Synthesis of Copper Oxide-Based Nanoformulations of Etoricoxib and Montelukast and Their Evaluation through Analgesic, Anti-Inflammatory, Anti-Pyretic, and Acute Toxicity Activities.

Copper oxide nanoparticles (CuO NPs) were synthesized through the coprecipitation method and used as nanocarriers for etoricoxib (selective COX-2 inhibitor drug) and montelukast (leukotriene product inhibitor drug) in combination therapy. The CuO NPs, free drugs, and nanoformulations were investigated through UV/Vis spectroscopy, FTIR spectroscopy, XRD, SEM, and DLS. SEM imaging showed agglomerated nanorods of CuO NPs of about 87 nm size. The CE1, CE2, and CE6 nanoformulations were investigated through DLS, and their particle sizes were 271, 258, and 254 nm, respectively. The nanoformulations were evaluated through in vitro anti-inflammatory activity, in vivo anti-inflammatory activity, in vivo analgesic activity, in vivo anti-pyretic activity, and in vivo acute toxicity activity. In vivo activities were performed on albino mice. BSA denaturation was highly inhibited by CE1, CE2, and CE6 as compared to other nanoformulations in the in vitro anti-inflammatory activity. The in vivo bioactivities showed that low doses (5 mg/kg) of nanoformulations were more potent than high doses (10 and 20 mg/kg) of free drugs in the inhibition of pain, fever, and inflammation. Lastly, CE2 was more potent than that of other nanoformulations.

An Advanced 'clickECM' That Can be Modified by the Inverse-Electron-Demand Diels-Alder Reaction.

The extracellular matrix (ECM) represents the natural environment of cells in tissue and therefore is a promising biomaterial in a variety of applications. Depending on the purpose, it is necessary to equip the ECM with specific addressable functional groups for further modification with bioactive molecules, for controllable cross-linking and/or covalent binding to surfaces. Metabolic glycoengineering (MGE) enables the specific modification of the ECM with such functional groups without affecting the native structure of the ECM. In a previous approach (S. M. Ruff, S. Keller, D. E. Wieland, V. Wittmann, G. E. M. Tovar, M. Bach, P. J. Kluger, Acta Biomater. 2017, 52, 159-170), we demonstrated the modification of an ECM with azido groups, which can be addressed by bioorthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we demonstrate the modification of an ECM with dienophiles (terminal alkenes, cyclopropene), which can be addressed by an inverse-electron-demand Diels-Alder (IEDDA) reaction. This reaction is cell friendly as there are no cytotoxic catalysts needed. We show the equipment of the ECM with a bioactive molecule (enzyme) and prove that the functional groups do not influence cellular behavior. Thus, this new material has great potential for use as a biomaterial, which can be individually modified in a wide range of applications.

Novel Electrophilic Warhead Targeting a Triple-Negative Breast Cancer Driver in Live Cells Revealed by "Inverse Drug Discovery".

The "inverse drug discovery" strategy is a potent means of exploring the cellular targets of latent electrophiles not typically used in medicinal chemistry. Cyclopropenone, a powerful electrophile, is generally used in bio-orthogonal reactions mediated by triarylphosphine or in photo-triggered cycloaddition reactions. Here, we have studied, for the first time, the proteome reactivity of cyclopropenones in live cells and discovered that the cyclopropenone warhead can specifically and efficiently modify a triple-negative breast cancer driver, glutathione S-transferase pi-1 (GSTP1), by covalently binding at the catalytic active site. Further structure optimization and signaling pathway validation have led to the discovery of potent inhibitors of GSTP1.

Stereodivergent Nitrocyclopropane Formation during Biosynthesis of Belactosins and Hormaomycins.

Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

Molecular Transformation Based on an Innovative Catalytic System.

Novel innovative catalytic systems such as hydrogen-bond donors and thiourea hybrid catalysts have been developed for the asymmetric synthesis of biologically important pharmaceuticals and natural products. Benzothiadiazines possess a stronger hydrogen-bond donor ability compared to thioureas and exhibit remarkable catalytic performance for the activation of α,ß-unsaturated amides. Hybrid thioureas (bearing an arylboronic acid and an ammonium salt) efficiently promote the hetero-Michael addition to α,ß-unsaturated carboxylic acids and the O-alkylation of keto enols with 5-chlorofuran-2(5H)-one. These hybrid catalysts enable the first total synthesis of non-racemic avenaol, a noncanonical strigolactone, as well as the asymmetric synthesis of several pharmaceuticals. In addition, this study discovers unique chemical phenomena (i.e., the dual role of benzoic acid as a boron ligand and a proton shuttle, the chirality switch of products by solvent used, and the dynamic kinetic resolution of a racemic electrophile in an SN2-type reaction).

[Synthetic Study of Polycyclic Natural Products Based on Development of New Strategy].

On the occasion of receiving the Pharmaceutical Society of Japan Award 2020, I explained our research activities on the total syntheses of polycyclic alkaloids as an invited review. The structure of lundurine B, which has an unstable cyclopropane fused indoline skeleton, was proved firstly by the total synthesis. I also describe the total syntheses of optically active lundurine B and rapidilectine B utilizing asymmetric desymmetrization of the spiro intermediate. We developed an intramolecular bond formation reaction between the 2-position of the furan ring to the iminium cation (furane-iminium cation cyclization) to synthesize manzamine alkaloids. The reaction was applied to the total synthesis of the core skeleton of nakadomarin A and ircinal A. A ring-closing metathesis reaction effectively applied to the synthesis of medium and large heterocyclic rings containing the cis double bond found in the structures of nakadomarin A and ircinal A. The total synthesis of schizocommunin, a metabolite of Schizophyllum commune isolated from a patient with human allergenic bronchopulmonary mycosis, was accomplished. We could correct the error in the proposed structure by total synthesis of the natural product. A part of the mechanism of cytotoxicity expression was clarified using newly synthesized shizocommunin.

Synthesis and Evaluation of Biological Activities of Bis(spiropyrazolone)cyclopropanes: A Potential Application against Leishmaniasis.

This work focuses on the search and development of drugs that may become new alternatives to the commercial drugs currently available for treatment of leishmaniasis. We have designed and synthesized 12 derivatives of bis(spiropyrazolone)cyclopropanes. We then characterized their potential application in therapeutic use. For this, the in vitro biological activities against three eukaryotic models-S. cerevisiae, five cancer cell lines, and the parasite L. mexicana-were evaluated. In addition, cytotoxicity against non-cancerous mammalian cells has been evaluated and other properties of interest have been characterized, such as genotoxicity, antioxidant properties and, in silico predictive adsorption, distribution, metabolism, and excretion (ADME). The results that we present here represent a first screening, indicating two derivatives of bis(spiropyrazolone)cyclopropanes as good candidates for the treatment of leishmaniasis. They have good specificity against parasites with respect to mammalian cells.

Asymmetric Radical Process for General Synthesis of Chiral Heteroaryl Cyclopropanes.

A highly efficient catalytic method has been developed for asymmetric radical cyclopropanation of alkenes with in situ-generated α-heteroaryldiazomethanes via Co(II)-based metalloradical catalysis (MRC). Through fine-tuning the cavity-like environments of newly-synthesized D2-symmetric chiral amidoporphyrins as the supporting ligand, the optimized Co(II)-based metalloradical system is broadly applicable to α-pyridyl and other α-heteroaryldiazomethanes for asymmetric cyclopropanation of wide-ranging alkenes, including several types of challenging substrates. This new catalytic methodology provides a general access to valuable chiral heteroaryl cyclopropanes in high yields with excellent both diastereoselectivities and enantioselectivities. Combined computational and experimental studies further support the underlying stepwise radical mechanism of the Co(II)-based olefin cyclopropanation involving α- and γ-metalloalkyl radicals as the key intermediates.

Repurposed and artificial heme enzymes for cyclopropanation reactions.

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed.

Layer-by-Layer Assembled Nanostructured Lipid Carriers for CD-44 Receptor-Based Targeting in HIV-Infected Macrophages for Efficient HIV-1 Inhibition.

Macrophages act as a cellular reservoir in HIV infection. Elimination of HIV from macrophages has been an unfulfilled dream due to the failure of drugs to reach them. To address this, we developed CD44 receptor-targeted, novel hyaluronic acid (HA)-coated nanostructured lipid carriers (NLCs) of efavirenz via washless layer-by-layer (LbL) assembly of HA and polyallylamine hydrochloride (PAH). NLCs were subjected to TEM analysis, size and zeta potential, in vitro release and encapsulation efficiency studies. The uptake of NLCs in THP-1 cells was studied using fluorescence microscopy and flow cytometry. The anti-HIV efficacy was evaluated using p24 antigen inhibition assay. NLCs were found to be spherical in shape with anionic zeta potential (-23.66 ± 0.87 mV) and 241.83 ± 5.38 nm particle size. NLCs exhibited prolonged release of efavirenz during in vitro drug release studies. Flow cytometry revealed 1.73-fold higher uptake of HA-coated NLCs in THP-1 cells. Cytotoxicity studies showed no significant change in cell viability in presence of NLCs as compared with the control. HA-coated NLCs distributed throughout the cell including cytoplasm, plasma membrane and nucleus, as observed during fluorescence microscopy. HA-coated NLCs demonstrated consistent and significantly higher inhibition (81.26 ± 1.70%) of p24 antigen which was 2.08-fold higher than plain NLCs. The obtained results suggested preferential uptake of HA-coated NLCs via CD44-mediated uptake. The present finding demonstrates that HA-based CD44 receptor targeting in HIV infection is an attractive strategy for maximising the drug delivery to macrophages and achieve effective viral inhibition.

A New Ligand Design Based on London Dispersion Empowers Chiral Bismuth-Rhodium Paddlewheel Catalysts.

Heterobimetallic bismuth-rhodium paddlewheel complexes with phenylglycine ligands carrying TIPS-groups at the meta-positions of the aromatic ring exhibit outstanding levels of selectivity in reactions of donor/acceptor and donor/donor carbenes; at the same time, the reaction rates are much faster and the substrate scope is considerably wider than those of previous generations of chiral [BiRh] catalysts. As shown by a combined experimental, crystallographic, and computational study, the new catalysts draw their excellent application profile largely from the stabilization of the chiral ligand sphere by London dispersion (LD) interactions of the peripheral silyl substituents.

Reaction of 3-Oxa-2-oxobicyclo[4.2.0]oct-4-ene-1-carboxylate with Dimethylsulfoxonium Methylide.

We have been interested in the reactivities of small-ring compounds and have reported reactions that proceed through cyclopropane intermediates starting from coumarin derivatives bearing an electron-withdrawing group at the 3-position or 2-oxo-2H-pyran-3-carboxylate derivatives and dimethylsulfoxonium methylide. This time, the reaction between 3-oxa-2-oxobicyclo[4.2.0]oct-4-ene-1-carboxylate and dimethylsulfoxonium methylide has been investigated. 3a,4,5,7a-Tetrahydro-7-hydroxybenzofuran-6-carboxylate and/or 2-hydroxybicyclo[4.1.0]hept-2-ene-3-carboxylate were obtained. The compounds were characterized using various spectral and X-ray crystallographic techniques. A plausible reaction mechanism has been discussed. This reaction was applied to some 3-oxa-2-oxobicyclo[4.2.0]oct-4-ene-1-carboxylate derivatives to clarify the generality.

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives.

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGloTM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Evaluation of 1,1-cyclopropylidene as a thioether isostere in the 4-thio-thienopyrimidine (TTP) series of antimalarials.

The 4-(heteroarylthio)thieno[2,3-d]pyrimidine (TTP) series of antimalarials, represented by 1 and 17, potently inhibit proliferation of the 3D7 strain of P. falciparum (EC50 70-100 nM), but suffer from oxidative metabolism. The 1,1-cyclopropylidene isosteres 6 and 16 were designed to obviate this drawback. They were prepared by a short route that features a combined Peterson methylenation / cyclopropanation transformation of, e. g., ketone 7. Isosteres 6 and 16 possess significantly attenuated antimalarial potency relative to parents 1 and 17. This outcome can be rationalized based on the increased out-of-plane steric demands of the latter two. In support of this hypothesis, the relatively flat ketone 7 retains some of the potency of 1, even though it appears to be a comparatively inferior mimic with respect to electronics and bond lengths and angles. We also demonstrate crystallographically and computationally an apparent increase in the strength of the intramolecular sulfur hole interaction of 1 upon protonation.

Danoprevir for the Treatment of Hepatitis C Virus Infection: Design, Development, and Place in Therapy.

On June 8, 2018, an NS3/4A protease inhibitor called danoprevir was approved in China to treat the infections of HCV genotype (GT) 1b - the most common HCV genotype worldwide. Based on phase 2 and 3 clinical trials, the 12-week regimen of ritonavir-boosted danoprevir (danoprevir/r) plus peginterferon alpha-2a and ribavirin offered 97.1% (200/206) of sustained virologic response at post-treatment week 12 (SVR12) in treatment-naïve non-cirrhotic patients infected with HCV genotype 1b. Adverse events such as anemia, fatigue, fever, and headache were associated with the inclusion of peginterferon alpha-2a and ribavirin in the danoprevir-based regimen. Moreover, drug resistance to danoprevir could be traced to amino acid substitutions (Q80K/R, R155K, D168A/E/H/N/T/V) near the drug-binding pocket of HCV NS3 protease. Despite its approval, the clinical use of danoprevir is currently limited to its combination with peginterferon alpha-2a and ribavirin, thereby driving its development towards interferon-free, ribavirin-free regimens with improved tolerability and adherence. In the foreseeable future, pan-genotypic direct-acting antivirals with better clinical efficacy and less adverse events will be available to treat HCV infections worldwide.

Pd(II)-Catalyzed Enantioselective γ-C(sp3)-H Functionalizations of Free Cyclopropylmethylamines.

Prized for their ability to reliably forge stereocenters with precise regiocontrol from simple and abundant starting materials, substrate-directable enantioselective reactions are widely used in modern organic synthesis. As such, enantioselective C(sp3)-H functionalization reactions directed by innate functional groups could provide new routes to introduce molecular complexity within the inert hydrocarbon moiety, but to date this approach has been met with little success. While free primary aliphatic amines are common, versatile intermediates in synthesis, they are traditionally unreactive in C(sp3)-H activation reactions. Herein we report the Pd-catalyzed enantioselective C(sp3)-H functionalization of free aliphatic amines (cyclopropylmethylamines) enabled by a chiral bidentate thioether ligand. This ligand's privileged bidentate coordination mode and thioether motif favor the generation of the requisite mono(amine)-Pd(II) intermediate, thus enabling the enantioselective C-H activation of free amines. The resulting C-Pd(II) species could engage in either Pd(II)/Pd(IV) or Pd(II)/Pd(0) catalytic cycles, enabling access to a diverse range of products through (hetero)arylation, carbonylation, and olefination reactions. Consequently, this versatile reactivity offers medicinal chemists a general strategy to rapidly prepare and functionalize biologically relevant amines.

Asymmetric trisalkylamine cyclopropenium derivatives with antimicrobial activity.

Cationic molecules are found in abundance as antimicrobial agents with a well-defined mechanism of action and significant therapeutic benefits. Quaternary ammonium-containing compounds are frequently employed due to their facile synthesis and tunable properties. Over time, however, bacterial resistance to these compounds has become a significant obstacle. We report here a series of asymmetric trisalkylamine cyclopropenium cationic derivatives as chemical isosteres of quaternary ammonium compounds, capable of strong antimicrobial activity and overcoming microbial resistance. These small molecules were prepared by one-pot reaction of tetrachlorocyclopropene (TCC) with unhindered secondary amines in the presence of Hünig's base. In this work we describe the synthesis, purification, and characterization of five trisamino-cyclopropenium derivatives and confirm their structures by spectral analysis and mass-spectrometry. Three of the compounds displayed considerable antimalarial activity (IC50 < 0.1 µM) without demonstrating significant toxic effects in vitro (TC50 > 1 µM). This class of cyclopropenium-based compounds provides an opening for the discovery of potent and non-toxic antimicrobial agents.

Ring-Opening Cyclization of Spirocyclopropanes Using Sulfoxonium Ylides.

Ring-opening cyclization of cyclohexane-1,3-dione-2-spirocyclopropanes using dimethylsulfoxonium methylide proceeded regioselectively to produce 2,3,4,6,7,8-hexahydro-5H-1-benzopyran-5-ones in good to high yields. The reactions of cycloheptane- and cyclopentane-1,3-dione-2-spirocyclopropanes could construct [7.6]- and [5.6]-fused ring systems. This reaction was also carried out using sulfoxonium ethylide, butylide, and benzylide, resulting in the formation of the corresponding 2,3-trans-disubstituted products in good to high yields, and it was shown that the dimethyl group can act as a dummy substituent. It was found that the 2- and 3-phenyhexahydrobenzopyran-5-ones can be readily converted into 5-hydroxyflavan and 5-hydroxyisoflavan, respectively.

Regio- and Enantioselective Bromocyclization of Difluoroalkenes as a Strategy to Access Tetrasubstituted Difluoromethylene-Containing Stereocenters.

Difluoromethylene-containing compounds have attracted substantial research interest over the past decades for their ability to mimic biological functions of traditional functional groups while providing a wide variety of pharmacological benefits bestowed by the C-F bond. We report a novel strategy to access RCF2Br-containing heterocycles by regio- and enantioselective bromocyclization of difluoroalkenes enabled by chiral anion phase-transfer catalysis. The utility of this methodology was highlighted through a synthesis of an analogue of efavirenz, a drug used for treating HIV. Additionally, the synthetic versatility of the CF2Br intermediates was showcased through functionalization to a variety of enantioenriched α,α-difluoromethylene-containing products.