2′-Chloro,2′-fluoro Ribonucleotide Prodrugs with Potent Pan-genotypic Activity against Hepatitis C Virus Replication in Culture

Article abstract of DOI:10.1021/acs.jmedchem.7b00067

Pan-genotypic nucleoside HCV inhibitors display a high genetic barrier to drug resistance and are the preferred direct-acting agents to achieve complete sustained virologic response in humans. Herein, we report, the discovery of a β-d-2′-Cl,2′-F-uridine phosphoramidate nucleotide 16, as a nontoxic pan-genotypic anti-HCV agent. Phosphoramidate 16 in its 5′-triphosphate form specifically inhibited HCV NS5B polymerase with no marked inhibition of human polymerases and cellular mitochondrial RNA polymerase. Studies on the intracellular half-life of phosphoramidate 16-TP in live cells demonstrated favorable half-life of 11.6 h, suggesting once-a-day dosing. Stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes make phosphoramidate 16 a prospective candidate for further studies to establish its potential value as a new anti-HCV agent.

Full text of DOI:10.1021/acs.jmedchem.7b00067

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Article
2’-Chloro,2’-Fluoro Ribonucleotide Prodrugs with Potent Pan-
genotypic Activity against Hepatitis C Virus Replication in Culture
Shaoman Zhou, Sawsan Mahmoud, Peng Liu, Longhu Zhou, Maryam Ehteshami,
Leda Bassit, Sijia Tao, Robert A. Domaoal, Ozkan Sari, Coralie De Schutter, Sheida
Amiralaei, Ahmed Khalil, Olivia Ollinger Russell, Tamara R McBrayer, Tony Whitaker,
Nageh Abou-Taleb, Franck Amblard, Steven J. Coats, and Raymond Felix Schinazi
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017
Downloaded from http://pubs.acs.org on June 8, 2017
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’-Chloro,2’-Fluoro Ribonucleotide Prodrugs
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with Potent Pan-genotypic Activity against
Hepatitis C Virus Replication in Culture
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#
1,2#
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Shaoman Zhou, Sawsan Mahmoud, Peng, Liu, Longhu Zhou, Maryam Ehteshami,
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Leda Bassit, Sijia Tao, Robert A. Domaoal, Ozkan Sari, Coralie De Schutter, Sheida
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Amiralaei, Ahmed Khalil, Olivia Ollinger Russell, Tamara McBrayer, Tony
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Whitaker, Nageh Abou-Taleb, Franck Amblard, Steven J. Coats,
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1*
Raymond F. Schinazi
1
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics,
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Emory University School of Medicine, Atlanta, USA; Department of Pharmaceutical Chemistry,
3
Faculty of Pharmacy, Helwan University, Helwan, Egypt; Cocrystal Pharma, Inc., Tucker, USA.
#
contributed equally – co-first authors
ABSTRACT
Pan-genotypic nucleoside HCV inhibitors display a high genetic barrier to drug resistance
and are the preferred direct acting agents to achieve complete sustained virologic
response in humans. Herein, we report, the discovery of a β-D-2’-Cl,2’-F-uridine
phosphoramidate nucleotide 16, as a non-toxic pan-genotypic anti-HCV agent.
Phosphoramidate 16 in its 5’-triphosphate form specifically inhibited HCV NS5B
polymerase with no marked inhibition of human polymerases and cellular mitochondrial
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RNA polymerase. Studies on the intracellular half-life of phosphoramidate 16-TP in live
cells demonstrated favorable half-life of 11.6 h suggesting once a day dosing. Stability in
human blood and favorable metabolism in human intestinal microsomes and liver
microsomes make phosphoramidate 16 a prospective candidate for further studies to
establish its potential value as a new anti-HCV agent.
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KEYWORDS: Nucleoside, prodrug, hepatitis C, antiviral
INTRODUCTION:
Hepatitis C virus (HCV) presents a global health problem with approximately 180 million
1
individuals infected worldwide with 80% of those progressing to chronic HCV infection.
Of those chronically infected individuals, approximately 30% will develop liver cirrhosis
2
and 10% will go on to develop hepatocellular carcinoma. Options comprising pegylated
interferon-α (PEG-IFN) in combination with ribavirin (RBV) was the treatment of choice
for HCV infection for many years; however, this treatment had demonstrated limited
3
, 4
efficacy and generally intolerable side effects.
Recently, two newly approved
once-daily combination hepatitis C drugs (Elbasvir + grazoprevir and ombitasvir +
5
paritaprevir + ritonavir + dasabuvir) have demonstrated improved safety and efficacy.
However, without nucleoside NS5B inhibitors as backbone, these new agents are only
approved for treating genotype 1 or 4 HCV-infected persons, thus excluding a large
patient population infected with HCV genotypes 2, 3, 5 and 6.
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The RNA-dependent RNA polymerase (RdRp) is essential to viral replication and has
been clinically validated as target for therapeutic intervention by design of specific
inhibitors. The HCV NS5B RdRp is part of a replication complex that is membrane
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,6
bound and is responsible for HCV RNA genome replication. Nucleoside inhibitors
(NIs), in their 5’-triphosphate form, interact with the HCV NS5B polymerase and are
attractive due to their high inhibitory potency, pan-genotypic profile, and high generic
7
barrier to resistance. So far, sofosbuvir is the only FDA approved nucleoside
phosphoramidate prodrug for HCV and is the backbone of the first all-oral,
pan-genotypic, single tablet regimen for the treatment of adults with genotype 1-6
chronic HCV infection (Combination of sofosbuvir with NS5A inhibitor, velpatasvir).
However, because of long duration of treatment (8-12 weeks), there is still an urgent need
to develop novel nucleoside analogs that are pan-genotypic and more efficacious, have an
improved safety profile and a high barrier to resistance, which could lead to a new
8
ultra-short combination therapy.
The 2’-methyl substitution on nucleoside analogs has played a unique role in HCV drug
9
discovery and clinical treatment. Even though sofosbuvir is the only FDA approved
nucleoside analog for HCV treatment so far, several 2’-methyl analogs have reached
1
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human clinical evaluation (Figure 1). Based on these structures, we envisioned new
anti-HCV nucleoside analogs by considering the isosteric replacement of the 2’-methyl
1
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group with groups or elements with similar van der Waals radius. Isosteres are groups
that exhibit some similarities in their chemical, physical and/or biological properties and
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as a result, they have the potential to exhibit similar or superior pharmacokinetic and/or
pharmacodynamic properties. Ultimately, we hypothesized that the replacement of the
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’-methyl group by a similarly sized chlorine atom could result in the formation of
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analogs with anti-HCV activity. Herein, we report the synthesis and biological evaluation
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of 2′-chloro,2’-fluoro ribonucleosides and their phosphate prodrugs as anti-HCV agents.
Figure 1. Selected clinical anti-HCV nucleoside analogs and targeted 2’-chloro, 2’-fluoro
nucleoside analogs.
RESULTS AND DISCUSSION:
Chemistry:
The key protected lactol 10 was prepared using the chemistry described in Scheme 1.
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Oxidation of commercially available 2-deoxy-D-ribose 4 with Br gave lactone 5 which
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was then reacted with tert-butyldiphenylsilyl choride (TBDPSCl) to give the
bis-protected lactone 6 in 83% yield. Reaction of 6 with fluorodibenezenesulfonimide
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(
NFSI) in presence of LiHMDS resulted in the formation of 7 in 24% yield.
Chlorination of compound 7 with N-chlorosuccinimide (NCS) in presence of LiHMDS
gave dihalogeno compounds 8 and 9 as a 4:1 mixture that were separated by flash
chromatography on silica gel. Finally, reduction of isomer 8 with lithium
tri-tert-butoxyaluminum hydride in THF gave the desired lactol 10 in 86% yield, as a α/β
mixture (1.5:1).
Scheme 1. Synthesis of protected lactone 10.
Reagents and conditions: a) Br , H O, rt, 5 d, 86%; b) TBDPSCl, imidazole, DMF, rt, 24 h,
2
2
o
o
8
5
3%; c) NFSI, LiHMDS, THF, -78 C, 1 h, 24%; d) NCS, LiHMDS, THF, -78 C, 30 min,
7%; e) Li(t-BuO) AlH, THF, 4 h, rt, 86%.
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With key lactol 10 in hand, various pyrimidine, purine and 7-deaza purine nucleosides,
along with their corresponding phosphoramidate prodrugs, were prepared as depicted in
Schemes 2-7. Thus, 1-OMs derivative 11, readily prepared from lactol 10 by mesylation,
was reacted under Vorbrüggen conditions with persilylated uracil in presence of TMSOTf
to give compound 12 as an inseparable mixture of α/β isomers (α/β ratio: 2:1).
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Desilylation with Et N.3HF in THF and subsequent chromatographic separation gave the
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desired uridine nucleoside 13 (β-isomer, 32%) along with its α-isomer 14 (50%). Finally,
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nucleoside 13 was reacted with phenyl L-isopropylalaninyl phosphorochloridate 15 in
presence of NMI in THF to give phosphoramidate prodrug 16 as an approximate 1:1
mixture of Rp/Sp isomers (Scheme 2).
Scheme 2. Synthesis of compound 13 and its corresponding phosphoramidate
prodrug 16.
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o
Reagents and conditions: a) MsCl, Et N, DCM, 0 C to rt; b) i) Uracil, BSA,
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1,2-dichloroethane, 80 C, 30 min; ii) TMSOTf, 1,2-dichloroethane, 80 C, 4 h, 67%; c)
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Et N.3HF, THF, 0 C to rt, 24 h, 82%; d) NMI, THF, rt, 4 h, 48%.
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Scheme 3. Synthesis of nucleoside 19 and its monophosphate prodrug 20.
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Reagents and conditions: a) i) N -benzoylcyctosine, BSA, 1,2-dichloroethane, 80 C, 30
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min; ii) TMSOTf, 1,2-dichloroethane, 80 C, 5 h, 63%; b) Et N.3HF, THF, 0 C to rt, 24 h;
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c) NH , MeOH, overnight, 88% over 2 steps; d) 15, t-BuMgCl, THF, 0 C to rt, 3 h, 18%.
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Coupling of 1-mesyl sugar 11 with persilylated N -benzoylcytosine in the presence of
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TMSOTf in 1,2-dichloroethane at 80 C gave compound 17a along with its α isomer 17b
(α/β ratio is 2:1). Desilylation of 17 with Et N.3HF and subsequent debenzoylation with
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a methanolic solution of ammonia gave cytosine analog 19 in 88% yield over 2 steps.
Cytosine phosphoramidate prodrug 20 was finally prepared by reaction of compound 19
with phenyl L-isopropylalaninyl phosphorochloridate 15 in presence of t-BuMgCl
(Scheme 3).
Scheme 4. Synthesis of adenine nucleoside 23 and its prodrug 24.
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Reagents and conditions: a) bis-N-Boc adenine, DIAD, PPh , THF, rt, 24 h, 32%; b)
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Et N.3HF, rt, 24 h, 86%; c) 15, NMI, THF, 0 C to rt, 4 h; d) 10% TFA-H O, 38% over two
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steps; e) 4N HCl in dioxane, DCM, rt, 1 h, 72%.
Because the Vorbrüggen type coupling (S 1) with the pyrimidine bases gave the
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corresponding α nucleosides as major compounds, we decided to prepare the purine and
7-deaza purine nucleosides under Mitsunobu conditions (S 2). Thus, coupling of lactol
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PPh3
gave a mixture of α/β anomers (ratio 1:1.5) that was separated by flash silica
gel column chromatography to give pure β−isomer 21 in 32% yield. Deprotection of
β-isomer 21 with Et N.3HF in THF resulted in bis-N-Boc protected adenine compound 22,
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which was converted to adenosine nucleoside 23 by treatment with 4M HCl in dioxane. On
the other hand, reaction of compound 22 with phenyl L-isopropylalaninyl
phosphorochloridate 15 in presence of NMI and finally, removal of the Boc group under
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acidic conditions (10% TFA in H O) afforded the corresponding phosphoramidate prodrug
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24 (Scheme 4).
Scheme 5. Synthesis of guanine nucleoside 27 and its prodrugs 28.
Reagents and conditions: a) 6-chloro-2-N-Boc purine, DIAD, PPh THF, rt, 24 h, 35%; b)
2
3,
o
Et N.3HF, rt, 24 h, 93%; c) 80% TFA in H O, rt, 34 h, 47%; d) 15, NMI, THF, 0 C to rt,
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.5 h, 64%; e) 80% TFA in H O, rt, 34 h, 33%.
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In a similar manner, coupling of 6-chloro-2-N-(Boc) purine with lactol 10 under
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separated by column chromatography. Deprotection of β isomer 25 with Et N.3HF in THF
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afforded compound 26 in 93% yield. One-pot hydrolysis and deprotection of compound 26
with 80% aqueous TFA gave guanine analog 27 in 47% yield. Reaction of compound 26
with L-isopropylalaninyl phosphorochloridate 15 in presence of NMI and subsequent
treatment with 80% aqueous TFA furnished guanine nucleoside prodrug 28 (Scheme 5).
Scheme 6. Synthesis of 7-deazapurine nucleosides 35-37 and their prodrugs 38-40.
Reagents and conditions: a) Ph P, DIAD, THF, rt, 2 d, 34-36%; b) aq. NH OH,
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,4-dioxane, 120 C, 20 h, 40-62%, c) Phenyl L-isopropylalaninyl phosphorochloridate 15,
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We then turned our attention to the non-canonical 7-deazapurines bases which are known
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to be tolerated by the HCV polymerase in other series. Thus, reaction of 7-iodo- or
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-fluoro- 6-chloro-7-deazapurine 29 and 30 or 6-chloro-7-deazapurine 31 with lactol 10 in
presence of DIAD and PPh in THF at rt provided a mixture of α:β isomers (ratio 1:1.2)
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that were separated by flash chromatography. Treatment of β-isomers 32a-34a with
NH OH resulted in 7-deazapurine nucleosides analogs 35-37, which were converted to
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their corresponding phosphoramidate prodrug 38, 39 and 40 by reaction with phenyl
L-isopropylalaninyl phosphorochloridate 15 in presence of NMI (Scheme 6).
Assignment of Absolute Configuration and α/β Isomerism
1
The 1’-stereochemistry of our final compounds were assigned by using H 2D-NOESY
experiments. For instance, in the case of β-isomer 13, NOE enhancements were observed
between H of the nucleobase and H as well as between H and H ; while interactions
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between the H and H and H and H were observed for α-isomer compound 14 (Figure
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4’
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Figure 2. Anomer assignment for compounds 13 and 14 via NOE experiments
Because stereochemistry at the 2’-position could not be clearly established through
common NMR experiments, we decided to use X-ray diffraction analysis. Unfortunately,
we were not able to grow suitable crystals from any of the intermediates described above
and only compound 41, obtained by reaction of uracil derivative 14 with TBDMSCl in
presence of imidazole (Scheme 7) gave us a single crystal and allowed us to confirm the
(
S)-configuration of C-2’ of our nucleosides (Figure 3).
Scheme 7. Synthesis of compound 41
Reagents and conditions: a) TBDMSCl, imidazole, DMF, rt, 16 h, 83%
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8
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0
1
2
3
4
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6
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8
9
0
Figure 3. X-ray crystal structure of 41 crystallized from i-PrOAc
Antiviral activity and cytotoxicity
Compounds 13, 19, 23, 27, 35, 36, 37 and their corresponding phosphoramidate prodrugs
6, 20, 24, 28, 38, 39, 40 were evaluated for inhibition of HCV genotype 1b RNA
1
1
8
replication in Huh-7 cells using a subgenomic HCV replicon system. Cytotoxicity in
Huh-7 cells was determined simultaneously by extraction and amplification of both HCV
1
9
RNA and cellular ribosomal RNA (rRNA). In addition, cytotoxicity was determined in
primary human peripheral blood mononuclear (PBM) cells, human lymphoblastoid CEM,
2
0,21
and African Green monkey Vero cells.
The results are summarized in Table 1. Uracil,
adenine and guanine nucleosides 13, 23 and 27 were devoid of anti-HCV activity at
concentration up to 10 ꢀM while they corresponding monophosphate prodrugs 16, 24 and
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2
8 were all active in the submicromolar range (EC ’s of 0.1, 0.7 and 0.3 ꢀM
5
0
respectively). Interestingly, both cytosine nucleoside 19 and its prodrug 20 showed
submicromolar activity in the HCV replicon assay. Compound 19 however, displayed
cytotoxicity in PBM and Vero cells in the low micromolar range (CC ’s of 12 and 8.4
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
5
0
ꢀM respectively). Finally, none of the 7-deazapurine analogs displayed anti-HCV activity.
Based on its potency and lack of cellular toxicity in the various cell line tested, we further
investigated the potential of compound 16.
Table 1. HCV genotype 1b replicon activity and cytotoxicity of nucleosides 13, 19, 23,
2
7, 35, 36, 37 and their phosphoramidate prodrugs 16, 20, 24, 28, 38, 39, 40
Compound Anti-HCV activity (µM)
Cytotoxicity, CC (µM)
5
0
EC50
EC90
Huh-7
PBM
CEM
Vero
1
3
> 100
> 100
> 100
>10
> 100
> 100
> 100
> 100
> 100
> 100
16
0.2 ± 0.1
0.4 ± 0.2
1
2
9
0
0.3 ± 0.03
0.1 ± 0.06
0.9 ± 0.02
0.3 ± 0.02
>10
>10
12
> 100
> 100
8.4
> 100
> 100
23
> 10
> 10
>10
> 33
> 33
> 100
84.8
> 100
36.3
> 100
53.9
24
0.6 ± 0.2
19 ± 2.9
2.1 ± 0.6
33 ± 0.5
27
> 100
> 100
> 100
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2
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3
3
3
8
5
8
6
9
7
0.3 ± 0.1
> 10
0.8 ± 0.2
> 10
>10
> 100
6.6
> 100
7.3
> 100
11.5
>
10
0
1
2
3
4
5
6
7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
> 10
> 10
> 10
> 100
82.6
99.7
ND
69.3
19.9
55.3
ND
>100
42.4
60.9
ND
> 10
> 10
>10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
40
> 10
> 10
> 100
69
> 100
2
’C-MeC
1.7 ± 0.8
0.2
6.5 ± 1.9
0.5 ± 0.3
> 10
>10
> 100
> 100
> 100
> 100
> 100
> 100
Sofosbuvir
ND: Not determined. All assays were performed in replicates. Only means are shown.
Activity profile:
Compound 16 was tested against a panel of genetically diverse HCV replicons and
showed anti-HCV activity in the submicromolar range (Table 2). Similar to SOF,
compound 16, which forms 16-triphosphate intracellularly, displayed a 2-3 fold decrease
in potency against genotype 1b polymerase mutations S96T (Table 3) and S282T (Table
4
) associated with drug resistance.
Table 2. Effect of compound 16 in culture against HCV Genotypes 1-4 (4 day assay)
Genotype (GT): IC derived from data using patient isolates
5
0
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Cmpd
16, µM
1a
1b
0.15
0.06
0.91
> 60
> 100
2a/k
0.05
0.02
1.0
2b
0.09
0.04
1.0
3a
4
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
0.13
0.06
0.93
> 60
0.15
0.09
0.95
> 60
> 100
0.17
0.07
0.95
> 60
> 100
SOF, µM
IFN, IU/mL
1
3, µM
> 60
> 100
> 60
> 100
a
PSI-6206, µM > 100
a
22
2
'-deoxy-2'-fluoro-2'-C-methyl-uridine
Table 3. 16-TP: IC with S96T mutant and various genotypes
5
0
Fold-change relative to NS5B
GT 1b WT
NS5B
IC50, µM
N
GT 1b WT
GT 1b S96T
GT 2a WT
GT 4a WT
5.5 ± 2.8
13 ± 2.2
6.1 ± 3.4
8.6 ± 7.0
1
3
2
2
2
2.4
1.1
1.6
Table 4. Effect of compound 16 in culture against HCV S282T mutant.
Wild-Type (ꢀM)
Cmpd S282T Fold Increase
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9
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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5
5
5
5
5
5
5
5
5
5
6
(
ꢀM)
EC50
EC90
EC50
EC90
EC50 EC90
1
6
0.02 ± 0.001 0.07 ± 0.005 0.07 ± 0.002 0.2 ± 0.03
0.09 ± 0.01 0.3 ± 0.05 0.4 ± 0.12 1 ± 0.13
3.5
4.4
2.9
3.0
0
1
2
3
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9
0
1
2
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4
5
6
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8
9
0
1
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0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
SOF
Toxicity profile: It has previously been reported that certain nucleoside analogs can
2
3
exhibit mitochondrial toxicity. In order to address this issue compounds 13 and 16
were evaluated for their effects on mitochondrial DNA levels. HepG2 cells were
propagated in the presence of nucleotide analogs (up to 50 ꢀM) for 14 days prior to
quantification of mitochondrial COXII DNA (mtDNA) and β-actin DNA using real-time
PCR. Lamivudine (3TC) and β-D-2’,3’-dideoxycytidine (ddC) (at 10 ꢀM) were included
as negative and positive controls, respectively (Table 5). Over a 14 day period, neither
the parent, nor the prodrug showed measurable mitochondrial toxicity up to 50 ꢀM in
HepG2 cell line (Table 5) whereas ddC, as anticipated, was highly toxic.
Table 5. Effect of compound 16 and 13 on mitochondrial (Mt), nuclear DNA levels and
lactic acid production in HepG2 cells (14-day assay)
Lactic
Conc
% Inhibition
IC , µM
5
MtDNA Content
0
Cmpd
acid
µM MtDNA /nDNA MtDNA / nDNA (% of control)
production
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(
% of control)
1
0
0
< 1 / < 1
27 / 13
130 (117-143)
60 ± 1.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
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35
36
37
38
39
40
41
42
43
44
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16
> 50 / > 50
5
84 (68-102)
56 ± 0.88
10
< 1 / 8.8
< 1 / <1
160 (113-230)
120 (77-175)
85 ± 7.3
1
3
> 50 / > 50
50
120 ± 5.8
10
< 1 / 7.3
<1 / 38.3
<1 / 38.8
176 (151 - 206)
187 (170 - 205)
255 (247 - 264)
SOF
25
>50 / >50
50
3
TC
10
10
16 / 38
96 / 53
> 10 / > 10
< 10 / < 10
140 (103-181)
9.5 (5.7-16)
120 ± 10
330 ± 27
ddC
No
drug
0 / 0
100 (95-105)
100 ± 2.6
control
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a
CC , values obtained from HepG2 cells were generated from two separate
50
experiments (± SD where applicable). mtDNA was measured and compared to nuclear
β-actin DNA.
0
1
2
3
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5
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8
9
0
1
2
3
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5
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9
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1
2
3
4
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9
0
b
Lactic acid measurements are the mean of triplicates from a single 96-well plate (±
SD).
Lactic acid levels were also measured in the culture supernatant after 14 days of
incubation with each drug. The total amount of lactic acid produced was determined for
each sample. Increased production of lactic acid (generally above 100% when normalized
2
4
to β-actin control) is associated with mitochondrial toxicity. We did not observe
increased lactic acid production with up to 50 ꢀM of compound 13 and its prodrug 16. As
expected, treatment with ddC resulted in increased lactic acid production (Table 5). In
addition, compound 16 was devoid of bone marrow toxicity at concentration up to 50 ꢀM
(
data not shown).
Cellular pharmacology: The intracellular metabolism of prodrug 16 in Huh-7 cells was
determined. Compound 16 was incubated in Huh-7 cells or primary human hepatocytes
(PHH) at 50 ꢀM for 4 h at 37 °C. Intracellular metabolites were extracted with 70%
ice-cold methanol in water and subsequently identified by LC-MS/MS. Compound 16
generated markedly higher levels of nucleoside 5’-triphosphate (2.7-fold and 2.3-fold in
Huh-7 cells and PHH, respectively) when compared to sofosbuvir (Figure 4).
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Figure 4. Intracellular 5’-triphosphate levels for compound 16 and 2’-F,2’-C-Me
nucleoside triphosphate derived from sofosbuvir in Huh-7 cells and PHH.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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35
36
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41
42
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50
51
52
53
54
55
56
57
58
59
60
Selectivity: the potential off-target impact of 16-TP on human DNA and RNA
2
5
polymerases was evaluated. Similar to 2’-F,2’-C-Me-UTP, the triphosphate metabolite
of sofosbuvir, 16-TP is not incorporated by RNA polII at concentration up to 100 ꢀM and
is only a weak substrate of POLRMT at this concentration (Table 6). In addition, 16-TP
has no major impact on either DNA polymerase α, β and γ (Table 7).
Table 6: Incorporation of 16-TP by the human nuclear polymerase (RNA polII) and the
mitochondrial RNA polymerase (POLRMT).
Inhibitor
RNA POL II IC50
POLRMT %
Incorporation
16-TP
> 100 ꢀM
35 ± 0.5
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
3
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5
5
5
5
5
5
5
6
2
’-F,2’-C-Me-UTP
> 100 ꢀM
3.1 ± 1.4
NA
Alpha-amanitin
+ve CTRL)
2.5 ± 1.7 nM
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
(
Table 7: Impact of 16-TP on human DNA polymerase activity
IC (ꢀM)
5
0
Inhibitor
DNA Pol α
20.2 ± 2.3
> 100
DNA Pol β
> 100
DNA Pol γ
16-TP
>
100
2’-F,2’-C-Me-UTP
> 100
> 100
NA
43
Aphidicolin
(+ve ctrl)
2.4 ± 1.0
NA
ddTTP
NA
19 ± 1.0
(+ve ctrl)
NA: Not Available
Stability and metabolism: Compound 16 was shown to be stable in both fresh human
blood for up to 2 h while the positive control, eucatropine, was at 19% remaining at the
same time point and in addition, human intestinal microsomes clearance was low (Table
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). However, as expected for a prodrug, compound 16 was unstable in human liver
microsomes. It had a short half-life (11.6 min) and high clearance with 4.2% remaining at
0 min (Table 9)
Table 8. Stability of compound 16 in intestinal microsomes.
Human Blood Human Intestinal
Microsomes
Verapamil
6
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
Compound
t (min)
16
Eucatropine
16
120
29
64
54
1
/2
CL
(µl/min/mg)
%
103 (2 h)
19 (2h)
79% (60 min) 56% (60 min)
Remaining
Table 9. Stability of compound 16 in human liver microsomes.
Human Liver Microsomes
Compound
16
Testosterone Diclofenac Propafenone
t (min)
1
1.6
15
92
8.9
5.3
1
/2
CL (µl/min/mg)
120
156
260
%
remaining at 60
min
4
.2%
6.3%
1.2%
0%
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1
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2
2
2
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5
6
PSI-7851 which is SOF except as the mixture of Rp/Sp phosphorous diastereomers,
analogues to compound 16, displayed a similar profile (human plasma t > 24 h; human
1
/2
liver S9 t = 0.57 h; simulated gastric fluid t = 22 h and simulated intestinal fluid t >
1
/2
1/2
1/2
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
2
6
2
4 h). Drug-drug interactions is an important issue in drug development and is often
connected to the inhibition of hepatic enzymes such as cytochrome P450’s (CYP450) by
one of the drug. Therefore, the potential drug-drug interaction liabilities of compound 16
was investigated in a CYP450 reversible inhibition assay. As shown in table 10,
compound 16 showed comparable inhibition to sofosbuvir with moderate inhibition of
CYP450 3A4 and 2C9.
Table 10. Inhibition of Major Human CYP P450 Enzymes at Physiological
Concentrations for 16.
Cytochrome P450 Inhibition IC (µM)
50
Compound
3A4
2C9
1A2
> 100
> 100
2D6
16
6.8 ± 0.9
8.4 ± 1.6
25 ± 4.4
60 ± 24
> 100
> 100
Sofosbuvir
Conclusions:
We report herein for the first time the synthesis and biological evaluation of a unique
series of 2’-fluoro,2’-chloro nucleosides. Among all the compounds synthesized, 16 is a
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potent and specific inhibitor of HCV. It does not inhibit replication of major DNA and
RNA viruses (At concentration up to 300 ꢀM for vaccinia virus or 113 ꢀM for rift valley
fever virus, dengue virus, west nile virus, yellow fever virus, japanese encephalitis virus,
chikungunya, influenza A H1N1 and respiratory syncytial virus; data not shown).
Compound 16 had excellent pan-genotypic activity with anti-HCV replicon activity
similar to SOF. Its nucleoside 5’-triphosphate was a specific inhibitor of HCV NS5B
polymerase with no significant inhibition of human beta, gamma DNA polymerase and
showed low incorporation by cellular mitochondrial RNA polymerase. Despite observing
an IC50 of 20 ꢀM versus polymerase alpha, we did not observe corresponding
cytotoxicity nor cytostatic behavior in five different cell lines (PBM, CEM, Vero, Huh7
and HepG2) at concentrations up to 100 ꢀM. No mitochondrial (MtDNA, nuclear DNA)
including lactic acid and bone marrow toxicities were observed up to 50 µM. Compound
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
1
6 was stable in human blood for up to 2 h, rapidly metabolized in human hepatocytes
and showed moderate to low metabolism in human intestinal microsomes. Moderate
inhibition of CYP450 3A4 and 2C9 was observed similar to that of observed with SOF.
The novel nucleotide analog 16 has an excellent preclinical profile suggesting further
development to establish its potential value as a novel anti-HCV nucleotide
phosphoramidate analog.
Experimental Section:
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6
Anhydrous solvents were purchased from Aldrich Chemical Company, Inc. (Milwaukee).
Reagents were purchased from commercial sources. Unless noted otherwise, the
materials used in the examples were obtained from readily available commercial
suppliers or synthesized by standard methods known to one skilled in the art of chemical
0
1
2
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TM
synthesis. H, C, F and P NMR spectra were taken on a Bruker Ascend 400
spectrometer at rt and reported in ppm downfield from internal tetramethylsilane (for
1
H-NMR). NMR processing was performed with MestReNova version 10.0.2-15465.
Deuterium exchange and decoupling experiments were utilized to confirm proton
assignments. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). All
J-values are in Hz and calculated by Mnova or MestReNova programs. Mass spectra
were determined on a Micromass Platform LC spectrometer using electrospray
ionization. Purity of final compounds was determined to be > 95%, using an UPLC
analyses performed on a Waters Acquity UPLC System with a Kinetex LC column (2.1
mm x 50 mm, 1.7 ꢀm, C18, 100 Å) and further supported by clean NMR spectra. Mobile
phase flow was 0.4 mL/min with, a 1.20 min gradient from 95% aqueous media (0.05%
formic acid) to 95% CH CN (0.05% formic acid), and a 4.5 min total acquisition time.
3
Photo diode array detection was from 190 to 360 nm. Analytic TLC was performed on
Analtech GHLF silica gel plates, and preparative TLC on Analtech GF silica gel plates.
Column chromatography was performed on Combiflash R 200 or via reverse-phase high
f
performance liquid chromatography.
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2-Deoxy-3,5-di-O-(tert-butyldiphenylsilyl)-D-ribonolactone (6)
To a solution of 2-deoxyribono-lactone 5 (8.95 g, 66.80 mmol) in 300 mL of anhydrous
DMF were added imidazole (22.7 g, 333 mmol, 5.0 eq) and tert-butyldiphenylsilyl
chloride (38.4 g, 140 mmol, 2.1 eq). The reaction was stirred at rt for 24 h and quenched
by addition of water. The water layer was extracted with hexanes (3 x 100 mL), and the
combined organic layers were washed with brine, and dried over anhydrous Na SO . The
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crude product was concentrated and purified by flash chromatography (hexanes/ethyl
acetate 50:1 to 30:1) to afford product 6 as a colorless oil (33.7 g, 83% yield).
1
H NMR (400 MHz, CDCl ) δ 7.68 (m, 4H), 7.64-7.60 (m, 2H), 7.57-7.34 (m, 14H),
3
4.70-3.59 (m, 1H), 4.43 (d, J = 1.7 Hz, 1H), 3.68 (dd, J = 11.6, 2.7 Hz, 1H), 3.23 (dd, J =
11.6, 2.4 Hz, 1H), 2.90 (dd, J = 17.9, 6.8 Hz, 1H), 2.63 (dd, J = 17.9, 1.8 Hz, 1H), 1.15 (s,
1
3
9
H), 1.01 (s, 9H). C NMR (101 MHz, CDCl ) δ 176.1, 135.7, 135.6, 135.6, 135.4,
3
132.9, 132.8, 132.6, 132.0, 130.2, 130.2, 130.0, 129.9, 128.0, 128.04, 127.9, 127.9, 88.1,
7.5, 77.2, 76.8, 71.0, 63.3, 39.2, 26.8, 26.7, 19.1, 19.0.
7
2-Deoxy-2-fluoro-3,5-di-O-(tert-butyldiphenylsilyl)-D-ribono-lactone (7).
In a 1000 mL round-bottom flask, compound 6 (39.7 g, 65.2 mmol) and NFSi (30.84 g,
9
7.8 mmol, 1.5 eq) were dissolved in 320 mL of anhydrous THF. The solution was cooled
to -78 °C, and 85 mL (85 mmol, 1.3 eq) of a 1 M solution of LiHMDS in THF was added
dropwise over a period of 35 min. The reaction was allowed to stir at -78 °C for an
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additional 1 h and was quenched with a saturated solution of NH Cl. Solid KMnO (4.0 g)
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4
o
was added portion wise at 0 C. After 1 h, the reaction mixture was filtrated through a pad
of silica gel and washed with EtOAc (350 mL). The water layer was then extracted with
hexanes (3 x 120 mL). The organic layers were finally combined, washed with a
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saturated solution of NaHCO , water, and brine, dried over anhydrous Na SO , and
3
2
4
concentrated in vacuo. The residue was purified by flash chromatography (hexanes/ethyl
acetate 100:0 to 20:1) to afford a crude product that was purified again by flash
chromatography (hexanes/DCM 10:1 to 3:1) to afford 7 (9.81 g, 24%) and starting
material 6 (8.7 g).
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H NMR (400 MHz, CDCl ) δ 7.71-7.62 (m, 4H), 7.60-7.55 (m, 2H), 7.53-7.30 (m, 14H),
3
5.39 (d, J = 7.0 Hz, 1H), 5.26 (d, J = 7.0 Hz, 1H), 4.87-4.77 (m, 1H), 4.44-4.35 (m, 1H),
1
9
3
.80-3.72 (m, 1H), 3.47 (dd, J = 12.2, 3.3 Hz, 1H), 1.12 (s, 9H), 0.93 (s, 9H). F NMR
1
3
(
377 MHz, CDCl ) δ -202.70 (dd, J = 51.1, 18.6 Hz). C NMR (101 MHz, CDCl3)
3
δ168.7 (d, J = 22.9 Hz), 135.8, 135.7, 135.67, 135.5, 132.7, 132.2, 131.6, 130.4, 130.3,
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29.9, 129.8, 128.0, 127.8, 127.7, 92.2 (d, J = 199.3 Hz), 81.95 (d, J = 9.8 Hz), 77.4, 77.2,
7.0, 76.76, 72.8 (d, J = 21.4 Hz), 61.2, 26.8, 26.6, 19.2, 19.1.
7
2
-Deoxy-2-fluoro-2-chloro-3,5-di-O-(tert-butyldiphenylsilyl)-D-ribono-lactone (8).
In a 250 mL round-bottom flask compound 7 (9.70 g, 15.47 mmol) and NCS (4.17 g,
31.2 mmol, 2.0 eq) were dissolved in 75 mL of anhydrous THF. The solution was
cooled to -78 °C, and 23.2 mL of a 1 M solution of LiHMDS in THF (23.2 mmol, 1.5 eq)
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was added dropwise over a period of 20 min. The reaction mixture was allowed to stir at
-
78 °C for an additional 45 min and was quenched with a saturated solution of NH Cl.
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The water layer was extracted with hexanes (3 x 70 mL) and the combined organic layers
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were washed with a saturated solution of NaHCO , water and brine. The solution was
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dried over anhydrous Na SO and concentrated under reduced pressure. The crude
2
4
product was purified by flash chromatography (hexanes/ethyl acetate 100:0 to 20:1) to
afford a 4/1 mixture of compounds 8 and 9 (5.83 g, 57%). Additional flash
chromatography on silica gel column (hexanes/ethyl acetate 100:0 to 20:1) afforded pure
compounds 8 （3.76 g， 37%）and 9（0.93g, 9%）.
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Compound 8: H NMR (400 MHz, CDCl ) δ 7.78-7.58 (m, 4H), 7.58-7.40 (m, 8H),
3
7.43-7.29 (m, 8H), 4.65-4.49 (m, 2H), 3.70 (dd, J = 12.0, 3.7 Hz, 1H), 3.65-3.51 (m, 1H),
19
1
.20-1.08 (s, 9H), 0.97-0.90 (s, 9H). F NMR (CDCl , 376.3 MHz) δ (ppm): δ -133.22 (d,
3
1
3
J = 7.7 Hz). C NMR (101 MHz, CDCl ) δ 165.2 (d, J = 26.6 Hz), 136.0, 135.7, 135.6,
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135.4, 132.4, 132.1, 132.0, 131.2, 130.4, 129.9, 129.9, 128.0, 127.9, 127.8, 101.3 (d, J =
2
61.6 Hz), 84.4, 74.8 (d, J = 15.6 Hz), 61.2, 29.7, 26.7, 26.6, 19.4, 19.0.
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Compound 9: H NMR (400 MHz, CDCl ) δ 7.73-7.61 (m, 4H), 7.56-7.48 (m, 2H),
3
7.47-7.40 (m, 6H), 7.34 (tt, J = 10.3, 6.6 Hz, 8H), 4.79 (dd, J = 13.9, 7.8 Hz, 1H),
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.35-4.26 (m, 1H), 3.76 (d, J = 12.5 Hz, 1H), 3.50 (dd, J = 12.5, 3.4 Hz, 1H), 1.13 (s, 9H),
19
13
0
.88 (s, 9H). F NMR (377 MHz, CDCl ) δ -128.25 (d, J = 13.9 Hz). C NMR (101
3
MHz, CDCl ) δ165.0 (d, J = 29.6 Hz), 136.0, 135.7, 135.6, 135.4, 132.6, 132.1, 131.9,
3
1
30.7, 130.6, 130.4, 129.9, 129.8, 128.0, 127.9, 104.2 (d, J = 267.1 Hz), 81.3 (d, J = 8.4
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