Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Chemistry and Properties of Indolocarbazoles.

The indolocarbazoles are an important class of nitrogen heterocycles which has evolved significantly in recent years, with numerous studies focusing on their diverse biological effects, or targeting new materials with potential applications in organic electronics. This review aims at providing a broad survey of the chemistry and properties of indolocarbazoles from an interdisciplinary point of view, with particular emphasis on practical synthetic aspects, as well as certain topics which have not been previously accounted for in detail, such as the occurrence, formation, biological activities, and metabolism of indolo[3,2- b]carbazoles. The literature of the past decade forms the basis of the text, which is further supplemented with older key references.

Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS.

The aim of this work was to synthesize morphine-3-O-sulfate and morphine-6-O-sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC-MS/MS method. Morphine-6-O-sulfate and morphine-3-O-sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed-phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5-500 ng/mL for morphine-3-O-sulfate and 4.5-454 ng/mL for morphine-6-O-sulfate in plasma. In urine, the measuring range was 50-5000 ng/mL for morphine-3-O-sulfate and 45.4-4544 ng/mL for morphine-6-O-sulfate. The intra-assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine-3-O-sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine-6-O-sulfate, while no detectable morphine-6-O-sulfate was found in plasma samples.

Tricyclic compounds containing nonenolizable cyano enones. A novel class of highly potent anti-inflammatory and cytoprotective agents.

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.

Synthesis and biological evaluation of fused thio- and selenopyrans as new indolocarbazole analogues with aryl hydrocarbon receptor affinity.

A series of thio- and selenopyrans having two fused indole units, structurally related to indolocarbazoles, have been prepared and evaluated for aryl hydrocarbon receptor (AhR) affinity, leading to the identification of several new significant AhR ligands. In particular, the parent thiopyrano[2,3-b:6,5-b']diindole and its derivative having a methyl group in the central ring, as well as the two corresponding selenopyrans, displayed the highest potencies of the compounds tested.

Synthetic studies of cephalandole alkaloids and the revised structure of cephalandole A.

A synthesis of the originally proposed 2-(1 H-indol-3-yl)-4 H-3,1-benzoxazin-4-one structure of the alkaloid cephalandole A (1) led to a structural revision, and the isolated natural product has now been identified as the previously known compound 3-(1 H-indol-3-yl)-2 H-1,4-benzoxazin-2-one (7). The structural assignment was corroborated by detailed NMR studies. A short synthesis of the related natural compound cephalandole B (2) has also been performed, confirming its structure. In addition some chemical transformations, involving, for example, the related synthetic molecule 2-(1 H-indol-3-yl)-3 H-quinazolin-4-one (9), are presented.

A new concise strategy for synthesis of dibenzo[b,f]thiepins and related fused symmetrical thiepin derivatives.

A new strategy for preparation of dibenzo[b,f]thiepins and related fused systems in good overall yields is described, featuring ortho-metalation of aromatic or heterocyclic aldehyde acetals followed by treatment with bis(phenylsulfonyl) sulfide for construction of the required bis(aryl)- or bis(heteroaryl) sulfide precursors, which were thereafter subjected to deacetalization, and finally McMurry coupling as the ring-forming step.

Synthetic applications of cyanoacetylated bisindoles: synthesis of novel cycloheptadiindoles, indolocarbazoles, and related aza analogues.

Cyclization reactions involving cyanoacetylated bisindoles have been studied, providing access to various novel cyclohepta[2,1-b:3,4-b']diindole derivatives as well as some related fused pentacyclic systems. Treatment of 3-cyanoacetyl-2,3'-diindolylmethane with methanesulfonic acid gave 6-(cyanomethyl)indolo[3,2-b]carbazole in a good yield.

Effects of analogs of indole-3-carbinol cyclic trimerization product in human breast cancer cells.

5,6,11,12,17,18-Hexahydrocyclonona[1,2-b:4,5-b*:7,8-b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERalpha), and activates expression of estrogen (E(2))-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12H-[1,2,5]trithionino[3,4-b:6,7-b*:9,8-b**]triindole (S(3)CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2-b:4,5-b*:7,8-b**]triindole (Me(3)CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me(3)CTr exhibited no detectable effect on cell growth, ERalpha binding to E(2), or ERalpha-responsive gene expression. S(3)CTr showed mixed ERalpha agonist activities. It bound to the ERalpha and activated receptor binding with DNA, weakly activated expression of transfected E(2)-responsive reporter gene constructs, and strongly inhibited the E(2)-induced activation of these reporter constructs. S(3)CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S(3)CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERalpha.

Design, synthesis, and biological evaluation of biotin conjugates of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid for the isolation of the protein targets.

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 1) and related compounds [for example, CDDO-Me (2) and CDDO-Im (3)] are potential anti-inflammatory, cancer chemopreventive, and chemotherapeutic agents. However, the mechanisms responsible for the multiple effects of CDDO are still unclear. Clarification of these mechanisms and particularly isolation of the protein targets are essential for the development of CDDO and its analogues as clinically useful drugs. Such knowledge would provide superior opportunities for designing new compounds with improved potency and selectivity. Therefore, to isolate protein targets using affinity chromatography with immobilized streptavidin as a carrier, we have designed and synthesized C-17 and C-23 biotin conjugates of CDDO (4, 5, and 6) on the basis of our established structure-activity relationships. For the synthesis of 6, a new important precursor, 23-hydroxy-CDDO-Me (29) was synthesized from 20 by a C-23 oxidation protocol, which involves cyclopalladation of the C-4 methyl group from a 3-one oxime. The inhibitory activity of C-23 conjugate 6 is only about 3 times less potent than the mother compound, CDDO, against the proliferation of MCF-7 breast cancer cells. Consequently, 6 may be a very promising tool for the isolation of the protein targets of CDDO.

Efficient synthesis of (-)- and (+)-tricyclic compounds with enone functionalities in rings A and C. A novel class of orally active anti-inflammatory and cancer chemopreventive agents.

Novel tricyclic compounds with enone functionalities in rings A and C [tricyclic-bis-enone (TBE) compounds] were designed on the basis of the structure of a synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)(1), which is a promising drug candidate for prevention and/or treatment of cancer and inflammatory diseases whose pathogenesis may involve excessive production of nitric oxide (NO) and/or prostaglandins. A series of TBE compounds in racemic form shows high inhibitory activity against production of NO induced by interferon-[gamma](IFN-[gamma]) in mouse macrophages. One of these compounds, (+/-)-(4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3,7-dicarbonitrile ((+/-)-3), is orally active at 15 mg kg(-1)(single administration) in a preliminary study using mouse peritoneal inflammation induced by thioglycollate and IFN-[gamma]. Therefore, we desired to synthesize optically active TBE compounds for a comparison of the biological potency of both enantiomers. We now describe the synthesis of both enantiomers of (4a[small beta],8a[small beta],10a[small alpha])-1,2,4a,6,8a,9,10,10a-octahydro-1,1,4a,8a-tetramethyl-2,6-dioxophenanthrene-3-carbonitrile (2) and 3 from commercially available simple compounds. Interestingly, (+)-3 having the same configuration as the CDDO antipode shows about 10 times higher inhibitory activity than (-)-3 on NO production in mouse macrophages. In contrast, (-)-3 inhibits proliferation of MCF-7 breast cancer cells, whereas (+)-3 does not.

Sulfur-rich heterocycles from 2-metalated benzo[b]thiophene and benzo[b]furan: synthesis and structure.

The reaction of 2-lithiated benzo[b]thiophene with 8 equiv of elemental sulfur was found to give pentathiepino[6,7-b]benzo[d]thiophene. In contrast, treatment of 2-lithiated benzo[b]furan with sulfur under similar conditions produced the interesting ring system bis(benzo[4,5]furo)[2,3-e:3',2'-g][1,2,3,4]tetrathiocine. Both of these new cyclic polysulfides were studied by X-ray crystallography. Two polymorphic forms of pentathiepino[6,7-b]benzo[d]thiophene were found, displaying similar conformations but different packing schemes, which was also evident from powder diffraction data.