Design, synthesis and anticancer activity of fluorocyclopentenyl-purines and – pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2018.06.003

Based on the potent anticancer activity of 6′-fluorocyclopentenyl-cytosine 2b in phase IIa clinical trials for the treatment of gemcitabine-resistant pancreatic cancer, we carried out a systematic structure-activity relationship study of 6′-fluorocyclopen

Full text of DOI:10.1016/j.ejmech.2018.06.003

Accepted Manuscript
Design, synthesis and anticancer activity of fluorocyclopentenyl-purines and –
pyrimidines
Ji-seong Yoon, Dnyandev B. Jarhad, Gyudong Kim, Akshata Nayak, Long Xuan
Zhao, Jinha Yu, Hong-Rae Kim, Ji Yun Lee, Varughese A. Mulamoottil, Girish
Chandra, Woong Sub Byun, Sang Kook Lee, Yong-Chul Kim, Lak Shin Jeong
PII:
S0223-5234(18)30493-8
10.1016/j.ejmech.2018.06.003
EJMECH 10473
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2018
Revised Date: 31 May 2018
Accepted Date: 1 June 2018
Please cite this article as: J.-s. Yoon, D.B. Jarhad, G. Kim, A. Nayak, L.X. Zhao, J. Yu, H.-R. Kim, J.Y.
Lee, V.A. Mulamoottil, G. Chandra, W.S. Byun, S.K. Lee, Y.-C. Kim, L.S. Jeong, Design, synthesis and
anticancer activity of fluorocyclopentenyl-purines and – pyrimidines, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.003.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Anticancer Activity of Fluorocyclopentenyl-purines and –
pyrimidines
Ji-seong Yoon,1 Dnyandev B Jarhad,1 Gyudong Kim,1 Akshata Nayak,1 Long
Xuan Zhao,2 Jinha Yu,1 Hong-Rae Kim,1 Ji Yun Lee,1 Varughese A.
Mulamoottil,1 Girish Chandra,1 Woong Sub Byun,1 Sang Kook Lee,1 Yong-Chul
Kim,3,* and Lak Shin Jeong1,*
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul
National University, Seoul, 151-742, Korea 2College of Chemistry and Chemical
Engineering, Liaoning Normal University, Dalian 116-029, China and 3School of
Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju,
500-712, Korea
Corresponding authors:
Lak Shin Jeong, Ph.D.
Research Institute of Pharmaceutical Sciences, College of Pharmacy
Seoul National University, Seoul 151-742, Korea, Tel) 82-2-880-7850
e-mail) lakjeong@snu.ac.kr
Yong-Chul Kim, Ph.D.
School of Life Sciences
Gwangju Institute of Science and Technology (GIST)
Gwangju, 500-712, Korea
e-mail) yongchul@gist.ac.kr
Key words: Fluorocyclopentenyl nucleoside; Structure-activity relationship;
Fluorination; Phosphorylation; Prodrug; Anticancer

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Abstract
Based on the potent anticancer activity of 6'-fluorocyclopentenyl-cytosine 2b in phase IIa
clinical trials for the treatment of gemcitabine-resistant pancreatic cancer, we carried out a
systematic structure-activity relationship study of 6'-fluorocyclopentenyl-pyrimidines 3a-i
and -purines 3j-o to discover novel anticancer agents. We also synthesized the
phosphoramidate prodrug 3p of adenine derivative 1b to determine if the anticancer activity
depended on the inhibition of DNA and/or RNA polymerase in cancer cells and/or on the
inhibition of S-adenosylhomocysteine (SAH) hydrolase. All of the synthesized pyrimidine
nucleosides exhibited much less potent anticancer activity in vitro than the cytosine
derivative 2b, acting as RNA and/or DNA polymerase inhibitor, indicating that they could
not be efficiently converted to their triphosphates for anticancer activity. Among all the
synthesized purine nucleosides, adenine derivative 1b and N6-methyladenine derivative 3k
showed potent anticancer activity, showing equipotent inhibitory activity as the positive
control, neplanocin A (1a) or Ara-C. However, the phosphoramidate prodrug 3p showed less
anticancer activity than 1b, indicating that it did not act as a RNA and/or DNA polymerase
inhibitor like 2b. This result also demonstrates that the anticancer activity of 1b largely
depends on the inhibition of histone methyltransferase, resulting from strong inhibition of
SAH hydrolase. The deamination of the N6-amino group, the addition of the bulky alkyl
group at the N6-amino group, or the introduction of the amino group at the C2 position almost
abolished the anticancer activity.

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1. Introduction
Neplanocin A (1a) [1], a naturally occurring nucleoside exhibits potent antiviral and
antitumor activities by inhibition of S-adenosylhomocysteine (SAH) hydrolase, which
catalyzes the interconversion of SAH into adenosine and
L-homocysteine (Figure 1) [2].
X
NH2
Y
N
N
N
F
X
N
O
N
N
HO
HO
HO OH
HO
OH
1a (X = H, Neplanocin A)
1b (X = F)
3a-i
X = OH or NH2
Y = F, Cl, Br, or I
SAR
NH2
X
N
N
N
F
X
N
N
N
O
Y
PO
HO
HO
OH
HO
OH
3j-p
2a (X = H)
2b (X = F)
X = NH2, NHMe, NH-cyclopropyl or OH
Y = H or NH2
P = H or phosphoramidate
Figure 1. The rationale for the design of target nucleosides 3a-p
Its 6'-fluoro analogue, fluoro-neplanocin A (1b) is also a potent inhibitor (IC50 = 0.48 µM) of
SAH hydrolase [3] and exhibits potent antiviral activity [3] and anticancer activity in
combination with 5-azacytidine [4]. On the other hand, the cyclopentenyl-cytosine (2a) [5]
exhibits potent antiviral and/or antitumor activities by reducing the cytidine-5'-phosphate
(CTP) pools and its 6'-fluoro analogue 2b also displays highly potent antitumor effects in a
broad range of tumor cell lines [6], but it shows different mechanisms of action [7] from that
of 2a. Compound 2b is intracellularly taken by a human equilibrative nucleoside transporter

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(hENT) and is phosphorylated by uridine-cytidine kinase (UCK) to its monophosphate and
subsequently to its triphosphate, which can be incorporated into RNA. Compound 2b is also
incorporated into DNA by the action of ribonucleotide reductase and/or it acts as a DNA
methyl transferase (DNMT) inhibitor [6] either by the incorporation of 2b into DNA by the
formation of a complex with the enzyme or directly by its triphosphate [7]. Compound 2b is
now undergoing phase IIa clinical trials for the treatment of gemcitabine-resistant pancreatic
cancer.
Inspired by the potent anticancer activity of 1b and 2b, it was of great interest to
synthesize novel 6'-fluorocyclopentenyl-pyrimidines 3a-i and –purines 3j-o for a systematic
structure-activity relationship (SAR) study and to evaluate them for anticancer activity. We
also synthesized the phosphoramidate prodrug 3p from the adenine analog 1b to determine if
the anticancer activity depended on the inhibition of SAH hydrolase or DNA and/or RNA
polymerase. Herein, we report the comprehensive structure-activity relationships of 6'-
fluorocyclopentenyl-pyrimidines 3a-i and -purines 3j-o and the phosphoramidate prodrug 3p
as novel antitumor agents.
2. Results and discussion
2.1. Chemistry
For the synthesis of the target nucleosides 3, the key fluoro intermediate 8a was first
synthesized from
D-ribose, as shown in Scheme 1. D-Ribose was converted to the key
intermediate 4, according to our previously published procedure [8]. The iodination of 4 with
iodine in the presence of pyridine afforded α-iodo compound 5 [9], which was reduced with
NaBH4 followed by the protection of the resulting alcohol 6 with the TBDPS group to yield 7
[9], which is the precursor for the α-fluorination. Treatment of 7 with N-
fluorobenzenesulfonimide (NFSI) in the presence of n-BuLi in the mixed solvents (THF,

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methyl tert-butyl ether (MTBE), heptane) at -78 °C yielded the desired 6-fluoro compound 8a
with concomitant formation of 6-H derivative 8b and TBDPS-migrated derivative 8c in a 2 :
1 : 1 ratio [3,9].
Scheme 1
I
I
TrO
TrO
TrO
[8]
b
a
O
D-Ribose
O
70%
OH
O
O
O
O
O
O
4
5
6
Tr =
76%
from 5
c
F
H
TBDPS
I
TrO
TrO
TrO
+
TrO
d
+
OTBDPS
O
OTBDPS
O
OH
O
OR
O
O
O
O
O
8a (45%)
8b (22%)
8c (18%)
7 (R = TBDPS)
o
Reagents and Conditions
: a) I2, pyridine, THF, rt, 4 h; b) NaBH4, CeCl3-7H2O, MeOH, 0 C, 30
o
min; c) TBDPSCl, imidazole, DMF, 40 oC, 12 h; d) NFSI, n-BuLi, THF/MTBE/heptane, -78 C, 30 min.
The key fluoro intermediate 8a was converted to the glycosyl donor 9 by treating it with
TBAF, which was subjected to the Mitsunobu condensation to synthesize the final pyrimidine
nucleosides 2b and 3a-i, as illustrated in Scheme 2. The condensation of 9 with N3-benzoyl-
pyrmidines under the standard Mitsunobu conditions followed by the removal of the N3-
benzoyl group, yielded the condensed products 10a-e. The treatment of 10a-e with 2 N HCl
afforded the final pyrimidine nucleosides 3a-e, respectively [6]. For the conversion of the
uracil derivatives 3a-e into the cytosine derivatives 2b and 3f-i, compounds 3a-e were
peracetylated to give 11a-e, which were treated with 1,2,4-triazole and POCl3 in the presence
of Et3N to give the N4-triazole derivatives 12a-e, respectively. Treatment of 12a-e with
ammonia water in 1,4-dioxane followed by the successive treatment with methanolic

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ammonia afforded the final cytosine derivatives 2b and 3f-i [6]. This conventional method
[10a] turned out to be superior to other methods using 2,4,6-triisopropylbenzenesulfonyl
chloride/NH3 [10b] or POCl3/NaN3/Pd-C [10c] in terms of overall yield.
The synthesis of the fluorocyclopentenyl-N6-substituted-purines 1b and 3j-l was
accomplished by condensing the glycosyl donor 9 with 6-chloropurine, as shown in Scheme 3.
The condensation of 9 with 6-chloropurine under the standard Mitsunobu conditions afforded
the 6-chloropurine derivative 13. To remove the acid-sensitive protecting groups such as

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trityl (Tr) and 2,3-isopropylidene, compound 13 was treated with 50% aqueous trifluoroacetic
acid (TFA) to yield the 6-chloropurine derivative 14 (75%), with the concomitant minor
formation of the N6-hydrolyzed hypoxanthine analog 3j (4%). It should be noted that higher
concentration of TFA, elevated temperature, and prolonged reaction time increased the
formation of 3j significantly and use of 2 N HCl instead of 50% TFA yielded the
hypoxanthine analog 3j as the major product. Treatment of 14 with ammonia, methylamine,
and cyclopropylamine afforded the final adenine analog 1b [3c], the N6-methyladenine
analog 3k, and the N6-cyclopropyladenine analog 3l, respectively.

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To prepare the 2,6-disubstituted-purine analogs 3m-o, the glycosyl donor 9 was condensed
with 2-amino-6-chloropurine under the standard Mitsunobu conditions to afford the 2-amino-
6-chloropurine derivative 15 (Scheme 4). Compound 15 was treated with 50% aqueous TFA
to yield the guanine analog 3m. Treatment of 15 with ammonia and cyclopropylamine
followed by treatment with 50% aqueous TFA, afforded the 2,6-diaminopurine derivative 3n
and the 2-amino-N6-cyclopropylaminopurine 3o, respectively.
In order to determine if the anticancer activity depended on inhibiting DNA and/or RNA
polymerase in cancer cells, we prepared the phosphoramidate prodrug 3p, as shown in
Scheme 5. The adenine derivative 1b was protected as 2,3-acetonide 16, which was treated
with di-tert-butyl dicarbonate (Boc2O) to give a mixture of mono-Boc- and di-Boc-adenine
derivatives 17 and 18. Treatment of the mixture of 17 and 18 with the phosphoramidate

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reagent (P) [11] in the presence of t-butylmagnesium chloride afforded the phosphoramidate
derivative 19, which was treated with 50% HCOOH to yield the final phosphoramidate
derivative 3p.
Scheme 5
NH2
N
NR1R2
NH2
N
N
N
N
N
N
N
N
N
N
F
N
F
F
a
HO
HO
HO
b
92%
O
O
O
O
HO
OH
1b
16
17 (R1 = H, R2 = Boc) (48%)
18 (R1 = R2 = Boc) (28%)
O
F
F
F
c
33%
O
O
HN
P
O
F
OPh
F
P
NHBoc
NH2
N
N
N
N
O
N
O
O
O
N
d
F
O
N
O
HN P O
N
F
85%
HN P O
OPh
OPh
O
O
HO
OH
3p
19
Reagents and Conditions: a) acetone, conc.H2SO4, 6 h; b) i. HMDS, DMAP, TMSOTf,
75 °C, 2 h; ii. Boc2O, THF, rt, 4 h; iii. MeOH:Et3N (5:1), 55 °C, 16 h; c) P, t-BuMgCl,
molecular seives, THF, 0 °C to rt, 48 h; d) 50% HCOOH, rt, 8 h.
2.2. Biological evaluation
All of the synthesized final nucleosides 1b, 2b, and 3a-p were assayed for the anticancer
activity in various human cancer cell lines including A549 (lung cancer cells), HCT-116
(colon cancer cells), SNU-638 (stomach cancer cells), MDA-MB-231 (breast cancer cells),

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SK-Hep-1 (liver cancer cells), and PC-3 (prostate cancer cells), using the SRB method (Table
1) [12,13].
Table 1. Anticancer activity of the 6'-fluorocyclopentenyl-pyrimidines 2b, 3a-i and –purines
1b, 3j-p in human cancer cell lines
IC50 (µM)
Compound No.
MDA-
MB-231
A549
HCT-116 SNU-638
SK-Hep-1 PC-3
2b (X = NH2, Y = H)a
3a (X = OH, Y = H)a
3b (X = OH, Y = F)
3c (X = OH, Y = Cl)
3d (X = OH, Y = Br)
3e (X = OH, Y = I)
0.76
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.17
0.18
>100
52.34
>100
33.09
84.5
0.31
>100
27.79
86.83
31.97
70.24
>100
>100
>100
83.65
1.88
0.48
0.62
>100
71.57
>100
74.99
>100
>100
>100
>100
>100
1.35
0.64
>100
43.73
61.88
32.05
46.4
>100
67.72
>100
44.01
55.15
>100
>100
>100
79.79
1.45
3f (X = NH2, Y = F)
3g (X = NH2, Y = Cl)
3h (X = NH2, Y = Br)
3i (X= NH2, Y = I)
>100
>100
>100
99.84
1.10
>100
>100
>100
82.89
1.52
1b (X= NH2, Y = H)c
3j (X= OH, Y = H)
3k (X= NHMe, Y = H)
3l (X= NHCPb, Y = H)
3m (X= OH, Y = NH2)
>100
2.39
>100
2.14
>100
2.28
>100
8.05
>100
2.76
>100
15.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100

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3n (X= NH2, Y = NH2)
3o (X= NHCPb, Y = NH2)
3p (P = phosphoramidate)
Neplanocin A (1a)d
Ara-Cd
85.43
>100
18.61
2.16
21.9
>100
4.57
1.45
4.4
32.19
>100
3.05
0.82
1.08
22.77
>100
18.37
0.86
>100
>100
23.96
2.36
1.8
>100
>100
10.54
2.14
1.75
10.49
48.4
aref 6; bCP = cyclopropyl; cref 3c; dpositive control
As shown in Table 1, the cytosine analog 2b exhibited the most potent anticancer activity
among the pyrimidine nucleosides. The introduction of a halogen at the C-5 position of the
pyrimidine nucleosides dramatically dropped the anticancer activity, indicating that they
could not be efficiently converted to their triphosphates by cellular kinases, unlike the
cytosine derivative 2b, which was efficiently converted by UCK, resulting in potent
anticancer activity. The 5-halouracil derivatives 3a-e generally exhibited slightly better
anticancer activity than the corresponding 5-halocytosine derivatives 3f-i, demonstrating that
the 5-halouracil moiety was a somewhat better substrate for cellular kinases than the 5-
halocytosine moiety in this series, although they exhibited weak anticancer activity. This
result was interesting in that the cytosine derivative 2b was very potent, while the
corresponding uracil derivative 3a was inactive up to 100 µM. Among the synthesized purine
nucleosides, the adenine derivative 1b (X = NH2, Y = H) exhibited potent anticancer activity,
and it was as equipotent as the positive control, neplanocin A (1a) or 2'-β-arabinofuranosyl-
cytosine (Ara-C), but its N6-deaminated compound 3j (X = OH, Y = H) was totally inactive
up to 100 µM, indicating that the adenine derivative 1b was not the substrate for adenosine
deaminase. The addition of a methyl group at the N6-amino group, giving 3k (X = NHMe, Y
= H), still maintained a similar anticancer activity, when compared to 1b. However, the
introduction of a bulkier group, like cyclopropyl, giving 3o, totally abolished the anticancer
activity. To our disappointment, the introduction of the amino group at the C2 position, such

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as 3m-n, resulted in abolished anticancer activity, indicating that no substitution at the C2
position favored anticancer activity. Surprisingly, the phosphoramidate analog 3p exhibited
less potent anticancer activity than the parent nucleoside 1b. The phosphoamidate prodrug
generally exhibits better biological activity than the parent drug because critical 5'-mono-
phosphorylation by cellular kinase is skipped [11a], which indicates that the inhibition of
DNA and/or RNA polymerase in cancer cells responsible for anticancer activity of 1b, is a
minor pathway, although it is converted to the triphosphate. It has been reported that
inhibition of SAH hydrolase increases the intracellular SAH concentration and reduction in
the level of S-adenosylmethionine (SAM), which results in the inhibition of enhancer of zeste
homolog 2 (EZH2) histone methyltransferase [14]. Inhibition of EZH2 activity induces
apotosis in different types of cancer cells [15]. The phosphoramidate prodrug 3p is not active
against SAH hydrolase up to 100 µM, but the parent compound 1b is a strong inhibitor (IC50
= 0.48 µM) of SAH hydrolase [3], which in turns inhibits EZH2 histone methyltransferase [4].
Similarly, the anticancer activity of 3k is attributed to the inhibition of SAH hydrolase (IC50 =
3.5 µM). Thus, it is suggested that the major pathway for the anticancer activity of 1b is the
inhibition of histone methyltransferase, resulting from strong inhibition (IC50 = 0.48 µM) of
SAH hydrolase.
3. Conclusions
Based on the potent anticancer activity of the 6'-fluorocyclopentenyl-cytosine 2b, we carried
out a structure-activity relationship study of the 6'-fluorocyclopentenyl-pyrmidines 3a-i and –
purines 3j-p, which were synthesized from -ribose via a vinyl fluorination as a key step.
D
From this study, the cytosine derivative 2b exhibited the most potent anticancer activity
among all of the tested compounds. The steric and electronic effects induced by halogen
atoms, which were substituted at the C5 position, were detrimental to cellular

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phosphorylation of the final nucleosides, resulting in weak anticancer activity. The adenine
derivative 1b and the N6-methyladenine derivative 3k demonstrated potent anticancer activity
comparable to the positive control, neplanocin A (1a) or Ara-C. It was quite surprising in that
only the cytosine derivative 2b without any substitution at the C5 position was acceptable for
anticancer activity in the pyrimidine series, whereas a small substituent such as H (1b) or Me
(3k) in the N6-substituted-aminopurine series was only tolerable for potent anticancer activity,
demonstrating a very narrow structural requirement for biological activity. The
phosphoramidate prodrug 3p of 1b did not improve the anticancer activity dramatically,
indicating that 1b did not seem to act as a DNA and/or RNA polymerase inhibitor after being
converted to its triphosphate, but acted as a histone methyltransferase inhibitor, resulting
from the inhibition of SAH hydrolase. All of these findings will contribute greatly to the
design of biologically active fluoro-carbocyclic nucleosides.
4. Experimental section
4.1.
Chemistry
The proton (1H), carbon (13C) NMR and fluorine (19F) NMR spectra were obtained on a
Jeol JNM-LA300 (300/75 MHz), Bruker AV 400 (400/376/100 MHz), Bruker AMX 500
(500/125 MHz), Jeol JNM-ECA600 (600/150 MHz), or Bruker AVANCE III 800 (800/200
MHz) spectrometer. The chemical shifts are reported in ppm units with Me4Si or CHCl3 as
the internal standard. All reactions were routinely carried out under an inert atmosphere of
dry nitrogen. The reactions were checked by thin layer chromatography (Kieselgel 60 F254,
Merck). The spots were detected by viewing them under a UV light, and by colorizing them
with charring after dipping them in a p-anisaldehyde solution or phosphomolybdic acid
solution. In aqueous work-up, all organic solutions were dried over anhydrous magnesium
sulfate and filtered prior to rotary evaporation at water pump pressure. The crude compounds
were purified by column chromatography on a silica gel (Kieselgel 60, 70-230 mesh, Merck).

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Unless otherwise noted, the materials were obtained from commercial suppliers and were
used without purification. All of the solvents were purified and dried by standard techniques
just before use. THF and Et2O were freshly distilled from sodium and benzophenone.
Methylene chloride, toluene, and benzene were purified by refluxing with CaH2. The hexanes
and ethyl acetate were purified by simple distillation. The mass spectra were obtained on a
Jeol JMS-700 (FAB) or Agilent 6530 Accurate-Mass Q-TOF (ESI). The optical rotations
were determined on JASCO P-2000 in the appropriate solvent. The UV spectra were recorded
on a Perkin Elmer Lambda 25. The melting points were determined on a Barnstead
Electrothermal IA9100.
4.1.1. tert-Butyl((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-dihydro-
3aH-cyclopenta[d][1,3]dioxol-4-yloxy)diphenylsilane (8a).
n-Butyllithium (11 mL, 17.6 mmol, 1.6 M solution in hexanes) was added dropwise to
a cooled (–78 °C) solution of 7 [9] (5.58 g, 7.04 mmol) and N-fluorobenzenesulfonimide
(3.33 g, 10.56 mmol) in anhydrous THF/tert-butyl methyl ether/n-heptane (3:1:1.5, 90 mL
total, 0.08 M) over a period of 1 h under N2. After stirring at the same temperature for 30
min, the reaction mixture was quenched with saturated aqueous NH4Cl (100 mL) and was
diluted with EtOAc (100 mL). Then the layers were separated and the aqueous layer was
extracted with EtOAc (2 × 50 mL). The combined organic layers were washed successively
with H2O and brine, dried over anhydrous MgSO4, filtered, and evaporated. Finally, the
residue was purified by medium pressure liquid chromatography (silica gel, hexanes/EtOAc =
25/1) to give vinyl fluoride 8a (2.17 g, 45%), 8b (1.03 g, 22%) and 8c (0.84 g, 18%).
8a: [α]D25 = −8.42 (c 0.19, MeOH); 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 6.6 Hz, 2 H),
7.72 (d, J = 6.6 Hz, 2 H), 7.35-7.44 (m, 13 H), 7.16-7.27 (m, 8 H), 4.93 (t, J = 6.6 Hz, 1 H),
4.34 (s, 1 H), 3.22-3.25 (m, 1 H), 3.89 (d, J = 11.8 Hz, 1 H), 3.77 (d, J = 12.3 Hz, 1 H), 1.44
(s, 3 H), 1.38 (s, 3 H), 1.07 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 157.3 (d, J = 288.0 Hz),

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143.8, 136.0 (d, J = 23.1 Hz), 133.5 (d, J = 21.2 Hz), 129.7 (d, J = 4.9 Hz), 127.7, 127.5 (d, J
= 9.2 Hz), 127.0, 115.6 (d, J = 4.2 Hz), 111.8, 87.0, 78.6 (d, J = 8.8 Hz), 75.3 (d, J = 7.2 Hz),
19
71.0 (d, J = 18.9 Hz), 56.3, 29.5 (d, J = 29.8), 27.9, 27.2, 26.7, 19.4; F NMR (376 MHz,
CDCl3) δ −128.2; MS (ESI+) found 707.2968 [calcd for C44H45FNaO4Si (M+Na)+ 707.2969].
1
8b: H NMR (400 MHz, CDCl3) δ 7.73-7.81 (m, 4 H), 7.34-7.45 (m, 13 H), 7.16-7.29 (m, 8
H), 5.76 (s, 1 H), 4.66 (d, J = 5.6 Hz, 1 H), 4.53-4.58 (m, 1 H), 4.42 (t, J = 5.5 Hz, 1 H), 3.84
(d, J = 14.2 Hz, 1 H), 3.58 (d, J = 14.2 Hz, 1 H), 1.42 (s, 3 H), 1.32 (s, 3 H), 1.09 (s, 9 H).
1
8c: H NMR (400 MHz, CDCl3) δ 7.72-7.81 (m, 4 H), 7.35-7.45 (m, 13 H), 7.22-7.30 (m, 8
H), 5.13 (m, 2 H), 4.80 (d, J = 13.6 Hz, 1 H), 4.19 (d, J = 14.4 Hz, 1 H), 3.73 (t, J = 5.2 Hz, 1
H), 1.31 (s, 3 H), 1.22 (s, 3 H), 1.12 (s, 9 H).
4.1.2. (3aS,4R,6aR)-5-Fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-dihydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (9).
Tetra-n-butylammonium fluoride (9.1 mL, 1.0 M solution in THF) was added
dropwise to a cooled (0 °C) solution of 8a (2.50 g, 3.65 mmol) in anhydrous THF (20 mL)
under N2. After stirring at room temperature for 2 h, the reaction mixture was evaporated.
Finally, the residue was purified by medium pressure liquid chromatography (silica gel,
25
hexanes/EtOAc = 4/1) to give 9 (1.417 g, 87%) as a white foam: [α]D = +20.77 (c 0.39,
1
MeOH); H NMR (400 MHz, CDCl3) δ 7.46-7.48 (m, 6 H), 7.21-7.31 (m, 9 H), 5.13 (t, J =
6.6 Hz, 1 H), 4.66-4.69 (m, 1 H), 4.40 (t, J = 6.6 Hz, 1 H), 3.92 (d, J = 12.0 Hz, 1 H), 3.77
13
(dd, J = 1.6, 12.0 Hz, 1 H), 2.81 (d, J = 9.3 Hz, 1 H), 1.42 (s, 3 H), 1.41 (s, 3 H); C NMR
(100 MHz, CDCl3) δ 157.7 (d, J = 287.8 Hz), 143.7, 128.6, 127.8, 127.0, 115.7 (d, J = 4.5
Hz), 112.3, 87.1, 78.7 (d, J = 8.9 Hz), 73.8 (d, J = 7.7 Hz), 69.1 (d, J = 21.0 Hz), 56.2, 27.7,
26.6; 19F NMR (376 MHz, CDCl3) δ −130.1; MS (ESI+) found 469.1779 [calcd for
C28H27FNaO4 (M+Na)+ 469.1791].

ACCEPTED MANUSCRIPT
4.1.3. General Procedure for Mistunobu Condensation
o
Diisopropyl azodicarboxylate (2.5 equiv) was added dropwise to a cooled (0 C)
suspension of triphenylphosphine (2.5 equiv) and 5-halo-N3-benzoyluracil (2.5 equiv) in
anhydrous THF (0.2 M) under N2. After stirring at room temperature for 15 min, the reaction
o
mixture was re-cooled to 0 C. Then, a solution of 9 (1.0 equiv) in anhydrous THF (0.2 M)
was added dropwise to the mixture. After stirring at room temperature for 24 h, the reaction
mixture was evaporated. Finally, the residue was purified by medium pressure liquid
chromatography (silica gel, hexanes/EtOAc = 3/1) to give the base condensed derivatives.
4.1.4. General Procedure for Debenzoylation
Saturated methanolic ammonia (5 mL) was added to the above generated base
condensed derivatives at room temperature. After stirring at room temperature for 15 h, the
reaction mixture was evaporated. Finally, the residue was purified by column
chromatography (silica gel, hexanes/EtOAc = 2/1) to give the debenzoylated derivatives 10a-
e as white foams.
4.1.4.1.
1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-
3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (10a).
25
1
Yield: 81%; [α]D = −37.04 (c 0.20, MeOH); UV (MeOH) λmax 263 nm; H NMR
(500 MHz, CDCl3) δ 9.43 (bs, 1 H), 7.46 (d, J = 7.4 Hz, 6 H), 7.46 (t, J = 7.2 Hz, 6 H), 7.22
(d, J = 7.3 Hz, 3 H, merged with CDCl3), 6.90 (d, J = 8.0 Hz, 1 H), 5.73 (dd, J = 1.5, 8.0 Hz,
1 H), 5.34 (t, J = 5.4 Hz, 1 H), 5.27 (s, 1 H), 4.56 (t, J = 5.0 Hz, 1 H), 3.97 (d, J = 12.7 Hz, 1
13
H), 3.83 (d, J = 12.7 Hz, 1 H), 1.44 (s, 3 H), 1.35 (s, 3 H); C NMR (125 MHz, CDCl3) δ

ACCEPTED MANUSCRIPT
163.1, 150.3, 149.9 (d, J = 228.4 Hz), 143.6, 141.2, 128.6, 127.9, 127.1, 122.6, 112.3, 103.0,
19
87.4, 80.3 (d, J = 5.3 Hz), 79.7 (d, J = 7.3 Hz), 65.7 (d, J = 19.1 Hz), 56.9, 27.4, 25.7; F
NMR (378 MHz, CDCl3) δ −130.5; HRMS (FAB+) found: 541.2132 [calcd for C32H30FN2O5
(M+H)+ 541.5594].
4.1.4.2.
5-Fluoro-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-
dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (10b)
Yield: 63%; [α]D25 = −16.94 (c 0.36, MeOH); UV (MeOH) λmax 268 nm; 1H NMR
(400 MHz, CDCl3) δ 9.14 (bs, 1 H), 7.45-7.48 (m, 6 H), 7.22-7.32 (m, 9 H), 6.97 (d, J = 5.4
Hz, 1 H), 5.31 (m, 2 H), 4.52 (t, J = 5.2 Hz, 1 H), 4.01 (d, J = 12.6 Hz, 1 H), 3.86 (d, J = 12.8
Hz, 1 H), 1.35 (s, 3 H), 1.44 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 156.3 (d, J = 26.8 Hz),
149.5 (d, J = 285.5 Hz), 148.9, 143.5, 140.8 (d, J = 238.7 Hz), 128.6, 127.9, 127.3, 125.2 (d,
J = 32.8 Hz), 123.4 (d, J = 3.1 Hz), 112.6, 87.5, 80.3 (d, J = 6.8 Hz), 79.5 (d, J = 9.2 Hz),
19
65.6 (d, J = 19.6 Hz), 56.9, 27.3, 25.7; F NMR (376 MHz, CDCl3) δ −128.2, −165.4; MS
(ESI−) found: 557.1891 [calcd for C32H27F2N2O5 (M-H)- 557.1888].
4.1.4.3.
5-Chloro-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-
dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (10c).
25
Yield: 62%; [α]D = −55.26 (c 0.19, MeOH); UV (MeOH) λmax 276 nm; 1H NMR
(400 MHz, CDCl3) δ 8.90 (bs, 1 H), 7.46 (d, J = 7.4 Hz, 7 H), 7.19-7.32 (m, 9 H), 5.32-5.35
(m, 2 H), 4.55 (t, J = 5.3 Hz, 1 H), 3.98 (d, J = 12.8 Hz, 1 H), 3.82 (d, J = 12.8 Hz, 1 H), 1.44
13
(s, 3 H), 1.35(s, 3 H); C NMR (100 MHz, CDCl3) δ 158.5, 149.6 (d, J = 285.7 Hz), 149.3,
143.5, 137.8, 128.6, 127.9, 127.2, 123.3 (d, J = 3.1 Hz), 112.6, 109.9, 87.5, 80.3 (d, J = 6.7

ACCEPTED MANUSCRIPT
Hz), 79.6 (d, J = 9.2 Hz), 66.0 (d, J = 20.2 Hz), 56.9, 27.3, 25.7; 19F NMR (376 MHz, CDCl3)
δ −128.2; MS (ESI+) found 597.1556 [calcd for C32H28ClFN2NaO5 (M+Na)+ 597.1568].
4.1.4.4.
5-Bromo-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-
dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H, 3H)-dione (10d).
25
Yield: 51%; [α]D = −52.50 (c 0.20, MeOH); UV (MeOH) λmax 279 nm; 1H NMR
(400 MHz, CDCl3) δ 8.90 (bs, 1 H), 7.46-7.48 (m, 6 H), 7.22-7.32 (m, 10 H), 5.34 (t, J = 6
Hz, 1 H), 5.30 (s, 1 H), 4.57 (t, J = 5.1 Hz, 1 H), 3.98 (d, J = 12.6 Hz, 1 H), 3.80 (d, J = 12.6
13
Hz, 1 H), 1.44 (s, 3 H), 1.35 (s, 3 H); C NMR (100 MHz, CDCl3) δ 158.6, 149.7 (d, J =
285.4 Hz), 149.6, 143.5, 140.4, 128.6, 127.9, 127.2, 123.2 (d, J = 2.9 Hz), 112.6, 97.6, 87.5,
19
80.3 (d, J = 6.6 Hz), 79.7 (d, J = 9.3 Hz), 66.2 (d, J = 20.2 Hz), 56.9, 27.3, 25.7; F NMR
(376 MHz, CDCl3) δ −128.2; MS (ESI+) found 619.1073 [calcd for C32H29BrFN2O5 (M+H)+
619.1244].
4.1.4.5.
1-((3aS,4S,6aR)-5-Fluoro-2,2-dimethyl-6-(trityloxymethyl)-4,6a-dihydro-3aH-
cyclopenta-[d][1,3] dioxol-4-yl)-5-iodopyrimidine-2,4(1H,3H)-dione (10e).
Yield: 63%; [α]D25 = −70.67 (c 0.15, MeOH); UV (MeOH) λmax 284 nm; 1H NMR
(400 MHz, CDCl3) δ 8.92 (bs, 1 H), 7.43-7.48 (m, 7 H), 7.22-7.33 (m, 9 H), 5.35 (t, J = 5.4
Hz, 1 H), 5.27 (s, 1 H), 4.58 (t, J = 5.1 Hz, 1 H), 3.96 (d, J = 12.5 Hz, 1 H), 3.78 (d, J = 12.5
Hz, 1 H), 1.43 (s, 3 H), 1.35(s, 3 H); 13C NMR (100 MHz, CDCl3) δ 159.7, 149.9, 149.8 (d, J
= 285.7 Hz), 145.6, 143.5, 128.6, 127.9, 127.2, 123.0 (d, J = 2.8 Hz), 112.5, 110.0, 87.4, 80.3
19
(d, J = 6.5 Hz), 79.7 (d, J = 9.2 Hz), 69.1, 66.3 (d, J = 21.2 Hz), 56.9, 27.3, 25.7; F NMR
(376 MHz, CDCl3) δ −128.2; MS (ESI+) found: 689.0916 [calcd for C32H28FIN2NaO5
(M+Na)+ 689.0925].

ACCEPTED MANUSCRIPT
4.1.5. General Procedure for Trityl and Isopropylidene Group Deprotection
A 2 N HCl solution (0.6 M) was added dropwise to a cooled (0 °C) solution of 10a-e
(1.0 equiv) in MeOH (0.6 M). After stirring at room temperature for 18 h, the reaction
mixture was co-evaporated with MeOH three times. The residue was diluted with MeOH and
was neutralized with saturated methanolic ammonia. The resulting reaction mixture was
allowed to stir at room temperature for 1 h, and was then evaporated. Finally, the crude
residue was purified by column chromatography (18C reverse-phase silica gel, H2O/MeCN =
99/1) to give the 5-halouracil derivatives 3a-e as white solids.
4.1.5.1.
1-((1S,4R,5S)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-
yl)pyrimidine-2,4(1H,3H)-dione (3a)[6]
Yield : 80%; mp 78-79 oC; [α]D27.2 = −93.5 (c 1.2, MeOH); UV (MeOH) λmax 264.0 nm; 1H
NMR (400 MHz, CD3OD) δ 4.12 (dt, J = 2.4, 13.2 Hz, 1 H), 4.19 (td, J = 1.2, 5.6 Hz 1 H),
4.37 (d, J = 12.8 Hz, 1 H), 4.68 (td, J = 1.2, 6.0 Hz, 1 H), 5.43 (bs, 1 H), 5.73 (d, J = 8.0 Hz,
13
1 H), 7.47 (dd, J = 1.2, 8.0 Hz, 1 H); C (100 MHz, CD3OD) δ 166.8, 155.4 (d, J = 284.6
Hz), 153.6, 144.7, 123.8 (d, J = 1.9 Hz), 104.1, 75.5 (d, J = 5.3 Hz), 71.6 (d, J = 8.8 Hz), 66.1
(d, J = 17.6 Hz), 55.1; 19F (376 MHz, CD3OD) δ -128.9; HRMS (ESI+) found 259.0722
[calculated for C10H12FN2O5 259.065 (M+H)+ ]; Anal. calcd for C10H11FN2O5: C, 46.52; H,
4.29; N, 10.85. Found: C, 46.55; H, 4.31; N, 10.91.
4.1.5.2.
5-Fluoro-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-
25
2-enyl)pyrimidine-2,4(1H,3H)-dione (3b). Yield: 52%; m.p: charred above 213 oC; [α]D
=
−10.00 (c 0.21, MeOH); UV (MeOH) λmax 272 nm; 1H NMR (400 MHz, CD3OD) δ 7.74 (dd,

ACCEPTED MANUSCRIPT
J = 0.8, 6.4 Hz, 1 H), 5.46 (bs, 1 H), 4.68 (td, J = 1.6, 6.0 Hz, 1 H), 4.37 (d, J = 13.2 Hz, 1 H),
4.19 (td, J = 1.6, 5.6 Hz, 1 H), 4.14 (t, J = 2.4 Hz, 1 H), 4.11 (t, J = 2.4 Hz, 1 H); 13C NMR
(100 MHz, CD3OD) δ 159.5 (d, J = 25.8 Hz), 154.5 (d, J = 284.9), 151.7, 143.6 (d, J = 233.2
Hz), 127.8 (d, J = 34.2 Hz), 123.6, 74.8 (d, J = 5.3 Hz), 70.9 (d, J = 8.3 Hz), 65.4 (d, J = 18.2
Hz), 54.4; 19F NMR (376 MHz, CD3OD) δ −126.9, −162.6 (d, J = 6.4 Hz); MS (FAB) found:
277.0645 [calcd for C10H11F2N2O5 (M+H)+ 277.0636]; Anal. Calcd for C10H11F2N2O5: C,
43.49; H, 3.65; N, 10.14. Found: C, 43.09; H, 3.61; N, 9.98.
4.1.5.3.
5-Chloro-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-
2-enyl)pyrimidine-2,4(1H,3H)-dione (3c). Yield: 50%; m.p: 124-126 oC; [α]D25 = −121.33 (c
0.15, MeOH); UV (MeOH) λmax 278 nm; 1H NMR (400 MHz, CD3OD) δ 7.82 (d, J = 0.8 Hz,
1 H), 5.46 (bs, 1 H), 4.69 (td, J = 1.6, 6.0 Hz, 1 H), 4.38 (d, J = 13.2 Hz, 1 H), 4.21 (td, J =
1.2, 6.0 Hz, 1 H), 4.14 (t, J = 2.4 Hz, 1 H), 4.11 (t, J = 2.4 Hz, 1 H); 13C NMR (100 MHz,
CD3OD) δ 161.5, 154.4 (d, J = 284.2 Hz), 152.1, 140.9, 123.5, 110.3, 74.9 (d, J = 5.3 Hz),
70.8 (d, J = 8.4 Hz), 65.8, 65.7, 54.4; 19F NMR (376 MHz, CD3OD) δ −126.9; MS (FAB+)
found 293.0345 [calcd for C10H11ClFN2O5 (M+H)+ 293.0341]; Anal. Calcd for
C10H11ClFN2O5: C, 41.04; H, 3.44; N, 9.57. Found: C, 41.34; H, 3.04, N, 9.17.
4.1.5.4.
5-Bromo-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-
enyl)pyrimidine-2,4(1H,3H)-dione (3d). Yield: 74%; m.p: 68-70 oC; [α]D25 = −163.68 (c 0.19,
MeOH); UV (MeOH) λmax 281 nm; 1H NMR (400 MHz, CD3OD) δ 7.89 (s, 1 H), 5.45 (bs,
1 H), 4.67 (t, J = 5.2 Hz, 1 H), 4.37 (d, J = 13.0 Hz, 1 H), 4.21 (t, J = 5.6 Hz, 1 H), 4.11 (d, J =
13.0 Hz, 1 H); 13C NMR (100 MHz, CD3OD) δ 161.7, 154.5 (d, J = 284.2 Hz), 152.4, 143.5,
123.4, 98.0, 74.9 (d, J = 5.4 Hz), 70.9 (d, J = 8.4 Hz), 65.8 (d, J =17.4 Hz), 54.4; 19F NMR
(376 MHz, CDCl3) δ −126.9; MS (FAB+) found 337.1014 [calcd for C10H10BrFN2O5 (M+H)+

ACCEPTED MANUSCRIPT
336.9835]; Anal. Calcd for C10H10BrFN2O5: C, 35.63; H, 2.99; N, 8.31. Found: C, 35.34; H,
2.67; N, 8.32.
4.1.5.5.
1-((1S,4R,5S)-2-Fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-enyl)-5-
iodopyrimidine-2,4(1H,3H)-dione (3e). Yield: 61%; m.p: 87-89 oC; [α]D25 = −153.24 (c 0.37,
MeOH); UV (MeOH) λmax 287 nm; 1H NMR (400 MHz, CD3OD) δ 7.90 (d, J = 0.8 Hz, 1 H),
5.43 (bs, 1 H), 4.68 (td, J = 1.2, 5.6 Hz, 1 H), 4.38 (d, J = 12.8 Hz, 1 H), 4.22 (td, J = 1.2, 5.6
Hz, 1 H), 4.12 (dt, J = 2.4, 12.8 Hz, 1 H); 13C NMR (100 MHz, CD3OD) δ 162.9, 154.6 (d, J
= 284.2 Hz), 152.7, 148.5, 123.2, 74.9 (d, J = 4.6 Hz), 70.8 (d, J = 8.3 Hz), 69.5, 65.7 (d, J =
17.5 Hz), 54.4; 19F NMR (376 MHz CD3OD) δ −126.8; MS (ESI+) found 406.9514 [calcd for
C10H10FIN2NaO5 (M+Na)+ 406.9516]; Anal. Calcd for C10H10FIN2NaO5: C, 31.27; H, 2.62;
N, 7.29. Found: C, 31.45; H, 2.23; N, 7.08.
4.1.6. General Procedure for the Conversion of the Uracil Derivatives 3a-e to the
Cytosine Derivatives 2b and 3f-i.
[Acetylation] Acetic anhydride (4.0 equiv) was added dropwise to a stirred solution of the
pyrimidine derivatives 3a-e (1.0 equiv) in anhydrous pyridine (0.17 M) under N2. After
stirring at room temperature for 18 h, the reaction mixture was evaporated. Then, the residue
was diluted with CH2Cl2 and H2O, and the organic layer was separated, dried over MgSO4,
filtered, and evaporated. Finally, the residue was purified by medium pressure liquid
chromatography (silica gel, hexanes/EtOAc = 9/1) to give the triacetates 11a-e as a thick
liquid.
o
[Introduction of 1,2,4-triazole] POCl3 (10.0 equiv) was added dropwise- to a cooled (0 C)
suspension of 1,2,4-triazole (10.0 equiv) in anhydrous CH3CN (1.1 M) under N2. After
o
stirring at room temperature for 1 h, the reaction mixture was cooled to 0 C and Et3N (10.0

ACCEPTED MANUSCRIPT
equiv) was added, followed by 11a-e (1.0 equiv) in anhydrous CH3CN (0.1 M). After stirring
for an additional 24 h at room temperature, the reaction mixture was evaporated to a half-
volume, diluted with CH2Cl2, and washed with H2O. The layers were separated, the organic
layer was dried over MgSO4, filtered, and evaporated to give 12a-e, which were used for the
next step without further purification.
[Amination with Deacetylation] Aqueous ammonia (0.2 M) was added to a stirred solution of
12a-e in 1,4-dioxane (0.13 M). After stirring at room temperature for 16 h in glass-sealed
tube, the reaction mixture was evaporated. A methanolic ammonia solution (5.0 mL) was
added to the above-generated reaction mixture. After stirring for an additional 15 h at room
temperature, the reaction mixture was evaporated. Finally, the residue was purified by
column chromatography (18C reverse-phase silica gel, H2O/MeCN = 99/1) to give 2b and 3f-i
as white solids.
4.1.6.1.
4-Amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-
en 1-yl)pyrimidin-2(1H)-one (2b) [6]. Yield : 40%; mp 101-102 oC; [α]D27.2 = −135.47 (c 5.3,
MeOH); UV (MeOH) λmax 274.5 nm; 1H NMR (400 MHz, CD3OD) δ 4.10 (dt, J = 2.4, 12.8
Hz, 1 H), 4.24-4.27 (m, 1 H), 4.36 (d, J = 12.8 Hz, 1 H), 4.68 (td, J = 1.2, 6.0 Hz 1H), 5.34
(bs, 1 H), 5.92 (d, J = 7.2 Hz, 1 H ), 7.47 (J = 6.8 Hz, 1 H); 13C (100 MHz, CD3OD) δ 167.6,
158.8, 155.6 (d, J = 284.9 Hz), 145.0, 122.0, 96.6, 74.6 (d, J = 3.9 Hz), 70.9 (d, J = 8.9 Hz),
67.1 (d, J = 17.5 Hz), 54.3; 19F (376 MHz, CD3OD) δ −128.1; HRMS (ESI+) found 258.0822
[calculated for C10H13FN3O4 (M+H)+ 258.081]; Anal. calcd for C10H12FN3O4: C, 46.59; H,
4.70; N, 16.34. Found: C, 46.63; H, 4.77; N, 16.40.

ACCEPTED MANUSCRIPT
4.1.6.2.
4-Amino-5-fluoro-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)
cyclopent-2-enyl)pyrimidin-2(1H)-one (3f). Yield: 21%; m.p: 121-123 C; [α]D25 = −60.00 (c
0.21, MeOH); UV (MeOH) λmax 276 nm; 1H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 3.0
Hz, 1 H), 5.82 (s, 1 H), 4.84 (bs, 2 H), 4.51 (s, 1 H), 4.02 (d, J = 7 Hz, 1 H), 3.87-3.93 (m, 2
H); 13C NMR (100 MHz, DMSO-d6) δ 159.8, 154.9 (d, J = 13.7 Hz), 154.1 (d, J = 282.6 Hz),
143.4, 141.0, 139.7 (d, J = 20.2 Hz), 121.9, 79.8 (d, J = 18.2 Hz), 73.4 (d, J = 6.3 Hz), 68.5 (d,
J = 9.3 Hz), 52.3; 19F NMR (376 MHz, CD3OD) δ −128.6, −157.3 (d, J = 8.6 Hz); MS (ESI+)
found 276.0793 [calcd for C10H12F2N3O4 (M+H)+ 276.0796]; Anal. Calcd for C10H12F2N3O4:
C, 43.32; H, 4.73; N, 15.16. Found: C, 43.45; H, 4.44; N, 14.98.
o
4.1.6.3.
4-Amino-5-chloro-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)
o
cyclopent-2-enyl)pyrimidin-2(1H)-one (3g). Yield: 42%; m.p: 135-137 C; [α]D25 = −166.19
(c 0.21, MeOH); UV (MeOH) λmax 289 nm; 1H NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1 H,
D2O exchangeable), 7.80 (s, 1 H), 7.25 (s, 1 H, D2O exchangeable), 5.31 (s, 1 H), 5.17 (d, J =
7.1 Hz, 1 H, D2O exchangeable), 4.98 (d, J = 5.9 Hz, 1 H, D2O exchangeable), 4.78 (dd, J =
6.2, 4.6 Hz, 1 H, D2O exchangeable), 4.47 (dd, J = 10.2, 4.8 Hz , 1 H), 4.10-16 (m, 2 H),
3.87-91 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 161.3, 154.1, 152.5 (d, J = 281.9 Hz),
141.4, 121.4, 99.1, 72.7 (d, J = 4.8 Hz), 68.9 (d, J = 9.1 Hz), 64.5 (d, J = 20.4 Hz), 52.6; 19F
NMR (376 MHz, DMSO-d6) δ −134.5; MS (ESI+) found 292.0492 [calcd for C10H12ClFN3O4
(M+H)+ 292.0500]; Anal. Calcd for C10H12ClFN3O4: C, 40.90; H, 4.46; N, 14.31. Found: C,
40.98; H, 4.06; N, 13.98.
4.1.6.4.
4-Amino-5-bromo-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)
o
cyclopent-2-enyl)pyrimidin-2(1H)-one (3h). Yield: 42%; m.p: 219-221 C; [α]D25 = −188.42
(c 0.19, MeOH); UV (MeOH) λmax 290 nm; 1H NMR (400 MHz, CD3OD) δ 7.85 (s, 1 H),

ACCEPTED MANUSCRIPT
5.37 (bs, 1 H), 4.69 (t, J = 5.2 Hz, 1 H), 4.37 (d, J = 12.8 Hz, 1 H), 4.28 (td, J = 1.2, 5.6 Hz, 1
H), 4.11 (dt, J = 2.4, 12.8 Hz, 1 H); 13C NMR (100 MHz, CD3OD) δ 164.9, 158.4, 157.4 (d, J
= 284.4 Hz), 146.4, 123.1, 90.0, 75.5 (d, J = 5.2 Hz), 71.6 (d, J = 8,8 Hz), 68.3 (d, J = 16.8
Hz), 55.1; 19F NMR (376 MHz, CD3OD) δ −133.8; MS (ESI+) found 335.9994 [calcd for
C10H12BrFN3O4 (M+H)+ 335.9995]; Anal. Calcd for C10H12BrFN3O4: C, 35.52; H, 3.88; N,
12.43. Found: C, 35.13; H, 4.23; N, 12.04.
4.1.6.5.
4-Amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-
enyl)-5-iodopyrimidin-2(1H)-one (3i). Yield: 38%; m.p: 228-230 oC; [α]D25 = −199.44 (c 0.18,
MeOH); UV (MeOH) λmax 297 nm; 1H NMR (500 MHz, CD3OD) δ 7.88 (s, 1 H), 5.32 (bs, 1
H), 4.67 (t, J = 5.5 Hz, 1 H), 4.36 (d, J = 13.0 Hz, 1 H), 4.27 (t, J = 6.0 Hz, 1 H), 4.09 (dt, J =
2.0, 13.0 Hz, 1 H); 13C NMR (100 MHz, DMSO-d6) δ 164.1, 154.7, 153.2 (d, J = 282.2 Hz),
149.8, 121.5, 73.1, 69.3 (d, J = 9.1 Hz), 65.0 (d, J = 17.9 Hz), 53.0, 49.0; 19F NMR (376
MHz, CD3OD) δ −133.7; MS (ESI+) found 384.1259 [calcd for C10H12FIN3O4 (M+H)+
383.9857]; Anal. Calcd for C10H12FIN3O4: C, 31.19; H, 3.40; N, 10.91. Found: C, 31.56; H,
3.80; N, 9.89.
4.1.7. 6-Chloro-9-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-3a,6a-
dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-9H-purine (13). Compound 9 was converted to
13, using a procedure similar to the one used in the preparation of 10: 1H NMR (300 MHz,
CDCl3) δ 8.76 (s, 1 H), 8.01 (s, 1 H), 7.43-7.50 (m, 6 H), 7.20-7.35 (m, 9 H), 5.53-5.62
(m, 2 H), 4.78 (t, J = 5.7 Hz, 1 H), 4.02 (d, J = 12.6 Hz, 1 H), 3.83 (d, J = 12.6 Hz, 1 H),
1.48 (s, 3 H), 1.37 (s 3 H); The resulting 13, contaminated with DIAD, was used in the next
step.

ACCEPTED MANUSCRIPT
4.1.8. (1S,2R,5S)-5-(6-Chloro-9H-purin-9-yl)-4-fluoro-3-(hydroxymethyl)cyclopent-3-
ene-1,2-diol (14) and 9-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-
en-1-yl)-1H-purin-6(9H)-one (3j).
A suspension of 13 (1.0 g, 1.72 mmol) in 50% TFA (10 mL, 0.17 M) was stirred at
room temperature for 15 h. The mixture was evaporated and the residue was purified by
column chromatography (silica gel, CH2Cl2/MeOH = 9/1 to 4/1) to give the 6-chloropurine
derivative 14 (387 mg, 75% from 9) as a pale yellow solid and 3j (19 mg, 4% from 9) as a
white solid.
Compound 14: m.p: 103-105 oC; [α]D20 = −12.52 (c 0.24, MeOH); UV (MeOH) λmax 265 nm;
1H NMR (600 MHz, CD3OD) δ 8.72 (s, 1 H), 8.62 (s, 1 H), 5.73 (s, 1 H), 4.83 (m, 1 H,
merged with H2O), 4.63 (t, J = 5.5 Hz, 1 H), 4.41 (d, J = 12.8 Hz, 1 H), 4.19-4.22 (m, 1 H);
13C NMR (150 MHz, CD3OD) δ 154.5 (d, J = 284.4 Hz), 154.1, 153.9, 152.4, 148.2, 133.6,
19
124.0, 76.1 (d, J = 4.3 Hz), 71.8 (d, J = 8.6 Hz), 64.8 (d, J = 18. Hz), 55.2; F NMR (376
MHz, CD3OD) δ −78.8, −134.7; MS (ESI+) found 301.0511 [calcd for C11H11ClFN4O3
(M+H)+ 301.0504].
Compound 3j: m.p: 280 oC (decomp.); [α]D20 = −3.23 (c 0.22, MeOH); UV (MeOH) λmax 250
nm; 1H NMR (800 MHz, CD3OD) δ 8.11 (s, 1 H), 8.02 (s, 1 H), 5.59 (s, 1 H), 4.78 (t, J = 5.0
Hz, 1 H), 4.52 (t, J = 5.6 Hz, 1 H), 4.39 (d, J = 13.2 Hz, 1 H), 4.17 (dt, J = 12.8, 2.2 Hz, 1 H);
13C NMR (200 MHz, CD3OD) δ 159.7, 155.0 (d, J = 284.4 Hz), 151.3, 147.5, 141.8, 126.5,
19
123.5, 76.6 (d, J = 4.7 Hz), 71.7 (d, J = 8.5 Hz), 64.3 (d, J = 18.1 Hz), 55.1; F NMR (376
MHz, CD3OD) δ −78.7, −132.4, −134.7; MS (ESI+) found: 283.0842 [calcd for C11H11FN4O4
(M+H)+ 283.0843]; Anal. Calcd for C11H11FN4O4: C, 46.81; H, 3.93; N, 19.85. Found: C,
46.98; H, 4.13; N, 19.65.

ACCEPTED MANUSCRIPT
4.1.9. (1S,2R,5S)-5-(6-Amino-9H-purin-9-yl)-4-fluoro-3-(hydroxymethyl)cyclopent-3-ene-
1,2-diol (1b) [3c].
Saturated ammonia in tert-butanol (10.0 mL) was added to a solution of 14 (69 mg, 0.23
o
mmol) in tert-butanol (1.0 mL, 0.23 M) in a steel bomb. After stirring at 70 C for 24 h, the
reaction mixture was evaporated. Finally, the residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH = 4/1) to give 1b (36 mg, 56%) as a white solid:
o
1
mp 181-184 C; [α]D25 = −181.1 (c 0.62, MeOH); UV (H2O) λmax 260.0 nm; H NMR (400
MHz, CD3OD) δ 8.17 (s, 1 H), 8.15 (s, 1 H), 5.56 (bs, 1 H), 4.79 (t, J = 5.2, 6.0 Hz, 1 H),
13
4.56 (t, J = 5.6 Hz, 1 H), 4.41 (d, J = 13.2 Hz, 1 H), 4.16 (td, J =2.4, 13.2 Hz, 1 H); C
NMR(100 MHz, CD3OD) δ 157.7, 154.7 (d, J = 285.3 Hz), 154.2, 151.2, 142.1, 122.8 (d, J =
2.3 Hz), 120.8, 75.7 (d, J = 4.6 Hz), 71.2 (d, J = 8.5 Hz), 63.6 (d, J = 18.4Hz), 54.7; 19F NMR
(376 MHz, CD3OD) δ −133.1; MS (FAB+) found 282 [calcd for C11H13FN5O3 282.2554
(M+H+)]; Anal. Calcd for C11H12FN5O3: C, 46.98; H, 4.30; N, 24.90. Found: C, 46.99; H,
4.28; N, 25.20.
4.1.10. (1S,2R,5S)-4-Fluoro-3-(hydroxymethyl)-5-(6-(methylamino)-1H-purin-9(6H)-
yl)cyclopent-3-ene-1,2-diol (3k).
Aqueous methylamine (40 wt%, 1 mL) was added to a stirred solution of 14 (57 mg,
0.19 mmol) in ethanol (2.0 mL, 0.1 M) in a glass-seal tube. After stirring for 20 h at room
temperature, the mixture was evaporated. Finally, the residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH = 9/1) to give 3k (24 mg, 43%) as a white solid:
o
m.p: 157-160 C; [α]D20 = −14.95 (c 0.36, MeOH); UV (MeOH) λmax 266 nm; 1H NMR (600
MHz, CD3OD) δ 8.23 (s, 1 H), 8.08 (s, 1 H), 5.55 (s, 1 H), 4.79 (t, J = 5.9 Hz, 1 H), 4.55 (t, J
= 6.0 Hz, 1 H), 4.41 (d, J = 12.8 Hz, 1 H), 4.14-4.19 (m, 1 H), 3.10 (bs, 3 H); 13C NMR (150

ACCEPTED MANUSCRIPT
MHz, CD3OD) δ 157.6, 155.3 (d, J = 284.4 Hz), 154.6, 150.6, 142.0, 123.2, 121.9, 76.2 (d, J
= 5.0 Hz), 71.7 (d, J = 8.7 Hz), 64.1 (d, J = 17.9 Hz), 55.2, 28.5; 19F NMR (376 MHz,
CD3OD) δ −78.7, −134.3; MS (ESI+) found: 296.1159 [calcd for C12H15FN5O3 (M+H)+
296.1159]; Anal. Calcd for C12H15FN5O3: C, 48.81; H, 4.78; N, 23.72. Found: C, 48.90; H,
4.48; N, 23.43.
4.1.11. (1S,2R,5S)-5-(6-(Cyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-
(hydroxymethyl)cyclopent-3-ene-1,2-diol (3l).
Cyclopropylamine (82 µL, 1.18 mmol) and triethylamine (230 µL, 1.65 mmol) were
successively added to a solution of 14 (71 mg, 0.24 mmol) in ethanol (1.5 mL, 0.16 M) in a
o
steel bomb. The reaction mixture was stirred at 67 C for 24 h, and was evaporated. The
residue was purified by column chromatography (silica gel, CH2Cl2/MeOH = 7/1) to give 3l
(20 mg, 25%) as a white solid: m.p: 93-95 oC; [α]D20 = −12.85 (c 0.11, MeOH); UV (MeOH)
λmax 270 nm; 1H NMR (600 MHz, CD3OD) δ 8.27 (s, 1 H), 8.11 (s, 1 H), 4.79 (t, J = 6.0 Hz,
1 H), 4.56 (t, J = 5.9 Hz, 1 H), 4.41 (d, J = 12.7 Hz, 1 H), 4.14-4.20 (m, 1 H), 2.96 (bs, 1 H),
0.87-0.92 (m, 2 H), 0.62-0.68 (m, 2 H); 13C NMR (150 MHz, CD3OD) δ 157.9, 155.2 (d, J =
285.1 Hz), 151.0, 149.5, 142.4, 123.3, 121.9, 76.2 (d, J = 4.3 Hz), 71.7 (d, J = 4.6 Hz), 64.2
(d, J = 18.7 Hz), 55.2, 25.3, 8.36; 19F NMR (376 MHz, CD3OD) δ −78.8, −134.4; MS (ESI+)
found 322.1326 [calcd for C14H17FN5O3 (M+H)+ 322.1315]; Anal. Calcd for C14H17FN5O3: C,
52.33; H, 5.02; N, 21.80. Found: C, 52.76; H, 4.98; N, 21.99.
4.1.12. 6-Chloro-9-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-3a,6a-
dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)-9H-purin-2-amine (15). Compound 9 (123 mg,
0.28 mmol) was converted to 15 (90 mg, 55%) as a white foam, using a procedure similar

ACCEPTED MANUSCRIPT
1
to the preparation of 10: [α]D20 = −1.23 (c 0.47, MeOH); UV (MeOH) λmax 255 nm; H
NMR (600 MHz, CDCl3) δ 7.65 (s, 1 H), 7.45-7.49 (m, 6 H), 7.22-7.31 (m, 9 H), 5.58 (t, J
= 4.56 Hz, 1 H), 5.33 (s, 1 H), 5.00 (s, 2 H), 4.72 (t, J = 5.46 Hz, 1 H), 4.04 (d, J = 11.8 Hz,
1 H), 3.77-3.82 (m, 1 H), 1.46 (s, 3 H), 1.38 (s, 3 H); 13C NMR (150 MHz, CDCl3) δ
159.0, 153.3, 151.8, 150.2 (d, J = 285.1 Hz), 143.7, 140.8, 128.6, 127.9, 127.2, 125.7,
121.5, 112.4, 87.3, 80.5 (d, J = 5.8 Hz), 80.2 (d, J = 9.3 Hz), 62.4 (d, J = 20.1 Hz), 56.5,
19
27.5, 26.0; F NMR (376 MHz, CDCl3) δ −129.0; MS (ESI+) found 598.2027 [cacld for
C33H30FN5O3 (M+H)+ 598.2021].
4.1.13. 2-Amino-9-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-
yl)-1H-purin-6(9H)-one (3m).
A suspension of 15 (65 mg, 0.11 mmol) in 50% TFA (6 mL, 0.018 M) was stirred for
15 h at room temperature. The reaction mixture was evaporated and the residue was purified
by column chromatography (silica gel, CH2Cl2/MeOH = 7/1) to give 3m (19 mg, 59%) as a
o
white solid: m.p: 223 C (decomp.); [α]D20 = −6.70 (c 0.29, MeOH); UV (MeOH) λmax 255
1
nm; H NMR (800 MHz, CD3OD) δ 7.73 (s, 1 H), 5.38 (s, 1 H), 4.76 (t, J = 4.8 Hz, 1 H),
4.49 (t, J = 5.6 Hz, 1 H), 4.39 (d, J = 12.9 Hz, 1 H), 4.13-4.17 (m, 1 H); 13C NMR (200 MHz,
CD3OD) δ 160.2, 156.1, 155.6 (d, J = 284.7 Hz), 154.3, 139.2, 123.0, 118.8, 76.2 (d, J = 4.9
Hz), 71.7 (d, J = 8.6 Hz), 63.7 (d, J = 18.1 Hz), 55.0; 19F NMR (376 MHz, CD3OD) δ −78.7,
−134.3; MS (ESI+) found 298.0950 [calcd for C11H13FN5O4 (M+H)+ 298.0952]; Anal. Calcd
for C11H13FN5O4: C, 44.45; H, 4.07; N, 23.56. Found: C, 44.12; H, 3.89; N, 23.16.
4.1.14. (1S,2R,5S)-5-(2,6-Diamino-9H-purin-9-yl)-4-fluoro-3-(hydroxymethyl)cyclopent-3-
ene-1,2-diol (3n).