Synthesis and Biological Evaluation of Naproxen Derivatives as Novel NLRP3 Inhibitors

Yonglian Li; Zonglin You; Vincent Kam Wai Wong; Min Chen; Kun Zhang; Wenfeng Liu; Suqing Zhao

doi:10.1248/cpb.c24-00465

Abstract

Naproxen, widely used to treat anti-inflammatory diseases, would cause serious of side effects. Based on the biological activities of cinnamic acid, naproxen derivatives containing cinnamic acid were designed, synthesized and used to enhance their anti-inflammatory activities and safeties. The results investigated that thirty novel naproxen derivatives had inhibitory effects on the nitric oxide (NO) release in RAW264.7 macrophage cells. A majority of naproxen derivatives showed the lower degree of cytotoxicity than that of naproxen. In vitro studies revealed that A22 (IC₅₀ = 7.38 ± 1.96 µM) blocked the activation of nuclear transcription factor κB (NF-κB) signaling pathway and pyrin domain containing protein 3 (NLRP-3) inflammasome in a concentration dependent manner, thereby down-regulating the expression of pro-inflammatory cytokines, such as interleukin (IL)-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Docking studies confirmed that A22 exhibited a well-fitting into the NLRP3 active site. Accordingly, A22 might be a novel NLRP3 inhibitor to treat inflammatory diseases.

Introduction

It is indicated that inflammation, caused by the exogenous or endogenous pathogens, was confirmed to be a defense reaction of the tissues to injury.¹⁾ In addition, inflammation is reported to result in the over-expression of pro-inflammatory factors, suggesting that it would play an important role in inflammatory diseases, including myocarditis, arthritis, Alzheimer’s disease, inflammatory bowel disease and even cancers.^2–6) Recent studies demonstrate that macrophages, closely related to the release of pro-inflammatory factors, were the important cell to regulate the inflammatory response.⁷⁾ Lipopolysaccharide (LPS), produced by Gram-negative bacteria, is a type of endotoxin and activator of innate immune responses.⁸⁾ It would stimulate macrophages to up-regulate various pro-inflammatory factors, including nitric oxide (NO), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).^9,10) As shown in Fig. 1, when the macrophages was activated with LPS, the secretion of pro-inflammatory factors was up-regulated and nuclear transcription factor κB (NF-κB) signaling pathway was activated.^11,12) Furthermore, pyrin domain containing protein 3 (NLRP-3) inflammasome, activated by LPS, was a key intracellular pattern recognition receptor. It could mediate the secretion of IL-1β.¹³⁾ Taken together, these provided an effective strategy to inhibit the activation of NF-κB signaling pathway and the production of NLRP3 inflammasome for the treatment of inflammation.

Fig. 1. Simplified View of the NF-κB and NLRP3 Inflammasome Activation Pathway

Naproxen, possessing a free carboxyl group, exhibited anti-inflammatory, analgesic, anti-pyretic and anti-cancer activity. It was currently used for the treatment of inflammation and inflammatory diseases.^14–18) However, long-term naproxen use caused serious adverse effects.¹⁹⁾ Recent studies provided several therapeutic strategies to treat inflammation and inflammatory diseases.^20–22) Structurally modifying naproxen with naturally active constituents was one of the effective strategies. It was revealed that naproxen and its derivatives containing curcumin or magnolol were conformed to suppress phorbol-12-myristate-13-acetate (TPA)-stimulated inflammation by inhibiting the mitogen-activated protein kinase (MAPK)-NF-κB signaling pathway, respectively.^20,21) Additionally, naproxen derivatives containing oleanolic acid was reported to be responsible for the anti-inflammatory effect of naproxen and oleanolic acid.²²⁾ Thiourea derivatives of naproxen had anti-inflammatory activity in vitro and in vivo.²³⁾ Furthermore, naproxen-based hydrazide derivatives are designed and synthesized. The results reveal that they might be potent COX-2 inhibitors.²⁴⁾ Hence, retaining the original skeletal structure of naproxen and modifying the structure of naproxen might produce novel bioactive compounds with great pharmacological potential.

Cinnamic acid was a naturally occurring aromatic fatty acid which extracted from cinnamon.²⁵⁾ It had low toxicity and good anti-inflammatory activity.^26,27) It was investigated that cinnamic acid derivatives blocked inflammatory response by suppressing the over-expression of pro-inflammatory cytokines, such as IL-6 and tumor necrosis factor α (TNF-α).²⁸⁾ Additionally, phenolic cinnamic acid derivatives were confirmed to show increased COX-2 inhibitory potency.²⁹⁾ It was reported that cinnamamide derivatives inhibited inflammation in sepsis and acute lung injury by targeting MD-2.³⁰⁾ Herein, cinnamic acid was selected as the natural-product-derived fragment to develop novel naproxen-derived compounds as NLRP3 inhibitors.

The aim of the present study is to obtain novel NLRP3 inhibitors, we synthesized 30 naproxen derivatives containing cinnamic acids. The in vitro anti-inflammatory activity of all target compounds was determined against NO production in RAW264.7 cells. These results were indicated that compound A22 showed a markedly inhibitory effect against NO production. Moreover, the anti-inflammatory mechanism of compound A22 was studied in present study. The experiments revealed that compound A22 could inhibit the over-expression of pro-inflammatory cytokines and suppress the activation of NF-κB signaling pathway and NLRP-3 inflammasome. This study identified naproxen derivatives having anti-inflammatory activity, establishing a theoretical foundation for the design of novel NLRP3 inhibitors.

Results and Discussion

Chemistry

Following the synthetic route showed in Chart 1, a series of naproxen derivatives (A1–A30) incorporating the cinnamic acids moiety were synthesized. Intermediate compound (1) was synthesized using slightly modified procedures.³⁰⁾ The target compounds A1–A30 were synthesized by using compound (1), different cinnamic acids, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) and 4-dimethylaminopyridine (DMAP) as the starting materials. The chemical structures of compounds A1–A30 were confirmed by melting points, NMR spectroscopy and LC-MS spectrometry. In present study, the inhibitory effect and mechanism of compounds A1–A30 were determined in vitro to access the potency of the target compounds.

Chart 1. Reagents and Conditions

(a) 48% hydrogen bromide, acetic acid, reflux, 3 h; (b) tert butyl alcohol, chlorotrimethylsilane, 25 °C, 2 h; (c) different aromatic aldehydes, pyridine, piperidine, propanedioic acid; (d) EDCI, DMAP, dichloromethane, 25 °C, 2 h.

The chemical structures of all target compounds were defined in present study. In the ¹H-NMR spectrum for compounds A1–A30, the olefinic protons appeared as doublet with coupling constant J = 16 Hz. The result indicated that the double bond in all target compounds should be trans double bond. The protons related to the CH showed as a quadruplet with the range of 3.89–3.80 ppm. The CH₃ proton signals were observed as doublets between 1.58 and 1.48 ppm. Moreover, in the ¹³C-NMR spectrum for compounds A1–A30, the O=C–O signals were detected between 175.79 and 163.62 ppm. In addition, the C(CH₃)₃ signals were detected as singlets with the range of 1.44–1.42 ppm in the ¹H-NMR spectrum for compounds A1–A30.

Biological Evaluation

Inhibitory Effect and Structure–Activity Relationships (SARs) of the Compounds on NO Production

It is investigated that once the RAW264.7 cells were activated by LPS, the production of NO and the expression of iNOS, COX-2 and IL-1β was up-regulated. Additionally, NF-κB signaling pathway and the NLRP-3 inflammasome were also activated under this condition.¹³⁾ Herein, we investigated whether the target compounds have any effect on the NO release in RAW264.7 cells. Naproxen, well-known as an anti-inflammatory drug, was used as the positive control in present study. As illustrated in Table 1, the initial biological screening showed that seventeen naproxen derivatives had different effects on the production of NO at 50 µM. Specifically, the inhibitory range of A1, A2, A5, A6, A7, A8, A9, A10, A11, A12, A14, A18, A20, A22, A24, A25, A26, and A28 is 21.48–99.52%, exceeding the inhibition of the control drug naproxen, which was 12.84%. The effects of two compounds (A22 and A24) were confirmed to be the greatest than that of other target compounds (up to 90% inhibition).

Table 1. Inhibitory Effects and IC₅₀ Values of Target Compounds on NO Release in RAW264.7 Cells


Compound	R	Inhibition (%, 50 µM)^a⁾	IC₅₀ (µM)^a⁾
A1	C₆H₅	44.99 ± 4.98	36.30 ± 7.73
A2	2-CH₃-C₆H₄	32.49 ± 1.53	89.65 ± 17.41
A3	3-CH₃-C₆H₄	NA^b⁾	NA^b⁾
A4	4-CH₃-C₆H₄	NA^b⁾	NA
A5	2-CH₃O-C₆H₄	60.20 ± 4.99	54.88 ± 2.05
A6	3-CH₃O-C₆H₄	64.08 ± 2.47	43.80 ± 2.06
A7	4-CH₃O-C₆H₄	77.37 ± 0.94	29.15 ± 0.98
A8	2-CF₃-C₆H₄	37.17 ± 1.19	78.09 ± 4.19
A9	3-CF₃-C₆H₄	45.87 ± 2.50	41.10 ± 1.69
A10	4-CF₃-C₆H₄	21.48 ± 4.90	NA
A11	2-F-C₆H₄	46.44 ± 2.32	45.15 ± 3.40
A12	3-F-C₆H₄	69.88 ± 4.57	35.69 ± 2.07
A13	4-F-C₆H₄	NA^b⁾	NA
A14	2-Cl-C₆H₄	43.34 ± 4.42	51.61 ± 0.98
A15	3-Cl-C₆H₄	NA^b⁾	NA
A16	4-Cl-C₆H₄	NA^b⁾	NA
A17	2-Br-C₆H₄	NA^b⁾	NA
A18	3-Br-C₆H₄	36.22 ± 6.40	75.90 ± 7.55
A19	4-Br-C₆H₄	NA^b⁾	NA
A20	2-NO₂-C₆H₄	52.50 ± 2.22	39.38 ± 4.28
A21	4-NO₂-C₆H₄	12.48 ± 2.01	NA
A22	2,4-NO₂-C₆H₃	99.52 ± 0.13	7.38 ± 1.96
A23	3,4,5-CH₃O-C₆H₂	8.35 ± 2.96	NA
A24	2-Furyl	90.06 ± 2.84	21.65 ± 1.53
A25	3-Thienyl	47.27 ± 3.42	39.59 ± 1.17
A26	5-CH₃-2-Thienyl	73.02 ± 4.59	31.29 ± 6.10
A27	1-Naphthyl	NA^b⁾	NA
A28	4-Pyridyl	77.08 ± 2.94	20.10 ± 1.62
A29	4-Quinolyl	NA^b⁾	NA
A30	6-CH₃O-2-naphthyl	NA^b⁾	NA
	Naproxen^c⁾	12.84 ± 1.27	> 100
	Cinnamic acid	15.34 ± 3.01	> 100

a) Data shown as mean ± standard error of the mean (S.E.M.) of three independent experiments. b) Exhibited no effect. c) The data showed in Ref. 31.

Among compounds A2–A21, these compounds with the phenyl substitution in ortho-position and meta-position showed stronger inhibition against the LPS-induced NO production than those of compounds with phenyl substitution in para-position. Moreover, the NO inhibition of these compounds with an electron-donating methoxyl group (A5, A6, and A7) was higher than that of the compound without the substituted group (A1). The compounds with the electron-withdrawing groups, such as the fluoro, nitro and trifluoromethyl groups (A9, A11, A12, A20, and A22), showed more potent inhibition against NO production than that of A1. However, the compounds with an electron-donating methyl group (A2, A3, and A4) and electron-withdrawing groups (A15, A16, A17, and A19) had a decrease in the NO production. The possible reason for this was that the compounds with an electron-donating methyl group and electron-withdrawing groups at the para-position on the benzene rings showed weak activity, attenuating hydrophobic substituents on the phenyl ring decreased the activity. These compounds with electron-withdrawing groups at ortho-position and meta-position exhibited good activities, strengthening hydrophobic substituents at ortho or meta position of phenyl ring enhances activity. However, the decreased activity and the mechanism of A15 with the chlor atom at meta-position and A17 with the bromo atom at ortho-position were unclear.

In addition, the compounds possessing furan-2-yl and pyridin-4-yl groups had pronounced inhibition on the production of NO, including A24 and A28. As shown in Table 1, compound A22 showed the strongest NO inhibition than those of other naproxen derivatives, suggesting that we provided a practical strategy for the design of novel naproxen-based drugs with high potency. The possible explanation for this phenomenon was that two nitro groups in A22 provided lone-pair electrons and the hydrogen-bond donors which caused the enhanced interaction between A22 with amino acid residues in active site. The SAR analysis revealed that eighteen compounds which had different O-substituted ester moiety (A1, A2, A5, A6, A7, A8, A9, A10, A11, A12, A14, A18, A20, A22, A24, A25, A26, and A28) exhibited the more potent inhibition than that of cinnamic acid. Accordingly, the cell viability of the target compounds was investigated in RAW264.7 cells.

Effect of Naproxen Derivatives on the RAW264.7 Cell Viability

The cytotoxicity and safety of the target compounds in RAW264.7 cells was assessed at a concentration of their IC₅₀ concentrations using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with naproxen serving as the positive control for comparison. Table 2 showed cell viability of A22 and A26 exceeded 80%, demonstrating that A22 and A26 had low cytotoxicity and high safety. The cell viability other naproxen derivates at their IC₅₀ concentrations was less than 80%, which is weaker than that of naproxen (84.57%).

Table 2. Effect of Target Compounds on the Cell Viability of the RAW264.7 Cells

Compound	Concentration (µM)	Cell viability (%)^a⁾	p-Value
Naporxen^b⁾	50	84.57 ± 3.19	0.001 < p < 0.01
A1	36.30	39.48 ± 2.61	p < 0.001
A2	89.65	73.09 ± 5.03	p < 0.001
A3	NA^c⁾	ND^d⁾	ND
A4	NA	ND	ND
A5	54.88	72.46 ± 3.50	p < 0.001
A6	43.80	30.94 ± 2.82	p < 0.001
A7	29.15	4.80 ± 0.66	p < 0.001
A8	78.09	63.31 ± 3.09	p < 0.001
A9	41.10	59.43 ± 9.30	p < 0.001
A10	NA	ND	ND
A11	45.15	7.37 ± 4.20	p < 0.001
A12	35.6	75.50 ± 5.67	0.001 < p < 0.01
A13	NA	ND	ND
A14	51.61	69.31 ± 5.01	p < 0.001
A15	NA	ND	ND
A16	NA	ND	ND
A17	NA	ND	ND
A18	75.90	56.39 ± 2.54	p < 0.001
A19	NA	ND	ND
A20	39.38	43.01 ± 2.14	p < 0.001
A21	NA	ND	ND
A22	7.38	99.76 ± 7.5	p < 0.05
A23	NA	ND	ND
A24	39.59	4.79 ± 1.62	p < 0.001
A25	22	2.15 ± 1.83	p < 0.001
A26	NA	ND	ND
A27	31	1.89 ± 2.03	p < 0.001
A28	20.10	81.63 ± 9.61	0.001 < p < 0.01
A29	NA	ND	ND
A30	NA	ND	ND

a) Data shown as mean ± S.E.M. of three independent experiments. b) The data showed in Ref. 31. c) Exhibited no activity. d) No determined.

Based on these results, the present study demonstrated that compound A22 had low cytotoxicity and high safety, and exhibited the strongest inhibitory effect on the LPS-induced NO production in comparison to those of the other naproxen derivatives. The result indicated that the high cytotoxicity of A24 caused the significantly inhibitory effect of A24 on the NO production in RAW264.7 cells. Therefore, the cell viability of compound A22 at different concentrations in LPS-induced RAW 264.7 cells was tested in order to investigate its cytotoxicity and safety profile. As presented in Fig. 2, it was revealed that compound A22 concentration-dependently affected the viability of the RAW264.7 cells. In addition, the cell viability of A22 exceeds 80% at concentrations below 25 µM. Hence, the highly bioactive representative A22 was selected for subsequent mechanistic investigation.

Fig. 2. Effect of Naproxen, A22 on the Viability of RAW264.7 Cells

Data shown as mean ± S.E.M. of three independent experiments.

Effects of A22 on iNOS, IL-1β and COX-2 Protein Expressions

LPS was confirmed to stimulate macrophages to up-regulate various pro-inflammatory cytokines and activate NF-κB signaling pathway and NLRP-3 inflammasome.^9,13) Thus, we determined the mechanism of A22 in LPS-induced RAW264.7 cells by Western blotting to explore the influence of A22 on pro-inflammatory cytokines and related signaling pathways. Figures 3A and 3B showed that after treated with A22 at different concentrations (6 and 13 µM) in LPS-induced RAW264.7 cells, the expression of NF-κB p65 had a decrease in a concentration-dependent manner. Once RAW264.7 cells were induced by LPS, we observed higher expression of NLRP3 than that of the blank group. Treatment with A22 displayed a concentration-dependent inhibitory activity against NLRP3 inflammasome in LPS-induced RAW264.7 cells (Fig. 3C). As illustrated in Figs. 3D–3F, the over-expression of iNOS, COX-2 and IL-1β were observed in LPS group, while treatment with A22 concentration-dependently inhibited the expression of pro-inflammatory cytokines, such as iNOS, COX-2 and IL-1β. Above all, our results indicate that A22 could suppress the over-expression of pro-inflammatory cytokines by blocking the activation of NF-κB signaling pathway and NLRP-3 inflammasome in LPS-RAW264.7 cells, outperforming that of naproxen.

Fig. 3. Effect of A22 on LPS-Induced iNOS, COX-2 and IL-1β by Blocking NF-κB and NLRP3 Pathways in RAW264.7 Cells

Values are expressed as mean ± standard deviation (n = 3); ^#p < 0.05, ^##p < 0.05, ^###p < 0.001 compared to the control group; * p < 0.05, ** p < 0.05, *** p < 0.001 compared to the LPS group, calculated by student’s t-test.

Molecular Docking Analysis of A22 with NLRP3

These results of molecular docking analysis indicated that A22 bounded well to the active site of NLRP3 (Protein Data Bank ID: 7ALV) with wellfitting mode. Figure 4 showed that naproxen and A22 fitted neatly into the pocket of NLRP3 and the specificity pocket becomes occupied. However, the interaction of amino acid residues with naproxen was not observed in Fig. 4A. As described in Fig. 4B, 2,4-dinitro groups and ester group from compound A22 provided the hydrogen-bond donors. Moreover, A22 bounded to NLRP3 by forming hydrogen bonds with amino acid residues THR-233, HIS-260, GLN-509, LYS232, GLY-231 and GLY-229. Consequently, A22 had good anti-inflammatory activity by forming hydrogen bonds with amino acid residues.

Fig. 4. Molecular Docking Experiment Showing the Binding of Naproxen and A22 (Blue) with NLRP3 Protein (Green) as Analyzed Using Pymol Software

Conclusion

In summary, 30 naproxen derivatives containing cinnamic acids are synthesized and their bioactivities are tested against LPS-induced inflammation. The potent compound A22 is further investigated for anti-inflammatory mechanism in vitro. Our results indicated that A22, which had low cytotoxicity and high safety, significantly inhibited NO production and the LPS-induced expression of pro-inflammatory cytokines. Mechanistically, these anti-inflammatory actions of compound A22 were mediated by directly inhibiting NF-κB and NLRP3 inflammasome activation. Molecular docking analysis indicated that THR-233, HIS-260, GLN-509, LYS232, GLY-231 and GLY-229 in the NLRP3 were key residues in the interaction between A22 and NLRP3 protein. Overall, the present study obtains a new NLRP3 inhibitor, A22, which deserved further development as an anti-inflammatory candidate. More importantly, this work provides a new strategy for the development of NLRP3 inhibitors.

Experimental

Materials

Cinnamic acids were obtained via the Knoevenagel condensation. LPS (Escherichia coli O111:B4) and MTT dye were supplied from Sigma-Aldrich (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS) and Dulbecco’s modified eagle medium (DMEM) were purchased from Gibco (Grand Island, NY, U.S.A.). Griess reagent kit was obtained from Biotium (CA, U.S.A.). Antibodies against IL-1β, COX-2, iNOS, NF-κB p65 and NLRP3 were supplied from Affinity Biosciences (OH, U.S.A.), respectively. RAW264.7 cell (p5–p9) line was cultured in our laboratory. To monitor whether the reactions have completed, TLC analysis was used in present study. We used a micro melting point apparatus (Shanghai, China) to measure the melting points of A1–A30. In addition, the micro melting point apparatus was not corrected. All ¹H-NMR and ¹³C-NMR spectra, reported as parts per million (ppm), were recorded on a Bruker 500 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. Mass spectrometry analysis of target compounds was analyzed by a LC-MS with an electrospray ionization source. Other chemicals, which used in present experiments, were bought from commercial vendors and used without further purifying unless otherwise stated.

General Procedure for the Preparation of Compound (1) and A1–A30

As previously described in the literature, compound (1) was synthesized in two steps.^31,32) The synthesis procedure of A1 was described as following: cinnamic acid (1.0 mmol), compound (1) (1.0 mol), EDCI (1.2 mmol)) and DMAP (0.1 mmol) were dissolved in dichloromethane and the mixture was stirred at room temperature for 2 h. After the reaction completed, the mixture was extracted by dichloromethane, and then the organic phase was washed twice with saturated sodium bicarbonate solution. Subsequently, the organic phase was dried on anhydrous magnesium sulfate and concentrated to obtain the crud product. The crude product was recrystallized in absolute ethanol to offer A1. Moreover, substituting cinnamic acid with other cinnamic acids as the reaction substrate, compounds A2–A30 were obtained in the same way presented in A1.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-cinnamate (A1). White solid, 76.1% yield, mp 128.3–130.1 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.95 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 1.8 Hz, 1H), 7.67–7.62 (m, 3H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.48–7.44 (m, 3H), 7.34 (dd, J = 8.8, 2.3 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 3.82 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 165.66, 148.36, 146.76, 138.64, 134.19, 132.84, 131.57, 130.79, 129.32, 129.05, 128.37, 127.93, 126.64, 125.90, 121.41, 118.40, 117.29, 80.72, 77.07, 46.56, 27.97, 18.50. High resolution (HR)-MS m/z: 403.1831 [M + H]⁺. Found 403.1825.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(o-tolyl)-acrylate (A2). White solid, 63.0% yield, mp 113.0–114.5 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.24 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.79–7.76 (m, 1H), 7.69 (dd, J = 7.7, 1.4 Hz, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.38–7.32 (m, 2H), 7.29 (s, 1H), 6.64 (d, J = 15.9 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 2.52 (s, 3H), 1.57 (d, J = 7.1 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 165.71, 148.39, 144.40, 138.62, 138.03, 133.14, 132.84, 131.56, 130.97, 130.51, 129.30, 127.91, 126.63, 126.49, 125.90, 121.42, 118.40, 118.26, 80.72, 77.06, 46.56, 27.97, 19.88, 18.50. HR-MS m/z: 417.1988 [M + H]⁺. Found 417.1980.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(m-tolyl-acrylate (A3). White solid, 87.2% yield, mp 114.7–116.0 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.93 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.79–7.77 (m, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.5, 1.8 Hz, 1H), 7.47–7.43 (m, 2H), 7.37–7.33 (m, 2H), 7.28–7.27 (m, 1H), 6.71 (d, J = 16.0 Hz, 1H), 3.82 (q, J = 7.2 Hz, 1H), 2.43 (s, 3H), 1.58 (d, J = 7.2 Hz, 3H), 1.44 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.78, 165.74, 148.40, 146.97, 138.73, 138.62, 134.15, 132.85, 131.65, 131.56, 129.31, 129.04, 128.93, 127.93, 126.62, 125.90, 125.57, 121.44, 118.40, 117.03, 80.72, 77.10, 46.57, 27.98, 21.37, 18.50. HR-MS m/z: 417.1988 [M + H]⁺. Found 417.1980.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(p-tolyl)-acrylate (A4). White solid, 87.7% yield, mp 128.6–129.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.92 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.55–7.52 (m, 2H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 7.27 (d, J = 7.9 Hz, 2H), 6.67 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 2.43 (s, 3H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.78, 165.87, 148.41, 146.79, 141.32, 138.59, 132.85, 131.54, 131.48, 129.78, 129.29, 128.38, 127.92, 126.60, 125.89, 121.46, 118.40, 116.13, 80.72, 77.07, 46.56, 27.97, 21.57, 18.49. HR-MS m/z: 417.1988 [M + H]⁺. Found 417.1981.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-methoxyphenyl)-acrylate (A5). White solid, 81.6% yield, mp 105.7–107.7 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.25 (d, J = 16.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.79–7.77 (m, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.62 (dd, J = 7.7, 1.7 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.45–7.40 (m, 1H), 7.35 (dd, J = 8.8, 2.3 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.98 (dd, J = 8.4, 1.0 Hz, 1H), 6.83 (d, J = 16.1 Hz, 1H), 3.94 (s, 3H), 3.81 (q, J = 7.1 Hz, 1H), 1.58 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.79, 166.20, 158.63, 148.53, 142.27, 138.54, 132.87, 132.02, 131.52, 129.40, 129.24, 127.92, 126.56, 125.89, 123.16, 121.58, 120.82, 118.44, 117.79, 111.25, 80.70, 77.09, 55.53, 46.56, 27.98, 18.51. HR-MS m/z: 433.1937 [M + H]⁺. Found 433.1929.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3-methoxyphenyl)-acrylate (A6). White solid, 71.9% yield, mp 107.6–109.2 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.91 (d, J = 15.9 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.78–7.76 (m, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 7.25–7.21 (m, 1H), 7.15 (t, J = 2.6 Hz, 1H), 7.03–7.00 (m, 1H), 6.70 (d, J = 16.0 Hz, 1H), 3.88 (s, 3H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.76, 165.60, 159.99, 148.34, 146.67, 138.65, 135.53, 132.83, 131.56, 130.05, 129.32, 127.92, 126.64, 125.89, 121.38, 121.06, 118.38, 117.58, 113.16, 80.71, 77.07, 55.36, 46.55, 27.96, 18.49. HR-MS m/z: 433.1937 [M + H]⁺. Found 433.1930.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-methoxyphenyl)-acrylate (A7). White solid, 76.4% yield, mp 117.2–118.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.93–7.85 (m, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.78–7.76 (m, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.60–7.57 (m, 2H), 7.49 (dd, J = 8.5, 1.8 Hz, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 6.99–6.95 (m, 2H), 6.58 (d, J = 15.9 Hz, 1H), 3.88 (s, 3H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.78, 166.00, 161.79, 148.46, 146.44, 138.56, 132.85, 131.52, 130.11, 129.26, 127.90, 126.94, 126.58, 125.88, 121.51, 118.40, 114.62, 114.48, 80.70, 77.07. HR-MS m/z: 433.1937 [M + H]⁺. Found 433.1930.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-(trifluoromethyl)phenyl)-acrylate (A8). White solid, 64.0% yield, mp 94.3–96.5 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.32 (dd, J = 15.9, 2.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.82 (t, J = 8.7 Hz, 2H), 7.79–7.75 (m, 2H), 7.68–7.62 (m, 2H), 7.58–7.52 (m, 1H), 7.50 (dd, J = 8.4, 1.8 Hz, 1H), 7.34 (dd, J = 8.8, 2.3 Hz, 1H), 6.69 (d, J = 15.8 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 164.73, 148.21, 142.09, 138.71, 133.09, 132.80, 132.23, 131.60, 130.02, 129.34, 128.96, 128.08, 127.93, 126.69, 126.29, 125.90, 125.00, 121.69, 121.24, 118.36, 80.74, 77.05, 46.56, 27.96, 18.48. HR-MS m/z: 471.1705 [M + H]⁺. Found 471.1698.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3-(trifluoromethyl)phenyl)-acrylate (A9). White solid, 56.0% yield, mp 94.4–96.1 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.95 (d, J = 16.0 Hz, 1H), 7.90–7.86 (m, 2H), 7.83–7.76 (m, 3H), 7.71 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 2.3 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 6.78 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.73, 165.11, 148.19, 144.81, 138.75, 134.96, 132.80, 131.74, 131.61, 131.48, 131.31, 129.62, 129.39, 127.92, 127.13, 127.07, 126.71, 125.90, 124.90, 124.85, 121.21, 119.33, 118.33, 80.73, 77.04, 46.55, 27.96, 18.48. HR-MS m/z: 471.1705 [M + H]⁺. Found 471.1699.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-(trifluoromethyl)phenyl)-acrylate (A10). White solid, 81.0% yield, mp 108.6–110.9 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.95 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.79–7.77 (m, 1H), 7.75–7.70 (m, 4H), 7.65 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.5, 1.8 Hz, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.73, 165.08, 148.18, 144.76, 138.78, 137.51, 132.79, 131.62, 129.41, 128.46, 127.92, 126.73, 126.02, 126.00, 125.91, 124.88, 122.71, 121.19, 119.91, 118.33, 80.73, 77.05, 46.55, 27.96, 18.49. HR-MS m/z: 471.1705 [M + H]⁺. Found 471.1701.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-fluorophenyl)-acrylate (A11). White solid, 87.4% yield, mp 93.6–95.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.07 (d, J = 16.3 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.78–7.76 (m, 1H), 7.67–7.62 (m, 2H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.46–7.41 (m, 1H), 7.34 (dd, J = 8.8, 2.4 Hz, 1H), 7.23 (td, J = 7.6, 1.1 Hz, 1H), 7.17 (ddd, J = 10.6, 8.3, 1.1 Hz, 1H), 6.82 (d, J = 16.2 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.76, 165.49, 162.56, 160.53, 148.31, 139.36, 139.34, 138.66, 132.83, 132.18, 131.58, 129.33, 127.93, 126.65, 125.90, 124.59, 121.35, 119.95, 118.37, 116.44, 116.27, 80.71, 77.07, 46.55, 27.97, 18.50. HR-MS m/z: 421.1737 [M + H]⁺. Found 421.1729.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3-fluorophenyl)-acrylate (A12). White solid, 77.6% yield, mp 93.4–94.4 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.06 (d, J = 16.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.78–7.76 (m, 1H), 7.67–7.61 (m, 2H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.47–7.40 (m, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.17 (ddd, J = 10.9, 8.3, 1.2 Hz, 1H), 6.82 (d, J = 16.2 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 165.51, 162.56, 160.53, 148.30, 139.35, 138.66, 132.82, 132.18, 131.57, 129.33, 127.92, 126.64, 125.89, 124.61, 121.34, 119.94, 118.37, 116.44, 116.27, 80.73, 77.05, 46.55, 27.96, 18.48. HR-MS m/z: 421.1737 [M + H]⁺. Found 421.1728.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-fluorophenyl)-acrylate (A13). White solid, 75.7% yield, mp 142.3–144.0 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.93–7.85 (m, 2H), 7.81 (d, J = 8.6 Hz, 1H), 7.78–7.77 (m, 1H), 7.66–7.58 (m, 3H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.33 (dd, J = 8.8, 2.3 Hz, 1H), 7.17–7.11 (m, 2H), 6.63 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.75, 165.53, 163.19, 148.32, 145.40, 138.68, 132.83, 131.57, 130.32, 129.34, 127.91, 126.67, 125.90, 121.36, 118.37, 117.04, 117.02, 116.32, 116.14, 80.72, 77.08, 46.55, 27.96, 18.50. HR-MS m/z: 421.1737 [M + H]⁺. Found 421.1730.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-chlorophenyl)-acrylate (A14). White solid, 75.1% yield, mp 97.4–99.6 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.36 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.78–7.77 (m, 1H), 7.74 (dd, J = 7.4, 2.0 Hz, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.52–7.47 (m, 2H), 7.40–7.33 (m, 3H), 6.71 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.2 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.76, 165.14, 148.28, 142.42, 138.68, 135.23, 132.82, 132.47, 131.59, 131.50, 130.33, 129.34, 127.93, 127.85, 127.22, 126.66, 125.90, 121.32, 119.97, 118.38, 80.72, 77.05, 46.55, 27.96, 18.49. HR-MS m/z: 437.1441 [M + H]⁺. Found 437.1435.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3-chlorophenyl)-acrylate (A15). White solid, 63.7% yield, mp 133.9–135.2 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.89–7.84 (m, 2H), 7.81 (d, J = 8.6 Hz, 1H), 7.78–7.76 (m, 1H), 7.65–7.61 (m, 2H), 7.52–7.48 (m, 2H), 7.45–7.37 (m, 2H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.71 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.74, 165.22, 148.23, 145.05, 138.71, 135.98, 135.08, 132.80, 131.59, 130.61, 130.29, 129.36, 128.06, 127.92, 126.69, 126.51, 125.90, 121.26, 118.80, 118.34, 80.72, 77.05, 46.55, 27.96, 18.49. HR-MS m/z: 437.1441 [M + H]⁺. Found 437.1436.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-chlorophenyl)-acrylate (A16). White solid, 83.7% yield, mp 148.1–150.6 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.89 (d, J = 7.5 Hz, 1H), 7.87 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.78–7.77 (m, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.57–7.53 (m, 2H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.44–7.40 (m, 2H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.68 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.2 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.75, 165.40, 148.28, 145.24, 138.70, 136.72, 132.82, 132.66, 131.58, 129.51, 129.36, 129.34, 127.92, 126.68, 125.91, 121.31, 118.36, 117.87, 80.72, 77.08, 46.55, 27.97, 18.50. HR-MS m/z: 437.1441 [M + H]⁺. Found 437.1436.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-bromophenyl)-acrylate (A17). White solid, 72.4% yield, mp 141.0–142.7 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.90–7.79 (m, 3H), 7.77 (t, J = 1.9 Hz, 2H), 7.65 (d, J = 2.3 Hz, 1H), 7.59–7.55 (m, 1H), 7.55–7.48 (m, 2H), 7.34–7.29 (m, 2H), 6.70 (d, J = 16.0 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.74, 165.19, 148.24, 144.94, 138.71, 133.52, 132.81, 131.59, 131.01, 130.54, 129.37, 127.93, 126.93, 126.69, 125.91, 123.17, 121.27, 118.82, 118.35, 80.72, 77.09, 46.55, 27.98, 18.51. HR-MS m/z: 481.0936 [M + H]⁺. Found 481.0928.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3-bromophenyl)-acrylate (A18). White solid, 70.1% yield, mp 109.7–111.5 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.32 (d, J = 15.9 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.79–7.76 (m, 1H), 7.73 (dd, J = 7.8, 1.7 Hz, 1H), 7.69–7.65 (m, 2H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.34 (dd, J = 8.8, 2.3 Hz, 1H), 7.32–7.29 (m, 1H), 6.66 (d, J = 15.9 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.76, 165.04, 148.28, 144.97, 138.68, 134.26, 133.60, 132.81, 131.65, 131.59, 129.33, 127.98, 127.93, 127.85, 126.66, 125.90, 125.61, 121.32, 120.16, 118.38, 80.72, 77.06, 46.55, 27.97, 18.49. HR-MS m/z: 481.0936 [M + H]⁺. Found 481.0930.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-bromophenyl)-acrylate (A19). White solid, 84.0% yield, mp 156.1–157.4 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.88 (s, 1H), 7.86 (d, J = 7.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.61–7.57 (m, 2H), 7.52–7.47 (m, 3H), 7.32 (dd, J = 8.8, 2.4 Hz, 1H), 6.70 (d, J = 16.0 Hz, 1H), 3.81 (d, J = 7.2 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.74, 165.39, 148.26, 145.31, 138.70, 133.09, 132.81, 132.31, 131.58, 129.70, 129.36, 127.91, 126.68, 125.90, 125.11, 121.29, 118.35, 117.99, 80.72, 77.06, 46.55, 27.96, 18.49. HR-MS m/z: 481.0936 [M + H]⁺. Found 481.0927.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2-nitrophenyl)-acrylate (A20). White solid, 73.1% yield, mp 117.9–119.2 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.39 (d, J = 15.8 Hz, 1H), 8.11 (dd, J = 8.1, 1.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.78–7.69 (m, 3H), 7.66 (d, J = 2.3 Hz, 1H), 7.63–7.59 (m, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.34 (dd, J = 8.8, 2.4 Hz, 1H), 6.63 (d, J = 15.8 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.74, 164.43, 148.35, 148.13, 142.04, 138.76, 133.71, 132.78, 131.62, 130.69, 130.42, 129.38, 129.31, 127.94, 126.71, 125.90, 125.08, 122.40, 121.19, 118.36, 80.73, 77.06, 46.55, 27.96, 18.49. HR-MS m/z: 448.1682 [M + H]⁺. Found 448.1678.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(4-nitrophenyl)-acrylate (A21). Brick yellow solid, 56.1% yield, mp 154.7–156.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.33–8.28 (m, 2H), 7.96 (d, J = 16.0 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.79–7.75 (m, 3H), 7.65 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.5, 1.8 Hz, 1H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.83 (d, J = 16.0 Hz, 1H), 3.81 (d, J = 7.2 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.71, 164.72, 148.75, 148.08, 143.62, 140.19, 138.86, 132.76, 131.65, 129.46, 128.92, 127.92, 126.81, 125.92, 124.29, 121.64, 121.06, 118.29, 80.75, 77.06, 46.54, 27.96, 18.49. HR-MS m/z: 448.1682 [M + H]⁺. Found 448.1679.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(2,4-dinitrophenyl)-acrylate (A22). Dark yellow solid, 51.0% yield, mp 155.7–158.1 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.95 (d, J = 2.3 Hz, 1H), 8.54 (dd, J = 8.5, 2.3 Hz, 1H), 8.37 (d, J = 15.9 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.79–7.76 (m, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.5, 1.8 Hz, 1H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.73 (d, J = 15.8 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.70, 163.62, 148.24, 148.11, 147.90, 139.69, 138.97, 136.16, 132.71, 131.68, 130.81, 129.50, 127.93, 127.81, 126.88, 125.92, 125.63, 120.88, 120.70, 118.26, 80.78, 77.05, 46.54, 27.96, 18.48. HR-MS m/z: 493.1533 [M + H]⁺. Found 493.1527.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(3,4,5-trimethoxyphenyl)-acrylate (A23). White solid, 74.1% yield, mp 125.0–126.5 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.90–7.75 (m, 4H), 7.64 (d, J = 2.3 Hz, 1H), 7.49 (dd, J = 8.5, 1.8 Hz, 1H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.86 (s, 2H), 6.62 (d, J = 15.9 Hz, 1H), 3.93 (s, 9H), 3.80 (q, J = 7.1 Hz, 1H), 1.56 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.75, 165.63, 153.52, 148.34, 146.70, 140.49, 138.66, 132.82, 131.55, 129.66, 129.33, 127.90, 125.89, 121.37, 118.36, 116.46, 105.48, 80.71, 77.07, 61.04, 56.20, 46.54, 27.96, 18.48. HR-MS m/z: 493.2148 [M + H]⁺. Found 493.2143.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(furan-2-yl)acrylate (A24). Gray solid, 42.8% yield, mp 81.2–81.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.86 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.77–7.75 (m, 1H), 7.67 (d, J = 15.7 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.49 (dd, J = 8.5, 1.8 Hz, 1H), 7.31 (dd, J = 8.9, 2.4 Hz, 1H), 6.73 (d, J = 3.5 Hz, 1H), 6.58 (d, J = 15.7 Hz, 1H), 6.54 (s, 1H), 3.80 (q, J = 7.1 Hz, 1H), 1.56 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 165.74, 150.78, 148.38, 145.31, 138.59, 132.82, 132.78, 131.53, 129.27, 127.90, 126.59, 125.88, 121.41, 118.36, 115.82, 114.79, 112.54, 80.70, 77.06, 46.54, 27.96, 18.48. ESI-MS m/z: 393.2635 [M + H] ⁺. Found 393.2634.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(thiophen-3-yl)-acrylate (A25). Brownish green solid, 50.9% yield, mp 122.5–123.7 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.92 (d, J = 15.9 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.78–7.76 (m, 1H), 7.64–7.61 (m, 2H), 7.49 (dd, J = 8.5, 1.8 Hz, 1H), 7.41 (d, J = 2.1 Hz, 2H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.76, 165.92, 148.37, 140.12, 138.62, 137.41, 132.83, 131.54, 129.31, 129.05, 127.91, 127.27. HR-MS m/z: 409.1395 [M + H]⁺. Found 409.1388.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(5-methylthiophen-2-yl)-acrylate (A26). Beige solid, 76.9% yield, mp 107.1–107.3 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 7.99–7.92 (m, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.77–7.76 (m, 1H), 7.63 (d, J = 2.3 Hz, 1H), 7.49 (dd, J = 8.5, 1.8 Hz, 1H), 7.32 (dd, J = 8.8, 2.3 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 6.77 (dq, J = 3.4, 1.1 Hz, 1H), 6.36 (d, J = 15.6 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 2.55 (d, J = 1.1 Hz, 3H), 1.57 (d, J = 7.1 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.77, 165.74, 148.43, 145.01, 139.50, 138.57, 137.38, 132.84, 132.57, 131.52, 129.26, 127.91, 126.79, 126.58, 125.88, 121.48, 118.38, 114.33, 80.69, 77.10, 46.55, 27.97, 18.50, 15.93. ESI-MS m/z: 423.3788 [M + H] ⁺. Found 423.3790.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(naphthalen-1-yl)-acrylate (A27). White solid, 71.0% yield, mp 145.7–148.2 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.80 (d, J = 15.8 Hz, 1H), 8.29 (dd, J = 8.5, 1.1 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.95–7.93 (m, 1H), 7.92–7.89 (m, 2H), 7.83 (d, J = 8.5 Hz, 1H), 7.80–7.78 (m, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.66–7.63 (m, 1H), 7.61–7.55 (m, 2H), 7.52 (dd, J = 8.5, 1.8 Hz, 1H), 6.82 (d, J = 15.7 Hz, 1H), 3.82 (d, J = 7.1 Hz, 1H), 1.58 (d, J = 7.2 Hz, 3H), 1.44 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.78, 165.55, 148.40, 143.71, 138.67, 133.74, 132.87, 131.60, 131.48, 131.05, 129.36, 128.85, 127.95, 127.11, 126.66, 126.39, 125.92, 125.54, 125.38, 123.35, 121.43, 119.83, 118.44, 80.73, 77.06, 46.57, 27.98, 18.52. HR-MS m/z: 453.1988 [M + H]⁺, 453.1982.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(pyridin-4-yl)-acrylate (A28). White solid, 52.8% yield, mp 136.8–137.9 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.74–8.70 (m, 2H), 7.90–7.79 (m, 3H), 7.78–7.76 (m, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.48–7.45 (m, 2H), 7.31 (dd, J = 8.8, 2.3 Hz, 1H), 6.86 (d, J = 16.1 Hz, 1H), 3.80 (q, J = 7.1 Hz, 1H), 1.56 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.71, 164.70, 150.73, 148.06, 143.70, 141.31, 138.84, 132.76, 131.64, 129.45, 127.92, 126.78, 125.91, 122.03, 121.95, 121.07, 118.30, 80.74, 77.07, 46.54, 27.96, 18.49. HR-MS m/z: 404.1784 [M + H]⁺. Found 404.1776.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(quinolin-4-yl)-acrylate (A29). White solid, 65.1% yield, mp 167.2–168.4 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 9.01 (d, J = 4.5 Hz, 1H), 8.66 (d, J = 15.9 Hz, 1H), 8.22 (t, J = 8.5 Hz, 2H), 7.90 (d, J = 8.9 Hz, 1H), 7.85–7.77 (m, 3H), 7.71–7.63 (m, 3H), 7.52 (dd, J = 8.5, 1.8 Hz, 1H), 7.36 (dd, J = 8.8, 2.3 Hz, 1H), 6.92 (d, J = 15.8 Hz, 1H), 3.82 (q, J = 7.1 Hz, 1H), 1.57 (d, J = 7.1 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.72, 164.66, 150.17, 148.76, 148.13, 141.12, 139.58, 138.86, 132.80, 131.67, 130.34, 129.93, 129.48, 127.95, 127.54, 126.80, 125.97, 125.94, 123.82, 123.29, 121.12, 118.35, 80.75, 77.06, 46.55, 27.97, 18.51. HR-MS m/z: 454.1940 [M + H]⁺. Found 454.1930.

(S)-6-(1-(Tert-butoxy)-1-oxopropan-2-yl)-naphthalen-2-yl-(E)-3-(6-methoxynaphthalen-2-yl)-acrylate (A30). White solid, 78.5% yield, mp 158.5–160.2 °C; ¹H-NMR (500 MHz, CDCl₃): δ (ppm) 8.07 (d, J = 15.9 Hz, 1H), 7.97–7.94 (m, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.84–7.71 (m, 5H), 7.67 (d, J = 2.3 Hz, 1H), 7.50 (dd, J = 8.5, 1.8 Hz, 1H), 7.36 (dd, J = 8.8, 2.3 Hz, 1H), 7.22 (dd, J = 8.9, 2.5 Hz, 1H), 7.18 (d, J = 2.5 Hz, 1H), 6.77 (d, J = 15.9 Hz, 1H), 3.97 (s, 3H), 3.82 (q, J = 7.1 Hz, 1H), 1.58 (d, J = 7.2 Hz, 3H), 1.43 (s, 9H). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) 173.78, 165.91, 159.06, 148.47, 147.02, 138.60, 135.99, 132.87, 131.55, 130.38, 130.30, 129.58, 129.30, 128.67, 127.93, 127.67, 126.61, 125.90, 124.21, 121.49, 119.63, 118.42, 116.13, 106.03, 77.08, 55.43, 46.56, 27.98, 18.50. HR-MS m/z: 483.2093 [M + H]⁺. Found 483.2084.

Cell Viability Assay

As previously described in the literature,³²⁾ the cytotoxicity of A1–A30 was tested in the RAW264.7 cells to assess the safety of naproxen derivatives containing cinnamic acids.

Measurement of NO in RAW264.7 Cells

As previously described in the literature,³²⁾ the Griess’ reagent kit was used to measure the NO production in LPS-induced RAW264.7 cells in order to investigate the effect of compounds A1–A30 on the NO production.

Protein Extraction and Western Blot Analysis

As previously described in the literature,³²⁾ the proteins, extracted from LPS-induced RAW264.7 cells, were tested by Western blot to assess the anti-inflammatory mechanism of A22 in vitro.

Molecular Docking

In the molecular docking, the chemical structure of A22 is drew by ChemDraw software and the NLRP3 structure is available (PDB code: 7ALV) from the Protein Data Bank. Firstly, the NLRP3 protein complex is corrected by AutoDock software. Secondly, all cocrystallized ligands and water molecules are removed before molecular docking. Subsequently, naproxen and A22 are docked to the NLRP3 protein structure in order to generate naproxen-NLRP3 or A22-NLRP3 complexes, respectively. Finally, the best docking results, involving in the binding interactions, are conducted using AutoDock Vina software and drew by Pymol software.

Data and Statistical Analysis

All data are represented as mean ± S.E.M. Statistical analysis is performed by two-way ANOVA method, followed by a Tukey test using Graphpad Prism software (GraphPad Software Inc., San Diego, CA, U.S.A.). p-Value less than 0.05 indicated that the difference is statistically significant. All experiments are repeated at least three independent experiments. The results are showed as a percentage of the control groups.

Funding

This work was supported by Natural Science Foundation of Wuyi University (No. 2019WGALH01).

Author Contributions

Wenfeng Liu designed the study and revised the manuscript. Yonglian Li and Zonglin You performed the in vitro experiment and drafted the manuscript. Zonglin You synthesized the target compounds. Yonglian Li and Min Chen carried out part of the in vitro experiment. Wenfeng Liu and Suqing Zhao co-wrote the manuscript. Yonglian Li, Vincent Kam Wai Wong and Kun Zhang advised on experimental design and provided technical assistance. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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