The parasite Trypanosoma brucei, ethiologic agent of human African trypanosomiasis (i.e. sleeping sickness), contains a kinetoplast with the mitochondrial DNA (kDNA) comprising of >70 % AT base pairs. Hence, DNA minor groove binding molecules have been investigated as antitrypanosomal agents. Diphenyl-based bis(2-iminoimidazolidines) are promising DNA minor groove binders that are curative in mouse models of stage 1 trypanosomiasis but devoid of activity in the late(CNS)-stage disease, possibly due to poor brain penetration caused by their dicationic nature.

As a strategy to reduce the pK_a of the basic 2-iminoimidazolidine groups, halogen atoms (R₁ = Cl, F) were introduced in the structure of lead compound 1 and the pK_a of the new compounds was determined . A reduction of 1–2 pK_a units for the imidazolidine group linked to the substituted phenyl ring was observed. In vitro activities (EC₅₀) against wild type and resistant strains of T. b. brucei were in the submicromolar range with four compounds being more active and selective than 1 (SI > 340).¹ The chloro-substituted derivative 5a, which was curative in vivo in a mouse model of stage 1 infection by T. b. rhodesiense, appeared as a new promising lead compound.²

Mechanistic studies were performed to identify the cellular target of these dicationic compounds. Altogether, our results show that 1 and 5a share the same mechanism of action against T. brucei, acting specifically on the integrity of the kinetoplast by altering the structure and replication of kDNA.²

Saquinavir mesylate (SQM) is subjected to forced degradation under conditions of hydrolysis, oxidation, dry heat, photolysis as recommended by International Conference on Harmonization guideline Q1A (R2). In total, (I-V) degradation products (DPs) were formed in acidic hydrolytic, alkaline hydrolytic and oxidative conditions. Successful separation of SQM and its DPs was achieved on C₁₈(4.6mm×75mm) 3.5µg column at ambient temperature (30˚C) with mobile phase A (10mM ammonium acetate in water), B100% acetonitrile at 2.0ml/min flow rate in the gradient mode. The injection volume was fixed at 20µl and detection wavelength at 238nm. The HPLC method was found to be linear, accurate, precise, sensitive, specific, rugged, and robust for quantification of SQM as well as degradation products. The major degradation products (DP-1) formed under hydrolytic acid conditions were identified and characterized by LC-MS/MS. Further, DP-1 were isolated through column chromatography and analyzed by ¹H NMR. In silico molecular docking studies on HIV protease (PDB: 4qgi) for DPs and SQM as well as prediction of toxicity and ADME properties were performed.

Systemic fungal infections are increasingly common, especially in immune compromised patients. Even with newly developed drugs, there remain issues of limited spectrum, side effects, and the development of resistance. Host defense peptides (HDPs) have been examined recently for their utility as therapeutic antifungals, especially due to the low levels of resistance that develop. Unfortunately, the peptides exhibit poor pharmacologic properties in vivo. We have demonstrated the potent activity of nonpeptidic compounds that mimic HDPs in both structure and function against clinical strains of Candida albicans associated with oral and invasive candidiasis in mouse models. However, to test numerous compounds in vivo requires large numbers of mice, with multiple time points, and requires immunosuppression of the mice using cyclophosphamide, which can influence pharmacological parameters. We have identified a strain of mouse that develops invasive candidiasis without the need for immunosuppressive drugs. When we infect these mice with a strain of C. albicans that constitutively expresses Red Fluorescent Protein, we can quantify the infection in real time by in vivo imaging. We can further observe the reduction in fluorescence in infected mice after treatment with an HDP mimetic. Together our results demonstrate a novel in vivo method for screening new antifungal drugs.

Oligonucleotides antiviral drugs have been actively implemented in medicine during the last decades nevertheless the molecular mechanism of their action is still unclear. As it was shown in our previous work, the combination of oligonucleotides with alcohol sugar D-mannitol leads to changes in their biological activity and efficiency. At this stage of our investigation, we studied the ability of oligoribonucleotides from total yeast RNA (ORNs) and oligoribonucleotides-D-mannitol complexes to affect the conformation and stability of interferon (IFN) α-2b – a key protein of the antiviral cell defense mechanism.

To investigate that interaction, conformational changes and stability of IFN protein in the presence and/or absence of the ligands were studied by fluorescence and CD (circular dichroism) spectroscopies. All experiments were performed on spectrofluorometer Jasco FP- 8200 and CD spectrometer Jasco J-815 with a Peltier temperature cell holder. Obtained thermal denaturation profiles of IFN α–2b alone and in the presence of ORNs and ORNs-D-mannitol complexes show that the addition of these ligands led to an increase of thermal stabilization of protein of 2 and 1.8 ⁰C respectively. The dissociation constant between INF and total yeast ORNs was Kd =2.88±1.14µM and between INF and ORNs with D-mannitol – Kd =0,92±0.23µM.

The analysis of IFN secondary structure changes by Bestsel shows that addition of ORNs or ORNs-D-mannitol complexes led to a decrease of α-helix components in the protein structure and to an increase of antiparallel β-stand, β-turn, and random coil components. At the same time, the analysis of the tertiary structure shows that adding ORNs-D-mannitol changes the architecture of the protein from the 2-layer sandwich to alpha-beta complex. On the other hand, adding ORNs did not cause any change in the tertiary structure.

We suppose that total yeast ORNs and ORNs-D-mannitol complexes act as compounds, altering the secondary and tertiary structures of the interferon and in this way can change its biological activity.

Cancer is a group of diseases that can affect any part of the body via an uncontrolled and anomalous cellular proliferation. In this research field, the tumor protein p53 is a widely-studied therapeutic target in cancer treatment, as this transcription factor is inactivated in all types of human cancers. In 50% of malignancies, p53 is found expressed in its wild-type form and generally inhibited by two major negative regulators, MDM2 and MDMX. In the remaining 50% of cases, p53 is inactivated by contact and conformational mutations principally on its DNA-binding site, thus not exercising its regulatory function. [1] In the last years, our research group has been actively involved in the synthesis of small molecules to reactivate the p53 pathway. Starting from the enantiopure aminoalcohol tryptophanol, we have recently developed several small molecules that reactivate p53 (Figure 1). Here we present our most updated results on the development of a chemical library of (S)- and (R)-tryptophanol-derived oxazoloisoindolinones. This class of compounds may be accessed by cyclocondensation reaction of enantiopure forms of tryptophanol and several achiral oxoacids. In this synthetic approach, the chiral inductor (tryptophanol) is responsible for the stereo-outcome of the final product and it is part of the main skeleton of the bioactive molecules. For those reasons, this asymmetric reaction is highly efficient/atom economic. Interestingly, this specific one-step synthetic strategy allows to the construction of a new chiral center. [2] From this work tryptophanol-derived bicyclic lactams SLMP53-1 and DIMP53-1 were identified as the most promising p53 reactivators. [3] Further hit-to-lead optimization is ongoing, and assessment of the antiproliferative activity of the optimized oxazoloisoindolinones against four different cancer cells lines highlights that this chemical family displays selectively potent antitumor activity towards p53 with no apparent toxic effects.

Acknowledgements

Fundação para a Ciência e a Tecnologia (FCT) through PTDC/DTP-FTO/1981/2014, PTDC/QUI-QOR/29664/2017, UID/DTP/04138/2013, PD/BI/135334/2017 and IF/00732/2013.

References

[1]. Espadinha M, Barcherini V, Lopes E A, Santos M M M (2018). Curr Top Med Chem 18: 647-60.

[2]. Dourado J, Pérez M, Griera R, Santos M M M (2016). RSC, Chapter 3.1.19, 198-201.

[3]. a) Soares J, Raimundo L, Pereira N A L, Monteiro A, Gomes S, Bessa C, Pereira C, Queiroz G, Bisio A, Fernandes J, Gomes C, Reis F, Gonçalves J, Inga A, Santos M M M, Saraiva L (2016). Oncotarget 7(4): 4326-43; b) Soares J, Espadinha M, Raimundo L, Ramos H, Gomes A S, Gomes S, Loureiro J B, Inga A, Reis F, Gomes C, Santos M M M, Saraiva L (2017). Molecular Oncology 11(6): 612-27.

The family of ionotropic glutamate receptors (iGluRs) is localized in the cell membrane of neurons and has crucial roles in the normal development of the central nervous system (CNS). Sustain healthy memory, learning, and cognitive processes are fundamental functions of these receptors. [1] N-Methyl-D-aspartate (NMDA) receptors belong to the family of iGluRs and its over-activation is associated to neuronal loss and, consequently, to major neurological disorders such as Parkinson and Alzheimer’s diseases. Recently, targeting the NMDA receptor was considered a promising strategy in the medicinal chemistry field and the development of effective NMDA receptor antagonists become an attractive therapeutic approach. [2]

In the last years, Santos’ group has been involved in the design and development of potent NMDA receptor antagonists, more precisely enantiopure bicyclic lactams. [3-5] To evaluate the activity of the potential NMDA receptor antagonists, was measured their capacity to inhibit NMDA-induced increase of intracellular Ca²⁺ levels in in vitro cultures of embryonary rat cortical neurons, using the Ca²⁺-sensitive fluorescent dye Fluo-4. The first molecule that showed some interesting results was a (S)-phenylalaninol oxazolopyrrolidone. [3] After, based on the oxazolopyrrolidone scaffold, a hit-to-lead optimization was carried out in the search for more potent NMDA receptor antagonists. A new library of enantiopure phenylalaninol bicyclic lactams was developed and most of the new compounds displayed NMDA receptor antagonism. It was even more interesting the significant difference in activities between the two enantiomers. The most promising compound showed an IC₅₀ value of 27 µM, on the same order of magnitude as that of memantine (47 µM), an NMDA receptor antagonist in clinical use for the treatment of Alzheimer’s disease. [5] More recently, we also extended our interest to more rigid molecules, also containing a bicyclic lactam core. Interestingly, this new family of compounds showed to be even more potent as NMDA receptor antagonists (4-fold more active than memantine). Additional biological tests indicated that the promising compounds can cross the blood-brain barrier (determined by an in vitro assay) and non-hepatotoxic, as well. Furthermore, the synthesis of the interesting aminoalchool-based libraries is easy to perform, resulting in moderate to good yields, and excellent stereoselectivities.

Acknowledgements:

We thank the Fundação para a Ciência e Tecnologia for financial support through iMed.ULisboa (UID/DTP/04138/2013), research project PTDC/QUI-QUI/111664/2009 and to FCT fellowship SFRH/BD/117931/2016 (M. Espadinha). We also want to thank for financial support through Instituto de Salud Carlos III, Spanish Ministry of Health (PI13/00789), and Spanish Ministry of Economy and Competitiveness (MINECO, grants SAF2012-31035 and SAF2015-64948-C2-1-R).

References:

[1] Stawski P., Janovjak H., Trauner D. Bioorg. Med. Chem. 2010, 18, 7759-7772.

[2] Morris R. Neuropharmacology, 2013, 74, 32-40.

[3] Pereira N. A. L., Sureda F. X., Turch m., Amat M., Bosch J., Santos M. M. M. Monatsh. Chem. 2013, 144, 473-477.

[4] Pereira N. A. L., Sureda F. X., Pérez M., Amat M., Bosch J., Santos M. M. M. Molecules, 2016, 21, 1027.

[5] Espadinha M., Dourado J., Lajarin-Cuesta R., Herrera-Arozamena C., Gonçalves L., Rodríguez-Franco I., de los Ríos C., Santos M. M. M. ChemMedChem. 2017, 12, 537-545.

Propranolol [(R,S)-1-isopropylamino-3-(1-naphthoxy)-2-propanol], is a well-known beta-adrenergic blocking agent used for treatment of arterial hypertension and other cardiovascular disorders [Prichard et al. BMJ-British Medical Journal. 1964, 2, 725-727], which is commercially available as a racemic mixture. However, it is also well proven that only the (S)-enantiomer has the desired therapeutic effect [Howe et al. J. Med. Chem. 1968, 11, 1118-1121]; therefore, many stereoselective synthetic protocols for the preparation of the (S)-eutomer can be found in literature, in most cases the introduction of chirality being mediated by an enzymatic resolution of the chemically-prepared racemate [Bevinakatti et al. J. Org. Chem. 1991, 56, 5372-5375; Pamies et al. J. Org. Chem. 2002, 67, 9006-9010]. Generally speaking, the resolution should preferentially be carried out in the first steps of the synthetic scheme, on a precursor of the desired target drug such as the racemic aryloxyhalohydrines, easily prepared by opening epychlorhydrine with an aromatic alcohol.

In this communication we will present the kinetic resolution of aryloxyhalohydrines (precursors of propranolol and other beta-adrenergic blockers) by lipase-catalysed stereoselective transterification with enol esters. A factorial design of experiments was undertaken to assess best reaction conditions (temperature, solvent, acyl donor, …) for the efficient separation of enantiomers, both of them useful for therapeutic purposes; in fact, besides the previously antihypertensive activity of (S)-propranolol, the correspondent (R)-antipode displays a stronger antiarrhythmic and membrane-stabilizing effect [Wang et al. Circ.-Arrhythmia Electrophysiol. 2008, 1, 370-378], and it is also useful as a vaginal contraceptive [Borumand et al. J. Drug Deliv. Sci. Technol. 2014, 24, 637-644]. Through this stereoselective enzymatic acylation, the corresponding halohydrine ester and remnant alcohol can be easily separated and efficiently transformed into both enantiomers of propranolol.

Cancer is the second leading cause of death worldwide, killing an estimated 1 in 6 people. Ellipticine (1) is a natural product which has potent anticancer activity and has been subject to extensive study since its discovery, in 1959, with the key aim of identifying derivatives with clinical application.

Functionalisation of the ellipticine pharmacophore is key to developing potent and selective analogues. For example, generation of quaternary ellipticine salts, helps to overcome issues surrounding solubility and can improve selectivity whereas the most potent anticancer ellipticine derivatives have a hydroxyl or methoxy substituent at the 9-position. This work outlines the synthesis of quaternary ellipticine salts and their subsequent biological evaluation. Alkyl groups were introduced at the 6-position, as well as formyl or hydroxy groups at the 9-position, as these substituents have been previously shown to improve activity.

Biological evaluation encompassed measurement of growth inhibition against twelve cancer cell lines and submission to the NCI 60 Cell Lines Screen. Substitution at the 9-position greatly improved activity, while increasing substituent size at the 6-position led to lower potency. A number of potent derivatives have been identified following biological evaluation, with long chain alkyl salts displaying sub-micromolar average GI₅₀ values.

Glycogen synthase kinase-3 (GSK-3) refers to a group of multifaceted serine/threonine protein kinases that, in mammals, exist as two isoforms (GSK-3a and GSK-3b). Both isoforms share very similar homology but represent contrasting pharmacology. The quest for targeted GSK-3 inhibition has recently become a mainstay for pharmaceutical companies due to the enzymes’ role in a multitude of under-addressed disease states including cancer, Alzheimer’s and bipolar disorder.

Herein, we describe the synthesis and evaluation of novel indole derivatives as anticancer agents. A bisindolyl template has been derived, starting from a substituted maleimide, through the introduction of an oxygen atom to the headgroup (hydroxymaleimide). Assessing the bioactivity of these derivatives through kinase assays allowed for the identification of substituent derived selectivity. Following on from this, indole substitution was completed and assessed with the identification of unique selectivity patterns in the GSK-3 and CDK kinase assays. Subsequent evaluation of anticancer activity utilising the NCI-60 cell screen showed growth inhibitory profiles towards a multitude of cell lines including: SNB-75 CNS cancer, A498 and UO-31 renal, MDA MB435 melanoma and a panel of leukemia cell lines. Achieving selective kinase inhibition through modulation of this bisindolyl template is evident and will inform future selective clinical candidates.

The 3,4,5-trimethoxyphenyl moiety is a common motif employed in anticancer drug discovery, due to its prevalence in a variety of important natural products such as Combretastatin. Work undertaken by our group and others has demonstrated that structural diversification of this template can lead to potent anticancer activity.

The synthesis and biological evaluation of a series of novel indole-trimethoxyphenyl derivatives are described herein. The consolidation of the combretastatin and bisindolyl templates towards the inclusion of a novel heterocyclic headgroup proffered a versatile pharmacophore with which to pursue chemical diversification. Rationalising the enhancement of existing H-bonding interactions or potential exploitation of new contacts, the introduction of substituted maleimides constituted an overarching theme. This allowed for the evaluation of the effects pertaining to oxygen insertion, extended maleimide substitution and N-functionalisation. Photo-mediated dehydrogenation of a key synthetic intermediate offered access to trimethoxyphenylcarbazoles, representing the first time a panel of such congeners has been reported with further derivatisation also possible.

Subsequent evaluation of anticancer activity of the indole-trimethoxyphenyl conjugates utilising the NCI-60 cell screen showed growth inhibitory profiles towards numerous cell lines including: A498 renal, IGROV1 ovarian, DU-145 prostate, SW-620 colon and MCF-7 breast cancer cell lines. The influence of structure on anticancer activity is described.