Synthesis and Evaluation of 2′-Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs

Article abstract of DOI:10.1080/15257770.2016.1202965

The preparation of 2′-deoxy-2′-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2′-position. Then, a series of novel 2′-deoxy-2′-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.

Full text of DOI:10.1080/15257770.2016.1202965

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Evaluation of 2′-Deoxy-2′-
Spirodiflurocyclopropyl Nucleoside Analogs
Xiao Liu, Xueliang Xia, Chenghai Sun, Cai Lin, Yiqian Zhou, Muzammal
Hussain, Fei Tang, Lu Liu, Xue Li & Jiancun Zhang
To cite this article: Xiao Liu, Xueliang Xia, Chenghai Sun, Cai Lin, Yiqian Zhou, Muzammal
Hussain, Fei Tang, Lu Liu, Xue Li & Jiancun Zhang (2016): Synthesis and Evaluation of 2′-
Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs, Nucleosides, Nucleotides and Nucleic
Acids, DOI: 10.1080/15257770.2016.1202965
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1202965
Published online: 11 Aug 2016.
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Date: 11 August 2016, At: 15:54

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –

http://dx.doi.org/./..
Synthesis and Evaluation of
ꢀ
ꢀ

-Deoxy- -Spirodiflurocyclopropyl Nucleoside Analogs
a,†
a,†
a
b
b
Xiao Liu , Xueliang Xia , Chenghai Sun , Cai Lin , Yiqian Zhou ,
b,c
b,c
b,c
a
b,d
Muzammal Hussain , Fei Tang , Lu Liu , Xue Li , and Jiancun Zhang
a
Institute of Pharmaceutical Research, South China Normal University, Guangzhou, Guangdong, PR China;
b
c
Guangzhou Institutes of Biomedicine and Heath, Chinese Academy of Sciences, Guangzhou, PR China;
d
University of Chinese Academy of Sciences, Beijing, PR China; State Key Laboratory of Respiratory
Disease, Guangzhou Institutes of Biomedicine and Health, Guangzhou, PR China
ABSTRACT
ARTICLE HISTORY
Received  April 
Accepted  June 
ꢀ
ꢀ
The preparation of 2 -deoxy-2 -siprodifluorocyclopropany-
lnucleoside analogs has been achieved from α-D-glucose in
several steps. The key step in the synthesis was the introduction
of the difluorocyclopropane through a difluorocarbene type
reaction at the 2 -position. Then, a series of novel 2 -deoxy-2 -
spirodifluorocyclopropanyl nucleoside analogs were synthesized
using the Vorbrüggen method. All the synthesized nucleosides
were characterized and subsequently evaluated against hepatitis
C and influenza A virus strains in vitro.
KEYWORDS
ꢀ
ꢀ
-deoxy- -

ꢀ
ꢀ
ꢀ
spirodifluorocyclopropanyl
nucleosides; hepatitis C;
influenza A
Introduction
Structurally diverse nucleoside analogs which act as selective inhibitors of poly-
merases and other enzymes have long been implicated for the treatment of human
[1]
viral diseases and cancer. Of the nucleosides that are currently used in clinical
settings, the majority have modified sugar moieties with naturally occurring nucle-
ꢀ
[2]
obases. In particular, the 2 -modified nucleoside derivatives, such as gemcitabine
[3]
and recently approved sofosbuvir (Figure 1A and B), have acquired great com-
mercial success. In this respect, fluorine
the metabolic stability, lipophilicity, and the binding affinity of many 2 -modified
[4,5]
substitutions have been shown to affect
ꢀ
[6]
nucleoside analogs .
ꢀ
ꢀ
[7]
In addition, 2 -deoxy-2 -spirocyclopropylcytidine
reported as a new member of the class of 2 -modified nucleoside deriva-
tives. This compound exhibits potent antiviral activity by inhibiting the hep-
(Figure 1C) has been
ꢀ
ꢀ
ꢀ
atitis C virus (HCV) NS5B RNA-dependent RNA polymerase. 2 -Deoxy-2 -
spirocyclopropylcytidine has been shown to display an EC of 7.3 μM measured
50
in the Huh7-Rep cell line and no associated cytotoxicity (CC50 > 98.4 μM). The
CONTACT JiancunZhang
zhang_jiancun@gibh.ac.cn
Academy of Sciences,  Kaiyuan Avenue, Science Park, Guangzhou , PR China.
GuangzhouInstitutesofBiomedicineandHealth,Chinese
†
These authors equally contributed to this work.
©
 Taylor & Francis Group, LLC

2
X. LIU ET AL.
ꢀ
Figure .  -modified nucleosides.
ꢀ
ꢀ
key structural feature of the 2 -deoxy-2 -spirocyclopropylcytidine is the previously
ꢀ
unreported 2 -cyclopropyl substitution, which makes it a useful surrogate in the
series of anti-HCV nucleosides. As part of our effort to identify novel nucleoside
derivatives for the treatment of viral infections, we sought a synthetic means to pre-
pare the diastereomers of the 2-difluorocyclopropane sugar that could be further
ꢀ
ꢀ
transformed to 2 -deoxy-2 -spirodifluorocyclopropanyl nucleosides. The results are
described here.
Chemistry
For the synthesis of the targeted nucleosides, we first designed and constructed a
series of 2-difluorocyclopropane ribosugars. The 2-difluorocyclopropane sugar 9a
and 9b were synthesized from α-ꢀ-glucose via 4a and 4b. The α-ꢀ-glucose was
treated with conc. H SO and acetone with recrystallization to give compound 1
2
4
in 65% yield. Compound 1 was subjected to oxidation and Wittig reaction to get the
alkenes 3. Then, different conditions were tried to generate difluocarbene, such as
[8]
[9]
[10]
[11]
and TFDA[11,12].
TMSCF3, TMSCF Br, ClF CCOONa,
TMSCl/MDFA,
2
2
The catalyst was chosen from NaI, NaF, KI, AIBN, and TBAB, and the solvent was
chosen from DMF, THF, CH CN, toluene and diglyme. The reaction temperature
3
ranged from room temperature to 180°C, and the reaction time varied from a few
hours to several days. However, all of these condensation conditions failed to gen-
erate detectable amounts of the desired product, except TMSCF with the catalyst
3
NaI in THF at 100°C that yielded difluorocyclopropanes 4a(R) and 4b(S) in 30%
(
R:S = 5:1). 4a was selectively deprotected and protected with a benzoyl group at
the primary hydroxyl group to give 6a, which was further deprotected to lead to 7a.
Compound 7a was then treated with NaIO and saturated sodium bicarbonate solu-
4
tion to give 8a, which was then protected by treatment with acetic anhydride and
trimethylamine in dichloromethane to give compound 9a. Compound 7b was also
subjected to the same conditions to yield compound 9b (Scheme 1).
For the preparation of targeted compounds, we first employed Vörbrügen[
13]
sugar-base condensations under various conditions. Two sugars 9a and 9b
ꢀ
and five bases (uracil, cytosine, 5 -fluorouracil, thymidine and adenine) were
used for the condensation reactions, while the silylating agent was either N,O-
bis(trimethylsilyl)acetamide (BSA) or hexamethyldisilazane (HMDS).[ Solvents
14]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme . Conditions: (a) acetone, conc. HSO,  °C; (b) Tempo, NaClO, NaHCO, TBAB, KBr, DCM;
(
(
c) Methyltriphenylphosphonium bromide, Potassium tert-butoxide, THF; (d) TMSCF, NaI, THF °C;
e) .% HSO, MeOH; (f) BzCl, TEA, − °C ∼  °C; (g) % CFCOOH, overnight; (h) NaIO, NaHCO,
DCM; (i) AcO, TEA, DMAP, DCM.

4
X. LIU ET AL.
Scheme . Conditions: (a) persilylated base, TMSOTf, , -DCE, r.t.; (b) NH in MeOH.
DCM, 1,2-DCE, and CH CN were analyzedin the presence or absence of a Lewis
3
[15]
acid catalyst trimethylsilyltrifluoromethanesulfonate (TMSOTf) or SnCl4 . The
reaction temperature ranged from room temperature to 50°C,and the reaction time
varied from a few hours to several days. Finally, it was found that dissolving the
mixture of sugar and silylated base in 1,2-DCE that was followed by the slow
addition of TMSOTf at room temperature can give the desired product in good
yields.
ꢀ
The modified uridine, 5 -fluorouridine and thymidine were prepared as depicted
in Scheme 2. Uridine 12(β) and 13(α) in 66% yield (over two steps) were obtained
ꢀ
as a 2:1 β:α mixture of the anomers. The 5 -fluorouridine 14(β) and 15(α) in 49%
yield (over two steps) were obtained as a 3:1 β:α mixture of anomers. The thymidine
1
6(β) and 17(α) in 69% yield (over two steps) were obtained as a 4:1 β:α mixture
of anomers.
With the intermediate 10ain hand, the synthesis of 19 was accomplished by con-
version of the uracil moiety to the corresponding cytidine derivative, using phos-
phorus oxychloride and 1 H-1,2,4-triazole as the reagents to obtain 18, which under
went aminolysis to give the target compound (Scheme 3).
Purine nucleosides were obtained using a similar synthetic strategy as described
in Scheme 2 to give intermediate 20, which was then subjected toaminolysis with
NH OH at 100°C to yield the adenosine nucleoside 21 (Scheme 4).
4
The other isomer 9b was also subjected to a similar synthetic route to afford
the corresponding target compounds. The desired uridines 22(β) and 23(α) were
Scheme . Conditions: (a) POCl,  H-,,-triazole, triethylamine, DCM, r.t,  h; (b) (i) NHOH, THF, r.t.,
 h; (ii) NH in MeOH, r.t., overnight.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme  Conditions: (a) persilylated-chloropurine, TMSOTf, ,-DCM, r.t; (b) NHOH, ,-dioxane,
 °C,  h.

ꢀ
obtained in 44% yield (over two steps) as a 1.8:1 β:α mixture of anomers. The 5 -
fluorouridine 24(β) and 25(α) were obtained in 43% yield (over two steps) as a
2.7:1 β:α mixture of anomers. The thymidines 26(β) and 27(α) were obtained in
54% yield (over two steps) as a 4:1 β:α mixture of anomers. The adenine nucleoside
30 was obtained in 24% yield (over two steps). These compounds are depicted in
Figure 2.
Figure . The chemical structures of compounds  to .

6
X. LIU ET AL.
The synthesized compounds were assigned based on the presence of a cross-
ꢀ
ꢀ
ꢀ
peak between H-1 ,H-3 , H-4 in 2D NOESY experiments to confirm the α and β
anomers.
Antiviral results
To gain some measure of biological potential, all the synthesized nucleoside deriva-
tives were evaluated in vitro against the HCV, and influenza A virus (H1N1, H3N2)
strains. All of the compounds had EC and IC values of >100 μM, and were nei-
50
50
ther active nor cytotoxic in the performed assays.
Conclusion
We have designed and syntheses a novel type of nucleoside analogs which bear
a difluorocyclopropane mioety at the C-2 position of the ribosugar. The 2-
difluorocyclopropane precursors 9a and 9b can be obtained from glucose, and the
nucleoside analogs were obtained through the conventional Vörbrügen method.
Experimental
Reactions requiring anhydrous conditions were conducted in oven-dried appara-
tus under a dry argon atmosphere, utilizing commercially available dry solvents
and reagents. All common reagents were purchased from commercial sources and
1
13
19
used without further purification. H, C, and F NMR spectra were recorded
on Bruker AV 400 MHz or 500 MHz Fourier transformation spectrometer. Spec-
tra were obtained from samples in CDCl , CD OD, or DMSO-d . Multiplicities are
3
3
6
as quoted: s = singlet,d = doublet, t = triplet, q = quartet, m = multiplet. Coupling
constants (J) are reported in Hz. Signal assignments are based on COSY, DEPT, spec-
tra. HRMS spectra were obtained using Agilent1200, MSD LC-MS.
(3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-
dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol(1)
Using a 1 L round bottom flask, 30 g (0.17 mol) of anhydrous α-ꢀ-glucose was added
in 0.6 L of acetone. The mixture was stirred vigorously in an ice-water bath. Concen-
trated H SO (30 mL) was added dropwise for 6 hours. After all the acid was added,
2
4
the solution was cooled to 0°C and neutralized by 50% aqueous potassium hydrox-
ide solution to maintain the pH of the solution near 7. After stirring overnight, the
solution was filtered, and the solvent was removed under reduced pressure. The
solid was dissolved in 100 mL of dichloromethane and the solution was washed with
1
00 mL of water. The aqueous phase was then extracted three times with 100 mL of
dichloromethane. The organic layers were combined and then concentrated under
reduced pressure. Recrystallization from petroleum ether gave 28 g of white crystals
1
(
64.7% yield). H NMR (500 MHz, Chloroform-d) δ: 5.94 (d, J = 3.6 Hz, 1 H), 4.53

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
(
d, J = 3.7 Hz, 1 H), 4.39–4.29 (m, 2H), 4.16 (dd, J = 8.7, 6.2 Hz, 1 H), 4.06 (dd,
J = 7.6, 2.8 Hz, 1 H), 3.98 (dd, J = 8.6, 5.4 Hz, 1 H), 2.65 (d, J = 3.7 Hz, 1 H), 1.49 (s,
13
3
H), 1.44 (s, 3H), 1.36 (s, 3H), 1.31 (s, 3H). C NMR (126 MHz, Chloroform-d) δ:
111.96, 109.79, 105.42, 85.22, 81.27, 75.34, 73.61, 67.80, 26.99, 26.90, 26.32, 25.28.
(3aR,5R,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-
dimethyldihydrofuro[2,3-d][1,3]dioxol-6(5H)-one (2)
To a solution of diacetone glucose 1 (30 g, 1.24 mol) in DCM (300 mL) were added
KBr (1.4 g, 11.5 mmol), TBAB (3,7 g, 11.5 mmol) and TEMPO (3.6 g, 23.1 mmol).
The resulting mixture was vigorously stirred, and an aqueous solution of NaClO
(
1.6 mol/L, 150 mL, 0.24 mol; pH adjusted with NaHCO to 9.5) was added drop-
3
wise. After addition of the bleach solution, the resulting reaction mixture was stirred
for 5 min, and the layers were separated. The aqueous phase was then extracted three
times with 100 mL of dichloromethane. The organic layers were combined, dried
(
Na SO ), and concentrated under reduced pressure. Product 2 was obtained as a
2 4
yellowish liquid and was used in the next step without additional purification.
(3aR,5S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-6-
methylene-tetrahydrofuro[2,3-d][1,3]dioxole (3)
Methyl triphenylphosphonium bromide (49.4g, 0.14 mol) and potassiumtert-
butoxide (15.5g, 0.14 mol) was added in anhydrous THF (300 mL) at 0°C. Then,
a solution of ketone (4.6 g, 17.8 mmol) in anhydrous THF (200 mL) was added
to the reaction mixture. After 1 hour of stirring at room temperature, the mixture
was partitioned between EA and water. The aqueous layer was washed 3 times with
EA, The combined organic layer was dried over MgSO , filtered, and evaporated.
4
The resulting residue was purified by silica gel column chromatography (PE:EA =
1
8
:1) to give compound 3 as a colorless oil (24.2g, two steps of 82% yield). H NMR
(
(
(
400 MHz, Chloroform-d) δ: 5.78 (d, J = 4.2 Hz, 1H), 5.48 (d, J = 1.9 Hz, 1H), 5.42
d, J = 2.2 Hz, 1H), 4.86 (d, J = 4.0 Hz, 1H), 4.63 (d, J = 5.8 Hz, 1H), 4.07–3.99
13
m, 2H), 3.95–3.86 (m, 1H), 1.49 (s, 3H), 1.41 (s, 3H), 1.34 (d, J = 4.8 Hz, 6H). C
NMR (126 MHz, Chloroform-d) δ: 146.76, 113.31, 112.51, 109.69, 104.45, 82.08,
9.17, 77.25, 66.66, 27.30, 27.00, 26.51, 25.35.
7
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
1R,3a R,5 S,6a R)-5 -((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-2 ,2 -
ꢀ
ꢀ
dimethyldihydro-5 H-spiro[cyclopropane-1,6 -furo[2,3-d][1,3]dioxole] (4a) and
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
1S,3a R,5 S,6a R)-5 -((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro-2 ,2 -
ꢀ
ꢀ
dimethyldihydro-5 H-spiro[cyclopropane-1,6 -furo[2,3-d][1,3]dioxole] (4b)
Anhydrous NaI (0.909 g, 6 mmol, 0.2 eq), 30 mL of freshly distilled THF as solvent,
and 3 (8 g, 31.2 mmol, 1 eq) were added sequentially in a 200 mL pressure tube
under inert atmosphere. Then TMSCF (24 mL, 156 mmol, 5 eq) was added. The
3
reaction vessel was sealed and heated to 100 °C in an oil bath for a period of 10 hours,
and then cooled to ambient temperature and was evaporated to give crude mixture

8
X. LIU ET AL.
of 4a and 4b. With carefully conducted column chromatography, (PE/EA = 12:1),
a (3R) (2.486 g, 26%) and 4b (3S) (0.478 g, 5%) were obtained as slightly yellow
4
oils, while 3 (4.423g, 55%) was recovered. Compound 4a [slightly more polar than
1
3
3
1
on TLC (PE/EA = 4:1)] had: H NMR (400 MHz, Chloroform-d) δ: 5.86 (d, J =
.6 Hz, 1H), 4.41–4.34 (m, 2H), 4.09–3.96 (m, 2H), 3.86 (ddd, J = 7.8, 6.3, 4.8 Hz,
H), 1.89 (ddd, J = 11.8, 8.1, 4.6 Hz, 1H), 1.57 (s, 3H), 1.35 (d, J = 8.7 Hz, 6H), 1.29
13
(
s, 3H). C NMR (126 MHz, Chloroform-d) δ: 113.22, 109.92, 104.56, 87.00, 77.83,
7
5.99, 75.98, 67.11, 27.24, 26.87, 26.39, 25.27, 17.82, 17.66. 19F NMR (471 MHz,
Chloroform-d) δ: −128.81 (ddd, J = 164.2, 13.9, 4.5 Hz), −134.56 (dd, J = 164.7,
1
1
(
(
1
1
1
0.6 Hz). Compound 4b [less polar than 3 on TLC (PE/EA = 4:1)] had: H NMR
500 MHz, Chloroform-d) δ: 5.84 (d, J = 3.9 Hz, 1H), 4.55 (d, J = 3.7 Hz, 1H), 4.27
t, J = 7.6 Hz, 1H), 4.22–4.14 (m, 1H), 4.08–4.01 (m, 1H), 3.90 (dd, J = 8.1, 5.0 Hz,
H), 1.80 (dt, J = 11.4, 7.4 Hz, 1H), 1.74–1.66 (m, 1H), 1.53 (s, 3H), 1.39 (s, 3H),
13
.30 (d, J = 15.2 Hz, 6H). C NMR (126 MHz, Chloroform-d) δ: 112.21, 111.44,
19
09.97, 104.79, 81.09, 79.34, 74.16, 68.04, 37.51, 27.11, 27.06, 26.94, 25.45, 15.55. F
NMR (471 MHz, Chloroform-d) δ: −127.74 (ddt, J = 158.7, 13.8, 6.9 Hz), −135.67
(dd, J = 158.7, 11.4 Hz).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
R)-1-((1R,3a R,5 S,6a R)-2,2-difluoro-2 ,2 -dimethyldihydro-5 H-
ꢀ
ꢀ
spiro[cycloprop-ane-1,6 -furo[2,3-d][1,3]dioxol]-5 -yl)ethane-1,2-diol (5a)
A solution of 4a (5 g, 16.33 mmol) in 75 mL methanol and 0.8% aqueous H SO4
2
(
75 mL) was added dropwise and stirred at room temperature. After the starting
material disappeared, the mixture was neutralized with aqueous NaHCO solution
3
and extracted three times with 150 mL of EA. The organic layers were concentrated
to give the crude product 5a (3.956 g, 76%, pale-yellow syrup) that was used directly
1
in the next step. A small portion was purified by column chromatography. H NMR
(
500 MHz, Methanol-d ) δ: 5.86 (s, 1H), 4.49–4.35 (m, 2H), 3.69 (d, J = 11.5 Hz, 1H),
4
3
1
1
.58 (dd, J = 11.1, 6.6 Hz, 1H), 3.31 (s, 1H), 1.98 (t, J = 9.6 Hz, 1H), 1.62–1.54 (m,
13
H), 1.52 (s, 3H), 1.31 (s, 3H). C NMR (126 MHz, Methanol-d ) δ: 113.76, 112.96,
4
19
05.66, 88.15, 78.41, 73.65, 64.63, 39.15, 27.43, 26.96, 18.05. F NMR (471 MHz,
Methanol-d ) δ: −129.59 (ddd, J = 165.2, 13.6, 4.0 Hz), −136.54 (dd, J = 164.9,
4
11.2 Hz).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
R)-1-((1S,3a R,5 S,6a R)-2,2-difluoro-2 ,2 -dimethyldihydro-5 H-
ꢀ
ꢀ
spiro[cycloprop-ane-1,6 -furo[2,3-d][1,3]dioxol]-5 -yl)ethane-1,2-diol (5b)
The compound 5b (yield 75%) was obtained using a similar synthetic strategy as
1
compound 5a. H NMR (500 MHz, Chloroform-d) δ: 5.76 (d, J = 4.2 Hz, 1H), 4.47
(
1
d, J = 4.2 Hz, 1H), 4.22 (t, J = 7.7 Hz, 1H), 3.79–3.63 (m, 4H), 3.50 (s, 1H), 1.85–
.77 (m, 1H), 1.66–1.58 (m, 1H), 1.43 (s, 3H), 1.24 (s, 3H). 13C NMR (126 MHz,
Chloroform-d) δ: 113.92, 112.07, 104.40, 80.76, 78.62, 70.38, 64.45, 60.61, 37.39,
19
2
6
6.77, 16.19. F NMR (471 MHz, Chloroform-d) δ: −127.24 (ddt, J = 159.2, 13.7,
.9 Hz), −136.05 (dd, J = 159.3, 11.3 Hz).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
R)-2-((1R,3a R,5 S,6a R)-2,2-difluoro-2 ,2 -dimethyldihydro-5 H-spiro[cyclo-
ꢀ
propane-1,6 -furo[2,3-d][1,3]dioxol]-5 -yl)-2-hydroxyethyl benzoate (6a)
To a cold (−5°C) stirred solution of 5a (3.956g, 14.86mmol) in dry DCM (70 mL)
was added TEA (2.5 mL, 17.84 mmol, 1.2 eq). Then a mixture of benzoyl chloride
(
1.71 mL, 14.86 mmol, 1.0 eq) in dichloromethane (20 mL) was added dropwise.
The reaction mixture was kept for an additional 4 h and stirred overnight at 0°C.
The solution was washed with a saturated NaHCO solution (3 ×150 mL) and water
3
(
2 ×150 mL), dried with sodium sulfate, and then evaporated to a colorless solid.
Finally, column chromatography was conducted with PE:EA = 3:1 to give 6a (3.22g,
1
5
9%, white solid). H NMR (500 MHz, Chloroform-d) δ: 8.07–7.98 (m, 2H), 7.58
(
t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.8 Hz, 2H), 5.92 (d, J = 3.6 Hz, 1H), 4.64 (dd,
J = 12.0, 2.4 Hz, 1H), 4.59 (d, J = 6.4 Hz, 1H), 4.44–4.38 (m, 2H), 3.86 (s, 1H),
2
1
1
1
.14 (ddd, J = 12.0, 8.5, 4.3 Hz, 1H), 1.58 (s, 3H), 1.51 (ddd, J = 12.4, 8.4, 3.4 Hz,
H), 1.35 (s, 3H). 13C NMR (126 MHz, Chloroform-d) δ: 167.39, 133.69, 130.05,
29.91, 128.80, 113.59, 111.35, 104.66, 87.21, 77.56, 71.34, 67.09, 38.01, 27.50, 27.17,
19
8.11. F NMR (471 MHz, Chloroform-d) δ: −128.91 (ddd, J = 167.5, 13.9, 4.4 Hz),
134.53–−135.35 (m).
−
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
R)-2-((1S,3a R,5 S,6a R)-2,2-difluoro-2 ,2 -dimethyldihydro-5 H-spiro[cycloprop-
ꢀ
ꢀ
ane-1,6 -furo[2,3-d][1,3]dioxol]-5 -yl)-2-hydroxyethyl benzoate (6b)
The compound 6b (yield 56%) was obtained using a similar synthetic strategy as
1
compound 6a. H NMR (500 MHz, Chloroform-d) δ: 8.01 (d, J = 8.3 Hz, 2H), 7.52
(
t, J = 7.4 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 5.86 (d, J = 4.1 Hz, 1H), 4.60 (dd, J
=
11.8, 2.3 Hz, 1H), 4.56 (d, J = 4.1 Hz, 1H), 4.43 (t, J = 7.8 Hz, 1H), 4.35 (dd,
J = 11.8, 6.3 Hz, 1H), 4.21–4.15 (m, 1H), 1.94–1.87 (m, 1H), 1.72 (ddd, J = 11.5,
13
8
1
6
.5, 4.4 Hz, 1H), 1.51 (s, 3H), 1.32 (s, 3H). C NMR (126 MHz, Chloroform-d) δ:
67.32, 133.42, 129.99, 129.95, 128.60, 113.93, 112.24, 104.51, 80.98, 78.23, 69.39,
19
7.59, 37.45, 26.99, 26.95, 16.39. F NMR (471 MHz, Chloroform-d) δ: −127.15
(
ddt, J = 160.3, 13.9, 7.0 Hz), −136.03 (dd, J = 160.1, 11.2 Hz).
(3R,6R,7S)-6-((benzoyloxy)methyl)-1,1-difluoro-5-oxaspiro[2.4]heptane-4,7-
diyl diacetate (9a)
The compound 6a was dissolved in 50mL CF COOH (50% in water), and stirred
3
overnight. After the starting material disappeared, the mixture was evaporated to
give crude product 7a, which was then used directly in the next step.
7
a was dissolved in 50 mL DCM, followed by the addition of 50 mL water,
NaHCO (1.09 g, 13.1 mmol, 1.5 eq), and NaIO (2.05 g, 9.6 mmol, 1.1 eq). The solu-
3
4
tion was stirred at room temperature. After the starting material disappeared, the
reaction mixture was filtered. Organic phase was evaporated, and the water phase
was extracted three times with 150 mL of EA. The organic layer was concentrated
to give the crude product 8a as slightly yellow oil.

1
0
X. LIU ET AL.
8
a was dissolved in 50 mL of dry DCM, which was followed by the addition
of dry TEA (2.5 mL, 18.2 mmol, 2.1 eq) and catalytic amount of DMPA at 0°C.
Then Ac O (1.7 mL, 17.3 mmol, 2.0 eq) was added dropwise, and reaction mix-
2
ture was stirred at room temperature for 5 h. Then sat. aq. NaHCO (50 mL) was
3
added, the mixture was partitioned, and the aqueous layer was washed three times
with DCM. The organic layers was concentrated, and recrystallization was per-
formed to give 9a (2.8 g, three steps of 84% yield) as white solid. Compound 9a:
1
H NMR (500 MHz, Chloroform-d) δ: 8.07 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 7.4 Hz,
1
(
H), 7.45 (t, J = 7.5 Hz, 2H), 6.41 (s, 1H), 5.39 (s, 1H), 4.55–4.44 (m, 3H), 2.08
s, 3H), 2.03 (s, 3H), 1.90–1.78 (m, 2H). 13C NMR (126 MHz, Chloroform-d) δ:
1
8
70.21, 169.43, 166.14, 133.50, 129.84, 129.72, 129.57, 129.59, 128.58, 110.28, 95.35,
4.46, 77.93, 63.40, 36.30, 20.97, 20.83, 18.74. 19F NMR (471 MHz, Chloroform-
d) δ: −132.77 (ddd, J = 164.5, 13.2, 6.0 Hz), −135.16 (ddd, J = 164.5, 12.8,
.5 Hz).
5
(3S,6R,7S)-6-((benzoyloxy)methyl)-1,1-difluoro-5-oxaspiro[2.4]heptane-4,7-
diyl diacetate (9b)
The compound 9b (with three steps of 80% yield) was obtained using a similar syn-
1
thetic strategy as compound 9a. H NMR (500 MHz, Chloroform-d) δ: 8.06–8.01
(
3
m, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 6.28 (s, 1H), 5.26 (d, J =
.1 Hz, 1H), 4.66 (p, J = 3.8 Hz, 1H), 4.58 (d, J = 3.6 Hz, 2H), 2.13 (d, J = 5.3 Hz,
13
6
H), 1.76 (ddd, J = 11.4, 9.1, 5.7 Hz, 1H), 1.69 (ddd, J = 12.0, 9.1, 6.0 Hz, 1H). C
NMR (126 MHz, Chloroform-d) δ: 170.87, 170.42, 166.51, 133.46, 130.03, 129.77,
19
1
28.62, 111.59, 96.95, 85.15, 72.97, 64.02, 38.78, 21.10, 15.28. F NMR (471 MHz,
Chloroform-d) δ: −134.65 (ddd, J = 154.2, 11.4, 6.0 Hz), −135.62 (ddd, J = 154.2,
2.0, 5.9 Hz).
1
1
-((3R,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-oxaspiro[2.4]hept-
an-4-yl)pyrimidine-2,4(1H,3H)-dione (12) and 1-((3R,4S,6R,7S)-1,1-difluoro-7hyd-
roxy-6-(hydroxymethyl)-5-oxaspiro[2.4]heptan-4-yl)pyrimidine-2,4(1H,3H)
-
dione (13)
Compound 9a (0.1g, 0.26 mmol) was dissolved in anhydrous 1,2-DCE (8 mL) and
added to the silylated uracil (1.04 mmol; see the general method for silylation of
uracil below), followed by the dropwise addition of TMSOTf (0.1 mL, 0.53 mmol,
2
eq; ∼2 min addition) at room temperature. The mixture was stirred for 2 hours
until the starting material disappeared, and then quenched with sat. aq. NaHCO3
10 mL). The aqueous layer was extracted with DCM (3 × 10 mL), and the com-
bined organic layers were dried over Na SO , filtered, and concentrated in vacuum.
(
2
4
The crude reaction mixture was purified by column chromatography to give com-
pound 10a and 11a77 mg in total (66% yield) of a 2:1 mixture of β:α anomers of the
nucleosides.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Compound 10a (50 mg, 0.11 mmol) was treated with 10 mL solution of NH3
in MeOH, and stirred overnight. After the starting material disappeared, the reac-
tion mixture was concentrated in vacuum and purified by column chromatography
(
DCM: isopropanol = 10:1) to give compound 12 as white solid (32 mg, 98%). The
similar synthetic strategy was used to give compound13 as white solid (15 mg, 87%).
1
Compound 12: H NMR (500 MHz, DMSO-d ) δ: 11.36 (s, 1H), 7.58 (d, J = 8.0 Hz,
6
1
H), 6.26 (s, 1H), 5.55 (d, J = 7.7 Hz, 2H), 5.02 (s, 1H), 3.87–3.64 (m, 3H), 3.57 (s,
13
1
H), 1.68 (d, J = 9.0 Hz, 1H), 1.46 (dd, J = 13.6, 9.1 Hz, 1H). C NMR (126 MHz,
Methanol-d ) δ: 165.80, 151.87, 143.34, 112.99, 103.25, 88.16, 84.72, 84.69, 76.32,
4
19
3
9.95, 19.32. F NMR (471 MHz, Methanol-d ) δ: −139.55 (dd, J = 170.8, 11.5 Hz),
4
+
−
142.59 (ddd, J = 171.0, 13.5, 5.5 Hz). MS (ESI) m/z: 291.1[M+H ], 325.0[M +
−
1
Cl ]. Compound 13: H NMR (400 MHz, DMSO-d ) δ: 11.30 (s, 1H), 8.29 (d, J
6
=
8.1 Hz, 1H), 6.19 (s, 1H), 6.14 (s, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.95 (t, J =
5
.5 Hz, 1H), 4.22 (t, J = 5.5 Hz, 1H), 4.14 (s, 1H), 3.45 (d, J = 4.7 Hz, 2H), 2.09–1.99
13
(
m, 1H), 1.99–1.88 (m, 1H). C NMR (126 MHz, Methanol-d ) δ: 166.01, 152.53,
44.08, 113.51, 102.99, 91.79, 90.53, 78.42, 63.16, 39.68, 24.62. F NMR (471 MHz,
4
19
1
Methanol-d ) δ: −132.74–−133.70 (m), −137.54 (ddd, J = 168.3, 13.5, 5.0 Hz). MS
4
(ESI) m/z: 291.1[M+H+], 325.0[M+Cl−].
General procedure for silylation of uracil
Uracil (0.233 g, 2.08 mmol) was treated with BSA (10 mL) and stirred at 80°C for
2
.5 h. Excess BSA was evaporated under reduced pressure at 50°C to give a cloudy
oil, which was directly used in the nucleoside glycosylation reaction.
1
-((3R,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (14) and
-((3R,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
1
oxaspiro[2.4]heptan-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (15)
The compounds 14 and 15 were obtained using a similar synthetic strategy as com-
pound 12 and 13 with compounds 14 (30 mg) and 15 (10 mg), two steps of 49% yield
1
and a 3:1 mixture of β:α anomers. Compound 14: H NMR (400 MHz, Methanol-
d ) δ: 8.28 (d, J = 6.8 Hz, 1H), 6.49 (d, J = 1.8 Hz, 1H), 4.36 (td, J = 4.3, 1.8 Hz,
4
1
1
3
1
H), 4.02 (dt, J = 4.7, 2.8 Hz, 1H), 3.87 (dd, J = 12.3, 2.6 Hz, 1H), 3.78 (dd, J =
2.2, 3.0 Hz, 1H), 1.78 (ddd, J = 13.0, 8.9, 6.3 Hz, 1H), 1.50 (ddd, J = 14.2, 8.9,
1
3
.6 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ: 159.22, 150.70, 141.82, 126.78,
4
1
9
13.00, 88.41, 85.03, 76.26, 62.21, 39.97, 19.35. F NMR (471 MHz, Methanol-d )
4
δ: −131.33 (dd, J = 165.1, 13.7 Hz), −137.64 (dd, J = 165.3, 12.9 Hz), −167.35 (d,
+
−
1
J = 6.9 Hz).MS (ESI) m/z: 309.1[M+H ], 343.0[M+Cl ]. Compound 15: H NMR
(
1
400 MHz, DMSO-d ) δ: 11.91 (s, 1H), 8.57 (d, J = 7.2 Hz, 1H), 6.30 (d, J = 3.8 Hz,
6
H), 6.18 (s, 1H), 4.95 (t, J = 5.9 Hz, 1H), 4.25 (t, J = 5.6 Hz, 1H), 4.15 (d, J =
13
3
.5 Hz, 1H), 3.44 (d, J = 5.5 Hz, 2H), 2.13–2.02 (m, 1H), 2.00–1.91 (m, 1H). C
NMR (126 MHz, DMSO-d ) δ: 157.97, 150.28, 140.84, 126.72, 113.44, 91.14, 89.35,
6

1
2
X. LIU ET AL.
1
9
7
6.71, 62.25, 38.37, 24.06. F NMR (471 MHz, DMSO-d ) δ: −129.78–−131.26 (m),
6
−
135.60 (ddd, J = 164.2, 13.9, 5.8 Hz), −166.53 (d, J = 7.2 Hz). MS (ESI) m/z:
+
−
3
09.1[M+H ], 343.0[M+Cl ].
1
-((3R,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (16) and 1-
(3R,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
(
oxaspiro[2.4]heptan-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (17)
The compounds 16 and 17 were obtained using a similar synthetic strategy as com-
pound 12 and 13 with compounds 16 (45 mg) and 17 (11 mg), two steps of 69% yield
1
and a 4:1 mixture of β:α anomers. Compound 16: H NMR (400 MHz, Methanol-
d ) δ: 7.78 (d, J = 1.3 Hz, 1H), 6.48 (s, 1H), 4.38 (dt, J = 6.5, 3.1 Hz, 1H), 4.00 (dt,
4
J = 4.7, 3.0 Hz, 1H), 3.86 (dd, J = 12.3, 2.8 Hz, 1H), 3.77 (dd, J = 12.3, 3.4 Hz,
1
1
1
H), 1.88 (d, J = 1.2 Hz, 3H), 1.76 (ddd, J = 13.0, 8.9, 6.4 Hz, 1H), 1.39 (ddd, J =
13
3.3, 8.8, 3.6 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ: 166.10, 152.08, 138.76,
4
19
12.99, 112.00, 88.00, 84.65, 76.18, 62.24, 39.80, 19.31, 12.40. F NMR (471 MHz,
Methanol-d ) δ: −131.20 (ddd, J = 165.6, 13.5, 5.9 Hz), −137.77 (dd, J = 164.9,
4
+
−
1
1
2.9 Hz).MS (ESI) m/z: 305.0[M+H ], 339.1[M+Cl ]. Compound 17: H NMR
(
4
7
400 MHz, Methanol-d ) δ: 8.23 (d, J = 1.4 Hz, 1H), 6.26 (d, J = 4.1 Hz, 1H),
4
.35 (t, J = 5.2 Hz, 1H), 4.18 (s, 1H), 3.62 (d, J = 5.2 Hz, 2H), 1.93 (td, J = 8.5,
13
.4, 3.2 Hz, 1H), 1.90 (d, J = 1.2 Hz, 3H), 1.85–1.75 (m, 1H). C NMR (126 MHz,
Methanol-d ) δ: 166.32, 152.74, 139.70, 111.52, 111.82, 91.55, 90.43, 78.46, 63.21,
4
19
3
−
9.52, 24.60, 12.53. F NMR (471 MHz, Methanol-d ) δ: −132.67–−133.49 (m),
4
+
−
137.27–−137.85 (m). MS (ESI) m/z: 305.0[M+H ], 339.1[M+Cl ].
4
-amino-1-((3R,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)pyrimidin-2(1H)-one (19)
The compound 10a(180 mg, 0.413 mmol) was dissolved in 8 mL dry DCM, followed
by the addition of 1, 2, 4-triazole (313 mg, 4.54 mmol, 11eq), and TEA (0.63 mL,
4
.54 mmol, 11eq). The reaction mixture was stirred at 0 °C, and the dry POCl3
(
0.1 mL, 1.1 mmol, 2.6 eq) was added. It was then placed at room temperature
for 4 hours, until the starting material disappeared, and the mixture was quenched
with cold water. The organic layer was partitioned and evaporated to give the crude
product 18.
The compound 18 was dissolved in THF (5 mL), followed by the addition of
NH OH (1 mL), and stirred at room temperature overnight. After the starting mate-
4
rial disappeared, it was evaporated and then a solution of NH in MeOH (5 mL) was
3
added. Stirred overnight, and then evaporated and purified by column chromatog-
raphy (DCM:ir-propanol = 4:1) to give compound 19 (25 mg, three steps of 21%
1
yield) as pale-yellow solid. H NMR (400 MHz, Methanol-d ) δ: 8.10 (d, J = 7.7 Hz,
4
1
H), 6.54 (s, 1H), 6.00 (d, J = 7.6 Hz, 1H), 4.37 (s, 1H), 4.02 (q, J = 3.6 Hz, 1H),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
3
.86 (dd, J = 12.3, 2.9 Hz, 1H), 3.77 (dd, J = 12.2, 3.4 Hz, 1H), 1.74 (dt, J = 14.0, 8.1
13
Hz, 1H), 1.47–1.33 (m, 1H). C NMR (126 MHz, Methanol-d ) δ: 165.38, 154.90,
4
19
145.06, 112.92, 96.41, 88.15, 85.59, 75.98, 62.18, 40.26, 19.00. F NMR (471 MHz,
Methanol-d ) δ: −130.67–−131.40 (m), −137.56 (dd, J = 165.2, 12.8 Hz). MS (ESI)
4
+
−
m/z: 291.1[M+H ], 325.0[M+Cl ].
(3R,4R,6R,7S)-4-(6-amino-9H-purin-9-yl)-1,1-difluoro-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-7-ol (21)
Compound 9a (0.1 g, 0.26 mmol) was dissolved in anhydrous 1,2-DCE (8 mL), and
the mixture was then added to the silylated 6-chloropurine (164 mg, 1.04 mmol, 4
eq; see the general method for silylation of uracil), followed by the dropwise addi-
tion of TMSOTf (0.1 mL, 0.53 mmol, 2eq; ∼2 min addition) at room temperature.
The mixture was stirred for 2 h until the starting material disappeared, and then
quenched with sat. aq. NaHCO (10 mL). The aqueous layer was extracted with
3
DCM (3 × 10 mL), and the combined organic layers were dried over Na SO , then
2
4
filtered, and finally concentrated in vacuum. The reaction mixture was purified by
column chromatography to give the crude product 20, which was then dissolved in
2
mL of 1,4-dioxane, followed by the addition of 5 mL of conc. NH OH. The reac-
4
tion vessel was sealed and heated to 100°C in an oil bath for 12 hours. It was then
evaporated and purified by column chromatography (DCM : ir-propanol = 3:1) to
1
give compound 21(20 mg, three steps of 24% yield) as pale-yellow solid. H NMR
(
400 MHz, DMSO-d ) δ: 8.50 (s, 1H), 8.23 (s, 1H), 6.86 (s, 2H), 6.42 (s, 1H), 6.17 (d,
6
J = 4.2 Hz, 1H), 4.97 (t, J = 5.9 Hz, 1H), 4.26 (s, 1H), 4.13 (t, J = 5.5 Hz, 1H), 3.56–
13
3
.46 (m, 3H), 2.25–2.14 (m, 1H), 2.15–2.06 (m, 1H). C NMR (126 MHz, DMSO-
d ) δ: 160.68, 153.38, 152.31, 145.15, 111.39, 91.63, 89.32, 76.53, 62.18, 52.97, 38.75,
6
19
2
3.81. F NMR (471 MHz, DMSO-d ) δ: −137.46 (dd, J = 162.2, 12.8 Hz), −139.58
6
+
−
(dd, J = 163.0, 12.3 Hz). MS (ESI) m/z: 314.0[M+H ], 348.1[M+Cl ].
1
-((3S,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)pyrimidine-2,4(1H,3H)-dione(22) and
-((3S,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
1
oxaspiro[2.4]heptan-4-yl)pyrimidine-2,4(1H,3H)-dione (23)
The similar synthetic strategy with compounds 12(β) and 13(α) was used to give
compounds 22(β) (22 mg) and 23(α) (12 mg) as white solids, two steps of 44%
1
yield and a 1.8:1 mixture of β:α anomers. Compound 22: H NMR (400 MHz,
Methanol-d ) δ: 7.72 (d, J = 8.1 Hz, 1H), 6.14 (d, J = 5.7 Hz, 1H), 5.72 (d, J =
4
8
7
1
1
.1 Hz, 1H), 4.49 (d, J = 7.4 Hz, 1H), 3.92 (dd, J = 12.0, 2.2 Hz, 1H), 3.85 (ddd, J =
.0, 4.3, 2.2 Hz, 1H), 3.79 (dd, J = 12.0, 4.3 Hz, 1H), 1.99 (ddd, J = 13.6, 8.3, 5.8 Hz,
13
H), 1.64 (ddd, J = 14.0, 8.3, 5.3 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ:
4
19
65.96, 152.10, 143.24, 130.84, 112.69, 103.11, 86.53, 68.61, 61.80, 38.57, 16.41. F
NMR (471 MHz, Methanol-d ) δ: −135.51 (d, J = 164.0 Hz), −138.83 (ddd, J =
4
+
−
163.9, 14.7, 5.9 Hz).MS (ESI) m/z: 291.1[M+H ], 325.0[M+Cl ]. Compound 23:

14
X. LIU ET AL.
1
H NMR (400 MHz, Methanol-d ) δ: 8.10 (d, J = 8.1 Hz, 1H), 6.34 (s, 1H), 5.74 (d, J
4
=
8.1 Hz, 1H), 4.36 (t, J = 5.0 Hz, 1H), 4.20 (d, J = 1.7 Hz, 1H), 3.61 (d, J = 4.4 Hz,
13
2
H), 1.90–1.81 (m, 1H), 1.47–1.39 (m, 1H). C NMR (126 MHz, Methanol-d ) δ:
4
19
1
64.62, 151.35, 142.53, 111.82, 101.87, 89.24, 85.28, 70.59, 61.79, 40.05, 14.46. F
NMR (471 MHz, Methanol-d ) δ: −135.61–−136.08 (m), −136.10–−136.55 (m).
4
+
−
MS (ESI) m/z: 291.1[M+H ], 325.0[M+Cl ].
1
-((3S,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione(24) and
-((3S,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
1
oxaspiro[2.4]heptan-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (25)
The similar synthetic strategy with compounds 12(β) and 13(α) was used to give
compounds 24(β) (27 mg) and 25(α) (10 mg) as white solids, two steps of 43% yield
1
and a 2.7:1 mixture of β:α anomers. Compound 24: H NMR (400 MHz, Methanol-
d ) δ: 7.99 (d, J = 6.5 Hz, 1H), 6.17 (s, 1H), 4.50 (d, J = 7.2 Hz, 1H), 3.96–3.90 (m,
4
1
5
H), 3.89–3.84 (m, 1H), 3.81 (dd, J = 12.1, 3.7 Hz, 1H), 1.99 (ddd, J = 13.6, 8.0,
.9 Hz, 1H), 1.66 (ddd, J = 14.0, 8.3, 5.5 Hz, 1H).
13
C NMR (126 MHz, Methanol-d ) δ: 176.37, 159.39, 150.82, 141.97, 126.73,
4
19
112.75, 86.71, 68.40, 61.55, 38.70, 16.35. F NMR (471 MHz, Methanol-d ) δ:
4
−
135.14 (d, J = 163.1 Hz), −138.75—139.58 (m), −168.42(s). MS (ESI) m/z:
+
−
1
3
09.1[M+H ], 343.0[M+Cl ]. Compound 25: H NMR (400 MHz, Methanol-d )
4
δ: 8.30 (d, J = 6.8 Hz, 1H), 6.34 (d, J = 1.5 Hz, 1H), 4.38 (t, J = 3.9 Hz, 1H), 4.19 (d,
J = 1.2 Hz, 1H), 3.60 (d, J = 4.5 Hz, 2H), 1.87 (ddd, J = 11.1, 9.1, 7.0 Hz, 1H), 1.48
13
(
1
ddt, J = 11.7, 5.9, 3.6 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ: 159.51, 151.43,
4
19
41.92, 127.39, 113.17, 90.71, 86.97, 71.99, 63.18, 41.40, 15.84. F NMR (471 MHz,
Methanol-d ) δ: −135.64 (ddd, J = 153.6, 11.5, 5.7 Hz), −136.22 (ddd, J = 153.3,
4
+
−
1
2.2, 6.8 Hz), −167.74 (d, J = 5.9 Hz). MS (ESI) m/z: 309.1[M+H ], 343.0[M+Cl ].
1
-((3S,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione(26) and
-((3S,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
1
oxaspiro[2.4]heptan-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (27)
The similar synthetic strategy with compounds 12(β) and 13(α) was used to give
compounds 26(β) (34 mg) and 27(α) (9 mg) as white solids, two steps of 54% yield
1
and a 4:1 mixture of β:α anomers. Compound26: H NMR (400 MHz, DMSO-d ) δ:
6
1
1
1
1.32 (s, 1H), 7.52 (d, J = 1.4 Hz, 1H), 6.04 (s, 1H), 5.68 (d, J = 6.6 Hz, 1H), 5.01 (s,
H), 4.36 (t, J = 7.1 Hz, 1H), 3.74 (qd, J = 5.6, 2.6 Hz, 2H), 3.63 (dt, J = 12.1, 5.4 Hz,
13
H), 1.89 (dt, J = 13.7, 7.8 Hz, 1H), 1.75–1.61 (m, 1H). C NMR (126 MHz, DMSO-
d ) δ: 164.57, 151.09, 138.57, 112.72, 110.47, 100.40, 86.08, 67.88, 61.30, 37.64, 16.32,
6
19
12.96. F NMR (471 MHz, Methanol-d ) δ: −135.53 (d, J = 161.9 Hz), −139.01
4

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
+
−
(
ddd, J = 163.4, 14.5, 5.8 Hz). MS (ESI) m/z: 305.0[M+H ], 339.1[M+Cl ]. Com-
1
pound27: H NMR (500 MHz, Methanol-d ) δ: 7.93 (d, J = 1.5 Hz, 1H), 6.33 (s, 1H),
4
4
.36 (td, J = 4.5, 1.9 Hz, 1H), 4.21 (d, J = 2.0 Hz, 1H), 3.61 (d, J = 4.4 Hz, 2H), 1.88 (d,
13
J = 1.4 Hz, 3H), 1.87–1.82 (m, 1H), 1.37 (ddd, J = 10.9, 9.1, 6.7 Hz, 1H). C NMR
(
7
1
126 MHz, Methanol-d ) δ: 166.24, 152.90, 139.50, 113.24, 112.08, 90.47, 86.58,
4
19
2.00, 63.25, 41.33, 15.95, 12.54. F NMR (471 MHz, Methanol-d ) δ: −135.66—
4
+
−
36.04 (m), −136.04—136.46 (m). MS (ESI) m/z: 305.0[M+H ], 339.1[M+Cl ].
4
-amino-1-((3S,4R,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)pyrimidin-2(1H)-one (28)
The similar synthetic strategy with compound 19 was used to give compound 28 as
1
white solid (10 mg, three steps of 32% yield). H NMR (400 MHz, Methanol-d ) δ:
4
7
.70 (d, J = 7.5 Hz, 1H), 6.23 (s, 1H), 5.90 (d, J = 7.5 Hz, 1H), 4.47 (d, J = 7.3 Hz,
1
H), 3.92 (dd, J = 11.9, 2.2 Hz, 1H), 3.91–3.73 (m, 2H), 1.98–1.87 (m, 1H), 1.68
13
(
1
dt, J = 14.1, 7.5 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ: 167.68, 158.22,
4
19
43.60, 112.84, 96.50, 86.32, 68.96, 66.88, 61.87, 38.98, 16.21. F NMR (471 MHz,
Methanol-d ) δ: −134.77 (d, J = 163.2 Hz), −138.86 (ddd, J = 162.7, 14.3, 5.6 Hz).
4
+
−
MS (ESI) m/z: 291.1[M+H ], 325.0[M+Cl ].
4
-amino-1-((3S,4S,6R,7S)-1,1-difluoro-7-hydroxy-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-4-yl)pyrimidin-2(1H)-one (29)
The similar synthetic strategy with compound 19 was used to give compound 29 as
1
white solid (8 mg, three steps of 29% yield). H NMR (400 MHz, Methanol-d ) δ:
4
8
.04 (d, J = 7.5 Hz, 1H), 6.35 (s, 1H), 5.92 (d, J = 7.5 Hz, 1H), 4.37 (q, J = 3.8 Hz,
1
H), 4.18 (d, J = 1.9 Hz, 1H), 3.61 (d, J = 4.6 Hz, 2H), 1.82 (q, J = 9.0 Hz, 1H), 1.37
13
(
1
dd, J = 16.3, 10.2 Hz, 1H). C NMR (126 MHz, Methanol-d ) δ: 167.46, 158.50,
4
19
44.70, 113.37, 96.64, 90.54, 72.15, 68.89, 63.13, 41.64, 15.76. F NMR (471 MHz,
Methanol-d ) δ: −134.78 (d, J = 159.0 Hz), −138.86 (dt, J = 162.3, 9.0 Hz). MS
4
+
−
(
ESI) m/z: 291.1[M+H ], 325.0[M+Cl ].
(3S,4R,6R,7S)-4-(6-amino-9H-purin-9-yl)-1,1-difluoro-6-(hydroxymethyl)-5-
oxaspiro[2.4]heptan-7-ol (30)
The similar synthetic strategy with compound 21 was used to give compound 30 as
1
pale-yellow solid (8 mg, three steps of 24% yield). H NMR (400 MHz, Methanol-d )
4
δ: 8.24 (s, 1H), 8.19 (s, 1H), 6.29 (d, J = 4.6 Hz, 1H), 4.94 (d, J = 7.5 Hz, 1H), 4.02–
3
.98 (m, 1H), 3.95 (dd, J = 12.3, 2.4 Hz, 1H), 3.84 (dd, J = 12.3, 4.4 Hz, 1H), 2.08
13
(
dt, J = 14.0, 7.3 Hz, 1H), 1.78–1.71 (m, 1H). C NMR (126 MHz, Methanol-d ) δ:
4
19
1
57.36, 153.80, 150.29, 141.32, 120.23, 112.37, 87.64, 68.61, 62.26, 39.42, 16.39. F
NMR (471 MHz, Methanol-d ) δ: −137.07 (ddt, J = 165.6, 11.6, 5.2 Hz), −137.95
4
+
−
(
ddd, J = 165.4, 14.2, 6.3 Hz). MS (ESI) m/z: 314.0[M+H ], 348.1[M+Cl ].

1
6
X. LIU ET AL.
Funding
The research was supported in part by Foshan Municipal Funds for Scientific and Tech-
nological Innovation Projects (2013HK100012), Guangzhou Science and Technology Project
(
2014J4100222), National Natural Science Foundation of China Grant (21402205). M.H. is spon-
ꢀ
sored by CAS-TWAS President s Fellowship for international PhD students.
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