Therapeutic Potential of Smurf-1 Inhibitors for the Treatment of Pulmonary Arterial Hypertension (PAH)

Patent Title:	Carboxamide Derivatives
Patent Number:	US 9,403,810 B2	Publication date:	Aug. 2, 2016
Priority Application:	EP 14168303	Priority date:	May 14, 2014
Inventors:	Bala, K. J.; Brearley, A.; Dale, J.; Edwards, A.-M.; Ahmed, M.; Porter, D.; Pulz, R. A.; Rooney, L. A.; Sandham, D. A.; Shaw, D.; Smith, N.; Taylor, J. L.; Taylor, R. J.; Troxler, T. J.; Wrigglesworth, J.
Assignee Company:	Novartis AG, Basel (CH)
Disease Area:	Pulmonary arterial hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), COPD, and asthma.	Biological Target:	Smad ubiquitination regulatory factor 1 (Smurf-1)

Summary:	The invention in this patent relates to isoxazole-3-carboxamide derivatives represented generally by formula (I). These compounds are selective Smurf-1 inhibitors and may thus provide useful treatment for a range of disorders, including, pulmonary arterial hypertension (PAH), glaucoma, hereditary hemorrhagic telangiectasia (HHT), proteinuria, wound healing, COPD, and asthma.
	Pulmonary arterial hypertension (PAH) is a devastating aggressive life-threatening disease. It is characterized by the remodeling of the small pulmonary arteries that may lead to progressive increase in pulmonary vascular resistance as well as the right ventricular hypertrophy and failure, a condition that may cause premature death in many cases. Current therapies are palliative and focus only on altering the vasoconstrictive elements of the disease but do not stop or reverse the progression of the disease. Transplantation (double lung or heart-lung) is the only available curative treatment. Therefore, there is a need for novel therapies that can target the causes of the pulmonary vascular remodeling of PAH.
	The protein degradation by the ubiquitin-dependent degradation system controls the intracellular concentrations of many regulatory proteins. The process includes conjugation of a protein substrate to ubiquitin through the sequential actions of three types of enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-carrier enzyme, and E3 ubiquitin-protein ligase. Smad ubiquitination regulatory factor 1 (Smurf-1) is a member of the HECT family of E3 ubiquitin ligase. Smurf-1 marks several specific protein substrates for proteolytic degradation via the ubiquitin-dependent proteolytic pathway including RhoA, bone morphogenetic protein receptors (BMPRs) I and II, smad1 and 5, TNFα receptor associated factor (TRAP) 6, and myD88. This diverse list of substrates indicates that Smurf-1 plays very important roles in regulating BMP signaling, neuronal cell polarity, cell migration, tumor cell invasion, mitochondrial autophagy mesenchymal stem cell proliferation, and epithelial–mesenchymal transition (EMT).
	The bone morphogenetic proteins (BMPs) are a group of growth factor signaling proteins that belong to the transforming growth factor β (TGF-β) superfamily. BMPs are synthesized and secreted from a variety of cell types, including pulmonary vascular smooth muscle cells and endothelial cells. They were first recognized for their role in the formation of bone and cartilage, but they were later found to play larger roles and perform multiple functions in regulating a wide spectrum of activities such as proliferation, differentiation, and apoptosis in a large variety of cell types, including osteoblasts, epithelial cells, neurons, immune cells, and smooth muscle cells. Researchers have identified >20 mammalian BMPs as well as three type I and three type II BMP receptors (BMPR-I and BMPR-II, respectively) that can bind to BMPs. Germline mutations in BMPR-II gene were found to be prevalent in 70% of heritable and in some sporadic forms of idiopathic PAH (IPAH). In addition, the lungs from patients with nonfamilial PAH display markedly reduced levels of vascular BMPR-I and -II implying a central role for disrupted BMP signaling in many forms of PAH. All these findings suggest that restoration of BMP signaling in the pulmonary vasculature of PAH patients may be a viable therapeutic target to develop novel antiremodeling therapeutics for the treatment of PAH.
	Smurf-1 has been shown to mediate the degradation of BMPR-I and -II and smad1 and 5 in a variety of cell types including osteoblasts, myoblasts, lung epithelium, neuronal tissue, and endocardial cells. In a recent study, researchers have observed significantly high levels of Smurf-1 in the chronic hypoxia and monocrotaline preclinical in vivo models of PAH. This elevation in Smurf-1 was associated with down-regulation of BMPR-I and -II and reduced expression of BMPR-II. It was concluded that Smurf-1 reduces the levels of BMPRs through ubiquitination and subsequent degradation. Elevation of Smurf-1 levels could be a main cause of downregulation of BMP signaling and the vascular cell proliferation and remodeling in PAH. Therefore, blocking and abrogating Smurf-1 function could be a viable strategy for developing effective PAH therapy. Thus, Smurf-1 inhibitors such as the compounds of formula (I) in this patent may potentially provide methods for treating, preventing, or ameliorating pulmonary arterial hypertension.
Important Compound Classes:
Key Structures:	The inventors listed 48 compounds of formula (I) including the following representative examples. Some of the examples were diastereomeric mixtures such as compounds 14 and 22c.
Biological Assay:	A panel of biochemical HECT E3 ligase autoubiquitinylation assays (Smurf-1, Smurf-2, WWP1, WWP2, ITCH, Nedd4, Nedd4L, and E6AP) was used to determine the HECT E3 ligase selectivity of the compounds of formula (I). The “DiscoverX PathHunter ProLabel Detection Kit” was used to determine the cellular activity at the Smurf-1 HECT domain by measuring the accumulation of Smurf-1 protein in HEK293 cells stably expressing Prolabel-tagged Smurf-1 under the control of a tetracycline-inducible promoter.
Biological Data:	The Smurf-1 IC50 values obtained from testing the above representative examples are shown in the following table:
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The author declares no competing financial interest.