Puerarin acid ester derivative with sleep promoting function as well as preparation method and application of puerarin acid ester derivative

Abstract

The invention discloses puerarin acid ester derivatives with a sleep promoting function as well as a preparation method and application of the puerarin acid ester derivatives. The puerarin acid ester derivative has the following molecular structural formula, the L-tryptophan is combined with puerarin through chemical modification, so that a plurality of original hydrophilic groups are esterified and blocked, the lipid solubility is greatly improved, the bioavailability is improved, meanwhile, the content regulation and control of three neurotransmitters, namely 5-hydroxytryptamine, dopamine and noradrenaline are synergistically enhanced, and the bioavailability of the puerarin-L-tryptophan-puerarin-L-tryptophan-puerarin-L-tryptophan-puerarin-L-tryptophan-puerarin-L-tryptophan is improved. Compared with limitation of a single natural component in the aspect of sleep promoting function, the traditional Chinese medicine composition has remarkably enhanced medicine effect and typical application advantages; .

Description

Technical Field

[0001] This invention relates to a derivative of puerarin, and more particularly to a puerarin ester derivative with sleep-promoting function, its preparation method and application, belonging to the pharmaceutical field. Background Technology

[0002] Sleep, an indispensable physiological process for the human body, is directly related to neuroendocrine homeostasis, immune function regulation, and cognitive function maintenance. Modern physiological research has confirmed that the core of sleep-wake cycle regulation lies in the dynamic balance of central neurotransmitters. Among them, serotonin (5-HT), as a key sleep-inducing factor, is one of the core mechanisms of insomnia due to insufficient levels or abnormal metabolism. On the other hand, excessive release of wakefulness neurotransmitters such as dopamine (DA) and norepinephrine (NE) can disrupt sleep balance, leading to symptoms such as difficulty falling asleep, shallow sleep, and frequent awakenings. With increasing social pressure, disordered work and rest schedules, and an aging population, the incidence of insomnia continues to rise, and the limitations of traditional treatments are becoming increasingly apparent. There is an urgent need to develop novel sleep-promoting agents with clear targets and high safety.

[0003] Currently, most clinically used sleep aids are chemically synthesized. While they can take effect quickly by directly inhibiting central nervous system excitation or regulating neurotransmitter receptors, long-term use has significant drawbacks: benzodiazepines are prone to addiction and dependence, while non-benzodiazepines may cause adverse reactions such as daytime sleepiness and cognitive decline, making it difficult to meet the long-term treatment needs of patients with chronic insomnia. Against this backdrop, sleep aids derived from natural products have become a research hotspot in the pharmaceutical field due to their advantages such as "mild effects, few side effects, and conditioning effects." In particular, the active ingredients of traditional Chinese medicine, which are both food and medicine, are more in line with modern health concepts due to their safety as both food and medicine.

[0004] As a classic medicinal and edible plant, kudzu root has attracted widespread attention for the development and application of its active ingredients. Puerarin, the most abundant flavonoid in kudzu root, not only has a clear sedative and tranquilizing effect, but also indirectly alleviates excessive excitation of the central nervous system caused by cerebral ischemia by improving cerebral microcirculation, dilating cerebral blood vessels, and enhancing oxygen and blood supply to brain tissue. Simultaneously, it can regulate central neurotransmitter metabolism, helping to increase 5-HT levels and inhibit excessive release of dopamine (DA) and norepinephrine (NE). Furthermore, puerarin's cardioprotective and antioxidant properties allow it to improve sleep while also supporting cardiovascular health, making it more suitable for middle-aged and elderly individuals or patients with insomnia and underlying cardiovascular diseases. However, when used alone, puerarin suffers from poor lipid solubility, low bioavailability, and limited sedative intensity, which restricts its clinical application. Summary of the Invention

[0005] To address the above technical problems, this invention proposes a puerarin ester derivative with sleep-promoting function, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: As a first aspect of the present invention, a puerarin ester derivative having a sleep-promoting function is provided, having the following molecular structural formula: .

[0007] When used alone as a sleep aid, puerarin suffers from poor lipid solubility, low bioavailability, and limited sedative potency. L-tryptophan is a direct precursor to 5-HT synthesis, but its absorption in vivo is easily inhibited by competition from other neutral amino acids (such as leucine and isoleucine), resulting in a limited effective dose reaching the central nervous system. Therefore, its sleep-promoting effect is limited when used alone, and it is difficult to achieve ideal therapeutic effects when used alone. This invention proposes a puerarin ester derivative through molecular structure design. This substance combines the precursor supply advantage of L-tryptophan with the activity-regulating advantage of puerarin, constructing a novel compound with both high bioavailability and strong sleep-promoting efficacy, demonstrating typical application advantages compared to the limitations of single natural components.

[0008] As a second aspect of the present invention, a method for preparing a puerarin ester derivative with sleep-promoting function is also provided, comprising an esterification reaction of L-tryptophan with puerarin; the reaction process is expressed as follows: After the reaction was completed, the product was separated and purified by alumina column chromatography, with the eluent being a mixture of dichloromethane and methanol in a volume ratio of (10-100):1.

[0009] As a preferred embodiment of the method of the present invention, the relative amounts of raw materials are as follows: the molar ratio of L-tryptophan to puerarin is (1.2-1.8):1.

[0010] As a preferred embodiment of the method of the present invention, the reaction steps include: 1) L-tryptophan reacts in the presence of an acyl chloride reagent to obtain an acyl chloride intermediate; 2) the acyl chloride intermediate is mixed with puerarin and the reaction continues in the presence of an acid-binding agent to generate the target product.

[0011] As a preferred embodiment of the method of the present invention, the acyl chloride reagent is one or more of thionyl chloride, oxalyl chloride, and phosphoryl chloride, and its amount is preferably 1-2 times the molar amount of L-tryptophan.

[0012] As a preferred embodiment of the method of the present invention, the acid-binding agent is selected from one or more of pyridine, triethylamine, and 4-dimethylaminopyridine, and its dosage is preferably 0.75-1.5 times the molar amount of puerarin.

[0013] As a preferred embodiment of the method of the present invention, steps 1) and 2) are both carried out under the protection of an inert atmosphere and anhydrous solvent; the solvent is selected from any one of N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMAc).

[0014] As a preferred embodiment of the method of the present invention, the reaction conditions in step 1) are: stirring at room temperature for 0.5-2 hours; Preferably, the reaction conditions in step 2) are: stirring at room temperature for 8-24 hours.

[0015] As a preferred embodiment of the method of the present invention, after the reaction is completed, the solvent is evaporated, the residue is dissolved in dichloromethane, and the product is then separated and purified by alumina column chromatography. Preferably, the eluent is a dichloromethane-methanol mixture with a volume ratio of (20-50):1.

[0016] As a third aspect of the present invention, an application is provided of a puerarin ester derivative with sleep-promoting function as described above or a puerarin ester derivative with sleep-promoting function prepared by the method described above, which is used to prepare a lipid-soluble improved bioactive preparation; the bioactive preparation includes sleep aid drug preparations, sleep-improving health foods or soothing sleep aid cosmetics.

[0017] The beneficial effects of this invention are as follows: By chemically modifying L-tryptophan and puerarin, multiple hydrophilic groups are esterified and blocked, which greatly improves its lipid solubility and is conducive to improving bioavailability. At the same time, it synergistically enhances the regulation of the content of three neurotransmitters, 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE). Compared with the limitations of single natural ingredients in promoting sleep, it has significantly enhanced efficacy and typical application advantages. Detailed Implementation

[0018] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0019] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention can be purchased commercially.

[0020] Example 1

[0021] 1.5 mmol L-tryptophan was dissolved in 50 mL of anhydrous DMF. Under nitrogen protection, 3 mmol SOCl2 was added dropwise, and the mixture was stirred at room temperature for 0.5 hours. After stirring, the solvent was evaporated to obtain an oily intermediate-acyl chloride. 1 mmol puerarin was mixed with the above oily intermediate, and 30 mL of anhydrous DMF and 1.5 mmol pyridine were added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was evaporated, and the residue was dissolved in 10 mL of dichloromethane. The residue was loaded onto the top of an alumina chromatography column using a wet loading method. Gradient elution was performed using a 20:1 volume ratio of dichloromethane to methanol mixture as the eluent. The elution process was monitored by thin-layer chromatography (TLC), the target component was collected, and the eluent was removed by vacuum evaporation to obtain the target product, namely puerarin ester derivative.

[0022] Example 2

[0023] 1.8 mmol L-tryptophan was dissolved in 50 mL anhydrous DMF. Under nitrogen protection, 2.7 mmol oxalyl chloride was added dropwise, and the mixture was stirred at room temperature for 2 hours. After stirring, the solvent was evaporated to obtain an oily intermediate-acyl chloride. 1 mmol puerarin was mixed with the above oily mixture, and 30 mL anhydrous DMF and 1.5 mmol triethylamine were added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated, and the residue was dissolved in 10 mL of dichloromethane. The residue was loaded onto the top of an alumina chromatography column using a wet loading method. Gradient elution was performed using a 50:1 volume ratio of dichloromethane to methanol mixture as the eluent. The elution process was monitored by thin-layer chromatography (TLC), the target component was collected, and the eluent was removed by vacuum evaporation to obtain the target product, namely the puerarin ester derivative.

[0024] Example 3

[0025] 1.2 mmol L-tryptophan was dissolved in 50 mL of anhydrous DMF. Under nitrogen protection, 1.5 mmol SOCl2 was added dropwise, and the mixture was stirred at room temperature for 1 hour. After stirring, the solvent was evaporated to obtain an oily intermediate-acyl chloride. 1 mmol puerarin was mixed with the above oily intermediate, and 30 mL of anhydrous DMF and 0.75 mmol 4-dimethylaminopyridine were added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, the solvent was evaporated, and the residue was dissolved in 10 mL of dichloromethane. The residue was loaded onto the top of an alumina chromatography column using a wet loading method. Gradient elution was performed using a dichloromethane-methanol mixture with a volume ratio of 30:1. The elution process was monitored by thin-layer chromatography (TLC), the target component was collected, and the eluent was removed by vacuum evaporation to obtain the target product, namely puerarin ester derivative.

[0026] 1H NMR (600 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.05 (s, 1H), 7.87 (d, J =9.5 Hz, 1H), 7.57 – 7.53 (m, 1H), 7.44 – 7.33 (m, 5H), 7.23 – 7.15 (m, 2H),7.10 (td, J = 7.3, 1.0 Hz, 1H), 6.87 (d, J = 9.7 Hz, 1H), 6.62 (s, 1H), 6.05(d, J = 6.8 Hz, 2H), 5.15 (dt, J = 8.8, 2.5 Hz, 1H), 4.91 – 4.86 (m, 2H),4.84 (d, J = 6.1 Hz, 1H), 4.66 (d, J = 5.7 Hz, 1H), 4.07 (p, J = 6.4 Hz, 1H), 3.98 – 3.91 (m, 1H), 3.85 (dt, J = 12.1, 4.6 Hz, 1H), 3.67 (dtd, J = 8.7,4.6, 2.3 Hz, 1H), 3.63 – 3.56 (m, 2H), 3.58 – 3.50 (m, 1H), 3.40 – 3.33 (m,1H), 3.11 (ddd, J = 15.4, 6.4, 0.8 Hz, 1H). Performance Evaluation (1) Prepare medicines Using the puerarin ester derivative prepared in Example 1 as the experimental drug, low, medium and high dose concentration drug groups, medium dose positive control group, and medium dose raw material control group (raw material is L-tryptophan and puerarin in a mass ratio of 1:1) were prepared with physiological saline. In addition, physiological saline was used as the blank control group solution.

[0027] (2) Animal modeling Several SPF-grade C57 female mice, weighing 20-25g, were acclimatized for one week (environmental conditions: temperature 22-25℃, humidity 50-60%, 12h light / 12h dark cycle, free access to food and water) in an SPF-grade animal room, with suitable temperature and humidity, and sufficient food and water throughout the process. The mice were randomly divided into 7 groups of 6 mice each, as follows: normal control group, blank control group, low-dose drug group, medium-dose drug group, high-dose drug group, positive control group, and raw material control group.

[0028] Except for the normal group, the other groups of mice were used to establish an insomnia model by intraperitoneal injection of PCPA. The specific operation was as follows: a PCPA (4-chloro-DL-phenylalanine) suspension with a concentration of 0.1 g / mL was prepared with physiological saline, and the mice in each group were injected intraperitoneally at a standard of 0.3 mL / 100 g body weight for 3 consecutive days; the normal group mice were injected intraperitoneally with the same volume of physiological saline for 3 consecutive days during the same period.

[0029] Criteria for successful modeling: Three days after PCPA injection, mice were observed to have significantly increased activity levels, increased food intake, and abnormal sensitivity to external stimuli such as sound and light, exhibiting restlessness. This indicated successful establishment of the insomnia model. All other groups, except the control group, continued experiments using mice that had successfully modeled the insomnia.

[0030] (3) Drug administration Mice in each group were administered the drug via gavage for 39 days, with administration time uniformly at 8:00 AM each day. The positive control group was administered estazolam solution via gavage at a dose of 120 mg / (kg·bw); the low, medium, and high dose groups were administered the corresponding concentrations of the experimental drug solution via gavage at doses of 100 mg / (kg·bw), 120 mg / (kg·bw), and 150 mg / (kg·bw), respectively; the raw material control group was administered the raw material solution via gavage at a dose of 120 mg / (kg·bw); and the normal group and blank control group were administered an equal volume of physiological saline via gavage.

[0031] (4) Experimental data collection and statistics Twelve hours after the last drug administration, all mice were fasted for 12 hours. Peripheral blood was then collected using the orbital blood sampling method. The blood samples were placed in centrifuge tubes and centrifuged at high speed (3000 r / min, 10 min, 4℃) to separate serum. The levels of 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in the serum were detected using an ELISA reader according to the instructions of the 5-HT, DA, and NE assay kits. The test data for each group were recorded and are shown in Table 1.

[0032] Table 1

[0033] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A puerarin ester derivative with sleep-promoting function, characterized in that, It has the following molecular structural formula: 。 2. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 1, characterized in that, This includes the process of esterifying L-tryptophan with puerarin. After the reaction was completed, the product was separated and purified by alumina column chromatography, with the eluent being a mixture of dichloromethane and methanol in a volume ratio of (10-100):

1.

3. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 2, characterized in that, The relative amounts of raw materials used were as follows: the molar ratio of L-tryptophan to puerarin was (1.2-1.8):

1.

4. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 2 or 3, characterized in that, The reaction steps include: 1) L-tryptophan reacts in the presence of an acyl chloride reagent to obtain an acyl chloride intermediate; 2) the acyl chloride intermediate is mixed with puerarin and the reaction continues in the presence of an acid-binding agent to generate the target product.

5. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 4, characterized in that, The acyl chloride reagent is one or more of thionyl chloride, oxalyl chloride, and phosphoryl chloride, and its amount is preferably 1-2 times the molar amount of L-tryptophan.

6. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 4, characterized in that, The acid-binding agent is selected from one or more of pyridine, triethylamine, and 4-dimethylaminopyridine, and its dosage is preferably 0.75-1.5 times the molar amount of puerarin.

7. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 4, characterized in that, Steps 1) and 2) are carried out under an inert atmosphere and with an anhydrous solvent; the solvent is selected from any one of N,N-dimethylformamide and N,N-dimethylacetamide.

8. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 4, characterized in that, The reaction conditions in step 1) are: stirring at room temperature for 0.5-2 hours; Preferably, the reaction conditions in step 2) are: stirring at room temperature for 8-24 hours.

9. The method for preparing the puerarin ester derivative with sleep-promoting function according to claim 2 or 3, characterized in that, After the reaction was completed, the solvent was evaporated, the residue was dissolved in dichloromethane, and the product was then separated and purified by alumina column chromatography. Preferably, the eluent is a dichloromethane-methanol mixture with a volume ratio of (20-50):

1.

10. The application of a puerarin ester derivative with sleep-promoting function as described in claim 1 or a puerarin ester derivative with sleep-promoting function prepared by the method of any one of claims 2-9, characterized in that, Used to prepare lipid-soluble, improved bioactive preparations; the bioactive preparations include sleep aid drug preparations, sleep-improving health foods, or soothing sleep aid cosmetics.

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