Discovery and structure-activity relationships of urea derivatives as potent and novel CCR3 antagonists

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist.     

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).     

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 of 6- or 4-functionalized indoles via a reductive cyclization approach and evaluation as aromatase inhibitors

Two new series of benzonitrile derivatives on position 6 or 4 of indole ring were successfully synthesized via a Leimgruber-Batcho reaction. All the compounds were evaluated in vitro on the inhibition of aromatase (CYP19) and 17alpha-hydroxylase-C17,20-lyase (CYP17). The racemate, 4-[(1H-imidazol-1-yl)(1H-indol-4-yl)methyl]benzonitrile 9, showed high level of inhibitory activity towards CYP19 (IC(50)=11.5 nM).     

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

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.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

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.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

Novel inhibitors of fatty acid oxidation as potential metabolic modulators

We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles, benzoxazoles and introducing small alkyl substituents into the piperazine ring resulted in analogues with enhanced inhibitory activity against 1-(14)[C]-palmitoyl-CoA oxidation in isolated rat heart mitochondria (6, IC(50)=70 nM; 25, IC(50)=23 nM).     

C(4)-alkyl substituted furanyl cyclobutenediones as potent, orally bioavailable CXCR2 and CXCR1 receptor antagonists

A novel series of cyclobutenedione centered C(4)-alkyl substituted furanyl analogs was developed as potent CXCR2 and CXCR1 antagonists. Compound 16 exhibits potent inhibitory activities against IL-8 binding to the receptors (CXCR2 Ki=1 nM, IC(50)=1.3 nM; CXCR1 Ki=3 nM, IC(50)=7.3 nM), and demonstrates potent inhibition against both Gro-alpha and IL-8 induced hPMN migration (chemotaxis: CXCR2 IC(50)=0.5 nM, CXCR1 IC(50)=37 nM). In addition, 16 has shown good oral pharmacokinetic profiles in rat, mouse, monkey, and dog.     

Discovery of potent CCR4 antagonists: Synthesis and structure-activity relationship study of 2,4-diaminoquinazolines

A new series of quinazolines that function as CCR4 antagonists were discovered during the screening of our corporate compound libraries. Subsequent compound optimization elucidated the structure-activity relationships and led the identification of 2-(1,4'-bipiperidine-1'-yl)-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 14a, which showed potent inhibition in the [(35)S]GTPgammaS-binding assay (IC(50)=18nM). This compound also inhibited the chemotaxis of human and mouse CCR4-expressing cells (IC(50)=140nM, 39nM).     

Synthesis, biological evaluation and molecular docking studies of novel 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives

In present study, a series of new 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives (6a-6x) as potential focal adhesion kinase (FAK) inhibitors were synthesized. The bioassay assays demonstrated that compound 6i showed the most potent activity, which inhibited the growth of MCF-7 and A431 cell lines with IC(50) values of 140 ± 10 nM and 10 ± 1 nM, respectively. Compound 6i also exhibited significant FAK inhibitory activity (IC(50)=20 ± 1 nM). Docking simulation was performed to position compound 6i into the active site of FAK to determine the probable binding model.     

Structure-Activity relationships of 2-substituted 5,7-Diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids as a novel class of endothelin receptor antagonists

Synthesis and structure-activity relationships of 2-substituted-5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids, a novel class of endothelin receptor antagonists, were described. Derivatization of a lead structure 1 (IC(50)=2.4nM, 170-fold selectivity) by incorporating a substituent such as an alkyl, alkoxy, alkylthio, or alkylamino group into the 2-position of the cyclopenteno[1,2-b]pyridine skeleton was achieved via the key intermediate 8. Introduction of an alkyl group led to the identification of potent ET(A)/ET(B) mixed receptor antagonists, a butyl (2d: IC(50)=0.21nM, 52-fold selectivity) and an isobutyl (2f: IC(50)=0.32nM, 26-fold selectivity) analogue. In contrast, installment of a primary amino group resulted in ET(A) selective antagonists, a propylamino 2p (IC(50)=0.12nM, 520-fold selectivity) and an isopropylamino 2q (IC(50)=0.10nM, 420-fold selectivity) analogue. These results suggested that a substituent at the 2-position of the 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids played a key role in the binding affinity for both ET(A) and ET(B) receptors.     

Synthesis, biochemical evaluation of a range of potent 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-Hydroxylase/17,20-Lyase (P45017alpha)

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of the cytochrome P-450 enzyme 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), i.e. 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the compounds synthesised are potent inhibitors, with 7-phenyl heptyl imidazole (11) (IC(50)=320 nM against 17alpha-OHase and IC(50)=100 nM against lyase); 1-[7-(4-fluorophenyl) heptyl] imidazole (14) (IC(50)=170 nM against 17alpha-OHase and IC(50)=57 nM against lyase); 1-[5-(4-bromophenyl) pentyl] imidazole (19) (IC(50)=500 nM against 17alpha-OHase and IC(50)=58 nM against lyase) being the most potent inhibitors within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components shows that all of the compounds tested are less potent towards the 17alpha-OHase in comparison to the lyase component, a desirable property in the development of novel inhibitors of P450(17alpha). From the modelling of these compounds onto the novel substrate heme complex (SHC) for the overall enzyme complex, the length of the compound, along with its ability to undergo interaction with the active site corresponding to the C(3) area of the steroidal backbone, are suggested to play a key role in determining the overall inhibitory activity.     

Novel retinoic acid 4-hydroxylase (CYP26) inhibitors based on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl scaffold

Retinoic acid (RA), the biologically active metabolite of vitamin A, is used medicinally for the treatment of hyperproliferative diseases including dermatological conditions and cancer. The antiproliferative effects of RA have been well documented as well as the limitations owing to toxicity and the development of resistance to RA therapy. RA metabolism inhibitors (RAMBAs or CYP26 inhibitors) are attracting increasing interest as an alternative method for enhancing endogenous levels of retinoic acid in the treatment of hyperproliferative disease. Here the synthesis and inhibitory activity of novel 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl derivatives in a MCF-7 CYP26A1 microsomal assay are described. The most promising inhibitor methyl 2,2-dimethyl-3-(4-(phenylamino)phenyl)-3-(1H-1,2,4-triazol-1-yl)propanoate (6) exhibited an IC(50) of 13 nM (compared with standards Liarozole IC(50) 540 nM and R116010 IC(50) 10 nM) and was further evaluated for CYP selectivity using a panel of CYP with >100-fold selectivity for CYP26 compared with CYP1A2, 2C9 and 2D6 observed and 15-fold selectivity compared with CYP3A4. The results demonstrate the potential for further development of these potent inhibitors.     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).