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

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.

Structures of the obtained 5-iodouracils 7 and 8 were established using 1 H-and 13 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 sharp peaks. The 1 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 13 C-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-C 4 H 9 and CH 2 C 6 H 11 , 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.

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 IC 50 >10 -5 M.

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-C 4 H 9 ), 7c (R = CH 2 C 6 H 11 ) and 7d (R = CH 2 C 6 H 5 ) 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 = CH 2 C 6 H 11 ). 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.

Synthesis of N-substituted 5-iodouracil analogs 7a-d and 8a-b
5-Iodouracil was dissolved in DMSO (5 mL), then K 2 CO 3 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).

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 3×10 9 cell/mL. The organisms were inoculated onto each plate and further incubated at 37 o 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. 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% CO 2 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.