Xanthorad-derived tetrahydroanthraquinones, methods of making and pharmaceutical compositions and uses thereof

Abstract

The application discloses tetrahydroanthraquinone compounds, a preparation method of the tetrahydroanthraquinone compounds, and a pharmaceutical composition and application of the tetrahydroanthraquinone compounds, in particular, 13 tetrahydroanthraquinone compounds (1-13) found in the root of the plant of Xanthoceras sorbifolia Bunge are disclosed, the compounds are prepared by a phytochemical method, the pharmaceutical composition containing the tetrahydroanthraquinone, and the application of the compounds in the treatment of pneumonia and pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to tetrahydroanthraquinone compounds extracted and isolated from the root of *Phellodendron amurense*, their preparation methods, and their use in the prevention and / or treatment of pneumonia and pulmonary fibrosis. Background Technology

[0002] *Prismatomeris connata* YZRuan is a plant belonging to the genus *Prismatomeris* in the Rubiaceae family. It is mainly distributed in Guangdong, Guangxi, and Hainan provinces. The root is primarily used medicinally, possessing properties such as cooling the blood and stopping bleeding, strengthening muscles and bones, promoting diuresis and relieving jaundice, and removing blood stasis and promoting tissue regeneration. It can be used to treat injuries from falls and blows, rheumatic bone pain, leukemia, hepatitis, gingival bleeding, thalassemia, and aplastic anemia.

[0003] Pneumonia refers to inflammation of the terminal airways, alveoli, and pulmonary interstitium, which can be caused by pathogenic microorganisms, physical and chemical factors, immune damage, allergies, and drugs. Before the use of antibiotics, bacterial pneumonia posed a significant threat to the health of children and the elderly. The advent and development of antibiotics once led to a significant decrease in the mortality rate of pneumonia. However, in recent years, despite the use of potent antibiotics and effective vaccines, the overall mortality rate of pneumonia has no longer decreased and has even increased. Traditional Chinese medicine uses a decoction of raw Astragalus membranaceus (Huang Gen) for internal use to prevent and treat pneumonia and pulmonary fibrosis. However, the active compounds involved in this treatment were previously unclear. Therefore, compounds 1-13, isolated and purified from the traditional Chinese medicine Astragalus membranaceus (Huang Gen), with a well-defined chemical structure and significant anti-inflammatory activity, show excellent clinical development potential for the prevention and treatment of pneumonia and pulmonary fibrosis. Summary of the Invention

[0004] The inventors discovered for the first time that the root extract of *Phellodendron chinense* has anti-inflammatory activity. Thirteen new tetrahydroanthraquinone compounds were extracted and isolated from the *Phellodendron chinense* root extract, and pharmacodynamic evaluation showed that they have good anti-inflammatory effects.

[0005] The technical problem solved by this invention is to provide 13 new tetrahydroanthraquinone compounds.

[0006] Another technical problem solved by this invention is that it provides 13 new methods for preparing tetrahydroanthraquinones.

[0007] Another technical problem solved by the present invention is to provide a pharmaceutical composition in which 13 tetrahydroanthraquinones have significant anti-inflammatory activity and can be used to treat pneumonia and pulmonary fibrosis.

[0008] Specifically, the structural formulas of compounds 1-13 involved in this invention are shown below.

[0009]

[0010] The present invention provides 13 novel methods for preparing tetrahydroanthraquinones, as detailed below.

[0011] The dried roots of *Rhizoma Scutellariae Radix* weighed 50 kg. After being pulverized, they were extracted three times by refluxing with 80% ethanol for 2 hours each time, to obtain a total extract of 80% ethanol from the roots of *Rhizoma Scutellariae Radix*. After concentration under reduced pressure, approximately 2500 g of extract was obtained. This extract was dispersed in 3 L of warm water and then extracted three times with ethyl acetate to obtain 132 g of ethyl acetate extract. The ethyl acetate extract was subjected to multiple column chromatography analyses (forward and reverse silica gel, gel chromatography) and finally purified by HPLC to obtain 13 new tetrahydroanthraquinones.

[0012] Another aspect of the present invention relates to pharmaceutical compositions in which the compounds of the present invention are active ingredients. These pharmaceutical compositions can be prepared according to methods known in the art. They can be formulated into any dosage form suitable for human or animal use by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The content of the compounds of the present invention in their pharmaceutical compositions is typically 0.1-95% by weight.

[0013] The compounds of this invention or pharmaceutical compositions containing them can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0014] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0015] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0016] To formulate the compounds of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannose, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0017] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0018] To formulate the drug delivery unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. Various diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0019] To prepare the compounds of this invention into injectable formulations, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc. pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing lyophilized powder injections, mannitol, glucose, etc., can also be added as supporting agents.

[0020] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0021] To achieve the purpose of medication and enhance the therapeutic effect, the drug or drug composition of the present invention can be administered using any known method of administration.

[0022] The dosage of the pharmaceutical compositions of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally, the suitable daily dose range of the compounds of the present invention is 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above doses can be administered as a single dose unit or divided into several dose units, depending on the physician's clinical experience and the administration regimen, including the use of other treatment methods.

[0023] The compounds or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compounds of the present invention have a synergistic effect with other therapeutic drugs, their dosage should be adjusted according to the actual situation.

[0024] This invention investigates the inhibitory activity of novel tetrahydroanthraquinone compounds on cellular inflammation using an in vitro pharmacological model of LPS-induced NO release from macrophages. Attached Figure Description

[0025] Figure 1 --Flowchart of Huang Gen Extraction

[0026] Figure 2 --Flowchart of separation of ethyl acetate extract from Sophora flavescens root

[0027] Figure 3- Cell proliferation of RAW264.7 cells treated with 0.05 μmol / mL monomer and 40 μg / mL component drugs for 12 h.

[0028] Figure 4- Cell proliferation of RAW264.7 cells treated with 0.2 μmol / mL monomeric drug for 12 h.

[0029] Figure 5- -Compound 1-Compound 13 treatment of LPS-induced NO release in RAW264.7 cells Detailed Implementation

[0030] Examples and pharmacological activity experiments further illustrate the present invention, but do not imply any limitation on the present invention. 1. Preparation of compounds 1-13 (see appendix) Figure 1-2 )

[0031] The dried roots of *Rhizoma Scutellariae* weighed 50 kg. After being crushed, they were refluxed with 80% ethanol three times, each time for 2 hours, to obtain a total extract of 80% ethanol from the roots of *Rhizoma Scutellariae*. After being concentrated under reduced pressure, an extract of about 2500 g was obtained. This extract was dispersed in 3 L of warm water and then extracted with ethyl acetate three times to obtain 132 g of the ethyl acetate extract.

[0032] Further separation was performed by silica gel column chromatography using dichloroethane / methanol (50:1-5:1). The elution fraction of 10:1 yielded 40g. This fraction was then subjected to gradient elution with petroleum ether-ethyl acetate (5:1-1:4) on silica gel column chromatography. After eluting for 40 column volumes, each fraction was combined into 8 elution fractions (denoted as Fr.1 to Fr.8) after 5 column volumes.

[0033] Fr.6 (10.2 g) was subjected to medium-pressure column chromatography (30% MeOH / H2O-100% MeOH / H2O, gradient elution for 9 hours). Approximately 500 ml of the sample was collected, yielding 45 fractions, named F1 to F45. F14 was prepared by C18 column HPLC (20% MeCN-H2O) to obtain compound 6 (1.8 mg). F16 was prepared by C18 column HPLC (55% MeOH-H2O) to obtain F16-1, and F16-1 was prepared by C18 column HPLC (30% MeCN-H2O) to obtain compound 5 (3.1 mg). F17 was prepared by cellulose column HPLC (60% MeCN-H2O) to obtain F17-1 and F17-2. F17-1 was then prepared by C18 column HPLC (35% MeCN-H2O) to obtain compound 4 (5.0 mg). F17-2 was then prepared by C18 column HPLC (40% MeCN-H2O) to obtain compound 3 (3.5 mg). F18 was prepared by cellulose column HPLC (80% MeOH-H2O) to obtain F18-1, F18-2, and F18-3. F18-1 was then prepared by cellulose column HPLC (60% MeCN-H2O) to obtain compound 9 (2.5 mg). F18-2 was then prepared by cellulose column HPLC (50% MeCN-H2O) to obtain compound 10 (2.8 mg). F18-3 was then prepared by C18 column HPLC (50% MeOH-H2O) to obtain compound 11 (3.3 mg). F19 was prepared by cellulose column HPLC (40% MeCN-H2O) to obtain F19-1, and F19-1 was prepared by cellulose column HPLC (80% MeOH-H2O) to obtain compound 12 (3.5 mg). F20 was prepared by C18 column HPLC (50% MeOH-H2O) to obtain F20-1, and F20-1 was prepared by cellulose column HPLC (90% MeOH-H2O) to obtain compound 13 (2.5 mg). F27 was prepared by cellulose column HPLC (85% MeCN-H2O) to obtain compound 8 (4.2 mg). F29 was prepared by C18 column HPLC (55% MeOH-H2O) to obtain compound 1 (1.1 mg). F30 was prepared by C18 column HPLC (60% MeOH-H2O) to obtain F30-1, and F30-1 was prepared by phenyl column HPLC (40% MeCN-H2O) to obtain compound 2 (3.8 mg). F34 was prepared by C18 column HPLC (60% MeOH-H2O) to obtain F34-1, and F34-1 was prepared by C18 column HPLC (40% MeCN-H2O) to obtain compound 7 (3.3 mg).

[0034] The physicochemical and spectroscopic data of compounds 1-14 are as follows:

[0035] The physicochemical and spectroscopic data of compound 1 are as follows:

[0036] Pale yellow powder; -46.0(c 0.10MeOH), (+)-HRESI-Ms m / z 395.1699[M+H] + ,(calcd.for C 20 H 27 O8, 375.1700). 1 H NMR data (CDCl3, 600MHz), δ H : 4.49 (1H, m, H-1), 3.54 (1H, dd, J = 7.8, 10.8Hz, H-2), 1.82 (1H, m, H-3), 2. 17 (1H, ddd, J = 1.2, 5.4, 19.2Hz, H-4), 2.85 (1H, ddd, J = 3.6, 11.4, 19.2Hz, H-4), 6.98 (1H, s, H-8), 1.19 (3H, d, J = 6.0Hz, H-11), 3.93 (3H, s, H-12), 3. 92(3H,s,H-13), 3.96(3H,s,H-14), 3.01(3H,s,H-15), 3.07(3H,s,H-16).

[0037] 13 C NMR data (CDCl3, 150MHz), δ C : 74.3(C-1), 77.6(C-2), 32.6(C-3), 31.7(C-4), 141.0(C-4a), 154.4(C-5), 144.2(C-6), 158.4(C-7), 104.5(C-8), 136.6(C-8a), 99 .3(C-9), 141.2(C-9a), 182.0(C-10), 120.7(C-10a), 17.5(C-11), 61.6(C-12), 61.3(C-13), 56.5(C-14), 51.9(C-15), 52.3(C-16).

[0038] The physicochemical and spectroscopic data of compound 2 are as follows:

[0039] Pale yellow powder; -45.0(c 0.10MeOH), (+)-HRESI-MS m / z 387.1408[M+Na] + ,(calcd.for C 19 H 24 O7Na, 387.1414). 1 H NMR data (CDCl3, 400MHz), δH : 5.16 (1H, d, J = 7.6Hz, H-1), 3.77 (1H, dd, J = 7.6, 10.8Hz, H-2), 2.10 (1H, m, H-3), 2.50 (1H, m, H-4), 3.31 (1H, m, H-4), 7. 26(1H,s,H-8), 1.43(3H,d,J=6.4Hz,H-11), 4.31(3H,s,H-12), 4.13(3H,s,H-13), 4.17(3H,s,H-14), 4.10(3H,s,H-15).

[0040] 13 C NMR data (CDCl3, 100MHz), δ C : 74.4(C-1), 78.2(C-2), 32.9(C-3), 31.6(C-4), 129.0(C-4a), 148.2(C-5), 140.3(C-6), 152.7(C-7), 97.0(C-8), 124.3(C-8 a), 145.3(C-9), 116.5(C-9a), 146.2(C-10), 112.7(C-10a), 17.8(C-11), 62.4(C-12), 61.3(C-13), 56.0(C-14), 60.8(C-15).

[0041] The physicochemical and spectroscopic data of compound 3 are as follows:

[0042] Pale yellow powder; -30.0(c 0.10MeOH), (+)-HRESI-MS m / z 349.1284[M+H] + ,(calcd.for C 18 H 21 O7, 349.1282). 1 H NMR data (CDCl3, 600MHz), δ H : 4.89 (1H, d, J = 4.2Hz, H-1), 3.54 (1H, m, H-2), 2.11 (1H, m, H-3), 2.17 (1H, m, H-4), 2.97 (1H, m, H-4), 7 .47(1H,s,H-8), 1.14(3H,d,J=6.6Hz,H-11), 3.93(3H,s,H-12), 3.96(3H,s,H-13), 4.01(3H,s,H-14).

[0043] 13 C NMR data (CDCl3, 150MHz), δ C: 64.3(C-1), 72.8(C-2), 29.9(C-3), 30.5(C-4), 147.1(C-4a), 154.5(C-5), 148.4(C-6), 157.5(C-7), 106.0(C-8), 129 .3(C-8a), 185.3(C-9), 139.2(C-9a), 183.3(C-10), 119.9(C-10a), 17.0(C-11), 61.5(C-12), 61.7(C-13), 56.6(C-14).

[0044] The physicochemical and spectroscopic data of compound 4 are as follows:

[0045] Pale yellow powder; -56.0(c 0.10MeOH), (+)-HRESI-MS m / z 349.1282[M+H] + ,(calcd.for C 18 H 21 O7, 349.1282). 1 H NMR data (CDCl3, 400MHz), δ H : 4.69 (1H, ddd, J = 1.2, 3.2, 8.0Hz, H-1), 3.50 (1H, dd, J = 8.0, 11.2Hz, H-2), 1.83 (1H, m, H-3), 2.15 (1H, ddd, J = 3.2, 11.2, 20.0Hz, H-4), 2.95 (1H, ddd, J=1.2, 5.2, 20.0Hz, H-4), 7.44 (1H, s, H-5), 1.20 (3H, d, J=6.4Hz, H-11), 4.00 (3H, s, H-12), 3.95 (3H, s, H-13), 3.92 (3H, s, H-14).

[0046] 13 C NMR data (CDCl3, 100MHz), δ C : 72.9(C-1), 77.4(C-2), 32.0(C-3), 31.8(C-4), 146.4(C-4a), 154.4(C-5), 157.5(C-6), 148.5(C-7), 106.1(C-8), 119 .7(C-8a), 182.9(C-9), 139.5(C-9a), 186.2(C-10), 129.4(C-10a), 17.4(C-11), 61.7(C-12), 56.6(C-13), 61.5(C-14).

[0047] The physicochemical and spectroscopic data of compound 5 are as follows:

[0048] Pale yellow powder; +15.0(c 0.10MeOH), (+)-HRESI-MS m / z 335.1127[M+H] + ,(calcd.for C 17 H 19 O7, 335.1125). 1 H NMR data (DMSO-d6, 400MHz), δ H : 2.51 (1H, mH-1), 2.72 (1H, d, J = 19.2Hz, H-1), 3.60 (1H, d, J = 4.0Hz, H-2), 2.46 (1H, m, H-4), 2.51(1H,m,H-4), 7.21(1H,s,H-5), 1.23(3H,s,H-11), 3.96(3H,s,H-12), 3.82(3H,s,H-13).

[0049] 13 C NMR data (DMSO-d6, 100MHz), δ C :29.7(C-1), 69.2(C-2), 68.6(C-3), 32.6(C-4), 142.2(C-4a), 103.6(C-5), 157.5(C-6), 140.4(C-7), 154.7(C-8 ), 110.4(C-8a), 188.8(C-9), 142.4(C-9a), 183.1(C-10), 127.5(C-10a), 25.7(C-11), 56.4(C-12), 60.3(C-13).

[0050] The physicochemical and spectroscopic data of compound 6 are as follows:

[0051] Pale yellow powder; +17.0(c 0.10MeOH), (+)-HRESI-MS m / z 259.0962[M+H] + ,(calcd.for C 15 H 15 O4, 259.0965). 1 H NMR data (MeOD, 400MHz), δ H: 2.72 (1H, mH-1), 2.95 (1H, m, H-1), 3.79 (1H, t, J = 4.0Hz, H-2), 2.66 (2H, m, H-4), 8.02 (2H, m, H-5 / 8), 7.74 (2H, m, H-6 / 7), 1.36 (3H, s, H-11).

[0052] 13 C NMR data (MeOD, 100MHz), δ C :30.5(C-1), 71.5(C-2), 70.8(C-3), 34.3(C-4), 143.4(C-4a), 127.0(C-5 / 8), 133.5(C-6 / 7), 134.6(C-8a), 185.9(C-9), 142.8(C-9a), 185.7(C-10), 134.6(C-10a), 25.3(C-11).

[0053] The physicochemical and spectroscopic data of compound 7 are as follows:

[0054] Pale yellow powder; -46.0(c 0.10MeOH), (+)-HRESI-MS m / z 319.1173[M+H] + ,(calcd.for C 17 H 19 O6, 317.1176). 1 H NMR data (CDCl3, 500MHz), δ H : 2.53 (1H, mH-1), 3.04 (1H, m, H-1), 3.78 (1H, m, H-2), 1.94 (2H, m, H-3), 2.32 (1H, m, H-4), 2.95 ( 1H, m, H-4), 7.30 (1H, s, H-8), 1.14 (3H, d, J = 7.0Hz, H-11), 4.02 (3H, s, H-12), 4.03 (3H, s, H-13).

[0055] 13 C NMR data (CDCl3, 100MHz), δ C: 31.1(C-1), 70.8(C-2), 33.9(C-3), 29.0(C-4), 143.4(C-4a), 155.7(C-5), 141.4(C-6), 157.9(C-7), 103.9(C-8 ), 111.1(C-8a), 183.4(C-9), 142.6(C-9a), 189.0(C-10), 127.9(C-10a), 17.4(C-11), 61.2(C-12), 56.6(C-13).

[0056] The physicochemical and spectroscopic data of compound 8 are as follows:

[0057] Pale yellow powder; +49.0(c 0.10MeOH), (+)-HRESI-MS m / z 319.1174[M+H] + ,(calcd.for C 17 H 19 O6, 317.1176). 1 H NMR data (MeOD, 400MHz), δ H : 2.68 (1H, mH-1), 2.89 (1H, t, J=4.0Hz, H-1), 3.78 (1H, m, H-2), 2.61 (1H, m, H-4), 7.85 (1H, d, J=8 .4Hz, H-5), 7.33 (1H, d, J = 8.4Hz, H-6), 1.34 (3H, s, H-11), 3.96 (3H, s, H-12), 3.84 (3H, s, H-13).

[0058] 13 C NMR data (MeOD, 100MHz), δ C :30.7(C-1), 71.7(C-2), 70.8(C-3), 34.1(C-4), 142.1(C-4a), 125.3(C-5), 117.0(C-6), 160.1(C-7), 150.0(C-8 ), 126.3(C-8a), 185.5(C-9), 144.1(C-9a), 184.9(C-10), 127.1(C-10a), 25.2(C-11), 56.8(C-12), 61.4(C-13).

[0059] The physicochemical and spectroscopic data of compound 9 are as follows:

[0060] Pale yellow powder; -52.0(c 0.10MeOH), (+)-HRESI-Ms m / z 289.1072[M+H] + ,(calcd.for C 16 H 17 O5, 289.1071). 1 H NMR data (CDCl3, 600MHz), δ H : 2.49 (1H, mH-1), 3.02 (1H, t, J=4.0Hz, H-1), 3.74 (1H, ddd, J=4.8, 7.2, 12.0Hz, H-2), 1.88 (1H, m, H-3), 2.28 (1H, m, H-4) , 2.92 (1H, m, H-4), 7.24 (1H, d, J = 8.4Hz, H-7), 7.88 (1H, d, J = 8.4Hz, H-8), 1.10 (3H, d, J = 6.6Hz, H-11), 3.96 (3H, s, H-12).

[0061] 13 C NMR data (CDCl3, 150MHz), δ C :31.2(C-1), 71.0(C-2), 34.0(C-3), 29.5(C-4), 142.5(C-4a), 146.1(C-5), 155.2(C-6), 119.6(C-7), 125 .0(C-8), 126.3(C-8a), 183.6(C-9), 142.5(C-9a), 184.1(C-10), 142.5(C-10a), 17.4(C-11), 62.3(C-12).

[0062] The physicochemical and spectroscopic data of compound 10 are as follows:

[0063] Pale yellow powder; -81.0(c 0.10MeOH), (+)-HRESI-MS m / z319.1178[M+H] + ,(calcd.for C 17 H 19 O6, 319.1176). 1 H NMR data (MeOD, 500MHz), δ H: 2.38 (1H, mH-1), 2.92 (1H, m, H-1), 3.61 (1H, m, H-2), 1.82 (1H, m, H-3), 2.21 (1H, m, H-4), 2.89 ( 1H, m, H-4), 7.33 (1H, s, H-8), 1.12 (3H, d, J = 7.0Hz, H-11), 3.90 (3H, s, H-12), 3.95 (3H, s, H-13).

[0064] 13 C NMR data (MeOD, 100MHz), δ C : 31.7(C-1), 71.4(C-2), 35.3(C-3), 31.2(C-4), 146.0(C-4a), 155.8(C-5), 157.4(C-6), 147.5(C-7), 111.8(C-8 ), 119.2(C-8a), 185.2(C-9), 141.2(C-9a), 184.3(C-10), 146.0(C-10a), 17.8(C-11), 61.8(C-12), 61.5(C-13).

[0065] The physicochemical and spectroscopic data of compound 11 are as follows:

[0066] Pale yellow powder; -66.0(c 0.10MeOH), (+)-HRESI-MS m / z 319.1176[M+H] + ,(calcd.for C 17 H 19 O6, 319.1176). 1 H NMR data (CDCl3, 400MHz), δ H :2.48(1H,mH-1), 3.01(1H,m,H-1), 3.72(1H,m,H-2), 1.88(1H,m,H-3), 2.25(1H,m,H-4), 2.88( 1H, m, H-4), 7.45 (1H, s, H-5), 1.09 (3H, d, J = 6.5Hz, H-11), 4.03 (3H, s, H-12), 3.95 (3H, s, H-13).

[0067] 13 C NMR data (CDCl3, 100MHz), δ C: 31.2(C-1), 71.1(C-2), 34.0(C-3), 29.4(C-4), 141.5(C-4a), 105.8(C-5), 151.1(C-6), 144.3(C-7), 146.9(C-8 ), 119.3(C-8a), 183.3(C-9), 142.8(C-9a), 183.9(C-10), 126.2(C-10a), 17.4(C-11), 56.7(C-12), 61.8(C-13).

[0068] 41.2(C-9a), 184.3(C-10), 146.0(C-10a), 17.8(C-11), 61.8(C-12), 61.5(C-13).

[0069] The physicochemical and spectroscopic data of compound 12 are as follows:

[0070] Pale yellow powder; -48.0(c 0.10MeOH), (+)-HRESI-MS m / z 289.1071[M+H] + ,(calcd.for C 16 H 17 O5, 289.1070). 1 H NMR data (MeOD, 500MHz), δ H : 2.36 (1H, mH-1), 2.92 (1H, m, H-1), 3.59 (1H, m, H-2), 1.79 (1H, m, H-3), 2.17 (1H, m, H-4), 2.84 ( 1H, m, H-4), 7.31 (1H, s, H-5), 7.46 (1H, s, H-8), 1.09 (3H, d, J = 7.0Hz, H-11), 3.98 (3H, s, H-12).

[0071] 13 C NMR data (MeOD, 100MHz), δ C : 32.0(C-1), 71.5(C-2), 35.3(C-3), 31.0(C-4), 144.0(C-4a), 113.2(C-5), 153.0(C-6), 153.4(C-7), 109 .1(C-8), 127.1(C-8a), 185.2(C-9), 142.7(C-9a), 185.4(C-10), 128.7(C-10a), 17.8(C-11), 56.7(C-12).

[0072] The physicochemical and spectroscopic data of compound 13 are as follows:

[0073] Pale yellow powder; -89.0(c 0.10MeOH), (+)-HRESI-MS m / z 259.0966[M+H] + ,(calcd.for C 15 H 15 O4, 259.6905). 1 H NMR data (MeOD, 400MHz), δ H : 2.21 (1H, mH-1), 2.85 (1H, m, H-1), 3.61 (1H, m, H-2), 1.81 (1H, m, H-3), 2.40 (1H, m, H-4), 2.93 (1H, m, H-4), 7.3 5 (1H, d, J = 2.4Hz, H-5), 7.07 (1H, dd, J = 2.48.4Hz, H-7), 7.90 (1H, d, J = 8.4Hz, H-8), 1.09 (3H, d, J = 6.4Hz, H-11).

[0074] 13 C NMR data (MeOD, 100MHz), δ C :30.9(C-1), 71.5(C-2), 35.2(C-3), 31.9(C-4), 144.7(C-4a), 112.9(C-5), 164.1(C-6), 121.3(C-7 ), 129.9(C-8), 125.8(C-8a), 184.9(C-9), 142.7(C-9a), 185.9(C-10), 135.7(C-10a), 17.8(C-11).

[0075] Pharmacological experiments

[0076] 1. Experimental Objective

[0077] We used an LPS-induced macrophage NO release model (inflammation model) to screen for novel components and monomeric compounds from *Phellodendron amurense* that inhibit cellular inflammation.

[0078] 2. Experimental Principle

[0079] The concentration of NO can be determined by a one-step method. NO reacts with oxygen and water to form nitrates and nitrites. The latter two react with nitrate colorimetric reagents to form a pale red azo compound. The concentration of NO can be indirectly determined by colorimetry.

[0080] 3. Experimental instruments and reagents

[0081] Experimental instruments: cell culture incubator, clean bench, enzyme-linked immunosorbent assay (ELISA) reader, cell counting chamber, cell culture flask.

[0082] Experimental reagents: DMEM / F12 cell culture medium, penicillin-streptomycin antibiotics, fetal bovine serum, lipopolysaccharide, CCK8 cell proliferation reagent, and nitric oxide assay kit.

[0083] 4. Experimental Procedure

[0084] 4.1 Cell Culture

[0085] RAW264.7 cells were cultured in complete medium (20% fetal bovine serum + 2% penicillin and streptomycin) at a concentration of 5.0 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 200 μL / mL in 96-well plates and cultured overnight at 37°C with 5% CO2.

[0086] 4.2 Cell proliferation

[0087] Working solutions were prepared using serum-free medium, consisting of 40 μg / mL component solutions and 0.05 μmol / mL and 0.2 μmol / mL monomer solutions. 200 μL of working solution was added to each well for 12 h. Blank wells containing only serum-free medium and cell-free zeroing wells containing only serum-free medium were also included. After 12 h, the medium in the wells was discarded, and CCK8 working solution (blank medium: CCK8 reagent = 10:1) was added. Cells were incubated at 37°C for 2 h, and absorbance was measured at 450 nm. The cell proliferation rate was calculated as: (measurement wells - zeroing wells) / (blank wells - zeroing wells) × 100%.

[0088] 4.3 Cell stimulation and intervention

[0089] LPS (1 μg / mL) solution, LPS (1 μg / mL) + drug component (40 μg / mL, 18 μg / mL, 8 μg / mL) solution, and LPS (1 μg / mL) + drug monomer (0.2 μmol / mL, 0.05 μmol / mL, 0.01 μmol / mL) solution were prepared using serum-free culture medium. A blank group, an LPS group, and an LPS + drug intervention group were set up. 200 μL of cells were cultured in each well for 12 h. After 12 h, the cell supernatant was aspirated for subsequent NO concentration determination.

[0090] 4.4 NO determination

[0091] Take 100 μL of cell supernatant, add 200 μL of NO reagent one, mix well, add 100 μL of reagent two, vortex thoroughly, let stand for 10 minutes, centrifuge at 4000 rpm for 15 minutes, and take 160 μL of supernatant for testing. Prepare NO chromogenic reagent according to the ratio of reagent three: reagent four: reagent five = 2.5:1:1, and set up blank wells (add 160 μL of double-distilled water), standard wells (add 160 μL of 20 μmol / L sodium nitrite standard solution), and assay wells. Add 80 μL of chromogenic reagent to each well, mix well, let stand for 15 minutes, and measure the absorbance at 550 nm. Calculate the NO content (μmol / L) = (analyte well - blank well) / (standard well - blank well) × standard well concentration × 4

[0092] 5. Experimental Results

[0093] like Figure 3 The results showed that, except for a significant difference in cell proliferation rate between the 0.05 μmol / mL compound 6 treatment group and the blank group, no significant cell proliferation inhibition was observed in the other drug treatment groups. The cell proliferation rate of the 0.05 μmol / mL compound 6 12h treatment group was 90.4487% (>90%), and this concentration is considered suitable for subsequent drug intervention.

[0094] like Figure 4 The results showed that, except for a significant difference in cell proliferation rate between the 0.2 μmol / mL compound 6 treatment group and the blank group, no significant cell proliferation inhibition was observed in the other drug treatment groups. The cell proliferation rate in the 0.2 μmol / mL compound 6 12h treatment group was 64.3556% (<90%), and this concentration is considered unsuitable for subsequent drug intervention.

[0095] Results of different drugs inhibiting LPS-induced NO release in RAW264.7 cells

[0096] Table 1 Results of different drugs inhibiting LPS-induced NO release in RAW264.7 cells

[0097]

[0098] like Figure 5The results showed that after 12 h of induction of RAW264.7 cells with 1 μg / mL LPS, NO release in the model group significantly increased. The 0.2 μmol / mL treatment groups of compounds 1, 3, 9, and 10 significantly decreased NO release; the 0.2 μmol / mL and 0.05 μmol / mL treatment groups of compounds 2, 4, and 8 significantly decreased NO release in a dose-dependent manner; the 0.2 μmol / mL, 0.05 μmol / mL, and 0.01 μmol / mL treatment groups of compounds 5 and 7 all significantly decreased NO release in a dose-dependent manner; and the 0.05 μmol / mL and 0.01 μmol / mL treatment groups of compound 6 significantly decreased NO release in a dose-dependent manner. The 0.2 μmol / mL treatment group of compound 11 significantly decreased NO release; the 0.2 μmol / mL and 0.05 μmol / mL treatment groups of compounds 12 and 13 significantly decreased NO release in a dose-dependent manner.

[0099] 6. Conclusion and Discussion

[0100] NO has a wide range of biological functions in the body. It can relax vascular smooth muscle, inhibit platelet aggregation, mediate cytotoxic effects and immune regulation. NO produced by macrophages is closely related to cellular inflammation and immune activity. Abnormal NO production by macrophages can lead to the occurrence and development of various diseases, such as cancer, lupus erythematosus, rheumatoid arthritis, pneumonia, COPD, and organ fibrosis.

[0101] All high-dose groups of compounds 1-13 significantly reduced LPS-induced NO release. In terms of NO release inhibition, compound 6 showed the strongest activity, while compounds 5, 7, 12, 13, 4, 2, and 8 all inhibited LPS-induced NO release.

Claims

1. Tetrahydroanthraquinone compounds or pharmaceutically acceptable salts thereof, as shown in formulas 2, 4-8, 12-13.

2. A pharmaceutical composition, characterized in that, It contains at least one compound as described in claim 1 or a pharmaceutically acceptable salt thereof and a pharmacodynamically acceptable carrier.

3. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of pneumonia.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis.

5. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of inflammation-related diseases.

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