Podocarpus macrophylrane type diterpenoid compound separated from pinus massoniana cone as well as preparation method and application of podocarpus macrophylrane type diterpenoid compound

Abstract

The invention relates to the technical field of natural product separation, in particular to a mataidane type diterpenoid compound separated from a pinus massoniana cone and a preparation method and application of the mataidane type diterpenoid compound. The masson pine cone is subjected to alcohol extraction, resin adsorption and column separation to obtain the mataidane diterpenoid compound. The podocarpus macrophyllus type diterpenoid compound disclosed by the invention can be used for resisting inflammation, and the podocarpus macrophyllus type diterpenoid compound has certain inhibitory activity on RAW264.7 cells.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, specifically to the separation of rosinane-type diterpenoids from the pine cones of Pinus massoniana, their preparation methods, and applications. Background Technology

[0002] The pine cones of the Masson pine (Pinus massoniana) are the mature fruits of the plant *Pinus massoniana*, belonging to the genus *Pinus* of the family Pinaceae. They are also known as pine burrs, pine cones, or pine balls. Research has found that their main chemical components include polysaccharides, terpenes, flavonoids, polyphenols, lignin, and volatile oils, making them a diverse and complex group of compounds. Masson pine cones have antitussive and antiasthmatic effects, and are used for coughs, asthma, phlegm, and chronic bronchitis. Clinical data shows that pine cones possess anti-inflammatory, antitumor, antibacterial, and antioxidant properties, and these potential benefits are increasingly attracting attention, with hopes to further explore their value.

[0003] The cones of *Pinus massoniana* have demonstrated various potential therapeutic effects in clinical treatment, such as relieving coughs, enteritis, and neurasthenia, as well as antiviral, antitumor, and anti-inflammatory properties (for treating chronic bronchitis). These effects make pine cones a highly anticipated natural medicinal resource. Furthermore, *Pinus massoniana* cones also possess immune-enhancing and antioxidant properties, and have been widely used in traditional medicine. Ancient texts such as the *Compendium of Materia Medica* record the effects of pine cones in relieving coughs, asthma, and dispelling wind and moistening the intestines. Modern research has also shown that the polyphenolic components in *Pinus massoniana* cones are associated with a lower cancer incidence rate, and their antitumor mechanisms may include activating apoptosis pathways and terminating the cell cycle. Therefore, the development and utilization of *Pinus massoniana* cones have broad prospects and deserve further in-depth research to fully realize their medicinal value.

[0004] Inflammation is a protective response of the body to tissue damage, infection, and abnormal states. It plays a role in clearing harmful substances, repairing damaged tissues, and regulating immune function. However, excessive inflammatory responses can lead to tissue damage and functional disorders in patients. RAW264.7 macrophages play a complex and crucial role in the occurrence, development, and homeostasis of inflammation by secreting pro-inflammatory cytokines and regulating polarization. Summary of the Invention

[0005] One object of the present invention is to provide a rosinane-type diterpenoid compound isolated from the pine cones of *Pinus massoniana*, and another object of the present invention is to provide a method for obtaining effective chemical components from the pine cones of *Pinus massoniana*, and to provide the application of the rosinane-type diterpenoid compound.

[0006] According to a first aspect of the invention, the invention provides a rosinane-type diterpenoid compound isolated from the pine cone of *Pinus massoniana*, said compound having a structure as shown in Formula 1:

[0007] .

[0008] According to a second aspect of the invention, the invention provides a pine resin-type diterpenoid compound isolated from the pine cone of *Pinus massoniana*, said compound having a structure as shown in Formula 2:

[0009] .

[0010] According to a third aspect of the invention, the invention provides a pine resin-type diterpenoid compound isolated from the pine cone of *Pinus massoniana*, said compound having a structure as shown in Formula 3:

[0011] .

[0012] According to a fourth aspect of the present invention, the present invention provides a method for preparing a rosinane-type diterpenoid compound isolated from the pine cone of *Pinus massoniana*, the method comprising: extracting *Pinus massoniana* pine cones with alcohol, adsorbing with macroporous resin and separating by column chromatography to obtain compounds 1-3;

[0013] Compounds 1-3 have structures as shown in Formulas 1 to 3:

[0014] .

[0015] Optionally, the extract of the pine cones of *Pinus massoniana* obtained by alcohol extraction is adsorbed onto a macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then separated by column chromatography to obtain 10 fractions Fr.1-10; Fr.4 is separated by column chromatography to obtain 7 fractions Fr.4.1-4.7; Fr.4.3 is separated by column chromatography to obtain compound 1; Fr.6 is separated by column chromatography to obtain 10 fractions Fr.6.1-6.10; Fr.6.5 is separated by column chromatography to obtain 7 fractions Fr.6.5.1-6.5.7; Fr.6.5.5 is separated by column chromatography to obtain compound 2; and Fr.6.5.4 is separated by column chromatography to obtain compound 3.

[0016] Optionally, obtaining compound 1 by column separation of Fr.4.3 includes eluting Fr.4.3 with methanol via a gel column to obtain two components Fr.4.3.1 and Fr.4.3.2, and eluting Fr.4.3.2 with methanol-water via a reversed-phase column and with methanol via a gel column to obtain compound 1.

[0017] Optionally, obtaining compound 2 by column separation of Fr.6.5.5 includes eluting Fr.6.5.5 with methanol-water via an MCI column to obtain two components Fr.6.5.5.1 and Fr.6.5.5.2, and eluting Fr.6.5.5.1 with methanol-water via a reversed-phase column and repeatedly eluting with methanol via a gel column to obtain compound 2.

[0018] Optionally, obtaining compound 3 by column separation of Fr.6.5.4 includes eluting Fr.6.5.4 with methanol-water via an MCI column only to obtain four fractions Fr.6.5.4.1-6.5.4.4, and repeatedly eluting Fr.6.5.4.1 with methanol via a gel column and with methanol-water via a reversed-phase column to obtain compound 3.

[0019] According to a fifth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-described rosinane-type diterpenoid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0020] According to a sixth aspect of the present invention, the present invention provides an inhibitor containing the above-mentioned rosinane-type diterpenoid compound.

[0021] According to a seventh aspect of the present invention, the present invention provides the use of the above-described rosinane-type diterpenoid compounds in the preparation of anti-inflammatory inhibitors.

[0022] According to an eighth aspect of the present invention, the present invention provides the use of the above-described rosinane-type diterpenoid compounds in the preparation of RAW264.7 cell inhibitors.

[0023] Optionally, the inhibitor may be in the form of tablets, pills, injections, granules, capsules, fat emulsions, droplets, powder for injection, ointments, gels, or sprays.

[0024] The present invention has the following beneficial effects:

[0025] This invention isolates and extracts phoenixolane-type diterpenoid compounds (formulas 1-3) from the pine cones of *Pinus massoniana*. Experimental testing shows that compounds 1-3 exhibit certain inhibitory activity against RAW264.7 cells, and can be used as a reference for the development of inflammation inhibitors and for the preparation of related drugs. The preparation method of this invention is simple and low-cost, promoting the development of *Pinus massoniana* pine cones in medicine, health products, food, and other fields, and playing an important role in promoting the sustainable development of the industry. Attached Figure Description

[0026] Figure 1 HR-ESI-MS of compound 1 of the present invention is shown;

[0027] Figure 2 Compound 1 of the present invention is shown. 1 H-NMR spectrum;

[0028] Figure 3 Compound 1 of the present invention is shown. 13 C-NMR spectrum;

[0029] Figure 4The nuclear magnetic resonance HSQC spectrum of compound 1 of the present invention is shown;

[0030] Figure 5 The nuclear magnetic resonance HMBC spectrum of compound 1 of the present invention is shown;

[0031] Figure 6 The nuclear magnetic resonance of compound 1 of the present invention is shown. 1 H- 1 H COSY spectrum;

[0032] Figure 7 The following is a DEPT 135 nuclear magnetic resonance spectrum of compound 1 of the present invention;

[0033] Figure 8 HR-ESI-MS of compound 2 of the present invention is shown;

[0034] Figure 9 Compound 2 of the present invention is shown. 1 H-NMR spectrum;

[0035] Figure 10 Compound 2 of the present invention is shown. 13 C-NMR spectrum;

[0036] Figure 11 The nuclear magnetic resonance HSQC spectrum of compound 2 of the present invention is shown;

[0037] Figure 12 The nuclear magnetic resonance HMBC spectrum of compound 2 of the present invention is shown;

[0038] Figure 13 The nuclear magnetic resonance of compound 2 of the present invention is shown. 1 H- 1 H COSY spectrum;

[0039] Figure 14 The DEPT 135 nuclear magnetic resonance spectrum of compound 2 of the present invention is shown;

[0040] Figure 15 HR-ESI-MS of compound 3 of the present invention is shown;

[0041] Figure 16 Compound 3 of the present invention is shown. 1 H-NMR spectrum;

[0042] Figure 17 Compound 3 of the present invention is shown. 13 C-NMR spectrum;

[0043] Figure 18 The nuclear magnetic resonance HSQC spectrum of compound 3 of the present invention is shown;

[0044] Figure 19 The nuclear magnetic resonance HMBC spectrum of compound 3 of the present invention is shown;

[0045] Figure 20 The nuclear magnetic resonance of compound 3 of the present invention is shown. 1 H- 1 H COSY spectrum;

[0046] Figure 21 The nuclear magnetic resonance DEPT135 spectrum of compound 3 of the present invention is shown. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of the invention, and not all of them.

[0048] Example 1

[0049] Step 1: Take 35kg of Masson pine cone medicinal material and reflux with 85% ethanol. Repeat the extraction 3 times, each extraction for 1.5h. The first extraction uses 4 times the amount of 85% ethanol, and the second and third extractions use 3 times the amount of 85% ethanol respectively. Filter the ethanol extract, combine the filtrates and recover the ethanol under reduced pressure. Directly heat and concentrate, continuously adding water to evaporate the ethanol until there is no obvious ethanol taste.

[0050] Step 2: After concentration under reduced pressure, the extract is obtained. After mixing, the extract is passed through a D101 macroporous adsorption resin column and eluted and concentrated with water and 80% ethanol, respectively, to obtain a water-eluted segment and an 80% ethanol-eluted segment.

[0051] Step 3: Pass the 80% ethanol eluent fraction through a polyamide column and elute with 50% ethanol to obtain an extract. Separate the extract using a normal-phase silica gel (200-300 mesh) column and perform gradient elution with petroleum ether-ethyl acetate (10:0-0:10) and ethyl acetate-methanol (10:0-3:7) sequentially. Detect the eluents by thin-layer chromatography, combine the colored eluent fractions, and analyze and combine them by TLC to obtain 10 fractions Fr.1-10. Concentrate the combined eluent fractions to dryness under reduced pressure for later use.

[0052] Step 4: Fr.4 from Step 3 was subjected to normal-phase silica gel chromatography with gradient elution using dichloromethane-ethyl acetate (10:0-0:10) to obtain seven fractions Fr.4.1-4.7. Among them, Fr.4.3 was eluted with methanol using a Toyopearl HW-40F gel column to obtain two fractions Fr.4.3.1 and Fr.4.3.2; Fr.4.3.2 was then eluted with 50%~55% methanol-water using an ODS reversed-phase chromatography column and with methanol using a Toyopearl HW-40F gel column to obtain compound 1.

[0053] Step 5: Fr.6 from Step 3 was passed through an MCI column and eluted with a gradient of 30%–95% ethanol-water to obtain 10 fractions Fr.6.1–6.10; among them, Fr.6.5 was passed through a normal-phase silica gel column and eluted with petroleum ether-ethyl acetate (10:0.3–6:4) to obtain 7 fractions Fr.6.5.1–6.5.7. Among them, Fr.6.5.5 was eluted with an MCI column and eluted with 35%–60% methanol-water to obtain 2 fractions Fr.6.5.5.1 and Fr.6.5.5.2; among them, Fr.6.5.5.1 was eluted with an ODS reversed-phase column and eluted with 25%–50% methanol-water and repeatedly eluted with methanol on a Toyopearl HW-40F gel column to obtain compound 2;

[0054] Step 6: Elute Fr.6.5.4 from Step 5 onto an MCI column with 35%~55% methanol-water to obtain four components Fr.6.5.4.1-6.5.4.4; among them, Fr.6.5.4.1 was repeatedly eluted with methanol on a Toyopearl HW-40F column and with 40%~80% methanol-water on an ODS reversed-phase column to obtain compound 3.

[0055] In this context, the above-mentioned petroleum ether-ethyl acetate ratio (10:0-0:10) is expressed as a ratio of petroleum ether to ethyl acetate of 10:0-0:10.

[0056] Structural identification of compounds 1-3 of this invention: using 1 H NMR, 13 C10 NMR spectroscopy, two-dimensional NMR spectroscopy, and high-resolution mass spectrometry are used to identify the structure of isolated monomeric compounds, such as... Figure 1-21 As shown.

[0057] Compound 1, Appearance (light yellow solid), Molecular formula: C 16 H 22 O2; HR-ESI-MS m / z: 247.1686 [M+H]+, calculated value 247.1693, 269.1521 [M+Na]+, calculated value 269.1512; its NMR data are shown in Table 1.

[0058] Compound 2, appearance (light yellow solid), molecular formula: C 17 H 22 O4; HR-ESI-MS m / z: 289.1445 [MH]-, calculated value 289.1434, its NMR data is in Table 2.

[0059] Compound 3, appearance (light yellow solid), molecular formula: C 17 H 22 O4; HR-ESI-MS m / z: 291.1587 [M+H]+, calculated value 291.1591, 289.1447 [MH]-, calculated value 289.1434, its NMR data are shown in Table 3.

[0060] The chemical formulas of the corresponding compounds 1-3 are shown in Formulas 1-3 below, wherein compound 1 is a 4,5-open ring-type Podophyllin-type diterpenoid and compound 3 is a 9,10-open ring-type Podophyllin-type diterpenoid.

[0061] .

[0062] Table 1. Compound 1 1 H (600 MHz) and 13 C(150 MHz) NMR

[0063]

[0064] Table 2. Compound 2 1 H (600 MHz) and 13 C (150 MHz) NMR

[0065]

[0066] Table 3. Compound 3 1 H (600 MHz) and 13 C (150 MHz) NMR

[0067]

[0068] Experimental Example 1

[0069] To achieve the above-mentioned objectives of this invention, this invention studies the anti-inflammatory activity of rosinane-type diterpenoids extracted from the pine cones of *Pinus massoniana*. LPS (lipopolysaccharide)-induced mouse macrophages RAW264.7 were used as an inflammation model. The effect of the compounds on the survival rate of RAW264.7 cells was tested using the CCK-8 assay to determine the safe dosage concentration. Furthermore, a NO detection kit was used to assess changes in the level of the inflammatory mediator nitric oxide (NO). This study has profound significance in preliminarily revealing the significant anti-inflammatory effects of rosinane-type diterpenoids extracted from the pine cones of *Pinus massoniana*, and lays a solid foundation for subsequent research on the drug's mechanism of action. The specific steps are as follows:

[0070] Step 1: Cell thawing: Quickly remove the frozen cells from the -80℃ freezer and thaw them rapidly in a 37℃ water bath. Spray the outside of the cryovials with medical alcohol for sterilization. Open the lid in a clean bench, aspirate the cell suspension using a pipette, and transfer it to a centrifuge tube. Slowly add 4-5 mL of culture medium dropwise, resuspending the cells by pipetting. Centrifuge at 1200 rpm for 3 minutes at room temperature. Aspirate the supernatant, add 1-2 mL of culture medium, resuspend, and transfer to a culture dish containing 3-4 mL of culture medium. Observe cell growth regularly.

[0071] Step 2: Cell Culture and Passaging: Observe cell growth daily. If cell growth is slow and has not yet reached 70-80% confluence, only a medium change is needed. Remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and then transfer it to a laminar flow hood for further processing. In the laminar flow hood, gently aspirate the culture medium from the flask or dish and wash the cells with physiological saline twice. After washing twice, add approximately 3 mL of complete culture medium and allow the cells to continue culturing. When the cells reach 70-80% confluence, passaging is necessary. Similarly, remove the cell culture flask or dish from the incubator, spray it with 75% medical alcohol, and then transfer it to a laminar flow hood. Next, gently aspirate the culture medium from the flask or dish and wash the cells with physiological saline twice. After washing twice, add approximately 1 mL of trypsin to digest the cells. The entire digestion process takes about 2 minutes. After trypsin digestion, the digestion solution needs to be transferred to a centrifuge tube. Use culture medium to wash the cells adhering to the walls of the culture flask or dish, and transfer them to the centrifuge tube as well. Centrifuge at 1200 rpm for 3 minutes. After centrifugation, discard the supernatant to obtain the cells adhering to the tube wall. At this point, add complete culture medium to the cells and repeatedly pipette to evenly disperse them before aliquoting and culturing.

[0072] Step 3: The experiment was divided into a control group and groups with different concentrations of LPS. The control group was given complete DMEM medium, and the groups with different concentrations of LPS were given medium with final LPS concentrations of 0.25, 0.5, 1, 2 and 4 μg / mL, respectively.

[0073] Step 4: Collect cells in the logarithmic growth phase and adjust the cell concentration to approximately 3 × 10⁻⁶. 5 Approximately 100 μL / mL was inoculated into each well of a 96-well plate and incubated at 37°C with 5% CO2 for 24 h to allow adhesion. After 24 h of incubation, the plate was washed once with PBS, and the OD value was measured using the CCK-8 assay. Each concentration was replicated in 5 wells, and the experiment was repeated 3 times.

[0074] Step 5: Preliminary screening of compounds from Pinus massoniana var. massoniana with anti-inflammatory activity: RAW264.7 cells in logarithmic growth phase were used, and the cell concentration was adjusted to 3 × 10⁻⁶ cells / year. 5 Cells were seeded at approximately 100 μL / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Cells were then divided into Control, Model, Positive, and Drug-treated groups. Control and Model groups were cultured in complete DMEM medium, the Positive group in medium containing DEX, and the Drug-treated groups in medium containing the compound. The final DEX concentration was 25 μmol / L, the final compound concentrations were 6.25, 12.5, 25, 50, and 100 μmol / L, and the final LPS concentration was 0.25 μg / mL. After 3 h of culture, the Control group was cultured in complete DMEM medium, the Model group in medium containing LPS, the Positive group in a mixture of DEX and LPS, and the Drug-treated group in a mixture of the compound and LPS. After 24 h of culture, the culture supernatant was collected, and the NO level in the supernatant was determined according to the NO detection kit instructions. Each concentration was tested in triplicate, and the experiment was repeated three times. The IC50 values ​​of compounds 1-3 for NO were... 50 The values ​​are shown in Table 3.

[0075] Table 3 IC50 of compounds 1-2 against NO 50 value

[0076]

[0077] Among them, IC 50 The concentration of the compound at which the proliferation inhibition rate is 50% is used to indicate its anti-inflammatory activity.

[0078] Table 6 shows that detecting changes in the level of the inflammatory mediator NO using a NO detection kit can directly reflect the degree of inhibition of the inflammatory response by the compounds. Based on the IC50 values ​​of compounds 1-3 against NO... 50The experimental results preliminarily revealed that the rosinane-type diterpenoids extracted from the pine cones of *Pinus massoniana* possess significant anti-inflammatory effects. Compound 1 exhibited the best inhibitory effect on the inflammatory response, providing strong evidence for further in-depth research into its anti-inflammatory mechanism. This indicates that the compounds of this invention can be used as a reference for the inhibitory use of inflammation and for the preparation of related drugs, laying the foundation for research on drug mechanisms of action.

[0079] This invention uses LPS-induced mouse macrophage RAW264.7 cells as an inflammation model. The CCK8 assay was used to test the effect of compounds on RAW264.7 cell survival, determining the appropriate dosage. A NO detection kit was used to detect the level of the inflammatory mediator nitric oxide (NO). The preliminary results of this invention not only validate the anti-inflammatory activity of the piperine-type diterpenoid compounds but also lay the foundation for subsequent research on their mechanism of action. Further investigation into the effects of these compounds on key aspects such as inflammatory signaling pathways, the synthesis and release of inflammatory mediators, and other key processes can gradually reveal their anti-inflammatory mechanism, providing theoretical support for the development of novel anti-inflammatory drugs. This research lays a solid foundation for further exploring the specific mechanisms of action of these piperine-type diterpenoid components in the treatment of inflammation-related diseases and provides important clues for the development of new drugs.

[0080] Uses, preparations, administration:

[0081] Medical uses and indications:

[0082] The biological data provided by this invention indicate that the compounds of this invention are beneficial for the treatment or prevention of diseases caused by abnormalities in RAW264.7 (macrophages). Therefore, the compounds of this invention are beneficial for the treatment of autoimmune diseases, including but not limited to: psoriasis, vitiligo, dermatitis, alopecia areata, rheumatoid arthritis, colitis, multiple sclerosis, systemic lupus erythematosus, and Crohn's disease. The compounds of this invention can be used as monotherapy or in combination therapy, and can be used in combination with other drugs besides those of this invention.

[0083] Pharmaceutical methods:

[0084] The pharmaceutical method of the present invention includes determining a therapeutically effective dose for a subject requiring the compounds of the present invention. The "therapeutically effective dose" varies depending on the stage, progression, or severity of the disease. The daily dose of the compounds and compositions of the present invention will depend on various factors of the patient, including the condition being treated, the severity of the condition, the efficacy of the specific compound used, the specific composition, age, weight, general health status, sex and diet, route and schedule of administration, metabolism and / or the rate of excretion of the compound, duration of treatment, etc. Furthermore, the required dose of the compounds of the present invention, after being formulated into a pharmaceutically acceptable carrier, can be administered to humans and other animals. Routes of administration include oral, rectal, parenteral, intracisional, intravaginal, intraperitoneal, topical (e.g., via transdermal patches, powders, ointments, or drops), sublingual, buccal, or nasal spray, etc. The effective dose of the compounds of the present invention is generally measured in terms of the amount administered per kilogram of patient body weight, preferably 0.1 to 125 mg / kg body weight, and generally 0.01 to 500 mg / kg body weight. Dosing can be done once or multiple times, daily, weekly, every other day or every few days, or on an intermittent schedule. For example, the compound can be administered daily, weekly (e.g., every Monday), indefinitely, or for several weeks (e.g., 4-10 weeks). The effective dose of the compounds of the present invention will vary depending on the compound used, the dosing regimen, the severity of the disease, the conditions being treated, and various physical factors associated with the patient. In most cases, satisfactory therapeutic effects are achieved when the daily dose of the preferred compounds of the present invention is about 0.01 to 500 mg / kg. The preferred dose is 0.1 to 125 mg / kg, and the more preferred dose is 1 to 25 mg / kg. Parenteral doses are typically at about 10%-20% of the oral dose level. When the compounds of the present invention are used as part of a combination therapy regimen, each component of the composition will be administered during a desired treatment period. Whether as a single dosage unit or as a single dosage form containing two components, the components in the composition can be administered simultaneously during the treatment period, or at different times during the treatment period, or one can be administered as a pretreatment of the other.

[0085] Regarding compounds:

[0086] The compounds of this invention can be used for treatment in their free form or, where appropriate, in the form of pharmaceutically acceptable salts or other derivatives. As used herein, "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this invention, which are suitable for humans and lower animals, without excessive toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, phosphonates, and other types of compounds are well known in the art. Such salts can be prepared by reacting the compounds isolated and purified in this invention with a suitable free base or acid.

[0087] Salts formed from pharmaceutically non-toxic acids, including but not limited to amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, or obtained by means well known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glycerophosphate, gluconate, hemisulfate, heptahydrate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, per-3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include suitable non-toxic ammonium, quaternary ammonium, and amino cations formed using ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0088] Furthermore, as used herein, the term "prodrug" refers to a compound that can be converted in vivo into the compound represented by Formula 1, Formula 2, or Formula 3 of this invention. This conversion occurs through the hydrolysis of the prodrug in the blood or through enzymatic action in the blood or tissues to the parent compound.

[0089] Composition:

[0090] The compositions described in this patent consist of any of the compounds described herein (or prodrugs, or pharmaceutically acceptable salts thereof, or other pharmaceutically acceptable derivatives thereof), and one or more pharmaceutically acceptable carriers or excipients. These compositions may optionally further comprise one or more additional therapeutic agents. The compounds of the present invention can be administered to the desired patient in combination with one or more other treatment regimens (e.g., tofacitinib or other kinase inhibitors, interferon, bone marrow transplantation, farnesyltransferase inhibitors, bisphosphonates, thalidomide administration combinations, cancer vaccines, hormone therapy, antibodies, radiation, etc.). The pharmaceutical composition of the compounds may be another one or more anti-inflammatory or anticancer agents.

[0091] As described herein, the compositions of the present invention comprise the compounds of the present invention and pharmaceutically acceptable carriers, including any and all solvents, diluents or other carriers, dispersants or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., to suit a particular dosage form. Examples of pharmaceutically acceptable carrier materials include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; ethylene glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solutions, as well as other non-toxic and compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and aromatizers, preservatives, and antioxidants may also be present in the composition.

[0092] formula:

[0093] This invention also covers a class of compositions (collectively referred to herein as “carrier” materials) using the active compounds of this invention in combination with one or more pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and, if desired, other active ingredients. The active compounds of this invention can be administered via any suitable route, preferably in the form of a pharmaceutical composition suitable for such route of administration at an effective dose required for the intended treatment. The compounds and compositions of this invention can be administered orally, via mucosal, topical, rectal, pulmonary, such as by inhalation spray, or parenterally, including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, and infusion techniques. Administration is in dosage form and contains a pharmaceutically acceptable carrier, adjuvant, and excipient. For oral administration, the pharmaceutical composition can be in the following forms, for example, tablets, capsules, suspensions, or liquids. Examples of dosage units are tablets or capsules. For example, they may contain an amount of active ingredient from 1 to 2000 mg, preferably from 1 to 500 mg, and more commonly from 5 to 200 mg. The appropriate daily dose for an individual or other mammal may vary depending on the patient and other factors, but can be determined again using conventional methods. As previously stated, the amount of compound in the administration and dosing regimens of the compounds and / or compositions involved in this invention depends on a variety of factors, including the subject's age, weight, sex, and medical condition, type of disease, severity of the disease, route and frequency of administration, and the specific compound used. Therefore, dosing regimens can vary considerably, but can be determined using standard methods. Typical daily doses are 0.01 to 500 mg / kg body weight, preferably 0.1 to 125 mg / kg body weight, and more preferably 1 to 25 mg / kg body weight.

[0094] The active compounds of the present invention are typically administered via a route of administration with one or more adjuvants, excipients, or carriers. If administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose esters, alkyl cellulose esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, farnesian gum, sodium alginate, polyvinylpyrrolidone / or polyvinyl alcohol, and then compressed into tablets or capsules for convenient administration. Such capsules or tablets may contain a controlled-release formulation that disperses the active compound in hydroxypropyl methylcellulose. Formulations suitable for topical administration include liquid or semi-liquid formulations (such as liniments, lotions, ointments, creams, or pastes) suitable for penetration through the skin and drops suitable for application to the eyes, ears, or nose. Suitable topical doses of the compounds of the present invention are 0.1 to 150 mg, once to four times daily, preferably once to twice daily. For topical administration, when using ointments, the active ingredient may be based on any paraffin or water-miscible ointment. Alternatively, the active ingredient can be formulated as a water-in-oil emulsion base cream. If desired, the aqueous phase of the cream base may include, for example, at least 30% by weight of, polyols such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, polyethylene glycol, and mixtures thereof. Topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogues. The compound may also be administered via a transdermal device. Transdermal administration is preferably achieved using patches containing a reservoir and a porous membrane or solid base. The oil phase of the emulsion of the present invention may be composed of known ingredients in a known manner, comprising at least one emulsifier in a mixture of fat or oil, or a mixture of both fat and oil. Preferably, the hydrophilic emulsifier may be used in combination with a lipophilic emulsifier as a stabilizer; additionally, it is preferred that it may also be used in combination with oil and fat. Suitable emulsifiers and emulsion stabilizers for use in the formulations of this invention include Tween 60, Span 80, cetearyl alcohol, myristicin, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone, or mixtures thereof with emulsifying waxes, or other materials known in the art. Creams should preferably be non-greasy, non-staining, and washable, and have a suitable consistency to prevent leakage from tubes or other containers. Straight-chain or branched, mono- or dialkyl esters such as diisohexadiate, isohexadecanoyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture of branched esters may also be used. Alternatively, high-melting-point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used. Formulations suitable for topical administration to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly for aqueous solvents of the active ingredient.The active ingredient in these formulations is preferably 0.5% to 20% by weight, more advantageously 0.5% to 10%, and most preferably about 1.5% concentration. For parenteral administration, the formulations can be in the form of aqueous or non-aqueous isotonic sterile injectable solutions or suspensions. These solutions and suspensions can be prepared from one or more sterile powders or granules, using the formulations mentioned herein for oral administration or using other suitable dispersants or wetting agents and suspending agents, carriers, or diluents. The compounds can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffer solutions. Other adjuvants and routes of administration are well known in the pharmaceutical industry.

[0095] The active ingredient can also be administered by injection, in combination with suitable carriers including saline, glucose, or water, or with cyclodextrin (Captisol), co-solvent solubilization (i.e., propylene glycol), or micellar solubilization (i.e., Tween 80). The formulation can also be a sterile injectable solution or a suspension in a non-toxic, parenteral acceptable diluent or solvent, such as 1,3-butanediol. Suitable solvents include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspension media. Any mild fixed oil used for this purpose can be used, including synthetic mono- or diglycerides.

[0096] For pulmonary administration, the pharmaceutical composition can be administered as an aerosol or via inhaler, including dry powder aerosols. Suppositories for rectal administration can be prepared by combining the drug with suitable non-irritating excipients, such as cocoa butter and polyethylene glycol, which are solids at room temperature but liquids at rectal temperature, thus melting and releasing the drug in the rectum. The pharmaceutical composition can be incorporated into conventional pharmaceutical processes such as sterilization and / or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can also be prepared with enteric coating. Such compositions may also contain adjuvants such as wetting agents, sweeteners, flavoring agents, and aromatizers.

[0097] The pharmaceutical compositions of the present invention comprise, as well as a pharmaceutically acceptable salt thereof, a kinase inhibitor (small molecule, peptide, antibody, etc.), an immunosuppressant, an anticancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an antiangiogenic compound; and any pharmaceutically acceptable carrier, adjuvant, or excipient. Alternative compositions of the present invention comprise compounds having a formula of 1, 2, or 3 as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or excipient. Such compositions may optionally comprise one or more additional therapeutic agents, including, for example, kinase inhibitors (small molecule, peptide, antibody, etc.), immunosuppressants, anticancer agents, antiviral agents, anti-inflammatory agents, antifungal agents, antibiotics, or antiangiogenic compounds.

[0098] The term (pharmaceutically acceptable carrier or adjuvant) refers to a carrier or adjuvant that can be administered to a patient together with the compound of the present invention, and which does not impair the drug activity and is non-toxic when the dose is sufficient to deliver a therapeutic dose. Pharmaceutically acceptable carriers, adjuvants, and excipients may be used in the pharmaceutical compositions of the present invention, including but not limited to ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-atocopHerol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, surfactants used in mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl, including 2- and 3-hydroxypropyl-cyclodextrins, or other soluble derivatives, may also be advantageously used to improve the delivery of compounds of the structural formula described herein. The pharmaceutical composition may be administered orally in any acceptable dosage form, including but not limited to capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally using aqueous suspensions and / or emulsions, the active ingredient may be suspended or dissolved in the oil phase with the emulsion and / or suspending agent. If desired, certain sweeteners, flavoring agents, and / or coloring agents may be added. The pharmaceutical composition may include the use of liposomes or microencapsulation techniques, various examples of which can be found in the literature. The pharmaceutical composition may be administered via nasal aerosol or inhalation. Such compositions are prepared according to known techniques in the field of pharmaceutical formulations and can be prepared into solutions in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons, and / or other solubilizers or dispersants, examples of which are well known in the prior art.

[0099] Combination therapy:

[0100] The compounds of the present invention can be used alone or in combination with one or more other compounds of the present invention or with one or more other pharmaceutical agents. When administered in combination, the therapeutic agents can be formulated for simultaneous or sequential administration at different times, or the therapeutic agents can be administered as a single composition. The term "combination therapy" refers to the use of the compounds of the present invention in conjunction with another pharmaceutical agent, administered either simultaneously or sequentially, with the aim of achieving optimal drug efficacy. Co-administration includes simultaneous delivery formulations as well as separate formulations for each compound. Therefore, the administration of the compounds of the present invention can be used concurrently with other known therapies in the art, such as in cancer treatment using radiotherapy or adjunctive therapies such as cell growth inhibitors, cytotoxic agents, or other anticancer agents to improve cancer symptoms. The present invention is not limited to the order of administration; the compounds of the present invention can be administered prior to, concurrently with, or after other anticancer agents or cytotoxic agents.

[0101] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A diterpenoid compound of the pine cone type isolated from *Pinus massoniana*, characterized in that, The compound has the structure shown in Formula 1: 。 2. A diterpenoid compound of the pine cone type isolated from *Pinus massoniana*, characterized in that, The compound has the structure shown in Formula 2: 。 3. A diterpenoid compound of the pine cone type isolated from *Pinus massoniana*, characterized in that, The compound has the structure shown in Formula 3: 。 4. A method for preparing pine resin-type diterpenoids isolated from the pine cones of *Pinus massoniana*, characterized in that, The preparation method includes: obtaining compounds 1-3 by alcohol extraction, macroporous resin adsorption and column separation of pine towers; Compounds 1-3 have structures as shown in Formulas 1 to 3: 。 5. The method for preparing the rosinane-type diterpenoid compounds isolated from the pine cones of *Pinus massoniana* as described in claim 4, characterized in that, The extract of the pine cones of *Pinus massoniana* obtained by alcohol extraction is adsorbed onto a macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then separated by column chromatography to obtain 10 fractions Fr.1-10; Fr.4 is separated by column chromatography to obtain 7 fractions Fr.4.1-4.7; Fr.4.3 is separated by column chromatography to obtain compound 1; Fr.6 is separated by column chromatography to obtain 10 fractions Fr.6.1-6.10; Fr.6.5 is separated by column chromatography to obtain 7 fractions Fr.6.5.1-6.5.7; Fr.6.5.5 is separated by column chromatography to obtain compound 2; and Fr.6.5.4 is separated by column chromatography to obtain compound 3.

6. A pharmaceutical composition comprising a rosinane-type diterpenoid compound as described in any one of claims 1-3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. An inhibitor containing a rosinane-type diterpenoid compound as described in any one of claims 1-3.

8. The use of the rosinane-type diterpenoid compound as described in any one of claims 1-3 in the preparation of anti-inflammatory inhibitors.

9. The use of the rosinane-type diterpenoid compound as described in any one of claims 1-3 in the preparation of RAW264.7 cell inhibitors.

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