阅读说明：本技术 一类生物碱化合物的制备方法 (Preparation method of alkaloid compound ) 是由 李丽梅 于 2021-09-09 设计创作，主要内容包括：本发明公开了中国水仙鳞茎中一类新生物碱化合物的制备方法。本发明方法制备得到的两个具有免疫抑制活性的生物碱类化合物,可用于该类化合物的提取纯化,所得化合物亦可用于进一步的免疫抑制研究,也可以作为中间体,继续反应得到更多更好的相关活性化合物。(The invention discloses a preparation method of a new alkaloid compound in Chinese narcissus bulb. The two alkaloid compounds with immunosuppressive activity prepared by the method can be used for extracting and purifying the compounds, and the obtained compounds can also be used for further immunosuppressive research and can also be used as intermediates to continuously react to obtain more better related active compounds.)

1. A process for the preparation of a compound of formula I, characterized in that:

wherein R is selected from H or methyl;

the method comprises the following steps:

(1) extracting Narcissus bulb with ethanol or methanol, removing solvent from the extractive solution, extracting with n-butanol in alkaline water solution, subjecting the n-butanol part to silica gel column chromatography, gradient eluting with chloroform-methanol at ratio of 1:0-0:1, and mixing similar components by thin layer tracking to obtain seven parts A-G;

(2) subjecting the part B to silica gel column chromatography, gradient eluting with petroleum ether-acetone 15:1-1:1, and thin layer tracking to combine similar components to obtain six parts B-1-B-6;

(3) performing ODS reversed phase column chromatography on part B-5, performing gradient elution with methanol-water of 0:1-1:1, and combining similar components by thin layer tracking to obtain five parts B-5-1-B-5-5;

(4) subjecting the upper half of B-5-2 to HPLC with C18 column, eluting with 20% methanol water to obtain compound of formula I with R as methyl;

(5) performing ODS reversed-phase column chromatography on the part G, performing gradient elution with methanol-water of 0:1-1:0, and combining similar components by thin-layer tracking to obtain four parts G-1-G-4; and (3) performing amino silica gel column chromatography on the G-1 part, and performing gradient elution by using chloroform-methanol (40: 1-20: 1) to obtain the compound of the formula I, wherein R is H.

2. The method of claim 1, wherein: in the content (1), the pH value of the alkaline aqueous solution is 9-11; chloroform-methanol gradient elution was performed with 2-4 column volumes per gradient.

3. The production method according to claim 1 or 2, characterized in that: in content (1), chloroform-methanol gradient elution was performed by eluting 3 column volumes per gradient.

4. The method of claim 1, wherein: in the content (2), when petroleum ether-acetone gradient elution is performed, each gradient elutes 2 to 4 column volumes, and further 3 column volumes are selected.

5. The method of claim 1, wherein: in the content (3), when methanol-water gradient elution is performed, 3 to 5 column volumes are eluted per gradient, and further 4 column volumes are selected.

6. The method of claim 1, wherein: in the content (4), 20% methanol water contains 1 to 3 ‰ trifluoroacetic acid.

7. The method of claim 1, wherein: in the content (4), the column size was 10X 250mm,10 μm; collecting the part with retention time of 8min by adopting 1mL/min to obtain the compound of the formula I with R as methyl.

8. The method of claim 1, wherein: in the content (5), when methanol-water gradient elution is performed, 3 to 5 column volumes are eluted per gradient, and further selected from 4 column volumes.

9. The method of claim 1, wherein: in the content (5), chloroform-methanol gradient elution is performed by eluting 2 to 3 column volumes per gradient, and further 2 column volumes are selected.

10. The method of claim 1, wherein: in content (5), the chloroform-methanol gradient was collected at 30:1 to give the compound of formula I where R is H.

Technical Field

The invention relates to a preparation method of a novel compound with immunosuppressive activity, belonging to a narcissus pseudonarcissus alkaloid compound.

Background

Chinese Narcissus (Narcissus tazetta var. chinensis) is a perennial herb of Narcissus (Narcissus) in Amaryllidaceae, and its bulb has the effects of clearing away heat and toxic material, and resolving hard mass and relieving swelling. Research shows that the bulb of Chinese narcissus mainly contains alkaloid, flavone, flavan and derivatives thereof, lignan and other components, wherein the lycoris alkaloid is a special secondary metabolite thereof, has rich structure types, and is divided into lycorine, tennis-ball hydramine, dorsalicin, narcissin, galanthamine and the like according to different ring types.

In order to improve the comprehensive utilization efficiency of the medicinal plant resources of the Chinese narcissus, the inventor carries out systematic research on alkaloid components of the bulb part of the Chinese narcissus so as to discover an extract or a compound with biological activity.

Disclosure of Invention

The inventor finally obtains the alkaloid compound with good biological immunosuppressive activity by extracting and purifying the alkaloid of the bulb part of the Chinese narcissus.

Specifically, the invention provides a preparation method of a compound shown in a formula I, which is characterized in that:

wherein R is selected from H or methyl;

the method comprises the following steps:

(1) extracting Narcissus bulb with ethanol or methanol, removing solvent from the extractive solution, extracting with n-butanol in alkaline water solution, subjecting the n-butanol part to silica gel column chromatography, gradient eluting with chloroform-methanol at ratio of 1:0-0:1, and mixing similar components by thin layer tracking to obtain seven parts A-G;

(2) subjecting the part B to silica gel column chromatography, gradient eluting with petroleum ether-acetone 15:1-1:1, and thin layer tracking to combine similar components to obtain six parts B-1-B-6;

(3) performing ODS reversed phase column chromatography on part B-5, performing gradient elution with methanol-water of 0:1-1:1, and combining similar components by thin layer tracking to obtain five parts B-5-1-B-5-5;

(4) taking the upper half part of the B-5-2 part for preparative HPLC, eluting with 20% methanol water to obtain a compound of formula I with R as methyl;

(5) performing ODS reversed-phase column chromatography on the part G, performing gradient elution with methanol-water of 0:1-1:0, and combining similar components by thin-layer tracking to obtain four parts G-1-G-4; and (3) performing amino silica gel column chromatography on the G-1 part, and performing gradient elution by using chloroform-methanol (40: 1-20: 1) to obtain the compound of the formula I, wherein R is H.

Wherein in the content (1), the pH value of the alkaline aqueous solution is 9-11; chloroform-methanol gradient elution is performed with 2-4 column volumes, e.g., 2, 3,4 column volumes per gradient.

Wherein the pH may be selected from 9, 10, 11 or other alkaline conditions.

In the content (2), when petroleum ether-acetone gradient elution is performed, 2 to 4 column volumes, for example, 2, 3,4 column volumes are eluted per gradient.

In the content (3), when methanol-water gradient elution is performed, 3 to 5 column volumes, for example, 3,4, 5 column volumes are eluted per gradient.

In the content (4), 20% methanol water contains 1-3 ‰ trifluoroacetic acid, such as 1, 2, 3 ‰.

In the content (4), 1mL/min is adopted, and the part with the retention time of about 8min is collected, so that the compound of the formula I with the R as the methyl is obtained. Of course, the retention time may vary if other flow rates are used, but the retention time may still be known according to conventional methods.

In the content (5), when methanol-water gradient elution is performed, 3 to 5 column volumes, for example, 3,4, 5 column volumes are eluted per gradient.

In the content (5), when chloroform-methanol gradient elution is performed, 2 to 3 column volumes, for example, 2, 3 column volumes are eluted per gradient.

In content (5), the chloroform-methanol gradient was collected at 30:1 to give the compound of formula I where R is H.

In the content (1) of the present invention, various modes such as heating reflux extraction, ultrasonic extraction, microwave-assisted extraction, leaching, etc. can be adopted.

The number of extraction is aimed at extracting the plant components as completely as possible, and generally 1, 2, 3, or 4 times of extraction can be used.

The two alkaloid compounds with immunosuppressive activity, which can be prepared by the method disclosed by the invention, can be used for extraction and purification of the compounds, and the obtained compounds can also be used for further immunosuppressive research and can also be used as intermediates to continuously react to obtain more better related active compounds.

The immunosuppressive activity and beneficial effects of the compounds of formula I are as follows:

the following was conducted to evaluate the effects of the two alkaloid compounds provided by the present invention in terms of immunosuppression in combination with cell assay.

1) T cell isolation and purification protocol

Peripheral blood of healthy blood donors is taken and PBMC is separated by Ficoll density gradient centrifugation. "unaltered" (intact) CD4+ T cells were obtained from PBMC by negative selection using immunomagnetic bead separation. For specific operation, refer to Miltenyi company Pan T Cell Isolation Kit II (Human) specification. Purified CD4+ T cells, as detected by flow cytometry, had a cell positivity of greater than 95% for subsequent testing.

2) T cell stimulation protocol

The anti-CD3/CD28 mAbs were used to stimulate T cells and the specific assay procedures were performed as per routine procedures.

3) Detection of immunosuppressive Activity

T cells were obtained from human Peripheral Blood Mononuclear Cells (PBMC) using immunomagnetic bead separation. Laying 96-well plate, 2X 10⁵A hole. Stimulating T activated proliferation by anti-CD3/CD28 mAbs, after 96hr, incubating the alkaloid compounds (1 and 2, concentration of 0.2,1,5,25 μ M in sequence) for 72hr, detecting the sample by CFSE staining combined with flow cytometry, and comparing with negative control (DMSO) to determine the inhibition effect of the sample on activated T cell proliferation.

4) Test results

The test result shows that the alkaloid compound shows good immunosuppressive activity and IC of the immunosuppressive activity on T cell proliferation₅₀26.3 and 21.5 mu M (table 1) respectively, and discloses that the alkaloid compound provided by the invention has good application prospect in the preparation of immunosuppressive drugs.

TABLE 1

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the NMR spectrum of Compound 1: (¹H NMR)；

FIG. 2 is a nuclear magnetic resonance carbon spectrum of Compound 1: (¹³C NMR)；

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of Compound 2: (¹H NMR)；

FIG. 4 is a nuclear magnetic resonance carbon spectrum of Compound 2: (¹³C NMR)；

FIG. 5 is the major HMBC correlation of compounds 1 and 2;

FIG. 6 is a graph relating the major NOESY of compounds 1 and 2.

Detailed Description

Example 1

Cutting 40 kg of fresh narcissus bulb by a vegetable cutter repeatedly, extracting for 2 times by methanol at 60 ℃, concentrating the extracting solution to obtain extract, dispersing by water, adjusting pH to 10 by ammonia water, extracting by n-butanol with the same volume to obtain total alkaloids (105G), carrying out normal phase silica gel column chromatography, carrying out gradient elution by chloroform-methanol (1:0-0:1) (each gradient has 3 column volumes), and combining similar components by TLC analysis to obtain A-G7 parts in sequence.

The fraction B (14g) was further subjected to normal phase silica gel column chromatography, gradient eluted with petroleum ether-acetone (15:1-1:1) (3 column volumes per gradient), concentrated and spotted to give 6 fractions B-1 to B-6, etc.

B-5(3.3g) was further subjected to ODS reverse phase column chromatography, eluting with methanol-water (0:1-1:1) gradients (4 column volumes per gradient), and after concentration, the similar fractions were combined on a dot-plate basis to give 5 fractions B-5-1 to B-5-5, etc.

B-5-2(55mg) was eluted with 20% methanol in water (1% TFA) and semi-preparative HPLC (C18 column 10 × 250mm,10 μm) gave compound 2(16mg,1mL/min, Rt 8 min).

The G fraction (40G) was subjected to ODS reverse phase column chromatography, eluted with methanol-water (0:1-1:0) gradient (4 column volumes per gradient), concentrated and spotted to give 4 fractions such as G-1 to G-4.

Wherein G-1(1.0G) was subjected to amino silica gel column chromatography eluting with a gradient of chloroform/methanol 40:1-20:1 (2 column volumes per gradient) to give compound 1(10mg) (compound 1 was obtained in a 30:1 gradient).

The physicochemical properties and spectral data of the obtained compounds 1, 2 are as follows:

compound 1: white amorphous powder, easily soluble in methanol and chloroform;UV(MeOH)λ_max(logε)：206(4.17),241(3.26),291(3.35)；¹H and¹³c NMR data are shown in Table 1; HRESIMS M/z 288.1236[ M ]+H]⁺(calcd for C₁₆H₁₈NO₄,288.1230). Is named as: 3-O-demethyl-6 a-deoxynorortazettine with chemical name of (3S,4aS,6aR,13bS) -3,4,4a,5,6,6a-hexahydro-8H- [1,3]dioxolo[4',5':6,7]isochromeno[3,4-c]indol-3-ol

Compound 2: white amorphous powder, easily soluble in methanol and chloroform;UV(MeOH)λ_max(logε)：207(4.28),241(3.36),293(3.47)；¹H and¹³c NMR data are shown in Table 1; HRESIMS M/z 302.1392[ M + H ]]⁺(calcd for C₁₇H₂₀NO₄,302.1387). Is named aS 6 a-deoxynorortazettine with the chemical name of (3S,4aS,6aR,13bS) -3-methoxy-3,4,4a,5,6,6a-hexahydro-8H- [1,3]dioxolo[4',5':6,7]isoc hromeno[3,4-c]indole。

The structural formulas of the compounds 1 and 2 are as follows:

TABLE 2 preparation of Compounds 1 and 2¹H NMR (400MHz) and¹³c NMR (100MHz) data (in CD)₃OD)

Structure confirmation of the novel compounds:

compound 1 is a white amorphous powder, dissolved in methanol; the reaction is positive with the improved bismuth potassium iodide, which indicates that the modified bismuth potassium iodide is probably a alkaloid compound; HR-ESI-MS gives 288.1236[ M + H ] as the peak of the excimer ion]⁺(calculated value is C)₁₆H₁₈NO₄288.1230), the molecular weight is determined to be 287. Deducing the molecular formula to be C by combining a hydrogen spectrum and a carbon spectrum₁₆H₁₇NO₄The unsaturation degree was 9.¹H-NMR(400MHz,CD₃OD) gives two single peaks delta in the aromatic proton region_H6.72(1H, s) and δ_H7.07(1H, s) indicating the presence of a tetra-substituted phenyl ring with a para-hydrogen; giving a pair of cis-substituted double bond protons delta_H6.38(1H, d, J ═ 10.0Hz) and δ_H6.29(dd, J ═ 10.0,4.8 Hz); giving the methylene proton signal delta_H5.98(s), combined carbon spectrum δ_C103.1, indicating that the compound has methylenedioxy. In addition to this, the present invention is,¹³C-NMR spectra (100MHz, CD)₃OD) and HMQC spectra showed that this compound also contained 3 aliphatic methylenes, 3 aliphatic methines, 4 aromatic quaternary carbons, 1 saturated quaternary carbon, which was initially assumed to be a narcissus alkaloid, and 1 less saturated quaternary carbon (delta. for The compound 1 compared to The nuclear magnetic data of The known compound nortazettine (The Journal of Organic Chemistry,1968,33(8):3105-3109.)_C101.4) addition of 1 aliphatic methine group (. delta.)_C 78.5,δ_H4.17), and 1 methoxy group is reduced, which indicates that the compound 1 is possibly 3-O-demethyl-6 a-deoxynorortazettine. In the HMBC spectra, H-4a is associated with C-6, C-6a, H-6a is associated with C-12a, H-1 is associated with C-3, C-4a, and H-3 is associated with C-4a, demonstrating the rationality of the above speculation (as shown in FIG. 5). In the NOESY spectrum, H-4a is related to H-6a and H-6 beta, and H-3 alpha is related to H-6 alpha, and the relative spatial configuration of the compound 1 is determined to be the same as that of nortazettine (shown in figure 6). Thus, Compound 1 was identified as 3-O-demethyl-6 a-deoxynorortazettine. It is composed of¹H NMR and¹³the C NMR data are shown in Table 2.

Compound 2 is a white amorphous powder, dissolved in methanol; the reaction is positive with the improved bismuth potassium iodide, which indicates that the modified bismuth potassium iodide is probably a alkaloid compound; HR-ESI-MS gives 302.1392[ M + H ] as the peak of the excimer ion]⁺(calculated value is C)₁₇H₂₀NO₄,302.1387). Deducing the molecular formula to be C by combining a hydrogen spectrum and a carbon spectrum₁₇H₂₀NO₄The unsaturation degree was 9. By careful comparison, compounds 2 and 1 were found to be nearly identical¹H NMR and¹³c NMR data, the only difference being that Compound 2 has one more methoxy group than 1. Suggesting that compound 2 may have 1 more methoxy group at the C-3 position than compound 1. The above conjectures are confirmed by the correlation of H-OMe with C-3 and H-3 with C-1 and C-4a in HMBC spectraNOESY correlation was consistent with compound 1 (as shown in fig. 5, 6), and thus compound 2 was identified as 6 a-deoxynorortazettine.

And (4) conclusion: the alkaloid compound provided by the invention is a new compound which is separated for the first time, is determined to be a polychlorinated narcissus type lycoris alkaloid compound by an optical rotation, ultraviolet, nuclear magnetic resonance and mass spectrometry determination method, and represents a specific structure. The result of an immunosuppressive activity detection test shows that the compound has obvious inhibitory activity on T cell proliferation, IC₅₀26.3 and 21.5 mu M respectively, thereby showing that the compound has better immunosuppressive activity, disclosing that the compound has good application prospect in preparing immunosuppressive drugs, and can also be used as a lead compound of immunosuppressive drugs, and being beneficial to the treatment of autoimmune diseases and immune rejection.