David Rennison

Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides

A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared and evaluated for their capacity to induce methemoglobinemia-with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb%=61.0±3.6) and 1,3,4-oxadiazole 10 (metHb%=52.4±0.9) demonstrating the greatest activity. Of the 5 candidates (compounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120mg/kg was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as the CYPs, was postulated as one reason for their failure.

Synthesis and methemoglobinemia-inducing properties of analogues of para-aminopropiophenone designed as humane rodenticides

A number of structural analogues of the known toxicant para-aminopropiophenone (PAPP) have been prepared and evaluated for their capacity to induce methemoglobinemia--with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed for alkyl analogues of PAPP (aminophenones 1-20; compound 6 metHb% = 74.1 ± 2). Besides lipophilicity, this structural sub-class suggested there were certain structural requirements for activity, with both branched (10-16) and cyclic (17-20) alkyl analogues exhibiting inferior in vitro metHb induction. Of the four candidates (compounds 4, 6, 13 and 23) evaluated in vivo, 4 exhibited the greatest toxicity. In parallel, aminophenone bioisosteres, including oximes 30-32, sulfoxide 33, sulfone 34 and sulfonamides 35-36, were found to be inferior metHb inducers to lead ketone 4. Closer examination of Hammett substituent constants suggests that a particular combination of the field and resonance parameters may be significant with respect to the redox mechanisms behind PAPPs metHb toxicity.

Two stereoisomers of the rat toxicant norbormide

The structures of two diastereoisomers of norbormide [systematic name: 5-[hydroxy(phenyl)(2-pyridyl)methyl]-8-[phenyl(2-pyridyl)methylene]-3a,4,7,7a-tetrahydro-4,7-methano-1H-isoindole-1,3(2H)-dione], viz. the unsolvated molecule, C(33)H(25)N(3)O(3), and the ethyl acetate hemisolvate, C(33)H(25)N(3)O(3).0.5C(4)H(8)O(2), have been determined unambiguously. They differ in the relative stereochemistry about the exocyclic double bond and the relative conformations of the aryl rings. Each compound exhibits both intra- and intermolecular hydrogen bonding.

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.

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.

An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging.

Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 μM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures.

Substituted Carbazoles – A New Class of Anthelmintic Agent

A series of novel carbazoles were synthesized based on structural modifications to lead carbazole 1 (EC100 = 2.5 μM against Haemonchus contortus in vitro), which was revealed in a small molecule screening program as a potentially promising platform for the development of new anthelmintic drugs. Subsequently, analogues 19, 21, 41, 42 (EC100 = 1.25 μM, all), and 39 (EC100 = 0.625 μM) were demonstrated to exhibit enhanced in vitro anthelmintic activity over the lead structure, with compound 39 also being shown to be active in vivo against Heligmosomoides polygyrus.

In vitro metabolism of norbormide in rat, mouse and guinea pig liver preparations.

Differences between species in response to norbormide (NRB) may arise through differential pharmacodynamic and/or pharmacokinetic properties. We hypothesise that species-selectivity is at least partly determined by differences in metabolism based on in vitro data generated in liver preparations from rats, mice and guinea pigs. HPLC separation and LC/MS identification revealed that NRB undergoes metabolism primarily to hydroxylated form that was tentatively identified in both rat and non-rat species with NADPH as the preferred cofactor. However, the metabolic profile and the rate are different between species. Gender differences are also reported in the metabolic rate in rats and we postulate that this may be responsible for different toxic sensitivities seen between sexes. Using this knowledge, we aim to develop pharmacological tool(s) for use in designing a new class of drugs that can be targeted in a tissue-selective manner. Further in vivo pharmacokinetic with receptor affinity studies are warranted.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.

‘Tethering’ fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110 μM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.