Denise Rageot

Recent Advances in Iridium-Catalyzed Asymmetric Hydrogenation : New Catalysts, Substrates and Applications in Total Synthesis

Iridium-catalyzed asymmetric hydrogenation has emerged as a highly efficient method for the synthesis of enantiomerically pure compounds. This account summarizes our recent efforts in this field. We have developed a new type of P,O-ligand that was successfully applied to the asymmetric hydrogenation of α,β-unsaturated carbonyl compounds. Furthermore we have demonstrated the potential of known iridium catalysts in the hydrogenation of α,β-unsaturated boronic esters. And finally we could demonstrate the utility of iridium-catalyzed asymmetric hydrogenation in the formal synthesis of the natural product Platensimycin.

5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compounds safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models. Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. ©2017 AACR.

The novel, catalytic mTORC1/2 inhibitor PQR620 and the PI3K/mTORC1/2 inhibitor PQR530 effectively cross the blood-brain barrier and increase seizure threshold in a mouse model of chronic epilepsy

Abstract The mTOR signaling pathway has emerged as a possible therapeutic target for epilepsy. Clinical trials have shown that mTOR inhibitors such as everolimus reduce seizures in tuberous sclerosis complex patients with intractable epilepsy. Furthermore, accumulating preclinical data suggest that mTOR inhibitors may have anti‐seizure or anti‐epileptogenic actions in other types of epilepsy. However, the chronic use of rapalogs such as everolimus is limited by poor tolerability, particularly by immunosuppression, poor brain penetration and induction of feedback loops which might contribute to their limited therapeutic efficacy. Here we describe two novel, brain‐permeable and well tolerated small molecule 1,3,5‐triazine derivatives, the catalytic mTORC1/C2 inhibitor PQR620 and the dual pan‐PI3K/mTOR inhibitor PQR530. These derivatives were compared with the mTORC1 inhibitors rapamycin and everolimus as well as the anti‐seizure drugs phenobarbital and levetiracetam. The anti‐seizure potential of these compounds was determined by evaluating the electroconvulsive seizure threshold in normal and epileptic mice. Rapamycin and everolimus only poorly penetrated into the brain (brain:plasma ratio 0.0057 for rapamycin and 0.016 for everolimus). In contrast, the novel compounds rapidly entered the brain, reaching brain:plasma ratios of ˜1.6. Furthermore, they significantly decreased phosphorylation of S6 ribosomal protein in the hippocampus of normal and epileptic mice, demonstrating effective mTOR inhibition. PQR620 and PQR530 significantly increased seizure threshold at tolerable doses. The effect of PQR620 was more marked in epileptic vs. nonepileptic mice, matching the efficacy of levetiracetam. Overall, the novel compounds described here have the potential to overcome the disadvantages of rapalogs for treatment of epilepsy and mTORopathies directly connected to mutations in the mTOR signaling cascade. HighlightsThe mTOR signaling pathway has emerged as a therapeutic target for epilepsy.However, clinically used mTOR inhibitors exhibit poor brain penetration and tolerability.Two novel inhibitors of mTORC1/2 or the PI3K/mTORC1/2 pathway are presented.Both compounds cross the blood‐brain barrier more effectively than everolimus.Both compounds increase seizure threshold in a mouse model of difficult‐to‐treat epilepsy.

Discovery and Preclinical Characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (PQR620), a Highly Potent and Selective mTORC1/2 Inhibitor for Cancer and Neurological Disorders

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, maximum concentration ( Cmax) in plasma and brain was reached after 30 min, with a half-life ( t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

Abstract 153: Tricyclic fused pyrimidinopyrrolo-oxazines reveal conformational preferences of morpholine for PI3K hinge region binding

Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases, produce PtdIns(3,4,5)P3 and trigger intracellular signaling pathways that are vital to cell growth, proliferation, survival and migration. Constitutive activation of PI3K is frequently observed in many tumor types, which defines PI3K as a valuable drug target in oncology.1 Numerous PI3K inhibitors in clinical development contain a morpholine moiety that mediates hinge region binding in the ATP pocket of PI3K by a hydrogen bond with the active site valine backbone nitrogen (Val851 in PI3Kα)2. We present here novel pyrimidinopyrrolo-oxazines related to the clinically advanced, pyridinylmorpholine and triazinylmorpholine derived pan-PI3K/mTOR inhibitors BKM120 and PQR309. The novel fused tricyclic core of these compounds contains two morpholine moieties of which one is conformationally restricted by the introduction of a methylene bridge that links the pyrimidine core with one of the two morpholine moieties. This modification leads to the generation of two regioisomers, each existing as a set of enantiomers. We investigated the influence of this conformational restriction on PI3K inhibitory activity and analyzed the distinct selectivity profiles and potencies of the respective stereo- and regio-isomers. The design and preparation of specific compounds in combination with biological assays (phosphorylation of PKB and S6, binding affinity to p110α), structure-activity relationship (SAR) and molecular modelling studies allowed us to understand the binding mode of these compounds and acquire valuable information that potentially lead to the development of derivatives with a distinct selectivity profile (e.g. PI3K versus mTOR). A selection of compounds demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values in the nanomolar range and high inhibitory potency of all PI3K isoforms (Ki(p110α) > 40 nM). Single p.o. administration of our lead compound to SD rats resulted in good oral bioavailability as well as excellent brain penetration. Furthermore, mechanism of action-based increases in glucose levels and insulin levels have been observed. In conclusion, we present here the development, optimization, preparation and biological evaluation of a novel class of potent, orally available and brain-penetrant pan-PI3K inhibitors that represent an innovative extension to known pyrimidinomorpholine derived PI3K inhibitors. Moreover, our results add to the understanding of how introducing specific structural and conformational modifications can lead to the development of optimized, selective PI3K and mTOR inhibitors. [1] Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. Nat. Rev. Cancer 2015, 15, 7-24. [2] Andrs, M.; Korabecny, J.; Jun, D.; Hodny, Z.; Bartek, J.; Kuca, K. J. Med. Chem. 2015, 58, 41-71. Citation Format: Alexander M. Sele, Denise Rageot, Florent Beaufils, Anna Melone, Thomas Bohnacker, Eileen Jackson, Jean-Baptiste Langlois, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Tricyclic fused pyrimidinopyrrolo-oxazines reveal conformational preferences of morpholine for PI3K hinge region binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2017-153

Abstract 1364: Novel 4-(pyrimidin-2-yl)morpholines targeting the colchicine-binding site of tubulin

Microtubules are dynamic polymers and integral components of the cytoskeleton. They play important roles in the regulation of cellular signaling, motility, maintenance of cell shape, secretion, intercellular transport and spindle formation during mitosis. 1 As a consequence, small molecules that interfere with the dynamics of microtubules have been recognized as powerful tools for the treatment of cancer. 2, 3 A systematic structure-activity relationship (SAR) study starting from morpholino-substituted biheteroaryls with moderate microtubule disrupting activities allowed the optimizations of biological activity, metabolic stability, and drug-like properties. Interestingly, the type of core (aryl, pyridine or pyrimidine) was of importance, as well as the regioisomeric arrangement of the pyrimidine nitrogens. Pyrimidines substituted with four- or five-membered N-heterocycles proved to be superior both in terms of biological activity and metabolic stability. Finally, optimization of the heteroaryl substituent of the pyrimidine derivatives culminated in the identification of a novel series of substituted 4-(pyrimidin-2-yl)morpholines targeting microtubule polymerization in the nM range. Selected compounds potently inhibit cellular microtubule polymerization with EC 50 values of 20-90 nM. This was confirmed by phosphorylation of Histone3, nuclear DNA condensation, and cell cycle arrest in G2/M or induction of cell death across multiple cell lines. Moreover, substituted 4-(pyrimidin-2-yl)morpholines were shown to be poor substrates for P-gp multi-drug resistance pumps, and therefore caused efficiently mitotic arrest and cell death in colchicine-resistant cells. The co-crystal structure of tubulin with selected compounds showed that 4-(pyrimidin-2-yl)morpholines bind to the colchicine pocket located between the α and β subunits of the αβ-tubulin dimer. Relevant inhibitor contact residues include Lys352, Met259, Ala316, Leu248, Val238, Tyr202 and Cys241 of β-tubulin. Moreover, two water molecules link the morpholine oxygen to the β-tubulin bound GTP. Conformational changes induced by inhibitor binding suggest that free or plus end β-tubulin is targeted by this compound series. Pre-clinical studies characterized a lead compound selection with excellent stability in human hepatocytes, and human, mouse and rat microsomes. Overall, these compounds qualify as a novel class of microtubule destabilizing agents that target the colchicine-binding site, and which warrant further investigations currently in progress (PK studies, xenograft tumor mouse models). 1) Walczak, C. E, Curr. Opin. Cell Biol. 2000, 12, 52-56. 2) Risinger, A. L.; Giles, F. J.; Mooberry, S. L, Cancer Treat. Rev. 2009, 35, 255-261. 3) Downing, K. H.; Nogales, E., Curr. Opin. Struct. Biol. 1998, 8, 785-791. Citation Format: Alexander M. Sele, Denise Rageot, Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, Michel O. Steinmetz, Matthias P. Wymann. Novel 4-(pyrimidin-2-yl)morpholines targeting the colchicine-binding site of tubulin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1364.

Abstract 1336: Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor

Mammalian target of rapamycin (mTOR) signaling pathway plays a fundamental role in cell proliferation, differentiation, growth and survival.[1] As a consequence, various tumors and central nervous system (CNS) disorders share aberrant activation of the mTOR pathway. Drugs targeting the mTOR pathway represent therefore a valuable path to address multiple therapeutic areas.[1-2] Here, we report the lead optimization of PQR620, a novel potent and selective brain penetrant inhibitor of mTORC1/2. The development of selective mTOR inhibitors is particularly challenging due to extensively conserved amino acid residues in the ATP binding pocket within the PI3K and PI3K-related protein kinase family. Here, we present a detailed ligand-based structure activity relationship study allowing selective targeting of mTOR kinase activity without the interference of other PI3K family members. Systematic variation of the hinge region and affinity binding motifs led to the identification of PQR620, a morpholino-triazinyl derivative, as potent and selective mTOR inhibitor. Substitution of the morpholine binding to the hinge region and introduction of a 2-aminopyridine, substituted with a difluoromethyl group, induced a >1000-fold selectivity towards mTOR over PI3Kα in enzymatic binding assays. In A2058 melanoma cells PQR620 demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values of 0.2 μM and 0.1 μM, respectively. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR620 demonstrated its potency to prevent cancer cell growth in an NTRC 44 cancer cell line panel, resulting in a 10log(IC50) of 2.86 (nM). Further pharmacological properties and in vivo efficacy of PQR620 are presented in detail in Ref. [3]. The preparation of PQR620 was optimized towards a robust synthetic route involving only 4 steps, allowing for a rapid access to quantities required for pre-clinical testing. In conclusion, PQR620 inhibits mTOR potently and selectively, and shows anti-tumor effects in vitro and in vivo. PQR620 is currently in pre-clinical development. [1] M. Laplante, D. Sabatini, Cell 2012, 149, 274-293. [2] Z. Z. Chong, Y. C. Shang, L. Zhang, S. Wang, K. Maiese, Oxid. Med. Cell. Longev. 2010, 3, 374–391. [3] F. Beaufils, D. Rageot, A. Melone, A. M. Sele, M. Lang, J. Mestan, R. A. Ettlin, P. Hillmann, V. Cmiljanovic, C. Walter, E. Singer, H. P. Nguyen, P. Hebeisen, D. Fabbro, M. P. Wymann, “Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620” presented at AACR Annual Meeting 2016, April 16-20, New Orleans, Louisiana, USA. Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Marc Lang, Jurgen Mestan, Vladimir Cmiljanovic, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1336.

Abstract 393A: Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620

Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD = 150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD = 30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t 1/2 ) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC 0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing. Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016 Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Alexander Sele, Marc Lang, Juergen Mestan, Robert A. Ettlin, Petra Hillmann, Vladimir Cmiljanovic, Carolin Walter, Elisabeth Singer, Hoa HP Nguyen, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393A.