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Made available in DSpace on 2022-04-28T19:47:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Dopamine is an important neurotransmitter that regulates numerous essential functions, including cognition and voluntary movement. As such, it serves as an important scaffold for synthesis of novel analogues as part of drug development effort to obtain drugs for treatment of neurodegenerative diseases, such as Parkinson's disease. To that end, similarity search of the ZINC database based on two known dopamine-1 receptor (D1R) agonists, dihydrexidine (DHX) and SKF 38393, respectively, was used to predict novel chemical entities with potential binding to D1R. Three compounds that showed the highest similarity index were selected for synthesis and bioactivity profiling. All main synthesis products as well as the isolated intermediates, were properly characterized. The physico-chemical analyses were performed using HRESIMS, GC/MS, LC/MS with UV–Vis detection, and FTIR, 1H NMR and 13C NMR spectroscopy. Binding to D1 and D2 receptors and inhibition of dopamine reuptake via dopamine transporter were measured for the synthesized analogues of DHX and SKF 38393. Laboratory of Fine Organic Chemistry Department of Chemistry and Biochemistry Faculty of Sciences and Technology of São Paulo State University, São Paulo Department of Pharmaceutical Chemistry Faculty of Life Sciences University of Vienna, Althanstraße 14 Mass Spectrometry Centre Faculty of Chemistry University of Vienna, Währinger Straße 38 Department of Analytical Chemistry Faculty of Chemistry University of Vienna, Währinger Straße 38 Centre for X-Ray Structure Analysis at Faculty of Chemistry University of Vienna, Währinger Straße 40-42 Department of Chemistry Faculty of Philosophy Sciences and Letters of Ribeirão Preto University of São Paulo, Avenida dos Bandeirantes, 3900, SP Department of Molecular Neurosciences Centre for Brain Research Medical University of Vienna, Spitalgasse 4 Department of Neuroproteomics Paracelsus Medical University Laboratory of Fine Organic Chemistry Department of Chemistry and Biochemistry Faculty of Sciences and Technology of São Paulo State University, São Paulo FAPESP: 2016/10149-0

Aging-related neurological deficits negatively impact mental health, productivity, and social interactions leading to a pronounced socioeconomic burden. Since declining brain dopamine signaling during aging is associated with the onset of neurological impairments, we produced a selective dopamine transporter (DAT) inhibitor to restore endogenous dopamine levels and improve cognitive function. We describe the synthesis and pharmacological profile of (S,S)-CE-158, a highly specific DAT inhibitor, which increases dopamine levels in brain regions associated with cognition. We find both a potentiation of neurotransmission and coincident restoration of dendritic spines in the dorsal hippocampus, indicative of reinstatement of dopamine-induced synaptic plasticity in aging rodents. Treatment with (S,S)-CE-158 significantly improved behavioral flexibility in scopolamine-compromised animals and increased the number of spontaneously active prefrontal cortical neurons, both in young and aging rodents. In addition, (S,S)-CE-158 restored learning and memory recall in aging rats comparable to their young performance in a hippocampus-dependent hole board test. In sum, we present a well-tolerated, highly selective DAT inhibitor that normalizes the age-related decline in cognitive function at a synaptic level through increased dopamine signaling.

Rationale: Atypical dopamine (DA) transport blockers such as modafinil and its analogs may be useful for treating motivational symptoms of depression and other disorders. Previous research has shown that the DA depleting agent tetrabenazine can reliably induce motivational deficits in rats, as evidenced by a shift towards a low-effort bias in effort-based choice tasks. This is consistent with human studies showing that people with major depression show a bias towards low-effort activities. Objectives: Recent studies demonstrated that the atypical DA transport (DAT) inhibitor (S)-CE-123 reversed tetrabenazine-induced motivational deficits, increased progressive ratio (PROG) lever pressing, and increased extracellular DA in the nucleus accumbens. In the present studies, a recently synthesized modafinil analog, (S, S)-CE-158, was assessed in a series of neurochemical and behavioral studies in rats. Results: (S, S)-CE-158 demonstrated the ability to reverse the effort-related effects of tetrabenazine and increase selection of high-effort PROG lever pressing in rats tested on PROG/chow feeding choice task. (S, S)-CE-158 showed a high selectivity for inhibiting DAT compared with other monoamine transporters, and systemic administration of (S, S)-CE-158 increased extracellular DA in the nucleus accumbens during the behaviorally active time course, which is consistent with the effects of (S)-CE-123 and other DAT inhibitors that enhance high-effort responding. Conclusions: These studies provide an initial neurochemical characterization of a novel atypical DAT inhibitor, and demonstrate that this compound is active in models of effort-related choice. This research could contribute to the development of novel compounds for the treatment of motivational dysfunctions in humans. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature. The authors would like to acknowledge Eurofins DiscoverX Corporation (Fremont, CA).

Atypical dopamine reuptake inhibitors, such as modafinil, are used for the treatment of sleeping disorders and investigated as potential therapeutics against cocaine addiction and for cognitive enhancement. Our continuous effort to find modafinil analogues with higher inhibitory activity on and selectivity toward the dopamine transporter (DAT) has previously led to the promising thiazole-containing derivatives CE-103, CE-111, CE-123, and CE-125. Here, we describe the synthesis and activity of a series of compounds based on these scaffolds, which resulted in several new selective DAT inhibitors and gave valuable insights into the structure-activity relationships. Introduction of the second chiral center and subsequent chiral separations provided all four stereoisomers, whereby the S-configuration on both generally exerted the highest activity and selectivity on DAT. The representative compound of this series was further characterized by in silico, in vitro, and in vivo studies that have demonstrated both safety and efficacy profile of this compound class.

Perinatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), usually first diagnosed in childhood, that are characterized by hyperactivity, impulsivity and learning and memory disability, among others. To test the hypothesis that dopamine signaling is one of the main factors underlying these impairments, a new atypical dopamine transporter (DAT) inhibitor, CE-123 (1, 3 or 10 mg/kg) was assessed for its potential to overcome the ethanol-induced behavioral effects in a rat model of FASD. In the present study, neonatal rats were exposed to alcohol intubations across the neonatal period (postnatal day (PND)4-9, the third trimester equivalent of human gestation) and, after weaning, the animals (male rats) were assigned randomly to three groups. The first group was tested at PND21 (hyperactivity test). A second group was tested at PND45 (anxiety test), at PND47 (locomotor activity test), at PND49 (spatial cognitive test in the Barnes maze) and PND50 (reversal learning in the Barnes maze). The third group was tested at PND50 (dopamine receptor mRNA expression). Our results support the hypothesis that dopamine signaling is associated with FASD because the dopamine (D1, D2 and D5) receptor mRNA expression was altered in the striatum, hippocampus and prefrontal cortex in adult rats exposed to ethanol during neonatal period. CE-123 (3 and 10 mg/kg) inhibited the hyperactivity and ameliorated (10 mg/kg) the impairment of reversal learning in alcohol-exposed rats. Thus, these findings provide support that CE-123 may be a useful intervention for same of the deficits associated with neonatal ethanol exposure.

Animal studies of effort-based choice behavior are being used to model effort-related motivational dysfunctions in humans. With these procedures, animals are offered a choice between high-effort instrumental actions leading to highly valued reinforcers vs. low effort/ low reward options. Several previous studies have shown that dopamine (DA) uptake inhibitors, including GBR12909, lisdexamfetamine, methylphenidate, and PRX-14040, can reverse the effort-related effects of the vesicular monoamine transport blocker tetrabenazine, which inhibits DA storage. Because many drugs that block DA transport act as major stimulants that also release DA, and produce a number of undesirable side effects, there is a need to develop and characterize novel atypical DA transport inhibitors. (S)-CE-123 ((S)-5-((benzhydrylsulfnyl) methyl)thiazole) is a recently developed analog of modafnil with the biochemical characteristics of an atypical DA transport blocker. The present paper describes the enantioselective synthesis and initial chemical characterization of (S)-CE-123, as well as behavioral experiments involving effort-based choice and microdialysis studies of extracellular DA. Rats were assessed using the fxed ratio 5/chow feeding choice test. Tetrabenazine (1.0 mg/kg) shifted choice behavior, decreasing lever pressing and increasing chow intake. (S)-CE-123 was coadministered at doses ranging from 6.0 to 24.0 mg/kg, and the highest dose partially but signifcantly reversed the effects of tetrabenazine, although this dose had no effect on fxed ratio responding when administered alone. Additional experiments showed that (S)-CE-123 signifcantly increased lever pressing on a progressive ratio/chow feeding choice task and that the effective dose (24.0 mg/kg) increased extracellular DA in nucleus accumbens core. In summary, (S)-CE-123 has the behavioral and neurochemical profle of a compound that can block DA transport, reverse the effort-related effects of tetrabenazine, and increase selection of high-effort progressive ratio responding. This suggests that (S)-CE- 123 or a similar compound could be useful as a treatment for effort-related motivational dysfunction in humans.

Background Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity. Results Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively. Conclusion This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.

Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to characterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked uptake of [3H] dopamine (IC50 = 4.606 μM) while effects on serotonin (SERT) and the norepinephrine transporter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval, represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1, CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory enhancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic for impairments of cognitive functions.

Reduced cognitive abilities are often characterized by an impairment of flexibility, i.e., the ability to switch from learned rules or categories that were important in certain contexts to different new modalities that rule the task. Drugs targeting the dopamine transporter (DAT) are widely used for their potential to enhance cognitive abilities. However, commercially available drugs are of limited specificity for DAT, blocking also noradrenaline and serotonine transporters, that can lead to unwanted side effects in healthy subjects. Therefore, we tested a newly synthetized compound (CE-123) with higher specificity for DAT in male rats in an attentional set-shifting task (ASST), that proves for cognitive flexibility and a 5-choice serial-reaction time task (5-CSRTT) assessing visuospatial attention and impulsivity. Treated rats at a dose of 0.3 and 1.0 but not 0.1 mg/kg bodyweight showed reduced extra-dimensional shifts in the ASST compared to controls indicating increased cognitive flexibility. Rats treated with R-Modafinil, a commercially available DAT inhibitor at a dose of 10 mg/kg bodyweight showed increased premature responses, an indicator of increased impulsivity, during a 10 s but not a 2.5, 5, or 7.5 s intertrial interval when compared to vehicle-treated rats in the 5-CSRTT. This was not found in rats treated with CE-123 at the same dose as for R-Modafinil. Visuospatial attention, except premature responses, did not differ between R-Modafinil and CE-123-treated rats and their respective controls. Thus, CE-123 increased cognitive flexibility with diminished impulsivity.

Made available in DSpace on 2022-04-28T19:07:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-22 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Modafinil is a wake promoting compound with high potential for cognitive enhancement. It is targeting the dopamine transporter (DAT) with moderate selectivity, thereby leading to reuptake inhibition and increased dopamine levels in the synaptic cleft. A series of modafinil analogues have been reported so far, but more target-specific analogues remain to be discovered. It was the aim of this study to synthesize and characterize such analogues and, indeed, a series of compounds were showing higher activities on the DAT and a higher selectivity toward DAT versus serotonin and norepinephrine transporters than modafinil. This was achieved by substituting the amide moiety by five- and six-membered aromatic heterocycles. In vitro studies indicated binding to the cocaine pocket on DAT, although molecular dynamics revealed binding different from that of cocaine. Moreover, no release of dopamine was observed, ruling out amphetamine-like effects. The absence of neurotoxicity of a representative analogue may encourage further preclinical studies of the above-mentioned compounds. Department of Pharmaceutical Chemistry Faculty of Life Sciences University of Vienna, Althanstraße 14 Ural Federal University Named after the First President of Russia B. N. Yeltsin, 19 Mira St. Department of Analytical Chemistry Faculty of Chemistry University of Vienna, Währinger Straße 38 X-ray Structure Analysis Centre Faculty of Chemistry University of Vienna, Währinger Straße 38 Department of Pediatrics Medical University of Vienna Laboratory of Fine Organic Chemistry Department of Chemistry and Biochemistry Faculty of Science and Technology University of Sao Paulo State, Roberto Simonsen 305 Institute of Pharmacology Centre of Physiology and Pharmacology Medical University of Vienna Neuroscience Laboratory Paracelsus Medical University FAPESP: 2016/10149-0

A series of compounds have been shown to enhance cognitive function via the dopaminergic system and indeed the search for more active and less toxic compounds is continuing. It was therefore the aim of the study to synthetise and test a novel heterocyclic compound for cognitive enhancement in a paradigm for working memory. Specific and effective dopamine re-uptake inhibition DAT (IC50 = 4,1 ± 0,8 μM) made us test this compound in a radial arm maze (RAM) in the rat. CE-125 (4-((benzhydrylsulfinyl)methyl)-2-cyclopropylthiazole), was tested for dopamine (DAT), serotonin and norepinephrine re-uptake inhibition by a well-established system. The working memory index (WMI) was evaluated in male Sprague Dawley rats that were intraperitoneally injected with CE-125 (1 or 10 mg/kg body weight). In order to evaluate basic neurotoxicity, the open field, elevated plus maze, rota rod studies and the forced swim test were carried out. Frontal cortex was taken at the last day of the RAM test and dopamine receptors D1R and D2R, DAT and phosphorylated DAT protein levels were determined. On the 10th day both doses were increasing the WMI as compared to the vehicle-treated group. In both, trained and treated groups, D1R levels were significantly reduced while D2R levels were unchanged. DAT levels were comparable between all groups while phosphorylated DAT levels were increased in the trained group treated with 1 mg/kg body weight. CE-125 as a probably non-neurotoxic compound and specific reuptake inhibitor was shown to increase performance (WMI) and modulation of the dopaminergic system is proposed as a possible mechanism of action.

Modafinil is a wake promoting drug approved for clinical use and also has cognitive enhancing properties. Its enantiomer R-Modafinil (R-MO) is not well studied in regard to cognitive enhancing properties. Hence we studied its effect in a spatial memory paradigm and its possible effects on dentate gyrus long-term potentiation (DG-LTP). Clinically relevant doses of R-MO, vehicle dimethyl sulfoxide (DMSO) or saline were administered for three days during the hole-board test and in in vivo DG-LTP. Synaptic levels of dopamine receptors D1R, D2R, dopamine transporter (DAT), and its phosphorylated form (ph-DAT) in DG tissue 4 h after LTP induction were quantified by western blot analysis. Monoamine reuptake and release assays were performed by using transfected HEK-293 cells. Possible neurotoxic side effects on general behaviour were also studied. R-MO at both doses significantly enhanced spatial reference memory during the last training session and during memory retrieval compared to DMSO vehicle but not when compared to saline treated rats. Similarly, R-MO rescues DG-LTP from impairing effects of DMSO. DMSO reduced memory performance and LTP magnitude when compared to saline treated groups. The synaptic DR1 levels in R-MO groups were significantly decreased compared to DMSO group but were comparable with saline treated animals. We found no effect of R-MO in neurotoxicity tests. Thus, our results support the notion that LTP-like synaptic plasticity processes could be one of the factors contributing to the cognitive enhancing effects of spatial memory traces. D1R may play an important regulatory role in these processes.

Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity.Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively.This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.