CN102225962A - Novel derivatives and preparation methods of berberine 9-amide-bonded cholic acid - Google Patents

Abstract

The invention relates to a new derivative bonded by berberine and cholic acid at the 9th position of berberine in formula (I) and a preparation method thereof. The invention also relates to a method for preparing a compound of the derivative and the application of the compound in the invention as medicine in treating tumors. In formula (I), R1 stands for hydroxy and carbonyl, while R2 and R3 stand for hydrogen, hydroxyl and carbonyl.

Description

Novel derivatives and preparation methods of berberine 9-amide-bonded cholic acid

technical field

The invention relates to a preparation method of a class of berberine derivatives and their application in antitumor therapy.

Background technique

Since M.-E. Chavalier and G. Peltin first extracted berberine from the bark of Xanthoxylonclava in 1826, pharmacologists have found that berberine has a wide antibacterial spectrum and has the functions of lowering blood fat, blood sugar, Anti-peptic ulcer and other pharmacological effects. Coptis chinensis, Cortex Phellodendron, Sangezhen, etc. are commonly used in traditional Chinese medicine as heat-clearing and detoxifying drugs, the main ingredient of which is berberine. In 1966, the Japanese Pharmacopoeia collected berberine hydrochloride and berberine tannic acid for the treatment of intestinal infections. With in-depth research, Japanese scholars in the 1970s proposed that berberine and its derivatives have anti-tumor effects. In recent years, many researchers have actively invested in the development and application of berberine. Studies have shown that berberine derivatives are hydrolyzed in vivo to generate quaternary ammonium salts, which can enter tumor cells and act as topoisomerase inhibitors to inhibit tumor cell DNA replication. Due to the poor intestinal absorption of berberine, it has a certain impact on its bioavailability

Berberine is soluble in water, hardly soluble in many organic solvents, and has little side effects. Due to different treatment methods, three different forms of berberine, quaternary ammonium, aldehyde and alcohol, can be obtained, among which the quaternary ammonium is the most stable. In order to improve the anti-tumor activity of berberine derivatives, enhance stability and fat solubility, increase bioavailability, and reduce their toxic and side effects as much as possible, the structural modification of berberine with strong water solubility can be effectively to deliver the desired amount of berberine to the target tissue.

Chemotherapeutic drugs are selectively delivered to the liver through liver-targeted drug delivery technology, which can reduce or avoid their systemic side effects. Bile acid is an endogenous hepatocyte-specific natural ligand. It is currently the only oral liver-targeted drug carrier with good biocompatibility. It is absorbed from the intestinal tract into the liver through active transport, and has a high degree of organ Therefore, using bile acid as a targeting carrier can not only achieve the liver targeting of drugs, reduce side effects, but also improve the bioavailability of drugs.

In recent years, research on liver-targeted drugs using bile acid as a carrier has continued to deepen. The literature reports include peptides, lipid-lowering drugs, antiviral drugs, anti-tumor drugs, hypoglycemic drugs, and nitrate drugs, etc. Conjugated liver-targeted drugs. Experimental studies on cells and animals have shown that after the drug is coupled with bile acid, the absorption of the drug by the liver increases to varying degrees, which reduces the toxic and side effects of the drug to a certain extent. Kramer et al. used chlorambucil as a model drug to couple with cholic acid, connected chlorambucil to the 3-hydroxyl of cholic acid, obtained cholic acid-chlorambucil conjugate, and investigated the hepatic activity of the conjugate. targeting and antitumor activity. The results showed that the original drug had only a weak effect on the hepatic bile acid transporter, while the corresponding cholic acid conjugate strongly inhibited the hepatic absorption of taurocholic acid, indicating that there was a strong interaction between the conjugate and the bile acid transporter. Monte et al. coupled glycinecholic acid with cisplatin to form a glycinecholic acid-cisplatin chelate. In vivo experiments showed that the chelate could be absorbed by tumor cells and the absorption amount was significantly higher than that of the original drug. Like natural bile acid, it can enter the enterohepatic circulation, increase the small intestine absorption and bile secretion of the original drug, and only a small amount is excreted through urine. At the same time, it can also effectively inhibit the growth of tumor cells in vitro and in vivo, and has certain antitumor activity, see [Chinese Journal of New Drugs, Volume 15, Issue 3, 2006].

In summary, the conjugate of the original drug and bile acid has the transport properties of bile acid, which can increase the hepatic absorption of the drug and achieve the purpose of targeting.

Contents of the invention

The object of the present invention is to provide a class of berberine derivatives that fully maintain the anti-tumor pharmacological activity of berberine, improve drug targeting, and solve the problems of poor fat solubility, poor targeting, and low bioavailability.

Another object of the present invention is to provide a preparation method of such berberine derivatives.

In order to accomplish the purpose of the present invention, the inventors have carried out a large number of creative researches, under the premise of filling and maintaining the antitumor pharmacological activity of berberine, to solve the problems of poor fat solubility, poor targeting, low bioavailability, etc., and finally Discovery of liver-targeted berberine derivatives by reacting berberine with cholic acid derivatives

The new berberine derivatives of the present invention are represented by the following general formula (I):

in

R ₁ is hydroxyl, carbonyl;

_R is hydrogen, hydroxyl, carbonyl;

R ₃ is hydrogen, hydroxyl, carbonyl;

The preparation method of new fat-soluble berberine derivative of the present invention:

It is characterized in that the berberine shown in formula (II)

Heating produces the intermediate berberine (III).

The cholic acid derivative shown in (IV) formula

in

The definition of _R1 is the same as claim 3;

The definition of R ₂ is the same as claim 4;

R The definition of ₃ is the same as claim 5;

Reaction with N-hydroxysuccinimide to generate cholic acid derivatives (V)

in

The definition of _R1 is the same as claim 3;

The definition of R ₂ is the same as claim 4;

R The definition of ₃ is the same as claim 5;

Reacts with glycine to generate cholic acid derivatives (VI)

in

The definition of _R1 is the same as claim 3;

The definition of R ₂ is the same as claim 4;

R The definition of ₃ is the same as claim 5;

Reaction with dihalohydrocarbons to generate cholic acid derivatives (VII)

in

The definition of _R1 is the same as claim 3;

The definition of R ₂ is the same as claim 4;

R The definition of ₃ is the same as claim 5;

The berberine derivative shown in (III) formula is reacted with the cholic acid derivative shown in (VII) formula to generate the berberine derivative (I) described in claim 1

in

The definition of _R1 is the same as claim 3;

The definition of R ₂ is the same as claim 4;

The definition of _R3 is the same as claim 5.

Advantages of the present invention: the berberine derivatives of the present invention not only maintain the stability of OO and N ⁺ on the five-membered ring of the active part of berberine, but also have good fat solubility, increase the bioavailability of the drug, and fully maintain the stability of small Antitumor pharmacological activity of berberine.

Detailed ways:

Embodiment 1: the synthesis of glycocholic acid-9-propoxyberberine: (C1)

(1) Synthesis of Berberine

Add 744mg (2mmol) of berberine into a 250mL round bottom flask, heat at 195-210°C for 30min at a vacuum of 20-30mmHg, the yellow solid gradually turns dark red, cool to room temperature in a vacuum desiccator, and perform silica gel column chromatography Purified to obtain a dark red powder with a yield of 73%, molecular weight: 322, and the structure as follows:

(2) Synthesis of cholic acid activated ester:

Add 204 mg (0.5 mmol) of cholic acid to a 50 mL round bottom flask, add 10 mL of dichloromethane, add 115 mg of N-hydroxysuccinimide, and add 1-ethyl-3-(3-dimethylaminopropyl) dropwise Carbodiimide 766mg (4mmol) dichloromethane solution 10mL, followed by TLC reaction. After the reaction is complete, add 30ml of chloroform to the reaction solution, extract with 50ml of saturated saline and 50ml of saturated _NaHCO3 solution for 3 times, dry over anhydrous _MgSO4 , filter under reduced pressure, remove _MgSO4 , concentrate under reduced pressure, perform silica gel column chromatography Separation and purification yielded a white powder. Yield 60%; molecular weight: 515; structural formula is as follows:

(3) Synthesis of glycocholic acid:

Add 257mg (0.5mmol) of cholic acid activated ester and 1mL DMSO to dissolve in a 25mL round bottom flask, add 45mg (0.6mmol) of glycine and 100μL triethylamine, follow the reaction by TLC, add 20mL anhydrous ether after the reaction is complete, and a white solid precipitates , suction filtration under reduced pressure, vacuum drying, yield 98%, molecular weight: 465, structure as follows:

(4) Synthesis of glycocholate-3-bromopropyl ester:

Add 233 mg (0.5 mmol) of glycocholic acid to a 25 mL round bottom flask, add 1 mL of DMF to dissolve, add 200 μL of 1,3-dibromopropane, add K ₂ CO ₃ 276 mg (2 mmol), follow the reaction by TLC, after the reaction is complete, reduce Pressure suction filtration, add 50mL of chloroform to the filtrate, extract with 50mL distilled water and 50mL saturated _NaHCO3 solution successively for 3 times, dry over anhydrous _MgSO4 , remove _MgSO4 by vacuum suction filtration, concentrate under reduced pressure, separate and purify by silica gel column chromatography, get White powder, yield 60%; molecular weight: 585, structure as follows:

(5) Synthesis of glycocholic acid-9-propoxyberberine: (C1)

Add 161mg (0.5mmol) of berberine to a 25mL round bottom flask, add 3mL of DMF to dissolve, add 468mg (0.8mmol) of glycocholate-3-bromopropyl, heat at 60°C, follow the reaction by TLC, after the reaction is complete Anhydrous ether was added to precipitate a yellow solid, which was filtered under reduced pressure, vacuum-dried, and subjected to silica gel column chromatography to obtain the target product with a yield of 34%; molecular weight: 827, and the structural formula is as follows:

Embodiment 2: the synthesis of glycodehydrocholic acid-9-propoxyberberine: (C2)

(1) Synthesis of Berberine

Same as (1) in Example 1.

(2) Synthesis of dehydrocholic acid activated ester:

Add 201 mg (0.5 mmol) of dehydrocholic acid to a 50 mL round bottom flask, add 10 mL of dichloromethane, add 115 mg of N-hydroxysuccinimide, and add 1-ethyl-3-(3-dimethylaminopropyl Base) carbodiimide 766mg (4mmol) dichloromethane solution 10mL, follow the reaction by TLC. After the reaction was complete, 30 mL of chloroform was added to the reaction solution, extracted three times with 50 mL of saturated saline and 50 mL of saturated _NaHCO3 solution, dried over anhydrous _MgSO4 , filtered under reduced pressure to remove _MgSO4 , concentrated under reduced pressure, and separated by silica gel column chromatography After purification, a white powder was obtained. Yield 98%; molecular weight: 509; structural formula is as follows:

(3) Synthesis of glycodehydrocholic acid:

Add 257mg (0.5mmol) of dehydrocholic acid activated ester in a 25mL round-bottomed flask, dissolve in 1mL DMSO, add 45mg (0.6mmol) of glycine, 100μL triethylamine, follow the reaction by TLC, add 20mL anhydrous ether after the reaction is complete, and precipitate White solid, suction filtration under reduced pressure, vacuum drying, yield 97%, molecular weight: 459, structure as follows:

(4) Synthesis of glycodehydrocholic acid-3-bromopropyl ester:

Add 230 mg (0.5 mmol) of glycodehydrocholic acid to a 25 mL round bottom flask, add 1 mL of DMF to dissolve, add 200 μL of 1,3-dibromopropane, add K ₂ CO ₃ 276 mg (2 mmol), follow the reaction by TLC, after the reaction is complete , filtered under reduced pressure, added 50 mL of chloroform to the filtrate, extracted three times with 50 mL of distilled water and 50 mL of saturated NaHCO ₃ solution successively, dried over anhydrous MgSO ₄ , removed MgSO ₄ by reduced pressure suction filtration, concentrated under reduced pressure, separated and purified by silica gel column chromatography , to get white powder, yield 93%; molecular weight: 579, the structure is as follows:

(5) Synthesis of glycodehydrocholic acid-9-propoxyberberine: (C2)

Add 161mg (0.5mmol) of berberine into a 25mL round bottom flask, add 3mL of DMF to dissolve, add 463mg (0.8mmol) of glycodehydrocholic acid-3-bromopropyl, heat at 60°C, follow the reaction by TLC, the reaction After completion, anhydrous ether was added to precipitate a yellow solid, which was filtered under reduced pressure, vacuum-dried, and subjected to silica gel column chromatography to obtain the target product with a yield of 41%; molecular weight: 821, and the structural formula is as follows:

Embodiment 3: Synthesis of glycodeoxycholic acid-9-propoxyberberine: (C3)

(1) Synthesis of Berberine

Same as (1) in Example 1.

(2) Synthesis of dehydrocholic acid activated ester:

Add 196 mg (0.5 mmol) of deoxycholic acid to a 50 mL round bottom flask, add 10 mL of dichloromethane, add 115 mg of N-hydroxysuccinimide, and add 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide 766mg (4mmol) dichloromethane solution 10mL, follow the reaction by TLC. After the reaction was complete, 30 mL of chloroform was added to the reaction solution, extracted three times with 50 mL of saturated saline and 50 mL of saturated _NaHCO3 solution, dried over anhydrous _MgSO4 , filtered under reduced pressure to remove _MgSO4 , concentrated under reduced pressure, and separated by silica gel column chromatography After purification, a white powder was obtained. Yield 98%; molecular weight: 509; structural formula is as follows:

(3) Synthesis of glycodeoxycholic acid:

Add 255 mg (0.5 mmol) of deoxycholic acid activated ester and 1 mL of DMSO into a 25 mL round bottom flask to dissolve, add 45 mg (0.6 mmol) of glycine and 100 μL of triethylamine, follow the reaction by TLC, add 20 mL of anhydrous ether after the reaction is complete, and white Solid, suction filtration under reduced pressure, vacuum drying, yield 95%, molecular weight: 449, structure as follows:

(4) Synthesis of glycodeoxycholic acid-3-bromopropyl ester:

Add 225 mg (0.5 mmol) of glycodeoxycholic acid to a 25 mL round bottom flask, add 1 mL of DMF to dissolve, add 200 μL of 1,3-dibromopropane, add K ₂ CO ₃ 276 mg (2 mmol), and track the reaction by TLC. After the reaction is complete, Suction filtration under reduced pressure, add 50mL of chloroform to the filtrate, extract with 50mL distilled water and 50mL saturated _NaHCO3 solution successively for 3 times, dry over anhydrous _MgSO4 , remove _MgSO4 by suction filtration under reduced pressure, concentrate under reduced pressure, separate and purify by silica gel column chromatography, Obtain white powder, yield 93%; Molecular weight: 569, structure is as follows:

(5) Synthesis of glycodeoxycholic acid-9-propoxyberberine: (C3)

Add 161mg (0.5mmol) of berberine into a 25mL round bottom flask, add 3mL of DMF to dissolve, add 455mg (0.8mmol) of glycodeoxycholic acid-3-bromopropyl, heat at 60°C, follow the reaction by TLC, and the reaction is complete After adding anhydrous diethyl ether, a yellow solid was precipitated, filtered under reduced pressure, vacuum-dried, and subjected to silica gel column chromatography to obtain the target product with a yield of 39%; molecular weight: 811, and the structural formula is as follows:

Example 4: Synthesis of glycochenodeoxycholic acid-9-propoxyberberine: (C4)

(1) Synthesis of Berberine

Same as (1) in Example 1.

(2) Synthesis of chenodeoxycholic acid activated ester:

Add 196 mg (0.5 mmol) of chenodeoxycholic acid to a 50 mL round bottom flask, add 10 mL of dichloromethane, add 115 mg of N-hydroxysuccinimide, and add 1-ethyl-3-(3-dimethylaminopropyl Base) carbodiimide 766mg (4mmol) dichloromethane solution 10mL, follow the reaction by TLC. After the reaction was complete, 30 mL of chloroform was added to the reaction solution, extracted three times with 50 mL of saturated saline and 50 mL of saturated _NaHCO3 solution, dried over anhydrous _MgSO4 , filtered under reduced pressure to remove _MgSO4 , concentrated under reduced pressure, and separated by silica gel column chromatography After purification, a white powder was obtained. Yield 90%; molecular weight: 509; structural formula is as follows:

(3) Synthesis of Glychenodeoxycholic Acid:

Add 255mg (0.5mmol) of chenodeoxycholic acid activated ester into a 25mL round bottom flask, dissolve in 1mL DMSO, add 45mg (0.6mmol) of glycine, 100μL triethylamine, follow the reaction by TLC, add 20mL anhydrous ether after the reaction is complete, and precipitate White solid, suction filtration under reduced pressure, vacuum drying, yield 95%, molecular weight: 449, structure as follows:

(4) Synthesis of glycochenodeoxycholic acid-3-bromopropyl ester:

Add 225 mg (0.5 mmol) of glycochenodeoxycholic acid into a 25 mL round bottom flask, add 1 mL of DMF to dissolve, add 200 μL of 1,3-dibromopropane, add K ₂ CO ₃ 276 mg (2 mmol), follow the reaction by TLC, after the reaction is complete , filtered under reduced pressure, added 50 mL of chloroform to the filtrate, extracted three times with 50 mL of distilled water and 50 mL of saturated NaHCO ₃ solution successively, dried over anhydrous MgSO ₄ , removed MgSO ₄ by reduced pressure suction filtration, concentrated under reduced pressure, separated and purified by silica gel column chromatography , to obtain a white powder with a yield of 86%; molecular weight: 569, and the structure is as follows:

(5) Synthesis of glycochenodeoxycholic acid-9-propoxyberberine: (C4)

Add 161mg (0.5mmol) of berberine into a 25mL round bottom flask, add 3mL of DMF to dissolve, add 455mg (0.8mmol) of glycochenodeoxycholic acid-3-bromopropyl, heat at 60°C, follow the reaction by TLC, the reaction After completion, anhydrous ether was added to precipitate a yellow solid, which was filtered under reduced pressure, vacuum-dried, and subjected to silica gel column chromatography to obtain the target product with a yield of 34%; molecular weight: 811, and the structural formula is as follows:

Example 5: Synthesis of Glycoursodeoxycholic Acid-9-propoxyberberine: (C5)

(1) Synthesis of Berberine

Same as (1) in Example 1.

(2) Synthesis of ursodeoxycholic acid activated ester:

Add 196 mg (0.5 mmol) of ursodeoxycholic acid into a 50 mL round bottom flask, add 10 mL of dichloromethane, add 115 mg of N-hydroxysuccinimide, and add 1-ethyl-3-(3-dimethylaminopropyl Base) carbodiimide 766mg (4mmol) dichloromethane solution 10mL, follow the reaction by TLC. After the reaction was complete, 30 mL of chloroform was added to the reaction solution, extracted three times with 50 mL of saturated saline and 50 mL of saturated _NaHCO3 solution, dried over anhydrous _MgSO4 , filtered under reduced pressure to remove _MgSO4 , concentrated under reduced pressure, and separated by silica gel column chromatography After purification, a white powder was obtained. Yield 84%; Molecular weight: 509; Structural formula is as follows:

(3) Synthesis of glycosylated ursodeoxycholic acid:

Add 255mg (0.5mmol) of ursodeoxycholic acid activated ester into a 25mL round bottom flask, dissolve in 1mL DMSO, add 45mg (0.6mmol) of glycine, 100μL triethylamine, follow the reaction by TLC, add 20mL anhydrous ether after the reaction is complete, and precipitate White solid, suction filtration under reduced pressure, vacuum drying, yield 95%, molecular weight: 449, structure as follows:

(4) Synthesis of glycosylated ursodeoxycholic acid-3-bromopropyl ester:

Add 225 mg (0.5 mmol) of glycoursodeoxycholic acid into a 25 mL round bottom flask, add 1 mL of DMF to dissolve, add 200 μL of 1,3-dibromopropane, add K ₂ CO ₃ 276 mg (2 mmol), and track the reaction by TLC. After the reaction is complete, Suction filtration under reduced pressure, add 50mL of chloroform to the filtrate, extract with 50mL distilled water and 50mL saturated _NaHCO3 solution successively for 3 times, dry over anhydrous _MgSO4 , remove _MgSO4 by suction filtration under reduced pressure, concentrate under reduced pressure, separate and purify by silica gel column chromatography, Obtain white powder, yield 86%; Molecular weight: 569, structure is as follows:

(5) Synthesis of glycoursodeoxycholic acid-9-propoxyberberine: (C5)

Add 161mg (0.5mmol) of berberine into a 25mL round-bottomed flask, add 3mL DMF to dissolve, add 455mg (0.8mmol) of glycoursodeoxycholic acid-3-bromopropyl, heat at 60°C, follow the reaction by TLC, the reaction After completion, anhydrous ether was added to precipitate a yellow solid, which was filtered under reduced pressure, vacuum-dried, and subjected to silica gel column chromatography to obtain the target product with a yield of 30%; molecular weight: 811, and the structural formula is as follows:

。

.

Claims (5)

1. described berberinc derivate is represented with following general formula (I):

Wherein

R ₁Be hydroxyl, carbonyl;

R ₂Be hydrogen, hydroxyl, carbonyl;

R ₃Be hydrogen, hydroxyl, carbonyl.

2. berberinc derivate according to claim 1, wherein R ₁Be hydroxyl, carbonyl; R ₂, R ₃Be hydrogen, hydroxyl, carbonyl.

3. the preparation method of the described berberinc derivate of claim 1:

It is characterized in that the Berberine shown in the formula (II)

Heating generates intermediate berberrubine (III).

With chlolic acid derivatives shown in (IV) formula

Wherein

R ₁Definition with claim 3;

R ₂Definition with claim 4;

R ₃Definition with claim 5;

React to generate chlolic acid derivatives (V) with N-hydroxy-succinamide

Wherein

R ₁Definition with claim 3;

R ₂Definition with claim 4;

R ₃Definition with claim 5;

With glycine reactant to generate chlolic acid derivatives (VI)

Wherein

R ₁Definition with claim 3;

R ₂Definition with claim 4;

R ₃Definition with claim 5;

With the dihalo hydrocarbon reaction to generate chlolic acid derivatives (VII)

Wherein

R ₁Definition with claim 3;

R ₂Definition with claim 4;

R ₃Definition with claim 5;

With berberinc derivate shown in (III) formula and the reaction of (VII) chlolic acid derivatives shown in the formula to generate the described berberinc derivate of claim 1 (I)

Wherein

R ₁Definition with claim 3;

R ₂Definition with claim 4;

R ₃Definition with claim 5.

4. the compound of claim 1-5 is as the purposes in the preparation antitumor drug.

5. the compound of claim 1-5 is used for the treatment of the application of activeconstituents of the medicine of tumour as preparation.