Pyridine-containing substrate analogs are restricted from accessing the human cytochrome P450 8B1 active site by tryptophan 281.

The human oxysterol 12α-hydroxylase cytochrome P450 8B1 (CYP8B1) is a validated drug target for both type 2 diabetes and nonalcoholic fatty liver disease, but effective selective inhibitors are not yet available. Herein, steroidal substrate-mimicking compounds with a pyridine ring appended to the C12 site of metabolism were designed as inhibitors, synthesized, and evaluated in terms of their functional and structural interactions with CYP8B1. While the pyridine nitrogen was intended to coordinate the CYP8B1 active site heme iron, none of these compounds elicited shifts in the CYP8B1 Soret absorbance consistent with this type of interaction. However, when CYP8B1 was cocrystallized with the pyridine-containing compound with the 3-keto-Δ4 steroid backbone most similar to the endogenous substrate, it was apparent that this ligand was bound in a channel leading to the active site, instead of near the heme iron. Inspection of this structure suggested that tryptophan 281 directly above the heme might restrict active site binding of potential inhibitors with this design. This hypothesis was supported when a CYP8B1 W281F mutation did allow all three compounds to coordinate the heme iron as designed. These results indicated that the design of next-generation CYP8B1 inhibitors should be compatible with the low-ceiling tryptophan immediately above the heme iron.

Synthesis of menarandroside A from dehydroepiandrosterone.

Menarandroside A, which bears a 12α-hydroxypregnenolone steroid backbone, was isolated from the plant, Cynanchum menarandrense. Treatment of extracts from this plant containing menarandroside A against secretin tumor cell line (STC-1) intestinal cells, resulted in an increased secretion of glucagon-like peptide 1 (GLP-1), a peptide that plays a role in the regulation of blood sugar levels. Increase in GLP-1 is beneficial for the treatment of type 2 diabetes. We disclose the synthesis of menarandroside A from dehydroepiandrosterone (DHEA). Key features of this synthesis include: (i) Wittig reaction of the C17-ketone of a 12-oxygenated DHEA derivative to introduce the C17-acetyl moiety, and (ii) the stereoselective reduction of a C12-keto intermediate bearing an sp2-center at C17 to yield the C12α-hydroxy group. In addition, an oxidation of a methyl enol ether derivative to an α-hydroxy methyl ester using tetrapropylammonium perruthenate (TPAP) and N-methyl-morpholine-N-oxide (NMO) was discovered.

Copper oxidation chemistry using a 19-iminopyridine-bearing steroidal ligand: (i) C5-C6 olefin difunctionalization and (ii) C1ß-hydroxylation/C19-peroxidation.

The Schönecker oxidation involves the 12beta-hydroxylation of 17-imino pyridine DHEA derivatives using copper and either molecular oxygen or hydrogen peroxide as the oxidant. In this study, a 19-imino pyridine DHEA derivative was synthesized and was treated with copper nitrate and hydrogen peroxide. Our results showed the difunctionalization of an olefin for delta-5 steroid substrates to yield a 5beta-hydroxylated 6alpha-nitrate ester product. In contrast, for 19-imino pyridine precursors with a 5alpha-androstane steroid backbone: a 1beta-hydroxylation and 19-peroxidation occurred to yield a 1beta-hydroxylated 19-imidoperoxoic acid product. In conclusion, new Schönecker oxidation chemistry was discovered (C5-C6 olefin difunctionalization and C1beta-hydroxylation/C19-peroxidation) when a 19-imino pyridine DHEA derivative was used as the substrate.

A synthesis of a rationally designed inhibitor of cytochrome P450 8B1, a therapeutic target to treat obesity.

Mice that lack the gene for expression of cytochrome P450 8B1 (P450 8B1) resist weight gain and improve glucose tolerance when fed a high-fat diet. Thus, the inhibition of P450 8B1 is a target to treat obesity-associated metabolic disorders. P450 8B1 is the enzyme that hydroxylates its substrate, 7α-hydroxy-cholest-4-en-3-one to 7α-,12α-dihydroxycholest-4-en-3-one, which ultimately results in the formation of cholic acid. Cholic acid is the 12α-hydroxylated bile acid implicated in enhanced absorption of cholesterol. The synthesis of a rationally designed inhibitor for P450 8B1 was achieved through the incorporation of a C12-pyridine in the C-ring of a steroid molecule. Seven days of new inhibitor treatment showed attenuation of glucose intolerance in mice that were fed a high fat and a high sucrose diet (HFHS) without affecting body weight. Taken together, these promising results will lead to a P450 8B1 inhibitor as a potential therapeutic strategy to treat obesity-associated insulin resistance.

Chemical synthesis of 7α-hydroxycholest-4-en-3-one, a biomarker for irritable bowel syndrome and bile acid malabsorption.

7α-Hydroxy-cholest-4-en-3-one is a biomarker for bile acid loss, irritable bowel syndrome, and other diseases associated with defective bile acid biosynthesis. Furthermore, 7α-hydroxy-cholest-4-en-3-one is the physiological substrate for cytochrome P450 8B1 (P450 8B1 or CYP8B1), the oxysterol 12α-hydroxylase enzyme implicated in obesity and cardiovascular health. We report the chemical synthesis of this physiologically important oxysterol beginning with cholesterol. The key feature of this synthesis involves a regioselective C3-allylic oxidation of a 3-desoxy-Δ4-7α-formate steroid precursor to form 7α-formyloxy-cholest-4-en-3-one, which was saponified to yield 7α-hydroxy-cholest-4-en-3-one.

Chemical synthesis of 7-oxygenated 12α-hydroxy steroid derivatives to enable the biochemical characterization of cytochrome P450 8B1, the oxysterol 12α-hydroxylase enzyme implicated in cardiovascular health and obesity.

Cholic acid is the endogenous 12α-hydroxylated bile acid, which possesses enhanced cholesterol absorption properties compared to its 12-desoxy counterpart, chenodeoxycholic acid. The oxysterol 12α-hydroxylase enzyme is cytochrome P450 8B1 (P450 8B1), which regioselectively and stereoselectively incorporates the 12α-hydroxy group in 7α-hydroxycholest-4-en-3-one, the biosynthetic precursor of cholic acid. Despite the vital role of P450 8B1 activity in cardiovascular health, research studies of other 12α-hydroxy steroid derivatives are rare. A synthetic route to incorporate a C12α-hydroxy group into the C12-methylene (-CH2-) in dehydroepiandrosterone derivatives is disclosed. The incorporation of the C12-oxygen was accomplished through a copper mediated Schönecker oxidation of an imino-pyridine intermediate, introducing the 12ß-hydroxy group. The resulting 12ß-hydroxy steroid derivative was oxidized to the C12-ketone, which was stereoselectively reduced with lithium tri-sec-butylborohydride to afford the 12α-hydroxy stereochemistry. The C7-position was oxidized to yield the various 7-keto, 7ß-hydroxy, and 7α-hydroxy derivatives. Furthermore, 7-ketodehydroepiandrosterone and 12 α-hydroxy-7-ketodehydroepiandrosterone both displayed NMDA receptor antagonistic activities at 10 µM concentrations. These C12α-hydroxy steroids will be used as tools to identify new biochemical properties of the enzymatic products of P450 8B1, the oxysterol 12α-hydroxylase.