Responsive prodrug as well as preparation method and application thereof

Abstract

The invention discloses a response type prodrug as well as a preparation method and application thereof, and relates to the technical field of triple negative breast cancer treatment drugs. The response type prodrug is an H2O2 response type drug and comprises CR8 and a boric acid ester fragment capable of responding to H2O2 splitting decomposition, and CR8 is molecular glue aiming at cyclin dependent kinase CDK12. The response type prodrug disclosed by the invention has the beneficial effects that the response type prodrug developed by the invention has the advantage of tumor selectivity, can respond to a hydrogen peroxide signal in vivo after tail vein injection, and specifically releases molecular gel CR8 in a tumor microenvironment, so that accurate treatment of triple-negative breast cancer is realized. The prodrug not only can effectively inhibit tumor growth, but also can activate T cell immune response. In addition, nano-clusters with the average size of 193 nm can be formed through the self-assembly characteristic, and targeted accumulation of drugs in a tumor area is promoted by enhancing permeability and retention effect.

Description

Technical Field

[0001] The present invention relates to the technical field of triple-negative breast cancer therapeutic drugs, and in particular to a responsive prodrug and a preparation method and application thereof. Background Art

[0002] Cancer remains the most common cancer among women worldwide. Current cancer treatment strategies are mainly determined by the expression of human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Targeted therapies aimed at blocking these receptors have been shown to improve overall survival. However, a specific subgroup of breast cancer, triple-negative breast cancer (TNBC), lacks the expression of HER2, PR, and ER and accounts for approximately 20% of all cancer cases. The lack of established therapeutic targets for TNBC poses a major challenge to its treatment. Therefore, the development of new therapeutic approaches is urgently needed.

[0003] Immune checkpoint inhibitors (ICIs), including PD-L1 inhibitors (aPD-L1), anti-PD-1, and anti-CTLA-4 antibodies, are currently considered the cornerstone of cancer immunotherapy, unleashing the body’s immune system to target and kill tumor cells. However, cancer has traditionally been considered an immune “cold” tumor with limited T cell infiltration. In TNBC, the anti-tumor immune response is primarily driven by CD4 + Helper T cells and CD8 + Cytotoxic T cells mediate this process. Therefore, activation of CD8 + T cell strategies are expected to enhance the efficacy of ICIs and improve the immune response against TNBC.

[0004] Molecular glues are a class of small molecules that can enhance or stabilize interactions between proteins. These compounds can target proteins that were previously considered undruggable, bypassing competition with natural ligands. And may convert "cold" tumors into "hot" tumors. To maximize the clinical efficacy of immunotherapy, combining ICI with methods to convert "cold" tumors into "hot" tumors may improve their response to ICI treatment. However, the potential toxicity of molecular glues remains a concern because they may degrade cancer tissue and normal tissue proteins, which greatly limits their clinical application.

[0005] Therefore, in order to solve the above problems, it is of great significance to prepare a responsive prodrug. Summary of the invention

[0006] The purpose of the present invention is to provide a responsive prodrug and a preparation method and application thereof to solve the problems raised in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A responsive prodrug, comprising CR8 and a responsive H 2 O 2 Cleavage of the boronate fragment.

[0009] More optimally, the CR8 is a molecular glue targeting the cyclin-dependent kinase CDK12.

[0010] Among them, CR8 is a molecular glue degrader that inhibits cyclin-dependent kinases by interacting with the cyclin-dependent kinase 12 (CDK12)-cyclin K (CCNK) complex. This interaction recruits the DDB1-CUL4-RBX1E3 ligase, promotes the ubiquitination of cyclin K, leads to the selective inactivation of CDK12, and ultimately induces tumor cell death. In the tumor microenvironment (TME), the death of tumor cells leads to the release of tumor antigens, which are processed and presented to the immune system, activating CD8 + T cells and may convert a "cold" tumor into a "hot" tumor.

[0011] More optimally, the boronate ester fragment is 4-(hydroxymethyl)phenylboronic acid pinacol ester.

[0012] Among them, due to the potential toxicity of CR8, the present invention introduces a borate fragment to combine with it to prepare H 2 O 2 Responsive drugs, because the active site of CR8 is masked, will not take effect until the molecules undergo enzymatic or chemical transformation in the living body, thereby effectively reducing its degradation of normal tissue proteins; and this design gives CR8 prodrug the advantage of tumor selectivity, allowing it to not only effectively inhibit tumor growth, but also reactivate T cell responses.

[0013] A method for preparing a responsive prodrug comprises the following steps: dissolving 4-(hydroxymethyl)phenylboronic acid pinacol ester in dichloromethane, adding 1,1'-carbonyldiimidazole dropwise, stirring at room temperature for 12-15 hours, washing, drying and concentrating to obtain a first intermediate; dissolving the first intermediate and CR8 in tetrahydrofuran, adding DUB dropwise, stirring at room temperature overnight, extracting and purifying to obtain a second intermediate; dissolving the second intermediate in an acetone-methanol mixed solvent, adding ammonium acetate and sodium periodate, stirring at room temperature overnight, post-treating to obtain a responsive prodrug.

[0014] More optimally, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to 1,1'-carbonyldiimidazole in the raw materials of the responsive prodrug is (0.05-0.15):(0.1-0.15).

[0015] More optimally, in the raw materials of the responsive prodrug, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to CR8 is (0.05-0.15): (0.01-0.1).

[0016] More optimally, the preparation method of CR8 comprises the following steps:

[0017] (1) Under room temperature, 4-(4-pyridyl)benzaldehyde, hydroxylamine hydrochloride, and sodium acetate are mixed in ethanol to react to obtain intermediate A; in a preferred embodiment, the ratio of 4-(4-pyridyl)benzaldehyde, hydroxylamine hydrochloride, sodium acetate, and ethanol is (0.08 mol to 0.12 mol): 0.11 mol: 0.11 mol: 25 mL;

[0018] (2) Dissolve the intermediate A in ethanol, use Pb / C catalyst under hydrogen atmosphere, and react at room temperature for 18 to 20 hours to obtain the intermediate B; in the preferred embodiment, the ratio of the intermediate A, ethanol, and Pb / C catalyst is (0.05 mol to 0.1 mol): 100 mL: 1 g; the content of the Pb / C catalyst is 10%;

[0019] (3) Dissolve intermediate B in tetrahydrofuran, cool to 0°C, add triethylamine and di-tert-butyl dicarbonate, stir at room temperature for 4-5 hours, concentrate in vacuo, and purify to obtain intermediate C; in a preferred embodiment, the ratio of intermediate B, tetrahydrofuran, triethylamine, and di-tert-butyl dicarbonate is (0.05 mol-0.1 mol): 75 mL: 0.06 mol: 0.06 mol;

[0020] (4) Dissolve the intermediate C in dichloromethane, cool to 0°C, add anhydrous trifluoroacetic acid, remove the ice bath, react at room temperature for 2-3 hours, concentrate in vacuo, and purify to obtain the intermediate D; in a preferred embodiment, the ratio of the amount of intermediate C, dichloromethane, anhydrous trifluoroacetic acid, and diethyl ether is (0.032 mol-0.035 mol): 40 mL: 40 mL: 20 mL;

[0021] (5) Dissolve the intermediate D in n-butanol, add primary amine and triethylamine, react at 111-115° C. for 3-4 hours, cool to 15-20° C., wash and dry to obtain the intermediate E; in a preferred embodiment, the ratio of the amount of the intermediate D, n-butanol, primary amine and triethylamine is (0.05 mol-0.1 mol): 100 mL: 0.06 mol: 0.25 mol;

[0022] (6) Dissolve the intermediate E in dimethyl sulfoxide, add potassium carbonate and part of 2-bromopropane, stir at room temperature for 5 to 6 hours, continue to add the remaining part of 2-bromopropane, continue to react for 5 to 6 hours, and post-treat to obtain the intermediate F; in the preferred embodiment, the ratio of the amount of intermediate E, dimethyl sulfoxide, potassium carbonate, and 2-bromopropane is (0.1 mol to 0.2 mol): 5 mL: 0.12 mol: 0.125 mol;

[0023] (7) Under nitrogen protection, the intermediate F is mixed with 2,6-dichloropurine, reacted at 160-170° C. for 8-9 h, cooled, and post-treated to obtain CR8; in a preferred embodiment, the ratio of 2,6-dichloropurine to intermediate F is 0.07 mol: (0.56 mol-0.6 mol).

[0024] An application of a responsive prodrug, wherein the responsive prodrug is used to prepare a drug for treating triple-negative breast cancer.

[0025] Compared with the prior art, the advantages of the present invention are as follows: the present invention successfully designs and synthesizes a prodrug, which is based on the molecular glue CR8 targeting the cyclin-dependent kinase CDK12 and has responsiveness to hydrogen peroxide.

[0026] This design gives the CR8 prodrug an advantage in tumor selectivity, allowing it to not only effectively inhibit tumor growth but also reactivate T cell responses. Administered by tail vein injection, the prodrug can respond to hydrogen peroxide signals in the in vivo environment, thereby releasing CR8 molecular glue in the tumor microenvironment and achieving precise treatment of triple-negative breast cancer. In addition, the prodrug has the ability to self-assemble, forming nanoparticles and further aggregated into nanoclusters with an average size of 193nm. This self-assembly property may promote its accumulation in the tumor area through enhanced permeability and retention effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The synthetic route of pCR8 in Example 1 is shown in FIG.

[0028] Figure 2 is the pCR8 response H in Example 1 2 O 2 The mechanism of release of quinone methide, boric acid and CR8;

[0029] Figure 3 is the HPLC spectrum of pCR8;

[0030] Figure 4 is the mass spectrometry spectrum of pCR8;

[0031] Figure 5 TEM results of CR8;

[0032] Figure 6 TEM results of pCR8;

[0033] Figure 7 The particle size results of pCR8;

[0034] Figure 8 For pCR8 with 10 mM HO 2 O 2 HPLC analysis after incubation in PBS at 37°C for different time points;

[0035] Fig. 9 For pCR8 with 10 mM HO 2 O 2 The remaining percentage after incubation in PBS at 37°C for different time points;

[0036] Fig.10 HPLC analysis of pCR8 after incubation in PBS at 37°C for different time points;

[0037] Fig.11 It is the remaining percentage of pCR8 after incubation in PBS at 37°C for different time points;

[0038] Fig.12 Survival rate of NIH3T3 cells after treatment with specific concentrations of CR8 and pCR8;

[0039] Fig.13 Specific concentrations of CR8 and pCR8 were used in the absence or presence of H 2 O 2 The survival rate of 4T1 cells after treatment with

[0040] Fig.14 In the presence or absence of H 2 O 2 The expression of CDK12 in 4T1 cells after treatment with specific concentrations of CR8 and pCR8;

[0041] Fig.15 In the presence or absence of H 2 O 2 The expression of CCNK in 4T1 cells after treatment with specific concentrations of CR8 and pCR8;

[0042] Fig.16 Scratch assay to evaluate the migration ability of 4T1 cells, scale bar is 500 μm;

[0043] Fig.17 For quantitative analysis of the healing area of ​​the scratch test;

[0044] Fig.18For the migration assay to assess the invasive ability of 4T1 cells, the scale bar is 200 μm;

[0045] Fig.19 4T1 cells were treated with specific concentrations of CR8 and pCR8 in the absence or presence of H 2 O 2 Dead and alive staining results in the case of;

[0046] Fig. 20 This is a photo of the tumor in BALB / C tumor-bearing mice after treatment;

[0047] Fig.21 The weight of tumor tissue in BALB / C tumor-bearing mice on the 8th day after drug treatment;

[0048] Fig. 22 This is the tumor growth curve of each group in the administration cycle of BALB / C tumor-bearing mice;

[0049] Fig.23 H&E stained sections of tumor tissues, scale bar 150 μm; CCNK, scale bar 250 μm and CDK12, scale bar 100 μm, immunohistochemical images;

[0050] Fig.24 For CD8 + Immunofluorescence images of T cells, scale bar is 250 μm;

[0051] Fig.25 The body weight of BALB / c tumor-bearing mice was measured during the 8-day treatment period;

[0052] Fig.26 It is the WBC index of whole blood of BALB / c tumor-bearing mice;

[0053] Fig. 27 It is the RBC index of whole blood of BALB / c tumor-bearing mice;

[0054] Fig.28 It is the HGB index of whole blood of BALB / c tumor-bearing mice;

[0055] Fig.29 It is the PLT index of whole blood of BALB / c tumor-bearing mice;

[0056] Fig.30 ALT is the liver function index of BALB / c tumor-bearing mice;

[0057] Fig.31 AST is the liver function index of BALB / c tumor-bearing mice;

[0058] Fig.32 ALP is the liver function index of BALB / c tumor-bearing mice;

[0059] Fig.33 BUN is the renal function index of BALB / c tumor-bearing mice;

[0060] Fig.34 CREA is the renal function index of BALB / c tumor-bearing mice;

[0061] Fig.35 H&E-stained sections of the heart, liver, spleen, lung, and kidney of each group of BALB / c tumor-bearing mice;

[0062] Fig.36 Flow cytometry shows CD8 + T cells and GzmB + CD8 + The percentage of T cells (middle) and CD8 + T cells (right);

[0063] Fig.37 CD8 + Quantitative analysis of T cell percentage;

[0064] Fig.38 GzmB in blood + CD8 + Quantitative analysis of the percentage of T cells;

[0065] Fig.39 CD8 + Quantitative analysis of T cell percentage;

[0066] Fig.40 GzmB + CD8 + Quantitative analysis of the percentage of T cells;

[0067] Fig.41 CD8 + Quantitative analysis of the percentage of T cells;

[0068] Fig.42 is the expression of IL-6 in BALB / c tumor-bearing tissues after treatment;

[0069] Fig.43 is the expression of IL-12 in BALB / c tumor-bearing tissues after treatment;

[0070] Fig.44 is the expression of IFN-γ in BALB / c tumor-bearing tissues after treatment;

[0071] Fig.45 The expression of TNF-α in BALB / c tumor-bearing tissues after treatment. DETAILED DESCRIPTION

[0072] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0073] It should be noted that the purchase manufacturers of all raw materials involved in the present invention are not subject to any special restrictions and exemplarily include: in the following examples, mouse embryonic fibroblasts (NIH3T3) and mouse breast cancer cells (4T1) are provided by Wuhan Pronosai Biotechnology Co., Ltd.; Transwell plates (Corning Co., Ltd., Lowell, MA, USA); 4-(4-pyridyl)benzaldehyde CAS is 99163-12-9; di-tert-butyl dicarbonate CAS is 24424-99-5; 2,6-dichloropurine CAS is 5451-40-1; 4-(hydroxymethyl)phenylboronic acid pinacol ester CAS is 302348-51-2.

[0074] Example 1: A method for preparing a responsive prodrug, comprising the following steps: Figure 1 Shown

[0075] Step 1: Synthesis of CR8:

[0076] (1) At room temperature, 0.1 mol of 4-(4-pyridyl)benzaldehyde ( Figure 1 1), 0.11 mol of hydroxylamine hydrochloride and 0.11 mol of sodium acetate were mixed in 25 mL of ethanol to react to obtain intermediate A ( Figure 1 2) in;

[0077] (2) 0.05 mol of intermediate A was dissolved in 100 mL of ethanol, and 1 g of 10% Pb / C catalyst was used under a hydrogen atmosphere to react at room temperature for 18 h to obtain intermediate B ( Figure 1 3) in;

[0078] (3) 0.05 mol of intermediate B was dissolved in 75 mL of tetrahydrofuran, cooled to 0°C, 0.06 mol of triethylamine and 0.06 mol of di-tert-butyl dicarbonate were added, stirred at room temperature for 4 h, concentrated in vacuo, and the residue was washed twice with 10 mL of ether and purified by column chromatography to obtain intermediate C ( Figure 1 4) in;

[0079] (4) 0.032 mol of intermediate C was dissolved in 40 mL of dichloromethane, cooled to 0°C, and then 40 mL of anhydrous trifluoroacetic acid was added. The ice bath was removed and the reaction was carried out at room temperature for 2 h. The residue was slurried with 20 mL of ether and 20 mL of ether was added. The precipitated product was intermediate D ( Figure 1 5) in;

[0080] (5) 0.05 mol of intermediate D was dissolved in 100 mL of n-butanol, 0.06 mol of primary amine and 0.25 mol of triethylamine were added, and the mixture was reacted at 111° C. for 3 h, then cooled to 20° C. The resulting solid was washed with 5 mL of glacial n-butanol and dried in vacuo to obtain intermediate E ( Figure 1 6);

[0081] (6) 0.1 mol of intermediate E was dissolved in 5 mL of DMSO, 0.12 mol of potassium carbonate and 0.1 mol of 2-bromopropane were added, and the mixture was stirred at room temperature for 5 h. Then 0.025 mol of 2-bromopropane was added, and the reaction was continued for 5 h. The solution was treated with 100 mL of ice water (5° C.) and extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, and concentrated in vacuo to obtain intermediate F ( Figure 1 7);

[0082] (7) Under nitrogen protection, 0.07 mol of 2,6-dichloropurine was reacted with 0.56 mol of intermediate F at 160° C. for 8 hours. After cooling, the reaction mixture was treated with 100 mL of water and extracted three times with 100 mL of ethyl acetate. The organic phase was washed with warm water, dried, and concentrated under vacuum to obtain CR8;

[0083] Step 2: Synthesis of responsive prodrug:

[0084] 4-(Hydroxymethyl)phenylboronic acid pinacol ester (234.1 mg, 0.1 mmol) was dissolved in 8 mL of dichloromethane, and 1,1'-carbonyldiimidazole (194.4 mg, 0.12 mmol) was added dropwise; the mixture was stirred at room temperature for 12 hours; then, the organic layer was washed three times with 10 mL of water, then washed with brine, dried over sodium sulfate, and concentrated under vacuum; the residue was directly dissolved in 10 mL of tetrahydrofuran with CR8 (216 mg, 0.05 mmol) without purification, DBU (1.2 eq) was added dropwise, and stirred at room temperature overnight; then water was added, and extracted with ethyl acetate, and the organic phase was washed with water and brine, dried over sodium sulfate, and concentrated under vacuum. The obtained product was purified using dichloromethane / methanol (100:3) as an eluent pair; subsequently, the product was dissolved in 8 mL of an acetone:methanol (1:1) mixture, and ammonium acetate and sodium periodate (2.3 equivalents each) were added, and the solution was stirred at room temperature overnight; the solvent was evaporated in vacuo, the mixture was extracted with ethyl acetate, and washed with water and brine, and then dried over sodium sulfate; the solution was concentrated under reduced pressure, and the residue was purified by column chromatography using dichloromethane / methanol (100:1) as an eluent to finally obtain the responsive prodrug (pCR8).

[0085] Performance test: The responsive prodrug prepared in Example 1 was subjected to relevant performance tests; the details are as follows:

[0086] (I) Hydrogen peroxide responsiveness

[0087] The responsive prodrug (pCR8) was dissolved in DMSO to prepare a 20 mg / mL stock solution, then diluted with PBS to a final concentration of 1 mg / mL of CR8, and hydrogen peroxide was added to a final concentration of 10 mM. The solution was incubated at 37°C, and samples were taken at determined time points for HPLC analysis.

[0088] The results are as follows Figure 8-Figure 11 As shown, the results show that in H 2 O 2 In the presence of H, pCR8 released CR8 with a half-life of 2.5 hours. 2 O 2 In the absence of stimulation, minimal cleavage of pCR8 was observed after 5 h, indicating slow degradation in the absence of stimulation. These findings suggest that H 2 O 2 The boronate fragment in pCR8 is efficiently cleaved, thereby releasing the active drug CR8.

[0089] (II) Evaluation of the in vitro antitumor activity of responsive prodrug (pCR8)

[0090] 1) Cell culture

[0091] 4T1 cells were grown in RPMI 1640 and NIH3T3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), both of which were supplemented with streptomycin-penicillin and 10% fetal bovine serum (FBS) from Gibco. Cells were maintained in an atmosphere of 5% CO. 2 / 95% air maintained at 37°C, all cell lines tested negative for mycoplasma, and the cells were cultured to 80% confluence, with a total of no more than 20 generations;

[0092] 2) Cell proliferation assay

[0093] The cell viability of 4T1 and NIH3T3 cells was determined using a cell counting kit (CCK-8). 3 The number of cells was seeded in a 96-well plate and incubated overnight at 37°C in complete medium. Each concentration was repeated three times. Then, different concentrations of CR8 and pCR8 (with or without different concentrations of H 2 O 2 ) for 72 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for one hour to measure their survival rate.

[0094] like Fig.12As shown, NIH3T3 cells were treated with specific concentrations of CR8 and pCR8, indicating that CR8 has a certain potential toxicity and will inhibit the survival of normal cells, while pCR8 is a responsive prodrug and will not affect the survival rate of normal cells to a certain extent; Fig.13 As shown, pCR8 has a lower toxicity to 4T1 cells compared with CR8, but 2 O 2 In the presence of , the inhibition of 4T1 cell survival rate was reduced.

[0095] 3) Western Blot

[0096] In cells lysed with RIPA buffer (50 mM Tris, pH = 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycholate; 0.1% SDS), supplemented with protease (cat#: P1045) and phosphatase inhibitors (cat#: P005), the lysate was centrifuged at 12000 rpm for 15 min at 4°C, the supernatant was separated by SDS-PAGE using 10% acrylamide gel, and then the protein was transferred to a PVDF membrane in 1× Tris / glycine buffer containing 20% ​​methanol at 80 V for 2 hours, blocked with 5% nonfat dry milk in TBST for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C, the membranes were washed three times in TBST for 10 minutes each, and incubated with HRP-conjugated secondary antibodies for 1 hour, and the detection of bound antibodies was performed using an ECL detection kit (ThermoFisher, cat#: A38554). Fig.14 and Fig.15 The results showed that CR8 could reduce the degradation of its target proteins CCNK and CDK12, but its prodrug had a lower degradation level of target proteins. 2 O 2 Afterwards, the level of target protein degradation was restored.

[0097] 4) Scratch healing experiment

[0098] 4T1 cells were plated at 8×10 per well 4 The cells were seeded in complete medium in 24-well plates and incubated for 24 h before scratches were introduced into the cell monolayer using a pipette tip. The scratches were rinsed with PBS to remove non-adherent cells and then treated with different concentrations of CR8 and pCR8, with or without H 2 O 2 , using a Nikon Ts microscope at 10× magnification to monitor cell migration to the scratch area and quantification using ImageJ software, all experiments were repeated three times; the measured results are shown in Figure 16-Figure 17As shown in Figure 5, pCR8 had a significantly lower ability to inhibit cell migration and proliferation than CR8. 2 O 2 In the presence of 5-6-nitropropene, the inhibitory ability of pCR8 on 4T1 cell migration was reduced.

[0099] 5) Cell migration assay

[0100] Migration assays were performed using 24-well Transwell plates with 6 μm membrane chambers. 4T1 cells (1×10 5 ) were seeded in serum-free medium, while the lower part of the chamber was in complete medium. After incubation for 24 h, the remaining cells on the top surface of the insert were removed with a cotton swab, and the migrated cells were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. The number of migrated cells was quantified at 10x magnification using a Nikon Ts microscope and analyzed using ImageJ software; the results measured are shown in Fig.18 As shown in Figure 2, compared with CR8, the ability of pCR8 to inhibit cell migration was significantly reduced. 2 O 2 In the presence of 5-6-nitropropene, the inhibitory ability of pCR8 on 4T1 cell migration was reduced.

[0101] 6) Live and dead staining experiment

[0102] The survival rate of 4T1 cells was assessed using a Calcein AM / Propidium Iodide (PI) double staining kit (DOJINDO LABORATORIES, cat#: C542). Cells were plated at 1 × 10 4 The amount of CR8 and pCR8 (500 nM) in 96-well plates was measured with or without H 2 O 2 The cells were then incubated for 24 hours in the presence of NSARC. The cells were then stained according to the kit protocol and the viability was assessed by confocal microscopy (AX / AX R with NSARC). Fig.19 As shown, pCR8 had significantly lower toxicity to 4T1 cells compared with CR8. 2 O 2 Significantly enhanced the cytotoxicity of pCR8.

[0103] (III) In vivo antitumor activity and biosafety evaluation of pCR8

[0104] 1) Animal modeling

[0105] Six-week-old female BALB / c mice obtained from SLAC (Shanghai, China) were housed in a specific pathogen-free (SPF) environment with free access to water and standard chow under a 12-h light / dark cycle. 4T1 cells (5 × 10 5 );

[0106] 2) Anticancer efficacy and safety evaluation

[0107] When the tumor volume reaches about 100 mm 3 Mice were randomly divided into five groups, each containing five individuals: (1) PBS (2) CR8 (3) pCR8 (4) aPD-L1 (5) pCR8 + aPD-L1. PBS, CR8 and pCR8 were injected through the tail vein every two days, while aPD-L1 was injected intraperitoneally every three days. Tumor diameter and body weight were recorded every two days, and tumor volume was determined using the following formula: width 2 × length × 0.5, Eight days after treatment, mice were euthanized, and tumors were collected for histological analysis, and blood samples were collected for liver and kidney function tests; the measured results were as follows Figure 20-Figure 34 As shown; during the 8 days of treatment, pCR8+aPD-L1 showed the most significant tumor inhibition, with the average tumor volume less than 50mm after treatment 3 ( Fig. 20 ); In addition, after treatment, the tumor weight and tumor volume of the pCR8+aPD-L1 group were also the smallest ( Fig.21 and Fig. 22 The mice's body weight remained stable throughout the treatment period, suggesting that the different interventions did not cause serious adverse effects ( Fig.23 ). Routine blood count results showed no significant differences in white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), or platelet count (PLT) between the PBS group and the treatment group ( Figure 24-Figure 27 However, biochemical analysis of liver function after treatment in the CR8 group showed that the levels of hepatocyte injury markers (alanine aminotransferase ALT, aspartate aminotransferase AST, and alkaline phosphatase ALP) and glomerular filtration rate indices (urea nitrogen BUN and creatinine CREA) were increased compared with the pCR8 and PBS groups ( Figure 28-Figure 32 ). These findings suggest that liver and kidney function was impaired in the CR8 group, whereas the biosafety of the pCR8 group was significantly improved. Histological examination of liver tissue in the CR8 group showed significant tissue damage, which further confirmed these observations. Overall, these results suggest that the biosafety of pCR8 is significantly better than that of CR8.

[0108] 3) Immunohistochemistry

[0109] Tumor tissues were removed, slides were fixed with 4% paraformaldehyde, and 5 μm thick sections were prepared for immunohistochemistry (IHC) staining of CCNK and CDK12 and observed using a 3DHISTECH panoramic MIDI slide scanner; the measured results Fig.33 As shown in Figure 2, the tumor tissues in the PBS group appeared to have increased cells, while the pCR8+aPD-L1 treatment group showed obvious tumor cell necrosis. In addition, immunohistochemical staining showed that the expression levels of CCNK and CDK12 in the tumor tissues of the CR8, pCR8, and pCR8+aPD-L1 groups were reduced compared with those in the PBS group, indicating the protein degradation effect of CR8. Fig.34 As shown, liver and kidney function was impaired in the CR8 group, while biosafety was significantly improved in the pCR8 group. Histological examination of liver tissue in the CR8 group showed significant tissue damage, which further confirmed these observations.

[0110] 4) Immunofluorescence

[0111] To prepare tumors for analysis, they were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The sections were incubated with anti-CD8 antibody at 4 °C overnight and then treated with secondary antibody for 30 min at room temperature in the dark. After DAPI staining for 5 min, the sections were sealed and CD8 in tumor tissues were observed under a confocal fluorescence microscope. + T cell expression; the results measured are as follows Fig.35 As shown, compared with the PBS group, the CD8 + A significant increase in T cells suggests that TME remodeling can alleviate CD8 + T cell exhaustion, thereby modulating the TME and enhancing antitumor responses. These findings suggest that CR8 plays a key role in eliciting antitumor immune responses and enhancing the effectiveness of ICIs.

[0112] (IV) In vivo tumor immune activation

[0113] 1) Streaming

[0114] After observing promising anticancer effects of pCR8 in vivo, we explored whether it might interact with T cells to enhance immune activation of T cells in the TME. + A key function of T cells is to recognize and eliminate infected or abnormal cells through a variety of mechanisms, including the direct release of cytotoxic molecules. + CD8 +T cells induce apoptosis of target cells by secreting granzyme B (GzmB). Therefore, after different treatments, tumor, blood and spleen tissues were collected for flow cytometric analysis. Tumors, spleen and blood were processed into single cell suspensions and then stained with PE anti-mouse CD3 (Biolegend, cat#:100307), KIRAVIABlue 520TM anti-mouse CD4 (Biolegnd, cat#:100477), PE / Cyanine7 anti-mouse CD8 (Biolegund, cat#:100721), APC anti-mouse CD45 (Biolegende, cat#:103111) and Granzyme B-BV421 (Biolegenda, cat#:423102) according to the manufacturer's instructions. Flow cytometry was performed using an LSR Fortessa analyzer (BD). Data analysis was performed using FlowJo software. The measured results are shown in the figure. Figure 36-Figure 41 As shown; after pCR8+aPD-L1 treatment, CD8 + T cells and GzmB + CD8 + The highest level of T cells ( Figure 6 AH).

[0115] 2)ELISA

[0116] Take whole blood from BLAB / c tumor-bearing mice and place it at room temperature for 30 minutes to 2 hours. Do not shake it violently to avoid hemolysis. After the whole blood coagulates naturally and the serum is separated, centrifuge it at about 1000-2000g for 10 minutes at 4℃. Take the yellow supernatant to get the serum. Add the sample and different concentrations of standard products to the corresponding wells at 90μL / well, add the sample dilution as the 0 concentration standard, and then add the prepared horseradish peroxidase estradiol at 10μL / well. Mix well for 10 seconds, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 120 minutes. If the sample contains estrogen, If the diol content is higher than 2000pg / mL, the sample should be diluted with zero-concentration sample diluent before measurement. Please remember to record the dilution multiple of the sample. Wash the plate 3 times, and pat it dry on thick absorbent paper for the last time. Add 100μL / well of the color developer TMB solution, seal the reaction well with a sealing film (white), and incubate at room temperature in the dark for 15-20 minutes. If the room temperature is low, the incubation time needs to be appropriately extended. At this time, you can incubate until the standard shows a very significant color change. If the sample concentration is high enough, a significant color change will also occur. Add 50μL / well of the stop solution, mix well and measure the A450 value immediately. The results are as follows Figure 42-45As shown, we observed upregulation of relevant biomarkers (TNF-α, IL-6, IL-12, and IFN-γ) after treatment compared with the PBS group, with the most significant upregulation in the pCR8+aPD-L1 group, indicating reprogramming of T cell translation.

[0117] Conclusion: The present invention designs and synthesizes a hydrogen peroxide-responsive prodrug based on the cyclin-dependent kinase CDK12 molecular glue CR8, thereby providing CR8 tumor selectivity, enabling it to effectively inhibit tumors and reprogram T cell responses, and ultimately achieving the effect of hydrogen peroxide responsive release and treatment of triple-negative breast cancer through tail vein administration.

[0118] Finally, it should be noted that the above description is only 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A responsive prodrug, characterized in that: The responsive prodrug includes CR8 and a boronate ester fragment that can be cleaved in response to H2O2.

2. A responsive prodrug according to claim 1, characterized in that: The CR8 is a molecular glue targeting the cyclin-dependent kinase CDK12.

3. A responsive prodrug according to claim 1, characterized in that: The boronate fragment is 4-(hydroxymethyl)phenylboronic acid pinacol ester.

4. The method for preparing a responsive prodrug according to claim 1, characterized in that: The following steps are involved: 4-(Hydroxymethyl)phenylboronic acid pinacol ester is dissolved in dichloromethane, 1,1'-carbonyldiimidazole is added dropwise, stirred at room temperature for 12 to 15 hours, washed, dried, and concentrated to obtain a first intermediate; the first intermediate and CR8 are dissolved in tetrahydrofuran, DUB is added dropwise, stirred at room temperature overnight, extracted and purified to obtain a second intermediate; the second intermediate is dissolved in an acetone-methanol mixed solvent, ammonium acetate and sodium periodate are added, stirred at room temperature overnight, and post-treated to obtain a responsive prodrug.

5. The method for preparing a responsive prodrug according to claim 4, characterized in that: The molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to 1,1'-carbonyldiimidazole in the raw materials of the responsive prodrug is (0.05-0.15): (0.1-0.15).

6. The method for preparing a responsive prodrug according to claim 4, characterized in that: In the raw materials of the responsive prodrug, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to CR8 is (0.05-0.15): (0.01-0.1).

7. The method for preparing a responsive prodrug according to claim 4, characterized in that: The preparation method of CR8 comprises the following steps: (1) Under room temperature, 4-(4-pyridyl)benzaldehyde, hydroxylamine hydrochloride, and sodium acetate are mixed in ethanol to react to obtain intermediate A; (2) Dissolve intermediate A in ethanol, use Pb / C catalyst under hydrogen atmosphere, and react at room temperature for 18 to 20 hours to obtain intermediate B; (3) Dissolve intermediate B in tetrahydrofuran, cool to 0°C, add triethylamine and di-tert-butyl dicarbonate, stir at room temperature for 4-5 hours, concentrate in vacuo, and purify to obtain intermediate C; (4) Dissolve intermediate C in dichloromethane, cool to 0°C, add anhydrous trifluoroacetic acid, remove the ice bath, react at room temperature for 2-3 hours, concentrate in vacuo, and purify to obtain intermediate D; (5) Dissolve intermediate D in n-butanol, add primary amine and triethylamine, react at 111-115° C. for 3-4 hours, cool to 15-20° C., wash and dry to obtain intermediate E; (6) Dissolve intermediate E in dimethyl sulfoxide, add potassium carbonate and part of 2-bromopropane, stir at room temperature for 5 to 6 hours, continue to add the remaining part of 2-bromopropane, continue to react for 5 to 6 hours, and post-treat to obtain intermediate F; (7) Under nitrogen protection, the intermediate F is mixed with 2,6-dichloropurine, reacted at 160-170°C for 8-9 hours, cooled, and post-treated to obtain CR8.

8. The use of a responsive prodrug according to claim 1, characterized in that: The responsive prodrug is used to prepare a drug for treating triple-negative breast cancer.

Images