Synthesis, characterization, and biological activities of 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole-2-oles

Article abstract of DOI:10.1007/s00044-014-0962-8

4-Imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3] benzothiazole-2-one (3) was synthesized by the reaction of 2-aminobenzothiazole with ethyl cyanoacetate in solvent free conditions at 150°C. A series of pyrimido benzothiazole-based azo dyes 4( a-m) were obtained by the coupling of carbocyclic amine-based diazonium chloride with compound (3). The synthesized dyes were purified and characterized by elemental analysis, FT-IR, 1 H NMR, and high-resolution mass spectral data. The solvatochromic behaviors of dyes in various solvents were examined. All the azo dyes exhibited pronounced in vitro antibacterial activities against Gram-positive and Gram-negative bacteria, as well as fungi. The results revealed that most of the compounds exhibited good levels of antibacterial activity. Compounds 4d and 4h showed excellent levels of antimicrobial activity with MIC values of 8.25 μg/mL. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0962-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0962-8
ORIGINAL RESEARCH
Synthesis, characterization, and biological activities of 4-imino-3-
arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole-2-oles
•
Selinay Eris¸kin Nesrin S¸ener Serkan Yavuz
•
•
˙
Izzet S¸ener
Received: 4 November 2013 / Accepted: 17 February 2014
Ó Springer Science+Business Media New York 2014
Abstract 4-Imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]
benzothiazole-2-one (3) was synthesized by the reaction
of 2-aminobenzothiazole with ethyl cyanoacetate in sol-
vent free conditions at 150 °C. A series of pyrimido
benzothiazole-based azo dyes 4(a–m) were obtained by
the coupling of carbocyclic amine-based diazonium
chloride with compound (3). The synthesized dyes were
purified and characterized by elemental analysis, FT-IR,
1H NMR, and high-resolution mass spectral data. The
solvatochromic behaviors of dyes in various solvents
were examined. All the azo dyes exhibited pronounced
in vitro antibacterial activities against Gram-positive and
Gram-negative bacteria, as well as fungi. The results
revealed that most of the compounds exhibited good
levels of antibacterial activity. Compounds 4d and 4h
showed excellent levels of antimicrobial activity with
MIC values of 8.25 lg/mL.
Introduction
Fused heterocyclic compounds are very important com-
pounds partially because of their pharmacogical properties
which include wide applications in medicinal chemistry
(Ellis, 2008). Pyrimido benzothiazoles are very important
class of heterocyclic because of their wide range of biolog-
ical pharmacological properties. Some of which have anti-
viral, antitumor, anti-inflammatory, antihypertensive, and
anti-allergy (Kappe, 1993; Atwal et al., 1989, 1991; Rovn-
yak et al., 1992; El-Sherbeny, 2000; Glennon et al., 1981;
Bartovic et al., 1995). Pyrimido benzothiazole derivatives
have also been known for their antimicrobial activities
(Wade et al., 1983; Alaimo, 1973; Gupta and Rawat, 2010;
Lanjewar et al., 2009; Heravi et al., 2008; Chang et al., 2008;
Shah et al., 2009; Kumar et al., 2009; Chaitanya et al., 2010;
Sahu et al., 2012). As a continuation of our previous work
¨
(Karcı et al., 2006, 2009; Oztu¨rk et al., 2012; S¸ener et al.,
Keywords Solvatochromism ꢀ Pyrimido benzothiazole ꢀ
Diazo-coupling reaction ꢀ Azo dyes ꢀ Antimicrobial activity
2006; Yavuz and Yildirim, 2013). 4-imino-3,4-dihydro-2H-
pyrimido[2,1-b][1,3]benzothiazole-2-one have been repor-
ted in the literature, by the cyclocondesation of benzothia-
zole with ethyl cyanoacetate in the presence of sodium
ethoxide/ethanol, or phosphoric acid (Tadakazu, 1991). We
wish to give new report the synthesis of 4-imino-3,4-dihy-
dro-2H-pyrimido[2,1-b][1,3]benzothiazole-2-one by the
reaction of 2-aminobenzothiazole with ethyl cyanoacetate in
solvent free conditions at 150 °C. Although a number of
paper have been published concerning the synthesis of
pyrimidine and azopyrimidine derivatives, those containing
an azo pyrimidobenzothiazole system have not yet been
reported. In the present study, we reported the synthesis of
4-imino-3,4-dihydro-2H-pyrimido[2,1-b][1,3]benzothiazole-2-
one and some 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]ben-
zothiazole-2-ol derivatives. The synthesized azo dyes were
evaluated biological and antimicrobial activities.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-0962-8) contains supplementary
material, which is available to authorized users.
˙
S. Eris¸kin ꢀ N. S¸ener ꢀ I. S¸ener (&)
Department of Chemistry, Faculty of Science-Arts, Pamukkale
University, 20017 Denizli, Turkey
e-mail: isener@pau.edu.tr; isener@pamukkale.edu.tr
S. Yavuz
Department of Chemistry, Faculty of Science, Gazi University,
06500 Ankara, Turkey
123

Med Chem Res
O
S
S
S
EtO-
150 oC
CN
N
NH
NH2
+
EtO
N
N
O
N
O
NC
2
1
NH
3
Scheme 1 Synthesis of compound (3)
13
10
7
S
S
8
12
1
2
N
NaNO2 / HCl
N
NH2
+
N
3
N
14
OH
O
11
6
9
X
4
5
16
NH
3
NH
N
N
18
15
17
22
19
X
4 (a-m)
20
21
a; X: H
e; X: p-CH3
f; X: m-NO2
g; X: m-OCH3
h; X: m-Cl
i; X: m-CH3
j; X: o-NO2
k; X: o-OCH3
l; X: o-Cl
m; X: o-CH3
b; X: p-NO2
c; X: p-OCH3
d; X: p-Cl
Scheme 2 Synthesis of compounds 4(a–m)
Results and discussion
spectra of synthesized compound (3) showed an intense
(–OH) band at 3375 cm-1, and a band (=NH) located at
3167–3133 cm-1. The other band at 1697 cm-1assigned to
(C=N). FT-IR spectra of compound (3) did not appear any
band for (C=O) group. The 1H NMR spectra of compounds
(3) exhibited singlet signal at d 4.15 ppm attributed to
(–CH2) protons and broad peak at d 12.75 ppm, which was
attributed to (=NH) proton. The IR spectra of dyes 4(a–
m) showed characteristic hydroxyl (–OH) bands at
3211–3385 cm-1, but did not showed any bands for car-
bonyl (C=O) group. We can suggest that these compounds
Chemistry
In this study, 4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]
benzothiazole-2-ol derivatives have been reported. The
structures of the synthesized dyes were determined by
analysis of their FT-IR, NMR, high-resolution mass spectra
for the molecular weights and elemental analysis. As
shown in Scheme 1, firstly, 4-imino-3,4-dihydro-2H-py-
rimido[2,1-b][1,3]benzothiazole-2-one was prepared by the
reaction of 2-aminobenzothiazole with ethyl cyanoacetate
in solvent free conditions at 150 °C.
Secondly, aniline derivatives were diazotized using
sodium nitrite in hydrochloric acid in ice bath at between 0
and 5 °C. The synthesized carbocyclic diazonium salts
were then coupled with 4-imino-3,4-dihydro-2H-pyrimi-
do[2,1-b][1,3]benzothiazole-2-one to give corresponding
4-imino-3-arylazo-4H-pyrimido[2,1-b][1,3]benzothiazole-
2-ol derivatives. This reaction route is depicted in
Scheme 2. By the purification of the reaction mixture, the
thirteen novel azo pyrimido[2,1-b][1,3]benzothiazole
derivatives have been obtained.
1
were only in imino-azo-enol form (T2) in solid state. H
NMR spectra of synthesized azo pyrimido[2,1-b][1,3]ben-
zothiazole derivatives 4(a–d) exhibit a broad peak at
12.01–12.28 ppm, which was attributed to =NH protons, a
broad peak at 12.58–13.25 ppm which was attributed
hydrazo (–NH) protons and showed a broad peak at
13.50–14.22 ppm attributed to tautomeric hydroxyl (–OH).
1H NMR spectra of dyes did not showed –NH2 protons
except for 4j. 1H NMR spectra of 4g, 4k, 4l, and 4m
showed only =NH proton, but did not showed hydrazo
–NH protons. 1H NMR spectra of 4e, 4f, 4h, and 4i showed
only =NH proton, and hydrazo –NH protons. 1H NMR
spectra of 4j showed only –NH2 protons. According to the
1H NMR results, suggest that dyes 4(a–d) a mixture of
predominantly in imino-azo-enol form (T2) and imino-
hydrazo-keto form (T3), the dyes of 4e, 4f, 4h, and 4i have
The obtained product 4(a–m) can exist in possible four
tautomeric forms namely amino-azo-keto form T1, imino-
azo-enol form T2, imino-hydrazo-keto form T3, imino-
azo-keto form T4 as shown in Scheme 3. The FT-IR
123

Med Chem Res
S
N
S
N
N
OH
N
N
O
NH
N
H
NH
N
N
X
imino-azo-enol
T2
KT
KT
X
S
imino-hydrazo-keto
T3
N
N
O
N
N
NH2
X
amino-azo-keto
T1
KT
S
N
N
O
NH
N
N
X
imino-azo-keto
T4
Scheme 3 The tautomeric form of azo pyrimido[2,1-b][1,3] benzothiazole derivatives
only in imino-hydrazo-keto form (T3), the dyes of 4g, 4k,
4l, and 4m have only in imino-azo-enol form (T2). The dye
of 4j has in amino-azo-keto (T1). All the synthesized dyes
did not have in imino-azo-keto (T4) tautomeric forms in
DMSO-d6 as showed in Scheme 3. The mass spectra of
dyes have molecular ion peak which corresponds to
molecular weight of respective compounds.
respect to the kmax in dimethyl sulfoxide (DMSO) and
dimethyl formamide (DMF) except for 4b. For example,
for the dye 4a kmax is 385 nm in DMSO, 394 nm in
chloroform. For the dye 4d kmax is 391 nm in DMSO 396
in chloroform. The explanation for this irregular behavior
may be due to the presence of nonbonding electron pairs of
carbonyl, oxygen, and nitrogen atoms in the molecule ring
(Harikrishnan and Menon, 2008). General observation
made is that most of the substituted dyes have higher
absorption maxima when compared to their unsubstituted
analogs. The absorption spectra of dye 4j showed two
absorbance in all solvent except for acetic acid. The other
spectra of the dyes showed single absorption peak with a
shoulder or two absorption peak with a shoulder. It can be
suggested that all dyes have a mixture of tautomer forms in
various solvent. The spectral shifts of the dye 4b in various
solvents are shown in Fig. 1.
Absorption spectra
The UV–Vis absorption spectra of the dyes 4(a–m) were
recorded over the range of k between 350 and 700 nm,
using variety of solvents in concentration 10-6 to 10-8 M.
The spectral data of the synthesized dyes are depicted in
Table 1.
The visible absorption spectra of the dyes did not cor-
relate with the polarity of solvent. The dyes showed color
ranging from dark red (kmax 357 nm) to yellow (kmax
592 nm). It was observed that kmax of p-substitute dyes
4(a,c,d) shifted bathochromically in chloroform with
The effects of the acid and base on the absorption
spectra of the dyes were investigated and these results are
given in Table 2.
123

Med Chem Res
Chloroform
Table 1 Influence of solvent on kmax (nm) of dyes
Dye no.
DMSO
DMF
Acetonitrile
Methanol
Acetic acid
4a
4b
4c
4d
4e
4f
385,325a
417,592,332a
403,319a
391,320a
389,328a
392,322a
386,320a
399,333a,487a
387,320a
365,425
387
403
419,584a
416,341a
404,330a
385
409,331a
422,334a
426,338a
413,336a
398,335a
389,330a
390,328a
407,332a
386,330a
359,420
420,338
405,334a
414,341a
392
394
414,552a
405
420,590
401
393,496a
411,333a
408,325a
396,323a
381,320a
383,322a
378,319a,478a
392,325
378,318a
409,345a
410,328a
389,328a
400,324a
396
395
385
393
387
387
4g
4h
4i
391
396,491a
380
403,485a
380
394
390
381
382
4j
363,416
413
405,491a
365,422
420
357,414
414
4k
4l
416,336a
405,327a
408,330a
405
401
4m
407
411
405
a
Shoulder
The absorption spectra of the dyes were sensitive in
methanol to the addition of acid and base. With the
exception of dyes 4c, 4k and 4m, when small amount of
potassium hydroxide (0.1 M) was added to methanol
solution, the kmax values showed bathochromic shift. When
hydrochloric acid (0.1 M) was added in methanol hypso-
chromic shifts were detected except for 4j. The spectral
shifts of 4b in acidic and basic solution are shown in Fig. 2.
variable inhibitory effects on the growth of the tested
Gram-positive and Gram-negative bacterial strains, and
also against antifungal strains. In general, the results
revealed that para phenyl substituted compounds exhibited
better activities than ortho and meta phenyl substituted
compounds. Furthermore, compound 4d having chlorine
substituent at para and 4h meta positions of the phenyl ring
exhibited potent antimicrobial activities against Gram-
positive and Gram-negative bacteria, as well as fungi. Most
of pyrimido benzothiazole derivatives have superior sig-
nificant antibacterial potency than antifungal potency. One
of the tested compound 4d exhibited good antimicrobial
activity against all tested organisms with respect to refer-
ence drugs. Compound 4d inhibited the growth of S. aureus
ATCC 29213, B. subtilis ATCC6633, and C. albicans NRRL
Y-477 with IZs 30, 32, and 31 mm, respectively. Also
compound 4h showed excellent activity against S. aureus
ATCC 29213 and E. coli ATCC 25922 with IZ 31 and
30 mm, respectively.
Antimicrobial activity
In the present study, a total of thirteen diazo derivatives
4(a–m) was evaluated for their in vitro antibacterial and
antifungal activities at 100 lg/mL concentration against
microbial strains such as two Gram-positive bacteria
(Staphylococcus aureus ATCC 29213 and Bacillus subtilis
ATCC 6633), two Gram-negative bacteria (Klebseilla
pneumonia ATCC13883 and Escherichia coli ATCC
25922), and two fungi (Saccharomyces cerevisiae and
Candida albicans NRRL Y-477). Agar-diffusion method
was used for the determination of the preliminary anti-
bacterial and antifungal activity. Ciprofloxacin and Keto-
conazole were used as standard antibacterial and antifungal
reference, respectively. The results were recorded for each
tested compound as the average diameter of inhibition
zones (IZ) of bacterial or fungal growth around the disks in
mm. Most of the newly synthesized compounds showed
excellent antimicrobial activities with respect to the control
drugs. The results in Table 3 revealed that the majority of
the synthesized compounds showed variable inhibition
activities against the tested strains. Data in Table 3
revealed that the most of tested compounds displayed
The minimum inhibitory concentration (MIC) of the
synthesized compounds against highly inhibited organisms
is reported in Table 4. Compound 4d exhibited low MIC
(8.25 lg/mL) against S. aureus ATCC 29213, B. subtilis
ATCC 6633, K. pneumonia ATCC13883, S. cerevisiae, and
C. albicans NRRL Y-477. In addition, compound 4h
showed MIC 8.25 lg/mL against S. aureus ATCC 29213,
K. pneumonia ATCC13883, E. coli ATCC 25922, S. ce-
revisiae, and C. albicans NRRL Y-477. Additionally,
compounds 4f and 4l exhibited MIC 8.25 lg/mL against S.
aureus ATCC 29213 and also compounds 4b and 4j
showed good inhibitory activities against B. subtilis ATCC
6633 (MIC 8.25 lg/mL).
123

Med Chem Res
MS) instruments (Waters Corporation, Milford MA, USA).
Elemental analyses were done on a Leco CHNS-932 ana-
lyzer (USA). UV–Vis absorption spectra were recorded on
an ATI (UK) Unicam UV-100 spectrophotometer over the
range of k between 300 and 700 nm. The wavelengths of
maximum absorption (kmax) were investigated in various
solvents such as DMSO, DMF, acetonitrile, methanol,
acetic acid, and chloroform at various concentrations
(1 9 10-6 M). Change of (kmax) was also investigated
when 0.1 mL of base (potassium hydroxide, 0.1 M) or
0.1 mL of acid (hydrochloric acid, 0.1 M) was added to
1 mL of the dye solution in methanol.
DMSO
DMF
Acetonitrile
Methanol
Acetic acid
Chloroform
1,0
0,8
0,6
0,4
0,2
0,0
400
500
600
700
Wavelength (nm)
Synthesis
Fig. 1 Absorption spectra of dye 4b in various solvents
Preparation of 4-imino-3,4-dihydro-2H-pyrimido[2,1-
b][1,3]benzothiazole-2-one (3)
Table 2 Absorption maxima of dyes in acidic and basic solutions
The starting compounds were synthesized by the reaction
of 2-aminobenzothiazole with ethyl cyanoacetate. 2-amin-
obenzothiazle 0.2 g (1.33 mmol) reacted with ethyl cya-
noacetate 0.7 mL (6.65 mmol) in solvent free conditions.
The reaction mixture was heated at 150 °C for 2 h, and
then cooled at room temperature and upon dilution with
100 mL ethanol:water mixture (1:1 by volume). The pre-
cipitated yellow products separated were filtered off,
washed with water several times, and dried. The obtained
product was crystalized from DMF:water mixture as yel-
Dye no.
Methanol
Methanol ? KOH
Methanol ? HCl
4a
4b
4c
4d
4e
4f
409,331a
422,334a
426,338a
413,336a
398, 335a
389,330a
390,328a
407,332a
386,330a
359,420
420,338
405,334a
414,341a
410
408a, 542
392
415
413
415
430
396
406
388
361a, 386
410
4g
4h
4i
416
445, 498a
381
391
low crystals, yield (90 %), mp: 236–238 °C; IR (cm-1
max: 3375 (OH), 3167–3133 (=NH), 3055 (Ar–H), 2944
)
406
365a, 466
411
385
360a, 418
t
4j
(Aliphatic C–H), 1697 (C=N); 1H NMR (DMSO-d6) d
(ppm): 4.15 (s, 2H, CH2), 7.31 (d, 1H, J = 7.47 Hz, H-10),
7.45 (1H, J = 7.47 Hz, H-13), 7.78 (1H, t, J = 7.47 Hz,
H-11), 8.05 (1H, t, J = 7.47, H-12), 12.75 (b, 1H, pyrim-
idone =NH); 13C NMR (DMSO-d6) d: 40.09 (CH2, C-5),
110.49 (CH, C-10), 122.05 (CH, C-12), 122.68 (CH, C-13),
124.8 (C, C-8), 127.11 (CH, C-11), 147.6 (C, C-2), 147.82
(C, C-9), 166.9 (C, C-6), 166.96 (C, C-2); HR-MS:
217.2482 [M?H]?, calcd. 217.2485. Anal. Cald. for
C10H7N3OS: C, 55.2 %; H, 3.25 %; N, 19.34 %. Found: C,
55.28 %; H, 3.26 %; N, 19.34 %.
4k
4l
416
424
404
4m
402
408
a
Shoulder
Experimental
General
All chemicals were purchased and were used without fur-
ther purification. Solvents were of spectroscopic grade.
Melting points of the synthesis dyes were determined using
Stuart smp 30 melting point apparatus (UK). Nuclear
magnetic resonance (NMR) spectra were recorded on a
Bruker (Germany) Spectrospin Avance DPX 400 Ultra-
Shield spectrometer at room temperature by using tetra-
methylsilane (TMS) as the internal standard. Chemical
shifts were (d) given in ppm. FT-IR spectra were recorded
on a Perkin Elmer (USA) spectrometer. MS analyses were
recorded on from Waters LCT Premier XE LTOF (TOF
General procedure for the synthesis of azo dyes 4(a–m)
Preparation of 4-imino-3-phenylazo-4H-pyrimido[2.1-
b][1,3]benzothiazole-2-ole (4a)
Aniline 0.93 g (10 mmol) was dissolved in hydrochloric
acid (7.5 mL) and water (15 mL) was added. The solution
was cooled to 0–5 °C. Sodium nitrite 0.69 g (10 mmol) in
water (10 mL) was then added to this cold solution drop
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Med Chem Res
Preparation of 4-imino-3-(40-nitrophenylazo)-4H-
Methanol
Methanol + HCl
Methanol + KOH
1,0
0,8
0,6
0,4
0,2
0,0
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4b)
Brown solid crystals, (82 %), mp: 232–234 °C. IR (cm-1
)
t
max: 3211 (–OH), 3104 (=NH), 3088 (Ar–H), 2987 (Ali-
phatic C–H), 1661 (C=N), 1598–1455 (N=N), 1514, 1336
(NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.37 (1H,
t, J = 8.1 Hz, H-12), 7.54 (1H, t, J = 8.1 Hz, H-11), 7.62
(1H, d, J = 8.74 Hz, H-13), 7.70 (1H, d, J = 8.75 Hz,
H-10), 8.01 (1H, d, J = 7.24 Hz, H-22), 8.04 (1H, d,
J = 7.24 Hz, H-18), 8.30 (2H, d, J = 8.74 Hz, H-19,
H-21), 12.01 (1H, b, =NH), 13.25 (1H, b, hydrazo-NH),
14.13 (s, tautomeric –OH). 13C NMR (100 MHz, DMSO-
d6) d: 104.90 (C, C-5), 108.71 (CH, C-10), 122.2 (CH,
C-12), 122.9 (CH, C-18, C-22), 123.27 (C, C-8), 125.18
(CH, C-13), 126.01 (CH, C-21), 127.46 (CH, C-11), 131.57
(C, C-4), 149.65 (C, C-20), 151.72 (C, C-6), 152.37 (C,
C-9), 155.28 (C, C-17), 168 (C, C-2); HR-MS: 366.3552
[M?H]?, calcd. 366.3553. Anal. Calcd. For C16H10N6O3S:
C, 52.45 %; H, 2.75 %; N, 22.94 %; Found: C, 52.46 %;
H, 2.76 %; N, 22.93 %.
400
500
600
700
Wavelength (nm)
Fig. 2 Absorption spectra of dye 4b in acidic and basic solutions
wise with vigorous stirring over 1 h. Excess nitrous acid
was destroyed by the addition of the urea. The result-
ing diazonium solution was then added in portion over
30 min. to a vigorously stirred solution of 4-imino-3,
4-dihydro-2H-pyrimido [2,1-b][1,3]benzothiazole-2-one
2.17 g (10 mmol), which was dissolved in potassium
hydroxide (20 mmol) and water (20 mL). The solution was
stirred at 0–5 °C for 2 h and the pH of the reaction mixture
was maintained at 6–7 by the simultaneous addition of
sodium acetate solutions. The precipitated product sepa-
rated upon dilution with water (50 mL) was filtered,
washed with water several times, dried and crystallized
from DMF:water (2:3 by volume) to give 4-imino-3-
phenylazo-4H-pyrimido[2.1-b][1,3]benzothiazole-2-ole as
a yellow crystals, yield (88 %), mp: 249–250 °C. IR
(cm-1) tmax: 3385 (–OH), 3167–3132 (=NH), 3039 (Ar–
H), 2924 (Aliphatic C–H), 1641 (C=N), 1510, 1453 (N=N);
1H NMR (400 MHz, DMSO-d6) d (ppm): 7.16 (1H, t,
J = 7.24 Hz, H-20), 7.45 (2H, t, J = 7.71 Hz, H-19,
H-21), 7.51 (2H, J = 6.95 Hz, H-11, H-12), 7.51 (1H, d,
J = 7.45 Hz, H-10), 7.57 (1H, d, J = 7.43 Hz, H-13), 8.03
(2H, d, J = 9.24 Hz, H-18, H-22), 12.25 (1H, b, =NH),
Preparation of 4-imino-3-(40-methoxyphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4c)
Orange solid crystals, (79 %), mp: 216–218 °C. IR (cm-1
max: 3251 (–OH), 3128 (=NH), 3028 (Ar–H), 2995 (Ali-
)
t
1
phatic C–H), 1637 (C=N), 1509–1455 (N=N); H NMR
(400 MHz, DMSO-d6) d (ppm): 3.79 (3H, s, –OCH3), 7.35
(1H, t, J = 7.79 Hz, H-12); 7.39 (1H, t, J = 7.79 Hz,
H-11); 7.47 (1H, d, J = 8.27 Hz, H-13); 7.64 (1H, d,
J = 7.79 Hz, H-10), 7.78 (1H, d, J = 7.79 Hz, H-21), 7.87
(2H, d, J = 7.79 Hz, H-18, H-22), 8.03 (1H, d,
J = 7.79 Hz, H-19), 12.20 (1H, b, =NH), 12.58 (1H, b,
hydrazo-NH), 14.06 (s, tautomeric –OH). 13C NMR
(100 MHz, DMSO-d6) d: 55.46 (CH3, C-25), 104.90 (C,
C-5), 108.71 (CH– C-10), 115.31 (CH, C-19, C-21), 122.2
(CH, C-12), 123.27 (C, C-8), 124.16 (CH, C-18, C-22),
125.18 (CH, C-13), 127.46 (CH, C-11), 131.57 (C, C-4),
147.46 (C, C-17), 151.72 (C, C-6), 152.37 (C, C-9), 160.04
(C, C-20), 168 (C, C-2); HR-MS: 351.3836 [M?H]?,
calcd. 351.3837. Anal. Calcd. for C17H13N5O2S: C,
58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.12 %; H,
32.74 %; N, 19.92 %.
12.72 (1H, b, hydrazo-NH), 14.22 (s, tautomeric –OH); 13
C
NMR (100 MHz, DMSO-d6) d: 104.90 (CH, C-5), 108.71
(CH– C-10), 112.19 (CH, C-18, C-22), 118.22 (CH, C-20),
122.20 (CH, C-12), 123.27 (C, C-8), 125.18 (CH, C-13),
127.46 (CH, C-11), 129.97 (CH, C-19, C-21), 131.57 (C,
C-4), 151.72 (C, C-6), 152.37 (C, C-9), 153.59 (C, C-17),
168 (C, C-2); HR-MS: 321.3576 [M?H]?, calcd.
321.3577. Anal. Cald. for C15H11N5OS: C, 59.80 %; H,
3.45 %; N, 21.79 %; Found: C, 59.81 %; H, 3.46 %; N,
21.78 %.
Preparation of 4-imino-3-(40-chlorophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4d)
Dark orange solid crystals, (81 %), mp: 248–250 °C. IR
(cm-1) tmax: 3260 (–OH), 3186 (=NH), 3062 (Ar–H), 2940
(Aliphatic C–H), 1637 (C=N), 1495–1454 (N=N); 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.41 (1H, t, J = 7.47 Hz,
H-12), 7.54 (1H, t, J = 7.47 Hz, H-11), 7.63 (1H, d,
The above procedure was also used to synthesize dye
4(b–g). The general route of synthesized dyes is outlined in
Scheme 1.
123

Med Chem Res
Table 3 Antimicrobial activity expressed as inhibition diameter zones in millimeters (mm) of chemical compounds against the pathological
strains based on well-diffusion assay
Compounds
Zone of inhibition (mm)
Bacteria
Fungi
Gram-positive
Gram-negative
S. aureus
B. subtilis
K. pneumoniae
E. coli
S. cerevisiae
C. albicans
4a
22 0.5
28 1.8
22 1.2
30 1.6
24 0.7
32 0.9
25 2.1
31 0.4
18 0.7
28 1.2
22 1.6
30 1.0
19 1.4
28 1.2
NT
19 1.7
32 1.3
22 0.8
32 0.9
22 1.5
28 1.4
27 1.5
28 0.9
16 0.3
30 1.3
24 2.4
24 1.8
18 1.4
29 0.6
NT
16 1.1
26 0.6
25 1.3
29 0.8
24 1.6
24 1.3
30 0.9
29 0.8
20 1.6
24 1.4
24 1.2
18 0.5
12 1.7
29 1.3
NT
18 0.7
16 0.9
28 2.0
28 1.9
20 1.3
25 1.5
31 1.7
30 1.8
19 0.8
22 1.3
26 0.6
22 1.4
12 1.1
24 0.7
NT
14 1.2
14 1.4
16 1.1
29 0.3
16 0.6
21 1.1
24 1.4
23 1.2
12 0.5
18 0.7
29 1.2
19 1.6
12 1.4
NT
15 1.6
18 1.4
16 1.2
31 0.3
15 2.1
19 1.4
28 1.6
29 0.8
13 0.7
18 1.5
28 0.6
23 1.2
14 2.2
NT
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
Ciprofloxacin
Ketoconazole
28 1.2
28 1.4
Zone of inhibition values are presented as the mean SEM from at least three separated experiments
NT not tested
J = 7.47 Hz, H-13), 7.65 (1H, d, J = 7.47 Hz, H-10), 7.85
(1H, d, J = 8.18 Hz, H-21), 8.97 (2H, d, J = 7.44 Hz, H-22,
H-18), 8.02 (1H, J = 7.43 Hz, H-19), 12.28 (1H, b, = NH),
hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d: 20.65
(CH3, C-25), 108.98 (CH, C-10), 117.05 (CH– C-18,
C-22), 123.18 (CH, C-12), 123.49 (CH, C-13), 124.3 (C,
C-8), 127.92 (CH, C-11), 130.51 (CH, C-19, C-21), 133.88
(C, C-4), 136.95 (C, C-20), 140.04 (C, C-17), 141.96 (C,
C-5), 147.11 (C, C-9), 153.35 (C, C-6), 168.9 (C, C-2);
HR-MS: 335.3842 [M?H]?, calcd. 335.3841. Anal. Calcd.
for C17H13N5OS: C, 60.88 %; H, 3.91 %; N, 20.88 %;
Found: C, 60.89 %; H, 3.92 %; N, 20.87 %.
12.79 (1H, b, hydrazo-NH), 13.50 (b, tautomeric –OH); 13
C
NMR (100 MHz, DMSO-d6) d: 104.90 (C, C-5), 108.71
(CH– C-10), 122.2 (CH, C-12), 123.27 (C, C-8), 123.75 (CH,
C-18, C-22), 125.18 (CH, C-13), 127.46 (CH, C-11), 130.4
(CH, C-21, C-19), 131.57 (C, C-4), 133.63 (C, C-20), 151.72
(C, C-6), 152.28 (C, C-17), 152.37 (C, C-9), 168 (C, C-2);
HR-MS: 355.8024 [M?H]?, calcd. 355.8023. Anal. Calcd.
For C16H10ClN5OS: C, 54.01 %; H, 2.83 %; N, 19.68 %;
Found: C, 54.01 %; H, 2.84 %; N, 19.67 %.
Preparation of 4-imino-3-(30-nitrophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4f)
Preparation of 4-imino-3-(40-methylphenylazo)-4H-
Burgundy red solid crystals, (79 %), mp: 250–251 °C. IR
(cm-1) tmax: 3290 (–OH), 3160 (=NH), 3065 (Ar–H), 2985
(Aliphatic C–H), 1665 (C=N), 1520–1455 (N=N), 1510,
1332 (NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.23
(1H, t, J = 6.52 Hz, H-21), 7.35 (1H, t, J = 7.73 Hz,
H-12), 7.39 (1H, t, J = 7.73 Hz, H-11), 7.47 (1H, d,
J = 7.32 Hz, H-13), 7.79 (1H, d, J = 7.33 Hz, H-10), 7.81
(1H, d, J = 7.33 Hz, H-22), 7.97 (1H, d, J = 7.33 Hz,
H-20), 8.03 (1H, s, H-18), 12.18 (1H, b, =NH), 12.63 (1H,
b, hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d:
109.98 (CH, C-10), 113.73 (CH, C-22), 115.14 (CH, C-20),
120.6 (CH, C18), 123.18 (CH, C-12), 123.48 (CH, C-13),
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4e)
Orange solid crystals, (83 %), mp: 251–252 °C. IR (cm-1
max: 3285 (–OH), 3168 (=NH), 3055 (Ar–H), 2930 (Ali-
)
t
1
phatic C–H), 1683 (C=N), 1428–1487 (N=N); H NMR
(400 MHz, DMSO-d6) d (ppm): 2.32 (3H, s, –CH3), 7.27
(2H, d, J = 7.08 Hz, H-19, H-21), 7.35 (1H, t,
J = 7.08 Hz, H-12), 7.57 (1H, t, J = 7.09 Hz, H-11), 7.79
(1H, d, J = 7.08 Hz, H-13), 7.81 (1H, d, J = 7.09 Hz,
H-10), 7.97 (1H, d, J = 7.09 Hz, H-22), 8.03 (1H, d,
J = 7.09 Hz, H-18), 12.18 (1H, b, =NH), 12.62 (1H, b,
123

Med Chem Res
Table 4 MIC (lg/mL) against the pathological strains based on two fold serial dilution technique
Compounds
MIC in lg/mL
Bacteria
Fungi
Gram-positive
S. aureus
Gram-negative
B. subtilis
K. pneumoniae
E. coli
S. cerevisiae
C. albicans
4a
33
66
132
33
66
132
132
132
8.25
132
66
132
66
4b
16.5
33
8.25
33
132
16.5
16.5
66
4c
33
132
8.25
132
66
4d
8.25
33
8.25
66
8.25
33
4e
4f
8.25
33
16.5
16.5
16.5
132
8.25
33
33
33
4g
8.25
8.25
66
8.25
8.25
66
33
16.5
8.25
132
132
16.5
33
4h
8.25
66
33
4i
132
132
16.5
66
4j
16.5
33
33
33
4k
33
16.5
33
4l
8.25
66
33
66
4m
66
132
8.25
NT
132
16.5
NT
132
NT
8.25
132
NT
Ciprofloxacin
Ketoconazole
8.25
NT
8.25
NT
16.5
NT not tested
Preparation of 4-imino-3-(30-chlorophenylazo)-4H-
124.3 (C, C-8), 127.92 (CH, C-11), 130.42 (CH, C-19),
133.88 (C, C-4), 141.96 (C, C-5), 146.37 (C, C-17), 147.11
(C, C-9), 149.6 (C, C-21), 153.35 (C, C-6), 168.9 (C, C-2);
HR-MS: 366.3552 [M?H]?, calcd. 366.3551. Anal. Calcd.
for C16H10N6O3S: C, 52.45 %; H, 2.75 %; N, 22.94 %;
Found: C, 52.43 %; H, 2.75 % N, 22.94 %.
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4h)
Light orange solid crystals, (84 %), mp: 215–217 °C. IR
(cm-1) tmax: 3212 (–OH), 3074 (=NH), 3006 (Ar–H), 2922
(Aliphatic C–H), 1639 (C=N), 1515–1456 (N=N); 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.42 (1H, t, J = 7.85 Hz,
H-21), 7.52 (2H, t, J = 7.85 Hz, H-11, H-12), 7.75 (1H, d,
J = 7.43 Hz, H-13), 7.83 (1H, d, J = 7.44 Hz, H-10), 7.97
(2H, d, J = 7.43 Hz, H-20, H-22), 9.49 (1H, s, H-18), 10.79
(1H, s, =NH), 11.93 (1H, b, hydrazo-NH,); 13C NMR
(100 MHz, DMSO-d6) d: 104.9 (C, C-5), 108.71 (CH,
C-10), 114.78 (CH, C-22), 115.02 (CH, C-18), 122.2 (CH,
C-12), 122.93 (CH, C-20), 123.27 (C, C-8), 125.18 (CH,
C-13), 127.46 (CH, C-11), 131.57 (C, C4), 131.67 (CH,
C21), 138.84 (C, C-19), 151.72 (C, C-6), 152.37 (C, C-9),
152.59 (C, C-17), 168 (C, C-2); HR-MS: 355.8024
[M?H]?, calcd. 355.8023. Anal. Calcd. for C16H10ClN5OS:
C, 54.01 %; H, 2.83 %; N, 19.68 %; Found: C, 54.02 %; H,
2.84 %; N, 19.69 %.
Preparation of 4-imino-3-(30-methoxyphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4g)
Yellow solid crystals, (83 %), mp: 219–221 °C. IR (cm-1
max: 3311 (–OH), 3175 (=NH), 3062 (Ar–H), 2993 (Ali-
)
t
1
phatic C–H), 1644 (C=N), 1514–1454 (N=N); H NMR
(400 MHz, DMSO-d6) d (ppm): 3.82 (3H, s, –OCH3), 6.76
(1H, t, J = 6.15 Hz, H-21), 7.08 (2H, t, J = 6.15 Hz,
H-11, H-12), 7.34 (1H, d, J = 6.77 Hz, H-13), 7.40 (1H, d,
J = 6.77 Hz, H-10), 7.62 (1H, d, J = 6.77 Hz, H-22), 7.97
(1H, d, J = 6.77 Hz, H-20), 8.00 (1H, s, H-18), 14.01 (1H,
b, =NH), 14.16 (1H, b, –OH); 13C NMR (100 MHz,
DMSO-d6) d: 55.71 (CH3, C-25), 104.9 (C, C-5), 107 (CH,
C-18), 108.71 (CH, C-10), 109.62 (CH, C-20), 111.72 (CH,
C-22), 122.2 (CH, C-12), 123.27 (C, C-8), 125.18 (CH,
C-13), 127.48 (CH, C-11), 131.57 (C, C-4), 133.32 (CH,
C-21), 151.72 (C, C-6), 152.15 (C, C-17), 152.37 (C, C-9),
162.88 (C, C-19), 168 (C, C-2); HR-MS: 351.3836
[M?H]?, calcd. 351.3837. Anal. Calcd. for C17H13N5O2S:
C, 58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.10 %;
H, 32.74 %; N, 19.91 %.
Preparation of 4-imino-3-(30-methylphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4i)
Pink solid crystals, (81 %), mp: 242–243 °C. IR (cm-1
max: 3221 (–OH), 3168 (=NH), 3035 (Ar–H), 2960 (Ali-
)
t
phatic C–H), 1683 (C=N), 1508–1489 (N=N); H NMR
1
(400 MHz, DMSO-d6) d (ppm): 2.38 (3H, s, –CH3), 7.01
123

Med Chem Res
(1H, d, J = 7.47 Hz, H-20), 7.31 (1H, t, J = 7.47 Hz, H-12),
7.36 (1H, t, J = 7.47 Hz, H-11), 7.49 (1H, t, J = 7.47 Hz,
H-21), 7.68 (1H, d, J = 7.47 Hz, H-13), 7.80 (1H, d,
J = 7.47 Hz, H-10), 7.96 (1H, s, H-18), 8.03 (1H, d,
J = 7.47 Hz, H-22), 12.15 (1H, b, 1H, =NH), 12.56 (1H, b,
hydrazo-NH); 13C NMR (100 MHz, DMSO-d6) d: 20.94
(CH3, C-24), 109.98 (CH, C-10), 113.19 (CH, C-18), 118.68
(CH, C-22), 123.18 (CH, C-12), 123.49 (CH, C-13), 124.3 (C,
C-8), 126.67 (CH, C-20), 127.92 (CH, C-11), 129.04 (CH,
C-19), 133.88 (C, C-4), 140.9 (C, C-21), 141.96 (C, C-5),
147.11 (C, C-9), 147.28 (C, C-17), 153.59 (C, C-6), 168.9 (C,
C-2); HR-MS: 335.3842 [M?H]?, calcd. 335.3841. Anal.
Calcd. for C17H13N5OS: C, 60.88 %; H, 3.91 %; N, 20.88 %;
Found: C, 60.88 %; H, 3.93 %; N, 20.89 %.
C-6), 151.37 (C, C-8), 152.37 (C, C-9), 168 (C, C-2); HR-
MS: 351.3836 [M?H]?, calcd. 351.3837. Anal. Calcd. for
C17H13N5O2S: C, 58.11 %; H, 3.73 %; N, 19.93 %; Found:
C, 58.12 %; H, 32.72 %; N, 19.92 %.
Preparation of 4-imino-3-(20-chlorophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4l)
Orange solid crystals, (80 %), mp: 159–162 °C. IR (cm-1
max: 3223 (–OH), 3123 (=NH), 3062 (Ar–H), 2979 (Ali-
)
t
1
phatic C–H), 1644 (C=N), 1500–1452 (N=N); H NMR
(400 MHz, DMSO-d6) d (ppm) 7.20 (1H, t, J = 7.50 Hz,
H-12), 7.41 (1H, t, J = 7.52 Hz, H-11), 7.45 (1H, t,
J = 7.52 Hz, H-20), 7.55 (1H, t, J = 7.50 Hz, H-21), 7.59
(1H, d, J = 7.50 Hz, H-10), 7.71 (1H, d, J = 7.50 Hz,
H-13), 7.97 (1H, d, J = 7.50 Hz, H-22), 8.00 (1H, d,
J = 7.50 Hz, H-19), 14.21 (1H, b, =NH), 14.48 (1H, b,
–OH); 13C NMR (100 MHz, DMSO-d6) d: 104.9 (C, C-5),
108.71 (CH, C-10), 117.26 (CH, C-22), 117.91 (C, C-18),
120.86 (CH, C-20), 122.2 (CH, C-12), 123.27 (C, C-8),
125.18 (CH, C-13), 127.46 (CH, C-11), 128.12 (CH, C-21),
130.82 (CH, C-19), 131.57 (C, C-4), 143.20 (C, C-17),
151.69 (C, C-6), 152.37 (C, C-9), 168 (C, C-2); HR-MS:
355.8024 [M?H]?, calcd. 355.8023. Anal. Calcd. for
C16H10ClN5OS: C, 54.01 %; H, 2.83 %; N, 19.68 %;
Found: C, 54.03 %; H, 2.82 %; N, 19.67 %.
Preparation of 4-imino-3-(20-nitrophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4j)
Red solid crystals, (81 %), mp: 264–265 °C. IR (cm-1
max: 3260 (–OH), 3150 (=NH), 3076 (Ar–H), 2935 (Ali-
)
t
phatic C–H), 1641 (C=N), 1506–1453 (N=N), 1515, 1337
(NO2); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.90 (2H,
s, –NH2), 7.35 (1H, t, J = 7.73 Hz, H-12), 7.43 (1H, t,
J = 7.73 Hz, H-11), 7.58 (2H, t, J = 7.73 Hz, H-21,
H-20), 7.86 (1H, d, J = 7.32 Hz, H-13), 7.97 (1H, d,
J = 7.32 Hz, H-10), 8.03 (1H, d, J = 7.32 Hz, H-22), 8.27
(1H, d, J = 7.32 Hz, H-19); 13C NMR (100 MHz, DMSO-
d6) d: 109.86 (CH, C-9), 116.68 (C, C-12), 119.98 (C, C-5),
121.53 (CH, C-6), 122.98 (CH, C-20), 124.71 (CH, C-7),
125.3 (CH, C-22), 125.88 (CH, C-19), 128.62 (CH, C-8),
133.3 (CH, C-21), 136.98 (C, C-18), 143.7 (C, C-4), 143.79
(C, C-17), 147.67 (C, C-11), 162.83 (C, C-2), 175.22 (C,
C-13); HR-MS: 366.3552 [M?H]?, calcd. 366.3551. Anal.
Calcd. for C16H10N6O3S: C, 52.45 %; H, 2.75 %; N,
22.94 %; Found: C, 52.44 %; H, 2.76 %; N, 22.95 %.
Preparation of 4-imino-3-(20-methylphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4m)
Light red solid crystals, (79 %), mp: 222–224 °C. IR (cm-1
max: 3228(–OH), 3120(=NH), 3037(Ar–H), 2974(Aliphatic
)
t
C–H), 1661 (C=N), 1497–1453 (N=N); 1H NMR (400 MHz,
DMSO-d6) d (ppm): 2.40 (3H, s, –CH3), 7.31 (1H, t,
J = 7.22 Hz, H-12), 7.35 (1H, t, J = 7.22 Hz, H-11), 7.40
(1H, t, J = 6.5 Hz, H-20), 7.54 (1H, t, J = 6.5 Hz, H-21),
7.58 (1H, d, J = 7.22 Hz, H-13), 7.97 (1H, d, J = 7.21 Hz,
H-10), 7.99 (1H, d, J = 7.22 Hz, H-22), 14.21 (1H, b, 1H,
=NH), 14.34 (1H, b, –OH); 13C NMR (100 MHz, DMSO-d6)
d: 16.7 (CH3, C-24), 104.9 (C, C-5), 108.71 (CH, C-10),
111.15 (CH, C-22), 121.22 (CH, C-20), 122.2 (CH, C-12),
123.27 (C, C-8), 125.18 (CH, C-13), 127.07 (CH, C-21),
127.46 (CH, C-11), 128.78 (C, C-18), 131.57 (C, C-4), 132.07
(C, C-19), 150.32 (C, C-17), 152.17 (C, C-6), 152.37 (C, C-9),
168 (C, C-2); HR-MS: 335.3842 [M?H]?, calcd. 335.3841.
Anal. Calcd. for C17H13N5OS: C, 60.88 %; H, 3.91 %; N,
20.88 %; Found: C, 60.87 %; H, 3.92 %; N, 20.87 %.
Preparation of 4-imino-3-(20-methoxyphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4k)
Dark red solid crystals, (81 %), mp: 163–165 °C. IR
(cm-1) tmax: 3210 (–OH); 3132 (=NH), 3042 (Ar–H), 2963
(Aliphatic C–H), 1651 (C=N), 1506–1454 (N=N); 1H NMR
(400 MHz, DMSO-d6) d (ppm): 3.98 (3H, s, –OCH3), 7.05
(1H, t, J = 7.66 Hz, H-12), 7.17 (1H, t, J = 7.66 Hz,
H-11), 7.19 (1H, t, J = 7.66 Hz, H-20), 7.40 (1H, t,
J = 7.95 Hz, H-21), 7.63 (1H, d, J = 7.66 Hz, H-13),7.67
(1H, d, J = 7.66 Hz, H-10), 7.98 (1H, d, J = 7.65 Hz,
H-19, H-22), 14.12 (1H, b, =NH), 14.25 (1H, b, –OH); 13
C
NMR (100 MHz, DMSO-d6) d: 56.1 (CH3, C-25), 104.9
(C, C-5), 108.71 (CH, C-10), 112.58 (CH, C-19), 117.72
(CH, C-22), 121.96 (CH, C-20), 122.2 (CH, C-12), 123.27
(C, C-8), 124.68 (CH, C-21), 125.18 (CH, C-13), 127.46
(CH, C-11), 131.57 (C, C-4), 138.03 (C, C-17), 150.91 (C,
Antimicrobial evaluation
Newly synthesized compounds 4(a–m) were individually
tested against a panel of Gram-positive and Gram-negative
bacterial pathogens, yeast, and fungi. The antimicrobial
123

Med Chem Res
activity of synthesized compounds was evaluated by the
agar well-diffusion method (Yavuz and Yildirim, 2013;
Scott, 1989) using 100 lL of suspension containing
1 9 106 CFU/mL of pathological tested bacteria and
1 9 106/mL of yeast spread on nutrient agar (NA) and
Sabourand dextrose agar (SDA) respectively. After the
media had cooled and solidified, wells (10 mm in diameter)
were made in the solidified agar and loaded with 100 lL of
tested compound solution prepared by dissolving 100 mg
of the chemical compound in 1 mL of DMSO. The incul-
cated plates were then incubated for 24 h at 37 °C for
bacteria and 48 h at 28 °C for fungi. Negative controls
were prepared using dimethyl sulphoxide employed for
dissolving the tested compound. Ciprofloxacin (50 lg/mL)
and Ketoconazole (50 lg/mL) were used as standard for
antibacterial and antifungal activity, respectively. After
incubation time, antimicrobial activity was evaluated by
measuring the zone of inhibition against the test organisms
and compared with that of the standard. Antimicrobial
activities were expressed as inhibition diameter zones in
millimeters (mm). The experiment was carried out in
triplicate and the average zone of inhibition was calculated.
compounds were evaluated and compared with standard
drugs. The results revealed that the most of the compounds
exhibited good levels of antibacterial activity against
Gram-positive bacteria, Gram-negative bacteria, as well as
fungi. In particular, compounds 4d and 4h showed excel-
lent levels of antimicrobial activity with MIC values of
8.25 lg/mL. Many of the synthesized motifs, possessing
electron withdrawing atom/group such as chlorine and
nitro were identified as the most potent antimicrobial
activity.
The mechanism of action of the compounds tested in
this study currently remains unknown. Thus, further studies
of related compounds in the context of their structure–
activity relationship, toxicity, and other biological effects
might be helpful in designing new antimicrobials for
therapeutic use.
Acknowledgments The authors are grateful to the Scientific
Research Projects Council of Pamukkale University (PAU.BAP,
2012FBE066, 2011FBE069).
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