Synthesis and evaluation of biological activity of rhodanine- pyrazoline hybrid molecules with a 2-(2,6-dichlorophenylamino)- phenylacetamide fragment

© 2020 Yu. L. Shepeta 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: 615.276:547.789:542.91


Chemistry
All materials were purchased from Merck and Sigma-Aldrich and were used without purification. Melting points were measured in open capillary tubes and are uncorrected. Elemental analyses were performed using Perkin-Elmer 2400 CHN analyzer. The 1 H NMR (400 MHz) spectra were recorded on Varian Mercury 400 (400 MHz) in DMSO-d 6 using tetramethylsilane as internal standard. Mass spectra were obtained using electrospray ionization (ESI) techniques on an Agilent 1100 Series LC/ MSD. The purity of all obtained compounds was checked by thin-layer chromatography (eluent benzene : EtOAc 1:1). (2). A mixture of 2-[2-(2,6-dichloro-phenylamino)phenyl]-N-(4-oxo-2-thioxothiazolidin-3-yl)acetamide (10 mmol) and triethyl orthoformate was heated at reflux for 2 h in acetic anhydride (10 mL (11). Yield 64 %, mp 171-172ºC. 1  Pharmacology Antitrypanosomal activity assay. Bloodstream forms of Trypanosoma brucei brucei (Tbb) strain 90-13 were cultured in HMI9 medium supplemented with 10 % FCS at 37°C under an atmosphere of 5 % CO 2 [22]. In all experiments, log-phase parasite cultures were harvested by centrifugation at 3,000 x g and im-mediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [23]. Drug stock solutions were prepared in pure DMSO. Trypanosoma brucei 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 µl. After a 72-h incubation, resazurin solution was added in each well at the final concentration of 45 µM and fluorescence was measured at 530 nm and 590 nm absorbance after a 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 control. Concentration inhibiting 50 % of parasite growth (IC 50 ) was determined from the dose-response curve with a drug concentrations ranging from 10 µg/ml to 0.625 µg/ml and presented in µM. IC 50 value is the mean +/ -the standard deviation of three independent experiments.

2-[2-(2,6-Dichlorophenylamino)-phenyl]-N-{5-[5-(4-fluorophenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-ylmethylene]-4-oxo-2-thioxothiazolidin-3-yl}-acetamide
In vitro anticancer assay. 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 [24][25][26]. 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. 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: Dose response parameters (GI 50 , TGI) were calculated for each compound. Growth inhibition of 50 % (GI 50 ) was calculated from [(Ti -Tz)/(C -Tz)] x 100 = 50, 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 as Ti = Tz. 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 structures of the synthesized compounds were confirmed by elemental analysis and spectroscopic data ( 1 H NMR and LCMS). In 1 H NMR spectra the characteristic secondary amine proton of diclofenac fragment appears as a singlet at δ ~10.50-11.35 ppm and methylene protons assigned as a singlet at δ ~3.65-3.85 ppm, respectively. The pyrazoline fragment of 3-11 shows characteristic patterns of an AMX system for CH 2

In vitro evaluation of the anticancer activity
The synthesized 5-(3,5-diaryl-4,5-dihydropyrazol-1-ylmethylene)-2-thioxothiazolidin-4-ones 7 and 9 were evaluated at the single concentration of 10 -5 M towards panel of approximately sixty cancer cell lines. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the US NCI protocol, which was described elsewhere (see e.g. http:// dtp.nci.nih.gov) [23][24][25][26]. The results of primary screening are reported as the percent cancer cell line growth (GP%) and presented in Table 1. The range of growth % shows the lowest and the highest growth % found among different cancer cell lines.
The most active 2- Finally, compound 9 possessed considerable activity against all tested human tumor cell lines and was selected in advanced assay against a panel of approximately sixty tumor cell lines at 10-fold dilutions of five concentrations (100 μM, 10 μM, 1 μM, 0.1 μM and 0.01 μM) [23][24][25][26]. 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. Three antitumor activity dose-response parameters were calculated for each cell line: GI 50 -molar concentra-tion 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 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. Compound 9 showed a broad spectrum of growth inhibition activity against tested human tumor cells with average GI 50 and TGI values 0.71/1.09 and 82.95/28.46μM, respectively ( Table 2). The selectivity index (SI) obtained by dividing the full panel MG-MID (μM) of the compound 9 by their individual subpanel MG-MID of cell line (μM) was considered as a measure of compound's selectivity. The compound 9 in the present study was found to be nonselective at

Antitrypanosomal activity
The antitrypanosomal activity of the novel 5 -( 3 , 5 -d i a r y l -4 , 5 -d i h y d r o p y r a z o l -1-ylmethylene)-2-thioxothiazolidin-4-ones 3, 4, 6, 7, 9-11 was studied in in vitro assay towards Trypanosome brucei brucei (Tbb). The IC 50 values were calculated based on at least three independent experiments. In general, the synthesized compounds displayed slight inhibition on growth of the tested parasites (Table 3). Hovewer, among the tested derivatives the most active compounds were found to be 3 and 6 with IC 50 values of 15.0 and 17.2 μM, respectively. The SAR study revealed that the level of antitrypanosomal activity of 5-(3,5-diaryl-4,5-dihydropyrazol-1-ylme thy lene)-2thioxothiazolidin-4-ones depends on substituent at N3 of thiazolidinone core. Noteworthy,      Continued Table 2 the diclofenac moiety in the N3 position of the basic heterocycle was adverse on activity against Trypanosoma species compared to previously described substituted pyra zolinethiazolidinone hybrid molecules [19,28]. Table 3. Antitrypanosomal activity of the tested compounds Compound Tbb IC 50 , µM 3 15.0 ± 0.12 4 29.0 ± 0.45 6 17.