Microwave assisted synthesis of some new thiazolopyrimidine, thiazolodipyrimidine and thiazolopyrimidothiazolopyrimidine derivatives with potential antioxidant and antimicrobial activity

Article abstract of DOI:10.3390/molecules17089652

Biginelli reaction of ethyl acetoacetate, thiourea and the appropriate aromatic aldehyde was used to produce ethyl 4-aryl-6-methyl-2-thioxo-1,2,3,4- tetrahydropyrimidine- 5-carboxylates, that reacted with bromomalononitrile to give ethyl 3-amino-5- aryl-2-cyano-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6- carboxylates rather than the isomeric 7H-thiazolo[3,2-a]pyrimidines. Thiazolopyrimidine derivatives reacted with carbon disulphide to yield ethyl 9-aryl-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1Hthiazolo[ 3,2-a:4,5-d'] dipyrimidine-8-carboxylates, that reacted with phenacyl bromide to produce ethyl 8-methyl-10-(4-methoxyphenyl)-3-substituted-5-thioxo-2(un)subatituted- 10H-thiazolo[3'',2'':1',2']pyrimido[4',5':4,5]thiazolo[3,2-a] pyrimidine-9-carboxylates. The aforementioned reactions were carried out using both conventional chemical methods and with the assistance of microwave irradaition. Comparison between both methods showed that the microwave assisted method is preferable because of the time reduction and yield improvements achieved. The new compounds were tested for their biological activity as antioxidants, antibacterial or antifungal agents. Some of the new compounds were found to have moderate to good antioxidant and antimicrobial activities.

Full text of DOI:10.3390/molecules17089652

Molecules 2012, 17, 9652-9667; doi:10.3390/molecules17089652
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave Assisted Synthesis of Some New Thiazolopyrimidine,
Thiazolodipyrimidine and Thiazolopyrimidothiazolopyrimidine
Derivatives with Potential Antioxidant and
Antimicrobial Activity
Mohamed M. Youssef 1,†,* and Mahmoud A. Amin 2,†
1
Chemistry Department, Faculty of Science, Cairo University, Cairo 12613, Egypt
2
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt
†
Current address: Chemistry Department, Faculty of Science, Taif University, Taif 21974, Saudi Arabia.
* Author to whom correspondence should be addressed; E-Mail: mmmyousef@yahoo.com;
Tel.: +966-596-072-593.
Received: 7 July 2012; in revised form: 3 August 2012 / Accepted: 7 August 2012 /
Published: 13 August 2012
Abstract: Biginelli reaction of ethyl acetoacetate, thiourea and the appropriate aromatic
aldehyde was used to produce ethyl 4-aryl-6-methyl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylates, that reacted with bromomalononitrile to give ethyl 3-amino-5-
aryl-2-cyano-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylates rather than the
isomeric 7H-thiazolo[3,2-a]pyrimidines. Thiazolopyrimidine derivatives reacted with
carbon disulphide to yield ethyl 9-aryl-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-
thiazolo[3,2-a:4,5-d']dipyrimidine-8-carboxylates, that reacted with phenacyl bromide to
produce ethyl 8-methyl-10-(4-methoxyphenyl)-3-substituted-5-thioxo-2(un)subatituted-
10H-thiazolo[3'',2'':1',2']pyrimido[4',5':4,5]thiazolo[3,2-a]pyrimidine-9-carboxylates. The
aforementioned reactions were carried out using both conventional chemical methods and
with the assistance of microwave irradaition. Comparison between both methods showed
that the microwave assisted method is preferable because of the time reduction and yield
improvements achieved. The new compounds were tested for their biological activity as
antioxidants, antibacterial or antifungal agents. Some of the new compounds were found to
have moderate to good antioxidant and antimicrobial activities.

Molecules 2012, 17
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Keywords: biginellireaction; 2-thioxopyrimidines; thiazolopyrimidines; thiazolo-dipyrimidines;
thiazolopyrimidothiazolopyrimidines; antioxidant activity; antimicrobial activity
1. Introduction
Thiazolopyrimidenes have been of interest due to their ability to inhibit 2-methylerythritol-2,4-
cyclodiphosphate synthase [1]. They have been also used as analgesic and antiparkinsonian agents [2],
modulators of Transient Receptor Potential Vanilloid–receptor 1 (TRPV1) [3], anticancer agents [4–6],
pesticides [7], phosphate inhibitors [8,9], acetylcholinesterase inhibitors [10] and antimicrobial
susbtances [11–13].
The microwave technique has several advantages over conventional methods of synthesis. Reduced
reaction times [14–17], less effects on the environment and better reaction yields are some of the common
advantages of using microwave irradaition. In the present research, we used both the microwave
technique as well as conventional methods to prepare some thiazolopyrimidine, thiazolodipyrimidine
and thiazolopyrimidothiazolopyrimidines derivatives with expected biological activity.
2. Results and Discussion
2.1. Chemistry
Prompted by the aforesaid biological and medicinal activities, samples of differently substituted
thiazolopyrimidines and thiazolodipyrimidines were synthesized, using both conventional chemical
methods and microwave irradiation assistance. The reaction of the precursors, ethyl 4-aryl-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 1a–d, with some bifunctional reagents seems to be
a facile and convenient route for the synthesis of such targets. The newly synthesized compounds were
tested for their antioxidant and antimicrobial activities.
The precursor pyrimidine derivatives 1a–d were prepared by the acid catalyzed condensation of
ternary mixtures of aromatic aldehydes, ethyl acetoacetate and thiourea in ethanol containing a
catalytic amount of hydrochloric acid, commonly known as Biginelli reaction [18–20] (Scheme 1).
Compounds 1a–d, prepared by Biginelli's method, showed correct values of elemental analyses, as
well as compatible spectroscopic data.
Treating each of 1a–d with bromomalononitrile (2) in an ethanol solution containing potassium
hydroxide yielded in each case a single product which could in principle be formulated to be
either the 5H-thiazolo[3,2-a]pyrimidine structure 3 or the isomeric 7H-thiazolo[3,2-a]pyrimidine
structure 4 (Scheme 1). Our preference for structure 3 over structure 4 was firstly based on comparison
1
1
of the H-NMR spectral data for compounds 1 and 3. Thus, the H-NMR spectrum of 3b showed, in
addition to the ethyl ester, methoxy, aromatic and NH2 proton signals, a singlet (3H) at δ 2.30 ppm
assigned to the CH3 protons and a singlet (1H) at δ 6.31 assigned to the pyrimidine H-5. The
appearance of the CH3 proton signal of 3b in the same position as that for the CH3 proton signal in 1b,
and also the downfield shift for the pyrimidine H-5 in 3b compared with the pyrimidine H-4 in 1b,
which appeared at δ = 5.12 ppm, indicates that the moiety around H-5 in 3b differs from that around

Molecules 2012, 17
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H-4 in 1b. Also, the moiety around CH3 at C-7 in 3b is almost similar to that around CH3 at C-6 in 1b.
Consequently, structure 3 could be tentatively assigned for the reaction products. Structure 4 would be
expected to show different δ values for the CH3 groups in 4b and 1b, and similar δ values for H-7 in
4b and H-4 in 1b. More conclusive evidence for structure 3 was based on carrying out a NOE
experiment on compound 3b.
Scheme 1. Synthesis of pyrimidinethiones, thiazolopyrimidines, thiazolodipyrimidines and
thiazolopyrimidothiazol opyrimidines.
Ar
NH
H
EtOOC
S
CH3COCH2COOEt
ArCHO
+
+
H2N
NH2
H3C
Br
N
S
H
1
CN
CN
alc. KOH
2
Heat or MWI
H
Ar
EtOOC
H3C
H
Ar
NH2
N
EtOOC
N
CN
N
S
S
H3C
N
H2N
3
CN
4
CS2
OCH3
He at or MWI
Ar
H
H
H
N
S
EtOO C
H3C
N
S
R2
EtOOC
5b
N
N
NH
N
R1COCHX
R2
Heat or MWI
N
H3C
N
R1
S
S
6
S
S
5
7
a, R1=CH3, R2=H
b, R1=C6H5, R2=H
c, R1=C H3, R 2=COCH3
a, R1=CH3, R2=H, X=Cl
b, R1=C6H5, R2=H, X=Br
c, R1=CH 3, R2=COCH3, R3=Cl
7
6
1,3,5 a, Ar=C6H4-N(CH3)2-p
b, Ar=C6H4-OCH3-p
c, Ar =C6H4-OH-o
d, Ar=
O
Structure 4, having a CH3 group at C-5 and NH2 at C-3 in close proximity, would show a change in
CH3 position signal due to NOE. Actually, upon performing the NOE experiment, the position of the
CH3 group was not affected, which indicates that CH3 and NH2 are not close to each other, thus
suggesting structure 3 for the reaction product.
Compounds 3, as typical enaminonitriles, could be used as precursors for the preparation of
thiazolodipyrimidines. Thus, heating under reflux a mixture of each of 3a–d with an excess of carbon
disulphide yielded, in each case, the corresponding 9-aryl-2,4-dithioxo-7-methylthiazolo[3,2-a:4,5-
d]dipyrimidine-8-carboxylate 5a–d (Scheme 1).

Molecules 2012, 17
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Finally, compound 5b reacted with α-halocarbonyl compounds, namely chloroacetone, phenacyl
bromide and 3-chloropentane-2,4-dione (compounds 6a–c), respectively, by heating in ethanolic
potassium hydoxide solution to produce the respective thiazolo[3'',2'':1',2']pyrimido[4',5':4,5]thiazolo-
[3,2-a]pyrimidine-9-carboxylate derivatives 7a–c (Scheme 1). Besides giving correct elemental
analyses and compatible spectral data (see the Experimental), structure 7 was assigned to the reaction
products based on the behaviour of a similar structure reported in literature by Hafez et al. [21].
Our research group has recently [22–25] been interested in performing synthesis of some
heterocyclic compounds under environmentally friendly, time saving microwave-assisted conditions.
Accordingly, we re-synthesized the previously described compounds 1a–d, 3a–d, 5a–d, and 7a–c
under microwave conditions, aiming to increase reaction yields and reduce the reaction times. The
results of these preparations indicated that reaction yields were increased by 17–23% compared to the
conventional conditions. Reaction times were also significantly reduced. Table 1 summarizes the
benefits of using microwave conditions for the synthesis of the above-mentioned compounds.
Table 1. Comparison between traditional methods and microwave assisted methods of
synthesis of compounds 1a–d, 3a–d, 5a–d, and 7a–c.
Reaction Yield %
Reaction Time
Compound
no.
Conventional
Conventional
Microwave
Microwave
Method
55
Method
3 h
1a
1b
1c
1d
3a
3b
3c
3d
5a
5b
5c
5d
7a
7b
7c
82
87
70
62
81
85
85
68
82
80
79
69
74
88
83
5 min
5 min
58
42
35
56
48
53
40
50
3 h
3 h
3 h
5 min
5 min
10 min
10 min
10 min
10 min
15 min
15 min
15 min
15 min
5 min
Overnight
Overnight
Overnight
Overnight
8 h
53
44
37
43
52
58
8 h
8 h
8 h
3 h
3 h
3 h
5 min
5 min
2.2. Biological Evaluation
2.2.1. Antioxidant Screening
The newly synthesized compounds were tested for antioxidant activity as reflected in their ability to
inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis. The
pro-oxidant activities of the aforementioned compounds were assessed by their effects on bleomycin-
induced DNA damage. Table 2 shows the antioxidant assays by erythrocyte hemolysis, which reveals
that compounds 3a and 3b showed interesting antioxidant activity in the lipid peroxidation assays and
considerable inhibitory activity in the hemolysis assay. Compounds 3c, 3d, 7a, 7b and 7c showed

Molecules 2012, 17
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moderate antioxidant and inhibitory activity. Table 3 shows the antioxidant assay by ABTS method.
Compounds 3a, 3b, 3c and 3d showed potent antioxidant activity.
Table 2. Antioxidant assays by erythrocyte hemolysis (A/B × 100).
Compounds
Absorbance of samples (A) Hemolysis (%)
Complete hemolysis
0.660
-
with distilled water (B)
Ascorbic acid
0.026
0.082
0.075
0.090
0.092
0.035
0.031
0.045
0.051
0.115
0.112
0.132
0.130
0.042
0.045
0.043
3.93
10.33
9.12
13.01
14.12
5.22
4.68
6.92
8.02
21.60
19.25
24.07
23.12
6.36
1a
1b
1c
1d
3a
3b
3c
3d
5a
5b
5c
5d
7a
7b
7c
6.81
6.51
Table 3. Antioxidant assays by ABTS method [Abs. (control) − Abs.(test)/Abs.(control) × 100].
Compounds
Absorbance of sample
Inhibition (%)
ABTS control
Ascorbic acid
0.54
0.06
0.20
0.23
0.29
0.28
0.10
0.12
0.15
0.13
0
88.8
63.0
57.4
46.3
48.1
81.5
77.7
72.2
75.9
1a
1b
1c
1d
3a
3b
3c
3d
5a
0.45
16.6
5b
5c
5d
7a
7b
7c
0.42
0.48
0.43
0.17
0.19
0.16
22.2
11.1
20.3
68.5
64.8
70.3
All compounds have been tested on bleomycin-dependent DNA damage. The results, shown in
Table 4, indicate that compounds 3a–d may have some protective activity towards DNA from the
damage induced by bleomycin.

Molecules 2012, 17
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Table 4. Assays for bleomycin-dependent DNA damage.
Compound
Absorbance of Samples
Ascorbic acid
0.020
0.026
0.029
0.037
0.033
3a
3b
3c
3d
2.2.2. Antimicrobial Evaluation
The newly synthesized heterocyclic compounds listed in Table 4 were tested for their antimicrobial
activity against the following microorganisms: Escherichia coli, Pseudomonas putida, Bacillus
subtilis, Streptococcus lactis, Aspergillus niger, Penicillium sp. and Candida albicans. The preliminary
screening of the investigated compounds was performed using the filter paper disc-diffusion method.
The most active compounds were 1a, 1b, 3a, 3b, 5a, and 5b, which showed moderate to slight
inhibitory action towards the microorganisms. The rest of compounds showed slight to no sensitivity at
all to the tested organisms, and the results are summarized in Table 5.
Table 5. Antimicrobial activities of the newly synthesized compounds.
Inhibition zone (mm)
Compd.
Gram-negative
Gram-positive
Fungi Yeast
No.
E. coli
P. putida
B. subtilis
S. lactis
A. niger
P. sp. C. albicans
1a
12
14
6
8
9
6
8
5
4
5
2
0
0
0
0
0
0
0
0
1b
6
0
7
0
1c
3
2
2
1d
3
2
0
0
0
0
3a
15
12
0
11
7
9
6
7
5
3b
7
5
7
5
3c
0
0
0
0
0
3d
2
2
0
0
0
0
5a
10
11
4
7
8
6
4
3
5b
8
9
6
5
2
5c
2
0
0
0
0
0
0
5d
3
0
0
0
0
0
7a
0
0
0
0
0
0
0
7b
7c
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Chloram-phenicol®
Ampicillin®
22
24
21
20
18
19
19
22
20
24
12
14
0
14
E. coli = Escherichia coli; P. putida = Pseudomonas putida; B. subtilis = Bacillus subtilis;
S. lactis = Streptococcus lactis; A. niger = Aspergillus niger; P. sp. = Penicillium sp.;
C. albicans = Candida albicans; The sensitivity of microorganisms to the tested compounds is
identified in the following manner *: Highly sensitive = Inhibition zone: 15–20 mm; Moderately
sensitive = Inhibition zone: 10–15 mm; Slightly sensitive = Inhibition zone: 1–10 mm; Not
sensitive = Inhibition zone: 0 mm; * each result represents the average of triplicate readings.

Molecules 2012, 17
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3. Experimental
3.1. General
Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus
and are uncorrected. IR spectra (KBr discs) were recorded on a Shimadzu FTIR-8201PC
Spectrophotometer. 1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury 300 MHz and
a Varian Gemini 200 MHz spectrometers using TMS as an internal standard and DMSO-d6, and as a
solvent. Chemical shifts were expressed as  (ppm) units. Mass spectra were recorded on a Shimadzu
GCMS-QP1000EX instrument using an inlet type sample injection at 70 eV. The Microanalytical
Center of Cairo University performed the microanalyses. Microwave reactions were performed with a
Millstone Organic Synthesis Unit (MicroSYNTH with touch control terminal) with a continuous
focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum
under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated
infrared temperature control under the reaction vessel, and control of the pressure was performed with
a pressure sensor connected to the septum of the vessel.
3.1.1. Ethyl 4-aryl-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 1a–d
Method A: A solution of thiourea (0.76 g, 0.01 mol), ethyl acetoacetate (1.30 g, 0.01 mol) and the
appropriate aromatic aldehyde (0.01 mol) in ethanol (50 mL) in the presence of conc. HCl (5 mL) was
heated under reflux for 3 h. The reaction mixture was then allowed to stand at room temperature
overnight whereby the solid precipitate so-formed was collected by filtration, washed with ethanol and
crystallized from ethanol.
Method B: The same reactants of Method A were heated in microwave oven at 140 °C for 5 min.
The reaction mixture was treated in a similar manner to Method A to obtain compounds 1a–d.
Ethyl 4-(4-(dimethylamino)phenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1a)
was obtained as pale green crystals, m.p. 201 °C. 1H-NMR:  (ppm) 1.10 (t, 3H, CH3, J = 4 Hz), 2.26
(s, 3H, CH3), 2.87 (s, 6H, N(CH3)2), 3.97 (q, 2H, CH2, J = 4 Hz), 5.02 (s, 1H, pyrimidine H-4), 6.65
(d, 2H, Ar-H, J = 5 Hz), 7.00 (d, 2H, Ar-H, J = 5 Hz), 9.55 (s, 1H, NH, D2O exchangeable) and 10.23
13
(s, 1H, NH, D2O exchangeable). C-NMR:  (ppm) 14.1 (CH3), 17.2 (CH3), 53.5 (pyrimidine C-4),
59.5 (N(CH3)2), 67.3 (CH2), 101.2, 112.2, 127.1, 131.2, 144.4, 150.0 (aromatic carbons + pyrimidine
C-5 and C-6), 165.3 (C=S) and 173.8 (C=O). IR (KBr) : 3268, 3188 (NH), 1718 (C=O), 1605, 1500 cm−1
(Aromatic C=C). MS (70 eV): (M+) m/z 319 (11.2%). Anal. Calcd. for C16H20N3O2S (319.42):
C(60.16%), H(6.63%), N(13.16%), S(10.04); Found: C(60.3%), H(6.8%), N(13.4%), S(10.0%).
Ethyl 4-(4-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1b) was
1
obtained as yellow crystals, m.p. 144 °C. H-NMR:  (ppm) 1.22 (t, 3H, CH3, J = 4 Hz), 2.26 (s, 3H,
CH3), 3.25 (s, 3H, OCH3), 3.95 (q, 2H, CH2, J = 4 Hz), 5.12 (s, 1H, pyrimidine H-4), 6.81 (d, 2H, Ar-H,
J = 6 Hz), 7.11 (d, 2H, Ar-H, J = 6 Hz), 9.50 (s, 1H, NH, D2O exchangeable) and 10.18 (s, 1H, NH,
13
D2O exchangeable). C-NMR:  (ppm) 14.0 (CH3), 17.4 (CH3), 53.5 (pyrimidine C-4), 63.9 (OCH3),
68.8 (CH2), 103.4, 113.1, 129.7, 134.2, 145.9 153.2 (aromatic carbons + pyrimidine C-5 and C-6),

Molecules 2012, 17
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165.6 (C=S) and 174.0 (C=O). IR (KBr) : 3272, 3185 (NH), 1718 (C=O), 1603, 1506 cm−1 (Aromatic
C=C). MS (70 eV): (M+) m/z 306 (8.5%). Anal. Calcd. for C15H18N2O3S (306.38): C(58.80%),
H(5.92%), N(9.14%), S(10.47); Found: C(59.0%), H(5.9%), N(9.3%), S(10.7%).
Ethyl 4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1c) was
1
obtained as beige crystals, m.p. 235 °C. H-NMR:  (ppm) 1.18 (t, 3H, CH3, J = 4 Hz), 2.19 (s, 3H,
CH3), 3.95 (q, 2H, CH2, J = 4 Hz), 5.10 (s, 1H, pyrimidine H-4), 6.73–7.07 (m, 4H, Ar-H), 8.40 (s,
1H, OH, D2O exchangeable), 9.55 (s, 1H, NH, D2O exchangeable) and 10.12 (s, 1H, NH, D2O
13
exchangeable). C-NMR:  (ppm) 13.5 (CH3), 16.8 (CH3), 53.5 (pyrimidine C-4), 67.6 (CH2), 102.7,
112.6, 115.3, 122.0 127.7, 131.9, 144.6 150.9 (aromatic carbons + pyrimidine C-5 and C-6), 165.2
(C=S) and 172.6 (C=O). IR (KBr) : 3280–3080 (broad, OH+NH), 1722(C=O), 1600, 1501 cm−1
(Aromatic C=C). MS (70 eV): (M+) m/z 292 (14.1%). Anal. Calcd. for C14H16N2O3S (292.09):
C(57.52%), H(5.52%), N(9.58%), S(10.97); Found: C(57.7%), H(5.8%), N(9.5%), S(11.1%).
Ethyl 4-(furan-2-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1d) was obtained
as brown crystals, m.p. 176 °C. 1H-NMR:  (ppm) 1.25 (t, 3H, CH3, J = 4 Hz), 2.22 (s, 3H, CH3), 4.20
(q, 2H, CH2, J = 4 Hz), 5.23 (s, 1H, pyrimidine H-4), 6.44 (d, 1H, furan-H), 6.56 (m, 1H, furan-H),
7.41 (d, 1H, furan-H), 9.34 (s, 1H, NH, D2O exchangeable) and 10.20 (s, 1H, NH, D2O exchangeable).
13C-NMR:  (ppm) 14.3 (CH3), 17.5 (CH3), 57.5 (pyrimidine C-4), 67.2 (CH2), 106.2, 110.9, 143.1,
145.3, 151.0 152.9 (furan carbons + pyrimidine C-5 and C-6), 165.8 (C=S) and 173.0 (C=O). IR (KBr)
: 3275, 3183 (NH), 1720 (C=O), 1607, 1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 266
(6.3%). Anal. Calcd. for C12H14N2O3S (266.32): C(54.12%), H(5.30%), N(10.52%), S(12.04); Found:
C(54.1%), H(5.4%), N(10.4%), S(12.0%).
3.1.2. Ethyl 3-amino-5-aryl-2-cyano-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylates (3a–d)
Method A: To a warm ethanolic potassium hydroxide solution [prepared by dissolving KOH
(0.56 g, 0.01 mol) in ethanol (50 mL)] of each of 1a–d (0.01 mol), bromomalononitrile 2 (1.45 g,
0.01 mol) was added portion-wise with stirring. The reaction mixture was then left overnight at room
temperature, whereby the solid product that separated upon dilution with water was filtered off and
crystallized from the proper solvent.
Method B: The same reactants of method A were heated at 140 °C in microwave oven for 10 min.
The reaction mixture was treated in a similar manner to method A to obtain compounds 3a–d.
Ethyl
3-amino-2-cyano-5-(4-(dimethylamino)phenyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-
carboxylate (3a) was crystallized from dil. dioxane as yellowish green crystals, m.p. 218 °C.
1H-NMR:  (ppm) 1.35 (t, 3H, CH3, J = 4 Hz), 2.34 (s, 3H, CH3), 2.95 (s, 6H, N(CH3)2), 4.15 (q, 2H,
CH2, J = 4 Hz), 6.22 (s, 1H, pyrimidine H-5), 6.60 (d, 2H, Ar-H, J = 5 Hz), 6.97 (d, 2H, Ar-H,
J = 5 Hz) and 8.78 (s, 2H, NH2, D2O exchangeable). 13C-NMR:  (ppm) 14.5 (CH3), 17.3 (CH3), 58.8
(pyrimidine C-5), 59.7 (N(CH3)2), 68.4 (CH2), 107.3 (CN), 112.2, 117.3, 127.1, 132.2, 149.4, 154.6,
157.3, 158.1, 158.9 (aromatic carbons + pyrimidine C-6 and C-7, C-8a + thiazole C-2, C-3), and 171.6
(C=O). IR (KBr) : 3310, 3244 (NH2), 2217 (CN), 1724 (C=O), 1605, 1500 cm−1 (aromatic C=C). MS

Molecules 2012, 17
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(70 eV): (M+) m/z 383 (7.3%). Anal. Calcd. for C19H21N5O2S (383.14): C(59.51%), H(5.52%),
N(18.26%), S(8.36); Found: C(59.6%), H(5.8%), N(18.3%), S(8.3%).
Ethyl
3-amino-2-cyano-5-(4-methoxyphenyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate
1
(3b) was crystallized from ethanol as yellow crystals, m.p. 220 °C. H-NMR:  (ppm) 1.30 (t, 3H,
CH3, J = 4 Hz), 2.30 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.18 (q, 2H, CH2, J = 4 Hz), 6.31 (s, 1H,
pyrimidine H-5), 6.80 (d, 2H, Ar-H, J = 6 Hz), 7.15 (d, 2H, Ar-H, J = 6 Hz), and 8.40 (s, 2H, NH2,
13
D2O exchangeable). C-NMR:  (ppm) 14.8 (CH3), 17.4 (CH3), 59.6 (pyrimidine C-5), 62.1 (OCH3),
67.4 (CH2), 108.1 (CN), 113.1, 118.8, 129.0, 134.1, 151.4, 155.1, 157.8, 158.7, 159.3 (aromatic
carbons + pyrimidine C-6 and C-7, C-8a + thiazole C-2, C-3), and 171.0 (C=O). IR (KBr) : 3300,
3230 (NH2), 2210 (CN), 1720 (C=O), 1605, 1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 370
(8.1%). Anal. Calcd. for C18H18N4O3S (370.43): C(58.36%), H(4.90%), N(15.12%), S(8.66); Found:
C(58.5%), H(4.8%), N(15.3%), S(8.5%).
Ethyl
3-amino-2-cyano-5-(2-hydroxyphenyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate
(3c) was crystallized from ethanol as yellow crystals, m.p. 277 °C. 1H-NMR:  (ppm) 1.32 (t, 3H, CH3,
J = 4 Hz), 2.34 (s, 3H, CH3), 4.05 (q, 2H, CH2, J = 4 Hz), 6.24 (s, 1H, pyrimidine H-5), 6.80–7.18 (m,
4H, Ar-H), 8.15 (s, 1H, OH, D2O exchangeable) and 8.53 (s, 2H, NH2, D2O exchangeable). 13C-NMR:
 (ppm) 14.3 (CH3), 17.1 (CH3), 58.8 (pyrimidine C-5), 67.1 (CH2), 107.3 (CN), 112.3, 116.1, 122.1,
126.3, 131.3, 137.1, 152.1, 157.1, 157.9, 158.8, 159.5 (aromatic carbons + pyrimidine C-6 and C-7,
C-8a + thiazole C-2, C-3), and 173.0 (C=O). IR (KBr) : 3320–3118 (broad, OH+NH2), 2210 (CN),
1716 (C=O), 1600, 1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 356 (12.5%). Anal. Calcd. for
C17H16N4O3S (356.40): C(57.29%), H(4.52%), N(15.72%), S(9.00); Found: C(57.5%), H(4.6%),
N(15.6%), S(9.1%).
Ethyl 3-amino-2-cyano-5-(furan-2-yl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate (3d) was
1
crystallized from dioxane as brown crystals, m.p. 345 °C. H-NMR:  (ppm) 1.27 (t, 3H, CH3,
J = 4 Hz), 2.21 (s, 3H, CH3), 4.25 (q, 2H, CH2, J = 4 Hz), 6.11 (s, 1H, pyrimidine H-5), 6.45 (d, 1H,
furan-H), 6.61 (m, 1H, furan-H), 7.43 (d, 1H, furan-H) and 8.87 (s, 2H, NH2, D2O exchangeable).
13C-NMR:  (ppm) 14.7 (CH3), 17.5 (CH3), 62.3 (pyrimidine C-5), 67.4 (CH2), 107.1 (CN), 112.6,
114.1, 127.3, 135.1, 139.6, 143.1, 145.3, 151.0 152.9 (furan carbons + pyrimidine C-6 and C-7,
C-8a + thiazole C-2, C-3) and 173.3 (C=O). IR (KBr) : 3310, 3244 (NH2), 2210 (CN), 1722 (C=O),
1605, 1510 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 330 (4.8%). Anal. Calcd. for C15H14N4O3S
(330.36): C(54.53%), H(4.27%), N(16.96%), S(9.71); Found: C(54.4%), H(4.4%), N(16.7%), S(9.8%).
3.1.3. Ethyl 9-aryl-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-thiazolo[3,2-a:4,5-d']dipyrimidine-8-
carboxylates 5a–d
Method A: Each of compounds 3a–d (0.01 mol) was heated under reflux with an excess of carbon
disulphide (10 mL) for 8 h. The reaction mixture was then cooled, and the solid that precipitated was
filtered at the pump and crystallized from the proper solvent.

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Method B: The same reactants of method A were heated at 140 °C in microwave oven for 15 min.
The reaction mixture was treated in a similar manner to method A to obtain compounds 5a–d.
Ethyl 9-(4-(dimethylamino)phenyl)-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-thiazolo-[3,2-a:4,5-
d']dipyrimidine-8-carboxylate (5a) was crystallized from dilute ethanol as grey crystals, m.p. 248 °C.
1H-NMR:  (ppm) 1.10 (t, 3H, CH3, J = 4 Hz), 2.26 (s, 3H, CH3), 2.84 (s, 6H, N(CH3)2), 3.95 (q, 2H,
CH2, J = 4 Hz), 5.88 (s, 1H, pyrimidine H-9), 6.60 (d, 2H, Ar-H, J = 5 Hz), 6.95 (d, 2H, Ar-H,
J = 5 Hz), 11.35 (s, 1H, NH, D2O exchangeable) and 12.12 (s, 1H, NH, D2O exchangeable). 13C-NMR:
 (ppm) 14.2 (CH3), 17.8 (CH3), 53.5 (pyrimidine C-9), 59.6 (N(CH3)2), 67.3 (CH2), 109.2, 111.3,
127.1, 132.2, 148.2, 154.3, 156.2 157.1, 161.9 (aromatic carbons + pyrimidine C-5a, C-7,
C-8 + thiazole C-4a, C-10a), 171.3 (C=S), 175.1 (C=O) and 181.4 (C=S). IR (KBr) : 3305, 3200
(NH2), 1712 (C=O), 1605, 1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 459 (3.2%). Anal. Calcd.
for C20H21N5O2S3 (459.61): C(52.26%), H(4.61%), N(15.24%), S(20.93); Found: C(52.3%), H(4.7%),
N(15.5%), S(20.8%).
Ethyl
9-(4-methoxyphenyl)-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-thiazolo[3,2-a:4,5-d']-
dipyrimidine-8-carboxylate (5b) was crystallized from ethanol as beige crystals, m.p. 243 °C.
1H-NMR:  (ppm) 1.13 (t, 3H, CH3, J = 4 Hz), 2.32 (s, 3H, CH3), 3.22 (s, 3H, OCH3), 4.10 (q, 2H,
CH2, J = 4 Hz), 5.95 (s, 1H, pyrimidine H-9), 6.80 (d, 2H, Ar-H, J = 6 Hz), 7.12 (d, 2H, Ar-H,
J = 6 Hz), 11.30 (s, 1H, NH, D2O exchangeable) and 12.10 (s, 1H, NH, D2O exchangeable). 13C-NMR:
 (ppm) 14.1 (CH3), 18.6 (CH3), 61.0 (pyrimidine C-9), 63.2 (OCH3), 67.3 (CH2), 110.4, 114.6, 130.0,
133.8 149.8, 155.2, 156.4, 157.9, 162.2 (aromatic carbons + pyrimidine C-5a, C-7, C-8 + thiazole C-4a,
C-10a), 171.1 (C=S), 175.0 (C=O) and 181.5 (C=S). IR (KBr) : 3310, 3200 (NH), 1718 (C=O), 1605,
1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 446 (4.0%). Anal. Calcd. for C19H18N4O3S3
(446.57): C(51.10%), H(4.06%), N(12.55%), S(21.54); Found: C(51.0%), H(4.2%), N(12.5%), S(21.8%).
Ethyl
9-(2-hydroxyphenyl)-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-thiazolo[3,2-a:4,5-d']-
dipyrimidine-8-carboxylate (5c) was crystallized from dioxane as pale green crystals, m.p. 295 °C.
1H-NMR:  (ppm) 1.28 (t, 3H, CH3, J = 4 Hz), 2.25 (s, 3H, CH3), 4.16 (q, 2H, CH2, J = 4 Hz), 6.11 (s,
1H, pyrimidine H-9), 6.80–7.20 (m, 4H, Ar-H), 8.22 (s, 1H, OH, D2O exchangeable), 11.40 (s, 1H,
13
NH, D2O exchangeable) and 12.25 (s, 1H, NH, D2O exchangeable). C-NMR:  (ppm) 14.3 (CH3),
17.1 (CH3), 58.8 (pyrimidine C-5), 67.1 (CH2), 107.3 (CN), 107.9, 112.4, 120.1, 127.4, 133.3, 138.1,
154.0, 158.1, 158.9, 159.4, 161.0 (aromatic carbons + pyrimidine C-5a, C-7, C-8 + thiazole C-4a,
C-10a), 171.4 (C=S), 174.8 (C=O) and 180.7 (C=S). IR (KBr) : 3300, 3230 (NH2), 1710 (C=O),
1600, 1500 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 432 (5.0%). Anal. Calcd. for C18H16N4O3S3
(432.54): C(49.98%), H(3.73%), N(12.95%), S(22.24); Found: C(49.8%), H(3.8%), N(13.1%), (22.1%).
Ethyl 9-(furan-2-yl)-7-methyl-2,4-dithioxo-2,3,4,9-tetrahydro-1H-thiazolo[3,2-a:4,5-d']dipyrimidine-
1
8-carboxylate (5d) was crystallized from dioxane as dark green crystals, m.p. 255 °C. H-NMR: 
(ppm) 1.25 (t, 3H, CH3, J = 4 Hz), 2.15 (s, 3H, CH3), 4.00 (q, 2H, CH2, J = 4 Hz), 6.11 (s, 1H,
pyrimidine H-9), 6.51 (d, 1H, furan-H), 6.78 (m, 1H, furan-H), 7.55 (d, 1H, furan-H), 11.40 (s, 1H,
NH, D2O exchangeable) and 12.25 (s, 1H, NH, D2O exchangeable). 13C-NMR:  (ppm) 14.5 (CH3),
17.1 (CH3), 62.1 (pyrimidine C-9), 66.8 (CH2), 106.6, 110.0, 123.3, 134.8, 138.7, 142.7, 144.9, 151.4

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157.9 (furan carbons + pyrimidine C-5a, C-7, C-8 + thiazole C-4a, C-10a) 171.8 (C=S), 173.9 (C=O)
and 181.2 (C=S). IR (KBr) : 3310, 3244 (2NH), 2210 (CN), 1722 cm−1 (C=O), 1605, 1510 (aromatic
C=C). MS (70 eV): (M+) m/z 406 (4.3 %). Anal. Calcd. for C16H14N4O3S3 (406.50): C(47.27%),
H(3.47%), N(13.78%), S(23.66); Found: C(47.0%), H(3.4%), N(13.8%), S(23.8%).
3.1.4. Ethyl 8-methyl-10-(4-methoxyphenyl)-3-substituted-5-thioxo-2-(un)substituted-10H-thiazolo-
[3'',2'':1',2']pyrimido[4',5':4,5]thiazolo[3,2-a]pyrimidine-9-carboxylates 7a–c
Method A: A solution of 5b (4.46 g, 0.01 mol) with each of chloroacetone (0.92 g, 0.1 mol),
phenacyl bromide (1.99 g, 0.01 mol) or 3-chloropentane-2,4-dione (1.34 g, 0.01 mol) in ethanolic
potassium hydroxide solution [prepared by dissolving KOH (0.56 g, 0.01 mol) in ethanol (50 mL)] was
heated under reflux for 3 h. A precipitate started to form on hot after 1 h. After cooling, the produced
precipitate was filtered off, dried and crystallized from dimethyl formamide.
Method B: The same reactants of method A were heated at 140 °C in microwave oven for 5 min.
The reaction mixture was treated in a similar manner to method A to obtain compounds 7a–c.
Ethyl
3,8-dimethyl-10-(4-methoxyphenyl)-5-thioxo-10H-thiazolo[3'',2'':1',2']pyrimido[4',5':4,5]-
thiazolo[3,2-a]pyrimidine-9-carboxylate (7a) was obtained as yellow crystals, m.p. 314 °C.
1H-NMR:  (ppm) 1.34 (t, 3H, CH3, J = 4 Hz), 2.45 (s, 3H, CH3), 3.11 (s, 3H, CH3), 3.91 (s, 3H,
OCH3), 4.53 (q, 2H, CH2, J = 4 Hz), 5.77 (s, 1H, pyrimidine H-10), 6.94 (s, 1H, thiazole H-2), 7.15 (d,
13
2H, Ar-H, J = 6 Hz), 7.68 (d, 2H, Ar-H, J = 6 Hz). C-NMR:  (ppm) 14.7 (CH3), 19.6 (CH3), 23.3
(CH3), 58.2 (OCH3), 63.1 (CH2), 67.5 (pyrimidine C-10), 101.6, 106.4, 112.3, 124.1, 128.0, 135.8
151.3, 155.0, 157.4, 158.3, 158.5, 160.1 (aromatic carbons + pyrimidine C-6a, C-7a, C-8, C-9, C-11a,
C-12a + thiazole C-2, C-3), 171.0 (C=O) and 182.5 (C=S). IR (KBr) : 1715 (C=O), 1600, 1508 cm−1
(Aromatic C=C). MS (70 eV): (M+) m/z 484 (3.6%). Anal. Calcd. for C22H20N4O3S3 (484.57):
C(54.52%), H(4.16%), N(11.56%), S(19.85); Found: C(54.34%), H(4.2%), N(11.8%), S(19.8%).
Ethyl 8-methyl-10-(4-methoxyphenyl)-3-phenyl-5-thioxo-10H-thiazolo[3'',2'':1',2']pyrimido-[4',5':4,5]-
thiazolo[3,2-a]pyrimidine-9-carboxylate (7b) was obtained as orange crystals, m.p. 335 °C. 1H-NMR:
 (ppm) 1.41 (t, 3H, CH3, J = 4 Hz), 3.17 (s, 3H, CH3), 3.95 (s, 3H, OCH3), 4.50 (q, 2H, CH2,
J = 4 Hz), 5.75 (s, 1H, pyrimidine H-10), 7.04 (s, 1H, thiazole H-2), 7.22 (d, 2H, Ar-H, J = 6 Hz),
13
7.41 (d, 2H, Ar-H, J = 6 Hz), 7.52–7.81 (m, 5H, Ar-H). C-NMR:  (ppm) 14.6 (CH3), 24.7 (CH3),
58.0 (OCH3), 63.4 (CH2), 67.2 (pyrimidine C-10), 103.2, 106.9, 112.8, 115.6, 124.9, 126.2, 128.0,
128.9, 129.3, 135.8, 152.2, 155.1, 157.2, 158.0, 160.4 (aromatic carbons + pyrimidine C-6a, C-7a, C-8,
C-9, C-11a, C-12a + thiazole C-2, C-3), 170.7 (C=O) and 183.0 (C=S). IR (KBr) : 1718 (C=O), 1600,
1504 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 546 (2.3%). Anal. Calcd. for C27H22N4O3S3
(546.68): C(59.32%), H(4.06%), N(10.25%), S(17.60); Found: C(59.2%), H(4.2%), N(10.6%), S(17.8%).
Ethyl
2-acetyl-3,8-dimethyl-10-(4-methoxyphenyl)-5-thioxo-10H-thiazolo[3'',2'':1',2']pyrimido-
[4',5':4,5]thiazolo[3,2-a]pyrimidine-9-carboxylate (7c) was obtained as yellow crystals, m.p. 308 °C.
1H-NMR:  (ppm) 1.35 (t, 3H, CH3, J = 4 Hz), 2.47 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.10 (s,3H,CH3),
3.95 (s, 3H, OCH3), 4.50 (q, 2H, CH2, J = 4 Hz), 5.68 (s, 1H, pyrimidine H-10), 7.00 (d, 2H, Ar-H,

Molecules 2012, 17
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J = 6 Hz), 7.55 (d, 2H, Ar-H, J = 6 Hz). 13C-NMR:  (ppm) 12.3 (CH3), 14.5 (CH3), 20.8 (CH3), 25.3
(CH3), 58.0 (OCH3), 63.1 (CH2), 65.5 (pyrimidine C-10), 105.6, 106.2, 112.6, 124.3, 127.2, 135.6
151.0, 154.3 155.6, 157.2, 158.1, 160.4 (aromatic carbons + pyrimidine C-6a, C-7a, C-8, C-9, C-11a,
C-12a + thiazole C-2, C-3), 170.4 (C=O) ), 175.3 (C=O) and 182.5 (C=S). IR (KBr) : 1715, 1695
(2 C=O), 1600, 1508 cm−1 (aromatic C=C). MS (70 eV): (M+) m/z 526 (5.5%). Anal. Calcd. for
C24H22N4O4S3 (526.08): C(54.73%), H(4.21%), N(10.64%), S(18.27); Found: C(54.5%), H(4.3%),
N(10.7%), S(18.1%).
3.2. Antioxidant Screening
3.2.1. Assay for Erythrocyte Hemolysis
Blood was obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes
were separated from plasma and the buffy coat and washed three times with 10 volumes of 0.15 M
NaCl. During the last washing, the erythrocytes were centrifuged at 2,500 rpm for 10 min to obtain a
constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this
assay system [26]. A 10% suspension of erythrocytes in pH 7.4 phosphate-buffered saline (PBS) was
added to the same volume of 200 mM 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) solution
(in PBS) containing samples to be tested at different concentrations. The reaction mixture was shaken
gently while being incubated at 37 °C for 24 h. The reaction mixture was then removed, diluted with
eight volumes of PBS and centrifuged at 2,500 rpm for 10 min. The absorbance A of the supernatant
was read at 540 nm. Similarly, the reaction mixture was treated with eight volumes of distilled water to
achieve complete hemolysis, and the absorbance B of the supernatant obtained after centrifugation was
measured at 540 nm. The percentage hemolysis was calculated by the equation (1 − A/B) × 100%. The
data were expressed as mean standard deviation. L-Ascorbic was used as a positive control.
3.2.2. Antioxidant Activity Screening Assay—ABTS Method
For each of the investigated compounds 2 mL of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid) (ABTS) solution (60 mM) was added to 3 M MnO2 solution (25 mg/mL) all prepared in
phosphate buffer (pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered, and the absorbance
(Acontrol) of the resulting green-blue solution (ABTS radical solution) was adjusted at ca. 0.5 at  734 nm.
Then, 50 L of 2 mM solution of the test compound in spectroscopic grade MeOH/phosphate buffer
(1:1) was added. The absorbance (Atest) was measured and the reduction in color intensity was
expressed as % inhibition. The % inhibition for each compound is calculated from the following
equation [27]:
ꢈꢉꢆꢁꢅꢊꢆꢋ ꢌ ꢈꢍꢎꢏꢐꢋꢑ
% ꢀꢁꢂꢃꢄꢃꢅꢃꢆꢁ ꢇ
ꢒ 100
ꢈꢉꢆꢁꢅꢊꢆꢋ
Ascorbic acid (vitamin C) was used as standard antioxidant (positive control). Blank sample was
run without ABTS and using MeOH/phosphate buffer (1:1) instead of sample. Negative control sample
was run with MeOH/phosphate buffer (1:1) instead of tested compound.

Molecules 2012, 17
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3.2.3. Bleomycin-Dependent DNA Damage
The assay was done according to Aeschlach et al. [28] with minor modifications. The reaction
mixture (0.5 mL) contained DNA (0.5 mg/ mL), bleomycin sulfate (0.05 mg/mL), MgCl2 (5 mM),
FeCl3 (50 M) and samples to be tested at different concentrations. L-Ascorbic acid was used as a
positive control. The mixture was incubated at 37 °C for 1 h. The reaction was terminated by addition
of 0.05 mL EDTA (0.1 M). The color was developed by adding 0.5 mL thiobarbituric acid (TBA) (1%,
w/v) and 0.5 mL HCl (25%, v/v) followed by heating at 80 °C for 10 min. After centrifugation, the
extent of DNA damage was measured by increase in absorbance at 532 nm.
3.3. Antimicrobial Screening
The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against
the following microorganisms: (a) Gram-negative: Escherichia coli and Pseudomonas putide; (b)
Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) Fungi: Aspergillus niger and Penicillium sp.;
(d) Yeast: Candida albicans.
Media: Three types of specific media were used in this study:
Medium (1): For bacteria (Nutrient Medium), consisting of (g/L distilled water): peptone, 5 and meat
extract, 3. pH was adjusted to 7.0.
Medium (2): For fungi (Potato Dextrose Medium), consisting of (g/L distilled water): Infusion from
potatoes, 4 and D(+)glucose, 20. pH was adjusted to 5.5.
Medium (3): For yeast (Universal Medium), consisting of (g/L distilled water): yeast extract, 3; malt
extract, 3; peptone, 5 and glucose, 10. pH was adjusted to 5.5.
For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min.
Procedure (Filter Paper Diffusion Method) [29]
Proper concentrations of microbial suspensions were prepared from 1 (for bacteria to 3 (for yeast
and fungi)-day-old liquid stock cultures incubated on a rotary shaker (100 rpm). In the case of fungi, 5
sterile glass beads were added to each culture flask. The mycelia were then subdivided by mechanical
stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a spectrophotometer
at 350 nm to give an optical density of 1.0 Appropriate agar plates were aseptically surface inoculated
uniformLy by a standard volume (ca. 1 mL) of the microbial broth culture of the tested microorganism,
namely E. coli, P. putide, B. subtilis, S. Lactis, A. Niger, Penicillium sp. and C. albicans.
Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at
121 °C. Test compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL.
The sterile discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs
have been air dried, they were placed on the agar surface previously seeded with the organism to be
tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs
were arranged per dish, suitably spaced apart, i.e., the discs should be separated by a distance that is
equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone.
Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to

Molecules 2012, 17
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permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at
30 °C for 72 h for yeast and fungi.
4. Conclusions
New thiazolopyrimidines have been synthesized using both conventional methods and microwave
assisted conditions. The latter methods proved very efficient in reducing reaction times as well as
increasing the overall yield of the reactions. The newly synthesized compounds were tested for their
antioxidant and antimicrobial activities. Some compounds showed good or moderate antioxidant
activity, whereas other compounds showed weak antimicrobial activity.
Acknowledgements
This research is financed by Al-Taif University, Al-Taif, Kingdom of Saudi Arabia. Project
Number 1-432-1282.
References
1. Geist, J.G.; Lauw, S.; Illarinova, V.; Fischer, M.; Gwawert, T.; Rohdich, F.; Eisenreich, W.;
Kaiser, J.; Groll, M.; Scheurer, C.; et al. Thiazolopyrimidine inhibitors of 2-methylerythritol
2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium
falciparum. ChemMedChem 2010, 5, 1092–1101.
2. Amr, A.-E.-G.; Maigali, S.S.; Abdulla, M.M. Synthesis, and analgesic and antiparkinsonian
activities of thiopyrimidine, pyrane, pyrazoline, and thiazolopyrimidine derivatives from 2-chloro-
6-ethoxy-4-acetylpyridine. Monatsh. Chem. 2008, 139, 1409–1415.
3. Branstetter, B.J.; Breitenbucher, J.G.; Lebsack, A.D.; Xiao, W. Thiaolopyrimidine Modulators of
TRPV1. U.S. Patent WO 005,303, 2008.
4. Flefel, E.E.; Salama, M.A.; El-Shahat, M.; El-Hashash, M.A.; El-Farargy, A.F. A novel synthesis
of some new pyrimidine and thiazolopyrimidine derivatives for anticancer evaluation. Phosphorus
Sulfur Silicon Relat. Elem. 2007, 182, 1739–1756.
5. Hammam, A.G.; Sharaf, M.A.; Abdel Hafez, N.A. Synthesis and anti-cancer activity of pyridine
and thiazolopyrimidine derivatives using ethylpiperidone as a synthon. Indian J. Chem. 2001 40B,
213–221.
6. Said, M.; Abouzid, K.; Mouneer, A.; Ahmedy, A.; Osman, A.-M. Synthesis and biological
evaluation of new thiazolopyrimidines. Arch. Pharm. Res. 2004, 27, 471–477.
7. Linder, W.; Brandes, W. Pesticidal Thiazolopyrimidine Derivatives. U.S. Patent 367,820, 1991.
8. Duval, R.; Kolb, S.; Braud, E.; Genest, D.; Garbay, C. Rapid discovery of triazolobenzylidene-
thiazolopyrimidines (TBTP) as CDC25 phosphatase inhibitors by parallel click chemistry and in
situ screening. J. Comb. Chem. 2009, 11, 947–950.
9. Kolb, S.; Mondésert, O.; Goddard, M.L.; Jullien, D.; Villoutreix, B.O.; Ducommun, B.;
Garbay, C.; Braud, E. Development of novel thiazolopyrimidines as CDC25B phosphatase
inhibitors. ChemMedChem 2009, 4, 633–648.

Molecules 2012, 17
9666
10. Zhi, H.; Chen, L.; Zhang, L.; Liu, S.; Wan, D.C.C.; Lin, H.; Hu, C. Design, synthesis, and
biological evaluation of 5H-thiazolo[3,2-a]pyrimidine derivatives as a new type of
acetylcholinesterase inhibitors. ARKIVOC 2008, xiii, 266–277.
11. Rashad, A.E.; Shamroukh, A.H.; Abdel-Megeid, R.E.; El-Sayed, W.A. Synthesis, reactions and
antimicrobial evaluation of some polycondensed thieno-pyrimidine derivatives. Synth. Commun.
2010, 40, 1149–1160.
12. El-Emary, T.I.; Abdel-Mohsen, S.A. Synthesis and antimicrobial activity of some new
1,3-diphenylpyrazoles bearing pyrimidine, Pyrimidinethione, thiazolopyrimidine, triazolopyrimidine,
thio- and alkylthiotriazolopyrimidinone moieties at the 4-position. Phosphorus Sulfur 2006, 181,
2459–2474.
13. Maddila, S.; Damu, G.L.V.; Oseghe, E.O.; Abafe, O.A.; Venakata, R.C.; Lavanya, P. Synthesis
and biological studies of novel biphenyl-3,5-dihydro-2H-thiazolo-pyrimidines derivatives.
J. Korean Chem. Soc. 2012, 56, 334–340.
14. Sosnowski, M.; Skulski, L. Microwave-accelerated iodination of some aromatic amines, using
urea-hydrogen peroxide addition compound (UHP) as the oxidant. Molecules 2002, 7, 867–870.
15. Gregg, B.; Golden, K.; Quinn, J. Indium(III) trifluoromethanesulfonate as an efficient catalyst for
the deprotection of acetals and ketals. J. Org. Chem. 2007, 72, 5890–5893.
16. Lerebours, R.; Wolf, C. Palladium(II)-catalyzed conjugate addition of arylsiloxanes in water.
Org. Lett. 2007, 9, 2737–2740.
17. Marion, N.; Gealageas, R.; Nolan, S. [(NHC)AuI]-catalyzed rearrangement of allylic acetates.
Org. Lett. 2007, 9, 2653–2656.
18. Biginelli, P. Synthesis of 3,4-dihydropyrimidin-2(1H)-ones. Gazz. Chem. Ital. 1893, 23, 360–372.
19. Kappe, C.O. 100 Years of the Biginelli dihydropyridine synthesis. Tetrahedron 1993, 49,
6937–6963.
20. Akbas, E.; Aslanoglu, F. Studies on reactions of pyrimidine compounds. Microwave-assisted
synthesis of 1,2,3,4-tetrahydro-2-thioxopyrimidine derivatives. Phosphorus Sulfur Silicon Relat.
Elem. 2008, 183, 82–89.
21. Hafez, H.N.; Hussein, H.A.R.; El-Gazzar, A.B.A. Synthesis of substituted thieno-[2,3-
d]pyrimidine-2,4-dithiones and their S-glycoside analogues as potential antiviral and antibacterial
agents. Eur. J Med. Chem. 2010, 45, 4026–4032.
22. Youssef, M.M.; Amin, M.A. Microwave assisted synthesis of some new heterocyclic
spiro-derivatives with potential antimicrobial and antioxidant activity. Molecules 2010, 15,
8827–8840.
23. Faty, R.M.; Youssef, M.M.; Youssef, A.M.S. Microwave assisted synthesis and unusual coupling
of some novel Pyrido[3,2-f][1,4]thiazepines. Molecules 2011, 16, 4549–4559.
24. Saad, H.A.; Youssef, M.M.; Mosselhi, M.A. Microwave assisted synthesis of some new fused
1,2,4-triazine bearing thiophene moiety of expected pharmacological activity. Molecules 2011,
16, 4937–4957.
25. Youssef, A.M.S.; Azab, M.E.; Youssef, M.M. Bromination and diazo-coupling of
pyridinethiones; microwave assisted synthesis of isothiazolopyridine, pyridothiazine and
pyridothiazepines. Molecules 2012, 17, 6930–6943.

Molecules 2012, 17
9667
26. Morimoto, Y.; Tanaka, K.; Iwakiri, Y.; Tokuhiro, S.; Fukushima, S.; Takeuchi, Y. Protective
effects of some neutral amino acids against hypotonic hemolysis. Biol. Pharm. Bull. 1995, 18,
1417–1422.
27. Lissi, E.; Modak, B.; Torres, R.; Escobar, J.; Urzua, A. Total antioxidant potential of
resinousexudates from Heliotropium species, and a comparison of ABTS and DPPH methods.
Free Radic. Res. 1999, 30, 471–477.
28. Aeschlach, R.; Loliger, J.; Scott, B.C.; Murciao, A.; Butler, J.; Halliwell, B.; Aruoma, O.
Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem.
Toxicol. 1994, 32, 31–36.
29. Coffen, D.L.; Korzan, D.G. Synthetic quinine analogs. III. Frangomeric and anchimeric processes
in the preparation and reactions of α,β-epoxy ketones. J. Org. Chem. 1971, 36, 390–395.
Sample Availability: Samples of the compounds are available from the authors.
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