Regioselectivity evaluation of the (Z)-5-(4-hydroxybenzylidene)-thiazolidine-2,4?dione alkylation in alkaline environment

Article abstract of DOI:10.1016/j.molstruc.2021.130629

Thiazolidine-2,4?dione represents a key heterocyclic core in medicinal chemistry because it has the ability to bind to a wide variety of protein targets and has been intensively studied for developing bioactive multitargeting agents. The N-alkylation of i

Full text of DOI:10.1016/j.molstruc.2021.130629

Journal of Molecular Structure 1241 (2021) 130629
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Regioselectivity evaluation of the (Z)-5-(4-hydroxybenzylidene)-
thiazolidine-2,4–dione alkylation in alkaline environment
Gabriel Marca,∗, Anca Stanaa, Adrian Pîrna˘ub,∗, Laurian Vlasec, Smaranda Onigad,
Ovidiu Onigaa
a Department of Pharmaceutical Chemistry “Iuliu Hațieganu” University of Medicine and Pharmacy, 41 Victor Babeș Street, RO-400012 Cluj-Napoca, Romania
b Department of Molecular and Biomolecular Physics, National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat
Street, RO-400293 Cluj-Napoca, Romania
c Department of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Hațieganu” University of Medicine and Pharmacy, 41 Victor Babeș Street,
RO-400012 Cluj-Napoca, Romania
d Department of Therapeuthic Chemistry “Iuliu Hațieganu” University of Medicine and Pharmacy, 12 Ion Creanga˘ Street, RO-400012 Cluj-Napoca, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiazolidine-2,4–dione represents a key heterocyclic core in medicinal chemistry because it has the
ability to bind to a wide variety of protein targets and has been intensively studied for developing
bioactive multitargeting agents. The N-alkylation of imides and O-alkylation of phenols are important
reactions frequently used in the synthesis of biologically active compounds, like the thiazolidine-2,4–
dione derivatives. Based on literature data, by treating (Z)-5-(4-hydroxybenzylidene)-thiazolidine-2,4–
dione with alkyl halides in a 1:1 molar ratio, either O-alkylation or N-alkylation reactions can take place.
Six (Z)-5-(4-hydroxybenzylidene)-thiazolidine-2,4–dione derivatives were synthesized by alkylating (Z)-5-
(4-hydroxybenzylidene)-thiazolidine-2,4–dione with aliphatic halogenated derivatives in alkaline environ-
ment, using a 1:1 molar ratio and then were physically and spectrally analyzed. The spectral data and the
results obtained from the theoretical evaluation of the parent compound’s acidity support the formation
of N-alkylated derivatives in the reaction conditions.
Received 19 March 2021
Revised 20 April 2021
Accepted 3 May 2021
Available online 16 May 2021
Keywords:
Alkylation
5-(4-hydroxybenzylidene)-thiazolidine-2,4-
dione
Alkyl halide
Alkaline
Spectroscopy
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
dione was the pharmacophore moiety in compounds acting as
PPARγ agonists/modulators, protein-tyrosine phosphatase 1B and
Of the five membered heterocyclic ring systems, thiazolidine-
2,4–dione is an extensively studied moiety for developing new
bioactive agents in the treatment of a broad spectrum of patholo-
gies like diabetes and its complications, microbial infections, in-
flammation, arthritis, cancer, melanoma, etc. [1]. Thiazolidine-
2,4–dione represents a privileged scaffold in medicinal chemistry
due to its vast biological profile with multifarious pharmacologi-
cal properties. Several thiazolidine-2,4–dione derivatives were re-
ported as potential antitumor agents because of their inhibitory
activity towards the phosphoinositide 3-kinase gamma, mitogen-
activated protein kinase, Pim kinase and histone deacetylase and
also as potential agonists of the nuclear peroxisome proliferator-
activated receptors (PPARs), which induce antiproliferative, an-
tiangiogenic and prodifferentiation pathways in many types of
tissue, thus downregulating carcinogenesis [2]. Thiazolidine-2,4–
aldose reductase-2 inhibitors, α-glucosidase inhibitors, free fatty
acid receptor agonists, β−3 agonists, effective in the treatment
of diabetes and its complications [3]. For the compounds bear-
ing the thiazolidine-2,4–dione nucleus were found applications in
the infectious diseases therapy like antifungal agents [4,5], antibac-
terial agents acting as autoinducer-2 quorum sensing, MurD lig-
ase or peptide deformylase inhibitors, antimalarial agents, antivi-
ral agents acting as HIV-1 reverse transcriptase inhibitors. Supple-
mentary, this scaffold was reported responsible for the pharmaco-
logical activity of compounds acting as tyrosinase inhibitors use-
ful for treating melanoma, antioxidant agents and xanthine oxidase
inhibitors, anti-inflammatory, COX-2 inhibitors and neuroprotective
agents [1,6].
Most thiazolidine-2,4–dione derivatives reported in the liter-
ature as biologically active compounds were developed through
structural modifications in positions 3 (the free –NH moiety) and 5
(the free –CH2 moiety) of the thiazolidine-2,4–dione nucleus, usu-
ally by N-alkylation and Knoevenagel condensation, respectively.
[1,6]
∗
Corresponding authors.
E-mail addresses: marc.gabriel@umfcluj.ro (G. Marc), apirnau@itim-cj.ro (A.
Pîrna˘u).
https://doi.org/10.1016/j.molstruc.2021.130629
0022-2860/© 2021 Elsevier B.V. All rights reserved.

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
The O-alkylation of phenols and N-alkylation of imides in al-
kaline environment are intensively studied by the researchers
in organic and medicinal chemistry [7-9] because they can be
widely used for obtaining bioactive compounds, like the ones
mentioned above. (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4–
dione has two potential acid groups, one being the phenol and the
other the imide. In alkaline environment, they will both manifest
their Brønsted-Lowry acidity by releasing the proton, thus creating
an anionic structure, the nucleophile. This one is capable of inter-
action with the methylene (-CH2-) group from an alkyl halide used
as alkylating agent, the electrophile, according to a SN2 mechanism
and following a two-step reaction. First, the anionic rich electronic
species attacks the methylene moiety of the electrophile in an ad-
dition reaction that is followed afterwards by an elimination re-
action of the halide anion [10]. The rate of the SN2 reaction de-
pends on both the concentration of electrophile and nucleophile
species in the reaction environment. The formation of the anionic
nucleophile species in alkaline environment is directly linked to
the solvent used and to the intrinsic properties of the substrate.
It is known that aprotic polar solvents like dimethylformamide
(DMF) or dimethylsulfoxide (DMSO) are able to expose the anionic
species, increasing its reactivity towards the electrophilic partner,
in order to perform the first step of the SN2 reaction [9].
synthesized compounds in the 8.5–14 ppm range. We have cho-
sen this spectral area because here are found the protons sig-
nals of the starting compound 2, given by the groups in its struc-
ture that could be alkylated: the signal characteristic to the phe-
nolic OH (broad, at 10.310 ppm) and the imide NH (broad, at
12.431 ppm). We focused on these broad signals present in the 1H
NMR spectrum of the parent molecule (2), the disappearance of
these signals, respectively the apparition of new signals, depend-
ing on the substituent that was introduced on the parent molecule.
Furthermore, it has been studied in the aromatic zone of the 1H
NMR spectrum how the benzylidene proton and benzyl protons
are shifted in compounds 5a-f, as a result of the substitution at
the level of the nitrogen or oxygen atom of the compound 2.
2. Results and discussion
All the compounds were synthesized and characterized.
2–chloro-N-((2-phenylthiazol-4-yl)methyl)acetamide (4f): white
solid; m.p. 105 °C; yield=77%; FT IR (KBr) νmax cm−1: 1661 (C O);
=
MS: m/z = 267.6 (M + 1).
(Z)−5-(4-hydroxybenzylidene)−3-((2-(phenylamino)thiazol-4-
yl)methyl)thiazolidine-2,4–dione (5b): yellow solid; m.p. 229–
230 °C; yield = 48%; 1H NMR (DMSO–d6, 500 MHz) δ: 10.370 (br,
1H, OH), 10.198 (s, 1H, NH), 7.899 (s, 1H, -CH=), 7.563–7.523 (m,
4H, Ar), 7.232 (t, J = 8.5 Hz, 2H, Ar), 6.946 (d, J = 8.5 Hz, 2H,
Ar), 6.902 (t, J = 7.5 Hz, 1H, Ar), 6.730 (s, 1H, Ar), 4.760 (s, 2H, -
CH2-); 13C NMR (DMSO–d6, 125 MHz) δ: 167.65, 165.985, 164.033,
160.694, 145.876, 141.544, 134.167, 133.124, 129.316, 124.375,
121.715, 117.298, 117.095, 116.934, 104.511, 41.783; FT IR (KBr) νmax
According to the literature, two possible types of compounds
can be obtained after alkylation of (Z)−5-(4-hydroxybenzylidene)-
thiazolidine-2,4–dione (compound 2) in alkaline environment with
aliphatic chlorinated derivatives, using a 1:1 molar ratio. Some pa-
pers reported the selective formation of ethers, by alkylation of the
phenolic oxygen, [11–16] while others suggested that N-alkylation
took place, when using equimolecular amounts of reagents [17-
21] (Fig. 1). The use of a reaction ratio of two equivalents of alky-
lating agent to one equivalent of compound 2, led to the loss of
regioselectivity, giving double O and N-alkylated derivatives [22].
In order to evaluate the veracity of one hypothesis or
the other, 6 compounds were synthesized, all being (Z)−5-(4-
hydroxybenzylidene)-thiazolidine-2,4–dione derivatives and were
analyzed spectrally (1H NMR, 13C NMR, MS, IR). The present study
is mostly focused on the analysis of the 1H NMR spectra of the
cm−1: 3414 (Ar-OH), 1725, 1675 (C O); MS: m/z = 410.3 (M + 1).
=
(Z)−2-(5-(4-hydroxybenzylidene)−2,4-dioxothiazolidin-3-yl)-N-
(4-methylbenzo[d]thiazol-2-yl) acetamide (5c): yellow solid; m.p.
273–274 °C; yield = 66%; 1H NMR (DMSO–d6, 500 MHz) δ: 12.986
(br, 1H, NH), 10.499 (br, 1H, OH), 8.138 (ds, 1H, Ar), 7.907 (s,
1H, -CH=), 7.769 (d, J = 9 Hz, 1H, Ar), 7.534 (d, J = 8.5 Hz, 2H,
Ar), 7.472 (dd, 1H, Ar), 6.932 (d, J = 8.5 Hz, 2H, Ar), 4.701 (s,
2H, -CH2-); 13C NMR (DMSO–d6, 125 MHz) δ: 167.749, 166.412,
165.824, 160.946, 158.846, 147.829, 134.811, 133.633, 133.215,
Fig. 1. The two possible types of alkylation products of (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4-dione, reported in the literature.
2

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
132.984, 128.350, 127.076, 124.172, 122.401, 122.023, 116.990,
116.647, 115.759, 44.044, 31.172; FT IR (KBr) νmax cm−1: 3408
its spectral analysis (Tables 1–3) allowed us to analyze this type of
signal and to observe how it can influence the interpretation of the
1H NMR spectrum in the spectral area of interest, 8.5–14 ppm. In
the case of compound 5a, it is obvious that the single broad sig-
nal given by a deshielded proton is given by the phenolic moiety
at 10.446 ppm. Because the broad signal characteristic to its much
deshielded proton at 12.431 ppm totally disappeared, it is obvious
that the alkylation took place at the nitrogen atom. A similar situ-
ation is found for the compound 5b, where the broad signal of the
deshielded proton of the phenol can be found at 10.370 ppm. The
supplementary peak given by the proton from the amino group is
found as a strong sharp signal at 10.198 ppm (Table 1). Guided by
the appearance and the chemical shift of this proton, this type of
proton cannot be confused with the proton of an amide or with
that of a thiazolidinedione.
=
(Ar-OH), 1734, 1708, 1672 (C O); MS: m/z = 426.1 (M + 1).
(Z)−2-(5-(4-hydroxybenzylidene)−2,4-dioxothiazolidin-3-yl)-N-
(thiazol-2-yl)acetamide (5d): yellow solid; m.p. carbonization >
280 °C; yield = 58%; 1H NMR (DMSO–d6, 500 MHz) δ: 12.705
(br, 1H, NH), 10.689 (br, 1H, OH), 7.892 (s, 1H, -CH2-), 7.524 (d,
J = 9 Hz, 2H, Ar), 7.501 (d, J = 3.5 Hz, 1H, Th), 7.270 (d, J = 3.5 Hz,
1H, Th), 7.009 (d, J = 9 Hz, 2H, Ar), 4.639 (s, 2H, -CH2-); 13C
NMR (DMSO–d6, 125 MHz) δ: 167.770, 165.859, 164.488, 163.221,
161.093, 138.219, 134.748, 133.152, 124.081, 117.032, 116.598,
114.499, 43.806; FT IR (KBr) νmax cm−1: 3426 (Ar-OH), 1735, 1699,
=
1467 (C O); MS: m/z = 362.1 (M + 1).
(Z)-ethyl 2-(2-(5-(4-hydroxybenzylidene)−2,4-dioxothiazolidin-3-
yl)acetamido)−4-methylthiazole-5-carboxylate (5e): yellow solid;
m.p. carbonization > 265 °C; yield=63%; 1H NMR (DMSO–d6,
500 MHz) δ: 13.073 (br, 1H, NH), 10.618 (br, 1H, OH), 7.884 (s, 1H,
-CH2-), 7.515 (d, J = 9 Hz, 2H, Ar), 6.997 (d, J = 8.5 Hz, 2H, Ar),
4.669 (s, 2H, -CH2-), 4.229 (q, J = 7.5 Hz, 2H, -CH2-), 2.551 (s, 3H,
-CH3), 1.264 (t, J = 7 Hz, 3H, Th); 13C NMR (DMSO–d6, 125 MHz)
δ: 167.728, 166.092, 165.789, 162.423, 161.058, 159.483, 156.571,
134.797, 133.159, 124.088, 117.011, 116.563, 115.052, 61.073, 43.946,
17.440, 14.626; FT IR (KBr) νmax cm−1: 3408 (Ar-OH), 1714, 1711,
This second type of alkylating agents used, the chloroacetamide
derivatives 4c-f aimed at obtaining the other final compounds (5c-
f), bearing an amide group. Based on these facts, the signals char-
acteristic to the proton from the amide group in the 8.5–14 ppm
spectral area were analyzed. In the compounds 5c-e, the aro-
matic amide gave the supplementary expected signals as broad,
between 12.705–13.078 ppm and the phenolic proton between
10.508–10.689 ppm. For compound 5f, the aliphatic amide gave the
supplementary expected signal as sharp at 8.899 ppm and the phe-
nolic proton at 10.372 ppm (Table 1).
=
1675, 1658 (C O); MS: m/z = 448.3 (M + 1).
(Z)−2-(5-(4-hydroxybenzylidene)−2,4-dioxothiazolidin-3-yl)-N-
((2-phenylthiazol-4-yl) methyl)acetamide (5f): yellow solid; m.p.
232–233 °C; yield = 61%; 1H NMR (DMSO–d6, 500 MHz) δ: 10.373
(br, 1H, OH), 8.899 (t, J = 5.5 Hz, 1H, NH), 7.947 (m, 2H, Ar), 7.882
(s, 1H, -CH=), 7.535–7.506 (m, 5H, Ar), 7.457 (s, 1H, Th), 6.945
(d, J = 8.5 Hz, 2H, Ar), 4.465 (d, J = 6 Hz, 2H, -CH2-), 4.377 (s,
2H, -CH2-); 13C NMR (DMSO–d6, 125 MHz) δ: 167.847, 167.665,
166.048, 165.845, 160.771, 155.192, 134.286, 133.481, 133.145,
130.758, 129.736, 126.572, 124.284, 117.039, 116.941, 116.130,
43.946, 39.774; FT IR (KBr) νmax cm−1: 3401 (Ar-OH), 1728, 1681,
The electrons conjugation effect found in the parent compound
2 between the benzylidene nucleus and thiazolidine-2,4–dione is
present in the final compounds 5a-f too. The alkylation of com-
pound 2 (whether N-alkylation or O-alkylation would take place)
would not make the conjugation of electrons disappear, but the
substitution on the nitrogen or oxygen atoms would influence the
conjugation of electrons and consequent the changes in the chem-
ical shift of protons can be found.
For a better analysis of the signals, we have used the spec-
tral data of a supplementary compound (6), previously reported
by our group [4,23]. This compound has a similar structure with
the parent compound 2, but has the oxygen atom substituted with
a carbon atom. Compound 6 can be considered an O-alkylated
derivative of compound 2, but it was obtained by another syn-
thetic route, independent of compound 2 and independent of
the hypothesis studied in the present paper, being obtained by
Knoevenagel condensation between p-methoxybenzaldehyde and
thiazolidin-2,4–dione in alkaline environment. Spectral data of
compound 6 was used in the present paper for spectral com-
parison, to strengthen the previously formulated conclusion, that
under the reaction conditions presented in the hypothesis, takes
place the N-alkylation of compound 2, not the O-alkylation.
The benzylidene proton from the parent compound 2 was found
at 7.699 ppm, while in compounds 5a-f it was found between
7.882–7.907 ppm. Comparing to the chemical shift of this proton
from the compound 2, in compounds 5a-f, the benzylidene pro-
ton appears with 0.208–0.183 ppm (average 0.192 ppm) downfield.
On the other hand, compared to the signal given by the benzyli-
dene proton in compound 6 (substituted at the oxygen atom sub-
stituted with methyl), it appears at 7.687 ppm, with a deviation of
only 0.012 ppm. Therefore, the benzylidene proton being negligi-
bly influenced by the presence of the carbon atom on the phe-
nolic oxygen, the shift to higher values is due to the substitu-
tion at the level of the nitrogen atom in the case of compounds
5a-f.
=
1663 (C O); MS: m/z = 452.3 (M + 1).
The molecular peaks found for all final compounds 5a-f sug-
gested that alkylation took place at just one out of the two possible
atoms (N or O) of the (Z)−5-(4-hydroxybenzylidene)-thiazolidine-
2,4–dione.
Analyzing the IR spectrum, the two peaks of the potential aryl-
alkyl ether, which could appear in case of the O-alkylation, given
by the symmetric stretch (near 1040 cm−1) and the asymmet-
ric stretch (near 1250 cm−1) of the etheric bonds C O C are not
found, suggesting that alkylation took place on the nitrogen atom
for all final compounds 5a-f and not on the phenolic oxygen.
Comparing the 1H NMR spectrum of the (Z)−5-(4-
hydroxybenzylidene)-thiazolidine-2,4–dione (compound 2) with
its parent compound thiazolidine-2,4–dione (compound 1), the
most deshielded proton gave a broad signal at 12.023 ppm in
the compound 1, respectively at 12.431 ppm in the compound 2,
by the NH proton. The other supplementary broad signal found
for compound 2 in the 1H NMR spectrum, corresponding to a
less deshielded proton at 10.310 ppm is given by the phenolic
hydrogen. After assignment of the protons from the structure of
compound 2 that gave these broad signals, further analysis of
the 1H NMR spectrum of the resulted 5a-f compounds can be
performed, to trace the appearance or disappearance of signals of
interest.
–
–
Structurally, compounds 4a and 4b are chloromethyl azoles.
These compounds have no amide groups in their structures. The
reason for choosing these two compounds was that they would
not give in the 1H NMR spectrum signals of the amide NH group,
which could interfere with the observation in the same spectral
zone of signal characteristic to the NH proton of thiazolidinedione
moiety or the phenolic OH proton. Compound 4b has, in addition
to 4a, a secondary aromatic amine. This compound’s synthesis and
The four benzylidene protons on the aromatic nucleus that ap-
pear as two coupled doublets (J = 8.5 Hz or J = 9 Hz), are found
in the spectrum of the parent compound 2 at 7.454 ppm and
6.919 ppm, respectively. Two benzylidene protons are found in
compounds 5a-f at 0.059–0.099 ppm (average 0.074 ppm) down-
field compared to the parent compound 2, while in compound
3

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
Table 1
The region 8.5–14 ppm from the 1H NMR spectrum of the studied compounds.
6 they are found with 0.187 ppm more deshielded compared to
the parent compound 2. The other two protons (meta-benzylidene)
are found in the compounds 5a-f at 0.017–0.090 ppm (average
0.048 ppm) downfield compared to the parent compound 2, while
in compound 6 they are found 0.164 ppm downfield.
NMR spectrum of the compounds 2, 5e and 6 was presented in
Table 3. Moreover, in Table 4 1H-1H COSY NMR spectrum for the
compounds 2, 5e and 6 are presented in comparison. The choice
to depict the 1H NMR spectrum of the compound 5e was made
because it has a simple structure, with no supplementary struc-
tural moieties such benzene rings which would give signals in the
analyzed spectral zone and the shift of the benzylidene and the
benzylic protons can be traced without difficulty. All spectral data
For a better observation of the numerical data presented in
the Table 2 and to understand better the shifting due to substi-
tution on the two different sites, a graphical depiction of the 1H
4

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
Table 2
Comparison of some distinctive signals from the 1H NMR spectrum of the compounds 2, 5a-f and 6.
NH
OH
Benzylidene
Benzylic
Compound
CS / DRC2 (ppm)
CS / DRC2 (ppm)
CS / DRC2 (ppm)
CS / DRC2 (ppm)
CS / DRC2 (ppm)
2
12.431 / 0
missing
missing
missing
missing
missing
missing
10.310 / 0
7.699 / 0
7.454 / 0
6.919 / 0
5a
5b
5c
5d
5e
5f
10.466 / 0.156
10.370 / 0.060
10.499 / 0.189
10.689 / 0.379
10.618 / 0.308
10.373 / 0.063
7.882 / 0.183
7.899 / 0.200
7.907 / 0.208
7.892 / 0.193
7.884 / 0.185
7.882 / 0.183
7.513 / 0.059
7.532 / 0.078
7.553 / 0.099
7.524 / 0.071
7.515 / 0.061
Overlapping
6.936 / 0.017
6.946 / 0.027
6.977 / 0.058
7.009 / 0.090
6.997 / 0.078
6.941 / 0.021
6
12.265 / 0.166
missing
7.687 / 0.012
7.641 / 0.187
7.083 / 0.164
CS: Chemical Shift.
DRC2: Deviation Relative to Compound 2.
Table 3
Comparison of some distinctive signals from the 1H NMR spectrum of the compounds 2, 5e and 6.
Proton
Compound
2
5e
6
of the compounds from this paper can be found in the Supplemen-
tary material.
O-alkylated compound, appears at the same value as in the case of
parent compound 2. The same shifting effect can be observed in
the case of benzyl protons. Due to N-alkylation, there is a shift ef-
fect towards higher values for those protons, compared to the par-
ent compound 2, depicted for compound 5e in Table 3. In the case
Significant shift to a higher value of the benzylidene proton
in compound 5e can be observed as a result of N-alkylation. The
same proton, in the case of compound 6 used as the reference of
5

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
Table 4
Comparison of 1H–1H COSY NMR spectrum of the compounds 2, 5e and 6 in the 6.5–8 ppm region.
2
5e
resented by the ionized thiazolidine-2,4–dione (Fig. 3- Species 2),
while at the same pH the phenol ionized species (Fig. 3- Species 3)
is only 0.7%. Comparing the two percentages, the species 2 is found
to be at that pH at about 122 fold more abundant then species 3.
Analyzing the data representing the four microspecies distribution
used for plotting Fig. 3, the phenoxide microspecies (species 3) can
be found in a negligible amount in the reaction medium. Through-
out the entire range of pH 7–10, species 2 is always found in quan-
tities over 100 times larger than species 3.
Fig. 2. The computed pKa of the two acidic groups of the (Z)−5-(4-
hydroxybenzylidene)-thiazolidine-2,4-dione.
The pKa obtained for the two acid protons, quite different from
each other and the consequent distribution between the resulted
microspecies, guides us from this point of view that in an alky-
lation reaction of the (Z)−5-(4-hydroxybenzylidene)-thiazolidine-
2,4–dione (compound 2), performed in alkaline medium, the pref-
erential product obtained will be due to N-alkylation, because the
rate of the SN2 reaction is dependent on the concentration of the
species. All data resulted from the microspecies calculation used
in depiction of Fig. 3 is provided in the Supplementary material
as percent of species through 0–14 pH range, as resulted from
www.chemicalize.org calculation.
of compound 6 used as a reference for an O-alkylated compound,
this effect is much more intense on benzyl protons, compared with
benzyl protons from compound 5e.
Therefore, if in the case of compounds 5a-f they had been ob-
tained by O-alkylation and not by N-alkylation, as established pre-
viously, a different pattern in terms of the chemical shift in the
signals given by those four protons on the aromatic nucleus and
by the benzylidene proton could have been observed, similar to
compound 6.
Theoretical in silico evaluation of the acidity of compound
2 provided the pKa of the two discussed moieties above. The
strongest acidic proton is the one from the thiazolidine-2,4–dione
ring, with a pKa = 7.20. The phenolic OH is computed to be less
acidic, with a pKa = 9.45 (Fig. 2). A low pKa of a group suggests
an increased acidity and a relatively higher amount of its conju-
gated base in the medium. A higher quantity of an acid’s conju-
gated base in the reaction medium (a potential nucleophile) will
lead to a higher amount of its derivative to be obtained in a reac-
tion with a nucleophile.
3. Conclusions
In order to evaluate how the alkylation reaction takes place
on (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4–dione, 6 com-
pounds were synthesized, 5 of which have not been reported pre-
viously in the literature, using different aliphatic chlorinated alky-
lating agents in alkaline environment.
All compounds were obtained by N-alkylation and none of
them were resulted by O-alkylation, based on the analysis of
the spectral data. A supplementary derived hypothesis evaluated
during the present study was if there could be some poten-
tial steric hindrance which could lead to O-alkylation instead on
N-alkylation, because some of the alkylating agents used were
chloroacetamide derivatives (compounds 4c-f), different to the rest
of alkylation agents that were non-chloroacetamide derivatives
(compounds 4a-b). Analyzing the resulted spectral data, there is no
difference between those two types of alkylating agents in terms
Analyzing the ionization steps of compound 2 from the initial
acidic structure to its correspondent conjugated bases, it can be
assumed that the first acidic proton to mobilize in the presence of
a base in the reaction medium will be that from the thiazolidine-
dione (the strongest acidic function in the molecule) and not from
the phenol (Fig. 3– Species 2 vs. Species 3). The highest percent
of monoionized compound 2 is found at pH=8.3, where 86.2% of
the four microspecies possible for the discussed compound is rep-
6

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
100
90
80
70
60
50
40
30
20
10
0
Species 1
Species 2
Species 3
Species 4
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
pH
Fig. 3. The four species computed and their relative abundance of (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4-dione in the pH range of 0–14.
of regioselectivity. In all final compounds 5a-f, independent on
the type of alkylating agent used, the broad signal of the pheno-
lic proton was found ranging between 10.310–10.689 ppm, prov-
ing that the substitution took place just at the nitrogen atom of
compound 2.
MS spectra were obtained in positive ionization with an Agi-
lent 1100 series with an Agilent Ion Trap SL mass spectrometer
(70 eV) instrument (Agilent Technologies, CA, USA). IR spectra were
recorded after compression of the solid powder samples in anhy-
drous KBr pellets under vacuum, using a FT/IR 6100 spectrometer
(Jasco, Cremella, Italy).
The 1H and 13C NMR spectra were recorded on an Avance NMR
spectrometer (Bruker, Karlsruhe, Germany) using DMSO–d6 as sol-
vent at 300 K. Chemical shift values are reported in δ units as
ppm (parts per million), relative to TMS as internal standard. The
pKa and the derived microspecies concentration calculations were
performed on www.chemicalize.com, a web application which pro-
vides structure-based calculations and predictions for molecules
(ChemAxon, Budapest, Hungary) [24–26].
Moreover, significant changes in shifting was identified for the
benzylidene protons and small differences in shifting were identi-
fied for the benzyl protons signals from the final compounds 5a-
f, compared with the signals of the corresponding protons from
compound 2. Using an O-alkylated derivative (compound 6) of
compound 2 as spectral reference, a cross-validation of the N-
alkylation hypothesis of compound 2, when obtaining the com-
pounds 5a-f in alkaline environment was made.
By correlating the results of the analysis of the spectral
data with those of the acidic character of the two acid groups
of the (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4–dione (com-
pound 2), results are congruent in concluding that the alkyla-
tion of (Z)−5-(4-hydroxybenzylidene)-thiazolidine-2,4–dione (com-
pound 2) in alkaline medium is regioselective, resulting only N-
substituted reaction products when using the reagents in a 1:1
molar ratio.
4.1. Chemical synthesis
The
parent
compound
(Z)−5-(4-hydroxybenzylidene)
thiazolidine-2,4–dione (compound 2) was obtained using our
previously reported protocol, starting from thiazolidine-2,4–dione
(compound 1) [22].
In order to validate one of the possible synthetic chemical
ways and to eliminate the other, (Z)−5-(4-hydroxybenzylidene)-
thiazolidine-2,4–dione (2) was subjected to alkylation in DMF
(dimethylformamide) in alkaline environment provided by K2CO3
using the alkylation agents 4a-f (Scheme 1). The reaction mixture
was stirred overnight at room temperature, using a previously re-
ported protocol [27]. In the synthesis of the compound 5c, the
amount of K2CO3 used was doubled, because the compound 4c
was used as hydrochloride, as isolated. Because the aim of the
4. Experimental section
All chemicals used for synthesis, purification and analysis of
the compounds were of analytical reagent grade purity and have
been purchased from local suppliers. The melting points of the
synthesized compounds were obtained by the open glass capil-
lary method, using an MPM-H1 melting point apparatus (Schorpp
Gerätetechnik, Überlingen, Germany) and are uncorrected.
7

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
Scheme 1. Reaction scheme used for obtaining the intermediate compounds 4a-f.
present work was to evaluate the possible products of the reac-
tion (O or N alkyl derivatives), the reaction conditions were not
changed in order to obtain better yields in final products. General
view of the potential final compounds’ structures presented in the
discussion of the two synthesis hypothesis (N or O alkylation) is
presented in Scheme 2.
on intermediate compound 3 g [33]. The obtained compound 3 h
was isolated as yellow oily liquid and was used without further
purification.
Synthesis of intermediate compound 4f was performed by
adapting
a previously reported protocol [14]. In a glass flask
equipped with a magnetic stirrer, immersed in an ice bath, 2.85 g
(15 mmol) of intermediate 3 h, 15 mL of dichloromethane and
3.03 g (30 mmol) of triethylamine were added to the reaction mix-
ture and 2.26 g (20 mmol) of chloroacetyl chloride were added
dropwise, for about 15 min. The reaction mixture was left stirring
in a closed vessel for 2 h, waiting to reach the room temperature.
The solvent was evaporated under reduced pressure. Water was
added over the resulted solid and the resulted precipitate was fil-
tered under reduced pressure. The impure solid was recrystallized
from a mixture of methanol-water and activated charcoal, in order
to obtain the pure white solid.
Copies of MS, IR, 1H NMR and 13C NMR spectra for the syn-
thesized compounds 1, 2, 3 h, 4c-f, 5a-f and 6 are provided in the
Supplementary Material. 1H–1H COSY NMR and 1H–13C HSQC NMR
spectra were recorded for compounds 2, 5d, 5e and 6 and are in-
cluded in the Supplementary Material.
The synthesis of the aliphatic chlorine intermediates 4a-f is
shown in Scheme 1. Intermediate compounds 4a-e and the fi-
nal compound 5a were obtained using previously reported proto-
cols [14,27,28]. The characterization data of the intermediate com-
pounds 4b-e, previously reported in the literature, is consistent
with the reported data [28–31].
The intermediate compound 3 g was synthesized using a previ-
ously reported protocol [32], involving in the first step a Hantzsch
cyclisation, in order to obtain the thiazole ring and next, the
formation of the urotropinium chloride salt in the presence of
the urotropine and the previously obtained 4-(chloromethyl)−2-
phenylthiazole. Later, the formation of the intermediate com-
pound 3 h ((2-phenylthiazol-4-yl)methanamine) was based on the
experimental protocol used previously in the synthesis of (2-
phenyloxazol-4-yl)methanamine, based on the Delépine reaction
8

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
Scheme 2. Reaction scheme for the synthesis of the final compounds 5a-f.
Declaration of Competing Interest
Stana: Funding acquisition, Methodology, Writing - original draft.
Adrian Pîrna˘u: Funding acquisition, Investigation, Resources, Vali-
dation. Laurian Vlase: Investigation, Validation. Smaranda Oniga:
Funding acquisition, Resources, Validation, Writing - review & edit-
ing. Ovidiu Oniga: Formal analysis, Project administration, Super-
vision.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Acknowledgments
Gabriel Marc: Conceptualization, Funding acquisition, Formal
analysis, Investigation, Methodology, Project administration, Re-
sources, Writing - original draft, Writing - review & editing. Anca
This research was financially supported by “Iuliu Hațieganu”
University of Medicine and Pharmacy, Cluj-Napoca, Roma-
9

G. Marc, A. Stana, A. Pîrna˘u et al.
Journal of Molecular Structure 1241 (2021) 130629
nia, through PCD 7690/68/15.04.2016, 5200/59/01.03.2017,
3067/4/01.02.2018 and 1529/39/18.01.2019 and by the Core
Program (Program Nucleu), grant PN 19 35 02 01.
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azolidinediones: synthesis, in vitro investigations of antiproliferative mecha-
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[17] P.A. Datar, S.B. Aher, Design and synthesis of novel thiazolidine-2,4-diones as
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