Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design, Synthesis, X-ray Structure and Therapeutic Potential.

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(-)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2',6'-dimethoxy-4'-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (Ki = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [35S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 2.59 nM, E(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.

New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

Fused Split Inteins: Tools for Introducing Multiple Protein Modifications.

The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.

Direct Vicinal Difunctionalization of Thiophenes Enabled by the Palladium/Norbornene Cooperative Catalysis.

Herein we report a direct vicinal difunctionalization of thiophenes via the palladium/norbornene (Pd/NBE) cooperative catalysis. A series of mono- and disubstituted thiophenes can be difunctionalized site-selectively and regioselectively at the C4 and C5 positions in good yields, enabled by an arsine ligand and a unique amide-based NBE. The synthetic utility has been shown in derivatizations of complex bioactive compounds and an open-flask gram-scale preparation. Preliminary results have been obtained in the difunctionalization of furans and a direct C4-selective arylation of 2-substituted thiophenes.

Near-infrared light-responsive alginate hydrogels based on diselenide-containing cross-linkage for on demand degradation and drug release.

A biodegradable, near-infrared (NIR) - responsive hydrogel is one of the most promising strategies as a remotely triggered drug carrier. In this study, novel NIR-responsive hydrogels based on alginate structures were prepared for controllable drug release. The hydrogels were formed rapidly by reacting norbornene-functionalized alginates and tetrazine cross-linkers containing diselenide bonds via inverse electron demand Diels-Alder click chemistry. In order to manipulate their properties, we prepared hydrogels with various cross-linking densities. NIR sensitive indocyanine green (ICG) and a drug, doxorubicin (DOX) were incorporated in the hydrogel matrix during gelation. The hydrogels showed a suppressed release profile under physiological conditions, while NIR light triggered a rapid release of DOX. Under NIR-light irradiation, ICG generated reactive oxygen species which could decompose diselenide bonds in the hydrogel matrix, inducing the gel-sol transition and release of entrapped DOX. The degradation of hydrogels could be also controlled by the ratio of the precursors.

Norbornadienes: Robust and Scalable Building Blocks for Cascade "Click" Coupling of High Molecular Weight Polymers.

Herein, we report the development of a scalable and synthetically robust building block based on norbornadiene (NBD) that can be broadly incorporated into a variety of macromolecular architectures using traditional living polymerization techniques. By taking advantage of a selective and rapid deprotection with tetrazine, highly reactive "masked" cyclopentadiene (Cp) functionalities can be introduced into synthetic polymers as chain-end groups in a quantitative and efficient manner. The orthogonality of this platform further enables a cascade "click" process where the "unmasked" Cp can rapidly react with dienophiles, such as maleimides, through a conventional Diels-Alder reaction. Coupling proceeds with quantitative conversions allowing high molecular weight star and dendritic block copolymers to be prepared in a single step under ambient conditions.

Addition Polyalkylnorbornenes: A Promising New Class of Si-Free Membrane Materials for Hydrocarbons Separation.

Nanoporous glassy polymers are perspective materials for the fabrication of gas separation membranes, especially for the application of gaseous hydrocarbon separation. However, the drawback of such materials is the pronounced physical aging resulting in the dramatic drop of gas transport properties due to relaxation of high-free-volume fraction in time. Herein, a novel and readily available group of such glassy polymers is reported based on 5-alkylnorbornenes. These polymers are easily synthesized from dicyclopentadiene and α-olefins by Diels-Alder reaction and vinyl (addition) polymerization of the formed cycloadducts in the presence of ([(η3 -C3 H5 )PdCl]2 /PCy3 /Na+ [B(3,5-(CF3 )2 C6 H3 )4 ]- catalyst. The obtained polymers display low-fraction free volume, stable gas permeability over time, and possess a unique feature for the glassy polymers-solubility controlled permeation of hydrocarbons and enhanced C4 H10 /CH4 selectivity.

Barbiturate derived amphiphilic homopolymers: synthesis, characterization, self-assembly and anticancer drug delivery.

Aim: Our aim was to synthesis and characterization of amphiphilic norbornene-derived thiobarbiturate homopolymers (NDTH 1-4) for drug delivery. Methods: Ring-opening metathesis polymerization technique was used to prepare a series of homopolymers. The hydrophobicity is introduced by increasing the number of carbon chains ([-CH2-]n; n = 1, 2, 3 & 4) in between norbornene backbone and thiobarbiturate species. Results: These vesicular aggregates have been used as anticancer Doxorubicin drug delivery vehicles at the acidic (5.5) and physiological (7.4) pHs. Confocal laser-scanning microscopy has demonstrated that the drug-loaded vesicles are easily internalized into living cells. Conclusion: Amphiphilic norbornene-derived thiobarbiturate homopolymer assemblies showed efficient nanocarrier for effective anticancer drug delivery.

New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3* Nicotinic Receptors.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4ß2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4ß2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4ß2, α7, and α3ß2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4ß2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3ß2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 µM). The primary amines described here represent new chemotypes for the α7 and α3* receptor subtypes.

Strategies toward Organic Carbon Monoxide Prodrugs.

Carbon monoxide is widely acknowledged as an important gasotransmitter in the mammalian system with importance on par with that of nitric oxide. It has also been firmly established as a potential therapeutic agent with a wide range of indications including organ transplantation, cancer, bacterial infection, and inflammation-related conditions such as colitis and sepsis. One major issue in developing CO based therapeutics is its delivery in a pharmaceutically acceptable form. Currently, there are generally five forms of deliveries: inhaled CO, photosensitive CO-releasing molecules, encapsulated CO, CO dissolved in drinks, and molecules that would release CO under physiological conditions without the need for light. For over a decade, the last category only included metal-based CO releasing molecules. What had been missing were organic CO prodrugs, which release CO under physiological conditions with tunable rates and in response to various exogenous and endogenous triggers such as water, chemical reagents, esterase, ROS, and changes in pH. This Account describes our work in this area as well as the demonstration for these organic prodrugs to recapitulate CO's pharmacological effects both in vitro and in vivo. Generally, two categories of CO prodrugs have been developed in our lab. Both can be considered as precursors of norbornadien-7-ones, which readily undergo cheletropic reaction under very mild conditions to extrude CO. The first category of CO prodrugs capitalizes on the inter- and intramolecular inverse electron demand Diels-Alder (DAinv) reaction to trigger CO release under physiological conditions. As for the bimolecular CO prodrugs, we proposed a new concept of "enrichment triggered CO release" by conjugating both components with a mitochondria-targeting moiety to achieve targeted CO delivery with improved biological outcomes in vitro and in vivo. As for the unimolecular CO prodrugs, the release half-lives can be readily tuned from minutes to days by varying the substituents on the dienone ring, the tethering linker, and the alkyne. Some significant structure-release rates relationships (SRRs) have been unveiled. An esterase-activated CO prodrug and a cascade prodrug system for co-delivery of CO and another payload have also been devised using such an intramolecular click and release strategy. The second category of CO prodrugs leverage on an elimination reaction to generate norbornadien-7-ones for CO release from norborn-2-en-7-ones. In the case of pH-sensitive ones, the CO release is triggered by ß-elimination, and the release rate can be quantitatively predicted using the Hammett constant of the substituents on the leaving group. The ROS-activated ones take advantage of ROS-induced selenoxide elimination to achieve targeted CO delivery to disease sites with elevated ROS level. We strongly believe that these CO prodrugs could serve as powerful tools for CO-associated biological studies and are promising candidates for ultimate clinical applications.

Coordination-Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

Designing well-defined photopolymerized synthetic matrices for three-dimensional culture and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Specifically, we selected a range of motifs inspired by iPSC binding to Matrigel, including laminin-derived IKVAV and YIGSR, α5ß1-binding PHSRNG10RGDS, αvß5-binding KKQRFRHRNRKG, and RGDS that is known to bind a variety of integrins for generally promoting cell adhesion. YIGSR and PHSRNG10RGDS resulted in the highest iPSC viability, where binding of ß1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells (NPCs) (decreased oct4 expression and increased pax6 expression) in response to soluble factors. The resulting NPCs formed clusters of different sizes in response to each peptide, suggesting that matrix biochemical cues affect iPSC proliferation and clustering in 3D culture. In summary, we have established photopolymerizable synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models.

New ROMP Synthesis of Ferrocenyl Dendronized Polymers.

First- and second-generation Percec-type dendronized ferrocenyl norbornene macromonomers containing, respectively, three and nine ferrocenyl termini are synthesized and polymerized by ring-opening metathesis polymerization using Grubbs' third-generation olefin metathesis catalyst with several monomer/catalyst feed ratios between 10 and 50. The rate of polymerization is highly dependent on the generation of the dendronized macromonomers, but all these ring-opening metathesis polymerization reactions are controlled, and near-quantitative monomer conversions are achieved. The numbers of ferrocenyl groups obtained are in agreement with the theoretical ones according to the cyclic voltammetry studies as determined using the Bard-Anson method.

HYBRIDS OF OLEANOLIC ACID WITH NORBORNENE-2,3-DICARBOXIMIDE-N- CARBOXYLIC ACIDS AS POTENTIAL ANTICANCER AGENTS.

The synthesis and cytotoxic activity of new oleanolic acid derivatives (8a-c and 9a-c) are presented. The obtained compounds are hybrids of oleanolic acid oximes and carboxylic acids containing short alkyl chains linked with nitrogen atom of norbomene-2,3-dicarboximide moieties via the nitrogen atom. The structures of the obtained new compounds (8a-c and 9a-c) were confinmed by spectral data. The derivatives 8a-c and 9a-c were subjected to the MTT assay in order to evaluate their cytotoxic activity towards HeLa, KB, MCF-7, HepG2 and HDF cell lines in comparison to mother compound (oleanolic acid, 1). Among the tested oximes acylated with carboxylic acids containing norbomene-imide moieties, the derivative 8b, with a propionoxyimino linker, exhibited the most advantageous level of cytotoxicity, with IC50 values from 2.75 pM (for MCF-7 cells) to 4.36 pM (for HDF cells).

Fatty Acid-Derived Pro-Toxicants of the Rat Selective Toxicant Norbormide.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB-derived pro-toxicants (3a - i, 4a - i, and 5a - i) were prepared in an effort to 'mask' this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague-Dawley, wild Norway, and wild ship rats is described. Most notably, pro-toxicant 3d was revealed to be free of all pre-cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two-choice bait-palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB-derived toxicants with enhanced palatability and efficacy profiles.

Synthesis and evaluation of cationic norbornanes as peptidomimetic antibacterial agents.

A series of structurally amphiphilic biscationic norbornanes have been synthesised as rigidified, low molecular weight peptidomimetics of cationic antimicrobial peptides. A variety of charged hydrophilic functionalities were attached to the norbornane scaffold including aminium, guanidinium, imidazolium and pyridinium moieties. Additionally, a range of hydrophobic groups of differing sizes were incorporated through an acetal linkage. The compounds were evaluated for antibacterial activity against both Gram-negative and Gram-positive bacteria. Activity was observed across the series; the most potent of which exhibited an MIC's ≤ 1 µg mL(-1) against Streptococcus pneumoniae, Enterococcus faecalis and several strains of Staphylococcus aureus, including multi-resistant methicillin resistant (mMRSA), glycopeptide-intermediate (GISA) and vancomycin-intermediate (VISA) S. aureus.

Norbornane-based nucleoside and nucleotide analogues locked in North conformation.

We report on the synthesis of novel conformationally locked nucleoside and nucleotide derivatives, which are structurally closely related to clinically used antivirals such as didanosine and abacavir. As a suitable conformationally rigid substitute of the sugar/pseudosugar ring allowing a permanent stabilization of the nucleoside in North conformation we employed bicyclo[2.2.1]heptane (norbornane) substituted in the bridgehead position with a hydroxymethyl group and in the C-3 position with a nucleobase. Prepared nucleoside derivatives were also converted into appropriate phosphoramidate prodrugs (ProTides) in order to increase delivery of the compounds in the cells. All target compounds were evaluated in a broad antiviral and cytostatic assay panel.

Synthesis and antimicrobial activity of new norbornyl system based oxadiazole thioglycosides and acyclic nucleoside analogs.

New sugar hydrazones linked to norbornyl ring system, their oxadiazole acyclic nucleoside analogs and the corresponding thioglycosides were synthesized. The synthesized compounds were tested for their antimicrobial activity and displayed different degrees of activities or inhibitory actions. Their oxadiazole acyclic nucleoside analogs and thioglycosides showed higher activities.

Time- and concentration-dependent reactivity of Cys, Hcy, and GSH on the Diels-Alder-grafted 1,3,5-tris conjugate of calix[6]arene to bring selectivity for Cys: spectroscopy, microscopy, and its reactivity in cells.

Herein we report the synthesis and characterization of 7-oxanorbornadiene (OND)-appended 1,3,5-tris conjugate of calix[6]arene (L2). L2 has been shown to exhibit selective reactivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH) under stoichiometric conditions. The selectivity of L2 is attributed to the steric crowding of three Diels-Alder centers possessing OND units present on the calix[6]arene platform, while a control molecular system possessing only one such unit without the calix[6]arene platform (L1) does not show any selectivity toward Cys. While L2 exhibited spherical particles, its reactivity with Cys resulted in flowerlike morphological features, as revealed by scanning electron microscopy. However, the reaction with GSH did not result in any such morphological features, a result that is in agreement with that observed from fluorescence studies in solution. L2 has been shown to react with Cys present in HeLa and Jurkat E6 cells by fluorescence microscopy.

From norbornane-based nucleotide analogs locked in South conformation to novel inhibitors of feline herpes virus.

A synthetic route toward a series of unique cyclic nucleoside phosphonates locked in South conformation is described. The desired conformation is stabilized by a substitution of the sugar moiety by bicyclo[2.2.1]heptane (norbornane) bearing a purine or pyrimidine nucleobase in the bridgehead position. Although the final phosphonate derivatives are devoid of any significant antiviral activity probably due to the unfavorable conformational properties, several intermediates and their analogs exhibit surprising activity against feline herpes virus. Since these compounds do not possess an appropriate hydroxymethyl function allowing phosphorylation and subsequent incorporation into the polynucleotide chain, it seems to be likely that these compounds act by a novel unknown mechanism of action and may represent a new possible alternative for nucleoside and nucleotide therapeutics of this widely spread feline infection. A number of derivatives exerted also a significant antiviral activity against Coxsackievirus B3 and B4.