3-NITROPROPIONIC ACID IS A SUICIDE INHIBITOR OF MITOCHONDRIAL RESPIRATION THAT, UPON OXIDATION BY COMPLEX II, FORMS A COVALENT ADDUCT WITH A CATALYTIC-BASE ARGININE IN THE ACTIVE SITE OF THE ENZYME*. Li-shar Huang 1 , Gang Sun 2± , David Cobessi 1§ , Andy Wang 1 , John T. Shen 1 , Eric Y. Tung 1 , Vernon E. Anderson 2 , and Edward A. Berry 1 From Lawrence Berkeley National Laboratory, Berkeley, CA; and , 2 Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH Running title: Structure of 3-nitropropionate - inactivated Complex II Address correspondence regarding the crystallographic work to EAB (EABerry@LBL.gov; telephone 1-510-486-4335), and correspondence regarding characterization of the tryptic fragments and 3-NP adduct to VEA (vea@cwru.edu). Current address: Div. of Bioorganic Chemistry and Mol. Pharm., Dept. of Int. Medicine, Wash. Univ. School of Medicine, Campus Box 8020, 660 South Euclid Avenue, St. Louis, MO 63110 Current address: Ecole Superieure de Biotechnologie de Strasbourg, UMR7100 CNRS, Boulevard Sebastien Brant, BP 10413, 67412 Illkirch, France We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 A resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg297 as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg297. The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg 297 to be increased by 83 Da and to have potential of losing 44 Da, consistent with decarboxylation, during fragmentation. The toxin 3-nitropropionic acid (3-NP 1 ) is produced by certain plants and fungi. It is a specific inhibitor of mitochondrial respiratory complex II. Fatalities after eating moldy sugarcane have been linked to 3-NP toxicity(1,2). Ruminants grazing in regions with 3-NP-producing plants acquire resistance due to reduction of the nitro group to an amine by ruminal bacteria(3). The effectiveness of 3-NP in vivo after injection or oral administration has made it useful in studies involving tissues or whole animals. Ingestion of 3-NP results in neurodegeneration with symptoms resembling those of Huntington's disease(4), and conversely Huntington's disease results in loss of complex II activity(5), thus 3-NP has been used to produce an animal model for study of Huntington's(6,7). Symptoms also include convulsions, and 3-NP is being looked at for inducing a model of epilepsy(8). Prior sub- acute 3-NP poisoning seems to provide resistance to ischemic damage to nervous tissue by a preconditioning effect(9) similar to that resulting from mild ischemia. The target of 3-NP is Complex II, which is both a member of the Krebs tricarboxylic acid cycle (oxidizing succinate to fumarate) and an entry-point for electrons into the respiratory chain at the level of ubiquinol. It consists of a large flavoprotein subunit (FP) containing covalently bound FAD, an iron-sulfur protein (IP) with three different iron-sulfur clusters, and two small membrane anchor subunits (chains C and D) ligating a single low-spin heme of type B. Human genetic defects in the IP subunits or chains C or D lead to

3-Nitropropionic Acid is a Suicide Inhibitor of Mitochondrial Respiration that, Upon Oxidation by Complex II, Forms a Covalent Adduct With a Catalytic Base Arginine in the Active Site of the Enzyme

Fluorinated pyrazoles play an important role in medicinal chemistry, drug discovery, agrochemistry, coordination chemistry, and organometallic chemistry. Since the early 1990s, their popularity has grown exponentially. Moreover, more than 50% of all contributions on the topic have been published in the last 5 years. In this review, analysis of novel synthetic approaches to fluorinated pyrazoles that appeared in recent years is performed. A particular emphasis is devoted to a detailed consideration of reaction mechanisms. In addition, the reasons that have led to the ever-increasing popularity of fluorinated pyrazoles in various areas of science are discussed. At the end of the review, several potentially interesting but yet mostly unknown classes of fluorinated pyrazoles are outlined.

Fluorinated Pyrazoles: From Synthesis to Applications.

BACKGROUND
Aedes aegypti is a major mosquito vector for the transmission of serious diseases, especially dengue and yellow fever. More than 1 billion people in developing countries are at risk. The widespread and continual use of pesticides can lead to resistant mosquitoes. In order to maintain mosquito control gains, it is critical to develop and evaluate novel bioactive molecules that differ in mode of action from currently used products.

RESULTS
A series of novel pyrimidine derivatives were designed and synthesized. Their structures were elucidated by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The biological activities of these compounds were tested against Ae. aegypti. Many of them exhibited insecticidal activity against adult and larval mosquitoes. Compound 4d displayed relatively good activity to reach 70% mortality at 2 µg mL−1. Furthermore, density functional theory calculations were established to study the structure–activity relationship of these novel compounds.

CONCLUSION
A practical synthetic route for pyrimidine derivatives is presented. This study suggests that these pyrimidine derivatives exhibit some activity against the yellow fever mosquito and, with further structure modification, could be novel lead compounds for the development of insecticides against mosquitoes. © 2016 Society of Chemical Industry

Synthesis and insecticidal activity of novel pyrimidine derivatives containing urea pharmacophore against Aedes aegypti.

Background In recent years, carboxamide fungicides, targeting succinate dehydrogenase (SDH), have shown highly efficient and broad spectrum fungicidal activity. Structure-activity relationship (SAR) results for these commercial fungicides show that the carboxamide group was a key active group. This is useful information for the discovery of new pyrazole carboxamide derivatives with fungicidal activity. Results Twenty-seven novel pyrazole carboxamides were designed and synthesized. Their fungicidal activities against Gibberella zeae, Phytophthora infestans, Phytophthora capsici, Rhizoctonia solani, Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Cercospora arachidicola, Sclerotinia sclerotiorum and Physalospora piricola were tested; derivatives possessed excellent inhibitory at 50 mg L-1 in particular. Furthermore, some pyrazole carboxamides exhibited remarkably high activities against Sclerotinia sclerotiorum in vitro with EC50 values of 2.04 to 15.2 μg mL-1 . In addition, some compounds also exhibited high activities against Physalospora piricola, Cercospora arachidicola and Phytophthora capsici. Inhibition activities against SDH proved that the designed analogues were effective at the enzyme level. The SAR of these pyrazole carboxamides was studied by using the docking method. Conclusion It is possible that pyrazole carboxamides, which exhibit good activity against Sclerotinia sclerotiorum, can be further optimized as a lead compounds of carboxamide fungicides. © 2019 Society of Chemical Industry.

Novel 4‐pyrazole carboxamide derivatives containing flexible chain motif: design, synthesis and antifungal activity

A novel simple 1,3,4-oxadiazole-2-carbohydrazide was reported to discover low-cost and versatile antifungal agents. Bioassay results suggested that a majority of the designed compounds were extremely bioactive against four types of fungi and two kinds of oomycetes. This extreme bioactivity was highlighted by the applausive inhibitory effects of compounds 4b, 4h, 5c, 5g, 5h, 5i, 5m, 5p, 5t, and 5v against Gibberella zeae, affording EC50 values ranging from 0.486 to 0.799 μg/mL, which were superior to that of fluopyram (2.96 μg/mL) and comparable to those of carbendazim (0.947 μg/mL) and prochloraz (0.570 μg/mL). Meanwhile, compounds 4g, 5f, 5i, and 5t showed significant actions against Fusarium oxysporum with EC50 values of 0.652, 0.706, 0.813, and 0.925 μg/mL, respectively. Pharmacophore exploration suggested that the N'-phenyl-1,3,4-oxadiazole-2-carbohydrazide pattern is necessary for the bioactivity. Molecular docking of 5h with succinate dehydrogenase (SDH) indicated that it can completely locate the inside of the binding pocket via hydrogen-bonding and hydrophobic interactions, revealing that this novel framework might target SDH. This result was further verified by the significant inhibitory effect on SDH activity. In addition, scanning electron microscopy patterns were performed to elucidate the anti-G. zeae mechanism. Given these features, this type of framework is a suitable template for future exploration of alternative SDH inhibitors against plant microbial infections.

Novel 1,3,4-Oxadiazole-2-carbohydrazides as Prospective Agricultural Antifungal Agents Potentially Targeting Succinate Dehydrogenase

Facile and efficient synthesis and herbicidal activity determination of novel 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one derivatives via microwave irradiation

Microwave Assisted Synthesis and Antifungal Activity of Some Novel Thioethers Containing 1,2,4-triazolo[4,3-a] pyridine Moiety

\n\nIn recent years, pyrazole carboxamide derivatives possessed excellent\nfungicidal activity. In the process of designing new fungicides, the carboxamide group was modified\nin order to find novel structure pyrazole carboxamide derivatives.\n\n\n\nTen novel trifluoromethyl pyrazole acyl thiourea derivatives were designed and synthesized.\nIn vivo fungicidal activities of these compounds were tested against Fusarium oxysporum,\nCorynespora mazei and Botrytis cinerea, respectively\n\n\n\nParticularly compounds exhibited significant control effective at 100 mg/L. More importantly,\nsome compounds showed the good control effective at 10 mg/L. Furthermore, docking\nwas established to study the structure-activity relationship of the title compounds.\n\n\n\n It is possible that trifluoromethylpyrazole acyl thiurea derivatives, which possess good\ncontrol effective against Botrytis cinerea, may become novel lead compounds for the development\nof fungicides with further structure modification.\n

Zhi-Wen Zhai

Papers

Novel 4‐pyrazole carboxamide derivatives containing flexible chain motif: design, synthesis and antifungal activity

Facile and efficient synthesis and herbicidal activity determination of novel 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one derivatives via microwave irradiation

Microwave Assisted Synthesis and Antifungal Activity of Some Novel Thioethers Containing 1,2,4-triazolo[4,3-a] pyridine Moiety

Novel Trifluoromethylpyrazole Acyl Thiourea Derivatives: Synthesis, Antifungal Activity and Docking Study

Novel trifluoromethylpyrazole acyl urea derivatives: Synthesis, crystal structure, fungicidal activity and docking study