BackgroundTransforming growth factor-β (TGF-β)/Smad signaling is well known to play a critical role in the pathogenesis of systemic sclerosis (SSc). We previously developed an artificial molecule, the histidine-pyridine-histidine ligand derivative HPH-15, which may have an antifibrotic effect. The purpose of the present study was to clarify the effects of this drug in human skin fibroblasts and in a preclinical model of SSc.MethodsThe effects of HPH-15 on expression of extracellular matrix components and TGF-β signaling in human dermal fibroblasts were analyzed. The antifibrotic properties of HPH-15 and its mechanisms were also examined in a bleomycin-induced skin fibrosis mouse model.ResultsHPH-15 suppressed the TGF-β-induced phosphorylation of Smad3 and inhibited the expression of collagen I, fibronectin 1, connective tissue growth factor, and α-smooth muscle actin induced by TGF-β in cultured human skin fibroblasts. In the bleomycin-induced skin fibrosis model, oral administration of HPH-15 protected against the development of skin fibrosis and ameliorated established skin fibrosis. Additionally, HPH-15 suppressed the phosphorylation of Smad3 in various cells, including macrophages in the bleomycin-injected skin. Further, in the treated mice, dermal infiltration of proinflammatory macrophages (CD11b+Ly6Chi) and M2 profibrotic macrophages (CD11b+CD204+ or CD11b+CD206+) was significantly decreased during the early and late stages, respectively. HPH-15 treatment resulted in decreased messenger RNA (mRNA) expression of the M2 macrophage markers arginase 1 and Ym-1 in the skin, whereas it inversely augmented expression of Friend leukemia integration 1 and Krüppel-like factor 5 mRNAs, the transcription factors that repress collagen synthesis. No apparent adverse effects of HPH-15 were found during the treatment.ConclusionsHPH-15 may inhibit skin fibrosis by inhibiting the phosphorylation of Smad3 in dermal fibroblasts and possibly in macrophages. Our results demonstrate several positive qualities of HPH-15, including oral bioavailability, a good safety profile, and therapeutic effectiveness. Thus, this TGF-β/Smad inhibitor is a potential candidate therapeutic for SSc clinical trials.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1534-y) contains supplementary material, which is available to authorized users.

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1β,3β,25-Dihydroxy-19-norvitamin D3 (4a) and 1α,3α,25-dihydroxy-19-norvitamin D3 (4b) were synthesized by employing a new A-ring synthon, (1R,3S)-3-((tert-butyldimethylsilyl)oxy)-5-oxocyclohexyl benzoate (19), which was derived from D-(-)-quinic acid in 12 steps. The A-ring was coupled with the circular dichroism (CD) ring by means of Julia-Kocienski olefination to construct the diene unit. The structures of the products were confirmed by (1)H-NMR and nuclear Overhauser effect (NOE) experiments.

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Fig. 1; also known as calcitriol) is the active hormonal form of vitamin D 3 (1); it regulates calcium and phosphorus homeostasis and exhibits potent antiproliferative activity.1-4) Its activity is regulated by the cytochrome oxidase P450 family member CYP24A1, which oxidizes 2 at the C24 and C23 positions to afford biologically inactive, side-chain-truncated, water-soluble end products.5-7) However, the metabolic stability of 2 can be increased by substitution of side-chain hydrogen with fluorine, which is very similar in size to hydrogen, but has different chemical bonding properties. The highly electron-withdrawing nature of fluorine means that the C-F bond has a marked ionic character, and this blocks the oxidation by CYP24A1. Indeed, 24,24-difluorinated 1α,25-dihydroxyvitamin D 3 showed high metabolic stability and almost 100% absorption in rats, although this enhanced metabolic stability did not yield increased biological activity.8) Many fluorinated derivatives of 2 have already been reported. Among them, falecalcitriol [9][10][11][12] is used to treat hypercalcemia, osteomalacia, and rickets, and is more efficient than calcitriol (2). Recently, DeLuca and colleagues reported 24,24-difluoro-1α,25-(OH) 2 -19-norvitamin D 3 (4), which showed stronger vitamin D receptor (VDR) binding affinity than compound 2 and promoted bone formation more efficiently.13) Thus, although 24-difluoro substitution has little effect on the biological potency of the natural hormone (2), it does alter the biological activity profile of 1α,25-(OH) 2 -19-norvitamin D 3 (3).With this background, we decided to extend our previously reported synthetic studies of 1,3-cis-25-dihydroxy-19-norvitamin D 3 (5a, b) 14) to obtain the 24-difluoro series (6a, b), to investigate the effects of fluoro substitution at the side chain on the biological activities of those ligands. Here, we describe the synthesis of 24,24-difluoro-1β,3β,25-dihydroxy-19-norvitamin D 3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D 3 (6b), and a comparison of their VDR-binding affinity with that of compounds 5a and b. RESULTS AND DISCUSSIONIn our previous study, we synthesized 1β,3β,25-dihydroxy-19-norvitamin D 3 (5a) and 1α,3α,25-dihydroxy-19-norvitamin D 3 (5b) by means of the Julia-Kocienski coupling with a 1,3-cis type A-ring synthon, followed by separation of the isomers 5a and b.14) Here we adopted similar methodology, using ketone 11 and sulfone 10. The sulfone 10 was synthesized from the Grundmann's ketone 7, which was reported by DeLuca and colleagues 13,15) (Chart 1). Compound 7 was subjected to Horner-Wittig olefination using triethyl phosphonoacetate in the presence of sodium hydride in tetrahydrofuran (THF) to give the α,β-unsaturated ester 8, and the ester was then reduced with diisobutylaluminium hydride (DIBAL)-H in toluene to give the allylic alcohol 9 in 68% yield (2 steps). The allylic alcohol 9 was subjected to the Mitsunobu reaction using 2-mercaptobenzothiazole in the presence of disopropyl azodicarboxylate (DIAD) and triphenylphosphi...