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

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 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, 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 μg/mL.

Full text

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 •
I
˙zzet S¸ener
Received: 4 November 2013 / Accepted: 17 February 2014
ÓSpringer 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,
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 lg/mL.
Keywords Solvatochromism Pyrimido benzothiazole 
Diazo-coupling reaction Azo dyes Antimicrobial activity
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
(Karcıet al.,2006,2009;O
¨ztu
¨rk et al.,2012;S¸ ener et al.,
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
DOI 10.1007/s00044-014-0962-8
MEDICINAL
CHEMISTR
Y
RESEARCH

Results and discussion
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.
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
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
-1
assigned to
(C=N). FT-IR spectra of compound (3) did not appear any
band for (C=O) group. The
1
H NMR spectra of compounds
(3) exhibited singlet signal at d4.15 ppm attributed to
(–CH
2
) protons and broad peak at d12.75 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
were only in imino-azo-enol form (T2) in solid state.
1
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).
1
H NMR spectra of dyes did not showed –NH
2
protons
except for 4j.
1
H NMR spectra of 4g,4k,4l, and 4m
showed only =NH proton, but did not showed hydrazo
–NH protons.
1
H NMR spectra of 4e,4f,4h, and 4i showed
only =NH proton, and hydrazo –NH protons.
1
HNMR
spectra of 4j showed only –NH
2
protons. According to the
1
H 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
S
N
NH2+
S
N
N
NH
O
EtO
O
CN
1
150 oC
S
N
NH
NC
O
23
EtO-
Scheme 1 Synthesis of compound (3)
S
N
N
NH
O
3
NH2
+
X
NaNO2 / HCl
S
N
N
NH
OH
NN
X
4 (a-m)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
a; X: H e; X: p-CH3 i; X: m-CH3 m; X: o-CH3
b; X: p-NO2 f; X: m-NO2 j; X: o-NO2
c; X: p-OCH3 g; X: m-OCH3 k; X: o-OCH3
d; X:
p
-Cl h; X: m-Cl l; X: o-Cl
Scheme 2 Synthesis of compounds 4(a–m)
Med Chem Res
123

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-d
6
as showed in Scheme 3. The mass spectra of
dyes have molecular ion peak which corresponds to
molecular weight of respective compounds.
Absorption spectra
The UV–Vis absorption spectra of the dyes 4(a–m) were
recorded over the range of kbetween 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 (k
max
357 nm) to yellow (k
max
592 nm). It was observed that k
max
of p-substitute dyes
4(a,c,d) shifted bathochromically in chloroform with
respect to the k
max
in dimethyl sulfoxide (DMSO) and
dimethyl formamide (DMF) except for 4b. For example,
for the dye 4a k
max
is 385 nm in DMSO, 394 nm in
chloroform
.
For the dye 4d k
max
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.
The effects of the acid and base on the absorption
spectra of the dyes were investigated and these results are
given in Table 2.
S
N
N
NH
OH
NN
X
imino-azo-enol
T2
X
S
N
N
NH
O
NN
X
S
N
N
NH
O
NN
H
X
S
N
N
O
NN
amino-azo-keto
T1
NH2
KT
KT
KT
imino-hydrazo-keto
T3
imino-azo-keto
T4
Scheme 3 The tautomeric form of azo pyrimido[2,1-b][1,3] benzothiazole derivatives
Med Chem Res
123

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 k
max
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.
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 3revealed 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
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.
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).
Table 1 Influence of solvent on k
max
(nm) of dyes
Dye no. DMSO DMF Acetonitrile Methanol Acetic acid Chloroform
4a 385,325
a
387 403 409,331
a
392 394
4b 417,592,332
a
420,590 419,584
a
422,334
a
411,333
a
414,552
a
4c 403,319
a
401 416,341
a
426,338
a
408,325
a
405
4d 391,320
a
393,496
a
404,330
a
413,336
a
396,323
a
396
4e 389,328
a
395 385 398,335
a
381,320
a
385
4f 392,322
a
393 387 389,330
a
383,322
a
387
4g 386,320
a
391 380 390,328
a
378,319
a
,478
a
380
4h 399,333
a
,487
a
396,491
a
403,485
a
407,332
a
392,325 394
4i 387,320
a
390 381 386,330
a
378,318
a
382
4j 365,425 363,416 365,422 359,420 409,345
a
357,414
4k 416,336
a
413 420 420,338 410,328
a
414
4l 405,327
a
405,491
a
405 405,334
a
389,328
a
401
4m 408,330
a
407 411 414,341
a
400,324
a
405
a
Shoulder
Med Chem Res
123

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
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 kbetween 300 and 700 nm. The wavelengths of
maximum absorption (k
max
) were investigated in various
solvents such as DMSO, DMF, acetonitrile, methanol,
acetic acid, and chloroform at various concentrations
(1 910
-6
M). Change of (k
max
) 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.
Synthesis
Preparation of 4-imino-3,4-dihydro-2H-pyrimido[2,1-
b][1,3]benzothiazole-2-one (3)
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-
low crystals, yield (90 %), mp: 236–238 °C; IR (cm
-1
)
t
max
: 3375 (OH), 3167–3133 (=NH), 3055 (Ar–H), 2944
(Aliphatic C–H), 1697 (C=N);
1
H NMR (DMSO-d
6
)d
(ppm): 4.15 (s, 2H, CH
2
), 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);
13
C NMR (DMSO-d
6
)d: 40.09 (CH
2
, 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
C
10
H
7
N
3
OS: C, 55.2 %; H, 3.25 %; N, 19.34 %. Found: C,
55.28 %; H, 3.26 %; N, 19.34 %.
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
Wavelen
g
th (nm)
400 500 600 700
Absorbance (Normalized)
0,0
0,2
0,4
0,6
0,8
1,0
DMSO
DMF
Acetonitrile
Methanol
Acetic acid
Chloroform
Fig. 1 Absorption spectra of dye 4b in various solvents
Table 2 Absorption maxima of dyes in acidic and basic solutions
Dye no. Methanol Methanol ?KOH Methanol ?HCl
4a 409,331
a
410 392
4b 422,334
a
408
a
, 542 415
4c 426,338
a
413 415
4d 413,336
a
430 396
4e 398, 335
a
406 388
4f 389,330
a
410 361
a
, 386
4g 390,328
a
416 381
4h 407,332
a
445, 498
a
391
4i 386,330
a
406 385
4j 359,420 365
a
, 466 360
a
, 418
4k 420,338 411 416
4l 405,334
a
424 404
4m 414,341
a
402 408
a
Shoulder
Med Chem Res
123

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
)t
max
: 3385 (–OH), 3167–3132 (=NH), 3039 (Ar–
H), 2924 (Aliphatic C–H), 1641 (C=N), 1510, 1453 (N=N);
1
H NMR (400 MHz, DMSO-d
6
)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),
12.72 (1H, b, hydrazo-NH), 14.22 (s, tautomeric –OH);
13
C
NMR (100 MHz, DMSO-d
6
)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 C
15
H
11
N
5
OS: C, 59.80 %; H,
3.45 %; N, 21.79 %; Found: C, 59.81 %; H, 3.46 %; N,
21.78 %.
The above procedure was also used to synthesize dye
4(b–g). The general route of synthesized dyes is outlined in
Scheme 1.
Preparation of 4-imino-3-(40-nitrophenylazo)-4H-
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
(NO
2
);
1
H NMR (400 MHz, DMSO-d
6
)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).
13
C NMR (100 MHz, DMSO-
d
6
)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 C
16
H
10
N
6
O
3
S:
C, 52.45 %; H, 2.75 %; N, 22.94 %; Found: C, 52.46 %;
H, 2.76 %; N, 22.93 %.
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
)
t
max
: 3251 (–OH), 3128 (=NH), 3028 (Ar–H), 2995 (Ali-
phatic C–H), 1637 (C=N), 1509–1455 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)d(ppm): 3.79 (3H, s, –OCH
3
), 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).
13
CNMR
(100 MHz, DMSO-d
6
)d: 55.46 (CH
3
, 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 C
17
H
13
N
5
O
2
S: C,
58.11 %; H, 3.73 %; N, 19.93 %; Found: C, 58.12 %; H,
32.74 %; N, 19.92 %.
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
)t
max
: 3260 (–OH), 3186 (=NH), 3062 (Ar–H), 2940
(Aliphatic C–H), 1637 (C=N), 1495–1454 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)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,
Wavelength (nm)
400 500 600 700
Absorbance (Normalized)
0,0
0,2
0,4
0,6
0,8
1,0
Methanol
Methanol + HCl
Methanol + KOH
Fig. 2 Absorption spectra of dye 4b in acidic and basic solutions
Med Chem Res
123

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),
12.79 (1H, b, hydrazo-NH), 13.50 (b, tautomeric –OH);
13
C
NMR (100 MHz, DMSO-d
6
)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 C
16
H
10
ClN
5
OS: C, 54.01 %; H, 2.83 %; N, 19.68 %;
Found: C, 54.01 %; H, 2.84 %; N, 19.67 %.
Preparation of 4-imino-3-(40-methylphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4e)
Orange solid crystals, (83 %), mp: 251–252 °C. IR (cm
-1
)
t
max
: 3285 (–OH), 3168 (=NH), 3055 (Ar–H), 2930 (Ali-
phatic C–H), 1683 (C=N), 1428–1487 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)d(ppm): 2.32 (3H, s, –CH
3
), 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,
hydrazo-NH);
13
C NMR (100 MHz, DMSO-d
6
)d: 20.65
(CH
3
, 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 C
17
H
13
N
5
OS: C, 60.88 %; H, 3.91 %; N, 20.88 %;
Found: C, 60.89 %; H, 3.92 %; N, 20.87 %.
Preparation of 4-imino-3-(30-nitrophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4f)
Burgundy red solid crystals, (79 %), mp: 250–251 °C. IR
(cm
-1
)t
max
: 3290 (–OH), 3160 (=NH), 3065 (Ar–H), 2985
(Aliphatic C–H), 1665 (C=N), 1520–1455 (N=N), 1510,
1332 (NO
2
);
1
H NMR (400 MHz, DMSO-d
6
)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);
13
C NMR (100 MHz, DMSO-d
6
)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),
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 19 ±1.7 16 ±1.1 18 ±0.7 14 ±1.2 15 ±1.6
4b 28 ±1.8 32 ±1.3 26 ±0.6 16 ±0.9 14 ±1.4 18 ±1.4
4c 22 ±1.2 22 ±0.8 25 ±1.3 28 ±2.0 16 ±1.1 16 ±1.2
4d 30 ±1.6 32 ±0.9 29 ±0.8 28 ±1.9 29 ±0.3 31 ±0.3
4e 24 ±0.7 22 ±1.5 24 ±1.6 20 ±1.3 16 ±0.6 15 ±2.1
4f 32 ±0.9 28 ±1.4 24 ±1.3 25 ±1.5 21 ±1.1 19 ±1.4
4g 25 ±2.1 27 ±1.5 30 ±0.9 31 ±1.7 24 ±1.4 28 ±1.6
4h 31 ±0.4 28 ±0.9 29 ±0.8 30 ±1.8 23 ±1.2 29 ±0.8
4i 18 ±0.7 16 ±0.3 20 ±1.6 19 ±0.8 12 ±0.5 13 ±0.7
4j 28 ±1.2 30 ±1.3 24 ±1.4 22 ±1.3 18 ±0.7 18 ±1.5
4k 22 ±1.6 24 ±2.4 24 ±1.2 26 ±0.6 29 ±1.2 28 ±0.6
4l 30 ±1.0 24 ±1.8 18 ±0.5 22 ±1.4 19 ±1.6 23 ±1.2
4m 19 ±1.4 18 ±1.4 12 ±1.7 12 ±1.1 12 ±1.4 14 ±2.2
Ciprofloxacin 28 ±1.2 29 ±0.6 29 ±1.3 24 ±0.7 NT NT
Ketoconazole NT NT NT NT 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
Med Chem Res
123

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 C
16
H
10
N
6
O
3
S: C, 52.45 %; H, 2.75 %; N, 22.94 %;
Found: C, 52.43 %; H, 2.75 % N, 22.94 %.
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
)
t
max
: 3311 (–OH), 3175 (=NH), 3062 (Ar–H), 2993 (Ali-
phatic C–H), 1644 (C=N), 1514–1454 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)d(ppm): 3.82 (3H, s, –OCH
3
), 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);
13
C NMR (100 MHz,
DMSO-d
6
)d: 55.71 (CH
3
, 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 C
17
H
13
N
5
O
2
S:
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-chlorophenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4h)
Light orange solid crystals, (84 %), mp: 215–217 °C. IR
(cm
-1
)t
max
: 3212 (–OH), 3074 (=NH), 3006 (Ar–H), 2922
(Aliphatic C–H), 1639 (C=N), 1515–1456 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)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,);
13
CNMR
(100 MHz, DMSO-d
6
)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 C
16
H
10
ClN
5
OS:
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-methylphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4i)
Pink solid crystals, (81 %), mp: 242–243 °C. IR (cm
-1
)
t
max
: 3221 (–OH), 3168 (=NH), 3035 (Ar–H), 2960 (Ali-
phatic C–H), 1683 (C=N), 1508–1489 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)d(ppm): 2.38 (3H, s, –CH
3
), 7.01
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 Gram-negative
S. aureus B. subtilis K. pneumoniae E. coli S. cerevisiae C. albicans
4a 33 66 132 66 132 132
4b 16.5 8.25 33 132 132 66
4c 33 33 33 16.5 132 132
4d 8.25 8.25 8.25 16.5 8.25 8.25
4e 33 66 33 66 132 132
4f 8.25 16.5 33 33 66 66
4g 33 16.5 8.25 8.25 33 16.5
4h 8.25 16.5 8.25 8.25 33 8.25
4i 66 132 66 66 132 132
4j 16.5 8.25 33 33 132 132
4k 33 33 33 16.5 16.5 16.5
4l 8.25 33 66 33 66 33
4m 66 66 132 132 132 132
Ciprofloxacin 8.25 8.25 8.25 16.5 NT NT
Ketoconazole NT NT NT NT 8.25 16.5
NT not tested
Med Chem Res
123

(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);
13
C NMR (100 MHz, DMSO-d
6
)d:20.94
(CH
3
, 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 C
17
H
13
N
5
OS: C, 60.88 %; H, 3.91 %; N, 20.88 %;
Found: C, 60.88 %; H, 3.93 %; N, 20.89 %.
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
)
t
max
: 3260 (–OH), 3150 (=NH), 3076 (Ar–H), 2935 (Ali-
phatic C–H), 1641 (C=N), 1506–1453 (N=N), 1515, 1337
(NO
2
);
1
H NMR (400 MHz, DMSO-d
6
)d(ppm): 2.90 (2H,
s, –NH
2
), 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);
13
C NMR (100 MHz, DMSO-
d
6
)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 C
16
H
10
N
6
O
3
S: 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-methoxyphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4k)
Dark red solid crystals, (81 %), mp: 163–165 °C. IR
(cm
-1
)t
max
: 3210 (–OH); 3132 (=NH), 3042 (Ar–H), 2963
(Aliphatic C–H), 1651 (C=N), 1506–1454 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)d(ppm): 3.98 (3H, s, –OCH
3
), 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-d
6
)d: 56.1 (CH
3
, 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,
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
C
17
H
13
N
5
O
2
S: 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
)
t
max
: 3223 (–OH), 3123 (=NH), 3062 (Ar–H), 2979 (Ali-
phatic C–H), 1644 (C=N), 1500–1452 (N=N);
1
HNMR
(400 MHz, DMSO-d
6
)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);
13
C NMR (100 MHz, DMSO-d
6
)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
C
16
H
10
ClN
5
OS: 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-methylphenylazo)-4H-
pyrimido[2.1-b][1,3]benzothiazole-2-ole (4m)
Light red solid crystals, (79 %), mp: 222–224 °C. IR (cm
-1
)
t
max
: 3228 (–OH), 3120 (=NH), 3037 (Ar–H), 2974 (Aliphatic
C–H), 1661 (C=N), 1497–1453 (N=N);
1
H NMR (400 MHz,
DMSO-d
6
)d(ppm): 2.40 (3H, s, –CH
3
), 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);
13
C NMR (100 MHz, DMSO-d
6
)
d:16.7(CH
3
, 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 C
17
H
13
N
5
OS: C, 60.88 %; H, 3.91 %; N,
20.88 %; Found: C, 60.87 %; H, 3.92 %; N, 20.87 %.
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
Med Chem Res
123

activity of synthesized compounds was evaluated by the
agar well-diffusion method (Yavuz and Yildirim, 2013;
Scott, 1989) using 100 lL of suspension containing
1910
6
CFU/mL of pathological tested bacteria and
1910
6
/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 lLof
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.
Determination of minimal inhibitory concentration
(MIC)
MIC is the lowest concentration of an antimicrobial com-
pound that will inhibit the visible growth of a microorganism
after overnight incubation. The micro dilution susceptibility
test in Mu
¨llereHinton Broth (Oxoid) was used for the
determination of antibacterial activity and Sabouraud Liquid
Medium (Oxoid) was used for the determination of anti-
fungal activity. Stock solutions of the tested compounds,
Ciprofloxacin and Ketoconazole were prepared in DMF at
concentration of 1,000 lg/mL. Twofold serial dilutions of
the tested compounds solutions were prepared using the
proper nutrient broth. The final concentration of the solutions
was 132, 66, 33, 16.5, and 8.25 lg/mL. The tubes were then
inoculated with the test organisms, grown in their suitable
broth at 37 °C for 24 h for bacteria (about 1 910
6
CFU/
mL), each 5 mL received 0.1 mL of the above inoculum and
incubated at 37 °C for 24 h. The lowest concentration
showing no growth was taken as the MIC. Control experi-
ments with DMF and uninoculated media were run parallel
to the test compounds under the same conditions. The MIC
(lg/mL) values are recorded in Table 4.
Conclusion
In summary, based on our previous work, we have syn-
thesized new series of azo pyrimido[2,1-b][1,3]benzothia-
zole derivatives. The antimicrobial activities of these
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).
References
Alaimo RJ (1973) Synthesis of some 4H-pyrimido[2,1-b]ben-
zothiazol-4-ones. J Heterocyclic Chem 10:769–772
Atwal KS, Rovnyak GC, O’Reilly BC, Schwartz J (1989) Substituted
1,4-dihydropyrimidines. 3. Synthesis of selectively functionalized
2-hetero-1,4-dihydropyrimidines. J Org Chem 54:5898–5907
Atwal KS, Swanson BN, Unger SE, Floyd DM, Moreland S, Hedberg
A, O’Reilly BC (1991) ihydropyrimidine calcium channel
blockers. 3. 3-Carbamoyl-4-aryl-1,2,3,4-tetrahydro-6-methyl-5-
pyrimidinecarboxylic acid esters as orally effective antihyper-
tensive agents. J Med Chem 34:806–811
Bartovic A, Ilavski D, Simo O, Zalibera L, Belicova
´A, Seman M
(1995) Synthesis of nitro-substituted 4-oxo-4H-pyrimido[2,1-
b]benzothiazole-3-carboxylic acids and their spectral character-
istics. Collect Czech Chem C 60:583–593
Chaitanya MS, Negendrappa G, Vaidya VP (2010) Synthesis,
biological and pharmacological activities of 2-methyl-4H-py-
rimido[2,1-b][1,3]benzothiazoles. J Chem Pharm Res 2:206–213
Chang S, Shun-Jun J, Yu L (2008) A novel, simple and efficient
synthesis of 3-amino-benzo[d]imidazo[2,1-b]thiazole derivatives
via a multicomponent procedure. J Chin Chem Soc 55:292–296
Ellis GP (2008) Chemistry of heterocyclic compounds: synthesis of
fused heterocycles, vol 47. Wiley, New York
El-Sherbeny MA (2000) Synthesis of certain pyrimido[2,1-b]benzo-
thiazole and benzothiazolo[2,3-b]quinazoline derivatives for
in vitro antitumor and antiviral activities. Arzneimittel-Forsch
50:848–853
Glennon RA, Gaines JJ, Rogers ME (1981) Benz-fused mesoionic
xanthine analogs as inhibitors of cyclic-AMP phosphodiesterase.
J Med Chem 24:766–769
Gupta A, Rawat S (2010) Synthesis and anti-inflammatory study of
novel fluorobenzothiazole derivatives. J Chem Pharm Res
2:244–258
Harikrishnan U, Menon SK (2008) The synthesis, characterization
and spectral properties of crown ether based disazo dyes. Dyes
Pigment 77:462–468
Heravi MM, Ranjbar L, Derikvand F, Alimadadi B, Oskooie HA,
Bamoharram FF (2008) A three component one-pot procedure
Med Chem Res
123

for the synthesis of [1,2,4]triazolo/benzimidazolo-quinazolinone
derivatives in the presence of H6P2W18O62.18H2O as a green
and reusable catalyst. Mol Divers 12:181–185
Kappe CO (1993) 100 years of the Biginelli dihydropyrimidine
synthesis. Tetrahedron 49:6937–6963
KarcıF, Demirc¸alıA, S¸ener I
˙, Tilki T (2006) Synthesis of 4-amino-1H-
benzo[4,5]imidazo[1,2-a]pyrimidin-2-one and its disperse azo
dyes. Part 1. Phenylazo derivatives. Dyes Pigments 71:90–96
KarcıF, S¸ ener N, Yamac¸M,S¸ ener I
˙, Demirc¸alıA (2009) Synthesis,
antimicrobial activity and absorption characteristics of some
novel heterocyclic disazo dyes. Dyes Pigments 80:47–52
Kumar R, Malik S, Chandra R (2009) Synthesis and antimicrobial
activity of 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl]-di-
hydropyridines and 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl]-3,4-dihydropyrimidin-2-ones. Indian J Chem B 48B:
718–724
Lanjewar KR, Rahatgaonkar AM, Chorghade MS, Saraf BD (2009)
Synthesis and antimicrobial activity of 5-(2-aminothiazol-4-yl)-
3,4-dihydro-4-phenylpyrimidin-2(1H)-one. Indian J Chem B
48B:1732–1737
O
¨ztu
¨rk F, Ac¸ıkL,S¸ ener I
˙, KarcıF, Kılıc¸ E (2012) Antimicrobial
properties and DNA interaction studies of 3-hetarylazoquinoline-
2,4-diol compounds. Turk J Chem 36:293–302
Rovnyak GC, Atwal KS, Hedberg A, Kimball SD, Moreland S,
Gougoutas JZ, O’Reillly BC, Schwartz J, Malley MF (1992)
Dihydropyrimidine calcium channel blockers. 4. Basic 3-substi-
tuted-4-aryl-1,4-dihydropyrimidine-5-carboxylic acid esters.
Potent antihypertensive agents. J Med Chem 35:3254–3263
Sahu PK, Sahu PK, Gupta SK, Thavaselvam D, Agarwal DD (2012)
Synthesis and evaluation of antimicrobial activity of 4H-
pyrimido[2,1-b]benzothiazole, pyrazole and benzylidene deriv-
atives of curcumin. Eur J Med Chem 54:366–378
Scott AC (1989) Laboratory control of antimicrobial therapy. In:
Collee JG, Duguid JP, Duguid JP, Fraser AG, Marmion BP (eds)
Mackie and Mac-Cartney practical medical microbiology, vol
13. Churchill Livingstone, Edinburgh, pp 161–181
S¸ ener I
˙, KarcıF, Ertan N, Kılıc¸ E (2006) Synthesis and investigations
of the absorption spectra of hetarylazo disperse dyes derived
from 2,4-quinolinediol. Dyes Pigments 70:143–148
Shah NK, Patel MP, Patel RG (2009) One-pot, multicomponent
condensation reaction in neutral conditions: Synthesis, charac-
terization, and biological studies of fused thiazolo[2,3-b]quinaz-
olinone derivatives. Phosphorus Sulfur 184:2704–2719
Tadakazu T (1991) Synthesis and reactions of [1,3-4]thiadiazole[3,2-
a]pyrimidinone derivatives. J Heterocyclic Chem 28:489–492
Wade JJ, Toso CB, Matson CJ, Stelzer VL (1983) Synthesis and
antiallergic activity of some acidic derivatives of 4H-pyrimi-
do[2,1-b]benzazol-4-ones. J Med Chem 26:608–611
Yavuz S, Yıldırım H (2013) Ferrocene derivatives carrying urea,
thiourea and sulfonamide moieties: synthesis and evaluation of
antibacterial and antifungal activities. J Chem 149693:1–7
Med Chem Res
123