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Chemical structures of compounds used in study

Supplementary Methods. Supplementary Figure 1. Somatic alterations in PPARG and RXRA are hallmarks of luminal bladder cancer. Supplementary Figure 2. PPARG pathway is activated by overexpression of RXRA S427F/S427Y, but not other mutant alleles in bladder cancer cells. Supplementary Figure 3. Representation of the effects of ligand-dependent modulation on the PPARG interactome. Supplementary Figure 4. Downregulation of FABP4 protein by treatment of PPARG-activated bladder cancer cell lines by inverse-agonist T0070907. Supplementary Figure 5. Genome engineering scheme for generating reporter cell line. Supplementary Figure 6. Basal expression of FABP4 is reduced by PPARG inverse agonists, but not antagonists. Supplementary Figure 7. Lipid metabolism genes are inhibited by PPARG inverse-agonists. Supplementary Figure 8. PPARG inverse-agonists inhibit proliferation of PPARG activated bladder cancer cell lines in clonogenic assays. Supplementary Figure 9. PPARG inverse-agonists, but not antagonists, inhibit proliferation of PPARG-activated bladder cancer cell lines. Supplementary Figure 10. Somatic alterations in RXRA and PPARG.

The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987–98. ©2017 AACR.

The crystal structure of the human selenocysteine-containing protein glutathione peroxidase 4 (GPX4) was determined at 1.0 Å resolution. A mass-spectrometry-based approach was developed to monitor the formation of adducts of the active-site selenocysteine Sec46 with covalent inhibitors. The crystal structure of Sec46-containing GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer] was determined at 1.54 Å resolution., Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Å resolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

Direct inhibition of GPX4 requires covalent modification of the active-site selenocysteine. While phenotypic screening has revealed that activated alkyl chlorides and masked nitrile oxides can inhibit GPX4 covalently, a systematic assessment of potential electrophilic warheads with the capacity to inhibit cellular GPX4 has been lacking. Here, we survey more than 25 electrophilic warheads across several distinct GPX4-targeting scaffolds. We find that electrophiles with attenuated reactivity compared to chloroacetamides are unable to inhibit GPX4 despite the expected nucleophilicity of the selenocysteine residue. However, highly reactive propiolamides we uncover in this study can substitute for chloroacetamide and nitroisoxazole warheads in GPX4 inhibitors. Our observations suggest that electrophile masking strategies, including those we describe for propiolamide- and nitrile-oxide-based warheads, may be promising for the development of improved covalent GPX4 inhibitors.

Direct inhibition of GPX4 requires covalent modification of the active-site selenocysteine. While phenotypic screening has revealed that activated alkyl chlorides and masked nitrile-oxides can inhibit GPX4 covalently, a systematic assessment of potential electrophilic warheads with the capacity to inhibit cellular GPX4 has been lacking. Here we survey more than 25 electrophilic warheads across several distinct GPX4-targeting scaffolds. Surprisingly, we find that electrophiles with attenuated reactivity compared to chloroacetamides are unable to target GPX4. The highly reactive propiolamide warheads we uncover in this study highlight the potential need for masking strategies similar to those we have described for nitrile-oxide-based GPX4 inhibitors. Finally, our observations that there are spatial requirements between warhead and scaffold for achieving optimal GPX4 targeting and that certain low-molecular-weight analogs inhibit GPX4 with selectivity suggest that rational design of GPX4 inhibitors may be a productive approach. The generation of ligand-bound crystal structures to facilitate such studies should therefore be prioritized by the field.

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4. Nitrile-oxide electrophiles were identified as covalent inhibitors of GPX4 that exhibit increased selectivity and reduced off-target effects relative to chloroacetamide-based inhibitors.

The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987–98. ©2017 AACR.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1–5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

The one-pot, three-component, coupling reaction of indoles/pyrroles, dimethyl malonate, and acetic acid was performed using Mn(III) acetate as an oxidant. In the presence of Mn(OAc)3, indole-2, and indole-3-carbonyl compounds were alkylated at the 3- and 2- positions, respectively, with subsequent oxidation and nucleophilic capture occurring at the newly formed benzylic carbon. In contrast, oxidation of 2- and 3-indole carboxylic acids afforded the corresponding 2-oxindol-3-ylidenes and 3-oxindol-2-ylidenes. The reaction conditions, scope, and mechanism are discussed herein.

The use of lithium bis-catechol borate (LiB(cat)(2)) as a reductive quencher for the photoredox mediated intermolecular C–H functionalization of various heteroaromatics with bromopyrroloindolines is described. LiB(cat)(2) offers a financial benefit over state-of-the-art quenchers currently in use while eliminating the side reactions that typically plague these couplings. The advantage of this methodology is highlighted by the synthesis of C3–C2′ (–) gliocladin C. Furthermore, additional examples of reactivity with various bromopyrroloindolines sets the stage for expedient routes towards other pharmaceutically active hexahydropyrroloindoline alkaloids and their analogues.

More and more new vehicle concepts are flooding the market in new areas worldwide. However, existing vehicle concepts cannot simply be transferred to other markets, due to the different user requirements and existing boundary conditions. For example, Africa is a continent with one billion people without its own established automobile market. This paper deals with the definition of load collectives of the battery and the electric motor for a customized vehicle development for the region Sub-Saharan Africa.

SUMMARYWe recently discovered that inhibition of the lipid peroxidase GPX4 can selectively kill cancer cells in a therapy-resistant state through induction of ferroptosis. Although GPX4 lacks a conventional druggable pocket, covalent small-molecule inhibitors are able to overcome this challenge by reacting with the GPX4 catalytic selenocysteine residue to eliminate enzymatic activity. Unfortunately, all currently-reported GPX4 inhibitors achieve their activity through reactive chloroacetamide groups. We demonstrate that such chloroacetamide-containing compounds are poor starting points for further advancement given their promiscuity, instability, and low bioavailability. Development of improved GPX4 inhibitors, including those with therapeutic potential, requires the identification of new electrophilic chemotypes and mechanisms of action that do not suffer these shortcomings. Here, we report our discovery that nitrile oxide electrophiles, and a set of remarkable chemical transformations that generates them in cells from masked precursors, provide an effective strategy for selective targeting of GPX4. Our results, which include structural insights, target engagement assays, and diverse GPX4-inhibitor tool compounds, provide critical insights that may galvanize development of improved compounds that illuminate the basic biology of GPX4 and therapeutic potential of ferroptosis induction. In addition, our discovery that nitrile oxide electrophiles engage in highly selective cellular interactions and are bioavailable in their masked forms may be relevant for targeting other currently undruggable proteins, such as those revealed by recent proteome-wide ligandability studies.

The one-pot, three-component, coupling reaction of indoles/pyrroles, dimethyl malonate, and acetic acid was performed using Mn(III) acetate as an oxidant. In the presence of Mn(OAc)3, indole-2, and indole-3-carbonyl compounds were alkylated at the 3- and 2- positions, respectively, with subsequent oxidation and nucleophilic capture occurring at the newly formed benzylic carbon. In contrast, oxidation of 2- and 3-indole carboxylic acids afforded the corresponding 2-oxindol-3-ylidenes and 3-oxindol-2-ylidenes. The reaction conditions, scope, and mechanism are discussed herein.

The photoredox-mediated direct intermolecular C-H functionalization of substituted indoles, pyrroles, and furans with diethyl bromomalonate is described, utilizing the visible light-induced reductive quenching pathway of Ru(bpy)(3)Cl(2). An analysis of reductive quenchers and mechanistic considerations has led to an optimized protocol for the heteroaromatic alkylations, providing products in good yields and regioselectivities, as well as successfully eliminating previously observed competitive side reactions. This methodology is highlighted by its neutral conditions, activity at ambient temperatures, low catalyst loading, functional group tolerance, and chemoselectivity.

This chapter describes cross-dehydrogenative-coupling (CDC) reactions mediated by visible light. The main focus is on transformations of amines through the generation of reactive intermediates such as iminium ions, azomethine ylides, or α-amino radicals through photo-oxidations. The chapter sections are organized by different photocatalytic systems based on transition metal complexes, dyes and porphyrins, and semiconductors. Also discussed are factors that influence the activation of C–H bonds as well as reaction mechanisms involving electron transfer and sensitization.

This account describes the successful application of a visible light-promoted radical coupling reaction as the key step in the total synthesis of the complex bisindole alkaloid gliocladin C. An initial approach involved the joining of a chiral triketopiperazine (TKP)-containing bromopyrroloindoline with an indole C2-aldehyde fragment using visible light photoredox catalysis to construct the C3 C3′ bisindole linkage. Removal of the indole C2 directing group was unsuccessful on this intermediate, and a second-generation approach was implemented such that directing group removal occurred in the absence of the TKP moiety. This also enabled the development of a catalytic decarbonylation reaction. Acylation and annulation of the pyrrolidine unit formed the TKP unit, but its subsequent oxidation proved challenging. By oxidizing the pyrrolidine first, the overall yield improved and the use of microwave conditions to effect the TKP formation shortened the number of steps.

[14323-06-9] Ruthenium(II), tris(2,2′-bipyridine-κN1, κN1′),1 chloride(1:2), (OC-6-11)-C30H24N6RuCl2 (MW 640.53) InChI = 1S/3C10H8N2.2ClH.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;;;/h3*1-8H;2*1H;/q;;;;;+2/p-2 InChIKey = SJFYGUKHUNLZTK-UHFFFAOYSA-L (cation) [15158-62-0] InChI = 1S/3C10H8N2.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;/h3*1-8H;/q;;;+2 InChIKey = HNVRWFFXWFXICS-UHFFFAOYSA-N (PF6 complex) [60804-74-2] InChI = S/3C10H8N2.2F6P.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;2*1-7(2,3,4,5)6;/h3*1-8H;;;/q;;;2*-1;+2 InChIKey = KLDYQWXVZLHTKT-UHFFFAOYSA-N (chloride hydrate) [50525-27-4] InChI = 1S/3C10H8N2.2ClH.6H2O.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;;;;;;;;;/h3*1-8H;2*1H;6*1H2;/q;;;;;;;;;;;+2/p-2 InChIKey = WHELTKFSBJNBMQ-UHFFFAOYSA-L (perchlorate) [15635-95-7] InChI = S/3C10H8N2.2ClHO4.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;2*2-1(3,4)5;/h3*1-8H;2*(H,2,3,4,5);/q;;;;;+2/p-2 InChIKey = BXKPAPTYLLPPEO-UHFFFAOYSA-L (BF4 complex) [63950-81-2] InChI = 1S/3C10H8N2.2BF4.Ru/c3*1-3-7-11-9(5-1)10-6-2-4-8-12-10;2*2-1(3,4)5;/h3*1-8H;;;/q;;;2*-1;+2 InChIKey = GWPUNVSVEJLECO-UHFFFAOYSA-N (visible light-active catalyst used in single-electron transfer reductions and oxidations1) Alternate Names: [Ru(bpy)3]2+, Ruthenium-tris(2,2′-bipyridyl). Physical Data: λmax = 452 nm, redox potentials2 (vs. SCE, saturated calomel electrode, as 0 V in CH3CN); −0.86 V (RuIII /RuII*), 1.29 V (RuIII/RuII), 0.84 V (RuII*/RuI), and −1.33 (RuII/RuI). Solubility: for X = Cl, sol in H2O, CH3OH, CH3CN, DMF, and DMSO; insoluble in nonpolar, aprotic solvents; for X = PF6, sol in CH3CN, acetone, DMF, and DMSO; insoluble in H2O. Form Supplied in: the dichloride is commercially available from Strem Chemicals, Inc. and Sigma-Aldrich as the hexahydrate. [Ru(bpy)3]2+ complexes appear as red to orange crystals or powder. Preparation Methods: for X = PF6, the catalyst is prepared from RuCl3, 2,2′-bipyridine, and NH4PF6 in EtOH.3 Purification: recrystallized in boiling H2O. Handling, Storage, and Precautions: containers should be stored under nitrogen atmosphere in the dark. Keep away from strong oxidizing agents. Upon visible light excitation of [Ru(bpy)3]2+ (bpy = 2,2′-bipyridine), the resulting metal-to-ligand charge transfer (MLCT) excited state species, *[Ru(bpy)3]2+, can be reductively or oxidatively quenched with the appropriate single-electron donor or acceptor, respectively. Reductive quenching of the excited state forms [Ru(bpy)3]+, which then performs a single-electron reduction to regenerate [Ru(bpy)3]2+. Alternatively, oxidative quenching forms [Ru(bpy)3]3+ , which then oxidizes a suitable donor to regenerate ground state [Ru(bpy)3]2+.

Hexahydropyrroloindoline alkaloids are a large class of natural products that are formally derived from two molecules of tryptophan.[1] A subset of this class, the C3–C3′ indole alkaloids, contain the 3a-(3-indolyl)-hexahydropyrrolo-[2,3-b]indole skeleton and include compounds such as gliocladin C,[2] gliocladine C,[3] leptosin D,[4] and the bionectins[5] (Figure 1). Aside from their interesting structural features, they exhibit a broad range of potent biological activities. For example, gliocladin C[2] and leptosin D[4] are cytotoxic against P-388 lymphocytic leukemia cell lines with ED50 values of 240 ng mL−1 and 86 ng mL−1, respectively, while bionectins A and B[5] exhibit antibacterial activity against MRSA (methicillin-resistant S. aureus) and QRSA (quinolone-resistant S. aureus) with MIC = 10–30 µm mL−1.

The use of 4-methoxy-N,N-diphenylaniline as an electron donor is crucial for the high yielding reaction.

2,6-Disubstituted dihydropyrans are a common feature in many bioactive polyketides, including the anticancer marine polyketide laulimalide. While much of the uncharacterized biosynthetic pathway for laulimalide can be confidently postulated, the biosynthetic origins of the trans 2,6-disubstituted dihydropyran cannot. We hypothesize that a transannular oxa-conjugate addition in a macrocyclic laulimalide precursor could be the origin of the 2,6-dihydropyran. To test this hypothesis, we constructed a model containing the key functional groups for oxa-conjugate addition-mediated dihydropyran formation. Under acid-mediated conditions, the model under went regiospecific oxa-conjugate addition producing a stable trans oxetane as the only regioisomer. The desired, more stable dihydropyran was not detected. This unprecedented regiospecificity is unexpected due to the ring strain of the oxetane and the anticipated facile ring opening retro-oxa-conjugate addition. The oxetane is stable to acid and basic conditions, as are a number of literature acyclic oxetanes that could undergo similar retro-oxa-conjugate addition. While the source of the oxetane kinetic stability is yet to be characterized, it may enable general oxetane construction via oxa-conjugate addition. The more stable dihydropyran regioisomer could not be generated due to poor geometrical orbital alignment and hard-soft incompatibility between the hard oxygen nucleophile and the soft activated polyenoate electrophile. These factors disfavor the breaking of conjugation by oxa-conjugate addition. Based on these results we propose that dihydropyran formation does not occur on completed polyketide macrocycles as we had proposed but rather during polyketide biosynthesis on the growing polyketide chain.

Nature's approach to biosynthesis often involves the rapid generation of advanced, enantiopure intermediates from simple starting materials. A new, highly efficient strategy adapts this approach, using organocascade catalysis to quickly construct a key intermediate that can be converted into several complex natural products.