Acyl phloroglucinol compound with 6 / 6 / 6 tricyclic skeleton and application of acyl phloroglucinol compound in preparation of anti-Alzheimer's disease drugs

Abstract

The invention discloses an acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton and application of the acyl phloroglucinol compound in preparation of an anti-Alzheimer's disease medicine, and relates to the technical field of medicines. The invention provides a method for preparing acyl phloroglucinol compounds with 6 / 6 / 6 tricyclic skeletons from hypericum plants through separation means such as silica gel column chromatography, ODS column chromatography and high performance liquid chromatography. The acylphloroglucinol compound has a direct binding effect with a nuclear receptor RXR alpha, regulates the expression of ABCA1 protein by targeting RXR alpha up-regulation, plays a role in promoting A beta removal, and can be applied to preparation of anti-Alzheimer's disease drugs taking the nuclear receptor RXR alpha as a target spot.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an acylphloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton and application thereof in the preparation of an anti-Alzheimer's disease drug. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment, which seriously endangers the health of the elderly. Its clinical treatment still faces severe challenges. Abnormal deposition of β-amyloid protein (Aβ) in the brain is one of the key factors in the pathogenesis of AD. Inhibiting Aβ production or promoting Aβ clearance is an important strategy for the treatment of AD. β-Secretase (BACE1) is the rate-limiting enzyme for Aβ production. Retinoid receptor-α (RXRα) agonists show great potential in promoting the clearance of Aβ. Retinoid X receptor-α (RXRα) is an ideal molecular target for anti-tumor drug research. (McConlogue L, Buttini M, Anderson JP, et al. Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice [J]. J Biol Chem, 2007, 282 (36): 26326-34.) LGD1069 (Targretin) targeting RXRα has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. Therefore, it is of great significance to search for highly effective and low-toxic anti-Alzheimer's disease drugs from natural products targeting RXRα.

[0003] Polycyclic Polyprenylated Acyl Phloroglucinols (PPAPs) are unique components of plants of the genus Hypericum in the family Garcinia. PPAPs are not only novel and diverse in structure, but also have a wide range of biological activities, such as anti-tumor, anti-depression, anti-virus, antioxidant, and antibacterial (Guo Jieru, Zhu Hucheng, Tang Fang, Zhang Jinwen. Research Progress on Natural Polycyclic Polyprenylated Acyl Phloroglucinol Derivatives [J]. Chinese Herbal Medicine, 2017(10):2129-2145.). The 6 / 6 / 6 tricyclic skeleton acylphloroglucinol compounds are PPAPs with pyran-fused 6 / 6 / 6 ring symmetry structures, and all exist in the form of enantiomers. They use mianmacid as the parent nucleus, and the C-5 position of the parent nucleus is all dimethyl substituted. Their characteristic structure is that two isopentenyl groups are cyclized to form a unique linear or angular 6 / 6 / 6 tricyclic symmetry structure. These compounds enrich the structural diversity of PPAPs. The chemical composition of Hypericum plants was studied in order to obtain more anti-Alzheimer's disease lead compounds targeting RXRα with novel skeleton structures. Summary of the invention

[0004] The first object of the present invention is to provide an acylphloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton.

[0005] The second object of the present invention is to provide a method for preparing acyl phloroglucinol compounds with a 6 / 6 / 6 tricyclic skeleton.

[0006] The third object of the present invention is to provide the use of acyl phloroglucinol compounds with a 6 / 6 / 6 tricyclic skeleton in the preparation of anti-Alzheimer's disease drugs.

[0007] The fourth object of the present invention is to provide an application of acyl phloroglucinol compounds with a 6 / 6 / 6 tricyclic skeleton as anti-Alzheimer's disease drugs targeting the nuclear receptor RXRα.

[0008] The structural formula of the acyl phloroglucinol compound of the 6 / 6 / 6 tricyclic skeleton is as follows:

[0009]

[0010] The preparation method of the acyl phloroglucinol compound of the 6 / 6 / 6 tricyclic skeleton comprises the following steps:

[0011] 1) Cut the whole herb of the Herba Achyranthis Bidens radiata into pieces and extract it with methanol for 2 to 4 times, each time for 12 to 24 hours, collect the filtrate and concentrate it under reduced pressure to obtain an extract, mix the sample with silica gel and then chromatograph it on a normal phase silica gel column, using dichloromethane, ethyl acetate and methanol as mobile phases to obtain three polar fractions;

[0012] In step 1), the cold soaking extraction can be performed 2 to 3 times, each time for 18 to 24 hours.

[0013] 2) The ethyl acetate fraction obtained in step 1) was subjected to MCI gel column chromatography, and gradient eluted with methanol and water to obtain 8 fractions (Fr.1 to Fr.8);

[0014] In step 2), the volume ratio of methanol to water can be 1:1, 6:4, 7:3, 8:2, 9:1, 1:0 respectively.

[0015] 3) The fraction Fr.5 in step 2) was subjected to silica gel column chromatography again, and gradient eluted with petroleum ether and ethyl acetate to obtain 10 fractions (Fr.5A to Fr.5J);

[0016] In step 3), the volume ratio of petroleum ether to ethyl acetate can be 20:1, 10:1, 5:1, 2:1, 1:1, respectively.

[0017] 4) The fraction Fr.5D in step 3) was subjected to silica gel column chromatography, eluted with petroleum ether and acetone in a gradient manner, to obtain 7 fractions (Fr.5D1 to Fr.5D7);

[0018] In step 4), the volume ratio of petroleum ether to ethyl acetate can be 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, respectively.

[0019] 5) The fraction Fr.5D4 in step 4) is separated by HPLC to obtain 4 fractions (Fr.5D4A to Fr.5D4D);

[0020] In step 5), the HPLC high performance liquid chromatography method can be ODS Rp-18, 5 μm, 250×10 mm, 47% ACN / H 2 O, 5mL / min.

[0021] 6) The fraction Fr.5D4B in step 5) was separated and prepared by HPLC to obtain compound 12.

[0022] In step 6), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H 2 O, 5mL / min.

[0023] 7) The fraction Fr.5D4D in step 5) was separated and prepared by HPLC to obtain compounds 10 and 11.

[0024] In step 7), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H 2 O, 5mL / min.

[0025] 8) The fraction Fr.5G in step 3) was subjected to silica gel column chromatography, eluted with dichloromethane and methanol gradient to obtain 8 fractions (Fr.5G1 to Fr.5G8)

[0026] In step 8), the volume ratio of dichloromethane to methanol can be 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, respectively.

[0027] 9) The fraction Fr.5G6 in step 8) was separated and prepared by HPLC to obtain compounds 5 and 6.

[0028] In step 9), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H 2 O, 5mL / min.

[0029] 10) The fraction Fr.5G7 in step 8) was separated by HPLC to obtain compounds 4 and 9.

[0030] In step 10), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H 2 O, 5mL / min.

[0031] 11) The fraction Fr.6 in step 2) was subjected to silica gel column chromatography again, and gradient eluted with petroleum ether and ethyl acetate to obtain 12 fractions (Fr.6A to Fr.6L);

[0032] In step 11), the volume ratio of petroleum ether to ethyl acetate can be 50:1, 20:1, 10:1, 5:1, 2:1 respectively.

[0033] 12) The fraction Fr.6G in step 11) was subjected to silica gel column chromatography, eluted with dichloromethane and methanol in a gradient manner, to obtain 6 fractions (Fr.6G1 to Fr.6G6);

[0034] In step 12), the volume ratio of dichloromethane to methanol can be 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 respectively.

[0035] 13) The fraction Fr.6G3 in step 12) is separated by HPLC to obtain Fr.6G3A-F.

[0036] In step 6), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 60% ACN / H 2 O, 5mL / min.

[0037] 14) The fraction Fr.6G3E in step 13) was separated and prepared by HPLC to obtain compounds 1, 2 and 3.

[0038] In step 14), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 55% ACN / H 2 O, 5mL / min.

[0039] 15) The fraction Fr.6H in step 11) was subjected to silica gel column chromatography, eluted with dichloromethane and methanol in a gradient manner, to obtain 9 fractions (Fr.6H1 to Fr.6H9;

[0040] In step 15), the volume ratio of dichloromethane to methanol can be 200:1, 50:1, 25:1, 10:1, 5:1, 2:1, respectively.

[0041] 16) fraction Fr.6H7 in step 15) was separated by HPLC to obtain compounds 7 and 8;

[0042] In step 16), the HPLC high performance liquid chromatography method can be COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 55% ACN / H 2 O, 5mL / min.

[0043] 17) performing chiral separation on the compound separated in the above step, IG column, 5μm, 250×10mm, 50%ACN / H 2 O, 5mL / min, to obtain compounds 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, 11b, 12a, and 12b.

[0044] The structural formula of the compound 1-12 may be:

[0045]

[0046] The acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton can be used in the preparation of anti-Alzheimer's disease drugs.

[0047] The acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton can be used as an anti-Alzheimer's disease drug targeting the nuclear receptor RXRα.

[0048] The present invention obtains acyl phloroglucinol compounds with a 6 / 6 / 6 tricyclic skeleton from plants of the genus Hypericum by separation methods such as silica gel column chromatography, ODS column chromatography, and high performance liquid phase preparative chromatography; the extraction and separation method is simple, and the raw materials are easily available. The acyl phloroglucinol compounds have a direct binding effect with the nuclear receptor RXRα, and the compounds can upregulate the expression of the regulated ABCA1 protein by targeting RXRα, thereby promoting the clearance of Aβ, and can be applied to anti-Alzheimer's disease drugs targeting the nuclear receptor RXRα or as lead compounds for the development of anti-Alzheimer's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The MTT method was used to detect the effects of compounds 1-12 at a concentration of 20 μM on the viability of BV2 cells (drug action for 24 hours).

[0050] Figure 2 The effects of compounds 1-12 on RXRα transcriptional activity were evaluated by luciferase dual reporter gene assay.

[0051] Figure 3 Western Blotting technique was used to detect the effects of compounds 1a-12b on the expression of ABCA1 protein in BV2 cells.

[0052] Figure 4 The figure shows the Western Blotting results of compounds 1a, 5a and 11b at different concentrations.

[0053] Figure 5 The fluorescence spectrum of compound 1a binding to RXRα-LBD. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] Embodiment 1:

[0056] Preparation and structural identification of an acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton

[0057] 1. Preparation of compounds:

[0058] Step 1: Air-dried Atractylodes macrocephala (7.5 kg) was soaked in methanol (3×75 L) three times at room temperature. The crude extract (800.0 g) was separated by silica gel column chromatography, eluted with dichloromethane, ethyl acetate and methanol, and three fractions (Fr.AC) were obtained.

[0059] Step 2: The ethyl acetate eluted fraction from step 1 was filtered through an MCI gel column (MeOH-H 2 O from 5:5 to 10:0, v / v), and Fr.B (160.9 g) was divided into 8 fractions (Fr.B1-8) by adjusting the ratio of methanol and water.

[0060] Step 3: Fr.B5 (19.63 g) from step 2 was fractionated on silica gel using a petroleum ether-ethyl acetate gradient elution system (20:1 to 1:1, v / v) to obtain 10 fractions: Fr.B5.1-5.10.

[0061] Step 4: Fr.B5.4 (0.3981 g) from step 3 was purified by pre-HPLC (ACN-H 2 O 47:53, v / v) was further separated to obtain Fr.B5.4.1-4.

[0062] Step 5: The fraction Fr.B5.4.2 (0.0127 g) from step 4 was chromatographed on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10×250 mm) column (ACN-H 2 O 50:50, v / v) to obtain compound 12.

[0063] Step 6: Fr.B5.4.4 (0.0200 g) from step 4 was purified by chromatography on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10 × 250 mm) column (ACN-H 2 O 50:50, v / v) to obtain compounds 10 and 11.

[0064] Step 7: Fr.B5.7 (2.9390 g) prepared in step 3 was fractionated on silica gel using a dichloromethane-methanol elution system (100:0 to 2:1, v / v) to obtain 8 fractions: Fr.B5.7.1-5.7.8.

[0065] Step 8: The fraction Fr.B5.7.6 (0.2800 g) from step 7 was purified by chromatography on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10×250 mm) column (ACN-H 2 O 50:50, v / v) to obtain compounds 5 and 6.

[0066] Step 9: B5.7.7 (0.1682 g) from step 7 was chromatographed on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10 × 250 mm) column (ACN-H 2 O 50:50, v / v) to afford compounds 4 and 9.

[0067] Step 10: Fr.B6 (22.45 g) prepared in step 2 was fractionated on silica gel using a petroleum ether-ethyl acetate gradient elution system (50:1 to 2:1, v / v) to obtain 12 fractions: Fr.B6.1-6.12.

[0068] Step 11: Fr.B6.7 (1.6743 g) prepared in Step 10 was fractionated on silica gel using a dichloromethane-methanol elution system (100:0 to 2:1, v / v) to obtain six fractions: Fr.B6.7.1-6.7.6.

[0069] Step 12: The fraction Fr.B6.7.3 (1.1620 g) from step 11 was purified by pre-HPLC (ACN-H 2 O60∶40, v / v) was further separated to obtain Fr.B6.7.3.1-6.

[0070] Step 13: The fraction Fr.B6.7.3.5 (0.1984 g) from step 12 was chromatographed on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10×250 mm) column (ACN-H 2 O 55:45, v / v) to obtain compounds 1, 2 and 3.

[0071] Step 14: Fraction Fr.B6.8 (0.5576 g) obtained in step 10 was fractionated on silica gel using a dichloromethane-methanol elution system (200:1 to 2:1, v / v) to obtain nine fractions: Fr.B6.8.1 to Fr.B6.8.9.

[0072] Step 15: The fraction Fr.B6.8.3.5 (0.0566 g) from step 14 was chromatographed on a COSMOSIL 5C18-AR-Ⅱ (5 μm, 10×250 mm) column (ACN-H 2 O 60:40, v / v) to afford compounds 7 and 8.

[0073] Step 16: Compound 1-12 in the above step is used Chiral analysis was performed on an IG column (ACN-H2O 40:60, v / v) to obtain 1a(6.0 mg), 1b(6.0 mg), 2a(10.0 mg), 2b(9.3 mg), 3a(9.1 mg) and 3b(10.0 mg), 4a(2.4 mg), 4b(1.8 mg), 5a(3.3 mg), 5b(3.1 mg), 6a(3.0 mg) and 6b(3.3 mg), 7a(1.2 mg), 7b(1.2 mg), 8a(1.2 mg), 8b(1.2 mg), 9a(4.0 mg) and 9b(4.4 mg), 10a(3.1 mg), 10b(3.0 mg), 11a(3.4 mg), 11b(3.4 mg), 12a(1.4 mg) and 12b(1.9 mg).

[0074] Figure 1 The MTT assay was used to detect the effects of compounds 1-12 on BV2 cell viability at a concentration of 20 μM (drug action for 24 h). Figure 2 The effects of compounds 1-12 on RXRα transcriptional activity were evaluated by luciferase dual reporter gene assay. Figure 3 The effects of compounds 1a-12b on the expression of ABCA1 protein in BV2 cells were detected by Western Blotting technique. Figure 4 The Western Blotting results of compounds 1a, 5a and 11b at different concentrations are shown. Figure 5 The fluorescence spectrum of compound 1a binding to RXRα-LBD is given.

[0075] 2. Structural identification of compounds:

[0076] 1: Colorless oil; UV(CH 3 OH)λ max (logε)=227(3.85),244(3.92),267(3.69),328(3.84)nm; for 1 H NMR (600 MHz) and 13 C NMR (150MHz) data see Table S1; HRESIMS[M+H] + m / z377.2317 (calcd for C 23 H 33 O 4 + 377.2323);

[0077] 1a: +122.8(c 5.00,CH 3 OH); ECD(CH3 OH)λ(Δε) 233 (+1.38), 252 (+0.47), 270 (+0.89), 292 (+0.71), 321 (+0.91) nm;

[0078] 1b: -116.8 (c 5.00, CH 3 OH); ECD(CH 3 OH)λ(Δε) 232 (-1.41), 254 (-0.46), 274 (-0.91), 294 (-0.71), 318 (-1.04) nm;

[0079] 2: Colorless oil; UV(CH 3 OH)λ max (logε) = 227 (3.79), 242 (3.81), 270 (3.67), 328 (3.87) nm; for 1 H NMR(600MHz) and 13 C NMR(150MHz) data see Table S1; HRESIMS[M + H] + m / z 391.2475 (calcd for C 23 H 33 O 4 + 391.2479);

[0080] 2a: +113.2 (c 5.00, CH 3 OH); ECD(CH 3 OH)λ(Δε) 231 (+1.59), 254 (+0.63), 274 (+1.29), 290 (+0.82), 321 (+1.13) nm;

[0081] 2b: -88.4 (c 5.00, CH 3 OH); ECD(CH 3 OH)λ(Δε) 228 (-1.16), 254 (-0.02), 273 (-0.31), 289 (-0.19), 323 (-0.59) nm;

[0082] 3: Colorless oil; UV(CH 3 OH)λ max (logε) = 227 (3.89), 242 (3.94), 270 (3.76), 328 (3.98) nm; for 11H NMR (600 MHz) and 13 13C NMR (150 MHz) data see Table S1; HRESIMS [M+H] + m / z 391.2475 (calcd for C 23 H 33 O 4 + 391.2479);

[0083] 3a: +128.4 (c 5.00, CH 3 OH); ECD (CH 3 OH) λ (Δε) 229 (+1.74), 252 (+0.94), 274 (+1.29), 299 (+0.93), 324 (+1.04) nm;

[0084] 3b: -121.2 (c 5.00, CH 3 OH); ECD (CH 3 OH) λ (Δε) 228 (-1.16), 251 (-0.04), 273 (-0.64), 295 (-0.37), 323 (-0.57) nm;

[0085] 4: Colorless oil; UV (CH 3 OH) λ max (log ε) = 227 (3.71), 242 (3.71), 275 (3.58), 327 (3.77) nm; for 1 1H NMR (600 MHz) and 13 13C NMR (150 MHz) data see Table S1; HRESIMS [M+H] + m / z 393.2265 (calcd for C 23 H 33 O 4 + 393.2272);

[0086] 4a: +81.78 (c 1.20, CH 3 OH); ECD (CH 3 OH) λ (Δε) 232 (+1.66), 253 (+0.93), 272 (+1.30), 298 (+0.99), 319 (+1.30) nm;

[0087] 4b: -62.5 (c 1.60, CH 3 OH); ECD (CH 3 OH) λ(Δε) 234 (-0.72), 252 (-0.41), 270 (-0.77), 296 (-0.63), 316 (-0.95) nm;

[0088] 5: Colorless oil; UV (CH 3 OH) λ max (logε) = 226 (3.88), 242 (3.86), 275 (3.74), 327 (3.96) nm; for 1 1H NMR (600 MHz) and 13 13C NMR (150 MHz) data see Table S1; HRESIMS [M + H] + m / z 407.2427 (calcd for C 23 21 33 H 4 + 407.2428);

[0089] 5a: +91.8 (c 2.20, CH 3 OH); ECD (CH 3 OH) λ(Δε) 232 (+1.86), 252 (+0.99), 274 (+1.66), 299 (+1.08), 318 (+1.31) nm;

[0090] 5b: -120.9 (c 2.20, CH 3 OH); ECD (CH 3 OH) λ(Δε) 232 (-1.81), 252 (-0.86), 273 (-1.86), 297 (-1.18), 317 (-1.42) nm;

[0091] 6: Colorless oil; UV (CH 3 OH) λ max (logε) = 226 (3.87), 242 (3.85), 275 (3.73), 327 (3.95) nm; for 1 1H NMR (600 MHz) and 13 13C NMR (150 MHz) data see Table S1; HRESIMS [M + H] + m / z 407.2427 (calcd for C 23 2133 O 4 + 407.2428);

[0092] 6a: +88.0 (c 2.00, CH 3 OH); ECD(CH 3 OH) λ(Δε) 237 (+1.92), 258 (+1.19), 274 (+1.31), 292 (+1.22), 320 (+1.80) nm;

[0093] 6b: -107.4 (c 2.07, CH 3 OH); ECD(CH 3 OH) λ(Δε) 238 (-1.51), 258 (-0.92), 277 (-1.07), 292 (-1.05), 324 (-1.82) nm;

[0094] 7: Colorless oil; UV(CH 3 OH) λ max (logε) = 224 (3.63), 243 (3.64), 325 (3.63) nm; for 1 1H NMR(600 MHz) and 13 13C NMR(150 MHz) data see Table S1; HRESIMS[M+H] + m / z 407.2427 (calcd for C 23 H 33 O 4 + 407.2428);

[0095] 7a: +62.5 (c 0.80, CH 3 OH); ECD(CH 3 OH) λ(Δε) 234 (+1.61), 256 (+0.97), 275 (+1.32), 308 (+0.71), 322 (+0.84) nm;

[0096] 7b: -40.0 (c 0.80, CH 3 OH); ECD(CH 3 OH) λ(Δε) 234 (-0.54), 250 (-0.45), 269 (-0.72), 322 (-0.46) nm;

[0097] 8: Colorless oil; UV(CH 3 OH)λ max (logε) = 224(3.60), 243(3.60), 325(3.53) nm; for 1 HNMR(600MHz) and 13 C NMR(150MHz) data see Table S1; HRESIMS[M+H] + m / z 407.2427 (calcd for C 23 H 33 O 4 + 407.2428);

[0098] 8a: +45.0 (c 0.80, CH 3 OH); ECD(CH 3 OH)λ(Δε) 244(+1.11), 259(+0.84), 268(+0.95), 289(+0.58), 323(+0.76) nm;

[0099] 8b: -55.0 (c 0.80, CH 3 OH); ECD(CH 3 OH)λ(Δε) 240(-0.69), 253(-0.57), 265(-0.59), 306(-0.38), 323(-0.81) nm;

[0100] 9: Colorless oil; UV(CH 3 OH)λ max (logε) = 225(3.79), 245(3.0), 274(3.58), 325(3.79) nm; for 1 H NMR(600MHz) and 13 C NMR(150MHz) data see Table S1; HRESIMS[M+H] + m / z393.2265 (calcd for C 23 H 33 O 4 + 393.2272);

[0101] 9a: +94.5 (c 2.67, CH 3 OH); ECD(CH 3OH) λ (Δε) 225 (+5.72), 253 (-0.27), 276 (+1.27), 302 (+0.35), 341 (+1.03) nm;

[0102] 9b: -112.5 (c 2.93, CH 3 OH); ECD (CH 3 OH) λ (Δε) 225 (-6.64), 252 (+1.00), 276 (-1.42), 309 (-0.33), 345 (-1.20) nm;

[0103] 10: Colorless oil; UV (CH 3 OH) λ max (logε) = 226 (3.92), 249 (3.84), 325 (4.03) nm; for 1 HNMR (600 MHz) and 13 C NMR (150 MHz) data see Table S1; HRESIMS [M + H] + m / z 407.2427 (calcd for C 23 H 33 O 4 + 407.2428);

[0104] 10a: +143.8 (c 2.27, CH 3 OH); ECD (CH 3 OH) λ (Δε) 225 (+9,73), 253 (-0.30), 275 (+2.27), 303 (+0.56) 344 (+1.55) nm;

[0105] 10b: -194.5 (c 2.07, CH 3 OH); ECD (CH 3 OH) λ (Δε) 225 (-9.33), 252 (+0.54), 275 (-2.07), 304 (-0.35), 344 (-1.28) nm;

[0106] 11: Colorless oil; UV (CH 3 OH) λ max (logε) = 226 (3.82), 249 (3.87), 325 (3.85) nm; for 1 HNMR (600 MHz) and 1313C NMR (150 MHz) data see Table S1; HRESIMS [M+H] + m / z 407.2427 (calcd for C 23 H 33 O 4 + 407.2428);

[0107] 11a: +131.0 (c 2.00, CH 3 OH); ECD (CH 3 OH) λ (Δε) 225 (+8.64), 253 (-0.16), 272 (+1.65), 300 (+0.57) 343 (+1.43) nm;

[0108] 11b: -144.7 (c 2.27, CH 3 OH); ECD (CH 3 OH) λ (Δε) 225 (-9.16), 253 (+0.10), 277 (-1.94), 301 (-0.51), 343 (-1.58) nm;

[0109] 12: Colorless oil; UV (CH 3 OH) λ max (logε) = 243 (3.62), 321 (3.62) nm; for 1 1H NMR (600 MHz) and 13 13C NMR (150 MHz) data see Table S1; HRESIMS [M+H] + m / z 409.2219 (calcd for C 23 H 33 O 4 + 409.2221);

[0110] 12a: +60.0 (c 0.93, CH 3 OH); ECD (CH 3 OH) λ (Δε) 229 (+4.03), 253 (+0.99), 277 (+1.35), 306 (+0.43) 336 (+0.93) nm;

[0111] 12b: -53.7 (c 1.27, CH 3 OH); ECD (CH 3OH)λ(Δε)229(-2.76),252(+0.63),274(-1.07),304(-0.09),335(-0.49)nm;

[0112] Compounds 1-4 1 H(600MHz)and 13 C NMR (150 MHz) data (in CDCl 3 )See Table 1.

[0113] Table 1

[0114]

[0115]

[0116] Compounds 5-8 1 H(600MHz)and 13 C NMR (150 MHz) data (in CDCl 3 )See Table 2.

[0117] Table 2

[0118]

[0119]

[0120] Compounds 5-8 1 H(600MHz)and 13 C NMR (150 MHz) data (in CDCl 3 )See Table 3.

[0121] Table 3

[0122]

[0123] Embodiment 2:

[0124] The compound involved in the present invention has an anti-Alzheimer's disease effect with nuclear receptor RXRα as a target.

[0125] 1 Materials and instruments

[0126] 1.1 Experimental cells: mouse microglial BV2 cells;

[0127] 1.2 Experimental instruments:

[0128] Inverted microscope: Olympus Leica, Japan;

[0129] Cell culture incubator: Thermo Corporation, USA;

[0130] Millipore ultrapure water instrument Millipore Corporation, USA;

[0131] High-speed refrigerated centrifuge, BeckMan Coulter Co., Ltd., USA;

[0132] Micropipette: Eppendorf, USA;

[0133] Protein vertical electrophoresis instrument, Bio-Rad, USA;

[0134] Disposable culture dishes Orange Scientific, Belgium;

[0135] 48-well culture plate Orange Scientific, Belgium;

[0136] 96-well culture plate Orange Scientific, Belgium;

[0137] Vertical automatic electric pressure steam sterilizer Shanghai Shen'an Medical Equipment Factory;

[0138] Electric constant temperature water bath Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0139] ELISA instrument Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0140] WH-3 Vortex Analyzer Shanghai Huxi Analytical Instrument Co., Ltd.;

[0141] Clean bench Suzhou Antai Air Technology Co., Ltd.;

[0142] 1.3 Main Reagents

[0143] DMEM (High glucose): Hyclone;

[0144] Fetal bovine serum: Hyclone;

[0145] Pancreatic digestion solution: Hydrone;

[0146] Opti-MEM:Hyclone;

[0147] BCAKit: Thermo Scientific, USA;

[0148] 1.4 Solution preparation

[0149] ①RIPA cell lysis buffer:

[0150] 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, 1mM PMSF, 5μg / mL Aprotenin, 5μg / mL Leupeptasin.

[0151] ②PBS buffer solution:

[0152] 137mMNaCl,2.7mMMKCl,10mMNa 2 HPO 4 ,2mM KH 2 PO 4 , adjust the pH to 7.4 with hydrochloric acid.

[0153] ③ Protein electrophoresis related reagents:

[0154] 1) 1.0M Tris-HCl buffer

[0155] Take 12.12 g of Tris, add hydrochloric acid dropwise to adjust the pH to 6.8, add deionized water, make up to 100 mL, adjust the pH to 6.8 again, sterilize at high temperature and store at room temperature.

[0156] 2) 1.5M Tris-HCl buffer

[0157] Take 18.2 g of Tris, add hydrochloric acid dropwise to adjust the pH to 8.8, add deionized water, make up to 100 mL, adjust the pH to 8.8 again, sterilize at high temperature and store at room temperature.

[0158] 3) Electrophoresis buffer

[0159] Take 3.03 g of Tris-HCl, 18.77 g of glycine, and 1 g of SDS and dissolve them in 1000 mL of distilled water.

[0160] 4) 2×SDS loading buffer

[0161] dd 2 O 6mL, 0.5M Tris (pH 6.8) 5mL, 20% SDS 4mL, bromophenol blue 4mg, 50% glycerol 4mL, β-mercaptoethanol (2-mercaptoethanol) 1mL.

[0162] 5) 1× TBST

[0163] 10mM Tris-HCl (pH 7.5), 150mM NaCl, 0.1% Tween-20.

[0164] 6) Blocking solution

[0165] 5% (W / V) skim milk powder was dissolved in 1× TBST.

[0166] 7) Electroporation

[0167] Dissolve 3.03 g of Tris and 14.4 g of glycine in 900 mL of distilled water, and finally add 100 mL of methanol solution and mix well.

[0168] 8) 10% SDS

[0169] 10g SDS, add distilled water to 100mL, dissolve in a 50℃ water bath, and store at room temperature.

[0170] 9) 10% ammonium persulfate (AP)

[0171] 0.1g ammonium persulfate, add 1.0mL ultrapure water, dissolve, and store at 4℃.

[0172] 2 Cytotoxic effects of compounds 1-12 on BV2 cells

[0173] Exogenous MTT can be reduced by succinate dehydrogenase in the mitochondria of living cells to water-insoluble blue-purple crystalline formazan and deposit in the cells, while dead cells do not have this function. The absorbance value at a wavelength of 490nm using an enzyme-linked immunosorbent assay can indirectly reflect the number of living cells.

[0174] 2.1 Experimental methods

[0175] ① Experimental cells: BV2 (mouse microglia)

[0176] ② Experimental operation:

[0177] 1) Collect cells in logarithmic growth phase and adjust the concentration of cell suspension to make a cell concentration of 10 5 Cell suspension of 10 cells / mL was prepared according to 4 The cells were inoculated into 96-well plates, 100 μL per well (the edge wells were filled with sterile PBS, 100 μL medium per well), and then placed in a 37°C incubator with 5% CO 2 Culture in an incubator.

[0178] 2) After confirming that the cells have adhered to the wall, discard the culture medium and add 100 μL of drug-containing culture medium to each well. Set up 3 replicate wells. Incubate at 37°C with 5% CO 2 Incubate in the incubator for 24 hours.

[0179] 3) Observe the cell status under an inverted microscope, add 10 μL MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well, and continue culturing for 4 h.

[0180] 4) Carefully discard the solution in the wells. Add 100 μL of dimethyl sulfoxide to each well and oscillate at a low speed on a shaker for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at a wavelength of 492 nm using an enzyme-linked immunosorbent assay. The experiment set up a blank group (culture medium, MTT, dimethyl sulfoxide) and a control group (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide). The formula for calculating tumor cell growth rate is as follows:

[0181] Growth rate (RT,%) = [(OD sample -OD blank ) / (OD DMSO -OD blank )]×100%

[0182] 2.2 Experimental Results

[0183] The experimental results of the MTT colorimetric assay showed that the 24 compounds detected by compounds 1-12 had no obvious toxic effects on BV2 cells at a concentration of 20 μM.

[0184] 3. Effects of compounds 1a-12b on RXRα transcriptional activity

[0185] 3.1 Experimental process

[0186] ① Cell culture: Human embryonic kidney 293T cells were revived and incubated in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO 2 Culture in an incubator; wash the cells twice with PBS and digest with trypsin for 2 minutes during passaging; pass the cells until they are in good condition.

[0187] ② Transfection: Take cells in the logarithmic growth phase and replace with 4 ml of fresh serum-containing DMEM medium 1 hour before transfection; prepare transfection solution, A 1 Tube: pGL3-Promoter-RXRE+RXRα+p-Relina; B 1 Tube: Liposome + Opti-MEM; A 2 Tube: pBind-RXRα-LBD+pG5luc+Opti-MEM; B 2 Tube: liposome + Opti-MEM. Mix tube A and tube B with an oscillator, let stand at room temperature for 5 minutes, then transfer tube A and tube B to the same tube, shake and mix, and let stand at room temperature for 20-30 minutes. Add the mixture to the culture dish of 293T cells, 10% fetal bovine serum DMEM culture medium, 37°C, 5% CO 2 Culture in an incubator.

[0188] ③ Plating: 24 hours after transfection, discard the culture medium, wash twice with PBS, add 2 ml of trypsin digestion solution to digest the cells for 2 minutes, terminate the trypsin digestion reaction with DMEM medium containing serum, repeatedly blow the cells with a pipette to make them detached from the wall, make a uniform single cell suspension, inoculate in 96-well plates, 100 μL per well, and place at 37°C with 5% CO 2 Culture in an incubator.

[0189] ④ Adding drugs: 6-8 hours after plating, observe whether the cells have attached to the wall under a microscope. Add different concentrations of compounds, set up single drug addition holes and 9-cis-RA drug addition holes, and the drug continues to act for 12 hours.

[0190] ⑤ Dual luciferase reporter gene detection analysis: Cell lysis: remove the culture medium in the cultured cells; wash the cultured cells with 1×PBS and remove the washing solution; add 50μL of 1×PLB to each well; gently shake the culture plate at room temperature for 30 minutes, take 20μL of lysis solution and place it in a light-proof test tube for testing. Use a fluorescent immunoassay analyzer to detect: add 20μL of LARⅡ solution, mix well, and detect the activity of firefly luciferase; then add 20μL Top& Reagent, mix well, and detect Renilla luciferase activity.

[0191] Fluorescence intensity value (%) = OD firefly luciferase / Renilla luciferase × 100%

[0192] 3.2 Experimental Results

[0193] Compounds 1a, 1b, 3a, 4a, 5a, 6a, 7b, 10b and 11b can all activate the transcriptional activity of RXRα, showing statistically significant differences compared with the control group.

[0194] 4. Western-blot detection of the effects of compounds 1-12 on the expression of ABCA1 protein

[0195] 4.1 Experimental methods

[0196] ① Experimental cells: BV2 (mouse microglia)

[0197] ② Experimental operation:

[0198] 1) Cell culture: After the cryopreserved BV2 cells were revived, they were placed in DMEM culture medium and incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator with constant humidity, and the culture medium was replaced after 24 hours of culture. The cells were subcultured when the cell density reached about 70%, and cells with stable morphology and growth status were obtained after 2-3 generations.

[0199] 2) Plate plating and drug addition: After subculturing, cells were evenly inoculated into 12-well plates. When the cells adhered to the wall and reached a density of about 70% (37°C, 5% CO 2 ), remove the original culture medium, add new culture medium containing the compound to be tested at a preset concentration (20 μM), incubate for 12 hours, then discard the culture medium and collect samples.

[0200] 3) Cell lysis: Remove the culture medium from the cultured cells, wash the cells twice with pre-cooled 1× PBS solution, then place the 6-well plate on an ice box, and add an appropriate amount of RIPA lysis buffer containing protease inhibitors to the 6-well plate. Gently shake the 6-well plate until the lysis buffer is evenly distributed on the cell surface. After lysis for 2 minutes, scrape the cells with a cell scraper, then transfer them to a pre-cooled 1.5mL EP tube, place it on an ice box and continue lysis for 30 minutes. Oscillate the cells in a vortex shaker for 2 seconds every 10 minutes. Finally, place the lysed cells in a pre-cooled 4℃ centrifuge, centrifuge at 12000rpm for 10 minutes, and take the centrifugal supernatant for later use.

[0201] 4) Determination of protein concentration: Take the centrifuged supernatant and determine the concentration of the protein sample using the BCA method.

[0202] 5) Protein gel electrophoresis: Take the same mass of protein samples, fill them up to the same volume with lysis buffer, add 1 / 4 volume of 5×SDS loading buffer (containing 5% β-mercaptoethanol), place in a constant temperature metal bath at 100℃ for 5 minutes, and centrifuge to remove the precipitate. Add equal amounts of protein samples to the comb holes of the prepared electrophoresis gel plate, control the total protein amount in each well to 20-60μg, and the volume does not exceed 30μL. Add 1μL protein maker at both ends and perform electrophoresis at 80V. When the sample enters the separation gel, the voltage can be increased to 120V and the electrophoresis is continued until the bromophenol blue band is 1-2cm away from the bottom of the gel and the electrophoresis is stopped.

[0203] 6) Transfer: After removing the gel from the gel plate, temporarily place it in the pre-cooled electrotransfer solution, then take a PVDF membrane of the same size (pre-treated with methanol for 1 minute) and filter paper and immerse them in the electrotransfer solution. Place the electrotransfer clip with the black side facing down, and lay the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in sequence. Remove the bubbles in the electrotransfer clip, install it into the electrotransfer tank in the order of the membrane facing the positive electrode, and perform electrotransfer in an ice box. At this time, the voltage is controlled at 90V, the current is controlled below 300mA, and the electrotransfer is carried out for 60-90min.

[0204] 7) Blocking: Place the electro-transferred PVDF membrane in TBST buffer containing 5% skim milk and block it at room temperature for 1 hour on a horizontal shaker.

[0205] 8) Primary antibody reaction: After taking out the PVDF membrane from the blocking solution, cut out the required protein bands, wash it twice with an appropriate amount of TBST buffer, put it in an antibody incubation box, add the corresponding primary antibodies, all primary antibodies are diluted with 5% BSA 1:1000 (anti-PARP, anti-GAPDH). Then shake the PVDF membrane in a shaker at 4°C overnight.

[0206] 9) Secondary antibody reaction: After recovering the primary antibody, the PVDF membrane was washed three times with TBTS buffer on a horizontal shaker for 10 min each time, and then the secondary antibody (1:5000) was added and incubated at room temperature for 1 h.

[0207] 10) ECL chemiluminescence detection: After removing the secondary antibody, wash the PVDF membrane with TBTS buffer on a horizontal shaker 4 times, 8 minutes each time. Place the PVDF membrane in a plastic film and mix the ECL chemiluminescent solution A and B at a ratio of 1:1 (V / V) for later use. In a dark room, apply an appropriate amount of the ECL chemiluminescent mixture evenly on the surface of the PVD film, expose in a dark room, and adjust the exposure time according to the signal strength. Finally, develop and fix the film in turn.

[0208] 4.2 Experimental Results

[0209] The results showed that compounds 1a, 5a, 7a, 8b, 9a, 9b, 10b, 11a, 11b, 12a and 12b could significantly upregulate the expression of ABCA1 protein in BV2 cells, and compounds 1a, 5a and 11b could upregulate the expression of ABCA1 protein in a concentration-dependent manner.

[0210] 5. Binding experiment of compound 1 and RXRα-LBD

[0211] Retinoic acid nuclear receptor α (RXRα) is a key member of the nuclear receptor superfamily. As a ligand-dependent transcription factor, RXRα can regulate a wide range of biological processes, including cell differentiation, proliferation, and apoptosis, by interacting with small molecule modulators. RXRα has the classic functional domains of nuclear receptors, including the DNA binding domain (DBD) and the ligand binding domain (LBD).

[0212] Fluorescence quenching experiments have been widely used in the study of interactions between proteins and small molecules. Tyrosine, tryptophan, and phenylalanine residues in proteins can make proteins have fluorescence absorption. If the protein interacts with its ligand small molecule, the fluorescence will be quenched.

[0213] 5.1 Experimental process:

[0214] ① The RXRα-LBD protein was prepared into a 5 μM protein solution with PBS, 1 mL of the protein solution was placed in a special cuvette for a fluorescence spectrophotometer, and the initial fluorescence absorption curve was measured in a fluorescence spectrophotometer Cary Eclipse Fluorescence spectrophotometer (Varian).

[0215] ②Gradual addition and determination of compounds: The compound to be tested is also prepared into a 1mM solution with PBS buffer. Then, 2μL of the prepared compound solution is added to the protein solution each time, and it is thoroughly mixed after each addition to ensure that the compound and the protein can fully interact with each other. After that, the fluorescence absorption curve is measured again using a fluorescence spectrophotometer. This process is repeated, and the fluorescence absorption curve is measured after each addition until the final concentration of the compound reaches 1μM, 2μM, 3μM, etc., increasing by 1μM each time until 15μM, and the fluorescence absorption curve at each concentration is recorded. Finally, the experimental data is processed using Origin software to fit the dissociation constant (Kd value) between the compound and RXRα-LBD. The Kd value (dissociation constant) can reflect the binding affinity between protein and small molecules. The smaller the Kd value, the stronger the affinity.

[0216] 5.2 Experimental Results

[0217] The compound has a direct binding interaction with RXRα-LBD, and its K D The value is 5.85μM ( Figure 5 ).

[0218] Through fluorescence quenching experiments, it was observed that there was a direct binding interaction between the compound and RXRα-LBD. The Kd value obtained by fitting was 5.85μM ( Figure 5 ). This result indicates that the compound has a strong binding affinity with RXRα-LBD, providing a basis for further biological function research.

[0219] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. An acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton, characterized in that Its structural formula is as follows:

2. A method for preparing an acyl phloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton as claimed in claim 1, characterized in that The following steps are involved: 1) Cut the whole herb of the Herba Achyranthis Bidens radiata into pieces and extract it with methanol for 2 to 4 times, each time for 12 to 24 hours, collect the filtrate and concentrate it under reduced pressure to obtain an extract, mix the sample with silica gel and then chromatograph it on a normal phase silica gel column, using dichloromethane, ethyl acetate and methanol as mobile phases to obtain three polar fractions; 2) The ethyl acetate fraction obtained in step 1) was subjected to MCI gel column chromatography, and gradient eluted with methanol and water to obtain 8 fractions (Fr.1-Fr.8); the volume ratios of methanol to water were 1:1, 3:2, 7:3, 4:1, 9:1, and 1:0 respectively; 3) The fraction Fr.5 in step 2) was subjected to silica gel column chromatography again, and gradient eluted with petroleum ether and ethyl acetate to obtain 10 fractions (Fr.5A to Fr.5J); the volume ratios of petroleum ether and ethyl acetate were 20:1, 10:1, 5:1, 2:1, and 1:1 respectively; 4) The fraction Fr.5D in step 3) was subjected to silica gel column chromatography, and gradient eluted with petroleum ether and acetone to obtain 7 fractions (Fr.5D1-Fr.5D7); the volume ratio of petroleum ether to ethyl acetate was 1:0, 100:1, 50:1, 25:1, 10:1, 5:1 respectively; 5) The fraction Fr.5D4 in step 4) was separated by HPLC to obtain four fractions (Fr.5D4A to Fr.5D4D); the HPLC method was ODS Rp-18, 5 μm, 250×10 mm, 47% ACN / H2O, 5 mL / min; 6) The fraction Fr.5D4B in step 5) was separated by HPLC to obtain compound 12; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H2O, 5mL / min; 7) The fraction Fr.5D4D in step 5) was separated by HPLC to obtain compounds 10 and 11; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H2O, 5mL / min; 8) The fraction Fr.5G in step 3) was subjected to silica gel column chromatography, and gradient eluted with dichloromethane and methanol to obtain 8 fractions (Fr.5G1-Fr.5G8); the volume ratio of dichloromethane to methanol was 1:0, 100:1, 50:1, 25:1, 10:1, 5:1 respectively; 9) The fraction Fr.5G6 in step 8) was separated by HPLC to obtain compounds 5 and 6; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H2O, 5mL / min; 10) The fraction Fr.5G7 in step 8) was separated by HPLC to obtain compounds 4 and 9; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 50% ACN / H2O, 5mL / min; 11) The fraction Fr.6 in step 2) was subjected to silica gel column chromatography again, and gradient eluted with petroleum ether and ethyl acetate to obtain 12 fractions (Fr.6A to Fr.6L), wherein the volume ratios of petroleum ether and ethyl acetate were 50:1, 20:1, 10:1, 5:1, and 2:1, respectively; 12) The fraction Fr.6G in step 11) was subjected to silica gel column chromatography, and gradient eluted with dichloromethane and methanol to obtain 6 fractions (Fr.6G1-Fr.6G6); the volume ratio of dichloromethane to methanol was 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 respectively; 13) The fraction Fr.6G3 in step 12) was separated by HPLC to obtain Fr.6G3A-F; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 60% ACN / H2O, 5mL / min; 14) The fraction Fr.6G3E in step 13) was separated by HPLC to obtain compounds 1, 2 and 3; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 55% ACN / H2O, 5mL / min; 15) The fraction Fr.6H in step 11) was subjected to silica gel column chromatography, eluted with dichloromethane and methanol in a gradient manner, to obtain 9 fractions (Fr.6H1 to Fr.6H9; 16) The fraction Fr.6H7 in step 15) was separated by HPLC to obtain compounds 7 and 8; the HPLC method was COSMOSIL 5C18-AR-Ⅱ, 5μm, 250×10mm, 55% ACN / H2O, 5mL / min; 17) performing chiral separation on the compound separated in the above step, IG column, 5 μm, 250 × 10 mm, 50% ACN / H2O, 5 mL / min, to obtain compounds 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, 11b, 12a, 12b; In step 17), the chiral separation method is IG column, 5 μm, 250×10 mm, 50% ACN / H2O, 5 mL / min. The structural formula of the compound is:

3. Application of an acylphloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton in the preparation of anti-Alzheimer's drugs.

4. An application of an acylphloroglucinol compound with a 6 / 6 / 6 tricyclic skeleton as an anti-Alzheimer's drug targeting the nuclear receptor RXRα.

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