Fig 1.
Examples of PDE4 inhibitors of first and second generations.
Fig 2.
Genesis concept of sulfonamides (5 and 6a-k) designed as PDE4 inhibitors.
Fig 3.
Synthesis sulfonamide derivatives.
Reagents and conditions: a) 1) H2SO4 /Ac2O /AcOEt, 0°C, 2 h; 2) AcOK / EtOH, 25°C, 30 min, 93%. b) SOCl2, DMF, 75°C, 4 h, 92%. c) CH2Cl2, Et3N, 2-(3,4-dimethoxyphenyl)ethanamine (6a) or 2-phenylethanamine (5), 25°C, 2–2.5 h, 70%-81% respectively. d) K2CO3, acetone, RX (X = I, Br), (6b-6e, 6g-6i and 6k), 40°C, 1.5 h, 34–96%. e) CH2Cl2, Et3N, 2-(3,4-dimethoxyphenyl)ethanamine (6f, 6j) 25°C, 2–2.5 h, 53%-83% respectively.
Table 1.
Physico-chemistry properties of rolipram (1), prototype 4 and its sulfonamide analogues 5 and 6a-k, calculated using the Program ACD/Percepta 14.0.
Table 2.
PDE4 Inhibition of rolipram and sulfonamides 5 and 6a-k.
Table 3.
PDE4 recombinant isoform inhibition (IC50, μM) for sulfonamide 6a (LASSBio-448) and rolipram.
Table 4.
Comparative ADME properties of rolipram (1) and LASSBio-448 predicted in silico using the Program ACD/Percepta 14.0.
Fig 4.
Effect of LASSBio-448 on ovalbumin (OVA)-induced infiltration of eosinophils in the BAL fluid (A) and lung tissue (B) from A/J mice. Animals were sensitized on days 0 and 7 and then challenged with OVA (25 μg/mouse) or saline on days 19 and 20. Treatment with LASSBio-448 (100 mg/Kg, oral), was given 1 h before each OVA challenge, and analyses were performed 24 h after the last stimulation. Values represent mean ± SEM from at least 3 animals. +P<0.05 as compared to saline-challenged group; *P<0.05 as compared to OVA-challenged group.
Fig 5.
Effect of rolipram and LASSBio-448 on ovalbumin (OVA)-induced infiltration of eosinophils in the lung tissue from A/J mice.
Animals were sensitized on days 0 and 7 and then challenged with OVA (25 μg/mouse) or saline on days 14, 21, 28 and 35. Animals were treated with rolipram (10 mg/Kg, oral) or LASSBio-448 (100 mg/Kg, oral) on days 26 and 22, 1 h before OVA challenge, and analyses performed 24 h after the last challenge. Values represent mean ± SEM from at least 7 animals. + P<0.05 as compared to saline-challenged group; *P<0.05 as compared to OVA-challenged group.
Fig 6.
Effect of rolipram and LASSBio-448 on ovalbumin (OVA)-induced mucus production (upper panels) and subepithelial fibrosis (lower panels) in the lungs from A/J mice. Animals were sensitized on days 0 and 7 and then challenged with OVA (25 μg/mouse) or saline on days 14, 21, 28 and 35. Animals were treated with rolipram (10 mg/Kg, oral) or LASSBio-448 (100 mg/Kg, oral) on days 26 and 22, 1 h before OVA challenge, and analyses performed 24 h after the last challenge. The analyses were performed in sensitized mice challenged with saline (A, D), OVA (B, E) and OVA treated with LASSBIo-448 (C, F). Morphometric analyses are seen in (G) mucus production and (H) subepithelial fibrosis. Slides were stained periodic acid-Schiff (upper panels) and Gomori trichrome (lower panels). Values represent mean ± SEM from at least 7 animals. + P<0.05 as compared to saline-challenged group; *P<0.05 as compared to OVA-challenged group.
Fig 7.
Effect of rolipram and LASSBio-448 on ovalbumin (OVA)-induced changes in lungs from A/J mice.
Airway responsiveness was measured by changes in airway resistance (A) and elastance (B) induced by increasing concentrations of methacholine, 24 h after the last ovalbumin or saline challenge. Animals were sensitized on days 0 and 7 and then challenged with OVA (25 μg/mouse) or saline on days 14, 21, 28 and 35. Animals were treated with rolipram (10 mg/Kg, oral) or LASSBio-448 (100 mg/Kg, oral) on days 26 and 22, 1 h before OVA challenge, and analyses performed 24 h after the last challenge. Values represent mean ± SEM from at least 7 animals. + P<0.05 as compared to saline-challenged group; *P<0.05 as compared to OVA-challenged group.
Fig 8.
Effect of rolipram and LASSBio-448 on ovalbumin (OVA)-induced cytokine production in the lungs of A/J mice.
IL-13 (A), IL-4 (B), IL-5 (C) and eotaxin-2 (D). Animals were sensitized on days 0 and 7 and then challenged with OVA (25 μg/mouse) or saline on days 14, 21, 28 and 35. Animals were treated with rolipram (10 mg/Kg, oral) or LASSBio-448 (100 mg/Kg, oral) on days 26 and 22, 1 h before OVA challenge, and analyses performed 24 h after the last challenge. Values represent mean ± SEM from at least 7 animals. + P<0.05 as compared to saline-challenged group; *P<0.05 as compared to OVA-challenged group.
Fig 9.
Effect of LASSBio-448 and cilomilast on lung pathological changes caused by LPS in mice.
LASSBio-448 (2.5 and 10 mg/kg, oral) or cilomilast (1 mg/kg, oral) were given 1 h before challenge (LPS, 25 μg/25 μL), and analyses on airway hyper-reactivity (A) and neutrophil infiltration, attested by MPO activity of lung tissue samples (B), were carried out 24 h post challenge. Values represent mean ± SEM from at least 7 animals. + P<0.05 as compared to vehicle-stimulated group; *P<0.05 as compared to LPS-stimulated mice.
Fig 10.
Reduction by LASSBio-448, rolipram or cilomilast in duration of the ketamine/xylazine anesthesia (%).
Mice of the strain A/J were injected with ketamine/ xylazine solution and then treated orally with LASSBio-448 (3, 10 and 30 mg/kg), rolipram (1, 3 and 10 mg/kg) or cilomilast (1 mg/kg). Values represent mean ± SEM from at least 7 animals. Figures in brackets shown in the top of each column are correspondent doses expressed in μmol/kg. *P<0.05 as compared to vehicle-treated group.
Fig 11.
Top poses of LASSBio-448 (orange carbon atoms) with PDE4A (A), PDE4B (B), PDE4C (C) and PDE4D (D) obtained with GOLD 5.2 software. Hydrogen atoms have been omitted for clarity. Hydrogen bonds are in dashed lines. PDE4D numbering has been used.
Fig 12.
Superimposition of the top poses of LASSBio-448 obtained by docking with PDE4A and PDE4C (light purple surface, PDE4A) (A), PDE4B and PDE4D (gold surface, PDE4D) (B). Hydrogen atoms have been omitted for clarity.