Synthesis of fused tetramate-oxazolidine and -imidazolidine derivatives and their antibacterial activity.

A chemoselective route which provides direct access to bicyclic tetramates, making use of Dieckmann cyclisation of functionalised oxazolidines and imidazolidines derived from an aminomalonate, is reported; calculations suggest that the observed chemoselectivity is kinetically controlled and leads to the thermodynamically most stable product. Some compounds in the library showed modest antibacterial activity against Gram-positive bacteria, and this activity is maximal in a well-defined region of chemical space (554 < Mw < 722 g mol-1; 5.78 < cLogP < 7.16; 788 < MSA < 972 Å2; 10.3 < rel. PSA < 19.08).

Tetramate derivatives by chemoselective Dieckmann ring closure of allo-phenylserines, and their antibacterial activity.

A general route which provides direct access to substituted bicyclic tetramates, making use of Dieckmann cyclisation of oxazolidine derivatives derived from allo-phenylserines, is reported. Of interest is the high level of diastereoselectivity observed for the N-acylation reaction of oxazolidines and the complete chemoselectivity of their ring closure in the Dieckmann cyclisation. Significantly, the sense of the chemoselectivity is different to earlier reported threo-phenylserine systems, showing the importance of steric bulk around the bicyclic ring system. The derived C7-carboxamidotetramates, but not C7-acyl systems, exhibited potent antibacterial activity against MRSA, with the most active compounds exhibiting well-defined physicochemical and structure-activity properties. This work clearly demonstrates that densely functionalised tetramates are both readily available and may exhibit high levels of antibacterial activity.

Tetramate Derivatives by Chemoselective Dieckmann Ring Closure of threo-Phenylserines and Their Antibacterial Activity.

A general route, which provides direct access to substituted bicyclic tetramates, making use of Dieckmann cyclization of oxazolidines derived from threo-arylserines, is reported; the latter were found to be available by an efficient aldol-like reaction of glycine with some substituted benzaldehydes under alkaline conditions. The tetramates were found to release chelated metal cations acquired during chromatographic purification by mild acid wash. Some compounds in the library showed good antibacterial activity against Gram-positive bacteria. Cheminformatic analysis demonstrates that the most active compounds were Ro5-compliant and occupy a narrow region of chemical space, distinct from that occupied by other known antibiotics, with the most potent compounds having 399 < Mw < 530 Da; 3.5 < cLogP < 6.6; 594 < MSA <818 Å2; 9.6 < rel. PSA <13.3%. MIC values were shifted to higher concentrations when tested in the presence of HSA or blood, but was not completely abolished, consistent with a plasma protein binding (PPB) effect.

Azepine-Indole Alkaloids From Psychotria nemorosa Modulate 5-HT2A Receptors and Prevent in vivo Protein Toxicity in Transgenic Caenorhabditis elegans.

Nemorosine A (1) and fargesine (2), the main azepine-indole alkaloids of Psychotria nemorosa, were explored for their pharmacological profile on neurodegenerative disorders (NDs) applying a combined in silico-in vitro-in vivo approach. By using 1 and 2 as queries for similarity-based searches of the ChEMBL database, structurally related compounds were identified to modulate the 5-HT2A receptor; in vitro experiments confirmed an agonistic effect for 1 and 2 (24 and 36% at 10 µM, respectively), which might be linked to cognition-enhancing properties. This and the previously reported target profile of 1 and 2, which also includes BuChE and MAO-A inhibition, prompted the evaluation of these compounds in several Caenorhabditis elegans models linked to 5-HT modulation and proteotoxicity. On C. elegans transgenic strain CL4659, which expresses amyloid beta (Aß) in muscle cells leading to a phenotypic paralysis, 1 and 2 reduced Aß proteotoxicity by reducing the percentage of paralyzed worms to 51%. Treatment of the NL5901 strain, in which α-synuclein is yellow fluorescent protein (YFP)-tagged, with 1 and 2 (10 µM) significantly reduced the α-synuclein expression. Both alkaloids were further able to significantly extend the time of metallothionein induction, which is associated with reduced neurodegeneration of aged brain tissue. These results add to the multitarget profiles of 1 and 2 and corroborate their potential in the treatment of NDs.

Tricyclic Fused Lactams by Mukaiyama Cyclisation of Phthalimides and Evaluation of their Biological Activity.

We report that phthalimides may be cyclized using a Mukaiyama-type aldol coupling to give variously substituted fused lactam (1,2,3,9b-tetrahydro-5H-pyrrolo[2,1-a]isoindol-5-one) systems. This novel process shows a high level of regioselectivity for o-substituted phthalimides, dictated by steric and electronic factors, but not for m-substituted phthalimides. The initial aldol adduct is prone to elimination, giving 2,3-dihydro-5H-pyrrolo[2,1-a]isoindol-5-ones, and the initial cyclisation can be conducted in such a way that aldol cyclisation-elimination is achievable in a one-pot approach. The 2,3-dihydro-5H-pyrrolo[2,1-a]isoindol-5-ones possess cross conjugation and steric effects which significantly influence the reactivity of several functional groups, but conditions suitable for epoxidation, ester hydrolysis and amide formation, and reduction, which provide for ring manipulation, were identified. Many of the derived lactam systems, and especially the eliminated systems, show low solubility, which compromises biological activity, although in some cases, antibacterial and cytotoxic activity was found, and this new class of small molecule provides a useful skeleton for further elaboration and study.

Metal Binding and Its Amelioration in Tetramates.

Metal chelation in tetramates may be ameliorated by changing the ligating group and by steric blocking, which in turn leads to a change in their antibacterial properties; the former was achieved by replacement of an amide with a C-9 C═N bond and the latter by the synthesis of cysteine-derived tetramates with functionalization at the C-6 or C-9 enolic groups. In both cases, the metal-chelating ability was weak, and a loss of antibacterial activity was observed. Tetramate alkylations with an extended tricarbonyl-conjugated system could be achieved under Mitsunobu conditions which led to regioisomers, distinguishable by careful heteronuclear multiple bond coherence correlation and carbonyl carbon chemical shift analysis. C-9 and C-6 O-alkylation were observed but not C-8 O-alkylation for tetramate carboxamides; interestingly, C-7 alkylation with allyl and prenyl derivatives was also observed, and this arose by the rearrangement of initially formed O-alkyl products. Only the C-7 alkylated tetramate derivatives 13a and 13d with no metal-chelating ability demonstrated promising antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with the most active analogue exhibiting a minimum inhibitory concentration of ≤ 1.95 µg/mL against MRSA, suggesting a mechanism of action independent of metal chelation. Otherwise, modifications at C-6/C-9 of tetramates led to a complete loss of metal-chelating ability, which correlated with the loss of antibacterial activity. This work further confirms that the metal-chelating capability is of fundamental importance in the biological activity of tetramates.

Mediation of metal chelation in cysteine-derived tetramate systems.

A study of bicyclic tetramates modified with a bulky ester, which leads to steric hindrance of distal chelating atoms as a route for the alteration of metal binding ability is reported. This approach required the development of a direct method for the synthesis of different esters of cysteine from cystine, which then provided access to bicyclic tetramates by Dieckmann cyclisation. Further derivation to ketones and carboxamides by Grignard addition and transamination reactions respectively provided rapid access to a chemical library of tetramates with diverse substitution. Of interest is that bicyclic tetramate ketones and carboxamides showed different tautomeric and metal binding behaviour in solution. Significantly, in both systems, the incorporation of bulky C-5 esters at the bridging position not only reduced metal binding, but also enhanced antibacterial potencies against Gram-positive MRSA bacteria. Those tetramates with antibacterial activity which was not metal dependent showed physiochemical properties of MSA of 559-737 Å2, MW of 427-577 Da, clogP of 1.8-6.1, clogD7.4 of -1.7 to 3.7, PSA of 83-109 Å2 and relative PSA of 12-15% and were generally Lipinski rule compliant. A subset of tetramates exhibited good selectivity towards prokaryotic bacterial cells. Given that the work reported herein is synthesis-led, without the underpinning detailed mechanistic understanding of biological/biochemical mechanism, that the most active compounds occupy a small region of chemical space as defined by MW, clogP, PSA and %PSA is of interest. Overall, the bicyclic tetramate template is a promising structural motif for the development of novel antibacterial drugs, with good anti-MRSA potencies and appropriate drug-like physiochemical properties, coupled with a potential for multi-targeting mechanisms and low eukaryotic cytotoxicity.

Synthetic Access to Hydrophilic Tetramate Derivatives of Cysteine.

The synthesis, structural, and antibacterial evaluation of bicyclic tetramate derivatives of cysteine rendered hydrophilic with pendant heterocyclic substituents is reported; effective synthetic protocols and antibacterial activity for a small library of polar derivatives were found, and direct evidence for strong metal chelation in these systems was obtained. A computational study has developed a detailed understanding of the controlling factors of the key Dieckmann cyclization step.

Prolongation of metallothionein induction combats Aß and α-synuclein toxicity in aged transgenic Caenorhabditis elegans.

Neurodegenerative disorders (ND) like Alzheimer's (AD), Parkinson's (PD), Huntington's or Prion diseases share similar pathological features. They are all age dependent and are often associated with disruptions in analogous metabolic processes such as protein aggregation and oxidative stress, both of which involve metal ions like copper, manganese and iron. Bush and Tanzi proposed 2008 in the 'metal hypothesis of Alzheimer's disease' that a breakdown in metal homeostasis is the main cause of NDs, and drugs restoring metal homeostasis are promising novel therapeutic strategies. We report here that metallothionein (MT), an endogenous metal detoxifying protein, is increased in young amyloid ß (Aß) expressing Caenorhabditis elegans, whereas it is not in wild type strains. Further MT induction collapsed in 8 days old transgenic worms, indicating the age dependency of disease outbreak, and sharing intriguing parallels to diminished MT levels in human brains of AD. A medium throughput screening assay method was established to search for compounds increasing the MT level. Compounds known to induce MT release like progesterone, ZnSO4, quercetin, dexamethasone and apomorphine were active in models of AD and PD. Thioflavin T, clioquinol and emodin are promising leads in AD and PD research, whose mode of action has not been fully established yet. In this study, we could show that the reduction of Aß and α-synuclein toxicity in transgenic C. elegans models correlated with the prolongation of MT induction time and that knockdown of MT with RNA interference resulted in a loss of bioactivity.

A robust and miniaturized screening platform to study natural products affecting metabolism and survival in Caenorhabditis elegans.

In this study a robust, whole organism screening based on Caenorhabditis elegans is presented for the discovery of natural products (NP) with beneficial effects against obesity and age-related diseases. Several parameters of the elaborated workflow were optimized to be adapted for probing multicomponent mixtures combining knowledge from traditional medicine and NP chemistry by generating optimized small-scale extracts considering scarcity of the natural source, solubility issues, and potential assay interferences. The established miniaturized assay protocol allows for in vivo probing of small amounts of even complex samples (~ 1 mg) to test their ability to increase the nematodes' survival time and the suppression of fat accumulation assessed by Nile red staining as hall marks of "healthy aging". The workflow was applied on 24 herbal and fungal materials traditionally used against symptoms of the metabolic syndrome and revealed promising results for the extracts of Gardenia jasminoides fruits and the sclerotia from Inonotus obliquus. Tested at 100 µg/mL they were able to significantly reduce the Nile red fluorescence and extend the 50% survival rate (DT50) compared to the control groups. This phenotype-directed in vivo approach opens up new horizons for the selection of natural starting materials and the investigation of their active principles as fast drug discovery tool with predictive value for human diseases.

Synthetic Access to 3-Substituted Pyroglutamic Acids from Tetramate Derivatives of Serine, Threonine, allo-Threonine, and Cysteine.

A general route which provides direct access to pyroglutamates from tetramates, making use of Suzuki coupling on an enol mesylate, followed by reduction, is reported. This work permits direct scaffold hopping from tetramate to substituted pyroglutamates. Some compounds in the library showed modest antibacterial activity against Gram-positive bacteria.

Functionalised bicyclic tetramates derived from cysteine as antibacterial agents.

Routes to bicyclic tetramates derived from cysteine permitting ready incorporation of functionality at two different points around the periphery of a heterocyclic skeleton are reported. This has enabled the identification of systems active against Gram-positive bacteria, some of which show gyrase and RNA polymerase inhibitory activity. In particular, tetramates substituted with glycosyl side chains, chosen to impart polarity and aqueous solubility, show high antibacterial activity coupled with modest gyrase/polymerase activity in two cases. An analysis of physicochemical properties indicates that the antibacterially active tetramates generally occupy physicochemical space with MW of 300-600, clog D7.4 of -2.5 to 4 and rel. PSA of 11-22%. This work demonstrates that biologically active 3D libraries are readily available by manipulation of a tetramate skeleton.

Antibacterial Mimics of Natural Products by Side-Chain Functionalization of Bicyclic Tetramic Acids.

Tetramic acids with unsaturated acyl chains are widely found in natural products possessing a range of biological activities, and bicyclic tetramates represent a suitable scaffold to prepare simple mimics of such complex molecules. An efficient route to functionalize the C(6)-acyl group of a bicyclic tetramate was developed and utilized to prepare a small chemical library with a range of saturated and unsaturated side-chains. The analogues with lipophilic residues possessed highly potent antibacterial activity, which was selective for Gram-positive bacteria, and the best compound was 37-fold more potent than the cephalosporin C control and with an appropriate therapeutic window.

Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB.

BACKGROUND: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells. METHODS: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays. RESULTS: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21(WAF1/Cip1)) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway. CONCLUSION: According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.

Antimicrobial and anti-inflammatory activities of endophytic fungi Talaromyces wortmannii extracts against acne-inducing bacteria.

Acne vulgaris is the most common skin disease, causing significant psychosocial problems such as anxiety and depression similar to a chronic illness for those afflicted. Currently, obtainable agents for acne treatment have limited use. Thus, development of novel agents to treat this disease is a high medical need. The anaerobic bacterium Propionibacterium acnes has been implicated in the inflammatory phase of acne vulgaris by activating pro-inflammatory mediators such as the interleukin-8 (IL-8) via the NF-κB and MAPK pathways. Talaromyces wortmannii is an endophytic fungus, which is known to produce high bioactive natural compounds. We hypothesize that compound C but also the crude extract from T. wortmannii may possess both antibacterial activity especially against P. acnes and also anti-inflammatory properties by inhibiting TNF-α-induced ICAM-1 expression and P. acnes-induced IL-8 release. Treatment of keratinocytes (HaCaT) with P. acnes significantly increased NF-κB and activator protein-1 (AP-1) activation, as well as IL-8 release. Compound C inhibited P. acnes-mediated activation of NF-κB and AP-1 by inhibiting IκB degradation and the phosphorylation of ERK and JNK MAP kinases, and IL-8 release in a dose-dependent manner. Based on these results, compound C has effective antimicrobial activity against P. acnes and anti-inflammatory activity, and we suggest that this substance or the crude extract are alternative treatments for antibiotic/anti-inflammatory therapy for acne vulgaris.