Synthesis of an Anti-hepatitis B Agent, 2′-Fluoro-6′-methylene-carbocyclic Adenosine (FMCA) and Its Phosphoramidate (FMCAP)

Article abstract of DOI:10.1021/acs.joc.8b02599

2′-Fluoro-6′-methylene-carbocyclic adenosine (FMCA, 12) and its phosphoramidate prodrug (FMCAP, 14) have been proven as a potential anti-HBV agent against both adefovir-resistant as well as lamivudine-resistant double (rtL180M/rtM204V) mutants. Furthermore, in vitro, these agents have demonstrated significant activity against lamivudine/entecavir triple mutants (L180M + S202G + M204V). These preliminary results encourage us for further biological evaluation of FMCA and FMCAP to develop as a potential clinical candidate as an anti-HBV agent, which may overcome the problem of drug resistance in HBV therapy. To support the preclinical exploration, a scalable synthesis of this molecule was needed. In this communication, a practical and scalable synthesis of FMCA, and its prodrug, is reported via ketone 1. The selective opening of the isopropylidene group of 2 led to compound 3. Protection of the allylic hydroxyl group of 3, followed by fluorination and deprotection, afforded the key intermediate 10, which was condensed with a Boc-protected adenine, followed by deprotection, furnished the target nucleoside FMCA (12) in high yield. Further coupling of phosphorochloridate of L-alanine isopropyl ester (13) with FMCA gave its phosphoramidate prodrug FMCAP (14) in good yield.

Full text of DOI:10.1021/acs.joc.8b02599

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Article
Synthesis of an Anti-hepatitis B Agent, 2#-Fluoro-6#-Methylene-
carbocyclic Adenosine (FMCA) and its Phosphoramidate (FMCAP)
Uma Sharan Singh, Varughese A. Mulamoottil, and Chung K. Chu
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02599 • Publication Date (Web): 27 Dec 2018
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Synthesis of an Anti-hepatitis B Agent, 2′-Fluoro-6′-
Methylene-carbocyclic Adenosine (FMCA) and its Phosphoramidate
(FMCAP)
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Uma S. Singh, Varughese A. Mulamoottil and Chung K. Chu*
The University of Georgia, College of Pharmacy
Athens, Georgia 30602
E-Mail: dchu@uga.edu
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NH2
N
N
N
O
O
O
F
HO
N
OH
F
8
O
O
O
9
OH
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1
10
FMCA
(12)
ABSTRACT
2′-Fluoro-6′-methylene-carbocyclic adenosine (FMCA, 12) and its phosphoramidate
prodrug (FMCAP, 14) (Figure 1) has been proven as a potential anti-HBV agent against both
adefovir-resistant as well as lamivudine-resistant double (rtL180M/rtM204V) mutants.
Furthermore, in vitro, these agents have demonstrated significant activity against
lamivudine/entecavir triple mutants (L180M+S202G+M204V). These preliminary results
encourage us for further biological evaluation of FMCA and FMCAP to develop as a potential
clinical candidate as an anti-HBV agent, which may overcome the problem of drug-resistance in
HBV therapy. To support the preclinical exploration, a scalable synthesis of this molecule was
needed. In this communication, a practical, and scalable synthesis of FMCA, and its prodrug are
reported via ketone 1. The selective opening of isopropylidene group of 2 led to compound 3.
Protection of the allylic hydroxyl group of 3 followed by fluorination, and deprotection afforded
the key intermediate 10, which was condensed with a Boc-protected adenine followed by
deprotection furnished the target nucleoside FMCA (12) in high yield. Further coupling of
phosphorochloridate of L-alanine isopropyl ester (13) with FMCA gave its phosphoramidate
prodrug FMCAP (14) in good yield.
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INTRODUCTION
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Hepatitis B virus (HBV) is one of the leading causes of morbidity, and mortality of
human population in the world. According to WHO, approximately 2 billion people have been
infected with HBV, out of them approximately 350 million people are suffering from chronic
HBV infection.1 Due to the chronic infection of HBV, worldwide 0.5–1.2 million deaths
annually are reported. The untreated HBV infection can develop into liver failure, cirrhosis, and
eventually hepatocellular carcinoma that will result in an urgent need for liver transplantation.
Although various drugs, and vaccines have been introduced for the treatment of the HBV
infection, none of them has been successful for its complete eradication.2 A class of
nucleos(t)ides are available for the treatment of chronic HBV infection.3 These nucleos(t)ides
inhibit viral reverse transcriptase (RT)/DNA polymerase which serves as an essential enzyme for
the DNA synthesis in HBV. Based on this mechanism, lamivudine was first introduced for the
treatment of chronic HBV infection. After a period of therapy, lamivudine-resistant HBV
(LVDr) was observed in a significant number of patients.4 Currently, entecavir and tenofovir
alafenamide are the most prescribed drugs for the treatment of HBV.5 A long-term therapy of
these drugs promotes double and triple mutations, and becomes drug-resistant HBV.6 Recently,
the reported triple mutation (L180M+M204V+S202G) limits the use of entecavir/lamivudine.7
These double and triple mutations of the virus have become a major challenge in the treatment of
HBV.8 Thus, agents effective against drug resistant HBV mutants in patients are critically
needed.
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For the last two decades, in search of novel antiviral agents, our group has been involved
in the discovery of 2ʹ-fluoro containing nucleosides.9-11 To overcome the current drug resistant
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problem of HBV, we discovered 2′-fluoro-6′-methylene carbocyclic adenosine (FMCA) and its
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phosphoramidate prodrug (FMCAP, Figure 1) as promising anti-HBV agents.12
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NH2
N
NH2
N
N
N
N
N
O
O
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H
N
HO
P O
OPh
N
N
O
F
F
CH3
OH
FMCA
(12)
OH
FMCAP
(14)
Figure 1. Structures of FMCA and its phosphoramidate prodrug FMCAP.
FMCA has demonstrated a significant activity against the wild-type as well as
lamivudine-, adefovir-, and lamivudine/entecavir-resistant mutants.13 Furthermore, it has also
been evaluated against lamivudine/entecavir-resistant clone (L180M+M204V+S202G), that has
become a challenge, as it confines the use of currently available drugs for the treatment of HBV.
Interestingly, FMCA has shown potential antiviral activity against resistant mutants. It is well
known that the first phosphorylation is the rate limiting step for the antiviral activity of the
parental nucleoside.14,15 Thus, to increase the phosphorylation, and thereby the antiviral activity,
the prodrug of FMCA was synthesized to bypass the initial phosphorylation step, and it has
indeed demonstrated a 12-fold increase in anti-HBV activity against the entecavir/lamivudine-
resistant triple mutant (L180M+M204V+S202G).16 Mitochondrial and cellular toxicity of
FMCA, and FMCAP have also been studied, and no significant toxicity was observed up to
100µM.16 Based on these results it was of great interest to examine further biological evaluation
of FMCA to determine its full potential as an anti-HBV agent, which requires a significant
amount of FMCA. Consequently, the development of a practical and cost effective synthesis of
FMCA/FMCAP was needed.
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In our previous communication, we have reported the synthesis of FMCA via Vince
lactam in 14 steps.17,18 However, the low yield of certain steps, limits the process for a large scale
synthesis. Furthermore, lack of commercial availability of carbocyclic intermediate 1 was also a
prime challenge. Our group and others have investigated the synthesis of carbocyclic
intermediate 1 from D-ribose, and convenient, but a small scale method has been previously
developed.19 Several commercial vendors adopted this synthesis, and currently the supply of
carbocyclic ketone 1 is readily available on demand. Therefore, herein we report a scalable,
practical synthesis of FMCA in 7 steps from the carbocyclic ketone 1. The straightforward
reactions as well as the use of readily available reagents make the process feasible for the
scalable synthesis of FMCA/FMCAP. During initial optimization of the synthesis, unexpected 2-
deoxy carbocylic moieties 6, and 7 were obtained. It is noteworthy to mention that the generation
of 2-deoxy carbocyclic moieties is interesting. The preparation of a 2-deoxy carbocyclic moiety
usually requires robust, and expensive synthesis,20 but generation of 6, and 7 were critical for this
approach as they influence the overall yield. If the process could be optimized to exclusively
obtain the 2-deoxy carbocyclic intermediate 6, this approach might be applied for the synthesis
of 2-deoxy cabocyclic nucleosides, which is not the current scope of this report.
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RESULTS AND DISCUSSION
Previously, we have reported a stereoselective synthesis of FMCA via Vince Lactam.17 However,
poor yields of the diazotization-elimination step of an amino group, as well as the inversion of
configuration of the hydroxyl group of the key intermediate makes the synthetic approach
challenging for a large scale synthesis of FMCA. In search of a more efficient method for the
synthesis of FMCA, herein we report a practical synthesis via the intermediate 1, which is a
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commercially available starting material. The new scheme may provide fewer steps, better
overall yield, and also can avoid the column chromatography in several steps.
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The synthesis of compound 2 was carried out by the introduction of an exocyclic
methylene group to ketone 1.16 In our previous report,21 the incorporation of the exocyclic double
bond at 6 position was done by the treatment of ketone 1 with Eschenmoser’s salt in the presence
of LDA, followed by Hoffman elimination with methyl iodide. The step was very challenging
and tedious, and furthermore an excess use of toxic methyl iodide was also not acceptable for a
large scale setting. In this report, to avoid these harmful and expensive reagents, the insertion of
an exocyclic double bond has been accomplished by the use of paraformaldehyde in the presence
of diisopropylamine, and TFA salt.22
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Thus, the ketone 1 was treated with paraformaldehyde in presence of diisopropyl
ammonium trifluoroacetate salt in THF to furnish an enone in 67% yield (scheme 1). Selective
reduction of the enone was carried out by using sodium borohydride/cerium chloride hydrate
complex (NaBH4/CeCl3.7H2O) via Luche reduction23 to obtain exclusively α-hydroxyl
compound 2 in 78% yield. A regioselective opening of the isopropylidene group of compound 2
was accomplished by the reported protocol of Ogasawara et al.24,25 The treatment of compound 2
with trimethylaluminum (2M solution in hexane) produced diol 3 with retention of the α-
configuration of hydroxyl groups in 76% yield. Selective protection of allylic alcohol 3 was
carried out with tert-butyldiphenylsilyl chloride (TBDPSCl) in presence of imidazole in DCM to
give the protected compound 4 in 92% yield. Due to the higher reactivity of the allylic alcohol,
and bulkier TBDPS group, compound 4 was exclusively obtained.
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Conversion of 2-α-hydroxy to 2-β-fluoro intermediate 8 was accomplished by treating the
alcohol 4 with diethylaminosulfur trifluoride (DAST) at - 30 ºC to room temperature for 30
minutes to give the 2-fluoro derivative 8 in 36% yield. During the course of the fluorination
reaction, an interesting observation was made that is noteworthy to report. The fluorination of
compound 4 was carried out by DAST in DCM. To complete the consumption of the starting
material 4, the reaction time was extended from which two additional polar spots appeared along
with the desired compound 8 on TLC. These results prompted us to isolate, and identify the two
polar spots from the fluorination reaction. The spectral data confirmed the formation of
compounds 6, and 7. It is noteworthy to mention that the preparation of 2-deoxy intermediates is
challenging, and often necessary in nucleoside chemistry. The ketone 6 can be selectively
reduced to an alcohol intermediate, which can be utilized for the synthesis of entecavir. 26,27
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Scheme 1. Synthesis of common key intermediate 10
O
O
O
O
O
(a)
(c)
(b)
OR
OH
OH
OH
52%
76%
O
O
O
O
OH
O
O
1
3
4
5
R = TBDPS (92%)
R = TBDMS (91%)
2
(d)
O
O
O
O
O
O
F
(e)
F
OR
OH
+
+
83%
O
O
O
10
7
6
8
9
R = TBDPS (36%)
R = TBDMS (62%)
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Reagents and Conditions: (a) i) (HCHO)n, i-Pr2NH.TFA, diisopropylamine, THF; ii) NaBH4,
CeCl3.7H2O, Methanol; (b) Al(Me)3 (2.0M in hexane), DCM; (c) TBDPSCl or TBDMSCl,
imidazole, DCM; (d) DAST, DCM; (e) TBAF, THF.
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1
1
The structure of compound 6 was validated by H NMR, H-1H COSY, DEPT-C NMR
and HMQC spectroscopy. The 1H NMR spectrum of compound 6 revealed double doublet of two
H-2 protons at  2.62, 2.26 and a quartet of H-3 proton at  4.07 ppm with a complete absence of
H-1 proton. H-1H COSY spectra of 6 showed the correlation of double doublet of H-2 protons
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with the quartet of H-3 protons. F-NMR of 6 showed a complete absence of a fluorine atom,
which confirmed the absence of fluorine atom, and the double doublets of H-2 protons proved
the formation of 2-deoxy sugar 6. For further confirmation, a DEPT experiment was performed
which showed three peaks of CH2 carbon at 118.4, 61.1, 47.5, and two peaks of the CH carbon at
68.1 and 50.4. The HMQC spectra also revealed that double doublet of two H-2 protons at 
2.62, 2.26 showed correlations with CH2 carbon at 47.5, which confirmed the structure of the
ketone 6.
Compound 7 was also confirmed by a similar spectral analysis. 1H NMR of compound 7
showed two doublets of H-2, and H-3 protons at  7.70, and 6.44 indicated the formation of
1
olefinic protons. The complete absence of 3-O- tert-butyl protons was detected by H-NMR.
Probably, in an acidic medium, the 3-O- tert-butyl group was eliminated to give the compound 7.
In the H-1H COSY spectrum, the olefinic H-2, and H-3 protons correlation confirmed the
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adjacent protons to each other. Furthermore, to confirm the structure of compound 7, DEPT and
HMQC experiments were performed. Wherein, in the DEPT experiment two CH2 were evident
at  117.7, 64.0, and three CH was obtained at  160.9, 135.7 and 45.8 along with single CH3 at
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27.5 ppm. In the HMQC spectrum, carbon at 160.9 showed a correlation with a doublet of H-3
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proton at  7.70, and carbon at 135.7, which showed the correlation with the doublet of H-2
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proton at  6.44, providing the evidence of the elimination of 3-O-tert-butyl group with
formation of conjugated alkene of ketone 7. A plausible mechanism of formation of compounds
6, and 7 is shown in scheme 2.
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Scheme 2. Plausible mechanism of the formation of compound 6 and 7
F
O
O
H
Ph
O Si
Ph
Ph
OTBDPS
O
O
O S
F
N
F
Path 1
Si F
Ph
O
O
O
H
OTBDPS
OH
OH
O
OH
O
O
O S
F
N
Path 2
F
O
H
O
O
O
O
O
H
O
H
6
7
O
However, due to formation of 6 and 7, the yield of the fluorination step from 4 to 8 was
not acceptable for a large scale synthesis of FMCA. Thus, to improve the yield of fluorination,
this reaction was re-examined on compound 5.
From these observations, it was concluded that the bulky TBDPS group of 4 is playing a
major role, and altering the conformation of cyclopentane ring that restricts the favorable
fluorine approach to replace the OH group. It is a possibility that the constrained orientation of
the carbocyclic ring pushes the elimination of TBDPS to generate 6 & 7 along with the desired
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compound 8. For our purpose, these results were not encouraging. Consequently, another
strategy was to change the C1-protecting group of 4, and then optimize the fluorination reaction
to increase the yield of desired fluorinated compound 8. However, the selective protection of the
1-allylic OH of 3 was also challenging, as the different selective protecting groups were tried,
but in each case the di-hydroxy protected compound was obtained as the major product. Hicks et
al. reported28 the selective hydroxy allylic protection with triethylsilane chloride (TESCl), but
our result were not encouraging, and with the ratio of 6/4 in favor of the desired-
mono/undesired-di-protected compound were obtained. Alternatively, the treatment of
compound 3 with tert-butyldimethylsilyl chloride yielded selectively allylic protected compound
5 in 91% yield. Furthermore, the fluorination of compound 5 with DAST furnished the
compound 9 in 62% yield with traces of compounds 6, and 7. To further improve the yield of the
fluorination, a variety of fluorinating reagents via SN2 type fluorination were also tried with
various reactions conditions, but none of them gave any improved yield of the compound 9. The
results are summarized in Table 1.
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Table 1. The optimization of fluorination of compound 5 with various fluorinating agents.
O
O
Reagents
F
O Si
OH
O Si
Conditions
O
O
5
9
Entry
Reagent
Additive
Solvent
T (°C)
Time
Yield (%)
(equiv)
1.
2.
3.
DAST
DAST
DAST
1.5
3
7
DCM
DCM
DCM
-20 °C to r.t.
-20 °C to r.t.
-20 °C to r.t.
0.5 h
0.5 h
10 min
38%
48%
62%
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4.
5.
Xtal Fluor-E, DBU29
1 : 1.5
DCM
DCM
-78 °C to r.t.
-78 °C to r.t.
4.0 h
10%
Deprotection of
TBDMS was
observed
Xtal Fluor-E, TEA.3HF, TEA29
1.5 : 2 : 1
20 min
35%
9
6.
7.
8.
Xtal Fluor-E, TEA.3HF, TEA
Xtal Fluor-E, TEA.3HF, TEA
Xtal Fluor-M, TEA.3HF, TEA29
3 : 4 : 2
DCM
Toluene
DCM
-78 °C to r.t.
-78 °C to r.t.
-78 °C to r.t.
30 min
1 h
30%
25%
25%
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1.5 : 2.0 : 1
1.5 : 2.0 : 1
1 h
9.
10.
11.
Fluolead30
Fluolead
PyFlour, DMAP31
2
4
DCM
DCM
Toluene
0 °C to r.t.
0 °C to r.t.
0 °C to r.t.
30 min
30 min
24 h
42%
25%
1 : 1.5
starting material was
as such
starting material was
as such
undesired spots were
formed (not isolated)
12.
13.
PyFlour, DMAP
1 : 1.5
3
Toluene
DCM
80 °C
4 h
3 h
Ishikawa’s Reagent
(N,N-diethyl-1,1,2,3,3,3-
hexafluoropropylamine)
Yarovenko reagent
(2-chloro-1,1,2-trifluoroethyl-
N,N-diethylamine)
0 °C to r.t.
14.
3
DCM
0 °C to r.t.
6 h
undesired spots were
formed (not isolated)
Note: The entry 3 was found the most suitable reaction condition for the fluorination of compound 5.
Scheme 3. Synthesis of FMCA and FMCAP from common intermediate 10
NH2
N (Boc)2
N
NH2
N
N
N
N
N
N
N
O
H
N
O
P
N
N
N
O
O
N
O
O
HO
(a)
(c)
(b)
CH3
O
F
F
F
F
61%
OH
74%
80%
O
CH3
HO
(FMCAP)
O
O
HO
(FMCA)
O
14
12
11
10
O
HN
13
P
Cl
OPh
Reagents and Conditions: (a) N, N-diBoc-adenine, DIAD, TPP, THF; (b) TFA, DCM; (c)
Compound 13, NMI, THF.
TBDMS deprotection of compound 9 was accomplished by tetrabutylammonium fluoride
(TBAF). Compound 9 was treated with a 2M solution of TBAF in THF at room temperature to
give the key intermediate compound 10 in 83% yield. The compound 10 was condensed with N,
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N-diBoc protected adenine under Mitsunobu coupling conditions using diisopropyl
azodicarboxylate (DIAD) and triphenylphosphine (TPP) in THF to give 11 in 74% yield (scheme
3). The tert-butyl, and Boc protecting groups of compound 11 were removed by using 2M
trifluoroacetic acid (TFA) in DCM at room temperature, affording the nucleoside 12 (FMCA) in
80% yield. The phosphoramidate prodrug (FMCAP) was synthesized by condensing FMCA with
compound 13. Compound 13 was furnished by treating phenyl dichlorophosphate with L-alanine
isopropyl ester in DCM at -78 ºC. Finally, FMCA was treated with 13 in the presence of N-
methyl imidazole (NMI) in THF at room temperature to obtain the target compound 14 in 61%
yield.
10
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CONCLUSION
To determine the full biological evaluation of FMCA and FMCAP, an efficient and scalable
synthetic method of FMCA has been developed via commercially available ketone 1. The
selective opening of acetonide of compound 2 followed by the allylic protection of 3 gave
compound 5. Fluorination of compound 5, and subsequent deprotection of TBDMS yielded the
key intermediate 10. Mitsunobu coupling with Boc-protected adenine followed by the
deprotection afforded the target compound 12 (FMCA) in 7 steps with approximately 8.9 %
overall yield via TBDMS protected compound 5. This process may serve as more efficient than
the previously reported method for scalable synthesis of FMCA. Further coupling of
phosphorochloridate 13 with FMCA produced phosphoramidate prodrug 14 (FMCAP). The
reduction of synthetic steps in comparison to the previous method, and the use of economical
reagents make this methodology amenable for a large scale preparation of FMCA.
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EXPERIMENTAL SECTION
5
6
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General Analytical Methods
8
9
Reagents and anhydrous solvents were purchased, and used without further purification.
Reactions were monitored by thin-layer chromatography plates (TLC silica gel GF 250 microns)
that were visualized using a UV lamp (254 nm), and developed with 15% solution of sulfuric
acid in methanol. Melting points were recorded on a digital melting point apparatus, and are
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1
uncorrected. Nuclear magnetic spectra were recorded on 500 MHz for H NMR, 470 MHz for
19F NMR and 125 MHz for 13C NMR with tetramethylsilane as an internal standard. CFCl3
19
(trichloro-fluoro methane was used as the internal standard (reference) for F NMR. Chemical
shifts (δ) are quoted as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (double doublet) and dt (double triplet). Optical rotations were measured on a
digital polarimeter. ESI high resolution mass spectra were recorded on a Q-TOF mass
spectrometer. Thin layer chromatography was performed on a glass plate coated with silica gel.
(3aS,4S,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-5-methylenetetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (2). To a stirred suspension of 1 (50.0 g, 206.6 mmol), and
paraformaldehyde (12.4 g, 413.2 mmol) in dry THF, diisopropyl ammonium trifluoroacetate
(44.4 g, 206.6 mmol), and diisopropylamine (29.0 mL, 206.6 mmol) were added. The suspension
was refluxed for 2 h. After that the mixture was cooled to room temperature, and additional
portion of paraformaldehyde (12.4 g, 413.2 mmol) was added. The reaction mixture was again
refluxed for 12 h. The mixture was concentrated under reduced pressure, and residue was diluted
with 1 L of ethyl acetate. The organic layer was washed with water (400 mL X 3), and dried over
Na2SO4, and concentrated in vacuo to afford the crude product that was used as such for the next
step. The crude material (52.0 g, 200.7 mmol) was dissolved in anhydrous methanol (500 mL),
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CeCl3.7H2O (98.7 g, 265.0 mmol) was added at -78 ºC, and stirred for 20 minutes. Then NaBH4
(9.7 g, 256.9 mmol) was added portion wise to the mixture. After 20 minutes stirring at the same
temperature, the reaction mixture was warmed to 0 ºC, and stirred for 30 minutes. A saturated
solution of NH4Cl (200 mL) was added, and stirred for additional 1 h. Excess organic solvent
was removed under reduced pressure, and 10% aqueous acetic acid solution (100 mL) was
added. The aqueous layer was extracted with ethyl acetate (200 mL X 2); combined organic
extracts were washed with brine (200 mL X 2), dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (5%
EtOAc/hexane) to give compound 2 as a white solid. (Yield 27 g, 52 % overall yield in 2 steps).
Mp 58-60 °C; []24D = -118.2° (c 0.42, CHCl3); 1H NMR (500 MHz, CDCl3)  5.25 (s, 1H), 5.11
(s, 1H), 4.50-4.53 (m, 3H), 3.46 (dd, J = 3.5 & 8.5 Hz, 1H), 3.27 (dd, J = 3.5 & 8.5 Hz, 1H), 2.63
10
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(t, J = 3.5 Hz, 1H), 2.25 (d, J = 11.0 Hz, 1H), 1.40 (s, 3H), 1.34 (s, 3H), 1.12 (s, 9H); C{1H}
NMR (125 MHz, CDCl3)  153.9, 110.2, 109.3, 81.6, 79.3, 73.8, 72.7, 64.3, 49.9, 27.3, 26.5,
24.7; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C14H24O4Na 279.1572; Found 279.1577.
(1S,2R,3S,4R)-3-(tert-Butoxy)-4-(tert-butoxymethyl)-5-methylenecyclopentane-1,2-diol (3).
Compound 2 (40.0 g, 156.2 mmol) was dissolved in 500 mL of DCM, and cooled to -78 ºC.
Trimethylaluminum solution (2M solution in hexane, 986.0 mL, 1562.5 mmol) was added, and
mixture was stirred at ambient temperature for 72 h. The mixture was cooled to -78 ºC, and
saturated ammonium chloride solution (200 mL) was added dropwise. The mixture was passed
through a celite bed, and was thoroughly washed with dichloromethane (250 mL X 2). Filtrate
was dried over Na2SO4, concentrated under reduced pressures. The crude was purified by silica
gel column chromatography (10% EtOAc/hexane) gave compound 3 as off-white solid. Yield
(32.4 g, 76 %). Mp 62-65 °C; []24D = -70.24 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.34
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(s, 1H), 5.13 (s, 1H), 4.26 (d, J = 10.0 Hz, 1H), 4.09 (s, 1H), 3.95-3.92 (m, 1H), 3.46 (dd, J = 4.0
& 8.5 Hz, 1H), 3.37 (dd, J = 5.0 & 8.0 Hz, 1H), 2.88 (d, J = 2.0 Hz, 1H), 2.65 (bs, 1H), 2.50 (d, J
= 11.0 Hz, 1H), 1.27 (s, 9H), 1.17 (s, 9H); 13C{1H} NMR (125 MHz, CDCl3)  152.4, 150.0,
109.9 (d, J = 6.0 Hz), 75.8, 74.9, 72.4, 62.2, 48.8, 28.5, 27.5; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C15H28O4Na 295.1885; Found 295.1882.
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(1S,2S,3R,5S)-2-(tert-Butoxy)-3-(tert-butoxymethyl)-5-((tert-butyldiphenylsilyl)oxy)-4-
methylenecyclopentan-1-ol (4). Compound 3 (20.0 g, 73.5 mmol), and imidazole (20.0 g, 294.1
mmol) were dissolved in anhydrous DCM (250 mL) at 0 ºC, and mixture was stirred for 15
minutes. tert-Butyldiphenylsilyl chloride (28.7 mL, 110.2 mmol) was added, and stirring was
continued for 4 h at room temperature. To the mixture 300 mL of water was added, and
separated organic layer was dried over Na2SO4, concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (3% EtOAc/Hexane) to give 4 as oil.
Yield: (34.6 g, 92%). []24D = -16.42 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.72 (d, J =
7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.35-7.28 (m, 6H), 5.11 (s, 1H), 4.93 (s, 1H), 4.28 (s, 1H),
3.73 (s 1H), 3.49 (s, 1H), 3.28-3.26 (m, 2H), 2.63 (s, 1H), 2.49 (bs, 1H), 1.06 (s, 9H), 1.04 (s,
9H), 0.96 (s, 9H); 13C{1H} NMR (125 MHz, CDCl3)  151.6, 135.8 (d J = 12.8 Hz), 135.8,
135.3, 134.8, 134.1, 133.6, 129.6, 127.7 (d, J = 20.0 Hz), 74.1, 72.0, 61.8, 28.4, 27.4, 26.9, 26.6,
19.4, 19.0, 14.1; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C31H46O4SiNa 533.3063; Found
533.3059.
(1S,2S,3R,5S)-2-(tert-Butoxy)-3-(tert-butoxymethyl)-5-((tert-butyldimethylsilyl)oxy)-4-
methylenecyclopentan-1-ol (5). Compound 3 (15.0 g, 55.1 mmol), and imidazole (15.0 g, 220.4
mmol) were dissolved in anhydrous DCM (300 mL), and the mixture was stirred for 15 minutes
at 0 ºC. tert-Butyldimethylsilyl chloride (13.2 g, 88.2 mmol) was added, and stirring was
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continued for 3 h at room temperature. The reaction mixture was diluted with water (300 mL),
and separated organic layer was dried over Na2SO4, concentrated under reduced pressure. The
crude was purified by silica gel column chromatography (4% EtOAc/Hexane) to give 5 as oil.
Yield: (19.5 g, 91 %). []24D = -59.42 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 4.99 (d, J =
2.0 Hz, 1H), 4.88 (s, 1H), 4.20 (t, J = 3.5 & 1.0 Hz, 1H), 3.87 (t, J = 7.5 & 3.5 Hz, 1H), 3.71 (s,
1H), 3.33-3.30 (m, 2H), 2.53 (s, 1H, OH), 2.46 (bs, 1H), 1.16 (s, 9H), 1.01 (s, 9H), 0.82 (s, 9H),
0.01 (s, 6H); 13C{1H} NMR (125 MHz, CDCl3)  156.6, 112.2, 78.9 (d, J = 18.6 Hz), 76.9, 76.0
(d, J = 20.0 Hz), 66.3, 52.6 (d, J = 20 Hz), 33.4 (d, J = 18.1 Hz), 32.4 (d, J = 14.7 Hz), 30.7 (d, J
= 12.5 Hz), 23.2, 0.13; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C21H43O4Si 387.2931; Found
387.2929.
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(((1S,2R,3S,4R)-3-(tert-Butoxy)-4-(tert-butoxymethyl)-2-fluoro-5-methylenecyclopentyl)
oxy)(tert-butyl)diphenylsilane (8). To a solution of compound 4 (20.0 g, 39.2 mmol) in
anhydrous DCM (250 mL), DAST (36.4 mL, 274.4 mmol) was added dropwise at -30 °C. The
mixture was warmed to room temperature, and stirred for 30 min. The mixture was cooled to -30
°C, and quenched with ice cold water. The aqueous layer was extracted with DCM (200 mL X
2). The combined organic extracts were washed with brine (100 mL X 2), and finally with water
(100 mL), dried over Na2SO4, concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (1% EtOAc/hexane) to give 8 as yellowish oil. Yield (7.2 g,
1
36 %). []24 = -30.17 (c 1.0, CHCl3); H NMR (500 MHz, CDCl3) δ 7.75-7.73 (m, 4H), 7.46-
D
7.38 (m, 6H), 5.12 (s, 1H), 4.94 (s, 1H), 4.68 (dt, J = 50.0 & 75.0 Hz, 1H), 4.64-4.60 (m, 1H),
4.04-3.99 (m, 1H), 3.42 (dd, J = 4.0 & 8.0 Hz, 1H), 3.32 (dd, J = 4.0 & 8.5 Hz, 1H), 2.50 (bs,
1H), 1.22 (s, 9H), 1.15 (s, 9H), 1.06 (s, 9H); 19F NMR (470 MHz, CDCl3)  -188.8 (dt, J = 16.5
& 53.1 Hz, 1F); 13C{1H} NMR (125 MHz, CDCl3)  148.1 (d, J = 9.5 Hz), 135.9 (d, J = 10.0
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Hz), 134.1, 133.6, 129.5 (d, J = 10. 0 Hz), 127.4, 109.1, 104.1, 73.9, 72.2, 61.9, 48.3, 31.6, 28.8,
27.3, 27.0, 22.6, 19.5; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C31H45FO3SiNa 535.3020;
Found 535.3017. The two polar compounds 6, and 7 formed in this reaction, were purified, with
an elevated polarity of eluent to 10-20%, EtOAc/hexane gave the purified compound 6 (2.39g,
24%), and 7 (1.41g, 20%) as an oil.
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(3R, 4R)-4-(tert-Butoxy)-3-(tert-butoxymethyl)-2-methylenecyclopentan-1-one (6). H NMR
(500 MHz, CDCl3) δ 6.02 (s, 1H), 5.33 (s, 1H), 4.07 (q, J = 6.0 &12.0 Hz, 1H), 3.45 (m, 2H),
2.76 (bs, 1H), 2.62 (dd, J = 7.0 &18.5 Hz, 1H), 2.26 (dd, J = 6.0 & 18.0 Hz, 1H), 1.13 (s, 9H),
1.10 (s, 9H); 13C{1H} NMR (125 MHz, CDCl3)  205.1, 1.45.4, 118.4, 74.2, 72.8, 68.3, 61.1,
50.4, 47.5, 28.6 (d, J = 15.2 Hz), 27.5; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C15H27O3
255.1960; Found 255.1956.
(R)-4-(tert-Butoxymethyl)-5-methylenecyclopent-2-en-1-one (7). 1H NMR (500 MHz, CDCl3)
δ 7.70 (d, J = 6.0 Hz, 1H), 6.44 (d, J = 5.5 Hz, 1H), 6.15 (s, 1H), 5.59 (s, 1H), 3.56 (bs, 2H), 3.37
13
(bs, 1H), 1.22 (s, 9H); C{1H} NMR (125 MHz, CDCl3)  196.8, 160.0, 143.6, 135.9, 117.8,
73.5, 64.1, 45.9, 27.7; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C11H17O2 181.1229; Found
181.1224.
(((1S,2R,3S,4R)-3-(tert-Butoxy)-4-(tert-butoxymethyl)-2-fluoro-5-methylenecyclopentyl)
oxy)(tert-butyl)dimethylsilane (9). To a solution of compound 5 (15.0 g, 38.8 mmol) in
anhydrous DCM (200 mL), DAST (35.8 mL, 272.0 mmol) was added dropwise at -30 °C. The
mixture was warmed to room temperature with stirring for 10 min. The mixture was cooled to -
30 °C, and quenched with ice cold water. The aqueous layer was extracted with DCM (200 mL
X 2). The combined organic extracts were dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (1.5% EtOAc/hexane)
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to give 9 as oil. Yield (9.3 g, 62.0 %). []24 = -68.32 (c 1.0, CHCl3); H NMR (500 MHz,
D
5
6
7
8
CDCl3) δ 4.99 (s, 1H), 4.84 (s, 1H), 4.39 (d, J = 4.5 Hz, 1H), 4.32 (dt, J = 8.0 & 45.0 Hz, 1H),
3.97 (dt, J = 6.0 & 17.0 Hz, 1H), 3.37 (dd, J = 4.0 & 9.0 Hz, 1H), 3.29-3.26 (m, 1H), 2.34 (bs,
1H), 1.10 (s, 9H), 1.04 (s, 9H), 0.82 (s, 9H), 0.00 (s, 6H); 19F NMR (470 MHz, CDCl3) δ -190.5
(dt, J = 16.5 & 52.6 Hz, 1F); 13C{1H} NMR (125 MHz, CDCl3)  153.4 (d, J = 10.5 Hz), 112.8,
108.3 (d, J = 191.0 Hz), 81.6 (m), 78.5, 67.0 (t, J = 8.6 & 18.1 Hz), 53.1 (d, J = 22.9 Hz), 33.8
(d, J = 15.8 Hz), 32.5 (d, J = 14.4 Hz), 30.8 (dd, J = 10.5 & 31.4 Hz ), 23.3, 0.27, 0.26, 0.17,
0.08; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C21H41FO3SiNa 411.2707; Found 411.2702.
(1S,2S,3S,4R)-3-(tert-Butoxy)-4-(tert-butoxymethyl)-2-fluoro-5-methylenecyclopentan-1-ol
(10). To a solution of compound 9 (9.2 g, 23.7 mmol) in THF (150 mL), tetrabutylammonium
fluoride (TBAF, 1M solution in THF) (35.5 mL, 35.5 mmol) was added, and the mixture was
stirred at room temperature for 3 h. The solvent was removed under reduced pressure, and
obtained residue was dissolved in ethyl acetate (250 mL). The organic layer was washed with
water (200 mL X 2), and finally with brine solution (100 mL), dried over Na2SO4, and
concentrated under reduced pressure. The crude was purified by silica gel column
chromatography (4% EtOAc/hexane) to afford compound 10 as white foam. Yield (5.4 g, 83%).
[]24D = -76.69 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.33 (s, 1H, 7-H), 5.15 (s, 1H, 7-
H), 4.55 (dt, J = 5.5 & 53.5 Hz, 1H, 2-H), 4.53-4.51 (m, 1H, 1-H), 4.19-4.16 (m, 1H, 3-H), 3.49-
3.47 (m, 1H, 5-H), 3.42-3.40 (m, 1H, 5-H), 2.64 (bs, 1H, 4-H), 2.28 (d, J = 8.0 Hz, 1H, 1-OH),
1.26 (s, 9H, 3-OC(CH3)3), 1.19 (s, 9H, 5-OC(CH3)3); 19F NMR (470 MHz, CDCl3)  -190.6 (dt J
9
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= 13.1 & 53.1 Hz, 1F); C{1H} NMR (125 MHz, CDCl3)  150.0, 149.0 (d, J = 6.6 Hz), 102.2
(d, J =189.2 Hz), 74.7, 72.6, 62.0, 48.9, 28.6, 27.4; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C15H27FO3Na 297.1842; Found 297.1839.
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9-((1R,3R,4R)-3ʹ-tert-Butoxy-4ʹ-(tert-butoxymethyl)-2ʹ-fluoro-5ʹ-methylenecyclopentyl)-
N,N-diboc-9H-purin-6-amine (11). To a stirred solution of triphenylphosphine (4.78 g, 18.24
mmol), in THF (50 mL) at -10 °C, was added DIAD (3.68 g, 18.24 mmol) dropwise, and mixture
was stirred at this temperature for 30 minutes. N, N-diBoc-protected adenine (3.7 g, 10.9 mmol)
solution in THF (20 mL) was added, and the mixture was stirred for 30 min at 0 °C. Compound
10 (2.0 g, 7.29 mmol) in THF (10 mL) was then added dropwise, and the reaction mixture was
stirred at room temperature for 1.5 h. The reaction mixture was quenched with methanol, and
solvent was removed under reduced pressure. The crude residue was purified by silica gel
column chromatography (5% EtOAc/ hexane) to give 11 as white foam. Yield (3.2 g, 74%).
[]24D = -51.47 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.91 (s, 1H, 8-H), 8.24 (s, 1H, 2-
H), 5.97 (d, J = 30.5 Hz, 1H, 1ʹ-H), 5.32 (s, 1H, 7ʹ-H), 4.90 (dd, J = 9.0 & 52.5 Hz, 1H, 2ʹ-H),
4.77 (s, 1H, 7ʹ-H), 4.33 (d, J = 14.0 Hz, 1H, 3ʹ-H), 3.62-3.60 (m, 1H, 5ʹ-H), 3.54-3.50 (m, 1H, 5ʹ-
H), 2.85 (bs, 1H, 4ʹ-H), 1.47 (s, 18H, 6-N(Boc)2), 1.28 (s, 9H, 3ʹ-OC(CH3)3), 1.27 (s, 9H, 5ʹ-
OC(CH3)3); 19F NMR (470 MHz, CDCl3)  -191.1 (ddd, J = 14.1, 32.9 & 46.0 Hz, 1F); 13C{1H}
10
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NMR (125 MHz, CDCl3)  153.9, 152.0, 150.3 (d, J = 20.0 Hz), 150.0, 146.4, 145.3, 128.1,
110.8 (d, J = 219.8 Hz), 83.7, 75.7, 73.2, 62.6, 49.8, 28.2, 27.8, 27.5; HRMS (ESI-TOF) m/z: [M
+ H]+ Calcd for C30H47FN5O6 592.3510; Found 592.3509.
(+)-9-[(1′R, 2′R, 3′R, 4′R)-2′-Fluoro-3′-hydroxy-4′-(hydroxymethyl)-5′-methylene-
cyclopentan-1′-yl]adenine (FMCA, 12). To a solution of compound 11 (3.3 g, 5.58 mmol) in
DCM (30 mL), trifluoroacetic acid (6 mL) was added, and the mixture was stirred at ambient
temperature for 16 h. The excess solvent was removed under reduced pressure, and residual TFA
was co-evaporated three times with methanol. The residue was diluted with methanol, and
neutralized with 28% aqueous ammonia solution, concentrated under reduced pressure. The
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crude was purified by column chromatography on silica gel (6% Methanol/DCM) to give 12 as
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white solid. Yield (1.2 g, 80%). Mp 215-218 °C; []24 = +152.10° (c 0.5, MeOH); UV (H2O)
D
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
max 259.0 nm ( 14000, pH 2), 260.0 nm ( 15600, pH 7), 260.0 nm ( 15600, pH 11); 1H NMR
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(500 MHz, CD3OD) δ 8.26 (s, 1H, 8-H), 8.10 (d, J = 2.5 Hz, 1H, 2-H), 5.90 (d, J = 26.0 Hz, 1H,
1ʹ-H), 5.46 (s, 1H, 7ʹ-H), 4.96 (dt, J = 2.5 & 52.5 Hz, 1H, 2ʹ-H), 4.95 (s, 1H, 7ʹ-H), 4.44 (dd, J =
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6.0 & 14.0 Hz, 1H, 3ʹ-H), 3.88-3.82 (m, 2H, 5ʹ-H), 2.81 (bs, 1H, 4ʹ-H); F NMR (470 MHz,
13
CD3OD) δ -195.95 (ddd, J = 13.2, 26.3 & 39.5 Hz, 1F); C{1H} NMR (125 MHz, CD3OD) δ
156.0, 152.5, 149.9, 146.0, 141.1, (d, J = 5.3 Hz), 117.9, 111.7, 95.9 (d, J = 184.0 Hz), 72.9 (d, J
= 23.6 Hz), 61.7, 57.5 (d, J = 17.4 Hz), 51.0; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C12H15FN5O2 280.1210; Found 280.1216.
{[(1R,3R,4R)-3-(6-Amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)
methoxy](phenoxyphosphoryl amino} propionic Acid Isopropyl Ester (14). To a solution of
phenyl dichlorophosphate (1.0 mol equiv), and the L-alanine isopropyl ester hydrochloride (1.0
mol) in anhydrous DCM at -78 ºC, triethylamine (2.0 mol) was added dropwise, and mixture
was stirred at same temperature for 1 h. The reaction mixture was slowly warmed to room
temperature, and stirred for 2 h. The solvent was removed under reduced pressure, and crude
residue was re-suspended in anhydrous ether, and filtered through a celite bed under nitrogen.
The filtrate was concentrated in vacuo to give compound 13, which was used as such for next
step.
To a stirring suspension of compound 12 (0.5 g, 1.79 mmol) in anhydrous THF, N-
methylimidazole, NMI (0.9 mL, 10.7 mmol) was added at 0 ºC. The phosphorochloridate 13 (2.2
g, 7.1 mmol) in THF (10 mL) was added dropwise, and the mixture was stirred for 12 h at
ambient temperature. The volatiles were removed under reduced pressure, and the residue was
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purified by silica gel column chromatography (2% Methanol/DCM) to give the compound 14 as
off-white solid. Yield (0.55 g, 61%). Mp 56-60 °C; 1H NMR (500 MHz, CDCl3) δ d 8.29 (s, 1H),
7.84 (d, J = 24.5 Hz, 1H), 7.28-7.10 (m, 5H), 5.88 (d, J = 30.0 Hz, 1H), 5.80 (bs, 2H), 5.18 (d, J
= 9.0 Hz, 1H), 4.96-4.76 (m, 3H), 4.39-4.34 (m, 2H), 4.17- 4.04 (m, 2H), 3.90-3.88 (m, 2H),
3.00 (bs, 1H), 1.31 (d, J = 6.5 Hz, 3H), 1.16 (dd, J = 6.0, & 14.0 Hz, 6H); 19F NMR (470 MHz,
CDCl3) δ -192.9-193.0 (m, 1F); 31P NMR (CDCl3, 202 MHz): δ 2.84, 2.37; 13C{1H} NMR (125
MHz, CDCl3) δ 173.3, 155.4, 153.1, 150.5, 144.5, 140.9, 129.8, 125.2, 120.2, 118.7 112.3, 95.6
(J = 169.2 Hz), 73.8 (d, J = 15.0 Hz), 69.5, 66.9, 57.1, 50.2 (d, J = 103.0 Hz), 21.6, 21.0; HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C24H31FN6O6P 549.2027; Found 549.2026.
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ASSOCIATED CONTENT
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Supporting Information: Copies of H NMR, F NMR and C NMR spectra of compounds 3-
14. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Email:dchu@uga.edu
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