Preparation method of gambogic acid-glucose conjugate with long connecting arm

Abstract

The invention provides a preparation process route of a gambogic acid-glucose conjugate with antitumor activity. According to the technical scheme, hemiacetal hydroxyl on glucose firstly reacts with a connecting chain with bifunctional groups, the obtained product serves as an initial raw material of the process route and then is coupled with carboxyl on gambogic acid, and the gambogic acid-glucose conjugate is prepared. The reaction raw material related to glucose is 2-(acetoxymethyl)-6-(bromo alkoxy) tetrahydro-2H-pyran-3, 4, 5-triacetate or 2-(azido alkoxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol, the reaction raw material is provided with an alkyl long chain, and the reaction raw material related to glucose is 2-(acetoxymethyl)-6-(bromo alkoxy) tetrahydro-2H-pyran-3, 4, 5-triacetate or 2-(azido alkoxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol or 2-(azido alkoxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol. The 2-(azido alkoxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triacetate can be directly connected with the carboxyl group of gambogic acid, and the 2-(azido alkoxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-3, 4, 5-triol can be connected with the carboxyl group of gambogic acid through an omega-bromo alkyl alkyne chain. The selected preparation process route is mild in reaction condition, high in yield, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of new drug research and development, and specifically relates to a method for preparing a gambogic acid-glucose conjugate designed with glucose transporter 1 (GLUT1) as a target. Background Art

[0002] Garcinia acid has excellent anti-tumor activity and has a good therapeutic effect on some major cancers (such as liver cancer, gastric cancer, lung cancer, etc.) [1] Currently, Garcinia has been approved for Phase II clinical trials as a drug for the treatment of lung cancer and other solid tumors. [2] However, the poor water solubility of gambogic acid and its toxic side effects in normal tissues limit its clinical application. [3-4] Existing solutions mainly use nanoparticles [5] , liposomes [6] , Microparticles [7] etc. to deliver garcinia acid to tumor cells to improve solubility, bioavailability and targeting. In solving this problem, we took a different approach and used garcinia acid as the lead compound to modify its chemical structure to reduce its toxicity to normal tissues.

[0003] Normal differentiated cells mainly rely on oxidative phosphorylation in mitochondria to generate the energy needed by cells, while tumor cells mainly obtain energy through glycolysis, even when there is sufficient oxygen supply. This phenomenon is called the Warburg effect. Aerobic glycolysis is a major characteristic of tumor cells. [8] Although aerobic glycolysis is an inefficient way to produce ATP, it prompts tumor cells to take in more nutrients and use these nutrients to produce new cell contents. By increasing the rate of glycolysis, tumor cells not only provide the energy required for the synthesis of cell contents, but also provide a large amount of raw materials (such as acetyl-CoA and NADPH) required for the synthesis of cell contents. Only when these two conditions are met at the same time can tumor cells proliferate rapidly. [9] Aerobic glycolysis is an inefficient way of energy production, which requires tumor cells to consume more glucose to produce enough energy. Because of this, glucose transporters (GLUTs) responsible for glucose absorption and transport are often highly expressed in tumor cells, especially in malignant tumors.

[10] This makes GLUTs one of the targets for tumor targeted therapy and drug resistance reversal.

[11] .

[0004] Based on the above considerations, garcinia acid can be used as an anticancer drug molecule, with glucose forming a guidance system, and a "molecular missile" can be formed through the connecting arm, namely garcinia acid-glucose conjugate, so that garcinia acid can enter the tumor tissue more, so as to improve the anti-tumor activity and reduce its toxicity. The activity test results show that garcinia acid-glucose conjugate has good inhibitory activity against laryngeal cancer cells such as Hep-2 and Tu212, and has low toxicity to normal liver cells such as L-02. For the preparation of this promising garcinia acid-glucose conjugate, it is urgent to develop a reasonable synthetic process route that can meet the requirements of industrial production. Summary of the invention

[0005] The purpose of the present invention is to couple gambogic acid and glucose molecules using a selected bifunctional connecting chain. The hemiacetal hydroxyl group on glucose first reacts with the bifunctional connecting chain, and the resulting product is used as the starting material of the process route, and then coupled with the carboxyl group on gambogic acid to prepare gambogic acid-glucose conjugates. The selected synthetic process route has mild reaction conditions, high yield, and low cost. The chemical structures of the two gambogic acid-glucose conjugates finally prepared are as follows: Figure 1 shown.

[0006] The preparation method of the garcinia acid-glucose conjugate disclosed by the present invention comprises the following steps:

[0007] 1. Preparation method of garcinia acid-glucose conjugate Ⅰ

[0008] (1) Garcinia acid and 2-(acetoxymethyl)-6-(bromoalkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester are dissolved in an organic solvent, reacted at 20-80° C. for 5-30 hours in the presence of a catalyst, and chromatographically separated using a silica gel column to obtain 2-(acetoxymethyl)-6-(((garcinia acyl)oxy)alkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester; wherein the organic solvent is acetonitrile or acetone, but is not limited to acetonitrile or acetone, and also includes inert solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc.; the catalyst is potassium carbonate, and may also be sodium carbonate.

[0009] (2) Dissolving 2-(acetoxymethyl)-6-(((gambogic acid yl)oxy)alkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester in an organic solvent, reacting at 0-70°C for 0.3-20 hours in the presence of a catalyst, and performing chromatography separation on a silica gel column to obtain a first gambogic acid-glucose conjugate; wherein the organic solvent is anhydrous ethanol or methanol; the catalyst is dibutyltin oxide, and also includes deprotection reagents such as sodium methoxide and sodium ethoxide; if the catalyst used is dibutyltin oxide, the reaction temperature is 60-70 degrees; if the catalyst used is sodium methoxide or sodium ethoxide, the reaction temperature is room temperature; the eluent used for chromatography separation is preferably a mixed solvent of dichloromethane and methanol.

[0010] The preparation process route I of the gambogic acid-glucose conjugate is as follows:

[0011]

[0012] 2. Preparation Method of Garcinia Acid-Glucose Conjugate Ⅱ

[0013] (1) In the presence of a catalyst, gambogic acid and ω-bromoalkylalkyne react in an organic solvent at 20-80° C. for 5-30 hours, and chromatographic separation is performed using a silica gel column to obtain gambogic acid alkylalkynyl ester; wherein the organic solvent is acetonitrile or acetone, but is not limited to acetonitrile or acetone, and also includes inert solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc.; the catalyst is sodium bicarbonate, but is not limited to sodium bicarbonate, and also includes sodium carbonate and potassium carbonate.

[0014] (2) dissolving a garcinia acid alkyl alkynyl ester in tetrahydrofuran, and dissolving 2-(azidoalkoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol in water, mixing the two solutions in equal volumes, and reacting at room temperature for 10-20 hours under the presence of a catalyst; then mixing ammonia water and saturated ammonium chloride solution in a ratio of 1:3, adding the mixture to the reaction solution, continuing to stir for 10-60 minutes, extracting with dichloromethane, taking the organic phase, drying, filtering, concentrating the filtrate under reduced pressure, and separating by chromatography on a silica gel column to obtain a second garcinia acid-glucose conjugate; wherein the catalyst is a combination of copper sulfate and sodium ascorbyl palmitate.

[0015] The preparation process route II of the garcinia acid-glucose conjugate is as follows:

[0016]

[0017] The beneficial effects of the present invention are: two different connecting chains are selected to couple the gamboge acid and glucose molecules, the chain length of the intermediate connecting chain can be adjusted, the obtained product has high purity and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the chemical structures of the two types of garcinia acid-glucose conjugates that were finally synthesized. The length of the connecting chain in the middle of each garcinia acid-glucose conjugate molecular structure can be adjusted. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail with reference to the following examples.

[0020] Example 1 Preparation of 3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)gambogic acid propyl ester

[0021] (1) Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3-((garcinia yl)oxy)propoxy)tetrahydro-2H-pyran-3,4,5-triacetate

[0022] In a 50 mL flask, add gambogic acid (1.5 mmol), potassium carbonate (1.5 mmol), and then add (2R, 3R, 4S, 5R, 6R)-2-(acetoxymethyl)-6-(3-bromopropoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (1.5 mmol), and dissolve with 12 mL acetone, react at 50 ° C for 22 hours, and then stop the reaction. Chromatographic separation is performed on a silica gel column, and the eluent is [V (petroleum ether): V (ethyl acetate) = 7:1] to obtain (2R, 3R, 4S, 5R, 6R)-2-(acetoxymethyl)-6-(3-((gambogic acyl) oxy) propoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester, with a yield of 80%.

[0023] (2) Synthesis of 3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)gambogic acid propyl ester

[0024] In a 50mL flask, (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3-((garcinia acyl)oxy)propoxy)tetrahydro-2H-pyran-3,4,5-triacetate (0.5mmol) was added, and dibutyltin oxide (1mmol) was added as a catalyst, and 8mL of anhydrous ethanol was added as a solvent. The reaction was stirred at 70°C for 15 hours and then stopped. Chromatographic separation was performed using a silica gel column, and the eluent was [V(dichloromethane):V(methanol)=20:1] to obtain 3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)garcinia acid propyl ester, with a yield of 71%.

[0025] Yellow solid; 1 H NMR (400MHz, CDCl3): δ = 11.94 (s, 1H), 6.65 (d, J = 10.4Hz, 1H), 6.57 (t, J = 6.8Hz, 1H), 5.46-5.43 (m, 1H), 5.11-4.99 (m, 2H ),4.34-4.33(m,1H),4.29-4.27(m,1H),4.23-4.18(m,2H),3.98-3.87(m,1H),3.80(s,4H),3.64-3.47(m,7H),3.36-3.27 (m,5H),3.21-3.20(m,1H),3.17-3.13(m,2H),2.85-2.82(m,1H),2.51-2.45(m,1H),2.09-2.01(m,2H),1.97-1.93(m,2H ),1.91(s,3H),1.73(s,3H),1.66(s,3H),1.63(s,3H),1.57(s,3H),1.35(s,3H),1.34(s,3H),1.26(s,3H); HRMS(ESI)m / z calcd for C 47 H 61 O 14 [M+H] + 849.4056, found 849.4056.

[0026] Example 2 Preparation of (1-(3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)gambogic acid methyl ester

[0027] (1) Synthesis of 1-prop-2-yn-1-yl garcinia acid ester

[0028] In a 50mL flask, add gambogic acid (2mmol), 3-bromo-1-propyne (2mmol), sodium bicarbonate (2mmol), and dissolve with 20mL acetone, react at 50°C for 22 hours, and then stop the reaction. Chromatographic separation is performed on a silica gel column, and the eluent is [V (petroleum ether): V (ethyl acetate) = 8:1] to obtain gambogic acid prop-2-yn-1-yl ester with a yield of 92%.

[0029] (2) Synthesis of (1-(3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)gambogic acid methyl ester

[0030] Weigh 1-yl prop-2-yn-1-yl garcinia ester (1mmol) in a 50mL flask and dissolve it in 12mL tetrahydrofuran. Then weigh (2R,3R,4S,5S,6R)-2-(3-azidopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (1.1mmol), dissolve it in 12mL water, draw the solution with a rubber dropper, and drop it into the above flask. Then add copper sulfate (2mmol) and sodium vitamin C (3mmol) in turn, stir at room temperature for 12 hours. After the reaction is completed, take ammonia water and saturated ammonium chloride solution, mix them in a ratio of 1:3, add them to the flask, and stir for 20 minutes. It was extracted with dichloromethane, and the organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and chromatographed on a silica gel column with an eluent of [V(dichloromethane):V(methanol)=20:1] to obtain (1-(3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)gambogic acid methyl ester with a yield of 82%.

[0031] Yellow solid; 1 H NMR (400MHz, CDCl3): δ = 12.84 (s, 1H), 7.68 (s, 1H), 7.55 (d, J = 6.4Hz, 1H), 6.61 (d, J = 10Hz, 1H), 6.06 (t, J = 6.8Hz, 1H), 5.43 (d, J = 10Hz, 1H) ),5.06-5.01(m,2H),4.96(s,2H),4.49(s,2H),4.32-4.30(m,2H),3.82-3.73(m,4H),3.58-3.48(m,5H),3.42-3.39(m,2H),3.31-3.29(m ,2H),3.19-3.06(m,2H),3.03-2.96(m,1H),2.87-2.80(m,1H),2.50(d,J=9.6Hz,1H),2.29-2.27(m,1H),2.12-2.08(m,2H),2.04-1.99(m ,2H),1.90(s,1H),1.80-1.76(m,1H),1.72(s,3H),1.67(s,3H),1.64-1.61(m,9H),1.54(s,3H),1.39(s,3H),1.24(s,3H); HRMS(ESI)m / z calcd for C 50 H 64 N3O 14 [M+H] + 930.4383, found 930.4388.

[0032] References

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[0043]

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Claims

1. Preparation method I of garcinia acid-glucose conjugate, characterized in that Follow these steps: (1) dissolving gambogic acid and 2-(acetoxymethyl)-6-(bromoalkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester in an organic solvent, reacting at 20-80° C. for 5-30 hours in the presence of a catalyst, and performing chromatography separation using a silica gel column to obtain 2-(acetoxymethyl)-6-(((gambogic acyl)oxy)alkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester; wherein the organic solvent is acetonitrile or acetone; and the catalyst is potassium carbonate; (2) dissolving 2-(acetoxymethyl)-6-(((gambogic acid yl)oxy)alkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester in an organic solvent, reacting at 0-70° C. for 0.3-20 hours in the presence of a catalyst, and performing chromatography separation using a silica gel column to obtain a first gambogic acid-glucose conjugate; wherein the organic solvent is anhydrous ethanol or methanol; and the catalyst is dibutyltin oxide; The structure of the first garcinia acid-glucose conjugate is shown in Formula I: 。 2. Preparation method II of garcinia acid-glucose conjugate, characterized in that Follow these steps: (1) In the presence of a catalyst, gambogic acid and ω-bromoalkylalkyne react in an organic solvent at 20-80° C. for 5-30 hours, and chromatographic separation is performed using a silica gel column to obtain gambogic acid alkylalkynyl ester; wherein the organic solvent is acetonitrile or acetone; and the catalyst is sodium bicarbonate; (2) dissolving a garcinia acid alkyl alkynyl ester in tetrahydrofuran, and dissolving 2-(azidoalkoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol in water, mixing the two solutions in equal volumes, and reacting at room temperature for 10-20 hours under the presence of a catalyst; then mixing ammonia water and saturated ammonium chloride solution in a ratio of 1:3, adding the mixture to the reaction solution, continuing to stir for 10-60 minutes, extracting with dichloromethane, taking the organic phase, drying, filtering, concentrating the filtrate under reduced pressure, and performing chromatography separation on a silica gel column to obtain a second garcinia acid-glucose conjugate; wherein the catalyst is a combination of copper sulfate and sodium ascorbyl palmitate; The structure of the second garcinia acid-glucose conjugate is shown in Formula II: 。 3. The preparation method I of the garcinia acid-glucose conjugate according to claim 1, characterized in that: The raw material 2-(acetoxymethyl)-6-(bromoalkoxy)tetrahydro-2H-pyran-3,4,5-triacetic acid triester selected in step (1) has a connecting chain in its structure.

4. The preparation method II of the garcinia acid-glucose conjugate according to claim 2, characterized in that: The connecting chain selected in step (1) is ω-bromoalkylalkyne, and the catalyst used is sodium bicarbonate.

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