The cysteine releasing pattern of some antioxidant thiazolidine-4-carboxylic acids

Article abstract of DOI:10.1016/j.ejmech.2016.03.019

Oxidative stress that corresponds to a significant increase in free radical concentration in cells can cause considerable damage to crucial biological macromolecules if not prevented by cellular defense mechanisms. The low-molecular-weight thiol glutathio

Full text of DOI:10.1016/j.ejmech.2016.03.019

Accepted Manuscript
The Cysteine Releasing Pattern of Some Antioxidant Thiazolidine-4-carboxylic acids
F. Esra Önen Bayram, Hande Sipahi, Ebru Türköz Acar, Reyhan Kahveci Ulugöl,
Kerem Buran, Hülya Akgün
PII:
S0223-5234(16)30193-3
10.1016/j.ejmech.2016.03.019
EJMECH 8444
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2015
Revised Date: 26 February 2016
Accepted Date: 7 March 2016
Please cite this article as: F.E. Önen Bayram, H. Sipahi, E.T. Acar, R.K. Ulugöl, K. Buran, H. Akgün, The
Cysteine Releasing Pattern of Some Antioxidant Thiazolidine-4-carboxylic acids, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.019.
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ACCEPTED MANUSCRIPT
Antioxidant activity
Cysteine release
or NOT ?

ACCEPTED MANUSCRIPT
The Cysteine Releasing Pattern of Some Antioxidant
Thiazolidine-4-carboxylic acids.
F. Esra Önen Bayram*a, Hande Sipahib, Ebru Türköz Acarc, Reyhan Kahveci Ulugöla, Kerem
Burana, Hülya Akgüna.
a) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey.
b) Department of Toxicology, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey.
c) Department of Analytical Chemistry, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey.
Corresponding author. Tel.: +90-216-578-0558; Fax: +90-216-578-1622; e-mail:
filizesraonen@gmail.com
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ABSTRACT.
Oxidative stress that corresponds to a significant increase in free radical concentration in cells can cause
considerable damage to crucial biological macromolecules if not prevented by cellular defense mechanisms. The
low-molecular-weight thiol glutathione (GSH) constitutes one of the main intracellular antioxidants. It is
synthesized via cysteine, an amino acid found only in limited amounts in cells because of its neurotoxicity. Thus,
to ensure an efficient GSH synthesis in case of an oxidative stress, cysteine should be provided extracellularly.
Yet, given its nucleophilic properties and its rapid conversion into cystine, its corresponding disulfide, cysteine
presents some toxicity and therefore is usually supplemented in a prodrug approach. Here, some thiazolidine-4-
carboxylic acids were synthesized and evaluated for their antioxidant properties via the DDPH and CUPRAC
assays. Then, the cysteine releasing capacity of the obtained compounds was investigated in aqueous and organic
medium in order to correlate the relevant antioxidant properties of the molecules with their cysteine releasing
pattern. As a result, the structures’ antioxidative properties were not only attributed to cysteine release but also to
the thiazolidine cycle itself.
KEYWORDS: oxidative stress, thiazolidine, cysteine, prodrug, heterocycle cleavage.
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1. Introduction
Continuous exposure of a cell to high levels of reactive oxygen or nitrogen species causes an imbalance in redox
homeostasis [1, 2]. Such a state is better known as an oxidative stress, that can cause various diseases such as
atherosclerosis [3-5], cancer [6, 7] or neurodegenerative disorders [8-10].
Thiols are compounds that present a sulfhydryl group on their structures and are largely described for their
antioxidant properties. Conversion into disulfides and scavenging radicals constitute the main mechanisms
through which thiols act as reducing agents [11-16]. Glutathione, the most abundant thiol-containing small
molecule of the cell helps to fight against oxidative stress when converted into its corresponding disulfide in the
presence of reactive species [17-19]. Glutathione biosynthesis requires cysteine [20]. Thus, this precursor’s
intracellular levels should be increased in case of an oxidative stress [21].
As free L-cysteine supplementation has been related to toxicity, [22-24] pro-drugs capable of releasing the
sulfur-containing amino acid directly or after enzymatic cleavage once administrated have been developed [25-
35].
Thiazolidines, five membered heterocycles resulting from the condensation of carbonyl compounds with
cysteine are already described for their cysteine releasing capacity and some 2-alkyl and aryl substituted
thiazolidine carboxylic acids were evaluated for their antioxidant properties [29-32, 34, 36]. In this study we
aimed to generate a series of thiazoldine-4-carboxylic acids as cysteine prodrugs. A series of substituted
benzaldehydes comprising diverse electron-withdrawing and electron attracting groups were chosen, so that the
impact of substitution on the cysteine releasing pattern and the antioxidant activity could be further discussed.
After being synthesized and characterized, the molecules’ antioxidative properties were evaluated using radical
scavenging methods.
2. Results
2.1 Chemistry
Thiazolidine carboxylic acid derivatives were synthesized with good yields by condensing L-cysteine with a
series of substituted benzaldehyde derivatives (Table 1). The typical cyclization reaction was carried out in basic
conditions in a water/ethanol mixture (50:50, v:v) as suggested in the literature for the preparation of these
compounds [5, 31, 37, 38].
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R
Yield (%) 2R,4R/2S,4R ratio
1
2
Ph-
89
65
70/30
40/60
o-CH3O-Ph
3
4
5
6
7
o-CH3-Ph-
o-CN-Ph-
o-Br-Ph-
m-Cl-Ph-
p-Cl-Ph-
75
62
86
74
75
60/40
95/5
70/30
90/10
95/5
8
9
p-CH3O-Ph-
p-CH3-Ph-
72
85
50/50
60/40
10
11
12
13
p-NO2-Ph-
80
75
80
84
5/95
40/60
40/60
40/60
2,6-difluoro-Ph-
2,3-difluoro-Ph
2,3-dichloro-Ph-
Table 1. Yields and diastereomeric ratios of compounds 1-13
1
The structures were analyzed by FT-IR, H NMR, 13C NMR and elemental analysis. The formation of the
thiazolidine heterocycle was confirmed through the typical signals corresponding to the second position of the
ring. While the hydrogen on C-2 gave a distinctive singlet around 5.5 ppm, the signal of the carbon atom
appeared as a peak at around 70 ppm.
The ring closure reaction results from two successive nucleophilic attacks of the aldehyde and leads to the
generation of a new chiral center in an uncontrolled manner [39, 40]. Thus, thiazolidine derivatives are obtained
as diastereomeric mixtures (Figure 1).
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Figure 1. Synthesis of substituted 2-phenyl-4-carboxylic acid thiazolidine derivatives Reagents: benzaldehyde
derivatives in C2H5OH/H2O (1/1, v/v)
Although clearly distinguishable on 1H NMR via the proton on the C-2 carbon of the heterocycle (Figure 2), the
isolation of the 2R, 4R and 2S, 4R isomers was not achieved since in fact, there is a rapid interconversion of one
diastereomer into the other through the corresponding Schiff base in physiological or basic pH values [41].
1
Figure. 2 H NMR of compound 9 diastereomeric ratios are determined via the C2 protons. The assignment to
2R,4R and 2S,4R diastereomers are given on the basis of NOE experiments detailed in the literature.
Interestingly, nevertheless, the diastereomeric mixture ratios varied according to the substituents of the aromatic
cycle (Table 1), the equilibrium being clearly in favor of one of the isomers in the case of 4, 6, 7 and 10. While
the ortho-cyano, meta- and para-chloro benzaldehydes definitely orient the reaction to the formation of the 2R,
4R isomer (distinction made through NOE data given in the literature [42]), the p-nitrobenzaldehyde led
preferably to the 2S, 4R thiazolidine (7) molecule. The exact reason for such displacement of the equilibrium
remains unknown since we could not precisely correlate the diastereomeric mixture ratios neither with the
electron-attracting or withdrawing properties of the substituent nor with its size or position.
2.2 Biology
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The synthesized thiazolidine compounds (1-13) were evaluated for their antioxidant properties using the classical
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and by determining the cupric reducing
antioxidant capacity (CUPRAC) of the compounds.
2.2.1 The DPPH assay
The DPPH assay, a very commonly used methodology for analyzing the antioxidant activity of molecules,
consists of the determination of the compound concentration that is capable of scavenging 50% of the DPPH
radicals in solution by monitoring the decrease of absorbance at 517 nm that corresponds to the maximum of
absorbance wavelength of the stable free DPPH radicals. To ensure a rapid and simultaneous screening of the
scavenging capacity of the synthesized thiazolidines, the existing procedure[43, 44] was modified and carried
out with 96-well plates.
Aqueous DMSO was chosen as a solvent as samples are not soluble in water at physiological pH. The DMSO
concentration was increased up to 5% (v/v) to dissolve a maximum number of thiazolidine carboxylic acids. Yet,
even at this elevated DMSO content, six samples (compound 4 and from 6 to 10) did not dissolve and could not
be evaluated. The DPPH radical scavenging ability of the analyzed structures was determined as indicated in the
experimental part and results are summarized in Table 2. The concentrations required for scavenging 50% of the
DPPH radicals were calculated and indicated as IC50 values expressed in micromolars as it is commonly done for
DPPH scavenging assays in the literature. The obtained values were compared to the antioxidant capacity of
butylated hydroxytoluene (BHT), the classical reference used for DPPH assays and cysteine since the
synthesized structures are meant to be cysteine prodrugs.
BHT
cysteine
1
2
3
4
5
11
12
13
IC50 in DMSO (µM)
not soluble 18.4 ±0.1 45.2 ±0.3 29.5 ±0.5 39.0 ±0.3 >1mM 37.7 ±0.1 38.3 ±0.4 46.8 ±0.8 61.7 ±2.3
IC50 in methanol (µM) 517 ±25 19.2±0.5 844.2±2.2 342.7±8.4 46.1±0.9 >1mM 39.1±1.3 343.4±12 >1mM 46.2±1.4
Table 2. DPPH scavenging capacity of compounds 1-13: IC50 values determined with samples dissolved either
in aqueous DMSO (%5, v/v) or in methanol
Cysteine, with an IC50 value of 18.4±0.1 µM gave the highest antioxidant capacity, an expected behavior that can
be attributed to the thiol function. The thiazolidine compounds were also found to have a greater antioxidant
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capacity when compared to BHT. The promising antioxidant property that thiazolidine molecules exhibited
strongly suggested a ring opening reaction in aqueous medium that leads to the antioxidant cysteine molecule.
To check that the antioxidant capacity is due to the ring opening reaction that easily occurs in aqueous medium,
the DPPH assay was also carried out by dissolving the samples in methanol. The significant increase of the IC50
values observed for compounds 1, 2, 11 and 12 while cysteines’s activity remained constant supported the
hypothesis that the high antioxidant activity observed in aqueous DMSO can be attributed to the release of
cysteine.
To confirm this hypothesis an HPLC analysis was also carried out with the tested compounds. The analysis was
performed either with samples being dissolved in aqueous DMSO or methanol before injection. Given that the
DPPH assay requires an incubation time of 50 minutes, HPLC analyses of the samples were also repeated 50
minutes after dissolution. Since the ring opening reaction corresponds to the reverse of the cyclization reaction,
cysteine release was controlled by monitoring the peak corresponding to the aldehyde obtained after ring
cleavage. Results are summarized in Table 3.
1
2
3
5
11
12
13
0 ' 20 ' 50 '
0 ' 20 ' 50 '
0 ' 20 ' 50 '
0 ' 20 ' 50 '
0 ' 20 ' 50 '
0 ' 20 ' 50 '
0 ' 20 ' 50 '
% Thiazolidine 76.3 27.8 13.8 82.8 43.9 26.8 6.8 5.9 5.6 19.0 16.2 14.3 53.8 39.2 38.2 83.7 50.0 28.7 17.8 16.6 15.7
5% aq DMSO
PBS
% Aldehyde
16.5 69.3 84.6 17.2 56.1 73.2 92.3 93.2 93.5 81.0 83.8 85.6 46.1 59.8 60.7 10.6 46.1 67.4 75.3 .76.5 77.4
% Thiazolidine 78.4 27.8 13,8 82.5 34.0 21.0 15.2 11.3 8.9 11.6 16.3 18.5 79.4 61.1 46.9 92.7 69.3 50.1 15.3 17.4 17.9
% Aldehyde 21.4 71.9 84.3 17.3 66.0 78.9 84.8 88.6 90.9 88.2 83.5 81.0 6.4 28.4 45.7 7.1 30.7 48.9 80.6 81.7 82.1
% Thiazolidine 85.0 62.4 49.9 92.9 78.9 67.6 12.9 11.3 10.1 14.1 11.5 9.5 96.2 72.3 54.8 87.3 64.6 43.5 21.7 17.5 14.2
% Aldehyde 14.7 37.2 49.2 6.5 20.7 30.2 86.4 87.4 88.8 84.3 86.2 88.1 3.1 27.0 44.4 9.0 33.1 54.1 76.7 80.4 83.5
pH=2.0
MeOH
% Thiazolidine 96.1 95.7 95.4 100 98.6 97.3 4.0 4.1 4.2 70.5 74 75.6 100 99.4 98.2 100 100 98.8 15.5 25.2 38.2
% Aldehyde 3.8 4.3 4.6 1.4 2.2 95.9 95.8 95.7 22.2 18.2 15.9 0.5 1.3 0.5 83.8 74.1 59.9
0
0
0
0
Table 3. HPLC analyses of thiazolidine compounds after dissolution in 5% aqueous DMSO, PBS, phosphoric
acid buffer or methanol at time zero, 20 minutes after dissolution and 50 minutes after dissolution
Chromatograms obtained for the samples were consistent with the IC50 values obtained with the DPPH assay.
First of all, in aqueous DMSO, all of the tested thiazolidines were shown to be progressively converted into
cysteine and benzaldehyde since HPLC analyses carried out 50 minutes after dissolution demonstrated a
remarkable increase in the benzaldehyde ratio when compared to the one observed at time zero. These results
strongly support that the significant antioxidant activity observed for all compounds with the DPPH assay in this
solvent is related to the release of cysteine in the medium.
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Concerning the results obtained when samples were dissolved in methanol, they indicate a ring opening for
compounds 3, 5 and 13. For the other tested compounds (1, 2, 11 and 12) benzaldehyde ratios remained
insignificant even for injections realized 50 minutes after compound dissolution, indicating the absence of
cleavage for the heterocycles in these conditions. The noticeable increase in the IC50 values obtained for
compounds 1, 2, 11 and 12 can thus be explained by the lack of immediate cysteine release in these conditions.
The reason of the differences in cysteine releasing ability of the synthesized thiazolidines observed in methanol
remain unexplained, since no obvious relationship between the structures and the ring cleavage phenomenon
could be found (Figure 3).
Figure. 3 (a) Structures capable of releasing cysteine in methanol (MeOH) (b) Structures that are stable and do
not open to release cysteine in MeOH
For instance, while the o,m-difluoro substituted derivative 12 was shown to be stable in methanol even 50
minutes after dissolution, its close analogue 13 (o,m-dichloro substituted thiazolidine) exhibited aldehyde peaks
very quickly.
As a result, it is possible to assume that the antioxidant activity determined via the DPPH assay is closely related
to the cysteine releasing capacity of the thiazolidine compounds.
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We also evaluated the cysteine releasing pattern of these compounds in physiological pH (using the PBS
phosphate buffer) and in acidic medium (0.1M phosphoric acid, pH=2.0) in order to analyze the stability of the
structures in conditions mimicking the stomach environment. For both media, the cysteine releasing pattern was
shown to be similar to the one obtained for the studies carried out in water even if a slower ring opening was
observed in acidic medium (Table 3).
These results suggested thus, that cysteine should be released when the thiazolidine molecule would be injected
in the body.
2.2.2The CUPRAC assay
The antioxidant capacities of the synthesized thiazolidines were also evaluated using the CUPRAC assay. This
method developed by Apak et al. consists of monitoring the Cu(I)-neocurpine complex formation at 450 nm, a
complex that results from the reduction of the Cu(II)-neocuprine complex in an ammonium acetate buffer in the
presence of a reducing agent. The antioxidant capacity is determined using the Trolox Equivalent Antioxidant
Capacity (TEAC) coefficient, a coefficient that corresponds to the ratio of the molar extinction coefficients of the
sample and trolox (TEAC=εsample/εTrolox) [45].
Samples were prepared in aqueous 5% DMSO and methanol and TEAC coefficient were determined for both
conditions (Table 4).
cysteine
TEACMeOH 0.63±0.09 1.54 ±0.12 0.96 ±0.06 0.57±0.05 0.80±0.07 1.50 ±0.07 1.52 ±0.01 1.22 ±0.06 0.74 ±0.04 0.83 ±0.09 0.83 ±0.06 0.93 ±0.05
TEACDMSO
1
2
3
5
6
7
9
10
11
12
13
0.49±0.03 0.99±0.05 0.79±0.04 0.75±0.04 1.00±0.05
ND
ND
ND
ND
1.05±0.1 0.84±0.05 0.99±0.05
Table 4. TEAC coefficients of samples. Samples were dissolved either in aqueous DMSO (%5, v/v) or in
methanol
The TEACMeOH of compound 4 and 8 and the TEACDMSO of compounds 4, 6-11 could not be determined due to
solubility issues. In both aqueous phase and methanol, the antioxidant capacities of all samples were found to be
greater than cysteine, a finding that supports that the antioxidant capacity of thiazolidines should not be related
only to cysteine release and that the thiazolidine moiety itself could reduce Cu (II). Moreover in methanol four
samples (1, 6, 7, 9) were found to have TEACMeOH coefficient values greater than one, indicating an antioxidant
capacity even better than trolox, a property that can again be attributed to the presence of the thiazolidine ring.
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3. Discussion
Thiazolidine-4-carboxylic acids have already been described in the literature for their antioxidant activity[29-32,
34, 36] but published studies did not present any data concerning the cysteine releasing capacity of these
molecules. In this work we aimed to develop some thiazolidine-4-carboxylic acids from a series of substituted
benzaldehydes and analyzed their antioxidant capacities via two methods, the DPPH and CUPRAC assays.
These assays were performed in both aqueous and organic media to analyze possible differences in cysteine
releasing pattern. The results indicated that in aqueous medium the thiazolidine heterocycle was converted into
its corresponding benzaldehyde and cysteine for at least 61% within 50 minutes, the required incubation time for
the DPPH assay suggesting strongly that the observed antioxidant activity is attributable to cysteine. This
hypothesis can be confirmed with the results obtained when derivatives are dissolved in methanol since then the
cysteine releasing capacities of the compounds are shown to be modified along with their antioxidant activity.
Our results demonstrated that cysteine releasing capacity depends on the substitution of the phenyl ring of 2-
phenyl-thiazolidine-4-carboxylic acid derivatives but that this ability is not a feature of the position of the
substitution since both the stable and unstable structures present substituents at their ortho- position. Also, the
stability of the heterocycle cannot be related to the electron-withdrawing or electron-donating properties of the
substituents since for instance 3 and 5 that are o-methyl (an electron-donating group) and o-bromo (an electron-
withdrawing group) derivatives respectively, are both shown to be unstable. Similarly, the o-methoxy 2(an
electron-donating group) and o,m-difluoro 12 (electron-withdrawing groups) tend to be stable and not release
cysteine when dissolved in methanol. Since the cysteine releasing capacity of thiazolidine rings is a key feature
for the development of cysteine prodrugs, we believe, given our results, that the nature of the aldehyde from
which the cycle derives should be considered when the prodrugs are designed since its structure can affect the
cysteine release pattern of the resulting thiazolidine.
Another finding of our antioxidant analyses results indicate that the antioxidant activity of thiazolidine rings
cannot be only attributed to their cysteine releasing property since our CUPRAC assay results demonstrated that
antioxidant activity can be observed with stable thiazolidine structures (compound 1, 6, 7 and 9). These data
suggest that the thiazolidine ring can be used in antioxidant drug design without being a cysteine prodrug.
4. Conclusion
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To conclude, in this work, 2-phenyl thiazolidine carboxylic acid derivatives were synthesized by condensing L-
cysteine with a series of mono or disubstituted benzaldehyde derivatives. DPPH scavenging and CUPRAC
assays were performed and the compounds were found to be capable of reducing the DPPH radical and Cu (II).
Also, the antioxidant capacity of the samples was shown to be related not only to the L-cysteine release but also
to the reducing capacity of the thiazolidine ring itself.
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5. Experimental
General procedure for the synthesis of compounds 1 to 13:
L-cysteine hydrochloride monohydrate (1.0 eq) and sodium hydroxide (1.0 eq) were dissolved in water. Then
aldehyde (1.0 eq) in ethanol was added and the mixture was stirred at room temperature (RT) for three hours.
Solid crystals of the expected product were filtered and washed with dichloromethane. The crystals were dried
under vacuo.
(2RS, 4R)-2-phenyl-1,3-thiazolidine-4-carboxylic acid (1)
Yield: %89. m.p.: 163 ᵒC. FT-IR (KBr), cm-1: 3436, 1575, 1435 1H-NMR (DMSO-d6) δ: 3.08 (t, J=8.8 Hz, 0.3H,
NCHCH2S); 3.14 (dd, J1=4.8 Hz, J2=10.4 Hz, 0.7H, NCHCH2S); 3.30 (dd, J1=7.6 Hz, J2=10.8 Hz, 0.7H,
NCHCH2S); 3.38 (dd, J1=7.6 Hz, J2=10.4 Hz, 0.3H, NCHCH2S); 3.90 (dd, J1=7.6 Hz, J2=8.8 Hz, 0.3H,
NCHCH2S), 4.23 (dd, J1=4.8 Hz, J2=7.2 Hz, 0.7H, NCHCH2S); 5.50 (s, 0.3H, NCHS); 5.67 (s, 0.7H, NCHS);
7.24 – 7.54 (m, 5H, Harom) 13C NMR (DMSO-d6) δ: 37.9, 38.3; 64.8, 65.3; 71.0, 71.7; 126.8, 127.2; 127.5,
128.1; 128.2, 128.4; 115.5, 155.7; 172.1, 172.9. Elemental analysis calcd for C10H11NO2S: C, 57.39; H, 6.300; N,
6.690; S, 15.32. Found: C, 57.01; H, 6.164; N, 6.776; S, 14.89.
(2RS,4R)-2-(2-methoxyphenyl)-1,3-thiazolidine-4-carboxylic acid (2)
1
Yield: %65. m.p.: 144.1 ᵒC. FT-IR (KBr), cm-1: 3147, 1639, 1490. H-NMR (DMSO-d6) δ: 2.94- 3.00 (m, 1H,
NCHCH2S); 3.18 (dd, J1=6.8 Hz, J2=10.4 Hz, 0.4H, NCHCH2S); 3.34 (dd, J1=6.8 Hz, J2=10.0 Hz, 0.6H,
NCHCH2S); 3.80 (s, 1.2H, OCH3); 3.81 (s, 1.8H, OCH3); 3.85 (dd, J=7.2 Hz, 9.2 Hz, 0.4H, NCHCH2S); 4.17 (t,
J=12.4 Hz 0.6H, NCHCH2S); 5.69 (s, 0.6H, NCHS); 5.86 (s, 0.4H, NCHS); 6.88–7.06 (m, 2H, Harom); 7.2–
7.34(m, 1H, Harom); 7.39 (dd, J1=1.2 Hz, J2=7.6 Hz, 0.4H, Harom); 7.51 (dd, J1=1.6 Hz, J2=8 Hz, 0.6H,
Harom). Elemental analysis calcd for C11H13NO3S: C, 55.21; H, 5.48; N, 5.85; S, 13.40. Found: C, 54.93; H,
5.434; N, 5.943; S, 13.06.
(2RS, 4R)-2-(2-methylphenyl)-1,3-thiazolidine-4-carboxylic acid (3)
Yield: %75. m.p.: 154.6 ᵒC. FT-IR (KBr), cm-1: 3427, 1666, 1590. 1H-NMR (DMSO-d6) δ: 2.31 (s, 1.8H, CH3);
2.35 (s, 1.2H, CH3); 3.05 (dd, J1=6.8 Hz, J2=10.2 Hz, 0.4H, NCHCH2S); 3.09 (dd, J1=5.2 Hz, J2=10.4 Hz, 0.6H,
NCHCH2S); 3.29 (dd, J1=6.8 Hz, J2=10.0 Hz, 0.4H, NCHCH2S); 3.38 (dd, J1=7.6 Hz, J2=10.4 Hz, 0.6H,
NCHCH2S); 3.90 (dd, J1=6.8 Hz, J2=7.2 Hz, 0.4H, NCHCH2S); 4.23 (dd, J1=5.6 Hz, J2=7.2 Hz, 0.6H,
NCHCH2S); 5.67 (s, 0.4H, NCHS); 5.85 (s, 0.6H, NCHS); 7.12 – 7.84 (m, 4H).
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(2RS-4R)-2-(2-cyanophenyl)-1,3-thiazolidine-4-carboxylic acid (4)
1
Yield: %62 . FT-IR (KBr), cm-1: 3400, 1613, 1487. H-NMR (DMSO-d6) δ: 3.74 (dd, J1=7.6 Hz, J2=12.4 Hz
0.95H, NCHCH2S); 3.80 (dd, J1=8.2 Hz, J2=12.0 Hz, 0.95H, NCHCH2S); 4.47 (t, J=7.6 Hz, 0.95H, NCHCH2S);
6.39 (s, 0.05H, NCHS) 6.42 (s, 0.95H, NCHS); 7.68–7.84 (m, 3H, Harom); 8.20 (d, J=8.0 Hz, 0.95H, Harom).
Elemental analysis calcd for C11H10N2O2S : C, 49.24; H, 4.51; N,10.44; S, 11.95. Found: C, 48.9; H, 4.61; N,
10.54; S, 12.17.
(2RS, 4R)-2-(2-bromophenyl)-1,3-thiazolidine-4-carboxylic acid (5)
1
Yield: %86. m.p.: 182.2 ᵒC FT-IR (KBr), cm-1: 3436, 1664 ,1515. H-NMR (DMSO-d6) δ: 2.97 (dd, J1=6.4 Hz,
J2=10.0 Hz, 0.7H, NCHCH2S); 3.06 (t, J=9.2 Hz, 0.3H, NCHCH2S); 3.25 (dd, J1=6.4 Hz, J2=10.0 Hz, 0.7H,
NCHCH2S); 3.35 (dd, J1=6.4 Hz, J2=9.6 Hz, 0.3H, NCHCH2S); 3.97 (dd, J1=6.8 Hz, J2=9.2 Hz, 0.4H,
NCHCH2S); 4.19 (t, J=6.8Hz, Hz 0.3H, NCHCH2S); 5.73 (s, 0.3H, NCHS); 5.91 (s, 0.7H, NCHS); 7.24-7.86 (m,
4H). 13C NMR (DMSO-d6) δ: 37.4, 37.5; 65.3, 65.5; 69.6, 69.8; 122.3, 122.5; 122.3, 127.7, 129.8, 132.4, 138,7,
141.8; 172.1, 172.5. Elemental analysis calcd for C10H10BrNO2S: C, 41.68; H, 3.50; N,4.680; S, 11.10. Found:
C, 36.38; H, 4.19; N, 4.938; S, 10.83.
(2RS, 4R)- 2-(3-chlorophenyl)-1,3-thiazolidine-4-carboxylic acid (6)
1
Yield: %74. m.p.: 160.4 ᵒC FT-IR (KBr), cm-1: 3438, 1574, 1477. H-NMR (DMSO-d6) δ: 3.09 (dd, J1=5.2 Hz,
J2=10.8 Hz, 0.9H, NCHCH2S); 3.28 (dd, J1=6.8 Hz, J2=10.4 Hz, 0.9H, NCHCH2S); 4.15 (dd, J1=4.8 Hz,
J2=6.4Hz, 0.9H, NCHCH2S); 5.55 (s, 0.1H, NCHS); 5.67 (s, 0.9H, NCHS); 7.28-7.37 (m, 3H, Harom), 7.48 (s,
1H, Harom). Elemental analysis calcd for C10H10ClNO2S: C, 49.28; H, 4.14; N,5.75; S, 13.01. Found: C, 48.98;
H, 4.002; N, 5.853; S, 12.90.
(2RS-4R)-2-(4-chlorophenyl)-1,3-thiazolidine-4-carboxylic acid (7)
1
Yield: %75. m.p.: 158.8 ᵒC. FT-IR (KBr), cm-1: 3435, 1579, 1491. H-NMR (DMSO-d6) δ: 3.09 (dd, J=5.2 Hz,
J2=10.8 Hz 0.95H, NCHCH2S); 3.27 (dd, J1=6.8 Hz, J2=10.0 Hz, 0.95H, NCHCH2S); 4.16 (dd, J1=4.8 Hz,
J2=6.4 Hz, 0.95H, NCHCH2S); 5.49 (s, 0.05H, NCHS); 5.66 (s, 0.95H, NCHS); 7.37 (d, J=8.8 Hz, 1.9H); 7.44
(d, J=8.4 Hz, 1.9H). LC-MS: ELS, rt = 3.80 min., m/z 244 [M + H]+, 285 [M + CH3CN]+. Elemental analysis
calcd for C10H10ClNO2S : C, 49.28; H, 4.14; N,5.750; S, 13.100. Found: C, 49.20; H, 4.046; N, 5.952; S, 12.91.
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(2RS-4R)- 2-(4-methoxyphenyl)-1,3-thiazolidine-4-carboxylic acid (8)
1
Yield: %72. m.p.: 159.0 ᵒC. FT-IR (KBr), cm-1: 3435,1586, 1463. H-NMR (DMSO-d6) δ: 3.07 (t, J=9.6 Hz,
0.5H, NCHCH2S); 3.15 (dd, J1=4.0 Hz, J2=10.0 Hz, 0.5H, NCHCH2S); 3.29 (dd, J1=7.2 Hz, J2=10.4 Hz, 0.5H,
NCHCH2S); 3.36 (dd, J1=7.2 Hz, J2=10.0 Hz, 0.5H, NCHCH2S); 3,74 (s, 1.5H, OCH3); 3.76 (s, 1.5H, OCH3);
3.87 (dd, J1=7.6 Hz, J2=9.2 Hz, 0.5H, NCHCH2S), 4.25 (dd, J1=4.0 Hz, J2=6.8 Hz, 0.5H, NCHCH2S); 5.46 (s,
0.5H, NCHS); 5.60 (s, 0.5H, NCHS); 6.89 (d, J=8.4 Hz, 1H, Harom); 6.93 (d, J=8.8 Hz, 1H, Harom); 7.37 (d,
J=8.4 Hz, 1H, Harom); 7.44 (d, J=8.4 Hz, 1H, Harom). LC-MS: ELSD, rt = 3.47 min., m/z 240 [M + H]+, 281
[M + CH3CN]+. Elemental analysis calcd for C11H13NO3S : C, 55.210; H, 5.480; N,5.850; S, 13.40. Found: C,
55.077; H, 5.213; N, 6.011; S, 13.20.
(2RS, 4R)- 2-(4-methylphenyl)-1,3-thiazolidine-4-carboxylic acid (9)
Yield: %85. m.p.: 161.8 ᵒC. FT-IR (KBr), cm-1: 3435, 1578, 1473. 1H-NMR (DMSO-d6) δ: 2.28 (s, 1.8H, CH3);
2.50 (s, 1.2H, CH3); 3.07 (t, J=9.2 Hz, 0.4H, NCHCH2S); 3.14 (dd, J1=4.4 Hz, J2=10.4 Hz, 0.6H, NCHCH2S);
3.28 (dd, J1=6.8 Hz, J2=10.0 Hz, 0.6H, NCHCH2S); 3.36 (dd, J1=6.8 Hz, J2=9.4 Hz, 0.4H, NCHCH2S); 3.88 (t,
J=8.4 Hz, 0.4H, NCHCH2S); 4.24 (dd, J1=4.4 Hz, J2=6.8 Hz, 0.6H, NCHCH2S); 5.46 (s, 0.4H, NCHS); 5.62 (s,
0.6H, NCHS); 7.14 (d, J=8.4 Hz, 0.4H, Harom); 7.18 (d, J=8.0 Hz, 0.4H, Harom); 7.32 (d, J=8.4 Hz, 0.6H,
Harom); 7.39 (d, J=8.0 Hz, 0.6H, Harom). 13C NMR (DMSO-d6) δ: 20.5, 20.6; 37.8, 38.6; 64.7, 65.3; 70.9, 71.6
; 126.8, 127.1; 124.8, 128.7, 128.9; 135.8, 136.8; 137.5, 137.9; 172.1, 172.9. Elemental analysis calcd for
C11H13NO2S : C, 59.17; H, 5.870; N,6.27; S, 14.36. Found: C, 59.40; H, 5.889; N, 6.405; S, 14.06.
(2RS, 4R)- 2-(4-nitrophenyl)-1,3-thiazolidine-4-carboxylic acid (10)
1
Yield: %80. m.p.: 110 ᵒC. FT-IR (KBr), cm-1: 3407, 1629, 1438. H-NMR (DMSO-d6) δ: 3.12 (dd, J1=8.8 Hz,
J2=9.6 Hz, 0.95H, NCHCH2S); 3.76 (dd, J1=6.8 Hz, J2=10.0 Hz, 0.95H, NCHCH2S); 3.98 (dd, J1=7.2 Hz, J2=8.8
Hz, 0.95, NCHCH2S); 5.67 (s, 0.95H, NCHS); 5.87 (s, 0.05H, NCHS); 7.8 (d, J=8.4 Hz, 1.9H, Harom); 8.2 (d,
J=8.4 Hz, 1.9H, Harom). LC-MS: ELSD, rt = 3.59 min., m/z 255 [M + H]+, 296 [M + CH3CN]+. Elemental
analysis calcd for C10H10N2O4S : C, 44.28; H, 4.090; N,10.33; S, 11.80. Found: C, 43.98; H, 4.315; N, 10.33; S,
11.26.
(2RS, 4R)-2-(2,6-difluorophenyl)-1,3-thiazolidine-4-carboxylic acid (11)
1
Yield: %75. m.p.: 140.6 ᵒC. FT-IR (KBr), cm-1: 3459, 1656, 1469. H-NMR (DMSO-d6) δ: 3.01 (t, J=9.2 Hz,
0.6H, NCHCH2S); 3.19 (dd, J1=3.6 Hz, J2=10.0 Hz, 0.4H, NCHCH2S); 3.33 (dd, J1=6.8 Hz, J2=10.4 Hz, 0.4H,
NCHCH2S); 3.46 (dd, J1=7.2 Hz, J2=10.4 Hz, 0.6H, NCHCH2S); 3,89 (t, J=8.8 Hz, 0.6H, NCHCH2S); 4.45 (dd,
14

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J1=3.6 Hz, J2=6.4 Hz, 0.4H, NCHCH2S), 5.78 (s, 0.6H, NCHS); 5.98 (s, 0.4H, NCHS); 7.11 (t, J=8.8 Hz, 0.8H,
Harom); 7.18 (t, J=9.0Hz, 1.2.H, Harom); 7.36-7.56 (m, 1H, Harom). Elemental analysis calcd for C11H10N2O2S
: C, 49.24; H, 4.51; N,10.44; S, 11.95. Found: C, 48.59; H, 4.641; N, 10.54; S, 12.57.
(2RS, 4R)-2-(2,3-difluorophenyl)-1,3-thiazolidine-4-carboxylic acid (12)
Yield: %80. m.p.: 133.5 ᵒC. FT-IR (KBr), cm-1: 3433, 1610, 1426. 1H-NMR (DMSO-d6) δ: 3.00 (dd, J1=6.0 Hz,
J2=10.4 Hz, 0.4H, NCHCH2S); 3.05 (t, J=9.2 Hz, 0.4H, NCHCH2S); 3.29 (dd, J1=6.4 Hz, J2=10.0 Hz, 0.6H,
NCHCH2S); 3.35 (dd, J1=6.4 Hz, J2=9.6 Hz, 0.6H, NCHCH2S); 3.94 (dd, J1=6.8 Hz, J2=8.8 Hz, 0.4H
NCHCH2S); 4.11 (t, J=6.0Hz, 0.6H NCHCH2S); 5.70 (s, 0.6H, NCHS); 5.93 (s, 0.4H, NCHS); 7.12-7.58 (m, 3H,
Harom). LC-MS: ELSD, rt = 3.60 min., m/z 246 [M + H]+, 287 [M + CH3CN]+. Elemental analysis calcd for
C11H10N2O2S : C, 48.89; H, 3.70; N,5.710; S, 13.010. Found: C, 48.77; H, 3.684; N, 5.822; S, 12.740.
(2RS, 4R)- 2-(2,3-dichlorophenyl)-1,3-thiazolidine-4-carboxylic acid (13)
1
Yield: %84. m.p.: 129.7 ᵒC. FT-IR (KBr), cm-1 3431, 1666, 1418. H-NMR (DMSO-d6) δ: 2.94 (dd, J1=7.6 Hz,
J2=10.8 Hz, 0.4H, NCHCH2S); 3.05 (t, J=9.2 Hz, 0.4H, NCHCH2S); 3.25 (dd, J1=6.4 Hz, J2=10.0 Hz, 0.6H,
NCHCH2S); 3.34 (dd, J1=6.8 Hz, J2=10.4 Hz, 0.6H, NCHCH2S); 4.02 (dd, J1=7.2 Hz, J2=9.2 Hz, 0.4H,
NCHCH2S), 4.13 (t, J=6.4 Hz, 0.6H, NCHCH2S); 5.80 (s, 0.4H, NCHS); 5.99 (s, 0.6H, NCHS); 7.36 (t, J=8.0
Hz, 0.6H, Harom); 7.41 (t, J=8.0 Hz, 0.4H, Harom); 7.53-7.55 (m, 1H, Harom); 7.61 (dd, J1=1.6 Hz, J2=7.6 Hz,
0.6H); 7.84 (dd, J1=1.6 Hz, J2=7.6 Hz, 0.4H). Elemental analysis calcd for C11H10N2O2S : C, 38.47; H, 3.55;
N,4.49; S, 10.20. Found: C, 38.04; H, 3.559; N, 4.770; S, 10.350.
HPLC assays
The Agilent 1260 series HPLC with a diode array detector and a 5 ꢀm Zorbax Eclipse XDB-C18 column (4.6
mm × 250 mm, 3.5µm) was used to record spectra. Water: acetonitrile (30:70) solvent system was used as the
mobile phase with a flow rate of 0.5 ml.min-1 at 25°C.Spectra were recorded at 254nm. The injection volume
was of 5 µL.
The DPPH assay
The DPPH radical scavenging abilities of the molecules 1-13 were evaluated with respect to the method of Akter
et al.[46] with few alterations [43]. The assay was performed in 96-well plates. The DPPH solution was prepared
(0.1 mM) and sample solutions were prepared by appropriate serial dilution within a range from 6.75 µM to
1000 µM. As a reference, a BHT solution was prepared and used at a concentration range varying between 125
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and 2000 µM. In each well 250 µL of DPPH solution and 50 µL of sample or reference solutions were added and
incubated in dark at RT for 50 minutes. Each sample was evaluated in triplicate. Absorbance was measured at
517 nm and radical scavenging ability of the molecules was calculated as follows:
DPPH radical - scavenging activity % = [1-(Aaverage – Ablank) / (Acontrol-Ablank)] ×100
The CUPRAC assay
The cupric ion reducing abilities of the molecules 1-13 were evaluated with respect to the method of Apak et
al.[44] with few alterations [43]. The assay was performed in 96-well plates. Reagent solutions were prepared
separately as 10mM CuSO4, 7.5 mM neocuproine and 1M ammonium acetate buffer (pH 7.0). Sample solutions
were prepared by appropriate serial dilution within a range from 6.75 µM to 1000 µM. Trolox, a water soluble
analogue of vitamin E, was used as reference in the concentration range of 10 µM-70µM. Into each well, equal
volumes of each reagent solution 85 µL, 51 µL of H2O and 43 µL of sample solution were added. Each sample
concentration was evaluated triplicate. The plate was incubated in 50ᵒC for 20 minutes and absorbance was
measured at 450 nm. Trolox coefficient values (TEAC) were calculated for each sample as TEAC = εsample
εtrolox
/
.
ACKNOWLEGMENTS
The authors thank Yeditepe University for financial support.
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Highlights
•
•
•
•
•
Thiazolidine carboxylic acids were synthesized with a series of benzaldehydes.
The antioxidant activity was evaluated via the DPPH and CUPRAC assays.
The cysteine releasing pattern of each thiazolidine compound was analyzed.
The antioxidant capacity was shown to be related not only to L-cysteine release.
Thiazolidine heterocycle itself can also be considered as antioxidant.