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Article

Structural Optimization and Structure–Activity Relationship of 4-Thiazolidinone Derivatives as Novel Inhibitors of Human Dihydroorotate Dehydrogenase

by
Fanxun Zeng
1,†,
Lina Quan
2,†,
Guantian Yang
1,
Tiantian Qi
2,
Letian Zhang
1,
Shiliang Li
2,
Honglin Li
2,
Lili Zhu
2,* and
Xiaoyong Xu
1,*
1
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
2
Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science & Technology, Shanghai 200237, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2019, 24(15), 2780; https://doi.org/10.3390/molecules24152780
Submission received: 23 June 2019 / Revised: 24 July 2019 / Accepted: 26 July 2019 / Published: 31 July 2019
(This article belongs to the Section Bioorganic Chemistry)
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Abstract

:
Human dihydroorotate dehydrogenase (hDHODH), one of the attractive targets for the development of immunosuppressive drugs, is also a potential target of anticancer drugs and anti-leukemic drugs. The development of promising hDHODH inhibitors is in high demand. Based on the unique binding mode of our previous reported 4-thiazolidinone derivatives, via molecular docking method, three new series 4-thiazolidinone derivatives were designed and synthesized as hDHODH inhibitors. The preliminary structure–activity relationship was investigated. Compound 9 of biphenyl series and compound 37 of amide series displayed IC50 values of 1.32 μM and 1.45 μM, respectively. This research will provide valuable reference for the research of new structures of hDHODH inhibitors.

1. Introduction

Pyrimidine, as a key precursor of RNA, DNA, glycoproteins, and phospholipids, plays a critical role in cellular metabolism and cell growth [1,2]. Human dihydroorotate dehydrogenase (hDHODH) is a mitochondrial enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis [3,4]. Unlike resting or fully differentiated cells which acquire pyrimidine mainly by the salvage pathways, rapidly proliferating cells, such as activated T cells, depend heavily on de novo nucleotide synthesis to meet their increased demand for nucleic acid precursors [5,6]. Thus, hDHODH would be an ideal drug target for the treatment of variety of diseases such as tumor, immunological disorders and acute myeloid leukemia [7,8,9,10,11,12,13,14].
Many hDHODH inhibitors have been developed and exhibited excellent therapeutic efficacy [15]. For example, leflunomide (1, Figure 1) was approved by FDA for the treatment of rheumatoid arthritis in 1998 [16,17]. Then this compound was found to be a prodrug, forming its active metabolite teriflunomide via a base caused ring-opening reaction in vivo [18,19]. Teriflunomide was developed to cure multiple sclerosis later [20]. Brequinar (2), one of the strongest inhibitors of hDHODH, only showed modest anticancer effects on a number of solid tumors in a phase II clinical trial [21,22]. Its effects on autoimmune diseases and viral diseases were also extensively studied [23,24,25]. Vidofludimus (3) was proven to be a promising pharmaceutical agent in systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and transplantation [7,26,27,28]. Vidofludimus also restored myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar [14]. The development of ML390 (4) that induced differentiation in acute myeloid leukemia (AML) demonstrated that the inhibition of hDHODH could overcome differentiation blockade in AML [29]. Recently compound 5 was reported to reduce tumor growth by increasing p53 synthesis [30]. Compound 6 showed brequinar-like hDHODH potency in vitro and was superior in terms of cytotoxicity and immunosuppression. In general, it is significant to develop promising hDHODH inhibitors for the severe side effects of leflunomide/teriflunomide and the lack of newly marketed hDHODH inhibitors.
Recently we had described 4-thiazolidinone derivatives 7 and 8 as novel hDHODH inhibitors [31]. It was found that the binding mode of 4-thiazolidinone with hDHODH was different from the classic inhibitors, that is, the thiazolidinone fragment locates at the outside of the bind pocket, cyano group contacts ALA-59 by a water-mediated hydrogen bond, carbonyl and TYR-38 forms hydrogen bond interaction, while the aromatic fragment contacts MET-43 by hydrophobic interaction (Figure 2A). But, it was found that compound 8 only occupies the hydrophobic subsite of the ubiquinone-binding site with its naphthalenyl group (Figure 2B,C) [31]. The binding mode of compound 8 suggested us that the replacement of its naphthalenyl group with a suitable longer aryl group could orient the new ligand towards the inner side of the binding site. An additional polar group, such as carboxyl and acetyl, would generate hydrogen bond or salt bridge interactions with residue Arg-136 in the hydrophilic subsite of the ubiquinone-binding site and may improve compounds’ binding affinity against hDHODH. Therefore, further structural optimization of 4-thiazolidinone derivatives that focused on the decoration of the aryl group to identify more potent hDHODH inhibitors was presented in this study.

2. Results and Discussion

2.1. Molecular Design Strategies

In order to probe the inner subsite of the ubiquinone-binding site of hDHODH, biphenyl derivatives were firstly designed and synthesized. The binding mode of 9 was simulated using the same molecular docking method reported previously [31,32]. It was shown that the introduction of 4-phenyl moiety could enhance the hydrophobic interaction between the biphenyl compounds and the binding pocket (Figure 3 and Figure 4). In addition, by observing the binding modes of 9, we found that there is some space for substitution at the ortho-position of the phenyl group to enhance the hydrophobic interactions with residues Met43, Leu46, Met111 and Pro364. Therefore, we tried to introduce a small fluorine atom into the structure and synthesized compounds 10-18. However, compared with brequinar, a para-phenyl might collide with the binding pocket, which would influence the binding affinity against the target hDHODH (Figure 3). Therefore, phenoxy (phenoxymethyl) and phenyl amide groups were introduced to improve the binding strength by adjusting the molecular configuration (Figure 5). After molecular docking simulations, it was found that the introduction of an ether bond could cause an angle (about 109°) to adjust the orientation of the phenyl group, which may be helpful to avoid the steric hindrance between the ligand and the receptor (Figure 5). If the linker was replaced by a longer amide bond, the whole molecule would also become longer, thus enabling the phenyl group to contact residues in the inner binding site. When substituting a carboxyl group to the meta-position of the phenyl group of the benzamide derivatives next, the carboxyl group would form hydrogen bond interactions with residues Gln47 and Arg136, which are beneficial for strengthening the binding affinity of the compounds (Figure 6). In addition, the amide bond could participate in a water-mediated hydrogen bond network, which may be favorable for molecular binding. The results of docking simulations suggested us that the design strategies may be feasible to improve the potency of the compounds. The detailed modification strategies are described in Figure 4 and Figure 7.

2.2. Chemistry

Key intermediates of compounds 9–26 were synthesized according to Scheme 1. Compounds 9c–18c were obtained by Suzuki cross-coupling reactions of compounds 9a–18a with 9b–18b. Etherification of compounds 19a–22a with 1-fluoro-4-nitrobenzene yielded compounds 19b–22b, which were reduced to afford compounds 19c–22c. Phenol was coupled with 1-fluoro-4-nitrobenzene to give compound 23a. Etherification of (bromomethyl) benzene with 4-nitrophenol afforded compound 24a, which was reduced to give compound 24b. Compounds 25b and 26b were obtained by etherification of compounds 25a, 26a with 4-nitrophenol, which were further reduced and hydrolyzed to give compounds 25d and 26d.
Compound IM and key intermediates of compounds 40–46 were obtained according to Scheme 2. 4-isothiocyanatobenzoic acid was prepared by reaction of 4-aminobenzoic acid with 1,1’-thiocarbonyldiimidazole (TCDI) in the presence of TEA. 4-Isothiocyanatobenzoic acid was treated with methyl 2-cyanoacetate and potassium hydroxide in DMF to provide ketene-N, S-acetal salt, which then reacted with 2-chloroacetyl chloride to give key intermediate IM.
Compounds 40c, 41c were obtained by reduction of compounds 40b, 41b, which were prepared by amidation reactions of compounds 40a, 41a with 4-nitrobenzoyl chloride. Compound 42b was synthesized by hydrazinolysis of compound 42a, which was prepared by reaction of phthalide with potassium phthalimide.
The important intermediate 43c was generated from starting material 3-cyanobenzoic acid via protection, reduction, and amidation. Compound 44e was prepared from 1-(tert-butyl)-2-methylbenzene via oxidation, nitration, reduction, amidation, and reduction.
Compound 45b was synthesized by amination of compound 45a, which was obtained by Friedel -Crafts acylation of 2-bromonaphthalene. Intermediate 46b was obtained by removing Boc group of compound 46a, which was obtained by amination of IM.
The synthetic route to obtain target compounds 9–46 was shown in Scheme 3. Isothiocyanates 9e–22e, 23c, and 24d were obtained from corresponding aryl amines by two-step reactions. Key intermediates 25e, 26e, 40d, 41d, 44f and compounds 9–26, 40, 41, 44 were prepared by the same route of 4-isothiocyanatobenzoic acid and IM, respectively. Compounds 27–39, 42, and 45 were generated by amination of intermediates 27a–39a, 42b, 45b with IM. Compound 43 was afforded by deprotection of compound 43. The imidization of 46b with 2-formylbenzoic acid was introduced to give compound 46.

2.3. Inhibitory Activities against hDHODH and SAR Study

In order to identify more potent compounds, biphenyl group was introduced for the size and hydrophobicity of the aryl group which is essential for the activities. As shown in Table 1, the activity of unsubstituted biphenyl derivative 9 was equal to that of compounds 7 and 8. Introducing a fluorine atom into the biphenyl group (compounds 10 and 11) decreased the inhibitory activities. 2′-Methyl analog 12 had slightly less activity than 10. Introduction of a methoxy group into the 2′-, 3′-, or 4′-positions of the biphenyl group (compounds 13–15) resulted in roughly 2-fold reduction in inhibitory activity compared to compound 10, amongst the three compounds, the 3′-methoxy derivative 14 was slightly more potent than compounds 13, 15. Introduction of electron-withdrawing groups (compounds 16–18) also failed to enhance the potency. Moreover, the results of the bioassay also showed that the ortho-substituted fluorine atom (compounds 10 and 11) did not make a positive contribution to the binding affinity, indicating that there may be steric clashes.
Since the biphenyl derivatives only showed moderate potency, biphenyl groups were replaced further by flexible aryl ethers further (Table 2). To our disappointment, diphenyl ether derivatives 19–23 displayed markedly diminished activity. The 3-phenoxyphenyl analog 23 was also inactive. Benzyl ether derivative 24 exhibited moderate activity with an IC50 value of 4.32 μM. The activity decreased when a carboxyl group (compounds 25 and 26) was introduced.
Then the moderate rigid and longer amide structure was introduced as a linker (compounds 27–46, Table 2). Phenyl derivative 28 showed a moderate activity, while the cyclohexyl analog 27 gave a dramatically decreased activity. It indicated that rigid aromatic ring is better than flexible cyclohexyl ring. Next we tried to introduce substituent groups into the phenyl group. A 4-tert-butyl substituent (compound 37) was tolerated, while the introduction of smaller substituent groups such as CH3, F, Cl, I, CF3, OCH3, CN etc. into the 2, 3 or 4-positions (compounds 29–36) was detrimental to the activity. The naphthalen-2-yl derivative 38 and benzyl derivative 39 were inactive. Subsequently, COOH and carbonyl groups were introduced to generate hydrogen bonds or salt bridge interactions with residue Arg136 in the hydrophilic subsite of the ubiquinone-binding site. However, the inhibition rates of the corresponding analogs 40–46 at 10 μM were still less than 50%. The bioassay results did not match our modeling results. It indicated that neither ether nor amide is an appropriate linker to extend the molecules to fit well in the pocket and eventually contact the inner residues Arg136 and Gln47.
Although the introduction of a biphenyl, diphenyl ether and amide structures into 4- thiazolidinone did not obviously increase the rate of inhibition of hDHODH, the attempts can provide some ideas for hDHODH inhibitor lead identification. We speculate that there may exist steric clashes caused by inappropriate biphenyl, ether and amide group linkers. Thus, we will try some other linkers like -C=N-N-, -C-C(=O)-, and -C-C(=O)-C- to replace the O of biphenyl ether or the CONH of the amide series in our future work to further investigate the SAR and to obtain more potent DHODH inhibitors. In addition, it was found that any changes to the structure connecting the N-terminus of thiazolidone have a weak influence on the bioactivities, which means that thiazolidone is important to increase the activity. In our future work, we will try to select other heterocycles and investigate the stability of thiazolidones under the determination conditions to further establish the factors influencing the bioactivity.

3. Materials and Methods

3.1. General Information

Unless otherwise indicated, all commercially available solvents and reagents were purchased directly from commercial suppliers and used as received without further purification. Melting points (m.p.) were recorded on Büchi B540 apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. 1H-NMR, 19F-NMR and 13C-NMR spectra were recorded on an AM-400 (1H at 400 MHz, 13C at 100 MHz, 19F at 376 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) with CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard. Chemical shifts are reported in δ (parts per million) values. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, coupling constant (Hz) and integration. High-resolution electron mass spectra (ESI-TOF) were performed on a Micromass LC-TOF spectrometer (Waters Co.,Ltd., Milford, MA, USA). High resolution mass spectra (HRMS) were recorded under electron impact (70 eV) conditions using a MicroMass GCT CA 055 instrument (Waters Co.,Ltd., Milford, MA, USA). Analytical thin-layer chromatography (TLC) was carried out on precoated plates (silica gel 60 F254) and spots were visualized with ultraviolet (UV) light.

3.2. Chemistry

3.2.1. Synthesis of Key Intermediates of Compounds 9–26.

General Procedure for the Synthesis of Intermediates 9c–18c

Substituted 4-iodoanilines 9b–18b (5 mmol), substituted phenylboronic acids 9a–18a (6 mmol), an orange solution of Pd(dba)2 (2 mol%) and triphenylphosphine (6 mol%) were added in a tube. The tube was evacuated and back-filled with argon. Potassium carbonate (20 mmol) solution (10 mL, 2 mol/L) and ethanol (10 mL) were added using syringe, and the mixture was stirred and refluxed for 3–5 h. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate. The combined organic phases were dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (Petroleum ether (PE)/Ethyl acetate (EA)=10:1, V/V), to give 9c–18c in 65%−95% yield. (Spectrums for target compounds 7–46 could be accessed in Supplementary Materials).

General Procedure for the Synthesis of Intermediates 19b–22b

K2CO3 (50 mmol) was carefully added to a solution of 19a–22a (12 mmol) and 1-fluoro-4-nitrobenzene (10 mmol) in DMF (8 mL). The mixture was stirred at heating condition (120 °C for 19b, 20b, and 22b, 60 °C for 21b) until the reaction was complete. The reaction mixture was diluted with water (20 mL), extracted with EA after cooling to room temperature. The combined organic layer was washed with 1 M NaOH, 1 M HCl and saturated salt water in order. Then the mixture was dried (Na2SO4), concentrated under reduced pressure to give 19b–22b in yield of 70–90%, which was used in the next step without further purification.

General Procedure for the Synthesis of Intermediates 19c–22c

Compounds 19b–22b (8 mmol) and Pd/C (10% w/w) were added to MeOH (40 mL). The resulted mixture was reacted under H2 atmosphere at room temperature for 2 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to give 19c–22c in yield of 95–98%, which was used in the next step without further purification.

Synthesis of Intermediate 23a

Phenol (12 mmol), 3-bromoaniline (10 mmol), K2CO3 (20 mmol), 1-butyl-1H-imidazole (5 mmol), and CuCl (4.5 mmol) were added to o-xylene (10 mL) under Ar atmosphere, and the mixture was heated to 140 °C for 20h. After cooling to room temperature, the mixture was filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (PE/EA=4:1, V/V) to give 23a in yield of 90%.

Synthesis of Intermediate 24a

K2CO3 (20 mmol) was added to a solution of (bromomethyl)benzene (12 mmol) and 4-nitrophenol (10 mmol) in DMF (10 mL). The resulted mixture was allowed to react at 90 °C for 2 h. The mixture was diluted with water (50 mL) after cooling to room temperature. The resulted mixture was filtered, washed with water. The filter cake was further crystallized from EtOH to give target compound in yield of 80%.

Synthesis of Intermediate 24b

Zinc powder (120 mmol) and AcOH (400 mmol) were added to a solution of 24a (8 mmol) in DCM (45 mL) in ice bath. The mixture was stirred at room temperature until the reaction was completed. The reaction mixture was filtered. The filtrate was washed with water, dried (Na2SO4) and concentrated under reduced pressure. The residue was further purified by column chromatography (PE/EA=4:1, V/V) to give 24b in yield of 80%. 1H-NMR (CDCl3): δ 7.39 – 7.31 (m, 4H) 7.27 (d, J = 6.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 4.27 (s, 2H), 4.16 (s, 2H) ppm; 13C-NMR (CDCl3): δ 147.79, 142.42, 139.61, 128.62, 127.60, 127.22, 116.21, 114.37, 49.36 ppm.

General Procedure for the Synthesis of Intermediates 25b and 26b

K2CO3 (15 mmol) was added to a solution of compounds 25a, 26b (10 mmol), and 4-nitrophenol (11 mmol) in DMF (30 mL) under Ar atmosphere, and the mixture was heated to 120 °C for 4 h. The mixture was diluted with water (50 mL) after cooling to room temperature. The resulted mixture was filtered, washed with water. The filter cake was dried to give target compounds 25b and 26b.
2-((4-Nitrophenoxy)methyl)benzonitrile (25b): 1H-NMR (CDCl3): δ 8.26–8.21 (m, 2H), 7.75 (d, J = 7.6 Hz, 1H), 7.70–7.64 (m, 2H), 7.52–7.47 (m, 1H), 7.11–7.06 (m, 2H). 5.35 (s, 2H) ppm; 13C-NMR (CDCl3): δ 162.97, 142.20, 138.97, 133.27, 133.15, 129.05, 128.63, 126.04, 116.85, 114.91, 111.50, 68.16 ppm.

General Procedure for Synthesis of Intermediates 25c and 26c

Reduced iron powder (100 mmol) and concentrated hydrochloric acid (0.5 mL) were carefully added to a mixture of compounds 25b, 26b, EtOH (60 mL) and water (6 mL). The reaction was reacted under reflux condition until the reaction was completed. The reaction mixture was filtered and the filter cake was washed with some EA. The filtrate was concentrated and used in the next step without further purification.
3-((4-Aminophenoxy)methyl)benzonitrile (26c): 1H-NMR (CDCl3): δ 7.68 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 5.18 (s, 2H) ppm; 13C- NMR (CDCl3): δ 151.30, 141.22, 140.89, 133.01, 132.80, 128.45, 128.21, 117.15, 116.37, 116.35, 111.04, 68.52 ppm.

General Procedure for Synthesis of Intermediates 25d and 26d

15% KOH (100 mL) was added to a mixture of compounds 25c, 26c and EtOH (25 mL) under Ar atmosphere, and the mixture was reacted under reflux condition for 36 h. The reaction mixture was washed with EA (30 mL), acidified with 1 N HCl, and extracted with EA. The combined organic layer was washed with saturated NaCl, dried (Na2SO4), concentrated and purified by column chromatography (DCM/MeOH=10:1, V/V) to give 25d, 26d in yield of about 40%.
2-((4-Aminophenoxy)methyl)benzoic acid (25d): 1H-NMR (DMSO-d6): δ 10.62 (s, 3H), 7.94 (d, J = 7.6 Hz, 1H), 7.66–7.56 (m, 2H), 7.48–7.43 (m, 1H), 7.33 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 5.47 (s, 2H) ppm; 13C-NMR (DMSO-d6): δ 168.05, 157.67, 137.86, 132.09, 130.47, 129.53, 127.96, 127.77, 124.64, 124.38, 115.60, 67.91 ppm.

Synthesis of Intermediate IM

4-Aminobenzoic acid (5 mmol) was slowly added to a solution of TCDI (6 mmol) and TEA (5.5 mmol) in DCM (7.5 mL) at 0 °C. The mixture was stirred for 2h at 0 °C and then added dropwise to 4M aqueous HCl (9 mL). The precipitation was filtered and washed with 1M aqueous HCl (1 mL×2). The resulting sold was dried to afford 4-isothiocyanatobenzoic acid in yield of 90%. Methyl 2-cyanoacetate (2 mmol) followed by a solution of 4-isothiocyanatobenzoic acid (2 mmol) in anhydrous DMF (2 mL) were added to a cold suspension of powdered KOH (4 mmol) in dry DMF (2 mL). The mixture was stirred at room temperature for 0.5 h, then cooled again to 0 °C, treated with a solution of 2-chloroacetyl chloride (3 mmol) in anhydrous DMF (2 mL) and stirred at room temperature overnight. The mixture was poured into ice-cold water, and the resulting precipitate was filtered off, dried, and crystallized from DCM-EtOH to give intermediate IM in yield of 68%. Mp 290.1-290.7 °C. 1H-NMR (DMSO-d6): δ 13.28 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 4.08 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.30, 172.30, 166.57, 165.31, 138.53, 132.45, 130.22, 129.71, 112.32, 75.76, 52.38, 32.27 ppm. HRMS (EI) calc. for C12H8N2O3S+ 318.0310; found 318.0312.

General Procedure for the Synthesis of Intermediates 40b and 41b

TEA (15 mmol) followed by 4-nitrobenzoyl chloride (10 mmol) was dropwise added to a solution of compound 40a, 41a in THF (40 mL) in ice bath. The mixture was allowed to stir at room temperature overnight. Then the mixture was diluted with water (50 mL), acidified with 1 M HCl, filtered. The filter cake was washed with water, crystallized from DCM-MeOH to give intermediate 40b, 41b.
3-(4-Nitrobenzamido)benzoic acid (41b): 1H-NMR (DMSO-d6): δ 10.80 (s, 1H), 8.43 (s, 1H), 8.23 (d, J = 8.8 Hz, 2H), 8.23 (d, J = 8.8 Hz, 2H), 8.06 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H) ppm; 13C-NMR (DMSO-d6): δ 167.08, 164.04, 149.19, 140.22, 138.91, 131.25, 129.25, 128.98, 124.92, 124.52, 123.53, 121.21 ppm.

General Procedure for the Synthesis of Intermediates 40c and 41c

Compounds 40c and 41c were prepared from 40b and 41b according the same procedure described for 19c–22c.

Synthesis of Intermediate 42a

Phthalide (10 mmol) and potassium phthalimide (11 mmol) was added to DMF (7 mL). The mixture was stirred under reflux condition until the reaction was completed. 35% AcOH (11 mL) was added after cooling to room temperature. The resulted mixture was filtered after stirring for 0.5 h. The filter cake was washed with water and EtOH in order, crystallized from DCM-MeOH to give intermediate 42a in yield of 56%.

Synthesis of Intermediate 42b

Compound 42a (5 mmol) was dissolved in DMF (10 mL) and EtOH (20 mL). The mixture was heated to 75 °C, added 80% hydrazine hydrate (0.6 mL) and stirred at this temperature overnight. The mixture was filtered after cooling to room temperature. The filtrate was concentrated to give 42b in yield of 80%.

Synthesis of Intermediate 43a

Concentrated H2SO4 (30 mmol) was added to a mixture of magnesium sulfate (120 mmol) and DCM (120 mL). Then 3-cyanobenzoic acid (30 mmol) and tert-butanol were added after stirring for 15 min. The mixture was stirred at room temperature for 24 h and filtered. The filtrate was neutralized with saturated NaHCO3, diluted with water, and extracted with DCM. The combined organic layer was washed with saturated NaCl, concentrated and purified by column chromatography (PE/EA=3:1, V/V) to give 43a in yield of 58%. 1H-NMR (DMSO-d6): δ 8.25 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 1.56 (s, 9H) ppm. 13C-NMR (DMSO-d6): δ 163.21, 136.23, 133.41, 132.47, 132.40, 130.04, 117.97, 111.91, 81.87, 27.62 ppm.

Synthesis of Intermediate 43b

A mixture of HCOOH and TEA (25 mL, V:V=5:1) followed by Pd/C (1.1 g) was slowly added to a solution of compound 43a in THF (25 mL) under Ar atmosphere. The mixture was stirred at 40 °C for 4 h and filtered. The filtrate was neutralized with saturated NaHCO3 and concentrated to remove THF. The residue was extracted with EA. The combined organic layer was washed with saturated NaCl, dried (Na2SO4), and concentrated to give compound 43b. GC-MS m/z 207 [M]+.

Synthesis of Intermediate 43c

Compound 43b (2 mmol), IM (2 mmol), and 4-dimethylaminopyridine (DMAP, 20 mg) was added to DCM (20 mL). Then 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride (EDCI, 4 mmol) was added. The mixture was stirred at room temperature until the reaction was completed, and concentrated. The residue was purified by column chromatography (PE/EA=4:3, V/V) to give 43c in yield of 68%. 1H-NMR (DMSO-d6): δ 9.28 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H), 7.89 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.09 (s, 2H), 3.71 (s, 3H), 1.54 (s, 9H) ppm.

Synthesis of Intermediate 44a

KMnO4 (60 mmol) was added to a solution of 1-(tert-butyl)-2-methylbenzene in tert-butanol (60 mL) and water (60 mL). The mixture was stirred under reflux condition until the reaction was completed and filtered. The filtrate was concentrated to remove tert-butanol, acidified with concentrated HCl, and filtered. The filter cake was dried to give 44a, which was directly used in the next step.

Synthesis of Intermediate 44b

Compound 44b was dissolved in concentrated H2SO4 (25 mL) and cooled to 0 °C. Then a solution of KNO3 (20 mmol) in concentrated H2SO4 (25 mL) was dropwise added to the mixture. The mixture was reacted at 0 °C until the reaction was completed, and dropwise added to ice water. The resulted mixture was filtered and washed with water. The filter cake was dried and crystallized from EtOH to give intermediate 44b in yield of 40% over two steps. 1H-NMR (DMSO-d6): δ 13.75 (s, 1H), 8.21 (dd, J = 8.8, 2.8 Hz, 1H), 8.10 (d, J = 2.8 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 1.43 (s, 9H) ppm; 13C-NMR (DMSO-d6): δ 170.88, 153.99, 144.99, 135.54, 128.88, 123.82, 122.76, 36.35, 30.62 ppm.

Synthesis of Intermediate 44c

Compound 44b (10 mmol) was added to a mixture of EtOH (60 mL) and water (15 mL). Then reduced iron powder (100 mmol) and concentrated HCl (10 drops) were added. The mixture was stirred under reflux condition until the reaction was completed, filtered. The filter cake was washed with EA. The filtrate was concentrated and directly used in the next step.

Synthesis of Intermediate 44d

4-Nitrobenzoyl chloride (6 mmol) was slowly added to a mixture of compound 44c, DCM (30 mL), and TEA (10 mmol). The obtained mixture was stirred at room temperature until the reaction was completed. The mixture was acidified with 1 M HCl and filtered. The filter cake was washed with water, dried and crystallized from DCM/MeOH to give intermediate 44d in yield of 40% over two steps. 1H-NMR (DMSO-d6): δ 13.15 (s, 1H), 10.61 (s, 1H), 8.42–8.35 (m, 2H), 8.23–8.17 (m, 2H), 7.81–7.76 (m, 2H), 7.49 (d, J = 9.2 Hz, 1H), 1.39 (s, 9H) ppm; 13C-NMR (DMSO-d6): δ 172.65, 163.77, 149.18, 141.93, 140.26, 136.09, 134.46, 129.16, 127.25, 123.54, 120.89, 119.73, 35.23, 31.10 ppm.

Synthesis of Intermediate 44e

Compound 44e was prepared from 44d in the same manner as described for 44c. 1H-NMR (DMSO-d6): δ 13.00 (s, 1H), 9.79 (s, 1H), 7.78–7.66 (m, 4H), 7.45–7.34 (m, 1H), 6.60 (d, J = 8.8 Hz, 2H), 5.76 (s, 2H), 1.37 (s, 9H) ppm; 13C-NMR (DMSO-d6): δ 165.20, 152.17, 140.36, 137.12, 129.31, 126.83, 120.79, 120.29, 119.28, 112.50, 79.15, 35.09, 31.18 ppm.

Synthesis of Intermediate 45a

2-Bromonaphthalene (50 mmol) was dissolved in dry DCM under Ar atmosphere and cooled to -10 °C. Then the mixture was added AlCl3 (150 mmol) and stirred until it turns green. Then acetyl chloride was added after cooling to -78 °C and stirred at this temperature for 3 h. The mixture was quenched with 1 M HCl, diluted with water, and extracted with DCM. The combined organic phases were washed with 1 M HCl and saturated NaCl in order, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (PE/EA=4:1, V/V) to give 45d in yield of 89%. 1H-NMR (CDCl3): δ 9.03 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 7.2, 1.0 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.62 (dd, J = 8.8, 2.0 Hz, 1H), 7.52 (dd, J = 8.4, 7.2 Hz, 1H), 2.74 (s, 3H) ppm; 13C-NMR (CDCl3): δ 201.06, 134.13, 133.08, 132.42, 131.13, 130.04, 129.84, 128.57, 124.76, 123.01, 29.76 ppm.

Synthesis of Intermediates 45b

Compound 45a (20 mmol), ammonia (100 mmol), CuI (4 mmol), L-proline (8 mmol), and K2CO3 (60 mmol) was suspended in DMSO (40 mL) under Ar atmosphere. The mixture was stirred at 85 °C for 24 h, and filtered after cooling to room temperature. The filter cake was washed with EA. The filtrate was diluted with water and extracted with EA. The combined organic phases were washed with saturated NaCl, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (PE/EA=3:1, V/V) to give 45b in yield of 52%.

Synthesis of Intermediates 46a

DMAP (0.5 mmol) and tert-butyl carbazate (6 mmol) were added to a mixture of compound IM and DCM (25 mL). Then the mixture was added EDCI (10 mmol) and stirred at room temperature until the reaction was completed. The solvent was removed under reduced pressure and the residue was purified by column chromatography (DCM/acetone=10:1, V/V) to give 46a in yield of 72%. 1H- NMR (CDCl3): δ 8.79 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.86 (s, 1H), 3.91 (s, 2H), 3.81 (s, 3H), 1.50 (s, 9H) ppm; 13C-NMR (CDCl3): δ 172.20, 170.07, 165.67, 165.62, 155.69, 137.12, 129.25, 129.15, 111.85, 81.99, 78.88, 52.90, 31.79, 28.34, 28.18 ppm.

Synthesis of Intermediate 46b

CF3COOH was added to a solution of compound 46a in DCM (45 mL). The mixture was stirred at room temperature overnight and concentrated to give 46b, which was directly used in the next step.

Synthesis of Aryl Isothiocyanates 9e–22e, 23c, 24d

A mixture of 1,4-diazabicyclo(2.2.2) octane (DABCO, 15 mmol), aromatic amines 9c–22c, 23a, 24b (5 mmol), and carbon disulfide (25 mL) in acetone (5 mL) was stirred overnight at room temperature. The precipitated solid was filtered. To a mixture of the solid and chloroform (20 mL) at 0 °C, was added dropwise a solution of triphosgene (2 mmol) in chloroform (10 mL) over 30 min. The reaction mixture was allowed to warm to room temperature and stirred overnight. After the resulting mixture was filtered, the filtrate was concentrated under reduced pressure and purified by column chromatography (100% PE) to give 9c–22c, 23a, 24b in 70%–95% yield as white solids or colorless oils.

Synthesis of Aryl Isothiocyanates 25e, 26e, 40d, 41d, 44f

Isothiocyanates 25e, 26e, 40d, 41d and 44f were prepared from 25d, 26d, 40c, 41c, 44e in the same manner as described for 4-isothiocyanatobenzoic acid.

3.2.2. Synthesis of Compounds 9–26, 40, 41, 44

Compounds 9–26, 40, 41 and 44 were prepared from 9e–22e, 23c, 24d, 25e, 26e, 40d, 41d, 44f in the same manner as described for compound IM.
Methyl (Z)-2-(3-([1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (9). Mp 235.1–235.5 °C. 1H-NMR (DMSO-d6): δ 7.82 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.51 (t, J = 8.0 Hz, 4H), 7.43 (t, J = 7.2 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. HRMS (ES+) calcd for C19H14N2O3S (M + H)+,351.0803; found, 351.0802.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (10). Mp 180.0–180.1 °C. 1H-NMR (DMSO-d6): δ 7.83–7.79 (m, 3H), 7.72 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.56–7.43 (m, 3H), 4.24 (ABq, Jgem = 18.8 Hz, 2H), 3.74 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −121.94 (t, J = 9.4 Hz) ppm. HRMS (ES+) calcd for C19H13FN2O3S (M + H)+,369.0703; found, 369.0709.
Methyl (Z)-2-cyano-2-(3-(3,5-difluoro-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (11). Mp 185.6–186.0 °C. 1H-NMR (DMSO-d6): δ 7.82 (dd, J1 = 16.8 Hz, J2 = 6.8 Hz, 4H), 7.55–7.47 (m, 3H), 4.40 (s, 2H), 3.76 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −118.44 (d, J = 11.1 Hz) ppm. HRMS (ES+) calcd for C19H12F2N2O3S (M + H)+,387.0615; found, 387.0621.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-2’-methyl-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (12). Mp 198.6–198.9 °C. 1H-NMR (400 MHz, DMSO-d6): δ 7.63 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 10.8 Hz, 1H), 7.33–7.26 (m, 5H), 4.25 (ABq, Jgem = 18.8 Hz, 2H), 3.75 (s, 3H), 2.26 (s, 3H) ppm. 19F NMR (376 MHz, DMSO-d6): δ −122.72 (t, J = 9.4 Hz) ppm. HRMS (ES+) calcd for C20H15FN2O3S (M + H)+, 383.0866; found, 383.0868.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-2’-methoxy-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (13). Mp 220.1–220.5 °C. 1H-NMR (DMSO-d6): δ 7.59 (t, J = 8.0 Hz, 2H), 7.50 (dd, J1 = 8.4 Hz, J2 = 1.6Hz, 1H), 7.43 (td, J1 = 8.0Hz, J2 = 1.6 Hz, 1H), 7.37 (dd, J1 = 7.2 Hz, J2 = 1.6Hz, 1H), 7.18 (d, J = 8.0 Hz,1H), 7.08 (t, J = 7.4 Hz, 1H), 4.24 (ABq, Jgem = 18.8 Hz, 2H), 3.82 (s, 3H), 3.75 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −123.08 (dd, J1 = 12.8 Hz, J2 = 8.5 Hz) ppm. HRMS (ES+) calcd for C20H15FN2O4S (M + H)+, 399.0815; found, 399.0814.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-3’-methoxy-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (14). Mp 193.1–193.6 °C. 1H-NMR (DMSO-d6): δ 7.84 (dd, J1 = 11.6 Hz, J2 = 1.6Hz, 1H), 7.73 (dd, J1 = 8.4 Hz, J2 = 1.6Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz,2H), 7.03 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H), 4.24 (ABq, Jgem = 18.8 Hz, 2H), 3.86 (s, 3H), 3.75 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −121.97 (dd, J1 = 12.8 Hz, J2 = 9.4 Hz) ppm. HRMS (ES+) calcd for C20H15FN2O4S (M + H)+, 399.0815; found, 399.0821.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-4’-methoxy-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (15). Mp 177.1–177.3 °C. 1H-NMR (DMSO-d6): δ 7.77–7.72 (m, 3H), 7.65 (d, J = 8.8 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 4.22 (ABq, Jgem = 18.6 Hz, 2H), 3.83 (s, 3H), 3.74 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −122.12 (dd, J1 = 13.2 Hz, J2 = 8.9 Hz) ppm. HRMS (ES+) calcd for C20H15FN2O4S (M + H)+, 399.0815; found, 399.0816.
Methyl (Z)-2-cyano-2-(3-(3’-cyano-3-fluoro-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene)acetate (16). Mp 191.2–191.3 °C. 1H-NMR (DMSO-d6): δ 8.33 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.95 (dd, J1 = 11.2 Hz, J2 = 7.6 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.72 (q, J = 7.6 Hz, 2H), 4.24 (ABq, Jgem = 18.8 Hz, 2H), 3.75 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −121.44 (td, J1 = 8.9 Hz, J2 = 3.4 Hz) ppm. HRMS (ES+) calcd for C20H12FN3O3S (M + H)+, 394.0662; found, 394.0668.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-3’-nitro-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene) acetate (17). Mp 179.2–179.9 °C. 1H-NMR (DMSO-d6): δ 8.57–8.56 (m, 1H), 8.32–8.26 (m, 2H), 8.02 (dd, J1 = 11.2 Hz, J2 = 2.0 Hz, 1H), 7.88–7.80 (m, 2H), 7.74 (t, J = 8.0 Hz, 1H), 4.24 (ABq, Jgem = 18.4 Hz, 2H), 3.75 (s, 3H). 19F-NMR (DMSO-d6): δ −121.31 (t, J = 10.6 Hz). HRMS (ES+) calcd for C19H12FN3O5S (M + H)+, 413.3851; found, 413.3853.
Methyl (Z)-2-cyano-2-(3-(3-fluoro-3’-(trifluoromethyl)-[1,1’-biphenyl]-4-yl)-4-oxothiazolidin-2-ylidene) -acetate (18). Mp 182.4–183.1 °C. 1H-NMR (DMSO-d6): δ 8.12 (s, 1H), 7.98 (d, J = 11.2 Hz, 1H), 7.83 (dd, J1 = 8.4 Hz, J2 = 3.2Hz, 2H), 7.78–7.70 (m, 2H), 4.18 (ABq, Jgem = 18.8 Hz, 2H), 3.75 (s, 3H) ppm. 19F-NMR (DMSO-d6): δ −61.03 (s), -121.55(dd, J1 = 12.3 Hz, J2 = 8.9 Hz) ppm. HRMS (ES+) calcd for C20H12F4N2O3S (M + H)+, 437.0583; found, 437.0591.
Methyl (Z)-2-cyano-2-(4-oxo-3-(4-phenoxyphenyl)thiazolidin-2-ylidene)acetate (19). White solid, Yield: 72%. Mp 167.6–168.3 °C; 1H-NMR (DMSO-d6): δ 7.46–7.42 (m, 2H), 7.39 (t, J = 8.0 Hz, 2H), 7.18–7.12 (m, 3H), 7.07 (d, J = 8.0 Hz, 2H), 4.07 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.40, 173.13, 165.44, 157.97, 156.72, 131.34, 130.08, 130.01, 123.56, 119.73, 118.35, 112.40, 75.73, 52.35, 32.17 ppm. HRMS (EI) calc. for C19H14N2O4S+ 366.0674; found 366.0675.
Methyl (Z)-2-cyano-2-(3-(4-(2-methoxyphenoxy)phenyl)-4-oxothiazolidin-2-ylidene)acetate (20). White solid, Yield: 74%. Mp 181.2–182.1 °C; 1H-NMR (DMSO-d6): δ 7.34 (d, J = 8.8 Hz, 2H), 7.25–7.16 (m, 2H), 7.05 (dd, J = 8.0, 1.2 Hz, 1H), 6.99–6.93 (m, 3H), 4.05 (s, 2H), 3.75 (s, 3H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.48, 173.02, 165.48, 159.03, 151.09, 143.57, 130.83, 128.87, 125.66, 121.13, 121.04, 117.08, 113.45, 112.28, 75.80, 55.61, 52.32, 32.08 ppm. HRMS (EI) calc. for C20H16N2O5S+ 396.0780; found 396.0779.
Methyl (Z)-2-cyano-2-(4-oxo-3-(4-(2-(trifluoromethyl)phenoxy)phenyl)thiazolidin-2-ylidene)acetate (21). White solid, Yield: 72%.Mp 159.7–161.3 °C; 1H-NMR (DMSO-d6): δ 7.61 (t, J = 8.2 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 6.4 Hz, 2H), 7.27 (d, J = 8.8 Hz, 2H), 4.08 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.35, 173.07, 165.42, 157.74, 156.76, 131.78, 131.23, 131.16, 130.77 (q, 2JCF = 31.9 Hz), 123.65 (q, 1JCF = 270.9 Hz), 121.75, 120.84, 119.73 (q, 3JCF = 3.8 Hz), 114.15 (q, 3JCF = 3.8 Hz), 112.42, 75.79, 52.35, 32.20 ppm. 19F-NMR (DMSO-d6): δ −61.13 (s, 3F) ppm. HRMS (EI) calc. for C20H13F3N2O4S+ 434.0548; found 434.0550.
Methyl (Z)-2-(3-(4-(4-(tert-butyl)phenoxy)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (22). White solid, Yield: 74%. Mp 212.9–213.9 °C; 1H-NMR (DMSO-d6): δ 7.41 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 4.07 (s, 2H), 3.71 (s, 3H), 1.28 (s, 9H) ppm. 13C-NMR (DMSO-d6): δ 173.40, 173.11, 165.45, 158.35, 154.28, 145.96, 131.24, 129.81, 126.67, 119.40, 118.05, 112.35, 75.76, 52.34, 34.03, 32.15, 31.21 ppm. HRMS (EI) calc. for C23H22N2O4S+ 422.1300; found 422.1299.
Methyl (Z)-2-cyano-2-(4-oxo-3-(3-phenoxyphenyl)thiazolidin-2-ylidene)acetate (23). White solid, Yield: 76%. Mp 183.4–184.3 °C; 1H-NMR (DMSO-d6): δ 7.52 (t, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 2H), 7.25–7.20 (m, 2H), 7.17–7.12 (m, 2H), 7.10 (d, J = 8.0 Hz, 2H), 4.01 (ABq, Jgem = 18.4 Hz, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.25, 172.68, 165.40, 157.06, 156.64, 136.07, 130.69, 130.04, 124.60, 123.63, 121.35, 119.87, 118.48, 112.51, 75.57, 52.36, 32.19 ppm. HRMS (EI) calc. for C19H14N2O4S+ 366.0674; found 366.0675.
Methyl (Z)-2-(3-(4-(benzyloxy)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (24). White solid, Yield: 75%. Mp 202.0–202.8 °C; 1H-NMR (DMSO-d6): δ 7.47 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.37–7.34 (m, 1H), 7.31 (dt, J = 8.8, 2.6 Hz, 2H), 7.11 (dt, J = 8.8, 2.6 Hz, 2H), 5.15 (s, 2H), 4.05 (s, 2H), 3.70 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.51, 173.12, 165.51, 159.73, 136.62, 130.54, 128.44, 127.91, 127.74, 127.48, 115.18, 112.24, 75.74, 69.49, 52.31, 32.00 ppm. HRMS (EI) calc. for C20H16N2O4S+ 380.0831; found 380.0834.
(Z)-2-((4-(2-(1-Cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)phenoxy)methyl)benzoic acid (25). White solid, Yield: 67%. Mp 210.6–211.5 °C; 1H-NMR (DMSO-d6): δ 13.13 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 5.52 (s, 2H), 4.05 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.50, 173.12, 168.05, 165.50, 159.68, 137.94, 132.17, 130.62, 130.50, 129.27, 127.80, 127.69, 127.57, 115.19, 112.22, 75.78, 67.88, 52.30, 32.02 ppm. HRMS (EI) calc. for C21H16N2O6S+ 424.0729; found 424.0728.
(Z)-3-((4-(2-(1-Cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)phenoxy)methyl)benzoic acid (26). White solid, Yield: 68%. Mp 233.2–234.1 °C; 1H-NMR (DMSO-d6): δ 13.02 (s, 1H), 8.06 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 5.24 (s, 2H), 4.05 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.48, 173.07, 167.08, 165.50, 159.58, 137.24, 131.97, 130.99, 130.58, 128.78, 128.38, 127.63, 115.23, 112.23, 75.78, 68.94, 52.30, 32.00 ppm. HRMS (EI) calc. for C21H16N2O6S+ 424.0729; found 424.0730.
(Z)-2-(4-(2-(1-Cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)benzoic acid (40). White solid, Yield: 70%. Mp 261.8–262.5 °C; 1H-NMR (DMSO-d6): δ 13.81 (s, 1H), 12.25 (s, 1H), 8.71 (d, J = 8.0 Hz, 1H), 8.14–8.06 (m, 3H), 7.74–7.63 (m, 3H), 7.24 (t, J = 7.4 Hz, 1H), 4.11 (s, 2H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.33, 172.24, 169.94, 165.31, 162.25, 140.80, 137.96, 136.10, 134.28, 131.26, 130.10, 128.01, 123.20, 120.06, 116.84, 112.36, 75.90, 52.37, 32.25, 30.73 ppm. HRMS (ESI) calc. for C21H15N3O6SNa+ (M + Na)+, 460.0579; found 460.0578. HRMS (EI) calc. for C21H16N2O6S+ 424.0729; found 424.0728.
(Z)-3-(4-(2-(1-Cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)benzoic acid (41). White solid, Yield: 68%. Mp 205.7–206.6 °C; 1H-NMR (DMSO-d6): δ 12.98 (s, 1H), 10.57 (s, 1H), 8.45 (s, 1H), 8.13 (d, J = 7.6 Hz, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 2H), 7.50 (t, J = 7.8 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.33, 172.24, 169.94, 165.31, 162.25, 140.80, 137.96, 136.10, 134.28, 131.26, 130.10, 128.01, 123.20, 120.06, 116.84, 112.36, 75.90, 52.37, 32.25, 30.73 ppm. HRMS (ESI) calc. for C21H15N3O6SNa+ (M + Na)+, 460.0579; found 460.0580.
(Z)-2-(tert-butyl)-5-(4-(2-(1-cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)benzoic acid (44). White solid, Yield: 69%. Mp 328.1–328.9 °C; 1H-NMR (DMSO-d6): δ 13.13 (s, 1H), 10.44 (s, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.80 (dd, J = 8.4, 2.4 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.4 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H), 1.39 (s, 9H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.71, 172.32, 165.35, 164.39, 141.59, 137.55, 136.35, 136.04, 134.42, 129.47, 128.66, 127.19, 120.88, 119.69, 112.38, 75.79, 52.40, 35.21, 32.23, 31.12 ppm. HRMS (EI) calc. for C25H23N3O6S+ 493.1308; found 493.1300.

3.2.3. Synthesis of Compounds 27–39, 45

General procedure for the synthesis of compounds 27–39, 45
Corresponding aromatic amines (1.2 mmol) and DMAP (0.1 mmol) were added to a mixture of compound IM (1 mmol) and DCM (5 mL). Then the mixture was added EDCI (2 mmol) and stirred at room temperature until the reaction was completed. The solvent was removed under reduced pressure and the residue was purified by column chromatography (DCM/acetone=10:1, V/V) to give compounds 27–39, 45.
Methyl (Z)-2-cyano-2-(3-(4-(cyclohexylcarbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (27). White solid, Yield: 78%. Mp 224.3–225.1 °C; 1H-NMR (DMSO-d6): δ 7.33 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 4.06 (s, 2H), 3.69 (s, 3H), 2.57 (t, J = 9.6 Hz, 1H), 1.80 (d, J = 9.6 Hz, 4H), 1.71 (d, J = 12.4 Hz, 1H), 1.48–1.31 (m, 4H), 1.30–1.18 (m, 1H) ppm. 13C-NMR (DMSO-d6): δ 173.46, 172.71, 165.50, 149.98, 132.44, 128.97, 127.46, 111.95, 75.90, 54.87, 52.30, 43.51, 33.80, 32.06, 26.22, 25.52 ppm. HRMS (EI) calc. for C20H21N3O4S+ 399.1253; found 399.1251.
Methyl (Z)-2-cyano-2-(4-oxo-3-(4-(phenylcarbamoyl)phenyl)thiazolidin-2-ylidene)acetate (28). White solid, Yield: 48%. Mp 194.9–195.6 °C; 1H-NMR (DMSO-d6): δ 10.40 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.41, 172.35, 165.37, 164.44, 138.93, 137.47, 136.33, 129.45, 128.67, 128.62, 123.87, 120.49, 112.39, 75.79, 52.41, 32.24 ppm. HRMS (EI) calc. for C20H15N3O4S+ 393.0783; found 393.0782.
Methyl (Z)-2-cyano-2-(4-oxo-3-(4-(o-tolylcarbamoyl)phenyl)thiazolidin-2-ylidene)acetate (29). White solid, Yield: 50%. Mp 274.1–275.0 °C; 1H-NMR (DMSO-d6): δ 10.05 (s, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.24 (t, J = 6.8 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H), 2.25 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.44, 172.35, 165.38, 164.28, 137.45, 136.17, 135.95, 133.82, 130.33, 129.47, 128.62, 126.70, 126.16, 126.03, 112.43, 75.78, 52.41, 30.67, 17.89 ppm. HRMS (EI) calc. for C21H17N3O4S+ 407.0940; found 407.0941.
Methyl (Z)-2-cyano-2-(3-(4-((4-fluorophenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (30). White solid, Yield: 43%. Mp 231.7–232.5 °C; 1H-NMR (DMSO-d6): δ 10.46 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.82 (dd, J = 8.8, 5.0 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.22 (t, J = 8.8 Hz, 2H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.40, 172.34, 165.36, 164.38, 158.39 (d, 1JCF = 239.1 Hz), 137.51, 136.17, 135.28 (d, 4JCF = 2.5 Hz), 129.47, 128.65, 122.33 (d, 3JCF = 7.8 Hz), 115.21 (d, 2JCF = 22.1 Hz), 112.40, 75.79, 52.40, 32.24 ppm. 19F-NMR (DMSO-d6): δ −118.51 – −118.61 (m, 1F) ppm. HRMS (EI) calc. for C20H14FN3O4S+ 411.0689; found 411.0690.
Methyl (Z)-2-cyano-2-(3-(4-((3-cyano-4-fluorophenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (31). White solid, Yield: 40%. Mp 264.2–265.1 °C; 1H-NMR (DMSO-d6): δ 10.75 (s, 1H), 8.31 (dd, J = 5.8, 2.6 Hz, 1H), 8.11 (d, J = 8.4 Hz, 3H), 7.63 (d, J = 8.4 Hz, 2H), 7.57 (t, J = 9.2 Hz, 1H), 4.10 (s, 2H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.30, 165.34, 164.80, 158.49 (d, 1JCF = 251.2 Hz), 137.85, 136.05 (d, 3JCF = 6.5 Hz), 135.57, 129.61, 128.76, 127.79 (d, 3JCF = 8.1 Hz), 124.38, 116.99 (d, 2JCF = 20.4 Hz), 113.95, 112.42, 99.85 (d, 2JCF = 16.1 Hz), 75.79, 52.41, 32.25 ppm. 19F-NMR (DMSO-d6): δ −114.31–−114.38 (m, 1F) ppm. HRMS (EI) calc. for C21H13FN4O4S+ 436.0642; found 436.0640.
Methyl (Z)-2-(3-(4-((4-chlorophenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (32). White solid, Yield: 44%. Mp 280.1–280.9 °C; 1H-NMR (DMSO-d6): δ 10.53 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.40, 172.33, 165.36, 164.57, 137.92, 137.60, 136.07, 129.50, 128.72, 128.55, 127.49, 121.96, 112.40, 75.79, 52.41, 32.24 ppm. HRMS (EI) calc. for C20H1435ClN3O4S+ 427.0394; found 427.0393; calc. for C20H1437ClN3O4S+ 429.0364; found 429.0369.
Methyl (Z)-2-(3-(4-((4-bromophenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (33). White solid, Yield: 42%. Mp 306.2–307.1 °C; 1H-NMR (DMSO-d6): δ 10.53 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 4.10 (s, 2H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.32, 165.36, 164.58, 138.35, 137.61, 136.07, 131.46, 129.50, 128.73, 122.32, 115.58, 112.40, 75.80, 52.41, 32.25 ppm. HRMS (EI) calc. for C20H1479BrN3O4S+ 470.9888; found 470.9887; calc. for C20H1481BrN3O4S + 472.9868; found 472.9863.
Methyl (Z)-2-cyano-2-(3-(4-((4-iodophenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (34). White solid, Yield: 45%. Mp 315.8–316.6 °C; 1H-NMR (DMSO-d6): δ 10.49 (s, 1H), 8.09 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.32, 165.36, 164.56, 138.82, 137.59, 137.30, 136.09, 129.49, 128.72, 122.56, 112.40, 87.64, 75.79, 52.42, 32.25 ppm. HRMS (EI) calc. for C20H14IN3O4S+ 518.9750; found 518.9751.
Methyl (Z)-2-cyano-2-(4-oxo-3-(4-((4-(trifluoromethyl)phenyl)carbamoyl)phenyl)thiazolidin-2-ylidene) acetate (35). White solid, Yield: 40%. Mp 264.2–265.1 °C; 1H-NMR (DMSO-d6): δ 10.75 (s, 1H), 8.13 (d, J = 8.0 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 4.11 (s, 2H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.40, 172.33, 165.36, 165.00, 142.61, 137.78, 135.87, 129.55, 128.85, 125.93 (q, 3JCF = 3.6 Hz), 124.35 (q, 1JCF = 269.5 Hz), 123.80 (q, 2JCF = 31.8 Hz), 120.23, 112.42, 75.78, 52.40, 32.25 ppm. 19F-NMR (DMSO-d6): δ −60.35 (s, 3F) ppm. HRMS (EI) calc. for C21H14F3N3O4S+ 461.0657; found 461.0658.
Methyl (Z)-2-cyano-2-(3-(4-((4-methoxyphenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (36). White solid, Yield: 55%. Mp 268.9–269.7 °C; 1H-NMR (DMSO-d6): δ 10.29 (s, 1H), 8.10 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 4.10 (s, 2H), 3.76 (s, 3H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.37, 172.31, 165.36, 163.96, 155.69, 137.32, 136.40, 131.97, 129.39, 128.53, 122.10, 113.76, 112.35, 75.83, 55.17, 52.38, 32.21 ppm. HRMS (EI) calc. for C21H17N3O5S+ 423.0889; found 423.0891.
Methyl (Z)-2-(3-(4-((4-(tert-butyl)phenyl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (37). White solid, Yield: 51%. Mp 256.1–257.0 °C; 1H-NMR (DMSO-d6): δ 10.34 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 4.10 (s, 2H), 3.72 (s, 3H), 1.29 (s, 9H) ppm. 13C-NMR (DMSO-d6): δ 173.42, 172.38, 165.37, 164.21, 146.19, 137.40, 136.35, 129.42, 128.63, 125.24, 120.23, 112.39, 75.74, 52.41, 34.06, 32.25, 31.18 ppm. HRMS (EI) calc. for C24H23N3O4S+ 449.1409; found 449.1410.
Methyl (Z)-2-cyano-2-(3-(4-(naphthalen-2-ylcarbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)acetate (38). White solid, Yield: 48%. Mp 285.0–286.0 °C; 1H-NMR (DMSO-d6): δ 10.61 (s, 1H), 8.49 (d, J = 1.2 Hz, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.95–7.84 (m, 4H), 7.63 (t, J = 8.4 Hz, 2H), 7.51 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 4.11 (s, 2H), 3.73 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.41, 172.35, 165.38, 164.66, 137.55, 136.57, 136.27, 133.27, 130.07, 129.50, 128.73, 128.18, 127.44, 126.40, 124.88, 120.96, 116.78, 112.41, 75.81, 52.41, 32.25 ppm. HRMS (EI) calc. for C24H17N3O4S+ 443.0940; found 443.0938.
Methyl (Z)-2-(3-(4-(benzylcarbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (39). White solid, Yield: 68%. Mp 117.3–118.1 °C; 1H-NMR (DMSO-d6): δ 9.22 (t, J = 5.6 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.37–7.32 (m, 4H), 7.27–7.22 (m, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.08 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.35, 165.37, 165.20, 139.46, 137.20, 135.82, 129.39, 128.28, 128.23, 127.27, 126.78, 112.33, 75.78, 52.39, 42.72, 32.22 ppm. HRMS (EI) calc. for C21H17N3O4S+ 407.0940; found 407.0939.
Methyl (Z)-2-(3-(4-((5-acetylnaphthalen-2-yl)carbamoyl)phenyl)-4-oxothiazolidin-2-ylidene)-2-cyanoacetate (45). White solid, Yield: 43%. Mp 275.0–275.8 °C; 1H-NMR (DMSO-d6): δ 10.73 (s, 1H), 9.10 (d, J = 1.6 Hz, 1H), 8.17 (d, J = 8.4 Hz, 2H), 8.12 (d, J = 7.2 Hz, 1H), 8.11–8.08 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.53 (t, J = 7.8 Hz, 1H), 4.11 (s, 2H), 3.73 (s, 3H), 2.74 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 201.53, 173.40, 172.34, 165.37, 164.72, 138.60, 137.59, 136.18, 134.45, 132.43, 130.57, 129.84, 129.60, 129.45, 128.85, 128.81, 123.62, 121.26, 115.06, 112.38, 75.82, 52.40, 32.25, 30.08 ppm. HRMS (EI) calc. for C26H19N3O5S+ 485.1045; found 485.1046.

3.2.4. Synthesis of Compounds 42, 43 and 46

Synthesis of (Z)-2-((4-(2-(1-cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)methyl)-benzoic acid (42)

TCDI (2.4 mmol) was added to a solution of compound IM in THF (10 mL). Then the mixture was stirred at room temperature for 3 h. Then the mixture was added compound 42b (2 mmol) and stirred at 50 °C for 2 h. The solvent was removed under reduced pressure after cooling to room temperature and the residue was purified by column chromatography (DCM/MeOH=20:3, V/V) to give 42 in a yield of 38%. White solid, Yield: 38%. Mp 217.3–218.1 °C; 1H-NMR (DMSO-d6): δ 13.08 (s, 1H), 9.14 (t, J = 5.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 7.6 Hz, 1H), 7.58–7.52 (m, 3H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 4.85 (d, J = 5.6 Hz, 2H), 4.09 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.93, 172.89, 168.93, 165.92, 165.87, 140.81, 137.76, 136.24, 132.51, 130.87, 129.94, 128.74, 127.76, 127.22, 112.86, 76.22, 52.90, 41.80, 32.73 ppm. HRMS (EI) calc. for C22H17N3O6S+ 451.0844; found 451.0838.

Synthesis of (Z)-3-((4-(2-(1-cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)methyl) benzoic acid (43)

CF3COOH (1.5 mL) was added to a solution of compound 43c (1 mmol) in DCM (15 mL). Then the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was crystallized from DCM/MeOH to give compound 43 in a yield of 85%.
White solid, Mp 205.7–206.6 °C; 1H-NMR (DMSO-d6): δ 12.92 (s, 1H), 9.26 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.94 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.47 (t, J = 8.0 Hz, 1H), 4.56 (d, J = 5.6 Hz, 2H), 4.09 (s, 2H), 3.71 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.34, 172.29, 167.22, 165.34, 165.25, 139.96, 137.26, 135.68, 131.88, 130.84, 129.42, 128.57, 128.24, 128.21, 127.81, 112.30, 75.81, 52.37, 42.53, 32.19 ppm. HRMS (ESI) calc. for C22H17N3O6SNa+ (M + Na)+, 474.0736; found 474.0737.

Synthesis of 2-(((E)-(4-((Z)-2-(1-cyano-2-methoxy-2-oxoethylidene)-4-oxothiazolidin-3-yl)benzamido)- methylene)amino)benzoic acid (46)

A drop of concentrated HCl was added to a solution of compound 46b (1 mmol) and 2-formylbenzoic acid in EtOH (30 mL). Then the mixture was stirred at room temperature until the reaction was completed. The solvent was removed under reduced pressure and the residue was crystallized from DCM/MeOH to give compound 46 in yield of 80%. White solid, Yield: 80%. Mp 222.8–223.5 °C; 1H-NMR (DMSO-d6): δ 13.26 (s, 1H), 12.25 (s, 1H), 9.24 (s, 1H), 8.12 – 8.05 (m, 3H), 7.92 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.55 (t, J = 7.6 Hz, 1H), 4.10 (s, 2H), 3.72 (s, 3H) ppm. 13C-NMR (DMSO-d6): δ 173.39, 172.33, 168.04, 165.36, 162.28, 147.08, 137.63, 134.78, 134.53, 132.00, 130.67, 130.30, 129.69, 129.51, 128.72, 126.68, 112.35, 75.80, 52.40, 32.24 ppm. HRMS (ESI) calc. for C22H16N4O6SNa+ (M + Na)+, 487.0688; found 487.0689.

3.3. In Vitro Assays

The plasmid pET-19b–hDHODH (Met30-Arg396) was transformed into BL21 (DE3) E. coli cells for protein production. Cells were grown at 37 °C in a rich medium, and were induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at an OD600 of 0.6–0.8. After incubation for an additional 18 h at 16 °C, the cells were harvested by centrifugation. The harvested cells were suspended in buffer A (50 mM HEPES pH 8.0, 400 mM NaCl, 10% glycerol) and disrupted by a high-pressure cracker at 4 °C. Triton X-100 was added to a final concentration of 1% into the lysate before centrifugation. The supernatant was loaded onto a HiTrap Chelating column (5 mL; GE Healthcare, Uppsala, Sweden). Pure hDHODH was eluted with buffer A, 0.1% Triton X-100 and 160 mM imidazole.
The purified hDHODH was diluted into a final concentration of 10 nM with the assay buffer contained 50 mM HEPES pH 8.0, 150 mM KCl, 0.1% Triton X-100. UQ0 and DCIP were added to the assay buffer to final concentrations of 100 and 120 μM, respectively. The dihydroorotate was added to a final concentration of 500 μM to initiate the reaction. Brequinar was measured as the positive control. Inhibition rate was calculated from (1-Vi/V0) × 100. For the determination of the IC50 values, eight to nine different concentrations were applied. Each inhibitor concentration point was tested in triplicate. IC50 values were calculated using the sigmoidal fitting option of the program Origin 8.0.

4. Conclusions

In conclusion, based on our previous work, three series of 4-thiazolidinone derivatives including biphenyl, diphenyl ether and amide groups were designed and synthesized as hDHODH inhibitors. The preliminary structure–activity relationships were investigated. The hDHODH inhibitory activities of several newly synthesized compounds and compounds 7 and 8 are at the same level, especially compounds 9 and 37 with IC50 values of 1.32 and 1.45 μM, respectively. Further modifications will be investigated to improve the activity of 4-thiazolidinone derivatives.

Supplementary Materials

Spectrums for target compounds 7–46 could be accessed in supplementary materials.

Author Contributions

Conceptualization, X.X., L.Z. (Lili Zhu) and F.Z.; investigation, F.Z. and L.Q.; methodology, F.Z., L.Q., X.X., G.Y., T.Q., L.Z. (Letian Zhang) and S.L.; software, L.Q., F.Z., T.Q., S.L. and H.L.; data curation, X.X., F.Z., L.Q., G.Y. and L.Z. (Lili Zhu); writing—original draft preparation, F.Z. and G.Y.; writing—review and editing, X.X. and L.Z (Lili Zhu).; supervision, X.X. and L.Z. (Lili Zhu).

Funding

X.X. and H.L. innovation Program of Shanghai Municipal Education Commission (201701070002E00037). The Fundamental Research Funds for the Central Universities.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 7–46 are available from the authors.
Molecules 24 02780 g001 550
Figure 1. Structures of representative inhibitors of hDHODH.
Figure 1. Structures of representative inhibitors of hDHODH.
Molecules 24 02780 g001
Molecules 24 02780 g002 550
Figure 2. Proposed binding modes of 4-thiazolidinone derivatives 7 (A) and 8 (B). The X-ray crystal structure of hDHODH with PDB ID of 4LS1 was used in molecular docking. Figure 2C is an overlay of compounds 7 and 8.
Figure 2. Proposed binding modes of 4-thiazolidinone derivatives 7 (A) and 8 (B). The X-ray crystal structure of hDHODH with PDB ID of 4LS1 was used in molecular docking. Figure 2C is an overlay of compounds 7 and 8.
Molecules 24 02780 g002
Molecules 24 02780 g003 550
Figure 3. Binding mode of 4-thiazolidinone derivative 9. Compound 9 is displayed as yellow sticks and brequinar is shown as cyan sticks.
Figure 3. Binding mode of 4-thiazolidinone derivative 9. Compound 9 is displayed as yellow sticks and brequinar is shown as cyan sticks.
Molecules 24 02780 g003
Molecules 24 02780 g004 550
Figure 4. Molecular design strategies of 4-thiazolidinones with biphenyl as hDHODH inhibitors.
Figure 4. Molecular design strategies of 4-thiazolidinones with biphenyl as hDHODH inhibitors.
Molecules 24 02780 g004
Molecules 24 02780 g005 550
Figure 5. Binding modes of 4-thiazolidinones with biphenyl ether structure (magenta sticks: compound 10, orange sticks: compound 19).
Figure 5. Binding modes of 4-thiazolidinones with biphenyl ether structure (magenta sticks: compound 10, orange sticks: compound 19).
Molecules 24 02780 g005
Molecules 24 02780 g006 550
Figure 6. Proposed binding mode of benzamide-substituted 4-thiazolidinones as hDHODH inhibitors (orange sticks: compound 41, gray sticks: compound 10).
Figure 6. Proposed binding mode of benzamide-substituted 4-thiazolidinones as hDHODH inhibitors (orange sticks: compound 41, gray sticks: compound 10).
Molecules 24 02780 g006
Molecules 24 02780 g007 550
Figure 7. Design and modification strategies of 4-thiazolidinones with biphenyl ether and amide as hDHODH inhibitors.
Figure 7. Design and modification strategies of 4-thiazolidinones with biphenyl ether and amide as hDHODH inhibitors.
Molecules 24 02780 g007
Molecules 24 02780 sch001 550
Scheme 1. Synthesis of key intermediates of compounds 9–26.
Scheme 1. Synthesis of key intermediates of compounds 9–26.
Molecules 24 02780 sch001
Molecules 24 02780 sch002 550
Scheme 2. Synthesis of IM and key intermediates of compounds 40–46.
Scheme 2. Synthesis of IM and key intermediates of compounds 40–46.
Molecules 24 02780 sch002
Molecules 24 02780 sch003 550
Scheme 3. Synthesis of compounds 9–46.
Scheme 3. Synthesis of compounds 9–46.
Molecules 24 02780 sch003
Table
Table 1. Structures and activities for 4-thiazolidinone analogs 9–18.
Table 1. Structures and activities for 4-thiazolidinone analogs 9–18.
Molecules 24 02780 i001
CompdR% Inhibition at 10 µMhDHODH IC50a (μM)
7Molecules 24 02780 i00279.11.75
8Molecules 24 02780 i00380.31.12
9Molecules 24 02780 i00473.81.32
10Molecules 24 02780 i00558.83.52
11Molecules 24 02780 i00633.9>10
12Molecules 24 02780 i00757.66.04
13Molecules 24 02780 i00854.76.64
14Molecules 24 02780 i00963.35.42
15Molecules 24 02780 i01055.46.86
16Molecules 24 02780 i01150.28.39
17Molecules 24 02780 i01250.74.35
18Molecules 24 02780 i01353.19.29
Brequinar 0.0084
a IC50 values were determined from three independent tests, and attempts to determine IC50 values were made if the inhibition rate at 10 μM was greater than 50%.
Table
Table 2. Structures and activities for 4-thiazolidinone analogs 19–46.
Table 2. Structures and activities for 4-thiazolidinone analogs 19–46.
Molecules 24 02780 i014
CompdR% Inhibition at 10 µMhDHODH IC50 (μM)
19Molecules 24 02780 i01552.09.43
20Molecules 24 02780 i01644.7>10
21Molecules 24 02780 i01729.7>10
22Molecules 24 02780 i01818.1>10
23Molecules 24 02780 i01934.4>10
24Molecules 24 02780 i02055.44.32
25Molecules 24 02780 i02129.9>10
26Molecules 24 02780 i02236.2>10
27Molecules 24 02780 i02341.7>10
28Molecules 24 02780 i02469.02.98
29Molecules 24 02780 i02547.0>10
30Molecules 24 02780 i02671.33.01
31Molecules 24 02780 i02753.68.56
32Molecules 24 02780 i02852.77.50
33Molecules 24 02780 i02942.0>10
34Molecules 24 02780 i03038.1>10
35Molecules 24 02780 i03124.5>10
36Molecules 24 02780 i03255.45.01
37Molecules 24 02780 i03375.61.45
38Molecules 24 02780 i03438.2>10
39Molecules 24 02780 i03539.9>10
40Molecules 24 02780 i03634.5>10
41Molecules 24 02780 i03723.5>10
42Molecules 24 02780 i03836.8>10
43Molecules 24 02780 i03944.0>10
44Molecules 24 02780 i04030.3>10
45Molecules 24 02780 i04142.2>10
46Molecules 24 02780 i04234.8>10

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Zeng, F.; Quan, L.; Yang, G.; Qi, T.; Zhang, L.; Li, S.; Li, H.; Zhu, L.; Xu, X. Structural Optimization and Structure–Activity Relationship of 4-Thiazolidinone Derivatives as Novel Inhibitors of Human Dihydroorotate Dehydrogenase. Molecules 2019, 24, 2780. https://doi.org/10.3390/molecules24152780

AMA Style

Zeng F, Quan L, Yang G, Qi T, Zhang L, Li S, Li H, Zhu L, Xu X. Structural Optimization and Structure–Activity Relationship of 4-Thiazolidinone Derivatives as Novel Inhibitors of Human Dihydroorotate Dehydrogenase. Molecules. 2019; 24(15):2780. https://doi.org/10.3390/molecules24152780

Chicago/Turabian Style

Zeng, Fanxun, Lina Quan, Guantian Yang, Tiantian Qi, Letian Zhang, Shiliang Li, Honglin Li, Lili Zhu, and Xiaoyong Xu. 2019. "Structural Optimization and Structure–Activity Relationship of 4-Thiazolidinone Derivatives as Novel Inhibitors of Human Dihydroorotate Dehydrogenase" Molecules 24, no. 15: 2780. https://doi.org/10.3390/molecules24152780

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