Preparation and anti-tumor application of novel 6-diazo-5-oxo-n-leucine derivative prodrug

Abstract

The invention provides preparation of a novel 6-diazo-5-oxo-n-leucine derivative prodrug, the prodrug connects DON and artesunate through a breakable amido bond, selective targeting on tumor-related macrophages can be realized, the safety is higher, anti-tumor immune response can be effectively activated through polarization of M1 type macrophages, and the prodrug has a good application prospect in the field of tumor treatment. The phenomenon of drug resistance in treatment of cancers such as colorectal cancer is overcome. According to the preparation of the novel 6-diazo-5-oxo-n-leucine derivative prodrug provided by the invention, the prodrug has the beneficial effects of strong targeting property, remarkable anti-tumor activity, clear immune metabolism regulation mechanism, high safety, good development prospect and the like, and can be used for preparing drugs for treating cancers such as breast cancer, melanoma and colorectal cancer.

Description

Technical Field

[0001] This invention relates to the fields of chemistry and medicine, and in particular to the preparation of novel 6-diazo-5-oxo-leucine derivative prodrugs and their antitumor applications. Background Technology

[0002] In recent years, the incidence of colorectal cancer has continued to rise, and it has become one of the major malignant tumors threatening human health worldwide. Although emerging treatment strategies such as immune checkpoint inhibitors have shown treatment potential in some patients, the response rate of colorectal cancer patients is limited and the treatment effect is still not ideal.

[0003] Numerous studies have shown that tumor cells construct a tumor microenvironment with highly immunosuppressive characteristics through metabolic reprogramming, which is a key factor leading to treatment tolerance and immune failure. Among these factors, glutamine metabolism, as a convergence pathway of tumor growth and immune regulation, has received increasing attention. Glutamine not only provides tumor cells with synthetic and energy support, but also regulates T cell activation, dendritic cell antigen presentation, and the polarization state of tumor-associated macrophages, and is widely involved in the maintenance of the immunosuppressive network.

[0004] Previous studies have attempted to improve tumor metabolic syndrome (TME) by intervening in this process with glutamine metabolism antagonists. 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, DON prodrug forms such as JHU-083 have improved efficacy and toxicity to some extent, but problems such as insufficient immunoselectivity and limited innate immune activation capacity still exist.

[0005] Therefore, it is necessary to provide a bifunctional immunometabolite prodrug of 6-diazo-5-oxo-leucine (DON) and artesunate to solve the above-mentioned technical problems. Summary of the Invention

[0006] This invention provides the preparation of a novel 6-diazo-5-oxo-leucine derivative prodrug, which solves the problems of insufficient immunoselectivity and limited innate immune activation ability of existing glutamine metabolism antagonists.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing a novel 6-diazo-5-oxo-ortholeucine derivative prodrug.

[0008] The chemical structure of the prodrug molecule is as follows:

[0009]

[0010] In the general formula:

[0011] Wherein R is selected from hydrogen atom, halogen, cyano, nitro, unsubstituted or substituted by 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 alkynyloxy, the following groups may be the same or different and selected from halogen, hydroxyl, cyano, nitro and amino; unsubstituted or substituted by the following groups, such as those 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 or C2-6 alkynylcarbonyl; an ester or amide group that is unsubstituted or substituted with the following groups, wherein the following groups of the hydroxyl or amino group in the substituted ester or amide group 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 or C2-6 alkynylcarbonyl;

[0012] Or optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown, as well as pharmaceutically acceptable carriers, excipients and excipients.

[0013] Preferably, in the general formula, R is selected from hydrogen atoms, C1-6 alkyl-substituted ester groups; or optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown, as well as pharmaceutically acceptable carriers, excipients and excipients, the dosage form of which is an oral tablet, capsule, injection, lyophilized powder for injection or sustained-release formulation.

[0014] Preferably, the compounds represented by the general formula are compounds T17, T27, T37, and T47, their optical isomers, diastereomers, and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients, and formulations, and their dosage forms are oral tablets, capsules, injections, lyophilized powder for injection, or sustained-release preparations.

[0015]

[0016] The application of the preparation of the novel 6-diazo-5-oxo-leucine derivative prodrug, as described in any one of claims 1-3, wherein the preparation of the novel 6-diazo-5-oxo-leucine derivative prodrug is used to prepare a cancer treatment drug.

[0017] Preferred, for use in the preparation of drugs against breast cancer, melanoma, and colorectal cancer.

[0018] The preparation of a novel 6-diazo-5-oxo-ortholeucine derivative prodrug, wherein DON and its derivatives are reacted with artesunate via amide condensation to obtain the compound represented by the general formula:

[0019]

[0020] Compared with related technologies, the preparation of the novel 6-diazo-5-oxo-ortholeucine derivative prodrug provided by this invention has the following beneficial effects:

[0021] This invention provides the preparation of a novel 6-diazo-5-oxo-ortholeucine derivative prodrug.

[0022] Highly targeted and selective: By covalently coupling the glutamine antagonist DON with artesunate, which has macrophage-targeting properties, selective delivery and activation of tumor-associated macrophages can be achieved, enabling spatially specific metabolic intervention in the tumor immune microenvironment, significantly improving therapeutic selectivity and reducing toxicity to normal tissues.

[0023] Significant antitumor activity: It exhibits superior tumor-suppressing effects compared to the existing glutamine antagonist JHU-083 in mouse models of colorectal cancer, breast cancer, and melanoma. It significantly inhibits tumor progression by inducing macrophage polarization towards the pro-inflammatory M1 type, activating the innate immune system, enhancing antitumor immune responses, and thus significantly suppressing tumor progression.

[0024] The mechanism of immune metabolic reprogramming has been clarified: it mediates the shift of macrophage metabolism from oxidative phosphorylation to aerobic glycolysis, induces lactate accumulation, and enhances the phagocytic capacity of macrophages, providing a theoretical basis for a deeper understanding of immune metabolic regulation.

[0025] Reduced toxicity exposure and increased safety window: It has higher tumor selectivity and significantly reduces toxicity exposure to non-target tissues such as the intestines and liver without reducing efficacy, resulting in better safety and broader clinical application potential;

[0026] With a clear chemical structure and promising development prospects, it has a well-defined synthetic route, stable structure, excellent potential for chemical modification and pharmacokinetic properties, making it suitable for further pharmacological evaluation and industrialization development. It can provide a new strategy for targeted therapy of glutamine-dependent tumors such as colorectal cancer. Attached Figure Description

[0027] Figure 1 A schematic diagram of the structure of a preferred embodiment for the preparation of the novel 6-diazo-5-oxo-ortholeucine derivative prodrug provided by the present invention;

[0028] Figure 2 For T17 13 C-NMR spectrum;

[0029] Figure 3 For T27 1 H-NMR spectrum;

[0030] Figure 4 For T27 13 C-NMR spectrum;

[0031] Figure 5 For T37 1 H-NMR spectrum;

[0032] Figure 6 For T37 13 C-NMR spectrum;

[0033] Figure 7 For T47 1 H-NMR spectrum;

[0034] Figure 8 For T47 13 C-NMR spectrum. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Please refer to the following: Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 ,in, Figure 1 A schematic diagram of the structure of a preferred embodiment for the preparation of the novel 6-diazo-5-oxo-ortholeucine derivative prodrug provided by the present invention; Figure 2 For T17 13 C-NMR spectrum; Figure 3 For T27 1 H-NMR spectrum; Figure 4 For T27 13 C-NMR spectrum; Figure 5 For T37 1 H-NMR spectrum; Figure 6 For T37 13 C-NMR spectrum; Figure 7 For T47 1 H-NMR spectrum; Figure 8 For T47 13 C-NMR spectrum. Preparation of a novel 6-diazo-5-oxo-ortholeucine derivative prodrug, the chemical structure of which is as follows:

[0037] In the general formula:

[0038] Wherein R is selected from hydrogen atom, halogen, cyano, nitro, unsubstituted or substituted by 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 alkynyloxy, the following groups may be the same or different and selected from halogen, hydroxyl, cyano, nitro and amino; unsubstituted or substituted by the following groups, such as those selected from C1-6 alkyl, C1-6 aminoalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C1-6 cyanoalkyl, C2- 6-Alkenyl, C2-6 Acynyl, C1-6 alkylsulfonyl, C1-6 alkylcarbonyl, C2-6 alkenylcarbonyl, or C2-6 alkynylcarbonyl; an ester or amide group that is unsubstituted or substituted with the following groups, wherein the hydroxyl or amino group in the substituted ester or amide group is 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, or C2-6 alkynylcarbonyl.

[0039]

[0040] Or optical isomers, diastereomers and pharmaceutically acceptable salts of the compound shown, as well as pharmaceutically acceptable carriers, excipients and excipients.

[0041] In the general formula, R is selected from hydrogen atoms, C1-6 alkyl-substituted ester groups; or optical isomers, diastereomers and pharmaceutically acceptable salts of the compound, as well as pharmaceutically acceptable carriers, excipients and excipients, in dosage forms such as oral tablets, capsules, injections, lyophilized powder for injection or sustained-release formulations.

[0042] The compounds represented by the general formula are T17, T27, T37, and T47 compounds and their optical isomers, diastereomers, and pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers, excipients, and formulations, in the form of oral tablets, capsules, injections, lyophilized powders for injection, or sustained-release preparations.

[0043]

[0044] Example 1: Isopropyl(S)-6-azido-5-oxo-2-(4-oxo-4-(((3R,5aS,6R,8aS,9R,10R,12R,12aS)-3,5a,6,9-tetramethyl-12H-decahydro-3,12-epoxy[1,2]dioxacyclopenta[4,3-i]isocyanene-10-yl)oxy)butamido)hexanoate (T17);

[0045] Artesunate (192.21 mg, 0.5 mmol, 1 eq), EDCI (180 mg, 0.94 mmol, 1.8 eq), and 2 mL of anhydrous dichloromethane were added sequentially to a 50 mL pear-shaped flask. The mixture was stirred and reacted at 0 °C for 30 min. Isopropyl (S)-2-amino-6-azo-5-oxohexanoate (100 mg, 0.469 mmol, 1 eq) was then added to the reaction mixture. After the addition was complete, the mixture was stirred at room temperature for 12 h. The solvent was removed by vacuum distillation, and 20 mL of water was added to the residue. The residue was extracted with dichloromethane (3 × 10 mL), and the organic phases were combined and washed successively with water (3 × 15 mL) and saturated brine (1 × 10 mL). The residue was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure to obtain crude T17. The crude T17 was purified by column chromatography (dichloromethane:methanol = 20:1) to obtain purified T17, a yellow solid, with a yield of 84 mg and a yield of 30.92%. 1 H NMR (500MHz, DMSO-d6) δ8.27(d,J=7.7Hz,1H),6.04(s,1H),5.65(d,J=9.7Hz,1H),5.54(s,1H),4.87(h,J=6.2Hz,1H),4.17(td,J=8 .5,5.2Hz,1H),2.66–2.52(m,2H),2.49–2.33(m,4H),2.31–2.25(m,1H),2.22–2.14(m,1H),2.01–1.91(m,2H),1.79(dddt,J=22.8, 19.6,8.7,4.8Hz,2H),1.62(tt,J=13.3,3.2Hz,2H),1.54(dt,J=13.7,4.2Hz,1H),1.47–1.38(m,2H),1.34(ddd,J=13.7,11.9,4.9H z,1H),1.28(s,3H),1.25–1.19(m,1H),1.18(dd,J=6.4,3.5Hz,6H),0.99–0.92(m,1H),0.88(d,J=6.3Hz,3H),0.75(d,J=7.1Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ194.43,171.71,171.58,171.27,104.03,92.12,91.06,80.32,68.42,51.90,51.60 ,45.06,36.45,36.37,34.19,32.10,29.75,29.33,26.62,25.97,24.67,21.96,21.91,21.48,20.51,12.20.

[0046] Example 2: Ethyl(S)-6-azido-5-oxo-2-(4-oxo-4-(((3R,5aS,6R,8aS,9R,10R,12R,12aS)-3,5a,6,9-tetramethyl-12H-decahydro-3,12-epoxy[1,2]dioxacyclopenta[4,3-i]isocyanene-10-yl)oxy)butamido)hexanoate (T27);

[0047] The compound in Example 2 was prepared in the same way as in Example 1, using ethyl (S)-2-amino-6-azo-5-oxohexanoate and artesunate as raw materials, and following the synthesis method of T17, T27 was obtained as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ8.30(d,J=7.9Hz,1H),6.04(s,1H),5.66(d,J=9.7Hz,1H),5.55(s,1H),4.22(ddd,J=9.2,7.6,5.3Hz,1H),4.0 8(qd,J=7.1,1.9Hz,2H),2.65–2.55(m,2H),2.49–2.42(m,2H),2.39(q,J=8.4,7.7Hz,2H),2.29(ddd,J=9.8,7.2,4.4Hz,1H),2.23–2.1 5(m,1H),2.03–1.95(m,2H),1.85–1.74(m,2H),1.62(td,J=13.5,3.4Hz,2H),1.55(dt,J=13.7,4.2Hz,1H),1.48–1.39(m,2H),1.36–1. 31(m,1H),1.29(s,3H),1.25(d,J=11.6Hz,1H),1.18(d,J=7.1Hz,3H),0.99–0.92(m,1H),0.89(d,J=6.4Hz,3H),0.76(d,J=7.1Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ194.45,172.22,171.57,171.28,104.03,92.12,91.06,80.32,60.96,51.77,51 .60,45.06,36.45,36.37,34.19,32.10,29.74,29.31,26.62,25.97,24.67,21.48,20.51,14.48,12.19.

[0048] Example 3: Methyl(S)-6-azido-5-oxo-2-(4-oxo-4-(((3R,5aS,6R,8aS,9R,10R,12R,12aS)-3,5a,6,9-tetramethyl-12H-decahydro-3,12-epoxy[1,2]dioxacyclopenta[4,3-i]isocyanen-10-yl)oxy)butamido)hexanoate (T37);

[0049] The compound in Example 3 was prepared in the same way as in Example 1, using methyl (S)-2-amino-6-azo-5-oxohexanoate and artesunate as raw materials, and following the synthesis method of T17, T37 was obtained as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ8.32(d,J=7.6Hz,1H),6.04(s,1H),5.66(d,J=9.8Hz,1H),5.55(s,1H),4.25(ddd,J=9.4,7.7,5.3Hz,1H), 3.61(s,3H),2.64–2.55(m,2H),2.49–2.42(m,2H),2.38(q,J=8.7,8.0Hz,2H),2.31–2.25(m,1H),2.22–2.14(m,1H),2.02–1.94(m ,2H),1.80(dddd,J=17.3,13.7,7.1,2.5Hz,2H),1.62(dddd,J=16.5,10.2,3.3Hz,2H),1.54(dt,J=13.5,4.2Hz,1H),1.45(qd,J=12 .7,3.5Hz,2H),1.36–1.30(m,1H),1.28(s,3H),1.21–1.16(m,1H),0.99–0.92(m,1H),0.88(d,J=6.4Hz,3H),0.75(d,J=7.0Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ194.42,172.72,171.57,171.29,104.04,92.13,91.06,80.33,52.32,51.67 ,51.60,45.05,36.45,36.37,34.19,32.10,29.72,29.29,26.61,25.98,24.67,21.48,20.51,12.18.

[0050] Example 4: tert-butyl(S)-6-azido-5-oxo-2-(4-oxo-4-(((3R,5aS,6R,8aS,9R,10R,12R,12aS)-3,5a,6,9-tetramethyl-12H-decahydro-3,12-epoxy[1,2]dioxacyclopenta[4,3-i]isocyanene-10-yl)oxy)butamido)hexanoate (T47);

[0051] The compound in Example 4 was prepared in the same way as in Example 1, using (S)-2-amino-6-azo-5-oxohexanoate tert-butyl ester and artesunate as raw materials, and following the synthesis method of T17, T47 was obtained as a yellow solid. 1 H NMR (500MHz, DMSO-d6) δ8.19(d,J=7.8Hz,1H),6.05(s,1H),5.66(d,J=9.7Hz,1H),5.55(s,1H),4.10(td,J=8.4,5.2Hz,1H),2.60(tq ,J=10.8,5.8,4.3Hz,2H),2.45(dt,J=14.4,6.9Hz,2H),2.40–2.25(m,3H),2.18(td,J=14.0,3.9Hz,1H),2.03–1.98(m,1H),1.92(dt ,J=13.3,5.9Hz,1H),1.83–1.71(m,2H),1.66–1.58(m,2H),1.57–1.51(m,1H),1.47–1.42(m,2H),1.39(s,9H),1.33(dd,J=12.4,4.7 Hz,1H),1.28(s,3H),1.17(dt,J=11.3,5.8Hz,1H),0.96(ddd,J=15.9,12.6,5.5Hz,1H),0.88(d,J=6.3Hz,3H),0.76(d,J=7.1Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ194.02,171.12,170.94,170.70,103.55,91.64,90.58,80.59,53.94,51.89,51.12,44.58,40.02,39.86,39 .69,39.52,39.35,39.19,39.02,36.25,35.97,35.89,33.71,31.62,29.31,28.89,27.60,26.30,25.50,24.19,21.01,20.04,11.73.

[0052] Example 5: Screening of in vivo antitumor activity of four prodrugs in an MC38 xenograft mouse model. In this example, an MC38 subcutaneous xenograft mouse model of colorectal cancer was constructed, and drug intervention and efficacy testing were conducted.

[0053] The experiment used 6–8 week old female C57BL / 6 SPF mice. Each mouse was subcutaneously inoculated with 1×10⁻⁶ mol / L spores in its right hind limb. 6 MC38 cells in logarithmic growth phase. When the tumor volume reaches approximately 80 mm... 3 Mice were randomly divided into 5 groups of 6 mice each. Treatment began as follows: Control group: PBS administered every other day from Day 7 to 21; T17, T27, T37, and T47 groups: 1 mg / kg, administered every other day from Day 7 to 21. Throughout the treatment process, the long axis (L) and short axis (W) of the tumor were measured every two days, and the result was calculated using the formula: V = (L × W) 2 ) / 2 to calculate tumor volume.

[0054] At the end of the experiment on day 21, all mice were euthanized by cervical dislocation. The tumor tissue was completely removed, weighed, and photographed. Samples were also retained for subsequent histopathological and immunostaining analysis. The results showed that among the four prodrug structures, T17 exhibited the most significant inhibitory effect on MC38 tumor growth, with a significantly smaller terminal tumor volume compared to the other three prodrug treatment groups.

[0055] Table 1: Comparison of terminal tumor volume in mice of different groups (unit: mm) 3 ).

[0056] Control T17 T27 T37 T47 Tumor volume 867.56± 144.36± 275.89± 463.90± 321.85± 58.95 29.74 38.87 67.02 35.95

[0057] Table 2: Comparison of terminal tumor weights in mice across different groups (unit: g)

[0058] Control T17 T27 T37 T47 Tumor weight 0.97± 0.22± 0.35± 0.48± 0.42± 0.06 0.04 0.61 0.15 0.14

[0059] Example 6: In vivo antitumor activity of T17 in MC38 xenograft mouse model. To systematically evaluate the in vivo antitumor effect of T17 and its efficacy advantages compared with single drugs (JHU083 and ART), this example constructed an MC38 colorectal cancer subcutaneous xenograft mouse model and conducted drug intervention and efficacy testing.

[0060] The experiment used 6–8 week old female C57BL / 6 SPF mice. Each mouse was subcutaneously inoculated with 1×10⁻⁶ mol / L spores in its right hind limb. 6 MC38 cells in logarithmic growth phase. When the tumor volume reaches approximately 80 mm... 3Mice were randomly divided into 5 groups of 6 mice each. Treatment began as follows: Control group: PBS administered every other day from Day 7 to 21; JHU083, ART, T17 group: 1 mg / kg, administered every other day from Day 7 to 21. Throughout the treatment process, the long axis (L) and short axis (W) of the tumor were measured every two days, and the result was calculated using the formula: V = (L × W) 2 ) / 2 to calculate tumor volume.

[0061] At the end of the experiment on day 21, all mice were euthanized by cervical dislocation. Tumor tissue was completely removed, weighed, and photographed. Samples were also retained for subsequent histopathological and immunostaining analysis. The results showed that T17 significantly inhibited the growth of MC38 tumors, with terminal tumor volumes significantly smaller than those in the JHU083 and ART treatment groups, demonstrating significantly better antitumor effects than single-drug and control groups.

[0062] Table 3: Comparison of terminal tumor volume in mice of different groups (unit: mm) 3 ).

[0063] Control JHU-083 ART T17 Tumor volume 834.01± 495.54± 532.09± 124.01± 105.33 89.49 145.97 30.57

[0064] Table 4: Comparison of terminal tumor weights in mice across different groups (unit: g)

[0065] Control JHU-083 ART T17 Tumor weight 0.85± 0.57± 0.63± 0.15± 0.16 0.27 0.11 0.22

[0066] Example 7: T17 regulates the tumor microenvironment of colorectal cancer

[0067] To systematically evaluate the regulatory effect of T17 on the tumor immune microenvironment, this embodiment constructed a mouse model of MC38 colorectal cancer subcutaneous xenograft, and performed drug intervention on the model and flow cytometry detection of various immune cells in the immune microenvironment after treatment.

[0068] Six- to eight-week-old female SPF-grade C57BL / 6 mice were used in the experiment. Each mouse was subcutaneously inoculated with 1×10⁻⁶ dredges in its right hind limb. 6 MC38 cells in the logarithmic growth phase, when the tumor volume reaches approximately 80 mm. 3 Mice were randomly divided into 5 groups of 6 mice each and administered the drug as follows: Control group: PBS treatment, Day 7–11; JHU083, ART, T17 group: 1 mg / kg, Day 7–11. After five consecutive treatments, the mice were euthanized by cervical dislocation, and the tumor tissue was completely removed. Single-cell suspensions were prepared using digestive enzymes for flow cytometry experiments.

[0069] Table 5: Proportion of immune cells changing in the immune microenvironment of mice in each group (unit: %)

[0070]

[0071]

[0072] Example 8: Effect of T17 on macrophage polarization state

[0073] To systematically evaluate the effect of T17 on macrophage polarization, this study extracted mouse bone marrow-derived macrophages and induced them to mature in vitro. These macrophages were then subjected to drug intervention, and the effect of T17 on macrophage polarization was detected by flow cytometry.

[0074] Mouse bone marrow-derived monocytes were induced to differentiate into macrophages in vitro. Treatment groups were treated with JHU-083, ART, and T17 at a final concentration of 10 μM, respectively, with DMSO serving as a control. After 36 hours of treatment, the culture medium was discarded, and macrophages were stained with F4 / 80-FITC, CD11b-percp-cy5.5, CD86-APC, and CD206-PE, and analyzed by flow cytometry.

[0075] Table 6: Percentage of changes in macrophage polarization state after T17 treatment (unit: %)

[0076] Control JHU-083 ART T17 M1 macrophages 6.17± 10.67± 18.56± 46.04± 1.41 2.89 4.32 4.51 M2 macrophages 14.63± 34.94± 10.56± 8.83± 4.73 3.73 3.85 2.78

[0077] Example 9: Effects of T17 on mouse serum cytokines

[0078] To further verify the regulatory role of T17 on macrophages, this experiment measured the changes in the levels of related cytokines (TNF-α, TGF-β, IL-12, and IL-10) secreted by macrophages in the serum of MC38 colorectal cancer subcutaneous xenograft mice.

[0079] Six- to eight-week-old female SPF-grade C57BL / 6 mice were used in the experiment. Each mouse was subcutaneously inoculated with 1×10⁻⁶ dredges in its right hind limb. 6 MC38 cells in the logarithmic growth phase, when the tumor volume reaches approximately 80 mm. 3 Mice were randomly divided into 5 groups of 6 mice each and administered the drug as follows: Control group: PBS control, Day 7–11; JHU083, ART, T17 group: 1 mg / kg, Day 7–11. After five consecutive treatments, whole blood was collected from mice via orbital sampling. After standing at room temperature for 2 hours, the blood was centrifuged at 12000 rpm for 20 minutes, and the supernatant was collected as mouse serum. Quantitative detection was performed using an ELISA kit (Thermo Fisher Scientific, 88-7324-88, 88-8350-22, 88-7121-88, 88-7105-88).

[0080] Table 7: Changes in serum cytokines in each group of mice (unit: pg / mL)

[0081] Control JHU-083 ART T17 TNF-α 20.41± 16.73± 25.84± 37.96± 1.43 0.56 3.60 5.68 IL-12 54.35± 45.89± 54.93± 75.95± 1.53 4.50 4.92 5.02 TGF-β 65.02± 61.95± 65.17± 49.58± 4.67 2.95 7.42 4.06 IL-10 800.34± 915.46± 758.65± 702.85± 34.56 43.94 27.35 45.92

[0082] Example 10: Effect of T17 on glutamine metabolism in macrophages

[0083] To further verify the regulatory effect of T17 on glutamine metabolism in macrophages, this experiment measured the changes in intracellular glutamine content in mouse bone marrow-derived macrophages after T17 treatment.

[0084] Mouse bone marrow-derived monocytes were induced to differentiate into macrophages in vitro. Treatment groups were treated with JHU-083, ART, and T17 at a final concentration of 10 μM, respectively, with DMSO serving as a control. After 36 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 10,000g, 4℃ for 15 minutes, and the supernatant was used for glutamine content determination. Quantitative detection was performed using a colorimetric method with an Elabscienc assay kit (E-BC-K853-M).

[0085] Table 8: Effect of T17 on intracellular glutamine concentration in macrophages (unit: μmol / 10^6)

[0086] Control JHU-083 ART T17 glutamine 1.80± 0.64± 1.54± 0.46± content 0.42 0.04 0.53 0.06

[0087] Compared with related technologies, the preparation of the novel 6-diazo-5-oxo-ortholeucine derivative prodrug provided by this invention has the following beneficial effects:

[0088] This invention provides the preparation of a novel 6-diazo-5-oxo-ortholeucine derivative prodrug.

[0089] Highly targeted and selective: By covalently coupling the glutamine antagonist DON with artesunate, which has macrophage-targeting properties, selective delivery and activation of tumor-associated macrophages can be achieved, enabling spatially specific metabolic intervention in the tumor immune microenvironment, significantly improving therapeutic selectivity and reducing toxicity to normal tissues.

[0090] Significant antitumor activity: It exhibits superior tumor-suppressing effects compared to the existing glutamine antagonist JHU-083 in mouse models of colorectal cancer, breast cancer, and melanoma. It significantly inhibits tumor progression by inducing macrophage polarization towards the pro-inflammatory M1 type, activating the innate immune system, enhancing antitumor immune responses, and thus significantly suppressing tumor progression.

[0091] The mechanism of immune metabolic reprogramming has been clarified: it mediates the shift of macrophage metabolism from oxidative phosphorylation to aerobic glycolysis, induces lactate accumulation, and enhances the phagocytic capacity of macrophages, providing a theoretical basis for a deeper understanding of immune metabolic regulation.

[0092] Reduced toxicity exposure and increased safety window: It has higher tumor selectivity and significantly reduces toxicity exposure to non-target tissues such as the intestines and liver without reducing efficacy, resulting in better safety and broader clinical application potential;

[0093] With a clear chemical structure and promising development prospects, it has a well-defined synthetic route, stable structure, excellent potential for chemical modification and pharmacokinetic properties, making it suitable for further pharmacological evaluation and industrialization development. It can provide a new strategy for targeted therapy of glutamine-dependent tumors such as colorectal cancer.

[0094] The preparation of a novel 6-diazo-5-oxo-ortholeucine derivative prodrug, wherein DON and its derivatives are reacted with artesunate via amide condensation to obtain the compound represented by the general formula:

[0095]

Claims

1. Preparation of a novel 6-diazo-5-oxo-norleucine derivative prodrug, characterized in that, the chemical structure of the prodrug molecule is as follows: in the general formula, wherein R is selected from the group consisting of a hydrogen atom, a halogen, a cyano group, a nitro group, a C1-6 alkyl group which is unsubstituted or substituted with at least one of the following groups, a C3-8 cycloalkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C2-6 alkynyloxy group, and the following groups which can be the same or different are selected from the group consisting of a halogen, a hydroxyl group, a cyano group, a nitro group and an amino group; an amino group which is unsubstituted or substituted with the following groups selected from the group consisting of a C1-6 alkyl group, a C1-6 aminoalkyl group, a C1-6 hydroxyalkyl group, a C1-6 alkoxyalkyl group, a C1-6 cyanoalkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6 alkylsulfonyl group, a C1-6 alkylcarbonyl group, a C2-6 alkenylcarbonyl group or a C2-6 alkynylcarbonyl group; an ester group or an amide group which is unsubstituted or substituted with the following groups which substitute the hydroxyl group or the amino group in the ester group or the amide group, and the following groups are selected from the group consisting of a C1-6 alkyl group, a C1-6 aminoalkyl group, a C1-6 hydroxyalkyl group, a C1-6 alkoxyalkyl group, a C1-6 cyanoalkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6 alkylsulfonyl group, a C1-6 alkylcarbonyl group, a C2-6 alkenylcarbonyl group or a C2-6 alkynylcarbonyl group; or an optical isomer, a diastereoisomer and a pharmaceutically acceptable salt of the compound, and a pharmaceutically acceptable carrier, an excipient and an adjuvant.

2. Preparation of novel 6-diazo-5-oxo-norleucine derivative prodrugs according to claim 1, characterized by, in the general formula, R is selected from the group consisting of a hydrogen atom, a C1-6 alkyl group substituted ester group; or an optical isomer, a diastereoisomer and a pharmaceutically acceptable salt of the compound, and a pharmaceutically acceptable carrier, an excipient and an adjuvant, and the dosage form is an oral tablet, a capsule, an injection solution, a freeze-dried powder injection or a sustained-release preparation.

3. Preparation of novel 6-diazo-5-oxo-norleucine derivative prodrugs according to claim 1, characterized by, the compound represented by the general formula is a T17, T27, T37 and T47 compound, and an optical isomer, a diastereoisomer and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, an excipient and an adjuvant, and the dosage form is an oral tablet, a capsule, an injection solution, a freeze-dried powder injection or a sustained-release preparation:

4. Anti-tumour use of the preparation of novel 6-diazo-5-oxo-norleucine derivative prodrugs, such as those described in any one of claims 1 to 3, characterised in that, the preparation of the novel 6-diazo-5-oxo-norleucine derivative prodrug is used for the preparation of a cancer treatment drug.

5. Anti-tumor application of the preparation of novel 6-diazo-5-oxo-norleucine derivative prodrugs according to claim 4, characterized by, a drug for the treatment of breast cancer, melanoma and colorectal cancer.

6. Preparation of novel 6-diazo-5-oxo-norleucine derivative prodrugs, characterized in that, the DON and its derivatives are subjected to amide condensation with artesunate to obtain the compound represented by the general formula:

Images