Forskolin derivative as well as preparation method and application thereof

Abstract

The invention discloses a forskolin derivative as well as a preparation method and application thereof, and relates to the technical field of biological medicine, the molecular formula of the forskolin derivative is C32H39N3O9, the molecular weight is 609.67600, and the specific structural formula is shown in the specification. The forskolin derivative prepared by the invention has excellent druggability and pharmacological activity, has a better neuroprotective effect, shows a stronger neuroprotective effect than donepezil and forskolin in an A beta25-35 induced PC12 cell injury model, and has lower toxicity to PC12 cells, so that the forskolin derivative can be used for preparing a neuroprotective drug. The compound can be applied to preparation of Alzheimer's disease medicines in the later period, and is of great significance to prevention and treatment of Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a forskolin derivative, a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's Disease (AD) was first discovered and reported by German doctor Alois Alzheimer in 1906. AD is a common neurodegenerative disease, accounting for about 70% of neurodegenerative disease cases, and has become a global health problem. Although there are drugs for treating AD in the clinic, their effects can only relieve early symptoms of patients and cannot prevent the development of the disease. Therefore, it is of great clinical significance to develop new compounds with neuroprotective effects.

[0003] Forskolin is a natural product with certain neuroprotective activity, but its physicochemical properties and pharmacological activity need to be further optimized. SUMMARY

[0004] The purpose of the present application is to provide a forskolin derivative, a preparation method and application thereof. By modifying the structure of forskolin, a forskolin derivative with excellent drug properties and pharmacological activity is obtained.

[0005] To achieve the above purpose, the present application provides a forskolin derivative, the molecular formula of which is C 32 H 39 N3O9, and the molecular weight is 609.67600; the structural formula of the forskolin derivative is: .

[0006] The present application also provides a preparation method of the forskolin derivative, comprising the following steps: S1, dissolving forskolin in a first organic solvent, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N,N-dimethylpyridine, and carrying out an overnight reaction at room temperature to obtain a first reaction liquid; S2, purifying the first reaction liquid by silica gel chromatography to obtain an intermediate A; S3, mixing the intermediate A, palladium acetate and a second organic solvent, and carrying out a catalytic cyclization reaction at room temperature to obtain a second reaction liquid; S4, the second reaction liquid is sequentially extracted with ethyl acetate, washed with saturated brine, filtered, dried and column chromatographed to obtain compound F10, i.e. the forskolin derivative.

[0007] Preferably, the first organic solvent in S1 is dichloromethane.

[0008] Preferably, the molar ratio of the forskolin, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and the N,N-dimethylpyridine in S1 is 1:1.5~2.5:0.4~0.6.

[0009] Preferably, the molar ratio of the intermediate A and the palladium acetate in S3 is 1:0.05~0.15.

[0010] Preferably, the second organic solvent in S3 is tetrahydrofuran.

[0011] Preferably, the time for the catalytic cyclization reaction in S3 is 8h~12h.

[0012] The application further provides a pharmaceutical composition comprising the above-mentioned forskolin derivative and one or more pharmaceutically acceptable carriers.

[0013] The application further provides an application of the above-mentioned pharmaceutical composition for preventing and / or treating neurodegenerative diseases.

[0014] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0015] In summary, the application provides a forskolin derivative, a preparation method and an application thereof, which have the following advantages compared with the prior art: the forskolin derivative in the application is obtained by modifying the structure of forskolin, has excellent drug properties and pharmacological activity, and has good neuroprotective effect, and shows stronger neuroprotective effect than donepezil and forskolin in an Aβ-induced PC12 cell damage model; and the forskolin derivative has less toxicity to PC12 cells, can be applied to the preparation of Alzheimer's disease drugs in the later stage, and is of great significance for preventing and treating Alzheimer's disease. 25-35 In summary, the application provides a forskolin derivative, a preparation method and an application thereof, which have the following advantages compared with the prior art: the forskolin derivative in the application is obtained by modifying the structure of forskolin, has excellent drug properties and pharmacological activity, and has good neuroprotective effect, and shows stronger neuroprotective effect than donepezil and forskolin in an Aβ-induced PC12 cell damage model; and the forskolin derivative has less toxicity to PC12 cells, can be applied to the preparation of Alzheimer's disease drugs in the later stage, and is of great significance for preventing and treating Alzheimer's disease.

[0016] The technical method of the application will be further described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a cell toxicity detection diagram of the compound F10 in the application based on Example 1 at concentrations of 5μM, 10μM, 20μM, 40μM and 80μM; Figure 2 FIG. 2 is a cell damage degree detection diagram of the compound F10 in the application based on Example 1 at concentrations of 5μM, 10μM and 20μM. DETAILED DESCRIPTION

[0018] The technical method of the application will be further described in detail below with reference to the accompanying drawings and examples.

[0019] The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application or its application and uses.

[0020] In all the examples shown and discussed herein, any specific value should be interpreted as merely exemplary and not as a limitation. Thus, other examples of the exemplary embodiments can have different values.

[0021] Unless otherwise defined, technical or scientific terms used in the present application shall have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] Embodiment 1 A method for preparing a forskolin derivative, comprising the following steps: S1, dissolving 1 mmol of forskolin in 2 mL of dichloromethane solution with a concentration of 1 mmol / mL, and adding 2 mmol of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and 0.5 mmol of N, N-dimethylpyridine (DMAP), and carrying out the reaction overnight at room temperature to obtain a first reaction solution.

[0023] S2, purifying the first reaction solution by silica gel chromatography to obtain an intermediate A with a yield of 83%.

[0024] S3, adding 1 mmol of the intermediate A and 0.1 mmol of palladium acetate to 3 mL of tetrahydrofuran, and carrying out a catalytic cyclization reaction for 8 h at room temperature to obtain a second reaction solution.

[0025] S4, the second reaction solution is sequentially extracted with ethyl acetate, washed with saturated brine, suction filtered, dried, and column chromatographed to obtain a compound F10, i.e., a forskolin derivative.

[0026] The nuclear magnetic resonance data of the compound F10 are as follows: M.p. 200-204°C; yield 46%. 1H NMR (300 MHz, CDC13) δ 8.30 (s, 1H), 7.69 - 7.62 (m, 2H), 7.08 - 7.00 (m, 2H), 5.73 (s, 1H), 5.27 (d, J = 3.8 Hz, 1H), 4.49 (s, 1H), 3.88 (s, 3H), 3.82 (d, J = 2.2 Hz, 1H), 3.59 (d, J = 2.2 Hz, 1H), 2.51 (d, J = 7.8 Hz, 2H), 2.42 - 2.26 (m, 2H), 2.15 (s, 3H), 1.79 (s, 1H), 1.74 (s, 3H), 1.69 - 1.58 (m, 2H), 1.53 (s, 3H), 1.37 (s, 3H), 1.29 (d, J = 4.7 Hz, 1H), 1.18 (d, J = 13.9 Hz, 6H).13C NMR (75 MHz, chloroform-d) δ 200.90, 169.69, 160.31, 159.72, 158.45, 141.35, 129.85, 125.16, 122.57, 114.97, 86.12, 80.03, 75.78, 73.73, 69.98, 69.55, 55.62, 49.16, 43.87, 43.70, 37.18, 34.70, 33.10, 29.71, 25.14, 21.17, 20.95, 19.39. Example 2 A method for preparing a derivative of forskolin, comprising the following steps: S1, dissolving 1 mmol of forskolin in 2 mL of dichloromethane solution with a concentration of 1 mmol / mL, and adding 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.5 mmol of N,N-dimethylpyridine (DMAP), and carrying out the reaction overnight at room temperature to obtain a first reaction solution.

[0027] S2, purifying the first reaction solution by silica gel chromatography to obtain an intermediate A with a yield of 73%.

[0028] S3, adding 1 mmol of the intermediate A and 0.1 mmol of palladium acetate to 3 mL of tetrahydrofuran, and carrying out catalytic cyclization reaction for 12 h at room temperature to obtain a second reaction solution.

[0029] S4, the second reaction liquid is extracted with ethyl acetate, washed with saturated brine, filtered, dried and column chromatography to obtain compound F10, i.e. a forskolin derivative.

[0030] Example 3 A preparation method of a forskolin derivative, comprising the following steps: S1, 1 mmol of forskolin is dissolved in 2 mL of dichloromethane solution with a concentration of 1 mmol / mL, and 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.5 mmol of N,N-dimethylpyridine (DMAP) are added, and the reaction is carried out at room temperature overnight to obtain a first reaction liquid.

[0031] S2, the first reaction liquid is purified by silica gel chromatography to obtain an intermediate A with a yield of 73%.

[0032] S3, 1 mmol of intermediate A and 0.1 mmol of palladium acetate are added to 3 mL of tetrahydrofuran, and a catalytic cyclization reaction is carried out at room temperature for 12 h to obtain a second reaction liquid.

[0033] S4, the second reaction liquid is extracted with ethyl acetate, washed with saturated brine, filtered, dried and column chromatography to obtain compound F10, i.e. a forskolin derivative.

[0034] The medical use of compound F10 in Example 1 is further proved by the following experiments: 1. MTT method for detecting cell toxicity PC12 cells are plated in a 96-well plate at a density of 1×10 4 After 12 hours of incubation, 10% of the complete medium is removed, 180 μL of 1% RPMI 1640 medium is added to each of the 5 drug administration groups, and then 20 μL of compound F10 with concentrations of 5 μM, 10 μM, 20 μM, 40 μM and 80 μM is added to each of the 5 drug administration groups, respectively, and 20 μL of 1% medium is added to the blank control group, and then the plate is placed in an incubator for 24 hours.

[0035] The next day, 20 μL of MTT solution with a concentration of 5 mg / mL is directly added to the 96-well plate of the drug administration group and the blank control group, and then the plate is placed in the dark for 4 hours, after which the medium is removed, and then 150 μL of dimethyl sulfoxide solution is added using a pipette. The plate is wrapped with tin foil and shaken on a shaker for about 10 min. The wavelength of the enzyme marker is set to 490 nm, and then the absorbance of each well is measured, and the cell survival rate of each well is calculated using Excel.

[0036] The results are as follows: Figure 1As shown, the forskolin derivative has less toxicity to PC12 cells, indicating that the forskolin derivative has excellent drug properties.

[0037] 2. MTT method for detecting cell damage degree PC12 cells were plated in 96-well plates at a density of 1×10 4 After 12 hours of incubation, 10% complete medium was removed from the 96-well plates, 178 μL of 1% medium was added to the drug administration group and the control group (drug administration control group, non-drug administration control group, and blank control group), and 20 μL of compound F10 at concentrations of 5 μM, 10 μM, and 20 μM was added to the three drug administration groups, respectively, and 20 μL of forskolin and donepezil at concentrations of 20 μM and 20 μM was added to the two drug administration control groups, respectively. 25-35 After 1 hour, 2 μL of β-amyloid protein (Aβ 25-35 ) at a concentration of 10 μM was added to the drug administration control group, non-drug administration control group, and drug administration group, and 20 μL of 1% medium was added to the blank control group, and the plates were incubated in an incubator for 24 hours.

[0038] The next day, 20 μL of MTT solution at a concentration of 5 mg / mL was directly added to the 96-well plates of the drug administration group and the control group, and the plates were placed in the dark for 4 hours, after which the medium was removed, 150 μL of dimethyl sulfoxide solution was added using a pipette, the plates were wrapped in tin foil, and the plates were shaken for about 10 minutes on a shaker. The wavelength of the enzyme marker was set to 490 nm, and the absorbance of each well was measured, and the cell survival rate of each well was calculated using Excel.

[0039] The results are shown in Table 1 and Figure 1. Figure 2 and Table 1, Figure 2 The statistical significance of the symbol#### in the table is described as follows: the difference between the current data and the blank control group (Control) is extremely statistically significant, and the probability value (p value) is less than 0.0001. The statistical significance of the symbol#### in the table is described as follows: the difference between the current data and the β-amyloid protein group (Aβ 25-35 ) is extremely statistically significant, and the probability value (p value) is less than 0.0001. Compound F10 at a concentration of 20 μM has good neuroprotective effects, and the cell survival rate of the drug administration group with compound F10 at a concentration of 20 μM is 86.79 ± 4.57%, which is higher than that of the drug administration control group.

[0040] Table 1 Cell survival rate of compound F10 at a concentration of 20 μM and the control group

[0041] The above experiments demonstrate that the forskolin derivative (compound F10) prepared in the present application has good neuroprotective effects on Aβ25-35 The compound F10 shows stronger neuroprotective effect than the administration control group (donepezil and forskolin) in the induced PC12 cell damage model, which proves the success of structural modification of the compound F10 and stronger pharmacological activity, and the forskolin derivative has less toxicity to PC12 cells, and can be applied to preparation of drugs for Alzheimer's disease in the later stage, and is of great significance for prevention and treatment of Alzheimer's disease.

[0042] Finally, it should be noted that the above examples are only used to illustrate the technical method of the present application, but not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present application can still be modified or equivalently replaced, and these modifications or equivalent replacements cannot make the modified technical method deviate from the spirit and scope of the technical method of the present application.

Claims

1. A derivative of forskolin, characterized in that, The molecular formula of the forskolin derivative is C 32 H 39 N3O9, and the molecular weight is 609.67600. The structural formula of the forskolin derivative is 。 2. A process for the preparation of a derivative of forskolin according to claim 1, characterized in that, The method comprises the following steps: S1, dissolving the forskolin in a first organic solvent, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N,N-dimethylpyridine, and performing an overnight reaction at room temperature to obtain a first reaction solution; S2, purifying the first reaction solution by silica gel chromatography to obtain an intermediate A; S3, mixing the intermediate A, palladium acetate and a second organic solvent, and performing a catalytic cyclization reaction at room temperature to obtain a second reaction solution; S4, the second reaction solution is sequentially extracted with ethyl acetate, washed with saturated brine, filtered, dried and column chromatographed to obtain the compound F10, i.e., the forskolin derivative.

3. A process for the preparation of a forskolin derivative according to claim 2, characterized in that, The first organic solvent in S1 is dichloromethane.

4. The method of claim 2, wherein the method is characterized by the steps of: The molar ratio of the forskolin, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and the N,N-dimethylpyridine in S1 is 1:1.5-2.5:0.4-0.

6.

5. The method of claim 2, wherein the method is characterized by the steps of: The molar ratio of the intermediate A and the palladium acetate in S3 is 1:0.05-0.

15.

6. The method of claim 2, wherein the method is characterized by the steps of: The second organic solvent in S3 is tetrahydrofuran.

7. The method of claim 2, wherein the method is characterized by the steps of: The catalytic cyclization reaction in S3 is performed for 8-12 hours.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the forskolin derivative as claimed in claim 1 and one or more pharmaceutically acceptable carriers.

9. Use of a pharmaceutical composition, characterized in that The pharmaceutical composition as claimed in claim 8 is used for preventing and / or treating neurodegenerative diseases.

10. The use of a pharmaceutical composition according to claim 9, characterized in that, The neurodegenerative disease is Alzheimer's disease.

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