Synthesis of 1′-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase Inhibitors

Article abstract of DOI:10.1080/15257770.2015.1075550

Ribose modified 1′-C-cyano pyrimidine nucleosides were synthesized. A silver triflate mediated Vorbrüggen reaction was used to generate the nucleoside scaffold and follow-up chemistry provided specific ribose modified analogs. Nucleosides and phosphoramid

Full text of DOI:10.1080/15257770.2015.1075550

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis of 1′-C-Cyano Pyrimidine Nucleosides
and Characterization as HCV Polymerase Inhibitors
Thorsten A. Kirschberg, Michael Mish, Neil H. Squires, Sebastian Zonte,
Evangelos Aktoudianakis, Sammy Metobo, Thomas Butler, Xie Ju, Aesop
Cho, Adrian S. Ray & Choung U. Kim
To cite this article: Thorsten A. Kirschberg, Michael Mish, Neil H. Squires, Sebastian Zonte,
Evangelos Aktoudianakis, Sammy Metobo, Thomas Butler, Xie Ju, Aesop Cho, Adrian
S. Ray & Choung U. Kim (2015): Synthesis of 1′-C-Cyano Pyrimidine Nucleosides and
Characterization as HCV Polymerase Inhibitors, Nucleosides, Nucleotides and Nucleic Acids,
DOI: 10.1080/15257770.2015.1075550
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1075550
Published online: 23 Sep 2015.
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Date: 25 September 2015, At: 06:14

Nucleosides, Nucleotides and Nucleic Acids, 00:1–23, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1075550
SYNTHESIS OF 1ꢀ-C-CYANO PYRIMIDINE NUCLEOSIDES AND
CHARACTERIZATION AS HCV POLYMERASE INHIBITORS
Thorsten A. Kirschberg,1 Michael Mish,1 Neil H. Squires,1 Sebastian Zonte,1
Evangelos Aktoudianakis,1 Sammy Metobo,1 Thomas Butler,1 Xie Ju,1
Aesop Cho,1 Adrian S. Ray,1 and Choung U. Kim2
1Medicinal Chemistry, Gilead Sciences, Foster City, CA, USA
2Kainos Medicine, Inc., Seoul, Republic of Korea
Ribose modified 1ꢁ-C-cyano pyrimidine nucleosides were synthesized. A silver triflate mediated
2
Vorbru¨ggen reaction was used to generate the nucleoside scaffold and follow-up chemistry provided
specific ribose modified analogs. Nucleosides and phosphoramidate prodrugs were tested for their
anti-HCV activity.
Keywords Nucleosides; 1ꢁ-cyano pyrimidine nucleosides; HCV
In the search for effective HCV treatments, nucleosides have occupied an
important role in the last decade.[1] Studies have focused on identifying the
best nucleo base, ribose modification, and choice of prodrug strategy.[2]
Especially, the ribose modifications were important for arriving at suitable
potency and selectivity to warrant clinical evaluation.[3]
In an earlier paper, a silver triflate mediated Vorbru¨ggen reaction par-
ticularly well suited for the synthesis of 1ꢁ-C-cyano substituted pyrimidines
with 2ꢁ-C-Me functionalized ribose was described.[4] The resultant pyrimi-
dine nucleosides and phosphoramidate prodrugs were found to be promis-
ing structural leads in the area of HCV. It has been shown that the viral
polymerase accepts the 1ꢁ-C-cyano substituted nucleoside triphosphates as
substrates and incorporates them into the growing viral RNA.[5] The 2ꢁ-C-Me
substitution caused chain termination that led to inhibition of viral replica-
tion. In this study, a series of 1ꢁ-C-cyano pyrimidine nucleosides with other
structural modifications at the ribose ring were prepared and evaluated for
anti-HCV polymerase activity. Certain synthetic limitation due to the pres-
ence of a 1ꢁ-C-cyano group had previously been described in the literature.[6]
Received 8 March 2014; accepted 20 July 2015.
Address correspondence to Thorsten A. Kirschberg, Medicinal Chemistry, Gilead Sciences, 333
Lakeside Drive, Foster City, CA 94404, USA. E-mail: tkirschberg@gilead.com
1

2
Thorsten A. Kirschberg et al.
Thus, screening of reactions suitable for 1ꢁ-C-cyano substituted pyrimidine
nucleosides was conducted.
Chemistry: In order to access alternative ribose modifications beyond
the 2ꢁ-C- Me derivatives, the 1ꢁ-C-cyano substituted uridine 3 and cytidine
5 were synthesized. The silver triflate mediated coupling was conducted
with the known ribosyl donor 1.[7] The reaction was high yielding, stereo
selective, and independent of starting material isomer composition. In the
absence of the steric encumbrance of the 2ꢁ-C-Me group, the reaction tem-
perature could be lowered to 80◦C without reduced conversion efficiency.
The uridine-to-cytidine conversion was best performed at the stage of the
tri-benzoate 2 as described in the literature.[8]
SCHEME 1 Reagents and conditions: (a) AgOTf, CH3CN / DCE (1,2-dichloroethane), TMS2-U, 80 ◦C,
82%; (b) NH3 MeOH (7N), rt, 79% (3); (c) 2, Tri-iso-propylbenzenesulfonyl chloride, 4-(dimethylamino)
pyridine (DMAP), Et3N, CH3CN, rt; (d) NH3 aq; 1,4-dioxane, rt; (e) NH3 MeOH (7N), rt, 36% (5) over
3 steps.
The 1ꢁ-C-cyano uridine 3 was used as the starting material for the syn-
thesis of 2ꢁ-O-Me analogs. Prior to this work, 1ꢁ unsubstituted 2ꢁ-O-Me cyti-
dine was reported to have anti-HCV activity.[9] Cyclic 3ꢁ-5ꢁ- silyl protection
represented a convenient way to isolate the 2ꢁ-hydroxyl group for derivatiza-
tion.[10] A temporary C4-O-ethylation set the stage for silver oxide catalyzed
2ꢁ-O methylation. The isolated yield was low with 26%. Global deprotection
with HCl yielded the nucleoside 6. A second 3ꢁ-5ꢁ bis-protection followed by
uridine-to-cytidine conversion led to target derivative 7. No chemical insta-
bility of the product or side reactions because of the presence of the cyano
group at the anomeric center was observed under these reaction conditions.
One additional area of interest was the 5ꢁ-C-alkyl substitutions. The
1ꢁ-C-cyano uridine was evaluated as a starting material for the synthesis of
such ribose modified pyrimidines. A conceptually intriguing approach was
the introducing of such a 5ꢁ-C-Me group after nucleoside assembly using
a 5ꢁ aldehyde. Based on the precedent established by the Syntex group, a
possible reaction between the C-2 oxygen and the 5ꢁ aldehyde during syn-
thesis and especially during isolation attempts was known.[11] The acetonide
protection of the 2ꢁ and 3ꢁ hydroxyl groups of 3 appeared sufficient for the
study at hand and was not further optimized. The oxidation was performed

1ꢁ-C-cyano Pyrimidine Nucleosides
3
SCHEME 2 Reagents and conditions: (a) 1,3-dichloro-1,1,3,3-tetra-iso-propyl disiloxane, pyridine, rt,
91%; (b) Tri-iso-propylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt, then EtOH, 79%; (c) MeI,
Ag2O, acetone, 26%; (d) HCl (1M) MeOH, rt, 70%; (e) 1,3-dichloro-1,1,3,3-tetra-iso-propyl disiloxane,
pyridine, rt, 55%; (f) Tri-iso-propylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt; NH3 aq; 1,4-
dioxane, rt, 52%; (g) HCl (1M) MeOH, rt, 24%.
with 2-Iodoxybenzoic acid (IBX) at elevated temperature, as this procedure
allowed for a simple reagent filtration at the end of the reaction and solvent
removal, eliminating the need for manipulations of the crude aldehyde.
This material was reacted with methyl magnesium bromide as obtained.[12]
The isolated yield of the isomeric secondary alcohol products 9 and 10 was
low due to the formation of multiple side products. The deprotection to
the nucleoside was performed with TFA in water. Thus, 10 was converted
to the corresponding nucleoside 11. Overall, the efficiency of this reaction
sequence and the possibility of synthesizing larger quantities of nucleoside
derivatives were deemed too low and an alternative was designed.
SCHEME 3 Reagents and conditions: (a) H2SO4, acetone, rt, 72%; (b) IBX, CH3CN, 80 ◦C, used
unpurified; (c) MeMgBr, THF, 0 ◦C to rt, 7% (9) (R), 3% (10) (S) over two steps; (d) 10, TFA/water, rt,
32% (11).
A simpler access was found by advancing the introduction of the eventual
5ꢁ-C- Me group into a ribosyl donor derived from 12.[13] As shown in Scheme

4
Thorsten A. Kirschberg et al.
4, single isomer 13 was thus synthesized in a highly efficient manner. This
route to 14 was used to assign the 5ꢁ stereocenter for the two isomers of 9/10
after deprotection of the acetonide. The conversion to the corresponding
cytidine derivative was performed as described earlier.
SCHEME 4 Reagents and conditions: (a) BF3-OEt2, TMSCN, rt; (b) NBS, CCl4 90 ◦C, 66% over two
steps; (c) AgOTf, CH3CN / DCE, TMS2-U, 135 ◦C, 68%; (d) NH3 MeOH (7N), rt, 71% (14); (e) 13,
tri-iso-propylbenzenesulfonyl chloride, DMAP, Et3N, CH3CN, rt; NH3 aq., 1,4-dioxane, rt, 60%; (f) NH3
MeOH (7N), rt, 67% (15).
In order to elucidate the effect of a 2ꢁ alpha hydroxyl stereochemistry
on anti-HCV properties in this series, an inversion at this center was tar-
geted.[14] The strategy relied on the successful formation of a C2 oxygen to
2ꢁ anhydro species (structure 18/Scheme 5). Using 13 as a starting material,
a selective deprotection of the 2ꢁ-O-benzoyl group was required. The use of
hydrazine in AcOH/pyr mixtures was condition uniquely applicable to the
system at hand.[15] No 2ꢁ–3ꢁ O-acyl transfer was observed.[16] Possibly the 1ꢁ-C-
cyano group acidified the neighboring 2ꢁ-hydroxyl group to a degree where
such transfer was no longer favorable. The mesylation and intramolecular
displacement were high yielding as was the acid catalyzed hydrolysis. Final
deprotection gave the uridine derivative 20. No conversion of the 1ꢁ-C-cyano
group to a primary amide was observed during the conversion of 18 to 19.[6]
A conversion to the cytidine derivative was performed after reprotection
of the 2ꢁ hydroxyl group in 19 with acetic anhydride. The acetate protecting
group was not completely stable under the reaction conditions required for
the nucleo base amination. Yet, this instability was inconsequential as the
resulting mixture was subjected to the final deprotection conditions to form
23.
2ꢁ-C-Me 2ꢁ-deoxy derivatives have been described as alternative anti-HCV
nucleosides.[17] The combination with the 1ꢁ-C-cyano group was realized
starting from the unprotected 2ꢁ-C-Me derivative 24. The 3ꢁ-, 5ꢁ- cyclic pro-
tection was chosen as it conveniently allows for the selective acylation of the
2ꢁ-OH group, and hence conversion to the corresponding methyl oxalate
ester. Radical deoxygenation with tributyl tin hydride gave the desired ma-
terial in high chemical yield.[18] It was observed that the deoxygenation was
moderately stereoselective under these conditions and led to a 78:22 mix-
ture (1H NMR) from the single isomer starting material. The conversion of

1ꢁ-C-cyano Pyrimidine Nucleosides
5
SCHEME 5 Reagents and conditions: (a) N2H4, pyr/HOAc, rt, 93% (crude); (b) MsCl, pyr, rt, 65%; (c)
Et3N, CH3CN, 70 ◦C, 87%; (d) HClaq, DMF, 40 ◦C, 93%; (e) NH3aq, MeOH, 27%.
SCHEME 6 Reagents and conditions: (a) Ac2O, pyr, rt, 90%; (b) tri-iso-propylbenzenesulfonyl chloride,
DMAP, Et3N, CH3CN, rt; NH3 aq., 1,4-dioxane, rt 68%; (c) NH3aq, 1,4-dioxane rt to 50 ◦C, 24% (over
two steps).
the single isomer 26 to the cytidine derivative 27 was high yielding over two
steps.
A more extensive analysis of a nucleoside towards its anti-HCV activity
includes the assessment of mono-phosphate prodrugs. Variable efficiencies
of enzymatic steps leading to the required mono-, di-, and triphosphate
species from modified nucleosides have been described.[19] Especially, the
intracellular conversion to the monophosphate has often been highlighted
as rate limiting.[20] In this study, a current and general method for the gener-
ation of monoamidate prodrugs was used.[21] Depicted below is the synthesis
of 30. The reaction was highly chemoselective for the primary 5ꢁ- hydroxyl
group and could be applied to otherwise unprotected nucleoside deriva-
tives.[22] Additionally, under the current conditions the reaction proceeded
with inversion at the phosphorus center permitting full chirality transfer
from homochiral reagents.[23]

6
Thorsten A. Kirschberg et al.
SCHEME 7 Reagents and conditions: (a) 1,3-dichloro-1,1,3,3-tetra-iso-propyl disiloxane, pyridine, rt,
96%; (b) MeO2CCOCl, DMAP, DCM / CH3CN, rt, 87%; (c) Bu3SnH, AIBN, toluene, 100 ◦C, 62%
(78:22); (d) 26, tri-iso-propyl benzenesulfonylchloride, DMAP, Et3N, CH3CN, rt; NH3 aq, 1,4-dioxane, rt,
75%; then (e) Tetrabutyl ammonium fluoride (TBAF), THF, rt, 86% (27); (f) 26, Tetrabutyl ammonium
fluoride (TBAF), THF, rt, 48% (28).
SCHEME 8 Reagents and conditions: (a) tBuMgCl, THF / NMP, 0 ◦C, then 29 and warm to rt, 48%.
For the derivatization of the 5ꢁ-C-Me derivatives, a 2ꢁ-, 3ꢁ- bis protected
starting material was used to ensure the desired chemoselectivity among
the three secondary alcohols of the ribose moiety. Because the final phos-
phoramidate was moderately sensitive to acid and base, the ortho-formate
protecting group with known high acid instability was chosen. The protec-
tion was achieved with trimethyl orthoformate in dioxane and p-toluene
sulfonic acid (p-TsOH) as a catalyst in 84% isolated yield. The product (31)
was an inconsequential mixture of two isomers with modest enrichment
of one versus the other. Prodrug synthesis and mild formic acid mediated
deprotection yielded the final product in good overall yield.
Biological activity: The nucleosides and their prodrugs were tested in
the genotype 1b replicon assay to measure anti-HCV activity.[24] All prodrugs
were single stereoisomers of the same configuration. Results of these studies
are summarized in Table 1.

7

8

1ꢁ-C-cyano Pyrimidine Nucleosides
9
SCHEME 9 Reagents and conditions: (a) tBuMgCl, THF / NMP, 0 ◦C, then 29 and warm to 50 ◦C, 83%;
(b) HCO2H, H2O, rt, 31%.
Table 1 summarizes the observed HCV activity. As already seen with other
1ꢁ-C-cyano substituted nucleosides, a moderate level of anti-HCV activity was
seen for a cytidine derivative 5.[25] Combining this structural modification
with either a 2ꢁ-O-Me modification or the 5ꢁ-C-Me group of either stereo-
chemistry rendered the compounds inactive.[26] This observation was un-
changed after applying a simple monophosphate prodrug strategy for either
series; e.g., 32. Only the 2ꢁ-C-Me, 2ꢁ-de-oxy cytidine derivative 27 was active in
the replicon assay. The potency was slightly enhanced when incubating with
the phosphoramidate prodrug arriving at low micromolar potency [EC50:
1.4 μM]. Yet, the compound also showed some toxicity in the replicon Huh
7 cell line [CC50: 4 μM].
Synthetic studies of 1ꢁ-C-cyano substituted pyrimidine nucleosides were
described. Structural modifications at the ribose moiety were introduced.
Overall, a high chemical stability of the 1ꢁ-C-cyano group to a variety of
reaction conditions was seen. Among the compounds presented here, the
2ꢁ-C-Me 2ꢁ-deoxy modification showed the highest level of anti-HCV potency.
EXPERIMENTAL SECTION
General Procedures
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian
Mercury Plus 400 MHz spectrometer at room temperature (rt). Chemical
shifts (δ) were reported in parts per million (ppm) and signals were reported
as singlet (s), doublet (d), triplet (t), and multiplet (m). Purities of the final
compounds were determined by analytical HPLC and were greater than
90% using an Agilent HPLC system, Phenominex Kinetex C18, 4.6 mm
× 100 mm (2.6 μm); gradient of 0.1% TFA in water (A) and 0.1% TFA
in CH3CN (B); flow rate 1.5 mL/min; wavelength UV 254 and 214 nM.
Preparative HPLC was performed on a Gilson system including two sets of
Gilson 332 pumps, a Gilson 156UV/vis detector, and a Gilson 215 injector
and fraction collector. The system was controlled by Trilution LC software.
A Synergi 150 mm × 30 mm 4u Hydro-RP 80 A column was used. The mobile

10
Thorsten A. Kirschberg et al.
phase was HPLC grade water (A) and HPLC grade CH3CN (B) LCMS was
conducted on a Thermo Finnigan MSQ Std using electron spray both positive
and negative [M+ H+ and M – H+], coupled with a Dionex Summit HPLC
system (model: P680A HPG) equipped with a Gemini 5 u C18 110A column
(30 mm × 4.6 mm) eluding with 0.05% formic acid in 1% CH3CN/water
(A) and 0.05% formic acid in 99% CH3CN/water (B). All solvents were dry
and used as obtained from commercial sources. All reagents were used as
obtained from commercial sources.
(2R,3R,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydro
xy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (3): Compound
1
(7.43 g, 13.5 mmol) was dissolved in 1,2-dichloroethane (DCE) (30 mL)
and CH3CN (30 mL). To this was added bis-TMS-uracil (5.2 g, 20.3 mmol)
and lastly Ag(OTf) (5.2g, 20.3 mmol). The reaction was heated at 80◦C for
60 minutes. The reaction mixture was then cooled to rt and precipitous
AgBr filtered off. Solvents were removed in vacuo and resulting residue
was redissolved in ethyl acetate (EtOAc) and washed with brine/aqueous
sat. NaHCO3 (1/1) and dried over Na2SO4. The solution was then filtered
and evaporated to dryness. The residue was purified by silica gel chro-
matography (eluent: EtOAc/hexanes) to afford 2 (6.4 g; yield 81.6%).
1H NMR (400 MHz, CD3OD): δ 8.15 (d, J = 8.4 Hz, 1H), 5.68 (d, J =
8.4 Hz, 1H), 4.52 (d, 1H), 4.26 (m, 1H), 4.06 (m, 1H), 3.97 (dd, J = 12.8,
2.0 Hz, 1H,), 3.73 (dd, J = 12.8, 2.0 Hz, 1H,). MS: [M + H+] = 581.9.
Compound 2 (4.5 g, 7.74 mmol) was dissolved and stirred in 7N–NH3
in MeOH (50 mL). After 16 hours, all volatiles were removed in vacuo.
The crude material was purified via silica gel chromatography (eluent:
1
MeOH/DCM) to yield compound 3 (1.62 g, 77.8%). H NMR (400 MHz,
DMSO-d6): δ 11.52 (m, 1H), 8.06 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 5.6 Hz,
1H), 5.61 (d, J = 8.4 Hz, 1H), 5.20 (m, 2H), 4.34 (m, 1H), 4.06 (m, 1H),
3.91 – 3.83 (m, 2H), 3.56 (m, 1H). MS: [M + H+] = 270.8. HRMS, calculated
for C10H12N3O6 (M+H)+, 270.0721; found 270.0726.
(2R,3R,4S,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5): To a stirred solution
of compound 2 (2 g, 3.44 mmol) in CH3CN (100 mL) was added Et3N
(0.96 mL, 6.88 mmol) and then 2,4,6 tri-iso-propylbenzenesulfonyl chlo-
ride (2.08 g, 6.88 mmol). 4-Dimethylamino pyridine (DMAP) (840 mg,
6.88 mmol) was added. After consumption of the starting material, conc.
aqueous ammonium hydroxide was added and the reaction was stirred at rt
under argon overnight. After 18 hours, the solvents were removed under
reduced pressure. The crude product was dissolved and stirred in NH3-7N
in MeOH (50 mL) at rt. After 6 hours, all volatiles were removed in vacuo
and the resulting crude product was dissolved in water and purified by
RP-HPLC (eluent: CH3CN/water) to give compound 5 (330 mg, 36% yield, 2
1
steps). H NMR (400 MHz, D2O): δ 7.84 (d, J = 7.6 Hz, 1H), 5.91 (d,

1ꢁ-C-cyano Pyrimidine Nucleosides
11
J = 7.6 Hz, 1H), 4.44 (m, 1H), 4.26 (m, 1H), 3.96 (m, 1H), 3.88 (dd,
J = 12.8/2.0 Hz, 1H), 3.67 (dd, J = 12.8/2.0 Hz, 1H). MS [M – H+] =
267.0.
(6aR,8R,9R,9aS)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-9-hydroxy
-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-8-
carbonitrile: Compound 3 (2.0 g, 7.4 mmol) was dissolved in dry pyridine
(24 mL) and 1,3-dichloro-1,1,3,3-tetra-iso-propyl disiloxane (2 mL. 1.3
eq) was added dropwise. The solution was stirred at rt for 16 hours. The
solvent was removed in vacuo and the resulting residue was dissolved in
EtOAc, washed with water and brine, and dried over Na2SO4. Filtration and
evaporation of solvents give crude material. The crude product was purified
by column chromatography on silica gel (eluent: EtOAc/hexanes to give
product (2.1 g, 55% yield). MS = 510 (M – H+).
(6aR,8R,9R,9aS)-8-(4-ethoxy-2-oxopyrimidin-1(2H)-yl)-9-hydroxy-
2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-8-
carbonitrile: Et3N (0.089 mL, 0.64 mmol) was added to a stirring solution
of starting material (300 mg, 0.58 mmol) in CH3CN (5 mL). 2,4,6-Tri-iso-
propylbenzenesulfonyl chloride (192 mg, 0.64 mmol) and DMAP (78 mg,
0.64 mmol) were added. The reaction mixture was stirred at rt for 1 hour.
Ethanol (25 mL) was added to the mixture, together with additional Et3N
(0.34 mL). The solution was stirred at rt for 5 hours. The solvent was
then removed under vacuum and the resulting residue was dissolved in
EtOAc, washed with concentrated NH4Cl, water, and brine, and dried over
Na2SO4. Filtration and evaporation of solvents in vacuo gave crude material.
The crude product was purified by column chromatography on silica gel
(eluent: EtOAc/hexanes) to give the product (250 mg, 79%). MS = 540
(M + H+).
(6aR,8R,9R,9aR)-8-(4-ethoxy-2-oxopyrimidin-1(2H)-yl)-2,2,4,4-tetrais
opropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-8-
carbonitrile: Starting material (900 mg, 1.8 mmol) was dissolved in dry
acetone (20 mL). Silver (I) oxide (3.3 g, 14 mmol) was added to the solution,
followed by methyl iodide (2.5 g, 18 mmol). The solution was stirred at
rt for 16 hours. The mixture was filtered to remove the suspended oxide,
concentrated under vacuum, and purified by column chromatography on
silica gel (eluent: EtOAc/hexanes) to give product (258 mg, 26% yield).
MS = 554 (M + H+).
(2R,3R,4R,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-
5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-carbonitrile (6): Starting
material (220 mg, 0.40 mmol) was dissolved in MeOH (6 mL) and the
resulting solution was cooled to 0◦C in an ice bath. Aqueous hydrochloric
acid (1M, 0.6 mL) was added dropwise. After the addition was complete,
the mixture was allowed to warm up to rt and was stirred for 12 hours. The
solvent was removed in vacuo and the residue was washed twice with DCM

12
Thorsten A. Kirschberg et al.
to remove protecting group derivatives. The crude was purified by column
chromatography on silica gel (eluent: MeOH/DCM) to give compound 6
1
(80 mg, 70% yield). H NMR (400 MHz, CD3OD): δ 7.99 (d, J = 7.7 Hz,
1H), 5.62 (d, J = 7.7 Hz, 1H), 4.18 (m, 2H), 3.97 (m, 2H), 3.78 (s, 3H),
3.73 (m, 1H). MS = 281.9 (M – H+). HRMS, calculated for C11H14N3O6
(M+H)+, 284.0877; found 284.0874.
(6aR,8R,9R,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-
tetraisopropyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilo-
cine-8-carbonitrile: Compound 6 (70 mg, 0.25 mmol) was dissolved in dry
pyridine (2 mL). 1,3-Dichloro-1,1,3,3-tetra-iso-propyl disiloxane (0.10 mL.
1.3 eq) was added dropwise. The solution was stirred at rt for 2 hours.
The solvent was then removed under vacuum and the resulting residue
was dissolved in EtOAc, and washed with water and brine. The organic
phase was dried over Na2SO4, filtered, and solvents were removed in vacuo.
The crude product was purified by column chromatography on silica gel
(eluent: EtOAc/hexanes) to give the product (120 mg, 91% yield). MS =
526 (M + H+).
(6aR,8R,9R,9aR)-8-(4-amino-2-oxopyrimidin-1(2H)-yl)-2,2,4,4-tetraiso-
propyl-9-methoxytetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-8-
carbonitrile: Et3N (60 mg, 0.60 mmol) was added to a stirring solution of
starting material (155 mg, 0.30 mmol) in CH3CN (1 mL). 2,4,6-Tri-iso-
propylbenzenesulfonyl chloride (179 mg, 0.60 mmol) and DMAP (72 mg,
0.060 mmol) were added. The reaction mixture was stirred at rt for 3 hours.
All volatiles were removed in vacuo. The residue was dissolved in CH3CN
(3 mL), and NH3 (28% aqueous solution, 6 mL) was added. The reaction
mixture was stirred at rt for 2 hours. The solvent was removed under
vacuum and the residue was dissolved in DCM and washed with brine. The
organic phase was dried over Na2SO4 and evaporated to dryness. The crude
product was purified by column chromatography on silica gel (eluent:
EtOAc/methanol 3:1 to give the product (80 mg, 52% yield). MS = 556 (M
+ H+).
(2R,3R,4R,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-5-
(hydroxymethyl)-3-methoxytetrahydrofuran-2-carbonitrile (7):
Starting
material (240 mg, 0.46 mmol) was dissolved in anhydrous THF (6 mL).
Tetrabutylammonium difluorodiphenylsilicate (TBAT) was added (540 mg,
1.01 mmol) and the resulting solution was stirred at rt for 20 minutes.
Acetic acid (0.06 mL, 1.0 mmol) was added. The mixture was concentrated
and the residue was dissolved in water. The insoluble material was removed
by filtration. The filtrate was purified by RP-HPLC (eluent, CH3CN/water)
1
to give compound 7 (30 mg, 24% yield). H NMR (400 MHz, CD3OD):
δ 8.14 (d, J = 7.7 Hz, 1H), 5.89 (d, J = 7.7 Hz, 1H), 4.16 (m, 2H), 3.99
(m, 2H), 3.80 (s, 3H), 3.73 (dd, J = 10.0/2.5 Hz, 1H). MS = 282.9 (M
– H+). HRMS, calculated for C11H15N4O5 (M+H)+, 283.1037; found
283.1038.

1ꢁ-C-cyano Pyrimidine Nucleosides
13
(3aR,4R,6R,6aR)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-
carbonitrile: Concentrated sulfuric acid (0.7 mL) was added to a solution
(turbid) of compound 3 (2.57 g, 9.5 mmol) in acetone (70 mL). The
resulting solution was stirred at rt for 3 hours. The reaction was neutralized
with Et3N (3.5 mL) and the solvent was removed under reduced pressure.
The resulting yellow oil was subjected to silica gel chromatography (eluent:
(20% MeOH in DCM) and DCM). The product containing fractions were
combined and the solvent was removed under reduced pressure providing
product (2.13 g, 72%).
(3aR,4R,6S,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-
formyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonitrile
(8):
2-Iodoxybenzoic acid (IBX) (3 g, 4.8 mmol, 45% wt) was stirred with EtOAc
(15 mL) for 15 minutes until a homogeneous mixture formed. The solid
was isolated by suction filtration and then added to a solution of the starting
material (500 mg, 1.6 mmol) in CH3CN (20 mL). The mixture was heated
at 80◦C for 30 minutes. The reaction mixture was cooled in an ice bath
and the mother liquor was isolated by suction filtration. The solvent was
removed under reduced pressure, and the white solid was azeotroped with
toluene. The aldehyde 8 was used immediately, and the yield was assumed
to be 100%.
(3aR,4R,6R,6aR)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-((R)-1-
hydroxyethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonitrile
(9)
and
(3aR,4R,6R,6aR)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
6-((S)-1-hydroxyethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-
carbonitrile (10): A solution of methylmagnesium bromide (2.69 mL,
8.1 mmol, 3M in diethyl ether) was added to a solution of 8 (495 mg,
1.6 mmol) in tetrahydrofuran (20 mL) at –20◦C under an atmosphere of
argon. After 5 minutes, the mixture was allowed to warm to rt. After 45
minutes, the mixture was cooled to 0◦C and the reaction was quenched with
saturated ammonium chloride (10 mL). The tetrahydrofuran was removed
under reduced pressure and the aqueous phase was extracted with EtOAc
(3 × 25 mL). The combined organic phases were dried over Na2SO4 and
filtered. The solvent was removed under reduced pressure. The resulting
residue was purified via reverse phase HPLC (eluent: CH3CN/water). The
product containing fractions were combined and the solvent was removed
by lyophilization providing 9 (35 mg, 7%) and 10 (15 mg, 3%).
(2R,3R,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-
dihydroxy-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-carbonitrile (11):
A
solution of 10 (15 mg, 0.046 mmol) and trifluoroacetic acid (1 mL) in
water (1 mL) was stirred at rt for 5.5 hours. The solution was diluted
with water (5 mL) and CH3CN (5mL), and the solvent was removed by
lyophilization. The resulting residue was subjected to reverse phase HPLC
(eluent: CH3CN/water). The product containing fractions were combined

14
Thorsten A. Kirschberg et al.
and the solvent was removed by lyophilization, providing 11 (4.2 mg, 32%).
1H NMR (400 MHz, D2O): δ 8.15 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 8.4 Hz,
1H), 4.42 (d, J = 4.4 Hz, m 1H), 4.00 (dd, J = 8.8/2.8 Hz, 1H), 3.94 (dd, J
= 8.4/4.0 Hz, 1H), 3.85 (dd, J = 6.4Hz/2.4 Hz, 1H), 1.27 (d, J = 6.4 Hz,
3H). MS = 281.8 (M – H+).
(2R,3R,4S)-2-((R)-1-(benzoyloxy)ethyl)-5-cyanotetrahydrofuran-3,4-diyl
dibenzoate: Compound 12 (1.0 g, 2.06 mmol) was dissolved in nitromethane
(20 mL) at rt. TMS-cyanide (296 mg, 2.97 mmol) was added followed by
BF3 etherate (0.246 mL). Stirring at rt was continued. After 45 minutes, the
volatiles were removed in vacuo. The crude material was taken into DCM
and the solution was washed with aqueous saturated sodium bicarbonate so-
lution. The organic layer was dried over Na2SO4. Filtration and evaporation
of solvents yielded the crude product (923 mg), which was used in the next
1
step without further purification. H NMR (400 MHz, CDCl3): δ 8.08 (m,
2H), 7.92 (m, 4H), 7.57 (m, 3H), 7.43–7.35 (m, 6H), 6.00–5.93 (m, 2H),
5.51 (m, 1H), 4.94 (d, J = 4.4 Hz, 1H), 4.53 (m, 1H), 1.52 (d, J = 6.8 Hz,
3H).
(3R,4R,5R)-5-((R)-1-(benzoyloxy)ethyl)-2-bromo-2-cyanotetrahydro-
furan-3,4-diyl dibenzoate: The starting material (760 mg, 1.53 mmol) and
N -bromo succinimide (NBS) (657 mg,3.0 mmol) were heated in CCl4 at
75◦C under constant UV irradiation for 5 hours. The reaction mixture
was cooled to rt, the solids were filtered off, and the product was purified
via flash chromatography on silica gel (eluent EtOAc/hexanes), affording
product (706.0 mg, 1.25 mmol). The product was obtained as a mixture of
1
two isomers. H NMR (400 MHz, CDCl3): δ 8.10–7.88 (m, 6H), 7.62–7.34
(m, 9H), 6.33–6.26 (m, 1H), 6.10 and 5.90 (m, 1H), 5.58 (m, 1H), 4.81–4.75
(m, 1H), 1.56 and 1.52 (d, J = 6.8 Hz, 3H).
(2R,3R,4R,5R)-5-((R)-1-(benzoyloxy)ethyl)-2-cyano-2-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3,4-diyl dibenzoate (13): Start-
ing material (703 mg, 1.24 mmol), bis-TMS uracil (630 mg, 2.46 mmol),
and silver(I)triflate (630 mg, 2.46 mmol) were placed into a microwave vial
under argon, and anhydrous DCE (5 mL) and CH3CN (5 mL) were added.
The mixture was heated at 135◦C for 30 minutes via use of a microwave
reactor. The reaction mixture was cooled to rt, filtered, and the volatiles
were removed in vacuo. The crude material was purified via chromatography
on silica gel (eluent: EtOAc/hexanes) affording 13 (504 mg, 0.845 mmol)
as a single isomer. 1H NMR (400 MHz, CDCl3): δ 8.08 (d, J = 7.2 Hz, 2H),
8.03–8.00 (m, 4H), 7.62–7.37 (m, 10H), 6.36 (d, J = 5.6 Hz, 1H), 6.11 (m,
1H), 5.66 (dq, J = 7.2/2.8 Hz, 1H), 4.50 (dd, J = 8.4/2.4 Hz, 1H), 4.90 (m,
1H), 1.57 (d, J = 6.8 Hz, 3H). MS = 596 (M + H+).
(2R,3R,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-
dihydroxy-5-((R)-1-hydroxyethyl)tetrahydrofuran-2-carbonitrile (14): To
a solution of 13 (65 mg, 0.109 mmol) in MeOH (1 mL) at rt was added
2 mL conc. aq. ammonia. The reaction mixture was stirred at rt for

1ꢁ-C-cyano Pyrimidine Nucleosides
15
8 hours. LC/MS analysis indicated that almost all the starting material was
consumed. The reaction was concentrated in vacuo. The crude reaction
product was dissolved in water, and purified via reverse phase HPLC (eluent:
CH3CN/water). The product containing fractions were combined, frozen,
1
and lyophilized to afford compound 14 (22.0 mg, 0.077 mmol). H NMR
(400 MHz, D2O): δ 7.93 (d, J = 8.4 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 4.50
(m, 1H), 4.15 (m, 3H), 1.16 (d, J = 6.8 Hz, 3H). MS = 282 (M – H +).
HRMS, calculated for C11H14N3O6 (M+H)+, 284.0877; found 284.0883.
(2R,3R,4R,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-5-((R)-1-
(benzoyloxy)ethyl)-2-cyanotetrahydrofuran-3,4-diyl
dibenzoate:
2,4,6-
Tri-iso-propylbenzene-1-sulfonyl chloride (1.58 g, 5.2 mmol) was added to
a solution of 13 (1.56 g, 2.6 mmol), DMAP (640 mg, 5.2 mmol), and Et3N
(0.73 mL, 5.2 mmol) in CH3CN (30 mL) and stirred at rt for 30 minutes.
Concentrated aqueous ammonium hydroxide (6 mL) was added. After 15
minutes, the CH3CN was removed under reduced pressure. The residue
was taken up in EtOAc (75 mL) and washed with water (20 mL), saturated
ammonium chloride (20 mL), and brine (20 mL). The organic phase was
dried over Na2SO4 and filtered. The solvent was removed under reduced
pressure and the residue was subjected to silica gel chromatography (eluent:
MeOH/DCM). The product containing fractions were combined and the
solvent was removed under pressure to provide product (920 mg, 60%). 1H
NMR (400 MHz, DMSO-d6): δ 8.0–7.36 (m, 16H), 6.13 (d, J = 5.6 Hz, 1H),
5.96 (t, J = 6.0 Hz, 1H), 5.57 (d, -J = 8 Hz, 1H), 5.51 (m, 1H), 5.08 (dd, J
= 6.8/4.4 Hz, 1H), 1.43 (d, J = 6.4 Hz, 3H). MS = 595.5 (M + H+).
(2R,3R,4S,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxy-5-
((R)-1-hydroxyethyl)tetrahydrofuran-2-carbonitrile (15):
Concentrated
ammonium hydroxide (30 mL) was added to a solution of starting material
(460 mg, 7.7 mmol) in 1,4-dioxane (15 mL). The solution was stirred at
50◦C in a sealed vessel. After 9 hours, the solution was cooled and the solvent
was concentrated to a volume of 10 mL. The mixture was cooled at 0◦C for
5 minutes and filtered. The filtrate was subjected to reverse phase HPLC
(eluent: CH3CN/water). The product containing fractions were combined.
The solvent was removed by lyophilization to provide 15 (148 mg, 67%). 1H
NMR (400 MHz, D2O): δ 7.88 (d, J = 7.6 Hz, 1H), 5.91 (d, J = 8.0 Hz, 1H),
4.42 (d, -J = 5.2 Hz, 1H), 4.16 (m, 1H), 4.09 (m, 2H), 1.16 (d, J = 7.2 Hz,
3H). MS = 283.1 (M + H+). HRMS, calculated for C11H15N4O5 (M+H)+,
283.1037; found 283.1051.
(R)-1-((2R,3S,4R,5R)-3-(benzoyloxy)-5-cyano-5-(2,4-dioxo-3,4-dihydro
pyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)ethyl benzoate (16): To a
solution of 13 (4.32 g, 7.25 mmol) in pyridine/glacial acid (55 mL/13.5 mL)
at rt was added hydrazine hydrate (598 mg, 10.87 mmol). The reaction
mixture was stirred at rt for 40 hours. Acetone (4 mL) was added and
stirring at rt was continued. After two additional hours, the solvent volume
was reduced in vacuo to ∼1/3 of the original volume. The mixture was

16
Thorsten A. Kirschberg et al.
diluted with EtOAc, washed with aqueous HCl (1N), and aqueous saturated
sodium bicarbonate solution/brine (1/1). The organic layer was dried
over Na2SO4. Filtration and evaporation of solvents afforded crude 16
(3.33 g, 6.77 mmol). The material was used in the next step without further
1
purification. H NMR (400 MHz, CDCl3): δ 8.43 (m, 1H), 8.17 (d, J =
6.8 Hz, 2H), 7.86 (d, J = 7.6 Hz, 2H), 7.61 – 7.41 (m, 7H), 6.04 (dd, J =
5.2/1.2 Hz, 1H), 5.63 (m, 2H), 4.99 (s, 1H), 4.94 (m, 1H), 4.79 (m, 1H),
1.54 (d, J = 6.8 Hz, 3H). MS = 489 (M – H +).
(S)-1-((2R,3R,4R,5R)-3-(benzoyloxy)-5-cyano-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-((methylsulfonyl)oxy)tetrahydrofuran-2-
yl)ethyl benzoate (17): To a solution of 16 (3.25 g, 6.61 mmol) in pyridine
(40 mL) at rt was added methylsulfonyl chloride (1.51 g, 13.22 mmol).
The reaction mixture was stirred at rt. After 3 hours, more methylsulfonyl
chloride (0.75 g, 6.61 mmol) was added and stirring at rt was continued.
After 5 hours, the volatiles were removed in vacuo. The crude reaction
mixture was diluted with EtOAc, and washed with aqueous HCl (1N) and
aqueous saturated sodium bicarbonate solution/brine (1/1). The organic
layer was dried over Na2SO4. Filtration and evaporation of solvents afforded
crude material that was purified by chromatography on silica gel (eluent:
1
EtOAc/hexanes) to afford 17 (2.53 g, 4.27 mmol). H NMR (400 MHz,
CDCl3): δ 9.22 (m, 1H), 8.07 (d, J = 7.6 Hz, 2H), 7.95 (d, J = 7.2 Hz,
2H), 7.61 (m, 2H), 7.47 – 7.39 (m, 5H), 5.81 – 5.72 (m, 3H), 5.29 (dd, J =
8.4/1.6 Hz, 1H), 4.87 (dd, J = 9.2/3.2 Hz, 1H), 3.36 (s, 3H), 1.45 (d, J =
7.2 Hz, 3H). MS = 570 (M + H +).
(S)-1-((2R,3R,3aS,9aR)-3-(benzoyloxy)-9a-cyano-6-oxo-3,3a,6,9a-
tetrahydro-2H-furo[2ꢁ,3ꢁ:4,5]oxazolo[3,2-a]pyrimidin-2-yl)ethyl
benzoate
(18): To a solution of 17 (2.45 g, 4.30 mmol) in CH3CN (40 mL) at rt was
added Et3N (2.45 g, 24.20 mmol). The reaction mixture was heated at 65◦C
(oil bath). After 2 hours, the reaction was cooled to rt and the volatiles were
removed in vacuo. The crude reaction mixture was diluted with EtOAc, and
washed with aqueous HCl (1N) and aqueous saturated sodium bicarbonate
solution/brine (1/1). The organic layer was dried over Na2SO4. Filtration
1
and evaporation of solvents afforded product 18 (1.78 g, 3.76 mmol). H
NMR (400 MHz, CDCl3): δ 8.07 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 7.2 Hz,
2H), 7.69–7.37 (m, 7H), 6.01 (d, J = 7.6Hz, 1H), 5.91 (m, 1H), 5.78 (d, J
= 0.8 Hz, 1H), 5.43 (m, 1H), 4.81 (m, 1H), 1.40 (d, J = 6.4 Hz, 3H). MS =
472 (M – H+).
(R)-1-((2R,3S,4S,5R)-3-(benzoyloxy)-5-cyano-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)ethyl benzoate
(19): To a solution of 18 (1.70 g, 3.59 mmol) in dimethyl formamide
(DMF) (10 mL) was added aqueous HCl (1N, 10 mL) at rt. To the resultant
suspension was added more DMF (10 mL). The reaction mixture was
heated at 40◦C (oil bath). After 30 minutes, the reaction was cooled to
rt, diluted with EtOAc, and washed with brine, aqueous LiCl (5%), and

1ꢁ-C-cyano Pyrimidine Nucleosides
17
aqueous saturated sodium bicarbonate solution/brine (1/1). The organic
layer was dried over Na2SO4. Filtration and evaporation of solvents afforded
crude reaction mixture that was purified via chromatography on silica gel
(eluent: EtOAc/hexanes) to afford the product 19 (1.65 g, 3.37 mmol). 1H
NMR (400 MHz, CDCl3): δ 10.08 (s, 1H), 8.13 (d, J = 7.2 Hz, 2H), 8.07 (d,
J = 7.2 Hz, 2H), 7.57–7.37 (m, 7H), 5.77 (m, 1H), 5.71 (m, 1H), 5.13 (s,
1H), 5.04 (s, 1H), 4.79 (d,d, J = 8.4/1.6Hz, 1H), 4.59 (m, 1H), 1.52 (d, J =
6.8 Hz, 3H). MS = 492 (M – H +).
(2R,3S,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-
dihydroxy-5-((R)-1-hydroxyethyl)tetrahydrofuran-2-carbonitrile (20): To
a solution of 19 (65.2 mg, 0.132 mmol) in MeOH (1 mL) at rt was added
conc. aq. ammonia (2 mL). The reaction mixture was stirred at rt for
12 hours. The reaction was concentrated in vacuo. The crude reaction
product was dissolved in water, and purified via reverse phase HPLC (eluent:
CH3CN/water). The product containing fractions were combined, frozen,
1
and lyophilized to afford 20 (10.3 mg, 0.036 mmol). H NMR (400 MHz,
D2O): δ 7.74 (d, J = 8.4 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 4.69 (m, 1H),
4.26 (m, 1H), 4.13 (m, 1H), 3.99 (m, 1H), 1.17 (d, J = 6.4 Hz, 3H). MS
= 284 (M + H +). HRMS, calculated for C11H14N3O6 (M+H)+, 284.0877;
found 284.0874.
(2R,3R,4S,5R)-4-acetoxy-2-((R)-1-(benzoyloxy)ethyl)-5-cyano-5-(2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl benzoate (21): To
a solution of 19 (189.2 mg, 0.385 mmol) in pyridine (2 mL) at rt was added
acetic anhydride (47.5 mg, 0.462 mmol). After 2 hours, the volatiles were
removed in vacuo. The crude reaction mixture was diluted with EtOAc and
washed with aqueous HCl (1N) and aqueous saturated sodium bicarbonate
solution/brine (1/1). The organic layer was dried over Na2SO4. Filtration
and evaporation of solvents afforded crude material that was purified
by chromatography on silica gel (eluent: EtOAc/hexanes) to afford 21
(185.5 mg, 0.348 mmol). 1H NMR (400 MHz, CDCl3): δ 8.15 (m, 3H), 8.04
(d, J = 7.6 Hz, 2H), 7.65–7.44 (m, 7H), 5.99 (s, 1H), 5.81–5.73 (m, 3H),
4.69 (m, 1H), 1.57–1.54 (m, 6H). MS = 533 (M + H+).
(2R,3R,4S,5R)-4-acetoxy-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-2-((R)-1-
(benzoyloxy)ethyl)-5-cyanotetrahydrofuran-3-yl benzoate (22): To a solution
of compound 21 (180.0 mg, 0.338 mmol), DMAP (82.4 mg, 0.676 mmol),
and Et3N (68.2 mg, 0.676 mmol) in CH3CN (8 mL) at rt was added
tri-iso-propylphenylsulfonyl chloride (204 mg, 0.676 mmol). Stirring at rt
was continued. After 30 minutes, the reaction was cooled to 0◦C. Aqueous
concentrated NH3 solution (2 mL) was added and the reaction was allowed
to warm to rt. After additional 30 minutes, the reaction mixture volume was
reduced in vacuo. The crude reaction mixture was diluted with EtOAc and
washed with aqueous HCl (1N) and aqueous saturated sodium bicarbonate
solution/brine (1/1). The organic layer was dried over Na2SO4. Filtration
and evaporation of solvents afforded crude material that was purified by

18
Thorsten A. Kirschberg et al.
chromatography on silica gel (eluent: EtOAc/hexanes) to afford a mixture
of compound 22 and its 2ꢁ hydroxy derivative (128.5 mg, combined). This
mixture was used in the next reaction.
(2R,3S,4S,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxy-5-
((R)-1-hydroxyethyl)tetrahydrofuran-2-carbonitrile (23): To a solution of 22
and its des-acetyl derivative (110.0 mg, ∼0.230 mmol) in 1,4-dioxane (1 mL)
at rt was added conc. aq. ammonia (2 mL). The reaction mixture was stirred
at rt for 1.5 hours and at 50◦C for 6 hours. The reaction was cooled to rt and
concentrated in vacuo. The crude reaction product was dissolved in water,
and purified via reverse phase HPLC (eluent: CH3CN/water). The product
containing fractions were combined, frozen, and lyophilized to afford 23
1
(22.7 mg, 0.081 mmol). H NMR (400 MHz, D2O): δ 7.68 (d, J = 7.6 Hz,
1H), 5.91 (d, J = 7.6 Hz, 1H), 4.70 (s, 1H), 4.23 (m, 1H), 4.10 (m, 1H), 3.98
(m, 1H), 1.17 (d, J = 6.4 Hz, 3H). MS = 283 (M + H +). HRMS, calculated
for C11H15N4O5 (M+H)+, 283.1037; found 283.1039.
(6aR,8R,9R,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-9-hydro-
xy-2,2,4,4-tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]-
trioxadisilocine-8-carbonitrile: To a dry, argon purged round bottom
flask (25 mL) was added 24 (980 mg) and anhydrous pyridine (4 mL).
1,3-Dichloro-1,1,3,3-tetra-iso-porpyl disiloxane (1.33 mL, 4.16 mmol) was
added dropwise and the reaction stirred for 2 hours at 0◦C. The ice bath was
removed and the mixture warmed to rt and continued to stir until complete
disappearance of the starting material. After 10 minutes of warming, the
reaction was diluted with water (10 mL) and the desired material was
collected via vacuum filtration. The material was placed under high vacuum
overnight for further drying. 1.90 g (96% yield) of the desired material was
collected. MS = 526.4 (M – H+)
(6aR,8R,9R,9aR)-8-cyano-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,
2,4,4-tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]
trioxadisilocin-9-yl methyl oxalate (25): To a dry, argon purged round
bottom flask (50 mL) was added starting material (1.7 g, 3.23 mmol),
anhydrous DCM (10 mL), and anhydrous CH3CN (10 mL). DMAP (1.19 g,
9.7 mmol) was then added portionwise followed by dropwise addition of
methyl chlorooxoacetate (0.89 mL, 9.7 mmol). The reaction was allowed
to stir at rt until complete disappearance of the starting material. After
1.5 hours, the crude reaction mixture was diluted with EtOAc followed
by washings with NaHCO3 (sat), water, and brine. The combined organic
layers were dried over Na2SO4 and the solvent was removed under reduced
pressure. The mixture was dried azeotropically using toluene and placed
under high vacuum overnight for completeness. 1.72 g (87% yield) of
1
the desired material, compound 25, was collected. H NMR (400 MHz,
CD3OD): δ 7.82 (d, J = 8.4 Hz, 1H), 5.78 (d, J = 8.4 Hz, 1H), 4.47 (m, 1H),
4.30 (m, 1H), 4.20 (m, 2H), 3.93 (s, 3H), 2.89 (s, 3H), 1.08 (m, 28H). MS =
609.8 (M – H +).

1ꢁ-C-cyano Pyrimidine Nucleosides
19
(6aR,8S,9S,9aS)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-
tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-
8-carbonitrile (26): To a dry, argon purged round bottom flask (500 mL)
was added compound 25 (1.75 g, 2.86 mmol) and anhydrous toluene
(120 mL). 2,2ꢁ-Azobis(2-methylpropionitrile (AIBN) (118.8 mg, 0.49 mmol)
and Bu3SnH (2.3 mL, 8.78 mmol) were then added and the flask was placed
into a heating block and set to 100◦C. After 3 hours, the solvent was removed
under reduced pressure and the crude reaction mixture was purified using
flash chromatography (eluent: EtOAc/hexanes). The desired material,
compound 26, was collected, along with alpha isomer (900 mg; 62%, 78:22
1
beta/alpha). Beta-isomer 26 after column separation; H NMR (400 MHz,
CD3OD): δ 7.82 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 8.4 Hz, 1H), 4.22 (m, 2H),
4.09 (m, 2H), 3.15 (m, 1H), 1.01 (m, 31H). MS = 507.9 (M – H −).
(6aR,8S,9S,9aS)-8-(4-amino-2-oxopyrimidin-1(2H)-yl)-2,2,4,4-
tetraisopropyl-9-methyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocine-
8-carbonitrile: To a dry, argon purged round bottom flask (100 mL) was
added 26 (500 mg, 1.0 mmol) and anhydrous CH3CN (13 mL). DMAP
(244 mg, 2.0 mmol) and Et3N (0.28 mL, 2.0 mmol) were then added to the
flask. Lastly, 2,4,6-tri-iso-propyl benzenesulfonyl chloride (0.28 mL, 2mmol)
was added to the mixture and the solution turned yellow. The reaction
continued to stir at rt until complete disappearance of the starting material.
After 45 minutes, ammonium hydroxide (2.6 mL, 20% by volume) was
added at 0◦C and the reaction was allowed to slowly warm to rt. After an
additional 20 minutes, the solvent was removed under reduced pressure.
The crude reaction mixture was diluted with EtOAc followed by washings
with NaHCO3 (sat), water, and brine. The combined organic layers were
dried over Na2SO4 and the solvent was removed under reduced pressure.
The crude reaction mixture was then purified using flash chromatography
(eluent: MeOH/DCM). 380 mg (75% yield) of the desired material was
collected. 1H NMR (400 MHz, CD3OD): δ 7.77 (d, J = 8.4 Hz, 1H), 5.93 (d,
J = 8.4 Hz, 1H), 4.23 (m, 2H), 4.07 (m, 2H), 3.17 (m, 1H), 1.25 (m, 1H),
1.09 (m, 27H), 0.96 (m, 3H). MS = 509.1 (M – H +).
(2S,3S,4S,5R)-2-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-5-
(hydroxymethyl)-3-methyltetrahydrofuran-2-carbonitrile (27): To a dry,
argon purged round bottom flask (50 mL) was added the starting material
(180 mg, 0.35 mmol) and anhydrous tetrahydrofuran (9 mL). Tetrabutyl
ammonium fluoride (TBAF) (0.23 mL, 0.778 mmol) was then added and
the reaction was allowed to stir at rt until complete disappearance of the
starting material. After 10 minutes, acetic acid was added to neutralize the
solution and then the solvent was removed under reduced pressure. The
crude reaction mixture was dissolved in water, filtered, and the filtrate
purified using RP-HPLC (eluent: CH3CN/water). 80 mg (86%) of the
desired material, compound 27, was collected. 1H NMR (400 MHz, CD3OD):
δ 7.77 (d, J = 8.4 Hz, 1H), 5.94 (d, J = 8.4 Hz, 1H), 4.24 (m, 1H), 3.91 (m,

20
Thorsten A. Kirschberg et al.
1H), 3.83 (m, 1H), 3.70 (m, 1H), 3.07 (m, 1H), 0.66 (d, J = 7.5 Hz, 3H). MS
= 267.1 (M + H +). HRMS, calculated for C11H15N4O4 (M+1)+, 267.1088;
found 267.1084.
(2S,3S,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-
(hydroxymethyl)-3-methyltetrahydrofuran-2-carbonitrile (28): Compound
28 was prepared under conditions similar to those described in preparation
of compound 27.
1H NMR (400 MHz, CD3OD): δ 7.83 (d, J = 8.4 Hz, 1H), 5.78 (d, J =
8.4 Hz, 1H), 4.24 (m, 1H), 3.94 (m, 1H), 3.84 (m, 1H), 3.71 (m, 1H), 3.07
(m, 1H), 0.76 (d, J = 7.5 Hz, 3H). MS = 268.1 (M + H +). HRMS, calculated
for C11H14N3O5 (M+H)+, 268.0928; found 268.0929.
Prodrug Synthesis
(S)-Isopropyl2-(((S)-(((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)
phosphoryl)amino)propanoate (30): A solution of compound 5 (50 mg,
0.188 mmol) in NMP (5 mL) was cooled to 0◦C under argon. To this
was added 3.5 eq. of t-BuMgCl dropwise. To the resulting suspension was
added 29 (218 mg, 0.47 mmol) predissolved in THF (3 mL) dropwise. The
reaction was then immediately heated to 50◦C and reaction progress was
monitored for completion by LCMS (45–60 minutes). When determined
to be complete, the reaction was cooled, 0.5 mL of each water and MeOH
were added to the reaction, the reaction was filtered, and purified by
1
RP-HPLC (eluent; CH3CN/water) to afford 30 (43 mg, 43% yield). H
NMR (400 MHz, CD3OD): δ 7.90 (d, J = 7.7 Hz, 1H), 7.42–7.30 (m, 2H),
7.27–7.13 (m, 3H), 5.83 (d, J = 7.8 Hz, 1H), 4.94 (t, J = 6.3 Hz, 2H),
4.53–4.38 (m, 3H), 4.30 (ddd, J = 12.6/5.7/3.8 Hz, 1H), 4.00 (dd, J =
8.0/4.7 Hz, 1H), 3.88 (dq, J = 9.9/7.1 Hz, 1H), 1.32 (dd, J = 7.2/1.1 Hz,
3H), 1.20 (dd, J = 6.3/2.9 Hz, 5H). 31P NMR (162 MHz, CD3OD): δ 3.86.
MS [M + H+] = 538.9. HRMS, calculated for C22H29N5O9P (M+H)+,
538.1697; found 538.1695.
(3aR,4R,6R,6aR)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-6-((R)-1-
hydroxyethyl)-2-methoxytetrahydrofuro[3,4-d][1,3]dioxole-4-carbonitrile
(31): A solution of compound 15 (148 mg, 0.52 mmol), p-toluenesulfonic
acid monohydrate (76 mg, 0.40 mmol), and trimethylortho formate (6 mL)
in 1,4-dioxane (6 mL) was stirred at rt for 1.5 hours. The reaction was
neutralized with Et3N (0.06 mL) and the solvent was removed under
reduced pressure. The residue was taken up in MeOH (8 mL) and stirred
at rt for 30 minutes. The solvent was removed under reduced pressure. The
residue was subjected to silica gel chromatography (eluent: MeOH/1%
Et3N in DCM). The product containing fractions were combined and the
solvent was removed under pressure to provide compound 31 (143 mg,
84%). 1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, J = 7.6 Hz, 1H), 7.41 (m,

1ꢀ-C-cyano Pyrimidine Nucleosides
21
2H), 6.04 (s, 1H), 5.74 (dd, J = 8/2 Hz, 1H), 5.12 (d, J = 4.8 Hz, 1H),
4.89 (m, 2H), 4.44 (m, 1H), 3.90 (m, 1H), 3.36 (s, 3H), 1.12 (d, J = 6.4Hz,
3H).
(2S)-Isopropyl2-(((S)-((1R)-1-((3aR,4S,6R,6aR)-6-(4-amino-
2-oxopyrimidin-1(2H)-yl)-6-cyano-2-methoxytetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)ethoxy)(phenoxy)phosphoryl)amino)propanoate:
A
solution of tert-butylmagnesium chloride (0.23 mL, 0.23 mmol 1.0 M)
in tetrahydrofuran was added to a solution of starting material (50 mg,
0.15 mmol) in tetrahydrofuran (2 mL) under argon. A white solid formed.
After 30 minutes, a solution of compound 29 (126 mg, 0.31 mmol) in
tetrahydrofuran (1 mL) was added and the mixture was heated to 50◦C.
After 20 minutes the solid had dissolved and the solution was yellow.
The reaction was cooled to 0◦C and quenched with methanol (1 mL).
The solvent was removed under reduced pressure and the residue was
subjected to silica gel chromatography (eluent: MeOH/DCM). The product
containing fractions were combined and the solvent was removed under
1
pressure to provide the product (76.8 mg, 83%). H NMR (400 MHz,
DMSO-d6): δ 7.78 (d, J = 7.6 Hz, 1H), 7.45 (s, 2H), 7.31 (t, J- = 8.4 Hz, 2H),
7.13 (m, 3H), 6.11 (s, 1H), 6.04 (dd, J = 13.2/10 5 Hz, 1H), 5.63 (d, J =
7.6 Hz, 1H), 4.89 – 4.76 (m, 3H), 4.57 (m, 1H), 3.75 (m, 1H), 3.37 (s, 3H),
1.38 (d, J = 6.0 Hz, 3H), 1.19 (d, J = 7.2 Hz, 3H), 1.14 (m, 6H). 31P NMR
(162 MHz, DMSO-d6): δ 3.06 (s). MS = 594.1 (M + H+).
(S)-Isopropyl2-(((S)-((R)-1-((2S,3S,4R,5R)-5-(4-amino-2-
oxopyrimidin-1(2H)-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-
yl)ethoxy)(phenoxy)phosphoryl)amino)propanoate (32): A solution of
starting material (76.8 mg, 0.13 mmol) in formic acid (5 mL, 95%) and
water (1 mL) was stirred for 20 minutes at rt. The solvent was removed
under reduced pressure and the residue was co-evaporated with EtOAc. The
resulting residue was subjected to reverse phase chromatography (eluent:
CH3CN/water). The product containing fractions were combined. The
1
solvent was removed by lyophilization to provide 32 (8.3 mg, 31%). H
NMR (400 MHz, DMSO-d6): δ 7.92 (d, J = 7.6 Hz, 1H), 7.45 (s, 1H), 7.39
(s, 1H), 7.35 (t, J- = 8.0 Hz, 2H), 7.16 (m, 3H), 6.72 (d, J = 5.2 Hz, 1H),
6.04 (dd, J = 13.2/10 Hz, 1H), 5.68 (d, J = 7.6 Hz, 1H), 5.27 (d, J = 7.2
HZ, 1H), 4.83 (m, 2H), 4.22 (m, 2H), 3.87 (m, 1H), 3.75 (m, 1H), 1.38
(d, J = 6.8 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H), 1.11 (m, 6H). 31P NMR
(162 MHz, DMSO-d6): δ 3.22 (s). MS = 552.0 (M + H+). HRMS, calculated
for C23H31N5O9P (M+H)+, 552.1854; found 552.1840.
ACKNOWLEDGMENTS
The authors thank Kathy Brendza and Loredana Serafini for performing
HRMS measurements for this study.

22
Thorsten A. Kirschberg et al.
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