapy is a revolutionary cancer treatment that relies on activating the immune system to eliminate cancer. Its clinical efficacy has garnered increasing attention in recent years. Immunotherapy is primarily categorized into immune checkpoint blockade (ICB), adoptive T cell therapy, and cancer vaccines. ICB therapy typically utilizes antagonists to block inhibitory pathways, such as the programmed cell death protein 1 (PD-1 / PD-L1) pathway and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). ICB has made significant clinical progress and has been applied to the treatment of various tumors, including melanoma, non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma, and classical Hodgkin's lymphoma. The most prominent checkpoint receptors are those involved in the programmed cell death protein 1 / programmed cell death ligand 1 (PD-1 / PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) pathways. Anti-PD-L1 agents atezolizumab and avelumab have been approved by the US Food and Drug Administration (FDA). PD-L1 is overexpressed on cancer cells and can provide inhibitory signals to PD-1. PD-1 is expressed on activated T cells and transduces inhibitory signals to antagonize the activated T cell receptor (TCR) and CD28 axis. Cancer cells use the immunosuppressive function of PD-L1 to avoid being killed.

[0006] Indoleamine 2,3-dioxygenase (IDO) is a key negative feedback regulatory protein that creates an immunosuppressive microenvironment for tumor cell growth. Overexpressed in some tumor cells, IDO catalyzes the degradation of the essential amino acid tryptophan (Trp), blocking the cell cycle and causing effector T cell death. Studies have shown that inhibiting IDO synthesis can effectively delay the conversion of tryptophan to the immunosuppressant kynurenine, thereby promoting a decrease in regulatory T cell levels and an increase in effector T cell levels, achieving a therapeutic immune-killing effect.

[0007] Immunotherapy has proven to be one of the most effective clinical treatments for various types of cancer. However, in most cases, immunotherapy targets lack specificity and may be expressed in normal tissues. The indirect release of immunotherapeutic agents into normal tissues can sometimes lead to serious side effects, such as fever, hypotension, skin reactions, and some hypersensitivity reactions. Therefore, improving tumor specificity is crucial. Summary of the Invention

[0008] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art (mainly targeting the adverse reactions of photothermal therapy alone and immunotherapy alone) by constructing a supramolecular photothermal agent and proposing a compound for combined photothermal and immunotherapy and its pharmaceutical composition.

[0009] This type of compound can self-assemble into micro-nano structures in water, and through the "passive targeting" of the EPR effect, the photothermal agent is enriched in the tumor tissue. Under the irradiation of the laser, the photothermal agent emits light and heat at the same time, realizing integrated diagnosis and treatment. It has the advantages of high photothermal conversion efficiency, excellent photothermal stability and easy degradation and high safety. At the same time, because the immune checkpoint inhibitor NLG919 used in this project can inhibit IDO-1 activity in the tumor, reverse the immunosuppressive microenvironment, activate the anti-tumor cascade immune effect, and induce immunogenic cell death (ICD) of cancer cells, and through NLG919-mediated IDO1 inhibition, it effectively delays the degradation of tryptophan into the immunosuppressant kynurenine, thereby promoting a decrease in the level of regulatory T cells and an increase in the level of effector T cells, achieving a therapeutic effect of immune killing.

[0010] The technical solution of the present invention is:

[0011] A compound for photothermal-immunotherapy combination therapy, wherein the compound has a structure represented by formula (III) or a micro-nano structure formed by self-assembly of its isomers, pharmaceutically acceptable salts, hydrates or solvates in aqueous solution:

[0012]

[0013] Or, a micro-nano structure formed by self-assembly in an aqueous solution having a structure represented by formula (I) or its isomers, pharmaceutically acceptable salts, hydrates or solvates:

[0014]

[0015]

[0016] Or, a micro-nano structure having a structure represented by formula (II) or its isomers, pharmaceutically acceptable salts, hydrates or solvates self-assembled in an aqueous solution:

[0017]

[0018] In the above formula (I), formula (II) and formula (III): A is an immunomodulatory group, which can be divided into three categories according to different targets: cytotoxic T lymphocyte antigen 4 (CTLA-4) monoclonal antibody, programmed death factor 1 (PD-1) monoclonal antibody and programmed death factor ligand 1 (PD-L1) monoclonal antibody. Other immune checkpoint inhibitory drugs include LAG-3, IDO inhibitors, CD137, CD134, etc.

[0019] In the formula (III):

[0020] X2 is selected from O, S or -CR 20 R 20 '-;

[0021] Y3, Y4, and Y5 are each independently selected from H, a hydroxyl group, a halogen atom, a substituted or unsubstituted amino group, and an oxyalkyl group;

[0022] t1, t2, and t3 are each independently selected from integers of 0 to 5;

[0023] R 13 、R 13 '、R 14 Each independently selected from -CN, -CF3, F, -SO2CF3, -NO2, -COOEt, -SO2ph,

[0024] R 15 -(CH2) m -、 m is an integer from 0 to 5;

[0025] R 16 and R 17 Together they form one of the following connections: or R 16 、R 17 Together with X2, we form the following connection Among them, R a 、R b 、R c 、R d 、R e 、R f 、R g Each is independently selected from H, halogen, substituted or unsubstituted hydrocarbon, substituted or unsubstituted carboxyl, substituted or unsubstituted hydroxyl and substituted or unsubstituted amino;

[0026] R 18 、R 18 '、R 19 、R 20 and R 20 'are each independently selected from H, a halogen atom, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted amide group, a substituted or unsubstituted ester group and a substituted or unsubstituted amino group;

[0027] In the formula (I):

[0028] B is a substituted or unsubstituted heterocyclic ring which is uncharged and contains one or more heteroatoms selected from N, O and S;

[0029] L is a substituted or unsubstituted conjugated carbon chain containing 2 to 5 double bonds;

[0030] X1 is O, N or -CR4R4'-;

[0031] n is 0 or 1;

[0032] R1, R1', and R2 are each independently selected from atoms and groups having electron-withdrawing ability;

[0033] R3 and R3', R4 and R4' are each independently selected from H, a halogen atom, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted acylamino group, a substituted or unsubstituted ester group and a substituted or unsubstituted amino group;

[0034] In the formula (II):

[0035] Y2 is Cl, Br, wherein q and q' are each independently selected from integers of 0 to 12;

[0036] R9 is -CN or

[0037] R 10 -(CH2) m -、 m is an integer from 0 to 5,

[0038] R 11 for

[0039] R 12 for

[0040] The q and q' are each independently selected from integers of 0 to 12;

[0041] In the above formula (I), formula (II) and formula (III), when the group is substituted, the substituent is monosubstituted or polysubstituted.

[0042] Further, the A is selected from

[0043] Furthermore, in formula (III), t1 and t2 are both 1, t3 is 0; m is 3;

[0044] The R 13 、R 13' are -CN, R 14 -CN or

[0045] The R 18 、R 18 '、R 19 Each independently selected from H, -(CH2) q CH3, -(CH2) q CF3, -(CH2) q CHCH2, -(CH2) q CCH, -(CH2) q OH, -(CH2) q COOH, -(CH2) q NH2, -(CH2) q CHO, -(CH2) q CO(CH2) q 'CH3, -(CH2) q O(CH2) r 'CH3, wherein q and q' are independently selected from integers of 0-12; preferably, the R 19 is -CH2CH3;

[0046] Said Y3 and Y5 are both H;

[0047] Y4 is Cl, Br or -NR 21 R 21 '-, where R 21 、R 21 ' are each independently selected from H, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted amide group, a substituted or unsubstituted ester group and a substituted or unsubstituted amino group; L1 is a carbon chain substituted or unsubstituted by an ester group.

[0048] Furthermore, in formula (I), the number of double bonds in the conjugated carbon chain is 2, 3, 4 or 5; X1 is O; n is 0; R1, R1', and R2 are each independently selected from -CN, -CF3, -F, -SO2CF3, -NO2, -COOEt, -SO2ph, Said R3, R3' are each independently selected from H, -(CH2) q CH3, -(CH2) q CF3, -(CH2) q CH=CH2、-(CH2)q C≡CH, -(CH2) q OH, -(CH2) q COOH, -(CH2) q NH2, -(CH2) q CHO, -(CH2) q CO(CH2) q 'CH3, -(CH2) q O(CH2) q 'CH3, Here, q and q' are each independently selected from integers of 0 to 12.

[0049] Preferably, R1 and R1' are both -CN; R2 is -CN or

[0050] In formula (I), specifically, L can be Wherein, Y1 is a halogen atom, a substituted or unsubstituted amino group or a hydrocarbonoxy group; m is an integer of 0-5, preferably, m is 3; R7 is independently selected from H, a halogen atom, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted acylamino group, a substituted or unsubstituted ester group and a substituted or unsubstituted amino group. Particularly preferably, m is 3, Y1 is Cl, Br, -NR8R8' or -OR8; and R7 is H, -CH3, R8 and R8' are each independently selected from H, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted acylamino group, a substituted or unsubstituted ester group and a substituted or unsubstituted amino group.

[0051] The micro-nanostructure formed by self-assembly of the compound of formula (I) in aqueous solution has a "photothermal"-"immune" combined therapeutic effect, and also has the advantages of high photothermal conversion efficiency, excellent photothermal stability, easy degradation and high safety.

[0052] Preferably, B is selected from a substituted or unsubstituted pyrrole or hydrogenated pyrrole ring, a substituted or unsubstituted furan or hydrogenated furan ring, a substituted or unsubstituted thiophene or hydrogenated thiophene ring, a substituted or unsubstituted pyrazole or hydrogenated pyrazole ring, a substituted or unsubstituted imidazole or hydrogenated imidazole ring, a substituted or unsubstituted oxazole or hydrogenated oxazole ring, a substituted or unsubstituted isoxazole or hydrogenated isoxazole ring, a substituted or unsubstituted thiazole or hydrogenated thiazole ring, a substituted or unsubstituted indole or hydrogenated indole ring, a substituted or unsubstituted benzofuran or hydrogenated benzofuran ring, a substituted or unsubstituted benzimidazole or hydrogenated benzofuran ring, a substituted or unsubstituted benzofuran ... a substituted or unsubstituted benzimidazole ring, a substituted or unsubstituted carbazole or hydrogenated carbazole ring, a substituted or unsubstituted pyridine or hydrogenated pyridine ring, a substituted or unsubstituted pyran or hydrogenated pyran ring, a substituted or unsubstituted thiopyran or hydrogenated thiopyran ring, a substituted or unsubstituted benzopyrazole or hydrogenated benzopyrazole ring, a substituted or unsubstituted pyridazine or hydrogenated pyridazine ring, a substituted or unsubstituted pyrimidineazine or hydrogenated pyrimidine ring, a substituted or unsubstituted pyrazine or hydrogenated pyrazine ring, a substituted or unsubstituted piperidine ring, a substituted or unsubstituted morpholine ring, a substituted or unsubstituted thiomorpholine ring, and a substituted or unsubstituted triazole ring;

[0053] More preferably, the B is Wherein, R5, R6, and R6' are each independently selected from H, a halogen atom, a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted alcohol group, a substituted or unsubstituted ether group, a substituted or unsubstituted aldehyde group, a substituted or unsubstituted carboxyl group, a substituted or unsubstituted acylamino group, a substituted or unsubstituted ester group, and a substituted or unsubstituted amino group.

[0054] Furthermore, in formula (II), R 10 is -CH2-, -(CH2)2-, -(CH2)3- or -(CH2)4-, preferably, R 10 It is -(CH2)3-.

[0055] The micro-nanostructure of Compound II is formed by self-assembly of Compound II (including Compound II-1 to Compound II-50 in Table 1) in aqueous solution. The particle size of the micro-nanostructure is 1 nm to 500 nm, preferably 10 nm to 200 nm, and more preferably 30 nm to 150 nm.

[0056] Because tumors (particularly solid tumors) are rich in blood vessels and lack a lymphatic drainage system, the micro-nanostructures described in this invention exhibit passive high permeability and retention at the tumor site. This high permeability and retention effect of micro-nanostructures in solid tumor tissue is known as the EPR effect. This passive tumor targeting ability gives these small molecule compounds, which can form micro-nanostructures through supramolecular assembly, a significant advantage over other reported small molecule photothermal conversion agents.

[0057] Further, the compound is compound II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23, II-24, II- 25, II-26, II-27, II-28, II-29, II-30, II-31, II-32, II-33, II-34, II-35, II-36, II-37, II-38, II-39, II-40, II-41, II-42, II-43, II-44, II-45, II-46, II-47, II-48, II-49 or II-50.

[0058] The present invention also provides a pharmaceutical composition comprising:

[0059] 1) A therapeutically effective dose of a compound having a structure represented by formula (I), formula (II) or formula (III) or its isomer, pharmaceutically acceptable salt, hydrate or solvate

[0060] 2) Pharmaceutically acceptable carrier.

[0061] Preferably, the pharmaceutically acceptable carrier comprises a diluent, a disintegrant, an excipient, a binder, a stabilizer or a combination thereof.

[0062] The compound provided by the present invention is used in the preparation of a drug for photothermal-immunotherapy combination therapy, or a drug for diagnosing and / or treating cancer. The combination therapy drug is a photothermal therapy drug or a photoacoustic therapy drug.

[0063] The cancer includes esophageal cancer, non-small cell lung cancer, biliary tract cancer, head and neck cancer, Barrett's esophagus, bladder cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain tumor, breast cancer or skin cancer, including melanoma.

[0064] The micro-nano structure of the present invention is a nano-disc structure formed by self-assembly of a compound having a structure represented by formula (I), formula (II) or formula (III), its isomer, pharmaceutically acceptable salt, hydrate or solvate in an aqueous solution.

[0065] The present invention also provides a method for preparing the micro-nano structure, comprising the following steps:

[0066] 1) dissolving a compound having a structure represented by formula (I), formula (II) or formula (III), an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof in an organic solvent;

[0067] The organic solvent selected is a mixture of one or more of alkanes, olefins, aromatic hydrocarbons, alcohols, ketones, aldehydes, carboxylic acids, esters or ethers; specifically, the organic solvent is a mixture of one or more of dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, ethylene glycol, n-propanol, isopropanol, propylene glycol, glycerol, n-butanol, isobutanol, butanediol or polyethylene glycol, acetone, dichloromethane or acetonitrile; preferably ethanol.

[0068] 2) adding the dissolved solution into water to obtain a compound solution with a final concentration of 1 nM to 1 M;

[0069] The final concentration is preferably 100 nM to 500 μM; most preferably 0.46 μM to 300 μM.

[0070] 3) The compound self-assembles in aqueous solution to form micro-nanostructures.

[0071] The preparation method is simple, convenient and suitable for large-scale production.

[0072] The present invention also provides a pharmaceutical composition of a micro-nanostructure, comprising: a therapeutically effective dose of the micro-nanostructure and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier comprises a diluent, a disintegrant, an excipient, a binder, a stabilizer, or a combination thereof.

[0073] The pharmaceutical composition provided by the present invention can be prepared as an injection, comprising a therapeutically effective dose of the micro-nanostructures and an injection solvent or additive, or a combination thereof; wherein the injection solvent is one or a mixture of two or more of water for injection, ethanol, propylene glycol, glycerol, and polyethylene glycol. Preferably, the pharmaceutical composition can be prepared as an injection solution.

[0074] The micro-nanostructure of the present invention is a nanodisc structure, and its pharmaceutical composition further includes an active agent encapsulated in the micro-nanostructure. The active agent is a therapeutic agent or a diagnostic agent, preferably a chemotherapeutic agent or a radiotherapeutic agent, including small molecule chemotherapeutic drugs, targeted therapy drugs, chemotherapeutic drugs, antibody drugs, etc. Furthermore, the micro-nanostructure also includes a targeting molecule, preferably an antibody, peptide, aptamer, or folic acid.

[0075] In another aspect, the present invention further provides the use of the micro-nanostructure or its pharmaceutical composition in the preparation of a phototherapy drug, and its use as a photosensitizer. The photosensitizer is used to prepare a phototherapy drug. The phototherapy drug is a photothermal therapy drug or a photoacoustic therapy drug.

[0076] The present invention also provides the use of the micro-nanostructure or a pharmaceutical composition thereof in the preparation of a drug for diagnosing and / or treating cancer. The cancers include esophageal cancer, non-small cell lung cancer, biliary tract cancer, head and neck cancer, Barrett's esophagitis, bladder cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain tumor, breast cancer, or skin cancer, including melanoma.

[0077] The present invention also provides a method for performing light therapy on a target area of ​​a subject, comprising:

[0078] 1) Providing the micro-nano structure;

[0079] 2) administering the micro-nanostructure to a subject;

[0080] 3) waiting for the micro-nanostructure to be enriched in the target area;

[0081] 3) Using light in the excitation wavelength band of the micro-nanostructure to irradiate the target area of ​​the subject, preferably, using 808 nm light waves for irradiation.

[0082] Beneficial effects of the present invention:

[0083] (1) The compounds provided by the present invention can self-assemble into micro-nano structures in aqueous solution, and can simultaneously achieve "passive" targeting of the EPR effect, so that the photothermal agent is more enriched in the tumor, with excellent tumor targeting effect, and thus have the advantages of high photothermal conversion efficiency, good photothermal stability, good photothermal effect, easy degradation, and high safety, and have broad prospects in cancer diagnosis and treatment.

[0084] (2) The compounds of formula (I), (II) or (III) provided by the present invention are useful for preparing combined phototherapy drugs, as well as drugs for diagnosing and treating cancer. They have good targeting effects, good therapeutic effects, and minimal trauma, and possess great market value and broad economic prospects.

[0085] (3) After the introduction of the NLG919 group in the present invention, the overall organic small molecule can still be constructed into a micro-nanostructure through supramolecular assembly. The micro-nanostructure can passively target tumors through the EPR effect, and at the same time, achieve precise release of immune checkpoint inhibitors under the stimulation of high glutathione in tumor tissue, avoiding adverse immune reactions and further improving the safety of immunotherapy. After the bond is broken, Cy7-TCF can achieve fluorescence enhancement, further improving the fluorescence imaging intensity and photothermal therapy effect. Moreover, after photothermal therapy, the immunogenicity is further improved, improving the immune efficacy, thereby achieving a "1+1>2" treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is a synthetic route diagram of compound II-1 provided by the present invention;

[0087] Figure 2 The binding pocket of compound II-1 in the protein and its interaction with the protein.

[0088] Figure 3 The projection electron micrograph of the compound provided by the present invention self-assembling into nanodiscs in aqueous solution and the particle size test results show that the entire molecule can self-assemble into a micro-nanostructure after chemically bonding the compound with the immunostimulatory group;

[0089] Figure 4 This is the temperature change diagram of 10 μmol compound II-1 under 808 nm laser irradiation;

[0090] Figure 5 This is a microscopic image of nanodiscs assembled from compound II-1 being engulfed by cells;

[0091] Figure 6 Fluorescence imaging of nanodiscs assembled from compound II-1 through "passive" targeting of HeLa cells;

[0092] Figure 7 The photoacoustic imaging images of the whole body of tumor-bearing mice at different time points after the nanodiscs assembled from compound II-1 were intravenously injected into the mice.

[0093] Figure 8 After the nanodiscs assembled with compound II-1 were intravenously injected into tumor-bearing mice, the fluorescence imaging of the tumor at different time points and the fluorescence intensity of the main organs of the mice after dissection

[0094] Figure 9 Photothermal imaging of compound II-1 during photothermal therapy in mice;

[0095] Figure 10 Changes in tumor volume after intravenous injection of nanodiscs assembled with compound II-1 and photothermal therapy in tumor-bearing mice;

[0096] Figure 11 H&E-stained images of heart, liver, spleen, lung, and kidney sections of tumor-bearing mice after intravenous injection of nanodiscs assembled with compound II-1 and photothermal therapy for 22 days.

[0097] Figure 12 Lungs and slices from treated and untreated mice;

[0098] Figure 13 Figure 2 shows the changes in body weight and tumor volume of mice in the treatment group and the control group;

[0099] Figure 14 24 hours after intravenous injection of nanodiscs assembled with compound II-1 into mice, the liver function and blood routine of the mice;

[0100] Figure 15 The expression levels of related cytokines near the metastatic tumors of the experimental group mice and the control group mice. DETAILED DESCRIPTION

[0101] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0102] In order to further understand the present invention, the present invention will be further described with reference to the accompanying drawings and embodiments.

[0103] The present invention provides certain specific examples of compounds, including compounds II-1 to II-50 shown in Table 1 below:

[0104] Table 1 Structural formulas of compounds II-1 to II-50

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] The above compounds can be synthesized by the following reaction formula:

[0115]

[0116] The main synthetic steps include:

[0117] (1) providing compounds a, b, c, and d respectively;

[0118] Synthesis of compound a:

[0119]

[0120] Compound 1', compound 2' and magnesium ethoxide were dissolved in ethanol and reacted at 60°C for 24 hours. The solvent was evaporated under vacuum and the obtained solid was purified by column chromatography to obtain the target compound a.

[0121] Synthesis of compound b:

[0122]

[0123] Add dichloromethane and compound 4' to a flask under ice-cooling and stir. Add compound 5' at constant pressure and stir. Then add compound 3' and react at 80°C for 3 hours. After the reaction is complete, pour the product into crushed ice to quench the reaction and refrigerate overnight. Evaporate the solvent under vacuum to obtain the crude product, compound b, which is used directly in the next reaction without purification.

[0124] Synthesis of compound c:

[0125]

[0126] Compound 6' and compound 7' were added to acetonitrile and heated to 110°C for 24 hours under reflux. The solvent was evaporated under vacuum and the resulting solid was washed with ether three times to obtain compound c.

[0127] Compound d was purchased directly.

[0128] (2) Compound a and compound b were dissolved in ethanol, heated to reflux, and then compound c was added, heated to reflux, and the solvent was evaporated under vacuum. The obtained solid was purified by column chromatography. The obtained product was dissolved in dichloromethane with compound d, and EDCHCl and DMAP were added. After reacting at room temperature overnight, the mixture was washed three times with saturated aqueous ammonium chloride solution and saturated aqueous sodium chloride solution. After removing water, the obtained solid was purified by column chromatography to obtain the product compound II.

[0129] Example 1 Synthesis of Compound II-1 and Its Fluorescence Properties

[0130] like Figure 1 As shown, the synthesis of compound II-1 includes the following steps:

[0131] 1) Synthesis of Compound 1: 3 g of Compound 6 and 3.89 g of iodoethanol were weighed and added to 15 mL of ethanol. The mixture was heated to 110° C. and refluxed for 18 hours. The mixture was washed with ether three times to obtain Compound 1.

[0132] 2) Synthesis of Compound 2: 0.97 g of malononitrile and 0.62 g of magnesium ethoxide were weighed and added to 10 mL of ethanol. 0.5 mL of 3-hydroxy-3-methylbutan-2-one was then added. The mixture was heated to 60° C. for 12 hours. The solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to yield the title compound 2.

[0133] 1 H NMR (400MHz, CDCl3): δ (ppm): 2.36 (s, 3H), 1.63 (s, 6H).

[0134] 3) Synthesis of Compound 3: 20 mL of dichloromethane and 20 mL of DMF were added to a flask under ice-cooling and stirred. 17.5 mL of phosphorus oxychloride was added at constant pressure and stirred. 5.3 mL of cyclohexanone was then added and heated to 80°C for 3 hours. After the reaction was complete, the product was poured into crushed ice to quench the reaction and placed in a refrigerator overnight. The solvent was evaporated under vacuum to obtain the crude product, Compound 3, which was used directly in the next reaction without purification.

[0135] 4) Synthesis of Compound 4: 1.00 g of Compound 2 and 1.27 g of Compound 3 were added to 50 mL of ethanol, heated to 90° C. and refluxed for 12 hours. After cooling to room temperature, the crude product Compound 4 was obtained by filtration and used directly in the next step without purification.

[0136] 5) Synthesis of compound Cy7-TCF-OH: 2.04 g of compound 1 and 2.00 g of compound 4 were dissolved in 100 mL of ethanol, heated to 100°C and refluxed for 10 hours. The solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to obtain the target compound Cy7-TCF-OH.

[0137] 6) Synthesis of Compound 5: 1 g of compound Cy7-TCF-OH and 0.53 g of compound 12 were dissolved in 50 mL of dichloromethane. 1.35 g of EDC·HCl and 0.25 g of DMAP were added dropwise under an ice bath and reacted at room temperature overnight. 50 mL of dichloromethane was then added, and the mixture was washed three times with 100 mL of saturated aqueous NH4Cl and saturated brine, respectively. After dehydration with anhydrous Na2SO4, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to yield the title compound, Compound 5.

[0138] 7) Synthesis of Compound II-1: 0.8 g of Compound 5 and 0.25 g of Compound 11 were dissolved in 50 mL of dichloromethane. 0.39 g of EDC·HCl and 0.09 g of DMAP were added dropwise under ice-cooling. The mixture was reacted at room temperature overnight. Subsequently, 50 mL of dichloromethane was added. The mixture was washed three times with 100 mL of saturated aqueous NH4Cl solution and saturated brine, respectively. After dehydration with anhydrous Na2SO4, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to obtain the target compound II-1.

[0139] 1 H NMR (400MHz, CDCl3): δ (ppm): 2.36 (s, 3H), 1.63 (s, 6H).

[0140] 1H NMR (400MHz, CDCl3): δ (ppm) 8.06 (d, 1H), 7.84 (d, 1H), 7.75 (s, 1H), 7.51–7.42 (m, 2H), 7.32–7.27 (m, 1H), 7.19 –7.15(m,2H),6.97(td,J=7.5,1H),6.79(d,1H),6.32(d,1H),5.69(d,1H),5.23(s,2H),5.13–5.04(m,1H),4.9 0(ddd,1H),4.34(t,2H),4.00(d,2H),2.60–2.47(m,9H),2.34(ddd,2H),2.25(dd,3H),2.17–2.09(m,2H),1.84 (dq,7H),1.69(s,6H),1.57(s,7H),1.43–1.35(m,2H),1.33–1.16(m,6H),1.13–0.99(m,3H),0.96–0.75(m,4H).

[0141] Experimental Example 2 Cell Imaging Experiment

[0142] The nuclear dye Hoechst33342 (100 nM), lysosomal dye Lyso-Green (75 nM) and II-1 (8 μM) were added to the cell culture medium for 30 minutes of cell staining. After staining, the cells were rinsed twice with PBS and observed and photographed under a confocal fluorescence microscope. Figure 5 As shown, when the photos of the three channels are combined, it is found that II-1 and the lysosome part are almost completely overlapped, indicating that compound II-1 is a dye that can target lysosomes.

[0143] Experimental Example 3: Intracellular Photothermal Effect Detection Experiment

[0144] HeLa cells were cultured in 96-well plates, with 104 cells per well. After 24 hours, different concentrations of II-1 (0.1μM to 100μM) were added. To test for dark toxicity, the plates were placed in an incubator for 24 hours and then stained for 20 minutes with the live cell dye Calcein-AM and the dead cell dye EthD-I. Subsequent observation under a fluorescence microscope revealed that almost all cells were alive, demonstrating that compound II-1 itself has minimal toxicity.

[0145] like Figure 6 As shown in the bar graph, the cells in the cell well plate for phototoxicity detection were irradiated with an 808nm laser for 6 minutes and observed and photographed under a fluorescence microscope. It was found that when the concentration of Ⅱ-1 was greater than 12.5μM, 100% of the cells were dead, proving that compound Ⅱ-1 has a strong photothermal killing ability on cancer cells under laser irradiation and has an excellent photothermal effect.

[0146] Combining the results of the laser irradiation group and the dark toxicity group shows that the compound itself has minimal toxicity, but its photothermal effect is particularly lethal to cancer cells, showing a bright future for the clinical application of photothermal therapy for cancer. Other compounds of the present invention also have similar photothermal therapeutic effects.

[0147] Experimental Example 4 Photoacoustic Imaging Test in Mice

[0148] First, a prethoracic tumor mouse model was constructed. 107 HeLa cells were injected into the chest of 6-week-old female nude mice, and the tumor volume was allowed to grow to 60 mm3. 200 μL of II-1 (200 μg) was then injected into the mouse via the tail vein. Three-dimensional photoacoustic tomography was used to monitor the tumor at different times. Figure 7 As shown, the difference in photoacoustic signals between the left and right tumor sites was most pronounced four hours after injection. Over time, photoacoustic signals were consistently present at the tumor site until 24 hours after injection. This example demonstrates the excellent tumor targeting properties of Compound II-1 and its superior photoacoustic signal quality. Furthermore, the nude mice showed no abnormalities such as spasms or convulsions within 24 hours, demonstrating the near-total lack of toxicity and the high safety of Compound II-1.

[0149] Experimental Example 5: Fluorescence Imaging Experiment in Photothermal Therapy in Mice

[0150] A prethoracic tumor mouse model was also constructed.

[0151] The experimental group mice were injected with 200 μL of II-1 (200 μg) through the tail vein. 107 HeLa cells were injected into the chest of 6-week-old female nude mice. The tumor volume was grown to 60 mm3. 200 μL of II-1 (200 μg) was injected into the mice through the tail vein. The fluorescence imaging system was used to detect at different time points. Figure 8 As shown, the fluorescence of the tumor in the experimental group gradually showed an increasing trend after 84 hours, indicating that the SS bond was broken. After the bond was broken, the fluorescence of the nanodisc was enhanced. This example proves that compound II-1 can respond to the high concentration of glutathione in the tumor tissue and release NLG919 while recombining the nanodisc to achieve fluorescence enhancement.

[0152] Experimental Example 5: Photoacoustic Imaging Experiment in Photothermal Therapy in Mice

[0153] The experimental group of mice were injected with 200 μL of II-1 (200 μg) through the tail vein, and the tumor site of the mice was irradiated with 808 nm laser for 10 minutes while taking pictures with a photothermal imager. Under the irradiation of laser, the tumor site was able to heat up to 57 ° C, from Figure 15 It can be seen that the temperature of the tissue around the tumor did not increase, indicating that II-1 has the advantage of causing less damage to the tissue near the tumor when used for photothermal therapy.

[0154] The experimental group of mice were injected with 200 μL of Ⅱ-1 (200 μg) through the tail vein.

[0155] Experimental Example 6: Photothermal therapy experiment in mice

[0156] Nude mice were divided into six groups. Group 1 received 200 μL of II-1 (200 μg) without laser irradiation; Group 2 received 200 μL of II-1 (200 μg) via the tail vein and irradiated the tumor site with an 808 nm laser for 10 minutes. Group 3 received 200 μL of II-2 (200 μg) without laser irradiation; Group 2 received 200 μL of II-2 (200 μg) via the tail vein and irradiated the tumor site with an 808 nm laser for 10 minutes; Group 5 received saline without laser irradiation; Group 6 received saline and irradiated the tumor site with a 10-minute laser. Tumor volume in each group was measured daily with a vernier caliper for 22 days.

[0157] like Figure 10 On the second day after photothermal treatment, the tumor of the mice ruptured. As time went on, there was no obvious tumor growth at the primary tumor site. The ruptured tumor site began to heal. On the 18th day, the ruptured site was completely healed with a small scar. The size of the distal tumor was significantly inhibited only by the injection of Ⅱ-1. The other groups all grew to varying degrees. Figure 13 The tumors of mice in the experimental group (Group 2) were eliminated after laser irradiation, while the tumor volumes of mice in the control group (Groups 1, 3, 4, 5, and 6) continued to increase. Figure 13 There was no abnormal change in the body weight of mice in both the experimental and control groups, and no obvious side effects of II-1 were observed. Figure 11 After 22 days of the experiment, mice in the experimental group were dissected, and the hearts, livers, spleens, and kidneys were sectioned and stained with H&E. Apoptosis of the tumor cells was observed, while no significant liver damage was observed. This demonstrates that II-1 has excellent photothermal therapeutic capabilities, does not damage internal organs, has minimal side effects, and is relatively safe and reliable.

[0158] Experimental Example 7 Evaluation of the Effect of Immunotherapy in Mice

[0159] By measuring the size of distal tumors, it can be clearly concluded that immunotherapy has a significant inhibitory effect on distal tumors. At the same time, by observing the lungs and slices of 6 different groups of mice, Figure 12 It can be seen that only the experimental group injected with II-1 and irradiated with laser had no obvious nodules in the lungs, while the lungs of mice in other groups all had tumor metastasis to varying degrees. Figure 12 This can also be observed significantly in lung tissue sections. Figure 13It can be seen that the levels of CD3, CD8, and Treg-T in the treatment group were significantly higher than those in the other control groups, while the level of INF-γ was significantly lower. This shows that II-1 can effectively exert its immunotherapy effect and inhibit tumor metastasis.

[0160] These experiments demonstrate that compound II-1 exhibits excellent photothermal tumor-killing efficacy and outstanding immunotherapy effects under 808nm laser irradiation, along with high safety. This suggests that it holds broad promise for clinical photothermal cancer treatment. Other compounds of the present invention have also demonstrated similar therapeutic effects.

[0161] Test Example 8 Safety test of compound II-1 in mice

[0162] Six Balb / c mice were injected and blood samples were collected for blood routine and liver function tests. Figure 14 As shown, blood counts for all six groups of mice, including lymphocytes, mean corpuscular volume, and red blood cell volume distribution width, were all within normal ranges. Liver function indicators, including albumin, cholesterol, alkaline phosphatase, and blood glucose, were also within normal ranges. These results indicate that Compound II-1 has a high safety profile, does not damage the liver in the short term, and is relatively safe and reliable.

[0163] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A compound having a combined photothermal and immune therapeutic effect, characterized in that: The compound is compound II-1, and the structure of compound II-1 is as follows:

2. A pharmaceutical composition, characterized in that The invention comprises the compound according to claim 1, and a pharmaceutically acceptable carrier.

3. Use of the compound according to any one of claims 1 in the preparation of drugs for dual-targeted phototherapy, or in the preparation of drugs for diagnosing and / or treating cancer.

4. The use according to claim 3, characterized in that The dual-targeted light therapy drug is a photothermal therapy drug or a photoacoustic therapy drug.

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