Sladjana Kostic-Rajacic

Antiglioma action of xanthones from Gentiana kochiana: Mechanistic and structure–activity requirements

The present study identifies xanthones gentiakochianin and gentiacaulein as the active principles responsible for the in vitro antiglioma action of ether and methanolic extracts of the plant Gentiana kochiana. Gentiakochianin and gentiacaulein induced cell cycle arrest in G(2)/M and G(0)/G(1) phases, respectively, in both C6 rat glioma and U251 human glioma cell lines. The more efficient antiproliferative action of gentiakochianin was associated with its ability to induce microtubule stabilization in a cell-free assay. Both the xanthones reduced mitochondrial membrane potential and increased the production of reactive oxygen species in glioma cells, but only the effects of gentiakochianin were pronounced enough to cause caspase activation and subsequent apoptotic cell death. The assessment of structure-activity relationship in a series of structurally related xanthones from G. kochiana and Gentianella austriaca revealed dihydroxylation at positions 7, 8 of the xanthonic nucleus as the key structural feature responsible for the ability of gentiakochianin to induce microtubule-associated G(2)/M cell block and apoptotic cell death in glioma cells.

Arylpiperazine-mediated activation of Akt protects SH-SY5Y neuroblastoma cells from 6-hydroxydopamine-induced apoptotic and autophagic death.

We investigated the ability of 19 recently synthesized arylpiperazine compounds to protect human SH-SY5Y neuroblastoma cells from the neurotoxin 6-hydroxydopamine (6-OHDA). The compound with the most potent neuroprotective action was N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), which reduced 6-OHDA-induced apoptotic death through stabilization of mitochondrial membrane and subsequent prevention of superoxide production, caspase activation and DNA fragmentation. 6-OHDA-triggered autophagic response was also reduced by 6b, which prevented inactivation of the main autophagy repressor mTOR, upregulation of proautophagic beclin-1, conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, as well as intracytoplasmic acidification induced by 6-OHDA. The inhibition of autophagy using LC3β gene silencing or pharmacological autophagy blockers 3-methyladenine or bafilomycin A1, mimicked the cytoprotective effect of 6b. While the treatment with 6b had no effect on the phosphorylation of proapoptotic MAP kinases ERK and JNK, it markedly increased the phosphorylation of the prosurvival kinase Akt in 6-OHDA-treated cells. Akt inhibitor DEBC or RNA interference-mediated Akt silencing reduced the ability of 6b to block 6-OHDA-triggered apoptotic and autophagic responses, thus confirming their dependency on Akt activation. The cytoprotective effect of 6b was also observed in 6-OHDA-treated neuronal PC12 cells, but not in SH-SY5Y or PC12 cells exposed to 1-methyl-4-phenylpyridinium, indicating that the observed neuroprotection was dependent on the cytotoxic stimulus. Because of the ability to prevent 6-OHDA induced apoptotic/autophagic cell death through activation of Akt, the investigated arylpiperazines could be potential candidates for treatment of neurodegenerative diseases.

Synthesis of Several Substituted Phenylpiperazines Behaving as Mixed D2/5HT1A Ligands

Twenty‐two different compounds have been synthesized with the aim of creating new, mixed D2/5HT1A ligands. For this purpose 1‐substituted phenylpiperazines attached by the N‐4 nitrogen to dopaminergic pharmacophores of the 2‐(5‐benzimidazole)ethyl‐, 2‐(5‐benztriazole)ethyl‐, 2‐[5‐(benzimidazole‐2‐thione)]ethyl‐ and 2‐[6‐(1,4‐dihydroquinoxaline‐2,3‐dione)]ethyl‐type were selected according to known structure‐affinity requirements of 1‐arylpiperazines.

Introduction of a methyl group in α- or β-position of 1-heteroarylethyl-4-phenyl-piperazines affects their dopaminergic/serotonergic properties

1‐(2‐Heteroarylalkyl)‐4‐phenylpiperazines containing methyl group in either the α‐ or the β‐position of the side alkyl chain were synthesized as racemic mixtures. They were evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5‐HT1A serotonin receptors using synaptosomal membranes of the bovine caudate nucleus and hippocampus, respectively, as a source of the corresponding receptors. Tritiated SCH 23390 (D1 receptor‐selective), spiperone (D2 receptor‐selective), and 8‐OH‐DPAT (5‐HT1A receptor‐selective) were employed as the radioligands. None of the new compounds expressed significant affinity for the D1 receptor. Introduction of the methyl group into the β‐position of the parent molecules increased the affinity for the D2 receptor (10b—13b), and decreased the affinity for the 5‐HT1A receptor with the exception of imidazole (11b) which was a rather efficient displacer of 8‐OH‐DPAT. Most potent of the newly synthesized compounds in [3H]spiperone assay were compounds (±)6‐[1‐methyl‐2‐(4‐phenylpiperazin‐1‐yl)‐ethyl]‐1,4‐dihydroquinoxaline‐2,3‐dione (10b), Kd = 6.0 nM and (±)5‐[1‐methyl‐2‐(4‐phenylpiperazin‐1‐yl)‐ethyl]‐1,3‐dihydrobenzoimidazol‐2‐thione (13b), Kd = 5.3 nM. However, compounds containing methyl group in α‐position (10a—13a) of the parent molecules expressed a decreased affinity for the D2 receptor, while the affinity for the 5‐HT1A receptor remained in the same range of concentrations as that of closely related achiral parent compounds ((14—17) run in the same binding assays as references.

Theoretical insight into sulfur–aromatic interactions with extension to D2 receptor activation mechanism

Contacts between aromatic rings and sulfur-containing amino acids are frequent in proteins. However, little is known about the nature of their interactions particularly if substituents are present on aromatic ring. In this paper, DFT quantum chemical calculations were used to study substituted benzenes in complex with hydrogen sulfide (H2S), methanethiol (CH3SH), and (Methylsulfanyl)methane (CH3SCH3). It was found that SH···π interaction is more stabilizing than the S···π interaction in the case of benzene, but this is changed with increasing electronegativity of the substituent on benzene ring. Although the change of energy of SH···π and S···π interaction follows the conventional model of substituent effect, where S···π interactions are maximized and SH···π interactions are diminished with electron-withdrawing substituent on benzene as a result of changes in the aryl π-system, it was found that it is mainly a consequence of direct electrostatic interaction between substituent and the sulfur-containing molecule. We also investigated the model system of Cys···Trp interaction, adjacent to a cluster of aromatic amino acids, in proteins, using explicit membrane molecular dynamics simulations results of D3 dopamine receptor crystal structure as starting point. It was found that fluorination in aromatic cluster enhances the Cys···Trp interaction. The effect is maximized when transferred through the rest of aromatic system suggesting possible explanation for frequent contacts between sulfur-containing and aromatic amino acids in proteins and their effects on protein folding and stabilization.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats.

Arylpiperazine‐based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2/5‐HT1A ligands, N‐{4‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl}‐phenyl]‐picolinamide (6a) and N‐{3‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate‐immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5‐HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0–7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL‐6, IL‐1, and GM‐CSF, TH1 cytokine IFN‐γ, TH17 cytokine IL‐17, as well as the signature transcription factors of TH1 (T‐bet) and TH17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti‐apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN‐93 and neuronal cell line PC12 from mitogen‐activated normal T cells or myelin basic protein‐activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation.

Arylpiperazine Dopamineric Ligands Protect Neuroblastoma Cells from Nitric Oxide (NO)-Induced Mitochondrial Damage and Apoptosis

The protective ability of novel arylpiperazine‐based dopaminergic ligands against nitric oxide (NO)‐mediated neurotoxicity is investigated. The most potent neuroprotective arylpiperazine identified during the study was N‐{4‐[2‐(4‐phenyl‐piperazin‐1‐yl)ethyl]‐phenyl}picolinamide, which protected SH‐SY5Y human neuron‐like cells from the proapoptotic effect of NO donor sodium nitroprusside (SNP) by decreasing oxidative stress, mitochondrial membrane depolarization, caspase activation and subsequent phosphatydilserine externalization/DNA fragmentation. The protective effect was associated with the inhibition of proapoptotic (JNK, ERK, AMPK) and activation of antiapoptotic (Akt) signaling pathways, in the absence of interference with intracellular NO accumulation. The neuroprotective action of arylpiperazines was shown to be independent of dopamine receptor binding, as it was not affected by the high‐affinity D1/D2 receptor blocker butaclamol. These results reported support the further study of arylpiperazines as potential neuroprotective agents.

Molecular Modeling of 5HT2A Receptor – Arylpiperazine Ligands Interactions

In this paper, we report the molecular modeling of the 5HT2A receptor and the molecular docking of arylpiperazine‐like ligands. The focus of the research was on explaining the effects the ligand structure has on the binding properties of the 5HT2A receptor and on the key interactions between the ligands and the receptor‐binding site. To see what the receptor–ligand interactions were, various substituents were introduced in one part of the ligand, keeping the rest unchanged. In this way, using a docking analysis on the proposed 5HT2A receptor model, we identified key receptor–ligand interactions and determined their properties. Those properties were correlated with experimentally determined binding affinities in order to determine the structure to activity relationship of the examined compounds.

Mixed Dopaminergic/Serotonergic Properties of Several 2-Substituted 4-[2-(5-Benzimidazole)ethyl]-1-arylpiperazines

A series of substituted 4‐[2‐(5‐benzimidazole)ethyl]‐arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4‐[2‐(N,N‐di‐n‐propyl‐amino)ethyl]‐1,2‐diaminobenzenes. They were evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5‐HT1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D1 receptor‐selective), spiperone (D2 receptor selective) and 8‐OH‐DPAT (5‐HAT1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D1 receptor, while the remaining ligands were inefficient or weak competitors of [3H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands. Also, compound 7 was an inefficient displacer of [3H]‐8‐OH‐DPAT. Compound 19 with a Ki value of 3.5 nM was the most potent competitor of [3H]spiperone and compound 13 (Ki = 3.3 nM) was the most efficient in displacing [3H]‐8‐OH‐DPAT from the 5‐HAT1A serotonin receptor. Ligands 5, 6, 8–11, and 13–20 expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.

Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas with dopamine D2 and 5-hydroxytryptamine 5HT1A receptors

It is suggested that the ratio of dopamine D(2) to 5-hydroxytryptamine 5-HT(1A) activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D(2) and 5-hydrohytryptamine 5-HT(1A) receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT(1A) receptors.