7‐Azidomethoxy‐Coumarins as Profluorophores for Templated Nucleic Acid Detection

Templated nucleic acid detection is an emerging bioanalytical method that makes use of the target DNA or RNA strand to initiate a fluorogenic reaction. The Staudinger reduction holds particular promise for templated sensing of nucleic acids because the involved functional groups are highly chemoselective. Here, the azidomethoxy group, which can be removed under Staudinger conditions, is used to cage 7‐hydroxycoumarin fluorophores. Reduction by phosphines and subsequent loss of the azidomethoxy substituent induce a significant bathochromic shift of the major absorbance band in the near UV region. When excited at the appropriate wavelength, this change in the absorbance spectrum translates into a substantial fluorescence turn‐on signal. The described profluorophores are readily conjugated to amino‐modified DNAs and are rapidly uncaged by a triphenylphosphine–DNA probe under the control of a DNA template. In addition, turnover of the probes on the target strand occurs and yields substantial signal amplification.     

Preparation of 1,4,5‐Trisubstituted 5‐Acyl‐1,2,3‐triazoles by Selective Acylation between Copper(I)‐Carbon(sp) and Copper(I)‐Carbon(sp2) Bonds with Acyl Chlorides

A one‐pot, three‐component method for preparation of 1,4,5‐trisubstituted 5‐acyl‐1,2,3‐triazoles has been developed based on a highly selective acylation of the copper(I)‐carbon(sp²) bond with acyl chlorides in the presence of the copper(I)‐carbon(sp) bond. The procedure is characterized by high efficiency and simple conditions.

     

1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA

In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.     

Potent Triple Helix Stabilization by 5′,3′‐Modified Triplex‐Forming Oligonucleotides

Anthraquinone and pyrene analogues attached to the 3′ and/or 5′ termini of triplex‐forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid‐phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 °C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 °C at pH 7 even though they do not contain any triplexstabilizing base analogues. These studies have implications for the design of triplex‐forming oligonucleotides for use in biology and nanotechnology.     

Discovery of 5′′‐Chloro‐N‐[(5,6‐dimethoxypyridin‐2‐yl)methyl]‐2,2′:5′,3′′‐terpyridine‐3′‐carboxamide (MK‐1064): A Selective Orexin 2 Receptor Antagonist (2‐SORA) for the Treatment of Insomnia

The field of small‐molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof‐of‐concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX₁R and OX₂R), termed dual orexin receptor antagonists (DORAs), affording late‐stage development candidates including Merck’s suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX₁R or OX₂R alone has been hampered by the dearth of suitable subtype‐selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2‐SORA) series to afford a potent, orally bioavailable 2‐SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5‐disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P‐glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2‐SORA clinical candidate, 5′′‐chloro‐N‐[(5,6‐dimethoxypyridin‐2‐yl)methyl]‐2,2′:5′,3′′‐terpyridine‐3′‐carboxamide (MK‐1064), in mouse, rat, dog, and rhesus sleep models.     

Functionalization of the 3′‐Ends of DNA and RNA Strands with N‐ethyl‐N‐coupled Nucleosides: A Promising Approach To Avoid 3′‐Exonuclease‐Catalyzed Hydrolysis of Therapeutic Oligonucleotides

The development of nucleic acid derivatives to generate novel medical treatments has become increasingly popular, but the high vulnerability of oligonucleotides to nucleases limits their practical use. We explored the possibility of increasing the stability against 3′‐exonucleases by replacing the two 3′‐terminal nucleotides by N‐ethyl‐N‐coupled nucleosides. Molecular dynamics simulations of 3′‐N‐ethyl‐N‐modified DNA:Klenow fragment complexes suggested that this kind of alteration has negative effects on the correct positioning of the adjacent scissile phosphodiester bond at the active site of the enzyme, and accordingly was expected to protect the oligonucleotide from degradation. We verified that these modifications conferred complete resistance to 3′‐exonucleases. Furthermore, cellular RNAi experiments with 3′‐N‐ethyl‐N‐modified siRNAs showed that these modifications were compatible with the RNAi machinery. Overall, our experimental and theoretical studies strongly suggest that these modified oligonucleotides could be valuable for therapeutic applications.     

Combining Designer Cells and Click Chemistry for a One‐Pot Four‐Step Preparation of Enantiopure β‐Hydroxytriazoles

The multistep catalytic process using designer cells, either added as freshly prepared suspensions or as stable lyophilized powder, and click reaction can be performed in one pot. The sequence of four reactions allows the production of both enantiomers of β‐hydroxytriazoles with high enantiomeric excess.     

Multimeric Lactoside “Click Clusters” as Tools to Investigate the Effect of Linker Length in Specific Interactions with Peanut Lectin,Galectin‐1, and ‐3

Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new “click clusters” toward biologically relevant galectins (gal‐1, gal‐3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme‐linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two‐site “sandwich” ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

Direct Synthesis of Partially Modified 2′‐O‐Pivaloyloxymethyl RNAs by a Base‐Labile Protecting Group Strategy and their Potential for Prodrug‐Based Gene‐Silencing Applications

An original and straightforward synthesis of partially modified 2′‐O‐pivaloyloxymethyl‐substituted (PivOM‐substituted) oligoribonucleotides has been achieved. The aim of this 2′‐enzymolabile modification was to enhance nuclease stability of RNA and transmembrane transport. To make these modified RNAs easily available we developed a base‐labile protecting group strategy with standard protections for nucleobases (acyl) and phosphates (cyanoethyl), a Q‐linker and two different acetalester protection groups for 2′‐OH: propionyloxymethyl (PrOM) and PivOM. Interestingly, orthogonal deprotection conditions based on anhydrous butylamine in THF were found to remove propionyloxymethyl groups selectively, while preserving PivOM groups. Duplex stability, circular dichroism studies and nuclease resistance, as well as the ability to inhibit gene expression of modified 2′‐O‐PivOM RNA, were evaluated.     

Microwave Heating Effects Rapid and Selective Decarboalkoxylation of Mono‐Alkylated Malonates and β‐Ketoesters

Brief microwave irradiation of mono‐alkylated malonates and β‐ketoesters at 160–200 °C in wet DMF induces smooth and selective decarboalkoxylation. Observations suggest that this reaction occurs by nucleophilic attack of water at the ester carbonyl carbon (hydrolysis) followed by decarboxylation of the resulting acid. The process occurs despite the absence of traditional acid, base or nucleophile catalysts or reagents.     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.     

Design and Synthesis of DNA‐Interactive β‐Carboline–Oxindole Hybrids as Cytotoxic and Apoptosis‐Inducing Agents

A new series of (E)‐3‐[(1‐aryl‐9H‐pyrido[3,4‐b]indol‐3‐yl)methylene]indolin‐2‐one hybrids were synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines, namely, HCT‐15, HCT‐116, A549, NCI‐H460, and MCF‐7, including HFL. Among the tested compounds, (E)‐1‐benzyl‐5‐bromo‐3‐{[1‐(2,5‐dimethoxyphenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl]methylene}indolin‐2‐one ( 10 s ) showed potent cytotoxicity against HCT‐15 cancer cells with an IC₅₀ value of 1.43±0.26 μm and a GI₅₀ value of 0.89±0.06 μm . Notably, induction of apoptosis by 10 s on the HCT‐15 cell line was characterized by using different staining techniques, such as acridine orange/ethidium bromide (AO/EB) and DAPI. Further, to understand the mechanism of anticancer effects, various assays such as annexin V‐FITC/PI, DCFDA, and JC‐1were performed. The flow cytometric analysis revealed that compound 10 s arrests the HCT‐15 cancer cells at the G0/G1 phase of the cell cycle. Additionally, western blot analysis indicated that treatment of 10 s on HCT‐15 cancer cells led to decreased expression of anti‐apoptotic Bcl‐2 and increased protein expression of both pro‐apoptotic Bax and caspase‐3, ‐8, and ‐9, and cleaved PARP with reference to actin. Next, a clonogenic assay revealed the inhibition of colony formation in HCT‐15 cancer cells by 10 s in a dose‐dependent manner. Moreover, upon testing on normal human lung cells (HFL), the compounds were observed to be safer with a low toxicity profile. In addition, viscosity and molecular‐docking studies showed that compound 10 s has typical intercalation with DNA.     

The Discovery and Development of the N‐Substituted trans‐3,4‐Dimethyl‐4‐(3′‐hydroxyphenyl)piperidine Class of Pure Opioid Receptor Antagonists

N‐Substituted trans‐3,4‐dimethyl‐4‐(3‐hydroxyphenyl)piperidines are a class of pure opioid receptor antagonists with a novel pharmacophore. This opioid receptor antagonist pharmacophore was used as a lead structure to design and develop several interesting and useful opioid receptor antagonists. In this review we describe: 1) early SAR studies that led to the discovery of LY255582 and analogues that are nonselective opioid receptor antagonists developed for the treatment of obesity; 2) the discovery and commercialization of LY246736 (alvimopan; ENTEREG®), a peripherally selective opioid receptor antagonist that accelerates the time to upper and lower GI recovery following surgeries that include partial bowel resection with primary anastomosis; and 3) the discovery and development of the potent and selective κ opioid receptor antagonist JDTic and analogues as potential pharmacotherapies for treating depression, anxiety, and substance abuse (nicotine, alcohol, and cocaine). In addition, the use of JDTic for obtaining the X‐ray structure of the human κ opioid receptor is discussed.     

Synthesis and Structure–Activity Relationship Studies of 2‐(1,3,4‐Oxadiazole‐2(3H)‐thione)‐3‐amino‐5‐arylthieno[2,3‐b]pyridines as Inhibitors of DRAK2

In recent years, DAPK‐related apoptosis‐inducing protein kinase 2 (DRAK2) has emerged as a promising target for the treatment of a variety of autoimmune diseases and for the prevention of graft rejection after organ transplantation. However, medicinal chemistry optimization campaigns for the discovery of novel small‐molecule inhibitors of DRAK2 have not yet been published. Screening of a proprietary compound library led to the discovery of a benzothiophene analogue that displays an affinity constant (K_d) value of 0.25 μM . Variation of the core scaffold and of the substitution pattern afforded a series of 5‐arylthieno[2,3‐b]pyridines with strong binding affinity (K_d=0.008 μM for the most potent representative). These compounds also show promising activity in a functional biochemical DRAK2 enzyme assay, with an IC₅₀ value of 0.029 μM for the most potent congener. Selectivity profiling of the most potent compounds revealed that they lack selectivity within the DAPK family of kinases. However, one of the less potent analogues is a selective ligand for DRAK2 and can be used as starting point for the synthesis of selective and potent DRAK2 inhibitors.