Ugi 5-center-4-component reaction of α-amino aldehydes and its application in synthesis of 2-oxopiperazines.

A synthetic route leading to densely functionalized 2-oxopiperazines is presented. The strategy employs a 5-center-4-component variant of Ugi multicomponent reaction followed by a deprotection/cyclization sequence. N-Boc-α-amino aldehydes were used for the first time as carbonyl components in a key Ugi 5-center-4-component reaction (U-5C-4CR). It is shown that the presented synthetic route can lead to rigid, heterocyclic scaffolds, as demonstrated by the synthesis of tetrahydro-2H-pyrazino[1,2-a]pyrazine-3,6,9(4H)-trione ß-turn mimetic and derivatives of 1,6-dioxooctahydropyrrolo[1,2-a]pyrazine and 3,8-dioxohexahydro-3H-oxazolo[3,4-a]pyrazine.

Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.

Synthesis and biological evaluation of novel 3-(5-substituted-1H-indol-3-yl)pyrrolidine-2,5-dione derivatives with a dual affinity for serotonin 5-HT1A receptor and SERT.

The serotonin 1A (5-HT1A) receptors and serotonin transporter (SERT) are important biological targets in the treatment of diseases of the central nervous system, especially for depression. In this study, new 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives linked with the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole moiety were synthesised and evaluated for their affinity for 5-HT1A receptor and serotonin reuptake inhibition. Selected compounds were then tested for their affinity for D2, 5-HT2A, 5-HT6 and 5-HT7 receptors, and also in in vitro metabolic stability assays in human microsomes. Finally, in vivo assays allowed us to evaluate the agonist-antagonist properties of pre- and postsynaptic 5-HT1A receptors. 3-(1-(4-(3-(5-methoxy-1H-indol-3-yl)-2,5-dioxopyrrolidin-1-yl)butyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole-5-carbonitrile (4f) emerged as the most promising compound from the series, due to its favourable receptor binding profile (Ki(5-HT1A) = 10.0 nM; Ki(SERT) = 2.8 nM), good microsomal stability and 5-HT1A receptor agonistic activity.

Computer-Aided Design and Synthesis of a New Class of PEX14 Inhibitors: Substituted 2,3,4,5-Tetrahydrobenzo[F][1,4]oxazepines as Potential New Trypanocidal Agents.

African and American trypanosomiases are estimated to affect several million people across the world, with effective treatments distinctly lacking. New, ideally oral, treatments with higher efficacy against these diseases are desperately needed. Peroxisomal import matrix (PEX) proteins represent a very interesting target for structure- and ligand-based drug design. The PEX5-PEX14 protein-protein interface in particular has been highlighted as a target, with inhibitors shown to disrupt essential cell processes in trypanosomes, leading to cell death. In this work, we present a drug development campaign that utilizes the synergy between structural biology, computer-aided drug design, and medicinal chemistry in the quest to discover and develop new potential compounds to treat trypanosomiasis by targeting the PEX14-PEX5 interaction. Using the structure of the known lead compounds discovered by Dawidowski et al. as the template for a chemically advanced template search (CATS) algorithm, we performed scaffold-hopping to obtain a new class of compounds with trypanocidal activity, based on 2,3,4,5-tetrahydrobenzo[f][1,4]oxazepines chemistry. The initial compounds obtained were taken forward to a first round of hit-to-lead optimization by synthesis of derivatives, which show activities in the range of low- to high-digit micromolar IC50 in the in vitro tests. The NMR measurements confirm binding to PEX14 in solution, while immunofluorescent microscopy indicates disruption of protein import into the glycosomes, indicating that the PEX14-PEX5 protein-protein interface was successfully disrupted. These studies result in development of a novel scaffold for future lead optimization, while ADME testing gives an indication of further areas of improvement in the path from lead molecules toward a new drug active against trypanosomes.

Identification of the Primary Structure of Selenium-Containing Polysaccharides Selectively Inhibiting T-Cell Proliferation.

We previously described the biosynthesis, isolation, and immunosuppressive activity of the selenium-containing polysaccharide fraction isolated from the mycelial culture of Lentinula edodes. Structural studies have shown that the fraction was a protein-containing mixture of high molar mass polysaccharides α- and ß-glucans. However, which of the components of the complex fraction is responsible for the immunosuppressive activity non-typical for polysaccharides of fungal origin has not been explained. In the current study, we defined four-polysaccharide components of the Se-containing polysaccharide fraction determined their primary structure and examined the effect on T- and B-cell proliferation. The isolated Se-polysaccharides, α-1,4-glucan (Mw 2.25 × 106 g/mol), unbranched ß-1,6-d-glucan, unbranched ß-1,3-d-glucan and ß-1,3-branched ß-1,6-d-glucan (Mw 1.10 × 105 g/mol), are not typical as components of the cell wall of L. edodes. All are biologically active, but the inhibitory effect of the isolated polysaccharides on lymphocyte proliferation was weaker, though more selective than that of the crude fraction.

Synthesis of new 4-butyl-arylpiperazine-3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives and evaluation for their 5-HT1A and D2 receptor affinity and serotonin transporter inhibition.

A series of novel 4-butyl-arylpiperazine-3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives were synthesized and evaluated for their 5-HT1A/D2 receptor affinity and serotonin reuptake inhibition. The compounds exhibited high affinity for the 5-HT1A receptor, (especially 4dKi = 0.4 nM) which depended on the substitution pattern at the phenylpiperazine moiety. From this series screen, compound 4c emerged with promising mixed receptor profiles for the 5-HT1A/D2 receptors and the serotonin transporter (Ki = 1.3 nM, 182 nM and 64 nM, respectively).

Structure-activity relationship and cardiac safety of 2-aryl-2-(pyridin-2-yl)acetamides as a new class of broad-spectrum anticonvulsants derived from Disopyramide.

A series of 2-aryl-2-(pyridin-2-yl)acetamides were synthesized and screened for their anticonvulsant activity in animal models of epilepsy. The compounds were broadly active in the 'classical' maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests as well as in the 6 Hz and kindling models of pharmacoresistant seizures. Furthermore, the compounds showed good therapeutic indices between anticonvulsant activity and motor impairment. Structure-activity relationship (SAR) trends clearly showed the highest activity resides in unsubstituted phenyl derivatives or compounds having ortho- and meta- substituents on the phenyl ring. The 2-aryl-2-(pyridin-2-yl)acetamides were derived by redesign of the cardiotoxic sodium channel blocker Disopyramide (DISO). Our results show that the compounds preserve the capability of the parent compound to inhibit voltage gated sodium currents in patch-clamp experiments; however, in contrast to DISO, a representative compound from the series 1 displays high levels of cardiac safety in a panel of in vitro and in vivo experiments.

Structural basis for ADP-dependent glucokinase inhibition by 8-bromo-substituted adenosine nucleotide.

In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo-substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.

Synthesis and biological investigations of 3ß-aminotropane arylamide derivatives with atypical antipsychotic profile.

This work is a continuation of our previous research, concentrating this time on lead structure modification to increase the 5-HT1A receptor affinity and water solubility of designed compounds. Therefore, the compounds synthesised within the present project included structural analogues of 3ß-acylamine derivatives of tropane with the introduction of a methyl substituent in the benzyl ring and a 2-quinoline, 3-quinoline, or 6-quinoline moiety. A series of novel 3ß-aminotropane derivatives was evaluated for their affinity for 5-HT1A, 5-HT2A, and D2 receptors, which allowed for the identification of compounds 12e, 12i, and 19a as ligands with highest affinity for the tested receptors; they were then subjected to further evaluation in preliminary in vivo studies. Selected compounds 12i and 19a displayed antipsychotic properties in the d-amphetamine-induced and MK-801-induced hyperlocomotor activity test in mice. Moreover, compound 19a showed significant antidepressant-like activity in the forced swim test in mice.

Comparison of enzymatic properties and small molecule inhibition of γ-glutamyltranspeptidases from pathogenic and commensal bacteria.

Helicobacter pylori infects the stomach of 50% of the population worldwide, thus causing chronic gastritis. Although this infection can be cured by antibiotic treatment, therapeutic options are increasingly limited due to the development of resistances. The γ-glutamyl-transpeptidase (gGT) of H. pylori (HpgGT) is a virulence factor important for colonization and contributes to bacterial immune evasion. Therefore, this enzyme is a potential target for developing new anti-infectives. As species specificity of such compounds is required in order to avoid off-target or adverse effects, comparative analysis of the gGTs from different organisms is a prerequisite for drug development. To allow detailed biochemical and enzymatic characterization, recombinant gGTs from five different bacteria as well as Homo sapiens were characterized and compared. Investigation of the enzymatic activity, the binding modes of known inhibitors to the catalytic center, and a high resolution X-ray structure of the HpgGT provided a starting point for the identification of new inhibitory substances targeting HpgGT. Inhibitors with Ki values in the nm to mm range were identified and their binding modes were analyzed by mass spectrometry. The results of this study provide a basis for the development of species-specific lead compounds with anti-infective potential by effectively inhibiting HpgGT.

Novel fluorinated pyrrolo[1,2-a]pyrazine-2,6-dione derivatives: synthesis and anticonvulsant evaluation in animal models of epilepsy.

A series of fluorinated analogs of the potent investigative anticonvulsant agent (4S,8aS)-4-phenylperhydropyrrolo[1,2-a]pyrazine-2,6-dione 1 was prepared and characterized by IR, 1H, 13C NMR and mass spectral data. The compounds have been evaluated in the in vivo rodent models of epilepsy. They displayed high activity in the 'classical' maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests as well as in the 6Hz model of pharmacoresistant limbic seizures. The results showed that incorporating fluorine atoms into the phenyl ring of 1 can be beneficial for the anticonvulsant potency. The most promising meta-trifluoromethyl and meta-trifluoromethoxy derivatives (4S,8aS)-5h and (4S,8aS)-5l, respectively, displayed very broad spectra of activity across the preclinical seizure models.

Expanding the substrate scope of Ugi five-center, four-component reaction U-5C-4CR): ketones as coupling partners for secondary amino acids.

Various symmetrical and unsymmetrical ketones were successfully coupled with secondary amino acids in the course of Ugi five-center, four-component reaction (U-5C-4CR), thus expanding the molecular diversity possible to be achieved by the reaction. The chemical yields depended on the degree of hindrance of the components employed and were satisfactory in view of possible steric interactions in the U-5C-4CR zwitterionic intermediate. The sense of diastereoinduction for reactions employing unsymmetrical ketones was examined by converting the resulting Ugi adducts into the corresponding rigid 2,6-diketopiperazine derivatives.

Synthesis of new perhydropyrrolo[1,2-a]pyrazine derivatives and their evaluation in animal models of epilepsy.

A series of novel stereochemically pure derivatives of the investigative broad-spectrum anticonvulsant ADD408003 was designed and synthesized. Five-center four-component (U-5C-4CR) and four-center three-component (U-4C-3CR) variants of Ugi reaction were used in the key step of the synthetic pathways. The compounds obtained were evaluated for the anticonvulsant activitiy in the maximal electroshock seizure (MES), subcutaneous Metrazole (scMET) and minimal clonic seizure (6 Hz) animal models of epilepsy. The efficacies of most derivatives in the 6 Hz model of pharmacoresistant partial seizures were markedly higher than in the 'classical' MES and scMET models. The most active compounds, (4R,8aR)-3a, and (4S,8aS)-6 displayed median effective doses (ED50) of 47.90 and 126.19 mg/kg, respectively, for the 6 Hz test.

Multicomponent synthesis and anticonvulsant activity of monocyclic 2,6-diketopiperazine derivatives.

In this study, a series of diastereomerically pure monocyclic 2,6-diketopiperazine (2,6-DKP) derivatives were synthesized. The key synthetic step involved a multicomponent Ugi five-center, four-component reaction which was used to generate the convertible tert-butylamidoesters with both good yields and high diastereoselectivity toward the desired bioactive (S,S) absolute configuration. In subsequent steps, selective tertbutyl cleavage by use of BF3·CH3COOH and base-induced intramolecular cyclocondensation gave the final 2,6-DKP derivatives. The relative stereochemistry of the target molecules was confirmed by 1H NMR experiments. The compounds obtained were submitted to in vivo screening in animal models of epilepsy. Some of them displayed good activity in maximal electroshock seizure and 6 Hz tests.

Structure-activity relationships of the aromatic site in novel anticonvulsant pyrrolo[1,2-a]pyrazine derivatives.

Novel, chiral derivatives of pyrrolo[1,2-a]pyrazine with aromatic substituents at carbon C-4 were synthesized by a short synthetic sequence involving Ugi multicomponent reaction. The compounds were evaluated for their in vivo efficacy in animal models of epilepsy within the Anticonvulsant Screening Program (ASP). High activity in 'classical' maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET) tests was characteristic for meta-substituted analogs. On the other hand, efficacy of compounds in the 6 Hz model of pharmacoresistant limbic seizures was only marginally affected by the orientation of substituents in the phenyl moiety. The most active derivative, 5a, displayed an ED50 value of 32.24 mg/kg and a protective index of 6.6 (PI) in the 6 Hz test. It was also active in a pilocarpine-induced status prevention model of pharmacoresistant status epilepticus.

Development of novel PEX5-PEX14 protein-protein interaction (PPI) inhibitors based on an oxopiperazine template.

Protein-protein interactions (PPIs) constitute an important but challenging class of molecular targets for small molecules. The PEX5-PEX14 PPI has been shown to play a critical role in glycosome biogenesis and its disruption impairs the metabolism in Trpanosoma parasites, eventually leading to their death. Therefore, this PPI is a potential molecular target for new drugs against diseases caused by Trypanosoma infections. Here, we report a new class of peptidomimetic scaffolds to target the PEX5-PEX14 PPI. The molecular design was based on an oxopiperazine template for the α-helical mimetics. A structural simplification along with modifications of the central oxopiperazine scaffold and addressing the lipophilic interactions led to the development of peptidomimetics that inhibit PEX5-TbPEX14 PPI and display cellular activity against T. b. brucei. This approach provides an alternative approach towards the development of trypanocidal agents and may be generally useful for the design of helical mimetics as PPI inhibitors.

Structure-based design, synthesis and evaluation of a novel family of PEX5-PEX14 interaction inhibitors against Trypanosoma.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

DYRK1A Kinase Inhibitors Promote ß-Cell Survival and Insulin Homeostasis.

The rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved. We demonstrate that our set of small molecule inhibitors of DYRK1A kinase potently promotes ß-cell proliferation, enhances long-term insulin secretion, and balances glucagon level in the organoid model of the human islets. Comparable activity is seen in INS-1E and MIN6 cells, in isolated mice islets, and human iPSC-derived ß-cells. Our compounds exert a significantly more pronounced effect compared to harmine, the best-documented molecule enhancing ß-cell proliferation. Using a body-like environment of the organoid, we provide a proof-of-concept that small-molecule-induced human ß-cell proliferation via DYRK1A inhibition is achievable, which lends a considerable promise for regenerative medicine in T1DM and T2DM treatment.

Relationship between selenium accumulation and mycelial cell composition in Lentinula edodes (Berk.) cultures.

It was postulated that fractions enriched in selenium (Se) isolated from Lentinula edodes mycelium polysaccharide might possess higher biological activity than the non-enriched fractions currently used to treat cancer. In order to obtain Se-enriched mycelial preparations, L. edodes cultures were cultivated in media enriched with sodium selenite. In order to determine whether the concentration of Se in the culture medium affected the biosynthesis and composition of cell wall and cell membrane, concentrations of the exopolysaccharide (EPS), chitin, and sterol (ergosterol) were measured in harvested mycelia. In addition, the relationship between Se accumulation and content of polyphenols and vitamin D(2) in L. edodes mycelium was examined. The effects of Se levels on the mycelium cell composition were determined in culture media enriched with Se at concentrations ranging from 0 to 30 microg/ml. In each culture mycelial growth, total Se and Se distribution were determined between mycelial fractions of different polarity. The EPS, polyphenolics, and ergosterol content in harvested mycelia rose in proportion to Se concentration in the culture medium. The chitin content in mycelia increased with Se concentrations in the range 0-5 microg/ml, but at higher concentrations chitin levels decreased. Data showed that Se in culture medium exerted potent effects on the composition of the mushroom cell wall and semipermeable membrane, and on the content of polyphenolics that are involved in detoxification processes. Our findings indicate the optimal concentration of Se required in the culture medium for maximal yield of immunostimulatory-active selenated exopolysaccharides.

Water envelope has a critical impact on the design of protein-protein interaction inhibitors.

We show that a water envelope network plays a critical role in protein-protein interactions (PPI). The potency of a PPI inhibitor is modulated by orders of magnitude on manipulation of the solvent envelope alone. The structure-activity relationship of PEX14 inhibitors was analyzed as an example using in silico and X-ray data.