Novel long alkyl side chain benzo[a]phenoxazinium chlorides: synthesis, photophysical behaviour and DNA interaction

Article abstract of DOI:10.1016/j.tet.2009.10.017

Several fluorescent benzo[a]phenoxazinium chlorides possessing a propyl-, octyl-, decyl-, dodecyl- or tetradecylamino at the 5-position of the heterocyclic moiety were efficiently synthesised. The absorption and emission maxima of all compounds lie in the

Full text of DOI:10.1016/j.tet.2009.10.017

Tetrahedron 65 (2009) 10441–10452
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel long alkyl side chain benzo[a]phenoxazinium chlorides: synthesis,
photophysical behaviour and DNA interaction
,
Carla M.A. Alves a, Sarala Naik a, Paulo J.G. Coutinho b, M. Sameiro T. Gonçalves a
*
a Centro de Qu´ımica, Universidade do Minho, Gualtar, 4710-057 Braga, Portugal
b Centro de F´ısica, Universidade do Minho, 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:
Several fluorescent benzo[a]phenoxazinium chlorides possessing a propyl-, octyl-, decyl-, dodecyl- or
tetradecylamino at the 5-position of the heterocyclic moiety were efficiently synthesised. The absorption
and emission maxima of all compounds lie in the range 627–638 nm and 654–678 nm, respectively, with
good fluorescence quantum yields. Studies of their photophysical properties in ethanol allowed for the
estimation of the acid–base dissociation constant, Ka, revealing an enhancement with the increase in the
alkyl side-chain length. It is in the aqueous medium only that the acid form is observed as coexisting
with H-aggregates. The solubility markedly decreased when the chain length increased. The residual
ethanol (0.2% v/v) used to facilitate the solubilisation of the benzo[a]phenoxazinium dyes allow for the
existence of the basic form in an aqueous solution, possibly through preferential solvation. Photophysical
studies in the presence of DNA revealed that the compounds with an alkyl side chain of up to eight
carbon atoms could intercalate between DNA nucleotides. Moreover, other forms of DNA binding were
found to be operative, involving also the basic form of benzo[a]phenoxazinium dyes.
Received 4 August 2009
Received in revised form
30 September 2009
Accepted 6 October 2009
Available online 12 October 2009
Keywords:
Long-wavelength fluorescent dyes
Benzo[a]phenoxazinium dyes
Fluorescent labelling
DNA probes
Acid–base probes
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
DNA on gold electrodes showed that the binding of NB with DNA in
solution contains both electrostatic and intercalative interactions,
Several molecules known as ligands interact with DNA through
covalent and electrostatic binding or intercalation.1 Intercalation
occurs when ligands of a suitable size and chemical nature fit
themselves in between base pairs of DNA. These ligands are usually
polycyclic, aromatic and planar structures, and consequently often
make good nucleic acid stains. Intensively studied DNA inter-
calators include ethidium bromide, proflavine, daunomycin, doxo-
rubicin and thalidomide. These compounds are used in
chemotherapeutic treatment to inhibit DNA replication in rapidly
growing cancer cells. Examples include doxorubicin (adriamycin)
and daunorubicin, both of which are used in the treatment of
Hodgkin’s lymphoma, as well as dactinomycin, which is used in
Wilm’s tumour, Ewing’s sarcoma and rhabdomyosarcoma.
and also suggested that NB can be used as an electrochemical in-
dicator in the preparation of DNA sensors.7 Furthermore, recently
electrochemical studies reported the use of Nile Blue as a DNA re-
dox active intercalator.8–10 Based on fluorescent spectroscopy
techniques, Mitra et al. clearly identified non-specific electrostatic
and intercalative modes of interaction of the probe with DNA at
lower and higher DNA concentrations, respectively.11 Comparison
of NB to the other conventional intercalators revealed that the use
of NB has the advantages of possessing low toxicity and comparable
sensitivity for DNA quantification.5
Another possibility of interaction of small organic molecules
with nucleic acids is groove binding, in which the molecules locate
themselves in the minor or major groove of the DNA.12 In this case,
binding occurs through hydrogen bonding, van der Waals and hy-
drophobic interactions while, in intercalation, the main associative
Studies on the interaction between DNA and ligands are ex-
tremely important for therapeutic2 and scientific reasons.3,4 Nile
Blue (NB),
a
well-known fluorescent cationic dye from the
interaction is p
–stacking.13
benzo[a]phenoxazinium family, possessing a planar and rigid
structure, has been used as a DNA probe.5 The interactions of Nile
Blue and DNA proved that this dye is a good intercalator of the DNA
double helix.6 Electrochemical studies on the interaction of NB with
To the best of our knowledge, only limited reports are available
on the use of NB despite its importance as a DNA probe, whereas
reports concerning the use of other members of the benzo[a]phe-
noxazinium dyes family are almost non-existent.
Our current research interests include the synthesis and
characterisation of fluorescent long-wavelength probes, namely
5,9-diaminobenzo[a]phenoxazinium dyes, which can be consid-
ered as Nile Blue analogues.14–20
* Corresponding author. Fax: þ351 253604382.
E-mail address: msameiro@quimica.uminho.pt (M.S.T. Gonçalves).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.10.017

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C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
Considering all these facts, we report herein the synthesis and
(NHCH2CH2, d 1.70–2.10 ppm) and as triplets (NHCH2CH2, d 3.29–
photophysics of several benzo[a]phenoxazinium dyes, possessing
long alkyl side chains, namely octyl-, decyl-, dodecyl- or tetrade-
cylamino at the 5-position of the tetracyclic system. Fundamental
photophysical characterisation of the synthesised compounds, in
ethanol and aqueous solutions with 0.2% v/v of ethanol, was carried
out with emphasis on an acid/base equilibrium, aggregation and
preferential solvation. Studies on the interaction of these fluo-
rochromophores with a self-organised system–DNA were also
carried out, as an extension of the previously preliminary research
3.39 ppm). In the 13C NMR spectra of compounds 3a–e, relevant
signals were assigned to the aliphatic carbons, namely the methyl
groups (d 11.75–14.11 ppm) and the methylenic groups next to the
nitrogen atom: NHCH2CH2 (d 22.33–29.37 ppm) and NHCH2CH2 (d
44.19–49.08 ppm).
By condensation of the N-alkylated-naphthylamines 3a–e with
5-alkylamino-2-nitrosophenol hydrochlorides 2a–c in an acidic
medium, benzo[a]phenoxazinium chlorides 1a–m were syn-
thesised (Scheme 1). The required 5-alkylamino-2-nitrosophenol
N
R
NO
OH
R
N
HCl / EtOH
reflux
R2
H
+
R2
R1
(CH2) CH3
n
N+
R1
O
N
Cl-
N
(CH2)nCH3
H
3
1
2
a R = Me, R1 = H, R2 = Et
b R = H, R1 = R2 = Et
c R = H, R1 = R2 = Me
a n = 2, R = Me, R1 = H, R2 = Et
b n = 2, R = H, R1 = R2 = Et
c n = 7, R = Me, R1 = H, R2 = Et
d n = 7, R = H, R1 = R2 = Et
e n = 7, R = H, R1 = R2 = Me
f n = 9, R = Me, R1 = H, R2 = Et
g n = 11, R = Me, R1 = H, R2 = Et
h n = 11, R = H, R1 = R2 = Et
i n = 11, R = H, R1 = R2 = Me
j n = 13, R = Me, R1 = H, R2 = Et
l n = 13, R = H, R1 = R2 = Et
m n = 13, R = H, R1 = R2 = Me
a n = 2
b n = 7
c n = 9
d n = 11
e n = 13
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 1a–m.
work using TritonÒ X-100 and cetyltrimethylammonium bromide
hydrochloride 2a–c was synthesised using the usual procedure,22
involving treatment of the corresponding 3-alkylaminophenol with
sodium nitrite in an acid solution.
micelles as biomimicking self-assembled systems.21
Reaction between nitrosophenol 2a and 2b with intermediate
3a, in ethanol, in the presence of concentrated hydrochloric acid,
gave compounds 1a and 1b. Similarly, by reacting nitroso com-
pounds 2a, 2b and 2c with 3b, benzo[a]phenoxazinium dyes 1c, 1d
and 1e, were, respectively, obtained. Intermediate 3c reacted with
compound 2a, resulting in benzo[a]phenoxazinium chloride 1f,
while the N-alkylated-naphthylamine derivatives 3d and 3e reac-
ted with nitrosophenols 2a, 2b or 2c, producing fluorophores
1g–m.
After silica gel dry chromatography purification, compounds
1a–m were obtained as blue solid materials in low to excellent
yields (Table 1) and were fully characterised by the usual analytical
techniques.
2. Results and discussion
2.1. Synthesis
The synthesis of benzo[a]phenoxazinium chlorides 1a–m was
initiated by the preparation of the N-alkylnaphthalen-1-amines
3a–e. The choice of these intermediates is crucial, since they are
responsible for the size and characteristics of the side chains of the
5-amino position of benzo[a]phenoxazinium dyes to be syn-
thesised and studied. Therefore, it was decided to start with the
synthesis of N-propylnaphthalen-1-amine 3a, with two methylenic
groups (n¼2) and then to vary the size of the alkyl chain of 1-
naphthylamine to seven (n¼7, 3b), nine (n¼9, 3c), eleven (n¼11, 3d)
and thirteen (n¼13, 3e) methylenic groups. Compounds 3a–e were
prepared by alkylation of 1-naphthylamine with the appropriate
bromo-derivative, by refluxing in ethanol (except for compound 3a,
where methanol was used).
The IR spectra of these benzo[a]phenoxazinium dyes showed, as
in the case of intermediates 3a–e, the expected bands, due to
stretching vibrations of the amine function (3450–3195 cmꢀ1), the
C–H linkage of the methyl and methylenic groups
(2968–2853 cmꢀ1), the C–C linkage of the aliphatic chains
(1201–816 cmꢀ1), as well as a strong band of the C]N bond (1643–
1588 cmꢀ1) as result of the oxazine ring.
After purification through silica gel dry chromatography, in-
termediates 3a–e were isolated as oils, solids or as an oily solid (3d),
in yields ranging from 44 (3d) to 88% (3c), and were fully charac-
terised by high resolution mass spectrometry, IR and NMR (1H and
13C) spectroscopy.
The 1H NMR spectra showed signals of the methyl group of the
aliphatic chains in the form of triplets (
methyl groups directly linked to the aromatic ring as singlets
2.38–2.45 ppm; 1a, 1c, 1f, 1g, 1j). In compounds 1e, 1i and 1m
with dimethylamine at 9-position of the benzo[a]phenoxazinium
ring, the methyl protons occurred as singlets ( w3.25 or 3.20 ppm).
The methyl protons of the ethyl groups (R1 and/or R2) appeared as
multiplets ( 1.10–1.50 ppm; 1c, 1f, 1g, 1h, 1j, 1l) or as triplets
1.30–1.42 ppm; 1a, 1b, 1d). For all compounds, the methylenic
groups closed to the nitrogen atom (NHCH2CH2) in 5-position of
heterocycles appeared as broad singlets ( 1.76–1.90 ppm; 1c,1f, 1g,
d 0.78–1.15 ppm) and the
The IR spectra of these compounds showed the expected bands,
due to stretching vibrations of the amine function (3428–
3389 cmꢀ1), the C–H linkage of the methylenic and methyl groups
(2958–2852 cmꢀ1) and the C–C linkage of the aliphatic chains
(1173–800 cmꢀ1).
(d
d
In addition to the signals of naphthalene protons, the 1H NMR
spectra showed the signals corresponding to aliphatic protons, in
particular those of methyl groups as triplets (
d
(d
d 0.91–1.09 ppm) and
d
methylenic groups close to nitrogen atom as multiplets

C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
10443
Table 1
Synthesis, UV–visible and fluorescence data in ethanol of benzo[a]phenoxazinium chlorides 1a–m
Compound (no.)
n
R
R1
R2
Yield [%]
lmax [nm] (
3
)
lem [nm]
VF
DSa [nm]
1a
1b
1c
1d
1e
1f
1g
1h
1i
2
2
7
7
7
9
11
11
11
13
13
13
Me
H
Me
H
H
Et
H
Et
Me
H
Et
Et
Et
Et
Me
Et
Et
Et
Me
Et
92
49
98
88
45
96
83
69
43
86
53
17
628 (46,465)
637 (43,471)
627 (67,486)
636 (68,015)
628 (45,378)
627 (44,978)
627 (63,128)
638 (46,339)
629 (55,551)
626 (90,934)
633 (20,860)
631 (38,760)
654
670
655
678
676
655
655
677
676
655
677
676
0.46
0.27
0.49
0.21
0.21
0.44
0.48
0.20
0.21
0.47
0.20
0.21
26
33
28
42
48
28
28
39
47
29
44
45
H
Me
Me
H
H
Me
H
H
Et
Me
H
Et
Me
1j
1l
1m
Et
Me
H
a
Stokes’ shift.
1h, 1j, 1l) or as multiplets (
protons directly linked to the nitrogen atom (NHCH2CH2) appeared
as multiplets ( 3.10–3.88 ppm; 1a–e,1g,1i,1m) or as broad singlets
3.18 or 3.71 ppm; 1f, 1h, 1j, 1m). Spectra also showed the
expected aromatic protons, in particular, H-10 as double doublets (
6.91–7.26 ppm; 1b, 1d, 1e, 1l) or as doublets ( 6.86 to w6.93 ppm;
1h, 1i, 1m), H-8 as singlets ( 6.08–6.50 ppm), meta doublets (J
2.4 Hz) ( 6.35 ppm; 1e, 6.87 ppm; 1b) and H-11, which appeared as
singlets ( 7.36–7.49 ppm; 1a, 1c, 1f, 1g, 1i), or as doublets ( 7.20–
d
1.60–2.0 ppm; 1a, 1d, 1e, 1i, 1l), and the
From our previously reported results it was found that, in ben-
zo[a]phenoxazinium dyes, the acid and basic forms coexist in
organic solvents possessing the ability to donate/accept a proton.16
In order to study the effect of the alkyl side chain on the acid–base
equilibrium, absorption spectra of compounds 1a, 1b, 1c, 1d, 1g, 1h,
d
(d
d
d
1j, 1l and 1m at concentrations between 2 mM and 50 mM were
d
measured. Figure 1 shows the molar absorptivity for compounds 1a
and 1b. A clear isosbestic point can be observed, which confirms
that only two molecular species are at equilibrium. The band at
around 500 nm corresponds to the basic neutral form and the band
around 630 nm corresponds to the cationic acid form. It was ob-
served that a higher fraction of basic form occurs when R1 and R2
are ethyl groups (R¼H), and thus in the acid form only one labile
proton is present in the amine function at 5-position. The absorp-
tion spectra of the acid and basic forms were obtained by adding
d
d
d
7.65 ppm; 1d, 1e, 1h, 1i, 1l, 1m).
The 13C NMR spectra showed the signals corresponding to the
aliphatic N-substituents in the heterocycle, namely the methyl
groups (
aromatic ring (
with dimethylamine at 9-position of the benzo[a]phenoxazinium
ring, the methyl carbons appeared at from 40.81 to 40.93 ppm. For
d
11.59–14.09 ppm) and for the R group attached directly to
d
17.72–18.60 ppm). In compounds 1e, 1i and 1m
d
10 mL of trifluoroacetic acid (TFA) and 20% methanolic solution of
all compounds, carbons of methylenic groups close to the nitrogen
atom (NHCH2CH2) in 5-position of heterocycles appeared between
22.19 and 29.18 ppm and the carbon atom directly linked to nitro-
gen (NHCH2CH2) occurred between 38.66 and 47.44 ppm. Spectra
tetraethylammonium hydroxide (TEAH), respectively, to a 2ꢁ10ꢀ5
benzo[a]phenoxazinium dye solution (Fig. 2A). This allowed for the
determination of the amount of acid and basic forms as a function
of total compound concentration by fitting the absorption spectra
with a weighed sum of spectra of the pure acid and basic forms.
Figure 3 illustrates the result of this procedure for compounds 1a
and 1b.
also showed the expected aromatic signals such as, C-6 (
d
92.23–
95.71 ppm), C-8
134.10 ppm).
(d 93.04–96.99 ppm) and C-11 (d 130.92–
In order to better compare the acid–base equilibrium of the
synthesised compounds in ethanol, an acid/base dissociation con-
stant, Ka, was estimated using a procedure similar to that used in
aqueous solutions (Eqs. 1–4):
2.2. Photophysical studies
Electronic absorption spectra of 10ꢀ5 or 10ꢀ6 M solutions of
benzo[a]phenoxazinium chlorides 1a–m in degassed absolute
ethanol were measured and data is summarised in Table 1. The
longest wavelength of maximum absorption (lmax) of all com-
pounds was located between 626 nm (1j) and 638 nm (1h). Con-
sidering benzo[a]phenoxazinium dyes 1c–e, with the same number
of methylenic groups (n¼7) and different substituents R, R1 and R2,
compound 1d (R¼H, R1¼R2¼Et) showed the higher wavelength of
maximum absorption (636 nm).
h
ih
i
EtOꢀ Hþ
h
i
KEt
¼
(1)
(2)
EtOH
h ih
i
A
Hþ
i
h
Ka
¼
AHþ
Similarly, comparison of other fluorophores with an equal alkyl
chain in the amine function of the 5-position, such as benzo[a]-
phenoxazinium chlorides 1g–i (n¼11) and 1j–m (n¼13), follows
the same trend, i.e., there is a bathochromic shift for the com-
pounds with the N-dialkylated amine at the 9-position of the
heterocycle R¼H, R1¼R2¼Me (2 or 5 nm) and R¼H, R1¼R2¼Et (7 or
11 nm) compared to compounds with the substituents R¼Me,
R1¼H, R2¼Et. Considering this behaviour, it was predicted that in-
creasing the size of the alkyl chain (a change in the values of n) of
the amine function at position 5 of the benzo[a]phenoxazinium
ring does not influence the values of lmax, but are dependent on the
amine substituent present in the opposite position.
h
i
h
i
h
i
h
i
Hþ þ AHþ
¼
EtOꢀ þ Clꢀ
(3)
(4)
h
i
h i
AHþ þ A ¼ CAHCl
where KEt is the ethanol dissociation constant, Ka is the benzo[a]-
phenoxazinium dye dissociation constant, A is the benzo[a]phe-
noxazinium dye in the
benzo[a]phenoxazinium dye in the acid form and CAHCl is the total
basic form, AHþ is
the

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C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
Figure 3. Decomposition of the absorption spectra of compounds 1a and 1b (solid
line) into a weighted sum of spectra of the corresponding pure acid and basic forms
(dotted line).
Figure 1. Molar absorptivity of compounds 1a and 1b in ethanol at concentrations 2, 5,
7.5, 10, 20 and 50 mM. The arrows point the direction of concentration increase.
values for Ka give an indication of the extension of acid–base
equilibrium in ethanolic solutions.
concentration of benzo[a]phenoxazinium dye. The value of KEt is
1.25ꢁ10ꢀ16 M.23 In Figure 4, the results of fitting the values of the
basic form fraction to the solution of Eqs. 1–4 are plotted for fluo-
rophores 1a and 1b. In some cases the amount of basic form should
decrease with compound concentration, much more than that
which was experimentally determined. Nevertheless, the obtained
Figure 5 shows the obtained pKa values plotted against the
number of carbon atoms in the alkyl side chain of the 5-amino
position of benzo[a]phenoxazinium dye. It was observed that pKa
decreases with the size of the alkyl chain, with an inversion from
C12 to C14. The fact that the same type of variation was observed for
Figure 2. Absorption (A) and fluorescence (B) spectra of compounds 1a/1b in its acid (3/4) or basic forms (1/2) of 2ꢁ10ꢀ5 M solutions (lex¼470 nm).

C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
10445
Figure 4. Fraction of basic form as a function of total compound concentration. The
line represents the fitting to Eqs. 1–4.
Figure 6. Molar absorptivity of compound 1b in water at concentrations 2, 5, 7.5, 10, 20
and 50 M. The arrows point the direction of concentration increase.
m
compounds with R¼H and R¼Me is an indication that the main site
of deprotonation is always the amine function in the 5-position of
benzo[a]phenoxazinium dye.
yields, Oxazine 1, used as a standard (FF¼0.11 in ethanol),24 was
excited at 590 nm for all compounds tested.
Compounds 1a–m have a wavelength of maximum emission
superior to 654 nm (1a) with the highest value of 678 nm (1d).
Fluorophores with the same combination of substituents R, R1 and
R2 and a different chain length displayed similar lem, which sug-
gested that these values are independent from the size of the alkyl
chain at the amine of the 5-position of the heterocycle. Thus, when
R¼Me, R1¼H, R2¼Et, lem is 655 nm (1c, 1f, 1g, 1j) or 654 nm (1a).
The higher values of lem arise with the combination R¼H,
R1¼R2¼Me, where lem was 676 nm (1e, 1i, 1m) and R¼H,
R1¼R2¼Et, where lem was 678 nm (1d) or 677 nm (1h, 1l).
Regarding Stokes’ shift, benzo[a]phenoxazinium chlorides with
the diethyl- or dimethylamine at the 9-position (R¼H, R1¼R2¼Et or
R¼H, R1¼R2¼Me; 1b, 1d, 1e, 1h, 1i, 1l, 1m) showed higher values
(33–48 nm) than fluorophores possessing the monoalkylated
amine (R1¼H, R2¼Et) and the methyl group at the 10-position of
the heterocycle (26–29 nm; 1a, 1c, 1f, 1g, 1j). These results showed
that benzo[a]phenoxazinium dyes with superior wavelengths of
maximum emission displayed the highest Stokes’ shifts.
For compounds with high water solubility (1a and 1b) the be-
haviour in aqueous solutions is completely different owing to non-
fluorescent H-aggregate formation.16 In Figure 6 the absorption
spectra of compound 1a in water at various concentrations is
plotted. An isosbestic point was again observed here, which in-
dicates that only one type of aggregates was formed and no basic
form is present. It is worth mentioning that in contrast to ethanolic
solutions, the main band now decreases with concentration. The
absence of the basic form can be understood by hydrogen bonding
of water molecules with the deprotonated form of benzo[a]phe-
noxazinium dyes. This interaction removes the lone electron pair
from the deprotonated amine and should essentially give the same
photophysical behaviour as the acid form.
Studies of the fluorescent properties of these compounds were
also carried out in ethanol; the maximum emission wavelengths
(lem) and relative fluorescence quantum yields (FF) were obtained
and are summarised in Table 1. For the determination of quantum
The acid–base equilibrium also influences the fluorescence
properties since the acid and basic forms of these compounds have
very different fluorescence spectra and relative fluorescence
quantum yields.16 Figure 2 shows the normalised spectra of fluo-
rescence of ethanolic solutions of compounds 1a and 1b in its acid
and basic forms. It can be observed that the basic form almost does
not show the red shift observed in the acid form when R changes
from Me to H. Although the fraction of the basic form can be as high
as 60%, the absorption at the excitation wavelength (590 nm) is
>99% due to the acid form (see Fig. 2), and the results of the fluo-
rescence quantum yields presented in Table 1 correspond to the
acid form.
Considering the fluorescence quantum yields of all compounds
(1a–m), it appears that values are located in two intervals, related
to the benzene ring substituents (R, R1 and R2) and independent
from the size of the alkyl chain of the amine in the naphthalene
ring. The values of VF with the combination R¼Me, R1¼H, R2¼Et
(1a, 1c, 1f, 1g, 1j) were between 0.44 and 0.49 and for other fluo-
rophores (1b, 1d, 1e, 1h, 1i, 1l, 1m), these occurred between 0.20
and 0.27.
From our previous study with long alkyl side-chain derivatives
of benzo[a]phenoxazinium chlorides21 it was found that they have
the ability to associate with positive and non-ionic micelles where
Figure 5. Recovered pKa values as
a function of total compound concentration.
ꢂdCompounds with R¼H, -dcompounds with R¼Me, Bdcompound with R¼H and
R1, R2¼Me.

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C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
the alkyl chain functions as an anchor. Following the evaluation of
these types of molecules as biological fluorescent probes, Figures 7
and 8 show the normalised absorption and fluorescence spectra of
compounds 1a, 1b, 1c, 1j, 1l and 1m at a fixed concentration of
2ꢁ10ꢀ6 M and various amounts of DNA corresponding to DNA–
phosphate/dye (p/d) molar ratios of 0, 1, 2, 5, 10, 50 and 100. The
corresponding normalisation factors (values of maximum
absorption and maximum emission intensity) are shown in the
insets.
Compounds with higher water solubility (1a and 1b) show
simpler absorption features with DNA. Compound 1b is the most
similar to Nile Blue with only an extra three carbon alkyl chain at 5-
position of the amine function of benzo[a]phenoxazinium ring. For
this compound, an enlargement of the blue side of the spectrum is
observed up to p/d¼10 and, after that point, the spectra regain its
original form but are shifted to the red. A slight enlargement in the
red side is already observed for p/d¼5, 10. The variations in the blue
side of the absorption spectra can be interpreted as variations in the
Figure 7. Normalised absorption spectra of compounds 1a, 1b, 1c, 1j, 1l and 1m in buffered DNA aqueous solutions after one day of equilibration. Curves 0–6 correspond to p/d¼0, 1,
2, 5, 10, 50, 100. The inset plots the normalisation factors, i.e., maximum absorption. Curve 0 corresponds to absorption spectrum immediately after preparation of the solution.
*

C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
10447
Figure 8. Normalised fluorescence spectra of compounds 1a, 1b, 1c, 1j, 1l and 1m in buffered DNA aqueous solutions after one day of equilibration. Curves 0–6 correspond to p/d¼0,
1, 2, 5, 10, 50, 100. The inset plots the normalisation factors, i.e., maximum fluorescence intensity (ꢂ) and this quantity divided by the absorbance at the excitation wavelength,
lex¼570 nm (B).The scale factor for maximum fluorescence intensity is indicated in the insets.
fraction of H-aggregates and corresponds to a decrease in fluores-
cence quantum yield, observed in the corresponding inset in Fig-
ure 8, while maintaining the fluorescence spectral shape. This can
result from electrostatic binding to DNA with neighbouring dye
molecules presenting a greater tendency of aggregating. The
amount of aggregates seems to be maximal for p/d¼5. The
absorption maximum first decreases with p/d, reaches a plateau
and then increases from p/d¼5 onwards. The decrease is again
explainable by an aggregation process (see Fig. 6). If the subsequent
increase in absorption is caused by a decrease in aggregate fraction,
then fluorescence intensity should increase with no change in the
spectral shape. As a significant red shift is observed for both fluo-
rescence (Fig. 8) and absorption (Fig. 7), it seems that a different
kind of dye binding to DNA is operative for high p/d values, which
can be groove binding or intercalation. However, using single
strand DNA, Mitra et al.11 interpreted similar results for Nile Blue in
DNA in terms of electrostatic and intercalative binding. The red
shift in absorption points to an intercalation process, also observed

10448
C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
in the case of ethidium bromide, is a typical intercalative binder
(the maximum absorbance of 480 nm in water shifts to 525 nm in
DNA25).
In the case of compounds 1a and 1c (easily soluble in water at
least up to 10 mM concentration), the same intercalation process
seems to occur at high p/d values as the same red shifted absorption
and emission are observed. For these compounds there is an ad-
ditional methyl group at the R position (10-position) and the amine
at the 9-position is monoalkylated. Additionally, for compound 1c
there is an eight carbon side chain in the amine at position 5. The
binding of compound 1c to DNA can be further confirmed by
the fact that the presence of DNA reduces its adsorption in glass. The
adsorption effect can be seen in the corresponding inset of Figure 7,
3. Conclusion
A series of long alkyl side chain benzo[a]phenoxazinium chlo-
rides were synthesised in good to excellent yields by the reaction
with corresponding 5-alkylamino-2-nitrosophenol hydrochloride
and N-alkylated-naphthylamines. The photophysical behaviour of
these molecules was confirmed to be dominated by acid–base
equilibria and H-aggregate formation. The acid/base dissociation
constant of the compounds in ethanol was estimated and a ten-
dency to increase with side-chain length was found.
The positive charge of these compounds, together with its
structure with four condensed aromatic rings, opens the possibility
of DNA binding. As some of the compounds have very little water
solubility, it was necessary to use a residual amount of ethanol to
help the solubilisation process and allow for the interaction with
DNA. The residual ethanol was found to favour the presence of the
basic form of the compound, probably through preferential solva-
tion. The photophysical behaviour in a DNA buffered solution
showed that these types of compounds intercalate in a DNA but
only if the side chain is up to eight carbon atoms long. Other forms
of DNA binding (electrostatic and groove) were also found to occur.
Overall, the new benzo[a]phenoxazinium chlorides synthesised
with maxima fluorescence emission in the near-infrared region are
promising labels for biomolecules.
where after one day of storage, the absorption of a 2 mM aqueous
solution decreases from 0.03 to 0.006. The DNA samples were
measured one day after their preparation; absorbance from 0.016 to
0.03 was observed in the case of compound 1c. The water solubility
of benzo[a]phenoxazinium chlorides is expected to decrease with
the increase of the alkyl side chain. As a result, for compounds 1c,
1j, 1l and 1m, the fraction of aggregates is much higher (absorption
band at 580–600 nm) and another absorption band appears near
520 nm. This can stem from preferential solvation by residual
ethanol (ethanol exists in a concentration of 0.2% v/v, i.e., 34 mM, as
a result of the preparation procedure). The preferentially ethanol
solvated molecules can be in their neutral basic form, which can
justify the absorbance around 520 nm. An additional absorption
band at 450 nm appears for compound 1m in water and can result
from H-aggregates of the neutral basic form. In Figure 7 one can see
that these forms appear in different relative amounts depending on
the R1, R2, R (compounds 1j, 1l and 1m) and especially on the size of
the alkyl side chain (compounds 1a, 1c, and 1j).
For compound 1c, the fluorescence intensity is approximately
constant up to p/d¼10 (Fig. 8). This indicates that electrostatic
binding, which enhances H-aggregate formation (also not evident
in absorption spectra of Fig. 7 up to p/d¼10), has not occurred and
the most probable main form of DNA interaction is groove binding
with the C8 chain laying along the DNA strand. The additional band
that appears for compounds 1c and 1j around 550 nm can thus
stem from compound molecules in the basic form that bind to DNA
grooves, possibly interacting via H-bond. The contribution of this
form to the emission spectra is seen for compound 1c as an en-
largement of the blue side of the emission band. For compound 1c
a sudden change is observed in absorption and fluorescence spectra
when p/d is above 10. This can be interpreted as binding of the acid
form resulting in H-aggregates (absorption at 580 nm) and in-
tercalation (red shifted absorption at w650 nm). The C8 alkyl chain
is also expected to be laying along the DNA strain.
4. Experimental
4.1. General
All melting points were measured on a Stuart SMP3 melting
point apparatus and are uncorrected. TLC analyses were carried
out on 0.25 mm thick precoated silica plates (Merck Fertigplatten
Kieselgel 60F254) and spots were visualised under UV light or
with the naked eye. Chromatography on silica gel was carried out
on Merck Kieselgel (230–240 mesh). IR spectra were determined
on a BOMEM MB 104 spectrophotometer, using KBr discs or neat
samples. NMR spectra were obtained on 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 ꢃC.
All chemical shifts are given in parts per million using dH
Me4Si¼0 ppm as a reference and J values are given in hertz. As-
signments were made by comparison of chemical shifts, peak
multiplicities and J values and were supported by spin decou-
pling-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–visible absorption
spectra (200–800 nm) were obtained using either a Shimadzu
UV/2501PC or Shimadzu UV/3101PC spectrophotometers. Fluo-
rescence spectra were collected using either FluoroMax-4 or
FluoroMax-3 spectrofluorometers. All chemical reagents were
used as received.
When the side chain is longer (C14 for compounds 1j, 1l and
1m), there is no evidence of intercalative binding. However, these
compounds clearly associate with DNA, as is seen in the variations
of both absorption maximum and absorption spectral shapes. For
compounds 1j and 1l, the absorption spectra in the presence of
DNA are dominated by H-aggregates and the acid form. In the case
of compound 1l, a small contribution from basic form at around
520 nm is observed increasing with p/d. This indicates that the
basic form binds to DNA (probably in the grooves) but differently
from the case of compounds 1c and 1j.
Natural double-stranded salmon sperm DNA was obtained from
Invitrogen. Mother solutions of salmon sperm DNA were made in
10 mM Tris–HCl buffer (pH¼7.4), with 1 mM EDTA. The purity of
DNA was checked by monitoring the absorption spectrum and the
ratio of the absorbance at 260 and 280 nm, A260/A280¼1.95 (good-
quality DNA has an A260/A280 ratio higher than 1.8).25 The DNA
concentrations in the number of bases (or phosphate groups) were
Compound 1m is similar to compound 1l with R1¼R2¼Me in-
stead of Et. Yet, its behaviour is different. The residual acid form
appears in the same spectral position (w650 nm) but the 600 nm
band, attributed to H-aggregates, shifts to 560 nm; the 520 nm
band, attributed to the basic form, goes to 450 nm. Thus, this
compound seems highly aggregated in aqueous solutions and re-
veals little interaction with DNA as is confirmed by a much lower
fluorescence emission, with almost no rational p/d variations in
spectral shape or intensity.
determined
from
the
molar
extinction
coefficient,
3
¼6600 Mꢀ1 cmꢀ1 at 260 nm.26,27 Appropriate amounts of 10ꢀ3 M
ethanolic solutions of the compounds were added to DNA solutions
at desired concentrations. The solutions were left at least 24 h to
stabilize.

C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
10449
4.2. General method for preparation of compounds 1a–m
NHCH2CH2), 3.57 (br s, 2H, NHCH2CH3), 6.30 (br s, 2H, 8-H and 6-
H), 7.49 (s, 1H, 11-H), 7.87 (br s, 2H, 2-H and 3-H), 8.80–8.90 (m,
1H, 1-H), 9.18 (br s, 1H, 4-H), 10.81 (br s, 1H, NH). 13C NMR (CDCl3,
75.4 MHz): dC¼13.95 (NHCH2CH3), 14.03 (NH(CH2)7CH3), 18.60
(CH3), 22.55 (CH2), 27.12 (CH2), 28.65 (NHCH2CH2), 29.14 (CH2),
29.24 (CH2), 31.71 (CH2), 38.72 (NHCH2CH2), 44.40 (NHCH2CH3),
92.44 (C-6), 93.22 (C-8), 123.46 (Ar-C), 123.99 (C-1), 125.54 (C-4),
127.06 (C-10), 129.66 (Ar-C), 129.94 (C-3), 130.57 (Ar-C), 130.98
(C-11), 131.61 (C-2), 133.48 (Ar-C), 146.72 (Ar-C), 150.57 (Ar-C),
To a cold solution (ice bath) of 5-(alkylamino)-2-nitrosophenol
hydrochloride 2a–c in ethanol (2 mL), N-alkylated-naphthylamine
(3a–e) and concentrated hydrochloride acid (5.0ꢁ10ꢀ2 mL) were
added. The mixture was refluxed during 2–25 h, and monitored by
TLC (dichloromethane/methanol or chloroform/methanol). After
evaporation of the solvent and dry chromatography purification on
silica gel, the required dye (1a–m) was obtained as a blue material.
154.03 (C-9), 156.71 (C-5) ppm. IR (KBr 1%, cmꢀ1):
n
¼3195, 2925,
4.2.1. N-[10-Methyl-5-(propylamino)-9H-benzo[a]phenoxazin-ylide-
ne]ethanaminium chloride (1a). The product of the reaction of 2a
(0.153 g, 8.51ꢁ10ꢀ4 mol) with 3a (0.150 g, 8.11ꢁ10ꢀ4 mol) (reflux
time 2 h) was chromatographed with dichloromethane/n-hexane
and dichloromethane/methanol, mixtures of increasing polarity, as
the eluent, to give compound 1a (0.28 g, 92%). Mp¼125.0–
126.2 ꢃC. TLC (dichloromethane/methanol, 6.5:0.5): Rf¼0.47. 1H
2853, 1642, 1592, 1561, 1544, 1519, 1498, 1451, 1435, 1315, 1295,
1259, 1232, 1185, 1163, 1129, 1085, 1054, 1010, 881, 817, 773, 733,
708, 666. HRMS (FAB): calcd for C27H34N3O [Mþ]: 416.2702;
found: 416.2708.
4.2.4. N-Ethyl-N-[5-(octylamino)-9H-benzo[a]phenoxazin-9-ylide-
ne]ethanaminium chloride (1d). The product of the reaction of 2b
(0.235 g; 1.21ꢁ10ꢀ3 mol) with 3b (0.309 g, 1.21ꢁ10ꢀ3 mol) (2 mL)
(reflux time 8 h) was chromatographed with chloroform/n-hexane
and chloroform/methanol, mixtures of increasing polarity, as the
eluent, to give compound 1d (0.46 g, 88%). Mp¼113.0–115.8 ꢃC. TLC
(chloroform/methanol, 9.5:0.5): Rf¼0.33. 1H NMR (CDCl3,
NMR (CDCl3, 400 MHz):
d¼1.07 (t, J¼5.4 Hz, 3H, NHCH2CH2CH3),
1.42 (t, J¼5.1 Hz, 3H, NHCH2CH3), 1.80–2.00 (m, 2H, NHCH2CH2),
2.41 (s, 3H, CH3), 3.20–3.35 (m, 2H, NHCH2CH2), 3.58 (br s, 2H,
NHCH2CH3), 6.30 (s, 1H, 8-H), 6.37 (s, 1H, 6-H), 7.49 (s, 1H, 11-H),
7.80–8.00 (m, 2H, 2-H and 3-H), 8.81 (d, J¼6.9 Hz, 1H, 1-H), 9.19
(br s, 1H, 4-H), 11.23 (br s, 1H, NH). 13C NMR (CDCl3, 100.6 MHz):
dC¼11.59 (NHCH2CH3), 14.03 (NHCH2CH3), 17.72 (CH3), 22.19
(NHCH2CH2), 38.83 (NHCH2CH2), 46.43 (NHCH2CH3), 93.04 (C-8),
93.80 (C-6), 124.11 (Ar-C), 124.21 (C-1), 125.70 (C-10), 126.12 (C-4),
129.09 (Ar-C), 129.87 (Ar-C), 130.67 (C-3), 131.29 (C-11), 133.52 (C-
2), 135.35 (Ar-C), 146.58 (Ar-C), 151.14 (Ar-C), 153.17 (C-9), 156.79
400 MHz):
d
¼0.84 (t, J¼7.2 Hz, 3H, CH3), 1.30–1.38 (m, 10H, 5ꢁCH2),
1.32 (t, J¼7.2 Hz, 6H, N(CH2CH3)2), 1.40–1.50 (m, 2H, CH2), 1.84–1.92
(m, 2H, NHCH2CH2), 3.56 and 3.60 (2ꢁd, J¼7.2 and 6.8 Hz, 2H,
N(CH2CH3)2), 3.72–3.82 (m, 2H, NHCH2CH2), 6.50 (s, 1H, 8-H), 6.51
(s, 1H, 6-H), 6.95 (dd, J¼9.4 and 2.8 Hz, 1H, 10-H), 7.65 (d, J¼9.2 Hz,
1-H, 11-H), 7.67–7.76 (m, 2H, 2-H and 3-H), 8.65 (d, J¼9.6 Hz, 1H, 1-
H), 9.26 (d, J¼8.8 Hz, 1H, 4-H). 13C NMR (CDCl3, 100.6 MHz):
dC¼12.60 (N(CH2CH3)2), 13.99 (CH3), 22.53 (CH2), 27.12
(NHCH2CH2), 28.98 (CH2), 29.13 (CH2), 29.26 (CH2), 31.70 (CH2),
44.96 (NHCH2CH2), 45.84 (N(CH2CH3)2), 93.21 (C-6), 95.58 (C-8),
113.71 (C-10), 123.91 (C-1), 124.16 (Ar-C), 126.21 (C-4), 128.35 (Ar-
C), 130.44 (C-3), 130.58 (Ar-C), 131.78 (C-2), 132.38 (C-11), 135.26
(Ar-C), 147.26 (Ar-C), 151.33 (Ar-C), 152.84 (C-9), 158.46 (C-5). IR
(C-5). IR (KBr 1%, cmꢀ1):
n
¼3201, 2968, 2931, 2874, 1642, 1592,
1561, 1544, 1520, 1498, 1451, 1435, 1384, 1343, 1318, 1258, 1234,
1186, 1165, 1144, 1129, 1086, 1056, 1014, 1004, 977, 882, 856, 818,
774. HRMS (FAB): calcd for C22H24N3O [Mþ]: 346.1919; found:
346.1914.
4.2.2. N-Ethyl-N-[5-(propylamino)-9H-benzo[a]phenoxazin-9-ylide-
ne]ethanaminium chloride (1b). The product of the reaction of 2b
(0.291 g, 1.5ꢁ10ꢀ3 mol) with 3a (0.185 g, 1ꢁ10ꢀ3 mol) (reflux time
5 h) was chromatographed with chloroform and chloroform/
methanol, mixtures of increasing polarity, as the eluent, to give
compound 1b (0.288 g, 49%). Mp¼235–237 ꢃC. TLC (chloroform/
(KBr 1%, cmꢀ1):
n
¼3424, 2956, 2925, 2853, 1640, 1588, 1547, 1498,
1454, 1435, 1383, 1330, 1273, 1255, 1164, 1123, 1073, 1012, 751, 707,
666. HRMS (EI): calcd for C28H36N3O [Mþ]: 430.28541; found:
430.28529.
methanol, 9.4:0.6): Rf¼0.19. 1H NMR (CD3OD, 400 MHz):
d
¼1.15 (t,
4.2.5. N-Methyl-N-[5-(octylamino)-9H-benzo[a]phenoxazin-9-ylide-
ne]methanaminium chloride (1e). The product of the reaction of 2c
(0.20 g; 1.31ꢁ10ꢀ3 mol) with 3b (0.34 g, 1.31ꢁ10ꢀ3 mol) (reflux
time 12 h) was chromatographed with chloroform/n-hexane and
chloroform/methanol, mixtures of increasing polarity, as the elu-
ent, to give compound 1e (0.24 g, 45%). Mp¼174.9–176.2 ꢃC. TLC
(chloroform/methanol, 9:1): Rf¼0.43. 1H NMR (CDCl3, 400 MHz):
J¼7.6 Hz, 3H, NHCH2CH2CH3), 1.37 (t, J¼6.8 Hz, 6H, 2ꢁNCH2CH3),
1.85–1.98 (m, 2H, NHCH2CH2CH3), 3.65–3.79 (m, 6H, 2ꢁNCH2CH3
and NHCH2CH2CH3), 6.87 (d, J¼2.4 Hz, 1H, 8-H), 6.90 (s, 1H, 6-H),
7.26 (dd, J¼9.4 Hz and 2.4 Hz, 1H, 10-H), 7.76–7.83 (m, 2H, 11-H and
3-H), 7.85–7.94 (m, 1H, 2-H), 8.32 (d, J¼8.0 Hz, 1H, 4-H), 8.79 (d,
J¼8.0 Hz, 1H, 1-H). 13C NMR (CDCl3, 100.6 MHz): dC¼11.77
(NHCH2CH2CH3), 12.98 (2ꢁNCH2CH3), 23.06 (NHCH2CH2CH3), 47.05
(2ꢁNCH2CH3), 47.44 (NHCH2CH2CH3), 94.46 (C-6), 96.99 (C-8),
116.64 (C-10), 123.85 (C-4), 124.71 (Ar-C), 125.54 (C-1), 130.90 (C-3),
131.53 (Ar-C), 132.51(Ar-C), 132.95(C-2), 134.10 (C-11), 134.91 (Ar-
C), 149.64 (Ar-C), 153.14 (Ar-C), 155.61 (C-9), 159.36 (C-5). IR (KBr
d¼0.86 (t, J¼5.1 Hz, 3H, CH3), 1.20–1.50 (m, 10H, 5ꢁCH2), 1.80–1.90
(m, 2H, NHCH2CH2), 3.24 (s, 6H, N(CH3)2), 3.70–3.85 (m, 2H,
NHCH2CH2), 6.35 (d, J¼2.4 Hz, 1H, 8-H), 6.46 (s, 1H, 6-H), 6.91 (dd,
J¼9.2 and 2.4 Hz, 1H, 10-H), 7.56 (d, J¼9.2 Hz, 1H, 11-H), 7.68–7.77
(m, 2H, 2-H and 3-H), 8.58–8.65 (m, 1H, 1-H), 9.20–9.30 (m, 1H, 4-
H). 13C NMR (CDCl3, 100.6 MHz): dC¼14.03 (NH(CH2)7CH3), 22.57
(CH2), 27.16 (CH2), 29.03 (CH2), 29.18 (NHCH2CH2), 29.30 (CH2),
31.74 (CH2), 40.89 (N(CH3)2), 45.06 (NHCH2CH2), 93.28 (C-6), 95.71
(C-8), 113.78 (C-10), 123.99 (C-1), 124.15 (Ar-C), 126.28 (C-4), 128.22
(Ar-C), 130.47 (C-3), 130.51 (Ar-C), 131.81 (C-11), 131.88 (C-2), 135.39
(Ar-C), 146.66 (Ar-C), 151.25 (Ar-C), 154.44 (C-9), 158.46 (C-5). IR
1%, cmꢀ1):
n
¼3378, 3169, 2966, 2929, 1639, 1586, 1546, 1452, 1434,
1384, 1327, 1276, 1257, 1196, 1166, 1148, 1122, 1073, 1015, 1002, 944,
884, 855, 821, 809, 766. HRMS (FAB): calcd for C23H26N3O [Mþ]
360.20657; found: 360.20704.
4.2.3. N-[10-Methyl-5-(octylamino)-9H-benzo[a]phenoxazin-9-yli-
dene]ethanaminium chloride (1c). The product of the reaction of
2a (0.216 g, 1.20ꢁ10ꢀ3 mol) with 3b (0.307 g, 1.20ꢁ10ꢀ3 mol)
(reflux time 4 h) was chromatographed with dichloromethane/
methanol, 59:1, as the eluent, to give compound 1c (0.49 g, 98%).
Mp¼147.5–149.0 ꢃC. TLC (dichloromethane/methanol, 5:1):
(KBr 1%, cmꢀ1):
n
¼3428, 2956, 2925, 2854, 1641, 1592, 1553, 1538,
1500, 1456, 1427, 1380, 1334, 1293, 1202, 1180, 1148, 1127, 1009, 906,
816, 784, 741, 716, 666. HRMS (TOF EI): calcd for C26H32N3O [Mþ]:
402.25403; found: 402.25399.
Rf¼0.45. 1H NMR (CDCl3, 300 MHz):
d
¼0.86 (t, J¼6.6 Hz, 3H,
4.2.6. N-[5-(Decylamino)-10-methyl-9H-benzo[a]phenoxazin-9-yli-
dene]ethanaminium chloride (1f). The product of the reaction of 2a
(0.18 g, 1.05ꢁ10ꢀ3 mol) with 3c (0.21 g, 7.3ꢁ10ꢀ3 mol) (reflux
NH(CH2)7CH3), 1.20–1.50 (2ꢁm, 13H, 5ꢁCH2 and NHCH2CH3), 1.85
(br s, 2H, NHCH2CH2), 2.45 (s, 3H, CH3), 3.20–3.30 (m, 2H,

10450
C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
time 8 h) was chromatographed with chloroform/n-hexane and
chloroform/methanol, mixtures of increasing polarity, as the
eluent, to give compound 1f (0.32 g, 96%). Mp¼168.8–171.8 ꢃC.
TLC (chloroform/methanol, 5:1): Rf¼0.40. 1H NMR (CDCl3,
(Ar-C), 152.85 (C-9), 157.92 (C-5). HRMS (EI): calcd for C32H44N3O
[Mþ]: 486.34800; found: 486.34789.
4.2.9. N-[5-(Dodecylamino)-9H-benzo[a]phenoxazin-9-ylidene]-N-
methylmethanaminium chloride (1i). The product of the reaction of
2c (0.22 g, 1.45ꢁ10ꢀ3 mol) with 3d (0.301 g, 9.67ꢁ10ꢀ4 mol) (reflux
time 25 h) was chromatographed with chloroform/n-hexane and
chloroform/methanol, mixtures of increasing polarity, as the elu-
ent, to give compound 1i (0.19 g, 43%). Mp¼163.0–163.8 ꢃC. TLC
(chloroform/methanol, 9.5:0.5): Rf¼0.57. 1H NMR (CDCl3,
300 MHz):
d
¼0.86 (t, J¼6.3 Hz, 3H, NH(CH2)9CH3), 1.18–1.46
(2ꢁm, 17H, 7ꢁCH2 and NHCH2CH3), 1.80 (br s, 2H, NHCH2CH2),
2.39 (s, 3H, CH3), 3.18 (br s, 2H, NHCH2CH2), 3.42 (br s, 2H,
NHCH2CH3), 6.08 (s, 1H, 8-H), 6.16 (s, 1H, 6-H), 6.78 (br s, 1H, NH),
7.36 (s, 1H, 11-H), 7.70–7.90 (m, 2H, 2-H and 3-H), 8.74 (d,
J¼8.7 Hz, 1H, 1-H), 8.91 (br s, 1H, 4-H), 10.59 (br s, 1H, NH). 13C
NMR (CDCl3, 75.4 MHz): dC¼13.74 (NHCH2CH3), 14.06
(NH(CH2)9CH3), 17.95 (CH3), 22.61 (CH2), 27.11 (CH2), 28.55
(NHCH2CH2), 29.24 (2ꢁCH2), 29.29 (CH2), 29.49 (CH2), 31.81
(CH2), 38.67 (NHCH2CH2), 44.69 (NHCH2CH3), 92.36 (C-6), 93.10
(C-8), 123.56 (C-1), 123.75 (Ar-C), 125.25 (C-4), 126.83 (C-10),
129.48 (Ar-C), 129.97 (C-3), 130.61 (Ar-C), 130.92 (C-11), 131.50
(C-2), 133.65 (Ar-C), 146.68 (Ar-C), 150.50 (Ar-C), 154.00 (C-9),
300 MHz):
d¼0.87 (t, J¼6.9 Hz, 3H, NH(CH2)11CH3), 1.12–1.60 (2ꢁm,
18H, 9ꢁCH2), 1.62–2.0 (m, 2H, NHCH2CH2), 3.25 (s, 6H, N(CH3)2),
3.70–3.88 (m, 2H, NHCH2CH2), 6.41 (s, 1H, 8-H), 6.49 (s, 1H, 6-H),
6.93 (d, J¼7.8 Hz, 1H, 10-H), 7.20 (d, J¼9.0 Hz, 1H, 11-H), 7.68–7.80
(m, 2H, 2-H and 3-H), 8.60–8.72 (m,1H,1-H), 9.21 (br s,1H, 4-H). 13C
NMR (CDCl3, 75.4 MHz): dC¼14.08 (NH(CH2)11CH3), 22.64 (CH2),
27.22 (CH2), 29.17 (CH2), 29.31 (CH2), 29.39 (CH2), 29.56 (CH2),
29.59 (2ꢁCH2), 29.62 (CH2), 31.87 (CH2), 40.93 (N(CH3)2), 45.24
(NHCH2CH2), 93.48 (C-6), 95.85 (C-8), 113.62 (C-10), 124.05 (C-1),
124.47 (Ar-C), 126.20 (C-4), 128.21 (Ar-C), 130.52 (C-3), 130.56 (Ar-
C), 131.83 (C-11), 131.90 (C-2), 135.84 (Ar-C), 146.74 (Ar-C), 151.19
156.93 (C-5). IR (KBr 1%, cmꢀ1):
1592, 1424, 1384, 1316, 1185, 1126, 1012, 666. HRMS (FAB): calcd
for C29H38N3O [Mþ]: 444.3015; found: 444.3021.
n
¼3440, 2956, 2925, 2853, 1643,
4.2.7. N-[5-(Dodecylamino)-10-methyl-9H-benzo[a]phenoxazin-9-
ylidene]ethanaminium chloride (1g). The product of the reaction of
2a (0.087 g, 4.82ꢁ10ꢀ4 mol) with 3d (0.151 g, 4.86ꢁ10ꢀ4 mol)
(reflux time 4 h) was chromatographed with dichloromethane/n-
hexane and dichloromethane/methanol, mixtures of increasing
polarity, as the eluent, to give compound 1g (0.19 g, 83%).
Mp¼93.1–96.0 ꢃC. TLC (dichloromethane/methanol, 9:1): Rf¼0.50.
(Ar-C), 154.38 (C-9), 158.35 (C-5). IR (KBr 1%, cmꢀ1):
2924, 2853, 1641, 1590, 1552, 1513, 1500, 1459, 1427, 1378, 1332,
1292, 1201, 1177, 1149, 1125, 1009, 905, 864, 817, 783, 715, 666.
HRMS (EI): calcd for C30H40N3O [Mþ]: 458.31711; found: 458.31659.
n
¼3448, 2955,
4.2.10. N-[10-Methyl-5-(tetradecylamino)-9H-benzo[a]phenoxazin-
9-ylidene]ethanaminium chloride (1j). The product of the reaction
of 2a (0.110 g, 6.13ꢁ10ꢀ4 mol) with 3e (0.200 g, 6.13ꢁ10ꢀ4 mol)
(reflux time 6 h) was chromatographed with chloroform/methanol,
mixtures of increasing polarity, as the eluent, to give compound 1j
(0.27 g, 86%). Mp¼113.9–115.5 ꢃC. TLC (chloroform/methanol, 9:1):
1H NMR (CDCl3, 300 MHz):
d¼0.88 (t, J¼6.9 Hz, 3H,
NH(CH2)11CH3), 1.20–1.50 (2ꢁm, 21H, 9ꢁCH2 and NHCH2CH3), 1.84
(br s, 2H, NHCH2CH2), 2.44 (s, 3H, CH3), 3.10–3.30 (m, 2H,
NHCH2CH2), 3.52 (br s, 2H, NHCH2CH3), 6.19 (s, 1H, 8-H), 6.27 (s,
1H, 6-H), 7.47 (s, 1H, 11-H), 7.80–7.90 (m, 2H, 2-H and 3-H), 8.76–
8.90 (m, 1H, 1-H), 9.10–9.24 (m, 1H, 4-H), 11.14 (br s, 1H, NH). 13C
NMR (CDCl3, 75.4 MHz): dC¼13.82 (NHCH2CH3), 14.04
(NH(CH2)11CH3), 18.27 (CH3), 22.60 (CH2), 27.12 (CH2), 28.62
(NHCH2CH2), 29.27 (CH2), 29.30 (CH2), 29.50 (CH2), 29.53 (CH2),
29.58 (CH2), 30.87 (CH2), 31.83 (CH2), 38.66 (NHCH2CH2), 44.51
(NHCH2CH3), 92.33 (C-6), 93.08 (C-8), 123.62 (Ar-C), 123.94 (C-1),
125.51 (C-4), 127.0 (C-10), 129.55 (Ar-C), 129.99 (C-3), 130.59 (Ar-
C), 130.92 (C-11), 131.54 (C-2), 133.53 (Ar-C), 146.71 (Ar-C), 150.58
Rf¼0.30. 1H NMR (CDCl3, 300 MHz):
d
¼0.87 (t, J¼6.9 Hz, 3H,
NH(CH2)13CH3), 1.20–1.50 (2ꢁm, 25H, 11ꢁCH2 and NHCH2CH3), 1.80
(br s, 2H, NHCH2CH2), 2.38 (s, 3H, CH3), 3.18 (br s, 2H, NHCH2CH2),
3.43 (br s, 2H, NHCH2CH3), 6.10 (s, 1H, 8-H), 6.18 (s, 1H, 6-H), 7.39 (s,
1H, 11-H), 7.81 (br s, 2H, 2-H and 3-H), 8.77 (d, J¼7.5 Hz, 1H, 1-H),
8.92 (br s, 1H, 4-H). 13C NMR (CDCl3, 75.4 MHz): dC¼13.77
(NHCH2CH3), 14.09 (NH(CH2)13CH3), 17.84 (CH3), 22.65 (CH2), 27.14
(CH2), 28.57 (NHCH2CH2), 29.32 (2ꢁCH2), 29.53 (CH2), 29.57 (CH2),
29.62 (2ꢁCH2), 29.66 (2ꢁCH2), 31.88 (CH2), 38.71 (NHCH2CH2),
44.74 (NHCH2CH3), 92.45 (C-6), 93.18 (C-8), 123.79 (Ar-C), 124.03
(C-1), 125.36 (C-4), 126.69 (C-10), 129.46 (Ar-C), 130.15 (C-3), 130.66
(Ar-C), 131.00 (C-11), 131.62 (C-2), 133.92 (Ar-C), 146.69 (Ar-C),
(Ar-C), 154.06 (C-9), 156.92 (C-5). IR (KBr 1%, cmꢀ1):
n
¼3450, 3210,
2956, 2923, 2852, 1643, 1592, 1561, 1544, 1520, 1451, 1436, 1384,
1317, 1295, 1262, 1233, 1185, 1163, 1129, 1085, 1010, 878, 816, 773,
733, 666. HRMS (FAB): calcd for C31H42N3O [Mþ]: 472.3328; found:
472.3335.
150.64 (Ar-C), 153.96 (C-9), 157.16 (C-5). IR (KBr 1%, cmꢀ1):
3230, 2954, 2919, 2852, 1644, 1592, 1562, 1546, 1520, 1453, 1436,
1384, 1316, 1261, 1185, 1164, 1129, 1087, 1010, 880, 774, 666. HRMS
(FAB): calcd for C33H46N3O [Mþ]: 500.3641; found: 500.3637.
n
¼3448,
4.2.8. N-[5-(Dodecylamino)-9H-benzo[a]phenoxazin-9-ylidene]-N-
ethylethanaminium chloride (1h). The product of the reaction of 2b
(0.121 g, 6.25ꢁ10ꢀ4 mol) with 3d (0.194 g, 6.25ꢁ10ꢀ4 mol) (reflux
time 7 h) was chromatographed with dichloromethane/n-hexane
and dichloromethane/methanol, mixtures of increasing polarity, as
the eluent, to give compound 1h (0.26 g, 69%). Mp¼130.3–131.4 ꢃC.
TLC (dichloromethane/methanol, 9.5:0.5): Rf¼0.42. 1H NMR (CDCl3,
4.2.11. N-Ethyl-N-[5-(tetradecylamino)-9H-benzo[a]phenoxazin-9-
ylidene]ethanaminium chloride (1l). The product of the reaction of
2b (0.195 g, 1.00ꢁ10ꢀ3 mol) with 3e (0.341 g, 1.00ꢁ10ꢀ3 mol)
(reflux time 10 h) was chromatographed with dichloromethane/
methanol 99:1, as the eluent, to give compound 1l (0.27 g, 53%).
Mp¼71.1–73.4 ꢃC. TLC (dichloromethane/methanol, 9:1): Rf¼0.68.
300 MHz):
d
¼0.78 (t, J¼6.6 Hz, 3H, NH(CH2)11CH3), 1.10–1.40 (3ꢁm,
24H, N(CH2CH3)2 and 9ꢁCH2), 1.82 (br s, 2H, NHCH2CH2), 3.40–3.60
(m, 4H, N(CH2CH3)2), 3.71 (br s, 2H, NHCH2CH2), 6.43 (s, 2H, 8-H
and 6-H), 6.92 (d, J¼9.3 Hz, 1H, 10-H), 7.56 (d, J¼9.0 Hz, 1H, 11-H),
7.58–7.70 (m, 2H, 2-H and 3-H), 8.48–8.58 (m, 1H, 1-H), 9.13 (br s,
1H, 4-H), 11.47 (br s, 1H, N–H). 13C NMR (CDCl3, 75.4 MHz):
dC¼12.51 (N(CH2CH3)2), 13.86 (NH(CH2)11CH3), 22.40 (CH2), 26.99
(CH2), 28.83 (NHCH2CH2), 29.07 (CH2), 29.19 (CH2), 29.35 (CH2),
29.38 (CH2), 30.70 (CH2), 31.63 (CH2), 44.74 (NHCH2CH2), 45.81
(N(CH2CH3)2), 92.90 (C-6), 95.41 (C-8), 113.93 (C-10), 123.68 (C-1),
123.79 (Ar-C), 125.74 (C-4), 128.38 (Ar-C), 129.91 (C-3), 130.33 (Ar-
C), 131.43 (C-2), 132.23 (C-11), 134.50 (Ar-C), 147.06 (Ar-C), 150.95
1H NMR (CDCl3, 400 MHz):
d¼0.81 (t, J¼7.2 Hz, 3H, NH(CH2)13CH3),
1.10–1.50 (3ꢁm, 28H, 11ꢁCH2 and N(CH2CH3)2), 1.80–2.0 (m, 2H,
NHCH2CH2), 3.54 and 3.58 (2ꢁd, J¼7.2 Hz, 4H, N(CH2CH3)2), 3.70–
3.80 (m, 2H, NHCH2CH2), 6.45 (s, 2H, 8-H and 6-H), 6.92 (dd, J¼9.2
and 2.4 Hz, 1H, 10-H), 7.58 (d, J¼9.2 Hz, 1H, 11-H), 7.60–7.70 (m, 2H,
2-H and 3-H), 8.50–8.60 (m, 1H, 1-H), 9.16 (br s, 1H, 4-H), 11.67 (br s,
1H, N–H). 13C NMR (CDCl3, 100.6 MHz): dC¼12.57 (N(CH2CH3)2),
13.95 (NH(CH2)13CH3), 22.50 (CH2), 27.08 (CH2), 28.92 (NHCH2CH2),
29.17 (CH2), 29.27 (CH2), 29.43 (CH2), 29.54 (2ꢁCH2), 29.47 (CH2),
29.49 (CH2), 29.51 (CH2), 31.73 (CH2), 44.93 (NHCH2CH2), 45.82
(N(CH2CH3)2), 93.05 (C-6), 95.51 (C-8), 113.80 (C-10), 123.79 (Ar-C),

C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
10451
124.02 (C-1), 125.94 (C-4), 128.35 (Ar-C), 130.15 (C-3), 130.45 (Ar-C),
131.56 (C-2), 132.29 (C-11), 134.85 (Ar-C), 147.15 (Ar-C), 151.11 (Ar-
3-H), 7.38–7.56 (m, 2H, 6-H and 7-H), 7.80–7.88 (m, 1H, 8-H), 7.90–
7.98 (m, 1H, 5-H). 13C NMR (CDCl3, 75.4 MHz): dC¼13.99 (CH3),
22.50 (2ꢁCH2), 26.86 (CH2), 27.13 (CH2), 29.03 (CH2), 31.62 (CH2),
49.08 (NHCH2CH2), 114.57 (C-4), 120.81 (C-5), 124.75 (C-7), 125.65
(C-2), 126.17 (C-4a), 126.47 (C-3), 126.57 (C-6), 128.73 (C-8), 134.33
C), 152.85 (C-9), 158.20 (C-5). IR (KBr 1%, cmꢀ1):
n¼3436, 2956,
2923, 2852, 1641, 1590, 1548, 1514, 1438, 1385, 1329, 1277, 1255,
1168, 1151, 1125, 1073, 1013, 705, 666. HRMS (EI): calcd for
C34H48N3O [Mþ]: 514.37906; found: 514.37919.
(C-8a), 136.21 (C-1). IR (neat, cmꢀ1):
n
¼3389, 3058, 2956, 2925,
2855, 2469, 1625, 1605, 1582, 1531, 1465, 1410, 1377, 1344, 1282,
1254, 1216, 1173, 1142, 1081, 1016, 953, 861, 800, 770, 724, 666.
HRMS (EI): calcd for C18H25N [Mþ]: 255.1987; found: 255.1985.
4.2.12. N-Methyl-N-[5-(tetradecylamino)-9H-benzo[a]phenoxazin-
9-ylidene]methanaminium chloride (1m). The product of the
reaction of 2c (0.15 g, 9.56ꢁ10ꢀ4 mol) with 3e (0.324 g,
9.56ꢁ10ꢀ4 mol) (reflux time 14 h) was chromatographed with
dichloromethane/n-hexane and dichloromethane/methanol, mix-
tures of increasing polarity, as the eluent, to give compound 1m
(0.079 g, 17%). Mp¼154.0–156.7 ꢃC. TLC (dichloromethane/metha-
4.3.3. N-Decylnaphthalen-1-amine (3c). The product of the
reaction of 1-naphthylamine (0.50 g, 3.5ꢁ10ꢀ3 mol) with 1-bro-
modecane (1.11 mL, 5.35ꢁ10ꢀ3 mol), in ethanol (2 mL) (reflux time
22 h 30 min) was chromatographed with chloroform/n-hexane,
mixtures of increasing polarity, as the eluent, to give compound 3c
as a brown oil (0.87 g, 88%). TLC (chloroform/n-hexane, 2:3):
nol, 9:1): Rf¼0.55. 1H NMR (CDCl3, 400 MHz):
¼0.84 (t, J¼5.2 Hz,
d
3H, NH(CH2)13CH3), 1.10–1.50 (3ꢁm, 22H, 11ꢁCH2), 1.76–1.90 (br s,
2H, NHCH2CH2), 3.20 (s, 6H, N(CH3)2), 3.71 (br s, 2H, NHCH2CH2),
6.0 (s,1H, 8-H), 6.40 (s,1H, 6-H), 6.86 (d, J¼8.0 Hz,1H,10-H), 7.48 (d,
J¼8.8 Hz, 1H, 11-H), 7.60–7.70 (m, 2H, 2-H and 3-H), 8.52 (d,
J¼7.6 Hz, 1H, 1-H), 8.95 (br s, 1H, 4-H). 13C NMR (CDCl3, 100.6 MHz):
dC¼13.99 (NH(CH2)13CH3), 22.54 (CH2), 27.11 (CH2), 28.89
(NHCH2CH2), 29.01 (CH2), 29.11 (CH2), 29.21 (CH2), 29.31 (CH2),
29.36 (CH2), 29.51 (CH2), 29.54 (CH2), 29.56 (CH2), 31.77 (CH2),
40.81 (N(CH3)2), 45.10 (NHCH2CH2), 93.09 (C-6), 95.65 (C-8), 113.94
(C-10), 123.87 (Ar-C), 123.92 (C-1), 125.44 (C-4), 128.36 (Ar-C),
130.15 (C-3), 130.40 (Ar-C), 131.60 (C-2), 131.83 (C-11), 139.13 (Ar-C),
146.59 (Ar-C), 150.99 (Ar-C), 154.51 (C-9), 158.17 (C-5). IR (KBr 1%,
Rf¼0.46. 1H NMR (CDCl3, 300 MHz):
¼0.91 (t, J¼6.9 Hz, 3H, CH3),
d
1.20–1.40 (2ꢁm, 14H, 7ꢁCH2), 1.80–2.0 (m, 2H, NHCH2CH2), 3.39 (t,
J¼7.8 Hz, 2H, NHCH2CH2), 7.22 (d, J¼7.5 Hz, 1H, 4-H), 7.41 (t,
J¼8.4 Hz, 1H, 3-H), 7.48–7.60 (m, 3H, 2-H, 6-H and 7-H), 7.85 (dd,
J¼7.5 and 1.8 Hz, 1H, 8-H), 8.09 (d, J¼8.1 Hz, 1H, 5-H). 13C NMR
(CDCl3, 75.4 MHz): dC¼14.06 (CH3), 22.61 (CH2), 27.00 (NHCH2CH2),
27.80 (CH2), 29.19 (CH2), 29.21 (2ꢁCH2), 29.44 (CH2), 31.81 (CH2),
47.65 (NHCH2CH2), 111.61 (C-4), 120.50 (C-5), 122.29 (C-2), 124.33
(C-4a), 125.94 (C-3), 126.0 (C-7), 126.21 (C-6), 128.69 (C-8), 134.31
(C-8a), 138.33 (C-1). IR (neat, cmꢀ1):
n
¼3428, 3055, 3010, 2954,
2924, 2853, 1624, 1583, 1527, 1478, 1466, 1409, 1376, 1344, 1281,
1253, 1170, 1141, 1114, 1088, 1018, 861, 843, 800, 783, 767, 722, 699,
cmꢀ1):
n
¼3449, 2956, 2923, 2853, 1640, 1590, 1552, 1459, 1428,
1384, 1332, 1292, 1178, 1125, 1009, 905, 818, 774, 715, 666. HRMS
(EI): calcd for C32H44N3O [Mþ]: 486.34727; found: 486.34789.
666. HRMS (EI): calcd for C20H29
283.2302.
N
[Mþ]: 283.2300; found:
4.3. General method for preparation of compounds 3a–e
4.3.4. N-Dodecylnaphthalen-1-amine (3d). The product of the
reaction of 1-naphthylamine (2.0 g, 1.40ꢁ10ꢀ4 mol) with 1-bro-
mododecane (3.55 mL, 1.469ꢁ10ꢀ3 mol), in ethanol (3 mL) (reflux
time 11 h) was chromatographed with dichloromethane/n-hexane,
1:6, as the eluent, to give compound 3d as a white oily solid (1.92 g,
44%). TLC (dichloromethane/n-hexane, 3:4): Rf¼0.63. 1H NMR
To a solution of 1-naphthylamine in methanol or ethanol, 1-
bromoalkane was added and the resulting mixture was refluxed for
11–33.5 h, and monitored by TLC (chloroform/n-hexane). After
evaporation of the solvent and dry chromatography purification on
silica gel, the required intermediate (1a–e) was obtained.
(CDCl3, 300 MHz):
d¼0.92 (t, J¼7.2 Hz, 3H, CH3), 1.20–1.55 (2ꢁm,
18H, 9ꢁCH2), 1.70–1.90 (m, 2H, NHCH2CH2), 3.30 (t, J¼7.5 Hz, 2H,
NHCH2CH2), 6.69 (d, J¼7.2 Hz, 1H, 4-H), 7.28 (d, J¼7.8 Hz, 1H, 2-H),
7.38 (t, J¼7.5 Hz,1H, 3-H), 7.42–7.50 (m, 2H, 6-H and 7-H), 7.78–7.88
(m, 2H, 8-H and 5-H). 13C NMR (CDCl3, 75.4 MHz): dC¼14.11 (CH3),
22.68 (CH2), 27.33 (NHCH2CH2), 29.28 (CH2), 29.35 (CH2), 29.47
(CH2), 29.61 (2ꢁCH2), 29.63 (CH2), 29.66 (CH2), 31.91 (CH2), 44.59
(NHCH2CH2), 104.89 (C-4), 117.56 (C-2), 119.83 (C-5), 123.41 (C-4a),
124.69 (C-7), 125.69 (C-6) 126.59 (C-3) 128.65 (C-8), 134.30 (C-8a),
4.3.1. N-Propylnaphthalen-1-amine (3a). The product of the re-
action of 1-naphthylamine (2.0 g, 1.4ꢁ10ꢀ2 mol) with 1-bromo-
propane (1.81 mL, 0.020 mol) in ethanol (2 mL) (reflux time 18 h)
was chromatographed with chloroform/n-hexane, mixtures of in-
creasing polarity, as the eluent, to give compound 3a as a dark
brown oil (1.55 g, 60%). TLC (chloroform/n-hexane, 5:2): Rf¼0.55. 1H
NMR (CDCl3, 300 MHz):
d
¼1.09 (t, J¼7.8 Hz, 3H, CH3), 1.80–1.90 (m,
2H, NHCH2CH2), 3.29 (t, J¼7.2 Hz, 2H, NHCH2CH2), 6.75 (d, J¼7.2 Hz,
1H, 4-H), 7.29 (d, J¼8.1 Hz,1H, 2-H), 7.38 (t, J¼7.8 Hz,1H, 3-H), 7.42–
7.50 (m, 2H, 6-H and 7-H), 7.78–7.82 (m, 1H, 8-H), 7.84–7.90 (m, 1H,
5-H). 13C NMR (CDCl3, 75.4 MHz): dC¼11.75 (CH3), 22.33
(NHCH2CH2), 46.65 (NHCH2CH2), 105.62 (C-4), 118.03 (C-2), 119.89
(C-5), 123.51 (C-4a), 124.83 (C-7), 125.73 (C-6), 126.52 (C-3), 128.66
143.09 (C-1). IR (Nujol, cmꢀ1):
n
¼3395, 3054, 3009, 2925, 2853,
1623, 1583, 1523, 1464, 1409, 1378, 1346, 1292, 1280, 1253, 1231,
1214, 1170, 1132, 1119, 1095, 1078, 1038, 1029, 1015, 956, 945, 768,
742, 723, 666. HRMS (EI): calcd for C22H33N [Mþ]: 311.2613; found:
311.2611.
(C-8), 134.30 (C-8a), 142.65 (C-1). IR (neat, cmꢀ1):
2923, 2852, 1624, 1583, 1475, 1463, 1455, 1409, 1379, 1345, 1280,
1142, 1080, 1016, 975, 858, 798, 768, 666. HRMS (EI): calcd for
C13H16N [Mþ]: 186.12823; found: 186.12773.
n
¼3427, 2958,
4.3.5. N-Tetradecylnaphthalen-1-amine (3e). The product of the
reaction of 1-naphthylamine (1.0 g, 6.98ꢁ10ꢀ3 mol) with
1-bromotetradecane (2.2 mL, 7.33ꢁ10ꢀ3 mol), in ethanol (3 mL)
(reflux time 12 h.) was chromatographed with chloroform/metha-
nol, mixtures of increasing polarity, as the eluent, to give compound
3e as a brown oil (0.790 g, 67%). TLC (chloroform/n-hexane, 6:4):
4.3.2. N-Octylnaphthalen-1-amine (3b). The product of the reaction
of 1-naphthylamine (1.0 g, 6.98ꢁ10ꢀ3 mol) with 1-bromooctane
(1.97 mL, 9.81ꢁ10ꢀ3 mol) in ethanol (2 mL) (reflux time 33 h
30 min) was chromatographed with chloroform/n-hexane, 1:1, as
the eluent, to give compound 3b (0.46 g, 51%) as a brown oil. TLC
(chloroform/n-hexane, 1:2): Rf¼0.61. 1H NMR (CDCl3, 300 MHz):
Rf¼0.68. 1H NMR (CDCl3, 300 MHz):
¼0.99 (t, J¼6.6 Hz, 3H, CH3),
d
1.26–1.60 (m, 22H, 11ꢁCH2), 1.76–1.86 (m, 2H, NHCH2CH2), 3.32 (t,
J¼6.9 Hz, 2H, NHCH2CH2), 6.68 (d, J¼7.2 Hz, 1H, 4-H), 7.31 (d,
J¼7.8 Hz, 1H, 2-H), 7.43 (t, J¼7.8 Hz, 1H, 3-H), 7.46–7.56 (m, 2H, 6-H
and 7-H), 7.87 (d, J¼8.6 Hz, 2H, 5-H and 8-H). 13C NMR (CDCl3,
75.4 MHz): dC¼14.11 (CH3), 22.69 (CH2), 27.35 (CH2), 29.37
(NHCH2CH2), 29.41 (CH2), 29.50 (CH2), 29.62 (CH2), 29.64 (CH2),
29.68 (CH2), 29.69 (2ꢁCH2), 29.71 (CH2), 31.93 (CH2), 44.19
d
¼0.97 (t, J¼7.2 Hz, 3H, CH3), 1.30–1.60 (m, 10H, 5ꢁCH2), 1.78–1.90
(m, 2H, NHCH2CH2), 3.33 (t, J¼7.5 Hz, 2H, NHCH2CH2), 6.85 (d,
J¼7.2 Hz, 1H, 4-H), 7.35 (d, J¼8.1 Hz, 1H, 2-H), 7.45 (t, J¼8.1 Hz, 1H,

10452
C.M.A. Alves et al. / Tetrahedron 65 (2009) 10441–10452
9. Gorodetsky, A. A.; Hammond, W. J.; Hill, M. G.; Slowinski, K.; Barton, J. K.
Langmuir 2008, 24, 14282–14288.
(NHCH2CH2), 104.15 (C-4), 117.01 (C-5), 119.74 (C-2), 123.30 (C-4a),
124.48 (C-7), 125.55 (C-6), 126.62 (C-3) 128.60 (C-8), 134.28 (C-8a),
10. Chen, Z.-W.; Balamurugan, A.; Chen, S.-M. Bioelectrochemistry 2009, 75, 13–18.
11. Mitra, R. K.; Sinha, S. S.; Pal, S. K. J. Fluoresc. 2008, 18, 423–432.
12. Yang, X.-L.; Wang, A. H.-J. Pharmacol. Ther. 1999, 83, 181–215.
13. Mahadevan, S.; Palaniandavar, M. Inorg. Chim. Acta 1997, 254, 291–302.
14. Frade, V. H. J.; Gonçalves, M. S. T.; Moura, J. C. V. P. Tetrahedron Lett. 2005, 46,
4949–4952.
143.56 (C-1). IR (neat, cmꢀ1):
n
¼3395, 3050, 3009, 2923, 2852,
2730, 1623, 1590, 1583, 1524, 1463, 1408, 1378, 1344, 1283, 1269,
1254, 1222, 1131, 1120, 1098, 1082, 1054, 1039, 1027, 1015, 955, 946,
925, 890, 876, 850, 768. HRMS (EI): calcd for C24H37N [Mþ]:
339.2926; found: 339.2923.
15. Frade, V. H. J.; Gonçalves, M. S. T.; Moura, J. C. V. P. Tetrahedron Lett. 2006, 47,
8567–8570.
16. Frade, V. H. J.; Gonçalves, M. S. T.; Coutinho, P. J. G.; Moura, J. C. V. P. J. Photo-
chem. Photobiol., A 2007, 185, 220–230.
Acknowledgements
17. Frade, V. H. J.; Coutinho, P. J. G.; Moura, J. C. V. P.; Gonçalves, M. S. T. Tetrahedron
2007, 63, 1654–1663.
18. Frade, V. H. J.; Barros, S. A.; Moura, J. C. V. P.; Gonçalves, M. S. T. Tetrahedron Lett.
2007, 48, 3403–3407.
19. Frade, V. H. J.; Barros, S. A.; Moura, J. C. V. P.; Coutinho, P. J. G.; Gonçalves, M. S. T.
Tetrahedron 2007, 63, 12405–12418.
20. Frade, V. H. J.; Sousa, M. J.; Moura, J. C. V. P.; Gonçalves, M. S. T. Tetrahedron Lett.
2007, 48, 8347–8352.
˜
ˆ
Thanks are due to Fundaçao para a Ciencia e Tecnologia (Portu-
gal) for its financial support of a BPD to Sarala Naik (SFRH/BPD/
´
´
37840/2007) and Centro de Quımica and Centro de Fısica (Uni-
versidade do Minho). The NMR spectrometer Bruker Avance II 400 is
part of the National NMR Network and was acquired with funds
from FCT and FEDER.
21. Alves, C. M. A.; Naik, S.; Coutinho, P. J. G.; Gonçalves, M. S. T. Tetrahedron Lett.
2009, 50, 4470–4474.
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