Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of KATP Channels

4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K_ATP channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin‐secreting pancreatic β‐cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis‐(1,3‐dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B‐type K_ATP channels using the radioligand [³H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide ( 1 a ). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B‐type K_ATP channels. The compound substituted with a bulky (1‐adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B‐type K_ATP channels without being able to activate the SUR1‐type K_ATP channels.     

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp‐rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp‐rich peptides derived from the C‐terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A₂ (PLA₂), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM‐2—was found to display improved activity against both Gram‐positive and Gram‐negative bacteria. To improve the antimicrobial activity of PEM‐2 for potential clinical applications further, we determined the solution structure of PEM‐2 bound to membrane‐mimetic dodecylphosphocholine (DPC) micelles by two‐dimensional NMR methods. The DPC micelle‐bound structure of PEM‐2 adopts an α‐helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM‐2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM‐2 could be due to interactions with bacterial membranes. Potent analogues of PEM‐2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.     

siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC₅₀ values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC₅₀ to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.     

Synthesis, σ Receptor Affinity,and Pharmacological Evaluation of 5‐Phenylsulfanyl‐ and 5‐Benzyl‐Substituted Tetrahydro‐2‐benzazepines

In accordance with a novel strategy for generating the 2‐benzazepine scaffold by connecting C6–C1 and C3–N building blocks, a set of 5‐phenylsulfanyl‐ and 5‐benzyl‐substituted tetrahydro‐2‐benzazepines was synthesized and pharmacologically evaluated. Key steps of the synthesis were the Heck reaction, the Stetter reaction, a reductive cyclization, and the introduction of diverse N substituents at the end of the synthesis. High σ₁ affinity was achieved for 2‐benzazepines with linear or branched alk(en)yl residues containing at least an n‐butyl substructure. The butyl‐ and 4‐fluorobenzyl‐substituted derivatives, (±)‐5‐benzyl‐2‐butyl‐2,3,4,5‐tetrahydro‐1H‐2‐benzazepine ( 19 b ) and (±)‐5‐benzyl‐2‐(4‐fluorobenzyl)‐2,3,4,5‐tetrahydro‐1H‐2‐benzazepine ( 19 m ), show high selectivity over more than 50 other relevant targets, including the σ₂ subtype and various binding sites of the N‐methyl‐D ‐aspartate (NMDA) receptor. In the Irwin screen, 19 b and 19 m showed clean profiles without inducing considerable side effects. Compounds 19 b and 19 m did not reveal significant analgesic and cognition‐enhancing activity. Compound 19 m did not have any antidepressant‐like effects in mice.     

Ahp Cyclodepsipeptides: The Impact of the Ahp Residue on the “Canonical Inhibition” of S1 Serine Proteases

S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small‐molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small‐molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small‐molecule inhibitors that simultaneously target the S and S′ subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β‐hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.     

Design and Synthesis of Trypanosoma brucei Active 1‐Alkyloxy and 1‐Benzyloxyadamantano 2‐Guanylhydrazones

Treating African trypanosomiasis : The synthesis and biological evaluation of novel 1‐alkyloxy and 1‐benzyloxyadamantano 2‐guanylhydrazones, their 1‐dioxa congeners and two 1‐benzyladamantano 2‐guanylhydrazones is reported. Preliminary structure–activity relationship data were elucidated and lead compounds suitable for further optimization were discovered.

     

Synthesis and Biological Characterization of (3R,4R)‐4‐(2‐(Benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol and Its Stereoisomers for Activity toward Monoamine Transporters

A novel series of optically active molecules based on a 4‐(2‐(benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.

     

Alanine and Lysine Scans of the LL‐37‐Derived Peptide Fragment KR‐12 Reveal Key Residues for Antimicrobial Activity

The human host defence peptide LL‐37 is a broad‐spectrum antibiotic with immunomodulatory functions. Residues 18–29 in LL‐37 have previously been identified as a minimal peptide (KR‐12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR‐12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad‐spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR‐12 is a tuneable template for antibiotic development.     

The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH‐like 2‐Heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) Biosynthesis Enzyme PqsBC

Pseudomonas aeruginosa is a bacterial pathogen that causes life‐threatening infections in immunocompromised patients. It produces a large armory of saturated and mono‐unsaturated 2‐alkyl‐4(1H)‐quinolones (AQs) and AQ N‐oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β‐ketoacyl‐acyl‐carrier protein synthase III (FabH)‐like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2‐heptyl‐4(1H)‐quinolone (HHQ), by condensing octanoyl‐coenzyme A (CoA) with 2‐aminobenzoylacetate (2‐ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium‐chain acyl‐CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono‐unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl‐binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH‐enzymes, for which such structural transitions have been postulated but have never been observed.     

Synthesis of N‐Substituted Iminosugar Derivatives and Evaluation of Their Immunosuppressive Activities

It is important to find more effective and safer immunosuppressants, because clinically used immunosuppressive agents have significant side effects. A series of N‐substituted iminosugar derivatives were designed and synthesized, and their immunosuppressive effects were evaluated by the CCK‐8 assay. The results revealed that iminosugars 10 e and 10 i , that is, (3R,4S)‐1‐(4‐heptyloxylphenylethyl)pyrrolidine‐3,4‐diol and (3R,4S)‐1‐[2‐(2‐chloro‐4‐(p‐tolylthio)‐phenyl‐1‐yl)ethyl]pyrrolidine‐3,4‐diol, respectively, exhibited the strongest inhibitory effects on mouse splenocyte proliferation (IC₅₀=2.16 and 2.48 μm , respectively), whereas the iminosugars containing an amide group near the hydrophilic head (compounds 10 j – n ) exhibited no inhibitory effects. Further studies revealed that the inhibitory effects on splenocyte proliferation may have come from the suppression of both IFN‐γ and IL‐4 cytokines. Our results suggest that synthetic iminosugars, especially compounds 10 e and 10 i , hold potential as immunosuppressive agents.     

A Novel Competitive Class of α‐Glucosidase Inhibitors: (E)‐1‐Phenyl‐3‐(4‐Styrylphenyl)Urea Derivatives

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo‐ and regiocontrolled. Here, we show that readily accessible achiral (E)‐1‐phenyl‐3‐(4‐strylphenyl)ureas are potent competitive α‐glucosidase inhibitors. A systematic synthesis study shows that the 1‐phenyl moiety on the urea is critical for ensuring competitive inhibition, and substituents on both terminal phenyl groups contribute to inhibition potency. The most potent inhibitor, compound 12 (IC₅₀=8.4 μM , K_i=3.2 μM ), manifested a simple slow‐binding inhibition profile for α‐glucosidase with the kinetic parameters k₃=0.005256 μM ^?1 min^?1, k₄=0.003024 min^?1, and ${K{{{\rm app}\hfill \atop {\rm i}\hfill}}}$ =0.5753 μM .     

(2,2‐Diphenyl‐[1,3]oxathiolan‐5‐ylmethyl)‐(3‐phenyl‐propyl)‐amine: a Potent and Selective 5‐HT1A Receptor Agonist

A selective 5‐HT _1A receptor agonist : A new series of ligands acting at 5‐HT_1A serotonin receptor were identified. Among them (2,2‐diphenyl‐[1,3]oxathiolan‐5‐yl‐methyl)‐(3‐phenyl‐propyl)amine (shown) possesses outstanding activity (pK_i=8.72, pD₂=7.67, E_max=85) and selectivity (5‐HT_1A/α_1D>150), and represents a new 5‐HT_1A agonist chemotype.

     

Synthesis and Structure–Activity Relationships of 1‐Benzyl‐4,5,6‐trimethoxyindoles as a Novel Class of Potent Antimitotic Agents

Combretastatin A‐4 derivatives : A series of combretastatin A‐4‐derived 1‐benzyl‐4,5,6‐trimethoxyindoles was designed and prepared as a novel class of potent antimitotic agents acting through the colchicine binding site on the microtubule.

     

Chemoenzymatic One‐Pot Synthesis of γ‐Butyrolactones

The synthesis of enantio‐ and diastereomerically pure γ‐butyrolactones is described using a one‐pot, two‐enzyme cascade. Ethyl 2‐methyl‐4‐oxopent‐2‐enoate ( 2 ) was reduced selectively first in a 1,4‐reduction using the old yellow enzyme (OYE1) [EC 1.6.99.1] and consecutively in a 1,2‐reduction by an alcohol dehydrogenase [EC 1.1.1.2].     

Evaluation of (4‐Arylpiperidin‐1‐yl)cyclopentanecarboxamides As High‐Affinity and Long‐Residence‐Time Antagonists for the CCR2 Receptor

Animal models suggest that the chemokine ligand 2/CC‐chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We previously described a new approach for the design of CCR2 antagonists by the use of structure–kinetics relationships (SKRs). Herein we report new findings on the structure–affinity relationships (SARs) and SKRs of the reference compound MK‐0483, its diastereomers, and its structural analogues as CCR2 antagonists. The SARs of the 4‐arylpiperidine group suggest that lipophilic hydrogen‐bond‐accepting substituents at the 3‐position are favorable. However, the SKRs suggest that a lipophilic group with a certain size is desired [e.g., 3‐Br: K_i=2.8 nM , residence time (t_res)=243 min; 3‐iPr: K_i=3.6 nM , t_res=266 min]. Alternatively, additional substituents and further optimization of the molecule, while keeping a carboxylic acid at the 3‐position, can also prolong t_res; this was most prominently observed in MK‐0483 (K_i=1.2 nM , t_res=724 min) and a close analogue (K_i=7.8 nM ) with a short residence time.