Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification

The present study is the first report demonstrating that ionic liquids consisting of cholinium cations and linear carboxylate anions ([Ch][CA] ILs) can be used for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Six variants of [Ch][CA] ILs were systematically prepared by combining cholinium cations with linear monocarboxylate anions ([C_nH_2n+1–COO]⁻, n = 0–2) or dicarboxylate anions ([HOOC–C_nH_2n+1–COO]⁻, n = 0–2). These [Ch][CA] ILs were analyzed for their toxicity to yeast cell growth and their ability to pretreat kenaf powder for subsequent enzymatic saccharification. When assayed against yeast growth, the EC₅₀ for choline acetate ([Ch][OAc]) was 510 mM, almost one order of magnitude higher than that for 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). The cellulose saccharification ratio after pretreatment at 110 °C for 16 h with [Ch][OAc] (100.6%) was almost comparable with that after pretreatment with [Emim][OAc]. Therefore, [Ch][OAc] is a biocompatible alternative to [Emim][OAc] for lignocellulosic material pretreatment.     

Improving the catalytic performance of porcine pancreatic lipase in the presence of [MMIm][MeSO4] with the modification of functional ionic liquids

Porcine pancreatic lipase (PPL) was chemically modified with various functional ionic liquids (ILs) to increase its catalytic performance in water-miscible IL. Catalytic activity and thermostability were tested with a p-nitrophenyl palmitate (pNPP) hydrolysis reaction. The native enzyme lost 18% of its initial activity in 0.4 M [MMIm][MeSO₄], whereas the activities of all the modified enzymes increased. The [HOOCBMIm][Cl] modification led to a 2-fold increase in activity in 0.3 M [MMIm][MeSO₄] than in aqueous. All the modified enzymes exhibited higher thermostability compared with the native enzyme at high temperature. In particular, the [HOOCBMIm][Cl] modification led to a 6-fold increase in thermostability at 60 °C. Conformational changes were confirmed by fluorescence spectroscopy and circular dichroism spectroscopy to elucidate the mechanism of catalytic performance alteration.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

Radiosynthesis and in vivo evaluation of [11C]MC80 for P-glycoprotein imaging

P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. The in vitro characterized modulator 6,7-dimethoxy-2-(6-methoxy-naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (MC80) of the P-gp pump was labelled with ¹¹C and evaluated in vivo for its potential to image P-gp function and expression. Radiochemical pure (>98%) [¹¹C]MC80 was obtained within 25 min starting from [¹¹C]methyl iodide with radiochemical yield of 26%. Biodistribution studies in FVB mice demonstrated a high baseline brain uptake (7.66 ± 1.38%ID/g at 1 min pi). Cerebral uptake was increased in mdr1a knock-out mice as well as after CsA pretreatment. Pre-administration of an excess of non-radioactive MC80 caused a reduced uptake in several target organs including brain, pancreas and intestines. The results indicate that [¹¹C]MC80 kinetics are modulated by P-gp. Reversed phase-HPLC analysis of brain revealed an excellent metabolic profile (>90% intact [¹¹C]MC80).     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

Design,modification and 3D QSAR studies of novel 2,3-dihydrobenzo[b][1,4]dioxin-containing 4,5-dihydro-1H-pyrazole derivatives as inhibitors of B-Raf kinase

Two series of novel 2,3-dihydrobenzo[b][1,4]dioxin-containing 4,5-dihydro-1H-pyrazole derivatives C1–C15 and D1–D15 have been synthesized and evaluated for their B-Raf inhibitory and anti-proliferation activities. Compound C14 ((3-(4-bromophenyl)-5-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanone) showed the most potent biological activity against B-Raf^V600E (IC₅₀ = 0.11 μM) and WM266.4 human melanoma cell line (GI₅₀ = 0.58 μM), being comparable with the positive control Erlotinib and more potent than our previous best compound, while D10 ((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)(5-(3-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone) performed the best in the D series (IC₅₀ = 1.70 μM; GI₅₀ = 1.45 μM). The docking simulation was performed to analyze the probable binding models and poses and the QSAR model was built for reasonable design of B-Raf inhibitors in future. The introduction of 2,3-dihydrobenzo[b][1,4]dioxin structure reinforced the combination of our compounds and the receptor, resulting in progress of bioactivity.     

Diverse magnetic and electrical properties of molecular solids containing the thiazyl radical BDTA

We have examined the crystal structures and the electrical and magnetic properties of the molecular compounds of a thiazyl radical, 1,3,2-benzodithiazolyl (abbreviated as BDTA). BDTA was found to be a useful building block for molecular conductors and magnets because it can operate as a counter cation, a donor or a ligand, depending on its charge. (i) A charge-transfer complex, [BDTA][TCNQ], crystallizes into a uniform segregated stacking structure with a short contact between the donor and acceptor columns. In spite of the partial charge transfer between the two components, this complex exhibits semiconductive behaviour, probably due to a large electron correlation on BDTA. (ii) The crystal structure of [BDTA][Ni(mnt)₂] (mnt = maleonitriledithiolate) consists of alternating stacking columns of S = 0 [BDTA]⁺ and S = 1/2 [Ni(mnt)₂]^?, in which a ferromagnetic coupling operates between the [Ni(mnt)₂]^? anions through the [BDTA] ⁺ cation. (iii) [BDTA]₂[Cu(mnt)₂] consists of an alternating stack of a head-to-head [BDTA]⁺ dimer and a planar [Cu(mnt)₂]^2? dianion. Short intermolecular S?S contacts between the stacks give rise to an ideal 1D Heisenberg antiferromagnetic chain of [Cu(mnt)₂]^2? with a coupling constant of J/k_B = 16–17 K. (iv) The crystal structure of [BDTA]₂[Co(mnt)₂] is similar to that of [BDTA]₂[Cu(mnt)₂] at 253 K, but this salt undergoes a phase transition at 190 K, below which a partial electron transfer occurs from [Co(mnt)₂]^2? to one of the [BDTA]⁺ cations along with formation of a coordination bond. (v) [BDTA][Ni(dmit)₂]₂ (dmit = 1,3-dithiol-2-thione-4,5-dithiolate) exhibits room-temperature conductivity of 0.1 S cm^?1 and semiconductive behaviour over the range 80–200 K, which can be interpreted in terms of multi-conducting bands.     

Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8 h) on survival before freezing at 4 °C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates = 6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (−2.2 °C/min) or MC (−0.3 °C/min) from 37 °C to 4 °C in separate aliquots and further equilibrated at 4 °C for 8 h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8 h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4 °C, of motility and PMI was observed 5 and 6 h respectively in RC group while >8 h in MC group. Rate of decline (slope) in motility and viability was higher (P < 0.05) in RC overtime during equilibration at 4 °C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P < 0.05) in MC when equilibrated for 2–8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2–8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.     

Isoamyl acetate synthesis in imidazolium-based ionic liquids using packed bed enzyme microreactor

The acylation of isoamyl alcohol with acetic anhydride catalyzed by immobilized Candida antarctica lipase B was studied in ionic liquids (ILs) based on quaternary imidazolium cations with alkyl, alkenyl, alkynyl, benzyl, alkoxyl or N-aminopropyl side chains. Among the tested ILs, the highest enzyme activity together with the highest isoamyl acetate yield were obtained in [C₇mmim][Tf₂N]. No loss of lipase B activity was observed during one-month incubation in this hydrophobic IL without the presence of substrates. Isoamyl acetate synthesis using [C₇mmim][Tf₂N] as solvent was further studied in a continuously operated miniaturized enzymatic packed bed reactor at various flow rates and temperatures. Up to 92% isoamyl acetate yield could be obtained within 15 min by using 0.5 M acetic anhydride and 1.5 M isoamyl alcohol inlet concentrations at 55 °C, corresponding to the volumetric productivity of 61 mmol l^?1 min^?1, which to the best of our knowledge is the highest reported so far for this reaction. No decrease in productivity was experienced during the subsequent runs of continuous microbioreactor operation performed within 14 consecutive days. The benefits of reactor miniaturization along with the green solvent application were therefore successfully exploited for the development of a sustainable flavour ester production.     

A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557

A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 μg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC₅₀ = 16.7 ± 0.34 μg/ml). This was the first report of spatozoate isolation from bacterial sources.     

Design,synthesis and biological evaluation of N-arylphenyl-2,2-dichloroacetamide analogues as anti-cancer agents

Our earlier research has shown that N-phenyl-2,2-dichloroacetamide analogues had much higher anti-cancer activity than the lead compound sodium dichloroacetate (DCA). In this current study, a variety of N-arylphenyl-2,2-dichloroacetamide analogues were synthesized via Suzuki coupling reaction and their anti-cancer activity was evaluated. The results showed that N-terphenyl-2,2-dichloroacetamide analogues had satisfactory anti-cancer activity. Among them, N-(3,5-bis(benzo[d][1,3]dioxol-5-yl)phenyl)-2,2-dichloroacetamide (6 k) had an IC₅₀ of 2.40 μM against KB-3-1 cells, 1.04 μM against H460 cells and 1.73 μM against A549 cells.     

Use of response surface methodology for the optimization of the lipase-catalyzed synthesis of mannosyl myristate in pure ionic liquid

The purpose of the present study is to find the conditions allowing to reach the highest 24 h-yield (24 h-η) for the synthesis of mannosyl myristate catalyzed by the immobilized lipase B from Candida antarctica (Novozym^® 435) in the ionic liquid (IL) [Bmpyrr][TFO] (1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate). A full factorial design (FFD) was used in order to study the influence of three variables (temperature, mannose/vinyl myristate ratio and total substrate quantity) on the 24 h-η. This design led to a model based on a second order polynomial response function. The resulting predicted contour plots have shown that the highest 24 h-η should be obtained with high temperatures, low sugar/vinyl ester molar ratio and intermediate total substrate quantities (mmol). The model has been successfully verified and experimentally confirmed at the optimal conditions of 80 °C, substrate molar ratio of 1/10 and total substrate quantity of 0.26 mmol leading to the highest predicted 24 h-η of 72.2%.     

N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide: A promising positron emission tomography ligand for fatty acid amide hydrolase

To visualize fatty acid amide hydrolase (FAAH) in brain in vivo, we developed a novel positron emission tomography (PET) ligand N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[¹¹C]methylphenyl)thiazol-2-yl]-1-carboxamide ([¹¹C]DFMC, [¹¹C]1). DFMC (1) was shown to have high binding affinity (IC₅₀: 6.1 nM) for FAAH. [¹¹C]1 was synthesized by C–¹¹C coupling reaction of arylboronic ester 2 with [¹¹C]methyl iodide in the presence of Pd catalyst. At the end of synthesis, [¹¹C]1 was obtained with a radiochemical yield of 20 ± 10% (based on [¹¹C]CO₂, decay-corrected, n = 5) and specific activity of 48–166 GBq/μmol. After the injection of [¹¹C]1 in mice, high uptake of radioactivity (>2% ID/g) was distributed in the lung, liver, kidney, and brain, organs with high FAAH expression. PET images of rat brains for [¹¹C]1 revealed high uptakes in the cerebellar nucleus (SUV = 2.4) and frontal cortex (SUV = 2.0), two known brain regions with high FAAH expression. Pretreatment with the FAAH-selective inhibitor URB597 reduced the brain uptake. Higher than 90% of the total radioactivity in the rat brain was irreversible at 30 min after the radioligand injection. The present results indicate that [¹¹C]1 is a promising PET ligand for imaging of FAAH in living brain.     

Catalytic performance of cross-linked enzyme aggregates of Penicillium expansum lipase and their use as catalyst for biodiesel production

Cross-linked enzyme aggregates (CLEAs) of lipase from Penicillium expansum (PEL) were prepared directly from fermentation broth, a more practical and economically viable procedure than the generally used methods that require purified or partially purified enzymes for CLEA preparation. A systematic study of the activity and stability of PEL-CLEAs was undertaken in aqueous solution, organic solvents, and ionic liquids (ILs). Immobilization of the enzyme resulted in a significantly enhanced stability in aqueous solution with regard to pH and temperature. PEL-CLEAs showed an improved activity in the IL [BMIm][PF₆] relative to that observed in hexane, both keeping increased with temperature (up to 90 °C in the IL and 60 °C in hexane). The effect of water content and water activity in these two nonaqueous media showed similar patterns as for the uncrosslinked enzyme. The half life of the CLEAs was higher in hydrophobic organic solvents (hexane and chloroform) than in aqueous solution, and presented a sigmoid relationship with the log P of the organic solvent tested. PEL-CLEAs catalyzed biodiesel production from microalgal oil in the IL [BMIm][PF₆] with a conversion of 85.7%, demonstrating that they can be taken as a promising catalyst for this application.     

Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

Differentiated influence of off-road and on-road cycling practice on balance control and the related-neurosensory organization

This study aimed to determine the sensorimotor strategies privileged by mountain bikers (MTB) and road cyclists (RC) for balance control. Twenty-four MTB and 24 RC (off-road Olympics, world, continental and national champions, Tour-de-France participants, on-road world cup race winner) volunteered to answer a questionnaire about the characteristics of cycling practice and perform a sensory organization test, aiming to evaluate balance control in 6 different sensory situations based upon visual and support surface perturbations (C1^ES to C6^ES). RC balance performances were better than those of MTB both during quiet stance eyes opened (C1^ES, p = 0.011) and when only somatosensory information is disrupted (C4^ES, p = 0.039), highlighting a higher use of vision to control balance in RC. Moreover, a positive correlation was shown in the whole population (MTB + RC) between the visual ratio (R^VIS = C4^ES/C1^ES) and the proportion of riding distance of on-road cycling (ρ = 0.28, p = 0.054). In MTB, the use of proprioception (somatosensory ratio: R^SOM = C2^{ES(eyes closed)}/C1^ES) was increased by a higher intensity of off-road cycling (ρ = 0.49, p = 0.018) and that of vision (R^VIS) by a higher intensity of on-road cycling (ρ = 0.41, p = 0.048). The difference in sensory organization between MTB and RC could be explained by adaptive processes elaborated from environmental stimulations and technical specificities of these disciplines.     

Synthesis and evaluation of 1-[2-(4-[11C]methoxyphenyl)phenyl]piperazine for imaging of the serotonin 5-HT7 receptor in the rat brain

1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT₇ receptor antagonist (K_i = 2.6 nM) with a low binding affinity for the 5-HT_1A receptor (K_i = 476 nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT₇ receptor, [¹¹C]4 was synthesized at high radiochemical yield and specific activity, by O-[¹¹C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [¹¹C]methyl iodide. Autoradiography revealed that [¹¹C]4 showed in vitro specific binding with 5-HT₇ in the rat brain regions, such as the thalamus which is a region with high 5-HT₇ expression. Metabolite analysis indicated that intact [¹¹C]4 in the brain exceeded 90% of the radioactive components at 15 min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [¹¹C]4 in the brain (1.2 SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT₇-selective antagonist SB269970 (3) did not decrease the uptake of [¹¹C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT₇ and higher in vivo stability in brain than 4.     

Coumarins from the roots of Prangos uloptera

Three new coumarins, 6-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-prenyletin, 3″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-oxypeucedanin hydrate and 2″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-oxypeucedanin hydrate, together with six known coumarins, 3″-O-[β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranosyl]-heraclenol, 3″-O-(β-d-glucopyranosyl)-heraclenol, tortuoside, 3″-O-(β-d-glucopyranosyl)-oxypeucedanin hydrate, heraclenol and oxypeucedanin hydrate, have been isolated from the roots of Prangos uloptera, and the structures of these coumarins were unequivocally determined by spectroscopic means, notably UV, HRESIMS, and 1D and 2D NMR spectroscopy.     

Radiosynthesis of N-(4-chloro-3-[11C]methoxyphenyl)-2-picolinamide ([11C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4)

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu₄ positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu₄ PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu₄ expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([¹¹C]3) as a PET radiotracer for mGlu₄, and characterized its biological properties in Sprague Dawley rats. [¹¹C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [¹¹C]CH₃I. Total synthesis time was 38 ± 2.2 min (n = 7) from the end of bombardment to the formulation. The radioligand [¹¹C]3 was obtained in 27.7 ± 5.3% (n = 5) decay corrected radiochemical yield based on the radioactivity of [¹¹C]CO₂. The radiochemical purity of [¹¹C]3 was >99%. Specific activity was 188.7 ± 88.8 GBq/mol (n = 4) at the end of synthesis (EOS).PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu₄ modulator (4) to investigate specificity and 3 studies blocking with an mGlu₅ modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [¹¹C]3 (peak activity between 1–3 min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu₄ modulator 4 showed 22–28% decrease of [¹¹C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu₅. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu₄, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.     

Synthesis of 2-(4-substituted benzyl-1,4-diazepan-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamides and their positive inotropic evaluation

Herein we describe the discovery of compound 3g, a potent positive inotropic agent compared with the standard drug, milrinone. Compound 3g was developed from a series of 2-(4-substitutedbenzyl-1,4-diazepan-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl) acetamides found in an evaluation of inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. Several compounds showed favorable activities, but 3g was the most potent, with 7.68 ± 0.14% increased stroke volume (milrinone 2.38 ± 0.05%) at 1 × 10^?5 M in our in vitro study. The chronotropic effects of compounds having significant inotropic effects were also evaluated.