CN114805138B - Prodrugs of 6-diazo-5-oxo-L-norleucine, preparation methods and applications thereof - Google Patents

Abstract

The invention provides a prodrug of 6-diazo-5-oxo-L-norleucine, as shown in formula (I); wherein, n is an integer from 0 to 5; R ₁ is C1 to C10 Alkyl group; R ₂ is nitro, substituted or unsubstituted azo group. Compared with the existing technology, the prodrug provided by the present invention is a hypoxia-activated prodrug, which can be selectively reduced to the original drug in the hypoxic part of the tumor, exerting the effect of inhibiting tumor growth, and at the same time reducing the effect of the original drug on other normal cells. Effect, reducing the toxic side effects of this type of drug on the gastrointestinal tract, and the combined treatment with vascular blockers has a significant effect. The combined treatment with aPD-1 and vascular blockers significantly inhibited the growth and metastasis of tumors in the 4T1 model. .

Description

Prodrugs of 6-diazo-5-oxo-L-norleucine, preparation methods and applications thereof

Technical field

The invention belongs to the field of pharmaceutical technology, and in particular relates to a prodrug of 6-diazo-5-oxo-L-norleucine, its preparation method and application.

Background technique

Cancer has become a major disease that affects people's quality of life. According to data released by the National Cancer Center, the incidence of cancer in our country is on the rise. Traditional cancer treatments include chemotherapy, radiotherapy and surgical resection. Chemotherapy uses chemical drugs to kill cancer cells and is currently the most effective treatment method. However, chemotherapy is often accompanied by serious side effects because chemotherapy drugs often lack selectivity. While killing cancer cells, they can also cause damage to normal cells. For example, some drugs can have toxic side effects on the heart, gastrointestinal system, liver and kidney tissues, skin, etc.

Hypoxic areas are common in solid tumors, and these areas can cause tumor drug resistance through various pathways, which is not conducive to tumor treatment. Under normal physiological conditions, there are no hypoxic areas in tissues, so these hypoxic areas bring opportunities for selective treatment of tumors. The unique property of solid tumors being hypoxic also brings different directions to the development of chemotherapy drugs. Currently, a variety of hypoxia-activated prodrugs (HAPs) have been developed clinically. Taking advantage of the hypoxic characteristics of tumors, HAPs can be selectively activated into active drugs at the tumor site, killing tumors and avoiding systemic toxicity caused by drugs. The strategy of modifying small molecule chemotherapy drugs into HAPs-like drugs not only reduces the systemic toxicity of the drug, but also converts the disadvantage of tumor hypoxia into the advantage of selective treatment.

DON is a type of glutamine antagonist that blocks glutamine metabolism in cells, thereby killing cells. However, the drug cannot distinguish between normal cells and tumor cells. In clinical trials, it has serious gastrointestinal side effects, and patients will suffer from diarrhea, vomiting and other symptoms. Ultimately, clinical trials were suspended. In recent years, Barbara Slucher's research group at Johns Hopkins University School of Medicine in the United States has chemically modified DON. The carboxyl site of DON is protected by ethyl ester, and the amino site is modified by leucine. A prodrug of JHU-083 was synthesized, and its synthesis route is as follows:

The prodrug is mainly reduced to DON through the action of aminopeptidase and esterase. Compared with DON prodrugs, JHU-083 reduces the gastrointestinal toxic side effects of DON. However, the reduction of the amino site of the prodrug mainly depends on the action of aminopeptidase, and aminopeptidase is widely present in various tissues. Therefore, the reduction selectivity of JHU-083 is insufficient and cannot efficiently achieve selective activation of tumor sites. , and there are many synthesis steps for JHU-083, which will lead to a low final overall reaction yield.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a hypoxia-activated 6-diazo-5-oxo-L-norleucine prodrug, its preparation method and application.

The invention provides a prodrug of 6-diazo-5-oxo-L-norleucine, as shown in formula (I):

Wherein, n is an integer from 0 to 5; R ₁ is a C1 to C10 alkyl group; R ₂ is a nitro group, a substituted or unsubstituted azo group.

Preferably, the substituents in the substituted azo group are selected from substituted or unsubstituted C1-C10 alkyl groups, substituted or unsubstituted C6-C20 aryl groups;

The substituents in the substituted C1-C10 alkyl group and the substituted C6-C20 aryl group are each independently selected from C1-C10 alkylamino groups.

Preferably, the R ₂ is nitro or a group represented by formula (1):

Among them, R ₃ and R ₄ are each independently a C1-C10 alkyl group.

Preferably, as shown in formula (II) or formula (III):

The invention also provides a method for preparing the above-mentioned 6-diazo-5-oxo-L-norleucine prodrug, including:

S1) React the compound represented by formula (IV) with the compound represented by formula (V) to obtain the compound represented by formula (VI);

S2) React the compound represented by formula (VI) with trimethylsilyldiazomethane and alkyllithium under low temperature conditions to obtain 6-diazo-5-oxo- represented by formula (I) Prodrugs of L-norleucine;

Or react the compound represented by formula (VII) with the compound represented by formula (VIII) to obtain the prodrug of 6-diazo-5-oxo-L-norleucine represented by formula (I);

Wherein, n is an integer from 0 to 5; R ₁ is a C1 to C10 alkyl group; R ₂ is a nitro group, a substituted or unsubstituted azo group.

The present invention also provides the use of the above-mentioned 6-diazo-5-oxo-L-norleucine prodrug in the preparation of drugs for treating tumors.

The present invention also provides a drug for treating tumors, including the above-mentioned prodrug of 6-diazo-5-oxo-L-norleucine.

Preferably, vascular blocking agents and/or immune checkpoint inhibitors are also included.

The invention provides a prodrug of 6-diazo-5-oxo-L-norleucine, as shown in formula (I); wherein, n is an integer from 0 to 5; R ₁ is C1 to C10 Alkyl group; R ₂ is nitro, substituted or unsubstituted azo group. Compared with the existing technology, the prodrug provided by the present invention is a hypoxia-activated prodrug, which can be selectively reduced to the original drug in the hypoxic part of the tumor, exert the effect of inhibiting tumor growth, and at the same time reduce the effect of the original drug on other normal cells. Effect, reducing the toxic side effects of this type of drug on the gastrointestinal tract, and the combined treatment with vascular blockers has a significant effect. The combined treatment with aPD-1 and vascular blockers significantly inhibited the growth and metastasis of tumors in the 4T1 model. .

Description of the drawings

Figure 1 shows the synthesis steps (a) of the prodrug of 6-diazo-5-oxo-L-norleucine in Example 1 of the present invention, the hydrogen nuclear magnetic spectrum characterization diagram of EOC-NBC (b), and the EOC- The NMR carbon spectrum characterization picture of NBC (c) and the mass spectrum characterization picture of EOC-NBC (d);

Figure 2 shows the hydrogen nuclear magnetic spectrum (a), carbon nuclear magnetic spectrum (b) and mass spectrum characterization of the prodrug HDON of 6-diazo-5-oxo-L-norleucine in Example 1 of the present invention. Figure (c) and HPLC spectrum (d);

Figure 3 shows the stability results of HDON, a prodrug of 6-diazo-5-oxo-L-norleucine in Example 1 of the present invention, in different buffers at 25°C (a), and in PBS- at 37°C. 7.4 Stability results in buffer (b), stability in plasma at 37°C (c), and mass spectrometry characterization of metabolites in plasma (d);

Figure 4 shows the reduction mechanism (a), the reduction efficiency curve (b) and the HPLC spectrum of the reduction product HDON, the prodrug HDON of 6-diazo-5-oxo-L-norleucine in Example 1 of the present invention. (c), reduction intermediate product DON-Et LC-MS characterization spectrum (d) and reduction product DON LC-MS characterization spectrum (e);

Figure 5 is the 4T1 cytotoxicity experiment graph (a), MC38 cytotoxicity experiment graph (b) and H22 cytotoxicity experiment graph (c) of DON and HDON in Example 1 of the present invention;

Figure 6 is a graph showing the body weight change curve of Kunming rats after continuous administration of HDON and DON for one week in Example 1 of the present invention;

Figure 7 shows the CD31 immunohistochemistry results (a), HIF-1α immunohistochemistry (b), and intracellular nitroreductase of the effect of CA4-NPs on the blood vessels of 4T1 breast cancer and MC38 colon cancer in Example 1 of the present invention. Content test chart (c) and tumor tissue nitroreductase content test chart (d);

Figure 8 is a tissue distribution diagram of single drug HDON and HDON+CA4-NPs at different times in Example 1 of the present invention;

Figure 9 is a schematic diagram of the treatment plan for H22 liver cancer in Example 1 of the present invention (a), a tumor volume change curve (b), a mouse weight change curve (c), a survival curve (d), and tumor size after treatment. Picture (e), tumor weight after treatment (f), HE section pictures of various organs and tumors of mice after treatment (g), tumor immune cell analysis picture (h);

Figure 10 is a schematic diagram of MC38 colon cancer treatment in Example 1 of the present invention (a), tumor volume change curve (b), mouse weight change curve (c), survival curve (d), and tumor size after treatment. Picture (e), tumor weight after treatment (f), glutamine content test picture of tumor tissue (g), analysis of mouse liver and kidney function indicators after treatment, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in serum , uric acid, urea nitrogen and creatinine content diagram (h);

Figure 11 is a HE section view of various organs and tumors in mice after MC38 model treatment in Example 1 of the present invention;

Figure 12 is the MC38 model tumor immune cell analysis chart (a) and serum cytokine analysis chart (b) in Example 1 of the present invention;

Figure 13 is a schematic diagram of the treatment plan for 4T1 breast cancer in Example 1 of the present invention (a), tumor volume change curve (b), mouse weight change curve (c), survival curve (d), and tumor after treatment. Weight picture (e), tumor size picture after treatment (f), India ink staining picture of mouse lung metastasis (g) and statistical picture of the number of mouse lung metastasis nodules (h);

Figure 14 shows the synthesis route diagram of Azo-DON (a), the hydrogen spectrum characterization diagram of Azo-NPC (b), the hydrogen spectrum characterization diagram of Azo-DON (c) and the ESI mass spectrum of Azo-DON in Example 2 of the present invention. Characterization diagram (d).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The invention provides a prodrug of 6-diazo-5-oxo-L-norleucine, as shown in formula (I):

Wherein, n is an integer from 0 to 5, preferably an integer from 1 to 3, more preferably 1 or 2; R ₁ is a C1 to C10 alkyl group, preferably a C1 to C8 alkyl group, more preferably C1 to C6 The alkyl group is more preferably a C2-C4 alkyl group, and most preferably a C2-C3 alkyl group.

R ₂ is a nitro group, a substituted or unsubstituted azo group; the substituent in the substituted azo group is preferably a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C6-C20 aryl group group, more preferably a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C6-C15 aryl group, still more preferably a substituted or unsubstituted C1-C4 alkyl group, or a substituted or unsubstituted C6 The ∼C10 aryl group is most preferably a substituted or unsubstituted C1-C2 alkyl group or a substituted or unsubstituted C6-C10 aryl group.

The substituents in the substituted C1-C10 alkyl group and the substituted C6-C20 aryl group are each independently preferably a C1-C10 alkylamino group, more preferably a C1-C8 alkylamino group, and even more preferably The C1-C6 alkylamino group is more preferably a C1-C4 alkylamino group, and most preferably a C1-C2 alkylamino group.

In the present invention, most preferably, the R ₂ is a nitro group or a group represented by formula (1):

Among them, R ₃ and R ₄ are each independently a C1-C10 alkyl group, preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, even more preferably a C1-C4 alkyl group, and most preferably C1～C3 alkyl group.

In the present invention, most preferably, the R ₂ is a nitro group or a group represented by formula (2):

Among them, R ₃ and R ₄ are each independently a C1-C10 alkyl group, preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, even more preferably a C1-C4 alkyl group, and most preferably C1～C3 alkyl group.

In the present invention, it is further preferred that the prodrug of 6-diazo-5-oxo-L-norleucine is represented by formula (II) or formula (III):

In the present invention, it is further preferred that the prodrug of 6-diazo-5-oxo-L-norleucine is represented by formula (II-1) or formula (III-1):

The prodrug provided by the invention is a hypoxia-activated prodrug, which can be reduced to the original drug in the hypoxic part of the tumor, exerting the effect of inhibiting tumor growth, while reducing the impact of the original drug on other normal cells, and reducing the impact of this type of drug on the stomach. In terms of intestinal toxic side effects, combined treatment with vascular blockers has a significant effect. Combined treatment with aPD-1 and vascular blockers significantly inhibited tumor growth and metastasis in the 4T1 model.

The prodrug provided by the invention can be reduced to DON by nitroreductase and esterase under hypoxic conditions, thereby reducing the toxic side effects of DON on the gastrointestinal tract and achieving selective reduction of the drug.

The invention also provides a method for preparing the above-mentioned 6-diazo-5-oxo-L-norleucine prodrug, which includes: S1) combining the compound represented by formula (IV) with the compound represented by formula (V) React with the compound represented by formula (VI) to obtain a compound represented by formula (VI); S2) react the compound represented by formula (VI) with trimethylsilyldiazomethane and alkyl lithium under low temperature conditions to obtain formula ( The prodrug of 6-diazo-5-oxo-L-norleucine shown in I).

The compound represented by formula (IV) is reacted with the compound represented by formula (V); in the present invention, the reaction is preferably carried out in an organic solvent; the compound represented by formula (IV) and the compound represented by formula (V) The molar ratio of the compounds shown is preferably 1: (1.8~2.2), more preferably 1:2; the organic solvent is an organic solvent well known to those skilled in the art, and is not particularly limited. In the present invention, it is preferably Acetonitrile; this reaction is preferably carried out in the presence of an aminopyridine compound and an organic amine; the aminopyridine compound is preferably 4-dimethylaminopyridine; the organic amine is preferably N,N-diisopropylethylamine ; The molar ratio of the compound represented by the formula (IV), the aminopyridine compound and the organic amine is preferably 1: (0.8～1.2): (1.8～2.2), more preferably 1:1:2; in the present invention In the method, the compound represented by formula (IV) and the compound represented by formula (V) are preferably first mixed in an ice bath, and then raised to room temperature for reaction; the mixing time is preferably 1 to 3 hours; in the present invention In this step, it is more preferred to mix and stir the compound represented by the formula (IV), the aminopyridine compound and the organic amine in an organic solvent and in an ice bath, and then add the compound represented by the formula (V) in an ice bath. Mix under the conditions, and then rise to room temperature for reaction; the mixing and stirring time is preferably 10 to 20 min, more preferably 15 min; the room temperature reaction time is preferably 30 to 60 h, more preferably 40 to 50 h, and still more preferably 45 ~48h.

After the reaction, it is preferably concentrated to obtain a solid, which is then dissolved in dichloromethane, washed with saturated brine, dried and concentrated, and then subjected to column chromatography to obtain a crude product; the eluent of the column chromatography is preferably ethyl acetate and hexyl acetate. alkane; the volume ratio of the ethyl acetate to hexane is preferably 1:20 to 2:1.

After dissolving the crude product in ethyl acetate and adding glacial ether for recrystallization, the compound represented by formula (VI) is obtained.

The compound represented by formula (VI) is reacted with trimethylsilyldiazomethane and alkyl lithium under low temperature conditions; the alkyl lithium is preferably n-butyllithium; the compound represented by formula (VI) , the molar ratio of trimethylsilyldiazomethane and alkyl lithium is preferably 1: (1～1.2): (1～1.5), more preferably 1:1.18:1.22; in the present invention, it is preferred to first Methylsilyl diazomethane and alkyl lithium are mixed and reacted under low temperature conditions, and then added to the organic solution of the compound represented by formula (VI) for reaction; the temperature of the mixed reaction is preferably -100°C to -90°C , more preferably -98°C; the mixing reaction time is preferably 20 to 40 min, more preferably 30 min; the solvent in the organic solution of the compound represented by formula (VI) is preferably tetrahydrofuran; the reaction temperature is preferably -80℃～-70℃, more preferably -78℃; the reaction time is preferably 20～40min, more preferably 30min.

After the reaction, preferably add water to quench the reaction, extract with ethyl acetate, and purify by column chromatography to obtain the precursor of 6-diazo-5-oxo-L-norleucine represented by formula (I). medicine; the eluent of the column chromatography is preferably chloroform and acetone; the volume ratio of chloroform to acetone is preferably 20:1.

Alternatively, the compound represented by formula (VII) is reacted with the compound represented by formula (VIII) to obtain the prodrug of 6-diazo-5-oxo-L-norleucine represented by formula (I).

The compound represented by formula (VII) is reacted with the compound represented by formula (VIII); the reaction is preferably carried out in an organic solvent; the organic solvent is preferably N,N-dimethylformamide; the reaction is preferably It is carried out in the presence of an organic amine; the organic amine is preferably triethylamine; in the present invention, it is preferred to mix the compound represented by formula (VII) and the compound represented by formula (VIII) with an organic solvent respectively to obtain respective solutions, and then mix the two solutions under low temperature conditions for reaction; the temperature of the reaction is preferably -10°C to 10°C, more preferably 0°C; the reaction time is preferably 1 to 3h, more preferably Preferably it is 2h.

After the reaction is completed, ethyl acetate is preferably added, washed with water and saturated brine in sequence, dried, and purified by column chromatography to obtain 6-diazo-5-oxo-L-zhengeide represented by formula (I). Prodrug of amino acid; the eluent of the column chromatography is preferably n-hexane and ethyl acetate; the volume ratio of n-hexane and ethyl acetate is preferably 3:1.

The preparation method of the 6-diazo-5-oxo-L-norleucine prodrug provided by the invention is simple and has a high yield.

The present invention also provides the use of the above-mentioned 6-diazo-5-oxo-L-norleucine prodrug in the preparation of drugs for treating tumors.

The present invention also provides a drug for treating tumors, including the above-mentioned prodrug of 6-diazo-5-oxo-L-norleucine.

Preferably, the drug for treating tumors also includes blood vessel blockers and/or immune checkpoint inhibitors.

Preferably, the blood vessel blocking agent is CA4-NPs; the immune checkpoint inhibitor is aPD-1.

Preferably, the mass ratio of the prodrug of 6-diazo-5-oxo-L-norleucine to the vascular blocking agent is 1:10-20, more preferably 1:(15-20), More preferably, it is 1: (18-20).

Preferably, the mass ratio of the prodrug of 6-diazo-5-oxo-L-norleucine to the immune checkpoint inhibitor is preferably 1: (0.05-0.2), more preferably 1: (0.08) ~0.12), more preferably 1:0.1.

In order to further illustrate the present invention, a 6-diazo-5-oxo-L-norleucine prodrug, its preparation method and application provided by the present invention are described in detail below in conjunction with the examples.

The reagents used in the following examples are all commercially available.

Example 1

The synthesis of 6-diazo-5-oxo-L-norleucine prodrug (HDON) is shown in Figure 1a.

1.1 Synthesis of intermediate EOC-NBC: Add EOC (5-oxypyrrolidine-2-carboxylic acid ethyl ester) (5g, 31.8mmol, 1equiv), DMAP (4-dimethylaminopyridine) (3.9 g, 31.8mmol, 1equiv), DIPEA (N,N-diisopropylethylamine) (8.2g, 63.6mmol, 2equiv) was dissolved in 90mL of anhydrous ACN (acetonitrile), stirred in an ice-water bath for 15min, and then slowly Add NBC (para-nitrobenzyl chloroformate) (2.96M in ACN, 13.7g, 63.6mmol, 2equiv) to the round-bottomed flask, continue stirring in the ice-water bath for 2 hours, then return to room temperature for 48 hours. After the reaction was completed, the mixture was concentrated by rotary evaporation to obtain a brown solid. Dissolve the solid with 200 mL of methylene chloride, wash with saturated brine (200 mL :10 2 column volumes, 1:2 5 column volumes, 1:1 4 column volumes, 2:1 2 column volumes) to obtain a crude product. The crude product was dissolved in an appropriate amount of ethyl acetate, and glacial ether was added for recrystallization to obtain white solid EOC-NBC. The structure was determined by ¹ H NMR and ¹³ C NMR using CDCl ₃ as the solvent, as shown in Figure 1b and c. ESI-MS (ESI ⁺ ) was also used to characterize EOC-NBC, [M+H] ⁺ =337.41, [M +Na] ⁺ =359.41, [M+NH ₄ ] ⁺ =354.46, as shown in Figure 1d.

1.2 Synthesis of HDON: Add EOC-NBC (450 mg, 1.34 mmol, 1.00 equiv) to a round-bottomed flask, dissolve it in 12 mL anhydrous tetrahydrofuran, and cool it to -116°C in a Dewar flask. In addition, TMS (trimethylsilyldiazomethane) (0.79 mL, 2M in hexane, 1.58 mmol, 1.18 equiv) was dissolved in 7 mL of anhydrous tetrahydrofuran, and cooled to -98°C. Slowly drop n-BuLi (0.65mL, 2.5M in hexane, 1.63mmol, 1.22equiv) into the TMS solution and react for 30 minutes. Then, the mixed solution of TMS and n-BuLi was added to the EOC-NBC solution, the temperature was slowly raised from -116°C to -78°C, and the reaction was continued for 30 minutes. After the reaction, 15 mL of aqueous solution was added to quench the reaction. Extract three times with ethyl acetate (10 mL×3). Then, it was washed three times with saturated brine (20 mL × 3), and the washed solution was dried over anhydrous magnesium sulfate. Purify by column chromatography (chloroform/acetone 20:1). The structure was determined by ¹ H NMR and ¹³ C NMR using CDCl ₃ as the solvent, as shown in Figure 2a and b; ESI-MS (ESI ⁺ ) was also used to characterize HDON, [M+Na] ⁺ =401.3, as shown in Figure 2c display; and the purity of HDON was detected using UV-high performance liquid chromatography (HPLC). The detection wavelength was 267nm, the mobile phase was acetonitrile:water (1/1, V/V), the flow rate was 1mL/min, and the chromatogram As shown in Figure 2d, it can be seen from Figure 2d that its purity is higher than 98%.

1.3 Evaluate the chemical stability and plasma stability of HDON. First, the chemical stability of HDON at 25°C was evaluated. 1mg HDON was dissolved in 1mL of acetonitrile, diluted to a concentration of 0.27mM with buffers of different pH values, placed under constant conditions of 25°C, and HPLC was used to detect 0h and 6h. , HDON concentration at 12h, 24h and 48h. The results show that HDON has good stability in PBS buffers with different pH values. As shown in Figure 3a, the remaining contents of HDON in PBS-8.5, 7.4, 6.8, and 5.5 at 48h are 95.80±0.33% and 98.76 respectively. ±1.17%, 98.77±1.42%, 99.33±0.99%. In order to simulate physiological conditions, a stability experiment was then conducted in a solution of PBS-7.4 at 37°C. As shown in Figure 3b, the remaining HDON content in the solution after 24 hours was 96.17±0.82%. Next, stability experiments were conducted in C57BL/6 mouse plasma at 37°C. Similarly, 1 mg/mL HDON acetonitrile solution was diluted to 0.2 mg/mL with PBS-7.4, and then mixed with equal volumes of mouse plasma. , the concentration of HDON in plasma was detected at 0h, 0.5h, 1h, and 3h, as shown in Figure 3c. HDON is rapidly metabolized in plasma, and its metabolized products were tested by ESI mass spectrometry, as shown in Figure 3d. It was found that HDON was not metabolized to DON, but a new intermediate product was generated, and the ethyl ester bond was hydrolyzed by the enzyme.

The reduction mechanism of HDON is shown in Figure 4a. First, the present invention tested the reduction of HDON under the action of nitroreductase at different concentrations at 37°C. The PBS-7.4 solution was deoxygenated using nitrogen, and 5 mg HDON was dissolved in 1 mL of ethanol castor oil (1/1, V/V ), then dilute with PBS-7.4 to obtain a 0.13mM solution. In a 1.5mL centrifuge tube, mix 200μL HDON (0.13mM) and 60μL NADH (2.35mM), and then add 40μL nitric acid to the sample solution under anoxic conditions. Hydrogen reductase (0.25mg/mL or 0.50mg/mL) solution, place the mixed sample in a constant temperature shaking chamber at 37°C, and detect the HDON content at 0h, 0.5h, 1h, 3h, 6h, and 12h. As shown in Figure 4b, as the enzyme concentration increases, the reduction rate of HDON also increases. Then the present invention tested the HPLC of HDON under the action of esterase and nitroreductase. In a 1.5mL centrifuge tube, 200 μL HDON (0.13mM) and 60 μL NADH (2.35mM) were mixed, and then added to the sample solution under anoxic conditions. Add 40 μL nitroreductase (0.50 mg/mL) solution to the above reaction mixture, add 400 μL esterase (5 U/mL) to the above reaction mixture after 1 hour, and continue to incubate for 3 hours. After the reaction, use a nitrogen blower to incubate the sample solution at room temperature. Concentrate, and use pre-column derivatization method to analyze the product (add 10 μL water, 50 μL pH=9 0.2M sodium bicarbonate buffer, and 100 μL 10mM DABS-Cl (dansyl chloride) to the concentrated sample tube. acetone solution, followed by 60°C water bath for 15 min). As shown in Figure 4c, the reduction product was verified by LC-MS (at 25°C, gradient elution with a flow rate of 1 mL/min, and the sample was detected by HPLC or LC-MS. The mobile phase was water + 0.1% Formic acid and acetonitrile + 0.1% formic acid. Acetonitrile + 0.1% formic acid gradient from 20% to 95% for 30 min), as shown in Figure 4d and Figure 4e. HDON can be reduced to the DON-Et intermediate under the action of nitroreductase (Figure 4d), and then reduced to DON under the action of esterase (Figure 4e). In vitro experiments have shown that HDON can be reduced to DON original drug under the action of nitroreductase and esterase.

The present invention conducted cytotoxicity experiments on different cell lines, evaluated the in vitro cytotoxicity of HDON and DON through CCK8 experiments, and inoculated H22 cells, 4T1 cells or MC38 cells (8.0×10 ³ cells per well) in a 96-well culture plate. , and add 180 μL of complete medium, and then incubate overnight. Dissolve DON in PBS-7.4 to obtain a 1 mM stock solution. HDON was dissolved in DMSO and then diluted with PBS-7.4 to obtain a 1 mM stock solution (containing 1% DMSO). Next, add 20 μL of drug solutions of different concentrations to each well. After 24, 48, or 72 hours of culture, 20 μL of CCK8 solution was injected into each well. Test the plate directly after 1-2 hours of incubation. Measure the absorbance of each well on a microplate reader at 450 nm. As can be seen from Figure 5, HDON significantly reduces the cytotoxicity of the drug to tumor cells compared with DON. Cytotoxicity experimental results show that the prodrug strategy can reduce the toxic and side effects of drugs.

The toxic and side effects of HDON were evaluated on Kunming rats. Female Kunming mice were randomly divided into 7 groups and received intraperitoneal injection of DON (0.66, 1.32, 2.64 μmol/kg) or HDON (0.00, 0.66, 1.32, 2.64 μmol/kg). Observe weight changes every day for 8 consecutive days for one week. As shown in Figure 6, HDON and DON were administered at different doses continuously for one week. The body weight of Kunming mice in the HDON group did not decrease. The relative body weights of the mice in the DON group were 93.35±16.15% (0.66μmol/kg) and 78.80±4.96% (1.32) respectively. μmol/kg) and 69.77±6.83% (2.64 μmol/kg), significant weight loss occurred in the two dose groups of DON. Experimental results show that in normal Kunming rats, HDON has no significant toxic or side effects compared with DON at the same dose.

At the cellular level and animal level, the present invention verified that HDON significantly reduced the toxic side effects of DON. In the present invention, it is used in combination with the blood vessel blocking agent CA4-NPs. First, the present invention detected the effect of CA4-NPs on blood vessels of 4T1 breast cancer (20 mg/kg, calculated as CA4) and MC38 colon cancer (18 mg/kg, calculated as CA4). After mice were injected with CA4-NPs into the tail vein, 24 h The tumor tissue was then sacrificed and stored in 4% paraformaldehyde solution. CD31 immunohistochemistry was used to determine the blood vessel density of the tumor tissue. As shown in Figure 7a, CA4-NPs can reduce the blood vessel density of the tumor, which is beneficial to deepening the tumor. hypoxic conditions. Next, the present invention confirmed the expression of HIF-1α using immunohistochemistry. As shown in Figure 7b, CA4-NPs can increase the degree of hypoxia in tumors and increase the expression of HIF-1α. At the same time, it was also verified that the degree of hypoxia in H22 liver cancer tumors is deeper than that of 4T1 breast cancer and MC38 colon cancer models. It is more conducive to the selective reduction of HDON. Next, the present invention verified that hypoxia can increase the expression of nitroreductase at the cellular level, as shown in Figure 7c. The same has been verified at the animal level. CA4-NPs can increase the expression of nitroreductase in tumor tissues of 4T1 breast cancer (20 mg/kg, calculated as CA4) and MC38 colon cancer (18 mg/kg, calculated as CA4), as shown in Figure 7d.

In order to verify the reduction of HDON in vivo, the present invention conducted a biodistribution experiment on 4T1 breast cancer. When the 4T1 tumors reached approximately 250 mm ³ , the mice were randomly divided into 6 groups. Three groups were given intraperitoneal injections of HDON (15 mg/kg) to mice alone, and the other three groups were given intraperitoneal injections of HDON (15 mg/kg) + intravenous injection of CA4-NPs (20 mg/kg, calculated as CA4). The mice were sacrificed at 1h, 4h and 8h respectively, and the main organs and tumor tissues were removed. The samples were pre-column derivatized using dansyl chloride, and the content of DON was detected by HPLC. As shown in Figure 8, it shows that HDON can be reduced to DON in the body. After combined with CA4-NPs for 1 hour, the content of DON in the tumor was 3.46 times higher than that of the single drug group and 37.3 times higher than that in the kidney. This shows that HDON has good ability to selectively activate tumors due to hypoxia. The reduction of HDON is positively related to the degree of hypoxia.

The H22 liver cancer model is a type of tumor model with deep hypoxia. The present invention first used HDON alone to conduct anti-tumor research. When the tumor volume reached ^about 100 mm, the mice were randomly divided into 2 groups: PBS-7.4, HDON (1 mg/kg). HDON was administered intraperitoneally every day until the tumor inhibition endpoint (Fig. 9a). On the 14th day, PBS-7.4 reached approximately 2000mm ³ . The TSR of HDON is 76.5% (Figure 9b). Ascites transfer occurred in the mice in the PBS group, and the weight of the mice increased, while HDON had no significant effect on the weight of the mice and had minimal toxic and side effects (Figure 9c). The present invention also observed the survival time of mice. As shown in Figure 9d, HDON treatment can prolong the survival time of mice. As shown in Figure 9e and Figure 9f, the HDON treatment group significantly inhibited tumor growth. Next, the present invention performed HE staining on various organs, tissues and tumors of mice (Fig. 9g). Compared with the PBS group, HDON had no obvious damage to the main organs and tissues of mice. Similarly, the present invention also conducted flow cytometric analysis of the mouse tumor immune microenvironment, as shown in Figure 9g. After HDON treatment, more CD4 ⁺ T cells (accounting for 0.37% of the total cell number) and CD8 ⁺ T cells (accounting for 0.23% of the total cell number) were detected within the tumor. Effectively activates anti-tumor immune response. The same MDSCs cells were significantly reduced, and the immune microenvironment of the tumor changed.

The present invention then studied the anti-tumor effect of hypoxia-activated glutamine antagonist (HDON) combined with CA4-NPs on the MC38 colon cancer model, as shown in Figure 10. When the tumor volume reaches ^about 100mm, the mice are randomly divided into 4 groups: PBS-7.4, HDON (1mg/kg), CA4-NPs (18mg/kg), HDON+CA4-NPs (1mg/kg+18mg/kg) kg). HDON was administered intraperitoneally every day until the tumor inhibition endpoint. CA4-NPs were injected into the tail vein once every four days for a total of three times (Fig. 10a). As shown in Figure 10b and Figure 10c, the PBS group grew the fastest, reaching 2000mm ³ on the 16th day. The tumor suppression rate (TSR) of the HDON+CA4-NPs group reached 98.3%, while the TSR of the HDON and CA4-NPs single-drug groups were 29.1% and 43.3% respectively, and the body weight after treatment was different from that of the PBS group. Not big. In addition to the anti-tumor effect, the present invention also monitored the survival rates of mice in different treatment groups and found that compared with other treatment groups, the HDON+CA4-NPs combined treatment group significantly prolonged the overall survival rate of mice (Figure 10d ). As shown in Figure 10e and Figure 10f, the HDON+CA4-NPs combined treatment group significantly inhibited tumor growth. Similarly, on the sixth day of tumor suppression, the glutamine content in the tumor tissue was tested. As shown in Figure 10g, the HDON and CA4-NPs combined treatment group significantly increased the glutamine content in the tumor tissue, which inhibited the tumor. Cellular uptake and utilization of glutamine. After the tumor suppression was completed, the liver and kidney function of the mouse serum was analyzed. The results showed that the treatment had no significant effect on the contents of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, uric acid, urea nitrogen and creatinine, indicating that the treatment had no significant effect. Caused damage to liver and kidney function in mice. Hematoxylin and eosin (H&E) staining was performed on various organs and tumor tissues of mice, and the HE section diagram of various organs and tumors of mice after MC38 model treatment was obtained, as shown in Figure 11. The results were the same as expected. Compared with the PBS group, the treatment group had no obvious effects on various organs and tissues of mice.

Next, the immune microenvironment of the MC38 colon cancer model was analyzed. As shown in Figure 12a, after combined treatment with HDON and CA4-NPs, more CD4 ⁺ T cells (accounting for 6.21% of the total cell number) were detected in the tumor cells. ) and CD8 ⁺ T cells (accounting for 3.05% of the total cell number), indicating that combined treatment of HDON and CA4-NPs activated the anti-tumor immune response. The same MDSCs cells were significantly reduced, and the immune microenvironment of the tumor changed. Consistent with immune cell analysis, serum was tested for pro-inflammatory cytokines (IL-6 and IFN-γ). As shown in Figure 12b, the combined treatment group of HDON and CA4-NPs significantly increased the contents of IL-6 and IFN-γ. The above experimental results show that under the action of CA4-NPs, HDON can be reduced to DON in tumor tissues, inhibiting the uptake of glutamine by tumor cells, and activating anti-tumor immune responses at the same time, without obvious toxic side effects on mice.

Similarly, the present invention conducted anti-tumor research on the 4T1 model by combining CA4-NPs and aPD-1, as shown in Figure 13. When the tumor volume reaches ^about 100mm, the mice are randomly divided into 8 groups: PBS-7.4, aPD-1 (100μg each), HDON (1mg/kg), HDON+aPD-1 (1mg/kg+100μg) , CA4-NPs (20mg/kg, calculated as CA4), CA4-NPs+aPD-1 (20mg/kg+100μg), HDON+CA4-NPs (1mg/kg+20mg/kg), HDON+CA4-NPs+ aPD-1(1mg/kg+18mg/kg+100μg). Among them, HDON was administered intraperitoneally every day until the end of tumor inhibition, CA4-NPs was injected into the tail vein once every four days, a total of three times, and aPD-1 was intraperitoneally administered three times, once every three days (Figure 13a). As shown in Figure 13b, the PBS group grew the fastest, and the tumor volume reached approximately 2000mm ³ on the 22nd day. The TSR of the HDON+CA4-NPs group was 98.07%, while the tumor inhibition rate of HDON+CA4-NPs+aPD-1 three-drug combination treatment was 99.80%, with significant effects. At the same time, there was no significant change in body weight after the treatment (Figure 13c). Next, a survival experiment was conducted, and the combined treatment of HDON and CA4-NPs could significantly improve the survival time of mice (Figure 13d). As shown in Figure 13e and Figure 13f, the combined treatment group of HDON and CA4-NPs significantly inhibited tumor growth. Lung metastasis of mice was stained with India ink, and the number of lung metastasis nodules was counted. Combined treatment of HDON and CA4-NPs can inhibit lung metastasis of tumors (Figure 13g, h).

Example 2

First, Azo-OH was synthesized according to the literature method. Dissolve Azo-OH (500 mg, 1.96 mmol, 1 equiv) in 5 mL of DCM (dichloromethane) and cool to 0°C. DMAP (23.95 mg, 0.196 mmol, 0.1 equiv) and triethylamine (327 μL, 2.352 mmol, 1.2 equiv) were added to the Azo-OH solution. Dissolve NPC (474.26 mg, 2.352 mmol, 1.2 equiv) in 5 mL of DCM, add the dissolved NPC solution dropwise to the Azo-OH solution, return to room temperature, and continue the reaction for 2 hours. After the reaction, wash once with saturated ammonium chloride (10 mL) and three times with water (10 mL × 3). Pure product Azo-NPC was obtained by recrystallization from ethyl acetate and n-hexane (Figure 14a). And the structure was determined by ¹ H NMR using CDCl ₃ as solvent (Fig. 14b).

Synthesis of Azo-DON: First, refer to the literature to synthesize DON-Et. Azo-NPC (116 mg, 0.276 mmol, 1.1 equiv) was dissolved in 5 mL of DMF (N, N-dimethylformamide) solution and cooled to 0°C. In addition, DON-Et (50 mg, 0.251 mmol, 1 equiv) was dissolved in 1 mL of DMF solution and quickly added to the Azo-NPC solution. Then TEA (triethylamine) (38.4 μL, 0.276 mmol, 1.1 equiv) was added. React for 2 hours at 0°C, then return to room temperature and react overnight. After the reaction, 50 mL of ethyl acetate was added, washed twice with water (50 mL × 2), washed twice with saturated brine (50 mL × 2), and dried over anhydrous magnesium sulfate. The product was purified by column chromatography (n-hexane:ethyl acetate 3:1). And the structure was determined by ¹ H NMR using _CDCl as solvent (Fig. 14c). Azo-DON was also characterized using ESI-MS (ESI ⁺ ), [M+H] ⁺ =481.5, [M+Na] ⁺ =503.5 (Figure 14d).

Claims (6)

1. A prodrug of 6-diazo-5-oxo-L-norleucine, characterized in that, as shown in formula (I): Wherein, n is an integer from 0 to 5; R ₁ is a C1 to C10 alkyl group; R ₂ is nitro group. 2. The prodrug according to claim 1, characterized in that, as shown in formula (II): 3. A method for preparing the prodrug of 6-diazo-5-oxo-L-norleucine according to claim 1, characterized in that it includes: S1) React the compound represented by formula (IV) with the compound represented by formula (V) to obtain the compound represented by formula (VI); S2) React the compound represented by formula (VI) with trimethylsilyldiazomethane and alkyllithium under low temperature conditions to obtain 6-diazo-5-oxo- represented by formula (I) Prodrugs of L-norleucine; Or react the compound represented by formula (VII) with the compound represented by formula (VIII) to obtain the prodrug of 6-diazo-5-oxo-L-norleucine represented by formula (I); Wherein, n is an integer from 0 to 5; R ₁ is a C1 to C10 alkyl group; R ₂ is nitro group. 4. Application of the prodrug of 6-diazo-5-oxo-L-norleucine according to any one of claims 1 to 2 in the preparation of drugs for treating tumors. 5. A drug for treating tumors, characterized by comprising the prodrug of 6-diazo-5-oxo-L-norleucine according to any one of claims 1 to 2. 6. The drug according to claim 5, further comprising a vascular blocker and/or an immune checkpoint inhibitor.