Versatile synthesis and biological evaluation of novel 3’-fluorinated purine nucleosides

A unified synthetic strategy accessing novel 3'-fluorinated purine nucleoside derivatives and their biological evaluation were achieved. Novel 3’-fluorinated analogues were constructed from a common 3’-deoxy-3’-fluororibofuranose intermediate. Employing Suzuki and Stille cross-coupling reactions, fifteen 3’-fluororibose purine nucleosides 1–15 and eight 3’-fluororibose 2-chloro/2-aminopurine nucleosides 16–23 with various substituents at position 6 of the purine ring were efficiently synthesized. Furthermore, 3’-fluorine analogs of natural products nebularine and 6-methylpurine riboside were constructed via our convergent synthetic strategy. Synthesized nucleosides were tested against HT116 (colon cancer) and 143B (osteosarcoma cancer) tumor cell lines. We have demonstrated 3’-fluorine purine nucleoside analogues display potent tumor cell growth inhibition activity at sub- or low micromolar concentration.


Introduction
Antimetabolites are extremely useful for the treatment of cancers and viral infections and are one of the largest classes of drugs. Most antimetabolite drugs are nucleoside derivatives that substitute for endogenous nucleosides and prevent DNA and protein replication [1]. Many of the drugs described by the World Health Organization as "essential medicines" are nucleo-side derivatives [2] and nearly 20% of all drugs for the treatment of cancers are nucleoside derivatives [3]. The design of new antimetabolites is an active field of research and several nucleoside derivatives have recently come to market such as gemcitabine, capecitabine, and decitabine [4][5][6]. Purine nucleoside analogues such as fludarabine, nelarabine, cladribine, and clofarabine are an important subset of nucleoside drugs [7][8][9][10]. Purine ribonucleosides substituted at position 6 have exhibited potent antimetabolite activity [11][12][13] and aryl or heterocyclic substituents have imparted cytostatic activities against various tumor cell-lines [14][15][16]. Moreover, some 6-heterocyclic substituted purine ribonucleosides also demonstrate strong antiviral activities [17]. Purine derivatives such as, 2'-β-C-methyl-6substituted purine nucleosides exhibit promising anti-HCV activity by blocking RNA dependent RNA polymerase [18][19][20]. Design and synthesis of purine-based nucleosides are still needed to enable new therapies for the treatment of drug-resistant tumors and viruses.
One of the first antimetabolite drugs rationally designed from biochemical data was 5-fluorouracil. A hydrogen atom on uracil was replaced with a fluorine atom for specific reasons. The fluorine atom possesses unique characteristics; it enhances the lipophilicity of organic compounds and C-F bonds have low chemical reactivity imparting high enzymatic stability and resistance to metabolic processes. The high electronegativity of fluorine and the lipophilicity it imparts improve the bioavailability of fluorine-containing drugs. Relative to the unfluorinated derivative, fluorinated drugs have demonstrated favorable pharmacological, physicochemical, pharmacokinetic, pharmacodynamic and safety profiles for a number of compounds [21][22][23]. Several blockbuster drugs such as Lipitor ® , Seretide ® , Crestor ® , Takepron ® , Sustiva ® , Celebrex ® , and the recently described fluorapacin and azvudine [24][25][26][27][28][29], all contain fluorine atom(s). Not surprisingly, 20% of marketed drugs contain fluorine atom(s). Gemcitabine, the 2'-deoxy-2',2'-difluorocytidine has been routinely utilized to treat solid tumors [30]. However, 3'-fluorine-modified nucleosides have not been wellstudied because of the challenges associated with the synthesis of modified carbohydrate moieties [31][32][33][34][35]. As a result, 3'-fluoro-6-heterocyclic-substituted purine nucleosides are not well represented in the literature. We chose to explore the biological potential of synthetic purine analogues combining substitutions at position 6 and a 3'-fluorine. Novel 6-heterocyclic substituted purine 3'-deoxy-3'-fluororibonucleosides were designed to discover more selective and potent novel antiviral and anticancer therapeutics. Various fluorine-modified ribonucleoside derivatives were designed, synthesized, and tested. The preliminary results are presented herein.

Results and Discussion
Current strategies to the synthesis of 3'-fluorine and 6-substituted purine require harsh conditions and laborious protecting group manipulation that results in low product yields. It was reported that 3'-deoxy-3'-fluoroadenosine (2) (Figure 1) was synthesized in 3.5% yield starting from adenosine [31,32]. This eight-step synthesis required harsh reaction conditions and HPLC purification of the final product. In addition, the product cannot be utilized for further derivatization to reach our objectives. This 3'-fluorine-modified adenosine 2 has also been synthesized starting from a well-protected xylofuranosyladenine derivative using a complicated strategy [33]. Similarly, 3'-deoxy-3'-fluoroguanosine (23, Figure 2) was isolated in 2% yield after protecting group manipulation from arabinoguano- sine [34]. Complicated orthogonally protected adenosine and guanosine derivatives with three or four different protecting groups have also been used for the synthesis of compounds 2 and 23, and the protocols required extensive manipulation of the protecting groups [35]. Compound 2 has also been synthesized starting from xylofuranoside by manipulating the protecting groups on the carbohydrate moiety [36]. De Clercq and co-workers [37] developed a protocol for the synthesis of 3'-fluororibofuranose in 10 steps, and it requires epoxide formation and ring opening as well as reversion of the hydroxy group on the sugar ring. Jeong and co-workers [38][39][40] synthesized fluorine-substituted ribofuranose but isolation required a challenging separation. Therefore, the synthetic challenges for  (24) was synthesized from D-xylose according to literature procedures [41]. Compound 24 was treated with iodine in methanol, and fluorinated with diethylaminosulfur trifluoride (DAST). The (25) was then obtained in 33.13% overall yield after further treatment with acetic anhydride-acetic acid-sulfuric acid system. More than 200 g scale was achieved for the synthesis, and high purity (98%) product was obtained. This key intermediate 25 can be used to synthesize a variety of 3'-fluoro-modified nucleoside derivatives, and it was utilized for the synthesis of all analogues reported herein. This strategy avoids tedious orthogonal protecting group manipulations previously reported in literature [31][32][33][34][35]. Our strategy provides the desired nucleoside intermediates and also opens up the opportunity for modification on any class of nucleosides with a 3'-fluorine atom to explore their biological and therapeutic potential. While this work is related with purine nucleosides, the strategy can be used for the synthesis of a variety of nucleosides with a wide range of heterocyclic moieties to investigate the impact of a 3'-fluorine atom on the biological activity of nucleosides. To construct the first series of fluorinated purine analogues, compound 26 was treated with a saturated solution of ammonia in methanol, which resulted in the amination at the 6-position and deprotection of the protecting groups to furnish 3'-deoxy-3'-fluoroadenosine (2) in 85% yield. Our synthetic strategy provided compound 2 in excellent yield (76%, 2 steps) compared to previously reported literature protocols (3.5%, 8 steps) [31,32]. The 6-chlorine of compound 26 was replaced by hydroxylamine, with concomitant removal of the protecting groups to yield N 6 -hydroxy-3'fluoro-3'-deoxyadenosine (3). Hydrogenation of compound 26 under hydrogen pressure (50 psi) over 10% Pd/C resulted in the de-chlorinated compound 27, which was further deprotected in a saturated solution of ammonia in methanol providing the desired 6-deaminated 3'-fluoro-adenosine 1 in 93% yield. We targeted 9-(3-deoxy-3-fluoro-β-D-ribofuranosyl)purine (1) in particular because it is the 3'-fluorine analogue of nebularine, a naturally occurring antibacterial and antineoplastic agent [42,43].  antitumor activities [44]. In order to explore the effect of fluorine on the biological activity of this pharmacophore, we synthesized 6-methylpurine-3'-deoxy-3'-fluoro-β-D-riboside (4) (Scheme 2). 6-Methylpurine (28) was synthesized from 6-chloropurine according to the reported protocol [44]. Compound 28 was silylated with BSA and glycosylated with the 3'-fluoro-sugar 25 to provide the desired compound 29 in 78% yield. Subsequent deprotection furnished the targeted novel fluorine modified 6-methylpurine riboside 4 in 80% yield.
Of particular importance, the 2-chlorine atom on the intermediate 42 was stable under the Stille and Suzuki reaction conditions and under ammonia deprotection conditions. However, the palladium-catalyzed cross coupling of dichloro-intermediate 42 with organostannane and organoboronic acid reagents resulted in lower yields when compared to the cross coupling of monochloro-intermediate 26.
From the amination studies of 2,6dichloropurines, the 6-position of the purine possesses higher reactivity towards nucleophiles than the 2-position. In addition, the selectivity for the 6-position is also higher for the Stille than for the Suzuki cross coupling. The lower yields obtained from the cross-coupling of 2,6-dichloropurines is most likely contributed to the 2-chlorine reducing the reactivity of the 6-chlorine, or undesired cross-coupled products at the 2-position of the purine that may have resulted, but were not isolated.

Biological evaluation
Newly synthesized compounds were tested against the human colon cancer cell line HCT-116 and the human osteosarcoma cancer cell line 143B (Table 2). The fluorinated nebularine    showed moderate levels of inhibitory activity, but other derivatives did not show detectable activity against the tested tumor cell lines. Antiviral and other biological evaluation of these 3'-fluorine modified nucleosides is in progress and will be reported in due course.

Supporting Information
Supporting Information File 1 Experimental procedures, characterization data, and