A facile green synthesis and in vitro antimicrobial activity 4H-pyrimido[2,1-b][1,3]benzothiazole derivatives using aluminum trichloride under solvent free conditions

Article abstract of DOI:10.1007/s00044-011-9908-6

Aluminum trichloride acts as readily available, inexpensive, and efficient catalyst for one-pot three-component condensation reaction of aldehydes, dicarbonyl, and 2-Amino benzothiazole under the solvent-free conditions to afford the 4H-pyrimido[2,1-b][1,

Full text of DOI:10.1007/s00044-011-9908-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3826–3834
DOI 10.1007/s00044-011-9908-6
ORIGINAL RESEARCH
A facile green synthesis and in vitro antimicrobial activity
4H-pyrimido[2,1-b][1,3]benzothiazole derivatives using
aluminum trichloride under solvent free conditions
•
Pramod K. Sahu Praveen K. Sahu
•
•
Jaggi Lal D. Thavaselvam D. D. Agarwal
•
Received: 13 June 2011 / Accepted: 16 November 2011 / Published online: 9 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Aluminum trichloride acts as readily available,
inexpensive, and efficient catalyst for one-pot three-com-
ponent condensation reaction of aldehydes, dicarbonyl, and
2-amino benzothiazole under the solvent-free conditions to
afford the 4H-pyrimido[2,1-b][1,3]benzothiazole deriva-
tives 4 with good yield. The compounds synthesized in this
study were evaluated for their antibacterial activities against
gram-positive and gram-negative bacteria, viz., Staphylo-
coccus aureus, Pseudomonas aeruginosa, Salmonella typhi,
Escherichia coli, Bacillus cereus, and Providencia rettegeri.
Compounds 4c, 4d, 4f, 4g, and 4h showed their good activ-
ities against tested bacterial species. Pyrimidine derivatives
4d, 4f, and 4g have shown good antifungal activities against
tested fungal strains, such as Aspergillus niger, Aspergillus
fumigates, Aspergillus flavus, etc.
Introduction
Ability for designing new and synthesis of potent, selective,
and lesstoxic anti-bacterial agents remains amajor challenge
for medicinal chemistry researchers. Pharmacologically,
pyrimidines have attracted considerable interest because of
their wide range of biological activities such as antihyper-
tensive activities (Atwal et al., 1991 and Rovnyak et al.,
1992), anti-tuberculosis (Khoshneviszadeh et al., 2009),
antimicrobial (Chitra et al., 2010), antifilarial activities
(Singh et al., 2008). Thiazoles are an important class of
pharmaceutical compounds which exhibit wide spectra of
biological activities (Vicini et al., 2003). Various substituted
thiazoles have been synthesized and examined for their
antifungal and antibacterial activities (Lanjewar et al., 2009;
Bansal, 2003, and Miwatashi et al., 2008). Multicomponent
reactions (MCRs) (Kappe, 2000, 2002; Zhu and Bienayme,
2005; Ramon and Yus, 2005, and Dallinger and Kappe,
2007) have attracted a large amount of attention of synthetic
organic chemists. Simple procedure, high bond-forming
efficiency, time and energy savings, and low expenditures
are among the advantages of these reactions. The use of
MCRs is the need of present century and is primarily driven
by pharmaceutical industries.
Keywords Antimicrobial activity Á Green synthesis Á
4H-pyrimido[2,1-b][1,3]benzothiazole derivatives Á
AlCl3
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-011-9908-6) contains supplementary
material, which is available to authorized users.
Shaabani et al. have synthesized 4H-pyrimido[2,1-b]
benzazoles using ionic liquid at 100°C in 5 h (Shaabani
et al., 2005). Ionic liquids suffer from limitations because
of poor solubility, toxicity to aquatic organism, as well as
being unable to be separated by distillation (Matzke et al.,
2007; Kralisch et al., 2005; Pham et al., 2010). The liter-
ature search shows that 2-amino benzothiazole containing
pyrimidine synthesis exhibits poor yield and long reaction
time, and hence, there is a need of rapid and higher
yielding process. The present article reports a rapid, effi-
cient, step with economic and green synthesis of biological
P. K. Sahu (&) Á J. Lal
Department of Industrial Chemistry, Jiwaji University, Gwalior
475011, Madhya Pradesh, India
e-mail: sahu.chemistry@gmail.com
P. K. Sahu Á D. D. Agarwal
Jagdishprasad Jhabarmal Tibrewala University, Rajasthan, India
D. Thavaselvam
Microbiology Division, Defence Research Development
Establishment, Jhansi Road, Gwalior 474002, India
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Med Chem Res (2012) 21:3826–3834
3827
active 4H-pyrimido[2,1-b]benzazoles derivatives by three-
component reaction of ethyl acetoacetate, 2-amino benzo-
thiazole, and aldehydes under solvent-free conditions,
using AlCl3 as a catalyst target to green approach.
3043 (C–Hstr), 2968 (C–Hstr in CH2CH3), 1670 (C=Ostr),
1589 (C=Nstr), 1462 (C=Cstr), 744 (C–Hdef); 1H NMR
(400 MHz, CDCl3): dH 1.27 (3H, t, JHH = 14.24 Hz,
CH2CH3), 2.46 (3H, s, CH3), 4.11–4.21 (2H, m, CH2CH3),
6.39 (1H, s, –CH), 7.07–7.43 (9H, m, ArH); 13C NMR
(100 MHz, DMSO): 165.44, 162.59, 154.04, 141.26,
137.43, 128.26, 126.79, 122.22, 111.65, 102.56, 59.35,
56.82, 23.17, 13.99; ESI–MS: m/z Calculated for
C20H18N2O2S 350.44, Found [M ? H]? 351.2.
Materials and methods
Experimental
The 1H NMR spectra were measured using BRUKER
AVANCE II 400 NMR spectrometer with tetramethylsil-
Ethyl-2-methyl-4-(4-hydroxy-3-methoxy phenyl)-4H-
pyrimido[2,1-b][1,3]benzothiazole-3-carboxylate (4b)
1
ane as an internal standard at 20–25°C; data for H NMR
are reported as follows: chemical shift (ppm), integration,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet, and br, broad), coupling constant (Hz). IR
spectra were recorded by SHIMADZU; IR spectrometer of
sample dispersed in KBr pellet or Nujol is reported in terms
of frequency of absorption (cm-1). E-Merck pre-coated
TLC plates, RANKEM silica gel G for preparative thin-
layer chromatography were used. Melting points were
determined in open capillaries and are uncorrected. Brain
heart infusionTM, Mueller–Hinton agar, Sabouraud dex-
trose agar, and Sabouraud dextrose broth were purchased
from OXOID LTD., Basingstoke, Hampshire, England. 96
Microwell with lid was purchased from IWAKI Brand
SCITECH DIV. Asahi Techno Glass, Japan. Ethyl aceto-
acetate, aldehydes, and aluminum trichloride were pur-
chased from Himedia Laboratory Ltd., Mumbai, India.
2-amino benzothiazole was purchased from Sigma Aldrich.
Reaction was carried out according to typical procedure
with vanillin (0.005), ethyl acetoacetate (0.005), and
2-amino benzothiazole (0.005) to give compound 4b. Pale-
yellow powder, mp 192–194°C, Rf = 0.53 (DCM:Toluene;
3:2); IR (KBr) (tmax, cm-1): 3059 (C–Hstr), 2983 (C–Hstr in
CH2CH3), 1703 (C=Ostr), 1597 (C=Nstr), 1504 (C=Cstr),
740 (C–Hdef); 1H NMR (400 MHz, CDCl3): dH 1.29 (3H, t,
JHH = 14.24 Hz, CH2CH3), 2.47 (3H, s, CH3), 3.8 (3H, s,
Ar–OCH3), 4.13–4.22 (2H, m, CH2CH3), 6.36 (1H, s,
–CH), 6.78 (1H, d, JHH = 5.6 Hz, ArH), 6.89–6.92 (2H, m,
ArH), 7.13–7.59 (4H, m, ArH), 9.82 (1H, s, OH); 13C NMR
(100 MHz, DMSO): 165.60, 162.34, 153.51, 147.12,
146.42, 137.61, 132.52, 126.37, 122.21, 115.18, 111.92,
110.88, 59.28, 55.43, 23.09, 14.08; ESI–MS: m/z Calcu-
lated for C21H20N2O4S 396.49, Found [M ? H]? 397.2.
Ethyl-2-methyl-4-(4-dimethylamino phenyl)-4H-
pyrimido[2,1-b][1,3]benzothiazole-3-carboxylate (4c)
One-pot three-component reaction
Reaction was carried out according to typical procedure
with p-dimethylamino benzaldehyde (0.005 mol), ethyl
acetoacetate (0.005 mol), and 2-amino benzothiazole
(0.005 mol) to give compound 4c. Pale-yellow powder, mp
175–178°C, Rf = 0.57 (DCM:Toluene; 3:2); IR (KBr)
(tmax, cm-1): 3059 (C–Hstr), 2897 (C–Hstr in CH2CH3),
1612 (C=Ostr), 1581 (C=Nstr), 1431 (C=Cstr), 815–754
Typical procedure A mixture of aldehydes (0.005 mol),
ethyl acetoacetate (0.005 mol), and 2-amino benzothiazole
(0.005 mol) were heated at 60–70°C under solvent-free
conditions using aluminum chloride as
a catalyst
(10 mol%). The reaction was monitored by TLC. After
completion of the reaction, the reaction mixture was cooled
to room temperature, and the content was transferred into
water with the help of methanol and filtered to obtain the
solid mass. The crude product was purified by column
chromatography with chloroform and methanol (1:1). Pure
compounds 4 were collected as pale yellow colored solid.
1
(C–Hdef); H NMR (400 MHz, CDCl3): dH 1.29 (3H, t,
JHH = 14.14 Hz, CH2CH3), 2.46 (3H, s, CH3), 3.06 (6H, s,
N(CH3)2), 4.12–422 (2H, m, CH2CH3), 6.66–7.92 (8H, m,
ArH); 13C NMR (100 MHz, DMSO): 189.35, 166.39,
165.44, 153.95, 152.63, 133.43, 127.71, 125.91, 124.45,
121.63, 120.57, 111.53, 111.29, 110.74, 23.13, 13.60; ESI–
MS: m/z Calculated for C22H23N3O2S 393.54, Found
[M ? H]? 394.2.
Ethyl-2-methyl-4-(phenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (4a)
Reaction was carried out according to typical procedure
with benzaldehyde (0.005 mol), ethyl acetoacetate
(0.005 mol), and 2-amino benzothiazole (0.005 mol) to
give compound 4a. Pale-yellow crystals, mp 178–180°C,
Rf = 0.47 (DCM:Toluene; 3:2); IR (KBr) (tmax, cm-1):
Ethyl-2-methyl-4-(4-nitro phenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (4d)
Reaction was carried out according to typical procedure
with p-nitro benzaldehyde (0.005 mol), ethyl acetoacetate
123

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Med Chem Res (2012) 21:3826–3834
Ethyl-2-methyl-4-(4-methoxy phenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (4g)
(0.005 mol), and 2-amino benzothiazole (0.005 mol) to
give compound 4d. Yellow powder, mp 170–172°C,
Rf = 0.52 (DCM:Toluene; 3:2); IR (KBr) (tmax, cm-1):
3348 (C–Hstr), 2933 (C–Hstr in CH2CH3), 1625 (C=Ostr),
1510 (C=Nstr), 1267 (C=Cstr), 962–812 (C–Hdef); 1H NMR
(400 MHz, CDCl3): dH 1.31 (3H, s, CH2CH3), 2.46 (3H, s,
CH3), 4.18–4.21 (2H, m, CH2CH3), 6.52 (1H, s, –CH),
7.03–8.12 (8H, m, ArH); 13C NMR (100 MHz, CDCl3):
166.25, 163.56, 155.83, 147.95, 147.62, 137.44, 128.06,
126.88, 124.43, 124.01, 123.73, 122.47, 111.36, 102.01,
60.42, 57.03, 23.97, 14.40; ESI–MS: m/z Calculated for
C20H17N3O4S 395.46, Found [M ? H]? 396.4.
Reaction was carried out according to typical procedure
with anisaldehyde (0.005 mol), ethyl acetoacetate
(0.005 mol), and 2-amino benzothiazole (0.005 mol) to
give compound 4g. Pale-yellow powder, mp 130–132°C,
Rf = 0.53 (DCM:Toluene; 3:2); IR (KBr) (tmax, cm-1):
2941 (C–Hstr in CH2CH3), 1627 (C=Ostr), 1508 (C=Nstr),
1280 (C=Cstr), 962–813 (C–Hdef); 1H NMR (400 MHz,
CDCl3): dH 1.28 (3H, t, CH3CH2, JHH = 14.24 Hz), 2.45
(3H, s, CH3), 3.71 (3H, s, Ar–OCH3), 4.11–4.21 (2H, m,
CH3CH2), 6.34 (1H, s, –CH), 6.78 (2H, d, ArH,
JHH = 8.64 Hz), 7.21–7.58 (6H, m, ArH); 13C NMR
(100 MHz, DMSO): 166.67, 163.27, 159.42, 154.51,
152.07, 138.06, 133.81, 128.51, 126.57, 123.88, 123.82,
122.14, 120.89, 119.04, 113.90, 111.80, 103.24, 60.09,
57.20, 55.18, 23.73, 14.40; ESI–MS: m/z Calculated for
C21H20N2O2S 380.47, Found [M ? H]? 381.3.
Ethyl-2-methyl-4-(2-hydroxy phenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (4e)
Reaction was carried out according to typical procedure
with salicylaldehyde (0.005 mol), ethyl acetoacetate
(0.005 mol), and 2-amino benzothiazole (0.005 mol) to
give compound 4e. Pale-yellow powder, mp 212–215°C,
Rf = 0.53 (DCM:Toluene; 3:2); IR (KBr) (tmax, cm-1):
3028 (C–Hstr), 2810 (C–Hstr in CH2CH3), 1668 (C=Ostr),
1575 (C=Nstr), 1485 (C=Cstr), 837–750 (C–Hdef); 1H NMR
(400 MHz, DMSO): dH 1.28 (3H, t, JHH = 14.16,
CH2CH3), 2.45 (3H, s, CH3), 4.10–4.19 (2H, m, CH2CH3),
6.33 (1H, s, –CH), 6.66–7.92 (8H, m, ArH), 9.89 (1H, s,
OH); 13C NMR (100 MHz, DMSO): 190.23, 171.88,
165.92, 162.99, 157.23, 151.33, 133.73, 128.14, 126.08,
122.92, 116.07, 155.70, 111.03, 59.26, 56.23, 23.11, 14.04;
ESI–MS: m/z Calculated for C20H18N2O3S 367.34, Found
[M]? 367.2.
Ethyl-2-methyl-4-(2, 6-dichloro phenyl)-4H-pyrimido
[2,1-b][1,3]benzothiazole-3-carboxylate (4h)
Reaction was carried out according to typical procedure
with 2,6-dichloro benzaldehyde (0.005 mol), ethyl aceto-
acetate (0.005 mol), and 2-amino benzothiazole
(0.005 mol) to give compound 4h. Pale-yellow powder, mp
150–152°C, Rf = 0.56 (DCM:Toluene; 3:2); IR (KBr)
(tmax, cm-1): 3012 (C–Hstr), 2922 (C–Hstr in CH2CH3),
1625 (C=Ostr), 1500 (C=Nstr), 1278 (C=Cstr), 960–812
1
(C–Hdef); H NMR (400 MHz, DMSO): dH 1.09 (3H, t,
JHH = 14.16 Hz, CH2CH3), 2.29 (3H, s, CH3), 4.01–4.05
(2H, m, CH2CH3), 7.01–7.66 (7H, m, Ar–H); 13C NMR
(100 MHz, DMSO): 171.88, 165.92, 162.99, 151.33,
137.57, 133.73, 132.41. 129.14, 126.08, 125.57, 124.52,
122.97, 121.69, 117.66, 116.07, 59.26, 23.11, 14.04;
ESI–MS: m/z Calculated for C20H16N2Cl2O2S 419.41,
Found [M]? 419.6.
Ethyl-2-methyl-4-(4-hydroxy phenyl)-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carboxylate (4f)
Reaction was carried out according to typical procedure
with p-hydroxy benzaldehyde (0.005 mol), ethyl acetoac-
etate (0.005 mol), and 2-amino benzothiazole (0.005 mol)
to give compound 4f. Pale-yellow powder, mp 210–212°C,
Rf = 0.59 (DCM:Toluene; 3:2); IR (KBr) (tmax, cm-1):
3288 (OHstr), 3059 (C–Hstr), 2897 (C–Hstr in CH2CH3),
1612 (C=Ostr), 1581 (C=Nstr), 1431 and 1377 (C=Cstr),
Antibacterial assay
Different concentrations 50, 25, 12.5, 6.25, 3.12, 1.56, and
0.78 lg/ml of the compounds (4a–4h) were prepared in
sterile microwell to determine minimum inhibitory con-
centration (MIC). Nutrient broth was adjusted to pH 7.0
used for the determination of MIC. The inoculum of the
test microorganisms was prepared by using 16-h-old cul-
tures adjusted by reference to the 0.5 McFarland standards
(1.5 9 108 CFU/ml). Brain heart infusion broth was pre-
pared; 150 ll of it was taken in each well and 10 ll of each
compound was added in broth with different concentra-
tions; then 10 ll of bacterial culture broth was added. The
plate was shaken to uniformly mix the inoculum with the
1
815–754 (C–Hdef); H NMR (400 MHz, CDCl3): dH 1.25
(3H, t, CH3CH2, JHH = 14.16 Hz), 2.35 (3H, s, CH3),
4.04–4.14 (m, 2H, CH3CH2), 6.30 (1H, s, –CH), 6.66 (2H,
d, ArH, JHH = 8.40 Hz), 7.13–7.29 (5H, m, ArH), 7.59
(1H, d, ArH, JHH = 7.76 Hz), 9.26 (1H, s, OH); 13C NMR
(100 MHz, DMSO): 165.55, 162.27, 157.22, 153.46,
137.56, 131.95, 128.13, 126.34, 123.57, 122.94, 122.20,
115.02, 111.80, 102.96, 59.27, 56.37, 23.09, 14.03; ESI–
MS: m/z Calculated for C20H18N2O3S 367.34, Found [M]?
367.2.
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Med Chem Res (2012) 21:3826–3834
3829
broth. Optical density was taken by photo spectrometer
(lQuant, Biotek Ltd. USA), and then incubated for 24 h at
37°C. Appearance of any turbidity shows that the com-
pound is not able to inhibit the growth of the bacteria,
while no turbidity indicates the inhibition of microorgan-
ism by the sample (Table 3).
4H-pyrimido[2,1-b]benzazole derivatives were character-
1
ized by mp, IR, H NMR, 13C NMR, and mass spectral
studies. IR spectra of all pyrimidine derivatives showed
peaks between 740 and 754 cm-1 due to C–Hdef, peak
around 1,500 cm-1 (C=Nben), band between 1,431 and
1,504 (C=Cstr in ring), strong band between 1,600 and
1,700 cm-1 (C=O), band between 1,240 and 1,274
(C–Hben) weak band between 2,800 and 3,000 cm-1
(C–Hstr of benzene ring). 1H NMR spectra of compound 4a
showed triplet at d 1.29 (OCH2CH3), singlet at d 2.47
(CH3), multiplet at d 4.17 (OCH2CH3), singlet at d 6.39
(C–H), multiplet at d 7.07–7.43 (benzene rings); compound
4b showed singlet at d 3.8 for methoxy group; 4c showed
singlet at d 3.06 for p-dimethylamino group; and 4f showed
singlet at d 8.9 for hydroxyl group. Mass spectra of all
compound showed corresponding molecular ion peaks in
mass spectra.
In vitro antifungal test
For antifungal testing, the pyrimidine derivatives and
fluconazole (standard drug) solution was prepared in
DMSO to make effective concentrations of 50, 25, 12.5,
6.25, 3.12, 1.56, and 0.78 lg/ml. Isolated fungal species
Aspergillus niger, Aspergillus fumigates, and Aspergillus
flavus were selected. Sabouraud dextrose agar was pre-
pared; 150 ll of it was taken in each well; 10 ll of each
compound was added in broth with different concentra-
tions; and then 10 ll of fungi culture broth was added. The
plate was shaken to uniformly mix the inoculum with the
broth and note the optical density. The well was incubated
for 72 h at 28°C. Appearance of any turbidity shows that
the compound is not able to inhibit the growth of the
bacteria, while no turbidity indicates the inhibition of
microorganism by the sample. To ensure that solvent had
no effect on fungal growth, a control test was performed
with test medium supplemented with DMSO at the same
dilutions as used in the experiment. The results are incor-
porated in Table 4.
To optimize the reaction conditions and catalyst loading,
a model reaction of benzaldehyde (0.005 mol), 2-amino
benzothiazole (0.005 mol), and ethyl acetoacetate
(0.005 mol) was carried out. The results show that in the
absence of catalyst, the reaction took a long time with poor
yield. The highest yield (79%) of 4H-pyrimido[2,1-b]
[1,3]benzazole was obtained when 10 mol% of aluminum
trichloride was used (Fig. 1). The reaction was extended to
perform the evaluation of various catalysts (Table 1). Out
of the various catalyst evaluations, AlCl3 showed the
highest yield with the shortest reaction time. Copper
chloride and lithium chloride gave moderate yield. Nickel
nitrate, cerium chloride, zinc chloride, acetic acid, and
silver nitrate gave slightly lower yields compared with
other catalysts. The study was further extended to other
substrate materials having electron-donating and electron-
withdrawing groups. The electronic effects due to sub-
stituent do not show any significant deviation. The highest
To determine zone of inhibition, sterilized filter disks
were dipped in these solutions and subsequently dried to
remove DMSO. Sabouraud dextrose agar was prepared and
allowed to solidify. One of these disks was kept free from
antifungal drug (fluconazole) and served as growth control.
Fungi selected were viz., A. niger, A. fumigates, A. flavus,
and 1 ml of each fungus culture was added in the Sabou-
raud dextrose agar plates and spread with the help of sterile
spreader. The filter paper disks soaked in the above men-
tioned dilutions of fluconazole and pyrimidine derivatives
were placed aseptically over the inoculated plates using
sterile forceps. The plates were incubated at 28°C for 72 h,
in upright position. The zone of inhibition was measured
using scale (Table 4).
CHO
O
O
N
S
R
NH2
+
+
CH3
C2H5O
3
1
2
(i)
Results and discussion
O
R
The present methodology involves the synthesis of 4H-
pyrimido[2,1-b]benzazole ring system using aldehydes 1
(0.005 mol) and ethyl acetoacetate 2 (0.005 mol) in the
presence of 2-amino benzothiazole 3 (0.005 mol) using
aluminum chloride as a catalyst under solvent-free
conditions (Scheme 1). Reaction was completed
within 70–120 min with good yield. The structures of
OC2H5
CH3
N
S
N
4
Scheme 1 Conditions: i heating at 60–65°C using AlCl3 under
solvent-free conditions
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Med Chem Res (2012) 21:3826–3834
yield was obtained with substituent –OH. Para-substituted
aldehydes gave better yield as compared with the ortho-
and meta-substituted aldehydes (Table 2); it may be due to
the steric hindrance. The results exhibit that this reaction is
better for those reactions carried out using solvents and
ionic liquids.
Table 1 Synthesis of triheterocyclic 4H-pyrimido[2,1-b]benzothia-
zole using different catalysts (10 mol %) under solvent-free condi-
tions at 60–65°C
Entry
Catalyst
Time (h)
Yield % of 4aa
1
2
3
4
5
6
7
8
9
10
a
SnCl2Á2H2O
ZnCl2
1.3
1.4
2
69
65
71
72
73
70
71
73
65
79
Ni(NO3)2
CeCl2
2
Antibacterial activity
CuCl2
1.5
2
CH3COOH
AgNO3
LiCl
All the synthesized compounds were screened for their
antibacterial activities based on micro dilution broth sus-
ceptibility test method. The stock solution of pyrimidine
derivatives (4a–4h) was prepared in DMSO. The stock
solution (lg/ml) of each of these pyrimidines was serially
diluted and added to Brain Heart Infusion broth, after which
a standardized bacterial suspension was added. The lowest
concentration of pyrimidine derivatives in lg/ml that pre-
vented in vitro growth of microorganism has been repre-
sented as MIC (minimum inhibitory concentration) shown
in Table 3. Susceptibility test in vitro was done on multi-
resistant bacteria Staphylococcus aureus (ATCC 11632),
Pseudomonas aeruginosa (ATCC 15499), Salmonella typhi
(ATCC 23564), Escherichia coli (ATCC 35218), Bacillus
cereus (MTCC 7350), and Providencia rettegeri isolated
from infected fish. The results have been tabulated in
2
1.2
1.3
1.2
MgCl2
AlCl3
Isolated yield
Table 3. Each was performed in triplicate, and the MICs
reported represent the best of at least two repetitions.
Compounds 4c, 4d, 4f, 4g, and 4h showed very promising
activities on multiresistant microorganism. The compound
4g having methoxy substitution at C-4 aryl ring has shown
better activity against all the tested bacterial strain (6.25
lg/ml) except S. typhi. Substitution at para position as
hydroxyl group (4f) shows good activity against E. coli
(6.25 lg/ml) and B. cereus (6.25 lg/ml). Introduction of
Fig. 1 Optimization of AlCl3
as catalyst in the synthesis of
4H-pyrimido[2,1-b]
[1,3]benzothiazole derivatives
90
12
10
80
70
60
50
40
30
20
10
0
8
AlCl3 (mol %)
Yield (%) 4a
Time (hours)
6
4
2
0
1
2
3
4
5
6
7
Entry
AlCl3 (mol %)
Time (hours)
Yield (%) 4aa
1
2
3
4
5
6
7
No catalyst
1
2
5
10
15
20
10
3
3
42
58
65
73
79
80
79
2
1.2
1.2
1.2
a isolated yield.
b Three component reactions of benzaldehyde, 2-amino benzothiazole and ethyl
acetoacetate carried out using AlCl3 under solvent-free conditions by heating at 60-65 °C.
123

Med Chem Res (2012) 21:3826–3834
3831
Table 2 Synthesis of triheterocyclic 4H-pyrimido[2,1-b]benzothiazole using AlCl3 under solvent-free conditions
Entry
1
Aldehydes
Product
Time (h)
1.2
Yield (%)a
M.P. (°C)
O
79
178–180
OC2H5
CH3
CHO
N
S
N
4a
OH
O
2
1.2
85
192–194
H3CO
HO
OC2H5
OCH3
CH3
N
S
CHO
N
4b
CH3
N
CH3
3
1.5
67
175–178
CH3
O
N
OC2H5
CH3
CH3
N
CHO
N
S
4c
NO2
O
4
1.8
70
170–172
O2N
OC2H5
CH3
N
S
CHO
N
4d
OH
O
5
2.0
89
212–215
OH
OC2H5
CH3
CHO
N
S
N
4e
123

3832
Med Chem Res (2012) 21:3826–3834
Table 2 continued
Entry
6
Aldehydes
Product
Time (h)
1.1
Yield (%)a
75
M.P. (°C)
OH
O
HO
210–212
OC2H5
CH3
N
S
CHO
N
4f
OCH3
CHO
O
7
2.0
89
130–132
H3CO
OC2H5
CH3
N
N
S
4g
O
8
1.5
60
150–152
Cl
OC2H5
CH3
Cl
Cl
CHO
Cl
N
S
N
4h
One-pot three component reactions of aldehydes, 2-amino benzothiazole and ethyl acetoacetate carried out
a
Isolated yield
the chloro group at ortho position of C-2 aryl ring (4f) also
has shown potential activity against E. coli and B. cereus
(6.25 lg/ml). Surprisingly, nitro group introduction at C-4
aryl ring shows potential activity against S. typhi and
B. cereus. Results show that all 4H-pyrimido[2,1-b]
[1,3]benzothiazole derivatives exhibited good activity
against B. cereus (Table 3). Most encouraging result was
found against P. aeruginosa, E. coli, B. cereus, and
P. rettegeri. MIC was compared with one of the best mar-
keted antibiotic, viz., ciprofloxacin. p-dimethylamino
benzaldehyde and p-nitro benzaldehyde have shown good
result. Derivatives 4f, 4g, and 4f have shown encouraging
results against all the tested bacteria strain. Pyrimidine
derivatives have shown encouraging results against
B. cereus. The literature survey suggests that pyrimidine
derivatives, synthesized by three-component reaction of
aldehydes, ethyl acetoacetate, and 2-amino benzothiazole,
have not been evaluated so far for their antibacterial activity.
Table 3 The in vitro antibacterial activity (MIC, lg/ml) of the syn-
thesized compounds
Entry
MIC (lg/ml)
S. P.
aureus aeruginosa typhi coli cereus rettegeri
S.
E.
B.
P.
4a
4b
4c
4d
4e
4f
-
25
25
-
12.5
12.5
-
25
-
-
25
25
-
12.5
12.5
25
12.5 12.5
6.25 12.5
6.25 12.5
6.25 25
25
-
-
12.5
6.25
6.25 12.5
6.25 12.5
6.25 6.25
6.25 12.5
1.62 1.62
12.5
6.25
12.5
12.5
6.25
12.5
1.62
25
4g
4h
12.5 6.25
25 6.25
3.25 1.62
Ciprofloxacin 3.25
- Resistant
MIC (lM) Minimum inhibitory concentration, i.e., the concentration
in the tube with highest dilution showing no turbidity
123

Med Chem Res (2012) 21:3826–3834
3833
Table 4 Cytotoxicity and antifungal activity of pyrimidine derivatives (zone of inhibition in mm) against fungal strains
Entry
Fungal strain
Cytotoxicity activity IC50
(lM/ml)
Zone of inhibition (in mm)
MIC(in lg/ml)
A. niger
A. fumigates
A. flavus
A. niger
A. fumigates
A. flavus
(L123 cell)
4a
-
10
-
-
-
-
-
11
-
18
-
25
-
6.25
-
-
[100
50
4b
10
-
6.25
-
-
4c
-
-
-
[100
[100
[100
50
4d
17
-
15
-
12.5
-
6.25
-
-
4e
12.5
-
4f
15
16
-
12
20
-
6.25
6.25
-
6.25
6.25
-
4g
12.5
-
[100
[100
4h
Fluconazole
18
22
1.62
1.62
1.62
- Resistant
Antifungal activity
treatment was performed in duplication. After the treat-
ment, drug containing media was removed and washed
with 200 ll of PBS. To each well of the 96 well plate,
100 ll of MTT reagent (Stock: 1 mg/ml in serum-free
medium) was added and incubated for 4 h at 37°C. After
4 h of incubation, the plate was inverted on tissue paper to
remove the MTT reagent. To solubilize formazan crystals
in the wells, 100 ll of 100% DMSO was added to each
well. The optical density was measured by microtiter plate
reader at 590 nm. The compound concentrations (lg)
required to reduce the viability of mock-infected cells by
50% as determined by MTT method are summarized in
Table 4.
The literature survey, however, suggests that these types of
pyrimidine derivatives have not been evaluated for their
antifungal activity so far. We have now evaluated the
antifungal activity of pyrimidine derivatives against fungi,
viz., A. niger, A. fumigates, and A. flavus. From the results
shown in Table 4, we can observe that pyrimidine deriv-
atives 4a (phenyl), 4b (4-hydroxy-3-methoxy), and 4e
(2-hydroxy) show moderate antifungal activities against
A. fumigates, A. niger, and A. flavus, respectively. Ana-
logues 4d (4-nitro) and 4f (4-hydroxy) derivatives show
good activities A. niger and A. fumigates; derivatives 4c
and 4h do not show any activity against tested fungal
strain. 4g (4-methoxy) derivative of pyrimidine has shown
excellent antifungal activity against all the tested fungal
strains. The antifungal activity of pyrimidine derivatives
was compared with the standard antifungal drugs fluco-
nazole. It was found that p-methoxy derivative (4g)
exhibits excellent activity against all the tested fungal
strains (Table 4). This has thrown open a new era for
exploring suitably designed, new scaffold in molecules as
potential antifungal/antibacterial drugs.
Conclusion
In conclusion, the present method employing AlCl3 is an
efficient, one-pot procedure for preparation of 4H-pyrimi-
do[2,1-b]benzazole derivatives in good yield. The assumed
structure of pyrimidines was confirmed by the IR, 1H
NMR, 13C NMR, and mass spectra interpretation. The
antibacterial activity study revealed that all the tested
compounds showed good to moderate antibacterial acti-
vities against P. aeruginosa, E. coli, B. cereus, and
P. rettegeri bacteria strain. Compounds 4c, 4d, 4f, 4g, and
4h have shown good activity against tested bacterial strain.
Pyrimidine derivatives 4a (phenyl), 4b (4-hydroxy-
3-methoxy), and 4e (2-hydroxy) show the moderately
antifungal activities against A. fumigates, A. niger, and
A. flavus, respectively. 4d (4-nitro) and 4f (4-hydroxy)
derivative show good activity against A. niger and
A. fumigates. Compound 4g has shown excellent antifungal
activity against all the tested fungal strains. The field is
further open for study of these compounds with respect to
toxicity, chronic toxicity, pharmacokinetics, and clinical
Cytotoxicity against L123 (human lung cells)
Cytotoxicity was performed by MTT assay method
(Mosman, 1983 and Heilmann et al., 2001). A 96-well flat
bottomed tissue culture plate was seeded with 2 9 103
cells in 0.1 ml of MEM medium supplemented with 10%
FBS and allowed to attach for 24 h. After 24 h of incu-
bation, cells were treated with test compounds to get a
concentration of 5, 10, 20, 50, and 100 lg/ml after being
incubated for 48 h. The cells in the control group received
only the medium containing the 0.2% DMSO. Each
123

3834
Med Chem Res (2012) 21:3826–3834
substitute and diethyl carbamoyl group as anti-tuberculosis
agents. Bioorg Med Chem 17:1579–1586
studies to establish these molecules as drugs in the market.
The operational simplicity, and easy availability of starting
material make it a rather better advanced alternative pro-
cedure than traditional multi-step methods. Green approach
of aluminum chloride makes it superior over organic acids
and hazardous metal-salt catalysts. The main advantages of
this methodology are the short reaction times, simple cat-
alyst system, higher yields, organic solvent-free reactions,
and ease of operational procedure.
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Synthesis and antimicrobial activity of 5-(2-aminothiazol-4-yl)-
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Acknowledgements We gratefully acknowledge to Mr. Yogesh
Sharma, Parabolic drugs Ltd., Chandigarh and SAIF Punjab Univer-
sity, Chandigarh for spectral analytical data.
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