Synthesis and study of the antimicrobial activity of novel tricyclic 2hpyrimido[ 2,1-b]benzothiazoles

Article abstract of DOI:10.2174/157018011796235211

Efficient synthesis of 4H-pyrimido[2,1-b]benzothiazoles by novel one-pot three-component reaction of an aldehyde, β-ketoester and 2-aminobenzothiazole using sulphamic acid as a catalyst is described. All synthesized compounds were evaluated for in vitro a

Full text of DOI:10.2174/157018011796235211

Letters in Drug Design & Discovery, 2011, 8, 633-639
633
Synthesis and Study of the Antimicrobial Activity of Novel Tricyclic 2H-
pyrimido[2,1-b]benzothiazoles
Ravindra M. Kumbhare* and Chinna Nagragu
Fluoroorganic Division, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received April 11, 2011: Revised May 19, 2011: Accepted May 24, 2011
Abstract: Efficient synthesis of 4H-pyrimido[2,1-b]benzothiazoles by novel one-pot three-component reaction of an
aldehyde, ꢀ-ketoester and 2-aminobenzothiazole using sulphamic acid as a catalyst is described. All synthesized com-
pounds were evaluated for in vitro antibacterial activity using Gram-positive bacteria and Gram-negative bacteria (Es-
cherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa , Bacillus subtilis, Proteus Valgaris). In vitro antifungal
activity was also determined against the five fungal species (Aspergillus flavus, Candida albicans Aspergillus fumigatus,
Penicillium mareneffei, Chrysosporium tropicum). Structure of the synthesized compounds was established by elemental
analysis and spectral data.
Keywords: Multicomponent condensation, 2-Aminobenzothiazole, 4H-Pyrimido[2,1-b]benzothiazoles, Sulphamic acid, An-
timicrobial activity, synthesis.
1. INTRODUCTION
was thought worthwhile to synthesize certain novel deriva-
tives of 4H-Pyrimido[2,1-b]benzothiazoles and screen them
for their biological activities particularly for their antibacte-
rial and antifungal activity. Representatives of some biologi-
cally important pyrimidobenzothiazolone compounds have
been illustrated in Fig. (1).
2-Aminobenzothiazole demonstrated interesting pharma-
cological activities including anticonvulsant [1], analgesic
[2], anti-tumor [3, 4], antibacterial [5, 6], antimicrobial [7, 8]
and muscle relaxant agents [9]. Particularly, these are highly
reactive compounds and extensively utilized as reactants or
reaction intermediates since the NH2 and endocyclic N func-
tions are suitably situated to enable reactions with common
bis electrophilic reagents to form a variety of fused hetero-
cyclic compounds [10]. In recent years, several attempts
were made for modifying the benzothiazole nucleus to im-
prove their biological activities. Modifications on the ben-
zothiazole nucleus have resulted in a large number of com-
pounds having diverse pharmacological activities. Among
them, substituted benzothiazole compounds have been
shown to be fungicidal agents [11], while tetrahydro deriva-
tives were tested in cancer chemotherapy [12].
N
S
Ph
N
Tetramisole
Anthelmintic and as
levamisole clinically used
anti-cancer agent
S
S
N
N
N
Thiazole and pyrimidine heterocyclic ring systems are
considered to be the typical privileged structures in pharma-
ceutical research as both the heterocyclic ring systems are
the active core of various bioactive molecules [13-18].
Pyrimidobenzothiazoles have been reported to exhibit a wide
spectrum of pharmacological activities such as antiviral, an-
titumor, anti-inflammatory, analgesic, anticonvulsant, mus-
cle relaxant, sedative etc. [19-22].
N
O
Cl
O
COOEt
OH
Pyrimido[2,1-b]benzothiazoles
Cytotoxic Agonist for benzodiazepine receptor
Fig. (1). Chemical structure of pyrimidobenzoyhiazole derivatives.
Moreover, pyrimido[2,1-b]benzothiazoles were evalu-
ated for their affinity at the central benzodiazepine receptor
[23]. Trapani and co-workers [24-26] have explored the
widespread activities of these compounds displaying anx-
iolytic, anticonvulsant, muscle relaxant and sedative proper-
ties. Hence in continuation of our efforts on the design of
novel antibacterial agents [27,28] and keeping in mind the
medicinal importance of pyrimidobenzothiazolone moiety, it
Methods for preparation of 4H-Pyrimido[2,1-b]benzothi-
azoles derivatives are scarce in literature. Earlier, Shaabani
et al. [29], have reported a multicomponent approach for
their preparation by one pot condensation of an aldehyde 1,
ꢀ-ketoester 2 and 2-aminobenzothiazole 3, using ionic liquid
1,1,3,3-tetramethylguanidinium trifluoroacetate as reaction
medium to obtain 4H-Pyrimido[2,1-b]benzothiazoles 4 in
75-85 % yield. This method has become expensive due to
ionic liquids. Here we report a simple and economical
method for this condensation producing derivatives 4 in im-
proved yields (80-89 %) using sulphamic acid as a catalyst
(Scheme 1).
*Address correspondence to this author at the Division of fluoroorganic,
Indian Institute of Chemical technology, Hyderabad-500607, India; Tel:
+91-040-27191776; Fax: +91-040-27193184;
E-mails: kumbhare@iict.res.in; rakumbhare@yahoo.com
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

634 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7
Kumbhare and Nagragu
COOC2H5
R
H
Ar
O
O
N
S
N
NH2SO3H
80 0C
N
NH2
Ar-CHO
C2H5O
R
S
1
2
3
4 a-o
R= Me, CF3
Ar = C6H5, 4-F-C6H5
4-OMe-C6H5, 4-Me-C6H5,
4-NO2-C6H5, 4-Cl-C6H5,
4-Br-C6H5,4-CN-C6H5, 4-CF3C6H5
Scheme 1.
2. RESULTS AND DISCUSSIONS
Chemistry
with 2-aminobenzothiazole in situ via intermediate 6, fol-
lowed by cyclization through Michael addition to afford 4H-
pyrimido[2,1-b] benzothiazole (Scheme 2) .
In our preliminary experiment we have studied
preparation of 4H-Pyrimido [2,1-b]benzothiazoles by multi-
component condensation using a variety of aryl aldehydes
with a 1,3-diketo-ester and 2-aminobenzothiazole by reflu-
xing in ethanol using sulphamic acid as a catalyst and
obtained the products in the range in 80-89% yield. The elec-
tron- withdrawing or electron-releasing functional groups on
benzaldehydes did not have any influence on the reaction
(Table 1).
3. MICROBIOLOGY
Newly synthesized target compounds 4a-4o were evalu-
ated for their in vitro anti-bacterial activity against various
pathogenic bacterial strains (Gram-negative and Gram-
positive) viz., Escherichia coli (ATCC–25922), Staphylo-
coccus aureus (ATCC–25923), Pseudomonas aeruginosa
(ATCC–27853), Bacillus subtilis (ATCC–6633), Proteus
Valgaris (ATCC–13315). Anti-fungal activity of the above
compounds 4a–o was evaluated against fungal strains viz.
Aspergillus flavus (NCIM No.524), Candida albicans
(NCIM No.3100), Aspergillus fumigatus (NCIM No.902),
Penicillium mareneffei (recultured) and Chrysosporium
tropicum (MTCC 2821). The anti-bacterial and antifungal
activities were evaluated by agar disc diffusion method as
per the guidelines of the National Committee for Clinical
Laboratory Standards (NCCLS, 1997) [30]. The solvent,
The products (entry 4a- 4o) were characterized based on
1
their IR, 13C NMR, H NMR and elemental analysis. The
mass spectra of these compounds displayed molecular ion
peaks at appropriate m/z values. This synthetic strategy is
more attractive than earlier methods due to easily recover-
able and reusable catalyst, easy workup and higher yields of
the product. The plausible reaction mechanism involves con-
densation of aldehyde with ꢀ-ketoester by Knoevenagel reac-
tion produces 3-arylidine-2,4-pentanedione 5, which reacts
Table 1. Sulphamic Acid Catalyzed Synthesis of Tricyclic 4H-pyrimido[2,1-b] benzothiazolesa (4a-o)
Compounds
Ar
Rb
Time (min)
Yield(%)c
4a
4b
4c
4d
4e
4f
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
50
55
45
60
45
70
60
50
45
70
55
60
55
45
55
86
90
83
88
84
90
85
80
83
84
84
82
89
82
87
4-OMe- C6H5
4-Me-C6H5
4-NO2-C6H5
4-F- C6H5
4-Cl- C6H5
C6H5
4g
4h
4i
4-OMe- C6H5
4-Me-C6H5
4-F- C6H5
4j
4k
4l
4-Cl- C6H5
4-Br- C6H5
4- NO2- C6H5
4- CF3 - C6H5
4-CN- C6H5
4m
4n
4o
aReaction was performed at 1mmol scale. bProducts were characterized by NMR,IR and Mass spectroscopy. cYield refers to pure products after column chromatography.

Synthesis and Study of the Antimicrobial Activity
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7 635
H
O
O
O
O
O
C2H5O
R
H2O
S
R
OC2H5
Ph
H
H2N
X
N
5
3
O
Ph
O
OH
OC2H5
R
N
H
S
R
C2H5O
N
S
N
N
Ph
H
6
Scheme 2.
DMSO used for the preparation of compounds did not show
inhibition against the tested organisms.
good activity against p. aeruginosa and B. subtilis, while
compounds 4d, 4j, and 4o showed moderate activity against
few bacterial strains.
3.1. Anti-bacterial Activity
3.2. Anti-Fungal Activity
The results of anti-bacterial screening of all the newly
synthesized compounds are presented in Table 2. Some of
them showed moderate to good activity with MIC value in
the range of 6.25–50 ꢀg/ml in DMSO. Particularly, com-
pound Ethyl-2-trifluoromethyl-4-(4-chlorophenyl)-4H-pyri-
mido[2,1-b][1,3] benzothiazole-3-carboxylate 4k showed
good activity (zone of inhibition 20 and 22 mm) against S.
aureus and P. aeruginosa. Compounds 4h, 4i, 4l showed
The results of anti-fungal screening of all the newly syn-
thesized compounds are presented in Table 3. Some of them
showed good to excellent activity with MIC value in the
range of 6.25–25 ꢀg/ml in DMSO. Compounds ethyl-2-
trifluoromethyl-4-(4-fluorophenyl)-4H-pyrimido[2,1-b][1,3]
benzothiazole-3-carboxylate 4j and Ethyl-2-trifluoromethyl-
4-(4-trifluorophenyl)-4H-pyrimido[2,1-b][1,3]benzothiazole-
Table 2. Antibacterial Activity of Compounds 4a-4o
Compounds
E. coil
S.aureus
P.aeruginosa
B. subtilis
P.valgaris
4a
14 (6.25)
14 (12.5)
12 (6.25)
21 (12.5)
15 (6.25)
16 (6.25)
14 (12.5)
20 (6.25)
23 (6.25)
17 (12.5)
20 (12.5)
20 (6.25)
16 (12.5)
18 (12.5)
17 (6.25)
27 (6.25)
12 (6.25)
14 (50)
14 (12.5)
14 (50)
16 (6.25)
16 (12.5)
12 (25)
16 (12.5)
14 (6.25)
16 (6.25)
16 (6.25)
17 (6.25)
16 (12.5)
16 (6.25)
17 (6.25)
16 (6.25)
16 (12.5)
18 (12.5)
16 (12.5)
18 (12.5)
16 (12.5)
14 (12.5)
22 (6.25)
4b
4c
12 (25)
14 (12.5)
22 (12.5)
18 (6.25)
14 (6.25)
16 (12.5)
20 (6.25)
21 (6.25)
17 (6.25)
22 (6.25)
20 (6.25)
16 (12.5)
17 (12.5)
19 (6.25)
28 (6.25)
4d
19 (6.25)
17 (12.25)
14 (12.5)
14 (25)
19 (12.5)
19 (6.25)
17 (12.5)
16 (12.5)
19 (6.25)
18 (6.25)
18 (6.25)
17 (6.25)
18 (6.25)
18 (6.25)
18 (12.5)
19 (12.5)
24 (6.25)
4e
4f
4g
4h
17 (6.25)
18 (6.25)
20 (12.5)
20 (6.25)
18 (6.25)
19 (12.5)
16 (6.25)
18 (12.5)
24 (6.25)
4i
4j
4k
4l
4m
4n
4o
Ciprofloxacin
MIC values are given in bracket. MIC (ꢀg/ml) = Minimum inhibitory concentration, i.e. the lowest concentration of drug which completely inhibit bacterial growth. Ciprofloxacin
was used as standard for anti-bacterial activity. Diameter of inhibition zone was measured in mm.

636 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7
Kumbhare and Nagragu
C.tropicum
Table 3. Antifungal Activity of Compounds 4a-4o
Compounds
A.flavus
C.albicans
A.fumigatus
P.mareneffei
4a
12 (12.5)
12 (6.25)
14 (6.25)
14 (6.25)
18 (6.25)
17 (6.25)
17 (6.25)
17 (6.25)
19 (12.5)
15 (12.5)
20 (6.25)
18 (6.25)
18 (6.25)
18 (12.5)
17 (6.25)
27 (6.25)
15 (6.25)
14 (25)
14 (12.5)
18 (25)
14 (6.25)
16 (6.25)
18 (6.25)
14 (6.25)
19 (6.25)
18 (12.5)
14 (6.25)
15 (6.25)
20 (6.25)
20 (6.25)
14 (25)
12 (12.5)
14 (12.5)
16 (12.5)
12 (12.5)
17 (6.25)
16 (12.5)
14 (25)
4b
4c
14 (25)
18 (25)
4d
15 (6.25)
20 (6.25)
14 (12.5)
17 (12.5)
18 (6.25)
18 (6.25)
22 (6.25)
20 (6.25)
16 (6.25)
18 (12.5)
20 (6.25)
18 (6.25)
24 (6.25)
18 (12.5)
16 (6.25)
14 (6.25)
18 (6.25)
17 (6.25)
21 (6.25)
21 (6.25)
21 (6.25)
19 (6.25)
18 (12.5)
22 (6.25)
20 (6.25)
28 (6.25)
4e
4f
4g
4h
19 (6.25)
14 (12.5)
16 (6.25)
19 (12.5)
15 (12.5)
18 (12.5)
19 (6.25)
18 (6.25)
22 (6.25)
4i
4j
4k
4l
18 (12.5)
18 (6.25)
18 (6.25)
19 (12.5)
24 (6.25)
4m
4n
4o
Fluconazole
MIC values are given in bracket. MIC (ꢂg/ml) = Minimum inhibitory concentration, i.e. the lowest concentration of drug which completely inhibit fungal growth. Fluconazole was
used as standard for anti-fungal activity. Diameter of inhibition zone was measured in mm.
3-carboxylate 4n, exhibited good activity against the four
fungal strains viz. C. albicans, A.fumigatus, p.mareneffei and
C.tropicum. Compound 4k showed excellent activity against
A.flavus, C. albicans and A. fumigates but no activity against
P.mareneffei . Compound 4i showed good activity against C.
albicans, A.fumigatus, P.mareneffei but no activity against
C.tropicum. Compounds 4g, 4l showed moderate activity
against A.flavus and A.fumigatus but no activity against
C.tropicum. Compounds 4e, 4o showed good activity against
A. flavus, C. albicans, A. fumigates and C.tropicum.
the compounds was checked by thin layer chromatography
(TLC) on Merck silica gel 60 F254 precoated sheets in n-
hexane/ ethyl acetate mixture and spots were developed us-
ing iodine vapor/ultraviolet light as visualizing agent.
4.1. The General Procedure for the Preparation of Com-
pounds 4a-o
A
mixture of ꢀ-ketoester (1 mmol), substituted
aryl aldehyde (1 mmol), 2-aminobenzothiazole (1 mmol) and
sulphamic acid (40mg, 8mol%) in ethanol (10 ml) was re-
fluxed and the progress of the reaction was followed by
TLC. After completion of the reaction, water ( 5 ml) was
added to reaction mixture and extracted with ethyl acetate
(2x15 ml). The organic layer was separated and dried over
Na2SO4, and solvent was removed under reduced pressure.
The crude product were purified by column chromatography
(60-120 mesh) using (ethyl acetate: n-hexane, 30:70) and
obtained 4a-o.
4. EXPERIMENTAL SECTION
All the chemicals and solvents used for this work were
obtained from Merck (Germany), Himedia (Mumbai) and
Aldrich chemical company (U.S.A.). The chemicals pur-
chased were of analytical reagent grade or were purified by
standard methods prior to use. Melting points of the synthe-
sized compounds were determined in open-glass capillaries
on Veego VMP-AM melting point apparatus and are uncor-
rected. IR absorption spectra were recorded on Shimadzu
FTIR-8400s using KBr pellets in the range of 4000–400cm-1;
1H NMR and 13C NMR spectra were recorded on Varian
Gemini 300 MHz spectrometer using tetramethylsilane
(TMS) as an internal standard. Chemical shifts are reported
in parts per million (ppm) down field from tetra methyl si-
lane. Spin multiplicities are described as s (singlet), d (dou-
blet), and m (multiplet). EI mass spectra were recorded on a
VG-7070H Micro mass spectrometer. IR, 1H NMR, 13C
NMR and Mass spectra were consistent with the assigned
structures. Elemental analyses (C, H, N) were done on a
CHN rapid analyzer. All the new compounds gave C, H and
N analysis within ± 0.03% of the theoretical values. Purity of
Ethyl-2-methyl-4-(phenyl)-4H-pyrimido [2, 1-b] [1, 3] ben-
zothiazole-3- carboxylate (4a)
0
Pale yellow solid, M.P. 174-176 C; IR (KBr): 3212,
3041, 1738, 1582, 1453, 1230, 740 cm-1; 1H NMR (300MHz,
CDCl3 + DMSO): ꢁ 1.28 (t, J = 6.58 Hz, 3H), 2.35 (s, 3H),
4.10-4.16 (q, 2H), 6.38 (s, 1H), 7.10-7.24 (m, 6H), 7.40-7.51
(m, 3H); 13C NMR (75 MHz, CDCl3+DMSO): ꢁ 166.37,
163.28, 141.14, 137.85, 128.70, 128.45, 127.20, 126.68,
123.10, 123.91, 122.23, 120.90, 111.90, 103.11, 60.17,
57.83, 23.45, 14.35; EI-MS: m/z 351( M+1); Anal. Calc. for
(C20H18N2O2S) C, 68.55; H, 5.18; N, 7.99; found; C, 68.56;
H, 5.16; N, 7.99.

Synthesis and Study of the Antimicrobial Activity
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7 637
Ethyl-2-methyl-4-(4-methoxyphenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4b)
102.83, 60.35, 57.37, 23.20, 14.37; EI-MS: m/z 385 (M+1);
Anal. Calc. for (C20H17ClN2O2S) C, 62.41; H, 4.45; N, 7.28;
found; C, 62.49; H, 4.43; N, 7.29.
Yellow Powder; M.P. 139-1410C; IR (KBr): 3207, 1742,
1508, 1242, 746 cm-1; 1H NMR (300MHz, CDCl3 + DMSO):
ꢀ 1.27 (t, J=7.34 Hz, 3H), 2.35 (s, 3H), 3.70 (s, 3H), 4.08-
4.17 (q, 2H), 6.35 (s, 1H), 6.74 (d, J= 8.81 Hz, 2H), 7.05-
7.14 (m, 5H), 7.43-7.51 (m, 1H); 13C NMR (75 MHz,
CDCl3+DMSO): ꢀ 164.55, 161.13, 157.39, 152.15, 135.96,
131.68, 126.50, 124.61, 121.93, 121.82, 120.15, 111.89,
109.83, 101.27, 58.03, 55.21, 53.17, 21.55, 12.38; EI-MS:
m/z 381( M+1); Anal. Calc. for (C21H20N2O3S) C, 66.29; H,
5.30; N, 7.36;found; C, 66.30; H, 5.27; N, 7.37.
Ethyl-2-trifluoromethyl-4-(phenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4g)
0
Yellow Powder; M.P.165-167 C; IR (KBr): 3202, 2978,
1737, 1520, 1446, 1335, 1170, 1084, 1080, 830, 746 cm-1;
1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.00 (t, J=7.18 Hz,
3H), 4.05-4.12 (q, 2H), 6.43 (s, 1H), 6.79-6.89 (m, 2H), 6.94
(t, J=7.55 Hz, 1H), 7.18-7.40 (m, 6H); 13C NMR (75 MHz,
CDCl3+DMSO): ꢀ 167.93, 162.54, 137.61, 135.22, 131.78,
128.76, 128.28, 127.70, 126.70, 125.62, 124.59, 121.69,
120.98, 112.29, 109.96, 60.24, 56.51, 12.88; EI-MS: m/z 405
(M+1); Anal. Calc. for (C20H15F3N2O2S) C, 59.40; H, 3.74;
N, 6.93; found; C, 59.39; H, 3.73; N, 6.93.
Ethyl-2-methyl-4-(4-methylphenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4c)
Yellow Powder; M.P. 143-145 0C; IR (KBr): 3208, 2973,
1742, 1603, 1465, 1335, 1235, 1105, 1075, 836, 695 cm-1;
1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.28 (t, J=7.35 Hz,
3H), 2.26 (s, 3H), 3.10 (s, 3H), 4.08-4.15 (q, 2H), 6.31 (s,
1H), 7.00-7.20 (m, 6H), 7.40-7.46 (m, 2H); 13C NMR (75
MHz, CDCl3+DMSO): ꢀ 164.60, 162.14, 157.45, 153.10,
134.98, 132.08, 126.30, 123.63, 121.98, 121.79, 120.45,
111.66, 109.63, 101.37, 58.13, 55.19, 53.27, 15.45, 12.38;
EI-MS: m/z 365 (M+1); Anal. Calc. for (C21H20N2O2S) C,
69.20; H, 5.53; N, 7.69; found; C, 69.21; H, 5.51; N, 7.69.
Ethyl-2-trifluoromethyl-4-(4-methoxyphenyl)-4H-
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (4h)
Yellow Powder; M.P. 170-1720C; IR (KBr): 3198, 2976,
1743, 1518, 1440, 1332, 1169, 1082, 1078, 834, 743 cm-1;
1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.04 (t, J=7.17 Hz,
3H), 3.79 (s, 3H), 3.95-4.05 (q, 2H), 6.15 (s, 1H), 6.70-6.98
(m, 5H), 7.14 (d, J=7.74 Hz, 2H), 7.34 (d, J=7.74 Hz, 1H);
13C NMR (75 MHz, CDCl3+DMSO): ꢀ 167.65, 162.48,
147.10, 138.18, 132.12, 128.95, 128.30, 126.54, 125.90,
124.85, 122.18, 121.45, 120.90, 112.60, 110.12, 60.40,
56.80, 55.12, 12.90; EI-MS: m/z 435 (M+1); Anal. Calc. for
(C21H17F3N2O3S) C, 58.06; H, 3.94; N, 6.45; found; C,
58.05; H, 3.93; N, 6.44.
Ethyl-2-methyl-4-(4-nitrophenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4d)
Pale yellow Powder; M.P.149-1510C; IR (KBr): 3215,
3110, 1740, 1503, 1335, 1230, 750 cm-1; 1H NMR (300MHz,
CDCl3 + DMSO): ꢀ 1.30 (t, J=7.34 Hz, 3H), 2.38 (s, 3H),
4.10-4.16 (q, 2H), 6.55 (s, 1H), 7.10-7.20 (m, 3H), 7.50-7.71
(m, 3H), 8.13 (d, J= 8.81Hz, 2H); 13C NMR (75 MHz,
CDCl3+DMSO): ꢀ 165.95, 163.23, 147.79, 147.35, 137.06,
128.10, 127.23, 124.96, 124.13, 124.09, 122.75, 111.69,
102.33, 60.73, 57.29, 23.05, 14.38; EI-MS: m/z 396 (M+1);
Anal. Calc. for (C20H17N3O4S) C, 60.75; H, 4.33; N, 10.63;
found; C, 60.79; H, 4.31; N, 10.60.
Ethyl-2-trifluoromethyl-4-(4methylphenyl)-4H-
pyrimido[2,1- b][1,3] benzothiazole-3- carboxylate (4i)
Yellow Powder; M.P. 181-1830C; IR (KBr): 3196, 2974,
1740, 1520, 1442, 1330, 1168, 1083, 1076, 834, 740 cm-1;
1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.02 (t, J=7.17 Hz,
3H), 2.40 (s, 3H), 3.95-4.14 (q, 2H), 6.19 (s, 1H), 6.80-7.00
(m, 6H), 7.35 (d, J=7.55 Hz, 2H); 13C NMR (75MHz, CDCl3
+ DMSO): ꢀ 167.62, 162.15, 146.62, 137.51, 131.93, 128.70,
128.14, 126.30, 125.32, 124.41, 121.40, 121.19, 120.73,
112.14, 109.30 59.92, 56.02, 20.07, 12.67; EI-MS: m/z 419
(M+1); Anal. Calc. for (C21H17F3N2O2S) C, 60.28; H, 4.10;
N, 6.69; found;C,60.27; H, 4.11; N, 6.70.
Ethyl-2-methyl-4-(4-fluorophenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4e)
Yellow Powder; M.P. 137-1390C; IR (KBr): 2925, 2854,
1744, 1694, 1585, 1505, 1372, 1241, 1201, 1079, 1011, 846,
1
739 cm-1; H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.27 (t,
Ethyl-2-trifluoromethyl-4-(4-fluorophenyl)-4H-
J=7.34 Hz, 3H), 2.35 (s, 3H), 4.10-4.16 (q, 2H), 6.40 (s, 1H),
6.95 (d, J=8.81 Hz, 2H), 7.15-7.28 (m, 4H), 7.53 (d, J=8.81
Hz, 2H); 13C NMR (75 MHz, CDCl3+DMSO): ꢀ 166.23,
163.15, 139.99, 137.55, 131.92, 128.96, 127.11, 124.65,
123.99, 122.60, 122.48, 111.89, 102.86, 60.39, 57.38, 23.22,
14.40; EI-MS: m/z 369 (M+1); Anal. Calc. for
(C20H17FN2O2S) C, 65.20; H, 4.65; N, 7.60; found; C, 65.21;
H, 4.68; N, 7.58.
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (4j)
0
Yellow Powder; M.P.174-176 C; IR (KBr): 3207, 2982,
2927, 1738, 1579, 1510, 1471, 1340, 1227, 1176, 1085,
1
1018, 850, 751 cm-1; H NMR (300MHz, CDCl3 + DMSO):
ꢀ 1.04 (t, J=6.98 Hz, 3H), 3.96-4.12 (q, 2H), 6.45 (s, 1H),
6.82-7.12 (m, 6H), 7.36 (d, J=7.74 Hz, 1H), 7.65-7.72 (m,
1H); 13C NMR (75 MHz, CDCl3+DMSO): ꢀ 167.98, 163.07,
138.59, 137.54, 131.29, 131.27, 127.64, 127.56, 124.76,
121.89, 121.64, 121.17, 115.56, 112.35, 109.94, 60.46,
55.93, 13.00; EI-MS: m/z 423 (M+1); Anal. Calc. for
(C20H14F4N2O2S) C, 56.87; H, 3.34; N, 6.63; found; C,
56.86; H, 3.33; N, 6.65.
Ethyl-2-methyl-4-(4-chlorophenyl)-4H-pyrimido[2,1-
b][1,3]benzothiazole-3- carboxylate (4f)
Pale yellow Powder; M.P. 133-1350C; IR (KBr): 3195,
2981, 1739, 1600, 1472, 1340, 1228, 1177, 1088, 1015, 841,
1
750 cm-1; H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.27 (t,
Ethyl-2-trifluoromethyl-4-(4-chlorophenyl)-4H-
J=6.98 Hz, 3H), 2.37 (s, 3H), 4.09-4.15 (q, 2H), 6.38 (s, 1H),
7.10-7.20 (m, 4H), 7.35-7.42 (m, 4H); 13C NMR (75 MHz,
CDCl3+DMSO): ꢀ 166.21, 163.13, 139.97, 137.52, 131.90,
128.82, 126.91, 124.51, 123.98, 122.57, 122.45, 111.87,
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (4k)
Yellow Powder; M.P. 167-1690C; IR (KBr): 3187, 2984,
1741, 1522, 1454, 1323, 1175, 1086, 1079, 838, 754 cm-1;

638 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7
Kumbhare and Nagragu
1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.06 (t, J=6.61 Hz,
3H), 3.98-4.05 (q, 2H), 6.39 (s, 1H), 6.79-6.98 (m, 4H),
7.17-7.36 (m, 4H); 13C NMR (75 MHz, CDCl3+DMSO): ꢀ
167.80, 162.45, 138.51, 137.40, 135.76, 134.10, 132.45,
128.53, 127.84, 124.77, 121.89, 121.55, 121.18, 112.21,
109.76, 60.48, 55.87, 12.95; EI-MS: m/z 439(M+1); Anal.
Calc. for (C20H14ClF3N2O2S) C, 54.74; H, 3.22; N, 6.38;
found; C, 54.78; H, 3.20; N, 6.39.
4.1. Experimental Procedure for Antimicrobial Activity
4.1.1. Disc Diffusion Method
The antimicrobial activity of newly synthesized com-
pounds was evaluated according to the guidelines of Na-
tional Committee for Clinical Laboratory Standards
(NCCLS, 1997) using the agar disc diffusion method [30].
Briefly, a 24/48 h-old culture of selected Bacteria/fungi was
mixed with sterile physiological saline (0.85%) and the tur-
bidity was adjusted to the standard inoculum of Mac- Far-
land scale 0.5 [106 colony forming units (CFU) per millili-
ter]. Petri plates containing 20 mL of Mueller Hinton Agar
(MHA, Hi- Media) were used for all the bacteria tested.
Fungi were cultured in Sabouraud’s dextrose agar
(SDA)/potato dextrose agar (PDA) (Hi- Media) and were
purified by single spore isolation technique [31]. The inocu-
lum was spread on the surface of the solidified media and
Whatman no. 1 filter paper discs (6 mm in diameter) im-
pregnated with the test compound (20 ꢁl/disc) were placed
on the plates. Ciprofloxacin (5 ꢁg/disc, Hi-Media) was used
as positive control for bacteria. Fluconazole (10 ꢁg/disc, Hi-
Media), was used as positive control for fungi. A paper disc
impregnated with dimethylsulfoxide (DMSO) was used as
negative control. Plates inoculated with the bacteria were
Ethyl-4-trifluoromethyl-2-(4-bromophenyl)-4H-
pyrimido[2,1-b][1,3]benzothiazole-3- carboxylate (4l)
Pale yellowish Powder; M.P.168-1700C; IR (KBr): 3186,
2982, 1740, 1520, 1456, 1324, 1176, 1085, 1078, 836, 756
cm-1; 1H NMR (300MHz, CDCl3 + DMSO): ꢀ 1.06 (t, J=7.18
Hz, 3H), 3.98-4.13 (q, 2H), 6.45 (s, 1H), 6.95-7.05 (m, 2H),
7.18-7.29 (m, 4H), 7.41 (d, J=8.31 Hz, 2H); 13C NMR
(75MHz, CDCl3 + DMSO): ꢀ 167.85, 160.67, 136.35,
134.69, 131.52, 130.82, 128.52, 127.59, 125.47, 124.80,
121.92, 121.60, 121.22, 112.23, 109.70, 60.53, 55.97, 12.97;
EI-MS: 484 m/z (M+1); Anal. Calc. for (C20H14BrF3N2O2S)
C, 49.70; H, 2.92; N, 5.80 found; C, 49.71; H, 2.89; 5.79.
Ethyl-2-trifluoromethyl-4-(4-nitrophenyl)-4H-
pyrimido[2,1-b][1,3] benzothiazole-3-carboxylate (4m)
Yellow Powder; M.P.124-1260C; 1H NMR (300MHz,
CDCl3 + DMSO) ꢀ: 1.08 (t, J=6.98 Hz, 3H), 3.90-4.10 (q,
2H), 6.48 (s, 1H), 6.82-7.15 (m, 6H), 7.36 (d, J=7.74 Hz,
1H), 7.60-7.72 (m, 1H); 13C NMR (75 MHz, CDCl3+DMSO)
ꢀ: 168.98, 164.07, 136.59, 137.54, 131.29, 131.27, 127.64,
127.56, 124.76, 121.89, 121.64, 121.17, 115.56, 112.35,
109.94, 60.46, 55.93, 13.00; IR (KBr)ꢀ: 3200, 2982, 2937,
1735, 1579, 1514, 1471, 1340, 1280, 1236, 1085, 1018, 850,
751 cm-1; EI-MS m/z 450 (M+1); Anal. Calc. for
(C20H14F3N3O4S) C, 53.45; H, 3.14; N, 9.35; found; C,
53.46; H, 3.10; N, 9.33.
0
incubated for 24 h at 37 C and the fungal culture was incu-
bated for 72 h at 250C. The inhibition zone diameters were
measured in millimeters. All the tests were performed in
triplicate and the average was taken as final reading.
4.1.2. Determination of MIC
Minimum inhibitory concentration (MIC) of any com-
pound is defined as the lowest concentration which com-
pletely inhibits visible growth (turbidity on liquid media).
MIC values were determined by testing performed according
to the guidelines of NCCLS document M27-A [30]. Solu-
tions of the test compounds, ciprofloxacin and fluconazole
were prepared in DMSO at a concentration of 100 ꢁg/ml.
From this stock solution, serial dilutions of the compounds
(50, 25… 3.12 ꢁg/ml) were prepared to determine the MIC.
All determinations were done in triplicates and the average
was taken as final reading. The standard antibiotic, cipro-
floxacin (100 ꢁg/ml) for bacteria and Fluconazole (100
ꢁg/ml) for fungi were used as positive controls and 100 ꢁl of
DMSO used as a negative control. At the end of the incuba-
tion period, the MIC values were determined.
Ethyl-2-trifluoromethyl-4-(4-trifluorophenyl)-4H-
pyrimido[2,1-b][1,3]benzothiazole- 3-carboxylate (4n)
1
Yellow Powder; M.P. 169-11720C; H NMR (300MHz,
CDCl3 + DMSO) ꢀ: 1.08 (t, J=6.61 Hz, 3H), 4.00-4.08 (q,
2H), 6.39 (s, 1H), 6.79-6.98 (m, 4H), 7.20-7.36 (m, 4H); 13
C
NMR (75 MHz, CDCl3+DMSO) ꢀ: 167.80, 162.45, 138.51,
137.40, 135.76, 134.10, 132.45, 128.53, 127.84, 125.45,
124.77, 121.89, 121.55, 121.18, 112.21, 109.76, 60.48,
55.87, 12.95; IR (KBr) ꢀ: 3180, 2984, 1741, 1522, 1404,
1323, 1240, 1086, 1079, 838, 754 cm-1; EI-MS m/z 473
(M+1); Anal. Calc. for (C21H14F6N2O2S) C, 53.39; H, 2.99;
N, 5.93; found; C, 53.40; H, 2.96; N, 5.90.
5. CONCLUSION
In conclusion, we have developed a novel and efficient
three-components condensation reaction of an aldehyde, ꢂ-
ketoester and 2-aminobenzothiazole producing 4H-
pyrimido[2,1-b]benzothiazoles ring systems. The results of
anti-bacterial screening reveal that among all the compounds
screened, compounds 4d, 4j,4k,4l showed good inhibition
toward all the bacteria tested. The structure activity relation-
ship studies (SAR) reveal that the compounds with CF3 sub-
stitution (4j,4k,4l) are very much active. The presence of
NO2 substituted 4H-pyrimido[2,1-b]benzothiazole derivative
4d also showed very good antibacterial activity against
S.aureus. Since almost all the compounds are effective.
Ethyl-2-trifluoromethyl-4-(4-cynophenyl)-4H-
pyrimido[2,1-b][1,3]benzothiazole-3-carboxylate (4o)
Pale yellowish Powder; M.P.132-1360C; 1H NMR
(300MHz, CDCl3 + DMSO) ꢀ: 1.10 (t, J=7.18 Hz, 3H), 3.96-
4.12 (q, 2H), 6.45 (s, 1H), 6.95-7.05 (m, 2H), 7.24-7.38 (m,
4H), 7.46 (d, J=8.31 Hz, 2H); 13C NMR (75MHz, CDCl3 +
DMSO) ꢀ: 167.85, 160.67, 136.35, 134.69, 131.52, 130.82,
128.52, 127.59, 125.47, 124.80, 121.92, 121.60, 121.22,
120.40, 112.23, 109.70, 60.53, 55.97, 12.97; IR (KBr)ꢀ:
3179, 2980, 1740, 1520, 1456, 1326, 1176, 1074, 1076, cm-1;
EI-MS m/z 430 (M+1); Anal. Calc. for (C21H14F3N3O2S) C,
58.74; H, 3.29; N, 9.79 found; C, 58.71; H, 3.28; 9.76.

Synthesis and Study of the Antimicrobial Activity
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7 639
2-Amino-benzothiazoles. Their Fungicidal Acitivity. Heterocycles,
1997, 45, 1579-1588.
In case of Antifungal activities C. albicans, A. fumigates,
and C. tropicum the compounds showing significant to ex-
cellent activity are 4j,4n and 4k. In the case of P. mareneffei,
all the compounds posses moderate to good inhibition with
reference to control. The structure activity relationship stud-
ied (SAR) revealed that 2-CF3 group of 4H-pyrimido[2,1-
b]benzothiazole 4g,4j,4k,4l,4n,4n at 2-CF3 group of 4H-
pyrimido[2,1-b]benzothiazole very much effectiveness
against these fungi. CF3 substituted 4H-pyrimido [2, 1-b]
benzothiazoles 4gꢀ4o (Table 1) have shown better
antibacterial and antifungal activity than corresponding non-
fluoro derivatives. In general, the antifungal activity of
compounds was better than their antibacterial activity.
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ACKNOWLEDGEMENTS
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We are thankful to Dr. J. S. Yadav, Director, IICT for
providing facilities and one of us (R.M.K) thanks S.E.R.C.,
Department of Science & Technology, Government of India
for financial assistance under the Fast Track Scheme for
young scientists (SR/FTP/CS-93/2006). Authors are thankful
to the Professor M. B. Patil and Dr. Harit Jha, Department of
Biochemistry, Nagpur University, Nagpur for determining
biological activity of compounds.
Cho, H.; Ueda, M.; Shima, K.; Mizuno, A.; Hayashimatsu, M.;
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