The Lathyrus excitotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid is a substrate of the L-cystine/L-glutamate exchanger system xc-

Beta-N-oxalyl-L-alpha-beta-diaminopropionic acid (beta-L-ODAP) is an unusual amino acid present in seeds of plants from the Lathyrus genus that is generally accepted as the causative agent underlying the motor neuron degeneration and spastic paraparesis in human neurolathyrism. Much of the neuropathology produced by beta-L-ODAP appears to be a direct consequence of its structural similarities to the excitatory neurotransmitter L-glutamate and its ability to induce excitotoxicity as an agonist of non-NMDA receptors. Its actions within the CNS are, however, not limited to non-NMDA receptors, raising the likely possibility that the anatomical and cellular specificity of the neuronal damage observed in neurolathyrism may result from the cumulative activity of beta-L-ODAP at multiple sites. Accumulating evidence suggests that system xc-, a transporter that mediates the exchange of L-cystine and L-glutamate, is one such site. In the present work, two distinct approaches were used to define the interactions of beta-L-ODAP with system xc-: Traditional radiolabel-uptake assays were employed to quantify inhibitory activity, while fluorometrically coupled assays that follow the exchange-induced efflux of L-glutamate were used to assess substrate activity. In addition to confirming that beta-L-ODAP is an effective competitive inhibitor of system xc-, we report that the compound exhibits a substrate activity comparable to that of the endogenous substrate L-cystine. The ability of system xc- to transport and accumulate beta-L-ODAP identifies additional variables that could influence its toxicity within the CNS, including the ability to limit its access to EAA receptors by clearing the excitotoxin from the extracellular synaptic environment, as well as serving as a point of entry through which beta-L-ODAP could have increased access to intracellular targets.     

Characterisation of sevoflurane effects on spinal somato-motor nociceptive and non-nociceptive transmission in neonatal rat spinal cord: an electrophysiological study in vitro

Sevoflurane is the latest halogenated ether introduced in clinical anaesthesia, and its effects at the spinal level are not fully characterised. The rat hemisected spinal cord preparation was used to test the effects of sevoflurane on spinal nociceptive and non-nociceptive synaptic transmission as well as on excitations produced by application of glutamate-receptor agonists. Sevoflurane was dissolved in artificial cerebrospinal fluid (ACSF) with a specific vaporiser, and its final concentration was assessed with gas chromatography. Sevoflurane reduced the mono-synaptic reflex (EC(50) approximately 219 microM) and the slow components of the dorsal root-ventral root potentials (EC(50) approximately 72 microM) elicited by single dorsal root stimulation as well as the cumulative depolarisation (CD) elicited by repetitive stimulation (EC(50) approximately 98 microM). AMPA- and NMDA-induced depolarisations were also reduced by sevoflurane (respective EC(50)s were 206 and 127 microM). Inhibition of NMDA-induced depolarisation was TTX resistant. However, complete blockade of NMDA receptors with d-AP5 did not prevent further reduction of the CD by sevoflurane. All the effects reported were concentration-dependent and reversible.We conclude that sevoflurane applied at clinically relevant concentrations induces a strong depression of nociceptive and non-nociceptive spinal systems, which may be partly mediated by interfering with excitatory amino acid transmission.     

N-Methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4- isoxazoleproprionate (AMPA) glutamate-receptor antagonists have different interactions with the discriminative stimuli of abused drugs

The effects of the AMPA-receptor antagonists NBQX and GYKI 52466 were compared with those of the NMDA-receptor channel blocker dizocilpine in two drug discrimination tests. In the first, rats were trained to discriminate morphine (2 mg/kg) from saline and in the second, to discriminate ketamine (7 mg/kg) from saline, using a two-lever food reinforced method. NBQX (1–6 mg/kg) did not substitute for either morphine or ketamine, even at a dose which reduced response rates (6 mg/kg). Likewise, the non-competitive antagonist GYKI 52466 (5 and 10 mg/kg) produced only saline lever responding in the ketamine trained rats. When tested in combination with the training drug, NBQX (4.5 mg/kg) did not alter the morphine generalisation gradient, and similarly, neither NBQX (3 mg/kg) nor GYKI 52466 (5 and 10 mg/kg) interacted with the ketamine cue. In contrast, dizocilpine (0.05 mg/kg) significantly disrupted discrimination of morphine and produced clear drug lever responding (0.0125–0.1 mg/kg) in ketamine trained rats. These results suggest that AMPA-receptor antagonists and non-competitive NMDA-antagonists have different stimulus properties. Received: 24 May 1996/Final version: 28 July 1996     

Decreased sensory responsiveness of noradrenergic neurons in the rat locus coeruleus following phencyclidine or dizocilpine (MK-801): role of NMDA antagonism

The effects of the schizophrenomimetic compound phencyclidine (PCP) on baseline activity and sensory-evoked responses of noradrenergic locus coeruleus neurons were studied with extracellular single-cell recording techniques in the chloral hydrate-anaesthetized male albino rat. PCP dose-dependently decreased firing rate, induced a more regular firing pattern of the neurons, and decreased neuronal responses to a peripheral sensory stimulus (electrical stimulation of the hindpaw). These effects of PCP were significantly decreased by pretreatment with reserpine or yohimbine, indicating that the effects of PCP were largely indirect and mediated through noradrenaline, i.e. by inhibition of its re-uptake, resulting in stimulation of ₂ autoreceptors. The effects of PCP were, however, mimicked by dizocilpine (MK-801), a selective non-competitive antagonist at excitatory amino acid receptors of the N-methyl-d-aspartate (NMDA) subtype, suggesting a role also for NMDA receptors in the suppression of sensory responsiveness of locus coeruleus neurons by PCP. In view of the purported physiological role of the locus coeruleus, this effect of PCP may well contribute to the psychotomimetic properties of the drug.     

Improved synthesis and resolution of β-(3-pyridyl)-DL-α-alanine

β-Benzamido-α-(3-pyridyl)-DL-α-alanine hydrochloride was synthesized from 3-pyridinecarboxyaldehyde via the azlactone which was hydrolyzed to the acrylic acid before hydrogenation. The methyl ester was effectively resolved with subtilisin. The optical purity of the D-isomer was established, since the D-isomer was used in synthesis of antagonists of the luteinizing hormone releasing hormone.     

Synthesis and antileukemic activity of novel 4-(3-(piperidin-4-yl) propyl)piperidine derivatives

To explore the anticancer effect associated with the piperidine framework, several (substituted phenyl) {4-[3-(piperidin-4-yl)propyl]piperidin-1-yl} methanone derivatives 3(a-i) were synthesized. Variation in the functional group at N-terminal of the piperidine led to a set of compounds bearing amide moiety. Their chemical structures were confirmed by (1) H NMR, IR and mass spectra analysis. Among these, compounds 3a, 3d and 3e were endowed with antiproliferative activity. The most active compound among this series was 3a with nitro and fluoro substitution on the phenyl ring of aryl carboxamide moiety, which inhibited the growth of human leukemia cells (K562 and Reh) at low concentration. Comparison with other derivative (3h) results shown by LDH assay, cell cycle analysis and DNA fragmentation suggested that 3a is more potent to induce apoptosis.     

Cysteic- and homocysteic acid-S-(aminoiminomethyl) amides: a new class of modified arginines useful in peptide synthesis

A novel, practical synthesis of the title compounds and their derivatives are described. The protecting groups for amino, guanidino and carboxylic functions of substituted amides of cysteic and homocysteic acid were selected with the aim of making the amino acid derivatives synthons for peptide synthesis both in solution and by the solid phase method. Studying the structure–activity relationship some new kyotorphin, [Leu¹] kyotorphin and MIF-1 analogues, containing the unusual amino acid cysteic acid-S-(aminoiminomethyl) amide (sArg) in position two, have been prepared. It is a very promising compound, a structural analogue of arginine and an efficient antagonist in its metabolism.     

Towards eradicating antibiotic-resistant bacteria: synthesis and antibacterial activities of substituted N-(2-nitrophenyl)pyrrolidine- and piperidine-2-carboxylic acids

Antibacterial resistance is a source of great concern in the effective prevention and treatment of infections caused by bacteria, making the development of requisite therapeutics a major challenge. N-(Nitrophenyl)cycloamino acids are important compounds in the synthesis of poly-condensed nitrogen-containing heterocycles with marked activities in many biological systems. A series of substituted N-(o-nitrophenyl)cycloamino-2-carboxylic acids 3a–3g were synthesized via the condensation reaction of substituted o-halogenonitrobenzenes with L-proline 2a and D,L-pipecolinic acid 2b, under refluxing alcoholic basic conditions in excellent yields. The synthesized compounds were characterised by FT-IR, (¹H & ¹³C) NMR, UV-Vis, mass spectroscopy and elemental analysis. Their antibacterial activities were evaluated against five Gram-positive and five Gram-negative bacterial strains using the broth micro-dilution procedure. The antibacterial activities of the synthesized compounds were compared with streptomycin and nalidixic acid as standard antibiotic drugs. The minimum inhibitory concentration (MIC) values of compounds 3a–3g revealed good antibacterial activities against the tested microorganisms. Compounds 3a–3g were more potent than nalidixic acid against Enterococcus faecalis, Mycobacterium smegmatis, Escherichia coli and Proteus vulgaris and also more potent than streptomycin against Enterobacter cloacae and Staphylococcus aureus. Compounds 3a, 3c and 3g displayed the highest antibacterial potency with an MIC value of 15.6 μg/mL against E. cloacae, E. faecalis and P. mirabilis, respectively. These results indicated that these aryl cycloamino acids with antibacterial activities had potential applications as substitutes for antimicrobial peptide antibiotics, which are not susceptible to bacterial resistance, to solve the problem of drug resistance.     

1-Benzylcyclopropylamine and 1-(phenylcyclopropyl)methylamine: an inactivator and a substrate of monoamine oxidase

1-Benzylcyclopropylamine (1) and 1-(phenylcyclopropyl)methylamine (2), cyclopropane analogues of phenethylamine, were tested as inactivators for monoamine oxidase (MAO). Compound 1 is a potent competitive reversible inhibitor of the oxidation of benzylamine and also is a mechanism-based inactivator. It requires 2.3 equiv of 1 to inactivate 1 equiv of MAO. The excess equivalents of 1 are converted into benzyl vinyl ketone. A one-electron mechanism of inactivation is proposed. Compound 2 is a substrate for MAO and is converted into 1-phenylcyclopropanecarboxaldehyde without inactivation of the enzyme. Mechanistic consequences are discussed as a result of this observation.     

Synthesis of 3-(3-pyridyl)- and 3-(3-benzo[b]thienyl)-D-alanine

The DL-arylamino acid ethyl ester derivatives of β-(3-pyridyl)-DL-alanine, and β-(3-benzo[b]thienyl)-DL-alanine were synthesized by diethyl acetamidomalonate condensation with the respective arylmethyl halides followed by partial hydrolysis to the monoethyl ester and decarboxylation. Each derivative was enzymatically resolved to a separable mixture of the corresponding N-acetyl-L-amino acid and the unchanged D amino acid derivative. Acidic hydrolysis of the latter gave the corresponding D-amino acid, the optical purity of which was established by HPLC analysis of the 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) derivative. The free D amino acids were converted to D-BOC derivatives by reaction with di-tert-butyldicarbonate in tert-butyl alcohol, water and sodium hydroxide.     

1-(Azolyl)-4-(aryl)-phthalazines: novel potent inhibitors of VEGF receptors I and II

Novel potent derivatives of phthalazine are described as an adenosine triphosphate-competitive inhibitors of vascular endothelial growth factor receptors I and II. A number of compounds display vascular endothelial growth factor receptor II inhibitory activity reaching that of Vatalanib A (IC(50): < 50 nm) in an homogenous time-resolved fluorescence enzymatic assay.     

HMG-CoA Reductase inhibitors: an updated review of patents of novel compounds and formulations (2011-2015)

Introduction: Statins are remarkably safe and efficient medications that are the mainstay of hypercholesterolemia treatment and have proven to be an invaluable tool to lower the risk of acute cardiovascular events. These compounds are inhibitors of 3-hydroxy-methylglutaryl CoA reductase (HMG-R), the rate-limiting enzyme in cholesterol biosynthesis. In spite of their success, they present undesirable side effects and are now loosing patent protection, which provides a great opportunity for the development of new and improved statins.

Areas covered: This review summarizes the new patents for HMG-R inhibitors for the 2011–2015 period. Combinations of existing statins with other drugs are also addressed, as well as novel applications of existing statins.

Expert opinion: Recent efforts for the discovery of HMG-CoA-R inhibitors has resulted in several new molecules. Most of these are based on commercially available statins, including sterol and terpenoid derivatives. A few peptides have also been patented. However, the origin of the side effects caused by previous statins continues to be, to a large extent, unknown. Although the patents published in the past 5 years are promising, and might result in new drugs, there is still no way to know if they will present reduced toxicity. Only future clinical trials will answer this question.     

A novel class of apical sodium--dependent bile salt transporter inhibitors: 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides

The apical sodium--dependent bile acid transporter (ASBT) is the main transporter to promote re-absorption of bile acids from the intestinal tract into the enterohepatic circulation. Inhibition of ASBT could increase the excretion of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. Therefore, ASBT is an attractive target for developing new cholesterol-lowering drugs. In this report, a series of 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides were designed as inhibitors of ASBT. Most of them demonstrated potency against ASBT transport of bile acids. In particular, compound 4a₁ was found to have the best activity, resulting in 80.1% inhibition of ASBT at 10 μmol/L.     

Phencyclidine (PCP)-like discriminative stimulus effects of metaphit and of 2-amino-5-phosphonovalerate in pigeons: generality across different training doses of PCP

Pigeons were trained to discriminate either a fixed dose of PCP (1 mg/kg; n=3) or a progressively decreasing dose (1-0.56–0.32 mg/kg; n=4) from saline. Lowering of the training dose shifted the dose-effect curve for PCP's discriminative stimulus effects about 5-fold to the left, in a parallel manner, but did not decrease the accuracy of the discrimination performance and did not significantly increase the extent to which pentobarbital and chlordiazepoxide produced PCP-appropriate responding. Dose-effect curves based on binary generalization data were evaluated statistically with new methods that may be more appropriate than those used previously. Metaphit, a proposed PCP-receptor acylator, and 2-amino-5-phosphonovalerate (AP5), an N-methyl-d-aspartate (NMDA) antagonist, produced complete PCP-appropriate responding in the high training dose group only at doses that suppressed the rate of responding and that produced ataxia. However, 4-fold lower doses of metaphit and AP5, which did not produce directly observable behavioral effects, were found to substitute completely for PCP in the low training dose group. These data support the notion that PCP, metaphit, and AP5 have a common discriminative effect in pigeons.     

N-methyl-d-aspartate (NMDA) receptor-mediated stimulation of noradrenaline release,but not release of other neurotransmitters,in the rat brain cortex: receptor location,characterization and desensitization

Summary Rat brain cortex slices and synaptosomes (in a few experiments also hippocampal synaptosomes) preincubated with ³H-noradrenaline, ³H-5-hydroxytryptamine, ³H-choline, ³H-glutamate or ³H--aminobutyric acid were used to investigate the ³H-transmitter release in response to exposure to N-methyl-d-aspartate (NMDA) and other excitatory amino acids. The slices and synaptosomes were superfused with Mg²⁺-free, otherwise physiologically composed salt solution.In cortical slices preincubated with ³H-noradrenaline, NMDA concentration-dependently stimulated ³H overflow, whereas no such effect occurred in slices preincubated with ³H-5-hydroxytryptamine, ³H-choline, ³H-utamate or ³H--aminobutyric acid. In cortical slices preincubated with ³H-noradrenaline, the NMDA-evoked ³H overflow was abolished by tetrodotoxin, presence of Mg²⁺ 1.2 mmol/l or absence of Ca²⁺. 2-Amino-5-phosphonovaleric acid produced a parallel shift to the right of the NMDA concentration-response curve, whereas (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801) not only shifted the concentration-response curve to the right but also reduced the maximum effect of NMDA. Other excitatory amino acid receptor agonists also stimulated ³H overflow, yielding the following rank order of potency: NMDA > l-glutamate > l-aspartate. Kainate and, in particular, quisqualate exhibited only low potencies and/or intrinsic activities. Prolonged (25 min) exposure of ³HNA-preincubated cortical slices to a high NMDA concentration produced a short-lasting peak of ³H overflow, followed by a second phase lasting as long as the compound was present; in this phase, ³H overflow was clearly less pronounced and gradually decreased with time. The stimulatory effect of a high NMDA concentration was concentration-dependently reduced by 20 min of pre-superfusion with NMDA or l-glutamate at concentrations which by themselves produced either no or, at the most, moderate increase in ³H efflux in the two 5-min periods before application of the NMDA stimulus; in contrast, the veratridine-evoked ³H overflow was increased by pre-exposure to these NMDA concentrations. Neither in 3H-noradrenaline-preincubated synaptosomes prepared from the cortex or hippocampus nor in cortical synaptosomes preincubated with ³H-5-hy-droxytryptamine did NMDA evoke ³H overflow. The veratridine-evoked ³H overflow from ³H-noradrenaline-preincubated cortical synaptosomes was not affected by simultaneous administration of NMDA.It is concluded that NMDA selectively stimulates noradrenaline release in the rat brain cortex via NMDA receptors which appear not to be located on the noradrenergic nerve terminals. The NMDA receptor is rapidly desensitized in response to continuous application of NMDA (tachyphylaxis) or l-glutamate (cross-tachyphylaxis). Send offprint requests to M. Göthert at the above address     

Effects of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and chlorpromazine on NO3- transport via anion exchanger in erythrocytes: inertness of DIDS in whole blood

We examined the effects of chlorpromazine on NO(3)(-) transport between erythrocytes (RBCs) and extracellular fluid. Chlorpromazine (10 microg/ml) did not influence NO(3)(-) movement in both whole blood and RBC suspension. Though an anion exchanger (AE1) inhibitor DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 100 microM) did not alter NO(3)(-) movement in whole blood, it inhibited the movement in a concentration-dependent manner in the RBC suspension. The inhibition was abrogated by plasma and albumin concentration-dependently. Our results indicated that chlorpromazine had no effect on NO(3)(-) transport through AE1 and that the inertness of DIDS on AE1 in whole blood is due to interference by albumin in plasma.     

非核苷类逆转录酶抑制剂的研究XVⅢ: 4-烯丙基和4-叠氮基取代的三嗪类化合物的合成及抗HIV活性

在已有工作基础上, 基于分子对接设计合成了8个新的4-烯丙基取代和4-叠氮基取代的二芳基三嗪类衍生物。抗HIV-1活性的测试结果表明所有新化合物均具有抗HIV-1活性。其中化合物7c不仅抑制HIV-1野生株的复制 (IC₅₀ = 0.034 μmol×L^-1, SI = 6 475), 且对Y181C和K103C双突变酶显示出较强的抑制活性, 其IC₅₀值为9.39 μmol×L^-1, 高于奈韦拉平。     

Proprotein convertase subtilisin / kexin 9 (PCSK9) inhibitors and the future of dyslipidemia therapy: an updated patent review (2011-2015)

Introduction: The identification by Abifadel et al. in 2003 of the first mutations of PCSK9 was the major breakthrough in the cholesterol field that led to a new therapeutic target. This discovery paved the way to new lipid lowering drugs reducing LDL-cholesterol levels through the inhibition of PCSK9. Two anti-PCSK9 monoclonal antibodies have received FDA and EMA approvals: Alirocumab and Evolocumab.

Areas covered: This article reviews the different strategies that are pursued to modulate the functional activity of PCSK9 for lowering LDL-cholesterol levels. It also provides a brief overview of the patents related to PCSK9 from 2011 until the end of 2015. This review is addressed to researchers from academia and pharmaceutical companies who are engaged in PCSK9 research/cholesterol regulation. Readers will gain an up-to-date overview of the different strategies that have been investigated to reduce PCSK9, focusing on anti-PCSK9 monoclonal antibodies and the related clinical trials.

Expert opinion: Anti-PCSK9 antibodies are a new class of lipid lowering drugs with promising results in reducing LDL-cholesterol. Long-term ongoing studies investigating on a large scale the efficacy and safety of the anti-PCSK9 antibodies and their cardiovascular outcomes are eagerly awaited.     

Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1): restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF(3) cells overexpressing bcl(2)

4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted derivatives of alpha, beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1). Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K(i) approximately 280 microM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min preincubation. ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA. In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA. DIMATE was a 30-100-fold more effective growth inhibitor than ATE. Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM. Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition. No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1. It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties. The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased.