• Abstract and Figures
• Public Full-text

4494 J. Med. Chem. 2004, 47, 4494-4506 Identification of 1,5-Naphthyridine Derivatives as a Novel Series of Potent and Selective TGF-â Type I Receptor Inhibitors Franc¸oise Gellibert,*,† James Woolven,‡ Marie-He´le`ne Fouchet,† Neil Mathews,§,| Helen Goodland,| Victoria Lovegrove,| Alain Laroze,† Van-Loc Nguyen,† Ste´phane Sautet,† Ruolan Wang,⊥ Cheryl Janson,#,× Ward Smith,∇,× Gae¨l Krysa,X Vale´rie Boullay,X Anne-Charlotte de Gouville,X Ste´phane Huet,X and David Hartley| Departments of Medicinal Chemistry and Biology, GlaxoSmithKline, 25-27 Avenue du Que´bec, 91951 Les Ulis, France, Discovery Research, Computational and Structural Science, and Department of Medicinal Chemistry, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom, Discovery Research, Assay Development and Compound Profiling, GlaxoSmithKline, Five Moore Drive, P.O. Box 13398, Research Triangle Park, North Carolina 27709, and Discovery Research, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406 Received January 30, 2004 Optimization of the screening hit 1 led to the identification of novel 1,5-naphthyridine aminothiazole and pyrazole derivatives, which are potent and selective inhibitors of the transforming growth factor-â type I receptor, ALK5. Compounds 15 and 19, which inhibited ALK5 autophosphorylation with IC50 ) 6 and 4 nM, respectively, showed potent activities in both binding and cellular assays and exhibited selectivity over p38 mitogen-activated protein kinase. The X-ray crystal structure of 19 in complex with human ALK5 is described, confirming the binding mode proposed from docking studies. Introduction Chart 1 Transforming growth factor (TGF-â) is a pluripotent cytokine involved in a variety of biological processes such as development, cell growth, differentiation, cell adhesion, migration, extracellular matrix (ECM) deposi- tion, and immune response regulation.1 This cytokine regulates not only the ECM deposition in response to tissue injury, thus contributing to the healing process, the GS domain. The GS domain is at the amino terminal 2 but is also involved in pathological fibrosis. Indeed, end of the intracellular kinase domain and is critical TGF-â overexpression has been implicated in multiple for activation by the type II receptor.9 Several studies disease states including pulmonary fibrosis, liver fibro- have shown that TGF-â signaling requires both the ALK 3-5 sis, renal glomerulosclerosis, and cancer. TGF-â and the type II receptors. Specifically, the binding of belongs to the TGF-â superfamily, which includes in TGF-â1 to the type II receptor causes phosphorylation humans the three TGF-â isoforms (TGF-â1, TGF-â2, of the GS domain of the TGF-â type I receptor, ALK5. and TGF-â3), the activins, and the bone morphogenetic The ALK5 receptor, in turn, phosphorylates the cyto- 6 proteins. TGF-â1 is the prototypic member of this plasmic proteins smad2 and smad3 at two carboxyl family of cytokines that signals through a family of terminal serines. The phosphorylated smad proteins transmembrane serine/threonine kinase receptors. These form heteromeric complexes of smad2, smad3, and receptors can be divided into two classes, the type I or smad4, after which the complex translocates into the activin like kinase (ALK) receptors and the type II nucleus to effect gene transcription.10 7,8 receptors. The ALK receptors are distinguished from A program was initiated to identify small molecules the type II receptors in that they lack the serine/ inhibiting the TGF-â signaling pathway. High through- threonine rich intracellular tail, possess serine/threo- put screening (HTS) of our corporate compound collec- nine kinase domains that are homologous between type tion was carried out using a TGF-â-dependent tran- I receptors, and share a common sequence motif called scriptional assay, comprising a HepG2 cell line stably transfected with a luciferase reporter gene driven by the * To whom correspondence should be addressed. Tel: +3316929 PAI-1 promoter. This screen identified the aminothia- 61 28. Fax: +33 1 69 07 48 92. E-mail: [email protected]. † Department of Medicinal Chemistry, GlaxoSmithKline, Les Ulis. zole derivative 1 as a potent inhibitor of TGF-â signaling ‡ Discovery Research, Computational and Structural Science, Glaxo- (IC50 ) 429 nM). We describe below the optimization of SmithKline. § Current address: Arrow Therapeutics, Britannia House, 7 Trinity 1 to give a series of high-affinity ALK5 inhibitors (Chart St., London, SE1 1DA, U.K. 1). A pyrazole compound 19 was cocrystallized with | Department of Medicinal Chemistry, GlaxoSmithKline, Stevenage. ALK5, and the structure of the complex was determined ⊥ Discovery Research, Assay Development and Compound Profiling, GlaxoSmithKline. by X-ray crystallography. # Current address: Shamrock Structures, 1440 Davey Road, Wood- ridge, IL 60517. Results and Discussion 3 Current address: Argonne National Laboratory, 9700 South Cass Consideration of the structure of 1 strongly suggested Avenue, Argonne, IL 60439. X Department of Biology, GlaxoSmithKline, Les Ulis. that it might inhibit a kinase, and ALK5 appeared a × GlaxoSmithKline, King of Prussia, PA 19406. likely candidate. Accordingly, an ALK5 autophospho- 10.1021/jm0400247 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/04/2004 Potent and Selective TGF-â Type I Receptor Inhibitors Journal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4495 The N3 of the aminothiazole scaffold may interact with the side chain of Lys-232. This amino acid is included in the catalytic segment, and an equivalent hydrogen bond had previously been seen between p38 MAP kinase inhibitors and Lys-53.12 As Lys-53 is a highly conserved residue in the ATP binding site, this interaction is unlikely to contribute to selectivity. The pyridyl moiety is directed into the interior of the protein toward Ser-280 in the so-called “selectivity pocket”, as established for p38 MAP kinase.13 This selectivity pocket is behind and orthogonal to the adenine binding site and is not utilized by ATP. Ser- 280 may be the key protein feature involved in inhibitor selectivity and is equivalent to Thr-106, the so-called “gate keeper” residue in p38 MAP kinase.12 These are the main residues governing the size and accessibility Figure 1. Schematic drawing of aminothiazole 1 potential of the selectivity pocket. Only a limited number of binding mode in the ATP binding site of the ALK5-FKBP12 complex. The interactions between inhibitor and ALK5 resi- kinases have threonine or serine as gate keeper resi- dues are shown with dotted lines. dues, most having larger residues at this position, which are believed to block the formation of the pocket. rylation assay was constructed using the purified hu- SARs. In an attempt to increase the potency of this man ALK5 kinase domain produced in Sf9 insect cells. novel series of ALK5 inhibitors, the three heterocyclic Aminothiazole 1 inhibited ALK5 activity in this assay subunits were modified in turn. To explore the selectiv- with an IC50 of 274 nM. While presenting some struc- ity pocket toward which the pyridin-2-yl is directed, the tural analogy with p38 MAP kinase inhibitors,11 this pyridine moiety was replaced by other aromatic groups compound did not exhibit p38 MAP kinase activity when (Table 1). The pyridin-3-yl analogue 2 showed a decrease tested in a relevant binding assay (IC50 > 16 µM). A in binding affinity, suggesting that the nitrogen atom chemistry program was initiated with the aim of of the 2-pyridyl ring might be involved in a hydrogen increasing potency. To support the study of structure- bonding interaction with the protein, even though this activity relationships (SAR) within the series, a binding was not evident from the docking studies. In support of assay was set up in which the ability of the test this supposition, the thiazol-2-yl compound 3 retained compounds to displace a rhodamine green-labeled ligand the potency of 1 in the binding assay. However, the from recombinant GST-ALK5 was measured. In this 2-fluorophenyl analogue 4 also showed a similar level assay, 1 exhibited an IC50 of 724 nM. The compounds of binding affinity, so, clearly, an appropriately posi- were evaluated in both the binding and the TGF-â- tioned nitrogen atom is not necessary for activity, dependent cellular assay, and the most potent com- although it remains possible that the 2-fluorophenyl pounds were also tested in the ALK5 autophosphory- group could participate in hydrogen bonding. Replace- lation assay. At the outset of this work, no structures ment of the 2-fluoro substituent by a 2-methoxy group of ALK5 inhibitors cocrystallized with the protein had 5 abolished the binding to ALK5. It was apparent from been published. However, the crystal structure of an the docking studies that the pyridin-2-yl of 1 and the inactive form of ALK5 complexed to FKBP129 was Ser-280 oxygen of the protein were in close proximity, available and this was used as a basis for docking with a distance of 3.21 Å measured between the pyridine studies. centroid and the Ser-280 oxygen. This was suggestive Binding Mode Hypothesis. On the basis of the of a possible electrostatic interaction, consistent with crystal structure of the ALK5-FKBP12 complex, we the observed activity of the 2-fluorophenyl analogue 4. carried out molecular modeling studies to support the To explore this idea further, the pyridin-2-yl ring was medicinal chemistry program. Knowing that compound substituted with electron-withdrawing and -donating 1 inhibited ALK5 by occupation of the ATP binding site, groups, some representatives of which are shown in the binding was ATP competitive (data not shown), and Table 1. The electron deficient 5-chloro (6) and 4-chloro comparison of ALK5 with a range of other kinase- (7) analogues retained the binding affinity to ALK5, inhibitor complexes for which structural information whereas the electron-rich 5-methyl derivative (8) showed was available enabled us to characterize residues that a significantly decreased affinity.

Load more

  14

Copy Link