CN112724135A - Hypoglycemic lupine derivative and preparation method thereof - Google Patents

Abstract

The invention discloses a lupine derivative for reducing blood sugar and a preparation method thereof, belonging to the technical field of drug synthesis and organic compound preparation; the preparation method comprises the following steps: taking lupine as a raw material, adding 3, 5-dimethoxy benzyl bromide in an organic solvent under the action of alkali, magnetically stirring at 90-130 ℃ for 10-24 hours to obtain a crude lupine derivative, separating the crude lupine derivative by silica gel column chromatography, and purifying by high performance liquid chromatography to obtain the lupine derivative; the pathological experiment shows that the new compound has better function of reducing blood sugar; toxicological experiments indicate that the LD of the novel compounds₅₀Is far more than 3.2g/kg, the maximum tolerance of the mouse reaches 24g/kg, and no obvious toxic or side effect is caused to internal organs.

Description

Hypoglycemic lupine derivative and preparation method thereof

Technical Field

The invention belongs to the technical field of drug synthesis and organic compound preparation, and relates to a lupine derivative for reducing blood sugar and a preparation method thereof.

Background

Lupeonine belongs to Lupeon alkaloid, and is mainly distributed in leaf and rhizome of Sophora pseudoacacia flower. Lupine has various biological activities, such as treating cardiovascular diseases, lowering blood sugar, etc. However, lupinus alkaloids have strong cytotoxicity and teratogenesis, and meanwhile, the alkaloids have great influence on the respiratory system, respiratory system symptoms caused by alkaloid poisoning mainly comprise respiratory tract stimulation, too fast and too slow respiratory tract frequency and the like, and severe patients can cause respiratory paralysis to die. In order to utilize the biological activity of lupine more efficiently and safely, it is necessary to modify its structure.

Disclosure of Invention

In order to solve the technical problem, the invention provides a hypoglycemic lupine derivative, which has a molecular structure shown in formula (I):

another object of the present invention is to provide a process for the preparation of hypoglycemic lupine derivatives comprising the steps of:

removing water in a reaction vessel, setting the atmosphere of the reaction vessel as an inert gas environment, dissolving lupine and 3, 5-dimethoxy benzyl bromide in an organic solvent, adding alkali, magnetically stirring at 90-130 ℃ for 10-24 hours, detecting by TLC (thin layer chromatography), determining that the reaction is finished, quenching the reaction with ice water, extracting for multiple times, sequentially washing with 10% hydrochloric acid solution, saturated salt solution and water, and drying with anhydrous sodium sulfate to obtain a crude product; performing silica gel column chromatography on the crude product, and performing high performance liquid chromatography purification to obtain a target product;

the feeding molar ratio of the lupine to the 3, 5-dimethoxy benzyl bromide is 1: 1.5-4.5;

the organic solvent is tetrahydrofuran, acetonitrile, diethyl ether, acetone, toluene, benzotrifluoride, dimethyl sulfoxide or ethyl acetate;

the alkali is sodium hydroxide, potassium hydride, sodium carbonate, potassium carbonate or lithium carbonate;

the filler in the silica gel column is alumina or silica gel, and the granularity of the silica gel is 100-400 meshes; gradient eluting the silica gel column eluent by using a petroleum ether-ethyl acetate mixed solvent according to the volume ratio of 20:1, 10:1, 5:1 and 1: 1;

the high performance liquid chromatography uses a C8 reversed phase chromatographic column, water is used as a mobile phase A, a mixed solution of water and acetonitrile with a volume ratio of 15:85 is used as a mobile phase B, and the flow rate is 5-100 mL/min.

According to a preferred embodiment of the above preparation method, the method comprises the following steps:

removing water in a reaction vessel, setting the atmosphere of the reaction vessel as an inert gas environment, dissolving 15g of lupine and 40.1g of 3, 5-dimethoxy benzyl bromide in acetone, adding sodium carbonate, stirring for 10 hours at 130 ℃, performing magnetic stirring, detecting by TLC (thin layer chromatography), determining that the reaction is finished, quenching the reaction by using ice water, extracting for multiple times, sequentially washing by using 10% hydrochloric acid solution, saturated salt solution and water, and drying by using anhydrous sodium sulfate to obtain a crude product; and (2) performing silica gel column chromatography on the crude product, wherein a filler in the silica gel column is silica gel, the particle size of the silica gel is 100-200 meshes, gradient elution is performed on silica gel column eluent by adopting a petroleum ether-ethyl acetate mixed solvent according to the volume ratio of 20:1, 10:1, 5:1 and 1:1, the eluent is collected, a C8 reverse phase chromatographic column is used, water is used as a mobile phase A, a mixed solution of water and acetonitrile according to the volume ratio of 15:85 is used as a mobile phase B, the flow rate is 5mL/min, and high performance liquid chromatography purification is performed to obtain the target product.

Compared with the prior art, the invention has the beneficial effects that:

the lupine derivative with a novel structure is obtained by taking lupine as a raw material through alkylation reaction; the pathological experiment shows that the compound has obvious blood sugar reducing effect on both alloxan diabetic rabbits and adrenergic hyperglycemia mice, and has no obvious influence on the blood sugar of normal mice; toxicological experiments indicate that the LD of the novel compounds₅₀The maximum tolerance of the mouse is up to 24g/kg and is far more than 3.2g/kg, the routine blood and liver and kidney functions of the rat in a toxicological experiment have no obvious influence, and the lupine derivative has no damage to organs of the experimental rat, so that the lupine derivative has no obvious toxic or side effect.

Drawings

FIG. 1: the hpc nmr hydrogen spectrum of the hypoglycemic lupine derivative of example 1.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1

Removing water in a reaction vessel, setting the atmosphere of the reaction vessel as an inert gas environment, dissolving 15g of lupine and 40.1g of 3, 5-dimethoxy benzyl bromide in 100mL of acetone, adding 6.5g of sodium carbonate, magnetically stirring at 130 ℃ for 10h, detecting by TLC (thin layer chromatography), determining that the reaction is finished, quenching the reaction by using ice water, extracting the reaction solution by using 20L of petroleum ether, then extracting by using 20L of ethyl acetate for 3 times, collecting ethyl acetate extract, then sequentially washing by using 10% hydrochloric acid solution, saturated salt water and water, and drying by using anhydrous sodium sulfate to obtain a crude product; and (2) performing silica gel column chromatography on the crude product, wherein a filler in the silica gel column is silica gel, the particle size of the silica gel is 100-200 meshes, gradient elution is performed on silica gel column eluent by adopting a petroleum ether-ethyl acetate mixed solvent according to the volume ratio of 20:1, 10:1, 5:1 and 1:1, the eluent is collected, a C8 reverse phase chromatographic column is used, water is used as a mobile phase A, a mixed solution of water and acetonitrile according to the volume ratio of 15:85 is used as a mobile phase B, the flow rate is 5mL/min, the ultraviolet detection wavelength is 286nm, and high performance liquid chromatography purification is performed at normal temperature to obtain the target product. The yield was 84.02%.

And (3) nuclear magnetic resonance hydrogen spectrum detection:

the sample was placed in a sample tube, and 0.5ml of DCL3 (deuterated chloroform) was injected into the sample tube with a syringe to dissolve the sample sufficiently. The sample and the reagent are required to be fully mixed, the solution is clear and transparent, and has no suspended matters or other impurities, and a nuclear magnetic resonance hydrogen spectrogram is obtained through nuclear magnetic resonance identification, and the result is shown in figure 1.

Experimental example 2 pharmacological study of the Compound of the present invention for lowering blood sugar

(1) Effect on blood glucose levels in adrenergic hyperglycemic mice

Healthy mice were selected, and some of the mice were randomly selected as normal control groups (control group 1), and the remaining mice were injected with 0.2mg/kg epinephrine. The adrenaline mice were further divided into a model control group (control group 2), a positive drug control group (control group 3), a lupine group (control group 4), and a lupine derivative group (experimental group). Normal group and model group were fed with physiological saline of equal volume, positive drug group was given acarbose, lupine group was given lupine, experimental group used lupine derivative of the present invention. Each group of mice was fed with the drug 1 time a day for 7 days continuously, and blood was collected from all the eye sockets half an hour after the last administration, and the blood glucose concentration was measured by the o-toluidine method. The results are shown in Table 1.

TABLE 1 Effect of Compounds of the invention on blood glucose levels in mice with adrenergic hyperglycemia

Group of	Dosage form	Blood sugar value mmoL/L
			Control group 1	0.25mL/10g	5.49±0.33
Control group 2	0.25mL/10g	15.08±0.59
			Control group 3	3.0g/kg	8.62±0.61
Control group 4	3.0g/kg	11.27±0.44
			Experimental group	3.0g/kg	9.03±0.51

The data in table 1 show that, compared with the model group, the lupine derivative of the invention can significantly reduce the blood sugar level of mice with adrenergic hyperglycemia, is equivalent to acarbose, but has better blood sugar reducing effect than lupine acting alone.

(2) Effect on alloxan diabetic rabbits and serum insulin release

Healthy rabbits are selected and fed in a laboratory for one week, and then are randomly grouped and fasted for 12 hours, 5% alloxan solution is injected into ear margin vein of the rabbits for 180mg/kg, and normal groups do not have any treatment. Modeling, fast taking blood from heart after fasting for 12h, and measuring fasting blood glucose by o-toluidine method. Rabbits with close blood sugar were randomly grouped as: the test method comprises the following steps of (1) feeding normal groups and model groups with physiological saline of the same volume, respectively feeding other groups of rabbits with corresponding medicines, continuously feeding for 4 weeks according to the dosage of 5mL per kilogram of body weight, feeding normal diet during the feeding period, and increasing the water drinking amount of the rabbits, wherein the normal groups and the model groups are fed with normal saline of the same volume, and the rest groups of rabbits are fed with corresponding medicines respectively. After 4 weeks of administration, the rabbits were fasted for 12 hours, and then all hearts were bled, and fasting blood glucose and serum insulin values of the rabbits were measured, and the results are shown in table 2.

TABLE 2 Effect of the Compounds of the present invention on fasting plasma glucose and serum insulin levels in alloxant diabetic rabbits

Group of	Blood sugar value mmoL/L	Serum insulin value (xu/mL)
			Control group 1	16.32±1.43	2.38±0.38
Control group 2	10.40±1.81	4.14±0.52
			Control group 3	12.44±1.37	3.28±0.41
Experimental group	9.18±1.76	4.55±0.46

The data in table 2 show that the compound of the present invention has a significant hypoglycemic effect on the alloxan diabetic rabbits, and also has an effect of promoting the release of serum insulin, compared to the model group.

(3) Influence on fasting plasma glucose in normal mice

Healthy mice were selected and randomly grouped into a normal control group (control group 1, fed with an equivalent amount of physiological saline), a positive drug control group (control group 2, administered acarbose), a lupine group (control group 3, administered lupine), and an experimental group (administered lupine derivative), all animals were fed with normal diet, administered 1 time a day, continuously administered for 1 week for fasting for 12 hours, blood was taken from the orbit, fasting blood glucose concentration was measured, and the results are shown in table 3.

TABLE 3 Effect of Compounds of the invention on fasting blood glucose levels in control Normal mice

Group of	Dosage form	Blood sugar value mmoL/L
			Control group 1	0.25mL/10g	7.72±0.60
Control group 2	3.0g/kg	7.70±0.53
			Control group 3	3.0g/kg	7.52±0.41
Experimental group	3.0g/kg	7.69±0.49

The data in table 3 show that the compounds of the present invention have no significant effect on fasting blood glucose levels in normal mice.

In conclusion, the compound has obvious blood sugar reducing effect on both alloxan diabetic rabbits and adrenergic hyperglycemia mice, has the effect of promoting the release of serum insulin on the alloxan diabetic rabbits, and has no obvious influence on the blood sugar of normal mice.

EXAMPLE 3 toxicological experiments with Compounds of the invention

(1) Half Lethal Dose (LD) of mouse drug₅₀) Measurement of

Healthy mice were taken and randomly grouped into 10 mice each. The compound of the invention is prepared into corresponding concentrations according to 0.1g/kg, 0.2g/kg, 0.4g/kg, 0.8g/kg, 1.6g/kg and 3.2g/kg respectively, the liquid dosage of each time is 0.25mL/10g, and all animals are subjected to intragastric administration for 1 time every day for 7 days continuously. The body weight, diet, appearance, behavior, secretion, excretion, etc. of the animals were observed during the administration period, and no abnormality was found in the above characteristics. On day 7, all animals were sacrificed and dissected, and visceral changes including heart, liver, spleen, lung and kidney were observed without any abnormality. Description of the Compounds of the invention for mouse LD₅₀Is much more than 3.2 g/kg.

(2) Determination of maximum tolerance of mouse

20 healthy mice were selected, half female and half male. Dissolving the compound of the invention in distilled water, dissolving the compound in the distilled water to 50% concentration, and performing intragastric administration after fasting all mice for 12h, wherein the administration is 0.5mL each time and 2 times a day, and the continuous intragastric administration is performed for 7 days. The observation of animal activities, diet, secretion, excreta and the like are normal, and the sacrifice of the anatomy and the observation of the change of internal organs including heart, liver, spleen, lung and kidney and other important organs is not abnormal. The maximum tolerance of the compound of the invention to mice is 24 g/kg.

(3) Toxicity test

Taking male and female halves of healthy rats, randomly dividing into 5 groups, including lupine derivative high, medium and low dosage groups, normal control group, and lupine group. Feeding lupine group with lupine of 7g/kg, 5g/kg and 3g/kg, feeding lupine group with lupine of 3g/kg, feeding normal control group with normal saline of 2mL/100g each time, 1 time per day, observing mouse activity, weighing 1 time per week, adjusting dosage according to weight, continuously administering for 12 weeks, killing part of rat neck after last administration for 4 hr, collecting blood, and testing blood routine, liver function and renal function. Dissecting after blood sampling, observing the shape of the main viscera by naked eyes, sequentially taking out and weighing, and calculating the index of the viscera; the bladder was removed and urine was collected from the bladder for routine examination. The major organs were fixed with formalin and sectioned for histological examination. After each group of remaining animals was observed for 2 weeks, each experimental data was obtained using the same procedure and method as described above.

The results show that the lupine group animals have reduced weight and poor spirit, the phenomena of dilatation and congestion of glomerular capillaries of the animals are found after debranning, and other organs and blood are normal and have no pathological changes; the weight gain of all animals in the lupine derivative group is in a normal state, the blood and urine routine and the liver and kidney functions of the animals are in a normal range, and no obvious difference exists; the macroscopic observation of each main organ has no abnormality; the morphological structure of cells is normal through histological examination, pathological change is avoided, and the visceral organ indexes have no significant difference among groups.

Toxicological experiments show that the mouse LD₅₀Far greater than 3.2g/kg, maximum tolerance of mouseThe receiving amount reaches 24g/kg, the routine blood and the liver and kidney functions of the rat in a toxicological experiment have no obvious influence, and the derivatives have no damage to the viscera of the experimental rat, thereby showing that the lupine derivatives have no obvious toxic or side effect.

Claims (3)

1. A hypoglycemic lupine derivative having the molecular structure of formula (I):

2. the preparation method of the lupine derivative for reducing the blood sugar is characterized by comprising the following steps:

removing water in a reaction vessel, setting the atmosphere of the reaction vessel as an inert gas environment, dissolving lupine and 3, 5-dimethoxy benzyl bromide in an organic solvent, adding alkali, magnetically stirring at 90-130 ℃ for 10-24 hours, detecting by TLC (thin layer chromatography), determining that the reaction is finished, quenching the reaction with ice water, extracting for multiple times, sequentially washing with 10% hydrochloric acid solution, saturated salt solution and water, and drying with anhydrous sodium sulfate to obtain a crude product; performing silica gel column chromatography on the crude product, and performing high performance liquid chromatography purification to obtain a target product;

the feeding molar ratio of the lupine to the 3, 5-dimethoxy benzyl bromide is 1: 1.5-4.5;

the organic solvent is tetrahydrofuran, acetonitrile, diethyl ether, acetone, toluene, benzotrifluoride, dimethyl sulfoxide or ethyl acetate;

the alkali is sodium hydroxide, potassium hydride, sodium carbonate, potassium carbonate or lithium carbonate;

the filler in the silica gel column is alumina or silica gel, and the granularity of the silica gel is 100-400 meshes; gradient eluting the silica gel column eluent by using a petroleum ether-ethyl acetate mixed solvent according to the volume ratio of 20:1, 10:1, 5:1 and 1: 1;

the high performance liquid chromatography uses a C8 reversed phase chromatographic column, water is used as a mobile phase A, a mixed solution of water and acetonitrile with a volume ratio of 15:85 is used as a mobile phase B, and the flow rate is 5-100 mL/min.

3. The process for the preparation of hypoglycemic lupine derivatives according to claim 2, comprising the following steps:

removing water in a reaction vessel, setting the atmosphere of the reaction vessel as an inert gas environment, dissolving 15g of lupine and 40.1g of 3, 5-dimethoxy benzyl bromide in acetone, adding sodium carbonate, stirring for 10 hours at 130 ℃, performing magnetic stirring, detecting by TLC (thin layer chromatography), determining that the reaction is finished, quenching the reaction by using ice water, extracting for multiple times, sequentially washing by using 10% hydrochloric acid solution, saturated salt solution and water, and drying by using anhydrous sodium sulfate to obtain a crude product; and (2) performing silica gel column chromatography on the crude product, wherein a filler in the silica gel column is silica gel, the particle size of the silica gel is 100-200 meshes, gradient elution is performed on silica gel column eluent by adopting a petroleum ether-ethyl acetate mixed solvent according to the volume ratio of 20:1, 10:1, 5:1 and 1:1, the eluent is collected, a C8 reverse phase chromatographic column is used, water is used as a mobile phase A, a mixed solution of water and acetonitrile according to the volume ratio of 15:85 is used as a mobile phase B, the flow rate is 5mL/min, and high performance liquid chromatography purification is performed to obtain the target product.

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