Macrocarpal terpene phenol derivatives, methods for their preparation and use

Abstract

The present application relates to a kind of Macrocarpal type terpene-phenol adduct derivative, preparation method, pharmaceutical composition and application in preparation anti-rheumatoid arthritis drug, the structure of this kind of derivative is as shown in general formula I.The present application obtains a kind of Macrocarpal type terpene-phenol adduct derivative by means of chemical structure modification, preparation process is simple and easy to operate, pharmacological result shows that the derivative of the present application shows the activity of inhibiting the proliferation of fibroblast-like synoviocyte of rheumatoid arthritis, can be used for preparing treatment anti-rheumatoid arthritis drug.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a macrocarpal-type terpene-phenol adduct derivative, its preparation method, pharmaceutical composition, and its use in the preparation of antirheumatoid arthritis drugs. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease primarily characterized by damage to the synovial membrane of joints. Its incidence rate in my country is 0.32%–0.36%, affecting approximately 1% of the global population. It is a chronic and difficult-to-treat disease with a high disability rate; the disability rate can reach 20% within one year of onset and 60% within ten years, earning it the nickname "the cancer that doesn't kill," and it falls under the category of modern intractable diseases as defined by the WHO. The basic pathological changes in rheumatoid arthritis include synovial cell proliferation, thickening of the synovial lining, infiltration of various inflammatory cells, pannus formation, and destruction of cartilage and bone tissue, ultimately leading to joint deformities and loss of function. Its high disability rate severely impacts people's physical and mental health.

[0003] Currently, the main treatment for RA relies on Western medicine, but due to its significant side effects, many patients cannot tolerate it, and the incidence of drug-induced diseases caused by inappropriate Western medicine treatment is even higher. Natural medicines, primarily based on traditional Chinese medicine, are not only widely available and diverse with fewer side effects, but also possess the characteristics of multi-link, multi-level, and multi-target comprehensive effects, giving them unique advantages in the treatment of RA. Therefore, finding and discovering the active ingredients in natural medicines is one of the important approaches for developing anti-RA drugs.

[0004] "Yi Kou Zhong" refers to the fruit of *Eucalyptus globulus* Labill., a plant in the Myrtaceae family. It is a traditional folk remedy in southern my country. The fruit's mature shape resembles a bell, hence the name "Yi Kou Zhong" (meaning "one-mouthed bell"). Immature fruits can be used to treat diarrhea or dysentery, while mature fruits can stimulate the immune system or treat joint pain, colds, dysentery, enteritis, and other ailments. Experiments have shown that macrocarpal A (MA), a terpene-phenol adduct isolated from *Yi Kou Zhong*, and its analogues possess good anti-RA activity. Based on this, derivatization synthesis was used to modify the structure of these compounds to obtain lead compounds with enhanced activity, providing a source of compounds for the development of anti-RA drugs.

[0005] There are currently no reports on the use of compounds of formula I or their pharmaceutical compositions in the preparation of drugs for treating rheumatoid arthritis. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a macrocarpal-type terpene-phenol adduct derivative, a pharmaceutical composition for treating rheumatoid arthritis containing the macrocarpal-type terpene-phenol adduct derivative as an active ingredient, a method for preparing the novel macrocarpal-type terpene-phenol adduct derivative, and tests the anti-RA activity of the derivative.

[0007] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution: The first aspect of the present invention provides a compound as shown in I or a pharmaceutically acceptable salt thereof.

[0008]

[0009] I

[0010] Among them, R1 is independently selected from: , , , , , , , , , , , ;

[0011] R2 is selected independently from: , , , , ;

[0012] R3 is selected independently from: , , , , ;

[0013] R4 is selected independently from: , , , , , , , , , , , , , ;

[0014] R5 is selected independently from: , , , ;

[0015] R6 is selected independently from: , , .

[0016] Compounds of Formula I are preferably those with the following specific structures, as shown in Formulas II, III, IV, and V:

[0017]

[0018] (1) Specifically II:

[0019]

[0020] Among them, R1 is independently selected from: , , , , , , , , , , , ;

[0021] R6 is selected independently from: , , .

[0022] Preferably, II is one of compounds 1-21, 43:

[0023]

[0024]

[0025]

[0026]

[0027]

[0028] .

[0029] (2) Specifically, III,

[0030]

[0031] Among them, R1 is independently selected from: , , ;

[0032] R2 is selected independently from: , , ;

[0033] R3 is selected independently from: , , , ;

[0034] Preferably, III is one of compounds 22-26:

[0035]

[0036] .

[0037] (3) Specifically IV,

[0038]

[0039] Among them, R4 is independently selected from: , , , , , , , , , , , , ;

[0040] R5 is selected independently from: , , .

[0041] Preferably, N is one of compounds 27-40:

[0042]

[0043] .

[0044] (3) Specifically V,

[0045]

[0046] R1 is selected from: ;

[0047] R2 is selected from: ;

[0048] R3 is selected from: ;

[0049] R4 is selected from: , ;

[0050] R5 is selected from: , .

[0051] Preferably, V is one of compounds 41-42.

[0052]

[0053] This invention also provides a method for preparing the above-mentioned Macrocarpal-type terpene-phenol adduct derivatives or their pharmaceutically acceptable salts, the preparation route of which is shown below: the starting materials for the derivative compound synthesis are MA, eucarobustol E, and MC, with the following structures:

[0054]

[0055] II, III, Compounds 1-26

[0056]

[0057] Synthetic routes of compounds 1-26

[0058] The Sol is a solvent selected from one or more of dichloromethane, dichloroethane, trichloromethane, tetrahydrofuran, methanol, ethanol, toluene, acetonitrile, ethyl acetate, N,N'-dimethylformamide, dimethyl sulfoxide, and water.

[0059] Synthetic route of compound 27 (IV)

[0060]

[0061] Synthetic route of compound 27

[0062] Synthetic routes of compounds 28, 29, and 30

[0063]

[0064] a: K2CO ３ a: CH3I, MeCN, 80℃; b: NaBH4, MeOH, rt; c: Pyrimidine isocyanicacid / pyrazine-2-carboxylic acid, DMAP, TEA, DCM, rt. Synthetic routes for compounds 28-30 IV. Synthetic routes for compounds 31 and 32.

[0065] a: K2CO ３a: CH3I, MeCN, 80℃; b: NaBH4, MeOH, rt; c: NBS, sol; d: 5-methyl-1,3,4-thia-diazole-2-amine, TEA, EtOH / H2O, rt. Synthetic routes for compounds 31 and 32. Synthetic routes for compounds 33-40.

[0066] a: K2CO ３ , CH3I, MeCN, 80℃; b: NaBH4, MeOH, rt; c: NBS, sol. c: K2CO3, RX, sol. Synthetic route of compounds 33-40 Compounds 33-40: Alkyl moiety R includes: alkyl, cycloalkyl, hydroxy-substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, arylalenyl, arylalkynyl, heterocyclic, trifluoromethyl, polyfluorosubstituted alkyl, cyanomethyl, acyl, carbamoyl, sulfonyl or aryloxyalkyl.

[0067] Synthetic routes for compounds V 41-42

[0068] a: K2CO3, CH3I, MeCN, 80℃; b: (i) Ms / Tf, K2CO3, sol; (ii) 10% Pd / C, Mg, NH4OAc, MeOH, Ar, rt; c: NBS, sol; c: NaBH4, MeOH, rt; d: (i) NBS, sol; (ii) 5-methyl-1,3,4-thia-diazole-2-amine, TEA, EtOH / H2O, rt. Synthetic routes of compounds 41-42; Compound 43: MC double bond addition.

[0069]

[0070] Synthetic route of compound 43

[0071] The inventors discovered that the aforementioned macrocarpal-type terpene-phenol adduct derivatives can be used in the preparation of drugs for treating RA, preventing RA, and / or improving RA symptoms. These drugs can serve as active ingredients in pharmaceutical compositions for treating RA, and the compositions may also include solvents or pharmaceutically acceptable carriers.

[0072] The present invention also provides a pharmaceutical composition comprising a compound of formula I as an active ingredient and a pharmaceutically acceptable carrier or other compound for treating rheumatoid arthritis.

[0073] When used as a medicine, the compounds of the present invention can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the compounds of the present invention, with the remainder being pharmaceutically acceptable, non-toxic, and inert pharmaceutically acceptable carriers and / or excipients for human and animal use, or in combination with other medicines for treating rheumatoid arthritis. The compositions of the present invention can be prepared as injections, tablets, capsules, etc.

[0074] The pharmaceutical carrier or excipient is one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients. The pharmaceutical composition of the present invention is used in a dose per unit body weight. All pharmaceutical compositions consisting of compounds as active ingredients are prepared into various dosage forms using methods recognized in the pharmaceutical industry, such as liquid preparations (injections, suspensions, emulsions, solutions, syrups, etc.), solid preparations (tablets, capsules, granules, powders, etc.), sprays, aerosols, etc. The drug of the present invention can be administered via injection (intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection), oral administration, sublingual administration, mucosal dialysis, and other routes of administration for the treatment of rheumatoid arthritis and its complications.

[0075] The beneficial effects of this invention are as follows: This invention obtains a novel Macrocarpal-type terpene-phenol adduct derivative through semi-synthetic modification. The preparation process is simple and easy. Pharmacological experiments have shown that the Macrocarpal-type terpene-phenol adduct derivative provided by this invention exhibits potent inhibitory activity against the proliferation of fibroblast-like synovial cells in rheumatoid arthritis, and has significant in vitro and in vivo anti-RA activity, making it suitable for the development of anti-RA drugs.

[0076] The advantages of this invention are:

[0077] 1. A structural modification method based on compound I is provided to prepare compounds with structures such as formula II, III, IV and V. The above methods are simple and safe to operate, and provide convenience for studying the biological activity of derivatives.

[0078] 2. Pharmacological activity verification showed that compounds of formula I, including compounds 1-43, exhibited strong anti-RA activity. Attached Figure Description

[0079] Figure 1 : Effect of compound 22 on body weight of CIA rats in each group;

[0080] Figure 2 : Effect of compound 22 on the width of the right hind limb ankle joint in CIA rats in each group;

[0081] Figure 3 : Effect of compound 22 on the thickness of the right hind limb ankle joint in CIA rats in each group;

[0082] Figure 4 : Effect of compound 22 on the circumference of the right hind limb ankle joint of CIA rats in each group;

[0083] Figure 5 : Pathological changes of synovial membrane in CIA rats after treatment with low dose of compound 22: synovial papillary hyperplasia covered with 6-10 layers of cells, and fibrinoid exudate can be seen in the synovial fissures (HE*100).

[0084] Figure 6 : Pathological changes of synovial membrane in CIA rats after treatment with medium dose of compound 22: No obvious hyperplasia of synovium, covered with 4-6 layers of cells, and a large number of lymphocytes infiltrated in the intercellular matrix of synovial cells (HE*100).

[0085] Figure 7 : Pathological changes of synovial membrane in CIA rats after treatment with high dose of compound 22: no obvious hyperplasia of synovium, tending to be normal, covered with 1-3 layers of cells (HE*100).

[0086] Figure 8 : Pathological changes in the synovial membrane of CIA rats in the blank control group: normal synovium (HE*100);

[0087] Figure 9 : Pathological changes in the synovium of the joints of CIA rats in the model group: synovial hyperplasia, with an increased number of lining cell layers (HE*100).

[0088] Figure 10 : Pathological changes in the synovium of CIA rats in the MTX group: minimal synovial hyperplasia and focal aggregation of multinucleated giant cells. Detailed Implementation

[0089] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0090] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0091] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0092] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The present invention can be further illustrated by the following experimental examples, but these are not intended to limit the invention. The structures of the compounds prepared in this invention have been confirmed by NMR and mass spectrometry analyses.

[0093] Example 1: Preparation of compounds 2, 12, 22, and 23

[0094] The structures of compounds 2, 12, 22, and 23 are as follows:

[0095]

[0096]

[0097] Its preparation process is as follows:

[0098] Weigh 1 eq of raw material MA into a 50 ml round-bottom double-necked flask and add 3-5 ml of acetonitrile as the reaction solvent. Stir and dissolve at room temperature. Add anhydrous potassium carbonate (1.2-6 eq) and stir at 45 °C for 0.5-2 h. Then cool to room temperature and add 1.2-5 eq of halogenation reagent. Stir for 0.5 h and then heat to reflux. Monitor the substrate conversion by TLC until complete. Concentrate the reaction solution to obtain the crude product. Add 30 ml of saturated saline solution to transfer the sample. Extract with ethyl acetate (30 ml × 3). Combine the organic layers, dry with anhydrous sodium sulfate and filter. Concentrate to obtain the crude product. Purify the product by silica gel column chromatography.

[0099] The NMR data for compounds 2, 12, 22, and 23 are as follows:

[0100] Compound 2, using compound MA as a starting material, with a reactant equivalence ratio of MA: iodoethane: potassium carbonate = 1:1.2:2, TLC monitoring conditions PE:EA = 4:1, silica gel column chromatography separation conditions PE:EA = 2:1, reaction time 4-6 h, yielded a white solid product. HRESI-MS m / z: [MH] - 499.3067 (calculated for C) 30 H 44 O6, 500.314). 1H-NMR (CDCl3, 400MHz) δH: 12.91 (1H, s), 12.85 (1H, s), 10.08 (2H, s), 4.24 (2H, q, J=7.0Hz), 3.52 (1H, dd, J=13.1Hz, 4.2Hz), 2.29 (1H, m), 2.00(1H, m), 1.83(3H, m), 1.74(2H, m), 1.66(3H, m), 1.51(1H, t, J=7.1Hz), 1.31(1H, m), 1.26(2H, t, J=7.5Hz), 1.15(3H, s), 1.09 (6H, s), 0.95 (1H, m), 0.78 (6H, d, J=6.0Hz), 0.65 (3H, s), 0.55 (2H, m); 13 C-NMR (CDCl3, 100MHz) δC192.11, 191.87, 169.16, 168.54,168.45, 114.86, 108.18, 108.12, 75.85, 60.37, 54.24, 48.25, 44.32, 44.05,39.26, 35.66, 35.16, 33.95, 28.92, 27.32, 27.15, 25.81, 24.56, 22.17, 21.63,20.44, 19.64, 17.84, 15.32, 14.19.

[0101] Compound 12, using eucarobustol E as a starting material, with a reactant equivalence ratio of eucarobustol E: iodoethane: potassium carbonate = 1:1.2:2, TLC monitoring conditions PE:EA = 4:1, silica gel column chromatography separation conditions PE:EA = 2:1, reaction time 4-6 h, yielded a white solid product. HR ESI-MS m / z: [MH] - 537.3187 (calculated for C) 31 H 46 O6,514.3316). 1H-NMR (CDCl3, 400MHz) δH: 12.90 (1H, s), 12.83 (1H, s), 10.07 (2H, s), 4.23 (2H, q, J=7.0Hz), 3.52 (1H, dd, J=13.1Hz, 4.2Hz), 3.18 (3H, s), 2.29 (1H, m), 2.16 (1H, m), 1.84 (2H, m), 1.64 (3H, m), 1.58 (2H, s), 1.51 (1H, t, J=7.04Hz), 1.39 (1H, m), 1.25 (2H, s), 1.09 (9H, s), 1.09 (6H, s), 0.96 (1H, m), 0.78 (6H, d, J=6.0Hz), 0.66 (3H, s), 0.55 (2H, m); 13 C-NMR (CDCl3, 100MHz) δC:192.11, 191.88, 169.20, 168.55, 168.46, 114.97, 108.19, 108.14, 79.88, 49.85,48.30, 48.12, 43.76, 37.26, 35.69, 35.20, 33.98, 29.70, 28.96, 27.46, 27.19,25.92, 24.58, 23.91, 22.20, 21.09, 20.15, 19.78, 18.43, 17.54, 15.34.

[0102] Compound 22, using compound MA as a starting material, was reactant in a reactant equivalence ratio of MA: iodomethane: potassium carbonate = 1:2.3:3. TLC monitoring conditions: PE:EA = 4:1; silica gel column chromatography separation conditions: PE:EA = 2:1; reaction time: 4-6 h, yielding a yellow solid product. HRESI-MS m / z: [MH] - 499.3067 (calculated for C) 30 H 44 O6, 500.3139). 1H-NMR (pyridine-d5, 400MHz) δH: 10.42 (1H, s), 10.32 (1H, s), 4.03 (3H, s), 3.97 (3H, s), 3.81 (1H, dd, J=12.7Hz, 4.4Hz), 2.33 (1H, m), 2.15 (2H, m), 2.04 (2H, m), 1.64 (1H, m), 1.52 (2H, m), 1.38 (3H, s), 1.24 (3H, s), 1.17 (6H, s), 0.94 (3H, d,J=6.5Hz), 0.87 (3H, d, J=6.5Hz), 0.81 (3H, t, J=6.8Hz), 0.74 (3H, s), 0.63 (2H, m); 13 C-NMR (pyridine-d5,100MHz) δC:192.80, 184.82, 168.28, 167.08, 166.34,113.22, 109.23, 107.00,73.19, 65.45, 62.10, 53.48, 47.38, 43.93, 43.03,34.97, 34.38, 33.84, 30.30, 27.74, 26.51, 26.02, 24.96, 23.77, 23.18, 21.43,19.49, 18.46, 16.47, 12.82.

[0103] Compound 23 was reacted with compound MA as a starting material, with a reactant equivalence ratio of MA: iodomethane: potassium carbonate = 1:5:6. TLC monitoring conditions: PE:EA = 4:1; silica gel column chromatography separation conditions: PE:EA = 2:1; reaction time: 4-6 h, yielding a yellow solid product. HRESI-MS m / z: [M+HCOO]- 559.3267 (calculated for C 31 H 46 O6, 514.3275). 1H-NMR (CDCl3, 400MHz) δH: 10.22 (1H, s), 10.21 (1H, s), 3.98 (3H, s), 3.89 (3H, s), 3.81 (3H, s), 3.53 (1H, dd, J=12.5, 4.1Hz), 1.93 (2H, m), 1.79 (1H, m), 1.68 (2H, m), 1.60 (2H, m), 1.45 (2H, m), 1.28 (1H, t, J=10.0Hz), 1.18 (3H, s), 1.09 (3H, s), 1.07 (3H, s), 1.03 (3H, s), 0.89 (1H, m), 0.77 (3H, d, J=6.5Hz), 0.66 (3H, d, J=6.5Hz), 0.51 (3H, s), 0.58 (2H, m); 13 C-NMR (CDCl3, 100MHz) δC:187.45, 187.29,167.92, 167.21, 167.02, 127.03, 117.41, 117.07, 75.82, 66.31, 64.64, 63.56,54.17, 48.56, 44.17, 44.07, 35.10, 34.64, 29.10, 27.35, 27.28, 25.79, 24.21,24.10, 22.64, 21.62, 20.37, 20.33, 19.49, 18.36, 17.50.

[0104] Example 2: Preparation of compound 28

[0105] The structure of compound 28 is as follows:

[0106]

[0107] The preparation process is as follows: Compound MA was used as a raw material, and compound 23 was prepared as a reaction substrate via phenolic hydroxyl etherification reaction. Sodium borohydride (5 eq) was placed in a 50 ml round-bottom flask, and 1.5 ml of methanol was added at 0 °C. The mixture was stirred for 0.5 h, and then compound 23 (1 eq) dissolved in 0.5 ml of methanol was added dropwise. The reaction was carried out at room temperature for 4.5 h. The substrate conversion was monitored by TLC until complete. After the reaction was completed, 50 ml of water was added to transfer the reaction system. Ethyl acetate was added for extraction, and the organic layers were combined. After washing with saturated brine, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was then separated by silica gel column chromatography.

[0108] The NMR data for compound 28 are as follows:

[0109] Compound 28, under TLC monitoring conditions of PE:EA = 1:1 and silica gel column chromatography separation conditions of PE:EA = 2:1, yielded a white solid product. HR ESI-MS m / z: [M+HCOO] - 563.3589 (calculated for C) 31 H 50 O6, 518.3608). 1 H-NMR (CDCl3, 400MHz) δH: 4.74 (4H, q, J=12.8Hz), 3.91 (6H, s), 3.85 (3H, s), 3.45 (1H, br, d, J=12.1Hz, 2.0Hz), 2.57 (2H, s), 1.97 (2H, m), 1.85 (1H, m), 1.74 (2H, m), 1.67 (1H, m), 1.61 (2H, m), 1.53 (1H, t, J=13.0Hz), 1.37 (1H, t, J=9.7Hz), 1.28 (3H, m), 1.16 (3H, s), 1.13 (3H, s), 1.11 (3H, s), 0.96 (1H, m), 0.91 (3H, d, J=5.7Hz), 0.74 (3H, d, J=5.8Hz), 0.58 (2H, m), 0.54 (3H, s); 13 C-NMR (CDCl3, 100MHz) δC: 59.81, 159.50, 157.69, 127.09, 123.07, 122.48, 75.91, 63.09, 62.95, 62.14, 56.63, 56.37, 54.53, 53.39, 42.13, 44.19, 44.17, 36.01, 34.64,29.15, 27.61, 27.39, 25.79, 24.26, 24.17, 23.14, 21.58, 20.46, 20.34, 19.44,17.49.

[0110] Example 3: Preparation of compound 33

[0111] The structure of compound 33 is as follows:

[0112]

[0113] The preparation process is as follows: Compound 28 (1 eq) was weighed into a 50 ml round-bottom double-necked flask and acetonitrile (3-5 ml) was added as a reaction solvent. The mixture was stirred and dissolved at room temperature. Anhydrous potassium carbonate (1.2-2 eq) was added and stirred at 45 °C for 0.5-2 h. The mixture was then cooled to room temperature and halogenated (1.2 eq) was added. After stirring for 0.5 h, the mixture was heated to reflux. The substrate was completely converted by TLC. The reaction solution was concentrated to obtain the crude product. 30 ml of saturated saline solution was added to transfer the sample. The sample was extracted with ethyl acetate (30 ml × 3). The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by silica gel column chromatography.

[0114] The NMR data for compound 33 are as follows:

[0115] Compound 33 was separated by silica gel column chromatography under conditions of PE:EA = 2:1, yielding a white solid product. HR ESI-MS m / z: [M+H] + 533.8632 (calculated for C) 32 H 52 O6, 532.7620). 1 H-NMR (CDCl3, 400MHz) δH: 4.74 (4H, q, J=12.8Hz), 3.91 (6H, s), 3.85 (3H, s), 3.45 (1H, br, d, J=12.1Hz, 2.0Hz), 3.28 (3H, s), 2.57 (1H, s), 1.97 (2H, m), 1.85 (1H, m), 1.74 (2H, m), 1.67 (1H, m), 1.61 (2H, m), 1.53 (1H, t, J=13.0Hz), 1.37 (1H, t, J=9.7Hz), 1.28 (3H, m), 1.16 (3H, s), 1.13 (3H, s), 1.11 (3H, s), 0.96 (1H, m), 0.91 (3H, d, J=5.7Hz), 0.74 (3H, d, J=5.8Hz), 0.58 (2H, m), 0.54 (3H, s); 13C-NMR (CDCl3, 100MHz) δC: 59.81, 159.50, 157.69, 127.09, 123.07, 122.48, 75.91, 63.09, 62.95, 62.14, 58.9, 56.63, 56.37, 54.53, 53.39, 42.13, 44.19, 44.17, 36.01,34.64, 29.15, 27.61, 27.39, 25.79, 24.26, 24.17, 23.14, 21.58, 20.46, 20.34,19.44,17.49.

[0116] Example 4: Preparation of compound 41

[0117] The structure of compound 41 is as follows:

[0118]

[0119] Its preparation process is as follows:

[0120] 1. Weigh compound 1 (1 eq) into a 50 ml round-bottom double-necked flask, add acetonitrile (3-5 ml) as the reaction solvent, stir to dissolve at room temperature, add anhydrous potassium carbonate (2 eq), stir at 45 ℃ for 0.5-2 h, then cool to room temperature, add p-toluenesulfonyl chloride (2.2 eq), heat to reflux reaction, monitor the substrate conversion by TLC, concentrate the reaction solution to obtain crude product, add 30 ml saturated saline to transfer the sample, extract with ethyl acetate (30 ml × 3), combine the organic layers, dry with anhydrous sodium sulfate and filter, concentrate to obtain crude product, separate and purify by silica gel column chromatography to obtain sulfonate product.

[0121] 2. Under normal pressure and 20°C, with Ar protection, a mixture of 0.25 mmol p-toluenesulfonate, 10 wt% Pd / C, magnesium (7.3 mg, 0.3 mmol), and ammonium acetate (0.25-7.5 mmol) was added to 1 mL of methanol and stirred. The substrate conversion was monitored by TLC until complete. The reaction mixture was filtered through a membrane filter, extracted with diethyl ether, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the intermediate product.

[0122] 3. Take 5 eq of sodium borohydride and place it in a 50 ml round-bottom flask. Add 1.5 ml of methanol at 0 °C and stir for 0.5 h. Add dropwise the intermediate product from step 2 dissolved in 0.5 ml of methanol (1 eq). React at room temperature. Monitor the substrate conversion by TLC until complete. After the reaction is complete, add 50 ml of water to transfer the reaction system. Extract with ethyl acetate, combine the organic layers, wash with saturated brine, dry to anhydrous sodium sulfate, filter and concentrate to obtain the crude product. Separate by silica gel column chromatography to obtain the product.

[0123] The NMR data for compound 41 are as follows:

[0124] Compound 41 was separated by silica gel column chromatography under conditions of PE:EA = 1:1, yielding a yellow solid product. HR ESI-MS m / z: [M+H] + 459.7462 (calculated for C) 29 H 46 O4, 458.6830). 1 H-NMR (CDCl3, 400MHz) 1 H-NMR (CDCl3, 400MHz) δH: 7.26 (1H, s), 7.24 (1H, s), 4.61 (4H, q, J=12.8Hz), 3.78 (3H,s), 3.45 (1H, dd, J=12.1Hz, 2.0Hz), 2.57 (2H, s), 1.97 (2H, m), 1.85 (1H, m), 1.74 (2H, m), 1.67 (1H, m), 1.61 (2H, m), 1.53 (1H, t, J=13.0Hz), 1.37 (1H, t, J=9.7Hz), 1.28 (3H, m), 1.16 (3H, s), 1.13 (3H, s), 1.11 (3H, s), 0.96 (1H, m), 0.91 (3H, d, J=5.7Hz), 0.74 (3H, d, J=5.8Hz), 0.58 (2H, m), 0.54 (3H, s); 13C-NMR (CDCl3, 100MHz) δC: 59.81, 159.50, 157.69, 129.7, 126.07, 122.48, 75.91,63.09, 62.95, 62.14, 56.63, 53.39, 42.13, 44.19, 44.17, 36.01, 34.64, 29.15,27.61, 27.39, 25.79, 24.26, 24.17, 23.14, 21.58, 20.46, 20.34, 19.44, 17.49.

[0125] Example 5: Preparation of compound 43

[0126] The structure of compound 43 is as follows:

[0127]

[0128] Its preparation process is as follows:

[0129] 5 ml of anhydrous ethanol was added to a round-bottom flask, MC (1 eq) was added, and the mixture was stirred until dissolved. HPW (4.24 mol%) was added, and the reaction was carried out at 65 °C for 2.5 h under argon atmosphere. The substrate conversion was monitored by TLC (PE:EA=2:1:3‰HAc) and found to be complete. After the reaction was completed, the reaction system was rapidly cooled to 0 °C to stop the reaction. The reaction solution was then concentrated, and 240 ml of n-hexane was added to transfer the sample. The n-hexane layer was concentrated to obtain the crude product, which was then separated by normal-phase preparative liquid chromatography (PE:EA=20:1:3‰HAc) to obtain the product.

[0130] The NMR data for compound 43 are as follows:

[0131] Compound 43, pale yellow solid. HR ESI-MS m / z: [M+Na] + 523.3031 (calculated for C) 30 H 44 O6, 500.313). 1H-NMR (CDCl3, 400MHz) δH: 13.50 (1H, s), 13.35 (1H, s), 10.12 (1H, s), 10.03 (1H, s), 3.65 (1H, m), 3.42-3.24 (2H, m), 2.47 (1H, m), 2.19 (1H, m), 1.82-1.78 (2H, m), 1.74 (2H, m), 1.64 (1H, s), 1.61 (3H, s), 1.37-1.30 (2H, m), 1.18 (6H, s), 1.06 (3H, s), 1.02 (3H, s), 0.81 (3H, t, J=6.4Hz), 0.75 (3H, s), 0.68 (3H, s), 0.61-0.55 (2H, m), 0.45 (1H, t, J=9.9Hz); 13 C-NMR (CDCl3, 100MHz) δC: 191.27, 190.91, 169.11, 167.01, 163.75, 106.13, 103.15, 102.67, 78.55,54.25, 48.72, 47.14, 43.02, 35.78, 34.13, 33.87, 33.09, 27.82, 26.10, 25.99,24.81, 23.38, 23.09, 21.01, 19.86, 19.12, 18.85, 17.82, 16.53, 15.26.

[0132] Example 7: In vitro pharmacological screening of derived compounds

[0133] The inhibitory effect of macrocarpal-type terpene-phenol adduct derivatives on the proliferation of fibroblast-like synovial cells in rheumatoid arthritis was detected by the MTT assay.

[0134] MTT colorimetric assay procedure: HFLS-RA cells in good growth condition, passaged to the 4th generation, were counted and then added to DMEM complete culture medium to adjust the cell density to 5 × 10⁴ cells / mL. 200 μL of each cell was seeded into a 96-well plate and transferred to an incubator. After 18-20 h, the plates were removed, and the test sample (adjusted to a concentration of 20 μmol / L) was added to each well. Simultaneously, a DMSO negative control group and a culture medium blank control group were set up in each well of the experimental group. The plates were then transferred to an incubator for further incubation. After 48 h, the plates were removed, and MTT solution (5 mg / mL, 20 μL / well) was added. The plates were incubated for 4 h. After removing the plates, the culture medium in the wells was aspirated, and DMSO (150 μL / well) was added with shaking to dissolve the formazan crystals. The absorbance (OD value) at 490 nm was measured using a microplate reader. Statistical analysis was performed using Graphpad software to calculate the half-maximal effective concentration (IC50). 50 (Unit: μM). The inhibition effect is shown in Table 1 below.

[0135] Table 1. Effects of Macrocarpal-type terpene-phenol adduct derivatives on HFLS-RA cell proliferation.

[0136] Compound numbering <![CDATA[IC 50 (μM)]]> Compound numbering <![CDATA[IC 50 (μM)]]> MA 13.62 24 8.97 1 1.17 25 1.65 2 1.49 26 9.22 3 9.44 27 20.03 4 9.01 28 7.76 5 9.18 29 21.03 6 9.09 30 20.70 7 9.03 31 10.32 8 5.03 32 0.48 9 8.97 33 10.81 10 1.75 34 10.74 11 7.89 35 11.35 12 6.14 36 10.81 13 8.99 37 10.74 14 8.89 38 10.61 15 9.05 39 10.69 16 8.99 40 11.58 17 8.99 41 5.63 18 9.01 42 8.19 19 8.81 43 16.43 20 8.73 21 2.54 22 0.74 23 8.07

[0137] In vitro pharmacological tests have demonstrated that the macrocarpal-type terpene-phenol adduct derivatives of the present invention all have good anti-RA activity. Among them, the derivative compounds 1, 2, 10, 21, 22, 25, 32, and 41 have significantly enhanced activity relative to the raw material MA and can be used to further prepare anti-RA drugs.

[0138] Example 8: In vivo pharmacological test results of the derived compounds

[0139] Using a rat model of collagen-induced arthritis (CIA), this study investigated the effects of macrocarpal-type terpene phenol adduct derivative 22 on the appearance, mobility, joint swelling, and joint pathological changes of CIA rats after treatment. The in vivo activity evaluation of the anti-RA effect of 22 was explored through measurement data and detection results.

[0140] Experimental Methods: A rat model of collagen-induced arthritis was established by injecting bovine type II collagen and complete Freund's adjuvant into rats. One week later, the rats were injected with bovine type II collagen and incomplete Freund's adjuvant to enhance immunization. The model status was observed after immunization to assess whether the model was successfully established. After successful model establishment, the rats were randomly divided into 5 groups: high-, medium-, and low-dose groups of compound 22, a model group, and a positive drug group. The rats in each group were treated by gavage with high-, medium-, and low-dose of compound 22 and methotrexate solution, respectively. The in vivo activity of compound 22 against RA was evaluated by measuring joint swelling, observing changes in appearance, and examining pathological tissue samples.

[0141] a. General condition observation and evaluation of treatment effect in CIA rats after treatment with compound 22

[0142] The results are as follows Figures 1-4 As shown.

[0143] After modeling, the body weight of rats in the model group all showed a decreasing trend. Due to the modeling, the rats' autoimmunity was reduced and they became lethargic, so their food intake was reduced accordingly, resulting in a decrease in body weight. After gavage treatment, the body weight of rats in each group began to increase significantly. The positive drug group showed a larger increase in body weight, and the high, medium and low dose groups of compound 22 showed a larger trend of body weight increase. The growth trends among the three groups were similar.

[0144] Meanwhile, compound 22 significantly reduced ankle joint width, thickness and circumference in CIA rats at extremely low doses (0.5, 1, 2 mg / kg), and its effect on improving the condition was superior to that of the positive control drug methotrexate (1 mg / kg). There were no adverse reactions in the rats in the 22 administration group, and the rats were in very good mental condition, with high activity and rapid response, which was no different from that in the blank control group.

[0145] b. Pathological changes in the synovial membrane of CIA rats after treatment with compound 22

[0146] The results are as follows Figures 5-10 As shown in the figure. After HE staining, microscopic observation revealed that the synovial lining layer in the blank group consisted of 1-2 layers, with neatly arranged synovial epithelial cells and no inflammatory cell infiltration or angiogenesis. In the model group, synovial epithelial cell proliferation was significant, with irregular arrangement and a large number of inflammatory cells infiltrating, a significant increase in the number of blood vessels, and pannus formation. In the MTX-positive control group, synovial tissue fibrosis was observed, with 1-3 layers of synovial lining. In the low-dose group, synovial epithelial and angiogenesis were significant, with a large number of inflammatory cells infiltrating, showing no significant difference compared to the model group. In the medium-dose group, synovial epithelial cell proliferation, the number of angiogenesis, the number of pannus, and the degree of inflammatory cell infiltration were reduced compared to the low-dose monosodium glutamate group. In the high-dose group, the number of synovial epithelial cells and angiogenesis was significantly reduced, the degree of inflammatory cell infiltration was significantly reduced, and the synovial tissue was close to normal.

[0147] In summary, this invention provides a macrocarpal-type terpene phenol adduct derivative, its preparation method, and its application. The raw materials for this derivative compound, MA, MB, or eucarobustol E, are abundant and simple to prepare from the fruit of *Eucalyptus globulus*. The obtained derivative compound exhibits significant inhibitory effects on the proliferation of fibroblast-like synovial cells in rheumatoid arthritis and can be widely used in the preparation of drugs for treating RA.

[0148] This invention also provides a pharmaceutical composition comprising the above-mentioned Macrocarpal-type terpene-phenol adduct derivative and pharmaceutically acceptable excipients, which can be used to treat RA.

[0149] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Compounds of Formula I and their pharmaceutically acceptable salts: Ⅰ in, R1 is independently selected from: , , , , , , , , , , , ; R2 is selected independently from: , , , , ; R3 is selected independently from: , , , , ; R4 is selected independently from: , , , , , , , , , , , , ; R5 is selected independently from: , , , ; R6 is selected independently from: , , .

2. The compound according to claim 1 and its pharmaceutically acceptable salt, having the following structure: in, R1 is independently selected from: , , , , , , , , , , , ; R6 is selected independently from: , , .

3. The compound according to claim 1 and its pharmaceutically acceptable salt, having the following structure: in, R1 is independently selected from: , , ; R2 is selected independently from: , , ; R3 is selected independently from: , , , .

4. The compound according to claim 1 and its pharmaceutically acceptable salt, having the following structure: in, R4 is selected independently from: , , , , , , , , , , , ; R5 is selected independently from: , , .

5. The compound according to claim 1 and its pharmaceutically acceptable salt, having the following structure: R1 is selected from: ; R2 is selected from: ; R3 is selected from: ; R4 is selected from: , ; R5 is selected from: , .

6. The compound according to any one of claims 1-5 and its pharmaceutically acceptable salt, selected from: 。 7. A method for preparing compounds 1-10 according to any one of claims 2 and 3, and their pharmaceutically acceptable salts: Reaction condition a: Halogenated alkane, K2CO 3， The reaction solvent; among which, The haloalkane is a chloroalkane, a bromoalkane, or an iodoalkane; the reaction solvent is selected from one or more of dichloromethane, dichloroethane, trichloromethane, tetrahydrofuran, methanol, ethanol, toluene, acetonitrile, ethyl acetate, N,N'-dimethylformamide, dimethyl sulfoxide, and water.

8. A method for preparing compound 27 of claim 6 and its pharmaceutically acceptable salt: Reaction condition a: K2CO 3， CH3I; reaction condition b contains Cu(OAC)2 and NH4OAc.

9. A method for preparing compounds 29 and 30 of claim 6 and their pharmaceutically acceptable salts: in, Reaction conditions a: K2CO3, CH3I, MeCN, 80℃; Reaction conditions b: NaBH4, MeOH, room temperature; Reaction conditions c: pyrimidine isocyanate / pyrazine-2-carboxylic acid, 4-dimethylaminopyridine, triethylamine, dichloromethane, room temperature.

10. A method for preparing compounds 31 and 32 of claim 6 and their pharmaceutically acceptable salts: in, Reaction conditions a: K2CO3, CH3I, MeCN, 80℃; Reaction conditions b: NaBH4, MeOH, room temperature; Reaction conditions c: NBS, wherein the reaction solvent is selected from one or more of the following: dichloromethane, dichloroethane, trichloromethane, tetrahydrofuran, methanol, ethanol, toluene, acetonitrile, ethyl acetate, N,N'-dimethylformamide, dimethyl sulfoxide, and water; Reaction conditions d: 5-methyl-1,3,4-thiadiazol-2-amine, triethylamine, ethanol / water, room temperature.

11. A method for preparing compounds 33-40 of claim 6 and their pharmaceutically acceptable salts: in, R is as shown in any one of compounds 33-40; reaction conditions a: K2CO3, CH3I, MeCN, 80℃; reaction conditions b: NaBH4, MeOH, room temperature; reaction conditions c: N-bromosuccinimide, wherein the reaction solvent is selected from one or more of the following: dichloromethane, dichloroethane, trichloromethane, tetrahydrofuran, methanol, ethanol, toluene, acetonitrile, ethyl acetate, N,N'-dimethylformamide, dimethyl sulfoxide, and water; K2CO3, RX.

12. A method for preparing compounds 41 and 42 of claim 6 and their pharmaceutically acceptable salts: in, Reaction conditions a: K2CO3, CH3I, MeCN, 80℃; Reaction conditions b in sequence: (i) Ms / Tf, K2CO3, sol; (ii) 10% Pd / C, Mg, NH4OAc, MeOH, Ar, room temperature; Reaction conditions c: NaBH4, MeOH, room temperature; Reaction conditions d: (i) N-bromosuccinimide; (ii) 5-methyl-1,3,4-thiadiazole-2-amine, triethylamine, ethanol / water, room temperature.

13. A method for preparing compound 43 of claim 6 and its pharmaceutically acceptable salt: in, Reaction conditions a: phosphotungstic acid, EtOH, Ar, 60℃, 0.5h.

14. Use of the compound of any one of claims 1-6 and its pharmaceutically acceptable salt in the preparation of a medicament for treating RA, preventing RA and / or improving RA symptoms.

15. A pharmaceutical composition, characterized in that, Includes the compounds of any one of claims 1-6 and their pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

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