Development of Oxygen-Bridged Pyrazole-Based Structures as Cannabinoid Receptor 1 Ligands.

In this work, the synthesis of the cannabinoid receptor 1 neutral antagonists 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole-3-carboxamide 1a and its deaza N-cyclohexyl analogue 1b has led to a deepening of the structure-activity studies of this class of compounds. A series of novel 4,5-dihydrobenzo-oxa-cycloheptapyrazoles analogues of 1a,b, derivatives 1c-j, was synthesized, and their affinity towards cannabinoid receptors was determined. Representative terms were evaluated using in vitro tests and isolated organ assays. Among the derivatives, 1d and 1e resulted in the most potent CB1 receptor ligands (KiCB1 = 35 nM and 21.70 nM, respectively). Interestingly, both in vitro tests and isolated organ assays evidenced CB1 antagonist activity for the majority of the new compounds, excluding compound 1e, which showed a CB1 partial agonist behaviour. CB1 antagonist activity of 1b was further confirmed by a mouse gastrointestinal transit assay. Significant activity of the new CB1 antagonists towards food intake was showed by preliminary acute assays, evidencing the potentiality of these new derivatives in the treatment of obesity.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Novel diazabicycloalkane delta opioid agonists.

Here we report the investigation of diazabicycloalkane cores as potential new scaffolds for the development of novel analogues of the previously reported diazatricyclodecane selective delta (δ) opioid agonists, as conformationally constrained homologues of the reference δ agonist (+)-4-[(αR)-α((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80). In particular, we have simplified the diazatricyclodecane motif of δ opioid agonist prototype 1a with bridged bicyclic cores. 3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane, 3,9-diazabicyclo[3.3.1]nonane, 3,9-diazabicyclo[4.2.1]nonane, and 3,10-diazabicyclo[4.3.1]decane were adopted as core motifs of the novel derivatives. The compounds were synthesized and biologically assayed as racemic (3-5) or diastereoisomeric (6,7) mixtures. All the novel compounds 3-7 showed δ agonism behaviour and remarkable affinity to δ receptors. Amongst the novel derivatives, 3,8-diazabicyclo[3.2.1]octane based compound 4 evidenced improved δ affinity and selectivity relative to SNC80.

Efficient cell transfection with melamine-based gemini surfactants.

Gemini surfactants consisting of two melamine scaffolds connected by a n-hexyl linker and functionalized with a 1-propylammonium polar head and a lipophilic chain having variable carbon length (from C8 to C16) were synthesized. These were then used successfully for the transfection of A549, U87 MG, and Bristol 8 cell lines with maxGFP expressing plasmid. The transfection protocol was optimized appropriately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions, the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required), improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA transfection.

NESS06SM reduces body weight with an improved profile relative to SR141716A.

We have recently synthesized a new series of 4,5-dihydrobenzo-oxa-cycloheptapyrazole derivatives with the aim to discover novel CB1 antagonist agents characterized by anti-obesity activity comparable to that of SR141716A but with reduced adverse effects such as anxiety and depression. Within the novel class, the CB1 antagonist 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta(1,2-c)pyrazole-3-carboxamide (NESS06SM) has been selected as lead compound. We found that NESS06SM is a CB1 neutral antagonist, characterized by poor blood-brain barrier permeability. Moreover, NESS06SM chronic treatment determined both anti-obesity effect and cardiovascular risk factor improvement in C57BL/6N Diet Induced Obesity (DIO) mice fed with fat diet (FD mice). In fact, the mRNA gene expression in Central Nervous System (CNS) and peripheral tissues by real time PCR, showed a significant increase of orexigenic peptides and a decrease of anorexigenic peptides elicited by NESS06SM treatment, compared to control mice fed with the same diet. Moreover, in contrast to SR141716A treatment, the chronic administration of NESS06SM did not change mRNA expression of both monoaminergic transporters and neurotrophins highly related with anxiety and mood disorders. Our results suggest that NESS06SM reduces body weight and it can restore the disrupted expression profile of genes linked to the hunger-satiety circuit without altering monoaminergic transmission probably avoiding SR141716A side effects. Therefore the novel CB1 neutral antagonist could represent a useful candidate agent for the treatment of obesity and its metabolic complications.

Synthesis and structure-activity relationship studies of novel tubulysin U analogues--effect on cytotoxicity of structural variations in the tubuvaline fragment.

Tubulysins are cytotoxic natural products with promising anti-cancer properties, originally isolated from myxobacterial cultures. Structurally, tubulysins are tetrapeptides, incorporating three unusual (Mep, Tuv and Tup) and one proteinogenic amino acid (Ile). Here we describe the synthesis and structure-activity relationship studies of novel tubulysin U and V analogues, with variations in the central Tuv fragment, which is known to be of paramount importance for tubulysins' potency and hence cytotoxicity, but has seldom been modified in previous studies. Specifically, we replaced the natural iso-propyl and acetoxy functionalities with other structurally related groups. In general, the new analogues showed much lower potency relative to native tubulysin U. However, one of the synthetic analogues (1f) having a MOM function replacing the acetyl group exhibited a 22 nM IC50 on the HT-29 cell line which is comparable to the IC(50) displayed by tubulysin U (3.8 nM). Furthermore, the synthetic methodology reported herein was found to be flexible enough to deliver different core-modified tubulysin analogues and hence may be regarded as a scalable and convenient strategy for the chemical generation of novel tubulysin analogues.

Effects of solar irradiance noise on a complex marine trophic web.

The analysis of experimental data of the solar irradiance, collected on the marine surface, clearly highlights the intrinsic stochasticity of such an environmental parameter. Given this result, effects of randomly fluctuating irradiance on the population dynamics of a marine ecosystem are studied on the basis of the stochastic 0-dimensional biogeochemical flux model. The noisy fluctuations of the irradiance are formally described as a multiplicative Ornstein-Uhlenbeck process, that is a self-correlated Gaussian noise. Nonmonotonic behaviours of the variance of the marine populations' biomass are found with respect to the intensity and the autocorrelation time of the noise source, manifesting a noise-induced transition of the ecosystem to an out-of-equilibrium steady state. Moreover, evidence of noise-induced effects on the organic carbon cycling processes underlying the food web dynamics are highlighted. The reported results clearly show the profound impact the stochastic environmental variables can have on both the populations and the biogeochemistry at the basis of a marine trophic network.

Low complexity model to study scale dependence of phytoplankton dynamics in the tropical Pacific.

We demonstrate that a simple model based on reaction-diffusion-advection (RDA) equation forced by realistic surface velocities and nutrients is skilled in reproducing the distributions of the surface phytoplankton chlorophyll in the tropical Pacific. We use the low-complexity RDA model to investigate the scale relationships in the impact of different drivers (turbulent diffusion, mean and eddy advection, primary productivity) on the phytoplankton chlorophyll concentrations. We find that in the 1/4^{∘} (∼25 km) model, advection has a substantial impact on the rate of primary productivity, while the turbulent diffusion term has a fairly negligible impact. Turbulent diffusion has an impact on the phytoplankton variability, with the impact being scale propagated and amplified by the larger scale surface currents. We investigate the impact of a surface nutrient decline and some changes to mesoscale eddy kinetic energy (climate change projections) on the surface phytoplankton concentrations. The RDA model suggests that unless mesoscale eddies radically change, phytoplankton chlorophyll scales sublinearly with the nutrients, and it is relatively stable with respect to the nutrient concentrations. Furthermore, we explore how a white multiplicative Gaussian noise introduced into the RDA model on its resolution scale propagates across spatial scales through the nonlinear model dynamics under different sets of phytoplankton drivers. The unifying message of this work is that the low-complexity (e.g., RDA) models can be successfully used to realistically model some specific aspects of marine ecosystem dynamics and by using those models one can explore many questions that would be beyond computational affordability of the higher-complexity ecosystem models.

Radiative Transfer Modeling With Biogeochemical-Argo Float Data in the Mediterranean Sea.

A radiative transfer model was parameterized and validated using Biogeochemical Argo float data acquired between 2012 and 2017 across the Mediterranean Sea. Fluorescence-derived chlorophyll a concentration, particulate optical backscattering at 700 nm, and fluorescence of chromophoric dissolved organic matter (CDOM) were used to parametrize the light absorption and scattering coefficients of the optically significant water constituents (such as pure water, non-algal particles, CDOM, and phytoplankton). The model was validated with in situ downwelling irradiance profiles and apparent optical properties derived both from irradiance profiles and satellite data, such as the diffuse attenuation coefficients and remote sensing reflectance. Results showed that by using regional parameterizations that are not only related to chlorophyll concentration and vertical distribution, the model was able to capture a more accurate spectral response in the examined wavelength range compared to chlorophyll-related (or Case 1) optical models. When using alternative models that incorporated also measurements of CDOM fluorescence or particulate optical backscattering, the model skill increased at all examined wavelengths. Finally, using a multi-spectral optical configuration also enabled the estimation of the relative contribution of separate water constituents in the examined spectral range. Simulations including non-algal particles and CDOM performed up to 61% and 79% better than when considering the optical properties of pure seawater alone. Moreover, a simulation including phytoplankton light absorption resulted in an error reduction of up to 42%, especially at 412 nm and with a more uniform response at the wavelengths considered.

bFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice.

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.

Synthesis and enzymatic evaluation of novel partially fluorinated thiol dual ACE/NEP inhibitors.

A novel family of peptidomimetics incorporating fluoroalkyl groups, mainly a trifluoromethyl, in alpha-position to a zinc(II)-binding thiol function, was synthesized in solution as well as in solid-phase. These compounds showed inhibitory potency in the nanomolar range against both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), whereas no inhibition of endothelin-converting enzyme-1 (ECE-1) was observed. The trifluoromethyl-derivatives were more potent than the parent unfluorinated analogues in the case of ACE, and less potent in the case of NEP.

Cumulative Impact Index for the Adriatic Sea: Accounting for interactions among climate and anthropogenic pressures.

Assessing and managing cumulative impacts produced by interactive anthropogenic and natural drivers is a major challenge to achieve the sustainable use of marine spaces in line with the objectives of relevant EU acquis. However, the complexity of the marine environment and the uncertainty linked to future climate and socio-economic scenarios, represent major obstacles for understanding the multiplicity of impacts on the marine ecosystems and to identify appropriate management strategies to be implemented. Going beyond the traditional additive approach for cumulative impact appraisal, the Cumulative Impact Index (CI-Index) proposed in this paper applies advanced Multi-Criteria Decision Analysis techniques to spatially model relationships between interactive climate and anthropogenic pressures, the environmental exposure and vulnerability patterns and the potential cumulative impacts for the marine ecosystems at risk. The assessment was performed based on spatial data characterizing location and vulnerability of 5 relevant marine targets (e.g. seagrasses and coral beds), and the distribution of 17 human activities (e.g. trawling, maritime traffic) during a reference scenario 2000-2015. Moreover, projections for selected physical and biogeochemical parameters (temperature and chlorophyll 'a') for the 2035-2050 timeframe under RCP8.5 scenario, were integrated in the assessment to evaluate index variations due to changing climate conditions. The application of the CI-Index in the Adriatic Sea, showed higher cumulative impacts in the Northern part of the basin and along the Italian continental shelf, where the high concentration of human activities, the seawater temperature conditions and the presence of vulnerable benthic habitats, contribute to increase the overall impact estimate. Moreover, the CI-Index allowed understanding which are the phenomena contributing to synergic pressures creating potential pathways of environmental disturbance for marine ecosystems. Finally, the application in the Adriatic case showed how the output of the CI-Index can provide support to evaluate multi-risk scenarios and to drive sustainable maritime spatial planning and management.

Novel pyrrolocycloalkylpyrazole analogues as CB1 ligands.

Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide-based compounds were designed and synthesized for cannabinoid CB1 and CB2 receptor interactions. Any of the new synthesized compounds showed high affinity for CB2 receptor with Ki values superior to 314 nm, whereas some of them showed moderate affinity for CB1 receptor with Ki values inferior to 400 nm. 7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-carboxamide (2j) exhibited good affinity for CB1 receptor (Ki CB1  = 81 nm) and the highest CB2 /CB1 selectively ratio (>12). Docking studies carried out on such compounds were performed using the hCB1 X-ray in complex with the close pyrazole analogue AM6538 and disclosed specific pattern of interactions related to the tricyclic pyrrolopyrazole scaffolds as CB1 ligands.

KEMTUB012-NI2, a novel potent tubulysin analog that selectively targets hypoxic cancer cells and is potentiated by cytochrome p450 reductase downregulation.

PURPOSE: There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to existing therapies. METHODS: A novel hypoxia-targeting agent, KEMTUB012-NI2, was synthesized by conjugating a 2-nitroimidazole hypoxia-targeting moiety to a synthetic tubulysin, a very potent antimitotic. Its hypoxic selectivity and mode of action were studied in breast cancer cell lines. RESULTS: KEMTUB012-NI2 exhibited a similar selectivity for hypoxic cells to that of tirapazamine, a well-established hypoxia-targeting agent, but was >1,000 times more potent in cell cytotoxicity assays. The hypoxia-targeting mechanism for both KEMTUB012-NI2 and tirapazamine was selective and mediated by one-electron reductases. However, while cytochrome p450 reductase (POR) downregulation could inhibit tirapazamine cytotoxicity, it actually sensitized hypoxic cells to KEMTUB012-NI2. CONCLUSION: KEMTUB012-NI2 is a potent new agent that can selectively target hypoxic cancer cells. The hypoxia selectivity of KEMTUB012-NI2 and tirapazamine appears to be differentially activated by reductases. Since reductases are heterogeneously expressed in tumors, the different activation mechanisms will allow these agents to complement each other. Combining POR downregulation with KEMTUB012-NI2 treatment could be a new treatment strategy that maximizes efficacy toward hypoxic tumor cells while limiting systemic toxicity.

Tricyclic pyrazoles. 4. Synthesis and biological evaluation of analogues of the robust and selective CB2 cannabinoid ligand 1-(2',4'-dichlorophenyl)-6-methyl-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide.

New analogues (2a-p) of the previously reported CB(2) ligands 6-methyl- and 6-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamides (1a,b) have been synthesized and evaluated for cannabinoid receptor affinity. One example, 1-(2',4'-dichlorophenyl)-6-methyl-N-cyclohexyilamine-1,4-dihydroindeno[1,2-c] pyrazole-3-carboxamide (2a) was shown to have single digit nanomolar affinity for cannabinoid CB(2) receptors. Furthermore, compounds 2a and 2b, as well as lead structures 1a,b, were also shown to be agonist in an in vitro model based on human promyelocytic leukemia HL-60 cells.

A critical review of both the synthesis approach and the receptor profile of the 8-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide and analogue derivatives.

8-Chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide 9a was discovered as potent and selective CB1 antagonist by part of our group few years ago. In particular it was reported to have an affinity towards the CB1 cannabinoid receptor (CB1R), expressed as Ki, of 0.00035 nM. Nevertheless significantly divergent data were reported for the same compound from other laboratories. To unequivocally define the receptor profile of 9a, we have critically reviewed both its synthesis approach and binding data. Here we report that, in contrast to our previously reported data, 9a showed a Ki value for CB1R in the order of nanomolar rather than of fentomolar range. The new determined receptor profile of 9a was also ascertained for analogue derivatives 9b-i, as well as for 12. Moreover, the structural features of the synthesized compounds necessary for CB1R were investigated. Amongst the novel series, effects on CB1R intrinsic activity was highlighted due to the substituents at the position 3 of the pyrazole ring of the 1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole scaffold. Although the cannabinoid receptor profile of 9a was reviewed in this work, the relevance of this compound in CB1R antagonist based drug discovery is confirmed.

Tricyclic pyrazoles. 3. Synthesis, biological evaluation, and molecular modeling of analogues of the cannabinoid antagonist 8-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide.

A series of analogues of 8-chloro-1-(2',4'-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide 4a (NESS 0327) (Ruiu, S.; Pinna, G. A.; Marchese, G.; Mussinu, J. M.; Saba, P.; Tambaro, S.; Casti, P.; Vargiu, R.; Pani, L. Synthesis and Characterization of NESS 0327: A Novel Putative Antagonist of CB1 Cannabinoid Receptor. J. Pharmacol. Exp. Ther. 2003, 306, 363-370) was synthesized and evaluated for their affinity to cannabinoid receptors. Depending on the chemical modification of the lead structure that was chosen, compounds 4b, 4c, 4i, 4l, and 4m still proved to be potent binders of the CB1 receptor. Moreover, several analogues (4c, 4d, 4e, and 4m) demonstrated superior CB2 receptor binding affinities compared to the parent ligand. Compounds 4b, 4c, 4i, and 4l displayed the most promising pharmacological profiles, having the highest selectivity for CB1 receptors with Ki(CB2) to Ki(CB1) ratios of 11,250, 2000, 3330 and 4625, respectively. Compound 4c increased the intestinal propulsion in mice and antagonized the effect induced by the CB1 receptor agonist WIN 55,212-2. Finally, molecular modeling studies were carried out on a set of tricyclic pyrazoles (2a-4a) and on rimonabant 1 (SR141716A), indicating that high CB1 receptors affinities were consistent for the tricyclic derivatives, both with a nonplanar geometry of the tricyclic cores and with a precise orientation of the substituent (chlorine) on this ring system.

Effect of 1-butanol on the microstructure of lecithin/water/tripalmitin system.

Warm microemulsions based on lipids characterized by a melting point over 50 degrees C have been successfully used as starting matrix in a quenching process to obtain solid lipid nanoparticles (SLN). In this work, we have investigated the effect of 1-butanol (B) on the phase behavior of the lecithin (LCT)/water (W)/tripalmitin (TP) system at 70 degrees C. The study has been carried out at LCT/B=1 (weight ratio). Emulsion and liquid crystalline phase regions have been observed in the ternary phase diagram, while the presence of 1-butanol in the LCT/W/B/TP system allows the formation of a wide area of liquid isotropic phase from the whole (LCT+B)/TP binary axis up to 37 wt% of water. The microstructure of this isotropic phase has been investigated by means of 1H NMR PGSE technique. The self-diffusion coefficients of the different components along oil and water dilution lines indicate a microstructural organization characterized by a highly connected water in oil domains.

The tubulysin analogue KEMTUB10 induces apoptosis in breast cancer cells via p53, Bim and Bcl-2.

PURPOSE: Tubulysins are natural tetrapeptides that inhibit tubulin polymerisation. Tubulysins are very potent inhibitors of mammalian cancer cell growth, but restricted availability has limited their characterisation and development as anti-cancer compounds. KEMTUB10 was recently developed as a synthetic analogue of natural tubulysins. METHODS: The cell cytotoxicity of KEMTUB10 was studied in cell lines that represent the main breast cancer sub-types. The KEMTUB10 pro-apoptotic mechanism of action was studied in MCF7 and MDAMB231 cells. RESULTS: KEMTUB10 exerts a potent cytotoxic effect in cells representing the main breast cancer sub-types. KEMTUB10 blocks cells in the G2/M phase of the cell cycle and is a strong stimulator of apoptosis/cell death. KEMTUB10-induced apoptosis involves p53 and Bim, and to some extent Bcl-2 phosphorylation. CONCLUSIONS: KEMTUB10 is a promising new anti-mitotic that exerts a potent cytotoxic effect in breast cancer cells, blocks cells in the G2/M phase of the cell cycle and stimulates apoptosis/cell death. KEMTUB10 has a distinct mode of action to taxol, appears to be sensitive to different molecular factors in cells and is likely subject to different mechanisms of acquired resistance. KEMTUB10 has the potential to be an important addition to the anti-cancer therapeutic armoury.

Design of novel 3,6-diazabicyclo[3.1.1]heptane derivatives with potent and selective affinities for α4ß2 neuronal nicotinic acetylcholine receptors.

New analogues (3a-l) of the previously described α4ß2 selective ligand 3-(6-halopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptanes (2a,b) have been synthesized and their binding activity for neuronal acetylcholine receptor subtypes α4ß2 and α7 were assayed. Six of these compounds (3a,b,c,j,k and l) showed high affinity and selectivity for α4ß2 receptors. The phenylpyridyl-diazabicycloheptane 3c displayed Ki value of 11.17 pM for α4ß2, in line with that of the halogenated homologues 3a,b, although it was characterized by an improved selectivity (Ki = 17 µM for α7 receptors). The influence of substitutions on the phenylpyridyl moiety on binding at both α4ß2 and α7 receptors has been examined through the Topliss decision tree analysis. Substitution with electron-donating groups (as CH3 and OCH3) resulted in a good affinity for α4ß2 receptors and substantially no affinity for α7. Amongst all the tested phenyl-substituted compounds, the p-NO2-phenyl substituted analogue 3j exhibited the highest α4ß2 affinity, with Ki value comparable to that of 3c. Intrinsic α4ß2 receptor mediated activity in [(3)H]-DA release assay was showed by compound 3a as well as by the reference analogue 2a, whereas phenyl substituted derivative 3c exhibited α4ß2 antagonist activity.