Cis-trans Diels-Alder adduct and application thereof in preparation of anti-Alzheimer disease medicine

Abstract

The invention belongs to the technical field of medicines, relates to a cis-trans Diels-Alder adduct extracted from white mulberry root-bark, and discloses application of the cis-trans Diels-Alder adduct in preparation of medicines for treating Alzheimer disease. The specific preparation process comprises the following steps: performing alcohol extraction, macroporous adsorption resin column adsorption chromatography and thin-layer chromatography detection on white mulberry root-bark coarse powder, collecting a fraction containing a target compound, and performing silica gel column reduced pressure chromatography, polyamide column chromatography, ODS column chromatography and high performance liquid chromatography on the fraction containing the target compound for separation and purification to obtain the white mulberry root-bark extract. The cis-trans Diels-Alder adduct is subjected to a reaction in the presence of a catalyst; according to the present invention, the Ellman method determination results show that the compound has significant inhibition effect on cholinesterase; the method for preparing the cis-trans Diels-Alder adduct is clear and controllable in operation, and the obtained compound is few in impurity and high in purity and has the potential of being developed into an anti-Alzheimer disease medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a cis-trans Diels-Alder adduct and application thereof in preparation of an anti-Alzheimer's disease drug. BACKGROUND

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, and its main clinical features are progressive memory decline, cognitive dysfunction and behavioral abnormalities, which seriously affect the quality of life of patients and bring heavy burden to families and society. At present, the number of AD patients in the world is continuously increasing, and there is no radical treatment method.

[0003] Studies have shown that the decline of acetylcholinergic system function is one of the important pathological mechanisms of AD, and the activity of butyrylcholinesterase (BChE) gradually increases in AD, which accelerates the hydrolysis of acetylcholine and further destroys cholinergic neurotransmission, aggravating cognitive dysfunction. Therefore, developing a drug that can effectively inhibit the activity of BChE has become one of the important strategies for treating AD.

[0004] Mulberry bark as a traditional Chinese medicinal material, its chemical composition is rich and has a variety of pharmacological activities. However, the components isolated from mulberry bark so far have less research on inhibiting BChE activity and treating AD. It is of great significance to develop new AD treatment drugs to further explore the potential components with BChE inhibitory activity in mulberry bark. SUMMARY

[0005] The present application aims to at least solve one of the problems in the related art. To this end, the main object of the present application is to provide a cis-trans Diels-Alder adduct and a preparation method thereof.

[0006] The present application also provides the use of the cis-trans Diels-Alder adduct in inhibiting the activity of butyrylcholinesterase and treating Alzheimer's disease.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] A cis-trans Diels-Alder adduct, characterized in that the molecular structure formula of the cis-trans Diels-Alder adduct is:

[0009]

[0010] As the same inventive concept, the present application also provides a preparation method of the cis-trans Diels-Alder adduct, comprising the following steps:

[0011] S1: extraction: the mulberry bark powder is placed in an extraction container, 3-10 times the amount of water-ethanol mixed solution is added, and reflux extraction is carried out, the extraction liquid is combined, and concentrated under reduced pressure until there is no alcohol taste, to obtain mulberry bark extract;

[0012] S2: the mulberry bark extract is dispersed and suspended with an appropriate amount of water or 5-15% ethanol, and subjected to macroporous adsorption resin column adsorption chromatography, and gradient elution is carried out with different mass concentrations of ethanol solution in sequence, and after being combined and concentrated respectively, each eluate is obtained;

[0013] S3: thin layer chromatography detection is carried out on each eluate, and the fractions containing the target compounds are combined; the fractions containing the target compounds are subjected to silica gel column chromatography under reduced pressure, and gradient elution is carried out with a dichloromethane-methanol system according to a certain volume ratio, and different elution fractions are collected;

[0014] S4: according to the thin layer chromatography detection results, similar elution fractions are combined, and the elution fractions containing the target compounds are further separated and purified by polyamide column chromatography, ODS column chromatography and high performance liquid chromatography, to obtain the cis-trans Diels-Alder adduct.

[0015] In some specific embodiments, the process conditions of the reflux extraction in step S1 are: reflux extraction at 50-80°C for 2-5 hours, repeated for 2-3 times.

[0016] In some specific embodiments, the mass concentrations of the ethanol solution in step S2 are 10% ethanol, 30% ethanol, 50% ethanol, 60% ethanol, 80% ethanol and 95% ethanol in sequence.

[0017] In some specific embodiments, the volume ratio of dichloromethane-methanol in step S3 is (70-100):(0-30).

[0018] In some specific embodiments, the polyamide column chromatography in step S4 is gradient eluted with a water-ethanol system, and the ODS column chromatography is gradient eluted with a methanol-water system; the high performance liquid chromatography uses a methanol-water system as the mobile phase.

[0019] As the same inventive concept, the present application also provides the use of the cis-trans Diels-Alder adduct and its pharmaceutically acceptable salt as a butyrylcholinesterase inhibitor.

[0020] As the same inventive concept, the present application also provides the use of the cis-trans Diels-Alder adduct and its pharmaceutically acceptable salt in the preparation of a drug for preventing or treating Alzheimer's disease.

[0021] Regarding the expression of the drug:

[0022] Preferably, the compounds of the present application further comprise a pharmaceutically acceptable carrier, such carriers include, but are not limited to, diluents, buffers, suspending agents, emulsifiers, granulating agents, excipients, fillers, binders, sprays, transdermal absorption agents, wetting agents, disintegrating agents, absorption accelerators, surface active agents, coloring agents, flavoring agents or adsorbing carriers.

[0023] The compounds of the present application can be administered alone or in the form of pharmaceutical compositions; the administration route can be oral, parenteral or topical; the pharmaceutical compositions can be formulated in various suitable forms according to the administration route.

[0024] The pharmaceutical compositions of the compounds of the present application can be administered in any of the following ways: orally, spray inhalation, rectally, nasally, buccally, topically, parenterally, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or by means of an implanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.

[0025] When administered orally, the compounds of the present application can be formulated in any orally acceptable formulation, including but not limited to tablets, capsules, aqueous solutions or aqueous suspensions; among them, the carriers used in tablets generally include lactose and corn starch, and lubricants such as magnesium stearate can also be added; the diluents used in capsule formulations generally include lactose and dried corn starch; the aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying agents and suspending agents; optionally, some sweeteners, fragrances or colorants can also be added to the above oral formulations.

[0026] In the present application, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable inorganic or organic salt. The compounds of the present application having acidic groups can form pharmaceutically acceptable salts with alkali metals or alkaline earth metals, preferably but not limited to sodium salt, potassium salt, magnesium salt or calcium salt.

[0027] In addition, it should be noted that the dosage and method of use of the compounds of the present application depend on many factors, including the age, weight, sex, natural health status, nutritional status of the patient, the activity intensity of the compound, the time of administration, the metabolic rate, the severity of the condition and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100 mg / kg body weight per day.

[0028] Compared with the prior art, the present application has at least the following advantages:

[0029] 1) The present application discovers and extracts cis-trans Diels-Alder adducts of formula I from mulberry bark, and experimentally verifies that they have significant butyrylcholinesterase inhibitory activity, thus having the potential to be used as anti-Alzheimer's disease drugs.

[0030] 2) The cis-trans Diels-Alder adduct of the present application inhibits the hydrolysis of acetylcholine by BChE by specifically binding to the active site of the enzyme, changing the spatial conformation of the enzyme, reducing the affinity of the enzyme for the substrate, thereby increasing the level of acetylcholine in the brain, improving cholinergic neurotransmission function, and further relieving the symptoms of cognitive dysfunction in patients with Alzheimer's disease. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present application, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced below.

[0032] Figure 1 HR-ESI-MS spectrum of the compound of formula I in the present application;

[0033] Figure 2 HR-ESI-MS spectrum of the compound of formula I in the present application; 1 H-NMR spectrum of the compound of formula I in the present application;

[0034] Figure 3 HR-ESI-MS spectrum of the compound of formula I in the present application; 13 C-NMR and DEPT spectrum of the compound of formula I in the present application;

[0035] Figure 4 HSQC spectrum of the compound of formula I in the present application;

[0036] Figure 5 HR-ESI-MS spectrum of the compound of formula I in the present application; 1 H- 1 H COSY spectrum of the compound of formula I in the present application;

[0037] Figure 6 HMBC spectrum of the compound of formula I in the present application;

[0038] Figure 7 UV spectrum of the compound of formula I in the present application;

[0039] Figure 8 ECD spectrum of the compound of formula I in the present application;

[0040] Figure 9 Lineweaver-Burk double-reciprocal plot of the compound of formula I in the present application inhibiting butyrylcholinesterase;

[0041] Figure 10 Docking conformation prediction and two-dimensional interaction diagram of the compound of formula I in the present application and butyrylcholinesterase;

[0042] Figure 11 Molecular structure of the compound of formula I in the present application. DETAILED DESCRIPTION

[0043] The application will be further described in conjunction with the accompanying drawings and examples. The following examples are only descriptive and not limiting, and cannot limit the protection scope of the application.

[0044] When expressing a quantity, concentration or other value or parameter either as an amount, a range, or a preferred range or as a preferred upper and lower limit, it is understood that any and every stated quantity, concentration or other value or parameter is also specifically contemplated unless otherwise stated. Numerical ranges include integers within the range, and fractions thereof. Unless otherwise stated, the numerical values listed in this document are stated inclusive of the recited endpoint and all integers and fractions within the range.

[0045] Unless otherwise indicated, all percentages, parts, ratios, etc. stated herein are by weight.

[0046] The materials, methods, and examples herein are illustrative only and, except as specifically stated, are not intended to be limiting.

[0047] Unless otherwise specified, the examples in the examples were carried out under conventional conditions or under the conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used were conventional products that can be purchased on the market.

[0048] In the following examples, the ethanol solution is an aqueous ethanol solution, and 80% ethanol refers to an aqueous ethanol solution with a mass concentration of 80%.

[0049] In the structural study of the following examples, HR-ESI-MS was determined by using a mass spectrometer of LCT 6200 series TOF / 6500 type from Agilent Company, USA, and NMR spectrum was determined by using a superconducting nuclear magnetic resonance instrument (500 MHz 1 H-NMR, 125 MHz 13 C-NMR) from Bruke Company, Switzerland.

[0050] Example 1:

[0051] This example provides a preparation method of a cis-trans Diels-Alder adduct, which comprises the following steps:

[0052] 1) Mulberry bark extraction

[0053] Take 9.3 kg of mulberry bark coarse powder (take dried mulberry bark on the market, crush into coarse powder, and reserve for use), soak in 3 times the amount of 80% ethanol for 1 hour, and then extract at 80°C for 3 times, each time for 3 hours. Filter and combine to obtain about 90 L of extract solution. Concentrate at 60°C under reduced pressure until there is no alcohol smell, and obtain mulberry bark extract extract;

[0054] 2) Extraction treatment and preparation of ethyl acetate extract

[0055] The extract of mulberry bark obtained in step 1) was dispersed and suspended with 10% ethanol to obtain a suspension (about 20 L, solid about 990 g), which was subjected to AB-8 macroporous adsorption resin column (column volume about 4 L) chromatography, and gradient elution was performed with 10% ethanol, 30% ethanol, 50% ethanol, 60% ethanol, 80% ethanol and 95% ethanol, respectively, 3 column volumes were eluted respectively, and after being combined and concentrated to dry weight, each eluate was obtained;

[0056] Each eluate was subjected to HPLC fingerprint analysis, and the 10% ethanol part was marked as component A (580 g), the 30% ethanol part was marked as component B (61.2 g), the 50% to 80% ethanol part was combined as component C (180 g), and the 95% ethanol part was marked as component D (50.8 g);

[0057] Component C (180 g) was subjected to silica gel vacuum column chromatography, and gradient elution was performed with dichloromethane-methanol (100:30) as the eluent, and according to the HPLC fingerprint analysis results, the fractions were combined to obtain components C1-C4;

[0058] Component C3 (93 g) was further subjected to polyamide column chromatography (gradient elution with water-ethanol system), and gradient elution was performed with 40% to 90% ethanol, and after HPLC fingerprint analysis, similar fractions were combined to obtain 6 components C31-C36;

[0059] Component C33 was separated by silica gel vacuum column chromatography (gradient elution with methanol-water system) to obtain four components C331-C334;

[0060] Component C332 was subjected to MPLC crude separation to obtain C332-D1-D14, and C332-D1 was purified by high performance liquid chromatography (HPLC, gradient elution with 45% methanol in 60 min) to obtain a Diels-Alder adduct (8 mg).

[0061] Test Example 1:

[0062] The Diels-Alder adduct prepared in Example 1 was subjected to structural identification by nuclear magnetic resonance (NMR) technology (including 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, etc.), high resolution mass spectrometry (HR-ESI-MS), ultraviolet spectroscopy (UV), circular dichroism (ECD) and other spectroscopic methods, specifically:

[0063] The Diels-Alder adduct is a light yellow amorphous powder, showing positive optical activity There are maximum absorption peaks at 220, 275, and 320 nm in the UV spectrum (e.g. Figure 7 High-resolution HR-ESI-MS gave a quasi-molecular ion peak of m / z 537.1768 [M+H] + (C 29 H 29 O 10 Calculated value 537.1761)(as Figure 1 As shown), its molecular formula is determined to be C 29 H 28 O 10 , the unsaturation is 16. Figure 2 As shown, 1 The H NMR spectrum (Table 1) shows two groups of ABX type aromatic proton signals [δ H 8.16(1H,d,J=8.9Hz,H-27), 6.26(1H,dd,J=8.9,2.3Hz,H-26), 6.13(1H,d,J=2.3Hz,H-24)] and [δ H 6.88 (1H, d, J = 8.4 Hz, H-20), 6.30 (1H, d, J = 2.4 Hz, H-17), 6.19 (1H, dd, J = 8.4, 2.5 Hz, H-19)], 1 isolated aromatic proton signal δ H 5.78 (1H, s, H-5), and related signals of trisubstituted methylcyclohexene ring [δ H 4.11 (1H, d, J = 6.4 Hz, H-8), 5.52 (1H, br s, H-9), 2.46 (1H, dd, J = 17.7, 4.8 Hz, Hα-11), H 2.20 (1H, br d, J = 17.2 Hz, Hβ-11), 3.77 (1H, q, J = 5.0 Hz, H-12), 4.45 (1H, t, J = 5.0 Hz, H-13), 1.86 (3H, br s, H-14)]. In addition, there is a group of vicinal oxygen ethyl signals in the hydrogen spectrum [δ H 4.44 (2H, q, 7.0, H-1′), 1.37 (3H, t, J=7.0 Hz, H-2′)]. Figure 5 As shown, the above hydrogen signal coupling relationship is 1 H- 1 The relevant signals in the H COSY spectrum were established and analyzed by HSQC spectrum (such as Figure 4 as shown); Figure 3 As shown in Table 1, in addition to the carbon signals corresponding to the above hydrogen signals, there are 14 quaternary carbon atoms, including 1 keto carbonyl carbon signal δ C209.6 (C-21), 1 ester carbonyl carbon signal δ C 171.3 (C-7), 7 contiguous oxygen sp 2 Hybrid carbon signals [δ C 156.9 (C-16), 157.6 (C-18), 161.8 (C-4), 162.3 (C-2), 164.5 (C-6), 166.3 (C-25), 166.7 (C-23)], and 5 normal sp 2 Hybrid carbon signals [δ C 94.2 (C-1), 108.5 (C-3), 114.5 (C-22), 123.4 (C-15), 134.4 (C-10)]. As Figure 6 shown in the HMBC spectrum, the HMBC correlation signals between H-14 and C-9, C-10 and C-11, and between H-11 and C-9 and C-10 further confirmed the presence of the methylcyclohexene moiety in the molecule. In addition, there were strong correlation signals between H-5 and C-1, C-3, C-4 and C-6, and weak correlation signals between H-5 and C-2 and C-7, and between H-1' and C-7, which indicated the presence of the ethyl 2,4,6-trihydroxy-substituted benzoate moiety in the molecule. Furthermore, there were HMBC correlation signals between H-17 and H-19 and C-15, between H-20 and C-16 and C-18, and between H-24 and C-22, C-23 and C-25, and between H-26 and C-22 and C-25, which assigned the relevant carbon and hydrogen signals in two 2,4-dihydroxy-trisubstituted benzene ring moieties. The following HMBC correlation signals analysis connected the above moieties together. Firstly, the correlation signals between H-12 and C-15, C-16 and C-20 connected one of the trisubstituted benzene rings to C-12 of the methylcyclohexene ring; secondly, there were strong HMBC correlation signals between H-13 and C-21, and weak correlation signals between H-24 and C-21, which suggested that the other trisubstituted benzene ring was connected to C-13 of the methylcyclohexene ring through the carbonyl C-21; in addition, there were strong correlation signals between H-13 and C-3, which indicated that the ethyl 2,4,6-trihydroxy-substituted benzoate moiety was connected to the methylcyclohexene moiety through the covalent bond between C-3 and C-8. Thus far, the planar structure of the compound was shown as Formula I, which was a Diels-Alder adduct, i.e., the Diels-Alder adduct in the present application was the compound shown as Formula I. Figure 11

[0064]

[0065] ​Diels-Alder type adducts are abundant in mulberry plants. According to the relative configuration of the substituents on the methylcyclohexene ring, they are divided into two main types: cis-trans and all-trans. In the compound of formula I, the coupling constants between H-8 / H-13 and H-12 / H-13 are both 5.0, indicating that the relative configuration of C-8, C-12 and C-13 is cis-trans. The compound of formula I has positive optical activity, and its ECD spectrum mainly presents a positive cotton effect (as shown in Figure 8 According to the principle of exciton chirality, the absolute configuration of the compound is determined to be 8S, 12S, 13R. After searching, the compound is a new compound that has not been reported in the literature.

[0066] Table 1. 500MHz 1 H and 125MHz 13 C NMR data (CD3OD) a

[0067]

[0068]

[0069] The compound of formula I is a light yellow amorphous powder; 158.3 (c 0.04, MeOH); UV (MeOH) λ max (logε): 220 (3.16), 275 (2.35), 320 (2.00) nm; ECD (c 0.0004, MeOH) mdeg (nm): -4.22 (328), +14.64 (269), +16.60 (225), +26.92 (205); HR-ESI-MS: positive ion mode found m / z 537.1768 [M+H] + (C 29 H 29 O 10 The calculated value is 537.1761, and the molecular formula of the compound is determined to be C 29 H 28 O 10 .

[0070] Application Example

[0071] This application example provides the effect of the compound of formula I in the present application on cholinesterase, in particular:

[0072] The Ellman method was used to test the inhibitory activity of the compound of formula I on acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8);

[0073] The test method is: prepare phosphate buffer solution (PBS, 0.1M, pH 8.0), prepare AChE and BChE solutions (0.2u / mL) with PBS, and 5-amino-1,3,4-thiadiazole-2-carboxaldehyde (ATCI), benzotriazole-1-carbon sulfonic acid S-benzyl ester (BTCI) and 5,5'-dithiobis (2-nitrobenzoic acid (DTNB) solution (concentration is 10mM);

[0074] Use methanol to prepare the stock solution (10mM) of the compound of formula I to be tested, and dilute each compound with methanol by a factor of two to prepare five series of concentrations for determining the half-inhibitory concentration (IC 50 ) of each compound. First, mix 160μL PBS, 2μL test sample, 20μL AchE or 20μL BChE and 10μL DTNB in a 96-well plate, and pre-incubate at 37℃ for 10 minutes;

[0075] Then, add 10μL ATCI or BTCI solution to initiate the reaction, and continue to incubate at 37℃ for 25 minutes; measure the absorbance at 412nm during the incubation period.

[0076] Calculate the inhibition rate using the following formula: IR% = [(Ac-As)] / (Ac-Ab)]x100%, where Ab represents the absorbance of the blank control (20μL PBS instead of enzyme solution), Ac represents the absorbance of the control (2μL methanol instead of sample solution), and As represents the absorbance of the sample (10μL PBS instead of ATCI or BTCI solution); all test settings for each sample are set in triplicate; calculate the IC 50 value of each compound by plotting the linear regression curve of the inhibition rate versus its logarithmic concentration.

[0077] The results show that the compound of formula I exhibits good selective inhibitory activity against BChE, with an IC 50 of 6.5μM, and does not show significant inhibition against AChE.

[0078] Further, using the same method as the activity determination, conduct a kinetic study of BChE inhibitory activity using a series of concentrations of substrate BTCI (0.1, 0.2, 0.3, 0.4 and 0.5mM). For each inhibitor, set three respective test concentrations according to its IC 50 value, with a normal group (inhibitor concentration is 0) operated in parallel. After adding the sample reaction, detect the absorbance at 10 minutes, 20 minutes and 25 minutes during the enzymatic reaction.

[0079] The speed of the enzymatic reaction (V) was expressed as the change in absorbance per minute; Lineweaver-Burk plots (double-reciprocal plots) were plotted for the normal group and the different concentration treatment groups (0 μΜ, 3 μΜ, 6 μΜ, 10 μΜ) using the reciprocal of the BTCI concentration (1 / [BTCI]) as the abscissa (x-axis) and the reciprocal of the speed (1 / V) as the ordinate (y-axis), and the Michaelis constant (Km) and maximum speed (Vmax) obtained therefrom were used to detect the inhibition pattern; subsequently, a quadratic curve was established using the inhibitor concentration [I] as the abscissa (x-axis) and the slope of the double-reciprocal line function of each experimental group as the ordinate (y-axis), and the intercept of the curve on the x-axis was equal to -Ki; a quadratic curve was established using the inhibitor concentration [I] as the abscissa and the intercept of the double-reciprocal line function of each experimental group as the ordinate (y-axis), and the intercept of the curve on the x-axis was equal to -aKi; the value of the thermodynamic factor a was calculated from the values of Ki and aKi.

[0080] The results show that, in the Lineweaver-Burk double-reciprocal plot (as shown in Figure 9 ), the compound of formula I gives a set of straight lines with different slopes, all intersecting in the second quadrant or the X-axis, indicating that it is a mixed inhibitor. For this type of interaction, the inhibitor can bind to both the free enzyme (E) and the enzyme-substrate (ES) complex, forming EI and ESI complexes, respectively. The Ki and aKi values of the compound of formula I for BChE are 4.2 and 5.8 μΜ, respectively, and the value of the thermodynamic factor a for BChE is further calculated to be 1.39.

[0081] We studied the interaction mode of the compound of formula I with the active site of the enzyme by means of the Autodock Vina software. The active site of BChE is a deep canyon, including the peripheral anionic site (PAS) located at the edge of the active pocket, the acyl binding pocket (Trp231, Leu286, Val288), the catalytic triad (Ser198, His438, Glu325) and the choline binding pocket (Trp82). According to the literature, the π-π interaction between the inhibitor molecule and the amino acid residues Trp82, Trp231 and Phe329, and the hydrogen bond interaction with His438 play a key role in inhibiting the activity of BChE. The results show that the compound of formula I can be successfully inserted into the binding pocket of BChE, forming different interactions with the residues of the enzyme (as shown in Figure 10The compound of Formula I forms π-π stacking and π-alkyl interactions with the amino residue Trp82, π-π interactions with Phe329 and Typ332, conventional hydrogen bonding interactions with Tyr128 and His438, and π-alkyl hydrophobic interactions with Trp430, among others, showing that it can occupy the catalytic triad and the choline binding pocket.

[0082] The above examples are only used to illustrate the technical solutions of the present application, but not limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. A cis-trans Diels-Alder adduct characterized in that, The molecular structure of the cis-trans Diels-Alder adduct is as follows:

2. A process for the preparation of the cis-trans Diels-Alder adduct of claim 1, characterized in that, The method comprises the following steps: S1: Extraction: Put mulberry bark powder into an extraction container, add 3-10 times of ethanol solution, and perform reflux extraction. Combine the extraction solutions, and concentrate under reduced pressure until there is no alcohol smell to obtain mulberry bark extract. S2: Disperse and suspend the mulberry bark extract in water or 5-15% ethanol solution, and perform macroporous adsorption resin column adsorption chromatography. Gradient elution is performed with ethanol solutions with different mass concentrations in sequence, and each eluate is concentrated after being combined. S3: Perform thin layer chromatography detection on each eluate, and combine the fractions containing target compounds; perform silica gel column chromatography on the fractions containing target compounds under reduced pressure, and gradient elution is performed with a dichloromethane-methanol system according to a certain volume ratio, and different elution fractions are collected. S4: According to the thin layer chromatography detection results, similar elution fractions are combined, and the elution fractions containing target compounds are further separated and purified by polyamide column chromatography, ODS column chromatography and high performance liquid chromatography, to obtain the cis-trans Diels-Alder adduct.

3. The process for the preparation of a cis-trans Diels-Alder adduct according to claim 2, characterized in that, The process conditions of the reflux extraction in step S1 are as follows: reflux extraction is performed at 50-80°C for 2-5 hours, and the extraction is repeated for 2-3 times.

4. The method for preparing the cis-trans Diels-Alder adduct according to claim 2, characterized in that: The mass concentrations of the ethanol solutions in step S2 are 10% ethanol, 30% ethanol, 50% ethanol, 60% ethanol, 80% ethanol and 95% ethanol in sequence.

5. The method for preparing the cis-trans Diels-Alder adduct according to claim 3, characterized in that: The volume ratio of dichloromethane-methanol in step S3 is (70-100):(0-30).

6. The method for preparing the cis-trans Diels-Alder adduct according to claim 2, characterized in that: The polyamide column chromatography in step S4 is gradient elution with a water-ethanol system, the ODS column chromatography is gradient elution with a methanol-water system, and the high performance liquid chromatography uses a methanol-water system as the mobile phase.

7. Use of the cis-trans Diels-Alder adduct and pharmaceutically acceptable salts thereof as claimed in claim 1 as a butyrylcholinesterase inhibitor.

8. Use of the cis-trans Diels-Alder adduct and pharmaceutically acceptable salts thereof as claimed in claim 1 in the preparation of a drug for preventing or treating Alzheimer's disease.

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