Bioavailability Studies and in vitro Profiling of the Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitor UCPH‐102

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH‐101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood–brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg^?1) of the closely related analogue UCPH‐102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH‐102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH‐102 (10 μm ) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH‐102 (20 mg kg^?1) did not induce acute effects or any visible changes in behavior.     

Novel Insights into Structure–Activity Relationships of N‐Terminally Modified PACE4 Inhibitors

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi‐Leu (ML) peptide, an octapeptide with the sequence Ac‐LLLLRVKR‐NH₂. Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N‐terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N‐terminal extension but by the protection of both ends with the d ‐Leu residue and 4‐amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile.     

Synthesis,Structure–Activity,and Structure–Stability Relationships of 2‐Substituted‐N‐(4‐oxo‐3‐oxetanyl) N‐Acylethanolamine Acid Amidase (NAAA) Inhibitors

N‐Acylethanolamine acid amidase (NAAA) is a cysteine amidase that preferentially hydrolyzes saturated or monounsaturated fatty acid ethanolamides (FAEs), such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), which are endogenous agonists of nuclear peroxisome proliferator‐activated receptor‐α (PPAR‐α). Compounds that feature an α‐amino‐β‐lactone ring have been identified as potent and selective NAAA inhibitors and have been shown to exert marked anti‐inflammatory effects that are mediated through FAE‐dependent activation of PPAR‐α. We synthesized and tested a series of racemic, diastereomerically pure β‐substituted α‐amino‐β‐lactones, as either carbamate or amide derivatives, investigating the structure–activity and structure–stability relationships (SAR and SSR) following changes in β‐substituent size, relative stereochemistry at the α‐ and β‐positions, and α‐amino functionality. Substituted carbamate derivatives emerged as more active and stable than amide analogues, with the cis configuration being generally preferred for stability. Increased steric bulk at the β‐position negatively affected NAAA inhibitory potency, while improving both chemical and plasma stability.     

Structure–Property Relationships of a Class of Carbamate‐Based Fatty Acid Amide Hydrolase (FAAH) Inhibitors: Chemical and Biological Stability

Cyclohexylcarbamic acid aryl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors, which includes the reference compound URB597. The reactivity of their carbamate fragment is involved in pharmacological activity and may affect their pharmacokinetic and toxicological properties. We conducted in vitro stability experiments in chemical and biological environments to investigate the structure–stability relationships in this class of compounds. The results show that electrophilicity of the carbamate influences chemical stability, as suggested by the relation between the rate constant of alkaline hydrolysis (log k_pH9) and the energy of the lowest unoccupied molecular orbital (LUMO). Introduction of small electron‐donor substituents at conjugated positions of the O‐aryl moiety increased the overall hydrolytic stability of the carbamate group without affecting FAAH inhibitory potency, whereas peripheral non‐conjugated hydrophilic groups, which favor FAAH recognition, helped decrease oxidative metabolism in the liver.     

Structure–Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates

Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8–13) have been studied as potential imaging probes and therapeutics for NT‐1‐receptor‐positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8–13) for radiotracer development, we herein report a side‐by‐side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [¹⁷⁷Lu]‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐labeled examples studied, analogues of NT(8–13) containing a short hydrophilic tetraethylene glycol (PEG₄) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro.     

Structure–Activity Relationship Study of Spider Polyamine Toxins as Inhibitors of Ionotropic Glutamate Receptors

The spider polyamine toxins Joro spider toxin‐3 (JSTX‐3) and Nephila polyamine toxins‐1 and ‐8 (NPTX‐1 and NPTX‐8) are isolated from the venom of the orb‐weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open‐channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid‐phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure–activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.     

Structure–Activity Relationships of 1,2‐Disubstituted Benzimidazoles: Selective Inhibition of Heme Oxygenase‐2 Activity

Devising ways to up‐ or down‐regulate heme oxygenase activity is attracting much interest as a strategy for the treatment of a variety of disorders. With a view of obtaining compounds that exhibit high potency and selectivity as inhibitors of the heme oxygenase‐2 (HO‐2) isozyme (constitutive) relative to the heme oxygenase‐1 (HO‐1) isozyme (inducible), several 1,2‐disubstituted 1H‐benzimidazoles were designed and synthesized. Specifically, analogues were synthesized in which the C2 substituent was the following: (1H‐imidazol‐1‐yl)methyl, (N‐morpholinyl)methyl, cyclopentylmethyl, cyclohexylmethyl, or (norborn‐2‐yl)methyl. Compounds with the cyclic system in the C2 substituent being a carbocyclic ring, especially cyclohexyl or norborn‐2‐yl, and the N1 substituent being a ring‐substituted benzyl group, especially 4‐chlorobenzyl or 4‐bromobenzyl, best exhibited the target criteria of high potency and selectivity toward inhibition of HO‐2. The new candidates should be useful pharmacological tools and may have therapeutic applications.     

Structure–Activity Relationships of SSAO/VAP‐1 Arylalkylamine‐Based Substrates

SSAO/VAP‐1 substrates may be valuable for the treatment or prevention of diabetes mellitus, as they show insulin‐mimetic properties. This review highlights the importance of studying the relevant steric and electronic features in the development of new ligands with better SSAO/VAP‐1 recognition, enhanced selectivity over other amine oxidases, and improved metabolic behavior.

     

Binding Mode and Structure–Activity Relationships around Direct Inhibitors of the Nrf2–Keap1 Complex

An X‐ray crystal structure of Kelch‐like ECH‐associated protein (Keap1) co‐crystallised with (1S,2R)‐2‐[(1S)‐1‐[(1,3‐dioxo‐2,3‐dihydro‐1H‐isoindol‐2‐yl)methyl]‐1,2,3,4‐tetrahydroisoquinolin‐2‐carbonyl]cyclohexane‐1‐carboxylic acid (compound (S,R,S)‐ 1 a ) was obtained. This X‐ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)‐ 1 a , as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.     

Novel Hinge‐Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure–Activity Relationship Study on Tofacitinib Bioisosteres

The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases crucially involved in cytokine signaling. JAKs have been demonstrated to be valid targets in the treatment of inflammatory and myeloproliferative disorders, and two inhibitors, tofacitinib and ruxolitinib, recently received their marketing authorization. Despite this success, selectivity within the JAK family remains a major issue. Both approved compounds share a common 7H‐pyrrolo[2,3‐d]pyrimidine hinge binding motif, and little is known about modifications tolerated at this heterocyclic core. In the current study, a library of tofacitinib bioisosteres was prepared and tested against JAK3. The compounds possessed the tofacitinib piperidinyl side chain, whereas the hinge binding motif was replaced by a variety of heterocycles mimicking its pharmacophore. In view of the promising expectations obtained from molecular modeling, most of the compounds proved to be poorly active. However, strategies for restoring activity within this series of novel chemotypes were discovered and crucial structure–activity relationships were deduced. The compounds presented may serve as starting point for developing novel JAK inhibitors and as a valuable training set for in silico models.     

Design,Synthesis and Structure–Activity Relationships Studies on the D Ring of the Natural Product Triptolide

Triptolide is a diterpene triepoxide natural product isolated from Tripterygium wilfordii Hook F, a traditional Chinese medicinal herb. Triptolide has previously been shown to possess antitumor, anti‐inflammatory, immunosuppressive, and antifertility activities. Earlier reports suggested that the five‐membered unsaturated lactone ring (D ring) is essential for potent cytotoxicity, however, to the best of our knowledge, systematic structure–activity relationship studies have not yet been reported. Here, four types of D ring‐modified triptolide analogues were designed, synthesized and evaluated against human ovarian (SKOV‐3) and prostate (PC‐3) carcinoma cell lines. The results suggest that the D ring is essential to potency, however it can be modified, for example to C18 hydrogen bond acceptor and/or donor furan ring analogues, without complete loss of cytotoxic activity. Interestingly, evaluation of the key series of C19 analogues showed that this site is exquisitely sensitive to polarity. Together, these results will guide further optimization of this natural product lead compound for the development of potent and potentially clinically useful triptolide analogues.     

Structure‐Based Design of Microsomal Prostaglandin E2 Synthase‐1 (mPGES‐1) Inhibitors using a Virtual Fragment Growing Optimization Scheme

A small library of 2,3‐dihydroxybenzamide‐ and N‐(2,3‐dihydroxyphenyl)‐4‐sulfonamide‐based microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) inhibitors was identified following a step‐by‐step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES‐1. During the virtual optimization process, the 2,3‐dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure‐guided replacement of the 2,3‐dihydroxybenzamide by the N‐(2,3‐dihydroxyphenyl)sulfonamide moiety led to the identification of N‐(2,3‐dihydroxyphenyl)‐4‐biphenylsulfonamide ( 6 ), the most potent small molecule of the series (IC₅₀=0.53±0.04 μm ). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES‐1‐inhibitory activity, with potential application as anti‐inflammatory and anticancer agents.     

Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure–Activity Relationships,Bioisosteric Imidazopyridine Nitriles,and X‐ray Crystal Structure Analysis with Human Cathepsin L

The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure‐based design and resulted in inhibitors with inhibition constants in the single‐digit nanomolar range. Comprehensive structure–activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high‐affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X‐ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub‐micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell‐based assays, the introduction of a basic side chain on the inhibitors resulted in a 35‐fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off‐target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quantitative in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.     

From Structure–Activity to Structure–Selectivity Relationships: Quantitative Assessment,Selectivity Cliffs,and Key Compounds

The exploration of structure–activity relationships (SARs) in chemical lead optimization is mostly focused on activity against single targets. Because many active compounds have the potential to act against multiple targets, achieving a sufficient degree of target selectivity often becomes a major issue during optimization. Herein we report a data analysis approach to explore compound selectivity in a systematic and quantitative manner. Sets of compounds that are active against multiple targets provide a basis for exploring structure–selectivity relationships (SSRs). Compound similarity and selectivity data are analyzed with the aid of network‐like similarity graphs (NSGs), which organize molecular networks on the basis of similarity relationships and SAR index (SARI) values. For this purpose, the SARI framework has been adapted to quantify SSRs. Using sets of compounds with differential activity against four cathepsin thiol proteases, we show that SSRs can be quantitatively described and categorized. Furthermore, local SSR environments are identified, the analysis of which provides insight into compound selectivity determinants at the molecular level. These environments often contain “selectivity cliffs” formed by pairs or groups of similar compounds with significantly different selectivity. Moreover, key compounds are identified that determine characteristic features of single‐target SARs and dual‐target SSRs. The comparison of compounds involved in the formation of selectivity cliffs often reveals chemical modifications that render compounds target selective.     

Anti‐Dengue‐Virus Activity and Structure–Activity Relationship Studies of Lycorine Derivatives

Dengue is a systemic viral infection that is transmitted to humans by Aedes mosquitoes. No vaccines or specific therapeutics are currently available for dengue. Lycorine, which is a natural plant alkaloid, has been shown to possess antiviral activities against flaviviruses. In this study, a series of novel lycorine derivatives were synthesized and assayed for their inhibition of dengue virus (DENV) in cell cultures. Among the lycorine analogues, 1‐acetyllycorine exhibited the most potent anti‐DENV activity (EC₅₀=0.4 μM ) with a reduced cytotoxicity (CC₅₀>300 μM ), which resulted in a selectivity index (CC₅₀/EC₅₀) of more than 750. The ketones 1‐acetyl‐2‐oxolycorine (EC₅₀=1.8 μM ) and 2‐oxolycorine (EC₅₀=0.5 μM ) also exhibited excellent antiviral activities with low cytotoxicity. Structure–activity relationships for the lycorine derivatives against DENV are discussed. A three‐dimensional quantitative structure–activity relationship model was established by using a comparative molecular‐field analysis protocol in order to rationalize the experimental results. Further modifications of the hydroxy group at the C1 position with retention of a ketone at the C2 position could potentially lead to inhibitors with improved overall properties.