New tetrazolic azo dyes linked to (thio)barbiturate and electron-rich aromatics as potential antimicrobial agents

Article abstract of DOI:10.3906/kim-1412-46

A series of new tetrazolic azo dyes based on (thio)barbiturate and electron-rich aromatics were synthesized in excellent yield. The electron-donor and tetrazole ring moieties were linked by a p-phenylazo bridge and the structural characterizations were achieved by FT IR, 1H and 13C NMR, and UV-visible spectrometry. The antibacterial activity of the synthesized compounds was tested against gram-positive and gram-negative bacterial strains, namely Acinetobacter calcoaceticus (ATCC23055), Escherichia coli (ATCC2592), Pseudomonas aeruginosa (ATCC27853), and Staphylococcus aureus (ATCC25923). As a result, potential antimicrobial effects were seen for some of the synthesized compounds.

Full text of DOI:10.3906/kim-1412-46

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 998 – 1011
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1412-46
Research Article
New tetrazolic azo dyes linked to (thio)barbiturate and electron-rich aromatics as
potential antimicrobial agents
Nader NOROOZI PESYAN1,∗, Davoud SOLEIMANI1, Nima HOSSEINI JAZANI2
1Faculty of Chemistry, Urmia University, Urmia, Iran
2Department of Microbiology, Immunology and Genetics, Faculty of Medicine,
Urmia University of Medical Sciences, Urmia, Iran
Received: 17.12.2014
•
Accepted/Published Online: 22.03.2015
•
Printed: 30.10.2015
Abstract: A series of new tetrazolic azo dyes based on (thio)barbiturate and electron-rich aromatics were synthesized
in excellent yield. The electron-donor and tetrazole ring moieties were linked by a p-phenylazo bridge and the structural
characterizations were achieved by FT IR, 1 H and 13 C NMR, and UV-visible spectrometry. The antibacterial activity of
the synthesized compounds was tested against gram-positive and gram-negative bacterial strains, namely Acinetobacter
calcoaceticus (ATCC23055), Escherichia coli (ATCC2592), Pseudomonas aeruginosa (ATCC27853), and Staphylococcus
aureus (ATCC25923). As a result, potential antimicrobial effects were seen for some of the synthesized compounds.
Key words: Tetrazole, azo dye, (thio)barbituric acid, antimicrobial effect
1. Introduction
Azo dye derivatives are considered important compounds from biological and medicinal viewpoints; some
examples are catechol diazo dyes as substrates for the enzyme catechol-O-methyltransferase (9);1 as potent
tyrosinase inhibitors (10);2 as potent and selective inhibitors of the tumor-associated isozymes IX and XII
over the cytosolic isoforms I and II (11);3 antimicrobial, anti-HCV, anti-SSPE, antioxidant, and antitumor
activities of arylazobenzosuberones (12);4 some novel arylazopyrazolodiazine and triazine analogs having an
antitumor effect (13 and 14);5 antimicrobial and cytotoxic arylazoenamines (15 and 16);6 antiviral and
cytotoxic activities (17);7 phenylimino-10H -anthracen-9-ones as novel antimicrotubule agents (18);8 novel
azo-resveratrol as a potent tyrosinase inhibitor (19);9 antifungal agents (20 and 21);10 (Figure 1), carbonic
anhydrase inhibitors;11β -aggregation inhibitors12 etc.
In addition, many other compounds containing the tetrazole functional group are also known for their
medicinal and biological effects. This functional group can take different roles including as a ligand in coordina-
tion chemistry, as a metabolically stable surrogate for a carboxylic acid group in medicinal chemistry,13,14 and
as a carboxylic acid isostere.15 5-Substituted-tetrazoles are reported to possess antibacterial,16 antifungal,17
antiviral,18 analgesic,19−23 anti-inflammatory,24,25 antiulcer,26 and antihypertensive activities;27 for instance,
irbesartan (22), valsartan (23), tasosartan (24), and losartan (25) (Figure 1). Moreover, it needs to be added
that the whole tetrazole function is metabolically stable.28
∗Correspondence: n.noroozi@urmia.ac.ir
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NOROOZI PESYAN et al./Turk J Chem
O
OH
O
OH
HO
N
N
SO2NH2
N
N
Ar
Ar
N
O
11
N
N
Ar'
10
9
Ar
O
H
H
N
N
N
HO
N
N
N
Ar
Ar
N
N
N
N
N
Ar
N
N
H2N
N
NH
15
12
14
N
H2N
13
NHX
R'
Ar
R1
R2
R4
R4
O
N
H
N N
N
N
N
R'
R'
N
16
17
18
OH
R
HO
N
R
N
N
N
N
N
Me
OH
OH
20
21
19
OH
N
Cl
O
CO2H
N
OH
H
N
N
N
N
N
N
O
O
NH
N
N
NH
N
NH
N
N
NH
N
N
N
N
N
N
N
Irbesartan (22)
Valsartan (23)
Tasosartan (24)
Losartan (25)
Figure 1. Structure of some drugs based on azo dyes and tetrazole.
In addition to medicinal applications, azo dyes are also used as colorimetric sugar sensors,29 MRI contrast
agents,30 and even in high technology fields such as electronic devices, linear and nonlinear optics, reprography,
and sensors.31−34 Based on these concepts, in this study we designed and colligated the tetrazole and azo
functional groups linked to an electron donor in the molecule in order to evaluate their antimicrobial effects.
2. Results and discussion
2.1. Chemistry
This article describes the synthesis of (E)-1-(4-(1H -tetrazol-5-yl)phenyl)-2-aryldiazenes (6a–6d), (E)-5-((4-
(1H -tetrazol-5-yl)phenyl)diazenyl)pyrimidine-2,4,6(1H ,3H ,5H)-trione and their sulfur analogues (6e–6h),
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NOROOZI PESYAN et al./Turk J Chem
and (E)-2-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)-5,5-dimethylcyclohexane-1,3-dione (6i) in the reaction of 4-
amino benzonitrile (1) as a starting material in three steps in good to excellent yield (Scheme 1; Table 1). In
these reactions, there is no need for separation or purification of 4-(1H -tetrazol-5-yl) benzenaminium chloride
(3), which is considered an advantage for the synthesis of tetrazole-based azo dyes in current studies. Due to
this advantage, the experimental work-up to determine azo dyes 6 will be very easy.
CN
CN
N N
NH
N N
N NH
N
1) NaN3
2) HCl
Ac2O
NaNO2
HCl
NH2
HN
1
O
2
NH
N2
Cl
3Cl
4
3
1) NaN3
2) HCl
+HCl
ED: Electron-
donor (5)
-HCl
N N
NH
N N
NH
N N
N N
N
N
N
N
N
NH
HN
NH2
NH3
N
7
8
6
N
O
ED
R1
Y
X
X
R5
R4
R2
R3
ED =
O
X-Y-X =
O
HN-CO-NH (e)
MeN-CO-NMe (f)
HN-CS-NH (g)
EtN-CS-NEt (h)
CH2-CMe2-CH2 (i)
R1 = R3 = R4 = OMe, R2 = R5 = H (a)
R1 = R3 = OMe, R2 = R4 = R5 = H (b)
R1 = R3 = OH, R2 = R4 = R5 = H (c)
R4 + R5 = C4H4, R2 = OH, R1 = R3 = H (d)
Scheme 1. Synthesis of azo dyes based on tetrazole and electron donors.
To determine 3 as a key material, the amino group in 1 should be protected initially. For this aim,
the reaction of 1 with acetic anhydride obtained N -(4-cyanophenyl) acetamide (2). The IR spectrum of 2
shows a peak in the frequency of 1671 cm−1 for carbonyl and the stretching vibration of NH of the amide
group appeared at the frequency of 3326 and 3256 cm−1 , while the stretching of the nitrile group appeared
at the frequency of 2221 cm−1 . The appearance of the carbonyl stretching of the acetamido group supports
the formation of 2. The 1 H NMR spectrum of 2 showed a singlet at δ 2.01 ppm (CH3 –CO–), a multiplet at
δ 7.75 ppm, and a singlet at δ 10.38 ppm (–NH–CO–). The 13 C NMR spectrum of this compound showed
seven distinct peaks, of which the ones at δ 24.2 and 169.2 ppm corresponded to methyl and carbonyl groups,
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NOROOZI PESYAN et al./Turk J Chem
respectively. The peaks at δ 143.5, 133.3, 119.1, and 118.9 ppm and at δ 104.7 ppm corresponded to phenyl
and CN groups, respectively (see Experimental part and Supplementary material; on the journal’s website).
The cycloaddition reaction of 2, with sodium azide and followed by concentrated hydrochloric acid, afforded 3
through compound N -(4-(1H -tetrazol-5-yl)phenyl)acetamide (8). Compound 8 was isolated for its structural
characterization. The IR spectrum of 8 showed a peak at the frequency of 1678 cm−1 for the carbonyl group
and the stretching vibration of the NH group appeared at the frequency of 3311 and 3267 cm−1 . The tetrazolic
35
NH stretching appeared at the frequencies of 2471–3195 cm−1
.
Compound 2 can also convert to 3 simultaneously in one step and without the need for separation of 8
(formation of tetrazole followed by deprotection reaction). The IR spectrum of 3 showed a peak at the frequency
of 2470 to 3134 cm−1 for the tetrazolic NH group and a peak at the frequency of 3383 cm−1 corresponded to
ammonium salt moiety, while some peaks of this group overlapped with the peaks of the tetrazolic NH group.
The 1 H NMR spectrum of 3 showed a broad singlet at δ 6.73 ppm that corresponded to the sum of tetrazolic
NH, while ammonium salt protons indicated two doublets at δ 7.29 and 8.04 ppm that corresponded to phenyl
protons. The 13 C NMR spectrum of 3, on the other hand, showed five distinct peaks (see Experimental part
and Supplementary material).
Compound 4-(1H -tetrazol-5-yl)aniline (7) can also be obtained from 3 under natural conditions (natu-
ralized by Na2 CO3). The IR spectrum of 7 showed two peaks at the frequencies of 3485 and 3385 cm−1 for
the primary amino group and the stretching vibration of the tetrazolic NH group appeared at the frequencies
of 3357 to 3213 cm−1 (see Experimental part and Supplementary material). Compound 7 can also be found in
zwitterionic form.
There is no need to separate and purify 3 from the crude reaction mixture in the azo dye synthesis.
This feature is the most favorable advantage of the reaction process (except for the spectroscopic analysis of
3). The salt of 3 converted to diazonium salt of 4 by the use of sodium nitrite added to the reaction mixture
at 0 ◦ C. Finally, azo dyes (6) were precipitated by dropwise addition of diazonium salt into the solution of
corresponding electron donors (ED, 5) at 0 ◦ C (Scheme 1 and see Experimental part). Representatively, the
IR spectrum of 6e showed peaks at the frequencies of 3478 cm−1 for NH/OH, 3200 and 3075 cm−1 for BA-NH
groups, 2479–2846 for tetrazolic NH, and 1743 and 1692 cm−1 for carbonyl groups of the barbituric acid ring
moiety. The 1 H NMR spectrum of this compound showed a singlet at δ 14.10 ppm for NH/OH and two singlets
at δ 11.55 and 11.32 ppm that corresponded to different chemical shifts of BA-NH groups. In addition, two
doublets at δ 7.77 and 8.09 ppm occurred in the phenyl ring. It seems that the peak of the tetrazole-NH
group overlaps with the DMSO-water peak at δ 3.58 ppm as a broad singlet. In many tetrazolic compounds,
the tetrazole-NH proton can be detected by adding a drop of D2 O (judging the appearance of the DOH peak
at 3.99 ppm (variable)).36 The 13 C NMR spectrum of 6e showed nine distinct peaks: two peaks at δ 161.9,
159.7, and 154.9 ppm corresponded to different chemical shifts of carbonyl groups and that at δ 149.7 ppm
corresponded to tetrazolic carbon atoms. The peaks at δ 143.5 and 121.1 are of quaternary carbon atoms, and
those at δ 128.4 and 117.2 are of phenyl CH carbons. Finally, the peak at δ 119.0 is of C=N and/or =C–N
carbon atom on the BA ring moiety (see Experimental part and Supplementary material).
Due to the formation of the intramolecular H-bond in azo-enol and/or keto-hydrazone forms and also
the restricted rotation about the C=N bond in keto-hydrazone form, the two carbonyl groups along with the
two substituents on the N, N -disubstituted (thio)barbituric acid ring moiety have been found in the results of
different chemical shifts (Scheme 2).37−39
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NOROOZI PESYAN et al./Turk J Chem
X
N
X
N
X
N
X
N
X
N
R
O
R'
O
R
R'
O
R
R'
O
R
R'
O
R
R'
O
N
N
N
N
N
N
N
N
N
N
N
N
H
N
N
N
H
O
O
H
O
O
H
H
H
N
NH
N
N
N
N
N
NH
N N
Azo-enol [II]
N
NH
N N
Azo-keto [I]
N N
N N
N N
Keto-hydrazone [III]
Zwitterionic forms
R = R' = H, X = O (6e)
R = R' = Me, X = O (6f)
R = R' = H, X = S (6g)
R = R' = Et, X = S (6h)
Scheme 2. Possible tautomeric forms of tetrazolic azo dyes based on symmetrical (thio)barbituric acids.
Representatively, the UV-visible spectra of azo dye 6c are shown in Figure 2. These spectra are recorded in
acetone (A) and ethanol (B) as aprotic and protic solvents, respectively, over the range of λ between 250 and 600
nm using two solvents in concentrations ≈10−4 –10−5 mol L−1 (M); for more information, see Experimental
part and Supplementary material. It was observed that despite there being some absorption spectra in acetone
and ethanol (6a, 6d, 6e, and 6i), they did not change significantly except for dyes 6b, 6c, 6f, 6g, and 6h.
Representatively, in 1.7 × 10−4 M , the λmax values of dye 6c in acetone and ethanol appeared at 331 (log ε
= 1.027) and 308 nm (log ε = 2.529), respectively. Obviously, the λmax values of dye 6c in ethanol as a protic
solvent hypsochromically shifted with respect to the λmax of acetone in higher concentrations (Figure 2A and
2B). In low concentrations of ethanol (8.7 × 10−6 to 3.5 × 10−5M), dye 6c showed two λmax at 290 and
332 nm, while from 1.0 × 10−5 to 3.5 × 10−4M it showed one distinct λmax that bathochromically shifted
Figure 2. UV-visible spectrum of 6c in acetone (A) and in ethanol (B) in various concentrations (M).
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NOROOZI PESYAN et al./Turk J Chem
with increasing concentration (Figure 2B). In contrast, in acetone as an aprotic solvent, 6c showed a distinct
λmax that bathochromically shifted slightly in higher concentrations (Figure 2A) (for more information, see
Experimental part and Supplementary material). The λmax values of dyes 6a–6i are summarized in Table 1.
Table 1. Structure, yields, and λmax of new tetrazolic azo dyes linked to (thio)barbiturate and electron-rich aromatics
(6a–6i).
Yielda (%)
max (nm), (EtOH*,
acetone**)
Entry
Electron-donor (ED, 5)
Tetrazolic azo dye (6)
OMe
MeO
N
H
N
307, 360 (*)
360, 458 (**)
1
OMe
50
N
N
MeO
OMe
OMe
(a)
N
N
MeO
(a)
313, 363 (*)
360 (**)
MeO
H
N
2
3
N
N
OMe
OH
70
60
N
N
OMe
(b)
N
N
(b)
(c)
OH
H
N
N
N
288, 331 (*)
332 (**)
N
N
H
O
OH (c)
363, 496 (*)
382, 479 (**)
OH
H
N
N
4
(d)
57
N
N
N
N
(d)
H O
O
O
NH
NH
H
N
HN NH
N
O
292, 330 (*)
333 (**)
5
6
55
60
N
N
O
O
(e)
N
N
O
O
(e)
O
Me
Me
O
Me
O
N
H
N
N
N
317, 408 (*)
391 (**)
N
O
N
N
N
(f)
N
N
O
O
(f)
Me
S
329, 366, 402, 465
(*)
NH
NH
H
N
HN NH
N
S
7
8
64
60
N
337, 366, 402, 455
(**)
N
O
O
(g)
N
N
O
O
(g)
S
Et
N
329, 360, 401, 456
(*)
Et
Et
H
N
N
N
N
N
S
N
342, 366, 402, 462
(**)
N
N
O
O
(h)
N
N
O
O
(h)
Et
288, 365, 397, 431
(*)
366, 402, 421 (**)
H
N
9
15
N
O
O
N
(i)
N
N
O
(i)
a
Isolated yields.
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NOROOZI PESYAN et al./Turk J Chem
2.2. Antimicrobial activities of 6a–6i
As outlined in Table 2, among all of the derivatives, compounds 6a, 6b, 6c, 6d, 6g, and 6h exhibited a good
and broad spectrum of antimicrobial activities against the four bacterial species Acinetobacter calcoaceticus
(ATCC23055), Escherichia coli (ATCC2592), Pseudomonas aeruginosa (ATCC27853), and Staphylococcus
aureus (ATCC25923), tested at the concentration of 100 µg/µL. For example, compounds 6c and 6e showed
potential inhibitory effects against the four above-mentioned bacterial strains. Compound 6b only inhibited
the growth of P. aeruginosa. Compounds 6a and 6h affected A. calcoaceticus and S. aureus while compound
6g only affected A. calcoaceticus (Table 2). Table 3 shows the antimicrobial activities against the four above-
mentioned bacterial species by six known standard antibiotics as a model test. The results derived from Table
2 are comparable with those from Table 3. Representatively, the image of antimicrobial test results for the
above-mentioned bacterial species is shown in Figure 3.
Figure 3. Representatively, antimicrobial test results for Acinetobacter calcoaceticus ATCC23055 (a and b), Escherichia
coli ATCC25922 (c and d), Pseudomonas aeruginosa ATCC27853 (e), and Staphylococcus aureus ATCC25923 (f). R in
parentheses (R) indicated resistant.
3. Experimental
3.1. General procedures
Melting points were measured by a digital melting point apparatus (Electrothermal) and were corrected. IR
spectra were determined in the region 4000–400 cm−1 on a NEXUS 670 FT IR spectrometer by preparing
KBr pellets. The 1 H and 13 C NMR spectra were recorded on a Bruker 400 FT NMR at 400 and 100 MHz,
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NOROOZI PESYAN et al./Turk J Chem
respectively (University of Tabriz, Tabriz, Iran). 1 H and 13 C NMR spectra were obtained in solution in DMSO-
d6 and/or in CDCl3 as solvent using TMS as internal standard. The data are reported as follows: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, bs = broad singlet, coupling constant(s) in
Hz integration. All reactions were monitored by TLC with silica gel-coated plates (EtOAc:n-hexane/8:10/v:v).
UV-visible spectra were recorded on a T80 UV-vis (PG instruments Ltd) spectrometer (Urmia University,
Urmia, Iran). Compounds 1, 5a–5i, sodium azide, sodium nitrite, hydrochloric acid, and the solvents used were
purchased from Merck and Aldrich without further purification.
Table 2. Antimicrobial activity of some potential tetrazolic azo dyes.
Acinetobacter Escherichia Pseudomonas Staphylococcus
calcoaceticusa colia
aeruginosaa
aureusb
ATCC25923
Compd.
ATCC23055
14 mmc
25
14
30
R
R
20
24
ATCC25922 ATCC27853
6a
6b
6c
6d
6e
6f
Rd
20
11
18
R
R
R
10
16
R
R
R
R
R
14
20
18
32
R
R
R
15
R
R
R
6g
6h
6i
a
R
R
R
b
c
d
Gram-negative. Gram-positive. Scale is based in millimeter radius. Resistant.
Table 3. Antimicrobial activities against the four bacterial species by six standard drugs as a model test.
Erythromycin
(5 µg)
Cephalothin
(30 µg)
Ampicillin
(10 µg)
Trimethoprim/
sulfamethoxazole
Imipenem
(10 µg)
Bacterial strains
Ciprofloxacin
Pseudomonas aeruginosa
ATCC27853
Acinetobacter calcoaceticus
ATCC23055
Escherichia coli
ATCC25922
NTa
Rb
R
R
R
31 mm
NT
NT
NT
18 mm
14 mm
11 mm
25 mm
24 mm
22 mm
R
NT
20 mm
27 mm
NT
NT
NT
Staphylococcus aureus
ATCC25923
20 mm
NT
b
aNot tested. Resistant.
3.2. General procedure for the preparation of 2
3.2.1. N -(4-Cyanophenyl)acetamide (2)
To a 50-mL round bottom flask equipped with a magnetic stirrer were added consecutively p-aminobenzonitrile
(2.36 g, 20 mmol) and acetic anhydride (dropwise 50 mmol) and then the reaction mixture was refluxed for
30 min. After cooling, the reaction mixture was poured into a beaker containing 30 mL of cool distilled water
and white solid precipitated. The mixture was boiled until decomposition of the acetic anhydride residue. The
precipitate was filtered out and washed with a mixture of cool ethanol and water (2.93 g, 92% yield).
Colorless solid, mp 206–208 ◦ C; FT IR (KBr) 3303 (NH), 3256, 3183, 3109, 3053 (CH-ar.), 2932 (CH-
aliph.), 2221 (CN), 1671 (CO) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 2.09 (s, 3H, CH3), 7.75 (s, 4H, C-ar.),
;
10.38 (s, 1H, NH); 13 C NMR (100 MHz, DMSO-d6) δ: 24.2 (CH3), 104.7 (CN), 118.9 (CH-ar.), 119.1 (C-ar.)
133.3 (CH-ar.), 143.5 (C-ar.), 169.2 (CO).
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NOROOZI PESYAN et al./Turk J Chem
3.3. General procedure for the preparation of 8
3.3.1. N -(4-(1H -tetrazol-5-yl)phenyl)acetamide (8)
In a 50-mL round bottom flask equipped with a magnetic stirrer and an oil-bath, a mixture of p-cyanoacetanilide
(2.93 g, 18.3 mmol), sodium azide (2.37 g, 36.5 mmol), and ammonium chloride (0.53 g) as a catalyst was
dissolved in 20 mL of DMF and refluxed for 12 h. The reaction progression was controlled by thin layer
chromatography (TLC) with the solvent mixture of EtOAc:cyclohexane:methanol/8:10:2 (V/V). The reaction
color turned pale yellow and the solvent was removed under reduced pressure by a rotary evaporator. The
viscous residue was put on an ice-bath, hydrochloric acid (2 M) was added dropwise until the pH was 1, and
the white solid was precipitated, filtered out, and recrystallized with the mixture of methanol and water (3.1 g,
84% yield).
Colorless solid, mp 289–300 ◦ C; FT IR (KBr) 3311, 3267 (NHCO), 3195, 3129, 3069, 2985, 2920, 2854,
2706, 2615, 2471 (N4 H, CH-ar.), 1678 (C=O) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 2.09 (s, 3H, CH3),
;
3.44 (bs, 1H), 7.78 (d, 2H, J = 8.3 Hz, CH-ar.), 7.96 (d, 2H, J = 8.3 Hz, CH-ar.), 10.26 (s, 1H, NHCO); 13 C
NMR (100 MHz, DMSO-d6) δ: 24.2 (CH3), 118.4 (C-ar.), 119.2 (CH-ar.), 127.7 (CH-ar.), 141.9 (C-ar.), 155.0
(C-tetrazole), 168.9 (C=O).
3.4. General procedure for the preparation of 7
3.4.1. 4-(1H -tetrazol-5-yl)aniline (7)
Colorless solid, mp 265–267 ◦ C; FT IR (KBr) 3485, 3385 (NH2), 3213, 3142, 3097, 3062, 3024, 3003, 2938,
2859, 2792, 2753, 2632, 2496, 2357 (N4 H, CH-ar.), 1622 cm−1 1 H NMR (400 MHz, DMSO-d6) δ 3.36 (bs,
;
2H, overlapped with the DMSO’s water peak), 5.77 (bs, 1H), 6.68 (d, 2H, J = 8.5 Hz, CH-ar.), 7.68 (d, 2H,
J = 8.5 Hz, CH-ar.); 13 C NMR (100 MHz, DMSO-d6) δ: 113.6 (CH-ar.), 128.2 (CH-ar), 142.0 (C-ar.), 151.6
(C-ar.), 160.0 (C-tetrazole).
3.5. General procedure for the preparation of 3
3.5.1. 4-(1H -tetrazol-5-yl)benzenaminium chloride (3)
In a 50-mL round bottom flask equipped with a magnetic stirrer and an oil bath, N -(4-(1H -tetrazol-5-
yl)phenyl)acetamide (2.6 g, 12.8 mmol) in 20 mL of hydrochloric acid (4 M) was refluxed for 5–6 h. After
cooling, the solvent was evaporated and white solid precipitated (2.04 g, 80% yield).
Colorless solid, mp 251–253 ◦ C; FT IR (KBr) 3383, 3263, 3044, 3007, 2979, 2922, 2854, 2769, 2758,
;
2470 (N4 H, NH+3 , CH-ar.), 1620, (C=C) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 7.29 (d, 2H, J = 8.4 Hz,
CH-ar.), 8.04 (d, 2H, J = 8.4 Hz, CH-ar.), 6.73 (bs, 4H); 13 C NMR (100 MHz, DMSO-d6) δ: 119.2 (C-ar.),
120.6 (CH-ar.), 128.4 (CH-ar.), 140.5 (C-ar.), 154.9 (C-tetrazole).
3.6. General procedure for the synthesis of tetrazolic azo dyes 6a–6d
In a 50-mL beaker equipped with an ice-salt bath, 4-(1H -tetrazol-5-yl)benzenaminium chloride (1.0 mmol) was
dissolved in 5 mL of distilled water at 0 ◦ C. Then the solution of sodium nitrite (2.0 mmol) in 10 mL of water
was added dropwise into the beaker over 30 min at 0 ◦ C and afterwards 5 mL of diluted HCl was added to the
reaction mixture and this made it a clear solution at 0 ◦ C. In the other vessel, 1 mmol of an electron donor
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NOROOZI PESYAN et al./Turk J Chem
was dissolved in 10 mL of 10% sodium hydroxide. Finally, the solution of diazonium salt was added dropwise
into the basic solution of electron donor. As a result, red solid dye was precipitated, filtered out, washed with
distilled water, recrystallized with methanol, and dried.
3.6.1. (E)-1-(4-(1H -tetrazol-5-yl)phenyl)-2-(2,4-dimethoxyphenyl)diazene (6b)
Red solid, mp 222–224 ◦ C (decomp.); FT IR (KBr) 3536, 3423, 3189, 3084, 3022, 2979, 2918, 2838, 2765, 2639
(N4 H, CH-ar.), 1605 cm−1 1 H NMR (400 MHz, DMSO-d6) δ 3.89 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.66
;
(dd, 1H, 3J = 9.9 Hz, 4J = 1.2 Hz, CH-ar.), 6.81 (d, 1H, J = 1.2 Hz, CH-ar.), 7.69 (d, 1H, J = 9.0 Hz, CH-
ar.), 7.98 (d, 2H, J = 7.5 Hz, CH-ar.), 8.22 (d, 2H, J = 7.5 Hz, CH-ar.); 13 C NMR (100 MHz, DMSO-d6) δ:
55.8 (OCH3), 56.2 (OCH3), 99.1 (CH-ar.), 106.6 (CH-ar.), 116.0 (C-ar.), 117.5 (CH-ar.), 123.1 (CH-ar.), 128.1
(CH-ar.), 130.0 (C-ar.), 135.9 (C-ar.), 153.8 (C-tetrazole), 159.3 (C-OCH3), 164.4 (C-OCH3); UV-visible data
(EtOH): λmax , (log εmax) = 313, 363 nm, (2.122, 1.793); UV-visible data (acetone): λmax , (log εmax) = 360
nm, (1.531).
3.6.2. (E)-4-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)benzene-1,3-diol (6c)
Red solid, mp 160–162 ◦ C (decomp.); FT IR (KBr) 3522 (OH), 3405 (OH), 3173, 2977, 2813, 2756, 2689 (N4 H,
CH-ar.), 1696, 1660 cm−1 1 H NMR (400 MHz, DMSO-d6) δ 6.37 (d, 1H, J = 10.8 Hz, CH-ar.), 6.48 (d, 1H, J
;
= 10.8 Hz, CH-ar.), 7.79 (d, 2H, J = 11.0 Hz, CH-ar.), 7.83 (d, 2H, J = 10.8 Hz, CH-ar.), 13.84 (bs, 2H, OH),
14.0 (bs, 1H, OH); 13 C NMR (100 MHz, DMSO-d6) δ: 128.6 (CH-ar.), 129.3 (CH-ar.), 130.7 (CH-ar.), 131.0
(CH-ar.), 145.0 (C-ar.), 146.8 (CH-ar.), 147.1 (C-ar.), 173.2 (C-ar.), 177.6 (C-ar.), 178.8 (C-ar.), 182.5 (C-ar.);
UV-visible data (EtOH): λmax , (log εmax) = 288, 331 nm, (1.291, 0.631, up to 3.5 × 10−5M); UV-visible data
(acetone): λmax , (log εmax) = 332 nm, (1.838).
3.6.3. (E)-1-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)naphthalen-2-ol (6d)
Red solid, mp 69–71 ◦ C; FT IR (KBr) 3433 (OH), 3062, 3029, 2926, 2860, 2759, 2623 (N4 H, CH-ar.), 1615
cm−1
;
1 H NMR (400 MHz, DMSO-d6) δ 6.82 (d, 1H, J = 9.5 Hz, CH-napht.), 7.46 (t, 1H, J = 9.8 Hz,
CH-napht.), 7.61 (t, 1H, J = 7.9 Hz, CH-napht.), 7.73 (d, 1H, J = 7.6 Hz, CH-napht.), 7.90 (d, 1H, J =
9.5 Hz, CH-napht.), 8.00 (d, 2H, J = 8.4 Hz, CH-ph), 8.15 (d, 2H, J = 8.4 Hz, CH-ph), 8.50 (d, 1H, J =
8.1 Hz, CH-napht.), 15.85 (s, 1H, OH); 13 C NMR (100 MHz, DMSO-d6) δ: 118.7 (CH-ar.), 121.7 (CH-ar.),
122.1 (C-ar.), 125.0 (CH-ar.), 126.6 (CH-ar.), 128.0 (C-ar.), 128.5 (CH-ar.), 129.1 (CH-ar.), 129.4 (C-ar.), 130.0
(C-ar.), 130.4 (C-ar.), 132.7 (CH-ar.), 141.7 (CH-ar.), 145.7 (C-tetrazole), 174 (C-OH); UV-visible data (EtOH):
λmax , (log εmax) = 363, 490 nm, (1.772, 1.273, up to 1.5 × 10−4M); UV-visible data (acetone): λmax , (log
εmax) = 382, 479 nm, (1.212, 0.933).
3.7. General procedure for the synthesis of tetrazolic azo dyes 6e–6h and 6i
In a 50-mL beaker equipped with an ice-salt bath, 4-(1H -tetrazol-5-yl)benzenaminium chloride (1.0 mmol) was
dissolved in 5 mL of distilled water at 0 ◦ C. Then the solution of sodium nitrite (2.0 mmol) in 10 mL of water
was added dropwise to the beaker over 30 min at 0 ◦ C and afterwards 5 mL of diluted HCl was added to the
reaction mixture and this made it a clear solution at 0 ◦ C. In the other vessel, 1 mmol of an electron-donor
(barbituric acid and its derivatives) was dissolved in 10 mL of water. Finally, the solution of diazonium salt
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NOROOZI PESYAN et al./Turk J Chem
was added dropwise to the solution of electron donor. As a result, yellow solid dye was precipitated, filtered
out, washed with distilled water, recrystallized with methanol, and dried.
3.7.1. (E)-5-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)pyrimidine-2,4,6(1H ,3H ,5H )-trione (6e)
Yellow solid, mp 245 ◦ C; FT IR (KBr) 3478 (OH/NH), 3323, 3200 (OH), 3075, 2846, 2769, 2687, 2629, 2574,
2507, 2479 (N4 H, CH-ar.), 1743, 1692, 1664 (C=O) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (d, 2H, J
;
= 8.3 Hz, CH-ar.), 8.09 (d, 2H, J = 8.3 Hz, CH-ar.), 11.40, 11.32 (2s, 1H, NH-BA), 11.55 (s, 1H, NH-BA),
14.10 (s, 1H, NH/OH); 13 C NMR (100 MHz, DMSO-d6) δ 117.2 (CH-ar.), 119.0 (C-BA), 121.1 (C-ar.), 128.4
(CH-ar.), 143.5 (C-ar.), 149.7 (C-tetrazole), 154.9 (CO), 159.7 (CO), 161.9 (CO); UV-visible data (EtOH):
λmax , (log εmax) = 292, 330 nm, (1.293, 0.870, up to 3.5 × 10−5M); UV-visible data (acetone): λmax , (log
εmax) = 333 nm, (1.852).
3.7.2. (E)-5-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)-1,3-dimethylpyrimidine-2,4,6(1H ,3H ,5H )-
trione (6f)
Yellow solid, mp 108–110 ◦ C; FT IR (KBr) 3430 (OH/NH), 3093, 3065, 2961, 2929, 2762, 2500 (N4 H, CH-ar.),
1724, 1676, 1645 (C=O) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 3.24 (s, 6H, 2NCH3), 7.82 (d, 2H, J = 8.5
;
Hz, CH-ar.), 8.12 (d, 2H, J = 8.5 Hz, CH-ar.), 14.16 (s, 1H, NH/OH); 13 C NMR (100 MHz, DMSO-d6) δ
27.3 (NCH3), 28.2 (NCH3), 117.4 (CH-ar.), 118.5 (C-BA), 121.9 (C-ar.), 128.4 (CH-ar.), 134.0 (C-ar.), 143.4
(C-tetrazole), 150.6 (CO), 158.6 (CO), 160.3 (CO); UV-visible data (EtOH): λmax , (log εmax) = 317, 408 nm,
(0.517, 0.200, up to 3.0 × 10−5M); UV-visible data (acetone): λmax , (log εmax) = 391 nm, (1.662).
3.7.3. (E)-5-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)-2-thioxo-dihydropyrimidine-4,6(1H ,5H )-
dione (6g)
Yellow solid, mp 178–180 ◦ C; FT IR (KBr) 3463 (OH/NH), 3328, 3212, 3152, 2923, 2875, 2769, 2725, 2685,
2626, 2569, 2530 (N4 H, CH-ar.), 1682, 1664 (C=O) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 7.82 (d, 2H, J =
;
8.6 Hz, CH-ar.), 8.12 (d, 2H, J = 8.6 Hz, CH-ar.), 12.50 (s, 1H, NH), 12.66 (s, 1H, NH), 14.18 (s, 1H, NH/OH);
13 C NMR (100 MHz, DMSO-d6) δ 117.7 (CH-ar.), 119.9 (C-TBA), 121.5 (C-ar.), 128.5 (CH-ar.), 143.4 (C-ar.),
155.0 (C-tetrazole), 158.3 (CO), 159.9 (CO), 177.7 (CS); UV-visible data (EtOH): λmax , (log εmax) = 329, 366,
402, 465 nm, (1.911, 2.077, 1.720, 1.804, up to 2.5 × 10−4M); UV-visible data (acetone): λmax , (log εmax) =
337, 366, 402, 455 nm, (1.660, 2.157, 1.728, 1.803).
3.7.4. (E)-5-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)-1,3-diethyl-2-thioxo-dihydropyrimidine-4,6
(1H ,5H )-dione (6h)
Yellow solid, mp 241–243 ◦ C; FT IR (KBr) 3441 (OH/NH), 3167, 3101, 2979, 2934, 2872, 2759, 2618, 2463 (N4 H,
CH-ar.), 1702, 1646 (C=O) cm−1 1 H NMR (400 MHz, DMSO-d6) δ 1.17 (t, 6H, J = 7.3 Hz, 2NCH2 CH3),
;
4.42 (q, 4H, J = 7.3 Hz, 2NCH2 CH3), 7.90 (d, 2H, J = 7.5 Hz, CH-ar.), 8.13 (d, 2H, J = 7.5 Hz, CH-ar.),
14.31 (bs, 1H, NH/OH); 13 C NMR (100 MHz, DMSO-d6) δ 12.1 (2NCH2 CH3), 35.3 (2NCH2 CH3), 112.0
(C-DETBA), 118.0 (CH-ar.), 119.4 (C-ar.), 120.1 (C-ar.), 128.5 (CH-ar.), 150.5 (C-tetrazole), 160 (CO), 164.0
(CO), 177.9 (CS); UV-visible data (EtOH): λmax , (log εmax) = 329, 360, 401, 456 nm, (1.975, 1.847, 1.811,
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NOROOZI PESYAN et al./Turk J Chem
1.795, up to 3.2 × 10−4M); UV-visible data (acetone): λmax , (log εmax) = 342, 366, 402, 462 nm, (1.790,
1.948, 1.728, 1.803, up to 1.1 × 10−4M).
3.7.5. (E)-2-((4-(1H -tetrazol-5-yl)phenyl)diazenyl)-5,5-dimethylcyclohexane-1,3-dione (6i)
Yellow solid, mp 240–242 ◦ C (decomp.); FT IR (KBr) 3435 (OH/NH), 3094, 2956, 2929, 2871, 2714, 2602, 2553,
2490, 2322 (N4 H, CH-ar.), 1673 (C=O), 1616 cm−1 1 H NMR (400 MHz, DMSO-d6) δ 1.04 (s, 6H, –C(CH3)2 –
;
), 2.59 (s, 2H, –CH2 –), 2.67 (s, 2H, –CH2 –), 3.40 (bs, 1H, NH-tetrazole, overlapped with the DMSO’s water
peak), 7.81 (d, 2H, J = 8.4 Hz, CH-ar.), 8.10 (d, 2H, J = 8.4 Hz, CH-ar.), 14.75 (s, 1H, NH/OH); 13 C NMR
(100 MHz, DMSO-d6) δ 28.0 (–C(CH3)2 –), 30.2 (–C(CH3)2 –), 51.8 (–CH2 –), 52.0 (–CH2 –), 117.7 (CH-ar.),
121.5 (C-ar.), 128.4 (CH-ar.), 130.8 (C-ar.), 143.7 (C-ar.), 154.9 (C-tetrazole), 192.8 (C=O), 197.2 (C=O);
UV-visible data (EtOH): λmax , (log εmax) = 288, 365, 397, 431 nm, (0.820, 2.979, 1.949, 1.816, up to 2.5 ×
10−4M); UV-visible data (acetone): λmax , (log εmax) = 366, 402, 421 nm, (2.971, 1.751, 1.793).
3.8. Biology
3.8.1. Materials and methods
3.8.2. Bacterial strains
The antibacterial activity of the synthesized compounds was tested against the gram-positive and gram-negative
bacterial strains Acinetobacter calcoaceticus (ATCC23055), Escherichia coli (ATCC2592), Pseudomonas aerug-
inosa (ATCC27853), and Staphylococcus aureus (ATCC25923).
3.8.3. Preparation of the test compound and antibacterial activity assays
The antibacterial activity of the compounds was assayed by Parekh et al.’s40 method with some modifications.
In brief, solutions with 100 µg/µL concentrations of each compound in DMSO (Merck) were prepared. A full
loop of the defined strain was inoculated in 25 mL of nutrient broth medium and incubated for 24 h in 37 ◦ C.
Mueller Hinton agar (MHA) (Merck) plates were prepared according to the manufacturer’s recommendations
by dissolving 34 g of the medium in 1000 mL of distilled water. Then 30 mL of autoclaved media were added
to a 10-cm plate. Inoculation of each strain was done by the pour-plate method. Next 200 µL of the activated
strain was added to the MHA medium at 45 ◦ C and after proper homogenization was distributed into a
petri dish. The complete microbiological procedures were performed in a laminar airflow in order to maintain
aseptic conditions. After solidification of the media, a well was made in the MHA with a sterile glass tube
(6 mm) and 50 µL of the drug compound was added to the well. Then 50 µL of DMSO was inoculated into
another well as a negative control. The antibacterial activities of the drug compounds were determined by
measuring the inhibition zone formed around each well against the defined bacterial strain. Erythromycin and
cephalothin were used as standard drugs for antibacterial effects against gram-positive bacteria while ampicillin,
trimethoprim/sulfamethoxazole, and ciprofloxacin were used against gram-negative bacteria and imipeneme was
used for P. aeruginosa.
4. Conclusion
In summary, new series of tetrazolic azo dyes based on (thio)barbiturates and electron-rich aromatics were
synthesized and their structures were characterized employing spectroscopic techniques. Their antimicrobial
properties were evaluated on gram-positive and gram-negative bacterial strains in detail.
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Acknowledgments
We gratefully acknowledge financial support from the Research Council of Urmia University and Urmia Uni-
versity of Medical Sciences.
Supplementary material
Full characterization data of compounds 6a–6i and antibacterial activity assays are available.
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