Synthesis of Boron-Containing Nucleoside Analogs

Over the last century, nucleoside-based therapeutics have demonstrated remarkable effectiveness in the treatment of a wide variety of diseases from cancer to HIV. In addition, boron-containing drugs have recently emerged as an exciting and fruitful avenue for medicinal therapies. However, borononucleosides have largely been unexplored in the context of medicinal applications. Herein, we report the synthesis, isolation, and characterization of two novel boron-containing nucleoside compound libraries which may find utility as therapeutic agents. Our synthetic strategy employs efficient one-step substitution reactions between a diverse variety of nucleoside scaffolds and an assortment of n-alkyl potassium trifluoroborate-containing electrophiles. We demonstrated that these alkylation reactions are compatible with cyclic and acyclic nucleoside substrates, as well as increasing alkyl chain lengths. Furthermore, regioselective control of product formation can be readily achieved through manipulation of base identity and reaction temperature conditions.


■ INTRODUCTION
−6 Boron has unique chemical characteristics; it does not follow the typical electron octet rule and can form three covalent bonds while retaining a reactive vacant p-orbital center.This distinct property gives boronic acids and their derivatives strong Lewis acid characteristics, giving rise to interesting and exceptional chemistry. 7Furthermore, this allows the boron center to convert between two molecular geometries; an uncharged, trigonal planar form and an anionic, tetrahedral state with an electron-rich ligand coordinated to the vacant p-orbital (Figure 1). 8urrently, there are five FDA-approved drugs on the market containing a boron atom center which is critical to therapeutic activity (Figure 2).These drugs include: Velcade, a peptidyl boronic acid treatment for multiple myeloma; 9 Kerydin, an oxaborole-containing antifungal agent; Ninlaro, a reversible proteasome inhibitor for the treatment of multiple myeloma; 10 Eucrisa, a nonsteroidal topical medication used for the treatment of eczema; 11 and Vaborbactam, a β-lactamase inhibitor used for the treatment of urinary tract infections. 12elcade and Ninlaro are alkyl boronic acids that form strong covalent adducts with threonine residues in the active site of proteasomes, 5 whereas Eucrisa is a benzoxaborole inhibitor that chelates to metal centers in the enzyme active site. 11,13hese diverse and unique interactions of boron drugs with their biological targets have significantly increased research efforts to further the use of boronic acids in medicinal chemistry. For example, nucleoside analogs such as azidothymidine (AZT) and adefovir have been powerful drugs for the treatment of HIV and Hepatitis B, respectively (Figure 3). 16In 1996, Chen and coworkers reported the first study of boronic acids being used as nucleoside analogs for therapeutic applications. 17The group synthesized a series of novel acyclic nucleoside boronic acid derivatives containing either a pyrimidine or purine base.These compounds were evaluated for their anti-HIV activity and found a few nucleoside analogs capable of exhibiting mild activity in vitro.They obtained EC 50 values in the low micromolar range for two acyclic borononucleoside compounds: 6-chloro-9-(4-dihydroxyborylbutyl) purine 1 and 2,6dichloro-9-(4-dihydroxyborylbutyl) purine 2 (Figure 3).These compounds exhibited EC 50 values for anti-HIV plaque reduction assay of 7.7 ± 1.5 μM and 0.99 ± 0.01 μM, for 1 and 2, respectively.However, they also observed significant cytotoxicity, with IC 50 values for CEM-SS cell lines of 43 ± 31.1 μM and 4.9 ± 2.2 μM, for 1 and 2, respectively.It was noted that they did not investigate the mechanism of action for their anti-HIV agents.
More recently in 2012, El Amri et al. synthesized and evaluated various borononucleoside analogs as 5′-monophosphate isosteres for human NMP kinases. 18The group screened eight borononucleoside compounds as substrates toward the five human NMP kinases: hAMPK, hCMPK, hTMPK, hUMPK, and hGMP.They observed that only one of the borononucleoside analogs, 3, acted as a weak substrate toward human thymidylate monophosphate kinase (TMP) (Figure 3).This result suggests that some borononucleosides can act as substrates for selected human NMP kinases.
These limited works largely represent the extent of research conducted toward synthesizing boronic acid-containing nucleosides for potential drug applications.In considering the above work by Chen et al. and El Amri et al., the synthesized compounds placed a boronic acid moiety near where one would find the phosphate group in a nucleoside monophosphate.−21 To this end, we set out to expand the portfolio of boron-containing nucleosides and develop synthetically rigorous methodology for the formation of nucleoside alkylated boronic acid compounds.

■ RESULTS AND DISCUSSION
First, we looked to expand the structural diversity present in Chen's borononucleoside compounds by incorporation of an ether linkage similar to that found in adefovir (Figure 3).These adefovir-boronic acid nucleoside analogs (naNuc) were synthesized with different nucleobases (6-chloropurine, 2,6dichloropurine, 2-amino-6-chloropurine, and adenine) and varied alkyl chain linkers (linker length 3−4 between the oxygen and boronic acid warhead).The synthesis of the naNuc series began with protecting the hydroxyl group on 2bromoethanol (4) as a tetrahydropyranyl (THP) ether to give 5 (Scheme 1). 22ext, nucleobases (6-chloropurine (CP), 2,6-dichloropurine (DCP), and 2-amino-6-chloropurine (ACP)) were used in substitution reactions with 5 to produce protected alcohol nucleoside compounds of series 6.After that, THP deprotection was performed to produce the corresponding primary alcohol nucleosides of series 7. 23 Synthesis of  The Journal of Organic Chemistry bromoalkyl potassium trifluoroborate electrophiles 8a−c (propyl, butyl, and pentyl, respectively) have been reported previously by the Tomsho group. 24,25Finally, boronic acidadefovir analogs were obtained via substitution reactions of bromoalkyl-potassium trifluoroborates (8a and 8b) with various alcohol nucleosides (7a, 7b, 7c, and commercially available 7d).In this substitution reaction, K 2 CO 3 was used as a base with tetrabutylammonium bromide (TBAB) as a phase transfer catalyst, and the reaction was heated to 90 °C overnight.After the substitution reaction, the trifluoroborate group was hydrolyzed to the free boronic acid. 26,27These final compounds were purified by silica gel flash column chromatography (3−10% MeOH in DCM) (Figure 4).
The crude 1 H NMR yields for adefovir-boronic acid nucleoside analogs range from 55 to 88%.However, the isolated yields for the compounds fall between 7 and 30%.These discrepancies between the 1 H NMR yields and the isolated yields occurred due to difficulties during column chromatography separation.The retention factor (Rf) of the alcohol compounds 7a−d and the desired final compounds are similar, making the separation challenging.In attempts to optimize these reactions, the reaction time was increased to 2 days to ensure complete conversion of the alcohol compounds 7a−d to the desired final compounds.However, increasing the reaction time resulted in decomposition of both the alcohol compounds 7a−d and the final adefovir-boronic acid nucleoside compounds, which suggests an inherent limitation of the stability of these compounds.
Synthesis of naNuc reactions generally proceeded well with high regio-and chemoselectivity.Some alcohol nucleobase analogs (7a−d) have more than one nucleophilic center with the potential to undergo alkylation following deprotonation (Scheme 1).For example, 2-amino-6-chloropurine and adenine intermediates 7c and 7d, respectively, have an exocyclic amine group which may alkylated.We have applied 2-D NMR methods (COSY, HSQC, and HMBC) to confirm the position of substitution on the nucleoside analogs (Figure 4).
In an effort to expand these types of syntheses to include nucleosides with more complex three-dimensional structures, we envisioned generating a second library of borononucleoside compounds with attached ribose-type rings.These include ubiquitous, natural, and medicinally relevant synthetic scaffolds such as thymidine, uridine, stavudine, lamivudine, and emtricitabine.We expected these new target nucleosides may be accessible via a similar one-step substitution reaction as described above, in which the nucleoside is alkylated using a trifluoroborate-containing halide electrophile 8a−c.This would give rise to a large number of synthetic analogs quickly and easily using variations on a single substitution reaction.We anticipated synthesizing an inaugural series of 5′-hydroxyl alkylated boron-containing thymidine compounds (3aT, 4aT, and 5aT).However, when designing the experimental conditions for substitution, we were cognizant that the nucleoside starting materials have multiple possible sites for substitution.In the example of thymidine, there are three nucleophilic positions on the molecule with the potential to undergo substitution, with a relative nucleophilicity of: 5′primary hydroxyl >3′-secondary hydroxyl > internal nucleobase amine.
Preliminary experimentation focused on using thymidine as a template nucleoside and 4-carbon trifluoroborate electrophile 8b as the alkylating agent (Table 1).These reactions were conducted in DMF due to the poor solubility of starting material in other polar aprotic organic solvents like tetrahydrofuran.Experiments were left to react for 6 h.Using sodium hydride (NaH) as a base for the initial deprotonation step, our first optimization experiment was performed using low temperatures (0 °C) with 1.05 equiv of thymidine substrate (Table 1, Exp 1).This resulted in the generation of only one substitution product as seen in the crude 1 H NMR spectrum, which was subsequently isolated using flash column chromatography (3% MeOH in DCM).Though confirmation of the product formed was nontrivial due to all potential regioisomers having similar physical and spectroscopic properties, we utilized 2D NMR experiments (COSY, HSQC, and HMBC) to elucidate the regioisomer formed.By analyzing the cross-correlation signals between protons and carbons on the alkyl side chain with those on the nucleoside scaffold in the heteronuclear multiple bond  The Journal of Organic Chemistry correlation (HMBC) NMR spectrum, we determined the product formed was nucleobase-substituted regioisomer, N4aT (172 mg, 0.55 mmol, 31%).This substitution pattern was rationalized by the relative pK a values of the thymidine labile protons (NH < 5′−OH < 3′−OH), giving rise to the nucleobase-deprotonated intermediate, which acts as a hindered, but viable nucleophile during the reaction.
Although we were pleased to have proof of concept and our first borononucleoside generated, we endeavored to continue developing and optimizing the reaction to provide access to the 5′-hydroxyl substituted analogs we had originally envisioned making.To this end, we studied the effects of base identity, stoichiometry, and reaction temperature on the regioselectivity of product formation (Table 1).First, we hypothesized that the use of 3 equiv of NaH base may be sufficient to globally deprotonate all positions on the thymidine nucleoside, which would allow the relative rates of nucleophilicity for each site to determine regioisomer formation (Table 1, Exp 2).By analyzing the crude reaction mixture by 1 H NMR spectroscopy, we were able to determine the relative ratio of regioisomer products formed.This was achieved with a high degree of confidence by comparing and contrasting the integration intensities of the product peaks corresponding to the 5′-CH 2 units.These signals resonate at different chemical shift values and have distinct J-coupling constants which are indicative of position of substitution.The product peak identities were also corroborated by tandem HMBC NMR analysis.While we expected the most nucleophilic 5′-hydroxyl location to dominate and give us the desired 4aT product, this was not observed, and the major isomer was once again N4aT (8:1).Next, we hypothesized that increasing the temperature of the nucleoside-base premix would shift the deprotonation event toward the 4aT product.To this end, we attempted reactions at room temperature (Table 1, Exp 3), as well as under mild heating (Table 1, Exp 4).While increases in the proportion of 4aT were observed, the predominant regioisomer formed was still the nucleobase-substituted, N4aT (6:1 and 4:1, respectively).
It was determined that the simple sodium hydride base was ultimately too small to deprotonate at any other site than the most acidic proton.As such, it was proposed that the use of a larger, bulkier base may prevent abstraction of the sterically hindered nucleobase proton, favoring the easily accessible 5′primary hydroxyl instead.To test this theory, sodium bis(trimethylsilyl)amide (NaHMDS) was employed as the base of choice, and all other conditions remained the same as the original experiment (Table 1, Exp 5).Indeed, the largest ratio of 4aT was observed using the bulkier amide base over sodium hydride (2:1).This gave us encouragement that we might be able to manipulate the reaction conditions further to push the deprotonation event toward the 5′-hydroxyl position.Therefore, we revisited using 3 equiv of base, this time with NaHMDS as the deprotonating agent (Table 1, Exp 6).However, in this instance, no consumption of thymidine was observed and starting material appeared to precipitate out of solution upon addition of base.We considered whether increasing the temperature with NaHMDS might have a similar effect at shifting product formation away from N4aT, as was observed in experiments 3 and 4. To examine this, we attempted the reaction at room temperature and at 60 °C (Table 1, Exp 7 and 8, respectively).To our delight, increasing the reaction temperature to room temperature gave a 1:1 ratio of products, and a further increase to 60 °C yielded 4aT as the major regioisomer (N4aT:4aT, 1:6).It was noted that increasing the temperature much beyond 60 °C caused degradation of the thymidine substrate, as was observed at 90 °C (Table Compound identification key: N = nucleobase-substituted, n = alkyl chain length, a = boronic acid, Nuc (nucleoside) = T (thymidine).
The Journal of Organic Chemistry nucleoside series of analogs under regioselective control of product formation (Scheme 2).
Isolated yields reported were often significantly lower than those determined by integration of the different compound peaks in the 1 H NMR of the crude products.Starting material conversion was high; however, a loss in recovered yield is attributed to the tight separation between compounds during preparative scale flash column chromatography.Improved isolated yields may be achieved on a small scale using HPLC separation.The methodology developed to generate the thymidine analog series was then successfully used to expand the scope of nucleoside series to include uridine, stavudine, lamivudine, and emtricitabine (Figure 5).The alkylation reactions showed similar regioselectivity for each nucleoside series under the optimized reaction conditions.Furthermore, these substitution reactions demonstrated functional group tolerance toward all five-nucleoside series employed.This firstgeneration library of boron-containing nucleoside analogs will be evaluated for their ability to act as potential therapeutic agents.

■ CONCLUSION
In summary, we have synthesized two libraries of boroncontaining nucleoside analogs, which may find utility toward therapeutic applications.We have expanded the scope of boron-containing nucleoside compounds to include alkylated boronic acids for a number of different nucleoside scaffolds, including 6-chloropurine, 2,6-dichloropurine, 2-amino-6-chloropurine, adenine, thymidine, uridine, stavudine, lamivudine, and emtricitabine.Furthermore, we have developed methodology to regioselectively access both nucleobase-and 5′-  The Journal of Organic Chemistry hydroxyl-substituted analogs using condition modifications on a single reaction.

■ EXPERIMENTAL SECTION
General Experimental Methods.All synthetic routes were performed according to the standard lab safety guidelines described in Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. 28All reactants and reagents were purchased and used without further purification.Almost all reactions were carried out under an inert atmosphere of argon using a glovebox or Schlenk line.Rotary evaporation was used to remove the solvents.Final compound drying was done under high vacuum (ca.0.01 Torr).Thin layer chromatography (TLC) was performed using silica gel 200 μM precoated polyester backed plates with a fluorescent indicator, and TLC plates were visualized with UV light (254 nm).Flash column chromatography was conducted with the indicated solvent system using normal phase silica gel 40−63 μM, 230−400 mesh.Structural assignments were made with additional information from gCOSY, gHSQC, and gHMBC experiments. 1H NMR spectra were recorded on a Bruker Avance at 400 MHz, 13 C NMR spectra were recorded at 100 MHz, and 11 B NMR spectra were recorded at 128 MHz.Chemical shifts are reported in δ values (ppm) relative to an internal reference of tetramethyl silane (TMS) or the residual solvent signal.Peak splitting patterns in the 1 H NMR are reported as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.Mass spectra were obtained on a Thermo-Fisher Exactive Orbitrap Mass Spectrometer using Electrospray Ionization.Compounds 8a−c were synthesized using previously reported procedures. 24,25Commercially available nucleosides: 6-chloropurine, 2,6-dichloropurine, 2-amino-6chloropurine, adenine, thymidine, uridine, stavudine, lamivudine, and emtricitabine were purchased from reputable chemical vendors with purity >99%.
2-Bromo-1-ethoxytetrahydropyran, 5. To an Ar-flushed and flame-dried rbf containing 2-bromoethanol (16.01 mmol, 1.0 eq, 2.00 g) was added dry DCM (80 mL).Then, p-TSA (1.60 mmol, 10%, 276 mg) was added quickly followed by the addition of DHP (20.8 mmol, 1.3 eq, 1.75 g) at room temperature.The mixture became homogeneous upon vigorous stirring, and the resulting solution was stirred overnight.The reaction was quenched with saturated aqueous NaHCO 3 and transferred to a separatory funnel.The aqueous layer was extracted with DCM (3 × 50 mL), and the combined organics were washed with water and brine.The organics were combined and concentrated to yield 5 as a yellow oil (3.16 g, 15.99 mmol, 95%). 1  General Procedure for the Synthesis of Nucleosidyl-9-(1ethoxytetrahydropyran), 6.A flame-dried Ar-flushed rbf was charged with nucleoside (1.0 equiv) and K 2 CO 3 (3.5 equiv).These were dissolved in 20 mL DMF and stirred at rt for 50 min.Next, 5 (1.0 equiv) and NaI (10 mol %) were dissolved in 20 mL dry DMF (from an Ar-flushed rbf) and were added to the reaction mixture.Then, the reaction mixture was stirred overnight at rt.The next day, the reaction mixture was filtered, and solids were rinsed with EtOAc.The combined organics were concentrated, and the product was purified by column chromatography (0−3% MeOH in EtOAc) to give nucleosidyl-9-(1-ethoxytetrahydropyran), 6.
6-Chloropurinyl-9-ethanol, 7a.Starting material; 6a (987 mg, 3.49 mmol): 1  General Procedure for the Synthesis of O-Substituted Adefovir-Boronic Acid Nucleoside Series, naNuc.An Ar-flushed, oven-dried rbf was charged with alcohol analogs 7 (1.0 equiv) and K 2 CO 3 (3.5 equiv).These were dissolved in 20 mL dry DMF and stirred at rt for 50 min.Next, 8a or 8b electrophile (1.0 equiv) was added to the reaction mixture followed by addition of TBAB (10%).The reaction was heated up to 90 °C in an oil bath and stirred overnight.The reaction mixture was then filtered and concentrated.The crude residue was dissolved in 20 mL MeOH before adding 3 mL H 2 O and (6.0 equiv) silica.The mixture was stirred for 4 h before it was filtered and concentrated.The crude boronic acid was purified by column chromatography, eluting with (4−10% MeOH in DCM) to give naNuc.
Compound Identification Key.
Compound Identification Key.

Figure 3 .
Figure 3. (i) Example of FDA-approved nucleoside analogs; anti-HIV and Hepatitis B drugs, including cyclic azidothymidine (AZT) and acyclic adefovir, respectively.(ii) Two acyclic borononucleoside analogs previously reported to exhibit anti-HIV properties (1 and 2).Thymidine borononucleoside analogue 3 behaves as a weak substrate toward human TMP kinase.

Scheme 2 .
Scheme 2. Formation of Boron-Containing Nucleoside Analogs Utilizing Base and Temperature-Dependent Regioselective Control a

Table 1 .
Model Reaction Optimization Studies of 4aT/N4aT Exploring Base Identity, Stoichiometry, and Temperature Effect on Regioselective Product Formation a 1, Exp 10).Having successfully developed and optimized two separate sets of conditions for the model substitution reaction, we have the ability to generate both the nucleobase-(Table 1, Exp 1) and 5′-hydroxyl-(Table 1, Exp 8) substituted analogs using modifications on a single reaction.This gives us the means to synthesize the entire thymidine