Synthetic Development of New 3-(4-Arylmethylamino)butyl-5-arylidene-rhodanines under Microwave Irradiation and Their Effects on Tumor Cell Lines and against Protein Kinases

A new route to 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-one 9 was developed in six steps from commercial 1,4-diaminobutane 1 as starting material. The key step of this multi-step synthesis involved a solution phase “one-pot two-steps” approach assisted by microwave dielectric from N-(arylmethyl)butane-1,4-diamine hydrochloride 6a–f (as source of the first point diversity) and commercial bis-(carboxymethyl)-trithiocarbonate reagent 7 for construction of the rhodanine platform. This platform was immediately functionalized by Knoevenagel condensation under microwave irradiation with a series of aromatic aldehydes 3 as second point of diversity. These new compounds were prepared in moderate to good yields and the fourteen synthetic products 9a–n have been obtained with a Z-geometry about their exocyclic double bond. These new 5-arylidene rhodanines derivatives 9a–n were tested for their kinase inhibitory potencies against four protein kinases: Human cyclin-dependent kinase 5-p25, HsCDK5-p25; porcine Glycogen Synthase Kinase-3, GSK-3α/β; porcine Casein Kinase 1, SsCK1 and human HsHaspin. They have also been evaluated for their in vitro inhibition of cell proliferation (HuH7 D12, Caco 2, MDA-MB 231, HCT 116, PC3, NCI-H727, HaCat and fibroblasts). Among of all these compounds, 9j presented selective micromolar inhibition activity on SsCK1 and 9i exhibited antitumor activities in the HuH7 D12, MDA-MBD231 cell lines.

Protein kinases represent an important class of enzymes that play an important role in the regulation of various processes. These enzymes catalyze protein-phosphorylation on serine, threonine and tyrosine residues, which are frequently deregulated in human diseases. Only the 518 human kinases have been investigated as potential therapeutic targets [13]. Consequently, the search of protein-kinase inhibitors represented interesting targets in the pharmaceutical industry for new therapeutic agents. Over the past decade, our research group have investigated the chemical development of five-membered heterocycle rings derived from marine alkaloid as low-molecular weight-inhibitors of dual specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and CLKs (cdc2-like kinases) [14][15][16], two families of kinases involved in various diseases including Alzheimer's disease (AD) [17], and also cancer [18][19][20].

Chemistry
Access to the planned 3-(4-arylphenylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-ones 9 is outlined in Scheme 1. For this study, we selected a diamino linker 1 with a butyl chain in order to obtain a good molecular flexibility between the 4-arylmethyl moiety and the 2-thioxo-1,3-thiazolidine-4-one platform. A molecule with the lowest number of carbons for the diamino linker is more conformationally restrained [25]. The 1,4-diamino-butane linker 1 was treated with di-tert-butyldicarbonate (Boc2O) in 1,4-dioxane at room temperature to afford mainly the mono-N-Boc protected amine 2 in good yield (84%) [26]. To obtain a sufficient number of compounds suitable for a preliminary biological screening, we privileged the transformation of N-Boc-1,4-diamino-butane 2 into mono protected N-arylmethyl diamine 5 by reductive amination in two steps. The (4-arylmethylamino)butyl chain appended on the N-3 position of the 2-thioxo-1,3-thiazolidine-4-one platform represent the first point of diversity for the desired target compounds 9. Preparation of 4 was easily realized by reaction between of appropriate arylaldehyde 3a-f with N-Boc diamine 2 in the presence of molecular sieves 3Å and, the condensation was conducted in a solution of diethyl ether Et2O at room temperature during 24 h. Then, transformation of arylaldimines 4 into mono protected N-arylmethyl diamines 5 could be readily accomplished in good yields (85% to 98%) using NaBH4 (5 equiv.) in MeOH at 50 °C during 24 h. For the cleavage of N-Boc group, the use of trifluoroacetic acid in dichloromethane is often efficient, but in our case, we observed the formation of impurities resulting from uncontrolled degradation of 5 in this strong acidic media. We thus preferred to use a more classical and practical approach by using a solution of 6M HCl. The deprotection was conducted in 1,4-dioxane at room temperature after 4 h of reaction time. As can be seen from the data presented in Table 1, the N-(arylmethyl)butane-1,4-diamine hydrochloride 6a-f were efficiently prepared with electron-rich and electron-poor aldehydes 3a-f in yields ranging from 72% to 98%. With the desired salts 6 in hand, we wished to examine the construction of the 2-thioxo-1,3thiazolidine-4-one platform followed by Knoevenagel condensation using arylaldehyde 3 under microwave irradiation for installation of the 5-arylidene moiety. The choice of arylaldehydes 3 in Knoevenagel condensation represent, in fact, the second point of diversity in the final structure of the targeted compounds 9. In a previous work issued from our laboratory [23], we have developed a "one-pot two-steps" method under microwave irradiation, which was applied for the synthesis of unsymmetrical linked bis-arylidene rhodanines from symmetric diamines.  3e  6b  3  30  30  10  9b  3b  6b  3  30  30  6  9c  3f  6b  3  30  30  5  9d  3g  6b  3  30  30  30  9e  3e  6d  3  15  15  21  9f  3f  6d  3  15  15  9  9g  3d  6d  3  30  30  5  9h  3e  6e  2  15  15  59  9i  3f  6e  2  15  15  59  9j  3h  6e  2  15  15  34  9k  3g  6e  3  30  30  46  9l  3e  6f  3  15  15  15  9m  3f  6f  3  15  Application of this methodology, based on a "modified Holmberg method", first involved, in our case, the reaction of the commercial bis-(carboxymethyl)-trithiocarbonate reagent 7 with the synthesized N-(arylmethyl)butane-1,4-diamine hydrochloride 6 to afford the intermediate 8 and secondly, Knoevenagel condensation to produce the desired 5-arylidene rhodanines 9. This "one-pot two-steps" methodology was realized under microwave dielectric heating because the use of commercial scientific laboratory microwave apparatus favoured higher product yields and significant rate enhancements compared to reactions which run with conventional heating (i.e., in oil bath) [27]. After several experiments, optimal reaction conditions for this "one-pot two-steps" synthesis involved reaction of a stoichiometric mixture of N-(arylmethyl)butane-1,4-diamine hydrochloride 6 and commercial reagent 7 solubilized in dimethoxyethane with one or two equivalents of triethylamine. This starting reaction mixture was placed in a commercial glass tube closed with a snap cap and was irradiated with appropriate reaction time (15 or 30 min.) at 90 °C. After this first period of microwave irradiation followed by a cooling down to room temperature, aromatic aldehyde 3 was directly added to the crude suspension and was submitted again to microwave dielectric heating at 110 °C during 15 or 30 min for condensation.
The desired compound 9 was obtained as precipitate after addition of methanol in the solventless crude reaction mixture (after elimination of the volatile compounds in vacuo) and triturated, followed by successive washings with deionized water, ethanol, ether and finally was recrystallized from absolute EtOH to increase the quality of the precipitated product 9. A set of 14 pure compounds 9a-n was prepared in 5%-59% yield ( Table 1) and all the products 9 were characterized by 1 H-, 13 C-NMR and HRMS before entering the biological tests. For the exocyclic double bond (CH=C) in C-5 position of all the 3-(4-arylmethylamino)butyl-2-thioxo-1,3-thiazolidine-4-one 9a-n, it's possible in theory to observe E-and/or Z-geometrical isomers. Examination of their 1 H-NMR spectra in DMSO-d6 showed only one signal for the methylene proton (CH=) in the range 7.54-7.74 ppm at lower field values than those expected for the E-isomers, which indicates that all the compounds 9a-n have the Z-configuration due to the high degree of thermodynamic stability of this isomer [28,29]. In 13 C-NMR, the signal of C-5 (C=) appears in the range 120.0-127.5 ppm, and we also observed only one signal for the exocyclic methylene proton (134.6 < δCH= < 135.9 ppm) that confirmed the presence of only the Z-geometrical isomer for the targeted compounds 9a-n.

General Section
Melting points were determined on a Kofler melting point apparatus and were uncorrected. Thin-layer chromatography (TLC) was accomplished on 0.2-mm precoated plates of silica gel 60 F-254 (Merck, Fontenay-sous-Bois, France). Visualization was made with ultraviolet light (254 and 365 nm) or with a fluorescence indicator. 1 H-NMR spectra were recorded on BRUKER AC 300 P (300 MHz) spectrometer, 13 C-NMR spectra on a BRUKER AC 300 P (75 MHz) spectrometer. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard. Data are given in the following order: δ value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of protons, coupling constants J is given in Hertz. The mass spectra (HRMS) were taken respectively on a MS/MS ZABSpec Tof Micromass (EBE TOF geometry) at an ionizing potential of 8 eV and on a VARIAN MAT 311 at an ionizing potential of 70 eV in the Centre Régional de Mesures Physiques de l'Ouest (CRMPO, Rennes, France). Reactions under microwave irradiations were realized in the Anton Paar Monowave 300 ® microwave reactor (Anton-Paar, Courtaboeuf, France) using borosilicate glass vials of 10 mL equipped with snap caps (at the end of the irradiation, cooling reaction was realized by compressed air). The microwave instrument consists of a continuous focused microwave power output from 0 to 800 W for this Monowave 300 ® apparatus. All the experiments in the microwave reactor were performed using a stirring option. The target temperature was reached with a ramp of 5 min and the chosen microwave power stayed constant to hold the mixture at this temperature. The reaction temperature is monitored using calibrated infrared sensor and the reaction time included the ramp period. The microwave irradiation parameters (power and temperature) were monitored by the Monowave software package of the Monowave 300 ® microwave reactor. Solvents were evaporated with a BUCHI rotary evaporator. All reagents and solvents were purchased from Acros, Sigma-Aldrich Chimie (Saint-Quentin Fallavier, France), TCI France and Fluka France and were used without further purification.
tert-Butyl (4-aminobutyl)carbamate (2). In a 250 mL two-necked round-bottomed flask, provided with magnetic stirrer and condenser, commercial 1,4-diaminobutane 1 (14.3 mL, 12.5 g, 0.14 mol) was solubilized in 69 mL of 1,4-dioxane at room temperature. To this mixture was added dropwise a solution of commercial di-tert-butyldicarbonate (6.5 g, 30 mmol) in 1,4-dioxane (85 mL) over a period of 3 h. After vigorous stirring at 25 °C during a 12 h period, the volatile compounds of the reaction mixture were eliminated in vacuo and into the crude reaction mixture was poured 150 mL of deionized water. The mixture was extracted with methylene chloride (5 × 50 mL), organic phases were collected and dried over magnesium sulfate. The filtrate was concentrated in a rotary evaporator under reduced pressure and was dried under high vacuum (10 −2 Torr) at 25 °C for 10 min. The desired carbamate 2 (1.58 g) was obtained as a colourless mobile oil in 84% yield and was further used without purification. 1  In a 100 mL two-necked round-bottomed flask, provided with magnetic stirrer and condenser, containing a solution of tert-butyl (4-aminobutyl)carbamate 2 (1.97 g, 10.5 mmol) in anhydrous ether (50 mL) was added dropwise during 30 min, a suspension of commercial aldehyde (10 mmol) in anhydrous ether (30 mL) and molecular sieves (2 g, 3 Å, 8-12 mesh). The crude reaction mixture is stirred vigorously for 16 h at room temperature until the disappearance of aromatic aldehyde 3 controlled by thin-layer chromatography on 0.2-mm precoated plates of silica gel 60 F-254 (Merck). The crude reaction mixture was filtered on filter paper and then concentrated in a rotary evaporator under reduced pressure. The crude residue was dried under high vacuum (10 −2 Torr) at 25 °C for 10 min. The desired aldimine 4 was obtained as yellowish viscous oil and was further used without purification.

Standard Procedure for Reduction of Aldimines 4a-f into N-Boc Monoprotected Diamines 5a-f
In a 50 mL two-necked round-bottomed flask, provided with a magnetic stirrer and reflux condenser, compound 4 (5 mmol) was dissolved in methanol p.a. (30 mL) under vigorous stirring and cooled at 0 °C. To this solution was added by small portions commercial sodium borohydride NaBH4 (0.95 g, 25 mmol) over a period of 20 min. The resulting suspension was stirred at 50 °C for 24 h (monitored by TLC on 0.2-mm precoated plates of silica gel 60 F-254, Merck). After cooling down to room temperature, the volatile compounds were removed in a rotary evaporator under reduced pressure, then deionized water (40 mL) was added in one portion to the crude residue. The mixture was transferred to a separating funnel and was extracted with dichloromethane (3 × 50 mL). The combined organic phases were dried over magnesium sulphate MgSO4, filtered on filter paper and the solvent was eliminated in vacuo. The crude residue was dried under high vacuum (10 −2 Torr) at 25 °C for 2 h. The desired compound 5 was obtained as yellowish viscous oil and was further used without purification.

Conclusions
In summary, we have developed, in this preliminary project, a new route to (5Z) 3-(4-arylmethylamino) butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-ones 9. Starting from commercial butane-1,4-diamine, the process involved six steps and the key step is a solution phase "one-pot two-steps" approach for the construction of the 2-thioxo-1,3-thiazolidine-4-one platform under microwave dielectric heating followed by Knoevenagel condensation for installation of the 5-arylidene moiety as second point of diversity. This methodology offered the possibility of preparing a library of fourteen new compounds in moderate to good yields and, the targeted compounds 9a-n have been built with a Z-geometry. The in vitro inhibition of cell proliferation was carried out on a panel of seven representative tumoral cell lines and the compounds 9a-n were also evaluated against four protein kinases. Among all of these compounds, the compound 9j turned out to be interesting because it presented selective micromolar inhibition activity on SsCK1 (IC50 1.4 μM). Molecules 9h and 9i were also bioactive on SsCK1 and HsCDK5-p25. The current results are the starting point of a new larger program within our group to investigate intensively the biological properties of these new inhibitors with potential application in Alzheimer's disease or in cancer.