Antitumor compounds, compositions and uses thereof

Abstract

The application discloses an anti-tumor compound, a composition and application thereof. The anti-tumor compound is a conjugate obtained by connecting pyrodermat-oxa a and doxorubicin through a ketone thiol bond, and is named as Ppa-TK-DOX. The composition containing Ppa-TK-DOX and a tumor stem cell differentiation agent can be prepared into nanoparticles with hyaluronic acid with a modified lipophilic fragment as a carrier, and is applied to preparation of an anti-tumor drug. The Ppa-TK-DOX has a simple synthesis method, good blood and tissue compatibility, and can play a synergistic treatment effect by combining chemotherapy and photodynamic therapy. The composition has stronger tumor cell stemness down-regulation effect and stronger specific tumor accumulation capacity, and shows good tumor penetration capacity. The composition in the form of nanoparticles further improves the anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a prodrug conjugate synthesized from a dihydroporphyrin-type photosensitizer—pyrophyllate a—and doxorubicin via a ketethiocarbamate bond, forming a composition with a tumor stem cell differentiation agent, and the application of the conjugate and composition in antitumor drugs. Background Technology

[0002] Doxorubicin (DOX) is a broad-spectrum antitumor drug that produces a wide range of biological effects on the body. It exhibits strong cytotoxicity to tumor cells, with mechanisms of action including insertion into tumor cell DNA disrupting gene expression, generating reactive oxygen species, and inhibiting topoisomerase II. It is a first-line treatment for several cancers, including breast cancer, ovarian cancer, bladder cancer, and lung cancer. The most serious adverse reaction is irreversible cardiomyopathy, and the toxicity is closely related to the total cumulative dose. Liposome administration can reduce the cardiotoxicity and other adverse reactions of doxorubicin; a liposomal doxorubicin formulation (DOXIL, ALZA) has been approved for marketing.

[0003] Single treatment methods, such as chemotherapy, are often insufficient to achieve optimal therapeutic effects. Combining chemotherapy with other treatments, such as photodynamic therapy, to exert synergistic therapeutic effects has become one of the current hot topics in clinical cancer treatment.

[0004] Prodrug strategies are crucial for innovative drug development. Structural modification is the most direct and effective method for developing prodrugs. Improving drug defects, optimizing physicochemical properties (such as lipophilicity and water solubility), and altering the route of administration can all be achieved through specific structural modifications. Furthermore, by designing prodrugs by linking the response characteristics of the tumor microenvironment (pH, redox, enzymes, etc.) to the prototype drug, the prodrug can be cleaved within the tumor, releasing the prototype drug to kill tumor cells while simultaneously reducing toxicity to normal tissues / cells.

[0005] Cancer stem cells are a subset of tumor cells capable of self-renewal, multi-lineage differentiation, distant metastasis, and resistance to radiotherapy and chemotherapy. The higher the proportion of cancer stem cells in a tumor, the higher its malignancy, the faster its growth rate, the stronger its resistance to treatment, the higher the probability of metastasis and recurrence, and the lower the patient's survival rate. Cancer stem cells are a determining factor in tumor occurrence, development, invasion, metastasis, and drug resistance. Finding highly effective and specific treatments targeting cancer stem cells is crucial for cancer treatment. Targeting and eliminating cancer stem cell subsets by reducing self-renewal signaling pathways, targeting molecular surface markers, and influencing the microenvironment, thereby weakening or even eliminating the stem cell characteristics of tumors, has become a promising clinical treatment approach. Summary of the Invention

[0006] This invention first provides a pyrophyllophosphophosphate a (CAS No.: 15664-29-6, molecular formula: C). 33 H 34 N4O3 (abbreviated as Ppa) and doxorubicin hydrochloride (CAS No.: 25316-40-9, molecular formula: C) 27 H 29 NO 11 The coupling of HCl is characterized by pyrophyllate a and doxorubicin via a ketethiocarbamate bond. connect.

[0007] The structural formula of the pyrophyllite α-ketothiolate-doxorubicin conjugate (abbreviated as Ppa-TK-DOX) provided by this invention is shown in Formula I:

[0008]

[0009] In formula I, It can be an anhydride amide Or ester

[0010]

[0011] It can be an amide Urea Or carbamate

[0012]

[0013] n1 is an integer from 1 to 10, preferably an integer from 1 to 5;

[0014] n2 is an integer from 1 to 10, preferably an integer from 1 to 5.

[0015] A further preferred embodiment of the pyrophyllite α-ketothiolate-doxorubicin conjugate provided by the present invention is characterized in that, in formula I:

[0016] for

[0017] for

[0018] This invention also provides a method for preparing the prodrug conjugate shown in Formula I, comprising:

[0019] Method 1:

[0020]

[0021] Step 1a: Dissolve pyrophyllite a in an organic solvent, add thionyl chloride and stir until homogeneous, then add a carboxyl-terminated ketethiocyanate linker (abbreviated as TK-COOH), stir for a period of time, add a precipitate solvent to precipitate the precipitate, filter, wash the precipitate, and dry to obtain carboxyl-ketethiocyanate-(anhydride bond)-pyrophyllite a (abbreviated as Ppa-TK-COOH);

[0022] Step 1b: Weigh an appropriate amount of Ppa-TK-COOH and carboxyl activator and dissolve them in an organic solvent. After stirring for a period of time, add an appropriate amount of doxorubicin (DOX) and react for a period of time. Dialyze the reaction solution with deionized water at room temperature and freeze-dry the dialysate to obtain pyrophyllite chlorophyll α-ketothiolate-doxorubicin (abbreviated as Ppa-TK-DOX).

[0023] In step a above, preferably, the molar ratio of pyrophyllite a to COOH-TK-COOH is 1:1 to 1:2; the reaction solvent is an organic solvent, such as tetrahydrofuran (THF) and N,N-dimethylformamide (DMF); the reaction is carried out at room temperature in the dark; the reaction time after adding COOH-TK-COOH is 12 to 48 hours; the preferred solvent for precipitation is cold n-hexane, and the solvent for washing the precipitate is n-hexane; the drying method is vacuum drying.

[0024] In step b above, preferably, the molar ratio of Ppa-TK-COOH to DOX is 3:1 to 1:3, more preferably 1:1 to 1:2; the reaction is carried out under the action of carboxyl activator 1-(3-dimethylaminopropyl)-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the molar ratio of Ppa-TK-COOH to EDC and NHS is 1:1.5:1.5; the reaction solvent is an organic solvent, such as N,N-dimethylformamide; the reaction is carried out at room temperature, and the reaction time after adding DOX is 24 to 48 hours; the dialysis time is 24 to 48 hours, and the molecular weight cutoff of the dialysis bag used for dialysis is 1000; the lyophilization conditions are a separator temperature set to -40 to -10°C and a vacuum degree set to 13 to 40 Pa.

[0025] Method 2:

[0026]

[0027] Step 2a: Dehydration condensation reaction is carried out between Ppa and the hydroxyl-terminated ketithiolide linker (TK-OH), followed by filtration and column chromatography purification to obtain hydroxy-ketithiolide-(ester bond)-pyromethesin a (abbreviated as Ppa-TK-OH);

[0028] Step 2b: Ppa-TK-OH and 4-nitrobenzene chloroformate undergo a dechlorination condensation reaction in the presence of an organic base catalyst, followed by filtration and column chromatography purification to obtain Ppa-TK-PNP;

[0029] Step 2c: Dissolve Ppa-TK-PNP and doxorubicin (DOX) in an organic solvent, stir and react in the presence of an organic base catalyst, dialyze the reaction solution in deionized water, filter to remove insoluble matter, freeze dry, and obtain pyrophyllite chlorophyll α-ketothiolate-doxorubicin conjugate.

[0030] In step 2a above, 1,3-dicyclohexylcarbodiimide (DCC) is preferably used as the activator, 4-dimethylaminopyridine (DMAP) as the catalyst, and dichloromethane (DCM) as the solvent for the condensation reaction. Specifically, Ppa is first activated with a dichloromethane (DCM) solution of 1,3-dicyclohexylcarbodiimide (DCC), and then added to an anhydrous DCM solution of 4-dimethylaminopyridine (DMAP). After stirring at room temperature for 0.5 to 2 hours, an anhydrous tetrahydrofuran (THF) solution containing TK-OH is added. After reacting at room temperature for 10 to 30 hours, the reaction mixture is filtered, the solvent is removed from the filtrate, and Ppa-TK-OH is obtained after purification by column chromatography.

[0031] In step 2b above, the organic base catalyst is preferably triethylamine (TEA), and the reaction solvent is preferably dichloromethane. The specific operation is as follows: Ppa-TK-OH and 4-nitrobenzene chloroformate are dissolved in DCM, anhydrous triethylamine (TEA) is added dropwise, and after reacting at room temperature for 10 to 30 hours, the reaction mixture is filtered, and the filtrate is concentrated and purified by column chromatography to obtain Ppa--TK-PNP.

[0032] In step 2c above, the organic base catalyst is preferably triethylamine (TEA), and the reaction solvent is preferably N,N-dimethylformamide. The specific operation is as follows: Ppa-TK-PNP and doxorubicin are dissolved in DMF, TEA is added dropwise, the reaction is stirred overnight, the reaction solution is dialyzed in deionized water for 48-72 hours, the insoluble matter is filtered off and lyophilized to obtain Ppa-TK-DOX.

[0033] Preferably, in Method 2 above, the molar ratio of Ppa to TK-OH is 1:2 to 1:8; the eluent for column chromatography is DCM:CH3OH = 60 to 100:1 (v / v); the molar ratio of Ppa-TK-OH to 4-nitrobenzene chloroformate is 1:2 to 1:10; the molar ratio of Ppa-TK-PNP, DOX, and TEA is 1:(1 to 2):(1.5 to 3); and the molecular weight cutoff of the dialysis bag used for dialysis is 1000.

[0034] Method 3:

[0035]

[0036] Steps 3a and 3b of Method 3 are the same as steps 2a and 2b of Method 2. The difference is in step 3c: first, Ppa-TK-PNP is reacted with hydrazine hydrate to obtain Ppa-TK-acylhydrazine, then Ppa-TK-acylhydrazine and DOX are dissolved in an organic solvent, and the reaction is carried out at room temperature with stirring in the presence of an organic base catalyst. The product is precipitated with a nonpolar solvent, purified, and dried to obtain pyrophyllite chlorophyll α-ketothiolate-doxorubicin conjugate.

[0037] In step 3c above, the organic base catalyst is preferably triethylamine (TEA), and the reaction solvent is preferably N,N-dimethylformamide. The specific operation is as follows: Ppa-TK-PNP and hydrazine hydrate are dissolved in DMF, TEA is added dropwise, and the reaction is stirred overnight. The reaction solution is dialyzed in deionized water for 48-72 hours, the insoluble matter is filtered off and lyophilized to obtain Ppa-TK-hydrazide. Ppa-TK-hydrazide and DOX are dissolved in an appropriate amount of anhydrous DMF, an appropriate amount of TEA is added, and the reaction is stirred at room temperature for 3 days. The product is precipitated and purified in a non-polar solvent (such as diethyl ether, petroleum ether, n-hexane, cyclohexane, etc.) and dried under vacuum.

[0038] The preferred molar ratio of Ppa-TK-hydrazide to DOX in the above method is 1:2 to 1:6.

[0039] The present invention further provides a composition comprising the above-mentioned pyrophyllite α-ketothiolate-doxorubicin conjugate, the composition comprising the pyrophyllite α-ketothiolate-doxorubicin conjugate and a tumor stem cell differentiation agent.

[0040] The tumor stem cell differentiation agent can be selected from: retinoic acid (All-Trans-Retinoic acid, Retinoic acid, CAS No.: 302-79-4, molecular formula: C). 20 H 28 O2), BRD7552 (CAS No.: 1137359-47-7, Molecular Formula: C 33 H 33 N3O 15 ), scalp protein, nicotinamide (CAS No.: 98-92-0, molecular formula: C6H6N2O), dexamethasone (CAS No.: 50-02-2, chemical formula C6H6N2O), acetaminophen, nicotinamide (CAS No.: 98-92-0, molecular formula: C6H6N2O), dexamethasone (CAS No.: 50-02-2, chemical formula: C6 22 H 29 FO5), Cytarabine (CAS No.: 147-94-4, molecular formula: C9H) 13 One of N3O5, etc., preferably retinoic acid (ATRA).

[0041] The composition comprising pyrophyllite chlorophyll α-ketothiolate-doxorubicin conjugate provided by the present invention is preferably prepared into nanoparticles.

[0042] The nanoparticles provided by the present invention use hyaluronic acid (HA) modified with lipophilic fragments as a carrier to load a composition containing pyrophyllite chlorophyll α-ketothiolate-doxorubicin conjugate.

[0043] The carrier provided by this invention uses hyaluronic acid as a hydrophilic fragment and couples a lipophilic fragment by grafting.

[0044] The lipophilic fragment provided by this invention is selected from C6-C24 medium- and long-chain fatty acids, medium- and long-chain fatty amines, medium- and long-chain fatty alcohols, as well as phospholipids and vitamin E succinate, preferably vitamin E succinate (TOS). The hyaluronic acid carrier modified with vitamin E succinate is abbreviated as HA-TOS.

[0045] In the HA-TOS provided by this invention, the molecular weight of hyaluronic acid is 6000-35000 Da, preferably 7000-15000 Da.

[0046] In the HA-TOS provided by this invention, the grafting rate of TOS is determined by nuclear magnetic resonance. 1 In the ¹H NMR spectrum, the ratio of the characteristic peaks at 2.5–2.9 ppm (methylene group of α-TOS) to 1.9–2.1 ppm (N-acetyl group of HA) is defined as the grafting rate of TOS. In the HA-TOS provided by this invention, the grafting rate of TOS is 2%–15%, preferably 5%–10%, and more preferably 6%–9%.

[0047] The present invention provides a method for preparing the above-mentioned nanoparticles, as follows:

[0048] An appropriate amount of HA-TOS was dissolved in water as the aqueous phase, and an appropriate amount of pyromethesin α-ketothiolate-doxorubicin (Ppa-TK-DOX) and retinoic acid were dissolved together in dichloromethane as the oil phase. After mixing the oil and water phases, the mixture was prepared into a primary emulsion by ultrasonication with a probe. The dichloromethane was removed by rotary evaporation to obtain the drug-loaded nanoparticles PTD / A-NPs.

[0049] The doxorubicin loading in the nanoparticles provided by this invention is 1%-10%, preferably 2%-10%, and more preferably 3%-10%. The doxorubicin loading is calculated as (mass of doxorubicin / total mass of nanoparticles) × 100%.

[0050] The retinoic acid loading in the nanoparticles provided by this invention is 2%-6%, preferably 3%-6%, and more preferably 4%-6%. Wherein, the retinoic acid loading = (mass of retinoic acid / total mass of nanoparticles) × 100%.

[0051] The nanoparticles provided by the present invention, after being dispersed with an appropriate amount of water, have an average particle size between 60-300 nm, preferably between 60-200 nm, and more preferably between 60-160 nm.

[0052] The pyrophyllite α-ketothiolate-doxorubicin conjugate provided by this invention can be used in the preparation of antitumor drugs. Preferably, the tumor is selected from breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and ovarian cancer.

[0053] The composition provided by the present invention can be used in the preparation of antitumor drugs. Preferably, the tumor is selected from breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and ovarian cancer.

[0054] This invention provides a synthetic route for conjugates of pyrophyllite a and doxorubicin. The compounds of this invention are synthesized using classical organic chemical reactions, and the molecular structures of the compounds provided by this invention are confirmed using common structural confirmation methods, such as nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry.

[0055] This invention provides a method for preparing a composition, which uses classic methods in the art to prepare nanoparticles. The in vitro properties of the nanoparticles are characterized using classic methods in the art, such as dynamic light scattering and column separation, and the characterization results are given.

[0056] In this invention, mouse breast cancer cells (4T1 cells) are used as a non-tumor stem cell model, and 4T1 side population stem cells (4T1 SP cells) are obtained by flow cytometry sorting using a classic method in the art as a tumor stem cell model. Both types of cells are recognized and well known in the art.

[0057] This invention employs recognized and advanced biological methods to evaluate the compounds, compositions, and nanoparticles of this invention in vitro and in vivo, and to study their mechanisms. Specifically, 9,10-anthratridimyl-bis(methylene)dimalonic acid (ABDA) is used to evaluate the ability to generate ROS in vitro. The release characteristics of the nanoparticles are studied using dialysis, with H2O2 added to simulate the oxidative cleavage of TK bonds by ROS to determine the release rate of functional molecules. Changes in the release rate after laser irradiation are used to infer details of the in vivo effects of the compounds or compositions. Taking advantage of the abundant CD44+ / CD24 population and high expression of stem cell-related factors in stem cells, PE-CD44 and FITC-CD24 antibodies are used to stain cells, and flow cytometry is used for analysis. The expression levels of tumor stem cell markers—Sox2, Nanog, and Oct4—were detected using Western blotting analysis. This experiment was used not only to identify cell stemness but also to evaluate the ability of the nanoparticles of the present invention to differentiate into tumor cell stemness. The cytotoxicity of the compounds and compositions of the present invention to tumor cells was evaluated using the Cell Counting Kit-8 (CCK-8) method. The distribution of the nanoparticles of the present invention in tumor sites and key organs of tumor-bearing mice was observed using in vivo small animal imaging. The in vivo anti-tumor activity and anti-tumor stem cell activity of the nanoparticles of the present invention in 4T1 tumor-bearing mice were evaluated using classic in vivo anti-tumor experiments, and the tumor inhibition rate and the downregulation ability of tumor stem cell factors were measured. In addition, the antigen presentation of DC cells and T cell activation within the tumor were also examined.

[0058] This invention has the following beneficial effects: 1) The pyromethoxyphylla α-TK-doxorubicin prodrug conjugate designed and synthesized in this invention has a simple synthesis method, a clear structure, and controllable quality; 2) The compounds provided by this invention have good blood and tissue compatibility, ensuring in vivo safety; 3) The compounds and their compositions provided by this invention generate more reactive oxygen species (ROS) upon laser irradiation; under the action of the oxidant, the TK bond in the structure of the compounds provided by this invention breaks rapidly, thereby releasing the chemotherapy drug DOX and the photosensitizer Ppa, and the combination of chemotherapy and photodynamic therapy exerts a synergistic therapeutic effect; 4) The compositions provided by this invention, after laser irradiation, show stronger tumor cell stemness reduction. 5) The composition provided by this invention has a stronger specific tumor accumulation ability and is widely distributed in tumor tissue, thus exhibiting good tumor penetration ability; 6) The composition nanoparticles provided by this invention have stronger in vivo anti-tumor activity, and the anti-tumor efficacy is further improved under laser irradiation; 7) The composition nanoparticles provided by this invention significantly downregulate tumor stem cell-related factors in tumor tissue, thereby exhibiting a stronger apoptosis effect and exerting a better tumor treatment effect; 8) The composition nanoparticles provided by this invention induce stronger antigen presentation of DC cells and T cell activation, promote the improvement of tumor suppression effect, and reveal the contribution of immune cells to combined anti-tumor therapy. Attached Figure Description

[0059] Figure 1 The following are the FT-IR spectra of the compounds in Example 1 (including Ppa, TK, PT, DOX, and PTD).

[0060] Figure 2 For example, the PTD in Example 1 1 H-NMR spectrum.

[0061] Figure 3 The image shows the ESI-MS spectrum of the PTD in Example 1.

[0062] Figure 4 The curve showing the relationship between the hemolysis rate and concentration of PTD / A-NPs in Example 2 is shown.

[0063] Figure 5 The curves showing the decrease in absorbance over time, measured at a wavelength of 380 nm, are shown for the conjugate PTD and its control free Ppa after laser irradiation.

[0064] Figure 6 The image shows the HPLC chromatograms of each compound in Example 4 after co-incubation with H2O2 solution for 24 hours.

[0065] Figure 7 The changes in the expression levels of Sox2, Nanog, and Oct4 in cells caused by different nanoparticles in Example 5 are shown.

[0066] Figure 8 The curves showing the relationship between cell viability and concentration under different nanoparticle conditions in Example 6, under no irradiation (-L) and laser irradiation (+L).

[0067] Figure 9 This is an in vivo imaging image showing the distribution of nanoparticles in an in situ tumor-bearing mouse in Example 7.

[0068] Figure 10 This is an in vitro fluorescence image of the tumor and major organs in an orthotopic tumor-bearing mouse 24 hours after nanoparticle injection in Example 7.

[0069] Figure 11 The values ​​are semi-quantitative values ​​of fluorescence intensity of tumors and major organs in orthotopic tumor-bearing mice 24 hours after nanoparticle injection in Example 7.

[0070] Figure 12 The image shows frozen sections (CLSM) of tumor tissue from the edge and core of the tumor-bearing mouse in situ 24 hours after nanoparticle injection in Example 7.

[0071] Figure 13 The curve shows the change in average fluorescence intensity of the nanoparticles in in situ tumor-bearing mice over time in Example 7.

[0072] Figure 14 This section describes the inhibitory efficacy of PTD / A-NPs on tumor-bearing mice (established by inoculation with 4T1 cells) in Example 8. a: Schematic diagram of the in vivo treatment regimen; b: Image of tumor tissue harvested from mice on day 29 after treatment initiation; c: Tumor growth curve of mice (mean ± SD, n = 6, *p < 0.05, ***p < 0.001, ****p < 0.0001); d: Tumor weight of tumor-bearing mice (mean ± SD, n = 6, *p < 0.05, ****p < 0.0001); e: H&E staining image of tumor sections (scale bar: 100 μm); f: Ki-67 staining image of tumor sections (scale bar: 100 μm); g: Representative TUNEL image of tumor sections (scale bar: 75 μm); h: CD44 extracted from tumor tissue. + / CD24 - Cell population ratio (mean ± SD, n = 3, *p < 0.05, **p < 0.01); i: CD4 extracted from tumor + CD25 + FoxP3 + Cell population ratio (mean ± SD, n = 3, *p < 0.05, **p < 0.01).

[0073] Figure 15 The nanoparticles in Example 8 act on CD3 in tumor cells within tumor tissue (established by seeding 4T1 cells) of tumor-bearing mice. + / CD8 + Flow cytometry analysis results.

[0074] Figure 16 The results are flow cytometry analysis of spleen cells extracted from the spleen of tumor-bearing mice (established by inoculation with 4T1 cells) in Example 8, where nanoparticles act on them. Detailed Implementation

[0075] The following examples are used to further illustrate the present invention but are not intended to limit the invention.

[0076] The raw materials and equipment used in the embodiments of this invention are all known products and were obtained by purchasing commercially available products.

[0077] Among them, D-α-tocopherol succinate was purchased from Sigma-Aldrich; hyaluronic acid was purchased from Shandong Freda Pharmaceutical Group Co., Ltd.; doxorubicin (DOX) (purity >98%) and retinoic acid were purchased from Dalian Meilun Biotechnology Co., Ltd.; pyromethesphalic acid-a (Ppa) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 3-mercaptopropionic acid was purchased from Shanghai Jingchun Biochemical Technology Co., Ltd.; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were purchased from Beijing Bailingwei Technology Co., Ltd.; anhydrous acetone, dimethylformamide (DMF) and tetrahydrofuran (THF) and other organic solvents were provided by Beijing Tongguang Fine Chemical Co., Ltd.; fetal bovine serum (FBS) was purchased from Gibco; culture medium, antibiotics, enzymes, paraformaldehyde, Hoechst 33342 and other materials were purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

[0078] The mouse 4T1 tumor cells used in this embodiment of the invention were purchased from the Shanghai Institute of Biological Sciences (SIBS, Shanghai, China). After co-incubating the 4T1 cells with Hoechst 33342, the 4T1 side population stem cells (4T1SP cells) were collected by flow cytometry.

[0079] In this embodiment of the invention, female Balb / c mice (18-20g, approximately 6 weeks old, SPF grade) were purchased from the Department of Laboratory Animal Science, Peking University School of Medicine. All animal experiments were conducted strictly in accordance with the "Peking University Laboratory Animal Care and Use Guidelines" and protocols approved by the Peking University Animal Ethics Committee.

[0080] Example 1: Synthesis of Ppa-TK-DOX (abbreviated as PTD)

[0081]

[0082] Ppa (0.1 mmol) was dissolved in 4 mL of tetrahydrofuran, thionyl chloride (0.1 mmol) and one drop of dimethylformamide were added, and the mixture was stirred for 24 hours. Then, propane-2,2-dimethylbis(thio)diacetic acid (0.1 mmol) was added, and the mixture was stirred overnight. Cold n-hexane was added to precipitate the precipitate, which was then filtered, washed, and dried to obtain Ppa-TK-COOH (PT). PT (0.1 mmol), EDC (0.15 mmol), and NHS (0.15 mmol) were weighed and dissolved in dimethylformamide. After stirring for 2 hours, DOX (0.1 mmol) was added, and the mixture was reacted at room temperature for 24 hours. The reaction solution was dialyzed against deionized water at room temperature for 48 hours, and the dialysate was lyophilized to obtain Ppa-TK-DOX (PTD).

[0083]

[0084] The synthesis method of Ppa-CC-DOX (abbreviated as PCD) is the same as that of PTD, except that propane-2,2-dimethylbis(thio)diacetic acid is replaced with an equimolar amount of pimelic acid. PCD is a control conjugate without TK bonds and is used to test the advantage of TK bond breaking in the compounds of the embodiments of the present invention.

[0085] The structure of Ppa-TK-DOX(PTD) in Example 1 was determined using nuclear magnetic resonance (NMR), infrared spectroscopy, and mass spectrometry. The solvent used for NMR was deuterated dimethyl sulfoxide (DMSO). The analytical results are as follows: Figures 1-3 As shown.

[0086] Infrared data: 3394.25cm -1 (NH), 2960.53cm -1 (-OH), 1744.23cm -1 (-C=O), 1683.54cm -1 (CN)

[0087] NMR data: 1 H NMR (400MHz, DMSO-d6) δ6.26 (s, 1H), 5.06 (dd, J = 12.8, 6.4Hz, 1H), 4.54 (dt, J = 12.9, 6.5Hz,12H),4.42(s,1H),4.08(d,J=3.5Hz,1H),3.92(s,1H),3.75-3.51(m,10H),3.0 0(p,J=1.8Hz,11H),2.50(s,5H),2.29(d,J=7.1Hz,1H),2.17-1.95(m,15H),1.88-1.7 4(m,12H),1.71(s,1H),1.67-1.58(m,1H),1.38(td,J=7.4,1.8Hz,18H),0.50(s,1H).

[0088] Example 2: Preparation and Characterization of Nanoparticles

[0089] 10 mg of HA-TOS was dissolved in 10 mL of deionized water as the aqueous phase, and 1 mg of PTD and 0.5 mg of ATRA were dissolved in 1 mL of dichloromethane as the oil phase. The oil and aqueous phases were mixed, and the mixture was ultrasonically prepared using a probe to form a primary emulsion. The dichloromethane was then removed by rotary evaporation to obtain drug-loaded nanoparticles PTD / A-NPs. Using the same process, PTD nanoparticles (PTD-NPs) and PCD nanoparticles (PCD-NPs) without ATRA, ATRA nanoparticles (ATRA-NPs, abbreviated as A-NPs) without PTD, and blank HA-TOS self-assembled nanoparticles (HAT-NPs) without PTD and ATRA were prepared.

[0090] The particle size and zeta potential of the nanoparticles were determined using a Malvern Zetasizer (Nano-ZS90, Malvern Instruments, UK), and a hemolysis assay was performed to evaluate the hemolysis rate of PTD / A-NPs.

[0091] The measurement results are as follows: The average DLS particle size of A-NPs is 109.93±2.02 nm, the PDI value is 0.224±0.01, and the Zeta potential is -25.47±3.8 mV; the average DLS particle size of PTD-NPs is 103.77±2.05 nm, the PDI value is 0.233±0.05, and the Zeta potential is -19.77±3.36 mV; the average DLS particle size of PCD-NPs is 168.77±3.84 nm, the PDI value is 0.175±0.02, and the Zeta potential is -19.67±2.72 mV; the average DLS particle size of PTD / A-NPs is 113.57±1.27 nm, the PDI value is 0.169±0.01, and the Zeta potential is -37.93±0.42 mV.

[0092] The hemolysis test results showed that the hemolysis rate was less than 5% when different concentrations of PTD / A-NPs were incubated with erythrocyte suspension. Figure 4 This indicates that the conjugate has good blood compatibility.

[0093] Example 3: Determination of in vitro ROS generation

[0094] The ability of the nanoparticles from Example 2 to generate ROS in vitro was evaluated using 9,10-anthratrium-bis(methylene)dimalonic acid (ABDA). 100 μL of PBS, free Ppa, PTD, and PTD / A-NPs solution (10 μg / mL, converted to Ppa concentration) were added to 1.9 mL of ABDA solution, shaken well, and then subjected to a laser (intensity set at 100 mV / cm). 2 The solution was irradiated with a wavelength set to 660 nm. The UV-Vis spectrum of the solution was scanned at predetermined time points, and the decrease in absorbance at a wavelength of 380 nm represented the level of ROS formation.

[0095] The reactive oxygen species (ROS) generation capacity of the conjugate Ppa-TK-DOX(PTD) was detected using the reactive oxygen species probe ABDA. Aqueous ABDA reacts with singlet oxygen to form endogenous oxidation products with no characteristic UV absorption; this reaction leads to a decrease in the height of ABDA's characteristic absorption peak. For example... Figure 5As shown, the absorbance of the PBS reaction solution at 380 nm did not change significantly with time. After laser irradiation, the absorbance of both the PTD solution and free Ppa at this wavelength decreased significantly, indicating that laser irradiation caused the PTD conjugate to generate reactive oxygen species, and the amount of ROS generated increased with the irradiation time.

[0096] Example 4: ROS response experiment of PTD

[0097] Measure 1 mL of PTD / A-NPs or PCD-NPs into their respective dialysis bags (molecular weight cutoff 7000 Da), seal them, and immerse them in a 50 mL EP tube containing 10 mL of release medium (PBS containing 10% ethanol, pH 7.4) to ensure the dialysis bag is completely submerged. Then, place the EP tube in a shaker and incubate at 80 rpm (37°C). Add different concentrations of H2O2 solution to the release medium, and use HPLC to determine the DOX concentration in the release medium to evaluate ROS responsiveness.

[0098] HPLC results are as follows Figure 6 As shown, no characteristic peaks were observed in the blank solution and free Ppa group. After treatment with H2O2, PTD showed a characteristic peak corresponding to DOX at 4.884 minutes, and the peak height of PTD was reduced compared with the untreated PTD group. In the HPLC chromatogram of the PCD group, no DOX characteristic peak appeared, indicating that the TK bond in the PTD structure was broken by reactive oxygen species, thereby releasing DOX.

[0099] Example 5: Study on the ATRA-induced stem cell differentiation effect

[0100] 4T1 SP cells were seeded in 12-well plates, and the nanoparticles described in the above example were added and incubated with the cells. The expression levels of classic stem cell-related transcription factors Sox2, Nanog, and Oct4 in the cells were detected using Western blot analysis.

[0101] Example 2: The synthesized nanoparticles were incubated with 4T1 SP cells (e.g., Figure 7 As shown in the figure, HAT-NPs and PTD-NPs did not show any downregulation of cell stem function, while PTD-NPs showed limited downregulation of cell stem function under laser irradiation conditions; formulations containing ATRA (A-NPs and PTD / A-NPs) showed significant downregulation of stem function regardless of whether irradiation was performed; the combination of ATRA, PTD, and laser irradiation showed a stronger downregulation effect on stem function, promoting the differentiation of stem cells into non-stem cells.

[0102] Example 6: In vitro cytotoxicity study

[0103] Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8). 4T1 SP cells were seeded in 96-well plates and cultured overnight to reach the appropriate density. After removing the serum-containing medium, serum-free medium (containing different concentrations of the nanoparticles synthesized in Example 2 above) was added to each well. After incubation for 24 hours, the cells were washed with PBS, and CCK-8 solution was added to each well for co-incubation for 1 hour. The absorbance of each well was measured at 450 nm using an iMark microplate reader (Bio-Rad 680, USA), and cell viability was calculated.

[0104] All nanoparticles maintained a high level of cell viability (above 60%) without irradiation (labeled "-L" in the figure). Figure 8 (Left figure) In the example containing Ppa, the cell viability of nanoparticles after laser irradiation (marked as "+L" in the figure) was significantly reduced, while the cell viability of the PBS group and A-NPs was not affected by laser irradiation. Figure 8 (Right figure); Compared with the PTD-NPs group, the unbroken PCD-NPs group showed weaker cell-killing ability, indicating that the carbon-carbon bond (CC) is difficult to break, thus preventing the release of DOX. Poorly differentiated cells such as SP cells are more resistant, while highly differentiated cells such as non-SP cells are more sensitive to chemotherapeutic drugs. Therefore, ATRA, which can induce the differentiation of tumor stem cells (as shown in the results of Example 5), combined with the conjugates provided in the embodiments of this invention, can exhibit unexpected cytotoxic effects.

[0105] Example 7: In vivo tumor targeting evaluation

[0106] To establish an orthotopic stem cell-related tumor model, 1×10 6 Four T1 SP cells were seeded into the mammary fat pads of female Balb / c mice. When the tumor volume increased to approximately 500 mm², 3 In this study, tumor-bearing mice were randomly divided into four groups, and PTD-NPs and PTD / A-NPs were administered via tail vein, respectively, with free Ppa serving as a control. Fluorescence signals were detected at 1, 2, 4, 8, 12, and 24 hours post-intravenous injection using an in vivo imaging system (IVIS spectroscopy, PerkinElmer, USA), and analyzed using LivingImage software. Subsequently, the mice were sacrificed, and the isolated tumors and major organs (heart, liver, spleen, lung, and kidney) were imaged and analyzed. Furthermore, the distribution of nanoparticles was observed using CLSM on frozen sections of tumor tissue.

[0107] Following intravenous injection, free Ppa is rapidly metabolized and excreted, while PTD-NPs and PTD / A-NPs show significant signals at the tumor site, originating from in vivo imaging systems. Figure 9), also from isolated tumors and major organs ( Figures 10-12 The fluorescence signals of the PTD-NPs and PTD / A-NPs groups reached their peak at 8 hours, while the signal of the free Ppa group reached its peak at approximately 4 hours. Figure 13 The stronger and more persistent fluorescence signal of PTD / A-NPs in tumor tissue indicates that PTD / A-NPs have a higher specific tumor accumulation capacity. This is because the hydrophilic hyaluronic acid shell of the nanoparticles has good active targeting ability, which helps to improve drug circulation time and specific accumulation. CLSM images of frozen tumor sections also demonstrate the accumulation of PTD / A-NPs at the tumor site. Figure 12 Furthermore, the fluorescence signal of PTD / A-NPs was widely and uniformly distributed in the tumor tissue, including the core region, indicating that PTD / A-NPs also exhibited good tumor penetration ability in vivo.

[0108] Example 8: Efficacy evaluation of in vivo anti-tumor stem cell orthotopic seeding for tumors

[0109] Inoculate female Balb / c mice with 1×10⁻⁶ mammary fat pads 6 A standard 4T1 tumor model was established using 4T1 cells, and the treatment regimen is as follows: Figure 14 As shown in Figure a. After mice were sacrificed on day 20, the remaining tumor tissue was extracted, cut into small pieces, and collagenized with 1 mg / mL collagenase. The mixture was then filtered twice through a 40 μm pore membrane to obtain a single-cell suspension. The cells were stained with appropriate antibodies and analyzed by flow cytometry. Furthermore, the extracted cells were seeded in ultra-low adsorption 96-well plates and incubated for 7 days in RPMI-1640 medium containing 10% FBS, and the number of tumor spheres was determined.

[0110] This study evaluated the antitumor efficacy of tumor-bearing mice established by implanting conventional 4T1 cells. The results showed that after drug administration, the tumor inhibition rate in the PTD / A-NPs plus laser irradiation (+L) group reached 96.77%, and some mice achieved complete tumor remission after treatment. Figure 14 (b-d) showed a significant advantage in inhibiting tumor growth; H&E staining ( Figure 14 (e) Ki-67 staining ( Figure 14 (f) and TUNEL analysis ( Figure 14 The results showed that the PTD / A-NPs(+L) group exhibited the best tumor cell proliferation inhibition and the strongest tumor cell apoptosis ability. After treatment with PTD / A-NPs(+L), CD44... + / CD24 - Cell population downregulated by 30.81% Figure 14In the middle (h), the expression of stem cell-related factors such as Sox2 and Oct4 was effectively inhibited.

[0111] This embodiment also detected CD8 in tumor tissue. + T cells and Treg cells and dendritic cells in the spleen. The results showed that treatment with PTD / A-NPs(+L) reduced CD8... + The proportion of T cell population increased ( Figure 15 CD4 + CD25 + FoxP3 + Cell population reduction ( Figure 14 This is beneficial for enhancing the anti-tumor effect. Furthermore, the expression levels of CD80, MHC-I, and MHC-II on the surface of DC cells in the PTD / A-NPs(+L) group of mice also showed an increasing trend. Figure 16 This indicates that administering the nanoparticles in the example induced stronger antigen presentation and T cell activation in mouse dendritic cells, thereby achieving a better tumor-suppressing effect. This example reveals the contribution of immune cells to the combined use of tumor stem cell differentiation, photodynamic therapy, and chemotherapy in anti-tumor treatment, which is an unexpected discovery.

Claims

1. A compound, characterized in that, The compound is a conjugate of pyrophyllate a and doxorubicin linked by a ketithiolide bond, and its structure is shown in Formula I: Its characteristic is that, in formula I for , for .

2. The method for preparing the compound according to claim 1, characterized in that: 1a) Dissolve pyrophyllite a in an organic solvent, add thionyl chloride and stir until homogeneous, then add carboxyl-terminated ketithyl thioglycol linker TK-COOH, stir for a period of time, add precipitant solvent cold n-hexane to precipitate the precipitate, filter, wash the precipitate, and dry to obtain compound PT. 1b) Compound PT and carboxyl activators 1-(3-dimethylaminopropyl)-ethylcarbodiimide and N-hydroxysuccinimide were dissolved in an organic solvent, and doxorubicin was added to react. The reaction solution was then dialyzed with deionized water. The molecular weight cutoff of the dialysis bag used for dialysis was 1000. The solution was lyophilized to obtain pyrophyllite α-ketothiolate-doxorubicin conjugate.

3. The preparation method according to claim 2, characterized in that, The organic solvents mentioned in step 1a) are tetrahydrofuran and N,N-dimethylformamide; the organic solvents mentioned in step 1b) are N,N-dimethylformamide, and the reaction is carried out for 24 to 48 hours after the addition of doxorubicin.

4. A composition, characterized in that, It comprises the pyrophyllite α-ketothiolate-doxorubicin conjugate and tumor stem cell differentiation agent shown in Formula I of claim 1.

5. The composition according to claim 4, characterized in that, The tumor stem cell differentiation agent is selected from one of the following: retinoic acid, BRD7552, cephalin, nicotinamide, dexamethasone, and cytarabine.

6. The composition according to claim 4, characterized in that, The composition is prepared into nanoparticles using hyaluronic acid modified with lipophilic fragments as a carrier, wherein the lipophilic fragments are selected from one of C6-C24 medium- and long-chain fatty acids, C6-C24 medium- and long-chain fatty amines, C6-C24 medium- and long-chain fatty alcohols, phospholipids, and vitamin E succinate, and the molecular weight of the hyaluronic acid is 6000~35000 Da.

7. The composition according to claim 6, characterized in that, The nanoparticles use hyaluronic acid modified with vitamin E succinate as a carrier to load the pyromethesin α-ketothiolate-doxorubicin conjugate and retinoic acid, wherein the loading of doxorubicin is 1%-10% and the loading of retinoic acid is 2%-6%.

8. The use of the compound of claim 1 or the composition of claim 4 in the preparation of an antitumor drug, characterized in that, The tumors were selected from breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and ovarian cancer.

Images