Chloroquine fumardiamides as novel quorum sensing inhibitors

Quorum sensing inhibitors (QSIs) that specifically interfere with bacterial cell-to-cell communication are considered as an alternative approach to conventional antibacterial therapy. In our study, a set of twenty-six fumardiamides with a quinoline head-group were evaluated as potential QSIs. Two strains of Gram-negative Chromobacterium violaceum (violacein-producing strain ATCC31532 and violacein-negative, mini-Tn5 mutant derivative CV026) were used as QS reporters for testing anti-QS and bactericidal activity of various quinoline fumardiamides. The initial screening of eighteen fumardiamides with primaquine, mefloquine and chloroquine scaffolds identified chloroquine derivatives as the most promising QSIs. Tail-group optimization of chloroquine fumardiamides led to the most active compounds 27, 29 and 30 bearing aminoethyl or piperidine moieties. At 400 mu M concentration, these compounds inhibited the QS of C. violaceum strains in a manner similar to quercetin (the model QSI), while at the 40 mu M concentration their inhibitory effect was twice less than that of quercetin. As none of the compounds displayed a bactericidal effect and that the QS inhibition was specific to the CV026 strain, our findings indicate that the structurally optimized chloroquine derivatives could function as quorum quenching (QQ) agents with a potential to block the signaling without entering the cell. In conclusion, our finding provides an important step toward the further design of agents targeting cell-to-cell communication.Peer reviewe

Synthesis and Biological Evaluation of Harmirins, Novel Harmine–Coumarin Hybrids as Potential Anticancer Agents

As cancer remains one of the major health burdens worldwide, novel agents, due to the development of resistance, are needed. In this work, we designed and synthesized harmirins, which are hybrid compounds comprising harmine and coumarin scaffolds, evaluated their antiproliferative activity, and conducted cell localization and cell cycle analysis experiments. Harmirins were prepared from the corresponding alkynes and azides under mild reaction conditions using Cu(I) catalyzed azide–alkyne cycloaddition, leading to the formation of the 1H-1, 2, 3-triazole ring. Antiproliferative activity of harmirins was evaluated in vitro against four human cancer cell lines (MCF-7, HCT116, SW620, and HepG2) and one human non-cancer cell line (HEK293T). The most pronounced activities were exerted against MCF-7 and HCT116 cell lines (IC50 in the single-digit micromolar range), while the most selective harmirins were 5b and 12b, substituted at C-3 and O-7 of the β-carboline core and bearing methyl substituent at position 6 of the coumarin ring (SIs > 7.2). Further experiments demonstrated that harmirin 12b is localized exclusively in the cytoplasm. In addition, it induced a strong G1 arrest and reduced the percentage of cells in the S phase, suggesting that it might exert its antiproliferative activity through inhibition of DNA synthesis, rather than DNA damage. In conclusion, harmirin 12b is a novel harmine and coumarin hybrid with significant antiproliferative activity and warrants further evaluation as a potential anticancer agent

Synthesis and antiplasmodial evaluation of novel mefloquine-based fumardiamides

The paper is focused on the synthesis and screening of the antiplasmodial activity of novel fumardiamides 5–10 with the mefloquine pharmacophore and a Michael acceptor motif. Multi-step reactions leading to the title compounds included two amide bond formations. The first amide bond was achieved by the reaction of (E)-ethyl 4-chloro-4-oxobut-2-enoate (1) and N1-(2,8-bis(trifluoromethyl)quinolin-4-yl)butane-1,4-diamine (2). The obtained ester 3 was hydrolysed and gave acid 4, which then reacted with the selected halogenanilines in the presence of HATU/DIEA and formed products 5–10. Title compounds showed marked, dose dependent activity in vitro against hepatic stages of Plasmodium berghei. IC50 values of the most active compounds 5, 7 and 9 bearing 3-fluoro, 3-chloro and 3-trifluoromethyl substituents were 3.04–4.16 µmol L–1, respectively. On the other hand, the compounds exerted only weak activity against the erythrocytic stages of two P. falciparum strains (Pf3D7 and PfDd2) in vitro, with the exception of compound 5 (IC50 = 2.9 µmol L–1)

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

Hazard assessment of nanomaterials using in vitro toxicity assays:Guidance on potential assay interferences and mitigating actions to avoid biased results

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles

Asymmetric Primaquine and Halogenaniline Fumardiamides as Novel Biologically Active Michael Acceptors

Novel primaquine (PQ) and halogenaniline asymmetric fumardiamides 4a–f, potential Michael acceptors, and their reduced analogues succindiamides 5a–f were prepared by simple three-step reactions: coupling reaction between PQ and mono-ethyl fumarate (1a) or mono-methyl succinate (1b), hydrolysis of PQ-dicarboxylic acid mono-ester conjugates 2a, b to corresponding acids 3a, b, and a coupling reaction with halogenanilines. 1- [bis(Dimethylamino)methylene]-1H-1, 2, 3- triazolo[4, 5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was used as a coupling reagent along with Hünig′s base. Compounds 4 and 5 were evaluated against a panel of bacteria, several Mycobacterium strains, fungi, a set of viruses, and nine different human tumor cell lines. p-Chlorofumardiamide 4d showed significant activity against Staphylococcus aureus, Streptococcus pneumoniae and Acinetobacter baumannii, but also against Candida albicans (minimum inhibitory concentration (MIC) 6.1–12.5 µg/mL). Together with p-fluoro and p-CF3 fumardiamides 4b, f, compound 4d showed activity against Mycobacterium marinum and 4b, f against M. tuberculosis. In biofilm eradication assay, most of the bacteria, particularly S. aureus, showed susceptibility to fumardiamides. m-CF3 and m- chloroaniline fumardiamides 4e and 4c showed significant antiviral activity against reovirus- 1, sindbis virus and Punta Toro virus (EC50 = 3.1–5.5 µM), while 4e was active against coxsackie virus B4 (EC50 = 3.1 µM). m-Fluoro derivative 4a exerted significant cytostatic activity (IC50 = 5.7–31.2 μM). Acute lymphoblastic leukemia cells were highly susceptible towards m-substituted derivatives 4a, c, e (IC50 = 6.7–8.9 μM). Biological evaluations revealed that fumardiamides 4 were more active than succindiamides 5 indicating importance of Michael conjugated system

Primaquine and Chloroquine Fumardiamides as Promising Antiplasmodial Agents

This paper describes a continuation of our efforts in the pursuit of novel antiplasmodial agents with optimized properties. Following our previous discovery of biologically potent asymmetric primaquine (PQ) and halogenaniline fumardiamides (1–6), we now report their significant in vitro activity against the hepatic stages of Plasmodium parasites. Furthermore, we successfully prepared chloroquine (CQ) analogue derivatives (11–16) and evaluated their activity against both the hepatic and erythrocytic stages of Plasmodium. Our results have shown that PQ fumardiamides (1–6) exert both higher activity against P. berghei hepatic stages and lower toxicity against human hepatoma cells than the parent drug and CQ derivatives (11–16). The favourable cytotoxicity profile of the most active compounds, 5 and 6, was corroborated by assays performed on human cells (human breast adenocarcinoma (MCF-7) and non-tumour embryonic kidney cells (HEK293T)), even when glucose-6-phosphate dehydrogenase (G6PD) was inhibited. The activity of CQ fumardiamides on P. falciparum erythrocytic stages was higher than that of PQ derivatives, comparable to CQ against CQ-resistant strain PfDd2, but lower than CQ when tested on the CQ-sensitive strain Pf3D7. In addition, both sets of compounds showed favourable drug-like properties. Hence, quinoline fumardiamides could serve as a starting point towards the development of safer and more effective antiplasmodial agents

Design, synthesis and cytostatic activity of sahaquines

U okviru ovog doktorskog rada dizajnirani su i sintetizirani sahakini, hibridni spojevi u kojima su spojeni dijelovi molekula antimalarika primakina, odnosno klorokina i citostatika SAHA-e (suberoilanilid hidroksamska kiselina). Sahakini sadrže tri ključna dijela: kinolinski prsten (dio molekule primakina ili klorokina), središnji dio (dikarboksilnu kiselinu: jantarnu, fumarnu, glutarnu, adipinsku, tereftalnu) te završni dio s dodatnom funkcionalnom skupinom (esterskom, karboksilnom, amidnom te O-benzil-, O-metil- ili nesupstituiranom hidroksamskom kiselinom). Sinteza sahakina polazila je od kinolinskog kraja. Primarna amino skupina primakina, odnosno analoga klorokina i monoester odgovarajućih dikarboksilnih kiselina povezani su amidnom vezom pomoću 1-[bis(dimetilamino)metilen]-1H-1,2,3-triazolo[4,5-b]piridinij 3-oksid heksafluorofosfata (HATU) u prisutnosti N,N-diizopropiletilamina (DIEA) ili preko kiselinskog klorida. Dobiveni su amido-esterski derivati koji su u sljedećem reakcijskom koraku hidrolizirani u amido-karboksilne kiseline te dalje amidirani s O-metil- ili Obenzilhidroksilaminima, odnosno halogenanilinima (s atomima fluora/klora ili trifluorometilnom skupinom u meta ili para položaju) uz HATU i DIEA. Sahakini sa slobodnom hidroksamskom skupinom dobiveni su katalitičkom hidrogenolizom O-benzilhidroksamskih derivata. Svi sahakini karakterizirani su uobičajenim analitičkim i spektroskopskim metodama (CHN, IR, 1H NMR, 13C NMR, MS). Antiproliferativno djelovanje sahakina ispitano je na humanim tumorskim stanicama: adenokarcinoma gušterače (Capan-1), akutne mijeloične leukemije (Hap1), karcinoma debelog crijeva (HCT-116), karcinoma pluća (NCI-H460), akutne limfoblastičke leukemije (DND-41), akutne promijelocitne leukemije (HL-60), kronične mijeloične leukemije (K-562), multiplog mijeloma (MM.1S), ne-Hodkinovog limfoma (Z-138), osteosarkoma (U2OS), hepatocelularnog karcinoma (HepG2), adenokarcinoma dojke (MCF-7), karcinoma pluća ne-malih stanica (H460) i glioblastoma (U251N). Najaktivniji derivati primakina bili su sahakini iz podskupine hidroksamskih kiselina te meta-supstituirani derivati halogenanilina s fumardiamidnom poveznicom. Sahakinu s najjačim citostatskim učinkom, N1-hidroksi-N5-(4-((6-metoksikinolin-8- il)amino)pentil)-glutaramidu, utvrđen je mehanizam djelovanja. Na staničnom modelu glioblastoma dokazano je da selektivno inhibira histon-deacetilazu 6, smanjuje ekspresiju matriks-metaloproteaze-2 i receptora za epidermalni faktor rasta.This doctoral thesis describes the design and synthesis of sahaquines, hybrid molecules in which molecular parts of antimalarial drug primaquine/chloroquine and cytostatic drug SAHA (suberoylanilide hydroxamic acid) are merged. Sahaquines constitute of three key parts: quinoline ring (part of primaquine or chloroquine molecule), linker (dicarboxylic acid: succinic, fumaric, glutaric, adipic, terephthalic) and the terminal part with an additional functional group (ester, carboxylic, amide, O-benzyl-, O-methyl- or unsubstituted hydroxamic acid). Sahaquine synthesis starts from the quinoline end. The primary amino group of primaquine/chloroquine is linked with an appropriate dicarboxylic monoester through an amide bond achieved with 1-[bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) in the presence of N,N-diisopropylethylamine (DIEA) or by previously activating carboxylic acid with thionyl chloride. Obtained amido-esters were hydrolysed to afford the corresponding amido-carboxylic acids, which were further coupled with O-benzyl- and Omethylhydroxylamine, or halogenanlines. HATU/DIEA was used again as the coupling system. Free hydroxamic acids were obtained by catalytic hydrogenation of O-benzyl derivatives. All new compounds were fully characterized by conventional spectroscopic and analytical methods (IR, 1H and 13C NMR, MS and elemental analyses). The cytostatic activity of sahaquines was tested on the following cancer cell lines: pancreatic adenocarcinoma (Capan-1), chronic myeloid leukemia (Hap1), colorectal carcinoma (HCT-116), lung carcinoma (NCI-H460), acute lymphoblastic leukemia (DND-41), acute myeloid leukemia (HL-60), chronic myeloid leukemia (K-562), multiple myeloma (MM.1S), non-Hodgkin lymphoma (Z-138), bone osteosarcoma (U2OS), hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF-7) and glioblastoma (U251N). The most active primaquine derivatives were sahaquines with free hydroxamic acid moiety and diamides with meta-substituted halogenanilines. The mechanism of action was determined for the most potent sahaquine, namely N1-hydroxy-N5- (4-((6-methoxyquinolin-8-yl)amino)pentyl)glutaramide. On the glioblastoma cell, it is shown that sahaquine selectively inhibits histone deacetylase 6, decreases the expression of matrix metalloproteinase-2 and the epidermal growth factor receptor

Endokrina terapija hormonski ovisnog karcinoma dojke

Estrogens are the key female hormones that induce the development of reproductive tissues and secondary sex characteristics. The effects of estrogens are mediated by binding to the estrogen receptors present in the cell cytosole which triggers the translocation of the receptor to the nucleus and transcription of DNA. Other beneficial effects include osteoclast inhibition, prevention of colon cancer, more favourable lipoprotein status, as well as protective cardiovascular effects. However, estrogens also promote the growth of breast cancer that expresses estrogen receptors. The therapeutic goal is the elimination of the estrogen effect on its receptors in the breast cancer tissue, which can be achieved through interference with estrogen biosynthesis or blockade of the estrogen binding to estrogen receptors. Drugs that interfere with estrogen biosynthesis are aromatase inhibitors, agonists and antagonists of gonadotropin-releasing hormone, and adrogens, whereas selective modulators of estrogen receptors and estrogen antagonists block the binding of estrogen. Inhibitors of cyclin-dependent kinases are also a valuable therapeutic option for estrogen-dependent breast cancer