Sinomenine derivatives and preparation methods and applications thereof

Abstract

The present invention belongs to the technical field of pharmaceutical chemistry and organic compound synthesis, and specifically relates to a sinomenine derivative and its preparation method and application. The sinomenine derivative has a structure shown in Formula I and Formula II, and is prepared by reacting sinomenine and a halogenated reagent as raw materials to obtain an intermediate I', and then the intermediate I' is oxidized with oxalyl chloride and DMSO to obtain a sinomenine derivative shown in Formula I, and then further reacted with p-toluenesulfonic acid to obtain a sinomenine derivative shown in Formula II. The sinomenine derivative exhibits excellent anti-rheumatoid arthritis activity, has few side effects, is highly safe, and has good application prospects.

Description

Technical Field

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

[0002] Sinomenine is an isoquinoline alkaloid extracted from the Chinese herbal medicine Sinomenium sinomenium. It has anti-inflammatory, anti-rheumatic, immunosuppressive, analgesic, sedative, and hypotensive pharmacological effects. It can act on the central nervous system and endocrine system, improve immunity and anti-inflammatory mechanisms through bidirectional regulation, eliminate pathogenic factors in the body, and repair tissue damage. Clinically, it is a therapeutic drug for rheumatic and rheumatoid arthritis with definite efficacy. However, during the use of the drug, it was found that Sinomenine has the disadvantages of causing rash, anaphylactic shock, leukopenia, severe gastrointestinal reactions, large required doses, and poor transdermal performance. Therefore, it is of great application significance to find new Sinomenine derivatives with higher efficacy and fewer side effects. 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 and their preparation method and application. The sinomenine derivatives have excellent anti-rheumatoid arthritis activity, small side effects, high safety, and their synthesis method has the advantages of mild reaction conditions, simple operation, high reaction product yield, and suitability for industrial production.

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

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

[0006]

[0007] Wherein, X is Br, Cl, F or I.

[0008] Furthermore, X is Br or Cl, preferably Br.

[0009] Furthermore, the sinomenine derivative shown in Formula I may have any of the following structures:

[0010]

[0011] In a second aspect, the present invention provides a method for preparing a sinomenine derivative represented by formula I, comprising the following steps:

[0012] Step 1, reacting sinomenine with a halogenating agent to obtain intermediate I';

[0013] Step 2: The intermediate I' undergoes an oxidation reaction with oxalyl chloride and DMSO to obtain the product.

[0014]

[0015] Further, the halogenating agent described in step 1 is POCl 3 or POBr 3 .

[0016] Furthermore, in step 1, the molar ratio of sinomenine to the halogenating agent is 1:1-10, preferably 1:4-5.

[0017] Furthermore, in step 1, the reaction solvent is at least one of dimethylformamide, alcohol, aromatic hydrocarbon, nitrile, and ester, preferably at least one of dimethylformamide, ethanol, toluene, xylene, acetonitrile, and ethyl acetate.

[0018] Furthermore, step 1 comprises adding a halogenating agent to a reaction solvent at -5°C to 5°C, then adding sinomenine, heating to 60-80°C for reaction for 4-16h, and post-treating to obtain intermediate I'.

[0019] Furthermore, in step 2, the molar ratio of intermediate I', oxalyl chloride and DMSO is 1:1.0-2.0:1.0-2.0, preferably 1:1.5:1.5.

[0020] Further, in step 2, the reaction solvent is at least one of halogenated hydrocarbons, alcohols, alkanes, aromatic hydrocarbons, nitriles, esters, ethers, and acetone, preferably at least one of dichloromethane, chloroform, methanol, ethanol, n-hexane, cyclohexane, benzene, toluene, xylene, acetonitrile, ethyl acetate, diethyl ether, and acetone.

[0021] Furthermore, step 2 is carried out in the presence of an organic base such as triethylamine or pyridine.

[0022] Furthermore, step 2 comprises adding a solution of DMSO in a reaction solvent dropwise to a solution of oxalyl chloride in a reaction solvent cooled to -78°C, then adding intermediate I', and then adding an organic base such as triethylamine and pyridine, and gradually heating the reaction to room temperature. After the reaction is completed, post-treatment is performed to obtain the product.

[0023] In a third aspect, the present invention provides a sinomenine derivative represented by formula II or a pharmaceutically acceptable salt thereof:

[0024]

[0025] Wherein, X is Br, Cl, F or I.

[0026] Furthermore, X is Br or Cl, preferably Cl.

[0027] Furthermore, the sinomenine derivative shown in formula II may have any of the following structures:

[0028]

[0029] In a fourth aspect, the present invention provides a method for preparing a sinomenine derivative represented by formula II, comprising the following steps: using a compound of formula I as a raw material, reacting with p-toluenesulfonic acid to obtain a product,

[0030]

[0031] Furthermore, the molar ratio of the compound of formula I to p-toluenesulfonic acid is 1:1.0-2.0, preferably 1:1.2.

[0032] Furthermore, the reaction solvent is at least one of halogenated hydrocarbons, alcohols, alkanes, aromatic hydrocarbons, nitriles, esters, ethers, and acetone, preferably at least one of dichloromethane, chloroform, methanol, ethanol, n-hexane, cyclohexane, benzene, toluene, xylene, acetonitrile, ethyl acetate, diethyl ether, and acetone.

[0033] Furthermore, the reaction temperature is 10-40°C, preferably room temperature.

[0034] Furthermore, the reaction time is 4-12 hours, preferably 6 hours.

[0035] In a fifth aspect, the present invention provides a pharmaceutical composition comprising (i) a sinomenine derivative of formula I or a pharmaceutically acceptable salt thereof, or a sinomenine derivative of formula II or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

[0036] In the sixth aspect, the present invention provides the use of the sinomenine derivative shown in formula I or a pharmaceutically acceptable salt thereof, the sinomenine derivative shown in formula II or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of drugs for anti-rheumatoid arthritis.

[0037] Unless otherwise specified, the terms used in this application have the following definitions. Definitions of terms not mentioned below are as commonly understood by those skilled in the art to which the present invention belongs.

[0038] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt prepared from a compound with a relatively nontoxic, pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When the compound contains relatively acidic and relatively basic functional groups, it can be converted into a base addition salt or an acid addition salt.

[0039] As used herein, the term "treat" refers to therapeutic therapy. When referring to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more 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 condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0040] The pharmaceutical composition can be prepared into various types of dosage unit forms for administration according to the therapeutic purpose.

[0041] The compounds of the present invention can be administered clinically using conventional administration methods.

[0042] Unless otherwise specified, the steps and conditions of the present application can refer to conventional operating steps and conditions in the art.

[0043] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0044] Unless otherwise specified, the abbreviations used in this application have the common meanings in the art. For example, POCl 3 Refers to phosphorus oxychloride, POBr 3 refers to phosphorus oxybromide, PTSA refers to p-toluenesulfonic acid, DMF refers to dimethylformamide, and DCM refers to dichloromethane.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The sinomenine derivatives or pharmaceutically acceptable salts thereof of the present invention can effectively inhibit a variety of inflammatory factors, significantly reduce the number of inflammatory cell migration, have the ability to significantly inhibit the proliferation of synovial fibroblasts, can significantly reduce the symptoms of joint swelling, improve the loss of bone density caused by arthritis, have a certain alleviating effect on the destruction of joint bone, have excellent anti-rheumatoid arthritis activity, have few side effects, and are highly safe.

[0047] (2) The synthesis method of the sinomenine derivative of the present invention has the advantages of mild reaction conditions, simple operation, high reaction product yield, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0049] Figure 1 The compound 3 synthesized in Example 2 1 H NMR spectrum;

[0050] Figure 2 The compound 3 synthesized in Example 2 13 C NMR spectrum;

[0051] Figure 3 The SX-27 synthesized in Example 3 1 H NMR spectrum;

[0052] Figure 4 The SX-27 synthesized in Example 3 13 C NMR spectrum;

[0053] Figure 5 The SX-29 synthesized in Example 5 1 H NMR spectrum;

[0054] Figure 6 The SX-29 synthesized in Example 5 13 C NMR spectrum;

[0055] Figure 7 The anti-inflammatory activity of sinomenine derivatives SX-27 / SX-29 was evaluated based on the RAW264.7 cell nitric oxide production inhibition model;

[0056] Figure 8 Show the effects of sinomenine derivatives SX-27 / SX-29 on the expression of different inflammatory factors in RAW264.7 cells;

[0057] Fig. 9Show the effect of sinomenine derivative SX-27 on the cell activity of rat synovial fibroblasts;

[0058] Fig.10 The microscopic observation shows the proliferation inhibitory activity of sinomenine derivative SX-27 on rat synovial fibroblasts;

[0059] Fig.11 Demonstrating the in vivo toxicity of the sinomenine derivative SX-27;

[0060] Fig.12 Demonstrating the in vivo toxicity of the sinomenine derivative SX-29;

[0061] Fig.13 It shows the changes in the toe volume of CIA rats administered SX-27 / SX-29 within 30 days after modeling, as well as the statistical graph of the toe volume of rats in each group on the 30th day of administration (46th day overall);

[0062] Fig.14 The foot of CIA rats was photographed on the 30th day after model establishment and administration of SX-27 / SX-29;

[0063] Fig.15 Shows micro CT images of CIA rats on the 30th day after modeling and administration of SX-27 / SX-29;

[0064] Fig.16 Shows HE staining of visceral tissues of CIA rats after administration of SX-27 / SX-29;

[0065] Fig.17 Shows HE staining of the ankles of CIA rats after administration of SX-27 / SX-29;

[0066] Fig.18 Shows Safranin O-fast green staining of the ankles of CIA rats after administration of SX-27 / SX-29;

[0067] Fig.19 The changes in clinical scores of CIA mice during the administration of SX-27 / SX-29 and the statistical graph of clinical scores of mice in each group on the 36th day of administration (78th day overall);

[0068] Fig. 20 The graph shows the changes in the thickness of the hind paws of CIA mice during the administration of SX-27 / SX-29 and the sum of the thickness of the hind paws of each group of mice on the 36th day of administration (78th day overall);

[0069] Fig.21 Photographs showing the hind paws of CIA mice on day 36 after administration of SX-27 / SX-29;

[0070] Fig. 22 Shows micro CT images of the ankles and knee joints of CIA mice on day 36 after administration of SX-27 / SX-29;

[0071] Fig.23 The gait analysis of CIA mice is shown after administration of SX-27 / SX-29;

[0072] Fig.24 Shows the organ index of CIA mice after administration of SX-27 / SX-29;

[0073] Fig.25 Shows routine blood tests of CIA mice after administration of SX-27 / SX-29;

[0074] Fig.26 Shown is the HE staining of visceral tissues of CIA mice after administration of SX-27 / SX-29. DETAILED DESCRIPTION

[0075] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the contents of the present invention and are only used to explain the present invention, but should not be understood as limiting the present invention.

[0076] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0077] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0078] Example 1: Synthesis of Compound 2

[0079]

[0080] Under argon protection, POCl was added dropwise to dry DMF (200 mL) cooled to 0 °C. 3(14mL, 147.36mmol, 4.0equiv.), stirred for 15 minutes and then added sinomenine (13.4g, 36.84mmol, 1.0equiv.). The temperature was then raised to 75°C, and after 8 hours of reaction, the solution turned dark red-brown. After TLC monitoring, the raw material was consumed, and the solvent was removed by vacuum rotary evaporation to obtain a brown-red residue. Subsequently, a saturated sodium acetate solution (500mL) cooled to 0°C was added for quenching, and a small amount of ammonia water was used to adjust the pH until the solution was alkaline. After three extractions with DCM (350mL), the organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. After the solvent was completely removed by vacuum rotary evaporation, column chromatography was used for separation using a solvent system of DCM / MeOH=20:1 to obtain compound 2 as a brown-yellow solid (7.3g, 18.8mmol, 51%).

[0081] 1 H NMR (400 MHz, CDCl 3 )δ6.64(d,J=8.3Hz,1H),6.56(d,J=8.3Hz,1H),4.69(s,1H),3.78(s,3H),3.50(s,3H),3.22–3.11(m,2H),2.96(d,J=18.1Hz,1H),2 .78(dd,J=18.1,5.2Hz,1H),2.61–2.55(m,1H),2.48–2.41(m,1H),2.39(s,3H),2.33–2.22(m,1H),2.09–2.01(m,1H),1.24(s,1H).

[0082] 13 C NMR (101 MHz, CDCl 3 )δ210.9,149.5,146.6,145.3,126.8,119.8,118.2,110.4,109.4,96.5 ,86.5,69.6,57.0,56.1,55.4,53.9,47.7,42.6,42.4,31.8,29.4,23.9.

[0083] Example 2: Synthesis of Compound 3

[0084]

[0085] Under argon protection, a solution of DMSO (4.7 mL, 66.00 mmol, 1.5 equiv.) in dry DCM (60 mL) was added dropwise to a solution of oxalyl chloride (5.6 mL, 66.00 mmol, 1.5 equiv.) in dry DCM (60 mL) cooled to -78 °C. After stirring for 5 minutes, a solution of compound 2 (16.5 g, 44.00 mmol, 1.0 equiv.) in dry DCM (60 mL) was added. After reacting for 0.5 h, Et 3 N (30.6mL, 220.00mmol, 5.0equiv.), and the reaction was gradually warmed to room temperature. After TLC monitoring, the raw material was consumed, and saturated ammonium chloride solution (500mL) was added for quenching. After three extractions with DCM (200mL), the organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. After the solvent was completely removed by vacuum rotary evaporation, column chromatography was used for separation with a solvent system of DCM / MeOH=40:1 to obtain compound 3 as a light yellow solid (12.6g, 33.9mmol, 77%).

[0086] 1 H NMR (400 MHz, CDCl 3 )δ6.84(d,J=8.4Hz,1H),6.76(d,J=8.4Hz,1H),4.65(d,J=2.3Hz,1H),3.81(s,3H),3.54(s,3H),3.32(m,1H) ,3.19(m,1H),2.99(d,J=17.9Hz,1H),2.75–2.62(m,2H),2.40(s,3H),2.15–1.96(m,2H),1.80–1.74(m,1H). Such as Figure 1 shown.

[0087] 13 C NMR (101 MHz, CDCl 3 )δ161.1,148.6,145.9,137.9,131.0,129.2,126.8,125.9,123.4,111.9,99.4,57.6,56.2,55.6,48.4,42.5,39.7(2C),35.6,22.7. Figure 2 shown.

[0088] Example 3: Synthesis of SX-27

[0089]

[0090] PTSA (1.6 g, 9.63 mmol, 1.2 equiv.) was added to a DCM (80 mL) solution of compound 3 (3.0 g, 8.02 mmol, 1.0 equiv.) and reacted at room temperature for 6 h. After the raw material was consumed by TLC monitoring, saturated sodium bicarbonate solution (500 mL) was added for quenching. After three extractions with DCM (50 mL), the organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. After the solvent was completely removed by vacuum rotary evaporation, column chromatography was used for separation using a solvent system of DCM / MeOH=40:1 to obtain compound SX-27 as a light yellow solid (2.7 g, 7.54 mmol, 94%).

[0091] 1 H NMR (400 MHz, CDCl 3 )δ6.91(d,J=8.5Hz,1H),6.80(d,J=8.5Hz,1H),3.77(s,3H),3.08–3.01(m,2H),2.61–2.48(m,4H),2.35– 2.25(m,1H),2.31(s,3H),2.14(td,J=12.2,3.6Hz,1H),1.85(td,J=12.3,4.7Hz,1H),1.72–1.65(m,1H). Such as Figure 3 shown.

[0092] 13 C NMR (101 MHz, CDCl 3 )δ188.9,159.3,145.9,141.6,138.4,132.1,126.2,124.3,122.9,112.4,56.1,56.1,47.2,42.5,39.5,38.3,37.5,35.1,21.5. Figure 4 shown.

[0093] Example 4: Synthesis of Compound 5

[0094]

[0095] Under argon protection, add CHCl cooled to 0°C 3 POBr (40 mL) was added 3(50.8g, 184.20mmol, 5.0equiv.), stirred for 5 minutes until the solid was completely dissolved. Then, dry DMF (160mL) was slowly added dropwise, and a white solid gradually precipitated out of the solution. After 15 minutes of reaction, sinomenine (13.4g, 36.84mmol, 1.0equiv.) was added, and then the temperature was raised to 75°C. After 8 hours of reaction, the solution turned dark red-brown. After TLC monitoring, the raw material was consumed, and the solvent was removed by vacuum rotary evaporation to obtain a brown-red residue. Then, a saturated sodium acetate solution (500mL) cooled to 0°C was added for quenching, and the pH was adjusted to alkaline with a small amount of ammonia water. After three extractions with DCM (350mL), the organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. After the solvent was removed by rotary evaporation under reduced pressure, the product was separated by column chromatography using a solvent system of DCM / MeOH=20:1 to obtain compound 5 as a brown-yellow solid (8.4 g, 19.89 mmol, 54%).

[0096] 1 H NMR (400 MHz, CDCl 3 )δ6.86(d,J=8.3Hz,1H),6.79(d,J=8.3Hz,1H),4.73–4.61(m,1H),3.80(s,3H),3.50(s,3H),3.25–3.16(m ,2H),2.97(d,J=18.3Hz,1H),2.86–2.78(m,1H),2.65–2.59(m,1H),2.48–2.30(m,6H),2.13–2.03(m,1H).

[0097] 13 C NMR (101 MHz, CDCl 3 )δ149.6,145.3,118.3,109.4,58.2,57.2,56.1,55.5,55.4,47.7,42.5,40.2,33.2,29.8,24.0.

[0098] Example 5: Synthesis of SX-29

[0099]

[0100] Under argon protection, a solution of DMSO (6.5 mL, 90.90 mmol, 1.5 equiv.) in dry DCM (40 mL) was added dropwise to a solution of oxalyl chloride (7.7 mL, 90.90 mmol, 3.0 equiv.) in dry DCM (40 mL) cooled to -78 °C. After stirring for 5 minutes, a solution of compound 5 (12.7 g, 30.30 mmol, 1.0 equiv.) in dry DCM (60 mL) was added. After reacting for 0.5 h, Et 3 N (21.1mL, 151.50mmol, 5.0equiv.), and the reaction was gradually warmed to room temperature. After TLC monitoring of the consumption of the raw materials, saturated ammonium chloride solution (200mL) was added for quenching. After three extractions with DCM (200mL), the organic phases were combined, washed with saturated brine, and dried with anhydrous sodium sulfate. After the solvent was completely removed by vacuum rotary evaporation, column chromatography was used for separation with a solvent system of DCM / MeOH=40:1 to obtain compound SX-29 as a light yellow solid (9.8g, 23.3mmol, 77%).

[0101] 1 H NMR (400 MHz, CDCl 3 )δ6.84(d,J=8.5Hz,1H),6.76(d,J=8.5Hz,1H),4.62(dd,J=17.7,2.4Hz,1H),3.81(s,3H),3.55–3.53(m,3H),3.33–3.2 9(m,1H),3.20–3.17(m,1H),3.02–2.95(m,1H),2.74–2.62(m,2H),2.40(s,3H),2.14–1.96(m,2H),1.80–1.74(m,1H). Such as Figure 5 shown.

[0102] 13 C NMR (101 MHz, CDCl 3 )δ162.0,161.2,148.8,148.7,146.0,138.0,137.9,132.9,131.1,129.4,127.0,127.0,126.1,123.5,122.2,112.0,99.6,99.1,57.8,57.7,56.3,55.8,55.7,48.5,48.5,42.6,41.2,40.8,39.9,39.8,35.7,35.7,22.8. Figure 6 shown.

[0103] Biological test example 1: in vitro cell activity and in vivo acute toxicity testing

[0104] The pathological manifestations of rheumatoid arthritis (RA) are mainly inflammation of the synovial membrane, proliferation of fibroblasts in the synovial membrane, and formation of new pannus, which eventually lead to erosion and destruction of bones and cartilage. In this process, the immune inflammatory microenvironment in the joint cavity can aggravate the proliferation of the synovial membrane by promoting the proliferation, migration and invasion of synovial fibroblasts, thereby recruiting a large number of inflammatory macrophages to infiltrate and release a large number of cytokines, leading to the continuous aggravation of RA. Therefore, the activity is mainly evaluated from the two aspects of anti-inflammatory and inhibition of synovial fibroblast proliferation.

[0105] Regarding anti-inflammatory activity, the potential anti-inflammatory effects of sinomenine derivatives SX-27 and SX-29 were evaluated using the lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibition model in macrophages (RAW264.7). The nitric oxide release was evaluated as follows: RAW264.7 cells in the logarithmic growth phase were cultured at 1.5×10 5 The cells were inoculated in a 48-well plate at a density of 100 μg / ml and cultured overnight until the cells reached the logarithmic growth phase. A blank control group, an inflammation model group, and three dose concentration groups of 2.5, 5, and 10 μM were set up: the blank group was replaced with fresh culture medium, and the other groups were first induced with LPS (1 μg / ml) to induce inflammation. The drug-treated group was treated with drugs of corresponding concentrations at the same time. After culturing for 24 hours, the supernatant was taken and operated according to the instructions of the NO detection kit (brand: Nanjing Jiancheng, item number A013-1-1), as follows:

[0106] (1) Set up blank tubes, standard tubes, and test tubes for each dosing group. After 24 hours of RAW264.7 cell dosing treatment, aspirate 300 μL of supernatant from each well as the test sample. In addition, aspirate an equal volume of distilled water as a blank tube and 300 μL of sodium nitrite solution (20 μM) as a standard tube.

[0107] (2) Add 200 μL of reagent 1 and 100 μL of reagent 2 in the kit to each of the above tubes, mix well, let stand for 10 minutes, centrifuge at 3500 r / min for 10 minutes, and aspirate 160 μL of the clarified supernatant into a new centrifuge tube.

[0108] (3) Prepare the color developer in the ratio of 2.5:1:1 using liquid 3:4:5. Add 80 μL of the color developer to the supernatant of each sample, mix well, and let stand for 15 minutes.

[0109] (4) Detect the OD value of each well at a wavelength of 570 nm using an ELISA reader. Calculate the NO release amount of each group and the NO inhibition rate of each drug-treated group in proportion based on the OD value of the standard tube solution (the formula is as follows). Detect the NO concentration at a wavelength of 570 nm using an ELISA reader.

[0110]

[0111] 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 8 statistical software. P < 0.05 was considered statistically significant. Figure 7 The results of the evaluation of the anti-inflammatory activity of sinomenine derivatives are shown. Compared with the control group: ### P<0.001; compared with the model group: *** P<0.001. The results showed that SX-27 and SX-29 had significant inhibitory effects on nitric oxide production at concentrations of 2.5, 5 and 10 μM.

[0112] In addition, the effects of SX-27 on the expression of inflammation-related proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), as well as the effects of SX-29 on the expression of inflammation-related proteins TNF-α and COX-2, were detected by Western Blot experiments. The Western blot experimental method was as follows: cells from each group were collected and lysed with RIPA lysis buffer (containing protease inhibitors) to obtain total protein. Then, 6%-15% SDS-PAGE was used to separate the protein, and the protein was transferred to a PVDF membrane by wet method, blocked with 5% skim milk powder for 20 minutes, and the primary antibody (1:1000-3000) was added, incubated at room temperature for 2 hours, washed thoroughly, and the secondary antibody (1:5000) was added, incubated at room temperature for 2 hours, washed thoroughly, and ECL reagent was added for color development, and photographed and recorded on the Tanon gel imaging system. Figure 8 The effects of sinomenine derivatives on different inflammatory factors were shown. The results showed that SX-27 / SX-29 had an inhibitory effect on multiple inflammatory factors at concentrations of 2.5, 5, and 10 μM.

[0113] Since the proliferation of synovial tissue is also one of the main symptoms of rheumatoid arthritis, the rat model of rheumatoid arthritis was established by inducing type II collagen, and the primary synovial fibroblasts were used for activity screening. MTT experiment and EdU experiment were carried out on the sinomenine derivative SX-27 to detect the activity of the compound in inhibiting the proliferation of synovial fibroblasts. The specific operation is as follows:

[0114] MTT assay: Synovial fibroblasts were seeded in 48-well plates at a seeding density of 2x10 4 The wells were placed in an incubator (37°C, 5% CO 2and 95% humidity) for 8 hours, a control group and a drug-treated group were randomly set up, with 4 replicates. The control group was not treated, and the drug-treated group was treated with SX-27 at concentrations of 2.5, 5, and 10 μM. After 24 hours of SX-27 treatment, the original culture medium in the well plate was aspirated, and DMEM high-glucose medium containing 50 μg / mL MTT was added, and incubated in the dark for 4 hours. Subsequently, the culture medium in the 48-well plate was aspirated, and 300 μL DMSO was added to dissolve the blue-purple formazan crystals. The absorbance of the sample was detected at a wavelength of 570 nm using an enzyme reader, and the experimental results were presented as mean ± standard deviation. The results showed that SX-27 had a certain inhibitory effect on the viability of rat synovial fibroblasts. For details, see Fig. 9 . Fig. 9 The results show that the sinomenine derivative SX-27 has an inhibitory activity on the cell viability of rat synovial fibroblasts. Compared with the control group: *** P<0.001.

[0115] EdU Experiments Follow BeyoClick TM EdU-488 Cell Proliferation Detection Kit Instructions:

[0116] Synovial fibroblasts were seeded in 6-well plates. After the cells grew for 8 hours, SX-27 was administered for 24 hours. EdU solution (10mM) was diluted with cell culture medium at a ratio of 1:500 to obtain 2× EdU working solution (20μM). Then, an equal volume of EdU working solution was added to the 6-well plate with cells to make the final concentration of EdU 1×, and the cells were continued to be incubated for labeling. After 12 hours, EdU labeling of cells was completed, the culture medium was discarded, and 1mL of 4% paraformaldehyde solution was added to each well and fixed at room temperature for 15 minutes. After that, the paraformaldehyde solution was removed, and the cells were washed with 500μL PBS per well for a total of 3 times, each time for 3-5 minutes. 1mL of PBS containing 0.3% Triton X-100 was added to each well and incubated at room temperature for 10-15 minutes, and then washed with PBS 1-2 times. 400μL of Click reaction solution (430μL Click Reaction Buffer, 20μL CuSO 4, 1μL Alexa Fluor 488 and 50μL ClickAdditive Solution), washed 3 times with PBS, and reacted at room temperature in the dark for 30 minutes. After discarding the Click reaction solution, wash again with PBS 3 times, add Hoechst 33342 diluted 1:1000 to each well and incubate at room temperature in the dark for 10 minutes, wash 3 times with PBS, use IX73 fluorescence microscope (Olympus) to observe the generation of cell fluorescence after administration at the wavelength corresponding to the dye, and take pictures and record (Hoechst33342 dye has a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460nm; Alexa Fluor 488 dye has a maximum excitation wavelength of 495nm and a maximum emission wavelength of 519nm). The results showed that after SX-27 administration, the number of proliferating rat synovial fibroblasts labeled with EdU decreased, indicating that SX-27 has a certain inhibitory effect on the proliferation of rat synovial fibroblasts. For details, see Fig.10 . Fig.10 The figure shows the proliferation inhibitory activity of sinomenine derivative SX-27 on rat synovial fibroblasts. EdU staining detection, scale bar = 100 μm.

[0117] In addition to the in vitro activity evaluation, the inventors also evaluated the safety and efficacy of the above-mentioned sinomenine derivatives at the in vivo animal level, and used ICR mice to conduct acute toxicity experiments on compounds SX-27 and SX-29 to explore the safe dosage for administration. For compound SX-27, a control group and a dosing group (doses set at 200, 276, 381, 525, 724, 1000 mg / kg) were set up, with 10 ICR mice in each group, half male and half female, and a 0.5% CMC-Na solution was used to prepare the solution and gavage each group of mice. The control group was given an equal volume of 0.5% CMC-Na solution, and the death and living conditions of the mice were observed for 7 days after one administration; for compound SX-29, a control group and a dosing group (doses set at 500, 1000, 2000, 4000, 5000 mg / kg) were set up, with 4 ICR mice in each group, half male and half female, and a 0.5% CMC-Na solution was used to prepare the solution and gavage each group of mice. The control group was given an equal volume of 0.5% CMC-Na solution, and the death and living conditions of the mice were observed for 7 days after one administration. After the experiment, the LD of compound SX-27 was calculated 50 The value is 428.8mg / kg (such as Fig.11 ), while compound SX-29 showed better safety, and the mortality rate of mice did not reach 100% at a dose of 5000 mg / kg (as shown in Fig.12 shown).

[0118] Biological Test Example 2: Evaluation of the efficacy of anti-rheumatoid arthritis drugs

[0119] 1) In vivo efficacy evaluation of SD rat CIA model of rheumatoid arthritis

[0120] For the efficacy experiment of SX-27 and SX-29 in rats with rheumatoid arthritis, 7-8 week old SD male rats were selected, 5 in each group, and equal volumes of Freund's complete adjuvant and bovine type II collagen solution were mixed and ground until the color became uniform milky white (no obvious oil droplets) to form injection emulsion I (modeling started on Day 1), and 100 μL of emulsion I was subcutaneously injected at the base of the tail of each rat; 7 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 base of the tail of each rat. After 7 days, the rats were evenly divided into groups according to the clinical scores, and the scores of each group were kept equal. The drug was administered on the 16th day after the first immunization.

[0121] The dosing settings were divided into groups as follows: normal group: 0.05% CMCNa / d; model group: 0.05% CMCNa / d; SX-27 low-dose group (10 mg / kg / d); SX-27 high-dose group (50 mg / kg / d); SX-29 low-dose group (10 mg / kg / d); SX-29 high-dose group (50 mg / kg / d); methotrexate group (1 mg / kg / 2d). The drugs were administered for a total of 30 days, and body weight and toe volume were measured every 5 days.

[0122] Fig.13 The changes in the toe volume of CIA rats within 30 days after drug administration after modeling and the statistical chart of the toe volume of rats in each group on the 46th day are shown. Compared with the model group, *P<0.05, **P<0.01.

[0123] Fig.14 The foot of CIA rats was photographed on the 30th day after modeling. After statistical analysis of the toe volume of CIA rats in each group, it was found that the toe volume of SX-27 (10 mg / kg), SX-29 (10 mg / kg) and methotrexate groups was reduced compared with the model group rats, which had a certain degree of efficacy in relieving arthritis foot swelling.

[0124] Fig.15 The micro CT images of CIA rats at day 30 after modeling are shown. Micro CT scans of the ankles of CIA rats at day 30 of drug administration showed that bone destruction in the ankle joints of rats was reduced after administration of SX-27 and SX-29, indicating that the above two derivatives have a certain alleviating effect on joint destruction.

[0125] After the administration, the legs of the experimental rats were fixed and used for HE staining to detect the tissue morphological changes in the joints; the liver, kidneys and lungs of each group of rats were fixed for HE staining to observe the tissue morphological changes and evaluate the safety of the drug.

[0126] Visceral tissue sections: Fig.16 The HE staining of the visceral tissues of CIA rats after the administration of the drug is shown, and from top to bottom are the HE staining of liver, lung, and kidney tissue sections, and the scale bar = 100 μm. Through HE staining, it was found that the liver, lung, and kidney cell morphology and arrangement of CIA rats after administration of SX-27 and SX-29 were normal, the alveolar expansion was normal, the kidney spherical cavity and renal tubules were arranged normally, and no drug-induced damage to the viscera, lungs, and kidneys was found. This shows that the sinomenine derivatives of the present invention have few side effects and high safety.

[0127] Fig.17 HE staining of the ankle of CIA rats after drug administration. Scale bar = 200 μm. HE staining analysis of rat ankle tissue sections is as follows: In the blank group, the rat ankle joint tissue structure remained normal, the synovial fibroblasts were arranged neatly, there was no abnormal proliferation of the synovium, and no inflammatory cell infiltration was observed in the joint cavity; the synovial tissue of the rats in the model group had synovial hyperplasia, the synovial cell arrangement was disordered, and there was a large number of lymphocytes and monocytes infiltration, indicating that an inflammatory response occurred. However, in each drug administration group, the degree of inflammatory cell infiltration in the synovium of the mouse joint cavity was reduced, and the arrangement of synovial fibroblasts tended to be normal. Fig.18 The Safranin O-fast green staining of the ankle of CIA rats after the end of drug administration is shown. Scale bar = 200 μm. The results of tissue Safranin O-fast green staining showed that the destruction of synovial cartilage and the proliferation of synovial fibroblasts in the joints of rats in the SX-27 administration group were significantly improved compared with those in the model group. The subchondral bone structure was clear and the calcified cartilage layer structure was visible.

[0128] 2) In vivo efficacy evaluation in the DBA / 1J mouse CIA model of rheumatoid arthritis

[0129] A pharmacological efficacy experiment on SX-27 and SX-29 in mice with rheumatoid arthritis was conducted. DBA / 1J male mice aged 6-8 weeks were selected, with 7 mice in each group. Equal volumes of Freund's complete adjuvant and bovine type II collagen solution were mixed and ground until the color became uniform milky white (without obvious oil droplets) to form injection emulsion I. 100 μL of emulsion I was subcutaneously injected at the base of the tail of each mouse. After 21 days, the protein solution and incomplete Freund's adjuvant were mixed and ground in a 1:1 ratio to form injection emulsion II. 100 μL of emulsion II was subcutaneously injected at the base of the tail of each mouse. 14 days after the second immunization, the mice were evenly divided into groups according to the scores and drug administration was started (carried out on the 36th day after the first immunization). The drug administration settings were as follows: normal group: 0.05% CMCNa / d; model group: 0.05% CMCNa / d; SX-27 low-dose group (10 mg / kg / d); SX-27 high-dose group (50 mg / kg / d); SX-29 low-dose group (10 mg / kg / d); SX-29 high-dose group (50 mg / kg / d); sinomenine group (50 mg / kg / d); methotrexate group (2 mg / kg / 3d). Body weight and toe volume were measured every 3 days, and clinical scores were performed to evaluate paw swelling. The scoring criteria are as follows.

[0130] Table 1 Clinical scoring criteria for arthritis

[0131] Score Disease 0 normal 1 Mild redness and swelling of the ankle or wrist 2 Moderate redness or swelling in the ankle or wrist 3 Severe redness and swelling of the paws, including the tips of the fingers 4 Maximum inflammation in the extremities, including multiple joints

[0132] Body weight and paw thickness were monitored every 3 days, and clinical scores were performed to evaluate paw swelling. The paw thickness and scores of mice were monitored.

[0133] Fig.19 The graph shows the changes in clinical scores of CIA mice during the drug administration process and the statistical graph of clinical scores of mice in each group on the 36th day of drug administration (78th day overall). Fig. 20 The graph shows the changes in the thickness of the hind paws of CIA mice during the drug administration process and the sum of the thickness of the hind paws of each group of mice on the 36th day of drug administration (the 78th day overall). Fig.21 The photo shows the hind paw of CIA mice on day 36 of drug administration. The monitoring results show that both the high- and low-dose groups of SX-27 and SX-29 can effectively reduce the clinical score of arthritis and the thickness of the paw of CIA mice, and alleviate the symptoms of joint swelling in mice.

[0134] Fig. 22 The micro CT images of CIA mice 36 days after modeling are shown. Micro CT scans of the ankles and knee joints of the front and back feet of CIA mice after drug administration show that the bone destruction in the joints is reduced after administration of SX-27 and SX-29, and the bone density loss caused by arthritis is improved, that is, the above two derivatives have a certain effect on alleviating the destruction of joint bones.

[0135] In addition, the behavior of mice after administration was evaluated using a gait analyzer. Fig.23 The gait analysis diagram of CIA mice with rheumatoid arthritis after drug administration is shown. The results show that the high and low dose groups of SX-27 can effectively reduce the average footprint area of ​​CIA mice, prolong the time of hind limb contact with the ground, and improve the gait pattern and behavioral coordination of mice.

[0136] Fig.24 Shows the organ index of CIA mice after drug administration. Compared with the normal group: ## P<0.005; compared with the model group: * P<0.05, ** P<0.01, NS, no statistical difference. The body weight and organ index of CIA experimental mice were counted, and it was found that the SX-27 high-dose group, SX-29 low-dose group, and high-dose group had no significant effect on the weight coefficients of the heart, liver, lung, and kidney. Compared with the model group, the adjustable spleen index was reduced, indicating that the above compounds may have an immunomodulatory effect.

[0137] Fig.25 The blood routine test of CIA mice after drug administration is shown. The figure shows the statistics of red blood cell count, white blood cell count, hemoglobin concentration, neutrophil count, lymphocyte count, eosinophil count and monocyte count. Compared with the normal group: ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01. After routine blood tests on DBA arthritis mice, the results of blood cell analysis showed that compared with the model group, the number of white blood cells and monocytes in the SX-27 and SX-29 groups was relatively reduced, tending to normal; there was no significant effect on lymphocytes.

[0138] Fig.26 HE staining of visceral tissues of CIA rats after drug administration. From top to bottom, HE staining of lung, liver, and kidney tissue sections, scale bar = 200μm. Visceral tissue sections: HE staining showed that the morphology and arrangement of liver, lung, and kidney cells of CIA mice after administration of SX-27 and SX-29 were normal, alveolar expansion was normal, and the arrangement of kidney spherical cavity and renal tubules was normal. No damage to the viscera, lungs, and kidneys caused by drugs was found.

[0139] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. Those skilled in the art may make various changes and modifications 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A sinomenine derivative or a pharmaceutically acceptable salt thereof represented by formula I: in, X is Br, Cl, F or I.

2. The sinomenine derivative represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Wherein X is Br or Cl.

3. The sinomenine derivative represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Wherein X is Br.

4. The method for preparing the sinomenine derivative represented by formula I according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, reacting sinomenine with a halogenating agent to obtain intermediate I'; Step 2: The intermediate I' undergoes an oxidation reaction with oxalyl chloride and DMSO to obtain the product.

5. The preparation method according to claim 4, characterized in that: Wherein the halogenating agent in step 1 is POCl3 or POBr3; and / or In step 1, the molar ratio of sinomenine to the halogenating agent is 1:1-10; and / or In step 2, the molar ratio of intermediate I', oxalyl chloride and DMSO is 1:1.0-2.0:1.0-2.

0.

6. The preparation method according to claim 5, characterized in that: In step 1, the molar ratio of sinomenine to the halogenating agent is 1:4-5; in step 2, the molar ratio of intermediate I', oxalyl chloride and DMSO is 1:1.5:1.

5.

7. A sinomenine derivative or a pharmaceutically acceptable salt thereof represented by formula II: in, X is Br, Cl, F or I.

8. The sinomenine derivative represented by formula II or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: Wherein X is Br or Cl.

9. The sinomenine derivative represented by formula II or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that: Wherein X is Cl.

10. The method for preparing the sinomenine derivative represented by formula II according to any one of claims 7 to 9, characterized in that: The following steps are involved: The compound shown in formula I is used as a raw material and reacted with p-toluenesulfonic acid to obtain a product.

11. The preparation method according to claim 10, characterized in that: wherein the molar ratio of the compound of formula I to p-toluenesulfonic acid is 1:1.0-2.0; and / or The reaction temperature is 10-40°C; and / or The reaction time is 4-12h.

12. The preparation method according to claim 11, characterized in that: The molar ratio of the compound of formula I to p-toluenesulfonic acid is 1:1.2; the reaction temperature is room temperature; and the reaction time is 6 hours.

13. A pharmaceutical composition, characterized in that It comprises (i) a sinomenine derivative represented by formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a sinomenine derivative represented by formula II according to any one of claims 7 to 9 or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

14. Use of the sinomenine derivative of formula I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3, or the sinomenine derivative of formula II or a pharmaceutically acceptable salt thereof as described in any one of claims 7 to 9, or the pharmaceutical composition as described in claim 13 in the preparation of a drug for treating rheumatoid arthritis.

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