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

Prodrugs of 6-diazo-5-oxo-L-norleucine, methods of making and uses thereof Download PDF

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CN114805138B
CN114805138B CN202210513291.9A CN202210513291A CN114805138B CN 114805138 B CN114805138 B CN 114805138B CN 202210513291 A CN202210513291 A CN 202210513291A CN 114805138 B CN114805138 B CN 114805138B
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prodrug
hdon
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norleucine
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CN114805138A (en
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刘芷麟
郑梦飞
许航
孙佳丽
汤朝晖
陈学思
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Changchun Institute of Applied Chemistry of CAS
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Abstract

The invention provides a prodrug of 6-diazonium-5-oxo-L-norleucine, which is shown as a formula (I); wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro, substituted or unsubstituted azo. Compared with the prior art, the prodrug provided by the invention is a hypoxia activated prodrug, can be selectively reduced to a raw drug at a tumor hypoxia part, plays a role in inhibiting tumor growth, reduces the influence of the raw drug on other normal cells, reduces the toxic and side effects of the drug on stomach and intestine, has obvious combined treatment effect with a vascular blocking agent, and remarkably inhibits the growth and metastasis of tumors on a 4T1 model by combined treatment with three drugs, namely, aPD-1 and the vascular blocking agent.

Description

Prodrugs of 6-diazo-5-oxo-L-norleucine, methods of making and uses thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a prodrug of 6-diazonium-5-oxo-L-norleucine, a preparation method and application thereof.
Background
Cancer has become a serious disease affecting the quality of life of people, and according to data issued by the national cancer center, the incidence of cancer in China is on an increasing trend. Traditional cancer treatments include chemotherapy, radiation therapy, and surgical resection. Chemotherapy is a treatment means which kills cancer cells by chemical drugs and is the most effective at present. However, chemotherapy is often associated with serious side effects, as chemotherapeutic agents tend to lack selectivity, killing cancer cells and, at the same time, damaging normal cells. For example, drugs can have toxic and side effects on heart, gastrointestinal system, liver and kidney tissues, skin and the like.
Hypoxic regions are prevalent in solid tumors and can cause tumor resistance through a variety of pathways, which is detrimental to tumor treatment. Under normal physiological conditions, however, the tissue does not have hypoxic regions, so these hypoxic regions present an opportunity for tumor-selective treatment. The unique property of solid tumor hypoxia brings different directions for the development of chemotherapeutic drugs. Currently, a variety of Hypoxia-activated prodrugs, hypoxia-Activated Prodrugs (HAPs), have been developed clinically. By utilizing the characteristics of tumor self hypoxia, HAPs can be selectively activated at the tumor site to be active medicines, so as to play a role in killing tumors and avoid systemic toxicity caused by the medicines. The strategy of modifying the small molecule chemotherapeutic drugs into HAPs not only reduces the systemic toxicity of the drugs, but also converts the disadvantage of tumor hypoxia into the advantage of selective treatment.
DON is a class of glutamine antagonists that block glutamine metabolism by cells, thereby killing the cells. However, the medicine cannot distinguish normal cells from tumor cells, and serious gastrointestinal toxic and side effects exist in clinical experiments, so that patients can suffer from symptoms such as diarrhea, vomiting and the like. Eventually, the clinical trial is suspended. In recent years, DON was chemically modified by the Japanese Lu Xieer subject group of the university of Johns Hopkins, U.S. and the carboxyl site of DON was protected by ethyl ester and the amino site was modified by leucine to synthesize a prodrug of JHU-083, the synthetic route of which is shown below:
the prodrug is reduced to DON primarily by aminopeptidase and esterase action. JHU-083 reduces the gastrointestinal toxic side effects of DON compared to DON prodrugs. However, the reduction of the amino site of the prodrug is mainly dependent on the action of aminopeptidase, and aminopeptidase is widely present in various tissues, so that the reduction selectivity of JHU-083 is insufficient, the selective activation of tumor sites cannot be efficiently realized, and the synthesis steps of JHU-083 are more, so that the final total reaction yield is low.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a prodrug of hypoxia activated 6-diazonium-5-oxo-L-norleucine, a preparation method and application thereof.
The invention provides a prodrug of 6-diazonium-5-oxo-L-norleucine, which is shown as a formula (I):
wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro, substituted or unsubstituted azo.
Preferably, the substituent in the substituted azo group is selected from the group consisting of a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C6-C20 aryl group;
the substituents in the substituted C1-C10 alkyl and substituted C6-C20 aryl are each independently selected from C1-C10 alkylamino.
Preferably, said R 2 Is nitro or a group of formula (1):
wherein R is 3 And R is R 4 Each independently is a C1 to C10 alkyl group.
Preferably, as shown in formula (II) or formula (III):
the invention also provides a preparation method of the prodrug of the 6-diazonium-5-oxo-L-norleucine, which comprises the following steps:
s1) reacting a compound shown in a formula (IV) with a compound shown in a formula (V) to obtain a compound shown in a formula (VI);
s2) reacting the compound shown in the formula (VI) with trimethylsilyl diazomethane and alkyl lithium under a low temperature condition to obtain a prodrug of 6-diazonium-5-oxo-L-norleucine shown in the formula (I);
or reacting a compound shown in a formula (VII) with a compound shown in a formula (VIII) to obtain a prodrug of 6-diazonium-5-oxo-L-norleucine shown in a formula (I);
wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro, substituted or unsubstituted azo.
The invention also provides application of the prodrug of the 6-diazonium-5-oxo-L-norleucine in preparing a tumor treatment drug.
The invention also provides a medicine for treating tumors, which comprises the prodrug of the 6-diazonium-5-oxo-L-norleucine.
Preferably, vascular blockers and/or immune checkpoint inhibitors are also included.
The invention provides a prodrug of 6-diazonium-5-oxo-L-norleucine, which is shown as a formula (I); wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro, substituted or unsubstituted azo. Compared with the prior art, the prodrug provided by the invention is a hypoxia activated prodrug, can be selectively reduced to a raw drug at a tumor hypoxia part, plays a role in inhibiting tumor growth, reduces the influence of the raw drug on other normal cells, reduces the toxic and side effects of the drug on stomach and intestine, has obvious combined treatment effect with a vascular blocking agent, and remarkably inhibits the growth and metastasis of tumors on a 4T1 model by combined treatment with three drugs, namely, aPD-1 and the vascular blocking agent.
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FIG. 1 is a chart showing the synthesis of a prodrug of 6-diazo-5-oxo-L-norleucine in example 1 of the present invention, step (a), nuclear magnetic resonance spectroscopy of EOC-NBC (b), nuclear magnetic resonance spectroscopy of EOC-NBC (c) and mass spectrometry characterization of EOC-NBC (d);
FIG. 2 shows a nuclear magnetic resonance spectrum (a), a nuclear magnetic resonance spectrum (b), a mass spectrum (c) and an HPLC (d) of the prodrug HDON of 6-diazo-5-oxo-L-norleucine in example 1 of the present invention;
FIG. 3 is a graph showing the stability of the prodrug of 6-diazo-5-oxo-L-norleucine, HDON, in example 1 of the present invention in different buffers at 25 ℃ (a), PBS-7.4 at 37 ℃ (b), plasma at 37 ℃ (c) and mass spectrum characterization of metabolites in plasma (d);
FIG. 4 is a graph showing the reduction mechanism (a), the reduction efficiency curve (b), the HPLC profile (c) of the reduction product, the DON-Et LC-MS characterization profile (d) of the reduction intermediate product and the DON LC-MS characterization profile (e) of the prodrug HDON of 6-diazo-5-oxo-L-norleucine in example 1 of the present invention;
FIG. 5 shows the 4T1 cytotoxicity test patterns (a), MC38 cytotoxicity test pattern (b) and H22 cytotoxicity test pattern (c) of DON and HDON in example 1 of the present invention;
FIG. 6 is a graph showing the change in body weight of Kunming mice after one week of continuous administration of HDON and DON in example 1 of the present invention;
FIG. 7 is a graph of the results of CD31 immunohistochemical treatment of vascular effects of CA4-NPs on 4T1 breast cancer and MC38 colon cancer in example 1 (a), HIF-1. Alpha. Immunohistochemical treatment (b), intracellular nitroreductase assay (c) and tumor tissue nitroreductase assay (d);
FIG. 8 is a graph showing tissue distribution of HDON and HDON+CA4-NPs of example 1 of this invention at different times;
fig. 9 is a schematic diagram (a), a tumor volume change graph (b), a mouse weight change graph (c), a survival time graph (d), a tumor size graph (e) after treatment, a tumor weight (f) after treatment, a tumor HE section graph (g) of organs and tumors of the mice after treatment, and a tumor immune cell analysis graph (H) according to the treatment plan of H22 liver cancer in example 1 of the present invention;
FIG. 10 is a graph showing MC38 colon cancer treatment schematic (a), tumor volume change graph (b), mouse weight change graph (c), survival graph (d), tumor size graph (e) after treatment, tumor weight (f) after treatment, glutamine content test graph (g) of tumor tissue, liver and kidney function index analysis of mouse after treatment, alkaline phosphatase in serum, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, uric acid, urea nitrogen and creatinine content graph (h) in example 1 of the present invention;
FIG. 11 is a graph showing HE sections of organs and tumors of mice treated by MC38 model in example 1 of the present invention;
FIG. 12 is a diagram of analysis of tumor immune cells and serum cytokine of MC38 model in example 1 (a) according to the present invention;
FIG. 13 is a schematic diagram (a), a graph (b) of tumor volume change, a graph (c) of mouse weight change, a graph (d) of survival time, a graph (e) of tumor weight after treatment, a graph (f) of tumor size after treatment, a graph (g) of ink staining of indian ink of lung metastasis of mice, and a graph (h) of node number statistics of lung metastasis of mice according to the embodiment 1 of the present invention;
FIG. 14 is a synthetic scheme (a) for Azo-DON, a hydrogen spectrum characterization (b) for Azo-NPC, a hydrogen spectrum characterization (c) for Azo-DON, and an ESI mass spectrum characterization (d) for Azo-DON according to the invention in example 2.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a prodrug of 6-diazonium-5-oxo-L-norleucine, which is shown as a formula (I):
wherein n is an integer of 0 to 5, preferablyAn integer of 1 to 3, preferably 1 or 2; r is R 1 The alkyl group is a C1-C10 alkyl group, preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, still more preferably a C2-C4 alkyl group, and most preferably a C2-C3 alkyl group.
R 2 Is nitro, substituted or unsubstituted azo; the substituent in the substituted azo group is preferably a substituted or unsubstituted C1 to C10 alkyl group, a substituted or unsubstituted C6 to C20 aryl group, more preferably a substituted or unsubstituted C1 to C6 alkyl group, a substituted or unsubstituted C6 to C15 aryl group, still more preferably a substituted or unsubstituted C1 to C4 alkyl group, a substituted or unsubstituted C6 to C10 aryl group, and most preferably a substituted or unsubstituted C1 to C2 alkyl group, a substituted or unsubstituted C6 to 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, still more preferably a C1-C6 alkylamino group, still 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 nitro or a group of formula (1):
wherein R is 3 And R is R 4 Each independently is a C1-C10 alkyl group, preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, still more preferably a C1-C4 alkyl group, and most preferably a C1-C3 alkyl group.
In the present invention, most preferably, the R 2 Is nitro or a group of formula (2):
wherein R is 3 And R is R 4 Each independently is a C1-C10 alkyl group, preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group, and still more preferably a C1-to-CC4 alkyl, most preferably C1-C3 alkyl.
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-diazonium-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, can be reduced to a raw drug at a tumor hypoxia part, plays a role in inhibiting tumor growth, reduces the influence of the raw drug on other normal cells, reduces the toxic and side effects of the drug on stomach and intestine, has obvious combined treatment effect with a vascular blocking agent, and remarkably inhibits the growth and metastasis of tumors on a 4T1 model by combined treatment with three drugs of aPD-1 and the vascular blocking agent.
The prodrug provided by the invention can be reduced into DON by nitroreductase and esterase under the condition of hypoxia, so that toxic and side effects of DON on stomach and intestine are reduced, and selective reduction of the drug is realized.
The invention also provides a preparation method of the prodrug of the 6-diazonium-5-oxo-L-norleucine, which comprises the following steps: s1) reacting a compound shown in a formula (IV) with a compound shown in a formula (V) to obtain a compound shown in a formula (VI); s2) reacting the compound shown in the formula (VI) with trimethylsilyl diazomethane and alkyl lithium under a low temperature condition to obtain the prodrug of the 6-diazonium-5-oxo-L-norleucine shown in the formula (I).
Reacting a compound represented by formula (IV) with a compound represented by formula (V); in the present invention, the reaction is preferably carried out in an organic solvent; the molar ratio of the compound represented by formula (IV) to the compound represented by formula (V) is preferably 1: (1.8 to 2.2), more preferably 1:2; the organic solvent is not particularly limited as long as it is an organic solvent well known to those skilled in the art, and acetonitrile is preferable in the present invention; the 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 shown in the formula (IV), the aminopyridine compound and the organic amine is preferably 1: (0.8-1.2): (1.8 to 2.2), more preferably 1:1:2; in the present invention, the compound represented by the formula (IV) and the compound represented by the formula (V) are preferably mixed under ice bath conditions and then allowed to react at room temperature; the mixing time is preferably 1 to 3 hours; in the invention, the step is more preferable to mix and stir the compound shown in the formula (IV), the aminopyridine compound and the organic amine in an organic solvent under the ice bath condition, then add the compound shown in the formula (V), mix under the ice bath condition, and then raise the temperature to room temperature for reaction; the mixing and stirring time is preferably 10-20 min, more preferably 15min; the reaction time at room temperature is preferably 30 to 60 hours, more preferably 40 to 50 hours, still more preferably 45 to 48 hours.
After the reaction, preferably concentrating to obtain a solid, dissolving the solid with dichloromethane, washing with saturated saline, drying, concentrating, and performing column chromatography to obtain a crude product; the eluent of the column chromatography is preferably ethyl acetate and hexane; the volume ratio of the ethyl acetate to the hexane is preferably 1: 20-2: 1.
after dissolving the crude product in ethyl acetate, adding glacial ethyl ether for recrystallization, and obtaining the compound shown in the formula (VI).
Reacting a compound shown in a formula (VI) with trimethylsilyl diazomethane and alkyl lithium under a low temperature condition; the alkyl lithium is preferably n-butyl lithium; the molar ratio of the compound of formula (VI), trimethylsilyl diazomethane to alkyllithium is preferably 1: (1-1.2): (1 to 1.5), more preferably 1:1.18:1.22; in the invention, it is preferable to mix and react trimethylsilyl diazomethane with alkyl lithium at low temperature, and then add the mixture into an organic solution of a compound shown in formula (VI) for reaction; the temperature of the mixing reaction is preferably-100 ℃ to-90 ℃, more preferably-98 ℃; the mixing reaction time is preferably 20 to 40min, more preferably 30min; the solvent in the organic solution of the compound represented by formula (VI) is preferably tetrahydrofuran; the temperature of the reaction is preferably-80 ℃ to-70 ℃, more preferably-78 ℃; the reaction time is preferably 20 to 40 minutes, more preferably 30 minutes.
After the reaction, preferably adding water to quench the reaction, extracting with ethyl acetate, and purifying by column chromatography to obtain a prodrug of 6-diazonium-5-oxo-L-norleucine shown in the formula (I); the eluent of the column chromatography is preferably chloroform and acetone; the volume ratio of chloroform to acetone is preferably 20:1.
or reacting the compound shown in the formula (VII) with a compound shown in the formula (VIII) to obtain the prodrug of the 6-diazonium-5-oxo-L-norleucine shown in the formula (I).
Reacting a compound represented by formula (VII) with a 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 carried out in the presence of an organic amine; the organic amine is preferably triethylamine; in the present invention, it is preferable that the compound represented by the formula (VII) and the compound represented by the formula (VIII) are mixed with an organic solvent to obtain respective solutions, and then the solutions of the two are mixed under low temperature conditions to react; the temperature of the reaction is preferably-10 ℃ to 10 ℃, more preferably 0 ℃; the reaction time is preferably 1 to 3 hours, more preferably 2 hours.
After the reaction, ethyl acetate is preferably added, and the mixture is washed by water and saturated saline water in turn, dried and purified by column chromatography to obtain the prodrug of the 6-diazonium-5-oxo-L-norleucine shown in the formula (I); the eluent of the column chromatography is preferably n-hexane and ethyl acetate; the volume ratio of the n-hexane to the ethyl acetate is preferably 3:1.
the prodrug of 6-diazonium-5-oxo-L-norleucine provided by the invention has the advantages of simple preparation method and higher yield.
The invention also provides application of the prodrug of the 6-diazonium-5-oxo-L-norleucine in preparing a tumor treatment drug.
The invention also provides a medicine for treating tumors, which comprises the prodrug of the 6-diazonium-5-oxo-L-norleucine.
Preferably, the medicament for treating tumors further comprises a vascular blocking agent and/or an immune checkpoint inhibitor.
Preferably, the vascular blocking agent is CA4-NPs; the immune checkpoint inhibitor is aPD-1.
Preferably, the mass ratio of the prodrug of the 6-diazonium-5-oxo-L-norleucine to the vascular blocking agent is 1:10 to 20, more preferably 1: (15 to 20), and more preferably 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 to 0.2), more preferably 1: (0.08 to 0.12), and more preferably 1:0.1.
to further illustrate the present invention, the following describes in detail a prodrug of 6-diazonium-5-oxo-L-norleucine, its preparation method and application provided in the present invention in connection with examples.
The reagents used in the examples below are all commercially available.
Example 1
Prodrug (HDON) synthesis of 6-diazo-5-oxo-L-norleucine is shown in FIG. 1 a.
1.1 Synthesis of intermediate EOC-NBC: to a round bottom flask was added EOC (5-oxopyrrolidine-2-carboxylic acid ethyl ester) (5 g,31.8mmol,1 equiv), DMAP (4-dimethylaminopyridine) (3.9 g,31.8mmol,1 equiv), DIPEA (N, N-diisopropylethylamine) (8.2 g,63.6mmol,2 equiv) was dissolved in 90mL anhydrous ACN (acetonitrile), stirred in an ice water bath for 15min, then NBC (p-nitrobenzyl chloroformate) (2.96M in ACN,13.7g,63.6mmol,2equiv) was slowly added to the round bottom flask, and the reaction was allowed to resume at room temperature for 48h after further stirring in an ice water bath. After completion of the reaction, rotary evaporation concentration gave a brown solid. The solid was dissolved with 200mL of methylene chloride, washed with saturated brine (200 mL. Times.3), dried over anhydrous magnesium sulfate, and rotary evaporated to give a crude product by column chromatography (ethyl acetate/hexane 1:20:2 column volumes, 1:10:2 column volumes, 1:2:5 column volumes, 1:1:4 column volumes, 2:1:2 column volumes). Dissolving the crude product in appropriate amount of ethyl acetate, adding glacial ethyl etherRecrystallization was performed to give EOC-NBC as a white solid. And use CDCl 3 As a solvent by 1 H NMR 13 C NMR to determine the Structure, as shown in FIGS. 1b and C, ESI-MS (ESI + ) Characterization of EOC-NBC, [ M+H ]] + ＝337.41，[M+Na] + ＝359.41，[M+NH 4 ] + = 354.46, as shown in fig. 1 d.
1.2 synthesis of HDON: EOC-NBC (450 mg,1.34mmol,1.00 equiv) was charged to a round bottom flask, dissolved in 12mL anhydrous tetrahydrofuran, and cooled to-116℃in a Dewar flask. TMS (trimethylsilyl diazomethane) (0.79mL,2M in hexane,1.58mmol,1.18equiv) was additionally dissolved in 7mL of anhydrous tetrahydrofuran and cooled to-98 ℃. n-BuLi (0.65mL,2.5M in hexane,1.63mmol,1.22equiv) was slowly added dropwise to the TMS solution and reacted for 30 minutes. The TMS and n-BuLi mixed solution was then added to the EOC-NBC solution, and the temperature was slowly raised from-116℃to-78℃and the reaction was continued for 30 minutes. After the reaction, 15mL of an aqueous solution was added to quench the reaction. Extraction was performed three times with ethyl acetate (10 mL. Times.3). Then, the solution was washed three times with saturated brine (20 mL. Times.3), and the washed solution was dried over anhydrous magnesium sulfate. Purification was performed by column chromatography (chloroform/acetone 20:1). And use CDCl 3 As a solvent by 1 H NMR 13 C NMR confirmed the structure as shown in FIGS. 2a and b; ESI-MS (ESI) was also used + ) Characterization of HDON, [ M+Na ]] + =401.3, as shown in fig. 2 c; the purity of the HDON is detected by ultraviolet-High Performance Liquid Chromatography (HPLC), the detection wavelength is 267nm, and the mobile phase is acetonitrile: the water (1/1, V/V) was used at a flow rate of 1mL/min, and the purity was higher than 98% as shown in FIG. 2 d.
1.3 chemical and plasma stability of HDON was evaluated. First, the chemical stability of HDON at 25℃was evaluated, 1mg of HDON was dissolved in 1mL of acetonitrile, diluted to a concentration of 0.27mM with buffers having different pH values, and the concentration of HDON was measured by HPLC at 25℃under constant conditions for 0h, 6h, 12h, 24h and 48h. The results show that the HDON has better stability in PBS buffer solutions with different pH values, and as shown in figure 3a, the residual content of the HDON in 48h PBS-8.5,7.4,6.8,5.5 is 95.80+/-0.33%, 98.76+/-1.17%, 98.77+/-1.42% and 99.33 +/-0.99% respectively. To simulate physiological conditions, stability experiments were then performed in a solution of PBS-7.4 at 37℃and after 24h the residual HDON content in the solution was 96.17.+ -. 0.82% as shown in FIG. 3 b. Next, stability experiments were performed in C57BL/6 mouse plasma at 37℃and 1mg/mL of HDON acetonitrile solution was diluted to 0.2mg/mL with PBS-7.4 and then mixed with mouse plasma in equal volumes, and the concentration of HDON in plasma was measured at 0h, 0.5h, 1h, 3h, as shown in FIG. 3C, and the HDON was rapidly metabolized in plasma and ESI mass spectrometry was performed on the post-metabolism products, as shown in FIG. 3d, to find that HDON was not metabolized to DON, but a new intermediate product was generated, the ethyl ester bond of which was enzymatically hydrolyzed.
The reduction mechanism of HDON is shown in fig. 4 a. First, the present invention tested the reduction of HDON by nitroreductase at different concentrations at 37℃by deoxygenating PBS-7.4 solution with nitrogen, dissolving 5mg of HDON in 1mL of ethanol castor oil (1/1, V/V), then diluting with PBS-7.4 to obtain 0.13mM solution, mixing 200. Mu.L of HDON (0.13 mM) and 60. Mu.L of NADH (2.35 mM) in a 1.5mL centrifuge tube, then adding 40. Mu.L of nitroreductase (0.25 mg/mL or 0.50 mg/mL) solution to the sample solution in an anoxic state, and placing the mixed sample in a constant temperature shaking chamber at 37℃to detect the content of HDON at 0h, 0.5h, 1h, 3h, 6h, 12 h. As shown in fig. 4b, as the enzyme concentration increases, the rate of HDON reduction also increases. Next, the present invention tested HPLC of HDON under the action of esterase and nitroreductase, 200. Mu.L of HDON (0.13 mM) and 60. Mu.L of NADH (2.35 mM) were mixed in a 1.5mL centrifuge tube, then 40. Mu.L of nitroreductase (0.50 mg/mL) solution was added to the sample solution in an anoxic state, 400. Mu.L of esterase (5U/mL) was added to the above reaction mixture after 1 hour, and incubation was continued for 3 hours, and the sample solution was concentrated at room temperature using a nitrogen blower after the end of the reaction and the product was analyzed by the method of pre-column derivatization (10. Mu.L of water, 50. Mu.L of 0.2M sodium bicarbonate buffer with pH=9, 100. Mu.L of 10mM DABS-Cl (dansyl chloride) acetone solution were added to the sample tube after concentration, followed by 15 minutes at 60℃in a water bath). As shown in fig. 4c, and its reduced products were verified by LC-MS (eluting at 25 ℃ c with a flow rate gradient of 1mL/min, samples were detected by HPLC or LC-MS. Mobile phase was water +0.1% formic acid and acetonitrile +0.1% formic acid gradient of 20% to 95% for 30 minutes), as shown in fig. 4d and fig. 4 e. HDON can be reduced to the DON-Et intermediate by nitroreductase (fig. 4 d) followed by esterase (fig. 4 e). In vitro experiments show that HDON can be reduced into DON original medicine under the action of nitroreductase and esterase.
The invention carries out cytotoxicity experiments of different cell lines, evaluates cytotoxicity of HDON and DON in vitro by CCK8 experiment, and uses H22 cells, 4T1 cells or MC38 cells (8.0X10 per well) 3 Cells) were inoculated into 96-well plates and 180 μl of complete medium was added followed by overnight incubation. DON was dissolved in PBS-7.4 to obtain 1mM stock solution. HDON was dissolved in DMSO and then diluted with PBS-7.4 to obtain a 1mM stock solution (containing 1% DMSO). Next, 20 μl of drug solution of different concentrations was added to each well. After 24 hours, 48 hours or 72 hours of incubation, 20 μl CCK8 solution was injected into each well. The well plate was directly tested after 1-2 hours of incubation. The absorbance of each well was measured at 450nm on a microplate reader. As can be seen from fig. 5, HDON significantly reduced the cytotoxicity of the drug to tumor cells compared to DON. The cytotoxicity experiment result shows that the prodrug strategy can reduce the toxic and side effects of the drug.
The toxic side effects of HDON were evaluated on Kunming mice, which were randomly divided into 7 groups, and DON (0.66,1.32,2.64. Mu. Mol/kg) or HDON (0.00,0.66,1.32,2.64. Mu. Mol/kg) was intraperitoneally injected for one week, respectively, and daily changes in body weight were observed for 8 consecutive days. As shown in FIG. 6, the HDON group of Kunming mice did not lose weight for one week with the DON group of mice with relative weights of 93.35 + -16.15% (0.66 μmol/kg), 78.80 + -4.96% (1.32 μmol/kg) and 69.77+ -6.83% (2.64 μmol/kg), respectively, showing significant weight loss in the DON two dose groups. Experimental results show that on normal Kunming mice, HDON has no obvious toxic or side effect compared with DON at the same dosage.
At the cellular and animal level, the invention verifies that HDON significantly reduces the toxic side effects of DON. The invention combines the application of the medicine with the vascular blocking agent CA 4-NPs. Firstly, the effect of CA4-NPs on blood vessels of 4T1 breast cancer (20 mg/kg, calculated as CA 4) and MC38 colon cancer (18 mg/kg, calculated as CA 4) is detected, tumor tissues are sacrificed after 24 hours after the intravenous injection of the CA4-NPs into the tail of a mouse, the tumor tissues are preserved in 4% paraformaldehyde solution, and the blood vessel density condition of the tumor tissues is determined by using CD31 immunohistochemistry, so that the CA4-NPs can reduce the blood vessel density of the tumor, which is beneficial to deepening the hypoxia condition of the tumor, as shown in figure 7 a. Next, the present invention confirmed the expression of HIF-1α by immunohistochemistry, as shown in FIG. 7b, indicating that CA4-NPs can elevate tumor hypoxia, increasing HIF-1α expression. Meanwhile, the hypoxia degree of the H22 liver cancer tumor is also verified to be deeper than that of 4T1 breast cancer and MC38 colon cancer model. And is more beneficial to the selective reduction of HDON. Next, the present invention verifies that hypoxia can increase nitroreductase expression at the cellular level, as shown in FIG. 7 c. Similarly, validation was performed at the animal level. CA4-NPs increased the expression of nitroreductase in tumor tissue of 4T1 breast cancer (20 mg/kg calculated as CA 4) and MC38 colon cancer (18 mg/kg calculated as CA 4), as shown in FIG. 7 d.
To verify the reduction of HDON in vivo, the present invention performed biodistribution experiments on 4T1 breast cancer. When the 4T1 tumor reaches about 250mm 3 Mice were randomly divided into 6 groups. Of these, three groups were given HDON (15 mg/kg) intraperitoneally alone and the other three groups were given HDON (15 mg/kg) +intravenous injection of CA4-NPs (20 mg/kg, calculated as CA 4). Mice were sacrificed at 1h,4h and 8h, and major organs and tumor tissues were removed, samples were pre-column derivatized with dansyl chloride, and the content of DON was detected by HPLC. As shown in FIG. 8, it was demonstrated that HDON could be reduced to DON in vivo, with a 3.46-fold higher intratumoral DON content than the single drug group and a 37.3-fold higher renal dose after 1h of combination with CA 4-NPs. This suggests that HDON has a good capacity for selective activation of tumor hypoxia. The reduction of HDON is positively correlated with the degree of hypoxia.
The H22 liver cancer model is a tumor model with deep hypoxia degree, and the invention firstly carries out anti-tumor research by independently using HDON. When the tumor volume reaches 100mm 3 On the left and right, mice were randomly divided into 2 groups of PBS-7.4, HDON (1 mg/kg). HDON daily intraperitoneal administration to inhibitEndpoint of tumor (fig. 9 a). On day 14, PBS-7.4 reached about 2000mm 3 . The TSR of HDON was 76.5% (fig. 9 b). Ascites metastasis occurred in PBS group mice, mice weight was increased, but HDON had no significant effect on mice weight, with little toxic side effect (fig. 9 c). The present invention also observed the survival time of mice, as shown in figure 9d, HDON treatment extended the survival time of mice. As shown in fig. 9e and 9f, the HDON treated group significantly inhibited tumor growth. Next, the present invention performed HE staining of various organ tissues and tumors of mice (FIG. 9 g), and HDON did not significantly damage the major organ tissues of mice compared to PBS group. Likewise, the present invention also performed a flow assay of the mouse tumor immunity microenvironment, as shown in fig. 9 g. After HDON treatment, more CD4 was detected in the tumor + T cells (0.37% of total cell number) and CD8 + T cells (0.23% of total cell number). Effectively activates the anti-tumor immune response. The same MDSCs cells are obviously reduced, and the immune microenvironment of the tumor is changed.
The invention next investigated the anti-tumor effect of hypoxia activated glutamine antagonist (HDON) in combination with CA4-NPs on MC38 colon cancer models, as shown in figure 10. When the tumor volume reaches 100mm 3 On the left and right, mice were randomly divided into 4 groups of PBS-7.4, HDON (1 mg/kg), CA4-NPs (18 mg/kg), HDON+CA4-NPs (1 mg/kg+18 mg/kg). HDON was intraperitoneally administered daily to the endpoint of tumor suppression. CA4-NPs were injected intravenously every four days at the tail three times (FIG. 10 a). The PBS group grew fastest and reached 2000mm at day 16 as shown in FIGS. 10b and 10c 3 . Tumor inhibition (tumor suppression rate, TSR) reached 98.3% in the HDON+CA4-NPs group, whereas TSR was 29.1% and 43.3% in the HDON and CA4-NPs single drug groups, respectively, and body weight was not significantly different from that in the PBS group after the end of treatment. In addition to the antitumor effect, the survival rate of mice in different treatment groups was also monitored, and it was found that the hdon+ca4-NPs combined treatment group significantly prolonged the overall survival rate of mice compared to other treatment groups (fig. 10 d). As shown in fig. 10e and 10f, the hdon+ca4-NPs combination treatment group significantly inhibited tumor growth. Also on the sixth day of tumor inhibition, the glutamine content in the tumor tissue was tested, as shown in FIG. 10g, for HDON in combination with CA4-NPsThe group obviously increases the glutamine content in tumor tissues, and plays a role in inhibiting the uptake and utilization of glutamine by tumor cells. After tumor inhibition, the serum of the mice is analyzed for liver and kidney functions, and the results show that the treatment has no obvious influence on the contents of alkaline phosphatase, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, uric acid, urea nitrogen and creatinine, and the treatment does not cause damage to the liver and kidney functions of the mice. Hematoxylin eosin (hematoxylin and eosin, H) was performed on various organs and tumor tissues of mice&E) The graph of the organs and tumor HE sections of the mice after MC38 model treatment is shown in fig. 11, and the treatment group has no significant effect on the organs and tissues of the mice compared to the PBS group as expected results.
Following analysis of the immune microenvironment of the MC38 colon cancer model, as shown in FIG. 12a, more CD4 was detected in tumor cells after HDON and CA4-NPs combination therapy + T cells (6.21% of total cell number) and CD8 + T cells (3.05% of total cell number) indicate that combination therapy of HDON with CA4-NPs activates anti-tumor immune responses. The same MDSCs cells are obviously reduced, and the immune microenvironment of the tumor is changed. Consistent with immune cell assays, the present invention tested for proinflammatory cytokines (IL-6 and IFN-gamma) in serum. As shown in FIG. 12b, the group treated with HDON in combination with CA4-NPs significantly increased the IL-6 and IFN-gamma levels. The experimental result shows that under the action of CA4-NPs, HDON can be reduced into DON in tumor tissues, so that the uptake of glutamine by tumor cells is inhibited, and meanwhile, the anti-tumor immune response is activated, so that no obvious toxic or side effect is caused to mice.
Similarly, the present invention performed anti-tumor studies in combination with CA4-NPs and aPD-1 on a 4T1 model, as shown in FIG. 13. When the tumor volume reaches 100mm 3 On the left and right, mice were randomly divided into 8 groups of PBS-7.4, aPD-1 (100. Mu.g each), HDON (1 mg/kg), HDON+aPD-1 (1 mg/kg+100. Mu.g), CA4-NPs (20 mg/kg, calculated as CA 4), CA4-NPs+aPD-1 (20 mg/kg+100. Mu.g), HDON+CA4-NPs (1 mg/kg+20 mg/kg), HDON+CA4-NPs+aPD-1 (1 mg/kg+18 mg/kg+100. Mu.g). Wherein HDON is administered intraperitoneally to the end of tumor suppression every day, CA4-NPs is injected intravenously every four days at the tail, and the total of three times is three times, and aPD-1 is administered intraperitoneallyOnce every three days (fig. 13 a). As shown in FIG. 13b, the PBS group grew fastest and tumor volume reached about 2000mm on day 22 3 . The TSR of the HDON+CA4-NPs group is 98.07%, and the tumor inhibition rate of the combined treatment of the HDON+CA4-NPs and aPD-1 is 99.80%, so that the effect is remarkable. At the same time, there was no significant change in body weight after the end of treatment (fig. 13 c). Next, a survival experiment was performed, and HDON combined treatment with CA4-NPs significantly improved the survival time of mice (FIG. 13 d). As shown in fig. 13e and 13f, the HDON and CA4-NPs combination treatment group significantly inhibited tumor growth. Mice were stained with indian ink for lung metastasis and counted for lung metastasis node number, and combined treatment with HDON and CA4-NPs inhibited tumor lung metastasis (fig. 13g, h).
Example 2
First, azo-OH was synthesized according to the literature method. Azo-OH (500 mg,1.96mmol,1 equiv) was dissolved in 5mL DCM (dichloromethane) and cooled to 0deg.C. DMAP (23.95 mg,0.196mmol,0.1 equiv) and triethylamine (327. Mu.L, 2.352mmol,1.2 equiv) were added to the Azo-OH solution. NPC (474.26 mg,2.352mmol,1.2 equiv) was dissolved in 5mL DCM, and the dissolved NPC solution was added dropwise to the Azo-OH solution, brought to room temperature and the reaction continued for 2h. After completion of the reaction, the reaction mixture was washed with saturated ammonium chloride 1 (10 mL) and water 3 times (10 mL. Times.3). Recrystallisation from ethyl acetate and n-hexane gives the pure Azo-NPC (FIG. 14 a). And use CDCl 3 As a solvent by 1 H NMR confirmed the structure (fig. 14 b).
Synthesis of Azo-DON: the DON-Et was synthesized by the first reference. Azo-NPC (116 mg,0.276mmol,1.1 equiv) was dissolved in 5mL of DMF (N, N-dimethylformamide) and cooled to 0deg.C. In addition, DON-Et (50 mg,0.251mmol,1 equiv) was dissolved in 1mL of DMF and added quickly to the Azo-NPC solution. TEA (triethylamine) (38.4. Mu.L, 0.276mmol,1.1 equiv) was then added. The reaction was carried out at 0℃for 2 hours, and after returning to room temperature, the reaction was carried out overnight. After the completion of the reaction, 50mL of ethyl acetate was added, followed by washing with water 2 times (50 mL. Times.2), saturated brine 2 times (50 mL. Times.2), and dried over anhydrous magnesium sulfate. Purification by column chromatography (n-hexane: ethyl acetate 3:1) afforded the product. And use CDCl 3 As a solvent by 1 H NMR confirmed the structure (fig. 14 c). ESI-MS (ESI) was also used + ) The Azo-DON was characterized and,[M+H] + ＝481.5，[M+Na] + =503.5 (fig. 14 d).

Claims (6)

1. A prodrug of 6-diazo-5-oxo-L-norleucine, characterized by the formula (I):
wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro.
2. The prodrug according to claim 1, wherein the prodrug is represented by formula (II):
3. a method of preparing a prodrug of 6-diazo-5-oxo-L-norleucine according to claim 1, comprising:
s1) reacting a compound shown in a formula (IV) with a compound shown in a formula (V) to obtain a compound shown in a formula (VI);
s2) reacting the compound shown in the formula (VI) with trimethylsilyl diazomethane and alkyl lithium under a low temperature condition to obtain a prodrug of 6-diazonium-5-oxo-L-norleucine shown in the formula (I);
or reacting a compound shown in a formula (VII) with a compound shown in a formula (VIII) to obtain a prodrug of 6-diazonium-5-oxo-L-norleucine shown in a formula (I);
wherein n is an integer of 0 to 5; r is R 1 Is C1-C10 alkyl; r is R 2 Is nitro.
4. Use of a prodrug of 6-diazo-5-oxo-L-norleucine according to any one of claims 1-2 in the preparation of a medicament for the treatment of a tumor.
5. A medicament for the treatment of tumors, characterized in that it comprises a prodrug of 6-diazonium-5-oxo-L-norleucine according to any one of claims 1 to 2.
6. The medicament of claim 5, further comprising a vascular blocking agent and/or an immune checkpoint inhibitor.
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CN108290827A (en) * 2015-07-31 2018-07-17 约翰霍普金斯大学 The prodrug of glutamine analogues
CN112807297A (en) * 2021-01-20 2021-05-18 深圳市福田区风湿病专科医院 Application of 6-diazo-5-oxo-L-norleucine in preparation of medicine for preventing and treating psoriasis
CN114177177A (en) * 2021-10-29 2022-03-15 中国科学院长春应用化学研究所 Preparation method of hypoxia tumor selective activation prodrug

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CN108290827A (en) * 2015-07-31 2018-07-17 约翰霍普金斯大学 The prodrug of glutamine analogues
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