Synthesis and Anti-HCV Activity of a Novel 2′,3′-Dideoxy-2′-α-fluoro-2′-β-C-methyl Guanosine Phosphoramidate Prodrug

Article abstract of DOI:10.1021/acsmedchemlett.7b00174

A novel 2′,3′-dideoxy-2′-α-fluoro-2′-β-C-methyl-6-methoxy guanosine (8) and its phosphoramidate prodrug (1) have been designed and synthesized. Their biological activity was evaluated in both cytotoxicity and cell-based HCV replicon assays. Neither compounds exhibited cytotoxicity up to the highest concentration tested (100 μM) in the Huh-7 cell line. The prodrug (1) displayed nanomolar level antiviral activity (EC₅₀ = 0.39-1.1 μM) against the HCV genotype (GT) 1a, 1b, 2a, and 1b S282T replicons.

Full text of DOI:10.1021/acsmedchemlett.7b00174

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Note
Synthesis and Anti-HCV Activity of a Novel 2',3'-Dideoxy-2'-
#-fluoro-2'-#-C-methyl Guanosine Phosphoramidate Prodrug
Wenquan Yu, Ertong Li, Zhigang Lv, Ke Liu, Xiaohe Guo, Yuan Liu, and Junbiao Chang
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 01 May 2017
Downloaded from http://pubs.acs.org on May 2, 2017
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 free 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 accessible to all
readers and 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.
ACS Medicinal Chemistry Letters 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 4
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Synthesis and AntiꢀHCV Activity of a Novel 2',3'ꢀDideoxyꢀ2'ꢀαꢀ
fluoroꢀ2'ꢀβꢀCꢀmethyl Guanosine Phosphoramidate Prodrug
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Wenquan Yu,1 Ertong Li,1 Zhigang Lv,1 Ke Liu,1 Xiaohe Guo,2 Yuan Liu,1 and Junbiao Chang*, 1
1College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan 450001, P. R. China
2High & New Technology Research Center of Henan Academy of Sciences, Zhengzhou, Henan 450002, P. R. China
KEYWORDS. 2',3'ꢀdideoxy guanosine; 2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethyl; phosphoramidate prodrug; antiꢀHCV activity; NS5B RdRp
inhibitor
ABSTRACT: A novel 2',3'ꢀdideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethylꢀ6ꢀmethoxy guanosine (8) and its phosphoramidate prodrug (1) have
been designed and synthesized. Their biological activity was evaluated in both cytotoxicity and cellꢀbased HCV replicon assays.
Neither of compounds exhibited cytotoxicity up to the highest concentration tested (100 ꢁM) in the Huhꢀ7 cell line. The prodrug (1)
displayed nanomolar level antiviral activity (EC50 = 0.39~1.1 ꢁM) against the HCV genotype (GT) 1a, 1b, 2a, and 1b S282T repliꢀ
cons.
Hepatitis C virus (HCV), a member of the Flaviviridea
family, is a positiveꢀsense singleꢀstranded RNA virus. HCV
infection affects over 170 million people worldwide1 and is a
major cause of hepatocellular carcinoma (HCC). So far, a seꢀ
ries of potential molecular targets have been indentified for
antiꢀHCV drug discovery, including NS2ꢀNS3 autoprotease,
the N3 protease, the N3 helicase, and the NS5B polymerase.
The NS5B, one of nonstructural proteins serving as the HCV
RNAꢀdependent RNA polymerase (RdRp), is essential for the
replication of the viral RNA genome and has attracted signifiꢀ
cant interest among medicinal chemists.2ꢀ5 Nucleosides active
against HCV are NS5B RdRp inhibitors, and they need to be
converted to the corresponding triphosphates that can incorpoꢀ
rate into viral RNA at the 3'ꢀterminal so as to stop viral RNA
elongation as chain terminators. Some nucleosides are weakly
active because they cannot be efficiently phosphorylated by
specific nucleoside kinases or are not substrates of the kinases
at all. On the other hand, nucleoside phosphates (nucleotides)
per se cannot be used as drugs very often because they are
chemically less stable and/or too polar to enter the cells.6, 7 In
order to bypass the rateꢀlimiting firstꢀstep phosphorylation as
well as to improve the chemical stability and biological activiꢀ
ty, various nucleoside phosphate prodrugs have been designed
and synthesized,7ꢀ10 Recently, the phosphoramidate prodrug
strategy has been demonstrated as an effective approach for
intracellular delivery of the monophosphorylated nucleosides.
The monophosphate can be further phosphorylated to diꢀ, and
then the biologically active triphosphate.
Figure 1. Representative 2'ꢀCꢀmethyl substituted nucleotide anaꢀ
logs with potent antiꢀHCV activity.
lack of the 3'ꢀhydroxy group. However, such analogs were
rarely reported in the literature with good antiꢀHCV activity, a
major reason for which could be riboꢀnucleosides with 3'ꢀOH
may not be good substrates of phosphorylation kinases.18
Herein, we designed and synthesized such a novel 2',3'ꢀ
dideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethyl nucleoside analog (8) and
its phosphoramidate prodrug (1) for the treatment of HCV
infection.19ꢀ20
The synthesis of the target 2',3'ꢀdideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀ
methylꢀ6ꢀmethoxy guanosine (8) is described in Scheme 1.
The starting material 4 was readily obtained according to a
previously reported synthetic route.13 Selective protection of
the 5'ꢀOH group in nucleoside 4 with tertꢀbutyldimethylsilyl
chloride in the presence of imidazole resulted in compound 5,
which was further converted into intermediate 6 by the treatꢀ
ment with 1,1ꢀthiocarbonyldiimidazole in acetonitrile.
Bu3SnH/AIBNꢀmediated 3'ꢀdeoxylation of compound 6 and
subsequent removal of the silyl protecting group with TBAF
afforded the 2',3'ꢀdideoxy guanosine 8.
Among various antiviral nucleos(t)ide compounds, the 2'ꢀCꢀ
methyl substituted analogs11ꢀ17 display potent antiꢀHCV activiꢀ
ty in vitro, in vivo, and in clinic (Figure 1). These nucleotides
act as nonꢀobligate chain terminators of HCV RdRp as they all
possess a 3'ꢀOH moiety. 2',3'ꢀDideoxy nucleosides, such as
Zidovudine and Emtricitabine exhibit good antiviral activity
against HIV and HBV because they cannot support the elongaꢀ
tion of the newly synthesized viral polynucleotide due to the
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 4
Scheme 1. Synthesis of 2',3'ꢀDideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀ
methylꢀ6ꢀmethoxy Guanosine 8
Table 1. Cytotoxicity and antiꢀHCV activity of guanosine 8
and its phosphoramidate prodrug 1a
1
2
3
4
5
OMe
N
OMe
CC50
(ꢁM)
HCV 1b
HCV 1a
HCV 2a
HCV 1b S282T
EC50 (ꢁM)
N
N
compd
N
N
a
b
EC50 (ꢁM)
EC50 (ꢁM)
EC50 (ꢁM)
O
O
N
N
NH2
N
NH2
HO
TBSO
8
1
> 100
> 100
> 100
0.58
ND
1.1
ND
ND
4
5
6
HO
F
HO
F
7
8
9
OMe
N
0.39
0.81
OMe
N
N
N
N
N
aEC50: 50% effective concentration; CC50: 50% cytotoxic conꢀ
centration.
O
c
N
NH2
O
TBSO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
NH2
TBSO
O
F
d
6
7
F
S
In summary, we have designed and synthesized a novel
2',3'ꢀdideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethylꢀ6ꢀmethoxy guanosine
(8) and its phosphoramidate prodrug (1). Neither of comꢀ
pounds is cytotoxic in the Huhꢀ7 cell line at the highest tested
concentration up to 100 ꢁM. Although guanosine 8 is inactive,
the corresponding prodrug (1) displayed potent antiꢀHCV acꢀ
tivity against the genotype (GT) 1a, 1b, 2a, and 1b S282T reꢀ
plicons with similar EC50s (between 0.39 and 1.1 ꢁM). For the
first time, we have discovered a 2',3'ꢀdideoxy nucleotide anaꢀ
log possessing potent antiviral activity against HCV infection.
These encouraging results warrant further investigation toꢀ
wards the development of compound 1 as a potential antiꢀ
HCV agent.
OMe
N
Im
N
N
O
N
NH2
HO
8
F
(a) TBDMSCl, imidazole, DMF, rt, 81%; (b) 1,1-thiocarbonyldiimidazole, MeCN,
rt, 69%; (c) (n-Bu)3SnH, AIBN, toluene, 80 ºC, 75%; (d) TBAF, THF, rt, 64%.
Scheme 2. Synthesis of the Phosphoramidate Prodrug 1
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedures, characterization data, and biological
assays (PDF)
AUTHOR INFORMATION
Corresponding Author
*changjunbiao@zzu.edu.cn.
To synthesize the phosphoramidate prodrug 1, the correꢀ
sponding perfluorophenyl phosphoramidate (12) was prepared
(Scheme 2). Treatment of phenyl dichlorophosphate (10) with
Lꢀalanyl cyclopentyl ester hydrochloride (9) and triethylamine
at ꢀ78 °C followed by reaction of the resulting monochloroꢀ
phosphate 11 with pentafluorophenol and triethylamine gave
the SPꢀcyclopentyl ester 12 after the recrystallization from a
mixture of ethyl acetate and hexanes. Reaction of nucleoside 8
with compound 12 in the presence of tertꢀbutyl magnesium
chloride afforded the desired prodrug 1 as the pure SPꢀ
enantiomer.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
Funding Sources
We thank the National Natural Science Foundation of China (Nos.
81302637 and 81330075) and Outstanding Young Talent Reꢀ
search Fund of Zhengzhou University (No. 1521316004) for fiꢀ
nancial support.
The biological evaluation was carried out in cytotoxicity
and cellꢀbased HCV replicon assays, respectively. As summaꢀ
rized in Table 1, neither of guanosine 8 and its phosphoramiꢀ
date prodrug (1) exhibited cytotoxicity at the highest concenꢀ
tration tested (100 ꢁM) in Huhꢀ7 cells. In the same cell line
containing replicating HCV genotype (GT) 1b replicon, altꢀ
hough the nucleoside 8 was inactive, its prodrug 1 inhibited
the replication of the replicon with an EC50 of 0.58 ꢁM. In
light of these encouraging results, the antiviral activity of
compound 1 was further profiled using different HCV replicon
cells. This prodrug also displayed nanomolar antiviral activity
(EC50 = 0.81 ꢁM) against the HCV GT 1a, 2a, and 1b S282T
replicons.
ABBREVIATIONS
HCV, hepatitis C virus; HCC, hepatocellular carcinoma; RdRp,
RNAꢀdependent
butyldimethylsilyl chloride; DMF, N,Nꢀdimethylformamide;
AIBN, azobisisobutyronitrile; TBAF, tetrabutylammonium fluoꢀ
ride; THF, tetrahydrofuran; GT, genotype; EC50, 50% effective
concentration; CC50, 50% cytotoxic concentration; PE, petroleum
ether; LTR, long terminal repeat; DMEM, Dulbecco's minimum
essential medium; FBS, fetal bovine serum.
RNA
polymerase;
TBDMSCl,
tertꢀ
REFERENCES
(1) Gower, E.; Estes, C.; Blach, S.; RazaviꢀShearer, K.; Razavi, H.
Global epidemiology and genotype distribution of the hepatitis
C virus infection. J. Hepatol. 2014, 61, S45−S57.
ACS Paragon Plus Environment

Page 3 of 4
ACS Medicinal Chemistry Letters
(2) Zhao, C.; Wang, Y. H.; Ma, S. T. Recent advances on the synꢀ
hepatitis C virus replication. J. Med. Chem. 2005, 48, 5504–
thesis of hepatitis C virus NS5B RNAꢀdependent RNA polyꢀ
merase inhibitors. Eur. J. Med. Chem. 2015, 102, 188−214.
5508.
1
2
3
4
5
6
7
8
9
(17) Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bera, S.; Bhat,
B.; Bhat, N.; Bosserman, M. R.; Brooks, J.; Burlein, C.; Carroll,
S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.; McMasters, D.
R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Toꢀ
massini, J. E.; Xia, J. Structureꢀactivity relationship of purine
ribonucleosides for inhibition of hepatitis C virus RNAꢀ
dependent RNA polymerase. J. Med. Chem. 2004, 47, 2283–
2295.
(18) Siddiqui, M. A.; Hughes, S. H.; Boyer, P. L.; Mitsuya, H.; Van,
Q. N.; George, C.; Sarafinanos, S. G.; Marquez, V. E. A 4'ꢀCꢀ
ethynylꢀ2',3'ꢀdideoxynucleoside analogue highlights the role of
the 3'ꢀOH in antiꢀHIV active 4'ꢀCꢀethynylꢀ2'ꢀdeoxy nucleosides.
J. Med. Chem. 2004, 47, 5041–5048.
(19) Chang, J.; Huang, Q.; Zhou, S. 2',3'ꢀDideoxyꢀ2'ꢀalphaꢀfluoroꢀ2'ꢀ
betaꢀCꢀmethylnucleosides and prodrugs thereof. CN Patent ZL
201280030844.X, July 19, 2012.
(20) Huang, Q.; Zhou, S.; Chang, J. 2',3'ꢀDideoxyꢀ2'ꢀalphaꢀfluoroꢀ2'ꢀ
betaꢀCꢀmethylnucleosides and prodrugs thereof. Pat. Appl. WO
2013013009 A3, January 24, 2013; US 20140315850 A1, Ocꢀ
tober 23, 2014.
(3) Zhao, F. B.; Liu, N.; Zhan, P.; Jiang, X. M.; Liu, X. Y. Discovꢀ
ery of HCV NS5B thumb 1 inhibitors: Coreꢀrefining from benꢀ
zimidazole to indole scaffold. Eur. J. Med. Chem. 2015, 94,
218−228.
(4) CailletꢀSaguy, C. Lim, S. P.; Shi, P. Y.; Lescar, J.; Bressanelli,
S. Polymerases of hepatitis C viruses and flaviviruses: structurꢀ
al and mechanistic insights and drug development. Antiviral
Res. 2014, 105, 8−16.
(5) Mayhoub, A. S. Hepatitis C RNAꢀdependent RNA polymerase
inhibitors: a review of structureꢀactivity and resistance relationꢀ
ships; different scaffolds and mutations. Bioorg. Med. Chem.
2012, 20, 3150−3161.
(6) Wagner, C. R.; Iyer, V. V.; McIntee, E. J. Pronucleotides: toꢀ
ward the in vivo delivery of antiviral and anticancer nucleoꢀ
tides. Med. Res. Rev. 2000, 20, 417−451.
(7) Pradere, U.; GarnierꢀAmblard, E. C.; Coats, S. J.; Amblard, F.;
Schinazi, R. F. Synthesis of nucleoside phosphate and phosꢀ
phonate prodrugs. Chem. Rev. 2014, 114, 9154−9218.
(8) Thornton, P. J.; Kadri, H.; Miccoli, A.; Mehellou, Y. Nucleoꢀ
side phosphate and phosphonate prodrug clinical candidates. J.
Med. Chem. 2016, 59, 10400−10410.
(9) Sofia, M. J. Nucleotide prodrugs for the treatment of HCV
infection. Adv. Pharmacol. 2013, 67, 39−73.
(10) Bobeck, D. R.; Schinazi, R. F.; Coats, S. J. Advances in nucleoꢀ
side monophosphate prodrugs as antiꢀHCV agents. Antivir.
Ther. 2010, 15, 935−950.
(11) Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.;
Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.
R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer,
H. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a βꢀDꢀ
2'ꢀdeoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethyluridine nucleotide prodrug
(PSIꢀ7977) for the treatment of hepatitis C virus. J. Med. Chem.
2010, 53, 7202–7218.
(12) McGuigan, C.; Madela, K.; Aljarah, M.; Bourdin, C.; Arrica,
M.; Barrett, E.; Jones, S.; Kolykhalov, A.; Bleiman, B.; Bryant,
K. D.; Ganguly, B.; Gorovits, E.; Henson, G.; Hunley, D.;
Hutchins, J.; Muhammad, J.; Obikhod, A.; Patti, J.; Walters, C.
R.; Wang, J.; Vernachio, J.; Ramamurty, C. V.; Battina, S. K.;
Chamberlain, S. Phosphorodiamidates as a promising new
phosphate prodrug motif for antiviral drug discovery: applicaꢀ
tion to antiꢀHCV agents. J. Med. Chem. 2011, 54, 8632−8645.
(13) Chang, W.; Bao, D.; Chun, B. K.; Naduthambi, D.; Nagaraꢀ
thnam, D.; Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Zhang,
H. R.; Bansal, S.; Espiritu, C. L.; Keilman, M.; Lam, A. M.;
Niu, C.; Steuer, H. M.; Furman, P. A.; Otto, M. J.; Sofia, M. J.
Discovery of PSIꢀ353661, a novel purine nucleotide prodrug for
the treatment of HCV infection. ACS Med. Chem. Lett. 2010, 2,
130–135.
(14) Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.;
Aktoudianakis, V.; Lew, W.; Ye, H.; Clarke, M.; Doerffler, E.;
Byun, D.; Wang, T.; Babusis, D.; Carey, A. C.; German, P.;
Sauer, D.; Zhong, W.; Rossi, S.; Fenaux, M.; McHutchison, J.
G.; Perry, J.; Feng, J.; Ray, A. S.; Kim, C. U. Discovery of the
first Cꢀnucleoside HCV polymerase inhibitor (GSꢀ6620) with
demonstrated antiviral response in HCV infected patients. J.
Med. Chem. 2014, 57, 1812−1825.
(15) Jonckers, T. H.; Vandyck, K.; Vandekerckhove, L.; Hu, L,
Tahri, A.; Van Hoof, S.; Lin, T. I.; Vijgen, L.; Berke, J. M.;
LachauꢀDurand, S.; Stoops, B.; Leclercq, L.; Fanning, G.; Samꢀ
uelsson, B.; Nilsson, M.; Rosenquist, Å.; Simmen, K.; Raboisꢀ
son, P. Nucleotide prodrugs of 2'ꢀdeoxyꢀ2'ꢀspirooxetane ribonuꢀ
cleosides as novel inhibitors of the HCV NS5B polymerase. J.
Med. Chem. 2014, 57, 1836−1844.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16) Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharꢀ
nish, P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R. F.;
Watanabe, K. A.; Otto, M. J.; Furman, P. A.; Stec, W. J.; Patꢀ
terson, S. E.; Pankiewicz, K. W. Design, synthesis, and antiviral
activity of 2'ꢀdeoxyꢀ2'ꢀCꢀmethylcytidine, a potent inhibitor of
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 4
Table of Contents (TOC) graphic
1
2
3
4
Synthesis and AntiꢀHCV Activity of a Novel 2',3'ꢀDideoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethyl Guanosine Phosphoramidate
Prodrug
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
ACS Paragon Plus Environment