E. Jeong et al. / Dyes and Pigments 87 (2010) 100e108
101
NH2
NH2
diazonium salt solution was checked with KI/starch paper for the
presence of HNO2 and solid sulfamic acid was added to destroy
excess HNO2. The solution was clarified by adding activated carbon
with stirring and the adsorbent was removed by filtration, with
simultaneous cooling of the filtrate in an ice bath.
R2
NH2
R1
1
NH2
2
Compound 11 (0.9 g, 5 mmol) was stirred with 10 mL H2O as HCl
(20%, 3 mL) was added slowly at 0 ꢀC. To this solution, NaOAc (1.8 g,
0.022 mol) was added with stirring. The cold solution of diazo
compound prepared above was added to this solution, keeping the
temperature at 0 ꢀC. After stirring for 24 h at 0 ꢀC, the yellow solid
was collected by filtration. The acetyl group was removed in
a solution of NaOH (1.4 g), 95% EtOH (14. mL), H2O (7 mL) by stirring
for 2 h at 100 ꢀC. The reaction mixture was cooled to RTand adjusted
to pH 7 using 20% HCl. The solvent was removed at reduced pressure
and the residue was extracted with DMF to remove salts. The brown
oil obtained after DMF removal was crystallized from acetone/H2O
to give 1 g (58%) dye 3 having m.p. 128 ꢀC (lit. [8] m.p. 132e134 ꢀC).
R
2 = OMe, OEt, OPr, OBu, O(CH2)2OH
R1 = CH(Me)2, Bu, OBu, O(CH2)2OH
Fig. 1. Phenylenediamines containing alkyl or alkoxy groups ortho to an amino group.
the role of the 40-N(CH2CH2OH)2 group in producing a mutagenic
response when an eOCH2CH2OH is incorporated into the 3-position.
The synthesis of dyes 4e7 (Fig. 4), arising from modifications of the
eN(CH2CH2OH)2 group, was conducted, and the resultant novel dyes
were characterized using 1H NMR, high-resolution mass spectrom-
etry, and combustion analyses. These new compounds permitted an
assessment of the importance of the number of N-hydroxyethyl
groups (dyes 4 and 5), the length of N-hydroxyalkyl chains (dye 6),
and the presence of free eOH groups (dye 7) in the mutagenicity of
parent dye 3. Mutagenicity of the target dyes was assessed using
three strains of Salmonella typhimurium.
1H NMR (DMSO-d6):
d
7.64e7.67 (d, 2H),
4.94e4.99 (t, 1H),
3.74e3.80 (q, 2H),
d 7.27e7.37 (m, 2H),
4.81e4.85 ppm (t, 2H),
d 3.51e3.71 (m, 8H), and
d
6.70e6.81 (m, 3H),
3.99e4.03 (t, 2H),
d
d
d
d
TLC: Rf ¼ 0.53 (2-Propanol:Toluene ¼ 1:3).
2.2.2. 4-((3-(2-Hydroxyethoxy)-4-amino)phenylazo)-N-
(2-hydroxyethyl)aniline (4)
2. Experimental
Compound 10 (1 g, 4.8 mmol) was diazotized and the resultant
solution clarified using activated carbon in the manner described
above for the synthesis of dye 3. Compound 12 (0.7 g, 5 mmol) was
suspended in H2O (10 mL) with stirring as HCl (20%, 3 mL) was added
slowly at 0 ꢀC. NaOAc (1.8 g, 0.022 mol) was added to this solution
with stirring and the coldsolution of diazo compound prepared in the
previous step was added to this solution, keeping the temperature at
0 ꢀC. After stirring for 24 h at 0 ꢀC, the yellow solid was collected by
filtration. Deacetylation, workup and dye isolation were the same as
outlined above for dye 3, to give 0.9 g (57%) dye 4 having m.p.
2.1. General
All of the chemicals used as starting materials in this study were
purchased from Aldrich Chemical Co., Milwaukee, WI. All of the
solvents used were obtained from Fisher Scientific, Pittsburgh, PA,
except for DMF, which was purchased from Aldrich Chemical Co. The
plates used for thin layer chromatography were Whatman PE silica
gel plates with UV254 indicator. 1H-NMR spectra were recorded on
a Brucker 500 MHz spectrometer and mass spectra were recorded on
an Agilent Technologies 6210 LCeTOF mass spectrometer. Visible
absorption spectra were recorded in MeOH, using a Varian Cary 3
UVeVisible spectrophotometer. The lmax values are those selected by
the software connected to spectrophotometer. The standard Salmo-
nella tests employing TA98, TA100, and TA1538 with and without S9
were conducted in this study [6]. Mutagenicity testing was conducted
at the US EPA, Research Triangle Park, North Carolina. Elemental
analyses were performed by the Analytical Service Laboratory in the
Soil Science Department of North Carolina State University.
106e107 ꢀC. 1H NMR (DMSO-d6):
7.23e7.24 (dd,1H), 6.65e6.70 (m, 3H),
2H), 4.94e4.97 (t,1H), 4.76 (s,1H), 3.98e4.00 (t, 2H),
(q, 2H), 3.55e3.60(q, 2H),
d
7.57e7.60 (d, 2H),
6.29e6.32 (t,1H),
3.75e3.77
d
7.26 (d, 1H),
d
d
d
d
5.56 (s,
d
d
d
d
d
d
3.16e3.20(q, 2H). TLC:Rf ¼ 0.33(EtOAc).
Calculated for C16H20N4O3: C, 60.75; H, 6.37; N,17.71. Found: C, 60.57;
H, 6.83; N, 17.19.
2.2.3. 4-((3-(2-Hydroxyethoxy)-4-amino)phenylazo) aniline (5)
Compound 16 (0.7 g, 2.3 mmol) was dissolved in a mixture of
H2O (5 mL) and DMF (1 mL). NaOH (1 N) was added to adjust the
mixture to pH 10 and the mixture was heated to 45 ꢀC. Na2S (0.36 g)
was added and the mixture was stirred for 30 min, adjusted to pH 4
with conc. HCl and filtered. The filtrate was neutralized using NaOH
(1 N) and EtOAc (30 mL) was added. The organic layer was sepa-
rated and stirred as HCl gas was added. The light purple solid was
collected to give 0.7 g dye 5 (87%) as the hydrochloride. The
hydrochloride (0.1 g) was stirred with EtOH (50 mL) and NH4OH
(3 mL) was added to the mixture until dissolution occurred. The
resultant solution was clarified using activated carbon and the
2.2. Synthesis of dyes and intermediates
2.2.1. 4-((3-(2-Hydroxyethoxy)-4-amino)phenylazo)-N,N-bis
(2-hydroxyethyl)-aniline (3)
Compound 10 (1 g, 4.8 mmol) was stirred with H2O (5 mL), as
a small amount of crushed ice was added and an ice bath was used
to cool the mixture to 0 ꢀC. HCl (2 N, 7 mL, 0 ꢀC) was added and the
phenylazoamine was diazotized by adding NaNO2 (1 N, 5.5 mL,
4.8 mmol) slowly at 0 ꢀC. After stirring at 0 ꢀC for 30 min, the
OR1
OCH3
O
NH2
H
N
NH2
N
N
R2
N
H2N
N
N
H3C
NaO3S
R1 = Me, Et, Pr, Bu, (CH2)2OH
R2 = H, NEt2, N(CH2CH2OH)2
C.I. Direct Black 17
Fig. 2. Substituted aminoazobenzenes evaluated to account for C.I. Direct Black 17 mutagenicity [4].