Synthesis, characterization and antimicrobial activity of some new azo dyes derived from 4-hydroxy-6-methyl-2H-pyran-2-one and its dihydro derivative

Article abstract of DOI:10.1016/j.dyepig.2020.109073

A series of new azo disperse dyes was synthesized by coupling 4-hydroxy-6-methyl-2H-pyran-2-one (triacetic acid lactone, TAL) or its hydrogenated derivative 4-hydroxy-6-methyl-5,6-dihydro-2H-pyran-2-one (dihydro triacetic acid lactone, DHTAL) with diazonium salts derived from aniline, 4-bromoaniline, 4-nitroaniline, 4-methoxyaniline, 2,4-dimethoxyaniline and 2,5-dimethoxyaniline. Spectroscopic data of these dyes dissolved in five organic solvents were measured. The effects of solvent polarity, nucleophilic component and substituent nature on the visible absorption spectra of the dyes are also reported. The structures of all compounds were confirmed by FT-IR, electronic absorption in UV and visible regions, ¹H, ¹³C and 2D NMR and high resolution mass spectroscopy (HRMS). In addition, in vitro antibacterial activity of the synthesized derivatives against Gram positive and Gram negative bacteria, both reference and clinical strains, was evaluated qualitatively and quantitatively by agar diffusion method.

Full text of DOI:10.1016/j.dyepig.2020.109073

Journal Pre-proof
Synthesis, characterization and antimicrobial activity of some new azo dyes derived
from 4-hydroxy-6-methyl-2H-pyran-2-one and its dihydro derivative
Soumaya Ben Mohamed-Smati, Fadhil Lafta Faraj, Imène Becheker, Hadjira
Berredjem, Franck Le Bideau, Maamar Hamdi, Françoise Dumas, Yahia Rachedi
PII:
S0143-7208(20)31770-8
DOI:
https://doi.org/10.1016/j.dyepig.2020.109073
DYPI 109073
Reference:
To appear in: Dyes and Pigments
Received Date: 1 December 2020
Accepted Date: 6 December 2020
Please cite this article as: Ben Mohamed-Smati S, Faraj FL, Becheker I, Berredjem H, Le Bideau F,
Hamdi M, Dumas F, Rachedi Y, Synthesis, characterization and antimicrobial activity of some new azo
dyes derived from 4-hydroxy-6-methyl-2H-pyran-2-one and its dihydro derivative, Dyes and Pigments,
https://doi.org/10.1016/j.dyepig.2020.109073.
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Author statement
Soumaya Ben Mohamed-Smati: Conceptualization, Investigation, Data Curation, Writing -
Original Draft. Fadhil Lafta Faraj: Investigation. Imène Becheker: Investigation. Hadjira
Berredjem: Supervision, Resources. Franck Le Bideau: Supervision, Resources. Maamar
Hamdi: Resources, Supervision. Françoise Dumas: Funding acquisition, Supervision, Data
Curation, Visualization, Writing - Original Draft, Writing - Review & Editing, Project
administration. Yahia Rachedi: Conceptualization, Writing - Original Draft, Funding
acquisition, Supervision, Project administration.

Graphical abstract

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Synthesis, characterization and antimicrobial activity of some new
azo dyes derived from 4-hydroxy-6-methyl-2H-pyran-2-one and
its dihydro derivative
[
a]
[a]
[b]
4
5
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7
8
9
0
1
2
3
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5
6
Soumaya Ben Mohamed-Smati,
Fadhil Lafta Faraj,
Imène Becheker,
Hadjira
and Yahia
[
b]
[c]
[a]
*[c]
Berredjem,
Franck Le Bideau, Maamar Hamdi, Françoise Dumas
*[a]
Rachedi
[a] Laboratory of Applied Organic Chemistry, Faculty of Chemistry, University of Science
and Technology Houari Boumediene, BP 32, El-Alia, 16111, Bab-Ezzouar, Algiers,
Algeria. E-mail: yrachedi@usthb.dz
1
1
1
1
1
1
1
[b] Laboratory of Applied Biochemistry and Microbiology, Faculty of Sciences, Department
of Biochemistry, University Badji Mokhtar, Annaba, Algeria
[c] Laboratory BioCIS, UMR CNRS 8076, CoSMIT, Université Paris Sud- CNRS, Université
Paris Saclay, Faculté de Pharmacie, 5, rue Jean-Baptiste Clément, 92296 Châtenay-
Malabry Cedex, France. E-mail: francoise.dumas@u-psud.fr
† Corresponding author
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Abstract: A series of new azo disperse dyes was synthesized by coupling 4-hydroxy-6-
methyl-2H-pyran-2-one (triacetic acid lactone, TAL) or its hydrogenated derivative 4-
hydroxy-6-methyl-5,6-dihydro-2H-pyran-2-one (dihydro triacetic acid lactone, DHTAL) with
diazonium salts derived from aniline, 4-bromoaniline, 4-nitroaniline, 4-methoxyaniline, 2,4-
dimethoxyaniline and 2,5-dimethoxyaniline. Spectroscopic data of these dyes dissolved in
five organic solvents were measured. The effects of solvent polarity, nucleophilic component
and substituent nature on the visible absorption spectra of the dyes are also reported. The
structures of all compounds were confirmed by FT-IR, electronic absorption in UV and
1
13
visible regions, H, C and 2D NMR and high resolution mass spectroscopy (HRMS). In
addition, in vitro antibacterial activity of the synthesized derivatives against Gram positive
and Gram negative bacteria, both reference and clinical strains, was evaluated qualitatively
and quantitatively by agar diffusion method.
Keywords:Azo dye, Pyran-2-one,Solvent effect, Substituent effect, Tautomerism,
Antimicrobial activity
1. Introduction
Azo dyes are the oldest and the largest used class of industrially synthesized organic dyes due
to their versatile application in various fields [1], such as dyeing textile, leather, paper, food
and cosmetic products [2-4], and for biomedical studies and applications [5]. They are also
used in high technology areas as laser [3-4], photodynamic therapy [6] and dye sensitized
solar cells [7].
Azo compounds contain at least a conjugated azo chromophore, (-N=N-), linking two
aromatic systems [8-13]. Generally, the synthesis of azo dyes involves two steps (Figure 1).
The first one is the conversion of an aromatic amine to a diazo compound, a process known as
diazotation, and the second step is the reaction of the titled diazo compound with a
nucleophilic component to produce the corresponding azo dye, a process known as diazo
2

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44
45
46
47
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50
51
52
53
54
55
56
57
coupling. This process is suitable for forming both azo dyes and pigments [8,9]. Among them,
the azo dyes derived from enol type coupling components constitute an interesting type of
compounds since they potentially have several tautomeric forms (enol-azo, keto-azo, hydrazo)
in solution and solid state [10,13-16]. Some important investigations have been carried out on
the synthesis and spectroscopic behavior of heteroarylazo dyes based on 4-hydroxycoumarin
[14-16], 4-hydroxyquinolone [13,17], 2-hydroxyacetophenone [11] and 4,6-
dihydroxypyrimidine [18] as an enol type coupling component, as well as other heteroarylazo
compounds [19-23].
In view of these encouraging reports, and in line with our interest to develop the chemistry of
bio-based resources [24], in this study we report on the synthesis of two series of some new
azo compounds and the investigation of their spectroscopic properties. In the first one, 4-
hydroxy-6-methyl-2H-pyran-2-one (1) (triacetic acid lactone, TAL) and in the second series
its hydrogenated derivative, 4-hydroxy-6-methyl-5,6-dihydro-2H-pyran-2-one (2) (dihydro
triacetic acid lactone, DHTAL) were chosen as coupling components in reaction with some
diazotized aromatic amines as diazo components (Figure 1). The structures of all products
1
13
were confirmed by IR, UV–Vis, H NMR, C NMR, 2D-NMR and HRMS.
3

R1
R1
R2
R2
OH
O
N
N
N
N
OH
O
O
O
5
6
7
8
9
: R = R = H
11: R = R = H
1 2
1
2
: R = H, R = p-Br
12: R = H, R = p-Br
1
2
1 2
: R = H, R = p-NO
1
2
2
13: R = H, R = p-NO
1 2 2
: R = H, R = p-OCH
14: R = H, R = p-OCH
1
2
3
1 2 3
: R = o-OCH R = p-OCH
15: R = o-OCH R = p-OCH3
1 3, 2
1
3,
2
3
1
0: R = o-OCH , R = m-OCH
1 3 2
3
OH
OH
N2+ Cl-
+
+
R1
O
O
R2
O
O
1
4 a-f
2
NH2
R1
R2
3 a-f
a: R = R = H
d: R = H, R = p-OCH
1 2 3
1
2
b: R = H, R = p-Br
e: R = o-OCH R = p-OCH3
1
2
1 3, 2
c: R = H, R = p-NO
f: R = o-OCH , R = m-OCH
1 3 2 3
1
2
2
5
8
9
5
Fig. 1. Synthetic strategy to azo dye structures 5 – 15.
60
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64
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69
Furthermore, infectious diseases caused by resistant microorganisms are responsible for
increased health costs as well as high morbidity and mortality. Hence, there is an urgent need
for discovery and development of novel antibiotics [25-28]. Therefore, the in vitro
antimicrobial activity of all synthesized dyes against six bacterial strains, either reference or
clinical, were examined in detail.
2. Results and Discussion
2.1. UV-Vis absorption spectra
2.1.1. Effects of solvent polarity on photo physical properties of dyes 5 – 15
The UV–Vis electronic spectra were recorded in the wavelength range 300 – 600 nm at
-5
-5
-1
concentration of approximately 10 to 2.5.10 mol.L depending on the solubility of the dye,
4

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the higher concentration corresponding to dye 11. To assess the effect of solvent polarities on
photophysical properties absorption of dyes, dyes 5 – 10 synthesized from TAL 1 as well as
disperse dyes 11 – 15 synthesized from DHTAL 2 were analyzed in five different organic
solvents having different polarity, dielectric constant (ε), and solvatochromic parameters (α,
*
β, and π values) such as n-hexane, ethanol, acetone, acetonitrile and dichloromethane.
From the presented values in table 1, it is apparent that practically no solvatochromism was
observed. The electronic absorption spectra showed absorption maxima in the region at 400 –
447 nm and 375 – 422 nm in n-hexane, 401 – 458 nm and 377 – 429 nm in acetone, 401 – 458
nm and 379 – 430 nm in acetonitrile, 417 – 463 nm and 387 – 436 nm in dichloromethane,
402 – 466 nm and 385 – 438 nm in ethanol for dyes 5 – 10 and 11 – 15, respectively as
illustrated for prototype compounds 5 (Figure 2a) and 11 (Figure 2b). In the UV–Vis spectra
of these compounds (5 – 15) two absorptions are noteworthy in all tested solvents. The first
one is a shoulder at 368 – 372 nm and the second one is a band between 400 – 466 nm for 5 –
10 and 375 – 438 nm for 11 – 15 (see Figure 2 for 5 and 11).
Table 1
Influence of solvent on maximum wavelength of absorption (λmax) of dyes 5 – 15.
λmax (nm)
Δ(λmax) (nm)
Structure
Dye
Hexane Acetone
CH CN
CH Cl
EtOH
3
2
2
5
6
7
8
9
400.6
423.2
-
401.6
418.4
405.3
437.8
458.6
454.8
401.6
415.8
401.6
436.4
458.2
453.8
418.6
424.4
417.2
443.8
462.6
461.0
413.4
421.2
402.2
442.4
466.4
442.4
18
8.6
5
6
7
8
9
: R = R = H
1 2
15.6
15.4
26.4
18.6
: R = H, R = p-Br
1
2
: R = H, R = p-NO
1
2
2
428.4
440.0
447.0
: R = H, R = p-OCH
3
1
2
: R1 = o-OMe,
R = p-OMe
2
1
0: R1 = o-OMe,
R2 = m-OMe
1
0
5

1
1
1
1
2
3
375.8
390.8
392.8
377.5
379.2
387.8
401.6
429.4
379.2
381.8
389.0
410.8
430.6
387.8
400.4
393.6
418.0
436.0
385.8
390.0
388.4
417.6
438.2
12.0
21.2
5.8
1
1
1
1
1
1: R = R = H
1 2
2: R = H, R = p-Br
1
2
3: R = H, R = p-NO
1
2
2
14 413.2
422.8
16.4
15.4
4: R = H, R = p-OMe
1
2
5: R = o-OMe,
1
1
5
R = p-OMe
2
86
87
88
89
90
91
92
It can be suggested that dyes may exist as a mixture of two tautomeric forms which are in
equilibrium. Furthermore, the spectra of unsubstituted derivatives 5 and 11 in various solvents
showed small changes in their maximum absorption wavelength λmax changing from 400 nm
in n-hexane to 418 nm in dichloromethane and from 375 nm in n-hexane to 385 nm in EtOH,
respectively (Table 1). This small variation, Δ(λmax) up to 26.4 nm (Table 1), suggests that the
dyes are involved in strong intramolecular hydrogen bond.
9
3
4
9
Fig.2 a. UV-vis absorption spectra of dye 5 (left) and b. dye 11 (right) in various solvents
(black: n-hexane; red: dichloromethane; blue: acetone; violin: ethanol; green: acetonitrile).
2.1.2. Nucleophilic component effects
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From typical spectroscopic data, a comparison of azo dyes 5 – 10 containing a 4-hydroxy-6-
methyl-2H-pyran-2-one moiety with the parallel series of azo dyes 11 – 15 containing a
hydrogenated derivative of 4-hydroxy-6-methyl-2H-pyran-2-one moiety revealed that dyes 5
6

1
00
01
– 10 showed a red shift as compared to dyes 11 – 15 which showed a blue shift in all used
solvents. For dye 5, δλmax = 40 nm relative to dye 11 λmax in dichloromethane (Table 1, Figure
3). For dye 6 δλmax = 39 nm relative to dye 12 λmax in acetone (Table 1).
1
1
02
03
1
1
04
05
1
Fig. 3. UV-vis absorption spectra of dyes 5 (blue) and 11 (red) in dichloromethane.
2.1.3. Substituent effects
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107
108
109
110
111
112
113
114
115
116
As is apparent in table 1, the introduction of electron-donating methoxy group into the
benzene ring results in bathochromic shifts in all solvents with respect to unsubstituted and
electron-withdrawing nitro substituted aryl groups. For instance, for dye 8, δλmax = 30 nm
relative to dye 5 and δλmax = 40 nm relative to dye 7 λmax in ethanol (Figure 4). For dye 14,
δλmax = 32 nm relative to dye 11 λmax and δλmax = 29 nm relative to dye 13 λmax in ethanol.
Similarly, the introduction of electron-donating o,p-dimethoxy group in the aryl ring increases
bathochromic shift in all solvents with respect to electron-donating p-methoxy and p-bromo
groups, electron-withdrawing nitro group and unsubstituted aryl. Thus in ethanol, δλmax = 24,
45, 54 and 64 nm for dye 9 relative to dyes 8, 6, 7 and 5 λmax, respectively (Figure 4), while
δλmax = 21, 48, 50 and 52 nm for dye 15 relative to dyes 14, 12, 13 and 11 λmax respectively.
7

1
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18
These results can be attributed to the electron withdrawing nature of the hydroxypyrane
moiety of the dyes.
1
5
: R = R = H
1 2
R1
R2
OH
O
6: R1 = H, R2 = p-Br
: R = H, R = p-NO
2
7
N
O
1
2
N
8: R = H, R = p-OCH
9
1 2 3
: R = o-OCH R = p-OCH
1 3, 2 3
1
0: R = o-OCH , R = m-OCH
1 3 2 3
1
19
20
1
Fig. 4. Absorption spectra of dyes 5 (violin), 6 (green), 7 (red), 8 (black) and 9 (blue) in
ethanol.
121
122
123
124
125
126
The azo-hydrazone tautomerism behavior of dyes formally derived from 1,3-dicarbonyl
compounds is now well understood [29-32]. As shown in scheme 1, the synthesized azo dyes
may exist in four possible tautomeric forms, tautomer T1 named enol-azo-keto (EAK), two
tautomers T2 and T3 as keto-hydrazo-keto (KHK1 and KHK2) forms, and T4, a keto-azo-
enol (KAE) tautomer.
8

1
27
28
1
Scheme 1. Four possible tautomeric forms of the prepared azo dyes.
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130
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133
134
135
136
137
138
139
140
141
142
143
2.2. IR absorption spectra
The infrared spectra of dyes 5 – 10 and new dyes 11 – 15 were recorded in KBr disks and
their characteristic IR absorption bands determined. The IR spectral data of the compounds
studied indicate that they exist in the keto-hydrazo-keto (KHK1 and KHK2) forms T2 and T3
and in the enol-azo-keto (EAK) T1 or keto-azo-enol (KAE) T4 ones. Thus all compounds
-
1
exhibit a weak and a broad NH stretching band in the region 3100 - 3000 cm , the low
frequency and the broadening of this group indicate that it is strongly involved in hydrogen
bonding in the solid state. In the carbonyl region, each of the compounds studied exhibits two
-
1
bands: the first one in the region 1600 – 1650 cm and the second one in the region 1700 –
-1
1750 cm due to their stretching vibrations of the 4-carbonyl and α-pyrone groups,
respectively. Such an assignment is substantiated by the IR spectra of azo dye
monophenylhydrazones of 4-hydroxycoumarine 16 and γ-phenyl-Δβ,γ-butenolide 17 (Figure
-1
5) which show their carbonyl stretching bands near 1620 and 1630 cm , respectively [14].
The observed downward shift of the carbonyl stretching band in the compounds 5 – 10 and 11
–
15,
by
analogy
to
4-hydroxycoumarine
and
γ-phenyl-Δβ,γ-butenolide
9

144
145
146
147
monophenylhydrazones, is due to the conjugation with the C=N double bond as observed in
the hydrazone tautomers T2 and T3 (KHK1 and KHK2) (Scheme 1). The IR spectra also
−
1
−1
showed bands in the region 2830 – 2970 cm and 1500 – 1520 cm which were assigned to
aromatic C–H and C=N vibrations, respectively.
1
48
49
1
Fig. 5. Molecular structures of monophenylhydrazones of 4-hydroxycoumarine 16 and γ-
phenyl-Δβ,γ-butenolide 17.
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
2. 3. NMR and HRMS analysis
1
13
Furthermore, structure elucidations of these two series were confirmed by H and C NMR,
2D-NMR and HRMS data as described in detail for representative compounds 10 and 11.
1
The H NMR spectrum of compound 10 in CDCl showed characteristic singlets for CH and
3
3
C5-proton of pyran ring at 2.23 and 5.83 ppm, respectively. The three aromatic protons
appeared as one doublet of doublet centered at 6.82 ppm and two doublets centered at 6.90
and 7.50 ppm, respectively. The broad peaks at 14.37 ppm and 16.34 ppm were assigned to
protons of the tautomeric hydrazone (=N–NH–) and hydroxyl (-O–H) motifs, respectively.
These results confirm that dye 10 may exist as a mixture of keto-hydrazo-keto (KHK1 and
KHK2) tautomers T2 and T3 and azo (EAK and KAE) tautomers T1 and T4 [30-32]. As
1
shown by H NMR spectra, dyes 5 – 9 may also exist as a mixture of keto-hydrazo-keto
(KHK1 and KHK2) tautomers T2 and T3 in CDCl solutions. The relative proportions of
3
1
these sets of tautomers were determined by H NMR spectroscopy of 5 – 10 by integration of
the hydrazone proton signal (between 14.08 and 14.62 ppm in CDCl ) relative to the
3
corresponding enol proton signal (between 16.26 and 16.50 ppm in CDCl ). These
3
1
0

166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
proportions were in the range of 88% to 92% thus showing that azo tautomers T1 and T4 are
predominant relative to hydrazo tautomers T2 and T3 in CDCl3.
1
3
The C NMR spectrum of compound 10 shows peaks at 20.6, 160.1 ppm for pyran methyl
and carbonyl carbons respectively, and at 101.4, 115.7 and 144.2 ppm for aromatic carbons.
Further support for the presence of azo motif (–N=N–) was provided by the signals at 125.5
and 130.3 ppm attributed to C and C , respectively. From the HSQC spectrum, we conclude
3
8
that protons resonance at σH 2.23, 3.83, 3.94, 5.83, 6.83, 6.91 and 7.50 ppm are bonded to the
carbons appearing at σC 20.5, 56.2, 56.6, 107.8, 115.7, 112.7 and 101.4 ppm, respectively
(Figure 6a). The HSQC spectrum allowed us to assign all protonated carbon resonances,
while the connectivities from the HMBC spectrum (e.g. 3.83 → 154.9, 5.83 → 20.5, 122.5
and 166.1; 6.82 → 101.4 and 144.2; 7.50 → 115.7, 130.3, 144.2 and 154.9) (Figure 6b)
support the structure of dye 10. ESI-HRMS for this formula (M =C H N O ) gave m/z (%)
1
4
15
2
5
+
+
values at 291.0990 (100) and 313.0806 (72) attributed to the cations [M+H] and [M+Na] ,
respectively. All together, these spectroscopic data are clearly consistent with the formation of
the target product 10.
O
154.9
.82-115.9
6.90-112.7
44.2
O
1
01.4-7.50
H
H
H
6
O
O
O
107.8-5.83
N
N
H
N
O
N
H
1
H
O
O
O
O
1
30.3
1
66.1
1
0
1
81
82
s
1
13
1
Fig. 6 a. Selected H and C NMR chemical shifts (right), b. HMBC correlations (left) of
183
184
185
186
187
compound 10.
The structure of the second series of new dyes was also confirmed on the basis of HRMS and
NMR data. Interestingly, the molecular ion peak of compound 11 was found in the mass
+
spectrum at m/z (%) 233.0921 (100) corresponding to [M+H] of molecular formula
1
C H N O . H NMR spectroscopic analysis of 11 confirms the presence of both
12
13
2
3
1
1

188
189
190
191
192
193
194
195
196
197
198
hydrogenated pyran ring and aromatic nucleus. The spectrum showed a triplet centered at
1.53 ppm, two multiplets at 2.71 – 2.89 ppm and 4.68 – 4.83 ppm which belong to
hydrogenated pyran protons. The aromatic protons resonated as three multiplets at 7.24 – 7.31
ppm, 7.41 – 7.46 ppm and 7.52 – 7.58 ppm. The broad peaks at 13.88 ppm and 15.16 ppm
were respectively assigned to the hydrazone (=N–NH–) and enol (=C–OH) protons of the
1
3
tautomers. In the C NMR spectra and from the HMBC charts of azo compound 11, it was
concluded that the peaks resonating at σC =21.2, 44.9 and 192.2 ppm correspond to methyl
C , methine C and carbonyl C carbon atoms of hydrogenated pyran, respectively, whereas
7
5
4
the phenolic carbon atoms appeared at σC =117.7 and 130.1 ppm. The signals at σC =122.9
and 141.1 ppm were attributed to C and C respectively which confirmed the presence of the
3
8
hydrazone motif (=N–NH–) (Figure 7).
1
99
00
1
13
2
Fig. 7 a. Selected H and C NMR chemical shifts (right), b. HMBC correlations (left) for
201
202
203
204
205
206
207
208
209
compound 11
The spectra of the studied compounds of the second series provide additional evidence that
they have the azo structures T1 and T4 (EAK and KAE) rather than the hydrazone structures
1
T2 and T3 (KHK1 and KHK2). For example H NMR spectrum of 11 in deuterated dimethyl
sulfoxide exhibits two signals at 13.88 and 15.16 ppm with nearly equal integration ratio.
Other compounds in these series show similar patterns.
2.4. Antimicrobial activity
Antibacterial activity was tested in vitro against Gram-positive and Gram-negative bacteria,
using established drugs as standards. Both series of azodyes, 5 – 10 and new products 11 – 15,
1
2

210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
were screened for antimicrobial activity qualitatively by measuring the diameter of the clear
inhibition zones (expressed in mm) around the paper discs using a transparent ruler, and
quantitatively by determination of the minimum inhibitory concentrations (MICs), which are
defined as the lowest concentration of compounds which visibly prevents the growth of
bacteria.
2.4.1. Determination of inhibition zones
All synthesized compounds 5 – 15 exerted significant inhibitory activity against the growth of
tested bacterial strains. As shown in Table 2, the diameter (mm) of inhibition zone values of
tested compounds 5 – 15 against different strains ranged between 10 and 25 mm. Values of
positive control, trimethoprim-sulfamethoxazole, were between 18 and 22 mm for both
clinical and reference strains of E. coli and S. aureus, while P. aeruginosa strains were
resistant to this antibiotic. The resistance shown by clinical P. aeruginosa to the synthesized
compounds 5 – 9 and 15, and to the selected reference antibiotic, is not surprising as these
bacteria are well known for being resistant to a wider range of antibiotics than most of other
bacteria. Interestingly, it is remarkable that this strain of P. aeruginosa was sensitive to other
dyes, exceptional those derived from hydrogenated derivative of the 4-hydroxy-6-methyl-2H-
pyran-2-one 11 – 14. (Table 2)
Table 2.
Antibacterial screening data for inhibition zones of compounds 5 – 10 and new synthesized
dyes 11 – 15 against different bacterial strains.
Antimicrobial activity (zone of inhibition in mm)
Reference strains
Clinical strains
Gram-
Gram+
Gram-
Gram+
Dye
S.a.
E.c. P.a.
S.a. 75
E.c. 10 P.a. 260
1
3

5
6
7
8
9
11
15
12
16
0
15 18
10 16
16
15
0
17
20
16
17
20
14
0
0
0
14
0
16 19
20 18
18 18
18
15
16
0
0
1
0
16
18
1
1
1
1
1
1
2
3
4
5
25
19
16
18
21
18 19
17 17
19 19
18 16
20 17
17
12
15
13
14
20
19
18
22
20
17
15
20
19
0
Control
22
20
0
20
18
0
230
231
232
233
234
235
236
237
238
239
240
241
242
S.a.: S. aureus; E.c.: E coli; P.a.: P. aeruginosa
It should be noted that the concentration of the dyes used in the assays ranged between 0.5
and 512 μg/mL, and all compounds exhibited good growth inhibition of bacteria at different
concentration as mentioned in Table 3. For example, compounds 9 and 15 have the best
inhibition of growth of E. coli reference strain at 8 μg/mL with an inhibition zone diameter of
20 mm, while compound 14 has the best activity against E. coli clinical strain at 2 μg/mL with
inhibition zone diameter of 22 mm. For reference strain of S. aureus, the best activity was
detected for compounds 10 and 11 at 128 and 256 μg/mL with inhibition zone diameters of 16
and 25 mm, respectively. It is also noteworthy that P. aeruginosa was much more sensitive to
compounds 8 and 13 for the reference strain at the same concentration (4 μg/mL) with
inhibition zone diameters of 19 and 20 mm respectively whereas the clinical strain was much
sensitive to 10 and 13 at 1 and 32 μg/mL with inhibition zone diameters of 18 and 20 mm,
respectively. It should be also noted that the concentrations of the dyes giving the best
1
4

243
244
245
246
247
248
249
250
251
252
253
254
activities were much lower than that of the standard antibiotic (25 μg/mL). Nevertheless, the
dyes at these lower concentrations seem to have similar levels of antibiotic activity (Table 3).
2.4.2. Determination of Minimum Inhibitory Concentrations
The data presented in Table 4 indicated that the substitution in phenyl ring exerted significant
influence on the antibacterial profile. Interestingly, compounds with methoxy-substituted ring
are found to be more active compounds in the respective series compared to the compounds
bearing other groups. It is apparent that different derivatives exhibit appropriate MIC values
against different strains of bacteria. For instance, compound 9 has excellent activity against
both reference and clinical strains of E. coli with MIC values of 2 and 32 μg/mL, respectively,
Table 3.
Concentration and diameter of inhibition zone of compounds displaying the best antibacterial
activity.
Strains
Dyes
Concentration
Inhibition zone
diameter(mm)
(μg/ml)
S. aureus
10
128
16
25
1
1
256
Control
25
8
22
20
20
20
19
20
0
Reference E. coli
9
1
5
8
Control
25
4
P. aeruginosa
S. aureus 75
8
1
3
4
Control
25
8
4
18
1
5

1
1
32
25
0.5
2
17
20
20
22
20
18
19
0
Control
Clinical
E. coli 10
9
1
4
Control
25
1
P. aeruginosa 260
10
1
4
256
25
Control
255
256
257
258
259
260
261
262
263
264
265
266
267
268
whereas product 10 showed significant inhibition against both reference and clinical S. aureus
and P. aeruginosa strains, with MIC values 128 μg/mL and 256 μg/mL, respectively. More
interesting results were obtained with the new synthesized series of compounds 11 – 15 with
lower MIC values compared with the ones obtained within the first series of dyes 5 – 10.
Exceptional activities against reference and clinical E. coli strains were observed for
compound 15 while for compound 14 only the clinical strain of E. coli was sensitive at the
same MIC value of 1 μg/mL. Excellent activities against reference S. aureus and P.
aeruginosa strains were observed for compounds 15 and 14 with MIC values of 16 and 32
μg/mL, respectively. Compared to methoxy-substituted compounds, both clinical S. aureus
and P. aeruginosa strains showed more sensitivity to dyes with unsubstituted and nitro-
substituted ring 11 and 13 with respective MIC values of 512 and 64 μg/mL.
Table 4.
In vitro antibacterial activity data in MIC (μg/ml) of tested compounds 5 – 10 and newly
synthesized dyes 11 – 15 against different microbial species.
Minimum Inhibitory Concentrations (μg/ml)
Reference strains
Clinical strains
1
6

Gram +
Compound S.a.
Gram - Gram +
Gram -
E.c. P.a. S.a. 75
E.c. 10 P.a. 260
5
6
7
8
9
11
15
12
16
0
128 256 512
512
256
128
32
64
512
4
R
R
R
2
512 512
512 R
R
R
512 512
256 512
128 256
R
8
512
256
64
64
64
256
512
1
0
1
2
3
4
5
16
25
19
16
18
21
8
1
1
1
1
1
128 128 512
2
8
64
R
8
64 512
32
1
16 32
64
R
R
1
1
269
270
271
272
273
274
275
276
277
278
279
280
S.a.: S. aureus; E.c.: E coli; P.a.: P. aeruginosa
3. Experimental
3.1. Measurements
Melting points were determined on a Stuart scientific SPM3 apparatus fitted with a
microscope and the values reported in °C are uncorrected. Absorption spectra were
determined on Jasco V-630 Bio UV spectrometer. FT-IR spectra were recorded on Magna-IR
550 spectrometer apparatus. NMR spectra were recorded with a Bruker Advance 1
1
13
spectrometer [400 MHz ( H) and 100 MHz ( C)] or a Bruker AH 300 FT spectrometer [300
1
13
MHz ( H) and 75 MHz ( C)]. Chemical shifts are expressed in parts per million (ppm)
downfield from TMS. Data are reported as follows: chemical shift [multiplicity (s: singlet, d:
1
doublet, t: triplet, m: multiplet), coupling constants (J) in Hertz (Hz), integration]. Shifts of H
1
3
and C NMR spectra were calibrated against the solvent residual isotopic peak as internal
1
7

1
13
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
reference. Reference peaks for the NMR spectra in (CD ) SO are 2.50 ( H) and 39.52 ( C)
3 2
1
13
and for CDCl 7.26 ( H) and 77.16 ( C). Mass spectra were determined on a Waters
3
Micromass LCT Premier Q-TOF Mass spectrometer coupled with a Waters Alliance HPLC.
3.2. Materials
All the chemicals and solvents used in this study were purchased from Fluka and Sigma
Aldrich. Grades of chemicals were as follow: 4-hydroxy-6-methyl-2-pyrone: 98%, aniline 3a:
≥99.5%, 4-bromoaniline 3b and 2,3-dimethoxyaniline 3f: for synthesis, 4-nitroaniline 3c and
p-anisidine 3d: ≥99%, 2,4-dimethoxyaniline 3e: 97%, sodium nitrite: ≥97.0%, hydrochloric
acid: 37%, sodium acetate: >99%.
The reference strains Staphylococcus aureus [ATCC 25923], Escherichia coli [ATCC-25922]
and Pseudomonas aeruginosa [ATCC-27853] used were obtained from Pasteur Institute,
Algiers. Clinical strains S. aureus, E. coli and P. aeruginosa used in this study were isolated
from different samples: pus, urine and blood. Identification of the bacterial strains was made
on cultural and biochemical characters.
3.3. Chemistry
Compounds 1 and 2 were prepared (Scheme 2) according to known procedures [24,33,34].
2
97
98
2
Scheme 2. Preparation of TAL 1 and DHTAL 2.
299
300
301
302
Azo derivatives 5 – 15 were synthesized by the azo-coupling reactions of substituted benzene
diazonium salts 4a – f with 4-hydroxy-6-methyl-2H-pyran-2-one (1) and 4-hydroxy-6-
methyl-5,6-dihydro-2H-pyran-2-one (2), respectively.
3.3.1. General procedure of diazotation
1
8

303
304
305
306
307
Aromatic amine 3a – f (6 mmol) was dissolved in a freshly prepared 10% solution of
concentrated hydrochloric acid (10 mL) then cooled to 0 – 5 °C using an ice-bath. A solution
of sodium nitrite (NaNO , 0.45 g, 6 mmol) in water (10 mL) was added dropwise while the
2
temperature was maintained below 5 °C. The resulting mixture was stirred for 30 min in an
ice bath (Scheme 3).
3
08
09
3
Scheme 3. Preparation of diazonium salts 4a – f
310
311
312
313
314
315
316
317
3.3.2. Preparation of dyes
When diazotation was complete, as described above, the resulting substituted aryl-diazonium
salts 4a – f were used to prepare the target molecules 5 – 15 as described below (Schemes 4
and 5). The two tautomeric forms are described as they appear from the spectra as a mixture
of Major (M) and minor (m) products. The selected physical properties of the synthesized
dyes 5 – 15 were measured and listed in Table 5.
Table 5.
Physical properties and yields of synthesized dyes
Dye
R
1
, R
2
m.p. (°C)
λmax (nm)
Color
Yield (%)
Ref
in CH Cl
2
2
Orange
Brick
83
50
5
[35]
this work
this work
[35]
5
6
7
8
H, H
183-185
235-237
245-247
192-194
418.6
424.4
417.2
443.8
H, 4-Br
H, 4-NO
Yellow
Orange
2
H, 4-OCH
75
3
1
9

9
2,4-OCH
3
3
218-220
175-177
462.6
461.0
Orange
Red
33
72
this work
this work
1
0
2,5-OCH
1
1
1
1
1
1
2
3
4
5
H, H
156
387.8
400.4
393.6
418.0
436.0
Yellow
Brown
Yellow
Purple
Brown
29
50
[36]
H, 4-Br
173
this work
this work
[36]
H, 4-NO
H, 4-OCH
2,4-OCH
201
37
2
150-153
180
30
3
100
this work
3
318
319
320
321
322
323
324
325
3.2.2.1. Synthesis of (E)-4-Hydroxy-6-methyl-3-(aryldiazenyl)-2H-pyran-2-ones (or
3-(2-(aryl)hydrazono)-6-methyl-3H-pyran-2,4-diones) (5 – 10)
The substituted aryl-diazonium salts (4a – f) were added dropwise to a mixture of 4-hydroxy-
6-methyl-2H-pyran-2-one (1) (TAL, 0.63 g, 5 mmol) in water (10 mL) and sodium acetate (1
g, 3 mmol). The reaction mixture was stirred for 2 h at 0 – 5 °C. The obtained precipitate was
filtered off, washed with cold water, dried and recrystallized from ethanol to give the pure
compounds 5 – 10 (Scheme 4).
3
26
2
0

327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
Scheme 4. Synthetic route for the preparation of azo dyes 5 – 10.
(E)-4-Hydroxy-6-methyl-3-(phenyldiazenyl)-2H-pyran-2-one
(or
6-methyl-3-(2-
phenylhydrazono)-3H-pyran-2,4-dione) (5): Orange solid (0.805g, 70%). FT-IR (KBr, υ
-1
1
cm ): 3030, 2980,1759, 1650, 1509. H NMR (300 MHz, CDCl ) δ ppm: 2.23 (s, 3H), 5.83
3
(s, 1H), 7.28 – 7.34 (m, 1H), 7.40 – 7.49 (m, 2H), 7.56 – 7.64 (m, 2H), 14.15 (bs, NHM),
1
3
16.31 (bs, OH ). C NMR (75 MHz, CDCl ) δ: 20.5 (C ), 107.6 (C ), 117.9 (C , C ), 122.0
M
3
7
5
9
13
(C ), 128.3 (C ), 129.8 (C , C ), 140.5 (C ), 159.6 (C ), 166.7 (C ), 181.0 (C ). HRMS
3
11
10
12
8
2
4
6
+
(ESI) m/z calcd for C H N O ([M+H] ): 231.0770; found: 231.0775 (100%) and calcd for
1
2
11
2
3
+
C H N O Na ([M+Na] ): 253.0589; found: 253.0598 (83%).
12
11
2
3
(E)-3-((4-Bromophenyl)diazenyl)-4-hydroxy-6-methyl-2H-pyran-2-one
(or
3-(2-(4-
bromophenyl)hydrazono)-6-methyl-3H-pyran-2,4-dione (6): Brick solid (1.228 g, 80%).
-1
1
FT-IR (KBr, υ cm ): 3103, 2960, 1738, 1625, 1512. H NMR (300 MHz, CDCl ) δ ppm: 2.24
3
(s, 1H), 5.85 (s, 1H), 7.45 – 7.52 (m, 2H), 7.54 – 7.59 (m, 2H), 14.08 (bs, NH ), 16.26 (bs,
M
1
3
OH ). C NMR (75 MHz, CDCl ) δ ppm: 20.7 (C ), 107.6 (C ), 119.5 (C , C ), 121.8 (C ),
M
3
7
5
9
13
3
122.5 (C ), 133.0 (C , C ), 139.7 (C ), 159.6 (C ), 167.2 (C ), 181.3 (C ). HRMS (ESI) m/z
1
1
10
12
8
2
4
6
+
79
calcd for C H BrN O ([M+H] , Br): 308.9875; found: 308.9872 (100%) and calcd for
1
2
10
2
3
+
79
C H BrN O Na ([M+Na] , Br): 330.9694; found: 330.9688 (50%).
12
10
2
3
(E)-4-Hydroxy-6-methyl-3-((4-nitrophenyl)diazenyl)-2H-pyran-2-one (or 6-methyl-3-(2-
(4-nitrophenyl)hydrazono)-3H-pyran-2,4-dione) (7): Yellow solid (1.031 g, 75%). FT-IR
-1
1
(KBr, υ cm ): 3084, 2935, 1758, 1650, 1501. H NMR (300 MHz, CDCl ) δ ppm: 2.21 (s,
3
3H), 5.98 (s, 1H), 7.83 – 7.90 (m, 2H), 8.25 – 8.31 (m, 2H), 14.35 (bs, NH ), 16.44 (bs,
m
+
OH ). HRMS (ESI) m/z calcd for C H N O ([M+H] ): 276.0620; found 276.0620 (33%)
M
12 10
3
5
+
and calcd for C H N O Na ([M+Na] ): 298.0440; found 298.0444 (5%).
1
2
9
3
5
(E)-4-Hydroxy-3-((4-methoxyphenyl)diazenyl)-6-methyl-2H-pyran-2-one (or 3-(2-(4-
methoxyphenyl)hydrazono)-6-methyl-3H-pyran-2,4-dione) (8): Orange solid (0.780 g,
2
1

-
1
1
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
60%).FT-IR (KBr, υ cm ): 3075, 2839, 1731, 1600, 1498. H NMR (300 MHz, CDCl ) δ,
3
ppm: 2.23 (d, J = 0.6 Hz, 3H), 3.85 (s, 3H), 5.83 (d, J = 0.6 Hz, 1H), 6.92 – 7.00 (m, 2H),
13
7.54 – 7.62 (m, 2H), 14.31 (bs, NH ), 16.48 (bs, OH ). C NMR (75 MHz, CDCl ) δ ppm:
m
M
3
20.4 (C ), 55.7 (OCH ), 107.5 (C ), 115.1 (C , C ), 119.7 (C , C13), 122.03 (C ), 134.1 (C ),
7
3
5
10
12
9
3
8
159.0 (C ), 160.0 (C ), 166.1 (C ), 181.2 (C ). HRMS (ESI) m/z calcd for C H N O
4
2
4
11
6
13 13
2
+
+
([M+H] ): 261.0875; found: 261.0876 (100%) and calcd for C H N O Na ([M+Na] ):
1
3
12
2
4
283.0695; found: 283.0693 (75%).
(E)-3-((2,4-dimethoxyphenyl)diazenyl)-4-hydroxy-6-methyl-2H-pyran-2-one (or 3-(2-
(2,4-dimethoxyphenyl)hydrazono)-6-methyl-3H-pyran-2,4-dione) (9): Orange solid (0.826
-1
1
g, 57%). FT-IR (KBr, υ cm ): 3050, 2940, 1739, 1620, 1511. H NMR (300 MHz, CDCl ) δ
3
ppm: 2.23 (s, 3H), 3.85 (s, 3H), 3.97 (s, 3H), 5.82 (s, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.58 (dd, J
1
3
= 9.0, 2.4 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H), 14.37 (bs, NH ), 16.34 (bs, OH ). C NMR (75
m
M
MHz, CDCl ) δ ppm: 20.8 (C ), 56.1 (OCH ), 56.6 (OCH ), 99.0 (C ), 106.6 (C ), 108.0
3
7
3
3
10
12
(C ), 118.9 (C ), 122.0 (C ), 124.2 (C ), 151.5 (C ), 161.0 (C ), 161.5 (C ), 165.8 (C ), 180.6
5
13
3
8
9
2
11
4
+
(C ). HRMS (ESI) m/z calcd for C H N O ([M+H] ): 291.0981; found 291.0980 (100%)
6
14 15
2
5
+
and calcd for C H N O Na ([M+Na] ): 313.0800; found 313.0793 (33%).
1
4
14
2
5
(E)-3-((2,5-Dimethoxyphenyl)diazenyl)-4-hydroxy-6-methyl-2H-pyran-2-one (or 3-(2-
(2,5-dimethoxyphenyl)hydrazono)-6-methyl-3H-pyran-2,4-dione (10): Red solid (0.681 g,
-
1
1
47%). FT-IR (KBr, υ cm ): 3072, 2841, 1749, 1610, 1498. H NMR (300 MHz, CDCl ) δ
3
ppm: 2.23 (s, 3H), 3.86 (s, 3H), 3.97 (s, 3H), 5.83 (d, J = 0.7 Hz, 1H ), 5.98 (s, 1H ), 6.82
M
m
(dd, J = 9.0, 2.9 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 2.9 Hz, 1H), 14.37 (bs, NHm),
1
3
16.34 (bs, OH ). C NMR (75 MHz, CDCl ) δ ppm: 20.6 (C ), 56.2 (OCH at C ), 56.6
M
3
7
3
12
(OCH at C ), 101.4 (C ), 107.8 (C ), 112.7 (C ), 115.7 (C ), 125.5 (C ), 130.3 (C ), 144.2
3
9
11
5
13
10
3
8
(C ), 154.9 (C ), 160.1 (C ), 166.1 (C ), 180.6 (C ). HRMS (ESI) m/z calcd for C H N O
5
9
12
2
4
6
14 15
2
2
2

+
+
376
377
378
379
380
381
382
383
384
([M+H] ): 291.0981; found: 291.0990 (100%) and calcd for C H N O Na ([M+Na] ):
14 14 2 5
313.0800; found: 313.0806 (72%).
3.2.2.2. Synthesis of (E)-4-hydroxy-6-methyl-3-(aryldiazenyl)-5,6-dihydro-2H-pyran-
2-one (or 3-(2-(aryl) hydrazono)-6-methyl-dihydro-3H-pyran-2,4-diones (11 – 15).
The obtained diazo liquor (4a – e) was added to a vigorously stirred mixture of 4-hydroxy-6-
methyl-5,6-dihydro-2H-pyran-2-one (2) (0.64 g, 5 mmol) in water (10 mL) and sodium
acetate (1 g, 3 mmol) in water (10 mL). The mixture was then stirred for 2 h at 0 – 5 °C. After
completion of the reaction, the resulting solid was filtered, washed with cold water and dried.
Recrystallization from ethanol gave pure solid (11 –15) (Scheme 5).
3
85
86
3
Scheme 5. Synthetic route for the preparation of azo dyes 11 – 15.
387
388
389
390
391
392
(E)-4-hydroxy-6-methyl-3-(phenyldiazenyl)-5,6-dihydro-2H-pyran-2-one (or 6-methyl-3-
(2-phenylhydrazono)-dihydro-3H-pyran-2,4-dione) (11): Yellow solid (0.986 g, 85%). FT-
-1
1
IR (KBr, υ cm ): 3070, 2940, 1710, 1600, 1511. H NMR (300 MHz, CDCl ) δ ppm: 1.53 (d,
3
J = 6.4 Hz, 3H), 2.71 – 2.89 (m, 2H), 4.68 – 4.83 (m, 1H), 7.24 – 7.31 (m, 1H), 7.43 (t, J =
1
3
7.9 Hz, 2H), 7.55 (t, J = 7.7 Hz, 2H), 13.88 (bs, 1H, NH ), 15.16 (bs, 1H, OH ). C NMR
m
M
(75 MHz, CDCl ) δ ppm: 21.2 (C ), 44.9 (C ), 45.2 (C ), 71.3 (C ), 72.9 (C ), 117.7
3
7M
5M
5m
6M
6m
2
3

393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
(C , C ), 117.8 (C , C ), 121.5 (C ), 122.9 (C ), 127.5 (C11m), 128.0 (C11M), 130.0
9M 13M 9m 13m 3m 3M
(C10M, C12M), 130.1 (C10m, C12m),141.0 (C ), 141.1 (C ), 163.7 (C ), 165.4 (C ), 188.5
8m
8M
2M
2m
+
(C ), 192.2 (C ). HRMS (ESI) m/z calcd for C H N O ([M+H] ): 233.0926; found:
4
m
4M
12 13
2
233.0921 (100%).
(E)-3-((4-Bromophenyl)diazenyl)-4-hydroxy-6-methyl-5,6-dihydro-2H-pyran-2-one (or
3-(2-(4-bromophenyl)hydrazono)-6-methyl-dihydro-3H-pyran-2,4-dione) (12): Brown
-1
1
solid (0.899 g, 58%). FT-IR (KBr, υ cm ): 3090, 2986, 1693, 1630, 1508. H NMR (300
MHz, DMSO) δ, ppm: 1.37 (d, J = 6.3 Hz, 3H), 2.73 (dd, J = 17.0, 3.2 Hz, 1H), 2.86 (dd, J =
17.0, 10.6 Hz, 1H), 4.74 – 4.92 (m, 1H), 7.55 – 7.67 (m, 4H), 13.25 (bs, 1H, NH ), 14.42 (bs,
m
13
1H, OH ). C NMR (75 MHz, DMSO) δ ppm: 20.3 (C ), 20.8 (C ), 44.5 (C ), 44.8
M
7m
7M
5M
(C ), 70.9 (C ), 72.3 (C ), 118.4 (C11m), 118.9 (C11M), 119.3 (C , C13M), 120.0 (C9m,
5
m
6M
6m
9M
C13m), 123.9 (C11M), 124.1 (C ), 132.9 (C10M, C12M), 133.1 (C10m, C12m), 141.1 (C ), 141.3
3M
8m
(C ), 162.9 (C ), 169.0 (C ), 189.0 (C ), 192.6 (C ). HRMS (ESI) m/z calcd for
8
M
2M
2m
4m
4M
+
79
C H BrN O ([M+H] , Br): 311.0031; found 311.0031 (100%).
12
12
2
3
(E)-4-Hydroxy-6-methyl-3-((4-nitrophenyl)diazenyl)-5,6-dihydro-2H-pyran-2-one (or 6-
methyl-3-(2-(4-nitrophenyl)hydrazono)-dihydro-3H-pyran-2,4-dione) (13): Yellow solid
-1
1
(1.149 g, 83%). FT-IR (KBr, υ cm ): 3060, 2960, 1715, 1625, 1504. H NMR (300 MHz,
CDCl ) δ ppm: 1.54 (d, J = 6.0 Hz, 3H), 2.71 – 2.90 (m, 2H), 4.68 – 4.85 (m, 1H), 7.40 – 7.57
3
13
(m, 4H), 13.82 (s, 1H, NH ), 15.08 (s, 1H, OH ). C NMR (75 MHz, CDCl ) δ ppm: 21.3
m
M
3
(C ), 21.6 (C ), 45.3 (C ), 45.6 (C ), 71.7 (C ), 73.4 (C ), 119.5 (C , C13M), 120.0
7
m
7M
5M
5m
6M
6m
9M
(C , C13m), 121.0 (C11m), 121.5 (C11M), 122.3 (C ), 123.6 (C ), 133.5 (C10M, C12M), 133.6
9
m
3m
3M
(C10m, C12m), 140.5 (C ), 140.6 (C ), 163.9 (C ), 165.6 (C ), 188.1 (C ), 192.9 (C ).
8m
8M
2M
2m
4m
4M
+
HRMS (ESI) m/z calcd for C H N O ([M+H] ): 278.0777; found: 278.0781 (100%).
1
2
12
3
5
(E)-4-Hydroxy-3-((4-methoxyphenyl)diazenyl)-6-methyl-5,6-dihydro-2H-pyran-2-one
(or 3-(2-(4-methoxyphenyl)hydrazono)-6-methyl-dihydro-3H-pyran-2,4-dione) (14):
2
4

-
1
1
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
Purple solid (0.589 g, 45%).FT-IR (KBr, υ cm ): 3078, 2909, 1708, 1630, 1510. H NMR
(300 MHz, CDCl ) δ, ppm: 1.53 (d, J = 6.2 Hz, 3H), 2.68 – 2.86 (m, 2H), 3.85 (s, 3H), 4.65 –
3
4.82 (m, 1H), 6.93 – 6.70 (m, 2H), 7.47 – 7.53 (m, 2H), 14.03 (s, 1H, NH ), 15.41 (s, 1H,
m
1
3
OH ). C NMR (75 MHz, CDCl ) δ ppm: 21.2 (C ), 44.7 (C ), 45.1 (C ), 56.0 (C ), 71.3
M
3
7
5M
5m
14
(C ), 72.9 (C ), 115.4 (C , C ), 119.4 (C , C ), 120.6 (C ), 122.1 (C ), 134.5 (C ),
6
M
6m
9
13
10
12
3m
3M
8M
134.6 (C ), 159.4 (C11m), 159.8 (C11M), 164.1 (C ), 165.7 (C ), 188.5 (C ), 191.7 (C ).
8
m
2M
2m
4m
4M
+
HRMS (ESI) m/z calcd for C H N O ([M+H] : 263.1032; found: 263.1028 (100%).
1
3
15
2
4
(E)-3-((2,4-Dimethoxyphenyl)diazenyl)-4-hydroxy-6-methyl-5,6-dihydro-2H-pyran-2-
one (or 3-(2-(2,4-dimethoxyphenyl)hydrazono)-6-methyl-dihydro-3H-pyran-2,4-dione
-1
1
(15): Brown solid (0.580 g, 40%). FT-IR (KBr, υ cm ): 3079, 2900, 1706, 1625, 1505. H
NMR (300 MHz, CDCl ) δ ppm: 1.51 (d, J = 6.4 Hz, 3H), 2.66 – 2.85 (m, 2H), 3.83 (s, 3H ),
3
m
3.84 (s, 3H ), 3.94 (s, 3H ), 3.96 (s, 3H ), 4.67 – 4.76 (m, 1H), 6.50 (s, 1H), 6.51 – 6.59 (m,
M
m
M
1H), 7.84 (d, J = 8.9 Hz, 1H ), 7.88 (d, J = 8.8 Hz, 1H ), 14.16 (bs, 1H, NH ), 15.47 (bs, 1H,
m
M
m
13
OH ). C NMR (75 MHz, CDCl ) δ, ppm: 21.3 (C ), 44.6 (C ), 45.0 (C ), 56.1 (C ), 56.4
M
3
7
5M
5m
14
(C ), 71.3 (C ), 72.7 (C ), 99.1 (C ), 106.1 (C12m), 106.1 (C12M), 118.2 (C13m), 118.4
1
5
6M
6m
10
(C13M), 120.1 (C ), 122.4 (C ), 124.1 (C ), 124.3 (C ), 150.7 (C ), 150.9 (C ), 160.3
3m
3M
8M
8m
9M
9m
(C11m), 160.7 (C11M), 164.6 (C ), 165.6 (C ), 188.8 (C ), 190.8 (C ). HRMS (ESI) m/z
2M
2m
4m
4M
+
calcdfor C H N O ([M+H] ): 293.1137; found: 293.1142 (41%) and calcd for
1
4
17
2
5
+
C H N O Na ([M+Na] ): 315.0957; found: 315.0952 (100%).
14
16
2
5
3.4. Biology
3.4.1. Bacterial strains
The antimicrobial activity of the synthesized compounds was evaluated in vitro versus three
reference strains: Staphylococcus aureus [ATCC-25923] (S.a.) as Gram positive bacterium;
Escherichia coli [ATCC-25922] (E.c.) and Pseudomonas aeruginosa [ATCC-27853] (P.a.) as
Gram negative bacteria and three clinical strains isolates from urine, blood and pus:
2
5

443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
Staphylococcus aureus 75 (S2), Escherichia coli 10 (S1), and Pseudomonas aeruginosa 260
(S3).
3.4.2. Antimicrobial activity determination
All compounds (5 –15) were dissolved in acetone and further serial ten-fold dilutions were
made in a concentration range from 0.5 to 512 μg/mL in order to prepare the working
solutions used for determination of inhibition zones as well as for determination of minimal
inhibitory concentrations (MIC). Trimethoprime-Sulfamethoxazole was used as reference
standard at a concentration of 25 μg/mL in order to validate methodology and also for
comparison of antimicrobial activities.
3.4.2.1 Determination of inhibition zones
All synthesized compounds (5 – 15) were examined for antibacterial activity against different
Gram-positive and Gram-negative strains of bacteria. Inhibition zones of the best compounds
(8 – 11, 13 – 15) were performed with the disk diffusion method using nutrient agar medium
[23,29]. The Mueller–Hinton agar medium was autoclaved for 30 min, poured under aseptic
conditions in each sterilized Petri dish, allowed to solidify and then inoculated with fresh
subculture of a bacterial inocula prepared in physiological sterile water. Sterile 6 mm filter
paper discs, impregnated with 20 μl of the different concentrations of tested compounds were
then placed on agar plates and the cultures were incubated at 37 °C for 24 h for bacterial
growth. Disks embedded with acetone were used as a negative control. Inhibition zones
formed on the medium were evaluated in millimeter (mm).
3.4.2.2 Determination of minimal inhibitory concentrations
The minimum inhibitory concentration (MIC) values of compounds 5 – 15 were determined
by broth micro-dilution method [22,29-30]. The microbial suspensions were prepared in
Mueller–Hinton broth from test organisms sub-cultured on nutrient agar. The serial prepared
solutions ranging from concentration 0.5 to 512 μg/mL were inoculated with fresh bacterial
2
6

468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
inoculums and incubated at 37 °C for 24 h. The results were recorded according to the
presence or absence of bacteria growth comparatively to the controls.
Conclusions
In this study, some known and unknown azo disperse dyes 5 – 15 were prepared by classical
method of azo coupling reaction using as nucleophilic partner TAL 1 and DHTAL 2. The
synthesized dyes were characterized by FT-IR, electronic absorption spectra in UV and
1
13
visible regions, H, C and 2D NMR and mass spectroscopy. The solvent polarities and
substituents effects on the photophysical properties of dyes were evaluated. The spectroscopic
data of the synthesized dyes revealed that these compounds exist as mixtures of two
tautomeric azo and hydrazo forms which are in equilibrium, the azo one being major.
Furthermore, measurement of antibacterial activity demonstrated that the dyes, at lower
concentration, showed similar activities against E. coli and S. aureus as those shown by the
control antibiotic, while P. aeruginosa strains were sensitive to synthesized molecules
compared to control antibiotic. The effect of these dyes on microbes therefore deserves
further examination.
Acknowledgements
The authors thank Karine Leblanc (Faculty of Pharmacy, Châtenay-Malabry, France) for high
resolution mass spectra and Camille Dejean for NMR spectra. The Ministry of Higher
Education and Scientific Research (Algeria), the Centre National de la Recherche Scientifique
(CNRS, France), the Ministry of Education, Research and Technology (France), IPSIT and
LabEx LERMIT (ANR-10-LABX-33) are gratefully acknowledged for financial support.
References
[1] Stead, CV. Azo Dyes (Chapter VI) in The Chemistry of Synthetic Dyes, Volume III,
Venkataraman K. Ed. Academic Press, New York and London, 1970, pp 303–369.
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7