An easy microwave-assisted synthesis of C8-alkynyl adenine pyranonucleosides as novel cytotoxic antitumor agents

We describe the synthesis of C8-alkynyl adenine pyranonucleosides 4, 5, and 8-phenylethynyl-adenine (II), via Sonogashira cross-coupling reaction under microwave irradiation. Compounds 4e and II were less cytostatic than 5-fluorouracil (almost an order of magnitude) against murine leukemia (L1210) and human cervix carcinoma (HeLa) cells, while the same compounds proved to be more active than 5-fluorouracil against human lymphocyte (CEM) cells.


Introduction
Synthetic nucleoside analogs with modified nucleobase moieties are of considerable importance in the search for promising lead candidates endowed with antiviral, anticancer, and antibacterial activities (Herdewijn, 2008;Manta et al., 2014). Among them, a number of purine and pyrimidine substituted nucleoside derivatives exhibited activity in both solid tumors and hematological malignancies, behaving as antimetabolites, competing with physiological nucleosides, and consequently, interacting with a large number of intracellular targets to induce cytotoxicity (Hatse et al., 1999).
Considering all the progress made toward this direction, we have recently embarked on the synthesis of C5-substituted uracil and cytosine glucopyranonucleosides bearing a variety of alkyne substituents, such as linear alkyl chains and aromatic rings substituted with linear and branched alkyl groups (Dimopoulou et al., 2013), which effectively inhibited the proliferation of a variety of tumor cell lines and they also proved as some of the most potent inhibitors of the active site of glycogen phosphorylase (Kantsadi et al., 2012). Among these agents, the C5-phenylethynyluracil pyranonucleoside showed appreciable cytotoxic activity (IC 50 of 5.2-6.2 µM), comparable to 5-fluorouracil (Dimopoulou et al., 2013).
As a continuation of our studies on the synthesis of basemodified pyranonucleosides and considering the interesting biological properties of substituted purines, it was speculated that the introduction of alkynyl modifications at the 8-position of either adenine pyranonucleosides or even adenine itself, could possibly lead to more efficacious therapeutic agents. This conjugation appeared to us as a challenge and the first biological results confirmed our hypothesis.

Antiproliferative Assay
Compounds 4e, 5a-d, and II, were evaluated for their cytostatic activity against human cervix carcinoma (HeLa) cells, human lymphocytes (CEM) as well as murine leukemia (L1210) cells. All assays were performed in 96-well microtiter plates. To each well (5-7.5) × 10 4 tumor cells were added and varying concentrations of the test compounds ranging from 250, 50, 10, 2, 0.4, to 0.08 µM. The tumor cells were allowed then to proliferate at 37 • C in a humidified CO 2 -controlled atmosphere. To obtain their optimal growth curves this is for 2 days of the murine leukemia (L1210) cells and for 3 days for the human lymphocytic (CEM) cells and the human cervix carcinoma (HeLa) cells. At the end of the incubation period, the cells were counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%. Experiments were repeated at least three times and these data are presented in Table 1.

Results and Discussion
Herein, we describe the synthesis of C8-alkynyl adenine pyranonucleosides and present their biological properties. The starting material of our synthesis was the commercially available per-O-acetylated D-glucose 1 which upon coupling, via two-step Vorbrüggen method (Vorbrüggen and Höfle, 1981), with N 6 -benzoyl adenine, gave selectively the N9-isomeric adenine nucleoside 2, under thermodynamically controlled conditions (SnCl 4 /CH 3 CN, reflux). Trans rule was followed (Baker, 1957) and the β-configured nucleoside 2 was solely obtained as deduced from 1 H NMR vicinal coupling data (J 1 ′ ,2 ′ = 9.4 Hz).
Since halogenated C8-purine nucleosides have proven useful intermediates for the efficient preparation of their corresponding C8-alkynyl derivatives (Agrofoglio et al., 2003), the adenine nucleoside 2 was directly brominated after treatment with Br 2 /AcOH/AcONa (Holmes and Robins, 1964), affording the 8-bromo analog 3, in approximately 60% yield after aqueous work-up and flash chromatography. The position of substitution of the bromine atom was further confirmed by the absence of the characteristic sharp absorption peak at 8.22 ppm due to H-8 of nucleoside 2, while the syn conformation for adenine was induced due to the bulky bromo substituent at C8 position (Sarma et al., 1974). 8-Alkynyl adenine nucleosides 4 were accessed, through Sonogashira cross-coupling reaction of intermediate 3 with several terminal acetylenes, under microwave irradiation (200 W) (Figure 1). In a typical experiment, an effective catalyst (Pd(PPh 3 ) 4 )/cocatalyst (CuI) combination proved to be (1:1) ratio (Meneni et al., 2007) affording 4a-e, in satisfactory yields (50-66%).
To provide a detailed structure-activity relationship studies, diverse alkyne substituents R were selected which include linear alkyl chains (4a, R = n-pentyl), aromatic rings (4b, R = phenyl, 4c, R = p-tolyl) and pyridine moieties (4d, R = 3pyridyl, 4e, R = 2-pyridyl). Finally, total deprotection of 4a-e by the action of saturated methanolic ammonia afforded only the target derivatives 5a-d, while attempts to remove all protecting groups from 4e either with sodium methoxide (Bozó et al., 1998) or potassium carbonate-methanol (Plattner et al., 1972) resulted in a mixture of intractable and inseparable materials. Based on the promising cytotoxic activity profile of our prior synthesized C5-phenylethynyl uracil glucopyranonucleoside (Dimopoulou et al., 2013) and in order to explore the impact of the glycosidic part on the biological activity as well as the potential inhibitory effects of adenine moiety, we sought to introduce phenylacetylene substituent in 8-position of adenine itself. Therefore, we investigated the development of the efficient Sonogashira alkynylation protocol for the cross-coupling of commercially available 8-bromoadenine (I) with phenylacetylene under microwave irradiation (200 W) (Figure 2). 8-Bromoadenine (I) was mixed with anhydrous DMF, phenylacetylene, triethylamine, Pd(PPh 3 ) 4 , CuI, irradiated with microwaves for 6 min at 60 • C and after removing volatiles in vacuo, the solid residue was purified by flash chromatography to provide compound II, in acceptable yield (40%).

Cytostatic Activity
The cytostatic activity of the novel C8-modified adenine pyranonucleosides 4e, 5a-d as well as 8-phenylethynyl-adenine (II) was determined against murine leukemia (L1210), human lymphocyte (CEM) and human cervix carcinoma (HeLa) cell cultures (Table 1). Compounds 4e and II were less cytostatic than 5-fluorouracil (almost an order of magnitude) against murine leukemia (L1210) and human cervix carcinoma (HeLa) cells, while the same compounds proved to be more active than 5fluorouracil against human lymphocyte (CEM) cells.

Conclusion
In summary, we have prepared several novel C8-alkynyl adenine nucleosides as well as 8-phenylethynyl-adenine, via Sonogashira coupling conditions under microwave irradiation. Among the compound series tested, the protected adenine pyranonucleoside 4e, as well as phenylethynyl adenine (II) showed significant cytotoxicity (IC 50 of 1.2-10.0 µM) against murine leukemia (L1210), human lymphocyte (CEM) and human cervix carcinoma (HeLa) cell cultures. Since the glucose derivative of phenylethynyl adenine, nucleoside 5b, showed no activity, it is clear that it is stable and not susceptible to hydrolysis. The replacement of glucose with ribo, arabino, and deoxyribose moieties as well as the introduction of functional substituents on the phenyl ring, such as halogens, nitro or amino and alkyl groups, could be explored in the future in an attempt to further increase the cytostatic potential of these lead compounds.