Design, synthesis, and biological activity of non-basic compounds as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N-oxide 6-6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1mg/kg of compound 75b.     

Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units

Based on the both of results for X-ray studies of tetrahydrothiazolopyridine derivative 1c and FXV673, we synthesized a series of thiazol-5-ylpyridine derivatives containing pyridine N-oxide and 2-carbamoylthiazole units to optimize the S4 binding element. N-Oxidation of thiazol-5-ylpyridine increased the anti-fXa activity more than 10-fold independent on the position of N-oxide. The 4-pyridine N-oxide derivatives 3a and 3d excelled over the tetrahydrothiazolopyridine 1b in potency. 2-Methylpyridine N-oxide 3d exhibited 49-fold selectivity over thrombin. Our modeling study proposed a binding mode that the pyridine N-oxide ring of 3a stuck into the "cation hole" , and the oxide anion of 3a occupied in the almost same space to that of FXV673. From observations of the SAR and modeling studies, we suggested the possibilities that the formation of hydrogen bond with the oxide anion in the "cation hole" and the affinity of cationic pyridine ring to S4 subsite were responsible for increase in anti-fXa activity.     

Design,synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part II: Exploration of 6–6 fused rings as alternative S1 moieties

A series of cis-1,2-diaminocyclohexane derivatives possessing a 6–6 fused ring for the S1 moiety were synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure–activity relationship (SAR), and physicochemical properties are reported herein, together with the discovery of compound 45c, which has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, including a reduced negative food effect.     

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.     

Stereoselective synthesis and biological evaluation of 3,4-diaminocyclohexanecarboxylic acid derivatives as factor Xa inhibitors

There have been few reports on synthetic methods for cis-1,2-diaminocyclohexane bearing a third ring substituent. Starting from 3-cyclohexenecarboxylic acid, we developed efficient methods for synthesizing the 3,4-diaminocyclohexanecarboxylic acid derivatives 2-5. We also evaluated their anti-Xa and anticoagulant activities. Among the compounds, acid 2a and amide 2b exhibited the most potent in vitro anti-fXa activity, indicating that the position and stereochemistry of a polar functional group on the cyclohexane ring greatly affected the in vitro anti-fXa activity.     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.     

Cycloalkanediamine derivatives as novel blood coagulation factor Xa inhibitors

This paper describes the synthesis of orally available potent fXa inhibitors 2 and 3 by modification of the piperazine part of lead compound 1. Carbonyl derivative 3 showed potent fXa activity but not sulfonyl derivative 2. Among the compounds synthesized, cyclohexane derivatives 3g and 3h and cycloheptane derivative 3j had potent anticoagulant activity as well as anti-fXa activity. Synthetic study of the optical isomers of 3g demonstrated that (-)-3g had more potent activity.     

Discovery of N-[(1R,2S,5S)-2-{[(5-chloroindol-2-yl)carbonyl]amino}-5-(dimethylcarbamoyl)cyclohexyl]-5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxamide hydrochloride: A novel,potent and orally active direct inhibitor of factor Xa

In the early 1990’s, we reported on the low-molecular selective fXa inhibitor DX-9065a having two amidino groups. However, it had poor oral bioavailability due to its strong basic amidino groups. To obtain fXa inhibitors with improved oral bioavailability, we investigated various non-amidino fXa inhibitors and finally discovered cis-1,2-diaminocyclohexane derivative 4c to have potent fXa inhibition, promising anticoagulant activity, and good oral bioavailability, compared with amidino compound DX-9065a. In addition, we will discuss the influence of the third substituent on the cyclohexane ring on anti-fXa activity, anticoagulant activity, PK profile, and lipophilicity.     

Sedentary Behavior,Physical Activity,and the Metabolic Syndrome among U.S. Adults

Objective: We examined the associations among physical activity, sedentary behavior, and metabolic syndrome in a representative sample of U.S. adults. Research Methods and Procedures: A total of 1626 men and women ≥20 years old from National Health and Nutrition Examination Survey 1999 to 2000 who attended the morning examination were evaluated. The metabolic syndrome was defined by using the definition from the National Cholesterol Education Program. Results: In unadjusted analysis, participants who did not engage in any moderate or vigorous physical activity during leisure time had almost twice the odds of having metabolic syndrome [odds ratio (OR), 1.90; 95% confidence interval (CI), 1.22 to 2.97] as those who reportedly engaged in ≥150 min/wk of such activity. Adjustment for age, sex, race or ethnicity, educational status, smoking status, and alcohol use attenuated the OR (OR, 1.46; 95% CI, 0.87 to 2.45). Compared with participants who watched television or videos or used a computer <1 h/d outside of work, the adjusted ORs for having metabolic syndrome were 1.41 (95% CI 0.80 to 2.51) for 1 h/d, 1.37 (95% CI 0.85 to 2.20) for 2 h/d, 1.70 (95% CI 0.92 to 3.14) for 3 h/d, and 2.10 (95% CI 1.27 to 3.47) for ≥4 h/d. Additional adjustment for physical activity or sedentary behavior minimally affected the ORs. Discussion: Sedentary behavior is an important potential determinant of the prevalence of the syndrome. Efforts to lessen the amount of time that U.S. adults spend watching television or videos or using a computer, especially if coupled to increases in physical activity, could result in substantial decreases in the prevalence of metabolic syndrome.     

Design, synthesis, and structure-activity relationships of novel spiro-piperidines as acetyl-CoA carboxylase inhibitors

Spiro-lactone (S)-1 is a potent acetyl-CoA carboxylase (ACC) inhibitor and was found to be metabolically liable in human hepatic microsomes. To remove one of the risk factors in human study by improving the metabolic stability, we focused on modifying the spiro-lactone ring and the benzothiophene portion of the molecule. Spiro-imide derivative 8c containing a 6-methylthieno[2,3-b]pyridine core exhibited potent ACC inhibitory activity and favorable pharmacokinetic profiles in rats.     

Design, synthesis and SAR of novel ethylenediamine and phenylenediamine derivatives as factor Xa inhibitors

We previously reported on a series of cyclohexanediamine derivatives as highly potent factor Xa inhibitors. Herein, we describe the modification of the spacer moiety to discover an alternative scaffold. Ethylenediamine derivatives possessing a substituent at the C1 position in S configuration and phenylenediamine derivatives possessing a substituent at the C5 position demonstrated moderate to strong anti-fXa activity. Further SAR studies led to the identification of compound 30h which showed both good in vitro activity (fXa IC₅₀ = 2.2 nM, PTCT2 = 3.9 μM) and in vivo antithrombotic efficacy.     

Riluzole prodrugs for melanoma and ALS: Design, synthesis, and in vitro metabolic profiling

Riluzole (1) is an approved therapeutic for the treatment of ALS and has also demonstrated anti-melanoma activity in metabotropic glutamate GRM1 positive cell lines, a mouse xenograft assay and human clinical trials. Highly variable drug exposure following oral administration among patients, likely due to variable first pass effects from heterogeneous CYP1A2 expression, hinders its clinical use. In an effort to mitigate effects of this clearance pathway and uniformly administer riluzole at efficacious exposure levels, several classes of prodrugs of riluzole were designed, synthesized, and evaluated in multiple in vitro stability assays to predict in vivo drug levels. The optimal prodrug would possess the following profile: stability while transiting the digestive system, stability towards first pass metabolism, and metabolic lability in the plasma releasing riluzole. (S)-O-Benzyl serine derivative 9 was identified as the most promising therapeutically acceptable prodrug.     

Synthesis, stability, and implications of phosphothioate agonists of sphingosine-1-phosphate receptors

Phosphothioates may provide metabolic stability when compared to their phosphate counterparts, while retaining the potency and efficacy as agonists at sphingosine-1-phosphate (S1P) G-protein coupled receptors. Unlike their phosphate precursors, phosphothioate compounds with S1P-receptor profiles similar to that of FTY720, an emerging immunomodulator, were shown to evoke prolonged lymphopenia in vivo. Analysis of mouse plasma concentrations for a series of related alcohol/phosphate/phosphothioate compounds showed the conversion of the phosphate to alcohol. These preliminary data highlight the importance of metabolic regulation of S1P receptor ligands.     

Mutagenicity of derivatives and metabolites of 1-nitropyrene: activation by rat liver S9 and bacterial enzymes

The mutagenicity and activation requirements of purified synthetic derivatives and potential metabolites of 1-nitropyrene have been characterized in the Ames plate incorporation assay with the Salmonella tester strains TA98, TA98NR and TA98/1,8-DNP6, in the presence or absence of exogenous metabolic activation provided by Aroclor-induced rat liver S9. All the compounds tested (1-aminopyrene, N-acetyl-1-aminopyrene, N-hydroxy-N-acetyl-1-aminopyrene, 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene) exhibited mutagenic activity under one or more assay conditions. 1-Nitropyrene was metabolized to 3-hydroxy-1-nitropyrene, 6- or 8-hydroxy-1-nitropyrene, 1-aminopyrene, N-acetyl-1-aminopyrene and other unidentified products (including some bound to protein) by an S9 preparation analogous to that used for exogenous metabolic activation in the Ames assay. 1-Nitropyrene and 3-hydroxy-1-nitropyrene were activated primarily by the 'classical' nitroreductase, while the other compounds, particularly in the presence of S9 metabolic activation, were dependent on transesterification for expression of their mutagenicity.     

西北汉族人群D10S1248、D2S441、D1S1677、D9S1122、D10S1435等5个miniSTR基因座的遗传多态性及其法医学应用

为了调查D10S1248、D2S441、D1S1677、D9S1122、D10S1435等5个miniSTR(mini short tandem repeats)基因座在西北汉族人群中的遗传多态性、遗传稳定性及其在陈旧降解检材中的法医学应用价值, 文章采用荧光PCR和基因分型技术对西北汉族154份无关个体血样、10个家系血液样本及10份陈旧降解检材进行片段长度分析。在西北汉族人群中, 5个miniSTR基因座分别检测出了8、7、7、6、7个等位基因, 等位基因频率分布符合Hardy-Weinberg平衡定律, 杂合度(Heterozygosity, H)为0.662~0.792, 个人识别率(Power of discrimination, PD)为0.869~0.915, 非父排除率(Power of exclusion, PE)为0.382~0.585, 多态信息含量(Polymorphism informa-tion content, PIC)为0.650~0.750。家系和陈旧降解检材的研究表明, 5个miniSTR基因座具有高度的遗传稳定性, 可对陈旧降解检材DNA进行有效的分型。5个miniSTR基因座适合作为西北汉族人群的遗传标记, 用于陈旧降解检材的法医学个人识别和亲权鉴定案件中。     

A novel L-isoleucine metabolism in Bacillus thuringiensis generating (2S,3R,4S)-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid

4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for L-isoleucine (L-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2S,3R,4S)-HIL. The (2S,3R,4S)-HIL was oxidized to (2S,3R)-AMKP by a NAD(+)-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B(12) antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP.     

Arylmethoxypyridines as novel, potent and orally active mGlu5 receptor antagonists

Optimisation of affinity, chemical stability, metabolic stability and solubility led from a chemically labile HTS hit 1 to mGlu5 receptor antagonists (24-26) with high affinity for the allosteric MPEP binding site, improved microsomal metabolic stability and anxiolytic-like activity in vivo as assessed by the Vogel conflict drinking test.     

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms

The growth and activity of introduced (S. lividans TK24 pIJ673 and S.lividans TK23) and indigenous (S.griseus CAG17) streptomycete strains in soil was studied, under controlled conditions. The effect of environmental parameters such as temperature, soil water content and nutrient availability on the growth and activity of these strains, was studied using a highly dynamic fed-batch soil microcosm system. Using this new system, repeated cycles of active streptomycete growth were achieved, allowing long-term investigation of metabolic activity, plasmid stability and conjugative plasmid transfer. In long-term experiments, respiration rates and enzyme activity patterns matched the pattern of germination/sporulation cycles of the inoculants. In situ hybridisation, using fluorescently labelled oligonucleotides, also proved the presence of metabolically active streptomycete mycelia in sterile soil. Plasmid stability under varying temperatures and selective pressure was studied using the above system. In both sterile and non sterile amended antibiotic containing soil, no intraspecific transfer of plasmid pIJ673 from S.lividans TK24 to S.griseus CAG17 was detected. The soil microcosm system used, though, permitted detection of intraspecific conjugative transfer of this plasmid from S.lividans TK24 to S.lividans TK23 in soil.     

Synthesis and anthelmintic activity of cyclohexadepsipeptides with (S,S,S,R,S,R)-configuration

The (S,S,S,R,S,R)-configurated cyclohexadepsipeptides (CHDPs) represent novel enniatin derivatives with strong in vivo activity against the parasitic nematode Haemonchus contortus Rudolphi in sheep. 2D NMR spectroscopic analysis revealed for the major conformation the asymmetric conformer, containing a cis-amide bond between C(alpha) protons of neighbouring 2-hydroxy-(S)-carboxylic acid and N-methyl-(S)-amino acid. The absolute configuration of the novel CHDPs was determined by X-ray crystallography. A correlation between the major conformer and its anthelmintic activity was found. Here, we report on a simple total synthetic pathway for this particular type of CHDPs.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.