Synthesis and fluorescence properties of aminocyanopyrrole and aminocyanothiophene esthers for biomedical and bioimaging applications

Article abstract of DOI:10.1016/j.molstruc.2020.127974

We prepared a series of substituted aminocyanopyrroles and another of aminocynaothiophenes. We describe an efficient new one-step synthetic strategy via the condensation of an alkyl sarcosinate and ethoxymethylenemalononitrile, through a Gewald-like reaction. The UV–visible absorption and steady-state and time resolved fluorescence properties of some representative compounds, as well as their acid-base behavior, is also presented. The compounds might be useful for medicinal applications and as bioimaging probes.

Full text of DOI:10.1016/j.molstruc.2020.127974

Journal Pre-proof
Synthesis and fluorescence properties of aminocyanopyrrole and
aminocyanothiophene esthers for biomedical and bioimaging applications
Oussama Cherif, Asma Agrebi, Sérgio Alves, Carlos Baleizão, José Paulo Farinha,
Fatma Allouche
PII:
S0022-2860(20)30299-4
DOI:
https://doi.org/10.1016/j.molstruc.2020.127974
MOLSTR 127974
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 November 2019
Revised Date: 9 February 2020
Accepted Date: 25 February 2020
Please cite this article as: O. Cherif, A. Agrebi, Sé. Alves, C. Baleizão, José.Paulo. Farinha, F.
Allouche, Synthesis and fluorescence properties of aminocyanopyrrole and aminocyanothiophene
esthers for biomedical and bioimaging applications, Journal of Molecular Structure (2020), doi: https://
doi.org/10.1016/j.molstruc.2020.127974.
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Synthesis and fluorescence properties of aminocyanopyrrole and
aminocyanothiophene esthers for biomedical and bioimaging
applications
Oussama Cherif a, Asma Agrebi a, Sérgio Alves b,*, Carlos Baleizão b,c, José Paulo
Farinha b,c, Fatma Allouche a,*
a Laboratory of Medicinal and Environmental Chemistry, Superior Institute of Biotechnology of Sfax, University of Sfax, 3018
Sfax – Tunisia.
b Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av Rovisco Pais, 1049-001 Lisboa,
Portugal.
c Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av Rovisco Pais, 1049-001 Lisboa,
Portugal
* Corresponding authors.
E-mail addresses: sergio.c.alves@tecnico.ulisboa.pt (S. Alves), fatmaallouch@yahoo.fr (F. Allouche)
Abstract
We prepared a series of substituted aminocyanopyrroles and another of aminocynaothiophenes. We describe an efficient new
one-step synthetic strategy via the condensation of an alkyl sarcosinate and ethoxymethylenemalononitrile, through a Gewald-
like reaction. The UV-visible absorption and steady-state and time resolved fluorescence properties of some representative
compounds, as well as their acid-base behavior, is also presented. The compounds might be useful for medicinal applications and
as bioimaging probes.
Keywords: Aminocyanothiophene · Aminocyanopyrrole · Sarcosinate · Gewald-like reaction · One-pot synthesis · Fluorescent
probes

1. Introduction
-O
H
O
H
N
Cl
HCl
N+
H
Pyrrole and thiophene derivatives are frequently found in
naturally occurring compounds, many of which exhibit useful
biological activity [1,2]. Polyfunctional derivatives of 3-
aminopyrroles are an important family of compounds [1],
with many applications as antibacterial, antiviral,
anticonvulsant, anti-inflammatory, analgesic and antipyretic
agents [3-6]. Pyrroles are also building blocks for porphyrins
[7], and polymers of pyrroles have found use as conducting
polymers and materials for non-linear optics [8]. Different
methods for the preparation of this heterocyclic system have
been proposed [9-13]. They can also further undergo a wide
variety of chemical reactions including electrophilic
substitution reactions, acylation and alkylation reactions at the
nitrogen [14].
OH
OH
R1
1
1'
R1OH
HCl
NC
R2
NC
R2
NH2
C C
O
H
NC
OEt
O
N+
H
N
OR1
DMF / K2CO3
- HCl
O
2
CH3
3
3b R1: CH3
R2: CH3
3a R1: CH3
R2: H
3c
R1: CH3
R2: C2H5
In the other hand, multi-substituted thiophene compounds
have demonstrated a broad spectrum of uses, including in
agrochemical applications [15], as potential antioxidant and
anti-inflammatory agents [16,17], anti-HIV PR inhibitors
[18], anti-breast cancer [19], anti-avian influenza virus
(H5N1) [20], multitarget kinase inhibitors [21], AMPK
activators [22], antitubercular agents [23], and as fluorescent
bioimaging dyes [24]. For this purpose there is also the
potential of enhancing the fluorescence properties through
aggregation-induced emission by immobilization of the dyes
in nanoparticles [25-43].
3d R1: CH3
R2: Ph
3e R1: C2H5 3f R1: C2H5
R2: CH3 R2: H
Scheme 1. One-pot strategy used for pyrroles synthesis.
with a halogen compound. In this work, the 3-amino-4-
cyanothiophene-2-carboxylates
4
(Scheme 2) were
synthesized via a single step process of a 3-alkyl-3-
alkoxyacrylonitrile malononitrile and a mercaptoacetic ester
in DMF and K2CO3 by a modified reaction of the Gewald
method [48-50]. The structures of the synthesized compounds
were confirmed by NMR and IR spectroscopies and by mass
spectrometry.
Hence, the development of substituted aminopyrroles and
aminothiophenes is of great interest in organic synthesis as
well as in photochemistry.
Here we aim to synthesize heterocyclic compounds with
pyrrole and thiophene groups functionalized with amine,
nitrile and esther groups at specific positions through simple
methods. These compounds are potentially biologically active
and might serve as building blocks for other useful molecules.
We also expect them to have interesting photophysical
properties that might make them useful for bioimaging,
especially with fluorescence-related microscopies including
confocal microscopy.
NC
NC
R2
C C
HS CH2 CO2R1
DMF / K2CO3
+
OEt
NC
R2
NH2
CO2R1
CN
S CH2 CO2R1
NC
R2
S
4
2. Results and discussion
4b R1: CH3
R1: C2H5
R2: CH3
4a R1: CH3
4c
2.1. Synthesis
Synthetic methods for preparing a series of substituted
pyrrole by amines at position 3 have rarely been reported [44-
46]. The synthesis of aminocyanopyrroles 3 was carried out,
using a multi-component reaction, through two steps. The first
step was an esterification reaction of an acetylchloride with an
alcohol which generated hydrochloric acid in the medium.
Sarcosine 1 was then added, and transformed into the
corresponding salt 1’, which in the presence of HCl and of an
excess of the alcohol underwent esterification to give the
corresponding hydrochloride of alkyl sarcosinate 2. In the
following steps, we have added to the mixture DMF and
K2CO3 as alkaline reagent (releasing one molecule of
hydrochloric acid and turning 2 into the corresponding alkyl
sarcosinate) and ethoxymethylenemalononitrile. The reaction
was heated under reflux for 6 hours at 60°C, yielding the
corresponding aminocyanopyrrole 3 (Scheme 1).
R2: H
R2: CH3
Scheme 2. Synthetic strategy for thiophenes 4.
2.2. Fluorescence, UV-Vis Absorption and Acid-base
Properties
The structure of the different compounds is very similar in
terms of ionizable groups, so we have studied one
representative compound of each of the pyrrole and thiophene
families, 3a and 4a, as well as the pyrrole derivative with a
conjugated phenyl group 3d.
In Figure 1 we present UV-Vis absorption (a), fluorescence
emission (b) and fluorescence excitation (c) spectra of 4a.
Other fluorescence emission spectra of 4a can be found in
Supplementary Material (fig. S1). The molar absorptivity
coefficients of the thiophene 4a (see Figures 1a, S2a and S3a
and table S1) and its respective fluorescence are more intense
than those of the pyrrole compounds 3a and 3d, and those of
3d (with the phenyl group) are more intense than those of 3a.
Several methods for the preparation of thiophenes have
been reported as well as their applications in the synthesis of
useful compounds [15-24,47,48]. Most thiophenes are formed
by reaction of a 3-thionate salt acrylic ester or acrylonitrile

in the absorption around 400-450 nm, and the corresponding
change in the fluorescence intensity when exciting at this
wavelength. The compound can still be excited at 460 nm,
making it usable in confocal microscopy. The titration curve
shown in Fig. 2a was obtained using this excitation
4
4
×
4 10
a
6
1
a
×
2 10
A
AH
pH
3
0.5
0
300
400
500
600
Wavelength / nm
1
b
0
0
2
4
6
6
6
8
10
10
10
12
pH
1.2
1
b
0.5
0
AH
A
pH
AH2
0.8
0.4
0.5
450
550
650
Wavelength / nm
1
0.5
0
c
0
1
0
12
2
4
8
pH
1
pH
A
c
AH
0.5
0
AH2
300
400
Wavelength / nm
Figure 1. UV-Visible absorption (a), fluorescence emission (λexc
=
435 nm) (b) and excitation (λem = 500 nm) (c) spectra of 4a (from red
to violet: pH 1.2; 1.8; 2.56; 3.13; 3.60; 4.20; 4.70; 5.23; 5.85; 6.47;
7.06; 7.73; 8.73; 9.53; 10.32; 1.02; 11.60).
0
2
4
8
12
pH
Figure 2. Titration curves overlapped with the calculated speciation
curves (thin solid lines) and titration fitting curves (thick dashed
lines): (a) 4a (Emission, λexc = 460 nm. Blue: I(λem = 480 nm)/I(λem
= 600 nm); (b) 3a (Emission, I(λem = 370 nm)/I(λem = 430 nm). Blue:
λexc = 270 nm; red: λexc = 340 nm; (c) 3d (Red: Normalized UV-vis
absorption, λ = 285 nm; blue: emission, λexc = 345 nm, I(λem = 550
nm)/I(λem = 450 nm). I: Intensity
In order to have a better view the behavior of the
absorption and fluorescence of the compounds depending on
the pH and to determine the ionization constants, we show
titration curves of the lone compounds at representative
wavelengths in Fig. 2. Bearing in mind the possibility of
ratiometric imaging [51,52], we show the fluorescence curves
as a ratio of fluorescence intensities at two wavelengths. We
also present in the plots the speciation curves which were
simulated with the HySS2009 software package [53]. By
fitting the experimental data with simulated curves of
absorption or fluorescence intensity, we obtain the ionization
constants (Table 1). In Table S1 we also present the
estimations for molar absorption coefficients resulting from
the fittings of the absorption spectra with the estimated pKa
values.
wavelength.
In Figure 2b and 2c we also present the titration curves for
the pyrrole compounds 3a and 3d at representative
wavelengths. Their UV-visible, fluorescence emission and
excitation spectra can be seen at Supplementary Material
figures S1 and S2. We propose possible acid-base equilibria
in Scheme S1.
The pyrrole compounds 3a, 3d have an extra ionization
constant when compared to the thiophene 4a, as expected due
The titration of compound 4a (Fig. 2a) shows the existence
of one acid-base equilibrium, which provokes a great change

to the nitrogen in the pyrrole ring. The presence of the
hydrophobic phenyl group makes compound 3d more acidic
than 3a, possibly in part due to the extra gain in stability in
water if the compound is protonated.
290 nm and collecting at 450 nm than when exciting at 335
nm due to a greater weight in the shortest component. The
average lifetime 〈τ〉 is also generally shorter for 3d than for
3a.
We conclude that 3d has a greater molar absorptivity and
fluorescence intensity when comparing with 3a due to the
presence of the phenyl group. This group also seems to lower
slightly the pKa values of 3d relatively to 3a, and its decays
become faster.
We have also measured the fluorescence lifetimes of 4a, 3a
and 3d at representative pH values. The decays were analyzed
with a sum of three exponential functions, yielding good
fitting results (see Supplementary Material Tables S3, S4 and
S5). An example of a decay is shown in Figure 3. The
remaining decays can be seen in Supplementary Material
figures S4 to S22.
Conclusions
Table 1. Ionization constants obtained by fitting of the
spectrofluorimetric and spectrophotometric titration curves
with the calculated speciation curves. Proposed acid-base
equilibria are depicted at Scheme S1 (Supplementary
Material)
In summary, we have developed an efficient new strategy
of
preparation
of
3-amino-4-cyano-1H-pyrrole-2-
carboxylates, in two steps from commercially available
inexpensive starting materials. We also describe an easy
protocol for the preparation of 2-aminothiophenes. These
products constitute building blocks useful in the access to
many nitrogen and sulphur heterocycles with potential
therapeutic interest or which could serve as precursors for
many useful biologically active molecules. They could also be
potentially useful fluorescent dyes for bioimaging
applications, especially in the case of thiophene derivatives,
which can be excited at 460 nm, making them usable in
confocal microscopy applications.
Compound
pKa1
pKa2
4a
3a
3d
3.1±0.2
2.5±0.2
1.3±0.2
-
6.5±0.2
4.3±0.2
As can be seen in Table S3 (Supplementary Material), in
the case of the thiophene compound 4a we observe that the
decays are relatively unchanged by the acid-base equilibria,
but are faster for the emission band appearing at 410 nm than
when collected at 350 nm. At pH ≈ 2, when exciting at 335
nm and collecting at 420 nm, the decay lifetime values of the
different components remain relatively unchanged from when
exciting at 285 nm, although the average lifetime is shorter
due to a greater weight of the shortest component.
Some representative compounds described herein were
tested for their UV-visible absorption and fluorescence
properties, as well as their acid-base behavior. We have seen
that the molar absorptivity and fluorescent intensity of the
thiophene compound 4a are significantly higher than those of
the pyrrole compounds 3a and 3d, and that the presence of the
phenyl group of 3d also affects the absorption and
fluorescence properties. The fluorescence and UV-visible
absorption capabilities of these compounds might enable
further studies of their biological activities by these
techniques.
Experimental
IR Spectra were determined for KBr on a Jasco Fourier
transform FT–IR 420 spectrometer with precision of 2 cm-1
covering the field of 400–4000 cm-1.
1
The H NMR and 13C NMR spectra were recorded in
solution in dimethyl sulfoxide (DMSO-d6) on a Bruker
spectrometer (1H at 400 MHz, 13C at 100 MHz). The chemical
shifts are expressed in parts per million (ppm). The
multiplicities of the signals are indicated by the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quadruplet;
and m, multiplet, and coupling constants are expressed in
hertz.
Figure 3. Fluorescence decay curve of compound 4a at pH 2.2, λexc
= 285 nm, λemi = 350 nm. Weighted residuals are plotted below the
decay curve. The fluorescence decay lifetimes were obtained by
fitting the decay curve with a sum of three exponential functions
(with weights of each decay component τ1, τ2 and τ3 being w1, w2 and
w3). The average decay lifetime is calculated as 〈τ〉 = w1 × τ1 + w2 ×
τ2 + w3 × τ3 . For 4a at pH 2.2, τ1 = 1.01 ns (w1 = 24%), τ2 = 2.49 ns
(w2 = 28%), τ3 = 10.82 ns (w3 = 48%), 〈τ〉 = 6.1 ns and χ2 = 1.09.
“Dec exp”: experimental decay; “Dec calc”: calculated decay; “L”:
Light source.
In the case of the pyrrole compound 3a, the longer
components τ2 and τ3 are also not too much affected by pH,
but there is a diminishing of the value of the shortest
component τ1 with pH, especially when passing from pH ≈ 3
to pH ≈ 9. There is also a decrease in the relative weight w1
with the corresponding increase in w2 and w3 .
Mass spectra were acquired in a Bruker Daltonics
micrOTOF-Q spectrometer with Electrospray ionization
(ESI).
The melting points were determined in an Electrotherma
l9100 apparatus and are not corrected.
The
reactions
were
monitored
by
thin-layer
chromatography (TLC) using aluminium sheets with silica gel
60F254 from Merck.
General method for the synthesis of methyl-3-amino-
4-cyano-1,5-dimetyhl-1H-pyrrole-2-carboxylate
In the pyrrole compound 3d, the decays also are not very
much affected by pH. There is, however, a diminishing in the
value of τ2 when exciting at 335 nm and passing from pH ≈ 3
to 6. The average lifetime 〈τ〉 is also shorter when exciting at

A
mixture
of
the
corresponding
3-alkyl-3-
2.25 g of sarcosine were added to a cold solution of 5 mL
of acetyl chloride and 22.5 mL of alcohol. The mixture was
then heated under reflux for 3 hours, leading to the formation
of sarcosine chlorohydrate. In the second step, sodium
carbonate (K2CO3) (10 mmol) was added to the mixture with
alkoxyacrylonitrile (10 mmol) and mercaptoacetic ester (10
mmol) in DMF (15 ml) with sodium carbonate (K2CO3) was
heated under reflux for 1h. The resulting precipitate was then
washed with water.
ethoxymethylenemalononitrile
(10 mmol)
and
Compound
4a:
methyl
3-amino-4-cyano-5-
dimethylformamide (DMF) (30 mL). The reaction was heated
for 6 hours at 60°C. The mixture was then poured into 100 ml
of water; the compound in the resulting precipitate was then
filtered and recrystallized from ethanol.
methylthiophene-2-carboxylate: 66%; mp: 240 ºC. FTIR:
ν
C=N: 2208 cm-1, νNH2: 3388-3421 cm-1, νC=O: 1665 cm-1. NMR
1H (DMSO-d6, 400 MHz): 6.81 (s, 2H, NH2), 3.74 (s, 3H)
2.51 (s, 3H). NMR 13C (DMSO-d6, 100 MHz): C6 162.9, C5
157.1, C3 153.4, C2 113.0, C4 101.3, C9 96.1, C7 51.3, C8 15.4.
+MS m/z 197.03 (M+1)
Compound 3a: methyl 3-amino-4-cyano-1-methyl-1H-
pyrrole-2-carboxylate: 62%; mp: 209 ºC. FTIR: νC=N: 2150
cm-1, νNH2: 3322-3413 cm-1, νC=O: 1668 cm-1. NMR 1H
(DMSO-d6, 400 MHz): 7.54 (s, 1H, H9), 5.79 (s, 2H, NH2);
3.74 (s, 3H, H7); 3.70 (s, 3H, H8). NMR 13C (DMSO-d6, 100
MHz): C6 160.9, C3 146.0, C5 133.9, C2 120.6, C10 115.0,
C4 79.6, C7 50.5, C8 37.7. +MS m/z 180.07 (M+1)
Compound 4b: methyl 3-amino-4-cyanothiophene-2-
carboxylate: 68%; mp: 250 ºC. FTIR: νC=N: 2215 cm-1, νNH2
:
3345-3446 cm-1, νC=O: 1669 cm-1. NMR 1H (DMSO-d6, 400
MHz): 7.7 (s, H5) 6.80 (s, 2H, NH2), 3.8 (s, 3H). NMR 13
C
(DMSO-d6, 100 MHz): C6 163.1, C5 158.3, C3 153.6, C2 113.1,
C4 101.9, C8 95.9, C7 51.8. +MS m/z 183.01 (M+1)
Compound 3b: methyl 3-amino-4-cyano-1,5-dimethyl-1H-
pyrrole-2-carboxylate: 92%; mp: 187 ºC. FTIR: νC=N: 2212
cm-1, νNH2: 3332-3423 cm-1, νC=O : 1661 cm-1. NMR 1H
(DMSO-d6, 400 MHz): 5.77 (s, 2H, NH2), 3.72 (s, 3H, H7);
3.63 (s, 3H, H8); 2.24 (s, 3H, H9). NMR 13C (DMSO-d6, 100
MHz): C6 160.9, C3 145.3, C5 142.4, C2 115.3, C10 104.2, C4
80.8, C7 50.4, C8 33.1, C9 11.2. +MS m/z 194.09 (M+1).
Compound 4c: ethyl 3-amino-4-cyano-5-methylthiophene-
2-carboxylate: 78%; mp: 205 ºC. FTIR: νC=N: 2192 cm-1,
νNH2: 3329-3425 cm-1, νC=O: 1680 cm-1. NMR 1H (DMSO-d6,
400 MHz): 6.79 (s, 2H, NH2), 4.22 (q, J=7.2 Hz, 2H), 2.51 (s,
3H), 1.24 (q, J=7.2 Hz, 3H). NMR 13C (DMSO-d6, 100
MHz): C6 162.6, C5 157.0, C3 153.4, C2 113.0, C4 101.2, C9
96.4, C7 60.0, C8 15.3, C10 14.2. +MS m/z 211.05 (M+1)
Compound 3c: methyl 3-amino-4-cyano-5-ethyl-1-methyl-
1H-pyrrole-2-carboxylate: 85%; mp: 165 ºC. FTIR: νC=N
:
2215 cm-1, νNH2: 3346-3446 cm-1, νC=O: 1668 cm-1. NMR 1H
(DMSO-d6, 400 MHz): 5.75 (s, 2H, NH2), 3.73 (s, 3H, H7);
3.66 (s, 3H, H8); 2.66 (q, J=7.6 Hz, 2H, H9); 1.13 (t, J=7.6Hz,
3H, H10). NMR 13C (DMSO-d6, 100 MHz): C6 161.0,
C3 147.2, C5 145.5, C2 115.1, C4 104.3, C7 79.8, C8 50.4, C11
32.9, C10 18.5, C9 12.7. +MS m/z 208.10 (M+1)
UV-visible Absorption and Fluorescence Measurements
Absorption, steady-state and time-resolved fluorescence
spectra of the different pyrrole and thiophene derivatives were
measured in aqueous solutions with sodium pyrophosphate
buffer 0.01 M. The pyrrole samples were prepared at the
concentration of 1 mM and the thiophene sample at 10 µM,
corresponding to an optical density of A ≈ 0.1 at 350 nm (3a,
3d) or 435 nm (4a). UV-visible absorption spectra were
obtained in a JASCO V-660 spectrometer, using quartz cells
(l = 1 cm), unless indicated otherwise. The fluorescence
spectra were recorded on a Horiba Jobin Yvon Fluorolog 3-22
spectrofluorimeter using quartz cells (l = 1 cm), excitation
slits with 3 nm bandwidth, emission slits with 1 nm
bandwidth, integration time of 0.1 s/point, maximum
sensitivity, and right angle mode.
Compound 3d: methyl 3-amino-4-cyano-1-methyl-5-
phenyl-1H-pyrrole-2-carboxylate: 82%; mp: 145 ºC. FTIR:
ν
C=N: 2211 cm-1, νNH2: 3342-3435 cm-1, νC=O: 1671 cm-1.
NMR 1H (DMSO-d6, 400 MHz): 7.5 (m, 5H, phenyl group);
5.88 (s, 2H, NH2), 3.78 (s, 3H, H7); 3.62 (s, 3H, H8).
NMR 13
C (DMSO-d6, 100 MHz): C6 161.1, C3 145.7,
C5 145.7, C10 143.8, C9 129.7, C12 128.8, C11 127.9, C2 115.2,
C13 105.6, C4 81.3, C7 50.71, C8 35.1. +MS m/z 256.10 (M+1)
Compound 3e: methyl 3-amino-4-cyano-1-ethyl-5-methyl-
1H-pyrrole-2-carboxylate: 87%; mp: 164 ºC. FTIR: νC=N
:
The fluorescence quantum yields were determined for 3a,
3d and 4a relative to standards using the procedure
recommended by IUPAC [54,55]. The compounds were
dissolved in phosphate buffer 0.01 M at neutral, acidic and
basic pH. For 3a and 3d quinine sulphate in H2SO4 0.5 M (ϕ =
0.546, λdem = 350−665 nm, λex=340 nm) was used as standard
2193 cm-1, νNH2: 3328-3425 cm-1, νC=O: 1668 cm-1. NMR 1H
(DMSO-d6, 400 MHz): 5.73 (s, 2H, NH2), 4.21 (q, J= 7.2 Hz,
2H, H7); 3.63 (s, 3H, H9); 2.25 (s, 3H, H10), 1.28 (t, J = 7.2
Hz, 3H, H8). NMR 13C (DMSO-d6, 100 MHz): C6 160.5,
C3 145.3, C5 142.3, C2 118.9, C11 104.3, C4 80.8, C7 58.9, C8
33.2, C9 14.4, C10 11.3. +MS m/z 208.10 (M+1)
and for 4a we used coumarin 153 in H2O (ϕ = 0.12, λdem
=
432−650 nm, λex=420 nm) as standard [54]. The absorption
and emission spectra of each compound and of the
corresponding standard (with excitation at the same
Compound 3f: methyl 3-amino-4-cyano-1-ethyl-1H-
pyrrole-2-carboxylate: 60%; mp : 192ºC. FTIR: νC=N: 2150
cm-1, νNH2: 3322-3402 cm-1, νC=O: 1669 cm-1. NMR 1H
(DMSO-d6, 400 MHz): 7.83 (s, 1H, H10), 5.07 (s, 2H, NH2);
3.51 (s, 3H, H9); 2.89 (q, J = 7.2 Hz, 2H, H7), 2.22 (t, J = 7.2
Hz, 3H, H8). NMR 13C (DMSO-d6, 100 MHz): C6 163.5,
C3 144.3, C5 138.4, C2 120.43, C10 114.2, C4 80.8, C7 50.4, C8
44.9, C9 28.8. +MS m/z 194.09 (M+1)
wavelength) were measured for at least
concentrations.
5 different
Fluorescence intensity decay curves with picosecond
resolution were obtained by the single-photon timing
technique using a laser excitation system that consists of a de-
locked Coherent Inova 440-10 argon ion laser synchronously
pumping a cavity dumped Coherent 701-2 dye laser
(Rhodamine 6G or DCM), which delivers 5-6 ps pulses at a
repetition rate of 460 kHz. Intensity decay measurements
General method for the synthesis of 3-amino-4-cyano-
1,5-dimetyhl-1H-thiophene-2-carboxylate

7. (a) Smith, K.M., Ed.; Porphyrins and Metalloporphyrins,
Elsevier: Amsterdam. 1975; (b) Franck. B.; Nonn, A. Novel
Porphyrinoids for Chemistry and Medicine by Biomimetic
Syntheses. Angew. Chem., Int. Ed. Engl. 1995, 34: 1795.
were made by alternate collection of excitation and decay
curves, using an emission polarizer set at the magic angle.
The excitation profile was recorded at the excitation
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Acknowledgments
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We wish to thank Aleksander Fedorov (IN, Instituto
Superior Técnico, Lisboa, Portugal) for his kind assistance in
the fluorescence decay measurements. This work was
partially supported by Fundação para a Ciência e a Tecnologia
(FCT-Portugal) and COMPETE (FEDER), within projects
UID/NAN/50024/2013 and PTDC/CTM-POL/3698/2014.
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Hegde, P. Synthesis and biological evaluation of thiophene
[3,2-b] pyrrole derivatives as potential anti-inflammatory
agents. Bioorg. Med. Chem. 2004, 12: 1221–1230.
S.A. wishes to thank FCT for
a postdoctoral grant
(SFRH/BPD/74654/2010). Centro de Química Estrutural
acknowledges the financial support of Fundação para a
Ciência e Tecnologia (UIDB/00100/2020).
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Supplementary Material
Supplementary Material related to this article can be found
at http://...

Table 1. Ionization constants obtained by fitting of the spectrofluorimetric and spectrophotometric
titration curves with the calculated speciation curves. Proposed acid-base equilibria are depicted at
Scheme S1 (Supplementary Material)
Compound
pKa1
pKa2
4a
3a
3d
3.1±0.2
2.5±0.2
1.3±0.2
-
6.5±0.2
4.3±0.2

Synthesis and fluorescence properties of aminocyanopyrrole and
aminocyanothiophene esthers for biomedical and bioimaging applications
Oussama Cherif, Asma Agrebi, Sérgio Alves, Carlos Baleizão, José Paulo Farinha, Fatma Allouche
Highlights:
• A series of substituted aminocyanopyrrole esthers has been synthesized;
• A series of substituted aminocyanotiophene esthers has also been synthesized;
• Efficient one-pot synthetic strategies are described;
• The fluorescence, UV-vis absorbance and acid-base behaviour of the compounds is
presented;
• These compounds might be useful for medicinal applications and as bioimaging probes.

Oussama Cherif: Investigation, Methodology, Resources, Writing
Asma Agrebi: Investigation, Methodology, Resources, Writing
Sérgio Alves: Investigation, Methodology, Resources, Writing
Carlos Baleizão: Resources, Writing, Funding acquisition
José Paulo Farinha: Resources, Writing, Funding acquisition
Fatma Allouche: Investigation, Methodology, Resources, Writing, Funding acquisition

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: