Synthesis of nucleoside analogues using acyclic diastereoselective reactions

The design of novel xylo-like nucleoside analogues bearing a C3’ all - carbon quaternary center and a C2’ - hydroxy substituent is described. Synthesis of this scaffold makes use of highly diastereoselective transformations on acyclic substrates. Central to the approach is formation of a 2,4-syn cyanohydrin from cyanide addition onto an aldehyde through a proposed seven-membered ring chelate using a bidentate Lewis acid. In addition, a highly diastereoselective Mukaiyama aldol reaction, an intramolecular radical atom cyclization, and thioaminal formation are used to generate this novel molecule. A series of related nucleoside analogues are being tested as antiviral and anticancer agents


Introduction
The implication of endogenous nucleosides and nucleotides in numerous biological pathways has, not surprisingly, inspired the development of various analogues as inhibitors of tumor growth and viral replication. 1,2,3Exploring the biological profiles of novel nucleoside analogues remains crucial, as shown by the recent approval of highly efficient antiviral drugs. 4We have initiated the syntheses of novel nucleoside analogues possessing an all-carbon quaternary stereogenic center at C3'. [5][6][7] It is proposed that this chiral center could enhance target specificity in addition to providing opportunities for the incorporation of different pharmacophores.As illustrated in Figure 1, the hydroxymethyl substituent at C3' can have either a xylo/lyxo-(β-face) or ribo/arabino-(α-face) like orientation.The syntheses of C2'-fluoro analogues in which the hydroxymethyl substituent is located on the α-face (ribo-like scaffolds) has been previously reported by our group. 8Herein, we describe the synthesis of xylo-like analogues with a C2'-hydroxy group.Incorporation of a C3'-hydroxymethyl substituent with a β-orientation is a feature of apio-nucleosides, a class of analogues in which the hydroxymethyl normally found at C4' is shifted to C3'. [9][10][11] Presently, our scaffolds are being tested for their antiviral and anticancer properties; however, this novel xylo-like scaffold may also show potential in the design of new fungicides 12 and insecticides, 13 as antimicrobial agents 14 and acetylcholinesterase inhibitors 15 all of which contain a xylo-furanoside core.To construct our novel scaffold (1), a series of diastereoselective acyclic transformations were developed (Scheme 1).Using our two-step acyclic approach for the synthesis of nucleoside analogues, 1',2'-trans furanoside 2, was formed from a kinetically controlled intramolecular cyclization of chiral thioaminal 3.This 1,2-syn thioaminal resulted from diastereoselective nucleobase addition onto dithioacetal 4. Scheme 1. Retrosynthetic analysis of xylo-like analogues.
Central to our synthetic approach was a Lewis acid controlled diastereoselective cyanide (TMSCN) addition onto aldehyde 6 through a proposed seven-membered ring chelate to generate the C2'-hydroxy substituent from formation of the acyclic cyanohydrin 5.An intramolecular silicon tethered free radical-based vinylation was used to construct the all-carbon stereogenic center in 7, the substrate of which was accessed from a Mukaiyama aldol reaction between aldehyde 9 and enoxysilane 10.

Results and Discussion
The reaction sequence to generate the targeted family of nucleoside analogues began with construction of the all-carbon quaternary center (Scheme 2).The key precursor 8a,b for the intramolecular radical atom transfer cyclization was efficiently accessed by a three-step sequence involving a Mukaiyama aldol reaction 16,17 between 2,3-isopropylidene-D-glyceraldehyde 9 and enoxysilanes 10 in the presence of MgBr2 .OEt2. 7 Treatment of the crude reaction mixture with HF-pyridine was necessary to remove undesired TMS protection of the oxygen at C3.The vinyldimethylchlorosilane moiety was then installed on the resulting free alcohols to provide the 3,4-anti (8a,b) and 3,4-syn (11a,b) bromides in a 10:1 ratio and excellent yield (52%) over the three steps.9][20][21] The steric hindrance imposed by the isopropylidene moiety of aldehyde 9 is likely to prohibit chelate formation between the aldehyde and α-oxygen.

Scheme 2. Synthesis of the all-carbon quaternary center in 12.
The free radical tandem cyclization/elimination reaction developed by our laboratory 22 was then performed on 8a,b in the presence of BEt3/O2 (Scheme 2).The reaction was shown to proceed through a fivemembered ring intermediate A (5-exo-trig) bearing a mixture of primary bromides. 22The stereochemistry of the newly formed quaternary center was proposed to originate from transition state A that minimizes intramolecular dipole-dipole interactions and allylic 1,3-strain. 23The five-membered silyloxy ether intermediates (A) were cleaved upon treatment with 3HF•NEt3 to give the 2,3-syn product 12 as the only observable diastereomer (>20:1) in 82% yield (see supporting information for stereochemical proofs).Derivatization of 12 towards aldehyde 6 was achieved by first carrying out silylation of the secondary alcohol (Scheme 3).Reduction of the methyl ester and subsequent benzoylation generated the protected primary alcohol 13 in 68% yield over three steps.Cleavage of the acetonide and oxidation of the 1,2-diol using periodic acid led to an aldehyde that was immediately reduced to the corresponding primary alcohol 14 (85% yield over two steps), which was then protected with a benzyl group to give 15.Deprotection of the primary benzoate with DIBAL-H followed by DMP oxidation provided aldehyde 6. Scheme 3. Synthesis of aldehyde 6 bearing an α-quaternary center and a β-hydroxy group.
The diastereoselective addition of cyanide 24,25 onto aldehyde 6 was next studied.The desired xylo-like scaffold of the targeted nucleoside analogue (1) required a syn stereochemistry between the substituents at C2 and C4 of the resulting cyanohydrin (5).1,2-Induction using a monodentate Lewis acid (TS B and TS C, Scheme 4) was expected to be poor, since two of the substituents of the quaternary center (methyl and vinyl) are sterically similar.The possibility of using the stereogenic secondary β-hydroxy group of the acyclic aldehyde was therefore considered.

Scheme 4. Transition states for diastereoselective cyanohydrin formation.
A bidentate Lewis acid could generate different reactive chelate intermediates.Chelation between the β-hydroxy group and the aldehyde is expected to give preference for the undesired 2,4-anti relationship through TS D, which avoids steric clash between the incoming nucleophile and the -CH2OBn found in TS E (2,4syn predictive transition state).However, the presence of the α-quaternary center could decrease the energy difference between TS D and TS E, the former having two gauche interactions between the substituents of the α-and β-stereogenic centers.Formation of a seven-membered ring intermediate (TS F and TS G) between the primary oxygen and the aldehyde could allow more flexibility for chelate formation and alleviate steric interactions between the incoming nucleophile and the quaternary center.Formation of the desired 2,4-syn cyanohydrin (TS G) in which nucleophilic attack occurs on the bottom face should be favored to avoid eclipsing interactions between the incoming nucleophile and the axial methyl of the quaternary center in TS G or with the β-hydroxy group in TS F.
To prevent potential competition between six-and seven-membered ring chelates, a bulky TBS group was incorporated onto the β-hydroxy of aldehyde 6. Precomplexation of aldehyde 6 with an excess of MgBr2•OEt2 24 was followed by addition of the cyanide source (TMSCN) at -15 o C (Scheme 5).Cyanohydrin 5 was formed in an 8:1 ratio in favor of the 2,4 syn isomer in excellent yield (83% over two steps).It is noteworthy that the reaction of TMSCN and the aldehyde alone in DCM resulted in recuperation of the starting material, thus highlighting the need for a source of Lewis acid.Cyanide addition did occur in the presence of a Lewis base (NEt3 or NH(i-Pr)2) to furnish OTMS protected cyanohydrins (results not shown), albeit with low diastereoselectivity. 26 These observations are in accordance with precedent examples demonstrating that a Lewis acidic or basic species is needed to activate the TMSCN through formation of a pentacoordinate siliconate ion. 27,28heme 5. Synthesis of 2,4-syn cyanohydrin.
To validate our initial hypotheses for 2,4-syn selectivity, a model study was undertaken at 0 o C (Table 1).As can be seen in entry 1, a 6:1 ratio in favor of the syn cyanohydrin was obtained at this temperature.When the reaction was performed with a monodentate Lewis acid, BF3 .OEt2, an expected low diastereoselectivity of 1.5:1 was observed in favor of the 2,4-syn product (entry 2).Interestingly, when the TBS β-hydroxy protecting group was replaced by a benzyl or p-methoxybenzyl group, (aldehydes 17 and 19), preference for the 2,4-syn cyanohydrin was maintained (entries 3 and 4) with MgBr2•OEt2, even though competing TS D could be at play.With aldehyde 21, bearing a free β-hydroxy group (entry 5), a reversal of selectivity in favor of the 2,4-anti product was indeed noted suggesting reaction through TS D.
Suppressing the possibility of creating a seven-membered ring chelate by replacing the primary benzyl protecting group with a bulky silyl ether, as in aldehyde 23, abolished the diastereoselection noted before (1.5:1, entry 6).Similarly, when the benzyl ether was replaced by its carbon equivalent as in 25, a decrease in selectivity was observed (2.5:1, entry 7).To ensure that this loss of selectivity was not the result of having an α-gem-dimethyl instead of the stereogenic center, we prepared 27 and observed a high syn preference (11:1 entry 8 versus 6:1 entry 1) which suggests a greater interaction between the incoming nucleophile and olefin in TS G. Taken together, these results support the intermediacy of a 7-membered ring chelate (TS G) [29][30][31] in inducing 2,4-syn diastereoselectivity for cyanohydrin formation in the presence of MgBr2•OEt2.
Interestingly, with the use of a titanium Lewis acid (Table 1, entries 9-13), formation of the 2,4-anti cyanohydrin could be favored.When aldehyde 19 bearing a β-PMB was reacted with 1.1 equivalents of TiCl3(OiPr), a 1:1 ratio of syn and anti cyanohydrins were formed (entry 9) in which the β-PMB was cleaved.Upon increasing the equivalents of TiCl3(OiPr) to 2.5 (entries 10 and 11) a 1:6 ratio was obtained in favor of the 2,4-anti diol 22b.Not surprisingly, with a β-OTBS, preference for the 2,4-syn cyanohydrin 5a was maintained (3:1 ratio, entry 12), favoring cyanation through 7-membered chelate TS G or monodentate activation.NMR spectra of the complexed aldehydes were acquired in CD2Cl2 (Figure 2).The first observation made was that both aldehydes resulted in similar 13 C spectra upon addition of 1.1 equivalents of TiCl3(OiPr), which indicates that the β-PMB of aldehyde 19 is cleaved before addition of the nucleophile. 32Complex spectra (not shown) were obtained with three new peaks in the carbonyl region.It is likely that these correspond to different intermediates that react unselectively to give mixtures of diastereomers (Table 1, entry 9).However, in the presence of 2.5 equivalents of TiCl3(OiPr) (Figure 2, upper spectrum) there is clearly only one complexed carbonyl species with a significant downfield (11.5 ppm) signal.In addition, the carbons corresponding to C3, C4 and C5 are also all shifted downfield, which supports their involvement in complex formation.
Although an X-ray structure of this titanium complex has yet to be determined, the above preliminary 13 C NMR spectra support formation of a complex as in intermediate E (Scheme 6).Formation of this intermediate is supported by Gau's studies of titanium complexes, 33 where he noted the prevalence of hexacoordinated species.In addition, these studies suggested that the binding ability of various chemical entities to titanium was -OiPr > Cl -> THF > Et2O > PhCHO > µ-Cl -> RCO2M.Based on the 13 C NMR data, the first step in the complexation of TiCl3(OiPr) with aldehydes 19 and 21 is formation of a covalent bond between the oxygen at C3 and the titanium resulting in intermediate B (Scheme 6).Following Gau's study, the second oxygen to coordinate would be the oxygen at C4 resulting in intermediate C in which the carbonyl is uncoordinated.Scheme 6. Transition state for 2,4-anti cyanohydrin formation with 2.5 equivalents of TiCl3(OiPr).
Although carbonyl activation is not necessary for cyanation to occur, 13 C NMR studies indicate that it is indeed complexed with the titanium.Upon addition of a second equivalent of TiCl3(OiPr), intermediate D could be formed, but monodentate activation is not likely to provide high diastereoselectivities, as observed with 1.1 equivalents of TiCl3(OiPr) (Table 1, entry 9).Formation of intermediate E in which both titaniums are coordinated to the oxygen at C3 would be consistent with the need for 2.5 equivalents of TiCl3(OiPr) to reach higher levels of diastereoselectivity. Various titanium complexes were examined by density functional theory (DFT) calculations in Gaussian 09 (D.01) with tight SCF convergence 34 using the M062X 35 /6-31G* level of theory in DCM with the polarizable continuum model (PCM). 36Intermediate E in which the isopropoxide ligands are located trans to the C1-aldehyde and C4-OBn group was of lowest energy.Through NBO analysis, a weak interaction was observed between the chloride ligand and the C4'-Ti thus both titanium centers exist as hexacoordinate species.Although, it is also possible to form a bicyclic [3.2.1]-type complex with 2.5 equivalents of TiCl3(OiPr), as previously proposed, 37 our preliminary 13 C NMR data is consistent with formation of intermediate E through initial displacement of a chloride ligand.Preferential attack of the cyanide opposite the β-alkyl chain in TS H would result in formation of the 2,4-anti cyanohydrin 22b.Interestingly, when aldehyde 17 bearing a β-OBn group was reacted in the presence of 2.5 equivalents of TiCl3(OiPr) (entry 13, Table 1), a 1:1 ratio of syn and anti cyanohydrins 18a,b was obtained.The presence of this benzyl protecting group results in a reaction pathway not involving intermediate E.
Investigation of this cyanation reaction has demonstrated that the choice of Lewis acid and β-protecting group are key to reach high levels of diastereoselectivity in favor of the 2,4-syn or 2,4-anti cyanohydrin allowing access to either the xylo-like (this manuscript) or the lyxo-like nucleoside analogues.In addition, this cyanation reaction highlights the potential for other stereoselective nucleophilic additions onto aldehydes possessing an α-stereogenic center for which there are few literature examples. 38,39o generate the novel nucleoside scaffold, the C2-protected 2,4-syn cyanohydrin 16 was first reduced with DIBAL-H and then transformed into di(ethylthio)acetal 4 in 70% yield (Scheme 7).Scheme 7. Acyclic approach for the synthesis of nucleoside analogues.At this point in the strategy, we made use of our novel two-step acyclic approach for the synthesis of nucleoside analogues, 40 the mechanism of which has been examined in detail using DFT calculations. 8,41We have recently reported the use of this strategy with a C3-quaternary center and a fluoride at C2, 8 however, this is the first report of using this strategy with a thioaminal bearing a C2-hydroxy and a C3-quaternary center.An essential aspect of this strategy is the diastereoselective synthesis of a 1,2-syn thioaminal (29) from a dithioacetal (4).Activation of this acyclic dithioacetal with iodine and addition of silylated nucleobase (thymine or uracil) provided the 1,2-syn thioaminals in excellent diastereoselectivity (only one isomer could be detected by 1 H NMR) and yield.Formation of the 1,2-syn thioaminal proceeds through a SN2-like mechanism in which the initially formed halothioether adopts a conformational preference orienting the C2-OTBS and the thioether moiety in close proximity to one another to maximize R−C2 and H−C2 sigma donation to the electron poor thiacarbenium intermediate in TS I.The presence of the counteranion (I -) stabilizes this transition state and prefers to be located on the opposite side of the incoming nucleobase. 8,41Selective removal of the less hindered C4-TBS protecting group provided the necessary thioaminals 30 and 31 in excellent yields.The next key step of our acyclic approach involves a stereospecific displacement of the activated sulfur of the thioaminal by the C4-hydroxy with inversion of configuration (O4'-C1 cyclization).This provided the D-1',2'-trans furanosides 32 (thymine, 71% yield) and 33 (uracil, 80% yield).Proof of structure for the nucleoside analogues was determined by 2D NOESY experiments (see supporting information).Transformation of the monosubstituted alkene of 32 and 33 into the desired primary alcohol was accomplished in five steps (Scheme 8).This monosubstituted alkene could serve as the starting point for a variety of chemical modifications to add heteroatoms and diverse functionalities.In this study, it was transformed into an hydroxymethyl group.Scheme 8. Synthesis of xylo-like nucleoside analogues.Dihydroxylation using osmium tetroxide followed by oxidative cleavage of the diol resulted in the corresponding aldehyde that was reduced using lithium borohydride providing the C3'-hydroxymethyl substituent.Removal of the C2'-TBS provided scaffolds 34 and 35 in 59% yield for these four steps.The desired xylo-like nucleoside analogues (36 and 37) were formed after removal of the primary benzyl protecting groups.

Conclusions
The synthesis of a novel class of xylo-like nucleoside analogues bearing a stereogenic quaternary center at C3' has been described.This acyclic synthetic route relies on highly diastereoselective chemical transformations investigated in our laboratory: an anti selective Mukaiyama aldol reaction, a syn selective intramolecular radical transfer cyclization, formation of a syn selective cyanohydrin followed by the synthesis and cyclization of a syn thioaminal.A key element of this route is the cyanide addition onto aldehydes activated through formation of proposed 7-membered ring chelates.These analogues and other molecules of this family are being tested for their antiviral and antiproliferative properties.The results of these tests will be reported in due course.

Experimental Section
General Comments.All reactions requiring anhydrous conditions were carried out under an atmosphere of nitrogen or argon in flame-dried glassware using standard syringe techniques.All anhydrous solvents were dried with 4 Å molecular sieves prior to use.The 4 Å molecular sieves (1-2 mm beads) were activated by heating at 180 o C for 48 hours under vacuum prior to adding to new bottles of solvent purged with argon.Commercially available reagents were used as received.Flash chromatography was performed on silica gel 60 (0.040 -0.063 mm) using forced flow (flash chromatography) of the indicated solvent system or an automated flash purification system Biotage Isolera One (version 1.3.6).Analytical thin-layer chromatography (TLC) was carried out on pre-coated (0.25 mm) silica gel aluminum plates.Visualization was performed with U.V. short wavelength and/or revealed with ammonium molybdate or potassium permanganate solutions. 1H NMR spectra were recorded at room temperature on a 500 MHz Varian Unity INOVA NMR spectrometer.The data are reported as follows: chemical shift in ppm referenced to residual solvent (CDCl3 δ 7.26 ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, t = triplet, td = triplet of doublets, m = multiplet, app = apparent), coupling constants (Hz), and integration. 13C NMR spectra were recorded at room temperature using 100.6 (Varian VXR 400 NMR) or 126 MHz.The data are reported as follows: chemical shift in ppm referenced to residual solvent (CDCl3 δ 77.16 ppm).Infrared spectra were recorded on a FTIR ABB Bomen (MB series) or a Bruker Platinum ATR (Alpha II series) spectrophotometer from a thin film of purified product and signals are reported in cm -1 .Mass spectra were recorded through electrospray ionization (ESI) positive ion mode using a Thermo Fischer LTQ Orbitrap XL.A Q exactive mass analyzer was used for HRMS measurements and were done by the Plateforme de découvertes en protéomique at l'Institut de Recherches Cliniques de Montréal (IRCM).Optical rotations were measured at room temperature from the sodium D line (589 nm) using a PerkinElmer 343 polarimeter and CDCl3 as solvent unless otherwise noted and calculated using the formula: [α]D =(100)αobs/(ℓ•(c)), where c = (g of substrate/100 ml of solvent) and ℓ = 1 dm.The characterization of chemical structures from X-ray data has been done at the Université de Montréal X-ray diffraction laboratory.Proofs of structure can be found in the supporting information.

General Procedure A: Reduction of ester
To a solution of ester in dry CH2Cl2 (67 mL, 0.20 M) at -40 o C, DIBAL-H (3.0 equiv.) was added.The solution was stirred until the ester (1.0 equiv.) was all consumed (1h30 at -40 o C) as determined by TLC.MeOH was added at -40 o C until gas formation ceased.An aqueous solution of potassium sodium tartrate was added, and the reaction mixture was warmed to room temperature.The aqueous layer was extracted with Et2O (3x).The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo.

General Procedure B: Oxidation of alcohol
To a solution of oxalyl chloride (1.3 equiv.) in anhydrous CH2Cl2 (0.10 M) at -78 o C, dimethyl sulfoxide (2.3 equiv.) was added dropwise.The solution was stirred for 20 minutes at which point the alcohol (1.0 equiv.), as a 0.40 M solution in anhydrous CH2Cl2, was added followed by stirring at -78 o C for 1h.Triethylamine (5.0 equiv.) was added and the reaction mixture was warmed to room temperature over 45 minutes.An aqueous solution of NH4Cl was added and the aqueous layer was extracted with Et2O (3x).The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo.
a Synthesis of racemic aldehydes 6,

17, 19, 21, 23, 25 and
27is described in the experimental section along with proofs of structure in the supporting information.The two cyanohydrin diastereomers were separated, deprotected to the corresponding diols and then protected as an acetonide.The relative stereochemistry of the syn and anti-acetonides was determined from 1D NOESY and the13C chemical shifts of the acetal carbon and the gem-dimethyl substituents.