Synthesis and anticancer activity of thiosubstituted purines

New thiopurines with the propargylthio, pyrrolidinobutynylthio, sulfenamide, and sulfonamide groups in the pyrimidine ring were synthesized. The anticancer activity of these compounds and previously obtained 2- or 6-substituted azathioprine analogs and dialkylaminoalkylthiopurines were tested in vitro against three cell lines: glioblastoma SNB-19, melanoma C-32, and human ductal breast epithelial tumor T47D. 2-Chloro-7-methyl-6-pyrrolidinobutynylthiopurine (5b) was the most potent compound against SBN-19 and C-32 cell lines with the activity similar to cisplatin (EC50 = 5.00 and 7.58 μg/ml, respectively). The dialkylaminoalkylthio derivatives (4b, 4c, 4e, and 4f) showed good activity against SBN-19 cell line (EC50 < 10 μg/ml). The azathioprine analogs (2a, 2b, and 3a) were more active than azathioprine against SBN-19 and C-32 cell lines. The sulfenamide and sulfonamide derivatives of purine were very weak active against tested cell lines. All studied thiopurines were less toxic than cisplatin.

However, the use of thiopurines is limited by their toxicities, which include hepatotoxicity, myelosuppression, pancreatitis, and allergic reactions (Prima et al., 2013). For this reason, many thiopurine derivatives and analogs have been synthesized for evaluation of their biological activities and reduced toxicity. Recently, 9-substituted derivatives of 6-(t-butoxycarbamylaminohexyl)thiopurine were obtained which showed moderate antibacterial activity (Rao et al., 2013); a series of 6-mercaptopurine analogs with 1,2,3-triazole or steroid rings exhibited promising antimalarial and antileishmanial activities (Corrales et al., 2011); and biologically active 6-allyldithiopurine and its riboside inhibited cell proliferation and induced apoptosis (Miron et al., 2009).
Heterocyclic sulfonamides are used as carbonic anhydrase inhibitors, antibacterial, anti-inflammatory, analgesic, hypoglycemic, antifungal, and antiviral agent (Joseph et al., 2010;Guzel et al., 2009;Azab et al., 2013;El-Sayed et al., 2011;Veisi et al., 2011). It is known that aryl and heteroaryl sulfonamides may act as antitumor agents through perturbation of cell cycle in the G 1 phase, distribution of microtubule assembly, or angiogenesis inhibition. They reverse the effect of tumor acidification, consequently inhibit the growth of cancer cell, and suppress tumor invasion mediated by the carbonic anhydrase active side (Zn 2? ) and so inhibit the catalytic ability of this enzyme (El-Sayed et al., 2011). Indisulam (E7070) is a novel sulfonamide anticancer agent currently in phase II clinical development for the treatment of several types of cancer. It was shown to act as a nanomolar inhibitor of carbonic anhydrase isoform IX (Sławiński et al., 2013;Mirian et al., 2011).
The sulfamoyl group has been extensively utilized as an activity-modifying substituent in many different classes of drugs. The synthesis of 2-substituted 7-or 9-methylpurines containing a sulfanilamide group at the positions 2, 6, or 8 of the purine ring with moderate antibacterial activity was reported by the reaction of chloropurines with sodium sulfanilamide (Beaman et al., 1966). Structural modification of the amino group at C-6 atom of adenosine leads to selective A 1 receptor agonists, while modification at C-2 atom confers high potency and selectivity on A 2A receptors. The synthesis of N-9-and 6-amino monosulfonyladenine and disulfonyladenine derivatives and its sulfonylated nucleoside with high potency and selectivity on A 1 and A 2A receptor agonists has been described (Zinic et al., 2001). A number of 6-sulfenamide, sulfinamide, and sulfonamide derivatives of 6-mercaptopurine, 6-thioguanine, and related ribonucleosides have been synthesized and evaluated for antileukemic activity in mice (Revankar et al., 1990). It is quite interesting that a subtle change to an oxidized sulfur atom in the form of sulfonamide resulted in a new group of purine derivatives possessing significant antitumor properties. Majority of the tested compounds exhibited very significant anti-L1210 cell activity (Revankar et al., 1990;Robins et al., 1991).
Oligonucleotides containing 1-alkynyl-6-thiopurine or 6-thioguanine bases demonstrate potent antiviral activity in several assays, including the human immunodeficiency virus reverse transcriptase enzyme assay (Broom et al., 1997). The purine derivatives possessing the alkynyl groups at position 6 are cytokinin analogs with profound plant growth-stimulating effect and have attracted considerable interest as potential anticancer compounds. All 6-phenylacetylenes and certain enyne of the purine are highly toxic against K-562 cells and exhibit cytotoxic activity against a human chronic myelogenous leukemia cell line comparable to well-known anticancer drugs (6-mercaptopurine and fludarabine) (Brathe et al., 2003). Many adenine and adenosine derivatives with 2-, 6-, or 8-alkynyl substituents are selective adenosine subtypes A 1 , A 2A , A 2B , and A 3 receptor antagonists. 2-Alkynyl-8-aryl adenine derivatives bear methyl or an amide moiety at the position 9, as A 2B receptor antagonists have hypoglycemic activity and may be used as antidiabetic agents (Harada et al., 2001). Stimulating A 2A receptor by 2-adenosine propargyl phenyl ether composition resulted in vasodilating activity and potential of using in coronary artery disease (Zablocki et al., 2001). Introduction of alkynyl chains on C-8 atom of adenosine led to very selective antagonists of the A 3 receptor (Volpini et al., 2001). Some 2-alkynyl and 2,(6-amino)dialkynyl adenosine derivatives show high affinity and different degrees of selectivity for A1, A 2A , and A3 receptor (Volpini et al., 2001).
Our previous research demonstrated the effective synthesis of thiosubstituted purines such as 2-or 6-substituted azathioprine analogs (Kowalska and Pluta, 2008;Kowalska et al., 2009) and dialkylaminoalkylthiopurines (Kowalska and Pluta, 2012). The aim of this study was to elaborate synthesis of new thiopurines with the propargylthio, pyrrolidinobutynylthio, sulfenamide, and sulfonamide groups in the pyrimidine ring, to examine anticancer activity of not only those compounds but also previously synthesized thiopurines and to discuss the structure-activity relationship.
The structure of all new compounds was determined by the spectral data analysis: 1 H and 13 C NMR, EI, CI or FAB MS, and HR MS. 1 H NMR spectra of new compounds revealed H-8, NCH 3 proton signals, and protons from C-2 (OCH 3 , SCH 3 ) and C-6 (CH 2 C 6 H 5 , NH 2 , N(C 2 H 5 ) 2 , NHC 6 H 5 , SCH 2 CCH, SCH 2 CCCH 2 NC 4 H 8 , SNH 2 , SO 2-NH 2 ) substituents. 13 C NMR spectra showed primary, secondary, tertiary, and quaternary carbon signals. In order to assign all of these signals, 2D NMR techniques (HSQC-the primary, secondary, and tertiary carbon atoms connected with the hydrogen atoms and HMBC-the primary, tertiary, and quaternary carbon atoms connected with the hydrogen atoms via two and mainly three bonds) for selected compounds (5a, 10e) were used. For compounds 5a, in the HSQC spectrum the H-8 proton at 8.59 ppm correlated with the signal at 151.09 ppm (C-8), the SCH 2 proton at 4.22 ppm correlated with the signal at 18.09 ppm (C, SCH 2 ), and the proton at 4.04 ppm (NCH 3 ) correlated with the signal at 34.84 ppm (C, NCH 3 ). The HMBC Scheme 1 Synthesis of 2-or 6-substituted azathioprine analogs 2, 3, dialkylaminoalkylthiopurines 4, propargylthio-and pyrrolidinobutynylthiopurines 5 Med Chem Res (2015) 24:3107-3116 3109 Scheme 2 Synthesis of amine 9, sulfenamide 10, and sulfonamide 8 derivatives of purines spectrum revealed correlation via three bonds between the H-8 proton at 8.59 ppm and the carbon signals at 34.84 ppm (C, NCH 3 ), at 123.16 ppm (C-5), and at 160.52 ppm (C-4). The proton signal at 4.22 ppm (H, SCH 2 ) correlated with the signals of carbon atoms via two bonds at 79.55 ppm (C, CCH) and via three bonds at 74.67 ppm (C, CH) and at 153.99 ppm (C-6). The proton signal at 4.04 ppm (H, NCH 3 ) correlated via three bonds with the signals at 123.16 ppm (C-5) and at 151.09 ppm (C-8). The proton-carbon correlation was presented in Scheme 3.
The anticancer activity of the obtained compounds was investigated in vitro using cultured glioblastoma SNB-19, melanoma C-32, and human ductal breast epithelial tumor T47D cell lines. Normal human fibroblasts (HFF-1) were used as a control, and azathioprine and cisplatin as reference drugs. Table 1 contains the activity of thiopurines as the EC 50 values.
The most active was 2-chloro-7-methyl-6-pyrrolidinobutynylthiopurine (5b) with the EC 50 values of 5.00 and 7.58 lg/ml against SNB-19 and C-32 cell lines, comparable to the cisplatin effect. This compound exhibited good selectivity and low toxicity, being weak active against T47D cell line and normal fibroblasts HFF-1. The introduction of the pyrrolidine ring to the alkynyl chain enhanced the activity (compound 5b vs. 5a).
Good activity against SNB-19 cell line was observed for the dialkylaminoalkylthio derivatives (4b, 4c, 4e, and 4f) with the EC 50 values below 10 lg/ml. The compounds with the ethyl chain were more active than those with the propyl chain. The most active was compound 4e with the two pyrrolidinoethylthio groups. The activity against other cell lines was lesser as well as their toxicity.
The azathioprine analogs with the imidazolylthio group in position 2 were more active than those with that group in position 6 with an exception of compound (3a) which exhibited good activity against C-32 cell line. The most active azathioprine analogs (2a, 3a) possessed the chlorine atom in position 2 or 6. Both compounds were more active than azathioprine (against first two cell lines) and compounds containing the methoxyl and thioalkyl groups (2b, 3b, 3c). Compound 3d with two imidazolylthio groups turned out to be unexpectedly inactive. In our opinion, such an effect was the result of the unusual spatial arrangement of those groups directed to N-1 atom with the donor-acceptor interaction between the imidazole rings, as it was observed in X-ray analysis of the monocrystal 3d (Kowalska et al., 2009).
The sulfenamide and sulfonamide derivatives are very weak active or inactive. The glioblastoma SNB-19 was most sensitive and breast tumor T47D least sensitive cell line for thiopurines. All studied compounds were less toxic than cisplatin.

Conclusion
In search for novel thiopurine derivatives, we described synthesis of 2-chloro-6-alkynylthio-7-methylpurines 5a, 5b from 2-chloro derivative and propargyl bromide and further via Mannich reaction with pyrrolidine. Other types of thiopurines bearing the sulfenamide 10a-e and sulfonamide 8a-d groups were obtained through S-amination of purinethiones 1a-d and oxidation with 3-chloroperoxybenzoic acid.
New thiopurines 5, 8, and 10, previously synthesized azathioprine analogs 2 and 3, and dialkylaminoalkylthiopurines 4 were investigated as antitumor agents. The most potent compound against SBN-19 and C-32 cell lines was 2-chloro-7-methyl-6-pyrrolidinobutynylthiopurine 5b with the activity similar to cisplatin. The dialkylaminoalkylthio derivatives 4b, 4c, 4e, and 4f showed good activity against SBN-19 cell line. The azathioprine analogs 2a, 2b, and 3a were more active than azathioprine against SBN-19 and C-32 cell lines. The sulfenamide and sulfonamide derivatives of thiopurine were very weak active against tested cell lines. All studied thiopurines were less toxic than cisplatin.
The melting points were determined in open capillary tubes on a Boetius melting point apparatus and were uncorrected. 1 H NMR spectra were recorded on a Bruker AVANS 300 spectrometer operating at 300 MHz and 75 MHz for 1 H and 13 C nuclei, respectively, in deuterochloroform and dimethyl sulphoxide-d 6 with tetramethylsilane as internal standard. Shifts were given in ppm, coupling constant (J) values were presented in hertz (Hz), and the abbreviations were as follows: s (singlet), d (doublet), t (triplet), and m (multiplet). Electron impact (EI MS), chemical ionization (CI MS), fast atom bombardment (FAB MS), and high-resolution (HR MS) (in the m-nitrobenzyl alcohol and glycerol matrix) mass spectra were run on a Finnigan MAT 95 spectrometer at 70 eV. The reactions were monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel 60 F254 (Merck) and chloroform-ethanol (9:1) as the solvents. Purity of the synthesized compounds was confirmed by TLC in the same way. Spots were detected by their absorption under UV light (k = 254 nm), and the chromatograms were further visualized by iodine vapor. Column chromatography separations were carried out with Merck Kieselgel 60 or aluminum oxide 90 (Merck) using a mixture of chloroform-ethanol (99:1, v/v) as an eluent.
Synthesis of 2-chloro-6-(prop-2-ynylthio)-7-methylpurine 5a Propargyl bromide (0.17 g, 1.44 mmol) was added to a solution generated from the reaction of 2-chloro-7methyl-6-thiopurine (0.2 g, 1 mmol) with t-BuOK (0.16 g, 1.44 mmol) in 10 ml of DMF at room temperature for 0.5 h. The reaction mixture was stirred for an additional 24 h at room temperature and then added to 25 ml of water. The resulted solid was filtered off and washed with water to give compound 5a. It was obtained as a pale yellow solid (  Synthesis of 2-chloro-6-(4-N-pyrrolidinylbut-2-ynylthio)-7methylpurine 5b To a mixture of propargyl derivative 5a (0.24 g, 1 mmol) and paraformaldehyde (0.06 g, 2 mmol) in 5 ml of dry dioxane, pyrrolidine (0.14 g, 2 mmol) and CuCl (0.01 g) were added. The reaction mixture was stirred at temperature 70°C for 2 h. After cooling, the resulting solid was filtered off and purified by column chromatography (silica gel, CHCl 3 , CHCl 3 -EtOH, 99:1 v/v) to give compound 5b. It was obtained as a pale yellow solid ( Synthesis of 2-chloro-6-benzylthio-7-methylpurine 6 2-Chloro-6-benzylthio-7-methylpurine 6 was prepared from 2-chloro-7-methyl-6-purinethione 1a (0.2 g, 1 mmol) by alkylation with benzyl chloride (0.25 g, 2 mmol) at room temperature in 4 % aqueous KOH solution (5 ml). After 30 min, the crude product was precipitated, filtered off, washed with water, and crystallized from benzene. It was obtained as a pale yellow solid ( Chlorination and amination of 2-chloro-6benzylthio-7-methylpurine 6 The chlorinolysis was carried out by passing chlorine gas into a stirred mixture composed of 2-chloro-6-benzylthio-7-methylpurine 6 (0.29 g, 1 mmol), 3 ml of chloroform, and 3 ml of 80 % acetic acid cooled at 5°C. The passage of chlorine gas was continued for 30 min, and then the mixture was poured into 10 ml of ice water. The chloroform layer was separated, and aqueous layer was extracted with chloroform (2 9 5 ml). The chloroform extracts were combined, washed with water, dried over anhydrous sodium sulfate, and evaporated in vacuo to give an oil residue. In order to remove benzyl acetate and benzyl chloride, the residue was triturated with ice-cold dry ether and the ether layer was separated. The mixture of obtained oil (0.26 g, 1 mmol), and 25 % aqueous ammonia (2.5 ml) or diethylamine (0.22 g, 3 mmol) in 10 % NaOH solution or aniline (0.19 g, 2 mmol) in 2.5 ml of benzene was stirred at 40-45°C for 2-6 h. The resulted solid was filtered off (in the case of the reaction with ammonia, the excess of ammonia was removed in vacuo and the residue was diluted with water up to volume 2.5 ml) to give compounds 9a-c. Products were purified by a column chromatography (aluminum oxide, CHCl 3 -EtOH, 99:1 v/v) to give: 2-Chloro-6-amine-7-methylpurine 9a was obtained as a white solid ( General synthesis of 2-substituted 7-methyl-6purinesulfenamide 10a-e A solution of commercial NaOCl (14.5 %, 1.7 ml) and 1.7 ml of water was cooled to 0°C in an ice-NaCl bath. Ammonium hydroxide (25 %, 2 ml) was similarly cooled and added with stirring to the bleach solution. The mixture was stirred at -5 to 0°C for 15 min and then allowed to cool to 0°C solution of 2-substituted 7-methyl-6-purinethione 1a-d (2 mmol) in 10 % KOH (5 ml) was dropped. The mixture was stirred in stoppered flask for 30 min at temperature -5 to 0°C. The reaction mixture was initially clear, light yellow solution, but after 30 min a solid began separating. After allowing the reaction mixture to stand at room temperature for an additional 1 h, the precipitated product was filtered off and washed with a small amount of cold water, followed by cold ethanol to yield purine 6-sulfenamide 10a-c, 10e. The crude products were purified by a column chromatography (silica gel, CHCl 3 , CHCl 3 -EtOH, 99:1 v/v). The aqueous filtrate separated after precipitation of product 10e was evaporated to dryness and extracted with absolute ethanol (3 9 5 ml). The ethanolic solvent was evaporated, and the residue was purified by column chromatography (aluminum oxide, CHCl 3 -EtOH, 10:1 v/v) to give compound 10d. General synthesis of 2-substituted 7-methyl-6purinesulfonamide 8a-c, 8e A solution of 3-chloroperbenzoic acid (MCPBA) (77 %, 0.86 g, 4 mmol) in 10 ml of ethanol was added dropwise for 10 min to a solution of sulfonamides 10a-c and 10e (1 mmol) in 79 ml of absolute ethanol. The mixture was stirred at room temperature for 1.5 h, and a precipitated solid was filtered off to give crude products 8a-c and 8e.
The ethanolic filtrate was evaporated to dryness and extracted with ether to remove 3-chlorobenzoic acid. The solid residue together with crude product was purified by a column chromatography (aluminum oxide, CHCl 3 , CHCl 3 -EtOH, 9:1 v/v) to give also compounds 8a-c, 8e.

Proliferation
In recent years, tetrazolium salts have been described which can be used for the measurement of cell proliferation and viability. The tetrazolium salts are cleaved to formazan by cellular enzymes. An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This augmentation in enzyme activity leads to an increase in the amount of formazan dye formed, which directly correlates with the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is quantified by a scanning ELISA reader by measuring the absorbance of the dye solution at appropriate wavelengths (k = 420-480 nm with a reference wavelength k = 600 nm).

WST-1 assay
Antiproliferative effect of compounds was determined using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). After exposure to tested compounds (at concentrations between 0 and 100 lg/ml) for 72 h, cells were incubated with WST-1 (10 ll) for 1 h, and the absorbance of the samples against a background control was read at 450 nm using with a reference wavelength k = 600 nm a microplate reader. Results are expressed as means of at least two independent experiments performed in triplicate.