α-[Amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile; Configurational equilibria in solution.

Inconsistent results have been reported for the effects of the mitogen-activating extracellular kinase (MEK) inhibitor α-[amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile (SL 327) on ethanol-induced conditioned place preference (EtOH-CPP). Since such inconsistencies may be due to the configurational composition of administered SL 327, the interconvertibility of the geometric isomers of this class of compounds has been investigated. This study provides conditions for determination of configurational composition of this class of compounds by HPLC and by 1H NMR and reports details of configurational equilibria as a function of medium and time in solution along with solubility data for SL 327 in aqueous DMSO. The results suggest that the apparently inconsistent results reported for CPP-EtOH may be due to the administration of suspension vs. solutions, as well as to different configurational compositions of SL 327.

Combination of SL327 and Sunitinib Malate leads to an additive anti-cancer effect in doxorubicin resistant thyroid carcinoma cells.

BACKGROUND: Receptor tyrosine kinases (RTKs) play crucial roles in numerous cancer cell processes including cell survival, proliferation, and migration. MEK1/2 MAPK kinases are very important for cancer survival and development. Anaplastic thyroid carcinoma (ATC) is a deadly type of thyroid cancer and there are no very effective systemic treatment strategies for ATC so far. Also, ATC can easily become resistant to therapy of traditional therapeutic drugs for ATC, such as doxorubicin. Drug combination treatment could be a promising therapeutic strategy for ATC, especially for drug resistant ATC. METHODS: We explored the combination effect between a MEK1/2 inhibitor SL327 and a multi-targeted RTK inhibitor Sunitinib Malate in doxorubicin resistant ATC cells using cell viability assay, cell migration assay, nuclei morphology and caspase-3 activity analysis, as well as in vivo tumor growth assay. RESULTS: There is a significant additive effect between SL327 and Sunitinib Malate in reducing viability, increasing apoptosis, and suppressing migration of doxorubicin-resistant ATC cells. Importantly, combination of SL327 and Sunitinib Malate induced significant additive suppression of in vivo doxorubicin-resistant ATC tumor growth. CONCLUSIONS: Our results suggest that the combination of MEK1/2 inhibitor and RTK inhibitor is promising for treatment of ATC especially doxorubicin-resistant ATC. The combination might not only enhance the anti-cancer efficacy, but also reduce the side effects and overcome drug resistance developed in ATC treatment. All these might provide useful information for clinical therapeutics of ATC.

Organometallic Derivatization of the Nematocidal Drug Monepantel Leads to Promising Antiparasitic Drug Candidates.

The discovery of novel drugs against animal parasites is in high demand due to drug-resistance problems encountered around the world. Herein, the synthesis and characterization of 27 organic and organometallic derivatives of the recently launched nematocidal drug monepantel (Zolvix® ) are described. The compounds were isolated as racemates and were characterized by 1 H, 13 C, and 19 F NMR spectroscopy, mass spectrometry, and IR spectroscopy, and their purity was verified by microanalysis. The molecular structures of nine compounds were confirmed by X-ray crystallography. The anthelmintic activity of the newly designed analogues was evaluated in vitro against the economically important parasites Haemonchus contortus and Trichostrongylus colubriformis. Moderate nematocidal activity was observed for nine of the 27 compounds. Three compounds were confirmed as potentiators of a known monepantel target, the ACR-23 ion channel. Production of reactive oxygen species may confer secondary activity to the organometallic analogues. Two compounds, namely, an organic precursor (3 a) and a cymantrene analogue (9 a), showed activities against microfilariae of Dirofilaria immitis in the low microgram per milliliter range.

Synthesis and Kinetic Characterisation of Water-Soluble Fluorogenic Acyl Donors for Transglutaminase†2.

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

Inhibition of endopeptidase and exopeptidase activity of cathepsin B impairs extracellular matrix degradation and tumour invasion.

Cathepsin B is a lysosomal cysteine protease that is implicated in a number of physiological processes, including protein turnover in lysosomes. Changes in its expression are associated with a variety of pathological processes, including cancer. Due to the structural feature, termed the occluding loop, cathepsin B differs from other cysteine proteases in possessing both, endopeptidase and exopeptidase activity. Here we investigated the impact of both cathepsin B activities on intracellular and extracellular collagen IV degradation and tumour cell invasion using new selective synthetic inhibitors, 2-{[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3). All three compounds (5 µM) reduced extracellular degradation of collagen IV by MCF-10A neoT cells by 45-70% as determined by spectrofluorimetry and they (50 µM) attenuated intracellular collagen IV degradation by 40-60% as measured with flow cytometry. Furthermore, all three compounds (5 µM) impaired MCF-10A neoT cell invasion by 40-80% as assessed by measuring electrical impedance in real time. Compounds 1 and 3 (5 µM), but not compound 2, significantly reduced the growth of MMTV-PyMT multicellular tumour spheroids. Collectively, these data suggest that the efficient strategy to impair harmful cathepsin B activity in tumour progression may include simultaneous and potent inhibition of cathepsin B endopeptidase and exopeptidase activities.

Isomerism of Cyanomethanimine: Accurate Structural, Energetic, and Spectroscopic Characterization.

The structures, relative stabilities, and rotational and vibrational parameters of the Z-C-, E-C-, and N-cyanomethanimine isomers have been evaluated using state-of-the-art quantum-chemical approaches. Equilibrium geometries have been calculated by means of a composite scheme based on coupled-cluster calculations that accounts for the extrapolation to the complete basis set limit and core-correlation effects. The latter approach is proved to provide molecular structures with an accuracy of 0.001-0.002 Å and 0.05-0.1° for bond lengths and angles, respectively. Systematically extrapolated ab initio energies, accounting for electron correlation through coupled-cluster theory, including up to single, double, triple, and quadruple excitations, and corrected for core-electron correlation and anharmonic zero-point vibrational energy, have been used to accurately determine relative energies and the Z-E isomerization barrier with an accuracy of about 1 kJ/mol. Vibrational and rotational spectroscopic parameters have been investigated by means of hybrid schemes that allow us to obtain rotational constants accurate to about a few megahertz and vibrational frequencies with a mean absolute error of ∼1%. Where available, for all properties considered, a very good agreement with experimental data has been observed.

Catalytic Enantioselective Reaction of α-Aminoacetonitriles Using Chiral Bis(imidazoline) Palladium Catalysts.

The catalytic enantioselective reaction of diphenylmethylidene-protected α-aminoacetonitriles with imines has been developed. Good yields and diastereo- and enantioselectivities were observed for the reaction of various imines using chiral bis(imidazoline)/Pd catalysts. The reaction of α-aminonitriles with di-tert-butyl azodicarboxylate afforded chiral α,α-diaminonitriles in high yields with high enantioselectivities.

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4.

Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4 was studied. Through using PEG as porogen, the environmental tolerance as well as the biocatalytic reaction efficiency of immobilized cells was greatly improved, i.e., Ca-alginate-PEG immobilized cells got better temperature and substrate concentration tolerance than Ca-alginate immobilized cells and showed similar efficiency with free cells, suggesting that the intrinsic mass transfer resistance of immobilization obviously decreased. It was also observed that the pore diameter and porosity of immobilization beads were related with the molecular weight of PEG. PEG400 was found to be a relatively suitable porogen for Ca-alginate-PEG immobilized cells catalyzed hydrolysis of glycinonitrile. It was noteworthy that the Ca-alginate-PEG immobilized cells could be reused more than 18 times with little loss of enzyme activity which had shown good operation ability and great application potential.

Determination of the new anthelmintic monepantel and its sulfone metabolite in milk and muscle using a UHPLC-MS/MS and QuEChERS method.

This is the first paper to report a method for the detection of the new anthelmintic monepantel and its sulfone metabolite in goat's milk and ovine muscle. Samples were extracted and purified using a modified QuEChERS method. A concentration step was included when analyzing in the low µg kg(-1) range. Analysis was carried out by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in a 13min run time using atmospheric pressure electrospray ionisation in the negative mode (ESI(-)) and multiple reaction monitoring (MRM) scanning. Monepantel (m/z 472) and monepantel-sulfone (m/z 504) both had product ions at m/z 186 and m/z 166. The method has been single-laboratory validated according to the 2002/657/EC guidelines. The mean recovery in milk was 108 and 106% for monepantel and monepantel-sulfone, respectively. The mean recovery in muscle was 109 and 108% for monepantel and monepantel-sulfone, respectively. The coefficients of variation for the within laboratory repeatability and reproducibility were ≤6.4% in milk and ≤14.2% in muscle. The decision limits (CCα) in milk were 2.20 and 2.08 µg kg(-1) for monepantel and monepantel-sulfone, respectively. The decision limits (CCα) in muscle were 771 and 746 µg kg(-1) for monepantel and monepantel-sulfone, respectively.

Discovery and development of veterinary antiparasitic drugs: past, present and future.

Despite investment in programs to manage the development of resistance to existing agents, this continues to drive the need for discovery of novel antiparasitic agents for veterinary medicine. Historically, antiparasitic drug discovery was driven by empirical screening, but technological advances have lead to an increased focus on mechanism-based approaches to drug discovery and this is projected to increase as our capabilities advance to improve both the throughput of assays and the quality of data generated. Investment in the development of combination products with novel agents is increasing and, despite regulatory hurdles in some regions, efforts to globally harmonize regulations will aid in delivering safe, efficacious drugs to help in resistance management and integrated parasite control programs.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus.

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

Deep-space glycine formation via Strecker-type reactions activated by ice water dust mantles. A computational approach.

A Strecker-type synthesis of glycine by reacting NH(3), H(2)C=O and HCN in presence of ice water (H(2)O-ice) as a catalyst has been theoretically studied at B3LYP/6-31+G(d,p) level within a cluster approach in order to mimic reactions occurring in the interstellar and circumstellar medium (ICM). Results indicate that, despite the exoergonic character of the considered reactions occurring at the H(2)O-ice surface, the kinetics are slow due to relatively high electronic energy barriers (ΔU(0)(≠)=15-45 kcal mol(-1)). Reactions occurring within H(2)O-ice cavities, in which ice bulk effects have been modeled by assuming a dielectric continuum (ε=78), show energy barriers low enough to allow NH(2)CH(2)OH formation but not NH=CH2 (ΔU(0)(≠)= 2 and 21 kcal mol(-1), respectively) thus hindering the NH(2)CH(2)CN formation, i.e. the precursor of glycine, through Strecker channels. Moreover, hydrolysis of NH(2)CH(2)CN to give glycine is characterized by high electronic energy barriers (ΔU(0)(≠)=27-34 kcal mol(-1)) and cannot readily occur at cryogenic temperatures. Nevertheless, the facts that NH=CH(2) formation can readily be achieved through the radical-radical HCN+2H - NH−−>CH2 reaction [D. E. Woon, Astrophys. J., 2002, 571, L177-L180], and that present results indicate that the Strecker step of NH=CH(2)+HCN−−>NH(2)CH(2)CN exhibits a relative low energy barrier (ΔU(0)(≠)=8­9 kcal mol(-1)), suggest that a combination of these two mechanisms allows for the formation of NH(2)CH(2)CN in the ICM. These results strengthen the thesis that NH(2)CH(2)CN could have been formed and protected by icy dust particles, and then delivered through micro-bombardments onto the early Earth, leading to glycine formation upon contact with the primordial ocean.

Ni(+) reactions with aminoacetonitrile, a potential prebiological precursor of glycine.

The gas-phase reactions between Ni(+) ((2)D(5/2)) and aminoacetonitrile, a molecule of prebiological interest as possible precursor of glycine, have been investigated by means of mass spectrometry techniques. The mass-analyzed ion kinetic energy (MIKE) spectrum reveals that the adduct ions [NC--CH(2)--NH(2), Ni(+)] spontaneously decompose by loosing HCN, H(2), and H(2)CNH, the loss of hydrogen cyanide being clearly dominant. The structures and bonding characteristics of the aminoacetonitrile-Ni(+) complexes as well as the different stationary points of the corresponding potential energy surface (PES) have been theoretically studied by density functional theory (DFT) calculations carried out at B3LYP/6-311G(d,p) level. A cyclic intermediate, in which Ni(+) is bisligated to the cyano and the amino group, plays an important role in the unimolecular reactivity of these ions, because it is the precursor for the observed losses of HCN and H(2)CNH. In all mechanisms associated with the loss of H(2), the metal acts as hydrogen carrier favoring the formation of the H(2) molecule. The estimated bond dissociation energy of aminoacetonitrile-Ni(+) complexes (291 kJ mol(-1)) is larger than those measured for other nitrogen bases such as pyridine or pyrimidine and only slightly smaller than that of adenine.

Protonation thermochemistry of aminoacetonitrile.

The gas-phase basicity (GB) of aminoacetonitrile (NH2CH2CN, 1) has been determined from measurement of proton transfer equilibrium constants in an ion cyclotron resonance mass spectrometer (GB(1) = 789.3 +/- 1.0 kJ x mol(-1)). Molecular orbital calculations up to the G2 level demonstrate that protonation occurs preferentially on the nitrogen atom of the NH2 group, and provide a theoretical proton affinity (PA(1)) of 824.0 kJ x mol(-1). Exact calculation of the entropy associated with hindered rotations and consideration of Boltzman distribution of conformers allow a theoretical estimate of the molar protonation entropy S degrees (1H+) - S degrees (1) = 8.6 J x mol(-1) x K(-1). Combining this value with experimental GB(1) leads to an 'experimental' proton affinity of 819.2 kJ x mol(-1), in close agreement with the G2 expectation.

Beta-substituted cyclohexanecarboxamide: a nonpeptidic framework for the design of potent inhibitors of cathepsin K.

A new series of nonpeptidic cathepsin K inhibitors that are based on a beta-substituted cyclohexanecarboxamide motif has been developed. Lead optimization yielded compounds with sub-nanomolar potency and exceptional selectivity profiles against cathepsins B, L, and S. Use of fluorine atoms to block metabolism on the cyclohexyl ring led to compounds with excellent pharmacokinetic properties. Considering the well-established role of cathepsin K in osteoclast-mediated bone turnover, compounds such as (-)-34a (hrab Cat K IC(50) 0.28 nM; >800-fold selectivity vs Cat B, L, and S; PK data in dogs: F 55%, t(1/2) = 15 h) exhibit great potential for development as an orally bioavailable therapeutic for treatment of diseases that involve bone loss.

Dialkylaminoacetonitrile derivatives as amide synthons. A one-pot preparation of heteroaryl amides via a strategy of sequential SNAr substitution and oxidation.

Dialkylamino acetonitrile derivatives were utilized as alternative to cyanohydrin synthons for preparation of the corresponding heteroaryl dialkyl amides via a strategy of sequential base-mediated coupling and oxidation. The most advantageous oxidant, NiO(2)-H(2)O, can readily oxidize 2-substituted aminoacetonitriles to the corresponding amides under both basic and neutral conditions by forming cyanohydrins in situ.

Acetonitrile derivatives as carbonyl synthons. One-pot preparation of diheteroaryl ketones via a strategy of sequential SNAr substitution and oxidation.

The anion of 2-aryl acetonitrile derivatives reacted with a variety of heteroaryl chlorides or bromides in an S(N)Ar manifold to afford intermediate anions which were susceptible to oxidation. The addition of sodium peroxide and aqueous NH(4)OAc solution effected oxidation to afford aryl heteroaryl ketones in good yields. Aryl acetonitrile derivatives are thus umpolung-type synthons of the corresponding aryl carbonyl functionality.

Density functional study on the mechanism of bicyclic guanidine-catalyzed Strecker reaction.

As a direct and viable synthesis of amino acids, the small organic molecule catalyzed asymmetric Strecker reactions have been explored successfully in recent years. For these catalysts, the active sites may be a guanidine group or similarly a urea group. In an effort to elucidate the reaction mechanism, we have investigated the bicyclic guanidine-catalyzed Strecker reaction of HCN and methanimine using density functional theory with the B3LYP method. Assisted by guanidine, two competitive pathways to aminoacetonitrile were rationalized. The aminoisoacetonitrile may not form due to the instability of the product.