Marc Nazare

Evidence for CCl/CBr⋅⋅⋅π Interactions as an Important Contribution to Protein–Ligand Binding Affinity†

Attractive chlorine: Noncovalent interactions between chlorine or bromine atoms and aromatic rings in proteins open up a new method for the manipulation of molecular recognition. Substitution at distinct positions of two factor Xa inhibitors improves the free energy of binding by interaction with a tyrosine unit. The generality of this motif was underscored by multiple crystal structures as well as high-level quantum chemical calculations (see picture).

A General and Mild Palladium‐Catalyzed Domino Reaction for the Synthesis of 2H‐Indazoles

Indazoles play an increasingly important role in drug discovery. They act as an efficient isostere for privileged structures such as indoles and benzimidazoles. Furthermore, this important scaffold is able to interact with a variety of diverse targets, as highlighted by the growing number of reports of biologically active indazole derivatives. However, only a limited number of approaches for the regioselective synthesis of N-substituted indazoles are available today. Most approaches afford the thermodynamically favored 1H-indazole or mixtures of 1Hand 2H-indazoles, whereas the regioselective formation of 2H-indazoles remains a very challenging task. The lack of a direct, efficient, and regioselective synthetic procedure for the construction of 2Hindazoles prevents their broader application in, for example, medicinal chemistry. Thus, there is an unmet need for the development of a simple and general synthesis of 2Hindazoles from readily available precursors. Herein we report a straightforward domino reaction sequence consisting of a regioselective coupling of monosubstituted hydrazines 2 with 2-halophenylacetylenes 1, followed by an intramolecular hydroamination through a 5-exo-dig cyclization and subsequent isomerization of the exocyclic double bond to give the aromatic 2H-indazole (Scheme 1). The first challenge in this strategy was the development of a regioselective transition-metal-catalyzed coupling of monosubstituted hydrazines 2 with 2-halophenylacetylenes 1 to afford the required N,N’-disubstituted hydrazines 4. Although a number of transition-metal-catalyzed coupling reactions of aryl halides with amides, amines, hydrazides, and hydrazones are known, only a few coupling reactions of hydrazines have been reported, and only one of the reported hydrazine couplings, for the formation of N,N-diaryl hydrazines, is regioselective. A second challenge in the development of our proposed strategy was the control of the hydroamination/ cyclization step to form the 1,2-dihydroindazole 5, as other possible cyclization pathways lead to other products, such as 1,2-dihydrocinnolines and N-azaindoles. The isomerization of the exocyclic double bond in dihydro-2H-indazole 5 to give the aromatic 2H-indazole 3 was expected to occur spontaneously under the reaction conditions and thus not to pose any problems. We initiated our investigation by screening for reaction conditions under which the coupling of 1-chloro-2-phenylethynylbenzene (1a) and phenylhydrazine (2a) would proceed efficiently to give the N,N’-diaryl hydrazine 4. Upon optimization of the reaction parameters (the transition metal, metal salt, ligand, base, solvent, and temperature), the desired coupling was found to proceed cleanly within just a few hours and with complete regioselectivity when the catalyst system [Pd2(dba)3]/PtBu3 (1:2) was used in toluene at 80 8C with NaOtBu as the base (dba = dibenzylideneacetone). This reaction is to our knowledge the first regioselective transition-metal-catalyzed coupling of monosubstituted hydrazines to give N,N’-disubstituted hydrazine products. We further optimized the reaction parameters to identify conditions that would promote the complete domino reaction of 1 a with 2a as a one-pot reaction (Table 1). We found that the use of polar solvents, such as DMF, NMP, or DMA, in combination with Cs2CO3 led to the formation of the desired 2H-indazole 3a in good yield (Table 1, entries 2–4), whereas Scheme 1. Proposed synthesis of 2H-indazoles.

Fragment Deconstruction of Small, Potent Factor Xa Inhibitors: Exploring the Superadditivity Energetics of Fragment Linking in Protein-Ligand Complexes.

Predictable thermodynamic additivity is one of the cornerstones of classical covalent chemistry, allowing accurate calculation of energy terms for complete processes by addition of terms for individual components. However this principle breaks down in complex noncovalent systems, such as biological systems, in which the energetics of individual components are not truly independent of each other. This complicates predicting protein structure and folding and, the focus of this work, the prediction of ligand binding to proteins. Molecular recognition in protein–ligand complexes predominantly occurs through multiple noncovalent interactions, whereas their contribution to the total free-energy of binding (DG) is often unevenly distributed over the contact interface. The identification of ligands as “molecular anchors” for high affinity regions in proteins (“hot spots”) is fundamental for fragment-based drug discovery, 3] indicating the similarity of ligandand protein-centric concepts. Often highaffinity ligands encompass more than one fragment in proximal protein sites; in a few cases, individual fragments in two neighboring sites could be linked to result in high binding affinity. Ideally, the DG of linked fragments should be significantly greater than the sum of DG increments from each fragment. This overproportional increase (“superadditivity”) is attributed to the fact that each fragment loses a significant part of its rigid body rotational and translational entropy upon complex formation. Thus, the sum of DG for two fragments includes two unfavorable rigid body entropy barrier terms, whereas the joined molecule is only affected by one of these terms. Any ligand has to overcome this barrier because of entropy loss upon association to its site. The nonadditivity for DG contributions is defined as linker coefficient E corresponding to the difference between the sums of fragment affinity and the final ligand [Eq. (1)]. DGfinal 1⁄4 DGfrag1 þ DGfrag2 þ DGlink with DGlink 1⁄4 R T ln E ð1Þ

N-[6-(4-Butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide (SAR216471), a Novel Intravenous and Oral, Reversible, and Directly Acting P2Y12 Antagonist

In the search of a potential backup for clopidogrel, we have initiated a HTS campaign designed to identify novel reversible P2Y12 antagonists. Starting from a hit with low micromolar binding activity, we report here the main steps of the optimization process leading to the identification of the preclinical candidate SAR216471. It is a potent, highly selective, and reversible P2Y12 receptor antagonist and by far the most potent inhibitor of ADP-induced platelet aggregation among the P2Y12 antagonists described in the literature. SAR216471 displays potent in vivo antiplatelet and antithrombotic activities and has the potential to differentiate from other antiplatelet agents.

Identification of High-Affinity P2Y12 Antagonists Based on a Phenylpyrazole Glutamic Acid Piperazine Backbone

A series of novel, highly potent P2Y₁₂ antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y₁₂ antagonists displaying not only low nanomolar binding affinity to the P2Y₁₂ receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC₅₀ values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

Synthesis of the (9S, 18R)-seco acid of the leukocyte adhesion inhibitor cyclamenol A

Abstract Cyclamenol A is a naturally occurring inhibitor of leukocyte adhesion to endothelial cells. The (9 S ,18 R ) diastereomer of cyclamenol A seco acid was synthesized by employing Sonogashira couplings and olefination reactions as the key steps.

Efficient enantioselective synthesis of a beta-hydroxyepoxide building block for the construction of macrocyclic natural products

The synthesis of a protected β-hydroxyepoxide macrolide building block in seven steps from commerically available (R)-3-hydroxybutyric acid methyl ester in 68% overall yield is described.

Modulation of Hexadecyl-LPA-Mediated Activation of Mast Cells and Microglia by a Chemical Probe for LPA5.

Mast cells and microglia play a critical role in innate immunity and inflammation and can be activated by a wide range of endogenous and exogenous stimuli. Lysophosphatidic acid (LPA) has recently been reported to activate mast cells and microglia. Using the human mast cell line HMC‐1 and the mouse microglia cell line BV‐2, we show that LPA‐mediated activation can be prevented by blockade of the LPA receptor 5 (LPA5) in both cell lines. The identification of new LPA5‐specific antagonists as tool compounds to probe and modulate the LPA5/LPA axis in relevant in vitro and in vivo assays should contribute to better understanding of the underlying role of LPAs in the development and progression of (neuro‐) inflammatory diseases.

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high-affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12 fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site-specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA-binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow-sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X-ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug-protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.