Fig 1.
General structure of bi-aryl amide compounds.
Fig 2.
The four strategies used for the synthesis of the library compounds (1–12)(a-f). Regents and reaction conditions: (i) N-(3-dimethylamino)-propyl-N’-ethyl-carbodiimide hydrochloride (EDC×HCl), 1-hydroxybenzotriazole (HOBt), N-ethyl-diisopropylamine (DIEA); (ii) arylboronic acid y1-12, base, palladium catalyst; (iii) aryl bromide x1-12, base, palladium catalyst; (iv) base, palladium catalyst; (v) secondary amine a-f, EDC×HCl, HOBt, DIEA. Bases and catalysts used in the Suzuki-Miyaura cross-coupling steps are detailed in the Materials and Methods section.
Fig 3.
Activity of synthesized compounds toward 11β-HSD1 and 11β-HSD2.
Enzyme activity was measured in lysates of HEK-293 cells expressing recombinant human 11β-HSD1 or 11β-HSD2 as described in Materials and Methods. Data represent mean from three independent experiments. nd: not determined.
Fig 4.
Inhibition of 11β-HSD1 activity in intact cells.
(A), Inhibition of 11β-HSD1-dependent cortisol formation in intact HEK-293 cells. HEK-293 cells stably co-expressing human 11β-HSD1 and H6PDH were incubated for 30 min in the presence of 200 nM radiolabeled cortisone and either 1 μM of the positive control inhibitor glycyrrhetinic acid (CTRL 1) or 100 nM and 1 μM of the respective test compounds. Formation of cortisol was determined by separation of steroids by TLC and scintillation counting. Data represent mean ± SD of three independent experiments, each performed in triplicate. (B), Inhibition of 11β-HSD1-dependent cortisol formation in intact human keratinocytes. Primary human keratinocytes were grown for two days, followed by incubation for 24 h with 1 μM of cortisone and various concentrations of inhibitor. Compound CAS 1009373-58-3 (Merck, CTRL 4) was used as positive control. An additional vehicle control was measured in the absence of exogenous cortisone (black bar). Formation of cortisol was determined by an enzyme immune assay. Data represent mean ± SD from three experiments. Shapiro-Wilk test was used to assess the normality of data. One-way analysis of variance (ANOVA) and Dunnett's multiple-comparison test were performed to evaluate differences between inhibitor treatments compared to the solvent control. All values were significantly different (p<0.01) compared to vehicle control in the presence of cortisone, except treatment of keratinocytes with 10 nM 2b.
Fig 5.
Reversal of cortisone-mediated decrease in dermal total collagen content upon treatment with selected compounds.
Human skin biopsies were treated topically with 4 μL of 10 μM or 100 μM of the selected compounds for 6 days. After the first 24 h of incubation with the inhibitors, the skin samples were simultaneously treated with inhibitor and 100 nM cortisone for the remaining 5 days. Dermal collagen content was semi-quantitatively assessed by Picrosirius Red histochemical staining. Data represent mean ± SEM from 6 human biopsies. Shapiro-Wilk test indicated that the data followed a normal distribution and unpaired t-test was used to test for significant differences. # p<0.01 compared to vehicle control in the absence of cortisone; * p<0.05, ** p<0.01, *** p<0.001 compared to cortisone treated vehicle control.
Fig 6.
Effect of selected compounds on dermal collagen III content in UV exposed human skin samples.
Human skin biopsies were treated topically with 4 μL of 10 μM or 100 μM of compound 3e (A,B), 2b (C,D), or 12e (E,F) and exposed to 3.0 J/cm2 (A,C,E) or 6.0 J/cm2 UV irradiation (B,D,F) for 6 days. Dermal collagen III expression was detected using a mouse monoclonal anti-collagen III antibody. Data represent mean ± SEM. Per treatment 12 skin samples were analyzed. The data set presented a normal distribution using Shapiro-Wilk test and an unpaired t-test was used to test for significant differences. ## p<0.01 for UV exposed vehicle control vs non-irradiated vehicle control. * p<0.05, ** p<0.01 for UV exposed inhibitor treated vs UV exposed vehicle control.