3-Propionyl-thiazolidine-4-carboxylic acid ethyl esters: A family of antiproliferative thiazolidines

Article abstract of DOI:10.1039/c4md00306c

Cancer results from unregulated cell growth. Reactivating the process of the programmed cell death, i.e. apoptosis, is a classical anticancer therapeutic strategy. The apoptosis-inducing property of the (2RS,4R)-2-phenyl-3-propionyl-thiazolidine-4-carboxylic acid ethyl ester (ALC 67) molecule has recently been discovered. We analyzed in this study the impact of the phenyl moiety of this molecule on its biological activity by synthesizing and evaluating analogues where this substituent was replaced by a series of aromatic and aliphatic groups. The results demonstrated that the molecule's antiproliferative property resisted such modifications. Thus, in addition to developing a family of thiazolidine compounds with promising anticancer properties; our investigation revealed that the second position of the thiazolidine ring can be used either to tune the physicochemical properties of ALC67 or to introduce a fluorescent tag to the structure in order to track it in cells and determine its exact molecular mechanism of action.

Full text of DOI:10.1039/c4md00306c

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3-Propionyl-thiazolidine-4-carboxylic acid ethyl
esters: a family of antiproliferative thiazolidines†
Cite this: DOI: 10.1039/c4md00306c
F. Esra O¨ nen-Bayram, a Kerem Buran,a Irem Durmaz,b Barkin Berkc and Rengul Cetin-
*
Atalayb
Cancer results from unregulated cell growth. Reactivating the process of the programmed cell death, i.e.
apoptosis, is a classical anticancer therapeutic strategy. The apoptosis-inducing property of the (2RS,4R)-
2-phenyl-3-propionyl-thiazolidine-4-carboxylic acid ethyl ester (ALC 67) molecule has recently been
discovered. We analyzed in this study the impact of the phenyl moiety of this molecule on its biological
activity by synthesizing and evaluating analogues where this substituent was replaced by a series of
aromatic and aliphatic groups. The results demonstrated that the molecule's antiproliferative property
resisted such modifications. Thus, in addition to developing a family of thiazolidine compounds with
promising anticancer properties; our investigation revealed that the second position of the thiazolidine
ring can be used either to tune the physicochemical properties of ALC67 or to introduce a fluorescent
tag to the structure in order to track it in cells and determine its exact molecular mechanism of action.
Received 14th July 2014
Accepted 14th September 2014
DOI: 10.1039/c4md00306c
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demonstrated the relevant cytotoxicity of
a propargylic
Introduction
compound, the ALC 67 molecule (Fig. 1A), on liver, colon, breast
and endometrial cancer cell lines,13 which was also proven to
induce apoptosis by activating caspase-9. However, the exact
mechanism of action of this compound remains unknown. As
2-phenylthiazolidine carboxylic acid (Fig. 1B) and the corre-
sponding ethyl ester (Fig. 1C) from which ALC 67 was synthe-
sized did not exhibit any biological activity, we assigned the
antiproliferative property of the molecule to its propargylic
group.
To analyze the impact of the phenyl moiety present at the
second position of the heterocycle on the bioactivity of ALC67,
we generated in this study 3-propionyl-thiazolidine-4-carboxylic
acid ethyl esters presenting a series of aromatic and aliphatic
moieties at this very position (Fig. 2). We then tested the
synthesized compounds for their biological activity to check if
the cytotoxicity resisted such modications.
Cancer is a disease that results from the abnormal proliferation
of normal cells. Cancer cells present mutations that allow them
to multiply rapidly, not only by escaping growth suppressors but
also by resisting apoptosis, the natural cell death mechanism.1–3
Reactivating apoptosis either by developing anti-apoptotic
protein inhibitors or pro-apoptotic protein agonists has
constituted a considerable anticancer strategy for two decades.4
Thiazolidines are ve-membered heterocycles containing a
sulfur and a nitrogen atom at their rst and third positions
respectively. They were rst described by Miller et al. for their
anticancer property in 2005.5,6 The authors, who rst generated
serine phosphate amides as lysophosphatidic acid analogues to
treat prostate cancer, noted their poor selectivity due to the
possible hydrolysis of the phosphate group present in their
structures.7 To circumvent this issue, they chose to work with 4-
thiazolidinone derivatives8 because this cycle is described as a
phosphate biomimetic.9 To optimize their cytotoxicity results the
authors then developed thiazolidine compounds, and subse-
quently discovered their promising apoptotic property.10–12
In a previous study based on these ndings, we prepared a
library of small molecules around a thiazolidine scaffold and
aDepartment of Pharmaceutical Chemistry, Yeditepe University, Faculty of Pharmacy,
Istanbul, Turkey. E-mail: esra.bayram@yeditepe.edu.tr; lizesraonen@gmail.com
bDepartment of Molecular Biology and Genetics, Faculty of Science, Bilkent University,
Ankara, Turkey
cDepartment of Pharmaceutical Chemistry, Medipol University, Faculty of Pharmacy,
Istanbul, Turkey
Fig. 1 (A) The molecular structure of the cytotoxic ALC67. (B) The
carboxylic acid precursor of ALC67, which exhibits no cytotoxicity. (C)
The ethyl ester precursor of ALC67, with no cytotoxic activity.
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00306c
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was conducted under basic conditions.15 As the ring-closure
generates a new chiral center in an uncontrolled manner,
thiazolidine compounds were obtained as diastereomeric
mixtures with satisfactory yields (59–92%).
The synthesized carboxylic acid molecules were analyzed by
1
FT-IR, H NMR, 13C NMR and mass spectrometry. The genera-
tion of the heterocyclic structure was conrmed by 1H NMR
since the spectra exhibited the expected characteristic signals of
a thiazolidine cycle. Indeed, in addition to the singlets at
around 5.2 ppm and 5.6 ppm that correspond to the signals of
C2–H of each diastereomer, a pair of doublet of doublets (dd) at
around 4.2 ppm and 3.80 ppm for the C4-H and a set of four dd
around 3.3 ppm for the unequivalent C5-Hs were also recorded
in the spectra (Fig. 3A). The successful formation of the thia-
zolidine ring was also conrmed by 13C NMR as the spectra
displayed the typical signals of C2, C4 and C5 at around 71 ppm,
65 ppm and 38 ppm respectively (Fig. 3B). The 2R, 4R and 2S, 4R
diastereomers were obtained in general in a 40 : 60 ratio. This
ratio was easily determined using the C2-H singlet signals of 1H
NMR. Interestingly the thiazolidines derived from o-uo-
robenzaldehyde and trimethylacetaldehyde (1d and 1m
respectively) gave diastereomers in 15 : 85 and 5 : 95 ratios,
probably due to the steric hindrance caused by the proximity of
the uorine atom or the tert-butyl group to the carboxylic acid
moiety in the 2R, 4R conguration. Moreover, 1k and 1l were
prepared from racemic mixtures of 3,5,5-trimethylhexanal and
2-methylpentanal respectively. For these reasons, each aldehyde
led to four different thiazolidines: the 1H NMR spectra of these
compounds exhibited four different doublets for the C2-H and
the 13C NMR showed sets of four peaks for each signal. Given
the difficulty of separation, the isomers were not isolated,
neither when proceeding to the esterication or acylation steps,
nor when biologically evaluated.
Fig. 2 Examination of the effect of phenyl moiety on the cytotoxicity
of ALC67.
Results and discussion
Compounds were synthesized according to pathways previously
described.13,14 Briey, rst an acetylation was carried out using
L-cysteine and a series of commercially available aromatic and
aliphatic aldehydes. Then, the resulting thiazolidine carboxylic
acids (1a–1m) were converted into their corresponding ethyl
esters (2a–2m) in ethanol in the presence of thionyl chloride.
Finally, the secondary amine of the heterocycle was acylated by
propiolic acid, which was previously activated by dicyclohexyl-
carbodiimide (DCC) to provide the terminal alkyne compounds
(3a–3m) (Scheme 1).
The rst step, which consisted of generating the thiazolidine
ring from the nucleophilic addition reaction of L-cysteine
(aminothiol) with a series of aliphatic and aromatic aldehydes,
Scheme 1 Preparation of 3-propionyl-thiazolidine-4-carboxylic acid
ethyl esters from a series of aliphatic and aromatic aldehydes. Fig. 3 (A) Typical 1H NMR signals of C2, C4 and C5 hydrogens of a
Reagents: (a) NaOH in ethanol–H2O (1/1); (b) thionyl chloride in diastereomeric mixture of thiazolidines. (B) Typical 13C NMR signals of
absolute ethanol; (c) DCC in dry dichloromethane.
C2, C4 and C5 carbons of a diastereomeric mixture of thiazolidines.
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Table 1 Synthesis yields and IC50 values of ALC67 on HUH7 and Mahlavu (MV) hepatocellular carcinoma cell lines determined by sulforhodamine
B assaya
R
Yield (%)
HUH7 IC50 Æ SEM (mM)
MV IC50 Æ SEM (mM)
Ph
ALC67
(3a)
(3b)
(3c)
(3d)
(3g)
(3h)
(3i)
5.3 Æ 0.9
1.4 Æ 0.1
0.7 Æ 0.2
1.4 Æ 0.4
1.7 Æ 0.4
2.6 Æ 0.6
1.8 Æ 0.4
0.5 Æ 0.1
1.7 Æ 0.3
1.6 Æ 0.4
0.6 Æ 0.3
0.8 Æ 0.1
0.1
0.4 Æ 0.5
0.7 Æ 0.2
0.4 Æ 0.2
1.7 Æ 2.0
1.7 Æ 0.6
2.4 Æ 2.3
2.0 Æ 1.4
0.4 Æ 0.1
1.6 Æ 0.2
1.1 Æ 0.6
0.5 Æ 0.1
0.9 Æ 0.1
<1
p-OCH3–Ph–
p-F–Ph–
m-F–Ph–
o-F–Ph–
p-CN–Ph–
–(CH2)4CH3
–(CH2)3CH3
–CH(CH2–CH3)–CH2CH2CH2CH3
–CH2–CH(CH3)–CH2–C(CH3)3
–CH(CH3)–CH2–CH2CH3
–C(CH3)3
CPT
89
37
58
30
24
55
87
30
55
63
52
(3j)
(3k)
(3l)
(3m)
5FU
30.7
10.0
a
CPT: camptothecin; 5FU: 5-uorouracil.
The conversion of carboxylic acid derivatives into ethyl ester
All obtained IC50 values were similar to ALC67's values (Table
moieties was achieved in the presence of thionyl chloride in 1): the bioactivity of the terminal alkyne molecule remained
absolute ethanol. The generation of the ethyl ester function was when the phenyl moiety was replaced either by aliphatic or
readily conrmed by FT-IR since the typical large band at aromatic groups suggesting that this position is not essential
around 3300 cmÀ1 corresponding to the hydroxyl of the for the molecule to be cytotoxic. Hence, a novel class of anti-
carboxylic acid function was no longer detected (data not proliferative thiazolidines was developed.
shown). NMR analyses were not performed on these molecules
The effect of substituting the phenyl moiety was analyzed
because of their instability: though pure compounds were through the 3a–3g compounds. The cytotoxic activity did not
obtained (checked on TLC), we noticed that they tended to vary, regardless of the electron donor or acceptor property of the
decompose when stored. This is why all obtained ester deriva- pending group: with the para-substituted molecules (3a, 3b and
tives were directly acylated without further analyses.
3g), the best activity was observed for the para-uorophenyl
To obtain ALC67 analogues, a propionyl group was intro- substituted derivative (smallest IC50 value) while the electron-
duced to the secondary amine of the thiazolidine ring of the 2a– donating methoxy and the electron-attracting cyano substitu-
2m molecules. The peptidic coupling reaction was carried out tions led to greater IC50 values (1.4 and 2.6 respectively). We also
by classically activating the carboxylic acid function of propiolic investigated the effect of the substitution position, preparing
acid by DCC. The presence of the terminal alkyne was analyzed the ortho- (3d), meta- (3c) and para-uorophenyl (3b) thiazoli-
by FT-IR, 1H NMR and 13C NMR. In fact in addition to the band dines. The results revealed better activity for the para-uo-
at 2100 cmÀ1 that corresponds to the stretching band of the rophenyl compound, indicating a possible impact of the
C^C bond, the IR spectra gave a strong and narrow band at substitution position on the biological activity due to the
3200 cmÀ1 for the H–C^C stretching. The presence of two generated steric hindrance.
singlets around 3.25 ppm, which are typical of terminal alkyne
Regarding the thiazolidines derived from alkyl aldehydes,
protons in 1H NMR, and the peaks of sp hybridized carbons the determined IC50 values showed that biological activity is
observed around 75 ppm and 81 ppm in 13C NMR also also maintained with both linear (3h, 3i) and branched
conrmed the successful synthesis of the 3a–3m molecules chains (3j–3m), with the highest activity observed for the 3i
(except for 3e and 3f for which we could not isolate pure prod- molecule.
ucts). Yields of the obtained compounds are given in Table 1.
The antiproliferative activity of the synthesized alkyne
compounds (3a–3m) was examined on two different hepato-
Conclusion
cellular carcinoma cell lines (HUH7 and Mahlavu cells) using All these results demonstrated that the phenyl moiety present
the sulforhodamine B assay.16 The derivatives were evaluated as on the second position of the thiazolidine ring of the ALC67
diastereomeric mixtures since we could not separate the molecule is not crucial for its biological activity. This observa-
isomers and also because thiazolidine compounds are tion allowed us to generate similarly cytotoxic novel molecules
commonly analyzed as diastereomeric mixtures in the litera- using a rapid and easy methodology. This investigation also
ture.5,6,10–13 The obtained cytotoxicity was compared to the suggested that the second position of the heterocycle can be
activity of ALC67, camptothecin and 5-uorouracil, two mar- used to tune the physicochemical properties of the cytotoxic
keted anticancer agents frequently used as positive controls in molecule or to further introduce a uorescent tag on this
cytotoxicity assays.17–21
position to elucidate its molecular mechanism of action.22
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