Design of Two New Sulfur Derivatives of Perezone: In Silico Study Simulation Targeting PARP-1 and In Vitro Study Validation Using Cancer Cell Lines.

Poly-ADP-Ribose Polymerase (PARP-1) is an overexpressed enzyme in several carcinomas; consequently, the design of PARP-1 inhibitors has acquired special attention. Hence, in the present study, three compounds (8-10) were produced through a Michael addition protocol, using phenylmethanethiol, 5-fluoro-2-mercaptobenzyl alcohol, and 4-mercaptophenylacetic acid, respectively, as nucleophiles and perezone as the substrate, expecting them to be convenient candidates that inhibit PARP-1. It is convenient to note that in the first stage of the whole study, the molecular dynamics (MD) simulations and the quantum chemistry studies of four secondary metabolites, i.e., perezone (1), perezone angelate (2), hydroxyperezone (3), and hydroxyperezone monoangelate (4), were performed, to investigate their interactions in the active site of PARP-1. Complementarily, a docking study of a set of eleven sulfur derivatives of perezone (5-15) was projected to explore novel compounds, with remarkable affinity to PARP-1. The molecules 8-10 provided the most adequate results; therefore, they were evaluated in vitro to determine their activity towards PARP-1, with 9 having the best IC50 (0.317 µM) value. Additionally, theoretical calculations were carried out using the density functional theory (DFT) with the hybrid method B3LYP with a set of base functions 6-311++G(d,p), and the reactivity properties were compared between the natural derivatives of perezone and the three synthesized compounds, and the obtained results exhibited that 9 has the best properties to bind with PARP-1. Finally, it is important to mention that 9 displays significant inhibitory activity against MDA-MB-231 and MCF-7 cells, i.e., 145.01 and 83.17 µM, respectively.

Interaction of MDIMP with the Voltage-Gated Calcium Channels.

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC50 values were found (in micromolars): >1000 (CaV2.3), 957 (CaV1.3), 656 (CaV1.2), 219 (CaV3.2), and 132 (CaV3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca2+ channels and a modest upregulation of CaV1.3 and CaV2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning CaV3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC50 value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (R)-MDIMP [methyl (R)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca2+ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

Synthesis, docking study and inhibitory activity of 2,6-diketopiperazines derived from α-amino acids on HDAC8.

Diketopiperazines (DKPs) have been regarded as an important scaffold from the viewpoint of synthesis due to their biological properties for the treatment of several diseases, including cancer. In this work, two novel series of enantiomeric 2,6-DKPs derived from α-amino acids were synthesized through nucleophilic substitution and intramolecular cyclization reactions. All the compounds were docked against histone deacetylase 8 (HDAC8), which is a promising target for the development of anticancer drugs. These compounds bound into the active site of HDAC8 in a similar way to Trichostatin A (TSA), which is an HDAC8 inhibitor. This study showed that the conformation of the 2,6-DKP ring, stereochemistry, and the type of substituent on the chiral center had an important role in the binding modes. The Gibbs free energies and dissociation constants values of HDAC8-ligand complexes showed that compounds (S)-4hBn, (S)-4m, (R)-4h, and (R)-4m were more stable and affine towards HDAC8 than TSA. The inhibitory activities of 4a, (S)-4h, (S)- and (R)-4(g, l, m) were evaluated in vitro on HDAC8. It was found that compounds (R)-4g (IC50 = 21.54 nM) and (R)-4m (IC50 = 10.81 nM) exhibited better inhibitory activities than TSA (IC50 = 28.32 nM). These results suggested that 2,6-DKPs derivatives may be promising anticancer agents for further biological studies.

Isoindoline Derivatives of α-Amino Acids as Cyclooxygenase 1 and 2 Inhibitors.

IC50 values were obtained for two series of isoindolines derived from α-amino acids over cyclooxygenase 1 and 2 (COX-1 and COX-2). In order to explain the biological activity observed, a structure-activity relationship (SAR) model was achieved for the tested compounds and 19 reference compounds with known selective inhibitory activity, through the correlation of the binding energies calculated from rigid docking of the best conformations into the catalytic sites of COX-1 and COX-2, as well as their molecular descriptors: Log P, molecular weight (MW), volume (V), and solvation energy (Esol) versus their experimental IC50 values by MLR and LS-SVM methods. The model probed whether the COX-1 and COX-2 inhibitory activities of the isoindolines correlate with steric, hydrophobic, and thermodynamic parameters. The correlation values with MLR for COX-1 and COX-2 (r(2) = 0.4193 and r(2) = 0.5929) were optimized with LS-SVM until r(2) = 0.6818 for COX-1 and r(2) = 0.8985 for COX-2, resulting in a good predictive ability for COX-1 and -2 inhibition with this model. In conclusion, the data suggests that the physicochemical descriptors evaluated have an impact on the inhibitory activity and selectivity of isoindolines over COX-1 and COX-2.

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase.

Three glutamic acid derivatives, two boron-containing and one imide-containing compound, were synthesized and tested for antimicrobial activity targeting glutamate-racemase. Antimicrobial effect was evaluated over Bacillus spp. Docking analysis shown that the test compounds bind near the active site of racemase isoforms, suggesting an allosteric effect. The boron derivatives had greater affinity than the imide derivative. In vitro assays shown good antimicrobial activity for the boron-containing compounds, and no effectiveness for the imide-containing compounds. The minimum inhibitory concentration of tetracycline, used as standard, was lower than that of the boron-containing derivatives. However, it seems that the boron-containing derivatives are more selective for bacteria. Experimental evidence suggests that the boron-containing derivatives act by inhibiting the racemase enzyme. Therefore, these test compounds probably impede the formation of the bacterial cell wall. Thus, the boron-containing glutamic acid derivatives should certainly be of interest for future studies as antimicrobial agents for Bacillus spp.

Synthesis of N-aminophalimides derived from α-amino acids: Theoretical study to find them as HDAC8 inhibitors by docking simulations and in vitro assays.

Phthalimides are valuable for synthesis and biological properties. New acetamides 3(a-c) and 4(a-c) were synthesized and characterized as precursors for novel N-aminophalimides 5(a-c) and 6(a-c). Structures of 4a, 5(a-b), and 6(a-b) were confirmed by single crystal X-ray. Docking studies identified compounds with favorable Gibbs free energy values for binding to histone deacetylase 8 (HDAC8), an enzyme targeted for anticancer drug development. These compounds bound to both the orthosteric and allosteric pockets of HDAC8, similar to Trichostatin A (TSA), an HDAC8 inhibitor. 6(a-c) contain hydroxyacetamide moiety as a zinc-binding group, a phthalimide moiety as a capping group, and aminoacetamide moiety as a linker group, which are important for ligand-receptor binding. ΔG values indicated that compounds 5b, 6b, and 6c had higher affinity for HDAC8 in the allosteric pocket compared to TSA. In vitro evaluation of inhibitory activities on HDAC8 revealed that compounds 3(a-c) and 5(a-c) showed similar inhibitory effects (IC50 ) ranging from 0.445 to 0.751 µM. Compounds 6(a-c) showed better affinity, with 6a (IC50 = 28 nM) and 6b (IC50 = 0.18 µM) showing potent inhibitory effects slightly lower than TSA (IC50 = 26 nM). These findings suggest that the studied compounds hold promise as potential candidates for further biological investigations.

9-Amino-acridinium nitrate monohydrate.

The pyridine N atom of the cation in the title hydrated salt, C(13)H(11)N(2) (+)·NO(3) (-)·H(2)O, is protonated; the N atom of the NH(2) group shows a planar conformation. The former N atom is hydrogen bonded to a water mol-ecule. The amino group is involved in three N-H⋯O hydrogen bonds with two neighboring nitrate anions. The water mol-ecule is hydrogen bonded to two adjacent nitrate anions. In the crystal, this results in a layered network.

Hydrogen-bond directionality and symmetry in [C(O)NH](N)2P(O)-based structures: a comparison between X-ray crystallography data and neutron-normalized values, and evaluation of reliability.

For [C(O)NH](N)2P(O)-based structures, the magnitude of the differences in the N-H...O, H...O=P and H...O=C angles has been evaluated when the N-H bond lengths, determined by X-ray diffraction, were compared to the neutron normalized values and the maximum percentage difference was obtained, i.e. about 3% for the angle even if the N-H bond lengths have a difference of about 30% (0.7 Šfor the X-ray and 1.03 Šfor the neutron-normalized value). The symmetries of the crystals are discussed with respect to the symmetry of the molecules, as well as to the symmetry of hydrogen-bonded motifs, and the role of the most directional hydrogen bond in raising the probability of obtaining centrosymmetric crystal structures is investigated. The work was performed by considering nine new X-ray crystal structures and 204 analogous structures retrieved from the Cambridge Structural Database.

p-Tolyl bis-(o-tolyl-amido)-phosphinate.

In the title compound, C(21)H(23)N(2)O(2)P, the P atom has a distorted tetra-hedral configuration. The O atom of the OC(6)H(4)-4-CH(3) group and the N atoms show sp(2) character. In the crystal, adjacent mol-ecules are linked by N-H⋯O hydrogen bonds into helical chains parallel to the b axis.

HPV16-E6 Oncoprotein Activates TGF-ß and Wnt/ß-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model.

OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. RESULTS: The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses' cell morphology were due to actin filaments reorganisation, activation of TGF-ß and Wnt/ß-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. CONCLUSION: The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

MDIMP, a novel cardiac Ca(2+) channel blocker with atrial selectivity.

In cardiac muscle cells both T-and L-type Ca(2+) channels (TTCCs and LTCCs, respectively) are expressed, and the latter are relevant to a process known as excitation-contraction coupling (ECC). Evidence obtained from docking studies suggests that isoindolines derived from α-amino acids bind to the LTCC CaV1.2. In the present study, we investigated whether methyl (S)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate (MDIMP), which is derived from L-leucine, modulates both Ca(2+) channels and ECC. To this end, mechanical properties, as well as Ca(2+) transients and currents, were all investigated in isolated cardiac myocytes. The effects of MDIMP on CaV1.2 (transiently expressed in 293T/17 cells) were also studied. In this system, evidence was found for an inhibitory action that develops and recovers in min, with an IC50 of 450µM. With respect to myocytes: atrial-TTCCs, atrial-LTCCs, and ventricular-LTCCs were also inhibited, in that order of potency. Accordingly, Ca(2+) transients, contractions, and window currents of LTCCs were all reduced more strongly in atrial cells. Interestingly, while the modulation of LTCCs was state-independent in these cells, it was state-dependent, and dual, on the ventricular ones. Furthermore, practically all of the ventricular LTCCs were closed at resting membrane potentials. This could explain their resistance to MDIMP, as they were affected in only open or inactivated states. All these features in turn explain the preferential down-regulation of the atrial ECC. Thus, our results support the view that isoindolines bind to Ca(2+) channels, improve our knowledge of the corresponding structure-function relationship, and may be relevant for conditions where decreased atrial activity is desired.

Hirshfeld surface analysis of new phosphoramidates.

Hirshfeld surfaces and two-dimensional fingerprint plots are used to visualize and analyze intermolecular interactions in six new phosphoramidate structures, [2,6-F2-C6H3C(O)NH]P(O)[X]2 {X = N(C2H5)2 (1), [X]2 = NHCH2C(CH3)2CH2NH and with one CH3OH solvated molecule (2)}, [C6H5O]2P(O)Y [Y = NC4H8O (3), NHC6H4(3-Br) (4)] and [Z]2P(O)OP(O)[Z]2 [Z = N(CH3)(CH2C6H5) (5), NHC6H4(4-CH3) (6)]. Study of the short intermolecular contacts in structures (1)-(6) by Hirshfeld surfaces demonstrate that the O atom of P=O is a better H-atom acceptor than the O atom of C=O for (1) and (2), and also relative to the O atom of the C6H5O group for (3) and (4), and relative to the bridge O atom of the P(O)OP(O) segment for (5) and (6). The results confirm that the crystal packing is related to the kind of substituent linked to the P atom. Compounds (1), (2), (4) and (6), with characteristic N-H···O hydrogen bonds, show a pair of intense spikes (including the intermolecular H···O contacts) in the fingerprint plots, summarizing the major features of each structure in the related two-dimensional plot. For (3) and (5), without any N-H unit, the two short spikes are observed for (3) but are absent for (5). The upper d(e) and d(i) values (distances to the Hirshfeld surfaces for the nearest atoms outside and inside) in the fingerprint plots are more compact in (3) than in (4), and in (5) than in (6), reflecting the more efficient packing in (3) and (5). The tertiary N atoms of (3) and (5) do not take part in any intermolecular contacts involving H atoms. Moreover, structures (3)-(6) show greater contribution from C···H contacts relative to O···H contacts. Finally, Hirshfeld surfaces and fingerprint plots are employed for a comparison of the two independent molecules in the asymmetric unit of (1) and also, for a comparison of (6), in the orthorhombic crystal system, with the previously reported monoclinic polymorph (Pourayoubi, Fadaei et al., 2012).

Analysis of N-H···O hydrogen bonds in new C(O)-NH-P(O)-based phosphoric triamides and analogous structures deposited in the Cambridge Structural Database.

Five new compounds belonging to the phosphoric triamide family have been synthesized: two of them with the formula XC(O)NHP(O)Y [X = CF3 (1) and CClF2 (2), Y = NHCH2C(CH3)2CH2NH] involving a 1,3-diazaphosphorinane ring part, and three 2,6-Cl2C6H3C(O)NHP(O)Z2 phosphoric triamides [Z = NHC(CH3)3 (3), N(CH3)(C6H11) (4) and N(CH3)(CH2C6H5) (5)]. The characterization was performed by (31)P{(1)H}, (1)H, (13)C NMR, IR spectroscopy besides (19)F NMR for fluorine containing compounds (1) and (2), and X-ray single-crystal structure analysis for (1), (3), (4) and (5). In each molecule the P atom has a distorted tetrahedral environment. The N atoms bonded to P atom have mainly sp(2) character with a very slight tendency to a pyramidal coordination for some amido groups. Different types of N-H···O hydrogen bonds have been analyzed for (1), (3), (4) and (5) and 118 other structures (including 194 hydrogen bonds) deposited in the Cambridge Structural Database, containing either C(O)-NH-P(O)[N(C)(C)]2 or C(O)-NH-P(O)[NH(C)]2. The participation of N(CP)-H···O=P [N(CP) = the nitrogen atom of the C(O)-NH-P(O) fragment], N-H···O=P, N-H···O=C and N(CP)-H···O=C hydrogen bonds in different hydrogen-bonded motifs are discussed. Moreover, the involvement of the O atoms of C=O or P=O in the [N(CP)-H][N-H]···O=P, [N-H]2···O=P, [N-H]2···O=C and [N-H]3···O=C groups are considered. A histogram of N···O distances, the distribution of N-H···O angles and the scatterplot of N-H···O angles versus N···O distances are studied.

Molecular modeling study of isoindolines as L-type Ca(2+) channel blockers by docking calculations.

Two series of isoindolines 1(a-g) and 2(a-g) were found by docking calculations to be possible L-type Ca(2+) channel (LCC) blockers. The theoretical 3-D model of the outer vestibule and the selective filter of the LCC was provided by Professor Lipkind; this model consists of transmembrane segments S5 and S6 and P-loops contributed by each of four repeats (I, II, III, and IV) of Ca(v) 1.2. Therefore, two well-known LCC blockers, nifedipine 3 and (R)-ethosuccinimide 4 were also evaluated, and their binding sites on the LCC were identified and compared with those obtained for 1(a-g) and 2(a-g). Analysis of the results shows that the target compounds tested probably could be LCC blockers, since they interact with or near the glutamic acid residues Glu393, Glu736, Glu1145 and Glu1446 (the EEEE locus), which belong to the LCC selectivity region. The G values for all of the Ca(2+) channel ligands are between-10.78 and -3.67 (kcal mol(-1)), showing that LCC-1b, -1e and -1f complexes are more stable than the other compounds tested. Therefore, theoretically calculated dissociation constants K(d) (microM) were obtained for all compounds. Comparing these values reveals that compounds 1b (0.0244 microM), 1e (0.0176 microM) and 1f (0.0125 microM) exhibit more affinity for the LCC than the other compounds. This screening shows that the two series of isoindolines probably could act as LCC blockers.