6-diazo-5-oxo-n-leucine prodrug as well as preparation method and application thereof

Abstract

The invention provides a 6-diazo-5-oxo-n-leucine prodrug and a preparation method and application thereof.According to the 6-diazo-5-oxo-n-leucine prodrug and a compound, a glutamine metabolism inhibitor JHU083 and an STING pathway agonist MSA-2 are connected through a cleavable amido bond, two action mechanisms are fused, the activity of the 6-diazo-5-oxo-n-leucine prodrug is improved, the activity of the 6-diazo-5-oxo-n-leucine prodrug is improved, and the activity of the 6-diazo-5-oxo-n-leucine prodrug is improved. On one hand, the metabolic advantage of tumor cells is limited, and the immunosuppressed tumor microenvironment is reversed; the STING pathway of dendritic cells is directly activated, immune factors such as type I interferon and tumor necrosis factor TNF-alpha are induced to release, and immune activation is synergistically enhanced; the dendritic cell function is improved, the antigen presentation capability is enhanced, the co-stimulatory molecule expression level of DC can be remarkably improved, the antigen presentation capability of the DC can be enhanced, the activation efficiency of T cells is improved, and the anti-tumor immune effect is enhanced; and the compound has good chemical stability and drug development potential, is suitable for further pharmacological research and preclinical development, and has good synthesizability and pharmaceutical properties.

Description

Technical Field

[0001] This invention belongs to the fields of chemistry and medicine, and particularly relates to a 6-diazo-5-oxo-ortholeucine prodrug, its preparation method and application. Background Technology

[0002] In recent years, tumor immunotherapy has become an important direction in cancer treatment. In particular, the strategy of activating the body's immune system to recognize and eliminate tumor cells has received widespread attention. Dendritic cells (DCs), as the most critical antigen-presenting cells, play a core role in stimulating the initial T cell response and establishing anti-tumor immune memory. However, in the immunosuppressive tumor microenvironment (TME) of solid tumors, DC function is often suppressed, manifested as impaired maturation and weakened antigen-presenting ability, ultimately leading to T cell activation disorders and immunotherapy tolerance.

[0003] Previous studies have shown that tumor cells competitively take up glutamine, limiting the utilization of this metabolic substrate by local immune cells, thereby further exacerbating the immunosuppressive state in the tumor microenvironment (TME). Currently, the representative glutamine antagonist is 6-diazo-5-oxo-leucine (DON), which can significantly block the glutamine utilization capacity of tumor cells. In addition, JHU083 is a glutamine antagonist in the form of a DON prodrug that can inhibit glutamine metabolism and reverse immunosuppression to some extent. However, its in vivo effects are highly dose-dependent, have significant side effects, and limited ability to activate the innate immune system. On the other hand, the STING (Stimulator of Interferon Genes) pathway, as an important mediating mechanism of innate immune response, plays an important role in anti-tumor immunity by inducing the production of inflammatory factors such as type I interferon. MSA-2 is a STING agonist that has shown the potential to activate dendritic cells (DCs) and enhance anti-tumor effects in various tumor models. However, due to its poor metabolic stability, poor solubility, and limited target, the therapeutic effect of MSA-2 when used alone is still not ideal.

[0004] Therefore, there are currently no novel compounds that can simultaneously intervene in metabolic inhibition and activate innate immune responses, and cannot synergistically enhance DC activity, improve the tumor immune microenvironment, or stimulate effective T cell responses, which is an important direction in current tumor immunotherapy research. Therefore, it is necessary to provide an immunometabolite prodrug of DON and STING agonists, along with its preparation method and application, to solve the aforementioned technical problems. Summary of the Invention

[0005] This invention provides a 6-diazo-5-oxo-leucine prodrug, its preparation method, and its application, which solves the problem that there are currently no novel compounds that can simultaneously intervene in metabolic inhibition and activate innate immune responses, and that cannot synergistically enhance DC activity, improve the tumor immune microenvironment, and stimulate effective T cell responses.

[0006] To solve the above-mentioned technical problems, the present invention provides a 6-diazo-5-oxo-leucine prodrug, comprising: the chemical structure of the prodrug molecule is as follows:

[0007]

[0008] In general formula (1):

[0009] Wherein R is selected from hydrogen atom, halogen, cyano, nitro, unsubstituted or substituted with at least one of the following groups: C1-6 alkyl, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkoxy, C2-6 alkenyloxy, C2-6 alkoxy, C2-6 alkenyloxy, etc., wherein the following groups may be the same or different and are selected from halogen, hydroxyl, cyano, nitro and amino; unsubstituted or substituted with the following groups: amino, wherein the following groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenylalkyl, etc. C1-6 alkyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl; unsubstituted and substituted ester and amide groups, wherein the hydroxyl and amino groups in the substituted ester and amide groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl;

[0010] The optical isomers, diastereomers, and pharmaceutically acceptable salts of the compounds shown, as well as pharmaceutically acceptable carriers, excipients, and excipients.

[0011] Preferably, in the general formula (1): R is selected from hydrogen atoms, C1-6 alkyl-substituted ester groups; optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown, and pharmaceutically acceptable carriers, excipients and formulations, characterized in that: its dosage form is oral tablets, capsules, injections, lyophilized powder for injection and sustained-release formulations.

[0012] Preferably, the compound represented by general formula (1) is the following compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients, characterized in that: its dosage form is oral tablets, capsules, injections, lyophilized powder injections and sustained-release preparations.

[0013]

[0014] The use of a 6-diazo-5-oxo-leucine prodrug, the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, and pharmaceutically acceptable carriers, excipients and excipients in the preparation of cancer therapeutic drugs.

[0015] Preferably, the use of the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients, in the preparation of drugs for treating breast cancer, melanoma and colorectal cancer.

[0016] A method for preparing a 6-diazo-5-oxo-leucine prodrug, wherein the DON and its derivatives are condensed with MSA-2 via amide condensation to obtain a compound of general formula (1).

[0017]

[0018] Compared with related technologies, the 6-diazo-5-oxo-ortholeucine prodrug provided by the present invention has the following beneficial effects:

[0019] This invention provides a 6-diazo-5-oxo-leucine prodrug. The compound combines two mechanisms of action by linking the glutamine metabolism inhibitor JHU083 with the STING pathway agonist MSA-2 via a cleavable amide bond. On one hand, it can limit the metabolic advantage of tumor cells, reversing the immunosuppressive tumor microenvironment; on the other hand, it directly activates the STING pathway of dendritic cells, inducing the release of immune factors such as type I interferon and tumor necrosis factor TNF-α, thereby significantly enhancing innate and adaptive immune responses. This dual mechanism of action synergistically enhances immune activation and improves dendritic cell function, enhancing antigen presentation capacity. It can significantly increase the expression levels of co-stimulatory molecules (such as CD80 and CD86) in dendritic cells, enhancing their antigen presentation capacity, thereby effectively improving T cell activation efficiency and enhancing anti-inflammatory effects. Tumor immune effects; targeting the tumor microenvironment and reducing systemic toxicity: Compared with monotherapy, this invention covalently links two pharmacodynamic components through chemical means, enabling them to achieve spatial co-localization and temporal synergistic release in the tumor microenvironment, reducing the risk of exposure to non-target tissues, and is expected to reduce systemic toxic side effects and improve treatment safety; traditional combination therapy of JHU083 and MSA-2 faces the problem of inconsistent pharmacokinetic characteristics and tissue distribution, which is overcome by a single-molecule unified release method, improving the consistency and controllability of combined therapy, and overcoming inconsistent drug distribution and pharmacokinetic differences; the compound structure of this invention is well-defined, the synthetic route is controllable, and it has good chemical stability and drug development potential, making it suitable for further pharmacological research and preclinical development, and possessing good syntheticability and pharmaceutical properties. Attached Figure Description

[0020] Figure 1 This invention provides a 6-diazo-5-oxo-leucine prodrug. 1 Schematic diagram of H-NMR;

[0021] Figure 2 This invention provides a 6-diazo-5-oxo-leucine prodrug. 13 Schematic diagram of C-NMR;

[0022] Figure 3 This is a schematic diagram of the HRMS of a 6-diazo-5-oxo-ortholeucine prodrug provided by the present invention;

[0023] Figure 4 HE staining of major organs of a mouse. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] First Embodiment

[0026] Please refer to the following: Figure 1 , Figure 2 , Figure 3 and Figure 4 ,in, Figure 1 This invention provides a 6-diazo-5-oxo-leucine prodrug. 1 Schematic diagram of H-NMR; Figure 2 This invention provides a 6-diazo-5-oxo-leucine prodrug. 13 Schematic diagram of C-NMR; Figure 3 This is a schematic diagram of the HRMS of a 6-diazo-5-oxo-ortholeucine prodrug provided by the present invention; Figure 4 HE staining of major organs of a mouse.

[0027] A 6-diazo-5-oxo-ortholeucine prodrug, comprising: the chemical structure of the prodrug molecule is as follows:

[0028]

[0029] In general formula (1):

[0030] Wherein R is selected from hydrogen atom, halogen, cyano, nitro, unsubstituted or substituted with at least one of the following groups: C1-6 alkyl, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkoxy, C2-6 alkenyloxy, C2-6 alkoxy, C2-6 alkenyloxy, etc., wherein the following groups may be the same or different and are selected from halogen, hydroxyl, cyano, nitro and amino; unsubstituted or substituted with the following groups: amino, wherein the following groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenylalkyl, etc. C1-6 alkyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl; unsubstituted and substituted ester and amide groups, wherein the hydroxyl and amino groups in the substituted ester and amide groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl;

[0031] The optical isomers, diastereomers, and pharmaceutically acceptable salts of the compounds shown, as well as pharmaceutically acceptable carriers, excipients, and excipients.

[0032] Preferably, in the general formula (1): R is selected from hydrogen atoms, C1-6 alkyl-substituted ester groups; optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown, and pharmaceutically acceptable carriers, excipients and formulations, characterized in that: its dosage form is oral tablets, capsules, injections, lyophilized powder for injection and sustained-release formulations.

[0033] Preferably, the compound represented by general formula (1) is the following compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients, characterized in that: its dosage form is oral tablets, capsules, injections, lyophilized powder injections and sustained-release preparations.

[0034]

[0035] The use of a 6-diazo-5-oxo-leucine prodrug, the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, and pharmaceutically acceptable carriers, excipients and excipients in the preparation of cancer therapeutic drugs.

[0036] Preferably, the use of the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients, in the preparation of drugs for treating breast cancer, melanoma and colorectal cancer.

[0037] A method for preparing a 6-diazo-5-oxo-leucine prodrug, wherein the DON and its derivatives are condensed with MSA-2 via amide condensation to obtain a compound of general formula (1).

[0038]

[0039] The following examples will provide a better understanding of the compounds of the present invention and their preparation. These examples are intended to illustrate, rather than limit, the scope of the invention.

[0040] Example 1: (S)-6-azido-2-(4-(5,6-dimethoxybenzo[b]thiophene-2-yl)-4-oxobutyrylamino)-5-oxohexanoic acid ethyl ester (T26)

[0041] MSA-2 (73.58 mg, 0.25 mmol, 1 eq), DIPEA (32.31 mg, 0.25 mmol, 1 eq), HATU (95.06 mg, 0.25 mmol, 1 eq), and 10 mL of anhydrous dichloromethane were added sequentially to a 50 mL round-bottom flask. The mixture was stirred and reacted at 0 °C for 30 min. DON (49.80 mg, 0.25 mmol, 1 eq) was then added to the reaction mixture. After the addition was complete, the mixture was stirred at room temperature for 24 h. The solvent was removed by vacuum distillation. 20 mL of water was added to the residue, and the mixture was extracted with dichloromethane (3 × 10 mL). The organic phases were combined and washed successively with water (3 × 15 mL) and saturated brine (1 × 10 mL). The mixture was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure to obtain crude T26. The crude T26 was purified by column chromatography (dichloromethane:methanol = 20:1) to obtain purified T26, a yellow solid, with a yield of 48 mg and a yield of 41%. 1 H-NMR (500MHz, CDCl3) δ7.89 (s, 1H), 7.24 (s, 2H), 6.60 (d, J=8.0Hz, 1H), 5.33 (s, 1H), 4.57 (td , J=8.3, 4.6Hz, 1H), 4.19 (qd, J=7.1, 1.2Hz, 2H), 3.97 (d, J=1.4Hz, 3H), 3.95 (d, J=1.4Hz, 3H), 3 .47(dt, J=17.6, 6.9Hz, 1H), 3.26(dt, J=17.7, 6.1Hz, 1H), 2.73–2.62(m, 2H), 2.50–2.34(m, 2H) , 2.24 (dtd, J=14.6, 7.5, 4.8Hz, 1H), 2.00 (dt, J=14.5, 7.4Hz, 1H), 1.28 (dd, J=7.7, 6.4Hz, 4H). 13C-NMR (126MHz, CDCl3) δ193.97, 192.65, 171.87, 171.79, 151.10, 148.91, 141.08, 136.80, 132.68, 129 .34, 105.96, 103.56, 61.67, 56.20, 56.08, 51.86, 36.39, 33.98, 30.20, 27.41, 14.14.HRMS (ESI): calcd for[M+H] + C 22 H 26 N3O7S, 476.1491; found, 476.1479.

[0042] Pharmacological studies of the present invention

[0043] Example 2: Evaluation of the inhibitory activity of T26 on the proliferation of various tumor cells

[0044] To evaluate the in vitro inhibitory effect of T26 on tumor cells from different sources, the MTT assay was used to detect its effects on mouse colorectal cancer cell line MC38, breast cancer cell line 4T1, and melanoma cell line B16. Cells were seeded at a density of 5,000 cells / well in 96-well cell culture plates. After adhering and stabilizing, different concentrations of T26 solution were added for treatment, with JHU083 and MSA-2 serving as control groups. All cells were incubated for 48 hours after treatment, followed by incubation for 5 hours with MTT solution (final concentration 0.5 mg / mL) added to each well. After discarding the culture medium, DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 490 nm to calculate cell viability for each treatment group.

[0045] Experimental results showed that T26 had a significant inhibitory effect on the proliferation of MC38 cells, 4T1 cells, and B16 cells, with an IC50 value of [missing information]. 50 The concentrations were 1.245 μM, 1.42 μM, and 1.67 μM, respectively, indicating that T26 improved antitumor efficacy while maintaining its glutamine metabolism antagonistic function.

[0046] Table 1: Inhibitory activity of T26 against the proliferation of various tumor cells (IC50, μM, mean ± standard deviation)

[0047]

[0048] Example 3

[0049] Inhibitory effect of prodrug T26 on tumor cell migration

[0050] The ability of tumor cells to migrate is one of the important biological characteristics that enables their invasive growth and distant metastasis. Therefore, inhibiting cell migration is considered an effective strategy to delay tumor progression. This study used a woundhealing assay to detect the effect of the prodrug T26 on tumor cell migration behavior.

[0051] The specific method is as follows: Draw parallel horizontal lines evenly on the back of a 6-well culture plate using a marker, spaced approximately 0.5–1 cm apart, ensuring at least 5 intersecting lines in each well. After digesting tumor cells in the logarithmic growth phase into a single-cell suspension using trypsin, administer 5 × 10⁵ cells per well. 4 Cells were seeded at a density of [number] cells. They were cultured at 37°C and 5% CO2 for 24 hours. Once the cell monolayer had reached confluence, a straight notch was drawn along a pre-drawn marker using a sterile pipette tip to create a simulated wound area. The cells were then gently washed twice with PBS to remove detached cells, and the medium was replaced with a medium containing different treatment drugs (T26, MSA-2, or JHU083), and cultured for another 72 hours. Images of the scratched area were acquired using an inverted microscope at 0 and 72 hours, and the change in scratch area was measured using image analysis software. Cell migration rate was calculated as (initial scratch area - area after 72 hours) / initial scratch area × 100%.

[0052] The experimental results showed that the T26 treatment group exhibited a significant decrease in migration rate in different tumor cell lines, and its inhibitory effect was superior to the control drugs MSA-2 and JHU083, suggesting that T26 has strong anti-migration potential (see Table 2).

[0053] Table 2: Inhibitory effect of T26 on the migration ability of various tumor cells (migration rate %)

[0054]

[0055] Example 4

[0056] Evaluation of the ability of the prodrug T26 to induce tumor cell death

[0057] To verify the cell death-inducing effect of the prodrug T26 on tumor cells, its cytotoxicity was evaluated by combining propidium iodide (PI) staining with fluorescence microscopy for observation and quantification.

[0058] The experimental procedure was as follows: Healthy tumor cells (e.g., MC38 cells, 4T1 cells, or B16 cells) were selected and seeded onto sterile round coverslips, which were then placed in 6-well culture plates. An appropriate amount of complete culture medium was added to each well, and the cells were cultured until a dense monolayer formed on the coverslip surface. Subsequently, culture medium containing a predetermined concentration of T26 was added to the experimental groups; the positive control group received an equimolar concentration of JHU083 or MSA-2; and the negative control group received no drug. All groups were incubated at 37°C and 5% CO2 for 72 hours.

[0059] After culture, remove the coverslip and excess culture medium, add 100 μL of PI working solution prepared according to the kit instructions, and gently shake to ensure the dye evenly covers the cell surface. Incubate at room temperature in the dark for 5–30 min, then aspirate the dye solution and rinse the cells 2–3 times with drug-free culture medium, 5 min each time. Finally, mount the coverslip on a slide and observe under a fluorescence microscope in the red channel (excitation wavelength approximately 535 nm, emission wavelength approximately 617 nm).

[0060] The experimental results showed that the proportion of PI-positive cells in the T26-treated group was significantly higher than that in the JHU083 and MSA-2 groups, indicating that T26 has a stronger effect in inducing tumor cell death. The results are shown in Table 3 below:

[0061] Table 3: Evaluation of T26's ability to induce tumor cell death (percentage of PI-positive cells)

[0062]

[0063]

[0064] Example 5

[0065] The prodrug T26 promotes dendritic cell maturation

[0066] This embodiment constructs a co-culture system of tumor cells and dendritic cells (DCs) and detects the expression levels of maturation markers CD80 and CD86 on the surface of DCs to evaluate the regulatory role of T26 on the maturation state of dendritic cells.

[0067] Dendritic cell line DC2.4 and mouse bone marrow-derived dendritic cells (BMDCs) were used as model cells in the experiment. The extraction method for BMDCs was as follows: femurs and tibias of C57BL / 6 mice were harvested, and bone marrow cells were isolated. These cells were then induced to differentiate for 7 days in RPMI-1640 complete medium containing 20 ng / mL recombinant mouse GM-CSF and 10 ng / mL recombinant mouse IL-4. Half of the medium was replaced and fresh cytokines were added every 3 days. Completely induced BMDCs were obtained on day 7. The dendritic cells were then transferred to the upper chamber of a Transwell system and pre-treated with 1×10⁻⁶ cytokines 24 hours in advance. 5 MC38 colon cancer cells were seeded at a density of 1 cell per well in the lower chamber of a 6-well plate and co-cultured at a ratio of 1:1 (by cell count). Co-culture lasted for 36 hours, during which different treatment groups were set up.

[0068] After culture, BMDCs and DC2.4 in the upper chamber were collected, and their surfaces were stained with anti-CD80-APC and anti-CD86-PE antibodies. CD80 was analyzed by flow cytometry. + With CD86 + The proportion of cells is used to assess the maturation status of dendritic cells.

[0069] Experimental results show that CD80 in BMDCs and DC2.4 of the T26 treatment group is significantly different. + With CD86 + The significantly increased proportion of cells suggests that T26 can significantly promote the maturation of dendritic cells.

[0070] Table 4: Effect of T26 on dendritic cell maturation capacity (CD80) + CD86 + Cell percentage %

[0071]

[0072] Example 6

[0073] The prodrug T26 enhances T cell activation.

[0074] To evaluate the effect of T26 on T cell activation, this embodiment uses an indirect co-culture system of BMDCs and T cells, and analyzes its immune-enhancing effect by detecting the number and activation ratio of T cells.

[0075] The experimental procedure is as follows: 6–8-week-old C57BL / 6 mice were selected, and their spleens were removed after cervical dislocation. T cells were purified using the magnetic bead negative selection method (Miltenyi Biotec) and resuspended in DMEM complete medium containing 10 ng / mL recombinant mouse IL-2 for later use.

[0076] MC38 cells were seeded at the bottom of a Transwell system 24 hours in advance and co-cultured with BMDCs in T26-containing medium for 24 hours. Subsequently, the treated BMDCs were collected and co-incubated with prepared T cells for 36 hours (37°C, 5% CO2). After co-culture, T cells were collected, and their activation status was detected by flow cytometry. Specifically, T cells were stained with anti-CD3-FITC, anti-CD8-PE, and anti-Granzyme B-APC, and CD3+ was analyzed. + CD8 + The number of T cells and Granzyme B + Percentage of positive cells.

[0077] Experimental results showed that the number of T cells and CD3+ in the T26 treatment group... + CD8 + Granzyme B of T cells + The positive rates were significantly higher than those in the blank control group and the control drug group, suggesting that T26 can enhance T cell proliferation and promote its activation, thereby improving immune response.

[0078] Table 5: Effect of T26 on T cell count (%)

[0079]

[0080] Table 6: Effect of T26 on T cell activation capacity (%)

[0081]

[0082] Example 7: Effect of T26 on glutamine metabolism in tumor cells

[0083] To further verify the intervention effect of T26 on glutamine metabolism in tumor cells, this experiment measured the changes in intracellular glutamine content in MC38 cells after T26 treatment.

[0084] MC38 cells in logarithmic growth phase were cultured under standard conditions. Treatment groups were treated with T26, JHU083, and MSA-2 at a final concentration of 5 μM, respectively, with DMSO serving as a control. After 24 hours of treatment, the culture medium was discarded, and the cells were resuspended in 0.9% physiological saline and homogenized. The cells were centrifuged at 10,000 g at 4°C for 15 minutes, and the supernatant was used for glutamine content determination. Quantitative detection was performed using a colorimetric method with an Elabscience assay kit (E-BC-K853-M).

[0085] The results are as follows Figure 2As shown in Table 2 below, the intracellular glutamine content in the T26 treatment group was significantly lower than that in the control group and the JHU083 and MSA-2 groups, indicating that T26 has a stronger glutamine deprivation effect and metabolic inhibition ability.

[0086] Table 7: Effect of T26 on intracellular glutamine concentration (intracellular glutamine content %)

[0087]

[0088] Example 8: In vivo antitumor activity of T26 in MC38 xenograft mouse model

[0089] To systematically evaluate the antitumor effect of T26 in vivo and its efficacy advantages compared with monotherapy (JHU083, MSA-2), this embodiment constructs an MC38 mouse subcutaneous xenograft model of colorectal cancer for drug intervention and efficacy evaluation.

[0090] Six- to eight-week-old female C57BL / 6 mice (SPF grade) were selected, and 1 × 10⁶ MC38 cells in logarithmic growth phase were subcutaneously injected into the right hind limb of each mouse. 6 One. Wait until the tumor grows to approximately 80mm in size. 3 Afterwards, drug treatment began, with administration via gavage. The experimental animals were randomly divided into the following five groups, with six animals in each group:

[0091] Control group: administered PBS for 21 days; JHU083 and MSA-2 group: 1 mg / kg, Day 7–11; 0.3 mg / kg, every other day, Day 12–21; T26 group: 1.5 mg / kg, Day 7–11; 0.45 mg / kg, every other day, Day 12–21. Throughout the administration process, the long axis (L) and short axis (W) of the tumor were measured every two days, and the tumor volume was calculated using the formula: V = (L × W²) / 2.

[0092] At the end of day 21, all mice were euthanized by cervical dislocation, and the tumor tissue was completely dissected for weighing and photography. Some samples were used for histopathological analysis and immunostaining. The results showed that T26 significantly inhibited the growth of MC38 tumors, and the tumor volume at the terminal stage was significantly lower than that in the JHU083 and MSA-2 groups, with better tumor inhibition effect than the single-drug and PBS groups.

[0093] Table 8: Comparison of terminal tumor volume in mice of different groups (unit: mm) 3 )

[0094]

[0095] Furthermore, HE staining of major mouse organs (heart, liver, spleen, lung, and kidney) showed no obvious inflammatory infiltration, necrosis, or structural damage, suggesting that T26 possesses good systemic tolerance and biocompatibility. Figure 4 As shown.

[0096] Compared with related technologies, the 6-diazo-5-oxo-ortholeucine prodrug provided by the present invention has the following beneficial effects:

[0097] This invention provides a 6-diazo-5-oxo-leucine prodrug, compound T26, which combines two mechanisms of action by linking the glutamine metabolism inhibitor JHU083 and the STING pathway agonist MSA-2 via a cleavable amide bond. On the one hand, it can limit the metabolic advantage of tumor cells, reversing the immunosuppressive tumor microenvironment; on the other hand, it directly activates the STING pathway of dendritic cells, inducing the release of immune factors such as type I interferon and tumor necrosis factor TNF-α, thereby significantly enhancing innate and adaptive immune responses. This dual mechanism of action synergistically enhances immune activation and improves dendritic cell function, enhancing antigen presentation capacity: T26 can significantly increase the expression levels of co-stimulatory molecules (such as CD80 and CD86) in dendritic cells, enhancing their antigen presentation capacity, thereby effectively improving the activation efficiency of T cells. Strong anti-tumor immune effect; targeting the tumor microenvironment and reducing systemic toxicity: Compared with monotherapy, this invention covalently links two pharmacodynamic components through chemical means, enabling them to achieve spatial co-localization and temporal synergistic release in the tumor microenvironment, reducing the risk of exposure to non-target tissues, and is expected to reduce systemic toxic side effects and improve treatment safety; Traditional combination therapy of JHU083 and MSA-2 faces the problem of inconsistent pharmacokinetic characteristics and tissue distribution. T26 overcomes this limitation through a single-molecule unified release method, improving the consistency and controllability of combined therapy, and overcoming inconsistent drug distribution and pharmacokinetic differences; The compound structure of this invention is well-defined, the synthetic route is controllable, and it has good chemical stability and drug development potential, making it suitable for further pharmacological research and preclinical development, and possessing good syntheticability and pharmaceutical properties.

Claims

1. A 6-diazo-5-oxo-leucine prodrug, characterized in that, include: The chemical structure of the prodrug molecule is as follows: In general formula (1): Wherein R is selected from hydrogen atom, halogen, cyano, nitro, unsubstituted or substituted with at least one of the following groups: C1-6 alkyl, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkoxy, C2-6 alkenyloxy, C2-6 alkoxy, C2-6 alkenyloxy, etc., wherein the following groups may be the same or different and are selected from halogen, hydroxyl, cyano, nitro and amino; unsubstituted or substituted with the following groups: amino, wherein the following groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenylalkyl, etc. C1-6 alkyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl; unsubstituted and substituted ester and amide groups, wherein the hydroxyl and amino groups in the substituted ester and amide groups are selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, and C2-6 alkynylcarbonyl; The optical isomers, diastereomers, and pharmaceutically acceptable salts of the compounds shown, as well as pharmaceutically acceptable carriers, excipients, and excipients.

2. A 6-diazo-5-oxo-ortholeucine prodrug according to claim 1, wherein in the general formula (1): R is selected from hydrogen atoms, C1-6 alkyl-substituted ester groups; optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown; and pharmaceutically acceptable carriers, excipients and excipients, characterized in that: Its dosage forms include oral tablets, capsules, injections, lyophilized powder for injection, and sustained-release preparations.

3. A 6-diazo-5-oxo-leucine prodrug according to claim 1, wherein the compound represented by general formula (1) is the following compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, and pharmaceutically acceptable carriers, excipients and excipients, characterized in that: Its dosage forms include oral tablets, capsules, injections, lyophilized powder for injection, and sustained-release preparations.

4. The application of a 6-diazo-5-oxo-leucine prodrug according to any one of claims 1-3, characterized in that, The use of the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients in the preparation of cancer therapeutics.

5. The application of the 6-diazo-5-oxo-ortholeucine prodrug according to claim 4, characterized in that, The use of the compound and its optical isomers, diastereomers and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients and excipients in the preparation of drugs for breast cancer, melanoma and colorectal cancer.

6. A method for preparing a 6-diazo-5-oxo-leucine prodrug according to any one of claims 1-5, characterized in that, The 6-diazo-5-oxo-leucine and its derivatives are reacted with MSA-2 via amide condensation to obtain a compound of general formula (1).

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