Sinomenine derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical chemistry and organic compound synthesis, and particularly relates to a sinomenine derivative as well as a preparation method and application thereof. The structural formula of the sinomenine derivative is as shown in a formula I. The sinomenine derivative is prepared by taking sinomenine and a halogenating reagent as raw materials, carrying out substitution reaction to obtain an intermediate I ', and then carrying out nucleophilic substitution reaction on the intermediate I' and triaryl phosphine halide metal to obtain the sinomenine derivative as shown in the formula I. The sinomenine derivative has remarkable anti-rheumatoid arthritis activity and anti-sepsis activity, is low in toxic and side effects and high in safety coefficient, and has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical fields of pharmaceutical chemistry and organic compound synthesis, and particularly relates to a class of sinomenine derivatives, a preparation method thereof, and an application thereof. Background Art

[0002] Sinomenine is an isoquinoline alkaloid extracted from Sinomenium acutum, a plant of the family Menispermaceae, and its pharmacological effects of anti-inflammatory, analgesic, immunosuppressive, and anti-allergic are clear. Sinomenine hydrochloride has been developed into a new drug - Zhengqingfengtongning injection and tablets, and is used as a clinical drug for the treatment of rheumatoid arthritis. However, existing clinical practices and pharmacodynamic studies have shown that sinomenine has significant limitations while exerting its therapeutic effects: firstly, the conventional therapeutic dose is relatively high (the daily dose range is 60 - 240 mg), which may cause adverse drug events such as anaphylactic shock, leukopenia, and severe gastrointestinal reactions; secondly, the low bioavailability limits the drug efficacy, and large-dose administration is required to maintain the blood drug concentration. Therefore, it is of important application significance to optimize the molecular structure of sinomenine and develop new derivatives with both high efficiency and low toxicity. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a class of sinomenine derivatives, a preparation method thereof, and an application thereof. The sinomenine derivatives have excellent anti-rheumatoid arthritis activity, small side effects, and high safety, and the synthesis method has the advantages of mild reaction conditions, simple operation, high yield of reaction products, and suitability for industrial production.

[0004] Specifically, the present invention is achieved through the following technical solutions:

[0005] In the first aspect, the present invention provides a sinomenine derivative represented by Formula I or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Wherein,

[0008] R1 - R4 are the same or different, and each independently selected from: hydrogen, halogen, cyano, nitro, hydroxyl, amino, C1 - C4 alkyl, halogenated C1 - C4 alkyl, C1 - C4 alkoxy, C1 - C4 alkylamino, C1 - C4 alkoxycarbonyl, C1 - C4 acyloxy;

[0009] L is selected from C1 - C10 alkylene;

[0010] Ar1 - Ar3 are the same or different, and each independently selected from: substituted or unsubstituted C6 - C20 aryl, substituted or unsubstituted 5 - 10 membered heteroaryl.

[0011] Further, R1-R4 are the same or different and each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, cyano, nitro, hydroxy, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetoxy.

[0012] Preferably, R1-R4 are selected from hydrogen.

[0013] Further, L is selected from -(CHR) n -, R being the same or different and each independently selected from hydrogen, methyl, ethyl, and n being selected from 1, 2, 3, 4, 5 or 6.

[0014] Preferably, L is selected from -(CH2) n -, n being selected from 1, 2, 3, 4, 5 or 6.

[0015] Further, the substituents of the "substituted or unsubstituted" in Ar1-Ar3 are selected from: halogen, cyano, nitro, hydroxy, amino, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, C1-C4 acyloxy.

[0016] Preferably, the "substituted or unsubstituted" substituents are selected from: fluorine, chlorine, bromine, iodine, cyano, nitro, hydroxy, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetoxy.

[0017] Further, the C6-C20 aryl in Ar1-Ar3 includes: phenyl, naphthyl, anthryl, phenanthryl, triphenylenyl, biphenyl.

[0018] Further, the 5-10 membered heteroaryl in Ar1-Ar3 includes: pyrrolyl, furyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyranyl, indolizinyl, indolyl, isoindolinyl, indazolyl, benzofuryl, benzothienyl, benzimidazolyl, benzothiazolyl, purinyl, quinolinyl, isoquinolinyl, thiazolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, naphthyridinyl.

[0019] Further, Ar1 - Ar3 are the same or different and each independently selected from phenyl, naphthyl, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrimidinyl which are substituted or unsubstituted by one or more of fluorine, chlorine, bromine, iodine, cyano, nitro, hydroxyl, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2 - trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetoxy.

[0020] Preferably, Ar1 - Ar3 are the same or different and each independently selected from phenyl, naphthyl, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl which are substituted or unsubstituted by one or more of fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, butyl, trifluoromethyl, methoxy, ethoxy, methylamino, ethylamino.

[0021] Further, the sinomenine derivatives are selected from:

[0022]

[0023] In a second aspect, the present invention provides a method for preparing the sinomenine derivative represented by formula I, comprising the following steps:

[0024] Step 1: React sinomenine with a halogenating reagent to obtain intermediate I';

[0025] Step 2: Perform a nucleophilic substitution reaction on intermediate I' with triarylphosphine gold halide to obtain the product,

[0026]

[0027] wherein X is independently selected from chlorine or bromine, and R1 - R4, L, Ar1 - Ar3 are as described herein.

[0028] Further, in Step 1, the reaction is carried out under the catalysis of sodium alkoxide or potassium alkoxide; preferably, the sodium alkoxide is sodium methoxide, sodium ethoxide, sodium tert - butoxide; the potassium alkoxide is potassium methoxide, potassium ethoxide, potassium tert - butoxide.

[0029] Further, in Step 1, the molar ratio of sinomenine to the halogenating reagent is 1:1 - 1.5, preferably 1:1.1 - 1.2.

[0030] Further, in Step 1, the molar ratio of sinomenine to sodium alkoxide or potassium alkoxide is 1:1.2 - 2, preferably 1:1.3 - 1.7.

[0031] Further, in Step 1, the reaction solvent is at least one of acetonitrile and DMF.

[0032] Further, Step 1 includes first placing sinomenine and sodium alkoxide or potassium alkoxide in an anhydrous solvent and stirring for reaction, and then slowly adding a halogenating reagent under the protection of an inert gas to obtain Intermediate I'.

[0033] Preferably, the inert gas is selected from at least one of nitrogen and argon.

[0034] Preferably, the reaction temperature is 20 - 40°C, preferably room temperature.

[0035] Preferably, the reaction time is 1 - 24 h, preferably 4 - 12 h.

[0036] Preferably, after the reaction, it is filtered with diatomaceous earth, the solvent is evaporated, and separated by column chromatography to obtain Intermediate I'.

[0037] Further, in Step 2, the reaction is carried out under the catalysis of sodium alkoxide or potassium alkoxide; preferably, the sodium alkoxide is sodium methoxide, sodium ethoxide, sodium tert-butoxide; the potassium alkoxide is potassium methoxide, potassium ethoxide, potassium tert-butoxide.

[0038] Further, in Step 2, the molar ratio of Intermediate I' to triarylphosphine gold halide is 1:1 - 1.8, preferably 1:1.1 - 1.3.

[0039] Further, in Step 2, the molar ratio of Intermediate I' to sodium alkoxide or potassium alkoxide is 1:1.2 - 2, preferably 1:1.3 - 1.7.

[0040] Further, in Step 2, the reaction solvent is at least one of alcohol, halogenated hydrocarbon, and acetone, preferably dichloromethane.

[0041] Further, Step 2 includes first placing Intermediate I' and sodium alkoxide or potassium alkoxide in an anhydrous solvent and stirring for reaction, and then slowly adding triarylphosphine gold halide under the protection of an inert gas to obtain the sinomenine derivative shown in Formula I.

[0042] Preferably, the inert gas is selected from at least one of nitrogen and argon.

[0043] Preferably, the reaction temperature is 20 - 40°C, preferably room temperature.

[0044] Preferably, the reaction time is 1 - 6 h, preferably 2 - 4 h.

[0045] Preferably, after the reaction, the solvent is evaporated, the residue is extracted with 0.05 - 0.3% ammonia water, filtered through diatomaceous earth, and then the filtrate is concentrated to dryness and recrystallized to obtain the sinomenine derivative shown in Formula I.

[0046] Preferably, the recrystallization solvent is a mixed solvent of dichloromethane and n-hexane.

[0047] In a third aspect, the present invention provides the use of the sinomenine derivative represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug.

[0048] Furthermore, the present invention provides the use of the sinomenine derivative represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-osteoarthritis drug.

[0049] Furthermore, the present invention provides the use of the sinomenine derivative represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for anti-rheumatoid arthritis and / or anti-sepsis.

[0050] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the sinomenine derivative represented by Formula I or a pharmaceutically acceptable salt thereof.

[0051] Unless otherwise specified, the terms used in this application have the following definitions, and the definitions of the terms not involved hereinafter are as commonly understood by those skilled in the art to which the present invention pertains.

[0052] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt prepared from a compound and a relatively non-toxic, pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, the base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When a compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When a compound contains both a relatively acidic and a relatively basic functional group, it can be converted into a base addition salt or an acid addition salt.

[0053] In this application, the term "treatment" refers to a therapeutic treatment. When referring to a specific disease or disorder, treatment means: (1) alleviating one or more biological manifestations of the disease or disorder, (2) interfering with (a) one or more points in the biological cascade that causes or gives rise to the disorder or (b) one or more biological manifestations of the disorder, (3) improving one or more symptoms, effects, or side effects associated with the disorder, or one or more symptoms, effects, or side effects associated with the disorder or its treatment, or (4) slowing the progression of the disorder or one or more biological manifestations of the disorder.

[0054] According to the treatment purpose, the pharmaceutical composition can be made into various types of dosage unit forms for administration.

[0055] The compounds of the present invention can be administered clinically by conventional administration methods.

[0056] Unless otherwise specified, the steps and conditions in this application can refer to the conventional operating steps and conditions in the art.

[0057] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0058] Unless otherwise specified, the abbreviations used in this application have their general meanings in the art. For example, SIN refers to sinomenine, MTX refers to methotrexate, DEX refers to dexamethasone, AF refers to auranofin, and TPPAuCl refers to triphenylphosphine gold chloride.

[0059] The present invention has the following beneficial effects compared with the prior art:

[0060] (1) The sinomenine derivative or its pharmaceutically acceptable salt of the present invention can effectively inhibit a variety of inflammatory factors. On the one hand, it can significantly reduce the symptoms of joint swelling, improve the bone density loss caused by arthritis, and have a certain alleviating effect on the destruction of joint bone quality; on the other hand, it can significantly relieve the serum inflammation level of septic mice, reduce the sepsis score, and improve the damage of various organs under pathological conditions. It has excellent anti-rheumatoid arthritis and anti-sepsis activities, with small side effects and high safety.

[0061] (2) The synthesis method of the sinomenine derivative of the present invention has the advantages of mild reaction conditions, simple operation, high yield of reaction products, and suitability for industrial production. Brief Description of the Drawings

[0062] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention.

[0063] Figure 1 1H NMR spectrum of compound 1 synthesized in Example 1 1 1H NMR spectrum;

[0064] Figure 2 1H NMR spectrum of compound 1 synthesized in Example 1 13 13C NMR spectrum;

[0065] Figure 3 1H NMR spectrum of SIN-Au synthesized in Example 2 1 1H NMR spectrum;

[0066] Figure 4 1H NMR spectrum of SIN-Au synthesized in Example 2 13 13C NMR spectrum;

[0067] Figure 5 31P NMR spectrum of SIN-Au synthesized in Example 2 31 31P NMR spectrum;

[0068] Figure 6 Showing the cytotoxicity IC of the sinomenine derivative SIN-Au based on RAW264.7 cells50 and nitric oxide inhibitory activity EC 50 Evaluation;

[0069] Figure 7 Showing the anti-inflammatory activity evaluation of sinomenine derivative SIN-Au in inhibiting the levels of inflammatory factors based on RAW264.7 and iBMDM cells;

[0070] Figure 8 Showing the effect of intragastric administration of sinomenine derivative SIN-Au on sepsis score in a sepsis mouse model;

[0071] Figure 9 Showing the effect of intragastric administration of sinomenine derivative SIN-Au in inhibiting the levels of serum inflammatory factors in a sepsis mouse model;

[0072] Figure 10 Showing the improvement of various organs of sepsis mice by intragastric administration of sinomenine derivative SIN-Au;

[0073] Figure 11 Showing the effect of intraperitoneal injection of sinomenine derivative SIN-Au on sepsis score in a sepsis mouse model;

[0074] Figure 12 Showing the effect of intraperitoneal injection of sinomenine derivative SIN-Au in inhibiting the levels of serum inflammatory factors in a sepsis mouse model;

[0075] Figure 13 Showing the improvement of various organs of sepsis mice by intraperitoneal injection of sinomenine derivative SIN-Au;

[0076] Figure 14 Showing the gross photograph of the foot of CIA mice on the 30th day after intraperitoneal injection of SIN-Au after modeling;

[0077] Figure 15 Showing the statistical chart of the effects of SIN-Au on arthritis score, footpad thickness, and body weight of CIA mice after modeling;

[0078] Figure 16 Showing the statistical chart of the effect of SIN-Au on the incidence of CIA mice after modeling;

[0079] Figure 17 Showing the statistical chart of the effect on foot pain threshold on the 30th day of administering SIN-Au;

[0080] Figure 18 Showing the micro CT images of the ankle joints of CIA mice after administering SIN-Au;

[0081] Figure 19Show the statistical charts of ankle bone density, trabecular bone separation, and bone surface area of CIA mice after the administration of SIN-Au ended;

[0082] Figure 20 Show the gait analysis diagram of CIA mice after the administration of SIN-Au ended;

[0083] Figure 21 Show the IgE and histamine levels of CIA mice after the administration of SIN-Au ended;

[0084] Figure 22 Show the organ indices of CIA mice after the administration of SIN-Au ended;

[0085] Figure 23 Show the liver function indices of CIA mice after the administration of SIN-Au ended;

[0086] Figure 24 Show the renal function indices of CIA mice after the administration of SIN-Au ended;

[0087] Figure 25 Show the HE staining diagrams of the visceral tissues of CIA mice after the administration of SIN-Au ended. Detailed implementation manners

[0088] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the content of the present invention and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0089] For those without specific technologies or conditions indicated in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0090] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available products unless otherwise specified.

[0091] Example 1: Synthesis of Compound 1

[0092]

[0093] Under argon protection, sinomenine (60 mg, 182.15 μmol, 1.0 equiv.) and potassium tert-butoxide (30.66 mg, 273.23 μmol, 1.5 equiv.) were added to 5 mL of anhydrous CH3CN. After stirring for 15 minutes, C9H7Br (39.97 mg, 204.92 μmol, 1.125 equiv.) dissolved in 1 mL of anhydrous CH3CN was added dropwise. The reaction was carried out at room temperature for 6 h, and the solution turned orange-yellow. After monitoring the completion of the reaction by TLC, it was filtered through diatomaceous earth and concentrated under reduced pressure. After removing all the solvents by rotary evaporation under reduced pressure, column chromatography was used for separation with a solvent system of DCM / MeOH = 25:1, and compound 1 was obtained as a yellowish-brown solid (40 mg, 91.0 μmol).

[0094] 1 H NMR (600 MHz, CDCl3) δ 7.54 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 5.49 (s, 1H), 5.26 (d, J = 11.6 Hz, 1H), 5.06 (d, J = 11.7 Hz, 1H), 4.05 (d, J = 16.0 Hz, 1H), 3.76 (s, 3H), 3.49 (s, 3H), 3.14 (t, J = 4.0 Hz, 1H), 3.06 (s, 1H), 2.99 (d, J = 17.9 Hz, 1H), 2.95 (m, 1H), 2.74 (dd, J = 18.0, 5.3 Hz, 1H), 2.50–2.44 (m, 2H), 2.40 (s, 3H), 1.96 (td, J = 11.5, 4.6 Hz, 1H), 1.85–1.80 (m, 2H).

[0095] 13 C NMR (151 MHz, CDCl3) δ 194.2, 152.6, 151.6, 147.4, 139.5, 132.2, 130.3, 130.3, 127.8, 123.2, 121.2, 115.6, 111.3, 83.9, 77.1, 72.8, 56.6, 55.8, 54.9, 50.0, 47.1, 46.2, 42.8, 41.1, 37.2, 24.8.

[0096] HRMS found: m / z: 444.2174 [M+H] + , calcd for C 28 H 30 NO4 + , 444.2170.

[0097] Example 2: Synthesis of Compound SIN-Au

[0098]

[0099] Under argon protection, compound 1 (88 mg, 198.4 μmol, 1.0 equiv.) and potassium tert-butoxide (33.4 mg, 297.6 μmol, 1.5 equiv.) were added to 5 mL of anhydrous DCM. After stirring for 15 minutes, AuCl(PPh3) (122.69 mg, 248 μmol, 1.25 equiv.) dissolved in 1 mL of anhydrous DCM was added dropwise over about 15 min. The reaction was carried out for 4 h, and the solution turned yellow. After monitoring the completion of the reaction by TLC, the solvent was removed under vacuum, and the residue was extracted with 0.1% ammonia water. After filtration through diatomaceous earth, the filtrate was concentrated to dryness. Recrystallization from CH2Cl2 / n-hexane (volume ratio about 1:20) gave a pale yellow solid, which was separated by filtration, washed with MeOH, and dried under vacuum to obtain the product SIN-Au (89 mg, 99.2 μmol).

[0100] 1 1H NMR (600 MHz, Chloroform-d) δ 7.59–7.42 (m, 19H), 6.74 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 5.48 (s, 1H), 5.21 (d, J = 11.3 Hz, 1H), 5.07 (d, J = 11.3 Hz, 1H), 4.13 (d, J = 16.0 Hz, 1H), 3.77 (s, 3H), 3.49 (s, 3H), 3.17 (t, J = 4.6 Hz, 1H), 2.99–2.95 (m, 2H), 2.97 (d, J = 4.7 Hz, 1H), 2.75 (dd, J = 17.8, 5.3 Hz, 1H), 2.50–2.43 (m, 2H), 2.41 (s, 3H), 1.96 (td, J = 11.7, 4.3 Hz, 1H), 1.86–1.78 (m, 2H).

[0101] 1313C NMR (151 MHz, CDCl3) δ 194.2, 152.7, 151.7, 147.7, 137.2, 134.5 (d, J = 13.8 Hz), 132.5, 131.7 (d, J = 2.5 Hz), 130.4, 130.3, 129.9 (d, J = 56.0 Hz), 129.3 (d, J = 11.3 Hz), 127.9, 124.2, 123.0, 115.6, 111.4, 104.4 (d, J = 25.4 Hz), 73.2, 56.6, 55.8, 54.9, 50.0, 47.2, 46.3, 42.9, 41.1, 37.2, 24.8。

[0102] 31 31P NMR (243 MHz, Chloroform-d) δ 42.34。

[0103] HRMS found: m / z: 902.2687 [M+H] + , calcd for C 46 H 44 AuNO4P + , 902.2668。

[0104] Biological Test Example 1: In Vitro Cytotoxicity and Activity Detection

[0105] First, the toxicity and anti-inflammatory activity of the synthesized compound SIN-Au were evaluated. Therefore, the MTT assay, NO assay, and qRT-PCR assay were used to test the toxicity and anti-inflammatory effect of SIN-Au, respectively.

[0106] MTT assay (showing IC 50 value): RAW264.7 cells were seeded at 1.0×10 4Inoculate at a density of cells per well into a 96-well plate and incubate at 37 °C in a 5% CO₂ incubator for 12 h. Subsequently, randomly divide the cells into a control group, a model group, and a drug administration group, with 3 replicate wells in each group. The control group is maintained under routine culture conditions. The model group is added with a medium containing 1 μg / mL LPS, and the drug administration group is added with different concentrations of SIN-Au, AF, TPPAuCl, and SIN (0.1, 0.4, 0.8, 1.6, 3.1, 6.2, 12.5, 25, 50, and 100 μM). After 24 h of drug intervention, remove the medium from each well and replace it with a high-glucose DMEM medium containing 50 μg / mL MTT reagent, and continue to incubate for 4 h in the dark. Remove the culture medium to terminate the reaction, add 300 μL of dimethyl sulfoxide (DMSO) to fully dissolve the formazan crystals, and finally measure the absorbance value of each well at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The experimental data were statistically analyzed in the form of mean ± standard deviation. The results showed that the toxicity of the sinomenine derivative SIN-Au (IC 50 = 47.1 μM) was less than that of the raw material TPPAuCl (IC 50 = 8.8 μM) and the positive drug AF (IC 50 = 4.5 μM). For specific reference, see Figure 6 . Figure 6 Figure shows the comparison of the IC 50 values of the sinomenine derivative SIN-Au and other drugs.

[0107] NO detection (showing the EC 50 value): After subculturing RAW264.7 cells in the logarithmic growth phase, inoculate them into a 48-well plate at a density of 7.5×10 4 cells per well and allow them to adhere overnight in an incubator at 37 °C and 5% CO₂. The experiment is divided into a control group, a model group, and a drug administration group (each drug concentration is 0.01, 0.1, 1, 4, 8, 10, 12, 12.5, 25, 50, and 100 μM). The control group is replaced with fresh complete medium, and the remaining groups are all added with a medium containing LPS (1 μg / mL) to establish an inflammation model. The drug intervention group is simultaneously given the corresponding concentration of the compound to be tested. After continuous intervention for 24 h, collect the culture supernatant, and strictly follow the instructions of the nitric oxide detection kit (brand: Nanjing Jiancheng, product number A013-1-1) to measure the NO content, as follows:

[0108] (1) Respectively set up a blank tube, a standard tube, and a measurement tube for each drug administration group. After 24 h of drug treatment of RAW264.7 cells, aspirate 300 μL of the supernatant from each well as the measurement sample. In addition, aspirate an equal volume of distilled water as the blank tube and 300 μL of sodium nitrite solution (20 μM) as the standard tube.

[0109] (2) Add 200 μL of Reagent 1 and 100 μL of Reagent 2 in the above-mentioned tubes in sequence. After mixing, let it stand for 10 minutes, centrifuge at 3500 r / min for 10 min, and aspirate 160 μL of the clear supernatant into a new centrifuge tube.

[0110] (3) Prepare the chromogenic reagent according to the ratio of Solution 3:Solution 4:Solution 5 at 2.5:1:1. Add 80 μL of the chromogenic reagent to each sample supernatant, mix well, and let it stand for 15 min.

[0111] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of each well at a wavelength of 570 nm. Calculate the NO release amount of each group and the NO inhibition rate of each administration group according to the OD value of the standard tube solution (the formula is as follows). Use an ELISA reader to detect the NO concentration at a wavelength of 570 nm and calculate the NO inhibition rate.

[0112]

[0113] The experimental data are expressed as mean ± standard deviation. The results show that the EC 50 value of SIN-Au is 2.6 μM, and the EC 50 value of SIN is greater than 100 μM, indicating that the activity of SIN-Au in inhibiting NO is significantly enhanced compared with SIN ( Figure 6 ).

[0114] qRT-PCR detection of changes in inflammatory factor levels: On iBMDM and RAW264.7 cells respectively, determine the mRNA levels of inflammatory factors iNOS, IL-1β, COX-2, and IFN-β by qRT-PCR. After subculturing the RAW264.7 cells in the logarithmic growth phase, inoculate them into a 12-well plate at a density of 4×10 5 cells / well and let them adhere overnight in an incubator at 37°C and 5% CO2. The experiment is divided into a control group, a model group, and an administration group. Among them, the control group maintains routine culture, the model group adds a medium containing 1 μg / mL LPS, and the administration group adds different concentrations of SIN-Au (1, 4, and 8 μM), AF (1 μM), and SIN (100 μM) respectively. After 6 hours of drug intervention, remove the medium in each well, wash it with PBS, extract RNA, and perform qRT-PCR reaction, as follows:

[0115] (1) Total RNA extraction from cells: The total RNA in cells was extracted using a total RNA extraction kit for cells. The specific operations were as follows: 500 μL of lysis buffer LB was added to the cells, and after pipetting until there were no cell clumps, it was added to DNA removal column A2. After centrifugation at 12,000 r / min for 2 min, the filtrate was taken. 800 μL of protein removal solution PL was added, immediately mixed well and then added to RNA adsorption column A2. After centrifugation at 12,000 r / min for 1 min, the filtrate was discarded. 500 μL of binding solution BD was added, and after centrifugation at 12,000 r / min for 1 min, the filtrate was discarded. 700 μL of wash solution W was added, and after centrifugation at 12,000 r / min for 1 min, the filtrate was discarded. After repeating the washing once, the empty tube was centrifuged at 12,000 r / min for 2 min, and then 50 μL of RNase-free H2O preheated at 60 °C was added. After standing for 2 min, it was centrifuged at 12,000 r / min to collect the RNA.

[0116] (2) RNA reverse transcription: After detecting the concentration of the RNA solution with a Q3000 ultraviolet spectrometer and diluting it with water to the same concentration, cDNA was obtained using the Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit. The specific operation was to add 3 μL of 5×gDNA digester Mix and 2 μg of total RNA to a PCR tube, and add RNase-free H2O to a total volume of 15 μL, and incubate at 42 °C for 2 min. 5 μL of Ⅲ SuperMix plus was added to the reaction system, and after mixing, the reverse transcription reaction was carried out according to the following program.

[0117]

[0118] (3) RT-PCR analysis: Using the qPCR SYBR Green Master Mix (Low Rox Plus) method to perform RT-PCR detection of the expression levels of iNOS, IL-1β, COX-2, and IFN-β. The specific operation was to mix 10 μL of qPCR SYBR Green Master Mix (Low Rox Plus), 1 μL each of 10 μM primer F and R, and 2 μL of cDNA, and add RNase-free H2O to a total volume of 20 μL. After mixing, the RT-PCR detection was carried out according to the following program:

[0119]

[0120] The qRT-PCR primer sequences of iNOS, IL-1β, COX-2, and IFN-β genes are as follows:

[0121]

[0122] The experimental data were expressed as mean ± standard deviation, and the results were analyzed by one-way analysis of variance (ANOVA) and t-test between groups using GraphPad Prism 10 statistical software. Figure 7 The inhibitory effect of sinomenine derivative SIN-Au on the levels of inflammatory factors was shown. The results indicated that SIN-Au had significant anti-inflammatory activity and could dose-dependently inhibit the mRNA levels of iNOS, IL-1β, COX-2, and IFN-β. Its effect was significantly better than that of SIN and was comparable to or slightly better than that of the positive drug AF. Compared with the control group, #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0123] Biological Test Example 2: Evaluation of Anti-sepsis Pharmacodynamics

[0124] To systematically evaluate the anti-inflammatory activity of sinomenine derivative SIN-Au, in this study, in vivo pharmacodynamic experiments were carried out using a sepsis systemic inflammation model through two routes of intragastric administration and intraperitoneal injection.

[0125] 1) Evaluation of the anti-sepsis pharmacodynamics of SIN-Au by intragastric administration

[0126] C57BL / 6 mice aged 7 - 8 weeks were selected and divided into Ctrl group, LPS group, and administration groups (SIN-Au 10 mg / kg and 50 mg / kg, Dex 2 mg / kg), with 5 mice in each group. The specific dosing and grouping settings were as follows: control group: normal saline / d; model group: normal saline / d; SIN-Au low-dose group (10 mg / kg / d); SIN-Au high-dose group (50 mg / kg / d); Dex (2 mg / kg / d), and the drugs were administered for 4 days. Before modeling, the above drugs were intragastrically pre-administered for 3 days. On the 4th day, 1 h after the intragastric administration was completed, LPS (5 mg / kg) was intraperitoneally injected for modeling. After 12 h, all mice were scored for sepsis in seven aspects: appearance, consciousness level, activity, response to stimuli, eyes, respiratory rate, and respiratory quality, and then the mice were sacrificed. Serum was taken for determination of inflammatory factor levels, and organs were taken for histological observation of the organs.

[0127] Figure 8The sepsis scores of mice in each group before sampling were shown. It was found that compared with the control group, the mice in the model group showed a series of symptoms of mouse sepsis, such as erected back hair, listlessness, slow movement, weakened arrest reaction, incomplete opening of eyes, increased secretions, and labored breathing. After treatment with sinomenine derivative SIN-Au, the above symptoms were significantly improved, indicating that SIN-Au significantly reduced the sepsis score in a dose-dependent manner. And the efficacy of the high-dose SIN-Au group was better than that of the positive drug dexamethasone group. Compared with the control group, #### P < 0.0001; compared with the model group, *P < 0.05, ***P < 0.001.

[0128] Figure 9 The levels of serum inflammatory factors in mice in each group measured by ELISA were shown. It was found that the sinomenine derivative SIN-Au significantly down-regulated the levels of inflammatory factors TNF-α and IL-6 in a dose-dependent manner. And the efficacy of the high-dose SIN-Au group was better than that of the positive drug dexamethasone group. Compared with the control group, #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01.

[0129] Figure 10 HE staining of the organs of mice in each group after sampling was shown, including the lung, liver, heart, kidney, and spleen, scale bar = 100 μm. The staining results showed that after LPS-induced modeling, atelectasis, pulmonary inflammatory infiltration, myocardial inflammatory cell hyperplasia, and unclear spleen structure disappearance occurred, but administration of SIN-Au could relieve the above phenomena and had an obvious anti-inflammatory effect. It can be seen from this that the sinomenine derivative of the present invention has good anti-sepsis efficacy after intragastric administration and no obvious toxicity was observed.

[0130] 2) Evaluation of the anti-sepsis efficacy of SIN-Au by intraperitoneal injection

[0131] Balb / c mice at 7-8 weeks of age were selected and divided into a Ctrl group, an LPS group, and dosing groups (SIN-Au at 1 mg / kg, 2.5 mg / kg, 5 mg / kg, and Dex at 2 mg / kg), with 8 mice in each group. The dosing vehicle was: 5% DMSO + 40% PEG400 + 55% normal saline. The specific dosing settings and groupings were as follows: control group: blank vehicle / d; model group: blank vehicle / d; SIN-Au low-dose group (1 mg / kg / d); SIN-Au medium-dose group (2.5 mg / kg / d); SIN-Au high-dose group (5 mg / kg / d); Dex (2 mg / kg / d), and the drugs were administered 2 times in total. First, the above drugs were pre-administered by intraperitoneal injection. 1 h later, LPS (5 mg / kg) was intraperitoneally injected to establish the model. 11 h later, the drugs were intraperitoneally injected again. 6 h later, all mice were scored for sepsis in seven aspects: appearance, consciousness level, activity, response to stimuli, eyes, respiratory rate, and respiratory quality, and then the mice were sacrificed. Serum was taken for determination of inflammatory factor levels, and organs were taken for histological observation of the organs.

[0132] Figure 11 The sepsis scores of mice in each group before sample collection were shown. It was found that compared with the control group, mice in the model group showed a series of symptoms of mouse sepsis, such as erected back hair, listlessness, slow movement, weakened arrest response, incomplete opening of eyes, increased secretions, and labored breathing. After treatment with the sinomenine derivative SIN-Au, the above symptoms were significantly improved, indicating that SIN-Au has an anti-sepsis effect. Compared with the control group, #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0133] Figure 12 The levels of inflammatory factors in the serum of mice in each group measured by ELISA were shown. It was found that the sinomenine derivative SIN-Au significantly down-regulated the levels of inflammatory factors TNF-α and IL-6 in a dose-dependent manner. Compared with the control group, #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0134] Figure 13 HE staining of the organs of mice in each group after sample collection was shown, including the lung, liver, heart, kidney, and spleen, scale bar = 100 μm. The staining results showed that after LPS-induced modeling, atelectasis, pulmonary inflammatory infiltration, myocardial inflammatory cell hyperplasia, unclear demarcation between white pulp and red pulp in the spleen and the appearance of vacuoles occurred, but administration of SIN-Au could relieve the above phenomena and significantly inhibit the occurrence and development of inflammation. It can be seen from this that the sinomenine derivative of the present invention has good anti-sepsis efficacy when administered by intraperitoneal injection, and no obvious toxicity was observed.

[0135] Biological Test Example 3: Evaluation of Anti-rheumatoid Arthritis Efficacy

[0136] For the anti-rheumatoid arthritis mouse efficacy experiment of SIN-Au, male DBA / 1J mice aged 6 - 8 weeks, 10 or 11 mice in each group, were selected. Equal volumes of Freund's complete adjuvant and bovine type II collagen solution were mixed and ground until the color became uniformly milky white (without obvious oil droplets) to form injection emulsion I, and 100 μL of emulsion I was subcutaneously injected at the root of the tail of each mouse; 21 days later, the protein solution and incomplete Freund's adjuvant were mixed and ground in a ratio of 1:1 to form injection emulsion II, and 100 μL of emulsion II was subcutaneously injected at the root of the tail of each mouse. After the second immunization, the mice were randomly grouped and drug administration treatment was started. The drug solvent was: 5% DMSO + 40% PEG400 + 55% normal saline. The specific drug administration settings and groups were as follows: Control group: blank solvent / 3d (i.p.); Model group: blank solvent / 3d (i.p.); SIN-Au low-dose group (1 mg / kg / 3d, i.p.); SIN-Au high-dose group (2.5 mg / kg / 3d, i.p.); SIN group (50 mg / kg / d, i.g.); MTX group (2 mg / kg / 3d, i.g.); AF group (10 mg / kg / 3d, i.p.). The incidence of arthritis was recorded every 3 days, and the arthritis score and ankle and foot sole thickness were measured. The arthritis scoring criteria are as follows.

[0137] Table 1 Clinical Scoring Criteria for Arthritis

[0138]

[0139]

[0140] Figure 14 Show the gross view of the lower limb ankle joints of CIA mice after the end of drug administration. Figure 15 Show the arthritis score, foot sole thickness and body weight changes of mice in each group during drug administration (recorded for 30 days after the second immunization). Figure 16 Show the changes in the incidence of arthritis in mice in each group during drug administration. It can be seen that the toe joints, ankle joints and foot soles of the mice in the model group were significantly swollen, while SIN-Au effectively inhibited the swelling of each joint, reduced the arthritis score, and significantly reduced the incidence of CIA. The most obvious was the SIN-Au high-dose group (2.5 mg / kg / 3d), and the incidence of arthritis was about 60% on the 30th day. In addition, the body weight monitoring data showed that in the initial stage of drug administration (days 0 - 12), drug administration of AF caused a significant decrease in the body weight of mice, suggesting that it may have a drug toxicity reaction. While drug administration of SIN-Au did not significantly cause a decrease in the body weight of mice, indicating that SIN-Au is safer than AF. #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01.

[0141] Figure 17 After the administration ended, a pain detector was used to measure the pain threshold of mice in each group. The measurement results showed that the mice in the CIA group were sensitive to pain stimuli, so their pain threshold was significantly lower than that of the control group. However, the administration of SIN-Au increased the pain threshold of CIA mice in a dose-dependent manner, and the effect of improving the pain threshold in the high-dose SIN-Au group was more obvious than that of SIN, AF, and MTX. The above data indicate that SIN-Au can effectively improve joint pain and reduce the pain threshold in CIA mice. Compared with the control group, #### P < 0.0001; compared with the model group, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0142] Figure 18 The micro-CT images of CIA mice after the administration ended were shown. Figure 19 The statistics of bone density, trabecular bone separation, and bone surface area obtained from the processing of micro-CT images were shown. By performing micro-CT scans on the lower limb ankle joints of CIA mice after the administration, it was found that the administration of SIN-Au significantly improved the injuries of the toe joints and ankle joints, and adjusted the parameters of bone density, trabecular bone separation, and bone surface area. It was shown that the sinomenine derivative SIN-Au effectively improved the bone density loss caused by arthritis, that is, it had a relieving effect on the destruction of joint bone quality. Compared with the control group, # P < 0.05, ## P < 0.01; compared with the model group, *P < 0.05.

[0143] Figure 20 The behavioral statistics of mice measured by a gait analyzer were shown, including gait symmetry, normal step sequence ratio, and number of steps per unit time. The results showed that SIN-Au improved the movement speed and abnormal step sequence of CIA mice in a dose-dependent manner, and enhanced the movement ability of CIA mice. Compared with the control group, # P < 0.05, ### P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0144] Figure 21 The serum histamine and IgE levels of CIA mice after the administration ended were shown. The levels of histamine and IgE reflected the degree of allergic reaction in mice. By measuring the serum histamine and IgE levels of CIA mice, it was found that SIN-Au had no obvious effect on both of them. Combining with the fact that no obvious phenomena such as hair loss, itching, and redness were found in mice during the administration process, so SIN-Au did not cause obvious allergic reactions. Compared with the control group, ###P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns represents no significant difference.

[0145] Figure 22 Show the organ indices of CIA mice after the administration ended, including lung index, liver index, spleen index, heart index, thymus index, and kidney index. It was found that the sinomenine-gold complex SIN-Au had certain improvement effects on the liver, thymus, and heart, while had no obvious effect on the lungs, spleen, and kidneys. Therefore, the sinomenine-gold complex does not cause obvious organ toxicity and may have immunomodulatory effects. Compared with the control group, ### P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ns represents no significant difference.

[0146] Figure 23 Show the effects of SIN-Au on the liver function of CIA mice, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Figure 24 Show the effects of SIN-Au on the kidney function of CIA mice, including creatinine and blood urea nitrogen. It was found that the sinomenine-gold complex SIN-Au had no obvious up-regulating effect on the levels of ALT, AST, and blood urea nitrogen in CIA mice, but could callback the creatinine level in CIA mice, indicating that SIN-Au had no significant toxicity to liver and kidney functions. Compared with the control group, # P < 0.05; compared with the model group, *P < 0.05, **P < 0.01, ns represents no significant difference.

[0147] Figure 25 Show the HE staining of the organs of mice in each group after sampling, including lungs, liver, heart, kidneys, spleen, and thymus, scale bar = 100 μm. The results showed that compared with the control group, the CIA model group had pulmonary inflammatory infiltration, unclear boundaries of glomeruli and renal tubules, slightly poor demarcation of the spleen medulla, and relatively higher thymic cortex / medulla region. After administration of SIN-Au, there was no obvious effect on the lungs, kidneys, and spleen, but it could improve the ratio of the thymic cortex region / medulla region. In addition, after administration of SIN-Au, the myocardial structure was normal and the hepatocytes were arranged normally, indicating that it had no obvious toxicity to the heart and liver. However, administration of AF caused myocardial damage, cytoplasmic vacuolization of hepatocytes, and unclear demarcation between red pulp and white pulp of the spleen; administration of MTX showed more inflammatory cell infiltration and further thickening of the alveolar wall. Therefore, SIN-Au has obvious anti-rheumatoid arthritis effect and no obvious toxic and side effects.

[0148] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. Sinomenine derivatives of formula I or pharmaceutically acceptable salts thereof: Wherein, R1-R4 are the same or different and each independently selected from: hydrogen, halogen, cyano, nitro, hydroxy, amino, C1-C4 alkyl, halo C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, C1-C4 acyloxy; L is selected from C1-C10 alkylene; Ar1-Ar3 are the same or different and each independently selected from: substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted 5-10 membered heteroaryl.

2. The sinomenine derivative represented by Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Said R1-R4 are the same or different and each independently selected from: hydrogen, fluorine, chlorine, bromine, iodine, cyano, nitro, hydroxy, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetyloxy.

3. The sinomenine derivative represented by Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Said L is selected from -(CHR) n -, R are the same or different and each independently selected from hydrogen, methyl, ethyl, and n is selected from 1, 2, 3, 4, 5 or 6.

4. The sinomenine derivative represented by Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Said Ar1-Ar3 are the same or different and each independently selected from phenyl, naphthyl, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrimidinyl which are substituted or unsubstituted by one or more of fluorine, chlorine, bromine, iodine, cyano, nitro, hydroxy, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetyloxy.

5. The sinomenine derivative represented by Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Said sinomenine derivatives are selected from:

6. The preparation method of the sinomenine derivative of formula I as described in claim 1, comprising the following steps: Step 1, reacting sinomenine with a halogenating reagent to obtain intermediate I'; Step 2, subjecting intermediate I' to a nucleophilic substitution reaction with triarylphosphine gold halide to obtain the product, wherein X is independently selected from chlorine or bromine, and R1-R4, L, Ar1-Ar3 are as described in claim 1.

7. Use of the sinomenine derivative of formula I or pharmaceutically acceptable salt thereof as described in any one of claims 1-5 in the preparation of anti-inflammatory drugs.

8. Use of the sinomenine derivative of formula I or pharmaceutically acceptable salt thereof as described in any one of claims 1-5 in the preparation of anti-osteoarthritis drugs.

9. Use of the sinomenine derivative of formula I or pharmaceutically acceptable salt thereof as described in any one of claims 1-5 in the preparation of drugs for anti-rheumatoid arthritis and / or anti-sepsis.

10. A pharmaceutical composition comprising the sinomenine derivative of formula I or pharmaceutically acceptable salt thereof as described in any one of claims 1-5.

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