Guanidinyl benzothiazole derivatives: Synthesis and structure activity relationship studies of a novel series of potential antimicrobial and antioxidants

Article abstract of DOI:10.1007/s11164-016-2454-6

Abstract: A series of N-phenyl-substituted and disubstituted guanidinyl benzothiazole derivatives 4a–4m were synthesized and characterized as novel antimicrobial and antioxidant agents. The in-vitro antioxidant activities of these compounds were evaluated and compared with commercial antioxidants, using ascorbic acid (AA) and employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assay. The results showed that IC₅₀ values of compounds 4a and 4m were similar to the IC₅₀ values of the standard. This assay indicates the good activities of these compounds. In addition, the in-vitro antimicrobial activities of these compounds were evaluated and the results demonstrated that the compounds 4i and 4l exhibited excellent antimicrobial activities. Compounds 4c and 4i having electron withdrawing groups at p-position and compound 4l and 4m having electron donating groups at o-position showed better antimicrobial activities compared to the standard. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-016-2454-6

Res Chem Intermed
DOI 10.1007/s11164-016-2454-6
Guanidinyl benzothiazole derivatives: Synthesis
and structure activity relationship studies of a novel
series of potential antimicrobial and antioxidants
1
1
Mahesh Bhat
•
S. L. Belagali
Received: 9 October 2015 / Accepted: 19 January 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A series of N-phenyl-substituted and disubstituted guanidinyl benzothiazole
derivatives 4a–4m were synthesized and characterized as novel antimicrobial and
antioxidant agents. The in-vitro antioxidant activities of these compounds were evalu-
ated and compared with commercial antioxidants, using ascorbic acid (AA) and
0
employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and 2,2 -azino-bis (3-ethyl-
benzthiazoline-6-sulphonic acid (ABTS) assay. The results showed that IC50 values of
compounds 4a and 4m were similar to the IC50 values of the standard. This assay
indicates the good activities of these compounds. In addition, the in-vitro antimicrobial
activities of these compounds were evaluated and the results demonstrated that the
compounds 4i and 4l exhibited excellent antimicrobial activities. Compounds 4c and 4i
having electron withdrawing groups at p-position and compound 4l and 4m having
electron donating groups at o-position showed better antimicrobial activities compared
to the standard.
Electronicsupplementarymaterial The onlineversionof thisarticle(doi:10.1007/s11164-016-2454-6)
contains supplementary material, which is available to authorized users.
&
Mahesh Bhat
maheshbhat08@gmail.com
1
Environmental Chemistry Laboratory, Department of Studies in Environmental Science,
University of Mysore, Manasagangothri, Mysore 570 006, India
123

M. Bhat, S. L. Belagali
Graphical Abstract
Keywords Guanidinyl benzothiazole Á Antioxidant activity Á DPPH assay Á ABTS
assay Á Antimicrobial
Introduction
One of the main aims of synthetic chemistry is the development of new
antimicrobial therapeutic agents. Even though many synthetic drugs are available,
they have limitations in treating new diseases. Guanidine functionalities are found
in a variety of natural products, either in cyclic form or as open-chain groups of
pendent substituents [1–4]. In the literature, a large number of antimicrobial agents
from both natural and synthetic origin have been found to possess guanidine
functionalities. Guanidines are physiologically active organic compounds possess-
ing a wide array of biological activities, including anticancer [5, 6], antidiabetic [7,
8
], antiviral, antiinflammatory [9], antibiotic [10, 11], antileishmanial [12–20],
antiprotozoal, antihistamine, and antihypertensive [21] properties. The three
nitrogen atoms of a guanidine moiety, which make it possible to bind to various
substituents, as a result of a guanidine derivative show a diverse range of
biochemical behavior that can be attributed to their flexible structure [22]. In the
case of drugs having low penetration through different membranes in the body, the
introduction of a guanidinium group into their molecular architecture increases the
ability of these molecules to cross biological barriers and thus enhance their
biological activities [23, 24]. Reactive oxygen species (ROS), produced as
byproducts of metabolic processes in living organisms, initiate toxic oxidative
reactions in bio-molecules, causing oxidative stress. A state of oxidative stress has
deleterious effects on almost all tissues and can initiate or enhance the rate of
pathological conditions such as neurodegeneration, inflammation, aging processes,
cancer, and cardiovascular diseases [25, 26]. Therefore, the synthesis of new
antioxidant and antimicrobial drugs today is gaining importance. The guanidine
derivatives have shown remarkable antioxidant characteristics for reducing
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
Fig. 1 The guanidine group
containing drugs, Guanadrel 1,
Guanoxan 2, Guanethidine 3 and
Creatine 4
HN
NH
NH2
NH
NH2
O
O
2
O
O
N
H
1
CH3
N
O
H
N
NH2
H N
2
OH
N
NH
NH
4
3
oxidative stress induced in blood serum and brain tissue by superoxide dismutase
and catalase enzymes during ischemia–reperfusion, causing restoration of blood
supply and showing a neuroprotective role [27].
Antimicrobial activity depends on the presence of protonated guanidine groups
and their resonance stabilization. Because of the diversity of their biological
applications, guanidine derivatives are considered to be one of the major functional
groups in organic compounds [28]. Some of the reported structures of the
biologically active guanidines are shown in Fig. 1, in which (1) guanadrel is a
postganglionic adrenergic blocking agent, (2) guanoxan is a sympatholytic drug, (3)
guanethidine is an antihypertensive drug, and (4) creatine is a guanidinyl compound
helping to supply energy to all cells in the body. In the past, very little effort has
been observed for the synthesis of benzothiazole-conjugated guanidine compounds.
Based on the above facts and the continuation of our drug development program
[
29–31], the present work involves the synthesis of a new series of open-chain
guanidine derivatives with benzothiazole substituents for the enhancement of
bioactivity.
Results and discussion
Chemistry
Synthesis of the desired compounds was achieved according to the steps illustrated
in Scheme 1. Benzoyl isothiocyanate (2) was synthesized by refluxing benzoyl
chloride with ammonium thiocyanate in acetone, and the obtained product was
refluxed with 6-amino benzothiazole, which gives the thiourea derivative of
benzothiazole (3). The thiourea intermediate of benzothiazole was mixed with
different substituted anilines in DMF with equimolar ratios [32] and also two
equivalents of triethylamine at 0 °C and allowed to remain at room temperature,
123

M. Bhat, S. L. Belagali
N
S
O
N
O
S
H N
O
2
NH SCN
4
NCS
S
N
H
N
H
Cl
Acetone, Reflux
Acetone
Reflux
2
3
1
NH2
HgCl , DMF
2
R
O
H
N
N
S
N
NH
R
4
a-m
4
4
4
4
4
a. R= p-OH
d. R = 3,4 dimethyl
g. R = p-F
j. R = H
m. R = o-OH
4b. R = p-Br
2
4e. R = p-CO H
4h. R = 3,5 dichloro
4k. R = p-NHCO Et
4c. R = p-OMe
4f. R = 3,4 dimethyl
4i. R = o- Cl, p-NO
4l. R = o-OH, p-Me
2
2
Scheme 1 Synthesis of guanidinyl benzothiazole derivatives
which gives the title compounds (4a–4m). The black precipitate (HgS) was removed
by ciliate filtration and the product was purified by recrystallization or by column
chromatography.
From the above method, we synthesized 13 guanidinyl derivatives in good yield,
and determined that the implemented method was simple and commercially viable.
The structures of all the newly synthesized compounds were confirmed by infrared
1
13
spectroscopy (IR), H, C NMR and mass spectral analysis (Supplementary
1
material). The presence of a singlet broad peak at 10.6 and 10.4 d in H NMR and a
1
3
weak peak at 158 d in C NMR spectrum shows the formation of the guanidine
1
13
product. The presence of all required peaks in the H NMR and C NMR spectra
confirms the assigned structures.
Biological evaluation
Antioxidant activity
In-vitro antioxidant activities of all the synthesized compounds were evaluated by
(
1) 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, which was a rapid and convenient
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
method for primary investigation of the antioxidant activity of the compounds, and
0
by (2) 2,2 -Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) cation radical
assay, which was a conventional and conformational model for assessing the
antioxidant activities of hydrogen transfer and chain-breaking antioxidants [33]. The
IC50 value was calculated graphically based on the capacity of the sample
concentration to scavenge 50 % of the free radicals. This is the net-effective
concentration of the drug molecule at which 50 % of the radicals were scavenged,
which refers to the concentration in which one half of the radicals are absorbed by the
antioxidants. The compounds having the IC50 values in the range of 10-20 lg/mL
were considered as the most efficient antioxidants. The IC50 values of ascorbic acid
and butylated hydroxytoluene (BHT) were also determined, and the values are
tabulated in Table 1.
The majority of the synthesized compounds showed potent to moderate
antioxidant activities. Compounds 4a, 4l, and 4m showed excellent radical
scavenging activities with IC50 values of 277.48, 7.68, and 18.87 lg/mL,
respectively. In the DPPH assay, the standard drug butylated hydroxytoluene
(
BHT) showed an IC50 value of 14.46 lg/mL. This value indicates that 4l and
m have comparable activities to the standard drug. In the ABTS radical
4
scavenging assay, the compounds 4a, 4f, 4k, 4l, and 4m showed good activities
with IC50 values of 79.46, 183.46, 407.04, 27.54, and 30.23 lg/mL, respectively,
which are comparable to the IC50 values of ascorbic acid 17.92 lg/mL. On the
basis of the above observation, compounds having –OH (phenolic groups on 4a,
4
l, and 4m), and –OCH3 (4c), –NHCO Et (4k) in the phenyl ring of the
2
guanidine moiety have excellent activity. In conclusion, the presence of the –OH
group increases the activity because of the increase in the acidic character of the
?
phenyl ring, which leads to the easy release of H and is responsible for the
scavenging action. The compound with the electron withdrawing group –NO2
(
4i) show the least activity.
Table 1 Antioxidant activity of synthesized benzothiazole guanidinyl derivatives
Compound
Antioxidant activity (lg/mL)
Compound
Antioxidant activity (lg/mL)
DPPH IC50
lg/mL)
ABTS IC50
(lg/mL)
DPPH IC50
(lg/mL)
ABTS IC50
(lg/mL)
(
4
4
4
4
4
4
4
a
b
c
d
e
f
277.48 ± 0.26
[500
79.41 ± 0.05
[500
4h
4i
494.83 ± 0.55
[500
460.13 ± 0.08
[500
497.89 ± 0.18
[500
462.28 ± 0.07
[500
4j
461.17 ± 0.02
449.95 ± 0.03
7.68 ± 0.06
18.87 ± 0.07
5.58 ± 0.06
496.07 ± 0.48
407.04 ± 0.02
27.50 ± 0.04
30.25 ± 0.01
17.92 ± 0.08
4k
4l
[500
[500
[500
183.40 ± 0.05
[500
4m
AA
g
[500
Italic letters indicate the standard drugs
AA ascorbic acid
123

M. Bhat, S. L. Belagali
Antimicrobial activity
All the synthesized compounds were evaluated for their antibacterial activity via the
in-vitro disc diffusion method against Escherichia coli (MTCC-40), Staphylococcus
aureus (MTCC-7443), Candida albicans (MTCC-183), and Aspergillus niger
(
MTCC-1344). Stock cultures of bacteria were maintained on nutrient agar (Himedia,
Mumbai) slants at 4 °C with periodical sub-culturing. The antimicrobial activities of
compounds were screened via disc diffusion [34]. In detail, 200-lg/L solutions of the
synthesized compounds were placed on the sterile disc and set in petri plates. The
plates were pre-incubated for 1 h at 4 °C. The bacterial petri plates were incubated at
3
7 °C for 24 h and the fungal plates for 5 days at 30 °C temperatures. The zone of the
compounds was tabulated, shown in Table 2. The results indicated that anti
Escherichia coli activity for 4e, 4i, and 4m show zones of inhibition of 26, 25, and
2
7 mm, respectively, which is more potent compared to the standard (chlorampheni-
col 24 mm). The compounds of 4e, 4f, 4i and 4m had zones of inhibition of 21, 20, 20,
and 21 mm, respectively, which are superior to the standard (19 mm) for the
Staphylococcus aureus pathogen. In a similar way, the compounds were also screened
for antifungal activities. Here, all the compounds except 4a and 4d show larger zones
of inhibition compared to the standard 14 mm. Compounds 4e and 4l show zones of
inhibition of 40 and 41 mm, which are more than three times that of the standard for
Table 2 Zone of inhibition of synthesized Guanidinyl benzothiazole derivatives
a
Compound
Zone of inhibition (mm) at the concentration of 200 lg/mL
Escherichia coli Staphylococcus aureus Candida albicans Aspergillus niger
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
14
13
13
12
26
17
20
24
25
16
15
15
27
24
–
14
–
13
16
18
12
40
35
22
25
36
27
22
41
37
–
23
18
15
19
21
29
22
34
41
18
15
25
17
–
10
11
21
20
–
g
h
i
11
20
11
10
13
21
19
–
j
k
l
m
b
Chloramphenicol
b
Bavistin
14
37
200-lg/mL dose was applied for each disc; Italicized values indicates superior activity compared to
respective control
a
Diameter of inhibition zone was measured in mm
b
Chloramphenicol and Bavistin were used as antibacterial and antifungal standards
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
Table 3 MIC values of synthesized benzothiazole guanidinyl derivatives for antimicrobial activity
Entry
Escherichia
a
coli
Staphylococcus
a
aureus
Candida
Aspergillus
a
niger
a
albicans
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
100
200
100
100
25
200
200
200
200
12.5
50
6.25
25
3.125
12.5
50
25
25
12.5
12.5
1.56
12.5
3.125
0.78
12.5
12.5
12.5
6.25
–
3.125
1.56
6.25
0.78
1.562
12.5
25
50
g
h
i
6.25
6.25
12.5
50
200
200
12.5
200
200
100
100
50
j
k
l
200
100
6.25
3.125
–
1.56
6.25
–
m
b
Chloramphenicol
b
Bavistin
–
1.55
0.195
a
Diameter of inhibition zone was measured in mm
b
Chloramphenicol and Bavistin were used as antibacterial and antifungal standards
Candida Albicans. Compound 4i was more (41 mm) active than the standard (37 mm)
for Aspergillus niger and the values are shown in Table 2.
The synthesized compounds have magnificent antimicrobial activities, so their
Minimum Inhibition Concentrations (MIC) were calculated by disc diffusion, as
shown in Table 3. Compounds 4g, 4h, and 4m have a 6.25-lg/mL MIC compared to
the standard (3.125 lg/mL) for Escherichia coli. For Staphylococcus aureus,
compound 4e (12.5 lg/mL), 4i (12.56 lg/mL), and 4f (50 lg/mL) have comparable
minimum inhibition concentrations with the standard (50 lg/mL). For Candida
albicans, compounds 4f (1.56 lg/mL), 4h (1.56 lg/mL), 4i (1.56 lg/mL), and 4l
(
1.56 lg/mL) showed similar MIC values to that of Bavistin (1.56 lg/mL). For
Aspergillus niger, compounds 4f (1.56 lg/mL), 4h (3.125 lg/mL),and 4i (0.78 lg/mL)
were comparable with the standard drug (Bavistin, with an MIC value 0.195 lg/mL).
From the structure activity relationship, it can be concluded that, the resonance
stabilization of the guanidine group was mainly responsible for the antimicrobial
activity. Additionally, the compounds bearing electron-withdrawing groups at p-
position (4e–CO H and 4i–NO ) and electron-donating groups at o-position (4l and
2
2
4m have -OH groups at p-position) are mainly responsible for several activities of
the synthesized guanidinyl benzothiazole derivatives.
123

M. Bhat, S. L. Belagali
Conclusion
In conclusion, we have designed and synthesized a new series of benzothiazole-
derived guanidine compounds with different substituents in a benzene ring (4a–4m,
as shown in Table 3). Compounds 4l and 4m have –OH (electron-donating) groups
at o-position, showing excellent antioxidant activities. Compounds 4e and 4i have
electron-withdrawing groups at p-position, and compounds 4l and 4m have
electron-donating groups at the o-position, showing better antimicrobial activities
compared to the standard. In the synthesized compounds, some of them show better
activities compared to the standard, so the benzothiazole guanidinyl compound
shows promise of being a better candidate in clinical trials.
Experimental
Reactions were performed in two necked, 100 mL round bottomed flasks. The
glassware was previously rinsed with acetone and dried in a hot air oven. The
starting materials were purchased from SD Fine Chemicals and the 6-Amino
benzothiazole from Aldrich with a purity greater than 99 %. The monitoring of
reactions was carried out by thin layer chromatography (TLC), using aluminium
plates coated with silica (Merck) and using appropriate solvent mixtures. The
melting points (mp) were determined by open capillary method and were
uncorrected. The products were confirmed by IR, Liquid Chromatography–Mass
1
Spectrometry (LCMS), and H NMR spectral studies. NMR spectra were obtained
on an Agilent 400 MHz NMR spectrometer with CDCl as solvent, and LCMS was
3
recorded by positive mode, using 0.1 % formic acid in acetonitrile as a solvent. The
antimicrobial activity study was performed with disc diffusion, using dextrose (for
antibacterial activity) and Czapek Dox agar (antifungal activity) as media.
All guanidines were synthesized by the reported method [32]. The thiourea
derivative of benzothiazole was mixed with different substituted anilines in DMF in
equimolar ratios, with two equivalents of triethylamine. The temperature was
maintained below 5 °C using an ice bath, and one equivalent of mercuric chloride
was added to the reaction mixture with vigorous stirring. The ice bath was removed
after 30 min and the mixture was stirred overnight. The progress of the reaction was
monitored by TLC. Chloroform (20 mL) was added to the reaction mixture and the
suspension was filtered through a silica bed funnel to remove HgS. The solvent from
the filtrate was evaporated under reduced pressure and the residue was redissolved
in CH Cl (20 mL), washed with water (4 9 50 mL), and the combined organic
2
2
phase was washed with brine (20 mL) and dried over anhydrous Na SO . The
2
4
solvent was evaporated, and the residue was purified by column chromatography
and recrystallized from ethanol. The schematic representation of the reaction
pathway is shown in Scheme 1.
Synthesis of N-{(1,3-benzothiazol-6-ylamino) [(4-hydroxy phenyl) amino] methyli-
1
dene} benzamide (4a) Off-white solid, 87.7 % yield, m.p. 175–176 °C. H NMR
(
400 MHz, DMSO-d6): d 10.70 (s, 1H), 10.02 (s, 1H), 9.37 (s, 1H), 9.22 (s, 1H)
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
8.13 (s, 1H), 7.97-7.90 (m, 3H), 7.55–7.53 (d, J = 7.6 Hz, 1H), 7.39–7.37 (d,
J = 7.2 Hz, 1H), 7.30–7.25 (m, 2H), 7.16–7.14 (d, J = 8.0 Hz, 2H), 6.71–6.69 (d,
1
3
J = 8.4 Hz, 2H). C NMR: (100 MHz, DMSO-d6): d 176.4, 157.8, 155.8, 150.64,
38.8, 135.9, 134.3, 131.5, 129.2, 129.1, 128.4, 128.3, 126.87, 126.7, 124.1, 123.9,
1
MS found: m/z 389.3 for [M ?1] peak and calculated for C H N O S is 388.4.
?
2
1 16 4 2
Elemental analysis calculated for C H N O S is C, 64.93; H, 4.15; N, 14.42 and
2
1 16 4 2
experimentally found as C, 64.75; H, 4.19; N, 14.15.
Synthesis of N-{(1,3-benzothiazol-6-ylamino)[(4-methoxy phenyl) amino] methyli-
1
dene} benzamide (4b) Off-white solid, 94.84 % yield, m.p. 198–200 °C. H NMR
(
400 MHz, DMSO-d6): d 10.57 (s, 1H, NH), 10.31 (s, 1H, NH), 9.41 (s, 1H H-5m),
7
.89–7.87 (d, J = 8.8 Hz, 2H Ar–H), 7.64–7.61 (d, J = 12.2 Hz, 2H, Ar–H),
.68–7.64 (t, J = 7.6, 13.6 Hz, 1H), 7.63–7.61 (d, J = 7.6 Hz, 2H), 7.50–7.47 (t,
7
J = 7.6, 14.4 Hz, 1H), 7.39–7.35 (m, 2H) 6.97–6.98 (d, J = 4.8 Hz, 1H), 6.56–6.54
1
3
(
d, J = 8.8 Hz, 1H). C NMR: (100 MHz, DMSO-d6): d 175.9, 158.5, 155.6,
1
1
4
1
43.0, 142.5, 138.9, 135.7, 134.6, 132.9, 130.1, 129.6, 125.4, 123.2, 116.8, 113.4,
?
05.7. MS found: m/z 451.0 for [M ?1] peak and calculated for C H BrN OS is
2
1
15
4
50.01. Elemental analysis calculated for C H BrN OS is C, 55.88; H, 3.35; N,
2
1
15
4
2.41 and experimentally found as C, 55.37; H, 3.42; N, 12.22.
Synthesis of N-{(1,3-benzothiazol-6-ylamino)[(4-methoxy phenyl) amino] methyli-
1
dene} benzamide (4c) Gray solid, 92.1 % yield, m.p. 197–198 °C. H NMR
(
J = 8.8 Hz, 2H), 8.04–8.00 (d, J = 15.6 Hz, 2H), 7.68–7.64 (t, J = 7.6, 13.6 Hz,
400 MHz, DMSO-d6): d 10.62 (s, 1H), 10.37 (s, 1H), 9.32 (s, 1H), 8.06–8.04 (d,
1
H), 7.62–7.60 (d, J = 7.6 Hz, 1H), 7.47–7.44 (t, J = 7.6, 14.4 Hz, 1H), 7.39-7.35
1
m, 5H) 6.97–6.95 (d, J = 8.8 Hz, 2H), 3.72 (s, 3H). C NMR: (100 MHz, DMSO-
3
(
d6): d 176.4, 157.6, 157.4, 156.0, 150.7, 138.7, 135.8, 134.4, 131.7, 130.3, 129.1,
?
1
28.4, 126.5, 123.9, 117.9, 114.6, 55.7. MS found: m/z 403.1 for [M ?1] peak and
calculated for C H N O S is 402.1. Elemental analysis calculated for C H N
22 18 4-
2
2
18
4
2
O S C, 65.65; H, 4.51; N, 13.92 and experimentally found as C, 65.39; H, 4.25; N,
2
1
4.11.
Synthesis of N-{(benzothiazol-6-ylamino) [(3,4-dimethyl phenyl) amino] methylene}
1
benzamide (4d) White crystalline solid, 95.23 % yield, m.p. 205–206 °C. H
NMR (400 MHz, DMSO-d6): d 10.64 (s, 1H), 10.40 (s, 1H), 8.21 (s, 1H), 8.05-7.99
(
q, 3H), 7.62-7.59 (q, 1H), 7.47–7.440 (t, J = 6.8, 14.4 Hz, 1H), 7.39–7.35 (t,
J = 7.6, 14.8 Hz, 2H), 7.24 (s, 1H), 7.18–7.12 (q, 2H), 2.25-1.96 (m, 6H). NMR:
100 MHz, DMSO-d6): d 176.2, 159.4, 156.7, 143.6, 143.0, 142.4, 137.2, 136.8,
35.1, 135.0, 130.2, 129.9, 126.9, 123.5, 123.2, 116.3, 114.1, 103.5, 19.1. MS
(
1
?
found: m/z 401.3 for [M ?1] peak and calculated for C H N OS is 400.14.
2
3 20 4
Elemental analysis, calculated for C H N OS is C, 68.98; H, 5.03; N, 13.99 and
3 20 4
2
experimentally found as C, 69.12; H, 5.14; N, 13.81.
Synthesis of 4-[3-(benzothiazol-6-yl)-2-benzoyl guanidino] benzoic acid (4e)
1
White solid, 60.60 % yield, m.p. 153–155 °C. H NMR (400 MHz, DMSO-d6) d
1
0.96 (s, 1H), 10.68 (s, 1H), 10.56 (s, 1H), 9.25 (s, 1H), 7.64–7.63 (d, J = 5.4 Hz,
H), 7.81–7.79 (d, J = 6.0 Hz, 2H), 7.89-7.88 (d, J = 5.6 Hz, 2H), 7.56–7.54 (d,
2
123

M. Bhat, S. L. Belagali
J = 7.6 Hz, 1H), 7.31–7.29 (m, 2H), 6.56–6.53 (m, 1H), 6.69–6.67 (d,
1
3
J = 8.76 Hz, 2H). C NMR: (100 MHz, DMSO-d6): d 175.8, 169.4, 158.7,
1
55.9, 144.1, 143.5, 142.2, 136.4, 134.8, 131.3, 129.9, 129.3, 123.2, 120.5, 117.6,
?
13.2, 105.1. MS found: m/z 417.0 for [M ?1] peak and calculated for
1
C H N O S is 416.09. Elemental analysis, calculated for C H N O S is C,
2
2
16
4
3
22 16 4 3
6
3.45; H, 3.87; N, 13.45 and experimentally found as C, 63.51; H, 3.79; N, 13.28.
Synthesis of N-{(benzothiazol-6-ylamino) [(3,5-dimethyl pheny)amino] methylene}
1
benzamide (4f) Off-white solid, 74.80 % yield, m.p. 150–151 °C. H NMR
(
400 MHz, DMSO-d6): d 10.62 (s, 1H), 10.38 (s, 1H), 8.43 (s, 1H), 8.06–7.97 (m,
3
H), 7.63–7.60 (q, 1H), 7.49–7.46 (t, J = 6.8, 14.4 Hz, 1H), 7.40–7.36 (t, J = 7.6,
1
4.8 Hz, 2H), 7.24 (s, 1H), 7.18–7.12 (q, 2H), 6.7 (s, 1H), 2.35 (s, 6H). C NMR:
3
1
(
100 MHz, DMSO-d6): d 176.2, 159.6, 156.3, 145.1, 144.2, 143.6, 142.8, 139.1,
1
34.6, 135.9, 130.2, 129.9, 126.9, 122.6, 120.9, 117.3, 112.8, 22.1. MS found: m/z
?
01.2 for [M ?1] peak and calculated for C H N OS is 400.14. Elemental
4
analysis calculated for C H N OS is C, 68.98; H, 5.03; N, 13.99 and
2
3 20 4
2
3 20 4
experimentally found as C, 69.05; H, 4.98; N, 13.65.
Synthesis of N-{(benzothiazol-6-ylamino) [(4-fluorophenyl) amino] methylene}
1
benzamide (4g) Pale brown solid, 85.60 % yield, m.p. 163–164 °C. H NMR
(
J = 4.8 Hz, 2H), 8.06–8.03 (d, J = 6.4 Hz, 2H), 7.66–7.63 (t, J = 6.0, 12.0 Hz,
400 MHz, DMSO-d6): d 10.59 (s, 1H), 10.48 (s, 1H), 9. 23 (s, 1H), 8.16–8.15 (d,
1
H), 7.62–7.60 (d, J = 7.6 Hz, 1H), 7.47–7.44 (t, J = 7.6, 14.4 Hz, 1H), 7.39–7.35
1
3
(
m, 4H) 6.97–6.95 (d, J = 8.8 Hz, 2H), C NMR: (100 MHz, DMSO-d6): d 175.9,
1
58.7, 155.8, 143.5, 142.2, 135.3, 134.7, 134.2, 131.7, 130.3, 129.1, 122.4, 120.8,
?
23.9, 116.3, 112.8, 105.7. MS found: m/z 391.2 for [M ?1] peak and calculated
1
for C H FN OS is 390.10. Elemental analysis calculated for C H FN OS is C,
2
1
15
4
21 15
4
6
4.60; H, 3.87; N, 14.35 and experimentally found as C, 64.48; H, 3.59; N, 14.68.
Synthesis of N-{(benzothiazol-6-ylamino) [(3,5-dichloro phenyl) amino]
1
methylene}benzamide (4h) H NMR (400 MHz, DMSO-d6): grayish solid,
7
8
7
6.88 % yield, m.p. 131–132 °C. d 10.62 (s, 1H), 10.38 (s, 1H), 8.43 (s, 1H),
.06–7.97 (m, 2H), 7.63–7.60 (q, 1H), 7.49-7.46 (t, J = 6.8, 14.4 Hz, 1H),
.40–7.36 (t, J = 7.6, 14.8 Hz, 2H), 7.23–7.22 (d, J = 5.6 Hz, 1H), 6.69–6.67 (d,
1
3
J = 8.8 Hz, 2 H), 6.7 (s, 1H), 6.57–6.56 (d, J = 4.4 Hz, 1 H). C NMR:
100 MHz, DMSO-d6): d 175.9, 158.7, 155.8, 148.1, 143.6, 141.5, 135.4, 134.8,
34.2, 129.8, 129.2, 122.6, 120.1, 115.6, 113.3, 106.4. MS found: m/z 441.1 for
(
1
?
[M ?1] peak and calculated for C H Cl N OS is 440.03. Elemental analysis,
21 14 2 4
calculated for C H Cl N OS is C, 57.15; H, 3.20; N, 12.69 and experimentally
2
1
14
2 4
found as C, 57.36; H, 3.15; N, 12.51.
Synthesis of N-{(benzothiazol-6-ylamino) [(2-chloro-4-nitrophenyl) amino] methy-
1
lene} benzamide (4i) H NMR (400 MHz, DMSO-d6): yellow solid, 53.52 %
yield, m.p. 156–158 °C. d 10.59 (s, 1H), 10.51 (s, 1H), 9.38 (s, 1H), 8.16–8.14 (d,
J = 8.8 Hz, 1H), 8.13–8.05 (m, 3H), 7.67–7.64 (t, J = 7.6, 12.0 Hz, 1H), 7.63–7.61
(
d, J = 8.0 Hz, 1H), 7.48–7.45 (t, J = 7.6, 14.4 Hz, 1H), 7.39-7.35 (m, 2H)
1
3
6
.97–6.95 (d, J = 8.8 Hz, 2H). C NMR: (100 MHz, DMSO-d6): d 176.4, 1587.9,
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
1
1
55.6, 150.1, 144.3, 142.6, 140.5, 136.4, 135.7, 129.9, 129.0, 126.4, 125.0, 122.4,
?
19.3, 112.3, 105.3. MS found: m/z 452.0 for [M ?1] peak and calculated for
C H ClN O S is 451.05. Elemental analysis calculated for C H ClN O S is C,
5 3
2
1
14
5
3
21 14
5
5.82; H, 3.12; N, 15.50 and found as C, 55.96; H, 3.11; N, 15.64.
Synthesis of N-[(benzothiazol-6-ylamino) (phenylamino) methylene] benzamide
(
1
4j) Off-white solid, 85.8 % yield, m.p. 158–160. H NMR (400 MHz, DMSO-
1
d6): d 10.68 (s, 1H), 10.45 (s, 1H), 9.36 (s, 1H), H NMR (400 MHz, DMSO-d6): d
1
0.59 (s, 1H), 10.48 (s, 1H), 9. 23 (s, 1H), 8.21–8.19 (d, J = 8.8 Hz, 2 H),
7
J = 5.6 Hz, 2H), 7.50–7.47(m, 1H), 7.22–7.19 (t, J = 7.6, 14.4 Hz, 1H), 6.83–6.81
.77–7.75 (d, J = 6.0 Hz, 2H), 7.73–7.71 (d, J = 7.6 Hz, 2H), 7.64–7.63 (d,
1
3
(
m, 2H), 6.56–6.55 (d, J = 4.8 Hz, 1H). C NMR: (100 MHz, DMSO-d6): d 175.9,
1
58.4, 156.1, 143.6, 142.4, 139.2, 135.5, 134.8, 134.4, 129.7, 129.2, 122.4, 121.7,
?
12.9, 106.1. MS found: m/z 373.2 for [M ?1] peak and calculated for
1
C H N OS is 372.10. Elemental analysis C, 67.72; H, 4.33; N, 15.04; O, 4.30;
S, 8.61.
2
1 16 4
Synthesis of (E)-ethyl {4-[3-(benzothiazol-6-yl)-2-benzoylguanidino] phenyl} car-
1
bamate (4k) Off-white solid, 87.62 % yield, m.p. 157–159 °C. H NMR
(
400 MHz, DMSO-d6) d 10.62 (s, 1H), 10.46 (s, 1H), 9.64 (s, 1H), 9.32 (s, 1H),
8
.23-8.20 (d, J = 1.6 Hz, 1H), 8.06-8.04 (d, J = 8.0 Hz, 1H), 8.00–7.98 (d,
J = 7.2 Hz, 2H), 7.68–7.60 (m, 1H), 7.53–7.44 (m, 3H), 7.39–7.34 (q, 5H),
1
3
4
.13–4.08 (q, 2H), 1.24–1.21 (t, J = 6.8, 14 Hz, 2H). C NMR: (100 MHz,
DMSO-d6): d 176.4, 157.4, 154.01, 150.7, 138.6, 137.1, 135.7, 134.5, 131.7, 129.1,
28.4, 125.3, 123.9, 123.2, 118.9, 117.9, 60.6, 14.9. MS found: m/z 460.3 for
1
?
[
M ?1] peak and calculated for C H N O S is 459.14. Elemental analysis
24 21 5 3
calculated for C, 62.73; H, 4.61; N, 15.24 and experimentally found as C, 62.95; H,
.73; N, 15.01.
4
Synthesis of N-{(1, 3-benzothiazol-6-ylamino) [(2-hydroxy-4-methyl phenyl) amino]
1
methylidene} benzamide (4l) Gray solid, 91.4 % yield, m.p. 150–151 °C. H NMR
(
400 MHz, DMSO-d6): d 10.95 (s, 1H), 9.81 (s, 1H), 9.31–9.29 (d, J = 12.8 Hz,
1
1
2
1
H), 8.28 (s, 1H), 8.05–7.96 (m, 3H), 7.71–7.65 (m, 1H), 7.48–7.44 (t, J = 7.2,
4.4 Hz, 1H), 7.40–7.36 (t, J = 7.2, 14.4 Hz, 2H), 7.27 (s, 1H), 6.86–6.80 (q, 2H),
1
3
.17 (s, 3H). C NMR: (100 MHz, DMSO-d6): d 175.9, 158.6, 155.8, 145.3, 144.2,
43.5, 135.3, 134.5, 134.2, 132.1, 129.8, 129.3, 122.4, 118.4, 116.5, 113.8, 106.1.
?
LCMS found: m/z 403.2 for [M ?1] peak and calculated for C H N O S is
2
2 18 4 2
402.12, Elemental analysis calculated C, 65.65; H, 4.51; N, 13.92 and experimen-
tally found as C, 65.92; H, 4.42; N, 13.74.
Synthesis of N-{(benzothiazol-6-ylamino) [(2-hydroxy phenyl) amino] methylene}
1
benzamide (4m) Brown solid, 76.1 % yield, m.p. 220–221 °C. H NMR
(
400 MHz, DMSO-d6): d 10.94 (s, 1H), 9.83 (s, 1H), 9.32-9.31 (d, J = 4.6 Hz,
1
1
6
H), 8.25 (s, 1H), 8.05-7.95 (m, 3H), 7.68–7.36 (m, 1H), 7.48-7.44 (t, J = 7.2,
4.4 Hz, 1H), 7.40–7.36 (t, J = 7.2, 14.4 Hz, 2H), 7.27 (s, 1H), 6.86–6.80 (q, 2H),
1
.62–6.59 (d, J = 10.4 Hz, 1H), 6.56–6.55 (d, J = 5.6 Hz, 1H). C NMR:
3
(
100 MHz, DMSO-d6): d 175.4, 158.3, 155.9, 144.1, 143.1, 142.3, 136.3, 135.1,
123

M. Bhat, S. L. Belagali
1
LCMS found: m/z 389.02 for [M ?1] peak and calculated for C H N O S is
34.3, 134.3, 129.9, 129.2, 123.6, 122.3, 120.1 117.8, 116.2, 115.4, 113.9, 102.7.
?
2
1 16 4 2
3
1
88.1. Elemental analysis, calculated for C H N O S is C, 64.93; H, 4.15; N,
21 16 4 2
4.42 and found as C, 64.68; H, 4.18; N, 14.61.
Antimicrobial activity
Microorganisms
Microbial cultures used in the study were procured from the Microbial Type Culture
Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh,
India. The cultures used were Escherichia coli (MTCC-40), Staphylococcus aureus
(
MTCC 7443), Candida albicans (MTCC-183), and Aspergillus niger (MTCC-
344). Stock cultures of bacteria were maintained on nutrient agar (Himedia,
1
Mumbai) slants at 4 °C with periodical sub-culturing.
Antimicrobial assay
The antimicrobial activities of the compounds were screened by the disc diffusion
method [34]. Briefly, the 200-lg solutions of the synthesized compounds were
placed on the pre-seeded disc on agar plates. Then, 100 lL of 24-h culture of the
test microorganisms in broth was used for the seeding. Chloramphenicol (200-lg
solution) and Bavistin (200 lg/mL solution) were used as positive controls for
bacteria and fungi, respectively, while the discs with only the solvent (200 lL/disc)
were used as negative controls. The plates were pre-incubated for 1 h at 4 °C. The
bacterial plates were incubated at 37 °C for 24 h and the fungal plates for 5 days at
3
0 °C. The diameters of the inhibition zones (in mm) were measured after the
incubation, and these values are shown in Table 2.
Determination of minimum inhibitory concentration (MIC)
A broth micro-dilution method was used to determine the MIC of the isolated
compounds [35]. A serial two-fold dilution of the compounds was prepared over the
range 200–0.08 lg. Each of the discs was inoculated with synthesized compounds.
The bacterial plates were incubated at 37 °C for 24 h and those of fungi at 28 °C for
7
2 h. The diameters of the inhibition zones (in mm) were measured and the
inhibition zone that appeared for the minimum concentration was taken as the MIC
of the compound. These values are shown in Table 3.
Antioxidant activity
Free radical scavenging ability by DPPH radical assay (1,1-diphenyl-2-picryl
hydrazyl)
The DPPH radical scavenging activity of the purified compounds was determined
by Raj et al [35]. Various concentrations of test samples in an aliquot of 100 lL
1
23

Guanidinyl benzothiazole derivatives: Synthesis and…
were mixed with 100 lL of 40 lM methanolic solutions of DPPH (Himedia,
Mumbai, India) in 96-well micro-titer plates. The decrease in absorbance at 517 nm
was recorded after the incubation of 15 min at room temperature. The absorbance of
the DPPH solution with only methanol and without sample was used as the control.
Ascorbic acid (AA, Himedia, Mumbai) was used as a standard to compare the
activity. Appropriate blank readings at 517 nm were recorded for each tested
dilution. The assay was carried out in triplicate. The percentage inhibition of the
DPPH radical by the samples was calculated according to the formula below.
Percentage of inhibition ¼ ½ðA À A Þ=A ފ  100
ð1Þ
where A is the absorbance of the control and A is the absorbance of the sam-
C
S
C
C
S
ple/standard after 15 min. The IC50 value was calculated graphically based on the
capacity of the compound concentration to scavenge 50 % of the free radicals.
0
ABTS [2,2 -azino-bis (3-ethylbenzthiazoline-6-sulphonic acid] radical scavenging
activity
ABTS assay was performed with modification of the Samaga et al. [36], method.
The ABTS stock solution was prepared by mixing equal volumes of 7.4 mM ABTS
(
Sigma-Aldrich, USA) solution and 2.6 mM potassium per sulfate solution followed
by incubation for 12 h at room temperature in the dark. The reaction mixture
consisted of 1 mL of the purified compound at different concentrations
(
0.5–500 lg/mL in respective solvents) and 3 mL of standardized ABTS solution.
The decrease in absorbance was measured at 734 nm after 15 min of incubation.
Data for each assay was recorded in triplicate. Ascorbic acid (NICE Chemicals Pvt.
Ltd., Cochin, India) was used as a positive control. The scavenging activity was
estimated based on the percentage of ABTS radicals scavenged by the following
formula.
Percentage of scavenging ¼ ½ðA À A Þ=A Š  100
ð2Þ
where A is absorbance of control, and A is absorbance of the tested sample/s-
C
S
C
C
S
tandard solution after 15 min. The IC50 value was calculated graphically based on
the capacity of the compound concentration to scavenge 50 % of the free radicals.
Acknowledgments One of the authors, Mahesh Bhat, gratefully acknowledges the Department of
Science and Technology, Ministry of Science and Technology, New Delhi, India, for providing an
INSPIRE Fellowship [IF130464], and to the Institute of Excellence, University of Mysore, for providing
instrumental facilities.
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