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; doi:10.3390/molecules18055482

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molecules

ISSN 1420-3049

www.mdpi.com/journal/molecules

Article

Synthesis, Antifungal and Antitumor Activity of Novel

(Z)-5-Hetarylmethylidene-1,3-thiazol-4-ones and

(Z)-5-Ethylidene-1,3-thiazol-4-ones

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 ²and 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;

E-Mail: braulio.insuasty@correounivalle.edu.co; Tel.: +57-315-484-6665; Fax: +57-2339-3248.

Received: 8 April 2013; in revised form: 2 May 2013 / Accepted: 8 May 2013 /

Published: 13 May 2013

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).

Molecules 2013, 18

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Keywords: antifungal activity; antitumor activity; hetarylmethylidenerhodanine derivatives;

piperidine; morpholine

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

1

the case of 3a, a yellow solid was obtained which after spectroscopic characterization (IR, H 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.

Molecules 2013, 18

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Compound 3a exhibited characteristic signals of its functional groups. The IR spectrum showed

absorption bands at 3,134, 1,684 and 1,213 cm⁻¹associated 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

13

methyl group of the pyrazole ring, respectively. The 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.

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)

294–295

307–309

315–317

230–231

145–147

279–281

254–256

263–265

232–234

Yield (%)

3a

3b

3c

3d

3e

4a

4b

4c

4d

86

91

86

85

64

81

53

92

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.

1

In the 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

13

ring, while the remaining signals corresponded to rest of compound 4a. In the 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 H₂S, as detected by its characteristic smell (Scheme 2).

Molecules 2013, 18

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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.

Compound

-X-

-CH₂-

-O-

m.p. (°C)

141–143

261–262

262–264

194–196

193–195

264–265

270–272

266–268

206–208

Yield (%)

5a

5b

5c

5d

5e

6a

6b

6c

6d

85

70

95

93

45

71

62

86

85

-O-

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

Molecules 2013, 18

5486

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.

Antifungal Activity

MIC/MFC (μg/mL)

Compound

Structure

Ca

Ct

Sc

Cn Afu Afl Ani

Mg

Tr

Tm

3a

3b

3c

3d

>250 >250 >250 >250 >250 >250 >250 >250 >250 >250

125/ 250/ 250/ 250/ 125/ 125/ 125/

>250 >250 >250

125

250

125

>250 >250 >250 >250 >250 >250 >250 <250 <250 <250

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Tm

Table 3. Cont.

Antifungal Activity

MIC/MFC (μg/mL)

Compound

Structure

Ca

7.8/

Ct

7.8/

Sc

3.9/ 15.6/ 31.2/ 31.2/ 62.5/ 7.8/

250 250 7.8

Cn

Afu

Afl

Ani

Mg

Tr

7.8/ 15.6/

15.6 15.6

3e

4a

31.2 31.2 15.6 62.5 250

125/ 62.5/ 62.5/

125 62.5 62.5

>250 >250 >250 >250 >250 >250 >250

4b

4c

4d

5a

5b

5c

>250 >250 >250 >250 >250 >250 >250 >250 >250 >250

5d

5e

>250 >250 >250 >250 >250 >250 >250 >250 >250 >250

6a

6b

6c

6d

>250 >250 >250 >250 >250 >250 >250 >250 >250 >250

amphotericin B

ketoconazole

terbinafine

-

0.78

1.56

0.50

0.50 0.25 0.50 0.50 0.50 0.12 0.07 0.07

3.12 0.39 0.78 0.78 1.56 0.04 0.01 0.02

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.

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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 LC₅₀found 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.

Compound 3c

Panel/Cell Line

GI₅₀^b(μM)

LC₅₀^c(μM)

Leukemia

CCRF-CEM

HL-60(TB)

K562

MOLT-4

RPMI-8226

SR

2.50

4.83

7.54

14.8

2.52

7.29

>100

Non Small Cell Lung

A549/ATCC

EKVX

5.88

3.03

22.7

0.62

2.03

2.68

7.63

5.50

2.96

>100

54.4

HOP-62

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

>100

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Table 4. Cont.

Compound 3c

Panel/Cell Line

GI₅₀^b(μM)

LC₅₀^c(μM)

Colon Cancer

COLO 205

HCC-2998

HCT-116

HCT-15

21.2

6.05

5.62

4.71

12.5

6.24

19.6

>100

70.2

96.2

>100

63.5

HT29

KM12

SW-620

>100

Prostate Cancer

PC-3

5.66

12.6

>100

DU-145

CNS Cancer

SF-268

SF-295

SF-539

SNB-19

SNB-75

17.2

3.33

5.53

6.14

17.8

5.54

>100

82.6

61.0

>100

64.8

U251

Melanoma

LOX IMVI

MALME-3M

M14 0.405

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

Renal Cancer

786-0

10.0

3.84

6.75

4.91

4.18

9.22

3.19

13.2

3.36

>100

6.11

>100

70.7

>100

58.4

>100

5.77

3.92

2.99

7.40

7.15

22.4

9.93

8.00

4.39

>100

94.4

52.1

>100

A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

Breast Cancer

MCF7

MDA-MB231/ATCC

HS 578T

BT-549

8.31

10.1

6.03

5.20

4.59

6.83

>100

86.5

>100

T-47D

MDA-MB-468

^aData obtained from NCI’s in vitro disease-oriented human tumor cell lines screen [15]; ^bGI₅₀was 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,

c

0.1 and 0.01 mM); LC₅₀is a parameter of cytotoxicity and reflects the molar concentration needed to kill

50% of the cells.

Molecules 2013, 18

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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

1

13

IRAffinity-1. The H and C-NMR spectra were run on a Bruker DPX 400 spectrometer operating at

400 and 100 MHz respectively, using DMSO-d₆and CDCl₃as 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

1

(86%), m.p. 294–295 °C; FT-IR (KBr), υ: (NH) 3134, (C=O) 1684 and (C=S) 1213 cm⁻¹; H-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,

13

1H, -NH-) ppm; C-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

1

(86%), m.p. 315–317 °C; FT-IR (KBr), υ: (NH) 3134, (C=O) 1695 and (C=S) 1223 cm⁻¹; H-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

13

(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; C-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'),

Molecules 2013, 18

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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)

1

1323 cm⁻¹; H-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%.

Molecules 2013, 18

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(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,

13

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; C-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

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(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

1

(93%), m.p. 194–196 °C; FT-IR (KBr), υ: (C=O) 1662 and (C=N, C=C) 1600, 1574 cm⁻¹; H-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

1

(KBr), υ: (C=O) 1699 and (C=N, C=C) 1639, 1554 cm⁻¹; H-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,

13

J = 7.12 Hz, 1H, H-6) ppm; C-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%.

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5494

(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

13

(s, 1H, H-6), 7.69 (d, J = 8.07 Hz, 2H, Ar-H_o), 8.01 (s, 1H, H-5') ppm; C-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),

13

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; C-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%.

Molecules 2013, 18

5495

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 ×10³colony 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].

Molecules 2013, 18

5496

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.

Conflict of Interest

The authors declare no conflict of interest.

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

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/3.0/).

Article Doi

Synthesis, antifungal and antitumor activity of novel (Z)-5- hetarylmethylidene-1,3-thiazol-4-ones and (Z)-5-ethylidene-1,3-thiazol-4-ones

DOI: 10.3390/molecules18055482

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