Bifunctional aryloxyphosphoramidate prodrugs of 2′-: C -Me-uridine: Synthesis and anti-HCV activity

Article abstract of DOI:10.1039/c6ob01189f

In an attempt to identify novel nucleoside phosphoramidate analogues for improving the anti-HCV activity of 2′-C-Me-uridine, we have synthesized for the first time a series of l-glutamic acid, l-serine, l-threonine and l-tyrosine containing aryloxyphosphoramidate prodrugs of 2′-C-Me-uridine. Evaluation of their activity against HCV revealed that they displayed very potent anti-HCV activity, with EC₅₀ values that are in the same range as of Sofosbuvir.

Full text of DOI:10.1039/c6ob01189f

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Bifunctional aryloxyphosphoramidate prodrugs of
2’-C-Me-uridine: synthesis and anti-HCV activity†
Cite this: DOI: 10.1039/c6ob01189f
Munmun Maiti,a Ling-Jie Gao,a Chunsheng Huang,b Roger G. Ptak,b
Michael G. Murray,b Steven De Jonghea and Piet Herdewijn*a
In an attempt to identify novel nucleoside phosphoramidate analogues for improving the anti-HCV activity
of 2’-C-Me-uridine, we have synthesized for the first time a series of L-glutamic acid, L-serine,
L-threonine and L-tyrosine containing aryloxyphosphoramidate prodrugs of 2’-C-Me-uridine. Evaluation
of their activity against HCV revealed that they displayed very potent anti-HCV activity, with EC50 values
that are in the same range as of Sofosbuvir.
Received 1st June 2016,
Accepted 22nd August 2016
DOI: 10.1039/c6ob01189f
www.rsc.org/obc
and thus negatively charged at physiological pH and hence,
are not able to penetrate the lipid-rich cell membrane. In
Introduction
Nucleosides and nucleotides have demonstrated wide-spread addition, phosphohydrolases (acid and alkaline phosphatases,
utility as antiviral agents.1 Antiviral nucleosides are essentially 5′-nucleotidases) rapidly convert the phosphates to the corres-
prodrugs since their antiviral activity depends upon their intra- ponding nucleosides.
cellular metabolism within virus-infected cells to form sequen-
In order to overcome these limitations, various prodrug or
tially the mono-, di- and triphosphates. It is these nucleotides, ‘pronucleotide’ approaches have been devised and investi-
and especially the triphosphates that are the pharmacologi- gated.2 In general, the goal of these approaches has been to
cally active species, as they are incorporated into a growing promote stability in the extracellular medium, passive
DNA or RNA strand by a DNA or RNA polymerase, resulting in diffusion through the lipophilic cell membranes and to liber-
chain termination and fraudulent DNA/RNA. The first phos- ate the parent nucleotide, where it can be further phosphory-
phorylation step leading to the formation of the nucleoside 5′- lated to the pharmacologically active species. As a result,
monophosphate is commonly catalyzed by a nucleoside kinase several prodrug approaches now exist. The synthetic derivatiza-
encoded by the host cell or the virus infecting the host cell. tion has been made by using various protecting groups to
Conversion of the nucleoside monophosphate to the corres- shield the phosphate charges. The development of the protect-
ponding 5′-diphosphate and triphosphates is carried out by ing groups has moved from using simple alkyl groups to more
nucleotidyl, and nucleoside diphosphate kinases, respectively. sophisticated structures that may efficiently deliver phosphory-
Hence, cellular kinases, as well as virally-encoded kinases play lated species into cells. One of the most promising approaches
a vital role in the activation of nucleoside drugs.
is the “aryloxyphosphoramidate” approach (also known as
In most cases, the first step of phosphorylation represents ProTide approach), pioneered by Jones et al. in the early 1980s,
the rate-limiting step of the bioactivation and its inefficiency and later developed by McGuigan et al. in the 1990s.3 The
may limit the therapeutic potential of the nucleoside ana- cleavage of this class of prodrugs is initiated by an esterase
logues. Bypassing this rate-limiting activation step may enzyme, then an intramolecular cyclization is believed to take
improve the biological activity of the nucleosides. In principle, place with displacement of the aryl moiety to form a short-
administration of nucleoside-5′-monophosphates would over- lived five-membered ring intermediate, which is hydrolyzed to
come the drawbacks. However, phosphates are strongly acidic, phosphoramidic acid. The cleavage of the monoamidate to the
active species may be catalyzed by a second enzyme, for
example, phosphoramidase or may result from simple hydro-
lysis in a more acidic subcellular compartment, releasing intra-
aKU Leuven, Rega Institute, Laboratory of Medicinal Chemistry,
Minderbroedersstraat 10, 3000 Leuven, Belgium.
cellularly nucleoside-monophosphate. Research on antivirally
active ProTides in the past 20 years led to the discovery of a
number of clinically efficacious ProTides (Fig. 1). Sofosbuvir
was the first ProTide that received marketing approval and it
has been licensed for the treatment of HCV infected patients.4
Tenofovir alafenamide (TAF) is an example of a phosphono-
E-mail: Piet.Herdewijn@kuleuven.be; Tel: +32 16 337387
bSouthern Research, Infectious Disease Research, 431 Aviation Way, Frederick,
MD 21701, USA
†Electronic supplementary information (ESI) available: NMR spectra of final
compounds. See DOI: 10.1039/c6ob01189f
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Fig. 1 Known ProTides.
amidate derivative.5 The first combination pill containing TAF L-alanine, using 2′-C-Me-uridine as prototype nucleoside. As
for the treatment of HIV was FDA approved for the treatment in previous reports on ProTides, the L-stereochemistry was
of HIV under the trade name Genvoya®. In addition, two other found to be superior when compared to the D-enantiomers,
TAF-based HIV treatments are currently under FDA review and we focused on the synthesis of ProTides with amino acids
in January 2016, Gilead requested FDA approval of stand-alone having the natural L-stereochemistry. In this manuscript, the
TAF for hepatitis B. GS-5734 is an example of a phosphorami- first synthesis of L-glutamic acid, L-serine, L-threonine and
date C-nucleoside derivative that exhibits antiviral activity L-tyrosine containing aryloxyphosphoramidate prodrugs of
against multiple variants of the Ebola virus in cell-based 2′-C-Me-uridine is presented, as well as the assessment of
assays.6 In a rhesus monkey model of Ebola virus infection, their anti-HCV activity.
treatment with GS-5734 protected the Ebola virus infected
animals from the lethal disease. A Phase 1 clinical trial in
healthy human volunteers is ongoing to determine its safety, Chemistry
tolerability and pharmacokinetics.
Synthesis of di-esters of L-glutamic acid
In nearly all reports on ProTides, L-alanine is the most
commonly used amino acid.7 While keeping the L-alanine
Although we previously used the thionyl chloride/isoamylalco-
hol system for the preparation of the amino acid esters from
motif intact, several structural modifications of the ester
the corresponding amino acids,12 we switched to the trimethyl-
functional group have been studied. Small ester moieties,
chlorosilane reagent for esterification of the carboxylic acid
such as methyl, ethyl and isopropyl esters, as well as bulkier
moiety of L-glutamic acid 1.13 The isoamylalcohol/TMSCl is a
groups such as cyclohexyl and benzyl esters have been syn-
facile system as compared to the classical methods (using an
thesized and studied for biological activities.8,9 On the other
acid and an appropriate alcohol) used for esterification,
hand, systematic structure–activity relationship (SAR) studies
because it is more convenient to use and measure but also
involving the amino acid side chain have not been per-
because it acts as a water scavenger so that the esterification is
formed and in most cases only glycine, leucine, valine,
leucine, and proline were systematically studied.8,9 From
faster and cleaner (Scheme 1).
these studies, it was concluded that L-alanine is optimal in
Synthesis of mono- and di-esters of L-serine
terms of antiviral activity of the corresponding nucleoside
phosphoramidate.7 The stereochemistry of the amino acid
TMSCl is added to a mixture of amylalcohol or isoamylalcohol
and O-benzyl-L-serine 3, affording compounds 4 and 5, respect-
ively. Similarly, esterification of the acid group of L-serine 6
affords compounds 7 and 8 (as their hydrochloride salts).
plays also an important role in the antiviral activity of the
ProTide. L-Alanine is preferred over D-alanine when evaluat-
ing the anti-HIV activity of phosphoramidate triester pro-
drugs of stavudine and zidovudine.10,11 Similarly, the
D-alanine analogue of Sofosbuvir completely lacks anti-HCV
activity.4 We recently reported the discovery of a novel series
of L-aspartic acid based ProTides of 2′-C-Me-uridine and 2′-C-
Me-cytidine with potent anti-HCV activity. In some cases,
compounds exhibited improved activity relative to the corres-
ponding L-alanine derived ProTides, depending on the ester
moiety of the two carboxylic acid moieties of L-aspartic
acid.12 Given these promising data, we have explored other
bifunctional amino acids (L-glutamic acid, L-serine,
L-threonine and L-tyrosine) as potential alternatives for ditions: (a) TMSCl, isoamyl alcohol, 35 °C, 30 h.
Scheme 1 Synthesis of L-glutamic acid di-ester. Reagents and con-
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Scheme 2 Synthesis of mono- and di-esters of L-serine. Reagents and conditions: (a) TMSCl, isoamyl/amyl alcohol, 35 °C, 30 h; (b) di-tert-butyl-
bicarbonate, TEA, 1,4-dioxane, rt, 1.5 h; (c) isobutyryl chloride, DMAP, TEA, dry DCM, rt, overnight; (d) HCl in iPrOH, DCM, 0 °C to rt, 10 h.
Protection of the amino group of compounds 7 and 8 as a Boc Synthesis of di-esters of L-tyrosine
furnished the esterified N-Boc-L-serine derivatives 10 and 11.
Esterification of the carboxylic acid of N-Boc-L-tyrosine 24, is
Boc-L-serine methyl ester
9
is commercially available.
followed by conversion of the phenolic hydroxyl group to an
isobutyryl ester moiety yielding intermediate 26. Finally, de-
protection affords the desired diester analogue of L-tyrosine 27
(Scheme 4).
Conversion of the primary hydroxyl group to an isobutyryl
ester by reaction with isobutyryl chloride, is followed by acidic
cleavage of the Boc group affording the desired di-esters of
L-serine 15–17 (Scheme 2).
Synthesis of mono- and di-esters of L-threonine
Synthesis of ProTides of 2′-C-uridine
The carboxylic acid moiety of O-benzyl-L-threonine 18 was The synthesis of the final nucleoside phenoxy phosphorami-
esterified using isoamyl alcohol and N,N,N′,N′-tetramethyl-O- date analogues is shown in Scheme 5.
(6-chloro-1H-benzotriazol-1-yl)uraniumhexafluorophosphate
Commercially available phenyl phosphorodichloridate 28
(HCTU) as coupling reagent (Scheme 3). The carboxylic acid was reacted with the amino acid esters 2, 4–5, 15–17, 19, 23
moiety of N-Boc-L-threonine 20 was esterified in the same way. and 27 in dichloromethane yielding the arylaminoacyl phos-
The secondary alcohol group was esterified with isobutyryl phorodichloridates 29. Coupling with 2′-C-methyl-2′,3′-O-iso-
chloride. Finally, acidic deprotection of the Boc group yielded propylidene-uridine, which was prepared according to
the desired di-ester derivative of L-threonine 23 as a hydro- literature protocol,12 furnished the 2′-C-Me-2′,3′-O-isopropyl-
chloride salt. idene-uridine ProTide analogues 30a–k. Finally, acidic
a
Scheme 3 Synthesis of mono- and di-esters of L-threonine. Reagents and conditions: (a) isoamyl alcohol, HCTU, TEA, CH2Cl2H, rt, 4 h; (b) isobu-
tyryl chloride, TEA, CH2Cl2, rt, 4 h; (c) TFA/CH2Cl2 (1/1), rt, 1 h, then Na2CO3 and HCl in iPrOH, rt.
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stable luciferase (Luc) reporter gene.15 This Luc reporter is used
as an indirect measure of HCV replication as its activity is
directly proportional to HCV RNA levels. Recombinant human
interferon alpha-2b (rIFNα-2b) and Sofosbuvir are included as
positive control. EC50, EC90 (compound concentrations reducing
HCV replication by 50% and 90% respectively) and CC50
(concentration decreasing cell viability by 50%) values were
determined. The results are summarized in Table 1.
The parent nucleoside 2′-C-Me-uridine shows only marginal
anti-HCV activity (EC50 = 6.31 µM) and all ProTides are
endowed with a more pronounced antiviral activity. When the
dimethyl ester of L-glutamic acid was used as amino acid motif
in the ProTide (compound 31a), only weak anti-HCV activity
was observed (EC50 = 1.52 µM). Increasing the chain length of
the ester moieties from a methyl ester (compound 31a) to an
isoamyl group (compound 31b) led to a 20-fold improvement
in anti-HCV activity. This might be related to the higher lipo-
philicity of the compound 31a (clog P = −1.28), when com-
pared to 31b (clog P = 2.69), and hence a better cellular
permeability. The choice of the isoamyl ester moiety is dictated
by the fact that these ester groups were found to be optimal to
confer antiviral activity to L-aspartic acid based aryloxyphos-
phoramidate nucleoside prodrugs.12
When the methyl ester of O-benzyl-L-serine is used in the
aryloxyphosphoramidate prodrugs (compound 31c), less than
optimal anti-HCV activity was obtained (EC50 = 0.18 µM).
When more lipophilic esters of O-benzyl-L-serine were used
(the amylester 31d and the isoamyl ester 31e), potent antiviral
activity was observed, displaying EC50 values of 0.03 µM and
<0.06 µM, respectively. In analogy with the diesters of
L-glutamic acid, a number of diesters of L-serine were syn-
thesized. The most polar derivative (compound 31f, clog P =
−0.12) was, as expected, the least active in the HCV replicon
assay with a EC50 value of 0.51 µM. Substitution of the methyl
ester group by an amyl or isoamyl group afforded compounds
Scheme 4 Synthesis of a di-ester of L-tyrosine. Reagents and con-
ditions: (a) isoamyl alcohol, HCTU, TEA, CH2Cl2, rt, 4 h; (b) isobutyryl
chloride, TEA, CH2Cl2, rt, 4 h; (c) TFA/CH2Cl2 (1/1), rt, 1 h and then
Na2CO3 and HCl in iPrOH, rt.
deprotection afforded the desired target compounds 31a–k.
Each ProTide was generated as a pair of diastereomers, due to
the chirality of the phosphorus centre, and compounds were
evaluated as isomeric mixtures.
Biological evaluation
The final ProTides 31a–k, as well as the parent 2′-C-Me-uridine, 31g and 31h, respectively, both endowed with a minor
were evaluated for anti-HCV activity as follows. The primary improvement in antiviral activity. Finally, L-threonine (com-
assay uses the genotype 1b (Con1 strain) HCV replicon to evalu- pounds 31i and 31j) and L-tyrosine (compound 31k) contain-
ate anti-HCV activity. The assay makes use of the cell line ET ing ProTides, as other examples of bifunctional amino acids,
(luc-ubi-neo/ET), which is a Huh7 human hepatoma cell line were prepared. Despite their favorable lipophilicity, these com-
harboring a subgenomic HCV replicon that incorporates a pounds exhibited lower anti-HCV activity in the range of
Scheme 5 Reagents and conditions: (a) N-methylimidazole, 2 or 4–5 or 15–17 or 19 or 23 or 27, CH2Cl2, −15 °C, 2 h; (b) 2’-C-methyl-2’,3’-O-iso-
propylidene-uridine, CH2Cl2 , −5 °C to rt, 4–6 h; (c) TFA : H2O (8 : 2, 0.1 M), 0 °C to rt, 3–6 h.
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Table 1 Luciferase-based HCV 1b replicon activity of ProTides 31a–k
Cmpd
R
Amino acid
EC50 a (µM)
EC90 a (µM)
CC50 a (µM)
SI50
clog P
2′-C-Me-uridine
31a
—
—
6.31
1.52
55.3
5.6
>100
>20
>15.8
>13
−1.69
−1.28
L-Glutamic acid
31b
L-Glutamic acid
0.08
0.21
>20
>250
2.69
31c
31d
L-Serine
L-Serine
0.18
0.03
0.6
>20
>111
196
0.65
2.77
0.14
5.89
31e
31f
L-Serine
L-Serine
<0.06
0.51
0.13
1.69
8
>127
>39
2.64
>20
−0.12
31g
31h
L-Serine
L-Serine
0.10
0.08
0.86
0.56
6.79
7.51
68
94
1.99
1.87
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Table 1 (Contd.)
Cmpd
R
Amino acid
L-Threonine
EC50 a (µM)
0.29
EC90 a (µM)
1.39
CC50 a (µM)
>20
SI50
>69
clog P
31i
2.18
31j
L-Threonine
0.47
1.83
11.46
24
2.94
31k
L-Tyrosine
0.40
1.47
>20
>50
2.82
IFNα-2b
Sofosbuvir
—
—
0.15
0.07
0.48
0.30
>10
>5.00
>67
>71
See Fig. 1
L-Alanine
a Results are from a single assay generated from quadruplicate assay wells at each concentration.
0.3–0.5 µM. These findings suggest that lipophilicity can be L-tyrosine (compound 31k) containing prodrugs are more potent
used as a predictive tool for HCV activity within a particular than Sofosbuvir, as evidenced by their very potent EC50 and
amino acid series, but is not useful to compare the biological EC90 values. The strong potency of these ProTides, in combi-
activities between different series. In general, the prodrugs nation with low cytotoxicity, gives rise to high selectivity indexes.
31a–k were not cytotoxic towards the Huh7 hepatoma cell line.
To exert their antiviral activity, the ProTides must be
This is similar as to what is observed for Sofosbuvir. Although metabolized in hepatocytes to release 2′-C-Me-uridine-mono-
in our hands, the highest tested concentration was 5 µM, it is phosphate intracellularly, which will then be further phos-
known from literature that Sofosbuvir is completely devoid of phorylated yielding the corresponding triphosphate congener,
cytotoxicity, up to 100 µM.4 For the most potent congeners that acts as an inhibitor of the HCV RNA-dependent RNA poly-
(compounds 31b, 31e and 31h), a selectivity index of at least merase NS5B. Human liver microsomes were chosen as a sur-
127 was calculated.
rogate in vitro model to test for hepatocyte stability. Prodrugs
To confirm the compounds anti-HCV activity, representative 31b, 31d, 31h, 31i and 31k were incubated at a concentration
examples were tested in a secondary assay. Briefly, a qRT-PCR of 0.1 µM and the parent compound was quantified at
(TaqMan) endpoint is used to directly measure HCV RNA different time points (0, 15, 30, 45 and 60 minutes). From
copies, rather than the luciferase read-out which is an indirect these data, the in vitro half-life and the intrinsic clearance
read-out. Similarly, cellular ribosomal RNA is measured as a values were calculated (Table 3). Extensive metabolism in
cytotoxicity control. The EC50, EC90 and CC50 values for each human liver microsomes leads to very short half lifes and high
compound is shown in Table 2. In addition, the potency of the intrinsic clearance values. Although no metabolite identifi-
compounds were compared with Sofosbuvir, the only FDA cation has been performed as part of this study, the intracellu-
approved nucleoside analogue for the treatment of HCV infected lar activation route for ProTides has extensively been studied
patients. The L-glutamic acid (compound 31b), the L-serine in literature,14 and it has been demonstrated that intracellular
(compounds 31e and 31h), the L-threonine (compound 31i) and metabolism of these prodrugs leads to formation of the
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Table 2 qRT-PCR based HCV 1b replicon activity of ProTides 31b and 31e
Cmpd#
R
EC50 a (µM)
0.01
EC90 a (µM)
0.13
CC50 a (µM)
9.98
SI50
998
31b
31e
31h
0.01
0.02
0.08
0.13
4.59
5.28
459
264
31i
0.04
0.69
7.27
182
31k
0.04
0.07
0.69
0.28
18.37
>5.00
459
>71
Sofosbuvir
Structure – Fig. 1
a Results are from a single assay generated from quadruplicate assay wells at each concentration.
Table 3 Metabolic stability of ProTides 31b, 31d, 31h, 31i and 31k
Conclusion
Clinta
a
T1/2
The aryloxyphosphoramidate prodrug technology is a well-
known strategy to improve the antiviral activity of nucleoside
analogues by bypassing the first and rate-limiting phosphoryl-
ation step. Previous research has demonstrated that lipophilic
diesters of L-aspartic acid instead of the commonly used
L-alanine esters in ProTides, afforded nucleoside analogues
with an improved antiviral activity. In this paper, the synthesis
of a novel series of ProTides of 2′-C-Me-uridine is described,
using L-glutamic acid, L-serine, L-threonine and L-tyrosine
Compound
(min)
(µl min−1 mg−1 protein)
31b
31d
31h
31i
8
14
9
10
10
844.60
496.80
763.70
699.00
679.60
31k
a Results are the mean of two independent experiments.
nucleoside-monophosphate. Taken together, these data indi- as amino acid motif. The most potent congeners display
cate that the bifunctional aryloxyphosphoramidate prodrugs of EC50 values in the low nanomolar range against HCV.
2′-C-Me-uridine are metabolized to their biologically active These data suggest that bifunctional amino acids can be con-
species.
sidered as an alternative to L-alanine in the design of antiviral
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aryloxyphosphoramidate nucleoside prodrugs. The advantage hexane was added and the white precipitate was filtered.
of using a bifunctional amino acid is that there are two sites Finally, the precipitate was washed several times with hexane
for structural variation.14 While the α-carboxyl acid group plays to obtain 5 as hydrochloride salt (90%). 1H NMR (300 MHz,
+
an important role in the displacement of the phenoxy group DMSO-d6): δ = 8.74 (br s, 3H, –NH3 ), 7.35–7.34 (m, 5H), 4.54
(via neighbouring group assistance), the remaining functional (dd, 2H), 4.33 (t, 1H), 4.23–4.08 (m, 2H), 3.87 (d, 2H),
group (the carboxylic acid of L-glutamic acid or the hydroxyl 1.59–1.52 (m, 2H), 1.30–1.25 (m, 4H), 0.87–0.82 (t, 3H) ppm.
group of L-serine, L-threonine and L-tyrosine) can be used as a 13C NMR (75 MHz, DMSO-d6): δ = 168.7, 138.3, 129.1, 128.6,
chemical handle to tune physicochemical properties, such as 128.5, 73.3, 68.3, 66.6, 53.3, 28.6, 28.2, 22.6, 14.7 ppm. HRMS
lipophilicity and aqueous solubility, by the attachment of suit- (ESI+) calcd for C15H24NO3 [M + H]+ 266.1751, found 266.1750.
able functional groups.
Isoamyl ester of Ser-(OBn)-OH (5)
To a suspension of Ser-(OBn)-OH 3 (1.0 g, 5 mmol) in anhy-
drous isoamyl alcohol (30 mL) was added trimethyl-
chlorosilane (4 mL, 30.7 mmol) dropwise at 0 °C under argon
atmosphere. The mixture was allowed to come to room temp-
erature and stirred for 72 h at 35 °C. After evaporation to
Experimental section
General
NMR spectra were recorded on a Bruker Avance II 300 MHz
with a 5 mm broad band probe, a 500 MHz spectrometer
dryness, hexane was added and the white precipitate was fil-
equipped with a TXI-HCP Z gradient probe or on a Bruker
Avance II 600 MHz spectrometer with a 5 mm TCI-HCN Z gra-
dient cryo-probe. The spectra were processed with Bruker
tered off. The precipitate was washed several times with
hexane to obtain 5 as hydrochloride salt (80%). 1H NMR
+
(300 MHz, DMSO-d6): δ = 8.69 (br s, 3H, –NH3 ), 7.40–7.29 (m,
Topspin 2.1 software. Chemical shifts (δ) were expressed in
5H), 4.54 (dd, 2H), 4.34 (t, 1H), 4.25–4.13 (m, 2H), 3.86 (d, 2H),
1
parts per million (ppm). The H and 13C NMR chemical shifts
1.69–1.58 (m, 1H), 1.52–1.43 (m, 2H), 0.88–0.84 (m, 6H) ppm.
were referenced relative to the TMS peak (δ = 0.00 ppm). 31P
NMR chemical shifts were referenced to an external 85%
H3PO4 standard (δ = 0.00 ppm). Mass spectra were acquired on
a quadrupole orthogonal acceleration time-of-flight mass
spectrometer (Synapt G2 HDMS, Waters, Milford, MA).
Samples were infused at 3 μL min−1 and spectra were obtained
in positive (or in negative) ionization mode with a resolution
of 15 000 (FWHM) using leucine enkephalin as the lock mass.
Chemicals of analytical and synthetic grade were obtained
from commercial sources and were used as such. Flash silica
column chromatography was performed on silica gel 60 A,
0.035–0.070 mm (Acros Organics).
13C NMR (75 MHz, DMSO-d6): δ = 168.7, 138.2, 129.1, 128.6,
128.5, 73.4, 68.3, 65.1, 53.3, 37.5, 25.1, 23.1, 23.0 ppm. HRMS
(ESI+) calcd for C15H24NO3 [M + H]+ 266.1751, found 266.1748.
Ser-(OH)-O-amyl ester hydrochloride (7)
To a suspension of L-serine 6 (2.0 g, 19 mmol) in anhydrous
amyl alcohol (50 mL) was added dropwise trimethyl-
chlorosilane (14.5 mL, 114.2 mmol) at 0 °C under argon atmo-
sphere. The mixture was allowed to come to room temperature
and stirred for 24 h at 40 °C. After evaporation to dryness,
hexane was added and the white precipitate was filtered off.
Finally, the precipitate was washed several times with hexane
1
and ether to obtain 7 as a hydrochloride salt (90%). H NMR
Diisoamyl ester of glutamic acid (2)
+
(300 MHz, DMSO-d6): δ = 9.49 (br s, 3H, –NH3 ), 6.52 (s, 1H),
To a suspension of L-glutamic acid 1 (2.0 g, 13.6 mmol) in
anhydrous isoamyl alcohol (60 mL) was added trimethyl-
chlorosilane (10.4 mL, 81.6 mmol) dropwise at 0 °C under
argon atmosphere. The mixture was allowed to come to room
temperature and stirred for 48 h at 35 °C. After evaporation to
dryness, hexane was added and the white precipitate was fil-
tered off. Finally, the precipitate was washed several times with
hexane to obtain 2 as hydrochloride salt (61%). 1H NMR
(300 MHz, DMSO-d6): δ = 8.71 (br s, 3H, –NH3+), 4.19, 4.06
(5H), 2.52 (2H), 2.08 (2H), 1.67, 1.50 (6H), 0.91 (12H). 13C NMR
(75 MHz, DMSO-d6): δ = 172.5, 170.0, 65.0, 63.5, 52.0, 37.7,
37.5, 30.0, 26.1, 25.4, 25.2, 23.2, 23.1 ppm. HRMS (ESI+) calcd
for C15H30NO4 [M + H]+ 288.2169, found 288.2166.
5.06–5.02 (m, 2H), 4.96 (1H), 4.72 (2H), 4.26 (br s, 2H), 2.21
(4H), 1.79, 1.77 (3H) ppm. 13C NMR (75 MHz, DMSO-d6): δ =
168.0, 65.5, 59.5, 54.4, 27.6, 27.3, 21.7, 13.8 ppm. HRMS (ESI+)
calcd for C8H18NO3 [M + H]+ 176.1281, found 176.1285. HRMS
(ESI+) calcd for C8H18NO3 [M + H]+ 176.1281, found 176.1281.
Ser-(OH)-O-isoamyl ester hydrochloride (8)
To a suspension of L-serine 6 (2.0 g, 19 mmol) in anhydrous
isoamyl alcohol (50 mL) was added trimethylchlorosilane
(14.5 mL, 114.2 mmol) dropwise at 0 °C under argon atmo-
sphere. The mixture was allowed to come to room temperature
and stirred for 30 h at 35 °C. After evaporation to dryness,
hexane was added and the white precipitate was filtered off.
Finally, the precipitate was washed several times with hexane
Amyl ester of Ser-(OBn)-OH (4)
and ether to obtain 8 as hydrochloride salt (90%). 1H NMR
+
To a suspension of Ser-(OBn)-OH 3 (1.0 g, 5 mmol) in anhy- (300 MHz, DMSO-d6): δ = 8.60 (br s, 3H, –NH3 ), 5.62 (s, 1H),
drous amyl alcohol (30 mL) was added trimethylchlorosilane 4.17–4.04 (m, 3H), 3.82 (s, 2H), 4.17–4.64 (m, 1H), 1.53–1.48
(4 mL, 30.7 mmol) dropwise at 0 °C under argon atmosphere. (m, 2H), 0.89, 0.87 (6H) ppm. 13C NMR (75 MHz, DMSO-d6):
The mixture was allowed to come to room temperature and δ = 168.0, 64.0, 59.5, 54.4, 36.7, 24.3, 22.3, 22.2 ppm. HRMS
stirred at 35 °C for 72 hours. After evaporation to dryness, (ESI+) calcd for C8H18NO3 [M + H]+ 176.1281, found 176.1285.
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Paper
Boc-Ser-(OH)-O-isoamyl ester (11)
ppm. 13C NMR (125 MHz, CDCl3): δ = 176.7, 170.0, 155.3, 80.3,
66.1, 53.2, 34.0, 28.4, 28.3, 28.0, 22.4, 19.1, 18.9, 14.0 ppm.
HRMS (ESI−) calcd for C17H30NO6 [M − H]− 344.2078, found
344.2082.
Compound 8 (3.0 g, 14.8 mmol) was dissolved in anhydrous
1,4-dioxane (60 mL). Di-tert-butyl-bicarbonate (4 g, 17.8 mmol)
followed by triethyl amine (3 mL, 22.3 mmol) were added and
the reaction mixture was stirred at room temperature for 1.5 h.
The reaction mixture was then evaporated to dryness and
diluted with ethyl acetate, washed with 0.5 N aq. HCl, followed
by aq. sat. sodium bicarbonate and brine. The organic layer
was dried over anhydrous MgSO4 and evaporated to dryness.
The crude product was purified by flash column chromato-
graphy by eluting with 20–30% EtOAc in hexane to obtain com-
pound 11 in 95% yield. Rf = 0.34 (hexane/EtOAc, 8 : 2). 1H NMR
(500 MHz, CDCl3): δ = 5.48 (br s, 1H), 4.36 (br s, 1H), 4.22–4.20
(m, 2H), 3.96–3.91 (m, 2H), 2.54 (m, 1H), 1.74–1.66 (m, 1H),
1.58–1.53 (m, 2H), 1.46 (s, 9H), 0.93, 0.92 (2 s, 6H) ppm. 13C
NMR (125 MHz, CDCl3): δ = 171.0, 155.9, 80.4, 64.6, 63.8, 56.0,
37.3, 28.4, 25.1, 22.5, 22.5 ppm. HRMS (ESI+) calcd for
C8H18NO3 [M + H]+ 176.1281, found 176.1285. HRMS (ESI+)
calcd for C13H25NO5Na [M + Na]+ 298.1625, found 298.1620.
Boc-Ser(iBu)-O-isoamyl ester (14)
A similar procedure as for the synthesis of 12 was used. Rf =
1
0.82 (hexane/EtOAc, 8 : 2). H NMR (500 MHz, CDCl3): δ = 5.28
(br d, 1H), 4.57–4.55 (m, 1H), 4.47–4.44 (m, 1H), 4.33–4.30 (m,
1H), 4.23–4.15 (m, 2H), 2.59–2.50 (m, 1H), 1.72–1.64 (m, 1H),
1.58–1.50 (m, 2H), 1.46 (s, 9H), 1.16–1.14 (m, 6H), 0.93–0.91
(m, 6H) ppm. 13C NMR (125 MHz, CDCl3): δ = 176.7, 170.0,
155.3, 80.4, 64.6, 64.3, 53.2, 37.3, 34.0, 28.4, 25.1, 22.5, 22.4,
19.1, 18.9 ppm. HRMS (ESI+) calcd for C17H31NO6Na [M + Na]+
368.2044, found 368.2036.
Ser(iBu)-OMe hydrochloride (15)
Compound 12 (2.65 g, 9.2 mmol) was dissolved in CH2Cl2.
A
solution of HCl in isopropanol (5–6 N, 10 mL) was added and
the reaction mixture was stirred at 30 °C for 6 h. Upon com-
pletion, the reaction mixture was evaporated to dryness to
obtain the product as white solid which was then washed with
ether and dried overnight to obtain 15 as hydrochloride salt in
Boc-Ser-(OH)-O-amyl ester (10)
A similar synthetic procedure as for the synthesis of com-
pound 11 is used. Rf = 0.34 (hexane/EtOAc, 8 : 2). 1H NMR
(500 MHz, CDCl3): δ = 5.47 (br s, 1H), 4.37 (br s, 1H), 4.19–4.16
(m, 2H), 3.96–3.92 (m, 2H), 2.52–2.49 (m, 1H), 1.68–1.65 (m,
1
95% yield. H NMR (300 MHz, DMSO-d6): δ = 8.95 (br s, 3H,
+
–NH3 ), 4.45 (3H), 3.75 (s, 3H), 2.64–2.52 (m, 1H), 1.10, 1.07 (2
d, 6H) ppm. 13C NMR (75 MHz, DMSO-d6): δ = 175.5, 167.5,
61.3, 53.0, 51.2, 33.0, 18.7, 18.4 ppm. HRMS (ESI+) calcd for
C8H16NO4 [M + H]+ 190.1074, found 190.1076.
2H), 1.46 (s, 9H), 1.35–1.32 (m, 4H), 0.92–0.89 (t, 3H) ppm. 13
C
NMR (125 MHz, CDCl3): δ = 171.0, 156.0, 80.4, 66.1, 63.9, 56.0,
28.4, 28.3, 28.1, 22.4, 14.0 ppm. HRMS (ESI+) calcd for
C13H25NO5Na [M + Na]+ 298.1625, found 298.1627.
Ser(iBu)-O-amyl ester hydrochloride (16)
This compound was made according to the procedure for the
synthesis of 15. H NMR (500 MHz, DMSO-d6): δ = 8.95 (br s,
Boc-Ser(iBu)-OMe (12)
1
3H, –NH3+), 4.47–4.43 (m, 3H), 4.23–4.18 (m, 1H), 4.15–4.11
(m, 1H), 2.59–2.53 (m, 1H), 1.61–1.59 (m, 2H), 1.30–1.28 (m,
4H), 1.10–1.07 (m, 6H), 0.87 (t, 3H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 175.5, 167.1, 63.9, 61.5, 51.3, 33.0, 27.6, 27.3,
21.7, 18.6, 18.4, 13.8 ppm. HRMS (ESI+) calcd for C12H24NO4
[M + H]+ 246.1700, found 246.1702.
To a solution of compound 9 (1 g, 4.56 mmol) in dry DCM
(50 ml) were added DMAP (56 mg, 0.5 mmol) and TEA (2 mL,
13.7 mmol). Isopropionyl chloride (1.2 mL, 11.4 mmol) was
added dropwise. The suspension was then stirred overnight at
room temperature. Upon completion, the reaction mixture was
diluted with DCM and washed with 0.5 N aq. HCl, sat. sodium
bicarbonate and with brine. The organic layer was dried over
anhydrous MgSO4 and evaporated to dryness. The crude
product was then purified in flash silica gel column chromato-
graphy eluting with 10–20% ether in hexane to obtain 12 in
95% yield. Rf = 0.38 (hexane/Et2O, 1 : 3). 1H NMR (300 MHz,
CDCl3): δ = 5.30 (d, 1H), 4.60–4.57 (m, 1H), 4.49–4.44 (m, 1H),
4.33–4.28 (m, 1H), 3.76 (s, 3H), 2.60–2.51 (m, 1H), 1.46 (s, 9H),
1.16 (d, 3H), 1.14 (d, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ =
176.7, 170.5, 155.3, 80.4, 64.1 52.8, 33.9, 28.4, 19.1, 18.9 ppm.
HRMS (ESI+) calcd for C13H23NO6Na [M + Na]+ 312.1418,
found 312.1413.
Ser(iBu)-O-isoamyl ester hydrochloride (17)
This compound was made according to the procedure for the
synthesis of 15.
1H NMR (500 MHz, DMSO-d6): δ = 8.82 (br s, 3H, –NH3+),
4.45–4.42 (m, 3H), 4.26–4.15 (m, 2H), 2.58–2.53 (m, 1H),
1.71–1.62 (m, 1H), 1.52–1.47 (m, 2H), 1.10–1.08 (m, 6H),
0.89–0.87 (m, 6H) ppm. 13C NMR (125 MHz, DMSO-d6): δ =
175.5, 167.1, 64.4, 61.5, 51.3, 36.6, 33.0, 24.2, 22.2, 22.1, 18.6,
18.4 ppm. HRMS (ESI+) calcd for C12H23NO4Na [M + Na]+
268.1519, found 268.1517.
Boc-Ser(iBu)-O-amyl ester (13)
Isoamyl Boc-L-Thr(Bzl) (17)
A similar procedure as for the synthesis of 12 was used. Rf = To a mixture Boc-Thr(Bzl)-OH (16) (1.55 g, 5.0 mmol) and
1
0.7 (hexane/EtOAc, 8 : 2). H NMR (500 MHz, CDCl3): δ = 5.29 isoamyl alcohol (3 ml) in anhydrous dichloromethane (30 ml)
(br d, 1H), 4.57–4.56 (m, 1H), 4.47–4.44 (m, 1H), 4.33–4.30 (m, at 0 °C, was added O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetra-
1H), 4.18–4.13 (m, 2H), 2.57–2.52 (m, 1H), 1.66–1.63 (m, 2H), methyluronium hexafluorophosphate (2.30 g, 5.5 mmol) and
1.46 (s, 9H), 1.34–1.32 (m, 4H), 1.16–1.14 (m, 6H), 0.90 (t, 3H) triethylamine (2 ml) respectively. The mixture was stirred and
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allowed to warm to room temperature. Stirring was continued by silica gel flash column chromatography (the mobile phase
till all starting material was consumed according to TLC ana- being a mixture of methanol in dichloromethane, in a gradient
lysis (about 4 h). The reaction mixture was then evaporated to gradually ranging from 0–20% methanol) to yield the title
1
dryness under reduced pressure, and the residue was purified compound 20 as colorless oil (3.0 g, 93%). H NMR (300 MHz,
by silica gel flash column chromatography (the mobile phase CDCl3): δ = 5.40 (m, 1H), 5.19 (m, 1H), 4.42 (m, 1H), 4.15 (t, J =
being a mixture of methanol in dichloromethane, in a gradient 7.0 Hz, 2H), 2.48 (m, J = 7.0 Hz, 1H), 1.72 (m, 1H), 1.65 (m,
gradually ranging from 0 to 10% methanol) to yield the title 2H), 1.48 (s, 9H), 1.30 (d, J = 6.4 Hz, 3H), 1.13 (d, J = 7.0 Hz,
compound 17 as a colorless oil (1.90 g, 99%). 1H NMR 6H), 0.93 (d, J = 6.5 Hz, 6H) ppm.
(300 MHz, CDCl3): δ = 7.32 (m, 5H), 5.32 (m, 1H), 4.58 (d, J =
11.7 Hz, 1H), 4.38 (d, J = 11.7 Hz, 1H), 4.30 (m, 1H), 4.13 (m,
Isoamyl O-isobutyryl-L-threonine hydrochloride (21)
3H), 1.63 (m, 1H), 1.47 (m, 2H), 1.45 (s, 9H), 1.27 (d, J = 6.3 Hz, To a solution of isoamyl-O-isobutyryl-Boc-L-threonine 20 (3.0 g,
3H), 0.90 (d, J = 6.6 Hz, 6H) ppm.
8.3 mmol) in dichloromethane (10 ml) was added slowly tri-
fluoroacetic acid (10 ml). The resulting mixture was stirred at
room temperature for 1 hour. The reaction mixture was evapor-
Isoamyl O-benzyl-L-threonine hydrochloride (18)
To a solution of isoamyl Boc-Thr(Bzl) 17 (1.9 g, 5.0 mmol) in ated to dryness under reduced pressure, and the residue was
dichloromethane (10 ml) was slowly added trifluoroacetic acid dissolved in dichloromethane (20 ml) and washed with a 5%
(10 ml). The resulting reaction mixture was stirred at room sodium carbonate solution. To the organic phase, 1.25 N HCl
temperature for 1 hour. The reaction mixture was evaporated in isopropanol (10 ml) was added. The mixture was concen-
to dryness under reduced pressure, and the residue was dis- trated under reduced to yield the title compound 21 as white
1
solved in dichloromethane (20 ml) and washed with a 5% solid (2.4 g, 98%). H NMR (300 MHz, DMSO-d6): δ = 8.85 (br.
sodium carbonate solution. To the organic phase, 1.25 N HCl s, 3H), 5.25 (m, 1H), 4.32 (m, 1H), 4.16 (m, 2H), 2.52 (m, 1H),
in isopropanol (5 ml) was added. The mixture was concen- 1.65 (m, 1H), 1.47 (m, 2H), 1.47 (d, J = 6.5 Hz, 3H), 1.08 (m,
trated under reduced to yield the title compound 18 as a white 6H), 0.89 (m, 6H) ppm.
1
solid (1.5 g, 95%). H NMR (300 MHz, DMSO-d6): δ = 8.64 (br.
s, 3H), 7.32 (m, 5H), 4.60 (d, J = 12.0 Hz, 1H), 4.42 (d, J = 12.0
Isoamyl Boc-L-tyrosine (23)
Hz, 1H), 4.15 (m, 4H), 1.64 (m, 1H), 1.43 (m, 2H), 1.31 (d, J = To a mixture of Boc-Tyr-OH 22 (1.41 g, 5 mmol) and isoamyl
6.4 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H) ppm.
alcohol (6 ml) in anhydrous dichloromethane (30 ml) at 0 °C,
was added O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (2.28 g, 5.5 mmol) and triethyl-
Isoamyl Boc-L-threonine (19)
To a mixture of Boc-Thr-OH 18 (2.19 g, 10 mmol) and isoamyl amine (2 ml) respectively. The mixture was stirred and allowed
alcohol (6 ml) in anhydrous dichloromethane (50 ml) at 0 °C, to warm to room temperature. Stirring was continued till all
was added O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyl- starting material was consumed according to TLC analysis
uronium hexafluorophosphate (4.56 g, 11 mmol) and triethyl- (about 4 hours). The reaction mixture was then evaporated to
amine (4 ml), respectively. The mixture was stirred and dryness under reduced pressure, and the residue was purified
allowed to warm to room temperature. Stirring was continued by silica gel flash column chromatography (the mobile phase
till all starting material was consumed according to TLC ana- being a mixture of methanol in dichloromethane, in a gradient
lysis (about 4 hours). The reaction mixture was then evapor- gradually ranging from 0 to 10% methanol) to yield the title
ated to dryness under reduced pressure, and the residue was compound 23 as white solid (1.23 g, 70%). 1H NMR (300 MHz,
purified by silica gel flash column chromatography (the DMSO-d6): δ = 9.21 (s, 1H), 7.20 (d, J = 7.8 Hz, 2H), 7.00 (d, J =
mobile phase being a mixture of methanol in dichloro- 7.8 Hz, 2H), 4.02 (m, 3H), 2.79 (m, 2H), 1.56 (m, 1H), 1.34
methane, in a gradient gradually ranging from 0 to 10% (s, 9H), 1.33 (m, 2H), 0.85 (m, 6H) ppm.
methanol) to yield the title compound 19 as a colorless sticky
Isoamyl O-isobutyryl-Boc-L-tyrosine (24)
solid (2.60 g, 90%). 1H NMR (300 MHz, CDCl3): δ = 5.31 (s,
1H), 4.24 (m, 1H), 4.22 (t, J = 6.9 Hz, 2H), 1.71 (m, 1H), 1.57 To a solution of isoamyl Boc-tyrosine 23 (351 mg, 1.0 mmol) in
(m, 2H), 1.47 (s, 9H), 1.26 (d, J = 6.4 Hz, 3H), 0.93 (d, J = anhydrous dichloromethane (5 ml) were added isobutyryl
6.6 Hz, 6H) ppm.
chloride (2.0 mmol) and N-methylimidazole (5 mmol), respect-
ively, at 0 °C. The resultant mixture was stirred and allowed to
room temperature, and the stirring was continued till the start-
Isoamyl O-isobutyryl-Boc-L-threonine (20)
To a solution of isoamyl Boc-L-threonine 19 (2.6 g, 9.0 mmol) ing material disappeared according to TLC analysis (about
in anhydrous dichloromethane (20 ml) at 0 °C were added iso- 4 h). The reaction mixture was then evaporated to dryness
butyryl chloride (13.5 mmol) and N-methylimidazole under reduced pressure, and the residue was purified by silica
(45 mmol) respectively. The resulting mixture was stirred and gel flash column chromatography (the mobile phase being a
allowed to room temperature, and stirring was continued till mixture of methanol in dichloromethane, in a gradient gradu-
all starting material disappeared according to TLC analysis ally ranging from 0–20% methanol) to yield the title com-
1
(about 4 hours). The reaction mixture was then evaporated to pound 24 as colorless oil (400 mg, 95%). H NMR (300 MHz,
dryness under reduced pressure, and the residue was purified CDCl3): δ = 7.16 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H),
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4.14 (t, J = 7.0 Hz, 2H), 3.09 (m, 2H), 2.80 (m, 1H), 1.63 (m,
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-methoxy-
1H), 1.51 (m, 2H), 1.44 (s, 9H), 1.32 (d, J = 7.0 Hz, 6H), 0.92 β-O-benzyl-L-serine)]phosphate (30c). Yield: 74%. Rf
= 0.8
(d, J = 6.5 Hz, 6H) ppm.
(CH2Cl2/MeOH, 9.5 : 0.5). 31P NMR (121 MHz, CDCl3): δ = 3.14,
2.77 ppm. HRMS (ESI+) calcd for C30H37N3O11P [M + H]+
646.2160, found 646.2170.
Isoamyl O-isobutyryl-L-tyrosine hydrochloride (25)
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-amyl-
β-O-benzyl-L-serine)]phosphate (30d). Yield: 62.5%. Rf = 0.58
(hexane/EtOAc, 1 : 9). 31P NMR (121 MHz, CDCl3): δ = 3.21,
2.87 ppm. HRMS (ESI-) calcd for C34H43N3O11P [M − H]−
700.2640, found 700.2646.
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-isoamyl-
β-O-benzyl-L-serine)]phosphate (30e). Yield: 20%. Rf = 0.53
(hexane/EtOAc, 1 : 9). 31P NMR (121 MHz, CDCl3): δ = 3.18,
2.87 ppm. HRMS (ESI+) calcd for C34H45N3O11P [M + H]+
702.2786, found 702.2770.
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-methoxy-
β-O-isobutyryl-L-serine)]phosphate (30f). Yield: 50%. Rf = 0.8
(CH2Cl2/MeOH, 9.5 : 0.5). 31P NMR (121 MHz, CDCl3): δ = 2.90,
2.71 ppm. HRMS (ESI−) calcd for C27H35N3O12P [M − H]−
624.1964, found 624.1969.
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-amyl-
β-O-isobutyryl-L-serine)]phosphate (30g). Yield: 85%. Rf = 0.47
(CH2Cl2/MeOH, 9.5 : 0.5). 31P NMR (121 MHz, CDCl3): δ =
2.65 ppm. HRMS (ESI−) calcd for C31H43N3O12P [M − H]−
680.2589, found 680.2593.
To
a solution of isoamyl O-isobutyryl-Boc-L-tyrosine 24
(400 mg, 0.95 mmol) in dichloromethane (5 ml) was slowly
added trifluoroacetic acid (5 ml). The resulting mixture was
stirred at room temperature for 1 hour. The reaction mixture
was evaporated to dryness under reduced pressure, and the
residue was dissolved in dichloromethane (10 ml) and washed
with a 5% sodium carbonate solution. To the organic phase,
1.25 N HCl in isopropanol (2 ml) was added. The mixture was
concentrated under reduced to yield the title compound 25 as
1
white solid (340 mg, 99%). H NMR (300 MHz, DMSO-d6): δ =
8.72 (br.s, 3H), 7.28 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H),
4.24 (m, 1H), 4.07 (m, 2H), 3.21 (m, 1H), 3.08 (m, 1H), 2.81 (m,
1H), 1.46 (m, 1H), 1.36 (m, 2H), 1.21 (d, J = 7.0 Hz, 6H), 0.83
(d, J = 6.5 Hz, 6H) ppm. 13C NMR (75 MHz, DMSO-d6): δ =
175.00, 169.17, 149.94, 132.35, 130.55, 121.87, 64.07, 53.28,
36.57, 35.51, 33.45, 24.20, 22.28, 22.24, 18.80 ppm.
General procedure for the synthesis of the 2′-C-methyl-2′,3′-O-
isopropyliden-uridine 5′-phosphoramidate analogues (30a–h)
Step 1
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenyl(α-isoamyl-
A solution/suspension of the appropriate amino acid ester β-O-isobutyryl-L-serine)]phosphate (30h). Yield: 87%. Rf = 0.67
hydrochloride (3.5 equiv.) in anhydrous dichloromethane (CH2Cl2/MeOH, 9.5 : 0.5). 31P NMR (121 MHz, CDCl3): δ = 3.20,
was cooled to −15 °C. Phenyl dichlorophosphate (2.5 equiv.) 3.00 ppm. HRMS (ESI−) calcd for C31H43N3O12P [M − H]−
was added slowly. After 10 minutes,
a
solution of 680.2589, found 680.2600.
N-methylimidazole (10 equiv.) in dry dichloromethane was
added dropwise. The mixture was allowed to reach room temp-
erature slowly and left to stir for 10–12 h.
Step 2
General procedure for the synthesis of the 2′-C-methyl-uridine
ProTides (31a–h)
The protected phosphoramidate 30a–h was dissolved in a solu-
In a separate flask, a solution/suspension of an appropri- tion of TFA–H2O (8 : 2, 0.1 M) and was stirred at room tempera-
ately protected nucleoside (1 equiv.) in anhydrous dichloro- ture until TLC shows no starting material (typically 3–6 h). The
methane was cooled to −5 °C. With stirring, the solution reaction mixture was evaporated to dryness and coevaporated
prepared in step 1 was added slowly over a period of 1 hour, three times with toluene. The crude residue was purified by
keeping the temperature around −5 °C. The cooling bath was silicagel flash column chromatography eluting with a mixture
removed, and the reaction was left to stir at room temperature of methanol and dichloromethane in different ratios (generally
(for nearly 4–6 h) until TLC indicates a reasonable amount of 2–5% methanol in dichloromethane) to obtain the title com-
product formation. The reaction mixture was then evaporated pounds 31a–h as a white solid. The overall yield of the reaction
to dryness under reduced pressure, and the residue was puri- is in the range from 65% to 85%.
fied by flash column chromatography eluting with CH2Cl2–
MeOH or EtOAc–hexane in different ratios. The overall yield of phate (31a). Yield: 77%. Rf = 0.28 (CH2Cl2/MeOH, 9.5 : 0.5).
the reaction is in the range of 30–90%.
1H NMR (500 MHz, MeOD): δ = 7.68–7.66 (2 d, 1H, H-6),
2′-C-Methyl-uridine-5′-[phenylbis(methoxy-L-glutamyl)]phos-
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenylbis(methoxy- 7.39–7.18 (a series of multiplets, 5H, OPh), 5.97, 5.96 (2 s, 1H,
L-glutamyl)]phosphate (30a). Yield: 15%. Rf = 0.39 (hexane/ H-1′), 5.65–5.61 (2 d, 1H, H-5), 4.56–4.35 (m, 2H, H-5′ & H-5″),
EtOAc, 2 : 8). 31P NMR (121 MHz, CDCl3): δ = 3.01, 2.96 ppm. 4.11–4.08 (m, 1H, H-4′), 4.00–3.93 (m, 1H, H-α-Glu), 3.80–3.77
HRMS (ESI-) calcd for C26H33N3O12P [M − H]− 610.1807, found (1H, H-3′), 3.69, 3.66, 3.62 (4 s, 6H, OMe), 2.44–2.26 (m, 2H, H-
610.1806.
β-Glu), 2.12–1.81 (m, 2H, H-γ-Glu), 1.16, 1.13 (2 s, 3H, –CH3-2′)
2′-C-Methyl-2′,3′-O-isopropyliden-uridine-5′-[phenylbis(isoamyl- ppm. 13C NMR (125 MHz, MeOD): δ = 175.5, 175.2 (–CO-α &
L-glutamyl)]phosphate (30b). Yield: 75%. Rf = 0.59 (CH2Cl2/ –CO-β), 166.7 (C-4), 153.2, 153.1, 153.0 (C-2 & phenyl C), 142.8,
MeOH, 9.5 : 0.5). 31P NMR (121 MHz, CDCl3): δ = 3.06, 142.7 (C-6), 131.8, 131.7 (phenyl C), 127.2, 127.1 (phenyl C),
3.04 ppm. HRMS (ESI+) calcd for C34H51N3O12P [M + H]+ 122.2–122.1 (phenyl C), 103.7 (C-5), 94.4, 94.3 (C-1′), 82.5, 82.4
724.3205, found 724.3226.
2
(C-4′), 80.5 (C-2′), 74.8, 74.6 (C-3′), 67.3 (d, JCP = 5.8 Hz, C-5′),
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2
66.8 (d, JCP = 4.9 Hz, C-5′), 56.2, 56.0 (C-α-Glu), 53.7, 53.0 CH2 ), 103.8, 103.7 (C-5), 94.3, 94.2 (C-1′), 82.4, 82.3 (C-4′),
3
(OMe), 31.5, 31.4 (C-γ-Glu), 30.8–30.6 (C-β-Glu), 21.0 (CH3-2′) 80.5, 80.4 (C-2′), 75.0 (
Ph), 74.8, 74.5 (C-3′), 73.3 (d, JCP =
ppm. 31P NMR (202 MHz, MeOD): δ = 3.95 ppm. HRMS (ESI−) 5.13 Hz, C-β-Ser), 73.2 (d, JCP = 6.23 Hz, C-β-Ser), 67.6
3
calcd for C23H29N3O12P [M − H]− 570.1494, found 570.1505.
(–O
CH2CH2CH2CH3), 67.2 (d, JCP = 5.06 Hz, C-5′), 66.7 (d,
4.70 Hz, C-5′), 57.3, 57.1 (C-α-Ser), 30.2–29.9
2
2′-C-Methyl-uridine-5′-[phenylbis(isoamyl-L-glutamyl)]phos- 2JCP
=
1
phate (31b). Yield: 66%. Rf = 0.39 (CH2Cl2/MeOH, 9.5 : 0.5). H (–OCH2
CH2CH3), 24.2 (–OCH2CH2CH2
CH3), 21.1
NMR (500 MHz, MeOD): δ = 7.68–7.66 (2 d, 1H, H-6), 7.38–7.18 (CH3-2′), 15.2 (–OCH2CH2CH2CH2
)
ppm. 31P NMR
(a series of multiplets, 5H, OPh), 5.97, 5.96 (2 s, 1H, H-1′), (202 MHz, MeOD): δ = 4.17 and 3.89 ppm. HRMS (ESI−) calcd
5.66–5.63 (2 d, 1H, H-5), 4.58–4.36 (m, 2H, H-5′ & H-5″), for C31H39N3O11P [M − H]− 660.2327, found 660.2322.
4.18–4.02 (m, 5H, H-4′ & –O
CH2CH(CH3)2), 4.00–3.92 (m,
2′-C-Methyl-uridine-5′-[ phenyl(α-isoamyl-β-O-benzyl-L-
1H, H-α-Glu), 3.80–3.76 (d, 1H, H-3′), 2.45–2.26 (m, 2H, serine)]phosphate (31e). Yield: 80%; Rf = 0.15 (CH2Cl2/MeOH,
H-γ-Glu), 2.09–1.81 (m, 2H, H-β-Glu), 1.70–1.61 (m, 2H, 9.7 : 0.3); 1H NMR (500 MHz, MeOD): δ = 7.70–7.65 (2 d, 1H,
–OCH2CH2 (CH3)2), 1.53–1.45 (m, 4H, –OCH2
1.16, 1.13 (2 s, 3H, –CH3-2′), 0.92–0.90 (m, 12H, –OCH2CH2CH 5.98, 5.95 (2 s, 1H, H-1′), 5.65–5.60 (2 d, 1H, H-5), 4.61–4.34
)2) ppm. 13C NMR (125 MHz, MeOD): δ = 175.1, 175.0, (m, 4H, H-5′, H-5″
Ph), 4.21–4.04 (m, 4H, H-4′,
174.9, 174.8 (–CO-α & –CO-β), 166.6 (C-4), 153.1, 153.0, 152.9 –O CH2CH(CH3)2 & H-α-Ser), 3.81–3.52 (m, 3H, H-3′ & H-
(C-2 & phenyl C), 142.7, 142.6 (C-6), 131.8, 131.7 (phenyl C), β-Ser), 1.68–1.59 (m, 1H, –OCH2CH2 (CH3)2), 1.51–1.44 (m,
CH(CH3)2), H-6), 7.36–7.17 (a series of multiplets, 10H, OPh & CH2 ),
(
&
127.2 (phenyl C), 122.2–122.1 (phenyl C), 103.7 (C-5), 94.4, 2H, –OCH2
CH(CH3)2), 1.13 (s, 3H, –CH3-2′), 0.88–0.86 (m,
94.2 (C-1′), 82.4, 82.3 (C-4′), 80.5, 80.4 (C-2′), 74.8, 74.5 (C-3′), 6H, –OCH2CH2CH(
)2) ppm. 13C NMR (125 MHz, MeOD):
2
2
3
3
67.3 (d, JCP = 5.2 Hz, C-5′), 66.7 (d, JCP = 5.2 Hz, C-5′), 65.9, δ = 173.5 (d, JCP = 4.96 Hz, –CO-α), 173.2 (d, JCP = 6.34 Hz,
65.2, 65.1 (–O
39.3–39.2 (–OCH2
CH2CH(CH3)2), 56.3, 56.1 (C-α-Glu), –CO-α), 166.6 (C-4), 153.1–152.9 (C-2 & phenyl C), 142.8, 142.6
CH(CH3)2), 31.8–31.7 (C-γ-Glu), 31.0–30.7 (C-6), 140.0, 139.9 (CH2 ), 131.8–122.2 (phenyl C & CH2 ),
(C-β-Glu), 27.1, 27.0 (–OCH2CH2 (CH3)2), 23.7–23.6 103.8, 103.7 (C-5), 94.3, 94.2 (C-1′), 82.4 (C-4′), 80.5, 80.4 (C-2′),
3
(–OCH2CH2CH(
)2), 21.1 (CH3-2′) ppm. 31P NMR (202 MHz, 75.1 (
Ph), 74.8, 74.6 (C-3′), 73.3 (d, JCP = 5.40 Hz, C-β-Ser),
3
2
MeOD): δ = 3.94 and 3.90 ppm. HRMS (ESI−) calcd for 73.2 (d, JCP = 6.48 Hz, C-β-Ser), 67.3 (d, JCP = 4.83 Hz, C-5′),
C31H45N3O12P [M − H]− 682.2746, found 682.2753.
66.7 (d, JCP = 4.83 Hz, C-5′), 66.0 (–O
CH2CH(CH3)2),
CH(CH3)2), 26.9
)2), 21.0 (CH3-
2
2′-C-Methyl-uridine-5′-[ phenyl(α-methoxy-β-O-benzyl-L- 57.3, 57.1 (C-α-Ser), 39.3, 39.2 (–OCH2
serine)]phosphate (31c). Yield: 87%. Rf = 0.38 (CH2Cl2/MeOH, (–OCH2CH2 (CH3)2), 23.6 (–OCH2CH2CH(
9.5 : 0.5). 1H NMR (500 MHz, MeOD): δ = 7.70–7.65 (2 d, 1H, 2′) ppm. 31P NMR (202 MHz, MeOD): δ = 4.15 and 3.88 ppm.
H-6), 7.36–7.17 (a series of multiplets, 10H, OPh & CH2 ), HRMS (ESI+) calcd for C31H41N3O11P [M + H]+ 662.2473, found
5.97, 5.95 (2 s, 1H, H-1′), 5.65–5.59 (2 d, 1H, H-5), 4.60–4.56 662.2488.
(m, 4H, H-5′, H-5″ &
Ph), 4.17–4.05 (m, 1H, H-4′ & H-α-Ser),
2′-C-Methyl-uridine-5′-[phenyl(α-methoxy-β-O-isobutyryl-L-
3.81–3.52 (m, 6H, H-3′, H-β-Ser & OCH3-Ser), 1.13 (s, 3H, –CH3- serine)]phosphate (31f). Yield: 75%. Rf = 0.46 (CH2Cl2/MeOH,
3
2′) ppm. 13C NMR (125 MHz, MeOD): δ = 173.9 (d, JCP = 4.77 9.5 : 0.5). 1H NMR (500 MHz, MeOD): δ = 7.67–7.65 (2 d, 1H,
3
Hz, –CO-α), 173.6 (d, JCP = 6.12 Hz, –CO-α), 166.7 (C-4), H-6), 7.39–7.19 (a series of multiplets, 5H, OPh), 5.97, 5.96 (2
153.1–152.9 (C-2 & phenyl C), 142.8, 142.7 (C-6), 140.0, 139.9 s, 1H, H-1′), 5.64–5.60 (2 d, 1H, H-5), 4.61–4.36 (m, 2H, H-5′,
(CH2 ), 131.7–122.2 (phenyl C & CH2 ), 103.7 (C-5), 94.3, H-5″), 4.31–4.08 (m, 4H, H-4′, H-α-Ser & H-β-Ser), 3.80–3.78
94.2 (C-1′), 82.4 (C-4′), 80.5, 80.4 (C-2′), 75.0 (
Ph), 74.8, 74.6 (1H, H-3′), 3.72, 3.68 (2 s, 3H, –OCH3-Ser), 2.55–2.47 (m, 1H,
3
3
(C-3′), 73.2 (d, JCP = 5.3 Hz, C-β-Ser), 73.0 (d, JCP = 6.32 Hz,
–
(CH3)2), 1.15–1.09 (m, 9H, –CH3-2′ & –CH(
)2) ppm. 13C
2
2
C-β-Ser), 67.2 (d, JCP = 5.21 Hz, C-5′), 66.7 (d, JCP = 4.91 Hz, NMR (125 MHz, MeOD): δ = 178.9 (– CH(CH3)2), 173.1 (d,
C-5′), 57.3, 57.1 (C-α-Ser), 53.8 (OCH3-Ser), 21.0 (CH3-2′) ppm. 3JCP = 5.07 Hz, –CO-α), 172.9 (d, JCP = 5.77 Hz, –CO-α), 166.7
3
31P NMR (202 MHz, MeOD): δ = 4.14 and 3.91. HRMS (ESI+) (C-4), 153.1–152.9 (C-2 & phenyl C), 142.8, 142.7 (C-6),
calcd for C27H33N3O11P [M + H]+ 606.1847, found 606.1859.
131.8–122.1 (phenyl C), 103.7 (C-5), 94.5, 94.3 (C-1′), 82.4, 82.3
2′-C-Methyl-uridine-5′-[phenyl(α-amyl-β-O-benzyl-L-serine)] (C-4′), 80.5 (C-2′), 74.9, 74.6 (C-3′), 67.4–66.8 (C-β-Ser & C-5′),
phosphate (31d). Yield: 77%. Rf = 0.53 (CH2Cl2/MeOH, 56.1, 56.0 (C-α-Ser), 54.0, 53.9 (OCH3-Ser), 35.8 (– (CH3)2),
9.5 : 0.5). 1H NMR (500 MHz, MeOD): δ = 7.70–7.64 (2 d, 1H, 21.1–20.0 (CH3-2′ & –CH(
)2) ppm. 31P NMR (202 MHz,
H-6), 7.36–7.17 (a series of multiplets, 10H, OPh & CH2 ), MeOD): δ = 3.90 and 3.65 ppm. HRMS (ESI+) calcd for
5.98, 5.96 (2 s, 1H, H-1′), 5.65–5.60 (2 d, 1H, H-5), 4.60–4.35 C24H32N3O12P [M + H]+ 586.1796, found 586.1793.
(m, 4H, H-5′, H-5″
–O CH2CH2CH2CH3 & H-α-Ser), 3.81–3.52 (m, 3H, H-3′ & serine)]phosphate (31g). Yield: 75%. Rf = 0.36 (CH2Cl2/MeOH,
H-β-Ser), 1.60–1.55 (m, 2H, –OCH2
&
Ph), 4.15–4.03 (m, 4H, H-4′,
2′-C-Methyl-uridine-5′-[ phenyl(α-amyl-β-O-isobutyryl-L-
1
CH2CH2CH3), 1.30–1.27 9.5 : 0.5). H NMR (500 MHz, MeOD): δ = 7.67–7.64 (1H, H-6),
(m, 4H, –OCH2CH2
0.88–0.85 (–OCH2CH2CH2CH2
MeOD): δ = 173.5 (d, JCP = 4.91 Hz, –CO-α), 173.2 (d, JCP
CH3), 1.13 (s, 3H, –CH3-2′), 7.38–7.18 (a series of multiplets, 5H, OPh), 5.98, 5.97 (2 s, 1H,
) ppm. 13C NMR (125 MHz, H-1′), 5.66–5.62 (1H, H-5), 4.62–4.38 (m, 2H, H-5′, H-5″),
3
3
=
4.34–4.05 (m, 6H, H-4′, –O
CH2CH2CH2CH3, H-α-Ser & H-
6.05 Hz, –CO-α), 166.6 (C-4), 153.1–152.9 (C-2 & phenyl C), β-Ser), 3.81, 3.79 (1H, H-3′), 2.54–2.47 (m, 1H, – (CH3)2 of
142.7, 142.6 (C-6), 139.9 (CH2 ), 131.8–122.2 (phenyl C & iBu), 1.64–1.59 (m, 2H, –OCH2 CH2CH2CH3), 1.33–1.30
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(m, 4H, –OCH2CH2
–CH( of iBu), 0.90–0.88 (t, 3H, –OCH2CH2CH2CH2
13C NMR (125 MHz, MeOD): δ = 178.8, 178.9 (– CH(CH3)2 of
CH3), 1.15–1.09 (m, 9H, –CH3-2′ & General procedure for the synthesis of the 2′-C-methyl-uridine
)
2
); ProTides (31i–k)
3
3
iBu), 172.6 (d, JCP = 5.39 Hz, –CO-α), 172.4 (d, JCP = 6.18 Hz, A solution of aforementioned 2′,3′-isopropylidene-protected
–CO-α), 166.6 (C-4), 153.1–152.8 (C-2 & phenyl C), 142.8, product in a mixture of TFA/H2O (4/1, 5 ml) was stirred at
142.6 (C-6), 131.8–122.1 (phenyl C), 103.8, 103.7 (C-5), room temperature for 2 hours. After concentration under the
94.4, 94.2 (C-1′), 82.4, 82.3 (C-4′), 80.5, 80.4 (C-2′), 74.9, reduced pressure, the residue was purified by silica gel flash
74.6 (C-3′), 67.8–66.8 (C-β-Ser, C-5′ & –O
CH2CH2CH2CH3), chromatography (the mobile phase being a mixture of metha-
56.2, 56.0 (C-α-Ser), 35.7 (– (CH3)2 of iBu), 30.2– nol in dichloromethane, in a ratio gradually ranging from
29.9 (–OCH2
21.1–20.0
CH2CH3), 24.1 (–OCH2CH2CH2
–CH( of iBu),
) ppm. 31P NMR (202 MHz, MeOD): δ = phosphate (31i). This compound was isolated in 46% yield as a
CH3), 0–20% MeOH) to yield the title compounds as white solids.
15.1 2′-C-Methyl-uridine-5′-[phenyl(isoamyl-O-benzyl-L-threonine)]
(CH3-2′
&
)
2
(–OCH2CH2CH2CH2
3.92 and 3.64 ppm. HRMS (ESI+) calcd for C28H41N3O12P white solid. 1H NMR (300 MHz, DMSO-d6): δ = 11.40 (br s, 1H),
[M + H]+ 642.2422, found 642.2429.
7.60 (m, 1H), 7.28 (m, 10H), 5.82 (m, 2H), 5.45 (m, 2H),
2′-C-Methyl-uridine-5′-[phenyl(α-isoamyl-β-O-isobutyryl-L- 5.25 (s, 2H), 4.44 (m, 4H), 3.97 (m, 5H), 3.65 (m, 1H), 1.60
serine)]phosphate (31h). Yield: 65%. Rf = 0.28 (CH2Cl2/MeOH, (m, 1H), 1.40 (m, 2H), 1.16 (m, 3H), 1.00 (m, 3H), 0.80 (m, 6H)
1
9.5 : 0.5). H NMR (500 MHz, MeOD): δ = 7.67–7.64 (1H, H-6), ppm. 13C NMR (150 MHz, DMSO-d6): δ = 171.36, 171.11,
7.38–7.18 (a series of multiplets, 5H, OPh), 5.98, 5.96 (2 s, 1H, 171.09, 163.00, 162.97, 150.94, 150.90, 150.86, 150.67, 129.72,
H-1′), 5.65–5.62 (1H, H-5), 4.62–4.37 (m, 2H, H-5′, H-5″), 129.68, 128.22, 127.59, 127.49, 124.71, 124.64, 120.28, 120.25,
4.33–4.09 (m, 6H, H-4′, –O
CH2CH(CH3)2, H-α-Ser & H- 120.21, 101.83, 101.79, 91.54, 79.98, 79.93, 78.02, 77.95, 75.35,
β-Ser), 3.80, 3.79 (1H, H-3′), 2.55–2.47 (m, 1H, – (CH3)2 of 75.30, 75.25, 72.60, 72.44, 70.12, 65.38, 64.72, 63.21, 63.17,
iBu), 1.69–1.61 (m, 1H, –OCH2CH2 (CH3)2), 1.55–1.47 (m, 59.07, 59.04, 24.40, 22.34, 22.28, 22.26, 19.96, 15.91,
2H, –OCH2
of iBu), 0.91–0.89 (m, 6H, –OCH2CH2CH(
CH(CH3)2), 1.15–1.09 (m, 9H, –CH3-2′ & –CH 15.70 ppm. 31P NMR (202 MHz, DMSO-d6): δ = 4.91, 4.83 ppm.
(
)
)2) ppm. HRMS (ES+) calcd for C32H42N3O11P [M + H]+ 676.2630, found
13C NMR (125 MHz, MeOD): δ = 178.9, 178.8 (– CH(CH3)2 of 676.2643.
2
3
3
iBu), 172.6 (d, JCP = 5.26 Hz, –CO-α), 172.4 (d, JCP = 6.07 Hz,
2′-C-Methyl-uridine-5′-[phenyl(isoamyl-O-isobutyryl-L-threo-
–CO-α), 166.6 (C-4), 153.1–152.9 (C-2 & phenyl C), 142.8, 142.6 nine)]phosphate (31j). This compound was isolated in 49%
1
(C-6), 131.8–122.1 (phenyl C), 103.8, 103.7 (C-5), 94.4, 94.2 yield as a white solid. H NMR (300 MHz, CDCl3): δ = 9.78 (br
(C-1′), 82.4, 82.3 (C-4′), 80.5, 80.4 (C-2′), 74.9, 74.6 (C-3′), s, 1H), 7.55 & 7.48 (d, J = 8.1 Hz, 1H), 7.20–7.35 (m, 5H), 6.00
67.5–66.3 (C-β-Ser, C-5′ & –O
α-Ser), 39.2 (–OCH2 CH(CH3)2), 35.8, 35.7 (– (CH3)2 of (m, 2H), 4.12 (m, 5H), 3.78 (m, 1H), 2.48 (m, 1H), 1.65 (m, 1H),
iBu), 26.9 (–OCH2CH2 (CH3)2), 23.6 (–OCH2CH2CH(
)2), 1.49 (m, 2H), 1.13 (m, 12H), 0.91 (m, 6H) ppm. 13C NMR
21.1–20.0 (CH3-2′ –CH(
of iBu) ppm. 31P NMR (150 MHz, DMSO-d6): δ = 175.34, 175.25, 170.54, 170.26,
CH2CH(CH3)2), 56.2, 56.1 (C- (d, J = 7.8 Hz, 1H), 5.67 (d, J = 8.1 Hz, 1H), 5.32 (m, 1H), 4.54
&
)
2
(202 MHz, MeOD): δ = 3.91 and 3.64 ppm. HRMS (ESI+) calcd 162.93, 162.90, 150.87, 150.84, 150.63, 140.12, 129.71, 129.28,
for C28H41N3O12P [M + H]+ 642.2422, found 642.2440.
129.01, 124.73, 124.66, 120.25, 120.23, 120.16, 120.13, 101.78,
101.73, 91.52, 79.92, 79.86, 77.94, 77.89, 72.65, 72.51, 70.28,
70.24, 70.16, 70.12, 65.52, 64.90, 63.40, 63.35, 58.12, 57.95,
36.82, 33.28, 30.78, 24.29, 22.32, 22.19, 19.94, 18.71, 18.66,
General procedure for the synthesis of 2′-C-methyl-2′,3′-O-
isopropyliden-uridine 5′-phosphoramidate analogues (30i–k)
To a mixture of the appropriate amino acid ester hydrochloride 16.20, 16.04 ppm. 31P NMR (202 MHz, DMSO-d6): δ = 4.21,
salt (1.0 mmol) in anhydrous dichloromethane (10 ml) was 3.64 ppm. HRMS (ES+) calcd for C29H42N3O12P [M + H]+
added dichlorophenyl phosphate (165 µl, 1.1 mmol) and 656.2579, found 656.2584.
N-methylimidazole (420 µl, 5 mmol) at −40 °C. The mixture
2′-C-Methyl-uridine-5′-[phenyl(isoamyl-O-isobutyryl-L-tyro-
was stirred and allowed to warm to room temperature. The stir- sine)]phosphate (31k). This compound was isolated in 50%
1
ring was continued for another 3 hours. The mixture was then yield as a white solid. H NMR (300 MHz, DMSO-d6): δ = 11.42
cooled to −40 °C again, and 2′,3′-isopropylidene-2′-C-methyl- (br s, 1H), 7.50 (m, 1H), 6.90–7.33 (m, 9H), 6.30 (m, 1H), 5.87
uridine (149 mg, 0.5 mmol) was added. The mixture was (m, 1H), 5.43 (m, 2H), 5.24 (m, 1H), 4.00 (m, 6H), 3.57 (m, 1H),
stirred and warmed to room temperature, and stirring was con- 2.93 (m, 1H), 2.80 (m, 2H), 1.6 (m, 1H), 1.46 (m, 2H), 1.22 (d, J
tinued till all starting material disappeared according to TLC = 7.0 Hz, 6H), 0.98 (d, J = 6.8 Hz, 3H), 0.80 (m, 6H) ppm. 13C
analysis. The reaction mixture was then evaporated to dryness NMR (150 MHz, DMSO-d6): δ = 175.07, 172.36, 162.94, 162.88,
under reduced pressure, and the residue was purified by flash 150.78, 150.73, 150.65, 149.44, 140.75, 139.83, 134.39, 134.37,
column chromatography (using a mixture of methanol in di- 130.34, 129.74, 129.72, 124.63, 124.56, 121.53, 120.07, 120.04,
chloromethane as mobile phase, in a gradient gradually 119.78, 119.75, 101.78, 101.72, 91.58, 79.90, 79.84, 79.78,
ranging from 0 to 10% methanol) to yield the 2′-C-methyl-2′,3′- 77.97, 77.86, 72.70, 72.35, 65.25, 64.25, 63.06, 56.27, 56.17,
O-isopropyliden-uridine-5′-phosphoramidate analogues 30i–k, 24.37, 24.33, 22.32, 22.28, 22.25, 19.94, 18.80 ppm. 31P NMR
which were used as such in next step, without any further (202 MHz, DMSO-d6): δ = 3.57, 3.05 ppm. HRMS (ES+) calcd
structural identification.
for C34H44N3O12P [M + H]+ 718.2735, found 718.2748.
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Biological evaluation
Metabolic stability assay (human liver microsomes)
The stability assay was contracted and carried out by Eurofins
(Eurofins ref. 0607). Pooled liver microsomes are preincubated
with NADPH-regenerating system (1 mM NADP, 5 mM G6P,
and 1 U mL−1 G6PDHase) in phosphate buffer (pH 7.4) con-
taining 3 mM MgCl2 and 1 mM EDTA in a 2 mL-block 96-well
plate for 10 min in a 37 °C shaking water bath. The reaction
was initiated by adding the test compound (final concen-
tration 0.1 μM) and incubated in a final volume of 700 μL in
the 37 °C shaking water-bath. At 0, 15, 30, 45, and 60 min after
reaction initiation, 100 μL of the incubation mixture is trans-
ferred to 100 μL of acetonitrile/methanol (50/50, v/v) in a
0.8 mL V-bottom 96-well plate. Samples are then mixed on a
plate shaker for 5 minutes and centrifuged at 2550g for 15 min
at room temperature. Each supernatant is transferred to a
clean cluster tube, followed by HPLC MS/MS analysis on a
Thermo Electron triplequadrupole system.
Primary HCV antiviral assay. For the primary HCV antiviral
screening assay, compounds were evaluated for anti-HCV
activity using a luciferase (Luc) reporter gene endpoint incor-
porated within a subgenomic HCV replicon. The Luc reporter
is used as an indirect measure of HCV replication as its activity
is directly proportional to HCV RNA levels. Assessment of cyto-
toxicity is conducted in parallel. Drug stocks are prepared in
DMSO and are diluted with tissue culture medium to the
desired high-test concentration. For each assay, the com-
pounds are then further diluted in tissue culture medium as
required. After incubation, the cells are processed to derive
EC50 and EC90 (compound concentrations reducing HCV repli-
cation by 50% and 90% respectively) values. In addition, CC50
(concentration decreasing cell viability by 50%) and SI50 (CC50/
EC50) values are determined and reported.
HCV replicon antiviral evaluation protocol. The genotype 1b
(Con1 strain) HCV replicon was used for primary screening
and uses the cell line ET (luc-ubi-neo/ET), which is an Acknowledgements
Huh7 human hepatoma cell line that contains an HCV repli-
This project has been funded in part with Federal funds from
con with a stable luciferase (Luc) reporter and three cell
culture-adaptive mutations.15 Recombinant human interferon
alpha-2b (rIFNα-2b) and the NS5B inhibitor Sofosbuvir is
included in each run as a positive control. Briefly, sub-conflu-
ent cultures of the ET cell line are plated into 96-well plates
that are dedicated for the analysis of cytotoxicity or antiviral
activity and the next day compounds are added to the appro-
priate wells. Cells are processed 72 h later when the cells are
still sub-confluent. Compounds are evaluated using 6 half-log
serial dilutions to derive where applicable EC50, EC90, CC50
and SI50 values. HCV replicon levels are assessed as HCV repli-
con-derived Luc activity (Steady-Glo® Luciferase Assay System,
Promega) and compound cytotoxicity is assessed on replicate
plates using a fluorometric method for determining cell viabi-
lity (CytoTox-ONE™ Homogeneous Membrane Integrity Assay,
Promega).
The antiviral and cytotoxicity data are the result of one
assay in which the compounds were tested in dose–response
with each concentration evaluated in quadruplicate. The
average values at each concentration were then graphed and
used to generate the reported EC50 and EC90 values. The %CV
for quadruplicate values are generally below 15%, and the
assay is robust and highly reproducible.
Secondary HCV antiviral assay. A qRT-PCR (TaqMan) end-
point is used to directly measure HCV RNA copies to confirm
compound activity. In the secondary assay, instead of assessing
luciferase activity at the assay endpoint, the cells are processed
for intracellular RNA extraction with an RNeasy 96 kit (Qiagen).
The level of HCV RNA is determined by qRT-PCR/TaqMan assay
using TaqMan® One-Step RT-PCR Master Mix Reagents
(Applied Biosystems, Foster City, CA) and an ABI Prism 7900
sequence detection system (Applied Biosystems). Compound
cytotoxic effects are measured from the same plate/samples
with TaqMan® Ribosomal RNA Control Reagents (Applied
Biosystems) as an indication of viable cell numbers.
the Division of Microbiology and Infectious Diseases, National
Institute of Allergy and infectious Diseases, National Institutes
of Health, Department of Health and Human Services, under
Contract No. HHSN272201100013I.
References
1 L. P. Jordheim, D. Durantel, F. Zoulim and C. Dumontet,
Nat. Rev. Drug Discovery, 2013, 12, 447.
2 U. Pradere, E. C. Garnier-Amblard, S. J. Coats, F. Amblard
and R. F. Schinazi, Chem. Rev., 2014, 114, 9154.
3 Y. Mehellou, J. Balzarini and C. McGuigan, ChemMedChem,
2009, 4, 1779.
4 M. J. Sofia, D. Bao, W. Chang, J. Du, D. Nagarathnam,
S. Rachakonda, G. Reddy, B. S. Ross, P. Wang, H. Zhang,
S. Bansal, C. Espiritu, M. Keilman, A. M. Lam,
H. M. Micolochick Steuer, C. Niu, M. J. Otto and
P. A. Furman, J. Med. Chem., 2010, 53, 7202.
5 A. S. Ray, M. W. Fordyce and M. J. Hitchcock, Antiviral Res.,
2016, 125, 63.
6 T. K. Warren, R. Jordan, M. K. Lo, A. S. Ray, R. L. Mackman,
V. Soloveva, D. Siegel, M. Perron, R. Bannister, H. C. Hui,
N. Larson, R. Strickley, J. Wells, K. S. Stuthman, S. A. Van
Tongeren, N. L. Garza, G. Donnelly, A. C. Shurtleff,
C. J. Retterer, D. Gharaibeh, R. Zamani, T. Kenny,
B. P. Eaton, E. Grimes, L. S. Welch, L. Gomba,
C. L. Wilhelmsen, D. K. Nichols, J. E. Nuss, E. R. Nagle,
J. R. Kugelman, G. Palacios, E. Doerffler, S. Neville, E. Carra,
M. O. Clarke, L. Zhang, W. Lew, B. Ross, Q. Wang, K. Chun,
L. Wolfe, D. Babusis, Y. Park, K. M. Stray, I. Trancheva,
J. Y. Feng, O. Barauskas, Y. Xu, P. Wong, M. R. Braun,
M. Flint, L. K. McMullan, S. Chen, R. Fearns,
S. Swaminathan, D. L. Mayers, C. F. Spiropoulou, W. A. Lee,
S. T. Nichol, T. Cihlar and S. Bavari, Nature, 2016, 531, 381.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Paper
7 D. Cahard, C. McGuigan and J. Balzarini, Mini-Rev. Med. 11 D. Saboulard, L. Naesens, D. Cahard, A. Salgado,
Chem., 2004, 4, 371.
R. Pathirana, S. Velazquez, C. Mcguigan, E. De Clercq and
8 M. Slusarczyk, M. H. Lopez, J. Balzarini, M. Mason,
J. Balzarini, Mol. Pharmacol., 1999, 56, 693.
W. G. Jiang, S. Blagden, E. Thompson, E. Ghazaly and 12 M. Maiti, M. Maiti, J. Rozenski, S. De Jonghe and
C. McGuigan, J. Med. Chem., 2014, 57, 1531. P. Herdewijn, Org. Biomol. Chem., 2015, 13, 5158.
9 C. McGuigan, P. Murziani, M. Slusarczyk, B. Gonczy, 13 J. Li and Y. Sha, Molecules, 2008, 13, 1111.
J. Vande Voorde, S. Liekens and J. Balzarini, J. Med. Chem., 14 E. Groaz and P. Herdewijn, Curr. Med. Chem., 2015, 22,
2011, 54, 7247.
3980.
10 C. McGuigan, A. Salgado, C. Yarnold, T. Y. Harries, E. De 15 T. Pietschmann, V. Lohmann, A. Kaul, N. Krieger, G. Rinck,
Clercq and J. Balzarini, Antiviral Chem. Chemother., 1996, 7,
184.
G. Rutter, D. Strand and R. Bartenschlager, J. Virol., 2002,
76, 4008.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.