An Efficient Synthesis and In vitro Cytostatic Activity of 5-Aminosulfonyl Uracil Derivatives

: Efficient synthesis of 5 -aminosulfonyl uracil derivatives 2 − 9 and results of their antiproliferative activity are provided. Sulfonylation of the amino group in 5 -aminouracil 1 with selected arylsulfonyl chlorides occurs regioselectively when the reaction is carried out in pyridine at room temperature. Simple isolation of the products by recrystallization of the crude product mixture from aqueous methanol provides good to excellent yields. The prepared 5 -aminosulfonyl uracil derivatives 2 − 9 were tested for the antiproliferative activity on a panel of seven tumor cell lines of different histological origin (HeLa, Caco- 2, NCI - H358, Raji, HuT78, Jurkat, K562) and normal MDCK I cells. Derivatives 2 − 9 were found more efficient t o lymphoma and leukemia cells compared to solid tumor and normal cells.

For sulfonylation of 1, the aromatic sulfonyl chlorides with different size and lipophilicity of aromatic groups with electron-donating or electron-withdrawing substituents on the aromatic system were selected. In this way, the molecules with variable electronic and lipophilic characteristics could be obtained, which may influence their chemical stability as well as physical and biological properties. Here we report the synthesis of 5-aminosulfonyl uracil derivatives 2−9 as well as their antiproliferative activity on normal MDCK I cells, and HeLa, Caco-2, NCI-H358, Raji, HuT78, Jurkat, and K562 tumor cell lines.

General
Chemicals and solvents were obtained from Sigma-Aldrich Chemical Company (St. Louis, USA) and used without further purification. Rf values refer to analytical TLC performed using pre-coated silica gel 60 F254 plates purchased from Sigma-Aldrich (Steinheim, Germany) and developed in the solvent system indicated. Compounds were visualized by use of UV light (254 and 365 nm). Melting points were determined on a Kofler hot-stage apparatus and were uncorrected. UV spectra were taken on a Philips PU8700 UV/VIS spectrophotometer (Philips Analytical, Cambridge, Great Britain). IR spectra were obtained in KBr pellets on a Perkin-Elmer 297 spectrophotometer (Perkin-Elmer, Waltham, MA, USA). NMR spectra were recorded on AV600 and AV300 MHz spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at 150.92 or 75. 47  General Procedure for the Condensation of 5-aminouracil 1 with sulfonyl chlorides A suspension of 5-aminouracil 1 (1 mmol) in dry pyridine (7.5 mL) was cooled to 0 °C and the appropriate sulfonyl chloride (1 mmol) was added. The reaction mixture was stirred at room temperature until the TLC showed the reaction was completed (1.5−20 h). The solvent was removed under reduced pressure and the crude product was recrystallized from methanol or aqueous methanol to afford the product.
Tested compounds were dissolved in dimethyl sulfoxide as a 1 × 10 -2 mol dm -3 stock solution. Working dilutions of derivatives 2−8 and 5FU were prepared in high pure water at a concentration range 10 -4 −10 -7 mol dm -3 and in the case of insoluble compound 9 at a concentration range 10 -5 -10 -7 mol dm -3 .
For the MTT test, the adherent cells, MDCK I, HeLa, and Caco-2, were seeded in 96 micro-well plates at concentration of 2×10 4 cells/cm 3 and allowed to attach overnight in the CO2 incubator. After 72 hours of the exposure to tested compounds, medium was replaced with 5 mg/cm 3 MTT solution and the resulting formazane crystals were dissolved in DMSO.
Leukemia cells (1 × 10 5 cells/cm 3 ) were plated onto 96 micro-well plates and after 72 hours of incubation, 5 mg/cm 3 MTT solution was added to each well and incubated 4 hours in the CO2 incubator. To each well, 10 % SDS with 0.01 mol dm -3 HCl was added to dissolve waterinsoluble MTT-formazane crystals. The microplate reader (iMark, BIO RAD, Hercules, CA, USA) was used for measurement of the absorbance at 595 nm.
All experiments were performed three times in triplicates. The percentage of treated tumor cells growth inhibition was calculated relative to the growth of untreated (control) cells.
Selectivity index (SI) was calculated for each compound using formula: SI = IC50 for normal cell line MDCK I / IC50 for respective tumor cell line. Higher values of SI indicate greater antitumor specificity and the SI > 1.00 identifies compounds with efficacy against tumor cells greater than toxicity against normal cells. [52][53][54]

Synthesis
Depending on the reaction conditions, alkylation of uracil with alkyl halides may result in N-1 or N-3, alkylated and  N-1,N-3 of uracil with a high excess of methyl iodide, in the presence of alkali, 1,3-dimethyl uracil was formed, [55] while the alkylation with a small excess alkyl halide in the presence of a base in DMF yields N-1-alkylated products with a small amount of N-1,N-3-dialkylated compounds. [56][57] On the other hand, 5-aminouracil 1 provides greater substitution possibilities due to the presence of a basic amino group at the C5 position of the ring. When potassium salt of 5-aminouracil is allowed to react with methyl iodide alkylation takes place in the N1 and N3 positions of the pyrimidine ring with formation of 1,3-dimethyl-5-aminouracil. [48] Treatment of 5-aminouracil 1 with p-toluenesulfonyl chloride in aq. sodium hydroxide gave 5-aminotosyl uracil 2 in 49 % yield, [48] while 5-sulfanilamidouracil [49] was synthesized by reacting 5-aminouracil 1 and N-acetylsulfanilyl chloride in pyridine, followed by deprotection of N-acetyl group in aqueous sodium hydroxide (52 % yield). Pecorari et al. [50] showed that in the reaction of 4-acetamido and 4-nitrobenzenesulfonyl chloride with 5-aminouracil 1 in aq. sodium hydroxide, mono C5 aminosulfonyl products 4 and 5 are formed in a mixture with different amounts of disubstituted products (N 5 ,N 5bis-sulfonyl), depending on pH.
We have previously described two methods for the preparation of N-1-sulfonylpyrimidine derivatives of general formula I ( Figure 2): a) condensation of silylated pyrimidine bases with different sulfonyl chlorides in acetonitrile; b) reaction of pyrimidine bases with sulfonyl chlorides in pyridine. [34,36] The above procedures worked well with aliphatic, aromatic, and heteroaromatic sulfonyl chlorides.
For the synthesis of the 5-aminosulfonyl uracil derivatives of general formula II (Figure 2), we used 5-aminouracil 1 and applied the method b) in pyridine for the regioselective introduction of the aromatic sulfonyl groups to the C5 amino group of uracil ring.
The substitution of the amino group in 1 proceeded well when equimolar amounts of 5-aminouracil 1 and p-toluenesulfonyl chloride were reacted in pyridine at room temperature. After simple isolation by recrystallization of the crude product mixture from aqueous methanol, 5-aminotosyl uracil 2 was isolated in 98 % yield (Scheme 1).
The structures of the synthesized compounds were confirmed by elemental analysis and by IR and NMR data. The 1 H NMR spectra of the products 2-9 confirm the respective structures of 5-aminosulfonyl uracil derivatives. Singlets, in the NMR spectra of 2-9 appearing in the range of δ 11.09-11.23 ppm, are attributed to the uracil N3 protons. The uracil N1 protons appear as broad singlets (2 and 5 at 10.88 and 11.03 ppm, respectively) or doublets in the range δ 10.86-11.02 ppm (J1,6 5−6 Hz), while the singlets of NH protons of C5 sulfonamido groups are shifted upfield and appear in the range δ 9.08-9.77 ppm. The multiplet signals within the δ 6.86-8.69 ppm region are assigned to the aromatic protons and H-6 protons of uracil moiety.

In comparison to effects on Raji cells, tested compounds demonstrated similar results on HuT78 cells (Supporting Information
Applied at the highest concentration range, compounds 2−6, and 8 inhibited more than 50 % of the growth of Jurkat cells (Figure 4, Supporting Information Figure S23). Also, 2,4,6-triisopropylbenzenesulfonamide 3 applied at c =10 -5 mol dm -3 , caused a 20 % reduction of Jurkat cell growth. Interestingly, most of the tested compounds applied at lower concentration range (10 -6 and 10 -7 mol dm -3 ) showed even a slight increase of Jurkat cell growth.

CONCLUSIONS
A small library of 5-aminosulfonyl uracil derivatives 2−9 with different aromatic substituents at C5-NH-SO2of uracil moiety was efficiently synthesized (yields 58−98 %). The products were prepared using equimolar amounts of 5aminouracil 1 and selected aromatic sulfonyl chloride in pyridine at room temperature.
The prepared compounds were tested for the antiproliferative activity on normal MDCK I cells and tumor HeLa, Caco-2, NCI-H358, Raji, HuT78, Jurkat, K562 cell lines. All of the newly synthesized compounds 2−9 showed only modest growth inhibition activity on normal (MDCK I) and solid tumor (HeLa, Caco-2, NCI-H358) cells. However, they were found more active on lymphoma (Raji, HuT78) and leukemia (Jurkat, K562) cells, showing statistically significant growth inhibition at the highest concentrations   (20−60 % growth inhibition at c =10 -4 mol dm -3 ). It should be noted that due to efficient synthetic protocol the sulfonyl group substituents of 5-aminosulfonyl uracil derivatives II could be easily varied providing the opportunity for diverse structural variations which may result with significant improvement of biological properties.