Sinomenine derivatives, and preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry and organic compound synthesis, and particularly relates to a sinomenine derivative, a preparation method and application thereof. The sinomenine derivative has a structural formula as shown in formula I. The sinomenine derivative is prepared by taking sinomenine and a halogenated reagent as raw materials, obtaining an intermediate I' through a substitution reaction, and then performing a nucleophilic substitution reaction on the intermediate I' and triaryl phosphine halogen gold to obtain the sinomenine derivative shown in formula I. The sinomenine derivative has significant anti-rheumatoid arthritis activity and anti-sepsis activity, low toxic side effects, high safety factor, and good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry and synthesis of organic compounds, and particularly relates to a kind of sinomenine derivatives and a preparation method and application thereof. BACKGROUND

[0002] Sinomenine is an isoquinoline alkaloid extracted from Sinomenium acutum, and its anti-inflammatory analgesic, immunosuppressive and anti-allergic pharmacological effects are clear. Sinomenine hydrochloride has been developed into a new drug, Zhengqingfengtongning injection and tablets, and is used as a clinical drug for treating rheumatoid arthritis. However, existing clinical practice and pharmacodynamic studies have shown that sinomenine has significant limitations in exerting therapeutic effects: first, the conventional therapeutic dose is relatively high (daily dose range 60-240 mg), which may cause anaphylactic shock, leukopenia and severe gastrointestinal reactions and other adverse drug events; second, the low bioavailability limits the drug efficacy, and a large dose of 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 high efficiency and low toxicity. SUMMARY

[0003] In view of the deficiencies of the prior art, the technical problem to be solved by the present application is to provide a class of sinomenine derivatives and a preparation method and 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 application is realized through the following technical solutions:

[0005] In a first aspect, the present application 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 is 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 and C1-C4 acyloxy;

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

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

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

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

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

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

[0015] Further, the "substituted or unsubstituted" substituents in Ar1-Ar3 are selected from the group consisting of: halogen, cyano, nitro, hydroxyl, amino, C1-C4 alkyl, haloC1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, C1-C4 acyloxy.

[0016] Preferably, the "substituted or unsubstituted" substituents are selected from the group consisting 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, acetyloxy.

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

[0018] Further, the 5-10 membered heteroaryl groups in Ar1-Ar3 include: pyrrolyl, furanyl, thiophenyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyranyl, indolizinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzothiazolyl, purinyl, quinolyl, isoquinolyl, thiazolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, naphthyridinyl.

[0019] Further, Ar1-Ar3 are the same or different, each independently selected from one or more substituted or unsubstituted: phenyl, naphthyl, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrimidinyl, with fluoro, chloro, bromo, iodo, cyano, nitro, hydroxyl, amino, methyl, ethyl, propyl, butyl, trifluoromethyl, 2,2,2-trifluoroethyl, methoxy, ethoxy, methylamino, ethylamino, methoxycarbonyl, ethoxycarbonyl, formyloxy, acetyloxy.

[0020] Preferably, Ar1-Ar3 are the same or different, each independently selected from one or more substituted or unsubstituted: phenyl, naphthyl, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, with fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, trifluoromethyl, methoxy, ethoxy, methylamino, ethylamino.

[0021] Further, the sinomenine derivative is selected from:

[0022]

[0023] In a second aspect, the present application provides a method for preparing a sinomenine derivative of Formula I, comprising the following steps:

[0024] Step 1, reacting sinomenine with a halogenating reagent to obtain an intermediate I’;

[0025] Step 2, carrying out a nucleophilic substitution reaction between the intermediate I’ and triarylphosphonium halogen to obtain a product,

[0026]

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

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

[0032] Further, step 1 comprises stirring and reacting the sinomenine with sodium or potassium alcoholate in anhydrous solvent, and then slowly adding halogenated reagent under the protection of inert gas to obtain the intermediate I'.

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

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

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

[0036] Preferably, after the reaction, the silica gel is used for filtration, the solvent is evaporated, and column chromatography is used for separation to obtain the intermediate I'.

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

[0038] Further, in step 2, the molar ratio of the intermediate I' to triarylphosphine halogen is 1:1-1.8, preferably 1:1.1-1.3.

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

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

[0041] Further, step 2 comprises stirring and reacting the intermediate I' with sodium or potassium alcoholate in anhydrous solvent, and then slowly adding triarylphosphine halogen under the protection of inert gas to obtain the sinomenine derivative of formula I.

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

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

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

[0045] Preferably, after the reaction, the solvent is evaporated, the residue is extracted with 0.05-0.3% ammonia water, and then the sinomenine derivative of formula I is obtained by filtering through silica gel and concentrating the filtrate to dryness and recrystallization.

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

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

[0048] Further, the present application provides use of the sinomenine derivative of Formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-osteoarthritis medicament.

[0049] Further, the present application provides use of the sinomenine derivative of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for anti-rheumatoid arthritis and / or anti-sepsis.

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

[0051] Unless otherwise indicated, the terms used in this application have the following meanings, and the definitions of terms not mentioned below are as generally understood by those skilled in the art to which the present application pertains.

[0052] In the present application, the term "pharmaceutically acceptable salt" refers to a salt prepared from a compound with a relatively non-toxic, pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When a compound of the present application contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the 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 therapeutic treatment. In reference to a particular condition, treatment refers to: (1) alleviating one or more of the biological manifestations of the disease or condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more of the biological manifestations of the condition, (3) ameliorating one or more symptoms, effects or side effects associated with the condition or one or more symptoms, effects or side effects associated with the treatment of the condition, or (4) slowing the development of the condition or one or more of the biological manifestations of the condition.

[0054] The pharmaceutical composition can be prepared into various types of administration unit dosage forms according to the purpose of treatment.

[0055] The compound of the present application can be clinically administered in a conventional administration manner.

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

[0057] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0058] Unless otherwise indicated, the abbreviations used in this application have their common general meaning in the art. For example, SIN refers to sinomenine, MTX refers to methotrexate, DEX refers to dexamethasone, AF refers to auranofin, TPPAuCl refers to triphenylphosphine chloroaurate.

[0059] Compared with the prior art, the present application has the following beneficial effects:

[0060] (1) The sinomenine derivative or pharmaceutically acceptable salt thereof of the present application can effectively inhibit various inflammatory factors, on the one hand, can significantly reduce the swelling of joints, improve the bone density loss caused by arthritis, and has a certain relieving effect on the destruction of joint bone quality; on the other hand, can significantly alleviate the serum inflammatory level of sepsis mice, reduce the sepsis score, and improve the damage of various organs under pathological state. It has excellent anti-rheumatoid arthritis and anti-sepsis activity, and has small side effects and high safety.

[0061] (2) The synthesis method of the sinomenine derivative of the present application has the advantages of mild reaction condition, simple operation, high yield of reaction product, and suitability for industrial production. BRIEF DESCRIPTION OF DRAWINGS

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

[0063] Figure 1 The H NMR spectrum of the compound 1 synthesized for Example 1 1 H NMR spectrum;

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

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

[0066] Figure 4 The H NMR spectrum of the SIN-Au synthesized for Example 2 13 C NMR spectrum;

[0067] Figure 5 The H NMR spectrum of the SIN-Au synthesized for Example 2 31 PNMR spectrum;

[0068] Figure 6 The IC of the sinomenine derivative SIN-Au based on RAW264.7 cell cytotoxicity50 and nitric oxide inhibitory activity EC 50 Evaluation;

[0069] Figure 7 Evaluation of anti-inflammatory activity of sinomenine derivative SIN-Au based on the inhibition of inflammatory factor levels in RAW264.7 and iBMDM cells;

[0070] Figure 8 Effect of sinomenine derivative SIN-Au administered by gavage on the sepsis score in a sepsis mouse model;

[0071] Figure 9 Effect of sinomenine derivative SIN-Au administered by gavage on the inhibition of serum inflammatory factor levels in a sepsis mouse model;

[0072] Figure 10 Improvement of sinomenine derivative SIN-Au administered by gavage on each organ of sepsis mice;

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

[0074] Figure 12 Effect of sinomenine derivative SIN-Au administered by intraperitoneal injection on the inhibition of serum inflammatory factor levels in a sepsis mouse model;

[0075] Figure 13 Improvement of sinomenine derivative SIN-Au administered by intraperitoneal injection on each organ of sepsis mice;

[0076] Figure 14 Photograph of the foot of a CIA mouse injected with SIN-Au 30 days after modeling was completed;

[0077] Figure 15 Statistical graph showing the effect of SIN-Au on the arthritis score, foot thickness, and body weight of a CIA mouse after modeling was completed;

[0078] Figure 16 Statistical graph showing the effect of SIN-Au on the incidence of a CIA mouse after modeling was completed;

[0079] Figure 17 Statistical graph showing the effect of SIN-Au on the foot pain threshold at 30 days after administration;

[0080] Figure 18 CIA mouse ankle micro CT image after administration of SIN-Au was completed;

[0081] Figure 19Figure 16 shows the statistical chart of bone density of CIA mice after administration of SIN-Au ended;

[0082] Figure 20 Figure 17 shows the gait analysis chart of CIA mice after administration of SIN-Au ended;

[0083] Figure 21 Figure 18 shows the IgE and histamine levels of CIA mice after administration of SIN-Au ended;

[0084] Figure 22 Figure 19 shows the organ index of CIA mice after administration of SIN-Au ended;

[0085] Figure 23 Figure 20 shows the liver function index of CIA mice after administration of SIN-Au ended;

[0086] Figure 24 Figure 21 shows the kidney function index of CIA mice after administration of SIN-Au ended;

[0087] Figure 25 Figure 22 shows the HE staining chart of organ tissues of CIA mice after administration of SIN-Au ended. DETAILED DESCRIPTION

[0088] The embodiments of the present application are described in detail below, the embodiments are used to better illustrate the content of the present application, and are used to explain the present application, and cannot be understood as a limitation of the present application.

[0089] The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased through a regular channel.

[0090] The experimental methods in the following embodiments are all conventional methods, if not specially mentioned. The experimental materials used in the following embodiments are all commercially available products, if not specially mentioned.

[0091] Example 1: Synthesis of compound 1

[0092]

[0093] To sinomenine (60 mg, 182.15 μmol, 1.0 equiv.) and potassium tert-butoxide (30.66 mg, 273.23 μmol, 1.5 equiv.) in 5 mL anhydrous CH3CN was added C9H7Br (39.97 mg, 204.92 μmol, 1.125 equiv.) dropwise in 1 mL anhydrous CH3CN under argon protection. The solution was orange yellow after stirring for 15 min and reacting at room temperature for 6 h. The reaction was monitored by TLC and completed after which the solution was filtered through celite and concentrated under reduced pressure. After the solvent was removed by rotary evaporation under reduced pressure, compound 1 was isolated as a brownish yellow solid (40 mg, 91.0 μmol) by column chromatography using a solvent system of DCM / MeOH = 25:1.

[0094] 1 H NMR (600 MHz, CDC13) 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, CDC13) 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] To compound 1 (88 mg, 198.4 μmol, 1.0 equiv.) and potassium tert-butoxide (33.4 mg, 297.6 μmol, 1.5 equiv.) in 5 mL of dry DCM was added AuCl(PPh3) (122.69 mg, 248 μmol, 1.25 equiv.) dropwise over 15 min under argon. The solution turned yellow after 4 h. After monitoring the reaction completion by TLC, the solvent was removed under vacuum and the residue was extracted with 0.1% aqueous ammonia. The filtrate was concentrated to dryness after passing through celite. The product was obtained as a light yellow solid after recrystallization from CH2Cl2 / n-hexane (about 1 :20 by volume), isolated by filtration, washed with MeOH and dried under vacuum (89 mg, 99.2 μmol).

[0100] 1 H 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] 13C NMR (151 MHz, CDC13) 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 PNMR (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, so MTT experiment, NO experiment and qRT-PCR experiment were used to test the toxicity and anti-inflammatory effect of SIN-Au, respectively.

[0106] MTT experiment (showing IC 50 value): RAW264.7 cells were seeded at 1.0 x 10 4Cells were seeded at a density of 1 / 2 well in 96-well plates and incubated at 37°C with 5% CO2 for 12 h. Cells were then randomly divided into control, model, and drug-treated groups, with three replicates per group. The control group maintained standard culture, the model group received medium containing 1 μg / mL LPS, and the drug-treated groups received 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 hours of drug intervention, the medium in each well was removed and replaced with DMEM high-glucose medium containing 50 μg / mL LMT reagent, and incubated for another 4 h in the dark. The culture medium was then removed to terminate the reaction, and 300 μL of dimethyl sulfoxide (DMSO) was added to fully dissolve the formazan crystals. Finally, the absorbance of each well was measured at 570 nm using a microplate reader. Experimental data were presented as mean ± standard deviation for statistical analysis. The results showed that the sinomenine derivative SIN-Au(IC) 50 =47.1μM) has less toxicity than the raw material TPPAPuCl (IC). 50 =8.8μM) and positive control drug AF (IC) 50 =4.5μM). See details. Figure 6 . Figure 6 The IC50 values ​​of sinomenine derivative SIN-Au and other drugs were shown. 50 Value comparison.

[0107] NO detection (displays EC) 50 Value): RAW264.7 cells in logarithmic growth phase were passaged and then subjected to a culture of 7.5 × 10⁻⁶ cells. 4 Inoculum was seeded at a density of 100 cells / well in 48-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The experiment was divided into a control group, a model group, and a drug treatment group (drug concentrations of 0.01, 0.1, 1, 4, 8, 10, 12, 12.5, 25, 50, and 100 μM). The control group received fresh complete culture medium, while the other groups received culture medium containing LPS (1 μg / mL) to establish an inflammation model. The drug intervention groups received the corresponding concentration of the test compound simultaneously. After 24 hours of continuous intervention, the culture supernatant was collected, and the NO content was determined strictly according to the instructions of the nitric oxide assay kit (brand: Nanjing Jiancheng, catalog number A013-1-1), as follows:

[0108] (1) Blank tubes, standard tubes, and test tubes for each drug-treated group were set up separately. After RAW264.7 cells were treated with the drug for 24 h, 300 μL of supernatant from each well was taken as the test sample. In addition, an equal volume of distilled water was taken as the blank tube, and 300 μL of sodium nitrite solution (20 μM) was taken as the standard tube.

[0109] (2) In each tube, add 200 μL of reagent one and 100 μL of reagent two in the kit, mix well, and stand for 10 minutes, centrifuge at 3500 r / min for 10 min, and take 160 μL of clear supernatant to a new centrifuge tube.

[0110] (3) Prepare the color developing agent according to the ratio of 2.5:1:1 of No. 3: No. 4: No. 5 liquid, add 80 μL of the color developing agent to each sample supernatant, mix well, and stand for 15 min.

[0111] (4) Detect the OD value of each well at 570 nm wavelength by using an enzyme marker. According to the OD value of the standard tube solution, calculate the NO release amount of each group and the NO inhibition rate of each administration group (formula as follows). Detect the NO concentration at 570 nm wavelength by using an enzyme marker, and calculate the NO inhibition rate.

[0112]

[0113] The experimental data is represented by 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: in iBMDM and RAW264.7 cells, the mRNA levels of inflammatory factors iNOS, IL-1β, COX-2 and IFN-β were determined by qRT-PCR. RAW264.7 cells in the logarithmic growth phase were subcultured, inoculated in a 12-well plate at a density of 4×10 5 cells / well, and adhered overnight in a 37℃, 5% CO2 incubator. The experiment was divided into a control group, a model group and a drug administration group. Among them, the control group was maintained in regular culture, the model group was added with a culture medium containing 1 μg / mL LPS, and the drug administration group was added with different concentrations of SIN-Au (1, 4 and 8 μM), AF (1 μM) and SIN (100 μM). After 6 hours of drug intervention, the culture medium in each well was removed, washed with PBS, and RNA was extracted and subjected to qRT-PCR reaction, as follows:

[0115] ​(1) Total RNA extraction: The total RNA in the cells was extracted by the total RNA extraction kit method, and the specific operation was as follows: 500 μL of lysis solution LB was added to the cells, and after blowing to remove the cell clumps, it was added to the DNA removal column A2, and after centrifugation at 12000 r / min for 2 min, the filtrate was taken, 800 μL of deproteinization solution PL was added, and immediately mixed, then added to the RNA adsorption column A2, centrifuged at 12000 r / min for 1 min, and the filtrate was discarded. 500 μL of binding liquid BD was added, and centrifuged at 12000 r / min for 1 min, and the filtrate was discarded. 700 μL of rinse solution W was added, and centrifuged at 12000 r / min for 1 min, and the filtrate was discarded. After repeating the rinse once, the empty tube was centrifuged at 12000 r / min for 2 min, and then 60°C preheated RNase-free H2O 50 μL was added, and after standing for 2 min, the RNA was collected by centrifugation at 12000 r / min.

[0116] (2) RNA reverse transcription: After the RNA solution was detected by Q3000 ultraviolet spectrum detector, it was diluted with water to the same concentration, and the cDNA was obtained by the method of Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR(gDNAdigester plus) kit method, and the specific operation was as follows: 5x gDNAdigester Mix 3 μL, total RNA 2 μg was added to the PCR tube, and RNase-free H2O was added to a total volume of 15 μL, and incubated 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 as follows.

[0117]

[0118] (3) RT-PCR analysis: The expression of iNOS, IL-1β, COX-2 and IFN-β was detected by RT-PCR using qPCR SYBR Green Master Mix(Low Rox Plus) method. The specific operation was as follows: qPCR SYBR Green Master Mix(Low Rox Plus) 10 μL, 10 μM primers F and R each 1 μL, cDNA 2 μL were mixed, and RNase-free H2O was added to a total volume of 20 μL, and mixed, and then the RT-PCR detection was carried out as follows:

[0119]

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

[0121]

[0122] The experimental data are expressed as mean ± standard deviation, and the results are analyzed by one-way ANOVA and t-test between groups using GraphPad Prism 10 statistical software. Figure 7 The inhibitory effect of sinomenine derivative SIN-Au on inflammatory factor levels is shown. The results show that SIN-Au has significant anti-inflammatory activity and can dose-dependently inhibit iNOS, IL-1β, COX-2 and IFN-β mRNA levels. Its effect is significantly better than that of SIN, and is comparable to or slightly better than that of AF, the positive drug. 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: anti-sepsis efficacy evaluation

[0124] To systematically evaluate the anti-inflammatory activity of sinomenine derivative SIN-Au, this study used a sepsis systemic inflammation model to carry out in vivo pharmacodynamic experiments by intragastrical administration and intraperitoneal injection.

[0125] 1) Anti-sepsis efficacy evaluation of SIN-Au by intragastrical administration

[0126] Select 7-8 week old C57BL / 6 mice, set Ctrl group, LPS group, drug administration group (SIN-Au 10 mg / kg and 50 mg / kg, Dex 2 mg / kg), 5 in each group. The specific drug administration setting groups are 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), a total of 4 days of drug administration. Before modeling, the above drugs were administered intragastrically for 3 days. On the 4th day, 1 h after the end of intragastrical administration, LPS (5 mg / kg) was injected intraperitoneally to model. 12 h later, all mice were scored for sepsis from seven aspects of appearance, level of consciousness, activity, response to stimulation, eyes, respiratory rate and respiratory quality, and the mice were sacrificed, serum was taken for determination of inflammatory factor levels, and organs were taken for histological observation of organs.

[0127] Figure 8The sepsis scores of each group of mice before sampling were displayed, and it was found that compared with the control group, the model group of mice showed a series of sepsis symptoms such as back hair standing up, listless, slow movement, weakened capture reaction, eyes unable to fully open, increased secretion, labored breathing, etc. After treatment with the sinactine 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 drug efficacy of the high-dose group of SIN-Au 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 serum inflammatory factor levels of each group of mice measured by ELISA were displayed, and it was found that the sinactine derivative SIN-Au significantly down-regulated the levels of inflammatory factors TNF-α and IL-6 in a dose-dependent manner. And the drug efficacy of the high-dose group of SIN-Au 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 The HE staining of the organs of each group of mice after sampling was displayed, including lung, liver, heart, kidney and spleen, and the scale = 100 μm. The staining results showed that after LPS modeling, alveolar atelectasis, lung inflammation infiltration, myocardial inflammatory cell proliferation, and spleen structure disappearance were observed, but the above phenomena could be alleviated by administration of SIN-Au, which had an obvious inhibitory effect on inflammation. Therefore, it was proved that the sinactine derivative of the application had good anti-sepsis efficacy by oral administration, and no obvious toxicity was observed.

[0130] 2) Anti-sepsis drug efficacy evaluation of SIN-Au by intraperitoneal injection

[0131] Select 7-8 week old Balb / c mice, set Ctrl group, LPS group, drug group (SIN-Au 1 mg / kg, 2.5 mg / kg, 5 mg / kg, Dex 2 mg / kg), 8 in each group. The drug solvent is: 5% DMSO+40% PEG400+55% normal saline. The specific drug setting grouping is as follows: control group: blank solvent / d; model group: blank solvent / 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), a total of 2 times of drug administration. First, the above drugs are injected intraperitoneally for pre-dosing, 1 hour later, LPS (5 mg / kg) is injected intraperitoneally for modeling. 11 hours later, drug intraperitoneal injection is performed again. 6 hours later, all mice are scored for sepsis from the following seven aspects: appearance, level of consciousness, activity, response to stimulation, eyes, respiratory rate and respiratory quality, and the mice are sacrificed, the serum is taken for determination of inflammatory factor level, and the organs are taken for histological observation of organs.

[0132] Figure 11 The sepsis scores of mice in each group before sampling are shown, and it is found that compared with the control group, the mice in the model group exhibit a series of sepsis symptoms such as back hair standing up, listlessness, slow movement, weakened catch reaction, eyes not fully open, increased secretion, and labored breathing. After treatment with the sinactine derivative SIN-Au, the above symptoms are 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 serum inflammatory factor levels of mice in each group measured by ELISA are shown, and it is found that the sinactine derivative SIN-Au significantly down-regulates 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 The HE staining of organs of mice in each group after sampling is shown, including lung, liver, heart, kidney and spleen, and the scale bar is 100 μm. The staining results show that after LPS modeling, alveolar atelectasis, lung inflammation infiltration, myocardial inflammatory cell proliferation, unclear boundary between white pulp and red pulp of the spleen and vacuoles appear, but administration of SIN-Au can alleviate the above phenomena and significantly inhibit the development of inflammation. Therefore, it is proved that the sinactine derivative of the application has good anti-sepsis efficacy after intraperitoneal administration, and no obvious toxicity is observed.

[0135] Bioassay Example 3: Anti-rheumatoid arthritis efficacy evaluation

[0136] For the rheumatoid arthritis mouse efficacy experiment, 6-8 week old DBA / 1J male mice were selected, 10 or 11 in each group, and equal volumes of Freund's complete adjuvant and bovine type II collagen solution were mixed and ground until the color changed to uniform milky white (no obvious oil droplets), forming injection emulsion I, 100 μL of emulsion I was injected subcutaneously at the base of the tail of each mouse; 21 days later, the protein solution was mixed with incomplete Freund's adjuvant at a ratio of 1:1 after grinding, forming injection emulsion II, 100 μL of emulsion II was injected subcutaneously at the base of the tail of each mouse. After the second immunization, the mice were randomly divided into groups and the administration was started. The administration vehicle was: 5% DMSO + 40% PEG400 + 55% normal saline. The specific administration setting groups were as follows: control group: blank vehicle / 3d (i.p.); model group: blank vehicle / 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 metatarsal thickness were measured, and the arthritis score criteria were as follows.

[0137] Table 1 Arthritis clinical score criteria

[0138]

[0139]

[0140] Figure 14 The general view of the ankle joint of the lower limbs of the CIA mice after administration is shown. Figure 15 The arthritis score, foot thickness and body weight changes of the mice in each group during administration (recorded for 30 days after the second immunization) are shown. Figure 16 The incidence of arthritis of the mice in each group during administration is shown. It can be seen that the toe joints, ankle joints and foot thickness of the mice in the model group were significantly swollen, while SIN-Au effectively inhibited the swelling of the joints, reduced the arthritis score, and significantly reduced the incidence of CIA. The high dose group of SIN-Au (2.5 mg / kg / 3d) was the most obvious, and the incidence of arthritis was about 60% on the 30th day. In addition, the body weight monitoring data showed that the administration of AF caused a significant decrease in the body weight of the mice in the early stage of administration (0-12 days), suggesting that it may have a drug toxicity reaction. The administration of SIN-Au did not significantly reduce the body weight of the 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 The pain threshold of each group of mice was detected by the pain tester after the end of administration. The determination results showed that the CIA group of mice was sensitive to pain stimuli, so the pain threshold was significantly lower than that of the control group. The administration of SIN-Au dose-dependently increased the pain threshold of CIA mice, and the high-dose group of SIN-Au improved the pain threshold more obviously than SIN, AF and MTX. The above data showed that SIN-Au could effectively improve the joint pain of CIA mice and reduce the pain threshold. 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 end of administration were shown. Figure 19 The bone density, bone trabecular separation and bone surface area statistics obtained by processing the micro CT images were shown. By micro CT scanning of the ankle joints of CIA mice after administration, it was found that administration of SIN-Au significantly improved the damage of toe joints and ankle joints, and restored the bone density, bone trabecular separation and bone surface area parameters. It was shown that the sinactine 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. Compared with the control group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05.

[0143] Figure 20 The behavior statistics of mice determined by the gait analysis instrument were shown, including gait symmetry, normal step sequence ratio and footstep number per unit time. The results showed that SIN-Au dose-dependently improved the movement speed and abnormal step sequence of CIA mice, and improved 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 end of administration were shown. The histamine and IgE levels reflected the degree of allergic reaction of mice. The determination of the serum histamine and IgE levels of CIA mice showed that SIN-Au had no obvious effect on the two, and no obvious phenomena such as mouse hair loss, itching and redness were found during administration, so SIN-Au would not cause obvious allergic reaction. Compared with the control group, ###P<0.001; compared with model group, *P<0.05, **P<0.01, ns represents no significant difference.

[0145] Figure 22 The organ index of CIA mice after administration was shown, 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 a certain improvement effect on liver, thymus, and heart, but had no obvious effect on lung, spleen, and kidney. Therefore, the sinomenine gold complex did not cause obvious organ toxicity and might have an immune regulation effect. Compared with the control group, ### P<0.001; compared with model group, *P<0.05, **P<0.01, ns represents no significant difference.

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

[0147] Figure 25 HE staining of organs of mice in each group after sampling was shown, including lung, liver, heart, kidney, spleen, and thymus, and the scale = 100 μm. The results showed that compared with the control group, the CIA model group had lung inflammation infiltration, unclear boundaries of glomerulus and renal tubule, slightly poor medulla division of spleen, and relatively high thymus cortex / medulla area. After administration of SIN-Au, there was no obvious effect on lung, kidney, and spleen, but the thymus cortex area / medulla area ratio could be improved. In addition, after administration of SIN-Au, the myocardial structure was normal, and the liver cell arrangement was normal, indicating that it had no obvious toxicity to heart and liver. However, administration of AF caused myocardial damage, hepatocyte cytoplasm vacuolization, and unclear boundary between red pulp and white pulp of spleen; administration of MTX caused more inflammatory cell infiltration and further thickening of alveolar wall. Therefore, SIN-Au had obvious anti-rheumatoid arthritis effect and no obvious side effects.

[0148] It is apparent that the above-mentioned embodiments are only examples for clearly illustrating the present application and are not intended to limit the embodiments of the present application. Those skilled in the art can make various modifications and variations without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, it is also intended to include these modifications and variations.

Claims

1. A sinomenine derivative represented by Formula I: ###0001### Formula I or a pharmaceutically acceptable salt thereof, wherein, R1-R4 are the same and are selected from the group consisting of: hydrogen; L is selected from the group consisting of: -CH2-; -CH2CH2-; -CH2CH2CH2-; -CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2 I ​ ​ said L is selected from -(CHR) n -, R is selected identically from hydrogen, n is selected from 1, 2 or 3; ​ 2. The sinomenine derivative of Formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, ​ 3. The sinomenine derivative of Formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein ​ 。 ​ ​ ​ ​ ​ ​ ​ ​

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