Nowadays, a significant number of antiepileptic drugs aimed at influencing the main inhibitory transmitter – gamma-aminobutyric acid (GABA). Compounds with various chemical structures, binding to different GABA_A sites, potentiate the action of amino acid. Recent studies have reported that terpenoids such as l-menthone and its derivatives were found to act as modulators of GABA_A receptors, thereby demonstrating anticonvulsant activity. On the other hand, neuroprotective and anticonvulsant potentialities were revealed in phenoxyacetic acid derivatives. Based on the foregoing, the combination of l-menthone and phenoxyacetic acid residues into one molecule is feasible for obtaining the pharmacological agents with antiseizure action. In order to achieve the above-mentioned goal, l-menthone hydrazones were synthesized via condensation of terpenoid with 4-R-phenoxyacetic acid hydrazides in the presence of a catalytic amount of glacial acetic acid. The structure of the target compounds has been established by FTIR-ATR, Raman, ¹H-NMR and ¹³C-NMR spectral analysis and EI/FAB/ESI mass spectrometry. Thermal properties of hydrazones were elucidated by DSC and their purity ‒ by HPLC coupled to mass spectrometry. Synthesized compounds were found to exist as Z/E geometrical isomers about C=N bond and cis/trans amide conformers. At the present study, the influence of obtained derivatives on the central nervous system was reliably confirmed by evaluating their anticonvulsant activity. The present findings indicate that all aforementioned compounds possess antiseizure action after oral administration on PTZ-induced convulsion and maximal electroshock-induced (MES) seizures.

A series of tetrahydropyrido[2,1-b][1,3,5]thiadiazine derivatives exhibited high antidepressant and adaptogen activities. We now report a more detailed analysis of the effects of those substances on dopamine metabolism. For that purpose, we decided to unite two techniques: evaluation of the impact of compounds with antidepressant activity on levodopa effects and the test of suspension by the tail. Caffeine-sodium benzoate, amitriptyline, and fluoxetine were chosen as references.

Citrus suhuiensis is a citrus fruit with greenish yellow peel. It is consumed fresh but the peels are discarded as waste whereas they might also contain biological potentials. This study aimed to determine the vitamin C and E contents as well as the antioxidant, antibacterial, and antiproliferative properties of Citrus suhuiensis peel crude extract. Vitamin C and E contents were measured following the AOAC method. The total phenolic content was evaluated using Folin-Ciocalteau assay. Capability of the extract to scavenge free radical was elucidated using DPPH assay. Agar disk diffusion method was used to evaluate its antibacterial effect against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The antiproliferative effects of Citrus suhuiensis crude extract on MCF-7 (estrogen-dependent breast cancer), MDA-MB-231 (non-estrogen-dependent breast cancer), T84 (colon cancer) and MCF10a (normal fibroblast) cell lines were assessed via MTS assay. Vitamin C (19.18±0.14 mg/100 g) in C. suhuiensis peel was significantly higher than vitamin E (0.018±0.00 mg/100 g) (p<0.001). The total phenolic content was 108.00±1.00 mg GAEs/g sample. However, Citrus suhuiensis crude extract was unable to scavenge 50% of DPPH free radicals. The extract did not have antimicrobial effect against the bacterial strains tested. It inhibited 50% MCF-7 estrogen-dependent breast cancer and T84 colon cancer cell growth at 90.03±0.18 and 50.11±1.46 µg/ml, respectively. No inhibition was observed on MDA-MB-231 non-estrogen-dependent breast cancer and MCF10a normal fibroblast cell growth. In conclusion, although C. suhuiensis peel has very limited antibacterial and DPPH radical scavenging activities, it exhibited antiproliferative effect against estrogen-dependent breast cancer and colon cancer cell growth, what could be due to its vitamin C and E and total phenolic contents. Nevertheless, further investigations are required to increase understanding on the underlying mechanisms.

The huge impact of cancer is a big concern nowadays. Protein Kinases (PKCs) are attractive anticancer targets due to their involvement in several processes of carcinogenesis. Particularly, the isoform δ (PKC-δ) acts as tumor suppressor in colon cancer, one of the most dominant cancers and cause of cancer mortality worldwide [1].

Promising bioactive molecules were found in Plectranthus genus, mainly diterpene royleanones [1, 2]. The 7α-acetoxy-6β-hydroxyroyleanone (Roy) is the major constituent of P. grandidentatus acetonic extracts. Several biological activities of Roy were reported in the literature, including antitumoral activity [3]. Moreover, the presence of two free hydroxyl groups (position C-6 and C12) in Roy structure drawn our attention to the possibility of preparing new derivatives with enhanced cytotoxic activity. In fact, in a previous work, the patented diterpene 6β-benzoyloxy-12-O-benzoylroyleanone (RoyBz) [3], shown selective activation of PKC-δ [5].

The aim of the present work is to prepare new potential PKC-δ activators from derivatization of Roy. Thus, Roy and RoyBz assisted the design of theoretical derivatives, through modification of C-12 and C-6 hydroxyl groups. Molecular docking simulations were carried out against the 3D structure of human PKC-δ regulatory domain, to identify the potential PKC-δ activators. The most promising compounds accepted by docking simulations are currently been prepared by hemi-synthesis using Roy as starting material for structure-activity relationships.

References:

[1]. Ladeiras D et al. (2016) Curr. Pharm. Des. 22 (12): 1682.[2]. Matias D et al. (2019) ACS Omega. 4: 8094.[3]. Bernardes CES et al. (2018) Mol. Pharm. 15 (4): 1412.[4]. Saraiva L et al., “Roy-Bz: a small molecule activator of Protein Kinase Cdelta (PKCDelta)” Patent Ref PCT/IB2017/050633.[5]. Bessa C et al., Cell Death Dis. 2018, Vol. 9.

This work showcased the first physicochemical investigation of Psoralen (PSO) binding to double stranded DNA (dsDNA) through electroanalytical methods, whose results were further explored through a cheminformatic study. Results evidenced that PSO presents one non-reversible anodic peak at E_pa ≈ 1.42 V which is associated with its oxidation and the formation of an epoxide derivative, moreover, PSO analytical signal (i.e. faradaic current) decreases linearly with the addition of dsDNA while the electric potential associated to PSO oxidation shifts towards more positive values, indicating therefore that dsDNA addition hinders PSO oxidation. These findings were corroborated by the chemoinformatic study, which evidenced that PSO intercalates noncovalently between base-pairs of the DNA duplex, and then irreversibly form adducts with both DNA strands, leading up to the formation of a cross-link which bridges DNA helix, what explains the linear dependence between the faradaic current generated by PSO oxidation and the concentration of dsDNA in the test-solution, as well as the dependence between E_p and the addition of dsDNA solution. Therefore, the findings herein reported evidence the applicability of electroanalytical approaches such as voltammetry in the study of DNA intercalating agents.

According to the last WHO report, the level of bacterial resistance is alarming all over the world. Drug resistant diseases cause at least 700 000 deaths a year [1]. The synthesis of new antibacterial compounds is an important step to overcome this problem.

Shiff bases are a privileged structure possessing a wide spectrum of pharmacological activities such as anticancer, antioxidant and antibacterial [2]. The synthesis of 3‑[(2‑hydroxy‑5‑nitrobenzylidene)amino]‑2‑(2‑hydroxy‑5‑nitrophenyl)‑2,3‑dihydroquinazolin‑4(1H)‑one was described, and its spectral analysis, antioxidant and cytotoxic activities were presented [3]. The aim of this work was to study the antibacterial activity of this compound and its Cu(II) complex [4] against a spectrum of bacterial strains as well as the eradication activity against S. aureus biofilm.

The compounds showed moderate antibacterial activity, which was slightly higher in the case of Cu(II) complex. The dynamics of activity against S. aureus was analyzed using the time-kill curve method, and the activity was considered as bacteriostatic. Minimum biofilm eradication activity (MBEC₉₀) against staphylococcal biofilm was only twofold higher than MIC against planktonic cells, which shows a great potential of these derivatives as anti-biofilm active drugs.

This study was supported by the Ministry of Education of the Czech Republic (LO1305) and by project VEGA 2/0022/18.

References:

• No time to wait–securing the future from drug-resistant infections. Report to the Secretary General of the Nations, 2019, https://www.who.int/docs/default-source/documents/no-time-to-wait-securing-the-future-from-drug-resistant-infections-en.pdf?sfvrsn=5b424d7_6.
• Sztanke, K.; Maziarka, A.; Osinka, A.; Sztanke, M. An insight into synthetic Schiff bases revealing antiproliferative activities in vitro. Med. Chem. 2013, 21(13):2648–3666.
• Hricoviniova, Z. Hricovini, M.; Kozics, K. New series of quinazolinone derived Schiff’s bases: synthesis, spectroscopic properties and evaluation of their antioxidant and cytotoxic activity. Pap. 2018, 72(4): 1041–1053.
• Hricoviniova, Z. et al. manuscript in preparation

Efflux pumps are transmembrane transport proteins, ubiquitous in bacteria, that can actively extrude several antimicrobial drugs from within cells into the external environment, allowing bacteria to develop multidrug resistance. Efforts have been put towards a selective, efficient efflux pump inhibitor (EPI), and although some progress has been achieved, no EPIs have been approved in the therapeutic scenario [1]. This problem leads to the inefficacy of a large amount of antimicrobial drugs, with antimicrobial resistance posing one of the most urgent threats concerning health problems of our days.

Thioxanthones are heterocyclic, privileged structures with a dibenzo-γ-thiopyrone scaffold [2]. Previous work by our group has demonstrated the potential of these compounds as human efflux pump modulators [3].

In this scope, a virtual library of approximately 1000 thioxanthones was designed, and in silico studies were performed. The compounds that displayed good docking scores for bacterial efflux pumps and lower scores for P-glycoprotein were selected to be obtained by synthesis.

The synthesis of thioxanthones was performed using a copper-catalysed Ullmann coupling. With this strategy we expect to obtain a library of novel thioxanthones with potential to inhibit efflux pumps and revert multidrug resistance. The structures of the synthetized compounds were determined by ¹H and ¹³C NMR and X-ray crystallography.

Antibacterial activity and synergism assays with antibacterial drugs were performed, with two compounds displaying promising results in combination with antibacterial drugs, although with no relevant antimicrobial activity. Future studies will involve insights into the mechanism of synergy of promising compounds.

Acknowledgements: This work was developed under the Strategic Funding UID/Multi/04423/2019 and Project No. POCI-01-0145-FEDER-028736, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds.

REFERENCES

[1] Durães, F.; Pinto, M.; Sousa, E., Curr Med Chem 2018, 25, 6030.6069.

[2] Palmeira, A.; Vasconcelos, M. H.; Paiva, A.; Fernandes, M. X.; Pinto, M.; Sousa, E. Biochem Pharmacol 2012, 83 (1), 57-68.

[3] Palmeira, A.; Rodrigues, F.; Sousa, E.; Pinto, M.; Vasconcelos, M. H.; Fernandes, M. X. Chem Biol Drug Des 2011, 78 (1), 57-72.

The composition of Hypericum maculatum herb contains a wide range of biologically active compounds: flavonoids (hyperoside, rutin, quercetin), tannins, carotenoids, essential oil, ascorbic acid and others.

Flavonoids and vitamin C have antioxidant, capillary strengthening and venotonic effects, carotenoids exhibit a pronounced regenerative effect.

Two-phase extraction by a system of solvents of different polarity allows for two extracts containing both polar and lipophilic bioactive compounds from one raw material in one technological cycle.

The authors investigated the effect of the non-polar extractant (hexane and almond oil) type and the emulsifier (Tween 80) on the effectiveness of bioactive substances two-phase extraction from the herb Hypericum maculatum. Tween 80 is a non-ionic surfactant from the group of polysorbates. The optimal content of Tween 80 in the extractant system was 1 ml per 1 g of raw material. The ratio of raw materials: polar phase: non-polar phase was 1: 10: 10. The quantitative content of flavonoids was determined in the studied polar phases by spectrophotometry, and carotenoids in non-polar phases.

It was found that flavonoids were more efficiently extracted with a system of extractants of 70% ethanol: almond oil. The addition of an emulsifier significantly increased the release of flavonoids and carotenoids during the extraction process. Tween 80 contributed to an increase in the solubility inside the particles of raw materials of both lipophilic and polar bioactive substances and facilitated their subsequent diffusion into the extractant.

Aim/Introduction:

Radiolabeled fluorescent dyes are crucial for bimodal imaging and currently in demand bimodal imaging (scintigraphic and optical imaging). Organic fluorescent dyes show unique optical properties such as high quantum yields, suitable stokes shifts and high extinction coefficients (1). Organic fluorophores as ICG or 5-ALA are well-known for fluorescent light-guided intraoperative surgery. The goal of this work is the development of new near-infrared (NIR) dyes for scintigraphic and optical imaging. The dyes are belonging to the silicon-rhodamine familiy. We have synthesized these first-in-class dyes for their subsequent conjugation to the SPECT-compatible radiometal technetium-99m with the aim to elucidate their potential as sentinel lymph node detecting agents. Moreover, PET-compatible Si-rhodamine conjugation to exemplary prominent tumor targeting binding vectors such as the PSMA-617-binding motif has also been performed. The introduced small molecule based dyes are intended to be used for noninvasive SPECT imaging (prestaging) followed by light-guided R0-tumor-resection.

Materials and Methods:

The combination of scintigraphic and optical imaging leads to new approaches in tumor imaging and its resection. This powerful strategy enables the differentiation of healthy and affected tumor tissues. We have developed dyes with absorption and emission properties in the near-infrared region of ca. 660 nm. We utilized the dyes for copper(I)-catalyzed alkyne-azide [3+2]-cycloaddition to receive respective 1,2,3-triazoles for complexing the well-known SPECT-radiometal technetium-99m using the click-to-chelate concept (2). Furthermore we have designed another DOTA-functionalized 1,2,3-triazole for complexing the PET-radiometal gallium-68. Finally, the dyes were characterized using NMR-, UV/VIS/NIR-spectroscopy and mass spectrometry.

Results:

The conventional synthesis of novel Si-rhodamines through xanthone building blocks provided novel amino- and azide-functionalized Si-rhodamines with overall yields of 60%. The azide-functionalized Si-rhodamines were converted with PSMA-inhibitor functionalized alkynes adapted from the PSMA-617 binding motif and L-propargylglycine to the corresponding 1,2,3-triazoles (3). Already determined extinction coefficients up to 120.000 M^-1cm^-1 and quantum yields of 0.45 show promising results, making them potentially useful for optical imaging. Furthermore the dyes were prepared as precursors for technetium-99m and gallium-68-labeling. For that purpose the prominent PET-active gallium-68 chelator DOTA was used. Corresponding rhenium-Si-rhodamines [used as “cold” technetium-surrogate] were characterized as well.

Conclusion:

A variety of novel NIR fluorescent dyes based on the Si-rhodamine lead structure were synthesized and chemically characterized. We successfully developed non-radioactive rhenium analogues of putative NIR rhodamine dyes for bimodal scintigraphic and optical imaging. Furthermore, our first-in-class radiolabeled silicon dyes are subject of current and future biological evaluation.

References:

(1) T. Nagano et al., J. Am. Chem. Soc. 2012, 134, 5029–5031.

(2) T. M. Mindt et al., J. Am. Chem. Soc. 2006, 47, 15096–15097.

(3) K. Kopka et al., J. Nucl. Med. 2015, 56, 914–920.

Combretastatin A-4 (CA-4), a natural stilbene is a potent microtubule-disrupting agent, which binds at the colchicine-binding site of tubulin. The design, synthesis and biochemical evaluation of a series of analogues of the microtubule-destabilising agent CA-4 is described. The monocyclic β-lactam CA-4 analogues containing halogen substituents at the C-3 position of β-lactam ring were synthesized using the Staudinger reaction. Previous investigations described two approaches for the construction of 3-fluoro-β-lactams using the ketene-imine condensation or the enolate-imine condensation method. In the present work, a ready synthesis of 3-fluoro- and 3,3-difluoro-substituted β-lactams was developed by a convenient microwave-assisted Reformatsky reaction using ethyl bromofluoroacetate and ethyl bromodifluoroacetate respectively. To the best to our knowledge, this is the first report of this new synthetic approach for 3-fluoro- and 3,3-difluoro-β-lactams as CA-4 analogues. The reaction was successful with short reaction time compared to the conventional Staudinger reaction, moderate yields and few steps. It is demonstrated to be a convenient and facile method for the preparation of a variety of 3-fluoro- and 3,3-difluoro-β-lactams. The 3-fluoro-β-lactams (1-8) and 3,3-difluoro-β-lactams (9-16) in this series contain the 3,4,5-trimethoxyphenyl ring A, (required for potent activity of CA-4), together with various ring B substituents. The structure of β-lactams 6 and 14 was confirmed by X-ray crystallography. Preliminary cell viability studies demonstrated the antiproliferative effects of these novel compounds, with IC₅₀ value of 0.153 µM for 6 in MCF-7 human breast cancer cell line.