Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

The α‐adrenoceptor antagonist,zolertine, inhibits α1D‐ and α1A‐adrenoceptor‐mediated vasoconstriction in vitro

1 The antagonist effect of zolertine (4‐phenyl‐1‐[2‐(5‐tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (α1D‐adrenoceptors), mesenteric (α1A/D‐adrenoceptors) and caudal arteries (α1A‐adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (α1B‐adrenoceptors), was investigated in endothelium‐denuded arterial rings.
2 The selective α1D‐adrenoceptor agonist, noradrenaline, elicited concentration‐dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid=aorta>>.Gt;mesenteric=rabbit aorta>caudal arteries.
3 The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48±0.18; SHR, 7.43±0.13 and WKY, 7.57±0.24; SHR, 7.40±0.08, respectively. Zolertine was a non‐competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98±0.16; SHR, 6.81±0.18 in the mesenteric artery, WKY, 5.73±0.11; SHR, 5.87±0.25 in the caudal artery and 6.65±0.09 in rabbit aorta.
4 Competition binding experiments using the α1‐adrenoceptor antagonist [³H]prazosin showed a zolertine pKi of 6.81±0.02 in rat liver (α1B‐adrenoceptors) and 6.35±0.04 in rabbit liver (α1A‐adrenoceptors) membranes.
5 Zolertine showed higher affinity for α1D‐adrenoceptors compared to α1A‐adrenoceptors, while it had an intermediate affinity for α1B‐adrenoceptors. The ability of the α1‐adrenoceptor antagonist zolertine to block α1D‐adrenoceptor‐mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency.     

Chronic Clorgyline Induces Selective Down‐Regulation of α2‐Adrenoceptor Agonist Binding Sites in Rat Brain

Inactivation of α₂‐adrenoceptors by N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) has been shown to induce an increase in brain regulatory Gα_i1/2 proteins, which was related to biphasic recovery of agonist binding sites. To investigate further the nature of this phenomenon, the chronic effects of clorgyline (a monoamine oxidase inhibitor antidepressant) on the recovery of α₂‐adrenoceptors after EEDQ and on EEDQ‐induced up‐regulation of Gα_i1/2 proteins were assessed in rat brain. Clorgyline (1 mg kg^?1 for 7–35 days) induced a time‐dependent down‐regulation (20% to 55%) of the density of cortical α₂‐adrenoceptor agonist sites ([³H]UK 14304/bromoxidine binding) but not of antagonist sites ([³H]RX 821002/2‐methoxy idazoxan binding). However, chronic clorgyline did not alter the immunoreactive levels of Gα_i1/2, Gα_i3, and Gα_o proteins in cortex. In clorgyline‐treated rats, the turnover functions for agonist and antagonist binding sites (receptor recovery after EEDQ) were different and indicated that the reduced density of α₂‐adrenoceptor agonist sites induced by clorgyline was due to a greater rate of receptor disappearance. The recovery of [³H]UK 14304 binding in clorgyline‐treated rats did not fit a biphasic recovery model and the turnover parameters were very similar to those obtained for the second phase of recovery (biphasic model) of agonist binding sites in naive rats. This suggested that clorgyline down‐regulated only the α₂‐adrenoceptors of rapid turnover which is associated with the increases in the expression of Gα_i1/2 proteins induced by EEDQ. In this context, clorgyline (1 mg kg^?1 for 7 days) fully prevented the up‐regulation (50%) of brain Gα_i1/2 proteins induced by EEDQ. The results indicate that one relevant mechanism involved in the in vivo desensitization of brain α₂‐adrenoceptors is an effective impairment of receptor‐G protein coupling.     

Microwave‐accelerated synthesis of psychoactive deuterated N,N‐dialkylated‐[α,α,β,β‐d4]‐tryptamines

A large number of N,N‐dialkylated tryptamines are known to induce psychoactive effects in humans. This has resulted in their increased attention within clinical and forensic communities. Deuterated tryptamines are ideal for use as internal standards during MS bioanalysis or of use in biochemical NMR studies. The present study reports on a microwave‐enhanced synthesis of 22 N,N‐dialkylated‐[α,α,β,β‐d4]‐tryptamines via the reduction with lithium aluminium deuteride of glyoxalylamide precursors obtained by the procedure of Speeter and Anthony. Syntheses were carried out using a single‐mode system under elevated pressure conditions where anhydrous tetrahydrofuran was used as the solvent at 150°C. Good yields were obtained within 5 min. Copyright © 2008 John Wiley & Sons, Ltd.     

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine Pretreatment Attenuates Methamphetamine‐Induced Dopamine Toxicity

Abstract: The effects of pretreatment with MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) on the acute and long‐term effects of methamphetamine on striatal dopamine were evaluated in BALB/c mice. Four subcutaneous injections of a non‐toxic dose of MPTP (8 mg/kg, at 2 hr intervals) were followed three days later by a toxic regimen of methamphetamine (four injections of 4 mg/kg, at 2 hr intervals) and mice were sacrificed immediately or three days later. Control mice received saline in place of the MPTP or methamphetamine and mice were observed for acute changes in body temperature, self‐injurious behaviour, and striatal dopamine metabolites, or long‐term changes in striatal dopamine levels, tyrosine hydroxylase immunoreactivity and glial fibrillary acidic protein. It was observed that pretreatment with MPTP protected mice against the acute increase in body temperature caused by the methamphetamine but, at the same time, delayed the occurrence of self‐injurious behaviour following the repeated injections of methamphetamine. Likewise, pretreatment with MPTP attenuated the long‐term depletion of striatal dopamine induced by the methamphetamine as well as the large increase in glial fibrillary acidic protein and the reduction in tyrosine hydroxylase immunoreactivity. The MPTP‐treatment itself did not alter any of these neurotoxic markers. Finally, the acute decrease in 3,4‐dihydroxyphenyacetic acid levels and increased ratio of 3‐methoxytyramine/dopamine observed 60 min. after a single injection of methamphetamine (4 mg/kg) were also attenuated in MPTP‐treated mice. These results are discussed in the context of the hypothesis that the low‐dose treatment with MPTP may modify exchange diffusion across the striatal cell membrane thereby altering the acute and long‐lasting effects of methamphetamine.     

A simple and efficient synthesis of [3α‐3H]5α‐androst‐16‐en‐3β‐ol

An efficient one step synthesis of [3α‐³H]5α‐androst‐16‐en‐3β‐ol by NaBT₄ reduction of a ketone precursor is described. The specific activity of the product was 21.6 Ci/mmol with a radiochemical purity >99%. Synthesis of the precursor, 5α‐androst‐16‐en‐3‐one, from commercially available 5α‐androst‐16‐en‐3α‐ol is also presented. Copyright © 2006 John Wiley & Sons, Ltd.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Influence of α‐Adrenoceptor Agonists and Antagonists on Imipramine‐Induced Hypothermia in Mice

Abstract: Effects of adrenoceptor agonists and antagonists on imipramine‐induced hypothermia in mice were studied. Intraperitoneal injection of imipramine (10–40 mg kg^?1), α₂‐adrenoceptor agonist clonidine (0.05–0.1 mg kg^?1), α₁‐adrenoceptor agonist phenylephrine (6 mg kg^?1) and α₁‐adrenoceptor antagonist prazosin (1–4 mg kg^?1) but not α₂‐adrenoceptor antagonist yohimbine (1–4 mg kg^?1) induced significant hypothermia. The hypothermic response induced by imipramine (10–30 mg kg^?1) was not altered by clonidine (0.05–0.1 mg kg^?1) or phenylephrine (2–6 mg kg^?1). The response of imipramine (10–30 mg kg^?1) was reduced significantly by yohimbine (2 mg kg^?1) and was potentiated by prazosin (1 mg kg^?1). The hypothermic effect of clonidine (0.1 mg kg^?1) and imipramine (20 mg kg^?1) were also decreased significantly by different doses of yohimbine (1–4 mg kg^?1). The hypothermia induced by different doses of prazosin (1–4 mg kg^?1) was not altered by yohimbine (2 mg kg^?1) or by low dose of imipramine (10 mg kg^?1). It is concluded that α₂‐adrenoceptor mechanism may be involved in the hypothermic effect of imipramine.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Peroxisome proliferator‐activated receptor‐γ coactivator‐1α in muscle links metabolism to inflammation

1. In higher eukaryotes, metabolism and immunity are tightly coupled. However, whereas in evolutionary terms a compromised immune response due to undernourishment has been the predominant problem, the inflammatory response to obesity and other lifestyle‐associated diseases has increased in relevance in Western societies in the past 100 years. 2. Traditionally, fat tissue has been considered as the major source of pro‐inflammatory secreted factors in these pathologies. However, in recent years the contribution of other tissues to disease‐causing chronic inflammation has been increasingly appreciated. 3. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) is one of the key regulatory factors in active skeletal muscle. Aberrant expression of PGC‐1α in inactive muscle fibres could be linked to a sedentary lifestyle, persistent systemic inflammation and a higher risk for many chronic diseases. Accordingly, modulation of PGC‐1α activity in skeletal muscle may have a broad range of therapeutic effects. Here, recent advances in the understanding of the role of muscle PGC‐1α in health and disease are reviewed.     

α1‐ and α2‐Adrenoceptor hyporesponsiveness in isolated bisected vas deferens of bile duct‐ligated rats

1 It has been suggested that cholestasis accompanied with changes in autonomic balance and hyporesponsiveness in muscarinic and adrenergic receptors of some organs, e.g. cardiovascular system. Increased plasma levels of epinephrine and norepinephrine has been shown during cholestasis suggesting augmented activity of sympathetic nervous system. In this study we evaluate both α₁ and α₂ responsiveness in isolated rat vas deferens, as a tissue with rich adrenergic innervations. 2 Epididymal and prostatic halves of vas deferens responsiveness have been studied to phenylephrine and clonidine respectively in three groups of un‐operated, sham‐operated (sham), and bile duct‐ligated (BDL) rats. 3 Our results indicate that in vas deferens of BDL animals, the concentration‐response curve of both phenylephrine and clonidine shifted to rightward compared to control group, while the position of concentration‐response curve of sham group did not change significantly (P > 0.05). EC₅₀ of phenylephrine and IC₅₀ of clonidine were increased showing a decreased responsiveness of tissue to phenylephrine (P < 0.05) and clonidine (P < 0.001) in BDL rats. 4 In this study, both subtype of α‐adrenoceptors (α₁ and α₂) has been studied in cholestatic rat vas deference. Our results showed that cholestasis induce hyporesponsiveness to phenylephrine and clonidine. These results are consistent with previous reports, suggesting the hyporesponsiveness of α₁‐adrenoceptors in pulmonary artery and papillary muscle and mesenteric beds. Our conclusion is that the cholestasis induces hyporesponsiveness to phenylephrine and clonidine in epididymal (α₁‐adrenoceptors) and prostatic (α₂‐adrenoceptors) halves of rat vas deferens respectively. Although the logical explanation to this hyporesponsiveness is the down regulation but it has been suggested that it is not because of down regulation.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT‐IR absorption, ¹H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

RS‐4252 Inhibits Amyloid β‐Induced Cytotoxicity in HeLa Cells

Abstract: Progressive deposition of amyloid β peptide in the senile plaques is a principal event in the neurodegenerative process of Alzheimer's disease. Several reports have demonstrated that amyloid β is cytotoxic using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) as an indicator of viability in cells. With the MTT assay, we screened an in‐house library to find compounds which suppress amyloid β‐induced inhibition of MTT reduction. We have previously reported that 6‐ethyl‐N,N'‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine (named RS‐0466), found in an in‐house library, was capable of significantly inhibiting amyloid β‐induced cytotoxicity in HeLa cells. From further screening hits, we newly focused on 4‐(7‐hydroxy‐2,2,4‐trimethyl‐chroman‐4‐yl)benzene‐1,3‐diol (named RS‐4252), which show comparable potency to RS‐0466 to ameliorate amyloid β‐induced cytotoxicity. Furthermore, RS‐4252 reversed the decrease in phosphorylated Akt by amyloid β. These results imply that RS‐4252 or one of its derivatives has the potential to be a therapeutic for Alzheimer's disease patients, and that activation of Akt is at least in part involved in the effect.     

Ambroxol Inhibits Peroxynitrite‐Induced Damage of α1‐Antiproteinase and Free Radical Production in Activated Phagocytic Cells

Abstract: The present study examined the effect of ambroxol on toxic action of peroxynitrite and the respiratory burst in activated phagocytic cells. Ambroxol decreased the inactivation or destruction of α₁‐antiproteinase induced by peroxynitrite (ONOO^?) or hypochlorous acid (HOCl), which was similar to penicillamine and glutathione and was greater than diclofenac sodium and naproxen sodium. Ambroxol significantly decreased ONOO^?–mediated tyrosine nitration and iron plus EDTA‐mediated degradation of 2‐deoxy‐D‐ribose. Ambroxol significantly attenuated the production of superoxide, hydrogen peroxide, HOCl, and nitric oxide in fMLP– or IL‐1‐activated phagocytic cells, while the inhibitory effects of antiinflammatory and thiol compounds were only observed in HOCl production. Ambroxol and antiinflammatory drugs did not show a cytotoxic effect on macrophages. The results suggest that ambroxol protects tissue components against oxidative damage by an action different from antiinflammatory drugs. Ambroxol may interfere with oxidative damage of α₁‐antiproteinase through a scavenging action on ONOO^? and HOCl and inhibition of the respiratory burst of phagocytic cells.     

Preparation of high specific activity tritium labeled 6α,9,‐difluoro‐11β,21‐dihydroxy‐16α,17‐[(1‐methylethylidene)bis(oxy)]pregna‐1,4‐diene‐3,20‐one,fluocinolone acetonide

Fluocinolone acetonide was tritiated by selective reduction of the 1,2‐double bond of the O‐protected analog under tritium, followed by re‐establishment of the 1,2‐double bond and deprotection. Protection of both hydroxyl functionalities was required. The product was obtained with specific activity 36.8 Ci/mmol. Copyright © 2009 John Wiley & Sons, Ltd.