N-(di)icosyl-substituted benzo[a]phenoxazinium chlorides: Synthesis and evaluation as near-infrared membrane probes

Article abstract of DOI:10.1002/ejoc.201001579

Five benzo[a]phenoxazinium chlorides containing alkyl chains with twenty carbon atoms at the 5- or 9-positions of the tetracyclic ring were efficiently synthesised and characterised by UV/Vis and NIR spectroscopy. The absorption and emission maxima in ethanol occur in the range 627-641 nm and 645-676 nm, respectively, with quantum yields varying from 0.14 to 0.38. Preliminary photophysical studies with these fluorochromophores in zwitterionic [2,3-bis(palmitoyloxy)propyl-2-(trimethylammonio)ethyl phosphate, DPPC] and cationic (N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide, DODAB) vesicles were carried out. The results showed that the new benzo[a]phenoxazinium derivatives are able to detect the gel to liquid-crystalline lipid phase transition through variations in either the extent of H-aggregation or in the acid-base equilibrium. Novel benzo[a]phenoxazinium chlorides possessing icosyl groups as substituents at the 5- or 9-positions of the tetracyclic ring were photophysically studied in both zwitterionic and cationic vesicles. The results revealed that these fluorochromophores are able to detect the gel to liquid-crystalline lipid phase transition through variations either in the extent of H-aggregation or in an acid-base equilibrium. Copyright

Full text of DOI:10.1002/ejoc.201001579

FULL PAPER
DOI: 10.1002/ejoc.201001579
N-(Di)icosyl-Substituted Benzo[a]phenoxazinium Chlorides: Synthesis and
Evaluation as Near-Infrared Membrane Probes
Sarala Naik,[a] Carla M. A. Alves,[a] Paulo J. G. Coutinho,[b] and
M. Sameiro T. Gonçalves*[a]
Keywords: Fluorescent probes / Membranes / Vesicles / Surfactants / Imaging agents
Five benzo[a]phenoxazinium chlorides containing alkyl
chains with twenty carbon atoms at the 5- or 9-positions of
the tetracyclic ring were efficiently synthesised and charac-
terised by UV/Vis and NIR spectroscopy. The absorption and
emission maxima in ethanol occur in the range 627–641 nm
and 645–676 nm, respectively, with quantum yields varying
from 0.14 to 0.38. Preliminary photophysical studies with
these fluorochromophores in zwitterionic [2,3-bis(palmitoyl-
oxy)propyl-2-(trimethylammonio)ethyl phosphate, DPPC]
and cationic (N,N-dimethyl-N-octadecyloctadecan-1-amin-
ium bromide, DODAB) vesicles were carried out. The results
showed that the new benzo[a]phenoxazinium derivatives are
able to detect the gel to liquid-crystalline lipid phase transi-
tion through variations in either the extent of H-aggregation
or in the acid–base equilibrium.
The variety of fluorescent markers available for studies
of the biophysical properties of lipid membranes includes
9-(dicyanovinyl) julolidine (DCVJ), 7-nitrobenz-2-oxa-1,3-
diazol-4-yl (NBD), Prodan, 7-(dialkylamino)coumarin,
styryl, 3-hydroxychromone (3HC), and their derivatives, as
well as Nile Red and Nile Blue; the last two are oxazine
dyes.[11–20,8,4]
The synthesis of novel fluorochromophores that are
based on the oxazine core, with substituents that allow for
their interaction with a variety of biological molecules, is
important for labelling purposes.[21] Most biological macro-
molecules and structures have hydrophobic and hydrophilic
zones, hence, the presence of a long alkyl chain in the fluo-
rescence label allows it to easily bind to the hydrophobic
parts of biomolecules or biomembranes, enabling the flu-
orophore to probe its environment.[22]
When these types of probes are incorporated into bio-
membranes, their oxazine core can report on the local
changes of the membrane properties, namely micropolarity,
the hydration level, charge and microviscosity. These prop-
erties are important to understand the structure, dynamics
and function of the biological membrane, as previously
mentioned. The cationic nature of benzophenoxazinium
fluorophores is expected to allow such compounds to probe
the charge density of the biomembrane.
Considering these facts, and as part of our current re-
search interests in the synthesis and characterisation of
fluorescence probes,[23–30] this work describes the synthesis
of five benzo[a]phenoxazinium chlorides with different
combinations of substituents at the 5-, 9- and 10-positions
of the polycyclic aromatic ring, as well as their photophysi-
cal behaviour in homogeneous media and in zwitterionic/
cationic vesicles.
Introduction
Cellular membranes, which are selective barriers that
separate inside and outside cellular volumes, are crucial for
interactions with the cell exterior by enabling the transfer
of many important compounds for cell metabolism and for
chemical and electrical signalling.[1–3] In all biomembranes,
the lipid bilayer constitutes the backbone in which various
proteins and glycan-containing membrane anchors are em-
bedded.
The understanding of biological functions of cell mem-
branes is directly connected to their fundamental physico-
chemical properties. Several parameters, such as membrane
electrostatics, phase state, hydration, and dynamics of the
constituent molecules, establish the membrane structure
and control the binding and transport of molecular and
ionic species. Furthermore, membranes can determine the
correct insertion, proper folding, and function of mem-
brane proteins.[4]
The use of fluorescent probes is one of the most suitable
methods for monitoring these parameters in situ, and such
environment-sensitive fluorophores are of extreme impor-
tance. These compounds provide information on the prop-
erties of the molecular environment through changes in
their photophysical characteristics.[5–10]
[a] Centre of Chemistry, University of Minho,
4710-057 Braga, Portugal
Fax: +351-253 604382
E-mail: msameiro@quimica.uminho.pt
[b] Centre of Physics, University of Minho,
4710-057 Braga, Portugal
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Results and Discussion
Synthesis
presence of hydrochloric acid, gave the benzo[a]phenoxazin-
ium chlorides 1a–c. Starting from 5-(icosylamino)-2-
nitrosophenol hydrochloride (2d) and 5-(diicosylamino)-2-
nitrosophenol hydrochloride (2e), and using N-propyl-
naphthalen-1-amine (3b), compounds 1d and 1e were ob-
tained, respectively. After purification by column
chromatography on silica gel, cationic dyes 1a–e were iso-
lated as blue solid materials in good to excellent yields
(Table 1), and were fully characterised by high resolution
mass spectrometry (HRMS), IR and NMR (1H and 13C)
spectroscopy.
Benzo[a]phenoxazinium chlorides 1a–e were synthesised
by condensation of 5-(alkylamino)-2-nitrosophenol hydro-
chlorides 2a–e with N-alkylnaphthalen-1-amines 3a or 3b
in acid media (Scheme 1). The required nitrosophenol 2a–e
was obtained by nitrosation of the corresponding 3-alkyl-
aminophenol derivative with sodium nitrite and hydro-
chloric acid in either water or a mixture of ethanol/water as
the solvent.[23–31] The 3-(icosylamino)phenol and 3-(di-
icosylamino)phenol, precursors of compounds 2d and 2e,
as well as N-icosylnaphthalen-1-amine (3a) or N-propyl-
naphthalen-1-amine (3b),[28,29] were obtained in moderate
to good yields by alkylation of 3-aminophenol and naphth-
alen-1-amine with 1-bromoicosane or 1-bromopropane (in
the case of compound 3b), in ethanol. Precursors of ni-
trosophenols 2a–c were commercial reagents.
Table 1. Synthesis and UV/Vis/NIR data for compounds 1a–e in
ethanol.
Yield [%] ε [M–1 cm–1] λabs [nm] λem [nm]
ΦF
1a
1b
1c
1d
1e
73
68
85
37
70
89888
56930
63961
27413
48553
627
637
631
627
641
645
671
670
647
676
0.38
0.23
0.14
0.36
0.14
The IR spectra of these benzo[a]phenoxazinium dyes
showed the expected bands due to stretching vibrations of
the amine function (3450–3402 cm–1), the C–H linkage of
the methyl and methylenic groups (2956–2850 cm–1), the C–
C linkage of the aliphatic chains (1186–817 cm–1), as well
as a strong band of the C=N bond (1657–1590 cm–1) as a
result of the oxazine ring.
1
The H NMR spectra showed signals from the methyl
groups of the aliphatic chains in the form of triplets or a
broad singlet (1e; δ = 0.83–1.09 ppm), and the methyl group
directly linked to the aromatic ring as a singlet (1a; δ =
2.40 ppm). In compound 1c, with dimethylamine at the 9-
position of the benzo[a]phenoxazinium ring, the methyl
protons resonated as singlets (δ = 3.24 ppm). The methyl
protons of the ethyl groups (R1 and/or R2) appeared as a
multiplet (1a) or a triplet (1b; δ = 1.19–1.40 ppm). For all
compounds, the methylenic groups close to the nitrogen
atoms (NHCH2CH2) in the 5- or 9-positions of the hetero-
cycle appeared as broad singlets or as multiplets (1c; δ =
1.71–1.95 ppm), and the protons directly linked to the ni-
trogen atoms (NHCH2CH2) appeared as broad singlets or
as multiplets (1c; δ = 3.27–3.81 ppm). The spectra also
showed the expected aromatic protons, in particular, H-10
resonated as doublets or as broad singlets (1d; δ = 6.92–
7.11 ppm), H-8 as singlets (1a and 1b), a meta doublet (1c;
J = 2.7 Hz) or broad singlets (1d and 1e; δ = 6.10–
6.48 ppm), and H-11, which appeared as a singlet (1a), a
multiplet (1e) or as doublets (δ = 7.37–7.90 ppm).
The 13C NMR spectra showed signals corresponding to
the aliphatic N-substituents in the heterocycle, namely the
methyl groups (δ = 11.55–14.05 ppm), and the methyl group
directly attached to the benzene ring (1a; δ = 18.29 ppm).
For compound 1c, with dimethylamine at the 9-position of
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 1a–e.
Condensation of 5-ethylamino-4-methyl-2-nitrosophenol the benzo[a]phenoxazinium ring, the methyl carbons ap-
hydrochloride (2a), 5-diethylamino-2-nitrosophenol hydro- peared at δ = 41.10 ppm. For all compounds, carbon atoms
chloride (2b) and 5-dimethylamino-2-nitrosophenol hydro- of methylenic groups close to the nitrogen atom
chloride (2c), with N-icosylnaphthalen-1-amine (3a) in the (NHCH2CH2) in the 5- and 9-positions of heterocycles ap-
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N-(Di)icosyl-Substituted Benzo[a]phenoxazinium Chlorides
peared between δ = 21.98 and 30.77 ppm, and the carbon
atoms directly linked to nitrogen (NHCH2CH2) occurred
between δ = 38.67 and 52.02 ppm. The spectra also showed
the expected aromatic signals, such as: C-6 (δ = 92.29–
93.44 ppm), C-8 (δ = 93.08–95.81 ppm) and C-11 (δ =
131.01–132.39 ppm).
Photophysical Studies
Electronic absorption and emission spectra of 10–6
m
solutions in degassed absolute ethanol were measured for
all the synthesised benzo[a]phenoxazinium chlorides 1a–e.
It was observed that the absorption maxima (λabs) for 1a–e
lie in the range of 627–641 nm, with molar absorptivities (ε)
of between 27413 and 89888 m–1 cm–1. The emission max-
ima (λem) were found to be in the range of 645–676 nm
(exciting at 570 nm). It can also be seen that 1a, with one
C20 alkyl chain at the 5-position and one methyl group at
the 10-position, has the same absorption and emission max-
ima as 1d, which has one C20 alkyl chain at the 9-position
but lacking the methyl group at the 10-position. The fluo-
rescence quantum yields measured with Oxazine 1 as a
standard (fluorescence quantum yield, ΦF = 0.11 in ethan-
ol)[32] for 1a and 1d are similar (0.38 and 0.36). Hence, it
can be assumed that similar substitutions at the 5- and 9-
positions reveal similar fluorescence parameters, irrespec-
tive of the substitution at the 10-position.
Figure 2. Absorption and fluorescence spectra of compound 1a in
DODAB (left) and DPPC (right) vesicles below (T = 25 °C) and
above (T = 55 °C) the gel to liquid-crystalline lipid phase transition.
Similarly, dyes 1b, 1c and 1e have comparable values for
absorption maxima, emission maxima and quantum yields.
In contrast to 1a and 1d, which are monoalkyl-substituted
at the 9-position, derivatives with dialkyl substitution at the
9-position showed a bathochromic shift in absorption and
emission maxima, the latter being superior (about 35 nm),
but presenting lower fluorescence quantum yields (0.14 or
0.23). The above results suggested that mono-substitution
at the 9-position displayed better fluorescence quantum
yields than di-substitution, irrespective of the chain length
and the substituent at the 10-position.
Figure 3. Absorption and fluorescence spectra of compound 1d in
DODAB (left) and DPPC (right) vesicles below (T = 25 °C) and
above (T = 55 °C) the gel to liquid-crystalline lipid phase transition.
As an initial photophysical study with biological model
systems, compounds 1a, 1d and 1e were incorporated into
zwitterionic (DPPC) and cationic (DODAB) vesicles (Fig-
ure 1). It was observed that, with the exception of com-
It was previously shown that, depending on the physico-
pound 1d in DPPC, these compounds are able to detect chemical environment, 5,9-disubstituted benzo[a]phenoxaz-
the gel to liquid-crystalline lipid phase transition by simple inium dyes can form H-aggregates (absorption at ca. 50 nm
absorption measurements, as well as by steady-state fluores- to the blue) and are involved in acid–base equilibria (ab-
cence emission (see Figures 2, 3 and 4).
sorption at ca. 100 nm to the blue).[23–30] Recently, we have
also provided evidence that the main site of acid–base equi-
libria is the amine at the 5-position.[29] An acid–base equi-
librium was not observed in aqueous solutions, probably
because the basic neutral form was hydrogen-bonded and
showed a similar photophysical behaviour to that of the
positive acid form.
In the DPPC vesicles, all the compounds seem to be well-
hydrated near the beginning of the membrane interface be-
cause the basic form is not observed. It can also be seen
that the icosylamino substituent at the 9-position (com-
pounds 1d and 1e) prevents the formation of H-aggregates,
Figure 1. Structures of DPPC and DODAB.
Eur. J. Org. Chem. 2011, 2491–2497
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M. S. T. Gonçalves et al.
FULL PAPER
ily be explained by the fact that, upon membrane insertion,
the 5-amino position will be buried within the aliphatic
chain, making deprotonation impossible. In addition, H-
aggregation is observed for this compound, which decreases
with the membrane phase transition, as observed for
DPPC.
Further confirmation that deprotonation occurs mainly
at the 5-amino position was obtained from ab initio molec-
ular quantum chemistry calculations, obtained with
Gaussian 09 software.[33] Optimised geometries were ob-
tained using a 3-21+G(d) basis set[34] and the DFT method
with the B3LYP functional. The molecular energy as well
as HOMO–LUMO energy levels were then obtained by
using a 6-311++G(d,p)[34] basis set with the same DFT
method. Results are shown in Table 2 for a benzo[a]pheno-
xazinium chloride with R = H and single methyl substitu-
tion at both the 5- and 9-amino positions in its acid and
two possible basic forms.
Figure 4. Absorption and fluorescence spectra of compound 1e in
DODAB (left) and DPPC (right) vesicles below (T = 25 °C) and
above (T = 55 °C) the gel to liquid-crystalline lipid phase transition.
Table 2. Quantum chemical calculations (in vacuo) using Gaussian
09. Energy values are given in Hartree units.
Acid
Base 1[a]
Base 2[b]
which are non-fluorescent. This aggregation process hap-
pens for compound 1a (Figure 2) when DPPC is present in
the gel phase, but is lost when the DPPC membrane un- Zero point energy
dergoes its phase transition. Temperature increases should
reduce the fluorescence intensity as the rate of the compet-
Molecular energy
–935.062
0.30499
–0.32094
–0.23211
–934.636
0.289868
–0.19267
–0.09159
–934.628
0.289954
–0.19176
–0.09974
HOMO energy
LUMO energy
[a] Base obtained by deprotonating at the 5-amino position. [b]
Base obtained by deprotonating at the 9-amino position.
ing non-radiative internal conversion process increases.
However, an increase in the fluorescence intensity is ob-
served for compound 1a, which can be explained by the
absence of non-fluorescent H-aggregates in the liquid-crys-
talline membrane phase. The 9-dialkylated-amino derivative
1e (Figure 4) detects the lipid phase transition with a shift
to the blue in both absorption and emission maxima. Con-
sidering that the hydration and fluidity of the interface in-
creases in the liquid-crystalline phase,[18] this can be ex-
plained by the relocation of the compound to the hydro-
phobic interior of the membrane; it has previously been
shown[23] that, for these types of compounds, a decrease in
polarity results in a blue spectral shift. The presence of the
double chain is important for this relocalisation process be-
cause it is not observed for the single-chain equivalent de-
rivative (compound 1d).
The behaviour of benzo[a]phenoxazinum chlorides 1a–e
in positive DODAB vesicles was completely different. In
this case, an acid–base equilibrium was clearly observed for
compounds 1d and 1e (Figures 3 and 4), which can be ex-
plained by their deeper position in the interior of the mem-
brane to avoid the positive charge of the DODAB mole-
cules. Although the basic form is dominant, the fluores-
From the HOMO and LUMO energies, the predicted
values of the maximum absorption wavelength are 513, 451
and 495 nm, respectively, for the acid, base 1 (deproton-
ation at 5-amino position) and base 2 (deprotonation at the
9-amino position). The experimentally observed maximum
absorption wavelength shift between the acid and basic
forms of compound 1d is 127 nm. This provides a first indi-
cation that deprotonation occurs at the 5-amino position,
because the predicted shift for base 1 is 62 nm whereas the
shift for base 2 is only 18 nm. From the calculated molecu-
lar energies and zero point vibrational energies, it is possible
to compute the proton affinities of the two bases (base 2 has
5 kcalmol–1 higher proton affinity than base 1). Because the
thermal energy at 25 °C is 0.89 kcalmol–1, it can be con-
cluded that the main deprotonation of benzo[a]phenoxazin-
ium chloride occurs at the 5-amino position.
Conclusions
5,9-Diaminobenzo[a]phenoxazinium dyes 1a–e, pos-
cence spectra is typical of the acid form, because its fluores- sessing (di)icosylamino side-chains at the 5- or 9-positions
cence quantum yield is much higher.[23,29] Upon the gel to of the polyaromatic system were efficiently synthesised.
liquid-crystalline phase transition, the proportion of the ba- These dyes displayed strong absorption, and fluorescence
sic form nearly goes to unity, and the fluorescence spec- emission in the near-infrared region with good fluorescence
trum, under these conditions, is dominated by a low inten- quantum yields. Results of photophysical behaviour studies
sity band that corresponds to the basic form. This effect is in biomembranes showed that the absorption and fluores-
greater for compounds with a single side-chain at the 9- cence spectra depended both on the charge of the biological
position of the benzo[a]phenoxazinum, such as 1d. The ab- membrane and on the lipid chain organisation. As a result,
sence of an acid–base equilibrium in compound 1a can eas- the synthesised molecules were able to report the gel to li-
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N-(Di)icosyl-Substituted Benzo[a]phenoxazinium Chlorides
NMR (CDCl3, 75.4 MHz): δ = 13.84 (NCH2CH3), 14.05 [N-
(CH2)19CH3], 18.29 (CH3), 22.60 (CH2), 27.10 (NHCH2CH2),
28.60 (CH2), 29.27 (CH2), 29.30 (CH2), 29.51 (CH2), 29.55 (CH2),
29.57 (CH2), 29.62 (CH2), 30.87 (CH2), 31.84 (CH2), 38.67
(NHCH2CH2), 44.48 (NHCH2CH3), 92.29 (C-6), 93.08 (C-8),
123.44 (Ar-C), 123.98 (C-1), 125.10 (C-4), 127.00 (C-10), 129.67
(Ar-C), 129.93 (C-3), 130.55 (Ar-C), 131.01 (C-11), 131.54 (C-2),
133.38 (Ar-C), 146.76 (Ar-C), 150.57 (Ar-C), 154.10 (C-9), 156.80
quid-crystalline lipid phase transition by simple absorption
and fluorescence measurements, as well as providing infor-
mation on the charge of the membranes.
Experimental Section
General Information: Melting points were measured with a Stuart
SMP3 melting point apparatus. TLC analyses were carried out with
0.25 mm thick precoated silica plates (Merck Fertigplatten Kiesel-
gel 60F254) and spots were visualised under UV light or with the
naked eye. Chromatography on silica gel was carried out with
Merck Kieselgel (230–240 mesh). IR spectra were recorded with a
BOMEM MB 104 spectrophotometer, using either KBr discs or
neat samples. NMR spectra were obtained with either a Varian
Unity Plus Spectrometer at an operating frequency of 300 MHz for
1H NMR and 75.4 MHz for 13C NMR, or a Bruker Avance III
400 at an operating frequency of 400 MHz for 1H NMR and
100.6 MHz for 13C NMR, using the solvent peak as an internal
reference at 25 °C. All chemical shifts are given in ppm using Me4Si
(δH = 0.0 ppm) as a reference; J values are given in Hz. Assign-
ments were made by comparison of chemical shifts; peak multi-
plicities and J values and were supported by spin decoupling-
double resonance and bidimensional heteronuclear HMBC and
HMQC correlation techniques. Mass spectrometry analyses were
performed at the “C.A.C.T.I. Unidad de Espectrometria de
Masas”, at the University of Vigo, Spain. UV/Vis/NIR absorption
spectra (200–800 nm) were obtained with either Shimadzu UV/
2501PC or Shimadzu UV/3101PC spectrophotometers. Fluores-
cence spectra were recorded with either FluoroMax-4 or Fluo-
roMax-3 spectrofluorometers. All chemical reagents were used as
received. Labelled vesicles were prepared by injection of an etha-
nolic solution containing the benzo[a]phenoxazinium chloride 1a–
e and either DODAB or DPPC into the required amount of an
aqueous pH7-buffered solution. The injection was made above the
gel to liquid crystalline phase transition and the DODAB/DPPC
to dye molar ratio was kept at 500:1. The dye concentration was
2.0ϫ10–6 m. The resulting solutions were allowed to equilibrate for
one day before absorption/fluorescence measurements were re-
corded.
(C-5) ppm. IR (KBr, 1%): ν = 3450, 3240, 2956, 2922, 2852, 1642,
˜
1591, 1562, 1544, 1520, 1500, 1451, 1436, 1348, 1343, 1315, 1295,
1260, 1234, 1185, 1163, 1130, 1054, 1086, 1010, 881, 817, 773, 734,
708, 666 cm–1. HRMS (EI): calcd. for C39H58N3O [M+] 584.45766;
found 584.45744.
N-Ethyl-N-[5-(icosylamino)-9H-benzo[a]phenoxazin-9-ylidene]-
ethanaminium Chloride 1b: The product of the reaction of 2b
(0.092 g, 4.73ϫ10–4 mol), with 3a (0.20 g, 4.73ϫ10–4 mol) (reflux
time 9 h) was purified by chromatography with dichloromethane/
n-hexane and dichloromethane/methanol mixtures of increasing
polarity as the eluent, to give compound 1b (0.19 g, 68%); m.p.
133.2–134.2 °C. TLC: Rf = 0.51 (dichloromethane/methanol, 9:1).
1H NMR (CDCl3, 300 MHz): δ = 0.84 [t, J = 6.6 Hz, 3 H,
NH(CH2)19CH3], 1.10–1.30 (m, 32 H, 16 ϫ CH2), 1.31 [t, J =
6.9 Hz, 3 H, N(CH2CH3)2], 1.45 (br. s, 2 H, CH2), 1.87 (br. s, 2 H,
NHCH2CH2), 3.50–3.62 [m, 2 H, N(CH2CH3)2], 3.77 (br. s, 2 H,
NHCH2CH2), 6.48 (s, 2 H, 8-H and 6-H), 6.92 (d, J = 9.0 Hz, 1
H, 10-H), 7.62 (d, J = 9.0 Hz, 1 H, 11-H), 7.65–7.75 (m, 2 H, 2-H
and 3-H), 8.55–8.65 (m, 1 H, 1-H), 9.26 (br. s, 1 H, 4-H) ppm. 13C
NMR (CDCl3, 75.4 MHz): δ = 12.58 [N(CH2CH3)2], 13.98
[NH(CH2)19CH3], 22.54 (CH2), 27.13 (CH2), 29.01 (NHCH2CH2),
29.21 (CH2), 29.30 (CH2), 29.47 (3ϫCH2), 29.51 (4ϫCH2), 29.56
(4 ϫ CH2 ), 31.77 (2 ϫ CH2 ), 45.05 (NHCH2 CH2 ), 45.80
[N(CH2CH3)2], 93.20 (C-6), 95.54 (C-8), 113.57 (C-10), 128.81 (C-
1), 124.28 (Ar-C), 126.22 (C-4), 128.19 (Ar-C), 130.28 (C-3), 130.50
(Ar-C), 131.60 (C-2), 132.25 (C-11), 135.25 (Ar-C), 147.14 (Ar-C),
151.15 (Ar-C), 152.71 (C-9), 158.29 (C-5) ppm. IR (KBr, 1%): ν =
˜
3440, 2955, 2921, 2851, 1640, 1588, 1548, 1494, 1461, 1436, 1384,
1328, 1276, 1257, 1166, 1123, 1075, 1013, 947, 865, 757, 700,
666 cm–1. HRMS (EI): calcd. for C40H60N3O [M+] 598.47259;
found 598.47309.
N-[5-(Icosylamino)-9H-benzo[a]phenoxazin-9-ylidene]-N-methyl-
methanaminium Chloride (1c): The product of the reaction of 2c
(0.072 g, 4.74ϫ10–4 mol) with 3a (0.201 g, 4.74ϫ10–4 mol) (reflux
time 7 h) was purified by chromatography with dichloromethane/
n-hexane and dichloromethane/methanol mixtures of increasing
polarity as the eluent, to give compound 1c (0.23 g, 85%); m.p.
171.2–173.7 °C. TLC: Rf = 0.37 (dichloromethane/methanol,
9.5:0.5). 1H NMR (CDCl3, 300 MHz): δ = 0.83 [t, J = 6.6 Hz, 3
H, NH(CH2)19CH3], 1.10–1.50 (2ϫm, 34 H, 17ϫCH2), 1.76–1.90
(m, 2 H, NHCH2CH2), 3.24 [s, 6 H, N(CH3)2], 3.60–3.76 (m, 2 H,
NHCH2CH2), 4.62 (br. s, 1 H, NH), 6.31 (d, J = 2.7 Hz, 1 H, 8-
H), 6.38 (s, 1 H, 6-H), 6.92 (dd, J = 9.0, 2.4 Hz, 1 H, 10-H), 7.49
General Method for the Preparation of Compounds 1a–e: To a cold
solution (ice bath) of 5-(alkylamino)-2-nitrosophenol hydrochloride
2a–e in ethanol (2–3 mL), N-alkylated naphthylamine 3a or 3b and
concentrated hydrochloride acid (5.0ϫ10–2 mL) were added. The
mixture was heated to reflux for 5 to 15 h, and the progress of
the reaction was monitored by TLC (dichloromethane/methanol or
chloroform/methanol). After evaporation of the solvent and col-
umn chromatography purification on silica gel, the required dye
1a–e was obtained as a blue material.
N-[5-(Icosylamino)-10-methyl-9H-benzo[a]phenoxazin-9-ylidene]-
ethanaminium Chloride (1a): The product of the reaction of 2a
(0.085 g, 4.73ϫ10–4 mol) with 3a (0.20 g, 4.73ϫ10–4 mol) (reflux (d, J = 9.3 Hz, 1 H, 11-H), 7.56–7.70 (m, 2 H, 2-H and 3-H), 8.51
time 5 h) was purified by chromatography with dichloromethane/
n-hexane and dichloromethane/methanol mixtures of increasing
polarity as the eluent, to give compound 1a (0.20 g, 73%); m.p.
125.0–126.2 °C. TLC: Rf = 0.79 (dichloromethane/methanol, 95:5).
1H NMR (CDCl3, 300 MHz): δ = 0.86 [t, J = 6.9 Hz, 3 H,
NH(CH2 )1 9 CH3 ], 1.19–1.40 (2 ϫ m, 37 H, 17 ϫ CH2 and
NHCH2CH3), 1.81 (br. s, 2 H, NHCH2CH2), 2.40 (s, 3 H, CH3),
3.27 (br. s, 2 H, NHCH2CH2), 3.47 (br. s, 2 H, NHCH2CH3), 6.16
(s, 1 H, 8-H), 6.22 (s, 1 H, 6-H), 6.71 (br. s, 1 H, NH), 7.37 (s, 1
(d, J = 8.1, 1.2 Hz, 1 H, 1-H), 8.95 (d, J = 7.8 Hz, 1 H, 4-H) ppm.
13C NMR (CDCl3, 75.4 MHz): δ = 13.95 [NH(CH2)19CH3], 22.51
(CH2), 27.07 (CH2), 28.95 (CH2), 29.18 (2ϫCH2), 29.30 (CH2),
29.48 (5 ϫ CH2), 29.53 (5 ϫ CH2), 30.77 (NHCH2CH2), 31.75
(CH2), 41.10 [N(CH3)2], 44.77 (NHCH2CH2), 93.02 (C-6), 95.68
(C-8), 114.35 (C-10), 123.62 (Ar-C), 123.88 (C-1), 125.36 (C-4),
128.62 (Ar-C), 129.93 (Ar-C), 130.30 (C-3), 131.61 (C-11), 131.90
(C-2), 134.39 (Ar-C), 146.63 (Ar-C), 150.88 (Ar-C), 154.63 (C-9),
157.67 (C-5) ppm. IR (KBr, 1%): ν = 3439, 2955, 2920, 2851, 1657,
˜
H, 11-H), 7.70–7.90 (br. s, 2 H, 2-H and 3-H), 8.73 (d, J = 7.2 Hz, 1642, 1589, 1552, 1537, 1513, 1494, 1463, 1427, 1383, 1330, 1291,
1 H, 1-H), 8.94 (br. s, 1 H, 4-H), 10.34 (br. s, 1-H, NH) ppm. 13C 1204, 1178, 1149, 1125, 1010, 905, 865, 850, 836, 773, 741, 715,
Eur. J. Org. Chem. 2011, 2491–2497
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2495

M. S. T. Gonçalves et al.
666 cm–1. HRMS (EI): calcd. for C38H56N3O [M+] 570.44321; of 20 min. The resulting mixture was stirred for 1 h and the reac-
FULL PAPER
found 570.44179.
tion was monitored by TLC (dichloromethane/methanol, 95:5). Af-
ter evaporation of the solvent, the 5-[(di)icosylamino]-2-nitroso-
phenol hydrochlorides 2d or 2e were obtained as yellow solids and
were used in the following step without further purification.
N-[5-(Propylamino)-9H-benzo[a]phenoxazin-9-ylidene]icosan-1-
aminium Chloride (1d): The product of the reaction of 2d (0.505 g,
1.13ϫ10–3 mol) with 3b (0.148 g, 8.0ϫ10–2 mol) (reflux time 12 h)
was purified by chromatography with chloroform and chloroform/
methanol mixtures of increasing polarity as the eluent, to give com-
pound 1d (0.185 g, 37%); m.p. 87–89 °C. TLC: Rf = 0.55 (chloro-
5-(Icosylamino)-2-nitrosophenol Hydrochloride (2d): From the reac-
tion of 3-(icosylamino)phenol (0.392 g, 1ϫ10–3 mol) in ethanol
(4 mL) and concentrated hydrochloric acid (0.2 mL) with sodium
nitrite (0.076 g, 1.1ϫ10–3 mol) in water (0.4 mL), compound 2d
was obtained (0.418 g).
1
form/methanol, 9:1). H NMR (CDCl3, 400 MHz): δ = 0.87 [t, J
= 7.2 Hz, 3 H, NH(CH2)19CH3], 1.07 (t, J = 7.2 Hz, 3 H,
NHCH2CH2CH3), 1.10–1.45 [m, 34 H, NHCH2CH2(CH2)17CH3],
1.73 [br. s, 2 H, NHCH2CH2(CH2)17CH3], 1.90 (br. s, 2 H,
NHCH2CH2CH3), 3.03 [br. s, 2 H, NHCH2CH2(CH2)17CH3], 3.49
(br. s, 2 H, NHCH2CH2CH3), 6.10 (br. s, 1 H, 8-H), 6.16 (br. s, 1
H, 6-H), 7.11 (br. s, 1 H, 10-H), 7.34 (d, J = 8.0 Hz, 1 H, 11-H),
7.74 (br. s, 3 H, 3-H, 2-H, N-H), 8.32 (br. s, 1 H, NH), 8.64 (d, J
= 6.8 Hz, 1 H, 4-H), 8.85 (br. s, 1 H, 1-H) ppm. 13C NMR (CDCl3,
100.6 MHz): δ = 11.59 (NHCH2CH2CH3), 14.09 [NH(CH2)19CH3],
21.98 (NHCH2CH2CH3), 22.66 (CH2), 27.17 (CH2), 28.48
[NHCH2CH2(CH2)17CH3], 29.33 (CH2), 29.59 (2ϫCH2), 29.63
( 4 ϫ C H 2 ) , 2 9 . 6 8 ( 4 ϫ C H 2 ) , 3 1 . 9 0 ( 4 ϫ C H 2 ) , 4 3 . 8 9
[NHCH2CH2(CH2)17CH3], 46.29 (NHCH2CH2CH3), 92.40 (C-6),
94.07 (C-8), 118.03 (C-10), 123.47 (Ar-C), 123.78 (C-4), 125.33 (C-
1), 129.52 (C-3), 130.13 (Ar-C), 130.69 (C-2), 131.35 (Ar-C), 131.65
(C-11), 132.51 (Ar-C), 147.80 (Ar-C), 150.43 (Ar-C), 155.93 (C-9),
5-(Diicosylamino)-2-nitrosophenol Hydrochloride (2e): From the re-
action of 3-(diicosylamino)phenol (0.300 g, 4.5ϫ10–4 mol) in eth-
anol (3 mL) and concentrated hydrochloric acid (0.2 mL) with so-
dium nitrite (0.040 g, 5.8ϫ10–4 mol) in water (0.1 mL), compound
2e was obtained (0.290 g).
Synthesis of 3-(Icosylamino)phenol and 3-(Diicosylamino)phenol: To
a solution of 3-aminophenol (1.0 g, 9.1ϫ10–3 mol) in ethanol
(5 mL), 1-bromoicosane (3.976 g, 1.08ϫ10–2 mol) was added and
the reaction mixture was heated to reflux for 44 h. After purifica-
tion by column chromatography on silica gel with chloroform and
chloroform/methanol as eluent, 3-(icosylamino)phenol was ob-
tained as a white solid (2.466 g, 70%); m.p. 81.7–83.7 °C. TLC: Rf
1
= 0.14 (dichloromethane). H NMR (CDCl3, 400 MHz): δ = 0.89
[t,
J = 6.8 Hz, 3 H, N(CH2)19CH3], 1.25–1.40 [m, 34 H,
NCH2CH2(CH2)17CH3], 1.56–1.65 [m, 2 H, NCH2CH2(CH2)17-
CH3], 3.08 [t, J = 7.2 Hz, 2 H, NCH2CH2(CH2)17CH3], 3.65 (t, J
= 7.2 Hz, 1 H, NH), 4.60 (br. s, 1 H, OH), 6.10 (t, J = 2.0 Hz, 1
H, 2-H), 6.16 (dd, J = 8.0, 2.0 Hz, 1 H, 4-H), 6.20 (dd, J = 8.4,
2.0 Hz, 1 H, 6-H), 7.01 (t, J = 8.0 Hz, 1 H, 5-H) ppm. 13C NMR
(CDCl3, 100.6 MHz): δ = 14.11 [N(CH2)19CH3], 22.68 (2ϫCH2),
27.15 [NCH2CH2(CH2)17CH3], 29.35 (2ϫCH2), 29.44 (2ϫCH2),
29.51 (2ϫCH2), 29.59 (2ϫCH2), 29.60 (2ϫCH2), 29.65 (2ϫCH2),
29.69 (2ϫCH2), 31.92 (CH2), 43.96 [NCH2CH2(CH2)17CH3], 99.35
(C-2), 103.98 (C-4), 105.90 (C-6), 130.12 (C-5), 150.15 (C-3), 156.69
157.12 (C-5) ppm. IR (KBr, 1%): ν = 3402, 3203, 2918, 2850, 1642,
˜
1590, 1547, 1495, 1467, 1432, 1384, 1321, 1283, 1256, 1237, 1186,
1161, 1122, 1012, 1000, 975, 823, 771, 718, 666 cm–1. HRMS
(FAB): calcd. for C39H58N3O [M + H]+ 584.45724; found
584.45744.
N-Icosyl-N-[5-(propylamino)-9H-benzo[a]phenoxazin-9-ylidene]-
icosan-1-aminium Chloride (1e): The product of the reaction of 2e
(0.500 g, 7.1ϫ10–4 mol) with 3b (0.88 g, 4.8ϫ10–4 mol) (reflux
time 15 h) was purified by chromatography with chloroform and
chloroform/methanol mixtures of increasing polarity as the eluent,
to give compound 1e (0.303 g, 70%); m.p. 140–142 °C. TLC: Rf =
(C-1) ppm. IR (KBr, 1%): ν = 3372, 3274, 2954, 2918, 2849, 1631,
˜
1594, 1518, 1505, 1486, 1473, 1464, 1391, 1374, 1333, 1252, 1239,
1205, 1191, 1165, 994, 946, 853, 846, 753, 729, 720, 688, 666 cm–1.
HRMS (FAB): calcd. for C26H48NO [M + H]+ 390.3718; found
390.37240.
1
0.26 (chloroform/methanol, 94:6). H NMR (CDCl3, 400 MHz): δ
= 0.87 [t, J = 7.2 Hz, 6 H, 2ϫN(CH2)19CH3], 1.09 (br. s, 3 H,
NHCH2CH2CH3), 1.20–1.50 [m, 68 H, 2ϫNCH2CH2(CH2)17-
CH3], 1.71 [br. s, 4 H, 2ϫNCH2CH2(CH2)17CH3], 1.95 (br. s, 2 H,
NHCH2CH2CH3), 3.49 [br. s, 4 H, 2ϫNCH2CH2(CH2)17CH3],
3.81 (br. s, 2 H, NHCH2CH2CH3), 6.55 (br. s, 1 H, 8-H), 6.61 (br.
s, 1 H, 6-H), 6.95 (d, J = 8.4 Hz, 1 H, 10-H), 7.70–7.90 (m, 3 H,
11-H, 3-H and 2-H), 8.73 (br. s, 1 H, 4-H), 9.30 (br. s, 1 H, 1-H),
11.68 (br. s, 1 H, NH) ppm. 13C NMR (CDCl3, 100.6 MHz): δ =
11.55 (NHCH2CH2CH3), 14.05 [2ϫN(CH2)19CH3], 22.33
(5ϫCH2), 22.62 (NHCH2CH2CH3), 26.97 (5ϫCH2), 27.40
[2ϫNCH2CH2(CH2)17CH3], 29.29 (4ϫCH2), 29.38 (4ϫCH2),
29.53 (4ϫCH2), 29.59 (4ϫCH2), 29.64 (4ϫCH2), 31.85 (4ϫCH2),
46.51 (NHCH2CH2CH3), 52.02 [2ϫNCH2CH2(CH2)17CH3], 93.44
(C-6), 95.81 (C-8), 113.69 (C-10), 124.03 (C-4), 124.23 (Ar-C),
126.33 (C-1), 128.41 (2ϫAr-C), 130.70 (C-3), 131.95 (C-2), 132.39
(C-11), 135.61 (Ar-C), 147.33 (Ar-C), 151.52 (Ar-C), 153.14 (C-9),
In addition to 3-(icosylamino)phenol, 3-(diicosylamino)phenol was
also obtained as a purple solid (1.582 g, 26%); m.p. 55.7–57.7 °C.
R = 0.20 (dichloromethane). IR (KBr, 1%): ν = 3496, 3383, 2918,
˜
f
2850, 1618, 1578, 1503, 1467, 1399, 1373, 1297, 1282, 1270, 1257,
1240, 1224, 1209, 1193, 1181, 1170, 1147, 1110, 1090, 1033, 999,
829, 720, 755, 689, 666 cm–1. 1H NMR (CDCl3, 400 MHz): δ =
0.89 [t, J = 6.8 Hz, 6 H, 2ϫN(CH2)19CH3], 1.10–1.50 [m, 68 H,
2ϫNCH2CH2(CH2)17CH3], 1.58 [br. s,
4 H, 2ϫNCH2CH2-
(CH2)17CH3], 3.22 [t, J = 8.0 Hz, 4 H, 2ϫNCH2CH2(CH2)17CH3],
4.56 (br. s, 1 H, OH), 6.09 (dd, J = 8.0, 2.4 Hz, 1 H, 4-H), 6.12 (t,
J = 2.0 Hz, 1 H, 2-H), 6.23 (dd, J = 8.4, 2.4 Hz, 1 H, 6-H), 7.04
(t, J = 8.0 Hz, 1 H, 5-H) ppm. 13C NMR (CDCl3, 100.6 MHz): δ
=
14.11
[2ϫN(CH2)19CH3],
22.69
(4ϫCH2),
27.19
[2ϫNCH2CH2(CH2)17CH3], 27.27 (4ϫCH2), 29.36 (4ϫCH2),
29.56 (4ϫCH2), 29.63 (4ϫCH2), 29.71 (12ϫCH2), 31.93
(2ϫCH2), 51.11 [2ϫNCH2CH2(CH2)17CH3], 98.49 (C-2), 102.00
(C-4), 104.65 (C-6), 130.01 (C-5), 149.82 (C-3), 156.66 (C-1) ppm.
HRMS (ESI): calcd. for C46H88NO [M + H]+ 670.68544; found
670.68604.
158.76 (C-5) ppm. IR (KBr, 1%): ν = 3430, 2955, 2918, 2850, 1638,
˜
1590, 1548, 1490, 1466, 1434, 1384, 1330, 1288, 1236, 1182, 1163,
1124, 1016, 1000, 721, 666 cm–1. HRMS (FAB): calcd. for
C59H98N3O [M + H]+ 864.76994; found 864.77044.
General Method for the Preparation of Compounds 2d and 2e: To
an ice-cold solution of the 3-[(di)icosylamino]phenol in ethanol,
concentrated hydrochloric acid was added and the mixture was
stirred until the reaction mixture became homogeneous. A solution
of sodium nitrite in water was then added dropwise over a period
N-Icosylnaphthalen-1-amine 3a: To a solution of naphthalen-1-
amine (1.01 g, 6.98ϫ10–3 mol) in ethanol (2 mL), 1-bromoicosane
(2.65 g, 7.33ϫ10–3 mol) was added and the resulting mixture was
heated to reflux for 17.5 h [the reaction was monitored by TLC
2496
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 2491–2497

N-(Di)icosyl-Substituted Benzo[a]phenoxazinium Chlorides
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(chloroform/n-hexane, 7:3)]. The solvent was evaporated and the
crude mixture was purified by column chromatography on silica
gel using chloroform/n-hexane mixtures of increasing polarity as
the eluent, to give compound 3a as a white oily solid (2.66 g, 90%).
R = 0.69 (chloroform/n-hexane, 7:3). IR (neat): ν = 3397, 2954,
˜
f
[15]
[16]
[17]
2917, 2849, 1620, 1583, 1523, 1466, 1409, 1380, 1283, 1253, 1140,
1120, 785, 768, 722, 666 cm–1. 1H NMR (CDCl3, 300 MHz): δ =
0.90 [t, J = 6.9 Hz, 3 H, NH(CH2)19CH3], 1.40–1.50 [m, 34 H,
NHCH2CH2(CH2)17CH3], 1.72–1.86 [m,
2 H, NHCH2CH2-
(CH2)17CH3], 3.30 [t, J = 6.9 Hz, 2 H, NHCH2CH2(CH2)17CH3],
6.73 (br. s, 1 H, 4-H), 7.28 (d, J = 7.8 Hz, 1 H, 2-H), 7.37 (t, J =
7.8 Hz, 1 H, 3-H), 7.42–7.50 (m, 2 H, 6-H and 7-H), 7.78–7.82
(m, 1 H, 8-H), 7.84–7.90 (m, 1 H, 5-H) ppm. 13C NMR (CDCl3,
75.4 MHz): δ = 14.11 [NH(CH2)19CH3], 22.68 (2ϫCH2), 26.63
(2ϫCH2), 27.14 (CH2), 27.94 (CH2), 28.31 [NHCH2CH2(CH2)17-
CH3], 29.22 (CH2), 29.32 (CH2), 29.35 (CH2), 29.53 (CH2), 29.56
(CH2), 29.69 (CH2), 30.91 (CH2), 31.91 (CH2), 43.19 (CH2), 46.57
(CH2), 48.82 (CH2), 53.00 [NHCH2CH2(CH2)17CH3], 117.85 (C-
4), 120.33 (C-5), 122.21 (C-2), 124.14 (C-4a), 126.05 (C-7), 126.17
(C-6), 126.71 (C-3), 128.69 (C-8), 134.34 (C-8a), 138.03 (C-1) ppm.
HRMS (EI): calcd. for C30H49N [M+] 423.3865; found 423.3866.
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Thanks are due to Fundação para a Ciência e Tecnologia (FCT)
for financial support (BPD to S. N., grant number SFRH/BPD/
37840/2007). The NMR spectrometer Bruker Avance III 400 is part
of the National NMR Network and was purchased in the frame-
work of the National Program for Scientific Re-equipment, con-
tract REDE/1517/RMN/2005, with funds from the Fundo Europeu
de Desenvolvimento Regional (FEDER), POCI 2010 and from
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Received: November 23, 2010
Published Online: March 16, 2011
Eur. J. Org. Chem. 2011, 2491–2497
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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