β-D-2′-C-methyl-2,6-diaminopurine ribonucleoside phosphoramidates are potent and selective inhibitors of hepatitis C virus (HCV) and are bioconverted intracellularly to bioactive 2,6-diaminopurine and guanosine 5′-triphosphate forms

Article abstract of DOI:10.1021/jm501874e

The conversion of selected β-d-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing for the generation of two bioactive nucleoside triphosphates (NTPs) in human hepatocytes. A variety of 2′-C-methyl DAPN-PDs were prepared and evaluated for inhibition of HCV viral replication in Huh-7 cells, cytotoxicity in various cell lines, and cellular pharmacology in both Huh-7 and primary human liver cells. The DAPN-PDs were pan-genotypic, effective against various HCV resistant mutants, and resistant variants could not be selected. 2′-C-Me-DAPN-TP and 2′-C-Me-GTP were chain terminators for genotype 1b HCV-pol, and single nucleotide incorporation assays revealed that 2′-C-Me-DAPN-TP was incorporated opposite U. No cytotoxicity was observed with our DAPN-PD when tested up to 50 μM. A novel, DAPN-PD, 15c, has been selected for further evaluation because of its good virologic and toxicologic profile and its ability to deliver two active metabolites, potentially simplifying HCV treatment.

Full text of DOI:10.1021/jm501874e

Article
pubs.acs.org/jmc
β‑D‑2′‑C‑Methyl-2,6-diaminopurine Ribonucleoside
Phosphoramidates are Potent and Selective Inhibitors of Hepatitis C
Virus (HCV) and Are Bioconverted Intracellularly to Bioactive 2,6-
Diaminopurine and Guanosine 5′-Triphosphate Forms
Longhu Zhou,† Hong-wang Zhang,† Sijia Tao,† Leda Bassit,†,‡ Tony Whitaker,§ Tamara R. McBrayer,‡,§
Maryam Ehteshami,† Sheida Amiralaei,† Ugo Pradere,† Jong Hyun Cho,† Franck Amblard,†,‡
Drew Bobeck,§ Mervi Detorio,†,‡ Steven J. Coats,§ and Raymond F. Schinazi*,†,‡
†Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of
Medicine, Atlanta, Georgia 30322, United States
‡Veterans Affairs Medical Center, Decatur, Georgia 30033, United States
§CoCrystal Pharma, Inc., Tucker, Georgia 30084, United States
ABSTRACT: The conversion of selected β-D-2,6-diamino-
purine nucleosides (DAPNs) to their phosphoramidate
prodrug (PD) substantially blocks the conversion to the G-
analog allowing for the generation of two bioactive nucleoside
triphosphates (NTPs) in human hepatocytes. A variety of 2′-
C-methyl DAPN-PDs were prepared and evaluated for
inhibition of HCV viral replication in Huh-7 cells, cytotoxicity
in various cell lines, and cellular pharmacology in both Huh-7
and primary human liver cells. The DAPN-PDs were pan-
genotypic, effective against various HCV resistant mutants, and resistant variants could not be selected. 2′-C-Me-DAPN-TP and
2′-C-Me-GTP were chain terminators for genotype 1b HCV-pol, and single nucleotide incorporation assays revealed that 2′-C-
Me-DAPN-TP was incorporated opposite U. No cytotoxicity was observed with our DAPN-PD when tested up to 50 μM. A
novel, DAPN-PD, 15c, has been selected for further evaluation because of its good virologic and toxicologic profile and its ability
to deliver two active metabolites, potentially simplifying HCV treatment.
Three major drug targets have reached evaluation in humans
for chronic HCV infection:8,9 the NS3/4A serine protease, the
large phosphoprotein NS5A, and NS5B RNA-dependent RNA
polymerase (RdRp) NS5B (nucleoside10 and non-nucleoside
inhibitors11). The HCV NS5B RNA-dependent RNA polymer-
ase (RdRp) is essential for HCV replication, and both
nucleoside and non-nucleoside antiviral agents targeting this
enzyme are in preclinical and clinical development. For a
nucleoside analog to be incorporated into a growing RNA chain
by a polymerase, it must first enter the cell by either passive or
active transport and undergo three separate phosphorylation
steps to ultimately provide the nucleoside analog in its
triphosphate form (NTP). It has been reported that many
nucleoside analogs are poor substrates for the phosphorylation
kinases, which convert the nucleosides to the corresponding
nucleoside monophosphate.12 The initial phosphorylation step
is often rate limiting en route to the formation of the active
NTP and in many cases does not occur with sufficient efficiency
to provide the active NTP form at therapeutically effective
concentrations. In order to bypass this rate-limiting step, many
INTRODUCTION
■
Over the past 5 decades nucleoside analogs have played an
ever-increasing role in the treatment of viral infections. To date
there are more than a dozen nucleoside analogs approved by
the FDA for the treatment of viral infections. Approximately
170 million people worldwide are infected with the seven major
genotypes of hepatitis C virus (HCV), and 3−4 million
individuals become newly infected each year.1 HCV infection
often leads to reduced liver function, cirrhosis, and cancer and
is the leading cause of liver transplantation.2 The first FDA-
approved treatment for chronic HCV was limited to pegylated
interferon-α alone or in combination with ribavirin,3 which was
effective in 40−60% of patients and is often associated with
significant side effects, such as fatigue, nausea, depression, and
thinning hair.4 In May 2011 the introduction of protease
inhibitors (PI) boceprevir and telaprevir to the above standard
of care marked the introduction of the first direct acting
antiviral agents (DAAs) for HCV.5,6 Unfortunately, these
treatments have limited efficacy for the genotype 1 virus, the
most prevalent in the U.S. and China. They are also associated
with various side effects, especially when combined with
pegylated interferon-α and ribavirin.7
Received: December 4, 2014
© XXXX American Chemical Society
A
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Figure 1. Chemical structures (where available) of nucleoside analog inhibitors that have entered human clinical trials for HCV therapy.
a
Scheme 1
a
Reagents and conditions: (a) 2,6-diaminopurine (3), DBU, TMSOTf, CH3CN, −40 to 65 °C, 5 h, 92%; (b) (Boc)2O, DMAP, THF, rt, 90%; (c)
NaOMe/MeOH, rt, 30 min, 92%; (d) NH3/MeOH, rt, 93%.
nucleoside prodrug analogs have been developed as more
lipophilic nucleoside monophosphate prodrugs (NMP-
PDs).10,13,14 NMP-PDs antiviral drugs have been reported to
increase the oral bioavailability, liver exposure, and brain
penetration of parent drugs. The most advanced nucleoside
NMP-PDs are the aryloxy phosphoramidates (“ProTide” or
“McGuigan PD”),15 1-aryl-1,3-propanyl esters (“HepDirect”),16
and lipid conjugates17 which have all have been evaluated in
humans. Pronucleotide (ProTide), first introduced in 1992,15 is
the most investigated NMP-PD and demonstrated improved
pharmacological activity of many parent nucleosides both in
vitro and in vivo.18 Currently the most advanced NMP-PDs
have the following limitations: (1) intracellular delivery of
potentially toxic agents; (2) a racemic phosphorus center; (3)
the nucleoside-MP phosphorus−oxygen bond being weak and
therefore prone to cleavage; (4) low to very low yields for the
preparation of NMP-PDs.14
across all genotypes in combination with pegylated interferon
and/or ribavirin or an NS5A inhibitor (e.g., Harvoni).
Sofosbuvir containing regimens with other DAAs can be
shorter in duration and achieve very high-sustained virologic
response (SVR). However, the continued high cost of DAAs
will preclude their prompt worldwide use especially when more
than one DAA is needed to cure an HCV-infected person. In
addition, the non-nucleoside inhibitors, unlike the nucleoside
analogs, are prone to have a low genetic barrier to resistance.
Therefore, there is an urgent need for more cost-effective
treatments that are pan-genotypic that can be globally
utilized.19
Early clinical trials of direct acting antiviral agents for HCV
treatment20 made clear the need for combination DAA therapy
in order to achieve high SVR and as the ultimate solution for
pan genotypic treatment of HCV. The ability to deliver more
than one active metabolite from a single drug could help
increase potency for inhibition of HCV replication and
potentially inhibit the formation of viral mutants.21 Like
Orthrus, the fearsome two-headed dog of Greek mythology
and guardian of Geryon’s herd of red cattle, an antiviral drug
that delivers two potent metabolites should increase potency
Nucleoside analog polymerase inhibitors are anticipated to
become the backbone of HCV therapy, and to date, at least 14
have entered clinical trials but only one has been approved
(Figure 1).10 Sofosbuvir, which received FDA approval in
December 2013, has highly potent clinical antiviral activity
B
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a
Scheme 2
a
Reagents and conditions: (a) ROH, cat. H2SO4, reflux, overnight, for 9a, 94%; for 9b, 89%; (b) POCl3, Et3N, diethyl ether, −70 °C to rt, overnight;
(c) amino acid esters hydrochloride (11), Et3N, DCM, −70 °C to rt, two steps for 12a, 72%; for 12b, 75%; for 12c, 70%; for 12d, 69%; for 12e,
70%; (d) Et3N, diethyl ether, −70 °C to rt, 35%.
and potentially suppress the formation of viral mutants. The
typical utilization of the aryloxy phosphoramidate prodrug
approach, such as in the case of sofosbuvir, involves the
intracellular delivery of phenol. While phenol is used in some
over-the-counter products as an oral anesthetic/analgesic and is
commonly used to temporarily treat pharyngitis (inflammation
of the throat), it has an LD50 of 270 mg/kg in mice and phenol
may cause harmful effects to the central nervous system and
heart, resulting in dysrhythmia, seizures, and coma. Chronic
exposure to phenol in humans has been linked to harmful
effects to the liver and kidneys.22 We sought to overcome these
potential liabilities by the introduction of a propyl ester side
chain at the ortho position of the phenol portion of our
prodrugs. Cellular pharmacology in human peripheral blood
mononuclear (PBM) cells showed that unmasking to the
monophosphate from this substituted phosphoramidate results
in the formation of hydroxyphenylpropanoic acid, which is a
known nontoxic metabolite23 of dihydrocoumarin, a common
flavoring agent widely used in food24 and cosmetics.25
of L-alanine esters 11 with readily prepared aryloxy
phosphorochloridates 10 (Scheme 2). Diaryloxy phosphoro-
chloridate 13 was freshly synthesized with the same reaction
conditions (Scheme 2). Since the phosphoryl chlorides 12 were
produced as an approximate 1 to 1 mixture of the two possible
diastereomers at the phosphorus center, the subsequent
phosphoramidates 15 (Scheme 3) were also obtained as a
diastereomeric mixture. This is due to the often clean inversion
of configuration at the phosphorus atom during the SNP
reaction (similar to the inversion at electrophilic carbon centers
in the more well-known SN2 reaction). The phosphorochlor-
idates 12 and 13 could be stored for extended periods when
diluted in dry THF to 1 M and stored in a well-sealed container
at −80 °C under nitrogen or argon.
Phosphoramidate prodrugs derived from the tetra-Boc
nucleoside 6 were prepared initially following literature
nucleoside phosphoryl chloride displacement methods, which
conveniently afforded the desired products as a mixture of two
diastereomers at the phosphorus center (14a−e).27 For
example, compound 15b was prepared by reacting 6 in the
presence of N-methylimidazole with the phosphoryl chloride
12b in THF/ACN to produce protected prodrug 14b.
Subsequent deprotection with 80% aqueous TFA gave
phosphoramidate 15b in high overall yield from this two-step
process (Scheme 3). Acetonitrile was used as a cosolvent to
improve the solubility of the reactants, which resulted in a more
complete reaction and much better isolated yields. Similar
reaction conditions were employed for the synthesis of prodrug
17, which contains an achiral phosphorus center. For the more
lipophilic and potentially better absorbed 2′,3′-carbonate
prodrug, 16,28 compound 13c was simply treated with
carbonyldiimidazole.
Herein we report the synthesis and biological evaluation of β-
D-2′-C-methyl-2,6-diaminopurine ribonucleoside phosphorami-
date prodrugs that intracellularly deliver two active nucleoside
triphosphates along with nontoxic prodrug metabolites.
CHEMISTRY
■
The 2,6-diaminopurine tribenzoylated nucleoside 4 was
synthesized from known sugar 226 and 2,6-diaminopurine 3
with TMSOTf in the presence of DBU. The unprotected 2,6-
diaminopurine nucleoside 7 was prepared by reaction of 4 with
ammonia in methanol. Boc protected nucleoside 6 was
necessary to significantly improve the yield of the phosphor-
amidate forming reaction and prepared by reaction of 4 with
(Boc)2O at rt, followed by careful debenzylation with sodium
methoxide at rt (Scheme 1). The crude debenzylation reaction
was cautiously neutralized with Dowex resin (H+ form) during
workup to avoid unwanted deprotection of the Boc groups.
Hydrolysis of dihydrocoumarin, 8, in anhydrous ethanol or
isopropanol gave esters 9a and 9b, respectively. The phenyl
dichlorophosphates 10a, 10b, and 10c were prepared from the
appropriate phenol and phosphorus oxychloride. Aryloxy
phosphorochloridates 12 were freshly prepared by treatment
The diastereomeric mixtures 15b−e were separated by
careful column chromatography on silica gel to obtain the two
single diastereomers. However, preparation of 15b(Rp) in larger
quantities required a more practical method. A single
diastereomeric phosphoramidating reagent containing a p-
nitrophenyl ester was used as an alternative to the chloridate
12b for the stereospecific synthesis of 15b(Rp).28,29 Accord-
ingly, the diastereomeric mixture 19(Rp) and 20(Sp) was
prepared and subjected to fractional crystallization to obtain the
C
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a
Scheme 3
a
Reagents and conditions: (a) 12a−e, N-methylimidazole, THF−CH3CN, rt; (b) 80% TFA, 0 °C to rt, two steps for 15a, 38%; for 15b, 56%; for
15c, 40%; for 15d, 41%; for 15e, 43%; (c) N,N′-carbonyl diimidazole, CH3CN, rt, 71%; (d) 13, N-methylimidazole, THF−CH3CN, rt; (e) 80%
TFA, 0 °C to rt, two steps for 22, 55%.
single isomers 19(Rp) and 20(Sp) (see Experimental Section)
(Scheme 4). Their absolute stereochemistry was determined by
single crystal X-ray analysis (Figure 2). Considering the change
in stereochemical nomenclature priorities, it is the reagent
having the Sp configuration that would lead, after inversion
through a nucleophilic displacement reaction with 6, to the
desired product 15b(Rp). Thus, nucleoside 6 was treated with
tert-butylmagnesium chloride in THF, and subsequent treat-
ment of the resulting suspension with 20(Sp) at ambient for 3
days afforded the single diastereomer Rp, which was
deprotected with aqueous TFA to afford 15b(Rp) (Scheme
4). The Rp reagent 19 gave the phosphoramidate product
15b(Sp) in similar yield (Scheme 4). The absolute config-
urations of the phosphorus diastereomers of 15c−e were
assigned by comparison to 15b(Sp) and 15b(Rp).
RESULTS AND DISCUSSION
■
Antiviral Activity and Cytotoxicity. 2′-C-Methyl-2,6-
diaminopurine phosphoramidate prodrugs (DAPN-PD) were
evaluated for inhibition of HCV RNA replication in Huh-7 cells
using a subgenomic HCV replicon system.30 Cytotoxicity in
Huh-7 cells were determined simultaneously by extraction and
amplification of both HCV RNA and cellular ribosomal RNA
(rRNA) (Table 1).31 DAPN-PD compounds showed greater
potency against HCV replication in Huh-7 cells when
compared to the parent DAPN nucleoside 7 (up to 14-fold;
e.g., EC50 = 0.2 for 15b(Rp) vs 5.4 μM for parent compound 7.
D
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a
Scheme 4
a
Reagents and conditions: (a) p-nitrophenol, Et3N, diethyl ether, rt, 85% (19 + 20, 1:1 ratio); (b) 6, t-BuMgCl, THF, −78 °C, then rt 3 days; (c)
80% TFA, 0 °C, then rt 4 h. 15b(Rp): 41% two steps. 15b(Sp): 39% two steps.
Figure 2. X-ray crystal structures of 19(Rp) and 20(SP).
The difference in activity between the various pairs of
phosphorus diastereomers could be due to different rates of
deesterification that lead to different levels of unmasked
monophosphate. In general one of the diastereomers was
producing the majority of the antiviral activity when compared
with the diastereomeric mixture. This was not the case for 15c,
as the two phosphorus diastereomers had very similar anti-
HCV potency. The 2′,3′-carbonate phosphoramidate 16
retained reasonable activity (EC50 = 2.8 μM), but later it was
found that the carbonate group falls off quickly in simulated
intestinal fluid (T1/2 < 5 h). The symmetrical phenolic ester 18
was inactive and presumed to be too stable in cell culture to
unmask to the monophosphate. 1 (BMS-986094) was included
as a positive control and, consistent with the literature,32
demonstrated nanomolar potency. Conversely, this compound
was found to be cytotoxic in Huh-7 replicon cells as indicated
by significant reductions in cellular rRNA levels (CC50 = 0.8
μM). No cytotoxic effect was observed with up to 10 μM with
the DAPN compounds in the same cell line.
DAPN compounds were further evaluated for cytotoxicity at
higher concentrations in human PBM cells, human lympho-
blastoid CEM, and African Green monkey Vero cells (Table
1).33 In contrast to 1 (BMS-986094) that showed cytotoxicity
in all cell types tested, we did not observe a cytotoxic effect with
the majority of DAPN compounds. Prototype DAPN-PD with
the highest antiviral activity, 15b(Rp), was also further evaluated
for toxicity in PC3 cells (a human prostate cancer cell line) as
well as its effect on cellular GAPDH levels and thymidine
uptake. At 100 μM 15b(Rp) we did not observe any toxicity,
effect on GAPDH levels, or effect on thymidine uptake (data
not shown).
It has previously been reported that some nucleotide analog
compounds may also inhibit Huh-7 cell growth34 and exhibit
mitochondrial toxicity.35 In order to address this issue,
compounds 7, 15a, 15b(Rp), and 15c were evaluated for
E
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Table 1. HCV Replicon Activity and Cytotoxicity of Phosphoramidate Prodrugs
a
b
replicon Huh-7 (μM)
cytotoxicity, CC50 (μM)
compd
EC50
5.4
EC90
>10
CC50
PBM
>100
CEM
Vero
>100
7
>33
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.8
>100
>100
>100
>100
>100
>100
>100
40
15a
0.9 0.3
2.3
4.0 2.8
7.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
64
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
90
15b
15b(Rp)
15b(Sp)
15c
0.2 0.1
2.7
0.7 0.3
8.7
0.7 0.2
1.0
2.5 0.2
2.7
15c(Rp)
15c(Sp)
15d
0.9
2.8
2.2
9.0
>100
>100
>100
>100
>100
>100
>100
25 9.9
8.7 9.1
15d(Rp)
15d(Sp)
15e
2.4
8.2
4.7
9.7
1.7
8.0
15e(Rp)
15e(Sp)
16
1.4
6.9
9.1
26
2.8
8.5
18
>10
>10
1
0.02 0.2
0.04 0.2
4.7 3.3
14 5.1
a
EC50, EC90, and CC50 values obtained from Huh-7 replicon were generated in triplicate from one or two separate experiments ( SD where
b
applicable). CC50 values of compounds reported for PBM, CEM, and Vero cells were generated from one to four separate experiments ( SD where
applicable).
Table 2. Mitochondrial Toxicity and Lactic Acid Production with DAPN Prodrugs
a
b
CC50 (μM)
lactic acid levels (% of β-actin control)
10 μM
compd
mtDNA
>50
β-actin DNA
1 μM
50 μM
7
>50
>50
>50
>50
110 0.9
100 13
150 1.7
90 5.1
190 20
170 3.7
140 2.0
120 5.6
81 0.1
110 6.1
90 4.5
110 1.9
80 3.0
15a
>50
15b(Rp)
15c
>50
>50
c
c
d
d
1
3.4, 5.9, <1
>10
<1, 2.3, 0.86
>10
UM
UM
e
e
3TC
ddC
ND
91 2.5
220 11
ND
e
e
<10
<10
ND
ND
a
CC50 values obtained from HepG2 cells were generated from two separate experiments ( SD where applicable). mtDNA was measured and
b
c
compared to nuclear β-actin DNA. Lactic acid measurements are the mean of triplicates from a single 96-well plate ( SD). Determined in three
separate experiments. UM: unmeasurable at 100 and 10 μM, as the percent of cells at these concentrations was <1. ND: not determined
d
e
their effects on mitochondrial DNA levels. HepG2 cells were
propagated in the presence of nucleotide analogs (up to 50
μM) 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
μM) were included as negative and positive controls,
respectively (Table 2). Neither the parent nor the DAPN
prodrug compounds tested showed measurable mitochondrial
toxicity up to 50 μM in HepG2 cell line. On the other hand, 1
(BMS-986094) showed mitochondrial toxicity with a CC50
value of 4.7 1.8 μM. Lactic acid levels were also measured in
the culture supernatant after 14 days of incubation with each
prodrug. The total amount of lactic acid produced was
determined for each sample. Increased production of lactic
acid (generally above 100% when normalized to β-actin
control) is associated with mitochondrial toxicity.36 We did
not observe increased lactic acid production with up to 50 μM
parent DAPN or DAPN-PD compounds. As expected,
treatment with ddC resulted in increased lactic acid production
(Table 2). We were unable to accurately measure lactic acid
levels with 1 (BMS-986094) because treatment with 10 μM of
this compound led to excessive cell death.
Finally, activity of candidate DAPN-PD compound 15b(Rp)
was also tested against a panel of genetically diverse HCV
replicons. We found that 15b(Rp) was equally active against all
HCV genotype replicons tested (Table 3). Furthermore, this
compound remained effective against a panel of HCV genotype
1b replicons harboring mutations associated with drug
resistance (Table 4).
Table 3. Pan-Genotypic Activity of DAPN pProdrug 15b(Rp)
Tested in Cell-Based Assays
Huh-7 replicon
GT 1a
EC50 (μM)
EC90 (μM)
0.35
0.26
0.26
0.29
0.23
0.39
0.32
0.74
0.39
0.70
0.42
0.39
0.89
0.64
GT 1b
GT 2a
a
GT 2b (chimera)
a
a
a
GT 3a (chimera)
GT 4a (chimera)
GT 5a (chimera)
a
Chimeric replicons harbored NS5B gene from indicated genotypes in
backbone of HCV GT 1b con1 strain.
F
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Table 4. Cell-Based Antiviral Activity of 15b(Rp) against
Mutant HCV GT 1b Strains Commonly Associated with
Drug Resistance
a
Huh-7 replicon
EC50 (μM)
EC90 (μM)
NS5A Y93H
NS3 R155K
NS5B S282T
NS5B C316Y
NS5B C445F
NS5B M414I
NS5B P495A
0.30
0.32
0.10
0.27
0.12
0.28
0.08
3.0
1.3
0.23
0.77
0.25
1.1
0.14
a
Values averaged from two to three experiments except GT 1a Y93H
(n = 1).
Cellular Pharmacology and Biochemical Evaluation. It
has been well established that 6-modified nucleosides can be
substrates for human deaminases.37 We therefore set out to
evaluate the intracellular metabolism of DAPN-PD in both
Huh-7 cells and primary human hepatocytes.38 Parent DAPN
compound 7 and DAPN-PD 15b(Rp) were incubated in Huh-7
cells at 50 μM for 4 h at 37 °C. Intracellular metabolites were
extracted with 70% ice-cold methanol in water and sub-
sequently identified by LC−MS/MS. Incubation of the parent
DAPN in Huh-7 revealed the generation of two triphosphate
metabolites, namely, 2′-C-Me-DAPN-TP and 2′-C-Me-GTP at
a 1:3 ratio (Figure 3A). Conversely, incubation of DAPN-PD
15b(Rp) increased the intracellular levels of 2′-C-Me-DAPN-
TP in Huh-7 cells over 13-fold compared to the levels achieved
with parent nucleoside DAPN (0.74 0.3 pmol/106 cells and
Figure 3. Intracellular metabolism of parent DAPN and DAPN-PD in
one donor hepatocytes. (A) An amount of 50 μM of either parent
DAPN (gray bars) or DAPN-PD (15b(Rp)) (white bars) was
incubated with Huh-7 cells for 4 h at 37 °C. Intracellular generation
of parent nucleotide, NI-MP, NI-DP, and NI-TP was analyzed using
LC−MS/MS. (B) An amount of 50 μM DAPN-PD (15b(Rp)) was
incubated with primary human hepatocytes (PHH, gray bars) cells for
4 h at 37 °C. Generation of intracellular metabolites was determined as
described above. Error bars represent standard deviation from three
separate replicates from a single 12-well plate.
9.9
5.5 pmol/106 cells, respectively). The same trend was
observed in primary human hepatocytes where 7-fold higher
triphosphate metabolites were observed when compared to
Huh-7 cells (Figure 3B). Very similar results were obtained in
Huh-7 cells and primary human hepatocytes for 15c. Together,
these data indicate that DAPN-PD was capable of delivering
two nucleoside triphosphate metabolites intracellularly.
In Vitro Inhibition of Viral RNA Synthesis by NTP
Metabolites Generated by DAPN-PD. 2′-C-Me-GTP is a
known inhibitor of HCV NS5B polymerase.39 On the other
hand, 2′-C-Me-DAPN-TP has not been previously examined
for its effect on RNA polymerization. In order to determine
whether 2′-C-Me-DAPN-TP alone can inhibit NS5B-mediated
RNA polymerization, we separately synthesized each of the 5′-
triphosphate analogs and evaluated them in cell-free assays
using purified recombinant NS5B enzyme (Figure 4). The IC50
values for 2′-C-Me-GTP and 2′-C-Me-DAPN-TP were
CONCLUSIONS
■
The chemical synthesis, antiviral activity, cytotoxicity, and
cellular metabolism of DAPN prodrug compounds were
described. The intracellular metabolism of DAPN-PD resulted
in the generation two NI-TP metabolites, namely, 2′-C-Me-
DAPN-TP and 2′-C-Me-GTP, both of which inhibited HCV
NS5B polymerase-mediated RNA synthesis in vitro. Further-
more, DAPN-PDs did not exhibit any of the in vitro toxicities
observed with 1 (BMS-986094) and appear to be differentiated
in their toxicity profile additionally with the release of nontoxic
prodrug metabolites. Further studies are underway to evaluate
to enzymatic properties of each of the nucleoside 5′-
triphosphate metabolites, as well as the intracellular metabolism
of DAPN-PD in various cell lines such as cardiomyocytes. On
the basis of these results, compound 15c has been selected for
further in depth preclinical studies.
determined to be 5.6
1.6 and 3.4
1.1, respectively.
Under the conditions tested, we found that both NTP
metabolites inhibited polymerization as indicated by the
reduction in full-length RNA product formation with increased
TP inhibitor concentrations. As expected, distinct pausing was
observed at sites of GMP incorporation for 2′-C-Me-GTP.
Conversely, incubation with 2′-C-Me-DAPN-TP resulted in the
appearance of distinct pausing bands at AMP incorporation
sites. These data suggest that 2′-C-Me-DAPN-TP acts as an A-
analog. This finding was consistent with previous observations
that nucleotides with the 2,6-diaminopurine modification are
incorporated opposite a template T or U.40 Further
biochemical characterization is underway to evaluate the kinetic
properties of 2′-C-Me-DAPN-TP in vitro.
EXPERIMENTAL SECTION
■
Anhydrous solvents were purchased from Aldrich Chemical Co., Inc.
(Milwaukee, WI). 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
G
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Journal of Medicinal Chemistry
Article
133.5, 133.6, 137.4, 151.3, 155.9, 159.7, 165.3, 165.4, 166.3. LC/MS
calcd for C32H29N6O7 (M + 1)+, 609.2; observed, 609.2.
(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(2,6-bis(bis(tert-
butoxycarbonyl)amino)-9H-purin-9-yl)-3-methyltetrahydro-
furan-3,4-diyl Dibenzoate, 5. A solution of 4 (1.4 g, 2.3 mmol),
Boc anhydride (3.0 g, 14 mmol), and DMAP (56 mg, 0.46 mmol) in
THF (12 mL) was stirred at rt for 30 h. After the reaction was
complete, the solvent was removed under reduced pressure and the
residue was purified by flash column chromatography (0−40% EtOAc
in hexane). 2.1 g of white solid 5 was obtained (90% yield). 1H NMR
(400 MHz, CD3OD) δ 8.39 (s, 1H, H8), 8.07−8.20 (m, 4H, Ar−H),
7.87−7.89 (m, 2H, Ar−H), 7.42−7.61 (m, 7H, Ar−H), 7.25−7.30 (m,
2H, Ar−H), 6.74 (s, 1H, H1′), 6.00 (d, 1H, J = 4.8 Hz), 4.95−4.96 (m,
2H), 4.72−4.76 (m, 1H), 1.58 (s, 3H), 1.44 (s, 18H), 1.38 (s, 18H).
13CNMR (100 MHz, CD3Cl) δ 17.8, 27.7, 27.7, 63.6, 76.0, 81.2, 83.3,
83.9, 84.2, 89.2, 127.7, 128.4, 128.5, 128.6, 128.7, 129.3, 129.4, 129.7,
129.75, 130.0, 133.4, 133.6, 133.7, 143.7, 150.0, 150.6, 151.4, 152.4,
153.3, 165.1, 165.2, 166.3.
Figure 4. Incorporation of NI-TPs by NS5B RNA-dependent RNA
polymerase. Increasing amounts of either 2′-C-Me-DAPN-TP or 2′-C-
Me-GTP were incubated with NS5B polymerase, RNA template,
radiolabeled GpG primer, and rNTP nucleotide mix. Amount of RNA
product formed was measured after a 2 h incubation and visualized on
a 20% denaturing polyacrylamide gel. The RNA template sequence is
indicated, and sites of either A analog or G analog incorporation are
highlighted with an asterisk. Arrow represents full length RNA product
formed in the absence of inhibitor.
Di-tert-butyl (9-((2R,3R,4R,5R)-3,4-Dihydroxy-5-(hydroxy-
methyl)-3-methyltetrahydrofuran-2-yl)-9H-purine-2,6-diyl)bis-
(tert-butoxycarbonylcarbamate), 6. To a solution of 5 (1.7 g, 1.7
mmol) in anhydrous methanol (50 mL) was added a solution of
sodium methoxide (4.4 M, 0.3 mL, 1.3 mmol) at rt for 30 min
(monitored by TLC and LC−MS). After the reaction was complete,
Dowex resin (H+ form) was added portionwise to adjust the pH to 7.0.
The resin was filtered and washed with methanol, the filtrate was
concentrated, and the residue was purified by flash column
chromatography (0−10% MeOH in CH2Cl2) to afford 1.1 g of
1
white solid 6 (92% yield). H NMR (400 MHz, CD3OD): 9.09 (s,
standard methods known to one skilled in the art of chemical
synthesis. 1H and 13C NMR spectra were taken on a Varian Unity Plus
400 spectrometer at rt and reported in ppm downfield from internal
tetramethylsilane. Deuterium exchange, decoupling experiments were
done 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. Purity of final compounds was determined to be
>95%, using analytical HPLC analyses performed on a Hewlett-
Packard 1100 HPLC instrument with a Phenomenex Gemini-NX
column (2 mm × 50 mm, 3 μm, C18, 110 Å) and further supported by
clean NMR spectra. Mobile phase flow was 0.5 mL/min with a 3.5 min
initial hold, a 6.5 min gradient from 96% aqueous media (0.05% formic
acid) to 96% CH3CN (0.05% formic acid), and a 15 min total
acquisition time. Photodiode array detection was from 190 to 360 nm.
Mass spectra were determined on a Micromass Platform LC
spectrometer using electrospray ionization. Analytic TLC was
performed on Whatman LK6F silica gel plates, and preparative TLC
was performed on Whatman PK5F silica gel plates. Column
chromatography was carried out on silica gel or via reverse-phase
high performance liquid chromatography.
(2R,3R,4R,5R)-5-((Benzoyloxy)methyl)-2-(2,6-diamino-9H-
purin-9-yl)-3-methyltetrahydrofuran-3,4-diyl Dibenzoate, 4.
To a stirred suspension of (3R,4S,5R)-5-((benzoyloxy)methyl)-3-
methyltetrahydrofuran-2,3,4-triyl tribenzoate 2 (2.9 g, 5 mmol) and
2,6-diaminopurine 3 (830 mg, 5.5 mmol) in anhydrous acetonitrile at
−78 °C was added DBU (2.3 mL, 15 mmol), followed by a slow
addition of TMSOTf (3.8 mL, 20 mmol). The reaction mixture was
stirred at −78 °C for 20 min and then raised to 0 °C. After it was
stirred for 30 min at 0 °C, the reaction mixture was heated gradually to
65 °C and stirred overnight. The reaction mixture was cooled, diluted
with CH2Cl2 (200 mL), and washed with saturated NaHCO3. The
layers were separated, and the resulting aqueous layer was extracted
with CH2Cl2 (2 × 20 mL). The combined organic layers were dried
over Na2SO4. After removal of the solvent, the residue was purified by
silica gel column chromatography (0−10% MeOH in EtOAc). 2.8 g of
compound 4 was obtained (92% yield). 1H NMR (400 MHz, CDCl3):
δ 8.15−8.17 (m, 2H), 7.97−8.02 (m, 4H), 7.77 (s, 1H), 7.45−7.61 (m,
5H), 7.32−7.37 (m, 4H), 6.62 (s, 1H), 6.53 (d, 1H, J = 6.6 Hz), 5.91
(s, 2H), 5.04−5.10 (m, 3H), 4.82−4.86 (m, 1H), 4.70−4.74 (m, 1H),
1.62 (s, 3H). 13C NMR (CDCl3): δ 17.5, 64.0, 76.0, 79.4, 85.8, 88.5,
114.9, 128.3, 128.4, 128.5, 128.9, 129.5, 129.7, 129.8, 129.9, 133.1,
1H), 6.19 (s, 1H), 4.22 (d, 1H, J = 8.8 Hz), 4.03−4.11 (m, 2H), 3.89
(dd, 1H, J = 2.8 Hz, J = 12.4 Hz), 1.41 (s, 18H), 1.40 (s, 18H), 0.92 (s,
3H). 13C NMR (CD3OD): 20.2, 27.9, 28.1, 60.9, 73.1, 80.2, 84.6, 84.9,
85.4, 93.3, 128.8, 147.0, 151.2, 151.9, 152.0, 152.9, 155.0. LC/MS
calcd for C31H49N6O12 (M + 1)+, 697.3; observed, 697.4.
(2R,3R,4R,5R)-2-(2,6-Diamino-9H-purin-9-yl)-5-(hydroxy-
methyl)-3-methyltetrahydrofuran-3,4-diol, 7. Nucleoside 4 (1.1
g, 1.7 mmol) was treated with saturated ammonia/methanol (100
mL). The reaction mixture was stirred at rt for 36 h to completion.
After removal of the solvent under reduced pressure, 150 mL of
dichloromethane was added to the residue. The white solid 7 was
collected and dried (500 mg, 93%). 1H NMR (400 MHz, DMSO-d6):
8.01 (s, 1H), 6.73 (s, 2H), 5.81 (s, 2H), 5.76 (d, J = 2.8 Hz, 1H),
5.18−5.22 (t, J = 6.8 Hz), 5.05 (s, 1H), 0.78 (s, 3H), 3.98−4.02 (m,
1H), 3.78−3.84 (m, 2H), 3.63−3.67 (m, 1H). LC/MS calcd for
C11H17N6O2 (M + 1)+, 297.1; observed, 297.2.
Ethyl 3-(2-Hydroxyphenyl)propanoate, 9a. Dihydrocoumarin
(52 g, 350 mmol) was added to 300 mL of anhydrous ethanol. H2SO4
(0.5 mL) was added, and the resulting solution was heated overnight
at reflux. To the solution was added Ambersep 900 OH resin with
stirring until approximately pH 7.0 was reached, and then the solution
was filtered. The filtrate was evaporated under reduced pressure, and
the residue was dissolved in ether acetate (300 mL), washed with
water (3 × 50 mL), brine (50 mL), and dried over MgSO4. The
solution was filtered, and the filtrate was evaporated under reduced
pressure. The residue was dried to give 64 g of solid product 9a (94%
1
yield). H NMR (400 MHz, CDCl3) δ 7.40 (s, 1H), 7.05−7.15 (m,
2H), 6.84−6.90 (m, 2H), 4.14 (q, J = 6.8 Hz, 2H), 2.90 (m, 2H), 2.72
(m, 2H), 1.23 (t, J = 6.8 Hz, 3H). LC/MS calcd for C11H15O3 (M +
1)+, 195.1; observed, 195.2.
Isopropyl 3-(2-Hydroxyphenyl)propanoate, 9b. Same proce-
dure was employed for the preparation of compound 9b in 89% yield.
1H NMR (400 MHz, CD3OD) δ 7.47 (brs, 1H), 7.09−7.27 (m, 2H,
Ar−H), 6.87−6.91 (m, 2H, Ar−H), 5.00−5.06 (m, 1H), 2.90−2.93
(m, 2H), 2.68−2.72 (m, 2H), 1.22 (d, 6H, J = 8.0 Hz). 13C NMR (100
MHz, CD3Cl) δ 21.7, 24.7, 35.5, 68.9, 117.1, 120.6, 127.4, 127.9,
130.5, 154.4, 175.2. LCMS calcd for C12H17O3 (M + 1)+, 209.1;
observed, 209.2.
Ethyl 3-(2-((Chloro(((S)-1-ethoxy-1-oxopropan-2-yl)amino)-
phosphoryl)oxy)phenyl)propanoate, 12b. To a solution of
phosphorus oxychloride (2.4 mL, 26 mmol) in 70 mL of anhydrous
H
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diethyl ether was added dropwise a solution of 9a (5.0 g, 26 mmol)
and triethylamine (3.6 mL, 26 mmol) in 80 mL of anhydrous diethyl
ether at −78 °C under an Ar atmosphere over 2 h. After stirring for 1 h
at −78 °C under Ar atmosphere, the solution was additionally stirred
for 15 h toward rt, and then the solids were removed by filtration
under a N2 atmosphere. The solid salt was washed with anhydrous
diethyl ether. The filtrate was concentrated under reduced pressure
and dried under high vacuum overnight at rt. To a mixture of the oil
10b and dried L-alanine ethyl ester hydrogen chloride 11 (3.9 g, 26
mmol) in 20 mL of anhydrous CH2Cl2 was added a solution of Et3N
(7 mL, 51 mmol) in 20 mL of anhydrous CH2Cl2 over 2 h at −78 °C
under Ar atmosphere. The solution was stirred for 16 h at rt, and the
solid was filtered. The filtrate was concentrated under reduced
pressure and purified on a silica gel column (EtOAc/hexane; gradient
0−50%, v/v) to give 9.5 g of compound 12b in 75% yield in two steps.
1H NMR (400 MHz, CDCl3) (1:1 mixture of P diastereomers): δ
7.14−7.49 (m, 4H), 4.70−4.80 (m, 1H), 4.09−4.27 (m, 5H), 2.92−
3.08 (m, 2H), 2.61−2.65 (m, 2H), 1.50−1.55 (m, 3H), 1.21−1.32 (m,
6H). 31P NMR (162 MHz, CDCl3) δ 8.88, 8.72.
neutralized by saturated NaHCO3. After solvent removal, the residue
was purified by flash column chromatography with a gradient of
MeOH (0−15% MeOH in CH2Cl2) to afford 225 mg of white solid
15a (71%) (38% yield over two steps). 1H NMR (400 MHz, CD3OD)
(1:1 mixture): 7.86 (s, 2H), 7.14−7.34 (m, 10H), 5.96 (s, 1H), 5.93 (s,
1H), 3.90−4.58 (m, 14H), 1.16−1.31 (m, 6H), 1.13−1.19 (m, 6H),
0.97 (s, 3H), 0.94 (s, 3H). 31P NMR (162 MHz, CD3OD): 4.77, 4.89.
LC/MS calcd for C22H31N7O8P (M + 1)+, 552.2; observed, 552.3.
Ethyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-
yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-
1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-
propanoate, 15b(Sp), and Ethyl 3-(2-(((R)-(((2R,3S,4R,5R)-5-(2,6-
Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-methyl-
tetrahydrofuran-2-yl)methoxy)(((S)-1-ethoxy-1-oxopropan-2-
yl)amino)phosphoryl)oxy)phenyl)propanoate 15b(Rp). A pro-
cedure similar to that used for 15a was employed for the synthesis of
prodrug 15b (56% over two steps). Two diasteroisomers were
separated and identified.
1
15b(Sp). Optical rotation [α]24 −7.08 (c 0.24, MeOH). H NMR
D
(400 MHz, CD3OD) δ 7.86 (s, 1H, H8), 7.10−7.39 (m, 4H, Ar−H),
5.93 (s, 1H, H1′), 3.96−4.51 (m, 9H), 2.99 (t, 2H, J = 8.0 Hz, CH2),
2.62 (t, 2H, J = 8.0 Hz, CH2), 1.34 (d, 3H, J = 7.2 Hz, CH3), 1.15−
1.17 (m, 6H, 2 × CH3), 0.97 (s, 3H, CH3). 31PNMR (162 MHz,
CD3OD) δ 5.03; LRMS calcd for C27H39N7O10P (M + 1)+ 652.25,
found 652.32.
The same procedure was employed for the preparation of
compounds 12a, 12c, 12d, and 12e in 72%, 70%, 69% and 70%
yields, respectively.
1
12a. H NMR (400 MHz, CDCl3): δ 7.17−7.40 (m, 1H, 5H),
4.10−4.50 (m, 4h), 1.50−1.53 (m, 3H), 1.28−1.33 (m, 3H). 31PNMR
(162 MHz, CDCl3): 8.13, 9.15.
15b(Rp). Optical rotation [α]24D +12.12 (c 0.13, MeOH). 1H NMR
(400 MHz, CD3OD) δ 7.85 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.26 (d,
J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.12 (t, J = 7.6
Hz, 1H), 5.91 (s, 1H), 4.51−4.48 (m, 2H), 4.25−3.95 (m, 7H), 2.99
(t, J = 8.0 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.34 (d, J = 6.8 Hz, 3H),
1.18 (t, J = 7.2 Hz, 3H), 1.16 (t, J = 7.2 Hz, 3H), 0.97 (s, 3H). 31P
NMR (162 MHz, CD3OD) δ 4.98. LCMS calcd for C27H39N7O10P (M
+ 1)+ 652.3, found 652.3.
12c:.1H NMR (400 MHz, CDCl3): δ 7.43−7.48 (m, 1H), 7.13−
7.26 (m, 3H), 4.97−5.12 (m, 2H), 4.50−4.53 (m, 1H), 4.08−4.23 (m,
1H), 2.93−3.02 (m, 2H), 2.58−2.62 (m, 2H), 1.18−1.29 (m, 12H).
31PNMR (162 MHz, CDCl3): 8.64, 8.87.
1
12d. H NMR (400 MHz, CDCl3): δ 7.44−7.48 (m, 1H), 7.14−
7.26 (m, 3H), 5.04−5.12 (m, 1H), 4.54−4.57 (m, 1H), 4.09−4.56 (m,
3H), 2.95−3.04 (m, 2H), 2.61−2.64 (m, 2H), 1.48−1.52 (m, 3H),
1.20−1.29 (m, 9H). 31PNMR (162 MHz, CDCl3): 7.35, 7.57.
Isopropyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-
9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-
(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)-
phenyl)propanoate 15c(Sp) and Isopropyl 3-(2-(((R)-
(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-
methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-ox-
opropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate
15c(Rp). A procedure similar to that used for 15a was employed for
the synthesis of prodrug 15c (40% over two steps). Two
diastereoisomers were separated and identified.
1
12e. H NMR (400 MHz, CDCl3): δ 7.44−7.48 (m, 1H), 7.16−
7.26 (m, 3H), 4.97−5.03 (m, 1H), 4.47−4.53 (m, 1H), 4.16−4.28 (m,
3H), 2.94−3.04 (m, 2H), 2.58−2.62 (m, 2H), 1.50−1.55 (m, 3H),
1.29−1.33 (m, 3H), 1.17−1.22 (m, 6H). 31PNMR (162 MHz,
CDCl3): 7.84, 8.08.
Diethyl 3,3′-(((Chlorophosphoryl)bis(oxy))bis(2,1-
phenylene))dipropanoate, 13. To a solution of phosphorus
oxychloride (0.47 mL, 5.0 mmol) in 30 mL of anhydrous diethyl
ether were added dropwise a solution of 9a (1.9 g, 10 mmol) and
triethylamine (1.4 mL, 10 mmol) in 50 mL of anhydrous diethyl ether
at −78 °C under an Ar atmosphere in 1.5 h. After stirring for 1 h at
−78 °C under Ar atmosphere, the solution was additionally stirred for
15 h toward rt and then the solid was removed by filtration under a N2
atmosphere. The solid salt was washed anhydrous diethyl ether. The
filtrate was concentrated under reduced pressure and purified on a
silica gel column (EtOAc/hexane; gradient 0−25%, v/v) to give 815
15c(Sp). 1H NMR (400 MHz, CD3OD) δ 7.86 (s, 1H), 7.37 (d, J =
8.4 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.17 (dt, J = 8.0 Hz, J = 1.6 Hz,
1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 5.05−4.84 (m, 2H), 4.62−
4.46 (m, 2H), 4.22 (s, 2H), 3.95−3.91 (m, 1H), 2.99 (t, J = 8.0 Hz,
2H), 2.60 (t, J = 8.0 Hz, 2H), 1.33 (d, J = 7.2 Hz, 3H), 1.20 (d, J = 6.4
Hz, 3H), 1.19 (d, J = 6.0 Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H), 1.14 (t, J =
6.4 Hz, 3H), 0.96 (s, 3H). 31P NMR (162 MHz, CD3OD) δ 5.08.
LRMS calcd for C29H43N7O10P (M + 1)+ 680.28, found 680.12.
15c(Rp). 1H NMR (400 MHz, CD3OD) δ 8.03 (s, 1H), 7.38 (d, J =
8.0 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 7.6 Hz, J = 1.6 Hz,
1H), 7.10 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.95−4.79 (m, 2H), 4.53−
4.46 (m, 2H), 4.18 (s, 2H), 3.95−3.91 (m, 1H), 2.99 (t, J = 8.0 Hz,
2H), 2.61 (t, J = 8.0 Hz, 2H), 1.35 (d, J = 7.2 Hz, 3H), 1.19−1.16 (m,
12H), 0.99 (s, 3H). 31P NMR (162 MHz, CD3OD) δ 5.02. LRMS
calcd for C29H43N7O10P (M + 1)+ 680.28, found 680.14.
1
mg of compound 13 as a colorless oil in 35% yield. H NMR (400
MHz, CDCl3): δ 7.44−7.47 (m, 2H), 7.18−7.31 (m, 6H), 4.09−4.14
(q, J = 12.0 Hz, J = 8.0 Hz, 4H), 3.00 (t, J = 6.0 Hz, 4H), 2.60 (t, J =
8.0 Hz, 4H), 1.22 (t, J = 8.0 Hz, 6H). 31P NMR (162 MHz, CDCl3) δ
−5.85.
(2S)-Ethyl 2-((((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-
(phenoxy)phosphorylamino)propanoate, 15a. To a solution of 6
(780 mg, 1.1 mmol) and N-methylimidazole (0.45 mL, 5.8 mmol) in
THF (5 mL) at 0 °C was added dropwise a 1 M solution of (2R)-ethyl
2-(chloro(phenoxy)phosphorylamino)propanoate1 (5.8 mL, 5.8
mmol). The resulting mixture was stirred overnight at rt. After
solvent removal under reduced pressure, the residue was purified by
flash column chromatography with a gradient of MeOH (0−10%
MeOH in CH2Cl2) to afford 580 mg of white solid 14a (54% yield). A
precold solution of TFA (80%, 23 mL) was added to a precold 14a
(550 mg, 0.58 mmol) in an ice bath. The solution was stirred from 0
°C to rt and stirred at rt for 4 h (monitored by TLC and LC/MS).
After the reaction was completed, the solvent was removed under
reduced pressure and the residue was coevaporated with methanol (4
× 15 mL). The residue was dissolved in methanol (20 mL) and
Ethyl 3-(2-(((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-
yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-
1-isopropoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)-
phenyl)propanoate, 15d(Sp), and Ethyl 3-(2-(((R)-
(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-4-
methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-ox-
opropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate,
15d(Rp). A procedure similar to that used for 15a was employed for
the synthesis of prodrug 15d (41% over two steps). Two
diasteroisomers were separated and identified.
15d(Sp). 1H NMR (400 MHz, CD3OD) δ 7.85 (s, 1H), 7.36 (d, J =
8.4 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.16 (dt, J = 8.0 Hz, J = 1.6 Hz,
1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 5.02−4.84 (m, 1H), 4.62−
4.49 (m, 2H), 4.21 (s, 2H), 4.07−3.91 (m, 3H), 2.99 (t, J = 8.0 Hz,
I
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1
2H), 2.62 (t, J = 8.0 Hz, 2H), 1.34−1.14 (m, 12H), 0.96 (s, 3H). 31P
NMR (162 MHz, CD3OD) δ 5.04. LRMS calcd for C28H41N7O10P (M
+ 1)+ 666.27, found 666.10.
CH2Cl2) to afford 270 mg of white solid 18 (77%). H NMR (400
MHz, CD3OD) δ 7.80 (s, 1H), 7.09−7.32 (m, 8H), 5.90 (s, 1H),
4.72−4.93 (m, 1H), 4.26−4.38 (m, 2H), 4.02−4.08 (m, 4H), 2.86−
2.92 (m, 4H), 2.46−2.54 (m, 4H), 1.16 (t, 6H), 1.00 (s, 3H). 31P
NMR (162 MHz, CD3OD) δ 12.21. LCMS calcd for C22H31N7O8P
(M + 1)+ 729.2; observed, 729.3.
15d(Rp). 1H NMR (400 MHz, CD3OD) δ 7.85 (s, 1H), 7.38 (d, J =
8.0 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 2.0 Hz,
1H), 7.12 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.62−4.48 (m, 4H), 4.25−
3.91 (m, 4H), 2.99 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.33
(d, J = 7.2 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.17 (d, J = 6.0 Hz, 3H),
1.14 (d, J = 6.4 Hz, 3H), 0.97 (s, 3H). 31P NMR (162 MHz, CD3OD)
δ 4.97. LRMS calcd for C28H41N7O10P (M + 1)+ 666.27, found 666.08.
Ethyl ((S)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-
dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(2-(3-iso-
propoxy-3-oxopropyl)phenoxy)phosphoryl)-L-alaninate,
15e(Sp), and Ethyl ((R)-(((2R,3S,4R,5R)-5-(2,6-Diamino-9H-
purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)-
methoxy)(2-(3-isopropoxy-3-oxopropyl)phenoxy)-
phosphoryl)-L-alaninate, 15e(Rp). A procedure similar to that used
for 15a was employed for the synthesis of prodrug 15e (41% over two
steps). Two diasteroisomers were separated and identified.
Ethyl 3-(2-(((R)-(((S)-1-Ethoxy-1-oxopropan-2-yl)amino)(4-
nitrophenoxy)phosphoryl)oxy)phenyl)propanoate, 19(Rp),
and Ethyl 3-(2-(((S)-(((S)-1-Ethoxy-1-oxopropan-2-yl)amino)(4-
nitrophenoxy)phosphoryl)oxy)phenyl)propanoate, 20(Sp). A
solution of Et3N in anhydrous diethyl ether (100 mL) was added
dropwise to a solution of 12b (10 g, 26 mmol) and p-nitrophenol (3.8
g, 27 mmol) in diethyl ether (200 mL) at 0 °C in 30 min. The reaction
mixture was stirred at 0 °C for 1 h and at rt for 15 h. The solids were
removed by filtration, and the filtrate was concentrated under reduced
pressure. The residue was purified on a silica gel column (EtOAc/
CH2Cl2, EtOAc gradient 0−10%, v/v) to give 10.8 g of a mixture of 19
and 20 in 85% yield in 1:1 ratio. The mixture was recrystallized in 2%
CH3CN in diisopropyl ether. Pure 19 was obtained by seeding the
recrystallization solution with pure 19 crystal seeds which were
obtained by column chromatography. The resulting solution was
stored for 18 h at 0 °C. Filtration of the resulting solid from the
mother liquor gave 2.2 g of isomer 19. 1H NMR (400 MHz, CDCl3) δ
8.22−8.24 (dd, J = 10 Hz, J = 2.0 Hz, 2H), 7.11−7.44 (m, 6H), 4.06−
4.20 (m, 6H), 2.88−3.00 (m, 2H), 2.54−2.59 (m, 2H), 1.40 (d, J = 6.8
Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). 31P NMR
(162 MHz, CDCl3) δ −2.01. LC−MS, m/z 495 (M + 1)+. A single
crystal of 19 was obtained by crystallization in 2% CH3CN in
diisopropyl ether and an X-ray structure of 19 was obtained to
unambiguously confirm the configuration of the phosphorus center as
Rp (Figure 2).
15e(Sp). 1H NMR (400 MHz, CD3OD) δ 7.85 (s, 1H), 7.37 (d, J =
8.0 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H), 7.17 (dt, J = 8.0 Hz, J = 2.0 Hz,
1H), 7.08 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 4.93−4.84 (m, 1H), 4.61−
4.49 (m, 2H), 4.22 (s, 2H), 4.18−3.91 (m, 3H), 2.99 (t, J = 8.0 Hz,
2H), 2.60 (t, J = 8.0 Hz, 2H), 1.34 (d, J = 6.4 Hz, 3H), 1.20 (t, J = 7.2
Hz, 3H), 1.15 (d, J = 6.0 Hz, 3H), 1.14 (d, J = 6.0 Hz, 3H), 0.97 (s,
3H). 31P NMR (162 MHz, CD3OD) δ 5.02; LRMS calcd for
C28H41N7O10P (M + 1)+ 666.27, found 666.19.
15e(Rp). 1H NMR (400 MHz, CD3OD) δ 7.85 (s, 1H), 7.38 (d, J =
8.4 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.18 (dt, J = 8.0 Hz, J = 2.0 Hz,
1H), 7.10 (t, J = 7.2 Hz, 1H), 5.91 (s, 1H), 4.93−4.84 (m, 1H), 4.51−
4.48 (m, 2H), 4.25−3.95 (m, 5H), 2.98 (t, J = 8.0 Hz, 2H), 2.60 (t, J =
8.0 Hz, 2H), 1.34 (d, J = 7.2 Hz, 3H), 1.18−1.49 (m, 9H), 0.97 (s,
3H). 31P NMR (162 MHz, CD3OD) δ 4.97. LRMS calcd for
C28H41N7O10P (M + 1)+ 666.27, found 666.09.
The filtrate was concentrated under reduced pressure to dryness,
and the mixture was stirred in diisopropyl ether (200 mL) with pure
20 seeds. An amount of 510 mg of pure 20 was collected. H NMR
1
Ethyl 3-(2-(((((3aS,4R,6R,6aR)-6-(2,6-Diamino-9H-purin-9-yl)-
6a-methyl-2-oxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-
methoxy)(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)-
oxy)phenyl)propanoate, 16. To a solution of compound 15b (110
mg, 0.17 mmol) in acetonitrile (10 mL) was added carbodiimidazole
(60 mg, 0.37 mmol), and the reaction mixture was stirred at rt
overnight. Water (0.2 mL) was added, and the reaction mixture was
stirred for 0.5 h. The mixture was concentrated and portioned between
ethyl acetate (100 mL) and water (15 mL). The organic layer was
washed with brine, dried over Na2SO4, concentrated, and the residue
was purified by silica gel column chromatography in a gradient of
MeOH (0−10% MeOH in CH2Cl2) to afford white solid 16 (84 mg,
71%). 1H NMR (400 MHz, CD3OD) δ 7.86 (s, 0.5H), 7.81 (s, 0.5H),
7.34−7.39 (m, 1H), 7.06−7.27 (m, 3H), 6.32 (s, 0.5H), 6.30 (s, 0.5H),
5.32−5.36 (m, 1H), 4.71−4.77 (m, 1H), 4.38−4.57 (2H), 3.97−4.16
(m, 5H), 2.93−3.00 (m, 2H), 2.57−2.65 (m, 2H), 1.32−1.39 (m, 6H),
1.13−1.23 (m, 6H). 31P NMR (162 MHz, CD3OD) δ 4.10, 3.89.
LCMS calcd for C28H37N7O11P (M + 1)+ 678.2, found 678.3.
Ethyl 3-(2-(((((2R,3S,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-
3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(2-(3-ethoxy-3-
oxopropyl)phenoxy)phosphoryl)oxy)phenyl)propanoate, 17.
To a solution of 6 (630 mg, 0.91 mmol) and N-methylimidazole
(0.35 mL, 4.5 mmol) in THF (3 mL) at 0 °C was added dropwise a 1
M solution of diethyl 3,3′-(((chlorophosphoryl)bis(oxy))bis(2,1-
phenylene))dipropanoate, 13 in THF (9 mL, 4.5 mmol). The
resulting mixture was stirred overnight at rt. After solvent removal
under reduced pressure, the residue was purified by flash column
chromatography in a gradient of MeOH (0−10% MeOH in CH2Cl2)
to afford 540 mg of white solid 17 (53% yield). A precold solution of
TFA (80%, 26 mL) was added to a precold 17 (540 mg, 0.58 mmol)
in an ice bath. The solution was stirred from 0 °C to rt and stirred at rt
for 4 h (monitored by TLC and LC/MS). After the reaction was
complete, the solvent was removed under reduced pressure and the
residue was coevaporated with methanol (4 × 15 mL). The residue
was dissolved in methanol (20 mL) and neutralized by saturated
NaHCO3. After solvent removal, the residue was purified by flash
column chromatography in a gradient of MeOH (0−15% MeOH in
(400 MHz, CDCl3) δ 8.22−8.24 (dd, J = 10 Hz, 2H), 7.11−7.43 (m,
6H), 4.00−4.18 (m, 6H), 2.93−2.98 (m, 2H), 2.55−2.60 (m, 2H),
1.43 (d, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz,
3H). 31P NMR (162 MHz, CDCl3) δ −2.07. LC−MS, m/z 495 (M +
1)+. A single crystal of 20 was obtained by crystallization in 2%
CH3CN in diisopropyl ether and an X-ray structure of 20 was obtained
to unambiguously confirm the configuration of the phosphorus center
as Sp (Figure 2).
Ethyl 3-(2-(((R)-(((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-
yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-
1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-
propanoate, 15b(RP). To a solution of 6 (100 mg, 0.14 mmol) in
THF (0.5 mL) was added 0.5 mL of t-BuMgCl solution (1 M, 0.5
mmol) at −78 °C under Ar atmosphere. The reaction mixture was
stirred for 30 min at this temperature and then warmed to rt. A
solution of 20 (210 mg, 0.42 mmol) in 1 mL of anhydrous THF was
added. The reaction mixture was stirred at rt for 3 days under Ar
atmosphere to completion. The solvent was evaporated under reduced
pressure, and to the residue was added a precooled 80% TFA solution
(10 mL) at 0 °C. The reaction mixture was stirred for an additional 4 h
toward rt to completion. After evaporation of the solvents under
reduced pressure, to the residue was added a small amount of
saturated solution of aqueous NaHCO3 to adjust to pH 7.0. The
mixture was concentrated under reduced pressure and then purified by
a silica gel column (MeOH/DCM, MeOH gradient 0−10%, v/v) to
afford 37.5 mg of 15b(RP) in 41% yield over two steps. Optical
rotation [α]24D −7.08 (c 0.24, MeOH). 1H NMR (400 MHz, CD3OD)
δ 0.97 (s, 3H, CH3), 1.15−1.20 (m, 6H, 2 x CH3), 1.34 (d, 3H, J = 7.2
Hz, CH3), 2.62 (t, 2H, J = 8.0 Hz, 2H, CH2), 2.99 (t, 2H, J = 8.0 Hz,
2H, CH2), 3.95−4.58 (m, 9H), 5.94 (s, 1H, H1′), 7.07−7.38 (m, 4H,
Ar−H), 7.86 (s, 1H, H8). 13C NMR (100 MHz, CD3OD) δ 14.5, 14.6,
20.4, 20.6, 26.8, 35.4, 51.7, 61.7, 62.5, 67.0, 74.4, 80.1, 81.9, 92.8,
114.4, 121.0, 126.2, 128.8, 131.8, 133.2, 137.5, 150.6, 152.7, 157.7,
162.0, 174.8, 175.1. 31P NMR (162 MHz, CD3OD): 5.03. LC/MS
calcd for C27H39N7O10P (M + 1)+, 652.2; observed, 552.2.
J
DOI: 10.1021/jm501874e
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Journal of Medicinal Chemistry
Article
Ethyl 3-(2-(((S)-(((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-
yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-
1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)-
propanoate 15b(SP). A procedure similar to that used for 15a was
employed for the preparation of 15b(SP) in 39% yield. Optical rotation
[α]24D +12.12 (c 0.13, MeOH). 1H NMR (400 MHz, CD3OD) δ 0.97
(s, 3H, CH3), 1.15−1.17 (m, 6H, 2 x CH3), 1.34 (d, 3H, J = 7.2 Hz,
CH3), 2.62 (t, 2H, J = 8.0 Hz, 2H, CH2), 2.99 (t, 2H, J = 8.0 Hz, 2H,
CH2), 3.96−4.51 (m, 9H), 5.93 (s, 1H, H1′), 7.10−7.39 (m, 4H, Ar−
H), 7.86 (s, 1H, H8). 13C NMR (100 MHz, CD3OD) δ 14.5, 14.6,
20.4, 20.8, 26.8, 35.4, 51.6, 61.6, 62.4, 67.7, 74.7, 80.0, 82.1, 93.0,
114.4, 121.1, 126.2, 128.8, 131.7, 133.1, 137.7, 150.5, 152.6, 157.6,
161.9, 174.7, 174.8. 31P NMR (162 MHz, CD3OD): 4.98. LC/MS
calcd for C27H39N7O10P (M + 1)+, 652.2; observed, 552.3.
((2R,3R,4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-3,4-dihydroxy-
4-methyltetrahydrofuran-2-yl)methyl Tetrahydrogen Triphos-
phate Tetratriethylammonium Salt (DAPN-TP) and
((2R,3R,4R,5R)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-
3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl Tetrahy-
drogen Triphosphate Tetratriethylammonium Salt (2′-Me-
GTP). A mixture of 2,6-diaminopurine ribonucleoside 7 (14 mg, 1
equiv), trimethyl phosphate (0.5 mL), and molecular sieves were
stirred at rt overnight. 2,4,6-Collidine (9 μL, 1.5 equiv) was added and
the mixture stirred for 10 min before phosphorus oxychloride (9 μL, 2
equiv) was added. The reaction solution was stirred for 1 h at rt. A
solution of tributylammonium pyrophosphate (0.26 mL of 1 M
solution in DMF, 5.5 equiv) and tributylamine (45 μL, 4 equiv) were
added simultaneously. The mixture was stirred for 30 min at rt. The
reaction was then quenched by addition of 2 mL of triethylammonium
bicarbonate buffer (0.25M) and stirred for 45 min. After extraction
with dichloromethane (2 × 3 mL), the aqueous phase was
concentrated under reduced pressure. The crude was subjected to
HPLC purification using ion-exchange DNAPac PA200 (9 mm × 250
mm) column (Dionex), with water and triethylammnoium bicarbonate
(0.5M) as eluent. LC/MS calcd for C11H18N6O13P3 (M − 1)+, 535.0;
observed, 535.0.
flipping and refined by least-squares minimization using SHELXL.42,43
A total of 8394 reflections were measured (5.12 ≤ 2θ ≤ 69.48), while
3469 unique data (Rint = 0.0159) were used in the refinements. The
final R1 was 0.0267 (I > 2σ(I)) and the weighted R value wR2 was
0.0728 (all data). All non-hydrogen atoms were refined anisotropically.
Hydrogen atoms were included but not refined. The maximum and
the minimum peak on the final difference Fourier map corresponded
to 0.239 and −0.255 e/Å3, respectively.
NS5B-Mediated RNA Polymerization Assay. C-terminal His-
tagged NS5BΔ21 enzyme was purified as previously described
(Powdrill et al., 2010). 1 μM NS5BΔ21 was incubated at 30 °C
with synthetic 1 μM 20-mer RNA templates (IDT) and 1 μM 32P-
radiolabeled GpG primer (Trilink) in a buffer containing 40 mM Tris,
pH 7.5, 6 mM NaCl, and 2 mM MgCl2. Reactions were initiated with
the addition of 10 μM NTP mix, 1 μM of competing NTP, and
varying concentrations of inhibitor ranging from 0 to 100 μM.
Reactions were allowed to proceed for 120 min and subsequently
stopped with the addition of 10 mM EDTA and formamide. Samples
were visualized on 20% denaturing polyacrylamide gel and quantified
using QuantityOne software. IC50 values were calculated using
KaleidaGraph software.
Intracellular Metabolism. Huh-7 cells and fresh plated human
primary hepatocytes (BioreclamationIVT, Baltimore, MD) were
seeded at 1 × 106 per well in 12-well plates. After attachment
(Huh-7 cells) or acclimatation overnight (hepatocytes), cells were
exposed to 50 μM compounds, respectively. At 4 h, medium was
removed from the cell layers and cells were washed twice with ice-cold
phosphate buffered saline (PBS) to remove any residual medium. Cells
were resuspended in 70% methanol containing 20 nM ddATP
overnight at −20 °C. The supernatants were dried under a flow of air
and dried samples stored at −20 °C until LC−MS/MS analysis.
Mitochondrial Toxicity Assa. Human hepatocarcinoma cells
(HepG2) were propagated at 37 °C and 5% CO2 atmosphere in
Dulbecco′s modified Eagle medium. Media contained 4.5 g/L D-
glucose and were supplemented with 10% heat-inactivated fetal bovine
serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. All
cell reagents were from Invitrogen Corporation (Carisbad, CA, USA),
Cellgro/Mediatech, Inc. (Manassas, VA, USA), and Hyclone (Logan,
UT, USA). Low-passage-number HepG2 cells were seeded at 5000
cells/well onto collagen-coated 24-well tissue culture plates. After 1
day of cell seeding, test compounds were added to the medium to
obtain final concentrations varying from 1 to 50 μM. Media and
compounds were replenished every 3−4 days until the end of 14 days.
All samples were performed in replicates of two in two independent
experiments. On culture day 14, cells and cell culture supernatants
were harvested. Total nucleic acid was isolated from cells by using
commercially available kits (MagNA Pure LC DNA Isolation Kit,
Roche). The mitochondrial cytochrome c oxidase subunit II (COXII)
gene and the rRNA gene were amplified in parallel by a real-time PCR
protocol that uses suitable primers and probes for both target and
reference amplifications (Stuyver et al., 2002).44 Real-time PCR was
performed by using a TaqMan 9700 HT sequence detection system
(Applied Biosystems, Foster City, CA, USA). The primers and probes
for the rRNA gene were purchased from Applied Biosystems. The
amount of target (mtDNA) was normalized to the amount of an
endogenous control (nuclear DNA) and was relative to the untreated
control. The 50% inhibitory concentration (IC50) of test compounds
was determined by using CalcuSyn program (Biosoft, Cambridge,
U.K.).
The same procedure was applied for 2′-C-Me-GTP, using 2′-C-Me
guanosine (20 mg, 1 equiv), trimethylphopsphate (0.65 mL), 2,4,6-
collidine (14 μL, 1.5 equiv), phosphorus oxychloride (14 μL, 2.1
equiv), tributylamine (63 μL, 4 equiv), tributylammonium pyrophos-
phate (0.37 mL of 1 M solution in DMF, 5.5 equiv),
triethylammonium bicarbonate buffer (2.5 mL, 0.25 M). LC/MS
calcd for C11H17N5O14P3 (M − 1)+, 536.0; observed, 535.9.
X-ray Crystallography of Diastereomers. Results from X-ray
structure determination of compound 19 are the following. Crystal
data for C22H27N2O9P (M = 494.43): 0.29 × 0.11 × 0.09, triclinic,
space group P1, a = 5.0028(3) Å, b = 8.9645(7) Å, c = 14.5700(14) Å,
β = 90.753(7)°, V = 603.56(8) Å3, Z = 1, μ(Cu Kα) = 1.484 mm−1,
Dcalc = 1.360 g/mm3, temperature 173 K. Intensity data were collected
on a Bruker APEX II CCD diffractometer with monochromated Cu
Kα radiation (λ = 1.541 78 Å) at 173 K in the 2θ range 3.18−64.34°.
The user interface Olex2 was used for the crystallographic calculations
and crystal structure visualization.41 The structure was solved with
Superflip by charge flipping and refined by least-squares minimization
using SHELXL.42,43 A total of 3668 reflections were measured (3.18 ≤
2θ ≤ 64.34), while 2214 unique data (Rint = 0.0273) were used in the
refinements. The final R1 was 0.0475 (I > 2σ(I)) and the weighted R
value wR2 was 0.1255 (all data). All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms were included but not refined. The
maximum and the minimum peak on the final difference Fourier map
corresponded to 0.46 and −0.27 e/Å3, respectively.
Results from the X-ray structure determination of compound 20 are
the following. Crystal data for C22H27N2O9P (M = 494.43): 0.28 ×
0.26 × 0.11, monoclinic, space group P2(1), a = 4.87400(10) Å, b =
28.8315(6) Å, c = 8.9118(2) Å, β = 104.3380(10)°, V = 1213.32(4)
Å3, Z = 2, μ(Cu Kα) = 1.477 mm−1, Dcalc = 1.353 g/mm3, temperature
173 K. Intensity data were collected on a Bruker APEX II CCD
diffractometer with monochromated Cu Kα radiation (λ = 1.541 78 Å)
at 173 K in the 2θ range 5.12−69.48°. The user interface Olex2 was
used for the crystallographic calculations and crystal structure
visualization.41 The structure was solved with Superflip by charge
Lactate Production Assay. Following a 14-day incubation with
compounds at various concentrations, lactic acid quantification from
cultured supernatant was performed in a 96-well plate using the D-
lactic acid/L-lactic acid test kit (Boehringer Mannheim, Indianapolis,
IN; USA/R-Biopharam, South Marshall, MI, USA/Roche). Extrac-
ellular levels of lactic acid were measured using a Multiscan Spectrum
(Thermo Electron Corporation). The total amount of lactic acid
produced and the fold change in lactic acid production (% of lactic
acid/% of nuclear DNA) were determined for each sample.
HCV Replicon Assay. Huh-7 clone B cells containing HCV
replicon RNA were seeded in a 96-well plate at 3000 cells/well, and
K
DOI: 10.1021/jm501874e
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Journal of Medicinal Chemistry
Article
the compounds were added in dose response in triplicate immediately
after seeding.45 Following 5 days of incubation (37 °C, 5% CO2), total
cellular RNA was isolated by using the RNeasy 96-well extraction kit
from Qiagen. Replicon RNA and an internal control (TaqMan rRNA
control reagents, Applied Biosystems) were amplified in a single step
multiplex real time RT-PCR assay. The antiviral effectiveness of the
compounds was calculated by subtracting the threshold RT-PCR cycle
of the test compound from the threshold RT-PCR cycle of the no-
drug control (ΔCt HCV). A ΔCt of 3.3 equals a 1 log reduction
(equal to 90% less starting material) in replicon RNA levels. The
cytotoxicity of the compounds was also calculated by using the ΔCt
rRNA values. 2′-C-Me cytidine was used as the positive control. To
determine EC90 and CC50 values, ΔCt values were first converted into
fraction of starting material and then were used to calculate the %
inhibition.
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a series of cytochrome P(450) 3A-activated prodrugs (HepDirect
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Toxicity Assays. For detailed descriptions of cytotoxicity assays,
see ref 33.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 404-727-1414. E-mail: rschina@emory.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by NIH CFAR Grant
2P30AI050409 and by the Department of Veterans Affairs.
R.F.S. is the Chairman and a major shareholder of CoCrystal
Pharma, Inc.
ABBREVIATIONS USED
■
DAPN, β-D-2,6-diaminopurine nucleoside; PD, prodrug; NTP,
nucleoside triphosphate; PI, protease inhibitor; DAA, direct
acting agent; RdRp, RNA-dependent RNA polymerase;
NMPD, nucleoside monophosphate prodrug; SVR, sustained
virologic response; PBM, peripheral blood mononuclear; SD,
standard deviation; mtDNA, mitochondrial DNA; ND, not
determined; NA, not applicable; GT, genotype; NI, nucleoside
inhibitor; DBU, 1,8-diazabicycloundec-7-ene; TMSOTf, trime-
thylsilyl trifluoromethanesulfonate; (Boc)2O, di-tert-butyl dicar-
bonate; DMAP, 4-dimethylaminopyridine; DCM, dichloro-
methane; THF, tetrahydrofuran
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