CN117924388A

CN117924388A - Raphanin precursor compound and preparation method and application thereof

Info

Publication number: CN117924388A
Application number: CN202410081697.3A
Authority: CN
Inventors: 贺贤然; 李少磊; 李晓龙
Original assignee: Shenzhen Fushan Biotechnology Co ltd
Current assignee: Shenzhen Fushan Biotechnology Co ltd
Priority date: 2024-01-19
Filing date: 2024-01-19
Publication date: 2024-04-26

Abstract

The invention discloses a sulforaphane precursor compound shown in a formula I. The compound of formula I can be converted to sulforaphane by myrosinase. The invention also provides a preparation method of the compound shown in the formula I.

Description

Raphanin precursor compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a sulforaphane precursor compound and a preparation method and application thereof.

Background

Sulforaphane (sulforaphane) is an isothiocyanate, which belongs to a sulfur-containing organic compound. Was first isolated from broccoli by Paul Talalay institution team at John Hopkins university, U.S. in 1992. Since discovery, researchers have carried out a series of researches on the sulforaphane, and found that the sulforaphane has obvious inhibition effect on liver cancer, breast cancer, colon cancer, prostate cancer and the like, and is also beneficial to achieving better treatment effect of the existing anticancer drugs. As the strongest inducer of Nrf2 (a protein for regulating the expression of antioxidant enzyme), the sulforaphane can not only induce cancer cell death and inhibit cancer cell proliferation, but also inhibit tumor metastasis and prevent cancer from worsening, and has high clinical research value.

Although the sulforaphane is widely used in crucifers, the sulforaphane is very low in content and unstable, and cannot be obtained from plants in large quantities directly. Whereas glucoraphanin is a precursor substance of glucoraphanin, which is produced by myrosinase. The glucoraphanin is stably enriched in broccoli seeds and sprouts, and is a main source for obtaining glucoraphanin at present. In 2017, the water extract of broccoli seeds (13% -20% of glucoraphanin) is approved as a new food raw material in China, and the demand for glucoraphanin at home and abroad is huge in growing space. However, the existing glucoraphanin is extracted from broccoli seeds or sprouts, and has limited extraction efficiency, high yield, and high cost.

Thus, there is an urgent need in the art for new sulforaphane precursor compounds. How to develop a method for obtaining sulforaphane without seed source control has more cost advantage and becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a sulforaphane precursor compound which is easy to obtain, stable and low in cost.

In order to achieve the above object, the present invention provides a sulforaphane precursor compound which is a compound represented by the following formula I

Or a stereoisomer, tautomer, hydrate or solvate thereof.

The invention also provides a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable stereoisomer, tautomer, hydrate, or solvate thereof, and a pharmaceutically acceptable carrier.

The invention also provides a preparation method of the sulforaphane precursor compound of the formula I, which comprises the following steps:

By reacting compound A

Bromine substitution reaction is carried out to obtain a compound B

The compound B and thiourea react under the action of a catalyst to obtain a compound C

Reacting said compound C with 5-methylsulfonylaminoxime under NCS, pyridine and triethylamine to obtain compound D

Reacting said compound D with a sulfur trioxide pyridine complex and an aqueous sodium bicarbonate solution to obtain compound E

Reacting said compound E with sodium methoxide to obtain a compound of formula I.

An exemplary overall reaction scheme is shown below:

Detailed Description

The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the examples of the present invention are commercially available or available by existing methods.

The embodiment of the invention provides a sulforaphane precursor compound, which has a structure shown in the following formula I:

the compounds may also be in the form of pharmaceutically acceptable stereoisomers, tautomers, hydrates or solvates thereof.

Typical compositions comprise a compound of formula I of the present invention and a pharmaceutically acceptable carrier. For example, the active compound is typically admixed with a carrier, either diluted by the carrier, or enclosed within a carrier which may be in the form of an ampoule, capsule, sachet (sachets), paper or other container. When the active compound is admixed with a carrier, or when the carrier acts as a diluent, the carrier may be a solid, semi-solid, or liquid material that acts as a carrier, excipient, or medium for the active compound. The active compound may be adsorbed on a particulate solid carrier (e.g. contained in a sachet). Some examples of suitable carriers are water, saline solution, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, lower alkyl ethers of stearic acid or cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethyl cellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent may comprise any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.

The formulations may be mixed with adjuvants which do not react adversely with the active compounds. These additives may include wetting agents, emulsifying and suspending agents, salts which influence osmotic pressure, buffers and/or coloring substances, preservatives, sweeteners or flavoring agents. The composition may also be sterilized, if desired.

The route of administration may be any route which effectively transports the compounds of formula F according to the invention to the appropriate or desired site of action, for example oral, nasal, pulmonary, buccal, subcutaneous, intradermal, transdermal or parenteral routes, for example rectal, depot (delivery), subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solutions or ointments, the oral route being preferred.

If a solid carrier is used for oral administration, the formulation may be tableted, placed in hard gelatin capsules as a powder or pellet, or it may be in the form of a lozenge (troche) or troche. If a liquid carrier is used, the formulation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid, such as an aqueous or non-aqueous liquid suspension or solution.

Injectable dosage forms typically include aqueous or oily suspensions which may be prepared using suitable dispersing or wetting agents and suspending agents. The injectable form may be in the solution phase or in the form of a suspension prepared with a solvent or diluent. Acceptable solvents or carriers include sterile water, ringer's solution, or isotonic saline solution. Alternatively sterile oil may be used as a solvent or suspending agent. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, monoglycerides, diglycerides or triglycerides.

For injection, the formulation may also be a powder suitable for reconstitution with a suitable solution as described above. Examples of these include, but are not limited to, freeze-dried, spin-dried or spray-dried powders, amorphous powders, granules, precipitates or microparticles. For injectable formulations, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations of these agents. The compounds may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. The unit dosage form for injection may be in an ampoule or in a multi-dose container.

According to another aspect of the present invention, there is provided a method for preparing a sulforaphane precursor compound represented by formula I, the method comprising:

Preferably, the preparation method specifically may employ the following steps.

S1, carrying out a bromine substitution reaction on a compound A and a brominating reagent to obtain a compound B;

As an alternative embodiment, the brominating reagent can be selected from hydrobromic acid, N-bromosuccinimide, and liquid bromine. The weight ratio of compound a to hydrobromic acid may be 1: (1-3), the reaction temperature can be 25-75 ℃ and the reaction time can be 2-24 h; the reaction condition is favorable for bromine substitution reaction to obtain B completely.

S2, enabling the compound B to react with thiourea and sodium metabisulfite to obtain a compound C;

The step S2 may be performed in an organic solvent, preferably selected from the group consisting of acetone, acetonitrile and methanol. Preferably, the weight ratio of the compound B to thiourea to sodium metabisulfite is 1: (1-3): (1-6), the reaction temperature is 0-85 ℃ and the reaction time is 1-16 h. The reaction conditions are favorable for obtaining the compound C.

S3, reacting the compound C with 5-methyl sulfoxide aldoxime under the condition of NCS (N-chlorosuccinimide) pyridine and triethylamine to obtain a compound D;

As an alternative implementation mode, firstly, dissolving 5-methyl sulfoxide aldoxime in methylene dichloride, adding 1-5 equivalents of pyridine and 1-5 equivalents of NCS under the protection of nitrogen, stirring for 1-6 hours, then adding a methylene dichloride solution of a compound C, stirring for 10-24 hours at room temperature after the addition is finished, adding 15-40 ml of 1mol/L sulfuric acid, carrying out liquid separation extraction, drying the combined organic phases by using anhydrous sodium sulfate or anhydrous magnesium sulfate, spin-drying, and purifying the crude product by a chromatographic column to obtain the compound D.

S4, reacting the compound D with a sulfur trioxide pyridine compound to obtain a compound E;

As an alternative implementation mode, firstly, dissolving the compound D in a certain amount of DMF, then adding 1-5 equivalents of sulfur trioxide pyridine compound, stirring for 1-16 hours at room temperature after the addition, then adding a certain amount of sodium bicarbonate solution with the concentration of 1mol/L, continuously stirring for 1-6 hours, directly spin-drying, and purifying the crude product through a chromatographic column to obtain the compound E.

S5, reacting the compound E with sodium methoxide in methanol to obtain a compound I.

The preparation method provided by the embodiment of the invention is simple, has higher yield, and can easily prepare the compound I.

The invention also provides application of the compound shown in the formula I or stereoisomers, tautomers, hydrates or solvates thereof in preparing the sulforaphane. In particular, the present invention provides a process for preparing sulforaphane using a compound of formula I, or a stereoisomer, tautomer, hydrate, or solvate thereof, as a precursor compound, comprising converting the compound of formula I, or a stereoisomer, tautomer, hydrate, or solvate thereof, to sulforaphane in the presence of myrosinase (e.g., horseradish powder), preferably simultaneously in the presence of vitamin C.

The process for preparing the compounds of formula I according to the application will now be described in detail with reference to the examples and experimental data.

EXAMPLE 1 preparation of Compounds of formula I

The compound of formula I is prepared according to the reaction equation shown below, and specifically includes:

1. synthesis of Compound of formula B

2G of alpha-D-pentaacetyl galactose was dissolved in 20 ml of dichloromethane at 0℃and after the addition of 12.4 g of HBr in acetic acid (mass fraction 33%) was completed, stirring was continued overnight, TLC showed complete reaction, the reaction solution was poured into a1 l beaker, the peripheral ice-bath was stirred with saturated aqueous sodium carbonate to adjust pH to around 10, the solution was separated, the aqueous phase was extracted 3 times with dichloromethane (15 ml. Times.3), the combined organic phases were dried over anhydrous sodium sulfate and dried by spinning to give 2g of yellow oily liquid B.

2. Synthesis of Compound of formula C

2G of B was dissolved in 10 ml of acetonitrile at room temperature, 1.11 g of thiourea was added, and after the addition was completed, the mixture was heated to 70℃and stirred for 3 hours, and TLC showed completion of the reaction. And (3) cooling the reaction liquid to room temperature, carrying out suction filtration, washing a filter cake with a small amount of acetonitrile, and spin-drying the filtrate to obtain a crude product. The crude product was then dissolved in 20 ml of dichloromethane and 20 ml of water, 6.2 g of sodium metabisulfite was added, after which the reaction mixture was heated to 65 ℃ and stirred at reflux overnight, TLC indicated complete reaction. After the reaction was cooled to room temperature, the solution was separated, the aqueous phase was extracted 3 times with dichloromethane (15 ml×3), the combined organic phases were dried over anhydrous sodium sulfate, dried by spin, and the crude product was purified by chromatography (petroleum ether/ethyl acetate=50:1 to 1:1). 1.45 g of colorless oily liquid C was obtained.

3. Synthesis of Compound of formula D

At room temperature, 0.65 g of Compound 3 (5-methylsulfonylmethane oxime) was dissolved in 15 ml of dichloromethane, 330 mg of pyridine and 558 mg of NCS (N-chlorosuccinimide) were added under nitrogen protection, stirring was continued for 2 hours after the addition was completed, 1.45 g of a solution of C in dichloromethane was further added, stirring was continued overnight after the addition was completed, 15 ml of a 1mol/L aqueous sulfuric acid solution was further added, stirring was continued for half an hour, TLC showed complete reaction, and MS detected the product. The solution was separated, the aqueous phase extracted 2 times with dichloromethane (20 ml×2), the combined organic phases dried over anhydrous sodium sulfate and the crude product purified by column chromatography (DCM/meoh=50:1 to 10:1) to give 760 mg of pale yellow oily liquid D.

MS [ ESI ] calculated (M+1) +,526.13; actual measurement value 526.15.

4. Synthesis of Compound of formula E

760 Mg of D was dissolved in 10 ml of DMF at room temperature, 690 mg of the sulfur trioxide pyridine complex was added, stirring was continued at room temperature for 3 hours after the addition was completed, and 12 ml of 1mol/L aqueous NaHCO ₃ solution was added, and stirring was continued for 3 hours after the addition was completed. MS showed complete reaction, direct spin-drying and purification of the crude product by column (DCM/meoh=10:1 to 1:1) afforded 430 mg of white solid E.

MS [ ESI ] calculated (M-23) -,604.07; actual measurement value 604.12.

5. Synthesis of Compounds of formula I

300 Mg of 5 is dissolved in 7 ml of methanol at room temperature, 100 mg of sodium methoxide is added, and after the addition is completed, the mixture is stirred at room temperature overnight, MS shows that the reaction is complete, and the mixture is directly dried by spin to obtain a crude product. The crude product is dissolved in 3 ml of methanol and 2 drops of water, heated to 50 ℃ and stirred for half an hour, and then the mixture is stood for cooling, suction filtration and mother liquor spin-drying are carried out to obtain 125 mg of I.

Nuclear magnetic resonance 1H NMR(400MHz,D2O)δ4.98(d,J＝9.2Hz,1H),4.01(s,1H),3.80(dd,J＝12.1,6.8Hz,1H),3.76-3.67(m,4H),3.03-2.89(m,2H),2.82(t,J＝6.7Hz,2H),2.70(s,3H),1.98-1.76(m,4H).

MS [ ESI ] calculated (M-23) -,436.03; actual measurement value 436.02.

Experimental example 2 production of glucoraphanin from a compound of formula I and detection thereof the present invention provides a method for preparing a glucoraphanin using a compound of formula I or a stereoisomer, tautomer, hydrate or solvate thereof as a precursor compound, comprising converting a compound of formula I or a stereoisomer, tautomer, hydrate or solvate thereof into a glucoraphanin in the presence of myrosinase (e.g. horseradish powder), preferably simultaneously in the presence of vitamin C.

In this example, 10.0mg of the compound of formula I was weighed into a volumetric flask, dissolved in distilled water, fixed to 25mL, and vitamin C was added to react with horseradish powder (1.0 mg, 10%) for 1 hour, so that the compound of formula I, which was a precursor compound of sulforaphane, was converted into sulforaphane.

The content of sulforaphane was 4.33mg by HPLC, and the enzymatic conversion of Compound I was calculated to be 43.3%.

10.0Mg of standard product of potassium glucoraphanin (Shenzhen Fushan organism, HPLC purity 98.0%) is weighed, dissolved in distilled water, fixed to 25mL, added with the same amount of vitamin C and horseradish powder as a control group for reaction for 1 hour, the content of the glucoraphanin is detected by HPLC and 3.92mg, and the enzymolysis conversion rate of the glucoraphanin is calculated to be 39.2%.

TABLE 1 comparison of the enzymatic conversion of the raphanin precursor Compound I and raphanin

Label name	Sample size/mg	Actual measurement of sulforaphane content/mg after enzymolysis
			Raphanin precursor compound I	10.0	4.33
Standard substance of potassium glucoraphanin	10.0	3.92

The molar conversion rate of the sulforaphane, which adopts the compound shown in the formula I, is increased by 22% compared with that of the sulforaphane which adopts the potassium glucoraphane. Such results are unexpected.

As shown in the experimental results of Table 1, the sulforaphane precursor compound I prepared by the invention can generate more sulforaphane under the same enzymolysis environment, and has higher sulforaphane enzymolysis conversion rate. Compared with the prior art in which the precursor substance glucoraphanin is obtained, the glucoraphanin precursor compound I is easier to prepare, has more advantages in cost and is beneficial to industrial production.

Claims

1. Compounds of formula I

Or a stereoisomer, tautomer, hydrate or solvate thereof.

2. A pharmaceutical composition comprising a compound of formula I according to claim 1 or a stereoisomer, tautomer, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.

3. A process for the preparation of a compound of formula I as defined in claim 1, comprising the steps of:

By reacting compound A

Bromine substitution reaction is carried out to obtain a compound B

And

4. A method of preparation according to claim 3, wherein the brominating reagent is selected from hydrobromic acid, N-bromosuccinimide and liquid bromine.

5. The process according to claim 3 or 4, wherein the catalyst is sodium metabisulfite and the weight ratio of compound B to sodium metabisulfite is 1 (0.1-1), preferably 1 (0.1-0.5), such as 1:0.1, 1:0.3 or 1:0.5.

6. Use of a compound of formula I as defined in claim 1 or a stereoisomer, tautomer, hydrate or solvate thereof for the preparation of sulforaphane.

7. Use of a compound of formula I as defined in claim 1 or a stereoisomer, tautomer, hydrate or solvate thereof for the manufacture of a medicament for use in the manufacture of an Nrf2 inducer.

8. Use of a compound of formula I as defined in claim 1 or a stereoisomer, tautomer, hydrate or solvate thereof for the manufacture of a medicament for the treatment or prophylaxis of cancer.

9. The use according to claim 8, wherein the cancer is selected from liver cancer, breast cancer, colon cancer and prostate cancer.