Investigation of anticancer and anti-parasitic activity of thiopyrano[2,3-d]thiazoles bearing norbornane moiety

A. P. Kryshchyshyn, D. V. Atamanyuk, D. V. Kaminskyy © 2017 A. P. Kryshchyshyn et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 615.012.1.076:547.789.6

A search for new anticancer agents among thiopyranothiazoles seems to be promising, and the target compounds of this work are shown in Fig. 1.
A number of hypotheses has been put forward in order to explain possible modes of antitumor action of the thiazolidinone derivatives and speculatively thiopyranothiazoles.For example, a mitochondria-depended proapoptic mode of action related with G 0 /G 1 arrest and an activation of ROS production; the caspase-depended and Bcl-depended pathways are the most discussed [13,22].
The present work is an extension of our ongoing efforts towards a search for new thiazolidinone-based anticancer agents.Another objective of the study was to discover whether there is any correlation between anticancer and antitrypanosomal activity as the latter was shown for a series of related thiopyranothiazoles [20].A repurposing approach is one of the currently used methods to discover new active antitrypanosomal agents [18,23].For example, anticancer drug Bortezomid showed excellent results in in vitro test against Trypanosome brucei inhibiting the parasites growth at nanomolar concentrations [24].The DNA topoisomerase inhibitors (aclarubicin, doxorubicin and mitoxantrone) were also tested against bloodstream forms of Trypanosoma brucei and their trypanocidal activities were comparable with those of commercial antitrypanosomal drugs [25].

Chemistry
All chemicals were of the analytical grade and commercially available.All reagents and solvents were used without further purification and drying.The starting 5-eneisorhodanines (I) [1] and 2,4-thiazolidinedione-5-acetic acid [26] were synthesized as described previously.NMR spectra were determined with Varian Mercury 400 (400 MHz) spectrometer, in DMSO-d 6 using tetramethylsilane as an internal standard.Elemental analyses (C, H, N) were performed on a Perkin-Elmer 2400 CHN analyzer and were within ± 0.4% from the theoretical values.Mass spectra were obtained using electrospray ionization (ESI) techniques on an Agilent 1100 Series LCMS.The purity of the compounds was checked by thin-layer chromatography performed with Merck Silica Gel 60 F254 aluminum sheets.

Pharmacology
Anticancer activity screening.In vitro cell line screening of anticancer activity of the synthesized compounds was carried out at the National Cancer Institute within Developmental Therapeutic Program (www.dtp.nci.nih.gov).Anticancer assays were performed according to the US NCI protocol, which was described elsewhere [27][28][29].
Antitrypanosomal activity screening.Bloodstream forms of Trypanosoma brucei brucei (Tbb) strain 90-13 were cultured in HMI9 medium supplemented with 10 % FCS at 37 °C in an atmosphere of 5 % CO 2 [30].In all experiments, log-phase cell cultures were harvested by centrifugation at 3000×g and immediately used.Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells [31].Drug stock solutions were prepared in DMSO.Tbb bloodstream forms (10 5 cells/ mL) were cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 ml.After the 72 h incubation, resazurin solution was added in each well at the final concentration of 45 mM and fluorescence was measured at 530 nm and 590 nm absorbance after further 4 h incubation.The percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that in the absence of drug.DMSO was used as a control.Concentration inhibiting 50% of parasite growth (IC 50 ) value was given as the mean +/-the standard deviation of three independent experiments.
Introduction of a variety of substituents at the N3 position of the thiazole ring is another efficient approach to the thiazolothiopyrane core modification in order to obtain new bio- logically active analogues [26,35].One more argument in favor of this approach is a decreased toxicity of the N-substituted structurally related 4-thiazolidinones [36].This goal was achieved via the alkylation reaction and further modification of the exocyclic functional groups.Synthetic protocol included in situ 9-aryl(heteryl)-3,7-dithia-5-azatetracyclo[9.2.1.0 2,10.0 4,8]tetradecen-4( 8  The purity and structure of synthesized compounds were confirmed by the analytical and spectral data. 1 H NMR spectra of the synthesized compounds showed classic system of doublets, triplets and multiplets at δ 1.10-3.30ppm characteristic for norbornane fragment.A signal of the CH-group's proton linked to the aromatic ring shows doublet at δ 3.36-3.98ppm and is often overlapped with the norbornane proton signals.In the most cases, signal of the N-CH 2 -CO group corresponds to two doublets of the diastereotopic methylene protons with the spin-spin coupling constant ~16.0 Hz that is caused by the diastereotopici ty of the protons of methylene group.The AMX-system in the form of 3 doublets of doublets is characteristic for CH 2 -CH group (compounds IIIp and IIi).

Anticancer activity
Compounds IIb, IIh, IIIh were selected for primary screening on three tumor cell lines: NCI-H460 (Lung cancer), MCF7 (Breast cancer), SF-268 (CNS cancer) at 10 -4 M concentra-tion.These compounds did not show significant growth inhibition of mentioned cancer cell lines.Moreover, the derivative with thiophene ring in the molecule even increased tumor cell growth (data not shown).
The compounds IIc, IIe, IIIn and IIIu were evaluated at one dose assay towards approximately 60 cell lines (concentration 10 -5 M).The human tumor cell lines represent all forms of cancer (such as, non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, leukemia, renal cancer, melanoma, prostate cancer).In the screening protocol, each cell line was inoculated and pre-incubated for 24-48 h on a microtiter plate.Test agents were then added at a single concentration and the culture was incubated for further 48 h.The end point determinations were made with a protein binding dye, sulforhodamine B. The results for each test agent were reported as the percent growth (GP) of the treated cells compared to the untreated control cells.The screening results are shown in Table 1.
All the tested compounds showed rather good growth inhibition levels against Leukemia cell lines.The best growth inhibition results against Leukemia panel were observed for IIe: cell lines CCRF-CEM (GI = 8.12 %) and HL-60(TB) (GI = -44.16%) and for IIc cell lines CCRF-CEM (GI = 7.01 %).These data cohere with our previously findings about leukemia selective action of the related thiazolidinones [13].The latter compound also inhibited Non-small cell lung cancer line NCI-H522 (GI = 8.12 %).Compound IIIn inhibited growth of Leukemia cell lines and showed cytostatic effect on HCT-15 line of Colon cancer (GI = -100 %).Complication of the basic core with triazole cycle did not influence the activity level, so IIIu showed only moderate anti-tumor activity with the best GI against renal cancer cell line UO-31 (GI = 29.35%).
Finally, the compounds IIIn and IIIu were selected for an advanced assay against a pa nel of approximately sixty tumor cell lines at 10fold dilutions of five concentrations (0.01-100 mM).Additionally, compounds IIa, IId, IIId, IIIg, were involved into the study without prescreening.The percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48-h exposure and using SRB protein assay to estimate cell viability or growth.The dose-response parameters were calculated for each cell line: GI 50 -molar concentration of the compound that inhibits 50 % net cell growth; TGI -molar concentration of the compound leading to the total inhibition; and LC 50 -molar concentration of the compound leading to 50 % net cell death.Furthermore, a mean graph midpoints (MG_MID) were calculated  Anticancer activity levels were also low for these compounds, but they were highly active towards selected cancer cell lines (IIf: log GI 50 = -7.01melanoma SK-MEL-2; IIj: log GI 50 = <-8.00renal cancer RXF-393).An interesting situation was observed for the compounds IIf with thiophene fragment, introduction of which led to the significant antitrypanosomal activity decrease; whilst its N-substituted derivatives inhibited the parasite growth at the concentrations 10 times lower.
Comparing anticancer profile of the tested compounds, it is worth to mention that IIg selectively inhibited growth of the Leukemia cell lines (log GI 50 = -5.16,-5.59) as well as its N-substituted derivative IIIg; and both have shown trypanocidal effects.

Conclusions
A series of novel thiopyranothiazole derivatives were synthesized and their anticancer activity was investigated.Being moderately active towards approximately 60 tumor cell lines, all tested compounds showed rather good growth inhibition against Leukemia cell lines.Chemical structures of the identified hit-com- pounds IId and IIIu indicate that the presence of the arylidene and heterocyclic fragments in the basic system enhances the antitumor effect of such compounds.To establish possible biochemical mechanisms of the action of novel compounds the COMPARE analysis was performed showing correlation at the GI 50 level for IId (PCC = 0.661) with alkylating agent fluorodopan.A number of 9-aryl(heteryl)-3,7dithia-5-azatetracyclo-[9.2.1.0 2,10.0 4,8]-tetradecen-4(8)-ones were tested in vitro against Trypanosoma brucei brucei.Thiopyra no thiazo les with different arylidene fragments in the N5 position showed higher inhibition le vel than their N-unsubstituted analogues.Inte resting is dual antitumor and antitrypanosomal effect observed for some compounds that may be used for establishing molecular modes of action for this class of compounds.