Total synthesis method and application of furanose ring pyrrole spiroketal alkaloid

Abstract

The invention discloses a total synthesis method and application of furanose ring pyrrole spiroketal alkaloid, and belongs to the technical field of chemical synthesis and biological medicine. According to the method, D-glucose is used as a raw material and reacts with dibenzylamine, 1-dibenzylamino-1-deoxy-D fructose is prepared through an Amadori rearrangement reaction, 1-amino-1-deoxy-D fructose is prepared through direct hydrogenation debenzylation, or 2-hydroxyl of 1-dibenzylamino-1-deoxy-D fructose is protected by adding methyl and then debenzylation protection is carried out, and 1-amino-1-deoxy-D fructose is obtained. The preparation method comprises the following steps: preparing 1-amino-1-deoxy-2-methoxyl-D-fructose; and respectively carrying out Maillard condensation reaction on the two obtained amino sugars and dihydropyrone, and then completing spiro reaction under an acidic condition to obtain acorine B and pinosine C. The acorine B and pinosine C synthesized by the invention can obviously reduce the weight of an aged mouse, reduce the fat index, reduce lipid droplets in the liver and fat in thymus, reduce the number of aged cells and improve the proportion of T cells in immune cells, and have the potentials of reducing fat and resisting immune aging.

Description

Technical Field

[0001] The present invention belongs to the technical fields of chemical synthesis and biomedicine, and specifically relates to a total synthesis method of furanose ring pyrrolidine spiroketal alkaloids and their applications in reducing fat and / or anti-immune aging. Technical Background

[0002] The process in which aging leads to a decline in immune system function is called immune aging. As people age, the structure and function of the immune system gradually degenerate, mainly affecting the composition, quantity, and function of immune organs, immune cells, and cytokines. It is one of the important signs of aging and is closely related to various senile diseases. Aging is also accompanied by changes in adipose tissue metabolism. The fat mass of both mice and humans increases with age. During the aging process, significant changes occur in adipose tissue, such as abundance, distribution, and cellular composition. These changes accelerate the onset of age-related diseases (Frasca D, Blomberg BB. Adipose tissue, immune aging, and cellular senescence. Semin Immunopathol. 2020. 42(5):573-587).

[0003] Since the initial discovery of pyrrolidine spiroketal alkaloids from Astragalus membranaceus var. mongholicus, the research on such alkaloids has been carried out for more than 20 years. The structures of such alkaloids can be divided into three parts: the sugar ring part, the morpholine ring part, and the pyrrole ring part. The main structural difference lies in the sugar ring part, which is divided into five-membered sugar ring (furan type) and six-membered sugar ring (pyran type) pyrrolidine spiroketal alkaloids. Secondly, it is the chiral configuration of the spiro carbon atom.

[0004] So far, researchers have successively extracted and discovered such alkaloids from traditional Chinese herbal medicines such as Acorus tatarinowii and Capparis spinosa, as well as rape pollen, whole wheat bread, Xylaria nigripes, and the Chinese patent medicine Shensong Yangxin Capsule. The plants, fungi, and Chinese patent medicines from which such alkaloids are derived have pharmacological effects such as antioxidant, anti-tumor, anti-arrhythmic, and reducing the risk of chronic diseases. The natural content of such compounds is extremely low and extraction is difficult. Cheng Yongxian et al. (Tong X, Zhou L, Wang Y, et al. Acortatarins A and B, Two Novel Antioxidative Spiroalkaloids with a Naturally Unusual Morpholine Motif from Acorus tatarinowii [J]. Organic Letters, 2010, 12(8): 1844-1847) extracted 7.3 mg of acortatarin A and 3.4 mg of acortatarin B from 50 kg of dried Acorus tatarinowii rhizomes. Yao Xinsheng et al. (Ding B, Dai Y, Hou Y L, et al. Spiroalkaloids from Shensong Yangxin capsule [J]. J Asian Nat Prod Res, 2015, 17(5): 559-566.) extracted 8.1 mg of shensongine A, 2.8 mg of shensongine B, and 14.8 mg of shensongine C from 2 kg of the powder of the Chinese patent medicine Shensong Yangxin Capsule.

[0005] Given the unique structure and potential biological activity of this class of compounds, the chemical synthesis of these compounds has become a topic of interest for many researchers. There have been successive reports on the total synthesis of pentose-ring pyrrolospiroketal alkaloids. Geng et al. (Geng H, Chen J L Y, Furkert D, et al. A convergent synthesis of the 2-formylpyrrolespiroketal natural product acortatarin A[J]. Synlett, 2012, 23(6):855-858.) started with cyclohexanone-protected dihydroxypropanal. First, the carbon chain was extended through an addition reaction, then the cyclohexanone protection was removed, the terminal hydroxyl group was protected with silyl, and the vicinal dihydroxy group was protected with isopropylidene; then the double bond was epoxidized, and sodium azide treatment gave a primary amine; then a Maillard condensation reaction was carried out with dihydropyranone, and cyclization occurred under acidic conditions. After removing the protection in a total of 11 steps, Acortatarin A (yield 5.28%) and Shensongine B (yield <1%) were obtained. Tan et al. (Wurst J M, Verano A L, Tan D S. Stereoselective synthesis of acortatarins A and B[J]. Org Lett, 2012, 14(17):4442-4445.) started with thymidine and obtained intermediates through a series of reactions. Three alkaloids could be obtained separately using different catalysts. Among them, with dimethyldioxirane catalysis, the double bond was first oxidized to an epoxy ring structure, then the formyl group on the pyrrole was reduced to a hydroxymethyl group, and nucleophilic attack opened the epoxy ring to obtain Acortatarin B; with mercury salt catalysis, starting from the same substrate thymidine, intermediates were obtained through a series of reactions to obtain Acortatarin A and Shensongine B. This method involved a total of 9 steps for the synthesis of Acortatarin A with a yield of about 30%, 8 steps for the synthesis of Acortatarin B with a yield of about 41%, and 9 steps for the synthesis of Shensongine B with a yield <1%.Aponick et al. (Borrero N V, Aponick A. Total Synthesis of Acortatarin A Using a Pd(II)-Catalyzed Spiroketalization Strategy[J]. The Journal of Organic Chemistry, 2012, 77(19):8410-8416.) developed a synthetic route with the spirocyclization reaction catalyzed by Pd(II) as the key step using δ-hydroxy esters as raw materials, and obtained Acortatarin A and Shensongine B. Starting from δ-hydroxy esters, a dithioalkyl group was introduced through bromination, methylation, and nucleophilic substitution, and then through ester group transformation, oxidative deprotection to form a ketone, bromination and other 7 steps to obtain an intermediate. After introducing a pyrrole group, 10% Pd-(PhCN)2Cl2 was used as a catalyst for cyclization to obtain a 1:1 intermediate, and Acortatarin A and Shensongine B were obtained respectively through 4 steps of oxidation, deprotection, etc. Acortatarin A was obtained with a yield of 7.5% and Shensongine B with a yield of <1% through 15 steps. Kuwahara et al. (Teranishi T, Kageyama M, Kuwahara S. Concise Total Synthesis of Acortatarin A[J]. Bioscience, Biotechnology, and Biochemistry, 2013, 77(3):676-678.) used mannitol as a raw material, introduced a pyrrole group through affinity substitution after 7 steps of reaction such as acetonide double protection, lead acetate carbon chain cleavage, and bromination treatment, catalyzed spirocyclization with boron tribromide, and deprotected to obtain Acortatarin A and Shensongine B. Utilizing the reaction characteristics of sodium periodate to oxidize vicinal diols under alkaline conditions, the six-membered sugar ring product generated during the spirocyclization process was removed. Acortatarin A was obtained with a yield of 4.8% and Shensongine B with a yield of 2.3% through 10 steps.Li et al. (Li M, Xiong J, Huang Y, et al. Xylapyrrosides A and B, two rare sugar-morpholine spiroketalpyrrole-derived alkaloids from Xylaria nigripes: isolation, complete structure elucidation, and total syntheses [J]. Tetrahedron, 2015, 71 (33): 5285-5295.) used dibenzyl ether as the raw material, changed the protection order, and then introduced the pyrrole group, spirocyclized under acidic conditions, and deprotected with titanium tetrachloride to obtain Acortatarin A and Shensongine B. After 14 steps of reaction, Acortatarin A was obtained with a yield of 32% and Shensongine B with a yield of 37%. Wimmer et al. (Wimmer E, Borghèse S, Blanc A, et al. Zeolite-Based Organic Synthesis (ZeoBOS) of Acortatarin A: First Total Synthesis Based on Native and Metal-Doped Zeolite-Catalyzed Steps [J]. Chemistry A European Journal, 2017, 23 (7): 1484-1489.) completed the synthesis of Acortatarin A and Shensongine B using zeolite catalysis, mainly using hydrogen protons or metal-doped zeolite catalysis; starting from 2-deoxyribose, they chose a similar method of Sudhakar et al., firstly selectively benzyl-protected, then nucleophilically substituted with the intermediate by Wittig reaction to grow the carbon chain, then spirocyclized under zeolite catalysis, and finally removed the protecting group in 11 steps to obtain the target products Acortatarin A and Shensongine B. The yield of Acortatarin A was 2%, and the yield of Shensongine B was <1%.

[0006] The reported synthetic routes to obtain pentacyclic pyrrole spiro ketal alkaloids require at least 8 steps and at most 15 steps. The synthetic processes are relatively complicated and the yields are generally low, which increases the synthetic cost. Research and development of new synthetic methods for pentacyclic pyrrole spiro ketal alkaloids has become an important topic that needs to be studied urgently. Summary of the invention

[0007] In view of the current situation of the synthesis of furanose-ring pyrrolidine spiroketals, the purpose of the present invention is to provide a concise total synthesis method of furanose-ring pyrrolidine spiroketal alkaloids and their applications in reducing fat and / or anti-immune aging.

[0008] Using D-glucose as a raw material, reacting with dibenzylamine, and preparing 1-dibenzylamino-1-deoxy-D-fructose through an Amadori rearrangement reaction. Then, directly hydrogenating to remove the benzyl group to obtain 1-amino-1-deoxy-D-fructose, or protecting the 2-hydroxy group of 1-dibenzylamino-1-deoxy-D-fructose with a methyl group and then removing the benzyl protection to obtain 1-amino-1-deoxy-2-methoxy-D-fructose. Subsequently, the two amino sugars are respectively subjected to Maillard condensation reaction with dihydropyranone, and then the spirocyclization reaction is completed under acidic conditions to obtain acorus alkaloid B and sanshool C. It only takes 4 steps to obtain acorus alkaloid B with a yield of 3.4%, and 5 steps to obtain sanshool C with a yield of 3.1%. Moreover, the synthetic route of the present invention can directly obtain five-membered sugar-ring pyrrolidine spiroketal alkaloids using a six-membered sugar ring as a raw material. The acorus alkaloid B and sanshool C synthesized by the present invention can significantly reduce the body weight of aging mice, reduce lipid droplets in liver tissue and fat in thymus tissue, reduce the number of aging cells and increase the proportion of naive T cells in immune cells, and have the potential for reducing fat and anti-immune aging.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention provides a total synthesis method of furanose-ring pyrrolidine spiroketal alkaloids, and the synthetic route is as follows:

[0011]

[0012] Based on the above technical solutions, further, the total synthesis method includes the following steps:

[0013] (1) Using D-glucose as a raw material, under acidic conditions, reacting with dibenzylamine through an Amadori rearrangement reaction,

[0014] The reaction temperature is 50 - 90 °C, and the reaction time is 2 - 5 h to obtain compound I;

[0015]

[0016] (2) Compound I is obtained under the catalytic reaction of anhydrous hydrogen chloride prepared in a system of lower alcohol and acetyl chloride or a system of lower alcohol and trimethylchlorosilane. The reaction temperature is 25 - 80 °C, and the reaction time is 5 - 48 h;

[0017]

[0018] (3) Using palladium on carbon as a catalyst, the benzyl group of compound I or IV is hydrogenated and reduced in an organic solvent under a hydrogen atmosphere to obtain compound II or V. The reaction temperature is 0 - 50 °C, and the reaction time is 4 - 90 h;

[0019]

[0020] (4) Under acidic conditions, compound II or V undergoes a Maillard condensation reaction with dihydropyranone to obtain compound III or VI. The reaction temperature is 15 °C - 100 °C, and the reaction time is 8 - 24 h;

[0021]

[0022] (5) Compound III or VI undergoes spirocyclization under acidic conditions to obtain acortatarin B or shensongine C. The reaction temperature is -10 - 50 °C, and the reaction time is 2 - 24 h;

[0023]

[0024] Based on the above technical solution, further, the acidic reagent providing the acidic condition in step (1) is an organic acid, including formic acid and acetic acid, and the solvents include methanol and ethanol.

[0025] Based on the above technical solution, further, the lower alcohols described in step (2) include methanol and ethanol.

[0026] Based on the above technical solution, further, the organic solvents described in step (3) include methanol and ethanol.

[0027] Based on the above technical solution, further, the acidic condition in step (4) is provided by a protonic acid or a Lewis acid. The protonic acid is acetic acid, and the Lewis acid is zinc chloride or zinc acetate. The solvents include methanol, ethanol, and dioxane.

[0028] Based on the above technical solution, further, the acidic reagents described in step (5) include p-toluenesulfonic acid, methanesulfonic acid, sulfamic acid, camphorsulfonic acid, or hydrochloric acid, and the solvents include tetrahydrofuran, dichloromethane, and chloroform.

[0029] In the second aspect, the present invention provides the furanosyl pyrrole spiroketal alkaloid obtained by the above total synthesis method.

[0030] In the third aspect, the present invention provides the application of the above furanosyl pyrrole spiroketal alkaloid in the preparation of drugs, health products, or functional foods for reducing fat.

[0031] Based on the above technical solution, further, drugs, health products or functional foods containing acorus alkaloid B and / or notopteropine C can reduce body weight, and reduce lipid droplets in liver tissue and fat in thymus tissue.

[0032] Fourthly, the present invention provides the application of the above-mentioned furanose ring pyrrolospiroketal alkaloid in the preparation of drugs, health products or functional foods for anti-immune aging.

[0033] Based on the above technical solution, further, drugs, health products or functional foods containing acorus alkaloid B and / or notopteropine C can delay the aging of thymus and spleen, and increase the proportion of naive T cells in peripheral blood, thymus and spleen immune cells.

[0034] The beneficial effects of the present invention compared with the prior art are as follows:

[0035] 1. The synthesis steps of acorus alkaloid B and notopteropine C of the present invention are simple, the raw materials are easily obtained and inexpensive, environmentally friendly, and easy to prepare in large quantities.

[0036] 2. The synthesized acorus alkaloid B and notopteropine C of the present invention can significantly reduce the body weight of aging mice, reduce lipid droplets in the liver and fat in the thymus, reduce the number of senescent cells and increase the proportion of naive T cells in immune cells, and have the potential for fat reduction and anti-immune aging. Description of the Drawings

[0037] In order to more clearly illustrate the embodiments of the present invention, the drawings involved in the embodiments will be briefly introduced below.

[0038] Figure 1 It is the HPLC diagram of acorus alkaloid B and notopteropine C. A: HPLC diagram of acorus alkaloid B, B: HPLC diagram of notopteropine C.

[0039] Figure 2 It is the curve graph of the body weight change of each group of mice. Compared with the blank group, ****p < 0.0001.

[0040] Figure 3 It is the HE staining diagram of the liver of each group of mice. A: blank group, B: metformin group, C: acorus alkaloid B group, D: notopteropine C group; scale bar: 100 μm.

[0041] Figure 4 It is the oil red O staining diagram of the liver of each group of mice. A: blank group, B: metformin group, C: acorus alkaloid B group, D: notopteropine C group; scale bar: 30 μm.

[0042] Figure 5 It is the diagram of thymus, spleen and fat index of each group of mice. A: thymus index, B: spleen index, C: fat index. Compared with the blank group, *p < 0.05.

[0043] Figure 6 HE staining images of the thymus of mice in each group (two samples in each group). A: blank group, B: metformin group, C: acorus alkaloid B group, D: salsolinol C group; scale bar: 600 μm.

[0044] Figure 7 Senescence-associated β-galactosidase staining images of the thymus tissues of mice in each group. A: blank group, B: metformin group, C: acorus alkaloid B group, D: salsolinol C group; scale bar: 30 μm.

[0045] Figure 8 Senescence-associated β-galactosidase staining images of the spleen tissues of mice in each group. A: blank group, B: metformin group, C: acorus alkaloid B group, D: salsolinol C group; scale bar: 30 μm.

[0046] Figure 9 Graph showing the changes in the content of peripheral blood T lymphocytes in mice in each group. A: proportion of CD3+CD4+ T cells in lymphocytes, B: proportion of CD3+CD8+ T cells in lymphocytes, C: proportion of naive T cells in CD3+CD4+ T cells, D: proportion of naive T cells in CD3+CD8+ T cells; compared with the blank group, *p < 0.05, **p < 0.01.

[0047] Figure 10 Graph showing the changes in the content of thymus T lymphocytes in mice in each group. A: proportion of CD3+CD4+ T cells in lymphocytes, B: proportion of CD3+CD8+ T cells in lymphocytes; compared with the blank group, *p < 0.05.

[0048] Figure 11 Graph showing the changes in the content of spleen T lymphocytes in mice in each group. A: proportion of CD3+CD4+ T cells in lymphocytes, B: proportion of CD3+CD8+ T cells in lymphocytes. Detailed implementation manners

[0049] The present invention will be further described in detail below in conjunction with the embodiments, but the implementation manners of the present invention are not limited thereto.

[0050] Example 1

[0051] This example provides a synthesis method of acorus alkaloid B and salsolinol C, and the synthesis route is as follows:

[0052]

[0053] It includes the following steps:

[0054] Preparation of Compound I: Weigh 10.0 g of D-glucose and add it to a 500 mL three-necked flask. Then add 120 mL of absolute ethanol in sequence and stir evenly. After that, weigh dibenzylamine (11.0 g, 55.56 mmol, 1 equiv) and add it to the reaction flask. Add 3 mL of glacial acetic acid, heat to 80 °C, reflux and stir for 3 h. After it cools to room temperature, filter, and wash the filter residue with ethanol multiple times to obtain 16.1 g of white needle-like solid with a yield of 82%.

[0055] 1 H NMR (400 MHz, DMSO) δ 7.30 (dt, J = 23.2, 7.2 Hz, 10H), 5.25 (s, 1H), 4.43 (d, J = 4.7 Hz, 1H), 4.38 (d, J = 2.7 Hz, 1H), 4.15 (t, J = 6.5 Hz, 1H), 3.81 (d, J = 12.0 Hz, 1H), 3.74 (d, J = 13.8 Hz, 2H), 3.63 (d, J = 13.8 Hz, 2H), 3.63 (1H), 3.58 (d, J = 2.3 Hz, 2H), 3.43 (d, J = 12 Hz, 1H), 2.68 (q, J = 13.3 Hz, 2H).

[0056] Preparation of Compound II: Take Compound I (7.18 g, 20 mmol), add 75 mL of glacial acetic acid, dissolve it by ultrasonic treatment, add 150 mL of absolute ethanol, displace with N2 three times, add 1.436 g of 10% Pd / C, and displace with H2 three times. React at room temperature for 72 h. Monitor by TLC (chloroform:methanol:water = 10:10:2) until the raw material spot disappears. Filter with diatomaceous earth as a filter aid, filter off Pd / C, evaporate the ethanol solvent, and recrystallize with ether to obtain 3 g of white solid with a yield of 63%. 1 H NMR (400 MHz, DMSO) δ 3.88–3.76 (m, 1H), 3.73–3.55 (m, 2H), 3.52–3.45 (m, 1H), 3.43–3.34 (m, 1H), 2.71 (t, J = 16.8 Hz, 2H), 1.82 (s, 3H).

[0057] Preparation of Compound III: Weigh Compound II (1.2 g, 5 mmol, 1.2 equiv), dissolve it by ultrasonic oscillation in 120 mL of 1,4-dioxane. Weigh dihydropyranone (0.6 g, 4.16 mmol, 1 equiv), dissolve it in 60 mL of 1,4-dioxane solvent. Mix the above solutions, place them in a 500 mL round-bottom flask, add 60 mL of glacial acetic acid, displace with nitrogen three times, stir and react at 50 °C. After about 20 hours, when TLC detection (chloroform:methanol:water = 10:3:1) shows that the raw materials disappear, stop the reaction. Rotate and evaporate to remove the dioxane solvent, and perform silica gel column chromatography (dichloromethane:methanol = 12:1) to obtain 0.35 g of a yellow oil, with a yield of 30%; ESI-MS calculated for C 12 H 17 NO7; [M-H] + 286.10, found 286.1.

[0058] Preparation of Compound IV: Weigh Compound I (10.77 g, 30 mmol), dissolve it in 2 mol / L hydrochloric acid, add an appropriate amount of methanol and spin-dry; transfer it to a 500 mL three-necked flask, successively add 150 mL of methanol and 3.12 mL of acetyl chloride, heat to 65 °C, reflux and stir; react for about 12 h, monitor the reaction by TLC (dichloromethane:methanol = 10:1) until the reaction is complete; add 100 mL of saturated NaHCO3 aqueous solution to neutralize to neutral, extract with 120 mL of ethyl acetate three times, dry over anhydrous sodium sulfate, and spin-dry the solvent; recrystallize with n-hexane, wash with n-hexane:dichloromethane = 1:1 to obtain 5.0 g of a white solid, with a yield of 43%; 1 H NMR (400 MHz, DMSO) δ 7.42 - 7.19 (m, 10H), 5.53 (d, J = 5.0 Hz, 1H), 4.97 (d, J = 5.7 Hz, 1H), 4.74 (t, J = 5.5 Hz, 1H), 3.83 (dd, J = 4.8, 2.6 Hz, 1H), 3.66 (d, J = 13.6 Hz, 2H), 3.62 (dd, J = 5.8, 2.8 Hz, 1H), 3.60 - 3.56 (m, 1H), 3.56 - 3.48 (m, 3H), 3.40 (dt, J = 11.4, 5.7 Hz, 1H), 3.13 (s, 3H), 2.72 (s, 2H).

[0059] Preparation of Compound V: Take Compound IV (4.5 g, 12.06 mmol), dissolve it in 135 mL of methanol, displace with N2 three times, add 0.9 g of 10% Pd / C, and displace with H2 three times; react at room temperature for 4 h, monitor by TLC (chloroform: methanol: water = 10:3:1) until the raw material spot disappears, filter with diatomaceous earth as a filter aid, filter out Pd / C, evaporate the methanol solvent, and obtain 2.6 g of a pale yellow oily substance with a crude yield of 110%.

[0060] Preparation of Compound VI: Weigh Compound V (0.8 g, 4.15 mmol, 1.2 equiv), dissolve it by ultrasonic oscillation in 80 mL of 1,4-dioxane, weigh dihydropyranone (0.48 g, 3.33 mmol, 1 equiv), dissolve it in 30 mL of 1,4-dioxane solvent, mix the above solutions, place them in a 250 mL round-bottom flask, add 15 mL of glacial acetic acid, displace with nitrogen three times, stir and react at room temperature. After about 24 h, monitor by TLC (chloroform: methanol: water = 10:3:1) until the raw materials disappear, stop the reaction, rotary evaporate to remove the dioxane solvent, and perform silica gel column chromatography (dichloromethane: methanol = 20:1) to obtain 0.44 g of a yellow oily substance with a yield of 44%. 1 HNMR (600 MHz, DMSO) δ 9.49 (s, 1H), 6.87 (d, J = 2.9 Hz, 1H), 6.18 (d, J = 4.0 Hz, 1H), 5.66 (d, J = 1.5 Hz, 1H), 5.25 (d, J = 1.5 Hz, 1H), 5.06 (td, J = 5.7, 1.5 Hz, 1H), 4.61 (d, J = 5.5 Hz, 1H), 4.57 (d, J = 6.1 Hz, 2H), 4.52 (d, J = 15.0 Hz, 1H), 4.08 (t, J = 6.1 Hz, 1H), 3.70 (q, J = 6.6 Hz, 1H), 3.65 (ddd, J = 8.3, 6.1, 2.6 Hz, 1H), 3.56 - 3.50 (m, 1H), 3.39 (dt, J = 11.8, 6.1 Hz, 1H), 3.17 (dd, J = 5.3, 1.5 Hz, 1H), 2.89 (s, 3H).

[0061] Preparation of Acorus alkaloid B: Weigh Compound III (640 mg, 2.21 mmol) and dissolve it in 100 mL of tetrahydrofuran, add 1 mL of 4M HCl, react at room temperature for 6 h, monitor by TLC (dichloromethane: methanol = 8:1) until the reaction is complete and the raw materials disappear, add saturated aqueous NaHCO3 to adjust to neutral, dry and rotary evaporate, then use a Buchner funnel to filter and remove salts, and load the sample onto a preparative high-performance liquid chromatography XB-C18 (21.2×250 mm, 5 μm), eluted with an eluent of methanol:water = 25:75 by volume ratio, to obtain 132 mg of Acortatarin B with a yield of 22%. 1 H NMR (500 MHz, CD3OD) δ 9.39 (s, 1H), 7.03 (d, J = 4.1 Hz, 1H), 6.09 (d, J = 4.1 Hz, 1H), 5.14 (d, J = 15.5 Hz, 1H), 4.88 (d, J = 15.4 Hz, 1H), 4.51 (d, J = 14.0 Hz, 1H), 4.31 (d, J = 14.0 Hz, 1H), 4.08 (t, J = 7.7 Hz, 1H), 3.94 - 3.84 (m, 2H), 3.73 (dd, J = 12.0, 3.3 Hz, 1H), 3.61 (dd, J = 12.0, 7.1 Hz, 1H).

[0062] Preparation of Shensongine C: Weigh 650 mg (2.2 mmol) of Compound VI, dissolve it in 125 mL of THF, add 0.25 mL of 4M HCl, monitor by TLC (dichloromethane:methanol = 8:1), react at room temperature for 6 h until the raw material is completely converted, adjust to neutral with saturated aqueous NaHCO3 solution, dry and evaporate to dryness, then filter to remove salts using a Buchner funnel, and load the sample onto preparative high-performance liquid chromatography XB-C18 (21.2×250 mm, 5 μm), eluted with an eluent of methanol:water = 25:75 by volume ratio, to obtain 116 mg of Shensongine C with a yield of 20%. 1 H NMR (400 MHz, CD3OD) δ 9.38 (s, 1H), 7.02 (d, J = 4.2 Hz, 1H), 6.08 (d, J = 4.2 Hz, 1H), 4.98 (d, J = 15.7 Hz, 1H), 4.86 (d, J = 15.7 Hz, 1H), 4.57 (d, J = 14.6 Hz, 1H), 4.28 (d, J = 14.6 Hz, 1H), 4.07 (d, J = 2.2 Hz, 1H), 3.94 (dd, J = 4.9, 2.5 Hz, 2H), 3.77 (dd, J = 12.1, 2.5 Hz, 1H), 3.66 (dd, J = 12.0, 4.9 Hz, 1H).

[0063] Example 2

[0064] Determination of the chromatographic conditions for Acorus tatarinowii alkaloid B and Shensongine C

[0065] Experimental method: The chromatographic detection conditions were as follows: The chromatographic column was Agilent XDB-C18 (4.6×150 mm, 5 μm), the mobile phase was methanol: water = 20:80 (volume ratio), the flow rate was 1 mL / min, isocratic elution was used, the column temperature was 30 °C, the injection volume was 10 μL, and the detection wavelength was 254 nm. The liquid chromatograms of acorus alkaloid B and chensong alkaloid C were measured on a Waters E2695 as shown in Figure 1 shown.

[0066] Example 3

[0067] Experimental animals

[0068] Experimental method: 36 18-month-old naturally aging female Kunming mice were randomly divided into 4 groups, with 9 mice in each group, namely the blank control group, the metformin positive control group, the acorus alkaloid B administration group, and the chensong alkaloid C administration group. The blank control group was intraperitoneally injected with normal saline every day, the positive control group was intragastrically administered 100 mg / kg metformin every day, and the acorus alkaloid B administration group and the chensong alkaloid C administration group were intraperitoneally injected with 20 mg / kg acorus alkaloid B and 20 mg / kg chensong alkaloid C respectively every day for 30 days. After 30 days, blood was taken from the orbital cavity, the mice were sacrificed by cervical dislocation, and the thymus, spleen, and adipose tissue were obtained by dissecting the mice for subsequent experiments.

[0069] Example 4

[0070] Body weight change of mice

[0071] Experimental method: During the experiment in Example 3, the body weight of the mice was weighed and recorded every 3 days.

[0072] The experimental results were as shown in Figure 2 shown. The body weight of the mice in the blank group decreased by 4.1%, the body weight of the mice in the metformin group decreased by 10.6%, and the body weights of the mice in the acorus alkaloid B and chensong alkaloid C groups decreased by 12.7% and 9.9% respectively, with no significant difference from the metformin group.

[0073] Example 5

[0074] Liver fat change

[0075] Experimental method: After the experiment in Example 3, 3 freshly obtained mouse livers were randomly selected from each group and fixed in 4% paraformaldehyde solution, and part of the liver was stored at -80 °C. Subsequently, Wuhan Sevier Biotechnology Co., Ltd. was commissioned to complete HE staining, oil red O staining, and detection.

[0076] The experimental results were as shown in Figure 3As shown, the overall morphology of the liver tissue after drug administration was significantly better than that of the blank group. The degree of steatosis decreased, the infiltration of inflammatory cells decreased, the nuclear morphology was regular, and almost no visible pathological changes were observed. Oil red O was used for fat staining in the tissue. After staining, the cell nuclei were blue, and the lipid droplets were orange or bright red. As Figure 4 shown, after drug administration, the lipid droplets in the livers of 18-month-old mice could be significantly reduced.

[0077] Example 6

[0078] Changes in thymus, spleen and fat indexes

[0079] Experimental method: After the experiment in Example 3 was completed, the thymus, spleen and adipose tissues of the mice were weighed, and the thymus index, spleen index and fat index were calculated. The calculation formulas are as follows: Organ or tissue index = Organ or tissue weight (mg) / Body weight (g)

[0080] The experimental results are as Figure 5 shown. The spleen indexes of the drug administration groups were all higher than those of the blank group, and the group of acorus alkaloid C was significantly higher than the blank group; the thymus indexes of the drug administration groups had an increasing trend compared with the blank group. It indicates that acorus tatarinowii alkaloid B and acorus alkaloid C have immunomodulatory effects. The fat indexes of the drug administration groups had a decreasing trend compared with the blank group, and the group of acorus alkaloid C was significantly lower than the blank group, indicating that acorus tatarinowii alkaloid B and acorus alkaloid C have fat-reducing effects.

[0081] Example 7

[0082] Changes in thymus tissue

[0083] Experimental method: After the experiment in Example 3 was completed, 3 freshly obtained mouse thymuses in each group were randomly taken and fixed in 4% paraformaldehyde solution, and some thymuses were stored at -80 °C. Subsequently, Wuhan Sevier Biotechnology Co., Ltd. was entrusted to complete HE staining.

[0084] The experimental results are as Figure 6 shown. In the blank group, the boundary between the cortex and medulla was blurred, a large amount of adipose tissue replaced, and the lymphocyte density decreased, indicating severe thymic involution. In contrast, in the drug administration groups, the cortex-medulla structure of the thymus was partially distinguishable and the degree of fatification was moderate, indicating that acorus tatarinowii alkaloid B and acorus alkaloid C have a delaying and protective effect on thymic involution to varying degrees.

[0085] Example 8

[0086] SA-β-Gal staining of thymus and spleen related to senescence

[0087] Experimental method: After the experiment in Example 3 was completed, 3 freshly obtained mouse thymus and spleen tissues in each group were quickly frozen with liquid nitrogen and stored at -80 °C. Wuhan Sevier Biotechnology Co., Ltd. was entrusted to perform SA-β-Gal staining.

[0088] One of the signs of aging is the increase of senescent cells. The senescence-related SA-β-Gal staining was used to detect the changes in senescent cells in the thymus and spleen of mice in each treatment group. Blue represents senescent cells. Figures 7 - 8 As shown in Table 1, compared with the blank control group, the groups treated with acorus calamine B and sphenanthelline C could reduce the number of senescent cells in the thymus and spleen of 18-month-old mice.

[0089] Table 1 Percentage of SA-β-Gal positive cells in thymus and spleen (Mean±SD)

[0090]

[0091] Example 9

[0092] Changes of peripheral blood T lymphocyte content

[0093] Experimental method: After the experiment of Example 3, peripheral blood was randomly collected from 3 mice in each group into EDTA anticoagulant tubes, and 1×10 6 Add cells to the corresponding flow cytometry tubes, add the pre-prepared antibodies, mix thoroughly, and incubate for 30 minutes in a dark environment. After incubation, centrifuge at 1500rpm for 3 minutes and discard the supernatant. Add 300μL PBS to each tube to resuspend the cells, mix well, and then perform flow cytometry detection.

[0094] Flow cytometry was performed on peripheral blood to analyze the changes of various immune cells, such as Figure 9 As shown, it was found that the proportions of CD3+CD4+T cells, CD3+CD8+T cells, initial CD3+CD4+T cells, and initial CD3+CD8+T cells in the peripheral blood of the Acorus alkaloid B and Panax pine C groups were all increased. Compared with the metformin group, the proportion of CD3+CD4+T cells in the Acorus alkaloid B and Panax pine C groups increased more, and the proportion of initial CD3+CD8+T cells increased similarly. Acorus alkaloid B and Panax pine C can improve the function of the immune system.

[0095] Example 10

[0096] Changes in thymic T lymphocyte content

[0097] Experimental method: After the experiment in Example 3 was completed, thymus tissues of 3 mice were randomly selected from each group. The thymus tissues were gently ground with a glass slide, and the thymus cells were rinsed with a small amount of PBS into a 70-μm cell strainer. The filtered cell suspension was transferred to a 15-mL centrifuge tube, centrifuged at 1200 rpm for 8 minutes, the supernatant was removed, the cells were resuspended with 3 mL of PBS, 1×10 cells were taken, added to the corresponding flow cytometry tubes, and the pre-prepared antibodies were added respectively. After thorough mixing, the mixture was incubated for 30 minutes under light-proof conditions. After the incubation was completed, it was centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. 300 μL of PBS was added to each tube to resuspend the cells, and after mixing, it was loaded onto a flow cytometer for detection.

[0098] Flow cytometry was performed on the thymus to analyze the changes in various immune cells. The results are as Figure 10 shown, indicating that the proportions of CD3+CD4+ T cells and CD3+CD8+ T cells in the groups administered with acorus alkaloid B and shensong alkaloid C were both increased, and both were higher than those in the metformin group. The results show that acorus alkaloid B and shensong alkaloid C can improve the function of the immune system.

[0099] Example 11

[0100] Changes in the content of splenic T lymphocytes

[0101] Experimental method: After the experiment in Example 3 was completed, spleen tissues of 3 mice were randomly selected from each group. The spleen tissues were gently ground with a glass slide, and the spleen cells were rinsed with a small amount of PBS into a 70-μm cell strainer. The filtered cell suspension was transferred to a 15-mL centrifuge tube, 8 mL of red blood cell lysate was added, centrifuged at 1200 rpm for 8 minutes, the supernatant was removed, the cells were resuspended with 3 mL of PBS, 1×10 6 cells were taken, added to the corresponding flow cytometry tubes, and the pre-prepared antibodies were added respectively. After thorough mixing, the mixture was incubated for 30 minutes under light-proof conditions. After the incubation was completed, it was centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. 300 μL of PBS was added to each tube to resuspend the cells, and after mixing, it was loaded onto a flow cytometer for detection.

[0102] Flow cytometry was performed on the spleen to analyze the changes in various immune cells. The results are as Figure 11 shown, indicating that the proportions of CD3+CD4+ T cells and CD3+CD8+ T cells in the groups administered with acorus alkaloid B and shensong alkaloid C were both increased. The results show that acorus alkaloid B and shensong alkaloid C can improve the function of the immune system.

[0103] In summary, the present invention designed and synthesized the furanosyl pyrrolospiroketal alkaloids acorus B and shensong alkaloid C, both of which can reduce the body weight of aging mice, reduce the fat content in the liver, reduce the number of senescent cells and increase the proportion of T cells in immune cells, and have the potential for fat reduction and anti-immune aging.

Claims

1. A method for the total synthesis of furanosyl pyrrolidine spiroketal alkaloids, characterized in that, The synthetic route is as follows:

2. The total synthesis method according to claim 1, characterized in that, The total synthesis method includes the following steps: (1) Using D-glucose as a raw material, under acidic conditions, reacting with dibenzylamine in an Amadori rearrangement reaction at a reaction temperature of 50-90 °C for 2-5 h to obtain Compound I; (2) Compound I is obtained under the catalytic reaction of anhydrous hydrogen chloride prepared in a system of lower carbon alcohol and acetyl chloride or a system of lower carbon alcohol and trimethylchlorosilane at a reaction temperature of 25-80 °C for 5-48 h; (3) Using palladium-carbon as a catalyst, hydrogenating and reducing the benzyl group of Compound I or IV in an organic solvent under a hydrogen atmosphere to obtain Compound II or V at a reaction temperature of 0-50 °C for 4-90 h; (4) Under acidic conditions, Compound II or V undergoes a Maillard condensation reaction with dihydropyranone to obtain Compound III or VI at a reaction temperature of 15 °C - 100 °C for 8-24 h; (5) Compound III or VI undergoes spirocyclization under acidic conditions to obtain Acortatarin B or Shensongine C at a reaction temperature of -10-50 °C for 2-24 h; 3. The total synthesis method according to claim 2, characterized in that, The acidic reagent providing the acidic conditions in step (1) is an organic acid, including formic acid and acetic acid, and the solvents include methanol and ethanol; the lower carbon alcohols mentioned in step (2) include methanol and ethanol; the organic solvents mentioned in step (3) include methanol and ethanol.

4. The total synthesis method according to claim 2, characterized in that, The acidic conditions in step (4) are provided by a protonic acid or a Lewis acid. The protonic acid is acetic acid, and the Lewis acid is zinc chloride or zinc acetate. The solvents include methanol, ethanol, and dioxane.

5. The total synthesis method according to claim 2, characterized in that The acidic reagents in step (5) include p-toluenesulfonic acid, methanesulfonic acid, sulfamic acid, camphorsulfonic acid, or hydrochloric acid, and the solvents include tetrahydrofuran, dichloromethane, and chloroform.

6. The furanosyl pyrrole spiroketal alkaloid obtained by the total synthesis method according to any one of claims 1-5.

7. Use of the furanosyl pyrrole spiroketal alkaloid according to claim 6 in the preparation of drugs, health products, or functional foods for reducing fat.

8. The application according to claim 7, wherein Drugs, health products, or functional foods containing Acortatarin B and / or Shensongine C can reduce body weight, reduce lipid droplets in liver tissue, and fat in thymus tissue.

9. Use of the furanosyl pyrrole spiroketal alkaloid according to claim 6 in the preparation of drugs, health products, or functional foods for anti-immune aging.

10. The application according to claim 9, wherein Drugs, health products, or functional foods containing Acortatarin B and / or Shensongine C can delay the aging of the thymus and spleen and increase the proportion of naive T cells in immune cells in peripheral blood, thymus, and spleen.

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