Quantitative determination of a novel dual PPAR alpha/gamma agonist using on-line turbulent flow extraction with liquid chromatography-tandem mass spectrometry

Turbulent flow chromatograph (TFC) is a technique for the direct and efficient analysis of drugs and metabolites in biological matrices. We report here TFC on-line with an HPLC-MS/MS assay for the determination of 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, MK-0767, KRP297, Fig. 1) in plasma. Samples were transferred using an automated system followed by the addition of internal standard (II), prepared in 0.1 M ammonium acetate (pH 4.0). The plasma samples were directly injected onto a C18 turbulent flow column on-line with an HPLC-MS/MS system, and the analytical column used was a ThermoHypersil Keystone C18. Detection was achieved by MS/MS, using positive ionization on a TurboIonSpray probe, operated in multiple reaction monitoring (MRM) mode. The linear range was 4-2000 ng/mL for I when using 50 microL of plasma. The method exhibited good linearity and reproducibility. The method also showed good selectivity and ruggedness when applied to clinical samples, and was successfully cross-validated with a conventional off-line SPE, LC-MS/MS method.     

Synthesis and biological evaluation of 5-(pyridin-2-yl)thiazoles as transforming growth factor-beta type1 receptor kinase inhibitors

A series of 5-(pyridin-2-yl)thiazoles (14a-l and 15a-l) has been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays. Among them, 3-[[5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)thiazol-2-ylamino]methyl]benzamide (15i) and 3-[[5-(6-ethylpyridin-2-yl)-4-(quinoxalin-6-yl)thiazol-2-ylamino]methyl]benzamide (15k) showed more than 95% inhibition at 0.1 microM in luciferase reporter assays using HaCaT cells transiently transfected with p3TP-luc reporter construct and ARE-luciferase reporter construct.     

Synthesis and structure-activity relationships of N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide derivatives as novel CCR3 antagonists

A novel class of potent CCR3 receptor antagonists were designed and synthesized starting from N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide (1),which was found by subjecting our chemical library to high throughput screening (HTS). The CCR3 inhibitory activity of the synthesized compounds against eotaxin-induced Ca(2+) influx was evaluated using CCR3-expressing preB cells. Systematic chemical modifications of 1 revealed that the 6-fluoro-2-naphthylmethyl moiety was essential for CCR3 inhibitory activity in this new series of CCR3 antagonists. Further structural modifications of the benzamide and piperidine moieties of 1 led to the identification of exo-N-{8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3- yl}biphenyl-2-carboxamide [corrected] (31) as a potent CCR3 antagonist with an IC(50) value of 0.020 microM.     

Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.     

Characterization of adducts formed in reactions of acrolein with thymidine and calf thymus DNA

Acrolein, an important industrial chemical and environmental contaminant, has been shown to interact with nucleic acids in vitro and in vivo. In this study, we examined the reactivity of acrolein towards thymidine and calf-thymus double- and single-stranded DNA in aqueous buffered solutions. LC-MS Analyses of the reaction mixture of acrolein with thymidine showed the formation of five structurally different adducts. The structures of the products were determined on the basis of mass spectrometry, UV absorbance, and (1)H- and (13)C-NMR spectroscopy. The adducts were identified as 3-(3-oxopropyl)thymidine (dT1), 3-[(tetrahydro-2,4-dihydroxypyran-3-yl)methyl]thymidine (dT2), 2-(hydroxymethyl)-5-(thymidin-3-yl)pent-2-enal (dT3), 3-hydroxy-2-methylidene-5-(thymidin-3-yl)pentanal (dT4), and 2-[(thymidin-3-yl)methyl]penta-2,4-dienal (dT5). The adducts dT2-dT5 were formed in reaction of dT1 with acrolein. In the reaction of acrolein with calf-thymus DNA, dT1 was the only adduct detected in the DNA hydrolysate.     

Synthesis and characterization of [¹²⁵I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.     

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.     

Dual neurokinin NK(1)/NK(2) antagonists: N-[(R,R)-(E)-1-arylmethyl-3-(2-oxo-azepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamides and 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-arylmethyl-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamides.

Based on the structure of N-[(R,R)-(E)-1-(4-chlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (1), attempts to improve the NK(2) affinity have resulted in the discovery of N-[(R,R)-(E)-1-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (9, DNK333) exhibiting a 5-fold improved affinity to the NK(2) receptor in comparison to 1. Simplification of the structure via elimination of a chiral centre led to 3-[N'-3,5-bis(trifluoromethyl)benzoyl-N-(3,4-dichlorobenzyl)-N'-methylhydrazino]-N-[(R)-2-oxo-azepan-3-yl]propionamide (22), a potent and fairly balanced NK(1)/NK(2) antagonist.     

Small-molecular inhibitors of Ca2+-induced mitochondrial permeability transition (MPT) derived from muscle relaxant dantrolene

A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized, 5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl]imidazolidin-1-yl}-2-furamide (17) possessed the most potent inhibitory activity against Ca²⁺-induced mitochondrial swelling. The structural development, synthesis and structure–activity relationship of these compounds are described.     

Quantitative determination of MK-0767, a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist, in human plasma by liquid chromatography-tandem mass spectrometry

5-[2,4-Dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl]methyl]benzamide (I, MK-0767 or KRP-297, Fig. 1), is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist. A LC-MS/MS method for the determination of I in human plasma has been successfully developed, validated and applied to clinical programs. The analyte and internal standard (II) are extracted from 0.05 mL plasma via solid phase extraction (SPE). HPLC is used for the separation of I and II from possible co-extracted endogenous and other compounds. Detection is by MS/MS in multiple reaction monitoring (MRM) mode using a TurboIonSpray probe. The whole sample preparation is automated by using a Packard Multiprobe liquid handling system. The linear range is 4-2000 ng/mL in plasma. Recoveries were 71.1% and 69.4% for I and II, respectively. The method exhibited good linearity, reproducibility and sensitivity, selectivity and robustness when used for the analysis of clinical samples.     

11C-Fallypride: radiosynthesis and preliminary evaluation of a novel dopamine D2/D3 receptor PET radiotracer in non-human primate brain

Fallypride [benzamide, 5-(3-fluoropropyl)-2,3-dimethoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]-, CAS RN 166173-78-0] is a selective dopamine D2/D3 receptor antagonist. Carbon-11 labeled fallypride may serve as a radiotracer for use in biomedical imaging technique positron emission tomography (PET). The precursor, 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]benzamide was synthesized from 2-hydroxy-3-methoxy-5-(2-propenyl)benzoic acid, methyl ester in seven steps with approximately 10% overall chemical yield. Using this precursor (11)C-fallypride was produced by radiolabeling with (11)C-methyl iodide in 25-40% radiochemical yields with specific activities of 200-1000 Ci/mmol. PET imaging studies in nonhuman primates with (11)C-fallypride showed radiotracer localization in dopaminergic brain regions such as caudate, putamen, thalamus and cortex. This regional localization of (11)C-fallypride is similar to that observed previously for (18)F-fallypride. The results suggest (11)C-fallypride is a useful PET radiotracer for imaging dopamine D2/D3 receptors.     

4-Hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl]piperidines: selective h5-HT1D agonists for the treatment of migraine

A series of 4-hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl] piperidines was investigated as potential selective h5-HT1D agonists for the treatment of migraine. The 4-[(N-benzyl-N-methyl)amino]methyl analog 12a was found to be a full agonist at the h5-HT1D receptor with good binding selectivity over the h5-HT1B receptor.     

Synthesis and biological evaluation of 5-[(aryl)(1H-imidazol-1-yl)methyl]-1H-indoles: potent and selective aromatase inhibitors

The synthesis and the aromatase (CYP19) inhibitory activity of 5-[(aryl)(imidazol-1-yl)methyl]-1H-indoles were reported. Among the tested racemate compounds, 5-[(4-chlorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole 8b emerged as a potent CYP19 inhibitor (IC(50)=15.3 nM). Chiral chromatography allowed isolation of the (+) enantiomer 8b2, which was about twice as active as the racemate (IC(50)=9 nM).     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

Synthesis of [18F]SU11248, a new potential PET tracer for imaging cancer tyrosine kinase

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-[18F]fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide, a new potential positron emission tomography tracer for imaging cancer tyrosine kinase, has been prepared by the nucleophilic substitution of the nitro-precursor N-[2-(diethylamino)ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide with K18F/Kryptofix 2.2.2 followed by a simple chromatography methodology combined solid-phase extraction with high-performance liquid chromatography purification procedures in 15-25% radiochemical yields.     

Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach

Abstract

Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5?R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K_2-C-MU = 4?±?0.03?×?10⁴M^?1, K_5-CM-MU = 1.95?±?0.03?×?10⁴ M^?1 and K_5-P-MU =1.56?±?0.03?×?10⁴ M^?1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases.

Communicated by Ramaswamy H. Sarma     

Independent synthesis of aminophospholipid-linked maillard products

Phospholipid-linked glycation products are supposed to play an important role in lipid oxidation in vivo. Independent syntheses and unequivocal structural characterization are reported for the phosphatidyl ethanolamine (PE)-derived Amadori compound 4-hydroxy-4-oxo-1-[(palmitoyloxy)methyl]-9-(2,3,4,5-tetrahydrox ytetrahydro-2H-pyran-2-yl)-3,5-dioxa-8-aza-4lambda5-ph osphanon-1-yl palmitate, pyrrolecarbaldehyde 2-[[[2-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]ethoxy](hydroxy)phosph oryl]oxy]-1-[(palmitoyloxy)methyl]ethyl palmitate, the carboxymethyl (CM) derivative 7-hydroxy-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa-3-aza-+ ++7lambda5-phosphaoctacosan-1-oic acid, and the carboxyethyl (CE) derivative 7-hydroxy-2-methyl-7,13-dioxo-10-(palmitoyloxy)-6,8,12-trioxa++ +-3-aza-7lambda5-phosphaoctacosan-l-oic acid. With these reference compounds, a liquid chromatography-mass spectrometry (LCMS) method for the determination of such PE-linked Maillard products has been developed.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Retinoic acid metabolism inhibition by 3-azolylmethyl-1H-indoles and 2, 3 or 5-(alpha-azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or alpha-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl) (1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 microM) ketoconazole exerted similar inhibitory effect (70% inhibition).