Brief Article pubs.acs.org/jmc
Discovery of Highly Isoform Selective Thiazolopiperidine Inhibitors of Phosphoinositide 3‑Kinase γ Philip N. Collier,* David Messersmith, Arnaud Le Tiran,† Upul K. Bandarage, Christina Boucher, Jon Come, Kevin M. Cottrell, Veronique Damagnez, John D. Doran, James P. Griffith, Suvarna Khare-Pandit, Elaine B. Krueger, Mark W. Ledeboer, Brian Ledford, Yusheng Liao, Sudipta Mahajan, Cameron S. Moody, Setu Roday, Tiansheng Wang, Jinwang Xu, and Alex M. Aronov Vertex Pharmaceuticals Inc., 50 Northern Avenue, Boston, Massachusetts 02210, United States S Supporting Information *
ABSTRACT: A series of high affinity second-generation thiazolopiperidine inhibitors of PI3Kγ were designed based on some general observations around lipid kinase structure. Optimization of the alkylimidazole group led to inhibitors with higher levels of PI3Kγ selectivity. Additional insights into PI3K isoform selectivity related to sequence differences in a known distal hydrophobic pocket are also described.
P
been made toward this goal.19−26 In this paper, we describe the design and synthesis of highly isoform selective inhibitors of PI3Kγ. In contrast to protein kinases, inhibitors with spatially more demanding hinge binding motifs are more frequently accommodated in the ATP binding site of lipid kinases. The prototypical pan-PI3K inhibitor LY294002, for example, possesses a morpholine hinge-binding unit. This is related to the observation that there is more space available deep within the ATP binding site in lipid kinases relative to protein kinases because of a slight shift in the orientation of the gatekeeper residue (Ile879 in PI3Kγ) relative to its counterpart in protein kinases (Figure 1a).27,28 Recently, we described a new family of potent and isoform selective benzothiazole inhibitors of phosphoinositide 3-kinase γ (PI3Kγ).29 On the basis of the above observations, we hypothesized that a scaffold modification from a benzothiazole to a spatially more demanding and less lipophilic thiazolopiperidine core should be accommodated and that this change should also lead to better druglike properties (Figure 1b).30,31 From our previous work on the benzothiazole scaffold, it was evident that one of the main drivers for high PI3Kγ affinity in that class was the C6 position substituent, so we were mindful of the fact that the proper positioning of the thiazolopiperidine R2 group in 2 would likely be a critical determinant of PI3Kγ affinity. Indeed, the key interactions of substituents on the benzothiazole C6 aryl group, specifically with Tyr867 and Lys883, would have to be maintained. A four-step synthetic sequence was used for the preparation of compounds described herein. The key step in the synthesis of the thiazolopiperidine scaffold was a condensation reaction
hosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that catalyze the phosphorylation of the inositol ring of phosphoinositides, key secondary messengers involved in a variety of cellular functions. Through this phosphorylation, PI3Ks control the growth, proliferation, and survival of cells. PI3Ks are divided into three classes (I, II, and III) based on substrate specificity, sequence homology, and types of regulatory subunits. Class I, the most extensively studied subfamily, is further divided into class Ia (α, β, δ) and class Ib (γ), the latter differentiating itself from class Ia members in that its activation is almost exclusively driven through GPCRs.1−5 The intricacies of PI3Kγ signaling are still being elucidated, but PI3Kγ is known to be an important player in a number of biological processes. For example, it has been reported that PI3Kγ activity is a requirement for optimal T-cell activation and differentiation.6 It also regulates the production of reactive oxygen species in neutrophils and plays a role in the migration of both neutrophils and macrophages.7,8 As a result, PI3Kγ has been investigated as a drug target for inflammation and autoimmune disease.9,10 There is also more recent evidence for the potential utility of PI3Kγ inhibitors for the treatment of cancer11 and cardiovascular disease.12−14 From a drug development perspective, any potential toxicity concerns of inhibiting an important signaling enzyme such as PI3Kγ are somewhat alleviated by the fact that PI3Kγ expression is mainly restricted to the hematopoietic system. The PI3Kγ knockout mouse is viable, fertile and shows no adverse phenotype.15−17 Class Ia PI3Ks have been implicated in insulin signaling; thus, selectivity over these, particularly the ubiquitously expressed α isoform, is a requirement in the treatment of chronic disease.18 Consequently, there has been much interest in the development of selective PI3Kγ inhibitors. This is a challenging task due to the highly conserved nature of the ATP binding sites in class I PI3K isoforms, but progress has © XXXX American Chemical Society
Received: March 27, 2015
A
DOI: 10.1021/acs.jmedchem.5b00498 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Brief Article
of the thiazolopiperidine scaffold manifested themselves in better druglike properties. For example, compound 4 had thermodynamic solubilities of 12.1 and 9.4 μg/mL in water and FaSSIF (pH 6.5), respectively, in comparison to values of 1.6 and 4
>4 >4
>4 3.2
>4 >4
>4 >4
>4 >4
>4 >4
3.6 >4
a Compound 16 shows >50% inhibition at 2 μM against 0/65 kinases from a wider panel. Compound 17 shows >25% inhibition at 2 μM against 0/50 kinases. a
Ki in units of μM. bFrom ref 29.
compounds also showed excellent selectivity against a wider panel of kinases (e.g., >50% inhibition at 2 μM against 0/65 kinases for compound 16) and no cross activity was observed for 16 against a diverse set of 34 non-kinase targets (see Supporting Information for full details). A typical thiazolopiperidine such as 17 inhibits MCP-1 induced chemotaxis of THP-1 cells with an IC50 of 185 nM (Table 4) and does not effect the viability or inhibit the proliferation of cells that have been shown to express other PI3K isoforms, in contrast to a less selective compound (22)29 from the earlier scaffold. Finally, moderate to good microsomal stability was observed with the thiazolopiperidine scaffold, e.g., compound 17, with 61%, 84%, and 100% remaining after 30 min in human, dog, and rat liver microsomal preparations, respectively, and this compared favorably with the benzothia-
selectivity observed with chain extended ureas based around PI3K sequence differences at residue Ala885.29 The high selectivity over the α isoform is driven by an unfavorable interaction of the difluoroethyl group of 16 with Asp798 in PI3Kα; in PI3Kγ, this residue is Gly829.29 We hypothesize that the general increase in affinity of the fluoroalkylimidazole subseries is driven by a weakly attractive orthogonal C−F···C O interaction of 3.6 Å with Thr827 in PI3Kγ (Figure 3). Multiple SAR examples have been reported where an increase in binding affinity was obtained when this interaction was present.32 An attractive interaction of the acidic methine protons of the fluoroalkyl and difluoralkyl groups with Glu814 (3.3 Å) may explain the observed isoform selectivity enhanceC
DOI: 10.1021/acs.jmedchem.5b00498 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Brief Article
(5) Okkenhaug, K.; Vanhaesebroeck, B. PI3K in lymphocyte development, differentiation and activation. Nat. Rev. Immunol. 2003, 3, 317−330. (6) Ladygina, N.; Gottipati, S.; Ngo, K.; Castro, G.; Ma, J.-Y.; Banie, H.; Rao, T. S.; Fung-Leung, W.-P. PI3Kγ kinase activity is required for optimal T-cell activation and differentiation. Eur. J. Immunol. 2013, 43, 3183−3196. (7) Laffargue, M.; Calvez, R.; Finan, P.; Trifilieff, A.; Barbier, M.; Altruda, F.; Hirsch, E.; Wymann, M. P. Phosphoinositide 3-kinase γ is an essential amplifier of mast cell function. Immunity 2002, 16, 441− 451. (8) Reif, K.; Okkenhaug, K.; Sasaki, T.; Penninger, J. M.; Vanhaesebroeck, B.; Cyster, J. G. Differential roles for phosphoinositide 3-kinases, p110γ, and p110δ, in lymphocyte chemotaxis and homing. J. Immunol. 2004, 173, 2236−2240. (9) Wymann, M. P.; Björklöf, K.; Calvez, R.; Finan, P.; Thomast, M.; Trifilieff, A.; Barbier, M.; Altruda, F.; Hirsch, E.; Laffargue, M. Phosphoinositide 3-kinase γ: a key modulator in inflammation and allergy. Biochem. Soc. Trans. 2003, 31, 275−280. (10) Ghigo, A.; Damilano, F.; Braccini, L.; Hirsch, E. PI3K inhibition in inflammation: toward tailored therapies for specific diseases. BioEssays 2010, 32, 185−196. (11) Falasca, M.; Maffucci, T. Targeting p110gamma in gastrointestinal cancers: attack on multiple fronts. Front. Physiol. 2014, 5, 391−400. (12) Smirnova, N. F.; Gayral, S.; Pedros, C.; Loirand, G.; Vaillant, N.; Malet, N.; Kassem, S.; Calise, D.; Goudounèche, D.; Wymann, M. P.; Hirsch, E.; Gadeau, A.-P.; Martinez, L. O.; Saoudi, A.; Laffargue, M. Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response. J. Exp. Med. 2014, 211, 1779−1792. (13) Gayral, S.; Garnotel, R.; Castaing-Berthou, A.; Blaise, S.; Fougerat, A.; Berge, E.; Montheil, A.; Malet, N.; Wymann, M. P.; Maurice, P.; Debelle, L.; Martiny, L.; Martinez, L. O.; Pshezhetsky, A. V.; Duca, L.; Laffargue, M. Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway. Cardiovasc. Res. 2014, 102, 118−127. (14) Chang, J. D.; Sukhova, G. K.; Libby, P.; Schvartz, E.; Lichtenstein, A. H.; Field, S. J.; Kennedy, C.; Madhavarapu, S.; Luo, J.; Wu, D.; Cantley, L. C. Deletion of the phosphoinositide 3-kinase p110γ gene attenuates murine atherosclerosis. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 8077−8082. (15) Sasaki, T.; Irie-Sasaki, J.; Jones, R. G.; Oliveira-dos-Santos, A. J.; Stanford, W. L.; Bolon, B.; Wakeham, A.; Itie, A.; Bouchard, D.; Kozieradzki, I.; Joza, N.; Mak, T. W.; Ohashi, P. S.; Suzuki, A.; Penninger, J. F. Function of PI3Kγ in thymocyte development, T cell activation, and neutrophil migration. Science 2000, 287, 1040−1046. (16) Hirsch, E.; Katanaev, V. L.; Garlanda, C.; Azzolino, O.; Pirola, L.; Silengo, L.; Sozzani, S.; Mantovani, A.; Altruda, F.; Wymann, M. P. Central role for G protein-coupled phosphoinositide 3-kinase γ in inflammation. Science 2000, 287, 1049−1053. (17) Li, Z.; Jiang, H.; Xie, W.; Zhang, Z.; Smrcka, A. V.; Wu, D. Roles of PLC-β2 and -β3 and PI3Kγ in chemoattractant-mediated signal transduction. Science 2000, 287, 1046−1049. (18) Ueki, K.; Yballe, C. M.; Brachmann, S. M.; Vicent, D.; Watt, J. M.; Kahn, C. R.; Cantley, L. C. Increased insulin sensitivity in mice lacking p85β subunit of phosphoinositide 3-kinase. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 419−424. (19) Ameriks, M. K.; Venable, J. D. Small molecule Inhibitors of phosphoinositide 3-kinase (PI3K) δ and γ. Curr. Top. Med. Chem. 2009, 9, 738−753. (20) Pomel, V.; Klicic, J.; Covini, D.; Church, D. D.; Shaw, J. P.; Roulin, K.; Burgat-Charvillon, F.; Valognes, D.; Camps, M.; Chabert, C.; Gillieron, C.; Françon, B.; Perrin, D.; Leroy, D.; Gretener, D.; Nichols, A.; Vitte, P. A.; Carboni, S.; Rommel, C.; Schwarz, M. K.; Rückle, T. Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase γ. J. Med. Chem. 2006, 49, 3857−3871.
Table 4. Cellular Selectivity for a Typical Thiazolopiperidinea THP-1
a
HUVEC
B cell
MCF7
MCP-1 stim.
CSF-1 stim.
compd
γ
α/β/δ
α/β
δ
α
22b 17
0.083 0.185
10 >20
ND 7.37
1.5 >20
6.4 >20
In units of μM. bFrom ref 29.
zole first generation scaffold (corresponding data for compound 22:29 51, 66, ND). In summary, we have discovered potent and highly isoform selective thiazolopiperidine inhibitors of PI3Kγ. Lipid kinase structural analysis was critical in guiding the scaffold design process. Insight into the origin of the high selectivity was gained from the combination of molecular modeling studies and analysis of PI3K sequence differences, information that may aid the discovery of new isoform selective agents. The compounds reported in this paper are among the most isoform selective, potent PI3Kγ inhibitors yet identified.
■
ASSOCIATED CONTENT
S Supporting Information *
Synthetic schemes and experimental procedures, characterization of organic molecules, biochemical and cellular assays, solubility assay, crystallographic information, and a csv file containing molecular formula strings. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00498.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-617-341-6921. E-mail:
[email protected]. Present Address †
A.L.T.: Institut de Recherche Servier, 3 Rue de la République, 92150 Suresnes, France. Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS The authors thank Drs. B. Davis and J. Williams for assistance in collecting some experimental data. ABBREVIATIONS USED FaSSIF, fasted state simulated intestinal fluid; GPCR, Gprotein-coupled receptor; ND, no data recorded; MCP-1, monocyte chemoattractant protein 1; HUVEC, human umbilical vein endothelial cell
■
REFERENCES
(1) Cantley, L. C. The phosphoinositide 3-kinase pathway. Science 2002, 296, 1655−1657. (2) Wymann, M. P.; Pirola, L. Structure and function of phosphoinositide 3-kinases. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1998, 1436, 127−150. (3) Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.; Bilanges, B. The emerging mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 2010, 11, 329−341. (4) Hawkins, P. T.; Anderson, K. E.; Davidson, K.; Stephens, L. R. Signalling through class 1 PI3Ks in mammalian cells. Biochem. Soc. Trans. 2006, 34, 647−662. D
DOI: 10.1021/acs.jmedchem.5b00498 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Brief Article
(21) Cushing, T. D.; Metz, D. P.; Whittington, D. A.; McGee, L. R. PI3Kδ and PI3Kγ as targets for autoimmune and inflammatory diseases. J. Med. Chem. 2012, 55, 8559−8581. (22) Bell, K.; Sunose, M.; Ellard, K.; Cansfield, A.; Taylor, J.; Miller, W.; Ramsden, N.; Bergami, G.; Neubauer, G. SAR studies around a series of triazolopyridines as potent and selective PI3Kγ inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 5257−5263. (23) Oka, Y.; Yabuuchi, T.; Fujii, Y.; Ohtake, H.; Wakahara, S.; Matsumoto, K.; Endo, M.; Tamura, Y.; Sekiguchi, Y. Discovery and optimization of a series of 2-aminothiazole-oxazoles as potent phosphoinositide 3-kinase γ inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 7534−7538. (24) Oka, Y.; Yabuuchi, T.; Oi, T.; Kuroda, S.; Fujii, Y.; Ohtake, H.; Inoue, T.; Wakahara, S.; Kimura, K.; Fujita, K.; Endo, M.; Taguchi, K.; Sekiguchi, Y. Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,4oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (TASP0415914) as an orally potent phosphoinositide 3-kinase γ inhibitor for the treatment of inflammatory dieases. Bioorg. Med. Chem. 2013, 21, 7578−7583. (25) Leahy, J. W.; Buhr, C. A.; Johnson, H. W. B.; Gyu Kim, B.; Baik, T.; Cannoy, J.; Forsyth, T. P.; Jeong, J. W.; Lee, M. S.; Ma, S.; Noson, K.; Wang, L.; Williams, M.; Nuss, J. M.; Brooks, E.; Foster, P.; Goon, L.; Heald, N.; Holst, C.; Jaeger, C.; Lam, S.; Lougheed, J.; Nguyen, L.; Plonowski, A.; Song, J.; Stout, T.; Wu, X.; Yakes, M. F.; Yu, P.; Zhang, W.; Lamb, P.; Raeber, O. Dicovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase γ inhibitors. J. Med. Chem. 2012, 55, 5467−5482. (26) Sunose, M.; Bell, K.; Ellard, K.; Bergamini, G.; Neubauer, G.; Werner, T.; Ramsden, N. Discovery of 5-(2-amino-[1,2,4]triazolo[1,5a]pyridin-7-yl)-N-(tert-butyl)pyridine-3-sulfonamide (CZC24758), as a potent, orally bioavailable and selective inhibitor of PI3K for the treatment of inflammatory disease. Bioorg. Med. Chem. Lett. 2012, 22, 4613−4618. (27) Aspel, B. Dual-Specificity Inhibitors of Lipid and Protein Kinases; ProQuest LLC: Ann Arbor, MI, 2008; p 14. (28) Knight, Z. A.; Shokat, K. M. Chemically targeting the PI3K family. Biochem. Soc. Trans. 2007, 35, 245−249. (29) Collier, P. N.; Martinez-Botella, G.; Cornebise, M.; Cottrell, K. M.; Doran, J. D.; Griffith, J. P.; Mahajan, S.; Maltais, F.; Moody, C. S.; Huck, E. P.; Wang, T.; Aronov, A. M. Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3kinase γ. J. Med. Chem. 2015, 58, 517−521. (30) Lovering, F.; Bikker, J.; Humblet, C. Escape from flatland: increasing saturation as an approach to improving clinical success. J. Med. Chem. 2009, 52, 6752−6756. (31) Ritchie, T. J.; Macdonald, S. J. F. The impact of aromatic ring count on compound developability − are too many aromatic rings a liability in drug design? Drug Discovery Today 2009, 14, 1011−1020. (32) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med. Chem. 2010, 53, 5061−5084.
E
DOI: 10.1021/acs.jmedchem.5b00498 J. Med. Chem. XXXX, XXX, XXX−XXX
