The occurrence and properties of the thiazole ring in various natural and synthetic products have been the interest of many research groups on account of its useful biological properties [1]. In this context, our research group is mainly invested in the synthesis of C,N,S-containing bioactive molecules able to modulate the activity of deregulated kinases (CDK5, GSK-3, CLK1, CK1 and the dual-specificity kinase DYRK1A) involved to some extent in Alzheimer’s disease (AD) [2].

A library of thirty eight novel thiazolo[5,4-f]quinazolin-9(8H)-one derivatives (series I, II,III and IV) was prepared via the Hügershoff reaction and a Cu catalyzed intramolecular C-S bond formation, helped by microwave-assisted technology when required. The efficient multistep synthesis of the key  6-amino-3-cyclopropylquinazolin-4(3H)-one has been reinvestigated and performed on a multigram scale from the starting 5-nitroanthranilic acid. The inhibitory potency of the final products was evaluated against five kinases involved in Alzheimer’s disease and showed that some molecules of the IV series described in this communication are particularly promising for the development of novel multi-target inhibitors of kinases, a new strategy for the development of powerful tools against neurodegenerative diseases [3].

References

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2. (a) Foucourt, A.; Hédou, D.; Dubouilh-Benard, C.; Désiré, L.; Casagrande, -S.; Leblond, B.; Loaëc, N.; Meijer, L.; Besson, T. Molecules 2014, 19, 15411; (b) Leblond, B.; Casagrande, A.-S.; Désiré, L.; Foucourt A.; Besson, T. WO 2013026806. Chem. Abst. 2013, 158, 390018; (c) Logé, C.; Testard, A.; Thiéry, V.; Lozach, O.; Blairvacq, M.; Robert, J.-M.; Meijer, L.; Besson, T.  J. Med. Chem. 2008, 43, 1469; (d) Testard, A.; Picot, L.; Fruitier-Arnaudin, I.; Piot, J.M.; Chabane, H.; Domon, L.; Thiéry, V.; Besson, T. . Enz. Inhib. Med. Chem. 2004, 19, 467.
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Antimicrobial Peptides (AMPs) represent one of the most durable and effective defence of multicellular organisms against bacterial infections. These cationic and amphipathic peptides represent promising leads for the development of antibiotics combatting the resistance of bacteria against antibiotics. However, their clinical applications have often been limited by an inadequate margin of safety [1].

A prodrug approach can overcome a toxicity barrier in drug delivery. Prodrugs of AMPs can be generated by transiently reducing or annulling their net positive charges by attaching a negative promoiety through a linker which can be degraded by an enzyme (bacterial or human) confined to sites of infection. For example, neutrophil elastase (NE), a human protease involved in chronic airway inflammation and infections associated with cystic fibrosis (CF), can restore the cationic property of AMPs modified with oligo-glutamate promoieties. Consequently, their bactericidal activities against the CF pathogen P. aeruginosa are restored by NE in CF bronchoalveolar lavage fluids. The potential of this prodrug approach in reducing the safety barrier in the clinical use of AMPs has also been demonstrated in vivo, in a murine model of lung delivery [2].

In parallel, a novel class of peptidomimetics with antimicrobial activities similar to AMPs, against Gram-positive bacteria, has been developed. Their spectrum of activity is currently extended to Gram-negative organisms.

[1] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002, 415(6870):389-395.

[2] Forde É, Schütte A, Reeves E, Greene C, Humphreys H, Mall M, Fitzgerald-Hughes D, Devocelle M. Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis. Antimicrob Agents Chemother. 2016, 60(5):2813-2821.

Many sulfonamides with the general formula R-SO₂NH₂ constitute an important class of inhibitors of the zinc enzyme carbonic anhydrase (CA) due to their use in antiglaucoma therapy. It is well established that a water-soluble sulfonamide, also possessing relatively balanced lipid solubility, would be an effective antiglaucoma drug via the topical route.

Design of new aromatic sulfonamides was carried out using computational methods of theoretical medicinal chemistry as described in our previous works.  Of particular interest are the molecular geometries of neutral and anionic species, acidities, and lipophilicities. Synthesis of the so-designed new aromatic sulfonamides was conducted according to published procedures. Antiglaucoma activity was evaluated in both in vitro and in vivo conditions. For determination of the intraocular pressure changes the experiment with adult male Chinchilla was used. In this lecture we present the design and synthesis of novel drug-like aromatic sulfonamides, namely (4-sulfamoyl-N-(3-morpholinopropyl) benzamide, N-(3-morpholinopropyl)benzene-1,4-disulfonamide, N-(4-diethylaminoethoxybenzyl)benzene-1,4-bis(sulfonamide) and their hydrochloride salts. They exhibited favorable biological, structural, physicochemical and some pharmacokinetic properties comparable to those obtained for therapeutically useful acetazolamide, dorzolamide and brinzolamide. The solid-state structure of novel aromatic sulfonamides has been examined by X-ray crystallography. Methods of theoretical medicinal chemistry were applied for structural characterization of these compounds in the gas phase and water solution. Of particular interest are the molecular geometries of neutral and anionic species, acidities, and lipophilicities.

Data obtained allows us to assume, that new aromatic sulfonamides may represent novel class of compounds for the discovery of new effective antiglaucoma drugs.

Based on an earlier suggested general pharnacophore approach, compounds active against tuberculosis virulent strain H37Rv were obtained. The main drawback of these compounds was an almost complete CYP 3A4 inhibition, which presumably could be overcome by macrocyclization. Three 15-16-membered macrocyclic scaffolds with pharmacophore groups located in appropriate positions were designed and synthetic schemes for their achievement were elaborated. Testing of these compounds against CYP450 enzymes confirmed generally lower inhibition of key cytochromes CYP 3A4 and CYP2D6 by the macrocyclic compounds compared to acyclic prototypes.

The popularity of nanodiamonds has risen over the last few years because they have proved to be safe, biocompatible and significantly less toxic than other well-known carbon nanomaterials [1]. According to some studies, diamond nanoparticles seem to be a good candidate for biomedical purposes and more precisely as a tool for therapeutic and diagnosis applications in the medical imaging context [2, 3].

The main goal of this work is to validate an effective bimodal diamond-based nanoprobe for medical imaging techniques and specific of apoptosis. The functionalization of the platform involves the grafting of an optical agent (an amine-derivative of rhodamine), an apoptosis-specific vector (TLVSSL or E3 peptide), and a paramagnetic contrast agent (an amine-derivative of Gd-DOTA). The benefit of this multifunctional platform is the alliance of properties of magnetic resonance imaging (MRI) and optical imaging (OI) in terms of high spatial resolution and high sensitivity.                                                                                    

In this work, an annealing process has been employed to saturate the surface in a uniform way with carboxylic acid groups. The carboxylated diamond platform (n-COOH) is the starting material which enables a high surface loading of specific molecules for biomedical applications. The atomic distribution of the n-COOH surface was analyzed by XPS to define the efficiency of the oxidation method. The quantitative determination of surface carboxyl groups added by the thermal treatment was performed by conductimetric titration measurements [4]. Parallel to the surface uniformity and the dispersion of nanoparticles in aqueous solution, a paramagnetic gadolinium complex (Gd-DOTA-NH₂) has been synthesized in order to make the nanosystem active in MRI. The intermediate products of the synthesis have been characterized by mass spectrometry and NMR techniques. The relaxivity properties of the final complex were studied at 20 and 60 MHz and compared to the commercial complex, the Gd-DOTA.

The successive couplings of the molecules of interest (fluorochrome, peptide, paramagnetic complex) with the carboxylated platform were performed in water via an EDC(/NHS)-coupling process. After intensive purification by dialysis, the efficiency of the new active and specific diamond nanoprobe as bimodal agent was assessed in terms of relaxometric properties and in vitro studies.

1. Zhang, X., et al., A comparative study of cellular uptake and cytotoxicity of multi-walled carbon nanotubes, graphene oxide, and nanodiamond. Toxicology Research, 2012. 1(1): p. 62-68.
2. Manus, L.M., et al., Gd (III)-nanodiamond conjugates for MRI contrast enhancement. Nano letters, 2009. 10(2): p. 484-489.
3. Chow, E.K., et al., Nanodiamond therapeutic delivery agents mediate enhanced chemoresistant tumor treatment. Science translational medicine, 2011. 3(73): p. 73ra21-73ra21.

      4. Schmidlin, L., et al., Identification, quantification and modification of detonation nanodiamond functional groups. Diamond and Related Materials, 2012. 22: p. 113-117.

Chirality is an interesting geometric property and it is meaningful to explore the interactions between chiral small molecules and stereoselective biomacromolecules, with pre-clinical and clinical significance. Since the first observation of enantioselective binding to human-derived P-glycoprotein (P-gp) by mefloquine enantiomers [1], sparse stereoselectivity studies with P-gp modulators emerged. Recently, we have shown that newly synthesized (thio)xanthonic derivatives protect against toxic P-gp substrates acting as potent inducers/activators of this transporter [2-3]. Now we aim to discover new P-gp modulators and enlightening the stereoselectivity of this ABC transporter face to this class of modulators.

Herein, we report the synthesis and characterization of a library of new chiral aminated thioxanthones in their enantiomeric pure form (Figure 1) and in silico and in vitro preliminary results concerning their P-gp modulation behavior.

In silico docking studies in P-gp rat model anticipated enantioselectivity for these new derivatives. Thioxanthones cytotoxicity was evaluated by the Neutral Red uptake assay, in order to select a non-cytotoxic working concentration. The compounds were assessed for their influence in P-gp ATPase assay. The investigation of P-gp expression and activity allowed to discover new P-gp modulators. Nevertheless, no significant differences between enantiomeric pairs of thioxanthones were observed.

[1] L. Lu, F. Leonessa, M.T. Baynham, R. Clarke, F. Gimenez, Y.T. Pham, F. Roux, I.W. Wainer, Pharmaceutical research, 18 (2001) 1327-1330.

 [2] R. Silva, A. Palmeira, H. Carmo, D.J. Barbosa, M. Gameiro, A. Gomes, A.M. Paiva, E. Sousa, M. Pinto, L. Bastos Mde, F. Remiao, Archives of toxicology, 89 (2015) 1783-1800.

[3] R. Silva, E. Sousa, H. Carmo, A. Palmeira, D.J. Barbosa, M. Gameiro, M. Pinto, L. Bastos Mde, F. Remiao, Archives of toxicology, 88 (2014) 937-951.

This work was partially supported through national funds provided by: FCT - Foundation for Science and Technology and European Regional, Development Fund (ERDF) and COMPETE under the projects PEst-C/MAR/LA0015/2013, PTDC/MAR-BIO/4694/2014, and INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR.

Aim: To evaluate the impact of rh-GH replacement therapy on neurotransmitters: gamma - amino butyric acid (GABA), dopamine (DA) and serotonin (5-HT) in growth hormone deficient children.                

Research design and methods: This retrospective study included 30 subjects with growth hormone deficit, clinically established: 20 boys (5-14 years) and 10 girls (6-14 years). All of them underwent GH replacement therapy from 9 months - 10.6 years. rh - GH dose varied in all subjects from 0.6-1.9 mg/day based on detailed clinical and anthropometric data. In 2015, all subjects in different phases of treatment were tested for plasma: GABA, DA, 5-HT(Elisa methods) and IGF-1(chemiluminescent method).                

Results: Median plasma GABA in boys vs girls was: 50.5 vs 46 ng/mL; median plasma DA in boys vs girls was: 43.34 vs 29.4 pg/mL; median 5-HT in boys vs girls was: 227.5 vs 208.7 ng/mL and median IGF-1 in boys vs girls was: 334 vs 357.25.7ng/mL. We established a statistically significant difference in plasma GABA and in DA values in boys vs. girls. High multiple regression coefficients were established between age and IGF-1, DA, GABA or between IGF1 and 5-HT, DA, GABA in boys vs. girls.                                    

Conclusion: This study established a link between brain neurotransmitters and the gain in height in GH-deficient children under replacement therapy in different phases of treatment.

Aim: Excess release of catecholamines is a characteristic for pheochromocytomas. The rate of catecholamine synthesis is determined by tyrosine hydroxylase (TH) enzyme. Usually, TH is detected by immunohistochemistry. In our study, we evaluated this enzyme in plasma from pheochromocytoma subjects establishing correlations with some metabolites.    

Subjects and Methods:10 subjects (9 women /1man aged 40-72 years ) clinically suspected of pheochromocytoma, were biochemically investigated for free plasma normetanephrines (NMNp)/ metanephrines (MNp), plasma chromogranin A (CgA), plasma TH and IGF-1.Comparison of tumoral metabolites values was done with our lab normal range. Statistical analysis used multiple regression to evaluate relationship between TH and all 3 parameters: NMNp/MNp/CgA. 

Results: All subjects showed excessive plasma NMNp (median: 1434 pg/mL); in 7 cases we observed an over-secretion of MNp (median: 441pg/mL) and CgA was higher in 9 cases (median: 668 ng/mL). TH was identified in all plasma samples with a median of: 2.08 ng/mL. Higher values of TH were detected in cases with an over-excess of metabolites. Good correlations were established between NMNp/TH (0.51), MNp/TH(0.81) and NMNp/CgA (0.71).    

Conclusion: Metabolites in excess are well correlated with TH values proving a great rate of  catecholamine synthesis in some cases. We could affirm TH could be used as an index of functionality in pheochromocytoma diagnosis.

Currently, significant interest in drug development is focused on obtaining drugs that simultaneously affect various pharmacological targets and thus exhibiting combined actions. Herein we demonstrate the possibility of development of novel drugs that are able to simultaneously modulate TRP-channels and bind to glycine receptors. For this purpose esters based on mono- and bicyclic terpenoids (menthol, thymol, carvacrol, guaiacol, eugenol, borneol) and an inhibitory amino acid (glycine) were synthesized via Steglich esterification. Their anticonvulsant action was evaluated by a PTZ-induced convulsion model and analgesic effect − by pharmacological models of thermal and chemical stimuli. All studied esters were found to produce antinociceptive effects and attenuate acute pain more than the reference drug benzocaine after topical application. The present findings indicate that glycine esters of abovementioned terpenoids are not classical prodrugs and possess their own pharmacological activity. Prolonged antiseizure action of the esters was revealed at 24 h after oral administration. Moreover, orally co-administered gidazepam (1 mg/kg) and glycine esters produce synergistic seizure prevention effects.

At present, about fifty percent of commercial pharmaceutical drugs are derived from natural sources. The abietane-type and related diterpenoids are a class of naturally occurring terpenoids in the plant kingdom, which have demonstrated a wide range of biological activities against cancer, and a variety of infectious diseases (viral and bacterial). Several research groups have explored the potential as chemotherapeutic agents of abietanes by means of semisynthetic derivatives from abietic acid-derived materials such as dehydroabietic acid (DHA), and dehydroabietylamine, also called leelamine. For example, DHA displays not only antiulcer and antimicrobial properties, but also antitumor effects. Recently, DHA was reported as a positive GABAA receptor modulator (potentiation of I_GABA 682.3% at 100 mM).

 DHA displays an equatorial carboxylic group located at C18 while in other natural congeners the carboxylic group adopts the axial configuration (C19) as in 4-epidehydroabietic acid or callitrisic acid. Recently, a series of related acids having a C19 carboxylic group have been isolated. For example, the jiadifenoic acids A-I. These have shown antiviral properties against Coxsackie virus. We have developed the synthesis of jiadifenoic acid C from callitrisic acid isolated from Sandarac resin. The ready availability of these acids from our work and the absence of their biological and pharmacological studies as well as chemical manipulation prompted us to carry out this research. In this study, we carried out the synthesis of several derivatives of callitrisic acid, including jiadifenoic acid C, methyl callitrisate and callitrisinol together with evaluation of their antiproliferative and modulating GABAA receptor activities. Thus, we were able to compare with the DHA series.

 Biological results showed that the studied callitrisic derivatives (functionalization at C19) were less potent GABAA-modulating molecules (potentiation of I_GABA 269-311% at 100 mM) than DHA (carboxyl at C18), of which jiadifenoic acid C having an allylic alcohol instead of an isopropyl moiety was practically inactive. On the other hand, functional group manipulation at C19 led to more potent antiproliferative derivatives (GI₅₀= 3.4-61 mM) than their corresponding C18-functionalized congeners.