Prodrug of 6-diazo-5-oxo-L-n-leucine of glutamine antagonist as well as preparation method and application of prodrug

Abstract

The invention discloses a prodrug of 6-diazo-5-oxo-L-n-leucine of a glutamine antagonist as well as a preparation method and application of the prodrug, and belongs to the field of chemical medicines. The invention provides a prodrug compound of 6-diazo-5-oxo-L-n-leucine as shown in a general formula (I) or a pharmaceutically acceptable salt thereof, the compound or the salt thereof has an excellent glutamine antagonism effect under an anoxic condition, and the purpose of effectively treating tumors can be achieved by inhibiting glutamine metabolism and destroying tumor cell metabolism; meanwhile, the compound GI is good in stability, and the occurrence rate of adverse events can be reduced. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and in particular relates to a prodrug of 6-diazo-5-oxo-L-norleucine, a glutamine antagonist, and a preparation method and application thereof. Background Art

[0002] The metabolic processes of cancer cells differ significantly from those of normal cells. Over 100 years ago, German scientists discovered that cancer cells, even in the presence of sufficient oxygen, continue to utilize active glycolysis to convert glucose into lactate (the Warburg effect). While this discovery revealed significant metabolic differences between cancer cells and healthy cells, it did not lead to effective anti-cancer therapies.

[0003] Normal cells stop proliferating when nutrients are deficient, but cancer cells, driven by oncogenes, continue to rely on nutrient supply. Therefore, in theory, targeting cancer metabolism can stimulate cell death in cancer cells.

[0004] Cellular metabolism can be divided into glucose, amino acids, lipids, etc. Glucose is the main source of energy for cells. Cancer cells utilize glucose at a rate dozens or even hundreds of times higher than normal cells. Due to the Warburg effect in tumor cells, in order to maintain normal mitochondrial function, tumor cells use the process of anaplerosis to replenish metabolites in the TCA cycle.

[0005] Glutamine, the amide of glutamic acid, is the most abundant circulating amino acid in the human body, with a concentration 10 to 100 times higher than other amino acids. Many tumors, including pancreatic, ovarian, and breast cancers, rely on glutamine as a primary energy source to fuel the TCA cycle, a condition known as "glutamine addiction" (Li T, Le A. Glutamine Metabolism in Cancer [J]. 2018).

[0006] The dependence of tumor cells on glutamine metabolism has made it a research hotspot as a potential anti-cancer drug target in recent years, but most of these compounds are still in the preclinical stage.

[0007] DON (6-diazo-5-oxo-L-norleucine) is a glutamine antagonist that blocks cellular glutamine metabolism, thereby killing cells. However, the drug cannot distinguish between normal and tumor cells. In clinical trials, it has been shown to cause severe gastrointestinal side effects, including diarrhea and vomiting. Ultimately, clinical trials were suspended. In recent years, Barbara Slusher's research group at the Johns Hopkins University School of Medicine chemically modified DON to synthesize a prodrug of DRP-104.

[0008]

[0009] This prodrug is primarily reduced to DON by aminopeptidases and esterases. Compared to DON, DRP-104 reduces DON's gastrointestinal toxicity. However, the reduction of the prodrug's amino group primarily relies on the action of aminopeptidases, which are widely present in various tissues. Therefore, DRP-104's reduction selectivity is insufficient, preventing efficient tumor-specific activation. Ultimately, the clinical trial was suspended. Summary of the Invention

[0010] In order to solve the above-mentioned problems existing in the prior art, the present invention provides a hypoxia-activated prodrug of 6-diazo-5-oxo-L-norleucine or a pharmaceutically acceptable salt thereof, which can treat tumors by inhibiting glutamine metabolism and destroying the vital activities of tumor cells.

[0011] The present invention also provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.

[0012] In addition, the present invention provides uses of the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0013] Solutions for solving problems

[0014] The present invention first provides a compound having the general formula (I) or a pharmaceutically acceptable salt thereof.

[0015]

[0016] wherein X is selected from -O- or -NHSO2-;

[0017] R1 is selected from:

[0018]

[0019] R2 is selected from H, C 1-5 Straight chain alkyl, C 3-7 Branched alkyl, C 3-7 Cycloalkyl, substituted C 1-5 Straight chain alkyl, substituted C 3-7 Branched alkyl, substituted C 3-7 Cycloalkyl; substituted groups are selected from amino, hydroxy, halogen (F, Cl, Br, I).

[0020] In one embodiment of the present invention, X is -O-, and R2 is preferably a substituted or unsubstituted tert-butyl group; the substituted group is selected from amino, hydroxyl, and halogen.

[0021] In one embodiment of the present invention, the above compound can be specifically selected from:

[0022]

[0023] In one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt. The inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate. Furthermore, the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, succinate, or methanesulfonate.

[0024] The present invention also provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent.

[0025] In one embodiment of the present invention, the pharmaceutical composition, when formulated, can be administered systemically or sequentially, and any effective route of administration for delivering the compound or pharmaceutical composition to the tumor site can be accomplished, including but not limited to, oral administration of the compound or composition, topical, transdermal, parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal injection, intranasal instillation, intracavitary or intravesical instillation, intraocular, intraarterial, intralesional, or by application to mucosa, such as the nasal, laryngeal, and bronchial mucosa tubes.

[0026] The present invention also provides use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting glutamine.

[0027] The present invention also provides use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting tumor cell metabolism.

[0028] The present invention also provides use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0029] In one embodiment of the present invention, the tumor is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, etc.

[0030] The present invention also provides an anti-tumor drug, characterized in that it comprises the above compound or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0031] Beneficial effects:

[0032] The present invention provides a prodrug of 6-diazo-5-oxo-L-norleucine or a pharmaceutically acceptable salt thereof, which can effectively treat tumors by inhibiting glutamine metabolism and disrupting the vital activities of tumor cells. The compound of the present invention has a strong glutamine antagonist effect and good GI stability, which can reduce the incidence of adverse events. DETAILED DESCRIPTION

[0033] The following examples illustrate, but are not intended to limit, the methods for synthesizing compounds of formula (I). All temperatures are in degrees Celsius. Unless otherwise stated, all evaporations were performed under reduced pressure. Unless otherwise stated, reagents were purchased from commercial suppliers and used without further purification. The structures of final products, intermediates, and starting materials were confirmed by standard analytical methods, such as elemental analysis and spectral characterization, e.g., MS and NMR. Abbreviations used are those conventional in the art.

[0034] Preparation of intermediate (S)-5-oxopyrrolidine-2-carboxylic acid isopropyl ester (Compound 1-2):

[0035]

[0036] At room temperature, L-pyroglutamic acid (compound 1-1, 1 g, 7.74 mmol, 1.0 eq) and isopropanol (0.56 g, 9.29 mmol, 1.2 eq) were dissolved in DCM. A catalytic amount of DMAP was added under a nitrogen atmosphere, and a solution of DCC in DCM was slowly added dropwise under an ice bath. After completion of the reaction, the reaction was filtered and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography to afford compound 1-2 (1.26 g, 95.4% yield).

[0037] MS-ESI (m / z): 172.10 [M+1] + .

[0038] Preparation of intermediate (S)-N-(methylsulfonyl)-5-oxopyrrolidine-2-carboxamide (Compound 13-2):

[0039]

[0040] At room temperature, L-pyroglutamic acid (compound 1-1, 1 g, 7.74 mmol, 1.0 eq) and methanesulfonamide (0.88 g, 9.29 mmol, 1.2 eq) were dissolved in DCM. A catalytic amount of DMAP was added under a nitrogen atmosphere, and a solution of DCC in DCM was slowly added dropwise under an ice bath. After completion of the reaction, the reaction was filtered and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography to afford compound 13-2 (1.46 g, 91.3% yield).

[0041] MS-ESI (m / z): 207.05 [M+1] + .

[0042] Example 1: (S)-6-diazo-2-((((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)carbonyl)amino)-5-oxohexanoic acid isopropyl ester

[0043]

[0044] Preparation of 2-isopropyl-1-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)(S)-5-oxopyrrolidine-1,2-dicarboxylate (Compound 1-3):

[0045] Compound 1-2 ((S)-5-oxopyrrolidine-2-carboxylic acid isopropyl ester) (1 g, 5.84 mmol, 1 eq), DMAP (4-dimethylaminopyridine) (0.71 g, 5.84 mmol, 1 eq), and DIPEA (N,N-diisopropylethylamine) (1.51 g, 11.7 mmol, 2 eq) were added to a round-bottom flask and dissolved in 30 mL of anhydrous ACN. The mixture was stirred in an ice-water bath for 15 min. Then, 1-methyl-2-nitro-1H-imidazole-5-methyl chloroformate (1.54 g, 7.01 mmol, 2 eq) was slowly added to the round-bottom flask. Stirring was continued in an ice-water bath for 2 h, and the mixture was returned to room temperature for reaction. After completion of the reaction, the mixture was concentrated by rotary evaporation to obtain a brown solid. The solid was dissolved in 50 mL of dichloromethane and washed with saturated brine (30 mL*3). The organic phase was dried, concentrated, and purified by column chromatography to obtain compound 1-3 (1.85 g, yield: 89.5%).

[0046] MS-ESI (m / z): 355.13 [M+1] + .

[0047] Preparation of (S)-6-diazo-2-((((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)carbonyl)amino)-5-oxohexanoic acid isopropyl ester (Example 1):

[0048] Dissolve compound 1-3 (500 mg, 1.41 mmol, 1.0 eq) in 5 mL of anhydrous tetrahydrofuran and cool to -116°C. Separately, dissolve trimethylsilyldiazomethane (TMS) (0.85 mL, 2 M in hexane, 1.69 mmol, 1.2 eq) in 5 mL of anhydrous tetrahydrofuran and cool to -98°C. Slowly add n-BuLi (0.68 mL, 2.5 M in hexane, 1.69 mmol, 1.2 eq) dropwise to the TMS solution and allow to react for 30 minutes. Next, add the TMS and n-BuLi mixture to the compound 1-3 solution, slowly raise the temperature from -116°C to -78°C, and continue the reaction for 30 minutes. After completion, quench the reaction by adding 5 mL of aqueous solution. Extract three times with ethyl acetate (15 mL x 3). The mixture was then washed three times with saturated brine (10 mL x 3), and the combined organic phases were dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography to obtain Example 1 (365.24 mg, yield: 65.3%).

[0049] MS-ESI (m / z): 397.15 [M+1] + .

[0050] 1 H NMR(400MHz, CDCl3)δ:7.45(s,1H),5.43(s,2H),5.00(m,1H),4.55(m,1H),4 .25(s,1H),3.80(s,3H),2.42-2.35(m,2H),2.25-2.17(m,2H),1.22(d,6H).

[0051] Examples 2 to 16 (see Table 1) were obtained by following the basic operation similar to that of Example 1.

[0052] Table 1: Structure and data of Examples 2 to 16

[0053]

[0054]

[0055]

[0056] Example 17:

[0057] Antiproliferative activity of P493B lymphoma cells under hypoxic (<0.01% O2) and / or normoxic (21% O2) conditions

[0058] The specific experimental steps are as follows:

[0059] (1) Prepare 1640 complete medium with the following proportions: 1640 basal medium 89%, fetal bovine serum 10%, and double antibody 1%.

[0060] (2) Prepare the test compound into a 5 mM stock solution with cell-grade DMSO and store in a 4°C refrigerator until use.

[0061] (3) The frozen P493B cells were quickly thawed in a constant temperature water bath at 37°C, transferred to a centrifuge tube containing 5 mL of complete culture medium, centrifuged at 1000 r for 5 min, suspended into a single cell suspension and cultured in a T25 culture flask. When the cell density reached 80%, the cells were passaged as follows: rinsed with PBS buffer, digested with 2 mL of trypsin for 3 min, added with 6 mL of complete culture medium to stop digestion and transferred to a 15 mL centrifuge tube, centrifuged at 1000 r for 5 min, discarded the supernatant, added with 5 mL of complete culture medium, blown evenly, and transferred to a T25 culture flask for continued culture.

[0062] (4) Cells in the logarithmic growth phase were prepared into a suspension and counted. The suspension was diluted to 20,000 cells / well and plated in a 96-well plate. 100 μL was added to each well and cultured in a 37°C constant temperature incubator for 24 h. After the cells adhered, the remaining medium in each well was aspirated and serial dilutions of the test example compound or the reference compound DRP-104 diluted in the medium were added to different concentrations (9 concentrations). A blank control group (complete medium only) and a positive control group (cells and medium without drug) were set up and cultured under hypoxic (<0.01% O2) and / or normoxic (21% O2) conditions for 72 h.

[0063] (5) Add 20 μL of Cell Titer 96 AQueous to each well and incubate for 2 hours. Vibrate the plate in a microplate reader (4 minutes) and measure the fluorescence (OD) value of each well at 490 nm. Calculate the relative cell viability based on the absorbance difference.

[0064] (6) Using Graphpad Prism 5 software, IC was calculated according to the following inhibition rate formula. 50 Value calculation:

[0065]

[0066] Table 2 lists the data on the cell anti-proliferative activities of the examples of the present invention and the reference compounds.

[0067] Table 2: Antiproliferative activity data of P493B lymphoma cells

[0068]

[0069] This series of compounds was tested for antiproliferative activity. Three parallel experiments were performed, and the results were averaged. The results are shown in Table 2. Under normoxic conditions, Examples 1-16 showed no inhibitory activity. Under hypoxic conditions, Examples 1-16 exhibited comparable or stronger inhibitory activity than the control, DRP-104.

[0070] Example 18: Compound Stability Evaluation

[0071] Intestinal tissue was homogenized with potassium phosphate buffer (0.1M) in a ratio of 1:9. DRP-104 or the 1640 basal culture medium solution of Examples 1 to 16 (final concentration of 10 μM) was added to 100 μl of plasma or tissue homogenate in triplicate for 0 and 60 minutes. At each time point, the reaction was terminated with 300 μL of methanol containing an internal standard (losartan, 0.5 μM). The mixture was vortexed for 30 seconds and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was used for subsequent analysis (the peak area ratio of the test substance and the internal standard was determined by liquid chromatography-mass spectrometry (LC-MS)), and the Plasma stability index or GI stability index was obtained accordingly.

[0072] Among them, the specific LC-MS determination conditions are:

[0073] Liquid chromatography column: Thermo BDS Hypersil C18 30X2.0mm, 3μm, with guard column MP, buffer: 25mM formic acid buffer, pH 3.5;

[0074] Aqueous phase (A): 90% water, 10% buffer;

[0075] Organic phase (B): 90% acetonitrile, 10% buffer;

[0076] Flow rate: 300 μl / min

[0077] Autosampler: injection volume 10 μl

[0078] The gradient program is shown in Table 3.

[0079] Table 3: Gradient program

[0080] Time (minutes) ％A ％B 0.0 100 0 1.5 0 100 2.0 0 100 2.1 100 0 3.5 100 0

[0081] The specific stability evaluation results are shown in Table 4.

[0082] Table 4: Compound stability results

[0083]

[0084]

[0085] As described herein, Examples 1-16 all exhibited good plasma stability, comparable to DRP-104. In particular, Examples 2, 4, 6-8, and 10-16 demonstrated good GI stability, significantly superior to DRP-104. Considering that the gastrointestinal tract is the primary site of toxicity in DON clinical studies, these excellent GI stability properties have the potential to reduce the incidence of adverse events.

[0086] From the stability data of the compounds of Examples 1 to 16, it can be seen that for the compounds of general formula (I), the linking group and the substituent group such as the R2 group have an important influence on the stability of the compound.

[0087] Although the present invention has been described by way of specific embodiments, it should not be construed as being limited thereto; rather, the present invention encompasses the general aspects disclosed hereinabove. Various modifications and embodiments are possible without departing from the spirit and scope of the present invention.

Claims

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof: in, X is selected from -O- or -NHSO2-; R1 is selected from: R2 is selected from H, C 1-5 Straight chain alkyl, C 1-5 Branched alkyl, C 3-6 Cycloalkyl or amino, substituted C 1-5 Straight chain alkyl, substituted C 3-7 Branched alkyl, substituted C 3-7 Cycloalkyl; substituted groups are selected from amino, hydroxy, halogen.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that X is -O-, R2 is a substituted or unsubstituted tert-butyl group; the substituted group is selected from amino, hydroxyl, and halogen.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The compound is selected from:

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt is an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; and the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, and ascorbate.

5. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

6. A glutamine antagonist, characterized in that The invention comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

7. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting glutamine metabolism.

8. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting tumor cell metabolism.

9. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

10. The use according to claim 8 or 9, characterized in that: The tumor is breast cancer, ovarian cancer, lung cancer, or pancreatic cancer.

Images