Palladium-Catalyzed Dearomatization of Benzothiophenes: Isolation and Functionalization of a Discrete Dearomatized Intermediate.

A Pd-catalyzed decarboxylative dearomatization reaction of a heterocyclic substrate enables access to an uncommon reaction intermediate that rearomatizes in the presence of amine bases in a net C-H functionalization sequence. The dearomatized benzo[b]thiophene intermediate bears an exocyclic alkene that can be functionalized through cycloaddition and halogenation reactions to deliver complex heterocyclic products.

Inhibition of SIRT2 promotes death of human cytomegalovirus-infected peripheral blood monocytes via apoptosis and necroptosis.

Peripheral blood monocytes are the cells predominantly responsible for systemic dissemination of human cytomegalovirus (HCMV) and a significant cause of morbidity and mortality in immunocompromised patients. HCMV establishes a silent/quiescent infection in monocytes, which is defined by the lack of viral replication and lytic gene expression. The absence of replication shields the virus within infected monocytes from the current available antiviral drugs that are designed to suppress active replication. Our previous work has shown that HCMV stimulates a noncanonical phosphorylation of Akt and the subsequent upregulation of a distinct subset of prosurvival proteins in normally short-lived monocytes. In this study, we found that SIRT2 activity is required for the unique activation profile of Akt induced within HCMV-infected monocytes. Importantly, both therapeutic and prophylactic treatment with a novel SIRT2 inhibitor, FLS-379, promoted death of infected monocytes via both the apoptotic and necroptotic cell death pathways. Mechanistically, SIRT2 inhibition reduced expression of Mcl-1, an Akt-dependent antiapoptotic Bcl-2 family member, and enhanced activation of MLKL, the executioner kinase of necroptosis. We have previously reported HCMV to block necroptosis by stimulating cellular autophagy. Here, we additionally demonstrate that inhibition of SIRT2 suppressed Akt-dependent HCMV-induced autophagy leading to necroptosis of infected monocytes. Overall, our data show that SIRT2 inhibition can simultaneously promote death of quiescently infected monocytes by two distinct death pathways, apoptosis and necroptosis, which may be vital for limiting viral dissemination to peripheral organs in immunosuppressed patients.

Discovery and Characterization of 2,5-Substituted Benzoic Acid Dual Inhibitors of the Anti-apoptotic Mcl-1 and Bfl-1 Proteins.

Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compound 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.

Tyr1-ψ[( Z)CF═CH]-Gly2 Fluorinated Peptidomimetic Improves Distribution and Metabolism Properties of Leu-Enkephalin.

Opioid peptides are key regulators in cellular and intercellular physiological responses, and could be therapeutically useful for modulating several pathological conditions. Unfortunately, the use of peptide-based agonists to target centrally located opioid receptors is limited by poor physicochemical (PC), distribution, metabolic, and pharmacokinetic (DMPK) properties that restrict penetration across the blood-brain barrier via passive diffusion. To address these problems, the present paper exploits fluorinated peptidomimetics to simultaneously modify PC and DMPK properties, thus facilitating entry into the central nervous system. As an initial example, the present paper exploited the Tyr1-ψ[( Z)CF═CH]-Gly2 peptidomimetic to improve PC druglike characteristics (computational), plasma and microsomal degradation, and systemic and CNS distribution of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Thus, the fluoroalkene replacement transformed an instable in vitro tool compound into a stable and centrally distributed in vivo probe. In contrast, the Tyr1-ψ[CF3CH2-NH]-Gly2 peptidomimetic decreased stability by accelerating proteolysis at the Gly3-Phe4 position.

Potent dialkyl substrate-product analogue inhibitors and inactivators of α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis by rational design.

Rational approaches for the design of enzyme inhibitors furnish powerful strategies for developing pharmaceutical agents and tools for probing biological mechanisms. A new strategy for the development of gem-disubstituted substrate-product analogues as inhibitors of racemases and epimerases is elaborated using α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis (MtMCR) as a model enzyme. MtMCR catalyzes the epimerization at C2 of acyl-CoA substrates, a key step in the metabolism of branched-chain fatty acids. Moreover, the human enzyme is a potential target for the development of therapeutic agents directed against prostate cancer. We show that rationally designed, N,N-dialkylcarbamoyl-CoA substrate-product analogues inactivate MtMCR. Binding greatly exceeds that of the substrate, (S)-ibuprofenoyl-CoA, up to ∼250-fold and is proportional to the alkyl chain length (4-12 carbons) with the N,N-didecyl and N,N-didodecyl species having competitive inhibition constants with values of 1.9 ±â€¯0.2 µM and 0.42 ±â€¯0.04 µM, respectively. The presence of two decyl chains enhanced binding over a single decyl chain by ∼204-fold. Overall, the results reveal that gem-disubstituted substrate-product analogues can yield extremely potent inhibitors of an epimerase with a capacious active site.

Synthesis and Opioid Activity of Tyr1 -ψ[(Z)CF=CH]-Gly2 and Tyr1 -ψ[(S)/(R)-CF3 CH-NH]-Gly2 Leu-enkephalin Fluorinated Peptidomimetics.

We describe the design, synthesis, and opioid activity of fluoroalkene (Tyr1 -ψ[(Z)CF=CH]-Gly2 ) and trifluoroethylamine (Tyr1 -ψ[(S)/(R)-CF3 CH-NH]-Gly2 ) analogues of the endogenous opioid neuropeptide, Leu-enkephalin. The fluoroalkene peptidomimetic exhibited low nanomolar functional activity (5.0±2 nm and 60±15 nm for δ- and µ-opioid receptors, respectively) with a µ/δ-selectivity ratio that mimics that of the natural peptide. However, the trifluoroethylamine peptidomimetics, irrespective of stereochemistry, did not activate the opioid receptors, which suggest that bulky CF3 substituents are not tolerated at this position.

Rational design and synthesis of substrate-product analogue inhibitors of α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis.

2,2-Bis(4-isobutylphenyl)propanoyl-CoA and 2,2-bis(4-t-butylphenyl)propanoyl-CoA are rationally designed, gem-disubstituted substrate-product analogues that competitively inhibit α-methylacyl-coenzyme A racemase from Mycobacterium tuberculosis with Ki values of 16.9 ± 0.6 and 21 ± 4 µM, respectively, exceeding the enzyme's affinity for the substrate by approximately 5-fold.

Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium.

Regioselective S-acylation of coenzyme A (CoA) is achieved under aqueous conditions using various aliphatic and aromatic carboxylic acids activated as their methyl acyl phosphate monoesters. Unlike many hydrophobic activating groups, the anionic methyl acyl phosphate mixed anhydride is more compatible with aqueous solvents, making it useful for conducting acylation reactions in an aqueous medium.

Inhibition of glutamate racemase by substrate-product analogues.

D-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of D-glutamate from L-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate-product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (Ki=3.1±0.6 mM, cf. Km=1.41±0.06 mM). The cyclic substrate-product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate-product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (Ki=18.4±1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate-product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.

Bioreduction of methyl heteroaryl and aryl heteroaryl ketones in high enantiomeric excess with newly isolated fungal strains.

Enantioenriched heteroaryl ethanols and aryl heteroarylmethanols are important intermediates and structural motifs in medicinal chemistry. Asymmetric biocatalytic reduction of corresponding ketones provides a straightforward approach for preparation of these compounds. Accordingly, three newly isolated fungal strains have been described, which produced the desired heteroaryl alcohols in high enantiomeric excess (ee). A broad substrate specificity was observed within these limited number of biocatalysts as demonstrated by preparation of a variety of heteroaryl alcohols, including (S)-5-(1-hydroxyethyl)furo[2,3-c]pyridine, a key intermediate for HIV-1 reverse transcriptase inhibitor, (S)-phenyl(pyridin-2-yl)methanol, an analgesic and (S,S)-2,6-bis(1-hydroxyethyl)pyridine, a chiral building block, mostly in >99% ee and 80-92% yield. Micro-morphologically, one of the isolate was found to be similar to Penicillium funiculosum. However, its ß-tubulin sequence showed only 88% sequence identity with the known ß-tubulin sequences of Penicillium. It may, therefore, represent a new species of Penicillium. The other biocatalysts were identified as Alternaria alternata and Talaromyces flavus.

A catalase-peroxidase for oxidation of ß-lactams to their (R)-sulfoxides.

In this communication we report for the first time a biocatalytic method for stereoselective oxidation of ß-lactams, represented by penicillin-G, penicillin-V and cephalosporin-G to their (R)-sulfoxides. The method involves use of a bacterium, identified as Bacillus pumilis as biocatalyst. The enzyme responsible for oxidase activity has been purified and characterized as catalase-peroxidase (KatG). KatG of B. pumilis is a heme containing protein showing characteristic heme spectra with soret peak at 406 nm and visible peaks at 503 and 635 nm. The major properties that distinguish B. pumilis KatG from other bacterial KatGs are (i) it is a monomer and contains one heme per monomer, whereas KatGs of other bacteria are dimers or tetramers and have low heme content of about one per dimer or two per tetramer and (ii) its 12-residue, N-terminal sequence obtained by Edman degradation did not show significant similarity with any of known KatGs.

Chiral solvating agents for cyanohydrins and carboxylic acids.

We have shown that a structure as simple as an ion pair of (R)- or (S)-mandelate and dimethylamminopyridinium ions possesses structural features that are sufficient for NMR enantiodiscrimination of cyanohydrins. Moreover, (1)H NMR data of cyanohydrins of known configuration obtained in the presence of the mandelate-dimethylaminopyridinium ion pair point to the existence of a correlation between chemical shifts and absolute configuration of cyanohydrins. Mandelate-DMAPH(+) ion pair and mandelonitrile form a 1:1 complex with an association constant of 338 M(-1) (DeltaG(0), -3.4 kcal/mol) for the (R)-mandelonitrile/(R)-mandelate-DMAPH(+) and 139 M(-1) (DeltaG(0), -2.9 kcal/mol) for the (R)-mandelonitrile/(S)-mandelate-DMAPH(+) complex. To understand the origin of enantiodiscrimination, the geometry optimization and energy minimization of the models of ternary complexes of (S)-mandelonitrile/(R)-mandelate/DMAPH(+) and (S)-mandelonitrile/(S)-mandelate/DMAPH(+) complexes was performed using DFT methodology (B3LYP) with the 6-31+G(d) basis set in Gaussian 3.0. Further, analysis of optimized molecular model obtained from theoretical studies suggested that (i) DMAP may be replaced with other amines, (ii) the hydroxyl group of mandelic acid is not necessary for stabilization of ternary complex and may be replaced with other groups such as methyl, (iii) the ion pair should form a stable ternary complex with any hydrogen-bond donor, provided its OH bond is sufficiently polarized, and (iv) alpha-H of racemic mandelic acid should also get resolved with optically pure mandelonitrile. These inferences were experimentally verified, which not only validated the proposed model but also led to development of a new chiral solvating agent for determination of ee of carboxylic acids and absolute configuration of aryl but not alkyl carboxylic acids.