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J. Liu et al.
S3B) compared to untreated mice. Compounds 93 and 156 also consistently enhanced hepatic hepcidin levels (P<0.05) (Online Supplementary Figure S3A) and diminished serum iron concentrations in mice 24 h after administra- tion (P<0.05) (Online Supplementary Figure S3B). Additionally, compounds 49, 93, 140, 156 and 165 increased hepcidin mRNA expression by more than 1.5 fold 24 h after administration, compared to the expression in untreated mice (P<0.05) (Online Supplementary Figure S3A). Serum hepcidin was significantly increased by approximately 1.5 fold, relative to that in untreated mice, at 6 h following administration of compounds 69, 93, 139, 156 and 165 (P<0.05) (Online Supplementary Figure S3B),
A
and remained higher than that in control mice at 24 h after treatment with compounds 93, 156 and 165, but not with compounds 69 and 139 (P<0.05) (Online Supplementary Figure S3B). However, only mice treated with compounds 93, 156 and 165 consistently showed concurrent reduc- tions in serum iron (P<0.05) (Online Supplementary Figure S3B), while mice treated with compounds 49 and 140 did not display such a consistent effect (Online Supplementary Figure S3B). Moreover, compounds 93, 156 and 165 were found to increase hepatic hepcidin expression in mice overall in a dose-dependent manner, from 2, to 10 and 30 mg/kg body weight (P<0.05) (Online Supplementary Figure S4). Considering these results together, compounds 93,
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C
Figure 1. Screening of the thiazolidinone library for compounds stimulating hep- cidin expression. (A) Synthesis of the thia- zolidinone library. A total of 18 anilines and 20 aromatic aldehydes were used as reactants, and 210 thiazolidinone com- pounds were obtained with a purity greater than 95%, as determined by liquid chromatography/mass spectrometry. Reagents and conditions: (i) R1-NH2, NH2SCN, H+, H2O, 80°C; (ii) ethyl chloroac- etate, NaOAC, EtOH, 60°C; (iii) aldehydes, piperdine, EtOH, 60°C. The bold letters (a- d) delineate the synthesis procedure, as described in the Methods section: primary thioureas (b) were constructed by reacting aniline (a) with ammonium thiocyanate, in the presence of acid; thioureas (b) react- ed with ethyl 2-chloroacetate to generate thiazolidinones (c) as a precipitate, which was filtered and washed with absolute ethanol to obtain the product; the final step of the reaction was carried out in piperidine and absolute ethanol at 60°C, and approximately 95% of the product (d) was formed as precipitate. (B) A heatmap diagram showing the average fold changes of hepcidin-promoter luciferase activity relative to that of untreated cells (n=4). (C) Endogenous hepcidin mRNA expression in SMMC-7721 cells upon administration of compounds at a concen- tration of 10 μM for 24 h (n=4). *P<0.05, #P<0.001, compared to untreated control (Ctrl).
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haematologica | 2019; 104(9)