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

Insuasty, Alberto; Ramírez, Juan; Raimondi, Marcela; Echeverry, Carlos; Quiroga, Jairo; Abonia, Rodrigo; Nogueras, Manuel; Cobo, Justo; Rodríguez, María Victoria; Zacchino, Susana A.; Insuasty, Braulio

doi:10.3390/molecules18055482

Open AccessArticle

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

by

Alberto Insuasty

¹,

Juan Ramírez

¹,

Marcela Raimondi

²,

Carlos Echeverry

¹,

Jairo Quiroga

¹,

Rodrigo Abonia

¹,

Manuel Nogueras

³

,

Justo Cobo

³,

María Victoria Rodríguez

²,

Susana A. Zacchino

² and

Braulio Insuasty

^1,*

¹

Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, A.A. 25360, Cali, Colombia

²

Pharmacognosy, Faculty of Biochemical and Pharmaceutical Sciences, Universidad Nacional de Rosario, Suipacha 531, (2000), Rosario, Argentina

³

Department of Inorganic and Organic Chemistry, Universidad de Jaén, 23071, Jaén, Spain

^*

Author to whom correspondence should be addressed.

Molecules 2013, 18(5), 5482-5497; https://doi.org/10.3390/molecules18055482

Submission received: 8 April 2013 / Revised: 2 May 2013 / Accepted: 8 May 2013 / Published: 13 May 2013

(This article belongs to the Section Medicinal Chemistry)

Download

Browse Figures

Versions Notes

Abstract

:

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).

Keywords:

antifungal activity; antitumor activity; hetarylmethylidenerhodanine derivatives; piperidine; morpholine

Graphical Abstract

1. 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.

2. Results and Discussion

2.1. 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, ¹H and ¹³C-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.

Reagents and Conditions: i = (R' = H), piperidine (catalytic amounts), reflux, 4 h; ii = (R' = CH₂COOH), MW (100 W, 100 °C, 30 psi), 5 min.

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 ¹H-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 ¹³C-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.

Table 1. Melting points and yields for the hetarylmethylidene rhodanine derivatives 3a–d, 3e and rhodanin-3-acetic acid derivatives 4a–d.

**Table 1.** Melting points and yields for the hetarylmethylidene rhodanine derivatives 3a–d, 3e and rhodanin-3-acetic acid derivatives 4a–d.
Compound	m.p. (°C)	Yield (%)
3a	294–295	86
3b	307–309	91
3c	315–317	86
3d	230–231	85
3e	145–147	64
4a	279–281	81
4b	254–256	53
4c	263–265	92
4d	232–234	63

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, ¹H and ¹³C-NMR and mass spectrometry to correspond to the desired compound 4a. It was obtained in 92% yield.

In the ¹H-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 ¹³C-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 H₂S, 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, ¹H, ¹³C-NMR and mass spectra. In the ¹H-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.

Scheme 2. General methodology for the synthesis of (Z)-5-(hetarylmethylidene)-2-(piperidin-1-yl)thiazol-4(5H)-ones and (Z)-5-(hetarylmethylidene)-2-morpholinothiazol-4(5H)-ones and their structures.

Reagents and Conditions: i = (R' = H), piperidine (2 equiv.), THF at reflux, 7–24 h.

In the ¹³C-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.

Table 2. Melting points and yields for the piperidine and morpholine derivatives 5a–d, 5e and 6a–d.

**Table 2.** Melting points and yields for the piperidine and morpholine derivatives 5a–d, 5e and 6a–d.
Compound	-X-	m.p. (°C)	Yield (%)
5a	-CH₂-	141–143	85
5b	-CH₂-	261–262	70
5c	-CH₂-	262–264	95
5d	-CH₂-	194–196	93
5e	-O-	193–195	45
6a	-O-	264–265	71
6b	-O-	270–272	62
6c	-O-	266–268	86
6d	-O-	206–208	85

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, ¹H and ¹³C-NMR and MS methods was characterized as the compound 5e (Table 2).

2.2. 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] against a panel of 10 fungal species comprising four yeasts (Candida albicans, C. tropicalis, Cryptococcus neoformans and Saccharomyces cerevisiae), three Aspergillus spp. (A. niger, A. fumigatus and A. flavus) and three dermatophytes (Trichophyton rubrum, T. mentagrophytes and Microsporum gypseum]. 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).

Compounds 3a, 3b, 3d, 4b–d, 5a–e and 6a–d, were inactive (Table 3). In contrast, compounds 3c, 3e and 4a showed varied activities, being 3e the one with the broadest and highest activity. An analysis of correlation between structure and activity showed that the most potent compound 3e possessed the simplest structure among the all tested compounds, possessing a thiazolidine ring and methyl substituent as the R moiety. The other structure with R = methyl (compound 5e) with a more complex structure containing a thiazole ring and morpholino substituent, did not possess activity up to 250 µg/mL.

Table 3. In vitro antifungal activities (MIC and MFC values in μg/mL, showed as MIC/MFC) of hetarylidenerhodanine derivatives.

**Table 3.** In vitro antifungal activities (MIC and MFC values in μg/mL, showed as MIC/MFC) of hetarylidenerhodanine derivatives.
Compound	Structure	Antifungal Activity MIC/MFC (μg/mL)
Compound	Structure	Ca	Ct	Sc	Cn	Afu	Afl	Ani	Mg	Tr	Tm
3a		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
3b		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
3c		>250	>250	>250	125/125	250/250	250/250	250/250	125/125	125/125	125/125
3d		>250	>250	>250	>250	>250	>250	>250	<250	<250	<250
3e		7.8/31.2	7.8/31.2	3.9/15.6	15.6/62.5	31.2/250	31.2/250	62.5/250	7.8/7.8	7.8/15.6	15.6/15.6
4a		>250	>250	>250	>250	>250	>250	>250	125/125	62.5/62.5	62.5/62.5
4b		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
4c		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
4d		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
5a		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
5b		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
5c		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
5d		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
5e		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
6a		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
6b		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
6c		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
6d		>250	>250	>250	>250	>250	>250	>250	>250	>250	>250
amphotericin B	-	0.78		0.50	0.25	0.50	0.50	0.50	0.12	0.07	0.07
ketoconazole	-	1.56		3.12	0.39	0.78	0.78	1.56	0.04	0.01	0.02
terbinafine	-	0.50		0.50	0.25	0.12	0.50	0.25	0.05	0.02	0.02

Antifungal activity was determined with the microbroth dilution assay following the CLSI guidelines. Fungi used: C.a.: Candida albicans ATCC10231, C.t.: Candida tropicalis C131; C.n.: Cryptococcus neoformans ATCC32264, S.c.: Saccharomyces cerevisiae ATCC9763, A.n.: Aspergillus niger ATCC9029, A.fl.: Aspergillus flavus ATCC 9170, A.fu.: Aspergillus fumigatus ATCC 26934, M.g.: Microsporum gypseum C 115, T.r.: Trichophyton rubrum C113, T.m.: Trichophyton mentagrophytes ATCC 9972.

An analysis of the effect of the substituents other than the methyl group showed that those with 4-methyl-1H-imidazol-5-yl [compounds named “b” (3, 4, 5 and 6)] or with 5-methylthiophen-2-yl [named “d” (3, 4, 5 and 6)] were inactive. On the other hand, among compounds with R = 1,3-diphenyl-1H-pyrazol-4-yl (named “c”), or R = 1-methyl-3-phenyl-1H-pyrazol-4-yl (named “a”), 3c showed low broad spectrum of activity and 4a displayed moderate activity (MICs between 62.5–125 μg/mL) respectively. Interestingly, the three active structures (3c, 3e and 4a) possess fungicidal rather than fungistatic activities, with MFC values between 7.8 and 250 µg/mL. Compound 3e showed the lowest MFC values against S. cerevisiae and the dermatophytes M. gypseum, T. rubrum and T. mentagrophytes.

2.3. 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) (GI₅₀: 2.50, 2.52 μM and LC₅₀ >100 μM) respectively.

It also exhibited activity against EKVX and NCI-H522 (Non-Small Cell Lung Cancer) (GI₅₀: 3.03, 2.96 μM and LC₅₀ >100 μM), the most sensitive cell line was HOP-92 (Non-Small Cell Lung Cancer) (GI₅₀: 0.62 μM and LC₅₀ >100 μM). These results although moderate, open the research on these compounds with the aim of finding new potential antitumor agents. The LC₅₀ found indicates a low toxicity of such compounds for normal human cell lines, as required for potential anti-tumor agents (see Table 4).

Table 4. In vitro testing expressed as growth inhibition of cancer cell lines for compound 3c ^a.

**Table 4.** In vitro testing expressed as growth inhibition of cancer cell lines for compound 3c ^a.
Panel/Cell Line	Compound 3c
Panel/Cell Line	GI₅₀ ^b (μM)	LC₅₀ ^c (μM)
Leukemia
CCRF-CEM	2.50	>100
HL-60(TB)	4.83	>100
K562	7.54	>100
MOLT-4	14.8	>100
RPMI-8226	2.52	>100
SR	7.29	>100
Non Small Cell Lung
A549/ATCC	5.88	>100
EKVX	3.03	>100
HOP-62	22.7	>100
HOP-92	0.62	>100
NCI-H226	2.03	>100
NCI-H23	2.68	>100
NCI-H322M	7.63	>100
NCI-H460	5.50	54.4
NCI-H522	2.96	>100
Colon Cancer
COLO 205	21.2	>100
HCC-2998	6.05	>100
HCT-116	5.62	70.2
HCT-15	4.71	96.2
HT29	12.5	>100
KM12	6.24	63.5
SW-620	19.6	>100
Prostate Cancer
PC-3	5.66	>100
DU-145	12.6	>100
CNS Cancer
SF-268	17.2	>100
SF-295	3.33	82.6
SF-539	5.53	61.0
SNB-19	6.14	>100
SNB-75	17.8	>100
U251	5.54	64.8
Melanoma
LOX IMVI	10.0	>100
MALME-3M	3.84	6.11
M14 0.405	6.75	>100
MDA-MB-435	4.91	>100
SK-MEL-2	4.18	70.7
SK-MEL-28	9.22	>100
SK-MEL-5	3.19	58.4
UACC-257	13.2	>100
UACC-62	3.36	5.77
Renal Cancer
786-0	3.92	>100
A498	2.99	94.4
ACHN	7.40	52.1
CAKI-1	7.15	>100
RXF 393	22.4	>100
SN12C	9.93	>100
TK-10	8.00	>100
UO-31	4.39	>100
Breast Cancer
MCF7	8.31	>100
MDA-MB231/ATCC	10.1	>100
HS 578T	6.03	>100
BT-549	5.20	86.5
T-47D	4.59	>100
MDA-MB-468	6.83	>100

^a Data obtained from NCI’s in vitro disease-oriented human tumor cell lines screen [15]; ^b GI₅₀ 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 LC₅₀ is a parameter of cytotoxicity and reflects the molar concentration needed to kill 50% of the cells.

3. Experimental

3.1. 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 ¹H and ¹³C-NMR spectra were run on a Bruker DPX 400 spectrometer operating at 400 and 100 MHz respectively, using DMSO-d₆ and CDCl₃ 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).

3.2. Synthesis

3.2.1. General Procedure for the Synthesis of (Z)-5-Hetarylmethylidene-2-thioxothiazolidin-4-ones 3a–d

Two drops of piperidine (0.01 equiv.) were added to an ethanolic solution of heterocyclic aldehyde (1a–d, 1.1 mmol) and rhodanine (2a, 1 mmol). The mixture was refluxed for 4–6 h and the solid formed was isolated by vacuum filtration and washed with cold ethanol.

(Z)-5-((3-Methyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-2-thioxothiazolidin-4-one (3a). Yellow solid (86%), m.p. 294–295 °C; FT-IR (KBr), υ: (NH) 3134, (C=O) 1684 and (C=S) 1213 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.40 (s, 3H, CH₃), 7.36 (t, J = 7.44 Hz, 1H, Ar-H_p), 7.39 (s, 1H, H-6), 7.51 (dd, J = 7.44 and 8.52 Hz, 2H, Ar-H_m), 7.93 (d, J = 8.52 Hz, 2H, Ar-H_o), 8.50 (s, 1H, H-5’), 13.71 (s, 1H, -NH-) ppm; ¹³C-NMR (DMSO-d₆), δ: 12.0 (-CH₃), 117.0 (C-4'), 119.4 (C_o), 122.3 (C-6), 123.4 (C-5), 127.5 (C_p), 128.0 (C-5'), 130.0 (C_m), 139.1 (C_i), 152.4 (C-3'), 169.4 (C=O), 195.5 (C=S) ppm. MS (EI, 70 eV) m/z (%): 301 (M⁺, 50), 214 (100), 213 (55), 129 (22), 109 (18), 107 (13), 104 (10), 102 (16), 96 (12), 77 (71), 70 (19), 69 (12). Anal. Calcd. for C₁₄H₁₁N₃OS₂ (301.03): C, 55.79%; H, 3.68%; N, 13.94%; found: C, 56.02%; H, 3.71%; N, 13.56%.

(Z)-5-((4-Methyl-1H-imidazol-5-yl)methylidene)-2-thioxothiazolidin-4-one (3b). Orange crystalline solid (91%), m.p. 307–309 °C; FT-IR (KBr), υ: (NH) 3558, (NH) 3225, (C=O) 1690 and (C=S) 1217 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.39 (s, 3H, CH₃), 7.49 (s, 1H, H-6), 7.83 (s, 1H, H-2'), 12.61 (s, 1H, NH-1'), 13.31 (s, 1H, NH-3) ppm; ¹³C-NMR (DMSO-d₆), δ: 9.7 (-CH₃), 120.2 (C-5), 123.5 (C-6), 132.7 (C-5' or C-4'), 135.4 (C-5' or C-4'), 137.6 (C-2'), 169.7 (C=O), 200.2 (C=S) ppm. MS (IE, 70 eV) m/z (%): 225 (M⁺, 38), 139 (10), 138 (70), 137 (100), 69 (25), 42 (13). Anal. Calcd. for C₈H₇N₃OS₂ (225.00): C, 46.25%; H, 3.13%; N, 18.65%; found: C, 46.09%; H, 3.10%; N, 18.98%.

(Z)-5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylidene)-2-thioxothiazolidin-4-one (3c). Yellow solid (86%), m.p. 315–317 °C; FT-IR (KBr), υ: (NH) 3134, (C=O) 1695 and (C=S) 1223 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 7.42 (t, J = 7.40 Hz, 1H, Ar-H_p), 7.46 (s, 1H, H-6), 7.51–7.59 (m, 5H, Ar-H_{m, m',p'}), 7.66 (d, J = 8.08 Hz, 2H, Ar-H_o'), 7.99 (d, J = 8.24 Hz, 2H, Ar-H_o), 8.63 (s, 1H, H-5') ppm; ¹³C-NMR (DMSO-d₆), δ: 116.3 (C-4'), 120.0 (C_o), 122.3 (C-6), 125.3 (C-5), 128.0 (C_p) 129.0 (C-5'), 129.1 (C_o'), 129.3 (C_m'), 129.4 (C_p'), 130.0 (C_m), 139.1 (C_i'), 139.4 (C_i), 154.3 (C-3'), 169.3 (C=O), 195.4 (C=S) ppm. MS (IE, 70 eV) m/z (%): 363 (M⁺, 76), 277 (21), 276 (100), 275 (39), 243 (10), 242 (10), 215 (17), 172 (13), 77 (65), 28 (10). Anal. Calcd. for C₁₉H₁₃N₃OS₂ (363.05): C, 62.79%; H, 3.61%; N, 11.56%; found: C, 62.57%; H, 3.89%; N, 11.32%.

(Z)-5-((5-Methylthiophen-2-yl)methylidene)-2-thioxo-thiazolidin-4-one (3d). Orange solid (85%), m.p. 230–231 °C; FT-IR (KBr), υ: (NH) 3143, (C=O) 1689 and (C=S) 1199 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.56 (s, 3H, CH₃), 7.02 (d, J = 3.71 Hz, 1H, H-4') 7.54 (d, J = 3.71 Hz, 1H, H-3'), 7.82 (s, 1H, H-6), 13.71 (s, 1H, NH) ppm; ¹³C-NMR (DMSO-d₆), δ: 16.1 (-CH₃), 121.9 (C-5), 125.6 (C-6), 128.6 (C-3'), 135.9 (C-5'), 136.6 (C-4'), 149.9 (C-2'), 169.5 (C=O), 195.1 (C=S) ppm. MS (IE, 70 eV) m/z (%): 241 (M⁺, 34), 155 (14), 154 (100), 153 (52), 121 (21), 97 (16), 77 (18), 69 (12), 59 (13). Anal. Calcd. for C₉H₇NOS₃ (240.97): C, 44.79%; H, 2.92%; N, 5.80%; found: C, 45.08%; H, 3.11%; N, 5.85%.

(Z)-5-Ethylidene-2-thioxothiazolidin-4-one (3e). Two drops of piperidine (0.01 equiv.) were added to an ethanolic solution of rhodanine (2a, 1 mmol) and paraldehyde (1.1 mmol). The mixture was refluxed for 7 h, the solution was cooled and crushed ice was added and the solid formed was isolated by vacuum filtration and washed with hexane and water. Purification was carried out by column chromatography silica gel using a mixture of CHCl₃–EtOAc (30:1) as eluent. The solvent was removed under reduced pressure. This compound was obtained as a yellow solid (64%), m.p. 145–147 °C; FT-IR (KBr), υ: (NH) 3161, (C=O) 1703 and (C=S) 1219 cm⁻¹; ¹H-NMR (CDCl₃), δ: 1.98 (d, J = 7.32 Hz, 3H, CH₃), 6.99 (q, J = 7.32 Hz, 1H, H-6) 9.75 (s, 1H, NH) ppm; ¹³C-NMR (CDCl₃), δ: 17.4 (–CH₃), 130.4 (C-5), 134.1 (C-6), 167.2 (C=O), 193.6 (C=S) ppm. MS (IE, 70 eV) m/z (%): 159 (M⁺, 100), 100 (25), 72 (66), 71 (36), 45 (7). Anal. Calcd. for C₅H₅NOS₂ (158.98): C, 37.72%; H, 3.17%; N, 8.80%; found: C, 37.65%; H, 3.22%; N, 8.43%.

3.2.2. General Procedure for the Synthesis of 2-(5Z)-(Hetarylmethylidene)-4-oxo-2-thioxothiazolidin-3-yl) Acetic Acids 4a–d

A mixture of heterocyclic aldehyde (1a–d, 0.4 mmol) and rhodanine-3-acetic acid (2b, 0.4 mmol) in DMF, was subjected to irradiation with microwaves for 5 min at 100 °C and 100 W. Then, a mixture of ethanol:water (1:1) was added and the solid formed was isolated by vacuum filtration and washed with ethanol.

(Z)-5-((3-Methyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-4-oxo-2-thioxothiazolidin-3-yl acetic acid (4a). Yellow solid (81%), m.p. 279–281 °C; FT-IR (KBr), υ: (–COOH) 3315, (C=O) 1715 and (C=S) 1323 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.43 (s, 3H, CH₃), 4.73 (s, 2H, –N–CH₂–COOH), 7.38 (t, J = 7.45 Hz, 1H, Ar-H_p), 7.52 (dd, J = 7.45 and 7.95 Hz, 2H, Ar-H_m), 7.61 (s, 1H, H-6), 7.95 (d, J = 7.95 Hz, 2H, Ar-H_o), 8.61 (s, 1H, H-5'), 13.45 (br. s., 1H, –COOH) ppm; ¹³C-NMR (DMSO-d₆), δ: 11.51 (–CH₃), 45.0 (–N–CH₂–COOH), 116.5 (C-4'), 119.0 (C_o), 124.1 (C_p), 127.1 (C-5), 128.1 (C-6), 129.4 (C-5'), 132.1 (C_m), 138.6 (C_i), 152.2 (C-3'),165.9 (–COOH), 167.2 (C=O), 192.6 (C=S) ppm. MS (IE, 70 eV) m/z (%): 359 (M⁺, 26), 214 (91), 129 (34), 117 (80), 72 (100). Anal. Calcd. for C₁₆H₁₃N₃O₃S₂ (359.04): C, 53.47%; H, 3.65%; N, 11.69%; found: C, 53.77%; H, 3.89%; N, 11.77%.

(Z)-5-((4-Methyl-1H-imidazol-5-yl)methylidene)-4-oxo-2-thioxothiazolidin-3-yl acetic acid (4b). Yellow solid (53%), m.p. 254–256 °C; FT-IR (KBr), υ: (COOH) 3368, (C=O) 1718 and (C=S) 1319 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.42 (s, 3H, CH₃), 4.68 (s, 2H, –N–CH₂–COOH), 7.69 (s, 1H, H-6), 7.86 (s, 1H, H-2'), 12.70 (s, 1H, NH), 13.71 (br. s., 1H, –COOH) ppm; ¹³C-NMR (DMSO-d₆), δ: 9.3 (–CH₃), 44.8 (–N–CH₂–COOH), 116.1 (C-5), 124.7 (C-6), 132.4 (C-5'), 135.8 (C-4'), 137.3 (C-2'), 166.3 (–COOH), 167.6 (C=O), 197.4 (C=S) ppm. MS (IE, 70 eV) m/z (%): 283 (M⁺, 51), 166 (9), 139 (13), 138 (100), 72 (13). Anal. Calcd. for C₁₀H₉N₃O₃S₂ (283.01): C, 42.39%; H, 3.20%; N, 14.83%; found: C, 42.55%; H, 4.10%; N, 14.44%.

(Z)-5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylidene)-4-oxo-2-thioxothiazolidin-3-yl acetic acid (4c). Yellow solid (92%), m.p. 263–265 °C; FT-IR (KBr), υ: (COOH) 3320, (C=O) 1715 and (C=S) 1320 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 4.67 (s, 2H, –N–CH₂–COOH), 7.42 (t, J = 8.00 Hz, 1H, Ar-H_p), 7.54–7.58 (m, 5H, Ar-H_{m, m',p'}), 7.64 (d, J = 7.90 Hz, 2H, Ar-H_o'), 7.66 (s, 1H, H-6), 8.04 (d, J = 8.03 Hz, 2H, Ar-H_o), 8.80 (s, 1H, H-5') 13.23 (br. s., 1H, –COOH) ppm; ¹³C-NMR (DMSO-d₆), δ: 44.79 (–N–CH₂–COOH), 115.4 (C-4'), 119.4 (C_o), 120.7 (C-6), 123.8 (C-5), 127.6 (C_o’), 128.7 (C-5'), 128.9 (C_p), 129.1 (C_m'), 129.5.0 (C_p'), 131.0 (C_m), 138.6 (C_i'), 139.6 (C_i), 154.0 (C-3'), 166.0 (–COOH), 167.2 (C=O), 192.6 (C=S) ppm. MS (IE, 70 eV) m/z (%): 421 (M⁺, 30), 276 (100), 72 (54). Anal. Calcd. for C₂₁H₁₅N₃O₃S₂ (421.06): C, 59.84%; H, 3.59%; N, 9.97%; found: C, 60.14%; H, 3.67%; N, 10.12%.

(5Z)-((5-Methylthiophen-2-yl)methylidene)-4-oxo-2-thioxothiazolidin-3-yl acetic acid (4d). Orange solid (63%), m.p. 232–234 °C; FT-IR (KBr), υ: (COOH) 3320, (C=O) 1712 and (C=S) 1321 cm⁻¹; ¹H-NMR (DMSO-d₆), δ: 2.53 (s, 3H, CH₃), 4.63 (s, 2H, –N–CH₂–COOH), 7.05 (d, J = 3.70 Hz, 1H, H-4') 7.61 (d, J = 3.70 Hz, 1H, H-3'), 8.04 (s, 1H, H-6), 13.42 (br. s., 1H, –COOH) ppm; ¹³C-NMR (DMSO-d₆), δ: 16.0 (–CH₃), 45.82 (–N–CH₂–COOH), 122.1 (C-5), 126.8 (C-6), 128.3 (C-3'), 136.9 (C-5'), 138.2 (C-4'), 147.1 (C-2'), 165.5 (–COOH), 167.9 (C=O), 191.8 (C=S) ppm. MS (IE, 70 eV) m/z (%): 299 (M⁺, 31), 154 (100), 121 (15), 7 (8), 45 (10). Anal. Calcd. for C₁₁H₉NO₃S₃ (421.06): C, 44.13%; H, 3.03%; N, 4.68%; found: C, 44.19%; H, 3.15%; N, 4.72%.

3.2.3. General Procedure for the Synthesis of 5-Hetarylmethylidene-2-(piperidin-1-yl)thiazol-4-ones, 5-hetarylmethylidene-2-morpholinothiazol-4-ones and (Z)-5-Ethylidene-2-morpholinothiazol-4(5H)-ones 5a–d, 5e and 6a–d

A mixture of piperidine or morpholine (2 mmol) and hetarylidene rhodanine derivatives 3a–b (1 mmol) or ethylene derivative 3e was refluxed in THF for 7–24 h. Crushed ice was added and the solid formed was isolated by vacuum filtration and washed with water and hexane.

(Z)-5-((3-Methyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-2-(piperidin-1-yl)thiazol-4(5H)-one (5a). White solid (85%), m.p. 141–143 °C; FT-IR (KBr), υ: (C=O) 1680 and (C=N, C=C) 1615, 1575, 1547 cm⁻¹; ¹H-NMR (CDCl₃), δ: 1.79 (br. s, 6H, –CH₂–CH₂–CH₂–), 2.47 (s, 3H, CH₃), 3.60 (br. s, 2H, N-CH₂), 4.03 (br. s, 2H, N-CH₂), 7.33 (t, J = 7.48 Hz, 1H, Ar-H_p), 7.49 (dd, J = 7.48 and 8.51 Hz, 2H, Ar-H_m), 7.66 (s, 1H, H-6), 7.69 (d, J = 8.51 Hz, 2H, Ar-H_o), 8.01 (s, 1H, H-5') ppm; ¹³C-NMR (CDCl₃), δ: 12.0 (–CH₃), 24.1 (–CH₂–CH₂–CH₂–), 49.6 (N-CH₂), 50.3 (N-CH₂), 118.0 (C-4'), 119.2 (C_o), 120.8 (C-6), 126.1 (C-5), 126.5 (C-5'), 126.9 (C_p), 129.5 (C_m), 139.5 (C_i), 152.0 (C-3'), 173.0 (C-2) 180.8 (C=O) ppm. MS (IE, 70 eV) m/z (%): 352 (M⁺, 20), 242 (7), 215 (15), 214 (100), 213 (29), 129 (6), 77 (8). Anal. Calcd. for C₁₉H₂₀N₄OS (352.14): C, 64.75%; H, 5.72%; N, 15.90%; found: C, 64.42%; H, 5.88%; N, 15.63%.

(Z)-5-((4-Methyl-1H-imidazol-5-yl)methylidene)-2-(piperidin-1-yl)thiazol-4(5H)-one (5b). Yellow solid (70%), m.p. 261–262 °C; FT-IR (KBr), υ: (NH) 3294, (C=O) 1663 and (C=N, C=C) 1609, 1557, 1539 cm⁻¹; ¹H-NMR (CDCl₃), δ: 1.74 (br. s, 6H, –CH₂–CH₂–CH₂–), 2.42 (s, 3H, CH₃), 3.62 (br. s, 2H, N-CH₂), 3.98 (br. s, 2H, N-CH₂), 7.66 (s, 1H, H-6), 7.72 (s, 1H, H-2'), 10.81 (s, 1H, NH) ppm; ¹³C-NMR (CDCl₃), δ: 9.7 (–CH₃), 26.1 (–CH₂–CH₂–CH₂–), 49.2 (N-CH₂), 50.1 (N-CH₂), 121.5 (C-6), 124.3 (C-5), 131.4 (C-5' o C-4'), 133.0 (C-5' o C-4'), 135.5 (C-2'), 177.1 (C-2), 182.3 (C=O) ppm. MS (IE, 70 eV) m/z (%): 276 (M⁺, 48), 166 (10), 139 (12), 138 (100), 137 (25). Anal. Calcd. for C₁₃H₁₆N₄OS (276.10): C, 56.50%; H, 5.84%; N, 20.27%; found: C, 56.32%; H, 5.51%; N, 20.32%.

(Z)-5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylidene)-2-(piperidin-1-yl)thiazol-4(5H)-one (5c). Yellow solid (95%), m.p. 262–264 °C; FT-IR (KBr), υ: (C=O) 1695 and (C=N, C=C) 1606, 1572, 1535 cm⁻¹; ¹H-NMR (CDCl₃), δ: 1.84 (br. s, 6H, –CH₂–CH₂–CH₂–), 3.61 (br. s, 2H, N-CH₂), 4.03 (br. s, 2H, N-CH₂), δ 7.38 (t, J = 7.38 Hz, 1H, Ar-H_p), 7.43–7.55 (m, 5H, Ar-H_{m, m',p'}), 7.70 (d, J = 8.20 Hz, 2H, Ar-H_o'), 7.82 (s, 1H, H-6), 7.83 (d, J = 8.64 Hz, 2H, Ar-H_o), 8.20 (s, 1H, H-5') ppm; ¹³C-NMR (CDCl₃), δ: 24.0 (–CH₂–CH₂–CH₂–), 25.4 (–CH₂–CH₂–CH₂–), 26.2 (–CH₂–CH₂–CH₂–), 49.6 (N-CH₂), 50.3 (N-CH₂), 117.5 (C-4'), 119.5 (C_o), 121.6 (C-6), 126.8 (C-5'), 127.3 (C_p) 127.8 (C-5), 128.7 (C_p'), 128.8 (C_m'), 128.9 (C_o'), 129.6 (C_m), 131.8 (C_i'), 139.5 (C_i), 154.5 (C-3'), 173.0 (C-2), 180.5 (C=O) ppm. MS (IE, 70 eV) m/z (%): 414 (M⁺, 6), 276 (17), 109 (15), 38 (37), 36 (100), 18 (10), 17 (22), 16 (15). Anal. Calcd. for C₂₄H₂₂N₄OS (414.15): C, 69.54%; H, 5.35%; N, 13.52%; found: C, 69.79%; H, 5.39%; N, 14.02%.

(Z)-5-((5-Methylthiophen-2-yl)methylidene)-2-(piperidin-1-yl)thiazol-4(5H)-one (5d). Orange solid (93%), m.p. 194–196 °C; FT-IR (KBr), υ: (C=O) 1662 and (C=N, C=C) 1600, 1574 cm⁻¹; ¹H-NMR (CDCl₃), δ: 1.72 (br. s, 6H, –CH₂–CH₂–CH₂–), 2.56 (s, 3H, CH₃), 3.59 (br. s, 2H, N-CH₂), 4.01 (br. s, 2H, N-CH₂), 6.82 (d, J = 3.40 Hz, 1H, H-4') 7.15 (d, J = 3.40 Hz, 1H, H-3'), 7.87 (s, 1H, H-6) ppm; ¹³C-NMR (CDCl₃), δ: 15.8 (–CH₃), 24.1 (–CH₂–CH₂–CH₂–), 25.4 (–CH₂–CH₂–CH₂–), 26.2 (–CH₂–CH₂–CH₂–), 49.6 (N-CH₂), 50.3 (N-CH₂), 124.3 (C-6), 125.5 (C-5), 127.0 (C-4'), 132.5 (C-3'), 137.3 (C-2'), 145.6 (C-5'), 173.8 (C-2), 180.9 (C=O) ppm. MS (IE, 70 eV) m/z (%): 292 (M⁺, 24), 155 (12), 154 (100), 153 (23). Anal. Calcd. for C₁₄H₁₆N₂OS₂ (292.07): C, 57.50%; H, 5.52%; N, 9.58%; found: C, 57.62%; H, 5.68%; N, 9.71%.

(Z)-5-Ethylidene-2-morpholinothiazol-4(5H)-one (5e). Brown solid (45%), m.p. 193–195 °C; FT-IR (KBr), υ: (C=O) 1699 and (C=N, C=C) 1639, 1554 cm⁻¹; ¹H-NMR (CDCl₃), δ: 2.00 (d, J = 7.12 Hz, 3H, CH₃), 3.56 (br. s, 2H, N-CH₂), 3.81 (br. s, 4H, –CH₂–O–CH₂–), 4.05 (br. s, 2H, N-CH₂), 6.99 (q, J = 7.12 Hz, 1H, H-6) ppm; ¹³C-NMR (CDCl₃), δ: 18.3 (–CH₃), 48.3 (N-CH₂), 48.7 (N-CH₂), 66.2 (–OCH₂–), 66.3 (–OCH₂–), 130.8 (C-6), 133.1 (C-5), 173.0 (C-2) 175.0 (C=O). ppm. MS (IE, 70 eV) m/z (%): 212 (M⁺, 100), 184 (9), 113 (27), 72 (86), 71 (23), 69 (12), 42 (11). Anal. Calcd. for C₉H₁₂N₂O₂S (212.06): C, 50.92%; H, 5.70%; N, 13.20%; found: C, 50.15%; H, 5.87%; N, 13.02%.

(Z)-5-((3-Methyl-1-phenyl-1H-pyrazol-4-yl)methylidene)-2-morpholinothiazol-4(5H)-one (6a). White solid (71%), m.p. 264–265 °C; FT-IR (KBr), υ: (C=O) 1683 and (C=N, C=C) 1616, 1559, 1502 cm⁻¹; ¹H-NMR (CDCl₃), δ: 2.47 (s, 3H, CH₃), 3.66 (br. s, 2H, N-CH₂), 3.85 (br. s, 4H, –CH₂–O–CH₂–), 4.10 (br. s, 2H, N-CH₂), 7.32 (t, J = 7.46 Hz, 1H, Ar-H_p), 7.47 (dd, J = 7.46 and 8.07 Hz, 2H, Ar-H_m), 7.68 (s, 1H, H-6), 7.69 (d, J = 8.07 Hz, 2H, Ar-H_o), 8.01 (s, 1H, H-5') ppm; ¹³C-NMR (CDCl₃), δ: 11.9 (–CH₃), 48.6 (N-CH₂), 48.8 (N-CH₂), 66.2 (–OCH₂–), 66.3 (–OCH₂–), 117.8 (C-4'), 119.2 (C_o), 121.8 (C-6), 125.6 (C-5), 126.1 (C-5'), 127.1 (C_p), 129.5 (C_m), 139.4 (C_i), 152.1 (C-3'), 174.0 (C-2) 180.3 (C=O) ppm. MS (IE, 70 eV) m/z (%): 354 (M⁺, 34), 215 (16), 214 (100), 213 (37), 129 (12), 109 (13), 77 (29), 18 (16). Anal. Calcd. for C₁₈H₁₈N₄O₂S (354.12): C, 61.00%; H, 5.12%; N, 15.81%; found: C, 60.73%; H, 5.32%; N, 15.42%.

(Z)-5-((4-Methyl-1H-imidazol-5-yl)methylidene)-2-morpholinothiazol-4(5H)-one (6b). Yellow solid (62%), m.p. 270–272 °C; FT-IR (KBr), υ: (NH) 3389, (C=O) 1664 and (C=N, C=C) 1605, 1540 cm⁻¹; ¹H-NMR (CDCl₃), δ: 2.44 (s, 3H, CH₃), 3.68 (br. s, 2H, N-CH₂), 3.81 (br. s, 4H, –CH₂–O–CH₂–), 4.06 (br. s, 2H, N-CH₂), 7.70 (s, 1H, H-2'), 7.71 (s, 1H, H-6), 10.23 (s, 1H, NH) ppm; ¹³C-NMR (CDCl₃), δ: 9.6 (–CH₃), 48.2 (N-CH₂), 48.5 (N-CH₂), 66.3 (–OCH₂–), 121.9 (C-6), 124.5 (C-5), 130.9 (C-5' o C-4'), 133.1 (C-5' o C-4'), 135.1 (C-2'), 178.3 (C-2), 181.6 (C=O) ppm. MS (IE, 70 eV) m/z (%): 278 (M⁺, 18), 138 (26), 85 (28), 73 (33), 69 (37), 60 (52), 57 (42), 55 (36), 44 (100), 43 (99). Anal. Calcd. for C₁₂H₁₄N₄O₂S (278.08): C, 51.78%; H, 5.07%; N, 20.13%; found: C, 52.03%; H, 5.15%; N, 20.28%.

(Z)-5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylidene)-2-morpholinothiazol-4(5H)-one (6c). White solid (86%), m.p. 266–268 °C; FT-IR (KBr), υ: (C=O) 1681 and (C=N, C=C) 1602, 1567, 1502 cm⁻¹; ¹H-NMR (CDCl₃), δ: 3.66 (br. s, 2H, N-CH₂), 3.86 (br. s, 4H, –CH₂–O–CH₂–), 4.10 (br. s, 2H, N-CH₂), 7.39 (t, J = 7.44 Hz, 1H, Ar-H_p), 7.43–7.55 (m, 5H, Ar-H_{m, m',p'}), 7.70 (d, J = 8.17 Hz, 2H, Ar-H_o'), 7.82 (d, J = 8.62 Hz, 2H, Ar-H_o), 7.85 (s, 1H, H-6), 8.19 (s, 1H, H-5') ppm; ¹³C-NMR (CDCl₃), δ: 48.6 (N-CH₂), 48.8 (N-CH₂), 66.2 (–OCH₂–), 66.3 (–OCH₂), 117.3 (C-4'), 119.5 (C_o), 122.6 (C-6), 126.8 (C-5'), 126.9 (C-5), 127.4 (C_p), 128.8 (C_p'), 128.9 (C_m'), 129.0 (C_o'), 129.6 (C_m), 131.7 (C_i'), 139.4 (C_i), 154.6 (C-3'), 174.0 (C-2), 180.1 (C=O) ppm. MS (IE, 70 eV) m/z (%): 416 (M⁺, 44), 277 (22), 276 (100), 275 (27), 215 (10), 77 (3). Anal. Calcd. for C₂₃H₂₀N₄O₂S (416.13): C, 66.33%; H, 4.84%; N, 13.45%; found: C, 66.12%; H, 5.03%; N, 13.62%.

(Z)-5-((5-Methylthiophen-2-yl)methylidene)-2-morpholinothiazol-4(5H)-one (6d). Orange solid (85%), m.p. 206–208 °C; FT-IR (KBr), υ: (C=O) 1673 and (C=N, C=C) 1599, 1574 cm⁻¹; ¹H-NMR (CDCl₃), δ: 2.57 (s, 3H, CH₃), 3.65 (br. s, 2H, N-CH₂), 3.84 (br. s, 4H, –CH₂–O–CH₂–), 4.08 (br. s, 2H, N-CH₂), 6.83 (d, J = 3.60 Hz, 1H, H-4') 7.18 (d, J = 3.60 Hz, 1H, H-3'), 7.91 (s, 1H, H-6) ppm; ¹³C-NMR (CDCl₃), δ: 15.9 (–CH₃), 48.6 (N-CH₂), 48.8 (N-CH₂), 66.2 (–OCH₂–), 66.4 (–OCH₂–), 124.6 (C-5), 125.2 (C-6), 127.1 (C-4'), 133.0 (C-3'), 137.0 (C-2'), 146.1 (C-5'), 174.8 (C-2), 180.5 (C=O) ppm. MS (IE, 70 eV) m/z (%): 294 (M⁺, 22), 156 (9), 155 (12), 154 (100), 153 (27), 121 (10), 97 (8). Anal. Calcd. for C₁₃H₁₄N₂O₂S₂ (294.05): C, 53.04%; H, 4.79%; N, 9.52%; found: C, 53.21%; H, 4.93%; N, 9.46%.

3.3. Antifungal Activity

Microorganisms and media: For the antifungal evaluation, reference strains from the American Type Culture Collection (ATCC, Rockville, MD, USA), and Culture Collection of Centro de Referencia en Micología-CEREMIC (CCC, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-(2000)-Rosario, Argentina), were used: C. albicans ATCC 10231, C. tropicalis CCC 191, S. cerevisiae ATCC 9763, C. neoformans ATCC 32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029, M. gypseum CCC 115, T. rubrum CCC 110, T. mentagrophytes ATCC 9972. Strains were grown on Sabouraud-chloramphenicol agar slants at 30 °C, maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid, Hampshire, UK), and subcultured every 15 days to prevent pleomorphic transformations. Inocula were obtained according to reported procedures and adjusted to 1–5 ×10³ colony forming units (CFU)/mL [13,14].

3.4. Antifungal Susceptibility Testing

Minimum Inhibitory Concentration (MIC) of each compound was determined by using broth microdilution techniques following the guidelines of the CLSI for yeasts [13] and for filamentous fungi [14]. MIC values were determined in RPMI-1640 (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with MOPS (Sigma). Microliter trays were incubated at 35 °C for yeasts and hyalohyphomycetes and at 28 °C for dermatophyte strains in a moist, dark chamber; MICs were recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the rest of fungi. The susceptibilities of the standard drugs ketoconazole, terbinafine, and amphotericin B (obtained from Sigma-Aldrich, St. Louis, MO, USA) were defined as the lowest concentration of drug which resulted in total inhibition of fungal growth. For the assay, compound stock solutions were two-fold diluted with RPMI-1640 from 250 to 0.24 μg/mL (final volume = 100 μL) and a final DMSO (Sigma) concentration <1%. A volume of 100 μL of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. MIC was defined as the minimum inhibitory concentration of the compound, which resulted in total inhibition of the fungal growth. Minimum Fungicide Concentration (MFC), the concentration of compound that kills fungi rather than inhibits the fungal growth, was determined by plating by duplicate 5 µL from each clear well of MIC determinations, onto a 150 mm SDA plate. After 48 h at 37 °C, MFCs were determined as the lowest concentration of each compound showing no growth3.5.

3.5. Antitumor Activity

All synthesized compounds were sent to the National Cancer Institute (NCI, Bethesda, MD, USA) to evaluate the cytotoxic activity. The process was performed in two stages. The first, consisted in evaluate the compounds at a single concentration of 1.0 μM. The second stage consisted of evaluating the compounds against 60 different cell lines (melanoma, leukemia, lung cancer, colon, brain, breast, ovary, kidney and prostate). The test consisted in a protocol of 48 h of continuous drug exposure using sulforhodamide B (SRB) protein assay to estimate cell growth [15].

4. 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 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 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 hetaryl-methylidenerhodanine antitumor derivatives. Due to these significant results, we have carried out chemical studies seeking structures that enhance the antifungal and antitumor activities.

Acknowledgments

The authors wish to credit The Developmental Therapeutics Program (DTP) of the National Cancer Institute of the United States (US) and Faculty of Biochemical and Pharmaceutical Sciences, Universidad Nacional de Rosario for performing the screening of compounds. This work was supported by Colciencias, Universidad del Valle, Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain), the Universidad de Jaen and ANPCyT (Argentina) to SZ (PICT 0608). The authors thank “Centro de Instrumentación Científico-Técnica de la Universidad de Jaén” and its staff for data collection.

Conflicts of Interest

The authors declare no conflict of interest.

References

Ramirez, M.A.; Borja, N.L. Epalrestat: An aldose reductase inhibitor for the treatment of diabetic neuropathy. Pharmacotherapy 2008, 28, 646–655. [Google Scholar] [CrossRef]
Ravi, S.; Chiruvella, K.K.; Rajesh, K.; Prabhu, V.; Raghavan, S.C. 5-Isopropylidene-3-ethyl rhodanine induce growth inhibition followed by apoptosis in leukemia cells. Eur. J. Med. Chem. 2010, 45, 2748–2752. [Google Scholar] [CrossRef]
Heerding, D.A.; Christmann, L.T.; Clark, T.J.; Holmes, D.J.; Rittenhouse, S.F.; Takata, D.T.; Venslavsky, J.W. New Benzylidenethiazolidinediones as Antibacterial Agents. Bioorg. Med. Chem. Lett. 2003, 13, 3771–3773. [Google Scholar] [CrossRef]
Dolezel, J.; Hirsova, P.; Opletalova, V.; Dohnal, J.; Marcela, V.; Kunes, J.; Jampilek, J. Rhodanineacetic acid derivatives as potential drugs: Preparation, hydrophobic properties and antifungal activity of (5-arylalkylidene-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)acetic acids. Molecules 2009, 14, 4197–4212. [Google Scholar] [CrossRef]
Orchard, M.G.; Neuss, J.C.; Galley, C.M.S.; Carr, A.; Porter, D.W.; Smith, P.; Scopes, D.I.C.; Haydon, D.; Vousden, K.; Stubberfield, C.R.; et al. Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1). Bioorg. Med. Chem. Lett. 2004, 14, 3975–3978. [Google Scholar] [CrossRef]
Sortino, M.; Delgado, P.; Juárez, S.; Quiroga, J.; Abonía, R.; Insuasty, B.; Nogueras, M.; Rodero, L.; Garibotto, F.M.; Enriz, R.D.; et al. Synthesis and antifungal activity of (Z)-5-arylidenerhodanines. Bioorg. Med. Chem. 2007, 15, 484–494. [Google Scholar]
Insuasty, B.; Gutiérrez, A.; Quiroga, J.; Abonia, R.; Nogueras, M.; Cobo, J.; Svetaz, L.; Raimondi, M.; Zacchino, S. Fungicide activity of 5-(4-chlorobenzylidene)-(Z)-2-dimethylamino-1,3-thiazol-4-one against cryptococcus neoformans. Arch. Pharm. 2010, 343, 48–53. [Google Scholar]
Insuasty, B.; Tigreros, A.; Martínez, H.; Quiroga, J.; Abonia, R.; Gutierrez, A.; Nogueras, M.; Cobo, J. An efficient two-step synthesis of novel thiazolo[2,3-b]pyrazolo[3,4-f][1,3,5]triazepines. J. Heterocycl. Chem. 2009, 46, 756–761. [Google Scholar] [CrossRef]
Xu, L.-L.; Zheng, C.-J.; Sun, L.-P.; Miao, J.; Piao, H.-R. Synthesis of novel 1,3-diaryl pyrazole derivatives bearing rhodanine-3-fatty acid moieties as potential antibacterial agents. Eur. J. Med. Chem. 2012, 48, 174–178. [Google Scholar] [CrossRef]
Chen, Z.-H.; Zheng, C.-J.; Sun, L.-P.; Piao, H.-R. Synthesis of new chalcone derivatives containing a rhodanine-3-acetic acid moiety with potential anti-bacterial activity. Eur. J. Med. Chem. 2010, 45, 5739–5743. [Google Scholar] [CrossRef]
Delgado, P.; Quiroga, J.; Cobo, J.; Low, J.N.; Glidewell, C. Supramolecular structures of four (Z)-5-arylmethylene-2-thioxothiazolidin-4-ones: Hydrogen-bonded dimers, chains of rings and sheets. Acta Crystallogr. C 2005, 61, o477–o482. [Google Scholar] [CrossRef]
Delgado, P.; Quiroga, J.; de la Torre, J.M.; Cobo, J.; Low, J.N.; Glidewell, C. Three substituted (Z)-5-benzylidene-2-thioxothiazolidin-4-ones: Hydrogen-bonded dimers that can be effectively isolated or linked into chains either by aromatic [pi]-[pi] stacking interactions or by dipolar carbonyl-carbonyl interactions. Acta Crystallogr. C 2006, 62, o382–o385. [Google Scholar] [CrossRef]
Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, approved standard-3rd ed.; CLSI Document M27-A3; CLSI: Wayne, PA, USA, 2008; Volume 28, Nº 14, pp. 1–25. [Google Scholar]
Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi, approved standard-2nd ed.; CLSI Document M38-A2; CLSI: Wayne, PA, USA, 2008; Volume 28, Nº 16, pp. 1–3. [Google Scholar]
Hubbard, W.C.; Alley, M.C.; Gray, G.N.; Green, K.C.; McLemore, T.L.; Boyd, M.R. Evidence for prostanoid biosynthesis as a biochemical feature of certain subclasses of non-small cell carcinomas of the lung as determined in established cell lines derived from human lung tumors. Cancer Res. 1989, 49, 826–832. [Google Scholar]

Sample Availability: Samples of the compounds are available from the authors.

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Share and Cite

MDPI and ACS Style

Insuasty, A.; Ramírez, J.; Raimondi, M.; Echeverry, C.; Quiroga, J.; Abonia, R.; Nogueras, M.; Cobo, J.; Rodríguez, M.V.; Zacchino, S.A.; et al. Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones. Molecules 2013, 18, 5482-5497. https://doi.org/10.3390/molecules18055482

AMA Style

Insuasty A, Ramírez J, Raimondi M, Echeverry C, Quiroga J, Abonia R, Nogueras M, Cobo J, Rodríguez MV, Zacchino SA, et al. Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones. Molecules. 2013; 18(5):5482-5497. https://doi.org/10.3390/molecules18055482

Chicago/Turabian Style

Insuasty, Alberto, Juan Ramírez, Marcela Raimondi, Carlos Echeverry, Jairo Quiroga, Rodrigo Abonia, Manuel Nogueras, Justo Cobo, María Victoria Rodríguez, Susana A. Zacchino, and et al. 2013. "Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones" Molecules 18, no. 5: 5482-5497. https://doi.org/10.3390/molecules18055482

APA Style

Insuasty, A., Ramírez, J., Raimondi, M., Echeverry, C., Quiroga, J., Abonia, R., Nogueras, M., Cobo, J., Rodríguez, M. V., Zacchino, S. A., & Insuasty, B. (2013). Synthesis, Antifungal and Antitumor Activity of Novel (Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-Ethylidene-1,3-thiazol-4-ones. Molecules, 18(5), 5482-5497. https://doi.org/10.3390/molecules18055482

Article Menu