ABSTRACT
The synthesis of a series of imidazole styrylindoles and sulfonyl styrylindoles derivatives is described. Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imidazole styrylindoles as potent inhibitors with activity greater or comparable with the standard ketoconazole. Flexible alignment and docking studies of the inhibitors in the CYP24A1 enzyme active site confirmed that complete occupation of the vitamin D access tunnel is essential to inhibitory activity, allowing exposure to multiple hydrophobic binding interactions and optimal conformation for the interaction of the imidazole nitrogen lone pair and the active site haem.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Indoles/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology , Styrene/chemistry , Vitamin D3 24-Hydroxylase/antagonists & inhibitors , Catalytic Domain , Humans , Models, Molecular , Molecular Structure , Protein Conformation , Structure-Activity RelationshipABSTRACT
The 20R- and 20S-isomers of 25-hydroxy-2-methylene-vitamin D3 and 3-desoxy-1α,25-dihydroxy-2-methylene-vitamin D3 have been synthesized. Two alternative synthetic routes were devised for preparation of the required A-ring synthons, starting from the chiral compound derived from the (-)-quinic acid and, alternatively, from the commercially available achiral precursor, monoprotected 1,4-cyclohexanedione. The A-ring dienynes were coupled by the Sonogashira process with the respective C,D-ring fragments, the enol triflates derived from the protected (20R)- or (20S)-25-hydroxy Grundmann ketones. All four compounds possessed significant in vivo activity on bone calcium mobilization and intestinal calcium transport. The presence of a 2-methylene group increased intestinal calcium transport activity of all four analogues above that of the native hormone, 1α,25-dihydroxyvitamin D3. In contrast, bone calcium mobilization was equal to that produced by 1α,25-dihydroxyvitamin D3 in compounds having a (20S)-configuration or diminished to one-tenth that of 1α,25-dihydroxyvitamin D3 in compounds with a (20R)-configuration.
Subject(s)
Calcifediol/chemistry , Calcium/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Animals , Bone and Bones/drug effects , Bone and Bones/metabolism , Calcifediol/pharmacology , Calcitriol/analogs & derivatives , Calcitriol/chemistry , Chemistry Techniques, Synthetic , Drug Evaluation, Preclinical/methods , HL-60 Cells/drug effects , Humans , Intestinal Mucosa/metabolism , Intestines/drug effects , Male , Molecular Structure , Rats, Sprague-Dawley , Receptors, Calcitriol/metabolism , Structure-Activity RelationshipABSTRACT
The synthesis of imidazole styrylbenzamide, tert-butyl styrylimidazole, and tert-butyl styrylsulfonate derivatives is described. Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imidazole styrylbenzamides as potent inhibitors of CYP24A1, having selectivity with respect to CYP27B1 comparable with or greater than that of the standard ketoconazole. Further evaluation of the 3,5-dimethoxy and 3,4,5-trimethoxy derivatives in chronic lymphocytic leukemia cells revealed that co-treatment of 1α,25-dihydroxyvitamin D3 plus inhibitor coordinately upregulated GADD45α and CDKN1A. Docking experiments on the inhibitors in the CYP24A1 enzyme active site suggest the compounds reach the active site through the vitamin D access tunnel and are exposed to multiple hydrophobic residues. The imidazole styrylbenzamides are optimally positioned to allow interaction of the imidazole with the heme, and, in the case of the methoxy derivatives, a hydrogen bond between the 3-methoxy group and Gln82 stabilizes the molecule in a favorable active conformation.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cytochrome P-450 Enzyme Inhibitors/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Vitamin D3 24-Hydroxylase/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Catalytic Domain , Cell Line, Tumor , Cell Proliferation/drug effects , Chemistry Techniques, Synthetic , Cytochrome P-450 Enzyme Inhibitors/chemistry , Cytochrome P-450 Enzyme Inhibitors/metabolism , Heme/metabolism , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/pharmacology , Inhibitory Concentration 50 , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Models, Molecular , Sequence Homology, Amino Acid , Structure-Activity Relationship , Vitamin D3 24-Hydroxylase/chemistryABSTRACT
Bioactivation of vitamin D consists of two sequential hydroxylation steps to produce 1α,25-dihydroxyvitamin D3. It is clear that the second or 1α-hydroxylation step is carried out by a single enzyme, 25-hydroxyvitamin D 1α-hydroxylase CYP27B1. However, it is not certain what enzyme or enzymes are responsible for the initial 25-hydroxylation. An excellent case has been made for vitamin D 25-hydroxylase CYP2R1, but this hypothesis has not yet been tested. We have now produced Cyp2r1 (-/-) mice. These mice had greater than 50% reduction in serum 25-hydroxyvitamin D3. Curiously, the 1α,25-dihydroxyvitamin D3 level in the serum remained unchanged. These mice presented no health issues. A double knockout of Cyp2r1 and Cyp27a1 maintained a similar circulating level of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Our results support the idea that the CYP2R1 is the major enzyme responsible for 25-hydroxylation of vitamin D, but clearly a second, as-yet unknown, enzyme is another contributor to this important step in vitamin D activation.
