Synthesis, characterization, antimicrobial activities and electrochemical behavior of new phenolic azo dyes from two thienocoumarin amines

Article abstract of DOI:10.24820/ARK.5550190.P010.994

The coupling reactions of diazotized thienocoumarin derivatives with p-acetaminophen, salicylic acid and acetylsalicylic acid gave four products in which the benzene ring of the phenolic reagents were primarily substituted by two 3-diazenyl-4H-thieno[3,4-

Full text of DOI:10.24820/ARK.5550190.P010.994

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Synthesis, characterization, antimicrobial activities and electrochemical behavior
of new phenolic azo dyes from two thienocoumarin amines
Kamal S. D. Djeukoua,a Emmanuel S. Fondjo,*a Jean-de-Dieu Tamokou,b Joseph Tsemeugne,a,f Peter F. W.
Simon,c Appolinaire Tsopmo,d Francis M. M. Tchieno,e Steve E. Ekom,b Chancellin N. Pecheu,e
Ignas K. Tonle,e and Jules-Roger Kuiateb
aLaboratory of Applied Synthetic Organic Chemistry, Department of Chemistry, Faculty of Science, University of
Dschang, P.O. Box 67 Dschang, Republic of Cameroon; bLaboratory of Microbiology and Antimicrobial
Substances, Department of Biochemistry, Faculty of Science, University of Dschang, PO Box 067 Dschang,
Republic of Cameroon; cPolymer Chemistry Laboratory, Faculty of Live Sciences, Rhine-Waal University of
Applied Sciences, Campus Kleve, Marie-Curie Strasse 1, D-47533 Kleve, Germany; dDepartment of Chemistry,
Carlton University, Room 207D, Steacie Building 1125 Colonel By Drive K1S 5B6, Ontario, Canada +1-613-520-
260 Ext 3122; e Laboratory of Electrochemistry and Chemistry of Materials, Department of Chemistry,
University of Dschang, P.O. Box 67 Dschang, Cameroon; fDepartment of Organic Chemistry, University of
Yaounde I, P.O. Box 812, Yaounde, Republic of Cameroon.
E-mail: sopbue@yahoo.fr
Received 06-07-2019
Accepted 01-13-2020
Published on line 02-11-2020
Abstract
The coupling reactions of diazotized thienocoumarin derivatives with p-acetaminophen, salicylic acid and
acetylsalicylic acid gave four products in which the benzene ring of the phenolic reagents were primarily
substituted by two 3-diazenyl-4H-thieno[3,4-c]chromen-4-one or 3-diazenyl-4-imino-4H-thieno[3,4-
c]chromene moieties. The newly prepared azo dyes were fully characterized based on their analytical and
spectroscopic data. The electrochemical analysis of the novel azo dyes carried out on a glassy carbon electrode
unequivocally further confirmed the assigned structures. The synthesized compounds tested for their
antimicrobial activity using broth microdilution method, showed good activities.
Keywords: Azo compounds, phenol derivatives, synthesis, characterization, redox studies, antimicrobial
activity
DOI: https://doi.org/10.24820/ark.5550190.p010.994
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Introduction
The synthesis of organic dyes as nonlinear optical materials is an increasingly prolific research axis in the
world.1-3 These materials are widely used in optical communication, information processing, frequency
doubling and integrated optics.4-6 Nonlinear optical materials (NLO) have several advantages over inorganic
materials used for the same reasons. We can thus name the great value of the nonlinear optical coefficient,
the easy methods of synthesis and preparation, and the low costs.7
Heterocyclic azo dyes are used as better coloring agents in the textile industry.8 The applications of azo
10,11
compounds in the electronic industry as colorimetric sensors,9 nonlinear optical (NLO) devices
and liquid
crystalline displays (LCDs)12 have been intensively investigated. This group of compounds has attracted much
attention for their use as potential sensitizers for photodynamic therapy (PDT).13
The azo dyes containing the thiophenic ring have several advantages over other dyes, in particular due to
their intense coloration effect.14,15
Electrochemical, photophysical and photochemical properties of azo molecules have been used to
predetermine their reactivity or to confirm their structures.16,17 In a recent work, our group reported the
electrochemical behavior of azo compounds containing hydroxyl group located at the ortho position of the azo
group.18 In the present study, we extend our investigations to diazo dyes possessing different substituents in
order to evaluate the influence of the substituent on the progress of the redox reactions. Indeed, another
work has shown that the type of substituent on the aromatic ring of some azo compounds has an influence on
the oxidation-reduction.19 The purpose of the present work was then to prepare and describe new azo
compounds, to determine their degradation under electrochemical conditions, and finally to determine their
antimicrobial properties.
Results and Discussion
Chemistry
The present investigation deals with the synthesis of novel azo compounds using
thienocoumarins 2 as starting materials. The general preparation procedure is displayed in Scheme 1.20-22
CH3
O
S
S
CN
X
NH2
S8/NH3
EtOH
NaNO2, H2SO4
0-5 ºC
HSO4
N2
O
2a, b
X
X
O
3a, b
1a, b
a: X = O
b: X = NH
a: X = O
b: X = NH
a: X = O
b: X = NH
Scheme 1. Reactions’ sequences to the in situ formed diazonium intermediates.
The thienyldiazonium ions 3 were readily synthesized by adding thienocoumarin 2 to nitrosyl sulfuric acid
at 0-5° C according to literature.23 Diazonium ion 3a was then coupled with three phenol derivatives namely
salicylic acid (4a), p-acetaminophen (4b) and acetylsalicylic acid (4c) to yield compounds 5a-c (Scheme 2).
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Scheme 2. Reactions’ sequences to compounds 5.
The coupling of the in situ formed diazonium ion 3b with 2-acetyl salicylic acid in similar reactions’
conditions gave compound 6 (Scheme 3) in good yield. It has to be pointed out, that compound 5d was found
in all reactions mentioned as an additional product. This compound resulting from the cyclotrimerization of
the intermediate 3-diazenyl-4H-thieno[3,4-c]chromen-4-one ion (3a), was previously reported.24 In the case
involving the intermediate ion 3b, the later might undergo oxidation of its imino group into the carbonyl
function prior to the cyclotrimerization process. Alternatively 3b might first cyclize before the oxidation of the
imino group into the carbonyl function occur.
O
NH
O
COOH
O
12%
5d
N
+
N
4c
3b +
S
6
41%
Scheme 3. Reaction sequence to compound 6.
The synthesized compounds 5 and 6 were characterized on the basis of their physical data and spectral
analysis.
The infrared spectra of all the synthesized dyes showed intense bands between 3621-3277 cm-1 due to
free and associated OH groups and other bands at 1733-1670 cm–1 which were assigned to the carbonyl
groups. The FT-IR spectra also showed bands at 1490 cm–1, assigned to the –N=N- linkage of the azo moieties.
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1
The H NMR spectrum of compound 5a showed five sets of aromatic Csp2-H protons characteristic of the
parent thiophene at 7.70 (2H, s, H-1’ and H-1’’); 7.35 (2H, dd, H-6’ and H-6’’, JHH 6.8 and 1.1 Hz); 7.69 (2H, ddd,
JHH 7.5, JHH 6.4 and 1.5 Hz, H-7’ and H-7’’); 7.39 (2H, ddd, JHH 7.3, JHH 6.9 and 1.6 Hz, H-8’ and H-8’’) and 8.80
(2H, dd, JHH 7.5 and 1.7 Hz, H-9’ and H-9’’). The signal of the salicylic acid group was found at 14.36 ppm. The
positions of both parent thiophenic moieties on the phenolic ring were assigned based on the coupling
constant observed between the only two remaining aromatic protons’ signals at 7.72 (1H, JHH 1.2 Hz, H-4) and
7.37 (1H, d, JHH 1.2 Hz, H-6).
13
The C NMR spectrum exhibited 22 signals in agreement with the assigned structure 5a. The DEPT-
spectrum indicates the presence of 12 hydrogenated carbons. Strong signals observed in this spectrum are
considered to belong to the benzenic cycles of the parent thiophene moiety, since the signal of two
hydrogenated thiophenic carbons were identified at 125.6 ppm.22-24 The signal of the carbonyl group of the
carboxylic group was found at 185.1 ppm.
The structure of compound 5a is further strongly supported by its HRMS ESI+ mode, which shows the
pseudo molecular ion peak at m/z 607 (1%) corresponding to [M-CO2+H2O+K+] and characteristic fragment ion
peaks at m/z 549 (1%, cleavage of CO2), m/z 493 (1%, cleavage of CO2, then losses of both azoic bridge). The
peaks at m/z 413 (1%), 360 (19) and 270 (100) were respectively attributed to the fragment ions (M+H-2S-2N2-
CO2-H2O), (M-N2-CO2-C7H6O3) and (C11H7O3N2SNa)+.
As previously mentioned, in the case of compounds 5c we noticed the opening of the coumarin rings.23, 26
We also noted that, as earlier described, these products result from multiple copulation of the diazonium
moiety on the phenol derivative.27
Similar analysis as above was conducted on the basis of the available elemental and spectroscopic data for the
structural assignments of compounds 5b, 5c and 6.
Redox behaviors of the azo dyes
The cyclic voltammograms obtained for compounds 5a-c and 6 showed a quasi-reversible system (peaks I and
II). This corresponds to the reduction of the pristine dye’s azo functions to hydrazo functions.28,29 Further
reduction is observed at peak III. This probably corresponds to the fragmentation of the hydrazo functions to
form amine functions. Peak IV corresponds to the further reduction of one of the latter fragmentation
products. Based on the foregoing discussion, the electrochemical mechanisms depicting the electrode
phenomena are shown in Schemes 4-6. Since one of the hydrazo functions of compound 5c is highly hindered,
its fragmentation probably takes place in two steps, with the less hindered function reduced first (scheme 5).
Electrochemical reaction mechanisms of the studied azo dyes
Compound 5a. Three distinct reduction peaks (II, III and IV) were observed for the electroreduction of azo
dyes 5a, the first one II due to the formation of the hydrazo product A1 (Scheme 4) which provide quasi
reversible oxidation–reduction peaks I during reverse and subsequent forward scans due to the formation of
oxidation product 5a and its subsequent reduction to compound A1. The second peak III, is due to the
reduction of hydrazo-compound A1 to yield two amines A2 and A3 (Scheme 4). The third peak can therefore
be attributed to the reduction of the carbonyl function of compound A2 to yield compound A4.
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S
H
N
S
H
N
OH
N
OH
N
N
COOH
N
COOH
O
+4e-, + 4H+
O
O
O
O
O
O
O
N
H
N
H
A1
S
5a
S
S
S
NH2
NH2
OH
+2e-, + 2H+
O
O
O
A4
A2
Scheme 4. Electrochemical reaction mechanism of 5a.
Compound 5b. The mechanistic details of the electrochemical oxidation and reduction of compound 5b are
similar to those of compound 5a.
Compound 5c. In the cyclic voltammograms of 5c, four peaks were recorded, of which three cathodic peaks (II,
III and IV) in the forward scan and one anodic peak (I) in the reverse scan, indicating the quasi-reversible
electrochemical nature of the dye. The anodic peak only appeared in the subsequent scan after the reduction
step.
The first peak can be attributed to the reduction of the two –N=N– bridges (Scheme 5) to yield the
hydrazo intermediate B1 which in turn is reduced to give compound 5c during reverse and subsequent
forward scan. The second peak III, is due to the reduction of one hydrazo bond of compound B1 particularly
the hydrazo bond adjacent to the electron-donating groups at the ortho position to the azo bridge to yield
amine B2 and hydrazo-compound B3. The last peak may be attributed to the reduction of the hydrazo-bond of
compound B3 to yields two amines B2 and B4.
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O
H
CH3
O
H
CH3
O
H H
N N
O
N
S
H2N
COOH
HO
COOH
HOOC
H2N
+2e-, + 2H+
+
COOH
HO
HOOC
N
S
N
HO
OH
HOOC
N
H
B2
H
S
S
B3
B1
O
CH3
O
CH3
HO
HOOC
S
O
O
N
+2e-, + 2H+
H2N
H2N
COOH
+
H2N
COOH
HO
HOOC
B2
NH2
B4
H
N
H
S
B3
Scheme 5. Electrochemical reaction mechanism of 5c.
Compound 6. The mechanistic details of the electrochemical oxidation and reduction of compound 6 is
indistinguishable with that of compound 5a.
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NH2
O
O
CH3
S
S
O
H
N
H
N
O
NH2
+2e-, + 2H+
H2N
COOH
COOH
+
O
NH
O
NH
C2
C3
C1
S
S
NH2
NH2
+2e-, + 2H+
O
NH
O
C4
NH2
C2
Scheme 6. Electrochemical reaction mechanism of 6.
Antimicrobial activity
The antimicrobial activity of azo compounds and phenol derivatives was performed against bacterial and
fungal species and their minimum inhibitory concentration (MIC) and minimum microbicidal concentration
(MMC) values are summarized in Table 1. The antimicrobial activities of compounds 1a, 1b, 2a, 2b, 5a, 5b, 5c
and 6 have been recorded on 25%(3/12), 16.66%(2/12), 100%(12/12), 100%(12/12), 100%(12/12),
100%(12/12) and 100%(12/12) of the tested microorganisms, respectively. The antibacterial and antifungal
activities of the prepared azo compounds 5 and 6 were higher than those of their starting materials as
compounds 1a and 1b were not active against all the tested bacterial species (MIC ˃ 256 µg/mL) whereas they
displayed weak antifungal activity (MIC = 64 - ˃ 256 µg/mL) when compared to that of their derivatives (MIC =
4 - 256ꢀµg/mL). Standard drugs ciprofloxacin and nystatin were used as controls for antibacterial and
antifungal activities, respectively performed better than most tested compounds (Table 1). Interestingly,
compounds 5a, 5b, 5c and 6 were in some cases equipotent with standard drugs against B. subtilis, S. aureus,
E. coli, V. cholerae NB2 and SG24 and C. neoformans. The activity of each compound varied depending of the
microorganism and compound 5a showed the lowest MIC value of 4 µg/mL against B. subtilis compared to 8
µg/mL for the control ciprofloxacin.
Compound 5a and 5b (MIC = 4 - 32ꢀµg/mL) were the most potent against all the bacterial and fungal strains
followed in decreasing order by compounds 6, 5c, 3a, 3b, 1a, and 1b. Compound 5a with two 3-diazenyl-4H-
thieno[3,4-c]chromen-4-one moieties at positions -3 and -5 of the phenolic ring, is equipotent with compound
5b against S. aureus ATCC25923, S. aureus, P. aeruginosa ATCC27853, V. cholerae, C. albicans, C. neoformans
and C. tropicalis. In contrast, compound 5c with opened coumarin rings of similar substituents at the same
positions on the phenolic ring exhibited similar potency as that of compounds 5a and 5b against C.
neoformans and C. tropicalis with MIC values of 4 and 8ꢀµg/mL respectively. A structure-activity-relationship
study from the results indicated the necessity of electron withdrawing and releasing groups at different
positions on the phenolic ring. So, a combination of electron withdrawing and releasing groups on the
phenolic ring can be synthesized and tested with a hope to get promising antimicrobial agents. A lower
MMC/MIC (≤4) value signifies that a minimum amount of tested compounds is used to kill the microbial
species, whereas, a higher values signifies the use of comparatively higher amount of compound for the
control of any microorganism.30 Hence, compounds 1a and 1b showed fungistatic effect on the sensitive fungal
strains while compound 2a displayed microbicidal activity against S. aureus ATCC25923, S. aureus, B. subtilis,
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P. aeruginosa ATCC27853 and E. coli and bactericidal effect against S. flexneri and V. cholerae. The other
compounds exhibited microbicidal effect on all the sensitive microorganisms. The results of the present
investigation are in agreement with those reported in the literature describing the antimicrobial activities of
some azo compounds and phenol derivatives against a wide range of microorganisms among which S. aureus,
31-35
36, 42-44
P. aeruginosa,31-33, 36 V. cholera,37-40 B. subtilis,37-40 C. albicans,31, 33, 36, 40 C. neoformans
and C.
tropicalis.45-48
.
Cytotoxic activity
The cytotoxic activity of azo compounds and phenol derivatives against red blood cells (RBCs) was investigated
using Triton X-100 as a positive control. The positive control showed about 100% lysis, whereas the phosphate
buffer saline (PBS) showed no lysis of RBCs. Interestingly, none of the tested compounds showed cytotoxic
activity against RBCs at concentrations up to 256 μg/mL (results not shown). This finding supports the
selective toxicity of the tested compounds towards the tested bacteria.
Table 1. Antibacterial and antifungal activities (in µg/ml) of azo compounds and phenol derivatives against
bacterial and yeast species
Microorga Inhibition
1a
1b
2a
2b
5a
5b 5c
6
Reference
drugs*
nisms
parameters
Bacteria
Staphyloc MIC
>256
>256
nd
>256
>256
nd
32
128
4
64
128
2
8
16
2
8
16
8
16
2
2
2
1
occus
MBC
16 32
2
aureus
ATCC2592
3
MBC/MIC
2
S. aureus
MIC
MBC
MBC/MIC
MIC
MBC
>256
>256
nd
>256
>256
nd
>256
>256
nd
>256
>256
nd
>256
>256
nd
>256
>256
nd
32
128
4
64
128
2
64
128
2
64
128
2
128
256
2
64
128
2
4
8
2
4
16
4
8
16
2
4
8
2
8
16
32
2
8
16
2
8
16
2
8
8
1
4
4
1
8
8
1
2
2
1
Bacillus
subtilis
16
16 32
2
8
16 32
2
MBC/MIC
2
16
Pseudomo MIC
nas MBC
aeruginos MBC/MIC
2
a
Escherichi MIC
>256
>256
nd
>256
>256
nd
>256
>256
nd
>256
>256
nd
64
128
2
128
256
2
128
256
2
128
>256
nd
4
16
4
8
16
2
8
16
16
32
2
16
32
2
4
4
1
4
4
1
a coli
MBC
32 16
4
16 32
32 64
2
MBC/MIC
MIC
MBC
1
Shigella
flexneri
MBC/MIC
MIC
2
Vibrio
cholerae
NB2
>256
>256
128
128
16
16 64
32 16
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MBC
MBC/MIC
V. chorae MIC
>256
nd
>256
>256
nd
>256
256
2
128
>256
nd
256
32
2
32
32 64
64 16
2
1
2
1
32 32
32 32
SG24
MBC
MBC/MIC
MIC
>256
nd
>256
>256
nd
>256
256
2
256
>256
nd
256
32
1
16
64 32
64 32
2
1
2
1
4
V.
16 16
16
cholerae
CO6
MBC
MBC/MIC
>256
nd
>256
nd
256
1
>256
nd
32
2
32 32
32
2
4
1
2
8
2
Yeasts
Candida
albicans
MIC
MFC
MFC/MIC
MIC
MFC
256
>256
nd
256
>256
nd
64
>256
nd
>256
>256
nd
256
>256
nd
128
>256
nd
32
64
2
32
32
1
16
32
2
32
64
2
32
64
2
16
64
4
8
16
2
8
16
2
4
8
2
16
16
32
2
8
16
2
4
16
4
4
4
1
4
4
1
4
4
1
32 32
4
8
2
16
C.
tropicalis
16 16
MFC/MIC
2
4
1
8
Cryptococ MIC
cus MFC
neoforma MFC/MIC
16 16
4
2
ns
*Ciprofloxacin for bacteria and nystatin for yeasts; nd : not determined. MIC: Minimum Inhibitory
Concentrations; MBC: Minimum Bactericidal Concentrations; MFC: Minimum Fungicidal Concentration
Conclusions
The study of the reactions of the title 2-aminothiophene substrates with the phenolic reagents has confirmed
the high reactivity of the intermediate thienyl diazonium ions 3 which coupling degree and regio-orientation
on the phenolic rings strongly depend on the nature of the substituents initially present. With salicylic acid the
electrophilic attack of the diazonium reagent 3a occurs at substitution free positions -3 and -5, whereas with
paraacetaminophen the electrophilic substitutions took place at positions -2 and -3 and with 2-acetylsalicylic
acid at positions -3 and -5. In contrast, 2-acetylsalicylic acid undergoes only one electrophilic substitution at
position -3 with the intermediate diazonium reagent 3b. All the newly prepared azo dyes where fully
characterized by their elemental and spectroscopic data. The electrochemical study on glassy carbon electrode
of the new dyestuffs brought further supporting evidences to the structural assignments. The antimicrobial
screenings revealed that all the four newly prepared azo dyes 5a, 5b, 5c and 6 have prominent antibacterial
and antifungal activities.
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Experimental Section
General. All Melting points are corrected and were determined with a STUART SCIENTIFIC Melting Point
Apparatus Model SMP3. The T.L.Cs were carried out on Eastman Chromatogram Silica Gel Sheets (13181;
6060) with fluorescent indicator. A mixture of ethyl acetate and methylene chloride (7:3) was used as eluent
and iodine was used as revelator for the chromatograms. The IR spectra were measured with a Fourier
Transform Infrared spectrometer Bruker Alpha. The UV spectra were recorded with a JENWAY 6715 UV-Vis
Spectrophotometer. Combustion analyses were carried out with a C, H, N, S Euro EA from Hekatech company,
their results were found to be in good agreement (±0.3%) with the calculated values. EIMS spectra were
1
recorded on a double focusing mass spectrometer (Varian MAT 311A). H-NMR spectra were recorded in
13
DMSO-d6 on a Bruker DRX spectrometer operating at 400 MHz. C-NMR spectra were recorded in DMSO-d6
on a Bruker DRX spectrometer operating at 100 MHz. TMS was used as internal reference. The chemical shifts
(δ) and coupling constants (J) are expressed in ppm and hertz respectively. Carbon attribution determined by
13C, HSQC and HMQC experiments.
All the reagents mentioned in this work were purchased from Aldrich and Fluka and were used without
further purification. Starting materials 2a and 2b have been prepared according to literature procedures as
published earlier.22
General procedure for the preparation of coupling products 5a-c and 6. Dry sodium nitrite (2.07 g, 3 mmol)
was slowly added over a period of 30 minutes to concentrated sulphuric acid (10 mL) with occasional stirring.
The solution was cooled to 0-5 °C. Compounds 2a and 2b was dissolved in DMSO (10 mL) and cooled to 0-5 °C.
The nitrosyl sulphuric acid solution was added to the solution of 2a and 2b and the temperature was
maintained to 0-5 °C. The clear diazonium salt solutions thus obtained consisting of the in situ formed
intermediates 3a and 3b, were used immediately in the coupling reactions.
The phenolic coupler (3 mmol) 4a-c was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5 °C.
The diazonium solutions of 3a and 3b previously prepared were added drop wise over 1 hour, and then 15 mL
of sodium acetate solution (10%) was added to the mixture. The solid precipitate was collected on a filter and
crystallised from water-ethanol (50% v/v) mixture to give the corresponding phenolic thienyl azo dyes.
2-Hydroxy-3,5-bis[2-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)diazenyl]benzoic acid (5a). Compound 5a was
obtained as black powder (1.57g, 56%). mp 192-194 °C; Rf 0.55 in CH2Cl2/AcOEt 80% v/v; UV (EtOH) λmax(Log ε)
235 (1.50), 297 (1.59), 335 (2.52), 430 (1.40) nm. IR (solid, KBr, νmax, cm-1): 3567 (free O-H), 3264 (ass O-H),
1731-1670 (C=O), 1637-1603 (CAr=CAr), 1544-1500 (thiophenic C=C), 1491 (-N=N-), 1268-1236 (C-N), 1163-1112
1
(C-O), 766-756 (=CAr-H) cm−1. H NMR (DMSO-d6, 400 MHz): δH 8.80 (2H, dd, JHH 7.5 and 1.7, Hz, H-9’, H-9’’),
7.72 (1H, d, JHH 1.2 Hz, H-6), 7.70 (2H, s, H-1’, H-1’’), 7.69 (2H, ddd, JHH 7.5 JHH 6.4 and 1.5 Hz, H-7’, H-7’’), 7.39
(2H, ddd, JHH 7.3 JHH 6.9 and 1.6 Hz, H-8’, H-8’’), 7.37 (1H, d, JHH 1.2 Hz, H-4), 7.35 (2H, dd, JHH 6.8, 1.1 Hz, H-6’,
13
H-6’’). C NMR (DMSO-d6, 100 MHz): δC 184.1(COOH), 162.8 (C-4’, C-4’’); 156.9 (C-3’, C-3’’); 155.4 (C-5a’, C-
5a’’); 153.6 (C-9b’, C-9b’’), 154,4 (C-2); 148,7 (C-3a’, C-3a’’); 135.5 (C-6); 135.4 (C-7’, C-7’’); 129.9 (C-9’, C-9’’),
126.1 (C-8’, C-8’’);125.6 (C-1’, C-1’’); 119.5 (C-5), 118.2 (C-9a’, C-9a’’); 117.7 (C-6’, C-6’’), 117.4 (C-4), 113,5 (C-
1), 102.4 (C-3). ESIMS m/z (%) 607 (1), 549 (1), 502 (8), 493(1), 413 (1), 384 (5), 360 (19), 322 (1), 320 (4), 292
(5), 270 (100), 248 (1), 228 (1), 208 (20). Anal. Calcd. for C29H14N4O7S2: C, 58.58; H, 2.37; N, 9.42; S, 10.79.
Found: C, 58.55; H, 2.39; N, 9.45; S, 10.81.
N-{4-Hydroxy-2.3-bis[2-(4-oxo-4H-thieno[3,4c]chromen-3-yl)diazenyl]phenyl} acetamide (5b). Compound 5b
was obtained as grey powder (0.93 g, 34%). mp 168-170 °C. Rf 0.57 in CH2Cl2/AcOEt 80% v/v; UV (EtOH)
λmax(Log ε) 235 (1.26), 296 (1.21), 334 (1.00), 355 (0.98), 360 (0.86), 420 (1.21) nm; IR (solid, KBr, νmax, cm-1):
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3545 (O-H), 3134 (N-H), 1726 (C=O), 1598 (C=C), 1290 (C-O) 1213 (C-N) 1490 (N=N) cm-1. 1H NMR (DMSO-d6,
400 MHz): δH 8.82 (2H, dd, JHH 8.1 and 1.2 Hz, H-9’, H-9’’), 7.74 (2H, s, H-1’, H-1’’), 7.71 (2H, ddd, JHH 7.5 JHH 7.2
and 1.6 Hz, H-7’, H-7’’), 7.53 (1H, d, JHH 7.2 Hz, H-6), 7.45 (2H, ddd, JHH 7.4 JHH 6.6 and 1.5 Hz, H-8’, H-8’’), 7.39
(2H, dd, JHH 8.9 and 1.4 Hz, H-6’, H-6’’), 7.37 (1H, d, JHH 7.3 Hz, H-5), 2.80 (3H, s, CH3). 13C NMR (DMSO-d6, 100
MHz): δC 162.9 (CH3C=O), 162.9 (C-4’, C-4’’); 157.0 (C-4), 155.4 (C-3’, C-3’’), 154.5 (C-1), 153.3 (C-9b’, C-9b’’),
155.1 (C-5a’, C-5a’’), 148,6 (C-3a’, C-3a’’), 135.4 (C-7’, C-7’’), 135.6 (C-6), 129.9 (C-9’, C-9’’), 126.1 (C-8’, C-8’’),
125.6 (C-1’’, C-1’), 118.2 (C-9a’, C-9a’’), 117.7 (C-6’, C-6’’), 117.4 (C-3), 113.5 (C-2), 102,3 (C-5), 18.4 (C(O)CH3).
ESIMS m/z (%) 607 (1), 552 (1), 521 (2), 517 (5), 493 (3), 413 (1), 384 (2), 360 (9), 284 (3), 270 (100), 228 (3),
208 (29), 189 (2). Anal. Calcd. for C30H17N5O6S2: C, 59.30; H, 2.82; N, 11.53; S, 10.55. Found: C, 59.18; H, 2.79;
N, 11.52; S, 10.52.
2-[2-(2-Acetyloxy)-3-carboxy-5-{2-carboxy-4-(2-hydroxphenyl)-2-thienyl]}phenyl)diazenyl]-4-(2-
hydroxyphenyl)-3-thiophenecarboxylic acid (5c). Compound 5c was obtained as black powder (1.79 g, 58%).
mp > 250 °C. Rf 0.55 in CH2Cl2/AcOEt 80% v/v; UV (EtOH) λmax(Log ε) 235 (1.41), 305 (1.39), 330 (1.42) nm. IR
(solid, KBr, νmax, cm-1): 3621-3500 (free O-H), 3500-3277 (ass O-H), 2291 (=C-H), 1706 (C=O), 1603-1590
(CAr=CAr), 1540-1523 (thiophenic C=C), 1490 (-N=N-), 1371-1234 (C-O), 1194-1110 (C-N), 752 (=CAr-H) cm−1. 1H
NMR (DMSO-d6, 400 MHz): δH 8.73 (1H, d, JHH 1.3 Hz, H-4’’), 8.01 (2H, ddd, JHH 1.7 JHH 6.9 and 8.6 Hz, H-4’, H-
4’’’’), 7.99 (1H, d, JHH 1.3 Hz, H-6’’), 7.83 (2H, dd, JHH 1.1 and 7.8 Hz, H-6’, H-6’’’’), 7.80 (2H, dd, JHH 7.8 and 8.5
Hz, H-5’, H-5’’’’), 7.73 (2H, dd, JHH 1.3 and 6.9 Hz, H-3’, H-3’’’’), 7.56 (2H, s, H-5, H-5’’’), 2.74 (3H, s, C(O)CH3). 13C
NMR (DMSO-d6, 100 MHz): δC 184.0 (3 COOH), 164.5 (COCH3), 154.9 (C-2’, C-2’’’’), 154.8 (C-2’’), 153.7 (C-2, C-
2’’’), 153.4 (C-1’’), 153.3 (C-1’, C-1’’’’) 147,7 (C-4, C-4’’’), 135.4 (C-3, C-3’’’), 135.1 (C-5’’), 128.8 (C-4’, C-4’’’’),
127.0 (C-4’’), 125.3 (C-3’’), 125.2 (C-5, C-5’’’), 120.0 (C-6’, C-6’’’’), 118.2 (C-6’’), 117.1 (C-5’, C-5’’’’); 114.8 (C-3’,
C-3’’’’), 18.1; (CH3). ESIMS m/z (%) 675 (1), 628 (3), 535 (3), 517 (3), 493 (3), 413 (1), 390 (6), 338 (6), 284 (5),
270 (100), 248 (16), 208 (7), 186 (15). Anal. Calcd. for C31H20N4O10S2: C, 55.35; H, 3.00; N, 8.33; S, 9.53; S,
14.05. Found: C, 55.38; H, 2.98; N, 8.30; S, 13.98.
2-(Acetyloxy)-3-[2-(4imino-4H-thieno[4,3-c]chromen-3-yl)diazenyl]benzoic acid (6). Compound 6 was
obtained as brown powder (1.35 g, 41%). mp 224-226 °C. Rf 0.55 in CH2Cl2/AcOEt 80%); UV (EtOH) λmax(Log ε)
235 (1.28), 275 (1.05), 335 (0.81), 360 (0.81), 420 (0.99) nm. IR (solid, KBr, νmax, cm-1): 3562 (O-H), 3324-3200
(N-H), 2640-2747 (=C-H)1733 (C=O), 1713 (C=N), 1605-1598 (C=C), 1547-1500 (thiophenic C=C), 1490 (-N=N-),
1273-1290 (N-H), 1060-1200 (C=O), 764 (=CAr-H) cm−1. 1H NMR (DMSO-d6, 400 MHz): δH 9.51 (2H; s; =NH); 8.82
(1H, dd, JHH 7.9 and 1.3 Hz, H-9’), 7.88 (1H, dd, JHH 7.9 and 1.3 Hz, H-6), 7,73 ( 1H, ddd, JHH 7.3 JHH 6.5 and 1.5
Hz, H-7’), 7.49 (1H, dd, JHH 7.0 and 1.1 Hz, H-6’), 7.40 (1H; s, H-1’), 7.39 (ddd, JHH 7.2 JHH 6.7 and 1.6 Hz, H-8’),
6.96 (1H, dd, JHH 7.9 and 8.3 Hz, H-5), 6.89 (1H, dd, JHH 8.3 and 0.9 Hz, 1H, H-4), 2.74 (3H, s, CH3). 13C NMR
(DMSO-d6, 100 MHz): δC 183.8 (COOH), 161.8 (COCH3), 157.3 (C-4’), 155.5 (C-5a’), 155.1 (C-9b’), 154.4 (C-3’),
154.2 (C-3a’), 148.9 (C-2), 135.8 (C-1), 135.6 (C-7’), 130.7 (C-6), 129.9 (C-9’), 125.4 (C-8’), 119.7 (C-1’), 119.2 (C-
4), 117.4 (C-6’), 117.3 (C-5), 115.8 (C-9a’), 112.4 (C-3), 18.2 (C(O)CH3). ms: (ESI+) m/z (%) 384 (17), 360 (100),
338 (21), 301 (8), 284 (14), 270 (68). Anal. Calcd. for C20H13N3O5S: C, 58.96; H, 3.22; N, 10.31; S, 7.87. Found: C,
58.98; H, 3.25; N, 10.28; S, 7.85.
Cyclic voltammetry. A PalmSens type 3 potenstiostat running with PSTrace 4.4 software, supplied by PalmSens
BV, Houten (The Netherlands) was used for the cyclic voltammetric (CV) measurements. A standard single
compartment three electrode cell was used with an Ag pseudo-reference electrode and a stainless steel rod
counter electrode. The working electrode was a glassy carbon electrode (GCE). All electrochemical
experiments were carried out without degassing the supporting electrolyte solution.
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Electrochemical procedure. The dyes used were dissolved in dimethylsulfoxide (DMSO). Cyclic
voltammograms were recorded in the potential range from –1.5 V to +0.5 V by transferring the GCE into a 0.05
M H2SO4 solution containing an azo dye at a given concentration. The potential scan rate was varied.
Tested microorganisms. The antimicrobial activity was performed against nine bacterial and three fungal
species. The selected microorganisms were the Gram-positive (Staphylococcus aureus ATCC25923, S. aureus,
Bacillus subtilis) and Gram-negative (Pseudomonas aeruginosa ATCC27853, Escherichia coli S2(1), Shigella
flexneri SDINT, Vibrio cholerae NB2, V. cholerae SG24, V. cholerae CO6) bacteria and yeast strains of Candida
albicans ATCC10231, C. tropicalis PK233 and Cryptococcus neoformans H99. These microorganisms were taken
from our laboratory collection. The fungal and bacterial strains were grown at 37 °C and maintained on
Sabouraud Dextrose Agar (SDA, Conda, Madrid, Spain) and nutrient agar (NA, Conda) slants respectively.
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration
(MMC). The antibacterial and antifungal activities were evaluated by determining the MICs and MMCs as
previously described.49 MICs of azo compounds and phenol derivatives were determined by broth micro
dilution method. Each test sample was dissolved in dimethylsulfoxide (DMSO) to give a stock solution. This
was serially diluted two-fold in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB)
for yeasts to obtain a concentration range of 512 to 0.25 μg/mL. Then, 100 µL of each sample concentration
was added to respective wells (96-well micro plate) containing 90 µL of SDB/ MHB and 10 µL of inoculum to
give final concentration ranges of 256 to 0.125 μg/mL. The final concentrations of microbial suspensions were
2.5x105 cells/mL for yeasts and 106 CFU/mL for bacteria. Dilutions of nystatin (Sigma-Aldrich, Steinheim,
Germany) and ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) were used as positive controls for yeasts and
bacteria respectively. Broth with 10 µL of DMSO was used as negative control. MICs were assessed visually and
were taken as the lowest sample concentration at which there was no growth or virtually no growth. The
lowest concentration that yielded no growth after the sub-culturing was considered as the MMCs.49 All the
tests were performed in triplicate.
Cytotoxicity assay. Whole blood (10 mL) from albino rats was collected by cardiac puncture in EDTA tubes.
The study was conducted according to the ethical guidelines of the Committee for Control and Supervision of
Experiments on Animals (Registration no. 173/CPCSEA, dated 28 January, 2000), Government of India, on the
use of animals for scientific research. Erythrocytes were harvested by centrifugation at room temperature for
10 min at 1000 xg and were washed three times in PBS buffer.50 The cytotoxicity was performed as previously
described.50
Acknowledgements
Emmanuel Sopbué Fondjo gratefully acknowledges the financial support from DAAD (grant N° 91691265).
Additional financial supports for the work were obtained from the University of Dschang’s research grant
committee and the Cameroonian Ministry of Higher Education special research allocation.
Supplementary Material
1
Electronic Supplementary Information (ESI) available: copies of the H and 13C NMR spectra and cyclic
voltammograms for all new compounds
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