Synthesis of some novel 2-amino-5-arylazothiazole disperse dyes for dyeing polyester fabrics and their antimicrobial activity

Article abstract of DOI:10.3390/molecules21010122

The present work describes the synthesis of a series of four novel biologically active 2-amino-5-arylazothiazole disperse dyes containing the sulfa drug nucleus. The structures of the synthesized thiazole derivatives are confirmed using UV-spectrophotomet

Full text of DOI:10.3390/molecules21010122

molecules
Article
Synthesis of Some Novel 2-Amino-5-arylazothiazole
Disperse Dyes for Dyeing Polyester Fabrics and Their
Antimicrobial Activity
Hatem E. Gaffer 1,†, Moustafa M. G. Fouda 1,2,† and Mohamed E. Khalifa 3,4,
*
Received: 20 December 2015 ; Accepted: 18 January 2016 ; Published: 21 January 2016
Academic Editor: Richard A. Bunce
1
Textile Research Division, National Research Center, Dokki, Giza, P. O. Box.12611, Egypt;
hatem197@yahoo.com (H.E.G.); m_gaballa@yahoo.com (M.M.G.F.)
Chemistry Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia
Chemistry Department, Faculty of Science, Taif University, P. O. Box 888, Taif 21974, Saudi Arabia
Department of Chemical Engineering, Higher Institute for Engineering and Technology, New Damietta,
P. O. Box 34518, Egypt
2
3
4
*
Correspondence: mohamedezzat200@hotmail.com or mohamedezzat@tu.edu.sa;
Tel.: +966-569-846-966 or +20-1096-5243-70
†
These authors contributed equally to this work.
Abstract: The present work describes the synthesis of a series of four novel biologically active
2-amino-5-arylazothiazole disperse dyes containing the sulfa drug nucleus. The structures of the
synthesized thiazole derivatives are confirmed using UV-spectrophotometry, infrared and nuclear
magnetic resonance techniques and elemental analysis. The synthesized dyes are applied to polyester
fabrics as disperse dyes and their fastness properties to washing, perspiration, rubbing, sublimation,
and light are evaluated. The synthesized compounds exhibit promising biological efficiency against
selected Gram-positive and Gram-negative pathogenic bacteria as well as fungi.
Keywords: azo dyes; 2-aminothiazole; sulfaguanidine; polyester; dyeing; antimicrobial activity
1. Introduction
Azo dyes are the most essential group of disperse dyes [1,2] and universally constitute more
than 50% of disperse dyes due to their strong tinctorial strength, ease of synthesis and low cost of
manufacture. Azo dyes have vivid colors in the yellow to blue-green region adjustable by varying
between azo components with electron withdrawing groups and coupling components with electron
donating groups. 2-Aminothiazoles have considerable interest from a therapeutic point of view because
of their utility as intermediates in the synthesis of antibiotics such as the well-known sulfa drugs [
They have been reported in the literature as antimicrobial [ ], antifungal [ ], anti-inflammatory
activity [ ], anesthetic [ ], anti-hypertesive [ ] and antiviral drugs [ ]. 2-Aminothiazole derivatives
have been also used as intermediates in the synthesis of various types of dyes [10 13]. In continuation
3].
4
5
6
7
8
9
–
of our interest in the synthesis of arylazothiazole derivatives [14–16], this paper reports the synthesis
of some 2-amino-5-arylazothiazoles and their application as disperse dyes on polyester fabrics.
In addition, the antibacterial activities of the synthesized dyes against various pathogenic bacteria and
fungi were also investigated.
Molecules 2016, 21, 122; doi:10.3390/molecules21010122
www.mdpi.com/journal/molecules

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2. Results and Discussion
2.1. Synthesis
Treatment of 2-aminothiazole derivatives
1
with the diazonium chloride derived from
sulfaguanidine
in Scheme 1. The structures of
spectral data. For example, the IR spectrum of
due to NH2 and NH functions, while the absorption band at 1610 cm´1 was attributed to the C=N
function. The 1H-NMR spectrum displayed a triplet signal at
7.05 ppm due to the NH proton, two
doublet signals at δ 7.74 and 8.04 ppm corresponding to the aromatic protons, a singlet at δ 8.16 ppm
2
afforded the corresponding 2-amino-5-arylazothiazole derivatives
and were established on the basis of their elemental analyses and
revealed absorption bands at 3346, 3232, 3173 cm´1
4 and 5 as shown
4
5
4
δ
characteristic for the C-4 thiazole-H, two signals at δ 8.51 and 8.67 due to two NH2 groups and a singlet
signal at δ 11.88 ppm due to NH proton.
Moreover, coupling of 2-aminothiazole derivatives
sulfapyrimidine furnished the corresponding 2-amino-5-arylazothiazole derivatives
in Scheme 1. The structures of these substituted thiazole dyes and were elucidated based on their
showed absorption peaks at 3364, 3286,
1
with the diazonium chloride derived from
3
6
and as shown
7
6
7
element´al1 analyses and spectral data. The IR of the dye
6
3142 cm due to the presence of NH2 and NH functions, in addition to the characteristic C=N group
absorption peak at 1612 cm´1. The 1H-NMR spectrum of dye
6 showed a triplet signal at 6.88 ppm
due to C-5 pyrimidine proton, a singlet at 7.13 ppm for the NH group, two doublets at 7.66 ppm and
8.04 ppm for four aromatic protons and a singlet at 8.22 ppm for the C-4 thiazole proton. The presence
of a doublet at 8.40 was attributed to the C-4 and C-6 pyrimidine-H protons, while the NH2 group
appeared shifted to 8.70 ppm.
R
O
S
O
S
HN
N
N
N
H
H
N
N
NH2
NH2
NH2
S
H2N
O
O
3
2
1
R
N
O
HN
NaNO /HCl
2
H
N
1)
2)
+
1
2
S
N
N
NH2
S
EtOH/NaOAc
H2N
O
=
=
4:
5:
R
R
H
Ph
N
R
O
S
N
N
NaNO /HCl
2
H
N
1)
2)
+
1
3
N
N
NH2
S
EtOH/NaOAc
O
=
=
7: R Ph
6: R
H
Scheme 1. Synthesis of 2-amino-4-arylazo-thiazole disperse dyes 4–7.
2.2. Dyeing and Fastness Properties
The functionalized 4-arylazo-3-methyl-2-substituted-thiophene disperse dyes
4–7 were applied
to polyester fabrics by 2% shade on the weight of fabric (o.w.f.), using the high-temperature pressure
˝
technique (130 C), and the visual color shades varied from red to reddish violet. Dyeing of polyester
fabrics was assessed in terms of their fastness properties (e.g., fastness to washing, perspiration,
rubbing, sublimation, and light) via a standard method [17]. All results, provided in Table 1, reveal
that these dyes have good fastness properties to washing, perspiration, rubbing and sublimation.
The light fastness of the synthesized dyes on polyester showed fading which is significantly affected
by the presence of the azo group, which decomposes by oxidation, reduction and/or photolysis [18].
The rate of this process depends on the chemical structure, treatment conditions, as well as the nature
of the substrate. The use of polyester fabric (i.e., non-proteinic fabric type), explained that the fading
process likely occurred by oxidation [19]. The ease of oxidation of azo linkages should be a function of
their electron density, thus the presence of electron-donating substituents should increase the fading
rate. This proposal was in agreement with the observed results (Table 1) which demonstrate that the
presence of the attached phenyl group decreased of light fastness to 4–5 (dyes 5 and 7).

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Table 1. Fastness properties of the synthetic dyes on polyester fabrics.
Perspiration 1
Acid Alkali
Rubbing 1
Sublimation 1
Staining Staining
at 180 ˝C at 210 ˝C
Light 2
(60 h)
Washing 1
Dye
Dry
Wet
4
5
6
7
4–5
4–5
4–5
4–5
4–5
3–4
4–5
4
4–5
4–5
4–5
4
3–4
3
3–4
4
4–5
3
4–5
4
4
5
5
3
3
4
4
3
5
4–5
5
4–5
1 These properties were evaluated using the grey scale (1—poor, 2—fair, 3—moderate, 4—good and 5—excellent).
2
This property was evaluated using the blue scale (1, 2—very poor, 3—poor, 4—fair, 5—good, 6, 7—very good,
8—excellent) [17].
2.3. Color Assessment
Colors of polyester fabrics is expressed in terms of CIELAB values (Table 2) and the following
˝
˝
CIELAB coordinates are assessed: lightness (L*), chroma (C*), hue angle from 0 to 360 (H), (a*) value
exemplifies the degree of redness (positive) and greenness (negative) and (b*) signifies the degree
of yellowness (positive) and blueness (negative). A reflectance spectrophotometer (Gretag Macbeth
CE 7000a) was utilized for the colorimetric assessments of the dyed samples. K/S values provided
by the reflectance spectrometer were calculated at
concentration on the dye substrate.
λ
max
and were directly associated with the dye
Table 2. Optical measurements of the synthetic dyes on polyester fabrics.
Dye
%Exhaustion
Absorption λmax/nm
K/S
L*
a*
b*
C*
H
4
5
6
7
65
71
69
66
446
444
463
458
9.26
12.67
11.33
9.37
81.09
73.23
74.22
89.35
3.35
20.42
40.58
43.88
48.71
43.51
80.71
76.75
76.21
85.84
´
1.48 23.13
8.17
1.78
37.20
3.14
´
Generally, the color hues of the thiazole dyes
5 and 7 on polyester fabric shifted to the greenish
directions, this was expressed by the negative values of a* (red-green axis). The positive values of b*
(yellow-blue axis) designated that the color hues of the thiazole dyes
to the yellowish directions as shown in Table 2.
4–7 on polyester fabric are shifted
Application of the synthesized compounds 4–7 as disperse dyes, showed the good affinity of
such dyes to polyester fabrics, as indicated from the satisfactory color yields and the acceptable K/S
values. Figure 1, illustrated the relationship between the dyeing bath concentrations and the strengths
of the synthesized disperse dyes 4–7, under high temperature dyeing conditions, where no direct
connection between the relative molecular weight of these dyes to the build-up was clear. These results
were in line with the previously reported by Müller [20] on the relation between substituents and
dye structure.
Figure 1. Comparison of K/S values for the synthesized disperse dyes 4–7 series.

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2.4. Antimicrobial Activity
Table 3 shows that all samples exhibited an acceptable antimicrobial activity against the tested
pathogenic bacteria at different concentrations 0.01, 0.5 and 1 g/mL, where all the results are mean of
µ
three replicates and significant difference at p < 0.05 by student’s test. At high concentrations, regarding
C. albicans; however, the extract exhibited an acceptable antifungal activity. The inhibition zone of all
synthetic products was at range 0.6 to 1.7 mm. The remarkable activity of the synthesized compounds
4–7 against Gram-negative bacteria, which were known to be more resistant against antibiotics than
Gram-positive bacteria because of their impenetrable cell wall, should be undergo further studies in
order to understand well the mechanism of action for each active compound.
2.5. Scanning Electron Microscopy
Scanning electron microscopy was used to observe the morphological alteration of the cells after
treatment with the synthesized compounds (e.g., the most active compound 7). As shown in Figures 2
and 3 the bacterial (S. aureus) and fungal (Candida albicans) cells treated with the synthesized compound
appeared to be shrink and the cell wall degradation was observed.
Figure 2. S. aureus in ethanol as control (left); S. aureus treated with synthesized compound 7 (right).
Figure 3. Candida albicans in ethanol as control (left); Candida albicans treated with synthesized
compound 7 (right).
2.6. Minimum Inhibitory Concentration (MIC)
A strong inhibition of the synthesized compounds
noticed (5 to 150 g/mL) as described in Table 4, to be vulnerable to all of them and their minimum
inhibition concentrations (MIC) values were ranged from 5–10 g/mL, with noticeably inhibitory
action. In this study, synthesized compounds and showed the highest antibacterial activity against
the tested bacteria with the lowest MIC value of 5
obviously clear that, all synthesized compounds
4–7 against the tested pathogenic bacteria was
µ
µ
4
7
µg/mL. According to the obtained results, it is
4–7 exhibited broad-spectrum activity against both
Gram-positive bacteria, Gram-negative bacteria as well as fungi.

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Table 3. Antimicrobial activity expressed as inhibition diameter zones (mm) of the synthesized compounds 4–7 at different concentrations (µg/mL).
Gram-Positive Bacteria
Gram-Negative Bacteria
P. aeruginosa K. pneumoniae
Fungi
Compd. No.
S. aureus
S. pyogenes
S. typhi
C. albicans
0.01
0.5
1.0
0.01
0.5
1
0.01
0.5
1
0.01
0.5
1
0.01
0.5
1.0
0.005
0.05
4
5
6
7
0.6
0.6
0.6
0.6
0.9
–
0.7
0.8
1
1.2
1.2
–
1
0.4
0.1
0.3
0.6
0.8
–
0.6
0.2
0.6
1.1
1.0
–
0.65
0.6
1.6
1.9
1.3
–
0.6
0.6
0.6
0.7
0.8
–
0.9
0.8
1
1.2
0.9
–
1.2
1
1.2
2
1.5
–
0.3
0.2
0.7
0.1
0.8
–
0.6
0.4
1
0.3
1.0
–
0.8
0.6
1.4
0.7
1.4
–
0.2
0.2
0.3
0.4
0.5
–
0.6
0.6
0.5
1
0.8
–
0.7
0.7
1.2
2
1.2
–
0.7
0.9
0.7
0.8
–
1.1
1.3
1.4
1.3
–
1.2
1.7
1.3
1.4
–
Ciprofloxacin
Ketoconazole
0.8
1.4
Table 4. Minimum Inhibitory Concentration (
different concentrations (µg/mL).
µg/mL) of the synthesized compounds 4–7 against Gram +ve (e.g., S. aureus and Gram -ve (e.g., S. typhi) bacteria at
S. aureus
S. typhi
Control
Conc. (%)
Control
Conc. (%)
Compd.#
5
10
60
150
5
10
60
150
49.32 ˆ 103
37.65 ˆ 103
31.44 ˆ 103
24.80 ˆ 103
19.39 ˆ 103
36.09 ˆ 103
34.22 ˆ 103
25.36 ˆ 103
22.71 ˆ 103
16.64 ˆ 103
4
5
6
7
(0 %)
(23.7 %)
(36.3 %)
(49.7 %)
(60.7 %)
(0 %)
(5.2 %)
(29.7 %)
(37.1 %)
(53.9 %)
49.32 ˆ 103
43.47 ˆ 103
26.16 ˆ 103
25.22 ˆ 103
17.32 ˆ 103
36.09 ˆ 103
36.56 ˆ 103
31.89 ˆ 103
22.24 ˆ 103
15.56 ˆ 103
(0 %)
(11.9 %)
(47.0 %)
(48.9 %)
(64.9 %)
(0 %)
(1.3 %)
(11.6 %)
(38.4 %)
(56.9 %)
49.32 ˆ 103
48.94 ˆ 103
48.19 ˆ 103
46.31 ˆ 103
28.24 ˆ 103
36.09 ˆ 103
32.67 ˆ 103
20.22 ˆ 103
19.44 ˆ 103
16.18 ˆ 103
(0 %)
(0.8 %)
(1.0 %)
(6.1 %)
(42.7 %)
(0 %)
(9.5 %)
(44.0 %)
(46.1 %)
(55.2 %)
49.32 ˆ 103
48.21 ˆ 103
37.65 ˆ 103
20.52 ˆ 103
19.20 ˆ 103
36.09 ˆ 103
31.89 ˆ 103
24.00 ˆ 103
21.78 ˆ 103
16.33 ˆ 103
(0 %)
(21.2 %)
(23.7 %)
(58.4 %)
(61.1 %)
(0 %)
(11.6 %)
(33.5 %)
(39.7 %)
(54.8 %)

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3. Experimental Section
3.1. Materials and Methods
3.1.1. Materials, Chemicals and Reagents
All the chemicals and solvents used in this study were obtained from Acros Organics, Thermo
Fisher Scientific Company (New Jersey, NJ, USA), and of pure grades. Polyester fabrics were kindly
supplied by Misr for Spinning and Weaving Company (Mahalla El-Kobra, Egypt). The dispersing agent
Setamol WS was supplied by BASF (Nienburg, Germany). The bacterial strains from the American
Type Culture Collection (ATCC) were purchased from MicroBioLogics Inc., (St. Cloud, MN, USA).
The tested microorganism strains in ethanol were used as controls for biological experiments.
3.1.2. Instrumentation
All melting points were measured by the capillary method on an electrothermal Gallenkamp
melting point apparatus (Gallenkamp Co., London, UK) and were uncorrected. Elemental analyses
were carried out at the Microanalytical Unit, National Research Centre, Giza, Egypt; the results
were in satisfactory agreement with the calculated values. The IR spectra (KBr) were recorded in
KBr disks by a Mattson 5000 FTIR spectrometer (Mattson Inst. Co., Madison, WI, USA) and not all
frequencies are reported. The NMR spectra (1H and 13C) were acquired using a WP 300 spectrometer
(Bruker Co., Billerica, MA, USA) at 300 MHz using TMS as an internal standard and DMSO-d6 as
solvent. The colorimetric measurements for the dyed polyester fabrics were carried out using a
reflectance spectrophotometer (GretagMacbeth CE 7000a, GretagMacbeth Co., Windsor, UK). Fastness
to washing was carried out using the automatic Rotadyer launder (sponsored by the British Standard
Institute—Society of Dyers and Colourists), fastness to perspiration was assessed according to the
test sponsored by the (BSS), fastness to rubbing was carried out according to the standard method
of testing (BSS) using a Crockmeter FD-17 apparatus (Electric Hungarian Co., Budapest, Hungary),
fastness to sublimation was carried out using scorch tester M247 A (Atlas Electric Devices Co., Chicago,
IL, USA) and fastness to light was carried out using the “Weather-o-meter” (Atlas Electric Devices Co.)
according to the AATCC standard test method.
3.2. Synthesis
General Procedure for the Synthesis of 2-Amino-4-arylazo-thiazole Disperse Dyes 4–7
A solution of aryl diazonium chloride was prepared by adding cold sodium nitrite solution (0.7 g
in 10 mL H2O) to a cold suspension of aryl amine
stirring. The freshly prepared solution was added with continuous stirring to a cold solution (0–5 C)
2 and/or 3 (0.01 mol) in 3 mL concentrated HCl with
˝
of 2-aminothiazol˝e
1 (1 g, 0.01 mol) in 30 mL ethanol and 4.0 g sodium acetate. The reaction mixture
was stirred at 0–5 C for 2 h, diluted with water, and the products collected by filtration. The obtained
2-amino-5-arylazothiazoles 4–7 were dried and recrystallized from ethanol.
˝
.
2-Amino-5-{[4-(carbamimidoyl-sulfamoyl)-phenyl]azo}thiazole (
4
). Red solid, yield 74%, m.p. 220–221 C
IR (KBr, cm´1): 3432, 3312, 3177 (NH2 and NH), 1640 (C=N). 1H-NMR (DMSO-d6,
δ
ppm): 7.05 (t,
1H, J = 7.15 Hz, NH), 7.59 (d, 2H, J = 7.80 Hz, Ar-H), 7.97 (d, 2H, J = 7.80 Hz, Ar-H), 8.36 (s, 1H, C-4
thiazole-H), 8.65 (d, 2H, J = 8.50 Hz, NH2), 9.40 (s, 2H, NH2), 11.35 (s, 1H, NH). 13C-NMR (DMSO-d6,
δ
ppm): 121.99 (2C), 129.51 (2C), 139.79, 144.88, 152.80, 155.20, 158.91, 173.40. Anal. Calcd. for
C10H11N7O2S2 (325.37): C, 36.91; H, 3.41; N, 30.13,Found: C, 36.83; H, 3.45; N, 30.20.
2-Amino-5-{[4-(carbamimidoyl-sulfamoyl)-phenyl] azo}-4-phenylthiazole ( ). Red solid, yield 77%, m.p.
241–241 C. IR (KBr, cm´1): 3433, 3393, 3321 (NH2 and NH), 1639 (C=N). 1H-NMR (DMSO-d6,
ppm):
6.94 (s, 1H, NH), 7.15–7.50 (m, 7H, Ar-H), 7.78 (d, 2H, J = 7.80 Hz, Ar-H), 8.48 (s, 2H, NH2), 9.18 (s,
2H, NH2), 11.37 (s, 1H, NH). 13C-NMR (DMSO-d6,
ppm): 121.46 (2C), 126.55 (2C), 128.21, 129.38
5
˝
δ
δ

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(2C), 130.86 (2C), 137.32, 140.64, 144.32, 151.08, 154.56, 160.27, 174.38. Anal. Calcd. For C16H15N7O2S2
(401.47): C, 47.87; H, 3.77; N, 24.42, Found: C, 47.73; H, 3.84; N, 24.50.
˝
2-Amino-5-{[4-(pyrimidin-2-yl-sulfamoyl)-phenyl] azo}thiazole (6). Red solid, yield 80%, m.p. 234–235 C.
IR (KBr, cm´1): 3351, 3254, 3146 (NH2 and NH), 1625 (C=N). 1H-NMR (DMSO-d6,
ppm): 6.80 (s,
δ
1H, NH), 7.17 (d, 1H, J = 7.50 Hz, C-5 pyrimidine-H), 7.32 (d, 2H, J = 7.80 Hz, Ar-H), 7.63 (d, 2H,
J = 7.80 Hz, Ar-H), 7.96 (d, 2H, J = 8.80 Hz, C-4 and C-6 pyrimidine-H), 8.25 (s, 1H, C-4 thiazole-H), ,
9.79 (s, 2H, NH2). 13C-NMR (DMSO-d6,
δ ppm): 112.82, 122.48 (2C), 127.75 (2C), 131.84, 138.79, 140.11,
142.68, 156.21 (2C), 167.86, 173.36. Anal. Calcd. For C13H11N7O2S2 (361.40): C, 43.20; H, 3.07; N, 27.13,
Found: C, 43.38; H, 3.15; N, 27.24.
2-Amino-4-phenyl-5-{[4-(pyrimidin-2-yl-sulfamoyl)-phenyl]azo}thiazole (
181–182 ˝C. IR (KBr, cm´1): 3463, 3372, 3348, 3234 (NH2 and NH), 1625 (C=N). 1H-NMR (DMSO-d6,
ppm): 6.84 (d, 1H, J = 7.50 Hz, C-5 pyrimidine-H), 7.11 (s, 1H, NH), 7.27–7.61 (m, 11H, Ar-H), 7.83
(d, 2H, J = 8.80 Hz, C-4 and C-6 pyrimidine-H), 8.62 (d, 2H, J = 8.50 Hz, NH2). 13C-NMR (DMSO-d6,
ppm): 113.64, 123.71 (2C), 127.06 (2C), 128.46, 129.18 (2C), 130.69 (2C), 135.74, 136.62 138.08, 141.26,
7). Red solid, yield 72%, m.p.
δ
δ
144.59, 157.27 (2C), 167.38, 172.91.Anal. Calcd. For C19H15N7O2S2 (437.50): C, 52.16; H, 3.46; N, 22.41,
Found: C, 52.02; H, 3.57; N, 22.57.
3.3. Disperse Dyeing Application
3.3.1. Dyebath Preparation Dyeing Procedure
The synthesized disperse dyes 4–7 under investigation were applied to polyester fabrics (0.04 g
dye/2 g fabric; o.w.f. 2%) by a convenient method for dyeing polyester fabrics in the laboratory
at 130 ˝C and high-pressure (24–30 psi). A dispersion of dye was formed by dissolving the proper
quantity of dye in 1 mL of acetone as solvent. 1% Setamol WS was added dropwise with continuous
stirring to the dye bath as anionic dispersing agent. The liquor ratio was 20:1, and the pH was adjusted
to 5.5 using aqueous acetic acid. Then, the wetted-out polyester fabrics were added to the dyebath.
D˝yeing process was accomplished by rising the dyebath temperature to 130 ˝C, at time intervals of
˝
3 C/min, the dyeing process was left for 60 min. After cooling to 50 C, the dyed fabrics were rinsed
with cold water and then reduction cleared using a mixture of 1 g/L sodium hydroxide and 1 g/L
sodium hydrosulfite for 10 min. at 80 ˝C. The dyed fabrics were finally rinsed again with hot water
followed by cold water and left for air drying.
3.3.2. Dyeing Characteristics on Polyester Fabrics
Color Fastness Tests
The color fastness properties of the dyed fabrics to washing, perspiration, rubbing, sublimation
and light were evaluated using standard methods [17]. The staining of adjacent cotton and nylon
fabrics was assessed using the grey scale: 1—poor, 2—fair, 3—moderate, 4—good and 5—excellent,
while the light fastness properties were scaled from 1–8 on the blue scale.
Color Properties of the Dyes on Polyester
˝
A GretagMacbeth CE 7000a reflectance spectrophotometer (D65 illumination, 10 observer) was
used for the colorimetric measurements on the dyed samples. K/S values given by the reflectance
spectrometer were calculated at λmax and were directly correlated with the dye concentration on the
dye substrate according to the Kubelka–Munk equation: K/S = (1
coefficient, S = scattering coefficient, R = reflectance ratio.
´
R)2/2R, where K = absorbance

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3.4. Biological Assessment
3.4.1. Test-Pathogens
The antibacterial activity tests were performed against five bacteria strains: Gram-positive
(Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 19615) and Gram-negative
(Salmonella typhi ATCC 6539, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC
700603), as well as Candida albicans ATCC 90028 for the antifungal activity test. The microorganisms
under test were grown onto Sabouraud agar. The microbial strains were conserved in Brain Heart
˝
Infusion medium (BHI) at
´
20 C. Three hundred
µ
L of each stock-culture was added to 3 mL of BHI
broth. Cultures were kept for 24 h at 37 ˝C
˘
1 ˝C and the purity of cultures was checked after 8 h of
incubation. After 24 h of incubation, bacterial suspension was diluted with sterile sodium chloride
solution, and bacterial suspension was diluted with BHI broth to a density of approximately 109 colony
forming unit/mL (CFU/mL) [21].
3.4.2. Antimicrobial and Antifungal Activity
The antimicrobial activities of the dyes 4–7 were assessed in vitro using the agar diffusion
method [22]. In this method, the medium was incubated with freshly prepared cells for each tested
bacteria or fungi. After solidification of the agar, a number of clean disks were immersed in˝to the
solvents (negative controls) or extract solutions and located on the plates. After incubation at 37 C for
24 h, the antimicrobial activity was assessed in term of diameter of the inhibition zone. Each test was
repeated three times and the average results (mm of zone of inhibition) is tabulated.
3.4.3. Minimum Inhibitory Concentration (MIC)
The selected bacteria for MIC were Staphylococcus aureus ATCC 25923 and Salmonella typhi ATCC
6539. MIC of four different samples was calculated by the two-fold serial dilution method [22
,23].
The levels dose of 5, 10, 60, 150 L/mL were utilized for MIC evaluation. 5 L of standardized
µ
µ
suspension of tested bacteria (108 CFU/mL) well was used for 1 mL of the broth. The test tubes were
incubated at 37 ˝C for 24 h. The growth was calculated by spectrophotometer at 600 nm. MIC for
bacteria was determined as the lowest concentration of the compound inhibiting the visual growth
of the test cultures on the agar plate. Each test in this experiment was repeated three times and the
average results in term of (mm of zone of inhibition) was tabulated.
3.4.4. Scanning Electron Microscopy
Scanning electron microscopy (SEM) was used to evaluate the susceptible bacteria that is subjected
to the synthetic products. A Small piece of agar is cut from the inhibition zone and fixed in a
glutaraldehyde solution 3% (v/v) buffered with 0.1 M sodium phosphate (pH 7.2) for 1 h at 25 ˝C.
Later on, the samples are washed with a buffer of sodium phosphate. Then, all pieces were then
post-fixed in 1 % (w/v) osmium tetroxide (OsO4) for 1 h and then washed again in buffer. Dehydration
steps were performed using propylene oxide. The specimens were left for drying and were mounted
onto stubs using double-sided carbon tape, and then were coated with a thin layer of gold by a Polaron
SC 502 sputter coater. All of them were observed on a JSM 6060 LV Scanning Electron Microscope (Jeol
Inc., Peabody, MA, USA) [24].
4. Conclusions
A series of novel 2-amino-5-arylazothiazole disperse dyes was synthesized. The newly
synthesized dyes were applied as disperse dyes for dyeing polyester fabric, where they exhibited good
dye ability and fastness properties. The novel compounds displayed broad-spectrum antimicrobial
efficiency against selected pathogenic Gram-positive and Gram-negative bacteria, as well as fungi.

Molecules 2016, 21, 122
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The remarkable activity against Gram-negative bacteria of the newly synthesized dyes will be subjected
to further study for the action mechanism of each compound.
The antimicrobial activity of the synthesized dyes does not affect the dyeing properties when
applied on fabrics but it is highly interesting to dye fabrics with effective antibacterial agents. Processes
utilizing textile engineering and chemical technologies to improve fabrics that function on human skin
have evolved over the centuries. Similar approaches are needed in fiber science to improve the quality
and performance of health care fabrics.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/1/122/s1.
Acknowledgments: This work was supported by King Saud University, Deanship of Scientific Research, College
of Science, Research Center.
Author Contributions: H.E.G., M.M.G.F. and M.E.K. conceived, designed the experiments, performed the
experiments, analyzed the data, contributed reagents/materials/analysis tools and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4–7 are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).