Psoralen derivative and application thereof in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention relates to the technical field of biological medicines, in particular to a psoralen derivative and application thereof in preparation of a medicine for treating rheumatoid arthritis. According to the derivative, through an ester prodrug strategy, psoralen C8 / C5 hydroxyl and a non-steroidal anti-inflammatory drug are coupled to obtain a double-pharmacophore molecule. According to the invention, the problems of poor lipid solubility and high hepatotoxicity of psoralen are solved through an ester prodrug design strategy, a high-efficiency and low-toxicity novel drug candidate is provided for RA treatment, and the prepared psoralen derivative can effectively treat rheumatoid arthritis and complications thereof, and can significantly improve arthropathy caused by RA.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a psoralen derivative and its application in the preparation of drugs for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation, articular cartilage and bone destruction. Its pathological process involves abnormal activation of Th1 / Th17 cells, excessive release of pro-inflammatory factors (such as TNF-α, IL-6, and IL-1β), and persistent activation of the NF-κB signaling pathway. The global incidence is approximately 0.5%-1% (data source: WHO 2022 report). Existing treatments generally have varying degrees of side effects; for example, nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids can cause gastrointestinal damage and immunosuppression.

[0003] Psoralen is a natural coumarin compound extracted from the seeds of Psorale acorylifolia. It has been shown to have anti-inflammatory effects (by inhibiting NF-κB nuclear translocation and downregulating the expression of IL-6 and TNF-α), immunomodulatory effects (reducing the ratio of Th1 / Th17 cells and increasing the number of regulatory T cells), and antioxidant effects (scavenging ROS and reducing oxidative damage to synovial cells).

[0004] However, psoralen drugs have the following significant limitations in the treatment of rheumatoid arthritis:

[0005] 1) Natural psoralen has poor lipid solubility (logP value 3.8), resulting in an oral bioavailability of only 22% in rats and a high risk of hepatotoxicity (rat serum ALT / AST levels increased to 2.5 times the normal value), which limits its clinical application. For example, Chinese patent CN119950493B discloses the isolation and purification of 3,4-dihydropsoralen from the roots of *Hypericum perforatum*. It is used to prepare drugs for the prevention or treatment of rheumatoid arthritis, but its poor lipid solubility affects its bioavailability. 2) While modifying the structure of psoralen to optimize drug activity, the structural modification targets are different, making it impossible to specifically regulate and treat rheumatoid arthritis. The esterification groups (such as substituted phenyl groups) in the existing technology CN106565734B are mainly used for catalyzing melanin synthesis or antibacterial purposes, without involving the targeted inhibition of inflammatory factors (such as TNF-α, IL-1β, IL-6) and signaling pathways specific to rheumatoid arthritis. Chinese patent CN106565734B discloses a series of 5-hydroxypsoralen ester derivatives, which introduce substituted benzoic acids (such as methylbenzoic acid, chlorobenzoic acid, etc.) into the psoralen molecule, mainly for promoting melanin production in mouse B16 cells (treatment of vitiligo) and inhibiting pathogenic microorganisms such as Candida albicans and Staphylococcus aureus (antibacterial use). The structural design of these derivatives focuses on enhancing melanin synthesis-related activities or antibacterial efficacy, and their indications are concentrated in skin depigmentation and infectious diseases. The mechanism of action is related to the activation of melanocyte tyrosinase and the destruction of pathogenic microorganism cell membranes. For example, Chinese patent CN106994145B discloses 8-geraniol psoralen for use in the manufacture of drugs for treating or alleviating lupus erythematosus.

[0006] In summary, current technologies have not yet achieved synergistic optimization of the activity, efficacy, safety, and pharmacokinetic properties of psoralen compounds in the treatment and regulation of RA. There is an urgent need to develop a novel structurally modified psoralen derivative for the efficient treatment of RA. Summary of the Invention

[0007] In view of this, the purpose of this invention is to propose a psoralen derivative and its application in the preparation of drugs for treating rheumatoid arthritis. A novel compound obtained by esterification modification of the psoralen structure is applied to the development of drugs for the treatment of rheumatoid arthritis (RA), which can significantly improve joint lesions and has higher bioavailability and lower risk of hepatotoxicity.

[0008] To achieve the above objectives, the present invention provides a psoralen derivative, wherein the psoralen derivative comprises a hydroxyl-containing psoralen monomer and a carboxylic acid-containing diphenyl monomer; and includes the following structural formula:

[0009]

[0010] This invention is based on a toxicity optimization strategy for psoralen derivatives. By modifying the targeted structure of psoralen, it achieves a dual improvement in anti-inflammatory activity and safety.

[0011] This invention uses 8-hydroxypsoralen or 5-hydroxypsoralen as a precursor for the synthesis of new drugs. The introduction of polar groups into 8-hydroxypsoralen and 5-hydroxypsoralen significantly improves metabolic characteristics, thereby improving the lipophilicity of psoralen derivatives, thus increasing bioavailability and reducing the risk of hepatotoxicity. Furthermore, it is esterified with flurbiprofen or diclofenac to form a prodrug system. Its lipophilicity (logP 2.8-3.1) allows the drug to be targeted and enriched in synovial tissue. After hydrolysis by esterase, local sustained release is achieved, which retains the synergistic anti-inflammatory effect of COX-2 inhibition, significantly reduces systemic exposure and the risk of ALT abnormalities, and thus greatly improves the efficacy of anti-RA.

[0012] Furthermore, the introduction of ester groups at the C8 and C5 positions makes the hydrophobicity and stereoconfiguration of psoralen derivatives more readily match the hydrophobic pockets of the esterase active site. For example, long-chain fatty ester groups can enhance the hydrophobic interaction with the catalytic site of esterases, while aromatic ester groups may bind stably through π-π stacking, promoting specific hydrolysis and thus achieving slow release after esterification hydrolysis.

[0013] Preferably, the structural formula of the psoralen derivative is:

[0014]

[0015] Preferably, the structural formula of the psoralen derivative is:

[0016]

[0017] The inventors discovered that the synthesized psoralen derivative has a good therapeutic effect on rheumatoid arthritis. Therefore, this invention further provides the application of psoralen derivative in the preparation of drugs for treating rheumatoid arthritis.

[0018] Preferably, the medicament contains an effective amount of a psoralen derivative and / or pharmaceutically acceptable excipients.

[0019] Preferably, the effective amount of psoralen derivative is a single component or a compound component of psoralen derivative.

[0020] Further preferably, the effective amount of psoralen derivative includes The composite components.

[0021] During the research, the inventors also unexpectedly discovered that the combination of 8-hydroxypsoralen derivatives and 5-hydroxypsoralen derivatives helps to synergistically improve the anti-RA efficacy and biosafety of the drug. The possible reason is:

[0022] 1) On the one hand, the introduction of a hydroxyl group at the C8 position of the psoralen core forms a stronger molecular polarity and hydrogen bond donor capacity, which may enhance anti-inflammatory activity by increasing polar interactions with target proteins. The substitution of the hydroxyl group at the C5 position changes the electron cloud distribution of the core, which may affect its binding conformation with key enzymes in the inflammatory signaling pathway. On the other hand, the combination of 8-hydroxyl and 5-hydroxy psoralen derivatives can cover the COX / LOX-IL-17 inflammatory network at multiple targets. The 8-hydroxy psoralen derivative may be related to the inhibition of 5-LOX and the blocking of the IL-17 pathway, while the 5-hydroxy psoralen derivative helps to optimize COX-2 selectivity and inhibit the phosphorylation level of NF-κB p65 protein, thereby achieving a synergistic effect in improving the anti-RA efficacy of the drug.

[0023] 2) The 8-hydroxypsoralen derivative, due to the introduction of the C8 hydroxyl group, exhibits increased polarity, promoting excretion. The hydroxyl group occupies the space of the CYP450 enzyme active site, hindering the entry of the parent psoralen into the catalytic site and weakening metabolic transformation. Simultaneously, the electron-donating properties of the hydroxyl group may alter the electron distribution of the parent structure, reducing the binding affinity to the CYP450 active site, decreasing metabolic inhibition, and thus promoting glucuronidation metabolism, significantly reducing intrahepatic accumulation. Conversely, the 5-hydroxypsoralen derivative, due to the introduction of the C5 hydroxyl group, alters the electron cloud distribution, interfering with binding to mitochondria, reducing ROS generation, and thus interfering with the selective inhibition of inflammatory signals in the ERK / MAPK pathway, reducing non-specific activation of hepatotoxicity-related pathways, thereby synergistically improving biocompatibility.

[0024] Preferably, the dosage of the psoralen derivative is 1-40 mg / kg / day.

[0025] Preferably, the pharmaceutically acceptable excipients include at least one of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorbents, surfactants, and lubricants.

[0026] Preferably, the drug dosage form includes one of the following: injection, tablet, capsule, powder, oral liquid, granule, pill, or granule.

[0027] Preferably, the drug administration route includes one of the following: intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0028] The beneficial effects of this invention are:

[0029] This invention, based on a toxicity optimization strategy for psoralen derivatives, achieves a dual enhancement of anti-inflammatory activity and safety through targeted structural modification of psoralen. The psoralen derivatives developed in this invention can treat rheumatoid arthritis, alleviating redness and swelling symptoms, reducing inflammatory cell infiltration in joint tissues, improving articular cartilage and synovial lesions, enhancing joint mobility, inhibiting the release of TNF-α, IL-6, and nitric oxide inflammatory factors, and reducing the phosphorylation levels of ERK1 / 2 and NF-κB p65 proteins.

[0030] In new drug development, the inventors used 8-hydroxypsoralen or 5-hydroxypsoralen as precursors for drug synthesis. The introduction of polar groups by 8-hydroxypsoralen and 5-hydroxypsoralen significantly improves metabolic characteristics, thereby improving the lipophilicity of psoralen derivatives, increasing bioavailability, and reducing the risk of hepatotoxicity. Furthermore, they are esterified with flurbiprofen or diclofenac to form a prodrug system. Its lipophilicity (logP 2.8-3.1) allows the drug to be targeted and enriched in synovial tissue. After hydrolysis by esterase, local sustained release is achieved, which retains the synergistic anti-inflammatory effect of COX-2 inhibition and significantly reduces systemic exposure and the risk of ALT abnormalities, thereby greatly improving the efficacy of anti-RA.

[0031] At the same time, the inventors also unexpectedly discovered that the combination of 8-hydroxypsoralen derivatives and 5-hydroxypsoralen derivatives helps to synergistically improve the anti-RA efficacy and biosafety of the drug. The possible reasons are:

[0032] 1) On the one hand, the introduction of a hydroxyl group at the C8 position of the psoralen core forms a stronger molecular polarity and hydrogen bond donor capacity, which may enhance anti-inflammatory activity by increasing polar interactions with target proteins. The substitution of the hydroxyl group at the C5 position changes the electron cloud distribution of the core, which may affect its binding conformation with key enzymes in the inflammatory signaling pathway. On the other hand, the combination of 8-hydroxyl and 5-hydroxy psoralen derivatives can cover the COX / LOX-IL-17 inflammatory network at multiple targets. The 8-hydroxy psoralen derivative may be related to the inhibition of 5-LOX and the blocking of the IL-17 pathway, while the 5-hydroxy psoralen derivative helps to optimize COX-2 selectivity and inhibit the phosphorylation level of NF-κB p65 protein, thereby achieving a synergistic effect in improving the anti-RA efficacy of the drug.

[0033] 2) The 8-hydroxypsoralen derivative, due to the introduction of the C8 hydroxyl group, exhibits increased polarity, promoting excretion. The hydroxyl group occupies the space of the CYP450 enzyme active site, hindering the entry of the parent psoralen into the catalytic site and weakening metabolic transformation. Simultaneously, the electron-donating properties of the hydroxyl group may alter the electron distribution of the parent structure, reducing the binding affinity to the CYP450 active site, decreasing metabolic inhibition, and thus promoting glucuronidation metabolism, thereby reducing intrahepatic accumulation. Conversely, the 5-hydroxypsoralen derivative, due to the introduction of the C5 hydroxyl group, alters the electron cloud distribution, interfering with binding to mitochondria, reducing ROS generation, and thus interfering with the selective inhibition of inflammatory signals in the ERK / MAPK pathway, reducing non-specific activation of hepatotoxicity-related pathways; thereby achieving a synergistic effect in improving biocompatibility. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The 1H NMR spectrum of the psoralen derivative synthesized in Example 1 of this invention is shown below.

[0036] Figure 2 The carbon NMR spectrum of the psoralen derivative synthesized in Example 1 of this invention;

[0037] Figure 3 The 1H NMR spectrum of the psoralen derivative synthesized in Example 2 of this invention is shown below.

[0038] Figure 4 The carbon NMR spectrum of the psoralen derivative synthesized in Example 2 of this invention;

[0039] Figure 5 The 1H NMR spectrum of the psoralen derivative synthesized in Example 3 of this invention is shown below.

[0040] Figure 6 The carbon NMR spectrum of the psoralen derivative synthesized in Example 3 of this invention;

[0041] Figure 7 The graph shows the statistical results of the efficacy of the derivatives FLXA, FLBE, FLXN, and FLXA+FLBE (m / m = 1:1) synthesized in Examples 1-4 of this invention and the comparative examples 1-7 on rat rheumatoid arthritis models.

[0042] Figure 8The diagram shows the liver toxicity of the derivatives FLXA, FLBE, FLXN, and FLXA+FLBE (m / m = 1:1) synthesized in Examples 1-4 of this invention and the comparative examples 1-7 to rats.

[0043] Figure 9 This is a comparison of the hind feet of RA rats before and after treatment with psoralen derivatives according to embodiments of the present invention.

[0044] Figure 8 Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0046] Example 1: Synthesis of psoralen derivative FLXA

[0047]

[0048] S1: Synthesis of the active intermediate FLCI: Flurbiprofen (5 g, 20.4 mmol) was dissolved in 30 mL of 1,2-dichloroethane and 0.1 mL of DMF in a 100 mL single-necked flask by stirring. Then, PCl3 (4.48 mL, 20.7 mmol) was added, and the mixture was heated to reflux at 35 °C for 8 h. After cooling to room temperature, the mixture was filtered, and the solvent was evaporated to give a yellow oily product in 95% yield.

[0049] S2: Synthesis of derivative FLXA: 8-hydroxypsoralen (50.0 mg, 0.24 mmol) was added to a 100 mL round-bottom flask at 25 °C, and 4 mL of dichloromethane was added dropwise to dissolve and stir. Triethylamine (10.0 mg, 0.1 mmol) was added dropwise, and finally the active intermediate FLCI (50.0 mg, 0.19 mmol) was added dropwise. The mixture was stirred at room temperature for 6 h, and the reaction was detected by TLC.

[0050] S3: After the reaction is complete, concentrate the sample, mix it with silica gel, and separate it by column chromatography. Use petroleum ether and ethyl acetate (volume ratio) as the eluent. Collect the separated product, freeze dry it to obtain a white powder with a yield of 86%.

[0051] 1 H NMR (400MHz, DMSO-d6) δ8.26–8.13(m,2H),7.96(s,1H),7.60(ddt,J=8.3,5.5,2.9Hz,3H),7.53–7.4 2(m,5H),7.19(d,J=2.3Hz,1H),6.50(d,J=9.6Hz,1H),4.53(q,J=7.1Hz,1H),1.69(d,J=7.1Hz,3H). 13C NMR(101MHz,DMSO-d6)δ171.53,160.73,159.36,158.28,148.96,147.85,145.45,143.50,141.77,141.69,135.29,131.52,131.49,129.26,129 .23,129.17,129.10,128.36,127.82,127.69,125.99,124.79,124.76, 122.36,118.56,116.66,116.09,115.86,114.88,107.80,44.09,19.19.

[0052] Example 2: Synthesis of psoralen derivative FLBE

[0053]

[0054] S1: Synthesis of the active intermediate FLCI: Same as in Example 1;

[0055] S2: Synthesis of derivative FLBE: 5-hydroxypsoralen (50.0 mg, 0.24 mmol) was added to a 100 mL round-bottom flask at 25 °C, and 4 mL of dichloromethane was added dropwise to dissolve and stir. Triethylamine (10.0 mg, 0.1 mmol) was added dropwise, and finally the active intermediate FLCI (50.0 mg, 0.19 mmol) was added dropwise. The mixture was stirred at room temperature for 6 h, and the reaction was detected by TLC.

[0056] S3: After the reaction is complete, concentrate the sample, mix it with silica gel, and separate it by column chromatography. Use petroleum ether and ethyl acetate (volume ratio) as the eluent. Collect the separated product, freeze dry it to obtain a white powder with a yield of 88%.

[0057] 1 H NMR(400MHz, DMSO-d6)δ8.12(d,J=2.3Hz,1H),7.93(dd,J=9.8,0.7Hz,1H),7.74(s,1H),7.66–7.59(m,3H),7.5 5–7.42(m,5H),6.67(dd,J=2.3,1.0Hz,1H),6.47(d,J=9.8Hz,1H),4.53(q,J=7.1Hz,1H),1.71(d,J=7.1Hz,3H). 13C NMR(101MHz,DMSO-d6)δ172.03,160.00,156.61,152.01,148.56,142.12,142.04,138.56,135.22,131.69,131.65,12 9.32,129.29,129.17,128.46,124.94,124.91,118.63,116.39,116.15,115.33,109.29,104.28,98.42,44.42,18.17.

[0058] Example 3: Synthesis of psoralen derivative FLXN

[0059]

[0060] S1: Synthesis of the active intermediate FLCII: Diclofenac (2 g, 8.8 mmol) was added to dichloromethane (20 mL) in a 100 mL single-necked flask, followed by the addition of oxaloyl chloride (2.34 mL, 26.4 mmol) and N,N-dimethylformamide (0.34 mL, 4.4 mmol). The reaction was carried out at room temperature for 2 h. After the reaction was completed, the dichloromethane was rotary evaporated under reduced pressure to give a white oily product with a yield of 98%.

[0061] S2: Synthesis of derivative FLXN: 5-hydroxypsoralen (50.0 mg, 0.24 mmol) was added to a 100 mL round-bottom flask at 25 °C, and 4 mL of dichloromethane was added dropwise to dissolve and stir. Triethylamine (10.0 mg, 0.1 mmol) was added dropwise, and finally the active intermediate FLCII (50.0 mg, 0.16 mmol) was added dropwise. The mixture was stirred at room temperature for 6 h, and the reaction was detected by TLC.

[0062] S3: After the reaction is complete, concentrate the sample, mix it with silica gel, and separate it by column chromatography. The eluent is petroleum ether: ethyl acetate (V / V) = 8:1. Collect the separated product, freeze dry it to obtain a white powder with a yield of 85%.

[0063] 1 H NMR (400MHz, DMSO-d6) δ = 7.78 (d, J = 8.5, 4H), 7.64 (dd, J = 8.8, 7.6, 2H), 7.41 (d, J = 7. 4,2H),7.23(td,J=7.7,1.3,2H),7.12(td,J=7.5,1.1,2H),6.40(s,1H),3.91(s,2H). 13C NMR (101MHz, DMSO) δ173.72,143.26,134.91,132.41,130.34,129.86,128.29,125.47,125.09,123.32,108.98,35.55.

[0064] Example 4

[0065] The psoralen derivative FLXA synthesized in Example 1 and the psoralen derivative FLBE synthesized in Example 2 were compounded in equal mass ratio.

[0066] Comparative Example 1

[0067] 3,4-Dihydropsoralen Comparative Example 2

[0068] Fluoropsoralen derivatives

[0069] Comparative Example 3

[0070] 50mg of 8-hydroxypsoralen + 50mg of flurbiprofen were combined for combined medication.

[0071] Comparative Example 4

[0072] 50mg of 8-hydroxypsoralen + 50mg of diclofenac were combined for combined medication.

[0073] Comparative Examples 5, 6, and 7 were flurbiprofen, diclofenac, and 8-hydroxypsoralen, respectively.

[0074] Animal Experiment 1: Evaluation of the efficacy of the drugs in the various examples and comparative examples on a rat model of rheumatoid arthritis.

[0075] This experiment used the internationally recognized type II collagen-induced arthritis (CIA) rat model and systematically evaluated the therapeutic effect of psoralen derivatives on rheumatoid arthritis (RA) using a multi-dimensional evaluation system (behavioral, imaging, histopathological, and molecular biological).

[0076] Animal strain: SPF-grade male SD rats (weight 180-220g), 10 rats per group.

[0077] CIA modeling: Bovine type II collagen (2 mg / mL) was emulsified with an equal volume of Freund's complete adjuvant (CFA), and 0.2 mL / rat was subcutaneously injected into the base of the tail. A booster immunization was performed 7 days later using the same method. Ankle diameter was measured using calipers; a swelling degree ≥30% was considered successful.

[0078] Experimental groups: blank control group (intraperitoneal injection of normal saline), model group (CIA modeling + normal saline), positive control group (CIA modeling + methotrexate, 2 mg / kg, orally once daily), and drug test groups of each example and comparative example (CIA modeling + drug, 10 mg / kg, orally once daily).

[0079] Dosage regimen and post-treatment: Dosing began on day 14 after the initial immunization (acute inflammatory phase) and continued for 21 days. Daily records of limb redness, swelling, deformity, and range of motion were recorded, graded on a scale of 0-4 (0 = no lesion, 4 = severe deformity). Patients fasted for 12 hours after the last dose. Blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 15 minutes to separate serum, and the serum sample was simultaneously collected and stored at -80℃.

[0080] Table 1 shows the limb grading scores on days 7, 14, and 21 after the start of medication:

[0081] Table 1

[0082]

[0083]

[0084] Indicator detection: Serum TNF-α and IL-6 levels were detected using ELISA. The experimental results are as follows: Figure 7 As shown.

[0085] Combining Table 1 and Figure 7 It can be seen that the treatment efficacy of Examples 1-4 is high, and the treatment effect in terms of the number of swollen joints and the level of inflammatory factors is significantly better than that of the control group and Comparative Examples 1-7 (P<0.001), indicating that the psoralen derivative of the present invention has a better therapeutic effect on RA. Meanwhile, according to Table 1 and... Figure 7 It can also be seen that the combination therapy of psoralen derivatives in Example 4 is significantly better than the single therapy of psoralen derivatives in Examples 1-3 in the treatment of RA. This indicates that the combination of 8-hydroxypsoralen derivatives and 5-hydroxypsoralen derivatives helps to synergistically improve the anti-RA efficacy of the drugs. The reason for this may be that, on the one hand, the introduction of a hydroxyl group at the C8 position of the psoralen core forms a stronger molecular polarity and hydrogen bond donor capacity, which may enhance anti-inflammatory activity by increasing the polar interaction with target proteins. The substitution of the hydroxyl group at the C5 position changes the electron cloud distribution of the core, which may affect its binding conformation with key enzymes in the inflammatory signaling pathway. On the other hand, the combination of 8-hydroxypsoralen derivatives and 5-hydroxypsoralen derivatives can cover the COX / LOX-IL-17 inflammatory network at multiple targets. The 8-hydroxypsoralen derivative may be related to the inhibition of 5-LOX and the blocking of the IL-17 pathway. The 5-hydroxypsoralen derivative helps to optimize COX-2 selectivity and inhibit the phosphorylation level of NF-κB p65 protein, thereby achieving a synergistic effect in improving the anti-RA efficacy of the drugs.

[0086] Animal Experiment 2: Determination of the distribution concentration of the drugs in rats for each example and comparative example.

[0087] This experiment used the internationally recognized rat model of type II collagen-induced arthritis (CIA).

[0088] Animal strain: SPF-grade male SD rats (weight 180-220g), 10 rats per group.

[0089] Experimental grouping: Drug administration for each example and comparative example: 10 mg / kg, orally once daily.

[0090] Dosing regimen and post-treatment: Dosing began on day 14 after the first immunization (acute inflammatory phase). Six hours after administration, rat paw edema fluid was collected, and the drug concentration in the edema fluid was determined by HPLC-MS / MS to compare the bioavailability of the drugs in rats in each example and comparative example.

[0091] Table 2 shows the drug concentrations in rats after administration of drugs in Examples 1-4 and Comparative Examples 1-4:

[0092] Table 2

[0093]

[0094] Note: *Compared with the comparative example, *p<0.05.

[0095] As shown in Table 2, compared with Comparative Examples 1-4, the present invention improves the target drug concentration through prodrug design, thereby improving drug bioavailability. The reasons may be that, on the one hand, the improved prodrug has enhanced lipophilicity (the ester bond increases hydrophobicity), which promotes penetration into inflamed tissue; on the other hand, the esterase activity at the inflamed site is increased, which specifically hydrolyzes the prodrug to release active molecules, achieving targeted therapy on RA inflammatory factors, thereby improving the bioavailability of psoralen derivatives and the therapeutic effect on RA.

[0096] Animal Experiment 3: Toxicity Assessment of Each Example and Comparative Example on a Hepatocyte Model

[0097] Human normal hepatocytes L02 were used for hepatotoxicity verification.

[0098] Grouping: blank control group (DMSO solvent control), drugs of each example and comparative example.

[0099] The MTT / CCK-8 assay was used to determine the cell viability of each example and comparative example after 24 hours of drug administration at a concentration of 100 μM, and the IC50 was calculated. The experimental results are as follows: Figure 8 As shown. By Figure 8It can be seen that, compared with Comparative Examples 1-7, Examples 1-4 have good biocompatibility and low hepatotoxicity. Among them, Example 4 has the lowest hepatotoxicity, indicating that the combination of 8-hydroxypsoralen derivative and 5-hydroxypsoralen derivative can synergistically improve the biocompatibility of the drug. The possible reason is that in FLXA, the introduction of the C8 hydroxyl group increases the polarity of 8-hydroxypsoralen, promoting excretion. Furthermore, the C8 hydroxyl group may inhibit the binding of the parent structure to the CYP450 active site, reducing metabolic inhibition and thus promoting glucuronidation metabolism, significantly reducing intrahepatic accumulation. In FLBE, the introduction of the C5 hydroxyl group alters the electron cloud distribution of 5-hydroxypsoralen, interfering with binding to mitochondria, reducing ROS generation, and thus interfering with the selective inhibition of inflammatory signals in the ERK / MAPK pathway, reducing non-specific activation of hepatotoxicity-related pathways, thereby achieving a synergistic effect in improving biocompatibility.

[0100] Animal Experiment 4: Verifying the therapeutic effect of the psoralen derivative prepared according to the embodiments of the present invention on RA.

[0101] This experiment used the internationally recognized rat model of type II collagen-induced arthritis (CIA).

[0102] Animal strain: SPF-grade male SD rats (weighing 180-220g), 2 in total.

[0103] Dosage regimen:

[0104] Example 1: FLXA, 2 mg / kg, orally once daily;

[0105] Drug administration began on day 21 after the initial immunization, and treatment continued for 14 days. The hind paw morphology of RA rats before and after treatment was compared. Figure 9 As shown.

[0106] Depend on Figure 9 It can be seen that before treatment, the hind feet of RA rats showed obvious symptoms of rheumatoid arthritis, with prominent joint redness, swelling, deformity and obvious inflammatory response. After treatment with the psoralen derivative FLXA in Example 1, the redness and swelling of the rat's hind paws significantly subsided, the joint morphology was close to normal, and the overall appearance and condition of the hind feet were greatly improved, clearly demonstrating the good therapeutic effect after treatment. This shows that the psoralen derivative of the present invention can effectively treat rheumatoid arthritis and its complications.

[0107] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in the details for the sake of brevity.

[0108] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A psoralen derivative, characterized in that, The psoralen derivative is composed of a hydroxyl-containing psoralen monomer and a carboxylic acid-containing diphenyl monomer; it includes the following structural formula:

2. The psoralen derivative according to claim 1, characterized in that, The structural formula of the psoralen derivative is:

3. The psoralen derivative according to claim 1, characterized in that, The structural formula of the psoralen derivative is:

4. The use of a psoralen derivative as described in any one of claims 1-3 in the preparation of a medicament for treating rheumatoid arthritis.

5. The application according to claim 4, characterized in that, The drug contains an effective amount of psoralen derivatives and / or pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The effective amount of psoralen derivative is a single component or a compound component of psoralen derivative; The composite components include:

7. The application according to claim 6, characterized in that, The dosage of the psoralen derivative is 1-40 mg / kg / day.

8. The application according to any one of claims 5-7, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, adsorbents, surfactants, and lubricants.

9. The application according to any one of claims 5-7, characterized in that, The drug dosage form includes one of the following: injection, tablet, capsule, powder, oral liquid, powder, pill or granule.

10. The application according to any one of claims 5-7, characterized in that, The drug administration route includes one of the following: intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

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