Synthesis, physical properties and cytotoxicity of stilbene-triazine derivatives containing amino acid groups as fluorescent whitening agents

Article abstract of DOI:10.1007/s10895-013-1239-1

A series of novel stilbene-triazine derivatives containing amino acid groups were synthesized and screened to evaluate their cytotoxicity. The UV absorptions of the derivatives were in the range of 240-450 nm. The absorption peaks of the cis-isomers and trans-isomers were in 281-291 nm and 353-361 nm, respectively. Their fluorescence emission peaks of the derivatives were located in the range of 400-650 nm. The whiteness data indicated that all the stilbene-triazine-amino acid derivatives showed excellent whitening effect on cotton fiber compared to untreated cotton. The preliminary cytotoxicity of these derivatives on a mouse fibroblast cell line (L-929 cells) was also investigated. The results showed that the compounds (7a-h) were nontoxic to L-929 cells as fluorescent whitening agents.

Full text of DOI:10.1007/s10895-013-1239-1

J Fluoresc
DOI 10.1007/s10895-013-1239-1
ORIGINAL PAPER
Synthesis, Physical Properties and Cytotoxicity of Stilbene-Triazine
Derivatives Containing Amino Acid Groups as Fluorescent
Whitening Agents
Maosheng Wan & Shaoli Zhou & Peifu Jiao &
Chengbo Cao & Jing Guo
Received: 4 March 2013 /Accepted: 14 May 2013
#
Springer Science+Business Media New York 2013
Abstract A series of novel stilbene-triazine derivatives
containing amino acid groups were synthesized and screened
to evaluate their cytotoxicity. The UV absorptions of the de-
rivatives were in the range of 240–450 nm. The absorption
peaks of the cis-isomers and trans-isomers were in 281–
291 nm and 353–361 nm, respectively. Their fluorescence
emission peaks of the derivatives were located in the range
of 400–650 nm. The whiteness data indicated that all the
stilbene-triazine-amino acid derivatives showed excellent
whitening effect on cotton fiber compared to untreated cotton.
The preliminary cytotoxicity of these derivatives on a mouse
fibroblast cell line (L-929 cells) was also investigated. The
results showed that the compounds (7a–h) were nontoxic to L-
929 cells as fluorescent whitening agents.
the electromagnetic spectrum, and re-emit light in the blue
region (typically 400–450 nm) [1–3]. Fluorescent activity is a
short term or rapid emission response, unlike phosphores-
cence, which is a delayed emission. These dyes are often used
to enhance the appearance of color of fabric and paper, caus-
ing a “whitening” effect, making materials look less yellow by
increasing the overall amount of blue light reflected. As a
result, FWA are often widely used for whitening paper and
textile [4–8]. Moreover, FWA have also been used as dyes in
biology [9], light emitting diodes [10], and chemosensors
[11]. However, FWA have long been well known for their
toxicities to human health and environment [12]. With higher
demands on healthy living conditions, it is very necessary to
develop the alternatives with low toxicity or better biocom-
patibility. At present, many functional compounds with low-
toxicity profile have been obtained by introducing amino acid
groups [13–15]. However, a search of the literature revealed
that stilbene-triazine derivatives (FWA) with amino acid
group have not been described. Therefore their synthesis and
properties study seemed to be an attractive task. Herein, we
would like to report the synthesis, structures, photo-physical
properties and cytotoxicity of the novel stilbene-triazine-
amino acid derivatives (7a–h).
.
.
Keywords Synthesis Stilbene-triazine derivatives
.
.
Amino acid Whiteness Cytotoxicity
Introduction
Fluorescent whitening agents (FWA) are dyes that absorb light
in the ultraviolet and violet region (usually 300–380 nm) of
Results and Discussion
:
M. Wan C. Cao (*)
School of Chemistry and Chemical Engineering, Shandong
University, Jinan, Shandong 250100, China
e-mail: cbcao@sdu.edu.cn
Synthesis of Derivatives
Reagents and conditions: (i) H2O, NaOH (aq), 0–5 °C, 2 h;
(ii) H2O, compound 4, NaOH (aq), 40–45 °C, 5 h, then HCl
(aq); (iii) H2O, R2H, NaOH (aq), 100 °C, 6 h, then HCl (aq)
at room temperature.
:
S. Zhou J. Guo (*)
School of Stomatology, Shandong University, Jinan, Shandong
250012, China
e-mail: guojing777@tom.com
The novel stilbene-triazine-amino acid derivatives (7a–h)
were synthesized in a three-step synthetic route as summa-
rized in Scheme 1. In brief, compound 3 was prepared from
P. Jiao
Department of Chemistry, Qilu Normal University, Jinan,
Shandong 250013, China

J Fluoresc
Scheme 1 Synthesis of the
compounds (7a–h)
the reaction of 4,4′-diaminostilbene-2,2′-disulfonic acid (1)
with 2,4,6-trichloro-1,3,5-triazine (2). This intermediate was
further condensed with aromatic amine (4) via nucleophilic
substitution reaction. Compounds 5 were isolated by acidify-
ing to pH 3–4 with 4 mol/L HCl solution and then collected
through vacuum filtration, and dried under vacuum overnight
at 40 °C. Compounds 5 were further reacted with different
amino acids (6), and the product of this reaction was precip-
itated by acidifying to pH 3–4 with 4 mol/L HCl solution, and
the reaction mixture was filtered by vacuum to isolate the
precipitate. The precipitate was washed with distilled water
to remove sodium chloride and dried overnight under vacuum
at 40 °C. The product was kept in a desiccator. The com-
pounds (7a–h) were isolated and characterized. The substitu-
ents and yields of the compounds are presented in Table 1.
can be seen from Fig. 1, the cis-isomer absorption peaks of
compounds 7c and 7g are much stronger compared to
others. It indicates that 7c and 7g are very easy to light-
induced to the cis-isomer because of the smaller substitu-
ents. The differences of UV absorption were due to the
different substituents.
Fluorescence Spectra
Figure 2 presents the emission spectra of the compounds (7a–
h). The emitting fluorescence of the compounds (7a–h) locat-
ed in the range of 400–650 nm although their intensity of
Table 1 Substituents and yields of the compounds (7a–h)
No
R1
R2
Yield (%)
UV–Vis Spectra
7a
7b
7c
7d
7e
7f
HO3S−
HO3S−
HO3S−
HO3S−
HO2C−
HO2C−
HO2C−
HO2C−
(H3C)2CHCH2(HO2C)CHNH−
HO2C(CH2)2(HO2C)CHNH−
HO2CCH2NH−
86
83
86
81
87
85
90
83
The UV–vis spectra of the compounds (7a–h) are shown in
Fig. 1. Their absorption bands ranged from 240 nm to
450 nm. The cis- and trans-isomers absorption peaks were
in 281–291 nm and 351–361 nm, respectively. Triazine-
stilbene compounds are well known for cis-trans isomeriza-
tion from the strongly fluorescent trans-isomer to the non-
fluorescent cis-isomer when they were exposed to light. As
(p)OHPhCH2CH(CO2H)NH−
(H3C)2CHCH2(HO2C)CHNH−
HO2CH(CH2)2(HO2C)CHNH−
HO2CCH2NH−
7g
7h
(p) HOPhCH2CH(CO2H)NH−

J Fluoresc
could not show considerable cytotoxicity to mouse fibroblast
cells and would be green as FWA.
Conclusion
A series of novel stilbene-triazine compounds containing ami-
no acid groups were synthesized through a three-step synthet-
ic route. The obtained compounds were characterized by the
1
analysis of the H-NMR, 13C-NMR and IR spectra. The
photo-physical characteristics of the molecules were also in-
vestigated by measuring the UV spectra and the fluorescence
spectra in aqueous solution. All compounds were applied to
pure cotton fiber as fluorescent whitening agents and their
performance were evaluated by measuring the degree of
whiteness. The results showed that the compounds (7a–h)
had a significant whitening effect on cotton. The maximum
whiteness value of treated cotton with the compound 7 g at a
concentration of 0.15 % (o.w.f) was found to be 120.7. The
cytotoxicity of all synthesized compounds was evaluated
using the MTT assay. Results demonstrated that the obtained
stilbene-triazine-amino acid derivatives showed excellent bio-
compatibility with mouse fibroblast cells. It is encouraging
that our compounds were not toxic to mouse fibroblast cells
and may serve as green light-emitting materials. Further ex-
periments are under way in our laboratory to confirm their
biocompatibility to human cells in vivo.
Fig. 1 UV absorption spectra of the compounds (7a–h)
fluorescence differed from each other. UV/visible absorption
maxima and fluorescence emission maxima of these com-
pounds were shown in Table 2. Because of the common parent
structure, the compounds (7a–h) had the similar shapes of
fluorescence emission spectra curve and UV absorption spec-
tra curve.
Whiteness Assessment
The compounds (7a–h) were applied to pure cotton fiber at a
concentration of 0.05 %, 0.1 %, 0.15 %, 0.20 %, and 0.25 %
(o.w.f). From Table 3, we learned that all of the compounds had
a significant whitening effect on cotton at different concentra-
tion. It is apparent that most of the compounds impart a high
degree of whiteness at the optimum dye concentration
(0.15 %). Among these compounds, 7 g was the most effective
molecular and the whiteness value of treated cotton was highest
(the whiteness=120.7 at the optimum dye concentration). The
data obtained are given in Table 3.
Experimental
Materials and Analysis
All reagents and materials were purchased from commercial
suppliers. 1H-NMR spectra were recorded in a Bruker
Cytotoxicity Evaluation
FWA should be safe to living organisms. Therefore, after
synthesizing the stilbene-triazine-amino acid derivatives and
studying their physical properties, we evaluated the cytotox-
icity of these derivatives to L-929 murine fibroblast cells.
Cells were treated with the compounds (7a–h) for 24 and
48 h at concentrations of 5, 10, 15, 20, 25 and 50 ug/mL.
Cell proliferation was evaluated by using MTTassay. The data
demonstrated that all the stilbene-triazine-amino acid deriva-
tives did not significantly inhibit the proliferation of L-929
cells (cell viability >95 %, Table 4) even if the concentration
increased up to 50 ug/mL (the concentration > the optimum
dye concentration) for 48 h. This result indicated that the
newly synthesized stilbene-triazine-amino acid derivatives
Fig. 2 Fluorescence emission spectra of the compounds (7a–h)

J Fluoresc
Table 2 UV absorbance and fluorescence characteristics of the com-
pounds (7a–h)
spectra were measured in aqueous solution in a quartz
cell (1.0 cm). The progress of all reactions was monitored
by the thin layer chromatography (TLC) technique, which
was performed on 2.0×5.0 cm, using a mixture of CH3OH,
CH2Cl2 and NH4OH as an eluent. The CIE Whiteness
values were measured using a Datacolor 600 reflectance
spectrophotometer.
No
UV absorbance
Fluorescence emission
max (nm)
Cis λmax (nm)
Trans λmax (nm)
λ
7a
7b
7c
7d
7e
7f
282
281
278
281
291
285
291
290
361
361
361
360
351
359
353
356
435
437
445
437
435
434
435
435
General Method for the Synthesis of the Compounds 5
To a 1,000 mL round-bottom flask, equipped with a stirring
bar, pH meter and thermometer, 400 mL distilled water and
100 g of ice were added. To this stirred mixture, 100 mmol
4,4′-diaminostilbene-2,2′-disulfonic acid (37.04 g) was
added and the pH was maintained 4–5 by drop-wise addi-
tion of 1 mol/L NaOH solution. Then 200 mmol 2,4,6-
trichloro-1,3,5-triazine (36.88 g) was gradually added to
the mixture while maintaining temperature at 0–5 °C with
external cooling. The reaction vessel was kept in an ice bath
with stirring for 2 h. Aromatic amine 200 mmol (4-amino-
benzoic acid and 4-amino-benzenesulfonic acid) was added
to the reaction mixture. The mixture was heated to 40–45 °C
and stirred for 5 h while maintaining pH 6–7 by drop-wise
7g
7h
Avance 400 (400 MHz) spectrometer, using TMS as internal
standard, DMSO-d6 or DMSO-d6+D2O as solvent and
chemical shifts were expressed in ppm. 13C-NMR spec-
tra were recorded in a Bruker Avance 400 (100 MHz,
DMSO-d6 as solvent). IR spectra were recorded with an
IR spectrophotometer Avtar 370 FT-IR. The UV spectra
were obtained on a Shimadzu UV-2401PC. Fluorescence
Table 3 Degree of whiteness and color data for cotton treated with the compounds (7a–h)
No
o.w.f (%)
Whiteness
L*
a*
b*
No
o.w.f (%)
Whiteness
L*
a*
b*
Untreated
0.05
75.0
108.9
110.6
112.4
110.5
110.2
103.1
108.7
110.1
109.2
108.4
103.6
105.2
106.2
105.8
103.8
111.4
112.1
113.7
113.4
111.7
94.78
95.58
95.69
95.72
95.58
95.42
95.11
95.72
95.85
95.53
95.93
95.51
95.61
95.65
95.76
95.80
95.49
95.62
95.71
95.85
95.63
−0.55
0.95
1.29
1.13
1.13
0.84
0.83
1.08
0.77
0.81
0.82
0.85
0.84
0.88
0.82
0.48
1.17
1.13
1.14
1.04
0.85
2.66
7a
7b
7c
7d
−4.44
−4.72
−4.73
−5.21
−4.71
−3.36
−4.30
−4.55
−4.51
−4.14
−3.27
−3.58
−3.78
−3.64
−3.17
−5.02
−5.11
−5.44
−5.30
−5.02
7e
0.05
0.10
0.15
0.20
0.25
0.05
0.10
0.15
0.20
0.25
0.05
0.10
0.15
0.20
0.25
0.05
0.1
104.7
104.8
104.2
104.1
102.8
98.3
95.73
95.67
95.63
95.53
95.57
95.61
95.53
95.46
95.70
95.6
0.74
−3.40
−3.47
−3.35
−3.37
−3.08
−2.05
−2.16
−2.76
−2.61
−2.17
−5.91
−5.93
−6.59
−6.85
−6.24
−1.21
−1.24
−1.64
−0.91
0.82
0.1
0.66
0.62
0.15
0.2
0.53
0.25
0.05
0.1
0.68
7f
0.47
98.6
0.40
0.15
0.2
101.1
101.0
95.8
0.41
0.39
0.25
0.05
0.10
0.15
0.20
0.25
0.05
0.1
0.23
7g
7h
116.2
116.5
120.7
119.7
117.8
93.9
95.90
95.98
96.10
96.05
95.92
95.36
95.69
95.66
95.38
95.66
1.55
1.42
1.51
1.41
1.44
−0.26
−0.53
−0.22
−0.64
−1.51
94.9
0.15
0.2
0.15
0.2
96.6
92.6
0.25
0.25
85.4
o.w.f, the weight ratio of the dye and the cotton fiber; L*, the lightness value; a*, the red/green value; b*, the yellow/blue value

J Fluoresc
Table 4 Effects of the compounds (7a–h) on mouse fibroblast L-929 cell viability
No L-929 cell viability (%) for 24 h
L-929 cell viability (%) for 48 h
5 ug/mL 10 ug/mL 15 ug/mL 20 ug/mL 25 ug/mL 50 ug/mL 5 ug/mL 10 ug/mL 15 ug/mL 20 ug/mL 25 ug/mL 50 ug/mL
7a 107.1
7b 108.2
103.6
97.4
106.3
99.2
102.3
97.1
99.1
96.1
98.7
96.8
100.3
102.1
104.3
104.2
106.3
108.8
102.5
100.1
104.2
101.8
95.9
107.4
100.9
96.9
100.8
97.9
95.5
102.3
99.3
98.5
100.1
105.7
104.1
104.1
98.8
7c
97.1
95.4
96.3
103.2
110.6
108.5
103.5
98.1
100.1
98.4
104.2
105.8
105.6
105.0
103.0
104.2
99.8
7d 105.7
7e 103.7
7f 111.4
7g 105.3
7h 101.8
98.3
102.2
100.7
100.4
99.4
99.5
102.8
98.1
104.9
98.4
104.1
107.2
108.5
96.9
97.2
107.6
95.7
100.5
98.2
97.1
111.2
97.1
104.1
95.2
102.1
101.9
104.1
104.4
100.3
97.7
97.6
103.3
104.1
103.2
95.3
97.1
106.5
addition of 1 mol/L NaOH solution. Compounds 5 were
isolated by aciding to pH 3–4 with 4 mol/L HCl solution
and collecting through vacuum filtration, and drying under
vacuum overnight at 40 °C, and the compounds 5 were
obtained in 96 % and 93 % yield.
Synthesis of 2,2′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(4-Sulfophenylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)Dipentanedioic
Acid (7b)
1H-NMR (400 MHz; DMSO-d6+D2O, δ): 1.98 (q, 4H, 2×
CH2), 2.37 (q, 4H, 2×CH2), 4.55 (t, 2H, 2×CH), 7.48 (d,
2H, ArH), 7.58 (d, 4H, ArH), 7.64 (d, 2H, ArH), 7.76 (s,
2H, CH=CH), 7.94 (s, 2H, ArH), 8.01 (d, 4H, ArH); IR
(KBr, cm−1): 3441–3028, 2970, 1621, 1472, 1400; 13C-
NMR (100 MHz, DMSO-d6): 26.6, 30.4, 67.8, 120.5,
120.9, 122.6, 123.1, 127.1, 127.5, 136.4, 143.9, 145.5,
173.7, 174.1.
General Method for the Synthesis of the Compounds (7a–h)
To a 250 mL reaction flask, equipped with a stirring bar and
a reflux condenser, 100 mL distilled water was added. Then
the flask was charged with 6 mmol compounds 5 and
12 mmol amino acid 6. The reaction mixture was stirred at
reflux for 6 h while maintaining pH 8–9 by drop-wise
addition of 1 mol/L NaOH solution. The disappearance of
compounds 5 were confirmed by TLC analysis. After 6 h,
the product was isolated by cooling and aciding to pH 3–4
with 4 mol/L HCl solution. The product was precipitated out
and filtered by vacuum, and the precipitate was washed with
distilled water (100 mL). The resulting compounds (7a–h)
were dried under vacuum overnight at 40 °C, obtained in
81–90 % yield, and stored in a desiccator.
Synthesis of 2,2′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(4-Sulfophenylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)Diacetic Acid (7c)
1H-NMR (400 MHz; DMSO-d6+D2O, δ): 4.01 (s, 4H, 2×
CH2), 7.53 (d, 2H, ArH), 7.57 (d, 4H, ArH), 7.40 (s, 2H,
ArH), 7.81 (s, 2H, CH=CH), 7.96 (d, 2H, ArH), 8.14 (d,
4H, ArH); IR (KBr, cm−1): 3441–3028, 2981, 1621, 1472,
1400; 13C-NMR (100 MHz, DMSO-d6): 42.9, 119.1,
120.2, 121.4, 126.5, 129.4, 138.7, 141.1, 143.5, 164.1,
165.9, 172.4.
Synthesis of 2,2′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(4-Sulfophenylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)bis(4-Methylpentanoic
Acid) (7a)
Synthesis of 2,2′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(4-Sulfophenylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)bis(3-
1H-NMR (400 MHz; DMSO-d6, δ): 0.91 (d, 12H,
2×(CH3)2), 1.57–1.73 (m, 6H, 2×CH2CH), 4.54 (t, 2H,
2×CH), 7.42 (d, 2H, ArH), 7.57–7.63 (d, 6H, ArH), 7.65
(s, 2H, ArH), 7.74 (s, 2H, CH=CH), 7.89–8.11(d, 4H, ArH),
10.15 (s, 2H, 2×NH), 10.49 (s, 4H, 4×NH); IR (KBr,
cm−1): 3445–3028, 2981, 1620, 1472, 1400; 13C-NMR
(100 MHz, DMSO-d6): 22.6, 24.8, 41.1, 62.9, 121.9,
126.8, 135.1, 136.4, 145.9, 150.4, 171.8, 173.6.
(4-Hydroxyphenyl)Propanoic Acid) (7d)
1H-NMR (400 MHz; DMSO-d6, δ): 2.97 (d, 4H, 2×CH2),
4.06 (t, 2H, 2×CH), 4.79 (s 2H, 2×OH), 6.70 (d, 2H, ArH),
7.02–7.56 (d, 12H, ArH), 7.57 (s, 2H, ArH), 7.67 (s, 2H,
CH=CH), 7.78 ( d, 2H, ArH), 8.14 (d, 4H, ArH), 9.60 (s,

J Fluoresc
2H, 2×NH), 10.39 (s, 4H, 4×NH); IR (KBr, cm−1): 3441–
3028, 2881, 1624, 1472, 1400; 13C-NMR (100 MHz, DMSO-
d6): 35.4, 70.9, 115.6, 122.6, 125.1, 126.7, 127.1, 130.9,
138.2, 145.9, 150.4, 156.6, 157.0, 170.8, 172.5.
2H, CH=CH), 7.60–8.05 (m, 20H, ArH), 9.29 (d, 2H,
2×NH), 10.51(s, 2H, 2×NH), 10.76 (s, 2H, 2×NH); IR
(KBr, cm−1): 3439–3139, 2929, 1590, 1513,1407; 13C-
NMR (100 MHz, DMSO-d6): 35.4, 71.9, 115.9, 120.9,
125.1, 126.7, 127.2, 131.0, 136.5, 150.4, 156.7, 157.0,
171.0, 172.5.
Synthesis of 4,4′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(1-Carboxy-3-
Methylbutylamino)-1,3,5-Triazine-6,2-Diyl))bis(Azanediyl)
Dibenzoic Acid (7e)
NMR Spectra
NMR spectra were performed on a Bruker AVANCE 400
(1H: 400 MHz, 13C: 100 MHz). Splitting patterns reported
here are: s (singlet), d (doublet), t (triplet), and m (multiplet).
1H-NMR (400 MHz; DMSO-d6, δ): 0.94 (d, 12H,
2×(CH3)2), 1.64 (m, 2H, 2×CH), 1.76(t, 4H, 2×CH2),
3.80 (t, 2H, 2×CH), 7.61 (d, 2H, ArH), 7.70 (d, 4H, ArH),
7.74 (s, 2H, CH=CH), 7.90 (s, 2H, ArH), 8.01 (d, 2H, ArH),
8.15 (d, 4H, ArH), 9.53 (s, 2H, 2×NH), 9.74 (s, 2H, 2×
NH), 10.46 (s, 2H, 2×NH); IR (KBr, cm−1): 3426–3028,
2960, 1623, 1489, 1405; 13C-NMR (100 MHz, DMSO-
d6):35.4, 36.2, 53.9, 56.1, 115.7, 115.9, 120.2, 127.1,
127.5, 138.2, 143.6, 145.7, 157.0, 165.5, 170,6, 172.5.
FT-IR Spectra
FT-IR spectra were recorded on a spectrophotometer Avtar
370 FT-IR. About 600 mg of KBr was ground in a mortar
with a pestle, and sufficient amounts of synthesized com-
pounds were ground with KBr to make a 0.5 wt% mixture.
After each sample was loaded, the sample chamber was
purged with nitrogen for a minimum of 10 min before data
collection.
Synthesis of 2,2′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(4-Carboxyphenylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)Dipentanedioic Acid (7f)
UV-Visible Absorption and Fluorescence Emission Spectra
1H-NMR (400 MHz; DMSO-d6+D2O, δ): 1.82–2.38 (m,
8H, 2×CH2CH2), 3.31 (t, 2H, 2×CH), 7.59 (d, 2H, ArH),
7.70 (d, 4H, ArH), 7.81 (d, 2H, ArH), 7.86 (s, 2H, CH=
CH), 7.94 (s, 2H, ArH), 8.10 (d, 4H, ArH); IR (KBr, cm−1):
3442–3028, 2930, 1592, 1489, 1408; 13C-NMR (100 MHz,
DMSO-d6): 26.8, 29.4, 57.3, 113.0, 117.6, 119.7, 126.5,
131.6, 143.2, 150.4, 153.5, 158.7, 168.2, 168.4, 172.6,
175.2.
Steady-state UV–vis is spectra of the monomers (10−5
M
solutions in deionized water at pH 8 with 1 mol/L
NaOH solution) were collected on a Shimadzu UV-2401PC
Spectrophotometer. Fluorescence data for the monomers
(10−7 M solutions in deionized water at pH 8 with 1 mol/L
NaOH solution) were collected on a Shimadzu RF-5301 PC
Spectrofluorophotometer.
Synthesis of 4,4′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(Carboxymethylamino)-1,
3,5-Triazine-6,2-Diyl))bis(Azanediyl)Dibenzoic Acid (7g)
Dyeing Procedure
Dyeing of pure cotton fiber that contained no FWAwas carried
out for 30 min using different concentration of a solution (pH 8
with 1 mol/L NaOH solution) of the compounds (7a–h) at
0.05 %, 0.10 %, 0.15 %, 0.20 % and 0.25 % (o.w.f). The liquor
to pure cotton fiber ratio was adjusted to 40:1; temperature was
increased from 20 °C to 50 °C at a rate of 2 °C/min within
15 min; Then the bath was maintained at 50 °C for 15 min.
Treated samples were removed from the treatment bath, rinsed
in cold water and dried at room temperature.
1H-NMR (400 MHz; DMSO-d6, δ): 4.05 (d, 4H, 2×CH2),
7.67 (d, 2H, ArH), 6.69–7.04 (d, 12H, ArH), 7.76 (s, 2H,
CH=CH), 7.88–7.89 (d, 8H, ArH), 9.22 (s, 2H, 2×NH),
10.30 (s, 2H, 2×NH), 10.70 (s, 2H, 2×NH); IR (KBr,
cm−1): 3425–3028, 2910, 1621, 1490, 1407; 13C-NMR
(100 MHz, DMSO-d6): 42.9, 120.1, 120.9, 121.7, 124.1,
126.8, 127.9, 133.8, 139.9, 142.5, 145.8, 167.2, 167.4,
169.3, 170.8.
Whiteness Measurements
Synthesis of 4,4′-(6,6′-(4,4′-(Ethene-1,2-Diyl)bis(3-Sulfo-4,
1-Phenylene))bis(Azanediyl)bis(4-(1-Carboxy-2-
(4-Hydroxyphenyl)Ethylamino)-1,3,5-Triazine-6,2-Diyl))
bis(Azanediyl)Dibenzoic Acid (7h)
The compounds (7a–h) were applied at a concentration of
0.05 %, 0.10 %, 0.15 %, 0.20 % and 0.25 % to pure cotton
fiber by the previously described dyeing procedure. The
average whiteness value of three measurements from each
sample at different locations was recorded and the L*a*b*
coordinates were determined.
1H-NMR (400 MHz; DMSO-d6, δ): 2.85 (d, 4H, 2×
CH2), 3.62 (t, 2H, 2×CH), 6.68 (d, 2H, ArH), 7.06 (d,

J Fluoresc
5. Alsins J, Björling M, Furó I, Egle V (1999) Dimer formation of a
stilbenesulphonic acid salt in aqueous solution. J Phys Org Chem
12:171–175
6. Lee J-K, Um S-I, Kang Y, Baek D-J (2005) The synthesis and
properties of asymmetrically substituted 4,4′-bis(1,3,5-triazin-6-
yl)diaminostilbene-2,2′-disulfonic acid derivatives as fluorescent
brighteners. Dye Pigment 64:25–30
7. Um S-I, Kang Y, Lee J-K (2007) The synthesis and properties of
triazine-stilbene fluorescent brighteners containing a monophenolic
antioxidant. Dye Pigment 75:681–686
Cell Culture
L-929 cells (mouse fibroblast cell line) were maintained in
RPMI-1640 medium supplemented with 10 % (v/v) bovine
calf serum and 80 U/ml gentamicin. Cells were cultured in a
5 % CO2, humidified incubator at 37 °C and detached by
0.05 % trypsin-EDTA.
8. Hussain M, Shamey R, Hinks D, El-Shafei A, Ali SI (2012)
Synthesis of novel stilbene-alkoxysilane fluorescent brighteners,
and their performance on cotton fiber as fluorescent brightening
and ultraviolet absorbing agents. Dye Pigment 92:1231–1240
9. Harrington BJ (2009) The staining of oocysts of cryptosporidium
with the fluorescent brighteners Uvitex 2B and calcofluor white.
Lab Med 40:219–223
10. Zhu W, Hu C, Chen K, Tian H (1998) Luminescent properties of
copolymeric dyad compounds containing 1,8-naphthalimide and
1,3,4-oxadiazole. Synth Met 96:151–154
Cell Viability Assay
Cells were grown on 96-well plates in culture media until
approximately 80 % confluency was reached. The compounds
(7a–h) in medium (5, 10, 15, 20, 25 and 50 ug/mL) were
added. After incubation (for 24 and 48 h), cell viability
was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium] assay. The absorbance of all samples
was measured at 499 nm using Spectra MAX 190 microplate
spectrophotometer (GMI Co., USA).
11. Bojinov VB, Konstantinova TN (2007) Fluorescent 4-(2,2,6,6-
tetramethylpiperidin-4-ylamino)-1,8-naphthalimide pH chemosensor
based on photoinduced electron transfer. Sensor Actuator B Chem
123:869–876
12. Field M, Wilhelm R, Quinlan J, Aley T (1995) An assessment of
the potential adverse properties of fluorescent tracer dyes used for
groundwater tracing. Environ Monit Assess 38:75–96
13. Zhao M, Bi L, Wang W, Wang C, Baudy-Floc’h M, Ju J, Peng S
(2006) Synthesis and cytotoxic activities of β-carboline amino
acid ester conjugates. Bioorg Med Chem 14:6998–7010
14. Saha AK, End DW (2005) Novel β-(imidazol-4-yl)-β-amino
acids: solid-phase synthesis and study of their inhibitory activity
against geranylgeranyl protein transferase type I. Bioorg Med
Chem Lett 15:1713–1719
References
1. Grabchev I (1995) The synthesis and properties of some triazine-
stilbene fluorescent brighteners. Dye Pigment 29:155–160
2. Konstantinova TN, Konstantinov HI, Betcheva RI (1999) The
synthesis and properties of some unsaturated triazinylstilbene
fluorescent brightening agents. Dye Pigment 43:197–201
3. Grabchev I, Philipova T (2000) Photophysical and photochemical
properties of some triazine-stilbene fluorescent brighteners. Dye
Pigment 44:175–180
15. Sassatelli M, Debiton É, Aboab B, Prudhomme M, Moreau P
(2006) Synthesis and antiproliferative activities of indolin-2-
one derivatives bearing amino acid moieties. Eur J Med Chem
41:709–716
4. Leaver IH, Milligan B (1984) Fluorescent whitening agents—a
survey (1974–82). Dye Pigment 5:109–144