Quinolone bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists.

Cyclopenta[g]quinolones of type 4 were designed with the aim to bioisosterically replace the phenol of potent GluN2B ligands such as ifenprodil and Ro 25-6981 by the quinolone system and to restrict the conformational flexibility of the aminopropanol substructure in a cyclopentane system. The designed ligands were synthesized in an eight-step sequence starting with terephthalaldehyde (5). Key steps pf the synthesis were the intramolecular Friedel-Crafts acylation of propionic acids 10 to yield the cyclopenta[g]quinolinediones 11 and the Mannich reaction of diketone 11a followed by conjugate addition at the α,ß-unsaturated ketone 12a. Although the quinolones 13a, 15a, and 16a contain an H-bond donor group (secondary lactam) as ifenprodil and Ro 25-6981, they show only moderate GluN2B affinity (Ki > 410 nM). However, the introduction of lipophilic substituents at the quinolone N-atom resulted in more than 10-fold increased GluN2B affinity of the benzyl and benzyloxymethyl derivatives cis-13c (Ko = 36 nM) and 13e (Ko = 27 nM). All compounds are selective over the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA) receptor. The benzyl derivative 13c showed six- and threefold selectivity over σ1 and σ2 receptors, respectively.

Discovery of Novel Aminocyclobutanecarboxylic Acid Derivatives as Succinate Dehydrogenase Inhibitors.

The conformational restriction switch concept has been adopted as a major tool for structural optimization of pharmaceuticals in order to expand the chemical structure scope and improve therapeutic activity against specific proteins. Several of the 1-aminocyclobutanecarboxylic acid derivatives produced in this way exhibited satisfactory antifungal activity in vitro compared with positive control boscalid. In vitro antifungal tests revealed that compound A21 had comparable, even higher antifungal activity against Rhizoctonia solani (R.s., EC50 = 0.03 mg/L) and Botrytis cinerea (B.c., EC50 = 0.04 mg/L) than fluxapyroxad (R.s., EC50 = 0.02 mg/L; B.c., EC50 = 0.20 mg/L) and boscalid (R.s., EC50 = 0.29 mg/L; B.c., EC50 = 0.42 mg/L). Furthermore, compound A20 was successfully screened and exhibited good inhibitory activity against porcine SDH, its IC50 value was 3.73 µM, which has considerable potency compared with fluxapyroxad (IC50 = 3.76 µM). The mode of action was determined using SEM and membrane potential research. The effects of the substituent steric hindrance, electrostatic property, hydrophobicity, and hydrogen-bond fields on structure-activity relationships were elaborated by the reliable models of comparative molecular field analysis and comparative molecular similarity index analysis. Furthermore, density functional theory simulations, molecule electrostatic potential, and molecular docking were also used to study the probable binding mode of target compounds with flexible fragments. The results showed that the scaffold of 1-aminocyclobutanecarboxylic acid derivatives could be used as lead for discovering new succinate dehydrogenase inhibitors.

Rapid discovery and crystallography study of highly potent and selective butylcholinesterase inhibitors based on oxime-containing libraries and conformational restriction strategies.

Butyrylcholinesterase is regarded as a promising drug target in advanced Alzheimer's disease. In order to identify highly selective and potent BuChE inhibitors, a 53-membered compound library was constructed via the oxime-based tethering approach based on microscale synthesis. Although A2Q17 and A3Q12 exhibited higher BuChE selectivity versus acetylcholinesterase, the inhibitory activities were unsatisfactory and A3Q12 did not inhibit Aß1-42 peptide self-induced aggregation. With A2Q17 and A3Q12 as leads, a novel series of tacrine derivatives with nitrogen-containing heterocycles were designed based on conformation restriction strategy. The results demonstrated that 39 (IC50 = 3.49 nM) and 43 (IC50 = 7.44 nM) yielded much improved hBuChE inhibitory activity compared to the lead A3Q12 (IC50 = 63 nM). Besides, the selectivity indexes (SI = AChE IC50 / BChE IC50) of 39 (SI = 33) and 43 (SI = 20) were also higher than A3Q12 (SI = 14). The results of the kinetic study showed that 39 and 43 exhibited a mixed-type inhibition against eqBuChE with respective Ki values of 1.715 nM and 0.781 nM. And 39 and 43 could inhibit Aß1-42 peptide self-induced aggregation into fibril. X-ray crystallography structures of 39 or 43 complexes with BuChE revealed the molecular basis for their high potency. Thus, 39 and 43 are deserve for further study to develop potential drug candidates for the treatment of Alzheimer's disease.

Cyclobutanes in Small-Molecule Drug Candidates.

Cyclobutanes are increasingly used in medicinal chemistry in the search for relevant biological properties. Important characteristics of the cyclobutane ring include its unique puckered structure, longer C-C bond lengths, increased C-C π-character and relative chemical inertness for a highly strained carbocycle. This review will focus on contributions of cyclobutane rings in drug candidates to arrive at favorable properties. Cyclobutanes have been employed for improving multiple factors such as preventing cis/trans-isomerization by replacing alkenes, replacing larger cyclic systems, increasing metabolic stability, directing key pharmacophore groups, inducing conformational restriction, reducing planarity, as aryl isostere and filling hydrophobic pockets.

Design, synthesis, and evaluation of novel 3-thiophene derivatives as potent fungistatic and fungicidal reagents based on a conformational restriction strategy.

Invasive fungal infections (IFIs) are emerging as serious infectious diseases worldwide, and due to the lack of effective antifungal agents and serious drug resistance, the limited efficacy of existing drugs has led to high morbidity and mortality in patients. We optimized the lead compound 7 by conformational restriction strategy to obtain a series of 3-thiophene phenyl compounds, of which compound 21b showed excellent inhibitory activity against pathogenic and drug-resistant fungi. In addition, the preferred compound 21b could prevent the formation of fungal biofilms and displayed satisfactory fungicidal activity. Furthermore, compound 21b was almost non-toxic to mammalian THLE2 and RAW264.7 cells and did not pose a risk of drug-drug interactions. These results strongly suggested that compound 21b is worthy of further study as a potential azole inhibitor.

Synthesis of tropane-based σ1 receptor antagonists with antiallodynic activity.

Following the concept of conformational restriction to obtain high affinity σ1 ligands, the piperidine ring of eliprodil was replaced by the bicyclic tropane system and an exocyclic double bond was introduced. The envisaged benzylidenetropanes 9 were prepared by conversion of tropanone 10 into the racemic mixture of (Z)-14 and (E)-14. Reaction of racemate (Z)-14/(E)-14 with enantiomerically pure (R)- or (S)-configured 2-phenyloxirane provided mixtures of diastereomeric ß-aminoalcohols (R,Z)-9 and (R,E)-9 as well as (S,Z)-9 and (S,E)-9, which were separated by chiral HPLC, respectively. X-ray crystal structure analysis of (S,Z)-9 allowed the unequivocal assignment of the configuration of all four stereoisomers. In receptor binding studies with radioligands, (R,E)-9 and (S,Z)-9 showed subnanomolar σ1 affinity with eudismic ratios of 8.3 and 40. In both compounds the 4-fluorophenyl moiety is oriented towards (S)-configured C-5 of the tropane system. Both compounds display high selectivity for the σ1 receptor over the σ2 subtype but moderate selectivity over GluN2B NMDA receptors. In vivo, (R,E)-9 (Ki(σ1) = 0.80 nM) showed high antiallodynic activity in the capsaicin assay. The effect of (R,E)-9 could be reversed by pre-administration of the σ1 agonist PRE-084 confirming the σ1 antagonistic activity of (R,E)-9.

Design and synthesis of novel orexin 2 receptor agonists based on naphthalene skeleton.

A novel series of naphthalene derivatives were designed and synthesized based on the strategy focusing on the restriction of the flexible bond rotation of OX2R selective agonist YNT-185 (1) and their agonist activities against orexin receptors were evaluated. The 1,7-naphthalene derivatives showed superior agonist activity than 2,7-naphthalene derivatives, suggesting that the bent form of 1 would be favorable for the agonist activity. The conformational analysis of 1,7-naphthalene derivatives indicated that the twisting of the amide unit out from the naphthalene plane is important for the enhancement of activity. The introduction of a methyl group on the 2-position of 1,7-naphthalene ring effectively increased the activity, which led to the discovery of the potent OX2R agonist 28c (EC50 = 9.21 nM for OX2R, 148 nM for OX1R). The structure-activity relationship results were well supported by a comparison of the docking simulation results of the most potent derivative 28c with an active state of agonist-bound OX2R cryo-EM SPA structure. These results suggested important information for understanding the active conformation and orientation of pharmacophores in the orexin receptor agonists, which is expected as a chemotherapeutic agent for the treatment of narcolepsy.

Synthesis of 8-aminomorphans with high KOR affinity.

2-Azabicyclo[3.3.1]nonanes (morphans) with a (3,4-dichlorophenyl)acetyl group at 2-position and a pyrrolidino moiety at 8-position were designed as conformationally restricted analogs of piperidine-based KOR agonists. The synthesis started with 4-oxopiperidine-2-carboxylic acid comprising 13 reaction steps. At first the ketone 10 was transformed into diester 7 bearing a propionate side chain. Dieckmann condensation of diester 7 to afford bicyclic enolester 14 and subsequent Krapcho deethoxycarbonylation represent the key steps of the synthesis. The enantiomeric pyrrolidines (1S,5R,8R)-5a and (1R,5S,8S)-5a were separated by chiral HPLC. The eutomer (1S,5R,8R)-5a showed high KOR affinity (Ki = 18 nM) and selectivity over MOR, DOR and σ2 receptors. It was concluded that the dihedral angle of the KOR pharmacophore N(pyrrolididine)-C-C-N(acyl) of (1S,5R,8R)-5a (68°) is close to the bioactive conformation of the flexible KOR agonist 3.

Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications*.

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

A general strategy to optimize the performance of aza-BODIPY-based probes for enhanced photoacoustic properties.

In this chapter, we describe a generalizable strategy to obtain a high PA output platform that is optimized for ratiometric imaging. Our approach entails conformationally restricting pendant aryl rings on the aza-BODIPY core to enhance orbital overlap which consequently increases the extinction coefficient. This strategy can potentially be applied to other dye platforms to enhance their signal intensity. We provide detailed protocols for the synthesis, in vitro characterization, and in vivo application.

[2.2]Paracyclophane-Based TCN-201 Analogs as GluN2A-Selective NMDA Receptor Antagonists.

Recent studies have shown the involvement of GluN2A subunit-containing NMDA receptors in various neurological and pathological disorders. In the X-ray crystal structure, TCN-201 (1) and analogous pyrazine derivatives 2 and 3 adopt a U-shape (hairpin) conformation within the binding site formed by the ligand binding domains of the GluN1 and GluN2A subunits. In order to mimic the resulting π/π-interactions of two aromatic rings in the binding site, a [2.2]paracyclophane system was designed to lock these aromatic rings in a parallel orientation. Acylation of [2.2]paracyclophane (5) with oxalyl chloride and chloroacetyl chloride and subsequent transformations led to the oxalamide 7, triazole 10 and benzamides 12. The GluN2A inhibitory activities of the paracyclophane derivatives were tested with two-electrode voltage clamp electrophysiology using Xenopus laevis oocytes expressing selectively functional NMDA receptors with GluN2A subunit. The o-iodobenzamide 12 b with the highest similarity to TCN-201 showed the highest GuN2A inhibitory activity of this series of compounds. At a concentration of 10 µM, 12 b reached 36 % of the inhibitory activity of TCN-201 (1). This result indicates that the [2.2]paracyclophane system is well accepted by the TCN-201 binding site.

Computational study on subfamilies of piperidine derivatives: QSAR modelling, model external verification, the inter-subset similarity determination, and structure-based drug designing.

A new subset of furan-pyrazole piperidine derivatives was used for QSAR model development. These compounds exhibit good Akt1 inhibitory activity; moreover, antiproliferative activities in vitro against OVCAR-8 (Human ovarian carcinoma cells) and HCT116 (human colon cancer cells), were confirmed for them. Based on the relevant three-dimensional (3D) and 2D autocorrelation descriptors, selected by genetic algorithm (GA), multiple linear regression (MLR) was established on half maximal-inhibitory concentration (IC50), in Akt1 and cancer cell lines independently. Robustness, stability, and predictive ability of the models were evaluated using external and internal validation (r2: 0.742-0.832, Q2LOO: 0.684-0.796, RMSE: 0.247-0.299, F: 32.283-57.578, and r2y-random: 0.049-0.080). Furthermore, in the new strategy, each of the evaluated models was generalized to two other subfamilies of piperidines to simultaneously compare the activities and structural similarity of these three subsets. Probably, structural similarity can be more considered as a criterion of similarity in the mechanism of action. Also, external verification of suggested predictive models was performed by another subset. Finally, by focusing on M64 as the most potent in vivo antitumor compound, 15 new derivatives were designed and six potent candidates were proposed for further investigation.

Dynamics-Function Analysis in Catalytic RNA Using NMR Spin Relaxation and Conformationally Restricted Nucleotides.

A full understanding of biomolecular function requires an analysis of both the dynamic properties of the system of interest and the identification of those dynamics that are required for function. We describe NMR methods based on metabolically directed specific isotope labeling for the identification of molecular disorder and/or conformational transitions on the RNA backbone ribose groups. These analyses are complemented by the use of synthetic covalently modified nucleotides constrained to a single sugar pucker, which allow functional assessment of dynamics by selectively removing a minor conformer identified by NMR from the structural ensemble.

RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function.

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived Kd values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.

Conformational Restriction of Histamine with a Rigid Bicyclo[3.1.0]hexane Scaffold Provided Selective H3 Receptor Ligands.

We designed and synthesized conformationally rigid histamine analogues with a bicyclo[3.1.0]hexane scaffold. All the compounds were selectively bound to the H3 receptor subtype over the H4 receptor subtype. Notably, compound 7 showed potent binding affinity and over 100-fold selectivity for the H3 receptors (Ki = 5.6 nM for H3 and 602 nM for H4). These results suggest that the conformationally rigid bicyclo[3.1.0]hexane structure can be a useful scaffold for developing potent ligands selective for the target biomolecules.

Synthesis, biological evaluation, and modeling studies of 1,3-disubstituted cyclobutane-containing analogs of combretastatin A4.

With the aim of circumventing the adverse cis/trans-isomerization of combretastatin A4 (CA4), a naturally occurring tumor-vascular disrupting agent, we designed novel CA4 analogs bearing 1,3-cyclobutane moiety instead of the cis-stilbene unit of the parent compound. The corresponding cis and trans cyclobutane-containing derivatives were prepared as pure diastereomers. The structure of the target compounds was confirmed by X-ray diffraction study. The title compounds were evaluated for their cytotoxic properties in human cancer cell lines HepG2 (hepatocarcinoma) and SK-N-DZ (neuroblastoma), and the overall activity was found in micromolar range. Molecular docking studies and molecular dynamics simulation within the colchicine binding site of tubulin were in good agreement with the obtained cytotoxicity data.

Conformational analysis of a cyclic AKH neuropeptide analog that elicits selective activity on locust versus honeybee receptor.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II ß-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI ß-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II ß turn exhibited by cycloAKH from the type VI ß turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations.

Saturated Bioisosteres of ortho-Substituted Benzenes.

Saturated bioisosteres of ortho-disubstituted benzenes (bicyclo[2.1.1]hexanes) were synthesized, characterized and validated. These cores were incorporated into the bioactive compounds Valsartan, Boskalid and Fluxapyroxad instead of the benzene ring. The saturated analogues showed a similar level of antifungal activity compared to that of Boskalid and Fluxapyroxad.

Structure-based design, synthesis and bioactivity evaluation of macrocyclic inhibitors of mutant isocitrate dehydrogenase 2 (IDH2) displaying activity in acute myeloid leukemia cells.

The enzymes involved in the metabolic pathways in cancer cells have been demonstrated as important therapeutic targets such as the isocitrate dehydrogenase 2 (IDH2). A series of macrocyclic derivatives was designed based on the marketed IDH2 inhibitor AG-221 by using the conformational restriction strategy. The resulted compounds showed moderate to good inhibitory potential against different IDH2-mutant enzymes. Amongst, compound C6 exhibited better IDH2R140Q inhibitory potency than AG-221, and showed excellent activity of 2-hydroxyglutarate (2-HG) suppression in vitro and its mesylate displayed good pharmacokinetic profiles. Moreover, C6 performed strong binding mode to IDH2R140Q after computational docking and dynamic simulation, which may serve as a good starting point for further development.

Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold.

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3'-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).