Synthesis and Characterization of Two Novel Oxazol-5-ones Derivatives and Their Multifunctional Properties; pH Sensitivity, Electropolymerizability and Antiproliferative Activity

Article abstract of DOI:10.1007/s10895-020-02552-9

The aim of this study is to synthesize oxazol-5-one derivatives, which have multi-functional properties. Nomenclatures of newly synthesized molecules are 4-(4-N,N-diethylaminophenylmethylene)-2-(3-thienyl)oxazol-5-one (4a) and 4-(4-(1,4,7,10-tetraoxa-13-azacyclopentadecyl)phenylmethylene)-2-(3-thienyl)oxazol-5-one (4b). These two novel derivatives contain pH sensitive and polymerizable groups. 3-Thienyl group was attached to position-2 of the oxazol-5-one ring to provide electrochemical polymerization capability. pH sensing properties were provided by attaching p-N,N-diethylaminophenylmethylene and p-aza-15-crown-5-phenylmethylene groups to the arylmethylene moiety at position-4 of the ring. Target molecules were synthesized by classical process known as Erlenmeyer-Pl?chl Azlactone Synthesis Erlenmeyer (Justus Liebigs Ann Chem 275:1-12, 1893), Rodrigues et al. (J Chem Educ 92:1543-1546, 2015). After structural characterization of 4a and 4b, absorption and emission characteristics were determined in solvents that have different polarities. Difference in maximum absorption and emission wavelengths of the molecules related to solvent polarities were observed at around 6–7 nm and 35–36 nm respectively. In pH studies of the target derivatives in PVC polymer matrix, ratiometric changes were observed at isosbestic point around 398 nm. Polymeric depositions of the molecules (4a, 4b) were proved by using cyclic voltammetry, electrochemical impedance spectrometry studies and scanning electron microscope images. MTT assay studies showed significant results like, 4b derivative’s strong cytotoxic activity on PC-3 (cancerous cell line) with IC₅₀ value of 12.57 ± 0.41 μg/ml without exhibiting any cytotoxic effect on HEK293 (healthy cell line).

Full text of DOI:10.1007/s10895-020-02552-9

Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02552-9
ORIGINAL ARTICLE
Synthesis and Characterization of Two Novel Oxazol-5-ones
Derivatives and Their Multifunctional Properties; pH Sensitivity,
Electropolymerizability and Antiproliferative Activity
İbrahim Hanif Nazlı1 & Serap Alp2 & Derya Topkaya2 & İmran Güney Afacan3 & Ayşe Nalbantsoy4
Received: 6 February 2020 /Accepted: 11 May 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The aim of this study is to synthesize oxazol-5-one derivatives, which have multi-functional properties. Nomenclatures of newly
synthesized molecules are 4-(4-N,N-diethylaminophenylmethylene)-2-(3-thienyl)oxazol-5-one (4a) and 4-(4-(1,4,7,10-tetraoxa-
13-azacyclopentadecyl)phenylmethylene)-2-(3-thienyl)oxazol-5-one (4b). These two novel derivatives contain pH sensitive and
polymerizable groups. 3-Thienyl group was attached to position-2 of the oxazol-5-one ring to provide electrochemical polymer-
ization capability. pH sensing properties were provided by attaching p-N,N-diethylaminophenylmethylene and p-aza-15-crown-
5-phenylmethylene groups to the arylmethylene moiety at position-4 of the ring. Target molecules were synthesized by classical
process known as Erlenmeyer-Plöchl Azlactone Synthesis Erlenmeyer (Justus Liebigs Ann Chem 275:1-12, 1893), Rodrigues
et al. (J Chem Educ 92:1543-1546, 2015) . After structural characterization of 4a and 4b, absorption and emission characteristics
were determined in solvents that have different polarities. Difference in maximum absorption and emission wavelengths of the
molecules related to solvent polarities were observed at around 6–7 nm and 35–36 nm respectively. In pH studies of the target
derivatives in PVC polymer matrix, ratiometric changes were observed at isosbestic point around 398 nm. Polymeric depositions
of the molecules (4a, 4b) were proved by using cyclic voltammetry, electrochemical impedance spectrometry studies and
scanning electron microscope images. MTT assay studies showed significant results like, 4b derivative’s strong cytotoxic activity
on PC-3 (cancerous cell line) with IC50 value of 12.57 0.41 µg/ml without exhibiting any cytotoxic effect on HEK293 (healthy
cell line).
.
.
.
Keywords Oxazol-5-one Thiophene Electropolymerization Cytotoxicity
Introduction
known as unsaturated oxazol-5-one class, are considered as
cyclic derivatives of the amino acids. Due to their favourable
optical properties, they have been used in many studies on
biosensors [6, 7], fluorophores [8], electro photographic photo-
receptors and in non-linear optical materials [8–10].
In this study, two novel oxazol-5-one derivatives, containing
ionophor group and polymerizable group were synthesized ac-
cording to Erlenmeyer Plöchl method [1–5]. These compounds,
As is known in the literature, aza-crown ethers are suscepti-
ble to specific cations [11–15]. For example, the
monoazacrown structures bound to fluorophores as an iono-
phore group were synthesized and their ability to complex with
various cations were investigated for many years [16–22].
In the articles published, studies on development of pH,
carbon dioxide and cation-sensitive optical sensors were per-
formed using different oxazol-5-one derivatives. For example,
transparent films of the sol-gel matrix / polyvinyl chloride
(PVC) matrix of 4-(p-N,N-dimethylaminophenylmethylene)-
2-phenyl-5-oxazolone were prepared and optical pH / carbon
dioxide sensors were developed [8–10, 23]. In addition, car-
bon dioxide sensor was developed in ethyl cellulose matrix by
* Serap Alp
serap.alp@deu.edu.tr
1
Graduate School of Natural and Applied Science, Dokuz Eylul
University, Izmir, Turkey
2
Faculty of Sciences, Department of Chemistry, Dokuz Eylul
University, Tınaztepe Campus, 35160 Izmir, Turkey
3
Graduate School of Natural and Applied Science, Ege University,
Izmir, Turkey
4
Faculty of Engineering, Department of Bioengineering,
EgeUniversity, Izmir, Turkey

J Fluoresc
using carbazole side group containing oxazole-5-one deriva-
tive [24]. Furthermore, the photo physical and photochemical
properties of the 4-(2-furylmethylene)-2-phenyl-5-oxazolone
derivative in different solvents and in solid matrix of polyvinyl
chloride (PVC) have been investigated. Determination of Fe3+
ion has been carried out by using this derivative [25]. In ad-
dition, biosensor applications with oxazole-5-one derivatives
bearing monoaza-15-crown-5 group were studied.
Determination of acetylcholine (ACh), glucose and
Donepezil concentrations were performed on PVC and sol-
gel matrices [6, 8].
Sensor applications generally based on process of doping
the molecules into a polymeric matrix contained plasticizer
[26–32]. The biggest challenge in sensor studies using
fluorophore organic dyes is the reduction in molecular re-
sponse capacity due to sensor preparation. The main causes
of matrix incompatibility are aggregation, damping by
energy-electron transfer to the matrix components and reduc-
tion of sensitivity of the formed matrix films.
neuroleptic [36–40] as they are ring derivatives of N-
acylaminoacids. Oxazol-5-one derivatives with antitumor
properties pose special interest and have been studied in recent
years [37]. Main importance of oxazol-5-one derivatives cy-
totoxic activity, although their lesser potency, is that unlike
most of commercial chemotherapy agents, oxazol-5-one de-
rivatives can have specific inhibition to only one kind of can-
cerous cell line and have very small or none cytotoxicity to-
wards healthy human cell line.
As a result, sensor probe properties against pH, responses
to electropolymerization and cytotoxic activities of 4a and 4b
had been examined. These multifunctional novel derivatives
have been shown in Fig. 1.
Experimental
Besides oxazol-5-ones’ sensor application in polymer ma-
trices, the properties of being super capacitor have also be-
come the focus of recent studies. Electropolymerization stud-
ies of polythiophene-based derivatives are dramatically in-
creasing due to their potential to be used as electrode material
for super capacitors. Polythiophenes, which are electrically
conductive polymers, have been modified with organic mol-
ecules to improve electrochemical energy storage properties
and to increase specific capacitances that are crucial to alter-
native energy storage systems. Organic molecular polymeri-
zation studies are also being carried out by using thiophene-
oxazol-5-one-thiophene conjugating systems for capacitance
enhancement studies [33–35].
Based on the studies mentioned above, synthesis of new,
polymerizable group containing oxazole-5-one derivatives
(4a, 4b) that can be used as pH probes has been decided in
this project. In this respect, disadvantages of polymer matrix
can be eliminated by direct polymerization of the molecules
without the need for any immobilization process in polymer
matrices was thought.
Material and Instrumentation
All chemicals and PVC membrane components were pur-
chased from Carlo Erba, Merck, Fluka and Sigma Aldrich.
Polyester support (Mylar type) provided by Du Pont. PVC
matrices were prepared according to the previous literature
[9]. All solvents used were at spectroscopic grade. Melting
points were determined by Barnstead Electrothermal 9,100
instrument. Mass spectra were carried out with
TripleTOF®4600 system. FT-IR spectra were recorded by
Perkin–Elmer Spectrum BX FTIR spectrometer using KBr
pellets. NMR data were collected by Varian 3.2 400 MHz
spectrometer in CDCl3 solutions and chemical shifts
expressed in ppm downfield from tetramethylsilane standard.
Absorption spectra and emission-excitation spectra were re-
corded by Shimadzu UV-1601 spectrophotometer and
Varian-Cary Eclipse spectrofluorometer, respectively. SEM
Image analyses were done by Carl Zeiss 300VP SEM instru-
mentation. Voltammetric analyses were done by Autolab
PGSTAT 204. Electrochemical impedance spectrometric
(EIS) measurements were carried out with the AUTOLAB
PGSTAT-30 electrochemical analysis system (Eco
Chemieans The Netherlands). The triple electrode system
Besides to widely studied sensor and spectroscopic appli-
cations, oxazole-5-one derivatives show important biological
activities such as antifungal, antimicrobial, anticancer,
Fig. 1 Structures of the
synthesized oxazol-5-one
derivatives

J Fluoresc
Fig. 2 Synthetic scheme of 1 and
2 i) SOCl2, ii) diluted
NaOH/NH2CH2COOH
was completed with the pencil graphite electrode, the Ag /
AgCl reference electrode and the Pt auxiliary electrode.
characterization of the compound 3 was carried out only by
1H NMR technique which is enough for identification of the
structure. 0.31 mL (3.4 mmol) POCl3 was added dropwise to
precooled (-10.0 oC), 1.0 g (3.4 mmol) of N-phenyl-(aza-15-
crown-5) dissolved in 3.0 mL (40 mmol) DMF. The mixture
was first stirred at room temperature, and then it was heated to
100.0 oC for four hours until the reaction completed (Fig. 3).
After work up process, the product was directly used in next
Synthesis
3-thienylcarbonylchloride (1) and N-(3-thienylcarbonyl)
glycine (2)
1
step. Yield 90.5%, m.p.: 85-87oC, H NMR (CDCl3, 400
N-(3-thienylcarbonyl)glycine, which is a derivative of
hippuric acid, was synthesized according to literature [41,
42]. (1): 3.0 g (23 mmol) 3-thiophenecarboxylic acid, 23 mL
toluene 1.73 mL (30 mmol) thionylchloride. Synthesized
crude product of 1 was directly used without purification to
synthesize 2 (Fig. 2). (2): 3-thienylcarbonylchloride (crude
product),23.0 mL 10.0% NaOH, 1.73 g (23 mmol) glycine,
2N HCl (for neutralisation). Product was collected as white
opaque crystals. Yield 54%, m.p.: 181–183 oC, FT-IR (KBr, υ
(cm-1)): 3400 − 2400 broad (-O-Hstretch), 3107 (= C-Hstretch),
MHz, δ, (ppm)): 9.65 (1H, s, aldehyde), 7.62–7.68 (2H,d,
J = 8.8 Hz, phenyl), 6.62–6.68, (2H, d, J = 9.2 Hz, phenyl),
3.6–3.65, (4H, m, crown ether), 3.71–3.76, (4H, m, crown
ether), 3.6–3.62, (8H, s, crown ether), 3.56–3.59, (4H, s,
crown ether).
Synthesis of 4 Derivatives (4a and 4b)
1
1681 (C = Ostretch), 1277 (O-C = Ostretch), H NMR (DMSO-
Aldehyde (1 eq.), glycine derivative (1 eq.), sodium acetate (1
eq.) and acetic anhydride (3 eq.) mixture was stirred at room
temperature for 10 min then at 85–90 oC for 4–6 h. After this
step, mixture cooled down to room temperature and products
were precipitated by using cold ethanol. Obtained precipitates
were filtered and washed with cold ethanol then with hot
water.
d6, CDCl3, 400 MHz, δ (ppm)): 12.25 (1H, br s, -COOH),
8.04 (1H, s, -NH), 7.97 (1H, dd, thiophenyl), 7.43 (1H, dd,
thiopheyl), 7.24 (1H, dd, thiophenyl), 3.97 (2H, d, J = 5.8 Hz,
-CH2), 13C NMR (DMSO-d6, CDCl3, 400 MHz, δ (ppm)):
176.49 (-COOH), 167.81 (-CONH-), 142.01 (thiophenyl-
C3), 133.55 (thiophenyl-C2), 131.69 (thiophenyl-C5), 130.72
(thiophenyl-C4), 46.01 (-CH2-).
4-(4-N,N-diethylaminophenylmethylene)-2-(3-thienyl)
oxazol-5-one (4a)
4-formylbenzo-aza-15-crown-5 (3)
0,72 g, (4.1 mmol) 4-N,N-dimethylaminobenzaldehyde,
0.75 g (4.1 mmol) 3-thienylcarbonyl glycine, 0.34 g (4.1
mmol) sodium acetate and 6 mL acetic anhydride (Fig. 4).
Product was collected as orange crystal. Yield 56%, m.p:
197–200 C, H NMR (DMSO-d6,-CDCl3, 400 MHz, δ,
(ppm)): 8.09 (1H, dd, J = 3.2 Hz, 1.2 Hz, thiophenyl), 8.07
The commercially available N-phenyl-aza-15-crown-5 with
phosphoroxychloride in dimethylformamide (DMF) was used
to attach the aldehyde group to the para position of the ben-
zene ring. This reaction was performed according to the article
that studied by Mateeva N. et al. [43]. Structural
o
1
Fig. 3 Synthetic scheme of 3 iii):
POCl3/DMF “Vilsmeier-Haake
Reaction”

J Fluoresc
Fig. 4 Synthetic scheme of 4a vi):
(CH3CO)2O/CH3COONa
(2H, d, J = 9.2 Hz, phenyl), 7.72 (1H, dd, J = 4.8 Hz, 1.2 Hz,
thiophenyl), 7.41 (1H, dd, J = 4.8 Hz, 1.2 Hz, thiophenyl),
7.17 (1H, s, =CH-, methine), 6.71 (2H, d, J = 9.2 Hz, phenyl),
3.45 (4H, q, J = 7.2 Hz, -CH2-CH3 ), 1.23 (6H, t, J = 7.2 Hz, -
CH2-CH3), 13C NMR (DMSO-d6,-CDCl3, 400 MHz, δ,
(ppm): 168.44 (-C5 oxazolone), 156.78 (-C2 oxazolone),
150.03 (-C4 oxazolone), 135.01 (phenyl), 133.10
(thiophenyl), 129.48 (thiophenyl), 128.95 (phenyl), 127.49
(phenyl), 126.86 (thiophenyl), 126.39 (thiophenyl), 111.34
(phenyl), 129.54 (= CH-) 44.60 (-CH2-CH3), 12.56 (-CH2-
CH3), LC-ESI-QTOF MS: m/z Found: [M + H]+ 327.1253;
‘molecular formula C18H18N2O2S’ requires [M + H]+
327.1167.
crown ether), 70.29 (2C, crown ether), 69.99 (2C, crown
ether), 68.25 (2C, crown ether), 52.83 (2C, crown ether),
LC-ESI-QTOF MS: m/z Found: [M + H]+ 473.1760; ‘molec-
ular formula C24H28N2O6S’ requires [M + H]+ 473.1746.
Spectrophotometric Studies
Absorption, emission and excitation spectra of synthesized
derivatives 4 in six different solvents were recorded and max-
imum wavelength values were determined. The combined ab-
sorption spectra of the derivative 4a in six different solvents
are given in Fig. 6. Stokes’ shift values, molar absorption
coefficients, fluorescence quantum yields, radiative lifetimes,
fluorescence lifetimes, radiative and non-radiative rate con-
stants were calculated (Table 1). Fluorescence quantum yields
(ΦF) were determined by the comparative method [44, 45]:
4-(4-(1,4,7,10-tetraoxa-13-azacyclopentadecyl)
phenylmethylene)-2-(3-thienyl)oxazol-5-one (4b)
1 . 0 g , ( 3 . 1 m m ol ) , 4 - ( 1 , 4, 7, 1 0- t et r a ox a- 13 -
azacyclopentadecyl) benzaldehyde, 0.57 g (3.1 mmol) 3-
thienyl carbonyl glycine, 0.25 g (3.1 mmol) sodium acetate
and 5 mL acetic anhydride (Fig. 5). Product has orange-red
ΦF ¼ Φstd  ðF Astdη2Þ=ðFstd A η std2Þ;
ð1Þ
where F and Fstd are the areas under the fluorescence emis-
sion curves of the samples and the standard, respectively. A
and Astd are the absorbances of the samples and standard at the
excitation wavelength and η and ηstd are the refractive indexes
of solvents used for the samples and standard, respectively.
Rhodamine 101 (in ethanol) (Φ = 1) [46, 47] was employed as
the standard.
The radiative lifetime, τ0, was estimated by the formula
[48, 49] assuming the lowest singlet state is the only fluores-
cent state:
o
crystal appearance. Yield 48%, m.p: 247–249 C, FT-IR
(KBr, υ,(cm− 1)): 1771 (C = Ostretch), 1602 (C = Nstretch),
1
1124 (-C-Ostretch), H NMR (DMSO-d6,-CDCl3, 400 MHz,
δ, (ppm)): 8.10 (1H, dd, thiophenyl), 8.07 (2H, d, phenyl ),
7.72 (1H, dd, thiophenyl), 7.42 (1H, dd, thiophenyl), 7.17
(1H, d, =CH-, methine), 6.72 (2H, d, phenyl), 3.80 (4H, t,
crown ether), 3.69 (4H, t, crown ether), 3.67 (8H, s, crown
ether), 3.63 (4H, s, crown ether), 13C NMR (DMSO-d6,-
CDCl3,400 MHz, δ, (ppm)): 168.35 (-C5 oxazolone), 157.14
(-C2 oxazolone), 150.13 (-C4 oxazolone), 134.82 (phenyl),
132.82 (thiophenyl), 128.89 (thiophenyl), 128.02 (phenyl-
C1), 121.74 (phenyl-C4), 126.92 (thiophenyl), 126.35
(thiophenyl), 111.72 (phenyl), 129.70 (= CH-), 71.31 (2C,
ꢀ0 ¼ 3:5 Â 108=ꢁmax2"max4ꢁ1=2
ð2Þ
Fig. 5 Synthetic scheme of 4b
vi): (CH3CO)2O/CH3COONa

J Fluoresc
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ACN
DCM
DMF
Ethanol
THF
Toluene
324
374
424
474
524
574
nm
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ACN
DCM
DMF
Ethanol
THF
Toluene
324
374
424
474
524
574
nm
Fig. 6 UV-Vis absorption spectra of 4a and 4b in six different solvents
where νmax is the wave number in cm− 1, εmax is the molar
extinction coefficient at the selected absorption wavelength,
and Δν1/2 is the half width of the selected absorption in wave-
number units of cm− 1. The fluorescence lifetime (τf) is calcu-
lated by the formula given below:
ΦF ¼ ꢀf =ꢀ0;
ð3Þ
The radiative (kr) and non-radiative (knr) rate constants
were calculated from the fluorescence quantum yields and
radiative lifetimes by using:

J Fluoresc
as a normal cell line HEK293 (human embryonic kidney cells)
were used for testing cytotoxicity of 4a and 4b derivatives. All
cell lines were purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA) and maintained in
Dulbecco’s modified Eagle’s medium F12 (DMEM/F12),
supplemented with 10% fetal bovine serum (FBS), 2 mM glu-
tamine, 100 U/mL of penicillin and 100 µg/mL of streptomy-
cin (Lonza, Visp, Switzerland). The cells were incubated at 37
oC in a humidified atmosphere of 5% CO2. The cells were
sub-cultured twice a week and cells in the exponential growth
phase were used in the experiments.
kr ¼ ΦF=ꢀf ¼ 1=ꢀ0 and knr ¼ ð1 À ΦFÞ=ꢀf;
ð4Þ
In this part of the work, polyvinylchloride (PVC) films
were prepared to determine the response of derivatives 4 to
hydrogen ion, according to previous conventional studies [9]
and the performance of the responses against pH was demon-
strated. The absorption studies of derivatives 4 prepared in
−
2−
PVC matrix in H2PO4 / HPO4 buffer at pH 1.0–7.0 are
given in Fig. 7.
Cytotoxicity of samples were determined using a modified
MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazoliumbromide)] assay, which detects the activity of mito-
chondrial reductase of viable cells [50]. In order to perform the
cytotoxicity assay, all cell lines were cultivated for 24 h in 96-
well microplates with an initial concentration of 1 × 105 cells/mL
Voltammetric Studies
Firstly, 0.14 g sodium perchlorate monohydrate was dissolved
in 10 mL acetonitrile. Thereafter 3.6 mg 4a was added to
mixture and dissolved. 2.5 mL of this solution was transferred
into the voltammetric cell where a pencil graphite electrode
(Faber castel, 2B, 0.7 mm) was the working electrode. The
three-electrode system was completed with Ag/AgCl as the
reference electrode platinum wire as the counter electrode.
Electropolymerization process was carried out by cycling the
potential in range of -0.5–1.5 V at a scan rate of 50 mV/s and
polymeric film depositions were inspected by electrochemical
impedance spectroscopic (EIS) measurements. SEM studies
were also carried out for further surface inspection of poly-
meric film deposition.
o
in a humidified atmosphere with 5% CO2 at 37 C. Then, the
cultured cells were treated with different dilutions of samples (4a
and 4b derivatives) (0.5, 5, 50 µg/ml) followed by incubation for
o
48 h at 37 C. Doxorubicin was used as positive control. The
optical density of the dissolved material was measured at 570 nm
spectrophotometer (Thermo Multiskan Spectrum). The viability
(%) was determined by the following formula:
% Viable cells= [(absorbance of treated cells) (absorbance of
blank)] / [(absorbance of control) – (absorbance of blank)] × 100.
Determination of half-minimal inhibitory concentration
(IC50), cytotoxicity was expressed as mean percentage increase
relative to the unexposed control SD. Control values was set
to 0% cytotoxicity. The IC50 values were reported at 95%
confidence intervals ( 95% CI). The analyses were performed
by using Graph Pad Prism 5 software (San Diego, ca., USA).
Cytotoxicity Studies
PC-3 (human prostate adenocarcinoma), A549 (human alve-
olar adenocarcinoma), SHSY-5Y (human neuroblastoma) and
Table 1
Maximum wavelengths (λ: nm,), Stokes’ shift values (Δλ:
(τf: ps), radiative (kr) and nonradiative (knr) rate constants obtained from
absorption, emission and excitation spectra of 4a and 4b in six different
solvents
nm), molar extinction coefficients (ε: mol-1dm3cm-1), fluorescence
quantum yields (ϕF), radiative lifetimes (τ0: ns), fluorescence lifetimes
abs
f
ex
Solvent
λmax
.
λmax
λmax
Δλ
(nm)
ɛ
ϕF
τ0(ns)
τf(ps)
kr
knr
(nm)
(nm)
(nm)
mol− 1dm3
cm− 1x(104)
4a
4b
THF
460
467
460
467
461
461
456
460
453
462
455
457
511
517
527
530
517
494
509
514
521
527
514
492
478
481
479
481
481
472
472
479
459
480
476
568
51
50
67
63
56
33
53
54
68
65
59
35
7.29
6.31
8.92
6.75
5.09
7.38
8.69
9.57
9.14
8.68
8.68
9.36
0.0065
0.0046
0.0041
0.0074
0.0044
0.0040
0.0052
0.0037
0.0040
0.0063
0.0032
0.0040
0.067
0.077
0.056
0.077
0.101
0.061
0.050
0.051
0.049
0.055
0.053
0.040
0.434
0.353
0.228
0.570
0.446
0.243
0.261
0.190
0.194
0.346
0.172
0.158
14.92
12.92
17.97
13.00
9.92
2287.2
2820.8
4375.6
1742.0
2230.4
4094.2
3815.1
5256.6
5134.2
2872.4
5811.6
6313.9
DCM
ACN
DMF
Ethanol
Toluene
THF
16.52
19.90
19.57
20.52
18.15
18.72
25.10
DCM
ACN
DMF
Ethanol
Toluene

J Fluoresc
0.9
0.7
0.5
0.3
0.1
-0.1
pH 1
pH 2
pH 3
pH 4
pH 5
pH 6
Buffer
321
371
421
471
nm
521
571
0.9
0.7
0.5
0.3
0.1
-0.1
pH 1
pH 2
pH 3
pH 4
pH 5
pH 6
Buffer
324
374
424
474
524
574
nm
Fig. 7 Absorption spectra of 4a and 4b at various pH values
Results and Discussions
solvent polarities were observed in extracted data.
Differences in maximum absorption and emission wave-
lengths of the molecules related to solvent polarities were
observed at around 6–7 nm and 35–36 nm respectively. The
longest absorption and emission wavelengths were detected in
DMF and the highest Stokes’ shifts were determined in ACN
(Table 1).
In pH studies of the target derivatives in PVC polymer
matrix, ratiometric changes were observed at isosbestic point
around 398 nm. While the absorption intensity at 450–480 nm
Within the scope of this study, at first pH sensitivity and probe
characteristics of the newly synthesized derivatives 4 have
been investigated by UV-Vis absorption and emission spec-
troscopies, secondly electropolymerization and copolymeriza-
tion studies have been carried out by cyclic voltammetry.
Moreover, biological activities have been determined by cy-
totoxicity tests. Since derivatives 4 contain groups with simi-
lar electronic effects, no significant changes dependent to

J Fluoresc
was decreasing, a new absorption band occurred at 330–390
nm depending on the pH value. With the results obtained from
pH studies it has been proven that these molecules can be used
in preparation of pH-sensor probe.
Voltammetric Behaviour of 4a Compound
Figure 8 shows voltammogram obtained by 7 consecutive
cycles. In these voltammograms, two oxidation peaks were
observed at 0.8 and 1.3 V in anodic scan. Small reduction
peaks were observed at 1.2 V, 0.9 and 0.4 V in cathodic scan
indicating a quasi-reversible redox mechanism is involved. In
addition, a polymeric deposition of the species can be
established from the successive increase in the peak currents.
Another proof of the polymeric film deposition can be ob-
tained by electrochemical impedance spectroscopic (EIS)
measurements. Deposition of a vast range of coatings includ-
ing polymeric structures on metallic or carbon based surfaces
can be tested via EIS measurements. The free ions in the
deposition solution penetrate into the polymeric structure
and alter the insulating structure of the polymer, which mod-
ifies the impedance characteristics of the overall conductive
surface/polymer element. In the present study, the EIS mea-
surements have utilized to monitor the charge transfer resis-
tance (Rct) of bare electrode before and after the
electropolymerization. The Rct value of bare electrode about
1299 ohm decreased to 106 ohm in the presence of 4a. This
can be interpreted as the conductive polymer deposited on the
surface significantly reduces the surface resistance. Similar
results have been obtained in the presence of thiophene (178
ohm) and EDOT (14 ohm) (Table 2).
Fig. 8 Cycling voltammogram of 4a compound
tests were done. Cytotoxic activities of 4a and 4b derivatives
were performed on PC-3 (human prostate adenocarcinoma),
A-549 (human lung carcinoma), SHSY5Y (human neuroblas-
toma cell line) and HEK293 (normal cell line) cells (Table 3;
Fig. 10) by MTT assay.
Significant results were obtained on SHSY5Y and PC-3
cells. The MTT assay results demonstrated that while deriva-
tive 4a did not show any cytotoxicity on A549 and PC-3, it
exhibited cytotoxic activity on SHSY5Y and HEK293 with an
IC50 values of 23.38 1.99 and 27.89 6.62 µg/ml, respec-
tively. Unlike 4a, the derivative 4b only showed strong cyto-
toxic activity on PC-3 with IC50 value of 12.57 0.41 µg/ml
without exhibiting any cytotoxic effect on HEK293. It is an
important result that the derivative 4b does not show cytotox-
icity on the HEK293 at tested maximum concentration (50 µg/
ml). As a result, derivative 4b can be used as fluorescent
biocompatible material in cell imaging systems and can be
considered as a promising anti-cancer agent for human pros-
tate adenocarcinoma to furtherly studied.
Close inspection of the surface has revealed the porous and
clustered polymeric structure of the deposit (Fig. 9a). In addi-
tion to the homopolymer of 4a, its copolymers with thiophene
and EDOT were synthesized electrochemically on a pencil
graphite electrode by applying the same procedure. SEM im-
ages of these co-polymers can be seen in Fig. 9b and c,
respectively.
Conclusions
For the former species, the 4a polymeric clusters can be
detected on the cauliflower-like porous structure of
polythiophene and for the latter, it was more apparent on the
finely distributed deposits of PEDOT co-polymers. Similar
studies were repeated for molecule 4b and negligible differ-
ences has been observed compared to the results of 4a.
Within the scope of this study, multifunctional dye molecules
have been synthesized, pH sensitivity of the target 4
Table 2 Electrochemical Impedance Spectroscopic (EIS)
Measurements
4a
Z (ohm)
4b
Z (ohm)
Cytotoxic Activity Studies
Bare Electrode
Thiophene
4a
1299
454
Bare Electrode
Thiophene
4b
1299
454
Since it’s also among the study targets that synthesized deriv-
atives 4 would have biological activities due to the functional
groups they contain (five membered heterocyclic ring, 4-N,N-
dialkylaminophenyl and thiophene moieties), cytotoxicity
106
388
4a + Thiophene
4a + EDOT
177,8
13,95
4b + Thiophene
4b + EDOT
222,6
305

J Fluoresc
Fig. 9 SEM images taken at the magnitude of 100x, 500x and 1000x respectively left to right of a) homopolymerization of 4a b) copolymerization of 4a
+ thiophene c) copolymerization of 4a + EDOT
derivatives in PVC polymer matrix has been revealed and pH
dependent ratiometric changes have been observed which are
extremely important in sensor applications. To avoid disad-
vantages of polymer matrices, electropolymerization and
copolymerization studies have been carried out on the thio-
phene group of target molecules, which mentioned as superi-
ority of the synthesized structures and it has been proved that
polymeric coating can be performed on the electrode. The
Fig. 10 The effect of 4a and 4b molecules on various cancer cell lines (A549, PC-3, SHSY5Y) and normal cell line (HEK293)

J Fluoresc
Table 3 Evaluation of the effect
of 4a, 4b and Doxorubicin on
various cancer cell lines (PC-3,
A-549, SHSY5Y) and normal cell
line (HEK293) by MTT assay
(IC50 values (µg/ml))
A549
PC3
HEK293
SHSY-5Y
4a
50 µg/ml<
50 µg/ml<
23.38 1.99 µg/ml
27.89 6.62 µg/ml
4b
-
12.57 0.41 µg/ml
5.52 0.21 µg/ml
-
-
Doxorubicin
4.96 1.54 µg/ml
1.19 0.92 µg/ml
5.77 2.82 µg/ml
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derivatives 4 can be directly coated on the electrode surface in
the form of polymer and copolymer films without the need of
a different polymer matrix or immobilization process.
As the electropolymerisability of these dye molecules is
proven by voltammetric studies, it can be concluded that pH
sensor applications are promising for voltammetric methods
as much as spectroscopic methods. The most important result
of studied derivatives as cytotoxic agents is that they are
promising molecules in anti-cancer application fields.
It is expected that some molecules may show selective
cytotoxicity on PC3 cells due to the difference in membrane
structure of PC3 cells compared to HEK293. Cytotoxic agents
may cause toxicity on cells via different mechanisms such as
destruction of cell membranes, prevention of protein synthe-
sis, irreversible binding to receptors, inhibition of
polydeoxynucleotide elongation or enzymatic reactions.
As the 4 molecules show selective cytotoxic activity on
cancer cells they need further investigation for elucidation of
their bioactive cytotoxic mechanisms and this study about
newly synthesized oxazole-5-one derivatives may shed light
on future studies in this field.
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Acknowledgements We gratefully acknowledge the financial support for
this project (116Z095) from The Scientific and Technological Research
Council of Turkey (TUBITAK).
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