Sci Pharm www.scipharm.at Research article Open Access Synthesis of New N,N'-Bis(5-arylidene-4-oxo- 4,5-dihydrothiazolin-2-yl)piperazine Derivatives Under Microwave Irradiation and Preliminary Biological Evaluation

New N,N’-bis(5-arylidene-4-oxo-4,5-dihydrothiazoline-2-yl)diamine derivatives 5 were prepared in two steps from rhodanine and piperazine, or 1,4-bis(3-amino-propyl)piperazine, under microwave reaction conditions with retention of configuration. Some of these compounds were tested for in vitro antiproliferative activities and for their kinase inhibitory potencies towards six kinases (CDK5/p25, GSK3α/β, DYRK1A, DYRK2, CLK1, and CLK2). The compound 5d showed nanomolar activity towards DYRK1A kinase (IC50 = 0.041 μM).


Introduction
The 2-amino-5-arylidene-5H-thiazol-4-ones and their 5-arylidene rhodanine precursors are a class of five-membered heterocyclic rings (FMHRs) considered as "privileged scaffolds" in the medicinal chemistry community [1]. Considerable work has been published over decades about their chemistry and biology. A number of compounds containing the 2-amino-5-arylidenethiazol-4(5H)-one moiety have been shown to exhibit antiinflammatory [2], antimicrobial [3], and antitumor [4] effects. Among these compounds, Darbufelone® A [5] (Figure 1) is orally active in animal models of inflammation [6] and DBPT B is under clinical trials for colon cancer [7]. 5-Arylidene rhodanines have also proven to be attractive for the discovery of new candidates. A series of rhodanine-based hits C (Figure 1) were found as potent and selective inhibitors of the "atypical" dualspecificity phosphatase (DSP) family member-JNK-stimulating phophatase-1 (JSP-1). Compounds of this class may be useful for the treatment of inflammatory and proliferative disorders [8]. As the last example, epalrestat D reduced the symptoms of diabetic neuropathy [9]. Protein kinases are the enzymes which control the phosphorylation of protein in cellular life [10] which is frequently deregulated in human diseases. For the pharmaceutical industry, the protein kinases represent interesting targets for new therapeutic agents [11] and this interest was boosted by the approval of the first marketed inhibitor Gleevec™ used in myeloid leukemia [12]. Due to the biological activity associated with the 2-aminothiazolidinone moiety, we decided in this paper to explore the synthesis of N,N'-bis (5-arylidene-4-oxo-4,5-dihydrothiazolidin-2-yl)diamines derived from piperazine or 1,4-bis(3aminopropyl)piperazine as linkers, and to study their effects on cells and protein kinases.

Chemistry
The strategy used for the synthesis of the symmetric derivatives 5 is outlined in Scheme 1. The reactions have been realized under microwave irradiation [13]. The main benefits of microwave irradiation technology are the significant rate-enhancements and sometimes elevated product yields enabling the rapid synthesis of molecules of potential value in medicinal chemistry [14]. The synthesis started by the solution-phase Knoevenagel condensation of aryl aldehydes 2 and commercial rhodanine 1a. The expected compounds 3a-c were prepared in yields ranging from 65 to 88% with a reaction time of 10 min. under microwave irradiation (MWI) at 65°C in the presence of sodium acetate. In a similar approach, the 5-arylidene rhodanine propanoic derivatives 3d-g were easily synthesized using solvent-less reaction conditions under microwave irradiation (130°C, 10 min.). The geometric double bond of 3 was attributed as being Z by the shielding effect of the carbonyl group C-4 on the olefinic proton H-5 (δ 7.5 ppm) in the 1 H NMR spectra. Sch. 1. a) for 3a-c: MeOH, AcONa 3 eq., MWI, 65°C, 10 min. and for 3d-g: MWI, 130°C, 10 min. b) MWI, 80-120°C, 30 min.
Transformation of 5-arylidene rhodanine 3 into 2-amino-5-arylidene-5H-thiazol-4-one after addition of a primary amine [15] usually involves activation of the C=S bond of rhodanine via the thioether intermediate that subsequently undergoes a thioalkyl/nitrogen displacement. In order to be able to carry out such sulfur/nitrogen displacement in a faster and more efficient way -avoiding the preparation of the thioether intermediate -we examined the influence of microwave irradiation on the reaction between compound 3 and the symmetric diamino linkers 4a,b. The experiments revealed that optimal reaction conditions were obtained at 80-120°C after 30 minutes. It is noteworthy that sulfur/nitrogen displacement reactions at 120°C under microwave irradiation have been realized in solution with hexane to avoid decomposition of compound 5. The desired compounds 5a-e were prepared in poor to moderate yields (13-26%) and their structures were substantiated by 1 H, 13 C NMR, and HRMS analyses and only the more thermodynamically stable Z-isomers were obtained.

Biology
To evaluate the potency of the compounds 3a-c, 5a, and 5c for their in vitro antiproliferative activities, we used six representative tumor cell lines of liver (Huh7), colon (Caco2, HCT 116), breast (MDA-MB 231), prostate (PC3), lung (NCI), and one normal cell line (fibroblats) and measured survival. Results are reported in Table 1. The 5-arylidene rhodanines 3a-c and two compounds 5a and 5c showed measurable, albeit very poor, cytotoxic activity. No clear tendency for higher activity of these compounds can be deduced from these results. Since the log P values of all tested compounds are in the same range, influences of lipophilicity on cytotoxic activity cannot be deduced as well. The kinase inhibitory potencies of compounds 3a-c and 5d were evaluated as IC 50 values towards six protein kinases (CDK5/p25, GSK3α/β, DYRK1A, DYRK2, CLK1 and CLK2) and the results are reported in Table 2. The 5-arylidene rhodanines 3a,b appeared to be inactive towards these six protein kinases, but interesting results were obtained with compounds 5d and 3c. The 1,4-bis[(5Z)-5-(4-methoxybenzylidene)methylene-4-oxo-4,5dihydrothiazol-2-yl]piperazine 5b and the 5-arylidene rhodanine 3c are very active on DYRK1A [16] and a noteworthy IC 50 = 41 nM was measured for 5b. The compound 5d has also shown submicromolar inhibition potencies towards DYRK2 (IC 50 = 0.6 μM) and CLK1 (IC 50 = 0.5 μM). Regarding these results, the potential of the (5Z) 5-arylidene-4-oxo-4,5-dihydro-thiazolidine-2-yl moiety appended on 1,4-bis(3-aminopropyl)piperazine 4b as a linker could be highly interesting in the development of a new class of inhibitors of DYRK1A kinase which is known to be involved in Alzheimer's disease/Down syndrome [17].

Conclusion
In conclusion, we worked out a short and practical synthesis under microwave irradiation of N,N'-(5-arylidene-4-oxo-4,5-dihydrothiazolidin-2-yl)diamines 5a-e derived from piperazine 4a [18,19] and 1,4-bis(3-aminopropyl)piperazine 4b. The in vitro antiproliferative activities are extremely weak and this could be due to a lower cellular penetration of compounds 3 and 5 or a lower interaction with the cellular targets. Surprisingly, the compound 5d has shown nanomolar inhibition potency towards DYRK1A and this interesting inhibition led us to expand our efforts in the synthesis of diversely disubstituted N,N'-bispiperazine derivatives with the 5-arylidene thiazolidinone moiety as new potential inhibitors of this kinase. Work is in progress to gain deeper insight into the structure-activity relationships (SAR) of this new interesting class of diamines.

General procedure I: Preparation of 5-arylidene-2-thioxo-thiazolidine-4-one 3a-c under microwave irradiation.
A mixture of rhodanine 1a (1 g, 7.5 mmol), aldehyde 2 (8.95-9 mmol), sodium acetate (1.85 g, 22.55 mmol), and methanol (5 ml) was placed in a borosilicate glass vial (10 ml) with a Teflon® magnetic stir bar and sealed with a snap cap. The glass tube was then introduced into the Monowave 300 Anton-Paar microwave cavity (P = 850 Watt) and the stirred mixture was irradiated at 65°C (with a power of 10 Watt) for 30 min. After microwave dielectric heating, the crude reaction mixture was allowed to cool down to room temperature, and then was filtered on a Buchner funnel and the insoluble compound 3 was washed with methanol (2x5 ml). The crude compound 3 was purified by recrystallization from methanol and further dried under high vacuum (10 −2 Torr) for 1 hour, which gave the desired compound 3 as a powder.

General procedure II: Preparation of 3-(5-arylidene-4-oxo-2-thioxo-thiazolidine-3yl)propanoic acid 3d-g under solvent-free microwave irradiation.
A mixture of rhodanine 1b (0.5 g, 2.44 mmol) and aldehyde 2 (2.44 mmol) was placed in a borosilicate glass vial (10 ml) with a Teflon® magnetic stir bar and sealed with a snap cap. The glass tube was then introduced into the Monowave 300 Anton-Paar microwave cavity (P = 850 Watt) and the stirred mixture was irradiated at 130°C for 10 min. After microwave dielectric heating, the crude reaction mixture was allowed to cool down to room temperature and 5 ml of a mixture of ethanol/hexane (1:1) were added directly to the glass vial. The resulting precipitated product 3 was filtered on a Buchner funnel and the insoluble compound 3 was washed with the same mixture (2x5 ml). The crude compound 3 was further dried under high vacuum (10 -2 Torr) for 1 hour, which gave the desired compound 3 as a powder.

General procedure III: Synthesis of N,N'-bis(5-arylidene-4-oxo-4,5-dihydrothiazolidine-2-yl)piperazine derivatives 5a-e under microwave irradiation.
A mixture of 5-arylidene-2-thioxo-thiazolidine-4-one 3 (4 mmol), piperazine 4a (172 mg, 2 mmol) or 1,4-bis(3-aminopropyl)piperazine 4b (400 mg, 2 mmol) was placed in a borosilicate glass vial (10 ml) with a Teflon® magnetic stir bar and sealed with a snap cap. The glass tube was then introduced into the Monowave 300 Anton-Paar microwave cavity (P = 850 Watt) and the stirred mixture was irradiated at 80-120°C (with a power of 5-200 Watt) for 30 min. After microwave dielectric heating, the crude reaction mixture was allowed to cool down to room temperature and 10 ml of cooled ethanol (4°C) were added directly in the glass vial. The resulting precipitated product 5 was filtered off, washed with 2 x 5 ml of ethanol and dried under high vacuum (10 −2 Torr) at room temperature for 1 hour. After 1 H NMR analysis, the product 5 was purified by recrystallization from methanol, which gave the desired compound 3 as a powder.

Cell culture and survival assays
Skin diploid fibroblastic cells were provided by BIOPREDIC International Company (Rennes, France). Caco-2 cells and Huh7 cells were obtained from the ECAC collection. Cells were grown according to ECAC recommendations. The RLEC-F1 clone was derived from an established rat biliary epithelial cell line as previously described [21]. The toxicity test of the compounds on these cells was as follows: 4 x 10 3 cells were seeded in 96 multiwell plates and left for 24 h for attachment, spreading, and growing. Then they were exposed for 48 h to increasing concentrations of the compounds, ranging from 0.1 to 25 μL in a final volume of 80 μL of culture medium. They were then fixed with 4% paraformaldehyde solution and the nuclei were stained with Hoechst 3342 and counted using automated imaging analysis (Simple PCI software).