Isolation of abietane diterpenoids from the pine cones of pinus massoniana and preparation method and application thereof

Abstract

This invention relates to the field of natural product separation technology, specifically to arosinane diterpenoids isolated from the pine cones of *Pinus massoniana*, their preparation methods, and applications. The preparation method of these arosinane diterpenoids includes alcohol extraction from the pine cones of *Pinus massoniana*, followed by macroporous resin adsorption separation and column separation. The arosinane diterpenoids disclosed in this invention can be used for anti-tumor purposes, particularly showing good inhibitory effects on human lung cancer cells, human liver cancer cells, human glioma cells, human colorectal adenocarcinoma cells, or human ovarian cancer cells.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and more specifically, to rosinane diterpenoids isolated 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 cones of a pine tree (Pinaceae family), also known as pine nut, pine cone, or pine ball. They are a abundant and widely distributed natural product in my country. Masson pine cones contain a variety of complex chemical components, mainly including polysaccharides, terpenes, flavonoids, polyphenols, lignin, and volatile oils. These components have significant medicinal value, offering possibilities for developing drugs to treat various diseases, including serious illnesses such as cancer. Currently, many drugs based on natural compounds are already in clinical use or undergoing clinical trials, and the medicinal potential of Masson pine cones is gradually attracting attention.

[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. 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. Summary of the Invention

[0004] One object of the present invention is to provide a rosinane 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 diterpenoid compound.

[0005] According to a first aspect of the invention, the invention provides a rosinane diterpenoid compound isolated from the pine cones of Pinus massoniana, said compound having a structure as shown in Formula 1:

[0006] .

[0007] According to a second aspect of the invention, the invention provides a rosinane diterpenoid compound isolated from the pine cone of *Pinus massoniana*, said compound having a structure as shown in Formula 2:

[0008] .

[0009] According to a third aspect of the present invention, the present invention provides a method for preparing rosinane diterpenoid compounds isolated from the pine cones 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 1-2;

[0010] The compounds 1-2 have structures as shown in Formula 1 to Formula 2:

[0011] .

[0012] Optionally, the extract of the pine cone of Pinus massoniana obtained by alcohol extraction is adsorbed by macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then subjected to a normal phase column and eluted sequentially with petroleum ether-ethyl acetate and ethyl acetate-methanol gradients to obtain 10 fractions Fr.1-10. Fr.4 is separated by a normal phase column to obtain compound 1, and Fr.7 is separated by a normal phase column to obtain compound 2.

[0013] Optionally, the separation of Fr.4 by normal-phase silica gel column chromatography to obtain compound 1 includes: eluting Fr.4 by a normal-phase silica gel column with a chloroform-ethyl acetate gradient to obtain 7 fractions Fr.4.1-Fr.4.7; separating Fr4.1 by a gel column to obtain 3 fractions Fr.4.1.1-4.1.3; separating Fr.4.1.3 by a gel column to obtain 5 fractions Fr.4.1.3.1-4.1.3.5; and separating Fr.4.1.3.2 by both normal-phase silica gel column chromatography and gel column chromatography to obtain compound 1.

[0014] Optionally, obtaining compound 2 by separating Fr.7 using a normal-phase silica gel column includes: eluting Fr.7 using a normal-phase silica gel column with a petroleum ether-ethyl acetate gradient to obtain 11 fractions Fr.7.1-7.11; separating Fr7.10 using a gel column to obtain 7 fractions Fr.7.10.1-7.10.7; and separating Fr.7.10.2 using a reverse-phase silica gel column and a gel column to obtain compound 2.

[0015] According to a fourth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-described rosinane diterpenoid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0016] According to a fifth aspect of the present invention, the present invention provides an inhibitor containing the above-mentioned rosinane diterpenoid compound.

[0017] According to a sixth aspect of the present invention, the present invention provides the use of the above-described rosinane diterpenoid compounds in the preparation of antitumor inhibitors.

[0018] Optionally, the tumor may be lung cancer, liver cancer, glioma, colorectal adenocarcinoma, or ovarian cancer.

[0019] According to a seventh aspect of the present invention, the present invention provides the use of the above-described rosinane diterpenoid compounds in the preparation of inhibitors for human lung cancer cells, human liver cancer cells, human glioma cells, human colorectal adenocarcinoma cells, or human ovarian cancer cells.

[0020] Optionally, the inhibitor may be in the form of tablets, pills, injections, granules, capsules, fat emulsions, droplets, powder for injection, ointments, gels, or sprays.

[0021] The present invention has the following beneficial effects:

[0022] This invention isolates and extracts rosinane diterpenoid compounds of formula 1-2 from the pine cones of *Pinus massoniana*. Experimental testing shows that compounds 1-2 exhibit inhibitory activity against human lung cancer cells, human liver cancer cells, human glioma cells, human colorectal adenocarcinoma cells, and human ovarian cancer cells. These compounds can be used as a reference for tumor inhibitors and for the preparation of related drugs. The preparation method of this invention is simple and low-cost, promoting the pharmaceutical development and application of *Pinus massoniana* pine cones and playing a significant role in promoting the sustainable development of the industry. Attached Figure Description

[0023] Figure 1 HR-ESI-MS of compound 1 of the present invention is shown;

[0024] Figure 2 Compound 1 of the present invention is shown. 1 H-NMR spectrum;

[0025] Figure 3 Compound 1 of the present invention is shown. 13 C-NMR spectrum;

[0026] Figure 4 The nuclear magnetic resonance HSQC spectrum of compound 1 of the present invention is shown;

[0027] Figure 5 The nuclear magnetic resonance HMBC spectrum of compound 1 of the present invention is shown;

[0028] Figure 6 The nuclear magnetic resonance of compound 1 of the present invention is shown. 1 H- 1 H COSY spectrum;

[0029] Figure 7 The following is a DEPT 135 nuclear magnetic resonance spectrum of compound 1 of the present invention;

[0030] Figure 8 HR-ESI-MS of compound 2 of the present invention in negative ion mode is shown;

[0031] Figure 9HR-ESI-MS of compound 2 of the present invention in positive ion mode is shown;

[0032] Figure 10 Compound 2 of the present invention is shown. 1 H-NMR spectrum;

[0033] Figure 11 Compound 2 of the present invention is shown. 13 C-NMR spectrum;

[0034] Figure 12 The nuclear magnetic resonance HSQC spectrum of compound 2 of the present invention is shown;

[0035] Figure 13 The nuclear magnetic resonance HMBC spectrum of compound 2 of the present invention is shown;

[0036] Figure 14 The nuclear magnetic resonance of compound 2 of the present invention is shown. 1 H- 1 H COSY spectrum; Figure 15 The nuclear magnetic resonance DEPT 135 spectrum of compound 2 of the present invention is shown. Detailed Implementation

[0037] 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.

[0038] Example 1

[0039] Step 1: Extract the Masson pine cone medicinal material (35kg) by reflux with 85% ethanol 3 times, each extraction for 1.5h. Use 4 times the amount of 85% ethanol for the first extraction, and 3 times the amount of 85% ethanol for the second and third extractions. 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.

[0040] Step 2: After concentration under reduced pressure, the extract is obtained. After mixing the extract, it is passed through a D101 macroporous adsorption resin column and eluted with water and 80% ethanol, respectively, to obtain a water elution segment and an 80% ethanol elution segment.

[0041] Step 3: The 80% ethanol fraction was passed through a polyamide column and eluted with 50% ethanol to obtain an extract (1115g). The extract was separated by a normal-phase silica gel (200-300 mesh) column, first eluted with petroleum ether-ethyl acetate (10:0-0:10), then eluted with ethyl acetate-methanol (10:0-3:7) in a gradient. The fractions were examined and combined by TLC to obtain 10 fractions (Fr.1-10). The combined eluted fractions were concentrated to dryness under reduced pressure for later use.

[0042] Step 4: Fr.4 from Step 3 was subjected to normal-phase silica gel column chromatography with gradient elution using dichloromethane-ethyl acetate (10:0-0:10) to obtain 7 fractions (Fr.4.1-4.7); among them, Fr.4.1 was eluted with a Sephadex LH-20 gel column with dichloromethane-methanol (1:1) to obtain 3 fractions (Fr.4.1.1-4.1.3); among them, Fr.4.1.3 was eluted with a Toyopearl HW-40F gel column with methanol to obtain 5 fractions (Fr.4.1.3.1-4.1.3.5); among them, Fr.4.1.3.2 was eluted with a normal-phase silica gel column with petroleum ether-ethyl acetate (10:0.1-5:5) and a Toyopearl HW-40F gel column with methanol. The obtained fractions were subjected to preparative HPLC (85% methanol-water) to obtain compound 1;

[0043] Step 5: Fr.7 from Step 3 was eluted by a normal-phase silica gel column with a gradient of petroleum ether-ethyl acetate (10:0.5-4:6) to obtain 11 fractions (Fr.7.1-7.11); among them, Fr.7.10 was eluted by a Toyopearl HW-40F gel column with methanol to obtain 7 fractions (Fr.7.10.1-7.10.7); among them, Fr.7.10.2 was eluted by an ODS reverse-phase silica gel column with 40%-100% methanol and water, and then eluted by a Toyopearl HW-40F gel column with methanol to obtain compound 2.

[0044] In the above example, dichloromethane-methanol (1:1) means that the ratio of dichloromethane to methanol is 1:1.

[0045] The structural identification performed in 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 1-14 As shown.

[0046] Compound 1, appearance (white solid), molecular formula: C 20 H 26 O; HR-ESI-MS m / z: 283.2070 [M+H]+ Calculated value: 283.2056; its NMR data are shown in Table 1.

[0047] Compound 2, properties (white crystals), molecular formula: C 20 H 30 O4; HR-ESI-MS m / z: 357.2047 [M+Na] + Calculated values: 357.2036, 333.2074 [MH] - Calculated value: 333.2060; its NMR data is in Table 2, and the corresponding chemical formulas with labels are shown in Formulas 1-2 below;

[0048]

[0049] Table 1. Compound 1 1 H (400 MHz) and 13 C(100 MHz) NMR

[0050]

[0051] Table 2. Compound 2 1 H (600 MHz) and 13 C (150 MHz) NMR

[0052]

[0053] Experimental Example 1

[0054] To achieve the above-mentioned objectives of this invention, this invention studies the antitumor activity of rosinane diterpenoids extracted from the pine cones of *Pinus massoniana*. This study is of profound significance in preliminarily revealing that rosinane diterpenoids extracted from the pine cones of *Pinus massoniana* possess certain antitumor effects, and lays a solid foundation for subsequent research on the drug's mechanism of action. The specific steps are as follows:

[0055] 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.

[0056] 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, they can be passaged. Similarly, first 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.

[0057] Step 3: Collect cells in logarithmic growth phase. The cell concentrations of human lung cancer cells (A549), human liver cancer cells (HepG2), human glioma cells (HS683), human colorectal adenocarcinoma cells (HCT-15), and human ovarian cancer cells (A2780) were adjusted to approximately 3 × 10⁴ cells / mL, and the concentration of human liver cancer cells (HepG2) was adjusted to approximately 5 × 10⁴ cells / mL. 100 μL of each cell was evenly seeded into 96-well cell culture plates. After 24 h of culture, the cells were divided into a control group, a positive control group, and a drug-treated group. The control group received complete culture medium, the drug-treated group received drug-containing culture medium with a final compound concentration of 50 μmol / L, and the positive control group received drug-containing culture medium with a final cisplatin (DDP) concentration of 50 μmol / L. After culturing cells for 72 hours, the liquid in the 96-well plate was aspirated, the cells were rinsed once with PBS, and 100 μL of 5% CCK8 solution was added to each well. After incubation for another 2 hours, the OD value at 450 nm was measured using a microplate reader. Each concentration was tested in parallel with 5 wells, and the experiment was repeated 3 times. The antitumor inhibition rate was calculated according to the formula shown in Table 3.

[0058] Table 3. Antitumor inhibition rate (%) of compounds 1-2

[0059]

[0060] Table 3 shows that the changes in cell viability detected by CCK8 assay can directly reflect the inhibitory effects of the compounds on tumors such as human lung cancer cells A549, human liver cancer cells HepG2, human glioma cells HS683, human colorectal adenocarcinoma cells HCT-15, and human ovarian cancer cells A2780. Compound 1 showed the best inhibitory effect on human ovarian cancer cells A2780, while compound 2 showed the best inhibitory effect on human lung cancer cells A549. The experimental results of compounds 1-2 preliminarily reveal that the rosinane diterpenoids extracted from pine cones of *Pinus massoniana* possess certain antitumor effects, providing strong evidence for further in-depth research on their antitumor mechanisms. This indicates that the compounds of this invention can be used for antitumor purposes and for the preparation of related drugs, laying the foundation for research on drug mechanisms of action.

[0061] The preliminary results of this invention not only validate the antitumor activity of rosinane diterpenoids, but also lay the foundation for subsequent research on their drug action mechanisms. This allows for the gradual elucidation of their antitumor mechanisms and provides theoretical support for the development of novel antitumor drugs. It also lays a solid foundation for further exploring the specific mechanisms of action of these rosinane diterpenoid components in the treatment of tumor-related diseases, providing important clues for the development of new drugs.

[0062] Uses, preparations, administration

[0063] Medical uses and indications

[0064] 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 tyrosine kinases (JAK1, JAK2, JAK3, TYK2). More than one-fifth of the compounds of this invention have been shown to strongly inhibit JAK tyrosine kinase activity, and the JAK kinase family is closely related to the occurrence and metastasis of autoimmune diseases and cancers. 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 are also beneficial for the treatment of cancers, including primary and metastatic cancers, including solid tumors. Such cancers include, but are not limited to: non-small cell lung cancer, small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal adenocarcinoma, melanoma, endometrial cancer, prostate cancer, bladder cancer, leukemia, gastric cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, chronic myeloid leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, esophageal cancer, brain tumor, B-cell and T-cell lymphoma, lymphoma, multiple myeloma, biliary carcinosarcoma, and bile duct cancer. The compounds of this invention also include treatments for cancers resistant to one or more other treatment methods. The compounds of this invention can also be used for diseases other than autoimmune diseases and cancers related to JAK1 kinase and / or JAK2 and / or JAK3 kinase, including but not limited to fundus diseases, pulmonary fibrosis, and liver fibrosis. The compounds of this invention can be used as monotherapy or in combination therapy, and can be used in combination with multiple compounds of this invention or with other drugs outside of this invention.

[0065] Pharmaceutical methods

[0066] 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.

[0067] About compounds

[0068] 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.

[0069] 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.

[0070] Furthermore, the term "prodrug" as used herein refers to a compound that can be converted in vivo into a compound represented by Formula 1 or Formula 2 of this invention. This conversion is achieved through the hydrolysis of the prodrug in the blood or through enzymatic action in the blood or tissues to the parent compound.

[0071] Composition

[0072] 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.

[0073] 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.

[0074] formula

[0075] 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.

[0076] 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. Optionally, the hydrophilic emulsifier may be used in conjunction with a lipophilic emulsifier as a stabilizer; additionally, it is preferred that it may also be used in conjunction 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.

[0077] 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.

[0078] 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.

[0079] The pharmaceutical compositions of the present invention comprise a compound of formula 1 or 2 as described herein, or a pharmaceutically acceptable salt thereof, a kinase inhibitor (small molecule, peptide, antibody, etc.), an immunosuppressant, an anticancer drug, 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 formula 1 or 2 as described herein, or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers, adjuvants, or excipients. 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.

[0080] 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.

[0081] combination therapy

[0082] 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.

[0083] Currently, standard treatment for primary tumors includes surgical resection, radiation, or chemotherapy. Typical chemotherapy includes any DNA alkylating agent, DNA intercalating agent, CDK inhibitor, or microtubule toxin. The chemotherapeutic agents used are just below the maximum tolerated dose.

[0084] The application of the compounds described in this invention in the field of anticancer treatment can be as a monotherapy or can also include conventional surgical or radiotherapy or chemotherapy or immunotherapy other than those described in this invention. Such treatment can be used simultaneously, sequentially or separately with the compounds of this invention, and can include one or more of the following classes of antitumor agents: (1) antiproliferative / antitumor drugs and combinations thereof, such as those used in medical oncology, such as alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolomide and nitrosourea); antimetabolites (e.g. gemcitabine and antifolate agents, such as fluorouracil and tegafur, raltitrexed, methotrexate, etc.). Pterin, cytarabine, hydroxyurea, and fluorouracil; antitumor antibiotics (e.g., anthracyclines like doxorubicin, bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, styracin, and sclerotinib); antimitotic agents (e.g., vinca alkaloids like vincristine, vinblastine, vinorelbine, and vinorelbine, and taxanes like paclitaxel and doxorubicin and polokinase inhibitors); and topoisomerase inhibitors (e.g., epipodophyllotoxins like etoposide and tinib). (1) Podophylline, acridine, topotecan and camptothecin; (2) Cell growth inhibitors such as anti-estrogenic (acting) drugs (e.g. tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), anti-androgens (e.g. bicalutamide, flutamide, nilumethoxazole and cyproterone acetate), LHRH antagonists or LHRH agonists (e.g. goserelin, leuprorelin and buserelin), progestins (e.g. megestrol acetate), aromatase inhibitors (e.g. (3) Anti-invasive agents, such as anastrozole, letrozole, vortexol and exemestane) and 5α-reductase inhibitors such as finasteride; (4) anti-invasive agents, such as c-Src protein kinase family inhibitors (dasatinib, BMS-354825; Journal of Pharmaceutical Chemistry, 2004, 47, 6658-6661) and bosutinib (SKI-606), and metalloproteinase inhibitors such as marimasitol, urokinase type plasminogen activator receptor function or antibody heparinase inhibitors.

[0085] 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 rosinane diterpenoid compound isolated from the pine cone of Pinus massoniana characterized in that, The compound has the structure shown in Formula 1: 。 2. A rosinane diterpenoid compound isolated from the pine cone of Pinus massoniana characterized in that, The compound has the structure shown in Formula 2: 。 3. A method for preparing rosinane diterpenoid compounds isolated from the pine cones of Pinus massoniana, characterized in that, The preparation method includes: obtaining compounds 1-2 by alcohol extraction, macroporous resin adsorption and column separation of pine towers from Masson pine; The compounds 1-2 have structures as shown in Formula 1 to Formula 2: 。 4. The method for preparing rosinane diterpenoid compounds isolated from the pine cones of *Pinus massoniana* as described in claim 3, characterized in that, The extract of the pine cone of Pinus massoniana by alcohol extraction is adsorbed by macroporous adsorption resin, eluted and concentrated with ethanol to obtain an ethanol fraction. The ethanol fraction is then passed through a normal phase column and eluted sequentially with petroleum ether-ethyl acetate and ethyl acetate-methanol gradients to obtain 10 fractions Fr.1-10. Fr.4 is separated by column to obtain compound 1, and Fr.7 is separated by column to obtain compound 2.

5. A pharmaceutical composition comprising a rosinane diterpenoid compound as described in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. An inhibitor containing a rosinane diterpenoid compound as described in claim 1.

7. An inhibitor containing a rosinane diterpenoid compound as described in claim 2.

8. The use of the rosinane diterpenoid compound as described in claim 1 or claim 2 in the preparation of antitumor inhibitors.

9. The use of the rosinane diterpenoid compound as described in claim 8 in the preparation of an antitumor inhibitor, wherein the tumor is lung cancer, liver cancer, glioma, colorectal adenocarcinoma, or ovarian cancer.

10. The use of the rosinane diterpenoid compound as described in claim 1 or claim 2 in the preparation of inhibitors for human lung cancer cells, human liver cancer cells, human glioma cells, human colorectal adenocarcinoma cells, or human ovarian cancer cells.

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