Pyrazole-based inhibitors of the transforming growth factor-beta type I receptor kinase domain (TbetaR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TbetaR-I receptor kinase domain.

Transforming growth factor-β (TGFβ) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFβ receptor I (TGFβRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFβRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFβ-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFβ-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects.Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFβ-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFβ pathway inhibitors.

Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.

We previously described the design and synthesis of rigid tricyclic phenylalanylleucine (PheLeu) mimetic 1 and its incorporation into 2, an inhibitor of angiotensin I-converting enzyme (ACE, EC 3.4.15.1).' Mimetic 1 was designed2 to closely resemble the anti orientation (XI = 180°, X2 = Oo) of the carboxy terminal histidylleucine (HisLeu) portion of angiotensin I (Chart I). The replacement of His by Phe in mimetic 1 was made with knowledge that His is not essential to ACE bindin? and that neutral endopeptidase 24.11 (NEP, EC 3.4.24.111, a related zinccontaining proteinase, cleaves the PheLeu dipeptide from Leu-enke~halin.~ The cleavage of bradykinin adjacent to Phe(8) by both ACE and NEP6 further suggests that these two metalloproteinases could have shared active-site characteristics. We speculated early on that side chain constrained peptidomimetics would be useful tools to study the conformational preferences in peptide-protein interactions. The similarities and biological significance of these two enzymes made them ideal choices for evaluation by this approach. More recently, NEP has been shown to play a role in the degradation of the natriuretic peptides! a family of hormones, some of which are secreted by the heart into the circulation in increased amounts in patienta with congestive heart failure (CHF).' Because the reninangiotensin-aldosterone system opposess the beneficial natriuretic and diuretic actions of atrial natriuretic peptide (ANP), inhibition of ACE during NEP inhibition should be advantageous. The feasibility of simultaneous ACE and NEP inhibition was investigated utilizing our dipeptide mimic approach. Gros et al. have described close analogs of thiorphan which exhibit equipotent nanomolar ACE and NEP inhibition in vitro and demonstrate enzyme occupation upon oral dosing of a prodrug form.Q We now report the design rationale and synthesis of a new class of subnanomolar dual ACE/NEP inhibitor, a member of which produces blood pressure lowering in animal models of both essential and salt dependent hypertension when orally administered in prodrug form.Derivatives of 1 were used to probe the active site requirements of both ACE and NEP (Chart 11). Whereas 2 was found to be an extremely selective inhibitor of ACE, epimeric tricyclic mercaptomethylene derivatives 3 and 4'0 gave the indication that simultaneous inhibition of ACE and NEP was feasible in spite of the intrinsic conformational constraints of mimetic 1. The mercaptoacetyl derivative of phenylalanylglycine had been reChart I Anglotensln I n Leu-Enkephalln TyrGlyGlj 1 , n Leu-Enkephalln U Chart I1 3 4 K,(ACE) nM 2 r7 2 130 KI(NEP) nM r10,OOO r300 45 5 2ported to inhibit NEP (Ki = 20 nM).I1 The mercaptoacetyl derivative 5 of mimetic 1 was found to be a potent inhibitor of ACE as well as NEP. This result, combined wfth the structural rigidity of mimetic 1, indicates that significant similarities in the SI' and S2' binding domains of these two enzymes exist and that an internal unsubstituted CO-NH function is not essential for binding to this ...

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