Table 2.
Summary of biophysical and biochemical properties of 3-indolyl-methanamines.
| Compound | Solubilit y (μM)a | Permeabilityb Log(Pe) (cm/s) | VDR–SRC2-3 Inhibition IC50 (μM)c | Rate constant k for the dissociation of SRC2-3 from VDR (10−5)d | Transcription Inhibition IC50 (μM)e | Toxicity LC50 (μM)f |
|---|---|---|---|---|---|---|
| 30a | 252.9 | −6.00 | 30.2±4.8 | 45.1 ± 1.6 | 10.9±2.8 | 15.3±2.9 |
| 30b | 93.9 | −6.03 | 31.7±4.3 | 34.2 ± 1.0 | 8.1±1.7 | 14.6±3.4 |
| 30c | 237.5 | −6.10 | 31.2±3.2 | 88.0 ± 4.7 | 12.1±1.8 | 17.2±2.5 |
| 30d | 175.1 | −6.34 | 43.6±7.8 | 35.7 ± 1.4 | 14.6±2.5 | 21.7±4.2 |
| 30e | 157.7 | −6.88 | n.s. | 2.0 ± 0.16 | 13.5±1.3 | 25.8±6.3 |
| 30f | 117.6 | −6.08 | 28.5±4.5 | 6.7 ± 0.12 | 12.2±2.4 | 16.2±2.5 |
| 30g | 189.0 | −6.40 | 104.8±15.2 | 1.8 ± 0.15 | 20.1±5.2 | 37.4±7.7 |
| 30h | 67.3 | −6.30 | 29.6±3.1 | 14.2 ± 0.28 | 15.0±2.4 | 20.8±3.5 |
| 30i | 503.4 | −6.83 | 58.6±8.1 | 2.3 ± 0.21 | 13.1±2.5 | 31.4±8.2 |
| 31a | 31.6 | −6.41 | 29.8±4.5 | 38.6 ± 0.89 | 8.5±1.8 | 12.6±2.2 |
| 31b | 68.0 | −6.24 | 36.7±5.1 | 4.5 ± 0.59 | 4.2±1.9 | 11.6±1.7 |
| 31c | 84.2 | −6.30 | 26.5±3.4 | 39.1 ± 1.3 | 5.8±2.1 | 12.1±2.2 |
| 31d | 35.9 | −6.45 | 17.7±3.2 | 23.7 ± 0.35 | 8.2±1.5 | 12.7±3.0 |
| 31e | 21.0 | −7.28 | n.s. | 0.87 ± 0.13 | 8.7±1.5 | 19.4±3.8 |
| 31f | 4.9 | −7.60 | n.s. | 1.2 ± 0.16 | 4.4±1.1 | 11.0±2.1 |
| 31g | n.d. | n.d. | n.s. | 2.5 ± 0.07 | 24.0±5.7 | 43.1±9.1 |
| 31 h | 59.7 | −6.22 | 24.3±4.4 | 25.2 ± 0.36 | 8.6±1.1 | 12.1±2.7 |
| 32a | 15.3 | −6.58 | n.o. | n.o. | >70 | >70 |
| 32b | 52.3 | −6.51 | 20.3±4.5 (partial) | 1.4 ± 0.12 | 21.6±3.3 | >70 |
| 32c | 11.8 | −6.89 | n.o. | n.o. | >70 | >70 |
Solubilities were determined in phosphate-buffered saline at pH 7.4;
Permeabilities were measured using the parallel artificial membrane permeation assay (PAMPA) at neutral pH (pH = 7.4). The following permeability standards were used (logPe): Ranitidine (−8.02 ± 0.074 cm/s) low permeability, Carbamazepine (−6.81 ± 0.0011 cm/s) medium permeability, and Verapamil (−5.93 ± 0.015 cm/s) high permeability. The solubility and permeability assay conditions reflect conditions required for activity in cell-based assays;
A fluorescence polarization competition assay was carried out using VDR-LBD (1μM) , Alexa Fluor-labeled peptide SRC2-3 (7 nM), VDR-agonist LG190178 (5 μM), and serial diluted small molecules. IC50 values were obtained by fitting data obtained after 3 hours to the following equation: Y=Bottom + (Top- Bottom)/(1+10^((LogIC50-X)*HillSlope)) using three independent experiments in quadruplet;
A fluorescence polarization assay described under c was monitored over time. Dissociation rate constants were obtained by linear fitting of ln(mP) (fluorescence polarization) against time (first order kinetics);
HEK293T cells were transfected with CMV-VDR, a CYP24A1 promoter driven luciferase expression vector, and a Renilla luciferase control vector. The data was normalized to Renilla luciferase activity and by signals observed for inactive and activated VDR (±1,25(OH)2D3);
Toxicity was determined based on signal observed for Renilla luciferase activity and normalized to signal observed for living and dead cells (± 100 μM of 3-dibutylamino-1-(4-hexyl-phenyl)-propan-1-one, CBT358); n.d. = not determined; n.s. = no saturation of signal at higher small molecule concentration (no reliable non-linear fitting possible); n.o. = not observed.