Optimization of spirocyclic proline tryptophan hydroxylase-1 inhibitors.

As a follow-up to the discovery of our spirocyclic proline-based TPH1 inhibitor lead, we describe the optimization of this scaffold. Through a combination of X-ray co-crystal structure guided design and an in vivo screen, new substitutions in the lipophilic region of the inhibitors were identified. This effort led to new TPH1 inhibitors with in vivo efficacy when dosed as their corresponding ethyl ester prodrugs. In particular, 15b (KAR5585), the prodrug of the potent TPH1 inhibitor 15a (KAR5417), showed robust reduction of intestinal serotonin (5-HT) levels in mice. Furthermore, oral administration of 15b generated high and sustained systemic exposure of the active parent 15a in rats and dogs. KAR5585 was selected for further pharmacological evaluation in disease models associated with a dysfunctional peripheral 5-HT system.

Tryptophan hydroxylase 1 Inhibition Impacts Pulmonary Vascular Remodeling in Two Rat Models of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH.

Discovery of acyl guanidine tryptophan hydroxylase-1 inhibitors.

An increasing number of diseases have been linked to a dysfunctional peripheral serotonin system. Given that tryptophan hydroxylase 1 (TPH1) is the rate limiting enzyme in the biosynthesis off serotonin, it represents an attractive target to regulate peripheral serotonin. Following up to our first disclosure, we report a new chemotype of TPH1 inhibitors where-by the more common central planar heterocycle has been replaced with an open-chain, acyl guanidine surrogate. Through our work, we found that compounds of this nature provide highly potent TPH1 inhibitors with favorable physicochemical properties that were effective in reducing murine intestinal 5-HT in vivo. Furthermore, we obtained a high resolution (1.90Å) X-ray structure crystal structure of one of these inhibitors (compound 51) that elucidated the active conformation along with revealing a dimeric form of TPH1 for the first time.

Discovery of spirocyclic proline tryptophan hydroxylase-1 inhibitors.

The central role of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) as a neurotransmitter with important cognitive and behavioral functions is well known. However, 5-HT produced in the brain only accounts for approximately 5% of the total amount of 5-HT generated in the body. At the onset of our work, it appeared that substituted phenylalanine derivatives or related aryl amino acids were required to produce potent inhibitors of TPH1, as significant losses of inhibitory activity were noted in the absence of this structural element. We disclose herein the discovery of a new class of TPH1 inhibitors that significantly lower peripherally 5-HT.

Discovery and characterization of the N-phenyl-N'-naphthylurea class of p38 kinase inhibitors.

An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.

Synthesis and SAR studies of indole-based MK2 inhibitors.

Chemistry has been developed to specifically functionalize two structurally similar classes of indole-based MK2 inhibitors at positions prompted by a combination of X-ray crystallographic and computer assisted drug design. A gain in molecular potency was obtained by introducing aminomethyl groups to the lactam rings of 6-arylcarbamoyl-tetrahydro-beta-carbolinone and 6-arylcarbamoyl-dihydropyrazino[1,2-a]indolone MK2 inhibitors. In addition, improvements in molecular potency were achieved by expansion of the lactam from a 6- to 7-membered ring leading to 7-arylcarbamoyl-tetrahydro-[1,4]diazepino[1,2-a]indolones.

Discovery and optimization of p38 inhibitors via computer-assisted drug design.

Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.

The discovery of carboline analogs as potent MAPKAP-K2 inhibitors.

The discovery of a series of potent, carboline-based MK2 inhibitors is described. These compounds inhibit MK2 with IC50s as low as 10 nM, as measured in a DELFIA assay. An X-ray crystal structure reveals that they bind in a region near the p-loop and the hinge region of MK2a.

Second-generation lymphocyte function-associated antigen-1 inhibitors: 1H-imidazo[1,2-alpha]imidazol-2-one derivatives.

A novel class of lymphocyte function-associated antigen-1 (LFA-1) inhibitors is described. Discovered during the process to improve the physicochemical and metabolic properties of BIRT377 (1, Figure 1), a previously reported hydantoin-based LFA-1 inhibitor, these compounds are characterized by an imidazole-based 5,5-bicyclic scaffold, the 1,3,3-trisubstituted 1H-imidazo[1,2-alpha]imidazol-2-one (i.e. structure 3). The structure-activity relationship (SAR) shows that electron-withdrawing groups at C5 on the imidazole ring benefit potency and that oxygen-containing functional groups attached to a C5-sulfonyl or sulfonamide group further improve potency. This latter gain in potency is attributed to the interaction(s) of the functionalized sulfonyl/sulfonamide groups with the protein, likely polar-polar in nature, as suggested by SAR data. X-ray studies revealed that these bicyclic inhibitors bind to the I-domain of LFA-1 in a pattern similar to that of compound 1.

Discovery and SAR of novel Naphthyridines as potent inhibitors of spleen tyrosine kinase (SYK).

The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the other positions is discussed in detail.

Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: orally active inhibitors of lck kinase.

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.