Synthesis and Anticancer Activity Evaluation of 5-[2-Chloro-3-(4-nitrophenyl)-2-propenylidene]-4-thiazolidinones

A series of novel 5-[(Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene]-thiazolidinones (Ciminalum–thiazolidinone hybrid molecules) have been synthesized. Anticancer activity screening toward the NCI60 cell lines panel, gastric cancer (AGS), human colon cancer (DLD-1), and breast cancer (MCF-7 and MDA-MB-231) cell lines allowed the identification of 3-{5-[(Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene]-4-oxo-2-thioxothiazolidin-3-yl}propanoic acid (2h) with the highest level of antimitotic activity with mean GI50/TGI values of 1.57/13.3 μM and a certain sensitivity profile against leukemia (MOLT-4, SR), colon cancer (SW-620), CNS cancer (SF-539), melanoma (SK-MEL-5), gastric cancer (AGS), human colon cancer (DLD-1), and breast cancers (MCF-7 and MDA-MB-231) cell lines. The hit compounds 2f, 2i, 2j, and 2h have been found to have low toxicity toward normal human blood lymphocytes and a fairly wide therapeutic range. The significant role of the 2-chloro-3-(4-nitrophenyl)prop-2-enylidene (Ciminalum) substituent in the 5 position and the substituent’s nature in the position 3 of core heterocycle in the anticancer cytotoxicity levels of 4-thiazolidinone derivatives have been established


Introduction
In recent years, one of successful directions in the structure design of "drug-like" molecules is the "hybrid-pharmacophore" approach that involves combining different fragments in one molecule that can be parts, biomimetics, and/or bioisosteres of biologically active molecules or drugs. This strategy allows potentiating the desired action or appearance of new effects [1][2][3] and can be relevant for the search for new highly active compounds based on 4-thiazolidinones as effective biophores. Thus, modern studies of the pharmacological potential of thiazolidinones have significantly expanded the range of their activity, including anticancer, antibacterial, antifungal, antiviral, antiparasitic, and anti-tuberculosis. Along with this, there is indisputable evidence of the affinity of these derivatives for biotargets involved in the biochemical processes of tumor cell growth (TNF-α-TNFRc-1, JSP-1, antiapoptotic complex Bcl-XL-BH3), the microorganisms life cycle Our research was aimed at optimization of anticancer activity profile of 5-[(Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene]-4-thiazolidinones and SAR analysis within these series in accordance with our systematic study of anticancer activity of thiazolidinonerelated derivatives [9,10].

In Vitro Evaluation of the Anticancer Activity
At the first stage of biological activity study, the antitumor activity screening of the selected compounds 2b, 2c, 2f, 2h, and 2j was performed according to the NCI DTP (USA) standard protocol at the concentrations ranging from 10 −4 to 10 −8 M toward 60 tumor cell lines [22][23][24][25]. 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. 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 50molar concentration of the compound leading to 50% net cell death. Furthermore, mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an average activity parameter over all cell lines for the tested compound. For the MG_MID calculation, insensitive cell lines were included with the highest concentration tested.
The obtained results of screening evaluation of Ciminalum-thiazolidinone hybrids confirmed their significant anticancer activity (Table 2). Thus, compounds 2f and 2h inhibited the growth of all tested cancer cell lines at submicromolar and micromolar concentrations. The average meanings of three dose-response parameters GI 50 , TGI, and LC 50 were 2.80/32.3/80.8 µM (2f) and 1.57/13.3/65.0 µM (2h), respectively. It is important to note that the most active compound 2h was active in the GI 50 concentration range of < 0.01-0.02 µM toward the following cell lines: MOLT-4, SR (Leukemia); SW-620 (Colon cancer); SF-539 (CNS cancer); SK-MEL-5 (Melanoma). Regarding the preliminary SAR analysis, it is worth mentioning that the presence of the (Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene moiety turned out to be a necessary requirement for achieving the anticancer effects. Moreover, the substituent nature at position 3 of the 4-thiazolidinone ring is important. Derivatives with carboxylic acids residues (2h, 2j) and p-hydroxyphenyl substituent (2f) proved to be the most effective. The absence of a substituent in position 3 (2b) or an additional fragment of the Cyminalum (2c) leads to the weakening of anticancer cytotoxicity.  The selectivity index (SI) obtained by dividing the full panel MG-MID (mM) of the tested compound by their individual subpanel MG-MID (mM) was considered as a measure of selectivity of anticancer activity (Table 3). Ratios between 3 and 6 mean moderate selectivity, ratios greater than 6 indicate high selectivity toward the corresponding cell line, while compounds not meeting either of these criteria are rated nonselective [26]. The most active compounds 2f and 2h in the present study were found to be high selective toward the leukemia subpanel at GI 50 levels (selectivity indices 9.89 and 10.73, respectively). Compound 2j possessed high selectivity toward the CNS cancer subpanel at both the TGI and LC 50 levels (selectivity index 11.53 and 10.25, respectively). In general, it is worth noting the selectivity of action against leukemia cell lines for the studied class of heterocyclic compounds.
In the second stage of the research, Ciminalum-thiazolidinone hybrids were investigated for antitumor activity on the lines of gastric cancer (AGS), human colon cancer (DLD-1), and breast cancers (MCF-7 and MDA-MB-231). The study was performed in the MTT assay according to the method described previously [27]. The studied cancer line was sensitive to the action of the studied compounds that inhibited its growth in micromolar ranges of GI 50 . The hit compounds that inhibited the growth of all four cancer lines with the lowest GI 50 values were [5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-rhodanines 2c, 2d, 2h, and 2i (Table 4). Moreover, it is important to note the high cytotoxic effect of rhodanine-3-carboxylic acid derivatives 2h and 2i toward breast cancer lines MCF-7 and MDA-MB-231 at the GI 50 level of 0.95-1.74 µM, which is consistent with previous data obtained according to DTP NCI protocol ( Table 2). Regarding the SAR analysis (Figure 4), the significant role of the 5-[2-chloro-3-(4nitrophenyl)prop-2-enylidene (Ciminalum) substituent in the anticancer cytotoxicity appearance was confirmed. Moreover, the presence of a thioxo group in position 2 of the core heterocycle is more important than the oxo group, as evidenced by the lower activity of the thiazolidinedione 2b compared to a structurally close rhodanine derivative 2a. The role of the substituents nature in position 3 of the rhodanine core on the level of anticancer cytotoxicity level is interesting and important for further in-depth research and the design of drug-like molecules. Thus, the most effective is the presence of carboxylic acids residues, among which fragments of propanoic (2h) and hexanoic (2i) acids are considered to be important for cytotoxicity toward AGS, DLD-1, MCF-7, and MDA-MB-231 cell lines. The introduction of an additional carboxylic group reduced the effect of derivatives by about 10 times (compounds 2k and 2l). Replacing the carboxyl group with a sulfo group had reduced the activity more significantly (compound 2g). In addition to the carboxylic acid residues, an additional Ciminalum fragment (2c) or 4,5,6,7-tetrahydrobenzo[b]thiophen-3ylcarboxamide moiety (2d) at position 3 of the rhodanine cycle were also important for the anticancer activity. Another part of our study was to determine the influence of compounds 2f, 2i, 2j, and 2h on normal human blood lymphocytes ( Figure 5). GI 50 values for compounds 2j and 2h were 48.97 µM and 54.54 µM correspondingly. Compounds 2i and 2f do not reach GI 50 up to 100 µM after 48 h incubation. The pure Ciminalum has the lowest IC 50 value (GI 50 = 10.4 µM) for human normal lymphocytes. Thus, normal blood lymphocytes are blood cells, as well as cells of leukemia cell lines, therapeutic index (TI) of compounds 2f, 2h, and 2j was calculated as GI 50 (normal blood lymphocyte)/GI 50 (leukemia cell line) ( Table 5).

General Information
All reagents and solvents were purchased from commercial suppliers and were used directly without further purification. NMR spectra were determined with Varian Unity Plus 400 (400 MHz) and Bruker 170 Avance 500 (500 MHz) spectrometers, in DMSO-d 6 using tetramethylsilane (TMS) as an internal standard. Melting points were measured on a Kofler hot-stage and are uncorrected. LC-MS was performed using a system with an Agilent 1100 Series HPLC equipped with diode-array detector and Agilent LC\MSD SL mass-selective detector using chemical ionization at atmospheric pressure (APCI). The NMR and LCMS spectra of compounds 2a-l are presented in Figures S1-S32.
Structure solution and refinement. The structure was solved by a dual space algorithm (SHELXT) [29] and refined against F 2 for all data (SHELXL) [30]. The position of the H atom bonded to the O atom was obtained from the difference Fourier map and was refined freely. The remaining H atoms were positioned geometrically and were refined within the riding model approximation: C-H = 0.98 Å (CH 3 ), 0.99 Å (CH 2 ), 0.95 Å (Csp 2 H), and U iso (H) = 1.2U eq (C) or 1.5U eq (C) for methyl H atoms. The methyl groups were refined as a rigid group, which were allowed to rotate. Final refinement converged with R = 0.0319 (for 4729 data with F 2 > 4σ(F 2 ), wR = 0.0864 (on F 2 for all data), and S = 1.052 (on F 2 for all data). The largest difference peak and hole was 0.281 and -0.275 eÅ 3 .

In Vitro Evaluation of the Anticancer Activity According DTP NCI Protocol
Primary anticancer assay was performed on a panel of approximately sixty human tumor cell lines derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [22][23][24][25]. Tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of human tumor cell lines at concentrations ranging from 10 −4 to 10 −8 M. A 48 h continuous drug exposure protocol was followed, and an SRB protein assay was used to estimate cell viability or growth.
Using absorbance measurements (time zero (Tz), control growth in the absence of drug (C), and test growth in the presence of drug (Ti)), the percentage growth was calculated for each drug concentration. Percentage growth inhibition was calculated as: [(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz (1) [(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz.
Dose-response parameters (GI 50 , TGI, LC 50 ) were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50 (1), which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC 50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz)/Tz] × 100 = −50 (2). Values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was excessive, the value for that parameter was expressed as more or less than the maximum or minimum concentration tested. The lowest values were obtained with the most sensitive cell lines. Compounds having GI 50 values ≤ 100 µM were declared to be active. The assay was performed by using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confluent cells, cultured for 24 h with 0.1, 1, 5, 10, 20, 30, and 100 µM concentrations of studied compounds in 24-well plates were washed with PBS. MTT was dissolved in PBS, and 25 µL were added to each well. Plates were incubated for 4 h at 37 • C in 5% CO 2 in an incubator. The medium with MTT was removed, and 1 mL of DMSO was added to the attached cells. Furthermore, cells were incubated for 5-10 min in RT and then 10 µl of Sorensen buffer was added to each well. The absorbance of converted dye in living cells was measured at a wavelength of 570 nm. The cell viability of breast cancer cells, gastric cancer cells, and human colon cancer cells cultured in the presence of ligands was calculated as percent of control cells.

Isolation of Human Blood Lymphocytes and Their Activation
First, 20 mL of venous blood was taken from volunteers (Ethical protocol number 2, 27 January 2019) and collected in the presence of 200 µL of undiluted fresh heparin (1/100). Sterile blood was diluted 2 times with 0.9% NaCl under the sterile conditions. Isolation of lymphocytes was performed in a density gradient of ficol-verografin using the protocol of the manufacturer (Lympoprep, NYCOMED PHARMA AS, Oslo Norway). The resulting lymphocytes were resuspended in the RPMI-1640 medium and cultured for several days (up to 10 days). To separate the lymphocytes from the monocytes, cell suspension was left for 24 h. After 24 h of culture, monocytes were attached, while lymphocytes were transferred to a fresh Falcon tube (15 mL). To stimulate the proliferation of lymphocytes, they were cultured on CD3+ antibody-coated plastic plate in the RPMI-1640 medium supplemented with 20% FBS.