Design, Synthesis, and Anti-RNA Virus Activity of 6′-Fluorinated-Aristeromycin Analogues

Article abstract of DOI:10.1021/acs.jmedchem.9b00781

The 6′-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6′-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N⁶-methyladenosine analogues 2a-e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6′,6′-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ～2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6′-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.

Full text of DOI:10.1021/acs.jmedchem.9b00781

Subscriber access provided by ALBRIGHT COLLEGE
Article
Design, Synthesis and Anti-RNA Virus Activity
of 6'-Fluorinated-aristeromycin Analogues
Ji-seong Yoon, Gyudong Kim, Dnyandev Jarhad, Hong-Rae Kim, Young-Sup Shin, Shuhao Qu, Pramod
Sahu, Hea Ok Kim, Hyuk Woo Lee, Su Bin Wang, Yun Jeong Kong, Tong-Shin Chang, Natacha
Ogando, Kristina Kovacikova, Eric J. Snijder, Clara Posthuma, Martijn van Hemert, and Lak Shin Jeong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00781 • Publication Date (Web): 07 Jun 2019
Downloaded from http://pubs.acs.org on June 9, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design, Synthesis and Anti-RNA Virus Activity of 6'-Fluorinated-aristeromycin
Analogues
1,#
1,2,#
1
1
Ji-seong Yoon, Gyudong Kim,
Dnyandev B. Jarhad, Hong-Rae Kim, Young-Sup
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
3
3
3
4
Shin, Shuhao Qu, Pramod K. Sahu, Hea Ok Kim, Hyuk Woo Lee, Su Bin Wang, Yun
Jeong Kong, Tong-Shin Chang, Natacha S. Ogando, Kristina Kovacikova, Eric J. Snijder,
4
4
5
5
5
5
5
1,*
Clara C. Posthuma, Martijn J. van Hemert, Lak Shin Jeong
1
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National
2
University, Seoul 151-742, Korea, College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju 500-757, Korea, Future Medicine
Co., Ltd, Seoul 06665, Korea, College of Pharmacy, Ewha Womans University, Seoul 120-
750, Korea and Department of Medical Microbiology, Leiden University Medical Center,
3
4
5
Albinusdreef 2, 2333ZA Leiden, The Netherlands
lakjeong@snu.ac.kr
#Contributed equally to this work
Keywords:6'-Fluorinated-ariseromycin, S-Adenosylhomocysteinehydrolase, Anti-RNAvirus
activity, Zika, MERS-CoV, Chikungunya, SARS-CoV
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
The 6-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at
inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-
adenosyl-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA.
The introduction of a fluorine at the 6-position enhanced the inhibition of SAH hydrolase and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
the activity against RNA viruses. The adenosine and N -methyladenosine analogues 2a-e
showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c
exhibited potent antiviral activity against all tested RNA viruses such as MERS-coronavirus,
SARS-coronavirus, chikungunya virus and/or Zika virus. 6,6-Difluoroaristeromycin (2c)
showed the strongest antiviral effect for MERS-CoV, with a ~2.5 log reduction in infectious
progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated
potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study
shows that 6-fluorinated aristeromycins can serve as starting points for the development of
broad-spectrum antiviral agents that target RNA viruses.
ACS Paragon Plus Environment

Page 3 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
■
Introduction
Over the past 15 years outbreaks of a number of emerging positive-stranded RNA (+RNA)
1
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
viruses, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle
3
4
East respiratory syndrome coronavirus (MERS-CoV), chikungunya virus (CHIKV), and Zika
5
virus (ZIKV) have seriously threatened human health and have had a substantial socio-
6
economic impact. SARS-CoV and MERS-CoV cause serious respiratory diseases that can be
fatal in approximately 10% and 35% of cases, respectively. CHIKV is transmitted by
7
mosquitoes and causes a painful arthritis that can persist for months. ZIKV is also transmitted
8
8
by mosquitoes, although sexual transmission occurs as well. This virus usually causes mild
disease, but can cause neurological complications in adults and fetal death or severe
complications, including microcephaly in infants when women are infected during pregnancy.9
CHIKV and ZIKV have caused massive outbreaks, totaling millions of infections over the past
decade. Currently, there are no effective chemotherapeutic agents or vaccines that can prevent
or cure infections of any of these four serious pathogens.
1
The aforementioned viruses belong to the +RNA virus group (Baltimore class IV), which
indicates that their genomic RNA has the same polarity as mRNA and can be directly translated
by host ribosomes upon release into the cytoplasm of a host cell. After infection, the genomes
of these viruses are translated into polyproteins that are subsequently cleaved into individual
proteins by viral and/or host proteases. The nonstructural proteins (nsps) of these viruses
harbour a variety of enzymatic activities that are required for the replication of the viral RNA,
10
and invariably include a RNA-dependent RNA polymerase (RdRp) , an enzyme which is not
present in uninfected cells. The RdRp transcribes the genomic RNA into a complementary
negative-stranded RNA that subsequently serves as the template for the synthesis of new
positive-stranded RNA.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Many +RNA viruses (including coronaviruses, CHIKV and ZIKV) also encode
methyltransferases (MTases)11 that are required for methylations of viral mRNA cap
1
2
structures. Since this capping is crucial for stability and translation of the viral RNA, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
evasion of the host innate immune response, the viral MTases are considered promising targets
1
2
for the development of antiviral therapy. Inhibition of MTases can be indirectly achieved by
1
3
the inhibition of S-adenosyl-L-homocysteine (SAH) hydrolase. The SAH hydrolase catalyzes
the interconversion of SAH into adenosine and L-homocysteine. Inhibition of this enzyme
leads to the accumulation of SAH in the cell, which in turn inhibits S-adenosyl-L-methionine
(
SAM)-dependent transmethylase reactions by feedback inhibition.13,14 Most of the viral
methyltransferases are dependent on SAM as the only methyl donor. Compounds that target
cellular proteins might exhibit a broader spectrum of activity, are less likely to lead to drug-
resistance, but have a higher likelihood of toxicity. Compounds that are specifically aimed at
viral proteins are expected to be less cytotoxic, but might have a more narrow spectrum of
14
antiviral activity and might have a lower barrier antiviral drug-resistance Thus, the approach
of targeting cellular proteins such as SAH hydrolase can be considered as a promising strategy
1
4
for the development of broad-spectrum antiviral agents. A number of compounds have been
14
reported to act as SAH hydrolase inhibitors. Type I inhibitors act through inactivation of the
+
NAD cofactor, and their inhibitory effect on the catalytic activity of the enzyme can be
+
14
reversed by the addition of excess NAD . Type II inhibitors are irreversible inhibitors of the
SAH hydrolase that form covalent bonds with amino acid residues in the active site of the
+
enzyme. This irreversible inhibition cannot be reversed by the addition of NAD or adenosine
or by dialysis.14
Since both the viral RdRp and host SAH hydrolase are critical for virus replication, we
aimed to design broad-spectrum nucleoside analogue inhibitors that could directly target RdRp
activity and/or indirectly inhibit the methylation of viral RNA through their effect on the host
ACS Paragon Plus Environment

Page 5 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
SAH hydrolase. Modified nucleosides are usually taken up by the cell via nucleoside
transporters, and can be successively converted into mono-, di-, and triphosphates by cellular
1
5
kinases. Then. these modified nucleoside triphosphates (NTPs) can compete with natural
NTPs during RNA synthesis or can be incorporated into the nascent viral RNA, leading to
chain termination or detrimental mutations.15
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
N
N
O
O
N
B
B
N
X Y
X Y
HO
HO
O
HN P O
O
HO OH
HO OH
HO OH
()-Aristeromycin (1)
2
3
X = F, Y = H
X = H, Y = F
X = Y = F
B = pyrimidines and purines
Figure 1. Rationale for the design of the target nucleosides 2 and 3.
()-Aristeromycin (1) is a naturally occurring carbocyclic nucleoside, that was originally
identified as a metabolite of Streptomyces citricolor in 1967.16a The first synthesis of 1 as
racemate was reported by Clayton and his co-worker,16b-d and its asymmetric syntheses have
since been reported.16e-h It is a type I SAH hydrolase inhibitor and exhibits potent antiviral
activity against many viruses.14a However, it could not be further advanced into clinical
development because of its cytotoxicity.17 Compound 1 was found to be toxic at low
+
-
+
concentrations in both adenosine kinase positive (AK ) and AK cells. AK cells were
-
presumably killed by the 5'-phosphorylated form of 1, while the toxicity in AK cells was
1
7
caused by 1 itself. However, this compound is also metabolized into a triphosphate form and
1
7
has been observed to exert a variety of metabolic effects. We aimed to use 1 as a prototype
for the design of dual-target compounds intended at directly inhibiting the viral RdRp and
indirectly inhibiting the capping process through targeting of cellular SAH hydrolase.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Since the introduction of a fluorine at the 6-position of carbocyclic nucleosides has been
1
8
known to affect biological activities to a significant extent, we aimed to synthesize the 6'-
fluorinated-aristeromycin analogues 2 by introducing fluorine at the 6-position of 1 (Figure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
). Prisbe and his co-workers18a have reported the synthesis of (±)-6'-- and (±)-6'--fluorinated
aristeromycins and their inhibitory activity on SAH hydrolase, but the synthesis and biological
activity of (±)-6',6'-difluoroaristeromycin was not reported, despite the fact that the structure
was claimed in the patent.18b Thus, we set out to synthesize the 6'-fluorinated-aristeromycin
analogues 2 in the optically pure D-forms since biological activity can generally be attributed
to one enantiomer, the D-isomer. Schneller and co-workers18c reported the elegant synthesis of
optically pure (–)-6ʹ-β-fluoro-aristeromycin, but its biological activity was not reported. Their
synthetic route involved the 6--fluoroazide as the key intermediate, which was synthesized
by employing S 2 fluorination of the 6--triflic azide with tris(dimethylamino)sulfur
N
1
9
(
trimethylsilyl)difluoride (TASF), whereas our current approach included the stereoselective
electrophilic fluorination of silyl enol ether with Selectfluor® as the fluorine source. In addition
to the adenosine analogues, aimed at inhibiting SAH hydrolase and/or RdRp, we have also
synthesized 6ʹ-fluorinated purine and pyrimidine nucleosides (changes in B of the structures in
Fiugure 1), which could interfere with viral RNA synthesis by targeting the viral RdRp after
their phosphorylation by cellular kinases.15 To bypass the first and rate-limiting 5-
monophosphorylation step, we have also synthesized a phosphoramidate prodrug 3 of
2
0
nucleoside 2, using the McGuigan ProTides. Herein, we report the synthesis of the 6-fluoro-
aristeromycin analogues 2 and 3 and a preliminary characterization of their effect on several
+
RNA viruses, which provided insight into structure-activity relationships (SARs).
ACS Paragon Plus Environment

Page 7 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
■
Results and Discussion
Chemistry. For the synthesis of the target nucleosides 2, the key fluorosugars 8a-c were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
synthesized from D-ribose via electrophilic fluorination, as shown in Scheme 1.
Scheme 1. Synthesis of 6--Fluoro-, 6--Fluoro-, and 6-Difluorosugar 8a-c
t-BuO
t-BuO
O
O
OTES
ref 21
a
b
D-Ribose
70%
98%
O
O
O
O
O
O
4
5
6
c
0%
0%
0.9%
0.24%
O
F F
5F
H
H
5 H F
t-BuO
F F
t-BuO
t-BuO
t-BuO
OH
OH
b, c
0%
H
6
O
6
O
+
7
2
2
O
O
O
O
O
O
O
O
7d
7
a (76%)
7b (15%)
7
c
d
70%
H F
e
74%
d
76%
F H
F F
t-BuO
t-BuO
t-BuO
OH
OH
OH
O
O
O
O
O
O
8c
8a
8b
o
Reagents and conditions: a. LiCu(CH Ot-Bu) ; b. TESCl, LiHMDS, THF, -78 C, 10 min; c. Selectfluor,
2
o
2
o
o
DMF, 0 C, 12 h; d. NaBH , MeOH, 0 C, 30 min. e. LiBH , MeOH, 0 C, 30 min.
4
4
D-Ribose was converted to D-cyclopentenone 4 according to our previously published
procedure.21 The 1,4-conjugated addition of 4 with Gilman reagent yielded the D-
2
2
cyclopentanone derivative 5. Treatment of 5 with lithium hexamethyldisilazide (LiHMDS)
followed by trapping with triethylsilyl chloride (TESCl) gave silylenol ether 6, which was
treated with (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate):
Selectfluor) in DMF at 0 °C to yield a 5:1 ratio of 6-β-fluorosugar 7a to 6-α-fluorosugar 7b.19
1
The stereochemistry of the fluorine in 7a and 7b was confirmed by H NOE experiments.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Irradiation of 6-H of 7b gave NOE effects on its 2-H and 5-H, indicating the 6-α-fluoro
configuration, but no NOE effects were observed on the same experiment in the case of 7a,
confirming the 6-β-fluoro configuration. The configuration of the fluorine in 7b was further
confirmed by the X-ray crystal structure obtained after it was converted to the final uracil
derivative 2g (Scheme 5). Further electrophilic fluorination of 6-β-fluorosugar 7a or 6-α-
fluorosugar 7b under the same conditions yielded the 6,6-difluorosugar 7c, which was
equilibrated to form a geminal diol due to the presence of electronegative fluorine atoms.
Electrophilic fluorinations with other electrophilic fluorines such as N-
fluorobenzenesulfonimide (NFSI) or N-fluoro-O-benzenedisulfonimide (NFOBS) were
problematic, resulting in low yields with many side spots. The reduction of 7a-c with sodium
borohydride (NaBH ) or lithium borohydride (LiBH ) in MeOH resulted in the production of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
the 1-hydroxyl derivatives 8a-c.
As the α-fluoro derivative 8b was obtained as the minor isomer, as shown in Scheme 1, we
23
wanted to improve the stereoselective synthesis of 8b, by using Rubottom oxidation as the
key step, as illustrated in Scheme 2. Rubottom oxidation of silylenol ether 6 with osmium
tetroxide (OsO ) and N-methylmorpholine-N-oxide (NMO) followed by trapping with t-
4
butyldimethylsilyl chloride (TBSCl) produced 6-β-alkoxyketone 9 as a single stereoisomer in
5
3% yield. The reduction of ketone 9 with NaBH gave alcohol 10, which was protected with
4
a benzyl group to give 11. Removal of the TBS group in 11 with tetra-n-butylammonium
fluoride (TBAF) yielded the 6-β-alcohol 12. To our disappointment, the treatment of 12 with
N,N-diethylaminosulfur trifluoride (DAST) gave the desired product, 6-α-fluoride 13a, but also
the undesired product 1-β-fluoride 13b at a 1:1 ratio. The formation of 13a (route I) resulted
from the direct S 2 reaction of 12a with fluoride, while 12a was readily converted into the
N
oxonium ion 12b (route II) via its participation of the neighboring benzyl group, which was
attacked exclusively by the fluoride at the less sterically hindered 1-position to yield the
ACS Paragon Plus Environment

Page 9 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
undesired product 13b (route III). However, the product via route IV was not formed because
of the steric effect of t-butyloxymethyl substituent.
Scheme 2. Synthetic Approach to 6--Fluorosugar 8b via Rubottom Oxidation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OTBS
OTBS
a
t-BuO
b
t-BuO
d
6
O
53%
88%
88%
OR
O
O
O
O
9
10 (R = H)
c
85%
11 (R = Bn)
NEt2
OH
F
t-BuO
F S F
t-BuO
I
F
O
6
e
route I
S 2
t-BuO
OBn
OBn
N
O
O
O
O
30%
OBn
II
12
13a
O
O
12a
13a:13b = 1:1
route II
F
IV
X
OBn
Bn
route IV
t-BuO
route III
F
No reaction
O
t-BuO
III
30%
1
O
O
O
O
13b
12b
Reagents and conditions: a. i. OsO , NMO  H O, THF, rt, 1 h, then NaHCO , MeOH,
4
2
3
rt, 3 h; ii. TBSCl, imidazole, DMF, rt, 3 h; b. NaBH , MeOH, rt, 1 h; c. BnBr, NaH,
4
o
o
DMF, 0 C to rt, 12 h; d. TBAF, THF, rt, 12 h; e. DAST, toluene, 0 C to rt, 2 h.
To avoid the participation of the neighboring group, we considered using a cyclic sulfate
substrate with electron-withdrawing property and conformational restraint to be the best choice.
Furthermore, cyclic sulfate has the advantage that it can be utilized as a surrogate for epoxide
during nucleobase condensation, as shown in Scheme 3. The regioselective cleavage of the 2,3-
acetonide in 10 with trimethylaluminum (AlMe ) followed by treatment of the resulting diol
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with thionyl chloride (SOCl ) yielded the 6-β-hydroxyl cyclic sulfite 14 after the removal of
2
the TBS group. The treatment of 14 with DAST yielded the desired 6-α-fluoro cyclic sulfite 15
as a single stereoisomer. The cyclic sulfite 15 was oxidized to form cyclic sulfate 16, which
was subsequently condensed with 6-chloropurine anion; however, this resulted in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
decomposition. Thus, we decided to synthesize the 6-α-fluoro derivative 8b according to
Scheme 1.
Scheme 3. Synthetic Approach to 6--Fluorosugar 8b via Cyclic Sulfate
OH
F
a,b,c
8%
t-BuO
t-BuO
d
10
4
O
S
O
S
t-BuO
O
t-BuO
O
14
15
O
O
e
Cl
N
F
N
N
t-BuO
N
f
O
F
t-BuO
t-BuO
O
S
O
O
16
t-BuO OH
17
Reagents and conditions: a. AlMe , CH Cl , -78 oC to rt, 12 h; b.
3
2
2
o
SOCl , Et N, CH Cl , 0 C, 10 min; c. TBAF, AcOH, THF, rt, 12 h; d.
2
3
2
2
o
DAST, CH Cl , 0 C to rt, 4 h; e. RuCl , NaIO , CCl :CH CN:H O
2
2
3
4
4
3
2
o
(
1/1/1.5), rt, 20 min; f. i. 6-chloropurine, 18-crown-6, NaH, THF, 65 C,
15 h; ii. 20% H SO , rt, 1 h.
2
4
Scheme 4 depicts the synthesis of the aristeromycin analogues 2a-e from the 6-β-fluoro-, 6-α-
1
9
fluoro-, and 6,6-difluorosugars 8a-c. Compounds 8a-c were treated with triflic anhydride
(
Tf O) followed by treatment with sodium azide to give azido derivatives 18a-c. The catalytic
2
hydrogenation of 18a-c yielded the amino derivatives 19a-c, respectively, which are starting
compounds for the base-building process. The treatment of 19a-c with 5-amino-4,6-
dichloropyrimidine18a-c,24 in the presence of N,N-diisopropylethylamine (DIPEA) under
ACS Paragon Plus Environment

Page 11 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
microwave radiation conditions yielded 20a-c, which were cyclized with diethoxymethyl
acetate18a-c,24 in the presence of microwave radiation to produce the 6-chloropurine derivatives
21a-c. The treatment of 21a-c with t-butanolic ammonia followed by the removal of protective
groups under acidic conditions yielded the 6-β-fluoro-, 6-α-fluoro-, and 6,6-
difluoroaristeromycins 2a-c, respectively. The structure of compound 2c was confirmed by a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
single-crystal X-ray analysis (see the Supporting Information). The treatment of 21a and 21c
with 40% aqueous methylamine followed by aqueous trifluoroacetic acid (TFA) resulted in N6-
methyl-aristeromycin analogues 2d and 2e, respectively.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4. Synthesis of -Fluoro-, -Fluoro-, and Difluoro-aristeromycin Analogues 2a-e
N3
NH2
X Y
X Y
t-BuO
t-BuO
a
b
8a-c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
a (X = F, Y = H)
b (X = H, Y = F)
c (X = Y = F)
18a (45%)
18b (88%)
18c (75%)
19a-c
c
Cl
N
Cl
N
H N
2
NH2
N
N
N
N
N
HN
X Y
N
X Y
t-BuO
t-BuO
N
e, g
d
N
X Y
HO
O
O
O
O
HO OH
21a (96%)
21b (76%)
21c (92%)
20a (66% from 18a)
20b (47% from 18b)
20c (67% from 18c)
2a (46%)
2b (65%)
2c (48%)
f, g
NHMe
N
N
N
N
X Y
HO
HO OH
d (X = F, Y = H, 69% from 21a)
2
2e (X = Y = F, 66% from 21c)
X-ray crystal structure of 2c
o
o
Reagents and conditions: a. i. Tf O, pyridine, 0 C, 30 min; ii. NaN , DMF, 60-100 C, 4-15 h; b.
2
3
o
Pd/C, H , MeOH, rt, 18 h; c. 5-amino-4,6-dichloropyrimidine, DIPEA, n-BuOH, 170-200 C, 4-7 h,
2
o
o
MW; d. CH C(O)OCH(OEt) , 140 C, 3 h, MW; e. NH /t-BuOH, 120 C, 15 h; f. NH Me/H O, (40
3
2
3
2
2
o
o
wt%), EtOH, 30 C, 2 h; g. 67% aq TFA, 50 C, 15 h.
ACS Paragon Plus Environment

Page 13 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 5. Synthesis of Fluorinated Pyrimidine Nucleoside Analogues 2f-j
O
O
MeO
NH
NH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19a (X = F, Y = H)
19b (X = H, Y = F)
19c (X = Y = F)
HN
X Y
O
a
b
N
O
t-BuO
X Y
HO
O
O
HO OH
22a (76%)
22b (88%)
22c (90%)
2f (X = F, Y = H, 56%)
2g (X = H, Y = F, 53%)
2h (X = Y = F, 52%)
c
O
NH
N
O
X Y
BzO
BzO OBz
2
2
3a (X = F, Y = H, 75% from 2f)
3b (X = Y = F, 61% from 2h)
X-ray crystal structure of 2g
d
NH2
N
N
O
X Y
HO
HO OH
2
2
i (X = F, Y = H, 24%)
j (X = F, Y = F, 33%)
X-ray crystal structure of 2h
Reagents and conditions: a. (E)-3-methoxy-2-propenoyl isocyanate, benzene, 4Å-MS,
o
DMF, -20 C to rt, 15 h; b. 2 M H SO , dioxane, reflux, 1.5 h; c. BzCl, pyridine, CH Cl ,
2
4
2
2
rt, 15 h; d. i. 1,2,4-triazole, POCl , Et N, CH CN, rt, 15 h. ii. NH OH, dioxane, rt, 15 h. iii.
3
3
3
4
NH /MeOH, rt, 15 h
3
The amino derivatives 19a-c were also converted into the pyrimidine nucleoside derivatives
2
f-j, as shown in Scheme 5. Treatment of 19a-c with (E)-3-methoxy-2-propenoyl isocyanate,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
which was prepared by reacting 3-methoxyacryloyl chloride with silver isocyanate,26 in
benzene produced 22a-c, respectively, which were cyclized with 2 M H SO to yield the
2
4
uridine derivatives 2f-h, respectively. The structures of 2g and 2h were confirmed by the X-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27
ray crystallography (see the Supporting Information) (Scheme 5). To synthesize the cytidine
derivatives 2i and 2j, compounds 2f and 2h were benzoylated to give 23a and 23b, respectively,
which were converted to the cytidine derivatives 2i and 2j using a conventional three step
procedures.28
ACS Paragon Plus Environment

Page 15 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 6. Synthesis of Phosphoamidate Prodrugs 3a-c
NH2
NR R
1
2
N
N
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
F F
F F
HO
HO
a
b
2c
O
O
O
O
24 (96%)
25a (R1 = Boc, R2 = H) (52%)
25b (R = R = Boc) (25%)
1
2
c
32%
NHBoc
NH2
N
N
N
N
N
N
O
O
N
O
O
O
d
O
F F
N
HN P O
OPh
F F
HN P O
OPh
O
O
HO OH
3a (82%)
26
O
N
O
NH
NH
O
O
O
N
O
X Y
HO
X Y
O
HN P O
OPh
a
c,d
2
2
f (X = F, Y = H)
h (X = Y = F)
O
O
HO OH
2
2
7a (98%)
7b (97%)
3b (X = F, Y = H) (30%)
3c (X = Y = F) (30%)
Reagents and Conditions: a. cH SO , acetone, rt, 4 h; b.
2
4
i. TMSOTf, DMAP, HMDS, 75 °C, 2 h; ii. Boc O, THF, rt,
2
O
O
F
F
F
F
4
h; iii. MeOH:Et N (5:1), 55 °C, 16 h; c. A, t-BuMgCl,
3
O
HN P O
OPh
o
4Å-MS, THF, 0 C to rt, 36 h; d. 50% HCOOH, rt, 8 h.
F
A
2
0
The uracil phosphoramidate analogue Sofosbuvir is used in the clinic as a powerful anti-
hepatitis C virus (HCV) agent. Therefore, we have also synthesized the uracil phosphoramidate
prodrugs 3b-c and the adenine phosphoramidate prodrug 3a derived from the purine and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pyrimidine nucleoside analogues 2a-j by using McGuigan’s ProTide prodrug methodology,20
as shown in Scheme 6. 6,6-Difluoro-aristeromycin (2c) was treated with acetone under acidic
conditions to give 2,3-acetonide 24. The treatment of 24 with di-tert-butyl dicarbonate (Boc O)
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yielded a mixture of 25a and 25b in a 2:1 ratio, which was converted to the phosphoramidate
prodrug 26 by treating with phosphoramiditing reagent (A)29 in the presence of t-
butylmagnesium chloride. The treatment of 26 with 50% formic acid produced the final product,
prodrug 3a. The monofluoro- and difluoropyrimidine derivatives 2f and 2h were similarly
converted to the final prodrugs 3b and 3c.
Table 1. Inhibition of SAH hydrolase and the replication of several +RNA viruses by all final
nucleoside analogues 2a-j and 3a-c
SAH
hydrolase
MERS-CoV
SARS-CoV
ZIKV
CHIKV
Compound
No.
IC50
EC50
(M) (M)
CC50
EC50
(M) (M)
CC50
EC50
(M) (M)
CC50
EC50
CC50
SI
SI
SI
SI
(M) (M)
0.8 6.3
>100 >100
(M)
1
1.32
0.37
>50
2
>50
ND
>5
0.64
ND
2.4
3.8
7.9
2
a
0.20
0.60
3
ND
ND
2b
9.70
1.06
ND
0.2
ND
3.2
ND
0.5
ND
5.9
2.54
0.26
3.97
>2.5
1.56
>9.6
0.53
0.13
1.32
2.49
2
c
16
11.8
>1.25 >9.6
2d
4.39
>50
>50
>50
12.5
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
2e
2f
0.76
>100
>100
>100
>100
>100
>100
>100 >100
>100 >100
2
g
2h
>50
>50
2i
>100 >100
2j
>50
9.3
>50
>50
3
a
6.8
>25
>3.7
1.75
>25
>14.3
1.95
>12.5 >6.4
ACS Paragon Plus Environment

Page 17 of 65
Journal of Medicinal Chemistry
1
2
3
4
3
b
>100
>100
>50
>50
>50
>50
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
c
ND: Not Determined; Selectivity Index (SI) = CC50/EC50
EC50: Effective concentration to inhibit the replication of the virus by 50%
CC50: Cytotoxic concentration to inhibit the replication of normal cells by 50%
EC50>100 indicates that no antiviral activity was observed at the highest concentration tested, either because there
was no protection or the compound was toxic.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition of SAH hydrolase. All compounds 1, 2a−j and 3a−c, were assayed for their ability
to inhibit recombinant human SAH hydrolase protein, expressed in E. coli JM109, using a 5,5′-
3
0
dithiobis-2-nitrobenzoate (DTNB) coupled assay as described by Lozada-Ramirez et al. As
expected, all adenosine derivatives 2a-e potently inhibited SAH hydrolase, but none of the
pyrimidine analogues 2f-j showed any inhibitory activity at concentrations up to 100 µM. None
of the prodrugs 3a-c exhibited inhibitory activity at concentrations up to 100 µM. This result
is not surprising because adenosine is the substrate for SAH hydrolase. Among the adenosine
analogues, 6-β-fluoroaristeromycin (2a) exhibited the most potent inhibitory activity (IC =
5
0
0.37 µM), which was 3.6-fold more potent than the control 1 (IC = 1.32 µM). However, 6-
5
0
α-fluoroaristeromycin (2b, IC = 9.70 µM) was 26-fold less potent than the corresponding 6-
5
0
β-fluoro analogue 2a and 7.4-fold less active than the 6-unsubstituted compound 1. This
indicates that the stereochemistry at the 6-position is important for inhibitory activity.
Interestingly, the introduction of two fluorines at the 6'-position, resulted in 2c (IC = 1.06
5
0
µM), which was slightly more potent than the control 1. The inhibitory activity of the 6-fluoro-
aristeromycin series can be ranked in the following order: 6-β-F > 6,6-F,F > 6-H > 6-α-F.
6
The introduction of a methyl group at the N -amino group of 2a, resulting in 2d, decreased the
inhibitory activity (IC = 4.39 µM) by 11.9-fold, while the addition of a methyl group to the
5
0
6
N -amino group of 2c, resulting in 2e, increased the inhibitory activity (IC = 0.76 µM) by
5
0
6
1
.7-fold. These results demonstrate that the N -mehyladenine and the adenine moieties do not
lead to a decrease in inhibitory activity.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Antiviral activity. The novel 6'-fluoro-aristeromycin analogues 2a-j and 3a-c were screened
for antiviral activity against a variety of +RNA viruses. The compounds were tested for
antiviral activity in cytopathic effect (CPE) reduction assays at 4 concentrations, i.e. 150, 50,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6.7, and 5.6 µM by preaparing 3-fold serial dilutions. Compounds that demonstrated antiviral
activity in this primary screen were further tested more extensively in dose response
experiments at 8 different concentrations to determine the EC . Cytotoxicity (CC ) was
5
0
50
determined in parallel in uninfected cells (Table 1).
As shown in Table 1, only the adenosine derivatives 2a-c exhibited potent antiviral
6
activities against +RNA viruses, while the other purine N -methyladenine derivatives 2d and
2
e and pyrimidine derivatives 2f-j did not show significant antiviral activities, not even at
100µM. This result suggests that the antiviral activity might be due to an (indirect) effect on
viral MTase activity through the inhibition of host SAH hydrolase. Inhibition of the viral RdRp
appears not to be important. The mechanism of action of these compounds has been studied in
more detail and results will be published elsewhere.
Compound 2a inhibited MERS-CoV replication with an EC of 0.20 µM; however, it was also
5
0
rather cytotoxic, resulting in a selectivity index (SI) of 3. Replacement of the remaining 6-H
in 2a with F, resulted in compound 2c, which exhibited a > 5-fold reduction in cytotoxicity,
while its antiviral activity remained unchanged, with an EC of ~0.20 µM and a SI of 15 for
5
0
MERS-CoV. This compound was also active against SARS-CoV with a SI of 12.5, suggesting
that it may be a broad-spectrum coronavirus inhibitor. In addition, it also inhibited ZIKV
replication with an EC of 0.26 µM (SI >10), and was active against CHIKV with an EC of
5
0
50
0
.13 µM. Compound 2b showed some inhibitory effects on CHIKV and ZIKV replication, but
this was likely due to pleiotropic cytotoxic effects, as the SI was <3. Among the
phosphoramidate prodrugs 3a-c, only the adenosine prodrug 3a exhibited significant broad-
ACS Paragon Plus Environment

Page 19 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
spectrum antiviral activities, demonstrating that it may inhibit the RdRp of RNA viruses after
conversion into the triphosphate form, although it remains to be determined in biochemical
2
0
assays whether the triphosphate form affects RdRp activity. Compound 3a had an EC of
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
.3 µM for MERS-CoV and 6.8 µM for SARS-CoV, but it also had a SI<10, and it was
therefore not considered a potent inhibitor of coronavirus replication. However, for CHIKV
and ZIKV, 3a had EC values of 1.95 µM and 1.75 µM, respectively with good selectivity
5
0
indices. Interestingly, the prodrug 3a was less potent, but also much less cytotoxic than the
parent compound 2c, which is unusual as regularly the phosphoamidate is more potent than the
2
0
parent drug. The phosphoamidate 3a might be slowly hydrolyzed to the 5-monophosphate
by metabolic enzymes, or to the parent drug 2c by a phosphatase, which could inhibit SAH
hydrolase, explaining the observed antiviral effect. Viral load reduction assays were performed
with compound 2c by infecting cells with CHIKV, ZIKV, SARS-CoV and MERS-CoV,
followed by treatment with different concentrations of 2c. At 30 hpi (CHIKV) or 48 hpi (ZIKV,
SARS- and MERS-CoV) infectious progeny titers in the medium were determined by plaque
assay (Figure 2). Treatment with concentrations higher than 1 μM of 2c reduced infectious
CHIKV titers by more than 2 log. The effect on ZIKV infectious progeny titers was limited
and showed a ~1 log reduction. For SARS-CoV the reduction in infectious progeny titer was
~1.5 log at 2c concentrations above 0.3 μM. The strongest antiviral effect was observed for
MERS-CoV, with a ~2.5 log reduction in infectious progeny titers when infected cells were
treated with 2c concentrations above 0.3 μM. Follow-up studies to gain more insight into the
mode of action of 2c and 3a and related compounds are currently ongoing and results will be
published elsewhere.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2: Effect of 2c on the infectious progeny of CHIKV, ZIKV, SARS-CoV and MERS-
CoV. Cells were infected with the virus indicated on the y-axis of the graph in medium with
various concentrations of 2c. Infectious progeny titers were determined by plaque assay (n =
4
) and viability of non-infected cells was monitored using the CellTiter 96®AQueous Non-
Radioactive Cell Proliferation Assay (Promega). Significant differences are indicated by *: *,
p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
Finally, we measured the logP of the most active compound 2c by pH-metric method,
using a T3 Sirius instrument, because the lipophilicity is a major determinant for compound
absorption, distribution in the body, penetration across biological barriers, metabolism and
excretion. The measured logP was 0.02, indicating that it is almost equally partitioned between
ACS Paragon Plus Environment

Page 21 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the lipid and aqueous phases. The relatively low logP of 2c is expected to be overcome by
converting it to the phosphoamidate 3a.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SAH Hydrolase Inhibitory Activity
1. 6'-fluorine (X,Y): -fluorine > difluorine > H,H > -fluorine
6
2
. Base (B): adenine > N -methyladenine >> pyrimidine
3. R: H >> P
NH2
N
N
B
X Y
N
RO
N
HO
HO OH
HO OH
2 and 3
(
)-Aristeromycin (1)
Anti-RNA Virus Activity
1
. MERS-CoV (X,Y/B/R): F,H/A/H = F,F/A/H > F,F/A/P >> ()-aristeromycin
2
. SARS-CoV (X,Y/B/R): F,F/A/H > F,F/A/P >> ()-aristeromycin
3
. ZIKA (X,Y/B/R): F,F/A/H > ()-aristeromycin > F,F/A/P > H,F/A/H
4
. CHIKV (X,Y/B/R): F,F/A/H > H,F/A/H > ()-aristeromycin > F,F/A/P
P = phosphoramidate
Figure 3. Summarized SAR of 6'-fluorinated aristeromycin analogues 2 and 3.
■
CONCLUSION
We have synthesized the 6-fluorinated aristeromycin analogues 2a-j, which were designed as
dual-target antiviral compounds aimed at inhibiting both the viral RdRp and the host SAH
hydrolase. The electrophilic fluorination of silyl enol ether with Selectfluor was the key step in
the synthesis. We have also synthesized the phosphoramidate prodrugs 3a-c to determine
whether these would inhibit virus replication through an effect on the viral RNA polymerase.
Figure 3 depicts the summarized SAR of the synthesized 6-fluorinated final nucleoside
analogues, 2a-j and 3a-c concerning the inhibition of human SAH hydrolase and the inhibition
of the replication of various +RNA viruses with capped genomes. It was discovered that the
introduction of fluorine at the 6-position increases the inhibitory activity on SAH hydrolase
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and the replication of selected +RNA viruses. Compared to the 6-unsubstituted compound 1,
the 6-fluorinated aristeromycin analogues 2a and 2c more potently inhibited SAH hydrolase
activity and the replication of MERS-CoV, SARS-CoV, ZIKV, and CHIKV. Among these
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds, 6-β-fluoroaristeromycin (2a) was the most potent with an IC of 0.37 µM for
50
SAH hydrolase activity and an EC of 0.20 µM for MERS-CoV replication. There was a
5
0
correlation between the inhibition of SAH hydrolase and the antiviral activity of the compounds,
suggesting the latter was mainly due to indirect targeting of viral methylation reactions. The
SAR studies and lack of antiviral effect of several purine and pyrimidine analogues suggests
that the antiviral effect of 1, 2a, and 2c is unlikely due to targeting of the viral RdRp. Compound
2
c appears to be an interesting compound for further development and evaluation as a broad-
spectrum antiviral agent, as it inhibited several coronaviruses, CHIKV, and ZIKV. More
detailed biological studies on the efficacy of these compounds in virus-infected cells and into
their mode of action are currently ongoing and will be published elsewhere.
■
Experimental section
1
13
Chemical Synthesis. General Methods. Proton ( H) and carbon ( C) NMR spectra were
obtained on a Bruker AV 400 (400/100 MHz), Bruker AMX 500 (500/125 MHz), Jeol JNM-
ECA600 (600/150 MHz), or Bruker AVANCE III 800 (800/200 MHz) spectrometer. Chemical
shifts are reported as parts per million (δ) relative to the solvent peak. Coupling constants (J)
are reported in hertz (Hz). Mass spectra were recorded on a Thermo LCQ XP instrument.
Optical rotations were determined on Jasco III in appropriate solvent. UV spectra were
recorded on U-3000 made by Hitachi in methanol or water. Infrared spectra were recorded on
FT-IR (FTS-135) made by Bio-Rad. Melting points were determined on a Buchan B-540
instrument and are uncorrected. The crude compounds were purified by column
ACS Paragon Plus Environment

Page 23 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
chromatography on a silica gel (Kieselgel 60, 70-230 mesh, Merck). Elemental analyses (C, H,
and N) were used to determine the purity of all synthesized compounds, and the results were
within ± 0.4% of the calculated values, confirming ≥ 95% purity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(((3aR,6R,6aR)-6-(tert-Butoxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)oxy)triethylsilane (6). To a cooled (–78 °C) solution of 5
(1568.0 mg, 6.470 mmol) in anhydrous THF (32.0 mL, 0.2 M) was dropwise added
chlorotriethylsilane (5.4 mL, 32.355 mmol), followed by addition of LiHMDS (19.0 mL, 1.0
M solution in THF, 19.0 mmol) under N . After being stirred at the same temperature for 10
2
min, the reaction mixture was quenched with saturated aqueous NH Cl (80 mL). The layers
4
were separated, and the aqueous layer was extracted with EtOAc (150 mL). The combined
organic layers were washed successively with H O and saturated brine, dried over anhydrous
2
MgSO , filtered, and evaporated. The residue was purified by column chromatography (silica
4
gel, hexanes/EtOAc, 100/1 to 30/1) to give 6 (2267.0 mg, 98%) as colorless oil: []D20 = +36.48
1
(
c 1.23, CHCl ); H NMR (400 MHz, CDCl )  4.73 (dd, J = 1.1, 6.0 Hz, 1 H), 4.58 (d, J = 2.1
3
3
Hz, 1 H), 4.36 (d, J = 6.1 Hz, 1 H), 3.27 (dd, J = 5.6, 8.6 Hz, 1 H), 3.15 (dd, J = 6.6, 8.6 Hz, 1
H), 2.72 (dd, J = 5.9, 5.9 Hz, 1 H), 1.42 (s, 3 H), 1.32 (s, 3 H), 1.12 (s, 9 H), 0.96 (t, J = 8.0
13
Hz, 9 H), 0.66-0.72 (m, 6 H); C NMR (100 MHz, CDCl )  154.1, 110.3, 104.4, 82.8, 79.7,
3
7
2.5, 63.9, 47.9, 27.4 (3 × CH -tert-butyl), 27.3, 25.8, 6.5 (3 × triethylsilyl), 4.6 (3 ×
3
triethylsilyl); IR (neat) 2973, 1648, 1363, 1262, 1204, 1056, 851, 748 cm-1; HRMS (FAB)
found 356.2388 [calcd for C H O Si+ (M+H)+ 356.2383].
1
9
36
4
(3aR,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyldihydro-3aH-
cyclopenta[d][1,3]dioxol-4(5H)-one (7a) and (3aR,5S,6R,6aR)-6-(tert-butoxymethyl)-5-
fluoro-2,2-dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (7b). To a cooled (0
oC) solution of silyl enol ether 6 (8.75 g, 24.548 mmol) in anhydrous DMF (123.0 mL, 0.20
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
M)
was
added
1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane
bis(tetrafluoroborate) (13.04 g, 36.824 mmol, Selectfluor) in one portion under N . After being
2
stirred at the same temperature for 12 h, the reaction mixture was quenched with saturated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aqueous NH Cl (130 mL), diluted with EtOAc (130 mL). The layers were separated and the
4
aqueous layer was extracted with EtOAc (2 × 100 mL). The combined organic layers were
washed successively with H O and saturated brine, dried over anhydrous MgSO , filtered, and
2
4
evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc,
1
4
0/1 to 20/1) to give 7a and 7b (5.80 g, 91%, total yield, 7a:7b = 5.2:1 by H NMR analysis).
Compound 7a: white solid; []D25 = –156.69 (c 0.735, CHCl ); H NMR (400 MHz, CDCl )
1
3
3

5.29 (dd, J = 8.2, 49.5 Hz, 1 H), 4.70 (t, J = 5.7 Hz, 1 H), 4.20 (dd, J = 2.4, 6.1 Hz, 1 H), 3.61
(dd, J = 1.6, 8.6 Hz, 1 H) 3.38-3.41 (m, 1 H), 2.75 (d, J = 8.2 Hz, 1 H), 1.41 (s, 3 H), 1.30 (s,
1
3
3
2
H), 1.06 (s, 9 H); C NMR (100 MHz, CDCl )  203.0 (d, J = 12.9 Hz), 111.4, 88.5 (d, J =
3
01.5 Hz), 78.2 (d, J = 6.9 Hz), 75.0 (d, J = 3.1 Hz), 74.3, 56.6 (d, J = 6.6 Hz), 40.5 (d, J =
1
9
15.5 Hz), 26.8 (3 × CH -tert-butyl), 26.2, 23.6; F NMR (376 MHz, CDCl )  –220.60~221.14
3
3
+
+
(m); LRMS (ESI+) found 283.13 [calcd for C H FO Na (M+Na) 283.1322]; Anal. Calcd
13 21 4
for C H FO : C, 59.98; H, 8.13. Found: C, 59.99; H, 8.53.
1
3
21
4
Compound 7b: white solid; []D25 = –83.72 (c 0.495, CHCl ); H NMR (600 MHz, CDCl ) 
1
3
3
5
.21-5.36 (ddd, J =1.3, 4.5, 50.8 Hz, 1 H), 4.55 (d, J = 5.9 Hz, 1 H), 4.50 (d, J = 5.9 Hz, 1 H),
1
3
3
.63 (d, J = 2.2 Hz, 2 H), 2.52-2.58 (m, 1 H), 1.41 (s, 3 H), 1.33 (s, 3 H), 1.13 (s, 9 H); C
NMR (150 MHz, CDCl )  207.8 (d, J = 12.9 Hz), 112.2, 91.9 (d, J = 192.4 Hz), 78.78 (d, J =
3
3
2
.5 Hz), 78.74, 73.6, 60.5 (d, J = 4.3 Hz), 45.0 (d, J = 17.9 Hz), 27.2 (3 × CH -tert-butyl), 26.8,
3
1
9
5.2; F NMR (376 MHz, CDCl )  –196.0~196.2 (m); HRMS (FAB) found 262.1679 [calcd
3
+
+
for C H FO (M+H) 261.1505]; Anal. Calcd for C H FO : C, 59.98; H, 8.13. Found: C,
1
3
22
4
13 21
4
5
9.77; H, 8.45.
ACS Paragon Plus Environment

Page 25 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
3aR,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyldihydro-3aH-
cyclopenta[d][1,3]dioxol-4(5H)-one (7c). Yield = 70% (mixture of 7c and 7d); white solid;
]D25 = –4.34 (c 0.21, MeOH); H NMR (7c and 7d mixture, 400 MHz, CDCl ; 7c and 7d
1
[
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mixture)  4.82 (s, 1 H), 4.72 (t, J = 6.1 Hz, 1 H), 4.52-4.57 (m, 1 H), 4.35-4.41 (m, 1 H), 4.25
(dd, J = 8.0, 4.0 Hz, 1 H), 3.74 (s, 1 H), 3.69 (d, J = 8.0 Hz, 1 H), 3.67-3.60 (m, 1 H), 3.54-
3
.59 (m, 1 H), 3.46 (d, J = 8.3 Hz, 1 H), 2.68 (d, J = 17.4 Hz, 1 H), 2.53-2.62 (m, 1 H), 1.48 (s,
3
H), 1.44 (s, 3 H), 1.34 (s, 3 H), 1.32 (s, 3 H), 1.21 (s, 9 H), 1.06 (s, 9 H).
General procedure for the synthesis of 8a-c. To a cooled (0 °C) solution of 7a-c (1 equiv) in
MeOH (0.18 M) sodium borohydride or lithium borohydride was added in a single portion in
a N atmosphere. After stirring for 30 min at the same temperature, the reaction mixture was
2
neutralized with acetic acid (2 mL) and evaporated. The residue was diluted with saturated
aqueous NH Cl, and the aqueous layer was extracted with EtOAc (2  100 mL). The combined
4
organic layers were dried over anhydrous MgSO , filtered, and evaporated. The residue was
4
purified by column chromatography (silica gel, hexanes/EtOAc, 20/1) to give 8a-c.
(3aS,4R,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (8a). Yield = 71%; colorless syrup; []D25 = –47.46 (c 0.395,
1
CHCl ); H NMR (400 MHz, CDCl )  4.91 (td, J = 6.6, 52.5 Hz, 1 H), 4.51-4.52 (m, 1 H),
3
3
4
.47 (ddd, J = 1.6, 6.3, 7.8 Hz, 1 H), 4.26-4.34 (m, 1 H), 3.52 (dd, J = 3.3, 8.8 Hz, 1 H), 3.36-
.39 (m, 1 H), 2.67 (d, J = 7.9 Hz, 1 H), 2.46 (bs, 1 H), 1.45 (s, 3 H), 1.32 (s, 3 H), 1.14 (s, 9
3
1
3
H); C NMR (100 MHz, CDCl )  111.1, 99.5 (d, J = 185.9 Hz), 81.2 (d, J = 4.4 Hz), 76.3 (d,
3
J = 9.0 Hz), 74.0 (d, J = 23.4 Hz), 73.0, 56.8 (d, J = 8.2 Hz), 44.6 (d, J = 18.1 Hz), ), 27.3 (3 ×
1
9
CH -tert-butyl), 26.1, 24.1; F NMR (376 MHz, CDCl ) –211.0~211.21 (m); HRMS (FAB)
3
3
+
found 263.1662 [calcd for C H FO (M+H)+ 263.1659]; Anal. Calcd for C H FO : C,
1
3
24
4
13 23
4
5
9.52; H, 8.84. Found: C, 59.32; H, 9.15.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
3aS,4R,5S,6R,6aR)-6-(tert-Butoxymethyl)-5-fluoro-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (8b). Yield = 67%; colorless syrup; []D25 = –40.42 (c 0.22,
1
MeOH); H NMR (500 MHz, CDCl )   dd J   52.4 Hz, 1 H), 4.46-4.53 (m, 2 H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
.13-4.24 (m, 1 H), 3.33-3.40 (m, 1 H), 2.81 (d, J = 11.4 Hz, 1 H), 2.50 (dt, J = 2.9, 22.9 Hz, 1
1
3
H), 1.46 (s, 3 H), 1.30 (s, 3 H), 1.08 (s, 9 H); C NMR (125 MHz, CDCl )    d, J
3
=
181.5 Hz,), 82.8, 79.3, 73.8(d, J = 16.3 Hz), 73.0, 60.6 (d, J = 12.1 Hz), 49.2 (d, J = 18.3
+
Hz), 27.1 (3 × CH -tert-butyl), 26.2, 24.2; HRMS (ESI ) found 285.1480 [calcd for
3
+
+
C H FNaO (M+Na) 285.1478]; Anal. Calcd for C H FO : C, 55.70; H, 7.91. Found: C,
1
3
23
4
13 23
4
5
5.40; H, 7.75.
(3aS,4R,6R,6aR)-6-(tert-Butoxymethyl)-5,5-difluoro-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (8c). Yield = 74%; colorless syrup; []D25 = 22.37 (c 0.28,
1
MeOH); H NMR (500 MHz, CDCl )  3 (t, J = 5.7 Hz, 1 H), 4.44 (ddd, J = 2.6, 6.4, 8.9
3
Hz, 1 H), 4.20-4.29 (m, 1 H), 3.55 (d, J = 8.7 Hz, 1 H), 3.39 (d, J = 8.8 Hz, 1 H), 2.76 (d, J =
1
3
11.5 Hz, 1 H), 2.43 (d, J = 17.2 Hz, 1 H), 1.46 (s, 3 H), 1.31 (s, 3 H), 1.12 (s, 9 H); C NMR
125 MHz, CDCl )  126.9 (dd, J = 252.3, 260.3 Hz), 110.9, 79.6 (d, J = 5.9 Hz), 75.5 (d, J =
(
3
1
2
1.3 Hz), 73.7 (dd, J = 18.5, 25.8 Hz), 73.4, 57.6 (dd, J = 4.6, 8.5 Hz), 48.7 (t, J = 20.8 Hz),
+
+
7.2 (3 × CH -tert-butyl), 25.9, 24.2; HRMS (ESI ) found 298.1834 [calcd for C H F NO
4
3
13 26
2
+
(M+NH ) 298.1830]; Anal. Calcd for C H F O : C, 55.70; H, 7.91. Found: C, 55.45; H, 7.56.
4 13 22 2 4
(3aR,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-((tert-butyldimethylsilyl)oxy)-2,2-
dimethyldihydro-3aH-cyclopenta[d][1,3]dioxol-4(5H)-one (9). To a cooled (0 °C) solution
of 6 (1275 mg, 3.57 mmol) in anhydrous THF (12 mL, 0.3 M) was added 4-methylmorpholine
N-oxide monohydrate (967 mg, 7.15 mmol, 2 equiv) and osmium tetroxide (1000 mg, 3.93
mmol, 1.1 equiv) under N atmosphere. After stirring for 30 min, the reaction mixture was
2
ACS Paragon Plus Environment

Page 27 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
added sodium thiosulfate pentahydrate (300 mg), sodium sulfite (300 mg) and acetone (30 mL)
and stirred additional 1 h at the same temperature. The layers were separated, and the aqueous
layer was extracted with EtOAc (100 mL). The combined organic layers were washed with
H O followed by saturated brine, dried over anhydrous MgSO , filtered, and evaporated. The
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
residue was used for the next step without further purification. To a solution of above generated
intermediate in anhydrous DMF (18 mL, 0.19 M) was added tert-butyldimethylsilyl chloride
(
1614 mg, 10.71 mmol) and imidazole (729 mg, 10.71 mmol) under N atmosphere. After
2
stirring for 3 h at room temperature, the reaction mixture was quenched with saturated aqueous
NH Cl (50 mL) and diluted with EtOAc (50 mL). The layers were separated, and the aqueous
4
layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed
successively with H O and saturated brine, dried over anhydrous MgSO , filtered, and
2
4
evaporated. The residue was purified by column chromatography (silica gel, hexanes/EtOAc,
4
0/1 to 20/1) to give 9 (705 mg, 53%) as a colorless syrup: []D25 = –103.19 (c 0.30, MeOH);
1
H NMR (400 MHz, CDCl ) δ 4.65 (d, J = 6.4 Hz, 1 H), 4.53 (d, J = 8.0 Hz, 1 H), 4.11 (d, J =
3
6
.3 Hz, 1 H), 3.61 (dd, J = 1.6, 8.0 Hz, 1 H), 3.30 (dd, J = 2.4, 8.1 Hz, 1 H), 2.41-2.46 (m, 1
1
3
H), 1.42 (s, 3 H), 1.30 (s, 3 H), 1.03 (s, 9 H), 0.88 (s, 9 H), 0.13 (s, 3 H), 0.05 (s, 3 H); C
NMR (100 MHz, CDCl ) δ 207.2, 110.9, 78.1, 75.8, 73.7, 71.3, 56.9, 42.3, 27.0 (3 × CH -tert-
3
3
+
butyl), 26.4, 25.7 (3 × CH -tert-butyl), 23.8, 18.3, -4.4, -5.6; HRMS (FAB ) (m/z) found
3
+
+
373.2398, [calcd for C H O Si (M+H) 373.2410]; Anal. Calcd for C H O Si: C, 61.25; H,
1
9
37
5
19 36
5
9
.74. Found: C, 61.26; H, 9.75.
3aS,4R,5R,6R,6aR)-6-(tert-Butoxymethyl)-5-((tert-butyldimethylsilyl)oxy)-2,2-
dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (10). To a cooled (0 °C) solution of
(
9
(471 mg, 1.26 mmol) in methanol (6.3 mL, 0.2 M) was added sodium borohydride (144 mg,
3
.79 mmol, 3 equiv) under N atmosphere. After being stirred at the same temperature for 1 h,
2
the reaction mixture was diluted with H O (20 mL) and EtOAc (20 mL). The layers were
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 65
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic
layers were washed successively with H O and saturated brine, dried over anhydrous MgSO ,
2
4
filtered, and evaporated. The residue was purified by column chromatography (silica gel,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hexanes/EtOAc, 30/1 to 20/1) to give 10 (415 mg, 88%) as a colorless syrup: []D25 = 40.39
1
(c 0.32, MeOH); H NMR (500 MHz, CDCl )  4.49 (d, J = 6.1 Hz, 1 H), 4.41 (t, J = 6.2 Hz,
3
1
3
H), 4.07 (t, J = 6.9 Hz, 1 H), 3.95 (dd, J = 6.8, 14.7 Hz, 1 H), 3.48 (dd, J = 3.9, 8.5 Hz, 1 H),
.32 (dd, J = 4.6, 8.5 Hz, 1 H), 2.43 (d, J = 8.4 Hz, 1 H), 2.12-2.18 (m, 1 H), 1.45 (s, 3 H), 1.32
1
3
(
s, 3 H), 1.12 (s, 9 H), 0.87 (s, 9 H), 0.09 (s. 3 H), 0.05 (s, 3 H); C NMR (125 MHz, CDCl )
3

110.4, 81.0, 78.8, 77.0, 76.1, 72.6, 57.3, 46.0, 27.4 (3 × CH -tert-butyl), 26.2, 25.8 (3 × CH -
3 3
+
+
tert-butyl), 24.0, 18.1, -4.5, -5.1; HRMS (FAB ) (m/z) found 375.2584, [calcd for C H O Si
1
9
39
5
+
(M+H) 375.2567]; Anal. Calcd for C H O Si: C, 60.92; H, 10.23. Found: C, 60.91; H, 10.25.
1
9
38
5
(((3aR,4R,5R,6R,6aR)-4-(Benzyloxy)-6-(tert-butoxymethyl)-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-5-yl)oxy)(tert-butyl)dimethylsilane (11). To a cooled (0 °C)
solution of 10 (193 mg, 0.515 mmol) in DMF (5.2 mL, 0.1 M) was added benzyl chloride (0.12
mL, 1.030 mmol, 2.0 equiv) and sodium hydride (41 mg, 1.030 mmol, 2.0 equiv) under N2
atmosphere. After being stirred at room temperature for 12 h, the reaction mixture was diluted
with H O (20 mL) and EtOAc (20 mL). The layers were separated, and the aqueous layer was
2
extracted with EtOAc (3 × 50 mL). The combined organic layers were washed successively
with H O and saturated brine, dried over anhydrous MgSO , filtered, and evaporated. The
2
4
residue was purified by column chromatography (silica gel, hexanes/EtOAc, 50/1) to give 11
(204 mg, 85%) as a colorless syrup: []D25 = 46.64 (c 0.66, MeOH); H NMR (400 MHz,
1
CDCl )  7.22-7.39 (m, 5 H), 4.76 (d, J = 12.4 Hz, 1 H), 4.59 (d, J = 12.4 Hz, 1 H), 4.45 (d, J
3
=
6.0 Hz, 1 H), 4.33-4.37 (m, 2 H), 3.83 (dd, J = 5.6, 8.8 Hz, 1 H), 3.39 (dd, J = 4.4, 8.8 Hz, 1
H), 3.32 (dd, J = 4.0, 8.4 Hz, 1 H), 2.05-2.11 (m, 1 H), 1.48 (s, 3 H), 1.29 (s, 3 H), 1.03 (s, 9
ACS Paragon Plus Environment

Page 29 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3
H), 0.88 (s, 9 H), 0.09 (s, 3 H), 0.05 (s, 3 H); C NMR (200 MHz, CDCl )  138.9, 128.4,
3
128.1, 127.9, 127.7, 127.2, 110.0, 82.1, 80.2, 76.0, 75.6, 72.4, 71.7, 57.5, 45.7, 27.3 (3 × CH3-
+
tert-butyl), 26.4, 25.8 (3 × CH -tert-butyl), 24.2, -4.7, -4.9; HRMS (FAB ) (m/z) found
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
4
65.3001, [calcd for C H O Si (M+H) 465.3029]; Anal. Calcd for C H O Si: C, 67.20; H,
26 45 5 26 44 5
9
.54. Found: C, 67.22; H, 9.55.
(3aR,4S,5R,6S,6aR)-4-(Benzyloxy)-6-(tert-butoxymethyl)-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-5-ol (12). To a cooled (0 °C) solution of 11 (179 mg, 0.385 mmol)
in anhydrous THF (3.8 mL, 0.1 M) was added tetra-n-butylammonium fluoride solution (1.2
mL, 1.0 M solution in THF, 1.2 mmol, 3.0 equiv) under N atmosphere. After being stirred at
2
room temperature for 12 h, the reaction mixture was diluted with H O (30 mL) and EtOAc (30
2
mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL).
The combined organic layers were washed successively with H O and saturated brine, dried
2
over anhydrous MgSO , filtered, and evaporated. The residue was purified by column
4
chromatography (silica gel, hexanes/EtOAc, 8/1) to give 12 (129 mg, 88%) as a colorless syrup:
[]D25 = 49.04 (c 0.28, MeOH); H NMR (400 MHz, CDCl )  7.39 (d, J = 7.2 Hz, 2 H), 7.29-
1
3
7
.35 (m, 2 H), 7.23-7.28 (m, 1 H), 4.85 (d, J = 12.4 Hz, 1 H), 4.62 (d, J = 12.4 Hz, 1 H), 4.51
(
t, J = 6.0 Hz, 1 H), 4.40-4.45 (m, 2 H), 3.81 (dd, J = 4.8, 7.2 Hz, 1 H), 3.58 (dd, J = 3.6, 8.8
Hz, 1 H), 3.44 (dd, J = 4.4, 8.8 Hz, 1 H), 2.70 (bs, 1 H), 2.26-2.32 (m, 1 H), 1.48 (s, 3 H), 1.31
13
(
s, 3 H), 1.08 (s, 9 H); C NMR (200 MHz, CDCl )  138.5, 128.3 (2 × CH-benzene), 128.0
3
(
2 × CH-benzene), 127.5, 111.1, 82.7, 80.6, 77.2, 76.7, 73.4, 71.9, 59.3, 45.4, 27.2 (3 × CH3-
tert-butyl), 26.5, 24.6; Anal. Calcd for C H O : C, 68.54; H, 8.63. Found: C, 68.52; H, 8.64.
2
0
30
5
(
3aR,4R,5S,6R,6aR)-4-(benzyloxy)-6-(tert-butoxymethyl)-5-fluoro-2,2-
dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxole (13a). To a cooled (0 °C) solution of 12
20 mg, 0.052 mmol) in anhydrous toluene (2.0 mL, 0.026 M) was dropwise added
(
diethylaminosulfur trifluoride (30 L, 0.210 mmol, 4.0 equiv) under N atmosphere. After
2
ACS Paragon Plus Environment