Ultrasound promoted synthesis of Nile Blue derivatives

Article abstract of DOI:10.1016/j.ultsonch.2013.05.010

Ultrasound irradiation was used for the first time towards the synthesis of new Nile Blue related benzo[a]phenoxazinium chlorides possessing isopentylamino, (2-cyclohexylethyl)amino and phenethylamino groups at 5-position of the heterocyclic system. The e

Full text of DOI:10.1016/j.ultsonch.2013.05.010

Ultrasonics Sonochemistry 21 (2014) 360–366
Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson
Ultrasound promoted synthesis of Nile Blue derivatives
B. Rama Raju a,b, Diogo M.F. Sampaio b, M.M. Silva b, Paulo J.G. Coutinho a, M. Sameiro T. Gonçalves 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:
Received 8 March 2013
Received in revised form 17 April 2013
Accepted 15 May 2013
Available online 29 May 2013
Ultrasound irradiation was used for the first time towards the synthesis of new Nile Blue related
benzo[a]phenoxazinium chlorides possessing isopentylamino, (2-cyclohexylethyl)amino and pheneth-
ylamino groups at 5-position of the heterocyclic system. The efficacy of sonochemistry was investigated
with some of our earlier reported synthesis of benzo[a]phenoxazinium chlorides. This newer protocol
proved competent in terms of reaction times and enhanced yields. Photophysical studies carried out in
ethanol, water and simulated physiological conditions, revealed that emission maxima occurred in the
range 644–656 nm, with high fluorescent quantum yields. Other attractive feature exhibited by these
materials includes good thermal stability. These properties might be useful in the development of fluo-
rescent probes for biotechnology.
Keywords:
Ultrasound irradiation
Sonochemistry
Nile Blue derivatives
Benzo[a]phenoxazines
NIR fluorescent probes
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
in improved yields, shorter reaction times, increased selectivities
or milder conditions with ultrasonic irradiation [26–35].
Fluorescence probes serve as an indispensable tool for cellular
imaging, determination of amino acids, proteins, DNA sequencing,
and fragment analysis in bioanalytical sciences [1–3]. Among
these, oxazine derivatives have been employed as standards in
fluorescence measurements of biological stains [4,5]. Phenoxazines
and its derivatives are also useful as tranquillisers [6], multidrug
resistance modulators in cancer cells [7], drugs for antitumor ef-
fects, disregulation of cell cycle, and apoptosis induction in
HTLV-I-positive leukaemia cells [8]. Benzo[a]phenoxazinium chlo-
rides belonging to the family of phenoxazines are quite attractive
due to their high fluorescent quantum yields at long wavelengths
(above 600 nm), low triplet yields and good photostabilities [9].
Moreover, the benzo[a]phenoxazinium chlorides can also function
as covalent probes for organic and biological molecules, namely
amino acids [10], proteins [11], peptides and DNA [12], as well as
in the non-covalent labelling of nucleic acids in monitoring protein
conformations and alterations for therapeutic purposes [13,14]. In
addition, these compounds function as potential photosensitizers
with cancer and broad antimicrobial photodynamic activities
[15,16], also act as antimicrobial and antimalarial agents [17–19].
Ultrasound irradiation is a well established tool which provides
specific activation based on a physical phenomenon: acoustic cav-
itation, and can be use as an alternative source for organic reac-
tions carried out under ordinary heating conditions [20–25].
Moreover, numerous organic transformations can be performed
In continuation of our research interests in the synthesis and
applications of oxazine based heterocycles, as well as in alternative
green synthetic protocols [10,17,36–42], the present work de-
scribes for the first time an efficient synthesis of benzo[a]phenox-
azinium chlorides by ultrasound-mediated condensation, in
comparison with conventional heating conditions. The new
benzo[a]phenoxazinium chlorides synthesized possesses isopen-
tylamino, (2-cyclohexylethyl)amino and phenethylamino groups
at 5-position of the heterocyclic system. Fundamental photophys-
ical studies of the synthesised cationic fluorophores were carried
out in ethanol, water and physiological pH. In addition, thermal
stability by differential scanning calorimetry was measured for
all compounds.
2. Experimental
2.1. 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 visualised under UV light. Chromatography on silica
gel was carried out on Merck Kieselgel (230–240 mesh). IR spectra
were determined on a BOMEM MB 104 spectrophotometer. UV–
Vis–NIR absorption spectra (200–700 nm) were obtained using a
Shimadzu UV/2501PC spectrophotometer. NMR spectra were ob-
tained on 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
⇑
Corresponding author. Tel.: +351 253604372; fax: +351 253604382.
E-mail address: msameiro@quimica.uminho.pt (M.S.T. Gonçalves).
1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2013.05.010

B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
361
internal reference at 25 °C. All chemical shifts are given in ppm and
J values are given in Hz. Assignments were carried out through the
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. – Unidad de Espectrometria de Masas’’, at the University
of Vigo, Spain. Fluorescence spectra were collected using a Spex
Fluorolog spectrofluorometer. Ultrassonic assisted reactions were
carried out in an ultrasonic instrument set-up (horn type),
‘‘Misonix S-4000 Sonicator’’, USA, on an operating frequency of
20 kHz, operating amplitude: 80; maximum power output:
600 W; bath temperature: 62–64 °C; horn of titanium alloy with
dimensions: 12.7 cm (length) Â 3.8 cm (diameter); ultrasound
irradiating face diameter: 5.1 cm. Differential scanning calorimet-
ric (DSC) analyses were performed in an 821e Mettler Differential
Scanning Calorimeter. Commercially available reagents were used
as received.
In the above reaction, along with 2a, N,N-bis(isopentyl)naphtha-
len-1-amine was also isolated as brown oil (0.162 g, 17%). TLC
(dichloromethane/methanol, 9.9:0.1): Rf = 0.71. 1H NMR (CDCl3,
300 MHz): dH = 0.85 (d, J = 6.6 Hz, 12H, N(CH2CH2CH(CH3)2)2),
1.35–1.49 (m, 4H, N(CH2CH2CH(CH3)2)2), 1.51–1.62 (m, 2H, N(CH2-
CH2CH(CH3)2)2), 3.10–3.20 (m, 4H, N(CH2CH2CH(CH3)2) 2), 7.16 (d,
J = 7.2 Hz, 2-H), 7.41 (t, J = 7.8 Hz, 1H, 3-H), 7.43–7.52 (m, 2H, 7-H
and 6-H), 7.55 (d, J = 7.8 Hz, 1H, 4-H), 7.78–7.88 (m, 1H, 8-H). 13C
NMR
(CDCl3,
100.6 MHz):
dC = 22.7 (N(CH2CH2CH(CH3)2)2),
26.3 N(CH2CH2CH(CH3)2)2), 36.0 (N(CH2CH2CH(CH3)2)2), 52.5
(N(CH2CH2CH(CH3)2)2), 117.7 (C-2), 123.0 (C-4), 124.2 (C-8),
125.0 (C-3), 125.5 (C-7), 125.6 (C-6), 128.1 (C-5), 131.0 (C-4a),
134.8 (C-8a), 148.6 (C-1). IR (KBr 1%, cmÀ1):
m
= 3056, 2956,
2926, 2868, 1624, 1583, 1527, 1477, 1409, 1379, 1344, 1286,
1225, 1144, 1108, 767, 570. HRMS: m/z (EI): calcd. for C20H29
[M+] 283.2300; found 283.2310.
N
2.2.2. Synthesis of N-(2-cyclohexylethyl)naphthalen-1-amine (2b)
5-(Ethylamino)-4-methyl-2-nitrosophenol hydrochloride (1)
was obtained by reaction of 3-(ethylamino)-4-methylphenol
(1.5 mmol) in ethanol (2 mL) and 12 M hydrochloric acid (0.4 mL)
with sodium nitrite (1.2 equiv.), under low temperature (ice-bath),
with stirring for 3 h, followed by evaporation of the reaction mix-
ture. This compound was used in the synthesis of benzo[a]phenox-
azinium chlorides 3a–g without any purification, as previously
reported [10,17,36–41,43].
Ethyl 4-(naphthalen-1-ylamino)butanoate (2d) and 3-(naph-
thalen-1-ylamino)propan-1-ol (2f) were prepared by reaction of
naphthalen-1-amine (2g) with ethyl-4-bromobutyrate (1.05 equiv.)
or 3-bromo-1-propanol (1.05 equiv.) in ethanol, under reflux con-
ditions during 15 h, followed by silica gel dry chromatography
purification as previously reported [10]. 4-(Naphthalen-1-ylami-
no)butanoic acid (2e) resulted from the reaction of compound 2d
with aqueous 1 M sodium hydroxide (1.8 equiv.) in 1,4-dioxane,
at room temperature with stirring for 8 h, followed by acidification
to pH 2–3 and extraction of the reaction mixture as previously de-
scribed [10].
Starting from naphthalen-1-amine (0.500 g, 3.50 mmol) in eth-
anol (2 mL), using (2-bromoethyl)cyclohexane (0.735 g,
3.84 mmol), and following the same procedure described before
for the preparation of 2a (reflux time 17 h), compound 2b was ob-
tained as brown solid (0.260 g, 28%), m.p. 84.7–86.7 °C. TLC (chlo-
roform/methanol, 9.5:0.5): Rf = 0.60. 1H NMR (CDCl3, 400 MHz):
dH = 1.04–1.17 (m, 2H, 2 Â CH Cy), 1.23–1.46 (m, 3H, 3 Â CH Cy),
1.50–1.63 (m, 1H, NHCH2CH2CH), 1.75 (q, J = 6.8 Hz, 2H, NHCH2-
CH2), 1.80–1.95 (m, 5H, 5 Â CH Cy), 3.36 (t, J = 7.6 Hz, 2H, NHCH2-
CH2), 4.33 (broad s, 1H, NH), 6.71 (d, J = 7.6 Hz, 2-H), 7.33 (d,
J = 8.4 Hz, 1H, 4-H), 7.46 (d, J = 7.8 Hz, 1H, 3-H), 7.49–7.57 (m,
2H, 6-H and 7-H), 7.86–7.93 (m, 2H, 8-H and 5-H). 13C NMR (CDCl3,
100.6 MHz): dC = 26.3 (2 Â CH2 Cy), 26.5 (CH2 Cy), 33.4 (NHCH2-
CH2CH), 35.7 (NHCH2CH2CH), 37.0 (2 Â CH2 Cy), 41.9 ((NHCH2CH2),
104.1 (C-2), 117.0 (C-4), 119.8 (C-8), 123.3 (C-8a), 124.5 (C-7),
125.6 (C-6), 126.6 (C-3), 128.6 (C-5), 134.3 (C-4a), 143.6 (C-1). IR
(KBr 1%, cmÀ1):
m = 3385, 3054, 3010, 2956, 2927, 2869, 2717,
2454, 1623, 1604, 1582, 1528, 1478, 1468, 1410, 1378, 1368,
1345, 1286, 1253, 1224, 1170, 1143, 1108, 1081, 1021, 951, 863,
800, 784, 768, 665. HRMS: m/z (EI): calcd. for C18H23N [M+]
253.1830; found 253.1838.
2.2. Preparation of precursors 2a–c
In the above reaction, along with 2b, N,N-bis(2-cyclohexyleth-
yl)naphthalen-1-amine was also isolated as colourless oil (0.089 g,
7%). TLC (dichloromethane/methanol, 9.5:0.5): Rf = 0.74. 1H NMR
(CDCl3, 400 MHz): dH = 0.83 (m, 4H, 4 Â CH Cy), 1.17–1.24 (m,
6H, 6 Â CH Cy), 1.25–1.34 (m, 2H, N(CH2CH2CH)2), 1.38–1.48 (m,
4H, N(CH2CH2CH)2), 1.60–1.74 (m, 10H, 10 Â CH Cy), 3.14–3.23
(m, 4H, N(CH2CH2CH)2), 7.17 (d, J = 8.4 Hz, 1H, 2-H), 7.42 (t,
J = 8.0 Hz, 1H, 3-H), 7.45–7.52 (m, 2H, 6-H and 7-H), 7.56 (d,
J = 8.4 Hz, 1H, 4-H), 7.81–7.87 (m, 1H, 5-H), 8.29–8.36 (m, 1H, 8-
H). 13C NMR (CDCl3, 100.6 MHz): dC = 26.3 (4 Â CH2 Cy), 26.6
(2 Â CH2 Cy), 33.4 (4 Â CH2 Cy), 34.5 (N(CH2CH2CH)2), 36.0 (N(CH2-
CH2CH)2), 52.0 (N(CH2CH2CH)2), 117.7 (C-2), 123.0 (C-4), 124.3 (C-
8), 124.9 (C-7), 125.5 (C-6), 125.6 (C-3), 128.1 (C-5), 131.1 (C-8a),
2.2.1. N-isopentyl-naphthalen-1-amine (2a)
To a solution of naphthalen-1-amine (0.500 g, 3.50 mmol) in
ethanol (2 mL), 1-bromo-3-methylbutane (0.580 g, 3.84 mmol)
was added, and the resulting mixture was refluxed for 14 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 chroma-
tography on silica gel using dichloromethane and dichlorometh-
ane/methanol 99:1, as the eluent. Compound 2a was obtained as
violet oil (0.401 g, 54%). TLC (dichloromethane/methanol,
9.9:0.1): Rf = 0.54. 1H NMR (CDCl3, 400 MHz): dH = 1.00 (d,
J = 6.4 Hz, 6H, NHCH2CH2CH(CH3)2), 1.70–1.86 (m, 3H, NHCH2CH2-
CH(CH3)2 and NHCH2CH2CH(CH3)2), 3.39 (t, J = 7.2 Hz, 2H, NHCH2-
CH2CH(CH3)2), 3.50 (br s, 1H, NH), 7.05 (dd, J = 6.2 and 2.4 Hz,
1H, 2-H), 7.40–7.46 (m, 2H, 3-H and 4-H), 7.49–7.56 (m, 2H, 7-H
and 6-H), 7.85 (dd, J = 7.0 and 2.0 Hz, 1H, 5-H), 8.02–8.08 (dd,
J = 7.2 and 2.0 Hz, 1H, 8-H). 13C NMR (CDCl3, 100.6 MHz):
dC = 22.3 (NHCH2CH2CH(CH3)2), 25.9 (NHCH2CH2CH(CH3)2), 36.9
(NHCH2CH2CH(CH3)2), 44.5 (NHCH2CH2CH(CH3)2), 108.8 (C-2),
120.1 (C-3), 120.3 (C-8), 123.8 (C-4a), 125.2 (C-7), 125.8 (C-6),
126.0 (C-4), 128.4 (C-5), 134.1 (C-8a), 140.0 (C-1). IR (KBr 1%,
134.9 (C-4a), 148.8 (C-1). IR (KBr 1%, cmÀ1):
m = 3436, 3046,
2922, 2849, 1576, 1526, 1506, 1448, 1401, 1381, 1344, 1263,
1147, 1119, 1082, 1052, 1016, 962, 886, 843, 774, 614. HRMS:
m/z (EI): calcd. for C26H37N [M+] 363.2926; found 363.2931.
2.2.3. Synthesis of N-(phenethyl)naphthalen-1-amine (2c)
Starting from naphthalen-1-amine (0.500 g, 3.50 mmol) in eth-
anol (2 mL), using (2-bromoethyl)benzene (0.710 g, 3.84 mmol),
and following the same procedure described before for the prepa-
ration of 2a (reflux time 15 h), compound 2c was obtained as violet
solid (0.268 g, 31%). TLC (chloroform/methanol, 9.5:0.5): Rf = 0.69.
m.p. 80.8–82.8 °C. 1H NMR (CDCl3, 400 MHz): dH = 3.13 (t,
J = 7.2 Hz, 2H, NHCH2CH2Ph), 3.62 (t, J = 6.9 Hz, 2H, NHCH2CH2Ph),
4.20 (br s, 1H, NH), 6.77 (d, J = 7.5 Hz, 1H, 2-H), 7.32–7.52 (m, 9H,
cmÀ1):
m = 3424, 3049, 2926, 2848, 2823, 1623, 1581, 1527, 1471,
1445, 1410, 1383, 1345, 1324, 1289, 1264, 1174, 1143, 1121,
1099, 1033, 784, 765. HRMS: m/z (EI): calcd. for C15H19N [M+]
213.1517; found 213.1519.

362
B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
5 Â Ar-H, 3-H, 4-H, 7-H and 6-H), 7.76 (d, J = 7.8 Hz, 1H, 5-H), 7.87
(d, 1H, J = 7.8 Hz, 8-H). 13C NMR (CDCl3, 100.6 MHz): dC = 35.2
(NHCH2CH2Ph), 45.2 (NHCH2CH2Ph), 104.6 (C-2), 117.5 (C-4),
119.7 (C-8), 123.4 (Ar-C), 124.6 (C-7), 125.6 (Ar-C), 126.5 (C-6),
126.6 (C-3), 128.6 (Ar-C), 128.6 (C-5), 128.8 (2 Â Ar-C), 134.3
39.7 (NHCH2CH3), 44.1 (NHCH2CH2CH(CH3)2), 93.9 (C-6), 94.5 (C-
8), 123.7 (C-1), 124.8 (Ar-C), 125.6 (C-4), 128.7 (Ar-C), 130.8 (C-
3), 132.1 (Ar-C), 132.5 (Ar-C), 132.7 (C-2), 132.8 (C-11), 134.5
(Ar-C), 149.4 (Ar-C), 153.0 (Ar-C), 156.7 (C-9), 158.6 (C-5). IR
(KBr 1%, cmÀ1):
m
= 3239, 2926, 1644, 1592, 1565, 1546, 1520,
(2 Â Ar-C), 139.2 (Ar-C), 143.0 (C-1). IR (KBr 1%, cmÀ1):
m = 3414,
1450, 1435, 1318, 1296, 1261, 1187, 1164, 1125, 1000, 876. HRMS:
3028, 2855, 2360, 1639, 1579, 1525, 1475, 1454, 1407, 1378,
1345, 1291, 1271, 1117, 1082, 1028, 846, 782, 769, 755, 697,
571. HRMS: m/z (EI): calcd. for C18H17N [M+] 247.1361; found
247.1359.
m/z (ESI): calcd. for
374.22224.
C
24H28N3O [M + H]+ 374.22269; found
In the above reaction, along with 2c, N,N-bis(phenethyl)naphtha-
len-1-amine was also isolated as colourless oil (0.045 g, 9%). TLC
(dichloromethane/methanol, 9.5:0.5): Rf = 0.78. 1H NMR (CDCl3,
400 MHz): dH = 2.81 (t, J = 7.6 Hz, 4H, N(CH2CH2Ph)2), 3.48 (t,
J = 8.0 Hz, 4H, N(CH2CH2Ph)2), 7.11–7.13 (m, 4H, 2-H, 3 Â Ar-H),
7.18–7.22 (m, 2H, 2 Â CH-Ph), 7.24–7.28 (m, 5H, 4-H, 4 Â Ar-H),
7.34 (d, J = 8.0 Hz, 1H, 1 Â Ar-H), 7.40–7.50 (m, 3H, 6-H, 7-H, 3-
H), 7.64 (d, J = 8.0 Hz, 1H, 5-H), 8.15 (d, J = 8.4 Hz, 1H, 8-H). 13C
NMR (CDCl3, 100.6 MHz): dC = 33.71 (N(CH2CH2Ph)2), 56.2 (N(CH2-
CH2Ph)2), 118.3 (C-2), 123.9 (C-4), 124.2 (C-8), 125.2 (2 Â Ar-C),
125.5 (C-7), 125.8 (C-6), 125.9 (2 Â Ar-C, C-3), 128.1 (2 Â Ar-C),
128.3 (2 Â Ar-C), 128.8 (2 Â Ar-C), 131.1 (C-5), 135.0 (Ar-C),
139.4 (2 Â Ar-C), 140.3 (Ar-C), 147.5 (C-1). IR (KBr 1%, cmÀ1):
2.3.4. N-(5-((2-Cyclohexylethyl)amino)-10-methyl-9H-
benzo[a]phenoxazin-9-ylidene) ethanaminium chloride (3b)
Blue solid (0.524 g, 93%). TLC (dichloromethane/methanol 9:1):
Rf = 0.87; m.p. 142.2–144.2 °C. 1H NMR (CD3OD, 400 MHz):
dH = 1.04–1.20 (m, 2H, 2 Â CH Cy), 1.22–1.36 (m, 4H, 4 Â CH Cy),
1.40 (t, J = 6.8 Hz, 3H, NHCH2CH3), 1.53 (br s, 1H, CH Cy), 1.70–
1.85 (m, 4H, NHCH2CH2 and 2 Â CH Cy), 2.36 (s, 3H, CH3), 3.50–
3.60 (m, 2H, NHCH2CH3), 3.74 (br s, 2H, NHCH2CH2), 6.86 (s, 1H,
8-H), 6.90 (s, 1H, 6-H), 7.68 (s, 1H, 11-H), 7.80–7.86 (m, 1H, 3-H),
7.92 (t, J = 7.0 Hz, 1H, 2-H), 8.33 (d, J = 7.2 Hz, 1H, 1-H), 8.91 (d,
J = 8.0 Hz, 1H, 4-H). 13C NMR (CD3OD, 100.6 MHz): dC = 14.2
(NHCH2CH3), 17.7 (CH3), 27.4 (2 Â CH2 Cy), 27.6 (CH2 Cy), 34.3
(2 Â CH2 Cy), 37.0 (CH Cy), 37.1 (NHCH2CH2), 39.7 (NHCH2CH3),
43.7 (NHCH2CH2), 93.9 (C-6), 94.6 (C-8), 123.7 (C-1), 124.8 (Ar-
C), 125.6 (C-4), 128.7 (Ar-C), 130.8 (C-3), 132.1 (Ar-C), 132.6 (Ar-
C), 132.7 (C-2), 132.9 (C-11), 134.6 (Ar-C), 149.4 (Ar-C), 153.0
m
= 3449, 3060, 3026, 2929, 2855, 1638, 1575, 1543, 1495, 1456,
1399, 1264, 1125, 1103, 1031, 1016, 801, 776, 747, 699, 575.
HRMS: m/z (EI): calcd. for C26H25N [M+] 351.1987; found 351.1987.
(Ar-C), 156.7 (C-9), 158.6 (C-5). IR (KBr 1%, cmÀ1):
m = 3454,
2.3. General method for the preparation of compounds 3a–g
3166, 2921, 2850, 1642, 1589, 1563, 1539, 1515, 1493, 1476,
1450, 1431, 1366, 1344, 1309, 1291, 1256, 1231, 1185, 1160,
1131, 1056, 1002, 928, 879, 859, 817, 809, 786, 776. HRMS: m/z
(ESI): calcd. for C27H32N3O [M + H]+ 414.25399; found 414.25370.
2.3.1. Conventional methodology
To a cold solution (ice bath) of 5-(ethylamino)-4-methyl-2-
nitrosophenol hydrochloride (1) (1.26 mmol) in ethanol (2 mL),
naphthalen-1-amine derivatives (2a–c) (0.63 mmol) and 12 M
hydrochloride acid (1.29 Â 10À2 mL) were added. The mixture
was refluxed during the time as indicated in Table 1, and moni-
tored by TLC (dichloromethane/methanol). After evaporation of
the solvent and column chromatography purification on silica
gel, with dichloromethane and dichloromethane/methanol, mix-
tures of increasing polarity, as the eluent, the required dye 3a–c
was obtained.
2.3.5. N-(10-Methyl-5-(phenethylamino)-9H-benzo[a]phenoxazin-9-
ylidene)ethanaminium chloride (3c)
Blue solid (0.503 g, 91%). Rf = (dichloromethane/methanol 9:1)
0.77; m.p. 131.9–133.9 °C. 1H NMR (CD3OD, 400 MHz): dH = 1.39
(t, J = 7.2 Hz, 3H, NCH2CH3), 2.29 (s, 3H, CH3), 3.15 (t, J = 7.6 Hz,
2H, NHCH2CH2Ph), 3.50 (q, J = 7.2 Hz, 2H, NCH2CH3), 3.91 (t,
J = 7.2 Hz, 2H, NHCH2CH2Ph), 6.73 (s, 1H, 8-H), 6.76 (s, 1H, 6-H),
7.20–7.29 (m, 1H, 1 Â Ar-H Ph), 7.30–7.37 (m, 4H, 4 Â Ar-H Ph),
7.52 (s, 1H, 11-H), 7.74 (t, J = 7.2 Hz, 1H, 3-H), 7.83 (t, J = 8.0 Hz,
1H, 2-H), 8.22 (d, J = 8.4 Hz, 1H, 1-H), 8.74 (dd, J = 8.8 and 0.8 Hz,
1H, 4-H). 13C NMR (CD3OD, 100.6 MHz): dC = 14.2 (NHCH2CH3),
17.7 (CH3), 35.8 (NHCH2CH2Ph), 39.8 (NHCH2CH3), 47.0 (NHCH2-
CH2Ph), 94.0 (C-6), 94.5 (C-8), 123.6 (Ar-C), 124.6 (C-3), 125.4 (C-
4), 128.0 (2 Â Ar-C), 128.8 (C-2), 129.8 (C-1), 130.1 (2 Â Ar-C),
130.7 (2 Â Ar-C), 132.1 (Ar-C), 132.3 (Ar-C), 132.6 (C-11), 132.8
(Ar-C), 134.1 (C-10), 139.5 (Ar-C), 149.2 (Ar-C), 152.6 (Ar-C),
2.3.2. Ultrasonic irradiation
To a cold solution (ice bath) of 5-(ethylamino)-4-methyl-2-
nitrosophenol hydrochloride (1) (1.26 mmol), in ethanol or
dimethylformamide (3e) (2 mL), naphthalen-1-amine or its deriv-
atives (2a–g) (0.63 mmol) and 12 M hydrochloride acid
(1.29 Â 10À2 mL) were added. The mixture was sonicated during
the time mentioned in Table 1, and monitored by TLC (dichloro-
methane/methanol). After evaporation of the solvent and column
chromatography purification on silica gel with dichloromethane
and dichloromethane/methanol, mixtures of increasing polarity,
as the eluent, the required compound 3a–g was obtained.
156.7 (C-9), 158.4 (C-5). IR (KBr 1%, cmÀ1):
m = 3416, 2922, 1642,
1591, 1562, 1544, 1520, 1478, 1448, 1317, 1295, 1261, 1186,
1162, 1133, 1054, 1009, 822, 785, 734, 701. HRMS: m/z (ESI): calcd.
for C27H26N3O [M + H]+ 408.20704; found 408.20548.
2.3.3. N-(5-(Isopentylamino)-10-methyl-9H-benzo[a]phenoxazin-9-
ylidene)ethanaminium chloride (3a)
Blue solid (0.46 g, 90%). TLC (dichloromethane/methanol 9:1):
Rf = 0.80; m.p. 214–216 °C. 1H NMR (CD3OD, 400 MHz): dH = 1.09
(d, J = 6.4 Hz, 6H, NHCH2CH2CH(CH3)2), 1.40 (t, J = 7.2 Hz, 3H,
NHCH2CH3), 1.73–1.90 (m, 3H, NHCH2CH2CH(CH3)2 and NHCH2-
CH2CH(CH3)2), 2.35 (s, 3H, CH3), 3.54 (q, J = 7.2 Hz, 2H, NHCH2CH3),
3.72 (t, J = 8.0 Hz, 2H, NHCH2CH2CH(CH3)2), 6.84 (s, 1H, 8-H), 6.90
(s, 1H, 6-H), 7.66 (s, 1H, 11-H), 7.81 (dt, J = 7.8 and 1.8 Hz, 1H, 3-
H), 7.91 (dt, J = 8.4 and 1.2 Hz, 1H, 2-H), 8.34 (d, J = 8.4 Hz, 1H, 1-
H), 8.89 (dd, J = 8.0 and 0.8 Hz, 1H, 4-H). 13C NMR (CDCl3,
100.6 MHz): dc = 14.2 (NHCH2CH3), 17.7 (CH3), 22.9 (NHCH2CH2-
CH(CH3)2), 27.4 (NHCH2CH2CH(CH3)2), 38.3 (NHCH2CH2CH(CH3)2),
2.4. Differential scanning calorimetry
A DSC 821e mettler differental scanning calorimeter was em-
ployed to determine the thermal behaviour of compounds 3a–g.
All samples were presented for analysis in 40 lL aluminium cans
with perforated lids to permit the release and removal of the
decomposition products. The can was hermetically sealed and
the thermogram of the sample was recorded. The sample was
heated from room temperature (25 °C) to 200 °C at 5 °C minÀ1 un-
der high purity argon atmosphere supplied at
a constant
35 mL minÀ1 flow rate.

B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
363
Table 1
Synthesis of benzo[a]phenoxazinium chlorides 3a–g under ultrasound irradiation (US) in comparison with the conventional heating.
Entry
Naphthalen-1-amine precursor
Product
Reaction time (min)
Without US
480
Yield (%)
Without US
96
US
90
US
1
90
N
H
N
N
H
O
3a
N
H
Cl-
2a
2
290
90
53
93
N
H
N
H
N
O
3b
N
H
Cl-
2b
3
4
270
480
90
78
75
91
91
N
H
H
N
N
O
3c
N
H
Cl-
H
2c
105
N
O
O
N
H
N
O
3d
N
H
Cl-
O
O
2d
5
6
7
450
180
540
300
64
40
97
80
NR
95
86
N
OH
OH
H
H
H
N
H
N
O
3e
N
H
Cl-
O
2e
O
N
N
H
OH
N
O
3f
N
H
OH
Cl-
2f
75
N
NH2
N
O
NH2
Cl-
2g
3g
NR – no reaction.
no)butanoic acid (2e) and 3-(naphthalen-1-ylamino)propan-1-ol
(2f) were obtained in accordance with the earlier reported proce-
dure [10].
3. Results and discussion
3.1. Synthesis
Initially, the condensation reaction of nitrosophenol 1 with
intermediate 2a under acidic conditions (12 M hydrochloric acid)
in ethanol at reflux was carried out. Benzo[a]phenoxazinium chlo-
ride 3a was obtained in excellent yield in 8 h. Taking into account
the advantages and convenience of sonochemistry, we attempted
to investigate the feasibility of the reaction under ultrasonic irradi-
ation in acidic reaction conditions. It was possible to obtain the ex-
pected benzo[a]phenoxazinium chloride 3a within dramatically
shorter time (1.5 h), also in excellent yield. With this encouraging
result, we became interested to synthesize a couple of new
benzo[a]phenoxazine derivatives, 3b and 3c, under the same reac-
tion conditions. Based on the results of the study and to extend the
scope of this newer protocol, we probed some of the earlier re-
ported reactions carried out in the conventional heating methods
[10] (entries 4–7 in Table 1). In all cases (except 3e), the experi-
mental results showed the prominent reduction of the reaction
times (from three to more than seven times) along with good to
excellent yields of the required benzo[a]phenoxazinium chlorides
under ultrasound reaction conditions (Scheme 1, Table 1). In this
The benzo[a]phenoxazinium chlorides 3a–g synthesis started
with the preparation of the required precursors, nitroso derivative
1 and N-alkylated naphthalen-1-amines 2a–f (2g is a commercial
reagent). The 5-(ethylamino)-4-methyl-2-nitrosophenol hydro-
chloride (1) was obtained by nitrosation of 3-(ethylamino)-4-
methylphenol with sodium nitrite in acid solution under ice cold
conditions [10,17,36–41,43]. N-isopentyl-naphthalen-1-amine
(2a), N-(2-cyclohexylethyl)naphthalen-1-amine (2b) and N-(phen-
ethyl)naphthalen-1-amine (2c) were obtained as an oil (2a) or
solids in moderate yields (28–54%), by the alkylation of naphtha-
len-1-amine with 1-bromo-3-methylbutane, (2-bromoethyl)cyclo-
hexane and (2-bromoethyl)benzene, respectively, in ethanol under
reflux conditions. In addition to the monoalkylated required com-
pounds, the related dialkylated products, N,N-bis(isopentyl)naph-
thalen-1-amine, N,N-bis(2-cyclohexylethyl)naphthalen-1-amine
and N,N-bis(phenethyl)naphthalen-1-amine were also obtained
in low yields (7–17%). The remaining precursors, namely ethyl 4-
(naphthalen-1-ylamino)butanoate (2d), 4-(naphthalen-1-ylami-

364
B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
EtOH, HCl
NO
OH
N
Conventional
heating
EtOH, HCl
US
HN
NHR
HN
Cl-
O
NHR
.HCl
3a-g
1
2a-g
R = (CH2)2CH(CH3)2, (CH2)2C6H11, (CH2)2C6H5, (CH2)3CO2C2H5, (CH2)3CO2H, (CH2)3OH, H
Scheme 1. Thermal and ultrasound assisted (US) synthesis of benzo[a]phenoxazinium chlorides 3a-g.
Table 2
condensation reaction, a polar protic solvent (like ethanol or meth-
anol) promotes the formation of benzo[a]phenoxazines. In the par-
ticular case of benzo[a]phenoxazinium chloride 3e, the use of polar
protic solvent is restricted due to the esterification of the carbox-
ylic side-chain terminal. Hence, in this reaction, polar aprotic sol-
vent (dimethylformamide) was used and it was found that under
these ultrasound conditions it does not favour for the formation
of benzo[a]phenoxazine 3e. The reduction in the reaction times
and the improvements in the yields obtained under ultrasound
conditions can probably be due to mechanical effects of shock
waves; the high temperatures and pressures developed locally by
cavitation, not reached in thermal assisted reactions.
Photophysical data of compounds 3a–c in ethanol, water and at pH 7.4 (kex 590 nm).
Solvent
Ethanol
3a
3b
3c
kabs (nm)
log
kem (nm)
UF
630
4.75
644
0.463
14
622
4.57
652
0.2
629
4.69
645
0.453
631
4.70
646
0.458
15
576(sh)/622
4.23/4.14
656
0.115
34
626
4.20
656
0.213
30
e
D
(nm)
kabs (nm)
log
16
Water
pH 7.4
550/627(sh)
4.13/3.89
652
0.061
e
kem (nm)
UF
D
(nm)
kabs (nm)
log
30
25
622
4.37
651
0.209
29
520(sh)/580(sh)/618
3.86/3.98/4.04
652
0.102
34
All of the new compounds obtained were characterised by high
resolution mass spectrometry, IR and NMR (1H and 13C) spectros-
copy, and the structures of the others compounds were confirmed
by comparison with earlier reported data [10].
e
kem (nm)
UF
D
(nm)
The 1H NMR spectra of compounds 3a–c displayed triplet or a
broad singlet (3b) (d 3.72–3.91 ppm) for the aliphatic protons of
the methylenic groups at positions 5, directly linked to the nitro-
gen atom, NHCH2CH2, as well as multiplets (d 1.70–1.90 ppm) or
a triplet (3c, d 3.15 ppm) for the groups closed to the same atom,
NHCH2CH2. The CH proton exhibited a multiplet (3a) or broad sin-
glet (3b) (d 1.53–1.90 ppm). The methyl protons of ethyl and iso-
sh – shoulder.
pentyl (3a) groups in amines of positions
5 and 9 of the
heterocycle appeared as triplets (d ꢀ1.40 ppm) and a doublet (iso-
pentyl group of 3a, d 1.09 ppm). There was also the presence of
protons of the methyl group directly linked to the aromatic ring
at position 10, which appeared as singlets (d 2.29–2.36 ppm). In
compound 3c, protons of the phenyl terminal of the lateral chain
at position 5 as multiplets occurred in the range 7.20–7.37 ppm.
In addition, spectra displayed the expected aromatic protons of
the polycyclic system, in particular H-8 (d 6.73–6.86 ppm), H-6 (d
6.76–6.90 ppm), and H-11 (d 7.52–7.68 ppm), which appeared in
the form of singlets.
The 13C NMR spectra showed the signals of the methylenic
groups of substituents of positions 5, directly linked to the nitrogen
atom NHCH2CH2 (d 43.70–47.0 ppm), as well as groups closed to
the same atom, NHCH2CH2 (d 35.80–38.33 ppm) and the CH carbon
(d 27.44 or 36.99 ppm). The methyl carbons of ethyl and isopentyl
(3a) groups in amines of positions 5 and 9 of the heterocycle also
appeared (d ꢀ14.15 or 22.85 ppm). In addition, there was the pres-
ence of carbons of the methyl group directly linked to the aromatic
ring at position 10 (d 17.66–17.72 ppm). Spectra showed the ex-
pected aromatic carbons, in particular C-8 (d 94.48–94.55 ppm);
C-6 (d 93.66–93.96 ppm) and C-11 (d 132.64–132.85 ppm).
Fig. 1. Normalized absorption and emission spectra of compound 3a in ethanol,
water and buffer at pH 7.4.
631 nm. Although for each solvent, the kabs are similar in the three
compounds, a hipsochromic shift (2–11 nm) was observed in aque-
ous solutions, in comparison with ethanol. Concerning the molar
absorptivities, benzo[a]phenoxazinium chlorides 3a–c displayed
excellent values in ethanol, and even though there was a decrease
in water and at pH 7.4, they are also very good in the latter sol-
vents. In aqueous environment both compounds 3b and 3c exhib-
ited significant aggregation as shown by either shoulders or bands
in the 550–580 nm spectral region [38]. This aggregation account
for the decrease in molar absorptivity and was seen to decrease
in pH 7.4 buffer with an appearance of an additional shoulder at
520 nm for compound 3c, which can be ascribed to the basic
deprotonated form [38].
3.2. Photophysical studies
Taking into account the potential of benzo[a]phenoxazinium
chlorides 3a–c to act as NIR fluorescent probes for various pur-
poses, including in biological contexts, fundamental photophysical
characterisation was carried out in ethanol, water and at simulated
physiological conditions (pH 7.4) (Table 2, Figs. 1–3).
Fluorescent emission properties of benzo[a]phenoxazinium
chlorides 3a–c were also measured in the organic and aqueous
media formerly mentioned. Maximum emission wavelengths
The longest wavelength of maximum absorption (kabs) of com-
pounds in the three solvents was located in the region 618–

B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
365
In all solvents, benzo[a]phenoxazinium salts displayed maxi-
mum emission wavelengths in the region of 644–656 nm, with
low to moderate Stokes’ shifts (14–34 nm), and relative fluores-
cence quantum yields at about 0.46 in ethanol and 0.06–0.21 in
water and at physiological simulated conditions. Comparison of
kem values in water and pH 7.4 with ethanol revealed a bathochro-
mic shift in aqueous media of 7–10 nm. This shift is compatible
with a
p
–pà type transition and the blue shift observed in absorp-
tion can be explained by the appearance of aggregates of H type
that absorb to the blue of the monomer and have negligible fluo-
rescence. This last fact also explains the significant decrease of
fluorescence quantum yield for compounds 3b and 3c in aqueous
media with some recovery in buffered pH 7.4 solution. Compound
3b showed more tendency to aggregate than compound 3c and this
can be explained by the fact that the cyclohexyl side group is more
apolar than the benzyl. The appearance of basic form also is ex-
pected to be more favourable for compound 3b as the basic form
allows the positive charge to move away from the nitrogen group
at the 5-amino position. The basic form emission is ꢀ10 times less
efficient that the acid cationic form and its emission maximum
wavelength occurs near 600 nm [38]. This can be seen in Fig. 2 as
a blue edge enlargement of the emission spectrum that do not oc-
cur for compounds 3a and 3c (Figs. 1 and 3).
Fig. 2. Normalized absorption and emission spectra of compound 3b in ethanol,
water and buffer at pH 7.4.
3.3. Thermal behaviour
The thermal behaviour of benzo[a]phenoxazinium chlorides
3a–g was studied during heating by Differential Scanning Calorim-
etry. The thermograms of DSC measurements were performed in
accordance with the programs described in the experimental sec-
tion and selected results are illustrated in Fig. 4. Observations
using DSC technique were consistent with a minimum thermal sta-
bility of 200 °C for all compounds. Peaks associated with melting
transitions of compounds were detected by the DSC analyses. Com-
pound 3b underwent fusion at an onset temperature (onset tem-
perature is taken as the point of intersection of the extrapolated
baseline with the tangent to the curve produced by the thermal
event) at 106.63 °C and an enthalpy of melting,
À100.32 mJ. Compound 3g displayed an onset melting tempera-
ture at 69.09 °C and an enthalpy of melting,
DHfus =
Fig. 3. Normalized absorption and emission spectra of compound 3c in ethanol,
water and buffer at pH 7.4.
D
Hfus = À36.22 mJ.
The other compounds were showed similar thermograms with
endothermic peaks.
(kem) and relative fluorescence quantum yields (UF) were obtained
and are summarised in Table 2. For the determination of quantum
yields, Oxazine 1, used as a standard (UF = 0.11 in ethanol) [44],
was excited at 575 nm for all compounds.
In commercial devices, the thermal performance is clearly of
critical importance, particularly in relation to the practical conse-
quences of severe devices abuse where overheating may be
expected.
Fig. 4. DSC thermograms of 3b and 3g (top).

366
B.R. Raju et al. / Ultrasonics Sonochemistry 21 (2014) 360–366
[10] V.H. Frade, P.J.G. Coutinho, J.C.V.P. Moura, M.S.T. Gonçalves, Tetrahedron 63
4. Conclusion
(2007) 1654–1663.
[11] B.S.B. Salomi, C.K. Mitra, L. Gorton, Synth. Met. 155 (2005) 426–429.
[12] A. Simmonds, J.N. Miller, C.J. Moody, E. Swann, M.S.J. Briggs, I. E. Bruce, Eur.
Patent WO97, 29154, 1997.
[13] X. Yan, S. Miragila, P.M. Yuan, U.S. Patent 6, 140, 500, 2000.
[14] J. Nakanishi, T. Nakajima, M. Sato, T. Ozawa, K. Tohda, Y. Umezawa, Anal.
Chem. 73 (2001) 2920–2928.
[15] R.S. Becker, S. Chakravorti, S. Das, Photochem. Photobiol. 51 (1990) 533–538.
[16] A. Juzeniene, Q. Peng, J. Moan, Photochem. Photobiol. Sci. 6 (2007) 1234–1245.
[17] V.H.J. Frade, M.J. Sousa, J.C.V.P. Moura, M.S.T. Gonçalves, Bioorg. Med. Chem. 16
(2008) 3274–3282.
The synthesis of benzo[a]phenoxazinium chlorides was effi-
ciently performed for the first time by ultrasound assisted conden-
sation of the required nitrosophenol with naphthalen-1-amine or
its N-alkylated precursors. The reduction of reaction times along
with excellent yields of the target compounds make this protocol
a remarkable alternative to conventional heating in the prepara-
tion of this type of polycyclic heterocycles.
[18] J.F. Ge, C. Arai, M. Yang, M.A. Bakar, J. Lu, N.S.M. Ismail, S. Wittlin, M. Kaiser, R.
Brun, S.A. Charman, T. Nguyen, J. Morizzi, I. Itoh, M. Ihara, Med. Chem. Lett. 1
(2010) 360–364.
[19] T. Küçükkilinç, I. Ozer, Arch. Biochem. Biophys. 461 (2007) 294–298.
[20] T.J. Mason, Chem. Soc. Rev. 26 (1997) 443–451.
[21] G. Cravotto, P. Cintas, Chem. Soc. Rev. 35 (2006) 180–196.
[22] R. Juarez, P. Concepcion, A. Corma, H. Garcia, Chem. Commun. 46 (2010) 4181–
4183.
[23] G. Cravotto, P. Cintas, Chem. Sci. 3 (2012) 295–307.
[24] B.S. Singh, H.R. Lobo, D.V. Pinjari, K.J. Jarag, A.B. Pandit, G.S. Shankarling,
Ultrason. Sonochem. 20 (2013) 633–639.
In view of the water-solubility, the photophysical characteris-
tics, namely absorption and fluorescent emission in the range
618–656 nm, with good relative fluorescent quantum yields (acid
form) and also high thermal stability, the new benzo[a]phenoxaz-
inium chlorides synthesized can be considered a promising addi-
tion to the family of long-wavelength fluorescent probes for
biotechnology applications.
[25] G. Cravotto, E.C. Gaudino, P. Cintas, Chem. Soc. Rev. (2013), http://dx.doi.org/
10.1039/c2cs35456j.
Acknowledgements
[26] A.R. Gholap, K. Venkatesan, T. Daniel, R.J. Lahoti, K.V. Srinivasan, Green Chem. 5
(2003) 693–696.
[27] R. Cella, H.A. Stefani, Tetrahedron 62 (2006) 5656–5662.
[28] S. Sadjadi, S. Sadjadi, R. Hekmatshoar, Ultrason. Sonochem. 17 (2010) 764–767.
[29] P. Cintas, G. Palmisano, G. Cravotto, Ultrason. Sonochem. 18 (2011) 836–841.
Thanks are due to Fundação para a Ciência e Tecnologia (FCT, Por-
tugal) 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 the Research Centres CQ/UM [Strategic Pro-
ject PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)]
and CFUM [Strategic Project PEst-C/FIS/UI0607/2011 (F-COMP-
´
[30] M. Baron, E. Metay, M. Lemaire, F. Popowycz, J. Org. Chem. 77 (2012) 3598–
3603.
[31] C. Cui, C. Zhu, X.-J. Du, Z.-P. Wang, Z.-M. Li, W.-G. Zhao, Green Chem. 14 (2012)
3157–3163.
[32] B. Dar, A. Singh, A. Sahu, P. Patidar, A. Chakraborty, M. Sharma, B. Singh,
Tetrahedron Lett. 53 (2012) 5497–5502.
[33] S. Sadjadi, M. Eskandari, Ultrason. Sonochem. 20 (2013) 640–643.
[34] N.G. Khaligh, F. Shirini, Ultrason. Sonochem. 20 (2013) 26–31.
[35] M.J.L. Fiego, M.A. Badajoz, C. Domini, A.B. Chopa, M.T. Lockhart, Ultrason.
Sonochem. 20 (2013) 826–832.
01-0124-FEDER-022711)].
A post-doctoral grant to B.R. Raju
(SFRH/BPD/62881/2009) is also acknowledged to FCT, POPH-QREN,
FSE.
[36] V.H.J. Frade, S.A. Barros, J.C.V.P. Moura, M.S.T. Gonçalves, Tetrahedron Lett. 48
(2007) 3403–3407.
[37] V.H.J. Frade, S.A. Barros, J.C.V.P. Moura, P.J.G. Coutinho, M.S.T. Gonçalves,
Tetrahedron 63 (2007) 12405–12418.
[38] C.M.A. Alves, S. Naik, P.J.G. Coutinho, M.S.T. Gonçalves, Tetrahedron 65 (2009)
10441–10452.
[39] C.M.A. Alves, S. Naik, P.J.G. Coutinho, M.S.T. Gonçalves, Tetrahedron Lett. 52
(2011) 112–116.
[40] A.D.G. Firmino, M.S.T. Gonçalves, Tetrahedron Lett. 53 (2012) 4946–4950.
[41] B.R. Raju, A.D.G. Firmino, A.L.S. Costa, P.J.G. Coutinho, M.S.T. Gonçalves,
Tetrahedron 69 (2013) 2451–2461.
[42] A.M. Piloto, A.M.S. Soares, S.P.G. Costa, M.S.T. Gonçalves, Amino Acids 42
(2012) 2275–2282.
[43] M.L. Crossley, P.F. Dreisbach, C.M. Hofmann, R.P. Parker, J. Am. Chem. Soc. 74
(1952) 573–578.
[44] R. Sens, K.H. Drexhage, J. Luminesc. 24 (1981) 709–712.
References
[1] M.S.T. Gonçalves, Chem. Rev. 109 (2009) 190–212.
[2] B. Dumat, G. Bordeau, A.I. Aranda, F. Mahuteau-Betzer, Y.E. Harfouch, G. Metgé,
F. Charra, C. Fiorini-Debuisschert, M.P. Teulade-Fichou, Org. Biomol. Chem. 10
(2012) 6054–6061.
[3] M. Saito, H. Saitoh, Biosci. Biotechnol. Biochem. 76 (2012) 1777–17780.
[4] A. Grofesik, M. Kubinyi, A. Ruzsinsky, T. Veszprémi, W.J. Jones, J. Mol. Struc.
(Theochem.) 555 (2000) 15–19.
[5] A. Chakraborty, R. Adhikari, S.K. Saha, J. Mol. Liq. 164 (2011) 250–256.
[6] H.M. Sobell, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5328–5331.
[7] K.N. Thimmaiah, J.B. Easton, G.S. Germain, C.L. Morton, S. Kamath, J.K.
Buolamwini, P.J. Houghton, J. Biol. Chem. 280 (2005) 31924–31935.
[8] N. Motohashi, L.A. Mitscher, R. Meyer, Med. Res. Rev. 11 (1991) 239–294.
[9] X. Song, D.S. Kassaye, J.W. Foley, J. Fluoresc. 18 (2008) 513–518.