Synthesis and structure-activity relationship of 2-thiopyrimidine-4-one analogs as antimicrobial and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2010.12.009

Considering that some thiopyrimidines were previously reported as potential therapeutics, the present study achieved novel analogs of bioactive 2-substituted thiopyrimidines-4-(3H)-ones via base catalyzed alkylation reaction of 2-thiouracil using alkyl an

Full text of DOI:10.1016/j.ejmech.2010.12.009

European Journal of Medicinal Chemistry 46 (2011) 738e742
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationship of 2-thiopyrimidine-4-one analogs
as antimicrobial and anticancer agents
,
b
,
,c
Supaluk Prachayasittikul a , Apilak Worachartcheewan c, Chanin Nantasenamat b
,
*
Maneekarn Chinworrungsee a, Nirun Sornsongkhram a, Somsak Ruchirawat d,
**
Virapong Prachayasittikul c
,
a Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand
b Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
c Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand
d Chulabhorn Research Institute and Chulabhorn Graduate Institute, Bangkok 10210, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Considering that some thiopyrimidines were previously reported as potential therapeutics, the present
study achieved novel analogs of bioactive 2-substituted thiopyrimidines-4-(3H)-ones via base catalyzed
alkylation reaction of 2-thiouracil using alkyl and aralkyl bromides. The title compounds were 2-(1-
butylthio)pyrimidine-4(3H)-one (5a), 2-(2-butylthio)pyrimidine-4(3H)-one (5b), 2-(cyclohexylmethylthio)
pyrimidine-4(3H)-one (5c), 2-(benzylthio)pyrimidine-4(3H)-one (5d) and 2-(1-adamantylthio)pyrimidine-
4(3H)-one (5e). Bioactivity tests revealed that thiopyrimidines 5a, 5c, 5d and 5e exhibited antimicrobial
activity. The thiopyrimidine-4-one (5c) showed complete inhibition against Streptococcus pyogenes and
Branhamella catarrhalis as well as antifungal action against Candida albicans. Significantly, the 1-ada-
mantylthiopyrimidine (5e) was shown to be the most potent cytotoxic compound against multidrug-
resistant small cell lung cancer (H69AR). Their structureeactivity relationships were discussed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 27 October 2010
Received in revised form
7 December 2010
Accepted 9 December 2010
Available online 16 December 2010
Keywords:
2-Thiopyrimidine-4-one
2-Thiouracil
Alkylation
Antimicrobial activity
Cytotoxicity
Structureeactivity relationship
1. Introduction
bioactive 2-thiouracil analogs could be of interest and potential
target compounds. To investigate for new lead candidates as
Pyrimidine constitutes an important component of nucleic acid
and it is used as building blocks in pharmaceutics for the synthesis
of antiviral, anticancer [1,2] antibacterial and antifungal [3] agents.
Similarly, the related thiouracil derivatives are potential thera-
peutics e.g. as antivirals and anticancers [4,5]. In particular, 6-n-
propyl-2-thiouracil (6-PTU, 1) is an antithyroid drug [2] where its
S-alkylation (2) and N3-alkylation (4) products (Fig. 1) have been
recently reported as novel antibacterial, antimalarial and cytotoxic
agents [6]. Thiouracils and their nucleoside analogs are found in
natural sources; 2-thiouracil (2-TU, 3) is generally present in t-RNA
of Escherichia coli [7], 4-TU and 2-thiocytidine in other sources [7]
including 4-thiouridine in bacterial and archaeal t-RNA [5]. In
fact, 2-TU is a thyroid drug recognized as a highly specific mela-
noma seeker [7]. We bear in mind that novel small molecular
medicinally important agents, therefore, a series of S-substituted 2-
thiouracils 5 was designed and synthesized via an alkylation of 2-
TU (Fig. 2) as well as tested for their antimicrobial, antimalarial and
cytotoxic activities. Their structure and activity relationship were
also examined.
2. Results and discussion
2.1. Chemistry
Title S-substituted thiouracils 5aee were achieved through the
alkylation of 2-TU with alkylating agents (ReBr) in base catalysis
(Et3N or K2CO3). The results showed that the S-alkylation products; 2-
(1-butylthio)pyrimidine-4(3H)-one (5a), 2-(2-butylthio)pyrimidine-
4(3H)-one (5b), 2-(cyclohexylmethylthio)pyrimidine-4(3H)-one (5c),
2-(benzylthio)pyrimidine-4(3H)-one (5d) and 2-(1-adamantylthio)
pyrimidine-4(3H)-one (5e) were obtained in 17.6e37.5%, where
R ¼ n-C4H9 gave the highest yield (37.5%) of n-butylthio analog 5a. It
is interested to note that bulky 1-adamantyl bromide (1-AdmBr,
* Corresponding author. Tel.: þ66 2 664 1000x8209; fax: þ662 259 2097.
** Corresponding author. Tel.: þ66 2 441 4376; fax: þ66 2 441 4380.
E-mail addresses: supaluk@swu.ac.th (S. Prachayasittikul), mtvpr@mahidol.ac.th
(V. Prachayasittikul).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.009

S. Prachayasittikul et al. / European Journal of Medicinal Chemistry 46 (2011) 738e742
739
Fig. 1. Chemical structures of 2-thiouracil derivatives.
R ¼ 1-Adm) produced 1-adamantylthiopyrimidine (5e) in a compa-
rable yield to that of 5d deriving from benzyl bromide (R ¼ CH2C6H5).
Similar S-alkylation was observed for 6-n-propyl-2-thiouracil, except
the reaction with benzyl bromide which furnished N-3 benzylation
product [6]. In addition, S-alkylation of 6-methyl-2-thiouracil was
alsoreported [8]. Previously, 2-TUreacted withchloroacetaldehydeor
ketone to give S-alkylation product [9]. However, steric hindered
t-butyl bromide did not react with 2-TU (as evidence by TLC chro-
matogram). Structures of the 2-thiopyrimidine-4-ones (5aee) were
determined by spectral data. The IR spectra showed strong CO
absorption at 1652e1731 cmꢀ1 and strong or sharp NH absorption in
range 3200e3287 cmꢀ1. The 2-thiopyrimidines (5aee) all displayed
Branhamella catarrhalis at concentration range 64e128
Significantly, B. catarrhalis was completely inhibited by cyclo-
hexylmethylthiopyrimidine analog 5c with a minimum inhibitory
m
g/mL.
concentration (MIC) of 64
exhibited antigrowth activity against S. pyogenes with the MIC of
128 g/mL. Complete inhibition of the B. catarrhalis was also noted
for n-butylthiopyrimidine 5a at 128 g/mL. Interestingly, only the
2-thiopyrimidine compound 5c showed growth inhibition (75%)
against the diploid fungus, Candida albicans at 128 g/mL. Previ-
mg/mL. In addition, the compound 5c also
m
m
m
ously, chlorothiouracil-hydroxamates were reported to exhibit
growth inhibition against C. albicans [14]. However, antimicrobial
activity of such 2-thiopyrimidines has not been reported. Thus,
these 2-thiopyrimidines 5a and 5cee represent a novel group of
antimicrobials.
doublets of H-5 (
constants of 6.4e6.6 Hz. 13C NMR spectra showed C-4 carbonyls at
162.73e164.69 ppm and C-2 of thioimines at 160.98e162.55 ppm.
d 6.20e6.22) and H-6 (d 7.83e7.88) with coupling
d
d
From HMBC spectra H-6 proton of 2-thiopyrimidine analogs 5aee
showed correlations with C-2, C-4 and C-5. On the other hand, H-10 of
the 2-thiopyrimidine analogs, except 1-adamantylthiopyrimidine 5e,
were also correlated with C-2, C-20 and ArC. Such behaviour was not
observed for the 2-thiopyrimidine 5e due to C-10 of 1-Adm group is
a quaternary carbon lacking of H-10. In addition, C-2 of the products
2.2.2. Antimalarial activity
2-Thiopyrimidine-4-one analogs 5aee were screened against
Plasmodium falciparum chloroquine resistant (T9.94). Results
revealed that the tested compounds were inactive antimalarials
showing an IC50 > 10ꢀ5 M, excepted 5b was insoluble in the testing
medium.
5aee appeared at d 160.98e162.55 ppm where C-2 of the starting 2-
TU displayed at 172.9 ppm. This confirms that S-alkylation reaction
took place ata thione group. The massspectra support the obtained S-
alkylated 2-thiopyrimidines 5aed which all showed molecular ions
[M]þ or [M þ H]þ as their base peaks. Except the analog 5e exhibited
a fragment ion at m/z 135 (1-Adm) as the base peak. Such fragmen-
tation is uniquely found in 1-adamantylthio analogs of pyridines
[10e12] and pyrimidines [6]. However, the 2-thiopyrimidines 5b, 5c
and 5e are new S-alkylated 2-thiouracils.
2.2.3. Cytotoxic activity
Cytotoxic activity of S-alkylated pyrimidine analogs 5aee was
performed against eleven cancer cell lines using a modified method
[15]. The results (Table 2) showed that s-butylthio- and benzylthio-
pyrimidines (5b and 5d) were inactive toward all the tested cancer
cells. No cytotoxic activity against HuCCA-1, MDA-MB 231, A549,
HeLa, HCC-S102 and HL-60 cell lines were observed for all the
tested analogs. Cyclohexylmethylthiopyrimidine analog 5c selec-
tively exhibited cytotoxic activity against P388 cell with the IC50 of
40.36
activity against many cancer cells; T47D, H69AR, HepG2 and P388
with the IC50 of 32.0, 35.0, 32.5 and 36.91 g/mL, respectively. It is
notable that 5e is the most potent cytotoxic agent against H69AR
with the IC50 of 35.0 g/mL where the IC50 of the control etoposide
is 30.0 g/mL. This is presumably due to a high lipophilicity of the
mg/mL 1-Adamantylthiopyrimidine-4-one (5e) exerted
2.2. Bioactivities
m
2.2.1. Antimicrobial activity
2-Thiouracil analogs 5aee were tested for antimicrobial activity
against twenty-seven strains of microorganisms using the agar
dilution method [13]. It was found that (Table 1) the tested analogs
all displayed growth inhibition against Streptoccus pyogenes and
m
m
1-Adm moiety which enhances its absorption to the cancer cells.
O
O
4
4
HN
HN
R-Br
Conditions
2
6
2
6
R
S
N
H
R'
S
N
R'
3
5
, R' = H
, R' = H
Conditions
a: DMF, Et3N, 140oC, 10 h
b: C2H5OH or H2O, K2CO3, reflux, 9-16 h
a
b
c
, R = n-C4H9
, R = s-C4H9
, R = CH2C6H11
d
, R = CH2C6H5
, R = 1-Adm
e
Fig. 2. S-alkylated thiouracil 5aee analogs.

740
S. Prachayasittikul et al. / European Journal of Medicinal Chemistry 46 (2011) 738e742
Table 1
(2c) at C-6 position [6] displayed the highest cytotoxic activity
against H69AR cell that was comparable to the activity of S-1-Adm
analog (5e, R0 ¼ H, absence of substituent at C-6). This is presum-
ably due to a hydrophobic effect of sterically hindered 1-Adm group
that enhances the penetration of compound 5e to the cancer cell.
Taken together of the S-alkylated (R) title compounds, the prom-
ising results were achieved when R ¼ CH2C6H11 and 1-Adm groups
as seen for 2-thiopyrimidine-4-one compounds 5c, 2c and 5e, 2d,
respectively. It was reported that methylcyclohexyl group when
substituted to pyrimidine-2,4-dione e.g. 5-iodouracil afforded N1
and N1,N3-alkylated products with good cytotoxic activity [13].
Similarly, 1-Adm group containing thiopyridine derivatives were
reported to be active antimicrobials [19], antioxidants [20], anti-
malarials, anticancers [21,22] and vasorelaxants [23]. In addition, 1-
Adm moiety constituting thiotetrahydropyridines also exhibited
antimicrobial and antioxidative activities [24,25]. From the results,
1-Adm and methylcyclohexyl are alkylated groups that could
possibly be functionalized to other related heterocyclic rings in
a search for seeking new bioactive compounds. Therefore, in
relating their structures (analogs 5 and 2) and bioactivities, it could
be seen that either 1-Adm or methylcyclohexyl group is likely to be
an important alkylated group for 2-thiopyrimidine-4-one analogs
as bioactive compounds. In particular, 1-Adm analog of 2TU (5e)
was shown to be the new and significant cytotoxic compound
against H69AR cell. Such cytotoxic activity was also noted for the
analog of 6PTU (2c) bearing methylcyclohexyl group at 2-thio
function. Up to this point, it could be concluded that S-alkylation of
the thyroid drugs (2TU and 6PTU) provided the analogs as the
promising cytotoxic agent as well as the antimicrobials.
Antimicrobial activitye of 2-thiopyrimidine-4-ones 5aee.
Compoundf
Activity
Inhibition (%)
64 g/mL
m
128 mg/mL
5a
5c
5d
5e
Active
Active
Active
Active
0
25a,c, 100b
25c, 75d, 100a
25a, 50b
100b
0
0
25a,c, 75b
Inhibition against aS. pyogenes, bB. catarrhalis, cN. mucosa, dC. albicans.
e
Ampicillin at 10 mg/mL was used as a control of the antibacterial testing system;
it showed 100% growth inhibition against S. aureus ATCC 25923, B. subtilis ATCC
6633, S. epidermidis ATCC 12228, S. pyogenes, E. tarda, N. mucosa and B. catarrhalis.
f
Compound 5b was not tested (insoluble in the testing medium).
This hydrophobic nature of 1-Adm moiety was found in a diverse
group of bioactive compounds [16e18]. In addition, n-butylth-
iopyrimidine analog (5a) displayed activity against KB and T47D
cells with the IC50 of 27.0 and 5.0 mg/mL, respectively. However,
cytotoxic activity of 5aee has not been reported in the literature.
Hence, the 2-thiopyrimidine analogs 5a, 5c and 5e are new series of
cytotoxic agents.
2.2.4. Structureeactivity relationship
According to the results of bioactivities, it is noted that S-alky-
lated (R) 2-thiopyrimidine-4-ones with R ¼ CH2C6H11 (5c) provides
better antimicrobial activity, against B. catarrhalis, than the other
active compounds (5a, 5d and 5e). Whereas R ¼ 1-Adm, the
compound 5e exerted cytotoxic action against H69AR cell with the
significant IC50 value as compared to the reference drug. In
comparison with the 2-thiopyrimidine-4-one analog 2 [6] bearing
substituent (R0 ¼ n-propyl) at C-6 position, the best antimicrobials
was observed when R ¼ 1-Adm (2d). The compound with an
absence of n-propyl group at C-6 (5e, R ¼ 1-Adm) also showed
partial inhibition (75%) against the same organism (B. catarrhalis) at
3. Conclusion
2-TU reacted with alkyl or aralkyl bromides in the presence of
Et3N or K2CO3 to furnish exclusively S-substituted 2-thiouracils
5aee from which 5b, 5c and 5e are new compounds. 2-Thiouracil
analogs 5a and 5cee exerted antigrowth activity against S. pyogenes
and B. catarrhalis in concentration range of 64e128 mg/mL. Signif-
icantly, the cyclohexylmethylthiopyrimidine 5c exhibited complete
inhibition against S. pyogenes and B. catarrhalis with the MICs of 128
128 mg/mL. This suggests that the best antimicrobial agent analog of
2-thiopyrimidine-4-one requires hydrophobic groups at both
2-thio and C-6 positions, like compound 2d [6]. In addition,
S-methylcyclohexyl derivative of 2-thiopyrimidine-4-one with
substituent (R0 ¼ n-C3H7, 2c) or without substituent (R0 ¼ H, 5c) at
C-6 provided the antimicrobial action against B. catarrhalis with the
same MIC value. Apparently, S-methylcyclohexyl derivative of 2-
thiopyrimidine-4-one analog containing substituent; R0 ¼ n-C3H7
and 64 mg/mL, respectively. Only the analog 5c that could show
antifungal action against C. albicans. 1-Adamantylthiopyrimidines
5e displayed cytotoxic activity against many cell lines; T47D,
H69AR, HepG2 and P388. Significant activity of 5e was observed
Table 2
Cytotoxic activity of 2-thiopyrimidine-4-ones 5aee.
against H69AR with the IC50 of 35.0
mg/mL, whereas the control
Cell linesa
IC50
(
m
g/mL)b,c
drug; etoposide showing the IC50 of 30.0
m
g/mL. All the tested 2-
thiopyrimidines were shown to be inactive antimalarials. Their
structureeactivity relationships were examined and found that 1-
Adm and CH2C6H11 were important alkylated groups for 2-thio-
pyrimidine-4-one analogs producing significant bioactivities.
In conclusion, the study leads to the identification of novel
antimicrobials (5a and 5cee) and cytotoxic agents (5a, 5c and 5e).
Significantly, 5c exhibits both antibacterial and antifungal actions,
whereas, 5e was found to be the most potent cytotoxic compound
against multidrug-resistant small cell lung cancer (H69AR). The
findings demonstrate a new potential for 2-thiopyrimidine-4-ones
as lead compounds for further development as medicinal agents.
5a
5b
5c
5d
5e
Etoposided
KB
27.00
>50
>50
5.0
>50
>50
>50
>50
>50
>50
>50
NA
>50
NA
NA
>50
NA
NA
>50
NA
NA
NA
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
32.00
>50
35.00
>50
32.5
0.25
4.00
0.24
0.05
0.60
30.00
0.38
21.00
6.00
0.85
0.12
HuCCA-1
MDA-MB 231
T47D
A549
H69AR
HeLa
HepG2
HCC-S102
HL-60
50.0
>50
>50
>50
>50
>50
>50
36.91
P388
40.36
NA: indicates not tested (insoluble in the testing medium).
a
Cancer cell lines were human epidermoid carcinoma of the mouth (KB), human
cholangiocarcinoma cancer cells (HuCCA-1), hormone-independent breast cancer
cell line (MDA-MB231), hormone-dependent breast cancer cell line (T47D), human
lung carcinoma cell line (A549), multidrug-resistant small cell lung cancer cell line
(H69AR), cervical adenocarcinoma cell line (HeLa), human hepatocellular liver
carcinoma cell line (HepG2), hepatocellular carcinoma cell line (HCC-S102), human
4. Materials and methods
4.1. General
Melting points were determined on an Electrothermal melting
point apparatus (Electrothermal 9100) and are uncorrected. 1H and
13C NMR spectra were recorded on a Bruker AVANCE 300 NMR
spectrometer (operating at 300 MHz for 1H and 75 MHz for 13C).
promyelocytic leukemia cell line (HL-60), murine leukemia cell line (P388).
b
When IC50 > 50 mg/mL denotes inactive compound.
The assays were performed in triplicate.
Etoposide was used as a reference drug.
c
d

S. Prachayasittikul et al. / European Journal of Medicinal Chemistry 46 (2011) 738e742
741
Infrared spectra (IR) were obtained on a Perkin Elmer System 2000
FTIR. Mass spectra were recorded on a Finnigan INCOS 50 and
a Bruker Daltonics (micro TOF). Elemental analysis was performed
on a Perkin Elmer Elemental analyzer 2400 CHN. Column chro-
matography was carried out using silica gel 60 (0.063e0.200 mm).
Analytical thin layer chromatography (TLC) was performed on silica
gel 60 PF254 aluminium sheets (cat. No. 7747 E., Merck).
(C-40),26.14 (C-50), 32.52 (C-30), 37.39 (C-20), 37.6 (C-10), 110.84 (C-
5),154.77 (C-6), 162.55 (C-2), 164.42 (C-4); LRMS(EI): m/z (%) ¼ 226
(13.09) [M þ H]þ, 225 (100.00) [M]þ, 178 (7.45), 129 (60.25); HRMS
(TOF): m/z [M þ H]þ: calcd for C11H17N2OS: 225.1056 found:
225.1050. Anal. Calcd. for C11H16N2OS: C, 58.90; H, 7.19; N, 12.49.
Found: C, 58.41; H, 6.99; N, 12.39.
Solvents were distilled prior to use. Chemicals for the synthesis
and bioactivity testings were of analytical grade. Reagents for cell
culture and assay were the following: RPMI-1640 (Rosewell Park
Memorial Institute medium, Gibco and Hyclone laboratories, USA),
HEPES (N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid),
4.2.4. 2-(Benzylthio)pyrimidine-4(3H)-one (5d)
2-TU (5 mmol) reacted with benzyl bromide (5 mmol) in DMF
(5 mL) and Et3N (2 mL) to give 2-(benzylthio)pyrimidine-4(3H)-one
(5d) 0.28 g (25.3%); mp 220e221 ꢁC (193e195 ꢁC [27], 174e175 ꢁ
C
[28]); IR(KBr) ymax: 3200, 3071, 1731, 1504 cmꢀ1 1H NMR(CDCl3):
;
L-glutamine, penicillin, streptomycin, sodium pyruvate and glucose
d
4.41 (s, 2H, H-10), 6.22 (d, 1H, J ¼ 6.5 Hz, H-5), 7.25e7.38 (m, 5H,
(Sigma, USA), Ham’s/F12 (Nutrient mixture F-12), DMEM (Dulbec-
co’s Modified Eagle’s Medium) and FBS (fetal bovine serum,
Hyclone laboratories, USA), gentamicin sulfate (Government Phar-
maceutical Organization, Thailand), MTT (3(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide, SigmaeAldrich, USA).
Ar-H), 7.88 (d, 1H, J ¼ 6.5 Hz, H-6); 13C NMR (CDCl3):
d
35.02(C-10),
111.07(C-5), 127.78e129.21 (Ar-C), 135.74 (C-20), 154.62 (C-6),
161.78 (C-2), 164.69 (C-4); LRMS (EI): m/z (%) ¼ 219 (12.44)
[M þ H]þ, 218 (100.00) [M]þ, 185 (56.56), 91 (26.94); HRMS (TOF)
m/z [M þ H]þ calcd. for C11H11N2OS: 219.0587 found: 219.0582.
Anal. Calcd. for C11H10N2OS: C, 60.53; H, 4.62; N, 12.83. Found: C,
61.44; H, 4.60; N, 12.74.
4.2. Synthesis of S-substituted 2-thiouracils 5aee
To a solution of 2-TU (3) and alkyl or aralkyl bromide in DMF (or
water or ethanol) was added triethylamine (or K2CO3) and then
heated under reflux for 10e16 h. The reaction was monitored by
TLC, extracted and worked up as usual. The products were purified
by silica gel column and recrystallized in methanol or dichloro-
methane/methanol.
4.2.5. 2-(1-Adamantylthio)pyrimidine-4(3H)-one (5e)
2-TU (10 mmol) reacted with 1-AdmBr (10 mmol) in DMF (8 mL)
and Et3N (2 mL). After chromatographic separation gave 2-(1-ada-
mantylthio)pyrimidine-4(3H)-one (5e) 0.59
g
(22.5%); mp
165e166 ꢁC; IR (KBr) ymax: 3287, 2915, 1667, 1565, 1526 cmꢀ1
;
1H
NMR (CDCl3):
d
1.67e2.26 (m,15H, 1-Adm-H), 6.22 (d, 1H, J ¼ 6.6 Hz,
H-5), 7.83 (d, 1H, J ¼ 6.6 Hz, H-6); 13C NMR (CDCl3):
d 111.54 (C-5),
4.2.1. 2-(1-Butylthio)pyrimidine-4(3H)-one (5a)
154.53 (C-6), 160.98 (C-2), 164.21 (C-4), 29.34e53.37 (1-Adm-C);
LRMS(EI): m/z (%) ¼ 263 (13.87) [M þ H]þ, 262 (49.62) [M]þ, 261
(70.46), 135 (100.00); HRMS (TOF): m/z [M þ H]þ calcd for
C14H19N2OS: 263.1213 found: 263.1218. Anal. Calcd. for C14H18N2OS:
C, 64.09; H, 6.91; N, 10.68. Found: C, 65.06; H, 6.68; N, 10.66.
2-TU (3 mmol) reacted with n-BuBr (6 mmol) and K2CO3
(3 mmol) in C2H5OH (30 mL) to furnish 2-(1-butylthio)pyrimidine-
4(3H)-one (5a) 0.21 g (37.5%); mp 89e90 ꢁC (98e99 ꢁC [26]), IR
(KBr) ymax: 3202, 2952, 1652, 1541 cmꢀ1; 1H NMR (CDCl3):
d 0.91 (t,
3H, J ¼ 7.2 Hz, H-40), 1.49 (sextet, 2H, J ¼ 7.2 Hz, H-30), 1.72 (quintet,
2H, J ¼ 7.2 Hz, H-20), 3.18 (t, 2H, J ¼ 7.2 Hz, H-10), 6.21 (d, 1H,
J ¼ 6.4 Hz, H-5), 7.85 (d, 1H, J ¼ 6.4 Hz, H-6); 13C NMR (CDCl3):
4.3. Bioactivities
d
12.93 (C-40), 21.58 (C-30), 28.92 (C-10), 31.21 (C-20), 110.30 (C-5),
4.3.1. Antimicrobial assay
153.69 (C-6), 162.04 (C-2), 162.73 (C-4); LRMS (EI): m/z (%) ¼ 185
(100.00) [M þ H]þ, 184 (6.60) [M]þ, 152 (11.45), 137 (23.17); HRMS
(TOF): m/z [M þ H]þ calcd for C8H13N2OS: 185.0743 found:
185.0749. Anal. Calcd. for C8H12N2OS: C, 52.15; H, 6.56; N, 15.20.
Found: C, 52.59; H, 6.26; N, 15.10.
Antimicrobial activity of the tested compounds was performed
using the agar dilution method as previously described [13]. In
brief, the tested compounds dissolved in DMSO were individually
mixed with 1 mL Müller Hinton (MH) broth while the negative
control was the MH broth without the tested compounds. The
solution was then transferred to the MH agar solution to yield the
4.2.2. 2-(2-Butylthio)pyrimidine-4(3H)-one(5b)
final concentrations of 32e128 mg/mL. Twenty-seven strains of
2-TU (5 mmol) reacted with s-BuBr (5 mmol) and K2CO3
(2.5 mmol) in H2O (20 mL) to give 2-(2-butylthio)pyrimidine-4
(3H)-one (5b) 0.16 g (17.6%); mp 90e91 ꢁC; IR (KBr) ymax: 3202,
microorganisms as shown below, cultured in MH broth at 37 ꢁC for
24 h, were diluted with 0.9% normal saline solution to adjust the
cell density of 3 ꢂ 109 cell/mL. The microorganisms were inocu-
lated onto each plate and further incubated at 37 ꢁC for 18e48 h.
Compounds which exerted high efficacy to inhibit cell growth of
the organisms were determined. The DMSO was tested in parallel
with the compounds and showed no effect on the tested organ-
isms. Twenty-seven strains of tested microorganisms were gram
negative bacteria: Escherichia coli ATCC 25922, Klebsiella pneu-
moniae ATCC 700603, Salmonella typhimurium ATCC 13311,
Salmonella choleraesuis ATCC 10708, Pseudomonas aeruginosa ATCC
15442, Edwardsiella tarda, Shigella dysenteriae, Citrobacter freundii,
Morganella morganii, Vibrio cholera, Vibrio mimicus, Aeromonas
hydrophila, Plesiomonas shigelloides, Xanthomonas maltophilia,
Neisseria mucosa, Branhamella catarrhalis; gram positive bacteria:
Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Enterococcus faecalis ATCC 29212, Micrococcus lutens
ATCC 10240, Corynebacterium diphtheriae NCTC 10356, Bacillus
subtilis ATCC 6633, Streptococcus pyogenes, Listeria monocytogenes,
Bacillus cereus, Micrococcus flavas and diploid fungus (yeast):
Candida albicans.
2969, 2923, 2871, 1653 cmꢀ1 1H NMR (CDCl3):
; d 1.02 (t, 3H,
J ¼ 6.9 Hz, H-40), 1.39 (d, 3H, J ¼ 6.9 Hz, H-10), 1.70 (quintet, 2H,
J ¼ 6.9 Hz, H-30), 3.91 (sextet, 1H, J ¼ 6.9 Hz, H-20), 6.20 (d, 1H,
J ¼ 6.6 Hz, H-5), 7.84 (d, 1H, J ¼ 6.6 Hz, H-6); 13C NMR (CDCl3):
d
11.32 (C-40), 20.61 (C-10), 29.23 (C-30), 43.31 (C-20), 110.78 (C-5),
154.7(C-6), 162.45 (C-2), 164.58 (C-4); LRMS (EI): m/z (%) ¼ 185
(100) [M þ H]þ, 184 (5.2) [M]þ, 152 (23.8), 137 (15.1), 124 (11.9);
HRMS(TOF): m/z [M þ H]þ calcd for C8H13N2OS: 185.0743 found:
185.0737. Anal. Calcd. for C8H12N2OS: C, 52.15; H, 6.56; N, 15.20.
Found: C, 52.46; H, 6.56; N, 15.57.
4.2.3. 2-(Cyclohexylmethylthio)pyrimidine-4(3H)-one (5c)
2-TU (5 mmol) reacted with cyclohexylmethyl bromide (5 mmol)
in DMF (5 mL) and Et3N (2 mL) to afford 2-(cyclohexylmethylthio)
pyrimidine-4(3H)-one (5c) 0.20 g (17.8%); mp 150e151 ꢁC; IR(KBr)
ymax: 3201, 2927, 2851, 1658 cmꢀ1; 1H NMR (CDCl3):
d 1.10e2.25 (m,
H-20, H-30, H-40, H-50), 3.10 (d, 2H, J ¼ 6.9 Hz, H-10), 6.21 (d, 1H,
J ¼ 6.6 Hz, H-5), 7.84 (d,1H, J ¼ 6.6 Hz, H-6); 13C NMR (CDCl3):
d 25.92

742
S. Prachayasittikul et al. / European Journal of Medicinal Chemistry 46 (2011) 738e742
4.3.2. Antimalarial assay
C.N. gratefully acknowledges support from the Science and Tech-
nology Research Grant of the Thailand Toray Science Foundation
and Mahidol University. A.W. is supported by the Royal Golden
Jubilee Ph.D. scholarship of the Thailand Research Fund under the
supervision of V.P.
Antimalarial activity of the tested compounds was evaluated
against P. falciparum chloroquine resistant (T9.94) using the liter-
ature method [29].
Human erythrocytes (type O) infected with P. falciparum chloro-
quine resistant (T9.94) were maintained in continuous culture,
according to the method described previously [30]. RPMI-1640
culture medium supplemented with 25 mM of HEPES, 40 mg/L
gentamicin sulfate and 10 mL of human serum was used in contin-
uous culture.
Initially, P. falciparum culture was synchronized by using sorbitol
induced hemolysis according to the method of Lambros and Van-
derberg [31] to obtain only ring stage-infected red blood cells and
then incubated for 48 h prior to the drug testing to avoid effect of
sorbitol. The experiments were started with synchronized
suspension of 0.5e1% infected red blood cell during ring stage.
Parasites were suspended with culture medium supplemented
with 15% human serum to obtain 10% cell suspension. The parasite
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This work was supported in part by the research grants of
Srinakharinwirot University (B.E. 2551) and Mahidol University
(B.E. 2551e2555). This project is supported by the Office of the
Higher Education Commission and Mahidol University under the
National Research Universities Initiative. We thank the Chulabhorn
Research Institute for the antimalarial and cytotoxic assays.
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