Selective inhibition of mammalian lanosterol 14 alpha-demethylase by RS-21607 in vitro and in vivo.

The discovery of selective lanosterol 14 alpha-demethylase inhibitors may lead to novel hypolipidemic drugs. RS-21607, (2S,4S)-cis-2[1H-imidazol-1-yl)methyl]-2-[2-(4-chlorophenyl)ethyl]-4- [[(4-aminophenyl)thio]methyl]-1,3-dioxolane, was characterized as a tight-binding, competitive inhibitor of lanosterol 14 alpha-demethylase purified from rat liver. The apparent Ki was determined to be 840 pM and found to be similar in hepatic microsomes from human, rat, and hamster. RS-21607, which contains two chiral centers, was a more effective lanosterol 14 alpha-demethylase inhibitor than its three stereoisomers. In vitro, RS-21607 had a greater affinity for lanosterol 14 alpha-demethylase than the other cytochromes P450 evaluated: CYP7, CYP27, CYP11A1, CYP19, CYP17, CYP11B1, CYP21, CYP3A4, CYP4A, CYP2D6, CYP1A2, CYP2C9, and 27-hydroxycholesterol 7 alpha-hydroxylase. The other stereoisomers were not as selective as RS-21607. Doses of 3-30 mg/kg RS-21607 given orally to hamsters caused a dose-dependent decrease in cholesterol biosynthesis with a corresponding accumulation of 24,25-dihydrolanosterol. RS-21607 inhibited the enzyme and cholesterol biosynthesis in hamster liver by 50% at 18 h following a 30 mg/kg oral dose. This was interpreted to indicate that RS-21607 is able to distribute to the site of action in hamsters and inhibit the target enzyme. In the same dose range, the plasma concentrations of testosterone, corticosterone, and progesterone, the endpoints for the cytochromes P450 involved in steroid biosynthesis, were relatively unaffected. These data show RS-21607 to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro. RS-21607 interacted with the purified enzyme to produce a type II binding spectrum, consistent with an interaction between the imidazole moiety and the heme. The electrostatic contribution of the imidazole binding was investigated using the desimidazole analog of RS-21607. The apparent Ki for the desimidazole compound (65 microM) was similar to the apparent Km for the substrate DHL (79 microM). Together, these data confirm that the ligand attached to the imidazole in RS-21607 is a good non-sterol substitute for DHL, i.e., binding to the enzyme with similar affinity, and that the coordination of the imidazole to the heme provides a major electrostatic contribution for the inhibition of lanosterol 14 alpha-demethylase by RS-21607. RS-21607 was also observed to increase the accumulation of 3 beta-hydroxy-24,25-dihydrolanost-8-en-32-al, the second intermediate in the multistep oxidation, but not the first intermediate. 24,25-dihydrolanost-8-ene-3 beta,32-diol.(ABSTRACT TRUNCATED AT 400 WORDS)

Stereoisomers of ketoconazole: preparation and biological activity.

The four stereoisomers of the antifungal agent ketoconazole (1) were prepared and evaluated for their selectivity in inhibiting a number of cytochrome P-450 enzymes. Large differences in selectivity among the isomers were observed for inhibition of the cytochromes P-450 involved in steroid biosynthesis, whereas little differences was observed for inhibition of those associated with hepatic drug metabolism. The cis-(2S,4R) isomer 2 was the most effective against rat lanosterol 14 alpha-demethylase, (2S,4R)-2 greater than (2R,4S)-4 much greater than (2R,4R)-3 = (2S,4S)-5, and progesterone 17 alpha,20-lyase, (2S,4R)-2 much greater than (2S,4S)-5 greater than (2R,4R)-3 = (2R,4S)-4, whereas the cis-(2R,4S) isomer 4 was more effective against cholesterol 7 alpha-hydroxylase, (2R,4S)-4 greater than (2S,4S)-5 greater than (2R,4R)-3, greater than (2S,4R)-2, and the trans-(2S,4S) isomer 5 was the most effective against aromatase, (2S,4R)-5 much greater than (2R,4R)-3 = (2R,4S)-4 greater than (2S,4R)-2. The cis-(2S,4R) and trans-(2R,4R) isomers 2 and 3 are equipotent in inhibiting corticoid 11 beta-hydroxylase and much more effective than their antipodes. Little selectivity was observed for inhibition of cholesterol side chain cleavage or xenobiotic hydroxylase. These data indicate that the affinity of azoles for cytochrome P-450 enzymes involved in steroid synthesis is highly dependent on the stereochemistry of the entire molecule, whereas binding to drug metabolizing enzymes is a less selective process.

Intrinsic potencies of novel thiol ester corticosteroids RS-85095 and RS-21314 as compared with clobetasol 17-propionate and fluocinonide.

The intrinsic potencies of two novel topical thiol ester corticosteroids, RS-85095 and RS-21314, were compared with the high potency corticosteroids clobetasol 17-propionate and fluocinonide, using human vasoconstriction assays. In these assays, four or five concentrations (0.03 to 3 mg/L) of the corticosteroids in 95% ethanol (alcohol, USP) were applied to predetermined sites on the forearm of volunteers and were occluded following evaporation of the alcohol. The responses were scored in terms of the presence or absence of vasoconstriction and the degree of vasoconstrictive intensity. No statistically significant difference was found in the intrinsic potencies of RS-85095, RS-21314, and clobetasol 17-propionate, and all three corticosteroids were significantly more potent than fluocinonide.

Platelet aggregation inhibitory activity and vasodepressor activity of a series of bicyclo[3.2.0]hept-6-ylidene iminoxy alkanoic acids with modified lower side chains.

Prostacyclin analogues derived from modification of the lower side chain of the bicyclo[3.2.0]hept-6-ylidene iminoxyacetic acid (1) were studied in inhibition of in vitro and ex vivo platelet aggregation and in the spontaneously hypertensive rat. Iminoxyacetic acids (13a), (13b), (13c) and iminoxypropionic acid (14b) were 2.9, 3.0, 1.9 and 2.0 times respectively more potent than PGE1 in inhibiting ADP-induced aggregation of human platelets in vitro. Following intravenous administration at a dose of 90-110 micrograms/kg in the guinea pig, iminoxyacetic acids (13a), (13c) and iminoxypropionic acid (14b) showed a maximum inhibition of 82-92% with a half life in the range of 14-22 min. Following oral administration at a dose of 1 mg/kg in the guinea pig, iminoxyacetic acids (13a) and (13b) inhibited heterologous platelet aggregation for 4.5 h. Following intravenous administration in spontaneously hypertensive rats, acids (13a)-(13c) and (14b) lowered the mean blood pressure in a dose dependent manner. At a dose of 100 micrograms/kg, the effect lasted for 20-40 min.

Synthesis and platelet aggregation inhibition activity of a series of enantiomeric bicyclo[3.2.0]heptane-6-oximinoacetic acids (1).

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.