New 1,3-diarylureas linked by CC Suzuki coupling to the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: Synthesis and fluorescence studies in solution and in lipid membranes

Article abstract of DOI:10.1016/j.jphotochem.2013.01.006

New six fluorescent 1,3-diarylureas linked by CC Suzuki coupling to the 6-position of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on the ortho or meta positions relative to the CC bond of the

Full text of DOI:10.1016/j.jphotochem.2013.01.006

Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
New 1,3-diarylureas linked by C C Suzuki coupling to the methyl
3-aminothieno[3,2-b]pyridine-2-carboxylate moiety: Synthesis and fluorescence
studies in solution and in lipid membranes
Maria-João R.P. Queiroza,∗, Daniela Peixotoa, Ana Rita O. Rodriguesb, Pedro M.F. Mendesb,
Cátia N.C. Costab, Paulo J.G. Coutinhob, Elisabete M.S. Castanheirab
a Departamento/Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b Centro de Física (CFUM), Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
New six fluorescent 1,3-diarylureas linked by C C Suzuki coupling to the 6-position of the methyl 3-
aminothieno[3,2-b]pyridine-2-carboxylate moiety were prepared by reaction of the amino groups on
the ortho or meta positions relative to the C C bond of the Suzuki coupling products, with different para-
substituted arylisocyanates (H, OMe, CN), in high to excellent yields. The fluorescence properties of the
1,3-diarylureas in solution and in lipid membranes of egg yolk phosphatidylcholine (Egg-PC), dipalmitoyl
phosphatidylcholine (DPPC), dipalmitoyl phosphatidylglycerol (DPPG) or dioctadecyldimethylammo-
nium bromide (DODAB), with or without cholesterol (Ch), were studied. The six 1,3-diarylureas have
reasonable fluorescence quantum yields in several solvents (0.02 ≤ ˚F ≤ 0.69) and present a moderately
solvent sensitive emission, but are not fluorescent in alcohols and water. The compounds bearing the ary-
lurea moiety in the meta position relative to the C C bond, especially with the OMe and CN substituents,
present the better solvatochromic properties.
Received 7 November 2012
Received in revised form 13 January 2013
Accepted 19 January 2013
Available online 28 January 2013
Keywords:
1,3-Diarylureas
Thieno[3,2-b]pyridines
Fluorescence
Lipid membranes
Fluorescence probes
Incorporation of the six compounds in lipid membranes indicates that all the compounds are deeply
located in the hydrophobic region of the lipid bilayers, feeling the transition between the rigid gel phase
and fluid phases.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Lipid membranes were composed of neutral/zwitterionic phos-
pholipids (DPPC – dipalmitoyl phosphatidylcholine; Egg-PC – egg
Unsymmetrical 1,3-diarylureas have attracted much attention
due to their diverse applications in agriculture, medicine, petro-
chemicals, supramolecular chemistry (anion receptors), biology
and as important intermediates and bifunctional organocatalysts
in organic synthesis [1–3].
Thienopyridines including their 1,3-diarylurea derivatives have
shown different biological activities, namely as antitumoral agents
[4] and receptor tyrosine kinase inhibitors [5].
yolk phosphatidylcholine), anionic phospholipids (DPPG – dipalmi-
toyl phosphatidylglycerol) or synthetic cationic lipids (DODAB –
dioctadecyldimethylammonium bromide). The incorporation of
cholesterol, an important component of most natural membranes,
may increase the stability of the lipid aggregates by modulating
the fluidity of the lipid bilayer, preventing crystallization of the
phospholipid acyl chains and providing steric hindrance to their
movement [7].
In this work, six new 1,3-diarylureas were prepared by reac-
tion of aminated compounds, resulting from Suzuki coupling of
methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate and
ortho or meta pinacolborane ester anilines [6], with different para-
substituted arylisocyanates (H, OMe, CN).
Fluorescence anisotropy measurements can give relevant infor-
mation about the compounds behavior and location in the lipid
membranes, namely if they are located deeply in the lipid bilayer,
feeling the differences between the rigid gel phase and the fluid
liquid-crystalline phase of the lipids.
Due to the potential biological activity of the new compounds,
their interaction with lipid membranes is of particular interest.
The photophysical properties of these thieno[3,2-b]pyridine 1,3-
diarylurea derivatives in solution and in lipid bilayers were studied.
2. Experimental
2.1. Synthesis
2.1.1. General remarks
∗
Melting points (◦C) were determined in a SMP3 Stuart appara-
tus and are uncorrected. 1H and 13C NMR spectra were recorded
Corresponding author. Tel.: +351 253604378; fax: +351 253604382.
E-mail address: mjrpq@quimica.uminho.pt (M.-J.R.P. Queiroz).
1010-6030/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2013.01.006

28
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
NH2
N
CO2Me
S
2a R3 = H, 90%
2b R3 = OMe, 90%
R3
HN
HN
O
2c R3 = CN, 80%
O
C
N
R3 = H, OMe or CN
R3
NH2
N
CH2Cl2/THF
rt, 16h
CO2Me
S
NH2
N
R2
CO2Me
R1
S
1a R1 =NH2, R2 =H
1b R2 =NH2, R1 = H
NH
3a R3 = H, 75%
3b R3 = OMe, 70%
3c R3 = CN, 75%
HN
O
R3
Scheme 1. Synthesis of 1,3-diarylureas 2 and 3 derivatives of thieno[3,2-b]pyridines by reaction of anilines with arylisocyanates.
on a Bruker Avance III at 400 and 100.6 MHz or on a Varian Unity
Plus at 300 and 75.4 MHz, respectively. Heteronuclear correlations,
1H–13C, HMQC or HSQC were performed to attribute some signals.
HRMS data were recorded using a method of direct injection by
ESI-TOF by the mass spectrometry service of the University of Vigo,
Spain.
The reactions were monitored by thin layer chromatography
(TLC) in aluminum plates covered with a layer of silica gel 60
(Macherey-Nagel) of 0.2 mm, with UV254 fluorescence indicator.
3.83 (s, 3H, OMe), 6.87 (2H, d, J = 9.2 Hz, 3ꢀꢀ and 5ꢀꢀ-H), 6.93 (2H,
br s, NH2), 7.36 (2H, d, J = 9.2 Hz, 2ꢀꢀ and 6ꢀꢀ-H), 7.38–7.50 (3H, m,
ArH), 7.89 (1H, br s, 2ꢀ-H), 8.55 (1H, br s, NH), 8.61 (1H, d, J = 2.0 Hz,
7-H), 8.73 (1H, broad s, NH), 8.92 (1H, d, J = 2.0 Hz, 5-H) ppm. 13C
NMR (100.6 MHz, DMSO-d6): ı 51.6 (OMe), 55.2 (OMe), 97.4 (C),
114.0 (3ꢀꢀ and 5ꢀꢀ-CH), 116.9 (2ꢀ-CH), 118.3 (CH), 120.2 (2ꢀꢀ and
6ꢀꢀ-CH), 120.8 (CH), 129.3 (7-CH), 129.7 (CH), 132.6 (C), 133.9 (C),
135.2 (C), 137.3 (C), 140.7 (C), 145.4 (C), 145.5 (5-CH), 147.9 (C),
152.8 (C O), 154.6 (C), 164.6 (C O) ppm. HRMS (ESI-TOF) Calcd.
for C23H21N4O4S [M+H]+ 449.1284; found 449.1284.
2.1.2. General procedure for the synthesis of 1,3-diarylureas
(Scheme 1)
2.1.2.3. Methyl
3-amino-6-{3-[3-(4-cyanophenyl)ureido]phenyl}
Compounds 1a–b [6] and different arylisocyanates (1 equiv.)
in 6 mL CH2Cl2:THF (1:1) were left stirring at room temperature
for 16 h. If a precipitate did not come out after this time, hex-
ane (3–5 mL) was added to the mixture to precipitate the product.
This was filtered under vacuum to give the corresponding 1,3-
diarylureas.
thieno[3,2-b]pyridine-2-carboxylate (2c). From compound 1a
(100 mg, 0.330 mmol) and 4-cyanophenylisocyanate (50.0 mg)
compound 2c was isolated as a yellow solid (120 mg, 80%), m.p.
264–265 ◦C. 1H NMR (300 MHz, DMSO-d6): ı 3.83 (3H, s, OMe),
6.94 (2H, br s, NH2), 7.45–7.47 (2H, m, ArH), 7.49–7.54 (1H, m,
ArH), 7.65 (2H, d, J = 9.0 Hz, 2ꢀꢀ and 6ꢀꢀ-H), 7.73 (2H, d, J = 9.0 Hz,
3ꢀꢀ and 5ꢀꢀ-H), 7.90 (1H, br s, 2ꢀ-H), 8.62 (1H, d, J = 2 Hz, 7-H), 8.92
(1H, d, J = 2.0 Hz, 5-H), 9.01 (1H, br s, NH), 9.30 (1H, br s, NH) ppm.
13C NMR (75.4 MHz, DMSO-d6): ı 51.6 (OMe), 97.4 (C), 103.4 (C),
117.4 (2ꢀ-CH), 118.1 (2ꢀꢀ and 6ꢀꢀ-CH), 118.7 (CH), 119.3 (C), 121.5
(CH), 129.4 (7-CH), 129.8 (CH), 133.3 (3ꢀꢀ and 5ꢀꢀ-CH), 133.9 (C),
135.0 (C), 137.4 (C), 140.0 (C), 144.1 (C), 145.5 (C), 145.6 (5-CH),
147.9 (C), 152.2 (C O), 164.6 (C O) ppm. HRMS (ESI-TOF) Calcd.
for C23H18N5O3S [M+H]+ 444.1125; found 444.1120.
2.1.2.1. Methyl 3-amino-6-[3-(3-phenylureido)phenyl]thieno[3,2-
b]pyridine-2-carboxylate (2a). From compound 1a (80.0 mg,
0.270 mmol) and phenylisocyanate (32.0 mg) compound 2a was
isolated as a yellow solid (100 mg, 90%), m.p. 226–226.5 ◦C. 1H
NMR (400 MHz, DMSO-d6): ı 3.83 (3H, s, OMe), 6.93 (2H, br s,
NH2), 6.95–6.99 (1H, m, ArH), 7.26–7.30 (2H, m, ArH), 7.40–7.52
(5H, m, ArH), 7.91 (1H, broad s, 2ꢀ-H), 8.62 (1H, d, J = 2 Hz, 7-H),
8.75 (1H, br s, NH), 8.82 (1H, br s, NH), 8.93 (1H, d, J = 2 Hz, 5-H)
ppm. 13C NMR (400 MHz, DMSO-d6): ı 51.5 (OMe), 97.4 (C), 117.0
(2ꢀ-CH), 118.3 (2 × CH), 118.4 (CH), 121.0 (CH), 121.9 (CH), 128.8
(2× CH), 129.3 (7-CH), 129.7 (CH), 133.9 (C), 135.1 (C), 137.3 (C),
139.6 (C), 140.5 (C), 145.4 (C), 145.5 (5-CH), 147.9 (C), 152.6 (C O),
164.6 (C O) ppm. HRMS (ESI-TOF) Calcd. for C22H19N4O4S [M+H]+
419.1172; found 419.1188.
2.1.2.4. Methyl 3-amino-6-[2-(3-phenylureido)phenyl]thieno[3,2-
b]pyridine-2-carboxylate (3a). From compound 1b (125 mg,
0.420 mmol) and phenylisocyanate (50.0 mg) compound 3a was
isolated as a yellow solid (120 mg, 75%), m.p. 226–227 ◦C. 1H NMR
(400 MHz, DMSO-d6): ı 3.83 (3H, s, OMe), 6.91–6.95 (m, 1H, ArH),
6.97 (2H, br s, NH2), 7.17–7.25 (3H, m, ArH), 7.35–7.44 (4H, m,
ArH), 7.86 (1H, br s, NH), 8.00 (1H, dd, J = 8.0 and 1.2 Hz, ArH),
8.43 (1H, d, J = 2 Hz, 7-H), 8.65 (1H, d, J = 2 Hz, 5-H), 8.79 (1H, br s,
NH) ppm. 13C NMR (100.6 MHz, DMSO-d6): ı 51.6 (OMe), 97.3 (C),
118.0 (2× CH), 121.8 (CH), 122.6 (CH), 123.5 (CH), 128.8 (2× CH),
128.9 (CH), 129.1 (C), 130.7 (CH), 132.1 (7-CH), 133.7 (C), 133.8
(C), 136.5 (C), 139.6 (C), 145.3 (C), 147.3 (5-CH), 148.0 (C), 152.6
2.1.2.2. Methyl 3-amino-6-{3-[3-(4-methoxyphenyl)ureido]phenyl}
thieno[3,2-b]pyridine-2-carboxylate (2b). From compound 1a
(150 mg, 0.540 mmol) and 4-methoxyphenylisocyanate (80.0 mg)
compound 2b was isolated as a yellow solid (217 mg, 90%), m.p.
243–244 ◦C. 1H NMR (400 MHz, DMSO-d6): ı 3.71 (s, 3H, OMe),

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
29
(C O), 164.6 (C O) ppm. HRMS (ESI-TOF) Calcd. for C22H19N4O3S
[M+H]+ 419.1172; found 419.1180.
For lipid vesicles preparation, the ethanolic injection method
was used [8–10]. For Egg-PC membranes preparation, defined vol-
ume of stock solution of lipid (86.2 mM) and each compound
(0.3 mM) in ethanol were injected together, under vigorous stir-
ring, to an aqueous buffer solution (10 mM Tris, pH = 7.4), at room
temperature. A similar procedure was adopted for DPPC, DODAB
and DPPG vesicles, but the injection of the required amounts of
stock solutions of lipid (50 mM for DPPC, 20 mM for DODAB and
26.8 mM for DPPG) and compounds in ethanol was done at 60 ◦C,
well above the melting transition temperature of each lipid (ca.
41 ◦C for DPPC [11], ca. 45 ◦C for DODAB [12], and 39.6 ◦C for DPPG
[13]). In all cases, the final lipid concentration was 1 mM, with a
compound/lipid molar ratio of 1:333.
2.1.2.5. Methyl 3-amino-6-{2-[3-(4-methoxyphenyl)ureido]phenyl}
thieno[3,2-b]pyridine-2-carboxylate (3b). From compound 1b
(100 mg, 0.330 mmol) with 4-methoxyphenylisocyanate (50.0 mg)
compound 3b was obtained (100 mg, 70%), m.p. 243–244 ◦C. 1H
NMR (400 MHz, DMSO-d6): ı 3.68 (3H, s, OMe), 3.83 (3H, s, OMe),
6.81 (2H, d, J = 9.2 Hz, 3ꢀꢀ and 5ꢀꢀ-H), 6.97 (2H, br s, NH2), 7.15–7.19
(1H, m, ArH), 7.25 (2H, d, J = 9.2 Hz, 2ꢀꢀ and 6ꢀꢀ-H), 7.32–7.35 (1H,
m, ArH), 7.38–7.42 (1H, m, ArH), 7.77 (1H, br s, NH), 7.99 (1H, br d,
J = 8.4 Hz, ArH), 8.42 (1H, d, J = 2.0 Hz, 7-H), 8.63 (1H, br s, NH), 8.64
(1H, d, J = 2.0 Hz, 5-H). 13C (100.6 MHz, DMSO-d6): ı 51.6 (OMe),
55.1 (OMe), 97.2 (C), 114.0 (3ꢀꢀ and 5ꢀꢀ-CH), 119.8 (2ꢀꢀ and 6ꢀꢀ-CH),
122.3 (CH), 123.2 (CH), 128.9 (CH), 130.7 (CH), 132.1 (7-CH), 132.6
(C), 133.8 (C), 136.7 (C), 145.3 (C), 147.3 (5-CH), 148.0 (C), 152.7
(C O), 154.4 (C), 164.6 (C O) ppm. HRMS (ESI-TOF) Calcd. for
C23H21N4O4S [M+H]+ 449.1284; found 449.1280.
2.3. Spectroscopic measurements
Absorption spectra were recorded in a Shimadzu UV-3101PC
UV–vis–NIR spectrophotometer. Fluorescence measurements were
performed using a Fluorolog 3 spectrofluorimeter, equipped with
double monochromators in both excitation and emission, Glan-
Thompson polarizers and a temperature controlled cuvette holder.
Fluorescence spectra were corrected for the instrumental response
of the system.
For fluorescence quantum yield determination, the solutions
were previously bubbled for 20 min with ultrapure nitrogen. The
fluorescence quantum yields (˚s) were determined using the
standard method (Eq. (1)) [14,15]. Quinine sulfate in H2SO4 0.05 M
was used as reference, ˚r = 0.546 at 25 ◦C [16].
2.1.2.6. Methyl
3-amino-6-{2-[3-(4-cyanophenyl)ureido]phenyl}
thieno[3,2-b]pyridine-2-carboxylate (3c). From compound 1b
(140 mg, 0.470 mmol) and 4-cyanophenylisocyanate (69.0 mg)
compound 3c was isolated as a yellow solid (120 mg, 75%), m.p.
210–211 ◦C. 1H NMR (400 MHz, DMSO-d6): ı 3.83 (3H, s, OMe),
6.96 (2H, br s, NH2), 7.22–7.26 (1H, m, ArH), 7.36–7.39 (1H, m,
ArH), 7.52–7.55 (1H, m, ArH), 7.53 (2H, d, J = 8.8 Hz, 2ꢀꢀ and 6ꢀꢀ-H),
7.67 (2H, d, J = 8.8 Hz, 3ꢀꢀ and 5ꢀꢀ-H), 7.93–7.95 (1H, m, ArH), 8.07
(1H, br s, NH), 8.44 (1H, d, J = 2 Hz, 7-H), 8.65 (1H, d, J = 2.0 Hz, 5-H),
9.28 (1H, br s, NH) ppm. 13C NMR (100.6 MHz, DMSO-d6): ı 51.6
(OMe), 97.3 (C), 103.3 (C), 117.9 (2ꢀꢀ and 6ꢀꢀ-CH), 119.3 (C), 123.2
(CH), 124.2 (CH), 129.0 (CH), 129.8 (C), 130.8 (CH), 132.1 (7-CH),
133.3 (3ꢀꢀ and 5ꢀꢀ-CH), 133.6 (C), 133.7 (C), 135.8 (C), 144.1 (C),
145.3 (C), 147.2 (5-CH), 147.9 (C), 152.3 (C O), 164.6 (C O) ppm.
HRMS (ESI-TOF) Calcd. for C23H18N5O3S [M+H]+ 444.1125; found
444.1129.
ꢀ
ꢁ
ArFsn2s
AsFrn2r
˚s =
˚
r
(1)
where A is the absorbance at the excitation wavelength, F is the inte-
grated emission area and n is the refraction index of the solvents
used. Subscripts refer to the reference (r) or sample (s) compound.
The absorbance value at excitation wavelength was always less
than 0.1, in order to avoid inner filter effects.
Solvatochromic shifts can be described by the Lippert–Mataga
equation (2), which relates the energy difference between absorp-
tion and emission maxima to the orientation polarizability [17,18]
2.2. Lipid membranes preparation
All the solutions were prepared using spectroscopic grade
solvents and ultrapure water (Milli-Q grade). 1,2-Dipalmitoyl-sn-
glycero-3-phosphocholine
(DPPC),
1,2-diacyl-sn-glycero-3-
1
4ꢁε0
2ꢂꢃ2
hcR3
ꢀabs − ꢀfl
=
ꢂf + const
(2)
phosphocholine from egg yolk (Egg-PC), 1,2-dipalmitoyl-sn-
glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPG) and
cholesterol were obtained from Sigma–Aldrich and dioctade-
cyldimethylammonium bromide (DODAB) from Tokyo Kasei (lipid
structures are shown below).
where ꢀabs is the wavenumber of maximum absorption, ꢀfl is the
wavenumber of maximum emission, ꢂꢃ = ꢃe − ꢃg is the differ-
ence in the dipole moment of solute molecule between excited
(ꢃe) and ground (ꢃg) states, R is the cavity radius (considering
the fluorophore a point dipole at the center of a spherical cavity
immersed in the homogeneous solvent), and ꢂf is the orientation
polarizability given by (Eq. (3)):
ε − 1
n2 − 1
ꢂf =
−
(3)
2n2 + 1
2ε + 1
where ε is the static dielectric constant and n is the refractive index
of the solvent.
An alternative expression, proposed by Bakhshiev, takes into
account the angle, ꢄ, between the ground and excited state dipole
moments of the fluorophore [19,20]:
1
4ꢁε0
2
hcR3
ꢀabs − ꢀfl
=
(ꢃ2g − 2ꢃgꢃe cos ꢄ + ꢃ2e)f (ε, n) + const (4)
2
where ꢃ2g − 2ꢃgꢃe cos ꢄ + ꢃe2 is equivalent to |ꢃe − ꢃg| and
ꢁ
ꢁ
ε − 1
ε + 2
n2 − 1
n2 + 2
f (ε, n) =
−
(5)

30
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
Fig. 1. Normalized fluorescence spectra of 3 × 10−6 M solutions of compound 2a in
several solvents (ꢅexc = 370 nm). Inset: absorption spectrum of 10−5 M solutions of
2a in dioxane and acetonitrile, as examples.
Fig. 2. Normalized fluorescence spectra of 3 × 10−6 M solutions of compound 2c in
several solvents (ꢅexc = 370 nm). Inset: absorption spectrum of 10−5 M solutions of
2c in dioxane and acetonitrile, as examples.
The steady-state fluorescence anisotropy, r, is calculated by
IVV − GIVH
fluorescence quantum yields obtained in chloroform, as the forma-
tion of hydrogen bonds between chloroform and proton acceptor
molecules has been already described [22].
r =
(6)
IVV + 2GIVH
The six compounds studied here are more fluorescent in polar
solvents like dimethylformamide and dimethylsulfoxide, which is
also a common behavior with the di(hetero)arylether derivatives
of thieno[3,2-b]pyridines studied earlier [21]. Also, the substituent
does not seem to have a significant influence in the fluorescence
quantum yield values (Table SD1).
where IVV and IVH are the intensities of the emission spectra
obtained with vertical and horizontal polarization, respectively
(for vertically polarized excitation light), and G = IHV/IHH is the
instrument correction factor, where IHV and IHH are the emission
intensities obtained with vertical and horizontal polarization (for
horizontally polarized excitation light).
For all compounds, significant red shifts are observed for
emission in polar solvents (34–36 nm between chloroform and
dimethylsulfoxide for compounds 2a–c and 20–25 nm for 3a–c).
In the absorption spectra, the red shifts are negligible (Table SD1),
indicating that solvent relaxation after photoexcitation plays an
important role, especially for compounds 2a–c. This indicates that
3. Results and discussion
3.1. Synthesis
New 1,3-diarylureas 2a–c and 3a–c were prepared by reaction of
the aromatic amino groups of the Suzuki coupling products 1a–b,
earlier obtained by us from methyl 3-amino-6-bromothieno[3,2-
b]pyridine-2-carboxylate and pinacolborane esters of anilines [6],
with arylisocyanates, in high to excellent yields (Scheme 1).
the 1,3-arylurea substituent in the meta position relative to C
C
bond contributes to increase the intramolecular charge transfer
(ICT) character of the excited state.
The solvatochromic plots for compounds 2a–c and 3a–c, shown
in Figs. 4 and 5, are reasonably linear, the slope being larger for
compound 2c. This predicts a higher ICT character of the excited
3.2. Fluorescence studies in several solvents
The absorption and fluorescence properties of compounds 2a–c
and 3a–c were studied in several solvents (Table SD1 in Supple-
mentary Data). The normalized fluorescence spectra of compounds
2a, 2c and 3b are shown in Figs. 1–3, respectively (examples of
absorption spectra are also shown as insets).
Supplementary material related to this article found,
in
the
online
version,
at
http://dx.doi.org/10.1016/
j.jphotochem.2013.01.006.
Compounds 2a–c and 3a–c exhibit reasonable fluorescence
emission in several solvents (the studied solvents do not include
cyclo- or n-alkanes, because the solubility of these compounds is
very poor in this kind of solvents). Fluorescence quantum yield
values are in the range of 6% in chloroform and 65% in DMSO
for compounds 2a–c and of 2% (chloroform) and 69% (DMSO) for
compounds 3a–c (Table SD1). However, no emission is observed in
protic solvents like water, methanol or ethanol.
This behavior, already observed for di(hetero)arylether deriva-
tives of thieno[3,2-b]pyridines recently synthesized [21], can be
due to specific solute–solvent interactions by hydrogen bonds
with protic solvents, namely by protonation of the nitrogen atom
of the pyridine ring. The same explanation can justify the low
Fig. 3. Normalized fluorescence spectra of 3 × 10−6 M solutions of compound 3b in
several solvents (ꢅexc = 370 nm). Inset: absorption spectrum of 10−5 M solutions of
3b in dioxane and acetonitrile, as examples.

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
31
Table 1
Dihedral angle ˛, N
C
N bond angle ˇ and C
S
C bond angle ꢄ in the ground and
first singlet excited state geometries for compounds 2a–c and 3a–c.
Compound
State
Dihedral angle ˛
N
C
N angle ˇ
C S C angle ꢄ
Ground −46.6◦
112.6◦
112.8◦
90.1◦
92.6◦
2a
Excited −36.8◦
Ground −46.9◦
Excited −36.6◦
112.5◦
112.8◦
90.1◦
92.6◦
2b
2c
3a
3b
3c
Ground −47.0◦
112.5◦
112.7◦
90.1◦
92.6◦
Excited −36.1◦
Ground
Excited
94.0◦
89.6◦
112.3◦
112.5◦
90.1◦
92.7◦
Ground
Excited
94.7◦
89.0◦
112.2◦
112.5◦
90.1◦
92.8◦
Ground
Excited
94.0◦
89.8◦
112.2◦
112.4◦
90.1◦
92.7◦
Fig. 4. Solvatochromic plots (Eq. (4)) for compounds 2a–c. Solvents: 1 – dioxane;
2 – chloroform; 3 – ethyl acetate; 4 – dichloromethane; 5 – dimethylsulfoxide; 6 –
N,N-dimethylformamide; 7 – acetonitrile (values of ε and n were obtained from Ref.
[23]).
basis set at the DFT (CAM-B3LYP/AUTO) level of theory [24,25] in
gas phase, the cavity radius (R) and the ground state dipole moment
(␮g) were determined for the six compounds (Table 2). The use of
CAM-B3LYP functional was needed, as initial trials with the simpler
B3LYP gave unrealistic underestimates of the HOMO–LUMO band
gap (<500 nm). This is a known problem with B3LYP functional in
the description of excited states with charge transfer character [25].
The optimized geometries of the ground state and the first excited
state were obtained with a smaller basis set (3-21G+*) and are sim-
ilar in the groups of compounds 2a–c and 3a–c (Figs. 6 and 7). In the
case of excited state calculations, a time dependent density func-
tional method was used (TD-SCF DFT). In Table 1, the dihedral angle
defined by the two molecular planes and the angles for the N
C N
and C C chemical bonds (in the thienopyridine moiety) are indi-
S
cated, evidencing notable differences in geometries between the
two sets of compounds. Comparing the ground and excited state
geometries, a small decrease (ca. 10◦ in 2a–c and 5◦ in 3a–c) of the
dihedral angle is observed in the excited state (Table 1), together
with a 2◦ increase in the C
S C bond angle for all compounds.
The directions of the calculated dipole moments in the ground
and excited states are also indicated in Figs. 6 and 7, evidencing an
increase in magnitude and a change of direction in the excited state
dipole moment vector relative to the ground state one. This clearly
indicates that the angle between the two dipole moment vectors
cannot be neglected and must be considered in the solvatochromic
plots (Bakhshiev’s equation (4)). Nevertheless, the change in the
dipole moment direction is very small for compounds 2c and 3a.
In the excited state, the dipole moment vectors for all compounds
point to the side of the thienopyridine moiety.
The absolute value of the difference in the excited and ground
state dipole moment vectors, estimated from the solvatochromic
plots (Figs. 4 and 5) and from molecular quantum mechanical cal-
culations, is presented in Table 2, for each compound. The obtained
values are very similar and, therefore, both methods point to the
presence of a charge transfer mechanism in the excited state,
more pronounced for compounds 2a–c. Compounds bearing an
Fig. 5. Solvatochromic plots (Eq. (4)) for compounds 3a–c. Solvents: 1 – dioxane;
2 – chloroform; 3 – ethyl acetate; 4 – dichloromethane; 5 – dimethylsulfoxide; 6 –
N,N-dimethylformamide; 7 – acetonitrile (values of ε and n were obtained from Ref.
[23]).
state for this compound, maybe related with the position of the
arylurea moiety together with the presence of a nitrile substituent.
The slopes of the solvatochromic plots are much lower for
compounds 3a–c, being similar for these three molecules. This
indicates that the charge transfer character of the excited state is
much lower for the arylureas in the ortho position relative to the
thieno[3,2-b]pyridine-2-carboxylate moiety.
From ab initio molecular quantum chemistry calculations,
obtained with Gaussian 09 software [24] and use of a 6-311+G(dp)
Table 2
ꢁ
ꢁ
Cavity radius (R), ground (ꢃg) and excited state (ꢃe) dipole moments obtained from theoretical calculations, and absolute value of the dipole moment difference (|ꢃe − ꢃg|),
from quantum mechanical calculations and from the solvatochromic plots.
˚
ꢁ
ꢁ
ꢁ
ꢁ
Compound
Cavity radius, R (A)
Ground state dipole
moment, ꢃg (D)
Excited state dipole moment, ꢃe
(D), from theoretical calculations
|ꢃe − ꢃg| (D) from
|ꢃe − ꢃg| (D) from
theoretical calculations
solvatochromic plots
2a
2b
2c
3a
3b
3c
5.6
6.2
5.9
5.7
6.0
5.9
3.1
4.7
4.9
2.2
3.3
7.4
6.1
7.3
10.1
6.7
7.8
10.2
5.1
5.2
5.3
4.6
4.5
4.7
5.2
5.8
6.2
4.1
4.4
4.2

32
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
Fig. 6. Optimized geometries of compounds 2a–c obtained by Gaussian 09 software (gray: C atoms; white: H atoms; red: O atoms; blue: N atoms; yellow: S atoms). Above:
ground state; below: lowest excited singlet state. The arrows indicate the direction of the dipole moment. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of the article.)
Fig. 7. Optimized geometries of compounds 3a–c obtained by Gaussian 09 software (gray: C atoms; white: H atoms; red: O atoms; blue: N atoms; yellow: S atoms). Above:
ground state; below: lowest excited singlet state. The arrows indicate the direction of the dipole moment. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of the article.)
electron-donating group
withdrawing group ( CN in 2c) have a higher ICT character
of the excited state than compound 2a.
Fig. 8 displays the representation of electronic density difference
between the lowest excited state and the ground state, for the low-
est excited state optimized geometry (relaxed S1 state). In general,
(
OCH3 in 2b) or an electron-
it can be observed that electron density variations reside mostly
on the thienopyridine-2-carboxylate moiety. This justifies that the
compounds do not exhibit a noticeable influence of the arylurea
substituent ( OCH3 or CN) in their photophysical properties,
namely in the fluorescence quantum yields. The more prominent
features in Fig. 8 are an electron density transfer from the amino
Fig. 8. Representation of the electronic density difference for compounds 2a–c and 3a–c (optimized geometry for the lowest excited singlet state) at an iso level of 0.0004;
green regions: loss of electronic density; red regions: enrichment of electronic density. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of the article.)

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
33
Fig. 9. Normalized fluorescence spectra of compounds 2a and 2c (3 × 10−6 M) in
Fig. 10. Normalized fluorescence spectra of compounds 3a and 3c (3 × 10−6 M) in
lipid aggregates of Egg-PC, Egg-PC:Ch, DPPC and DPPC:Ch, at 25 ◦C and 55 ◦C.
lipid aggregates of Egg-PC, Egg-PC:Ch, DPPC and DPPC:Ch, at 25 ◦C and 55 ◦C.
group linked to the thiophene ring and its sulfur atom to the nitro-
gen atom in the pyridine moiety and to the carboxylate group. In
the ␲-electron system, alternating increases and decreases of elec-
tronic density are observed. This confirms the ICT character of the
first excited state of these compounds.
r increases with the rotational correlation time of the fluorescent
molecule (and, thus, with the viscosity of the fluorophore environ-
ment) [29].
Steady-state anisotropy relates to both the excited-state life-
time and the rotational correlation time of the fluorophore [29],
The photophysical behavior of the six compounds shows that
they can be considered as solvatochromic probes, especially com-
pounds 2a–c. The sensitivity of the fluorescence emission to the
fluorophore environment can be very useful when probing the loca-
tion/behavior of these compounds in lipid membranes.
ꢂ
ꢃ
1
r
1
r0
=
1 +
(7)
c
where r0 is the fundamental anisotropy, is the excited-state life-
time and
is the rotational correlation time.
c
The fluorescence steady-state anisotropies of compounds 2a–c
and 3a–c in lipid membranes are shown in Table 3. Anisotropy
values in glycerol at room temperature were also determined for
comparison.
3.3. Fluorescence studies in lipid membranes
Fluorescence experiments of the six compounds incorporated in
lipid membranes of several compositions were carried out. These
lipid aggregates were composed either by neat phospholipids, or
by phosphatidylcholines with cholesterol (Ch), for a better simula-
tion of the biological membranes. In fact, the vesicles composed of
70% Egg-PC and 30% cholesterol (Egg-PC:Ch (7:3)) are often used as
models of the biological membranes [26,27].
Lipid membranes of neat DPPC (zwitterionic), DPPG (anionic),
DODAB (cationic), Egg-PC (zwitterionic, composed of a phos-
phatidylcholine mixture), Egg-PC:Ch (7:3) and DPPC:Ch (7:3), with
incorporated compounds, were prepared and the fluorescence
emission was monitored in both gel (below the main transi-
tion temperature, Tm) and liquid-crystalline (above Tm) phases of
the phospholipids. At room temperature, the phospholipids DPPC,
DODAB and DPPG are in ordered gel phase, where the hydrocarbon
chains are fully extended and closely packed. The melting transi-
tion temperature of Egg-PC is very low [28] and this lipid is in the
fluid liquid-crystalline phase at room temperature.
Fluorescence spectra of compounds incorporated in these lipid
aggregates are presented in Figs. 9 and 10. All the six compounds
exhibit reasonable fluorescence emission when incorporated in
lipid membranes, indicating that they are mainly located in the
region of the lipid bilayer, as they are not fluorescent in alcohols or
water. The maximum emission wavelengths in lipid membranes
generally point to a hydrophobic medium for all compounds in
these lipid aggregates, feeling an environment with polarity near
dioxane or less polar than dioxane (Table 3). A slightly more
hydratedenvironmentis anticipatedforthesixcompoundsin DPPG
vesicles at 25 ◦C, considering the values of the maximum emission
wavelengths.
Steady-state fluorescence anisotropy results (Table 3) allow
concluding that all compounds are mainly located in the inner
region of the lipid membrane. The transition from the rigid gel
phase to the fluid liquid-crystalline phase is clearly detected by
a notable decrease in anisotropy at 55 ◦C. An increase of the
steady-state anisotropy is predicted from a decrease of the excited-
state lifetime (Eq. (7)). Upon rising temperature (from 25 ◦C to
55 ◦C), the excited-state lifetime decreases due to the increase
of non-radiative deactivation pathways (mainly the rate constant
for internal conversion S1→S0). Instead of an expected rise in
anisotropy (Eq. (7)), a decrease is observed, which can only be
attributed to a diminution of the rotational correlation time of the
fluorophore, that arises from the decrease of membrane microvis-
cosity upon changing from the gel to the liquid-crystalline phase.
Fluorescence anisotropy values for the compounds incorporated
in lipid membranes exhibit also a significant decrease when lipid
aggregate fluidity increases by addition of cholesterol. Simulta-
neously, a blue shift in emission spectra (Figs. 9 and 10 and Table 3)
is observed when fluidity increases, either by phase transition to the
liquid-crystalline phase or by cholesterol addition. These changes
are much more pronounced for compounds 2a–c and may indicate
a relocation of the fluorophores in a less hydrated environment. The
decrease in local microviscosity can facilitate a deeper penetration
of these molecules in lipid bilayers. The results obtained here point
to a promising utility of the new diarylureas to monitor changes in
fluidity of lipid membranes, especially compounds 2a–c.
Microviscosity in hydrophobic domains of microheterogeneous
systems has been measured using the widely known fluorescence
probe 1,3-bis-(1-pyrenyl)propane (BPP) [30–35]. This probe can
form intramolecular excimers and is highly sensitive to constraints
imposed by its environment, reported by variations in the ratio
Fluorescence anisotropy (r) measurements can give relevant
information about the location of the compounds in liposomes, as

34
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 255 (2013) 27–35
Table 3
Steady-state fluorescence anisotropy (r) values and maximum emission wavelengths (ꢅem) for compounds 2a–c and 3a–c in lipid aggregates, below (25 ◦C) and above (55 ◦C)
transition temperature of the lipids. Anisotropy values in glycerol at room temperature are also shown for comparison.
Lipid
T (◦C)
2a
2b
2c
3a
3b
3c
ꢅem (nm)
452
r
ꢅem (nm)
449
r
ꢅem (nm)
451
r
ꢅem (nm)
449
r
ꢅem (nm)
451
r
ꢅem (nm)
449
r
Egg-PC
25
25
0.221
0.197
0.205
0.187
0.210
0.190
0.248
0.228
0.249
0.221
0.240
0.231
Egg-PC:Ch (7:3)
443
440
443
446
446
445
25
55
458
443
0.238
0.168
459
441
0.232
0.167
452
443
0.262
0.175
452
449
0.256
0.163
451
448
0.259
0.122
452
449
0.251
0.180
DPPC
DPPC:Ch
(7:3)
25
55
434
430
0.172
0.143
438
432
0.175
0.136
439
431
0.192
0.155
450
446
0.199
0.104
449
445
0.171
0.118
450
446
0.194
0.127
25
55
457
446
0.229
0.182
457
447
0.200
0.167
453
446
0.243
0.131
448
445
0.230
0.172
456
444
0.251
0.180
451
444
0.247
0.183
DODAB
25
55
467
457
0.230
0.181
466
458
0.223
0.190
466
457
0.250
0.195
461
457
0.233
0.149
464
460
0.226
0.138
457
454
0.209
0.102
DPPG
Glycerol
25
474
0.325
465
0.295
469
0.308
464
0.330
462
0.327
459
0.324
between excimer and monomer emission intensities. The new
diarylurea derivatives 2a–c and 3a–c showed in this work an inter-
esting potential as fluidity probes for lipid membranes (especially
2a–c) through their intrinsic photophysical properties, rather than
by the relative efficiency of a dynamic conformational change lead-
ing to an intramolecular pyrene excimer formation, as observed
in BPP. However, further studies are needed to assess the utility
of these new probes for microviscosity determinations of other
types of microheterogeneous systems already studied using BPP,
like micelles, liquid crystals, lipoproteins and synthetic polymers
[30,33–35].
PTDC/QUI-QUI/111060/2009 (F-COMP-01-0124-FEDER-015603)
(organic synthesis).
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