Synthesis of novel 2-(Substituted amino)alkylthiopyrimidin-4(3H)-ones as potential antimicrobial agents

Article abstract of DOI:10.3390/molecules19010279

5-Alkyl-6-(substituted benzyl)-2-thiouracils 3a,c were reacted with (2-chloroethyl) diethylamine hydrochloride to afford the corresponding 2-(2-diethylamino)ethylthiopyrimidin- 4(3H)-ones 4a,b. Reaction of 3a-c with N-(2-chloroethyl)pyrrolidine hydrochlor

Full text of DOI:10.3390/molecules19010279

Molecules 2014, 19, 279-290; doi:10.3390/molecules19010279
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel 2-(Substituted amino)alkylthiopyrimidin-
4(3H)-ones as Potential Antimicrobial Agents
Mohamed I. Attia 1, Ali A. El-Emam 1, Abdulghafoor A. Al-Turkistani 1, Amany L. Kansoh 2 and
Nasser R. El-Brollosy 1,3,
*
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P. O. Box 2457, Riyadh 11451, Saudi Arabia
2
3
Microbial Chemistry Department, Genetic Engineering and Biotechnology Division,
National Research Centre, Giza 12622, Egypt
Department of Chemistry, Faculty of Science, Tanta University, Tanta 31527, Egypt
* Author to whom correspondence should be addressed; E-Mail: brollosy@yahoo.com;
Tel: +966-114-673-765, Fax: +966-114-676-220.
Received: 29 November 2013; in revised from: 13 December 2013 / Accepted: 16 December 2013/
Published: 27 December 2013
Abstract: 5-Alkyl-6-(substituted benzyl)-2-thiouracils 3a,c were reacted with (2-chloroethyl)
diethylamine hydrochloride to afford the corresponding 2-(2-diethylamino)ethylthiopyrimidin-
4(3H)-ones 4a,b. Reaction of 3a–c with N-(2-chloroethyl)pyrrolidine hydrochloride and/or
N-(2-chloroethyl)piperidine hydrochloride gave the corresponding 2-[2-(pyrrolidin-1-
yl)ethyl]-thiopyrimidin-4(3H)-ones 5a–c and 2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-
4(3H)-ones 6a,b, respectively. Treatment of 3a–d with N-(2-chloroethyl)morpholine
hydrochloride under the same reaction conditions formed the corresponding
2-[2-(morpholin-4-yl)ethyl]thiopyrimidines 6c–f. On the other hand, 3a,b were reacted
with N-(2-bromoethyl)phthalimide and/or N-(3-bromopropyl)phthalimide to furnish the
corresponding 2-[2-(N-phthalimido)ethyl]-pyrimidines 7a,b and 2-[3-(N-phthalimido)-
propyl]pyrimidines 7c,d, respectively. Compounds 3a–d, 4a,b, 5a–c, 6a–f and 7a–d were
screened against Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus
subtilis NRRL 4219 and Bacillus cereus), yeast-like pathogenic fungus (Candida albicans
ATCC 10231) and a fungus (Aspergillusniger NRRL 599). The best antibacterial activity
was displayed by compounds 3a, 3b, 4a, 5a, 5b, 6d, 6f, 7b and 7d, whereas compounds
4b, 5b, 5c, 6a, 6b and 6f exhibited the best antifungal activity.

Molecules 2014, 19
280
Keywords: 2-thiouracils; pyrimidin-4(3H)-ones; alkylation; antibacterial activity;
anti-fungal activity
1. Introduction
In chemotherapy pyrimidines are considered as privileged structures with a large spectrum of biological
activities. They are known very widely in Nature since they are components of RNA and DNA.
The chemotherapeutic efficacy of pyrimidines may be due to their ability to inhibit vital enzymes
responsible for nucleic acid biosynthesis such as reverse transcriptase, dihydrofolate reductase,
uridine and thymidine phosphorylase, as well as thymidylate synthetase. Several pyrimidine
derivatives exhibit diverse pharmacological activities as antiviral [1–9], anti-inflammatory [10–12],
and antimalarial agents [13–15]. Many pyrimidines have been demonstrated to possess anticancer [16–21],
antituberculosis [22] and anti-allergic [23] activities. Moreover, several pyrimidine derivatives have
been reported as antithyroid [24] and antimicrobial agents [25–30], as well as human thymidine and
uridine phosphorylase inhibitors [31–33].
In a previous study [34], we synthesized a series of 2-(substituted amino)ethylthiopyrimidines
analogues of S-DABOs to be screened as reverse transcriptase inhibitors against human
immunodeficiency virus (HIV-1). We found it of interest to evaluate the antimicrobial activity for such
pyrimidine derivatives. In the present work, and as a part of our continuing interest in the chemistry of
pyrimidines [30,34–42], the synthesis and antimicrobial evaluation of some novel 2-(substituted
amino)alkylthiopyrimidin-4(3H)-one derivatives have been investigated.
2. Results and Discussion
2.1. Chemistry
5-Alkyl-6-(substituted benzyl)-2-thiouracils 3a–d were prepared, as described in our previous
work [34,42], by reaction of (substituted phenyl)acetonitrile 1 with the appropriate ethyl 2-bromoesters 2
in anhydrous THF in the presence of zinc dust, followed by treatment of the β-ketoesters thus formed
with thiourea in the presence of sodium ethoxide. Compounds 3a and 3c were reacted with
(2-chloroethyl) diethylamine hydrochloride in DMF in the presence of anhydrous potassium carbonate to
afford 6-(4-chlorobenzyl)-2-(2-diethylamino)ethylthio-5-methylpyrimidin-4(3H)-one (4a) [34] and 6-(3,4-
dimethoxybenzyl)-2-(2-diethylamino)ethylthio-5-ethylpyrimidin-4(3H)-one (4b) in good yields (Scheme 1).
6-(4-Chlorobenzyl)-5-methyl-2-[2-(pyrrolidin-1-yl)ethyl]thiopyrimidin-4(3H)-one
(5a)
[34]
(5c)
[34],
and
6-(4-chlorobenzyl)-5-ethyl-2-[2-(pyrrolidin-1-yl)ethyl]thiopyrimidin-4(3H)-one
(5b)
6-(3,4-dimethoxybenzyl)-5-ethyl-2-[2-(pyrrolidin-1-yl)ethyl]thiopyrimidin-4(3H)-one
were
obtained, respectively, in good yields, on reaction of compounds 3a, 3b and/or 3c with
N-(2-chloroethyl)pyrrolidine hydrochloride in the presence of anhydrous potassium carbonate in DMF
(Scheme 2). Alkylation of 3b and/or 3c with N-(2-chloroethyl)piperidine hydrochloride in DMF
containing potassium carbonate gave 6-(4-chlorobenzyl)-5-ethyl-2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-
4(3H)-one (6a) [34] and 6-(3,4-dimethoxybenzyl)-5-ethyl-2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-

Molecules 2014, 19
281
4(3H)-one (6b) in 77% and 72% yields, respectively. Reaction of compounds 3a–d with
N-(2-chloroethyl)morpholine hydrochloride under the same reaction conditions formed the
corresponding 2-[2-(morpholin-4-yl)ethyl]thiopyrimidines 6c–f in 63%–74% yields (Scheme 2).
Scheme 1. Synthesis of compounds 3a–d and 4a,b.
Br
O
X
CN
O
R
+
R
HN
O
X
S
N
H
1
2
3
R
X
a
Me
Et
Et
4-Cl
4-Cl
3,4-(OCH3)2
4-CH3
b
c
d
O
N
Et
X
Cl
R
N
HN
. HCl
N
S
K2CO3 / DMF
4
R
X
a
b
Me
Et
4-Cl
3,4-(OCH3)2
Scheme 2. Synthesis of compounds 5a–c, 6a–f and 7a–d.
O
O
X
X
Cl
.HCl
R
N
R
HN
HN
N
S
N
H
S
N
5
3
R
X
Cl
.HCl
N
a
b
c
Me
Et
Et
4-Cl
4-Cl
3,4-(OCH3)2
O
Y
N
Br
n
O
O
N
O
N
X
R
Cl
R
HN
HN
O
N
Y
N
S
S
n
O
6
7
6
R
X
Y
7
a
b
c
R
n
2
2
3
3
a
b
c
d
e
f
Et
Et
4-Cl
CH2
Me
Et
3,4-(OCH3)2 CH2
Me
Et
4-Cl
4-Cl
O
O
O
O
Me
Et
Et
4-CH3
d
Et
3,4-(OCH3)2
On the other hand, compounds 3a and 3b were treated with N-(2-bromoethyl)phthalimide and/or
N-(3-bromopropyl)phthalimide in the presence of potassium carbonate in DMF to furnish the
corresponding 2-[2-(N-phthalimido)ethyl]pyrimidines 7a,b and 2-[3-(N-phthalimido)propyl]-pyrimidines
7c,d in 69%, 71% and 62%, 64% yields, respectively (Scheme 2).

Molecules 2014, 19
282
2.2. Antimicrobial Testing
The antimicrobial activities of the synthesized compounds, 3a–d, 4a,b, 5a–c, 6a–f and 7a–d
(200 µg/10 mm disc) as well as the reference drugs, ampicillin and clotrimazole, were screened against
yeast-like pathogenic fungus (Candida albicans ATCC 10231), fungus (Aspergillus niger NRRL 599)
and Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis NRRL 4219 and
Bacillus cereus) which are important human pathogenic microorganisms. A Diameter of Inhibition Zone
(DIZ) assay [43] was performed to evaluate the preliminary antimicrobial potential of the test compounds
against the test organisms and the results are given in Table 1.
Table 1. Antimicrobial activity of compounds 3a–d, 4a,b, 5a–c, 6a–f and 7a–d, the broad
spectrum antibacterial drug ampicillin and the antifungal drug clotrimazole against
Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis NRRL
4219 and Bacillus cereus), yeast-like pathogenic fungus (Candida albicans ATCC 10231)
and fungus (Aspergillus niger NRRL 599).
Diameter of Growth Inhibition Zone (mm) a
Comp. No.
Staphylococcus
Bacillus
Bacillus
Candida
Aspergillus
aureus
22
30
-
subtilis
cereus
albicans
niger
3a
17
18
-
21
18
-
15
22
-
-
-
3b
3c
-
3d
-
-
-
-
-
4a
30
-
12
-
12
-
-
13
25
-
4b
21
13
32
35
30
20
-
5a
30
27
-
15
13
-
13
15
-
5b
13
31
21
25
-
5c
6a
23
23
24
27
-
-
-
6b
-
-
6c
-
-
6d
18
12
15
-
12
12
13
-
15
-
-
6e
-
6f
21
21
25
-
25
22
12
13
12
24
-
7a
7b
7c
13
-
12
-
-
18
-
7d
24
35
13
38
16
35
Ampicillin
Clotrimazole
38
40
a (-): Inactive (inhibition zone < 10 mm).
The synthesized compounds showed varying degrees of inhibition zones against the tested
microorganisms. The antibacterial results revealed that compounds 3a, 3b, 4a, 5a, 5b, 6a–d, 6f, 7a, 7b
and 7d showed strong activity (growth inhibition zones > 18 mm against one or more of the tested
microorganisms), compound 6e exhibited weak activity (growth inhibition zone 10–13 mm), while

Molecules 2014, 19
283
compounds 3c, 3d, 4b, 5c and 7c showed no antibacterial activity (growth inhibition zones < 10 mm).
Concerning the antifungal results, compounds 3b, 4b, 5b, 5c, 6a, 6b, 6f and 7a exhibited strong
activity, compounds 3a, 6d and 7c showed moderate activity (growth inhibition zones 14–18 mm),
compounds 4a, 5a, 7b and 7d showed weak activity, whereas no antifungal activity was noticed for
compounds 3c, 3d, 6c and 6e. In general, the best antibacterial activity was displayed by compounds
3a, 3b, 4a, 5a, 5b, 6d, 6f, 7b and 7d. Compounds 4b, 5b, 5c, 6a, 6b and 6f exhibited the best
antifungal activity, whereas compounds 3c and 3d showed no activity against the test organisms.
Gram-positive bacteria, Staphylococcus aureus, and the yeast-like, Candida albicans, are considered
the most sensitive among the tested microorganisms. The synthesized test compounds showed no
activity against Gram negative pathogens, Escherichia coli and Pseudomonas aeruginosa.
Although several compounds showed strong antibacterial and antifungal activities, none of them were
found to be superior to the reference drugs. Compounds 3a, 3b, 4a, 5a, 5b, 6b, 6d, 6f, 7b and 7d
displayed a relatively broad spectrum activity, accordingly, their MIC values were determined. The
MIC values for compounds 3a, 3b, 4a, 5a, 5b, 6b, 6d, 6f, 7b and 7d against the most sensitive tested
microorganisms, Staphylococcus aureus and Candida albicans are represented in Table 2.
Table 2. The minimal inhibitory concentration (MIC, µg/mL) values for compounds 3a,
3b, 4a, 5a, 5b, 6b, 6d, 6f, 7b and 7d against the most sensitive tested microorganisms,
Staphylococcus aureus and Candida albicans.
The minimal inhibitory concentration (MIC µg/mL) a
Compound No.
Staphylococcus aureus
Candida albicans
3a
100
25
100
25
3b
4a
25
ND
100
25
5a
25
5b
25
6b
100
25
50
6d
100
50
6f
7b
50
50
100
100
7d
50
Ampicillin
Clotrimazole
6.0
6.0
a
The lowest concentration of the test compound that inhibits the growth of microorganism (μg/mL).
ND: not determined.
According to the above results, the antimicrobial activity seemed to be dependent on the nature of
substituents. Compounds containing a 4-chlorobenzyl substituent at C-6 of the pyrimidine ring showed
the best antibacterial activity, whereas, the best antifungal results were given by compounds containing
2-(pyrrolidin-1-yl)ethylthio and 2-(piperidin-1-yl)ethylthio substituents at C-2 of the pyrimidine ring.
Concerning compounds 7a–d, the ethyl group at C-5 of the ring was found to improve the
antimicrobial activity.

Molecules 2014, 19
284
3. Experimental
3.1. General
Melting points (°C) were measured in open glass capillaries using a Branstead 9100 Electrothermal
melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra
Shield NMR spectrometer (Fällanden, Switzerland) operating at 500.13 MHz for 1H and 125.76 MHz
for 13C, the chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) as
internal standard; coupling constants (J) are expressed in Hz and signals are expressed as s (singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). Electrospray ionization mass spectra (ESI-MS) were
recorded on an Agilent 6410 Triple Quad tandem mass spectrometer (Santa Clara, CA, USA) at 4.0 kV
for the positive ions. The progress of reactions was monitored by TLC (DC-alufolio 60 F254) from
Merck, and visualization with ultraviolet light (UV) at 365 and 254 nm. For column chromatography
Merck silica gel (0.040–0.063 mm) was used. The tested microorganisms were obtained from MIRCIN
Cairo, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Bacteria, fungi and yeast-like fungi
were cultivated on agar media of nutrient, Czapek’sdox and malt–extract, respectively. The reference
drugs ampicillin trihydrate (CAS 7177-48-2) and clotrimazole (CAS 23593-75-1) were obtained from
Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Compounds 3a–d, 4a, 5a,b and 6a were
reported in our previous studies [34,42].
3.2. General Procedure for Preparation of 2-(Substituted amino)ethylthiopyrimidines 4b, 5c and 6b–f
To a solution of the appropriate compound 3a–d (1 mmol) in anhydrous DMF (5 mL), was added
anhydrous potassium carbonate (0.304 g, 2.2 mmol) followed by the appropriate 2-chloroethyl
substituted amine hydrochloride (1.1 mmol). The mixture was stirred at room temperature for 24 h,
then was diluted with H2O (100 mL) and extracted with diethyl ether (3 × 50 mL). The combined
organic extract was washed with H2O (3 × 50 mL), dried (MgSO4) and evaporated under reduced pressure.
The residue was chromatographed on silica gel column with CHCl3 to afford the target compounds.
6-(3,4-Dimethoxybenzyl)-2-[2-(diethylamino)ethy]lthio-5-ethylpyrimidin-4(3H)-one (4b) White solid.
1
M.p.: 119–120 °C, Yield: 0.287 g (71%). H-NMR (CDCl3): δ = 0.89 (t, 3H, J = 7.0 Hz, CH3),
1.00–1.04 (m, 6H, 2 × CH3), 2.41 (q, 2H, J = 7.0 Hz, CH2), 2.71–2.74 (m, 4H, 2 × CH2),
2.89–2.91 (m, 2H, CH2), 3.03–3.06 (m, 2H, CH2), 3.67 (s, 2H, CH2), 3.77 (s, 3H, OCH3), 3.78 (s, 3H,
OCH3), 6.70 (bs, 2H, Harom.), 6.77 (s, 1H, Harom.), 11.71 (bs, 1H, NH). 13C-NMR (CDCl3):
δ = 10.06 (CH3), 10.16 (CH3), 13.49 (CH3), 18.89 (CH2), 36.27 (CH2), 39.78 (CH2), 47.05 (2 × CH2),
54.62 (CH2), 55.88 (2 × OCH3), 120.68 (C-5), 111.09, 112.22, 119.14, 130.93, 147.63, 148.84 (Carom.),
157.59 (C-6), 161.32 (C-4), 164.69 (C-2). ESI-MS, m/z (Rel. Int.): 406 (M + H+, 78).
6-(3,4-Dimethoxybenzyl)-5-ethyl-2-[2-(pyrrolidin-1-yl)ethyl]thiopyrimidin-4(3H)-one (5c) White solid.
1
M.p.: 143–145 °C, Yield: 0.274 g (68%). H-NMR (DMSO-d6): δ = 0.92 (t, 3H, J = 7.5 Hz, CH3),
1.68–1.71 (m, 4H, 2 × CH2), 2.37 (q, 2H, J = 7.5 Hz, CH2), 2.52–2.55 (m, 4H, 2 × CH2),
2.68 (t, 2H, J = 6.0 Hz, CH2), 3.20 (t, 2H, J = 6.0 Hz, CH2), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3),
13
3.84 (s, 2H, CH2), 6.72–6.88 (m, 3H. Harom.). C-NMR (DMSO-d6): δ = 13.40 (CH3), 18.14 (CH2),

Molecules 2014, 19
285
23.08 (CH2), 28.72 (CH2), 35.11 (CH2), 53.21 (CH2), 54.96 (CH2), 55.39 (OCH3), 55.48 (OCH3),
120.52 (C-5), 111.83, 112.89, 120.57, 131.06, 147.29, 148.50 (Carom.), 159.97 (C-6), 161.20 (C-4),
164.01 (C-2). ESI-MS, m/z (Rel. Int.): 404 (M + H+, 90).
6-(3,4-Dimethoxybenzyl)-5-ethyl-2-[2-(piperidin-1-yl)ethyl]thiopyrimidin-4(3H)-one (6b) White solid.
1
M.p.: 127–129 °C, Yield: 0.301 g (72%). H-NMR (CDCl3): δ = 0.88 (t, 3H, J = 7.5 Hz, CH3),
1.45–1.47 (m, 2H, CH2), 1.78–1.80 (m, 4H, 2 × CH2), 2.39 (q, 2H, J = 7.5 Hz, CH2),
2.50–2.52 (m, 4H, 2 × CH2), 2.73 (t, 2H, J = 5.0 Hz, CH2), 2.99 (t, 2H, J = 5.0 Hz, CH2), 3.75 (s, 2H, CH2),
3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 6.70 (s, 2H, Harom.), 6.79 (s, 1H, Harom.), 11.73 (s, 1H, NH).
13C-NMR (CDCl3): δ = 13.23 (CH3), 18.91 (CH2), 24.01, 24.43, 55.44 (Cpiperidin.), 36.21 (CH2), 39.80
(CH2), 55.90 (2 × OCH3), 61.57 (CH2), 122.64 (C-5), 111.09, 112.23, 120.71, 130.88, 147.33, 148.52
(Carom.), 157.07 (C-6), 162.85 (C-4), 164.51 (C-2). ESI-MS, m/z (Rel. Int.): 418 (M + H+, 85).
6-(4-Chlorobenzyl)-5-methyl-2-[2-(morpholin-4-yl)ethyl]thiopyrimidin-4(3H)-one (6c) White solid.
M.p.:158–159 °C, Yield: 0.280 g (74%).1H-NMR (DMSO-d6): δ = 1.95 (s, 3H, CH3), 2.33 (t, 4H,
J = 4.5 Hz, 2 × CH2), 2.46 (t, 2H, J = 7.0 Hz, CH2), 3.13 (t, 2H, J = 7.0 Hz, CH2), 3.54 (t, 4H,
J = 4.5 Hz, 2 × CH2), 3.84 (s, 2H, CH2), 7.24, 7.32 (2 × d, 4H, J = 8.5 Hz, Harom.), 12.61 (s, 1H, NH).
13C-NMR (DMSO-d6): δ = 10.31 (CH3), 26.70 (CH2), 34.17 (CH2), 52.86, 65.97 (Cmorpholin.), 57.35
(CH2); 115.19 (C-5), 128.13, 130.00, 130.58, 137.24 (Carom.), 157.15 (C-6), 159.80 (C-4), 163.34
(C-2). ESI-MS, m/z (Rel. Int.): 380 (M + H+, 100).
6-(4-Chlorobenzyl)-5-ethyl-2-[2-(morpholin-4-yl)ethyl]thiopyrimidin-4(3H)-one (6d) White solid.
1
M.p.: 133–135 °C. Yield: 0.271 g (69%). H-NMR (DMSO-d6): δ = 0.94 (t, 3H, J = 7.5 Hz, CH3),
2.31 (t, 4H, J = 4.5 Hz, 2 × CH2), 2.43–2.46 (m, 4H, 2 × CH2), 3.12 (t, 2H, J = 7.0 Hz, CH2), 3.53 (t, 4H,
J = 4.5 Hz, CH2), 3.85 (s, 2H, CH2), 7.25, 7.33 (2 × d, 4H, J = 8.5 Hz, Harom.), 12.79 (s, 1H, NH).
13C-NMR (DMSO-d6): δ = 13.16 (CH3), 18.02 (CH2), 26.64 (CH2), 30.59 (CH2), 52.82, 65.96
(Cmorpholin.), 57.32 (CH2), 121.27 (C-5), 128.09, 130.64, 130.78, 137.55 (Carom.), 157.20 (C-6), 159.44
(C-4), 162.77 (C-4). ESI-MS, m/z (Rel. Int.): 394 (M + H+, 100).
5-Ethyl-6-(4-methylbenzyl)-2-[2-(morpholin-4-yl)ethyl]thiopyrimidin-4(3H)-one (6e) White solid.
1
M.p.: 137–139 °C.Yield: 0.236 g (63%). H-NMR (DMSO-d6): δ = 0.90 (t, 3H, J = 7.5 Hz, CH3),
2.25 (s, 3H, CH3), 2.31–2.39 (m, 6H, 3 × CH2), 2.58 (t, 2H, J = 7.0 Hz, CH2), 3.15 (t, 2H, J = 7.0 Hz, CH2),
3.51 (t, 4H, J = 4.5 Hz, 2 × CH2), 3.79 (s, 2H, CH2), 7.07, 7.11 (2 × d, 4H, J = 8.0 Hz, Harom.),
12.49 (s, 1H, NH). 13C-NMR (DMSO-d6): δ = 13.14 (CH3), 18.08 (CH2), 20.51 (CH3), 26.62 (CH2),
52.83, 65.98 (Cmorpholin.), 57.38 (CH2), 120.98 (C-5), 128.56, 128.76, 135.04, 135.41 (Carom.),
157.93 (C-6), 162.20 (C-4), 164.63 (C-2). ESI-MS, m/z (Rel. Int.): 374 (M + H+, 100).
6-(3,4-Dimethoxybenzyl)-5-ethyl-2-[2-(morpholin-4-yl)ethyl]thiopyrimidin-4(3H)-one (6f) White solid.
1
M.p.: 151–152 °C, Yield: 0.276 g (66%). H-NMR (DMSO-d6): δ = 0.90 (t, 3H, J = 7.5 Hz, CH3),
2.30–2.39 (m, 6H, 3 × CH2), 2.58 (t, 2H, J = 7.0 Hz, CH2), 3.21 (t, 2H, J = 7.0 Hz, CH2), 3.57 (t, 4H,
J = 4.5 Hz, 2 × CH2), 3.70 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.76 (s, 2H, CH2), 6.72–6.87 (m, 3H,
13
H
arom.), 12.54 (s, 1H, NH). C-NMR (DMSO-d6): δ = 13.32 (CH3), 18.06 (CH2), 26.68 (CH2), 52.83,
65.98 (Cmorpholin.), 55.36 (OCH3), 55.42 (OCH3), 57.44 (CH2), 117.23 (C-5), 111.76, 112.85, 120.57,

Molecules 2014, 19
286
131.29, 147.27, 148.45 (Carom.), 158.34 (C-6), 162.20 (C-4), 162.43 (C-2). ESI-MS, m/z (Rel. Int.):
420 (M + H+, 100).
3.3. General Procedure for Preparation of 2-[2-(N-Phthalimido)ethyl]thiopyrimidin-4(3H)-ones 7a,b
and 2-[3-(N-Phthalimido)propyl]thiopyrimidin-4(3H)-ones 7c,d
Anhydrous potassium carbonate (0.152 g, 1.1 mmol) was added to a solution of the appropriate
compound 3a,b (1 mmol) in DMF (5 mL), followed by addition of N-(2-bromoethyl)phthalimide
and/or N-(3-bromopropyl)phthalimide (1.1 mmol). The reaction mixture was stirred at room temperature
for 24 h and worked up as described above for the preparation of compounds 4–6.
6-(4-Chlorobenzyl)-5-methyl-2-[2-(N-phthalimido)ethyl]thiopyrimidin-4(3H)-one (7a) White solid.
1
M.p.: 251–253 °C, Yield: 0.302 g (69%). H-NMR (DMSO-d6): δ = 1.90 (s, 3H, CH3), 3.34 (t, 2H,
J = 6.0 Hz, CH2), 3.82 (s, 2H, CH2), 3.87 (t, 2H, J = 6.0 Hz, CH2), 7.32–7.33 (m, 4H, Harom.),
13
7.83–7.88 (m, 4H, Harom.), 12.69 (s, 1H, NH). C-NMR (DMSO-d6): δ = 10.27 (CH3), 28.12 (CH2),
30.59 (CH2), 36.61 (CH2), 115.11 (C-5), 123.00, 128.15, 130.64, 130.83, 131.41, 134.36, 137.18 (Carom.),
159.13 (C-6), 162.21 (C-4), 163.11 (C-2), 167.60 (CO). ESI-MS, m/z (Rel. Int.): 440 (M + H+, 18).
6-(4-Chlorobenzyl)-5-ethyl-2-[2-(N-phthalimido)ethyl]thiopyrimidin-4(3H)-one (7b) White solid.
1
M.p.: 203–204 °C, Yield: 0.322 g (71%). H-NMR (DMSO-d6): δ = 0.87 (t, 3H, J = 7.5 Hz, CH3),
2.37 (q, 2H, J = 7.5 Hz, CH2), 3.35 (t, 2H, J = 6.0 Hz, CH2), 3.80 (s, 2H, CH2), 3.86 (t, 2H, J = 6.0 Hz,
CH2), 7.33–7.34 (m, 4H, Harom.), 7.83–7.89 (m, 4H, Harom.), 12.71 (s, 1H, NH). 13C-NMR (DMSO-d6):
δ = 13.03 (CH3), 18.00 (CH2), 28.07 (CH2), 30.60 (CH2), 36.71 (CH2), 115.19 (C-5), 123.00, 128.12,
130.70, 130.83, 131.40, 134.36, 137.50 (Carom.), 162.34 (C-4), 163.22 (C-2), 167.60 (CO). ESI-MS,
m/z (Rel. Int.): 454 (M + H+, 31).
6-(4-Chlorobenzyl)-5-methyl-2-[3-(N-phthalimido)propyl]thiopyrimidin-4(3H)-one (7c) White solid.
1
M.p.: 234–235 °C, Yield: 0.282 g (62%). H-NMR (DMSO-d6): δ = 1.85–1.91 (m, 2H, CH2),
1.93 (s, 3H, CH3), 3.02 (t, 2H, J = 6.5 Hz, CH2), 3.60 (t, 2H, J = 6.5 Hz, CH2), 3.74 (s, 2H, CH2), 7.19,
7.25 (2 × d, 4H, J = 8.0 Hz, Harom.), 7.80–7.85 (m, 4H, Harom.), 12.72 (s, 1H, NH). 13C-NMR (DMSO-d6):
δ = 10.29 (CH3), 26.97 (CH2), 28.13 (CH2), 30.59 (CH2), 36.37 (CH2), 115.32 (C-5), 122.88, 128.03,
130.53, 130.74, 131.55, 134.22, 137.18 (Carom.), 157.86 (C-6), 162.71 (C-4), 163.67 (C-2), 167.87
(CO). ESI-MS, m/z (Rel. Int.): 454 (M + H+, 20).
6-(4-Chlorobenzyl)-5-ethyl-2-[3-(N-phthalimido)propyl]thiopyrimidin-4(3H)-one (7d) White solid,
1
M.p.: 197–198 °C, Yield: 0.298 g (64%). H-NMR (DMSO-d6): δ = 0.93 (t, 3H, J = 7.5 Hz, CH3),
1.84–1.89 (m, 2H, CH2), 2.41 (q, 2H, J = 7.5 Hz, CH2), 3.02 (t, 2H, J = 6.5 Hz, CH2), 3.59 (t, 2H,
J = 6.5 Hz, CH2), 3.76 (s, 2H, CH2), 7.21, 7.26 (2 x d, 4H, J = 8.0 Hz, Harom.), 7.80–7.85 (m, 4H, Harom.),
13
12.74 (s, 1H, NH). C-NMR (DMSO-d6): δ = 13.11 (CH3), 18.01 (CH2), 26.97 (CH2), 28.09 (CH2),
30.59 (CH2), 36.34 (CH2), 115.69 (C-5), 122.88, 128.00, 130.60, 130.74, 131.55, 134.21, 137.47 (Carom.),
158.98 (C-6), 161.92 (C-2), 163.43 (C-4), 167.87 (CO). ESI-MS, m/z (Rel. Int.): 468 (M + H+, 17).

Molecules 2014, 19
287
3.4. Determination of the Antimicrobial Activity by the Agar Disc-Diffusion Method [43]
Sterile nutrient, Czapek’s dox and malt extract agar media were inoculated, separately, with 100 µL
cell suspension of the chosen microorganism, bacteria, fungi and yeast-like fungi, respectively, and
poured into Petri-dishes (20 cm diameter). The test compounds (200 µg/10 mm diameter disc) were
placed onto the surface of the agar Petri-dishes. The antimicrobial activities were expressed as the
diameter of the growth inhibition zone in mm.
3.5. Determination of Minimal Inhibitory Concentration (MIC) [44]
The minimal inhibitory concentrations (MICs) of the test compounds were determined using serial
dilutions technique. Different concentrations ranging 50.0–200.0 µg/mL for each compound in
dimethyl sulphoxide (DMSO) were placed on filter paper disc (1 cm diameter). The discs were
deposited on the surface of inoculated agar plates and kept at low temperature before incubation which
favours diffusion over microbial growth to detect the inhibition zone clearly. The plates were
incubated at 30 °C for 24 h for bacteria and yeast and for 48 h for fungi.
4. Conclusions
In the present study, several 2-(substituted amino)alkylthiopyrimidin-4(3H)-ones were synthesized
and screened against Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Bacillus subtilis
NRRL 4219 and Bacillus cereus), yeast-like pathogenic fungus (Candida albicans ATCC 10231) and
fungus (Aspergillus niger NRRL 599) which are important human pathogenic microorganisms. Most
of the test compounds showed good antimicrobial activities.
Acknowledgments
The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through the Research Group Project
No. RGP-VPP-274.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Mitsuya, H.; Yarchoan, R.; Broder, S. Molecular targets for AIDS therapy. Science 1990, 249,
1533–1544.
2. Miyasaka, T.; Tanaka, H.; Baba, M.; Hayakawa, H.; Walker, R.T.; Balzarini, J.; de Clercq, E.
A novel lead for specific anti-HIV-1 agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine.
J. Med. Chem. 1989, 32, 2507–2509.
3. Hopkins, A.L.; Ren, J.; Tanaka, H.; Baba, M.; Okamato, M.; Stuart, D.I.; Stammers, D.K.
Design of MKC-442 (emivirine) analogues with improved activity against drug-resistant HIV
mutants. J. Med. Chem. 1999, 42, 4500–4505.

Molecules 2014, 19
288
4. Malik, V.; Singh, P.; Kumar, S. Unique chlorine effect in regioselective one-pot synthesis of
1-alkyl-/allyl-3-(o-chlorobenzyl) uracils: Anti-HIV activity of selected uracil derivatives.
Tetrahedron 2006, 62, 5944–5951.
5. Gazivoda, T.; Raic-Malic, S.; Marjanovic, M.; Kralj, M.; Pavelic, K.; Balzarini, J.; de Clercq, E.;
Mintas, M. The novel C-5 aryl, alkenyl and alkynyl substituted uracil derivatives of L-ascorbic acid:
Synthesis, cytostatic, and antiviral activity evaluations. Bioorg. Med. Chem. 2007, 15, 749–758.
6. Novikov, M.S.; Buckheit, R.W., Jr.; Temburnikar, K.; Khandazhinskaya, A.L.; Ivanov, A.V.;
Seley-Radtke, K.L. 1-Benzyl derivatives of 5-(arylamino)uracils as anti-HIV-1 and anti-EBV
agents. Bioorg. Med. Chem. 2010, 18, 8310–8314.
7. Novikov, M.S.; Valuev-Elliston, V.T.; Babkov, D.A.; Paramonova, M.P.; Ivanov, A.V.;
Gavryushov, S.A.; Khandazhinskaya, A.L.; Kochetkov, S.N.; Pannecouque, C.; Andrei, G.; et al.
N1,N3-disubstituted uracils as nonnucleoside inhibitors of HIV-1 reverse transcriptase.
Bioorg. Med. Chem. 2013, 21, 1150–1158.
8. Sakakibara, N.; Hamasaki, T.; Baba, M.; Demizu, Y.; Kurihara, M.; Irie, K.; Iwai, M.; Asada, E.;
Kato, Y.; Maruyama, T. Synthesis and evaluation of novel 3-(3,5-dimethylbenzyl)uracil analogs
as potential anti-HIV-1 agents. Bioorg. Med. Chem. 2013, 21, 5900–5906.
9. Novikov, M.S.; Babkov, D.A.; Paramonova, M.P.; Khandazhinskaya, A.L.; Ozerov, A.A.;
Chizhov, A.O.; Andrei, G.; Snoeck, R.; Balzarini, J.; Seley-Radtke, K.L. Synthesis and anti-HCMV
activity of 1-[ω-(phenoxy)alkyl]uracil derivatives and analogues thereof. Bioorg. Med. Chem.
2013, 21, 4151–4157.
10. Isobe, Y.; Tobe, M.; Inoue, Y.; Isobe, M.; Tsuchiya, M.; Hayashi, H. Structure and activity
relationships of novel uracil derivatives as topical anti-inflammatory agents. Bioorg. Med. Chem.
2003, 11, 4933–4940.
11. Evaldsson, C.; Ryden, I.; Uppugunduri, S. Anti-inflammatory effects of exogenous uridine in an
animal model of lung inflammation. Inter. Immunopharmcol. 2007, 7, 1025–1032.
12. Keche, A.P.; Hatnapure, G.D.; Tale, R.H.; Rodge, A.H.; Birajdar, S.S.; Kamble, V.M. A novel
pyrimidine derivatives with aryl urea, thiourea and sulfonamide moieties: Synthesis,
anti-inflammatory and antimicrobial evaluation. Bioorg. Med. Chem. Lett. 2012, 22, 3445–3448.
13. Agarwal, A.; Srivastava, K.; Puri, S.K.; Chuahan, P.M. Synthesis of 2,4,6-trisubstituted
pyrimidines as antimalarial agents. Bioorg. Med. Chem. 2005, 13, 4645–4650.
14. Agarwal, A.; Srivastava, K.; Puri, S.K.; Chuahan, P.M. Antimalarial activity of
2,4,6-trisubstituted pyrimidines. Bioorg. Med. Chem. Lett. 2005, 15, 1881–1883.
15. Singh, K.; Kaur, H.; Chibale, K.; Balzarini, J. Synthesis of 4-aminoquinoline-pyrimidine hybrids
as potent antimalarials and their mode of action studies. Eur. J. Med. Chem. 2013, 66, 314–323.
16. Xie, F.; Zhao, H.; Zhao, L.; Lou, L.; Hu, Y. Synthesis and biological evaluation of novel 2,4,5-
substituted pyrimidine derivatives for anticancer activity. Bioorg. Med. Chem. Lett. 2009, 19, 275–278.
17. El-Deeb, I.M.; Lee, S.H. Design and synthesis of new anticancer pyrimidines with multiple-kinase
inhibitory effect. Bioorg. Med. Chem. 2010, 18, 3860–3874.
18. Prachayasittikul, S.; Worachartcheewan, A.; Nantasenamat, C.; Chinworrungsee, M.;
Sornsongkhram, N.; Ruchirawat, S.; Prachayasittikul, V. Synthesis and structure-activity
relationship of 2-thiopyrimidine-4-one analogs as antimicrobial and anticancer agents. Eur. J.
Med. Chem. 2011, 46, 738–742.

Molecules 2014, 19
289
19. Tsoukala, E.; Agelis, G.; Dolinsek, J.; Botic, T.; Cencic, A.; Komiotis, D. An efficient synthesis
of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil and 5-fluorouracil as potential
antitumor or/and antiviral agents. Bioorg. Med. Chem. 2007, 15, 3241–3247.
20. Manta, S.; Tsoukala, E.; Tzioumaki, N.; Kiritsis, C.; Balzarini, J.; Komiotis, D. Synthesis of
4,6-dideoxy-3-fluoro-β-D-glucopyranosyl analogues of 5-fluorouracil, N6-benzyl adenine, uracil,
thymine, N4-benzoyl cytosine and evaluation of their antitumor activities. Bioorg Chem. 2010, 38,
48–55.
21. Lauria, A.; Patella, C.; Abbate, I.; Martorana, A.; Almerico, A.M. An unexpected Dimroth
rearrangement leading to annelated thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidines with potent
antitumor activity. Eur. J. Med. Chem. 2013, 65, 381–388.
22. Matyugina, E.; Khandazhinskaya, A.; Chernousova, L.; Andreevskaya, S.; Smirnova, T.;
Chizhov, A.; Karpenko, I.; Kochetkov, S.; Alexandrova, L. The synthesis and antituberculosis
activity of 5'-nor carbocyclic uracil derivatives. Bioorg. Med. Chem. 2012, 20, 6680–6686.
23. Tobe, M.; Isobe, Y.; Goto, Y.; Obara, F.; Tsuchiya, M.; Matsui, J.; Hirota, K.; Hayashi, H.
Synthesis and biological evaluation of CX-659S and related compounds for their inhibitory effects
on the delayed-type hypersensitivity reaction. Bioorg. Med. Chem. 2000, 8, 2037–2047.
24. Bhabak, K.P.; Bhowmick, D. Synthesis and structural characterization of some trisulfide analoges
of thiouracil-based antithyroid drugs. J. Mol. Struct. 2012, 1022, 16–24.
25. Sriharsha, S.N.; Satish, S.; Shashikanth, S.; Raveesha, K.A. Design, synthesis and antibacterial
activity of novel 1,3-thiazolidine pyrimidine nucleoside analogues. Bioorg. Med. Chem. 2006, 14,
7476–7481.
26. Semenov, V.E.; Voloshina, A.D.; Toroptzova, E.M.; Kulik, N.V.; Zobov, V.V.; Giniyatullin, R.K.;
Mikhailov, A.S.; Nikolaev, A.E.; Akamsin, V.D.; Reznik, V.S. Antibacterial and antifungal
activity of acyclic and macrocyclic uracil derivatives with quaternized nitrogen atoms in spacers.
Eur. J. Med. Chem. 2006, 41, 1093–1101.
27. Svenstrup, N.; Kuhl, A.; Ehlert, K.; Habich, D. Improved synthesis of antibacterial 3-substituted
6-aniliouracils. Bioorg. Med. Chem. Lett. 2008, 18, 3215–3218.
28. Al-Abdullah, E.S.; Al-Obaid, A.M.; Al-Deeb, O.A.; Habib, E.E.; El-Emam, E.E. Synthesis of
novel 6-phenyl-2,4-disubstituted pyrimidine-5-carbonitriles as potential antimicrobial agents.
Eur. J. Med. Chem. 2011, 46, 4642–4647.
29. Krim, J.; Grunewald, C.; Taourirte, M.; Engels, J.W. Efficient microwave-assisted synthesis,
antibacterial activity and high fluorescence of 5-benzimidazolyl-2'-deoxyuridines. Bioorg. Med.
Chem. 2012, 20, 480–486.
30. Al-Deeb, O.A.; Al-Turkistani, A.A.; Al-Abdullah, E.S.; El-Brollosy, N.R.; Habib, E.E.;
El-Emam, A.A. Pyrimidine-5-carbonitriles-part III: Synthesis and antimicrobial activity of novel
6-(2-substituted propyl)-2,4-disubstituted pyrimidine-5-carbinitriles. Heterocycl. Commun. 2013,
19, 411–419.
31. Orr, G.F.; Musso, D.L.; Boswell, G.E.; Kelly, J.L.; Joyner, S.S.; Davis, S.T.; Baccanari, D.P.
Inhibition of uridine phosphorylase: Synthesis and structure-activity relationships of
aryl-substituted 5-benzyluracils and 1-[(2-hydroxyethoxy)methyl]-5-benzyluracils. J. Med. Chem.
1995, 38, 3850–3856.

Molecules 2014, 19
290
32. Murray, P.E.; McNally, V.A.; Lockyer, S.D.; Williams, K.J.; Stratford, I.J.; Jaffar, M.;
Freeman, S. Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as
novel inhibitors of thymidine phosphorylase. Bioorg. Med. Chem. 2002, 10, 525–530.
33. Focher, F.; Ubiali, D.; Pregnolato, M.; Zhi, C.; Gambino, J.; Wright, G.E.; Spadari, S.
Novel nonsubstrate inhibitors of human thymidine phosphorylase, a potential target for
tumor-dependent angiogenesis. J. Med. Chem. 2000, 43, 2601–2607.
34. El-Brollosy, N.R.; Al-Omar, M.A.; Al-Deeb, O.A.; El-Emam, A.A.; Nielsen, C. Synthesis of
novel uracil non-nucleosides analogues of 3,4-dihydro-2-alkylthio-6-benzyl-4-oxopyrimidines
and 6-benzyl-1-ethyloxymethyl-5-isopropyluracil. J. Chem. Res. 2007, 263–267.
35. El-Brollosy, N.R.; Jorgensen, P.T.; Dahan, B.; Boel, A.M.; Pedersen, E.B.; Nielsen, C.
Synthesis of Novel N-1 (allyloxymethyl) Analogues of 6-Benzyl-1-(ethoxymethyl)-5-
isopropyluracil (MKC-442, Emivirine) with Improved Activity Against HIV-1 and its Mutants.
J. Med. Chem. 2002, 45, 5721–5726.
36. El-Brollosy, N.R.; Pedersen, E.B.; Nielsen, C. Synthesis of novel MKC-442 analogues with
potent activities against HIV-1. Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241.
37. El-Essawy, F.A.; El-Brollosy, N.R.; Pedersen, E.B.; Nielsen, C. Synthesis of new uracil non-
nucleoside derivatives as potential inhibitors of HIV-1. J. Heterocyl. Chem. 2003, 40, 213–217.
38. Wamberg, M.; Pedersen, E.B.; El-Brollosy, N.R.; Nielsen, C. Synthesis of 6-arylvinyl analogues
of the HIV drugs SJ-3366 and Emivirine. Bioorg. Med. Chem. 2004, 12, 1141–1149.
39. El-Brollosy, N.R.; Nielsen, C.; Pedersen, E.B. Synthesis of N-1-(indanyloxymethyl) and
N-1-(4-Hydroxybut-2-enyloxymethyl) analogues of the HIV drug Emivirine and GCA-186.
Monatsh. Chem. 2005, 136, 1247–1254.
40. Sorensen, E.R.; El-Brollosy, N.R.; Jorgensen, P.T.; Pedersen, E.B.; Nielsen, C. Synthesis of
6-(3,5-dichlorobenzyl) derivatives as isosteric analogues of the HIV drug 6-(3,5-dimethylbenzyl)-
1-(ethoxymethyl)-5-isopropyluracil (GCA-186). Arch. Pharm. Chem. Life Sci. 2005, 338, 299–304.
41. El-Brollosy, N.R.; Sorensen, E.R.; Pedersen, E.B.; Sanna, G.; LaColla, P.; Loddo, R.
Synthesis and antiviral evaluation of 6-(trifluoromethylbenzyl) and 6-(fluorobenzyl) analogues of
HIV drugs emivirine and GCA-186. Arch. Pharm. Chem. Life Sci. 2008, 341, 9–19.
42. El-Brollosy, N.R.; Al-Deeb, O.A.; El-Emam, A.A.; Pedersen, E.B.; LaColla, P.; Collu, G.;
Sanna, G.; Loddo, R. Synthesis of novel uracil non-nucleoside derivatives as potential reverse
transcriptase inhibitors of HIV-1. Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670.
43. Penna, C.A.; Marino, S.G.; Gutkind, G.O.; Clavin, M.; Ferraro, G.; Martino, V. Antimicrobial
activity of Eupatorium species growing in Argentina. J. Herbs. Spices Med. Plants 1998, 5, 21–28.
44. Wilkins, T.D.; Holdeman, J.J.; Abramson, I.J.; Moore, W.E.C. Standardized single-disc method for
antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemother. 1972, 1, 451–455.
Sample Availability: Samples of the compounds 3a–d, 4a,b, 5a–c, 6a–f and 7a–d are available from
the corresponding author.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).