Structural insights of a PI3K/mTOR dual inhibitor with the morpholino-triazine scaffold

Stimulation of the PI3K/Akt/mTOR pathway, which controls cell proliferation and growth, is often observed in cancer cell. Inhibiting both PI3K and mTOR in this pathway can switch off Akt activation and hence, plays a powerful role for modulating this pathway. PKI-587, a drug containing the structure of morpholino-triazines, shows a dual and nano-molar inhibition activity and is currently in clinical trial. To provide an insight into the mechanism of this dual inhibition, pharmacophore and QSAR models were developed in this work using compounds based on the morpholino-triazines scaffold, followed by a docking study. Pharmacophore model suggested the mechanism of the inhibition of PI3Kα and mTOR by the compounds were mostly the same, which was supported by the docking study showing similar docking modes. The analysis also suggested the importance of the flat plane shape of the ligands, the space surrounding the ligands in the binding pocket, and the slight difference in the shape of the binding sites between PI3Kα and mTOR.     

Preparation and characterization of particles with small differences in polydispersity

Colloidal particles are widely used both in fundamental research and in materials science. One important parameter influencing the physical properties of colloidal materials is the particle size distribution (polydispersity) of the colloidal particles. Recent work on colloidal crystallization has demonstrated that even subtle changes in polydispersity can have significant effects. In this study we present centrifugation techniques for subtly manipulating the width and the shape of the particle size distribution, for polydispersities less than 10%. We use scanning electron microscopy as well as dynamic and static light scattering to characterize the particle size distributions. We compare the results and highlight the difficulties associated with the determination of accurate particle size distributions.     

Kinetic Analysis of the Light-induced Fluorescence Quenching in Light-harvesting Chlorophyll a/b Pigment-Protein Complex of Photosystem II

We carried out a kinetic analysis of the light-induced fluorescence quenching (AF) of the light-harvesting chlorophyll a/b pigment-protein complex of photosystem II (LHCII) that was first observed by Jennings et at (Pho-tosynth. Res. 27, 57–64, 1991). We show that during a 2 min light, 2 min dark cycle, both the light and dark phases exhibit biexponential kinetics; this is tentatively explained by the presence of two types of light-induced quenchers in different domains of aggregated LHCII. Quantitative analysis could be carried out on the faster kinetic component; the slower component that was not completed during the measurement was not amenable for quantitative analysis. Our analysis revealed that the rate of the light-induced decrease of the fluorescence yield depended linearly on the light intensity, which shows that the generation of the quencher originates from a reaction that is first order with respect to the concentration of the excited domains. As shown by the estimated rate constant, pho-togeneration of the quencher is a fast reaction that can compete with other excitation-relaxation pathways. Both the decay and the recovery time constants of AF depended strongly on the temperature. Thermodynamic analysis showed that the fast light-induced decline in the fluorescence was determined by a low fraction of the excited states. Recovery was associated with large decrease in the entropy of activation that indicated the involvement of large structural rearrangements. Macroaggregated LHCII exhibited larger ΔF than small aggregates, which is consistent with the proposed role of aggregated LHCII in thy-lakoid membranes in nonphotochemical quenching.     

Bis(m-phenylene)-32-crown-10-based cryptands, powerful hosts for paraquat derivatives

Four new bis(m-phenylene)-32-crown-10-based cryptands with different third bridges were prepared. Their complexes with paraquat derivatives were studied by proton NMR spectroscopy, mass spectrometry, and X-ray analysis. It was found that these cryptands bind paraquat derivatives very strongly. Specifically, a diester cryptand with a pyridyl nitrogen atom located at a site occupied by either water or a PF(6) anion in analogous complexes exhibited the highest association constant K(a) = 5.0 x 10(6) M(-1) in acetone with paraquat, 9000 times greater than the crown ether system. X-ray structures of this and analogous complexes demonstrate that improved complexation with this host is a consequence of preorganization, adequate ring size for occupation by the guest, and the proper location of the pyridyl N-atom for binding to the beta-pyridinium hydrogens of the paraquat guests. This readily accessible cryptand is one of the most powerful hosts reported for paraquats.     

Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument

Bioanalytical support of plasma pharmacokinetic (PK) studies for drug discovery programs primarily involves the quantitative analysis of dosed compounds using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring (SRM) mode. However, there is a growing need for information on the metabolism of new chemical entities (NCEs), in addition to the time-concentration profiles from these studies. In this paper, we present a novel approach to not only quantify parent drugs with SRM, but also simultaneously screen for metabolites using a hybrid triple quadrupole/linear ion trap (QqQ(LIT)) instrument. This was achieved by incorporating both the conventional SRM-only acquisition of parent compounds and the SRM-triggered information-dependent acquisition (IDA) of potential metabolites within the same scan cycle during the same LC/MS/MS run. Two test compounds were used to demonstrate the applicability of this approach. Plasma samples from PK studies were processed by simple protein precipitation and the supernatant was diluted with water before injection. The fast scanning capability of the linear ion trap allowed for the information-dependent acquisition of metabolite MS/MS spectra (<1 s/scan), in addition to the collection of adequate data points for SRM-only channels. The MS/MS spectra obtained from potential metabolites in post-dose samples correlated well with the spectra of the parent compounds studied, therefore providing additional confirmatory structure information without the need for repetitive analyses. Relative quantitative time-concentration profiles of identified metabolites were also obtained. Furthermore, this articulated SRM+SRM-IDA approach generated equivalent quantitative results for parent compounds to those obtained by conventional SRM-only analysis. This approach has been successfully used to support discovery PK screening programs.     

Studies on the mechanism of 1,2-dihydropyrazin-2-one ring formation from dipeptidyl chloromethyl ketone and its chemical properties: immediate deamination during catalytic hydrogenation

1,2-Dihydropyrazin-2-one derivatives, which have two aminoalkyl groups at the positions 3 and 6, were found to be efficient tools for the construction of potent, selective and long-acting opioid mimetics. During the course of preparation, we found that the catalytic hydrogenation of 3,6-bis(benzyloxycarbonylaminomethyl)-5-methyl-1,2-dihydropyrazin-2-one to remove the benzyloxycarbonyl groups resulted in a side reaction. By MS and NMR studies and by preparation of additional 1,2-dihydropyrazin-2-one derivatives, the structure of the by-product was identified as 3-aminomethyl-5,6-dimethyl-1,2-dihydropyrazin-2-one. Preparation of additional compounds substituted with deuterium provided us with sufficient information to confirm the structure of the product and to support a cyclization mechanism in its formation.     

Polyrotaxanes by free‐radical polymerization of acrylate and methacrylate monomers in the presence of a crown ether

Poly[(methyl acrylate)‐rotaxa‐(30‐crown‐10)] ( 5 ) and poly[(methyl methacrylate)‐rotaxa‐(30‐crown‐10)] ( 6 ) were synthesized by azobisisobutyronitrile‐initiated free‐radical bulk polymerizations of the respective monomers in the presence of 30‐crown‐10 ( 1 ; equimolar; 5 times the monomer mass). For 5 , 3.8 mass % (0.81 mol % with respect to the monomer) of the crown was incorporated versus 1.7 mass % (0.39 mol % with respect to the monomer) for 6 . Control reactions with 18‐crown‐6, which is to small to be threaded, showed that chain transfer to the crown ethers was detectable only for the acrylate but was relatively negligible and spectroscopically distinct. The threading yields were much higher with these systems than with polystyrene, most likely because of the greater compatibility of the crown ether with these polar monomers and polymers and the consequent ability to carry out the polymerizations homogeneously in the absence of added solvent; however, the threading process was still essentially statistical. Therefore, the polymerization of methacrylate monomers 8a – 8c based on tetraarylmethane moieties connected via diethyleneoxy or triethyleneoxy spacers was examined in the presence of 1 in the belief that the supramolecular semirotaxane monomer 9 formed statistically in situ could be captured more efficiently and produce higher threading yields, presumably of side‐chain polyrotaxanes, than the simple (meth)acrylate monomers. Azobisisobutyronitrile‐initiated polymerizations either neat or in toluene produced polyrotaxanes 10 with up to about 1.6 mass % and 2 mol % threaded crown ether, presumably trapped on the pendant stoppered side chains. Although primarily statistical in nature, the latter rotaxane syntheses afforded on a molar basis 3–7 times more efficient incorporation of 1 than styrene (0.33 mol %), methyl acrylate (0.81 mol %), or methyl methacrylate (0.39 mol %) monomers for the preparation of main‐chain polypseudorotaxanes and indeed even surpassed the 60‐crown‐20/polyacrylonitrile system (1.5 mol %). This was presumed to be due to the fact that the loss of the crown ether, once it was threaded onto the monomer to form 9 and the latter was polymerized, was either retarded (by the tetraphenylmethyl stopper in 10a ) or prevented completely [by tris(p‐t‐butylphenyl)phenylmethyl stoppers in 10b and 10c ]. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1978–1993, 2001     

Removal of the PsaF Polypeptide Biases Electron Transfer in Favor of the PsaB Branch of Cofactors in Triton X‐100 Photosystem I Complexes from Synechococcus sp. PCC 7002†

Continuous wave (CW) and transient electron paramagnetic resonance studies have implied that when PsaF is removed genetically, the double reduction of A_1A is facile, the lifetime of A_1A^? is shorter and the ratio of fast to slow kinetic phases increases in PS I complexes isolated with Triton X‐100 (Van der Est, A., A. I. Valieva, Y. E. Kandrashkin, G. Shen, D. A. Bryant and J. H. Golbeck [2004] Biochemistry 43 , 1264–1275). Changes in the lifetimes of A_1A^? and A_1B^? are characteristic of mutants involving the quinone binding sites, but changes in the relative amplitudes of A_1A^? and A_1B^? are characteristic of mutants involving the primary electron acceptors, A_0A and A_0B. Here, we measured the fast and slow phases of electron transfer from A_1B^? and A_1A^? to F_X in psaF and psaE psaF null mutants using time‐resolved CW and pump‐probe optical absorption spectroscopy. The lifetime of the fast kinetic phase was found to be unaltered, but the lifetime of the slow kinetic phase was shorter in the psaF null mutant and even more so in the psaE psaF null mutant. Concomitantly, the amplitude of the fast kinetic phase increased by a factor of 1.8 and 2.0 in the psaF and psaE psaF null mutants, respectively, at the expense of the slow kinetic phase. The change in ratio of the fast to slow kinetic phases is explained as either a redirection of electron transfer through A_1B at the expense of A_1A, or a shortening of the lifetime of A_1A^? to become identical to that of A_1B^?. The constant lifetime and the characteristics of the near‐UV spectrum of the fast kinetic phase favor the former explanation. A unified hypothesis is presented of a displacement of the A‐jk(1) α‐helix and switchback loop, which would weaken the H‐bond from Leu₇₂₂ to A_1A, accounting for the acceleration of the slow kinetic phase, as well as weaken the H‐bond from Tyr₆₉₆ to A_0A, accounting for the bias of electron transfer in favor of the PsaB branch of cofactors.