Design,synthesis and biological evaluation of 2-mercapto-3-phenethylquinazoline bearing anilide fragments as potential antitumor agents: Molecular docking study

A novel series of 2-(3-phenethyl-4(3H)quinazolin-2-ylthio)-N-substituted anilide and substituted phenyl 2-(3-phenethyl-4(3H) quinazolin-2-ylthio)acetate were designed, synthesized and evaluated for their in-vitro antitumor activity. Compound 15 possessed remarkable broad-spectrum antitumor activity which almost sevenfold more active than the known drug 5-FU with GI₅₀ values of 3.16 and 22.60 μM, respectively. Compound 15 exhibited remarkable growth inhibitory activity pattern against renal cancer (GI₅₀ = 1.77 μM), colon cancer (GI₅₀ = 2.02 μM), non-small cell lung cancer (GI₅₀ = 2.04 μM), breast cancer (GI₅₀ = 2.77 μM), ovarian cancer (GI₅₀ = 2.55 μM) and melanoma cancer (GI₅₀ = 3.30 μM). Docking study was performed for compound 15 into ATP binding site of EGFR-TK which showed similar binding mode to erlotinib.     

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F₂-isoprostanes (F₂-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F₄-NPs), phospholipase A₂ (PLA₂) and platelet activating factor–acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F₂-IsoPs, HETEs, 7β-and 27-hydroxycholesterol, 7-ketocholesterol, F₄-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA₂ and PAF-AH activities were lower, in PD patients compared to controls (p <  0.05). The levels of plasma F₂-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend <  0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r =  ?0.305, p =  0.023) and plasma total HETEs (r =  ?0.285, p =  0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

Selective inhibition of human acetylcholinesterase by xanthine derivatives: In vitro inhibition and molecular modeling investigations

The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀ = 6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀ = 7.25 μM) < pentoxifylline (hAChE IC₅₀ = 6.60 μM) ? propentofylline (hAChE IC₅₀ = 6.40 μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC₅₀ = 0.04 μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC₅₀ > 50 μM) relative to the reference agent donepezil (hBuChE IC₅₀ = 13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.     

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

BackgroundAnalysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.MethodsThe method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (¹³C₃-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n = 6), brain tumour (n = 2), leukaemia (n = 5), and Salla disease (n = 1).ResultsLimit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n = 10) and 10.4% (n = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n = 10) and 12.8% (n = 40) respectively. Tested patients showed values of TSA above established reference value.ConclusionThe validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

Cytotoxic glucosydic iridoids from Veronica americana

Fractionation guided by the cytotoxic activity of the methanolic extract of Veronica americana led to the isolation of two new iridoids identified as 4β-hydroxy-6-O-(p-hydroxybenzoyl)-tetrahydrolinaride (1) and 10-O-protocatechuyl-catalpol (2), together with four known aromatic acids, veratric acid (3) p-methoxybenzoic acid (4), p-hydroxybenzoic acid (5) and protocatechuic acid (6). The structure of these compounds was determined by spectroscopic analysis. Iridoid glycosides 1 and 2 showed selective cytotoxic activity against the human cancer cell lines HF-6 (IC₅₀ = 0.031 and 0.066 μM, respectively) and PC-3 (IC₅₀ = 0.721 and 0.801 μM, respectively), with less sensitivity in normal MRC-5 cells (IC₅₀ = 77.103 and 1451.562 μM, respectively). Compound 1 was 9.9 times more potent than camptothecin against HF-6 cell line, while compound 2 was 4.7 times, more potent, against the same cell line, than camptothecin. The found biological efficacy of 1 and 2 allow us to propose these compounds as candidates for the development of effective anticancer therapeutic agents.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.     

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at α4β21 nicotinic acetylcholine receptors, has moderate affinity (K_i = 5.46 μM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2–20 of lobeline were synthesized and evaluated for interaction with α4β21 and α71 neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at α71 nAChRs. Similar to lobeline (K_i = 4 nM), sulfonic acid esters had high affinity at α4β21 (K_i = 5–17 nM). Aromatic carboxylic acid ester analogs of lobeline (2–4) were 100–1000-fold less potent than lobeline at α4β21 nAChRs, whereas aliphatic carboxylic acid ester analogs were 10–100-fold less potent than lobeline at α4β21. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the ³⁶Rb⁺ efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC₅₀ values of 0.85 μM and 1.60 μM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13–45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K_i = 1.98–10.8 μM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at α4β21 nAChRs (K_i = 19.3 μM) and was equipotent with lobeline, at VMAT2 (K_i = 2.98 μM), exhibiting a 6.5-fold selectivity for VMAT2 over α4β2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.     

Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC₅₀ = 0.045 μM, HIV RT RNase H; 13 μM, HIV RT-polymerase; 24 μM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC₅₀ = 0.19 μM) with a modest window with respect to cytotoxicity (CC₅₀ = 3.3 μM).     

Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE₂ in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE₂ reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R¹ = H, R² = p-CH₃O) exhibited the most potent activity in cells (EC₅₀ = 0.02 μM) and minimal inhibition of COX-2 activity (3% at 5 μM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.     

Comparing anti-HIV,antibacterial, antifungal,micellar, and cytotoxic properties of tricarboxylato dendritic amphiphiles

Three series of homologous dendritic amphiphiles—RCONHC(CH₂CH₂COOH)₃, 1(n); ROCONHC(CH₂CH₂COOH)₃, 2(n); RNHCONHC(CH₂CH₂COOH)₃, 3(n), where R = n-C_nH_2n+1 and n = 13–22 carbon atoms—were assayed for their potential to serve as antimicrobial components in a topical vaginal formulation. Comparing epithelial cytotoxicities to the ability of these homologues to inhibit HIV, Neisseria gonorrhoeae, and Candida albicans provided a measure of their prophylactic/therapeutic potential. Measurements of the ability to inhibit Lactobacillus plantarum, a beneficial bacterium in the vagina, and critical micelle concentrations (CMCs), an indicator of the potential detergency of these amphiphiles, provided additional assessments of safety. Several amphiphiles from each homologous series had modest anti-HIV activity (EC₅₀ = 110–130 μM). Amphiphile 2(18) had the best anti-Neisseria activity (MIC = 65 μM), while 1(19) and 1(21) had MICs against C. albicans of 16 and 7.7 μM, respectively. Two measures of safety showed promise as all compounds had relatively low cytotoxic activity (EC₅₀ = 210–940 μM) against epithelial cells and low activity against L. plantarum, 1(n), 2(n), and 3(n) had MICs ? 490, 1300, and 940 μM, respectively. CMCs measured in aqueous triethanolamine and in aqueous potassium hydroxide showed linear dependences on chain length. As expected, the longest chain in each series had the lowest CMC—in triethanolamine: 1(21), 1500 μM; 2(22), 320 μM; 3(22), 340 μM, and in potassium hydroxide: 1(21), 130 μM; 3(22), 40 μM. The CMC in triethanolamine adjusted to pH 7.4 was 400 μM for 1(21) and 3900 μM for 3(16). The promising antifungal activity, low activity against L. plantarum, relatively high CMCs, and modest epithelial cytotoxicity in addition to their anti-Neisseria properties warrant further design studies with dendritic amphiphiles to improve their safety indices to produce suitable candidates for antimicrobial vaginal products.     

Synthesis and anticancer activity of sclerophytin-inspired hydroisobenzofurans

Three structurally related sets of hydroisobenzofuran analogs of sclerophytin A were prepared in three or four steps from (S)-(+)-carvone via an aldol-cycloaldol sequence. The most potent members of each set of analogs exhibited IC₅₀’s of 1–3 μM in growth inhibitory assays against KB3 cells. The NCI 60-cell line 5-dose assay for analog 6h revealed a GI₅₀ = 0.148 μM and LC₅₀ = 9.36 μM for the RPMI-8226 leukemia cell line, and a GI₅₀ = 0.552 μM and LC₅₀ = 26.8 μM for the HOP-92 non-small cell lung cancer cell line.     

Acylated iridoid glycosides and acylated rhamnopyranoses from Gmelina arborea flowers

Nine acylated iridoid glycosides (1–9), five acylated rhamnopyranoses (10–14) and verbascoside (15) were isolated from Gmelina arborea flowers, including 5 new compounds (1, 2, and 10–12). The cytoprotective activity of 11 selected compounds (1–8, 10, 11, and 15) against CCl₄-induced cytotoxicity on liver was determined. Compounds 1, 2, 4, 7, 8 and 15 displayed hepatoprotective activity. 6-O-α-l-(2″, 3″-di-O-trans-p-hydroxycinnamoyl)rhamnopyranosylcatalpol (2) exhibited the most potent cytoprotective effect with an EC₅₀ value of 42.5 μM (SI = 19.3) compared with biphenyldimethylesterate (DDB, EC₅₀ = 277.3 μM, SI = 9.8) and bicylo-ethanol (EC₅₀ = 279.2 μM, SI = 12.2). Among the acylated iridoid glycosides, the compounds (2 and 8) containing phenolic hydroxy groups were more active than were those lacking them.     

Relationship between zinc levels and plasma leptin in hemodialysis patients

Recent evidences suggested a possible relationship between zinc deficiency and leptin levels in pathogenesis of anorexia in chronic kidney disease. The present study addressed the relationship between zinc and leptin in hemodialysis (HD) patients.MethodsFifty HD patients (54.3 ± 12.7 years old, 62% men) were studied and compared to 21 healthy volunteers (50.7 ± 15.7 years old, 43% men). Biochemical data, serum zinc, plasma leptin, IL-6, TNF-α and C-Reactive Protein levels were determined. Anthropometric parameters, food intake and appetite score were also assessed.ResultsThe leptin levels were higher in HD patients (16.1 μg/mL (0.21–118.25) vs 6.0 μg/mL (0.50–23.10)) in healthy volunteers (p = 0.04), whereas serum zinc levels were lower (54.5 ± 16.3 μg/dL) compared to healthy volunteers (78.4 ± 9.4 μg/dL) (p = 0.0001). The plasma leptin was correlated negatively with plasma zinc (r = ?0.33; p = 0.007), energy (r = ?0.38; p = 0.002) and protein intake (r = ?0.34; p = 0.006) and, positively correlated with BMI (r = 0.54; p = 0.0001), % body fat (r = 0.70; p = 0.0001) and conicity index (r = 0.46; p = 0.001). Plasma zinc was associated with hemoglobin (r = 0.30; p = 0.04) and negatively associated with TNF-α (r = ?0.37; p = 0.002) and C-Reactive Protein (r = ?0.37; p = 0.004). There was no correlation among Zn, leptin and appetite score in these patients.ConclusionThis study showed that low plasma zinc levels are negatively associated with high leptin levels in HD patients.     

Regiospecific and conformationally restrained analogs of melphalan and dl-2-NAM-7 and their affinities for the large neutral amino acid transporter (system LAT1) of the blood–brain barrier

Regiospecific and conformationally restrained analogs of melphalan and dl-2-NAM-7 have been synthesized and their affinities for the large neutral amino acid transporter (LAT1) of the blood–brain barrier have been determined to assess their potential for accessing the CNS via facilitated transport. Several analogs had K_i values in the range 2.1–8.5 μM with greater affinities than that of either l-phenylalanine (K_i = 11 μM) or melphalan (K_i = 55 μM), but lower than dl-2-NAM-7 (K_i = 0.08 μM). The results indicate that regiospecific positioning of the mustard moiety on the aromatic ring in these analogs is very important for optimal affinity for the large neutral amino acid transporter, and that conformational restriction of the dl-2-NAM-7 molecule in benzonorbornane and indane analogs leads to 25- to 60-fold loss, respectively, in affinity.     

Inhibitory effect of novel tetrahydropyrimidine-2(1H)-thiones on melanogenesis

The series of imidazoldine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3 were discovered as inhibitor of α-MSH-induced melanin production in melanoma B16 cells. The primary bioassay showed that 1-(4-ethylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3e (>100% inhibition at 10 μM, IC₅₀ = 1.2 μM) and 1-(4-tert-butylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3f (>100% inhibition at 10 μM, IC₅₀ = 0.76 μM) exhibited potent inhibitory effect against α-MSH-induced melanin production. Compounds 3 inhibit the biosynthesis of tyrosinase without affecting its catalytic activity in melanogenesis.     

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2′-deoxycytidine for uridine and 2′-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn²⁺-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K_m = 1004 μM and k_cat = 4.8 s^?1 for cytidine, and K_m = 1059 μM and k_cat = 3.5 s^?1 for 2′-deoxycytidine. The pH dependence of k_cat and k_cat/K_M for cytidine indicate that protonation of a single ionizable group with apparent pK_a value of 4.3 abolishes activity, and protonation of a group with pK_a value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 Å resolution. Analysis of the crystallographic structure indicated the presence of a Zn²⁺ coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.     

Tyrosine phosphorylation of cytochrome c as a signaling event in frozen thawed buffalo spermatozoa at the cross-roads of capacitation and apoptosis

Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4 h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H₂O₂) and osmotic (180 mM NaCl) for 4 h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p < 0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.     

Relationships between the structure of 6-allyl-6,8-diazabicyclo[3.2.2]nonane derivatives and their σ receptor affinity and cytotoxic activity

A series of bridged piperazine derivatives was prepared and the affinity toward σ₁ and σ₂ receptors by means of radioligand binding assays as well as the inhibition of the growth of six human tumor cell lines was investigated. All possible stereoisomers of the 2-hydroxy, 2-methoxy, 2,2-dimethoxy, 2-oxo, and 2-unsubstituted 6,8-diazabicyclo[3.2.2]nonanes were prepared in a chiral pool synthesis starting with (S)- and (R)-glutamate. A Dieckmann analogous cyclization was the key step in the synthesis of the bicyclic framework. The configuration in position 2 was established by a diastereoselective LiBH₄ reduction and subsequent Mitsunobu inversion. Structure–affinity relationships demonstrate that substituents in position 2 decrease σ₁ receptor affinity which might be due to unfavorable interactions with the σ₁ receptor protein. Without a substituent in position 2 high σ₁ affinity was obtained (23a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane): K_i = 11 nM). Experiments with six human tumor cell lines showed a weak but selective growth inhibition of the human small cell lung cancer cell line A-427 by the methyl ethers ent-16b (IC₅₀ = 18.9 μM), 21a (IC₅₀ = 16.4 μM), ent-21a (IC₅₀ = 20.4 μM), and 21b (IC₅₀ = 27.1 μM) and the unsubstituted compounds 23a and 23b (42% inhibition at 20 μM).