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Article

Synthesis of N-Substituted 5-Iodouracils as Antimicrobial and Anticancer Agents

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.
Molecules 2009, 14(8), 2768-2779; https://doi.org/10.3390/molecules14082768
Submission received: 6 July 2009 / Revised: 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 μg/mL. These 5-iodouracil analogs represent a new group of anticancer and antibacterial agents with potential for development for medicinal applications.

Graphical Abstract

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 .
Figure 1. Structures of substituted uracil analogs 1-4 .
Molecules 14 02768 g001
It is known that substituted uracils (especially at the 5-position) play a vital role in many metabolic processes [7,8,9]. 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.
Figure 2. Title substituted uracils 5 and 6.
Molecules 14 02768 g002

Results and Discussion

Chemistry

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: 7a7c > 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 [10,11,12,13]. 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.
Structures of the obtained 5-iodouracils 7 and 8 were established using 1H- and 13C-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 sharp peaks. The 1H-NMR spectra showed singlets of H-6 at δ 7.53-8.17 ppm, while the C-6 peak appeared at δ 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.
Table 1. Alkylation products from 5-iodouracil with alkyl and aralkyl bromides.
Table 1. Alkylation products from 5-iodouracil with alkyl and aralkyl bromides.
Molecules 14 02768 i001
EntryRSubstitution Products (%)
N1-N1, N3-
1 Molecules 14 02768 i0027a (28.0)8a (6.7)
2 Molecules 14 02768 i0037b (6.1)
3 Molecules 14 02768 i0047c (28.1)8b (7.5)
4 Molecules 14 02768 i0057d (11.4)
5 t-C4H9
6 1-Adm
7 CH2CH2−OH
Table 2. Selected spectral data of N1- and N1,N3-substituted uracils 7 and 8.
Table 2. Selected spectral data of N1- and N1,N3-substituted uracils 7 and 8.
Compoundδ (ppm)υmax (cm-1)Mass spectra (m/z)
H-6C-6C=ONHMolecular ionBase peak
7a7.59148.871715,16673022294294
7b7.53145.211716, 17003159294237
7c7.54149.361701, 16603159334238
7d8.17149.481714, 1669311232891
8a7.60146.741698, 1651°350333
8b7.53147.291701, 1653°430238

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 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.
Table 3. Antibacterial activity* of substituted 5-iodouracils 7 and 8.
Compound**ActivityInhibition (%)
7aActive50a25b,c
7cActive50a25b,c
7dActive50a25b,c
8bInactive00
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 μg/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 μg/mL, respectively. In addition, 8a inhibited the growth of MOLT-3 with IC50 of 37.53 μg/mL. The activity of T47D was also inhibited by 7d, showing IC50 of 43.0 μg/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 μg/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.
Table 4. Anticancer activity of substituted 5-iodouracils 7 and 8.
Table 4. Anticancer activity of substituted 5-iodouracils 7 and 8.
Cell lineIC50 (μg/mL)a,b
7a7b7c7d8a8bEtoposide(Doxorubicin)
HepG2>50>5036.00>50>5016.5012.00
HuCCA-1>50>50>50>50>5049.00(0.50)
A549>50>50>50>50>5033.000.60 (0.45)
MOLT-3NA>50NANA37.53>500.019
KB>50NA35.00>50NANA0.25
HCC-S102>50NA>50>50NANA6.00
HL60>50NA>50>50NANA0.85
P388>50NA41.47>50NANA0.12
HeLa>50NA46.00>50NANA0.38
MDA-MB231>50NA>50>50NANA0.24
T47D>50NA20.0043.00NANA0.05
H69AR>50NA50.00>50NANA30.00
NA = not tested. a: When IC50 >50 μg/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 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): υmax 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, H-1), 7.59 (s, 1H, H-6), 8.86 (br, 1H, NH-3); 13C-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): υmax 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): υmax3159, 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.
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): υmax3159, 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): υmax 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, 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); 13C-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 209-210°C (lit. mp 210-213°C [18]); IR (KBr): υmax 3112, 3011, 1714, 1669, 1452, 1426, 733 cm-1; 1H- 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 at 37 °C for 24 h, were diluted with 0.9% normal saline solution to adjust the cell density of 3×109 cell/mL. The organisms were inoculated onto each plate and further incubated at 37 oC 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.
Table 5. The twenty-seven strains of microorganisms used for antimicrobial activity testing.
Microorganisms
Gram-negative bacteria
Escherichia coli ATCC 25922Morganella morganii
Klebsiella pneumoniae ATCC 700603Vibrio cholera
Salmonella typhimurium ATCC 13311Vibrio mimicus
Salmonella choleraesuis ATCC 10708Aeromonas hydrophila
Pseudomonas aeruginosa ATCC 15442Plesiomonas shigelloides
Edwardsiella tardaXanthomonas maltophilia
Shigella dysenteriaeNeisseria mucosa
Citrobacter freundiiBranhamella catarrhalis
Gram-positive bacteria
Stapphylococcus aureus ATCC 25923Bacillus subtilis ATCC 6633
Stapphylococcus epidermidis ATCC 12228Streptococcus pyogenes
Enterococcus faecalis ATCC 29212Listeria monocytogenes
Micrococcus lutens ATCC 10240Bacillus cereus
Corynebacterium diphtheriae NCTC10356Micrococcusflavas
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 μL in each well and then add 50 μL 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.

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 μL) per well in 96-well plate, and incubated in humidified atmosphere, 95% air, 5% CO2 at 37 °C. After 24 h, additional medium (100 μL) containing the test compound and vehicle was added to a final concentration of 50 μg/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.
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)Hormone-dependent breast cancer cell line (T47D)
Hepatocellular carcinoma cell line (HCC-S102)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.
  • Samples Availability: Contact the authors.

References

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MDPI and ACS Style

Prachayasittikul, S.; Sornsongkhram, N.; Pingaew, R.; Worachartcheewan, A.; Ruchirawat, S.; Prachayasittikul, V. Synthesis of N-Substituted 5-Iodouracils as Antimicrobial and Anticancer Agents. Molecules 2009, 14, 2768-2779. https://doi.org/10.3390/molecules14082768

AMA Style

Prachayasittikul S, Sornsongkhram N, Pingaew R, Worachartcheewan A, Ruchirawat S, Prachayasittikul V. Synthesis of N-Substituted 5-Iodouracils as Antimicrobial and Anticancer Agents. Molecules. 2009; 14(8):2768-2779. https://doi.org/10.3390/molecules14082768

Chicago/Turabian Style

Prachayasittikul, Supaluk, Nirun Sornsongkhram, Ratchanok Pingaew, Apilak Worachartcheewan, Somsak Ruchirawat, and Virapong Prachayasittikul. 2009. "Synthesis of N-Substituted 5-Iodouracils as Antimicrobial and Anticancer Agents" Molecules 14, no. 8: 2768-2779. https://doi.org/10.3390/molecules14082768

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