CN113717188B

CN113717188B - Alkaloid compound and application thereof

Info

Publication number: CN113717188B
Application number: CN202111056784.6A
Authority: CN
Inventors: 李丽梅
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2022-07-19
Anticipated expiration: 2041-09-09
Also published as: CN113717188A

Abstract

The invention discloses a new alkaloid compound in Chinese narcissus bulb. In vitro cell activity research shows that the novel compound separated from the bulb of Chinese narcissus has better inhibitory activity on T cells, and the compound can be developed and prepared into immunosuppressive drugs.

Description

Alkaloid compound and application thereof

Technical Field

The invention relates to a new compound with immunosuppressive activity, belonging to a polychlorinated narcissus 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 alkaloid compounds with good biological activity by extracting and purifying alkaloids in bulb parts of the Chinese narcissus.

Specifically, the invention provides a compound as shown in formula I, a pharmaceutically acceptable salt or a deuterated compound thereof, wherein the structural formula of the compound is as follows:

wherein R is selected from H, C1-C5 alkyl, -C (O) -R1, glycosyl;

the R1 is selected from saturated or unsaturated alkyl of C5-C11 and C13-C23.

In the present invention, the C1-C5 alkyl group may be selected from methyl, ethyl, propyl, butyl, etc.; can be further selected from C1-C3 alkyl.

In the present invention, the glycosyl may be selected from glycosyl groups commonly found in natural extracts, such as xylosyl, mannosyl, glucosyl, galactosyl, arabinosyl, glucuronyl, galacturonyl, rhamnosyl, and the like.

When R is selected from glycosyl, the compound is a glucoside structure, and the glycosidic bond can be naturally present in plant species or can be prepared by conventional glycosylation reaction.

In the present invention, the structure of the group-C (O) -is a carbonyl group.

Wherein, when R1 is selected from C5-C11 saturated or unsaturated alkyl groups, the-C (O) -R1 group can be derived from a medium chain fatty acid, such as caproic acid, caprylic acid, capric acid, lauric acid.

When R1 is selected from C13-C23 saturated or unsaturated alkyl groups, the-C (O) -R1 group can be derived from a long chain fatty acid, such as palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid.

When R is selected from-C (O) -R1, the compound can be obtained by isolation and extraction from plants, and can also be prepared by conventional esterification reaction.

The immunosuppressant in the invention is a medicine with an inhibitory effect on the immune response of an organism, can inhibit the proliferation and functions of cells (macrophages such as T cells, B cells and the like) related to the immune response, and can reduce the immune response of antibodies.

T lymphocytes (T lymphocytes), abbreviated as T cells, are bone marrow-derived lymphoid stem cells that, after differentiation and maturation in the thymus, are distributed through lymph and blood circulation to immune organs and tissues throughout the body to exert an immune function.

The alkaloid compounds have immunosuppressive activity, and can be used for preparing immunosuppressive drugs. Therefore, the invention also provides a pharmaceutical composition, and the active ingredients of the pharmaceutical composition comprise the alkaloid compounds and pharmaceutically acceptable auxiliary materials.

The pharmaceutical composition contains various pharmaceutically common additives (such as an excipient and the like) so as to prepare a pharmaceutical preparation. The pharmaceutical composition can be formulated into various types of administration unit dosage forms, such as tablets, pills, powders, liquids, suspensions, gels, emulsions, creams, granules, hard capsules, suppositories, and injections (solutions and suspensions, generally injections), etc., according to the therapeutic purpose. Preferably, the pharmaceutical composition is in the form of injection for local injection (e.g., subcutaneous, perinervous, intra-articular cavity, etc.); alternatively, the pharmaceutical composition is in the form of gel, lotion, cream, etc., and can be applied topically; or the dosage form of the pharmaceutical composition is tablets, pills, powder, granules, hard capsules and the like, and the immunosuppressive effect is achieved through oral administration.

For shaping the pharmaceutical composition in tablet form, any excipient known and widely used in the art may be used. For example, carriers such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and the like; binders such as water, ethanol, propanol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose and potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents such as dry starch, sodium alginate, agar powder and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyethylene sorbitan, sodium lauryl sulfate, monoglyceride stearate, starch, lactose and the like; disintegration inhibitors such as white sugar, glycerol tristearate, coconut oil and hydrogenated oil; adsorption promoters such as quaternary ammonium hydroxide and sodium lauryl sulfate; humectants such as glycerin, starch, and the like; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; and lubricants such as pure talc, stearate, boric acid powder, polyethylene glycol, and the like. If desired, the tablets can also be made as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layer film tablets and multilayer tablets using customary coating materials.

For shaping the pharmaceutical composition in the form of a pill, any of the excipients known and widely used in the art may be used, for example, carriers such as lactose, starch, coconut oil, hardened vegetable oil, kaolin, talc and the like; binders such as gum arabic powder, xanthan powder, gelatin, ethanol, and the like; disintegrating agents, such as agar and kelp powder.

For shaping the pharmaceutical composition in the form of suppositories, any excipient known and widely used in the art may be used, for example, polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like.

For preparing pharmaceutical compositions in the form of injection solutions, the solutions and suspensions may be sterilized and, preferably, suitable amounts of sodium chloride, glucose or glycerol, etc., may be added to make injection solutions which are isotonic with blood. In the preparation of ampoules, any of the carriers commonly used in the art may be used, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol and fatty acid esters of polyethylene sorbitan, and the like. In addition, conventional lytic agents, buffers, analgesics, and the like may be added.

The content of the alkaloid compound and the pharmaceutically acceptable salt thereof in the pharmaceutical composition provided by the invention is not particularly limited, and can be selected within a wide range, and generally can be 0.1-99.9% by mass, preferably 1-70% by mass, and more preferably 1-30% by mass.

In the present invention, the method of administration of the pharmaceutical composition is not particularly limited. The formulation of various dosage forms can be selected for administration according to the age, sex and other conditions and symptoms of the patient. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally; the injection can be simply used for intramuscular, intradermal, subcutaneous or intraabdominal injection; the suppository is administered to the rectum.

In the present invention, the administration dose can be appropriately selected depending on the administration method, the age, sex and other conditions of the patient and the symptoms. Typical dosages administered may be: about 0.01 to 300mg of the pharmaceutically active ingredient per kg body weight per day. Generally, each unit dosage form for administration may contain 1 to 200mg of the pharmaceutically active ingredient. The above preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

Other immunosuppressive agents may also be included in the combinations of the invention, for example (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites such as cyclosporine, tacrolimus, and the like; (3) antimetabolites such as azathioprine and 6-mercaptopurine, and the like; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT 3; (5) alkylating agents such as cyclophosphamide. One or more of (a).

Unless otherwise indicated, the terms and abbreviations disclosed in the present application have their standard meanings.

In the present invention, the deuterated compound refers to a compound obtained by substituting one or more hydrogens in the molecule of the alkaloid compound of the present invention with deuterium as an isotope.

Deuterium is a naturally occurring hydrogen isotope. Replacement of hydrogen with deuterium makes it possible to improve the pharmacokinetic parameters of the drug while ensuring the therapeutic effect.

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 the NMR spectrum of Compound 2 (C¹³C 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

40 kg of fresh narcissus bulb is repeatedly chopped by a vegetable cutter, extracted for 2 times by methanol at 60 ℃, the extract is concentrated to obtain extract, dispersed by water, the pH value is adjusted to 10 by ammonia water, extracted by n-butanol with the same volume to obtain total alkaloid (105G), normal phase silica gel column chromatography is carried out, gradient elution is carried out by chloroform-methanol (1:0-0:1) (each gradient has 3 column volumes), and similar components are combined by TLC analysis to obtain A-G7 parts in sequence.

The B fraction (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 combine similar fractions to give 6 fractions B-1 to B-6, etc.

B-5(3.3g) was further subjected to ODS reverse phase column chromatography, eluted with methanol-water (0:1-1:1) gradient (4 column volumes per gradient), concentrated and spot-plated to combine similar components to obtain 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 and ¹³c 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 the 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 and ¹³c 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]isochromeno[3,4-c]indole。

The structural formulas of the

compounds

1 and 2 are as follows:

TABLE 1 combination of ¹H of

objects

1 and 2 ¹³NMR (400MHz) and C NMR (100MHz) data (i)n CD₃OD)

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 number 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 para positionA tetra-substituted benzene ring of 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, in the case of the present invention,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 provided with¹H NMR and¹³the C NMR data are shown in Table 1.

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 number 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 that other HMBC and NOESY associations were consistent with Compound 1, as demonstrated by the correlation of H-OMe with C-3 and H-3 with C-1 and C-4a in the HMBC spectra (see FIGS. 5 and 5)6) and thus compound 2 was identified as 6 a-deoxynorortazettine.

Example 2:

immunosuppressive activity and beneficial effects of the novel compounds:

the effects of the two alkaloid compounds provided by the present invention in terms of immunosuppression are evaluated in conjunction with cell assays as follows.

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 2) respectively, and discloses that the alkaloid compound provided by the invention has good application prospects in preparation of immunosuppressive drugs.

TABLE 2

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 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, can be used as a lead compound of immunosuppressive drugs, and is beneficial to the treatment of autoimmune diseases and immune rejection.

Claims

1. A compound according to formula I, having the formula:

wherein R is selected from H or methyl.

2. The compound of claim 1, having the formula: