Synthesis and anticancer activity of novel 3,6-disubstituted 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives

AbstractThe development of new antitumor agents is one of the most pressing research areas in medicinal chemistry and medicine. The importance of triazole and thiadiazole rings as scaffolds present in a wide range of therapeutic agents has been well reported and has driven the synthesis of a large number of novel antitumor agents. The presence of these heterocycles furnishes extensive synthetic possibilities due to the presence of several reaction sites. Prompted by these data we designed, synthesized and evaluated a series of novel 3,6-disubstituted 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives as potential anticancer agents. We emphasized in the strategy of combining two chemically different but pharmacologically compatible molecules (the 1,2,4-triazole and 1,3,4 thiadiazole) in one frame. Several of the newly synthesized 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole derivatives showed substantial cytostatic and cytotoxic antineoplastic activity invitro, while they have produced relatively low acute toxicities invivo, giving potentially high therapeutic ratios. Insilico screening has revealed several protein targets including apoptotic protease-activating factor 1 (APAF1) and tyrosine-protein kinase HCK which may be involved in the biological activities of active analogues

Novel Hit Compounds as Putative Antifungals: The Case of Aspergillus fumigatus

The prevalence of invasive fungal infections has been dramatically increased as the size of the immunocompromised population worldwide has grown. Aspergillus fumigatus is characterized as one of the most widespread and ubiquitous fungal pathogens. Among antifungal drugs, azoles have been the most widely used category for the treatment of fungal infections. However, increasingly, azole-resistant strains constitute a major problem to be faced. Towards this direction, our study focused on the identification of compounds bearing novel structural motifs which may evolve as a new class of antifungals. To fulfil this scope, a combination of in silico techniques and in vitro assays were implemented. Specifically, a ligand-based pharmacophore model was created and served as a 3D search query to screen the ZINC chemical database. Additionally, molecular docking and molecular dynamics simulations were used to improve the reliability and accuracy of virtual screening results. In total, eight compounds, bearing completely different chemical scaffolds from the commercially available azoles, were proposed and their antifungal activity was evaluated using in vitro assays. Results indicated that all tested compounds exhibit antifungal activity, especially compounds 1, 2, and 4, which presented the most promising minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values and, therefore, could be subjected to further hit to lead optimization

Thermal, dynamic and structural properties of drug AT1 antagonist olmesartan in lipid bilayers

It is proposed that AT1 antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT1 receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC), 13C magicangle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles. 13C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as 13C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche:trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile

Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

AbstractDrug–membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution 1H and 13C nuclear magnetic resonance (NMR) and solid state 13C and 31P (NMR) spectroscopies. In addition, 31P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT1 receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques

The use of physicochemical methodologies to study the conformational analysis of peptide analogs against multiple sclerosis and their interactions with lipid bilayers

The aims of this dissertation are to: (a) study the phenomenon of partial interdigitation for two synthetic peptides possessing different biological activity against Autoimmune Encephalomyelitis (EAE), namely agonistic and antagonistic respectively and (b) analyze the conformational characteristics of two synthetic peptides analogues of Μyelin Βasic Protein and their stereoelectronic interactions with their active site in order to understand their biological response. Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS) characterized by discrete areas of inflammation and demyelination that can occur in multiple anatomical locations in the CNS. Although the antigenic components of myelin in MS have not been identified with certainty yet, Myelin Basic Protein (MBP) is believed to be one of the main autoantigens. MBP87-99 epitope is encephalitogenic in Experimental Autoimmune Encephalomyelitis (EAE), a prototypic model for induced autoimmune diseases, considered as an instructive model for MS in humans. A promising approach in the treatment of MS is the use of Altered Peptide Ligands (APLs) derived from the proteins of myelin sheath, mainly from the MBP. The myelin-specific T-cells induce paralysis and demyelination when they are triggered by the native MBP. When these T cells recognize the APL, they produce interleukin IL-4, which can reverse EAE. Clinical signs of EAE induced by MBP72–85 were completely suppressed by the linear APL [Arg91, Ala96] MBP87–99 . MBP87-99 epitope peptide and its antagonist [Arg91, Ala96] MBP87-99 interactions with lipid bilayers of dipalmitoylphosphatidylcholine (DPPC) bilayers were investigated using Differential Scanning Calorimetry (DSC) and high resolution solid state 13C and 31P NMR spectroscopy in an attempt to explore the role of the lipid bilayers in their physiological action and more specifically their ability to exert partial interdigitation. For the Differential Scanning Calorimetry results molar ratios from 1% up to 10% have been used in order to explore the role of the concentration in the activity. The greatest thermal modifications are observed for the agonist and at the molar ratio of 5%. The two peptides caused partial interdigitaion in the lipid bilayers at concentrations ? 5% molar ratio with the agonist to exert a greater degree of partial interdigitation. From the two peptides studied only agonist’s effect is saturated as it is depicted from the observed two discrete areas when a molar ratio of 10% is used. In the case of antagonist, only a significant broadening in the breadth of the phase transition is observed. ....................................................................................................Στόχοι της διδακτορικής διατριβής είναι να μελετηθούν: (α) το φαινόμενο της μερικής ενδοεπιχώρησης σε λιπιδικές διπλοστιβάδες που προκαλούν δύο συνθετικά πεπτίδια τα οποία παρουσιάζουν αγωνιστική και ανταγωνιστική δράση αντίστοιχα κατά της Πειραματικής Αυτοάνοσης Εγκεφαλομυελίτιδας (Experimental Autoimmune Encephalomyelitis, EAE), ένα μοντέλο της Σκλήρυνσης Κατά Πλάκας σε ζώα και (β) η αλληλεπίδραση δύο συνθετικών πεπτιδικών αναλόγων της Βασικής Πρωτεΐνης της Μυελίνης (ΜBP), στο ενεργό κέντρο δράσης τους ώστε να κατανοηθούν οι στερεοηλεκτρονιακές αλληλεπιδράσεις που οδηγούν στη βιολογική τους δράση. Η Σκλήρυνση Κατά Πλάκας (ΣΚΠ) είναι μία χρόνια ασθένεια του Κεντρικού Νευρικού Συστήματος (ΚΝΣ) που επηρεάζει τους νευρώνες, τα κύτταρα του εγκεφάλου και το νωτιαίο μυελό, τα οποία διαβιβάζουν πληροφορίες, δημιουργούν τη σκέψη, την αντίληψη και επιτρέπουν στον εγκέφαλο να ελέγξει το σώμα. Τα νευρικά κύτταρα περιβάλλονται από ένα λεπτό περίβλημα, το οποίο αποτελείται από μυελίνη, μια ουσία που βοηθά στην προστασία των νεύρων καθώς και στη μετάδοση των νευρικών ώσεων. Η ΣΚΠ προκαλεί σταδιακή καταστροφή της μυελίνης (απομυελίνωση), με αποτέλεσμα οι νευρώνες να μην μπορούν να μεταδώσουν αποτελεσματικά τα ηλεκτρικά τους σήματα. Μία ελπιδοφόρα προσέγγιση στην αντιμετώπιση της ΣΚΠ περιλαμβάνει τη σύνθεση σταθερών πεπτιδικών αναλόγων τα οποία εμφανίζουν αυξημένη ανταγωνιστική βιολογική δράση. Τα τροποποιημένα πεπτίδια (Altered Peptide Ligands – APLs) σχεδιάζονται και συντίθενται με βάση ανοσοκυρίαρχες περιοχές πρωτεϊνών της μυελίνης όπως της MBP, PLP και MOG. Στη παρούσα μελέτη διερευνήθηκαν οι αλληλεπιδράσεις του πεπτιδικού επιτόπου της Βασικής Πρωτεΐνης της Μυελίνης (Myelin Basic Protein, MBP) MBP87-99, ο οποίος παρουσιάζει αγωνιστική δράση, και του ανταγωνιστή MBP87-99 (Arg91, Ala96) με τις λιπιδικές διπλοστιβάδες. Πραγματοποιήθηκαν πειράματα Διαφορικής Θερμιδομετρίας Σάρωσης, Πυρηνικού Μαγνητικού Συντονισμού (NMR) στερεής κατάστασης και Φασματοσκοπίας Raman. Το λιπίδιο που χρησιμοποιήθηκε για τη δημιουργία των διπλοστιβάδων είναι η διπαλμιτική φωσφατιδυλοχολίνη (DPPC). Στα πειράματα Διαφορικής Θερμιδομετρίας Σάρωσης χρησιμοποιήθηκαν μοριακές αναλογίες πεπτιδίου ως προς DPPC από 1-10%. Σε όλο το εύρος των συγκεντρώσεων που μελετήθηκαν, και ειδικά σε συγκέντρωση 5%, προκύπτει ότι ο αγωνιστής προκαλεί μεγαλύτερες μεταβολές στις διπλοστιβάδες της DPPC, σε σχέση με τον ανταγωνιστή. Υπάρχουν ισχυρές ενδείξεις ότι τα δύο πεπτίδια προκαλούν μερική ενδοεπιχώρηση στις λιπιδικές διπλοστιβάδες σε συγκεντρώσεις ? 5%, με τον αγωνιστή να προκαλεί μερική ενδοεπιχώρηση σε μεγαλύτερο βαθμό. ................................................................................................

Structure-Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of denovo SBD and DCC constitutes an efficient starting point for hit identification and optimization

Polymerization of Higher a-Olefins Using a Cs-Symmetry Hafnium Metallocene Catalyst. Kinetics of the Polymerization and Microstructural Analysis

The Cs-symmetry hafnium metallocene [(p-Et3Si)C6H4]2C(2,7-di-tert-BuFlu) (C5H4)Hf(CH3)2 and tetrakis(pentafluorophenyl) borate dimethylanilinium salt ([B(C6F5)4] [Me2NHPh]þ) were used as the catalytic system for the polymerization of higher a-olefins (from hexene-1 to hexadecene-1) in toluene at 0 C. The evolution of the polymerization was studied regarding the variation of the molecular weight, molecular weight distribution and yield with time. The effect of the monomer structure on the polymerization kinetics was established. The role of trioctylaluminum in accelerating the polymerization was investigated. 13C NMR spectroscopy was used to study the microstructure of the poly(a-olefins) by the determination of the pentad monomer sequences. The thermal properties of the polymers were obtained by differential scanning calorimetry, DSC. The results were discussed in connection with the polymer microstructur

Probing for missing links in the binary and ternary V(V)-citrate-(H 2O 2) systems: Synthetic efforts and in vitro insulin mimetic activity studies

In a pH-specific fashion, V 2O 5 and citric acid in the absence and presence of H 2O 2 reacted and afforded, in the presence of NaOH and (CH 6N 3) 2CO 3, two new dinuclear V(V) binary non-peroxo (CH 6N 3) 6[V 2O 4 (C 6H 4O 7) 2] · 2H 2O (1) and ternary peroxo (CH 6N 3) 4[V 2O 2 (Ο 2) 2(C 6H 5O 7) 2] · 6Η 2Ο (2) species, respectively. Complexes 1 and 2 were further characterized by elemental analysis, UV/Vis, FT-IR, NMR (solution and solid state Cross Polarization-Magic Angle Spinning (CP-MAS)) and Raman spectroscopies, cyclic voltammetry, and X-ray crystallography. Both 1 and 2 are members of the family of dinuclear V(V)-citrate species bearing citrate with a distinct coordination mode and degree of deprotonation, with 2 being the missing link in the family of pH-structural variants of the ternary V(V)-peroxo-citrate system. Given that 1 and 2 possess distinct structural features, relevant binary V(III), V(IV) and V(V), and ternary V(V) species bearing O- and N-containing ligands were tested in in vitro cell cultures to assess their cellular toxicity and insulin mimetic capacity. The results project a clear profile for all species tested, earmarking the importance of vanadium oxidation state and its ligand environment in influencing further binary and ternary interactions of vanadium arising with variable mass cellular targets, ultimately leading to a specific (non)toxic phenotype and glucose uptake abilit

Insights into AT₁ Receptor Activation through AngII Binding Studies

This study investigates the binding of angiotensin II (AngII) to the angiotensin II type 1 receptor (AT₁R), taking into consideration several known activation elements that have been observed for G-protein-coupled receptors (GPCRs). In order to determine the crucial interactions of AngII upon binding, several MD simulations were implemented using AngII conformations derived from experimental data (NMR ROEs) and <i>in silico</i> flexible docking methodologies. An additional goal was to simulate the induced activation mechanism and examine the already known structural rearrangements of GPCRs upon activation. Performing MD simulations to the AT₁R – AngII – lipids complex, a series of dynamic changes in the topology of AngII and the intracellular part of the receptor were observed. Overall, the present study proposes a complete binding profile of AngII to the AT₁R, as well as the key transitional elements of the receptor and the agonist peptide upon activation through NMR and <i>in silico</i> studies

Insights into AT₁ Receptor Activation through AngII Binding Studies

This study investigates the binding of angiotensin II (AngII) to the angiotensin II type 1 receptor (AT₁R), taking into consideration several known activation elements that have been observed for G-protein-coupled receptors (GPCRs). In order to determine the crucial interactions of AngII upon binding, several MD simulations were implemented using AngII conformations derived from experimental data (NMR ROEs) and <i>in silico</i> flexible docking methodologies. An additional goal was to simulate the induced activation mechanism and examine the already known structural rearrangements of GPCRs upon activation. Performing MD simulations to the AT₁R – AngII – lipids complex, a series of dynamic changes in the topology of AngII and the intracellular part of the receptor were observed. Overall, the present study proposes a complete binding profile of AngII to the AT₁R, as well as the key transitional elements of the receptor and the agonist peptide upon activation through NMR and <i>in silico</i> studies