Synthesis, photophysical characterisation and photostability studies of NIR probes with aliphatic, aromatic and chlorinated terminals in 5- and 9-amino positions of benzo[a]phenoxazines

Article abstract of DOI:10.1016/j.dyepig.2016.04.049

A new series of benzo[a]phenoxazinium chlorides possessing mono and disubstituted amines with 3-chloropropyl groups at the 9-position, isopropyl, cyclohexyl and phenyl groups as terminals at 5-postion was synthesised. Photophysical studies were performed

Full text of DOI:10.1016/j.dyepig.2016.04.049

Dyes and Pigments 132 (2016) 204e212
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, photophysical characterisation and photostability studies of
NIR probes with aliphatic, aromatic and chlorinated terminals in 5-
and 9-amino positions of benzo[a]phenoxazines
,
b
a
a
b
B. Rama Raju a , Marcello M.T. Carvalho , Maria Ines P.S. Leitao , Paulo J.G. Coutinho ,
^
~
M. Sameiro T. Gonçalves a
,
*
a Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
b Centre of Physics, 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:
A new series of benzo[a]phenoxazinium chlorides possessing mono and disubstituted amines with 3-
chloropropyl groups at the 9-position, isopropyl, cyclohexyl and phenyl groups as terminals at 5-
postion was synthesised. Photophysical studies were performed in dry ethanol and aqueous solutions.
The terminals at the 5-amino position were found to influence the acid-base equilibrium. The presence of
hydroxyl functionality at 2-position was found to introduce an additional basic form that is the one in
equilibrium with the cationic acid form in dry ethanol solutions. The photostability of these compounds
in different media was also investigated and a high resistance to photobleaching in model biological
membranes was observed. In proteins a moderate of 20% photobleaching occurs in 1 h 30 min, while in
water more than 60% of the compound molecules are photodegraded during the same time interval.
© 2016 Elsevier Ltd. All rights reserved.
Received 15 March 2016
Received in revised form
28 April 2016
Accepted 29 April 2016
Available online 30 April 2016
Keywords:
Nile blue
Benzo[a]phenoxazines
Near-infrared fluorescent probes
Photostability
Fluorochromophores
1. Introduction
with substituted phenyl rings to the phenoxazinium skeleton [10].
Benzo[a]phenoxazinium chlorides can also function as covalent
Synthetic fluorescent labels are indispensable tools for quanti-
tative detection in various fields of modern sciences [1]. These are
employed in several separation techniques, and are quite remark-
able for their fluorescence detection even to the single molecular
level as in case of laser-assisted fluorometry [2]. Fluorescence im-
aging in longer wavelength region (600e1000 nm) is particularly
appealing as biomolecules display less background fluorescence
and deeper penetration into the substances [3e6]. Several fluo-
rescent dyes reported in the literature suffer from serious draw-
backs such as their cumbersome synthesis, limited availability and
biological interference [7]. Among the polycyclic oxazines, benzo[a]
phenoxazinium salts, such as Nile Blue (NB) derivatives are
attractive fluorophores due to their sensitivity, photostable nature,
high fluorescence quantum yield and in general favourable pho-
tophysical properties in the near-infrared spectral region [8]. These
compounds are used as fungicide agents [9] and exhibit potent
antiprotozoal activities, being even quite effective when attached
probes for organic and biological molecules, namely amino acids,
proteins, peptides and DNA, as well as in the non-covalent labelling
of nucleic acids for use in monitoring changes in protein confor-
mation [11,12], labelling of lysosomes during cell division and
apoptosis [13] and imaging tumors that over-express cyclo-
oxygenase-2 [14].
Considering the importance and as a continuation of our current
research interest towards the design, synthesis and application of
NB derivatives with different substituents [15e25], the present
work is focused in the preparation of a new set of benzo[a]phe-
noxazinium chlorides with aliphatic, aromatic and chlorinated
terminals in 5- and 9-amino positions of the heteroaromatic sys-
tem. The photophysical behaviour of these compounds in anhy-
drous ethanol and in aqueous solutions was investigated along
with photostability studies in water and in biological medium.
These measurements are important for evaluating the suitability of
the synthesised compounds as biological markers in imaging
applications.
* Corresponding author.
E-mail address: msameiro@quimica.uminho.pt (M.S.T. Gonçalves).
http://dx.doi.org/10.1016/j.dyepig.2016.04.049
0143-7208/© 2016 Elsevier Ltd. All rights reserved.

B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
205
2. Experimental section
different polarity, as the eluent, the expected benzo[a]phenox-
azines 4aed and 5 were isolated as blue solids in moderate yields.
2.1. Material and instruments
2.3.1. 3-Chloro-N-(2-hydroxy-5-(isopentylamino)-9H-benzo[a]
phenoxazin-9-ylidene)propan-1-aminium chloride 4a
The product of the reaction of 5-((3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 1 (0.158 g, 0.629 mmol) in ethanol
(1 mL) and concentrated hydrochloric acid (0.017 mL) with 5-
(isopentylamino)naphthalen-2-ol 3a (0.086 g; 0.377 mmol) (reflux
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analysis was carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualised 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. UVeVis
absorption spectra (200e800 nm) were obtained using Shimadzu
UV/3101PC spectrophotometer and fluorescence spectra with Spex
Fluorolog spectrofluorometer. 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 and 100.6 MHz for 13C
using the solvent peak as internal reference at 25 ꢀC. All chemical
shifts are given in ppm using dH Me4Si ¼ 0 ppm as reference and J
values are given in Hz. Assignments were made by comparison of
chemical shifts, peak multiplicities and J values and were supported
by spin decoupling-double resonance and bidimensional hetero-
nuclear correlation techniques. Low and high resolution mass
spectrometry analysis were performed at the “C.A.C.T.I. e Unidad
de Espectrometria de Masas”, at University of Vigo, Spain. Photo-
stability studies were carried out using a xenon arc lamp (OSRAM
HBO 200 W) equipped with a bandpass filter centered 600 10 nm
(ThorLabs, New Jersey, USA). Commercially available reagents were
used as received.
time
3 h) was chromatographed with dichloromethane and
dichloromethane/methanol 9.0:1.0 to give compound 4a as a blue
solid (0.166 g, 49%). mp 200e202 ꢀC. Rf ¼ 0.50 (dichloromethane/
methanol, 9:1). IR (KBr 1%): nmax 3400, 3200, 2955, 2925, 2854,
1588, 1546, 1464, 1324, 1270, 1221, 1155, 1126, 1035, 817, 720 cmꢁ1
.
1H NMR (CD3OD, 400 MHz): dH 1.09 (d, J ¼ 6.4 Hz, 6 H,
NHCH2CH2CH(CH3)2), 1.70e1.85 (m, 4 H, NHCH2CH2CH(CH3)2 and
NHCH2CH2CH(CH3)2), 2.18 (quint, J ¼ 6.4 Hz, 2 H, NHCH2CH2CH2Cl),
3.51 (t, J ¼ 6.8 Hz, 2 H, NHCH2CH2CH(CH3)2), 3.60e3.72 (m, 2 H,
NHCH2CH2CH2Cl), 3.75 (t, J ¼ 6.4 Hz, 2 H, NHCH2CH2CH2Cl), 6.64 (s,
1 H, 6-H), 6.67 (d, J ¼ 1.6 Hz, 1 H, 8-H), 6.99 (d, J ¼ 9.2 Hz, 1 H, 10-H),
7.17 (dd, J ¼ 8.8 and 2.0 Hz, 1 H, 3-H), 7.63 (d, J ¼ 8.8 Hz, 1 H, 11-H),
8.0 (t, J ¼ 3.2 Hz,1 H,1-H), 8.11 (d, J ¼ 8.8 Hz,1 H, 4-H) ppm. 13C NMR
(CD3OD, 101.6 MHz): dC 22.89 (NHCH2CH2CH(CH3)2), 23.12
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH2Cl),
(NHCH2CH2CH(CH3)2),
27.45
38.57
43.06
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH2Cl),
32.62
41.60
44.21
(NHCH2CH2CH2Cl), 93.27 (C-6), 95.85 (C-8), 110.03 (C-1), 117.17 (C-
10), 117.29 (AreC), 119.51 (AreC), 120.12 (C-3), 126.32 (C-4), 130.69
(C-11), 135.07 (2 ꢂ AreC), 135.47 (AreC), 153.28 (C-9), 157.38
(AreC), 159.45 (C-5), 162.77 (AreC) ppm. HRMS: m/z (ESI): Found
[M]þ: 424.17803; C24H27ClN3O2 requires [M]þ: 424.17863.
2.2. Synthetic method for the preparation of precursor 3a
2.2.1. 5-(Isopentylamino)naphthalen-2-ol 3a
To a solution of 5-aminonaphthalen-2-ol (0.477 g, 3.0 mmol) in
ethanol (2 mL), 1-bromo-3-methylbutane (0.644 g, 3.3 mmol) 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 evapo-
rated and the mixture was purified by column chromatography on
silica gel using mixture of dichloromethane and methanol (9.5:0.5)
as the eluent. Compound 3a was obtained as colourless solid
(0.528 g, 77%). mp 111e113 ꢀC. Rf ¼ 0.55 (dichloromethane/meth-
anol 9.5:0.5). IR (KBr 1%): nmax ¼ 3353, 3062, 2956, 2927, 2869,
1623, 1581, 1531, 1469, 1407, 1368, 1339, 1270, 1219, 1171, 1151, 1027,
996, 957, 862, 813, 795, 772, 743 cmꢁ1. 1H NMR (CD3OD, 400 MHz):
dH 1.01 (d, J ¼ 6.4 Hz, 6H, NHCH2CH2CH(CH3)2), 1.70 (m, 2H,
NHCH2CH2CH(CH3)2), 1.76e1.91 (m, 1H, NHCH2CH2CH(CH3)2), 3.28
(t, J ¼ 7.6 Hz, 2H, NHCH2CH2CH(CH3)2), 6.52 (d, J ¼ 7.2 Hz, 1H, 6-H),
7.0e7.06 (m, 2H, 8-H, 3-H), 7.07 (d, J ¼ 2.6 Hz, 1H, 1-H), 7.32 (t,
J ¼ 7.6 Hz, 1H, 7-H), 7.71 (d, J ¼ 9.2 Hz, 1H, 4-H) ppm. 13C NMR
(CD3OD, 101.6 MHz): dC 22.64 (NHCH2CH2CH(CH3)2), 26.19
2.3.2. 3-Chloro-N-(5-(isopentylamino)-9H-benzo[a]phenoxazin-9-
ylidene)propan-1-aminium chloride 4b
The product of the reaction of 5-((3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 1 (0.114 g, 0.454 mmol) in ethanol
(1 mL) and concentrated hydrochloric acid (0.012 mL) with N-iso-
pentylnaphthalen-1-amine 3b (0.058 g; 0.60 mmol) (reflux time
8 h) was chromatographed with dichloromethane and dichloro-
methane/methanol 9.0:1.0 to give compound 4b as a blue solid
(0.148 g, 66%). mp 168e170 ꢀC. Rf ¼ 0.58 (dichloromethane/meth-
anol, 9.5:0.5). IR (KBr 1%): nmax 3440, 2958, 1641, 1590, 1548, 1497,
1454, 1433, 1321, 1282, 1186, 1158, 1122, 999, 773 cmꢁ1 1H NMR
.
(CD3OD, 400 MHz): dH 1.08 (d, J ¼ 6.4 Hz, 6 H, NHCH2CH2CH(CH3)2),
1.76e1.89 (m, 3 H, NHCH2CH2CH(CH3)2 and NHCH2CH2CH(CH3)2),
2.17e2.26 (m, 2 H, NHCH2CH2CH2Cl), 3.61 (t, J ¼ 6.8 Hz, 2 H,
NHCH2CH2CH(CH3)2), 3.74e3.83 (m, 4 H, NHCH2CH2CH2Cl and
NHCH2CH2CH2Cl), 6.90 (s, 1 H, 8-H), 7.03 (s, 1 H, 6-H), 7.15 (d,
J ¼ 8.4 Hz, 1 H, 10-H), 7.82e7.92 (m, 2 H, 11-H and 3-H), 7.97 (t,
J ¼ 7.6 Hz, 1 H, 2-H), 8.39 (d, J ¼ 8.4 Hz, 1 H, 4-H), 8.98 (d, J ¼ 7.6 Hz,
(NHCH2CH2CH(CH3)2),
38.41
(NHCH2CH2CH(CH3)2),
42.47
1
H, 1-H) ppm. 13C NMR (CD3OD, 100.6 MHz): dC 22.82
(NHCH2CH2CH(CH3)2), 102.78 (C-6), 110.51 (C-1), 116.01 (C-8 and C-
3), 118.74 (C-4a), 121.85 (C-4), 127.49 (C-7), 135.70 (C-8a), 143.73 (C-
5), 153.32 (C-2) ppm.
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH(CH3)2),
22.91
32.63
41.64
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH2Cl),
(NHCH2CH2CH(CH3)2),
27.42
38.35
42.95
(NHCH2CH2CH2Cl), 44.30 (NHCH2CH2CH2Cl), 92.39 (C-6), 95.86 (C-
8), 123.84 (C-4), 125.03 (C-10), 125.76 (C-1), 131.11 (C-3), 132.02
(AreC), 132.79 (2 ꢂ AreC), 133.09 (C-2), 134.02 (C-11), 135.39
(2 ꢂ AreC), 153.53 (C-9), 158.0 (AreC), 159.63 (C-5) ppm. HRMS: m/
z (ESI): Found [M]þ: 408.18308; C24H27ClN3O requires [M]þ:
408.18372.
2.3. General procedure for the synthesis of benzo[a]phenoxazines
4aed and 5
To a cold solution (ice bath) of nitrosophenols 1 or 2 (1.5 mmol)
in ethanol (2 mL), 5-aminonaphthalen-2-ol or naphthalen-1-amine
derivatives 3aed (1.0 mmol) and concentrated hydrochloride acid
(40
m
L) were added. The mixture was refluxed during the time
2.3.3. 3-Chloro-N-(5-((2-cyclohexylethyl)amino)-9H-benzo[a]
phenoxazin-9-ylidene)propan-1-aminium chloride 4c
The product of the reaction of 5-((3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 1 (0.136 g, 0.541 mmol) in ethanol
mentioned below, and monitored by TLC. After evaporation of the
solvent and column chromatography purification on silica gel with
dichloromethane and dichloromethane/methanol, mixtures of

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B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
(1 mL) and concentrated hydrochloric acid (0.013 mL) with N-(2-
cyclohexylethyl)naphthalen-1-amine 3c (0.082 g; 0.325 mmol)
(reflux time 3 h) was chromatographed with dichloromethane and
dichloromethane/methanol 9.0:1.0 to give compound 4c as a blue
solid (0.118 g, 52%). mp 248e250 ꢀC. Rf ¼ 0.43 (dichloromethane/
methanol, 9:1). IR (KBr 1%): nmax 3444, 2926, 1640, 1588,1548,1493,
1448, 1431, 1317, 1268, 1202, 1157, 1122, 998, 819, 773 cmꢁ1. 1H NMR
dH (CD3OD, 400 MHz), 1.0e1.60 (3 ꢂ m, 9H, CH Cy), 1.62e1.80 (m,
2H, NHCH2CH2C6H11), 1.81e1.90 (m, 2H, CH Cy), 2.10e2.25 (m, 2H,
NHCH2CH2CH2Cl), 3.40e3.60 (m, 2H, NHCH2CH2CH2Cl), 3.62e3.85
(m, 4H, NHCH2CH2CH2Cl and NHCH2CH2C6H11), 6.82 (d, J ¼ 2.0 Hz,
1 H, 8-H), 6.92 (s, 1 H, 6-H), 7.11 (dd, J ¼ 9.0 and 1.6 Hz, 1 H, 10-H),
7.77e7.86 (m, 2H, 11-H and 3-H), 7.93 (t, J ¼ 8.0 Hz, 1 H, 2-H), 8.34
(d, J ¼ 8.0 Hz,1 H, 4-H), 8.90 (d, J ¼ 8.0 Hz,1 H,1-H) ppm. 13C NMR dC
(CD3OD, 100.6 MHz), 27.33 (CH Cy), 27.41 (CH Cy), 27.55 (CH2 Cy),
32.61 (CH2 Cy), 33.08 (NHCH2CH2CH2Cl), 34.31 (CH2 Cy), 36.98
(NHCH2CH2C6H11 and CH Cy), 41.68 (NHCH2CH2CH2Cl), 43.00
(NHCH2CH2CH2Cl), 43.88 (NHCH2CH2C6H11), 94.34 (C-6), 95.60 (C-
8), 123.84 (AreC), 124.25 (AreC), 124.92 (C-4), 125.67 (C-1), 126.10
(AreC), 131.07 (C-3), 131.83 (AreC), 131.91 (AreC), 132.62 (C-2),
133.04 (AreC), 133.69 (AreC), 133.96 (C-11), 135.20 (AreC), 153.29
(AreC), 157.93 (C-9), 159.40 (C-5) ppm. HRMS: m/z (ESI): Found
[M]þ: 448.21449; C27H31ClN3O requires [M]þ: 448.21502.
1.75e1.90 (m, 3 H, NHCH2CH2CH(CH3)2 and NHCH2CH2CH(CH3)2),
2.19e2.30 (m, H, N(CH2CH2CH2Cl)2), 3.73e3.90 (m, 10 H,
4
NHCH2CH2CH(CH3)2, N(CH2CH2CH2Cl)2) and N(CH2CH2CH2Cl)2,
7.04 (s, 2 H, 8-H and 6-H), 7.34 (d, J ¼ 8.8 Hz, 1 H, 10-H), 7.87 (t,
J ¼ 7.2 Hz, 1 H, 3-H), 7.92e8.02 (m, 2 H, 2-H and 11-H), 8.40 (d,
J ¼ 8.0 Hz, 1 H, 1-H), 8.96 (d, J ¼ 8.0 Hz, 1 H, 4-H) ppm. 13C NMR
(CD3OD, 100.6 MHz): dC 22.82 (NHCH2CH2CH(CH3)2), 27.41
(NHCH2CH2CH(CH3)2),
(NHCH2CH2CH(CH3)2),
31.36
43.15
(N(CH2CH2CH2Cl)2),
(NHCH2CH2CH(CH3)2),
38.39
44.49
(N(CH2CH2CH2Cl)2), 44.30 (N(CH2CH2CH2Cl)2), 94.88 (C-6), 97.60
(C-8), 116.20 (C-10), 124.01 (C-1), 125.11 (AreC), 125.91 (C-4), 131.17
(AreC), 131.40 (C-3), 132.71 (AreC), 133.34 (C-2), 133.99 (C-11),
136.60 (AreC), 149.44 (AreC), 153.76 (C-9), 155.62 (AreC), 160.11
(C-5) ppm. HRMS: m/z (ESI): Found [M]þ: 484.19154; C27H32Cl2N3O
requires [M]þ: 484.19169.
2.4. Typical procedure for the photostability studies with benzo[a]
phenoxazinium chlorides 4b and 5
Solutions of the compounds 4b and 5 (C ¼ 1 ꢂ 10ꢁ5 M) was
prepared in ethanol, water, buffered solutions of BSA and mem-
branes (soya lecithin) in a 5 mL flask. Then a volume of 2 mL was
pipetted into a quartz cuvette and placed directly in the path of
incident light with an irradiance of 10 mWcmꢁ2 (Xenon arc lamp,
OSRAM HBO 200 W) equipped with a band pass filter centered on
2.3.4. 3-Chloro-N-(5-(phenethylamino)-9H-benzo[a]phenoxazin-
9-ylidene)propan-1-aminium 4d
600
10 nm (ThorLabs, New Jersey, USA). The absorbance was
The product of the reaction of 5-((3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 1 (0.116 g, 0.462 mmol) in ethanol
(1 mL) and concentrated hydrochloric acid (0.011 mL) with N-
phenethylnaphthalen-1-amine 3d (0.068 g; 0.277 mmol) (reflux
recorded from the initial (t ¼ 0 min) to the maximum (t ¼ 110 min)
irradiation with various time intervals in between.
3. Results and discussion
time
3 h) was chromatographed with dichloromethane and
dichloromethane/methanol 9.0:1.0 to give compound 4c as a blue
solid (0.092 g, 40%). mp 220e222 ꢀC. Rf ¼ 0.52 (dichloromethane/
methanol, 9:1). IR (KBr 1%): nmax 3420, 2925, 2855,1639,1583,1543,
1492, 1450, 1432, 1313, 1287, 1259, 1172, 1154, 1124, 994, 828, 773,
3.1. Synthetic methods
The synthesis of benzo[a]phenoxazinium chlorides 4aed and 5
was initiated with the preparation of N-alkylated derivatives of 3-
aminophenol, 5-aminonaphthalen-2-ol and naphthalen-1-amine.
The reaction of 3-aminophenol with 1-bromo-3-chloropropane
followed by silica gel column chromatography purification affor-
ded 3-(3-chloropropylamino)phenol and 3-(bis(3-chloropropyl)
amino)phenol, respectively. The nitrosation of these compounds
was carried out with sodium nitrite in hydrochloric acidic solution
to obtain 5-(3-chloropropylamino)-2-nitrosophenol hydrochloride
1 and 5-(bis(3-chloropropyl)amino)-2-nitrosophenol hydrochlo-
ride 2 [15].
738 cmꢁ1 1H NMR dH (CD3OD, 400 MHz), 1.36 (m,
. 2 H,
NHCH2CH2CH2Cl), 3.17 (t, J ¼ 7.2 Hz, 2 H, NHCH2CH2C6H5), 3.58 (t,
J ¼ 6.8 Hz, 2 H, NHCH2CH2C6H5), 3.76 (t, J ¼ 6.4 Hz, 2 H,
NHCH2CH2CH2Cl), 3.99 (t, J ¼ 7.2 Hz, 2 H, NHCH2CH2CH2Cl), 6.82 (s,
1 H, H-8), 6.90 (s, 1 H, 6-H), 7.12 (dd, J ¼ 9.2 and 2.0 Hz, 1 H, 10-H),
7.20e7.24 (m, 1 H, AreH), 7.28e7.36 (m, 4 H, 4 ꢂ AreH), 7.79e7.89
(m, 2 H, 11-H and 3-H), 7.92 (t, J ¼ 7.6 Hz, 1 H, 2-H), 8.29 (d,
J ¼ 8.0 Hz, 1 H, 4-H), 8.90 (d, J ¼ 7.2 Hz, 1 H, 1-H) ppm. 13C NMR dC
(CD3OD,
(NHCH2CH2C6H5),
100.6
MHz),
41.68
32.61
(NHCH2CH2CH2Cl),
(NHCH2CH2C6H5),
35.86
42.97
The 5-(isopentylamino)naphthalen-2-ol 3a was obtained by the
N-alkylation reaction of 5-aminonaphthalen-2-ol with 1-bromo-3-
methylbutane in ethanol under reflux conditions. Similarly, the
(NH2CH2CH2CH2Cl), 47.12 (NHCH2CH2CH2Cl), 94.53 (C-6), 95.53 (C-
8), 123.75 (C-4), 124.86 (C-10), 125.68 (C-1), 129.82 (AreC), 129.86
(AreC), 130.09 (2 ꢂ AreC), 130.77 (AreC), 131.05 (C-3), 132.08
(AreC), 132.40 (1 ꢂ AreC), 132.65 (AreC), 133.03 (C-2), 133.59
(AreC), 134.04 (C-11), 135.10 (AreC), 139.39 (AreC), 153.20 (C-9),
158.03 (AreC), 159.51 (C-5) ppm. HRMS: m/z (ESI): Found [M]þ:
442.16734; C27H25ClN3O requires [M]þ: 442.16807.
precursors
N-isopentyl-naphthalen-1-amine
3b,
N-(2-
cyclohexylethyl)naphthalen-1-amine 3c and N-(phenethyl)naph-
thalen-1-amine 3d were synthesised by the reaction of naph-
thalen-1-amine with 1-bromo-3-methylbutane, (2-bromoethyl)
cyclohexane and (2-bromoethyl)benzene in ethanol following the
procedure described [16]. After purification by silica gel column
chromatography, intermediates 3aed were isolated and spectro-
scopic 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-
alkylated derivatives of 5-aminonaphthalen-2-ol and naphthalen-
1-amine 3aed in an acidic medium afforded the corresponding
benzo[a]phenoxazinium chlorides 4aed and 5, respectively
(Scheme 1). Thus, reaction of nitrosophenol 1 with intermediates
3aed in ethanol, in the presence of concentrated hydrochloric acid,
and after silica gel column chromatography purification gave the
benzo[a]phenoxazinium chlorides 4aed, possessing the isopropyl,
cyclohexyl and phenyl terminals at 5-position, monosubstituted
2.3.5. 3-Chloro-N-(3-chloropropyl)-N-(5-(isopentylamino)-9H-
benzo[a]phenoxazin-9-ylidene) propan-1-aminium chloride 5
The product of the reaction of 5-(bis(3-chloropropyl)amino)-2-
nitrosophenol hydrochloride 2 (0.113 g, 0.345 mmol) in ethanol
(1 mL) and concentrated hydrochloric acid (0.008 mL) with N-iso-
pentylnaphthalen-1-amine 3b (0.044 g, 0.60 mmol) (reflux time
3 h) was chromatographed with dichloromethane and dichloro-
methane/methanol 9.0:1.0 to give compound 5 as a blue solid
(0.133 g, 58%). mp 221e223 ꢀC. Rf ¼ 0.14 (dichloromethane/meth-
anol, 9.5:0.5). FTIR (KBr 1%): nmax 2956, 1639, 1587, 1546, 1491, 1456,
1434, 1330, 1279, 1220, 1178, 1159, 1123, 999, 775 cmꢁ1
.
1H NMR
(CD3OD, 400 MHz): dH 1.08 (d, J ¼ 6.0 Hz, 6 H, NHCH2CH2CH(CH3)2),

B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
207
Scheme 1. Synthesis of benzo[a]phenoxazinium chlorides 4aed and 5.
with the 3-chloropropyl group at the amine of 9-position. Similarly,
the nitrosophenol 2 reacted with precursors 3a, producing the
cationic dye 5, but with the amino of 9-position of the polycyclic
system disubstituted with 3-chloropropyl groups. All these com-
pounds were obtained as blue solids in moderate yields and were
fully characterised by high resolution mass spectrometry, IR and
NMR (1H and 13C) spectroscopy (Table 1).
excitation, of the compounds in water (panels A and B), ethanol
(panels C and D) and basified (addition of a small amount of tet-
raethylammonium hydroxide) or acidified (addition of a small
amount of trifluoroacetic acid) ethanol (panels E and F).
Previous studies of benzo[a]phenoxazinium chloride derivatives
[27,28] showed that the absorption spectra in ethanol media is
composed of two contributions. The acidic form (BzHþ) and other,
that is the neutral form (Bz), which is ~100 nm blue shifted. This is
clearly seen in Fig. 1C and from the distinct behaviour in basified or
acidified ethanol (Fig. 1E).
3.2. Physical studies
In ethanol media, compound 5 which is di-alkylated at 9-amino
position, shows a higher fraction of basic form. Globally, when
comparing with similar compounds without a bulky group at the
terminal of 5-amino position [15], it is possible to conclude that the
bulky groups used in this study, significantly increase the fraction of
basic form. The absorption spectra of compound 4a, possessing OH
functionality at position 2, shows a very distinct behaviour. It can be
seen that in basified ethanol the usual neutral form appears
Electronic absorption and fluorescence spectra of solutions of
fluorophores 4aed and 5 in absolute ethanol and water were
measured at various concentrations. Summarised data of this study
is presented in Table 1.
The fluorescence quantum yields FF were evaluated using oxa-
zine 1 in ethanol as standard (FF ¼ 0.11) [26] at 575 nm or 470 nm
excitation.
Fig. 1 shows absorption and fluorescence spectra, at 470 nm
Table 1
Yields and photophysical data of compounds 4aed and 5 in dry ethanol, acidified ethanol with TFA, basified ethanol with TEAH and in aqueous solution (C ¼ 1 ꢂ 10ꢁ5 M).
Compound
Yield (%)
4a
4b
4c
4d
5
66
49
52
40
58
Solvent
Ethanol
labs (nm)
616
2.5
644//55
0.11
28
620
0.8
645//50
0.36
25
621
1.1
645//50
0.38
24
625
1.6
645//52
0.45
20
630
0.4
666//56
0.40
36
ε (104 Mꢁ1 cmꢁ1
lem//fwhm (nm)
)
a
a
FF
D
(nm) a
Ethanol acidified with TFA
Ethanol basified with TEAH
Water
labs//fwhm (nm)
ε (104 Mꢁ1 cmꢁ1
lem//fwhm (nm)
617//75
5.2
650//69
0.38
33
482//94
2.6
622
0.0004
140
604//166
1.03
650//57
0.089
46
619//72
3.7
650//61
0.46
31
493//96
1.7
611//97
0.021
118
600//116
1.48
653//56
0.10
53
620//69
4.9
650//62
0.47
30
493//92
2.1
610//98
0.027
117
612//106
1.43
653//58
0.16
41
623//66
5.2
650//64
0.45
27
497//91
2.5
611//100
0.027
114
618//94
1.99
656//60
0.18
38
628//74
4.3
667//64
0.44
39
499//88
1.9
611//110
0.019
112
631//95
1.64
677//62
0.098
46
)
a
a
FF
D
(nm) a
labs//fwhm (nm)
ε (104 Mꢁ1 cmꢁ1
lem//fwhm (nm)
)
b
b
FF
D
(nm) b
labs//fwhm (nm)
ε (104 Mꢁ1 cmꢁ1
lem//fwhm (nm)
)
a
a
FF
D
(nm) a
a
Emission spectra obtained with excitation at 575 nm
Emission spectra obtained with excitation at 470 nm.
b

208
B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
Fig. 1. Normalised absorption/emission spectra of compounds 4aed and 5 (C ¼ 1 ꢂ 10ꢁ5 M) in water (A/B), ethanol (C/D) and either acidified or basified ethanol with respectively
TFA or TEAH (E/F).
(Fig. 1E) with a slight blue shift whereas in normal ethanol what
would be the contribution from that form appears deviated more
than 50 nm in comparison with the other compounds (Fig. 1C). This
deviation can be understood by considering an additional form that

B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
209
corresponds to deprotonation of the OH group with possible
interaction, by H-bond, with solvent molecules. In that basic form
form TFA was added to the ethanolic solution. However, two types
of emission bands were observed (Fig. 1F) upon excitation with
470 nm wavelength. From previous studies [15,27,28], the band
centered between 644 and 666 nm with a high quantum yield
corresponds to the acid form emission. The other, near 540 nm, has
recently been attributed to a tautomeric form (proton displacement
resulting in localization of the positive charge in one of the 5- or 9-
amino positions) with the corresponding excitation spectrum
showing absorption at ~510 nm and ~480 nm [15]. These bands are
not seen in the absorption spectra in acidified ethanol (Fig. 1E) so
that the impact of the tautomerization process on the quantifica-
tion of the acid-base equilibria (Table 2) should be very low. Only in
compounds 4a and 5 the tautomeric forms are absent so that the
tabulated values of the quantum yields of these compounds are 38%
and 44% respectively, which should be close to the actual values. For
the other compounds, the calculated quantum yields with an
excitation wavelength of 575 nm at which only the acid form
emission is recorded, increases from ethanol to acidified ethanol.
This is expected as the fraction of basic form which has low
quantum yield reduces practically to zero upon acidification with
TFA (Fig. 1E). Those quantum yields lies between 45% and 47% and
expected to be very close to the actual values as the tautomeric
forms were not detected in the absorption spectra in acidified
ethanol.
In water, only small amounts of tautomer emission was
observed for compounds 4b and 4a (Fig. 1B). Considering that no
basic form is observed in the corresponding absorption spectrum
(Fig. 1A) and also the existing H-aggregates are non-emissive, the
obtained values of fluorescence quantum yield are the actual values
for the acid form multiplied by its fractional contribution to the
absorption at the excitation wavelength. Assuming similar fluo-
rescence quantum yield values to those obtained in acidified
ethanol it is possible to estimate the fraction of absorption due to
aggregates at 575 nm as being 76%, 78%, 66%, 61% and 78%,
respectively, for compound 4aed and 5. As H-aggregates absorp-
tion occurs towards the blue of the monomer, these high aggre-
gation levels explains the observed blue shift of the absorption
spectra from ethanol to water whereas the opposite trend is
observed in emission data (Table 1). The same reasoning explains
the higher values of Stokes shifts observed in water when
compared in ethanol.
the
p-conjugation system would not be significantly changed
resulting in a less blue shifted absorption than when the deproto-
nation occurs at the 5-position. As in previous studies [15,17],
compound 5 shows that double alkylation at 9-position results in
red shift of the acid form absorption.
In water media, as observed in our previous studies for similar
compounds [15,27,28], non-emissive H-aggregates of the acidic
form are observed as a blue shoulder around 600 nm or as a flat
absorption band (Fig. 1A). The aggregation fraction increases with
compound concentration resulting in corresponding spectral
changes. This has been seen in previous studies on this type of
compounds and also occurs for these compounds 4aed and 5 (data
not shown).
Variations in the absorption spectra of these compounds in
anhydrous ethanol with its concentration corresponds to changing
ratios of the acid and basic forms of the compounds as can be
concluded from Fig. 2. Typical variations are shown in Fig. 2B being
that, as stated previously, compound 4a behaves differently
(Fig. 2A).
With the exception of compound 4a, the experimental spectra
were fitted to a weighted sum of the spectra obtained in acidified
and basified ethanol. For compound 4a, the spectrum of the basic
form was defined by a sum of two Gaussian functions. Fig. 2A and B
shows the result of this fitting procedure together with the used
spectra of acid and basic forms.
Considering that acid and basic forms are at chemical equilib-
rium, the following equation can be derived for the fraction of basic
form, fb:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
ð1 ꢁ fbÞ
fbCT
2
fbCT
2
Ka ꢁ
þ
2 þ KEtOH ¼ 0
(1)
fb
where CT is the compound total concentration, Ka, is the dissocia-
tion constant and KEtOH is ethanol self-dissociation constant. This
model was previously and successfully applied in compounds of the
same family [17,28]. In Fig. 2C it is shown that the fitting of the
fraction of basic form, obtained from spectral deconvolution pro-
cedure, with eq. (1). In Table 2 the resulting equilibrium constants
are reported for an ethanol self-dissociation constant of
7.97 ꢂ 10ꢁ13 M2, obtained globally for all data in Fig. 2C. As in a
previous study [17] the value of the ionic product corresponding to
the ethanol self-dissociation equilibrium is much higher than ex-
pected [29] and, as stated therein, can probably be accounted from
specific interactions of the studied compound with ethanol sol-
vating molecules. For compounds without substitution at the 2-
position and monosubstituted at the 9-amino position, the ob-
tained Ka values follows the order cyclohexyl < benzyl < isopropyl
which seems related to decreasing order of steric hindrance in the
terminal at the 5-amino position. Disubstitution at the 9-amino
position has already been reported to increase the dissociation
equilibrium constant [17].
The fluorescence spectra in ethanol (Fig. 1D and F) shows that,
with the exception of compound 4a, it is possible to completely
displace the acid-base equilibrium into the basic form by the
addition of TEAH. As already reported for similar compounds
[27,28], the basic form has a broad fluorescence band centered at
~610 nm. The calculated quantum yields are located between 0.019
and 0.027 (Table 1). For compound 4a a very low and broad emis-
sion is observed indicating that the hydroxyl group at the 2-
position has a profound effect on the emission of the neutral
basic form of this family of compounds.
In order to utilize the studied compounds either as dyes, fluo-
rescent probes or biomarkers in fluorescence imaging it is impor-
tant to evaluate their photostability in various conditions. Fig. 3
shows the results of variations of absorption spectra of com-
pounds 4b and 5 in water, BSA protein and soybean lecithin lipid
membranes, upon irradiation with light at 600 nm obtained from a
200 W Xe arc-lamp filtered by an interference filter with ~20%
transmission at 600 nm and 10 nm fwhm.
The photostability depends on the medium where the com-
pound
resides
and
increases
in
the
order
membranes > BSA > water. Compound 5, which is disubstituted at
the 9-amino position, is marginally more photostable than com-
pound 4b (Fig. 3E). In water, the compounds shows less photo-
stability reaching 59% and 61% photodegradation for compounds 5
and 4b respectively, after 90 min irradiation. The H-aggregates are
more photolabile as evident from the absorption spectra in water
which show a red shift with an increase of the irradiation time
(Fig. 3A and B). In the studied models of biological media (Fig. 3C
and D), no significant spectral changes are observed. Interestingly,
the fraction of aggregation is higher in BSA than in membranes and
is much higher for compound 4b than for compound 5. In BSA
medium, the basic form is clearly seen at ~510 nm only for com-
pound 5 (Fig. 3C). Both previous features and the slightly higher
stability of compound 5 are probably a consequence of di-alkylation
In order to fully displace the acid-base equilibrium into the acid

210
B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
Fig. 2. Absorption spectra of compounds 4a (A) and 5 (B) in dried ethanol media at concentrations from 7 ꢂ 10ꢁ6 M to 26 ꢂ 10ꢁ6 M (solid lines - experimental; grey lines - fitted
spectrum); the dotted line is the spectrum of 1 ꢂ 10ꢁ5 M concentration in acidified ethanol; the dashed line is the spectrum correspondent to the basic form that, in the case of
compound 4a (A) an additional spectrum represented by dash-dot-dot line is the fitted spectrum of the basic form that is involved in the acid-base equilibria. Panel C shows the
obtained fraction of basic form of compounds 4aed and 5 in anhydrous ethanol media.
Table 2
no photodegradation was observed for compound 5 within 120 min
of irradiation. Considering that light intensity used in the photo-
stability experiment was 1.65 mW with a beam diameter of 8 mm,
120 min exposure corresponds to 59.4 s of irradiation with a
594 nm 2 mW HeNe laser with 0.8 mm beam diameter. Assuming
1 s acquisition time per pixel it would be possible to image a 8 mm
Equilibrium dissociation constants of compounds 4aed and 5 in dried ethanol.
Compound
4aa
4b
4c
4d
5
Ka (10ꢁ5 M)
1.1
4.5
2.7
3.2
7.2
a
Obtained by fitting the absorption spectrum of the basic form.
circular zone with 800
mm spatial resolution for more than 50
times, without significant photobleaching of compound 5 when
incorporated in biological membranes. The spatial resolution can
be improved by focusing the laser, but the field of view as well as
the time per pixel needs to be reduced in order to not exceed the
maximum number of photons per molecule during the acquisition
at 9-amino position. In BSA the photodegradation reaches only 19%
for compounds 5 and 4b, respectively, after 50 min of irradiation. In
soybean lecithin membranes, the photostability is very high
reaching only 5% for compound 4b upon 110 min of irradiation, and

B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
211
Fig. 3. Photostability experiments with 600 nm irradiation light. Normalised absorption spectra of compounds 4b and 5 in water (A, B), in BSA proteins (C) and in soybean lecithin
membranes (D). Panel E shows the remaining fraction of molecules as a function of irradiation time with the inset representing the initial absorption of each studied system.
of each picture. Considering the used experimental conditions in
the photostability experiments, 120 min irradiation time corre-
sponds to 5.95 ꢂ 10ꢁ5 mol of photons. Each molecule within the
irradiated cylinder, with 8 mm diameter and 1 cm length, was thus
exposed, in average, to 1.2 ꢂ 104 photons per molecule without
suffering appreciable photodegradation.
4. Conclusion
Four new benzo[a]phenoxazinium chlorides possessing mono-
or disubstituted amines with 3-chloropropyl groups at the 9-
position, isopropyl, cyclohexyl and phenyl groups as terminals at
5-amino position of the polycyclic system were efficiently

212
B.R. Raju et al. / Dyes and Pigments 132 (2016) 204e212
fluorescence imaging. Chem Soc Rev 2013;42:622e61.
synthesised. The photophysical behaviour of these compounds was
evaluated in dry ethanol and water. Acid base equilibria in ethanolic
medium were found to be influenced by the presence of bulky
groups in the 5-amino position. A hydroxyl group at the 2-position
introduced an additional deprotonation site giving different pho-
tophysical behaviour for that type of compounds. The dialkylation
at the 9-amino position mainly induced a red shift on the absorp-
tion and emission spectra and originated a higher percentage of the
neutral basic form. Photostability studies showed that dialkylation
at the 9-amino position induces a slightly higher resistance to
photobleaching, H-aggregates showed more lability than mono-
mers in water. Overall, based on the results obtained, namely the
absorption and fluorescence emission above 600 nm
(600e700 nm) and the good photostability particularly in biolog-
ical media, the benzo[a]phenoxazinium chlorides described are
potential interesting alternative probes for bioapplications.
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Acknowledgments
~
^
Thanks are due to the Fundaçao para a Ciencia e a Tecnologia
(FCT, Portugal) for financial support to the NMR portuguese
network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and
FEDER (European Fund for Regional Development)-COMPETE-
QREN-EU for financial support to Research Centres CQ/UM [PEst-
C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302)] and CFUM
[PEst-C/FIS/UI0607/2013 (F-COMP-01-0124-FEDER-022711)]. Post-
doctoral grant to B. R. Raju (SFRH/BPD/62881/2009) is also
acknowledged to FCT, POPH-QREN, FSE.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2016.04.049.
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