Viktor A. Zapol'skii

Chemistry of polyhalogenated nitrobutadienes, 14: Efficient synthesis of functionalized (Z)-2-allylidenethiazolidin-4-ones

Summary The reaction of mercaptoacetic acid esters with pentachloro-2-nitro-1,3-butadiene (1) provides an appropriate precursor for the synthesis of special thiazolidin-4-ones. Applying different anilines as the second constituent for the requisite cyclization step, a series of (Z)-2-allylidenethiazolidin-4-ones was obtained in yields up to 81%. Some subsequent reactions have been examined too, such as the formation of perfunctionalized 1H-pyrazoles upon treatment with hydrazine. Thiazolidinones are as well known for their physiological activities as for their application in optoelectronics.

Identification of Multiple Druggable Secondary Sites by Fragment Screening against DC-SIGN

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using 19 F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and 1 H-15 N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors.

Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

trans-Bis(perchlorato-κO)tetra­kis(1H-pyrazole-κN2)copper(II)

The title compound, [Cu(ClO4)2(C3H4N2)4], was obtained unexpectedly by the reaction of copper(II) perchlorate hexahydrate with equimolar amounts of 1-chloro-1-nitro-2,2,2-tripyrazolylethane in methanol solution. The crystal structure comprises octahedrally coordinated Cu2+ ions, located on an inversion centre, with four pyrazole ligands in the equatorial plane. The average Cu—N distance is 2.000 (1) Å. Two perchlorate ions are coordinated to copper in trans positions [Cu—O = 2.4163 (11) Å].

Corrigendum: Identification of Multiple Druggable Secondary Sites by Fragment Screening against DC-SIGN

DC-SIGN is a cell surface receptor for several pathogenic threats such as HIV, Ebola virus or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate–protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely owed to its hydrophilic, solvent-exposed carbohydrate binding site. Here, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using F NMR and chemical fragment microarrays. Hit validation including SPR and HN HSQC NMR revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbour drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors. Many members of the C-type lectin (CLR) family are expressed as transmembrane receptors on cells of the innate immune system and serve as pattern recognition receptors regulating the immune response. Recognition of surface carbohydrates by these receptors is mediated via a central Ca co-factor and promotes immune cell signaling as well as pathogen uptake resulting in antigen processing and presentation on MHC molecules. DC-SIGN is a prominent member of the CLR family that recognizes mannoseand fucose-type ligands on viruses such as HIV, Ebola virus, Dengue virus, Phlebovirus as well as bacteria such as Mycobacterium tuberculosis. In particular, DC-SIGN-expressing cells are amongst the first immune cells to encounter HIV and promote transinfection of T cells. These findings have generated significant interest in the development of inhibitors of DC-SIGN. The extracellular domain of DC-SIGN forms homotetramers which each consists of a neck and a carbohydrate recognition domain (CRD). This increases the avidity of the low-affinity monovalent carbohydrate recognition (Kd (mannose) = 3.5 mM) into the nanomolar range for multivalent interactions (Kd (gp120) = 1-2 nM). Utilizing the multivalent display of simple carbohydrates on various supports lowered the inhibitory constants of several reported inhibitors into the nanoto picomolar affinity range. On the other hand, low-molecular weight carbohydrate-derived inhibitors show only modest affinity in the medium to high micromolar regime. Moreover, noncarbohydrate, drug-like inhibitors for DC-SIGN are limited to quinoxalinones, which display high potency in cell-based assays but are electrophiles with partially limited stability . In general, the development of inhibitors for mammalian glycanbinding proteins is challenging owing to their shallow and hydrophilic binding sites. Starting from the primary carbohydratebinding site elaborating on the original carbohydrate as a scaffold has proven to be a promising route for some targets. However, such approaches capitalize on the presence of extended binding sites that allow for affinity maturation of the carbohydrate scaffold by the attachment of hydrophobic substituents. Often such secondary sites are neither well described nor directly accessible from the analysis of crystallographic protein structures as they might stem from minor alterations of the protein geometry. Previous reports indicated the presence of binding sites for drug-like molecules to DC-SIGN . As these drug-like inhibitors lack functional groups to directly interact with the primary Ca ion, an allosteric mechanism was proposed, but the nature of their pockets remained elusive. Here, we applied fragment-based screening to identify low molecular weight ligands and their respective binding sites for DC-SIGN. A library of 986 fragments was screened in parallel by surface plasmon resonance (SPR) and a reporter displacement assay (RDA). Along with the results from our previously reported screening of a sublibrary of 281 fluorinated fragments using F NMR and chemical fragment microarrays, a large set of orthogonal screening data was available for DC-SIGN . A detailed analysis of the entire parallel screening dataset is available in the Supplementary Information (Figures 1, S2, S3). Most importantly, SPR and F NMR screening resulted in 49 (5.0% hit rate) and 46 hits (16.3%), respectively (Figure 1 a,b). These 95 compounds were counter-screened by SPR and RDA (Figure 1 c,d), resulting in 61 (64%) hits validated by SPR and 4 (4%) hits validated by SPR and RDA. The best affinity of 0.3 mM was estimated for compound 6, the ligand with the highest efficiency that has been reported to date for DC-SIGN (LE = 0.34 kcal HA mol). Moreover, these results suggest a moderate druggability of DC-SIGN, which is in accordance to our previous results. It is noteworthy that only four compounds showed activity in the RDA with IC50 values above 1 mM. This assay directly probes for competitors rather than binders and consequently implies a low druggability of the carbohydrate binding site. Taken together, the large discrepancy between binders and competitors indicates potential druggable secondary sites. ______________________________________________ Supporting information can be found under https://doi.org/10.1002/anie.201701943 [a] J. Aretz, H. Baukmann, E. Shanina, Dr. J. Hanske, Dr. R. Wawrzinek, Dr. P. H. Seeberger, Dr. C. Rademacher Department of Biomolecular Systems Max Planck Institute of Colloids and Interfaces Am Mühlenberg 1, 14476 Potsdam (Germany) E-mail: christoph.rademacher@mpikg.mpg.de [b] J. Aretz, H. Baukmann, Dr. J. Hanske, Dr. P. H. Seeberger, Dr. C. Rademacher Department of Biology, Chemistry, and Pharmacy Freie Universität Berlin Takustraße 3, 14195 Berlin (Germany) [c] Dr. V. A. Zapol’skii, Dr. D. E. Kaufmann Institute of Organic Chemistry Clausthal University of Technology Leibnizstraße 6, 38678 Clausthal-Zellerfeld (Germany) M ax P la nc k In st itu te o f C ol lo id s an d In te rf ac es · A ut ho r M an us cr ip t Figure 1. Examples from fragment screening and counter-screening against DC-SIGN. a) Scatter plot of relative response versus compound ID from SPR screening. A response (RU) larger than 3.0 or a calculated LE > 0.5 were chosen for hit identification (black) whereas compounds exceeding the calculated RUmax, showing negative response, or slow or irreversible kinetics were excluded as potential false positives. b) Example from the F NMRbased fragment screening described previously. The expansion from a T2filtered F NMR spectrum (T = 1 s, νCPMG = 50 Hz) shows three compounds that were increased in signal intensity after mannan addition. c) SPR sensorgram and one-site-binding model fit of 8 from the counter-screen. The apparent dissociation constant was estimated to be 500 μM (n=3). d) Second counter-screening using a FITC-dextran-based RDA. Compounds were measured in triplicates at 1 mM concentration and compared to a DMSO control. To expand our insight into the presence of secondary sites, we applied H-N HSQC NMR analysis using N isotope-labeled DC-SIGN CRD (Figure 2 a) and computational pocket predictions. For the NMR analysis, we first tested the DC-SIGN ligand mannose, which is a millimolar binder (Figure S6, S7). Here, mannose addition to DC-SIGN CRD induced chemical shift perturbations (CSPs) and reduced resonance intensities of residues close to the primary site as already observed earlier for the carbohydrate Lewis X(Figure 2 b,c). Then, we analyzed 28 fragments to identify their potential binding sites (Figure 2). These fragments were chosen based on the aforementioned RDA and SPR results. In total, 14 of 19 (74%) validated fragments from the SPR screening and all ten (100%) from the F NMR screening induced changes in the HSQC spectra. As expected from compounds passing a cascade of orthogonal techniques, the validation rates obtained by H-N HSQC NMR increased compared to the first and second counter-screen. Most importantly, all fragments induced CSPs or altered peak intensities of subsites compared to mannose (Figure 2 b,c). In addition, these changes were distinct between individual fragments (Figure 2 b, S8-15), indicating the presence of several secondary binding sites. Figure 2. Hit validation and binding site mapping by H-N HSQC NMR. a) Example from a H-N HSQC NMR spectrum of DC-SIGN CRD in the presence of 1% DMSO (black) and 1 mM 17 (orange). Changes in chemical shift and peak intensity are indicated by a red and blue arrow, respectively. Unassigned peaks were indexed. b) CSP mapping of mannose, 1, and 7. CSPs exceeding a threshold (0.025 ppm, red) and intensities decreasing by more than 50% (blue) as well as data for unassigned residues (grey bars and left) were utilized for mapping the binding site of fragments. c) CSPs above and intensities below the threshold (red and blue, respectively) induced by mannose mapped on the structure of DC-SIGN CRD (PDB: 2XR6) in complex with pseudo trimannoside (green). In parallel, three computational pocket prediction algorithms were applied to the structure of DC-SIGN CRD. In addition to our previously reported results employing DoGSiteScorer , hot spots for binding of drug-like molecules were identified by computational solvent mapping using FTMap and potentially inducible sites were predicted by CryptoSite. We determined five pockets with FTMap which locate in front and in the back of the primary carbohydrate binding site (site I and II, respectively), two pockets close to the neck domain (site III and IV), and one cluster of hot spots between the α1 and α2 helix opposite the primary site (site V; Table S3, Figure 3, S16). Among these five binding sites, site II to V also contain potentially inducible residues according to CryptoSite. Site I, III, IV, and V were also identified by DoGSiteScorer (Table S4, S5). Of note, the primary carbohydrate binding site was not identified by any of these methods, which is in agreement with our H-N HSQC NMR fingerprinting. Furthermore

Chemistry of Polyhalogenated Nitrobutadienes, Part 11: ipso-Formylation of 2-Chlorothiophenes under Vilsmeier-Haack Conditions

The regioselective ipso-formylation of electron-rich, 3,4-push-pull-substituted 2-chlorothiophenes under Vilsmeier-Haack conditions was performed in good yields. The synthetic scope of this new reaction was explored using various halothiophenes, chloroanilines, and 1-methyl-3-chloroindole. In comparison with their structural C-H analogs the chlorinated thiophenes, anilines, and the indole proved to be less reactive toward electrophilic attack by chloromethyleniminium salts. Graphical Abstract Chemistry of Polyhalogenated Nitrobutadienes, Part 11: ipso-Formylation of 2-Chlorothiophenes under Vilsmeier-Haack Conditions