Vps34 is a phosphoinositide 3-kinase (PI3K) class III isoform that has attracted major attention over the recent years because of its role in autophagy. Herein we describe the biological characterization of SAR405, which is a low-molecular-mass kinase inhibitor of Vps34 (KD 1.5 nM). This compound has an exquisite protein and lipid kinase selectivity profile that is explained by its unique binding mode and molecular interactions within the ATP binding cleft of human Vps34. To the best of our knowledge, this is the first potent and specific Vps34 inhibitor described so far. Our results demonstrate that inhibition of Vps34 kinase activity by SAR405 affects both late endosome-lysosome compartments and prevents autophagy. Moreover, we show that the concomitant inhibition of Vps34 and mTOR, with SAR405 and the US Food and Drug Administration-approved mTOR inhibitor everolimus, results in synergistic antiproliferative activity in renal tumor cell lines, indicating a potential clinical application in cancer.

Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimidopyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology.

Indoles are ubiquitous motifs in pharmaceuticals as well as in important natural products. New and straightforward methods to access these substrates are thus always highly desirable. [1] In this context, the metal-catalyzed cycloisomerization of polyunsaturated precursors is an ideal process to be explored. One of the main strategies has consisted of a 5-endo-dig metal-catalyzed [2,3] cyclization of acetylenic derivatives (Scheme 1). Ortho-Halogenoanilines constitute valuable starting materials for the synthesis of substrates 1 and can even be used to generate in situ the akynylaryl species by a Sonogashira-type coupling reaction prior to cyclization.[2d] A recent variant based on imines has also been reported. [4] To the best of our knowledge, the alternative 5-exo-dig isomerization approach from precursors 2 has received much less attention, [5] and we decided to examine this potentially new route.Platinum(II)-based catalysis has recently witnessed a tremendous development which has led to new synthetic methods [6] as well as versatile applications in the total synthesis [7] of natural products and asymmetric catalysis.[8]Recently, we reported on the use of allenyne and enynamide partners, [9] and showed that the substituent at the propargylic position had a dramatic influence on the course of the PtCl 2 -catalyzed cycloisomerization of various enyne systems.[10] To examine the scope of the reaction and to generate diverse platforms we have thus investigated flexible propargylic precursors of type 3 [11,12] (Scheme 1) on which we can easily vary the oxygen, nitrogen, and alkyne substituents (X, R 1 , and R 2 ).Our initial studies involving substrate 3 a (Scheme 2, Eq. (1) were highly encouraging, and enabled indole 4 a to be isolated in 91 % yield. We next examined more challenging precursors. Gratifyingly, N,N-diallyl precursor 3 b also underwent the transformation [Scheme 2, Eq. (2)]. In this case, an additional transfer of an allyl group from the nitrogen to the terminal alkyne carbon atom occurred (Scheme 2, entry 1). This formally constitutes an aminoallylation of the triple bond followed by an isomerization of the unsaturated bond. An analogous allyl transfer has been previously described by Fürstner et al. in the synthesis of furan derivatives. However, in this case, stabilization of the vinyl-metal intermediate via an enolate species seems necessary since only acetylenic Scheme 1. Transition-metal-catalyzed formation of indoles from o-alkynylanilines and o-propargylanilines. M = metal.Scheme 2. Indole formation and allyl transfer.

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a pro-survival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents, including acquired resistance. We aimed to identify small-molecule autophagy inhibitors using a HTS/HCA approach through a phenotypic, cell image-based assay, in order to screen multiple biological targets simultaneously and to screen compounds in a physiologically relevant environment. LC3 is a component of the autophagosome, which undergoes a cytoplasmic redistribution from diffuse to punctate dots during autophagy. We employed HeLa cells stably expressing EGFP-LC3 in a primary phenotypic screen. As a first step, a “Validation Library” of about 8,000 pre-selected compounds, about 25% of which had known biological activity and the others representing a range of chemical structures, was run in duplicate both to assess screening suitability and likely hit rate, and to give a valuable preview of possible active structures or biological targets. The primary screen of about 0.25 million compounds yielded around 10,500 positive compounds. These were tested in a suite of further cellular assays designed to eliminate unwanted positives, together with the application of chemi- and bioinformatics to pick out compounds with known biological activity. These processes enabled the selection of compounds that were the most promisingly active and specific. The screening “tree” identified, amongst others with as yet unidentified targets, chemical series active against autophagy-relevant biological targets ULK or Vsp34, validating the phenotypic screening methods selected. Finally, about 400 compounds were fully qualified after following this triage. The development of the assays, compound screening process and the compound triage is described.

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