Effects of H2S-donor ascorbic acid derivative and ischemia/reperfusion-induced injury in isolated rat hearts.

Hydrogen sulfide (H2S), a gasotransmitter, plays a crucial role in vasorelaxation, anti-inflammatory processes and mitigating myocardial ischemia/reperfusion-induced injury by regulating various signaling processes. We designed a water soluble H2S-releasing ascorbic acid derivative, BM-164, to combine the beneficial cardiovascular and anti-inflammatory effects of H2S with the excellent water solubility and antioxidant properties of ascorbic acid. DPPH antioxidant assay revealed that the antioxidant activity of BM-164 in the presence of a myocardial tissue homogenate (extract) increased continuously over the 120 min test interval due to the continuous release of H2S from BM-164. The cytotoxicity of BM-164 was tested by MTT assay on H9c2 cells, which resulted in no cytotoxic effect at concentrations of 10 to 30 µM. The possible beneficial effects of BM-164 (30 µM) was examined in isolated 'Langendorff' rat hearts. The incidence of ventricular fibrillation (VF) was significantly reduced from its control value of 79 % to 31 % in the BM-164 treated group, and the infarct size was also diminished from the control value of 28 % to 14 % in the BM-164 treated group. However, coronary flow (CF) and heart rate (HR) values in the BM-164 treated group did not show significantly different levels in comparison with the drug-free control, although a non-significant recovery in both CF and HR was observed at each time point. We attempted to reveal the mechanism of action of BM-164, focusing on the processes of autophagy and apoptosis. The expression of key autophagic and apoptotic markers in isolated rat hearts were detected by Western blot analysis. All the examined autophagy-related proteins showed increased expression levels in the BM-164 treated group in comparison to the drug-free control and/or ascorbic acid treated groups, while the changes in the expression of apoptotic markers were not obvious. In conclusion, the designed water soluble H2S releasing ascorbic acid derivative, BM-164, showed better cardiac protection against ischemia/reperfusion-induced injury compared to the untreated and ascorbic acid treated hearts, respectively.

Amphiphilic Sialic Acid Derivatives as Potential Dual-Specific Inhibitors of Influenza Hemagglutinin and Neuraminidase.

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

Mannich-type modifications of (-)-cannabidiol and (-)-cannabigerol leading to new, bioactive derivatives.

(-)-Cannabidiol (CBD) and (-)-cannabigerol (CBG) are two major non-psychotropic phytocannabinoids that have many beneficial biological properties. However, due to their low water solubility and prominent first-pass metabolism, their oral bioavailability is moderate, which is unfavorable for medicinal use. Therefore, there is a great need for appropriate chemical modifications to improve their physicochemical and biological properties. In this study, Mannich-type reaction was used for the synthetic modification of CBD and CBG for the first time, and thus fifteen new cannabinoid derivatives containing one or two tertiary amino groups were prepared. Thereafter the antiviral, antiproliferative and antibacterial properties of the derivatives and their effects on certain skin cells were investigated. Some modified CBD derivatives showed remarkable antiviral activity against SARS-CoV-2 without cytotoxic effect, while synthetic modifications on CBG resulted in a significant increase in antiproliferative activity in some cases compared to the parent compound.

Pharmacological Evaluation of Newly Synthesized Cannabidiol Derivates on H9c2 Cells.

Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid that can be found in Cannabis sativa and possesses numerous pharmacological effects. Due to these promising effects, CBD can be used in a wide variety of diseases, for instance cardiovascular diseases. However, CBD, like tetrahydrocannabinol (THC), has low bioavailability, poor water solubility, and a variable pharmacokinetic profile, which hinders its therapeutic use. Chemical derivatization of CBD offers us potential ways to overcome these issues. We prepared three new CBD derivatives substituted on the aromatic ring by Mannich-type reactions, which have not been described so far for the modification of cannabinoids, and studied the protective effect they have on cardiomyocytes exposed to oxidative stress and hypoxia/reoxygenation (H/R) compared to the parent compound. An MTT assay was performed to determine the viability of rat cardiomyocytes treated with test compounds. Trypan blue exclusion and lactate dehydrogenase (LDH) release assays were carried out to study the effect of the new compounds in cells exposed to H2O2 or hypoxia/reoxygenation (H/R). Direct antioxidant activity was evaluated by a total antioxidant capacity (TAC) assay. To study antioxidant protein levels, HO-1, SOD, catalase, and Western blot analysis were carried out. pIC50 (the negative log of the IC50) values were as follows: CBD1: 4.113, CBD2: 3.995, CBD3: 4.190, and CBD: 4.671. The newly synthesized CBD derivatives prevented cell death induced by H/R, especially CBD2. CBD has the largest direct antioxidant activity. The levels of antioxidant proteins were increased differently after pretreatment with synthetic CBD derivatives and CBD. Taken together, our newly synthesized CBD derivatives are able to decrease cytotoxicity during oxidative stress and H/R. The compounds have similar or better effects than CBD on H9c2 cells.

In vitro and in vivo antiplasmodial evaluation of sugar-modified nucleoside analogues.

Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing antimalarial therapies has complicated the problem, and the emergence of resistance to artemisinin derivatives is even more concerning. It is increasingly difficult to cure malaria patients due to the limited availability of effective antimalarial drugs, resulting in an urgent need for more efficacious and affordable treatments to eradicate this disease. Herein, new nucleoside analogues including morpholino-nucleoside hybrids and thio-substituted nucleoside derivatives were prepared and evaluated for in vitro and in vivo antiparasitic activity that led a few hits especially nucleoside-thiopyranoside conjugates, which are highly effective against Pf3D7 and PfRKL-9 strains in submicromolar concentration. One adenosine derivative and four pyrimidine nucleoside analogues significantly reduced the parasite burden in mouse models infected with Plasmodium berghei ANKA. Importantly, no significant hemolysis and cytotoxicity towards human cell line (RAW) was observed for the hits, suggesting their safety profile. Preliminary research suggested that these thiosugar-nucleoside conjugates could be used to accelerate the antimalarial drug development pipeline and thus deserve further investigation.

Reperfusion-induced injury and the effects of the dithioacetate type hydrogen sulfide donor ibuprofen derivative, BM-88, in isolated rat hearts.

Hydrogen sulfide (H2S) plays an important role in cardiac protection by regulating various redox signalings associated with myocardial ischemia/reperfusion (I/R) induced injury. The goal of the present investigations is the synthesis of a newly designed H2S-releasing ibuprofen derivative, BM-88, and its pharmacological characterization regarding the cardioprotective effects in isolated rat hearts. Cytotoxicity of BM-88 was also estimated in H9c2 cells. H2S-release was measured by an H2S sensor from the coronary perfusate. Increasing concentrations of BM-88 (1.0 to 20.0 µM) were tested in vitro studies. Preadministration of 10 µM BM-88 significantly reduced the incidence of reperfusion-induced ventricular fibrillation (VF) from its drug-free control value of 92% to 12%. However, no clear dose dependent reduction in the incidence of reperfusion-induced VF was observed while different concentrations of BM-88 were used. It was also found that 10 µM BM-88 provided a substantial protection and significantly reduced the infarct size in the ischemic/reperfused myocardium. However, this cardiac protection was not reflected in any significant changes in coronary flow and heart rates. The results support the fact that H2S release plays an important role mitigating reperfusion-induced cardiac damage.

Triaza-tricyclanos - synthesis of a new class of tricyclic nucleoside analogues by stereoselective cascade cyclocondensation.

Herein, we report a stereoselective synthesis of a novel type of conformationally constrained nucleoside analogue in which the sugar part is replaced by a new symmetrical tricycle consisting of a morpholine ring condensed with two imidazolidines. 1,5-Dialdehydes obtained from trityl- and dimethoxytrityl-protected uridine, ribothymidine, inosine, cytidine, adenosine and guanosine by metaperiodate oxidation were reacted with N1,N3-dibenzyl-1,2,3-triaminopropane; the latter reactant was produced using a new method that avoids explosive intermediates. Reactions of dialdehydes with propane-triamine via cascade tricyclization resulted in the corresponding triaza-tricyclic derivatives bearing three new stereogenic centers in high yields. Out of the eight possible diastereoisomers, one stereoisomer was formed in each case due to the chiral control of the starting nucleoside-dialdehydes and the steric constraint of the condensed ring system. The absolute configuration of the new stereotriad was determined by X-ray diffraction and NMR experiments. A mechanistic study performed under reductive conditions to trap the presumed bicyclic intermediate showed that the triamine reactant first attacks the 2'-aldehyde group, followed by a rapid bicyclization to form the imidazolidino-morpholine unit.

N-Fluoroalkylated Morpholinos - a New Class of Nucleoside Analogues.

The first concise and efficient synthesis of some fluorine-containing morpholino nucleosides has been developed. One synthetic strategy was based on the oxidative ring cleavage of the vicinal diol unit of uridine, cytidine adenosine and guanosine derivatives, followed by cyclisation of the dialdehyde intermediates by double reductive amination with fluorinated primary amines to obtain various N-fluoroalkylated morpholinos. Another approach involved cyclisation of the diformyl intermediates with ammonia source, followed by dithiocarbamate formation and desulfurization-fluorination with diethylaminosulfur trifluoride yielding the corresponding morpholine-based nucleoside analogues with a N-CF3 element in their structure.

Synthesis of an amphiphilic vancomycin aglycone derivative inspired by polymyxins: overcoming glycopeptide resistance in Gram-positive and Gram-negative bacteria in synergy with teicoplanin in vitro.

Gram-negative bacteria possess intrinsic resistance to glycopeptide antibiotics so these important antibacterial medications are only suitable for the treatment of Gram-positive bacterial infections. At the same time, polymyxins are peptide antibiotics, structurally related to glycopeptides, with remarkable activity against Gram-negative bacteria. With the aim of breaking the intrinsic resistance of Gram-negative bacteria against glycopeptides, a polycationic vancomycin aglycone derivative carrying an n-decanoyl side chain and five aminoethyl groups, which resembles the structure of polymyxins, was prepared. Although the compound by itself was not active against the Gram-negative bacteria tested, it synergized with teicoplanin against Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, and it was able to potentiate vancomycin against these Gram-negative strains. Moreover, it proved to be active against vancomycin- and teicoplanin-resistant Gram-positive bacteria.

Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

Semisynthetic teicoplanin derivatives with dual antimicrobial activity against SARS-CoV-2 and multiresistant bacteria.

Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.

Synthesis of Four Orthogonally Protected Rare l-Hexose Thioglycosides from d-Mannose by C-5 and C-4 Epimerization.

l-Hexoses are important components of biologically relevant compounds and precursors of some therapeuticals. However, they typically cannot be obtained from natural sources and due to the complexity of their synthesis, their commercially available derivatives are also very expensive. Starting from one of the cheapest d-hexoses, d-mannose, using inexpensive and readily available chemicals, we developed a reaction pathway to obtain two orthogonally protected l-hexose thioglycoside derivatives, l-gulose and l-galactose, through the corresponding 5,6-unsaturated thioglycosides by C-5 epimerization. From these derivatives, the orthogonally protected thioglycosides of further two l-hexoses (l-allose and l-glucose) were synthesized by C-4 epimerization. The preparation of the key intermediates, the 5,6-unsaturated derivatives, was systematically studied using various protecting groups. By the method developed, we are able to produce highly functionalized l-gulose derivatives in 9 steps (total yields: 21-23%) and l-galactose derivatives in 12 steps (total yields: 6-8%) starting from d-mannose.

Investigation of the Role and Effectiveness of Chitosan Coating on Probiotic Microcapsules.

Microencapsulation and coating are preferred methods to increase the viability of the probiotic strains. The effect of microencapsulation technologies and materials used as microcapsule cores on viability is being investigated during development. In the present study, chitosan-coated and Eudragit L100-55-coated alginate microspheres were produced to encapsulate Lactobacillus plantarum probiotic bacteria. After the heat loading and simulated gastrointestinal juice dissolution study, the differences in viability were compared based on the CFU/mL values of the samples. The kinetics of the bacterial release and the ratio of the released live/dead cells of Lactobacillus plantarum were examined by flow cytometry. In all cases, we found that the CFU value for the chitosan-coated samples was virtually zero. The ratio of live/dead cells in the 120 min samples was significantly reduced to less than 20% for chitosan, while it was nearly 90% in the uncoated and Eudragit L100-55-coated samples. In the case of chitosan, based on some published MIC values and the amount of chitosan coating determined in the present study, we concluded the reason for our results. It was the first time to determine the amount of the released chitosan coat of the dried microcapsule, which reached the MIC value during the dissolution studies.

The First Dimeric Derivatives of the Glycopeptide Antibiotic Teicoplanin.

Various dimeric derivatives of the glycopeptide antibiotic teicoplanin were prepared with the aim of increasing the activity of the parent compound against glycopeptide-resistant bacteria, primarily vancomycin-resistant enterococci. Starting from teicoplanin, four covalent dimers were prepared in two orientations, using an α,ω-bis-isothiocyanate linker. Formation of a dimeric cobalt coordination complex of an N-terminal L-histidyl derivative of teicoplanin pseudoaglycone has been detected and its antibacterial activity evaluated. The Co(III)-induced dimerization of the histidyl derivative was demonstrated by DOSY experiments. Both the covalent and the complex dimeric derivatives showed high activity against VanA teicoplanin-resistant enterococci, but their activity against other tested bacterial strains did not exceed that of the monomeric compounds.

The Very First Modification of Pleuromutilin and Lefamulin by Photoinitiated Radical Addition Reactions-Synthesis and Antibacterial Studies.

Pleuromutilin is a fungal diterpene natural product with antimicrobial properties, semisynthetic derivatives of which are used in veterinary and human medicine. The development of bacterial resistance to pleuromutilins is known to be very slow, which makes the tricyclic diterpene skeleton of pleuromutilin a very attractive starting structure for the development of new antibiotic derivatives that are unlikely to induce resistance. Here, we report the very first synthetic modifications of pleuromutilin and lefamulin at alkene position C19-C20, by two different photoinduced addition reactions, the radical thiol-ene coupling reaction, and the atom transfer radical additions (ATRAs) of perfluoroalkyl iodides. Pleuromutilin were modified with the addition of several alkyl- and aryl-thiols, thiol-containing amino acids and nucleoside and carbohydrate thiols, as well as perfluoroalkylated side chains. The antibacterial properties of the novel semisynthetic pleuromutilin derivatives were investigated on a panel of bacterial strains, including susceptible and multiresistant pathogens and normal flora members. We have identified some novel semisynthetic pleuromutilin and lefamulin derivatives with promising antimicrobial properties.

Natural Apocarotenoids and Their Synthetic Glycopeptide Conjugates Inhibit SARS-CoV-2 Replication.

The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and ß-apo-8'carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.

Tightly linked morpholino-nucleoside chimeras: new, compact cationic oligonucleotide analogues.

The polyanionic phosphodiester backbone of nucleic acids contributes to high nuclease sensitivity and low cellular uptake and is therefore a major obstacle to the biological application of native oligonucleotides. Backbone modifications, particularly charge alterations is a proven strategy to provide artificial oligonucleotides with improved properties. Here, we describe the synthesis of a new type of oligonucleotide analogues consisting of a morpholino and a ribo- or deoxyribonucleoside in which the 5'-amino group of the nucleoside unit provides the nitrogen of the morpholine ring. The synthetic protocol is compatible with trityl and dimethoxytrityl protecting groups and azido functionality, and was extended to the synthesis of higher oligomers. The chimeras are positively charged in aqueous medium, due to the N-alkylated tertiary amine structure of the morpholino unit.

Systematic Study of Regioselective Reductive Ring-Opening Reactions of 4,6-O-Halobenzylidene Acetals of Glucopyranosides.

Reductive openings of cyclic acetals are widely used in modern synthetic organic chemistry for the regioselective introduction of protecting groups. A systematic study was performed on the applicability and efficacy of various hydride donor and protic or Lewis acid reagent combinations in the reductive ring opening of glucosidic 4,6-halobenzylidene acetals bearing an ortho-, meta-, and para-chloro- or -bromo substituent on the benzene ring. Most of the reagent combinations tested cleaved the 4,6-O-halobenzylidene acetal rings at O4 or O6 efficiently and with the expected regioselectivity. The LiAlH4-AlCl3 and the BH3·THF-TMSOTf combinations produced the 4-O-halobenzyl ether/6-OH products with complete regioselectivity and high yields. The use of Me3N·BH3-AlCl3 reagent system in toluene was also effective in cleaving the acetal ring at O6 but was accompanied by Al-chelation-assisted debenzylation side reactions. The NaCNBH3-HCl and the Et3SiH-BF3·Et2O combinations were highly effective in yielding the 6-halobenzyl ether/4-OH derivatives. Et3SiH, in combination with TfOH, produced the 6-O-ether/4-OH products in rapid reactions but also triggered silylation and reductive halobenzylation as secondary transformations. Reductive opening of the 1,3-dioxane ring of pyranosidic 4,6-O-halobenzylidene acetals by the proper reagent combination was found to be an efficient method for the regioselective introduction of versatile halobenzyl protecting groups onto the pyranose ring.

Basic Pharmacological Characterization of EV-34, a New H2S-Releasing Ibuprofen Derivative.

BACKGROUND: Cardioprotective effects of H2S are being suggested by numerous studies. Furthermore, H2S plays a role in relaxation of vascular smooth muscle, protects against oxidative stress, and modulates inflammation. Long-term high-dose use of NSAIDs, such as ibuprofen, have been associated with enhanced cardiovascular risk. The goal of the present work is the synthesis and basic pharmacological characterization of a newly designed H2S-releasing ibuprofen derivative. METHODS: Following the synthesis of EV-34, a new H2S-releasing derivative of ibuprofen, oxidative stability assays were performed (Fenton and porphyrin assays). Furthermore, stability of the molecule was studied in rat serum and liver lysates. H2S-releasing ability of the EC-34 was studied with a hydrogen sulfide sensor. MTT (3-(4,5-dimethylthiazol 2-yl)-2,5-(diphenyltetrazolium bromide)) assay was carried out to monitor the possible cytotoxic effect of the compound. Cyclooxygenase (COX) inhibitory property of EV-34 was also evaluated. Carrageenan assay was carried out to compare the anti-inflammatory effect of EV-34 to ibuprofen in rat paws. RESULTS: The results revealed that the molecule is stable under oxidative condition of Fenton reaction. However, EV-34 undergoes biodegradation in rat serum and liver lysates. In cell culture medium H2S is being released from EV-34. No cytotoxic effect was observed at concentrations of 10, 100, 500 µM. The COX-1 and COX-2 inhibitory effects of the molecule are comparable to those of ibuprofen. Furthermore, based on the carrageenan assay, EV-34 exhibits the same anti-inflammatory effect to that of equimolar amount of ibuprofen (100 mg/bwkg). CONCLUSION: The results indicate that EV-34 is a safe H2S releasing ibuprofen derivative bearing anti-inflammatory properties.

MTT Test and Time-lapse Microscopy to Evaluate the Antitumor Potential of Nucleoside Analogues.

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.