The synthesis and the biological evaluation of new thiazolidin-4-one derivatives containing a xanthine moiety

Article abstract of DOI:10.3390/molecules18089684

Starting from theophylline (1,3-dimethylxanthine) new thiazolidin-4-one derivatives 7a_1-7, 7b_1-7 have been synthesized as potential antidiabetic drugs. The structure of the new derivatives was confirmed using spectral methods (FT-IR, ¹H-NMR, ¹³C-NMR). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the ferric reducing power, the total antioxidant activity and the DPPH and ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are considered to be implicated in a variety of pathological events, such as diabetes mellitus and its micro- and macrovascular complications. The results of chemical modulation of the thiazolidin-4-one intermediaries 6a, 6b through condensation with several aromatic aldehydes is the improvement of the antioxidant effect. All benzylidenethiazolidin-4-one derivatives 7a _1-7, 7b_1-7 are more active than their parent thiazolidin-4-ones. The most active compounds are the ones obtained by reaction of condensation with 4-hydroxybenzaldehyde (compounds 7a₅, 7a ₆), 4-dimethylaminobenzaldehyde (compounds 7a₆, 7b ₆) and 2-nitrobenzaldehyde (compounds 7a₇, 7b ₇).

Full text of DOI:10.3390/molecules18089684

Molecules 2013, 18, 9684-9703; doi:10.3390/molecules18089684
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Synthesis and the Biological Evaluation of New
Thiazolidin-4-one Derivatives Containing a Xanthine Moiety
Florentina Geanina Lupascu 1, Oana Maria Dragostin 1, Liliana Foia 2, Dan Lupascu 1,* and
Lenuta Profire 1,*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of
Medicine and Pharmacy, 16 University Street, Iasi 700115, Romania;
E-Mails: geani0407@yahoo.com (F.G.L.); farmacist_oanamaria@yahoo.com (O.M.D.)
2
Department of Biochemistry, Faculty of Dentistry, “Grigore T. Popa” University of Medicine and
Pharmacy, 16 University Street, Iasi 700115, Romania; E-Mail: lilifoia@yahoo.co.uk
* Authors to whom correspondence should be addressed; E-Mails: lenuta.profire@umfiasi.ro (L.P.);
dan.lupascu@umfiasi.ro (D.L.); Tel.: +40-232-412-375; Fax: +40-232-211-818.
Received: 10 July 2013; in revised form: 2 August 2013 Accepted: 5 August 2013 /
Published: 13 August 2013
Abstract: Starting from theophylline (1,3-dimethylxanthine) new thiazolidin-4-one
derivatives 7a1–7, 7b1–7 have been synthesized as potential antidiabetic drugs. The structure
of the new derivatives was confirmed using spectral methods (FT-IR, 1H-NMR,
13C-NMR). The in vitro antioxidant potential of the synthesized compounds was evaluated
according to the ferric reducing power, the total antioxidant activity and the DPPH and
ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are
considered to be implicated in a variety of pathological events, such as diabetes mellitus
and its micro- and macrovascular complications. The results of chemical modulation of the
thiazolidin-4-one intermediaries 6a, 6b through condensation with several aromatic
aldehydes is the improvement of the antioxidant effect. All benzylidenethiazolidin-4-one
derivatives 7a1–7, 7b1–7 are more active than their parent thiazolidin-4-ones.
The most active compounds are the ones obtained by reaction of condensation
with 4-hydroxybenzaldehyde (compounds 7a5, 7a6), 4-dimethylaminobenzaldehyde
(compounds 7a6, 7b6) and 2-nitrobenzaldehyde (compounds 7a7, 7b7).
Keywords: thiazolidin-4-one; xanthine; synthesis; spectral methods; antioxidant effect

Molecules 2013, 18
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1. Introduction
The thiazolidine ring system represents a very important structural unit in drug discovery [1].
A survey of the literature reveals that extensive studies have been carried out on the synthesis of
thiazolidines. These compounds are known to exhibit interesting biological activity such as
antimicrobial [2–5], antitubercular [6], antipsihotic [7], anticancer [8,9], antiviral [10], anti-HIV [11],
anti-inflammatory [12,13] and antihyperglycemic [14–17] effects. As antidiabetic drugs,
thiazolidinediones (pioglitazone—Actos® and rosiglitazone—Avandia®, Figure 1) have an important
place in type 2 diabetes mellitus therapy [18].
Figure 1. The structure of pioglitazone and rosiglitazone.
C2H5
CH3
N
O
O
S
S
O
(CH2)2
CH2
O
(CH2)2
CH2
NH
NH
Rosiglitazone
Pioglitazone
O
O
Diabetes mellitus, a chronic metabolic disorder, is a major health problem that is approaching
pandemic proportions worldwide. It is characterized by absolute or relative deficiencies in insulin
secretion and/or insulin action [19]. An inadequate glycaemic control can be the leading cause of
cardiovascular disorders, blindness, renal failure and amputations [19,20]. Type 2 diabetes mellitus
(T2DM) accounts for over 90% of the disease cases and it is characterized by high levels of glucose
resulting from progressive pancreatic β-cell dysfunction caused by chronic insulin resistance [19]. As
agonists of nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ),
thiazolidinediones (TZD) reduce insulin resistance in the liver and peripheral tissues; increase the
intake of insulin-dependent glucose and decrease withdrawal of glucose from the liver [14,18]. On the
other hand, the thiazolidine derivatives have been reported to have antioxidant activities that could be
effective in the T2DM therapy [21–23].
A relationship between reactive oxygen species and many diseases including diabetes, myocardial
infarction, neurological disorders, asthma, cancer, rheumatoid arthritis has been observed [24], so in
case of diabetic patients, elevated levels of blood glucose is the main cause which leads to an increased
production of free radicals, especially reactive oxygen species resulted from glucose auto-oxidation,
lipid peroxidation and protein glycosylation. Endothelial cells chronically exposed to oxidative stress
lead to diabetes long term complications (micro- and macrovascular) [19].
The latest research regarding antidiabetic drugs has focused on dipeptidyl peptidase IV inhibitors
that act by increasing the concentrations of the endogenous incretins GLP-1 and GI, and consequently
reducing of fasting post-prandial hyperglycaemia [25]. The most recently marketed inhibitor is
linagliptin (Ondero®, Trajenta®, Figure 2), a nonpetidommimetic drug with a xanthine structure, which
received approval in 2011 [26]. At the same time xanthine derivatives are known to have antioxidant
and radical scavenging activity that contributes to their pharmacological properties [27,28].

Molecules 2013, 18
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Figure 2. The structure of linagliptin.
O
N
N
N
N
N
N
O
N
NH2
CH3
Linagliptin
CH3
Based on the arguments presented above, we are reporting here the design, synthesis and
antioxidant effects of new heterocyclic compounds which combine a thiazolidine structure with a
xanthine one, as potential antidiabetic agents.
2. Results and Discussion
2.1. Chemistry
The thiazolidine derivatives 7a1–7; 7b1–7 were prepared using the method summarized in Scheme 1.
Scheme 1. Synthetic procedure for compounds 7a1–7, 7b1–7.
CH2CONHNH2
N
O
O
O
O
CH2COOC2H5
H
N
-Na+
H3C
O
H3C
O
H3C
O
H3C
O
N
N
N
N
N
N
N
a
c
b
N
N
N
N
N
N
N
CH3
2
CH3
CH3
CH3
1
4
3
R1
C6H4
C6H4
NH
O
R1
O
N
H2C C NH N
C O
CH2
O
NH NH C
C
H2C
N
O
N
S
N
S
e
.
H3C N
O
H3C N
d
N
N
R1
23
N
O
CH3
6a, 6b
CH3
5a, 5b
24
25
22
21
20
8
9
N
17
R2
16
18
H2C C NH
N
12
10
C O
O
11
19
CH
S
C
O
N
1
f
6
13
H3C N
5
4
7
14 15
2
N
O
N
3
CH3
7a1-7
7b1-7
R1 = H, Cl;
R2 = H, F(4), Cl(4), Br(4), OH(4), N(CH3)2(4), NO2(2)
Reagents and conditions: (a) sodium, absolute methanol; (b) ethyl chloroacetate, ethanol:DMFA, heating 7 h;
(c) hydrazine hydrate, ethanol, heating 6 h; (d) phenyl isothiocyanate/4-chlorophenyl isothiocyanate,
1,4-dioxane:DMFA, heating 6 h; (e) chloroacetyl chloride, methanol:chloroform, heating 10 h; (f) aromatic
aldehydes, 1,4-dioxane, piperidine, heating 6 h.

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Through the esterification of theophylline (1,3-dimethylxanthine, 1) sodium salt 2 with
ethylcloroacetate, (1,3-dimethylxanthin-7-yl)ethyl acetate (3) was obtained, which was turned into
hydrazide derivative 4 [29]. During the next step this intermediate was reacted with phenylisothiocyanate
and 4-chlorophenylisothiocyanate leading to the corresponding thiosemicarbazide derivatives 5a, 5b.
By cyclization of 5a, 5b with chloracetyl chloride the corresponding thiazolidin-4-one derivatives 6a, 6b
were obtained. The last step of the synthesis consisted in the condensation of the thiazolidin-4-one
derivatives with several aromatic aldehydes—benzaldehyde, 4-bromo-/4-fluoro-/4-chloro-/4-hydroxy-
/4-dimethylamino-/2-nitrobenzaldehyde.
1
The structure of the compounds was assigned on the basis of spectral data (IR, H-NMR,
13C-NMR). In the IR spectra of the thiosemicarbazide derivatives 5a, 5b the characteristic absorption
band of the thione group (C=S) was observed at 1160 cm−1 (5a), and 1188 cm−1 (5b), respectively. The
valence vibration of the bond C-Cl (5b) produced a narrow absorption band of medium intensity at
825 cm−1. In the 1H-NMR spectra of the compounds the aromatic protons resonated as multiplets in the
7.19–7.47 ppm region (5a), or the 7.31–7.51 ppm region (5b), respectively, confirming the success of
the reaction between hydrazide derivative 4 and the phenyl isothiocyanates. The protons of the
xanthine structure were also identified in the spectra of the thiosemicarbazides. The protons of the two
methyl groups appeared as two singlets at 3.09 ppm and 3.45 ppm (5a), or at 3.22 ppm and 3.57 ppm (5b),
respectively. The proton of the methine group resonated as a singlet at 8.09 ppm, while the protons of
the methylene group also resonated as a singlet in the 5.08–5.09 ppm region.
In the IR spectra of thiazolidin-4-ones derivatives 6a, 6b the characteristic band of the thione group
(C=S) disappeared and the appearance of the C-S bond characteristic of the thiazolidin-4-one it was
observed in the range of 690–695 cm−1. The 1H-NMR spectra of the compounds showed the methylene
protons of the thiazolidin-4-one ring at 3.67 ppm (6a) and at 3.78 ppm (6b) resonating as a singlet.
That confirmed the formation of the thiazolidin-4-one ring.
The structure of the benzylidenethiazolidin-4-one derivatives 7a1–7, 7b1–7 was confirmed by the
1
appearance in the H-NMR spectra of the proton signal characteristic for the methine group
from the benzilidene thiazolidin-4-one ring as a sing let in the range of 7.28–7.38 ppm (7a1–7) and at
7.24–7.51 ppm (7b1–7). In the 13C-NMR spectra the characteristic signal for this methine group
appeared in the 140.35–143.07 ppm range (7a1–7), or 138.51–142.84 (7b1–7), respectively.
2.2. Biological Evaluation
2.2.1. Ferric Reducing Power
The measurement of the reducing power defines an important aspect of the compounds’ antioxidant
activity. In the presence of the electron-donating compounds, the potassium ferric/ferricyanide
complex is reduced to its ferrous form (Fe2+). The amount of Fe2+ is then quantitatively monitored by
measuring the intensity of the Perls Prussian blue colour complex formed by reaction with ferric
chloride, at 700 nm [30]. The reaction between the ferrous form and the ferric chloride is:
4FeCl3 + 3K4[Fe(CN)6] → Fe[Fe(CN)6]3 + 12KCl

Molecules 2013, 18
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The absorbance value of the samples at different concentrations (28.4 µg/mL, 56.8 µg/mL,
113.63 µg/mL and 170.45 µg/mL) are presented in Figures 3 and 4. The results expressed as EC50
values (mg/mL) are shown in Table 1. Low values of EC50 demonstrate a higer ferric reducing power.
As we expected the absorbance of the sample increased with the concentration, thus proving the fact
that reducing power of the tested compounds is concentration-dependent. The analysis of the data
presented in the Table 1, revealed that the chemical modulation of the thiazolidin-4-one ring through
condensation with several aromatic aldehydes had a good influence on the antioxidant potential; all
tested compounds being more active than the corresponding thiazolidin-4-ones 6a, 6b. It was also
observed that both series of compounds 7a1–7, 7b1–7 have a comparable reducing power. In the series of
3-phenylthiazolidin-4-one derivatives 7a1–7 the most active compounds were those which resulted
from condensation with 4-hydroxybenzaldehyde (compound 7a5) and 4-dimethlaminobenzaldehyde
(compound 7a6). For these compounds the EC50 values were 0.033 ± 0.0019 (for 7a5) and
0.033 ± 0.0012 (for 7a6), which means that they are about four times more active than the
corresponding thiazolidin-4-one (6a, EC50 = 0.137 ± 0.0036). Regarding the compounds 7b1–7 obtained
through condensation of 3-(4-chlorophenyl)-thiazolidin-4-one derivative 6b with several aromatic
benzaldehydes, we could observe that the most active compounds were 7b5 and 7b6, the corresponding
analogues of 7a5 and 7a6. These compounds were about 5.7 times (7b5, EC50 = 0.027 ± 0.0013) and
6.7 times (7b6, EC50 = 0.023 ± 0.0011) more active than the corresponding thiazolidin-4-one derivative
(6b, EC50 = 0.155 ± 0.0038). The chemical modulation of the parent thiazolidin-4-one derivatives
improved their ferric reducing power, all tested compounds being more active than
3-phenlythiazolidin-4-one, respectively the 3-(4-chlorophenyl)thiazolidin-4-one but they are less active
than the ascorbic acid (AA) at the same concentration.
Figure 3. The absorbance of 7a1–7 in ref. to 6a.

Molecules 2013, 18
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Figure 4. The absorbance of 7b1–7 in ref. to 6b.
Table 1. The ferric reducing power (EC50 mg/mL) of the derivatives6a, 6b, 7a1–7, 7b1–7.
Sample
6a
EC50 mg/mL
0.137 ± 0.0036
0.109 ± 0.0014
0.097 ± 0.0018
0.055 ± 0.0036
0.096 ± 0.0023
0.033 ± 0.0019
0.033 ± 0.0012
0.058 ± 0.0041
0.0075 ± 0.0002
Sample
6b
EC50 mg/mL
0.155 ± 0.0038
0.109 ± 0.0045
0.102 ± 0.0024
0.046 ± 0.0015
0.066 ± 0.0017
0.027 ± 0.0013
0.023 ± 0.0011
0.041 ± 0.0010
7a1
7b1
7a2
7b2
7a3
7b3
7a4
7b4
7a5
7b5
7a6
7b6
7a7
7b7
AA
Data are mean ± SD (n = 3, p < 0.005).
2.2.2. Total Antioxidant Activity
The total antioxidant activity was determined by the formation of phosphomolybdenum blue
complex by the reduction of Mo(VI) to Mo(V) under the action of electron donating compounds. The
maximum absorption of the complex was recorded at 695 nm [31]. The absorbance values of samples
at different concentrations (23, 34, 45, 68, 91 and 136 µg/mL) are presented in Figures 5 and 6.
The EC50 values (mg/mL) are shown in Table 2. Low values of EC50 indicate a higer effectiveness in
antioxidant properties.

Molecules 2013, 18
9690
Figure 5. The absorbance of 7a1–7 in ref. to 6a.
Figure 6. The absorbance of 7b1–7 in ref. to 6b.
Table 2. The total antioxidant activity (EC50 mg/mL) of the derivatives 6a, 6b, 7a1–7, 7b1–7.
Sample
6a
EC50 mg/mL
0.133 ± 0.0045
0.105 ± 0.0025
0.099 ± 0.0038
0.048 ± 0.0023
0.076 ± 0.0025
0.025 ± 0.0012
0.033 ± 0.0014
0.037 ± 0.0007
0.0067 ± 0.0003
Sample
6b
EC50 mg/mL
0.124 ± 0.0009
0.090 ± 0.0018
0.092 ± 0.0008
0.043 ± 0.0026
0.099 ± 0.0024
0.022 ± 0.0013
0.026 ± 0.0018
0.043 ± 0.0028
7a1
7b1
7a2
7b2
7a3
7b3
7a4
7b4
7a5
7b5
7a6
7b6
7a7
7b7
AA
Data are mean ± SD (n = 3, p < 0.05).

Molecules 2013, 18
9691
The data of this study also support the conclusion that the antioxidant activity of the tested
compounds increases with concentration and that all benzylidenethiazolidin-4-one derivatives
7a1–7, 7b1–7 are more active than parent thiazolidin-4-one derivatives (6a, 6b). It was observed that the
presence of chloro radical on the phenyl ring at N3 of thiazolidin-4-one has a good influence on the
antioxidant properties, the derivatives (7b1–7) being generally more active than their analogues from
the 7a1–7 series. In both series the most active compounds were hydroxyl- (7a5, 7b5) and
dimethylamino- (7a6, 7b6) analogues. The hydroxyl derivatives, 7a5 (EC50 = 0.025 ± 0.0012) and 7b5
(EC50 = 0.022 ± 0.0013) were 5.3 times and 5.6 times more active than corresponding thiazolidin-4-one,
6a (EC50 = 0.133 ± 0.0045), respectivelly 6b (EC50 = 0.124 ± 0.0009). The antioxidant effects of the
dimethylamino analogues (7a6, 7b6) were similar with of hydroxyl derivatives, being 4 times
(7a6, EC50 = 0.033 ± 0.0014), respectivelly 4.8 times (7b6, EC50 = 0.026 ± 0.0018), more active than
parent compounds (6a, 6b). The tested compounds were less active than ascorbic acid (AA) at the
same concentration.
2.2.3. DPPH Radical Scavenging Assay
DPPH (1,1-diphenyl-2-picryl-hydrazyl) is a stable free radical with a violet colour that, under the
action of proton donating compounds, is reduced to a light yellow colour that this change can be
monitored at 517 nm [32]. The DPPH radical scavenging ability (%) of samples at different
concentrations (37 µg/mL, 55.55 µg/mL, 111.11 µg/mL and 144.80 µg/mL) are presented in
Figures 7 and 8. The EC50 values (mg/mL) are shown in Table 3. Low values of EC50 indicate a higer
effectiveness in DPPH scavenging ability.
Figure 7. The scavenging ability (%) of 7a1–7 in ref. to 6a.

Molecules 2013, 18
9692
Figure 8. The scavenging ability (%) of 7b1–7 in ref. to 6b.
Table 3. The DPPH scavenging ability (EC50 mg/mL) of the derivatives 6a, 6b, 7a1–7, 7b1–7.
Sample
6a
EC50 mg/mL
0.136 ± 0.0006
0.094 ± 0.0013
0.107 ± 0.0018
0.066 ± 0.0011
0.077 ± 0.0005
0.038 ± 0.0044
0.039 ± 0.0008
0.048 ± 0.0001
0.0065 ± 0.0031
Sample
6b
EC50 mg/mL
0.127 ± 0.0013
0.106 ± 0.0011
0.101 ± 0.0044
0.049 ± 0.0006
0.085 ± 0.0017
0.038 ± 0.0024
0.037 ± 0.0037
0.038 ± 0.0014
7a1
7b1
7a2
7b2
7a3
7b3
7a4
7b4
7a5
7b5
7a6
7b6
7a7
7b7
AA
Data are mean ± SD (n = 3, p < 0.05).
The chemical modulation of the thiazolidin-4-one derivatives 6a, 6b through condensation with
several aromatic benzaldehydes determines the improvement of the DPPH radical scavenging ability,
as both series of compounds 7a1–7 and 7b1–7 were more active than their parents. In reference to
compound 6a, its benzylidene analogues 7a1–6 are 1.5–3.5 times more active. Under similar conditions
the compounds 7b1–7 are slightly less active, being 1.2–3.4 times more active than the corresponding
thiazolidin-4-one 6b. The most active compounds are hydroxyl- (compounds 7a5, 7b5),
dimethylamino- (compounds 7a6, 7b6) and nitro- (compounds 7a7, 7b7) analogues.
2.2.4. ABTS Radical Scavenging Assay
The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation decolourisation
assay is based on the ability of the hydrogen donating antioxidants to scavenge the long-life radical
cation ABTS+. The ABTS+ which is a blue chromophore is generated by the reaction between
2.2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and ammonium persulfate. The antioxidant

Molecules 2013, 18
9693
compound produces a discoloration of the solution with a decrease in the absorbance measured at
734 nm [33]. The ABTS radical scavenging ability (%) of the samples at different concentrations
(12.5 µg/mL, 25 µg/mL, 50 µg/mL and 75 µg/mL) are presented in Figures 9 and 10. The EC50 values
(mg/mL) are presented in Table 4. Low values of EC50 indicate a higer effectiveness in ABTS
scavenging ability.
Figure 9. The scavenging ability (%) of 7a1–7 in ref. with 6a.
Figure 10. The scavenging ability (%) of 7b1–7 in ref. with 6b.

Molecules 2013, 18
Table 4. The ABTS scavenging ability (EC50 mg/mL) of the derivatives 6a, 6b, 7a1–7, 7b1–7.
9694
Sample
6a
EC50 mg/mL
0.074 ± 0.0039
0.047 ± 0.0032
0.049 ± 0.0026
0.037 ± 0.0015
0.043 ± 0.0028
0.027 ± 0.0017
0.025 ± 0.0012
0.036 ± 0.0098
0.0055 ± 0.0024
Sample
6b
EC50 mg/mL
0.069 ± 0.0031
0.048 ± 0.0014
0.041 ± 0.0041
0.031 ± 0.0035
0.040 ± 0.0029
0.024 ± 0.0047
0.021 ± 0.0041
0.030 ± 0.0017
7a1
7b1
7a2
7b2
7a3
7b3
7a4
7b4
7a5
7b5
7a6
7b6
7a7
7b7
AA
Data are mean ± SD (n = 3, p < 0.05).
From the data presented in Table 4 it is obvious that the ABTS radical scavenging ability of the
benzylidenethiazolidin-4-one derivatives 7a1–7, 7b1–7 is higher than that of the thiazolidin-4-ones 6a, 6b.
As in the DPPH radical scavenging assay the most active compounds are the derivatives obtained by
condensation of thiazolidin-4-ones with 4-hydroxy-, 4-dimethylamino- and 2-nitrobenzaldehydes. The
hydroxy- (compounds 7a5, 7b5) and dimethylamino- (compounds 7a6, 7b6) analogues are about three
times more active, while the nitro- (compounds 7a7, 7b7) are two times more active than the
corresponding thiazolidin-4-ones 6a, 6b, but they are less active than ascorbic acid (AA) at the
same concentration.
3. Experimental
3.1. General Procedures
The melting points were measured using a Buchi Melting Point B-540 apparatus and they are
uncorrected. The FT-IR spectra were recorded on an ABB Bomen MB3000 spectrometer, over a
500–4,000 cm−1 range, after 32 scans at a resolution of 4 cm−1. The spectra processing was carried out
1
with the Horizon MBTM FTIR Software. The H-NMR and 13C-NMR spectra were obtained on a
Bruker Avance DRX-300 spectrometer using tetramethylsilane as internal standard and DMSO-d6 as
solvent. The chemical shifts were shown in δ values (ppm). The progress of the reaction was
monitored on TLC, using pre-coated Kieselgel 60 F254 plates (Merck) and the compounds were
visualized by using UV light.
3.2. Synthetic Procedures
3.2.1. General Procedure for the Preparation of 1-[2-(1,3-Dimethylxanthin-7-yl)acetyl]-4-(4-R-phenyl)-
thiosemicarbazides 5a, 5b
4-R-phenyl isothiocyanate (R = H, Cl; 23 mmol) was added to a solution of (1,3-dimethylxanthin-7-
yl)acetyl hydrazine (7 g, 23 mmol) in mixture of 1,4-dioxane (250 mL) and DMFA (100 mL), and then
heated under reflux for 6 hours. After cooling at room temperature the white solid was filtered off,
dried and recrystallized from ethanol: 1,4-dioxane (3:1).

Molecules 2013, 18
9695
1-[2-(1,3-Dimethylxanthin-7-yl)acetyl]-4-(phenyl)thiosemicarbazides (5a). Yield 89%, m.p. 230–231 °C;
FT-IR (KBr, cm−1): 3,238, 3,098 (-NH-), 3,012, 1,599, 1,520 (phenyl ring), 1,749 (-C=O amide), 1,680
(C=O), 1,645 (-C=N), 1,470 (-CH2-), 1,206 (-C-N), 1,160 (-C=S); 1H-NMR (DMSO-d6) δ in ppm: 8.09
(s, 1H, -CH=N); 8.03 (d, 1H, -NH-); 7.47, 7.35, 7.19 (dm, 5H, Ar-H); 5.09 (s, 2H, -CH2-); 3.45, 3.09
(s, 6H, 2CH3); 2.00 (s, 2H, -NH-C(=S)-NH-).
1-[2-(1,3-Dimethylxanthin-7-yl)acetyl]-4-(4-chlorophenyl)thiosemicarbazides (5b). Yield 84%, m.p.
240–242 °C; FT-IR (KBr, cm−1): 3,285, 3,115 (-NH-), 3,040, 3,013, 1,591, 1,531 (phenyl ring), 1,693
1
(C=O), 1,645 (-C=N), 1,474 (-CH2), 1,225 (-C-N), 1,188 (-C=S), 825 (C-Cl); H-NMR (DMSO-d6) δ in
ppm: 8.09 (s, 1H, -CH=N); 7.99 (d, 1H, -NH-); 7.51, 7.31 (d, 4H, Ar-H), 5.08 (s, 2H, -CH2-); 3.57,
3.22 (s, 6H, 2CH3); 2.04 (s, 2H, -NH-C(=S)-NH-).
3.2.2. General Procedure for the Preparation of 2-{2-[2-(1,3-Dimethylxanthin-7-yl)acetyl]hydrazono}-
3-(4-R-phenyl) Thiazolidin-4-ones (R = H, Cl) 6a, 6b
Chloroacetyl chloride (39 mmol) was added slowly at room temperature to a solution of
1-[2-(1,3-dimethylxanthin-7-yl)acetyl]-4-(4-R-phenyl)thiosemicarbazides 5a, 5b (13 mmol) in a
mixture of methanol-chloroform (1:1, 250 mL). After 10 h of heating under reflux a yellow clear
solution was obtained. The solvent was removed under reduced pressure and the residue was washed
with cold water, (100 mL), filtered off, dried and recrystallized from ethanol 96%. The progress of the
reaction was monitored by silica gel coated TLC plates.
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(phenyl)thiazolidin-4-one (6a). Yield 52%, m.p.
239–241 °C; FT-IR (KBr, cm−1): 3,310, 3,201, 3,145 (-NH-), 3,048, 1,604, 1,553 (phenyl ring), 1,701
1
(-C=O), 1,642 (C=N), 1,471 (-CH2-), 1,234 (C-N), 690 (C-S); H-NMR (DMSO-d6) δ in ppm: 8.28
(s, 1H, -CH=N); 7.55, 7.33, 6.99 (dm, 5H, Ar-H); 7.01 (s, 1H, -NH-); 5.17 (s, 2H, -CH2-); 3.67 (s, 2H,
S-CH2-); 3.44, 3.23 (s, 6H, 2CH3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorphenyl)thiazolidin-4-one (6b). Yield
50%, m.p. 248–249 °C; FT-IR (KBr, cm−1): 3,312, 3,223, 3,179 (-NH-), 3,051, 1,603, 1,547 (phenyl
1
ring), 1,696 (-C=O), 1,644 (C=N), 1,466 (-CH2-), 1,216 (C-N), 812 (C-Cl), 695 (C-S); H-NMR
1H-NMR (DMSO-d6) δ in ppm: 7.99 (s, 1H, -CH=N ); 7.04 (s, 1H, -NH-); 7.52, 7.45 (d, 4H, Ar-H);
3.78 (s, 2H, S-CH2-); 5.08 (s, 2H, -CH2-); 3.41, 3.17 (s, 6H, 2CH3).
3.2.3. General Procedure for the Preparation of 2-{2-[2-(1,3-Dimethylxanthin-7-yl)acetyl]hydrazono}-
3-(4-R1-phenyl-5-(R2-benzyliden)thiazolidin-4-ones 7a1-7, 7b1-7
The aromatic aldehydes (79 mmol) and piperidine (79 mmol) as catalyst were added slowly to a
solution of 2-{2-[2-(1,3-dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-R1-phenyl)thiazolidine-4-one
6a, 6b (79 mmol) in 1,4-dioxane (100 mL). The mixture of reaction was heated under reflux for 6 h
and then the solvent was removed under reduced pressure. The crude product was precipitated in cold
water, filtered off, washed with cold water and recrystallised from 1,4- dioxane.

Molecules 2013, 18
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2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(benzyliden)thiazolidin-4-one (7a1).
Yield 72%, m.p. 260–262 °C; FT-IR (KBr, cm−1): 3,255, 3,191 (-NH-), 3,082, 1,598, 1,547 (phenyl
ring), 1,696 (-C=O), 1,641 (C=N), 1,376 (-CH2-), 1,229 (C-N), 680 (C-S-C); 1H-NMR (DMSO-d6) δ in
ppm: 8.27 (s, 1H, -CH=N-); 7.64–6.99 (dm, 10H, Ar-H); 7.35 (s, 1H, C=CH-); 6.92 (s, 1H, -NH-);
13
5.79 (s, 2H, -CH2-); 3.44, 3.23 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm: 173.70 (C9), 165.63
(C12), 154.44, 150.95 (C6, C2), 154.05 (C10), 148.41 (C4), 146.60 (C7), 142.94 (C13), 135.27 (C19),
132.19 (C20), 129.08 (C22, C24), 128.68 (C15, C17), 127.99 (C16), 127.52 (C14, C18), 124.77 (C23), 121.97
(C21, C25), 117.37 (C11), 105.77 (C5), 30.65 (C8), 29.47, 27.5 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(4-fluorobenzyliden)thiazolidin-4-
one (7a2). Yield 70%, m.p. 263–265 °C; FT-IR (KBr, cm−1): 3,254, 3,138 (-NH-), 3,082, 1,599, 1,547
(phenyl ring), 1,696 (-C=O), 1,642 (C=N), 1,352 (-CH2-), 1,229 (C-N), 1,153 (C-F), 680 (C-S-C);
1H-NMR (DMSO-d6) δ in ppm: 8.18 (s, 1H, -CH=N-); 7.50–6.96 (dm, 9H, Ar-H); 7.28 (s, 1H, C=CH-);
13
6.89 (s, 1H, -NH-); 5.73 (s, 2H, -CH2-); 3.40, 3.18 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm:
172.29 (C9), 166.36 (C12), 163.12 (C16), 155.11, 154.96 (C6, C2), 151.63 (C10), 148.75 (C4), 146.67
(C7), 143.07 (C13), 133.24 (C19), 129.65 (C20), 129.26 (C22, C24), 128.83 (C14, C18), 124.19 (C23),
122.71 (C21, C25), 118.05 (C11), 117.85 (C15, C17), 106.36 (C5), 31.06 (C8), 30.04, 28.09 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(4-chlorobenzyliden)thiazolidin-4-
one (7a3). Yield 74%, m.p. 235–237 °C; FT-IR (KBr, cm−1): 3,254, 3,140 (-NH-), 3,082, 1,598, 1,547
(phenyl ring), 1,696 (-C=O), 1,641 (C=N), 1,350 (-CH2-), 1,229 (C-N), 804 (C-Cl), 680 (C-S-C);
1H-NMR (DMSO-d6) δ in ppm: 8.24 (s, 1H, -CH=N-); 7.55-6.99 (dm, 9H, Ar-H); 7.34 (s, 1H, C=CH-);
13
6.94 (s, 1H, -NH-); 5.77 (s, 2H, -CH2-); 3.42, 3.21 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm:
174.41 (C9), 166.53 (C12), 154.95, 154.82 (C6, C2), 151.52 (C10), 148.86 (C4), 145.41 (C7), 140.76
(C13), 133.87 (C19), 134.02 (C16), 133.35 (C20), 129.61 (C22, C24), 129.26 (C15, C17), 127.88 (C14, C18),
125.47 (C23), 122.66 (C21, C25), 117.93 (C11), 106.24 (C5), 30.01 (C8), 29.03, 28.04 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(4-bromobenzyliden)thiazolidin-4-
one (7a4). Yield 62%, m.p. 258–260 °C; FT-IR (KBr, cm−1): 3,254, 3,139 (-NH-), 3,081, 1,599, 1,547
(phenyl ring), 1,696 (-C=O), 1,642 (C=N), 1,352 (-CH2-), 1,229 (C-N), 680 (C-S-C), 660 (C-Br);
1H-NMR (DMSO-d6) δ in ppm: 8.27 (s, 1H, -CH=N-); 7.55–6.99 (dm, 9H, Ar-H); 7.38 (s, 1H, C=CH-);
13
6.92 (s, 1H, -NH-); 5.84 (s, 2H, -CH2-); 3.48, 3.26 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm:
172.81 (C9), 165.63 (C12), 155.29, 154.44 (C6, C2), 151.22 (C10), 148.41 (C4), 146.87 (C7), 140.35
(C13), 134.63 (C19), 133.45 (C20), 131.57 (C15, C17), 129.06 (C22, C24), 128.44 (C14, C18), 125.87 (C23),
123.54 (C21, C25), 121.83 (C16), 117.38 (C11), 106.88 (C5), 31.07 (C8), 29.48, 27.51 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(4-hydroxybenzyliden)thiazolidin-
4-one (7a5). Yield 78%, m.p. 243–245 °C; FT-IR (KBr, cm−1): 3,267 (OH), 3,253, 3,138 (-NH-),
3,081, 1,598, 1,547 (phenyl ring), 1,695 (-C=O), 1,641 (C=N), 1,350 (-CH2-), 1229 (C-N), 1152 (C-O),
1
680 (C-S-C); H-NMR (DMSO-d6) δ in ppm: 8.28 (s, 1H, -CH=N-); 7.75–6.99 (dm, 9H, Ar-H), 7.35
(s, 1H, C=CH-); 6.93 (s, 1H, -NH-); 5.79 (s, 2H, -CH2-); 3.32, 3.23 (s, 6H, 2CH3); 13C-NMR (DMSO-d6)
δ in ppm: 173.33 (C9), 165.64 (C12), 158.33 (C16), 154.44, 154.21 (C6, C2), 150.95 (C10), 148.41 (C4),

Molecules 2013, 18
9697
146.47 (C7), 140.36 (C13), 132.06 (C20), 129.05 (C22, C24), 127.99 (C14, C18, C19), 125.27 (C23), 123.37
(C21, C25), 117.37 (C11), 115.79 (C15, C17), 105.77 (C5), 31.44 (C8), 29.47, 27.51 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(4-N-dimethylaminobenzyliden)
thiazolidin-4-one (7a6). Yield 88%, m.p. 255–257 °C; FT-IR (KBr, cm−1): 3,256, 3,139 (-NH-), 1,599,
1,549 (phenyl ring), 1,699 (-C=O), 1,643 (C=N), 1,353 (-CH2-), 1,230 (C-N), 681 (C-S-C);
1H-NMR (DMSO-d6) δ in ppm: 8.19 (s, 1H, -CH=N-); 7.52-6.96 (dm, 9H, Ar-H); 7.35 (s, 1H, C=CH-);
6.92 (s, 1H, -NH-); 5.74 (s, 2H, -CH2-); 3.41, 3.19 (s, 6H, 2CH3); 2.52, 2.49 (s, 6H, 2CH3, N(CH3)2);
13C-NMR (DMSO-d6) δ in ppm: 173.23 (C9), 166.29 (C12), 154.95, 154.65 (C6, C2), 151.59 (C10),
148.79 (C4), 148.46 (C16), 147.26 (C7), 140.63 (C13), 132.07 (C20), 129.63 (C14, C18), 129.05 (C22, C24),
127.26 (C23), 124.64 (C19), 122.85 (C21, C25), 117.98 (C11), 115.62 (C15, C17), 106.33 (C5), 39.87 (2C,
N(CH3)2), 33.45 (C8), 30.02, 28.07 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-phenyl-5-(2-nitrobenzyliden)thiazolidin-4-
one (7a7). Yield 91%, m.p. 247–250 °C; FT-IR (KBr, cm−1): 3,255, 3,139 (-NH-), 3,082, 1,598, 1,547
(phenyl ring), 1,696 (-C=O), 1,641 (C=N), 1,499 (NO2), 1,350 (-CH2-), 1,229 (C-N), 680 (C-S-C);
1H-NMR (DMSO-d6) δ in ppm: 8.22 (s, 1H, -CH=N-); 8.21–6.99 (dm, 9H, Ar-H), 7.34 (s, 1H, C=CH-);
13
6.91 (s, 1H, -NH-); 5.76 (s, 2H, -CH2-); 3.42, 3.21 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm:
170.61 (C9), 166.22 (C12), 155.08, 154.85 (C6, C2), 151.54 (C10), 148.84 (C4), 145.40 (C18), 144.45 (C7),
140.99 (C13), 133.91 (C20), 131.53 (C15), 130.12 (C19), 129.61 (C22, C24), 128.41 (C16), 127.63 (C14),
126.37 (C23), 122.72 (C21, C25), 121.11 (C17), 117.94 (C11), 106.31 (C5), 31.11 (C8), 30.01, 28.05 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(benzyliden)thiazolidin-
4-one (7b1). Yield 68%, m.p. 265–267 °C; FT-IR (KBr, cm−1): 3,297, 3,118 (-NH-), 2,994, 1,591,
1,545 (phenyl ring), 1,699 (-C=O), 1,639 (C=N), 1,348 (-CH2-), 1,214 (C-N), 777 (C-Cl), 710 (C-S-C);
1H-NMR (DMSO-d6) δ in ppm: 8.06 (s, 1H, -CH=N-); 7.51-7.31 (dm, 9H, Ar-H); 7.24 (s, 1H, C=CH-);
13
7.01 (s, 1H, -NH-); 5.17 (s, 2H, -CH2-); 3.32, 3.20 (s, 6H, 2CH3); C-NMR (DMSO-d6) δ in ppm:
173.73 (C9), 167.63 (C12), 156.18 (C10), 154.39, 150.97 (C6, C2), 148.24 (C4), 146.52 (C7), 142.81
(C13), 135.18 (C19), 132.41 (C20), 128.57 (C22, C24), 128.02 (C15, C17), 127.03 (C16), 126.87 (C14, C18),
120.84 (C23), 119.76 (C21, C25), 117.08 (C11), 106.29 (C5), 29.45 (C8), 29.01, 27.42, (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(4-fluorobenzyliden)
thiazolidin-4-one (7b2). Yield 70%, m.p. 255–257 °C; FT-IR (KBr, cm−1): 3,229, 3,186 (-NH-), 3,000,
1,605, 1,545 (phenyl ring), 1,694 (-C=O), 1,647 (C=N), 1,344 (-CH2-), 1,225 (C-N), 1,149 (C-F), 759
1
(C-Cl), 747 (C-S-C); H-NMR (DMSO-d6) δ in ppm: 8.28 (s, 1H, -CH=N-); 7.62-7.36 (d, 8H,
Ar-H); 7.36 (s, 1H, C=CH-); 7.02 (s, 1H, -NH-); 5.80 (s, 2H, -CH2-); 3.44, 3.23 (s, 6H, 2CH3); 13C-NMR
(DMSO-d6) δ in ppm: 173.38 (C9), 165.31 (C12), 163.83 (C16), 155.93 (C10), 154.44, 150.95 (C6, C2),
148.42 (C4), 145.43 (C7), 142.84 (C13), 132.68 (C20), 130.64 (C19), 128.87 (C22, C24), 125.35 (C14, C18),
121.32 (C21, C25), 120.41 (C23), 118.88 (C11), 116.16 (C15, C17), 105.76 (C5), 30.43 (C8), 29.47,
27.51 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(4-chlorobenzyliden)
thiazolidin-4-one (7b3). Yield 59%, m.p. 250–252 °C; FT-IR (KBr, cm−1): 3,296, 3,118 (-NH-), 2,994,

Molecules 2013, 18
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1,591, 1,547 (phenyl ring), 1,699 (-C=O), 1,641 (C=N), 1,348 (-CH2-), 1,214 (C-N), 777 (C-Cl), 712
(C-S-C); 1H-NMR (DMSO-d6) δ in ppm: 8.28 (s, 1H, -CH=N-); 7.63-7.36 (d, 8H, Ar-H); 7.35 (s, 1H,
C=CH-); 7.01 (s, 1H, -NH-); 5.84 (s, 2H, -CH2-); 3.48, 3.29 (s, 6H, 2CH3); 13C-NMR (DMSO-d6) δ in
ppm: 173.62 (C9), 165.31 (C12), 154.66 (C10), 154.32, 150.87 (C6, C2), 151.78 (C4), 148.41 (C7), 142.07
(C13), 139.23 (C19), 133.35 (C16), 131.36 (C20), 129.19 (C15, C17), 128.84 (C14, C18), 125.36 (C22, C24),
123.99 (C21, C25), 121.25 (C23), 118.87 (C11), 105.76 (C5), 30.32 (C8), 29.44, 27.48 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(4-bromobenzyliden)
thiazolidin-4-one (7b4). Yield 69%, m.p. 245–247 °C; FT-IR (KBr, cm−1): 3,297, 3,117 (-NH-), 2,994,
1,589, 1,545 (phenyl ring), 1,699 (-C=O), 1,635 (C=N), 1,348 (-CH2-), 1,213 (C-N), 775 (C-Cl), 710
(C-S-C), 635 (C-Br); 1H-NMR (DMSO-d6) δ in ppm: 8.04 (s, 1H, -CH=N-); 7.49–7.33 (d, 8H, Ar-H), 7.24
(s, 1H, C=CH-); 7.03 (s, 1H, -NH-); 5.17 (s, 2H, -CH2-); 3.30, 3.20 (s, 6H, 2CH3); 13C-NMR (DMSO-d6)
δ in ppm: 173.32 (C9), 167.61 (C12), 163.57 (C16), 154.58 (C10), 154.39, 154.39 (C6, C2), 150.97 (C4),
147.95 (C7), 142.04 (C13), 131.89 (C20), 130.53 (C19), 128.58 (C14, C18), 128.02 (C22, C24), 122.87 (C21,
C25), 120.84 (C23), 119.77 (C11), 117.32 (C15, C17), 106.29 (C5), 33.78 (C8), 29.45, 27.42 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(4-hydroxybenzyliden)
thiazolidin-4-one (7b5). Yield 58%, m.p. 248–250 °C; FT-IR (KBr, cm−1): 3,318 (OH), 3,229,
3,187 (-NH-), 3,000, 1,607, 1,547 (phenyl ring), 1,695 (-C=O), 1,650 (C=N), 1,344 (-CH2-), 1,227 (C-N),
1,150 (C-O), 799 (C-Cl), 748 (C-S-C); 1H-NMR (DMSO-d6) δ in ppm: 8.28 (s, 1H,-CH=N-); 7.60-7.34
(d, 8H, Ar-H); 7.28 (s, 1H, C=CH-); 7.01 (s, 1H, -NH-); 5.77 (s, 2H, -CH2-); 3.42, 3.21 (s, 6H, 2CH3);
13C-NMR (DMSO-d6) δ in ppm: 173.33 (C9), 165.33 (C12), 157.43 (C16), 154.80 (C10), 151.03 (C4),
154.43, 150.26, (C6, C2), 148.36 (C7), 142.72 (C13), 131.82 (C20), 128.87 (C14, C18), 127.03 (C19),
125.50 (C22, C24), 124.76 (C21, C25), 123.53 (C23), 118.67 (C11), 117.08 (C15, C17), 105.79 (C5), 30.61
(C8), 29.49, 27.52 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(4-N-dimethylamino-
benzyliden)thiazolidin-4-one (7b6). Yield 65%, m.p. 258–260 °C; FT-IR (KBr, cm−1): 3,294, 3,118
(-NH-), 2,994, 1,590, 1,548 (phenyl ring), 1,699 (-C=O), 1,638 (C=N), 1,348 (-CH2-), 1,214 (C-N),
776 (C-Cl), 711 (C-S-C); 1H-NMR (DMSO-d6) δ in ppm: 8.06 (s, 1H, -CH=N-); 7.70-7.31 (d, 8H, Ar-H),
7.16 (s, 1H, C=CH-); 7.02 (s, 1H, -NH-); 5.17 (s, 2H, -CH2-); 3.32, 3.20 (s, 6H, 2CH3); 3.05, 2.97 (s,
6H, 2CH3, N(CH3)2); 13C-NMR (DMSO-d6) δ in ppm: 173.54 (C9), 167.60 (C12), 154.39 (C10), 150.97
(C4), 155.28, 150.33, (C6, C2), 150.04 (C16), 147.95 (C7), 138.51 (C13), 131.41 (C20), 128.71 (C14, C18),
125.45 (C22, C24), 124.92 (C19), 124.12 (C21, C25), 123.11 (C23), 119.78 (C11), 113.88 (C15, C17), 105.94
(C5), 40.32 (2C, N(CH3)2), 30.56 (C8), 29.45, 27.43 (C1, C3).
2-{2-[2-(1,3-Dimethylxanthin-7-yl)acethyl]hydrazono}-3-(4-chlorophenyl)-5-(2-nitrobenzyliden)
thiazolidin-4-one (7b7). Yield 75%, m.p. 235–237 °C; FT-IR (KBr, cm−1): 3,296, 3,118 (-NH-), 2,994,
1,591, 1,547 (phenyl ring), 1,699 (-C=O), 1,641 (C=N), 1,499 (NO2), 1,348 (-CH2-), 1,214 (C-N), 777
(C-Cl), 712 (C-S-C); 1H-NMR (DMSO-d6) δ in ppm: 8.28 (s, 1H, -CH=N-); 8.06-7.38 (d, 8H, Ar-H); 7.18
(s, 1H, C=CH-); 7.02 (s, 1H, -NH-); 5.80 (s, 2H, -CH2-); 3.44, 3.23 (s, 6H, 2CH3); 13C-NMR (DMSO-d6)
δ in ppm: 173.96 (C9), 165.27 (C12), 154.72 (C10), 154.44, 148.42 (C6, C2), 150.95 (C4), 147.82 (C7),
147.42 (C18), 139.63 (C13), 137.86 (C15), 131.74 (C20), 129.64 (C19), 129.38 (C16), 128.82 (C22, C24),

Molecules 2013, 18
9699
127.51 (C14), 122.97 (C21, C25), 120.83 (C23), 120.6 (C17), 119.03 (C11), 105.76 (C5), 33.64 (C8), 29.46,
27.49 (C1, C3).
3.3. Biological Evaluation
3.3.1. Antioxidant Assays
The antioxidant activity was estimated using in vitro tests: ferric reducing power, total antioxidant
capacity, DPPH radical scavenging assay and ABTS radical scavenging ability.
3.3.2. Ferric Reducing Power
The ferric reducing power of the compounds was quantified by the method described by [30] with
slight modifications. phosphate buffer (1 mL, 0.2 M, pH 6.6) and potassium ferricyanide (1 mL, 1%
w/v) were added in a test tube to the sample (50 µL, 100 µL, 200 µL, 300 µL) obtained from a stock
solution (5 mg/mL in DMSO). The mixture was incubated at 50 °C for 20 min in a water bath and then
the reaction was stopped by adding trichloroacetic acid (1 mL, 10% w/v). After centrifugation at
4,500 rpm for 15 min, the upper layer (1 mL) was collected and diluted with deionised water (1 mL)
and then ferric chloride (0.2 mL, 0.1% w/v) was added. The mixture was left at room temperature for
10 min and then the absorbance was measured at 700 nm against a blank solution (DMSO mixed with
the reagents). All the tests were performed in triplicate. For each sample the effective concentration
(EC50) was calculated and the ascorbic acid (AA) was used as positive control.
3.3.3. Total Antioxidant Activity
The total antioxidant activity of tested compounds was evaluated using the phosphomolybdenum
method according to the procedure of [31] with minor modifications. The method is based on the
reduction of Mo (VI) to Mo (V) by the tested compounds followed by the formation of a green
phosphate/Mo(V) complex at acid pH. There were used different sample volumes (10, 15, 20, 30, 40,
60 µL) obtained from a stock solution (5 mg/mL in DMSO). The samples were mixed with the reagent
solution (2 mL, 28 mM sodium phosphate; 4 mM ammonium molybdate; 0.6 M sulphuric acid),
incubated at 95 °C for 90 min, cooled at room temperature and then centrifuged at 4,500 rpm for 15 min.
The upper layer was collected and the absorbance was measured at 695 nm against a blank solution
(DMSO mixed with the reagents). For each sample the effective concentration (EC50) was calculated
and the ascorbic acid (AA) was used as positive control. All tests were performed in triplicate.
3.3.4. DPPH Radical Scavenging Assay
The radical scavenging activity of the tested compounds towards the radical 1,1-diphenyl-2-
picrylhydrazyl (DPPH) was measured as described by [32] with slight modifications. Various
concentrations of the tested compounds (50 µL, 75 µL, 150 µL, 200 µL) from a stock solution
(5 mg/mL in DMSO) were mixed with DMSO to obtain 200 μL sample, and then DPPH in methanol
(2,500 μL, 0.1 mM) was added. The mixture was left for 1 hour at room temperature, in the dark, and
after that the absorbance was measured at 517 nm against a blank solution. The radical scavenging capacity

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was calculated according to the following equation: Scavenging activity % = [(Ao − At)/A0] × 100.
A0 is the absorbance of DPPH methanol solution. At is the absorbance of the sample after 1 h. For each
sample the effective concentration (EC50) was calculated and the ascorbic acid (AA) was used as
positive control. All tests were performed in triplicate.
3.3.5. ABTS Radical Scavenging Assay
The ABTS+ radicals were activated by reacting of ABTS (7 mM) with ammonium persulphate
(2.45 mM). The mixture was left at room temperature for 16 hours in the dark as described by [33].
The intensely coloured ABTS+ radical cation solution was diluted with ethanol to obtain an absorbance
value of 0.7 ± 0.02 at 734 nm. Different sample volumes (5 µL, 10 µL, 20 µL, 25 µL) from a stock
solution (5 mg/mL in DMSO) were mixed with DMSO to 25 µL and then ABTS solution (1975 µL)
was added. After 6 min the absorbance was measured and the radical scavenging capacity was
calculated according to the following equation: Scavenging activity % = [(Ao − At)/A0] × 100. A0 is
the absorbance before adding the sample. At is the absorbance after 6 min of reaction. For each sample
the effective concentration (EC50) was calculated and the ascorbic acid (AA) was used as positive
control. All tests were performed in triplicate.
3.3.6. Statistical Analysis
All antioxidant assays were carried out in triplicate. Data were analyzed by an analysis of variance
(ANOVA) (p < 0.05) and were expressed as means ± SD. The EC50 values were calculated by linear
interpolation between the values registered above and below 50% activity.
4. Conclusions
In this study new heterocyclic compounds that combine a xanthine structure with a thiazolidine one
have been synthesized. The structures of all new compounds were proven using spectral methods.
The compounds were evaluated for their antioxidant activity using in vitro assays: ferric
reducing power, total antioxidant activity, ABTS and DPPH radical scavenging ability. The all
benzylidenethiazolidin-4-one derivatives 7a1-7, 7b1-7 showed improved antioxidant effects in reference
to the corresponding thiazolidin-4-ones 6a, 6b. The encouraging preliminary results support the
antioxidant potential of the synthesized compounds and their possible applications in several diseases
mediated by reactive oxygen species (ROS), including diabetes mellitus, and motivate our next
research focused on their potential antidiabetic effects.
Acknowledgments
This work was supported by the project “Doctoral Scholarships for increasing competitiveness in
the medical and pharmaceutical field” (POSDRU/88/1.5/S/58965). The authors thank Polymer
Research Group, Department of Organic Chemistry, University of Ghent, Belgium for their help to
perform IR and NMR analyses.
Conflict of Interest

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9701
The authors declared no conflict of interest.
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Sample Availability: Samples of the compounds 7a1–7, 7b1–7 are available from the authors.
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