Skip to main content
NCBI home page
RSC Medicinal Chemistry logoLink to RSC Medicinal Chemistry
. 2024 Apr 4;15(6):2002–2017. doi: 10.1039/d4md00011k

Identification of chalcone analogues as anti-inflammatory agents through the regulation of NF-κB and JNK activation†

Die Zhang a,‡, Wenping Wang a,‡, Huiping Ou a,b,‡, Jinhua Ning a, Yingxun Zhou a, Jin Ke a, Anguo Hou a, Linyun Chen a, Peng Li b,c,✉, Yunshu Ma a,✉, Wen Bin Jin a,c,✉
PMCID: PMC11187561  PMID: 38911149

Abstract

To develop new anti-inflammatory agents with improved pharmaceutical profiles, a series of chalcone analogues were designed and synthesized. In vitro anti-inflammatory activity of these compounds was evaluated by screening their inhibitory effects on NO production in RAW264.7 cell lines. The most promising compounds 3h and 3l were selected for further investigation by assessment of their dose-dependent inhibitory activity against cytokines such as TNF-α, IL-1β, and IL-6 and PGE2 release. The further study also indicated that 3h and 3l could significantly suppress the expression of iNOS and COX-2 through the NF-κB/JNK signaling pathway. Furthermore, compounds 3h and 3l could also remarkably inhibit the mRNA expression of inflammation-related genes. Meanwhile, 3h could also down-regulate ROS production. Docking simulation was conducted to position compounds 3h and 3l into the iNOS binding site to predict the probable binding mode. In conclusion, this series of chalcone analogues with reasonable drug-likeness obtained via in silico rapid prediction can be used as promising lead candidates.

To develop new anti-inflammatory agents with improved pharmaceutical profiles, a series of chalcone analogues were designed and synthesized. The figure was drawn by Figdraw (http://www.figdraw.com).graphic file with name d4md00011k-ga.jpg

1. Introduction

Inflammation refers to the defense response of living tissues with blood vessels for the recognition, removal, and repair of infection or non-infection, which is manifested as redness, swelling, heat, pain, and dysfunction.1,2 It is accompanied by pathological processes such as infection, trauma, shock, rheumatoid arthritis and atherosclerosis.3,4 The overexpression of dominant cytokines, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), at both mRNA and protein levels is responsible for inflammation via cytokine signaling pathways.5 Moreover, the overexpression of inflammatory mediators, including prostaglandin E2 (PGE2) and nitric oxide (NO), could be also induced by cytokines such as TNF-α, IL-6 and IL-1 upon inflammation stimulation.6 Therefore, the inhibition of the release of these aforementioned cytokines and inflammatory mediators has been considered a potential target for the regulation of inflammatory reactions in diseases mentioned above.7–9

Resina Draconis (Chinese name: Longxuejie) is the resin of the lily family plant Dracaena cochinchinensis (Lour.) SC Chen, mainly produced in southern Yunnan, China.10 Pharmacological studies have illustrated that it has obvious bioactive effects such as analgesic, anti-inflammatory, antifungal, antibacterial, antiplatelet aggregation, and reducing NOS activity.11–14 As shown in Fig. 1, loureirin A, loureirin B, and cochinchinenin A, belonging to dihydrochalcones, are important bioactive natural products (BNPs) found in Resina Draconis.15 Recently, numerous studies have reported that both naturally occurring and synthetic chalcones and dihydrochalcones as anti-inflammatory agents are involved in the inhibition of cytokines and inflammatory mediator production in inflammatory disorders.13,16–20 However, limited studies are reported on evaluating the anti-inflammatory activity of chalcones and dihydrochalcones or their structure–activity relationships (SARs).

Fig. 1. Structures of active compounds isolated from Resina Draconis.

Fig. 1

Open in a new tab

In this study, inspired by the structures of loureirin derivatives, we report the synthesis and the in vivo anti-inflammatory effect screening of two series of chalcones 3a–n and dihydrochalcones 4a–n (Scheme 1) for the inhibition of NO production in LPS-induced RAW264.7 cell lines compared to celecoxib and loureirin A as positive controls. Furthermore, the most promising chalcones or dihydrochalcones were screened out and selected for the investigation of the anti-inflammatory mechanism at the transcriptional level and mRNA level. Meanwhile, the levels of reactive oxygen species (ROS) production in single living cells inhibited by the most promising compound were also measured using a real-time single cell multi-mode analyzer.

Scheme 1. Reagents and conditions: (i) EtOH, 20% NaOH, 0 °C, and 8 h; (ii) EtOH, acetone, H2, 10% Pd/C, and HCO2NH2.

Scheme 1

Open in a new tab

2. Materials and methods

Chemistry

All commercial reagents and solvents were purchased from Aladdin Holdings Group Co., Ltd (Shanghai, China) and directly used without further purification unless otherwise stated. The anhydrous solvents were injected into the solutions via a syringe. All reactions were monitored by TLC by using UV light at the wavelength of 254 nm. Chromatographic purifications were performed on silica gel (160–200 mesh) by using gradient mixtures of petroleum ether and ethyl acetate as the eluent. All 1H NMR and 13C NMR spectra were recorded on a Bruker Avance spectrometer. High-resolution mass spectra (HRMS) were obtained on an Agilent 6545 by quadrupole time-of-flight (Q-TOF) mode. The structures of compounds other than 3e, 3j, 3n, 4d, 4e, 4i, 4j, 4m, and 4n were elucidated previously by NMR and the data were well in accordance with the paper published before.21–24 All tested compounds were illustrated to have >95% purity according to the HPLC assay (Agilent 1220, Germany). Comparison of FT-IR spectroscopy (Zhongke Ruijie Great 20 FT-IR spectrometer, China) of compounds 3 and 4 showed that the peak at 984 cm−1 disappeared, further confirming that the trans conformation of chalcone has been converted to dihydrochalcone (please refer to the ESI†). The melting points were measured on a Shanghai Material-light Microscopic Melting Point Instrument SGW®X-5.

General procedure for the preparation of chalcones 3

A mixture of substituted propiophenones (1 mM) and the appropriate substituted benzaldehyde (0.5 g, 3.6 mM) in dry ethyl alcohol (25 mL) was stirred for 30 min. Then, 20% NaOH (5.5 mL) was slowly added to the solution and stirred for 6 h at 0 °C. A yellow solid was precipitated. Then, the solution was adjusted to pH 3–4 with 20% HCl, filtered, and the residue was collected as a desired compound, followed by crystallization in 95% ethanol.

(E)-1,3-Bis(4-methoxyphenyl)prop-2-en-1-one (3a)

Yield, 69%; mp 134.9–135.6 °C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.85 (s, 3 H) 3.82 (s, 3 H) 6.88–7.01 (m, 4 H) 7.42 (d, J = 15.65 Hz, 1 H) 7.54–7.63 (m, 2 H) 7.77 (d, J = 15.65 Hz, 1 H) 7.99–8.08 (m, 2 H); 13C NMR (101 MHz, chloroform-d) δ 188.7, 163.3, 161.5, 143.8, 131.3, 130.7, 130.1, 127.8, 119.5, 114.4, 113.8, 77.5, 76.8, 55.5, 55.4; HRMS (Q-TOF) m/z calculated for C17H16O3 [M + Na]+: 291.1099 found: 291.0991.

(E)-1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3b)

Yield, 90%; mp 122.0–122.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.80 (d, J = 15.41 Hz, 1H), 7.68 (dd, J = 1.83, 8.44 Hz, 1H), 7.57–7.66 (m, 3H), 7.45 (d, J = 15.65 Hz, 1H), 6.94 (dd, J = 4.40, 8.56 Hz, 3H), 3.97 (d, J = 2.69 Hz, 6H), 3.86 (s, 3H); 13C NMR (101 MHz, chloroform-d) δ 188.6, 161.5, 153.1, 149.2, 143.8, 131.6, 130.1, 127.8, 122.8, 119.4, 114.4, 110.8, 110.0, 77.4, 77.0, 76.7, 56.1, 56.1, 55.4; HRMS (Q-TOF) m/z calculated for C18H18O4 [M + H]+: 299.1205 found: 299.1035.

(E)-3-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3c)

Yield, 87%; mp 138.3–139.1 °C; 1H NMR (400 MHz, chloroform-d) δ 7.80 (d, J = 15.41 Hz, 1H), 7.58–7.66 (m, J = 8.80 Hz, 2H), 7.37 (d, J = 15.65 Hz, 1H), 7.24–7.31 (m, 2H), 6.86–7.02 (m, J = 8.80 Hz, 2H), 3.96 (s, 6H), 3.94 (s, 3H), 3.86 (s, 3H); 13C NMR (101 MHz, chloroform-d) δ 189.3, 161.7, 153.1, 144.6, 133.9, 130.3, 127.6, 119.4, 114.5, 106.0, 77.4, 77.1, 76.7, 61.0, 56.4, 55.4; HRMS (Q-TOF) m/z calculated for C19H20O5 [M + H]+: 329.1311 found: 329.1381.

(E)-1-(3,5-Difluorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3d)

Yield, 83%; mp 142.3–143.1 °C; 1H NMR (400 MHz, methanol-d4) δ 7.84 (d, J = 15.41 Hz, 1H), 7.73–7.79 (m, J = 8.80 Hz, 2H), 7.67–7.73 (m, 2H), 7.60 (d, J = 15.65 Hz, 1H), 7.20–7.32 (m, 1H), 6.97–7.06 (m, J = 8.80 Hz, 2H), 3.88 (s, 3H); 13C NMR (151 MHz, chloroform-d) δ 187.8, 163.8, 162.2, 162.1, 146.3, 141.6, 130.5, 127.2, 118.5, 114.5, 111.4, 111.4, 111.3, 111.2, 107.9, 107.8, 107.6, 77.2, 77.0, 76.8, 55.5; HRMS (Q-TOF) m/z calculated for C16H12F2O2 [M + H]+: 275.0805 found: 275.0896.

(E)-1-(4-Fluoro-3-methylphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3e)

Yield, 90%; mp 86.7–87.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.81–7.91 (m, 2H), 7.77 (d, J = 15.65 Hz, 1H), 7.54–7.64 (m, 2H), 7.38 (d, J = 15.65 Hz, 1H), 7.08 (t, J = 8.80 Hz, 1H), 6.88–6.96 (m, 2H), 3.84 (s, 3H), 2.29–2.37 (m, 3H); 13C NMR (101 MHz, chloroform-d) δ 189.0, 165.4, 162.9, 161.7, 144.6, 134.5, 134.5, 132.3, 132.2, 130.3, 128.3, 128.2, 127.5, 125.5, 125.3, 119.3, 115.3, 115.1, 114.4, 77.4, 77.1, 76.8, 55.4, 14.6, 14.6; HRMS (Q-TOF) m/z calculated for C17H15FO2 [M + H]+: 271.1056 found: 271.1127.

(E)-3-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3f)

Yield, 73%; mp 130.1–130.8 °C; 1H NMR (400 MHz, chloroform-d) δ 8.00–8.08 (m, 2H), 7.76 (d, J = 15.65 Hz, 1H), 7.41 (d, J = 15.41 Hz, 1H), 7.23 (dd, J = 1.96, 8.31 Hz, 1H), 7.16 (d, J = 1.96 Hz, 1H), 6.94–7.03 (m, 2H), 6.89 (d, J = 8.31 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.88 (s, 3H); 13C NMR (101 MHz, chloroform-d) δ 188.7, 163.3, 151.3, 149.2, 144.1, 131.3, 130.7, 128.1, 123.0, 119.8, 113.8, 111.1, 110.1, 77.4, 77.1, 76.8, 56.0, 56.0, 55.5; HRMS (Q-TOF) m/z calculated for C18H18O4 [M + Na]+: 321.1205 found: 321.1095.

(E)-1,3-Bis(3,4-dimethoxyphenyl)prop-2-en-1-one (3g)

Yield, 81%; mp 123.5–124.3 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 3.90–3.97 (m, 12 H) 6.83–6.94 (m, 2 H) 7.17 (d, J = 1.71 Hz, 1 H) 7.20–7.26 (m, 1 H) 7.43 (d, J = 15.65 Hz, 1 H) 7.62 (d, J = 1.96 Hz, 1 H) 7.68 (dd, J = 8.44, 2.08 Hz, 1 H) 7.76 (d, J = 15.65 Hz, 1 H); 13C NMR (101 MHz, chloroform-d) δ 188.6, 153.1, 151.3, 149.2, 144.1, 131.5, 128.0, 122.9, 122.9, 119.6, 111.1, 110.8, 110.2, 109.9, 77.5, 77.2, 76.8, 56.0, 56.0; HRMS (Q-TOF) m/z calculated for C19H20O5 [M + H]+: 329.1311 found: 329.1382.

(E)-3-(3,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3h)

Yield, 93%; mp 159.8–160.5 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 3.90–3.96 (m, 15 H) 6.89 (d, J = 8.31 Hz, 1 H) 7.16 (d, J = 1.71 Hz, 1 H) 7.25 (dd, J = 8.31, 1.96 Hz, 1 H) 7.27 (s, 2 H) 7.36 (d, J = 15.41 Hz, 1 H) 7.76 (d, J = 15.65 Hz, 1 H); 13C NMR (101 MHz, CHLOROFORM-d) δ 189.2, 189.2, 153.1, 151.4, 149.2, 144.8, 142.3, 133.7, 127.8, 122.9, 119.7, 111.1, 110.5, 106.1, 77.6, 77.2, 76.9, 60.9, 56.3, 55.9; HRMS (Q-TOF) m/z calculated for C19H22O5 [M + H]+: 359.1416 found: 359.1487.

(E)-1-(3,5-Difluorophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3i)

Yield, 91%; mp 140.8–141.5 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 3.94 (s, 3 H) 3.95–3.97 (m, 3 H) 6.91 (d, J = 8.31 Hz, 1 H) 7.02 (tt, J = 8.50, 2.26 Hz, 1 H) 7.16 (d, J = 1.96 Hz, 1 H) 7.22–7.29 (m, 2 H) 7.47–7.54 (m, 2 H) 7.79 (d, J = 15.65 Hz, 1 H); 13C NMR (101 MHz, chloroform-d) δ 187.8, 164.3, 164.2, 161.8, 161.7, 151.9, 149.4, 146.6, 141.5, 127.4, 123.7, 118.7, 111.4, 111.4, 111.3, 111.2, 110.2, 108.0, 107.8, 107.5, 77.4, 77.1, 76.7, 56.0, 56.0; HRMS (Q-TOF) m/z calculated for C17H14F2O3 [M + H]+: 305.0911 found: 305.1088.

(E)-3-(3,4-Dimethoxyphenyl)-1-(4-fluoro-3-methylphenyl)prop-2-en-1-one (3j)

Yield, 73%; mp 143.7–144.5 °C; 1H NMR (400 MHz, chloroform-d) δ 7.83–7.92 (m, 2H), 7.76 (d, J = 15.65 Hz, 1H), 7.36 (d, J = 15.65 Hz, 1H), 7.25 (dd, J = 1.83, 8.19 Hz, 1H), 7.16 (d, J = 1.96 Hz, 1H), 7.11 (t, J = 8.93 Hz, 1H), 6.91 (d, J = 8.31 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 2.30–2.40 (m, 3H); 13C NMR (101 MHz, chloroform-d) δ 189.2, 162.9, 151.5, 149.3, 145.0, 134.5, 134.5, 132.3, 132.2, 128.3, 128.2, 127.8, 125.5, 125.3, 123.2, 119.7, 115.3, 115.1, 111.1, 110.2, 77.4, 77.0, 76.7, 56.0, 14.6, 14.6; HRMS (Q-TOF) m/z calculated for C18H17FO3 [M + H]+: 301.1162 found: 301.1234.

(E)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3k)

Yield, 91%; mp 170.8–171.7 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 3.86 (s, 3 H) 3.88–3.94 (m, 9 H) 6.86 (s, 2 H) 6.92–7.01 (m, 2 H) 7.44 (d, J = 15.65 Hz, 1 H) 7.70 (d, J = 15.65 Hz, 1 H) 7.99–8.09 (m, 2 H); 13C NMR (101 MHz, chloroform-d) δ 188.5, 163.4, 153.4, 144.1, 140.3, 131.1, 130.8, 130.6, 121.2, 113.8, 105.6, 77.5, 77.2, 76.9, 60.9, 56.2, 55.4; HRMS(Q-TOF) m/z calculated for C19H20O5 [M + H]+: 329.1311 found: 329.1381.

(E)-1-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3l)

Yield, 76%; mp 163.0–163.5 °C; 1H NMR (400 MHz, chloroform-d) δ 7.67–7.76 (m, 2H), 7.62 (d, J = 1.96 Hz, 1H), 7.45 (d, J = 15.65 Hz, 1H), 6.85–6.94 (m, 3H), 3.88–3.98 (m, 15H); 13C NMR (101 MHz, chloroform-d) δ 188.4, 153.4, 153.2, 149.2, 144.0, 140.3, 131.3, 130.5, 123.0, 121.0, 110.8, 110.0, 105.6, 77.5, 77.2, 76.9, 60.9, 56.2, 56.0; HRMS (Q-TOF) m/z calculated for C20H22O6 [M + H]+: 359.1416 found: 359.1487.

(E)-1,3-Bis(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3m)

Yield, 92%; mp 100.0–100.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.72 (d, J = 15.41 Hz, 1H), 7.36 (d, J = 15.41 Hz, 1H), 7.26 (s, 2H), 6.87 (s, 2H), 3.89–3.97 (m, 19H); 13C NMR (101 MHz, chloroform-d) δ 189.3, 153.5, 153.1, 144.9, 142.5, 140.5, 133.6, 130.4, 121.3, 106.3, 105.8, 77.5, 77.2, 76.9, 60.9, 60.9, 56.4, 56.2; HRMS (Q-TOF) m/z calculated for C21H24O7 [M + H]+: 389.1522 found: 389.1434.

(E)-1-(4-Fluoro-3-methylphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3n)

Yield, 61%; mp 107.0–107.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.82–7.91 (m, 2H), 7.71 (d, J = 15.41 Hz, 1H), 7.37 (d, J = 15.41 Hz, 1H), 7.11 (t, J = 8.80 Hz, 1H), 6.87 (s, 2H), 3.88–3.95 (m, 10H), 2.36 (d, J = 1.71 Hz, 3H); 13C NMR (101 MHz, chloroform-d) δ 189.0, 165.5, 162.9, 153.5, 145.0, 140.6, 134.3, 134.3, 132.3, 132.2, 130.3, 128.4, 128.3, 125.6, 125.4, 121.1, 115.3, 115.1, 105.8, 77.4, 77.3, 77.1, 76.7, 61.0, 56.3, 14.6, 14.6; HRMS (Q-TOF) m/z calculated for C19H19FO4 [M + Na]+: 353.1267 found: 353.1178.

General procedure for the preparation of chalcones 4

A mixture of compound 3 (1 mol), ammonium formate (0.13 g, 2 mmol) and 10% Pd/C (0.01 g, 0.09 mmol) in dry ethyl alcohol (9 mL) and propiophenone (9 mL) was stirred at room temperature overnight. The solution was filtered, then the organic solvent was evaporated under reduced pressure to give a crude mixture and re-dissolved in acetone (10 mL), the mixture was re-filtered, and the organic layers were concentrated under reduced pressure to afford the desired product as a clear oil. Subsequent purification of the mixture was performed by using flash column chromatography on silica gel with gradient elution to afford the desired compound 4.

1,3-Bis(4-methoxyphenyl)propan-1-one (4a)

Yield, 50%; mp 72.5–73.1 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 2.89–3.05 (m, 2 H) 3.15–3.27 (m, 2 H) 3.76 (s, 3 H) 3.84 (s, 3 H) 6.78–6.87 (m, 2 H) 6.87–6.95 (m, 2 H) 7.11–7.19 (m, 2 H) 7.88–7.96 (m, 2 H); 13C NMR (101 MHz, chloroform-d) δ 198.0, 163.5, 158.0, 133.5, 130.3, 130.1, 129.4, 114.0, 113.7, 77.4, 77.1, 76.8, 55.5, 55.3, 40.4, 29.5; HRMS (Q-TOF) m/z calculated for C17H18O3 [M + Na]+: 293.1256 found: 293.1146.

1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)propan-1-one (4b)

Yield, 52%; mp 92.0–92.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.58 (dd, J = 1.96, 8.56 Hz, 1H), 7.53 (d, J = 1.96 Hz, 1H), 7.12–7.23 (m, 2H), 6.75–6.94 (m, 3H), 3.93 (s, 3H), 3.95 (s, 3H), 3.79 (s, 3H), 3.23 (t, J = 7.70 Hz, 2H), 3.00 (t, J = 7.70 Hz, 2H); 13C NMR (101 MHz, chloroform-d) δ 198.0, 158.0, 153.2, 149.0, 133.5, 130.2, 129.4, 122.6, 113.9, 110.2, 110.0, 77.3, 77.0, 76.7, 56.1, 56.0, 55.3, 40.3, 29.6; HRMS (Q-TOF) m/z calculated for C18H20O4 [M + H]+: 301.1362 found: 301.2615.

3-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)propan-1-one (4c)

Yield, 52%; mp 126.7–127.3 °C; 1H NMR (400 MHz, chloroform-d) δ 7.14–7.22 (m, 3 h), 6.81–6.92 (m, 2H), 3.88–3.93 (m, 9H), 3.79 (s, 3H), 3.15–3.28 (m, 2H), 2.93–3.07 (m, 2H); 13C NMR (101 MHz, chloroform-d) δ 198.2, 158.1, 153.1, 133.3, 132.2, 129.4, 114.0, 105.6, 77.3, 77.0, 76.7, 61.0, 56.3, 55.3, 40.6, 29.5; HRMS (Q-TOF) m/z calculated for C19H22O5 [M + Na]+: 353.1467 found: 353.1359.

1-(3,5-Difluorophenyl)-3-(4-methoxyphenyl)propan-1-one (4d)

Mp 81.0–81.9 °C; yield, 43%; 1H NMR (400 MHz, chloroform-d) δ 7.38–7.49 (m, 2H), 7.12–7.19 (m, 2H), 7.00 (tt, J = 2.32, 8.44 Hz, 1H), 6.81–6.87 (m, 2H), 3.79 (s, 3H), 3.16–3.24 (m, 2H), 2.97–3.03 (m, 2H); 13C NMR (101 MHz, chloroform-d) δ 196.7, 196.7, 164.4, 164.2, 161.9, 161.8, 158.2, 139.8, 132.7, 129.4, 114.0, 111.1, 111.0, 110.9, 110.9, 108.5, 108.3, 108.0, 77.4, 77.1, 76.7, 55.3, 40.8, 29.0; HRMS (Q-TOF) m/z calculated for C16H14F2O2 [M + Na]+: 299.0962 found: 299.1111.

1-(4-Fluoro-3-methylphenyl)-3-(4-methoxyphenyl)propan-1-one (4e)

Yield, 45%; mp 50.2–51.3 °C; 1H NMR (400 MHz, chloroform-d) δ 7.72–7.89 (m, 2H), 7.12–7.21 (m, 2H), 7.04 (t, J = 8.80 Hz, 1H), 6.77–6.91 (m, 2H), 3.78 (s, 3H), 3.15–3.28 (m, 2H), 2.91–3.06 (m, 2H), 2.30 (d, J = 1.71 Hz, 3H); 13C NMR (101 MHz, chloroform-d) δ 198.1, 163.1, 158.0, 133.3, 133.1, 133.0, 131.9, 131.9, 129.4, 128.0, 127.9, 125.4, 125.3, 115.4, 115.1, 113.9, 77.4, 77.1, 76.8, 55.3, 40.7, 29.3, 14.6, 14.6; HRMS (Q-TOF) m/z calculated for C17H17FO2 [M + H]+: 273.1213 found: 273.1284.

3-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)propan-1-one (4f)

Yield, 36%; mp 82.3–82.9 °C; 1H NMR (400 MHz, chloroform-d) δ 7.91–7.99 (m, 2H), 6.89–6.98 (m, J = 8.80 Hz, 2H), 6.73–6.83 (m, 3H), 3.85–3.88 (m, 9H), 3.23 (t, J = 7.70 Hz, 2H), 3.01 (t, J = 7.70 Hz, 2H); 13C NMR (101 MHz, chloroform-d) δ 198.0, 163.5, 134.1, 130.3, 130.0, 120.2, 113.7, 111.9, 111.3, 77.4, 77.0, 76.7, 56.0, 55.9, 55.5, 40.4, 30.0; HRMS (Q-TOF) m/z calculated for C18H20O4 [M + H]+: 301.1362 found: 301.1230.

1,3-Bis(3,4-dimethoxyphenyl)propan-1-one (4g)

Yield, 59%; mp 121.3–122.1 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 2.87–2.97 (m, 2 H) 3.11–3.20 (m, 2 H) 3.78 (s, 3 H) 3.77 (s, 3 H) 3.82–3.88 (m, 6 H) 6.66–6.73 (m, 3 H) 6.79 (d, J = 8.31 Hz, 1 H) 7.44 (d, J = 1.96 Hz, 1 H) 7.49 (dd, J = 8.44, 2.08 Hz, 1 H); 13C NMR (101 MHz, chloroform-d) δ 198.0, 153.3, 149.1, 148.9, 147.4, 134.1, 130.2, 122.7, 120.2, 111.9, 111.4, 110.2, 110.0, 77.4, 77.1, 76.7, 56.1, 56.0, 56.0, 55.9, 40.2, 30.2; HRMS (Q-TOF) m/z calculated for C19H22O5 [M + Na]+: 353.1467 found: 353.1362.

3-(3,4-Dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)propan-1-one (4h)

Yield, 39%; mp 136.2–137.1 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 2.93 (t, J = 7.58 Hz, 2 H) 3.17 (t, J = 7.70 Hz, 2 H) 3.74–3.86 (m, 15 H) 6.67–6.77 (m, 3 H) 7.12 (s, 2 H); 13C NMR (101 MHz, chloroform-d) δ 197.1, 152.1, 147.9, 146.5, 141.6, 132.9, 131.2, 119.2, 111.0, 110.4, 104.6, 76.4, 76.0, 75.7, 59.9, 55.3, 54.9, 54.9, 39.6, 29.0; HRMS (Q-TOF) m/z calculated for C20H24O6 [M + Na]+: 383.1573 found: 383.1465.

1-(3,5-Difluorophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one (4i)

Yield, 30%; mp 94.9–96.1 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 2.95–3.06 (m, 2 H) 3.18–3.28 (m, 2 H) 3.87 (s, 3 H) 3.86 (s, 3 H) 6.72–6.84 (m, 3 H) 7.00 (tt, J = 8.38, 2.38 Hz, 1 H) 7.40–7.49 (m, 2 H); 13C NMR (101 MHz, chloroform-d) δ 196.7, 164.4, 164.2, 161.9, 161.8, 149.0, 147.6, 139.9, 139.8, 139.7, 133.3, 120.2, 111.9, 111.4, 111.1, 111.0, 110.9, 110.8, 108.6, 108.3, 108.1, 77.4, 77.1, 76.7, 55.9, 55.9, 40.8, 29.5; HRMS (Q-TOF) m/z calculated for C17H16F2O3 [M + K]+: 345.1068 found: 345.2155.

3-(3,4-Dimethoxyphenyl)-1-(4-fluoro-3-methylphenyl)propan-1-one (4j)

Yield, 50%; mp 90.2–90.8 °C; 1H NMR (400 MHz, chloroform-d) δ 7.74–7.87 (m, 2H), 7.06 (t, J = 8.80 Hz, 1H), 6.75–6.82 (m, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.14–3.34 (m, 2H), 2.89–3.11 (m, 2H), 2.31 (d, J = 1.96 Hz, 3H); 13C NMR (101 MHz, chloroform-d) δ 198.1, 165.6, 163.1, 149.0, 147.5, 133.9, 133.1, 133.1, 131.9, 131.8, 128.0, 127.9, 125.4, 125.3, 120.2, 115.3, 115.1, 111.9, 111.4, 77.4, 77.0, 76.7, 56.0, 55.9, 40.6, 29.8, 14.6, 14.5; HRMS (Q-TOF) m/z calculated for C18H19FO3 [M − H]−: 301.1318 found: 301.1311.

1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propan-1-one (4k)

Yield, 49%; mp 128.6–129.1 °C; 1H NMR (400 MHz, chloroform-d) δ ppm 2.86–3.07 (m, 2 H) 3.17–3.29 (m, 2 H) 3.78–3.89 (m, 12 H) 6.47 (s, 2 H) 6.85–6.98 (m, 2 H) 7.87–7.98 (m, 2 H); 13C NMR (101 MHz, chloroform-d) δ 197.8, 163.5, 153.2, 137.3, 130.3, 130.0, 113.7, 105.4, 77.4, 77.1, 76.8, 60.8, 56.1, 55.5, 55.3, 54.1, 40.2, 32.0, 30.8; HRMS (Q-TOF) m/z calculated for C19H22O5 [M + H]+: 331.1467 found: 331.1380.

1-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propan-1-one (4l)

Yield, 59%; mp 134.9–135.7 °C; 1H NMR (400 MHz, chloroform-d) δ 7.41–7.53 (m, 2H), 6.79 (d, J = 8.31 Hz, 1H), 6.39 (s, 2H), 3.84 (s, 3H), 3.85 (s, 3H), 3.68–3.81 (m, 9H), 3.17 (t, J = 7.58 Hz, 2H), 2.92 (t, J = 7.46 Hz, 2H); 13C NMR (101 MHz, chloroform-d) δ 197.8, 153.3, 153.2, 149.0, 137.2, 130.1, 122.7, 110.2, 110.1, 105.5, 77.5, 77.2, 76.9, 60.8, 56.1, 56.0, 56.0, 40.1, 30.9; HRMS (Q-TOF) m/z calculated for C20H24O6 [M + H]+: 361.1573 found: 361.1644.

1,3-Bis(3,4,5-trimethoxyphenyl)propan-1-one (4m)

Yield, 59%; mp 152.6–153.3 °C; 1H NMR (400 MHz, chloroform-d) δ 7.21 (s, 2H), 6.47 (s, 2H), 3.91 (d, J = 3.91 Hz, 9H), 3.77–3.88 (m, 9H), 3.25 (d, J = 8.07 Hz, 2H), 3.01 (s, 2H); 13C NMR (101 MHz, chloroform-d) δ 198.0, 153.3, 153.1, 142.7, 137.1, 136.5, 132.2, 105.6, 105.5, 77.4, 77.1, 76.7, 60.9, 60.9, 56.4, 56.1, 40.5, 30.9; HRMS (Q-TOF) m/z calculated for C21H26O7 [M + H]+: 391.1679 found: 391.1749.

1-(4-Fluoro-3-methylphenyl)-3-(3,4,5-trimethoxyphenyl)propan-1-one (4n)

Yield, 54%; mp 79.1–79.8 °C; 1H NMR (400 MHz, chloroform-d) δ 7.73–7.85 (m, 2H), 7.06 (t, J = 8.80 Hz, 1H), 6.46 (s, 2H), 3.79–3.88 (m, 9H), 3.22–3.29 (m, 2H), 2.95–3.04 (m, 2H), 2.31 (d, J = 1.96 Hz, 3H); 13C NMR (101 MHz, chloroform-d) δ 197.9, 165.6, 163.1, 153.3, 153.1, 137.1, 136.4, 133.0, 131.9, 131.8, 128.0, 127.9, 125.5, 125.3, 115.4, 115.1, 105.5, 105.4, 105.3, 77.4, 77.1, 76.8, 60.8, 60.7, 56.1, 55.9, 40.5, 40.3, 30.6, 14.6, 14.5; HRMS (Q-TOF) m/z calculated for C19H21FO4 [M + Na]+: 355.1424 found: 355.1315.

Biological evaluation

Cell line and culture

The mouse macrophage cell line RAW264.7 was purchased from Pricella. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, Viva Cell) and 1% penicillin/streptomycin (Viva Cell). The cells were incubated in a humidified incubator in 5% CO2 at 37 °C.

Nitric oxide (NO) assay

RAW264.7 macrophages were cultured in a 24-well plate with a density of 100 000 cells per well for 36 h and then pre-treated with various chalcone analogues (20 μM) for 2 h.25 Afterwards they were further incubated with LPS (1 μg mL−1) for 24 h before NO production assay. Then, the supernatant was mixed with the Griess reagent (S0021M, Beyotime, China) to detect NO production. Celecoxib and loureirin A were used as a positive control. LPS served as a negative control. The optical density of the mixture was determined at 540 nm by using an ELISA plate reader and the NO concentration was calculated according to the sodium nitrite standard curve.

Enzyme-linked immunosorbent assay (ELISA)

After treatment of RAW264.7 macrophages with synthetic compounds and LPS stimulation, the corresponding cytokine levels were measured with TNF-α ELISA kit (EMC102a, Neobioscience, China), IL-6 ELISA kit (EK306/3-96, Multi Sciences, China), IL-1β ELISA kit (514010, Multi Sciences), and PGE2 ELISA kit (514010, Cayman Chemical, USA) according to the manufacturer's instructions, respectively.26 Loureirin A served as the positive control.

Western blot assay

After treatment with synthetic compounds and LPS stimulation, RAW264.7 macrophages were washed twice with cold phosphate buffered saline (PBS) and lysed in 1% Triton X-100 buffer containing phosphatase and protease inhibitors. After incubating the lysate on ice for 30 min, debris was removed by centrifugation at 1000 rpm for 10 min at 4 °C in order to afford the supernatant. The protein concentration was detected by using a BCA protein assay kit (PK10026, Proteintech, China). The total proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred to polyvinylidene fluoride (PVDF) membranes for incubation with primary antibody at 4 °C overnight. The next day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h. Immunoreactive bands were visualized by using a high-sensitivity ECL chemiluminescence detection kit (PK10003, Proteintech, China) and analyzed using ImageJ software.

Real-time quantitative PCR

Cells were homogenized in total RNA extraction kit (Solarbio, Beijing, China) for extraction of RNA according to the manufacturer's instruction. The cDNA was synthesized with PrimeScript RT Master Mix (Takara, Tokyo, Japan) on a Biometra Tadvanced 96 SG (Analytik Jena, Jena, Germany). Quantitative PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher, Waltham, USA) with GOTaq qPCR Master Mix (Promega, Madison, USA). The primers of genes including TNF-α, IL-6, iNOS, IL-1β, COX-2, and GAPDH were synthesized by Sangon Biotech. The primer sequences of mouse genes used are shown as follows:

TNF-α sense primer, 5′-CTTCTCATTCCTGCTTGTG-3′;

TNF-α antisense primer, 5′-ACTTGGTGGTTTGCTACG-3′;

IL-6 sense primer, 5′-GTCCTTCCACCCCAATTTCCA-3′;

IL-6 antisense primer, 5′-TAACGCACTAGGTTTGCCGA-3′;

COX-2 sense primer, 5′-TGAGTACCGCAAACGCTTCTC-3′;

COX-2 antisense primer, 5′-TGGACGAGGTTTTTCCACCAG-3′;

iNOS sense primer, 5′-AGCAACTACTGCTGGTGGTG-3′;

iNOS antisense primer, 5′-TCTTCAGAGTCTGCCCATTG-3′;

IL-1β sense primer, 5′-CCTGGGCTGTCCTGATGAGAG-3′;

IL-1β antisense primer, 5′-TCCACGGGAAAGACACAGGTA-3′;

GAPDH sense primer, 5′-CACTCACGGCAAATTCAACGGCA-3′;

GAPDH antisense primer, 5′-GACTCCACGACATACTCAGCAC-3′.

The amount of each gene was determined and normalized by the amount of GAPDH.

ROS measurements

2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA, CM01378, Proteintech, China), a cell-permeable ROS-sensitive probe, was used to detect the single intracellular ROS levels. RAW264.7 macrophages were plated on confocal petri dishes with a density of 100 000 cells per well. After 36 h, the cells were pre-treated with different concentrations (10, 20, and 40 μM) of 3h for 2 h and then stimulated with LPS (1 μg mL−1) for 24 h. H2DCFDA was further diluted in DMSO before use. RAW264.7 cells were then mixed with 50 μM H2DCFDA solution in PBS prior to immediate incubation in the dark for 30 min. Eventually, the ROS level of the mixture was measured using a live-time single cell multi-mode analyzer (Ruiming Biotech, China).

Cytotoxicity assay

Briefly, according to the published paper,27–29 the antiproliferative assay was based on the reduction of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) to yellow formazan dye by succinate dehydrogenase in living cells. RAW 264.7 cells in 100 μL of culture media were seeded in a 96-well plate at a density of 20 000 cells per well and incubated for 24 h at 37 °C. Then, the cells were treated with various concentrations of the compound for 4 h before CCK-8 assay. Blank culture medium was used as a negative control. Medium without cells was used as the blank control. After treatment, CCK-8 reagent (C6005, NCM Biotechnology, China) in medium was added to each well and the cells were further incubated for 50 min at 37 °C according to the manufacturer's instructions. The optical density was measured at 450 nm using a microplate reader (Synergy HTX, Bio-Tek). The percentage of surviving cells was calculated using the following formula: (corrected reading from the test well-corrected reading from the blank well)/(corrected reading from negative well-corrected reading from the blank well) × 100%. IC50 was calculated by using the software IC50 Calculator.

Computational docking studies

Autodock Vina software was applied for virtual docking. The 3D structures of compounds 3h and 3l were generated and energy optimized using ChemDraw 22.0 software before they were imported into the Autodock software. The X-ray crystal structure of iNOS protein (PDB ID: 1r35) downloaded from the Protein Data Bank (https://www.rcsb.org/) was investigated for docking experiments after deletion of the unnecessary water molecules, metal ions, and small ligands attached to the protein. The substrate-binding pocket of iNOS protein has been defined according to the published paper.18 The potential binding results were visualized using PyMOL version 2.5.2 software.

In silico prediction of physicochemical properties and pharmacokinetic profile

Molsoft website (https://www.molsoft.com/servers.html) was used to predict Lipinski's rule of compounds. PreADMET website (https://preadmet.bmdrc.kr/) was also utilized for Pre-ADMET prediction of compounds.

Statistical analysis

The results were presented as means ± SD. In our analysis, statistical analyses were performed using the single factor ANOVA test or Student's t test.

3. Results and discussion

Chemistry

The chalcones 3 were prepared in good yields using the Claisen–Schmidt condensation (Scheme 1). This method for the preparation of chalcones was productive since it predominantly generated the trans conformer from simple building blocks. A large number of substituted benzaldehydes and acetophenones were adopted as they were commercially available and inexpensive.

A series of dihydrochalcones 4 were prepared by base-promoted reaction of chalcones 3 with a palladium-carbon system under a hydrogen atmosphere. These products proved easy to separate by flash chromatography (Scheme 1).

Biological evaluation

Griess assay for nitric oxide (NO) production and cytotoxicity determination

It is well known that NO, also known as endothelium-derived relaxing factor, plays an essential role in the pathogenesis of inflammation-associated disorders. Additionally, several studies have demonstrated that the production of NO as a biomarker was highly associated with the severity of the diseases.30,31 Therefore, inhibiting NO release would be a useful method in the therapy of inflammation. Initially, to verify the anti-inflammatory effects of chalcone analogues, the LPS-stimulated inflammation macrophage RAW264.7 cell line was utilized. As shown in Fig. 2A, compound 3h with an IC50 value of 7.6 ± 1.6 μM could inhibit the NO production of the LPS-stimulated RAW264.7 cell line in a concentration-dependent manner. When the concentration of compound 3h was set to 20 μM, the anti-inflammatory effect was suggested to be the most promising. Meanwhile, we also evaluated the cytotoxic effects of compound 3h on proliferation. As illustrated in Fig. 2B and C, treatment of RAW 264.7 cells with 20 μM 3h or 3l prior to induction with LPS showed no cytotoxicity. Hence, the concentration of 20 μM of chalcone analogues for anti-inflammatory activity screening could be more preferred.

Fig. 2. (A) NO production in the supernatant of RAW264.7 macrophages after the administration of compound 3h using the NO assay. The results were shown as the percent of LPS control. Data were expressed as mean ± SD (n = 3). Statistical significance relative to the LPS group was illustrated: ***p < 0.001. (B) Cytotoxicity evaluation of different concentrations of compound 3h on RAW 264.7 cells using the CCK-8 assay. (C) Cytotoxicity evaluation of different concentrations of compound 3l on RAW 264.7 cells using the CCK-8 assay. Data were indicated as mean ± SD (n = 3).

Fig. 2

Open in a new tab

Next, LPS-induced NO production assay confirmed that nearly all of our compounds were supposed to be NO inhibitors with the exception of compounds 4f and 4n as shown in Fig. 3. Surprisingly, the parent compound loureirin A did not inhibit the NO production of the RAW264.7 cell line induced by LPS. Fourteen loureirin analogues at a concentration of 20 μM displayed 65–86% inhibition rate of NO production, comparable to or more promising than that of celecoxib at the same dosage. Eight loureirin analogues were indicated to be even more potent than celecoxib in inhibiting LPS-induced NO production. Importantly, we found that compounds 3h and 3l showed the most potent anti-inflammatory activity among all synthetic loureirin analogues.

Fig. 3. Loureirin analogues inhibit NO production in LPS-induced RAW264.7. The results were presented as the percent of LPS control. Data were obtained by at least three independent experiments, and each was performed in triplicates. ***p < 0.001, **p < 0.01 compared with LPS-stimulated cells.

Fig. 3

Open in a new tab

Compounds 3h and 3l inhibited the TNF-α, IL-1β and IL-6 production in a dose-dependent manner

The most promising compounds 3h and 3l were selected for further determination of their dose-dependent inhibitory activity against LPS-stimulated TNF-α, IL-1β and IL-6 production. RAW264.7 macrophages were pretreated with compounds 3h or 3l (2.5, 5, 10, 20 and 40 μM) for 2 h, followed by subsequent incubation with LPS at a concentration of 1 μg mL−1 for 24 h. Finally, the production of TNF-α, IL-1β and IL-6 was assessed by enzyme-linked immunosorbent assay (ELISA), respectively. The results were summarized and are shown in Fig. 4, illustrating that TNF-α, IL-1β and IL-6 release was inhibited by compounds 3h and 3l in a dose-dependent manner. Accordingly, the IC50 values of compound 3h against the expression of TNF-α, IL-1β and IL-6 were 7.1 ± 0.4 μM, 10.0 ± 1.25 μM, and 8.4 ± 0.25 μM, respectively. The IC50 values of compound 3l against the expression of TNF-α, IL-1β and IL-6 were 7.9 ± 0.6 μM, 12.3 ± 1.5 μM, and 2.1 ± 1.0 μM, respectively. Therefore, inhibition of TNF-α, IL-1β and IL-6 production by compounds 3h and 3l in a dose-dependent manner suggested their potential as anti-inflammatory agents.

Fig. 4. RAW264.7 macrophages were treated with different concentrations of 3h (A) and 3l (B), respectively. The expression of TNF-α, IL-1β and IL-6 in the supernatant was detected using ELISA. Statistical data are expressed as mean ± SD and were calculated using the single factor ANOVA test. #p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the control group. *p < 0.05, **p < 0.01, and ****p < 0.001, compared with the LPS group.

Fig. 4

Open in a new tab

Compounds 3h and 3l inhibited iNOS, PGE2 and COX-2 production

Inducible nitric oxide synthase (iNOS) as a key mediator of inflammation could produce NO from l-arginine, followed by triggering of PGE2 induction.32,33 Besides cytokines TNF-α, IL-1β and IL-6 evaluated in Fig. 4, cyclooxygenase 2 (COX-2) has also been discovered as a core inflammatory mediator involved in the inflammation.34,35 Therefore, compounds 3h and 3l with potent inhibitory effects of NO production were selected for further IC50 determination of inhibition on iNOS, PGE2 and COX-2 by ELISA and Western blot. As shown in Fig. 3 and 5, we confirmed that LPS-induced RAW264.7 macrophages could overexpress COX-2 and iNOS to increase PGE2 and NO release. Treatment with compounds 3h and 3l displayed a distinct inhibition of iNOS activity with IC50 values of 11.9 ± 1.1 μM and 4.6 ± 1.7 μM, and PGE2 production with IC50 of 9.5 ± 2.4 μM and 4.0 ± 0.4 μM, respectively. In accordance with the inhibition of NO release, compounds 3h and 3l could significantly reduce the production of iNOS and COX-2. Western blotting analysis showed that the protein levels of COX-2 and iNOS in LPS-induced RAW264.7 macrophages were significantly decreased after administration of different concentrations of 3h and 3l, respectively (10, 20, and 40 μM). This study proved that compounds 3h and 3l exerted potential inhibitory effects on the LPS-induced inflammatory response of the RAW264.7 cell line.

Fig. 5. RAW264.7 macrophages induced by LPS were treated with different concentrations of 3h and 3l. (A and B) The expression of PGE2 in the supernatant was detected using ELISA. The results are presented as the percent of LPS control. Data were obtained by at least three independent experiments, and each was performed in triplicates. ***p < 0.001 compared with LPS-stimulated cells. (C–H) iNOS and COX-2 expressions were determined via Western blotting. β-Actin served as the loading control. Statistical data are expressed as mean ± SD (n = 3) and were calculated using the single factor ANOVA test. ###p < 0.001, compared with the control group. **p < 0.01 and ***p < 0.001, compared with the LPS group.

Fig. 5

Open in a new tab

Compounds 3h and 3l inhibited LPS-induced mRNA expression of inflammatory genes

To further evaluate the inhibitory activity of compounds 3h and 3l against pre-inflammatory mediators at the level of mRNA, the mRNA expressions of inflammation-related genes including TNF-α, IL-1β, IL-6, iNOS and COX-2 were determined by real-time quantitative PCR (RT-qPCR) in LPS-induced RAW264.7 macrophages. Briefly, the LPS-induced RAW264.7 cell line was incubated with compounds 3h and 3l at a series of concentrations of 5, 10 and 20 μM, respectively. Subsequently, total RNA was extracted and specific mRNAs were assessed by using the RT-qPCR technique. As shown in Fig. 6, LPS at a concentration of 1 μg mL−1 could significantly increase the mRNA expression level of TNF-α, IL-1β, IL-6, iNOS and COX-2 compared to those of the vehicle control, suggesting that the LPS could successfully induce the inflammation. However, incubation with compounds 3h or 3l (5, 10 and 20 μM) could remarkably down-regulate the expression of these aforementioned mRNAs with a statistical significance (p < 0.05 or p < 0.01) in a dose-dependent manner, indicating that our compounds could attenuate the inflammation. As shown in Fig. 6, these data implied that compounds 3h and 3l could influence the inflammatory mediators including TNF-α, IL-1β, IL-6, iNOS, COX-2, etc. at the level of mRNA.

Fig. 6. Cells were pretreated with different concentrations (5, 10 and 20 μM) of compounds 3h (A) and 3l (B) or vehicle control for 2 h, followed by the addition of LPS (1 μg mL−1) for 6 h. The mRNA levels of TNF-α, iNOS, COX-2, IL-1β, and IL-6 were quantified using RT-qPCR technique. The mRNA values for each gene were normalized to the internal control β-actin mRNA. Data are expressed as mean ± SD of three independent experiments. Statistical significance relative to the LPS group is indicated as follows: *, p < 0.05; **, p < 0.01; ***P < 0.001.

Fig. 6

Open in a new tab

Molecular docking of iNOS with compounds 3h and 3l

Autodock Vina was applied to explore and better understand the potency and the ability of the evaluated compounds to fit nicely into the active site of iNOS protein. The most active compounds 3h and 3l were selected to investigate their binding with iNOS protein whose crystal structure (PDB ID: 1r35) was obtained from the Protein Data Bank (https://www.rcsb.org/). The docking structures of compounds 3h and 3l with the minimum lowest energy were created by using Chem 3D 20.0. Docking results suggested that both compounds 3h and 3l could nicely fit into the binding pocket of iNOS (PDB ID: 1r35), indicating that these in silico predictions were highly in accordance with the aforementioned in vitro iNOS evaluation results.

Cartoon representations of the binding pocket of the structure of mouse iNOS (PDB ID: 1r35) in complex with compounds 3h and 3l are shown in Fig. 7. The protein–ligand interaction profiler (PLIP) website pointed out that 3h formed five hydrogen bonds with residues ARG260, ARG375, GLN381, and ARG382, as well as pi–alkyl interactions with ALA276 and GLN381 amino acid residues. Additionally, the PLIP website also predicted that 3l showed three hydrogen-bonding interactions with residues GLN-381 and ARG375. The length of the hydrogen bonds ranged from 2.99 Å to 3.96 Å, showing strong physicochemical force. Interestingly, the phenyl moiety of compound 3l also engaged in a hydrophobic interaction such as pi–sigma interaction with GLN381. Meanwhile, the binding affinity values of compounds 3h and 3l were −6.2 kcal mol−1 and −6.9 kcal mol−1, respectively, indicating that our compounds and iNOS protein could moderately bind to each other under natural conditions.

Fig. 7. Binding mode of the iNOS (PDB code: 1r35) pocket in complex with compounds 3h (A) and 3l (B).

Fig. 7

Open in a new tab

Compounds 3h and 3l suppress the LPS-induced JNK/NF-κB signaling

Since compounds 3h and 3l showed the most promising inhibitory effects on LPS-induced inflammation, we investigated which LPS-induced downstream signals of inflammation were inhibited by 3h and 3l. There recently has been increasing evidence that iNOS, IL-1β, and TNF-α are positively regulated in inflammation by the NF-κB pathway. As anticipated, NF-κB p65 phosphorylation is a key step in the activated NF-κB pathway, which leads to the upregulation of the above inflammation-related proteins. Therefore, the effects of compounds 3h and 3l on LPS-induced NF-κB activation were investigated. The results displayed in Fig. 8A and B suggested that compounds 3h and 3l could significantly down-regulate LPS-triggered NF-κB p65 phosphorylation in a dose-dependent manner. In addition, numerous studies indicated that JNK signaling is supposed to be an upstream co-regulator of NF-κB signaling. These data shown in Fig. 8C and D further implied that compounds 3h and 3l could also suppress LPS-triggered JNK phosphorylation in a concentration-dependent manner. Taken together, all of these results displayed in Fig. 8 suggested that the anti-inflammatory mechanism of compounds 3h and 3l may be highly associated with the JNK/NF-κB signaling pathway.

Fig. 8. Compounds 3h and 3l suppressed LPS-triggered JNK/NF-κB signaling activation. RAW264.7 macrophages were pre-treated with vehicle (DMSO), 3h, or 3l at 10, 20, or 40 μM for 2 h and then stimulated with LPS (1 μg mL−1) for 2 h. The levels of p-p65 (A and B) and p-JNK (C and D) were evaluated using specific antibodies with β-actin and p65, as well as β-actin and JNK as the loading control. The corresponding column figures further indicated the normalized optical density as a percentage of the control group. Data are represented as mean ± SEM of at least three independent experiments: *, p < 0.05, **, p < 0.01, and ***, p < 0.001, compared with the LPS only group. ###, p < 0.001, compared with the control group.

Fig. 8

Open in a new tab

ROS measurements

Numerous studies have confirmed that ROS, key signaling molecules produced by mitochondria, are involved in the progression of inflammatory disorders.36,37 As a result, we also measured the levels of ROS in single living cells regulated by the most promising compound 3h. It was suggested that 3h at a concentration of 40 μM exerted a mild inhibitory activity of ROS production by using the real-time single cell multi-mode analyzer, but in a dose-dependent manner (Fig. 9).

Fig. 9. RAW264.7 macrophages induced by LPS were treated with 3h at different concentrations. The levels of ROS were analyzed using a real-time single cell multi-mode analyzer. A. Different concentrations of drugs to treat cells before we measured ROS. B. Statistical data are expressed as mean ± SD, and were calculated using the single factor ANOVA test. ###, p < 0.001 compared with the control group. ***p < 0.001 compared with the LPS group.

Fig. 9

Open in a new tab

In silico rapid prediction of drug-likeness and ADME/Tox data

The ADMET profiles of drug candidates become the bottleneck in drug discovery. The reason why only nearly 50% of the IND (investigational new drug) could enter the phase I clinical trials could be attributed to ADME/Tox deficiencies. Therefore, in silico rapid prediction of the drug-likeness and ADME/Tox data of biologically active agents is supposed to be an essential tool in drug discovery.38 The results shown in Table 1 revealed that our synthetic compounds 3h and 3l are in full compliance with Lipinski's rule of five without any violation. In addition, the above-mentioned compounds had tPSA values lower than 140 Å2, which is supposed to be suitable for oral absorption.

Calculated parameters of Lipinski's rule of five for synthetic compounds 3h and 3l, loureirin A and celecoxib.

Compound MW log P HBD HBA Rule of five tPSA
Lipinski's rule ≤500 ≤5 ≤5 ≤10 — ≤140
Celecoxib 381.08 3.77 2 4 Suitable 63.66 A2
Loureirin A 286.12 3.35 1 4 Suitable 46.01 A2
3h 358.14 3.36 0 6 Suitable 51.45 A2
3l 358.14 3.16 0 6 Suitable 51.45 A2
Open in a new tab

Furthermore, as shown in Table 2, compounds 3h and 3l were predicted to be non-carcinogenic in mouse. Both of them with HIA values approximately close to 1 were supposed to have excellent intestinal absorption. From these results in Table 2, we could draw the conclusion that compounds 3h and 3l have reasonable drug-likeness, and hence, warrant the development of new anti-inflammatory agents.

Predicted ADMET data of 3h, 3l, loureirin A and celecoxib.

Compound HIA MDDR-like rule PPB BBB Carcino-mouse
Celecoxib 96.69 Mid-structure 91.08 0.03 Negative
Loureirin A 95.54 Mid-structure 91.28 0.14 Negative
3h 98.33 Mid-structure 88.08 0.03 Negative
3l 98.33 Mid-structure 87.37 0.03 Negative
Open in a new tab

4. Conclusions

In this study, we have designed and synthesized 28 loureirin analogues by using the Claisen–Schmidt condensation, followed by palladium-on-carbon-catalyzed facile hydrogenative reduction.39 The screening results of anti-inflammatory activity against LPS-induced RAW264.7 macrophages confirmed that loureirin analogues at a concentration of 20 μM could reduce the production of NO to an extent. Importantly, half of them displayed 65–86% inhibition rate of NO production, much more potent than celecoxib at the same dosage. Furthermore, the most promising compounds 3h and 3l could down-regulate the expression of pro-inflammatory factors such as IL-1β, IL-6, TNF-α, PEG2, COX-2, and iNOS through the NF-κB/JNK signaling pathway in a dose-dependent manner, suggested by ELISA and Western blot assay. Furthermore, compounds 3h and 3l could also remarkably down-regulate the mRNA expressions of inflammation-related genes including TNF-α, IL-1β, IL-6, iNOS, and COX-2 by RT-qPCR in LPS-induced RAW264.7 macrophages. In addition, CCK-8 assay using RAW 264.7 cells implied that our compounds at a concentration of 20 μM exhibited negligible cytotoxicity. These results indicated that loureirin analogues may become a hit compound for the treatment of inflammation.

Disclosure statement

All authors disclosed no relevant relationships.

Author contributions

Conceptualization, Wenbin Jin; data curation, Die Zhang, Wenping Wang; investigation, Die Zhang, Wenping Wang, Huiping Ou, Jinhua Ning, Yingxun Zhou, Jin Ke, Anguo Hou and Linyun Chen; project administration, Wenbin Jin; resources, Wenbin Jin and Yunshu Ma; supervision, Wenbin Jin, Peng Li and Yunshu Ma; writing – original draft, Wenbin Jin; writing – review & editing, Wenbin Jin, Peng Li and Yunshu Ma.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-015-D4MD00011K-s001
MD-015-D4MD00011K-s002

Acknowledgments

We acknowledge the support from the National Natural Science Foundation of China (Grant No. 82104067), Bioactive Ethnopharmacol Molecules Chemical Conversion and Application Innovation Team of Department of Education of Yunnan Province (2022), Yunnan Provincial Joint Project of Traditional Chinese Medicine (202001AZ070001-091, 202301AZ070001-038), Scientific Research Startup Fund for Shenzhen High-Caliber Personnel of SZPT, No. 6023330007K, and Shenzhen Science and Technology Program, No. 20231127011819001.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00011k

References

  1. Medzhitov R. Origin and physiological roles of inflammation. Nature. 2008;454(7203):428–435. doi: 10.1038/nature07201. [DOI] [PubMed] [Google Scholar]
  2. Tracey K. J. The inflammatory reflex. Nature. 2002;420(6917):853–859. doi: 10.1038/nature01321. [DOI] [PubMed] [Google Scholar]
  3. A current view on inflammation Nat. Immunol. 2017;18(8):825–825. doi: 10.1038/ni.3798. [DOI] [PubMed] [Google Scholar]
  4. Liu C. Chu D. Kalantar-Zadeh K. George J. Young H. A. Liu G. Cytokines: From Clinical Significance to Quantification. Adv. Sci. 2021;8(15):2004433. doi: 10.1002/advs.202004433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kany S. Vollrath J. T. Relja B. Cytokines in Inflammatory Disease. Int. J. Mol. Sci. 2019;20(23):6008. doi: 10.3390/ijms20236008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Abdulkhaleq L. A. Assi M. A. Abdullah R. Zamri-Saad M. Taufiq-Yap Y. H. Hezmee M. N. M. The crucial roles of inflammatory mediators in inflammation: A review. Vet. World. 2018;11(5):627–635. doi: 10.14202/vetworld.2018.627-635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Z. Bozec A. Ramming A. Schett G. Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis. Nat. Rev. Rheumatol. 2019;15(1):9–17. doi: 10.1038/s41584-018-0109-2. [DOI] [PubMed] [Google Scholar]
  8. Chen Y. Fang Z.-M. Yi X. Wei X. Jiang D.-S. The interaction between ferroptosis and inflammatory signaling pathways. Cell Death Dis. 2023;14(3):205. doi: 10.1038/s41419-023-05716-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mohamed M. F. A. Marzouk A. A. Nafady A. El-Gamal D. A. Allam R. M. Abuo-Rahma G. E.-D. A. El Subbagh H. I. Moustafa A. H. Design, synthesis and molecular modeling of novel aryl carboximidamides and 3-aryl-1,2,4-oxadiazoles derived from indomethacin as potent anti-inflammatory iNOS/PGE2 inhibitors. Bioorg. Chem. 2020;105:104439. doi: 10.1016/j.bioorg.2020.104439. [DOI] [PubMed] [Google Scholar]
  10. Fan J. Y. Yi T. Sze-To C. M. Zhu L. Peng W. L. Zhang Y. Z. Zhao Z. Z. Chen H. B. A systematic review of the botanical, phytochemical and pharmacological profile of Dracaena cochinchinensis, a plant source of the ethnomedicine “dragon's blood”. Molecules. 2014;19(7):10650–10669. doi: 10.3390/molecules190710650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Tian Y. Wang L. Chen X. Zhao Y. Yang A. Huang H. Ouyang L. Pang D. Xie J. Liu D. Tu P. Li J. Hu Z. DHMMF, a natural flavonoid from Resina Draconis, inhibits hepatocellular carcinoma progression via inducing apoptosis and G2/M phase arrest mediated by DNA damage-driven upregulation of p21. Biochem. Pharmacol. 2023;211:115518. doi: 10.1016/j.bcp.2023.115518. [DOI] [PubMed] [Google Scholar]
  12. Guo S. C. Yu B. Jia Q. Yan H. Y. Wang L. Q. Sun F. F. Ma T. H. Yang H. Loureirin C extracted from Dracaena cochinchinensis S.C. Chen prevents rotaviral diarrhea in mice by inhibiting the intestinal Ca(2+)-activated Cl(−) channels. J. Ethnopharmacol. 2024;318(Pt B):117077. doi: 10.1016/j.jep.2023.117077. [DOI] [PubMed] [Google Scholar]
  13. Kamal A. Reddy V. S. Santosh K. Bharath Kumar G. Shaik A. B. Mahesh R. Chourasiya S. S. Sayeed I. B. Kotamraju S. Synthesis of imidazo[2,1-b][1,3,4]thiadiazole–chalcones as apoptosis inducing anticancer agents. MedChemComm. 2014;5(11):1718–1723. doi: 10.1039/C4MD00228H. [DOI] [Google Scholar]
  14. Rücker H. Al-Rifai N. Rascle A. Gottfried E. Brodziak-Jarosz L. Gerhäuser C. Dick T. P. Amslinger S. Enhancing the anti-inflammatory activity of chalcones by tuning the Michael acceptor site. Org. Biomol. Chem. 2015;13(10):3040–3047. doi: 10.1039/C4OB02301C. [DOI] [PubMed] [Google Scholar]
  15. Zou Y. Zhao Q. Zhang X. Yu H. Zhou Y. Li Z. Xiao M. Xiang Q. Zhang L. Shi W. Tao H. Chen L. Han B. Yin S. The immunosuppressive effects and mechanisms of loureirin B on collagen-induced arthritis in rats. Front. Immunol. 2023;14:1094649. doi: 10.3389/fimmu.2023.1094649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ur Rashid H. Xu Y. Ahmad N. Muhammad Y. Wang L. Promising anti-inflammatory effects of chalcones via inhibition of cyclooxygenase, prostaglandin E(2), inducible NO synthase and nuclear factor κb activities. Bioorg. Chem. 2019;87:335–365. doi: 10.1016/j.bioorg.2019.03.033. [DOI] [PubMed] [Google Scholar]
  17. Nowakowska Z. A review of anti-infective and anti-inflammatory chalcones. Eur. J. Med. Chem. 2007;42(2):125–137. doi: 10.1016/j.ejmech.2006.09.019. [DOI] [PubMed] [Google Scholar]
  18. Ibrahim T. S. Moustafa A. H. Almalki A. J. Allam R. M. Althagafi A. Md S. Mohamed M. F. A. Novel chalcone/aryl carboximidamide hybrids as potent anti-inflammatory via inhibition of prostaglandin E2 and inducible NO synthase activities: design, synthesis, molecular docking studies and ADMET prediction. J. Enzyme Inhib. Med. Chem. 2021;36(1):1067–1078. doi: 10.1080/14756366.2021.1929201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smith N. M. Soh P. Asokananthan N. Norret M. Stewart G. A. Raston C. L. Immunomodulatory effects of functionalised chalcones on pro-inflammatory cytokine release from lung epithelial cells. New J. Chem. 2009;33(9):1869–1873. doi: 10.1039/B909172F. [DOI] [Google Scholar]
  20. Ciupa A. Griffiths N. J. Light S. K. Wood P. J. Caggiano L. Design, synthesis and antiproliferative activity of urocanic-chalcone hybrid derivatives. MedChemComm. 2011;2(10):1011–1015. doi: 10.1039/C1MD00155H. [DOI] [Google Scholar]
  21. LeBlanc R. Dickson J. Brown T. Stewart M. Pati H. N. VanDerveer D. Arman H. Harris J. Pennington W. Holt H. L. Lee M. Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins. Bioorg. Med. Chem. 2005;13(21):6025–6034. doi: 10.1016/j.bmc.2005.06.028. [DOI] [PubMed] [Google Scholar]
  22. Pinto P. Machado C. M. Moreira J. Almeida J. D. P. Silva P. M. A. Henriques A. C. Soares J. X. Salvador J. A. R. Afonso C. Pinto M. Bousbaa H. Cidade H. Chalcone derivatives targeting mitosis: synthesis, evaluation of antitumor activity and lipophilicity. Eur. J. Med. Chem. 2019;184:111752. doi: 10.1016/j.ejmech.2019.111752. [DOI] [PubMed] [Google Scholar]
  23. Yu W. Ping Cheng L. Pang W. Ling Guo L. Design, synthesis and biological evaluation of novel 1, 3, 4-oxadiazole derivatives as potent neuraminidase inhibitors. Bioorg. Med. Chem. 2022;57:116647. doi: 10.1016/j.bmc.2022.116647. [DOI] [PubMed] [Google Scholar]
  24. Ramprasad J. Nayak N. Dalimba U. Yogeeswari P. Sriram D. Peethambar S. K. Achur R. Kumar H. S. S. Synthesis and biological evaluation of new imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole derivatives. Eur. J. Med. Chem. 2015;95:49–63. doi: 10.1016/j.ejmech.2015.03.024. [DOI] [PubMed] [Google Scholar]
  25. Dzoyem J. P. Donfack A. R. Tane P. McGaw L. J. Eloff J. N. Inhibition of Nitric Oxide Production in LPS-Stimulated RAW 264.7 Macrophages and 15-LOX Activity by Anthraquinones from pentas schimperi. Planta Med. 2016;82(14):1246–1251. doi: 10.1055/s-0042-104417. [DOI] [PubMed] [Google Scholar]
  26. Wu J. Li J. Cai Y. Pan Y. Ye F. Zhang Y. Zhao Y. Yang S. Li X. Liang G. Evaluation and Discovery of Novel Synthetic Chalcone Derivatives as Anti-Inflammatory Agents. J. Med. Chem. 2011;54(23):8110–8123. doi: 10.1021/jm200946h. [DOI] [PubMed] [Google Scholar]
  27. Yang W. Wang W. Cai S. Li P. Zhang D. Ning J. Ke J. Hou A. Chen L. Ma Y. Jin W. Synthesis and In Vivo Antiarrhythmic Activity Evaluation of Novel Scutellarein Analogues as Voltage-Gated Nav1.5 and Cav1.2 Channels Blockers. Molecules. 2023;28(21):7417. doi: 10.3390/molecules28217417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hankittichai P. Buacheen P. Pitchakarn P. Na Takuathung M. Wikan N. Smith D. R. Potikanond S. Nimlamool W. Artocarpus lakoocha Extract Inhibits LPS-Induced Inflammatory Response in RAW 264.7 Macrophage Cells. Int. J. Mol. Sci. 2020;21(4):1355. doi: 10.3390/ijms21041355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jin W. Xu C. Dong N. Chen K. Zhang D. Ning J. Li Y. Zhang G. Ke J. Hou A. Chen L. Chen S. Chan K.-F. Identification of isothiazolones analogues as potent bactericidal agents against antibiotic resistant CRE and MRSA strains. BMC Chem. 2023;17(1):183. doi: 10.1186/s13065-023-01100-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tripathi P. Tripathi P. Kashyap L. Singh V. The role of nitric oxide in inflammatory reactions. FEMS Immunol. Med. Microbiol. 2007;51(3):443–452. doi: 10.1111/j.1574-695X.2007.00329.x. [DOI] [PubMed] [Google Scholar]
  31. Ali H. Khan A. Ali J. Ullah H. Khan A. Ali H. Irshad N. Khan S. Attenuation of LPS-induced acute lung injury by continentalic acid in rodents through inhibition of inflammatory mediators correlates with increased Nrf2 protein expression. BMC Pharmacol. Toxicol. 2020;21(1):81. doi: 10.1186/s40360-020-00458-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mohamed M. F. A. Marzouk A. A. Nafady A. El-Gamal D. A. Allam R. M. Abuo-Rahma G. E. A. El Subbagh H. I. Moustafa A. H. Design, synthesis and molecular modeling of novel aryl carboximidamides and 3-aryl-1,2,4-oxadiazoles derived from indomethacin as potent anti-inflammatory iNOS/PGE2 inhibitors. Bioorg. Chem. 2020;105:104439. doi: 10.1016/j.bioorg.2020.104439. [DOI] [PubMed] [Google Scholar]
  33. Du Q. Luo J. Yang M. Q. Liu Q. Heres C. Yan Y. H. Stolz D. Geller D. A. iNOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice. Mol. Med. 2020;26(1):56. doi: 10.1186/s10020-020-00182-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Minghetti L. Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol. 2004;63(9):901–910. doi: 10.1093/jnen/63.9.901. [DOI] [PubMed] [Google Scholar]
  35. Kaur B. Singh P. Inflammation: Biochemistry, cellular targets, anti-inflammatory agents and challenges with special emphasis on cyclooxygenase-2. Bioorg. Chem. 2022;121:105663. doi: 10.1016/j.bioorg.2022.105663. [DOI] [PubMed] [Google Scholar]
  36. Mittal M. Siddiqui M. R. Tran K. Reddy S. P. Malik A. B. Reactive oxygen species in inflammation and tissue injury. Antioxid. Redox Signaling. 2014;20(7):1126–1167. doi: 10.1089/ars.2012.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Morris G. Gevezova M. Sarafian V. Maes M. Redox regulation of the immune response. Cell. Mol. Immunol. 2022;19(10):1079–1101. doi: 10.1038/s41423-022-00902-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lamie P. F. Philoppes J. N. 2-Thiopyrimidine/chalcone hybrids: design, synthesis, ADMET prediction, and anticancer evaluation as STAT3/STAT5a inhibitors. J. Enzyme Inhib. Med. Chem. 2020;35(1):864–879. doi: 10.1080/14756366.2020.1740922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jin W. B. Wang Z. Yang W. Zhang D. Ning J. H. Ke J. Hou A. G. Chen L. Y. Ma Y. S. Selective [3 + 2] cycloaddition reaction of isothiazol-3(2h)-ones with in situ formed azomethine ylide to thiazolidines and oxazolidines. J. Heterocyclic Chem. 2023;60(8):1383–1393. doi: 10.1002/jhet.4668. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-015-D4MD00011K-s001
MD-015-D4MD00011K-s001.pdf (2.5MB, pdf)
MD-015-D4MD00011K-s002
MD-015-D4MD00011K-s002.pdf (560.6KB, pdf)

Articles from RSC Medicinal Chemistry are provided here courtesy of Royal Society of Chemistry

RESOURCES