Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones

New hetaryl- and alkylidenerhodanine derivatives 3a–d, 3e, and 4a–d were prepared from heterocyclic aldehydes 1a–d or acetaldehyde 1e. The treatment of several rhodanine derivatives 3a–d and 3e with piperidine or morpholine in THF under reflux, afforded (Z)-5-(hetarylmethylidene)-2-(piperidin-1-yl)thiazol-4(5H)-ones and 2-morpholinothiazol-4(5H)-ones 5a–d, 6a–d, and (Z)-5-ethylidene-2-morpholinothiazol-4(5H)-one (5e), respectively, in good yields. Structures of all compounds were determined by IR, 1D and 2D NMR and mass spectrometry. Several of these compounds were screened by the U.S. National Cancer Institute (NCI) to assess their antitumor activity against 60 different human tumor cell lines. Compound 3c showed high activity against HOP-92 (Non-Small Cell Lung Cancer), which was the most sensitive cell line, with GI50 = 0.62 μM and LC50 > 100 μM from the in vitro assays. In vitro antifungal activity of these compounds was also determined against 10 fungal strains. Compound 3e showed activity against all fungal strains tested, but showed high activity against Saccharomyces cerevisiae (MIC 3.9 μg/mL).


Introduction
In recent years, the synthesis and pharmacological properties of several rhodanine derivatives have been reported [1,2]. Among them, the literature highlights the antibacterial activity of 5-arylidene rhodanine derivatives [3], antimicrobial activity of 5-hetarylidene rhodanine derivatives [4], and antifungal activity of 5-arylidene rhodanine-3-acetic acid [5] and 5-arylidene rhodanines [6]. The substitution of rhodanine derivatives at C-2 (C=S) of the ring has produced compounds with important biological activity [7]. This type of compounds has been used as precursors for the synthesis of new fused heterocyclic systems [8]. Recently, new hetarylmethylidene derivatives were synthesized by Xu and co-workers [9] from the reaction of 1,3-diarylpyrazole-4-carbaldehyde with rhodanine-3-acetic acid. These compounds showed important antimicrobial activity. Herein, we report the synthesis of some new hetarylmethylidene rhodanine derivatives and their antitumor and antifungal activities.

Chemistry
New rhodanine derivatives were prepared from heterocyclic aldehydes 1a-d by different pathways, leading to the hetarylmethylidenerhodanine 3a-d and the rhodanine-3-acetic acid derivatives 4a-d. To obtain the expected compounds 3a-d, a mixture of rhodanine 2a with the respective heterocyclic aldehyde 1a-d and catalytic amounts of piperdine was heated for 4 h at reflux in absolute ethanol. In the case of 3a, a yellow solid was obtained which after spectroscopic characterization (IR, 1 H and 13 C-NMR and mass spectrometry) was confirmed to be the proposed compound. It was obtained in 86% yield (Scheme 1).

Scheme 1.
General methodology for the synthesis of rhodanine and rhodanine-3-acetic acid derivatives and their structures. Compound 3a exhibited characteristic signals of its functional groups. The IR spectrum showed absorption bands at 3,134, 1,684 and 1,213 cm −1 associated with the -NH, C=O and C=S functionalities, respectively. In the 1 H-NMR spectrum, a broad singlet at δ = 13.71 ppm was assigned to the -NH group and singlets at 7.39 and 2.40 ppm were assigned to the vinylidenic proton and to the methyl group of the pyrazole ring, respectively. The 13 C-NMR spectrum showed signals at δ = 169.4 and 195.5 ppm assigned to the (C=O) and (C=S) functionalities, respectively. All signals agree with the proposed structure 3a. Finally, the mass spectrum, showed a peak (m/z 301) corresponding to the molecular ion. Similar results were observed for compounds 3b-d, obtained in good yields, as shown in Table 1. Chen and co-workers have previously reported the synthesis of rhodanine-3-acetic acid derivatives in acetic acid under reflux and using sodium acetate as catalyst [10]. Here we propose the use of microwave irradiation for the synthesis of these compounds with shorter reaction times and easier works-up.
In this sense, a mixture of heterocyclic aldehyde 1a and rhodanine-3-acetic acid was subjected to microwave irradiation (CEM-focused microwave reactor) using DMF as solvent at 100 °C and 100 W of power for 5 min, leading to the formation of a yellow solid which was characterized by IR, 1 H and 13 C-NMR and mass spectrometry to correspond to the desired compound 4a. It was obtained in 92% yield.
In the 1 H-NMR spectrum, we observed a broad singlet at 13.45 ppm assigned to the acid proton (-COOH) and a signal at 4.73 ppm assigned to methylene protons between the acid group and thiazole ring, while the remaining signals corresponded to rest of compound 4a. In the 13 C-NMR spectrum, a signal at 167.2 ppm corresponding to a carbonyl carbon (-COOH) was observed. With the help of DEPT-135 at 45.0 ppm the signal assigned to the methylene carbon between the -COOH group and the rhodanine ring was discerned.
The same procedure was followed to obtain compounds 4b-d in good yields (Scheme 1, Table 1), which highlights the efficiency of the microwave radiation for the synthesis of these compounds. The Z-configuration of compounds 3a-d and 4a-d was deduced based on the previously reported crystal structure of compounds of the (Z)-5-arylidenerhodanine type [11,12].
Subsequently, compound 3a upon reflux during 18 h with an excess of piperidine (2 equiv.) in THF afforded a white solid accompanied by the loss of H 2 S, as detected by its characteristic smell (Scheme 2). This solid corresponded to (Z)-5-(hetarylmethylidene)-2-(piperidin-1-yl)thiazol-4(5H)-one (5a, 85% yield), as confirmed by its IR, 1 H, 13 C-NMR and mass spectra. In the 1 H-NMR spectrum of compound 5a, a singlet at 8.1 ppm corresponding to the proton of the pyrazole ring, a singlet at 7.66 ppm corresponding to the vinylidenic proton, and two broad singlets (2H each one) at 4.03 and 3.60 ppm, assignable to the adjacent methylenes to nitrogen of the piperidine ring, were observed. In the 13 C-NMR spectrum, the disappearance of the characteristic signal of the (C=S) carbon atom, along with the appearance of aliphatic signals at 50.3, 49.6, 26.1, 25.4 and 24.0 ppm (corresponding to the piperidine moiety), confirmed the structure proposed for compound 5a. The mass spectrum showed a peak with (m/z 352) which is in accordance with the expected molecular ion for a structure like 5a. The same procedure was followed for hetarylmethylidenic derivatives 3b-d, with similar results, affording compounds 5b-d, as shown in Table 2. Based on these results; we decided to extend the same methodology to the hetarylmethylidenic derivatives 3a-d but using morpholine instead of piperidine. This approach led to the synthesis of the (Z)-5-(hetarylmethylidene)-2-morpholinothiazol 4(5H)-ones 6a-d, Table 2. In a further experiment, the synthesis of the (Z)-5-ethylidene-2-thioxothiazolidin-4-one (3e) was achieved by refluxing during 7 h an ethanolic solution of rhodanine, paraldehyde and catalytic amounts of piperidine. A yellow solid was obtained in 64% yield. This compound was subjected to reaction with morpholine as described above for compounds 6a-d, thereby obtaining a brown solid in 45% yield, which, by IR, 1 H and 13 C-NMR and MS methods was characterized as the compound 5e (Table 2).

In Vitro Antifungal Activity
Minimum Inhibitory Concentration (MIC) of compounds 3a-e, 4a-d, 5a-e and 6a-d were determined with the microbroth dilution methods M27-A3 and M38-A2 of CLSI [13,14]  Compounds with MICs > 250 μg/mL were considered inactive. MICs between 250-125 μg/mL were indicative of low activity; between 62.5-31.25 μg/mL, moderate activity; MICs  15.6 μg/mL, high activity. Among the last ones, compounds displaying MICs ≤ 10 μg/mL were considered of great interest for further development. In addition to MIC, active compounds (MICs  250 μg/mL) were tested for its capacity of killing fungi rather than inhibiting them through the determination of the Minimum Fungicidal Concentration (MFC). It was determined by plating an aliquote from each clear well of MIC determinations, onto a plate containing clear culture medium. After incubation, MFCs were determined as the lowest concentration of each compound showing no growth, which clearly indicated that fungi were dead rather than inhibited (the detailed methodology is explained in the Experimental section).

In Vitro Antitumor Activity
All compounds synthesized were sent to the U.S. National Cancer Institute (NCI) to evaluate antitumor activity. The results showed that only compound 3c had an interesting antitumor activity and therefore was evaluated against 60 different cell lines (melanoma, leukemia, lung cancer, colon, brain, breast, ovary, kidney and prostate). In order to determine its cytostatic activity compound 3c was evaluated at five concentrations (100, 10, 1.0, 0.1 and 0.001 μM). Compound 3c shows an interesting activity against CCRF-CEM and RPMI-8226 (leukemia) (GI 50 : 2.50, 2.52 μM and LC 50 >100 μM) respectively.
It also exhibited activity against EKVX and NCI-H522 (Non-Small Cell Lung Cancer) (GI 50 : 3.03, 2.96 μM and LC 50 >100 μM), the most sensitive cell line was HOP-92 (Non-Small Cell Lung Cancer) (GI 50 : 0.62 μM and LC 50 >100 μM). These results although moderate, open the research on these compounds with the aim of finding new potential antitumor agents. The LC 50 found indicates a low toxicity of such compounds for normal human cell lines, as required for potential anti-tumor agents (see Table 4).  6.83 >100 a Data obtained from NCI's in vitro disease-oriented human tumor cell lines screen [15]; b GI 50 was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) compared to control cells during the drug incubation; Determined at five concentration levels (100, 10, 1.0, 0.1 and 0.01 mM); c LC 50 is a parameter of cytotoxicity and reflects the molar concentration needed to kill 50% of the cells.

General
Reagents and solvents used below were obtained from commercial sources. Melting points were measured using a Stuart SMP3 melting point device. IR spectra were obtained with a Shimadzu IRAffinity-1. The 1 H and 13 C-NMR spectra were run on a Bruker DPX 400 spectrometer operating at 400 and 100 MHz respectively, using DMSO-d 6 and CDCl 3 as solvents and TMS as internal standard. The mass spectrum was obtained on a Shimadzu-GCMS-QP2010 spectrometer operating at 70 eV. Microwave experiments were carried out on a focused microwave reactor (300W CEM Discover) Thin layer chromatography (TLC) was performed on a 0.2-mm pre-coated plates of silica gel 60GF254 (Merck, Darmstadt, Germany).

Conclusions
New hetaryl-and alkylidenerhodanine derivatives 3a-e, and 4a-d, 5a-d and 6a-d were prepared from heterocyclic aldehydes 1a-d or acetaldehyde 1e. The compounds were screened by the US National Cancer Institute (NCI) to assess their antitumor activity against 60 different human cancer cell lines. Compound 3c showed high activity against HOP-92 (Non-Small Cell Lung Cancer), which was the most sensitive cell line, with GI 50 = 0.62 μM and LC 50 > 100 μM from the in vitro assays. In vitro antifungal activity of these compounds was also determined against 10 fungal strains. Compound 3e showed high activity against yeasts and dermatophyte strains, displaying the lowest MIC against Saccharomyces cerevisiae (MIC = 3.9 μg/mL). It is worth to take into account that we have found two interesting compounds: 3e, that appears to be an antifungal candidate for future research, and compound 3c, that could be an interesting molecule for the design of new hetarylmethylidenerhodanine antitumor derivatives. Due to these significant results, we have carried out chemical studies seeking structures that enhance the antifungal and antitumor activities.