Amide alkaloid compound extracted from pepper roots as well as preparation method and application of amide alkaloid compound

Abstract

The invention belongs to the technical field of medicines, and relates to 19 amide alkaloids, including 5 pairs of amide alkaloid enantiomers, extracted and separated from roots of Piper nigrum L. of Piperraceae plant. A crude pepper root extract is separated through silica gel column chromatography, HP20 column chromatography, ODS (octadecylsilyl) column chromatography and HPLC (high performance liquid chromatography) separation in sequence, and finally 19 amide alkaloids including 5 pairs of amide alkaloid enantiomers are obtained. The invention also relates to investigation on the biological activity of the compounds. The preparation method is simple, the reproducibility is good, the purity of the obtained compound is high, and the obtained compound has acetylcholin esterase inhibitory activity, butyrylcholin esterase inhibitory activity and beta-amyloid protein aggregation inhibitory activity.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to 19 amide alkaloids separated from the root of the medicinal plant pepper, and studies the uses of the 19 amide alkaloids in terms of acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and beta-amyloid protein aggregation inhibitory activity. Background Art

[0002] Pepper (Pipernigrum L.) is a woody climbing vine of the Piperaceae family. It is widely distributed in tropical and subtropical regions and is cultivated in Taiwan, Fujian, Guangdong, Guangxi, Yunnan and other provinces in my country. Pepper fruits (white pepper and black pepper) are the most popular spices in the world and are also traditional Chinese medicines. They have the effects of warming the middle and dispelling cold, and removing qi and eliminating phlegm.

[0003] Alzheimer's disease is a neurodegenerative disease with a certain degree of heredity, which manifests as forgetfulness and other cognitive impairments. It is the most common cause of dementia in the middle-aged and elderly population. Currently, the cholinergic hypothesis and the amyloid-β-synergistic endothelial and neuronal toxicity hypothesis (ABSENT) are the most studied. The cholinergic hypothesis believes that Alzheimer's disease is caused by cholinergic neuron damage caused by neurotransmitter defects in the brains of Alzheimer's patients. On the other hand, the ABSENT hypothesis will attempt to prove that amyloid-β itself produces all free radicals, leading to vascular dysfunction and neuronal damage seen in Alzheimer's disease. Summary of the invention

[0004] The present invention aims to provide 19 amide alkaloids isolated from the root of Piper nigrum L., a plant of the Piperaceae family, including 5 pairs of enantiomeric amide alkaloid compounds, and to provide extraction and preparation methods of such amide alkaloid compounds, and also to provide their uses in acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and β-amyloid aggregation inhibitory activity. The structure of the amide alkaloid compound is shown below:

[0005]

[0006] The present invention includes the following preparation technology scheme:

[0007] The method for preparing amide alkaloid compounds using pepper root specifically comprises the following steps:

[0008] The dried roots of pepper were extracted with 70% ethanol, the extracts were combined and concentrated to obtain an extract, the extract was extracted with dichloromethane and n-butanol, and the obtained components were subjected to silica gel vacuum chromatography, and gradient elution with dichloromethane:methanol in a volume ratio of 10:0 to 3:1 was performed to obtain Fr.1 and Fr.2.

[0009] Fr.2 was subjected to silica gel vacuum chromatography, with petroleum ether:acetone in a volume ratio of 5:1 to 1:1 and dichloromethane:methanol in a volume ratio of 30:1 to 1:1, and HP-20 / ODS column chromatography with a gradient elution of 20%-90% ethanol water to obtain fraction Fr.AL.

[0010] Fr.D was subjected to silica gel vacuum column chromatography with a gradient elution of petroleum ether:acetone in a volume ratio of 8:1 to 1:1 and dichloromethane:methanol in a volume ratio of 30:1 to 1:1, and purified by RP-HPLC with acetonitrile-water to obtain compound 2, compound 3, compound 4, compound 5, compound 7, compound 9, compound 12, compound 13, and compound 14, which were separated by Daicel Chiralpak IC chiral column to obtain compound 1a / compound 1b, compound 6a / compound 6b, compound 8a / compound 8b, compound 10a / compound 10b, and compound 11a / compound 11b.

[0011] The extraction and preparation method of the invention is applicable to the plant Pipernigrum L. of the genus Piper in the family Piperaceae.

[0012] Preferably, in the preparation method, the dried pepper root is extracted with 70% industrial ethanol by reflux for 3 times, each time for 3 hours.

[0013] Preferably, in the preparation method, RP-HPLC is performed with an acetonitrile-water mobile phase of 60:40-10:90.

[0014] The chiral separation conditions in the present invention are applicable to compound 1, compound 6, compound 8, and compounds 10-11.

[0015] The structural identification results of the compounds obtained by the present invention are as follows:

[0016] The structures of compounds 1-14 were identified by high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, computed NMR, and computed ECD. The corresponding spectra are shown in Figures 1 to 28 As shown, the corresponding main structural characterization data are shown in Tables 1 to 3.

[0017] Compound 1: yellow oil; UV (MeOH) λ max (log E)264(2.45)nm; HRESIMS ion[M+H] + atm / z 238.1814 (calcd for C 14 H 24 NO2,238.1802), the molecular formula is determined to be C 14 H 23 NO2; by analyzing its 1 HNMR,13 C NMR, HSQC spectrum, HMBC spectrum, Mosher method and calculated ECD confirmed the structure of compound 1 as a new compound. (+)-1a: ECD(CH3OH)(Δε)216(+0.18),258(-0.21)nm. (-)-1b: ECD(CH3OH)(Δε)231(+0.23),250(-0.04),268(+0.08),300(-0.26)nm.

[0018] Compound 2: yellow oil; UV (MeOH) λ max (log E)265(2.11),206(1.77)nm; HRESIMSion[M+H] + at 238.1806(calcd for C 14 H 24 NO2,238.1802), and its molecular formula is determined to be C 14 H 23 NO2; ECD (CH3OH) (Δε) 230 (+0.35), 282 (-0.14), 298 (+0.18), 320 (-0.25) nm. 1 HNMR, 13 C NMR, HSQC spectrum, HMBC spectrum and bulkiness rule confirmed the structure of compound 2 as a new compound.

[0019] Compound 3: yellow oil; UV (MeOH) λ max (log E)265(1.92),203(0.42)nm; HRESIMSion[M+Na] + at m / z 260.1632 (calcd for C 14 H 23 NNaO2,260.1621), its molecular formula is determined to be C 14 H 23 NO2; ECD (CH3OH) (Δε) 236 (+0.09), 265 (-0.44), 309 (+0.13), 330 (-0.22) nm. 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum and bulkiness rule confirmed the structure of compound 3 as a new compound.

[0020] Compound 4: yellow oil; UV (MeOH) λ max (log E)259(1.96),206(2.28)nm; 238.1797[M+H] + (calcd for C 14 H 24 NO2,238.1802), and its molecular formula is determined to be C 14 H 23 NO2; ECD (CH3OH) (Δε) 218 ​​(+0.30), 326 (-0.19) nm. 1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 4 as a new compound.

[0021] Compound 5: yellow oil; UV (MeOH) λ max (log E)253(1.75),205(2.07)nm; 252.1969[M+H] + (calcd for C 15 H 23 NO2,252.1958), and its molecular formula is determined to be C 15 H 255 NO2; ECD (CH3OH) (Δε) 222 (+0.20), 267 (-0.06), 286 (+0.15), 328 (-0.17) nm. 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum and bulkiness rule confirmed the structure of compound 5 as a new compound.

[0022] Compound 6: colorless oil; UV (MeOH) λ max (log E)261(1.20)nm; HRESIMS at m / z246.1479[M+Na] + (calcd 246.1465for C 13 H 21 NNaO2), the molecular formula is determined to be C 13 H 21 NO2. By analyzing its 1 HNMR, 13 C NMR, HSQC spectrum, HMBC spectrum, and bulkiness rule confirmed the structure of compound 6 as a new compound. (–)-6a: ECD(CH3OH)(Δε)207(-0.33),223(+0.21),233(-0.20)nm. (+)-6b: ECD(CH3OH)(Δε)208(-0.37),215(+0.03),230(-0.43)nm.

[0023] Compound 7: yellow oil; UV (MeOH) λ max (log E)256(1.14),209(1.35)nm; HRESIMSion at m / z 252.1946[M+H] + (calcd for C 15 H 26 NO2,252.1958), and its molecular formula is determined to be C 15 H 25 NO2,; ECD (CH3OH) (Δε) 206 (+0.12), 225 (-0.27), 243 (+0.02), 280 (-0.80), 317 (+0.11) nm. 1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 7 as a new compound.

[0024] Compound 8: colorless oil; UV (MeOH) λ max (log E)284(1.44),205(1.80)nm; HRESIMSpeak at m / z 304.1599[M+H]+(calcd for C 17 H 22 NO4,304.1543), whose molecular formula is C 17 H 21 NO4; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum, bulkiness rule and calculated ECD confirmed the structure of compound 8 as a new compound. (+)-8a: ECD(CH3OH)(Δε)249(+0.39),277(-0.02),294(+0.12)nm. (–)-8b: ECD(CH3OH)(Δε)233(+0.29),260(-1.02)nm.

[0025] Compound 9: yellow oil; UV (MeOH) λ max (log E)287(1.14),209(1.40),205(2.23)nm; HRESIMS ion at m / z 306.1698[M+H] + (calcd for C 17 H 24 NO4,306.1700), its molecular formula is; ECD (CH3OH) (Δε) 240 (+0.47), 290 (-0.27) nm. 1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 9 as a new compound.

[0026] Compound 10: colorless oil; UV (MeOH) λ max (log E)286(1.28),238(1.42),205(2.20)nm; HRESIMS peak at m / z356.1481([M+Na] + )(calcd for C 18 H 23 NNaO5,,356.1468), the molecular formula is determined to be C 18 H 23 NO5; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum, bulkiness rule, calculated NMR and calculated ECD confirmed the structure of compound 10 as a new compound. (+)-10a: ECD(CH3OH)(Δε)239(-0.29)259(+0.85),281(-0.21)nm. (–)-10b: ECD(CH3OH)(Δε)252(-1.29)nm.

[0027] Compound 11: colorless oil; UV (MeOH) λ max (log E)286(1.45),206(2.29)nm; HRESIMSpeak at m / z 356.1489([M+Na] + )(calcd for C 18 H 23NNaO5,,356.1468); the molecular formula is determined to be C 18 H 23 NO5; By analyzing its 1 H NMR, 13 C NMR, HSQC spectrum, HMBC spectrum, bulkiness rule, calculated NMR and calculated ECD confirmed the structure of compound 11 as a new compound. (+)-11a: ECD(CH3OH)(Δε)244(-0.66),255(+2.72),288(-0.49)nm. (–)-11b: ECD(CH3OH)(Δε)231(+0.63),255(-2.83)nm.

[0028] Compound 12: yellow oil; UV (MeOH) λ max (log E)253(1.67),203(1.97)nm; HRESIMSion at m / z 240.1967[M+H] + (calcd for C 14 H 26 NO2,240.1958), and its molecular formula is determined to be C 14 H 25 NO2; ECD (CH3OH) (Δε) 211 (+0.10), 283 (-0.34) nm. The molecular formula was determined to be C 17 H 23 NO4; By analyzing its 1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 12 as a new compound.

[0029] Compound 13: yellow oil; UV (MeOH) λ max (log E)307(0.49),203(1.78)nm; HRESIMSion at m / z 240.1973[M+H] + (calcd for C 14 H 26 NO2,240.1958), and its molecular formula is determined to be C 14 H 25 NO2; ECD (CH3OH) (Δε) 220 (+0.43), 271 (-0.29) nm.1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 13 as a new compound.

[0030] Compound 14: Colorless needle-shaped crystals; UV (MeOH) λ max (log E)286(1.68),233(1.72),206(2.29)nm; HRESIMS ion at m / z 294.1720[M+H] + (calcd for C 16 H 24 NO4,294.1700), and its molecular formula is determined to be C 16 H 23 NO4; ECD (CH3OH) (Δε) 216 (+0.09), 288 (-0.29) nm. 1 HNMR, 13 C NMR, HSQC, HMBC and bulkiness rule confirmed the structure of compound 14 as a new compound.

[0031]

[0032]

[0033]

[0034] The present invention also provides the use of the amide alkaloid compounds in pepper extracted and separated by the present invention or their pharmaceutically acceptable salts in the preparation of drugs having acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and / or β-amyloid protein aggregation inhibitory activity.

[0035] The present invention also provides the use of the amide alkaloid compounds or pharmaceutically acceptable salts thereof extracted and separated from pepper in the present invention in the preparation of anti-Alzheimer's disease drugs. The 19 new amide alkaloid compounds of the present invention were investigated for their anti-acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and β-amyloid protein aggregation inhibitory activity.

[0036] The results showed that compounds 2-3, compound 11a and compound 11b had significant inhibitory activity on acetylcholinesterase; compound 2, compound 4, compound 10b, compound 11a, compound 11b and compound 12 had significant inhibitory activity on butyrylcholinesterase; compound 1a, compound 4, compound 7, compound 8a, compound 10a, compound 10b and compound 12 had significant β-amyloid protein aggregation inhibitory activity.

[0037] Beneficial effects of the present invention:

[0038] It is expected that the new amide alkaloid compounds described in the present invention have anti-Alzheimer's disease effects. The present invention provides new amide alkaloid compounds for the field of anti-Alzheimer's disease. The advantages are that the compounds are all new compounds, the compounds are distinctly characterized as amide alkaloids, easy to separate and purify, and have good anti-acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and β-amyloid protein aggregation inhibitory activity, which provides a new type of compound for anti-Alzheimer's disease and has the value of further development. In addition, the method of the present invention is reasonably designed, has the characteristics of simple process, low cost and industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 HMBC spectrum of compound 1;

[0040] Figure 2 Compound 1 1 H- 1 H COSY spectrum;

[0041] Figure 3 HMBC spectrum of compound 2;

[0042] Figure 4 Compound 2 1 H- 1 H COSY spectrum;

[0043] Figure 5 HMBC spectrum of compound 3;

[0044] Figure 6 Compound 3 1 H- 1 H COSY spectrum;

[0045] Figure 7 HMBC spectrum of compound 4;

[0046] Figure 8 Compound 4 1 H- 1 H COSY spectrum;

[0047] Fig. 9HMBC spectrum of compound 5;

[0048] Fig.10 Compound 5 1 H- 1 H COSY spectrum;

[0049] Fig.11 HMBC spectrum of compound 6;

[0050] Fig.12 Compound 6 1 H- 1 H COSY spectrum;

[0051] Fig.13 HMBC spectrum of compound 7;

[0052] Fig.14 Compound 7 1 H- 1 H COSY spectrum;

[0053] Fig.15 HMBC spectrum of compound 8;

[0054] Fig.16 Compound 8 1 H- 1 H COSY spectrum;

[0055] Fig.17 HMBC spectrum of compound 9;

[0056] Fig.18 Compound 9 1 H- 1 H COSY spectrum;

[0057] Fig.19 HMBC spectrum of compound 10;

[0058] Fig. 20 Compound 10 1 H- 1 H COSY spectrum;

[0059] Fig.21 HMBC spectrum of compound 11;

[0060] Fig. 22 Compound 11 1 H- 1 H COSY spectrum;

[0061] Fig.23 HMBC spectrum of compound 12;

[0062] Fig.24 Compound 12 1 H- 1H COSY spectrum;

[0063] Fig.25 HMBC spectrum of compound 13;

[0064] Fig.26 Compound 13 1 H- 1 H COSY spectrum;

[0065] Fig. 27 HMBC spectrum of compound 14;

[0066] Fig.28 Compound 14 1 H- 1 H COSY spectrum;

[0067] Fig.29 AchE inhibition rate;

[0068] Fig.30 BchE inhibition rate;

[0069] Fig.31 Inhibition rate of anti-Aβ aggregation activity. DETAILED DESCRIPTION

[0070] Example 1

[0071] The method for preparing amide alkaloid compounds using pepper roots specifically comprises the following steps: taking dried pepper roots (50.0 kg) and extracting them with 70% ethanol, combining the extracts and concentrating them to obtain an extract, extracting the extract with dichloromethanol and n-butanol, and subjecting the obtained components to silica gel vacuum chromatography, and gradient eluting with dichloromethane:methanol (10:0 to 3:1, v / v) to obtain Fr.1 (395 g) and Fr.2 (819 g).

[0072] Fr.2 was subjected to silica gel vacuum chromatography with petroleum ether:acetone (5:1 to 1:1, v / v) and dichloromethane:methanol (30:1 to 1:1, v / v) and HP-20 / ODS column chromatography with a 20%-90% ethanol-water gradient elution to obtain fraction Fr.AL.

[0073] Fr.D (10.0 g) was purified by silica gel vacuum column chromatography with gradient elution of petroleum ether:acetone from 8:1 to 1:1 by volume and dichloromethane:methanol from 30:1 to 1:1 by volume, and purified by RP-HPLC with acetonitrile-water to give compounds 2 (9.3 mg), 3 (6.0 mg), 4 (19.3 mg), 5 (7.5 mg), 7 (26.3 mg), 9 (5.7 mg), 12 (5.5 mg), 13 (4.6 mg), and 14 (12.9 mg). Compounds 1a (6.8 mg) / 1b (3.7 mg), 6a (3.1 mg) / 6b (1.9 mg), 8a (2.8 mg) / 8b (2.8 mg), 10a (1.8 mg) / 10b (1.8 mg), and 11a (3.5 mg) / 11b (3.1 mg) were obtained by separation on a Daicel Chiralpak IC chiral column.

[0074] In the method, the pepper used is Pipernigrum L., a plant of the genus Piper in the family Piperaceae.

[0075] In the method, dried pepper roots are extracted with 70% industrial ethanol under reflux for 3 times, each time for 3 hours.

[0076] In the method, RP-HPLC was performed with an acetonitrile-water mobile phase of 60:40-10:90.

[0077] Example 2

[0078] Investigation of the inhibitory activity of compounds 1-14 on acetylcholinesterase

[0079] Donepezil was used as a positive control. The concentration of the test compound was prepared into a gradient of 100, 40, 20, 10 and 1 μmol / L using PBS (pH = 7.4). The entire reaction system contained 25 μL of the test compound, 12.5 μL of enzyme, 125 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which was refrigerated overnight. Finally, 50 μL of iodinated acetylthiocholine (ATCI) was added to terminate the reaction. After 5 minutes of reaction, the absorbance was measured at a wavelength of 412 nm. The results are shown in Figure 2. Fig.29 As shown, it was shown that compounds 2-3, compound 11a and compound 11b had significant anti-acetylcholinesterase activity.

[0080] Example 3

[0081] Investigation of the inhibitory activity of compounds 1-14 on butyrylcholinesterase

[0082] Donepezil was used as a positive control. The concentration of the test compound was prepared into a gradient of 100, 40, 20, 10 and 1 μmol / L using PBS (pH = 7.4). The entire reaction system contained 25 μL of the test compound, 10.0 μL of enzyme, 125 μL of DTNB, and was refrigerated overnight. Finally, 50 μL of iodinated butyrylcholine (BTCI) was added to terminate the reaction. After 5 minutes of reaction, the absorbance was measured at a wavelength of 412 nm. The results are shown in Figure 2. Fig.30 As shown, it was shown that compound 2, compound 4, compound 10b, compound 11a, compound 11b and compound 12 had significant inhibitory activity against butyrylcholinesterase.

[0083] Example 4

[0084] Investigation of the inhibitory activity of compounds 1-14 on β-amyloid protein aggregation

[0085] Curcumin was used as a positive control. The concentration of the test compound was adjusted to 20 μmol / L using PBS (pH = 7.4). The entire reaction system contained 10.0 μL of the test compound, 10.0 μL Aβ, 60 μL PBS, incubated at 37°C for 24 hours, and finally added 80 μL of thioflavin T (Th-T). After reacting for 5 minutes, the absorbance was measured at a wavelength of 485 nm. The results are shown in Fig.31 As shown, it is shown that compound 1a, compound 4, compound 7, compound 8a, compound 10a, compound 10b and compound 12 have significant inhibitory activity against β-amyloid protein aggregation.

[0086] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An amide alkaloid compound in pepper, characterized in that: It is any of the following structures:

2. The amide alkaloid compound in pepper according to claim 1, characterized in that The pepper is a plant of the genus Piper in the family Piperaceae [Pipernigrum L.].

3. A method for preparing the amide alkaloid compounds in pepper according to claim 1 or claim 2, characterized in that: The steps include: The dried root of pepper was extracted with 70% ethanol, and the combined extracts were concentrated to obtain an extract. The extract was extracted with dichloromethane and n-butanol, and the obtained components were subjected to silica gel vacuum chromatography, and gradient elution with dichloromethane:methanol in a volume ratio of 10:0 to 3:1 was performed to obtain Fr.1 and Fr.

2. Fr.2 was subjected to silica gel vacuum chromatography, with petroleum ether:acetone in a volume ratio of 5:1 to 1:1 and dichloromethane:methanol in a volume ratio of 30:1 to 1:1, and HP-20 / ODS column chromatography with a gradient elution of 20%-90% ethanol water to obtain fraction Fr.AL. Fr.D was subjected to silica gel vacuum column chromatography, with petroleum ether:acetone in a volume ratio of 8:1 to 1:1 and dichloromethane:methanol in a volume ratio of 30:1 to 1:

1. It was purified by RP-HPLC with acetonitrile-water to obtain compound 2, compound 3, compound 4, compound 5, compound 7, compound 9, compound 12, compound 13, and compound 14. Chiralpak IC chiral column was used to separate compound 1a / compound 1b, compound 6a / compound 6b, compound 8a / compound 8b, compound 10a / compound 10b and compound 11a / compound 11b.

4. The method for preparing the amide alkaloid compounds in pepper according to claim 3, characterized in that: The pepper used is Pipernigrum L., a plant of the genus Piper in the family Piperaceae.

5. The method for preparing the amide alkaloid compounds in pepper according to claim 3, characterized in that: The dried pepper roots were extracted with 70% industrial ethanol under reflux for 3 times, each time for 3 h.

6. The method for preparing the amide alkaloid compounds in pepper according to claim 3, characterized in that: RP-HPLC was performed with a mobile phase of acetonitrile-water in a range of 60:40-10:

90.

7. A pharmaceutical composition, characterized in that The invention comprises the amide alkaloid compound in pepper according to claim 1 or claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of an amide alkaloid compound in pepper according to claim 1 or claim 2 or a pharmaceutically acceptable salt thereof in the preparation of a drug having acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and / or β-amyloid protein aggregation inhibitory activity.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of a drug having acetylcholinesterase inhibitory activity, butyrylcholinesterase inhibitory activity and / or β-amyloid protein aggregation inhibitory activity.

10. Use of the amide alkaloid compound in pepper according to claim 1 or claim 2 or its pharmaceutically acceptable salt or the pharmaceutical composition according to claim 7 in the preparation of an anti-Alzheimer's disease drug.

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