Synthesis and Evaluation of Azole-substituted 2-Aryl-6-methoxy-3,4-dihydronaphthalenes and -naphthalenes as Inhibitors of 17α-Hydroxylase-C17,20-Lyase (P450 17

The synthesis and biological evaluation of azole-substituted 3,4-dihydronaphthalenes ( 7a, 7b, 14a, 14b ) and naphthalenes ( 12a, 12b, 16a, 16b ) as nonsteroidal inhibitors of 17α-hydroxylase-C17,20-lyase (P450 17, CYP 17) are described. In the case of the dihydronaphthalenes, introduction of the phenyl substituent into the 2-position was accomplished by coupling 2-hydroxy-3,4-dihydronaphthalene-2-trifluoromethanesulfonate 1 with the corresponding aryl-Zn-bromides 4a and 4b in the presence of Pd(PPh₃)₄ as catalyst yielding 5a and 5b as key intermediates. In the case of the naphthalenes, 2-bromonaphthalene 9 was reacted with the corresponding Grignard reagents yielding 10a and 10b . After transformation of the intermediate acetals 5a, 5b, 10a, 10b into the corresponding aldehydes, the latter compounds were reacted with tosylmethyl isocyanide and K₂CO₃ to give the oxazoles 7a, 7b, 12a , and 12b . The imidazoles 14a, 14b, 16a , and 16b were prepared by heating the corresponding 4-tosyloxazolines in ammonia, which were prepared by reacting the aldehydes with tosylmethyl isocyanide and NaCN. Using a microsomal fraction of human testicular enzyme, the title compounds did not inhibit the target enzyme.     

Synthesis of Novel Oximes of 2-Aryl-6-methoxy-3,4-dihydronaphthalene and Their Evaluation as Inhibitors of 17α-Hydroxylase-C17,20-Lyase (P450 17)

The synthesis and biological evaluation of oximes of 2-aryl-6-methoxy-3,4-dihydronaphthalene ( 7a, 7b, 14a, 14b ) as nonsteroidal inhibitors of 17α-hydroxylase-C17,20-lyase (P450 17, CYP 17) is described. The target compounds were synthesized and identified by ¹H NMR and MS. The preparation of the key intermediates 5a and 5b was accomplished by coupling 4a and 4b with 1 (2-hydroxy-6-methoxy-3,4-dihydronaphthalene-2-trifluoromethanesulfonate) using the palladium complex Pd(PPh₃)₄ as catalyst. Hydrolysis of 5a and 5b in THF-HCl solution at room temperature gave the corresponding keto compounds 6a and 6b . The other important intermediates – the substituted (E)-2-methylene-1-tetralones 10a and 10b – were obtained by condensation of 1-tetralone with the corresponding aromatic aldehydes ( 9a and 9b ). Hydrogenation (H₂), followed by reduction (NaBH₄), and subsequent hydrolysis and elimination led to the keto compounds 13a and 13b . The title compounds, the oximes 7a, 7b and 14a, 14b were formed by reaction of hydroxylamine hydrochloride with the corresponding keto compounds. Using a microsomal fraction of human testicular enzyme, 7a, 7b, 14a, and 14b inhibited the target enzyme only marginally.     

Synthesis of Z- and E-1-Methyl-2-(1-hydroximinoethyl)-6-methoxy-3,4-dihydronaphthalene and Evaluation as Inhibitors of 17α-Hydroxylase-C17,20-Lyase (P450 17)

The synthesis and biological evaluation of Z- and E-1-methyl-2-(1-hydroximinoethyl)-6-methoxy-3,4-dihydro-naphthalene ( Z-1 and E-1 ) as nonsteroidal inhibitors of 17α-hydroxylase-C17,20-lyase (P450 17, CYP17) is described. Z-1 and E-1 were separated by column chromatography and identified by ¹H NMR. The synthesis of the key compound 3 was accomplished by a new reaction acetylating the 1-methyl-6-methoxy-3,4-dihydronaphthalene compound 2 under Friedel-Crafts conditions. Compound 2 was obtained from the 1-tetralone via Wittig reaction. Using a microsomal fraction of human testicular enzyme, Z-1 and E-1 inhibited the target enzyme only marginally.     

Synthesis and In Vitro Evaluation of 3-(1-Azolylmethyl)-1H-indoles and 3-(1-Azolyl-1-phenylmethyl)-1H-indoles as Inhibitors of P450 arom

In the challenge to develop potent inhibitors of aromatase for reducing the levels of estrogens, we found that azolyl-substituted indoles inhibit aromatase activity. 3-(1-Azolylmethyl)-1H-indoles 9–15 and 3-(1-azolyl-1-phenylmethyl)-1H-indoles 22–25 were prepared, and tested on their ability to inhibit P450 arom. Analysis of the inhibitory effect exerted by several derivatives ( 11, 12, 22 , and 23 ) on microsomal aromatase in vitro activity indicates that azolyl-substituted indoles containing an imidazole moiety are more potent inhibitors than triazole derivatives. In the first series, the introduction of the N-benzyl moiety has been found to enhance the inhibitory profile of these 3-(1-azolylmethyl)-1H-indole derivatives. The corresponding 4-fluoro derivative 12 displays the highest inhibitory activity (IC₅₀ = 0.0718 μM) of all investigated compounds; thus, 12 is 258 times as potent as aminoglutethimide (AG). The presence of a chloro grouping in para position of the phenyl ring in compounds 22 and 24 exerts a positive effect only in the triazol-1-yl sub-series: compound 25 is 4-fold more potent than 24 .     

Metabolism of bilirubin by human cytochrome P450 2A6

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K_i of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme.     

CYP17 inhibitors. Annulations of additional rings in methylene imidazole substituted biphenyls: synthesis, biological evaluation and molecular modelling

Twenty-one novel compounds originating from two classes of annulated biphenyls were synthesized as mimetics of the steroidal A- and C-rings and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of the hepatic CYP enzyme 3A4. Potent CYP17 inhibitors were found for each class, compound 9 (17 and 71% at 0.2 and 2 microM, respectively) and 21 (591 nM). Compound 21 showed only weak inhibition of CYP3A4 (32 and 64% at 2 and 10 microM, respectively). Both compounds, however, exhibited moderate to strong inhibition of the glucocorticoid-forming enzyme CYP11B1. The most interesting compounds were docked into our protein model. They bound into one of the modes which we have previously published. New interaction regions were identified.     

Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity

Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug–food or drug–herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver–Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver–Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC₅₀ values of 0.07 and 0.03 μM, respectively. The K_i value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.     

Oxidation of 1-chloropyrene by human CYP1 family and CYP2A subfamily cytochrome P450 enzymes: catalytic roles of two CYP1B1 and five CYP2A13 allelic variants

1.?1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined.

2.?CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation.

3.?Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower k_cat value than other CYP2A13.1 variants.

4.?CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene.

5.?Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2?A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.     

Synthesis and Evaluation of the Metabolites of AMG 221, a Clinical Candidate for the Treatment of Type 2 Diabetes

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Functional Significance of Cytochrome P450 1A2 Allelic Variants,P450 1A2*8, *15, and *16 (R456H,P42R,and R377Q)

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ∼ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (k_cat) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (k_cat/K_m) increased up to 2.5 fold with a slight increase of its K_m value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.     

Inducible bilirubin oxidase: a novel function for the mouse cytochrome P450 2A5

We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic “BR oxidase”. A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible “BR oxidase” where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced.     

人细胞色素P450 1A2 cDNA克隆及鉴定

为建立人细胞色素 P450转基因细胞 ,从人肝组织提取总 RNA,RT- PCR扩增人细胞色素 P4501 A2基因的 c DNA( CYP1 A2 c DNA) ,将其连接到p GEM- T载体上 ,并对 CYP1 A2 c DNA进行全序列测定 .结果与 Jaiswal等发表的 CYP1 A2 c DNA序列相比 ,所克隆的 c DNA,codon51 5AAT→ AAC都编码天冬酰胺 ,而 codon 51 0后面插入了一个CTG(亮氨酸 ) ,则与 Quattrochi等报道的相同 .本实验室克隆的 CYP1 A2 c DNA可能是一种新的CYP1 A2 c DNA,有可能来自于一种新的 CYP1 A2等位基因的转录 .     

Cryopreserved precision-cut rat liver slices: morphology and cytochrome P450 isoforms expression after prolonged incubation

Precision-cut liver slices are an accepted in vitro system for toxicological investigations. However, cryopreservation of slices would make a more efficient utilisation, particularly of human liver tissue possible. In the present study sections of cryopreserved male rat liver slices were examined immunohistochemically for cytochrome P450 (CYP) isoforms expression after prolonged incubation and after exposure to typical inducers. Morphologically, with just thawed slices no major alterations were seen, but remarkable cell damage was observed even after 2 h of incubation mainly in the middle of the slices and in the periportal and intermediate regions of the lobules. After 24 h of incubation, viable cells were only observed at the edges of the slices or around bigger vessels. In the viable cells of the cryopreserved liver slices after 2 h of incubation CYP expression pattern was similar to that in normal liver specimens: a low CYP1A1, but a strong CYP2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. After 24 h, the immunostaining for CYP2B1 and 3A2 in the viable cells was reduced, but that for CYP1A1 was increased. Incubation with beta-naphthoflavone further elevated CYP1A1 and 2B1 expression. Phenobarbital caused an enhanced CYP2B1 and 3A2 and dexamethasone and pregnenolone 16 alpha-carbonitrile an increased CYP3A2 immunostaining. These results show that also in cryopreserved liver slices and after a prolonged incubation, a distinct expression pattern and an in vitro induction of phase I enzymes can be demonstrated immunohistochemically.     

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase,glutathione S-transferase,and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.     

Design,Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of β‐amino Acids

A series of novel di‐ and tripeptidyl epoxyketone derivatives composed of β‐amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti‐proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI‐H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC₅₀ values ranging from 0.97 ± 0.05 to 1.85 ± 0.11 μm . In addition, all the four compounds showed anti‐proliferation activities with IC₅₀ values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e . All the experimental results validated that the β‐amino acid building block has the potential for the development of proteasome inhibitors.     

Design,Synthesis, and Evaluation of 3‐Aryl‐4‐pyrrolyl‐maleimides as Glycogen Synthase Kinase‐3β Inhibitors

A series of 3‐aryl‐4‐pyrrolyl‐maleimides were designed, synthesized, and evaluated for their glycogen synthase kinase‐3β (GSK‐3β) inhibitory activity. Most compounds exhibited potent activity against GSK‐3β. Among them, compounds 11a , 11c , 11h , 11i , and 11j significantly reduced Aβ‐induced Tau hyperphosphorylation, showing the inhibition of GSK‐3β at the cellular level. Structure–activity relationships were discussed based on the experimental data obtained.     

Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies,linkage disequilibrium and influence on caffeine metabolism

AIMS: Several single nucleotide polymorphisms (SNPs) of the cytochrome P450 enzyme 1A2 gene (CYP1A2) have been reported. Here, frequencies, linkage disequilibrium and phenotypic consequences of six SNPs are described. METHODS: From genomic DNA, 114 British Caucasians (49 colorectal cancer cases and 65 controls) were genotyped for the CYP1A2 polymorphisms -3858G-->A (allele CYP1A2*1C), -2464T-->delT (CYP1A2*1D), -740T-->G (CYP1A2*1E and *1G), -164A-->C (CYP1A2*1F), 63C-->G (CYP1A2*2), and 1545T-->C (alleles CYP1A2*1B, *1G, *1H and *3), using polymerase chain reaction-restriction fragment length polymorphism assays. All patients and controls were phenotyped for CYP1A2 by h.p.l.c. analysis of urinary caffeine metabolites. RESULTS: In 114 samples, the most frequent CYP1A2 SNPs were 1545T-->C (38.2% of tested chromosomes), -164A-->C (CYP1A2*1F, 33.3%) and -2464T-->delT (CYP1A2*1D, 4.82%). The SNPs were in linkage disequilibrium: the most frequent constellations were found to be -3858G/-2464T/-740T/-164A/63C/1545T (61.8%), -3858G/-2464T/-740T/-164C/63C/1545C (33.3%), and -3858G/-2464delT/-740T/-164A/63C/1545C (3.51%), with no significant frequency differences between cases and controls. In the phenotype analysis, lower caffeine metabolic ratios were detected in cases than in controls. This was significant in smokers (n = 14, P = 0.020), and in a subgroup of 15 matched case-control pairs (P = 0.007), but it was not significant in nonsmokers (n = 100, P = 0.39). There was no detectable association between CYP1A2 genotype and caffeine phenotype. CONCLUSIONS: (i) CYP1A2 polymorphisms are in linkage disequilibrium. Therefore, only -164A-->C (CYP1A2*1F) and -2464T-->delT (CYP1A2*1D) need to be analysed in the routine assessment of CYP1A2 genotype; (ii) in vivo CYP1A2 activity is lower in colorectal cancer patients than in controls, and (iii) CYP1A2 genotype had no effect on phenotype (based on the caffeine metabolite ratio). However, this remains to be confirmed in a larger study.