Competing Domino Knoevenagel-Cyclization Sequences with N-Arylcinnamylamines.

Domino Knoevenagel-cyclization reactions of N-arylcinnamylamines were carried out with active methylene reagents, which took place with five competing cyclization mechanisms: intramolecular hetero Diels-Alder reaction, stepwise polar [2 + 2] cycloaddition, styryl or aza-Diels-Alder reactions followed by rearomatization, and [1,5]-hydride shift-6-endo cyclization. In the stepwise aza-Diels-Alder reaction, the N-vinylpyridinium moiety acted as an azadiene, producing a condensed heterocycle with tetrahydroquinolizinium and tetrahydroquiniline subunits. Antiproliferative activity with low micromolar IC50 values was identified for some of the novel scaffolds.

Visible-Light-Induced Synthesis of Branched Ethers via Multicomponent Reactions.

The Spin-Center Shift (SCS) elimination is a specific way for the generation of radicals with relevance in synthetic and biochemical pathways. The combination of SCS-mediated radical chemistry and atom-transfer radical addition (ATRA) offers new directions in diversity-oriented chemical synthesis. Herein, we report a photoredox three-component reaction of α-acyloxy-N-heterocycles as radical precursors, styrene derivatives as radical trapping agents, and alcohols as nucleophilic quenchers. The novel radical-polar crossover reaction provides access to a diverse set of branched ethers possessing high structural complexity. The utility of the transformation was also demonstrated by the synthesis of a complex drug derivative and it was easily scalable to the multigram level. The scope and limitations were also explored and a plausible mechanism was proposed.

Intertwining Olefin Thianthrenation with Kornblum/Ganem Oxidations: Ene-type Oxidation to Furnish α,ß-Unsaturated Carbonyls.

A widely applicable, practical, and scalable synthetic method for efficient ene-type double oxidation of alkenes is reported via a two-step alkenyl thianthrenium umpolung/Kornblum-Ganem oxidation strategy. This chemo- and stereoselective procedure allows easy access to various α,ß-unsaturated carbonyls that may be otherwise difficult or cumbersome to synthesize by conventional methods. For α-olefins, this metal-free transformation can be tuned according to synthetic needs to produce either the elusive (Z)-unsaturated aldehydes or their (E) counterparts. Moreover, this strategy has enabled streamlined synthesis of distinct butadienyl pheromones and kairomones.

Isotope Ratio Encoding of Sequence-Defined Oligomers.

Information storage at the molecular level commonly entails encoding in the form of ordered sequences of different monomers and subsequent fragmentation and tandem mass spectrometry analysis to read this information. Recent approaches also include the use of mixtures of distinct molecules noncovalently bonded to one another. Here, we present an alternate isotope ratio encoding approach utilizing deuterium-labeled monomers to produce hundreds of oligomers endowed with unique isotope distribution patterns. Mass spectrometric recognition of these patterns then allowed us to directly readout encoded information with high fidelity. Specifically, we show that all 256 tetramers composed of four different monomers of identical constitution can be distinguished by their mass fingerprint using mono-, di-, tri-, and tetradeuterated building blocks. The method is robust to experimental errors and does not require the most sophisticated mass spectrometry instrumentation. Such isotope ratio-encoded oligomers may serve as tags that carry information, but the method mainly opens up the capability to write information, for example, about molecular identity, directly into a pure compound via its isotopologue distribution obviating the need for additional tagging and avoiding the use of mixtures of different molecules.

Enantioselective Cyclopropanation Catalyzed by Gold(I)-Carbene Complexes.

The formation of polysubstituted cyclopropane derivatives in the gold(I)-catalyzed reaction of olefins and propargylic esters is a potentially useful transformation to generate diversity, therefore any method in which its stereoselectivity could be controlled is of significant interest. We prepared and tested a series of chiral gold(I)-carbene complexes as a catalyst in this transformation. With a systematic optimization of the reaction conditions, we were able to achieve high enantioselectivity in the test reaction while the cis:trans selectivity of the transformation was independent of the catalyst. Using the optimized conditions, we reacted a series of various olefins and acetylene derivatives to find that, although the reactions proceeded smoothly and the products were usually isolated in good yield and with good to exclusive cis selectivity, the observed enantioselectivity varied greatly and was sometimes moderate at best. We were unable to establish any structure-property relationship, which suggests that for any given reagent combination, one has to identify individually the best catalyst.

Targeting non-alcoholic fatty liver disease: Design, X-ray co-crystal structure and synthesis of 'first-in-kind' inhibitors of serine/threonine kinase25.

We describe the synthesis of a series of 3-t-butyl 5-aminopyrazole p-substituted arylamides as inhibitors of serine-threonine25 (STK25), an enzyme implicated in the progression of non-alcoholic fatty liver disease (NAFLD). Appending a p-N-pyrrolidinosulphonamide group to the arylamide group led to a 'first-in kind' inhibitor with IC50 = 228 nM. A co-crystal structure with STK 25 revealed productive interactions which were also reproduced using molecular docking. A new series of triazolo dihydro oxazine carboxamides of 3-t-butyl 5-aminopyrazole was not active against STK25.

Synthesis and Vibrational Circular Dichroism Analysis of N-Heterocyclic Carbene Precursors Containing Remote Chirality Centers.

VCD analysis of 16 diastereomeric pairs of NHC precursors containing two isolated chirality centers and different substitution patterns identified VCD transitions characteristic of the chirality center in the imidazolium ring or in the side chain, which, in contrast to ECD and OR, could be utilized to assign the two chirality centers separately by simple comparison, regardless of the type and position of achiral aromatic substituents. While the ECD and OR data showed great dependence on the position of an achiral substituent such as a methoxy group, characteristic experimental VCD transitions remained consistent and they could be used to determine the absolute configuration of all the regio- and stereoisomers and substituted analogues. VCD, ECD and OR approaches were evaluated, and several carbene precursors were found, for which only the VCD method could distinguish the four stereoisomers. With t-butyl, phenyl or 2-naphthyl substituents at the C-1' chirality center, the ECD spectra of the C-1' epimers were near-identical, and hence it was only the VCD approach that showed distinct differences suitable for the configurational assignment. The chiroptical characterization of our diastereomeric pairs of NHC precursors enables the future application of related derivatives having different substitution patterns in stereoselective transformations.

Targeting DYRK1A/B kinases to modulate p21-cyclin D1-p27 signalling and induce anti-tumour activity in a model of human glioblastoma.

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.

The Effect of Core Replacement on S64315, a Selective MCL-1 Inhibitor, and Its Analogues.

Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

The Chemistry Behind ADCs.

Combining the selective targeting of tumor cells through antigen-directed recognition and potent cell-killing by cytotoxic payloads, antibody-drug conjugates (ADCs) have emerged in recent years as an efficient therapeutic approach for the treatment of various cancers. Besides a number of approved drugs already on the market, there is a formidable follow-up of ADC candidates in clinical development. While selection of the appropriate antibody (A) and drug payload (D) is dictated by the pharmacology of the targeted disease, one has a broader choice of the conjugating linker (C). In the present paper, we review the chemistry of ADCs with a particular emphasis on the medicinal chemistry perspective, focusing on the chemical methods that enable the efficient assembly of the ADC from its three components and the controlled release of the drug payload.

Synthesis and Photochemical Application of Hydrofluoroolefin (HFO) Based Fluoroalkyl Building Block.

A novel fluoroalkyl iodide was synthesized on multigram scale from refrigerant gas HFO-1234yf as cheap industrial starting material in a simple, solvent-free, and easily scalable process. We demonstrated its applicability in a metal-free photocatalytic ATRA reaction to synthesize valuable fluoroalkylated vinyl iodides and proved the straightforward transformability of the products in cross-coupling chemistry to obtain conjugated systems.

Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

Copper-Catalyzed Trifluoromethylation of Alkoxypyridine Derivatives.

The trifluoromethylation of aromatic and heteroaromatic cores has attracted considerable interest in recent years due to its pharmacological relevance. We studied the extension of a simple copper-catalyzed trifluoromethylation protocol to alkoxy-substituted iodopyridines and their benzologs. The trifluoromethylation proceeded smoothly in all cases, and the desired compounds were isolated and characterized. In the trifluoromethylation of 3-iodo-4-methoxyquinoline, we observed a concomitant O-N methyl migration, resulting in the trifluoromethylated quinolone as a product. Overall, the described procedure should facilitate the broader use of copper-catalyzed trifluoromethylation in medicinal chemistry.

Aromatic Foldamer Helices as α-Helix Extended Surface Mimetics.

Helically folded aromatic oligoamide foldamers have a size and geometrical parameters very distinct from those of α-helices and are not obvious candidates for α-helix mimicry. Nevertheless, they offer multiple sites for attaching side chains. It was found that some arrays of side chains at the surface of an aromatic helix make it possible to mimic extended α-helical surfaces. Synthetic methods were developed to produce quinoline monomers suitably functionalized for solid phase synthesis. A dodecamer was prepared. Its crystal structure validated the initial design and showed helix bundling involving the α-helix-like interface. These results open up new uses of aromatic helices to recognize protein surfaces and to program helix bundling in water.

Establishing Drug Discovery and Identification of Hit Series for the Anti-apoptotic Proteins, Bcl-2 and Mcl-1.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a K d of 40 µM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Structure-Guided Discovery of a Selective Mcl-1 Inhibitor with Cellular Activity.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using nuclear magnetic resonance and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compounds, and cellular activity was brought on scale by offsetting the negative charge of the anchoring carboxylate group. The obtained compounds described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compound, the chemical series leading to the identification of our more advanced compounds S63845 and S64315.

Structure-Based Design and Synthesis of Harmine Derivatives with Different Selectivity Profiles in Kinase versus Monoamine Oxidase Inhibition.

Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is an emerging biological target with implications in diverse therapeutic areas such as neurological disorders (Down syndrome, in particular), metabolism, and oncology. Harmine, a natural product that selectively inhibits DYRK1A amongst kinases, could serve as a tool compound to better understand the biological processes that arise from DYRK1A inhibition. On the other hand, harmine is also a potent inhibitor of monoamine oxidase A (MAO-A). Using structure-based design, we synthesized a collection of harmine analogues with tunable selectivity toward these two enzymes. Modifications at the 7-position typically decreased affinity for DYRK1A, whereas substitution at the 9-position had a similar effect on MAO-A inhibition but DYRK1A inhibition was maintained. The resulting collection of compounds can help to understand the biological role of DYRK1A and also to assess the interference in the biological effect originating in MAO-A inhibition.

Computational Prediction and Rationalization, and Experimental Validation of Handedness Induction in Helical Aromatic Oligoamide Foldamers.

Metadynamics simulations were used to describe the conformational energy landscapes of several helically folded aromatic quinoline carboxamide oligomers bearing a single chiral group at either the C or N terminus. The calculations allowed the prediction of whether a helix handedness bias occurs under the influence of the chiral group and gave insight into the interactions (sterics, electrostatics, hydrogen bonds) responsible for a particular helix sense preference. In the case of camphanyl-based and morpholine-based chiral groups, experimental data confirming the validity of the calculations were already available. New chiral groups with a proline residue were also investigated and were predicted to induce handedness. This prediction was verified experimentally through the synthesis of proline-containing monomers, their incorporation into an oligoamide sequence by solid phase synthesis and the investigation of handedness induction by NMR spectroscopy and circular dichroism.