Thio Analogs of Pyrimidine Bases: Synthesis, Spectroscopic Study, and In Silico Biological Activity Evaluation of New 2-o-(m- and p-)Chlorobenzylthio-6-Methyl-5-Piperidino-(Morpholino-)Methyluracils

Six new 2-o-(m- and p-)chlorobenzylthio-6-methyl-5-piperidino-(or morpholino-) methyluracils have been prepared. The structures of these compounds were confirmed by spectroscopic (FT-IR, UV-Vis, 1H NMR, 13C NMR, and HMBC) and elemental analyses. Estimation of pharmacotherapeutic potential has been made for synthesized compounds on the basis of prediction of activity spectra for substances (PASS).


Introduction
Thio derivatives of pyrimidine bases have remarkably contributed to biological and medicinal chemistry. Chemical modification of these compounds have led to a large number of mono-and di-S and N-substituted analogs showing therapeutic properties, especially antivirial, antithyroid, and antitumor activities [1][2][3][4][5][6][7]. The antimetabolites of 5,6dimethyluracil 5-morpholinomethyl-6-methyl-2-thiouracil [8] and 5-piperidinomethyl-6-methyl-2-thiouracil [9] have been synthesized via the Mannich reaction. However, to the best of our knowledge no work has been published on the synthesis as well as on physicochemical properties of the monochlorobenzylthio-substituted derivatives of these compounds. This fact has stimulated us to investigate the reaction of chlorobenzylation of 2thio-5-piperidinomethyl-6-methylthiouracil and 2-thio-5morpholinomethyl-6-methylthiouracil as well as the Mannich reaction of 2-o-(m-and p-)chlorobenzylthio-6-methylthiouracils, formaldehyde, and piperidine (or morpholine). Recently novel pharmacological action of 2,4-di-o-(m-and p-)bromo-(chloro-and nitro-)benzylthio-5-bromouracils (and 6-methyluracils) [ [11] has been found on the basis of the computeraided drug discovery approach with the compounds program Prediction of Activity Spectra for Substances (PASS) [12][13][14][15]. Since only the structural formula of the chemical compound is necessary to obtain a PASS prediction, this approach was used in the present work. This paper deals with the synthesis and physicochemical properties of 1-6. Additionally, the analysis of biological activity spectra prediction for 1-6 made in this paper is a good example of in silico studies of chemical compounds.
The 13 CNMR spectra of compounds 1-6 in DMSO d6 showed characteristic signals in the range of 31.55-32.81 ppm and 53.04-53.71 ppm assigned to S-CH 2 and N-CH 2 , respectively. The thiouracil ring exhibited signals in the range 110.01-114.71 ppm and 158.81-162.11 ppm assigned to C 5 =C 6 , respectively. The 13 C NMR spectra of compounds 1-6 showed the presence of a methyl group from the 6methyl-2-thiouracil ring at 20.90-21.30 ppm. Table 1: FT-IR, UV-Vis, and 1 H NMR data of compounds 1-6.

Comp.
UV/Vis (CH 3 OH) λ max nm (log ε)     The HMBC spectrum clearly shows the connectivities of all hydrogen and carbon atoms involved, including quaternary carbons. The HMBC results allow an unequivocal assignment of S-substitution of benzyl group at uracil ring of 1-6 ( Table 3). The HMBC experiment is conducted without 13 C decoupling so that correlations via one or more bond can be discerned and one-bond correlation affords double cross-peaks in the 1 H dimension. For compounds 1-6 the 1 H NMR spectrum exhibits three singlets at 4.29-4.46,  3.28-3.58, and 2.22-2.33 ppm ascribed to protons of S-CH 2 , C 5 -CH 2 and C 6 -CH 3 , respectively. The HMBC of 1-6 shows peaks corresponding to two-bond correlations for C 6 -CH 3 /C 6 (158.81-162.11 ppm) and three-bond correlations for S-CH 2 /C 2 (163.68-168.05 ppm) and C 5 -CH 2 /C 4 (161.82-163.19 ppm). The FT-IR spectra of 1-6 show absorption bands of medium intensities in the region 809-820 cm −1 assigned to ν C-Cl vibration as well as in the region 1035-1091 cm −1 assigned to δ C-Cl vibration ( Table 1). The FT-IR spectra of 1-6 show also absorption bands in the region 2852-2864 cm −1 assigned to ν CH 2 -S as well as in the region 1444-1447 cm −1 assigned to δ CH 2 -S vibration ( Table 1). The UV/Vis spectra of 1-6 show λ max in the range 245-250 nm (Table 1).

ISRN Organic Chemistry
In the present paper the biological activity spectra were predicted for all six synthesized compounds (1-6) using PASS [12][13][14][15]. We have also selected the types of activities that were predicted for a potential compound with the highest probability (focal activities). They are presented in Table 5. According to these data the most frequently predicted types of biological activities are antiviral (Influenza), antiseborrheic, and prolyl aminopeptidase inhibitor. It ought to be pointed out that in the series of compounds 1, 3 and 6 such activity as mucomembranous protector has also been predicted, as well as in the series of compounds 2, 3, 5, and 6 such activity as prolyl aminopeptidase inhibitor.

Experiment
The purity of all described compounds was checked by melting points, TLC, and elemental analyses. Melting points (uncorrected) were determined on a Boetius microscope hot stage. Rf values refer to silica gel F 254 TLC plates (Merck) developed with CHCl 3 : CH 3 OH (10 : 1) and observed under UV light (λ = 254 and 366 nm). UV/Vis spectra were recorded with a SPECORD UV/Vis Spectrophotometer in methanol. IR spectra were recorded with FT-IR Bruker IFS-113 Spectrophotometer in KBr pellets. The 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were determined with Varian Gemini 300 spectrometer in DMSO d6 solution at a concentration between 0.25 and 0.40 M in the 5 mm sample tubes at ambient temperature. Chemical shifts are given in δ scale (ppm). Elemental analyses were performed with a Vector Euro EA 3000 analyzer.
General procedure A: 0.2 g (0.8 mmole) of 2-thio-5piperidino-(morpholino-)methyl-6-methyluracils [8,9] was dissolved with stirring in room temperature in 2.5 mL of 3 N NaOH in methanol. Next, to the solution 0.116 mL (0.9 mmole) of m-chlorobenzyl chloride was added. The reaction mixture was stirred in room temperature for 24 hours and after that time 5 mL distilled water was added. The obtained crude product was collected by filtration, washed with distilled water, and dried in the exicator. The obtained dry solid was dissolved in 10 mL of CHCl 3 and separated by silica gel column chromatography (Merck 203-400 mesh) using the following solvent mixtures: CHCl 3 : CH 3 OH 50 : 1 (40 mL), 40 : 1 (40 mL), 30 : 1 (30 mL), 20 : 1 (30 mL), and 10 : 1 (20 mL). The fractions of 20 mL were collected. On the basis of analytical TLC fractions of product desired were obtained by combining 20 mL fractions. They were concentrated on a rotary evaporator. Compounds 2 and 5 were shown to by analytically pure.