Synthesis and enzymatic inhibition effects of thiazolidinedione 3C-like protease inhibitors

Abstract

The 3C-like protease (also known as M^pro) plays a key role in SARS-CoV-2 replication and has similar substrates across mutant coronaviruses, making it an ideal drug target. We synthesized 19 thiazolidinedione derivatives via the Knoevenagel condensations and Mitsunobu reactions as potential 3C-like protease inhibitors. The activity of these inhibitors is screened in vitro by employing the enzymatic screening model of 3C-like protease using fluorescence resonance energy transfer. Dithiothreitol is included in the enzymatic reaction system to avoid non-specific enzymatic inhibition. Active inhibitors with diverse activity are found in this series of compounds, and two representative inhibitors with potent inhibitory activity are highlighted.

Keywords

3C-like protease inhibitor anti-coronavirus compound SARS-CoV-2 thiazolidinedione derivatives

New thiazolidinedione derivatives were designed and synthesized. Two of these compounds were proved to have potent inhibitory activity against SARS-CoV-2 M^pro.

Introduction

At the end of 2019, COVID-19, caused by SARS-CoV-2, led to a global health crisis, with more than six hundred million people cumulatively diagnosed with SARS-CoV-2 and more than six and a half million deaths according to the World Health Organization (WHO). SARS-CoV-2 is an enveloped, positive single-stranded RNA virus belonging to the subgenus Sarbecovirus of the genus Betacoronavirus,¹ the genome of which consists of about 30,000 nucleotides.² SARS-CoV-2 has high homology with SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). After the infection of host cells by this virus, the ORF1a/b of SARS-CoV-2 is first translated and expressed with two polyprotein precursors (pp1a and pp1ab) with the help of the host cell.³ The polyprotein precursors undergo intramolecular cleavage by the action of the main protease (M^pro for short). M^pro, a papain-like protease, is also known as a 3C-like protease (3CL^pro),⁴ because its cleavage site specificity is similar to that of the 3C protease of picornavirus, and both are referred to as M^pro/3CL^pro. The non-structural proteins produced by the polyprotein precursors are involved in the production of viral subgenomic RNA and four structural proteins (envelope/E, membrane/M, spike/S, and nucleocapsid/N), which, in turn, are responsible for the reproduction and release of the offspring virus. Since the M^pro protease plays a crucial role in the virus's life cycle and there is no homologous protein in the human body, the M^pro master protease is an ideal target for developing antiviral drugs.⁵

Since the SARS outbreak in 2003, several anti-SARS drugs based on the M^pro structure have been investigated. Anti-HIV drugs such as lopinavir and ritonavir also act on similar protease targets and have been well-validated in clinical practice.⁶ Meanwhile, the screened M^pro inhibitors have a certain degree of broad-spectrum anti-coronavirus ability and may even be used for the treatment of other animal diseases caused by coronaviruses (porcine coronavirus, for example), because M^pro is highly conserved in beta coronavirus.⁷ SARS-CoV-2 M^pro/3CL^pro differs from SARS-CoV M^pro/3CL^pro by only 12 amino acids, and the homology is greater than 96%.⁸ Also according to the Global Initiative on Sharing Avian Influenza Data (GISAID), SARS-CoV-2 M^pro is highly conserved. The mutation rate of its binding domain is less than 0.001.⁹ Therefore, mutations do not broadly affect the efficacy of SARS-CoV-2 M^pro inhibitors. As its key roles are in mediating viral replication and transcription, M^pro is viewed as an ideal drug target for viruses.¹⁰

Jin et al.¹¹ screened about 10,000 compounds using fluorescence resonance energy transfer (FRET) analysis and selected seven compounds with IC₅₀ values in the range of 0.67–21.4 µM, including tideglusib and TDZD-8 (Figure 1). Tideglusib and TDZD-8 are novel 3CL protease inhibitors possessing a thiadiazolidinedione structure. However, recent research results suggest that the thiadiazolidinedione structure of tideglusib suffers from instability. Inhibition of 3CL protease activity by tideglusib is significantly reduced when dithiothreitol (DTT) is added to the enzymatic assay of cysteine protease. The results of this experiment show that tideglusib is sensitive to DTT and is a non-specific inhibitor of SARS-CoV-2 M^pro.¹² We hypothesize that the sulfur-nitrogen bond in the structure of the thiadiazolidinedione is a high-energy bond. The addition of DTT breaks the sulfur-nitrogen bond leading to ring opening. Thus, tideglusib has significant issues as a drug.

Figure 1.

Novel 3CL protease inhibitors possessing a thiadiazolidinedione structure.

Computer simulations show that when tideglusib is bound to the substrate protease, the two carbonyl groups of the thiadiazolidinedione structure form hydrogen bonds with the protease. Two side chains occupy the pocket to dock COVID-19 virus M^pro.¹¹ Thus, we designed their bioisostere structures. Thiazolidinediones also have two carbonyl groups and two side chains, and one of the nitrogen atoms has been replaced with a methylene group to improve structural stability. Thiazolidines can be used as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, which are potential agents for the treatment of type 2 diabetes mellitus and its complications,¹³ as tyrosinase inhibitors.¹⁴ It also has an inhibitory effect on RNA viruses,¹⁵ so it was hypothesized that it might have an inhibitory effect on 3CL protease.

The enzyme activity was measured by inhibition assays using FRET (Figure 2). The MCA-AVLQSGFR-Lys(Dnp)-Lys-NH₂ substrate peptide was derived from residues P4-P5’ of the SARS-CoV-2 M^pro N-terminal autoprocessing site with the AVLQSGFRK sequence. The enzymatic cleavage between Q4 and S5 of AVLQSGFR-Lys(Dnp)-Lys-NH2 releases a highly fluorescent 7-methoxycoumarin (MCA) moiety that is affected by the internal bursting agent of 2,4-dinitrophenol (DNP), resulting in a large increase in fluorescence intensity. The substrate is cleaved by 3CL/M^pro to produce MCA fluorescent group, which has a maximum excitation at 320 nm and a maximum emission wavelength of 405 nm. The activity of 3CL M^pro is detected by measuring the fluorescence intensity. A potent 3CL M^pro inhibitor, GC376 serves as a positive control for the experiments.¹⁶

Figure 2.

Principle of FRET.

Results and discussion

The intermediates 1–5 (Figure 3) were synthesized by the Knoevenagel condensation of thiazolidinedione and five different substituted benzaldehydes (Scheme 1). TZD has a range of tautomers. The S_N2 substitution reactions involving halogenated hydrocarbons under alkaline conditions are likely to occur in both N and O. Therefore, a highly selective Mitsunobu reaction (Scheme 2) was used to synthesis 19 compounds Y-01–Y-19 (Table 1) by replacing the hydrogen on the nitrogen atom with an alcohol.

Figure 3.

Structures of intermediates 1–5.

Scheme 1.

Synthesis of intermediates 1–5 (a) Piperidine, AcOH, EtOH, 90 °C, overnight.

Scheme 2.

Synthesis of Y-01–Y-19 (a) Triphenylphosphine, diethyl azodicarboxylic acid. THF, ice bath, overnight.

Table 1.

Compounds Y-01–Y-19.

Compound	R¹	R²	Yield
Y-01			76%
Y-02			73%
Y-03			94%
Y-04			78%
Y-05			83%
Y-06			47%
Y-07			77%
Y-08			66%
Y-09			83%
Y-10			84%
Y-11			85%
Y-12			62%
Y-13			50%
Y-14			72%
Y-15			48%
Y-16			42%
Y-17			29%
Y-18			37%
Y-19			66%

In vitro tests of the inhibition activity were carried out on the 19 compounds synthesized (final concentration of the reaction system 50 µM) and the positive control GC376 (final concentration of the reaction system 0.2 μM). Of these, 64.2% inhibition was achieved for Y-10, 61.9% for Y-15, and 41.2% for Y-06, while 17.2% inhibition was achieved for Y-04% and 15.9% inhibition was achieved for Y-05 at 50 µM (Table 2). The most potent compounds, Y-10 and Y-15, were selected for further IC₅₀ assay studies.

Table 2.

Inhibition rate relative to the negative control.

Compound	Concentration	Time	Inhibition rate
Y-10	50 µM	2–8 min	64.2%
Y-15	50 µM	2–8 min	61.9%
Y-06	50 µM	2–8 min	41.2%
Y-04	50 µM	2–8 min	17.2%
Y-05	50 µM	2–8 min	15.9%

The IC₅₀ values of the selected compounds inhibiting SARS-CoV-2 3CL M^pro were calculated and plotted. Reference group GC376 has an IC₅₀ value of 60.04 nM. Compound Y-10 has an IC₅₀ value of 75 µM, and compound Y-15 has an IC₅₀ value of 58.61 µM (Figure 4). The remaining compounds have IC₅₀ values higher than 100 µM.

Figure 4.

IC₅₀ values of compounds Y-10 and Y-15.

Conclusion

All the 1,2,4-thiazolidine-3,5-dione derivatives synthesized demonstrated inhibitory effects against recombinant 3CL M^pro. With the addition of DTT, Y-15 and Y-10 had the most potent and specific enzyme inhibition activity against the recombinant 3CL M^pro. The results of this study provide novel and important reference to design SARS-CoV-2 M^pro targeted inhibitors.

Experimental

Materials and reagents

All reagents used were analytically pure reagents from commercially available companies such as Lakin, Adamas, and Titan, and reactions were monitored by thin-layer chromatography (silica gel HSGF254 from Yantai Jiangyou Silicone Development Co., Ltd., China). Spots were observed by ultraviolet (UV) irradiation (254 nm), and compounds were purified by silica gel column chromatography (100–200 mesh). Melting points were determined on a melting point determination apparatus RY-1 (Tianjin, China). ¹H NMR, ¹³C NMR, and ¹⁹F NMR spectra were recorded using a Bruker 600 MHz NMR spectrometer with DMSO-d₆ (D = 99.9% + TMS = 0.03%) as the solvent. Mass spectra were obtained on an Agilent 6120 Ion Trap LC/MS 500 analysis system (Santa Clara, CA, USA). Elemental analyses were obtained on an Elementar Vario EL III elemental analyzer (Langenselbold, Germany).

MCA-AVLQSGFR-Lys (Dnp)-Lys-NH₂ and SARS-CoV-2 Main Protease M^pro were obtained from Beyotime Biotechnology Inc. (China). GC376 was purchased from TargetMol (Wellesley Hills, MA, USA). Costar^® 96-well assay plates (Cat no. 3925) was purchased from Coring Inc. (Corning, NY, USA).

Enzymatic activity assays

Briefly, the 3CL M^pro of 30 nM was mixed with serial dilutions of each compound in reaction buffer (50 mM Tris-HCl, pH 7.3, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.01% TritonX-100; 1 mM DTT), and incubated at 37 °C for 10 min. Next, a final concentration of 20 μM fluorogenic substrate MCA-AVLQSGFR-Lys(Dnp)-Lys-NH₂ was added. The fluorescence intensity was immediately measured at the wavelengths of 320 nm (excitation) and 405 nm (emission) every 3 min for 30 min using a TECAN infinite 200PRO plate reader (Männedorf, Switzerland). GC376 (100 nM) was used as a positive control.

The compounds with potent inhibition observed in preliminary inhibition assays were selected for further IC₅₀ assays. The IC₅₀ values against 3CL M^pro were measured with 9–11 concentration sets, and three independent experiments were performed. Data were analyzed with GraphPad Prism software.

Intermediates 1–5 general procedure

To a solution of 2,4-thiazolidinedione (20 mmol) and 3-fluorobenzaldehyde (20 mmol) in anhydrous ethanol (20 mL), added dropwise piperidine (4 mL) and glacial acetic acid (2 mL). The resulting mixture was heated to 110 °C for 3 h and stirred overnight at 90 °C. After cooling to room temperature, the reaction mixture was poured into ice water (100 mL), and a large amount of yellow solid precipitated. The crude product was filtered off, recrystallized from ethanol, and dried in vacuo to afford intermediate 1 as a light yellow solid.

(Z)-5-(3-Fluorobenzylidene)thiazolidine-2,4-dione (intermediate 1). Light yellow solid, 73% yield.¹⁷

(Z)-5-(2-Fluorobenzylidene)thiazolidine-2,4-dione (intermediate 2). Light yellow solid, 62% yield.¹⁸

(Z)-5-(4-Fluorobenzylidene)thiazolidine-2,4-dione (intermediate 3). Yellow solid, 87% yield.¹⁹

(Z)-5-(4-(Dimethylamino)benzylidene)thiazolidine-2,4-dione (intermediate 4). Orange solid, 79% yield.²⁰

(Z)-5-(4-Hydroxy-3-methoxybenzylidene)thiazolidine-2,4-dione (intermediate 5). Yellow solid, 60% yield.²¹

Compounds Y-01–Y-06

To a stirred mixture of intermediate 1 (2 mmol) and 2-phenoxyethanol (2.4 mmol) in tetrahydrofuran (THF) (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, diethyl azodicarboxylate (DEAD) (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after thin layer chromatography (TLC) showed complete consumption of intermediate 1 (PE/EA = 9:1). The crude product was purified by column chromatography on silica gel eluting with PE/EA = 9:1 to give compound Y-01. Products Y-02–Y-06 were prepared in a similar manner.

(Z)-5-(3-Fluorobenzylidene)-3-(2-phenoxyethyl)thiazolidine-2,4-dione (Y-01): Light yellow solid, 76% yield. m.p. 102 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.95 (s, 1H), 7.59 (dd, J = 14.2, 8.0 Hz, 1H), 7.48 (d, J = 10.0 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.34 (td, J = 8.4, 2.3 Hz, 1H), 7.27 (dd, J = 8.6, 7.4 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 6.90 (d, J = 7.8 Hz, 2H), 4.21 (t, J = 5.7 Hz, 2H), 4.04 (t, J = 5.7 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.4 (s), 165.9 (s), 162.7 (d, J = 245.0 Hz), 158.4 (s), 135.7 (d, J = 8.2 Hz), 132.2 (d, J = 2.6 Hz), 131.9 (d, J = 8.5 Hz), 130.0 (s), 126.1 (d, J = 2.7 Hz), 123.2 (s), 121.4 (s), 117.9 (d, J = 21.2 Hz), 117.4 (d, J = 22.5 Hz), 115.0 (s), 64.1 (s), 41.4 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.7 (s). ESIMS: m/z (relative intensity) 366.0 [M + Na]⁺ (100). Anal. calcd for C₁₈H₁₄FNO₃S: C, 62.96%; H, 4.11%; N, 4.08%; found: C, 63.16%; H, 4.09%; N, 3.98%.

(Z)-5-(3-Fluorobenzylidene)-3-(2-(pyridin-2-yl)ethyl)thiazolidine-2,4-dione (Y-02): White solid, 73% yield. m.p. 112 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.46 (d, J = 4.6 Hz, 1H), 7.90 (s, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.59 (dd, J = 14.9, 7.2 Hz, 1H), 7.48 (d, J = 10.1 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 8.5 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.22 (t, 1H), 4.00 (t, J = 7.3 Hz, 2H), 3.05 (t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.8 (s), 162.7 (d, J = 245.1 Hz), 158.2 (s), 149.6 (s), 137.2 (s), 135.8 (d, J = = 8.2 Hz), 131.9 (d, J = 8.4 Hz), 131.8 (d, J = 2.5 Hz), 126.1 (d, J = 2.6 Hz), 123.8 (s), 123.4 (s), 122.3 (s), 117.9 (d, J = 21.2 Hz), 117.4 (d, J = 22.5 Hz), 41.9 (s), 35.2 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.7 (s). ESIMS: m/z (relative intensity) 329.0 [M + H]⁺ (100). Anal. calcd for C₁₇H₁₃FN₂O₂S: C, 62.18%; H, 3.99%; N, 8.53%; found: C, 62.42%; H, 3.95%; N, 8.40%.

(Z)-5-(3-Fluorobenzylidene)-3-(pyridin-3-ylmethyl)thiazolidine-2,4-dione (Y-03): White solid, 94% yield. m.p. 128 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.58 (d, J = 1.8 Hz, 1H), 8.51 (dd, J = 4.7, 1.2 Hz, 1H), 7.96 (s, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.58 (dd, J = 14.6, 7.5 Hz, 1H), 7.48 (d, J = 9.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.38 (dd, J = 7.8, 4.8 Hz, 1H), 7.34 (t, J = 8.4 Hz, 1H), 4.88 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.6 (s), 165.8 (s), 162.7 (d, J = 245.2 Hz), 149.7 (s), 149.5 (s), 136.1 (s), 135.7 (d, J = 8.2 Hz), 132.5 (d, J = 2.6 Hz), 131.9 (d, J = 8.5 Hz), 131.6 (s), 126.1 (d, J = 2.6 Hz), 124.2 (s), 123.3 (s), 117.9 (d, J = 21.2 Hz), 117.4 (d, J = 22.4 Hz), 42.9 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.6 (s). ESIMS: m/z (relative intensity) 315.0 [M + H]⁺ (100). Anal. calcd for C₁₆H₁₁FN₂O₂S: C, 61.14%; H, 3.53%; N, 8.91%; found: C, 61.26%; H, 3.63%; N, 9.25%.

(Z)-5-(3-Fluorobenzylidene)-3-(2-methoxyethyl)thiazolidine-2,4-dione (Y-04): Light yellow solid, 78% yield. m.p. 71.2 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.93 (s, 1H), 7.59 (td, J = 8.0, 6.3 Hz, 1H), 7.48 (dd, J = 10.1, 1.9 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.35 (td, J = 8.5, 2.3 Hz, 1H), 3.83 (t, J = 5.6 Hz, 2H), 3.54 (t, J = 5.6 Hz, 2H), 3.23 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.8 (s), 162.7 (d, J = 245.2 Hz), 135.7 (d, J = 8.2 Hz), 132.1 (d, J = 2.6 Hz), 131.9 (d, J = 8.4 Hz), 126.1 (d, J = 2.7 Hz), 123.2 (s), 117.9 (d, J = 21.2 Hz), 117.3 (d, J = 22.4 Hz), 68.2 (s), 58.3 (s), 41.4 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.7 (s). ESIMS: m/z (relative intensity) 304.0 [M + Na]⁺ (100). Anal. calcd for C₁₃H₁₂FNO₃S: C, 55.51%; H, 4.30%; N, 4.98%; found: C, 55.68%; H, 4.28%; N, 4.88%.

(Z)-3-(2-Ethoxyethyl)-5-(3-fluorobenzylidene)thiazolidine-2,4-dione (Y-05): Yellow solid, 83% yield. m.p. 78.1 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.91 (s, 1H), 7.58 (dd, J = 14.9, 7.1 Hz, 1H), 7.50-7.41 (m, 2H), 7.33 (t, J = 8.5 Hz, 1H), 3.82 (t, J = 5.8 Hz, 2H), 3.57 (t, J = 5.8 Hz, 2H), 3.43 (q, J = 7.0 Hz, 2H), 1.05 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.8 (s), 162.7 (d, J = 245.1 Hz), 135.7 (d, J = 8.0 Hz), 132.0 (d, J = 2.4 Hz), 131.9 (d, J = 8.3 Hz), 126.1 (d, J = 2.6 Hz), 123.2 (s), 117.8 (d, J = 21.3 Hz), 117.3 (d, J = 22.5 Hz), 66.0 (s), 65.7 (s), 41.6 (s), 15.4 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.7 (s). ESIMS: m/z (relative intensity) 318.0 [M + Na]⁺ (100). Anal. calcd for C₁₄H₁₄FNO₃S: C, 56.94%; H, 4.78%; N, 4.74%; found: C, 57.14%; H, 4.74%; N, 4.66%.

(Z)-3-(2-(2-Ethoxyethoxy)ethyl)-5-(3-fluorobenzylidene)thiazolidine-2,4-dione (Y-06): White solid, 47.1% yield. m.p. 97 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.93 (s, 1H), 7.58 (dd, J = 14.2, 7.9 Hz, 1H), 7.47 (d, J = 9.9 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.34 (td, J = 8.5, 2.0 Hz, 1H), 3.83 (t, J = 5.7 Hz, 2H), 3.62 (t, J = 5.7 Hz, 2H), 3.50 (t, 2H), 3.41 (t, 2H), 3.37 (q, J = 7.0 Hz, 2H), 1.04 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.8 (s), 162.7 (d, J = 245.1 Hz), 135.7 (d, J = 8.1 Hz), 132.0 (d, J = 2.3 Hz), 131.9 (d, J = 8.4 Hz), 126.1 (d, J = 2.5 Hz), 123.3 (s), 117.9 (d, J = 21.2 Hz), 117.3 (d, J = 22.4 Hz), 70.0 (s), 69.6 (s), 66.6 (s), 66.0 (s), 41.6 (s), 15.5 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −111.7 (s). ESIMS: m/z (relative intensity) 362.0 [M + Na]⁺ (100), 340.0 [M + H]⁺ (80). Anal. calcd for C₁₆H₁₈FNO₄S: C, 56.63%; H, 5.35%; N, 4.13%; found: C, 56.48%; H, 5.30%; N, 3.97%.

Compounds Y-07–Y-13

To a stirred mixture of /intermediate 2 (2 mmol) and 2-phenoxyethanol (2.4 mmol) in THF (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, DEAD (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after TLC showed complete consumption of intermediate 2 (PE/EA = 9:1). The crude product was purified by column chromatography on silica gel eluting with PE/EA = 9:1 to give compound Y-07. Products Y-08–Y-13 were prepared in a similar manner.

(Z)-5-(2-Fluorobenzylidene)-3-(2-phenoxyethyl)thiazolidine-2,4-dione (Y-07): White solid, 77% yield. m.p. 105 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.92 (s, 1H), 7.57 (td, J = 7.7, 1.8 Hz, 2H), 7.40 (t, J = 13.0, 5.2 Hz, 2H), 7.27 (t, J = 8.6, 7.4 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.9 Hz, 2H), 4.22 (t, J = 5.7 Hz, 2H), 4.04 (t, J = 5.7 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.4 (s), 165.8 (s), 161.0 (d, J = 252.3 Hz), 158.4 (s), 133.5 (d, J = 8.9 Hz), 130.0 (s), 129.5 (s), 125.9 (d, J = 3.3 Hz), 124.6 (d, J = 6.1 Hz), 124.4 (s), 121.5 (s), 121.3 (d, J = 12.0 Hz), 116.8 (d, J = 21.3 Hz), 115.0 (s), 64.1 (s), 41.5 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −113.9 (s). ESIMS: m/z (relative intensity) 366.0 [M + Na]⁺ (100). Anal. calcd for C₁₈H₁₄FNO₃S: C, 62.96%; H, 4.11%; N, 4.08%; found: C, 63.26%; H, 4.11%; N, 3.77%.

(Z)-5-(2-Fluorobenzylidene)-3-(2-(pyridin-2-yl)ethyl)thiazolidine-2,4-dione (Y-08): White solid, 66% yield. m.p. 114 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.46 (d, J = 4.7 Hz, 1H), 7.86 (s, 1H), 7.70 (t, J = 8.4, 6.9 Hz, 1H), 7.60-7.50 (m, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.25-7.19 (m, 1H), 4.00 (t, J = 7.3 Hz, 2H), 3.05 (t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.2 (s), 165.7 (s), 161.0 (d, J = 252.2 Hz), 158.1 (s), 149.6 (s), 137.1 (s), 133.4 (d, J = 8.8 Hz), 129.4 (s), 125.9 (s), 124.5 (s), 124.2 (d, J = 6.1 Hz), 123.8 (s), 122.3 (s), 121.3 (d, J = 11.7 Hz), 116.7 (d, J = 21.4 Hz), 41.9 (s), 35.2 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −114.0 (s). ESIMS: m/z (relative intensity) 329.0 [M + H]⁺ (100). Anal. calcd for C₁₇H₁₃FN₂O₂S: C, 62.18%; H, 3.99%; N, 8.53%; found: C, 62.36%; H, 3.96%; N, 8.26%.

(Z)-5-(2-Fluorobenzylidene)-3-(pyridin-3-ylmethyl)thiazolidine-2,4-dione (Y-09): White solid, 83% yield. m.p. 130.2 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.58 (d, J = 2.0 Hz, 1H), 8.52 (dd, J = 4.8, 1.5 Hz, 1H), 7.93 (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.59-7.54 (m, 2H), 7.41-7.36 (m, 3H), 4.88 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.6 (s), 165.7 (s), 161.0 (d, J = 252.3 Hz), 149.7 (s), 149.5 (s), 136.2 (s), 133.5 (d, J = 8.9 Hz), 131.5 (s), 129.5 (s), 125.9 (d, J = 3.4 Hz), 124.9 (d, J = 6.1 Hz), 124.5 (s), 124.2 (s), 121.3 (d, J = 12.0 Hz), 116.8 (d, J = 21.4 Hz), 43.0 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −113.8 (s). ESIMS: m/z (relative intensity) 315.0 [M + H]⁺ (100). Anal. calcd for C₁₆H₁₁FN₂O₂S: C, 61.14%; H, 3.53%; N, 8.91%; found: C, 61.09%; H, 3.80%; N, 8.90%.

(Z)-5-(2-Fluorobenzylidene)-3-(2-methoxyethyl)thiazolidine-2,4-dione (Y-10): White solid, 84% yield. m.p. 73.5 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.89 (s, 1H), 7.60-7.51 (m, 2H), 7.38 (t, J = 8.1 Hz, 2H), 3.83 (t, J = 5.6 Hz, 2H), 3.54 (t, J = 5.6 Hz, 2H), 3.24 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.7 (s), 161.0 (d, J = 252.3 Hz), 133.4 (d, J = 8.9 Hz), 129.4 (s), 125.9 (d, J = 3.3 Hz), 124.5 (d, J = 6.2 Hz), 124.3 (s), 121.3 (d, J = 11.8 Hz), 116.7 (d, J = 21.4 Hz), 68.2 (s), 58.3 (s), 41.4 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −113.9 (s). ESIMS: m/z (relative intensity) 304.0 [M + Na]⁺ (100). Anal. calcd for C₁₃H₁₂FNO₃S: C, 55.51%; H, 4.30%; N, 4.98%; found: C, 55.73%; H, 4.23%; N, 5.02%.

(Z)-3-(2-Ethoxyethyl)-5-(2-fluorobenzylidene)thiazolidine-2,4-dione (Y-11): White solid, 85% yield. m.p. 80.5 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.89 (s, 1H), 7.59-7.53 (m, 2H), 7.38 (t, J = 8.1 Hz, 2H), 3.82 (t, J = 5.8 Hz, 2H), 3.57 (t, J = 5.8 Hz, 2H), 3.43 (q, J = 7.0 Hz, 2H), 1.05 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.7 (s), 161.0 (d, J = 252.3 Hz), 133.4 (d, J = 8.9 Hz), 129.4 (s), 125.9 (d, J = 3.5 Hz), 124.5 (d, J = 6.2 Hz), 124.3 (s), 121.3 (d, J = 11.8 Hz), 116.7 (d, J = 21.4 Hz), 66.0 (s), 65.7 (s), 41.6 (s), 15.4 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −113.9 (s). ESIMS: m/z (relative intensity) 318.1 [M + Na]⁺ (100), 296.0 [M + H]⁺ (60). Anal. calcd for C₁₄H₁₄FNO₃S: C, 56.94%; H, 4.78%; N, 4.74%; found: C, 57.12%; H, 4.75%; N, 4.72%.

(Z)-5-(2-Fluorobenzylidene)-3-(2-morpholinoethyl)thiazolidine-2,4-dione (Y-12): White solid, 62% yield. m.p. 124 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.91 (s, 1H), 7.61-7.54 (m, 2H), 7.39 (t, J = 7.9 Hz, 2H), 3.78 (t, J = 6.4 Hz, 2H), 3.55-3.46 (m, 4H), 2.52 (t, J = 6.4 Hz, 2H), 2.40 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.4 (s), 165.8 (s), 161.0 (d, J = 252.1 Hz), 133.5 (d, J = 8.9 Hz), 129.5 (s), 125.9 (d, J = 3.5 Hz), 124.4 (d, J = 5.6 Hz), 124.4 (s), 121.3 (d, J = 11.9 Hz), 116.8 (d, J = 21.4 Hz), 66.7 (s), 55.1 (s), 53.6 (s), 39.2 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −114.0 (s). ESIMS: m/z (relative intensity) 337.0 [M + H]⁺ (100). Anal. calcd for C₁₆H₁₇FN₂O₃S: C, 57.13%; H, 5.09%; N, 8.33%; found: C, 57.26%; H, 5.09%; N, 8.25%.

(Z)-3-(2-(2-Ethoxyethoxy)ethyl)-5-(2-fluorobenzylidene)thiazolidine-2,4-dione (Y-13): White solid, 50% yield. m.p. 95 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.88 (s, 1H), 7.58-7.51 (m, 2H), 7.40-7.34 (m, 2H), 3.83 (t, J = 5.7 Hz, 2H), 3.63 (t, J = 5.7 Hz, 2H), 3.51 (t, J = 5.7, 3.8 Hz, 2H), 3.41 (t, J = 5.7, 3.9 Hz, 2H), 3.37 (q, J = 7.0 Hz, 2H), 1.04 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3 (s), 165.7 (s), 161.0 (d, J = 252.2 Hz), 133.4 (d, J = 8.9 Hz), 129.4 (s), 125.9 (d, J = 3.5 Hz), 124.4 (s), 124.3 (d, J = 3.4 Hz), 121.2 (d, J = 11.9 Hz), 116.7 (d, J = 21.4 Hz), 70.0 (s), 69.6 (s), 66.6 (s), 66.0 (s), 41.6 (s), 15.5 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −114.0 (s). ESIMS: m/z (relative intensity) 340.0 [M + H]⁺ (100), 362.0 [M + Na]⁺ (70). Anal. calcd for C₁₆H₁₈FNO₄S: C, 56.63%; H, 5.35%; N, 4.13%; found: C, 56.61%; H, 5.25%; N, 4.01%.

Compounds Y-14–Y-16

To a stirred mixture of intermediate 3 (2 mmol) and 2-pyridineethanol (2.4 mmol) in THF (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, DEAD (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after TLC showed complete consumption of intermediate 3 (PE/EA = 7:3). The crude product was purified by column chromatography on silica gel eluting with PE/EA = 8:2 to give compound Y-14. Products Y-15 and Y-16 were prepared in a similar manner.

(Z)-5-(4-Fluorobenzylidene)-3-(2-(pyridin-2-yl)ethyl)thiazolidine-2,4-dione (Y-14): Yellow solid, 72% yield. m.p. 132 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.45 (dd, J = 4.8, 0.8 Hz, 1H), 7.90 (s, 1H), 7.74-7.66 (m, 3H), 7.38 (t, J = 8.7 Hz, 2H), 7.28 (d, J = 7.7 Hz, 1H), 7.22 (t, J = 7.3, 5.0 Hz, 1H), 4.00 (t, J = 7.3 Hz, 2H), 3.04 (t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.40 (s), 165.9 (s), 163.4 (d, J = 251.0 Hz), 158.2 (s), 149.6 (s), 137.1 (s), 133.1 (d, J = 8.9 Hz), 132.1 (s), 130.1 (d, J = 3.1 Hz), 123.8 (s), 122.3 (s), 121.4 (d, J = 2.3 Hz), 117.0 (d, J = 22.0 Hz), 41.8 (s), 35.2 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −108.5 (s). ESIMS: m/z (relative intensity) 329.0 [M + H]⁺ (100). Anal. calcd for C₁₇H₁₃FN₂O₂S: C, 62.18%; H, 3.99%; N, 8.53%; found: C, 62.41%; H, 3.94%; N, 8.62%.

(Z)-5-(4-Fluorobenzylidene)-3-(2-morpholinoethyl)thiazolidine-2,4-dione (Y-15): Yellow solid, 48% yield. m.p. 143 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.94 (s, 1H), 7.69 (dd, J = 8.5, 5.5 Hz, 2H), 7.39 (t, J = 8.7 Hz, 2H), 3.77 (t, J = 6.4 Hz, 2H), 3.50 (s, 4H), 2.52 (t, J = 6.5 Hz, 2H), 2.40 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.5 (s), 166.0 (s), 163.4 (d, J = 251.0 Hz), 133.1 (d, J = 8.9 Hz), 132.3 (s), 130.1 (d, J = 3.0 Hz), 121.3 (s), 117.0 (d, J = 22.0 Hz), 66.7 (s), 55.2 (s), 53.6 (s), 39.1 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −108.5 (s). ESIMS: m/z (relative intensity) 337.1 [M + H]⁺ (100). Anal. calcd for C₁₆H₁₇FN₂O₃S: C, 57.13%; H, 5.09%; N, 8.33%; found: C, 57.25%; H, 5.09%; N, 8.60%.

(Z)-5-(4-Fluorobenzylidene)-3-(2-(pyrrolidin-1-yl)ethyl)thiazolidine-2,4-dione (Y-16): Yellow solid, 42% yield. m.p. 107 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.95 (s, 1H), 7.73-7.67 (m, 2H), 7.44-7.36 (m, 2H), 3.76 (t, J = 6.5 Hz, 2H), 2.63 (t, J = 6.5 Hz, 2H), 2.47 (s, 4H), 1.64 (dt, J = 6.4, 3.0 Hz, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.5 (s), 166.0 (s), 163.4 (d, J = 250.8 Hz), 133.1 (d, J = 8.9 Hz), 132.4 (s), 130.1 (d, J = 3.1 Hz), 121.4 (s), 117.0 (d, J = 22.0 Hz), 54.0 (s), 52.8 (s), 41.2 (s), 23.7 (s). ¹⁹F NMR (565 MHz, DMSO-d₆): δ −108.5 (s). ESIMS: m/z (relative intensity) 321.1 [M + H]⁺ (100). Anal. calcd for C₁₆H₁₇FN₂O₂S: C, 59.98%; H, 5.35%; N, 8.74%; found: C, 59.77%; H, 5.32%; N, 8.55%.

Compound Y-17

To a stirred mixture of intermediate 4 (2 mmol) and 2-phenoxyethanol (2.4 mmol) in THF (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, DEAD (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after TLC showed complete consumption of intermediate 4 (PE/EA = 7:3). The crude product was purified by column chromatography on silica gel eluting with PE/EA = 9:1 to give compound Y-17.

(Z)-5-(4-(Dimethylamino)benzylidene)-3-(2-phenoxyethyl)thiazolidine-2,4-dione (Y-17): Yellow solid, 29% yield. m.p. 112 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.80 (s, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.30-7.22 (m, 2H), 6.92 (t, J = 7.3 Hz, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 4.19 (t, J = 5.7 Hz, 2H), 4.01 (t, J = 5.7 Hz, 2H), 3.00 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.9 (s), 166.2 (s), 158.4 (s), 152.1 (s), 134.7 (s), 132.9 (s), 130.0 (s), 121.4 (s), 120.2 (s), 115.0 (s), 113.4 (s), 112.5 (s), 64.2 (s), 41.0 (s), 40.0 (s). ESIMS: m/z (relative intensity) 369.0 [M + H]⁺ (100). Anal. calcd for C₂₀H₂₀N₂O₃S: C, 65.20%; H, 5.47%; N, 7.60%; found: C, 65.32%; H, 5.40%; N, 7.49%.

Compound Y-18

To a mixture of intermediate 4 (2 mmol) and 2-pyridineethanol (2.4 mmol) in THF (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, DEAD (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after TLC showed complete consumption of intermediate 4 (PE/EA = 7:3). The crude product was recrystallized from acetone, filtered off, and dried in vacuo to afford compound Y-18.

(Z)-5-(4-(Dimethylamino)benzylidene)-3-(2-(pyridin-2-yl)ethyl)thiazolidine-2,4-dione (Y-18): Yellow solid, 37% yield. m.p. 118 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.46 (d, J = 4.1 Hz, 1H), 7.76 (s, 1H), 7.70 (td, J = 7.6, 1.7 Hz, 1H), 7.45 (d, J = 8.9 Hz, 2H), 7.27 (d, J = 7.7 Hz, 1H), 7.22 (dd, J = 7.0, 5.2 Hz, 1H), 6.82 (d, J = 8.9 Hz, 2H), 3.98 (t, J = 7.3 Hz, 2H), 3.04 (d, J = 7.4 Hz, 2H), 3.02 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.8 (s), 166.1 (s), 158.3 (s), 152.1 (s), 149.6 (s), 137.1 (s), 134.3 (s), 132.8 (s), 123.7 (s), 122.3 (s), 120.2 (s), 113.7 (s), 112.5 (s), 41.6 (s), 40.1 (s), 35.4 (s). ESIMS: m/z (relative intensity) 354.1 [M + H]⁺ (100). Anal. calcd for C₁₉H₁₉N₃O₂S: C, 64.57%; H, 5.42%; N, 11.89%; found: C, 64.89%; H, 5.38%; N, 11.77%.

Compound Y-19

To a stirred mixture of intermediate 5 (2 mmol) and 2-pyridineethanol (2.4 mmol) in THF (12 mL), added triphenylphosphine (4 mmol). When the triphenylphosphine had dissolved completely, DEAD (4 mmol) was added dropwise at ice bath temperature. After stirring for 10 min, the ice bath was removed, and the mixture was stirred for a further 12 h at room temperature. The solvent was removed under vacuum after TLC showed complete consumption of intermediate 5 (PE/EA = 6:4). The crude product was purified by column chromatography on silica gel eluting with PE/EA = 7:3 to give compound Y-19.

(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-3-(2-(pyridin-2-yl)ethyl)thiazolidine-2,4-dione (Y-19): Yellow solid, 66% yield. m.p. 134 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 10.01 (s, 1H), 8.46 (d, J = 4.1 Hz, 1H), 7.79 (s, 1H), 7.69 (td, J = 7.6, 1.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.22 (dd, J = 6.9, 5.1 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.08 (dd, J = 8.3, 1.9 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 3.98 (t, J = 7.3 Hz, 2H), 3.82 (s, 3H), 3.03 (t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.7 (s), 166.1 (s), 158.2 (s), 150.1 (s), 149.6 (s), 148.5 (s), 137.1 (s), 134.0 (s), 124.8 (s), 124.7 (s), 123.7 (s), 122.3 (s), 117.3 (s), 116.7 (s), 114.8 (s), 56.1 (s), 41.7 (s), 35.3 (s). ESIMS: m/z (relative intensity) 357.0 [M + H]⁺ (100). Anal. calcd for C₁₈H₁₆N₂O₄S: C, 60.66%; H, 4.53%; N, 7.86%; found: C, 60.68%; H, 4.51%; N, 7.66%.

Supplemental Material

sj-docx-1-chl-10.1177_17475198231152556 – Supplemental material for Synthesis and enzymatic inhibition effects of thiazolidinedione 3C-like protease inhibitors

Supplemental material, sj-docx-1-chl-10.1177_17475198231152556 for Synthesis and enzymatic inhibition effects of thiazolidinedione 3C-like protease inhibitors by Xin Ye, Yuhua Li, Lina Guo, Yiling Yao, Rouyu Zhu, Shuang Wei, Hua Diao and Zhiyu Shao in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Commission of Shanghai Municipality (grant no. 22S11901000).

ORCID iD

Zhiyu Shao

Supplemental material

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