Steroidal C-21 heteroaryl thioethers (Part 2): discovery of orally bioavailable selective glucocorticoid receptor modulators (dissociated steroids).

The prednisolone C-21 heteroaryl thioethers have been synthesized and evaluated in cell based transrepression and transactivation assays. Most of the compounds demonstrated weak transactivational activity in both human and rat tyrosineaminotransferase functional assay while keeping potent anti-inflammatory activity. The benzimidazole thioether 7 exhibited comparable anti-inflammatory activity and improved safety profile compared to the classical oral steroid prednisolone.

Steroidal C-21 heteroaryl thioethers. Part 3: pregn-4-eno-[3,2-c]pyrazole fused A ring modified steroids as selective glucocorticoid receptor modulators (dissociated steroids).

The introduction of A ring pyrazole modification to the hydrocortisone C-21 heteroaryl thioethers generated compounds with excellent transrepression potency (IL-8 inhibition) compared to their hydrocortisone analogs. However, the transcriptional transactivation activity of these compounds were considerably higher than the corresponding hydrocortisone analogs. Among all the compounds evaluated, a quinoxaline thioether modification demonstrated the best overall in vitro separation.

Pharmacological characterization of a novel α2C-adrenoceptor agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N'-methylurea (compound A).

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Effect of mometasone furoate (MF)/formoterol fumarate (F) combination (MF/F) on late-phase responses in allergen-challenged Brown Norway rats.

Mometasone furoate (MF)/formoterol fumarate (F) combination is a new inhaIed corticosteroid/long-acting ß2-adrenergic agonist (ICS/LABA). The purpose of this study was to evaluate the effects of different dose combinations of MF/F on a variety of late-phase responses to aerosolized antigen challenge in ovalbumin sensitized Brown Norway rats. Late-phase responses were assessed by reductions in lung function, measured by forced vital capacity (FVC) and increased numbers of inflammatory cells and pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid of ovalbumin challenged rats. Intratracheal administration of MF/F 5 h before aerosolized ovalbumin challenge inhibited the increase in inflammatory cells, including eosinophils and levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor-α (TNF-α) appearing in the bronchoalveolar lavage fluid 24 h after the antigen challenge. The combination index for inhibition of both inflammatory cells and cytokines was consistently <1 suggesting a synergistic interaction between MF and F. Intratracheal MF/F given 24 h after the aerosolized ovalbumin challenge reversed the reduction in FVC with statistically significant effects seen over a 24 h period after drug whereas MF and F alone reversed the antigen-induced reduction in FVC at selected times only. At 5 h after drug administration, when both MF and F were partially active, the combination index for MF/F was <1 suggesting a synergistic interaction between MF and F for reversal of the lung function. These results demonstrate that MF/F combination inhibits a variety of late-phase responses induced by allergen challenge and it is likely that MF/F will have a significant benefit in clinical asthma to suppress lung inflammation and improve lung function.

Steroidal C-21 mercapto derivatives as dissociated steroids: discovery of an inhaled dissociated steroid.

A series of C-21 mercapto derivatives of hydrocortisone have been synthesized and evaluated in cell based transrepression and transactivation assays. The benzothiazole derivative, compound 6 not only showed a dissociated profile in vitro functional assays but also a pharmacological profile in a Brown-Norway rat therapeutic index model of asthma that dissociated side effects (thymolysis) while maintaining efficacy against pulmonary inflammation and lung function.

The synthesis and structure-activity relationship of 4-benzimidazolyl-piperidinylcarbonyl-piperidine analogs as histamine H3 antagonists.

A structure-activity relationship study of the lead piperazinylcarbonylpiperidine compound 3 resulted in the identification of 4-benzimidazolyl-piperidinylcarbonyl-piperidine 6h as a histamine-3 (H(3)) receptor antagonist. Additional optimization of 6h led to the identification of compounds 11i-k with K(i)

Spiro-piperidine azetidinones as potent TRPV1 antagonists.

A series of spiro-piperidine azetidinone were synthesized and evaluated as potential TRPV1 antagonists. An important issue of plasma stability was investigated and resolved. Further focused SAR study lead to the discovery of a potent antagonist with good oral pharmacokinetic profile in rat.

Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety.

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in the guinea pig models.

The discovery of tropane derivatives as nociceptin receptor ligands for the management of cough and anxiety.

The discovery of 1 as a high-affinity ligand for the nociceptin receptor has led to the synthesis of a series of tropane (8-methyl-8-azabicyclo[3.2.1]octane) derivatives as optimized ligands. These compounds exhibit high affinity for the nociceptin receptor, moderate to excellent selectivity over the opioid mu receptor, and behave as full agonists. In this Letter, we present the synthesis and highlight the structure-activity relationship of tropane derivatives culminating in the identification of 24 and 32 as potent and orally active antitussive and anxiolytic agents. The in vitro and in vivo activities, pharmacokinetic profile, and the hPXR activity, which predicts the potential 3A4 induction in human, are disclosed.

Structure-activity relationships of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the treatment of cough.

A series of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs have been synthesized and identified to bind to the nociceptin receptor with high affinity. Many of these analogs showed high binding selectivity over classic opioid receptors such as mu receptor. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. Selected compounds with potent oral antitussive activity in the guinea pig model are disclosed.

Characterization of adenosine receptors in the human bladder carcinoma T24 cell line.

The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A1, A2A and A2B receptors but not for the adenosine A3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca2+]i) with the following order of potency and EC50 values: 5'-N-Ethylcarboxamidoadenosine (NECA) (1153+/-214)>5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436+/-186)>adenosine (4823+/-932). This rank order of potency is typical of adenosine A2B receptors. In addition, select adenosine receptor antagonists N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A2B receptors. The select adenosine A2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca2+]i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A2B receptor in the T24 cell line.

New asthma targets: recent clinical and preclinical advances.

Current asthma therapy is directed at the relief of chronic inflammation or improving lung function through bronchodilation. These approaches treat the overt symptoms of asthma but do not approach underlying causes of the disease. Such therapies have limited efficacy and for a number of patients the disease remains poorly controlled. The short-term future of asthma therapy will probably focus on the treatment of multiple symptoms to provide improved lung function. Long-term approaches to asthma will have to focus on modulation of the mechanisms that are the underlying causes of the various asthmatic pathophysiologies. These targets include a number of TH2-type T-cell-generated cytokines and chemokines, G-protein-coupled receptors, TH2-related transcription factors, neurotrophins and adhesion molecules. Additional new targets and understanding of asthma may also arise from genetic analysis.

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1).

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.

Development and potential utility of dual and triple NK receptor antagonists.

The mammalian tachykinin (TK) peptides and their three neurokinin (NK) receptors represent an effector system with wide-ranging actions on neuronal, airway smooth muscle, mucosal, endothelial, immune, inflammatory and remodeling cell function. Recent clinical and preclinical data suggests pathophysiological relevance for TKs in various diseases including asthma, emesis and depression. The promiscuous TK-NK receptor interactions and incompletely overlapping functions mediated by each NK receptor may indicate added therapeutic benefit of using multiple NK receptor blockade. Consequently, there has been substantial pharmaceutical effort in projects to develop nonpeptide dual and triple NK receptor antagonists. This review identifies the chemical and biological approach used to develop a TK antagonist active at the three NK receptors. Clinical activity has been observed using single and/or dual NK receptor antagonists in asthma, depression/anxiety and, most notably, emesis trials but no compound with mono or multiple NK receptor antagonist activities has cleared all the development and regulatory hurdles to commercialization. Current experience indicates that potent dual and triple NK receptor-selective antagonists possessing appropriate affinity and pharmacokinetic properties can be developed. As an example, the biological and pharmacokinetic profiles of a new representative of this class of agent, SCH 206272, is detailed in the present review. Whether such agents will fulfill researchers' expectations must await further clinical trials.

Biochemical characterization of desloratadine, a potent antagonist of the human histamine H(1) receptor.

We have characterized desloratadine (5H-benzo[5,6]cyclohepta[1,2-b]pyridine, 8-chloro-6,11-dihydro-11-(4-piperidinylidene), CAS 100643-71-8) as a potent antagonist of the human histamine H(1) receptor. [3H]Desloratadine bound to membranes expressing the recombinant human histamine H(1) receptor in Chinese hamster ovary cells (CHO-H(1)) in a specific and saturable manner with a K(d) of 1.1+/-0.2 nM, a B(max) of 7.9+/-2.0 pmol/mg protein, and an association rate constant of 0.011 nM(-1) x min(-1). The K(d) calculated from the kinetic measurements was 1.5 nM. Dissociation of [3H]desloratadine from the human histamine H(1) receptor was slow, with only 37% of the binding reversed at 6 h in the presence of 5 microM unlabeled desloratadine. Seventeen histamine H(1)-receptor antagonists were evaluated in competition-binding studies. Desloratadine had a K(i) of 0.9+/-0.1 nM in these competition studies. In CHO-H(1) cells, histamine stimulation resulted in a concentration-dependent increase in [Ca(2+)](i) with an EC(50) of 170+/-30 nM. After a 90-min preincubation with desloratadine, the histamine-stimulated increase in [Ca(2+)](i) was shifted to the right, with a depression of the maximal response at higher concentrations of antagonist. The apparent K(b) value was 0.2+/-0.14 nM with a slope of 1.6+/-0.1. The slow dissociation from the receptor and noncompetitive antagonism suggests that desloratadine may be a pseudoirreversible antagonist of the human histamine H(1) receptor. The mechanism of desloratadine antagonism of the human histamine H(1) receptor may help to explain the high potency and 24-h duration of action observed in clinical studies.

SCH 206272: a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist.

Experiments were performed to characterize the pharmacology of SCH 206272 [(R,R)-1'[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl]-N-methyl-2-oxo-[1,4'bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK(1), NK(2), and NK(3) receptors. SCH 206272 inhibited binding at human tachykinin NK(1), NK(2), and NK(3) receptors (K(i) = 1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca(2+)](i) mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK(1), NK(2), or NK(3) receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (pK(b) = 7.7 +/- 0.3) induced by the tachykinin NK(1) receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (pK(b = 8.2 +/- 0.3) induced by the tachykinin NK(2) receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (pK(b = 7.6 +/- 0.2), NKA-induced contraction of the bronchus (pK(b) = 7.7 +/- 0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1-10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1-3 mg/kg, p.o.) inhibited NK(1) and NK(2) activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease.

Cloning and pharmacological characterization of mouse TRPV1.

The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX) = arvanil > capsaicin = olvanil > OLDA > PPAHV) (EC50 values of 0.15+/-0.04 nM, 0.27+/-0.07 nM, 9.1+/-1.2 nM, 3.7+/-0.3 nM, 258+/-105 nM, and 667+/-151 nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC50 (I-RTX) = 0.35+/-0.12 nM, 1.9+/-0.7 nM, and 0.80+/-0.68 nM, IC50 (BCTC) = 1.3+/-0.36 nM, 0.59+/-0.16 nM, and 0.37+/-0.15 nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC50 of 1426+/-316 nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.

Human cord blood-derived mast cells synthesize and release I-309 in response to IgE.

Mast cells are the central mediating cells of allergic reactions. Binding of allergen specific IgE to high affinity IgE receptor (Fcepsilon RI) and subsequent binding of allergen by the IgE causes receptor cross-linking and activation. In a study examining the differential gene expression in human cord blood-derived mast cells (CBMCs) mediated by activation of Fcepsilon RI both with IgE and IgE followed by cross-linking with alpha-IgE, the chemokine I-309 was found to be upregulated. I-309 is the ligand for the CCR8 receptor and is responsible for chemoattraction of TH2 type T-cells. Interestingly, I-309 RNA and protein levels were elevated not only in response to IgE/alpha-IgE activation but also by IgE alone. In addition, the I-309 levels were augmented by growth of the CBMCs in the presence of the proinflammatory cytokine IL-4. GM-CSF and MIP-1alpha secretion was also induced by IgE. These results suggest that IgE, through the production and release of cytokines such as I-309, GM-CSF and MIP-1alpha could promote an inflammatory reaction in the absence of antigen stimulation of mast cells.

Mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models.

Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348 ± 5451, which was blocked by spironolactone (0.3-60 mg/kg, p.o.) and eplerenone (0.3-30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55 ± 5, 122 ± 9, and 83 ± 8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10-60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.

Discovery of orally active 3-pyridinyl-tropane as a potent nociceptin receptor agonist for the management of cough.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.