Synthesis and antiviral evaluation of cyclopentyl nucleoside phosphonates

Article abstract of DOI:10.1016/j.ejmech.2018.03.008

The synthesis of both 2?-hydroxy-3?-deoxy and 2?-deoxy-3?-hydroxy cyclopentyl nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine and guanine from a single precursor has been performed. The guanine containing analogues showed antiviral activity. Especially the 3?-deoxy congener 23 was active, displaying an EC₅₀ of 5.35 μM against TK⁺ VZV strain and an EC₅₀ of 8.83 μM against TK^? VZV strain, besides lacking cytotoxicity. However, the application of phosphonodiamidate prodrug strategy did not lead to a boost in antiviral activity.

Full text of DOI:10.1016/j.ejmech.2018.03.008

Accepted Manuscript
Synthesis and antiviral evaluation of cyclopentyl nucleoside phosphonates
Mengmeng Wang, Puneet Srivastava, Chao Liu, Robert Snoeck, Graciela Andrei,
Steven De Jonghe, Piet Herdewijn
PII:
S0223-5234(18)30243-5
10.1016/j.ejmech.2018.03.008
EJMECH 10273
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 January 2018
Revised Date: 28 February 2018
Accepted Date: 1 March 2018
Please cite this article as: M. Wang, P. Srivastava, C. Liu, R. Snoeck, G. Andrei, S. De Jonghe, P.
Herdewijn, Synthesis and antiviral evaluation of cyclopentyl nucleoside phosphonates, European Journal
of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.008.
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ACCEPTED MANUSCRIPT
Graphical Abstract
O
N
O
NH
NH2
O
B
phosphonodiamidate
prodrug
HO
P
O
HO
P
O
N
N
OH
no antiviral activity
OH
R2
R1
OH
TK+ VZV: EC50 of 5.35
TK- VZV: EC50 of 8.83
CC50 > 289
M
B=A, G, C, T
R1=H or OH
R2=OH or H
M

ACCEPTED MANUSCRIPT
Synthesis and Antiviral Evaluation of Cyclopentyl Nucleoside Phosphonates
Mengmeng Wang,† Puneet Srivastava,† Chao Liu,† Robert Snoeck,‡ Graciela Andrei,‡ Steven De Jonghe,‡
Piet Herdewijn*,†
†Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000 Leuven,
Belgium
‡Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat
49, 3000 Leuven, Belgium
ABSTRACT
The synthesis of both 2
with the natural nucleobases adenine, thymine, cytosine and guanine from a single precursor has been
performed. The guanine containing analogues showed antiviral activity. Especially the 3 -deoxy congener
23 was active, displaying an EC50 of 5.35 M against
M against TK+ VZV strain and an EC50 of 8.83
-hydroxy-3ʹ-deoxy and 2ʹ-deoxy-3 -hydroxy cyclopentyl nucleoside phosphonates
ʹ ʹ
ʹ
µ
µ
TK- VZV strain, besides lacking cytotoxicity. However, the application of phosphonodiamidate prodrug
strategy did not lead to a boost in antiviral activity.
Keywords
Carbocyclic nucleoside, nucleoside phosphonate, antiviral, prodrug.
1. Introduction
The synthesis of modified nucleosides is an ongoing research theme in antiviral drug discovery. A
prominent class of structurally modified nucleoside analogues are carbocyclic nucleosides, in which the
oxygen of the furanose ring of the natural nucleosides is replaced by a methylene group [1,2]. The
absence of the labile glycosidic bond makes carbocyclic nucleosides chemically and enzymatically more
stable than their natural counterparts. A number of biologically active carbocyclic nucleosides have been
isolated from natural sources. Examples include Aristeromycin and Neplanocin A (Figure 1), which are
both carbocyclic adenosine analogues [3], that act as inhibitors of S-adenosyl-L-homocysteine (SAH)
hydrolase and exhibit broad spectrum antiviral activity but with concomitant cytotoxicity on the host cell
[4,5]. It inspired medicinal chemists to synthesize novel carbocyclic nucleosides as selective antiviral
agents [6,7]. It led to the marketing approval of Abacavir [8] and Entecavir [9] as drugs for the treatment
of HIV- and HBV-infected patients, respectively.

ACCEPTED MANUSCRIPT
Figure 1. Examples of biologically active carbocyclic nucleosides.
The antiviral activity of these nucleoside analogues depends upon their intracellular metabolism within
virus-infected cells to form sequentially the mono-, di- and triphosphates. Nucleoside triphosphates are
the pharmacologically active species, as they are incorporated into a growing DNA or RNA strand
resulting in chain termination. The first phosphorylation step is usually the rate-limiting step in the
conversion to the nucleoside-5ʹ-triphosphate. The synthesis of a nucleoside phosphonate, in which the P-
O-CH2 group of the naturally occurring nucleoside monophosphate is replaced by a P-CH2-O moiety,
allows the first phosphorylation step to be bypassed. Nucleoside phosphonates are essentially nucleoside
monophosphate analogues, having the advantage of being chemically and metabolically stable. The
success of this motif is due to the fact that it is isopolar and isosteric with the phosphate group [10].
Hence, nucleoside phosphonates can undergo enzymatic phosphorylations furnishing the phosphonate
diphosphates, which act as analogues of the natural nucleoside triphosphates.
Despite the promising characteristics of carbocyclic nucleosides and nucleoside phosphonates as
antivirals, carbocyclic nucleoside phosphonates, combining the two aforementioned approaches, have
been described to a much lesser extent (Figure 2). A phosphonate analogue of carbocyclic brivudine
(compound 1) lacked antiviral activity against herpes viruses [11]. A carbocyclic nucleoside phosphonate
bearing a 4
′-ethynyl group (compound 2) displayed in vitro anti-HIV activity [12]. 2′-Deoxyguanosine-5ʹ-
phosphonate (compound 3) was endowed with cytotoxic activity against HEp-2 cells [13].
Figure 2. Known carbocyclic nucleoside phosphonates.
As only a limited number of carbocyclic nucleoside phosphonates are known, we embarked on the
synthesis of a series of 2 -deoxy-3 -hydroxy and 2 -hydroxy-3 -deoxy carbocyclic nucleoside
′
′
′
′
phosphonates with the natural nucleobases adenine, thymine, cytosine and guanine. These compounds
were evaluated for antiviral activity against different DNA viruses from the herpesvirus family.
2. Chemistry

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2.1. Synthesis of cyclopentyl nucleoside phosphonates with adenine and guanine bases. The synthesis of
both series of compounds is shown in Scheme 1. Reaction of the commercially available starting material
4 with 4,6-dichloro-5-formamidopyrimidine 5 and 2-amino-4,6-dichloro-5-formamidopyrimidine 6,
afforded the 6-chloro-purine and 2-amino-6-chloro-purine containing nucleosides 7 and 8, respectively
[14]. Alkylation of the secondary hydroxyl group under basic conditions afforded the protected
nucleoside phosphonates 9 and 10. Substitution of the chlorine of compound 9 by an amino group was
achieved by treatment of compound 9 with a 7N NH3 solution in MeOH, yielding the adenine derivative
11. Acidic deprotection of the isopropylidene moiety of 10 and 11 afforded compounds 12 and 13.
Reductive removal of a hydroxyl group under standard Barton deoxygenation conditions gave a mixture
of compounds 14/15 and 16/17 in a ratio of 5:3. These regioisomeric mixtures were used for the
following step without separation. Lewis acid-mediated cleavage of the phosphonate esters gave the
nucleoside phosphonic acids 18/19 and 20/21. At this stage of the synthesis, isomers 18 and 19 were
separated by reversed phase HPLC, whereas the 2-amino-6-chloropurine moiety of the mixture 20/21 was
converted to the guanine base by refluxing 20/21 in a 4N sodium hydroxide solution, affording the
guanine cyclopentyl phosphonates 22/23. The 2′-deoxy derivative 22 and the 3′-deoxy analogue 23 were
successfully separated by reversed phase HPLC. The assignment of the correct regiochemistry of isomers
18/19 and 22/23 was based on H-H COSY NMR spectroscopy. As a representative example, the key
COSY correlations for compounds 18 and 19 are shown in Figure 3. The observation of COSY
correlations of H-1
deoxy analogue. Correlations of H-1
deoxy regiochemistry to compound 19.
ʹ
with H-2a
ʹ
and H-2b
ʹ
, and H-4
ʹ
with H-3
ʹ
allowed to identify compound 18 as the 2
′-
ʹ
with H-2
ʹ
and H-4
ʹ
with H-3a
ʹ
and H-3b allowed to assign the 3′-
ʹ

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of adenine and guanine containing cyclopentyl nucleoside phosphonates. Reagents
and conditions: (a) 5 or 6, DIPEA, n-butanol, overnight, 64% for 7 and 66% for 8; (b) (iPrO)2POCH2OTf,
NaH, THF, 0 °C to rt, 20 min, 92% for 9 and 76% for 10; (c) 7N NH3 in MeOH, 65 °C, overnight, 70%;
(d) PTSA, MeOH, 5 h, 86% for 12 and 80% for 13; (e) (1) TCDI, DMAP, CH2Cl2, overnight; (2) AIBN,
Bu3SnH, toluene, reflux, 20 min, 70% for 2 steps (14:15 = ratio 5:3; 16:17 = ratio 5:3); (f) TMSBr,
CH3CN, rt, overnight, 65–80%; (g) for 20/21, 4N NaOH, reflux, 4 h, 77%.
Figure 3. H-H COSY correlations of compounds 18 and 19.
2.2. Synthesis of cyclopentyl nucleoside phosphonates with a thymine base. In the first step, reaction of
compound 4 with ethyl [(2E)-3-ethoxy-2-methylprop-2-enoyl] carbamate 24, was followed by an
intramolecular cyclization in aqueous ammonia yielding thymine derivative 25 (Scheme 2). Introduction
of the phosphonate moiety, reductive removal of the hydroxyl groups and final deprotection proceeded
analogously as in Scheme 1, affording a mixture of the 2
′-deoxy and 3′-deoxy thymine derivatives that
were separated by RP-HPLC, yielding pure compounds 30 and 31.
Scheme 2. Synthesis of thymine containing cyclopentyl nucleoside phosphonates. Reagents and
conditions: (a) (1) 24, dioxane, 100 °C, 3 h; (2) aq. NH4OH, 90 °C, overnight, 85%; (b)
(iPrO)2POCH2OTs, NaH, THF, 0 °C to rt, 10 h, 84%; (c) PTSA, MeOH, 3 h, 85%; (d) (1) TCDI, DMAP,
CH2Cl2, overnight; (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 85% for 2 steps (28:29 = ratio 5:3); (e)
TMSBr, CH3CN, rt, overnight, 85%.

ACCEPTED MANUSCRIPT
2.3. Synthesis of cyclopentyl nucleoside phosphonates with a cytosine base. Similarly as for the thymidine
congeners, the formation of the cytosine nucleobase is also a two-step, one-pot reaction that included the
addition of amine 4 to intermediate (E)-3-ethoxyacryloyl isocyanate 32 and a subsequent cyclization with
aq. ammonia furnishing uridine analogue 33 (Scheme 3). Insertion of the phosphonate functionality and
acidic cleavage of the acetonide protecting group yielded compound 35. Deoxygenation of compound 35
using standard Barton deoxygenation reaction conditions gave a mixture of compounds 36 and 37, which
was used for further reactions as a regioisomeric mixture. Prior to conversion of uracil to cytosine, the 3
ʹ-
hydroxyl (of compound 36) or the 2 -hydroxyl (of compound 37) were protected as a tert-
ʹ
butyldimethylsilyl ether. The resulting derivatives 38 and 39 were then treated with TIPSCl, followed by
treatment with an aqueous ammonia solution, yielding the protected cytosine derivatives 40 and 41.
Deprotection of the silyl group and cleavage of phosphonate esters gave access to the nucleoside
phosphonic acids 42 and 43, that were separated by reversed phase HPLC.
Scheme 3. Synthesis of cytosine containing cyclopentyl nucleoside phosphonates. Reagents and
conditions: (a) (1) 32, DMF, −15 to 0 °C, overnight; (2) aq. NH4OH, 90 °C, overnight, 80%; (b)
(iPrO)2POCH2OTs, NaH, THF, 0 to rt, 10 h, 92%; (c) PTSA, MeOH, 3 h, 85%; (d) (1) TCDI, DMAP,
CH2Cl2, overnight; (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 79% for 2 steps (36:37 = ratio 5:3); (e)
TBSCl, imidazole, DMF, rt, overnight, 80%; (f) (1) TIPSCl, DMAP, Et3N, CH3CN, rt, overnight; (2) aq.
NH4OH, 0.5 h, 70%; (3) Et3N·3HF, THF, rt, overnight, 80%; (g) TMSBr, CH3CN, rt, overnight, 79%.
2.4. Synthesis of the prodrugs of cyclopentyl nucleoside phosphonates containing a guanine base.
Because of the promising antiviral activity of the parent nucleoside phosphonates 22 and 23, they were
converted to their corresponding prodrugs. Initial attempts were focused on the synthesis of
phenoxyphosphonoamidate prodrugs with
applying standard reaction conditions [15] with a mixture of phenol and
using 2,2 -dithiodipyridine and triphenylphosphine as activating agents yielded the desired compounds in
very low yield. This was due to the low solubility of phosphonates 22 and 23 in pyridine as well as to the
L-alanine isopropylester as amino acid motif. However,
L
-alanine isopropyl ester HCl and
ʹ

ACCEPTED MANUSCRIPT
concomitant formation of the corresponding phosphonodiamidates. Therefore, we switched to the
synthesis of phosphonodiamidate rather than phenoxyphosphonoamidates (Scheme 4). In addition,
phenoxyphosphonoamidate prodrugs exist as diasteromeric mixtures due to the chirality of the
phosphorus atom, while phosphonodiamidates are isolated as single isomer. The phosphonodiamidate
prodrugs were prepared from the parent phosphonates (compounds 22 and 23) and
ester HCl using 2,2 -dithiodipyridine and triphenylphosphine as activating agents affording the desired
prodrugs 44 and 45 in moderate yields.
L-alanine isopropyl
ʹ
Scheme 4. Synthesis of cyclopentyl phosphonodiamidate prodrugs with a guanine base. Reagents and
conditions: (a) -alanine isopropyl ester HCl salt, Et3N, PPh3, pyridine, 60 °C, overnight, 32–36%.
L
3. Results and discussion
All cyclopentyl nucleoside phosphonates were evaluated for antiviral activity against different DNA
viruses from the Herpesviridae family. These included varicella-zoster virus (VZV, strains TK+ and TK−),
human cytomegalovirus (HCMV, strains AD-169 and Davis) and herpes simplex virus 1 and 2 (HSV-1
KOS, HSV-2 G, HSV-1 KOS ACVr). The antiviral assays are based on inhibition of virus-induced
cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts. Antiviral activity is
expressed as the EC50 or compound concentration required reducing virus-induced cytopathogenicity or
viral plaque formation by 50%. At the same time, the cytostatic activity of the compounds was also
investigated. The cytostatic concentration is calculated as the CC50, or the compound concentration
required to reduce cell proliferation by 50% relative to the number of cells in the untreated controls.
Alternatively, cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration
(MCC) or the compound concentration that caused a microscopically detectable alteration of cell
morphology. As can be derived from the data in Table 1, the adenine (compounds 18/19) and the cytosine
(compounds 42/43) containing congeners were completely devoid of antiviral activity. The 2
′-deoxy
thymidine analogue (compound 30) displayed weak antiviral activity, whereas the 3 -deoxy thymidine
′

ACCEPTED MANUSCRIPT
derivative (compound 31) completely lacked antiviral activity. The most promising antiviral activity
within the carbocyclic nucleoside phosphonates was found within the guanosine series. The 2
analogue (compound 22) showed weak antiviral activity against VZV, HCMV and HSV, with EC50
values in the 8-34 µM range. The 3 -deoxy guanosine analogue (compound 23) was especially active
′-deoxy
′
against VZV (EC50’s in the range of 5-8 µM), whereas its activity against HCMV and HSV was less
pronounced. The high polarity of the nucleoside phosphonates hampers their cellular permeability, which
has a negative impact on the antiviral activity. Phosphonoamidate prodrugs are well-known to increase
the antiviral activity of nucleoside phosphonates against HIV and HBV [16-19]. We applied this strategy
to compounds 22 and 23 by the synthesis of the corresponding phosphonodiamidate prodrugs 44 and 45,
respectively, to evaluate the phosphonoamidate prodrug strategy against VZV, HCMV and HSV. To our
surprise, both prodrugs completely lacked antiviral activity. This suggested that the diamidate prodrugs
are not recognized by specific cellular enzymes in HEL cells, which are necessary to release the
biologically active nucleoside phosphonate.
Table 1. Antiviral activity and cytotoxicity of cyclopentyl nucleoside phosphonates and their prodrugs
against HSV, HCMV and VZV in HEL cells
Compound
Antiviral activity EC50a (µM)
Cytotoxicity (µM)
VZV
HCMV
HSV
Cell
morphology
(MCC)b
HEL
Cell
growth
(CC50)c
HEL
TK+
VZV
strain
TK-
VZV
strain
AD-
169
Davis
strain
HSV-1
KOS
HSV-2
HSV-1
KOS ACVr
strain
G strain
strain
strain
258
>303
17.14
5.35
79
>303
>303
18.73
14.97
13.98
>247
>327
>327
24.65
>175
NDd
>303
>303
14.74
37.98
25.92
>247
>327
>327
13.54
>175
NDd
>303
>303
22.72
7.57
>303
132.04
33.94
57.92
92.27
>247
>327
>327
85.55
>175
0.29
>303
161.44
14.10
99.05
9.89
>303
>303
>289
>289
>247
>247
>327
>327
>175
>175
>444
>300
>391
>358
NDd
NDd
18
>303
8.38
19
>289
>289
69.42
NDd
NDd
NDd
22
23
8.83
23.14
>247
>327
>327
34.99
66.60
3.37
5.73
117.32
>247
>327
>327
34.99
>175
0.30
30
>247
>327
>327
29.79
>175
14.12
3.39
>247
>327
>327
34.99
>175
>88
31
42
43
>175
>175
>444
>300
>391
>358
44
45
Acyclovir
Brivudin
Ganciclovir
Cidofovir
0.021
NDd
NDd
NDd
NDd
0.045
0.045
3.18
>30
>30
NDd
NDd
6.46
4.83
0.019
2.27
3.91
0.79
0.98
1.75
aEffective concentration required to reduce virus plaque formation by 50%. bMinimum cytotoxic concentration that

ACCEPTED MANUSCRIPT
causes a microscopically detectable alteration of cell morphology. cCytotoxic concentration required to reduce cell
growth by 50%. dNot determined.
4. Conclusion
In this manuscript, the synthesis of eight cyclopentyl nucleoside phosphonates with a adenine, thymine,
cytosine and guanine base was described. Both 2′-deoxy and 3′-deoxy analogues were prepared. As the
guanosine congeners showed limited antiviral activity, the synthesis of their corresponding
phosphonodiamidate prodrugs was also effected. Both prodrugs were completely devoid of antiviral
activity, which is a rare exception to the general accepted theory that synthesis of phosphonoamidate
prodrugs of nucleoside analogues leads to an increase of antiviral activity. This is most likely due to the
fact that these phosphonoamidates are intracellularly not metabolized to the parent nucleoside
phosphonates.
5. Experimental section
5.1. General information
Moisture sensitive reactions were carried out under an argon atmosphere using oven-dried glassware. All
reagents and solvents were obtained from commercial sources and used as received. Meting points were
1
determined on a Buchi 530 melting point apparatus and were uncorrected. H, 13C and 31P NMR spectra
were recorded on a 300, 500 or 600 MHz Bruker Avance spectrometer and using tetramethylsilane as an
internal standard or referenced to the residual solvent signal and 85% H3PO4 for 31P NMR. 2D NMR (H-
COSY, HSQC, and HMBC) was used for the assignment of proton and carbon signals of intermediates
and final compounds. High-resolution mass spectra (HRMS) were recorded on a quadrupole orthogonal
acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were
infused at 3 µ
L min−1, and spectra were obtained in positive or negative ionization mode with a resolution
of 15 000 (fwhm) using leucine enkephalin as a lock mass. Column chromatography was carried out on
silica gel 60 Å, 0.035–0.070 mm (Acros Organics). Preparative high performance liquid chromatography
(HPLC) purifications were performed on a Phenomenex Gemini 110A column (C18, 10 µm, 21.2 mm ×
250 mm) using CH3CN/0.05 M TEAB buffer or H2O/CH3CN as eluent gradient. Purities of all of the
tested compounds were above 95% by HPLC analysis.
5.1.1. 9-[(1′R,2′S,3′R,4′S)-4′-Hydroxy-2′,3′-O-isopropylidene-cyclopentan-1′-yl]-6-chloro-9-H-purine (7)
[20]. To a solution of compound 4 (0.35 g, 2,02 mmol) and 4,6-dichloro-5-formamidopyrimidine (0.422
mg, 2.22 mmol) in n-butanol (20 mL) was added DIPEA (1.41 mL, 8.08 mmol). The mixture was
refluxed at 120 °C overnight. The solvent was removed under reduced pressure, and the residue was

ACCEPTED MANUSCRIPT
purified by column chromatography (1:1, hexane/EtOAc) to afford compound 7 (0.40 g, 64% yield) as a
1
yellowish amorphous solid. m.p. 134-136 °C. H NMR (500 MHz, CDCl3):
δ
8.77 (s, 1H, H-2), 8.58 (s,
), 4.75 (d, J = 5.4 Hz, 1H, H-3 ),
), 2.29 (d, J = 15.5 Hz, 1H, H-5b ), 1.54 (s, 3H,
CH3), 1.32 (s, 3H, CH3) ppm. 13C NMR (125 MHz, CDCl3):
151.7 (C-2), 151.4 (C-4), 151.0 (C-6),
145.7 (C-8), 131.6 (C-5), 111.8 [C(CH3)2], 87.0 (C-3
), 86.2 (C-2 ), 76.1 (C-4 ), 62.5 (C-1 ), 36.9 (C-5 ),
26.7 (CH3), 24.2 (CH3) ppm. HRMS: [M+H]+ calcd for C13H16N4O3Cl, 311.0905; found, 311.0903.
5.1.2. 9-[(1 R,2 S,3 R,4 S)-4 -Hydroxy-2 ,3 -O-isopropylidene-cyclopentan-1 -yl]-2-amino-6-chloro-9-H-
1H, H-8), 5.13 (d, J = 9.0 Hz, 1H, H-1
ʹ
), 4.93 (d, J = 5.5 Hz, 1H, H-2
ʹ
ʹ
4.58 (d, J = 4.8 Hz, 1H, H-4 ), 2.93-2.85 (m, 1H, H-5a
ʹ
ʹ
ʹ
δ
ʹ
ʹ
ʹ
ʹ
ʹ
′
′
′
′
′
′
′
′
purine (8). This compound was prepared as described for 7. Obtained from compound 4 (0.50 g, 2.89
mmol), 2-amino-4,6-dichloro-5-formamidopyrimidine (0.71 g, 3.46 mmol) and DIPEA (2.01 mL, 11.55
1
mmol) as a yellowish amorphous solid (0.62 g, 66% yield). m.p. 137-139 °C. H NMR (500 MHz,
DMSO):
1H, H-2
H-5 ), 2.11-2.07 (m, 1H, H-5
159.7 (C-2), 154.0 (C-4), 149.4 (C-6), 142.3 (C-8), 123.3 (C-5), 110.8 [C(CH3)2], 86.3 (C-3
), 74.8 (C-4 ), 59.7 (C-1 ), 36.9 (C-5
), 26.7 (CH3), 24.4 (CH3) ppm. HRMS: [M+H]+ calcd for
δ
8.25 (s, 1H, H-8), 6.92 (brs, 2H, NH2), 5.50 (d, J = 3.4 Hz, 1H, OH), 4.92 (dd, J = 1.8, 6.1 Hz,
), 4.73-4.70 (m, 1H, H-1 ), 4.56 (d, J = 6.1 Hz, 1H, H-3 ), 4.17 (m, 1H, H-4 ), 2.50 (1H overlap
), 1.41 (s, 3H, CH3), 1.23 (s, 3H, CH3) ppm. 13C NMR (125 MHz, DMSO):
), 84.5 (C-
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
ʹ
2ʹ
ʹ
ʹ
ʹ
C13H17N5O3Cl, 326.1014; found, 326.1011.
5.1.3. 9-[(1 R,2 S,3 R,4 S)-4 -Diisopropylphosphonomethoxy-2′,3′-O-isopropylidene-cyclopentan-1′-yl]-6-
′
′
′
′
′
chloro-9-H-purine (9). To a solution of compound 7 (0.40 g, 1.29 mmol) and triflate
diisopropylphosphonomethanol (0.55 g, 1.67 mmol) in anhydrous THF (10 mL) was added sodium
hydride (60% in mineral oil, 0.062 g, 1.54 mmol) at 0 °C. The mixture was stirred for 20 min at room
temperature. It was quenched with sat. aq. NH4Cl and was concentrated. The residue was partitioned
between H2O and EtOAc. The organic layer was washed with brine and dried over Na2SO4. After
filtration and concentration, the residue was purified by column chromatography (30:1, DCM/MeOH) to
1
afford compound 9 (0.58 g, 92% yield) as a white amorphous solid. H NMR (300 MHz, CDCl3):
δ
8.77
), 4.81 (d, J =
), 3.90-3.74 (m, 2H, PCH2),
), 1.53 (s, 3H, CH3), 1.36-1.29 (m, 15H, CH3) ppm.
151.9 (C-6), 151.8 (C-2), 150.9 (C-4), 144.8 (C-8), 131.6 (C-5), 112.2
), 85.3 (C-2
), 83.4 (C-3 ), 71.4 [CH(CH3)2], 71.3 [CH(CH3)2],
), 35.0 (C-5 ), 26.5 (CH3), 24.1(CH3), 24.0 (CH3), 23.9 (CH3),
18.1 ppm. HRMS: [M+H]+ calcd for C20H31N4O6ClP,
(s, 1H, H-2), 8.47 (s, 1H, H-8), 5.13 (d, J = 7.0 Hz, 1H, H-1
6.0 Hz, 1H, H-3 ), 4.79-4.70 [m, 2H, CH(CH3)2], 4.21 (d, J = 3.1 Hz, 1H, H-4
2.86-2.77 (m, 1H, H-5 ), 2.39 (d, J = 15.3 Hz, 1H, H-5
ʹ), 4.93 (d, J = 6.0 Hz, 1H, H-2ʹ
ʹ
ʹ
ʹ
ʹ
13C NMR (75 MHz, CDCl3):
δ
3
[C(CH3)2], 86.2 (d, JP,C = 10.6 Hz, C-4
′
ʹ
ʹ
64.5 (d, 1JP,C = 168.6 Hz, PCH2), 60.9 (C-1
ʹ
ʹ
23.9 (CH3) ppm.31P NMR (121 MHz, CDCl3):
δ
489.1664; found, 489.1661.

ACCEPTED MANUSCRIPT
5.1.4. 9-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-O-isopropylidene-cyclopentan-1′-yl]-2-
amino-6-chloro-9-H-purine (10). This compound was prepared as described for 9. Obtained from
compound 8 (0.35 g, 1.07 mmol) and triflate diisopropylphosphonomethanol (0.53 g, 1.61 mmol) as a
yellowish amorphous solid (0.41 g, 76%). m.p. 56-58 °C. 1H NMR (300 MHz, CDCl3):
8), 5.51 (brs, 2H, NH2), 4.86-4.81 [m, 2H, CH(CH3)2], 4.77-4.69 (m, 3H, H-1
and H-2
4.13 (m, 1H, H-4 ), 2.34-2.29 (m, 1H, H-5
), 3.88-3.71 (m, 2H, PCH2), 2.74-2.64 (m, 1H, H-5
158.9 (C-2), 153.6 (C-4), 151.0
δ
8.01 (s, 1H, H-
and H-3 ), 4.15-
), 1.48 (s,
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
3H, CH3), 1.32-1.27 (m, 15H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ
3
(C-6), 141.6 (C-8), 125.0 (C-5), 111.9 [C(CH3)2], 86.1 (d, JP,C = 10.9 Hz, C-4
′), 84.7 (C-2
ʹ
), 83.6 (C-3
ʹ),
1
71.2 [CH(CH3)2], 65.4 (d, JP,C = 169.4 Hz, PCH2), 59.9 (C-1
ʹ
), 34.9 (C-5
ʹ), 26.5 (CH3), 24.1 (CH3), 23.9
(CH3), 23.8 (CH3), 23.8 (CH3) ppm.31P NMR (121 MHz, CDCl3):
δ
18.2 ppm. HRMS: [M+H]+ calcd for
C20H32N5O6ClP, 504.1773; found, 504.1774.
5.1.5.
9-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-O-isopropylidene-cyclopentan-1′-
yl]adenine (11). A solution of compound 9 (0.50 g, 1.02 mmol) in methanolic ammonia (35 mL) was
heated at 65 °C in a sealed bomb overnight. The solvent was removed under reduced pressure, and the
residue was purified by column chromatography (20:1, DCM/MeOH) to afford compound 11 (0.34 g, 70%
1
yield) as a white amorphous solid. m.p. 60-61 °C. H NMR (300 MHz, CDCl3):
δ
8.36 (s, 1H, H-2), 8.16
(s, 1H, H-8), 6.19 (br, 2H, NH2), 5.08-5.05 (m, 1H, H-1
and CH(CH3)2], 4.17-4.14 (m, 1H, H-4
(m, 1H, H-5
), 1.53 (s, 3H, CH3), 1.36-1.28 (m, 15H, CH3) ppm. 31P NMR (121 MHz, CDCl3):
HRMS: [M+H]+ calcd for C17H29N5O6P, 430.1849; found, 430.1837.
ʹ), 4.91-4.89 (m, 1H, H-2ʹ), 4.80-4.73 [m, 3H, H-
3ʹ
ʹ
), 3.91-3.74 (m, 2H, PCH2), 2.82-2.73 (m, 1H, H-5
ʹ
), 2.39-2.33
18.2 ppm.
ʹ
δ
5.1.6.
9-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-dihydroxy-cyclopentan-1′-yl]adenine
(12). To the solution of compound 11 (0.30 g, 0.64 mmol) in MeOH (10 mL) was added PTSA (0.13 g,
0.70 mmol). The mixture was stirred at room temperature for 5 h. The solvent was removed under
reduced pressure, and the residue was purified by column chromatography (10:1, DCM/MeOH) to afford
1
compound 12 (0.24 g, 86% yield) as a white amorphous solid. m.p. 136-138 °C. H NMR (300 MHz,
CDCl3):
4.57-4.50 (m, 1H, H-1
2.92-2.84 (m, 1H, H-5
CDCl3):
18.9 ppm. HRMS: [M+H]+ calcd for C17H29N5O6P, 430.1849; found, 430.1837.
δ
8.15 (s, 1H, H-2), 7.93 (s, 1H, H-8), 6.28 (br, 2H, NH2), 4.81-4.68 [m, 3H, H-3
), 4.27-4.25 (m, 1H, H-2 ), 4.03-3.99 (m, 1H, H-4 ), 3.84-3.80 (m, 2H, PCH2),
), 2.16-2.08 (m, 1H, H-5
), 1.31-1.27 (m, 12H, CH3) ppm. 31P NMR (121 MHz,
ʹ and CH(CH3)2],
ʹ
ʹ
ʹ
ʹ
ʹ
δ
5.1.7. 9-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-dihydroxy-cyclopentan-1′-yl]-2-amino-
6-chloro-9-H-purine (13). Compound 13 was prepared as described for compound 12. Obtained from
compound 7 (0.31 g, 0.61 mmol) as a white amorphous solid (0.23 g, 80%). m.p. 95-96 °C. 1H NMR (300
MHz, CDCl3):
δ 7.88 (s, 1H, H-8), 5.97 (brs, 2H, NH2), 5.62 (brs, 1H, OH), 4.77-4.69 [m, 5H, CH(CH3)2

ACCEPTED MANUSCRIPT
and H-1
ʹ
and H-2
ʹ
and H-3ʹ], 4.29 (brs, 1H, OH), 4.01-3.96 (m, 1H, H-4ʹ), 3.89-3.74 (m, 2H, PCH2), 2.78-
2.71 (m, 1H, H-5
ʹ
), 2.24-2.20 (m, 1H, H-5
159.0 (C-2), 153.6 (C-4), 150.8 (C-6), 141.8 (C-8), 124.7 (C-5), 84.5 (d, 3JP,C = 12.6 Hz, C-4
), 74.5 (C-2
), 71.7 [CH(CH3)2], 71.6 [CH(CH3)2], 65.4 (d, 1JP,C = 170.8 Hz, PCH2), 59.2 (C-1
), 26.5 (CH3), 24.0 (CH3), 23.9 (CH3), 23.9 (CH3) ppm.31P NMR (121 MHz, CDCl3):
ʹ
), 1.33-1.30 (m, 12H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
), 75.6 (C-
δ
3
5
′
ʹ
ʹ
ʹ
ʹ), 32.9 (C-
δ
19.8 ppm.
HRMS: [M+H]+ calcd for C17H28N5O6ClP, 464.1460; found, 464.1456.
5.1.8. 9-[(1
9-[(1 R,2 R,4
solution of compound 12 (0.34 g, 0.8 mmol) and DMAP (30 mg, 0.24 mmol) in anhydrous CH2Cl2 (20
mL) was added 1,1 -thiocarbonyldiimidazole (0.28 g, 1.58 mmol) at room temperature. The mixture was
′
S,3
′
S,4
′
S)-4
′
-Diisopropylphosphonomethoxy-3
′
-hydroxy-cyclopentan-1′-yl]adenine (14) and
′
′
′
S)-4′-Diisopropylphosphonomethoxy-2
′
-hydroxy-cyclopentan-1
′
-yl]adenine (15). To
a
ʹ
stirred overnight. It was washed with brine, dried over Na2SO4, and concentrated under reduced pressure.
To the residue in toluene (20 mL) was added azobis(isobutyronitrile) (0.052 mg, 0.316 mmol) and
tributyltin hydride (0.85 mL, 3.16 mmol). The mixture was refluxed for 20 min. The solvent was removed
under reduced pressure, and the residue was purified by silica gel column chromatography (30:1,
DCM/MeOH) to afford a mixture of compounds 14 and 15 (0.23 g, 70%) as a white foam. 31P NMR (121
MHz, CDCl3):
δ
19.5, 18.9 ppm. HRMS: [M+H]+ calcd for C17H29N5O5P, 414.1900; found, 414.1898.
5.1.9. 9-[(1
9-H-purine (16) and 9-[(1
amino-6-chloro-9-H-purine (17). The mixture of compounds 16 and 17 (70%) was prepared as described
′
S,3
′
S,4
′
S)-4
′
-Diisopropylphosphonomethoxy-3
′
-hydroxy-cyclopentan-1
′
-yl]-2-amino-6-chloro-
′
R,2 R,4 S)-4 -Diisopropylphosphonomethoxy-2
′
′
′
′
-hydroxy-cyclopentan-1
′
-yl]-2-
for compounds 14 and 15. 31P NMR (121 MHz, CDCl3):
δ
19.9, 19.8 ppm. HRMS: [M+H]+ calcd for
C17H28ClN5O5P, 448.1510; found, 448.1510.
5.1.10. 9-[(1
′
S,3
′
S,4
′
S)-4
′
-Phosphonomethoxy-3′-hydroxy-cyclopentan-1
′-yl]adenine (18) and 9-
[(1 R,2 R,4 S)-4
′
′
′
′
-Phosphonomethoxy-2
′
-hydroxy-cyclopentan-1′
-yl]adenine (19). To a solution of a
mixture of compounds 14 and 15 (0.15 g, 0.36 mmol) in anhydrous CH3CN (5 mL) was added
bromotrimethylsilane (0.38 mL, 2.90 mmol) at 0 °C. The mixture was stirred at room temperature
overnight and quenched with 1.0 M TEAB buffer (1 mL). The organic solvent was removed under
reduced pressure and the water layer was lyophilized. The residue was first purified by silica gel column
chromatography (10:1:0 to 10:5:1, DCM/MeOH/1.0 M TEAB) to give the crude product. Further
purification using preparative reversed phase HPLC with a gradient of CH3CN in 0.05 M TEAB solution
(gradually ranging from 2% to 30% CH3CN) gave compounds 18 (57 mg, 48% yield) and 19 (39 mg, 32%
yield), both as a white amorphous solid.

ACCEPTED MANUSCRIPT
5.1.10.1. compound 18: m.p. 150-153 °C. 1H NMR (600 MHz, D2O):
2), 5.00-4.97 (m, 1H, H-1 ), 4.46-4.44 (m, 1H, H-3 ), 4.00-3.97 (m, 1H, H-4
PCH2), 2.77-2.72 (m, 1H, H-5 ), 2.32-2.28 (m, 2H, H-2 ), 2.04-2.00 (m, 1H, H-5
MHz, D2O):
154.8 (C-6), 151.5 (C-2), 148.2 (C-4), 140.4 (C-8), 117.9 (C-5), 86.5 (d, 3JP,C = 11.9 Hz, C-
), 74.2 (C-3 ), 38.0 (C-2 ), 35.7 (C-5
), 66.0 (d, 1JP,C = 156.8 Hz, PCH2), 51.4 (C-1 ) ppm. 31P NMR (121
14.3 ppm. HRMS: [M-H]- calcd for C11H15N5O5P, 328.0816; found, 328.0812.
δ
8.22 (s, 1H, H-8), 8.05 (s, 1H, H-
), 3.68 (d, J = 9.4 Hz, 2H,
) ppm. 13C NMR (150
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
4
′
ʹ
ʹ
ʹ
ʹ
MHz, D2O):
δ
5.1.10.2. compound 19: m.p. 170-172 °C. 1H NMR (600 MHz, D2O):
2), 4.72-4.70 (m, 1H, H-2 ), 4.67-4.65 (m, 1H, H-1 ), 4.27-4.23 (m, 1H, H-4
2.80-2.77 (m, 1H, H-3 ), 2.37-2.33 (m, 1H, H-5 ), 2.17-2.13 (m, 1H, H-3 ), 1.98-1.93 (m, 1H, H-5
13C NMR (150 MHz, D2O):
3JP,C = 11.7 Hz, C-4
), 74.8 (C-2
δ
8.35 (s, 1H, H-8), 8.15 (s, 1H, H-
), 3.46-3.44 (m, 2H, PCH2),
) ppm.
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
155.1 (C-6), 151.9 (C-2), 148.9 (C-4), 140.8 (C-8), 118.2 (C-5), 77.1 (d,
1
′
ʹ
), 66.9 (d, JP,C = 151.6 Hz, PCH2), 58.6 (C-1
ʹ
), 37.2 (C-3
ʹ
), 35.7 (C-5
ʹ
)
ppm. 31P NMR (121 MHz, D2O):
δ
15.9 ppm. HRMS: [M-H]- calcd for C11H15N5O5P, 328.0816; found,
328.0813.
5.1.11.
9-[(1
′
S,3
′
S,4
′
S)-4
′
′
-Phosphonomethoxy-3
′
-hydroxy-cyclopentan-1
′
-yl]-2-amino-6-chloro-9-H-
-yl]-2-amino-6-chloro-
purine (20) and 9-[(1
′
R,2
′R,4
S)-4 -Phosphonomethoxy-2
′
′-hydroxy-cyclopentan-1′
9-H-purine (21). The mixture of compounds 20 and 21 (71%) was prepared as described for 18 and 19,
butwithout separation for next step. HRMS: [M-H]- calcd for C11H14N5O5P, 362.0426; found, 362.0429.
5.1.12. 9-[(1
′
S,3
′
S,4
′
S)-4
′
-Phosphonomethoxy-3′-hydroxy-cyclopentan-1
′-yl]guanine (22) and 9-
[(1 R,2 R,4 S)-4
′
′
′
′
-Phosphonomethoxy-2
′
-hydroxy-cyclopentan-1′
-yl]guanine (23). To a solution of the
mixture of 20 and 21 (0.20 g, 0.54 mmol) in dioxane (20 mL) was added 4N NaOH (10 mL). The mixture
was refluxed for 4 h. It was neutralized by adding 4N HCl after cooling to room temperature. The organic
solvent was removed under reduced pressure and the water layer was lyophilized. The first purification by
silica gel flash chromatography and further purification by RP-HPLC followed the same method as
described for compounds 18 and 19. It furnished the pure compounds 22 (90 mg, 48%) and 23 (54 mg,
29%), both as a white amorphous solid.
1
5.1.12.1. compound 22: m.p. 185-187 °C. H NMR (500 MHz, D2O):
δ
7.73 (s, 1H, H-8), 4.51-4.45 (m,
1H, H-1
ʹ
), 4.01-3.95 (m, 1H, H-3
ʹ
), 3.81-3.75 (m, 1H, H-4
ʹ
), 3.46-3.43 (m, 2H, PCH2), 2.55-2.46 (m, 1H,
H-5 ), 2.05-2.00 (m, 2H, H-2
ʹ
ʹ
), 1.72-1.64(m, 1H, H-5
ʹ
) ppm. 13C NMR (125 MHz, D2O):
δ
158.8 (C-6),
), 66.4 (d,
13.2 ppm.
3
153.7 (C-2), 150.9 (C-4), 137.7 (C-8), 115.6 (C-5), 86.5 (d, JP,C = 10.5 Hz, C-4
1JP,C = 151.1 Hz, PCH2), 50.8 (C-1
′), 74.1 (C-3
ʹ
ʹ
), 37.6 (C-2
ʹ
), 35.9 (C-5
ʹ
) ppm. 31P NMR (121 MHz, D2O):
δ
HRMS: [M-H]- calcd for C11H15N5O6P, 344.0765; found, 344.0765.
1
5.1.12.2. compound 23: m.p. 220-222 °C. H NMR (500 MHz, D2O):
δ 7.81 (s, 1H, H-8), 4.53-4.49 (m,

ACCEPTED MANUSCRIPT
1H, H-2
H-5 ), 2.25-2.20 (m, 1H, H-3
MHz, D2O):
)), 74.6 (C-2
(121 MHz, D2O):
5.1.13. 1-[(1 R,2
ʹ
), 4.34-4.29 (m, 1H, H-1
ʹ
), 4.15-4.14 (m, 1H, H-4
ʹ
), 3.49-3.46 (m, 2H, PCH2), 2.66-2.60 (m, 1H,
) ppm. 13C NMR (125
ʹ
ʹ
), 1.98-1.93(m, 1H, H-5
ʹ
), 1.87-1.81 (m, 1H, H-3ʹ
δ
158.4 (C-6), 153.3 (C-2), 151.4 (C-4), 138.0 (C-8), 115.6 (C-5), 77.6 (d, 3JP,C = 13.4 Hz, C-
1
4
′
′
), 66.4 (d, JP,C = 153.5 Hz, PCH2), 60.3 (C-1
′), 37.2 (C-3
′
), 35.6 (C-5
′
) ppm. 31P NMR
δ
13.6 ppm. HRMS: [M-H]- calcd for C11H15N5O6P, 344.0765; found, 344.0768.
′
′
S,3 R,4 S)-4 -Hydroxy-2 ,3 -O-isopropylidene-cyclopentan-1 -yl]thymine (25). To a
′
′
′
′
′
′
solution of compound 4 (0.40 g, 2.31 mmol) in dioxane (30 mL) was added ethyl [(2E)-3-ethoxy-2-
methylprop-2-enoyl] carbamate 24 (0.46 g, 2.31 mmol). The mixture was heated at 100 °C for 3 h. The
solvent was removed under reduced pressure, and the residue was used for cyclization without further
purification. Aq. ammonia (20 mL) was added to the residue in a sealed bomb at 90 °C overnight. The
solvent was removed under reduced pressure, and the residue was purified by column chromatography
(30:1, DCM/MeOH) to afford compound 25 (0.56 g, 85%) as a white amorphous solid. m.p. 88-90 °C. 1H
NMR (300 MHz, CDCl3):
1H, H-2 ), 4.65 (d, J = 5.9 Hz, 1H, H-1
2.05-2.00 (m, 1H, H-5
), 1.89 (s, 3H, T CH3), 1.48 (s, 3H, CH3), 1.30 (s, 3H, CH3) ppm. 13C NMR (75
MHz, CDCl3): 164.3 (C-4), 151.2 (C-2), 140.3 (C-6), 111.6 [C(CH3)2], 110.8 (C-5), 87.2 (C-3 ), 84.3
(C-2 ), 76.1 (C-4 ), 66.1 (C-1 ), 37.8 (C-5
), 26.7 (CH3), 24.3 (CH3), 12.2 (T CH3) ppm. HRMS: [M+H]+
calcd for C13H19N2O5, 283.1288; found, 283.1288.
δ
10.0 (brs, 1H, NH), 7.31 (d, J = 1.1 Hz, 1H, H-6), 4.90 (dd, J = 2.1, 5.9 Hz,
ʹ
ʹ
), 4.56 (m, 1H, H-3 ), 4.33 (s, 1H, H-4 ), 2.76-2.66 (m, 1H, H-5 ),
ʹ
ʹ
ʹ
ʹ
δ
ʹ
ʹ
ʹ
ʹ
ʹ
5.1.14.
1-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-O-isopropylidene-cyclopentan-1′-
yl]thymine (26). Compound 26 was prepared as described for compound 9. Obtained from compound 25
(0.60 g, 2.13 mmol) and tosylate diisopropylphosphonomethanol (1.49 g, 4.25 mmol) as a yellowish
amorphous solid (0.82 g, 84%). m.p. 45-47 °C. 1H NMR (300 MHz, CDCl3):
1H, H-6), 4.88-4.83 (m, 1H, H-2 ), 4.79-4.71 (m, 3H, H-3 and CH(CH3)2), 4.67-4.65 (m, 1H, H-4
4.01 (m, 1H, H-1 ), 3.91-3.73 (m, 2H, PCH2), 2.67-2.57 (m, 1H, H-5 ), 2.13-2.05 (m, 1H, H-5 ),1.95 (s,
163.9 (C-4),
), 83.9 (C-3 ), 83.6
), 34.9 (C-5
δ
9.70 (brs, 1H, NH), 7.31 (s,
), 4.05-
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
3H, T CH3), 1.49 (s, 3H, CH3), 1.34-1.29 (m, 15H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ
3
150.9 (C-2), 138.2 (C-6), 112.2 [C(CH3)2], 110.8 (C-5), 85.8 (d, JP,C = 12.8 Hz, C-4
′
ʹ
(C-2
ʹ
), 71.2 [CH(CH3)2], 71.1 [CH(CH3)2], 65.5 (d, 1JP,C = 169.0 Hz, PCH2), 61.8 (C-1
ʹ
ʹ), 26.6
(CH3), 24.3(CH3), 23.9 (CH3), 23.9(CH3), 12.3 (T CH3) ppm. 31P NMR (121 MHz, CDCl3):
δ
18.9 ppm.
HRMS: [M+H]+ calcd for C20H34N2O8P, 461.2047; found, 461.2047.
5.1.15. 1-[(1
(27). Compound 27 was prepared as described for compound 12. Obtained from compound 27 (0.4 g,
1.18 mmol) as a white amorphous solid (0.3 g, 85%). m.p. 64-67 °C. 1H NMR (300 MHz, CDCl3):
10.4
(brs, 1H, NH), 7.31 (s, 1H, H-6), 5.07-4.71 [m, 5H, CH(CH3)2 and H-1 and H-2 and H-3 ], 4.28 (brs, 1H,
′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-dihydroxy-cyclopentan-1′-yl]thymine
δ
ʹ
ʹ
ʹ

ACCEPTED MANUSCRIPT
OH), 4.17 (brs, 1H, OH), 3.92-3.75 (m, 3H, H-4
1.68-1.61 (m, 1H, H-5
), 1.49 (s, 3H, CH3), 1.34-1.31 (m, 12H, CH3) ppm. 31P NMR (121 MHz, CDCl3):
19.8 ppm. HRMS: [M+H]+ calcd for C17H30N2O8P, 421.1734; found, 421.1735.
and PCH2), 2.75-2.64 (m, 1H, H-5 ), 1.89 (s, 3H, T CH3),
ʹ ʹ
ʹ
δ
5.1.16. 1-[(1
1-[(1 R,2 R,4
mixture of compounds 28 and 29 was prepared as described for compounds 14 and 15. Obtained from
′
S,3
′
S,4
′
S)-4
′
-Diisopropylphosphonomethoxy-3
′
-hydroxy-cyclopentan-1
′
-yl]thymine (28) and
′
′
′
S)-4′-Diisopropylphosphonomethoxy-2
′
-hydroxy-cyclopentan-1
′
-yl]thymine
(29). The
compound 27 (0.40 g, 1.18 mmol) as a white foam (0.34 g, 85%). 31P NMR (121 MHz, CDCl3):
δ 19.8,
19.6 ppm. HRMS: [M+H]+ calcd for C17H30N2O7P, 405.1785; found, 405.1783.
5.1.17. 1-[(1
′
S,3
′
S,4
′
S)-4
′
-Phosphonomethoxy-3′-hydroxy-cyclopentan-1
′-yl]thymine (30) and 1-
[(1 R,2 R,4 S)-4
′
′
′
′
-Phosphonomethoxy-2
′
-hydroxy-cyclopentan-1′
-yl]thymine (31). Compounds 30 and 31
were prepared as described for compounds 18 and 19. Obtained from compounds 28 and 29 (0.25 g, 0.61
mmol) as a white amorphous solid (0.17 g, 85%).
1
5.1.17.1. compound 30: m.p. 180-182 °C. H NMR (500 MHz, D2O):
δ
7.63 (s, 1H, H-6), 5.04-5.00 (m,
1H, H-1
H-5 ), 2.14-2.08 (m, 1H, H-2
ppm. 13C NMR (125 MHz, D2O):
10.2 Hz, C-4 ), 74.2 (C-3
), 68.3 (d, 1JP,C = 151.2 Hz, PCH2), 52.4 (C-1
ʹ
), 4.37-4.34 (m, 1H, H-3
), 2.06-2.01 (m, 1H, H-2
168.6 (C-4), 152.4 (C-2), 139.9 (C-6), 111.3 (C-5), 86.2 (d, JP,C
), 36.1 (C-2 ), 34.4 (C-5
13.4 ppm. HRMS: [M-H]- calcd for C11H16N2O7P, 319.0700;
ʹ
), 3.87-3.84 (m, 1H, H-4
ʹ
), 3.58-3.46 (m, 2H, PCH2), 2.57-2.51 (m, 1H,
ʹ
ʹ
ʹ
), 1.87 (s, 3H, T CH3), 1.80-1.74 (m, 1H, H-5
ʹ
)
3
δ
=
′
′
′
′
′), 11.4 (T
CH3) ppm. 31P NMR (121 MHz, D2O):
δ
found, 319.0697.
1
5.1.17.2. compound 31: m.p. 170-172 °C. H NMR (500 MHz, D2O):
overlap, H-1 ), 4.49-4.44 (m, 1H, H-2 ), 4.13 (brs, 1H, H-4 ), 3.42 (d, J = 9.4 Hz, 2H, PCH2), 2.58-2.52 (m,
1H, H-5 ), 2.28-2.24 (m, 1H, H-3 ), 1.87 (s, 3H, T CH3), 1.86-1.76 (m, 2H, H-3
(125 MHz, D2O):
73.5 (C-3
), 67.0 (d, 1JP,C = 151.1 Hz, PCH2), 61.1 (C-1
NMR (121 MHz, D2O):
13.5 ppm. HRMS: [M-H]- calcd for C11H16N2O7P, 319.0700; found, 319.0702.
δ 7.64 (s, 1H, H-6), 5.04-5.00 (1H
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
and H-5ʹ
) ppm. 13C NMR
3
δ
167.9 (C-4), 153.9 (C-2), 139.2 (C-6), 111.5 (C-5), 76.8 (d, JP,C = 11.5 Hz, C-4
′),
′
′
), 37.1 (C-3
′
), 34.5 (C-5
′
), 11.5 (T CH3) ppm. 31P
δ
5.1.18. 1-[(1′R,2′S,3′R,4′S)-4′-Hydroxy-2′,3′-O-isopropylidene-cyclopentan-1′-yl]uracil (33). To
a
solution of 3-ethoxyacrylic acid (2.0 g, 17.22 mmol) in DCM (70 mL) was added thionyl chloride (1.89
mL, 25.8 mmol) at 0 °C. The mixture was refluxed for 3 h and then concentrated to get the residue (E)-3-
ethoxyacryloyl chloride for next step. To a solution of the residue in toluene (10 mL) was added silver
cynate (3.89 g, 25.98 mmol). The mixture was refluxed for 30 min and cooled to room temperature. The
supernatant liquor was added to a solution of compound 4 (1.5 g, 8.66 mmol) in DMF (15 mL) at −15 °C.
The reaction was stirred at −15 °C for 2 h and then stirred at room temperature overnight. The solvent

ACCEPTED MANUSCRIPT
was removed under reduced pressure and the residue was used for cyclization without further purification.
Aq. ammonia (20 mL) was added to the residue in a sealed bomb at 90 °C overnight. The solvent was
removed under reduced pressure and the residue was purified by column chromatography (20:1,
1
DCM/MeOH) to afford compound 33 (2.2 g, 80%) as a white amorphous solid. m.p. 185-187 °C. H
NMR (600 MHz, DMSO-d6):
1H, H-5), 5.46 (d, J = 3.1 Hz, 1H, OH), 4.73-4.72 (m, 1H, H-2
2.1, 6.5 Hz, 1H, H-3 ), 4.04 (s, 1H, H-4
CH3), 1.21 (s, 3H, CH3) ppm. 13C NMR (150 MHz, DMSO-d6):
111.1 [C(CH3)2], 101.1 (C-5), 86.2 (C-3 ), 83.4 (C-2
24.6 (CH3) ppm. HRMS: [M+H]+ calcd for C12H17N2O5, 269.1131; found, 269.1125.
δ
11.26 (brs, 1H, NH), 7.81 (d, J = 8.0 Hz, 1H, H-6), 5.58 (d, J = 8.0 Hz,
), 4.68-4.65 (m, 1H, H-1 ), 4.43 (dd, J =
), 1.87-1.83 (m, 1H, H-5 ), 1.39 (s, 3H,
163.3 (C-4), 151.1 (C-2), 143.6 (C-6),
), 61.2 (C-1 ), 36.9 (C-5 ), 26.8 (CH3),
ʹ
ʹ
ʹ
ʹ
), 2.36-2.32 (m, 1H, H-5
ʹ
ʹ
δ
ʹ
ʹ
), 74.5 (C-4
ʹ
ʹ
ʹ
5.1.19.
1-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-O-isopropylidene-cyclopentan-1′-
yl]uracil (34). Compound 34 was prepared as described for compound 9. Obtained from compound 33
(0.40 g, 1.49 mmol) and tosylate diisopropylphosphonomethanol (1.0 g, 2.98 mmol) as a yellowish
1
amorphous solid (0.61 g, 92%). m.p. 56-58 °C. H NMR (300 MHz, CDCl3):
δ
9.63 (brs, 1H, NH), 7.56
), 4.80-4.66 (m, 4H,
), 3.87-3.72 (m, 2H, PCH2), 2.69-2.59 (m, 1H, H-5
),
), 1.49 (s, 3H, CH3), 1.35-1.30 (m, 12H, CH3) ppm. 31P NMR (121 MHz, CDCl3):
18.9 ppm. HRMS: [M+H]+ calcd for C17H32N2O8P, 446.18178; found, 447.1886.
(d, J = 8.1 Hz, 1H, H-6), 5.72 (dd, J = 2.3, 8.1 Hz, 1H, H-5), 4.94-4.89 (m, 1H, H-2
H-3 and H-1 and CH(CH3)2), 4.06-4.03 (m, 1H, H-4
2.09-2.03 (m, 1H, H-5
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
5.1.20.
(35). Compound 35 was prepared as described for compound 12. Obtained from compound 34 (0.5 g,
1.12 mmol) as a white foam (0.39 g, 85%). 1H NMR (300 MHz, CDCl3):
7.50 (d, J = 8.1 Hz, 1H, H-6),
1-[(1′R,2′S,3′R,4′S)-4′-Diisopropylphosphonomethoxy-2′,3′-dihydroxy-cyclopentan-1′-yl]uracil
δ
5.72 (d, J = 8.1 Hz, 1H, H-5), 4.93-4.85 (m, 1H, H-1
ʹ
), 4.80-4.70 (m, 2H, CH(CH3)2), 4.29-4.25 (m, 1H,
H-3
H-5
ʹ
ʹ
), 4.19-4.17 (m, 1H, H-4
), 1.74-1.70 (m, 1H, H-5
ʹ), 3.95-3.91 (m, 1H, H-2ʹ), 7.56 (d, J = 9.1 Hz, 2H, PCH2), 2.73-2.64 (m, 1H,
ʹ
), 1.35-1.31 (m, 12H, CH3) ppm. 13C NMR (75 MHz, CDCl3):
δ 163.6 (C-4),
3
152.0 (C-2), 142.4 (C-6), 103.4 (C-5), 84.5 (d, JP,C = 10.2 Hz, C-4
′
), 74.4 (C-3
ʹ), 72.0 [CH(CH3)2], 71.9
1
(C-2
ʹ
), 71.8 [CH(CH3)2], 66.1 (d, JP,C = 164.8 Hz, PCH2), 60.5 (C-1
ʹ
), 33.4 (C-5ʹ
), 24.3 (CH3) ppm. 31P
NMR (121 MHz, CDCl3):
407.1569.
δ
19.8 ppm. HRMS: [M+H]+ calcd for C16H28N2O8P, 407.1577; found,
5.1.21. 1-[(1
′
R,3
′
R,4
′
S)-4
′
-Diisopropylphosphonomethoxy-3′-hydroxy-cyclopentan-1′-yl]uracil (36) and
1-[(1 R,2 S,4 S)-4
′
′
′
′
-diisopropylphosphonomethoxy-2
′
-hydroxy-cyclopentan-1′
-yl]uracil (37). Compounds
36 and 37 were prepared as described for compounds 14 and 15. Obtained from compound 35 (0.1 g, 0.24
mmol) as a white foam (75 mg, 79%). HRMS: [M+H]+ calcd for C16H28N2O7P, 491.1628; found,
391.1628.

ACCEPTED MANUSCRIPT
5.1.22.
yl]uracil (38) and
cyclopentan-1 -yl]uracil (39). To a mixture of compounds 36 and 37 (0.30 g, 0.76 mmol) in DMF (10 mL)
1-[(1
′
R,3
′
R,4
′
S)-4
′
-Diisopropylphosphonomethoxy-3
′
-O-tert-butyldimethylsily-cyclopentan-1
′-
1-[(1
′
R,2 S,4 S)-4 -Diisopropylphosphonomethoxy-3
′
′
′
′-O-tert-butyldimethylsily-
′
was added imidazole (0.15 g, 2.31 mmol) and tert-butyldimethylchlorosilane (0.24 g, 1.61 mmol). The
reaction mixture was stirred at room temperature overnight. It was partitioned between H2O and EtOAc.
The organic layer was dried over Na2SO4 and concentrated. The residue was purified by column
chromatography (30:1, DCM/MeOH) to afford a mixture of compounds 38 and 39 (0.31 g, 80%) as a
white solid. HRMS: [M-H]- calcd for C22H42N2O7PSi, 505.2493; found, 505.2492.
5.1.23. 1-[(1
′
S,3
′
S,4
′
S)-4
′
-Phosphonomethoxy-3′-hydroxy-cyclopentan-1′-yl]cytosine (40) and 1-
[(1 R,2 R,4 S)-4
′
′
′
′
-Phosphonomethoxy-2
′
-hydroxy -cyclopentan-1′-yl]cytosine (41). To a solution of a
mixture of compounds 38 and 39 (0.25 g, 0.49 mmol) in CH3CN (15 mL) was added 2,4,6-
triisopropylbenzenesulfonyl chloride (0.375 g, 1.24 mmol) and Et3N (0.34 mL, 2.48 mmol). The mixture
was stirred at room temperature overnight. Aq. ammonia was added to the above solution when
compound 38 and 39 disappeared according to TLC analysis. The mixture was stirred for 0.5 h. The
solvent was removed under reduced pressure and the residue was purified by column chromatography
(20:1, DCM/MeOH). The product was used for the next step. To a solution of the product (0.20 g, 0.39
mmol) in THF (7 mL) was added triethylamine trihydrofluoride (0.13 mL, 0.79 mmol). The mixture was
stirred at room temperature overnight. TMSOMe (0.2 mL) was added to quench the reaction. The solvent
was removed under reduced pressure and the residue was partitioned between H2O and EtOAc. The
organic layer was dried over Na2SO4 and concentrated. The residue was purified by column
chromatography (10:1, DCM/MeOH) to afford a mixture of compounds 40 and 41 (0.125 g, 80%) as a
white solid. HRMS: [M+H]+ calcd for C16H29N3O6P, 390.1788; found, 390.1782.
5.1.24. 1-[(1
′
S,3
′
S,4
′
S)-4
′
-Phosphonomethoxy-3′-hydroxy-cyclopentan-1′-yl]cytosine (42) and 1-
[(1 R,2 R,4 S)-4
′
′
′
′
-Phosphonomethoxy-2
′
-hydroxy-cyclopentan-1′
-yl]cytosine (43). Compounds 42 and 43
were prepared as described for compounds 18 and 19. Obtained from compounds 40 and 41 (0.10 g, 0.25
mmol) as a white amorphous solid (63 mg, 79%).
5.1.24.1. compound 42: m.p. 158-160 °C. 1H NMR (500 MHz, D2O):
6), 6.09-6.07 (d, J = 7.53 Hz, 1H, H-5), 5.20-5.17 (m, 1H, H-1 ), 4.42-4.41 (m, 1H, H-3
1H, H-4 ), 3.71-3.68 (m, 2H, PCH2), 2.63-2.58 (m, 1H, H-5 ), 2.20-2.16 (m, 1H, H-2 ), 2.09-2.04 (m, 1H,
H-2 164.7 (C-4), 157.2 (C-2), 144.4 (C-6),
), 1.84-1.80 (m, 1H, H-5
) ppm. 13C NMR (125 MHz, D2O):
), 65.9 (d, 1JP,C = 156.8 Hz, PCH2), 53.9 (C-1
), 37.4
15.5 ppm. HRMS: [M-H]- calcd for C10H15N3O6P,
δ
7.89-7.88 (d, J = 7.56 Hz, 1H, H-
), 3.93-3.91 (m,
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
96.3 (C-5), 86.4 (d, 3JP,C = 12.1 Hz, C-4
′), 74.4 (C-3
′
′
31
(C-2
′
), 34.8 (C-5
′
) ppm. P NMR (121 MHz, D2O):
δ
304.0703; found, 304.0708.

ACCEPTED MANUSCRIPT
5.1.24.2. compound 43: m.p. 167-169 °C. 1H NMR (500 MHz, D2O):
6), 6.11-6.10 (d, J = 7.47 Hz, 1H, H-5), 5.04-5.00 (1H overlap, H-1 ), 4.51-4.47 (m, 1H, H-2
(m, 1H, H-4 ), 3.44-3.43 (d, J = 9.50 Hz, 2H, PCH2), 2.63-2.58 (m, 1H, H-5
),1.88-1.84 (m, 1H, H-3 ) 1.82-1.79(m, 1H, H-5
) ppm. 13C NMR (125 MHz, D2O):
(C-2), 143.8 (C-6), 99.5(C-5), 77.7 (d, 3JP,C = 11.9 Hz, C-4
), 74.8 (C-2
62.0 (C-1 ), 37.5 (C-3 ), 35.2 (C-5
) ppm. 31P NMR (121 MHz, D2O):
C10H15N3O6P, 304.0703; found, 304.0695.
δ 7.89-7.88 (d, J = 7.45 Hz, 1H, H-
ʹ
ʹ), 4.15-4.13
ʹ
ʹ), 2.31-2.27 (m, 1H, H-
3ʹ
ʹ
ʹ
δ
165.6 (C-4), 158.7
′
′
), 66.8 (d, 1JP,C = 151.3 Hz, PCH2),
δ
13.5 ppm.HRMS: [M-H]- calcd for
′
′
′
5.1.25.
cyclopentan-1
isopropyl ester HCl (68 mg, 0.4 mmol) in anhydrous pyridine (4 mL) was added Et3N (0.065 mL, 0.47
mmol). The mixture was stirred at 60 °C under nitrogen for 20 min. Another solution of 2,2
9-[(1
′
S,3
′
S,4
′
S)-4
′
-O-(N,N
′
-Bis(isopropyl-L-alaninate)methylphosphonodiamidate)-3
′
-hydroxy-
′
-yl]guanine (44). To a solution of compound 42 (30 mg, 0.067 mmol) and
L
-alanine
′-
dithiodipyridine (103 mg, 0.47 mmol) and PPh3 (123 mg, 0.47 mmol) in anhydrous pyridine (1 mL) was
made and stirred for 15 min to give a clear light yellow solution. This solution was added to the above
solution, and the combined reaction mixture was stirred at 60 °C for 12 h. The solvent was removed under
reduced pressure and the residue was partitioned between H2O and EtOAc. The organic layer was washed
with sat. NaHCO3 and brine and dried over Na2SO4. After filtration and concentration, the residue was
purified by column chromatography (10:1, DCM/MeOH). Further purification using preparative reversed
phase HPLC with a gradient of CH3CN in water (ranging from 40% to 90% CH3CN) gave compound 44
(14 mg, 36%) as a white amorphous solid. m.p. 90-92 °C. 1H NMR (500 MHz, DMSO-d6):
H-8), 6.47 (brs, 2H, NH2), 5.09 (s, 1H, OH), 4.91-4.84 (m, 3H, CH(CH3)2 and H-1
), 4.58-4.54 (m, 1H,
NH), 4.45-4.42 (m, 1H, NH), 4.18 (s, 1H, H-3 ), 3.87-3.79 (m, 2H, Ala-CH), 3.78-3.77 (m, 1H, H-4 ),
3.67-3.65 (m, 2H, PCH2), 2.50 (1H overlap H-5 ), 2.13-2.09 (m, 1H, H-2 ), 2.05-2.01 (m, 1H, H-2 ), 1.88-
1.84 (m, 1H, H-5
), 1.27 (dd, J = 2.4, 7.1 Hz, 6H, Ala-CH3 ), 1.20-1.16 (m, 12H, CH(CH3)2) ppm. 13C
NMR (125 MHz, DMSO-d6): 173.62, 173.60 (Ala-CO), 157.1 (C-6), 153.6 (C-2), 151.1 (C-4), 135.6
(C-8), 116.6 (C-5), 87.0 (3JP,C = 11.7 Hz, C-4 ), 66.0 (1JP,C = 135.3 Hz, PCH2),
), 73.6 (C-3 ), 67.8 (C-1
50.7 [CH(CH3)2], 48.3 (Ala-CH), 40.1 (C-2 ), 36.6 (C-5
), 21.5 [CH(CH3)2], 20.8 (Ala-CH3) ppm. 31P
NMR (121 MHz, D2O):
20.6 ppm. HRMS: [M+H]+ calcd for C23H39N7O8P, 572.2592; found, 572.2609.
δ 7.75 (s, 1H,
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
ʹ
ʹ
ʹ
ʹ
ʹ
δ
5.1.26.
cyclopentan-1
from compound 43 (24 mg, 0.053 mmol) as a white amorphous solid (10 mg, 33%). m.p. 115-118 °C. 1H
NMR (500 MHz, DMSO-d6): 7.74 (s, 1H, H-8), 6.42 (brs, 2H, NH2), 5.26 (s, 1H, OH), 4.92-4.86 (m,
2H, CH(CH3)2), 4.58-4.54 (m, 1H, NH), 4.50-4.44 (m, 1H, H-2 ), 4.43-4.40 (m, 1H, NH), 4.34-4.30(m,
1H, H-1 ), 4.07-4.05 (m, 1H, H-4 ), 3.87-3.80 (m, 2H, Ala-CH),3.67-3.65 (d, J = 8.5 Hz, 2H, PCH2), 2.50
9-[(1′R,2′R,4′S)-4′-O-(N,N′-Bis(isopropyl-L-alaninate)methylphosphonodiamidate)-2′-hydroxy-
′
-yl]guanine (45). Compound 45 was prepared as described for compound 44. Obtained
δ
ʹ
ʹ
ʹ

ACCEPTED MANUSCRIPT
(1H overlap H-5 ʹ), 1.99-1.95 (m, 1H, H-5ʹ), 1.79-1.75 (m, 1H, H-3ʹ), 1.28 (dd, J
ʹ), 2.09-2.05 (m, 1H, H-3
= 1.7, 7.1 Hz, 6H, Ala-CH3 ), 1.21-1.18 (m, 12H, CH(CH3)2) ppm. 13C NMR (125 MHz, DMSO-d6):
173.63, 173.60 (Ala-CO), 156.9 (C-6), 153.3 (C-2), 151.4 (C-4), 136.1 (C-8), 116.8 (C-5), 77.8 (3JP,C
δ
=
12.8 Hz, C-4
ʹ
), 73.9 (C-2
ʹ
), 67.8 [CH(CH3)2], 66.0 (1JP,C = 135.1 Hz, PCH2), 60.1 (C-1
ʹ), 48.3 (Ala-CH),
38.8 (C-3 ), 36.1 (C-5
ʹ
ʹ
), 21.5 [CH(CH3)2], 20.7 (Ala-CH3) ppm. 31P NMR (121 MHz, D2O):
δ
20.6 ppm.
HRMS: [M-H]- calcd for C23H37N7O8P, 570.2446; found, 570.2448.
5.2. Antiviral evaluation
The compounds were evaluated against the following viruses: herpes simplex virus 1 (HSV-1) strain KOS,
thymidine kinase-deficient (TK−) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus 2
(HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK− VZV strain 07−1, human
cytomegalovirus (HCMV) strains AD-169 and Davis. The antiviral assays are based on inhibition of
virus-induced cytopathicity (HSV and HCMV) or plaque formation (VZV) in human embryonic lung
(HEL) fibroblasts. Confluent cell cultures in microtiter 96-well plates are inoculated with 100 CCID50 of
virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units
(PFU) (VZV). After 2 hours of adsorption, the viral inoculum is removed and the cultures are further
incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque
formation is recorded after 2-3 (HSV), 5 (VZV) or 6-7 (HCMV) days post-infection. Antiviral activity is
expressed as the EC50 or compound concentration required inhibiting virus-induced cytopathicity or viral
plaque formation by 50%.
The cytostatic activity measurements are based on the inhibition of cell growth. HEL cells are seeded at a
rate of 5 x 103 cells/well into 96-well microtiter plates and allow proliferating for 24 hours. Then, medium
containing different concentrations of the test compounds is added. After 3 days of incubation at 37 °C,
the cell number is determined with a Coulter counter. The cytostatic concentration is calculated as the
CC50, or the compound concentration required reducing cell proliferation by 50% relative to the number
of cells in the untreated controls. CC50 values are estimated from graphic plots of the number of cells
(percentage of control) as a function of the concentration of the test compounds. Alternatively,
cytotoxicity of the test compounds is expressed as the minimum cytotoxic concentration (MCC) or the
compound concentration that causes a microscopically detectable alteration of cell morphology.
AUTHOR INFORMATION
Corresponding Author
*Phone: +32 16 322657. E-mail: Piet.Herdewijn@kuleuven.be.
Notes

ACCEPTED MANUSCRIPT
The authors declare no competing financial interest.
Acknowledgments
Mengmeng Wang acknowledges the China Scholarship Council (CSC) for funding (grant number:
201407040089). We thank Prof. Jef Rozenski (KU Leuven) for providing HRMS data. We thank Luc
Baudemprez for technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article was uploaded.
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
Highlights
ò The synthesis of eight cyclopentyl nucleoside phosphonates was carried out.
ò The 3ʹ-deoxy guanosine analogue displayed promising activity against VZV.
ò The 3ʹ-deoxy guanosine analogue lacked cytotoxicity.
ò The corresponding phosphonodiamidate prodrugs was devoid of antiviral activity.