SyntheSiS and anti-leukemic activity of pyrrolidinedione-thiazolidinone hybridS

A series of novel 2-(5-ylidene-4-oxo-2-thioxo-thiazolidin-3-yl)-succinimides and 5-ylidene-3-(1-arylpyrrolidine-2,5-dione)-thiazolidine-2,4-diones were synthesized. An efficient simple protocol for rhodaninepyrrolidinedione hybrids synthesis which allows avoiding the step of anhydride formation was proposed. Following the previous data on antileukemic properties of related thiazolidinone derivatives, the activity of 19 target compounds was investigated towards four leukemia cell lines: Dami, hl-60, Jurkat, and K562. Among the tested compounds, 3-[5-(4-chloro-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-1-phenyl-pyrrolidine-2,5-dione (compound 1) possessed good and selective antiproliferative action against Dami and hl-60 cell lines and satisfactory toxicity level (acute toxicity evaluated in vivo in mice).

The crucial impact of the C5-(ylid)ene fragment on the level of anti-cancer activity was confirmed by numerous studies [1][2][3][4][5]. But recently 5-ene-4-thiazolidinones (especially 5-ene-rhodanines -"ene_rhod_A") are treated as frequent hitters or pan assay interference compounds (PAINS) that may easily bind to various proteins, and thus possess low selectivity [36][37][38] in high throughput screening campaigns. This is due to possible Michael acceptor (MA) functionality that, however, often is not confirmed in experimental studies [39][40][41]. The PAINS concept remains controversial and such compounds should not be excluded from the drug development process per se [4]. Moreover, the possibility of targeting various proteins can also be considered as advantageous within the polypharmacological approach [37] or concept of multi-target drugs [42]. Such compounds being the examples of privileged scaffolds can be the structures that provide baseline affinity for a whole protein family [37,43]. Additiona lly, the 5-ene-4-thiazolidinone scaffold offers great possibilities for molecular optimization that aims to increase selectivity and to transform it into other chemical classes to avoid functionalities of MA [4,5,44,45].
Since there is a tendency of prevalent antileukemic action observed when analyzing the data on 5-ene-4-thiazolidinone anticancer activity [1,2,6,25,31,46,47], the study presented in this manuscript became the continuation of the search for small drug-like 4-thiazolidinone-3-carboxylic acids and their derivatives with anti-proliferative properties against leukemic cell lines.

materials and methods
All reagents and materials were purchased from commercial sourses and used without purifica-tion. Melting points were measured in open capillary tubes on a BUCHI B-545 melting point apparatus (Flawil, Switzerland) and are uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer (Waltham, Massachusetts, USA) and were within ±0.4% of the theoretical values. The 1 H NMR spectra were recorded on Varian Gemini 400 MHz (Palo Alto, California, USA) and 13 C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d 6 using tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units with use of δ scale.

General procedure for 5-ylidene-3-(1-arylpyrrolidine-2,5-dione)-rhodanines synthesis (Compounds 1-6, 9, 10, 13-19)
method A. A mixture of 5-ylidene-4-oxo-2-thioxothiazolidin-3-succinic acid (10 mmol) and 5 ml of thionyl chloride in 15 ml of a/h dioxane was refluxed for 1 h, cooled and precipitated by hexane. Formed anhydride was filtered off and used for further transformations without additional purification. A mixture of appropriate anhydride (5 mmol) and aromatic amine (5 mmol) in 10 ml of acetic acid was heated under reflux for 3 h. After cooling the reaction mixture, the obtained solid product was filtered off and recrystallized. method B. The mixture of 5-ylidene-rhodanine-3-succinic acid (5 mmol) and aromatic amine (5 mmol) in 10 ml of acetic acid was heated under reflux for 12-14 h. The progress of the reaction was monitored by TLC. After cooling, the formed precipitate was filtered off and recrystallized.     3       General procedure for 5-ylidene-3-(1-arylpyrrolidine-2,5-dione)-2,4-thiazolidinediones synthesis (compounds 7, 8, 11 and 12). A mixture of 5-ylidene-2,4-thiazolidinedione (10 mmol) and potassium hydroxide (10 mmol) in ethanol was heated under reflux for 1 h. To the obtained salt (5 mmol) N-arylchlorosuccinimide (5 mmol) in 15 ml of EtOH:DMF mixture, catalytic amounts of potassium iodide and potassium carbonate were added and heated under refluxed for 4 h. The reaction mixture was cooled and poured into water, the formed solid product was filtered off and recrystallized from the appropriate solvent.  Anti-leukemic activity screening. In vitro screening of anticancer activity of the synthesized 19 compounds towards four leukemia cell lines (Dami, HL-60, Jurkat, K562) was measured by the MTT test [48]. Tumor cells were seeded for 24 h in 96-well microtiter plates at a concentration of 2000 cells/well or 10,000 suspension cells/well (100 µl/well). After that cells were incubated for 72 h with additions of the synthesized compounds. MTT which is converted to dark blue, water insoluble MTT formazan by the mitochondrial dehydrogenases, was used to determine viable cells according to the manufacturer's protocol (Sigma Aldrich, St. Louis, Missouri, USA).

results
Synthesis of rhodanine derivatives was based on the modification of rhodanine-3-succinic acid and involved several steps: synthesis of 5-ylidenerhodanine-3-succinic acids which were converted into target imides via the stage of cyclic anhydride formation (Scheme). To avoid this stage, the alternative one-step method for target 5-ylidene-3-(1-arylpyrrolidine-2,5-dione)-rhodanines synthesis was worked out: long term heating of 5-ylidene-rhodanine-3-succinic acid with aromatic amine (1:1) in the acetic acid medium (method B). 2,4-Thiazolidinedione was used as a starting compound for the synthe- The tested compounds were evaluated at one dose (10 µM) in the MTT assay (Table). The most sensitive cell line to the tested compounds turned out to be the DAMI line. Four compounds from the rhodanine-based group (1, 2, 9, and 14) inhibited its growth by more than 50%. In general, compounds bearing the 2-thioxo-4-thiazolidone core showed higher inhibition rates than their analogs with the thiazolidine-2,4-dione cycle.

discussion
The project involved the design of the target compound structures based on our previous study results and the literature data; development of the synthetic protocols and synthesis of thiazolidinonepyrrolidinedione conjugates; and in vitro screening towards leukemic cell lines and acute toxicity evaluation in vivo in mice for the most active compounds.

Conflict of interest.
Authors have completed the Unified Conflicts of Interest form at http://ukrbiochemjournal.org/wp-content/uploads/2018/12/ coi_disclosure.pdf and declare no conf lict of interest .