Trigliane type diterpenoid compound as well as preparation method and application thereof

Abstract

The invention discloses a trilliane type diterpenoid compound as well as a preparation method and application thereof. The preparation method of the trilliane type diterpenoid compound comprises the following steps: obtaining monomeric compounds 12-dexyphorbol-13-underdecanoate-17-hydroxyl and 12-dexyphorbol-13-tridecanoate-17-hydroxyl from the root of Euphorbiaceae plant Berchemia lanceolata by adopting a separation technology, and carrying out a reaction on the monomeric compounds 12-dexyphorbol-13-underdecanoate-17-hydroxyl and 12-dexyphorbol-13-tridecanoate-17-hydroxyl and 12-dexyphorbol-13-tridecanoate-17-hydroxyl to obtain a compound I; through anti-inflammatory activity screening of the two, it is found that the two have good anti-inflammatory activity, and the two are used for preparing an anti-inflammatory pharmaceutical composition. The result not only provides a powerful scientific basis for the type of compound as a potential anti-inflammatory drug, but also reveals an important development value of the compound as an anti-inflammatory activity lead compound.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical applications, and particularly relates to a class of tigliane-type diterpenoids, a preparation method thereof, and uses thereof. Background Art

[0002] Inflammation is an important biological process by which the body responds to harmful stimuli, and it participates in pathogen clearance, tissue repair, and immune regulation through a complex cellular signaling network; recent studies have shown that a persistent inflammatory microenvironment can promote the occurrence and development of tumors through abnormal cell signal transduction. In the mechanism of the transformation of inflammation to tumors, the differentiation of monocytes mediated by chemokines (such as tumor-associated macrophages TAM) and their M2-type polarization process have been proven to promote tumor immune escape by constructing an immunosuppressive microenvironment; in particular, the inflammatory mediator nitric oxide (NO), as a key signaling molecule, can form a positive feedback loop of pro-inflammatory and pro-cancer by regulating pathways such as NF-κB.

[0003] Lipopolysaccharide (LPS) is the main pathogenic component that induces endotoxin shock, and it can induce the generation of iNOS in monocytes and macrophages in vivo, produce a large amount of NO, and further release a variety of inflammatory cytokines; inducing cells to activate iNOS to produce excessive NO and measuring and calculating the NO generation inhibitory activity of drugs by the Griess method is an effective means for the preliminary screening of anti-inflammatory active compounds.

[0004] Baliospermum solanifolium (Burman) Suresh is an important medicinal resource in the traditional Ayurvedic medical system in Sri Lanka. It shows the effects of purging and removing accumulation, expelling water and reducing swelling in clinical applications, and has been verified through hundreds of years of practice. Modern pharmacological research shows that the ethanol extract of this plant has a variety of biological activities, including anti-allergic, anti-inflammatory, and cytotoxicity, etc.

[0005] Currently, clinically used anti-inflammatory drugs (non-steroidal anti-inflammatory drugs and glucocorticoids) generally have problems of insufficient safety during long-term use; for example, gastrointestinal damage, immunosuppression, etc. Natural diterpenoids have become an important direction for the research and development of anti-inflammatory drugs due to their structural diversity, low toxicity, high biocompatibility, and significant biological activities; among them, tigliane-type diterpenoids mainly focus on anti-tumor and antioxidant effects, etc. A class of tigliane-type diterpenoids proposed in the present invention has obvious anti-inflammatory activity through biological activity tests, and has important development value as a leading anti-inflammatory active compound. Summary of the Invention

[0006] In view of the above-mentioned defects or deficiencies in the prior art, it is desired to provide a class of tigliane-type diterpenoids, their preparation methods and uses.

[0007] A class of tigliane-type diterpenoids provided by the present invention has the following structural formula I:

[0008]

[0009] Wherein, R1, R2, R3 and R4 in formula I are independently selected from the following substituents: hydrogen, hydroxyl, methyl, carbamoyl, -C 2-16 linear or branched alkyl, -C 2-16 linear or branched alkanoyl, -C 2-16 alkenyl, -C 3-8 cycloalkyl, -C 4-8 cycloalkanoyl, -C 2-16 alkenoyl, -C 4-8 aryl, -C 4-8 heteroaryl, -C 2-16 aliphatic amino, -C 2-16 aliphatic aminoacyl, cycloalkylamino, benzoyl, phenylacetyl, glucosyl, xylosyl.

[0010] Preferably, the specific structural formulas of the tigliane-type diterpenoids after R1, R2, R3 and R4 in formula I are selected as substituents are the following formulas II and III:

[0011]

[0012] A preparation method of a class of tigliane-type diterpenoids includes the following steps:

[0013] 1) Add the dried roots of Croton tiglium to 60%-95% ethanol and reflux for extraction 3 times, 2 hours each time, to obtain an ethanol extract. The ethanol extract is concentrated under reduced pressure to recover the solvent and obtain an extract;

[0014] 2) Elute the extract with D101 macroporous resin using 20% ethanol, 30% ethanol, 70% ethanol, 90% ethanol and pure ethanol. Concentrate the eluate of the 70% ethanol fraction to obtain a 70% ethanol component, and then elute it with dichloromethane / methanol at 25:1, 10:1, 5:1, 1:1 to obtain 6 fractions Fr.C1-Fr.C6;

[0015] 3) Fr.C5 is eluted by ODS column chromatography with a methanol / water gradient to obtain 5 fractions Fr.C5a-Fr.C5e;

[0016] 4) Fr.C5d was eluted with a petroleum ether / ethyl acetate gradient to obtain three fractions, Fr.C5d1 - Fr.C5d3. Fr.C5d2 was used as the mobile phase with a methanol / water ratio of 85:15 and a flow rate of 10 ml / min, and the tigliane - type diterpenoid compounds shown in Formula II and Formula III in Claim 2 were obtained by HPLC method.

[0017] Use of a class of tigliane - type diterpenoid compounds, one or more of the tigliane - type diterpenoid compounds as defined in Claim 1 or Claim 2 are used as active ingredients in a pharmaceutical composition.

[0018] Furthermore, the pharmaceutical composition is an anti - inflammatory drug.

[0019] Furthermore, the pharmaceutical composition includes pharmaceutically acceptable pharmaceutical excipients.

[0020] Furthermore, it is a use in the preparation of a drug having an anti - inflammatory effect.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] A class of tigliane - type diterpenoid compounds of the present invention provides a strong scientific basis as potential anti - inflammatory drugs, and also reveals its important development value as an anti - inflammatory active lead compound; its preparation method is to obtain monomeric compounds with medicinal activity from Blumea balsamifera, expanding the source of drugs; an in vitro activity screening system was used for activity evaluation, and it was found that the compounds and their compositions of the present invention have good anti - inflammatory activity. Compared with current clinical anti - inflammatory drugs, they have the advantages of low toxicity and high biocompatibility, and also enrich the research on monomeric compounds of Blumea balsamifera.

[0023] It should be understood that the content described in the invention content part is not intended to limit the key or important features of the embodiments of the present invention, nor to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] By reading the detailed description of the non - restrictive embodiments with reference to the following drawings, other features, objectives and advantages of the present invention will become more obvious:

[0025] Figure 1 It is the structural formula of a class of tigliane - type diterpenoid compounds;

[0026] Figure 2 For the new compound 1 (Formula II) of the present invention 1 1H - NMR (proton nuclear magnetic resonance) spectrum;

[0027] Figure 3 For the new compound 1 (Formula II) of the present invention 13 13C-NMR (Carbon-13 Nuclear Magnetic Resonance) spectrum;

[0028] Figure 4 For the new compound 2 (Formula III) of the present invention 1 1H-NMR (Proton Nuclear Magnetic Resonance) spectrum;

[0029] Figure 5 For the new compound 2 (Formula III) of the present invention 13 13C-NMR (Carbon-13 Nuclear Magnetic Resonance) spectrum. Detailed implementation manners

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than limiting the invention. Additionally, it should be noted that for the convenience of description, only parts related to the invention are shown in the drawings.

[0031] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and embodiments.

[0032] Please refer to Figures 1 to 5 , the embodiments of the present invention provide a class of tigliane-type diterpenoid compounds, and their structural formula is as follows: Formula I:

[0033]

[0034] Among them, R1, R2, R3 and R4 in Formula I are each independently selected from the following substituents: hydrogen, hydroxyl group, methyl group, carbamoyl group, -C 2-16 linear or branched alkyl group, -C 2-16 linear or branched alkanoyl group, -C 2-16 alkenyl group, -C 3-8 cycloalkyl group, -C 4-8 cycloalkanoyl group, -C 2-16 alkenoyl group, -C 4-8 aryl group, -C 4-8 heteroaryl group, -C 2-16 aliphatic amino group, -C 2-16 aliphatic aminoacyl group, cycloalkylamino group, benzoyl group, phenylacetyl group, glucosyl group, xylosyl group.

[0035] Preferably, the specific structural formulas of the tigliane-type diterpenoid compounds after R1, R2, R3 and R4 in Formula I are selected as substituents are as follows: Formula II and Formula III:

[0036]

[0037] A preparation method of a class of tigliane - type diterpenoids, comprising the following steps:

[0038] 1) Add the dried roots of *Croton tiglium* to 60% - 95% ethanol and reflux extract 3 times, each time for 2 hours, to obtain an ethanol extract. The ethanol extract is concentrated under reduced pressure to recover the solvent and obtain an extract.

[0039] 2) Elute the extract with D101 macroporous resin using 20% ethanol, 30% ethanol, 70% ethanol, 90% ethanol, and pure ethanol. Concentrate the eluate of the 70% ethanol fraction to obtain a 70% ethanol component, and then elute it with dichloromethane / methanol at ratios of 25:1, 10:1, 5:1, and 1:1 to obtain 6 fractions, Fr.C1 - Fr.C6.

[0040] 3) Fr.C5 is chromatographed on an ODS column with a methanol / water gradient elution to obtain 5 fractions, Fr.C5a - Fr.C5e.

[0041] 4) Fr.C5d is eluted with a petroleum ether / ethyl acetate gradient to obtain 3 components, Fr.C5d1 - Fr.C5d3. Fr.C5d2 is used as the mobile phase with methanol / water (85∶15, 10 ml / min) and the tigliane - type diterpenoids shown in Formula II and Formula III in Claim 2 are obtained by HPLC method.

[0042] In summary, obtaining monomeric compounds with medicinal activity from *Croton tiglium* expands the sources of drugs.

[0043] The use of a class of tigliane - type diterpenoids is for use in the preparation of a drug with anti - inflammatory effects, wherein one or more tigliane - type diterpenoids are used as active ingredients in a pharmaceutical composition.

[0044] Among them, the pharmaceutical composition is an anti - inflammatory drug; the pharmaceutical composition includes pharmaceutically acceptable pharmaceutical excipients.

[0045] An in vitro anti - inflammatory experiment is carried out on the diterpenoids of the present invention.

[0046] Inflammation includes arthritis, gastritis, periodontitis, pneumonia, tonsillitis, bronchitis, dermatitis, etc.

[0047] A class of tigliane - type diterpenoid compounds of the present invention are applied in a pharmaceutical composition, which contains compounds of Formula I - III or their pharmaceutically acceptable salts, or after being combined with compatible drugs and pharmaceutically acceptable carriers, are prepared into any suitable pharmaceutical composition, and can be suitable for forms such as oral administration, inhalation, implantation, and injection. For example, tablets, capsules, granules, injections, oral liquids, infusion preparations, dripping pills, or ointments, etc.

[0048] The carrier is a conventional carrier in the pharmaceutical field, such as diluents, excipients, binders, fillers, disintegrants, and adjuvants, etc. The diluent can be water, alcohol, etc., the excipient can be cyclodextrin, etc., the binder can be polyvinylpyrrolidone, gelatin, cellulose derivatives, etc., the filler can be starch, etc., the disintegrant can be calcium hydrogencarbonate, etc., and the adjuvant can be sweeteners, etc.

[0049] Example 1 Separation and Identification of Tigliane - type Diterpenoids in Blumea balsamifera

[0050] Separation process of the diterpenoid compounds of the present invention

[0051] (1) For the dried roots of Blumea balsamifera, add ethanol at 60% - 95% and reflux for extraction 3 times, 2 hours each time, to obtain an ethanol extract. The extract is concentrated under reduced pressure to recover the solvent and obtain an extract paste.

[0052] (2) Use D101 macroporous resin for the extract paste, and elute with 20% ethanol, 30% ethanol, 70% ethanol, 90% ethanol, and pure ethanol. Concentrate the eluate of the 70% ethanol part to obtain a 70% ethanol component, and then elute with dichloromethane / methanol at ratios of 25:1, 10:1, 5:1, and 1:1 to obtain 6 fractions Fr.C1 - Fr.C6.

[0053] (3) Fr.C5 is subjected to ODS column chromatography and eluted with a methanol / water gradient to obtain 5 fractions Fr.C5a - Fr.C5e.

[0054] (4) Fr.C5d is eluted with petroleum ether / ethyl acetate at gradients of 25:1, 10:1, 5:1, and 1:1 to obtain 3 components Fr.C5d1 - Fr.C5d3. Fr.C5d2 uses methanol / water (85∶15, 10 ml / min) as the mobile phase and is analyzed by HPLC to obtain Compound 1 (Formula II) and Compound 2 (Formula III).

[0055] Structure identification of the tigliane - type diterpenoid compounds of the present invention

[0056] Compound 1 (Formula II)

[0057] Yellow oil, HRESIMS: 533.3477[M + H] + ,Molecular formula: C31 H 48 O7. Its 1 H NMR and 13 C NMR data assignments are shown in Table 1.

[0058] [400 MHz ( 1 H), 100 MHz ( 13 C), solvent: CDCl3].

[0059] Table 1 13 C (100 MHz) and 1 H (400 MHz) NMR data of Compound 1

[0060]

[0061]

[0062] a Assignments are interchangeable.

[0063] Compound 2 (Formula III)

[0064] Yellow oil, HRESIMS: 561.3784 [M + H] + , molecular formula: C 33 H 52 O7. Its 1 H NMR and 13 C NMR data assignments are shown in Table 2.

[0065] [400 MHz ( 1 H), 100 MHz ( 13 C), solvent: CDCl3].

[0066] Table 2 13 C (100 MHz) and 1 H (400 MHz) NMR data of Compound 2

[0067]

[0068] a Assignments are interchangeable.

[0069] Example 2 Anti - inflammatory Activity Experiment

[0070] 1. Experimental Method

[0071] Take RAW264.7 cells in the logarithmic growth phase (6×10 per well 4Cells (number of cells not specified) were seeded into a 96-well cell culture plate containing 10% fetal bovine serum;

[0072] After overnight culture, different concentrations of Compound 1, Compound 2, and the positive control drug (L-NMMA) diluted with DMEM medium were added respectively (3 replicates were set for each group);

[0073] After 1 hour of culture, 1 μg / mL of LPS was added to treat the cells and the culture was continued for 16 hours;

[0074] Subsequently, the supernatant was aspirated and transferred to a new 96-well plate. 50 μL of Griess reagent A and 50 μL of Griess reagent B were added. After thorough mixing, the absorbance value was measured at a wavelength of 546 nm on an enzyme-linked immunosorbent assay (ELISA) reader;

[0075] Finally, the inhibition rate of NO production of different concentrations of drugs was calculated based on the measurement results, and the IC 50 value was calculated using GraphPad Prism according to the inhibition rate.

[0076] 2. Results

[0077] In this study, N-Monomethyl-L-arginine was used as the positive control drug to evaluate the inhibitory activity of Compound 1 and Compound 2 obtained in this invention on nitric oxide (NO) production. The results showed that Compound 1 and Compound 2 exhibited strong inhibitory activity on NO production, as shown in Table 3.

[0078] Table 3. Anti-inflammatory activity data of the compounds of this invention

[0079] Compound <![CDATA[IC 50 (μM)]]> Compound 1 19.14±1.28 Compound 2 16.06±1.21 N-Monomethyl-L-arginine 33.62±1.53

[0080] The results indicated that Compound 1 and Compound 2 had obvious anti-inflammatory activity, and this invention provided candidate compounds for the research and development of new anti-inflammatory drugs.

[0081] In the description of this specification, terms such as "connection", "installation", "fixation", etc. should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection or an indirect connection through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to the specific circumstances.

[0082] In the description of this specification, the descriptions of the terms "one embodiment", "some embodiments", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0083] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A class of tigliane diterpenoids, characterized in that, Its structural formula is as follows: Formula I: Among them, R1, R2, R3 and R4 in Formula I are each independently selected from the following substituents: hydrogen, hydroxyl, methyl, carbamoyl, -C 2-16 linear or branched alkyl, -C 2-16 linear or branched alkanoyl, -C 2-16 alkenyl, -C 3-8 cycloalkyl, -C 4-8 cycloalkanoyl, -C 2-16 alkenoyl, -C 4-8 aryl, -C 4-8 heteroaryl, -C 2-16 aliphatic amino, -C 2-16 aliphatic amido, cycloalkylamino, benzoyl, phenylacetyl, glucosyl, xylosyl.

2. A class of tigliane diterpenoid compounds according to claim 1, characterized in that, The specific structural formulas of the tigliane - type diterpenoid compounds with R1, R2, R3, and R4 in Formula I selected as substituted groups are as follows: Formula II and Formula III:

3. A method for preparing a class of tigliane diterpenoids, characterized in that, It includes the following steps: 1) Add the dried roots of Croton tiglium to 60% - 95% ethanol and reflux for extraction 3 times, 2 hours each time, to obtain an ethanol extract. The ethanol extract is concentrated under reduced pressure to recover the solvent and obtain an extract. 2) Use D101 macroporous resin to elute the extract with 20% ethanol, 30% ethanol, 70% ethanol, 90% ethanol, and pure ethanol. Concentrate the eluate of the 70% ethanol fraction to obtain a 70% ethanol component, and then elute it with dichloromethane / methanol at ratios of 25:1, 10:1, 5:1, and 1:1 to obtain 6 fractions Fr.C1 - Fr.C6. 3) Fr.C5 is subjected to ODS column chromatography and eluted with a methanol / water gradient to obtain 5 fractions Fr.C5a - Fr.C5e. 4) Fr.C5d is eluted with a petroleum ether / ethyl acetate gradient to obtain 3 components Fr.C5d1 - Fr.C5d3. Fr.C5d2 uses a mobile phase with a ratio of methanol to water of 85∶15 and a flow rate of 10 ml / min, and the tigliane - type diterpenoid compounds shown in Formula II and Formula III in Claim 2 are obtained by HPLC.

4. Use of a class of tigliane diterpenoids, characterized in that, One or more of the tigliane - type diterpenoid compounds described in Claim 1 or Claim 2 are used as active ingredients in a pharmaceutical composition.

5. Use of a class of tigliane diterpenoid compounds according to claim 4, characterized in that, The pharmaceutical composition is an anti - inflammatory drug.

6. Use of a class of tigliane diterpenoid compounds according to claim 4, characterized in that, The pharmaceutical composition includes pharmaceutically acceptable pharmaceutical excipients.

7. Use of a class of tigliane diterpenoid compounds according to claim 4, characterized in that, It is for use in the preparation of a drug with anti - inflammatory effects.

Images