Different chemical proteomic approaches to identify the targets of lapatinib.

The process of identifying the protein targets and off-targets of a biologically active compound is of great importance in modern drug discovery. Various chemical proteomics approaches have been established for this purpose. To compare the different approaches, and to understand which method would provide the best results, we have chosen the EGFR inhibitor lapatinib as an example molecule. Lapatinib derivatives were designed using linkers with motifs, including amino (amidation), alkyne (click chemistry) and the diazirine group (photo-affinity). These modified lapatinib analogues were validated for their ability to inhibit EGFR activity in vitro and were shown to pull down purified recombinant EGFR protein. In all of the approaches evaluated here, we identified EGFR as the main protein target from the lysate of immortalised cell line expressing EGFR, thus validating its potential use to identify unknown protein targets. Taken together, the results reported here give insight into the cellular activities of lapatinib.

Benzoxaboroles-Novel Autotaxin Inhibitors.

Autotaxin (ATX) is an extracellular enzyme that hydrolyses lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which has a role in the mediation of inflammation, fibrosis and cancer. ATX is a drug target that has been the focus of many research groups during the last ten years. To date, only one molecule, Ziritaxestat (GLPG1690) has entered the clinic; it is currently in Phase 3 clinical trials for idiopathic pulmonary fibrosis. Other small molecules, with different binding modes, have been investigated as ATX inhibitors for cancer including compounds possessing a boronic acid motif such as HA155. In this work, we targeted new, improved inhibitors of ATX that mimic the important interactions of boronic acid using a benzoxaborole motif as the acidic warhead. Furthermore, we aimed to improve the plasma stability of the new compounds by using a more stable core spacer than that embedded in HA155. Compounds were synthesized, evaluated for their ATX inhibitory activity and ADME properties in vitro, culminating in a new benzoxaborole compound, 37, which retains the ATX inhibition activity of HA155 but has improved ADME properties (plasma protein binding, good kinetic solubility and rat/human plasma stability).

Macrolactonolides: a novel class of anti-inflammatory compounds.

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17ß-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.

Sulphur cycling between terrestrial agroecosystem and atmosphere.

Central gas station of the natural gas borehole system Podravina is located near the village Molve. It delivers more than a quarter of total energy used in Croatia to its consumers. Over the years, adapting technology to increasingly demanding and rigorous standards in environmental protection has become paramount. Yet, despite all the industry has undertaken to address the risk of harmful substances entering the food chain, a multidisciplinary research team of independent scientists monitors the content of specific substances in all components of the ecosystem. This paper presents measurements of total sulphur contents in soil surface [(0 to 3) cm] and subsurface [(3 to 8) cm] layers (study period: autumn 2006 - spring 2010) and in plants (study period: spring 2000 - spring 2010), and the concentration of gaseous sulphur compounds in the air. Concentrations of hydrogen sulphide (H2S) and mercaptans (RSH) were measured from the summer of 2002 until the autumn of 2010, while concentrations of sulphur dioxide (SO2) were measured from the spring of 2008 until the autumn of 2010. The paper also shows total annual atmospheric sulphur (S-SO4) deposition at Bilogora measuring station (study period: 2001 - 2010). Average monthly concentrations of H2S in air varied between 0.2 µg m-3 and 2.0 µg m-3, RSH between 0.1 µg m-3 and 24.5 µg m-3, and SO2 between 0.4 µg m-3 and 2.8 µg m-3 depending on the location and the season of sampling. Mean values of total sulphur in soil and in Plantago lanceolata plant ranged between 610 mg kg-1 and 1,599 mg kg-1 and between 3,614 mg kg-1 and 4,342 mg kg-1, respectively, depending on the soil type, location, and sampling depth. Average values of total sulphur mass ratio for all examined single soil samples (n=80) were 1,080 mg kg-1 for both studied layers, and 4,108 mg kg-1 for all analysed plant samples (n=85). Average total annual atmospheric sulphur deposition at Bilogora measuring station was 6.3 kg of S-SO4 per hectare.

Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin.

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

A conjugate of pyridine-4-aldoxime and atropine as a potential antidote against organophosphorus compounds poisoning.

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.

Arsenic in air and soil in the vicinity of the central gas station Molve, Croatia.

Spatial and temporal distribution of arsenic levels in soil during the 9 year monitoring period was investigated on four different soil types in the area of the gas borehole system Podravina in Croatia. Arsenic levels in the PM(10) particle fraction were measured periodically at the same locations for 3 years. Arsenic levels in soil significantly depended on soil types. Elevated levels were found on gleysol vertic, at two sampling sites, with values exceeding 30 mg/kg of arsenic in soil. Arsenic levels in air were low and they were not significantly different between sampling sites, suggesting that gas borehole activities have no influence on arsenic levels in the environment.

Determination of aqueous stability and degradation products of series of coumarin dimers.

The stability in aqueous solution of five classes of coumarin dimers (I-V, compounds 1-29) was studied by HPLC-MS/MS at various pH values. The relationship between chemical structure and stability is discussed. It was found that dimeric compounds with strong electron withdrawing groups (EWGs) on the α-carbon to the bridging C-atom are stable at all pH values, whereas other derivatives undergo retro-Michael addition at rates which are also affected by the substituents on the aromatic rings. In some cases formation of stable isomers or oxidation products was observed. In order to evaluate their developability and potential for progression to in vivo studies, representative compounds were tested in an in vitro microsomal stability assay.

Novel spirotetracyclic zwitterionic dual H(1)/5-HT(2A) receptor antagonists for the treatment of sleep disorders.

Histamine H(1) and serotonin 5-HT(2A) receptors mediate two different mechanisms involved in sleep regulation: H(1) antagonists are sleep inducers, while 5-HT(2A) antagonists are sleep maintainers. Starting from 9'a, a novel spirotetracyclic compound endowed with good H(1)/5-HT(2A) potency but poor selectivity, very high Cli, and a poor P450 profile, a specific optimization strategy was set up. In particular, we investigated the possibility of introducing appropriate amino acid moieties to optimize the developability profile of the series. Following this zwitterionic approach, we were able to identify several advanced leads (51, 65, and 73) with potent dual H(1)/5-HT(2A) activity and appropriate developability profiles. These compounds exhibited efficacy as hypnotic agents in a rat telemetric sleep model with minimal effective doses in the range 3-10 mg/kg po.

Applicability of an ultrasonic nebulization system for the airways delivery of beclomethasone dipropionate in a murine model of asthma.

PURPOSE: We have assessed the use of an ultrasonic nebulization system (UNS), composed of ultrasonic nebulizer and diffusion dryer filled with charcoal, for the effective delivery of beclomethasone to the airways in a murine asthma model. METHODS: Solution of beclomethasone in ethanol was aerosolized using an ultrasonic nebulizer. Passage of the aerosol through a drying column containing charcoal and deionizer produced dry beclomethasone particles. Particles were delivered to BALB/c mice placed in a whole-body exposition chamber 1 h before intranasal challenge with ovalbumine. Efficacy of beclomethasone delivery was evaluated by examining bronchoalveolar lavage fluid (BALF) cytology. RESULTS: Effect of three UNS system parameters on aerosol particle size was investigated. The critical parameter affecting the size of dry particles was beclomethasone concentration in aerosolized solution and solution flow rate while power level of ultrasonic nebulizer generator had no effect. Administration of beclomethasone at calculated dose of 150 microg/kg to mice significantly decreased total cell number and relative eosinophil number in BALF. CONCLUSIONS: The UNS system produces a monodisperse aerosol that can be used for inhalative delivery of poorly water soluble substances to experimental animals. The UNS system minimizes formulation requirements and allows rapid and relatively simple efficacy and toxicity testing in animals.

Evidence for the Formation of alpha-Hydroxydialkylnitrosamines in the pH-Independent Solvolysis of alpha-(Acyloxy)dialkylnitrosamines.

A kinetic study of the decay of alpha-(acyloxy)dialkylnitrosamines in aqueous solutions, at 25 degrees C, ionic strength 1 M (NaClO(4)), and 4% acetonitrile by volume, is reported. Rate constants for disappearance of the N-NO chromophore or appearance of benzaldehyde product increase with the introduction of electron-withdrawing groups into the acyloxy moiety. For some compounds, in some regions of pH, the kinetics are non-first-order. For these compounds, in other regions of pH, the rate constants are coincident with those previously reported for the decay of the corresponding alpha-hydroxydialkylnitrosamines. These data provide direct evidence that alpha-hydroxydialkylnitrosamines are intermediates in the pH-independent decomposition of the alpha-(acyloxy)dialkylnitrosamines studied.