Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Pharmacokinetics and pharmacodynamics of AR9281, an inhibitor of soluble epoxide hydrolase, in single- and multiple-dose studies in healthy human subjects.

AR9281, a potent and selective inhibitor of soluble epoxide hydrolase (s-EH), is in clinical development targeting hypertension and type 2 diabetes. The safety, pharmacokinetics, and pharmacodynamics of AR9281 were evaluated in double-blind, randomized, placebo-controlled, ascending, single oral dose (10-1000 mg) and multiple dose (100-400 mg every 8 hours for 7 days) studies in healthy subjects. AR9281 was well tolerated, and no dose-related adverse events were observed during either study. The drug was rapidly absorbed with a mean terminal half-life ranging from 3 to 5 hours. The area under the plasma concentration-time curve increased in an approximately dose-proportional manner up to the 500-mg dose and exhibited a greater than dose linearity at higher doses. AR9281 directly and dose-dependently inhibited blood s-EH activity with 90% inhibition or greater over an 8-hour period at the 250-mg dose and over a 12-hour period at the 500-mg dose. Multiple doses of AR9281 ranging from 100 to 400 mg every 8 hours resulted in a sustained inhibition of s-EH activity at 90% or greater during the trough. The current studies provide proof of safety and target inhibition of AR9281 in healthy subjects. AR9281 pharmacokinetic and pharmacodynamic characteristics support a twice-daily or thrice-daily dosing regimen in patients.

1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia.

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.

Non-urea functionality as the primary pharmacophore in soluble epoxide hydrolase inhibitors.

Inhibition of soluble epoxide hydrolase has been proposed as a promising new pharmaceutical target for diseases involving hypertension and vascular inflammation. The most potent sEH inhibitors reported to date contain a urea or amide moiety as the central or 'primary' pharmacophore. We evaluated replacing the urea pharmacophore with other functional groups such as thiourea, sulfonamide, sulfonylurea, aminomethylene amide, hydroxyamide, and ketoamide to identify novel and potent inhibitors. The hydroxyamide moiety was identified as a novel pharmacophore affording potency comparable to urea.

Design and synthesis of thiazole-5-hydroxamic acids as novel histone deacetylase inhibitors.

We have designed and synthesized a series of structurally novel hydroxamic acid-based histone deacetylase (HDAC) inhibitors characterized by a zinc chelating head group attached directly to a thiazole ring. The thiazole ring connects to a piperazine spacer, which is capped with a sulfonamide group. These novel molecules potently inhibit an HDAC enzyme mixture derived from HeLa cervical carcinoma cells and show potent antiproliferative activity against the breast cancer cell line MCF7.

Synthesis and structure-activity studies of antibacterial oxazolidinones containing dihydrothiopyran or dihydrothiazine C-rings.

A new series of antimicrobial oxazolidinones bearing unsaturated heterocyclic C-rings is described. Dihydrothiopyran derivatives were prepared from the saturated tetrahydrothiopyran sulfoxides via a Pummerer-rearrangement/elimination sequence. Two new synthetic approaches to the dihydrothiazine ring system were explored, the first involving a novel trifluoroacetylative-detrifluoroacetylative Pummerer-type reaction sequence and the second involving direct dehydrogenation of tetrahydrothiopyran S,S-dioxide intermediates. Final analogs such as 4 and 13 represent oxidized congeners of recent pre-clinical and clinical oxazolidinones.

Conformational constraint in oxazolidinone antibacterials. Synthesis and structure-activity studies of (azabicyclo[3.1.0]hexylphenyl)oxazolidinones.

The oxazolidinones are a new class of synthetic antibacterials effective against a broad range of pathogenic Gram-positive bacteria, including multi-drug-resistant strains. Linezolid is the first drug from this class to reach the market and has become an important new option for the treatment of serious infections, particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enteroccocus faecium (VRE). In the search for novel oxazolidinones with improved potency and spectrum, we have prepared and evaluated the antibacterial properties of conformationally constrained analogues in which the morpholine ring of linezolid is replaced with various substituted azabicyclo[3.1.0]hexyl ring systems. Several classes of azabicyclic analogues were identified with activity comparable or superior to that of linezolid. These include analogues bearing hydroxyl, amino, amido, or carboxyl groups on the azabicyclic ring. The azabicyclic acid analogue 50 was 4 times more potent than linezolid against key Gram-positive and fastidious Gram-negative pathogens (S. aureus, Streptococcus pneumoniae, and E. faecalis MICs < or = 1 microg/mL; Haemophilus influenzae MIC = 4 microg/mL).

Mercaptoamide-based non-hydroxamic acid type histone deacetylase inhibitors.

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. A mercaptoamide functionality was designed as a bidentate zinc chelator and incorporated into the hydroxamic acid based SAHA (1) scaffold in order to identify non-hydroxamate compounds as potential inhibitors of histone deacetylases. Two sets of mercaptoamides 2 and 3 with varying spacer length were synthesized and their HDAC inhibitory activity was evaluated. Low micromolar inhibition was observed for mercaptoamides 2e, 3b, and 3d.

Conformationally restricted analogs of deoxynegamycin.

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones.

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.

Novel oxazolidinone-quinolone hybrid antimicrobials.

Antimicrobial compounds incorporating oxazolidinone and quinolone pharmacophore substructures have been synthesized and evaluated. Representative analogues 2, 5, and 6 display an improved potency versus linezolid against gram-positive and fastidious gram-negative pathogens. The compounds are also active against linezolid- and ciprofloxacin-resistant Staphylococcus aureus and Enterococcus faecium strains. The MOA for these new antimicrobials is consistent with a combination of protein synthesis and gyrase A/topoisomerase IV inhibition, with a structure-dependent degree of the contribution from each inhibitory mechanism.

N- and C-terminal modifications of negamycin.

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected beta-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein synthesis (IC(50)=2.3 microM), has antibacterial activity (Escherichia coli, MIC=16 microgram/mL), and is efficacious in an E. coli murine septicemia model (ED(50)=16.3mg/kg).

N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity.

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

Solid-Phase Synthesis of beta-Sultams.

Solid-phase synthesis of beta-sultams amenable for construction of sulfonyl beta-lactam analogue combinatorial libraries is reported. Imine intermediates generated from polymer-immobilized amino acids and aldehydes are reacted with (chlorosulfonyl)acetates in the presence of pyridine to afford the solid-phase-tethered beta-sultam products. The latter can be released from support by acidic cleavage (TFA) or photocleavage, depending on the nature of the linker employed (acid-labile or photolabile linkers). Immobilized 4-(9-fluorenyl)methoxycarbonyl beta-sultams are further functionalized on supports to afford, upon cleavage, the respective carboxy and amido thiazetidine derivatives. The method can be employed in production of beta-sultam libraries for identification of new antibacterial agents.