Synthesis of N-substituted 5-iodouracils as antimicrobial and anticancer agents

Article abstract of DOI:10.3390/molecules14082768

This study reports the synthesis of some substituted 5-iodouracils and their bioactivities. Alkylation of 5-iodouracils gave predominately N1-substituted-(R)-5-iodouracil compounds 7a-d (R = n-C₄H ₉, s-C₄H₉, CH<

Full text of DOI:10.3390/molecules14082768

Molecules 2009, 14, 2768-2779; doi:10.3390/molecules14082768
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of N-Substituted 5-Iodouracils as Antimicrobial and
Anticancer Agents
Supaluk Prachayasittikul 1,*, Nirun Sornsongkhram 1, Ratchanok Pingaew 1,
Apilak Worachartcheewan 2, Somsak Ruchirawat 3 and Virapong Prachayasittikul 2,*
1
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110,
Thailand
2
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University,
Bangkok 10700, Thailand
3
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
* Authors to whom correspondence should be addressed; E-mail: supaluk@swu.ac.th (S.P.),
mtvpr@mahidol.ac.th (V.P.); Tel. +662-664-1000 ext 8209 (S.P.), +662-441-4376 (V.P.);
Fax: +662-259-2097 (S.P.); +662-441-4308 (V.P.)
Received: 6 July 2009; in revised form: 15 July 2009 / Accepted: 27 July 2009 /
Published: 27 July 2009
Abstract: This study reports the synthesis of some substituted 5-iodouracils and their
bioactivities. Alkylation of 5-iodouracils gave predominately N1-substituted-(R)-5-
iodouracil compounds 7a-d (R = n-C4H9, s-C4H9, CH2C6H11, CH2C6H5) together with
N1,N3-disubstituted (R) analogs 8a-b (R = n-C4H9, CH2C6H11). Their antimicrobial
activity was tested against 27 strains of microorganisms using the agar dilution method.
The analogs 7a, 7c and 7d displayed 25-50% inhibition against Branhamella catarrhalis,
Neisseria mucosa and Streptococcus pyogenes at 0.128 mg/mL. No antimalarial activity
was detected for any of the analogs when tested against Plasmodium falciparum (T9.94).
Their anticancer activity was also examined. Cyclohexylmethyl analogs 7c and 8b
inhibited the growth of HepG2 cells. Significantly, N1,N3-dicyclohexylmethyl analog 8b
displayed the most potent anticancer activity, with an IC50 of 16.5 g/mL. These 5-
iodouracil analogs represent a new group of anticancer and antibacterial agents with
potential for development for medicinal applications.

Molecules 2009, 14
Keywords: 5-iodouracil analogs; N-alkylation; antibacterial; antimalarial; anticancer
2769
activities
Introduction
A number of pyrimidine bases have been shown to possess antiviral and anticancer activities [1],
particularly uracils possessing halogens at the 5-position e.g. 5-fluorouracil; a well known anticancer
drug [2], and its N1-substituted derivative 1 [3], as well as nucleoside analogs 2 and 3 of 5-iodouracil
and 5-trifluoromethyluracil, which are antivirals [4]. Furthermore, acyclic-nucleoside analogs acting as
anti HIV-1 agents have been reported. Examples are acyclic 5,6-disubstituted uracils 4a-g (Figure 1)
[4,5]. In addition, N1,N3-disubstituted uracils were reported to exhibit antibacterial and antifungal
activities [6]. These N1- or N1,N3-substituted uracils were synthesized via alkylation of the
corresponding uracils [1,6].
Figure 1. Structures of substituted uracil analogs 1-4.
It is known that substituted uracils (especially at the 5-position) play a vital role in many metabolic
processes [7-9]. So far substituted 5-iodouracil and 5-hydroxymethyluracil analogs are quite rare in the
literature, so there is considerable interest in searching for novel bioactive uracils with substituents at
the N1 and or N1,N3 positions. The title molecules are substituted 5-iodo- and 5-hydroxymethyluracils
5 and 6 where R = alkyl and aralkyl (Figure 2). We report herein the synthesis of analogs 5 and 6 and
their evaluation for antibacterial, antimalarial and anticancer actions.
Figure 2. Title substituted uracils 5 and 6.

Molecules 2009, 14
Results and Discussion
Chemistry
2770
The title compounds 5 were synthesized by reacting 5-iodouracil with alkyl bromides (RBr) in
dimethyl sulfoxide at 80 °C for 48 h in the presence of potassium carbonate. Results are given in Table
1. It was found that alkylation of 5-iodouracil with RBr took place predominately at the N1 position
when R was derived from a primary or secondary alkyl bromide to give the products 1-(1-butyl)-5-
iodopyrimidine-2,4(1H, 3H)-dione (7a, R = n-C4H9, 28%), 1-(2-butyl)-5-iodopyrimidine-2,4(1H, 3H)-
dione (7b, R = s-C4H9, 6.1%), 1-(cyclohexylmethyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (7c, R =
CH2C6H11, 28.1%) and 1-benzyl-5-iodopyrimidine-2,4(1H, 3H)-dione (7d, R = CH2C6H5, 11.4%),
respectively. It was noted that primary alkyls (n-butyl and cyclohexylmethyl) give comparable or
higher yields than aralkyl (benzyl) and higher than secondary alkyl (s-butyl) as follows: 7a  7c > 7d >
7b. In addition, minor N1,N3-dialkylation products: 1,3-di(1-butyl)-5-iodopyrimidine-2,4(1H, 3H)-
dione (8a) and 1,3-bis(cyclohexylmethyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (8b) were observed in
comparable yields when R = n-butyl and cyclohexylmethyl, respectively. Such dialkylation of 5-
iodouracil was not observed in the reaction with benzyl bromide. The reactions of 5-iodouracil with
sterically hindered RBr such as R = t-C4H9 and 1-adamantyl (1-Adm) failed to give the products under
the same conditions or when the reaction was carried out in N,N-dimethylformamide containing
triethylamine at 140 °C for 10 h, as noted by TLC. This suggets that the N-alkylation proceeds via a
SN2 reaction. Unfortunately, 2-bromoethanol did not react with 5-iodouracil in the presence of K2CO3
or Et3N as observed by TLC. N-Functionalizations of uracils at the N1- and N1,N3-positions were
previously reported [10-13]. O-Alkylation of hydroxypyrimidines were also reported, e.g. 4,6-
dihydroxypyrimidines gave a mixture of O4,O6- and N1,O4-disubstituted products [14]. Attempts
were made to synthesize the title compound 6 under similar conditions as used for compound 5, but
this was unsuccessful.
1
13
Structures of the obtained 5-iodouracils 7 and 8 were established using H- and C-NMR, IR and
mass spectra. The IR spectra showed strong CO stretching bands in the 1,651-1,716 cm-1 range, while
the characteristic NH peak of N1 substituted uracils 7a-d appeared in the 3,022-3,159 cm-1 range as
1
sharp peaks. The H-NMR spectra showed singlets of H-6 at  7.53-8.17 ppm, while the C-6 peak
appeared at  145.21-149.48 ppm in the 13C-NMR spectra. The HMBC spectra exhibited relationships
between the H-6 proton and the carbons C-1, C-2, C-5 and C-4 and conversely, of H-1 with C-2, C-6
and C-2. Such C-H connectivity indicated that in the uracil analogs 7a-d the N1 position contained
alkyl or aralkyl group substituents. Similar correlations were also observed for H-1 with C-2, C-4 and
C-2, suggesting that in the case of analogs 8a and 8b additional substitution took place at N3. Both
N1- and N1, N3-substitution patterns were in evidence when R = n-C4H9 and CH2C6H11, as found in
uracils 7a, 7c and 8a, 8b, respectively. The mass spectra of analogs 7a-d and 8a-b all exhibited their
molecular ions and base peaks resulting from fragmentations of alkyl or aralkyl at the N1- and/or
N1,N3-positions, except for the analog 7a, which showed the molecular ion as the base peak (Table 2).
Based on 2D-NMR spectra (COSY, DEPT90, DEPT135, HMQC and HMBC), IR and mass spectra,
the substitution patterns of the N1- and N1,N3-alkylation products were clearly identified.

Molecules 2009, 14
Table 1. Alkylation products from 5-iodouracil with alkyl and aralkyl bromides.
2771
Substitution Products (%)
Entry
R
N1-
N1, N3-
1'
1
2
7a (28.0)
8a (6.7)
CH2CH2CH2CH3
1'
7b (6.1)

CH3CHCH2CH3
3
4
7c (28.1)
8b (7.5)
7d (11.4)

5
6
7
t-C4H9






1-Adm
CH2CH2OH
Table 2. Selected spectral data of N1- and N1,N3-substituted uracils 7 and 8.
 (ppm)
H-6 C-6

max (cm-1)
C=O
Mass spectra (m/z)
Compound
7a
NH
Molecular ion Base peak
7.59 148.87
1715,1667 3022
294
294
7b
7c
7d
8a
8b
7.53 145.21
7.54 149.36
8.17 149.48
7.60 146.74
7.53 147.29
1716, 1700 3159
1701, 1660 3159
1714, 1669 3112
294
334
328
350
430
237
238
91
1698, 1651
1701, 1653


333
238
Antibacterial activity
Antibacterial activity of the analogs 1-(1-butyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (7a), 1-(cyclo-
hexylmethyl)-5-iodopyrimidine-2,4(1H,3H)-dione (7c) and 1-benzyl-5-iodopyrimidine-2,4(1H,3H)-
dione (7d) and 1,3-bis(cyclohexylmethyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (8b) compounds and
was evaluated against 27 strains of microorganisms using the agar dilution method [15]. The results
(Table 3) showed that the analogs 7a, 7c and 7d inhibited 50% growth of B. catarrhalis and 25%
growth of N. mucosa and S. pyogenes at 0.128 mg/mL. However, 8b was found to be inactive against

Molecules 2009, 14
2772
all the tested microorganisms. The activity of such compounds has not been reported in the literature,
therefore, these analogs 7a, 7c and 7d are new antibacterial leads.
Table 3. Antibacterial activity* of substituted 5-iodouracils 7 and 8.
Compound**
Activity
Inhibition (%)
7a
7c
7d
8b
Active
Active
Active
Inactive
50a
50a
50a
0
25b,c
25b,c
25b,c
0
Inhibition against aB. catarrhalis, bN. mucosa, cS. pyogenes, *Ampicillin at 0.01 mg/mL was used as a control of
the antibacterial testing system; it showed 100% inhibition on selected microorganisms (S. aureus ATCC 25923
and B. subtilis ATCC 6633). **Concentration of 0.128 mg/mL was used.
Antimalarial activity
The activity of analogs 7a-d and 8a-b was tested as described [16] against Plasmodium falciparum
chloroquine resistant (T 9.94) using chloroquine hydrochloride as a reference drug. It was found that
all the tested compounds were inactive as antimalarials with IC50 >10-5 M.
Anticancer activity
Anticancer activity assays [17] against 12 cell lines using etoposide and/or doxorubicin as positive
controls were carried out. The results (Table 4) revealed that 1,3-bis(cyclohexylmethyl)-5-
iodopyrimidine-2,4(1H, 3H)-dione (8b) was active against HepG2, A549 and HuCCA-1 with IC50
values of 16.5, 33.0 and 49.0 g/mL, respectively. 1-(Cyclohexylmethyl)pyrimidine analog 7c
exhibited activity against T47D, KB, HepG2, P388 and HeLa cells with IC50 of 20.0, 35.0, 36.0, 41.47
and 46.0 g/mL, respectively. In addition, 8a inhibited the growth of MOLT-3 with IC50 of 37.53
g/mL. The activity of T47D was also inhibited by 7d, showing IC50 of 43.0 g/mL. It is notable that
the growth of HepG2 is selectively inhibited by 1- and 1,3-substituted cyclohexylmethyl analogs 7c
and 8b, respectively. However, the 1,3-bis(cyclohexylmethyl) analog 8b exhibited higher activity than
1-cyclohexylmethyl analog 7c. This perhaps due to higher lipophilicity of 1,3-disubstituted analog 8b
which enhances its absorption by the cancer cells. Significantly, the analog 8b was the most active
against HepG2 with IC50 of 16.5 g/mL. These compounds 7c-d and 8a-b are new potential anticancer
agents. Compounds 7a and 7b were inactive against all the tested cell lines.

Molecules 2009, 14
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Table 4. Anticancer activity of substituted 5-iodouracils 7 and 8.
IC50 (g/mL)a,b
Cell line
Etoposide
(Doxorubicin)
7a
7b
7c
7d
8a
8b
HepG2
HuCCA-1
A549
>50
>50
>50
NA
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
NA
NA
NA
NA
NA
NA
NA
36.00
>50
>50
>50
>50
NA
>50
>50
>50
>50
>50
>50
>50
>50
>50
37.53
NA
NA
NA
NA
NA
NA
NA
NA
16.50
49.00
33.00
>50
NA
12.00
(0.50)
0.60 (0.45)
0.019
0.25
>50
MOLT-3
KB
NA
35.00
>50
HCC-S102
HL60
NA
6.00
>50
NA
0.85
P388
41.47
46.00
>50
NA
0.12
HeLa
NA
0.38
MDA-MB231
T47D
NA
0.24
20.00 43.00
50.00 >50
NA
0.05
H69AR
NA
30.00
NA = not tested. a: When IC50 >50 g/mL denotes inactive for anticancer activity. b: The assays were
performed in triplicate.
Conclusions
Alkylation of 5-iodouracil furnished mainly N1-substituted uracils 7a-d, together with minor
amounts of the N1,N3-disubstituted analogs 8a-b, when the substituent groups (R) were primary and
secondary. Among these, 7b-c and 8a-b are new analogs. The analogs 7a (R = n-C4H9), 7c
(R = CH2C6H11) and 7d (R = CH2C6H5) showed 25-50% growth inhibition against B. catarrhalis,
N. mucosa and S. pyogenes at 0.128 mg/mL. No antifungal and antimalarial activities were observed
for any of the tested compounds. It is notable that anticancer activity was seen for the analogs 7c and
8b bearing a cyclohexylmethyl group (R = CH2C6H11). Significantly, the N1,N3-dicyclohexylmethyl
uracil analog 8b exhibited the most potent anticancer activity, but was inactive as an antibacterial. It
can be concluded that these 5-iodouracil analogs represent a new group of anticancer and antibacterial
agents with potential to be further developed for medicinal applications.
Experimental
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). Infrared spectra (IR) were obtained

Molecules 2009, 14
2774
on Perkin Elmer System 2000 FTIR. Mass spectra were recorded on a Finnigan INCOS 50 and Bruker
Daltonics (micro TOF) instruments. Column chromatography was carried out using silica gel 60
(0.063–0.200 mm). Analytical thin layer chromatography (TLC) was performed on silica gel 60 PF254
aluminium sheets (cat. No. 7747 E., Merck). Solvents were distilled prior to use. Chemicals used for
the syntheses were of analytical grade. Reagents for cell culture and assays were the following: RPMI-
1640 (Gibco and Hyclone Laboratories, USA), HEPES, L-glutamine, penicillin-streptomycin, sodium
pyruvate and glucose (Sigma, USA), Ham’s/F12, DMEM and fetal bovine serum (Hyclone
Laboratories, USA), Gentamicin sulfate (Government Pharmaceutical Organization, Thailand), 3(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, USA).
Synthesis of N-substituted 5-iodouracil analogs 7a-d and 8a-b
5-Iodouracil was dissolved in DMSO (5 mL), then K2CO3 was added and the mixture stirred at
80 C for 15 min. Alkylating agent was added dropwise (5 min) to the solution then stirred for 48 h at
80 C. Products were collected by filtration or by solvent extractions. Purification by silica gel column
using hexane-ethyl acetate (8:2) as eluting solvent gave the required compounds. The products were
recrystallized from methanol or dichloromethane-methanol (1:1).
1-(1-Butyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (7a) and 1,3-di(1-butyl)-5-iodopyrimidine-2,4(1H,
3H)-dione (8a): 5-Iodouracil (0.476 g, 2.0 mmol), K2CO3 (0.138 g, 1.0 mmol) and 1-butyl bromide
(0.274 g, 2.0 mmol) gave 7a (0.165 g, 25.01%) and 8a (0.047 g, 6.67%). Compound 7a; mp 175-176
°C; IR (KBr): max 3,022, 2,949, 1,715, 1,667, 1,606 cm-1; 1H-NMR (CDCl3): δ 0.95 (t, 3H, J = 7.2 Hz,
H-4), 1.35 (sextet, 2H, J = 7.2 Hz, H-3), 1.66 (quintet, 2H, J = 7.5 Hz, H-2), 3.73 (t, 2H, J = 7.2 Hz,
13
H-1), 7.59 (s, 1H, H-6), 8.86 (br, 1H, NH-3); C-NMR (CDCl3): δ 13.58 (C-4), 19.62 (C-3), 31.19
(C-2), 48.98 (C-1), 67.46 (C-5), 148.87 (C-6), 150.34 (C-2), 160.31 (C-4); LRMS (EI): m/z (%) = 295
(10.97) [M + H]+, 294 (100.00) [M]+, 238 (97.07), 167 (46.60); HRMS (TOF) m/z [M + H]+ calcd for
C8H12IN2O2: 294.9938 found: 294.9940. Compound 8a; mp 70-71 °C; IR (KBr): max 3,051, 1,698,
1,651, 594 cm-1; 1H-NMR (CDCl3): δ 0.93-1.01 (m, 6H, H-4, H-4), 1.32-1.44 (m, 4H, H-3, H-3),
1.57-1.74 (m, 4H, H-2, H-2), 3.76 (t, 2H, J = 7.4 Hz, H-1), 4.00 (t, 2H, J = 7.5 Hz, H-1), 7.60 (s, 1H,
H-6); 13C-NMR (CDCl3): δ 13.57 (C-4), 13.69 (C-4), 19.68 (C-3), 20.13 (C-3), 29.50 (C-2). 31.20
(C-2), 42.88 (C-1), 49.89 (C-1), 67.57 (C-5), 146.74 (C-6), 150.97 (C-2), 160.05 (C-4); LRMS (EI):
m/z (%) = 351 (20.33) [M + H]+, 350 (69.42) [M]+, 333 (100.00), 308 (32.97), 293 (58.48), 252
(72.27), 238 (30.76); HRMS (TOF): m/z [M + H]+ calcd for C12H20IN2O2: 351.2018 found: 351.0567.
1-(2-Butyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (7b): 5-Iodouracil (2.38 g (10.0 mmol), K2CO3 1.382
g (10.0 mmol) and 2-butyl bromide 1.372 g (10.0 mmol) furnished compound 7b 0.18 g (6.12%).
Compound 7b; mp 194-195 °C; IR (KBr): max3159, 3034, 2962, 1716, 1700, 1654, 1599, 612 cm-1;
1H-NMR (CDCl3): δ 0.9 (t, 3H, J = 7.2 Hz, H-4), 1.31 (d, 3H, J = 6.9 Hz, H-1), 1.57-1.68 (m, 2H, H-
3), 4.59 (sextet, 1H, J = 6.9 Hz H-2), 7.53 (s, 1H, H-6), 8.73 (br, 1H, NH-3); 13C-NMR (CDCl3): δ
10.52 (C-4), 19.64 (C-1), 28.69 (C-3), 53.78 (C-2), 67.87 (C-5), 145.21 (C-6), 150.61(C-2),
159.67(C-4); LRMS (EI): m/z (%) = 295 (11.18) [M + H]+, 294(47.12) [M]+, 237 (100), 167 (37.4);
HRMS (TOF) m/z [M + H]+ calcd for C8H12IN2O2: 294.9938 found: 294.9941.

Molecules 2009, 14
2775
1-(Cyclohexylmethyl)-5-iodopyrimidine-2,4(1H, 3H)-dione (7c) and 1,3-bis(cyclohexylmethyl)-5-iodo-
pyrimidine-2,4(1H, 3H)-dione (8b): 5-Iodouracil 0.476 g (2.0 mmol), K2CO3 0.138 g (1.0 mmol) and
(bromomethyl)cyclohexane 0.354 g (2.0 mmol) gave 7c 0.182 g (28.09%) and 8b 0.065 g (7.5%).
Compound 7c; mp 240-241°C; IR (KBr): max3159, 3021, 2920, 1701, 1660, 1606, 622 cm-1; 1H-NMR
(CDCl3): δ 0.92-1.75 (m, 11H, H-2, H-3, H-4, H-5), 3.54 (d, 2H, J = 7.2 Hz, H-1), 7.54 (s, 1H, H-6),
9.04 (br, 1H, NH-3); 13C-NMR (CDCl3): δ 25.47 (C-4), 26.07 (C-5), 30.30 (C-3), 37.35 (C-2), 55.13
(C-1), 67.14 (C-5), 149.36 (C-6), 150.58 (C-2), 160.38 (C-4); LRMS (EI) : m/z (%) = 335 (12.84) [M
+ H]+, 334 (76.68) [M]+, 252 (11.45), 238 (100.00), 208 (12.14); HRMS (TOF) m/z [M + H]+ calcd for
C11H16IN2O2: 335.0251 found: 335.0252. Compound 8b; m.p. 138-139°C; IR (KBr): max 3075, 2924,
1701, 1653, 1617 cm-1; 1H-NMR (CDCl3): δ0.89-1.81 (m, 22H, H-2, H-2, H-3, H-3,H-4, H-4, H-5,
13
H-5), 3.56 (d, 2H, J = 7.01 Hz, H-1), 3.84 (d, 2H, J = 7.2 Hz, H-1), 7.53 (s, 1H, H-6); C-NMR
(CDCl3): δ 25.51 (C-4), 25.77 (C-4), 26.11 (C-5), 26.29 (C-5), 30.39 (C-3), 30.76 (C-3), 36.19 (C-
2), 37.37 (C-2), 48.80 (C-1), 56.17 (C-1), 67.28 (C-5), 147.29 (C-6), 151.33 (C-2), 160.33 (C-4);
LRMS (EI): m/z (%) = 431 (9.87) [M + H]+, 430 (58.49) [M]+, 333 (64.21), 238 (100), 97 (79.25);
HRMS (TOF) m/z [M + H]+ calcd for C18H28IN2O2: 431.3237 found: 431.3242.
1-Benzyl-5-iodopyrimidine-2,4(1H, 3H)-dione (7d): 5-Iodouracil 0.476 g (2.0 mmol), K2CO3 (0.138 g,
1.0 mmol) and benzyl bromide (0.342 g, 2.0 mmol) gave compound 7d (0.075 g, 11.44%); mp 209-
1
210°C (lit. mp 210-213°C [18]); IR (KBr): max 3112, 3011, 1714, 1669, 1452, 1426, 733 cm-1; H-
NMR (CDCl3): δ 5.01 (s, 2H, H-1), 7.30-7.42 (m, 5H, ArH), 8.17 (s, 1H, H-6), 10.46 (br, 1H, NH-3);
13C-NMR (CDCl3): δ 50.86 (C-1), 67.17 (C-5), 136.63 (C-2),149.48 (C-6),150.79 (C-2), 160.28 (C-
4), 127.88,127.97,128.73 (Ar-C); LRMS (EI): m/z (%) = 329 (5.82) [M + H]+, 328 (48.13) [M]+, 91
(100.00); HRMS (TOF): m/z [M + H]+ calcd for C11H10IN2O2: 328.9781 found: 328.9791.
Chloroquine resistant Plasmodium falciparum (T9.94)
Human erythrocytes (type O) infected with chloroquine resistant P. falciparum (T9.94) were
maintained in continuous culture, according to the method described previously [16]. RPMI-1640
culture medium supplemented with 25mM HEPES, 40 mg/L gentamicin sulfate and 10 mL of human
serum was used in continuous culture.
Cancer cells
Cells were grown in Ham’s/F12 medium containing 2 mM L-glutamine supplemented with
100 U/mL penicillin-streptomycin and 10% fetal bovine serum. Except HepG2 and MOLT-3 cells
were grown in DMEM and RPMI-1640 medium, respectively.
Antimicrobial assay
Antimicrobial activity of the tested compounds was performed using agar dilution method as
previously described [15]. Briefly, the tested compounds dissolved in DMSO were individually mixed
with 1 mL Müller Hinton (MH) broth while the negative control was the MH broth with omission of
the tested compounds. The solution was then transferred to the MH agar solution to yield the final
concentrations of 0.032-0.256 mg/mL. Twenty seven strains of microorganisms, cultured in MH broth

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at 37 C for 24 h, were diluted with 0.9% normal saline solution to adjust the cell density of
o
3×109 cell/mL. The organisms were inoculated onto each plate and further incubated at 37 C for
18-48 h. Compounds which possessed high efficacy to inhibit bacterial cell growth were analyzed. The
microorganisms used for the activity testing are listed in Table 5.
Table 5. The twenty-seven strains of microorganisms used for antimicrobial activity testing.
Microorganisms
Gram-negative bacteria
Escherichia coli ATCC 25922
Klebsiella pneumoniae ATCC 700603
Salmonella typhimurium ATCC 13311
Salmonella choleraesuis ATCC 10708
Pseudomonas aeruginosa ATCC 15442
Edwardsiella tarda
Morganella morganii
Vibrio cholera
Vibrio mimicus
Aeromonas hydrophila
Plesiomonas shigelloides
Xanthomonas maltophilia
Neisseria mucosa
Shigella dysenteriae
Citrobacter freundii
Branhamella catarrhalis
Gram-positive bacteria
Stapphylococcus aureus ATCC 25923
Bacillus subtilis ATCC 6633
Streptococcus pyogenes
Listeria monocytogenes
Bacillus cereus
Stapphylococcus epidermidis ATCC 12228
Enterococcus faecalis ATCC 29212
Micrococcus lutens ATCC 10240
Corynebacterium diphtheriae NCTC10356
Micrococcus flavas
Diploid fungus (Yeast)
Candida albicans
Antimalarial assay
Antimalarial activity of the tested compounds was evaluated against Plasmodium falciparum
chloroquine resistant (T9.94) using the literature method [16,19]. The experiments were started with
synchronized suspension of 0.5% to 1% 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 suspension was put into 96-well microculture plate; 50 L in each well and then add
50 L of various tested drug concentrations. These parasite suspensions were incubated for 48 h in the
atmosphere of 5% CO2 at 37 C. The percents parasitemia of control and drug-treated groups were
examined by microscopic technique using methanol-fixed Giemsa stained of thin smear
blood preparation.

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Cytotoxicity assays
Cytotoxicity assays were performed using the modified method described previously [17]. Briefly,
cell lines suspended in RPMI-1640 containing 10% FBS were seeded at 1104 cells (100 L) per well
in 96-well plate, and incubated in humidified atmosphere, 95% air, 5% CO2 at 37 C. After 24 h,
additional medium (100 L) containing the test compound and vehicle was added to a final
concentration of 50 g/mL, 0.2% DMSO, and further incubated for 3 days. Cells were subsequently
fixed with 95% EtOH, stained with crystal violet solution, and lysed with a solution of 0.1 N HCl in
MeOH, after which absorbance was measured at 550 nm. Whereas HuCCA-1, A549 and HepG2 cells
were stained by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and for
MOLT-3 cell was stained by XTT. The cell lines used for the assay are listed in Table 6. IC50 values
were determined as the drug and sample concentration at 50% inhibition of the cell growth.
Table 6. Twelve cell lines used for the cytotoxicity assays.
Cell lines
Human hepatocellular liver carcinoma cell line
(HepG2)
Human promyelocytic leukemia cell line
(HL-60)
Human cholangiocarcinoma cancer cells (HuCCA-1) Murine leukemia cell line (P388)
Human lung carcinoma cell line (A549)
Cervical adenocarcinoma cell line (HeLa)
T-lymphoblast (MOLT-3, acute lymphoblastic
leukemia)
Hormone-independent breast cancer cell line
(MDA-MB231)
Human epidermoid carcinoma of the mouth (KB)
Hepatocellular carcinoma cell line (HCC-S102)
Hormone-dependent breast cancer cell line
(T47D)
Multidrug-resistance small cell lung cancer cell
line (H69AR)
Acknowledgements
This project was in part supported by a research grant from Mahidol University (B.E. 2551-2555).
We thank the Chulabhorn Research Institute for the antimalarial and anticancer testings. A.W. is
supported by the Royal Golden Jubilee (Ph.D.) scholarship of the Thailand Research Fund under
supervision of V.P.
References
1. Ogilvie, K.K.; Hamilton, R.G.; Gillen, M.F.; Radatus, B.K.; Smith, K.O.; Galloway, K.S. Uracil
analogues of the acyclonucleoside 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]- methyl]guanine
(BIOLF-62). Can. J. Chem. 1984, 62, 16-21.

Molecules 2009, 14
2778
2. Zenker, N. Thyroid function and thyroid drugs. In: Principles of Medicinal Chemistry. Foye
W.O., editor, 3rd ed.; Lea & Febiger: Philadelphia, London, UK, 1990. p. 603-621.
3. del Carmen Núñeza, M.; Entrena, A.; Rodríguez-Serrano, F.; Marchal, J.A.; Aránega, A.; Gallo,
M.A.; Espinosa, A.; Campos, J.M. Synthesis of novel 1-(2,3-dihydro-5H-4,1-benzoxathiepin-3-
yl)-uracil and -thymine, and their corresponding S-oxidized derivatives Tetrahedron 2005, 61,
10363-10369.
4. De Clercq, E. Antiviral drugs in current clinical use. J. Clin. Virol. 2004, 30, 115-133.
5. Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Nitta, I.; Baba, M.; Shigeta, S.; Walker,
R.T.; De Clercq, E.; Miyasaka, T. Synthesis and antiviral activity of deoxy analogs of 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents.
J. Med. Chem. 1992, 35, 4713-4719.
6. 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.
7. Kamalakannan, P.; Venkappayya, D.; Balasubramanian, T. A new antimetabolite, 5-
morpholinomethyl-2-thiouracil—spectral properties, thermal profiles, antibacterial, antifungal and
antitumour studies of some of its metal chelates. J. Chem. Soc., Dalton Trans. 2002, 3381-3391.
8. Motawia, M.S.; Abdel-Megied, A.E.S.; Pedersen, E.B.; Nielsen, C.M.; Ebbesen, P. Synthesis of
5-alkoxymethyl derivatives of 3'-amino-2',3'-dideoxyuridine and evaluation of their activity
against HIV and cancer. Acta Chem. Scand. 1992, 46, 77-81.
9. Fabrissin, S.; de Nardo, M.; Nisi, C.; Morasca, L.; Dolfini, E.; Franchi, G. Synthesis and
anticancer activity of 5-diethylaminomethyl derivatives and nitrogen mustards of uracil and 2-
thiouracils. J. Med. Chem. 1976, 19, 639-642.
10. Gondela, A.; Walczak, K. N-Functionalization of uracil derivatives: synthesis of chiral 2-(3-
methyl-5-nitro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)alkanoic acids and their methyl esters
Tetrahedron: Asymmetry 2005, 16, 2107-2112.
11. Malik, V.; Singh, P.; Kumar, S. Regioselective synthesis of 1-allyl- and 1-arylmethyl uracil and
thymine derivatives Tetrahedron 2005, 61, 4009-4014.
12. Frieden, M.; Giraud, M.; Reese, C.B.; Song, Q. Synthesis of 1-[cis-3-(hydroxymethyl)-
cyclobutyl]-uracil, -thymine and -cytosine. J. Chem. Soc., Perkin Trans. 1998, 1, 2827-2832.
13. Kundu, N.G.; Das, P.; Balzarini, J.; De Clercq, E. Palladium-catalyzed synthesis of [E]-6-(2-
acylvinyl)uracils and [E]-6-(2-acylvinyl)-1-[(2-hydroxyethoxy)methyl]uracils-their antiviral and
cytotoxic activities. Bioorg. Med. Chem. 1997, 5, 2011-2018.
14. Dolakova, P.; Dracinsky, M.; Masojidkova, M.; Solinova, V.; Kasicka, V.; Holy, A. Acyclic
nucleoside bisphosphonates: synthesis and properties of chiral 2-amino-4,6-bis[(phosphono-
methoxy)alkoxy]pyrimidines. Eur. J. Med. Chem. 2009, 44, 2408-2424.
15. Prachayasittikul, S.; Worachartcheewan, A.; Lawang, R.; Ruchirawat, S.; Prachayasittikul, V.
Activities of thiotetrahydropyridines as antioxidant and antimicrobial agents. EXCLI J. 2009, 8,
107-114.
16. Trager, W.; Jensen, J.B. Human malaria parasites in continuous culture. Science 1976, 193,
673-675.

Molecules 2009, 14
2779
17. Tengchaisri, T.; Chawengkirttikul, R.; Rachaphaew, N.; Reutrakul, V.; Sangsuwan, R.; Sirisinha,
S. Antitumor activity of triptolide against cholangiocarcinoma growth in vitro and in hamsters.
Cancer Lett. 1998, 133, 169-175.
18. Weaver, D.F.; Guillain, B.M.; Carran, J.R.; Jones, K. Preparation of pyrimidine derivatives as
anti-ictogenic and or/ anti-epileptogenic agents. WO Patent 2002083651, 2002 [Chem. Abstr.
2002, 137, 325-426].
19. Lambros, C.; Vanderberg, J.P. Synchronization of Plasmodium falciparum erythrocytic stages in
culture. J. Parasitol. 1979, 65, 418-420.
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