Synthesis and characterization of novel color chemosensors based on azo dyes for possible application in opioid pharmacology

Article abstract of DOI:10.1002/jhet.436

(Chemical Equation Presented) An applicable strategy of chemical labeling of morphine (M) and 6-acetyl morphine (6-AM) using diazonium salts is described. M or 6-AM was coupled with aryl diazoinum salts to give morphine azo dyes. The coupling of the diazotized 4,4′-diaminostilbene with M or 6-AM in ratio 1:2 gave stilbene-based azo dyes containing two M or 6-AM units, respectively. Diazotization of 5-(p-aminophenyl)-10,15,20-triphenylporphyrin and subsequent azo coupling of the diazoniun salt with M and with 6-AM was our route to get highly fluorescent morphine dyes. The resulting dyes can exist in two possible tautomeric structures, azo and hydrazone, stabilized to a significant extent by intramolecular H-bonding. It was shown that these dyes exists predominantly or exclusively in their hydrazone form. This conclusion is drawn from the lack of a distinct band in the 380-420 nm region of the absorption spectra (a region in which the long wavelength absorption band for the azo-form is observed), together with results of NMR studies in deuterated DMSO. The tautomeric properties of the compounds were judged by density functional calculations at the B3LYP/6-31G* and B3LYP/6-31G** levels. The analysis of spiked compounds in human urine samples was studied by capillary electrophoresis (CE) with UV-fluorescence photo-diode array detectors.

Full text of DOI:10.1002/jhet.436

1134
Synthesis and Characterization of Novel Color Chemosensors
Based on Azo Dyes for Possible Application in Opioid
Pharmacology
Vol 47
Afaf R. Genady*
Department of Chemistry, Faculty of Science, University of Tanta, 31527 Tanta, Egypt
*E-mail: afafgenady@hotmail.com
Received November 7, 2009
DOI 10.1002/jhet.436
Published online 19 July 2010 in Wiley Online Library (wileyonlinelibrary.com).
An applicable strategy of chemical labeling of morphine (M) and 6-acetyl morphine (6-AM) using di-
azonium salts is described. M or 6-AM was coupled with aryl diazoinum salts to give morphine azo
dyes. The coupling of the diazotized 4,40-diaminostilbene with M or 6-AM in ratio 1:2 gave stilbene-
based azo dyes containing two M or 6-AM units, respectively. Diazotization of 5-(p-aminophenyl)-
10,15,20-triphenylporphyrin and subsequent azo coupling of the diazoniun salt with M and with 6-AM
was our route to get highly fluorescent morphine dyes. The resulting dyes can exist in two possible tau-
tomeric structures, azo and hydrazone, stabilized to a significant extent by intramolecular H-bonding. It
was shown that these dyes exists predominantly or exclusively in their hydrazone form. This conclusion
is drawn from the lack of a distinct band in the 380–420 nm region of the absorption spectra (a region
in which the long wavelength absorption band for the azo-form is observed), together with results of
NMR studies in deuterated DMSO. The tautomeric properties of the compounds were judged by density
functional calculations at the B3LYP/6-31G* and B3LYP/6-31G** levels. The analysis of spiked com-
pounds in human urine samples was studied by capillary electrophoresis (CE) with UV-fluorescence
photo-diode array detectors.
J. Heterocyclic Chem., 47, 1134 (2010).
INTRODUCTION
of organic synthesis (Fig. 1) [4,5]. Hence, a number of
morphine derivatives have been reported to date [6].
Heroin, which is obtained synthetically from the acetyla-
tion of M, has an analgesic potency two to three times
that of the parent drug and, due to the two acetyl
groups, has better penetration across the blood-brain bar-
rier [7]. Heroin itself is rarely present in detectable
quantities in body fluids. The drug hydrolyses rapidly to
6-acetylmorphine (6-AM), which in turn hydrolyses to
M. Therefore, heroin consumption can be confirmed by
identifying its two primary metabolites [8,9]. In addi-
tion, heroin is different from most other opioids in that
it has little or no affinity for opioid receptors in the
brain. The analgesic effects of the drug are attributed to
the combined effect of 6-AM and M [2].
A great amount of attention continues to be devoted
to the development of synthetic molecular receptors
with the ability to recognize neutral organic species,
including abused drugs. The morphine alkaloids com-
prise a family of structurally related natural products of
unique clinical importance in medicine [1]. Morphine is
a fascinating compound that has been used as an effi-
cient analgesic and is indispensable in treating pains
associated with cancer [2]. Morphine (M) is also found
in normal brain, blood, and liver tissue [3]. However, it
is strictly controlled by authorities due to its addictive
nature. On the other hand, the unusual architecture of M
has offered a continuing challenge to the art and science
C
V
2010 HeteroCorporation

September 2010
Synthesis and Characterization of Novel Color Chemosensors Based on
Azo Dyes for Possible Application in Opioid Pharmacology
1135
addition, the products of these reactions are unstable,
light sensitive, and give many components that seem to
be associated with the reagent blank.
The most important problems for development of a
new morphine detector are tedious and time-consuming
reaction steps. In an effort to develop novel morphine
derivatives that are effective as chemosensors for heroin
use at very low concentrations, herein we report fast,
economic, and simple approaches to the synthesis of a
novel series of highly fluorescence azo-morphine dyes.
Compared with the previously reported methods [20–
27], the present test produces an intense color which is
not affected by the presence of any diluents or adulter-
ants and which is easily adapted to field use. Diaminos-
tilbene and porphyrin related dyes are strongly light
absorbing and highly luminescent [28,29]. These dyes
are covalently attached to proteins and other biological
and nonbiological materials to make these materials flu-
orescent so that they can be detected. The binding
advantage of trans-4,40-bis-diazostilbene or diazopor-
phyrin over diazobenzene is production of highly fluo-
rescent dyes that can be detected even at very low mor-
phine concentration. The developed method was used
for determination of highly diluted M and 6-AM in
spiked human urine sample with very high accuracy and
precision.
Figure 1. Schematic structures of some abused drugs [heroin, codeine,
6-acetylmorphine (6-AM), and morphine (M)].
It is generally accepted that two sites, the basic nitro-
gen and the phenol moiety, are necessary for analgesic
binding to its receptors [10,11]. The phenolic hydroxyl
group is recognized as a requisite for the formation of a
hydrogen bond with a dipolar site on the receptor and
for good antinociceptive activity [11,12]. However, the
free hydroxyl group is also a potential site for metabo-
lism, conjugation, and excretion resulting in low oral bi-
oavailability and short duration of action [13,14]. One
of the approaches to improve the pharmacological prop-
erties of analogues is to modify this phenolic hydroxyl
function. Several potent compounds have been synthe-
sized and identified by replacing the hydroxyl moiety of
morphinans with other functional groups (amino, car-
boxamido, 2-aminothiazole) [15,16].
EXPERIMENTAL
Materials. All chemicals and reagents were commercially
available and were used as received. Most of solvents were at
least of reagent grade and were used without further purifica-
tion. M, 6-AM, and codeine were obtained from Lipomed Inc.
(One Broaway, Cambridge, MA, USA). 5-(p-Aminophenyl)-
10,15,20-triphenylporphyrin was prepared according to the lit-
erature [30]. Analytical thin layer chromatography (TLC) was
performed on a glass plates of silica gel 60 GF254 (Merck).
Visualization was accompanied by UV light (254 nm). Column
chromatography was conducted on silica gel 60 (Merck 70–
The use of dyes in chemistry, biology, and medicine
is growing continuously, with many new applications in
the diagnosis and treatment of disease [17–19]. More-
over, azo dyes have been known for over forty years,
where they were used for investigations in cancer treat-
ment [20]. Numerous fluorescent probes for monosac-
charides based on azo dyes have been described in the
literature. Moreover, many abused drugs (i.e., heroin,
codine, 6-AM and M) are tertiary amines (Fig. 1) and
are not compatible with the most commonly utilized
amine reactive fluorescent dyes. These dyes include
compounds such as fluorescein isothiocyanate isomer I
(FITC) [21], 4-(4,6-dichloro-s-triazin-2-ylamino)fluores-
cein (DTAF) [22], 4-fluoro-7-nitrobenzofurazan (NBD-F)
[23], and 3-(4-carboxybenzoyl)-2-quinolinecarboxalde-
hyde (CBQCA) [24]. Other fluorogenic reagents specifi-
cally made for derivatization of the tertiary amine group
such as the malonic acid/acetic anhydride system [25]
and the aconitic acid method [26] result in a deteriorat-
ing effect on the fluorescence of the reaction product. In
1
230 mesh). H NMR and 13C NMR spectra were measured on
JEOL JNM-AL 300 (300 MHz) and VARIAN UNITY-INOVA
400 (400 MHz) spectrometers. Chemical shifts of 1H NMR
and 13C NMR were expressed in parts per million (ppm, d
units), and coupling constant was expressed in units of Hertz
(Hz). Elemental analyses were performed by a Perkin-Elmer
2400 automatic elemental analyzer. All compounds gave ele-
mental analysis within 60.4%. Electrospray ionization (ESI)
mass spectra were recorded on a Shimadzu LCMS-2010 eV
spectrometer in CH3OH. The UV–vis data were measured on
Shimadzu 3101 PC instrument. The fluorescence (excitation
and emission) spectra were determined with Perkin Elmer
Lambada 50 PC spectrophotometer: excitation slit width ¼ 5
nm, emission slit width ¼ 5 nm. AP/ACE MDQ CE system
coupled with photo-diode array detectors (PAD) supplied from
Beckman (Fullerton, CA, USA) was used throughout the
experiments. Separation was carried out in a 50.2 cm long ꢀ
50 lm (10 cm to the detector, short way). After each
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1136
A. R. Genady
Vol 47
experiment, the capillary was washed with 0.1 mol dmꢁ3 so-
dium hydroxide for 2.0 min, distilled water for 1.0 min, and
the separation electrolyte for 2.0 min. Hydrodynamic injection
mode was applied for sample loading. 32 Karat version 7.0
supplied from Beckman (Fullerton, CA, USA) was used for
controlling the CE system as well as data acquisition and
processing.
General procedure for the synthesis of compounds
1–6. Diazotization. A 0–5ꢂC solution of substituted aniline
(0.5 mmol) and 1N HCl (2 mL) in deionized (DI) water (5
mL) was treated with a 0–5ꢂC solution of NaNO2 (100 mg,
1.5 mmol) in DI water (5 mL) and the diazotization continued
for 10 min.
Coupling. The resulting diazonium salt solution was poured
into a 0–5ꢂC solution of M or 6-AM (0.5 mmol) in NaOH (50
mg, 1.25 mmol, 5 mL). The mixture was stirred at 0–5ꢂC for
another 10 min. The resulting precipitate was filtered off,
washed with NaCl, DI water, and dried in vacuo. The products
were purified by flash column chromatography using hexane
and ethylacetate in ratio 2:1 as eluent. Upon storage of the azo
coupling products 1–6 at ambient temperature for several
months neither change in their UV–vis spectra nor appearance
of foreign signals in their 1HNMR spectra were observed,
which provides evidence of their stability.
z(%): 433 (85) [Mþ]; Anal. Calcd. for C24H23N3Os (433.46):
C 66.50, H 5.35, N 9.69; found: C 66.42, H 5.29, N 9.61.
2-(p-Methoxy-phenylazo)morphine (3). Yield: 193 mg
(92%), mp ¼ 167–169ꢂC, Rf ¼ 0.39 as red solid; IR (KBr) m:
3382, 3375 (OH), 1635 (C¼¼C, alkene), 1624 (C¼¼O, hydra-
zone), 1610 (C¼¼N), 1523 (C¼¼C, aromatic), 1287, 1094
1
(CAOAC) cmꢁ1; HNMR (300 MHz, DMSO): d ¼ 12.75 (br
s, 1H, NHO), 7.79 (d, J ¼ 8.12 Hz, 2H, aromatic H), 7.54 (d,
J ¼ 8.12 Hz, 2H, aromatic H), 6.97 (s, 1H, aromatic H), 5.42
(d, 1H, J ¼ 9.25 Hz, CH¼¼CH), 5.25 (d, 1H, J ¼ 9.25 Hz,
CH¼¼CH), 4.68 (d, 1H, J ¼ 7.51 Hz), 4.22–4.25 (m, 1H),
3.41–3.55 (m, 1H), 3.02 (d, 1H, J ¼ 17.32 Hz), 2.58–2.65 (m,
1H), 2.57 (d, 1H, J ¼ 7.19 Hz), 2.45 (dd, 1H, J ¼ 5.35 Hz),
2.36 (s, 3H, NCH3), 2.27 (dd, 1H, J ¼ 4.17 Hz), 2.05 (td, 1H,
J ¼ 9.05 Hz, J ¼ 5.6 Hz), 1.92 (d, 1H, J ¼ 10.51 Hz);
13CNMR (75 MHz, DMSO) d ¼ 183.52 (C¼¼O), 158.45,
156.79, 146.45, 138.95, 131.57, 126.87, 42.45, (C), 132.98,
128.45, 127.96, 125.34, 119.12, 118.07, 91.35, 67.85, 58.96,
40.84 (CH), 45.56, 35.05, 21.06 (CH2), 60.13 (OACH3), 42.69
(NACH3). (ESI): m/z(%) 419 (93) [Mþ]; Anal. Calcd. for
C24H25N3O4 (419.47): C 68.72, H 6.01, N 10.02; Found: C
68.67, H 6.00, N 10.00.
2-(p-Nitro-phenylazo)morphine (4). Yield: 182 mg (84%),
mp ¼ 192–194ꢂC, Rf ¼ 0.41 as a red solid. IR (KBr) m: 3380,
3375 (OH), 1638 (C¼¼C, alkene), 1620 (C¼¼O, hydrazone),
1600 (C¼¼N), 1520 (C¼¼C, aromatic), 1517, 1334, (NO2),
4-(Morphine-2-yl-azo)benzenesulfonic acid (1). Yield: 200
mg (87%), mp ¼ 156–158ꢂC, Rf ¼ 0.48 as a red solid; IR
(KBr) m: 3385, 3379 (OH), 1641 (C¼¼C, alkene), 1620 (C¼¼O,
hydrazone), 1605 (C¼¼N), 1525 (C¼¼C, aromatic), 1350, 1150
1282, 1091 (CAOAC) cmꢁ1 1HNMR (300 MHz, DMSO): d
;
¼ 13.52 (br s, 1H, NHO), 7.82 (d, J ¼ 6.52 Hz, 2H, aromatic
H), 7.65 (d, J ¼ 6.52 Hz, 2H, aromatic H), 7.05 (s, 1H, aro-
matic H), 5.62 (d, 1H, J ¼ 9.07 Hz, CH¼¼CH), 5.19 (d, 1H, J
¼ 9.07 Hz, CH¼¼CH), 4.72 (d, 1H, J ¼ 6.95 Hz), 4.37–4.32
(m, 1H), 3.35–3.29 (m, 1H), 3.0 (d, 1H, J ¼ 16.98 Hz), 2.55–
2.6 (m, 1H), 2.52 (d, 1H, J ¼ 7.0 Hz), 2.45 (dd, 1H, J ¼ 5.1
Hz), 2.35 (s, 3H, NCH3), 2.24 (dd, 1H, J ¼ 4.56 Hz), 1.94 (td,
1H, J ¼ 8.79 Hz, J ¼ 5.12 Hz), 1.85 (d, 1H, J ¼ 10.44 Hz);
13CNMR (75 MHz, DMSO) d ¼ 182.54 (C¼¼O), 157.55,
155.05, 147.15, 139.05, 132.02, 126.52, 42.63, (C), 132.85,
128.49, 128.02, 125.65, 119.22, 118.47, 91.05, 67.75, 58.72,
40.79 (CH), 45.34, 34.79, 22.12 (CH2), 42.77 (NACH3).
(ESI): m/z(%) 434 (91) [Mþ]; Anal. Calcd. for C23H22N4Os
(434.44): C 63.59, H 5.10, N 12.90; found: C 63.54, H 5.06, N
12.86.
(SO2), 1282, 1091 (CAOAC) cmꢁ1 1H NMR (300 MHz,
;
DMSO): d ¼ 12.75 (br s, 1H, NHO), 7.78 (d, J ¼ 7.85 Hz,
2H, aromatic H), 7.56 (d, J ¼ 7.85 Hz, 2H, aromatic H), 7.02
(s, 1H, aromatic H), 5.53 (d, 1H, J ¼ 9.12 Hz, CH¼CH), 5.28
(d, 1H, J ¼ 9.12 Hz, CH¼¼CH), 4.76 (d, 1H, J ¼ 7.5 Hz),
4.23–4.27 (m, 1H), 3.37–3.32 (m, 1H), 3.01 (d, 1H, J ¼ 17.5
Hz), 2.59–2.64 (m, 1H), 2.56 (d, 1H, J ¼ 7.05 Hz), 2.42 (dd,
1H, J ¼ 5.24 Hz), 2.36 (s, 3H, NCH3), 2.25 (dd, 1H, J ¼ 4.2
Hz), 1.96 (td, 1H, J ¼ 9.32 Hz, J ¼ 5.12 Hz), 1.87 (d, 1H, J
¼ 10.54 Hz); 13C NMR (75 MHz, DMSO) d ¼ 183.23
(C¼¼O), 156.59, 153.45, 147.23, 139.75, 131.17, 126.5, 42.95
(C), 133.05, 129.89, 128.12, 125.15, 118.23, 117.76, 91.25,
68.45, 59.34, 40.86 (CH), 45.56, 35.05, 21.06 (CH2), 42.69
(NACH3); MS (ESI), m/z(%): 469 (100) [Mþ]; Anal. Calcd.
for C23H23N3O6S (469.51): C 58.84, H 4.94, N 8.95; found: C
58.81, H 4.89, N 8.92.
4-(6-Acetylmorphine-2-yl-azo)benzenesulfonic acid (5). Yield:
230 mg (90%), mp ¼ 145–147ꢂC, Rf ¼ 0.5 as a red solid; IR
(KBr) m: 3382, 3375 (OH), 1722 (C¼¼O, acetyl), 1640 (C¼¼C,
alkene), 1619 (C¼¼O, hydrazone), 1602 (C¼¼N), 1521 (C¼¼C,
2-(m-Carboxy-phenylazo)morphine (2). Yield: 165 mg
(76%), mp ¼ 184–186ꢂC, Rf ¼ 0.32 as an orange solid; IR
(KBr) m: 3382, 3375 (OH), 1712 (C¼¼O, carboxylic), 1642
(C¼¼C, alkene), 1618 (C¼¼O, hydrazone), 1595 (C¼¼N), 1521
aromatic), 1350, 1150 (SO2), 1285, 1095 (CAOAC) cmꢁ1
;
1HNMR (300 MHz, DMSO): d ¼ 13.27 (br s, 1H, NHO), 7.77
(d, J ¼ 7.9 Hz, 2H, aromatic H), 7.58 (d, J ¼ 7.9 Hz, 2H, aro-
matic H), 7.0 (s, 1H, aromatic H), 5.52 (d, 1H, J ¼ 9.02 Hz,
CH¼¼CH), 5.25 (d, 1H, J ¼ 9.02 Hz, CH¼¼CH), 4.75 (d, 1H, J
¼ 7.32 Hz), 4.25–4.29 (m, 1H), 3.35–3.4 (m, 1H), 3.04 (d,
1H, J ¼ 17.56 Hz), 2.59–2.64 (m, 1H), 2.53 (d, 1H, J ¼ 7.0
Hz), 2.40 (dd, 1H, J ¼ 5.24 Hz), 2.35 (s, 3H, NCH3), 2.26
(dd, 1H, J ¼ 4.12 Hz), 2.15 (s, 3H, COCH3), 2.0 (td, 1H, J ¼
8.75 Hz, J ¼ 5.25 Hz), 1.9 (d, 1H, J ¼ 10.26 Hz); 13CNMR
(75 MHz, DMSO) d ¼ 181.76, 172.56 (C¼¼O), 157.21, 154.24,
147.05, 138.95, 131.55, 126.65, 42.31, (C), 132.46, 129.44,
128.75, 125.19, 118.25, 117.86, 91.25, 68.45, 59.34, 40.86
(CH), 45.56, 35.05, 22.06 (CH2), 42.69, 21.17 (NACH3,
COCH3). (ESI): m/z(%) 511 (96) [Mþ]; Anal. Calcd. for
1
(C¼¼C, aromatic), 1280, 1087 (CAOAC) cmꢁ1; H NMR (300
MHz, DMSO): d ¼ 13.25 (br s, 1H, NHO), 7.86–7.45 (m, 4H,
aromatic H), 6.85 (s, 1H, aromatic H), 5.57 (d, 1H, J ¼ 9.0
Hz, CH¼¼CH), 5.32 (d, 1H, J ¼ 9.0 Hz, CH¼¼CH), 4.69
(d, 1H, J ¼ 7.42 Hz), 4.2–4.25 (m, 1H), 3.35–3.3 (m, 1H),
3.04 (d, 1H, J ¼ 15.9 Hz), 2.54–2.59 (m, 1H), 2.51 (d, 1H, J
¼ 7.0 Hz), 2.43 (dd, 1H, J ¼ 4.86 Hz), 2.35 (s, 3H, NCH3),
2.25 (dd, 1H, J ¼ 4.12 Hz), 1.97 (td, 1H, J ¼ 9.67 Hz, J ¼
5.52 Hz), 1.91 (d, 1H, J ¼ 10.05 Hz); 13C NMR (75 MHz,
DMSO) d ¼ 181.83, 177.56 (C¼¼O), 155.69, 145.36, 138.45,
131.65, 126.85, 42.64, (C), 133.21, 126.37, 126.05, 125.87,
125.12, 124.86, 119.45, 118.69, 91.86, 67.75, 58.94, 40.25
(CH), 45.12, 34.68, 21.46 (CH2), 42.85 (NACH3); (ESI): m/
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2010
Synthesis and Characterization of Novel Color Chemosensors Based on
Azo Dyes for Possible Application in Opioid Pharmacology
1137
C25H25N3O7S (511.55): C 58.7, H 4.93, N, 8.21; found: C
58.57; H, 4.91; N, 8.15.
tion of 5-(p-aminophenyl)-10,15,20-triphenylporphyrin (785
mg, 1.25 mmol) in 1N HCl (5 mL). The mixture was stirred at
5ꢂC for 15 min. A solution of sodium acetate (0.14 g, 1.71
mmol) in water (5 mL) and M or 6-AM (1.11 mmol) in 3%
aqueous NaOH (5 mL) were added to the diazonium salt solu-
tion. Then, the reaction mixture was stirred at room tempera-
ture for 15 min and diluted to 100 mL with water and filtered.
The filtrate was neutralized with HCl to pH 7, the porphyrin
filtered off, washed with aqueous ammonia solution (10%),
then with water, and dried to constant weight at room tempera-
ture. For purification, the porphyrin was dissolved in boiling
ether (50 mL) and chromatographed on a column (2.5 cm ꢀ
60 cm) of silica gel eluting with ether. The elute was evapo-
rated to 5 mL and porphyrin (10 or 11) was precipitated with
hexane (20 mL).
2-(m-Carboxy-phenylazo)-6-acetylmorphine (6). Yield: 202
mg (85%), mp ¼ 177–179ꢂC, Rf ¼ 0.35 as an orange solid; IR
(KBr) m: 3377 (OH), 1725 (C¼¼O, acetyl), 1718 (C¼¼O, car-
boxylic), 1639 (C¼¼C, alkene), 1619 (C¼¼O, hydrazone), 1592
(C¼¼N), 1525 (C¼¼C, aromatic), 1282, 1085 (CAOAC) cmꢁ1
;
1HNMR (300 MHz, DMSO): d ¼ 13.39 (br s, 1H, NHO),
7.82–7.5 (m, 4H, aromatic H), 6.89 (s, 1H, aromatic H), 5.55
(d, 1H, J ¼ 8.7 Hz, CH¼¼CH), 5.34 (d, 1H, J ¼ 8.7 Hz,
CH¼¼CH), 4.7 (d, 1H, J ¼ 7.75 Hz), 4.24–4.27 (m, 1H), 3.36–
3.32 (m, 1H), 3.03 (d, 1H, J ¼ 15.35 Hz), 2.55–2.61 (m, 1H),
2.51 (d, 1H, J ¼ 7.4 Hz), 2.45 (dd, 1H, J ¼ 4.85 Hz), 2.3 (s,
3H, NCH3), 2.25 (dd, 1H, J ¼ 4.6 Hz), 2.09 (s, 3H, CH3),
1.93 (td, 1H, J ¼ 9.11 Hz, J ¼ 5.25 Hz), 1.89 (d, 1H, J ¼
10.29 Hz); 13CNMR (75 MHz, DMSO) d ¼ 185.05, 176.98,
172.89 (C¼¼O), 155.43, 145.35, 138.42, 131.62, 126.25, 42.65,
(C), 133.27, 126.57, 126.11, 125.23, 125.34, 124.67, 119.49,
118.21, 91.28, 67.45, 58.75, 40.36 (CH), 45.16, 34.59, 21.61
(CH2), 42.85, 21.99 (NACH3, COCH3); (ESI): m/z(%): 475
(88) [Mþ]; Anal. Calcd. for C26H25N3O6 (475.49): C 65.67, H
5.30, N 8.84; found: C 65.51, H 5.29, N 8.79.
5-(Morphine-2-yl-azophenyl)10,15,20-triphenylporphyrin
(10). Yield: 950 mg, (82%), mp ¼ 230–232ꢂC, Rf ¼ 0.37 as a
voilet solid; IR (KBr) m: 3384, 3377 (OH), 3310 (CH), 2989,
2927 (NH), 1638 (C¼¼C, alkene), 1625 (C¼¼O, hydrazone),
1604(C¼¼N), 1525 (C¼¼C, aromatic), 1280, 1087 (CAOAC)
cmꢁ1
;
1HNMR (300 MHz, DMSO): d ¼ 13.52 (br s, 2H,
NHO), 8.65–8.96 (m, 8H, b-pyrrole), 7.02–8.21 (m, 19H,
Harom), 5.64 (d, 1H, J ¼ 9.05 Hz, CH¼¼CH), 5.2 (d, 1H, J ¼
9.05 Hz, CH¼¼CH), 4.75 (d, 1H, J ¼ 7.0 Hz), 4.35–4.25 (m,
1H), 3.35–3.29 (m, 1H), 3.02 (d, 1H, J ¼ 17.0 Hz), 2.55–2.6
(m, 1H), 2.52 (d, 1H, J ¼ 8.42 Hz), 2.45 (dd, 1H, J ¼ 5.5 Hz),
2.35 (s, 3H, NCH3), 2.25 (dd, 1H, J ¼ 4.5 Hz), 1.99 (td, 1H, J
¼ 8.9 Hz, J ¼ 5.3 Hz), 1.92 (d, 1H, J ¼ 10.52 Hz), ꢁ2.79 (s,
2H, NH); 13CNMR (75 MHz, DMSO) d ¼ 181.94 (C¼¼O),
155.43, 153.56, 149.37, 148.82, 147.05, 139.05, 136.92, 135.8,
132.05, 126.63, 42.83, (C), 133.83, 133.24, 130.82, 129.69,
128.37, 128.23, 125.32, 125.17, 118.23, 117.8, 117.46, 115.03,
112.01, 108.75, 91.54, 67.43, 58.78, 52.46, 40.82 (CH), 45.62,
35.45, 22.65 (CH2), 42.52 (NACH3); (ESI): m/z(%) 925 (100)
[Mþ]; Anal. Calcd. for C61H47N7O3 (926.07): C 79.11, H 5.12,
N 10.59; Found: C 79.03, H 5.01, N 10.49.
General procedure for the synthesis of compounds 7 and
8. These compounds were prepared from M or 6-AM (0.5
mmol) and trans, 4,40-diaminostilbene (407 mg, 1.2 mmol),
using the procedure described for 1–6.
Trans-4,40-bis(morphine-2-yl-azo)stilbene (7). Yield: 365
mg (91%), mp ¼ 199–201ꢂC, Rf ¼ 0.45 as a red solid; IR
(KBr) m: 3385, 3376 (OH), 1642 (C¼¼C, alkene), 1624 (C¼¼O,
hydrazone), 1605 (C¼¼N), 1527 (C¼¼C, aromatic), 1284, 1087
1
(CAOAC) cmꢁ1; HNMR (300 MHz, DMSO): d ¼ 13.45 (br
s, 2H, NHO), 7.80 (d, 2H, aromatic H), 7.54 (t, 2H, aromatic
H), 7.44 (s, 2H, CH¼¼CH), 4.55 (m, 2H), 4.21–4.26 (m, 2H),
3.38–3.33 (m, 2H), 3.0 (m, 2H), 2.59–2.64 (m, 1H), 2.55 (m,
2H), 2.39 (m, 2H), 2.32 (s, 6H, NCH3), 2.21 (m, 2H), 1.99 (m,
2H), 1.89 (m, 2H); 13CNMR (75 MHz, DMSO) d ¼ 182.47
(C¼¼O), 155.98, 146.75, 138.85, 131.27, 126.35, 42.56, (C),
133.12, 129.59, 128.19, 125.16, 118.29, 117.77, 91.45, 68.86,
59.51, 52.43, 40.86 (CH), 45.52, 35.12, 22.12 (CH2), 42.47
(NACH3); (ESI): m/z(%) 802 (85) [Mþ]; Anal. Calcd. for
C48H46N6O6 (802.92): C 71.80, H 5.77, N 10.47; Found: C
71.76, H 5.73, N 10.44.
5-(6-Acetylmorphine-2-yl-azophenyl)10,15,20-triphenylpor-
phyrin (11). Yield: 983 mg (85%), mp ¼ 219–221ꢂC, Rf ¼
0.42 as a voilet solid; IR (KBr) m: 3384, 3377 (OH), 3310
(CH), 2989, 2927 (NH), 1722 (C¼¼O acetyl), 1638 (C¼¼C,
alkene), 1625 (C¼¼O hydrazone), 1604(C¼¼N), 1525 (C¼¼C, ar-
omatic), 1280, 1087 (CAOAC) cmꢁ1 1HNMR (300 MHz,
;
Trans-4,40-bis(6-acetylmorphine-2-yl-azo)stilbene (8). Yield:
365 mg (91%), mp ¼ 212–214ꢂC, Rf ¼ 0.52 as a red solid; IR
(KBr) m: 3384, 3377 (OH), 1730 (C¼¼O, acetyl), 1640 (C¼¼C,
alkene), 1625 (C¼¼O, hydrazone), 1602 (C¼¼N), 1522 (C¼¼C, aro-
DMSO): d ¼ 13.35 (br s, 2H, NHO), 8.95–8.60 (m, 8H, b-pyr-
role), 8.25–6.94 (m, 19H, Harom), 6.83 (s, 1H, aromatic H),
5.56 (d, 1H, J ¼ 8.5 Hz, CH¼¼CH), 5.37 (d, 1H, J ¼ 8.5 Hz,
CH¼¼CH), 4.52 (d, 1H, J ¼ 7.8 Hz), 4.32–4.27 (m, 1H), 3.38–
3.33 (m, 1H), 3.04 (d, 1H, J ¼ 15.35 Hz), 2.59–2.64 (m, 1H),
2.52 (d, 1H, J ¼ 7.4 Hz), 2.45 (dd, 1H, J ¼ 4.85 Hz), 2.32 (s,
3H, NCH3), 2.25 (dd, 1H, J ¼ 4.6 Hz), 2.14 (s, 3H, CH3),
1.95 (td, 1H, J ¼ 9.11 Hz, J ¼ 5.25 Hz), 1.87 (d, 1H, J ¼
10.29 Hz), ꢁ2.79 (s, 2H, NH); 13CNMR (75 MHz, DMSO) d
¼ 183.67, 172.54 (C¼¼O), 155.45, 154.06, 149.49, 148.85,
147.25, 139.15, 136.87, 135.82, 132.35, 127.75, 42.65, (C),
133.73, 133.26, 130.89, 129.73, 128.25, 128.21, 125.37,
125.36, 118.22, 117.85, 117.29, 115.11, 112.14, 108.76, 91.55,
67.45, 58.77, 52.49, 40.85 (CH), 45.64, 35.46, 22.68 (CH2),
42.22, 23.19 (NACH3, COCH3); (ESI): m/z(%) 967 (93) [Mþ];
Anal. Calcd. for C63H49N7O4 (968.11): C 78.16, H 5.10, N
10.13; Found: C 78.02, H 5.07, N 10.09.
;
matic), 1281, 1085 (CAOAC) cmꢁ1 1HNMR (300 MHz,
DMSO): d ¼ 13.49 (br s, 2H, NHO), 7.78 (d, 2H, aromatic H),
7.61 (t, 2H, aromatic H), 7.46 (s, 2H, CH¼¼CH), 4.52 (m, 2H),
4.22–4.27 (m, 2H), 3.4–3.36 (m, 2H), 3.01 (m, 2H), 2.62–2.64
(m, 1H), 2.52 (m, 2H), 2.35 (m, 2H), 2.27 (s, 6H, NCH3), 2.23
(m, 2H), 2.19 (s, 6H, COCH3), 1.97 (m, 2H), 1.86 (m, 2H);
13CNMR (75 MHz, DMSO) d ¼ 181.94, 171.67 (C¼¼O), 155.43,
147.05, 139.05, 132.05, 126.63, 42.83, (C), 133.24, 129.69,
128.23, 125.17, 118.23, 117.46, 91.69, 68.57, 59.38, 52.46, 40.82
(CH), 45.62, 35.45, 22.61 (CH2), 42.49 (NACH3); (ESI): m/z(%)
886 (90) [Mþ]; Anal. Calcd. for C52H50N6O8 (886.99): C 70.41,
H 5.68, N 9.47; Found: C 70.39, H 5.67, N 9.45.
General procedure for the synthesis of compounds 10
and 11. A 0–5ꢂC solution of sodium nitrite (0.12 g, 1.74
mmol) in water (2 mL) was added dropwise to a stirred solu-
Biological studies. A 500 mg Bond Elut SPE column was
used for the extraction. The SPE columns were conditioned by
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1138
A. R. Genady
Vol 47
Scheme 1
the sequential passage of 2 ꢀ 3 mL of methanol, 2 ꢀ 3 mL of
water, and 2 ꢀ 5 mL of water adjusted to pH 9.5 with
NH4OH. Ten millilitres of human urine sample adjusted to pH
9.5 with NH4OH was vortex, centrifuged, and applied to the
SPE columns at a rate of 1.0 mL/min. The columns were
washed with 2 ꢀ 5 mL of distilled water and left to dry for 10
min. The drugs were eluted with a solution consisting of a sin-
gle phase mixture of dichloromethane/acetone (50/50) and col-
lected in glass tubes. The elution solvent was evaporated to
dryness under a nitrogen stream. The dried residues were then
reconstituted in slightly warm water, and derivatization was
carried out and then the samples were analyzed using AP/ACE
MDQ CE system coupled with photo-diode array detectors
(PAD).
dihydrochloride with M or 6-AM in 1:2 stoichiometric
ratio to give stilbene based azo dyes containing two M
(7) or two 6-AM (8) moieties as shown in Scheme 2.
The resulting bis-azo dye are belong to the class of
direct dyes [31,32]. Furthermore, we have established
that 5-(p-aminophenyl)-10,15,20-triphenylporphyrin (9)
[30] is readily diazotized with sodium nitrite in aqueous
mineral acid solution. The diazonium salt obtained is
fairly stable; it ꢂdecomposes significantly at temperature
greater than 25 C. The reaction of porphyrin diazonium
salt with M or 6-AM leads to porphyrins containing res-
idues of azo dyes in meso position of 10 or 11, respec-
tively (Scheme 3). The resulting colored compounds
were purified by flash column chromatography using
hexane/ethyl acetate (2:1) as eluent to produce azo-M
(1–4, 7, and 10) and azo-6-AM (5, 6, 8, and 11) with
excellent yields. Azo coupling reactions of morphines
occur predominately ortho to the electron donating
hydroxyl group of the morphine aromatic ring. Hence,
the inclusion of this design motif in the target dyes
avoids potentially difficult separation of isomers.
RESULTS AND DISCUSSION
Synthesis. Our straightforward synthesis of morphine
azo dyes (1–6) is outlined in Scheme 1. In a first step,
the diazonium ions of aniline derivatives were generated
with sodium nitrite in 1N HCl. The diazonium ions
were then coupled by nucleophilic substitution with the
corresponding substrates M or 6-AM. Azo coupling
reactions were performed using the diazonium salts of
4-aminosulfonic acid, 3-aminobenzoic acid, 4-methoxya-
niline, and 4-nitroaniline to yield 2-(arylazo)morphines
1–6, respectively, (Scheme 1). No reaction was found,
however, to occur with codeine under the same reaction
conditions.
Spectroscopic studies. Overall characterization of
dye structures was carried out by elemental analysis,
NMR, UV–vis, IR, and mass spectrometry (see experi-
mental section for details). The NMR spectra of com-
pounds 1–8, 10, and 11 are consistent with proposed
structures, showing the expected features with correct
1
integration ratios. Both H and 13C NMR spectra indi-
Synthesis of fluorescent azostilbene morphine dyes
was achieved by reaction of trans-4,40-diazostilbene
cated the appearance of new signals corresponding to
the aryl moiety of each azo-compound (Fig. 2). Spectral
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Synthesis and Characterization of Novel Color Chemosensors Based on
Azo Dyes for Possible Application in Opioid Pharmacology
1139
Scheme 2
NMR spectroscopy is established as an effective tech-
nique to study tautomer composition [33–35]. The intra-
molecular hydrogen bond ring is essentially planar and
coplanar with its adjacent phenyl ring, which stabilized
the hydrazone form. 2-Arylazomorphine derivatives
(dyes 1–8, 10, and 11) exist predominantly in the hydra-
zone form via intramolecular hydrogen bonds, which
result in the linearity and coplanar conformation of the
dyes [28]. The proton peaks involved in hydrogen bonds
appear at much lower field than normal proton peak
of hydroxyl group and these (12.75–13.52 ppm for dyes
1–8, 10, and 11) were confirmed by 1H NMR. The
downfield position of the resonance from the hydrazone
proton is attributed to internal hydrogen bonding in
which the carbonyl oxygen is hydrogen bonded to this
proton [33]. Dyes that occur as the azo tautomer show a
13C resonance at ca. 160 ppm from the carbon attached
to the phenolic hydroxyl group, whereas those that occur
as the hydrazone tautomer show a resonance at ca. 180
ppm for the same carbon atom within a carbonyl group
(Fig. 2). Dyes that occur as both tautomers show a sin-
gle resonance between these limits, due to rapid tauto-
merisation, with the position determined by the relative
concentrations of the two tautomers [34–36]. 13C NMR
spectra from DMSO samples of 1–8, 10, and 11 con-
firmed the hydrazone structure by detecting a new car-
bonyl peak at 181–183 ppm assigned to C3 of M or
6-AM and, thus, morphine dyes 1–8, 10 and 11 are pres-
ent as the hydrazone tautomer (ca. 100%).
properties of synthesized dyes were affected by intramo-
lecular hydrogen bond between the phenolic hydroxyl
group of morphine moiety and the central nitrogen atom
of the azo bridge of the azo dye residues. Azo dyes in
which the azo group is conjugated with a hydroxyl
group can exhibit azo-hydrazone tautomerism, and
According to DFT calculations at the B3LYP/6-31G*
level, the hydrazono toutomers of 1–8, 10, and 11 are
Scheme 3
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1140
A. R. Genady
Vol 47
13
Figure 2. C NMR spectrum of compound 3 in DMSO(d6) at 20ꢂC.
favored over the azo toutomers by 2.4–3.6 kcal moleꢁ1
.
The electronic absorption spectra (EAS) of the inves-
tigated morphine dyes 1–8, 10, and 11 in ethanolic solu-
tions were studied. There is no visual evidence for a
band around 380–420 nm, which could be assigned to
the azo form. The compounds comprised two to three
bands in the UV region and one band in the visible
region (Fig. 4). The band of shortest wavelength appear-
ing in the range 210–255 nm was best ascribed to p–p*
transition of the benzenoid system of the compounds.
The second band observed in UV region, in the wave-
length range 270–285 nm was attributed to p–p* transi-
tion within the furan heterocyclic moiety of the com-
pounds. The third band observed in the UV region at
285–290 nm was assigned to n–p* electronic transition
As major entropy differences and crystal lattice effects
are not to be expected here, these results should secure
the hydrazone formulas. Consequently, it can be consid-
ered that morphine azo dyes (1–8, 10, and 11) have
such structures as shown in Schemes 1–3.
IR spectra assigned with the aid of the NMR data,
provide fingerprints for hydrazone form and hydrogen
bonding. The IR spectra of all the resulting colored
compounds confirmed the presence of a C¼¼O bond
which resonates at 1625–1618 cmꢁ1. IR spectrum of
compound 4 showed absorption bands at 1517 and 1334
cmꢁ1 due to the presence of a NO2 group, whereas the
SO2 group of compound 1 and 5 has two vibrational fre-
quencies at 1350 and 1150 cmꢁ1. Moreover, the vibra-
tional frequency of aliphatic OH band m(OAH) of M or
the COCH3 band m(C¼¼O) of 6-AM was not found to be
sensitive to the connection of M or 6-AM with the azo
derivatives. For compounds 1–4 and 7 m(OAH) lies in
the range of 3385–3375 cmꢁ1, and for compounds 5, 6
and 8 m(C¼¼O) from 1730 to 1718 cmꢁ1, comparable to
the frequencies of M m(OAH) at 3373 cmꢁ1, and 6-AM
m(C¼¼O) at 1713 cmꢁ1, indicating that the intra bonding
of the morphine moiety was not perturbed by substitu-
tion on the phenyl ring.
Evidence in support of structures 1–8, 10, and 11 is
presented by mass spectrometry. The ESI-MS spectra of
these compounds in MeOH showed molecular ion peaks
(Mþ) corresponding to the formula of each compound.
Mass spectra of all compounds showed molecular ion
peaks corresponding to their expected pattern of abun-
dance ranging from 85 to 100% (Fig. 3).
Figure 3. ESI-MS of compound 7 in CH3OH.
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Synthesis and Characterization of Novel Color Chemosensors Based on
Azo Dyes for Possible Application in Opioid Pharmacology
1141
the azo dye interacts with the p system of porphyrin
ring. The Soret band of tetraphenyporphyrin (kmax
ꢃ
400 nm; e ꢃ 4.75 ꢀ 105 dm3 molꢁ1 cmꢁ1) is found
alongside with the broad absorption band of azo dye res-
idue (kmax ꢃ 590 nm; e ꢃ 3.35 ꢀ 104 dm3 molꢁ1
cmꢁ1), which does not permit a confident judgment to
be made on whether transfer of p electron density from
the azo dye residue to the porphyrin ring has taken
place. However, the sharp reduction in intensity of the
Soret band and the growth in intensity of the electronic
transition bands and also their bathochromic shift indi-
cate the existence of such interactions.
The compounds (1–8) exhibited emission fluorescence
peak even at very low concentration (5–8 ꢀ 10ꢁ9 mol
dmꢁ3) in aqueous solution as indicated by capillary
electrophoresis (CE) with UV-fluorescence photo diode-
array detectors. However, compounds 10 and 11 showed
highly intense fluorescence peaks at 665 and 670 nm,
respectively. The synthesis of 10 and 11 will provide
new insight into the role of morphine determination
using simple and fast chemistry as well as highly sensi-
tive techniques [e.g., CE with laser induced fluorescence
detector (LIF)].
Figure 4. Electronic absorption spectra of 2 ꢀ 10-5 mol dm-3 of 1, di-
azonium salt of sulfanilic acid and M.
of OH groups. The long wavelength band at about 510
nm corresponds to the hydrazone form [37] (Fig. 4).
This band was capable of being assigned to p–p* transi-
tion involving the whole electronic system of the com-
pounds with a considerable charge-transfer (CT) charac-
ter. Such a CT originated mainly from the aryl azo to
the morphine moiety, i.e., this band was due to intramo-
lecular CT transition. When analyzing EAS of the por-
phyrin azo dyes (10 and 11), it was difficult to draw an
unambiguous conclusion as to whether the p system of
Biology. The determination of M in biological sam-
ples has become almost a routine assay in many toxicol-
ogy laboratories owing to the spread of the abuse of
Figure 5. Electropherograms of 5 ꢀ 10-6 mol dm-3 diazonium salt of sulfanilic acid in water under the optimized conditions: 10.0 s injection time,
applied voltage 25 kV, 25ꢂC, 100 mmol dm-3 borate electrolyte concentration, and pH 9.0.
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1142
A. R. Genady
Vol 47
Figure 6. Electropherograms of human urine sample spiked with 5 ꢀ 10-9 mol dm-3 of M coupled with 5 ꢀ 10-6 mol dm-3 diazonium salt of sulfa-
nilic acid under the optimized conditions: 10.0 s injection time, applied voltage 25 kV, 25ꢂC, 100 mmol dm-3 borate electrolyte concentration, and
pH 9.0.
heroin, which is mainly biotransformed into M. In this
experiment, the coupling reaction of diazonium salt of
sulfanilic acid was carried out with drug-free urine sam-
ple and with urine sample spiked with M and 6-AM. No
remarkable change was observed for the drug-free urine
sample as indicated by CE (data not shown). For urine
sample spiked with the M, a deep red color appeared at
once which was measured by CE, giving two peaks after
45 s and 65 s corresponding to azo-M (1) and diazo-
nium salt of sulfanilic acid, respectively, (Figs. 4 and 5).
The extraction recoveries were found to be >99.5% and
RSD values of the recovery did not exceed 0.92% indi-
cating good repeatability of the adopted method.
central N atom intramolecular proton transfer exists
with the hydrazone form being major component. The
compounds existed in hydrazone forms exclusively,
being stabilized by the intramolecular hydrogen bonds.
The resulting azo compounds are highly fluorescent in
ethanol and water. Low detection limit was obtained
ranging from 5–8 nmol dmꢁ3 for M or 6-AM coupled
with freshly prepared diazonium salts. Consequently,
this method is characterized by simple, rapid, and eco-
nomic determination of abused drugs in forensic cases
as an initial test and clinical analysis to prevent over-
dose-induced toxicity.
Acknowledgments. The author is grateful to Professor Hir-
oyuki Nakamura, Department of Chemistry, Faculty of
Science, Gakushuin University, Japan, for providing facili-
ties for NMR and mass spectra measurements. They also
thank Dr. Mohamed E. El-Zaria, Department of Chemis-
try, Faculty of Science, Tanta University, Egypt, for HPLC
measurements.
CONCLUSIONS
A number of M and 6-AM azo dyes were synthesized
and the possibility of using these dyes as color chemo-
sensors of abused drugs is reported. The synthesis starts
from commercially available aniline derivatives and can
be completed in one step with an overall yield of 76–
92%. Trans-4,40-diaminostilbene or 5-(p-aminophenyl)-
10,15,20-triphenylporphyrin in azo dye reaction gave
highly fluorescent morphine dyes which could be easily
detected at very low concentrations using CE techni-
ques. It is found that between the phenolic OH and the
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