Optimization and metabolic stabilization of a class of nonsteroidal glucocorticoid modulators

The optimization of a series of nonsteroidal glucocorticoid modulators is reported. Potent selective GR ligands that have improved metabolic stability were discovered typified by the subnanomolar acid 12 (GR binding IC(50)=0.6 nM).     

N-Alkylidenearylcarboxamides as new potent and selective CB(2) cannabinoid receptor agonists with good oral bioavailability

A novel series of N-alkylidenearylcarboxamides 4, a CB(2) receptor agonist, were synthesized and evaluated for activity against the human CB(2) receptor. In a previous paper, we reported that sulfonamide derivative 1 acted as a potent CB(2) receptor agonist (IC(50)=65 nM, EC(50)=19 nM, E(max)=90%). However, compound 1 also exhibited poor metabolic stability in human liver microsomes. During the structural modification of 1, we found that a novel series of N-alkylidenearylcarboxamide, 4-1, had a moderate affinity for the CB(2) receptor (IC(50)=260 nM, EC(50)=86 nM, E(max)=100%) and good metabolic stability in human liver microsomes. We explored its analogues to discover compounds with a high affinity for the CB(2) receptor and with good oral bioavailability. Among them, compounds 4-9 and 4-27 had high affinities for the human CB(2) receptor (CB(2) IC(50)=13 nM and 1.2 nM) and a high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=270 and 1600); furthermore, significant plasma levels were observed following oral administration in rats (C(max)=233 ng/mL and 148 ng/mL, respectively, after a dose of 10 mg/kg). Furthermore, compound 4-9 had good oral bioavailability (F=52%, 3mg/kg).     

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.     

Isoquinoline derivatives as potent CRTH2 receptor antagonists: synthesis and SAR

Synthesis and structure-activity relationship of a novel series of isoquinoline CRTH2 receptor antagonists are described. One of the most potent compounds, TASP0376377 (6m), showed not only potent binding affinity (IC(50)=19 nM) but also excellent functional antagonist activity (IC(50)=13 nM). TASP0376377 was tested for its ability of a chemotaxis assay to show the effectiveness (IC(50)=23 nM), which was in good agreement with the CRTH2 antagonist potency. Furthermore, TASP0376377 showed sufficient selectivity for binding to CRTH2 over the DP1 prostanoid receptor (IC(50)>1 μM) and COX-1 and COX-2 enzymes (IC(50)>10 μM).     

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.     

Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal derivatives possessing potent diversified pharmacological properties

As a part of our investigations into the structural-activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC50=0.18 μM and IC50=0.168 μM, respectively, approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007 μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity K(i)=25 nM, approximately 115-fold higher in comparison to standard drug galanthamine (K(i)=2.9 μM).     

Small molecule somatostatin receptor subtype-2 antagonists

The first potent small molecule sst2 antagonists are reported. Altering known sst2 agonist molecules yielded compounds with high sst2 binding affinity and full antagonist activity. Compound 7a, for example, displaced somatostatin binding to the sst2 receptor with an IC(50)=2.9 nM and antagonized somatostatin action with an IC(50)=29nM.     

Design and synthesis of piperidine farnesyltransferase inhibitors with reduced glucuronidation potential

The design and synthesis of a novel piperidine series of farnesyltransferase (FTase) inhibitors with reduced potential for metabolic glucuronidation are described. The various substitution and exchange of the phenyl group at the C-2 position of the previously described 2-(4-hydroxy)phenyl-3-nitropiperidine 1a (FTase IC(50)=5.4nM) resulted in metabolically stable compounds with potent FTase inhibition (14a IC(50)=4.3nM, 20a IC(50)=3.0nM, and 50a IC(50)=16nM). Molecular modeling studies of these compounds complexed with FTase and farnesyl pyrophosphate are also described.     

Synthesis and evaluation of cis-3,4-disubstituted piperidines as potent CC chemokine receptor 2 (CCR2) antagonists

A series of cis-3,4-disubstituted piperidines was synthesized and evaluated as CC chemokine receptor 2 (CCR2) antagonists. Compound 24 emerged with an attractive profile, possessing excellent binding (CCR2 IC(50)=3.4 nM) and functional antagonism (calcium flux IC(50)=2.0 nM and chemotaxis IC(50)=5.4 nM). Studies to explore the binding of these piperidine analogs utilized a key CCR2 receptor mutant (E291A) with compound 14 and revealed a significant reliance on Glu291 for binding.     

Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.     

Sulfonamide derivatives as new potent and selective CB2 cannabinoid receptor agonists

A novel series of sulfonamide derivatives 3, the CB(2) receptor agonists, was synthesized and evaluated for activity against the human CB(2) receptor. We first identified sulfonamide 3a, which was obtained by random screening of our in-house chemical library as a moderately active (CB(2) IC(50)=340nM) CB(2) receptor agonist. We then attempted to test its analogues to identify compounds with a high affinity for the CB(2) receptor. One of these, compound 3f, exhibited high affinity for the human CB(2) receptor (IC(50)=16nM) and high selectivity for CB(2) over CB(1) (CB(1) IC(50)/CB(2)IC(50)=106), and behaved as a full CB(2) receptor agonist in the [(35)S]GTPgammaS binding assay (CB(2) EC(50)=7.2nM, E(max)=100%).     

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).     

Benzyl vinylogous amide substituted aryldihydropyridazinones and aryldimethylpyrazolones as potent and selective PDE3B inhibitors

Aryldihydropyridazinones and aryldimethylpyrazolones with 2-benzyl vinylogous amide substituents have been identified as potent PDE3B subtype selective inhibitors. Dihydropyridazinone 8a (PDE3B IC(50)=0.19 nM, 3A IC(50)=1.3 nM) was selected for in vivo evaluation of lipolysis induction, metabolic rate increase, and cardiovascular effects.     

Structure-activity relationships of seco-prezizaane terpenoids in gamma-aminobutyric acid receptors of houseflies and rats

Thirteen seco-prezizaane terpenoids isolated from star anise species (Illcium floridanum, Illcium parviflorum, and Illcium verum) were investigated for their ability to inhibit the specific binding of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of gamma-aminobutyric acid (GABA) receptors, to housefly-head and rat-brain membranes. Veranisatin A was found to be the most potent inhibitor in both membranes, with an IC(50)(fly) of 78.5 nM and an IC(50)(rat) of 271 nM, followed by anisatin (IC(50)(fly)=123 nM; IC(50)(rat)=282 nM). Six of the other 11 tested compounds were effective only in housefly-head membranes. Pseudoanisatin proved to display a high (>26-fold) selectivity for housefly versus rat GABA receptors (IC(50)(fly)=376 nM; IC(50)(rat) >10,000 nM). Although pseudoanisatin does not structurally resemble EBOB, Scatchard plots indicated that the two compounds bind to the same site in housefly receptors. Anisatin and pseudoanisatin exhibited moderate insecticidal activity against German cockroaches. Comparative molecular field analysis (CoMFA), a method of three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis, demonstrated that seco-prezizaane terpenoids can bind to the same site as do picrotoxane terpenoids such as picrotoxinin and picrodendrins, and the CoMFA maps allowed us to identify the parts of the molecules essential to high activity in housefly GABA receptors.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 3: Further optimization of the 2-, 6-, and 7'-substituents and initial pharmacokinetic assessments

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.     

A three-step protocol for lead optimization: quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2 (MAPKAPK2) inhibitors

A three-step protocol for SAR development was introduced and applied to the SAR studies of the MK2 inhibitor program. Following this protocol, key conformational features and functional groups for improving MK2 inhibitor activity were quickly identified. Through this effort, the initial gap observed between in vitro binding activity and cellular activity in the lead identification stage was very much reduced. Compound 28 was identified with single digit binding activity (IC(50)=8 nM) and good cellular activity (EC(50)=310 nM). This provides further evidence that non-ATP-competitive binding MK2 inhibitors are feasible by targeting the outside ATP pocket.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

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.     

Design, synthesis, and biological evaluation of N-acetyl-2-carboxybenzenesulfonamides: a novel class of cyclooxygenase-2 (COX-2) inhibitors

N-Acetyl-2-carboxybenzenesulfonamide (11), and a group of analogues possessing an appropriately substituted-phenyl substituent (4-F, 2,4-F(2), 4-SO(2)Me, 4-OCHMe(2)) attached to its C-4, or C-5 position, were synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 inhibition studies showed that 11 is a more potent inhibitor (COX-1 IC(50)=0.06microM; COX-2 IC(50)=0.25microM) than aspirin (COX-1 IC(50)=0.35microM; COX-2 IC(50)=2.4microM), and like aspirin [COX-2 selectivity index (S.I.)=0.14], 11 is a nonselective COX-2 inhibitor (COX-2 S.I.=0.23). Regioisomers having a 2,4-difluorophenyl substituent attached to the C-4 (COX-2 IC(50)=0.087microM; COX-2 S.I. >1149), or C-5 (COX-2 IC(50)=0.77microM, SI>130), position of 11 exhibited the most potent and selective COX-2 inhibitory activity relative to the reference drug celecoxib (COX-1 IC(50)=33.1microM; COX-2 IC(50)=0.07microM; COX-2 S.I.=472). N-Acetyl-2-carboxybenzenesulfonamide (11, ED(50)=49 mg/kg), and its C-4 2,4-difluorophenyl derivative (ED(50)=91 mg/kg), exhibited superior antiinflammatory activity (oral dosing) in a carrageenan-induced rat paw edema assay compared to aspirin (ED(50)=129 mg/kg). These latter compounds exhibited comparable analgesic activity to the reference drug diflunisal, and superior analgesic activity compared to aspirin, in a 4% NaCl-induced abdominal constriction assay. A molecular modeling (docking) study indicated that the SO(2)NHCOCH(3) substituent present in N-acetyl-2-carboxy-4-(2,4-fluorophenyl)benzenesulfonamide, like the acetoxy substituent in aspirin, is suitably positioned to acetylate the Ser(530) hydroxyl group in the COX-2 primary binding site. The results of this study indicate that the SO(2)NHCOCH(3) pharmacophore present in N-acetyl-2-carboxybenzenesulfonamides is a suitable bioisostere for the acetoxy (OCOMe) group in aspirin.     

Potent 4-aminopiperidine based antimalarial agents

A series of compounds with potent activity against a multi-drug-resistant strain of Plasmodium falciparum, the causative agent of the deadliest strain of malaria, is described. These compounds were also tested for cytotoxicity in human foreskin fibroblast assays, evaluated to determine their logD, and assayed for metabolism by human and murine hepatocytes. This work resulted in the development of compounds 9e and 10d, which showed good potency (IC(50)=75 nM and <60 nM, respectively, against Dd2), acceptable logD values, and reasonable metabolic stability.