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Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Electron donor–acceptor (EDA) complexes can controllably generate radicals under mild conditions through selective photoexcitation events. However, unproductive reactivity from fast deactivation of...

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

Electron donor-acceptor (EDA) complexes can controllably generate radicals under mild conditions through selective photoexcitation events. However, unproductive reactivity from fast deactivation of the photoexcited complexes through back electron transfer has slowed the development of EDA complexes in synthetic methodology. Here, we disclose the study of EDA complexes derived from 2-methoxynaphthalene donor and acylated ethyl isonicotinate N-oxide acceptor that undergo a fast N–O bond fragmentation event upon photoexcitation. This reaction design not only overcomes the limitations of back electron transfer but also enables the regeneration of the donor species, representing a rare example EDA photochemistry in a catalytic regime. The synthetic utility is demonstrated through visible light-driven radical trifluoromethylation and Minisci alkylation reactions. The scalability of the EDA complex promoted reaction evidenced by the successful multigram-scale trifluoromethylation of methyl N-Boc pyrrole-2-carboxylate in a continuous flow manifold.

A net redox-neutral method for the decarboxylative alkylation of heteroarenes using photoredox catalysis is reported. Additionally, this method features the use of simple, commercially available carboxylic acid derivatives as alkylating agents, enabling the facile alkylation of a variety of biologically-relevant heterocyclic scaffolds under mild conditions.

Cells in the solid tumor microenvironment are frequently exposed to hypoxic conditions, necessitating molecular adaptations for survival. Of particular importance are transcriptional changes mediated by heterodimeric Hypoxia-Inducible Factor (HIF) proteins that consist of an oxygen-regulated α monomer (either HIF-1α, -2α, and -3α) coupled to a constitutively expressed β monomer (HIF-1β/ARNT). In normal oxygen conditions, HIF-2α is degraded following ubiquitination by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex. Exposure to hypoxia, VHL mutation, or epigenetic silencing of pVHL leads to HIF-2α stabilization and transcription of pro-tumorigenic gene sets in both cancer and non-cancer cells. Inhibition of HIF-2α has been shown clinically to be an effective strategy to mitigate tumor growth, particularly in patients suffering from VHL disease or clear cell renal cell carcinoma (ccRCC), a cancer that has a particularly high prevalence of pVHL dysfunction. Applying a pharmacophore mapping and structure-based design approach, we identified a novel and potent small molecule HIF-2α inhibitor, AB521. AB521 avidly binds the HIF-2α PAS-B domain, preventing HIF-2α-mediated gene transcription. AB521 is characterized by a favorable preclinical pharmacokinetic profile and is projected to be suitable for once-daily dosing in humans. When delivered orally in mice, AB521 significantly regressed established 786-O xenograft tumors and decreased pharmacodynamic markers associated with HIF-2α in a dose-dependent manner. In vitro, AB521 potently inhibited HIF-2α-specific luciferase reporter transcription under high-serum conditions, VEGF protein secretion, colony formation in soft agar, and did not exhibit off-target cytotoxicity in 786-O cells. AB521 selectively inhibited HIF-2α-, but not HIF-1α-, mediated gene expression in hypoxic Hep3B hepatocellular carcinoma cells. AB521 also inhibited the transcriptional activity of endogenous HIF-2α in relevant human primary cell types, including endothelial cells and pro-tumorigenic M2-polarized macrophages. Importantly, inhibiting HIF-2α did not impact functionality of activated hypoxic human T cells, suggesting that AB521 would be favorable combination partner for I-O therapeutic agents. Indeed, expression of CD73, the primary enzyme responsible for synthesis of the immunosuppressive metabolite adenosine, was highly correlated with hypoxic signatures across several indications in publicly available bioinformatic datasets, suggesting combinations with adenosine pathway antagonists in ccRCC and beyond. In summary, AB521 is a novel and selective HIF-2α inhibitor with potent anti-tumor activity. Clinical evaluation of this molecule is expected to begin in the latter part of 2021. Citation Format: Kelsey E. Sivick Gauthier, Dana Piovesan, Soonweng Cho, Kenneth V. Lawson, Patrick G. Schweickert, Alejandra Lopez, Suan Liu, Timothy Park, Artur Mailyan, Jeremy T. A. Fournier, Joel W. Beatty, Samuel L. Drew, Jarek Kalisiak, Balint Gal, Guillaume Mata, Zhang Wang, Brandon R. Rosen, Clayton Hardman, Matthaw P. Epplin, Kai Yu, Karl T. Haelsig, Lixia Jin, Elaine Ginn, Jennie Au, Cesar A. Meleza, Joel Tencer, Amber Pham, Hyock J. Kwon, Stephen W. Young, Manmohan Leleti, Jay P. Powers, Matthew J. Walters. AB521 potently and selectively inhibits pro-tumorigenic gene transcription by Hypoxia-Inducible Factor (HIF)-2α in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P206.

A hypoxic environment is a common characteristic of solid tumors. Cancer cells adapt to hypoxia and become more aggressive by up-regulation of genes associated with metabolism, growth, proliferation, angiogenesis, and erythropoiesis. Hypoxia-inducible factors (HIFs) are the central driving force for the cellular response to hypoxia and regulate a vast array of these genes. HIFs are heterodimers composed of an oxygen-sensitive HIF-α subunit (HIF-1α, HIF-2α, and HIF-3α) and a constitutively expressed HIF-1β subunit. HIF-2α is constitutively synthesized and regulated in an oxygen-dependent manner. Proline residues present in the oxygen-dependent degradation domain of the HIF-2α subunit are hydroxylated and subject to ubiquitination via the von Hippel-Lindau (pVHL) E3 ligase complex for subsequent proteasomal degradation. Under hypoxia or pseudohypoxia, caused by loss of VHL function, this process is inhibited, allowing HIF-2α translocation to the nucleus where, in complex with HIF-1β/ARNT, it promotes transcription of various pro-tumorigenic gene sets. Inhibition of HIF-2α has been demonstrated to be an effective strategy to mitigate tumor growth in the clinic, particularly in clear cell renal cell carcinoma (ccRCC). Applying a pharmacophore mapping and structure-based design approach, we identified multiple novel series of small molecule HIF-2α inhibitors which avidly bind the HIF-2α PAS-B domain and disrupt dimerization with HIF-1β, preventing HIF-2α-mediated gene transcription. Interrogation of structure activity relationships and pharmacokinetic trends yielded highly optimized inhibitors, including AB521, which potently inhibits HIF-2α reporter transcription and VEGF secretion in a human ccRCC line. AB521 was confirmed to bind HIF-2α via multiple biochemical assays and inhibited HIF-2α-, but not HIF-1α-, mediated gene expression in a hepatocellular carcinoma cell line. Furthermore, AB521 is characterized by a favorable preclinical pharmacokinetic profile with high stability to human hepatocytes and no significant direct or time-dependent inhibition of the major CYP450 drug metabolizing enzymes. AB521 exhibits high bioavailability across preclinical species and is projected to possess a human pharmacokinetic profile suitable for once-daily dosing. In conclusion, we have discovered and extensively characterized a series of novel HIF-2α inhibitors, exemplified by AB521, which potently and selectively inhibits HIF-2α and possesses a favorable preclinical pharmacokinetic profile. The data described herein provides further support for the development of novel HIF-2α inhibitors for cancer therapy. Citation Format: Kenneth V. Lawson, Kelsey E. Sivick Gauthier, Artur K. Mailyan, Jeremy T. Fournier, Joel W. Beatty, Samuel L. Drew, Jarek Kalisiak, Balint Gal, Guillaume Mata, Zhang Wang, Xuelei Yan, Lixia Jin, Elaine Ginn, Dana Piovesan, Jennifer Au, Cesar A. Meleza, Stephen W. Young, Matthew J. Walters, Manmohan Leleti, Jay P. Powers. Discovery and characterization of AB521, a novel, potent, and selective hypoxia-inducible factor (HIF)-2α inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1206.

A method for the radical chlorodifluoromethylation of (hetero)arenes using chlorodifluoroacetic anhydride is reported. This operationally simple protocol proceeds under mild photochemical conditions with high functional group compatibility and complements the large body of literature for the trifluoromethylation of (hetero)arenes. Introduction of the chlorodifluoromethyl motif enables rapid diversification to a wide array of aromatic scaffolds. This work showcases the chlorodifluoromethyl group as an attractive entryway to otherwise synthetically challenging electron-rich difluoromethyl-(hetero)arenes. Furthermore, facile conversion of the CF(2)Cl moiety into the corresponding aryl-esters, gem-difluoroenones, and β-keto-esters is demonstrated.

Natural product modification with photoredox catalysis allows for mild, chemoselective access to a wide array of related structures in complex areas of chemical space, providing the possibility for novel structural motifs as well as useful quantities of less abundant congeners. While amine additives have been used extensively as stoichiometric electron donors for photocatalysis, the controlled modification of amine substrates through single-electron oxidation is ideal for the synthesis and modification of alkaloids. Here, we report the conversion of the amine (+)-catharanthine into the natural products (−)-pseudotabersonine, (−)-pseudovincadifformine, and (+)-coronaridine utilizing visible light photoredox catalysis.

The direct trifluoromethylation of (hetero)arenes is a process of high importance to the pharmaceutical industry. Many reagents exist for this purpose and have found widespread use in discovery efforts; however, the step-intensive preparation of these reagents and their corresponding cost have resulted in minimal use of these methods in large-scale applications. For the ready transition of direct trifluoromethylation methodologies to large-scale application, the further development of processes utilizing inexpensive CF3 sources available on a metric ton scale is highly desirable. We report the use of pyridine N-oxide derivatives in concert with trifluoroacetic anhydride to promote a high-yielding and scalable trifluoromethylation reaction. Key mechanistic insights include the observation of electron donor-acceptor complexes in solution as well as a high dependence on photon flux. These observations have culminated in the application of this chemistry on a kilogram scale, demonstrating the utility of this reagent combination for preparative applications.

The large number of reagents that have been developed for the synthesis of trifluoromethylated compounds is a testament to the importance of the CF3 group as well as the associated synthetic challenge. Current state-of-the-art reagents for appending the CF3 functionality directly are highly effective; however, their use on preparative scale has minimal precedent because they require multistep synthesis for their preparation, and/or are prohibitively expensive for large-scale application. For a scalable trifluoromethylation methodology, trifluoroacetic acid and its anhydride represent an attractive solution in terms of cost and availability; however, because of the exceedingly high oxidation potential of trifluoroacetate, previous endeavours to use this material as a CF3 source have required the use of highly forcing conditions. Here we report a strategy for the use of trifluoroacetic anhydride for a scalable and operationally simple trifluoromethylation reaction using pyridine N-oxide and photoredox catalysis to affect a facile decarboxylation to the CF3 radical., Trifluoromethylation is a key transformation, particularly for pharmaceuticals, but many reagents are expensive and difficult to scale up. Here, the authors show that trifluoroacetic anhydride can act as a CF3 source, allowing the radical reactions to be easily and inexpensively carried out at scale.

Conspectus While the use of visible light to drive chemical reactivity is of high importance to the development of environmentally benign chemical transformations, the concomitant use of a stoichiometric electron donor or acceptor is often required to steer the desired redox behavior of these systems. The low-cost and ubiquity of tertiary amine bases has led to their widespread use as reductive additives in photoredox catalysis. Early use of trialkylamines in this context was focused on their role as reductive excited state quenchers of the photocatalyst, which in turn provides a more highly reducing catalytic intermediate. In this Account, we discuss some of the observations and thought processes that have led from our use of amines as reductive additives to their use as complex substrates and intermediates for natural product synthesis. Early attempts by our group to construct key carbon–carbon bonds via free-radical intermediates led to the observation that some trialkylamines readily behave as efficient hydrogen atom donors under redox-active photochemical conditions. In the wake of in-depth mechanistic studies published in the 1970s, 1980s and 1990s, this understanding has in turn allowed for a systematic approach to the design of a number of photochemical methodologies through rational tuning of the amine component. Minimization of the C–H donicity of the amine additive was found to promote desired C–C bond formation in a number of contexts, and subsequent elucidation of the amine’s redox fate has sparked a reevaluation of the amine’s role from that of reagent to that of substrate. The reactivity of tertiary amines in these photochemical systems is complex, and allows for a number of mechanistic possibilities that are not necessarily mutually exclusive. A variety of combinations of single-electron oxidation, C–H abstraction, deprotonation, and β-scission result in the formation of reactive intermediates such as α-amino radicals and iminium ions. These processes have been explored in depth in the photochemical literature and have resulted in a firm mechanistic grasp of the behavior of amine radical cations in fundamental systems. Harnessing the synthetic potential of these transient species represents an ongoing challenge for the controlled functionalization of amine substrates, because these mechanistic possibilities may result in undesired byproduct formation or substrate decomposition. The presence of tertiary amines in numerous alkaloids, pharmaceuticals, and agrochemicals lends credence to the potential utility of this chemistry in natural product synthesis, and herein we will discuss how these transformations might be controlled for synthetic purposes.

The alkaloids (-)-pseudotabersonine (I), (-)-pseudovincadifformine (II) and (+)-coronaridine (III) are synthesized from commercially available (+)-catharanthine by Ir-catalyzed photoredox reaction.

[207802-45-7] C36H24N6RuCl2 (MW 712.59 anhydrous) InChI = 1S/3C12H8N2.2ClH.Ru/c3*1-3-9-5-6-10-4-2-8-14-12(10)11(9)13-7-1;;;/h3*1-8H;2*1H;/q;;;;;+2/p-2 InChIKey = UWXWBVKIJZGXQL-UHFFFAOYSA-L (visible light-absorbing photocatalyst used for single-electron transfer reactions)1 Alternative Names: [Ru(phen)3]Cl2, ruthenium-tris(1,10-phenanthroline), tris(1,10-phenanthroline)ruthenium(II) chloride, dichlorotris(1,10-phenanthroline)ruthenium(II). Physical Data: λmax = 447 nm,2, 3 redox potentials1 (vs. saturated calomel electrode (SCE), as 0 V in CH3CN); −0.87 V (RuIII/RuII*), +1.26 V (RuIII/RuII), +0.82 V (RuII*/RuI), and −1.36 V (RuII/RuI). Solubility: soluble in H2O, CH3CN, CH2Cl2, CH3OH, CH3CH2OH. Form Supplied in: [Ru(phen)3]Cl2 is a red to dark red or red-orange crystalline powder. The hydrate is commercially available from Sigma-Aldrich. Preparative Methods: the complex can be prepared from Ru(DMSO)4Cl2 and 1,10-phenanthroline,4 or alternatively can be obtained directly from RuCl3, 1,10-phenanthroline, and NaPO2H2 in 0.1 N aqueous HCl.5 Purification: recrystallized from distilled water. Handling, Storage, and Precautions: the catalyst should be stored in the dark under an inert atmosphere.

Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi , and current drugs are lacking in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have explored initial structure-activity relationships around a class of imidazole-based compounds. This profiling has uncovered compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits the T. cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of 4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for further optimization.