A convenient ultrasound-promoted synthesis of some new thiazole derivatives bearing a coumarin nucleus and their cytotoxic activity

Article abstract of DOI:10.3390/molecules17089335

Successful implementation of ultrasound irradiation for the rapid synthesis of a novel series of 3-[1-(4-substituted-5-(aryldiazenyl)thiazol-2-yl) hydrazono)ethyl]-2Hchromen- 2-ones 5a-h, via reactions of 2-(1-(2-oxo-2H- chromen-3-yl)ethylidene) thiosemicarbazide (2) and the hydrazonoyl halides 3(4), was demonstrated. Also, a new series of 5-arylidene-2-(2-(1-(2-oxo-2H-chromen- 3-yl)ethylidene)hydrazinyl)thiazol- 4(5H)-ones 10a-d were synthesized from reaction of 2 with chloroacetic acid and different aldehydes. Moreover, reaction of 2-cyano-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene)- acetohydrazide (12) with substituted benzaldehydes gave the respective arylidene derivatives 13a-c under the conditions employed. The structures of the synthesized compounds were assigned based on elemental analyses and spectral data. Also, the cytototoxic activities of the thiazole derivative 5a was evaluated against HaCaT cells (human keratinocytes). It was found that compound 5a possess potent cytotoxic activity.

Full text of DOI:10.3390/molecules17089335

Molecules 2012, 17, 9335-9347; doi:10.3390/molecules17089335
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A Convenient Ultrasound-Promoted Synthesis of Some New
Thiazole Derivatives Bearing a Coumarin Nucleus and Their
Cytotoxic Activity
Sobhi M. Gomha 1,* and Khaled D. Khalil 1,2
1
Department of Chemistry, Faculty of Science, University of Cairo, Giza 12613, Egypt
2
Chemistry Department, Faculty of Science, University of Kuwait, P.O. Box 5969, Safat 13060,
Kuwait; E-Mail: khd.khalil@yahoo.com
* Author to whom correspondence should be addressed; E-Mail: s.m.gomha@hotmail.com;
Tel.: +20-237-400-304; Fax: +20-225-685-799.
Received: 25 June 2012; in revised form: 18 July 2012 / Accepted: 24 July 2012 /
Published: 3 August 2012
Abstract: Successful implementation of ultrasound irradiation for the rapid synthesis of a
novel series of 3-[1-(4-substituted-5-(aryldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-
chromen-2-ones 5a–h, via reactions of 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
thiosemicarbazide (2) and the hydrazonoyl halides 3(4), was demonstrated. Also, a new
series of 5-arylidene-2-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)thiazol-
4(5H)-ones 10a–d were synthesized from reaction of 2 with chloroacetic acid and different
aldehydes. Moreover, reaction of 2-cyano-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-
acetohydrazide (12) with substituted benzaldehydes gave the respective arylidene
derivatives 13a–c under the conditions employed. The structures of the synthesized
compounds were assigned based on elemental analyses and spectral data. Also, the
cytototoxic activities of the thiazole derivative 5a was evaluated against HaCaT cells
(human keratinocytes). It was found that compound 5a possess potent cytotoxic activity.
Keywords: thiosemicarbazides; thiazoles; hydrazonoyl halides; ultra-sound irradiation;
cytotoxic activity

Molecules 2012, 17
9336
1. Introduction
The synthesis of coumarins and their derivatives has attracted considerable attention from organic
and medicinal chemists for many years as a large number of natural and synthetic products contain this
heterocyclic nucleus [1–5]. A number of natural and synthetic coumarin derivatives have been reported
to exert notable antimicrobial [1,2], antifungal [3,4] and cytotoxic [5] activity.
Thiazole derivatives have also attracted increasing attention due to their numerous pharmacological
applications and biological activities, such as anti-inflammatory, analgesic, antimicrobial, anti-HIV,
antihypertensive and herbicidal activity [6–10].
Ultrasonic-assisted organic synthesis (UAOS) is a powerful and green approach which is being used
more and more to accelerate synthesis of organic compounds [11]. Increases in reaction rate and yields
occur on application of ultrasound waves [12–16].
In view of these observations and in continuation of our previous work on the synthesis of
heterocyclic systems for biological evaluation [17–19], we report herein a facile route to various
thiazole derivatives incorporating coumarin moieties using the ultra-sound irradiation technique.
Additionally we have found that one of the synthesized compounds has shown high cytotoxic activity.
2. Results and Discussion
2.1. Chemistry
2-(1-(2-Oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (2) was previously prepared by
refluxing 3-acetyl-2H-chromen-2-one (1) and thiosemicarbazide in absolute ethanol in the presence of
catalytic amounts of HCl [20] (Scheme 1).
The target compounds 5a–h, 3-[1-(4-substituted-5-(aryldiazenyl)thiazol-2-yl)hydrazono) ethyl]-2H-
chromen-2-ones, were synthesized in a one pot reaction of thiosemicarbazide 2 and hydrazonoyl
halides 3(4) in the presence of TEA under ultrasonic irradiation (Scheme 1).
The structural elucidation of the compounds was based on spectral evidence and microanalyses. The
mass spectra of these products 5a–c showed the molecular ion peaks at the expected m/z values. Their
IR spectra showed the disappearance of the NH2 group, and revealed in each case one band at
1568–1558 cm−1, assignable to the N=N group (see Experimental).
The thiazole derivatives 8(9) were synthesized in good yields by the treatment of thiosemicarbazide
derivative 2 with chloroacetone (6) or phenacyl bromide (7) in dioxane under ultrasonic irradiation
following the Hantzsch thiazole synthesis [21]. Upon coupling the thiazole derivatives 8(9) with
diazotized aniline, in presence of sodium acetate trihydrate, the azo derivatives 5a–h were obtained.
The structures of the latter products were confirmed by the appearance of a N=N band in the IR spectra
1
and the lack of signals due to the C-5 proton of the thiazole ring in their H-NMR spectra (see
Experimental). The azo derivatives of similar thiazoles have found wide applications in the dyeing of
synthetic fibers [22,23] and the azo derivatives described in the present work may find similar
applications.
4-Thiazolidinone compound 11 was obtained by reaction of thiosemicarbazide 2 with chloroacetic
acid in glacial acetic acid and in the presence of anhydrous sodium acetate. Reaction of the latter
product 11 with substituted aldehydes afforded the corresponding arylidines 10a–d.

Molecules 2012, 17
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Scheme 1. Synthesis of 5-arylazothiazole derivatives 5a–h.
CH3
O
O
O
X
1
CH3
R
N
S
RCOC
H
NNHAr
N
3(4)
N
NH2NHCSNH2
N
Dioxan / Et3N, )))
Ethanol / HCl, )))
N
Ar
O
O
5a-h
CH3
N
H
N
NH2
ArN2Cl /
CH3COONa.3H2O
S
O
O
CH3
R
2
N
S
H
N
RCOCH2X
6(7)
5a-h
Ar
N
R
C6H5
CH3
a
b
c
d
e
f
Dioxan, )))
O
O
4-MeC6H4
4-ClC6H4
CH3
CH3
CH3
Ph
8(9)
3,6 and 8: R = CH3, X = Cl ;
4,7 and 9: R = C6H5, X = Br
4-NO2C6H4
C6H5
4-MeC6H4
4-ClC6H4
4-NO2C6H4
Ph
g
h
Ph
Ph
The one pot synthesis of products 10a–d has been carried out via reaction of thiosemicarbazide 2
with chloroacetic acid and aldehydes in glacial acetic acid in presence of excess anhydrous sodium
1
acetate (Scheme 2). The H-NMR spectra data were also consistent with the assigned structures;
thiazolidinone CH2 protons of 11 appeared at δ 3.97 ppm, arylidiene CH proton of 10a–d was observed at
8.60–8.67 ppm (see Experimental).
In addition, the hydrazide-hydrazone derivative 12 was prepared by ultrasonic irradiation of
3-acetyl-2H-chromen-2-one (1) and 2-cyanoacetohydrazide in absolute ethanol in the presence of
catalytic amounts of HCl (Scheme 3). The structure of compound 12 was established on the basis of
analytical and spectral data. Thus its 1H-NMR spectrum showed the presence of a singlet at δ 4.25 ppm
for the CH2 group, and a singlet at δ 11.19 ppm for an NH group. Its mass spectrum revealed a
molecular ion peak at m/z 269 (see Experimental).

Molecules 2012, 17
Scheme 2. Synthesis of 5-arylidenethiazolinone derivatives 10a–d.
9338
CH3
CH3
N
H
N
H
ClCH2COOH \ ArCHO
AcOH / AcONa, )))
N
N
NH2
N
O
O
S
O
S
O
O
2
10a-d
Ar
ClCH2COOH
AcOH / AcONa, )))
CH3
H
N
N
ArCHO
N
O
AcOH / AcONa, )))
O
O
S
11
Ar = YC6H4 ; Y : a, H; b, CH3 ; c, 4-Cl; d, 4-NO2.
Scheme 3. Reaction of acetohydrazide 12 with aromatic aldehydes.
CH3
O
O
O
1
CH3
NH2NHCOCH2CN
Ethanol / HCl, )))
Ar
ArCHO
NNHCOC
EtOH, Pipridine, )))
CN
O
O
CH3
13a-c
Ar = YC6H4 ; Y : a, H; b, CH3 ; c, 4-Cl.
NNHCOCH2CN
HN
O
CH3
O
O
Salicyaldehyde
EtOH, Pipridine, )))
12
NNHCO
O
O
14
Furthermore, treatment of the acetohydrazide 12 with substituted benzaldehydes, under ultrasonic
irradiation, afforded the benzylidene derivatives 13a–c on the basis of their spectral data (Scheme 3)
which confirmed the structures of the products by the appearance of a C=CH signal at δ 9.34 ppm and
the lack of the characteristic signal due to methylene protons (see Experimental). The reaction of 12
with salicylaldehyde gave the coumarin derivative 14 (Scheme 3), in analogy with the reported
literature [24,25]. The IR spectrum of compound 14 showed the lack of absorption bands
corresponding to a C≡N group and presence of bands at 3,198 cm−1 due to the NH group.

Molecules 2012, 17
9339
2.2. In Vitro Cytotoxicity Assay
The effect of compound 5a on cellular viability was studied using the MTT Assay. The HaCaT cells
are plated and cultured in 12-well cell culture plates for 24 h (four plates represent the four days
incubation with 5a, each plate divided into 6 wells as control and 6 wells as a test) (Figure 1).
Figure 1. Represents the MTT assay results of healthy cells HaCat cells incubated with
50 μL 0.5 mol 5a compared to control one. The used concentration of 5a does not induce
significant cytotoxic effect on the healthy HaCaT cells.
3. Experimental
3.1. Chemistry
3.1.1. General
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus.
The IR spectra were recorded in potassium bromide discs on a Pye Unicam SP 3300 or a Shimadzu FT
IR 8101 PC infrared spectrophotometers. The NMR Spectra were recorded at 300 MHz on a Varian
1
Mercury VX-300 NMR spectrometer. H-NMR (300 MHz) and 13C-NMR (75 MHz) were run in
deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass
spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. Elemental
analyses of the products were carried out at the Microanalytical Centre of Cairo University, Giza,
Egypt. All reactions were followed by TLC (Silica gel, Merck). Irradiation was done in an
ultrasonicator, (Electric supply: 230 v, A.C. 50 Hz, 1phase; Ultrasonic frequency: 36 KHz; Ultrasonic
power: 100 W). In vitro cytotoxicity assay was performed at Regional Center for Food & Feed,
Agricultural Research Center, Giza, Egypt, using the MTT Assay. 2-(1-(2-oxo-2H-chromen-3-
yl)ethylidene)thiosemicarbazide (2) [20] and hydrazonoyl halides 3 [26,27] were prepared as reported
in the literature.

Molecules 2012, 17
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3.1.2. Synthesis of 3-[1-(4-Substituted-5-(aryldiazenyl)thiazol-2-yl)hydrazono) ethyl]-2H-chromen-2-
ones 5a–h
3.1.2.1. Method A: General Procedure
A mixture of 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene) thiosemicarbazide (2, 0.522 g, 2 mmol) and
appropriate hydrazonoyl halides 3(4) (2 mmol) in dioxane (30 mL) containing triethylamine (0.2 g,
2 mmol) was irradiated by an ultrasonic generator in a water-bath at 50–60 °C for 30 min. (monitored
by TLC). The formed yellow precipitate was isolated by filtration, washed with ethanol, dried and
recrystallized from dioxane to give compounds 5a–h.
3-[1-(2-(4-Methyl-5-(phenyldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5a). Yield
72%; yellow solid; mp = 142 °C; IR (KBr): v 1559 (N=N), 1624 (C=N), 1724 (C=O), 3408 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.20 (s, 3H, CH3), 2.38 (s, 3H, CH3), 7.12–7.89 (m, 9H, ArH), 8.37 (s, 1H,
13
Coumarin-H4), 10.60 (s, 1H, D2O exchangeable, NH); C-NMR (DMSO-d6): δ 14.61 (CH3), 9.52
(CH3), 112.15, 114.45, 120.22, 120.55, 121.85, 123.25, 124.33, 125.75, 127.16, 127.31, 129.73,
131.61, 133.73, 138.61 (Ar-C and Ar-CH), 153.13(C=N), 157.32(C=N), 167.51 (C=O); MS m/z
(%):404 (M+ + 1, 7), 403 (M+, 100), 106 (75), 91 (82), 77 (27). Anal. Calcd for C21H17N5O2S (403.11): C,
62.52; H, 4.25; N, 17.36. Found C, 62.42; H, 4.18; N, 17.12%.
3-[1-(2-(4-Methyl-5-(p-tolyldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5b). Yield
74%; yellow solid; mp = 230 °C. IR (KBr): v 1558 (N=N), 1623 (C=N), 1724 (C=O), 3408 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.20 (s, 3H, CH3), 2.38 (s, 3H, CH3), 2.58 (s, 3H, CH3), 7.12–7.88 (m, 8H, ArH),
8.35 (s, 1H, coumarin-H4), 10.63 (s, 1H, D2O exchangeable, NH); MS m/z (%): 417 (M+, 4), 106
(100), 91 (82), 65 (27). Anal. Calcd for C22H19N5O2S (417.13): C, 63.29; H, 4.59; N, 16.78. Found C,
63.22; H, 4.47; N, 16.53%.
3-[1-(2-(5-((4-Chlorophenyl)diazenyl)-4-methylthiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5c).
Yield 76%; yellow solid; mp = 188 °C; IR (KBr): v 1564 (N=N), 1626 (C=N), 1732 (C=O), 3410 (NH)
1
cm−1; H-NMR (DMSO-d6): δ 2.20 (s, 3H, CH3), 2.38 (s, 3H, CH3), 7.10–7.98 (m, 8H, ArH), 8.38
(s, 1H, coumarin-H4), 10.66 (s, 1H, D2O exchangeable, NH); MS m/z (%): 438 (M+ + 1, 8), 437 (M+, 16),
127 (100), 73 (68). Anal. Calcd for C21H16ClN5O2S (437.07): C, 57.60; H, 3.68; N, 15.99. Found C,
57.51; H, 3.62; N, 15.71%.
3-[1-(2-(4-Methyl-5-((4-nitrophenyl)diazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5d).
Yield 73%; yellow solid; mp = 195 °C; IR (KBr): v 1568 (N=N), 1629 (C=N), 1702(C=O), 3404 (NH)
1
cm−1; H-NMR (DMSO-d6): δ 2.22 (s, 3H, CH3), 2.40 (s, 3H, CH3), 7.12–7.95 (m, 8H, ArH), 8.39 (s,
1H, coumarin-H4), 10.68 (s, 1H, D2O exchangeable, NH); MS m/z (%): 448 (M+, 13), 201(54), 127
(100), 82 (94), 67 (100). Anal. Calcd for C21H16N6O4S (448.10): C, 56.24; H, 3.60; N, 18.74. Found C,
56.14; H, 3.65; N, 18.42%.
3-[1-(2-(4-Phenyl-5-(phenyldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5e). Yield
72%; yellow solid; mp = 182 °C; IR (KBr): v 1558 (N=N), 1626 (C=N), 1744 (C=O), 3413 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.44 (s, 3H, CH3), 7.12–7.95 (m, 14H, ArH), 8.30 (s, 1H, coumarin-H4), 10.56

Molecules 2012, 17
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13
(s, 1H, D2O exchangeable, NH); C-NMR (DMSO-d6): δ 10.31 (CH3), 112.35, 115.35, 119.43, 119.55,
121.35, 123.29, 123.52, 124.26, 125.12, 125.65, 126.34, 127.22, 129.28, 130.29, 132.33, 132.29,
136.67, 138.34 (Ar-C and Ar-CH), 154.27 (C=N), 155.98 (C=N), 167.29 (C=O); MS m/z (%): 465
(M+, 10), 359 (100), 134 (83), 89 (60), 77 (31). Anal. Calcd for C26H19N5O2S (465.13): C, 67.08; H,
4.11; N, 15.04. Found C, 67.00; H, 4.02; N, 14.86%.
3-[1-(2-(4-Phenyl-5-(p-tolyldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5f). Yield
72%; yellow solid; mp = 178 °C; IR (KBr): v 1562 (N=N), 1628 (C=N), 1743 (C=O), 3415 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.20 (s, 3H, CH3), 2.44 (s, 3H, CH3), 7.12–7.95 (m, 13H, ArH), 8.31 (s, 1H,
coumarin-H4), 10.59 (s, 1H, D2O exchangeable, NH); MS m/z (%): 480(M+ + 1, 7), 479 (M+, 100),
134 (83), 89 (82), 77 (31). Anal. Calcd for C27H21N5O2S (479.14): C, 67.62; H, 4.41; N, 14.60. Found
C, 67.42; H, 4.51; N, 14.54%.
3-[1-(2-(5-((4-Chlorophenyl)diazenyl)-4-phenylthiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5g).
Yield 72%; yellow solid; mp = 187 °C; IR (KBr): v 1565 (N=N), 1626 (C=N), 1742 (C=O), 3412 (NH)
cm−1; 1H-NMR (DMSO-d6): δ 2.44 (s, 3H, CH3), 7.02–7.98 (m, 13H, ArH), 8.34 (s, 1H, coumarin-H4),
10.65 (s, 1H, D2O exchangeable, NH; MS m/z (%): 500 (M+ + 1, 8), 499 (M+, 28), 359 (100), 134 (64),
77 (18). Anal. Calcd for C26H18ClN5O2S (499.09): C, 62.46; H, 3.63; N, 14.01. Found C, 62.44; H,
3.56; N, 13.91%.
3-[1-(2-(5-((4-Nitrophenyl)diazenyl)-4-phenylthiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (5h).
Yield 72%; brown solid; mp = 178 °C; IR (KBr): v 1566 (N=N), 1628 (C=N), 1742(C=O), 3414 (NH)
cm−1; 1H-NMR (DMSO-d6): δ 2.28 (s, 3H, CH3), 7.12–7.95 (m, 13H, ArH), 8.34 (s, 1H, coumarin-H4),
10.69 (s, 1H, D2O exchangeable, NH); MS m/z (%): 511(M+ + 1, 14), 510 (M+, 46), 417 (65), 257 (24),
107 (1), 67 (100). Anal. Calcd for C26H18N6O4S (510.11): C, 61.17; H, 3.55; N, 16.46. Found C, 61.11;
H, 3.48; N, 16.34%.
3.1.2.2. Method B
3.1.2.2.1. Synthesis of 3-[1-((4-Substituted thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-ones 8,9
A mixture of 2 (0.27 g, 1 mmol) and chloroacetone (6) or phenacyl bromide (7) (1 mmol) in
absolute ethanol (30 mL) was irradiated with an ultrasonic generator in a water-bath at 50–60 °C for
20 min. (monitored by TLC). The product started to separate out during the course of reaction. The
crystalline solid was filtered, washed with water, dried and recrystallized from DMF to give the
corresponding compounds 8 and 9, respectively.
3-[1-(4-Methylthiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (8). Yield 79%; yellow solid;
1
mp = 170 °C; IR (KBr): v 1721 (C=O), 3267 (NH) cm−1; H-NMR (DMSO-d6): δ 2.24 (s, 3H, CH3),
2.51 (s, 3H, CH3), 7.07–8.02 (m, 5H, ArH and thiazole-H5), 8.22 (s, 1H, coumarin-H4), 9.31 (s, 1H,
D2O exchangeable, NH); MS m/z (%): 299 (M+, 6), 129 (29), 77 (12), 60 (100). Anal. Calcd for
C15H13N3O2S (299.07): C, 60.18; H, 4.38; N, 14.04. Found C, 60.10; H, 4.28; N, 13.84%.

Molecules 2012, 17
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3-[1-((4-Phenylthiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-one (9). Yield 84%; yellow solid;
1
mp = 232 °C; IR (KBr): v 1744 (C=O), 3418 (NH) cm−1; H-NMR (DMSO-d6): δ 2.44 (s, 3H, CH3),
7.10–8.02 (m, 10H, ArH and thiazole-H5), 8.26 (s, 1H, coumarin-H4), 9.28 (s, 1H, D2O exchangeable,
13
NH); C-NMR (DMSO-d6): δ 10.11 (CH3), 112.11, 116.28, 119.42, 120.36, 122.29, 123.01, 124.33,
125.75, 127.16, 127.31, 129.73, 134.33, 135.64, 139.83 (Ar-C and Ar-CH), 154.24 (C=N), 158.12
(C=N), 167.21 (C=O); MS m/z (%): 362 (M+ + 1, 12), 361 (M+, 48), 359 (100), 133 (61), 89 (81) 77
(32). Anal. Calcd for C20H15N3O2S (361.09): C, 66.46; H, 4.18; N, 11.63. Found C, C, 66.53; H, 4.10;
N, 11.43%.
3.1.2.2.2. Coupling of 8(9) with Arenediazonium Chlorides
To a solution of 8 or 9 (1 mmol) in ethanol (20 mL) was added sodium acetate trihydrate (0.138 g,
1 mmol), and the mixture was cooled to 0–5 °C in an ice bath. To the resulting cold solution was added
portionwise a cold solution of arenediazonium chloride [prepared by diazotizing aniline derivatives
(1 mmol) dissolved in hydrochloric acid (6 M, 1 mL) with a solution of sodium nitrite (0.07 g, 1 mmol)
in water (2 mL)]. After complete addition of the diazonium salt, the reaction mixture was stirred for a
further 30 min in an ice bath. The solid that separated was filtered off, washed with water and finally
recrystallized from ethanol to give product proved to be identical in all respects (mp, mixed mp and IR
spectra) with compounds 5a–h which obtained from method A.
3.1.3. Synthesis of 5-Arylidene-2-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)thiazol-4(5H)-
ones 10a–d
3.1.3.1. Method A
A mixture of 2 (0.261 g, 1 mmol), chloroacetic acid (0.1 g, 1 mmol) and appropriate aldehyde (1
mmol) in glacial acetic acid (20 mL) containing anhydrous sodium acetate (0.33 g, 4 mmol) was irradiated
with an ultrasonic generator in a water-bath at 50–60 °C for 30 min. (monitored by TLC). The reaction
mixture was left to cool and the formed solid was filtered off, washed with water, dried and
recrystallized from ethanol to give 10a–d.
5-Benzylidene-2-[2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl]thiazol-4(5H)-one (10a). Yield
84%; yellow solid; mp = 176 °C; IR (KBr): v 1678, 1725 (2C=O), 3360 (NH) cm−1; 1H-NMR (DMSO-d6):
δ 2.24 (s, 3H, CH3), 6.91–7.88 (m, 9H, ArH), 8.19 (s, 1H, coumarin-H4), 8.63 (s, 1H, N=CH), 10.87
13
(s, 1H, D2O exchangeable, NH); C-NMR (DMSO-d6): δ 9.97 (CH3), 112.15, 114.45, 121.23, 121.62,
121.89, 122.42, 124.19, 124.84, 126.46, 128.43, 129.87, 130.41, 134.43, 146.73 (Ar-C and Ar-CH),
154.19 (C=N), 158.12 (C=N), 167.11 (C=O), 181.56 (C=O); MS m/z (%): 302 (M+ + 1, 9), 301
(M+, 62), 218 (65), 172 (40), 130 (100), 77 (43). Anal. Calcd for C21H15N3O3S (389.08): C, 64.77; H,
3.88; N, 10.79. Found C, 64.58; H, 3.78; N, 10.48%.
5-[4-Methylbenzylidene)-2-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)thiazol-4(5H)-one (10b).
1
Yield 79%; yellow solid; mp = 172 °C; IR (KBr): v 1678, 1721 (2C=O), 3353 (NH) cm−1; H-NMR
(DMSO-d6): δ 2.24 (s, 3H, CH3), 2.51 (s, 3H, CH3), 6.91–7.88 (m, 8H, ArH), 8.19 (s, 1H, coumarin-H4),
8.60 (s, 1H, N=CH), 10.83 (s, 1H, D2O exchangeable, NH); MS m/z (%): 404 (M+ + 1, 15), 403

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(M+, 46), 235 (100), 146 (64), 130 (18), 77 (39). Anal. Calcd for C22H17N3O3S (403.10): C, 65.49; H,
4.25; N, 10.42. Found C, 65.43; H, 4.11; N, 10.12%.
5-(4-Chlorobenzylidene)-2-[2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl]thiazol-4(5H)-one (10c).
1
Yield 82%; yellow solid; mp = 186 °C; IR (KBr): v 1679, 1722 (2C=O), 3353 (NH) cm−1; H-NMR
(DMSO-d6): δ 2.26 (s, 3H, CH3), 6.91–7.96 (m, 8H, ArH), 8.19 (s, 1H, coumarin-H4), 8.62 (s, 1H,
N=CH), 10.86 (s, 1H, D2O exchangeable, NH); MS m/z (%): 424 (M+ + 1, 13), 423 (M+, 46), 235
(100), 172 (36), 130 (58), 63 (69). Anal. Calcd for C21H14ClN3O3S (423.04): C, 59.50; H, 3.33; N,
9.91. Found C, 59.37; H, 3.13; N, 9.61%.
5-(4-Nitrobenzylidene)-2-[2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl]thiazol-4(5H)-one (10d).
1
Yield 76%; yellow solid; mp = 170 °C; IR (KBr): v 1679, 1723 (2C=O), 3366 (NH) cm−1; H-NMR
(DMSO-d6): δ 2.26 (s, 3H, CH3), 6.91–7.96 (m, 8H, ArH), 8.16 (s, 1H, coumarin-H4), 8.67 (s, 1H,
N=CH), 10.86 (s, 1H, D2O exchangeable, NH); MS m/z (%): 435 (M+ + 1, 7), 434 (M+, 44), 235 (100),
218 (66), 130 (70), 63 (58). Anal. Calcd for C21H14N4O5S (434.07): C, 58.06; H, 3.25; N, 12.90. Found
C, 57.86; H, 3.26; N, 12.79%.
3.1.3.2. Method B
3.1.3.2.1. Synthesis of 2-[2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl]thiazol-4(5H)-one (11)
A mixture of 2 (0.261 g, 1 mmol) and chloroacetic acid (0.1 g, 1 mmol) in glacial acetic acid
(30 mL) containing anhydrous sodium acetate (0.33 g, 4 mmol) was irradiated with an ultrasonic
generator in a water-bath at 50–60 °C for 30 min. (monitored by TLC). The reaction mixture was
cooled and the resulting precipitate was filtered off and recrystallized from ethanol to give 11. Yield
84%; yellow solid; mp = 220 °C; IR (KBr): v 1667, 1717 (2C=O), 3167 (NH) cm−1; 1H-NMR (DMSO-d6):
δ 2.26 (s, 3H, CH3), 3.97 (s, 2H, CH2), 6.91–8.19 (m, 4H, ArH), 8.63 (s, 1H, coumarin-H4), 10.87 (s,
13
1H, D2O exchangeable, NH); C-NMR (DMSO-d6): δ 10.46 (CH3), 39.35 (CH2), 118.45, 120.18,
122.23, 124.33, 129.73, 131.61, 133.73, 138.61 (Ar-C and Ar-CH), 153.43 (C=N), 156.56 (C=N),
167.12 (C=O), 176.33 (C=O); MS m/z (%): 303 (M+ + 2, 3), 302 (M+ + 1, 9), 301 (M+, 36), 235 (73),
146 (36), 130 (100), 77 (59). Anal. Calcd for C14H11N3O3S (301.05): C, 55.80; H, 3.68; N, 13.95.
Found C, 55.68; H, 3.60; N, 13.75%.
3.1.3.2.2. Reaction of 8 with Aromatic Aldehydes
General procedure: To a solution of 5-thiazolidinone 11 (0.30 g, 1 mmol) and appropriate aldehyde
(1 mmol) in glacial acetic acid (20 mL), anhydrous sodium acetate (0.33 g, 4 mmol) was irradiated
with an ultrasonic generator in a water-bath at 50–60 °C for 30 min (monitored by TLC). The product,
so separated, was filtered, washed with water, dried and recrystallized from ethanol to give compounds
which proved to be identical in all respects (mp, mixed mp and IR spectra) with the
hydrazonothiazolidinones 10a–d which obtained from method A.

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3.1.4. Synthesis of 2-Cyano-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene)acetohydrazide (12)
To a solution of 2-cyanoacetohydrazide (1.0 g, 10 mmol) and 3-acetyl-2H-chromen-2-one (1, 1.88 g,
10 mmol) in absolute ethanol (30 mL) three drops of conc. HCl were added and the reaction mixture
was irradiated with an ultrasonic generator in a water-bath at 50–60 °C for 20 min. then left to cool.
The solid product formed was collected by filtration, dried and recrystallized from ethanol to give 12.
Yield 86%; yellow microcrystals; mp = 172 °C; IR (KBr): v = 1690, 1724 (2C=O), 2230 (CN), 3186
1
(NH) cm−1; H-NMR (DMSO-d6): δ 2.17 (s, 3H, CH3), 4.25 (s, 2H, CH2), 7.39–8.30 (m, 4H, ArH),
8.95 (s, 1H, coumarin-H4), 11.19 (s, 1H, D2O exchangeable, NH); MS m/z (%): 270 (M+ + 1, 5), 269
(M+, 33), 229 (82), 115 (100), 89 (60), 63 (54). Anal. Calcd for C14H11N3O3 (269.08): C, 62.45; H,
4.12; N, 15.61. Found C, C, 62.41; H, 4.02; N, 15.38%.
3.1.5. Reaction of 11 with Aromatic Aldehydes
General procedure: Equimolecular mixture of 2-cyano-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
acetohydrazide (12, 2.69 g, 0.01 mol) and appropriate aldehyde (0.01 mol), in anhydrous ethanol (20 mL)
containing piperidine (0.50 mL) was irradiated with an ultrasonic generator in a water-bath at
50–60 °C for 30 min (monitored by TLC). The formed solid was collected by filtration and
recrystallized from the proper solvent to give compounds 13a–d.
2-Cyano-N'-[1-(2-oxo-2H-chromen-3-yl)ethylidene]-3-phenylacrylohydrazide (13a). Yield 80%; yellow
solid (from ethanol); mp = 198 °C; IR (KBr): v 1672, 1728 (2C=O), 2210 (CN), 3336 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.19 (s, 3H, CH3), 7.24–8.21(m, 9H, ArH ), 8.87 (s, 1H, coumarin-H4), 9.34
(s, 1H, C=CH), 11.22 (s, 1H, D2O exchangeable, NH); 13C-NMR (DMSO-d6): δ 10.74 (CH3), 112.36,
115.26, 117.26, 119.42, 121.65, 122.86, 123.43, 124.16, 124.86, 127.67, 127.99, 129.04, 133.43,
134.87, 139.37 (Ar-C, Ar-CH and C≡N), 154.94 (C=N), 168.13 (C=O), 174.14 (C=O); MS m/z (%):
358 (M+ + 1, 4), 357 (M+, 40), 185 (100), 129 (73), 109 (73), 55 (91). Anal. Calcd for C21H15N3O3
(357.11): C, 70.58; H, 4.23; N, 11.76. Found C, 70.49; H, 4.21; N, 11.46%.
2-Cyano-N'-[1-(2-oxo-2H-chromen-3-yl)ethylidene]-3-p-tolylacrylohydrazide (13b). Yield 82%;
yellow solid (from DMF); mp = 187 °C. IR (KBr): v 1672, 1728 (2C=O), 2216 (CN), 3336 (NH) cm−1;
1H-NMR (DMSO-d6): δ 2.19 (s, 3H, CH3), 2.49 (s, 3H, CH3), 7.20–8.23 (m, 8H, ArH), 8.89 (s, 1H,
coumarin-H4), 9.34 (s, 1H, C=CH), 11.26 (s, 1H, D2O exchangeable, NH); MS m/z (%): 372 (M+ + 1, 6),
371 (M+, 45), 229 (75), 186 (55), 115 (100), 63 (62). Anal. Calcd for C22H17N3O3(371.13): C, 71.15;
H, 4.61; N, 11.31. Found C, C, 71.05; H, 4.36; N, 11.11%.
3-(4-Chlorophenyl)-2-cyano-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene)acrylohydrazide (13c). Yield
78%; yellow solid (from ethanol ); mp = 202 °C; IR (KBr): v 1674, 1732 (2C=O), 2218 (CN), 3334
(NH) cm−1; 1H-NMR (DMSO-d6): δ 2.19 (s, 3H, CH3), 7.21–8.23(m, 8H, ArH), 8.89 (s, 1H, coumarin-H4),
9.35 (s, 1H, C=CH), 11.34 (s, 1H, D2O exchangeable, NH); MS m/z (%): 392 (M+ + 1, 7), 391 (M+, 22),
273 (47), 219 (100), 84(94). Anal. Calcd for C21H14ClN3O3 (391.07): C, 64.37; H, 3.60; N, 10.72.
Found C, C, 64.07; H, 3.62; N, 10.32%.

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2-Imino-N'-[1-(2-oxo-2H-chromen-3-yl)ethylidene]-2H-chromene-3-carbohydrazide (14). Yield 78%;
yellow solid (from DMF); mp = 240 °C; IR (KBr): v 1669, 1712 (2C=O), 3198, 3325 (2NH) cm−1;
1H-NMR (DMSO-d6): δ 2.19 (s, 3H, CH3), 7.10–8.36 (m, 9H, ArH ), 8.44 (s, 1H, D2O exchangeable, NH),
8.89 (s, 1H, coumarin-H4), 11.38 (s, 1H, D2O exchangeable, NH); MS m/z (%): 374 (M+ + 1, 13), 373
(M+, 13), 201 (51), 171 (100), 115 (89), 62 (58). Anal. Calcd for C21H15N3O4 (373.11): C, 67.56; H,
4.05; N, 11.25. Found C, C, 67.44; H, 4.15; N, 11.05%.
3.2. Cytotoxic Activity
The method applied is similar to that reported by Skehan et al. using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Healthy HaCaT epithelial primary cell line
(human keratinocytes) was cultured on 18 mm diameter glass cover slips in a 12-well tissue culture
plate in DMEM plus 5% FBS at 37–38 °C under 5% CO2. The cover slips were coated with collagen
type I (Roche) in advance for optimum cell growth. The HaCaT cells are cultured in 12-well cell
culture plates for 24 h, (four plates represent the four days incubation with 5a, each plate divided into
6-wells as control and 6-wells as a test). Rinsing the old medium and adding new one then 50 μL
0.5 mol 5a are added to test wells (not to the control one) and returned to incubator for 8 h washing the
excess 5a by DPBS buffer and add 1 mL medium to each well then incubated for the designated
periods for each plate (Figure 1).
4. Conclusions
In summary, we have developed a new green methodology and synthesized several 3-[1-(4-
substituted-5-(aryldiazenyl)thiazol-2-yl)hydrazono)ethyl]-2H-chromen-2-ones by ultrasound irradiation.
The cytototoxic activity of one of them was also evaluated against healthy HaCaT cells.
Acknowledgements
The authors wish to thank Taher Salah of Nanotechnology Center, Regional Center for Food &
Feed, Agricultural Research Center for his support to carry out the antimicrobial activity.
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Sample Availability: Samples of the synthesized compounds are available from the authors.
© 2012 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
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