New potential antitumoral di(hetero)arylether derivatives in the thieno[3,2-b]pyridine series: Synthesis and fluorescence studies in solution and in nanoliposomes

Article abstract of DOI:10.1016/j.jphotochem.2012.04.007

New fluorescent methoxylated di(hetero)arylethers in the thieno[3,2-b]pyridine series were prepared by a copper-catalyzed Ullmann-type CO coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate with ortho, meta and para-methoxyphenols, using N,N-dimethylglycine as the ligand and Cs₂CO₃ as the base. The compounds obtained were tested for their inhibitory growth activity in three human tumor cell lines MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-small cell lung cancer). The di(hetero)arylethers bearing a methoxy group in the ortho and meta positions showed very low GI₅₀ values (1.1-2.5 μM) in all the three tumor cell lines. Their fluorescence properties in solution and when encapsulated in different nanoliposome formulations, composed either by egg-yolk phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylglycerol (DMPG), dioctadecyldimethylammonium bromide (DODAB), distearoyl phosphatidylcholine (DSPC), with or without cholesterol (Ch), or distearoyl phosphatidylethanolamine-(polyethylene glycol)2000 (DSPE-PEG), were studied. All compounds can be carried in the hydrophobic region of the liposome membrane. The liposomes with incorporated compounds are nanometric in size (diameter lower than 150 nm) and have generally low polydispersity.

Full text of DOI:10.1016/j.jphotochem.2012.04.007

Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
New potential antitumoral di(hetero)arylether derivatives in the
thieno[3,2-b]pyridine series: Synthesis and fluorescence studies
in solution and in nanoliposomes
Maria-João R.P. Queiroza,∗, Sofia Diasa, Daniela Peixotoa, Ana Rita O. Rodriguesb, Andreia D.S. Oliveirab,
Paulo J.G. Coutinhob, Luís A. Vale-Silvac,d, Eugénia Pintoc,d, 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
c Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
d CEQUIMED-UP, Centro de Química Medicinal da Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
New fluorescent methoxylated di(hetero)arylethers in the thieno[3,2-b]pyridine series were prepared by
a copper-catalyzed Ullmann-type C O coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-
2-carboxylate with ortho, meta and para-methoxyphenols, using N,N-dimethylglycine as the ligand and
Cs2CO3 as the base. The compounds obtained were tested for their inhibitory growth activity in three
human tumor cell lines MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-small
cell lung cancer). The di(hetero)arylethers bearing a methoxy group in the ortho and meta positions
showed very low GI50 values (1.1–2.5 ␮M) in all the three tumor cell lines. Their fluorescence properties
in solution and when encapsulated in different nanoliposome formulations, composed either by egg-yolk
phosphatidylcholine (Egg-PC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylglyc-
erol (DMPG), dioctadecyldimethylammonium bromide (DODAB), distearoyl phosphatidylcholine (DSPC),
with or without cholesterol (Ch), or distearoyl phosphatidylethanolamine-(polyethylene glycol)2000
(DSPE-PEG), were studied. All compounds can be carried in the hydrophobic region of the liposome
membrane. The liposomes with incorporated compounds are nanometric in size (diameter lower than
150 nm) and have generally low polydispersity.
Received 29 February 2012
Accepted 4 April 2012
Available online 12 April 2012
Keywords:
Ullmann-type C O coupling
Di(hetero)arylethers
Thieno[3,2-b]pyridines
Fluorescence
Nanoliposomes
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The incorporation of Ch may increase the stability by modulating
the fluidity of the lipid bilayer preventing crystallization of the
Thienopyridine derivatives including di(hetero)arylethers have
attracted much attention because of their potential different bio-
logical activities namely as antitumoral agents [1], nonreceptor Src
kinase inhibitors [2] and receptor tyrosine kinase inhibitors [3–5].
Nanoliposomes are new technological developments for the
encapsulation and delivery of bioactive agents. Because of their
biocompatibility and biodegradability, along with their size,
nanoliposomes have potential applications in a vast range of fields,
including nanotherapy. Nanoliposomes are able to enhance the
performance of bioactive agents by improving their bioavailability,
in vitro and in vivo stability, as well as preventing their unwanted
interactions with other molecules [6]. They may contain, in addi-
tion to phospholipids, other molecules such as cholesterol (Ch)
which is an important component of most natural membranes.
phospholipid acyl chains and providing steric hindrance to their
movement. Further advances in liposome research found that
polyethylene glycol (PEG), which is inert in the body, allows longer
circulatory life of the drug delivery system [7].
In this work we describe the synthesis of new fluorescent
di(hetero)arylethers in the thieno[3,2-b]pyridine series, by
copper-catalyzed Ullmann-type
3-amino-6-bromo-thieno[3,2-b]pyridine-2-carboxylate,
C
O
coupling of the methyl
earlier
prepared by us [8], with o-, m- and p-methoxyphenols. The effects
of the three coupling products obtained in the growth inhibition of
human tumor cell lines, MCF-7 (breast adenocarcinoma), A375-C5
(melanoma), NCI-H460 (non-small cell lung cancer), were eval-
uated and the ortho and meta-methoxydi(hetero)arylethers were
shown to be the most promising antitumor compounds, presenting
very low GI50 values in all the cell lines studied. For the latter
compounds, the fluorescence properties were evaluated in differ-
ent solvents and when encapsulated in different nanoliposome
formulations, with or without cholesterol and PEG. Fluorescence
∗
Corresponding author. Tel.: +351 253604378; fax: +351 253678983.
E-mail address: mjrpq@quimica.uminho.pt (M.-J.R.P. Queiroz).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.04.007

72
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
with petroleum ether. 1H NMR (CDCl3, 400 MHz): ı 3.81 (3H, s,
OMe), 3.90 (3H, s, OMe), 6.21 (2H, s largo, NH2), 6.63–6.66 (2H,
m, ArH), 6.74–6.77 (1H, m, ArH), 7.27–7.32 (1H, m, ArH), 7.53
(1H, d, J = 2.4 Hz, HetArH), 8.45 (1H, d, J = 2.4 Hz, HetAr) ppm. 13C
NMR (CDCl3, 100.6 MHz): ı 51.56 (OMe), 55.44 (OMe), 98.60 (C),
105.54 (CH), 110.28 (CH), 111.38 (CH), 118.41 (CH), 130.61 (CH),
135.18 (C), 139.89 (CH), 142.02 (C), 147.34 (C), 153.48 (C), 156.48
(C), 161.23 (C), 165.24 (C O) ppm. MS (EI) m/z (%): 330.07 (M+,
100), 298.04 (M+ OMe, 67), HRMS M+ calcd. for C16H14N2O4S:
330.0674, found 330.0675.
anisotropy measurements can give relevant information about the
compound behavior and location in the several liposomes, namely
if they are located in the lipid bilayer, feeling the differences
between the gel and the liquid-crystalline phases of phospho-
lipids. These studies are important keeping in mind future drug
delivery applications of these new potential anticancer drugs.
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
on a Bruker Avance III at 400 and 100.6 MHz, respectively. Het-
eronuclear correlations, 1H–13C, HMQC or HSQC were performed
to attribute some signals.
2.1.2.3. Methyl
b]piridine-2-carboxylate (2c). Thieno[3,2-b]pyridine
6-(4-methoxyphenoxy)-3-aminothieno[3,2-
(100 mg,
1
0.363 mmol) and 4-methoxyphenol (1.0 equiv., 44.0 mg,
0.363 mmol), were heated and the reaction mixture was treated
according the general procedure. Column chromatography using
50% diethylether/petroleum ether gave compound 2c as a yellow
solid (79.0 mg, 65%), m.p. 129–131 ◦C, after some washes with
petroleum ether. 1H NMR (CDCl3, 400 MHz): ı 3.84 (3H, s, OMe),
3.90 (3H, s, OMe), 6.27 (2H, s largo, NH2), 6.95 (2H, d, J = 9.2 Hz,
2 × ArH), 7.05 (2H, d, J = 9.2 Hz, 2 × ArH), 7.44 (1H, d, J = 2.4 Hz,
HetArH), 8.44 (1H, d, J = 2.4 Hz, HetArH) ppm. 13C NMR (CDCl3,
100.6 MHz): ı 51.55 (OMe), 55.68 (OMe), 98.29 (C), 115.29 (2 × CH),
116.64 (CH), 121.21 (2 × CH), 135.29 (C), 139.31 (CH), 141.44 (C),
147.41 (C), 148.65 (C), 154.90 (C), 158.84 (C), 165.30 (C O) ppm.
MS (EI) m/z(%): 330.07 (M+, 100), 298.04 (M+ OMe, 68), HRMS M+
calcd. for C16H14N2O4S: 330.0674, found 330.0671.
MS and HRMS data were recorded using a method of direct
injection by EI (70 eV) and 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.
Column chromatography was performed on Macherey-Nagel silica
gel 230–400 mesh, using a solvent gradient, increasing the polarity
in mixtures of diethylether/petroleum ether in portions of 10% of
diethylether until product isolation. Petroleum ether refers to the
boiling range 40–60 ◦C.
2.1.2. General procedure for the copper-catalyzed C
(Scheme 1)
O
2.2. Biological activity
In a Schlenk tube, dry dioxane (3 mL), CuI (10 mol%), N,N-
dimethylglycine (30 mol%), the corresponding phenol (1.3 equiv.),
Cs2CO3 (2.0 equiv.) and methyl 3-amino-6-bromothieno[3,2-
b]pyridine-2-carboxylate (1), were added under argon, and the
mixture was heated with stirring at 110 ◦C for 8 h. After cooling,
NaOH aqueous (30%, w/v) and ethyl acetate were added. The phases
were separated, the organic phase was dried (MgSO4) and filtered.
The solvent was evaporated under reduced pressure giving a solid
which was submitted to column chromatography.
2.2.1. Reagents
Fetal bovine serum (FBS), l-glutamine, phosphate buffered
saline (PBS) and trypsin were from Gibco Invitrogen Co. (Scot-
land, UK). RPMI-1640 medium was from Cambrex (NJ, USA). Acetic
acid, dimethyl sulfoxide (DMSO), doxorubicin, penicillin, strepto-
mycin, ethylenediaminetetraacetic acid (EDTA), sulforhodamine B
(SRB) and trypan blue were from Sigma Chemical Co. (Saint Louis,
USA). Trichloroacetic acid (TCA) and Tris were sourced from Merck
(Darmstadt, Germany).
2.1.2.1. Methyl
b]pyridine-2-carboxylate (2a). Thieno[3,2-b]pyridine
0.545 mmol) and 2-methoxyphenol (1.3 equiv.,
6-(2-methoxyphenoxy)-3-aminothien[3,2-
1
(150 mg,
0.1 mL,
2.2.2. Solutions of the compounds
Stock solutions of the tested compounds were prepared in
DMSO and kept at −70 ◦C. Appropriate dilutions were freshly pre-
pared in the test medium just prior to the assays. The effect of
the vehicle solvent (DMSO) on the growth of the cell lines was
evaluated by exposing untreated control cells to the maximum con-
centration of DMSO used in the assays (0.25%). No influence was
found (data not shown).
0.709 mmol), were heated and the reaction mixture was treated
according to the general procedure. Column chromatography using
60% diethylether/petroleum ether gave compound 2a as a yellow
solid (67.0 mg, 40%), m.p. 137–139 ◦C, after some washes with
petroleum ether. 1H NMR (CDCl3, 400 MHz): ı 3.81 (3H, s, OMe),
3.89 (3H, s, OMe), 6.21 (2H, s largo, NH2), 6.99–7.01 (1H, m, Ar H),
7.06 (1H, dd, J = 8.4 and 1.6 Hz, ArH), 7.12 (1H, dd, J = 8.0 and 1.6 Hz,
ArH), 7.24–7.38 (1H, m, Ar H), 7.34 (1H, d, J = 2.4 Hz, HetAr), 8.46
(1H, d, J = 2.4 Hz, HetAr) ppm. 13C NMR (CDCl3, 100.6 MHz): ı 51.55
(OMe), 55.85 (OMe), 98.17 (C), 113.07 (CH), 116.11 (CH), 121.41
(CH), 122.07 (CH), 126.49 (CH), 135.33 (C), 138.54 (CH), 141.22 (C),
143.15 (C), 147.38 (C), 151.45 (C), 154.46 (C), 165.30 (C O) ppm.
MS (EI) m/z (%): 330.07 (M+,100), 298.04 (M+ OMe, 69). HRMS M+
calcd. for C16H14N2O4S: 330.0674, found 330.0680.
2.2.3. Cell cultures
Three human tumor cell lines, MCF-7 (breast adenocarcinoma),
NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma)
were used. MCF-7 and A375-C5 were obtained from the Euro-
pean Collection of Cell Cultures (ECACC, Salisbury, UK), and
NCI-H460 was kindly provided by the National Cancer Insti-
tute (NCI, Bethesda, USA). They were routinely maintained as
adherent cell cultures in RPMI-1640 medium supplemented
with 5% heat-inactivated FBS, 2 mM glutamine and antibiotics
(penicillin 100 U/mL, streptomycin 100 ␮g/mL), at 37 ◦C in a
humidified atmosphere containing 5% CO2. Exponentially grow-
ing cells were obtained by plating 1.5 × 105 cells/mL for MCF-7 and
0.75 × 105 cells/mL for A375-C5 and NCI-H460, followed by a 24 h
incubation.
2.1.2.2. Methyl
b]pyridine-2-carboxylate (2b). Thieno[3,2-b]pyridine
0.545 mmol) and 3-methoxyphenol (1.3 equiv.,
6-(3-methoxyphenoxy)-3-aminothieno[3,2-
1
(150 mg,
0.1 mL,
0.709 mmol), were heated and the reaction mixture was treated
according to the general procedure. Column chromatography
using 40% diethylether/petroleum ether gave compound 2b as a
yellow solid (96.0 mg, 53%), m.p. 111–112 ◦C, after some washes

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
73
2.2.4. Cell growth inhibition assay
2.3. Liposomes preparation
The effects on the in vitro growth of human tumor cell lines
were evaluated according to the procedure adopted by the NCI
(USA) in their “In vitro Anticancer Drug Discovery Screen”, using the
protein-binding dye sulforhodamine B to assess cell growth [9,10].
Briefly, exponentially growing cells were exposed for 48 h, in 96-
well microtiter plates, to five serial dilutions of each test compound,
starting from a maximum concentration of 150 ␮M (if possible).
Following the exposure period adherent cells were fixed in situ,
washed and stained with SRB. The bound stain was solubilized and
the absorbance was measured at 492 nm in a plate reader (Biotek
Instruments Inc., Powerwave XS, Wincoski, USA). Dose-response
curves were obtained for each test compound and cell line, and the
growth inhibition of 50% (GI50), corresponding to the concentration
of the compounds that inhibited 50% of the net cell growth was cal-
culated as described elsewhere [10]. Doxorubicin was tested in the
same manner to be used as a positive control.
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-
distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-diacyl-
sn-glycero-3-phosphocholine from egg yolk (egg-PC), 1,2-
dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt)
(DMPG) and cholesterol (Ch) were obtained from Sigma–Aldrich.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(poly-ethylene glycol)-2000] (ammonium salt) (DSPE-PEG) was
obtained from Avanti Polar Lipids, while dioctadecyldimethylam-
monium bromide (DODAB) was from Tokyo Kasei (lipid structures
are shown below).
O
O
O
H
P
O
O
O
N
O
O
DPPC
O
O
P
O
O
H
O
O
O
O
O
N
O
O
DSPC
O
O
P
R1
H
R1, R2 = fatty acid residues
O
R2
N
O
O
egg-PC
O
O
P
OH
O
H
HO
O
O
O
O
O
Na
DMPG
H3C
H3C
N
Br
DODAB
O
O
P
O
O
H
O
O
O
O
H3CO(H2CH2CO)45
N
H
O
NH4
DSPE-PEG

74
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
NH2
N
CO2Me
S
O
HO
OMe
OMe
i
2a, 40%
i
NH2
NH2
CO2Me
N
HO
OMe
MeO
N
CO2Me
S
O
S
Br
2b, 53%
1
OMe
i
HO
NH2
CO2Me
MeO
N
S
O
i) CuI (10mol%), N,N-dimethylglycine (30mol%),
Cs2CO3 (2 equiv.), dry dioxane, 110 oC, Ar, 8h.
2c, 65%
Scheme 1. Synthesis of di(hetero)arylethers 2a–c by copper-catalyzed C O coupling.
Nanoliposomes were prepared by evaporation of a mixture of
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 [18,19],
lipids and compound in chloroform under vacuum for 12 h. The
lipid/compound film was then hydrated with an aqueous buffer
solution (10 mM Tris–HCl buffer, pH = 7.4), above the lipids melt-
ing transition temperature (ca. 41 ◦C for DPPC [11], 23 ◦C for DMPG
[12], 55 ◦C for DSPC [13] and 45 ◦C for DODAB [14]), followed by six
extrusion cycles (using a LIPEXTM extruder) through 100 nm poly-
carbonate membranes. Between the extrusion cycles, the solutions
were allowed to equilibrate for 1 h. The final total lipid concentra-
tion was 1 mM, with a compound/lipid molar ratio of 1:333.
DLS and zeta potential measurements: Liposomes mean diameter
and size distribution (polydispersity index) were measured using
a Dynamic Light Scattering (DLS) equipment (NANO ZS Malvern
Zetasizer), at 25 ◦C, using a He–Ne laser of 633 nm and a detector
angle of 173◦. Five independent measurements were performed for
each sample. Malvern Dispersion Technology Software (DTS) was
used with multiple narrow mode (high resolution) data processing,
and mean size (nm) and error values were considered.
1
4ꢁε0
2ꢂꢃ2
hcR3
ꢀabs − ꢀfl
=
ꢂf + const
(2)
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 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 [20,21]:
2.4. 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 stan-
dard method (Eq. (1)) [15,16]. Quinine sulfate in H2SO4 0.05 M was
used as reference, ˚r = 0.546 at 25 ◦C [17].
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
f (ε, n) =
−
(5)
(6)
n2 + 2
The steady-state fluorescence anisotropy, r, is calculated by
IVV − GIVH
r =
IVV + 2GIVH
ArFsn2s
AsFrn2r
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).
˚s =
˚
r
(1)
where A is the absorbance at the excitation wavelength, F the inte-
grated emission area and n the refraction index of the solvents used.
Subscripts refer to the reference (r) or sample (s) compound. The

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
75
Table 1
Growth inhibitory activity of the di(hetero)arylethers 2a–c on the three human
tumor cell lines in study.
Compounds
GI50 (␮M)a
MCF-7
A375-C5
NCI-H460
2a
2b
2c
1.4 0.1
2.5 0.1
46.8 4.0
1.4 0.1
2.5 0.3
83.5 5.8
1.1 0.1
2.3 0.1
45.0 1.4
a
The lowest concentrations causing 50% of cell growth inhibition (GI50) after a
continuous exposure of 48 h, expressed as means SEM of three independent exper-
iments performed in duplicate. Doxorubicin was used as positive control (GI50:
MCF-7 = 43.3 2.6 nM; A375-C5 = 130.2 10.1 nM; NCI-H460 = 35.6 1.6 nM).
3. Results and discussion
3.1. Synthesis
Fig. 1. Normalized fluorescence spectra of 3 × 10−6 M solutions of compound 2a in
several solvents (ꢅexc = 360 nm). Inset: Absorption spectrum of 2 × 10−5 M solutions
of 2a in acetonitrile and dimethylformamide, as examples.
New
copper-catalyzed
di(heteroaryl)ethers
2a–c
were
prepared
by
C
O
coupling of the methyl 3-amino-6-
bromothieno[3,2-b]pyridine-2-carboxylate (1), earlier prepared
by us [8], with ortho, meta and para-methoxyphenols, using CuI as
the catalyst, N,N-dimethylglycine as the ligand and Cs2CO3 as the
base, in moderate to good yields (Scheme 1).
future drug delivery purposes was monitored using the intrinsic
fluorescence of the compounds.
The Ullmann ether synthesis has been extensively used for the
formation of diarylethers [22,23]. However, the harsh reaction con-
ditions (125–220 ◦C in neat phenol or solvents such as pyridine
collidine or N,N-dimethylformamide), the usual requirement for
stoichiometric (or higher) quantities of copper, and the fact that
unactivated aryl halides usually react in low yields have limited
the utility of this reaction [22]. Thus, development of methods for
the synthesis of diarylethers under relatively mild conditions is
receiving increasing interest. Ma and Cai reported for the first time
in 2003 that, under the action of N,N-dimethylglycine as the lig-
and, CuI-catalyzed coupling reaction of aryl halides and phenols
using Cs2CO3 as the base, could be carried out at 90 ◦C to give the
corresponding diarylethers [24]. As described for the amino acid
promoted CuI-catalyzed C N bond formation from aryl halides and
amines or N-containing heterocycles, the mechanism for the forma-
tion of the diarylethers (C O bond formation) may occur through
two pathways: (1) oxidative addition/reductive elimination; (2) ␲-
complex mechanism. Based on the fact that copper ions can form
chelates with amino acids through the carboxyl and amino groups,
Ma et al. put forward two possible catalytic cycles [25].
3.3. Fluorescence studies in several solvents
The absorption and fluorescence properties of compounds 2a
and 2b were studied in several solvents (Table 2). The normalized
fluorescence spectra of both compounds are shown in Figs. 1 and 2,
respectively. Examples of absorption spectra are shown as insets.
Compounds 2a and 2b exhibit reasonable fluorescence in sev-
eral solvents. Fluorescence quantum yield values are in the range
of 3% in chloroform and 34% in DMSO for compound 2a and of 2%
(chloroform) and 29% (DMSO) for compound 2b (Table 2). However,
no emission is observed in protic solvents, like water or ethanol.
This behavior can be due to specific solute-solvent interactions by
hydrogen bonds with protic solvents, namely by protonation of
the N atom of the pyridine ring. The same explanation can justify
the low fluorescence quantum yields obtained in chloroform, as
the formation of hydrogen bonds between chloroform and proton
acceptor molecules is known since a long time [29].
For both compounds, significant red shifts are observed for
emission in polar solvents (50 nm between cyclohexane and
dimethylsulfoxide for compound 2a and 52 nm for 2b). In
the absorption spectra, the red shifts are negligible (Table 2),
3.2. Biological activity
The effects of the di(hetero)arylethers 2a–c on the human tumor
cells growth were evaluated in three human tumor cell lines MCF-7
(breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-
small cell lung cancer) and the results are presented in Table 1. The
di(hetero)arylethers bearing a methoxy group in the ortho and meta
positions 2a and 2b, respectively, are the most promising antitumor
compounds presenting very low GI50 values (1.1–2.5 ␮M) in the
tumor cell lines in study. The position of the methoxy group is very
important for the activity, since compound 2c with the methoxy
group in the para position relative to the ether function, presented
very much higher GI50 values.
Doxorubicin, used as positive control, presents very high cyto-
toxicity in these cell lines but it is also very toxic for the human body
and nowadays is used in a liposomal pegylated formulation that
decreases its toxicity and adverse secondary effects and increases
the captation by the tumor [26–28].
Due to the antitumoral potential of the di(hetero)arylethers 2a
and 2b and as both are fluorescent, their photophysical proper-
ties were studied. Also, their encapsulation in nanoliposomes for
Fig. 2. Normalized fluorescence spectra of 3 × 10−6 M solutions of compound 2b in
several solvents (ꢅexc = 355 nm). Inset: Absorption spectrum of 2 × 10−5 M solutions
of 2b in acetonitrile and dimethylformamide, as examples.

76
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
Table 2
Maximum absorption (ꢅabs) and emission wavelengths (ꢅem), molar absorption coefficients (␧) and fluorescence quantum yields ( F) for compounds 2a and 2b in several
solvents.
ꢅabs (nm) (ε/104 M−1 cm−1
)
ꢅem (nm)
˚
F
a
Solvent
2a
2b
2a
2b
2a
2b
Cyclohexane
Dioxane
359 (0.37), 310 (0.54), 288 (3.07)
360 (0.55); 289 (3.69)
355 (0.50), 309 (0.77), 287 (3.40)
354 (0.58), 310 (0.89), 288 (3.49)
358 (0.56), 289 (3.39)
360 (0.39), 311 (0.61), 289 (3.06)
361 (0.46), 289 (3.00)
361 (0.45), 312 (0.71), 288 (3.02)
355 (0.45), 311 (0.69), 288 (2.80)
357 (0.59), 290 (3.02)
425
440
448
447
465
475
459
445
–
427
441
453
451
470
479
461
448
–
0.26
0.27
0.07
0.13
0.20
0.34
0.14
0.03
–
0.29
0.28
0.09
0.16
0.12
0.29
0.12
0.02
–
Ethyl acetateb
Dichloromethane
N,N-dimethylformamideb
Dimethylsulfoxideb
Acetonitrile
362 (0.62), 290 (3.42)
364 (0.47), 291 (2.71)
354 (0.55), 309 (0.84), 286 (3.46)
354 (0.57), 311 (0.91), 289 (3.50)
359 (0.56), 310 (0.88), 288 (3.72)
358 (0.56), 310 (0.89), 287 (3.69)
355 (0.38), 310 (0.60), 287 (2.48)
361 (0.46), 313 (0.87), 290 (2.88)
361 (0.41), 311 (0.77), 289 (2.83)
361 (0.46); 288 (3.06)
Chloroformb
Ethanol
Methanol
–
–
–
–
aRelative to quinine sulfate in H2SO4 0.05 M ((F = 0.546 at 25 ◦C [16]). Error about 10%.
bSolvents cut-off: dimethylsulfoxide: 270 nm; N,N-dimethylformamide: 275 nm; ethyl acetate: 265 nm; chloroform: 250 nm.
indicating that solvent relaxation after photoexcitation plays an
important role. In polar solvents, a clear band enlargement in emis-
sion is also observed (Figs. 1 and 2), which is usually related to an
intramolecular charge transfer (ICT) mechanism and/or to specific
solvent effects [18].
The absolute value of the difference in the excited and ground
state dipole moment vectors, estimated from the solvatochromic
plots (Fig. 3) and from molecular quantum mechanical calcula-
tions, are presented in Table 3. The obtained values are similar
and, therefore, both methods point to the presence of a significant
charge transfer mechanism in the excited state, more pronounced
for compound 2b.
Fig. 5 displays the representation of HOMO and LUMO molecu-
lar orbitals for the two compounds, obtained with the calculated
optimized geometries for the ground and the lowest excited
singlet state. It can be observed that the HOMO and LUMO
molecular orbitals are mainly located in the thieno[3,2-b]pyridine
system, with a small contribution of the ether oxygen atom. The
HOMO–LUMO transition (for both geometries) of these compounds
shows a significant charge transfer from the amine group linked to
the thiophene ring and from its S atom to the pyridine moiety. A
charge density decrease in the oxygen atom of the carbonyl group
is also observed in the HOMO–LUMO transition. This confirms the
CT character of the excited state for both compounds.
The solvatochromic plots for compounds 2a and 2b, shown in
Fig. 3, are reasonably linear, the slope being larger for compound
2b. This shows that the presence of the methoxy group in the meta
position causes an enhance of the ICT character of the excited state.
From ab initio molecular quantum chemistry calculations,
obtained with Gaussian 09 software [31] and use of a 6-311 + G(dp)
basis set at the TD-SCF DFT (B3LYP) level of theory [32] in gas
phase, the cavity radius (R) and the ground state dipole moment
(ꢃg) were determined for the two compounds (Table 3). The opti-
mized geometry of the ground state of the di(hetero)arylethers 2a
and 2b shows that the methoxyphenyl moiety is completely out of
the plane of the thienopyridine system. In the lowest excited state
a distortion occurs, which is much more pronounced in compound
2b, where the two moieties are further apart (Fig. 4). The directions
of the calculated dipole moments in the ground and excited state
are also indicated in Fig. 4, evidencing a change in the excited state
dipole moment vector to almost the opposite direction 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)).
The photophysical behavior of both compounds shows that they
can be considered as solvatochromic probes. The significant sensi-
tivity of the fluorescence emission to the fluorophore environment
can be very useful when probing the location/behavior of these
compounds in liposomes.
3.4. Fluorescence studies in nanoliposomes
Fluorescence experiments of both compounds encapsulated
in liposomes of several compositions were carried out. These
liposomes were composed either by neat phospholipids, or by
phospholipid mixtures, with or without cholesterol (Ch) and PEG.
Several different lipid compositions were studied, keeping in mind
future drug delivery applications of 2a and 2b as potential anti-
cancer drugs.
First, liposomes of neat DPPC (zwitterionic), DMPG (anionic),
DODAB (cationic) and Egg-PC (zwitterionic, composed of a phos-
phatidyl choline mixture) with encapsulated compounds were
prepared and fluorescence emission was monitored in both gel
(below the melting temperature, Tm) and liquid-crystalline (above
Tm) phases of the lipid. At room temperature, the phospholipids
DPPC and DODAB are in ordered gel phase, where the hydro-
carbon chains are fully extended and closely packed. DMPG has
a melting transition temperature very near room temperature
(Tm = 23 ◦C). The melting transition temperature of Egg-PC is very
low [33] and this lipid is in the fluid liquid-crystalline phase at
room temperature. Fluorescence spectra of compounds 2a and b
incorporated in these liposomes are presented in Fig. 6. The maxi-
mum emission wavelengths in Egg-PC and DPPC lipid membranes
Fig. 3. Solvatochromic plots (Eq. (4)) for compounds 2a and 2b. Solvents: 1 – cyclo-
hexane; 2 – dioxane; 3 – chloroform; 4 – ethyl acetate; 5 – dichloromethane; 6 –
dimethylsulfoxide; 7 – N,N-dimethylformamide; 8 – acetonitrile (values of ε and n
were obtained from ref. [30]).

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
77
Table 3
ꢀ
ꢀ
ꢁ
ꢂ
ꢀꢀ
ꢀ
ꢀ
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 (Å)
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
5.4
5.8
2.8
4.7
4.7
3.4
7.1
7.7
6.5
7.2
Fig. 4. Optimized geometries of compounds 2a and 2b obtained by Gaussian 09 software (gray: C atoms; white: H atoms; red: O atoms; blue: N atoms; yellow: S atoms).
Left: ground state; Right: 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.)
point to a hydrophobic environment for both compounds in lipid
membranes (Fig. 6, Table 4). In DODAB and DMPG aggregates, a
band enlargement and a shift to higher emission wavelengths are
observed, indicating a more hydrated environment for the com-
pounds in these aggregates. The red shift is more significant for
both compounds in DMPG aggregates, which can be explained by
the formation of DMPG unilamellar vesicles with perforations [34],
or leaky vesicles [35], where the compounds may feel more pene-
tration of water molecules.
the fluorescent molecule (and, thus, with the viscosity of the
fluorophore environment) [36]. Anisotropy values in a viscous
solvent (glycerol) were also determined, for comparison. Steady-
state fluorescence anisotropy results (Table 4) allow concluding
that both compounds are mainly located in the inner region of
the lipid membrane, feeling the penetration of water molecules,
especially in DMPG aggregates. The transition from the rigid gel
phase to the fluid liquid-crystalline phase is clearly detected by
a notable decrease in anisotropy at 50 ◦C. The r values in Egg-
PC liposomes at 25 ◦C (fluid phase) are also significantly lower
than those observed in lipids which are in the gel phase at this
temperature.
Fluorescence anisotropy (r) measurements (Table 4) can give
relevant information about the location of the compounds in
liposomes, as r increases with the rotational correlation time of
Fig. 5. Representation of HOMO and LUMO molecular orbitals of the di(hetero)arylethers 2a (above) and 2b (below). Left: optimized geometry for the ground state; right:
optimized geometry for the lowest excited singlet state.

78
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
Fig. 6. Normalized fluorescence spectra of compounds 2a and 2b (3 × 10−6 M) in lipid aggregates of Egg-PC, DPPC, DMPG and DODAB, below (17 ◦C or 25 ◦C) and above (50 ◦C)
the melting temperature of the lipids.
Table 4
Previous studies have shown that DODAB-based liposomes
exhibit very large sizes (diameter larger than 250 nm) [39–41] and,
Steady-state fluorescence anisotropy (r) values and maximum emission wave-
lengths (ꢅem) for compounds 2a and 2b in lipid aggregates, below (17 ◦C or 25 ◦C)
for this reason, this cationic lipid was not used in the liposome for-
mulations prepared. On the contrary, most of the nanoliposomes
contain DMPG, as the presence of pores across this lipid membrane
has a promising biological relevance in applications for controlled
release from nanocompartments [34].
and above (50 ◦C) transition temperature of the lipids. Anisotropy values in glycerol
at room temperature are also shown for comparison.
Lipid
T (◦ C)
Compound 2a
ꢅem (nm)
Compound 2b
ꢅem (nm)
r
r
The size and size distribution (polydispersity index) of the pre-
pared nanoliposomes with encapsulated compounds 2a and 2b
were obtained by DLS. All the liposomes have a mean hydrody-
namic diameter lower than 140 nm and generally low polydis-
persity (Table 5). The Egg-PC:Ch (7:3), Egg-PC:Ch:DMPG (7:3:1)
and DSPC:Ch:DSPE-PEG2000 (1:1:0.75) formulations allowed us to
obtain the smaller nanoliposome diameters, which are lower than
100 nm for both compounds. The Egg-PC:Ch formulation is the one
with a lower polydispersity.
Egg-PC
DPPC
25
25
50
25
50
17
50
25
433
432
432
439
438
443
442
420
0.121
0.196
0.095
0.179
0.115
0.147
0.105
0.295
436
433
434
437
436
447
446
418
0.120
0.186
0.075
0.155
0.112
0.154
0.108
0.309
DODAB
DMPG
Glycerol
Standard deviations (SD) were calculated from the mean of the
data of a series of five experiments conducted using the same
parameters.
The two compounds show significant fluorescence emission
when incorporated in nanoliposomes (Fig. 7) and steady-state fluo-
rescence anisotropy values are generally high at room temperature
(Table 6).
Considering future developments of these compounds delivery
using nanoliposomes as drug carriers, several different liposome
formulations were prepared [37–39], some of them containing
cholesterol (Ch) and polyethylene glycol (PEG). The incorporation
of Ch may increase the stability by modulating the fluidity of the
lipid bilayer, preventing crystallization of the phospholipid acyl
chains and providing steric hindrance to their movement. Further
advances in liposome research found that PEG, which is inert in the
body, allows longer circulatory life of the drug delivery system [7].
Upon increasing temperature, both compounds exhibit
a
significant decrease in fluorescence anisotropy (Table 6), feeling
an increase in fluidity of the nanoliposome membranes at high
Table 5
Mean hydrodynamic diameter and polydispersity of several nanoliposome formulations with encapsulated compounds 2a and 2b, at 25 ◦C.
Formulation
Compound 2a
Compound 2b
Hydrodynamic diameter
(nm) (mean SD)
Polydispersity
(mean SD)
Hydrodynamic diameter
(nm) (mean SD)
Polydispersity
(mean SD)
Egg-PC:Ch (7:3)
Egg-PC:Ch:DMPG (7:3:1)
DPPC:DMPG (1:1)
DPPC:DMPG (1:2)
DPPC:DMPG (2:1)
84.4 0.4
81.2 0.5
133.4 0.6
131.7 0.6
125.6 0.5
117.4 0.5
83.6 0.4
0.19 0.01
0.26 0.01
0.34 0.01
0.43 0.02
0.39 0.02
0.34 0.01
0.31 0.01
87.8 0.4
90.2 0.5
116.2 0.6
141.0 0.4
114.1 0.4
136.3 0.6
71.7 0.3
0.21 0.01
0.36 0.02
0.42 0.02
0.24 0.01
0.34 0.01
0.35 0.01
0.28 0.01
DSPC:DMPG (5:1)
DSPC:Ch:DSPE-PEG2000 (1:1:0.75)

M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
79
Fig. 7. Normalized fluorescence spectra of compounds 2a and 2b (3 × 10−6 M) in nanoliposomes of different compositions at room temperature.
Table 6
Both compounds have reasonable fluorescence quantum yields
and can be considered as solvatochromic probes.
Steady-state fluorescence anisotropy (r) values and maximum emission wave-
lengths (ꢅem) for compounds 2a and 2b encapsulated in nanoliposomes, at 25 ◦
C
Our experimental results in nanoliposomes suggest that both
compounds can be transported in the hydrophobic region of
the lipid bilayer. The Egg-PC:Ch (7:3), Egg-PC:Ch:DMPG (7:3:1)
and DSPC:Ch:DSPE-PEG2000 (1:1:0.75) nanoliposomes are the best
formulations for encapsulation of these thienopyridines, consid-
ering the size (diameter lower than 100 nm) and polydispersity.
These results may be important for future drug delivery applica-
tions of these new potential antitumoral aryletherthienopyridines,
using nanoliposomes as drug carriers.
and 50 ◦C.
Formulation
Compound 2a
Compound 2b
ꢅem (nm)
r
ꢅem (nm)
r
Egg-PC:Ch (7:3)
Egg-PC:Ch:DMPG (7:3:1)
25
25
50
25
50
25
50
25
50
25
50
25
435
434
433
445
444
444
443
447
437
458
458
440
0.117
0.176
0.121
0.239
0.144
0.220
0.147
0.213
0.143
0.215
0.200
0.216
439
441
442
452
443
445
446
447
437
457
457
448
0.109
0.149
0.109
0.184
0.108
0.176
0.125
0.176
0.116
0.210
0.196
0.185
DPPC:DMPG (1:1)
DPPC:DMPG (1:2)
DPPC:DMPG (2:1)
DSPC:DMPG (5:1)
Acknowledgements
To the Foundation for the Science and Technology (FCT,
Portugal) for financial support to the NMR Portuguese network
(PTNMR, Bruker Avance III 400-Univ. Minho). FCT and FEDER (Euro-
pean Fund for Regional Development) for financial support to
the Research Centers, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-
01-0124-FEDER-022716)] and CFUM [PEst-C/FIS/UI0607/2011
(F-COMP-01-0124-FEDER-022711)], and to the research projects
PTDC/QUI/81238/2006 (FCOMP-01-0124-FEDER-007467) (photo-
physical studies) and PTDC/QUI-QUI/111060/2009 (F-COMP-01-
0124-FEDER-015603) (organic synthesis and biological studies),
which are also financed by COMPETE/QREN/EU.
DSPC:Ch:DSPE-PEG2000
(1:1:0.75)
50
437
0.133
449
0.136
temperature. Therefore, both compounds are mainly located in the
nanoliposome membranes. The decrease in r values at 50 ◦C is low
in DSPC:DMPG (5:1) vesicles, due to the higher melting transition
temperature of DSPC (Tm = 55 ◦C [13]), as this phospholipid is
still in the gel phase at 50 ◦C. This fact can also contribute to the
more hydrated environment reported by these compounds in
DSPC:DMPG liposomes, as the rigidity of these membranes can
prevent the compounds to penetrate more deeper in the lipid
membranes. Overall, these results indicate that both compounds
can be transported in the hydrophobic region of the lipid bilayers.
References
[1] I. Hayakama, R. Shioya, T. Agatsuma, H. Furokawa, Y. Sugano, Thienopyri-
dine, Benzofuran derivatives as potent anti-tumor agents possessing different
structure-activity relationship, Bioorganic and Medicinal Chemistry 14 (2004)
3411–3414.
[2] D.H. Boschelli, B. Wu, A.C. Barrios Sosa, H. Durutlic, J.J. Chen, Y. Wang, J.M. Golas,
J. Lucas, F. Boscheli, Synthesis and Src kinase inhibitory activity of 2-phenyl-
and 2-thienyl-7-phenylaminothieno[3,2-b]pyridine-6-carbonitriles, Journal of
Medicinal Chemistry 48 (2005) 3891–3902.
[3] S. Claridge, F. Raeppel, M.-C. Granger, N. Bernstein, O. Saavedra, L. Zhan, D.
Llewellyn, A. Wahhab, R. Deziel, J. Rahil, N. Beaulieu, H. Nguyen, I. Dupont, A.
Barsalou, C. Beaulieu, I. Chute, S. Gravel, M.-F. Robert, S. Lefebvre, M. Dubay, R.
Pascal, J. Gillespie, Z. Jin, J. Wang, J.M. Besterman, A.R. MacLeod, A. Vaisburg, Dis-
covery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of
c-Met and VEGFR2 tyrosine kinase, Bioorganic and Medicinal Chemistry Letters
18 (2008) 2793–2798.
4. Conclusions
New fluorescent methoxylated di(hetero)aryl ethers in the
thieno[3,2-b]pyridine series, 2a–c, were prepared by a copper-
catalyzed Ullmann-type C O coupling in moderate to good yields,
using an amino acid as the ligand.
Cell growth inhibition assays showed that compounds 2a and
2b are promising as antitumoral drugs, presenting very low GI50
values in the three human tumor cell lines tested.

80
M.-J.R.P. Queiroz et al. / Journal of Photochemistry and Photobiology A: Chemistry 238 (2012) 71–80
[4] S. Raeppel, S. Claridge, O. Saavedra, F. Gaudette, L. Zhan, M. Mannion, N. Zhou,
[26] M.R. Ranson, J. Carmichael, K. O’Byrne, S. Stewart, D. Smith, A. Howell,
Treatment of advanced breast cancer with sterically stabilized liposomal dox-
orubicin: results of a multicenter phase II trial, Journal of Clinical Oncology 15
(1997) 3185.
[27] O. Lyass, B. Uziely, R. Ben-Yosef, D. Tzemach, N.I. Heshing, M. Lotem, G. Bruf-
man, A. Gabizon, Correlation of toxicity with pharmacokinetics of pegylated
liposomal doxorubicin (Doxil) in metastatic breast carcinoma, Cancer 89 (2000)
1037–1047.
[28] M.E.R. O’Brien, N. Wigler, M. Inbar, R. Rosso, E. Grischke, A. Santoro, R. Catane,
D.G. Kieback, P. Tomczak, S.P. Ackland, F. Orlandi, L. Mellars, L. Alland, C. Tendler,
Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated
liposomal doxorubicin HCl (CAELYXTM/Doxil®) versus conventional doxoru-
bicin for first-line treatment of metastatic breast cancer, Annals of Oncology
15 (2004) 440–449.
[29] (a) K.C. James, P.R. Noyce, Hydrogen bonding between testosterone propionate
and solvent in chloroform-cyclohexane solutions, Spectrochimica Acta Part A
27 (1971) 691–696;
F. Raeppel, M.-C. Granger, L. Isakovick, R. Deziel, H. Nguyen, N. Beaulieu, C.
Beaulieu, I. Dupont, M.-F. Robert, S. Lefebvre, M. Dubay, J. Rahil, J. Wang, H. Ste-
Croix, A.R. MacLeod, J. Besterman, A. Vaisburg, N-(3-fluoro-4-(2-arylthieno[3,2-
b]pyridine-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: a
novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors, Bioor-
ganic and Medicinal Chemistry Letters 19 (2009) 1323–1328.
[5] F. Gaudette, S. Raeppel, H. Nguyen, N. Beaulieu, C. Beaulieu, I. Dupont, A. Robert
Macleod, J.M. Besterman, A. Vaisburg, Identification of potent and selective
VEGFR receptor tyrosine kinase inhibitors having new amide isostere head-
groups, Bioorganic and Medicinal Chemistry Letters 20 (2010) 848–852.
[6] R. Banerjee, Liposomes, Applications in medicine, Journal of Biomaterials Appli-
cations 16 (2001) 3–21.
[7] Y. Malam, M. Loizidou, A.M. Seifalian, Liposomes and nanoparticles: nanosized
vehicles for drug delivery in cancer, Trends in Pharmacological Sciences 30
(2009) 592–599.
[8] R.C. Calhelha, M.-J.R.P. Queiroz, Synthesis of new thieno[3,2-b]pyridine
derivatives by palladium-catalyzed couplings and intramolecular cyclizations,
Tetrahedron Letters 51 (2010) 281–283.
[9] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. War-
ren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for
anticancer-drug screening, Journal of the National Cancer Institute 82 (1990)
1107–1112.
(b) G.R. Wiley, S.I. Miller, Thermodynamic parameters for hydrogen-bonding
of chloroform with Lewis bases in cyclohexane – proton magnetic-resonance
study, Journal of the American Chemical Society 94 (1972) 3287–3293.
[30] D.R. Lide (Ed.), Handbook of Chemistry and Physics, 83rd ed., CRC Press, Boca
Raton, 2002.
[31] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M.
Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M.
Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C.
Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth,
P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman,
J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A. 02, Gaussian, Inc.,
Wallingford, CT, 2009.
[32] F. Jensen, Introduction to Computational Chemistry, John Wiley & Sons, West
Sussex, England, 1999.
[33] D. Papahadjopoulos, N. Miller, Phospholipid model membranes. I. Structural
characteristics of hydrated liquid crystals, Biochimica et Biophysica Acta 135
(1967) 624–638.
[34] K.A. Riske, L.Q. Amaral, H.-G. Döbereiner, M.T. Lamy, Mesoscopic structure in
the chain-melting regime of anionic phospholipid vesicles: DMPG, Biophysical
Journal 86 (2004) 3722–3733.
[35] R.P. Barroso, K.R. Perez, I.M. Cuccovia, M.T. Lamy, Aqueous dispersions of DMPG
in low salt contain leaky vesicles, Chemistry and Physics of Lipids 165 (2012)
169–177.
[10] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J.
Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo,
M. Boyd, Feasibility of a high-flux anticancer drug screen using a diverse panel
of cultured human tumor-cell lines, Journal of the National Cancer Institute 83
(1991) 757–776.
[11] B.R. Lentz, Membrane fluidity as detected by diphenylhexatriene probes, Chem-
istry and Physics of Lipids 50 (1989) 171–190.
[12] M.T. Lamy-Freund, K.A. Riske, The peculiar thermo-structural behavior of the
anionic lipid DMPG, Chemistry and Physics of Lipids 122 (2003) 19–32.
[13] M. Ueno, S. Katoh, S. Kobayashi, E. Tomoyama, S. Ohsawa, N. Koyama, Y. Morita,
Evaluation of phase transition temperature of liposomes by using the tau-
tomerism of ␣-benzoylacetoanilide, Journal of Colloid and Interface Science
134 (1990) 589–592.
[14] E. Feitosa, P.C.A. Barreleiro, G. Olofsson, Phase transition in dioctade-
cyldimethylammonium bromide and chloride vesicles prepared by different
methods, Chemistry and Physics of Lipids 105 (2000) 201–213.
[15] J.N. Demas, G.A. Crosby, Measurement of photoluminescence quantum yields
– review, Journal of Physical Chemistry 75 (1971) 991–1024.
[16] S. Fery-Forgues, D. Lavabre, Are fluorescence quantum yields so tricky to mea-
sure? A demonstration using familiar stationery products, Journal of Chemical
Education 76 (1999) 1260.
[17] S.R. Meech, D. Phillips, Photophysics of some common fluorescence standards,
Journal of Photochemistry 23 (1983) 193–217.
[18] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Aca-
demic/Plenum Press, New York, 1999.
[36] B. Valeur, Molecular Fluorescence – Principles and Applications, Weinheim,
Wiley-VCH, 2002.
[19] N. Mataga, T. Kubota, Molecular Interactions and Electronic Spectra, Marcel
Dekker, New York, 1970.
[37] M.R. Mozafari, V. Hasirci, Mechanism of calcium ion induced multil-
amellar vesicle DNA interaction, Journal of Microencapsulation 15 (1998)
55–65.
[38] Y. Ran, S.H. Yalkowsky, Halothane, a novel solvent for the preparation of
liposomes containing 2-4ꢁ-amino-3ꢁ-methylphenyl benzothiazole (AMPB), an
anticancer drug: A technical note, AAPS Pharmacetical Science and Technology
4 (2003) (article 20).
[39] N. Berger, A. Sachse, J. Bender, R. Schubert, M. Brandl, Filter extrusion of lipo-
somes using different devices: comparison of liposome size, encapsulation
efficiency, and process characteristics, International Journal of Pharmaceutics
223 (2001) 55–68.
[20] N.G. Bakhshiev, Universal molecular interactions and their effects on the posi-
tion of the electronic spectra of molecules in two component solutions. I. Theory
(liquid solutions), Optics and Spectroscopy 10 (1961) 379–384.
[21] N.G. Bakhshiev, Universal molecular interactions and their effects on the posi-
tion of the electronic spectra of molecules in two component solutions, Optics
and Spectroscopy 12 (1962) 309–313, Optics and Spectroscopy 13 (1962)
24–29.
[22] J. Lindley, Tetrahedron report number 163: copper assisted nucleophilic sub-
stitution of aryl halogen, Tetrahedron 40 (1984) 1433–1456.
[23] L.D. Boger, A.M. Patane, J. Zhou, Total synthesis of bouvardin, O-
methylbouvardin, and O-methyl-N9-desmethylbouvardin, Journal of the
American Chemical Society 116 (1994) 8544–8556.
[40] L.R. Tsuruta, A.M. Carmona-Ribeiro, Counterion effects on colloid stability of
cationic vesicles and bilayer-covered polystyrene microspheres, Journal of
Physical Chemistry 100 (1996) 7130–7134.
[24] D. Ma, Q. Cai, N,N-dimethyl glycine-promoted Ulmann coupling reaction of
phenols and aryl halides, Organic Letters 5 (2003) 3799–3802.
[25] H. Zhang, Q. Cai, D. Ma, Amino acid promoted CuI-catalyzed C N bond forma-
tion between aryl halides and amines or N-containing heterocycles, Journal of
Organic Chemistry 70 (2005) 5164–5174.
[41] E.M.S. Castanheira, M.S.D. Carvalho, A.R.O. Rodrigues, R.C. Calhelha, M.-J.R.P.
Queiroz, New potential antitumoral fluorescent tetracyclic thieno[3,2-
b]pyridine derivatives: interaction with DNA and nanosized liposomes,
Nanoscale Research Letters 6 (2011) (article 379).