Synthesis, structural investigations and in vitro biological evaluation of N, N-dimethyl aniline derivatives based azo dyes as potential pharmacological agents

Article abstract of DOI:10.1016/j.molstruc.2019.03.042

Three novel bioactive azo dyes were synthesized by a classical diazo-coupling method using sulfamethazine with three different N, N-dimethyl aniline derivatives as coupling components under appropriate experimental conditions. The structural features of t

Full text of DOI:10.1016/j.molstruc.2019.03.042

Accepted Manuscript
Synthesis, structural investigations and in vitro biological evaluation of N, N-
dimethyl aniline derivatives based azo dyes as potential pharmacological agents
Vinodkumar, J. Keshavayya
PII:
S0022-2860(19)30312-6
10.1016/j.molstruc.2019.03.042
MOLSTR 26311
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
13 November 2018
13 March 2019
Please cite this article as: Vinodkumar, J. Keshavayya, Synthesis, structural investigations and in
vitro biological evaluation of N, N-dimethyl aniline derivatives based azo dyes as potential
pharmacological agents, Journal of Molecular Structure (2019), doi: 10.1016/j.molstruc.
2019.03.042
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ACCEPTED MANUSCRIPT
Synthesis, structural investigations and in vitro biological evaluation of N, N-dimethyl
aniline derivatives based azo dyes as potential pharmacological agents
Vinodkumar and J Keshavayya*
Department of Chemistry, School of Chemical Sciences,
Jnanasahyadri, Kuvempu University,
Shankaraghatta - 577 451, Shimoga, Karnataka, India
Email: jathikeshavayya1959@gmail.com
Abstract
Three novel bioactive azo dyes were synthesized by a classical diazo-coupling
method using sulfamethazine with three different N, N-dimethyl aniline derivatives as
coupling components under appropriate experimental conditions. The structural features of
the newly synthesized compounds were explored by using various analytical and
spectroscopic techniques. Further, these compounds were assessed for the different
pharmacological activities such as antimicrobial, anti-tuberculosis, antioxidant and anti-
inflammatory properties and results indicated that, these azo compounds exhibited effective
pharmacological activities.
Keywords: Sulfamethazine; Azo dye; Biocidal activity; Anti-tuberculosis; Anti-
inflammatory; DPPH assay.
1. Introduction
Azo dyes are the dynamic class of organic compounds which have received great
attention and have been widely used in the various fields of science and technology such as in

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pharmaceuticals, polymers, electronics, electrochemical, biological and analytical
investigations [1-5]. These dyes are highly coloured and due to their tremendous ability to
impart entire spectrum of colours, they have been used for the past decades as significant
dyes and pigments and they represent nearly 60-70% of the commercial dyes worldwide [6-
7]. Further, these compounds are being utilized as separating agents in a mixture of metal
ions [8]. The azo dye containing heterocyclic ring has received much attention in
pharmaceuticals industry compared to those with the simple carbocyclic aromatic system due
to their extraordinary colouring properties, tinctorial strength, brightness, fastness properties
and distinct bathochromic effect [9-10]. Among the various heterocyclic azo dyes, those
containing nitrogen atom are actively involved in the different pharmacological activities
such as antitumor [11-13], anti-inflammatory, antimycotic [14], antimicrobial as well as
antioxidant [15-17].
Although, various drugs are available in the market for treating most of the dreadful
diseases, many of such drugs are having still innumerable undesirable properties such as
toxicity, drug resistance etc. Hence, there is a need to discover potent drugs to mitigate the
impending clinical challenges. Additionally, sulfamethazine drugs are well-known antibiotics
for the treatment of bacterial infections and they have shown potent pharmacological
activities because of their non-toxicity and specific action against target diseases [18].
In the recent years, a good lot of work has been carried out in the field of azo dyes and
their metal complexes in our laboratory. Further these compounds have been characterized by
using various physicochemical methods and tested for various biological activities [19-22]. In
continuation of the above work, the present study has been carried out in aiming at the
synthesis of novel bioactive compounds derived from sulfamethazine moiety and their
structural investigations by various analytical techniques. Further, these compounds have also

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assessed for various pharmacological applications such as in vitro biocidal, anti-tuberculosis,
anti-inflammatory as well as antioxidant activities.
2. Experimental
2.1. Materials and Methods
All the chemicals, solvents and reagents were of analytical grade and were used as received
without further purification. Progress of the reactions was monitored by thin layer
chromatography performed on silica gel coated aluminium sheets. Melting points were taken
in an open capillary tube using electro thermal instrument without any correction. The UV-
Visible spectra of the newly synthesized dyes were recorded on Shimadzu UV Probe
spectrophotometer in the range of 200-800 nm with two solvents DMSO
(dimethylsulphoxide) and DMF (dimethylformamide) at a concentration of 10-6 mol L-1.
Infrared spectra of the azo compounds were obtained by using Bruker FT-IR
spectrophotometer instrument with KBr disc method and absorption bands were recorded in
the range of 400- 4000 cm-1. The 1H NMR spectra were recorded in DMSO-d6 solvent using
a Bruker Ascend 400 MHz instrument and their chemical shifts were expressed as δ values.
Mass spectra of the dye molecules were recorded at 70 eV of electron energy using Waters
Model-SynaptG2 USA instrument.
2.2. Synthesis of Sulfamethazine azo dyes 1(S1-S2):
Heterocyclic amine (2 mmol) was dissolved in dilute hydrochloric acid (2 mL HCl in
3 mL of H2O) and cooled to 0-5 ℃ in an ice bath. To this reaction mixture, previously
prepared a solution of sodium nitrite (2 mmol) in 2 mL sulphuric acid was added dropwise
and the mixture was stirred for about 2 h. The resulting diazonium salt solution was utilized
for coupling reaction by adding to the ethanolic solution of N, N-dimethyl aniline (2 mmol)

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derivatives at the same temperature and stirring was continued for another 2 h. A neutral
condition of the reaction mixture was maintained by the simultaneous addition of required
volume of saturated sodium bicarbonate solution and progress of the reaction was monitored
by TLC. The crude product was filtered off, washed several times with water, dried and
recrystallized from ethanol and corresponding azo derivatives were obtained in order to good
yields. The overall reaction path for the synthesis of azo dyes was depicted in the Scheme-1.
N2+Cl-
NH2
NaNO2 / H2SO4
0 - 5 °C, 2 h
O
N
N
S
O
O
NH
N
N
S
O
NH
Coupling at neutral pH
0 - 5 °C, 2 h
NH
N
N
c
a
b
N
N
N
O
S
N
N
NH
N
NH
S
O
O
NH
O
N
O
S
N
N
N
HN
O
N
N
N N
S1
S3
S2
Scheme-1: Synthetic route for the preparation of azo dyes, S1-S3

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2.2.1. Synthesis of N-(4, 6-dimethylpyrimidin-2-yl)-4-{(E)-[4, 4- (dimethylamino) phenyl]
diazenyl} benzenesulfonamide (S1)
0
1
The dye was orange colour solid with 65 % yield, m.p. 192-195 C. H NMR (DMSO-d6): δ
2.26 (s, 6H, CH3 attached to pyridine ring), 3.08 (s, 6H, CH3 attached to phenyl ring), 6.76 (s,
1H, Ar-H), 6.84 (d, 2H, Ar-H, J=8.8 Hz), 7.80-7.87 (m, 4H, Ar-H), 8.08 (d, 2H, Ar-H, J=8.4
Hz), 11.69 (s, 1H, NH). FT-IR (KBr): N=N (1597cm-1), Ar-C-H (2967 cm-1), -N-H (3229 cm-
1). LC-MS (m/z) = 410 [M+H]+. Anal.cal (%) for C20H22N6O2S: C (58.52), H (5.40), N
(20.47), O (7.80), S (7.81). Found: C (58.47), H (5.32), N (20.35), S (7.76).
2.2.2. Synthesis of N-(4, 6-dimethylpyrimidin-2-yl)-4-{(E)-[4, 4- (diethylamino) phenyl]
diazenyl} benzenesulfonamide (S2)
0
1
The dye was reddish-orange colour solid with 62 % yield, m.p. 194-198 C. H NMR
(DMSO-d6): δ 1.15 (t, 6H, CH3, J=6.8 Hz), 2.26 (s, 6H, CH3 attached to pyridine ring), 3.46
(q, 4H, CH2, J=6.8 Hz), 6.76 (s, 1H, Ar-H), 6.76 (s, Ar-H), 6.81 (d, 2H, Ar-H, J=9.2 Hz),
7.78-7.84 (m, 4H, Ar-H), 8.08 (d, 2H, Ar-H, J=8.4 Hz), 11.59 (s, 1H, NH). FT-IR (KBr):
N=N (1598 cm-1), Ar-C-H (2971 cm-1), NH (3230 cm-1). LC-MS (m/z) = 438 [M+H]+.
Anal.cal (%) for C22H26N6O2S: C (60.25), H (5.98), N (19.16), O (7.30), S (7.31). Found: C
(60.12), H (5.79), N (18.98), S (7.28).
2.2.3.Synthesis of 4-{(Z)-[4-(phenylamino) phenyl] diazenyl}-N-(4, 6-dimethylpyrimidin-2-yl)
benzenesulfonamide (S3)
0
1
The dye was dark orange colour solid with 60 % yield, m.p. 203-208 C. H NMR (DMSO-
d6): δ 2.26 (s, 6H, CH3 attached to pyridine ring), 6.79 (s, 1H, Ar-H), 6.81-9.03 (m, 13H, Ar-
H), 11.64 (s, 2H, NH). FT-IR (KBr): N=N (1586 cm-1), Ar-C-H (3032 cm-1), NH (3361 cm-1).

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LC-MS (m/z) = 459 [M+H]+. Anal.cal (%) for C24H22N6O2S: C (62.86), H (4.84), N (18.33),
O (6.98), S (6.99). Found: C (62.74), H (4.76), N (18.12), S (6.87).
2.3. Antimicrobial activity
In the present study, the novel heterocyclic azo dyes (S1-S3) were tested for growth
inhibitory effect against pathogenic microorganisms by tube dilution technique [23-24]. The
organisms used for the antimicrobial investigation are Escherichia faecalis (Gram positive
bacteria), Pseudomonas aeruginosa and Escherichia coli (Gram negative bacteria) and fungal
strains are Candida albicans, Aspergillus flavus and Aspergillus niger. Briefly, 20 μL of the
azo compound was added into the 380 μL of BHI (Brain heart infusion) broth and from the
above mixture, 200 μL was taken and added into the tube containing 200 μL of fresh BHI
broth. From the above, 200 μL of the test solution was taken and serially diluted to next 9
tubes separately of each samples in order to get the final concentration of the test compounds
in the range of 100-0.4 mg mL-1. From the previously cultured respective microorganisms, 5
μL was taken and added into 200 μL BHI broth and this culture suspension was transferred
0
into the serially diluted tubes and incubated for 24 h at 37 C. At the end of the incubation,
the tubes were checked for the appearance of turbidity. For antibacterial and antifungal
activities, Ciprofloxacin and Fluconazole were used as positive controls respectively and
DMSO as a negative control. Further, the results of the above activities were recorded in
terms of minimum inhibitory concentration (MIC).
2.4. Anti-tuberculosis activity
The anti-tuberculosis activities of the synthesized azo compounds were investigated against
Mycobacterium Tuberculosis (H37 RV strain, ATCC-27294) by employing the microplate
Alamar Blue assay (MABA) technique. This method is non-hazardous, thermally stable and

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shows good correlation with proportional and BACTEC radiometric method. Briefly, 200 μL
of sterile deionised water was added to the sterile 96 wells plate in order to minimize the
evaporation of medium in the test wells during incubation. To this added 100 μL of the
Middlebrook 7H9 broth and serial dilution of the compounds were made directly on the plate.
Finally, different azo dyes were studied in the concentrations range of 100-0.2 μg mL-1.
Plates were enclosed and preserved with parafilm and incubated for five days at 37 ºC. After
completion of this period, 25 μL of freshly prepared 1:1 mixture of Alamar Blue reagent and
10% tween 80 was added to the plate and incubated for 24 h. The development of blue color
in the wells indicates the bactericidal nature of the synthesized compounds while pink color
exhibits inertness nature of the dye compound towards the bacterial growth. MIC method was
defined as lowest drug concentration which prevents the color change from blue to pink [25].
2.5. Anti-inflammatory activity
Anti-inflammatory activity of the target compounds was investigated by Gelatin Zymography
electrophoresis method [26-27]. The non-continuous electrophoresis gels were prepared and
50 µL of matrix metalloproteinase (MMP) sample was mixed with 50 µL of the azo
compounds and incubated for 1 h. Further, the non-reducing buffer was mixed with equal
quantity of negative control (MMP) as well as positive control (MMP + tetracycline
hydrochloride). The electrophoresis was run at 50 V for 15 min and later increased to 100 V.
After completion of the experiment, gels were washed with SDS (sodium dodecyl sulfate)
surfactant followed by zymogram renaturing buffer for 1 h to remove the remaining
surfactant and allow the gels for renaturing of the proteins. The gels were further incubated in
0
zymogram incubation buffer for overnight at 37 C. Finally, the gels were stained with
Coomassie blue R-250 and the development of white bands indicated the presence of

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gelatinases with the lower bands representing gelatinases-A (MMP-2) which are about 72
kDa while the upper bands are gelatinases-B (MMP-9) which are about 95 kDa.
2.6. DPPH radical scavenging activity
The antioxidant activities of azo compounds were studied by using DPPH (2, 2-diphenyl-1-
picrylhydrazyl) radical scavenging assay [28]. An aliquot of 25 µL of different concentrations
(40 µg, 80 µg and 120 µg) of azo compounds in DMSO was added to a microplate separately
and mixed with 175 µL of 0.086 mM ethanolic solution of DPPH. The mixture was incubated
at ambient temperature for 30 minutes and scavenging effect of the azo dyes was analyzed at
517 nm while taking ascorbic acid as a standard. In this test, a solution of radical was
decolourized after reduction with an antioxidant (AH) or a radical (R•) in accordance with the
proposed reaction [29].
DPPH• + AH → DPPH-H + A•, DPPH• + R• → DPPH-R
The DPPH test is based on the capability of the stable free DPPH radical to react with
hydrogen donors. The percentage of inhibition activity was calculated by using the formula
given below and IC50 (Inhibitory concentration) was also calculated.
% inhibition of DPPH radical= [(Abr-Aar)/ Abr× 100]
Where Abr is the absorbance before reaction and Aar is the absorbance after reaction
has taken place.

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3. Results and Discussion
We have reported the three novel azo dyes having sulfamethazine moiety and their synthetic
route is depicted in Scheme 1. The target compounds S1-S3 were synthesized from facile,
efficient and economical reactants by classical diazotization technique. Physical and
analytical data of the prepared azo compounds were summarized in Table-1. Structural
confirmations of the azo dyes were carried out by various physicochemical methods such as
1
FT-IR, H NMR, LC-MS, elemental analysis and electronic spectroscopic methods. Spectral
data were found to be in good agreement with the proposed structure of the prepared azo
compounds.
<Table-1>
3.1. IR spectral data
Infrared spectra of the azo compounds S1-S3 were given in Figs. 1-3 and all the compounds
have shown a strong absorption peak in the region of 2971-3032 cm-1 are because of aromatic
CH stretching bonds respectively. The peaks at 1586-1598 cm-1 are due to C=C stretching
and 1231-1268 cm-1 are due to aliphatic CH stretching. The absorption peaks at 1597-1721
cm-1 are assigned to C=N stretching. Strong absorption peaks present at 3229-3361 cm-1,
1497-1521 cm-1 are due to the NH and azo groups respectively.
< Fig-1>
< Fig-2>

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< Fig-3>
3.2. 1H NMR spectral data
Proton NMR spectra of the synthesized compounds were recorded in DMSO-d6 using Bruker
spectrophotometer and spectra are given in Figs. 4-6. It is an additional support for the
structural confirmation of azo compounds. The number of protons present in the azo
compounds was achieved from chemical shift (δ in ppm). The spectra resonated a triplet at δ
1.13-1.17 ppm for methyl proton, a singlet at δ 2.26 ppm corresponding to methyl group
attached to pyrimidine ring, a singlet resonated at δ 3.08 ppm with respect to dimethylaniline
proton, a quartet at δ 3.45-3.50 ppm for methylene proton, a singlet at δ 6.76 ppm related to
aromatic proton, a doublet at δ 6.81-6.83 ppm corresponding to aromatic protons, a multiplet
at δ 7.78-7.87 ppm corresponding to aromatic protons, a doublet at δ 8.08-8.10 ppm
corresponding to aromatic protons, a singlet at δ 11.59-11.64 ppm related to NH proton, but
compound S3 shows multiplet at δ 6.81-9.03 ppm which is related to aromatic protons.
< Fig-4>
< Fig-5>
< Fig-6>
3.3. Mass spectral data
Mass spectra of the prepared azo dyes shows that the molecular ion peaks are in good
agreement with their suggested empirical formula given in Table-1 and spectra are given in
Figs. 7-9 The synthesized azo compounds S1, S2 and S3 showed [M+H]+ peaks at m/z 410,
438 and 459 respectively which were related to the molecular weight of the dyes.

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< Fig-7>
< Fig-8>
< Fig-9>
3.4. Electronic absorption spectra
<Table-2>
The electronic absorption spectra of the synthesized azo dyes S1-S3 were recorded in
two solvents (DMSO, DMF) in the range of 200-800 nm and the results are summarized in
Table-2. All the dyes exhibit main absorption band in the range of 444 - 468 nm which can be
assigned as n→π* electronic transition of an azo chromophore. This is due to the interaction
between a solvent molecule and nitrogen atom of the prepared azo compounds having non-
bonding electrons (Figs.10-11) [30]. From the findings given in Table-2, it is revealed that
substituents effect also plays a very important role in the absorption spectra of dyes S1-S3 and
also introducing the electron donating substituents like CH3, C2H5, and C6H5 on the diazo
component, the bathochromic shift is facilitated as an account of the interaction between the
hydrogen atom of dyes with solvent molecules and increasing polarity of the dye system in
the excited state [31]. Furthermore, all these azo dyes exhibited the molar absorpitivity it the
range of 3.979 - 4.270 which account for the intense colour of the dyes in solution.
< Fig-10>
< Fig-11>

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3.5. Antimicrobial activity
A multidrug resistance of bacterial strains continues to be a great challenge due to their
biochemical and morphological modifications and need for the novel drug has motivated
much attention. Therefore, the azo group containing heterocyclic moiety have received
significant interest due to their wide spectrum of biological and pharmacological applications
[32-33]. In the present study, the in vitro antibacterial activities of synthesized dyes were
studied by MIC method against three pathogenic bacterial strains while taking ciprofloxacin
drug as a positive control. The results are summarised in the Table-3, and it is evident that all
the compounds S1-S3 shown efficient antibacterial activity against tested bacterial strains,
except for P.aeruginosa which exhibited resistant in all the azo dyes, whereas E.coli showed
mild sensitiveness towards dyes, S3 displayed excellent antibacterial activity in E. fecalis.
Similarly, compounds S1-S3 exhibited potential antifungal properties against
C.albicans, A.flavus and A.niger strains and results are as shown in Table-3. The antifungal
activities of azo compounds are in the order, S1 > S3 > S2, with S1 exhibiting high activity
compared to the other compounds which showed moderate effects. From the results, it is
clear that tested azo compounds have efficient bactericidal and fungicidal properties.
<Table-3>
3.6. Anti-tuberculosis activity
Tuberculosis is one of the challenging infectious diseases for therapeutic field usually
infected by the Mycobacterium tuberculosis which affects lungs and other parts of the body
and even sometimes it may lead to death. To overcome these problems new drugs were

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developed in modern years but, the utility is limited due to their adverse effects [34]. In the
present study, the synthesized azo dyes were screened for their anti-tuberculosis activity
against M. tuberculosis and the results are shown in Table-4. The results reveal that all the
compounds exhibited significant resistant against M.tuberculosis. Among these dyes, S3
displayed good anti-tuberculosis activity than the other two compounds which are shown
moderate activity with anti-Tb drugs such as pyrazinamide, ciprofloxacin and compatible
with streptomycin as shown in Fig-12.
<Table-4>
< Fig-12>
3.7. Anti-inflammatory activity
Inflammatory is a complex biological reaction of body tissues to harmful stimuli, such as a
pathogen, damaged cells or irritant and protective nature of the body. It indicates to remove
the initial cause of cell injury and initiate the tissue repair. Nowadays, large numbers of
medicines are developed in related to anti-inflammatory agents some of them are aspirin,
ibuprofen, and diclofenac [35]. The above investigation expected to through delight in
understanding the impact of azo compounds on anti-inflammatory activity against two
enzymes MMP-2, MMP-9 and results are represented in Fig-13 and Fig-14. From the
obtained data all the compounds have shown significant inhibition effect against MMP-2 and
MMP-9 enzymes. Out of these studied dyes, S2 shows highly potent anti-inflammatory
activity against MMP-2 enzyme and others exhibits reasonable activity. In case of MMP-9
enzyme S3, S1 and S2 compounds shows good to moderate anti-inflammatory activity.

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< Fig-13>
< Fig-14>
3.8. Antioxidant activity
Antioxidants are the molecules that consume the free radicals produced by the oxidation
process thereby prevents the living tissues from injury and abnormal metabolic system and
some of the antioxidants are ascorbic acid, thiols etc. In recent years, synthetic antioxidants
are found to be more advantageous than natural ones. But, usage of the synthetic antioxidants
is constrained because of their toxicity and resistance. Hence in this point of view, there is a
need to develop a new class of organic compounds which are less toxic, stable, cheap and do
not show any side effect [36]. Therefore, in the present investigation, azo compounds are
assessed for their scavenging activity with the ease of DPPH assay. The radical activity of
target compounds were tested at different concentrations 40 µg, 80 µg and 120 µg is shown
in Fig-15. From the results, it is obvious that activity is completely concentration dependent
and among these compounds S2 displayed good activity, while S1 and S3 exhibit moderate
activity compared with standard ascorbic acid.
< Fig-15>

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4. Conclusions
In summary, we have successfully synthesized three novel bioactive azo compounds by the
well known diazo-coupling method and their structural features were accomplished on the
basis of spectral data. Electronic absorption spectral study specifies that, the prepared azo
compounds containing excellent colouring properties due to the presence of chromophores in
their structures. Further, these azo dyes proved to have significant antibiotic property against
tested pathogens and among the tested dyes S3 exhibits significant bactericidal activity than
S1 and S2, while in case of fungicidal activity S1 shows efficient inhibition effect towards
antifungal nature. All the synthesized compounds displayed promising anti-tuberculosis
activity against M. Tuberculosis, among them S3 shown higher activity than the remaining
compounds and compatible with the standard drug Streptomycin. Compound S2 displayed
excellent anti-inflammatory effect against MMP-2 enzyme and S3 also exhibited noticeable
anti-inflammatory action against MMP-9 enzyme. Antioxidant property of the synthesized
compounds was examined by using DPPH scavenging assay, among the tested compounds
S2 shows good antioxidant activity. From the present study, we can develop an advanced
strategy by the combination of two or more pharmacological properties in a single compound
which may be useful in the treatment of multiple diseases. Hence, these synthesized azo
compounds were found to be interesting lead molecules for curing multiple diseases enabling
to build a platform for the designing of novel drugs with multiple therapeutic properties.

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Therefore, the results herein reported may be used in the pharmaceutical area for developing
potential drugs in the future.
Acknowledgement
One of the author, Mr. Vinodkumar very thankful to the Department of Post Graduate Studies
and Research in Chemistry, Kuvempu University, Shimoga for furnishing laboratory
amenities. Authors are also grateful to Manipal University, Manipal for allocating spectral
information and Maratha Mandal’s central research laboratory, Belgaum for providing
biological studies.

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Fig-1 IR spectrum of compound S1
Fig-2 IR spectrum of compound S2

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Fig-3 IR spectrum of compound S3
Fig-4 1H NMR spectrum of compound of S1

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Fig-5 1H NMR spectrum of compound of S2
Fig-6 1H NMR spectrum of compound of S3

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Fig-7 LC-MS spectrum of compound S1
Fig-8 LC-MS spectrum of compound S2

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Fig-9 LC-MS spectrum of compound S3
Fig-10. UV-Visible spectra of the dyes S1-S3 in DMSO
Fig-11. UV-Visible spectra of the dyes S1-S3 in DMF

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Fig-12. Anti-tuberculosis activity of the dyes, S1-S3
Fig-13. Electrophoresis bands of the dyes S1-S3 showing anti-inflammatory activity.

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MMP-2
MMP-9
100
80
60
40
20
0
98
70
60
85
65
80
100
78
0
10
S1
S2
S3
PC
NC
Fig-14. Anti-inflammatory activity of the dyes, S1-S3
Fig-15. DPPH activity of the dyes, S1-S3

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Research Highlights
 Synthesis of azo compounds by a conventional diazo-coupling method which is facile
and effective.
 Sulfamethazine compounds were known for their antimicrobial properties.
 Sulfamethazine incorporated azo dyes are also exhibited effective pharmacological
properties.
 In future, these kinds of dyes can be used in the development of multiple therapeutic
drugs by inserting multiple properties in a single compound.

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Table 1: Physical and analytical data of the dyes S1-S3
Compound
Mol. Formula. Mol.
Colour
Yield (% )
M.P (℃)
Mass.
409
438
S1
S2
S3
C13H11N5O4S
C22H26N6O2S
C24H22N6O2S
192-195
194-198
203-208
Orange
Reddish-orange
Dark orange
65
62
60
458
Table 2: UV-Visible spectral data of the dyes S1-S3
Compounds
λmax(nm)
DMSO
453
log ε Compounds
λmax(nm)
DMF
444
log ε
S1
S2
S3
4.270
4.120
3.979
S1
S2
S3
3.979
4.413
3.659
457
468
459
451
Table 3: Antibacterial and antifungal activity of the dyes S1-S3
Samples
E.fecalis
100
50
25
12.5
6.25
3.12
1.6
0.8
0.4
mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL
S1
S2
S3
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
R
S
S
R
R
S
E.coli
S1
S2
S
S
S
S
S
S
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S3
P.aeruginosa
S1
S2
S3
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

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C.albicans
S1
S2
S3
S
S
S
S
S
S
S
R
S
S
R
R
S
R
R
S
R
R
R
R
R
R
R
R
R
R
S
A.flavus
S1
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
R
R
S
R
R
R
S2
S3
A.niger
S1
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
R
R
S2
S3
S- Sensitive
R- Resistant
Table 4: Anti-tuberculosis activity of the dyes, S1-S3
Compounds
100
50
25
12.5
6.25
3.12
1.6
0.8
µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
S1
S2
S
S
S
S
S
S
R
R
S
R
R
S
R
R
S
R
R
R
R
R
R
R
R
R
S3
S-Sensitive
R- Resistant