Synthesis and photophysical properties of side-chain chlorinated benzo[a]phenoxazinium chlorides

Article abstract of DOI:10.1016/j.tet.2013.01.035

Novel water-soluble benzo[a]phenoxazinium chlorides mono- or disubstituted with 3-chloropropyl groups at the amine of position 9, and also at the hydroxyl function at position 2, were synthesized. These compounds possessing one, two or three chlorinated t

Full text of DOI:10.1016/j.tet.2013.01.035

Tetrahedron 69 (2013) 2451e2461
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Synthesis and photophysical properties of side-chain chlorinated
benzo[a]phenoxazinium chlorides
,
B. Rama Raju a b, A. Daniela G. Firmino b, A. Luísa S. Costa b, Paulo J. G. Coutinho a,
,
M. Sameiro T. Gonc¸ alves b
*
a Centre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
b Centre of Chemistry, University of Minho, Campus of 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:
Novel water-soluble benzo[a]phenoxazinium chlorides mono- or disubstituted with 3-chloropropyl
groups at the amine of position 9, and also at the hydroxyl function at position 2, were synthesized.
These compounds possessing one, two or three chlorinated terminals displayed fluorescence emission in
ethanol and water in the range 644e666 nm and 649e676 nm, respectively, with fluorescence quantum
yields from 0.26 to 0.38.
Received 5 November 2012
Received in revised form 7 January 2013
Accepted 9 January 2013
Available online 18 January 2013
It was found that this type of molecules is involved in acidebase equilibrium and tautomerization with
localized positive charge. Ab-initio ground and excited state DFT calculations were made for the various
possible tautomers of a model compound. Preliminary studies of the photophysical behaviour of these
compounds in AOT/cyclohexane reverse micelles with low water content (uo¼[Water]/[AOT]ꢀ5) showed
that they interact strongly with the head groups in the interface feeling the presence of an increased
amount of water. At very low uo values, AOT molecules promote the tautomerization process allowing it
to follow both in absorption and emission spectra.
Keywords:
Chlorinated compounds
Benzo[a]phenoxazines
Nile Blue derivatives
Fluorescent labels
AOT reverse micelles
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
been extensively used as a medium to study chemical and bi-
ological reactions.1 The entrapped water is heterogeneous in nature
The synthesis of novel fluorochromophores based on the oxa-
zine core, such as benzo[a]phenoxazines possessing substituents,
that originate interesting photophysical properties could be im-
portant for labelling purposes. This type of probes through its
oxazine core can report on local changes of physicoechemical
properties namely micropolarity, hydration level, local charge and
microviscositydvery important properties to understand the
structure, dynamics and functions of self-organized media, such as
micelles, microemulsions, liposomes, etc. The cationic nature of
these fluorophores is expected to locate themselves at interfaces
constituted by anionic surfactants allowing the study of their
structure and physicoechemical properties.
When small amounts of water are added to solutions of ap-
propriate surfactants in water immiscible solvents nanometer-
sized water pools surrounded by the polar end of the surfactants
are formed. These are called reversed micelles and the ratio of
water to surfactant concentration (u0) is an important parameter as
its value determines the average size of the resulting water pools in
the surfactant/organic solvent water in oil (w/o) microemulsion.
The water pool entrapped in reversed micelles/microemulsions has
and its properties gradually change as a function of u0 and depend
on the distance from the polar head layer.2
The behaviour of water molecules in the nanopool has been the
subject of extensive investigation.3e7 The use of techniques, such as
ultrafast infrared vibrational echo spectroscopy,3 infrared pump-
probe spectroscopy,4 NMR,5 calorimetry,6 neutron scattering7 and
molecular dynamics simulation,8 allowed the identification of
various types of water in reverse micelles. The water physically
trapped between surfactant head groups, i.e., water directly bound
(by H-bonding) to the head groups and relatively unperturbed bulk
like water at the centre of the nanopools. There are still some
controversy on the size (u0 values) of the reverse micelles at which
water behaviour in the core becomes similar to that of bulk wa-
ter9,10 with the limiting value in dispute being 0 >10.9
u
Due to various applications of reverse micelles, such as drug
delivery systems11 and as templates for the synthesis of semi-
conductor12 or metallic nanoparticles,13 they have been intensively
used in recent years. Previous studies of commercial benzo[a]
phenoxazinium perchlorate, Nile Blue, in reverse micelles mainly
showed varying amount of normal and deprotonated forms with
water content.14e16
Considering these facts and as part of our research interests in
* Corresponding author. Fax: þ351 253604382; e-mail address: msameiro@qui-
the synthesis and characterization of fluorescence probes,17 this
mica.uminho.pt (M.S.T. Gonc¸ alves).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.035

2452
B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
work describes the preparation of a new set of benzo[a]phenox-
azinium chlorides possessing chlorinated terminals at substituents
of the side-chain hydroxyl and/or the amino groups of positions 2-
and 9- of the polycyclic system. These terminals can offer the
possibility of covalent labelling of the analytes through nucleophilic
substitution, in addition to the inherent non-covalent capability of
benzo[a]phenoxazinium chlorides synthesized. The study of the
variation in photophysical behaviour, in homogeneous media and
in AOT reverse micelles was performed.
hydroxyl and amine groups as terminals of the aliphatic chains was
achieved by alkylation of naphthlen-1-amine with 1-
bromopropane, 3-bromopropan-1-ol and 3-bromopropan-1-
amine hydrobromide, respectively, in ethanol under reflux con-
ditions.17b,c After purification by silica gel column chromatography,
intermediates 3aed were isolated as solid and oils (3c) in moderate
to good yields, and spectroscopic data were in accordance with the
expected structures.
The reaction of 3-(3-chloropropylamino)phenol hydrochloride 1
or 3-(bis(3-chloropropyl)amino)phenol hydrochloride 2 with N- or
N- and O-alkylated 5-aminonaphthalen-2-ol and naphthalen-1-
amine derivatives 3aed in an acidic medium afforded the corre-
sponding benzo[a]phenoxazinium chloride 4aed or 5a,b, re-
spectively (Scheme 1). Thus, reaction between nitrosophenol 1 with
intermediates 3aed in ethanol, in the presence of concentrated
hydrochloric acid, and after silica gel column chromatography pu-
rification gave the benzo[a]phenoxazinium chlorides 4aed, pos-
sessing the amine of 9-position monosubstituted with the 3-
chloropropyl group. Similarly, the nitrosophenol 2 reacted with
precursors 3a and 3b, producing the cationic dyes 5a,b, but with the
9-position of the polycyclic system disubstituted with 3-
chloropropyl groups. All these compounds were obtained as blue
solids in moderate yields and were fully characterized by high res-
olution mass spectrometry, IR and NMR (1H and 13C) spectroscopy.
2. Results and discussion
2.1. Synthesis
The synthesis of benzo[a]phenoxazinium chlorides 4aed and
5a,b was initiated by the preparation of N- or N- and O-alkylated
derivatives of 3-aminophenol, naphthalen-1-amine and 5-
aminonaphthalen-2-ol. The choice of these precursors is essential,
since they determined the substituents present at 2-, 5- and 9-po-
sitions of the tetracyclic aromatic system of the target compounds.
The reaction of 3-aminophenol with 1-bromo-3-chloropropane
followed by silica gel column chromatography purification
afforded the mono- and di-N-alkylated derivatives, namely 3-
(3-chloropropylamino)phenol and 3-(bis(3-chloropropyl)amino)
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 4aed and 5a,b.
phenol, respectively. By nitrosation of these compounds with
sodium nitrite in hydrochloric acid solution the required 5-
(3-chloropropylamino)-2-nitrosophenol hydrochloride 1 and 5-
(bis(3-chloropropyl)amino)-2-nitrosophenol hydrochloride 2 were
obtained.18
In the IR spectra of benzo[a]phenoxazines 4 and 5 we high-
lighted the bands of the amine and hydroxyl groups
(3427e3184 cmꢂ1), as well as a strong band of the C]N bond
(1640e1588 cmꢂ1) due to the fused oxazine ring.
The 1H NMR spectra showed the methyl groups for the terminal
The synthesis of 6-(3-chloropropoxy)-N-propylnaphthalen-1-
amine 3a was carried out by the O-alkylation of 5-(propylamino)
naphthalen-2-ol with 1-bromo-3-chloropropane in the presence of
cesium carbonate by heating at 60 ꢁC in acetonitrile. The starting
reagent was obtained from the reaction of 5-aminonaphthalen-2-ol
with 1-bromopropane by refluxing in methanol, as previously
reported.17f
of the aliphatic chains as a triplet (d 1.12e1.14 ppm), the methylene
protons directly linked to the nitrogen atom at 5- and 9-positions
(NHCH2 or NCH2) as multiplets or triplets (in case of
NHCH2CH2CH2Cl or NCH2CH2CH2Cl) (
the adjacent methylene groups, NHCH2CH2 or NCH2CH2 as multi-
plets ( 1.80e2.30 ppm), and NHCH2CH2CH2 or NCH2CH2CH2, which
appeared as multiplets or triplets (4c, 5a) ( 3.54e3.85 ppm). The
d 3.54e4.26 ppm), as well as
d
d
Similarly, the synthesis of N-propylnaphthalen-1-amine 3b, 3-
(naphthalen-1-ylamino)propan-1-ol 3c and N1-(naphthalen-1-yl)
propane-1,3-diamine hydrobromide 3d, having the methyl,
remaining methylene protons attached to oxygen atom (com-
pounds 4a and 5a), namely OCH2 appeared as a triplet (4a) or
a multiplet (5a) (d 4.38e4.43 ppm), groups close to the same atom,

B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
2453
OCH2CH2, which appeared as multiplets (
OCH2CH2CH2, in the form of a triplet (4a) or a multiplets (5a) (
3.72e3.87 ppm). In addition to the signals corresponding to the
aliphatic N- and O-substituents in the heterocycle, the 13C NMR
spectra displayed the expected aromatic carbons, that we high-
d
2.30e2.41 ppm) and
authors in similar compounds17g,h (Table 1). Substitution at 2-
position seems to have the same effect even without di-alkylation
at 9-position (compound 5a). Also, noteworthy is the much lower
molar absorptivity of compound 4d. In ethanol media, this is ex-
plainable by a predominance of the basic form, which always shows
lower absorptivity (Table 1) especially for this compound. However,
in water where neither basic form nor H-aggregate formation is
prominent, other specific interaction arising from the NH2$HBr
group must be involved. Substitution at the 2-position shows
a slight decrease in the molar absorptivity in water and ethanol
media.
d
lighted C-8 (d 94.13e97.56 ppm), C-6 (d 94.18e97.47 ppm) and C-11
(d
133.57e135.0 ppm).
2.2. Photophysical studies
Electronic absorption and fluorescence spectra of solutions of
fluorophores 4aed and 5a,b in degassed absolute ethanol and
distilled water were measured. Summarized data of this study is
presented in Table 1. The fluorescence quantum yields, VF, were
evaluated using oxazine 1 in ethanol as standard (VF¼0.1119) at
575 nm or 470 nm excitation. Figs. 1 and 2 show absorption and
fluorescence spectra, at 470 nm excitation, of the compounds in
water (panel A), ethanol (panel B) and basic ethanol (addition of
a small amount of tetraethylammonium hydroxide, panel C).
Fluorescence data from Table 1 exhibit that the maximum
emission wavelength of the basic form is between 605 and 626 nm,
whereas for the acid form the maximum lies in the region
644e666 nm in ethanol and 649e676 nm in aqueous media. This
red shift is consistent with a
pep* type transition and was ob-
served previously.17g,j With the exception of compound 4d all
fluorescence quantum yields in water are between two and seven
times lower than in ethanol. Results from earlier studies17g,h show
Table 1
Photophysical data of compounds 4aed and 5a,b in ethanol, basic ethanol and water (C¼2ꢃ10ꢂ6 M)
Cpd
Ethanol
Basic ethanol
Water
labs (nm)
lem (nm)
D
labs (nm)
lem (nm)
D
labs (nm)
lem (nm)
D
ε (104 Mꢂ1 cmꢂ1
)
FF
ε (104 Mꢂ1 cmꢂ1
)
FF
ε (104 Mꢂ1 cmꢂ1
)
FF
a
(nm)
b
(nm)
a
(nm)
a
FF, Ac
4a
4b
4c
4d
5a
5b
627
12.6
663
0.34
0.40
646
0.35
0.43
647
0.37
0.41
644
0.26
0.29
665
0.27
0.33
666
0.38
0.44
36
26
37
31
35
39
505
6.5
614
0.011
109
115
118
120
120
126
606c
4.6
663
0.048
57
49
37
41
45
48
620
19.2
498
10
613
0.049
613
11.9
652
0.16
620
23.1
498
13.1
616
0.051
615
16
652
0.17
613/493
1.1/1.6
485
1.6
605
0.017
608
2.2
649
0.27
630
12.8
504
6.4
624
0.027
627
5.4
672
0.062
627
15.1
500
12.3
626
0.055
628
14.2
676
0.075
a
Excitation at 575 nm.
Excitation at 470 nm.
1% Variation between 598 and 624 nm.
b
c
As in previous studies of other family of benzo[a]phenox-
that changing mono to di-alkylation at the 9-amino position results
in a red shifted emission and also a decrease in fluorescence
quantum yield. In the case of compounds reported in this work, it is
possible to conclude that the existence of oxy-substituents at po-
sition 2 of the heterocycle originates the same red shift but without
a decrease in the fluorescence efficiency (compound 4a). However,
it seems that di-alkylation with chlorine terminated side chains in
the absence of substitution in 2-position also maintains the high
quantum yield (compound 5b). It is noteworthy that the reported
fluorescence quantum yields at 575 nm excitation are less than the
corresponding true cationic form quantum yield due to the residual
absorbance of light by the basic neutral form. An attempt to exclude
the basic form by acidifying ethanol with tetrafluoroacetic acid
(data not shown) was not successful as additional fluorescence
bands appeared near 540 nm. Assuming that in ethanol in presence
of the base (TEAH), only the deprotonated Bz form exists it is
possible to estimate the contribution of BzHþ form to the absorp-
tion spectra in neat ethanol. This fraction depends on the com-
pound and lies between 0.81 (compound 4b) and 0.91 (compound
azinium chlorides,17g,h the absorption spectra in ethanol media is
dominated by an acidic form (BzHþ) and a w100 nm blue shifted
neutral form (Bz), which is clearly observed in ethanol medium in
presence of base tetraethylammonium hydroxide (TEAH) (Fig. 1B
and C). Among the compounds without substitution at heterocycle
position 2, compound 4c has the higher fraction of acidic form. This
could be due to an intramolecular interaction between OH and the
N atom at position 5 as this is the main site of deprotonation as
shown by ab-initio calculations in a previous study.17i On the con-
trary compound 4d displayed a huge fraction of basic form even in
normal ethanol. This can be explained by the positive charge in
NHþ3 , which facilitates the exit of the proton in the 5-amino
position.
In aqueous media (Fig. 1A) the basic form is almost absent and
the spectra reveals a blue shoulder/enlargement especially for the
compounds substituted at the 2-position (4a and 5a). This has been
shown to be due to the formation of H-aggregates.17g It can also be
seen that the di-alkylation at 9-position (compounds 5a and 5b)
originates a red shift of absorbance as already reported by the
4d). In Table
1 the corresponding estimation of the true

2454
B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
A
B
C
1.0
0.5
4a
4b
4c
4d
5a
5b
0.0
1.5
4a
4b
4c
4d
5a
5b
1.0
0.5
0.0
1.0
4a
4b
4c
4d
5a
5b
0.5
0.0
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 1. Normalized absorption spectra of compounds 4aed and 5a,b in water (A), ethanol (B) and basic ethanol (C) (C¼2ꢃ10ꢂ6 M).
fluorescence quantum yield of the cationic BzHþ form in ethanol is
also shown (FF,Ac). Compound 4d shows a lower fluorescence
quantum yield and this must again be an effect of the positive
charge in NHþ3 that arises from the NH2$HBr substituent.
Fluorescence spectra at 470 nm excitation (Fig. 2) reveals the
emission of both acid and basic forms, but the latter is pre-
dominantly excited (Fig. 1). Although the basic form has far greater
absorption at 470 nm, the emission spectra is dominated by the
acidic form. This is accountable by the fact that the quantum yield is
10 times greater for the acidic form than the basic form (Table 1).
Again an additional band near 540 nm is clearly observed for some
of the compounds (4b, 4c and 4d) both in water and ethanol. This
band could arise from tautomerization equilibrium with localized
optimized geometries at the DFT-MPW1PW91 level of theory
with a 3-21Gþ* basis are shown for the tautomers of a model benzo
[a]phenoxazine without substitution at position 2 of the hetero-
cycle and with single methyl groups at both 5- and 9-amino posi-
tions. The calculations were done both in the presence and absence
of the chlorine counter-anion and were followed by a frequency
calculation at the same level of theory.
In a final step, excited state information was obtained by a TD-
DFT calculation with
a MPW1PW91 functional and a 6-
31Gþþ(d,p) basis. The results of the various ab-initio calculations
are shown in Table 2. From the optimized geometries in the absence
of counter-anion, it is seen that there is full positive charge de-
localization as the structure is completely planar (all N atoms in sp2
hybridization). The transfer of a proton from one N position to the
other (from structure in the left to that in the right in Scheme 2)
originates a decrease in the planarity of the N atoms in relation to
the heterocycle as one of those changes hybridization from nearly
sp2 to sp3. Interestingly, the chloride anion seems to allow a partial
recovery of the sp2 hybridization through the formation of CleHeN
hydrogen-bridge. This interaction is not observed when there is full
positive charge and a slight loss of resonance among the
system (Scheme 2).
p-electron
Tautomer A allows full positive charge delocalization. Tautomer B
has a positive charge localized in the 9-amino position, whereas
tautomer C has the positive charge localized in the 5-amino position.
To confirm this hypothesis ab-initio calculations were per-
formed using Gaussian 09 Rev A.02 software package.20 In Fig. 3

B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
2455
A
1.0
0.5
4a
4b
4c
4d
5a
5b
0.0
1.0
B
4a
4b
4c
4d
5a
5b
0.5
0.0
1.0
C
4a
4b
4c
4d
5a
5b
0.5
0.0
500
550
600
650
700
750
Wavelength (nm)
Fig. 2. Normalized fluorescence spectra of compounds 4aed and 5a,b in water (A), ethanol (B) and basic ethanol (C) at 470 nm excitation (C¼2ꢃ10ꢂ6 M).
positive charge delocalization (tautomer A) and the chlorine anion
locates at 2.48 A from the heterocycle and in closer distance to one
tautomer C at w538 nm. Thus, it is possible to get some more
confidence on the assignment of the 540 nm emission band to
a tautomer form of the BzHþClꢂ molecule.
Fig. 4 shows the absorption spectra of compounds 5b and 4d in
AOT/cyclohexane reverse micelles. An absorption band is clearly
seen at w460 nm, which decreases as the water content (uo pa-
rameter) increases.
This variation is in correspondence with a decrease in a fluo-
rescence band near 550 nm (Fig. 5). It is then possible to conclude
that the tautomerization process is occurring in this microemulsion
media and its importance decreases with an increase in the water
content. Further, the absorption and emission maxima of the band
corresponding to the tautomer A (BzHþ with fully delocalized
positive charge) is seen shifted to lower energies with the increase
in uo but does not reach the values observed in aqueous media
(Table 1). The fluorescence quantum yield at 575 nm excitation is
much higher when uo¼0 and decreases as the water content in-
creases. All the studied quantities (see the insets of Figs. 4 and 5)
ꢀ
of the methyl groups that slightly bends itself towards chlorine.
From Table 2, it is possible to conclude that tautomers B and C are
higher in energy than tautomer A when chloride anion is absent.
However, this change when the counter-anion is close to the Bz
heterocycle with tautomers B having less energy than tautomer A.
Tautomer C is slightly more energetic but the excess energy is lower
than the thermal energy at 25 ꢁC (3.72 kcal molꢂ1).
Thus, in situations where ion-pairing or, presumably, associa-
tion with proton accepting molecules is possible, the tautomeri-
zation process can occur. The latter possibility certainly can happen
in the studied ethanolic or aqueous media.
The excited state calculations were done in vacuum and for the
ground state geometry. As result, the tabulated HOMOeLUMO gaps
should correspond to absorption maximum. Assuming a Stokes’s
shift halfway between the acidic and basic form (w75 nm), it is
possible to estimate that tautomers B emit at w511 nm and

2456
B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
Scheme 2. Tautomerization equilibrium of benzo[a]phenoxazinium chlorides.
Fig. 3. Optimized ground-state geometries of tautomers A, B and C of a model benzo[a]phenoxazine in the absence or presence of chloride counter-anion. In B1 and B2 the chloride
anion was initially placed in different sides of the heterocycle.

B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
2457
Table 2
Ab-initio calculation with structures in Fig. 3
DFT calculations (MPW1PW91 functional)
Without chlorine counter-anion
With chlorine counter-anion
A
B
C
A
B1
B2
C
ꢀ
Cl distance to nearest N or C atom (A)
Cl distance to H atom (A)
Dihedral angle Cle1e2e3 (ꢁ)
d
d
d
2.48
d
2.90
1.41
ꢂ112.1
0.310559
ꢂ1395.052
ꢂ1.102
2.90
1.41
111.5
0.311528
ꢂ1395.051
ꢂ0.456
2.83
1.61
7.04
0.310021
ꢂ1395.048
0.929
ꢀ
d
d
d
d
d
d
ꢂ89.1
0.312896
ꢂ1395.052
d
Zero point Vibrational energy (Hartree)
Ground state energy (Hartree)
Molecular energy relative to tautomer A
0.309904
ꢂ934.663
d
0.309226
ꢂ934.594
179
0.308607
ꢂ934.590
188
(kcal molꢂ1
)
HOMOeLUMO gap (nm)
494.2
453.4
471.7
593.8
438.2
434.67
463.12
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.3
610
600
590
x10 0.3
A
B
x10
620
610
0.2
0.1
0.0
0.2
0.1
0.0
o
o
0
2
4
0
2
o
4
o
o
o
400
500
600
700
800
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 4. Absorbance spectra of compound 5b (A) and 4d (B), C¼4ꢃ10ꢂ6 M, in AOT/cyclohexane microemulsions as a function of water content (uo) with PEtOH¼0.34. The inset shows
the absorbance at 575 nm (open squares, right axis) and the maximum absorption wavelength of the red side band (filled squares, left axis).
practically stabilizes at uo¼3. Previous studies placed the uo limit
above which water in reverse micelles behaves as bulk water be-
tween 7 and 10.9,21
This indicates that the studied compounds are seeing differ-
ences in the hydration shell of the AOT layer in the reverse micelles.
The maximum fluorescence quantum yield reaches 0.67 for both
compounds and it should correspond to a close association be-
tween BzHþ and the negative charged group of the AOT surfactant
with diminishing water content. For compound 5b, the quantum
yield stabilizes at a value lower than that observed in ethanol
670
0.7
0.6
0.5
0.4
0.3
650
640
630
0.7
0.6
0.5
0.4
0.3
B
A
o
o
1.0
660
0.8
650
0.6
4
0
2
4
0
2
o
o
0.4
0.2
0.0
500
600
700
800
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 5. Normalized fluorescence spectra of compound 5b (A) and 4d (B), C¼4ꢃ10ꢂ6 M, in AOT/cyclohexane microemulsions as a function of water content (uo) with PEtOH¼0.34. The
inset shows the fluorescence quantum yield at 575 nm excitation (open squares, right axis) and the maximum emission wavelength (filled squares, left axis).

2458
B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
whereas the reverse situation occurs for compound 4d. This must
be due to the NH2$HBr substituent that favours the location of the
compound close to the AOT head groups even in high water
content.
water content. AOT molecules promote the tautomerization pro-
cess with the presence of water molecules having the reverse effect.
Association with AOT molecules originates a higher fluorescence
quantum yield of the molecular form with delocalized cationic
charge. It was also concluded that small traces of ethanol affect the
properties of the reverse micelle interface to a point that instead of
tautomerization the usual deprotonation process occurred.
Solutions of compounds 4d and 5b in AOT/cyclohexane micro-
emulsions contained small amounts of ethanol. This small polar
molecule can either localize on the micelle interface working as
a co-surfactant, or be on the water pool. In order to better follow
the effect of the presence of ethanol molecules on the AOT w/o
microemulsion an equivalent parameter to uo is defined:
PEtOH¼[EtOH]/[AOT], and varied maintaining uo fixed at 0 or 2
(Fig. 6). It can be seen that up to PEtOH¼0.34 a slight increase of
tautomerization is observed. However, when ethanol content is 10
times higher the predominant phenomena is the formation of the
basic Bz neutral form (broad emission band near 600 nm), and the
fluorescence quantum yield of the remaining acid form markedly
decreases for both compounds when uo¼0, but only for compound
4d when uo¼2. This appears to indicate that the stronger associa-
tion with AOT head groups prompted by the NH2$HBr side-chain
substituent is perturbed by the presence of ethanol molecules in
the reverse micelle interface.
4. Experimental
4.1. General
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualized under UV light. Chromatography on silica gel
was carried out on Merck Kieselgel (230e240 mesh). IR spectra
were determined on a BOMEM MB 104 spectrophotometer. NMR
spectra were obtained on a Varian Unity Plus Spectrometer at an
operating frequency of 300 MHz for 1H and 75.4 MHz for 13C or
a Bruker Avance III 400 at an operating frequency of 400 MHz for 1H
400
0.20
450
500
550
600
650
400
B
450
500
550
600
650
700
A
0.15
0.10
0.05
x5
0.00
1.0
C
D
0.6
0.6
0.5
0.4
0.3
0.8
0.6
0.4
0.2
0.0
0.4
0.2
0
1
2
3
0
1
2
3
PEtOH
PEtOH
500
600
700
800
500
600
700
800
Wavelength (nm)
Fig. 6. Absorbance and normalized fluorescence spectra at 470 nm excitation of compound 5b (A, C) and 4d (B, D), C¼4ꢃ10ꢂ6 M in AOT/cyclohexane microemulsions at uo¼0 (black
lines) and uo¼2 (grey lines) with various ethanol content: PEtOH¼0 (thin line), PEtOH¼0.34 (thick line) and PEtOH¼3.4 (dashed line). The inset shows the fluorescence quantum yields
at 575 nm both for uo¼0 (black squares) and uo¼2 (grey squares).
3. Conclusion
and 100.6 MHz for 13C using the solvent peak as internal reference
at 25 ꢁC. All chemical shifts are given in parts per million using dH
Me4Si¼0 ppm as 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 decoupling-
double resonance and bidimensional heteronuclear correlation
techniques. Low and high resolution mass spectrometry analyses
were performed at the ‘C.A.C.T.I. e Unidad de Espectrometria de
Masas’, at University of Vigo, Spain.
Water-soluble fluorescent benzo[a]phenoxazinium chlorides
with one, two or three chlorine atoms as terminals of their sub-
stituents at 2- and 9-positions were obtained. In addition, two of
these cationic fluorophores possess the hydroxyl or amine func-
tions as terminals in the aliphatic chains of position 5, providing
them extra potential for covalent labelling beyond their inherent
capabilities for non-covalent labelling of (bio)molecules. These
molecules were shown to be involved in tautomerization equilib-
rium leading to localization of the positive charge at one of the N
atoms in 5- or 9-positions with chloride counter-anion (or other
proton accepting molecule) making an H-bond that allows partial
recovery of the molecule’s resonance possibilities. The use of these
molecules as fluorescence probes in AOT reverse micelles allowed
following changes in the hydration shell of the surfactant layer with
UVevis absorption spectra (200e800 nm) were obtained using
Shimadzu UV/3101PC Spectrophotometers and fluorescence spec-
tra with Spex Fluorolog spectrofluorometer. Commercially available
reagents were used as received. Reverse micelles in cyclohexane
were prepared by injection of a needed amount of water into
a cyclohexane 0.2 M solution of AOT (dioctyl sodium sulfosucci-
nate) into which a given volume of either 1ꢃ10ꢂ4 M or 1ꢃ10ꢂ3
M

B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
2459
ethanolic solution of the studied compounds was added in order to
obtain a final concentration of 4ꢃ10ꢂ6 M. For preparations without
ethanol, the required volume of ethanolic mother solution was
evaporated with a stream of nitrogen prior to the addition of AOT
solution in cyclohexane.
4.2.2.3. 6-(3-Chloropropoxy)-N-propylnaphthalen-1-amine
3a. To a solution of 5-(propylamino) naphthalen-2-ol17f (0.070 g,
3.48ꢃ10ꢂ4 mol) in acetonitrile (2 mL), 1-bromo-3-chloropropane
(0.038 mL, 3.83ꢃ10ꢂ4 mol) and cesium carbonate (0.554 g,
1.70ꢃ10ꢂ3 mol) were added, and the resulting mixture was heated at
60 ꢁC for 1.5 h. The progress of the reaction was monitored by TLC
(ethyl acetate/light petroleum 1:5). The excess of base was filtered out,
the solvent was evaporated and the crude mixture was purified by
column chromatography on silica gel using ethyl acetate/light petro-
leum 1:5, as the eluent. Compound 3a was obtained as a light brown
solid (0.09 g, 93%). Mp¼97.1e100.2 ꢁC. TLC (ethyl acetate/light petro-
leum 1:5): Rf¼0.67. 1H NMR (DMSO-d6, 400 MHz): dH¼0.96 (t,
J¼7.6 Hz, 3H, NHCH2CH2CH3), 1.64e1.76 (m, 2H, NHCH2CH2CH3),
2.17e2.28 (m, 2H, OCH2CH2CH2Cl), 3.15 (t, J¼7.6 Hz, 2H,
NHCH2CH2CH3), 3.82 (t, J¼6.4 Hz, 2H, OCH2CH2CH2Cl), 4.18 (t,
J¼6.0 Hz, 2H, OCH2CH2CH2Cl), 6.48 (broad s, 1H, H-2), 7.00e7.13 (m,
2H, H-4 and H-7), 7.20 (d, J¼2.4 Hz,1H, H-5), 7.24 (t, J¼8.0 Hz,1H, H-3),
8.09 (d, J¼9.2 Hz, 1H, H-8). 13C NMR (DMSO-d6, 100.6 MHz): dC¼11.67
(NHCH2CH2CH3), 21.20 (NHCH2CH2CH3), 31.73 (OCH2CH2CH2Cl), 42.04
(OCH2CH2CH2Cl), 45.92 (NHCH2CH2CH3), 64.19 (OCH2CH2CH2Cl),
103.00 (C-2), 107.36 (C-5), 116.00 (C-4), 116.21 (C-7), 118.52 (C-8a),
123.48 (C-8), 127.34 (C-3), 135.57 (C-4a), 143.06 (C-1), 156.14 (C-6). IR
4.2. Synthesis of precursors 1e3
4.2.1. 5-((3-Chloropropyl)amino)-2-nitrosophenol
hydrochloride
1. To an ice-cold solution of 3-(3-chloropropylamino)phenol
(0.155 g, 8.37ꢃ10ꢂ4 mol) in ethanol (1 mL), concentrated hydro-
chloric acid (0.28 mL) was added and stirred for 5 min. The solution
of sodium nitrite (0.058 g, 9.20ꢃ10ꢂ4 mol) in water (0.1 mL) was
then added drop-wise within an interval of 20e25 min. The
resulting mixture was stirred for 3 h and monitored by TLC
(dichloromethane/methanol, 1:9 and 2:8). After evaporation of the
reaction, compound 1 was obtained as an orange oily solid (0.180 g)
and was used in the following step without any purification.
4.2.2. 5-(Bis(3-chloropropyl)amino)-2-nitrosophenol hydrochloride
2. Starting from 3-(bis(3-chloropropyl)amino)phenol (0.100 g,
3.81ꢃ10ꢂ4 mol) in ethanol (1 mL) and concentrated hydrochloric
acid (0.105 mL), using sodium nitrite (0.03 g, 4.19ꢃ10ꢂ4 mol) in
water (0.1 mL), and following the same procedure as described
before for the preparation of 1, compound 2 was obtained as an
orange oily solid (0.113 g) and was used in the following step
without any purification.
(KBr 1%, cmꢂ1):
n¼3405, 2959, 2926, 2872, 1623, 1587, 1529, 1476,
1460,1432,1378,1341,1284,1267,1224,1181,1151,1070,1047, 974, 912,
865, 837, 818, 779, 743. HRMS: m/z (EI): calcd for C16H20NO35Cl [Mþ]
277.1233; found 277.1235; calcd for C16H20NO37Cl [Mþ] 279.1204;
found 279.1208.
4.3. General procedure for preparation of compounds 4 and 5
4.2.2.1. 3-(3-Chloropropylamino)phenol. To
a solution of 3-
aminophenol (1.4 g, 1.28ꢃ10ꢂ2 mol) in ethanol (2 mL), 1-bromo-
3-chloropropane (1.27 mL, 1.28ꢃ10ꢂ2 mol) was added, and the
resulting mixture was refluxed for 5 h. The progress of reaction was
monitored by TLC (dichloromethane/methanol, 9.5:0.5). After
completion of the reaction, the solvent was evaporated and the
mixture was purified by column chromatography on silica using
dichloromethane/methanol, mixtures of increasing polarity, as the
eluent. The expected product was obtained as brown oil (0.688 g,
29%). TLC (dichloromethane/methanol, 9.5:0.5): Rf¼0.54. 1H NMR
(CDCl3, 400 MHz): dH¼2.00e2.10 (m, 2H, NHCH2CH2CH2Cl), 3.31 (t,
J¼6.4 Hz, 2H, NHCH2CH2CH2Cl), 3.64 (t, J¼6.3 Hz, 2H,
NHCH2CH2CH2Cl), 4.25 (broad s, 2H, NH and OH), 6.14 (t, J¼2.1 Hz,
1H, 2-H), 6.18e6.26 (m, 2H, 4-H and 6-H), 7.03 (t, J¼8.0 Hz,1H, 5-H).
13C NMR (CDCl3, 100.6 MHz): dC¼31.77 (NHCH2CH2CH2Cl), 41.02
(NHCH2CH2CH2Cl), 42.57 (NHCH2CH2CH2Cl), 99.86 (C-2), 104.81 (C-
6), 106.06 (C-4), 130.29 (C-5), 149.25 (C-3), 156.73 (C-1). IR (KBr 1%,
To a cold solution (ice bath) of 5-((3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 1 (2 equiv) or 5-(bis(3-chloropropyl)
amino)-2-nitrosophenol hydrochloride 2 (2 equiv), in ethanol
(2e3 mL), precursors 3aed (1 equiv) and concentrated hydro-
chloride acid (0.25 equiv) were added. The mixture was refluxed
during the time mentioned below, and monitored by TLC. After
evaporation of the solvent and column chromatography purifica-
tion on silica gel with dichloromethane and dichloromethane/
methanol, mixtures of different polarity, as the eluent, the required
dye 4 or 5 was obtained as a blue solid.
4.3.1. 3-Chloro-N-(2-(3-chloropropoxy)-5-(propylamino)-9H-benzo
[a]phenoxazin-9-ylidene)propan-1-aminium chloride 4a. The prod-
uct of the reaction of 1 (0.055 g, 2.2ꢃ10ꢂ4 mol) in ethanol (3 mL)
and concentrated hydrochloric acid (2.26ꢃ10ꢂ3 mL) with 6-(3-
chloropropoxy)-N-propylnaphthalen-1-amine
3a
(0.031
g,
cmꢂ1):
n
¼3198, 2947, 2587, 1615, 1505, 1488, 1470, 1372, 1316, 1284,
1.1ꢃ10ꢂ4 mol) (reflux time 1 h), was chromatographed with
dichloromethane and dichloromethane/methanol 8.5:1.5, to give
compound 4a (0.039 g, 37%). Mp¼128.3e130.8 ꢁC. Rf¼0.33
(dichloromethane/methanol, 9:1). 1H NMR (CD3OD, 400 MHz):
dH¼1.14 (t, J¼7.2 Hz, 3H, NHCH2CH2CH3), 1.85e1.95 (m, 2H,
NHCH2CH2CH3), 2.10e2.24 (m, 2H, NHCH2CH2CH2Cl), 2.30e2.41
(m, 2H, OCH2CH2CH2Cl), 3.64e3.80 (m, 4H, NHCH2CH2CH2Cl and
NHCH2CH2CH3), 3.87 (t, J¼6.4 Hz, 2H, OCH2CH2CH2Cl), 4.26 (t,
J¼6.8 Hz, 2H, NHCH2CH2CH2Cl), 4.38 (t, J¼6.0 Hz, 2H,
OCH2CH2CH2Cl), 6.70 (d, J¼2.0 Hz, 1H, H-8), 6.90 (s, 1H, H-6), 7.11
(dd, J¼7.8 Hz and 1.6 Hz, 1H, H-3), 7.32e7.38 (m, 1H, H-10), 7.75 (d,
J¼9.2 Hz, 1H, H-11), 8.18 (d, J¼2.4 Hz, 1H, H-1), 8.24 (d, J¼9.2 Hz,
1H, H-4). 13C NMR (CD3OD, 100.6 MHz): dC¼11.73 (NHCH2CH2CH3),
23.31 (NHCH2CH2CH3), 31.21 (NHCH2CH2CH2Cl), 33.19 (OCH2-
CH2CH2Cl), 41.59 (NHCH2CH2CH2Cl), 42.21 (OCH2CH2CH2Cl), 43.17
(NHCH2CH2CH2Cl), 47.53 (NHCH2CH2CH3), 66.47 (OCH2CH2CH2Cl),
94.18 (C-6), 97.39 (C-8), 108.00 (C-1), 115.60 (C-3), 118.50 (CeAr),
120.31 (C-10),126.33 (C-4), 130.31 (CeAr),133.79 (CeAr),134.96 (C-
11), 136.42 (CeAr), 149.16 (CeAr), 153.63 (CeAr), 155.16 (C-9),
1233,1172,1084,1020,1000, 859, 828, 780, 735, 693, 655. HRMS: m/
z (EI): calcd for C9H1235ClNO [Mþ] 185.0607; found 185.0602. Calcd
for C9H1237ClNO [Mþ] 187.0578; found 187.0585.
4.2.2.2. 3-(Bis(3-chloropropyl)amino)phenol. In the above re-
action, this compound was also isolated as brown oil (0.222 g, 7%).
TLC (dichloromethane/methanol, 9.5:0.5): Rf¼0.71. 1H NMR (CDCl3,
400 MHz): dH¼1.93e1.99 (m, 4H, N(CH2CH2CH2Cl)2), 3.38 (t,
J¼7.2 Hz, 4H, N(CH2CH2CH2Cl)2), 3.49 (t, J¼6.0 Hz, 4H,
N(CH2CH2CH2Cl)2), 5.40 (s, 1H, OH), 6.14e6.26 (m, 3H, 2-H, 6-H and
4-H), 7.00 (t, J¼8.4 Hz, 1H, 5-H). 13C NMR (CDCl3, 100.6 MHz):
dC¼29.79 (N(CH2CH2CH2Cl)2), 42.94 (N(CH2CH2CH2Cl)2), 49.59
(N(CH2CH2CH2Cl)2), 100.01 (C-2), 102.58 (C-6), 105.53 (C-4), 130.34
(C-5), 148.68 (C-3), 156.81 (C-1). IR (KBr 1%, cmꢂ1):
n¼3189, 2956,
2653, 2552, 1615, 1578, 1505, 1488, 1464, 1451, 1360, 1316, 1288,
1235, 1169, 1117, 1082, 1055, 1011, 998, 945, 928, 879, 828, 785, 733,
698. HRMS: m/z (EI): calcd for C12H1735Cl2NO [Mþ] 261.0687;
found 261.0689. C12H1735Cl37ClNO [Mþ] 263.0658; found
263.0659. Calcd for C12H1737Cl2NO [Mþ] 265.0628; found 265.0617.
159.93 (C-5), 163.33 (C-2). IR (KBr 1%, cmꢂ1):
n
¼3389, 2924, 2853,

2460
B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
1640, 1590, 1548, 1461, 1419, 1331, 1281, 1222, 1151, 1127, 1036, 909,
815, 781, 717, 666. HRMS: m/z (ESI): calcd for C25H2835Cl2N3O2
[Mþþ1] 472.15531; found 472.15354. C25H2835Cl37ClN3O2 [Mþþ1]
474.15252; found 474.15016. Calcd for C25H2837Cl2N3O2 [Mþþ1]
476.14951; found 476.15235.
(0.202 g, 37%). TLC (dichloromethane/methanol, 9:1): Rf¼0.15. 1H
NMR
(CD3OD,
400
MHz):
dH¼2.11e2.20
(m,
2H,
NHCH2CH2CH2NH2$HBr), 2.21e2.30 (m, 2H, NHCH2CH2CH2Cl), 3.13
(t, J¼7.6 Hz, 2H, NHCH2CH2CH2NH2$HBr), 3.60e6.69 (m, 2H,
NHCH2CH2CH2NH2$HBr), 3.72 (t, J¼6.4 Hz, 2H, NHCH2CH2CH2Cl),
3.77 (t, J¼6.0 Hz, 2H, NHCH2CH2CH2Cl), 6.96 (s, 1H, 8-H), 7.15 (s, 1H,
6-H), 7.85e7.93 (m, 4H, 10-H, 11-H, 3-H and 2-H), 8.51 (d, J¼7.2 Hz,
1H, 4-H), 8.80 (d, J¼6.4 Hz, 1H, 1-H). 13C NMR (CD3OD, 100.6 MHz):
dC¼27.67 (NHCH2CH2CH2NH2$HBr), 29.86 (NHCH2CH2CH2Cl), 38.39
(NHCH2CH2CH2NH2$HBr), 42.20 (NHCH2CH2CH2NH2$HBr), 42.97
(NHCH2CH2CH2Cl), 43.28 (NHCH2CH2CH2Cl), 95.53 (C-6), 96.17 (C-
8), 122.46 (C-4), 124.69 (C-10), 125.91 (C-1), 130.16 (C-3), 131.76
(AreC), 131.88 (C-2), 132.15 (AreC), 133.57 (C-11), 137.36 (AreC),
148.34 (AreC), 152.04 (AreC), 153.60 (C-9), 160.54 (C-5). IR (KBr 1%,
4.3.2. 3-Chloro-N-(5-(propylamino)-9H-benzo[a]phenoxazin-9-
ylidene)propan-1-aminium chloride 4b. The product of the reaction
of 1 (0.139 g, 6.49ꢃ10ꢂ4 mol) in ethanol (2 mL) and concentrated
hydrochloric acid (0.170 mL), with N-propylnaphthalen-1-amine 3b
(0.060 g, 3.24ꢃ10ꢂ4 mol)(refluxtime 7 h)waschromatographed with
dichloromethane and dichloromethane/methanol, mixtures of in-
creasing polarity, as the eluent, to give compound 4b (0.083 g, 31%).
Mp¼184.9e185.3 ꢁC. TLC (dichloromethane/methanol, 9.5:0.5):
Rf¼0.20. 1H NMR (CD3OD, 400 MHz): dH¼1.13 (t, J¼7.6 Hz, 3H,
NHCH2CH2CH3),1.89e1.99 (m, 2 H, NHCH2CH2CH3), 2.16e2.25(m, 2H,
NHCH2CH2CH2Cl), 3.59 (t, J¼6.8 Hz, 2H, NHCH2CH2CH2Cl), 3.70e3.82
(m, 4H, NHCH2CH2CH3 and NHCH2CH2CH2Cl), 6.86 (d, J¼2.4 Hz,1H, 8-
H), 7.04 (s,1H, 6-H), 7.12 (d, J¼9.2 Hz,1H,10-H), 7.82e7.87 (m, 2H,11-
H, 3-H), 7.96 (t, J¼8.0 Hz,1H, 2-H), 8.38 (d, J¼8.4 Hz,1H, 4-H), 8.95 (d,
J¼8.0 Hz, 1H, 1-H). 13C NMR (CD3OD, 100.6 MHz): dC¼11.70
(NHCH2CH2CH3), 23.02 (NHCH2CH2CH3), 32.63 (NHCH2CH2CH2Cl),
cmꢂ1):
n
¼3396, 3210, 2924, 2853,1638,1587,1545,1493,1459,1436,
1324, 1277, 1260, 1152, 1123, 1008, 826, 770, 704, 665. HRMS: m/z
(ESI): calcd for C22H2435ClN4O [Mþþ1] 395.16332; found 395.16332.
Calcd for C22H2437ClN4O [Mþþ1] 397.16037; found 397.16082.
4.3.5. 3-Chloro-N-(2-(3-chloropropoxy)-5-(propylamino)-9H-benzo
[a]phenoxazin-9-ylidene)-N-(3-chloropropyl)propan-1-aminium
chloride 5a. The product of the reaction of 2 (0.14 g, 4.32ꢃ10ꢂ4 mol)
in ethanol (3 mL) and concentrated hydrochloric acid (4.44ꢃ
10ꢂ3 mL), with 6-(3-chloropropoxy)-N-propylnaphthalen-1-amine
3a (0.060 g, 2.16ꢃ10ꢂ4 mol) (reflux time 7 h), was chromato-
graphed with dichloromethane and dichloromethane/methanol
9.5:0.5, to give compound 5a (0.071 g, 30%). Mp¼97.8e99.1 ꢁC. TLC
(dichloromethane/methanol, 9:1): Rf¼0.52. 1H NMR (CD3OD,
400 MHz): dH¼1.12 (t, J¼7.6 Hz, 3H, NHCH2CH2CH3), 1.80e1.93 (m,
2H, NHCH2CH2CH3), 2.10e2.30 (m, 4H, N(CH2CH2CH2Cl)2),
2.31e2.40 (m, 2H, OCH2CH2CH2Cl), 3.60e3.70 (m, 2H,
NHCH2CH2CH3), 3.71 (t, J¼6.0 Hz, 6H, N(CH2CH2CH2Cl)2), 3.72e3.82
(2H, m, OCH2CH2CH2Cl), 3.87 (t, J¼6.0 Hz, 4H, N(CH2CH2CH2Cl)2),
4.38e4.43 (m, 2H, OCH2CH2CH2Cl), 6.82 (s, 1H, H-8), 6.86 (d,
J¼2.4 Hz, 1H, H-6), 7.15e7.28 (m, 1H, H-3), 7.29 (dd, J¼9.0 and
2.4 Hz, 1H, H-10), 7.75 (d, J¼9.6 Hz, 1H, H-11), 8.11 (d, J¼2.8 Hz, 1H,
H-1), 8.22 (d, J¼9.2 Hz, 1H, H-4). 13C NMR (CD3OD, 100.6 MHz):
41.63
(NHCH2CH2CH2Cl),
42.96
(NHCH2CH2CH2Cl),
47.44
(NHCH2CH2CH3), 94.50 (C-6), 96.21 (C-8),123.81 (C-10), 124.28 (C-4),
125.0 (AreC),125.76 (C-1),131.10 (C-3),131.96 (AreC),132.79 (AreC),
132.08 (C-2), 134.05 (C-11), 135.39 (AreC), 150.34 (AreC), 153.52
(AreC), 157.97 (C-9), 159.76 (C-5). IR (KBr 1%, cmꢂ1):
n¼3206, 2962,
2930, 2873,1640,1588,1547,1494,1455,1433,1384,1322,1278,1237,
1185,1159,1123,1012,1000, 976, 855, 827, 774. HRMS: m/z (ESI): calcd
for C22H2335ClN3O [Mþþ1] 380.15242; found 380.15218. Calcd for
C22H2337ClN3O [Mþþ1] 382.14945; found 382.14947.
4.3.3. 3-Chloro-N-(5-(3-hydroxypropylamino)-9H-benzo[a]phenox-
azin-9-ylidene)propan-1-aminium chloride 4c. The product of the
reaction of 1 (0.180 g, 8.4ꢃ10ꢂ5 mol) in ethanol (2 mL) and concen-
trated hydrochloric acid (0.236 mL), with 3-(naphthalen-1-ylamino)
propan-1-ol 3c (0.084 g; 4.2ꢃ10ꢂ5 mol) (reflux time 3.5 h) was
chromatographed with dichloromethane, dichloromethane/metha-
nol, mixtures of increasing polarityasthe eluent, to give compound 4c
(0.151 g, 42%). Mp¼228.0e228.9 ꢁC. TLC (dichloromethane/methanol,
9:1): Rf¼0.33. 1H NMR (CD3OD, 400 MHz): dH¼2.0e2.14 (m, 2H,
NHCH2CH2CH2OH), 2.15e2.25 (m, 2H, NHCH2CH2CH2Cl), 3.54 (t,
J¼6.8 Hz, 2H, NHCH2CH2CH2Cl), 3.75e3.85 (m, 6H, NHCH2CH2CH2OH,
NHCH2CH2CH2OH and NHCH2CH2CH2Cl), 6.68 (s,1H, 8-H), 6.87 (s,1H,
6-H), 7.05 (d, J¼9.2 Hz, 1H, 10-H), 7.66 (d, J¼9.2 Hz, 1H, 11-H), 7.77 (t,
J¼7.6 Hz,1H, 3-H), 7.86 (t, J¼8.0 Hz,1H, 2-H), 8.22 (d, J¼8.4 Hz,1H, 4-
H), 8.72 (d, J¼8.0 Hz,1H,1-H).13C NMR (CD3OD,100.6 MHz): dC¼32.14
(NHCH2CH2CH2OH), 32.61 (NCH2CH2CH2Cl), 41.72 (NHCH2CH2CH2Cl),
43.06 (NHCH2CH2CH2OH), 43.40 (NHCH2CH2CH2Cl), 60.40
(NHCH2CH2CH2OH), 94.29 (C-6), 95.95 (C-8), 123.76 (C-4 and C-10),
124.67 (AreC), 125.52 (C-1), 131.0 (C-3), 131.79 (AreC), 132.33 (C-2),
132.95 (AreC), 133.87 (C-11), 134.81 (AreC), 149.95 (AreC), 152.95
dC¼11.74
(NCH2CH2CH2Cl), 31.18 (NCH2CH2CH2Cl), 33.18 (OCH2CH2CH2Cl),
42.18 (N(CH2CH2CH2Cl)2), 42.27 (OCH2CH2CH2Cl), 43.28
(NHCH2CH2CH3),
23.33
(NHCH2CH2CH3),
30.20
(N(CH2CH2CH2Cl)2), 47.50 (NHCH2CH2CH3), 66.45 (OCH2CH2CH2Cl),
94.13 (C-8), 97.47 (C-6), 107.83 (C-1), 115.71 (C-3), 118.43 (CeAr),
120.22 (C-10), 126.39 (C-4), 130.27 (CeAr), 133.79 (C-11), 134.81
(CeAr), 136.22 (CeAr), 149.13 (CeAr), 153.50 (CeAr), 155.39 (C-9),
159.78 (C-5), 163.22 (C-2). IR (KBr 1%, cmꢂ1):
n
¼3427, 2958, 2926,
1640, 1591, 1548, 1514, 1485, 1461, 1420, 1384, 1332, 1300, 1278,
1222, 1155, 1130, 1039, 909, 820, 665 cmꢂ1. HRMS: m/z (ESI): calcd
for C28H3335Cl3N3O2 [Mþþ1] 548.16329; found 548.16515. Calcd
for C28H3335Cl237ClN3O2 [Mþþ1] 550.16049; found 550.16361.
Calcd for C28H3335Cl37Cl2N3O [Mþþ1] 552.15753; found 552.16035.
Calcd for C28H3337Cl3N3O2 [Mþþ1] 554.15448; found 554.16155.
(AreC), 157.88 (C-9), 159.26 (C-5). IR (KBr 1%, cmꢂ1):
n¼3207, 3084,
4.3.6. 3-Chloro-N-(3-chloropropyl)-N-(5-(propylamino)-9H-benzo
[a]phenoxazin-9-ylidene) propan-1-aminium chloride 5b. The
product of the reaction of 2 (0.113 g, 3.88ꢃ10ꢂ4 mol) in ethanol
(1.5 mL) and concentrated hydrochloric acid (0.105 mL), with 3b
(0.036 g, 1.94ꢃ10ꢂ4 mol) (reflux time 9 h) was chromatographed
with dichloromethane and dichloromethane/methanol, mixtures
of increasing polarity, as the eluent, to give compound 5b (0.074 g,
39%). Mp¼244.7e246.1 ꢁC. TLC (dichloromethane/methanol,
9.5:0.5): Rf¼0.32. 1H NMR (CD3OD, 400 MHz): dH¼1.14 (t, J¼7.2 Hz,
3H, NHCH2CH2CH3), 1.86e1.98 (m, 2H, NHCH2CH2CH3), 2.22e2.30
(m, 4H, N(CH2CH2CH2Cl)2), 3.74e3.80 (m, 6H, N(CH2CH2CH2Cl)2
and NCH2CH2CH3), 3.85 (t, J¼7.2 Hz, 4H, N(CH2CH2CH2Cl)2), 7.02 (s,
1H, 8-H), 7.08 (s, 1H, 6-H), 7.33 (d, J¼8.8 Hz, 1H, 10-H), 7.87 (t,
J¼7.6 Hz, 1H, 3-H), 7.90e8.0 (m, 2H, 11-H and 2-H), 8.40 (d,
2940, 1641, 1592, 1549,1524,1498,1455,1435,1372, 1328,1999,1279,
1256, 1279, 1237, 1219, 1198, 1188, 1162, 1129, 1089, 1129, 1055, 1005,
951, 923, 871, 838, 779, 744, 706, 665. HRMS: m/z (ESI): calcd for
C22H2335ClN3O2 [Mþþ1] 396.14733; found 396.14756. Calcd for
C22H2337ClN3O2 [Mþþ1] 398.14438; found 398.14473.
4.3.4. N-(5-((3-Aminopropyl)amino)-9H-benzo[a]phenoxazin-9-
ylidene)-3-chloropropan-1-aminium chloride 4d. The product of the
reaction of 1 (0.232 g, 1.07ꢃ10ꢂ3 mol) in ethanol (2 mL) and con-
centrated hydrochloric acid (0.287 mL), with N1-(naphthalen-1-yl)
propane-1,3-diamine hydrobromide 3d (0.151 g, 5.39ꢃ10ꢂ4 mol)
(reflux time 9 h) was chromatographed with dichloromethane,
dichloromethane/methanol, as the eluent, to give compound 4d

B.R. Raju et al. / Tetrahedron 69 (2013) 2451e2461
2461
J¼8.0 Hz, 1H, 4-H), 8.96 (d, J¼8.0 Hz, 1H, 1-H). 13C NMR (CD3OD,
100.6 MHz): dC¼11.70 (NHCH2CH2CH3), 23.10 (NHCH2CH2CH3),
31.15 (N(CH2CH2CH2Cl)2), 43.14 (N(CH2CH2CH2Cl)2), 47.64
(NHCH2CH2CH3), 50.04 (N(CH2CH2CH2Cl)2), 95.01 (C-6), 97.56 (C-
8), 116.11 (C-10), 123.98 (C-4), 125.20 (AreC), 125.93 (C-1), 131.11
(AreC), 131.39 (C-3), 133.31 (C-2), 133.97 (AreC), 135.00 (C-11),
136.70 (AreC), 149.42 (AreC), 153.74 (AreC), 155.58 (C-9), 160.32
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(C-5). IR (KBr 1%, cmꢂ1):
n
¼3185, 2963,1640,1589,1546,1495,1460,
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Acknowledgements
ˇ
~
Thanks are due to the Fundac¸ ao para a Ciencia e Tecnologia (FCT,
Portugal) for financial support to the NMR portuguese network
(PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (Eu-
ropean Fund for Regional Development)-COMPETE-QREN-EU for
financial support to the Research Centres CFUM [PEst-C/FIS/
UI0607/2011 (F-COMP-01-0124-FEDER-022711)] and CQ/UM [PEst-
C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)].
A post-
doctoral grant to B.R.R. (SFRH/BPD/62881/2009) is also acknowl-
edged to FCT, POPH-QREN, FSE.
18. Crossley, M. L.; Turner, R. J.; Hofmann, C. M.; Dreisbach, P. F.; Parker, R. P. J. Am.
Chem. Soc. 1952, 74, 578e584.
19. Sens, R.; Drexhage, K. H. J. Lumin. 1981, 24, 709e712.
Supplementary data
20. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.;
Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.;
Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers,
E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J.
M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.;
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.01.035.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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