Design and Synthesis of Novel Double-Ring Conjugated Enones as Potent Anti-rheumatoid Arthritis Agents

Abstract

Rheumatoid arthritis (RA) is a chronic and systemic disease of inflammatory synovitis with unknown etiology. In previous studies, we found that the double-ring conjugated enone structure has anti-rheumatoid arthritis activity and could effectively inhibit the proliferation of rat synovial cells in vitro and has good anti-inflammatory activity in vivo. Herein, we further modified the structure, which was a novel double-ring conjugated enone, to study its anti-rheumatoid arthritis activity. Results showed that the most potent compound 32 could effectively inhibit the proliferation of rat synovial cells in vitro and has better anti-inflammatory activity compared with that of the positive control methotrexate, as shown by in vivo activity evaluation. More interestingly, compound 32 could effectively inhibit the increase of TNF-α, IL-1β, and IL-6 induced by LPS and regulate the expression of TLR4, MyD88, NF-κB, and IκB in the signaling pathway of TLR4/NF-κB. Our results provided a promising starting point for the development of highly effective small molecules for the treatment of RA.

1. Introduction

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammatory synovitis.¹ It is characterized by multiple joints, symmetry, and invasive joint inflammation of hand and foot facet joints and usually accompanied with the involvement of extra-articular organs and positive serum rheumatoid factor, which may lead to joint deformity and loss of function.²⁻⁴ The pathogenesis of RA is still unclear and may be related to heredity, infection, sex hormones, and other factors. RA is a recurrent disease with a high disability rate and poor prognosis. According to the World Health Organization (WTO), the average incidence of RA in the world’s total adult population is 0.5–1%.⁵⁻⁸ By the end of 2018, the number of RA patients had exceeded 30 million, and it is increasing year by year. The disease incidence is different with race and gender, which is significantly higher in females than that of males. During the RA treatment to reduce the inflammatory response of the joints, the drug treatment plays a crucial role.⁹⁻¹¹ So far, the therapeutic drugs for RA treatment could be divided into four categories, namely nonsteroidal anti-inflammatory drugs, glucocorticoids, anti-rheumatism symptom-improving drugs and biological antibodies, and botanical drugs.¹²⁻¹⁴ Searching for effective components from traditional botanical drugs has become an important method of RA research.¹⁵ It has been found that the active components of plant drugs such as triterpenoids, phenols, flavonoids, and polysaccharides have anti-inflammatory, analgesic, and immunomodulatory effects, showing the prospect of research and development of plant drugs in RA.¹⁶ Further separation of monomers and small-molecule compounds from active components of phytodrugs is an important part of plant pharmaceutical chemistry.¹⁷ As for the lead compound, further structural optimization and modification could be used to obtain more ideal drugs of anti-rheumatoid arthritis treatment.¹⁸Fissistigma oldhamii is a traditional Chinese medicine, the root of which is mainly used to treat arthritis, rheumatism, bone pain, and other diseases.¹⁸ In the process of screening the anti-rheumatoid arthritis activity of the extracted compounds, it is found that Dysodensiol K has good biological activity (IC₅₀ = 11.8 ± 0.23 μM) and could inhibit the growth of synovial cells to a certain extent.¹⁹ In the previous work, Dysodensiol K was used as the lead compound to design and synthesize four series of double-ring conjugated enones, the anti-rheumatoid arthritis activity of which was evaluated. The results of anti-rheumatoid arthritis activity evaluation showed that compounds 1 and 2 were identified as potential drug candidates for the treatment of RA (Figure 1).¹⁹ In the screening of rat synovial cell formation in vitro, compounds 1 and 2 showed ideal inhibitory activity, among which the IC₅₀ values were 2.68 and 2.71 μM, respectively.^19,20 Meanwhile, the double-ring conjugated enones showed therapeutic and ameliorative effects on RA in vivo in the CFA rat model, achieving the same therapeutic level compared with that of methotrexate. In this project, the lead compound 1 is taken as the skeleton structure for structural modification, and 16 target compounds are designed according to the basic principle of structural optimization of lead compounds (Figure 2). In this study, we describe the synthesis of novel 6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione and evaluate the anti-rheumatoid arthritis activity of the derivatives. The purpose of this study is to further optimize the structure of the lead compound and obtain the best target compound for RA treatment. The structure–activity relationship (SAR) is studied by comparing the anti-rheumatoid arthritis biological activities of the derivatives, on the basis of which the binding conformation of the target compounds and receptor TLR4 was further studied through molecular docking simulation.

Figure 1.

Chemical structure of double-ring conjugated enones.

Figure 2.

Chemical structure design of target compounds.

2. Results and Discussion

2.1. Design and Synthesis

Double-ring conjugated enones have been confirmed to have good anti-rheumatoid arthritis activity, among which lead compound 1 showed the best performance in vitro and in vivo.^19,20 However, the anti-rheumatoid arthritis activity evaluation results of compound 1 showed that its activity was not superior to that of the positive control methotrexate. Therefore, further structural optimization was needed to obtain the ideal target compound. In this study, we used compound 1 as the lead compound to design 16 target compounds with double-ring conjugated enone structures (Figure 2). Based on the previous studies of anti-rheumatoid arthritis activity (19), we found that the double-ring conjugated enone structure was a necessary skeleton structure with anti-rheumatoid arthritis activity. Meanwhile, the SAR study showed that the substituent at position 4 in the six-membered ring had a great influence on the activity. Therefore, in the process of designing the structure of the target compounds, we retained the double-ring conjugated enone structure to retain its anti-rheumatoid arthritis activity to the maximum extent. At the same time, the electron-withdrawing group and electron-releasing group were introduced into position 4 of the six-membered ring to change its anti-rheumatoid arthritis activity, so as to further study its SAR. In the synthesis process of the target compounds, we used ethyl acetoacetate (compound 3) as the starting material to prepare the target compounds through four steps: carbonyl protection, condensation, hydrolysis, and cyclization (Scheme 1). First, compound 3 and ethylene glycol reacted in anhydrous benzene with p-toluenesulfonic acid (p-TsOH) as a catalyst by reflux treatment for 48 h to obtain compound 4. Next, compound 4 and 4-substituted cyclohexanone were placed in anhydrous ethanol with sodium glycolate as a basic substance. After 12 h of reflux treatment, the solvent ethanol was removed. Then, the product of the above-mentioned reaction was hydrolyzed with 2 mol/L HCl at 60 °C for 2 h, in the process of which the key intermediate compounds 5–20 were obtained. Finally, compounds 5–20 were placed in anhydrous ethanol using potassium hydroxide as a basic substance. After 12 h of reflux treatment, the target compounds 21–36 were prepared. The structures of the target compounds 21–36 were confirmed by ¹H NMR, ¹³C NMR, HR-ESI-MS, and elemental analysis. To determine the absolute configuration of these target compounds, we performed ECD calculations and compared them with experimental ECD. The results showed that the calculated ECD was consistent with the experimental ECD, so the target compounds were confirmed to have the R configuration. The synthesis route has mild reaction conditions, simple operation, cheap raw materials, and high overall yield. The synthesis route can provide an experimental basis for small-scale or pilot-scale experiments and has the potential of small-scale production.

Scheme 1. Synthetic Route of the Compounds 21–36.

Reagents and conditions: (a) ethanediol, benzene, p-TsOH, and reflux for 48 h; (b) 4-substituted cyclohexanone, C₂H₅OH, C₂H₅ONa, and reflux for 12 h; (c) HCl, H₂O, 60 °C, and 2 h, and (d) KOH, C₂H₅OH, and reflux for 12 h.

2.2. In Vitro Anti-rheumatoid Arthritis Activity

RA is an autoimmune disease characterized by inflammation of the synovium,¹ which is a chronic disease and mainly affects joints. It is one of the main causes of labor loss and disability. Many countries have listed RA as one of the key diseases to tackle. Based on the previous studies of activity,¹⁹ we found that double-ring conjugated enones (compound 1 and compound 2) have anti-rheumatoid arthritis activity and the potential to be used in the treatment of this disease. The activity of the candidate compounds obtained in the previous studies was not better than that of the positive control, which restricts the further research and development of double-ring conjugated enones. In this study, compound 1 was used as the lead compound and 16 double-ring conjugated enones are designed based on bioelectronic isoarrangement and other drug design principles. In vitro anti-rheumatoid arthritis activity was evaluated by a semi-inhibitory concentration test (IC₅₀), cell morphology, LDH release test, and TLR4 inhibition test. Results of the inhibition rate of rat synovial cells showed that the designed and synthesized compounds had a good inhibitory activity with IC₅₀ ranging from 1.36 ± 0.08 to 6.76 ± 0.31 μM (Table 1). Compared with lead compound 1 and the positive control (methotrexate, IC₅₀ = 2.38 ± 0.20 μM), compounds 23, 24, 28, 32, and 36 exhibited similar or even superior inhibitory activity. Compounds 24 and 32 showed the best inhibitory activity with IC₅₀ values of 1.53 ± 0.13 and 1.36 ± 0.08 μM, respectively. Combined with the results of the anti-rheumatoid arthritis activity test in vitro, we obtained the SAR of some double-ring conjugated enones. The double-ring conjugated enone structure was the necessary parent structure for the anti-rheumatoid arthritis activity of these compounds, and the substituents introduced at position 4 of the six-membered ring in the parent structure have a great influence on the activity. In general, the introduction of hydrophilic groups (such as compound 32) provides relatively good activity. The introduced substituents were the electron-withdrawing group rather than the electron-releasing group. According to the SAR analysis, except that the structure of compound 32 was different from that of compound 24, the space structure of compound 24 occupied a relatively large volume. According to the analysis of binding activity data, compound 24 (6.76 ± 0.31 μM) was significantly different from compound 25 (1.53 ± 0.13 μM). Structurally, two F atoms were introduced into the benzene ring of compound 25. This may change its spatial structure after the introduction of the F atom, so that it cannot bind well with the receptor, thus leading to the reduction of activity. The difference between compound 26 and compound 27 was that the length of the introduced chain is different, but there was no significant difference in the activity result, which indicated that the length of the chain may not affect its anti-rheumatoid arthritis activity. These results also provided a reference for the further optimization of its structure in the future. To intuitively observe the inhibitory activity of the target compounds on rat synovial cells, we further studied the morphology of rat synovial cells (Figure 3). The observation results showed that compared with the normal group, the morphology of synovial cells in the DMSO group was not abnormal, while the synovial cells in the methotrexate group were significantly damaged, the refraction of cells was significantly reduced, the cytoplasm became narrow and shriveled, fibroblasts were significantly reduced, some cells burst, and cell fragments increased. Compound 23, 24, 28, 32, and 36 groups showed significant concentration dependence on synovial cell injury. Morphological results showed that the refractive index of the cells decreased to different degrees, the cytoplasm became narrow and shriveled, fibroblasts decreased significantly, some cells burst, and cell fragments increased. Among these, compounds 24 and 32 had the most significant effect. To study the cytotoxicity of the target compound to rat synovial cells, damage to the cell membrane of rat synovial cells, we tested the LDH release value of rat synovial cells (Table 2). The test results showed that compared with the normal group, the content of LDH in the supernatant of rat synovial cells in the DMSO group was basically unchanged, while the content of LDH in the supernatant of rat synovial cells in the methotrexate positive group was significantly increased. This indicates that methotrexate had certain ability to damage the synovial cell membrane of rats and showed certain cytotoxicity. Compounds 21, 23, 24, 27, 28, 32, 33, and 36 had the best inhibitory effect on LDH release, suggesting that they exerted little damage to synovial cells in rats. Studies have confirmed that Toll-like receptor 4 (TLR4) was an important target of RA.² To further investigate the anti-rheumatoid arthritis activity of these target compounds, we tested the inhibitory activity of TLR4 of compounds 23, 24, 32, and 36in vitro (Table 3). The results showed that the target compounds had a good inhibitory effect on TLR4, and its IC₅₀ value was at a micromolar level. Among the tested target compounds, compounds 24 and 32, with IC₅₀ values of 0.53 ± 0.09 and 0.41 ± 0.05 μM, respectively, showed superior inhibitory activity against TLR4 compared to that of the positive control (methotrexate, IC₅₀ = 1.07 ± 0.21 μM).

Table 1. Inhibitory Activity of Rat Synovial Cells In Vitro.

compounds	IC50 ± SD (μM)a	compounds	IC50 ± SD (μM)a
1 (lead compound)	2.68 ± 0.16	29	4.70 ± 0.32
21	3.03 ± 0.25c	30	3.73 ± 0.28
22	4.11 ± 0.24	31	4.25 ± 0.29
23	2.42 ± 0.17c	32	1.36 ± 0.08c
24	1.53 ± 0.13c	33	3.01 ± 0.19c
25	6.76 ± 0.31	34	4.42 ± 0.26
26	3.42 ± 0.20	35	4.48 ± 0.22
27	3.43 ± 0.22c	36	2.54 ± 0.16
28	2.94 ± 0.21	methotrexateb	2.38 ± 0.20c

^aValues were average of three independent experiments run in triplicate.

^bMethotrexate was used as the positive control.

^cThe significance symbol was p < 0.01.

Figure 3.

Morphology of rat synovial cells in vitro. (a) normal group; (b) DMSO group; (c) methotrexate group; (d) compound 23 group; (e) compound 24 group; (f) compound 28 group; (g) compound 32 group; and (h) compound 36 group. In the cell imaging analysis system, the morphology of rat synovial cells was observed after 150 times (×150) magnification.

Table 2. Release Values of LDH.

compounds	concentration	LDH activity (U/L)a±SD)
21	1 μM	297.79 ± 49.6
23	1 μM	342.78 ± 22.74c
24	1 μM	334.46 ± 21.04*
27	1 μM	274.90 ± 54.00
28	1 μM	375.29 ± 17.87c
32	1 μM	307.02 ± 30.86c
33	1 μM	264.24 ± 27.71
36	1 μM	292.07 ± 31.56
methotrexateb	1 μM	438.75 ± 11.50c
DMSO	5%	233.29 ± 16.00
normal		228.09 ± 35.17

^aValues were average of three independent experiments run in triplicate.

^bMethotrexate was used as the positive control.

^cThe significance symbol was p < 0.01.

Table 3. TLR4 In Vitro Inhibition Data.

compounds	TLR4 IC50 (μM)a ±SD
23	1.15 ± 0.16
24	0.53 ± 0.09c
32	0.41 ± 0.05c
36	1.26 ± 0.23
methotrexateb	1.07 ± 0.21

^aValues were average of three independent experiments run in triplicate.

^bMethotrexate was used as the positive control.

^cThe significance symbol was p < 0.05.

2.3. Anti-rheumatoid Arthritis Activity In Vivo

Activity evaluation in vivo was an important content in the study of medicinal chemistry.²¹ All the target compounds synthesized in this study showed a good activity evolution against rheumatoid arthritis in vitro. Compounds 24 and 32 were selected to further study their anti-rheumatoid arthritis activity in vivo, and complete Freund’s adjuvant (CFA)-induced arthritis in rats was used as an animal model for evaluation. In this activity evaluation, the paw volume changes were first measured before and after treatment. Finally, the levels of the inflammatory cytokines IL-6 and TNF-α after treatment in rat serum were measured (Figures 4 and 5). Figure 4a shows the change of paw volume before and after treatment at a dose of 50 mg·kg^–1·d^–1 for 14 day.¹⁹ Results showed that the paw volume of rats in the blank group changed significantly and the positive control group and compound 24 group also showed significant volume change, while the compound 32 group showed little change. Figure 4b shows the change of paw volume before and after treatment with 100 mg·kg^–1·d^–1 for 14 days. It can be seen from Figure 4b that the paw volume of rats in the blank group changed significantly, while that of the positive control changed little, and the values of the compound 24 and compound 32 groups were hardly changed before and after treatment, indicating that the target compound 24 and compound 32 could exert good swelling suppressive effects in vivo. Figure 4c shows the test result of the IL-6 content in the serum of the rat abdominal artery after treatment. It can be seen that the IL-6 content in the blank group was abnormally increased, while the positive control group, compound 24 group, and compound 32 group were basically at a low level after a high-dose (100 mg·kg^–1. d^–1) and low-dose (50 mg·kg^–1·d^–1) treatments. These results indicated that compound groups could effectively inhibit the inflammatory factor IL-6. Figure 4d shows the content of the inflammatory factor TNF-α in the serum of the rat abdominal artery after treatment. It can be seen that the content of TNF-α in the blank group was abnormally increased, and the positive control group, compound 24 group, and compound 32 group were also decreased after high-dose (100 mg·kg^–1·d^–1) and low-dose (50 mg·kg^–1·d^–1) treatments. Among them, the decrease was more obvious after high-dose treatment, which also indicated that the compound group had the effect of inhibiting the inflammatory factor TNF-α. Results of the anti-rheumatoid arthritis activity evaluation showed that compounds 24 and 32 could effectively inhibit the paw swelling of rats. At the same time, they could effectively inhibit the content of the inflammatory factors IL-6 and TNF-α and exert a good anti-inflammatory effect in vivo. To study the toxicology of the target compounds in vivo, compounds 16, 23, 24, 32, and 36 were selected for the acute toxicity test, results of which showed that the target compounds were of low toxicity (Table 4).

Figure 4.

In vivo anti-rheumatoid arthritis activity. (a) Change of paw volume in rats at a dose of 50 mg·kg^–1·d^–1; (b) change of paw volume in rats at a dose of 100 mg·kg^–1·d^–1; (c) IL-6 concentrations; and (d) TNF-α concentrations. Compared with the normal group, the significance symbol was * = p < 0.05; compared with the blank group, the significance symbol was # = p < 0.05.

Figure 5.

Contents of the inflammatory factors TNF-α, IL-1β, and IL-6 in rat synovial cells are determined. (a) Content of the inflammatory factor TNF-α; (b) content of the inflammatory factors IL-1β; and (c) content of the inflammatory factors IL-6. Compared with the control group, the significance symbol was ** = p < 0.01; compared with the LPS group, the significance symbol was ## = p < 0.01.

Table 4. LD₅₀ Test of Results.

compounds	administration	LD50 (mg/kg)a ± SD
16	ig	2713.76 ± 9.28
23	ig	2307.43 ± 7.37
24	ig	2359.04 ± 7.95
32	ig	2657.65 ± 9.06
36	ig	2708.70 ± 8.63
methotrexateb	ig	1368.43 ± 5.34

^aValues were average of three independent experiments run in triplicate.

^bMethotrexate was used as the positive control.

2.4. Mechanism of Action of Anti-rheumatoid Arthritis

The mechanism of drug action is a theory that explains why or how drugs work, and it is an important content of pharmacodynamics research.²¹ Studying the mechanism of drug action is helpful to understand and master the effect of drug and guide rational drug use in clinic. With the development of modern science and technology, the level of understanding and research on the mechanism of drug action has been deepened and improved. As most drugs interfere with various physiological and biochemical processes by interacting with macromolecular components of tissues and organs, their mechanisms of action are diverse. The TLR4/NF-κB signaling pathway is an important pathway of inflammatory response and apoptosis, which plays a key role in the occurrence and development of RA, cerebral ischemia, tumor, and other diseases. TLR4 induces downstream NF-κB activation through the MyD88-dependent pathway and ultimately induces the expression of inflammatory cytokines and cytokines. NF-κB is a key transcription factor in inflammatory response. The NF-κB p65/p50 isodimer is the most common subunit form of NF-κB, which is usually inactivated by binding to its inhibitory protein IκB. Once stimulated, NF-κB p65 is phosphorylated to degrade IκB, leading to NF-κB activation. Activation of NF-κB not only aggravates inflammatory injury by stimulating the production of TNF-α, IL-6, and IL-1β but also induces synovial cells to regulate the expression levels of pro-apoptotic proteins such as TLR4, MyD88, NF-κB, and IκB. To investigate the anti-rheumatoid arthritis mechanism of the target compounds, we selected compound 32 for the relevant tests, including the determination of the inflammatory factors TNF-α, IL-1β, and IL-6 in the supernatant of rat synovial cells. The expression levels of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, NF-κB, and IκB in rat synovial cells were measured. As shown in Figure 5, compared with the normal group, the contents of the inflammatory factors TNF-α, IL-1β, and IL-6 in the supernatant of synovial cells of rats in the LPS group are significantly increased (p < 0.01). Compared with the LPS group, the contents of the inflammatory factors TNF-α, IL-1β, and IL-6 in the supernatant of 1 μM methotrexate and 1 μM compound 32 groups are significantly decreased (p < 0.01), suggesting that compound 32 could significantly inhibit the LPS-induced increase of TNF-α, IL-1β, and IL-6 in synovial cells. As shown in Figure 6, compared with the normal group, the expression of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, and NF-κB in the LPS group is significantly increased (p < 0.01), while the expression of the IκB protein is significantly decreased (p < 0.01). Compared with the LPS group, the expression of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, and NF-κB in 1 μM methotrexate and 1 μM compound 32 groups are significantly downregulated (p < 0.01), while the expression of the IκB protein is significantly upregulated (p < 0.01), suggesting that compound 32 can effectively regulate the expression levels of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, NF-κB, and IκB. This may be an important mechanism of inhibiting the LPS-induced inflammatory response of rat synovium cells, inhibiting abnormal cell proliferation and promoting apoptosis. In conclusion, compound 32 can effectively inhibit the LPS-induced abnormal increase of TNF-α, IL-1β, and IL-6 in rat synovial cells and regulate the expression of the TLR4/NF-κB signaling pathway-related proteins TLR4, MyD88, NF-κB, and IκB. This may be an important molecular mechanism for improving the abnormal proliferation of synovial cells in rats with rheumatoid arthritis and promoting inflammation and apoptosis.

Figure 6.

TLR4/NF-κB signaling pathway-related proteins expression. (a) Contents of TLR4; (b) contents of MyD88; (c) contents of NF-κB; and (d) contents of IκB. Compared with control the group, the significance symbol was ** = p < 0.01; compared with the LPS group, significance symbol was ## = p < 0.01.

2.5. Molecular Docking Study

Molecular docking of the TLR4-MD2 (PDB ID: 3FXI, Figure 7a) receptor was studied using AutoDock 4.2 modeling software.²² The docking results were processed with Pymol 2.1 to obtain molecular chimerism, and the positions of hydrogen bonds and binding bags were analyzed.²³Figure 7b shows that compound 32 binded to the TLR4-MD2 receptor and could enter the active pockets formed by ASN 114, VAL 113, and ARG 106. At the same time, compound 32 could also form hydrogen bonds with the residues ASN 114 and ARG 106 to increase its binding ability to the TLR4-MD2 receptor. The lowest binding free energy between compound 32 and the TLR4-MD2 receptor was −6.8 kcal/mol.²⁴ To study the difference between compounds 32 and 24, we performed molecular docking simulation on compound 24 (Figure 7c). The results of its docking showed that the hydrogen bond between compound 24 and the TLR4 receptor was very small, but the vacancy position was suitable to enter its pocket, which may be the reason for its good activity.

Figure 7.

Binding modes between TLR4-MD2 and compound 32. (a) Structure of TLR4-MD2 (PDB ID: 3FXI); (b) docking and binding pattern of compound 32 into the TLR4-MD2 active site; and (c) docking and binding pattern of compound 24 into the TLR4-MD2 active site.

3. Conclusions

In summary, the aim of this study was to identify the anti-rheumatoid arthritis activity of novel double-ring conjugated enones. The target compound 32 exhibited better anti-rheumatoid arthritis activity than that of the positive control methotrexate and showed less cytotoxicity and acute toxicity. In addition, results of the action mechanism showed that compound 32 could effectively inhibit the increase of TNF-α, IL-1β, and IL-6 induced by LPS and regulate the expression of TLR4, MyD88, NF-κB, and IκB in the signaling pathway of TLR4/NF-κB, which may be an important molecular mechanism for improving the abnormal proliferation of synovial cells in rats with rheumatoid arthritis and promoting inflammation and apoptosis. Molecular docking simulation showed that compound 32 could enter the pocket of the active site of the TLR4-MD2 receptor. Due to its excellent anti-rheumatoid arthritis activity, compound 32 can be used as a candidate drug for the treatment of RA, which needs further research and development.

4. Experimental Section

4.1. Chemistry Section

Reagents used in this research did not require further purification. Nuclear magnetic resonance (NMR) spectroscopy was performed using a Bruker AMX-400 spectrometer (IS as TMS). An Agilent 6460 system was used for mass spectrometry analysis. All final biotested compounds were analyzed by high-performance liquid chromatography (HPLC) using an Angelon 1260 series HPLC system. The purity was determined by reversed-phase HPLC, and the purity of all biotested compounds was ≥95%.

4.1.1. General Method for the Synthesis of Compound 4

Ethyl acetoacetate (compound 3, 12.6 mL, 0.10 mol) and ethanediol (6.6 mL, 0.12 mol) were reacted in a 50 mL solvent of benzene with a certain amount of p-methylbenzene sulfonic acid (p-TsOH, 0.5% mol) as a catalyst in a 250 mL round-bottom flask. The reaction was carried out under refluxing for 48 h. After that, the mixture was distilled in vacuum to remove the reaction solvent benzene and obtain a crude product of ethyl 2-(2-methyl-1, 3-dioxolan-2-yl) acetate (compound 4), which was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 3:1 was used as an eluent; the Rf value was 0.26) to acquire a colorless liquid of compound 4.

4.1.2. General Method for the Synthesis of Compounds 5–20

In a 250 mL round-bottom flask, compound 4 (0.10 mol) and 4-oxocyclohexanecarboxylic acid (0.10 mol) were reacted in a solvent of 50 mL of absolute ethyl alcohol, in which sodium ethanolate (0.10 mol) used as an alkaline substance for the reaction was added. The reaction lasted for 12 h under refluxing. After that, the mixture was distilled under vacuum to remove the solvent ethyl alcohol and then dissolved in 50 mL of 2 mol/L hydrochloric acid (HCl) solution and kept at the temperature of 60 °C for 2 h. The solution was extracted with 20 mL of ethyl acetate after cooling, and the organic layer was separated and washed with 20 mL of saturated sodium bicarbonate (NaHCO₃). After washing, the organic layer was dried with anhydrous sodium sulfate (Na2SO4) and filtered to collect the liquid. After evaporation under reduced pressure, the crude product compound 5 was obtained, which was then purified by silica gel column chromatography (petroleum ether/ethyl acetate = 4:1 was used as an eluent; the R_f value was 0.35) to acquire the pure product as a yellow oily liquid. Compounds 6–20 synthesized by the above-mentioned method were also yellow oily liquids.

4.1.3. General Method for the Synthesis of Compounds 21–36

In a 250 mL round-bottom flask, compound 5 (0.10 mol) and anhydrous potassium hydroxide (KOH, 0.10 mol) were reacted in 50 mL of absolute ethyl alcohol for 12 h under refluxing. After the reaction, the solution was distilled under reduced pressure to remove the solvent ethanol. The mixture was dissolved with 50 mL of deionized water, and its pH was adjusted to neutral with 2 mol/L hydrochloric acid (HCl). The solution was extracted with 20 mL of ethyl acetate and washed with 20 mL of saturated sodium bicarbonate (NaHCO3), the organic layer of which was collected and dried with anhydrous sodium sulfate (Na2SO4). Then, the organic solution was filtered and evaporated to obtain a crude product (2R)-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalene-2-carboxylic acid (compound 21). The residue after evaporation was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 4:1 was used as an eluent; the R_f value was 0.35), and the elution product named as compound 21 was a yellow oily liquid. The compounds 22–36 synthesized by the above-mentioned method were yellow oily liquids or light-yellow solids.

(2R)-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalene-2-carboxylic acid (Compound 21), yellow oily liquid, yield 53.5%; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.90–2.43 (4H, 2.00 (dd, J = 10.2, 3.4 Hz), 2.10 (dd, J = 10.3, 2.5 Hz), 2.27 (dt, J = 13.8, 3.0 Hz), 2.39 (dd, J = 13.6, 3.3 Hz)), 2.48–2.69 (2H, 2.58 (dd, J = 13.9, 2.3 Hz), 2.62 (dd, J = 10.2, 3.3 Hz)), 2.91–3.05 (2H, 2.97 (d, J = 13.3 Hz), 3.02 (tt, J = 3.1, 2.5 Hz)), 3.10–3.35 (2H, 3.17 (dd, J = 10.3, 3.0 Hz), 3.25 (d, J = 13.3 Hz)), 5.93 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ: 25.5, 28.6, 33.1, 43.5, 49.6, 57.4, 124.2, 171.9, 179.9, 201.8, 208.0; HR-ESI-MS m/z: calcd for C₁₁H₁₂O₄ {[M + H]⁺} 208.2130, found, 208.2131; Anal. calcd for C₁₁H₁₂O₄: C, 63.45; H, 5.81; O, 30.74; found: C, 63.44; H, 5.80; O, 30.73%.

(2R)-methyl-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalene-2-carboxylate (Compound 22), yellow oily liquid, yield 67.9%; ¹H NMR (400 MHz, DMSO-d₆) δ: 1.95–2.20 (2H, 2.02 (ddt, J = 13.5, 3.8, 2.0 Hz), 2.10 (dd, J = 13.5, 3.6 Hz)), 2.20–2.58 (4H, 2.30 (dd, J = 13.4, 5.7 Hz), 2.35 (dd, J = 10.3, 8.6 Hz), 2.46 (dd, J = 15.0, 2.0 Hz), 2.53 (dd, J = 10.1, 3.8 Hz)), 2.74 (1H, dd, J = 10.3, 2.0 Hz), 2.94–3.13 (2H, 3.03 (d, J = 13.5 Hz), 3.04 (d, J = 13.5 Hz)), 3.28 (1H, dd, J = 8.6, 5.7 Hz), 3.67 (3H, s), 6.01 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ: 20.8, 24.7, 27.2, 38.5, 48.5, 60.3, 65.4, 126.1, 171.5, 191.0, 209.9; HR-ESI-MS m/z: calcd for C₁₂H₁₄O₄ {[M + H]⁺} 222.2400, found, 222.2402; Anal. calcd for C₁₂H₁₄O₄: C, 64.85; H, 6.35; O, 28.80; found: C, 64.84; H, 6.34; O, 28.82%.

(2R)-ethyl-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalene-2-carboxylate (Compound 23), yellow oily liquid, yield 64.4%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.16 (3H, t, J = 7.1 Hz), 1.95–2.23 (2H, 2.03 (ddt, J = 13.5, 3.8, 2.0 Hz), 2.12 (dd, J = 13.5, 3.6 Hz)), 2.21–2.57 (4H, 2.30 (dd, J = 13.4, 2.6 Hz), 2.35 (dd, J = 10.3, 8.6 Hz), 2.47 (dd, J = 15.0, 3.6, 2.0 Hz), 2.52 (dd, J = 10.1, 3.8 Hz)), 2.72 (1H, dd, J = 10.3, 2.0 Hz), 2.94–3.12 (2H, 3.01 (d, J = 13.5 Hz), 3.04 (d, J = 13.5 Hz)), 3.31 (1H, dd, J = 8.6, 5.7 Hz), 4.04–4.18 (2H, 4.11 (q, J = 7.1 Hz), 4.12 (q, J = 7.1 Hz)), 6.01 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 14.7, 27.7, 28.5, 33.9, 44.7, 50.1, 56.2, 60.0, 121.9, 172.6, 175.7,201.1, 208.7; HR-ESI-MS m/z: calcd for C₁₃H₁₆O₄ {[M + H]⁺} 236.2670, found, 236.2668; Anal. calcd for C₁₃H₁₆O₄: C, 66.09; H, 6.83; O, 27.09; found: C, 66.10; H, 6.84; O, 27.07%.

(7R)-7-((1S,4S)-4-(p-tolyl) cyclohexyl)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 24), yellow oily liquid, yield 85.8%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.31–2.06 (14H, 1.35 (dd, J = 10.3, 2.8 Hz), 1.52 (dq, J = 12.7, 2.8 Hz), 1.55 (dd, J = 13.5, 2.8 Hz), 1.64 (dd, J = 13.9, 5.6 Hz), 1.73 (dq, J = 13.5, 2.8 Hz), 1.90 (dd, J = 13.3, 5.8 Hz), 1.95 (dd, J = 13.3, 6.3 Hz)), 2.27 (3H, s), 2.34–2.58 (2H, 2.45 (dd, J = 13.5, 5.6 Hz), 2.49 (dd, J = 13.5, 5.7 Hz)), 2.71 (1H, tt, J = 10.3, 2.8 Hz), 3.01 (1H, d, J = 13.2 Hz), 3.13–3.37 (2H, 3.23 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.2 Hz)), 5.97 (1H, s), 7.05 (2H, dd, J = 8.0, 1.3 Hz), 7.16 (2H, dd, J = 8.0, 1.1 Hz); ¹³C NMR (100MHz, DMSO-d₆) δ: 14.3, 20.8, 28.1, 30.8, 33.15, 34.27, 36.2, 41.9, 44.7, 50.4, 60.0, 124.2, 126.3, 129.0, 135.1, 144.9, 171.6, 180.4, 201.0, 207.7; HR-ESI-MS m/z: calcd for C₂₃H₂₈O₂ {[M + H]⁺} 336.4750, found, 336.4748; Anal. calcd for C₂₃H₂₈O₂: C, 82.10; H, 8.39; O, 9.51; found: C, 82.11; H, 8.37; O, 9.52%.

(7R)-7-((1S, 4S)-4-(3, 4-difluorophenyl) cyclohexyl)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 25), yellow oily liquid, yield 83.2%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.30–2.06 (14H, 1.35 (dd, J = 10.3, 2.8 Hz), 1.52 (dq, J = 12.7, 2.8 Hz), 1.55 (dd, J = 13.5, 2.8 Hz), 1.64 (dd, J = 13.9, 5.6 Hz), 1.73 (dq, J = 13.5, 2.8 Hz), 1.90 (dd, J = 13.3, 5.8 Hz), 1.95 (dd, J = 13.3, 6.3 Hz)), 2.27 (3H, s), 2.34–2.58 (2H, 2.45 (dd, J = 13.5, 5.6 Hz), 2.49 (dd, J = 13.5, 5.7 Hz)), 2.71 (1H, tt, J = 10.3, 2.8 Hz), 3.01 (1H, d, J = 13.2 Hz), 3.13–3.37 (2H, 3.23 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.2 Hz)), 5.97 (1H, s), 7.20–7.43 (3H, 7.28 (dd, J = 1.0, 0.5 Hz), 7.31 (dd, J = 8.4, 1.0 Hz), 7.35 (dd, J = 8.4, 0.5 Hz)); ¹³C NMR (100MHz, DMSO-d₆) δ: 23.9, 28.1, 30.3, 32.7, 34.5, 35.9, 42.8, 43.9, 60.4, 66.9, 115.3,116.9, 122.6, 145.0, 146.5, 151.5, 171.1, 191.0, 209.6; HR-ESI-MS m/z: calcd for C₂₂H₂₄F₂O₂ {[M + H]⁺} 358.4288, found, 358.4289; Anal. calcd for C₂₂H₂₄F₂O₂: C, 73.72; H, 6.75; F, 10.60; O, 8.93; found: C, 73.70; H, 6.76; F, 10.60; O, 8.94%.

(7R)-7-((1R, 4R)-4-pentylcyclohexyl)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 26), yellow oily liquid, yield 88.3%; ¹H NMR (400MHz, DMSO-d₆) δ: 0.83 (3H, s), 1.16–1.69 (14H, 1.25 (dd, J = 6.9, 3.6 Hz), 1.31 (dd, J = 10.3, 2.8 Hz), 1.38 (dd, J = 10.3, 2.8 Hz), 1.47 (dd, J = 13.0, 2.8 Hz), 1.52 (dd, J = 10.3, 1.9 Hz), 1.59 (dd, J = 3.6, 2.8 Hz)), 1.75–2.13 (3H, 1.87 (dd, J = 13.9, 5.7 Hz), 1.91 (dd, J = 13.2, 5.8 Hz), 2.04 (dd, J = 13.2, 6.3 Hz)), 2.33–2.58 (2H, 2.46 (dd, J = 13.5, 8.9 Hz), 2.49 (dd, J = 13.5, 1.4 Hz)), 3.04 (1H, d, J = 13.2 Hz), 3.11–3.38 (2H, 3.23 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.2 Hz)), 5.98 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 11.8, 26.5, 29.4, 30.8, 31.2, 34.5, 35.1, 36.3, 38.6, 41.6, 48.8, 56.6, 124.1, 163.2, 191.3, 207.1; HR-ESI-MS m/z: calcd for C₁₈H₂₆O₂ {[M + H]⁺} 274.4040, found, 274.4037; Anal. calcd for C₁₈H₂₆O₂: C, 78.79; H, 9.55; O, 11.66; found: C, 78.78; H, 9.56; O, 11.67%.

(7R)-7-((1R, 4R)-4-ethylcyclohexyl)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 27), yellow oily liquid, yield 86.8%; ¹H NMR (400MHz, DMSO-d₆) δ: 0.84 (3H, s), 1.16–1.69 (20H, 1.22 (tt, J = 6.9, 6.5 Hz), 1.26 (tt, J = 6.8, 6.5 Hz), 1.28 (dd, J = 6.9, 6.6 Hz), 1.31 (dtd, J = 12.9, 10.3, 2.8 Hz), 1.31 (dtd, J = 12.9, 10.3, 2.8 Hz), 1.31 (dd, J = 10.3, 2.8 Hz), 1.38 (dd, J = 10.3, 2.8 Hz), 1.47 (dd, J = 13.0, 2.8 Hz), 1.52 (dd, J = 10.3, 1.9 Hz), 1.59 (dd, J = 3.6, 2.8 Hz)), 1.75–2.13 (3H, 1.87 (dd, J = 13.9, 5.7 Hz), 1.91 (dd, J = 13.2, 5.8 Hz), 2.04 (dd, J = 13.2, 6.3 Hz)), 2.33–2.58 (2H, 2.46 (dd, J = 13.5, 8.9 Hz), 2.49 (dd, J = 13.5, 1.4 Hz)), 3.04 (1H, d, J = 13.2 Hz), 3.11–3.38 (2H, 3.23 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.2 Hz)), 5.98 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 14.3, 17.8, 20.8, 22.8, 26.7, 28.1, 30.1, 32.5, 33.5, 37.3, 37.8, 41.9, 57.4, 126.5, 171.5, 191.8, 208.3; HR-ESI-MS m/z: calcd for C₂₁H₃₂O₂ {[M + H]⁺} 316.4850, found, 316.4853; Anal. calcd for C₂₁H₃₂O₂: C, 79.70; H, 10.19; O, 10.11; found: C, 79.71; H, 10.20; O, 10.08%.

(7R)-7-(4-oxocyclohexyl)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 28), yellow oily liquid, yield 56.8%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.40–1.74 (5H, 1.50 (dd, J = 10.2, 2.5 Hz), 1.58 (dd, J = 10.2, 11.9 Hz), 1.64 (dd, J = 13.9, 1.4 Hz)), 1.79–2.09 (5H, 1.89 (dd, J = 13.9, 5.7 Hz), 1.98 (dd, J = 13.2, 6.3 Hz)), 2.23–2.57 (6H, 2.32 (dd, J = 14.4, 2.3 Hz), 2.35 (dd, J = 14.4, 2.3 Hz), 2.37 (dd, J = 10.2, 3.3 Hz), 2.46 (dd, J = 13.5, 8.9 Hz)), 3.01 (1H, d, J = 13.2 Hz), 3.12–3.36 (2H, 3.20 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 16.2 Hz)), 5.98 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 31.2, 32.5, 34.5, 35.1, 35.8, 41.4, 41.7, 48.8, 56.6, 124.1, 163.2, 191.3, 207.1, 210.7; HR-ESI-MS m/z: calcd for C₁₆H₂₀O₃ {[M + H]⁺} 260.3330, found, 260.3327; Anal. calcd for C₁₆H₂₀O₃: C, 73.82; H, 7.74; O, 18.44; found: C, 73.80; H, 7.74; O, 18.46%.

(7R)-7-phenyl-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 29), yellow oily liquid, yield 76.9%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.75 (1H, dd, J = 14.2, 1.4 Hz), 1.89–2.28 (3H, 1.97 (dd, J = 14.2, 5.7 Hz), 2.02 (dd, J = 13.5, 5.8 Hz), 2.16 (dd, J = 13.5, 6.3 Hz)), 2.40–2.57 (2H, 2.47 (dd, J = 13.5, 5.6 Hz), 2.54 (dd, J = 13.5, 1.4 Hz)), 2.92–3.14 (2H, 2.98 (dd, J = 10.0, 1.9 Hz), 3.09 (d, J = 13.2 Hz)), 3.20–3.48 (2H, 3.31 (d, J = 13.2 Hz), 3.43 (dd, J = 8.1, 6.3 Hz)), 5.96 (1H, s), 7.09–7.33 (5H, 7.12 (dd, J = 7.8, 0.5 Hz), 7.17 (tt, J = 7.7, 1.3 Hz), 7.26 (dd, J = 7.8, 1.8 Hz)); ¹³C NMR (100MHz, DMSO-d₆) δ: 33.7, 34.5, 37.1, 39.2, 48.3, 56.6, 124.1, 126.1, 128.2, 128.7, 141.1, 163.2, 191.3, 207.1; HR-ESI-MS m/z: calcd for C₁₆H₁₆O₂ {[M + H]⁺} 240.3020, found, 240.3018; Anal. calcd for C₁₆H₁₆O₂: C, 79.97; H, 6.71; O, 13.32; found: C, 79.95; H, 6.73; O, 13.31%.

4-((2R)-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) benzonitrile (Compound 30), yellow oily liquid, yield 74.1%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.77 (1H, dd, J = 14.2, 1.4 Hz), 1.89–2.28 (3H, 1.97 (dd, J = 14.2, 5.7 Hz), 2.02 (dd, J = 13.5, 5.8 Hz), 2.16 (dd, J = 13.5, 6.3 Hz)), 2.40–2.57 (2H, 2.47 (dd, J = 13.5, 5.6 Hz), 2.54 (dd, J = 13.5, 1.4 Hz)), 2.92–3.14 (2H, 2.98 (dd, J = 10.0, 1.9 Hz), 3.09 (d, J = 13.2 Hz)), 3.20–3.48 (2H, 3.31 (d, J = 13.2 Hz), 3.43 (dd, J = 8.1, 6.3 Hz)), 5.96 (1H, s), 7.31 (2H, dd, J = 8.8, 0.5 Hz), 7.84 (2H, dd, J = 8.8, 1.9 Hz); ¹³C NMR (100MHz, DMSO-d₆) δ: 33.7, 34.5, 37.1, 39.2, 48.3, 56.6, 109.8, 118.5, 124.1, 128.7, 132.4, 145.4, 163.2, 191.3, 207.1; HR-ESI-MS m/z: calcd for C₁₇H₁₅NO₂ {[M + H]⁺} 265.3120, found, 265.3123; Anal. calcd for C₁₇H₁₅NO₂: C, 76.96; H, 5.70; N, 5.28; O, 12.06; found: C, 76.95; H, 5.71; N, 5.29; O, 12.05%.

(7R)-7-heptyl-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 31), yellow oily liquid, yield 79.6%; ¹H NMR (400MHz, DMSO-d₆) δ: 0.85 (3H,s), 1.13–1.38 (10H, 1.23 (tt, J = 6.9, 6.5 Hz), 1.26 (dd, J = 6.8, 4.3 Hz), 1.29 (dd, J = 7.0, 6.9 Hz)), 1.49–2.11 (5H, 1.58 (dd, J = 13.7, 1.4 Hz), 1.69 (dd, J = 10.0, 1.9 Hz), 1.88 (dd, J = 13.7, 5.7 Hz), 2.03 (dd, J = 13.4, 6.3 Hz)), 2.35–2.58 (2H, 2.46 (dd, J = 13.5, 5.6 Hz), 2.49 (dd, J = 13.5, 1.4 Hz)), 3.01 (1H, d, J = 16.2 Hz), 3.10–3.37 (2H, 3.19 (dd, J = 8.1, 6.3 Hz), 3.27 (d, J = 13.2 Hz)), 5.96 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 14.5, 21.2, 22.2, 29.3, 31.7, 36.1, 38.4, 50.6, 60.1, 125.3, 170.2, 192.5, 207.0; HR-ESI-MS m/z: calcd for C₁₇H₂₆O₂ {[M + H]⁺} 262.3930, found, 262.3928; Anal. calcd for C₁₇H₂₆O₂: C, 77.82; H, 9.99; O, 12.19; found: C, 77.80; H,10.00; O, 12.20%.

(7R)-7-hydroxy-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 32), yellow oily liquid, yield 51.8%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.67 (1H, dd, J = 10.5, 1.4 Hz), 1.82–2.26 (3H, 1.96 (dd, J = 10.5, 5.7 Hz), 2.07 (dd, J = 14.2, 5.8 Hz), 2.13 (dd, J = 14.2, 6.3 Hz)), 2.24–2.49 (2H, 2.36 (dd, J = 13.2, 5.6 Hz), 2.39 (dd, J = 13.2, 1.4 Hz)), 3.01 (1H, d, J = 13.1 Hz), 3.22–3.38 (2H, 3.28 (dd, J = 8.1, 6.3 Hz), 3.31 (d, J = 13.1 Hz)), 3.85 (1H, dd, J = 10.0, 1.9 Hz), 5.96 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 330.1, 32.4, 37.0, 42.8, 57.5, 68.9, 124.6, 172.6, 191.1, 211.1; HR-ESI-MS m/z: calcd for C₁₀H₁₂O₃ {[M + H]⁺} 180.2030, found, 180.2029; Anal. calcd for C₁₀H₁₂O₃: C, 66.65; H, 6.71; O, 26.63; found: C, 66.64; H, 6.73; O, 26.62%.

N-((2R)-6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) acetamide (Compound 33), yellow oily liquid, yield 59.4%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.73 (1H, dd, J = 10.9, 1.4 Hz), 1.82–2.07 (4H, 1.88 (s), 1.97 (dd, J = 10.9, 5.7 Hz)), 2.16 (1H, dd, J = 13.3, 5.8 Hz), 2.30–2.58 (3H, 2.39 (dd, J = 13.3, 6.3 Hz), 2.42 (dd, J = 13.1, 5.6 Hz), 2.49 (dd, J = 13.1, 1.4 Hz)), 3.01 (1H, d, J = 13.1 Hz), 3.11–3.37 (2H, 3.19 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.1 Hz)), 3.88 (1H, dd, J = 10.0, 1.9 Hz), 5.96 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 23.5, 26.4, 29.5, 31.4, 45.6, 56.6, 58.3, 124.1, 163.2, 170.5, 191.3, 207.1; HR-ESI-MS m/z: calcd for C₁₂H₁₅NO₃ {[M + H]⁺} 221.2560, found, 221.2558; Anal. calcd for C₁₂H₁₅NO₃: C, 65.14; H, 6.83; N, 6.33; O, 21.69; found: C, 65.13; H, 6.84; N, 6.32; O, 21.70%.

(7R)-7-(benzyloxy)-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 34), yellow oily liquid, yield 68.3%; ¹H NMR (400MHz, DMSO-d₆) δ: 1.74 (1H, dd, J = 14.5, 1.4 Hz), 1.90–2.45 (5H, 2.03 (dd, J = 14.5, 5.7 Hz), 2.17 (dd, J = 14.2, 5.8 Hz), 2.34 (dd, J = 13.2, 5.6 Hz), 2.40 (dd, J = 13.2, 1.4 Hz)), 3.01 (1H, d, J = 13.1 Hz), 3.18–3.38 (2H, 3.26 (dd, J = 8.1, 6.3 Hz), 3.29 (d, J = 13.1 Hz)), 3.95 (1H, dd, J = 10.0, 1.9 Hz), 4.50–4.65 (2H, 4.55 (s), 4.63(s)), 5.96 (1H, s), 7.25–7.53 (5H, 7.33 (tt, J = 7.5, 1.3 Hz), 7.39 (dd, J = 7.8, 0.5 Hz), 7.43 (dd, J = 7.8, 10.5 Hz)); ¹³C NMR (100MHz, DMSO-d₆) δ: 30.5, 31.1, 32.2, 44.8, 56.6, 72.6, 81.4, 124.1, 127.5, 127.7, 128.5, 137.5, 163.2, 191.3, 207.1; HR-ESI-MS m/z: calcd for C₁₇H₁₈O₃ {[M + H]⁺} 270.3280, found, 270.3283; Anal. calcd for C₁₇H₁₈O₃: C, 75.53; H, 6.71; O, 17.76; found: C, 75.51; H, 6.70; O, 17.78%.

7,7-dimethyl-6, 7, 8, 8a-tetrahydronaphthalene-1, 3 (2H, 5H)-dione (Compound 35), yellow oily liquid, yield 77.8%; ¹H NMR (400MHz, DMSO-d₆) δ: 0.90–1.05 (6H, 0.96 (s)), 1.43 (1H, dd, J = 14.4, 1.4 Hz), 1.55–1.78 (2H, 1.64 (dd, J = 14.4, 5.7 Hz), 1.69 (dd, J = 13.0, 8.1 Hz)), 1.89 (1H, dd, J = 13.0, 6.3 Hz), 2.30–2.56 (2H, 2.38 (dd, J = 13.2, 5.6 Hz), 2.49 (dd, J = 13.2, 1.4 Hz)), 3.01 (1H, d, J = 13.2 Hz), 3.20–3.45 (2H, 3.29 (d, J = 13.2 Hz), 3.39 (dd, J = 8.1, 6.3 Hz)), 5.96 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 14.0, 29.7, 30.8, 31.6, 40.8, 60.4, 126.1, 171.5, 191.4, 209.9; HR-ESI-MS m/z: calcd for C₁₂H₁₆O₂ {[M + H]⁺} 192.2580, found, 192.2577; Anal. calcd for C₁₂H₁₆O₂: C, 74.97; H, 8.39; O, 16.64; found: C, 74.98; H, 8.37; O, 16.65%.

7,7-difluoro-6, 7, 8, 8a-tetrahydronaphthalene-1,3 (2H, 5H)-dione (Compound 36), yellow oily liquid, yield 69.2%; ¹H NMR (400MHz, DMSO-d₆) δ: 2.00–2.28 (2H, 2.09 (dd, J = 14.1, 1.4 Hz), 2.16 (dd, J = 14.1, 5.7 Hz)), 2.33–2.67 (4H, 2.42 (dd, J = 13.3, 8.1 Hz), 2.45 (dd, J = 13.5, 5.6 Hz), 2.53 (dd, J = 13.5, 1.4 Hz), 2.57 (dd, J = 13.3, 6.3 Hz)), 3.01 (1H, d, J = 13.1 Hz), 3.20–3.37 (2H, 3.29 (d, J = 13.1 Hz), 3.33 (dd, J = 8.1, 6.3 Hz)), 5.96 (1H, s); ¹³C NMR (100MHz, DMSO-d₆) δ: 22.7, 34.3,35.4, 45.4, 60.0, 115.3, 122.3, 171.5, 192.3, 208.4; HR-ESI-MS m/z: calcd for C₁₀H₁₀F₂O₂ {[M + H]⁺} 200.1848, found, 200.1845; Anal. calcd for C₁₀H₁₀F₂O₂: C, 60.00; H, 5.04; F, 18.98; O, 15.98; found: C, 60.01; H, 5.05; F, 18.97; O, 15.97%.

4.2. Biology Activity Section

4.2.1. Determination of the Inhibitory Effect of Rat Synovial Cells

Rat synovial cells were taken from the logarithmic phase of the growth cycle, and the cell density was adjusted. It was inoculated into 96-well plates (10⁴ cells/well) of 100 μL of medium, which was placed in an incubator at 37 °C with 5% CO₂ for 24 h. Then, the cultured cells were randomly divided into the normal group, DMSO group, positive control group (methotrexate group), and compound groups. The normal group was given no substance, and the DMSO group was given 20 μL of 5‰ dimethyl sulfoxide. The positive control and target compound groups were given 20 μL of the test substance with different concentration gradients. The four groups of plates were incubated for 48 h. After adding 20 μL of CCK8 solution, each orifice plates were incubated for 3–4 h, the OD value of which was measured using a microplate reader at a wavelength of 450 nm. The cell inhibition rate of each group was calculated as follows: cell inhibition rate (%) = [(OD_{normal group} – OD_{sample group})/OD_{normal group}] × 100%.

4.2.2. Morphology of Rat Synovial Cells In Vitro

When the rat synovial cells of rats were at 70–80% fusion in the logarithmic growth phase, the cell density was adjusted after digestion with 0.25% trypsin. On a 24-well culture plate, 5000 cells per well were inoculated and randomly divided into the normal group, DMSO group, control group (methotrexate group), and compound group. Methotrexate (1 μM) and the test compound (1 μM) were added to each group except the normal one. Each group with the same amount of medium was continued to be cultured for 48 h, the morphological change of which was observed by cell imaging analysis.

4.2.3. Determination of the Viability Effect of Synovial Cells with LDH Activity Assay In Vitro

The density of rat synovial cells from the logarithmic phase of the growth cycle was adjusted, which was then inoculated into 100 μL of medium in a 96-well plate with 2 × 10⁴ cells/well. The orifice plates were cultivated in an incubator at 37 °C with 5% CO₂ for 48 h. Then, the cultured cells were randomly divided into the normal group, DMSO group, positive control group (methotrexate group), and compound groups. The normal group was given no substance, and the DMSO group was given 5% dimethyl sulfoxide (20 μL). The positive control group and the target compound group were given 20 μL of the test substance with different concentration gradients. After incubation for 48 h, the orifice plates were centrifuged at 2000r for 10 min, the supernatant of which was collected to detect the activity using a lactate dehydrogenase (LDH) assay kit. The main steps were referred to the kit instruction. The calculation formula of LDH activity was as follows: LDH activity (U/L) = [(OD_{sample group} – OD_{control group})/(OD_{normal group} – OD_blank group)] × 0.2 mM ×1000.

4.2.4. In Vitro TLR4 Inhibition Test

Before the test, all reagents should be recovered to room temperature and gently mixed with the sample to avoid foaming. The TLR4 test kit (USCN) provided blank holes, control holes, and sample holes. In addition to the blank hole, 100 μL of the positive control (methotrexate) or sample was added to the remaining holes with different concentrations. The sample was added to the bottom of the substrate without contacting the wall of the hole and mixed by gentle shaking. The plates were sealed and incubated at 37 ° C for 90 min. After discarding the liquid, the plates were added with 100 μL of biotinic antibody working liquid and incubated at 37 °C for 60 min. The liquid was discarded again. Then, the plates were washed by soaking 1–2 min three times. Next, the plates were clapped slightly on the absorbent paper to remove the liquid in the hole. Then, each hole was added with 100 μL of enzyme conjugate working solution (HRP enzyme conjugate) and incubated at 37 °C for 30 min with film mulching. After discarding the liquid again, the plates were soaked five times (each time for 1–2 min) and then clapped slightly on the absorbent paper to remove the liquid. After that, the enzyme plate, each hole of which was added with 90 μL of substrate solution, was incubated at 37 °C for about 15 min with film covering. Each hole was added with end liquid of 50 μL to terminate the reaction, and its color changed from blue to yellow. The light density was measured immediately using an enzyme marker at 450 nm wavelength (OD value).

4.2.5. Complete Freund’s Adjuvant Induced Arthritis in Rats

Complete Freund’s adjuvant (CFA) induced using Freund’s adjuvant was tested in SPF-grade adult rats (200 ± 20 g, China). The rats were fed with conventional feed and free water in a feeding room with a temperature of 22 ± 3 °C, relative humidity of 40–70%, and no convection wind. After 2 weeks of acclimatization, rats in the blank control group, positive control group, and compound group were injected with 0.1 mL of complete Freund’s adjuvant (CFA) at 2 cm from the tail root to induce inflammation, and the normal group was injected with 0.1 mL of normal saline. After 14 days of inflammation, rats in the positive control and compound group were orally given high (100 mg·kg^–1·d^–1) and low (50 mg·kg^–1·d^–1) doses of the test substance daily for 14 days, while the blank control and normal groups were not given the treatment drug. Before inflammation and on the 1st and 14th day of administration, the paw volume of rats was measured using an SA701 toe volume meter. After the 14th day of administration, all rats used for the test were killed and dissected. Blood samples from the rat abdominal aorta were collected, and the levels of the inflammatory cytokines IL-6 and TNF-α in serum were determined using an ELISA kit (RAB0307).

4.2.6. Acute Toxicity Test in Mice

Mice with a body weight of 18–22 g and specific pathogen-free (SPF) characteristics were selected and reared in a feeding chamber with a temperature of 22 ± 2 °C, relative humidity of 40–70% and no convection air. Mice were exposed to 12 h of lighting (from 6 a.m. to 6 p.m.) and 12 h of darkness and fed with conventional laboratory feed and free water. After one week of acclimatization, the mice were randomly divided into 10 groups with half male and half female. According to the requirements of the experiment, the isodose groups were divided into six groups. Before gavage, the mice were fasted for 8 h. After a one-time gavage of 0.4 mL of the test substance, the mice were fasted for 2 h and then fed normally. The mortality, death time, and poisoning of all mice were observed within 14 days. The lethal dose (LD₅₀) was used to indicate the acute toxicity.

4.3. Mechanism Section

4.3.1. Determination of Inflammatory Cytokines in the Rat Synovial Cell Supernatant

When the rat synovial cells of rats had grown to 70–80% fusion in the logarithmic growth phase, the cell density was adjusted after digestion with 0.25% trypsin. Twenty thousand cells per well were inoculated on a 24-well culture plate in black, which were randomly divided into the normal group, LPS group, LPS+methotrexate group, and LPS+compound group. After 24 h of incubation, the rat synovial cells were injected with LPS (1 μg·mL^–1) for 24 h. With the same amount of medium, methotrexate (1 μM) and the test compound (1 μM) were added in the other groups except for the normal group and LPS group. After continuous intervention for 24 h, the top layer of each group was collected and centrifuged at 3000 rpm for 15 min. Then, the levels of TNF-α, IL-1β, and IL-6 in the supernatant of cells in each group were detected according to the instructions of the ELISA kit (RAB0307 Sigma-Aldrich).

4.3.2. Determination of the TLR4/NF-κB Signaling Pathway in Rat Synovial Cells

When the rat synovial cells of rats had grown to 70–80% fusion in logarithmic growth phase, the cell density was adjusted after digestion with 0.25% trypsin. Five thousand cells per well were inoculated on a 96-well culture plate in black, which were randomly divided into the normal group, LPS group, LPS+methotrexate group, and LPS+compound group. The cells were incubated for 24 h and then injected with LPS (1 μg·mL^–1) for 24 h. With the same amount of medium, methotrexate (1 μM) and the test compound (1 μM) were added in the other groups except for the normal group and LPS group. After continuous intervention treatment for 24 h, the fluid in the hole was discarded, cooled with 0.01 M PBS, and moistened twice. The fluid was fixed with 4% cell fixation solution for 45 min, treated with 0.25% Triton-100 for 15 min, and sealed with 5% BSA for 10 min. Then, the rabbit anti-rat TLR4, MyD88, NF-κB, and IκB primary antibody diluent (1:100 or 1:200) was added to the fluid and incubated overnight in the dark at 4 °C. After discarding the diluent, the plate was rinsed with 0.01 M PBS five times and added with the FITC-labeled goat anti-rabbit secondary antibody diluent (volume ratio: 1:400), which was incubated for 30 min in the dark at 37 °C. Then, the solution was discarded, and the plate was cooled with 0.01 M PBS and moistened four times. The DAPI diluent (volume ratio: 1:800) was added and incubated for 10 min at room temperature without light. After discarding the solution, it was cooled with 0.01 M PBS again and moistened three times. The relative expression levels of TLR4, MyD88, NF-κB, and I-κB were determined by the mean fluorescence intensity of the cells.

4.4. Molecular Docking Simulation

Based on the semiflexible principle, AutoDock 4.2 software was used for molecular docking simulation. The active site was located in the TLR4-MD2 dimer protein structure (PDB ID: 3FXI). Before the docking simulation with Autodock 4.2 software, the ligands were dehydrated and dehydrogenated using PyMOL 2.1. The mesh frame centered on the active site of TLR4-MD2 was 30 Å in size. The validity of the docking method was verified by the position and pose retrieval of the cocrystal ligand of the TLR4-MD2 dimer structure. Meanwhile, Discovery Studio 4.5 and Pymol 2.1 were used to analyze the polarity interaction of the complexes with the lowest binding free energy.

4.5. Data Analysis

SPSS 20.0 software (methods: Duncan’s multiple range test) was used to analyze all the trial data, and the data results were expressed as mean ± standard deviation (SD). One-way ANOVA was used for statistical analysis to determine the significance of the difference between the two groups. The significance level of the analysis results was set as a probability value less than 0.05 (p < 0.05).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c05492.

• ¹H NMR and ¹³C NMR spectra of compounds 21–25, 27, 31, 32, 35, and 36, HPLC diagram of the compounds 24 and 32, and ECD plots of the compounds 24 and 32 (PDF)

The authors declare no competing financial interest.