Idebenone-aspirin splicing compound as well as preparation method and application thereof

Abstract

The invention discloses an idebenone-aspirin splicing compound as well as a preparation method and application of the idebenone-aspirin splicing compound. The idebenone-aspirin splicing compound IDE-4 provided by the invention has good proliferation inhibition activity on rheumatoid arthritis effector cell synovial fibroblasts, and the rheumatoid arthritis effector cell synovial fibroblasts can be used for inhibiting the proliferation of the rheumatoid arthritis effector cell synovial fibroblasts. Pharmacological experiment research shows that the splicing compound IDE-4 can obviously inhibit proliferation of human primary rheumatoid arthritis synovial fibroblasts and human immortalized rheumatoid arthritis synovial fibroblast lines MH7A and migration of human synovial sarcoma cells SW982; the inhibition effect is obviously superior to that of a positive control drug methotrexate and parent nucleus idebenone and aspirin thereof, the good development value is achieved, and a new thought and approach are provided for synthesis of aspirin derivatives and research on rheumatoid arthritis resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an idebenone-aspirin splice and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease with joint inflammation and pain as the main manifestations. It can affect the normal function of joints and lead to joint deformities in severe cases. The main pathological change of RA is synovitis, which can invade the joint bones and cartilage, causing destruction of joint bones, cartilage and joint capsules, leading to joint deformities and loss of function, seriously endangering human health and placing a heavy burden on patients' families and society. At present, the pathogenesis of RA is still unclear. Studies have shown that in the synovial tissue of RA, synovial fibroblasts (Fibroblast-like synoviocytes, FLSs) are the main effector cells of synovial hyperplasia, with abnormal proliferation, migration, invasion and other biological characteristics of tumor cells. FLSs play a key role in the formation of pannus by invading bone and cartilage, play an important role in the occurrence and development of RA, and are considered to be the target of RA treatment. Western medicine is a commonly used treatment for RA. Although it can relieve clinical symptoms to a certain extent, it has problems such as low cure rate, long time consumption, repeated illness, unsatisfactory overall efficacy, and large toxic side effects.

[0003] Idebenone has the effects of activating mitochondrial respiratory activity, increasing myocardial cell ATP production, reducing oxidative stress, and inhibiting inflammatory responses. It is one of the most effective antioxidants in clinical use and is used to treat patients with brain function damage and mental and behavioral disorders related to cerebrovascular disease, and has low toxic side effects. At present, there are few reports on the anti-RA research of idebenone and its derivatives. Aspirin is a non-steroidal drug with a long history and widely used antipyretic analgesic. It has anti-inflammatory, analgesic and antipyretic effects, which can relieve RA symptoms, but cannot cure inflammation. At present, there are no literature reports on idebenone-aspirin splices. Summary of the invention

[0004] The purpose of the present invention is to provide an idebenone-aspirin conjugate and its preparation method and application in view of the deficiencies in the prior art. The conjugate exhibits good proliferation inhibition activity on RA effector cells FLSs, which is significantly stronger than the clinical first-line drug methotrexate, has good development value, and also provides new ideas and approaches for the synthesis of aspirin derivatives and anti-RA research, and the preparation process is simple.

[0005] The technical solution of the present invention is as follows:

[0006] 1. An idebenone-aspirin conjugate, characterized in that its structural formula is as follows:

[0007]

[0008] 2. A method for preparing the idebenone-aspirin conjugate as described above, comprising the following steps:

[0009] (1) Take N,N'-dicyclohexylcarbodiimide, add acetonitrile and stir to dissolve, and set aside;

[0010] (2) Take a clean flask, add a magnet, evacuate the flask and replace the gas with argon three times, then add aspirin and acetonitrile in sequence, and stir at room temperature to dissolve;

[0011] (3) adding idebenone and 4-dimethylaminopyridine to the solution of step (2) in sequence, and stirring in an ice-water bath to dissolve;

[0012] (4) adding the solution obtained in step (1) to step (3), removing the ice water bath, and stirring the reaction at room temperature;

[0013] (5) Take the reaction solution obtained in step (4), add water, then add ethyl acetate for extraction, combine the organic phases, dry with Na2SO4, filter, and concentrate the filtrate to dryness;

[0014] (6) The concentrate obtained in step (5) is applied to a silica gel column for elution, the eluate is collected, and evaporated to dryness to obtain a yellow solid, which is the idebenone-aspirin conjugate.

[0015] Furthermore, in the above-mentioned method for preparing the idebenone-aspirin conjugate, the amount of N,N'-dicyclohexylcarbodiimide used in step (1) is 91 mg, and the amount of acetonitrile added is 1 mL.

[0016] Furthermore, in the method for preparing the idebenone-aspirin conjugate described above, the amount of aspirin added in step (2) is 53 mg, and the amount of acetonitrile added is 2 mL.

[0017] Furthermore, in the method for preparing the idebenone-aspirin conjugate described above, the amount of idebenone added in step (3) is 100 mg, and the amount of 4-dimethylaminopyridine added is 4 mg.

[0018] Furthermore, in the above-mentioned method for preparing the idebenone-aspirin conjugate, the stirring reaction time in step (4) is 2.5 hours.

[0019] Furthermore, in the above-mentioned method for preparing the idebenone-aspirin conjugate, the amount of water added in step (5) is 10 mL, and the extraction with ethyl acetate is performed by adding ethyl acetate for extraction three times, each time with 10 mL.

[0020] Furthermore, in the method for preparing the idebenone-aspirin conjugate described above, the eluent in step (6) is a solution with a volume ratio of petroleum ether:ethyl acetate = 4:1.

[0021] The application of the idebenone-aspirin conjugate as described above is in the preparation of a drug for treating rheumatoid arthritis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides a class of novel structural compounds, namely, idebenone-aspirin conjugate IDE-4. Pharmacological experimental studies have shown that the compound IDE-4 can significantly inhibit the proliferation of human primary RA-FLSs, the proliferation of human immortalized RA-FLSs line MH7A, and the migration of human synovial sarcoma cells SW982, and the inhibitory activity is significantly stronger than that of the clinical first-line drug methotrexate and its parent nucleus idebenone and aspirin. It has great development value and also provides new ideas and approaches for the synthesis of aspirin derivatives and anti-RA research.

[0024] 2. The present invention uses aspirin and idebenone, currently commonly used drugs in clinical practice, as the parent core. These two drugs have low toxicity and side effects and good safety, which effectively ensures the safety of the product of the present invention.

[0025] 3. The present invention modifies aspirin, a commonly used drug for clinical analgesia, and the product has good safety and drugability. It also provides more novel modification methods for the chemical modification of compounds containing carboxyl functional groups such as aspirin, as well as new ideas and approaches for the structural modification research and activity research of such drugs.

[0026] 4. The preparation method of the present invention is simple, has no special requirements on production equipment, is easy to operate, has low cost, and is easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the experimental results of the effect of IDE-4 on the migration of human synovial sarcoma cells SW982, where A is the control group; B is the methotrexate group; C is the idebenone group; D is the aspirin group; and E is the IDE-4 group. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope and application scope of the present invention are not limited:

[0029] 1. Preparation of an idebenone-aspirin conjugate

[0030] Example 1

[0031] A preparation method of an idebenone-aspirin conjugate comprises the following steps:

[0032]

[0033] (1) Take 91 mg of N,N'-dicyclohexylcarbodiimide, add 1 mL of acetonitrile and stir to dissolve, and set aside;

[0034] (2) Take a clean flask, add a magnetic rod, evacuate the flask and replace the gas with argon three times, then add 53 mg of aspirin and 2 mL of acetonitrile, and stir at room temperature to dissolve;

[0035] (3) Add 100 mg of idebenone and 4 mg of 4-dimethylaminopyridine to the solution of step (2) in sequence, and stir in an ice-water bath to dissolve;

[0036] (4) adding the solution obtained in step (1) to step (3), removing the ice water bath, and stirring the reaction at room temperature for 2.5 hours;

[0037] (5) Take the reaction solution obtained in step (4), add 10 mL of water, and then add ethyl acetate to extract 3 times, 10 mL each time, combine the organic phases, dry with Na2SO4, filter, and concentrate the filtrate to dryness;

[0038] (6) The concentrate obtained in step (5) was loaded onto a silica gel column and eluted with a solution having a volume ratio of petroleum ether:ethyl acetate = 4:1. The eluate was collected and evaporated to dryness to obtain a yellow solid idebenone-aspirin conjugate IDE-4 (101 mg, yield 68%). f =0.5(petroleum ether / EtOAc,4:1).

[0039] The structure confirmation results of the prepared compound IDE-4 are as follows:

[0040] 1H NMR (400MHz, CDCl3) δ8.00 (dd, J=7.8, 1.7Hz, 1H), 7.57-7.49 (m, 1H), 7.29 (td, J=7 .6,1.2Hz,1H),7.08(dd,J=8.1,1.2Hz,1H),4.24(t,J=6.8Hz,2H),3.97(d,J=1.2Hz ,6H),2.42(t,J=7.4Hz,2H),2.33(s,3H),1.99(s,3H),1.75-1.67(m,2H),1.41-1. 37(m,2H),1.34(d,J=8.6Hz,2H),1.31(s,2H),1.28(s,2H),1.25(d,J=13.7Hz,6H); 13 C NMR (100MHz, CDCl3) δ184.64,184.08,169.62,164.47,150.57,144.19,142.98,138.59,133.67,131.66,125.93,123.71,123 .40,65.22,61.07,29.73,29.38,29.33,29.25,29.15,28.64,28.54,26.30,25.85,20.97,11.84.MS(ESI)m / z=523.1([M+Na] + ).HPLC purity(99.13%).

[0041] Comparative Example 1: Screening of different solvents

[0042] This comparative example investigates the preparation method of the idebenone-aspirin conjugate, using the following solvent to replace the acetonitrile in Example 1, and the dosage and reaction conditions are consistent with those in Example 1. The results are as follows:

[0043] 1,4-Dioxane: yield 40%;

[0044] Tetrahydrofuran (THF): yield 35%;

[0045] Dichloromethane (DCM): yield 30% (idebenone reaction was not complete);

[0046] Toluene: yield 40% (idebenone was not completely reacted).

[0047] When acetonitrile was used as the solvent in Example 1, the yield was 68%, which shows that acetonitrile is better to be used as the solvent.

[0048] Comparative Example 2: Investigation of the dosage of aspirin and N,N'-dicyclohexylcarbodiimide

[0049] This comparative example investigates the preparation method of the idebenone-aspirin conjugate based on comparative example 1. In view of the fact that the idebenone reaction is not complete when dichloromethane and toluene are used as solvents in comparative example 1, an attempt is made to adjust the amount of aspirin and N,N'-dicyclohexylcarbodiimide when toluene is used as solvent to improve the reaction and see whether the idebenone reaction is complete. Results:

[0050] Toluene solvent, aspirin 80 mg, other ingredients unchanged, yield 46% (idebenone reaction was not complete);

[0051] Toluene solvent, 155 mg of N,N'-dicyclohexylcarbodiimide, other ingredients unchanged, yield 44% (idebenone reaction was not complete).

[0052] It can be seen that when toluene is used as the solvent, the reaction of idebenone cannot be completed by changing the equivalents of aspirin and N,N'-dicyclohexylcarbodiimide, and the yield cannot be improved.

[0053] 2. Pharmacology and efficacy tests

[0054] Example 2: Effect of IDE-4 on the proliferation of primary human RA-FLSs

[0055] 1. Experimental Materials

[0056] 1.1 Cell line: primary human RA-FLSs

[0057] 1.2 Main reagents and instruments: fetal bovine serum (BI, Israel), DMEM medium (Gibco, USA), penicillin-streptomycin (Gibco, USA), dimethyl sulfoxide (Sigma), CCK-8 kit (Jiangsu Addison Biotechnology Co., Ltd.), trypsin (Gibco), Synergy TM H1 full-function microplate reader (BioTek, USA), low-speed centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd.), constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), clean bench (Singapore Yisi High-tech Co., Ltd.), cell culture incubator (Singapore Yisi High-tech Co., Ltd.).

[0058] 2. Experimental Methods

[0059] 2.1 Cultivate primary RA-FLSs. Digest the cells with 0.25% trypsin, collect the cells and transfer them to a centrifuge tube and centrifuge them at 1200 rpm for 5 minutes. Resuspend the cells in cell culture medium (10% fetal bovine serum + DMEM basal medium + 1% penicillin-streptomycin), inoculate the cells at a density of 4000 cells / well in a 96-well cell culture plate, and then culture them in a cell culture incubator at 37°C, 5% CO2 and saturated humidity.

[0060] 2.2 Set up 3 replicate wells in each group, add drugs to each well according to the following conditions, and then continue to culture for 48 hours.

[0061] Control group: the group without medication.

[0062] Positive control group: The methotrexate group, idebenone group and aspirin group were divided into 3 groups each, for a total of 9 groups:

[0063] Positive control group 1: methotrexate was added at a final concentration of 10 μM.

[0064] Positive control group 2: methotrexate was added at a final concentration of 20 μM.

[0065] Positive control group 3: methotrexate was added at a final concentration of 40 μM.

[0066] Positive control group 4: Idebenone was added at a final concentration of 10 μM.

[0067] Positive control group 5: Idebenone was added at a final concentration of 20 μM.

[0068] Positive control group 6: Idebenone was added at a final concentration of 40 μM.

[0069] Positive control group 7: Aspirin was added at a final concentration of 10 μM.

[0070] Positive control group 8: Aspirin was added at a final concentration of 20 μM.

[0071] Positive control group 9: Aspirin was added at a final concentration of 40 μM.

[0072] IDE-4 Group: Divide the IDE-4 group into 3 groups:

[0073] IDE-4 group 1: Add IDE-4 at a final concentration of 10 μM.

[0074] IDE-4 group 2: Add IDE-4 at a final concentration of 20 μM.

[0075] IDE-4 Group 3: Add IDE-4 at a final concentration of 40 μM.

[0076] After intervening RA-FLSs under the above conditions, 10 μL CCK-8 was added to each well and cultured in the incubator for 1.5 hours. The absorbance of each group at a wavelength of 450 nm was then measured using an enzyme marker. The cell proliferation inhibition rate of the control group was 0%, and the proliferation inhibition rates of RA-FLSs in other groups were calculated.

[0077] 3. Experimental results

[0078] Table 1 Effect of IDE-4 on the proliferation of primary human RA-FLSs

[0079]

[0080] As shown in Table 1, IDE-4 can significantly inhibit the proliferation of primary human RA-FLSs, and its proliferation inhibition effect increases with increasing concentration, that is, it is concentration-dependent; and its proliferation inhibition effect is significantly better than that of the positive control drug methotrexate and its parent core idebenone and aspirin.

[0081] Example 3: Effect of IDE-4 on the proliferation of human immortalized RA-FLSs line MH7A

[0082] 1. Experimental Materials

[0083] 1.1 Cell line: Human immortalized RA-FLSs line MH7A

[0084] 1.2 Main reagents and instruments: fetal bovine serum (BI, Israel), DMEM medium (Gibco, USA), penicillin-streptomycin (Gibco, USA), dimethyl sulfoxide (Sigma), CCK-8 kit (Jiangsu Addison Biotechnology Co., Ltd.), trypsin (Gibco), Synergy TM H1 full-function microplate reader (BioTek, USA), low-speed centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd.), constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), clean bench (Singapore Yisi High-tech Co., Ltd.), cell culture incubator (Singapore Yisi High-tech Co., Ltd.).

[0085] 2. Experimental Methods

[0086] 2.1 Cultivate the human immortalized RA-FLSs line MH7A, digest the cells with 0.25% trypsin, collect the cells and transfer them to a centrifuge tube and centrifuge them at 1200 rpm for 5 minutes, resuspend the cells in cell culture medium (10% fetal bovine serum + DMEM basal medium + 1% penicillin-streptomycin), inoculate the cells at a density of 4000 cells / well in a 96-well cell culture plate, and then culture them in a cell culture incubator at 37°C, 5% CO2 and saturated humidity.

[0087] 2.2 Set up 3 replicate wells in each group, add drugs to each well according to the following conditions, and then continue to culture for 48 hours.

[0088] Control group: the group without medication.

[0089] Positive control group: The methotrexate group, idebenone group and aspirin group were divided into 3 groups each, for a total of 9 groups:

[0090] Positive control group 1: methotrexate was added at a final concentration of 10 μM.

[0091] Positive control group 2: methotrexate was added at a final concentration of 20 μM.

[0092] Positive control group 3: methotrexate was added at a final concentration of 40 μM.

[0093] Positive control group 4: Idebenone was added at a final concentration of 10 μM.

[0094] Positive control group 5: Idebenone was added at a final concentration of 20 μM.

[0095] Positive control group 6: Idebenone was added at a final concentration of 40 μM.

[0096] Positive control group 7: Aspirin was added at a final concentration of 10 μM.

[0097] Positive control group 8: Aspirin was added at a final concentration of 20 μM.

[0098] Positive control group 9: Aspirin was added at a final concentration of 40 μM.

[0099] IDE-4 Group: Divide the IDE-4 group into 3 groups:

[0100] IDE-4 group 1: Add IDE-4 at a final concentration of 10 μM.

[0101] IDE-4 group 2: Add IDE-4 at a final concentration of 20 μM.

[0102] IDE-4 Group 3: Add IDE-4 at a final concentration of 40 μM.

[0103] After intervening MH7A under the above conditions, 10 μL CCK-8 was added to each well and cultured in the incubator for 1.5 hours. The absorbance of each group at a wavelength of 450 nm was then measured using an ELISA reader. The cell proliferation inhibition rate of the control group was 0%, and the proliferation inhibition rates of the other groups on MH7A cells were calculated.

[0104] 3. Experimental results

[0105] Table 2 Effect of IDE-4 on MH7A proliferation

[0106]

[0107] As shown in Table 2, IDE-4 can significantly inhibit the proliferation of MH7A, and its proliferation inhibition effect increases with increasing concentration, that is, it is concentration-dependent; and its proliferation inhibition effect is significantly better than the positive control drug methotrexate and its parent core idebenone and aspirin.

[0108] Example 4: Effect of IDE-4 on the migration of human synovial sarcoma cells SW982

[0109] 1. Experimental Materials

[0110] 1.1 Cell line: human synovial sarcoma cell line SW982

[0111] 1.2 Main reagents and instruments: fetal bovine serum (BI, Israel), DMEM medium (Gibco, USA), penicillin-streptomycin (Gibco, USA), dimethyl sulfoxide (Sigma), trypsin (Gibco), Synergy TM H1 full-function microplate reader (BioTek, USA), low-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.), constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), clean bench (Singapore Yisi High-tech Co., Ltd.), cell culture incubator (Singapore Yisi High-tech Co., Ltd.).

[0112] 2. Experimental Methods

[0113] 2.1 Culture human synovial sarcoma cells SW982, digest the cells with 0.25% trypsin, collect the cells and transfer them to a centrifuge tube and centrifuge them at 1200 rpm for 5 minutes, resuspend the cells in cell culture medium (10% fetal bovine serum + DMEM basal medium + 1% penicillin-streptomycin), inoculate the cells at a density of 300,000 per well in a 6-well cell culture plate, and then culture them in a cell culture incubator at 37°C, 5% CO2 and saturated humidity.

[0114] 2.2 Each group was treated with drugs according to the following conditions and then cultured for 48 hours.

[0115] Control group: the group without medication.

[0116] Positive control group: The methotrexate group, idebenone group and aspirin group were each divided into 1 group, for a total of 3 groups:

[0117] Positive control group 1: methotrexate was added at a final concentration of 40 μM.

[0118] Positive control group 2: Idebenone was added at a final concentration of 40 μM.

[0119] Positive control group 3: Aspirin was added at a final concentration of 40 μM.

[0120] IDE-4 group: IDE-4 was added at a final concentration of 40 μM.

[0121] After the SW982 cells were treated under the above conditions, the cells were collected and resuspended in serum-free medium, and inoculated into the Transwell chamber at a density of 50,000 / well. The culture medium in the lower chamber was DMEM medium containing 10% serum. After 24 hours, the chamber was taken out, fixed with methanol for 15 minutes, and then stained with crystal violet for 30 minutes. The migration of cells in each group was recorded under a microscope.

[0122] 3. Experimental results

[0123] like Figure 1 As shown, IDE-4 can significantly inhibit the migration of SW982, and the inhibitory effect is significantly better than that of the positive control drug methotrexate and its parent drugs idebenone and aspirin.

Claims

1. An idebenone-aspirin conjugate, characterized in that: Its structural formula is as follows:

2. The method for preparing the idebenone-aspirin conjugate according to claim 1, wherein: The following steps are involved: (1) Take N,N'-dicyclohexylcarbodiimide, add acetonitrile and stir to dissolve, and set aside; (2) Take a clean flask, add a magnet, evacuate the flask and replace the gas with argon three times, then add aspirin and acetonitrile in sequence, and stir at room temperature to dissolve; (3) adding idebenone and 4-dimethylaminopyridine to the solution of step (2) in sequence, and stirring in an ice-water bath to dissolve; (4) adding the solution obtained in step (1) to step (3), removing the ice water bath, and stirring the reaction at room temperature; (5) Take the reaction solution obtained in step (4), add water, then add ethyl acetate for extraction, combine the organic phases, dry with Na2SO4, filter, and concentrate the filtrate to dryness; (6) The concentrate obtained in step (5) is applied to a silica gel column for elution, the eluate is collected, and evaporated to dryness to obtain a yellow solid, which is the idebenone-aspirin conjugate.

3. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: In step (1), the amount of N,N'-dicyclohexylcarbodiimide used is 91 mg, and the amount of acetonitrile added is 1 mL.

4. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: In step (2), the amount of aspirin added is 53 mg, and the amount of acetonitrile added is 2 mL.

5. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: In step (3), the amount of idebenone added is 100 mg, and the amount of 4-dimethylaminopyridine added is 4 mg.

6. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: The stirring reaction time in step (4) is 2.5 hours.

7. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: The amount of water added in step (5) is 10 mL, and the amount of ethyl acetate added for extraction is 3 times, 10 mL each time.

8. The method for preparing the idebenone-aspirin conjugate according to claim 2, characterized in that: The eluent in step (6) is a solution with a volume ratio of petroleum ether:ethyl acetate = 4:

1.

9. The use of the idebenone-aspirin conjugate according to claim 1, characterized in that: The application is application in preparing medicine for treating rheumatoid arthritis.

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