Amide alkaloid compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly discloses an amide alkaloid compound as well as a preparation method and application thereof.The amide alkaloid compound is prepared by extracting fructus piperis longi with an organic solvent, extracting, concentrating and drying. And then separating by three to four methods of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography and chiral high performance liquid chromatography to obtain fourteen new amide alkaloid monomeric compounds. Fourteen compounds are determined to be new amide alkaloid compounds through high-resolution mass spectrum, nuclear magnetic resonance spectrum, circular dichroism spectrum and quantum chemistry means, and structural identification is carried out on the amide alkaloid compounds. In addition, in-vitro hypoglycemic, anti-tumor and anti-inflammatory activity determination is carried out on the fourteen amide alkaloid compounds, and results show that the amide alkaloid compounds have hypoglycemic, anti-tumor and anti-inflammatory activity and can be used for hypoglycemic, anti-tumor and anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an amide alkaloid compound, its preparation method, and its uses. Background Technology

[0002] Piper longum Linn. is a perennial herbaceous vine belonging to the genus Piper in the family Piperaceae. Also known as Piper pumila, Piper longum, Piper pumila ...

[0003] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and impaired insulin utilization. It is mainly classified into three types: type 1, type 2, and gestational diabetes. Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that plays a crucial role in cell signal transduction, particularly in the insulin and leptin signaling pathways. PTP1B is closely related to type 2 diabetes and is one of the important targets in current diabetes drug development. Alpha-glucosidase is a digestive enzyme located at the brush border of the small intestine, responsible for breaking down complex carbohydrates (such as starch and oligosaccharides) into monosaccharides (mainly glucose) for intestinal absorption. Due to its key role in postprandial blood glucose regulation, α-glucosidase has become an important target for the treatment of type 2 diabetes. Previous findings have shown that a few amide alkaloids in Piper longum can effectively inhibit the activity of PTP1B and α-glucosidase, providing a research basis for this patent.

[0004] Modern research indicates that amide alkaloids are the most abundant and pharmacologically active chemical components in Piper longum, such as piperlongumine, which has attracted considerable attention due to its ability to selectively induce apoptosis in cancer cells. With the advancement of modern separation techniques and pharmacological research, these compounds are expected to become an important source for new drug development. Summary of the Invention

[0005] The purpose of this invention is to provide an amide alkaloid compound, its preparation method, and its uses, in order to solve the problem of the preparation of amide alkaloid compounds and their non-use in the preparation of hypoglycemic, antitumor, and anti-inflammatory drugs.

[0006] To achieve the above objectives, the basic solution provided by this invention is: an amide alkaloid compound, the structural formula of which is: The compound of formula (1) is (2E,4E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (2) is (2E,4E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (3) is (2E,4E,10S,11S)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (4) is (2E,4E,10R,11R)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (5) is (2E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (6) is (2E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (7) is (2E,4S,5R)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (8) is (2E,4R,5S)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (9) is (1R,2S,4aR,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (10) is (1S,2S,4aR,8aR)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (11) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (12) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-piperidinyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (13) is (3R,3a,S,4S,7R,7aR)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; The compound of formula (14) is (3S,3aR,4R,S,7aS)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one.

[0007] The beneficial effects of this invention are as follows: Amide alkaloids are extracted from the fruit of Piper longum, and then separated using three to four of the following methods: normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel permeation chromatography, and chiral high-performance liquid chromatography, yielding fourteen new amide alkaloid monomeric compounds. High-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, and quantum chemical methods were used to confirm that the fourteen compounds are new amide alkaloids, and their structures were identified. Furthermore, the in vitro inhibitory activity of these fourteen amide alkaloids on protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, human cervical cancer cells (HeLa), human colon cancer cells (HT-29), human breast cancer cells (MCF-7), and lipopolysaccharide-induced mouse macrophages RAW264.7 was determined. The results show that the amide alkaloids can significantly inhibit the activity or content of these enzymes, tumor cell lines, and nitric oxide, and can be used as hypoglycemic, antitumor, and anti-inflammatory drugs.

[0008] Option 2, a method for preparing an amide alkaloid compound, comprising the following steps: a. Take Piper longum fruit as raw material, crush it, and then extract it by percolation or cold soaking at room temperature with 5-12 times the volume of ethanol aqueous solution with a volume concentration of 50%-100% or methanol aqueous solution with a volume concentration of 50%-100%. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum fruit. b. Disperse the crude extract obtained in step a with water, add ethyl acetate, dichloromethane or trichloromethane for extraction 3-5 times, concentrate the ethyl acetate, dichloromethane or trichloromethane extract to obtain ethyl acetate, dichloromethane or trichloromethane extract paste. c. Separate the ethyl acetate, dichloromethane or trichloromethane extract obtained in step b using three or four of the following methods: normal phase silica gel column chromatography, reversed phase silica gel column chromatography, gel column chromatography and chiral high performance liquid chromatography, to obtain compounds of formula (1) to (14).

[0009] Option 3, which is the preferred option of Option 2, involves the following three separation methods in step c: The ethyl acetate, dichloromethane, or chloroform extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether-acetone or dichloromethane-methanol at a volume ratio of 100:1-0:1. The fraction F of 50:1-10:1 is collected and subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution or acetone-water solution at a concentration of 10%-100% to obtain the racemic mixtures of compounds (1) to (14). The racemic mixtures are then subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-isopropanol or n-hexane-ethanol solution at a concentration of 99:1-50:50 to obtain the compounds (1) to (14).

[0010] Option 4, a preferred option of Option 2, involves the following four separation methods in step c: The ethyl acetate, dichloromethane, or chloroform extract obtained in step b is subjected to normal-phase silica gel column chromatography, with a gradient elution of petroleum ether-ethyl acetate or dichloromethane-methanol at a volume ratio of 100:1-0:1. The 50:1-10:1 fraction F is collected. Fraction F is then subjected to reverse-phase silica gel column chromatography, with a gradient elution of 10%-100% methanol-water solution or 10%-100% acetone-water solution, and the 40%- The elution fraction F40-90 was obtained by eluting with 90% methanol-water solution or acetone-water solution. The F40-90 fraction was subjected to gel column chromatography and eluted with a gradient of 50%-100% methanol-water solution or 50%-100% dichloromethane-methanol solution to obtain the racemic mixtures of the compounds (1) to (14). The racemic mixtures were then subjected to chiral high performance liquid chromatography and eluted with a gradient of n-hexane-isopropanol or n-hexane-ethanol solution with a concentration of 99:1-50:50 to obtain the compounds (1) to (14).

[0011] Option 5, which is a preferred option of Option 2, involves normal-phase silica gel column chromatography in step c, which is atmospheric or pressurized column chromatography. The packing material used is normal-phase silica gel, and the eluent used is a mixture of two solvents, namely petroleum ether or dichloromethane, ethyl acetate or methanol, using isocratic elution or gradient elution.

[0012] Option 6, which is a preferred option of Option 2, in step c, the reversed-phase silica gel column chromatography is atmospheric pressure column chromatography, and the eluent is a gradient elution of 10%-100% methanol-water solution or 10%-100% acetone-water solution.

[0013] Option 7, which is a preferred option of Option 2, in step c, the gel column chromatography is atmospheric pressure column chromatography, the packing material is Sephadex LH-20 gel, and the eluent is a gradient elution of 50%-100% methanol-water solution or 50%-100% dichloromethane-methanol solution.

[0014] Option 8, which is a preferred option of Option 2, in step c, the chiral high performance liquid chromatography is pressurized column chromatography, and the eluent is a gradient elution of n-hexane-isopropanol or n-hexane-ethanol solution with a concentration of 99:1-50:50.

[0015] Option 9: The use of an amide alkaloid compound in the preparation of hypoglycemic, antitumor, and anti-inflammatory drugs. Attached Figure Description

[0016] Figure 1 For the compound of formula (1 / 2) of this invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 2 For the compound of formula (1 / 2) of this invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 3 For the compound of formula (3 / 4) of this invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 4 For the compound of formula (3 / 4) of this invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 5 For the compound of formula (5 / 6) of this invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 6 For the compound of formula (5 / 6) of this invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 7 For the compound of formula (7 / 8) of this invention 1 1H NMR (600 MHz, CDCl3) spectrum; Figure 8 For the compound of formula (7 / 8) of this invention 13 C NMR (150 MHz, CDCl3) spectrum; Figure 9 For the compound of formula (9) of the present invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 10For the compound of formula (9) of the present invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 11 The compound of formula (10 / 11) of this invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 12 The compound of formula (10 / 11) of this invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 13 For the compound of formula (12) of the present invention 1 1H NMR (600 MHz, CD3OD) spectrum; Figure 14 For the compound of formula (12) of the present invention 13 C NMR (150 MHz, CD3OD) spectrum; Figure 15 For the compound of formula (13 / 14) of the present invention 1 1H NMR (500 MHz, CD3OD) spectrum; Figure 16 For the compound of formula (13 / 14) of the present invention 13 C NMR (125 MHz, CD3OD) spectrum. Detailed Implementation

[0017] The present invention will be further described in detail below through specific embodiments: Example 1 like Figures 1 to 16 As shown: An amide alkaloid compound, the structural formula of which is: The compound of formula (1) is (2E,4E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (2) is (2E,4E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (3) is (2E,4E,10S,11S)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (4) is (2E,4E,10R,11R)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (5) is (2E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (6) is (2E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (7) is (2E,4S,5R)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (8) is (2E,4R,5S)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (9) is (1R,2S,4aR,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (10) is (1S,2S,4aR,8aR)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (11) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (12) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-piperidinyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (13) is (3R,3a,S,4S,7R,7aR)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; The compound of formula (14) is (3S,3aR,4R,S,7aS)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one.

[0018] The structures of the aforementioned amide alkaloids were determined using high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, and quantum chemical calculations. The structural identification process is as follows: The compound of formula (1) is (2E,4E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one, a white powder with a specific rotation [α]. 20 D +42.3 (concentration 0.15, methanol); UV (methanol) 199.7, 267.7 nm; Circular dichroism (methanol) 200 (Δε+3.36), 214 (Δε+1.94), 234 (Δε+1.89), 262 (Δε+0.78), 285 (Δε+1.73), 316 (Δε+0.54); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. + The calculated value of m / z is 396.1781, which determines its molecular formula to be C. 21 H 27 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythroline configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,4E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0019] The compound of formula (2) is (2E,4E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one, a white amorphous powder with a specific rotation [α]. 20 D +37.8 nm (concentration 0.16, methanol); UV (methanol) 199.7 nm, 267.7 nm; Circular dichroism (methanol) 200 (Δε+3.03), 213 (Δε+1.86), 234 (Δε+1.88), 264 (Δε+0.64), 285 (Δε+1.75), 315 (Δε+0.47); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. +The calculated value of m / z is 396.1781, which determines its molecular formula to be C. 21 H 27 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythroline configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,4E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0020] The compound of formula (3) is (2E,4E,10S,11S)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one, a pale yellow oil with a specific rotation of [α]. 20 D +45.3 (concentration 0.14, methanol); UV (methanol) 200.0, 267.3 nm; Circular dichroism (methanol) 203 (Δε+8.09), 232 (Δε+1.32), 285 (Δε+1.11); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. + The m / z value is 424.2091 (calculated value is 424.2094), which determines its molecular formula to be C. 23 H 31 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure exhibiting a threotype configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,4E,10S,11S)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundecyl-2,4-dien-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0021] The compound of formula (4) is (2E,4E,10R,11R)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundecyl-2,4-dien-1-one, a pale yellow oil with a specific rotation of [α]. 20 D -39.8 (concentration 0.18, methanol); UV (methanol) 200.0, 267.3 nm; Circular dichroism (methanol) 203 (Δε+7.36), 232 (Δε+1.26), 285 (Δε+1.17); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. + The m / z value is 424.2091 (calculated value is 424.2094), which determines its molecular formula to be C. 23 H 31 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure exhibiting a threotype configuration. Based on a comparison of circular dichroism (CD) spectral data with quantum chemical calculations, its absolute configuration was determined, and it was named (2E,4E,10R,11R)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundecyl-2,4-dien-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0022] The compound of formula (5) is (2E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one, a colorless oil with a specific rotation of [α]. 20 D +69.7 (concentration 0.16, methanol); UV (methanol) 201.1, 238.4, 283.8 nm; Circular dichroism (methanol) 206 (Δε+1.46), 224 (Δε+0.75), 244 (Δε+0.62), 280 (Δε+0.49); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. + The calculated value of m / z is 412.2094, which determines its molecular formula to be C. 22 H 31 NO5; According to 1 H, 13C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythroline configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0023] The compound of formula (6) is (2E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one, a colorless oil with a specific rotation of [α]. 20 D +77.6 (concentration 0.11, methanol); UV (methanol) 201.1, 238.4, 283.8 nm; Circular dichroism (methanol) 205 (Δε+1.53), 223 (Δε+0.83), 243 (Δε+0.68), 282 (Δε+0.53); via the quasi-molecular ion peak [M+Na] in its high-resolution mass spectra. + The calculated value of m / z is 412.2094, which determines its molecular formula to be C. 22 H 31 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythroline configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0024] The compound of formula (7) is (2E,4S,5R)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one, a colorless oil with a specific rotation of [α]. 20 D+28.3 (concentration 0.16, methanol); UV (methanol) 199.5, 238.2, 284.3 nm; Circular dichroism (methanol) 206 (Δε+11.80), 227 (Δε+3.12), 307 (Δε+1.06); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The m / z value is 334.1648 (calculated value is 334.1649), which determines its molecular formula to be C. 18 H 23 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythroline configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,4S,5R)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one. 1 H and 13 The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated chloroform.

[0025] The compound of formula (8) is (2E,4R,5S)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one, a colorless oil with a specific rotation of [α]. 20 D +33.1 (concentration 0.12, methanol); UV (methanol) 199.5, 238.2, 284.3 nm; Circular dichroism (methanol) 204 (Δε+13.31), 228 (Δε+3.52), 308 (Δε+1.22); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The m / z value is 334.1648 (calculated value is 334.1649), which determines its molecular formula to be C. 18 H 23 NO5; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound, with the vicinal diol in its structure having an erythrocyanine configuration. Based on a comparison of circular dichroism spectral data with quantum chemically calculated data, its absolute configuration was determined, and it was named (2E,4R,5S)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one. 1 H and 13The C NMR attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), deuterated chloroform.

[0026] The compound of formula (9) is (1R,2S,4aR,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide, a colorless crystal with a specific rotation [α]. 20 D +73.3 (concentration 0.12, methanol); UV (methanol) 198.9, 233.8 nm; Circular dichroism (methanol) 212 (Δε+8.17); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 356.2221 (calculated value is 356.2220). 20 H 29 NO3; According to 1 H, 13 CNMR and two-dimensional nuclear magnetic resonance data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data and quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (1R,2S,4aR,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; 1 H and 13 The C NMR attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0027] Compound (10) is (1S,2S,4aR,8aR)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide, a white powder with a specific rotation [α]. 20 D +59.7 (concentration 0.12, methanol); UV (methanol) 201.9, 234.6, 289.6 nm; Circular dichroism (methanol) 203 (Δε+23.1); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 356.2221 (calculated value is 356.2220). 22 H 29 NO3; According to 1 H, 13C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data with quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (1S,2S,4aR,8aR)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; 1 H and 13 The C NMR attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0028] The compound of formula (11) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide, a white powder with a specific rotation [α]. 20 D +63.8 nm (concentration 0.08, methanol); UV (methanol) 201.9, 234.6, 289.6 nm; Circular dichroism (methanol) 203 nm (Δε +24.16); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 356.2221 (calculated value is 356.2220). 20 H 29 NO3; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data and quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; 1 H and 13 The C NMR values ​​are the same as those of the compound in formula (6), and their assignments are shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0029] The compound of formula (12) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-piperidinyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; a white powder with a specific rotation [α]. 20 D+63.3 (concentration 0.18, methanol); UV (methanol) 199.3, 234.6, 288.1 nm; Circular dichroism (methanol) 204 (Δε +13.17); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 368.2211 (calculated value is 368.2220). 23 H 30 NO3; According to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data with quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-piperidinyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; 1 H and 13 The C NMR attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), deuterated methanol.

[0030] The compound of formula (13) is (3R,3aS,4S,7R,7aR)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidin-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; a white powder with a specific rotation [α]. 20 D +79.1 nm (concentration 0.13, methanol); UV (methanol) 201.8, 240.1, 287.2 nm; Circular dichroism (methanol) 200 (Δε+14.63), 220 (Δε+3.26), 240 (Δε+1.51); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 441.2382 (calculated value 441.2384). 25 H 34 N2O5; according to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data and quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (3R,3aS,4S,7R,7aR)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidin-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; 1 H and 13The C NMR assignments are shown in Table 2 [500 MHz ( 1 H), 125 MHz 13 C), deuterated methanol.

[0031] The compound of formula (14) is (3S,3aR,4R,S,7aS)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidin-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; a white powder with a specific rotation [α]. 20 D +63.7 (concentration 0.19, methanol); UV (methanol) 201.8, 240.1, 287.2 nm; Circular dichroism (methanol) 200 (Δε+13.78), 220 (Δε+4.67), 240 (Δε+1.36); via the quasi-molecular ion peak [M+H] in its high-resolution mass spectra. + The molecular formula is determined to be C based on m / z 441.2382 (calculated value 441.2384). 25 H 34 N2O5; according to 1 H, 13 C10 NMR and 2D NMR data confirmed its structural framework as an amide alkaloid compound; based on a comparison of circular dichroism spectral data and quantum chemically calculated circular dichroism spectral data, its absolute configuration was determined, and it was named (3S,3aR,4R,S,7aS)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidin-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; 1 H and 13 The C NMR assignments are shown in Table 2 [500 MHz ( 1 H), 125 MHz 13 C), deuterated methanol.

[0032] Table 1. Equations (1) to (8) 1 H and 13 C NMR data [δ(ppm), J(Hz)] a: Overlapping NMR signals Table 2. Equations (9) to (14) 1 H and 13 C NMR data [δ(ppm), J(Hz)] a: Overlapping NMR signals Example 2 A method for preparing an amide alkaloid compound includes the following steps: a. Take Piper longum fruit as raw material, crush it, and then extract it by percolation or cold soaking at room temperature with 5-12 times the volume of ethanol aqueous solution with a volume concentration of 50%-100% or methanol aqueous solution with a volume concentration of 50%-100%. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum fruit. b. Disperse the crude extract obtained in step a with water, add ethyl acetate, dichloromethane or trichloromethane for extraction 3-5 times, concentrate the ethyl acetate, dichloromethane or trichloromethane extract to obtain ethyl acetate, dichloromethane or trichloromethane extract paste. c. The ethyl acetate, dichloromethane, or trichloromethane extract obtained in step b is separated by three to four of the following methods: normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, gel column chromatography, and chiral high-performance liquid chromatography, to obtain compounds of formulas (1) to (14); normal-phase silica gel column chromatography is atmospheric or pressurized column chromatography, the packing material is normal-phase silica gel, and the eluent is a mixture of two solvents, namely petroleum ether or dichloromethane, ethyl acetate or methanol, using isocratic elution or gradient elution; reversed-phase silica gel column chromatography is atmospheric column chromatography, and the eluent is a gradient elution of 10%-100% methanol-water solution or 10%-100% acetone-water solution; gel column chromatography is atmospheric column chromatography, and the packing material is Sephadex gel. LH-20, eluent 50%-100% methanol-water solution or 50%-100% dichloromethane-methanol solution gradient elution; chiral high performance liquid chromatography is pressurized column chromatography, eluent 99:1-50:50 hexane-isopropanol or hexane-ethanol solution gradient elution.

[0033] Example 3 (One of the three separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by cold soaking in 5 times the volume of 50% ethanol-water solution at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with chloroform three times, combine the chloroform extracts, and evaporate to dryness under reduced pressure to obtain chloroform extract paste. c. The chloroform extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether-acetone in a ratio of 100:1 to 0:1, and the fraction F in a ratio of 50:1 to 10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution in a ratio of 10% to 100% to obtain racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-isopropanol solution in a ratio of 99:1 to 50:50 to obtain compounds (1) to (14).

[0034] Example 4 (one of three separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by percolation with 12 times the volume of 100% ethanol at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with dichloromethane 5 times, combine the dichloromethane extracts, and evaporate to dryness under reduced pressure to obtain dichloromethane extract paste. c. The dichloromethane extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane-methanol in a concentration of 100:1-0:1, and the fraction F in a concentration of 50:1-10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of acetone-water solution in a concentration of 10%-100% to obtain racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-tetrahydrofuran solution in a concentration of 99:1-50:50 to obtain compounds (1) to (14).

[0035] Example 5 (One of the three separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by cold soaking in 10 times the volume of 50% methanol-water solution at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with ethyl acetate three times, combine the ethyl acetate extracts, and evaporate to dryness under reduced pressure to obtain ethyl acetate extract paste. c. The ethyl acetate extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether-acetone in a ratio of 100:1 to 0:1, and the fraction F in a ratio of 50:1 to 10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution in a ratio of 20% to 99% to obtain racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-isopropanol solution in a ratio of 99:1 to 50:50 to obtain compounds (1) to (14).

[0036] Example 6 (One of the four separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by cold soaking in 4 times the volume of 100% methanol at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with ethyl acetate 4 times, combine the ethyl acetate extracts, and evaporate to dryness under reduced pressure to obtain ethyl acetate extract paste. c. The ethyl acetate extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane-methanol in a concentration of 100:1-0:1, and the fraction F in a concentration of 50:1-10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution in a concentration of 10%-100%, and the fraction F40-90 in a concentration of methanol-water solution in a concentration of 40-90% is collected. The fraction F40-90 is subjected to gel column chromatography with a gradient elution of dichloromethane-methanol solution in a concentration of 50%-100%, to obtain the racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-tetrahydrofuran solution in a concentration of 99:1-50:50 to obtain compounds (1) to (14).

[0037] Example 7 (One of the four separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by percolation with 12 times the volume of 50% ethanol-water solution at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with chloroform 5 times, combine the chloroform extracts, and evaporate to dryness under reduced pressure to obtain chloroform extract paste. c. The chloroform extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane-methanol at a concentration of 100:1-0:1, and the fraction F of 50:1-10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of acetone-water solution at a concentration of 10%-100%, and the fraction F40-90 of acetone-water solution at a concentration of 40-90% is collected. The fraction F40-90 is subjected to gel column chromatography with a gradient elution of methanol-water solution at a concentration of 50%-100%, to obtain the racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-tetrahydrofuran solution at a concentration of 99:1-50:50 to obtain compounds (1) to (14).

[0038] Example 8 (One of the four separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by cold soaking in 9 times the volume of 50% methanol-water solution at room temperature. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with ethyl acetate 4 times, combine the ethyl acetate extracts, and evaporate to dryness under reduced pressure to obtain ethyl acetate extract paste. c. The ethyl acetate extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether-acetone in a concentration of 50:1-1:1, and the fraction F in the concentration of 50:1-10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution in a concentration of 10%-90%, and the fraction F40-90 in the concentration of methanol-water solution in a concentration of 40-90% is collected. The fraction F40-90 is subjected to gel column chromatography with a gradient elution of methanol-water solution in a concentration of 50%-100%, to obtain the racemic mixtures of the compounds of formula (1) to (14). These racemic mixtures are subjected to chiral high performance liquid chromatography with a gradient elution of n-hexane-tetrahydrofuran solution in a concentration of 90:10-50:50 to obtain the compounds of formula (1) to (14).

[0039] Example 9 (One of the four separation methods) A method for preparing an amide alkaloid compound includes the following steps: a. Take 5 kg of dried Piper longum fruit, crush it, and extract it by cold soaking in 8 times the volume of 100% ethanol at room temperature. Then, evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum. b. Disperse the crude extract obtained in step a with water, extract with dichloromethane 5 times, combine the dichloromethane extracts, and evaporate to dryness under reduced pressure to obtain dichloromethane extract paste. c. The dichloromethane extract obtained in step b is subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane-methanol at a concentration of 100:1-0:1, and the fraction F at a concentration of 50:1-10:1 is collected. The fraction F is subjected to reverse-phase silica gel column chromatography with a gradient elution of acetone-water solution at a concentration of 10%-90%, and the fraction F40-90 eluted by acetone-water solution at a concentration of 40-90% is collected. The fraction F40-90 is subjected to gel column chromatography with a gradient elution of dichloromethane-methanol solution at a concentration of 50%-100%, to obtain the racemic mixtures of compounds (1) to (14). These racemic mixtures are subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-isopropanol solution at a concentration of 80:20-50:50 to obtain compounds (1) to (14).

[0040] Example 10 The present invention relates to the application of amide alkaloids isolated from Piper longum fruit in the preparation of drugs for treating diabetes, taking protein tyrosine phosphatase 1B (PTP1B) as an example; Materials and reagents: p-Nitrophenyl phosphate disodium salt (pNPP), dimethyl sulfoxide (DMSO), protein tyrosine phosphatase 1B (PTP1B), and 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethyl-N-(4-(thiazol-2-yl)aminosulfonyl)phenyl)benzofuran-6-sulfonamide were all purchased from Sigma.

[0041] Experimental methods: Using pNPP as a substrate, the activity of PTP1B was determined based on the color reaction generated by the hydrolysis of the phosphate groups of pNPP by PTP1B. 179 μL of enzyme-containing phosphate-buffered saline (PBS buffer) was added to a 96-well plate, followed by 1 μL of test sample or 1 μL of DMSO. After mixing, 20 μL of 35 mmol / L PNPP was added after 5 minutes, and the plate was incubated at room temperature in the dark for 30 minutes. An enzyme-free system was used as a blank, and 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethyl-N-(4-(thiazo-2-yl)aminosulfonyl)phenyl)benzofuran-6-sulfonamide was used as a positive control. The absorbance at 405 nm was measured using a microplate reader, and the inhibition rate at different concentrations was calculated. The inhibition rate (%) was calculated using the formula: [Enzyme activity group OD - (Drug group OD - Drug control group OD) / (Enzyme activity group OD - Enzyme activity control group OD)] × 100%. The experimental data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed between the drug-treated group and the model group using SPSS 19.0. *P<0.05 and **P<0.01 were considered statistically significant.

[0042] Experimental results: The compound of formula (14) obtained in this invention can significantly inhibit the activity of PTP1B, and its half-inhibition rate (IC50) for PTP1B is [value missing]. 50 The values ​​were 5.33-0.30 μmol / L.

[0043] Example 11 The present invention relates to the application of amide alkaloids isolated from Piper longum fruit in the preparation of drugs for treating diabetes, taking α-glucosidase as an example; Materials and reagents: disodium p-nitrophenyl phosphate (pNPP), dimethyl sulfoxide (DMSO), and α-glucosidase were all purchased from Sigma-Aldrich. Acarbose was purchased from Merck.

[0044] Experimental methods: Using pNPG as a substrate, the activity of α-glucosidase was determined by detecting changes in the product's response to pNPG. The total reaction volume was 100 μL, including 2 μL of the test sample, 71.5 μL of phosphate buffer solution at pH 6.8, and 1.5 μL of α-glucosidase. After incubation at room temperature for 10 minutes, 25 μL of 10 mmol / L pNPG was added, the mixture was shaken and incubated at 37°C for 30 minutes. The absorbance at 405 nm was measured using a microplate reader, and the inhibition rate at different concentrations was calculated. An enzyme-free solution was used as a blank, and acarbose was used as a positive control. The inhibition rate (%) was calculated using the formula: [OD of enzyme activity group - (OD of drug group - OD of drug control group) / (OD of enzyme activity group - OD of enzyme activity control group)] × 100%. The experimental data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was performed between the drug-treated group and the model group using SPSS 19.0. *P<0.05 and **P<0.01 were considered statistically significant.

[0045] Experimental results: The compounds of formulas (10), (11), and (14) obtained in this invention can significantly inhibit the activity of α-glucosidase, and their half-inhibition rate (IC50) against α-glucosidase is [value missing]. 50 The values ​​were 102.14-1.71, 157.73-9.93, and 36.79-3.97 μmol / L.

[0046] Example 12 This invention relates to the application of amide alkaloids isolated from Piper longum fruit in the preparation of drugs for treating tumors, using human cervical cancer cells (HeLa) as an example; Experimental methods: The MTT assay was used. Cells in the logarithmic growth phase were selected, the culture medium was aspirated, and the cells were washed once with phosphate-buffered saline (PBS). Cells were digested with trypsin, and culture medium was added to stop the digestion. After gentle pipetting, the cells were counted. Cells were seeded in 96-well plates (100 μL per well) and cultured overnight. The test compound (20 μL per well) was added, with a concentration gradient for each compound and three replicates for each concentration. The cells were then cultured at 37°C in a CO2 incubator for 48 hours. The old culture medium was discarded, and 100 μL of MTT was added. The cells were then cultured for another 2 hours. After incubation at 37°C for 2 hours, the absorbance (OD) at 570 nm was measured using a microplate reader.

[0047] Cell viability percentage % = (compound OD - blank OD / control group OD - blank OD) × 100%.

[0048] Cell inhibition rate % = 1 - Cell viability % = [1 - (Compound OD - Blank OD / Control group OD - Blank OD)] × 100%.

[0049] Experimental results: The compounds of formulas (6), (9), (12), (13) and (14) obtained in this invention can inhibit the proliferation activity of human cervical cancer cells (HeLa), and their half-inhibition rates (IC50) against HeLa are 21.71 ± 1.84, 15.97 ± 1.08, 48.19 ± 2.50, 15.60 ± 0.92 and 27.32 ± 1.66 μmol / L, respectively.

[0050] Example 13 The present invention relates to the application of amide alkaloids isolated from Piper longum fruit in the preparation of drugs for treating tumors, using human colon cancer cells (HT-29) as an example; The experimental method is the same as in Example 12.

[0051] Experimental results: The compounds of formulas (3), (4), (5), (6), (9), and (14) obtained in this invention can inhibit the proliferation activity of human colon cancer cells (HT-29), and their half-inhibition rate (IC50) against HT-29 is [value missing]. 50 The values ​​were 35.98±2.68, 31.89±2.26, 24.39±1.61, 8.28±0.41, 7.13±0.54 and 10.22±0.53 μmol / L, respectively.

[0052] Example 14 The present invention relates to the application of amide alkaloids isolated from Piper longum fruit in the preparation of drugs for treating tumors, using human breast cancer cells (MCF-7) as an example; The experimental method is the same as in Example 12.

[0053] Experimental results: The compounds of formulas (6), (10), and (11) obtained in this invention can inhibit the proliferation activity of human breast cancer cells (MCF-7), and their half-inhibition rate (IC50) against MCF-7 is [value missing]. 50 The values ​​were 34.40 ± 2.78, 36.90 ± 1.98 and 42.82 ± 3.34 μmol / L, respectively.

[0054] Example 15 The present invention relates to the application of amide alkaloid compounds isolated from Piper longum fruit in the preparation of anti-inflammatory drugs, using lipopolysaccharide-induced mouse macrophages RAW264.7 as an example; Materials and reagents: 15mL centrifuge tubes were purchased from Thermo Fisher Scientific (batch number: 339605), 24-well plates were purchased from Corning Incorporated (batch number: 3524), NO detection kits were purchased from Beyotime Biotechnology Co., Ltd. (batch number: S0023), Dulbecco's modified eagle medium (DMEM) containing 1% penicillin and streptomycin was used, and high-glucose medium was purchased from Hyclone Pharmaceuticals, Inc.

[0055] Experimental methods: First, the effect of the compound on cell viability was tested using the MTT assay, the same method as in Example 12. After confirming that the drug concentration had no significant effect on cell viability, different concentrations of the sample were added and incubated for 1 hour, followed by the addition of 1 μg / mL lipopolysaccharide (LPS) for a total incubation of 16 hours. After incubation, the cell supernatant was collected, and the nitric oxide content in the cell supernatant was determined using the Beyotime assay. The absorbance was measured at 540 nm using a microplate reader to create a standard curve. The nitric oxide content in the culture supernatant was calculated based on the standard curve, and the nitric oxide inhibition rate was calculated.

[0056] Inhibition rate (%) = [(model group OD)] - Sample group OD) / Model group OD ]×100% Experimental results: The compounds of formulas (1), (2), (3), (4), (5), (6), (10), and (12) obtained in this invention can inhibit the activity of nitric oxide in lipopolysaccharide-induced mouse macrophages, and their half-maximum inhibitory rate (IC50) for nitric oxide is [value missing]. 50The values ​​were 79.87 ± 0.92, 95.33 ± 6.57, 96.55 ± 8.20, 91.36 ± 3.10, 81.70 ± 5.43, 101.61 ± 4.24, 72.97 ± 5.45 and 16.30 ± 2.15 μmol / L, respectively.

[0057] The above results indicate that the compounds of formulas (1) to (14) can effectively inhibit the activity of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, human cervical cancer cells (HeLa), human colon cancer cells (HT-29), human breast cancer cells (MCF-7), and lipopolysaccharide-induced mouse macrophages, and have therapeutic effects on diabetes, tumors, and inflammation. They can be used as pharmaceuticals for the treatment of diabetes, tumors, and inflammation.

[0058] In summary, all reagents used in the above examples were of analytical grade. Methanol, acetonitrile, ethanol, and n-hexane in the high-performance liquid chromatography (HPLC) were of HPLC grade (Merck, Germany). The column chromatography equipment included: normal-phase silica gel (100-200 mesh): produced by Qingdao Marine Chemical Plant; reversed-phase silica gel (ODS): gel: Sephadex LH-20 dextran gel (Switzerland); and HPLC (Dionex, USA) with the following setup: P680 HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, and Chameleon chromatography workstation. The preparative high-performance liquid chromatography (HPLC) setup (Dionex Corporation, USA) was as follows: P680 HPLC pump, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, and Chameleon chromatography workstation; mass spectrometry was performed using a quadrupole-time-of-flight hybridization mass spectrometer (Applied Biosystems, USA); nuclear magnetic resonance (NMR) was performed using a VARIAN VNMRS 600 MHz NMR spectrometer; and ECD spectral data were obtained using a Chirascan circular dichroism spectrometer (UK).

[0059] The long pepper fruit was harvested from the Qinzhou area of ​​Guangxi.

[0060] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An amide alkaloid compound, characterized in that, The structural formula of this compound is: The compound of formula (1) is (2E,4E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (2) is (2E,4E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8,9-dihydroxy-1-piperidin-1-ylnon-2,4-dien-1-one; The compound of formula (3) is (2E,4E,10S,11S)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (4) is (2E,4E,10R,11R)-11-(1,3-benzodioxolane-5-yl)-10,11-dihydroxy-1-piperidin-1-ylundec-2,4-dien-1-one; The compound of formula (5) is (2E,8S,9R)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (6) is (2E,8R,9S)-9-(1,3-benzodioxolane-5-yl)-8-hydroxy-9-methoxy-1-piperidin-1-ylnon-2-en-1-one; The compound of formula (7) is (2E,4S,5R)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (8) is (2E,4R,5S)-5-(1,3-benzodioxolane-5-yl)-4-hydroxy-5-methoxy-1-piperidin-1-ylpent-2-en-1-one; The compound of formula (9) is (1R,2S,4aR,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (10) is (1S,2S,4aR,8aR)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (11) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-isobutyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (12) is (1R,2R,4aS,8aS)-1-(1,3-benzodioxolane-5-yl)-N-piperidinyl-1,2,4a,5,6,7,8,8a-octahydronaphthalene-2-carboxamide; The compound of formula (13) is (3R,3a,S,4S,7R,7aR)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one; The compound of formula (14) is (3S,3aR,4R,S,7aS)-4-(1,3-benzodioxolane-5-yl)-2-isobutyl-7-(piperidine-1-carbonyl)-2,3,3a,4,7,7a-hexahydro-1H-2-isoindol-1-one.

2. The method for preparing an amide alkaloid compound according to claim 1, characterized in that, Includes the following steps: a. Take Piper longum fruit as raw material, crush it, and then extract it by percolation or cold soaking at room temperature with 5-12 times the volume of ethanol aqueous solution with a volume concentration of 50%-100% or methanol aqueous solution with a volume concentration of 50%-100%. Evaporate the solvent under reduced pressure at 45℃ to obtain crude extract of Piper longum fruit. b. Disperse the crude extract obtained in step a with water, add ethyl acetate, dichloromethane or trichloromethane for extraction 3-5 times, concentrate the ethyl acetate, dichloromethane or trichloromethane extract to obtain ethyl acetate, dichloromethane or trichloromethane extract paste. c. Separate the ethyl acetate, dichloromethane or trichloromethane extract obtained in step b using three or four of the following methods: normal phase silica gel column chromatography, reversed phase silica gel column chromatography, gel column chromatography and chiral high performance liquid chromatography, to obtain compounds of formula (1) to (14).

3. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, The three separation methods in step c are: The ethyl acetate, dichloromethane, or chloroform extract obtained in step b was subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether-acetone or dichloromethane-methanol at a volume ratio of 100:1-0:

1. The fraction F of 50:1-10:1 was collected and subjected to reverse-phase silica gel column chromatography with a gradient elution of methanol-water solution or acetone-water solution at a concentration of 10%-100% to obtain racemic mixtures of compounds (1) to (14). The racemic mixtures were then subjected to chiral high-performance liquid chromatography with a gradient elution of n-hexane-isopropanol or n-hexane-ethanol solution at a concentration of 99:1-50:50 to obtain compounds (1) to (14).

4. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, The four separation methods in step c are: The ethyl acetate, dichloromethane, or trichloromethane extract obtained in step b was subjected to normal-phase silica gel column chromatography, with a gradient elution of petroleum ether-ethyl acetate or dichloromethane-methanol at a volume ratio of 100:1 to 0:

1. The 50:1 to 10:1 fraction F was collected. Fraction F was then subjected to reverse-phase silica gel column chromatography, with a gradient elution of 10% to 100% methanol-water solution or 10% to 100% acetone-water solution, and the 40% to 90% methanol-water solution or acetone-water solution was collected. The aqueous solution eluent fraction F40-90 was subjected to gel column chromatography and eluted with a gradient of 50%-100% methanol-water solution or 50%-100% dichloromethane-methanol solution to obtain the racemic mixtures of compounds (1) to (14). The racemic mixtures were then subjected to chiral high performance liquid chromatography and eluted with a gradient of 99:1-50:50 hexane-isopropanol or hexane-ethanol solution to obtain the compounds (1) to (14).

5. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, In step c, the normal-phase silica gel column chromatography is atmospheric or pressurized column chromatography, the packing material is normal-phase silica gel, and the eluent is a mixture of two solvents, namely petroleum ether or dichloromethane, ethyl acetate or methanol, using isocratic elution or gradient elution.

6. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, In step c, the reversed-phase silica gel column chromatography is atmospheric pressure column chromatography, and the eluent is a gradient elution of 10%-100% methanol-water solution or 10%-100% acetone-water solution.

7. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, In step c, the gel column chromatography is atmospheric pressure column chromatography, the packing material is Sephadex LH-20 gel, and the eluent is a gradient elution of 50%-100% methanol-water solution or 50%-100% dichloromethane-methanol solution.

8. The method for preparing an amide alkaloid compound according to claim 2, characterized in that, In step c, the chiral high-performance liquid chromatography is pressure column chromatography, and the eluent is a gradient elution of n-hexane-isopropanol or n-hexane-ethanol solution with a concentration of 99:1-50:

50.

9. Use of the amide alkaloid compound of claim 1 in the preparation of hypoglycemic, antitumor and anti-inflammatory drugs.

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