Gold(I)-Triphenylphosphine Complexes with Hypoxanthine-Derived Ligands: In Vitro Evaluations of Anticancer and Anti-Inflammatory Activities

A series of gold(I) complexes involving triphenylphosphine (PPh₃) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HL_n) of the general composition [Au(L_n)(PPh₃)] (1–9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4–6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4–6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC₅₀ ≈ 1–30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study.     

Highly and Broad-Spectrum In Vitro Antitumor Active cis-Dichloridoplatinum(II) Complexes with 7-Azaindoles

The cis-[PtCl₂(naza)₂] complexes (1–3) containing monosubstituted 7-azaindole halogeno-derivatives (naza), showed significantly higher activity than cisplatin towards ovarian carcinoma A2780, its cisplatin-resistant variant A2780R, osteosarcoma HOS, breast carcinoma MCF7 and cervix carcinoma HeLa cell lines, with the IC₅₀ values of 3.8, 3.5, 4.5, 2.7, and 9.2 μM, respectively, obtained for the most active complex 3. As for 4 and 5 having disubstituted 7-azaindoles in their molecule, the significant cytotoxicity was detected only for 4 against A2780 (IC₅₀ = 4.8 μM), A2780R (IC₅₀ = 3.8 μM) and HOS (IC₅₀ = 4.3 μM), while 5 was evaluated as having only moderate antiproliferative effect against the mentioned cancer cell lines with IC₅₀ = 33.4, 24.7 and 46.7 μM, respectively. All the studied complexes 1–5 effectively avoided the acquired resistance of ovarian carcinoma cell line. On the other hand, the complexes did not reveal any inhibition activity on the purified 20S proteasome from the A2780 cells. The representative complexes 3 and 5 showed low ability to be hydrolysed, but their stability was markedly lowered in the presence of physiological sulphur-containing biomolecule glutathione (GSH), as proved by the ¹H NMR spectroscopy and mass spectrometry studies. A rate of interaction of the studied complexes with GSH was affected by an addition of another mechanistically relevant biomolecule guanosine monophosphate. The differences in interactions of 3 and 5 with GSH correlate well with their different cytotoxicity profiles.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Organometallic Half-Sandwich Dichloridoruthenium(II) Complexes with 7-Azaindoles: Synthesis,Characterization and Elucidation of Their Anticancer Inactivity against A2780 Cell Line

A series of organometallic half-sandwich dichloridoruthenium(II) complexes of the general formula [Ru(η ⁶-p-cym)(naza)Cl₂] (1–8; p-cym = p-cymene; naza = 7-azaindole or its derivatives) was synthesised and fully characterized by elemental analysis, mass spectrometry, and infrared and multinuclear NMR spectroscopy. A single-crystal X-ray structural analysis of [Ru(η ⁶-p-cym)(2Me4Claza)Cl₂] (6) revealed a typical piano-stool geometry with an N7-coordination mode of 2-methyl-4-chloro-7-azaindole (2Me4Claza). The complexes have been found to be inactive against human ovarian cancer cell line A2780 up to the highest applied concentration (IC₅₀ > 50.0 μM). An inactivity of the complexes is caused by their instability in water-containing solvents connected with a release of the naza N-donor ligand, as proved by the detailed ¹H NMR, mass spectrometry and fluorescence experiments.     

New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC₅₀ = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC₅₀ values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC₅₀ < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC₅₀ = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.     

Natural inspired piperine-based ureas and amides as novel antitumor agents towards breast cancer

In this work, the natural piperine moiety was utilised to develop two sets of piperine-based amides (5a–i) and ureas (8a–y) as potential anticancer agents. The anticancer action was assessed against triple negative breast cancer (TNBC) MDA-MB-231, ovarian A2780CP and hepatocellular HepG2 cancer cell lines. In particular, 8q stood out as the most potent anti-proliferative analogue against TNBC MDA-MB-231 cells with IC₅₀ equals 18.7 µM, which is better than that of piperine (IC₅₀ = 47.8 µM) and 5-FU (IC₅₀ = 38.5 µM). Furthermore, 8q was investigated for its possible mechanism of action in MDA-MB-231 cells via Annexin V-FITC apoptosis assay and cell cycle analysis. Moreover, an in-silico analysis has proposed VEGFR-2 as a probable enzymatic target for piperine-based derivatives, and then has explored the binding interactions within VEGFR-2 active site (PDB:4ASD). Finally, an in vitro VEGFR-2 inhibition assay was performed to validate the in silico findings, where 8q showed good VEGFR-2 inhibitory activity with IC₅₀ = 231 nM.     

Cross Dimerization of Amyloid-β and αSynuclein Proteins in Aqueous Environment: A Molecular Dynamics Simulations Study

Self-assembly of the intrinsically unstructured proteins, amyloid beta (Aβ) and alpha synclein (αSyn), are associated with Alzheimer’s Disease, and Parkinson’s and Lewy Body Diseases, respectively. Importantly, pathological overlaps between these neurodegenerative diseases, and the possibilities of interactions between Aβ and αSyn in biological milieu emerge from several recent clinical reports and in vitro studies. Nevertheless, there are very few molecular level studies that have probed the nature of spontaneous interactions between these two sequentially dissimilar proteins and key characteristics of the resulting cross complexes. In this study, we have used atomistic molecular dynamics simulations to probe the possibility of cross dimerization between αSyn_1–95 and Aβ _1–42, and thereby gain insights into their plausible early assembly pathways in aqueous environment. Our analyses indicate a strong probability of association between the two sequences, with inter-protein attractive electrostatic interactions playing dominant roles. Principal component analysis revealed significant heterogeneity in the strength and nature of the associations in the key interaction modes. In most, the interactions of repeating Lys residues, mainly in the imperfect repeats ‘KTKEGV’ present in αSyn_1–95 were found to be essential for cross interactions and formation of inter-protein salt bridges. Additionally, a hydrophobicity driven interaction mode devoid of salt bridges, where the non-amyloid component (NAC) region of αSyn_1–95 came in contact with the hydrophobic core of Aβ _1–42 was observed. The existence of such hetero complexes, and therefore hetero assembly pathways may lead to polymorphic aggregates with variations in pathological attributes. Our results provide a perspective on development of therapeutic strategies for preventing pathogenic interactions between these proteins.     

Domain specific interaction in the XRCC1-DNA polymerase beta complex

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase β (β-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain–domain interaction in the XRCC1-β-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I–linker–BRCT-II C-terminal fragment and the linker–BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact β-Pol and the β-Pol 31 kDa domain. The XRCC1-NTD_1–183 (residues 1–183) was found to bind β-Pol, the β-Pol 31 kDa domain and the β-Pol C-terminal palm-thumb (residues 140–335), and the interaction was further localized to XRCC1-NTD_1–157 (residues 1–157). The XRCC1-NTD_1–183-β-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36–355 and residues 1–159, were found to interact with β-Pol, the β-Pol 31 kDa domain, and the β-Pol C-terminal thumb-only domain polypeptides expressed from the respective β-Pol constructs. Neither the XRCC1-NTD_1–159, nor the XRCC1_36–355 polypeptide was found to interact with a β-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75–212) showed no interaction with β-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD_1–183 was found to bind β-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K_d of 0.4–2.4 µM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD_1–159 and β-Pol that is of an affinity comparable to other binding interactions involving β-Pol.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.     

Structure–activity relationship,in vitro and in vivo evaluation of novel dienyl sulphonyl fluorides as selective BuChE inhibitors for the treatment of Alzheimer's disease

To discover novel scaffolds as leads against dementia, a series of δ-aryl-1,3-dienesulfonyl fluorides with α-halo, α-aryl and α-alkynyl were assayed for ChE inhibitory activity, in which compound A10 was identified as a selective BuChE inhibitor (IC₅₀ = 0.021 μM for eqBChE, 3.62 μM for hBuChE). SAR of BuChE inhibition showed: (i) o- > m- > p-; –OCH₃ > –CH₃ > –Cl (–Br) for δ-aryl; (ii) α-Br > α-Cl, α-I. Compound A10 exhibited neuroprotective, BBB penetration, mixed competitive inhibitory effect on BuChE (K_i = 29 nM), and benign neural and hepatic safety. Treatment with A10 could almost entirely recover the Aβ_1-42-induced cognitive dysfunction to the normal level, and the assessment of total amount of Aβ_1-42 confirmed its anti-amyloidogenic profile. Therefore, the potential BuChE inhibitor A10 is a promising effective lead for the treatment of AD.     

Immunotherapy of Tumors with α2-Macroglobulin-Antigen Complexes Pre-Formed In Vivo

The cell surface receptor CD91/LRP-1 binds to immunogenic heat shock proteins (HSP) and α₂M ligands to elicit T cell immune responses. In order to generate specific immune responses, the peptides chaperoned by HSPs or α₂M are cross-presented on MHC molecules to T cells. While the immunogenic HSPs naturally chaperone peptides within cells and can be purified as an intact HSP-peptide complex, the peptides have had to be complexed artificially to α₂M in previous studies. Here, we show that immunogenic α₂M-peptide complexes can be isolated from the blood of tumor-bearing mice without further experimental manipulation in vitro demonstrating the natural association of tumor antigens with α₂M. The naturally formed immunogenic α₂M-peptide complexes are effective in prophylaxis and therapy of cancer in mouse models. We investigate the mechanisms of cross-presentation of associated peptides and co-stimulation by APCs that interact with α₂M. These data have implications for vaccine design in immunotherapy of cancer and infectious disease.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ_25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ_25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ_25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ_25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ_25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ_25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ_25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ_25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

Development of naringenin-O-alkylamine derivatives as multifunctional agents for the treatment of Alzheimer’s disease

In this study, a series of naringenin-O-alkylamine derivatives were designed and obtained by introducing an alkylamine fragment into the naringenin skeleton. The in vitro biological activity results revealed that compounds 5f and 7k showed good antioxidant activity with ORAC values of 2.3eq and 1.2eq, respectively. Compounds 5f and 7k were reversible and excellent huAChE inhibitors with IC₅₀ values of 0.91 μM and 0.57 μM, respectively. Moreover, compounds 5f and 7k could inhibit self-induced Aβ_1–42 aggregation with 62.1% and 43.8% inhibition rate, respectively, and significantly inhibited huAChE-Aβ_1–40 aggregation with 51.7% and 43.4% inhibition rate, respectively. In addition, compounds 5f and 7k were selective metal chelators and remarkably inhibited Cu²⁺-induced Aβ_1–42 aggregation with 73.5% and 68.7% inhibition rates, respectively. Furthermore, compounds 5f and 7k could cross the blood-brain barrier in vitro and displayed good neuroprotective effects and anti-inflammatory properties. Further investigation showed that compound 5f did not show obvious hepatotoxicity and displayed a good hepatoprotective effect by its antioxidant activity. The in vivo study displayed that compound 5f significantly improved scopolamine-induced mice memory impairment. Therefore, compound 5f was a potential multifunctional candidate for the treatment of AD.     

Fighting experience alters brain androgen receptor expression dependent on testosterone status

Contest decisions are influenced by the outcomes of recent fights (winner–loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner–loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner–loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/β (ER_α/β), glucocorticoid (GR) and serotonin (5-HT_1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner–loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ER_α/β, 5-HT_1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly.