Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit.

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50<0.1µM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.

Construction of a 3D-shaped, natural product like fragment library by fragmentation and diversification of natural products.

A fragment library consisting of 3D-shaped, natural product-like fragments was assembled. Library construction was mainly performed by natural product degradation and natural product diversification reactions and was complemented by the identification of 3D-shaped, natural product like fragments available from commercial sources. In addition, during the course of these studies, novel rearrangements were discovered for Massarigenin C and Cytochalasin E. The obtained fragment library has an excellent 3D-shape and natural product likeness, covering a novel, unexplored and underrepresented chemical space in fragment based drug discovery (FBDD).

The chemical behavior of terminally tert-butylated polyolefins.

The chemical behavior of various oligoenes 2 has been studied. The catalytic hydrogenation of diene 3 yielded monoene 4. Triene 7 was hydrogenated to diene 8, monoene 9 and saturated hydrocarbon 10. Bromine addition to 3 and 7 yielded the dibromides 17 and 18, respectively, i.e., the oligoene system has been attacked at its terminal olefinic carbon atoms. Analogously, the higher vinylogs 19 and 20 yielded the 1,8- and 1,10-bromine adduts 23 and 24, respectively, when less than 1 equivalent of bromine was employed. Treatment of tetraene 19 with excess bromine provided tetrabromide 25. In epoxidation reactions, both with meta-chloroperbenzoic acid (MCPBA) and dimethyldioxirane (DMDO) two model oligoenes were studied: triene 7 and tetraene 19. Whereas 7 furnished the rearrangement product 31 with MCPBA, it yielded the symmetrical epoxide 32 with DMDO. Analogously, 19 was converted to mono-epoxide 33 with MCPBA and to 34 with DMDO. Diels-Alder addition of 7 with N-phenyltriazolinedione (PTAD) did not take place. Extension of the conjugated π-system to the next higher vinylog, 19, caused NPTD-addition to the symmetrical adduct 37 in good yield. Comparable results were observed on adding NPTD (equivalent amount) to pentaene 20 and hexaene 21. Using 36 in excess provided the 2:1-adduct 40 from 21 and led to a complex mixture of adducts from heptaene 22. With tetracyanoethylene (TCNE) as the dienophile, tetraolefin 19 yielded the symmetrical adduct 43, although the reaction temperature had to be increased. Pentaene 20 and hexaene 21 led to corresponding results, adducts 44 and 45 being produced in acceptable yields. With nonaene 42 and TCNE the 2:1-adduct 48 was generated according to its spectroscopic data. Exploratory photochemical studies were carried out with tetraene 19 as the model compound. On irradiation this reacted with oxygen to the stable endo-peroxide 52.

Preparation, structural properties and thermal isomerization of hex-3-ene-1,5-diyne bridged [2.2]paracyclophanes.

The [2.2]paracyclophane moiety is used as a spacer to connect the ends of a hex-3-ene-1,5-diyne unit, a π-system that on thermolysis usually cycloaromatizes to a benzene ring (Bergman cyclization). For the preparation of the pseudo-geminally-bridged system 9, the diacetylene 3 was chain-extended to the diol 16, which after conversion to the pseudo-geminal dibromide 17 was ring-closed by treatment with LiHMDS/HMPA to the [2.2]paracyclophane enediyne 9. Whereas the McMurry coupling of the pseudo-ortho bisaldehyde 24 resulted in the formation of the hexadienyne-bridged cyclophane 27, the pseudo-ortho-bridged hydrocarbon 11 was obtained by preparing first the diol 28 from 24, converting the latter into the dioxolane 29, which in the last step furnished the olefin 11 by treatment with Tf2 O/EtN(iPr)2 . The authentic Bergman product 10 of the pseudo-gem-bridged hexenediyne 9 was synthesized by a conventional sequence starting from the ethynyl formyl substrate 18. Since the pseudo-ortho-enediyne-bridged hydrocarbon 11 is thermally labile, its benzannelated derivate 34 was prepared. No classical Bergman cyclization reactions could be observed for any of the [2.2]paracyclophane-bridged hexenediynes prepared here. In the pseudo-gem-series the fulvenes 14 and 15 were the only products that could be identified under thermal conditions (McMurry coupling); the benzannelated substrate 34 gave the benzofulvene-bridged cyclophane 36 on photolysis. Bergman cyclizations yielding fulvene derivatives are extremely rare. The mechanism of the cyclization of 9 and 34 is discussed, using compliance constants. The structure assignments of the hydrocarbons synthesized in this study are based on spectroscopic studies as well as X-ray structural analyses for 9, 10, 11, 27, and 34.

Building complex carbon skeletons with ethynyl[2.2]paracyclophanes.

Ethynyl[2.2]paracyclophanes are shown to be useful substrates for the preparation of complex, highly unsaturated carbon frameworks. Thus both the pseudo-geminal- 2 and the pseudo-ortho-diethynylcyclophane 4 can be dimerized by Glaser coupling to the respective dimers 9/10 and 11/12. Whereas the former isomer pair could not be separated so far, the latter provided the pure diastereomers after extensive column chromatography/recrystallization. Isomer 11 is chiral and could be separated on a column impregnated with cellulose tris(3,5-dimethylphenyl)carbamate. The bridge-extended cyclophane precursor 18 furnished the ring-enlarged cyclophanes 19 and 20 on Glaser-Hay coupling. Cross-coupling of 4 and the planar building block 1,2-diethynylbenzene (1) yielded the chiral hetero dimer 22 as the main product. An attempt to prepare the biphenylenophane 27 from the triacetylene 24 by CpCo(CO)2-catalyzed cycloisomerization resulted in the formation of the cyclobutadiene Co-complex 26. Besides by their usual spectroscopic and analytical data, the new cyclophanes 11, 12, 19, 20, 22, and 26 were characterized by X-ray structural analysis.

Bromine-bromine interactions in the packing of cis,trans,cis-1,2,3,4-tetrakis(bromomethyl)cyclobutane.

The title compound, C8H12Br4, displays crystallographic inversion symmetry, so that the cyclobutane ring is exactly planar. The ring C-C bond with eclipsed substituents is lengthened somewhat to 1.572 (5) Å. The packing can be described in terms of three Br...Br contacts; two of these combine to form layers of molecules parallel to the ac plane, while the third crosslinks the layers in the third dimension. A simple topological descriptor for systems involving Br···Br contacts is proposed.

The preparation of several 1,2,3,4,5-functionalized cyclopentane derivatives.

With the goal of eventually synthesizing [5]radialene (3), the still missing member of the parent radialene hydrocarbons, we have prepared the pentaacetates 21 and 31, the pentabromide 29 and the hexabromide 32. In principle these should be convertible by elimination reactions to the desired target molecule.

Structural studies on the full-length LysR-type regulator TsaR from Comamonas testosteroni T-2 reveal a novel open conformation of the tetrameric LTTR fold.

LysR-type transcriptional regulators (LTTRs) constitute the largest family of regulators in prokaryotes. The full-length structures of the LTTR TsaR from Comamonas testosteroni T-2 and its complex with the natural inducer para-toluensulfonate have been characterized by X-ray diffraction. Both ligand-free and complexed forms reveal a dramatically different quaternary structure from that of CbnR from Ralstonia eutropha, or a putative LysR-type regulator from Pseudomonas aeruginosa, the only other determined full-length structures of tetrameric LTTRs. Although all three show a head-to-head tetrameric ring, TsaR displays an open conformation, whereas CbnR and PA01-PR present additional contacts in opposing C-terminal domains that close the ring. Such large differences may be due to a broader structural versatility than previously assumed or either, reflect the intrinsic flexibility of tetrameric LTTRs. On the grounds of the sliding dimer hypothesis of LTTR activation, we propose a structural model in which the closed structures could reflect the conformation of a ligand-free LTTR, whereas inducer binding would bring about local changes to disrupt the interface linking the two compact C-terminal domains. This could lead to a TsaR-like, open structure, where the pairs of recognition helices are closer to each other by more than 10 A.

Use of Intramolecular 1,5-Sulfur-Oxygen and 1,5-Sulfur-Halogen Interactions in the Design of N-Methyl-5-aryl-N-(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,4-thiadiazol-2-amine SMN2 Splicing Modulators.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.

A general route to fully terminally tert-butylated linear polyenes.

Starting from the readily available α,ß-unsaturated ketone, 3-tert-butyl-4,4-dimethyl-2-pentenal, higher vinylogues, and fully terminally tert-butylated polyolefins with up to 13 consecutive conjugated double bonds have been prepared by either McMurry dimerization or Wittig chain-elongation routes. The highly unsaturated conjugated π systems, which show a remarkable stability, have been characterized by spectroscopic methods and, in many cases, by X-ray structural analysis. The yields are high enough to allow for thorough chemical reactivity studies.

Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution.

Shewanella oneidensis is an environmentally versatile Gram-negative gamma-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 A resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30-1.60 A resolution, thus providing a rare opportunity to dissect the structure-function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride-Meisenheimer complex.

Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

Preparation and NMR spectra of four isomeric diformyl[2.2]paracyclophanes (cyclophanes 66).

Four isomeric dialdehydes 4, readily available from cycloaddition of propiolic aldehyde (2) to 1,2,4,5-hexatetraene (1), were separated by chromatography and recrystallization, and were characterized by their spectroscopic data. The individual isomers can now be easily identified from their ¹H NMR spectra even if only one of them is present.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

Sr(5)(VOF(5))(3)F(H(2)O)(3) refined from a non-merohedrally twinned crystal.

The title compound, penta-strontium tris-[penta-fluorido-oxido-vanadate(IV)] fluoride trihydrate, was obtained under hydro-thermal conditions. Its crystal structure has been refined from intensity data of a non-merohedrally twinned crystal. Two domains in almost equal proportions are related by a -180° rotation along the reciprocal [101]* vector. The structure may be considered as a derivative of the fluorite structure type, adopted here by SrF(2). In the title compound, fluorite-like large rods are recognized, built up from a group of 16 Sr atoms of which 6 are substituted by V atoms, leading to [Sr(10)V(6)](∞) units. These rods extend infinitely along the b axis and are inter-connected by the three water mol-ecules. Each of the water mol-ecules is shared by two different Sr atoms belonging to two different rods. The rods are also inter-connected by an 'independent' F atom in a distorted triangular [FSr(3)] coordination and by hydrogen-bonding inter-actions via donor water mol-ecules. The acceptors are either F atoms or the O atoms of the vanadyl ion, VO(2+), that is part of the [VOF(5)] isolated octa-hedron.

Crystallographic characterization and identification of a minor isomer of C(84) fullerene.

We report the synthesis and single crystal X-ray analysis of C(84) ().AgTPP (Ag tetraphenylporphyrin) cocrystal-the first ordered crystal structure containing a pristine higher fullerene.

Bulky N-substituted 1,3-benzazaphospholes: access via Pd-catalyzed C-N and C-P cross coupling, lithiation, and conversion to novel P=C-PtBu2 hybrid ligands.

The syntheses of novel bulky N-substituted 1,3-benzazaphospholes are presented, together with their reactions with tert-butyllithium and coupling with tBu 2PCl to novel P, P'-hybrid ligands that combine the highly basic and bulky di- tert-butylphosphanyl group with pi-acidic low-coordinated phosphorus. The syntheses start with the preparation of new N-secondary 2-bromoanilines 1 by reduction of N-acyl 2-bromoanilides or more generally by Pd-catalyzed selective monoamination of o-dibromobenzene, followed by Pd-catalyzed C-P coupling with P(OEt) 3 to the respective 2-anilino-phosphonates 2. The next steps are reduction to 2-phosphanylanilines 3 and condensation with Me 2NCH(OMe) 2, which leads via phosphaalkenes 4 to the corresponding N-substituted benzazaphospholes 5. The reaction with tBuLi depends on the steric demand of the N substituent. Methyl, neopentyl-, and mesityl-derivatives were converted to P=C Li species 6 and coupled with tBu 2PCl to novel P=C-P tBu 2 ligands 7, whereas N-adamantyl and N-2,6-diisopropylphenyl-derivatives prefer addition of tBuLi at the PC bond to form dihydroderivatives. The chemical shifts of the low-coordinated phosphorus of 5 and 7 were found to reflect electronic and steric effects of the N substituents. The comparison of the crystal structures of N-neopentyl-1,3-benzazaphospholes 5 and 7 gives evidence of steric repulsion between the adjacent di- tert-butyl and neopentyl groups by the preferred anti orientation of the P- tert-butyl groups and moderate deviations of C2 and P3 of 7b from the ring plane.

Bioinspired co-crystals of Imatinib providing enhanced kinetic solubility.

Realizing the full potential of co-crystals enhanced kinetic solubility demands a comprehensive understanding of the mechanisms of dissolution, phase conversion, nucleation and crystal growth, and of the complex interplay between the active pharmaceutical ingredient (API), the coformer and co-existing forms in aqueous media. One blueprint provided by nature to keep poorly water-soluble bases in solution is the complexation with phenolic acids. Consequently, we followed a bioinspired strategy for the engineering of co-crystals of a poorly water-soluble molecule - Imatinib - with a phenolic acid, syringic acid (SYA). The dynamics of dissolution and solution-mediated phase transformations were monitored by Nuclear Magnetic Resonance (NMR) spectroscopy, providing mechanistic insights into the 60 fold-increased long lasting concentrations achieved by the syringate co-crystals as compared to Imatinib base and Imatinib mesylate. This lasting effect was linked to SYA's ability to delay the formation and nucleation of Imatinib hydrate - the thermodynamically stable form in aqueous media - through a metastable association of SYA with Imatinib in solution. Results from permeability studies evidenced that SYA did not impact Imatinib's permeability across membranes while suggesting improved bioavailability through higher kinetic solubility at the biological barriers. These results reflect that some degree of hydrophobicity of the coformer might be key to extend the kinetic solubility of co-crystals with hydrophobic APIs. Understanding how kinetic supersaturation can be shaped by the selection of an interactive coformer may help achieving the needed performance of new forms of poorly water-soluble, slowly dissolving APIs.

Discovery of the First Potent, Selective, and Orally Bioavailable Signal Peptide Peptidase-Like 2a (SPPL2a) Inhibitor Displaying Pronounced Immunomodulatory Effects In Vivo.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1'-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.

A triazine-based three-directional rigid-rod tecton forms a novel 1D channel structure.

A large, symmetrically substituted triazine-based molecule, synthesized by a copper-free Sonogashira coupling procedure self-assembles to form a novel 1D channel structure that hosts chlorobenzene molecules as guests.