The Recurring Roles of Chlorine in Synthetic and Biological Studies of the Lissoclimides.

Halogenated natural products number in the thousands, but only in rare cases are the evolutionary advantages conferred by the halogens understood. We set out to investigate the lissoclimide family of cytotoxins, which includes several chlorinated members, because of our long-standing interest in the synthesis of chlorinated secondary metabolites.Our initial success in this endeavor was a semisynthesis of chlorolissoclimide (CL) from the commercially available sesquiterpenoid sclareolide. Featuring a highly selective and efficient-and plausibly biomimetic-C-H chlorination, we were able to access enough CL for collaborative studies, including X-ray cocrystallography with the eukaryotic ribosome. Through this experiment, we learned that CL's chlorine atom engages in a novel halogen-π dispersion interaction with a neighboring nucleobase in the ribosome E-site.Owing to the limitations of our semisynthesis approach, we established an analogue-oriented approach to access numerous lissoclimide compounds to both improve our understanding of structure-activity relationships and to learn more about the halogen-π interaction. In the course of these studies, we made over a dozen lissoclimide-like compounds, the most interesting of which contained chlorine-bearing carbons with unnatural configurations. Rationalizing the retained potency of these compounds that appeared to be a poor fit for the lissoclimide binding pocket, we came to realize that the chlorine atoms would engage in these same halogen-π interactions even at the expense of a chair to twist-boat conformational change, which also permitted the compounds to fit in the binding site.Finally, because neither of the first two approaches could easily access the most potent natural lissoclimides, we designed a synthesis that took advantage of rarely used terminal epoxides to initiate polyene cyclizations. In this case, the chlorine atom was incorporated early and helped control the stereochemical outcome of the key step.Over the course of this project, three different synthesis approaches were designed and executed, and our ability to access numerous lissoclimides fueled a range of collaborative biological studies. Further, chlorine played impactful roles throughout various aspects of both synthesis and biology. We remain inspired to learn more about the mechanism of action of these compounds and to deeply investigate the potentially valuable halogen-π dispersion interaction in the context of small molecule/nucleic acid binding. In that context, our work offers an instance wherein we might have gained a rudimentary understanding of the evolutionary importance of the halogen in a halogenated natural product.

Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL.

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family.

Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1 conjugates.

Twelve double fatty chains and Aib8-Arg34-GLP-1 (7-37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib8-Arg34-GLP-1 (7-37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure-activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

Understanding the role of intermolecular interactions between lissoclimides and the eukaryotic ribosome.

Natural products that target the eukaryotic ribosome are promising therapeutics to treat a variety of cancers. It is therefore essential to determine their molecular mechanism of action to fully understand their mode of interaction with the target and to inform the development of new synthetic compounds with improved potency and reduced cytotoxicity. Toward this goal, we have previously established a short synthesis pathway that grants access to multiple congeners of the lissoclimide family. Here we present the X-ray co-crystal structure at 3.1 Å resolution of C45, a potent congener with two A-ring chlorine-bearing stereogenic centers with 'unnatural' configurations, with the yeast 80S ribosome, intermolecular interaction energies of the C45/ribosome complex, and single-molecule FRET data quantifying the impact of C45 on both human and yeast ribosomes. Together, these data provide new insights into the role of unusual non-covalent halogen bonding interactions involved in the binding of this synthetic compound to the 80S ribosome.

New Succinimides with Potent Anticancer Activity: Synthesis, Activation of Stress Signaling Pathways and Characterization of Apoptosis in Leukemia and Cervical Cancer Cells.

Based on previously identified dicarboximides with significant anticancer and immunomodulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels-Alder reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia (K562, MOLT4), cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray gene profiling demonstrated upregulation of proapoptotic genes involved in receptor-mediated and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF-kB signaling. Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for further development as anticancer drugs.

BF3-OEt2 Catalyzed C3-Alkylation of Indole: Synthesis of Indolylsuccinimidesand Their Cytotoxicity Studies.

A simple and efficient BF3-OEt2 promoted C3-alkylation of indole has been developed to obtain3-indolylsuccinimidesfrom commercially available indoles and maleimides, with excellent yields under mild reaction conditions. Furthermore, anti-proliferative activity of these conjugates was evaluated against HT-29 (Colorectal), Hepg2 (Liver) and A549 (Lung) human cancer cell lines. One of the compounds, 3w, having N,N-Dimethylatedindolylsuccinimide is a potent congener amongst the series with IC50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively, and compound 3i was most active amongst the series with IC50 value 1.5 µM against A549 cells. Molecular docking study and mechanism of reaction have briefly beendiscussed. This method is better than previous reports in view of yield and substrate scope including electron deficient indoles.

Establishment of a 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) based lectin microarray for efficiently detecting serum glycans in gastric cancers.

Development of cancers is involved in changes of a variety of glycans. Lectin microarray is one of the most powerful methodologies for investigation of glycan alterations in biological samples with its advantages of high through-put, selectivity and specificity of the technique. However, utilization of lectin microarrays available commercially keeps of great challenges. In this study, we took use of the molecular self-assembled monolayer technique to modify a gold surface with the reagent 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) in combination with 16-amino-1-hexadecanethiol hydrochloride. Cross-linking effect of DOTA-NHS-ester is brought about via activating three -OH ends to three terminals of succinylimidines, making selective binding of the terminal amino groups in proteins possible. We immobilized ten commercial lectins on the platform and measured changes of serum lectin-matched glycans in patients with gastric cancer. The results demonstrated that this biochip modification platform conferred impressive chemical surface stabilization, sensitivity and geometric images. We observed that all the serum glycans tested in the patients were significantly higher than those in the controls (P < 0.05). The biochip would provide a versatile platform for investigation of potential glycan biomarkers in making tumor diagnosis decision and analyzing escape of tumors from immunity.

Mechanism of Photoredox-Initiated C-C and C-N Bond Formation by Arylation of IPrAu(I)-CF3 and IPrAu(I)-Succinimide.

Herein, we report on the photoredox-initiated gold-mediated C(sp2)-CF3 and C(sp2)-N coupling reactions. By adopting gold as a platform for probing metallaphotoredox catalysis, we demonstrate that cationic gold(III) complexes are the key intermediates of the C-C and C-N coupling reactions. The high-valent gold(III) intermediates are accessed by virtue of photoredox catalysis through a radical chain process. In addition, the bond-forming step of the coupling reactions is the reductive elimination from cationic gold(III) intermediates, which is supported by isolation and crystallographic characterization of key Au(III) intermediates.

Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo antidiabetic and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives.

α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus (DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-dione derivatives and evaluated for their ability to inhibit α-Glucosidase. Pyrrolidine-2,5-dione derivatives (11a-o) showed moderate to poor α-glucosidase inhibition. Compound 11o with the IC50 value of 28.3 ±â€¯0.28 µM emerged as a good inhibitor of α-glucosidase. Thiazolidine-2,4-dione and dihydropyrimidine (TZD-DHPM) hybrids (22a-c) showed excellent inhibitory activities. The most active compound 22a displayed IC50 value of 0.98 ±â€¯0.008 µM. Other two compounds of this series also showed activity in low micromolar range. The in-vivo antidiabetic study of three compounds 11n, 11o and 22a were also determined using alloxan induced diabetes mice model. Compounds 11o and 22a showed significant hypoglycemic effect compared to the reference drug. In-vivo acute toxicity study showed the safety of these selected compounds. In-silico docking studies were carried out to rationalize the in-vitro results. The binding modes and bioassay results of TZD-DHPM hybrids showed that interactions with important residues appeared significant for high potency.

Construction of Multivalent Homo- and Heterofunctional ABO Blood Group Glycoconjugates Using a Trifunctional Linker Strategy.

The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

Photochromic Indolyl Fulgimides as Chromo-pharmacophores Targeting Sirtuins.

Sirtuins are involved in epigenetic regulation, the pathogenesis of cancer, and several metabolic and neurodegenerative diseases. Despite being a promising drug target, only one small molecule passed class II clinical trials to date. Deriving a better mechanistic understanding is hence crucial to find new modulators. We previously reported on a series of dithienyl maleimides as photochromic tool compounds. However, their photochromic behavior was limited. To improve the interconversion and stability of both photoisomers, we replaced the dithienyl maleimide with a fulgimide as a photochromic core to result in biologically active compounds reversibly addressable with purple and orange light. We characterize the obtained compounds regarding their spectroscopic properties, their photostability, and binding characteristics toward sirtuins resulting in a fully remote-controllable Sirtuin modulator using visible light as the external stimulant.

Phosphate-Catalyzed Succinimide Formation from Asp Residues: A Computational Study of the Mechanism.

Aspartic acid (Asp) residues in proteins and peptides are prone to the non-enzymatic reactions that give biologically uncommon l-ß-Asp, d-Asp, and d-ß-Asp residues via the cyclic succinimide intermediate (aminosuccinyl residue, Suc). These abnormal Asp residues are known to have relevance to aging and pathologies. Despite being non-enzymatic, the Suc formation is thought to require a catalyst under physiological conditions. In this study, we computationally investigated the mechanism of the Suc formation from Asp residues that were catalyzed by the dihydrogen phosphate ion, H2PO4-. We used Ac-l-Asp-NHMe (Ac = acetyl, NHMe = methylamino) as a model compound. The H2PO4- ion (as a catalyst) and two explicit water molecules (as solvent molecules stabilizing the negative charge) were included in the calculations. All of the calculations were performed by density functional theory with the B3LYP functional. We revealed a phosphate-catalyzed two-step mechanism (cyclization-dehydration) of the Suc formation, where the first step is predicted to be rate-determining. In both steps, the reaction involved a proton relay mediated by the H2PO4- ion. The calculated activation barrier for this mechanism (100.3 kJ mol-1) is in reasonable agreement with an experimental activation energy (107 kJ mol-1) for the Suc formation from an Asp-containing peptide in a phosphate buffer, supporting the catalytic mechanism of the H2PO4- ion that is revealed in this study.

Phosphate-Catalyzed Succinimide Formation from an NGR-Containing Cyclic Peptide: A Novel Mechanism for Deammoniation of the Tetrahedral Intermediate.

Spontaneous deamidation in the Asn-Gly-Arg (NGR) motif that yields an isoAsp-Gly-Arg (isoDGR) sequence has recently attracted considerable attention because of the possibility of application to dual tumor targeting. It is well known that Asn deamidation reactions in peptide chains occur via the five-membered ring succinimide intermediate. Recently, we computationally showed by the B3LYP density functional theory method, that inorganic phosphate and the Arg side chain can catalyze the NGR deamidation using a cyclic peptide, c[CH2CO⁻NGRC]⁻NH2. In this previous study, the tetrahedral intermediate of the succinimide formation was assumed to be readily protonated at the nitrogen originating from the Asn side chain by the solvent water before the release of an NH3 molecule. In the present study, we found a new mechanism for the decomposition of the tetrahedral intermediate that does not require the protonation by an external proton source. The computational method is the same as in the previous study. In the new mechanism, the release of an NH3 molecule occurs after a proton exchange between the peptide and the phosphate and conformational changes. The rate-determining step of the overall reaction course is the previously reported first step, i.e., the cyclization to form the tetrahedral intermediate.

Photochromic histone deacetylase inhibitors based on dithienylethenes and fulgimides.

Histone deacetylases (HDACs) play a crucial role in numerous biological processes and therefore are targeted in anticancer research and in the field of epigenetics. Dithienylethenes (DTEs) and fulgimides were functionalized with hydroxamic acids, which is a known moiety binding to zinc dependent HDACs, to gain photoswitchable HDAC inhibitors. The new DTE based inhibitors showed moderate photochromic properties in polar solvents and the inhibitory activity changes up to a factor of four. The photochromic performance of the prepared fulgimide inhibitors was very good, even in aqueous buffer. They achieved a maximum three-fold difference in inhibitory activity. Docking experiments using the crystal structures of the tested enzymes gave a rationale for the observed moderate differences in the activities of the inhibitors.

Efficient synthesis of [18 F]FPyME: A new approach for the preparation of maleimide-containing prosthetic groups for the conjugation with thiols.

An improved high yielding radiosynthesis of the known thiol-reactive maleimide-containing prosthetic group1-[3-(2-[18 F]fluoropyridine-3-yloxy)propyl]pyrrole-2,5-dione ([18 F]FPyME) is described. The target compound was obtained by a two-step one-pot procedure starting from a maleimide-containing nitro-precursor that was protected as a Diels-Alder adduct with 2,5-dimethylfurane. Nucleophilic radiofluorination followed by heat induced deprotection through a Retro Diels Alder reaction yielded, after chromatographic isolation, [18 F]FPyME with a radiochemical yield of 20% in about 60 min overall synthesis time. A variety of other [18 F]fluoropyridine based maleimide-containing prosthetic groups should be accessible via the described synthetic strategy.

Synthesis and evaluation of anticonvulsant properties of new N-Mannich bases derived from 3-(1-phenylethyl)- and 3-benzyl-pyrrolidine-2,5-dione.

Two series of new derivatives of pyrrolidine-2,5-dione were synthesized and evaluated for their anticonvulsant properties. Initial screening for their anticonvulsant properties was performed in mice after intraperitoneal administration, using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6-Hz seizure tests. Quantitative pharmacological research revealed that the highest level of protection was demonstrated by compound N-[{4-methylpiperazin-1-yl}-methyl]-3-(1-phenylethyl)-pyrrolidine-2,5-dione monohydrochloride (22) which was effective both in the scPTZ test (ED50=39 mg/kg) and in the 6-Hz test (ED50=36 mg/kg). This molecule showed higher potency than reference antiepileptic drugs such as ethosuximide, lacosamide and valproic acid. With the aim of explaining the possible mechanism of action of the selected molecule, its influence on sodium and calcium channels as well as NMDA and GABAA receptors binding properties were evaluated in vitro.

N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([(18)F]SFBTMGMB): a residualizing label for (18)F-labeling of internalizing biomolecules.

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the (18)F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([(18)F]SFBTMGMB-Boc2) was synthesized by a click reaction of an azide precursor and [(18)F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with (18)F using [(18)F]SFBTMGMB ([(18)F]RL-I), obtained by the deprotection of [(18)F]SFBTMGMB-Boc2, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62-80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with (18)F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

NHC-Catalyzed Asymmetric Synthesis of Functionalized Succinimides from Enals and α-Ketoamides.

The efficient asymmetric synthesis of highly substituted succinimides from α,ß-unsaturated aldehydes and α-ketoamides via NHC-catalyzed [3+2] cycloaddition has been developed. The new scalable protocol significantly expands the utility of NHC catalysis for the synthesis of heterocycles and provides easy access to assemble a wide range of succinimides from simple starting materials.