Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as Dual Inhibitors of HIV and HBV Replication

Article abstract of DOI:10.1021/acs.jmedchem.6b01260

The synthesis of four l-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displ

Full text of DOI:10.1021/acs.jmedchem.6b01260

Article
pubs.acs.org/jmc
Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as
Dual Inhibitors of HIV and HBV Replication
Chao Liu,† Shrinivas G. Dumbre,† Christophe Pannecouque,‡ Chunsheng Huang,§ Roger G. Ptak,§
Michael G. Murray,§ Steven De Jonghe,† and Piet Herdewijn*,†
†Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
‡Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
§Infectious Disease Research, Southern Research, 431 Aviation Way, Frederick, Maryland 21701, United States
S
* Supporting Information
ABSTRACT: The synthesis of four L-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine,
cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent
antiviral activity displaying an EC50 of 4.69 μM against HIV-1 and an EC50 value of 0.5 μM against HBV, whereas completely
lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led
to a boost in antiviral potency. The most potent congeners were a L-aspartic acid diisoamyl ester phenoxy prodrug and a L-
phenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low
nanomolar range and selectivity indexes of more than 300.
treatment of hepatitis B (HBV) infections.4 The substrate
binding site of the reverse transcriptase enzyme of the human
immunodeficiency virus (HIV) has proved to be an attractive
INTRODUCTION
■
The currently licensed antiviral medications are based on a
wide variety of chemical structures (e.g., heterocycles,
target as seven nucleoside analogues have been licensed as anti-
peptidomimetics). However, nucleosides are especially attrac-
HIV drugs.5 Several of these nucleoside analogues (lamivudine
tive as antiviral drugs for a number of reasons.1 Nucleoside
and emtricitabine) received also marketing approval for the
analogues target the viral polymerase active sites, which are
treatment of HBV infections.4 Ribavirin and sofosbuvir are the
validated as targets for antiviral drug discovery. Moreover, the
only marketed nucleoside analogues for RNA viral infections.
active sites of these polymerases are often conserved and
Ribavirin is being used for the treatment of respiratory syncytial
nucleoside analogues have a high barrier of resistance
emergence compared to other classes of inhibitors. In addition,
nucleoside based inhibitors usually display equivalent potency
against different serotypes/genotypes of a particular virus and
virus (RSV) infections and in combination with interferon-α for
the treatment of hepatitis C (HCV) infections. It is also
licensed for the use in treatment of Lassa virus infections.6
Sofosbuvir is an uridine nucleotide analogue inhibitor
even related viruses and are therefore excellent candidates for
(developed as a phosphoramidate prodrug) of the HCV
the development of broad-spectrum antivirals.
At present, all antiviral agents available for the treatment of
NS5B RNA polymerase.7
The antiviral activity of these “classical” nucleosides depends
upon their intracellular metabolism within virus-infected cells to
form sequentially the mono-, di-, and triphosphates. It is these
herpesvirus infections are nucleoside analogues: either acyclic
guanosine analogues (acyclovir, penciclovir, ganciclovir)2 or
thymidine analogues, such as (E)-5-(2-bromovinyl)-2′-deoxy-
uridine (BVDU).3 All of these compounds target the viral DNA
polymerase. DNA polymerase inhibitors based on a nucleoside
Received: August 19, 2016
scaffold (telbivudine, entecavir) have been licensed for the
© XXXX American Chemical Society
A
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Figure 1. Examples of biologically active nucleoside phosphonates.
Figure 2. Prodrugs of nucleoside phosphonates.
nucleotides, and especially the triphosphates that are the
pharmacologically active species, as they are incorporated into a
growing DNA or RNA strand by a DNA or RNA polymerase,
resulting in chain termination or fraudulent DNA/RNA. The
first phosphorylation step leading to the formation of the
nucleoside 5′-monophosphate is commonly catalyzed by a
nucleoside kinase encoded by the host cell or the virus infecting
the host cell. Conversion of the nucleoside monophosphate to
the corresponding 5′-diphosphate and triphosphate is carried
out by nucleoside, nucleotide, and nucleoside diphosphate
kinases, respectively. Hence, cellular kinases, as well as virally
encoded kinases, play a vital role in the activation of nucleoside
drugs.
The first phosphorylation step in the generation of the
biologically active nucleoside triphosphate analogue is very
often inefficient. To bypass this rate-limiting step in the
conversion to the nucleoside-5′-triphosphate, nucleoside
phosphonates have been synthesized, allowing the first
phosphorylation step required for nucleoside activation to be
skipped. Nucleoside phosphonates are essentially nucleoside
monophosphate analogues, having the advantage of being
metabolically stable. Several phosphate isosteres have been
adopted to prepare nucleoside phosphonates. The phospho-
nomethoxy functionality (P−C−O) emerged as the most
promising isostere of the naturally occurring phospho-oxy-
methyl (P−O−C) moiety in nucleoside monophosphate. This
has successfully been used in the field of HIV, with the
discovery of cyclic phosphonates [5-(6-amino-purin-9-yl)-2,5-
dihydro-furan-2-yloxymethyl]-phosphonic acid (d4AP)8 and its
fluorinated counterpart 2′Fd4AP (also known as GS-9148)9
(Figure 1). The same phosphonate motif is also present in the
acyclic nucleoside phosphonates cidofovir, adefovir, and
tenofovir.10 The success of this motif is due to the fact that it
is isopolar and isosteric with the phosphate group. Hence, they
can undergo enzymatic phosphorylation that converts them
into the corresponding phosphonate diphosphates, which act as
analogues of the natural nucleoside triphosphates. Our group
previously described before a series of L-2′-deoxythreose
nucleoside phosphonate analogues, from which two congeners
showed excellent activity against HIV. Both phosphonomethox-
ydeoxythreosyl adenine (PMDTA) and phosphonomethox-
ydeoxythreosyl thymine (PMDTT) displayed low μM EC50
values against HIV-1 and HIV-2 and lacked cellular toxicity.11
Despite the favorable characteristics of the nucleoside
phosphonates (metabolic stability and bypassing of the first
phosphorylation step), the fact that they are acidic implies that
they are negatively charged at physiological pH and hence are
not able to easily penetrate the lipid-rich cell membrane, which
hampers their antiviral activity. Therefore, various prodrug or
pronucleotide approaches have been investigated to promote
the passive diffusion through the lipophilic cell membranes and
to liberate the parent nucleotide intracellularly where it can be
further phosphorylated to the pharmacologically active
species.12 The (acyloxy)alkyl esters have been successfully
used as prodrug moieties. Adefovir dipivoxil is a pivaloylox-
ymethylester prodrug, and tenofovir disoproxil fumarate is the
fumarate salt of a isopropyloxycarbonyloxymethyl prodrug.
More recently, the aryloxyphosphonoamidate prodrug ap-
proach13 has been applied to nucleoside phosphonates. A
well-known example is tenofovir alafenamide (TAF), which
recently received marketing approval as a component in
different single tablet regimens for the treatment of HIV
infected patients (Figure 2).14
Although the aforementioned PMDTA and PMDT are
endowed with promising anti-HIV activity, prodrugs of these
phosphonates have never been described in literature. In this
paper, the design and synthesis of phosphonomonoamidate and
phosphonodiamidate prodrugs of L-α-2′-deoxythreosyl phos-
phonate nucleosides are presented. In addition, to complete the
structure−activity relationship (SAR) study of deoxythreosyl
phosphonate nucleosides as antiviral agents, the synthesis of a
previously unknown guanosine containing L-2′-deoxythreose
nucleoside phosphonate and a 2′-fluoro-2′-deoxy modified
PMDTA analogue was effected. Moreover, the compounds
were subjected to a broader antiviral profiling, including HIV
and HBV.
B
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a
Scheme 1. Synthesis of the 1′,2′-Diacylated L-Threose Derivatives 3 and 6
a
Reagents and conditions: (a) TBSCl, cat. DMAP, imidazole, 0 °C to rt, overnight, 97%; (b) DIBAL-H, dry THF, −70 to −60 °C, 3 h; (c) acetic
anhydride, Et3N, dry CH2Cl2, 0 °C to rt, overnight, 73% for 2 steps; (d) acetic chloride, MeOH, rt, 2 h, 83% for 2 steps; (e) (iPrO)2POCH2OTf,
NaH, THF, −5 to 0 °C, 15 min, 87%; (f) acetic anhydride, H2SO4, CH2Cl2, rt, overnight, 83%.
a
Scheme 2. Synthesis of PMDTA and PMDTT
a
Reagents and conditions: (a) BSA, TMSOTf, CH3CN, 65 °C, 16 h for 7a (56%) and 2 h for 7b (83%); (b) Et3N·3HF, THF, rt, 24 h, 97% for 8a
and 98% for 8b; (c) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 80% for 9a and 85% for 9b; (d) NaOH, THF/MeOH/H2O, 0 °C, 20 min, 80% for
10a, LiOH, CH3CN/MeOH/H2O, 0 °C, 30 min, 85% for 10b; (e) (1) TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux,
20 min, 98% for 11a and 97% for 11b; (f) satd NH3 in MeOH, rt, overnight, 92%; (g) TMSBr, 2,6-lutidine, CH3CN, rt, overnight, 60% for 12a,
TMSI, 2,6-lutidine, CH3CN, rt, overnight, 65% for 12b.
phosphonomethyl triflate and NaH in THF to afford 3′-O-
phosphonomethylated 5 as a mixture of diastereoisomers in
87% yield. This methyl threonoside was converted into the
1′,2′-diacyl glycosyl donor 6 in 83% yield.
Synthesis of PMDTA and PMDTT. The synthesis of PMDTA
and PMDTT has been described earlier starting from 2′-O-
TBDMS-3′-O-benzoyl-L-threose lactone, using a lengthy
procedure involving multiple protection and deprotection
steps.11 The synthetic approach shown in Scheme 2 allows to
reduce the number of steps. The 1′,2′-diacylated α/β mixture 3
is a suitable glycosyl donor for the preparation of threose
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of the 1′,2′-Diacylated L-Threose
Building Blocks. 2′-O-Benzoyl-L-threonolactone 1 was synthe-
sized from L-ascorbic acid following a literature procedure
(Scheme 1).15,16 Although silylation of the 3′-hydroxyl group of
1 has been performed before using the bulky and lipophilic tert-
butyldiphenylsilyl chloride, we opted to use a tert-butyldime-
thylsilyl protecting group. DIBAL-H mediated reduction of the
lactone 2 to the corresponding lactol was followed by
acetylation, affording key intermediate 3 as a mixture of α/β
anomers in 73% yield.17 To introduce a phosphonate group
early on in the synthetic sequence, lactone 2, after reduction,
was treated with methanol in the presence of acetic chloride,
furnishing methyl threonoside 4 in 83% yield. The
phosphonate function was then introduced using diisopropyl-
nucleoside phosphonates. The Vorbruggen glycosylation with
̈
silylated benzoyl adenine and thymine using trimethylsilyl
trifluoromethanesulfonate (TMSOTf) at 65 °C in acetonitrile
gave compounds 7a and 7b in 56% and 83% yield, respectively.
Deprotection of the TBS group by treatment with Et3N·3HF in
C
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a
Scheme 3. Synthesis of PMDTC
a
Reagents and conditions: (a) BSA, TMSOTf, CH3CN, 70 °C, 1.5 h, 84%; (b) Et3N·3HF, THF, rt, 24 h, 97% for 14 and 79% for 20; (c)
(iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 45 min, 72%; (d) BSA, TMSOTf, CH3CN, 0 °C, overnight, 40%; (e) NaOH, THF/MeOH/H2O, 0
°C, 20 min, 80% for 16 and 71% for 18; (f) (1) TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 70% for 17
and 91% for 19; (g) satd NH3 in MeOH, rt, overnight, 88%; (h) TMSI, 2,6-lutidine, CH3CN, rt, overnight, 48%.
THF afforded nucleosides 8a and 8b. Introduction of the
phosphonate functionality using diisopropylphosphonomethyl
triflate and NaH in THF afforded nucleosides 9a and 9b in 80%
and 85% yield, respectively. Removal of the 2′-benzoyl group
under basic conditions afforded nucleosides 10a and 10b. 2′-
Deoxygenation under standard Barton deoxygenation con-
ditions gave nucleosides 11a and 11b. The benzoyl group,
protecting the exocyclic amino group of the adenine moiety of
compound 11a, was removed with ammonia in methanol, and
hydrolysis of the phosphonate ester groups of 11b and 11c was
carried out with TMSBr or TMSI in the presence of 2,6-
lutidine at room temperature to furnish target compounds
PMDTA 12a and PMDTT 12b.
the N-alkylated product as the major compound. Therefore, an
alternative strategy, starting from phosphonomethylated L-
threose sugar 6, was envisioned. Vorbruggen glycosylation of
̈
presilylated N4-benzoylcytosine using trimethylsilyl trifluoro-
methanesulfonate (TMSOTf) as the Lewis acid at 0 °C in
anhydrous acetonitrile gave cytosine threonucleoside 15 in 40%
yield. Deprotection of the benzoyl group, followed by a Barton
deoxygenation reaction, yielded nucleoside derivative 17.
Alternatively, alkaline deprotection of the 2′-benzoyl group of
13, followed by 2′-deoxygenation, yielded the 3′-O-TBS-
protected threose nucleoside derivative 19. Fluoride-mediated
cleavage of the silyl protecting group and 3′-O-phosphonome-
thylation afforded the protected 2′-deoxy-threose cytidine
analogue 17. Finally, alkaline deprotection of the benzoyl
group and Lewis acid mediated cleavage of the phosphonate
esters afforded PMDTC (compound 22).
Synthesis of PMDTC. PMDTC has been synthesized before
in literature starting from the corresponding uridine analogue.11
We opted for the direct introduction of the cytosine nucleobase
on the threose sugar moiety, as shown in Scheme 3. Coupling
of N4-benzoylcytosine (CBz) with the protected L-threose sugar
Synthesis of PMDTG. In an initial attempt of the synthesis of
PMDTG, the N2-acetyl-O6-diphenyl-carbamoyl protected
guanine derivative (GDPCAc) was selected as a coupling partner
derivative 3 under Vorbruggen reaction conditions afforded the
̈
desired L-threose nucleoside derivative 13. Fluoride-mediated
deprotection of the 3′-hydroxyl group afforded 14. Phospho-
nomethylation of the nucleosides 14 afforded only minor
amounts of 15, as evidenced from TLC analysis, due to the low
solubility of 14 in tetrahydrofuran. However, switching the
reaction solvent to N,N-dimethylformamide led to formation of
in the Vorbruggen glycosylation condensation, yielding
̈
compound 22a in 66% yield (Scheme 4). Deprotection of
the 3′-hydroxyl group furnished intermediate 23a.18 Extensive
efforts to phosphonomethylate nucleoside 23a under various
reaction conditions (e.g., sodium hydride, silver oxide mediated
alkylation) remained unsuccessful. Similarly, Vorbruggen
̈
D
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a
Scheme 4. Synthesis of PMDTG
a
Reagents and conditions: (a) BSA, TMSOTf, toluene, 80 °C, 2 h, 66% for 22a, DBU, TMSOTf, CH3CN, 70 °C, 1.5 h, 67% for 22b; (b) Et3N·3HF,
THF, rt, 24 h, 96% for 23a and 97% for 23b; (c) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 67%; (d) 2 N NH3 in MeOH, rt, 2 h, 75%; (e) (1)
TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 68%; (f) (1) TMSBr, 2,6-lutidine, CH3CN, rt, overnight, (2)
2-mercaptoethanol, NaOMe, MeOH, reflux, 19 h, 49%.
a
Scheme 5. Synthesis of 2′-Fluoro-PMDTA
a
Reagents and conditions: (a) DMP oxidation (15% in CH2Cl2), 0 °C to rt, 1 h; (b) NaBH4, MeOH, 0 °C to rt, 1 h, 86% for two steps; (c) DAST,
pyridine, CH2Cl2, −10 °C to rt, overnight, 63%; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 24 h, 62%.
nucleosidation of the diacylated L-threose devivative 6 with
silylated N2-acetyl-O6-diphenyl-carbamoylguanine using
TMSOTf or SnCl4 as the Lewis catalyst, which was successful
for the synthesis of PMDTC, failed for the guanine-containing
congener.
phosphonate function was introduced using diisopropylphos-
phonomethyl triflate and NaH in THF to afford nucleoside 24
in 67% yield. The benzoyl group at the 2′ position was
hydrolyzed with 2 N NH3 in methanol, which upon Barton’s
reductive 2′-deoxygenation gave the 2′-deoxy nucleoside 26 in
68% yield. After the hydrolysis of the phosphonate ester
function in 26, the 6-chloro group was transformed to hydroxyl
group by refluxing with 2-mercaptoethanol and NaOMe in
methanol to afford PMDTG (compound 27) in 49% yield.
Synthesis of 2′-fluoro-PMDTA. The synthesis of 2′-deoxy-
2′-fluoro modified PMDTA analogue 31 is shown in Scheme 5.
Prior to the nucleophilic fluorination of the 2′ hydroxyl group,
Therefore, 2-amino-6-chloropurine was selected as coupling
partner in the glycosylation reaction. The Vorbruggen
̈
glycosylation of the threose derivative 3 with 2-amino-6-
chloropurine using trimethylsilyl trifluoromethanesulfonate
(TMSOTf) as the Lewis acid at 70 °C in anhydrous acetonitrile
gave compound 22b in 67% yield. The TBS group was
removed by treatment with Et3N·3HF in THF, and the
E
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a
Scheme 6. Synthesis of Prodrugs of PMDTA, PMDTT, PMDTC, and PMDTG
a
Reagents and conditions: (a) amino acid ester, PhOH, Et3N, 2,2′-dithiodipyridine, PPh3, pyridine, 13−66% for 32a−b, 33a−b, 34a, and 35a, amino
acid ester, Et3N, 2,2′-dithiodipyridine, PPh3, pyridine, 23−73% for 32c, 33c, 34d, 35c, and 35d.
epimerization of the 2′-hydroxyl of compound 10a from a
threose to an erythrose is needed. This was successfully
accomplished via Dess−Martin periodinane (DMP) oxidation
of 10a, followed by sodium borohydride reduction to afford
selectively compound 29. Fluorination of 29 was carried out
with diethylaminosulfur trifluoride (DAST) from −10 °C to rt
to furnish 2′-fluorinated nucleoside 30 in 63% yield, with
inversion of configuration. Under these reaction condition,
concomitantly, benzoyl deprotection took place. Finally,
hydrolysis of the phosphonate ester function of 30 with
TMSBr in the presence of 2,6-lutidine afforded the target
nucleoside 31.
Table 1. Anti-HIV Activity and Cytotoxicity of
Deoxythreosyl Nucleoside Phosphonates and Their
Prodrugs
a
EC50 (μM)
SI
b
HIV-1
(IIIB)
HIV-2
(ROD)
CC50
compd
(μM)
HIV-1 HIV-2
12a (PMDTA)
12b (PMDTT)
22 (PMDTC)
27 (PMDTG)
4.69
27.07
>343
>302
≥222
5.23
23.55
>343
≥315
297
≥67
11
≥60
13
>343
>302
262
>302
31
≥214.0
(2′-F-PMDTA)
Synthesis of Prodrugs of PMDTA, PMDTT, PMDTC, and
PMDTG. The phosphonomonoamidate prodrugs (32a−b,
33a−b, 34a, and 35a) were prepared from the parent
nucleoside phosphonate using 2,2′-dithiodipyridine and
triphenylphosphine as activating agents and utilizing a mixture
of phenol and the appropriate amino acid ester (Scheme 6).9
This method affords diasteromeric mixtures of compounds due
to the chirality of the phosphorus atom. The phosphonobisa-
midate prodrugs (32c, 33c, 34d, 35c, and 35d) were prepared
in a similar way, using the desired amino acid ester.9
Anti-HIV Activity. The α-L-2′-deoxythreose nucleoside
phosphonates 12a, 12b, 22, 27, and 31 were assessed for
antiviral activity using the MTT method in MT-4 cells infected
with a wild-type HIV-1 strain IIIB and a wild-type HIV-2 strain
(ROD). In parallel, cytotoxicity of the compounds in the MT-4
cell line was assessed (Table 1).19,20 Lamivudine (2′,3′-dideoxy-
3-thiacytidine, commonly called 3TC) was included as positive
control as it has dual anti-HIV (Table 1) and anti-HBV (Table
2) activity.
32a
32b
32c
33a
33b
33c
34a
34d
35a
35c
35d
3TC
0.103
0.0216
0.0033
0.565
0.0173
0.0292
>160
>193
>112
>62
0.0535
0.0036
0.0020
0.424
0.0110
0.0204
>160
61.8
16.9
597
792
1148
4812
570
1.12
347
127.3
42.2
223
295
2353
1971
3862
2781
56.6
>160
≥193
≥112
>62
>193
>112
>62
>97.6
2.22
>97.6
8.81
≥97.6
>87.2
>39
>10
a
EC50: compound concentration required to achieve 50% protection
b
of MT-4 cells against HIV-induced cytopathicity. CC50: compound
concentration required to reduce the viability of mock-infected cells by
50%.
corresponding thymidine analogue PMDTT (compound 12b)
is slightly less active and displayed an EC50 value of 27.07 μM
against HIV-1 and 23.55 μM against HIV-2. The cytosine
PMDTA (compound 12a) is endowed with an EC50 value of
4.69 μM against HIV-1 and 5.23 μM against HIV-2. The
F
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(PMDTC, compound 22) and guanosine (PMDTG, com-
pound 27) containing congeners completely lack anti-HIV
activity. The 2′-F analogue of PMDTA (compound 31) is also
completely devoid of anti-HIV activity.
or they are not recognized by the HIV reverse transcriptase.
This is in sharp contrast with PMDTA for which it has been
demonstrated that the diphosphate metabolite is an efficient
substrate for HIV-1 reverse transcriptase (functioning as a chain
terminator), whereas it is only a very poor substrate for human
DNA polymerase α.11
Anti-HBV Activity. Acyclic nucleoside phosphonates
adefovir and tenofovir are endowed with potent anti-HIV as
well as anti-HBV activity. Therefore, the ability of the α-L-2′-
deoxythreose nucleoside phosphonates to inhibit the repli-
cation of HBV was assessed in the HepG2 2.2.15 cell line,
which is a stable human hepatoblastoma cell line that contains
two copies of the HBV wild-type strain ayw1 genome and
constitutively produces high levels of HBV.22 Real-time qPCR
(TaqMan) was used to measure extracellular HBV DNA copy
number associated with virions released from HepG2 2.2.15
cells. A tetrazolium dye uptake assay was employed to measure
cell viability and to calculate CC50 values.23,24
Despite the only mediocre anti-HIV activity of PMDTT and
especially PMDTA, their anti-HIV activity still warrants further
investigation, as it is known that the application of a suitable
prodrug strategy can boost antiviral activity. A noteworthy
example is tenofovir alafenamide (also known as TAF), which
is 1000-fold more active against HIV-1 (EC50 = 5 nM) when
compared to the parent nucleoside tenofovir (EC50 = 5 μM).14
Different prodrugs of the α-L-2′-deoxythreose nucleoside
phosphonates were designed. As L-alanine is often the preferred
amino acid motif in ProTides, an alanine containing
aryloxyphosphonomonoamidate prodrug of each nucleoside
phosphonate was therefore prepared (series a in Scheme 6).
Recently, we discovered that appropriately substituted L-
aspartic acid diester phosphoramidate prodrugs confer high
anti-HCV activity to 2′-C-Me-uridine.21 In analogy, L-aspartic
acid di-isoamylesters of α-L-2′-deoxythreose nucleoside phos-
phonates were also synthesized (series b in Scheme 6) and
studied for antiviral activity. The main drawback of these
phosphonomonoamidate prodrugs is that diastereomeric
mixtures are generated due to the chirality of the phosphorus
atom. In the early phase of drug discovery, this usually is not an
issue as these compounds are biologically evaluated as mixtures.
However, upon further development, separation of both
diastereomers is necessary imposing an additional hurdle in
the development of these prodrugs. Therefore, we also
envisioned the synthesis of a phosphono-diamidate prodrug,
avoiding the problem of diastereomeric mixtures (series c and d
in Scheme 6).
The alanine containing prodrug of PMDTA (compound
32a) shows a 50-fold increased activity against HIV-1 and a
100-fold improvement of activity against HIV-2. The
phosphono-amidate prodrug of PMDTA carrying a di-
isoamylester L-aspartic acid as amino acid motif (compound
32b) is endowed with potent antiviral activity displaying EC50
values of 0.022 and 0.0036 μM against HIV-1 and HIV-2,
respectively. The phosphonodiamidate analogue 32c is the
most potent compound with EC50 values of 0.0033 μM (HIV-
1) and 0.002 μM (HIV-2). The same prodrug strategy was
applied to PMDTT (compound 12b), and similar trends as in
the PMDTA series were observed. The L-alanine phosphonoa-
midate prodrug 33a is endowed with a 50-fold increased anti-
HIV activity as compared to the parent PMDTT (compound
12b). The L-aspartic acid containing phenoxyphosphonomo-
noamidate prodrug (compound 33b) and the phosphonodia-
midate prodrug (compound 33c) are endowed with com-
parable anti-HIV activities, with EC50 values in the range of
11−30 nM.
The biological data obtained in the HBV assay are quite
similar to the anti-HIV data (Table 2). Among the parent
Table 2. Anti-HBV Activity and Cytotoxicity of
Deoxythreosyl Nucleoside Phosphonates and Their
Prodrugs
a
b
c
compd
EC50 (μM) EC90 (μM) CC50 (μM)
SI50
12a (PMDTA)
0.50
40.2
>100
>100
>100
>100
>100
>10
>100
>100
>100
>100
>100
>10
>200
12b (PMDTT)
>2.49
22 (PMDTC)
>100
>100
79.58
0.03
27 (PMDTG)
31 (2′-F-PMDTA)
32a
32b
32c
33a
33b
33c
34a
34d
35a
35c
35d
3TC
>333
>1250
>1000
>38
0.01
0.98
>10
0.01
4.20
>10
0.26
>10
>100
34.03
>10
0.25
>100
>10
136
0.28
>36
47.89
>100
>100
>100
>100
0.01
>100
>100
>100
>100
>100
>2.0
>100
>100
>100
>100
>100
>2.0
>2
>200
a
b
c
EC50: compound concentration that reduces viral replication by 50%.
EC90: compound concentration that reduces viral replication by 90%.
CC50: compound concentration that reduces cell viability by 50%.
phosphonates, PMDTA (compound 12a) is the most potent
anti-HBV compound, with an EC50 value of 0.5 μM. PMDTT
(compound 12b) is 10-fold less potent (EC50 value 40.2 μM),
whereas PMDTC (compound 22) and PMDTG (compound
27) are completely devoid of activity against HBV (EC50 values
greater than 100 μM). The fluorinated counterpart of PMDTA
(compound 31) shows very weak activity against HBV (EC50:
79 μM).
To see whether, analogously to the anti-HIV activity, the
prodrug had an improved inhibitory activity against HBV, the
prodrugs were also evaluated. The different prodrugs of
PMDTA (compounds 32a−c) are approximately 50 times
more potent than PMDTA. Although the EC50 values of the
different prodrugs are in the same range (EC50 values of 0.01−
0.03 μM), a close inspection of the EC90 values reveals that the
phosphonodiamidate prodrug 32c (EC90 = 4.20 μM) and
In general, the PMDTA (compounds 32a−c) and PMDT
prodrugs (compounds 33a−c) do show an increased
cytotoxicity in the MT-4 cell line when compared to the free
phosphonates. However, because of their excellent antiviral
activity, selectivity indexes of 200 or more are still obtained.
Anti-HIV evaluation of the different prodrugs of PMDTC
(compounds 34a and 34d) and PMDTG (compounds 35a,
35c, and 35d) did not reveal an improvement in antiviral
activity. This suggests that the limited cellular permeability of
the parent phosphonates is not the reason for the lack of
antiviral activity. Either PMDTC and PMDTG are not
phosphorylated to their corresponding diphosphophosphonates
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especially the L-aspartic acid di-isoamylester prodrug 32b (EC90
= 0.98 μM) are much more potent than the classical L-alanine
containing PMDTA prodrug 32a (EC90 > 10 μM). The CC50
values of the different prodrugs in the hepatoma cell line
exceeds 10 μM, resulting in SI values of more than 300 for the
alanine prodrug 32a, whereas the more potent prodrugs 32b
and 32c are endowed with SI over 1000.
The prodrugs of PMDTT (compounds 33a−c) all have very
similar potencies in the HBV assay, as they all are endowed
with an EC50 value of 0.25 μM, making them about 150 times
more potent than PMDTT itself.
To determine whether reductions in extracellular HBV DNA
copy number that were observed in the primary assay (Table 2)
correlate to a concomitant reduction in intracellular HBV DNA
copy number, a secondary HBV assay was performed. This
assay was carried out in a manner similar to the primary assay,
but at the end of the assay, the cells were processed to isolate
total intracellular DNA. Real-time TaqMan qPCR assay was
then performed using the isolated DNA to measure the
reductions in intracellular HBV DNA copy number. The two
most potent prodrugs from the primary anti-HBV assay (32b
and 32c) were tested in the secondary assay (Table 3). The
vitro chemical and enzymatic hydrolysis of representative
PMDTA prodrugs was investigated.
Chemical Stability at pH 1 and pH 8. The chemical stability
of prodrug 32b was evaluated in phosphate buffer at pH values
of biological relevance (i.e., pH 1 and 8). The disappearance of
the two peaks in the phosphorus NMR spectrum (due to the
existence of diastereomers) was monitored for 14 h by 31P
NMR at 37 °C. Under acidic conditions (pH 1, see Supporting
Information, Figure S1), the prodrug 32b started to degrade
after 1 h and new peaks arose in the 31P NMR spectrum (with
chemical shift values of 24.60, 23.84, and 25.05, 24.31 ppm).
Although these degradation products have not been unambig-
uously identified, these might arise from epimerization at the
anomeric carbon or opening of the sugar ring under the acidic
conditions. After 2 h, almost no original prodrug 32b was left
and a clear signal at 15.31 ppm emerged, which gradually
increased over time. The signals at the chemical shift 15.31
ppm may correspond to a product with cleavage of the phenol
moiety.
On the other hand, prodrug 32b is reasonably stable under
mild basic conditions (pH 8, see Supporting Information,
Figure S2), as after 14 h, 43% of the prodrug 32b was still
present. A single peak at 15.48 ppm became apparent after 30
min, and it gradually increased over time. At the end of the
experiment, HPLC and mass spectral analysis of the NMR
sample (see Supporting Information, Figure S3) revealed that
this peak in 31P NMR can be assigned to intermediate A
(Figure 3), which was formed by cleavage of the phenol moiety
of 32b.
Table 3. Anti-HBV Activity of Compounds 32b and 32c:
Secondary Assay
a
b
c
compd
EC50 (μM)
EC90 (μM)
CC50 (μM)
SI
32b
32c
<0.01
0.03
0.36
6.37
35.24
>100
>2.0
8810
>3333
>254
3TC
<0.01
>2.0
Incubations with Carboxypeptidase Y. To exert their
antiviral activity, the prodrugs need to be metabolized
intracellularly to their corresponding nucleoside-phosphonates.
On the basis of the proposed intracellular activation route
reported for other ProTide families,13 a hypothetical metabolic
route is shown in Figure 4. It starts with the hydrolysis of the α-
carboxylic acid ester group, mediated by a carboxyesterase type
of enzyme. Spontaneous cyclization and concomitant release of
the phenoxy moiety yields intermediate C. The cyclic mixed
anhydride intermediate is then hydrolyzed, affording inter-
mediate D. The final step involves a phosphoramidase type of
enzyme, which cleaves off the amino acid, generating the
nucleoside phosphonate.
To investigate whether enzymatic cleavage of the ester group
is sufficient to trigger the following steps, an enzymatic
experiment incubating the prodrug 32b with carboxypeptidase
Y enzyme in acetone-d6 and Trizma buffer (pH = 7.6) at 25 °C
was performed, whereby 31P NMR spectra were recorded at
specific time intervals. To improve the visualization of results,
the spectra were processed using the Lorentz−Gauss
deconvolution method (Figure 5). A difference in the rate of
a
b
c
EC50: compound concentration that reduces viral replication by 50%.
EC90: compound concentration that reduces viral replication by 90%.
CC50: compound concentration that reduces cell viability by 50%.
results from the primary assay were confirmed. Both prodrugs
display pronounced anti-HBV activity, and especially the L-
aspartic acid based prodrug 32b is highly potent based on the
calculated EC50 and EC90 values. The anti-HBV activity of both
compounds is in the same range as the activity of 3TC and with
a selectivity index better than that of the control drug.
Stability Studies. Although we observe very potent in vitro
antiviral activity against HIV and HBV with the PMDTA
prodrugs, in vivo, the prodrugs can only reach the target cells
(the hepatocytes in case of HBV infection and lymphoid cell for
HIV infected patients), on the condition that they are resistant
to hydrolysis by extracellular and intracellular carboxylesterases
during the absorption and distribution process. Partial or full
conversion of these prodrugs to intermediates or the free
nucleoside monophosphate will result in a lower cell
penetration and a reduced antiviral potency. Therefore, the in
Figure 3. Degradation of compound 32b at pH 8.
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Figure 4. Proposed activation pathway for prodrug 32b.
Figure 5. Deconvoluted 31P NMR spectra of prodrug 32b over time after carboxypeptidase Y digestion.
hydrolysis of the two diastereomers was observed. After
incubation for 16 h, diastereomer (δ = 23.63 pm) was
completely metabolized, whereas still substantial amounts of
the other diastereomer (δ = 24.71 ppm) were still present.
After 3 h, a new single peak (δ = 17.13 ppm) appeared in the
31P NMR spectrum, which was assigned to metabolite D
(Figure 4), as confirmed by HPLC and mass spectral analysis of
the NMR sample at the end of the experiment (see Supporting
Information, Figure S4). Mass spectroscopy does not allow
discrimination whether the α- or β-carboxyl ester moiety is
cleaved. However, recent research demonstrated that in the
case of L-aspartate acid diester based nucleoside phosphor-
amidates, the β-carboxyl ester of the L-aspartate moiety was the
first to be hydrolyzed. On the other hand, considering the fact
that the α-carboxyl group is responsible for the nucleophilic
attack at the phosphorus center to displace the aryl group, the
α-carboxyl ester of the aspartate moiety should then be
hydrolyzed first.
be metabolized intracellularly to release the L-2′-deoxythreose
nucleoside phosphonate, which will then be further phosphory-
lated, yielding the corresponding diphosphophosphonate
analogue that acts as an inhibitor of the HIV reverse
transcriptase or HBV DNA polymerase. The stability of
prodrugs 32a−c in the human liver S9 fraction and human
liver microsomes was investigated (Table 4). The L-alanine
containing monoamidate prodrug 32a is stable against
degradation in the human liver S9 fraction and human liver
microsomes. It suggests that this type of prodrug will survive
first-pass metabolism, and they are suitable to be developed as
Table 4. Stability of Prodrugs 32a−c in Human Liver S9
Fraction and Human Liver Microsomes
compd
human liver S9 t1/2 (min)
human liver microsomes t1/2 (min)
32a
32b
32c
>60
12.1
<10
>60
8
Stability in Human Liver S9 Fraction and Human Liver
Microsomes. To exert their antiviral activity, the ProTides must
7
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Article
acid (0.05 mmol, 1 equiv), appropriate amino acid ester hydrochloride
(0.10 mmol, 2 equiv), and phenol (0.25 mmol, 5 equiv) in anhydrous
pyridine (1.0 mL). The resultant mixture was stirred at 60 °C under
argon atmosphere. In a separate flask, 2,2′-dithiodipyridine (0.42
mmol, 8.3 equiv) and PPh3 (0.30 mmol, 6 equiv) were dissolved in
anhydrous pyridine (1.0 mL) and the resultant yellow solution was
stirred at rt for 15 min. The solution was then added to the solution of
phosphonic acid in one portion. The combined mixture was stirred at
60 °C for overnight. The mixture was then concentrated under
reduced pressure. The resultant residue was first purified by silica gel
chromatography (60:1 to 10:1, DCM/MeOH) and then purified by
RP-HPLC (linear gradient, 5−95% CH3CN in water) to give the pure
product as a mixture of P(R) and P(S) isomers.
anti-HIV drugs, as the target cells for anti-HIV medication are
lymphocytes, which are present in the systemic circulation. On
the other hand, the L-aspartic acid based phosphonomonoami-
date prodrug 32b and the bisamidate prodrug 32c are both
metabolically labile in liver S9 fraction and human liver
microsomes with half-lives of less than 10 min. This type of
prodrug is well suited for further development as an anti-HBV
drug, as fast metabolism in the liver will quickly generate high
levels of the nucleoside phosphonate (the pharmacologically
active metabolite) in hepatocytes, which are the target cells for
HBV infection.
General Procedure for Preparation of Bisamidate Prodrugs
(32c, 33c, 34d, 35c, and 35d). Anhydrous trimethylamine (0.70
mmol, 12 equiv) was added to a solution of phosphonic acid (0.05
mmol, 1 equiv) and appropriate amino acid ester hydrochloride (0.30
mmol, 6 equiv) in anhydrous pyridine (1.0 mL). The resultant mixture
was stirred at 60 °C under argon atmosphere. In a separate flask, 2,2′-
dithiodipyridine (0.35 mmol, 7 equiv) and PPh3 (0.35 mmol, 7 equiv)
were dissolved in anhydrous pyridine (1.0 mL) and the resultant
yellow solution was stirred at rt for 15 min. The solution was then
added to the solution of phosphonic acid in one portion. The
combined mixture was stirred at 60 °C for overnight. The mixture was
then concentrated under reduced pressure. The resultant residue was
first purified by silica gel chromatography (60:1 to 10:1, DCM/
MeOH) and then purified by RP-HPLC (linear gradient, 5−95%
CH3CN in water) to give the pure product.
2-O-Benzoyl-3-O-tert-butyldimethylsilyl-L-threonolactone
(2). To a solution of lactone 1 (29.8 g, 134 mmol), cat. DMAP (0.1 g),
and imidazole (15.8 g, 231 mmol) in dry acetonitrile at 0 °C, a
solution of tert-butyldimethylchlorosilane (22.2 g, 148 mmol) in
anhydrous acetonitrile was added dropwise. The reaction mixture was
stirred at room temperature for overnight. The acetonitrile was
evaporated under reduced pressure, the residue was taken up into 500
mL of EtOAc, and 300 mL of water was added. The resulting mixture
was sequentially washed with ice-cold 1 M HCl solution, water, satd aq
NaHCO3, and brine. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by column chromatography
(4:1 hexane/EtOAc) to afford 2 (44.0 g, 97% yield) as a colorless oil.
1H NMR (300 MHz, CDCl3): δ 8.06−8.09 (m, 2H, Ph), 7.58−7.63
(m, 1H, Ph), 7.44−7.49 (m, 2H, Ph), 5.59 (d, J = 7.3 Hz, 1H, H-2′),
4.80 (q, J = 7.2 Hz, 1H, H-3′), 4.53 (dd, J = 9.1, 7.0 Hz, 1H, H-4a′),
4.09 (dd, J = 9.1, 7.3 Hz, 1H, H-4b′), 0.86 (s, 9H, CH3), 0.07 (s, 3H,
SiCH3), 0.06 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 170.4
(C-1′), 165.2 (PhCO), 133.9, 130.1, 128.7 (Ph), 75.1 (C-2′), 71.7 (C-
3′), 70.6 (C-4′), 25.6 [C(CH3)3], 18.0 [C(CH3)3], −4.7, −4.9
(SiCH3). HRMS: [M + H]+ calcd for C17H25O5Si, 337.1466; found,
337.1460.
1-O-Acetyl-2-O-benzoyl-3-O-tert-butyldimethylsilyl-L-threo-
furanose (3).17 Prepared according to the literature17 and obtained as
a colorless oil. Yield 73%. α-Anomer: 1H NMR (300 MHz, CDCl3): δ
8.01−8.04 (m, 2H, Ph), 7.57−7.62 (m, 1H, Ph), 7.43−7.48 (m, 2H,
Ph), 6.23 (s, 1H, H-1′), 5.29 (d, J = 1.7 Hz, 1H, H-2′), 4.44−4.49 (m,
1H, H-3′), 4.28 (dd, J = 9.3, 5.7 Hz, 1H, H-4a′), 4.01 (dd, J = 9.3, 4.2
Hz, 1H, H-4b′), 2.12 (s, 3H, CH3CO), 0.91 (s, 9H, CH3), 0.15 (s, 3H,
SiCH3), 0.10 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 169.8
(CH3CO), 165.3 (PhCO), 133.5, 129.8, 129.1, 128.5 (Ph), 99.9 (C-
1′), 83.2 (C-2′), 75.4 (C-4′), 74.9 (C-3′), 25.6 [C(CH3)3], 21.1
(CH3CO), 17.9 [C(CH3)3], −4.8, −5.1 (SiCH3). β-Anomer: 1H NMR
(300 MHz, CDCl3): δ 8.02−8.05 (m, 2H, Ph), 7.54−7.60 (m, 1H,
Ph), 7.41−7.46 (m, 2H, Ph), 6.49 (d, J = 4.4 Hz, 1H, H-1′), 5.26 (t, J
= 4.7 Hz, 1H, H-2′), 4.68−4.73 (m, 1H, H-3′), 4.27 (dd, J = 9.1, 6.5
Hz, 1H, H-4a′), 3.78 (dd, J = 9.1, 4.7 Hz, 1H, H-4b′), 1.94 (s, 3H,
CH3CO), 0.87 (s, 9H, CH3), 0.09 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3).
13C NMR (75 MHz, CDCl3): δ 169.4 (CH3CO), 165.4 (PhCO),
CONCLUSION
■
The synthesis of four L-2′-deoxy-threose nucleoside phospho-
nates containing the natural nucleobases adenine, thymine,
cytosine, and guanosine has been performed, using an
improved synthetic method. In addition, a fluorinated congener
of PMDTA has been synthesized. PMDTT, and especially
PMDTA, are both endowed with antiviral activity against HIV
and HBV, whereas other congeners within this series
completely lack antiviral activity. Different phosphonomonoa-
midate and phosphonobisamidate prodrugs of these nucleoside
phosphonates were also prepared. The application of these
prodrug technologies to PMDTA and PMDTT led to a boost
in antiviral potency. The anti-HIV potency increased by a factor
of 100 to 1000, depending on the prodrug moiety, whereas 50−
150-fold enhancements for anti-HBV activity were observed.
The prodrugs of PMDTA are reasonably stable in pH 8 buffer
but can be activated by carboxypeptidase Y type enzyme and
human liver enzymes. The promising antiviral data of the
phosphono-amidate prodrugs of PMDTA and PMDTT suggest
that they deserve further investigation as dual anti-HIV and
anti-HBV drugs.
EXPERIMENTAL SECTION
■
Chemistry. NMR spectra were recorded on a Bruker Advance 300
MHz (1H NMR, 300 MHz; 13C NMR, 75 MHz; 31P NMR, 121 MHz)
or 500 MHz (1H NMR, 500 MHz; 13C NMR, 125 MHz; 31P NMR,
202 MHz) or 600 MHz (1H NMR, 600 MHz; 13C NMR, 150 MHz)
spectrometer with tetramethylsilane as internal standard or referenced
to the residual solvent signal and 85% H3PO4 for 31P NMR
experiments. Two-dimensional NMRs (H-COSY, NOESY, HSQC,
and HMBC) were used for the assignment of the intermediates and
final compounds. High resolution mass spectra were measured on a
quadrupole orthogonal acceleration time-of-flight mass spectrometer
(Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3
μL/min, and spectra were obtained in positive or negative ionization
mode with a resolution of 15000 (fwhm) using leucine enkephalin as
lock mass. Precoated aluminum sheets (Fluka silica gel/TLC-cards,
254 nm) were used for TLC. Purities of the prodrugs were verified to
be >95% by HPLC analysis. HPLC conditions to assess purity were as
follows: Shimadzu HPLC equipped with a LC-20AT pump, a DGU-
20A5 degasser, and a SPD-20A UV detector; Inertsil ODS-3 (4 μm,
4.6 mm × 100 mm) or Symmetry C18 column (5 μm, 4.6 mm × 150
mm); gradient elution of H2O/CH3CN or H2O/MeOH from 95/5 to
5/95 in 15 min; flow rate, 1 mL/min; wavelength, UV 254 nm.
Preparative HPLC purifications were performed on a Phenomenex
Gemini 110A column (C18, 10 μm, 21.2 mm × 250 mm). Column
chromatography was performed on silica gel 60 Å, 0.035−0.070 mm
(Acros Organics). All reagents and solvents were obtained from
commercial sources and were used as received. Moisture sensitive
reactions were performed under an argon atmosphere using oven-
dried glassware.
133.4, 129.7, 129.2, 128.5 (Ph), 94.65 (C-1′), 80.0 (C-2′), 73.4 (C-3′),
72.5 (C-4′), 25.6 [C(CH3)3], 20.9 (CH3CO), 17.9 [C(CH3)3], −4.7,
−5.0 (SiCH3). HRMS: [M + Na]+ calcd for C19H28O6SiNa, 403.1548;
found, 403.1548.
General Procedure for Preparation of Monoamidate
Podrugs (32a−b, 33a−b, 34a, and 35a). Anhydrous trimethyl-
amine (0.70 mmol, 12 equiv) was added to a solution of phosphonic
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1-O-Methyl-2-O-benzoyl-L-threose (4). To a solution of lactol
(8.04 g, 23.75 mmol) in anhydrous methanol (59 mL) was added
acetyl chloride (1.69 mL, 23.75 mmol). After stirring at rt for 2 h, Et3N
(4 mL) was added and the mixture was concentrated under reduced
pressure. The residue was partitioned between water and EtOAc, and
the organic layer was washed with satd aq NaHCO3 and brine, dried
over Na2SO4, and concentrated under reduced pressure. The residue
was purified by column chromatography (3:1 to 2:1, hexane/EtOAc)
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-O-tert-butyldi-
methylsilyl-L-threose (7a). A suspension of 3 (0.51g, 1.35 mmol)
and N6-benzoyladenine (0.36 g, 1.49 mmol) in anhydrous CH3CN (15
mL) was treated with BSA (0.73 mL, 3.0 mmol) and heated to 65 °C.
Stirring and heating was continued until a clear solution was formed
(ca. 1 h), and TMSOTf (0.49 mL, 2.70 mmol) was added. After
heating at 65 °C for 16 h, the mixture was cooled to rt and poured into
an ice-cold 100 mL of satd aq NaHCO3/EtOAc 1:1. The organic layer
was separated, the water layer was extracted with EtOAc. The
combined organic layers were washed with brine and dried over
Na2SO4. Evaporation of the solvent and column chromatography (5:2
1
to afford 4 (4.7 g, 83% yield) as a colorless oil. H NMR (300 MHz,
CDCl3): δ 8.00−8.06 (m, 2H, Ph), 7.56−7.62 (m, 1H, Ph), 7.42−7.48
(m, 2H, Ph), 5.14 (brs, 1H, H-1′), 5.11 (s, 1H, H-2′), 4.30−4.38 (m,
2H, H-3′ and H-4a′), 3.96−4.02 (m, 1H, H-4b′), 3.45 (s, 3H, OMe).
13C NMR (75 MHz, CDCl3): δ 166.1 (PhCO), 133.5, 129.8, 129.1,
128.5 (Ph), 106.5 (C-1′), 83.6 (C-2′), 75.2 (C-3′), 73.8 (C-4′), 55.0
(OMe). HRMS: [M + Na]+ calcd for C12H14O6Na, 261.0734; found,
261.0738.
1
CH2Cl2/EtOAc) gave 7a (0.42 g, 56% yield) as a white foam. H
NMR (300 MHz, CDCl3): δ 9.60 (s, 1H, NH), 8.67 (s, 1H, H-2), 8.37
(s, 1H, H-8), 7.94−8.00 (m, 4H, Ph), 7.47−7.55 (m, 2H, Ph), 7.36−
7.40 (m, 4H, Ph), 6.45 (s, 1H, H-1′), 5.56 (s, 1H, H-2′), 4.48 (d, J =
3.2 Hz, 1H, H-3′), 4.20−4.31 (m, 2H, H-4′), 0.77 [s, 9H, C(CH3)3],
0.07 (s, 3H, SiCH3), −0.02 (s, 3H, SiCH3). 13C NMR (75 MHz,
CDCl3): δ 164.7 (PhCO), 152.3 (C-2), 151.2 (C-4), 149.2 (C-6),
141.6 (C-8), 133.5, 133.5, 132.2, 129.5, 128.3, 128.3, 127.6 (Ph), 122.9
(C-5), 87.8 (C-1′), 82.3 (C-2′), 76.3 (C-4′), 74.8 (C-3′), 25.3
[C(CH3)3], 17.6 [C(CH3)3], −5.2, −5.6 (SiCH3). HRMS: [M + H]+
calcd for C29H34N5O5Si, 560.2324; found, 560.2330.
1′α-(Thymin-1-yl)-2′-O-benzoyl-3′-O-tert-butyldimethylsilyl-
L-threose (7b). This compound was prepared using a similar
procedure as described for 7a. Obtained from 3 (10.4 g, 27.3
mmol), thymine (3.44 g, 27.3 mmol), BSA (13.3 mL, 54.5 mmol), and
TMSOTf (14.8 mL, 81.7 mmol) as a white foam, yield 83% (10.2 g).
1H NMR (300 MHz, CDCl3): δ 9.50 (s, 1H, NH), 8.02−8.05 (m, 2H,
Ph), 7.56−7.62 (m, 1H, Ph), 7.52 (d, J = 1.2 Hz, 1H, H-6), 7.42−7.47
(m, 2H, Ph), 6.19 (d, J = 0.9 Hz, 1H, H-1′), 5.23 (s, 1H, H-2′), 4.36
(d, J = 2.6 Hz, 1H, H-3′), 4.15−4.23 (m, 2H, H-4′), 1.92 (d, J = 1.2
Hz, 3H, T CH3), 0.90 [s, 9H, C(CH3)3], 0.16 (s, 3H, SiCH3), 0.15 (s,
3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 165.0 (PhCO), 164.3 (C-
4), 150.4 (C-2), 136.5 (C-6), 133.7, 129.9, 129.8, 128.9, 128.5 (Ph),
110.2 (C-5), 89.6 (C-1′), 82.7 (C-2′), 76.5 (C-3′), 74.7 (C-4′), 25.6
[C(CH3)3], 17.9 [C(CH3)3], 12.6 (T CH3), −4.8, −5.3 (SiCH3).
HRMS: [M + H]+ calcd for C22H31N2O6Si, 447.1946; found,
447.1941.
1-O-Methyl-2-O-benzoyl-3-O-diisopropylphosphonometh-
yl-L-threose (5). To a solution of 4 (2.23 g, 9.36 mmol) and triflate
diisopropylphosphonomethanol (4.61 g, 14.04 mmol) in anhydrous
THF (50 mL) was added NaH (60% in mineral oil, 0.45 g, 11.23
mmol) at −5 °C. The reaction mixture was warmed to 0 °C and
stirred for 15 min. The reaction was quenched with satd aq NH4Cl and
was subsequently concentrated. The residue was partitioned between
water and EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography (2:1 to 1:1, hexane/EtOAc) to
afford 5 (3.43 g, 87% yield) as a colorless oil containing about 10% of
β-anomer. α-Anomer: 1H NMR (600 MHz, CDCl3): δ 8.02−8.05 (m,
2H, Ph), 7.57−7.61 (m, 1H, Ph), 7.44−7.47 (m, 2H, Ph), 5.29 (s, 1H,
H-2′), 5.04 (s, 1H, H-1′), 4.71−4.81 [m, 2H, CH(CH3)2], 4.38 (dd, J
= 9.8, 7.1 Hz, 1H, H-4a′), 4.28−4.30 (m, 1H, H-3′), 3.95 (dd, J = 9.6,
5.1 Hz, 1H, H-4b′), 3.86−4.07 (m, 2H, PCH2), 3.40 (s, 3H, OMe),
1.32−1.34 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz, CDCl3): δ
165.5 (PhCO), 133.5, 129.8, 129.3, 128.5 (Ph), 107.0 (C-1′), 84.8 (d,
3JP,C = 10.4 Hz, C-3′), 80.9 (C-2′), 71.4, 71.1 [CH(CH3)2], 71.1 (C-
4′), 65.0 (d, 1JP,C = 168.4 Hz, PCH2), 54.7 (OMe), 24.0 [CH(CH3)2].
31P NMR (121 MHz, CDCl3): δ 18.5. β-anomer: 1H NMR (600 MHz,
CDCl3): δ 8.06−8.08 (m, 2H, Ph), 7.58−7.61 (m, 2H, Ph), 7.45−7.48
(m, 2H, Ph), 5.27 (d, J = 4.5 Hz, 1H, H-1′), 5.10 (t, J = 4.6 Hz, 1H, H-
2′), 4.71−4.78 [m, 2H, CH(CH3)2], 4.56−4.58 (m, 1H, H-3′), 4.22
(dd, J = 9.9, 6.7 Hz, 1H, H-4a′), 3.89 (dd, J = 9.9, 3.6 Hz, 1H, H-4b′),
3.76−3.89 (m, 2H, PCH2), 3.33 (s, 3H, OMe), 1.29−1.34 [m, 2H,
CH(CH3)2]. 13C NMR (150 MHz, CDCl3): δ 165.8 (PhCO), 133.4,
129.8, 129.3, 128.5 (Ph), 101.8 (C-1′), 83.2 (d, 3JP,C = 12.5 Hz, C-3′),
79.5 (C-2′), 71.4, 71.3 [CH(CH3)2], 68.5 (C-4′), 64.6 (d, 1JP,C = 169.9
Hz, PCH2), 55.4 (OMe), 24.0 [CH(CH3)2]. 31P NMR (121 MHz,
CDCl3): δ 18.1. HRMS: [M + H]+ calcd for C19H30O8P, 417.1673;
found, 417.1673.
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-L-threose (8a).16
To a solution of compound 7a (11.6 g, 20.7 mmol) in 200 mL of
anhydrous THF at rt was added triethylamine trihydrofluoride. The
reaction mixture was stirred at rt for 24 h and found to be complete.
The solvent was removed under reduced pressure. The residue was
taken up into 300 mL of EtOAc, and 200 mL of water was added. The
organic layer was separated and washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
column chromatography (50:1 CH2Cl2/MeOH) to obtain 8a (9.0 g,
1
97% yield) as a white solid. H NMR (300 MHz, CDCl3): δ 9.45 (s,
1-O-Acetyl-2-O-benzoyl-3-O-diisopropylphosphonomethyl-
L-threose (6). To a solution of 5 (0.20 g, 0.48 mmol) in anhydrous
CH2Cl2 (5 mL) was added acetic anhydride (0.18 mL, 1.92 mmol)
and a catalytic amount of sulfuric acid. After stirring at room
temperature for overnight, the reaction mixture was neutralized by
Et3N (3 mL) at 0 °C and concentrated under reduced pressure. The
crude product was diluted by EtOAc (20 mL), washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography (1:1, hexane/EtOAc)
to afford 6 (0.18 g, 83% yield) as a colorless oil containing about 10%
1H, NH), 8.74 (s, 1H, H-2), 8.32 (s, 1H, H-8), 7.99−8.02 (m, 4H,
Ph), 7.43−7.63 (m, 6H, Ph), 6.13 (d, J = 1.3 Hz, 1H, H-1′), 5.63 (s,
1H, H-2′), 4.64 (d, J = 3.0 Hz, 1H, H-3′), 4.36 (d, J = 10.1 Hz, 1H, H-
4a′), 4.27 (dd, J = 10.1, 3.8 Hz, 1H, H-4b′). 13C NMR (75 MHz,
CDCl3): δ 165.6, 164.8 (PhCO), 152.0 (C-2), 150.4 (C-4), 150.0 (C-
6), 143.1 (C-8), 133.9, 133.4, 132.8, 129.8, 128.7, 128.6, 128.5, 127.9
(Ph), 123.5 (C-5), 90.3 (C-1′), 84.1 (C-2′), 76.1 (C-4′), 74.6 (C-3′).
HRMS: [M + H]+ calcd for C23H20N5O5, 446.1459; found, 446.1451.
1′α-(Thymin-1-yl)-2′-O-benzoyl-L-threose (8b).15 This com-
pound was prepared as described for 8a. Obtained from 7b (11.6 g,
26.0 mmol) and triethylamine trihydrofluoride (8.5 mL, 52.0 mmol) as
a white solid. Yield 98% (8.5 g). 1H NMR (300 MHz, CDCl3): δ 9.93
(s,1H, NH), 7.96−7.98 (m, 2H, Ph), 7.52−7.57 (m, 1H, Ph), 7.47 (d,
J = 0.9 Hz, 1H, H-6), 7.37−7.42 (m, 2H, Ph), 5.99 (d, J = 1.2 Hz, 1H,
H-1′), 5.48 (s, 1H, H-2′), 4.77 (brs, 1H, OH), 4.46 (brs, 1H, H-3′),
4.31 (d, J = 10.0 Hz, H-4a′), 4.16 (dd, J = 10.0, 3.6 Hz, H-4b′), 1.79 (s,
3H, T CH3). 13C NMR (75 MHz, CDCl3): δ 165.5 (PhCO), 164.4
(C-4), 150.5 (C-2), 137.2 (C-6), 133.6, 129.7, 128.6, 128.4 (Ph),
109.9 (C-5), 90.7 (C-1′), 82.6 (C-2′), 75.51 (C-4′), 73.9 (C-3′), 12.2
(T CH3). HRMS: [M + H]+ calcd for C16H17N2O6, 333.1081; found,
333.1082.
1
of β-anomer, and the major α-anomer was characterized. H NMR
(600 MHz, CDCl3): δ 8.02−8.04 (m, 2H, Ph), 7.59−7.62 (m, 1H,
Ph), 7.45−7.48 (m, 2H, Ph), 6.31 (s,1H, H-1′), 5.42 (d, J = 0.9 Hz,
1H, H-2′), 4.74−4.80 [m, 2H, CH(CH3)2], 4.44 (dd, J = 10.1, 6.7 Hz,
H-4a′), 4.30−4.32 (m, 1H, H-3′), 4.12 (dd, J = 10.1, 4.3 Hz, 1H, H-
4b′), 3.86−4.10 (m, 2H, PCH2), 2.12 (s, 3H, OCH3), 1.33−1.35 [m,
12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 169.7 (CH3CO),
165.3 (PhCO), 133.7, 129.8, 128.9, 128.6 (Ph), 99.8 (C-1′), 84.3 (d,
3JP,C = 11.6 Hz, C-3′), 80.3 (C-2′), 73.1 (C-4′), 71.3, 71.2
1
[CH(CH3)2], 65.2 (d, JP,C = 168.6 Hz, PCH2), 24.0 [CH(CH3)2],
21.1 (CH3CO). 31P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M +
Na]+ calcd for C20H29O9PNa, 467.1442; found, 467.1444.
K
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-O-diisopropyl-
phosphonomethyl-L-threose (9a).11 This compound was prepared
as described for 5. Obtained from 8a (0.345 g, 0.77 mmol), triflate
diisopropylphosphonomethanol (0.254 g, 0.77 mmol), and NaH (60%
in mineral oil, 0.102 g, 2.56 mmol) as a colorless oil. Yield 80% (0.385
CDCl3): δ 164.6 (C-4), 151.0 (C-2), 136.4 (C-6), 110.0 (C-5), 93.0
(C-1′), 85.2 (d, 3JP,C = 10.7 Hz, C-3′), 78.6 (C-2′), 73.6 (C-4′), 71.3,
1
71.2 [CH(CH3)2], 64.3 (d, JP,C = 168.4 Hz, PCH2), 24.0, 23.9
[CH(CH3)2], 12.4 (T CH3). 31P NMR (121 MHz, CDCl3): δ 18.3.
HRMS: [M + H]+ calcd for C16H28N2O8P, 407.1578; found, 407.1582.
1′α-(N6-Benzoyladenin-9-yl)-2′-deoxy-3′-O-diisopropyl-
phosphonomethyl-L-threose (11a). To a solution of 10a (137 mg,
0.26 mmol) and DMAP (10 mg, 0.08 mmol) in andydrous CH2Cl2
(2.70 mL) was added 1,1′-thiocarbonyldiimidazole (TCDI) (94 mg,
0.53 mmol) at rt. The reaction mixture was stirred at 40 °C for
overnight. The mixture was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. To the residue in toluene (5
mL) was added azobis(isobutyronitrile) (AIBN) (17 mg, 0.11 mmol)
and tributytin hydride (0.28 mL, 1.05 mmol). The reaction mixture
was refluxed for 20 min. The solvent was removed under reduced
pressure, and the residue was purified by column chromatography
(30:1 CH2Cl2/MeOH) to afford 11a (130 mg, 98% yield) as a white
1
g). H NMR (300 MHz, CDCl3): δ 9.32 (brs, 1H, NH), 8.80 (s, 1H,
H-2), 8.51 (s, 1H, H-8), 8.03−8.08 (m, 1H, Ph), 7.47−7.66 (m, 6H,
Ph), 6.56 (s, 1H, H-1′), 5.80 (s, 1H, H-2′), 4.71−4.82 [m, 2H,
CH(CH3)2], 4.49−4.51 (m, 2H, H-3′ and H-4a′), 4.35−4.40 (m, 1H,
H-4b′), 3.95−4.01 (m, 2H, PCH2), 1.31−1.36 [m, 12H, CH(CH3)2].
13C NMR (75 MHz, CDCl3): δ 165.1, 164.6 (PhCO), 152.7 (C-2),
151.5 (C-4), 149.5 (C-6), 141.9 (C-8), 133.9, 133.7, 132.6, 129.9,
128.7, 128.6, 128.4, 127.9 (Ph), 122.7 (C-5), 87.8 (C-1′), 83.7 (d, 3JP,C
= 9.5 Hz, C-3′), 80.2 (C-2′), 73.5 (C-4′), 71.5, 71.4 [CH(CH3)2], 64.6
(d, 1JP,C = 169.4 Hz, PCH2), 23.9 [CH(CH3)2]. 31P NMR (121 MHz,
CDCl3): δ 17.8. HRMS: [M + H]+ calcd for C30H35N5O8P, 624.2218;
found, 624.2217.
1′α-(Thymin-1-yl)-2′-O-benzoyl-3′-O-diisopropylphospho-
nomethyl-L-threose (9b).11 This compound was prepared as
described for 5. Obtained from 8b (2.0 g, 6.02 mmol), triflate
diisopropylphosphonomethanol (1.1 g, 6.62 mmol), and NaH (60% in
mineral oil, 0.794 g, 19.86 mmol) as a white solid. Yield 85% (2.6 g).
1H NMR (300 MHz, CDCl3): δ 9.25 (brs, 1H, NH), 8.01−8.04 (m,
1
solid. H NMR (500 MHz, CDCl3): δ 9.15 (s, 1H, NH), 8.79 (s, 1H,
H-2), 8.49 (s, 1H, H-8), 8.00−8.02 (m, 2H, Ph), 7.57−7.59 (m, 1H,
Ph), 7.50−7.53 (m, 2H, Ph), 6.55 (dd, J = 7.7, 2.0 Hz, 1H, H-1′),
4.69−4.75 [m, 2H, CH(CH3)2], 4.51−4.53 (m, 1H, H-3′), 4.38 (d, J =
10.5 Hz, 1H, H-4a′), 4.08 (dd, J = 10.5, 4.1 Hz, 1H, H-4b′), 3.67−3.75
(m, 2H, PCH2), 2.62−2.74 (m, 2H, H-2′). 1.28−1.33 [m, 12H,
CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 164.5 (PhCO), 152.5
(C-2), 151.5 (C-4), 149.3 (C-6), 142.1 (C-8), 133.8, 132.6, 128.8,
2H, Ph), 7.58−7.60 (m, 1H, Ph), 7.43−7.48 (m, 3H, Ph and H-6),
6.26 (d, J = 1.5 Hz, 1H, H-1′), 5.39 (s, 1H, H-2′), 4.74−4.81 [m, 2H,
CH(CH3)2], 4.39 (d, J = 10.7 Hz, 1H, H-4a′), 4.26 (d, J = 3.6 Hz, H-
3′), 4.13 (dd, J = 10.7, 3.7 Hz, H-4b′), 3.88−4.06 (m, 2H, PCH2), 1.97
(d, J = 0.9 Hz, 3H, T CH3), 1.32−1.35 [m, 12H, CH(CH3)2]. 13C
NMR (75 MHz, CDCl3): δ 165.2 (PhCO), 163.8 (C-4), 150.4 (C-2),
136.0 (C-6), 133.8, 129.8, 128.5 (Ph), 111.3 (C-5), 89.0 (C-1′), 83.7
3
127.8 (Ph), 122.8 (C-5), 83.7 (C-1′), 80.2 (d, JP,C = 9.5 Hz, C-3′),
73.9 (C-4′), 71.3, 71.3 [CH(CH3)2], 64.0 (d, 1JP,C = 169.2 Hz, PCH2),
38.6 (C-2′), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.2.
HRMS: [M + H]+ calcd for C23H31N5O6P, 504.2006; found, 504.2023.
1′α-(Thymin-1-yl)-2′-deoxy-3′-O-diisopropylphosphono-
methyl-L-threose (11b).11 This compound was prepared as
described for 11a. Obtained from 10b (1.53 g, 3.8 mmol) as a
3
(d, JP,C = 10.9 Hz, C-3′), 80.2 (C-2′), 72.7 (C-4′), 71.4, 71.3
1
[CH(CH3)2], 64.5 (d, JP,C = 168.6 Hz, PCH2), 23.9 [CH(CH3)2],
1
12.5 (T CH3). 31P NMR (121 MHz, CDCl3): δ 17.9. HRMS: [M +
H]+ calcd for C23H32N2O9P, 511.1840; found, 511.1853.
white solid. Yield 97% (1.43 g). H NMR (300 MHz, CDCl3): δ 8.97
(brs, 1H, NH), 7.55 (d, J = 1.2 Hz, 1H, H-6), 6.24 (dd, J = 8.1, 2.6 Hz,
1H, H-1′), 4.69−4.80 [m, 2H, CH(CH3)2], 4.30−4.36 (m, 2H, H-4a′
and H-3′), 3.84 (dd, J = 10.8, 3.6 Hz, 1H, H-4b′), 3.71 (d, J = 9.2 Hz,
2H, PCH2), 2.50−2.60 (m, 1H, H-2a′), 2.16 (d, J = 15.3 Hz, 1H, H-
2b′), 1.97 (d, J = 1.1 Hz, 3H, T CH3), 1.31−1.35 [m, 12H,
CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 163.8 (C-4), 150.6 (C-
2), 136.4 (C-6), 110.7 (C-5), 84.8 (C-1′), 80.2 (d, 3JP,C = 11.4 Hz, C-
1′α-(N6-Benzoyladenin-9-yl)-3′-O-diisopropylphosphono-
methyl-L-threose (10a). To a suspension of 9a (8.65 g, 13.87 mmol)
in 280 mL of THF/MeOH/H2O 5:4:1 at 0 °C was added 6.94 mL
(13.87 mmol) of 2 N aq NaOH. After 20 min, the solution was
neutralized with acetic acid. The mixture was concentrated under
reduced pressure. The residue was partitioned between water and
EtOAc. The organic layer was washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography (30:1 CH2Cl2/MeOH) to afford
10a (5.7 g, 80% yield) as a white solid. 1H NMR (300 MHz, CDCl3):
δ 9.53 (brs, 1H, NH), 8.66 (s, 1H, H-2), 8.36 (1H, H-8), 7.98−8.00
(m, 2H, Ph), 7.44−7.58 (m, 3H, Ph), 6.21 (d, J = 1.7 Hz, 1H, H-1′),
4.80 (s, 1H, H-2′), 4.63−4.73 [m, 2H, CH(CH3)2], 4.28−4.34 (m,
3H, H-4′ and H-3′), 3.82 (d, J = 8.8 Hz, 2H, PCH2), 1.24−1.31 [m,
12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 164.8 (PhCO),
152.1 (C-2), 151.2 (C-4), 149.1 (C-6), 142.1 (C-8), 133.4, 132.5,
128.5, 127.8 (Ph), 122.7 (C-5), 90.5 (C-1′), 85.9 (d, 3JP,C = 9.7 Hz, C-
3′), 78.9 (C-2′), 72.5 (C-4′), 71.4 [CH(CH3)2], 64.4 (d, 1JP,C = 169.5
Hz, PCH2), 23.8, 23.7 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ
18.4. HRMS: [M + H]+ calcd for C23H31N5O7P, 520.1955; found,
520.1961.
1
3′), 73.4 (C-4′), 71.2 [CH(CH3)2], 64.0 (d, JP,C = 169.8 Hz, PCH2),
38.3 (C-2′), 24.0 [CH(CH3)2], 12.5 (T CH3). 31P NMR (121 MHz,
CDCl3): δ 18.2. HRMS: [M + H]+ calcd for C16H28N2O7P, 391.1629;
found, 391.1628.
1′α-(Adenin-9-yl)-2′-deoxy-3′-O-diisopropylphosphono-
methyl-L-threose (11c).11 A solution of 11a (2.17 g, 4.32 mmol) in
satd NH3 in MeOH (300 mL) was stirred at rt for overnight. After
removing the volatiles, the crude residue was purified by column
chromatography (20:1 CH2Cl2/MeOH) to afford 11c (1.58 g, 92%
yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ 8.35 (s, 1H, H-
8), 8.31 (s, 1H, H-2), 6.48 (dd, J = 7.5, 2.4 Hz, 1H, H-1′), 6.10 (brs,
2H, NH2), 4.71−4.78 [m, 2H, CH(CH3)2], 4.45−4.48 (m, 1H, H-3′),
4.36 (d, J = 10.5 Hz, 1H, H-4a′), 4.06 (dd, J = 10.5, 4.3 Hz, 1H, H-
4b′), 3.71−3.75 (m, 2H, PCH2), 2.57−2.73 (m, 2H, H-2′), 1.28−1.35
[m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 155.5 (C-6),
153.0 (C-2), 149.7 (C-4), 139.5 (C-8), 119.5 (C-5), 83.4 (C-1′), 80.5
1′α-(Thymin-1-yl)-3′-O-diisopropylphosphonomethyl-L-
threose (10b).11 A solution of 9b (0.186 g, 0.364 mmol) in 0.4 mL of
acetonitrile was treated with LiOH (8.7 mg, 0.364 mmol) in 0.2 mL of
water and 0.4 mL of MeOH. The mixture was stirred at rt for 0.5 h,
and the reaction mixture was neutralized with acetic acid. The solvent
was removed under reduced pressure, and the residue was partitioned
between water and EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography (20:1 CH2Cl2/
MeOH) to afford 10b (126 mg, 85% yield) as a white foam. 1H NMR
(300 MHz, CDCl3): δ 10.59 (brs, 1H, NH), 7.41 (d, J = 1.2 Hz, H-6),
5.84 (s, 1H, H-1′), 5.67 (brs, 1H, OH), 4.64−4.77 [m, 2H,
CH(CH3)2], 4.39 (s, 1H, H-2′), 4.26−4.36 (m, 2H, H-4′), 4.14 (d,
J = 2.4 Hz, H-3′), 3.75 (d, J = 9.1 Hz, 2H, PCH2), 1.93 (d, J = 1.0 Hz,
3H, T CH3), 1.28−1.33 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz,
3
(d, JP,C = 11.2 Hz, C-3′), 73.7 (C-4′), 71.4, 71.3 [CH(CH3)2], 64.1
(d, 1JP,C = 169.6 Hz, 2H, PCH2), 38.5 (C-2′), 24.0, 23.9 [CH(CH3)2].
31P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M + H]+ calcd for
C16H27N5O5P, 400.1744; found, 400.1746.
1′α-(Adenin-9-yl)-2′-deoxy-3′-O-phosphonomethyl-L-
threose Triethylammonium Salt (PMDTA, 12a).11 To a solution
of 11c (30 mg, 0.075 mmol) and 2,6-lutidine (0.07 mL, 0.60 mmol) in
dry CH3CN (3 mL) was added bromotrimethylsilane (0.08 mL, 0.6
mmol) at 0 °C. The reaction mixture was stirred at rt overnight and
quenched with 1.0 M TEAB solution (1 mL). The solvent was
removed under reduced pressure. The residue was partitioned between
water and EtOAc/ether (1:1), the water layer was lyophilized, and the
residue was first purified by chromatography on a silica gel column
(10:1:0 to 10:5:1, CH2Cl2/MeOH/1 M TEAB) to give the crude
L
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
product. Further purification using preparative reverse phase HPLC
with gradient CH3CN in 0.05 M TEAB solution from 2% to 30% gave
12a (19 mg, 60%) as a white foam. 1H NMR (500 MHz, D2O): δ 8.55
(s, 1H, H-8), 8.15 (s, 1H, H-2), 6.35 (dd, J = 8.2, 2.2 Hz, 1H, H-1′),
4.51−4.53 (m, 1H, H-3′), 4.31 (d, J = 10.4 Hz, 1H, H-4a′), 4.06 (dd, J
= 10.3, 4.1 Hz, 1H, H-4b′), 3.45−3.53 (m, 2H, PCH2), 2.75−2.81 (m,
1H, H-2a′), 2.64 (d, J = 15.1 Hz, 1H, H-2b′). 13C NMR (125 MHz,
D2O): δ 155.0 (C-6), 152.0 (C-2), 148.2 (C-4), 140.9 (C-8), 117.9
(d, J = 10.0 Hz, H-4a′), 4.30−4.33 (m, 2H, H-4b′ and H-3′), 3.85−
3.95 (m, 2H, PCH2), 1.31−1.34 [m, 12H, CH(CH3)2]. 13C NMR
(125 MHz, CDCl3): δ 165.1 (PhCO), 162.4 (C-4), 155.0 (C-2), 145.1
(C-6), 133.7, 133.2, 129.9, 129.0, 128.8, 128.5, 127.5 (Ph), 96.2 (C-5),
90.9 (C-1′), 83.3 (3JP,C = 9.6 Hz, C-3′), 79.6 (C-2′), 74.5 (C-4′), 73.9
(C-3′), 71.3 [CH(CH3)2], 64.6 (1JP,C = 168.5 Hz, PCH2), 24.0
[CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.9. HRMS: [M +
H]+ calcd for C29H35N3O9P, 600.2105; found, 600.2130.
1′α-(N4-Benzoylcytosin-1-yl)-3′-O-diisopropylphosphono-
methyl-L-threose (16). This compound was prepared as described
for 10a. Obtained from 15 (260 mg, 0.43 mmol) as a white solid. Yield
3
(C-5), 82.9 (C-1′), 79.0 (d, JP,C = 10.3 Hz, C-3′), 73.3 (C-4′), 66.6
(d, 1JP,C = 151.3 Hz, PCH2), 36.5 (C-2′). 31P NMR (121 MHz, D2O):
δ 13.1. HRMS: [M − H]− calcd for C10H13N5O5P, 314.0660; found,
314.0657.
1
80% (72 mg). H NMR (500 MHz, CDCl3): δ 9.31 (brs, 1H, NH),
8.02 (d, J = 7.5 Hz, 1H, H-6), 7.93−7.95 (m, 2H, Ph), 7.57−7.60 (m,
3H, H-5 and Ph), 7.49 (t, J = 8.0 Hz, 2H, Ph), 5.87 (s, 1H, H-1′), 5.52
(brs, 1H, OH), 4.64−4.72 [m, 2H, CH(CH3)2], 4.52 (s, 1H, H-2′),
4.34−4.39 (m, 2H, H-4′), 4.18 (s, 1H, H-3′), 3.73−3.87 (m, 2H,
PCH2), 1.26−1.30 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz,
CDCl3): δ 166.7 (PhCO), 162.7 (C-4), 155.8 (C-2), 144.8 (C-6),
1′α-(Thymin-1-yl)-2′-deoxy-3′-O-phosphonomethyl-L-
threose Triethylammonium Salt (PMDTT, 12b).11 This com-
pound was prepared as described for 12a. Obtained from 11b (205
mg, 0.53 mmol), 2,6-lutidine (0.49 mL, 4.20 mmol), and
iodotrimethylsilane (0.60 mL, 4.20 mmol) as a white foam. Yield
65% (138 mg). 1H NMR (300 MHz, D2O): δ 7.70 (s, 1H, H-6), 6.11
(d, J = 6.3 Hz, 1H, H-1′), 4.30 (brs, 2H, H-4a′ and H-3′), 3.86 (d, J =
7.5 Hz, H-4b′), 3.51 (d, J = 9.1 Hz, PCH2), 2.50−2.55 (m, 1H, H-2a′),
2.20 (d, J = 15.1 Hz, 1H, H-2b′), 1.82 (s, 3H, T CH3). 13C NMR (75
MHz, D2O): δ 166.3 (C-4), 151.4 (C-2), 138.2 (C-6), 110.7 (C-5),
85.1 (C-1′), 79.4 (d, 3JP,C = 11.5 Hz, C-3′), 73.4 (C-4′), 64.6 (d, 1JP,C
= 157.6 Hz, PCH2), 36.4 (C-2′), 11.4 (T CH3). 31P NMR (121 MHz,
D2O): δ 15.3. HRMS: [M − H]− calcd for C10H14N2O7P, 305.0544;
found, 305.0555.
133.1, 132.9, 128.8, 127.8 (Ph), 96.3 (C-5), 94.6 (C-1′), 85.5 (3JP,C
=
10.4 Hz, C-3′), 78.7 (C-2′), 74.0 (C-4′), 71.2 [CH(CH3)2], 64.2 (d,
1JP,C = 168.8 Hz, PCH2), 23.9 [CH(CH3)2]. 31P NMR (121 MHz,
CDCl3): δ 18.5. HRMS: [M + H]+ calcd for C22H31N3O8P, 496.1843;
found, 496.1856.
1′α-(N4-Benzoylcytosin-1-yl)-2′-deoxy-3′-O-diisopropyl-
phosphonomethyl-L-threose (17). This compound was prepared
as described for 11a or 5. Obtained from 16 (60 mg, 0.12 mmol) or 20
(137 mg, 0.46 mmol) as a white foam, Yield 70% (41 mg) and 72%
(156 mg), respectively. 1H NMR (300 MHz, CDCl3): δ 8.63 (brs, 1H,
NH), 8.09 (d, J = 7.2 Hz, 1H, H-6), 7.91 (d, J = 7.6 Hz, 2H, Ph),
7.60−7.66 (m, 1H, Ph), 7.51−7.56 (m, 3H, H-5 and Ph), 6.21 (dd, J =
7.1 Hz, 1.8 Hz, 1H, H-1′), 4.66−4.79 [m, 2H, CH(CH3)2], 4.49 (dd, J
= 10.5, 1.2 Hz, 1H, H-4a′), 4.39 (t, J = 4.0 Hz, 1H, H-3′), 4.06 (dd, J =
10.5, 3.5 Hz, 1H, H-4b′), 3.57−3.70 (m, 2H, PCH2), 2.52−2.61 (m,
1H, H-2a′), 2.46 (d, J = 15.2 Hz, 1H, H-2b′), 1.28−1.36 [m, 12H,
CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 166.1 (PhCO), 162.1
(C-4), 155.4 (C-2), 145.3 (C-6), 133.1, 129.1, 127.4 (Ph), 95.6 (C-5),
87.8 (C-1′), 80.2 (3JP,C = 9.7 Hz, C-3′), 75.1 (C-4′), 71.2
1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-3′-O-tert-butyldi-
methylsilyl-L-threose (13). This compound was prepared using a
similar procedure as described for 7a. Obtained from 3 (3.17 g, 8.3
mmol), N4-benzoylcytosine (2.15 g, 10.0 mmol), BSA (5.09 mL, 20.8
mmol), and TMSOTf (4.52 mL, 25.0 mmol) as a white solid. Yield
84% (3.76 g). 1H NMR (500 MHz, CDCl3): δ 8.67 (s, 1H, NH), 8.11
(d, J = 7.4 Hz, H-6), 8.06−8.08 (m, 2H, Ph), 7.90−7.92 (m, 2H, Ph
and H-5), 7.45−7.62 (m, 7H, Ph), 6.26 (s, 1H, H-1′), 5.37 (s, 1H, H-
2′), 4.41 (d, J = 2.8 Hz, 1H, H-3′), 4.30−4.35 (m, 2H, H-4′), 0.85 [s,
9H, C(CH3)3], 0.16 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3). 13C NMR
(125 MHz, CDCl3): δ 165.0 (PhCO), 162.3 (PhCO), 154.9 (C-2),
145.5 (C-6), 133.6, 133.2, 129.9, 129.0, 128.5 (Ph), 127.5 (C-5), 91.1
(C-1′), 82.0 (C-2′), 77.6 (C-4′), 74.4 (C-3′), 25.6 [C(CH3)3], 17.9
[C(CH3)3]. HRMS: [M + H]+ calcd for C28H34N3O6Si, 536.2211;
found, 536.2225.
1
[CH(CH3)2], 63.8 (d, JP,C = 169.2 Hz, PCH2), 38.7 (C-2′), 24.0
[CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.3. HRMS: [M +
H]+ calcd for C22H31N3O7P, 480.1894; found, 480.1889.
1′α-(N4-Benzoylcytosin-1-yl)-3′-O-tert-butyldimethylsilyl-L-
threose (18). This compound was prepared as described for 10a.
Obtained from 13 (2.65 g, 4.95 mmol) as a white foam. Yield 71%
(1.52 g). 1H NMR (300 MHz, CDCl3): δ 9.25 (brs, 1H, NH), 8.05 (d,
J = 7.5 Hz, 1H, H-6), 7.92 (d, J = 7.6 Hz, 2H, Ph), 7.44−7.58 (m, 4H,
Ph and H-5), 5.88 (s, 2H, H-1′ and OH), 4.40 (dd, J = 9.4, 3.0 Hz, 1H,
H-4a′), 4.30 (s, 1H, H-2′), 4.25 (s, 1H, H-3′), 4.16 (d, J = 9.4 Hz, 1H,
H-4b′), 0.75 [s, 9H, C(CH3)3], 0.03 (s, 3H, SiCH3), −0.07 (s, 3H,
SiCH3). 13C NMR (75 MHz, CDCl3): δ 166.5 (PhCO), 162.4 (C-2),
155.7 (C-4), 145.3 (C-6), 13 2.9, 132.8, 128.7, 127.6 (Ph), 95.8 (C-5),
94.5 (C-1′), 80.8 (C-2′), 77.6 (C-4′), 76.3 (C-3′), 25.4 [C(CH3)3],
17.7 [C(CH3)3], −5.2, −5.3 (SiCH3). HRMS: [M + H]+ calcd for
C21H30N3O5Si, 432.1949; found, 432.1949.
1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-L-threose (14).18
This compound was prepared as described for 8a. Obtained from
13 (3.8 g, 7.0 mmol) and triethylamine trihydrofluoride (2.3 mL, 14
1
mmol) as a white solid. Yield 97% (2.9 g). H NMR (600 MHz,
DMSO-d6): δ 11.28 (s, 1H, NH), 8.22 (d, J = 7.5 Hz, 1H, H-6), 8.01−
8.05 (m, 4H, Ph), 7.70−7.73 (m, 1H, Ph), 7.63 (t, J = 7.4 Hz, 1H, Ph),
7.56−7.60 (m, 2H, Ph), 7.51−7.54 (m, 2H, Ph), 7.42 (d, J = 7.7 Hz,
1H, H-5), 6.00 (d, J = 0.5 Hz, 1H, H-1′), 5.83 (d, J = 2.8 Hz, 1H, OH),
5.40 (s, 1H, H-2′), 4.33−4.36 (m, H-3′ and H-4a′), 4.29 (dd, J = 9.9
Hz, 3.9 Hz, H-4b′). 13C NMR (150 MHz, DMSO-d6): δ 167.4
(PhCO), 164.6 (PhCO), 163.5 (C-4), 154.6 (C-2), 145.8 (C-6), 134.0,
133.2, 132.8, 129.6, 129.0, 128.5 (Ph), 95.7 (C-5), 91.0 (C-1′), 81.6
(C-2′), 76.7 (C-4′), 72.5 (C-3′). HRMS: [M + H]+ calcd for
C22H20N3O6, 422.1347; found, 422.1346.
1′α-(N4-Benzoylcytosin-1-yl)-2′-deoxy-3′-O-tert-butyldime-
thylsilyl-L-threose (19). This compound was prepared as described
for 11a. Obtained from 18 (1.24 g, 2.87 mmol) as a white foam. Yield
91% (1.1 g). 1H NMR (300 MHz, CDCl3): δ 8.98 (brs, 1H, NH), 8.10
(d, J = 7.5 Hz, 1H, H-6), 7.91 (d, J = 7.2 Hz, 2H, Ph), 7.56−7.61 (m,
1H, Ph), 7.46−7.51 (m, 3H, Ph and H-5), 6.15 (d, J = 6.6 Hz, 1H, H-
1′), 4.47 (t, J = 3.7 Hz, 1H, H-3′), 4.20 (d, J = 9.6 Hz, 1H, H-4a′), 4.16
(dd, J = 9.6, 3.3 Hz, 1H, H-4b′), 2.23−2.53 (m, 2H, H-2′), 0.79 [s, 9H,
C(CH3)3], 0.04 (s, 3H, SiCH3), −0.05 (s, 3H, SiCH3). 13C NMR (75
MHz, CDCl3): δ 166.6 (PhCO), 162.1 (C-4), 155.0 (C-2), 145.5 (C-
6), 132.9, 128.8, 128.5, 127.5 (Ph), 95.4 (C-5), 88.1 (C-1′), 78.8 (C-
4′), 71.0 (C-3′), 41.9 (C-2′), 25.5 [C(CH3)3], 17.7 [C(CH3)3], −5.1,
−5.2 (SiCH3). HRMS: [M + H]+ calcd for C21H30N3O4Si, 416.2000;
found, 416.2003.
1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-3′-O-diisopropyl-
phosphonomethyl-L-threose (15). A suspension of N6-benzoylcy-
tosine (97 mg, 0.45 mmol) in anhydrous CH3CN (2 mL) was treated
with BSA (0.25 mL, 1.01 mmol) and heated to 65 °C. After stirring for
1 h, a solution of 6 (100 mg, 0.23 mmol) in anhydrous CH3CN (1
mL) was added followed by TMSOTf (0.12 mL, 0.68 mmol) at 0 °C.
The mixture was stirred at 0 °C for overnight. The reaction mixture
was poured into an ice-cold 50 mL of satd aq NaHCO3 soln/EtOAc
1:1. The organic layer was washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by column chromatography
(50:1, DCM/MeOH) gave 15 (54 mg, 40% yield) as a white foam. 1H
NMR (500 MHz, CDCl3): δ 8.72 (brs, 1H, NH), 8.04−8.06 (m, 3H,
H-6 and Ph), 7.90 (d, J = 7.4 Hz, Ph), 7.59−7.63 (m, 3H, H-5 and
Ph), 7.52 (t, J = 7.9 Hz, 2H, Ph), 7.47 (t, J = 8.1 Hz, 2H, Ph), 6.32 (s,
1H, H-1′), 5.51 (s, 1H, H-2′), 4.70−4.76 [m, 2H, CH(CH3)2], 4.52
1′α-(N4-Benzoylcytosin-1-yl)-2′-deoxy-L-threose (20). This
compound was prepared as described for 8a. Obtained from 19
1
(1.14 g, 2.74 mmol) as a white foam. Yield 79% (0.65 g). H NMR
M
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
(300 MHz, CDCl3): δ 9.09 (brs, 1H, NH), 8.09 (d, J = 7.5 Hz, 1H, H-
6), 7.88 (d, J = 7.2 Hz, 2H, Ph), 7.55−7.60 (m, 1H, Ph), 7.40−7.50
(m, 3H, Ph and H-5), 6.00 (d, J = 6.6 Hz, 1H, H-1′), 4.61 (t, J = 3.7
Hz, 1H, H-3′), 4.33 (d, J = 9.6 Hz, 1H, H-4a′), 4.09 (dd, J = 9.6, 3.3
Hz, 1H, H-4b′), 2.71 (d, J = 14.9 Hz, 1H, H-2a′), 2.42−2.50 (m, 1H,
H-2b′). 13C NMR (75 MHz, CDCl3): δ 166.4 (PhCO), 162.2 (C-4),
155.2 (C-2), 145.6 (C-6), 132.9, 128.8, 127.7 (Ph), 95.6 (C-5), 89.1
(C-1′), 78.5 (C-4′), 70.0 (C-3′), 41.3 (C-2′). HRMS: [M + H]+ calcd
for C15H16N3O4, 302.1135; found, 302.1140.
(0.30 g, 67%) as a white foam. H NMR (300 MHz, CDCl3): δ 8.22
(s, 1H, H-8), 8.03 (d, J = 7.2 Hz, 2H, Ph), 7.60 (t, J = 7.3 Hz, 1H, Ph),
7.45 (t, J = 7.5 Hz, 2H, Ph), 6.29 (s, 1H, H-1′), 5.54 (s, 1H, H-2′),
5.50 (brs, 2H, NH2), 4.52 (s, 1H, H-3′), 4.33 (dd, J = 9.8, 3.7 Hz, 1H,
H-4a′), 4.25 (d, J = 9.8 Hz, 1H, H-4b′). 0.88 [s, 9H, C(CH3)3], 0.15
(s, 3H, SiCH3), 0.11 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ
164.9 (PhCO), 159.1 (C-2), 153.2 (C-4), 151.0 (C-6), 141.0 (C-8),
133.7, 129.7, 128.6, 128.5 (Ph), 125.0 (C-5), 87.7 (C-1′), 82.5 (C-2′),
76.5 (C-4′), 75.1 (C-3′), 25.5 [C(CH3)3], 17.8 [C(CH3)3], −5.0, −5.3
(SiCH3). HRMS: [M + H]+ calcd for C22H29ClN5O4Si, 490.1672;
found, 490.1681.
1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-diisopropylphosphono-
methyl-L-threose (21).11 This compound was prepared as described
for 11c. Obtained from 17 (177 mg, 0.37 mmol) as a white foam.
Yield 88% (122 mg). 1H NMR (300 MHz, CDCl3): δ 7.68 (d, J = 7.4
Hz, 1H, H-6), 6.19 (dd, J = 7.4, 1.9 Hz, 1H, H-1′), 5.78 (d, J = 7.5 Hz,
1H, H-5), 4.61−4.76 [m, 2H, CH(CH3)2], 4.35 (dd, J = 10.4, 1.3 Hz,
1H, H-4a′), 4.27−4.30 (m, 1H, H-3′), 3.93 (dd, J = 10.4, 3.6 Hz, 1H,
H-4b′), 3.63 (d, J = 9.2 Hz, 2H, PCH2), 2.44−2.54 (m, 1H, H-2a′),
2.89 (d, J = 15.1 Hz, 1H, H-2b′), 1.26−1.33 [m, 12H, CH(CH3)2].
13C NMR (75 MHz, CDCl3): δ 165.8 (C-4), 156.1 (C-2), 141.7 (C-
1′α-(N2-Acetyl-O6-diphenylcarbamoyl-9-yl)-2′-O-benzoyl-L-
threose (23a).18 This compound was prepared as described for 8a.
Obtained from 22a (6.0 g, 8.5 mmol) and triethylamine trihydro-
fluoride (2.76 mL, 16.9 mmol) as a white solid. Yield 96% (4.8 g). 1H
NMR (500 MHz, CDCl3): δ 8.45 (s, 1H, NH), 8.06 (s, 1H, H-8),
8.02−8.04 (m, 2H, Ph), 7.58−7.62 (m, 1H, Ph), 7.34−7.48 (m, 10H,
Ph), 7.36 (t, J = 7.8 Hz, 1H, Ph), 7.25 (brs, 1H, Ph), 5.93 (d, J = 1.7
Hz, 1H, H-1′), 5.86 (s, 1H, H-2′), 5.58 (brs, 1H, OH), 4.71−4.73 (m,
1H, H-3′), 4.44 (dd, J = 9.7, 4.3 Hz, 1H, H-4a′), 4.33 (dd, J = 9.7, 5.6
Hz, 1H, H-4b′), 2.24 (s, 3H, CH3CO). 13C NMR (125 MHz, CDCl3):
δ 168.6 (CH3CO), 166.2 (PhCO), 156.3, 154.0 (C-4), 151.4, 150.2,
144.5 (C-8), 141.6, 133.9, 129.9, 129.2, 128.7, 128.6, 121.9 (C-5), 91.0
(C-1′), 84.5 (C-2′), 75.5 (C-4′), 75.4 (C-3′), 24.88 (CH3CO).
HRMS: [M + H]+ calcd for C31H27N6O7, 595.1936; found, 595.1943.
1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-L-threose
(23b). This compound was prepared as described for 8a. Obtained
from 22b (220 mg, 0.45 mmol) and triethylamine trihydrofluoride
(0.15 mL, 0.90 mmol) as a white foam. Yield 97% (165 mg). 1H NMR
(300 MHz, CDCl3): δ 7.98 (s, 1H, H-8), 8.01 (d, J = 7.2 Hz, 2H, Ph),
7.63 (t, J = 7.3 Hz, 1H, Ph), 7.47 (t, J = 7.5 Hz, 2H, Ph), 5.92 (d, J =
1.7 Hz, 1H, H-1′), 5.58 (s, 1H, H-2′), 5.42 (brs, 2H, NH2), 4.57 (d, J
= 3.2 Hz, 1H, H-3′), 4.33 (d, J = 9.8 Hz, 1H, H-4a′), 4.22 (dd, J = 9.8,
3.7 Hz, 1H, H-4b′). 13C NMR (75 MHz, CDCl3): δ 165.7 (PhCO),
158.4 (C-2), 152.5 (C-4), 151.9 (C-6), 142.1 (C-8), 134.0, 129.8,
128.6, 128.4 (Ph), 126.0 (C-5), 90.4 (C-1′), 83.8 (C-2′), 75.8 (C-4′),
74.6 (C-3′). HRMS: [M + H]+ calcd for C16H15ClN5O4, 376.0807;
found, 376.0808.
3
6), 93.6 (C-5), 86.6 (C-1′), 80.3 (d, JP,C = 11.6 Hz, C-3′), 74.1 (C-
4′), 71.3, 71.1 [CH(CH3)2], 63.7 (d, 1JP,C = 170.2 Hz, PCH2), 38.6 (C-
2′), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.4. HRMS:
[M + H]+ calcd for C15H27N3O6P, 376.1632; found, 376.1629.
1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-phosphonomethyl-L-
threose Triethylammonium Salt (PMDTC, 22).11 This compound
was prepared as described for 12a. Obtained from 21 (120 mg, 0.32
mmol), 2,6-lutidine (0.30 mL, 2.56 mmol), and iodotrimethylsilane
(0.36 mL, 2.56 mmol) as a white foam. Yield 48% (60 mg). 1H NMR
(600 MHz, D2O): δ 7.95 (d, J = 7.5 Hz, 1H, H-6), 6.19 (dd, J = 8.0,
2.0 Hz, 1H, H-1′), 6.04 (d, J = 7.6 Hz, 1H, H-5), 4.40 (d, J = 10.2 Hz,
1H, H-4a′), 4.36−4.38 (m, 1H, H-3′), 3.95 (dd, J = 10.4, 3.6 Hz, 1H,
H-4b′), 3.45−3.52 (m, 2H, PCH2), 2.52−2.58 (m, 1H, H-2a′), 2.26
(d, J = 15.2 Hz, 1H, H-2b′). 13C NMR (150 MHz, D2O): δ 165.8 (C-
4), 157.3 (C-2), 142.4 (C-6), 95.6 (C-5), 85.9 (C-1′), 79.2 (d, 3JP,C
=
11.6 Hz, C-3′), 73.6 (C-4′), 64.9 (d, 1JP,C = 155.3 Hz, PCH2), 36.6 (C-
2′). 31P NMR (121 MHz, D2O): δ 14.6. HRMS: [M − H]− calcd for
C9H13N3O6P, 290.0547; found, 290.0545.
1′α-(N2-Acetyl-O6-diphenylcarbamoyl-9-yl)-2′-O-benzoyl-3′-
O-tert-butyldimethylsilyl-L-threose (22a). A suspension of N2-
acetyl-O6-(diphenylcarbamoyl)guanine (9.94 g, 25.6 mmol) in 60 mL
of (CH2Cl)2 was treated with BSA (13.77 mL, 56.3 mmol) and heated
to 80 °C for 1 h. The solvent was evaporated and replaced with 30 mL
of dry toluene and TMSOTf (5.09 mL, 28.2 mmol), and a solution of
3 (4.87 g, 12.8 mmol) in 30 mL of dry toluene was added. The mixture
was heated to 80 °C for 2 h, cooled to rt, and poured into an ice-cold
mixture of EtOAc and aq satd NaHCO3 with stirring. The organic
layer was separated and washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
column chromatography (2:1 to 3:2, hexane/EtOAc) gave 22a (6.0 g,
1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-3′-O-diiso-
propylphosphonomethyl-L-threose (24). This compound was
prepared using a similar procedure as described for 5. Obtained from
23b (85 mg, 0.23 mmol), triflate diisopropylphosphonomethanol (148
mg, 0.45 mmol), and NaH (60% in mineral oil, 18 mg, 0.45 mmol) as
1
a white foam. Yield 67% (84 mg). H NMR (300 MHz, CDCl3): δ
8.12 (s, 1H, H-8), 8.05 (d, J = 7.2 Hz, 2H, Ph), 7.64 (t, J = 7.4 Hz, 1H,
Ph), 7.47 (t, J = 7.9 Hz, 2H, Ph), 6.22 (d, J = 1.3 Hz, 1H, H-1′), 5.85
(s, 1H, H-2′), 5.33 (brs, 2H, NH2), 4.71−4.82 [m, 2H, CH(CH3)2],
4.42−4.45 (m, 2H, H-3′ and H-4a′), 4.33 (dd, J = 10.7, 4.7 Hz, 1H, H-
4b′), 3.86−4.06 (m, 2H, PCH2), 1.30−1.37 [m, 12H, CH(CH3)2]. 13
C
1
NMR (75 MHz, CDCl3): δ 165.1 (PhCO), 159.2 (C-2), 153.4 (C-4),
66% yield) as a white foam. H NMR (500 MHz, CDCl3): δ 8.34 (s,
151.4 (C-6), 141.1 (C-8), 134.0, 129.9, 128.7, 128.6 (Ph), 125.3 (C-
1H, H-8), 8.05−8.07 (m, 2H, Ph), 7.95 (brs, 1H, NH), 7.65−7.24 (m,
13H, Ph), 6.31 (s, 1H, H-1′), 5.55 (s, 1H, H-2′), 4.54 (brs, 1H, H-3′),
4.34 (dd, J = 9.9, 3.7 Hz, 1H, H-4a′), 4.30 (d, J = 9.8 Hz, 1H, H-4b′),
2.53 (s, 3H, CH3CO), 0.88 [s, 9H, C(CH3)3], 0.16 (s, 3H, SiCH3),
0.09 (s, 3H, SiCH3). 13C NMR (125 MHz, CDCl3): δ 171.4
(CH3CO), 165.0 (PhCO), 156.2, 154.4(C-4), 152.2, 150.2, 142.6 (C-
8), 134.0, 129.8, 129.2, 128.7, 127.2 (Ph), 120.6 (C-5), 88.4 (C-1′),
82.7 (C-2′), 76.6 (C-4′), 75.1 (C-3′), 25.6 [C(CH3)3], 21.0 (CH3CO),
14.2 [C(CH3)3]. HRMS: [M + H]+ calcd for C37H41N6O7Si, 709.2800;
found, 709.2806.
1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-3′-O-tert-bu-
tyldimethylsilyl-L-threose (22b). To a solution of 3 (0.35g, 0.92
mmol), 2-amino-6-chloro-9H-purine (0.17 g, 1.01 mmol), and DBU
(0.41 mL, 2.76 mmol) in dry MeCN (9 mL) was added dropwise
TMSOTf (0.66 mL, 3.68 mmol) at 0 °C. The resulting clear-brown
solution was stirred for 1.5 h at 70 °C, after which it was cooled to
room temperature and aq satd NaHCO3 (20 mL) was added. The
aqueous phase was extracted with EtOAc, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was purified by
column chromatography (10:1 to 5:1, hexane/EtOAc) to afford 22b
3
5), 88.2 (C-1′), 84.1 (d, JP,C = 10.6 Hz, C-3′), 79.8 (C-2′), 73.3 (C-
1
4′), 71.5 [CH(CH3)2], 64.7 (d, JP,C = 168.5 Hz, PCH2). 23.9
[CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.8. HRMS: [M +
H]+ calcd for C23H30ClN5O7P, 554.1566; found, 554.1568.
1′α-(2-Amino-6-chloropurin-9-yl)-3′-O-diisopropylphospho-
nomethyl-L-threose (25). A solution of 24 (5.0 g, 9.03 mmol) in 2
N NH3 in MeOH (150 mL) was stirred at rt for 2 h. The mixture was
concentrated under reduced pressure, and the residue was purified by
column chromatography (20:1 CH2Cl2/MeOH) to give compound 25
1
(3.0 g, 75% yield) as a white foam. H NMR (300 MHz, CDCl3): δ
8.02 (s, 1H, H-8), 5.91 (d, J = 2.7 Hz, 1H, H-1′), 5.72 (brs, 3H, NH2
and OH), 4.76 (s, 1H, H-2′), 4.62−4.73 [m, 2H, CH(CH3)2], 4.25−
4.29 (m, 3H, H-3′ and H-4′), 3.76−3.91 (m, 2H, PCH2), 1.26−1.33
[m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 159.2 (C-2),
153.2 (C-4), 151.1 (C-6), 141.1 (C-8), 125.0 (C-5), 90.4 (C-1′), 86.4
(3JP,C = 8.9 Hz, C-3′), 78.9 (C-2′), 72.3 (C-4′), 71.8 [CH(CH3)2],
64.8 (1JP,C = 167.8 Hz, PCH2). 23.9 [CH(CH3)2]. 31P NMR (121
MHz, CDCl3): δ 18.6. HRMS: [M + H]+ calcd for C16H26ClN5O6P,
450.1304; found, 450.1301.
N
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1′α-(2-Amino-6-chloropurin-9-yl)-2′-deoxy-3′-O-diisopro-
pylphosphonomethyl-L-threose (26). This compound was pre-
pared as described for 11a. Obtained from 25 (0.61 g, 1.35 mmol) as a
white foam. Yield 68% (0.40 g). 1H NMR (300 MHz, CDCl3): δ 8.20
(s, 1H, H-8), 6.30 (dd, J = 6.8, 3.1 Hz, 1H, H-1′), 5.30 (brs, 2H, NH2),
4.65−4.83 [m, 2H, CH(CH3)2], 4.50 (brs, 1H, H-3′), 4.33 (d, J = 10.5
Hz, 1H, H-4a′), 4.04 (dd, J = 10.4, 4.2 Hz, 1H, H-4b′). 3.67−3.80 (m,
2H, PCH2), 2.56−2.70 (m, 2H, H-2′), 1.29−1.36 [m, 12H,
CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 158.7 (C-2), 153.2
(C-4), 151.1 (C-6), 141.0 (C-8), 125.1 (C-5), 83.1 (C-1′), 80.0 (d,
mmol) in 4.5 mL of anhydrous CH2Cl2 at −78 °C was treated with
diethylaminosulfur trifluoride (DAST) (0.12 mL, 0.908 mmol). The
reaction mixture was allowed to warm to rt and was stirred overnight.
The resulting solution was then carefully poured into an ice-cold satd
aq sodium bicarbonate (10 mL) and extracted with CH2Cl2 (3 × 10
mL). The combined organic extracts were washed with brine, dried
over NaSO4, filtered, and concentrated. The residue was purified by
column chromatography (30:1 to 15:1, DCM/MeOH) to obtain 28
1
(60 mg, 63% yield) as a white foam. H NMR (500 MHz, CDCl3): δ
2
8.38 (s, 1H, H-2), 8.11 (s, 1H, H-8), 6.38 (d, JF,H = 19.5 Hz, 1H, H-
3JP,C = 10.0 Hz, C-3′), 73.4 (C-4′), 71.1 [CH(CH3)2], 63.8 (d, 1JP,C
=
1
1′), 5.77 (brs, 2H, NH2), 5.48 (d, JF,H = 49.0 Hz, 1H, H-2′), 4.71−
167.8 Hz, PCH2), 37.8 (C-2′), 23.7 [CH(CH3)2]. 31P NMR (121
MHz, CDCl3): δ 18.1. HRMS: [M + H]+ calcd for C16H26ClN5O5P,
434.1354; found, 434.1354.
4.75 [m, 2H, CH(CH3)2], 4.43−4.48 (m, 2H, H-3′ and H-4a′), 4.31−
4.34 (m, 1H, H-4b′), 3.76−3.82 (m, 2H, PCH2), 1.29−1.34 [m, 12H,
CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 155.4 (C-6), 153.3 (C-
1
1′α-(Guanin-9-yl)-2′-deoxy-3′-O-phosphonomethyl-L-
threose Triethylammonium Salt (PMDTG, 27). To a solution of
26 (184 mg, 0.42 mmol) in anhydrous CH3CN (9 mL) was added 2,6-
lutidine (0.40 mL, 3.39 mmol) and bromotrimethylsilane (0.45 mL,
3.39 mmol) at 0 °C. After stirring at room temperature for overnight,
the solvent was removed under reduced pressure and coevaporated
three times with anhydrous methanol (3 × 3 mL). The residue was
dissolved in anhydrous MeOH (4 mL), and 2-mercaptoethanol (0.15
mL, 2.12 mmol) and NaOMe (5.4 M in MeOH, 0.39 mL, 2.12 mmol)
were added. The mixture was refluxed for 19 h, cooled, quenched with
2 mL of 1 M TEAB buffer, and evaporated. The residue was
partitioned between water and EtOAc, and the aqueous phase was
lyophilized and the residue was purified by RP-HPLC running a
gradient of CH3CN in 0.1 M TEAB buffer solution from 2% to 30% to
2), 149.6 (C-4), 139.2 (C-8), 119.6 (C-5), 96.2 (d, JF,C = 186.4 Hz,
C-2′), 87.9 (d, 2JF,C = 36.7 Hz, C-1′), 83.1 (C-3′, 3JP,C = 9.8 Hz, 2JF,C
27.3 Hz), 72.8 (C-4′), 71.6 [CH(CH3)2, 2JP,C = 6.0 Hz] 64.9 (d, 1JP,C
=
=
169.0 Hz, PCH2), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ
17.4. HRMS: [M + H]+ calcd for C16H26FN5O5P, 418.1650; found,
418.1647.
(1′α,2′R)-1′-(Adenin-9-yl)-2′-deoxy-2′-fluoro-3′-O-phospho-
nomethyl-L-threose Triethylammonium Salt (2′-F-PMDTA, 31).
This compound was prepared as described for 12a. Obtained from 30
(60 mg, 0.14 mmol), 2,6-lutidine (0.13 mL, 1.2 mmol), and
bromotrimethylsilane (0.15 mL, 1.2 mmol) as a white foam. Yield
1
62% (48 mg). H NMR (600 MHz, D2O): δ 8.38 (s, 1H, H-8), 8.16
(s, 1H, H-2), 6.33 (d, 2JF,H = 19.5 Hz, 1H, H-1′), 5.57 (d, 1JF,H = 49.0
Hz, 1H, H-2′), 4.54−4.56 (m, 1H, H-3′), 4.46 (d, J = 12.0 Hz, H-4a′),
4.33−4.35 (m, 1H, H-4b′), 3.54−3.61 (m, 2H, PCH2). 13C NMR (150
MHz, D2O): δ 155.44 (C-6), 152.68 (C-2), 148.41 (C-4), 140.81 (C-
1
afford 27 (90 mg, 49%) as a yellowish foam. H NMR (300 MHz,
D2O): δ 8.05 (s, 1H, H-8), 6.06 (dd, J = 7.9, 2.0 Hz, 1H, H-1′), 4.39
(brs, 1H, H-3′), 4.23 (d, J = 10.4 Hz, 1H, H-4a′), 3.94 (dd, J = 10.4,
4.0 Hz, 1H, H-4b′). 3.55 (d, J = 9.3 Hz, 2H, PCH2), 2.60−2.70 (m,
1H, H-2a′), 2.49 (d, J = 15.4 Hz, H-2b′). 13C NMR (75 MHz, D2O): δ
158.3 (C-6), 153.4 (C-2), 150.7 (C-4), 138.1 (C-8), 115.4 (C-5), 82.8
(C-1′), 79.6 (3JP,C = 12.0 Hz, C-3′), 73.2 (C-4′), 65.2 (1JP,C = 155.2
Hz, PCH2), 36.6 (C-2′). 31P NMR (121 MHz, D2O): δ 15.1. HRMS:
[M − H]− calcd for C10H13N5O6P, 330.0609; found, 330.0604.
1′α-(N6-Benzoyladenin-9-yl)-3′-O-diisophosphonomethyl-L-
erythrose (29). To a solution of 10a (137 mg, 0.264 mmol) in 6 mL
of dichloromethane was added a solution of Dess−Martin periodinane
(15% in CH2Cl2, 0.82 mL, 0.396 mmol) at 0 °C. The reaction mixture
was stirred at rt for 1 h (a white solid formed). To the reaction mixture
was added a solution of satd aq sodium thiosulfate and satd aq
NaHCO3 (1:1, 10 mL), and the mixture was stirred at rt for 20 min.
After separation, the organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated to afford the crude ketone 28 (134
mg, 98%) as a white foam. The crude ketone was used for the next
step without further purification.
1
2
8), 118.19 (C-5), 96.67 (d, JF,C = 184.0 Hz, C-2′), 87.36 (d, JF,C
=
3
2
37.7 Hz, C-1′), 82.12 (C-3′, JP,C = 10.6 Hz, JF,C = 27.2 Hz), 73.08
(C-4′), 67.32 (d, JP,C = 150.9 Hz, PCH2). 31P NMR (121 MHz,
1
D2O): δ 12.7. HRMS: [M-H]− calcd for C10H12FN5O5P, 332.0565;
found, 332.0558.
1′α-(Adenin-9-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-alaninate)]-
(phenoxy)methylphosphonoamidate]}-L-threose (32a). Ob-
tained from PMDTA (28 mg, 0.067 mmol, Et3N salt), L-alanine
isopropyl ester hydrochloride (23 mg, 0.134 mmol), and PhOH (32
mg, 0.336 mmol) as a white solid. Yield 47% (16 mg). 1H NMR (500
MHz, acetone-d6): δ 8.44 (s, 1H, H-8), 8.20 (s, 1H, H-2), 7.16−7.36
(m, 5H, Ph), 6.45−6.47 (m, 1H, H-1′), 4.89−4.95 [m, 1H,
CH(CH3)2], 4.66−4.85 (m, 1H, Ala-NH), 4.52−4.56 (m, 1H, H-3′),
4.37−4.42 (m, 1H, H-4a′), 4.02−4.08 (m, 4H, H-4b′, PCH2 and Ala-
CH), 2.76−2.84 (m, 1H, H-2a′), 2.56−2.63 (m, 1H, H-2b′), 1.25−
1.28 (m, 3H, Ala-CH3), 1.15−1.19 [m, 6H, CH(CH3)2]. 13C NMR
(125 MHz, acetone-d6): δ 174.1, 173.7 (Ala-CO), 157.0 (C-6), 153.7,
153.6 (C-2), 151.7, 151.6 (Ph), 150.62, 150.59 (C-4), 140.6, 140.5 (C-
8), 130.4, 130.31, 125.32, 125.3, 121.8, 121.73, 121.67, 121.6 (Ph),
120.02, 119.98 (C-5), 84.2, 83.9 (C-1′), 81.88, 81.85 (3JP,C = 13.4, 13.0
Hz, C-3′), 74.36, 73.90 (C-4′), 69.14, 69.08 [CH(CH3)2], 65.99, 65.91
(1JP,C = 156.5, 155.7 Hz, PCH2), 50.5, 50.4 (Ala-CH), 39.10, 39.07 (C-
2′), 21.84, 21.81 [CH(CH3)2], 21.3, 21.0 (Ala-CH3). 31P NMR (121
MHz, acetone-d6): δ 21.6, 20.6. HRMS: [M + H]+ calcd for
C22H30N6O6P, 505.1959; found, 505.1965.
1′α-(Adenin-9-yl)-2′-deoxy-3′-O-{[N-(diisoamyl-L-
aspartate)](phenoxy)methylphosphonoamidate}-L-threose
(32b). Obtained from PMDTA (26 mg, 0.062 mmol, Et3N salt), L-
aspartic acid isoamyl ester hydrochloride (39 mg, 0.125 mmol), and
PhOH (29 mg, 0.312 mmol) as a white solid. Yield 66% (27 mg). 1H
NMR (500 MHz, acetone-d6): δ 8.40 (s, 1H, H-8), 8.19 (s, 1H, H-2),
7.14−7.37 (m, 5H, Ph), 6.70 (brs, 2H, NH2), 6.44−6.47 (m, 1H, H-
1′), 4.73−4.87 (m, 1H, Asp-NH), 4.51−4.57 (m, 1H, H-3′), 4.36−
4.42 (m, 2H, H-4a′ and Asp-CH), 3.97−4.12 [m, 7H, H-4b′, PCH2
and OCH2CH2CH(CH3)2], 2.53−2.84 (m, 4H, Asp-CH2, H-2′),
1.58−1.68 [m, 2H, OCH2CH2CH(CH3)2], 1.42−1.50 [m, 4H,
OCH2CH2CH(CH3)2], 0.86−0.89 [m, 12H, OCH2CH2CH(CH3)2].
13C NMR (125 MHz, acetone-d6): δ 172.7, 172.4, 171.1 (Asp-CO),
Crude ketone was taken up in 7.5 mL of dry methanol and treated
with sodium borohydride (20 mg, 0.527 mmol) at 0 °C. The reaction
mixture was stirred at rt for 1 h and quenched by the dropwise
addition of satd aq NaHCO3 (2 mL). The reaction mixture was
extracted with EtOAc, and the organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography (20:1 DCM/MeOH) to obtain
29 (118 mg, 86% yield) as a white foam. 1H NMR (600 MHz,
CDCl3): δ 9.18 (brs, 1H, NH), 8.77 (s, 1H, H-2), 8.46 (s, 1H, H-8),
8.04−8.05 (m, 2H, Ph), 7.60−7.61 (m, 1H, Ph), 7.52−7.54 (m, 2H,
Ph), 6.48 (d, J = 4.8 Hz, 1H, H-1′), 5.05 (brs, 1H, OH), 4.71−4.81 [m,
2H, CH(CH3)2)], 4.38 (brs, 1H, H-2′), 4.35−4.38 (m, 1H, H-3′),
4.28−4.31 (m, 1H, H-4a′), 4.11−4.19 (m, 1H, H-4b′), 3.77−4.07 (m,
2H, PCH2), 1.26−1.36 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz,
CDCl3): δ 164.6 (PhCO), 152.6 (C-2), 151.8 (C-4), 149.0 (C-6),
143.5 (C-8), 134.0, 132.6, 128.8, 127.9 (Ph), 122.1 (C-5), 83.9 (C-1′),
3
81.6 (d, JP,C = 6.5 Hz, C-3′), 72.1, 71.9 [CH(CH3)2], 71.1 (C-2′),
1
70.1 (C-4′), 66.0 (d, JP,C = 168.6 Hz, PCH2), 24.0 [CH(CH3)2]. 31P
NMR (121 MHz, CDCl3): δ 19.2. HRMS: [M + H]+ calcd for
C23H31N5O7P, 520.1955; found, 520.1959.
157.0, 156.9 (C-6), 153.7, 153.6 (C-2), 151.68, 151.66 (Ph), 150.63
(C-4), 140.6, 140.5 (C-8), 130.4, 130.3, 125.40, 125.38, 121.82,
(1′α,2′R)-1′-(Adenin-9-yl)-2′-deoxy-2′-fluoro-3′-O-diiso-
phosphonomethyl-L-threose (30). A solution of 29 (118 mg, 0.227
O
DOI: 10.1021/acs.jmedchem.6b01260
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Journal of Medicinal Chemistry
Article
121.78, 121.72, 121.68 (Ph), 120.1, 120.0 (C-5), 84.2, 83.9 (C-1′),
acetone-d6): δ 22.2, 21.2. HRMS: [M + H]+ calcd for C30H45N3O10P,
638.2837; found, 638.2820.
81.9 (3JP,C = 13.5 Hz, C-3′), 74.4, 73.9 (C-4′), 65.94, 65.90 (1JP,C
=
1′α-(Thymin-1-yl)-2′-deoxy-3′-O-[N, N′-bis(n-propyl-L-
phenylalaninate)methylphosphonobisamidate]-L-threose
(33c). Obtained from PMDTT (16 mg, 0.038 mmol, Et3N salt) and L-
phenylalanine-O-n-Pr ester hydrochloride (56 mg, 0.23 mmol) as a
156.6, 155.9 Hz, PCH2), 64.6, 64.5, 63.8 [OCH2CH2CH(CH3)2],
51.54, 51.46 (Asp-CH), 39.6, 39.1 (Asp-CH2), 37.93, 37.90, 37.87
[OCH2CH2CH(CH3)2], 25.64, 25.62, 25.55, 25.53 [OCH2CH2CH-
(CH3)2], 22.7, 22.6 [OCH2CH2CH(CH3)2]. 31P NMR (121 MHz,
acetone-d6): δ 22.2, 21.2. HRMS: [M + H]+ calcd for C30H44N6O8P,
647.2953; found, 647.2953.
1
white solid. Yield 34% (9 mg). H NMR (500 MHz, acetone-d6): δ
9.92 (s, 1H, NH), 7.61−7.62 (m, 1H, H-6), 7.20−7.29 (m, 10H, Ph),
6.16, 6.15 (d, J = 2.8 Hz, 1H, H-1′), 4.09−4.28 (m, 4H, H-3′, H-4a′
and Phe-CH), 3.98−4.05 (m, 4H, OCH2CH2CH3), 3.74−3.80 (m,
2H, NH and H-4b′), 3.44−3.49 (m, 1H, NH), 3.38 (dd, J = 8.7, 1.1
Hz, 2H, PCH2), 2.82−3.08 (m, 4H, Phe-CH2), 2.52−2.57 (m, 1H, H-
2a′), 2.00−2.06 (m, 1H, H-2b′), 1.84 (d, J = 1.2 Hz, 3H, T CH3),
1.57−1.66 (m, 4H, OCH2CH2CH3), 0.86−0.93 (m, 6H,
OCH2CH2CH3). 13C NMR (125 MHz, acetone-d6): δ 173.6, 173.3
(Phe-CO), 164.2 (C-4), 151.3 (C-2), 138.0, 137.8 (Ph), 137.2 (C-6),
130.4, 130. 3, 128.90, 128.86, 127.30, 127.27 (Ph), 110.5 (C-5), 85.1
(C-1′), 80.9 (3JP,C = 12.1 Hz, C-3′), 73.4 (C-4′), 66.94, 66.92
(OCH2CH2CH3), 66.6 (1JP,C = 136.9 Hz, PCH2), 54.9, 54.6 (Phe-
CH), 41.2, 41.0 (Phe-CH2), 38.3 (C-2′), 22.4, 22.3 (OCH2CH2CH3),
12.4 (T CH3), 10.5, 10.4 (OCH2CH2CH3). 31P NMR (121 MHz,
acetone-d6): δ 19.6. HRMS: [M + H]+ calcd for C34H46N4O9P,
685.2997; found, 685.3006.
1′α-(Adenin-9-yl)-2′-deoxy-3′-O-{[N,N-bis(n-propyl-L-phe-
nylalaninate)] methylphosphonobisamidate}-L-threose (32c).
Obtained from PMDTA (23 mg, 0.055 mmol, Et3N salt) and L-
phenylalanine-O-n-Pr ester hydrochloride (81 mg, 0.331 mmol) as a
1
white solid. Yield 73% (28 mg). H NMR (500 MHz, acetone-d6): δ
8.47 (s, 1H, H-8), 8.20 (s, 1H, H-2), 7.18−7.29 (m, 10H, Ph), 6.42−
6.44 (m, 1H, H-1′), 4.2−4.35 (m, 3H, Phe-CH, H-3′ and H-4a′),
4.04−4.15 (m, 1H, Phe-CH), 3.95−4.03 (m, 5H, OCH2CH2CH3 and
H-4b′), 3.85−3.95 (m, 1H, Phe-NH), 3.47−3.49 (m, 2H, PCH2),
3.35−3.45 (m, 1H, Phe-NH), 2.80−3.15 (m, 4H, Ph-CH2), 2.68−2.75
(m, 1H, H-2a′), 2.40−2.45 (m, 1H, H-2b′), 1.55−1.64 (m, 4H,
OCH2CH2CH3), 0.90−0.91 (m, 6H, OCH2CH2CH3). 13C NMR (125
MHz, acetone-d6): δ 173.9, 173.4 (Phe-CO), 157.1 (C-6), 153.7 (C-
2), 150.6 (C-4), 140.7 (C-8), 138.3, 137.9, 130.6, 130.6, 129.1, 129.0,
3
127.4, 127.3 (Ph), 119.9 (C-5), 83.9 (C-1′), 81.4 (d, JP,C = 13.8 Hz,
C-3′), 74.1 (C-4′), 67.1, 67.1 (OCH2CH2CH3), 66.9 (d, 1JP,C = 136.3
Hz, PCH2), 55.3, 54.7 (Phe-CH), 41.3, 41.2 (Ph-CH2), 38.9 (C-2′),
22.6 (OCH2CH2CH3), 13.8 (OCH2CH2CH3). 31P NMR (121 MHz,
acetone-d6): δ 19.7. HRMS: [M + H]+ calcd for C34H45N7O7P,
694.3112; found, 694.3120.
1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-
alaninate)](phenoxy)methylphosphonoamidate}-L-threose
(34a). Obtained from PMDTC (17 mg, 0.043 mmol, Et3N salt), L-
alanine isopropyl ester hydrochloride (14 mg, 0.087 mmol), and
1
PhOH (20 mg, 0.22 mmol) as a white solid. Yield 15% (3 mg). H
NMR (300 MHz, CD3OD): δ 7.83, 7.79 (d, J = 7.5 Hz, 1H, H-6),
7.32−7.38 (m, 2H, Ph), 7.14−7.21 (m, 3H, Ph), 6.07−6.12 (m, 1H,
H-1′), (5.80, 5.79) (d, J = 7.5 Hz, 1H, H-5), 4.85−4.99 [m, 1H,
CH(CH3)2], 4.38−4.44 (m, 1H, H-4a′), 4.27−4.35 (m, 1H, H-3′),
3.80−3.99 (m, 4H, H-4b′, PCH2 and Ala-CH), 2.48−2.58 (m, 1H, H-
2a′), 2.19−2.24 (m, 1H, H-2b′), 1.18−1.29 [m, 9H, Ala-CH3 and
CH(CH3)2]. 13C NMR (150 MHz, CD3OD): δ 174.9, 174.5 (Ala-
CO), 167.7 (C-4), 158.41, 158.38 (C-2), 151.6, 151.4 (Ph), 143.2,
143.0 (C-6), 130.8, 126.2, 126.1, 122.0, 121.8 (Ph), 95.50, 95.48 (C-
5), 88.1 (C-1′), 82.3 (3JP,C = 13.1 Hz, C-3′), 75.2, 75.0 (C-4′), 70.2
[CH(CH3)2], 65.8, 65.7 (1JP,C = 158.3, 156.8 Hz, PCH2), 51.0 (Ala-
CH), 39.7 (C-2′), 21.9 [CH(CH3)2], 21.1, 20.7 (Ala-CH3). 31P NMR
(121 MHz, CD3OD): δ 23.9, 22.7. HRMS: [M + H]+ calcd for
C21H30N4O7P, 481.1846; found, 481.1848.
1′α-(Thymin-1-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-
alaninate)](phenoxy)methylphosphonoamidate}-L-threose
(33a). Obtained from PMDTT (18 mg, 0.044 mmol, Et3N salt), L-
alanine isopropyl ester hydrochloride (15 mg, 0.088 mmol), and
1
PhOH (21 mg, 0.22 mmol) as a white solid. Yield 41% (9 mg). H
NMR (500 MHz, acetone-d6): δ 9.91 (s, 1H, NH), 7.68−7.69 (m, 1H,
H-6), 7.16−7.35 (m, 5H, Ph), 6.20−6.23 (m, 1H, H-1′), 4.92−4.94
[m, 1H, CH(CH3)2], 4.59−4.71 (m, 1H, NH), 4.39−4.44 (m, 1H, H-
3′), 4.34−4.38 (m, 1H, H-4a′), 3.96−4.05 (m, 3H, Ala-CH and
PCH2), 3.82−3.87 (m, 1H, H-4b′), 2.60−2.66 (m, 1H, H-2a′), 2.14−
2.20 (m, 1H, H-2b′), 1.80−1.81 (m, 3H, T CH3), 1.25−1.30 (m, 3H,
Ala-CH3), 1.18−1.20 [m, 6H, CH(CH3)2]. 13C NMR (125 MHz,
acetone-d6): δ 174.0, 173.7 (Ala-CO), 164.4 (C-4), 151.6, 151.5 (C-2),
137.5, 137.4 (C-6), 130.34, 130.30, 125.36, 125.33, 121.78, 121.75,
121.64, 121.60 (Ph), 110.64, 110.60 (C-5), 85.4, 85.3 (C-1′), 81.6 (d,
3JP,C = 12.6 Hz, C-3′), 73.9, 73.6 (C-4′), 69.2, 69.1 [OCH(CH3)2],
1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-{[N,N′-bis(n-propyl-L-
alaninate)]methylphosphonobisamidate}-L-threose (34d). Ob-
tained from PMDTC (18 mg, 0.046 mmol, Et3N salt) and L-alanine
isopropyl ester hydrochloride (46 mg, 0.28 mmol) as a white solid.
1
66.8, 65.8 (PCH2, JP,C = 155.8, 156.4 Hz), 50.5, 50.4 (Ala-CH), 38.5
1
(C-2′), 21.87, 21.85, 21.80 [OCH(CH3)2], 21.3, 21.2 (Ala-CH3), 12.6
(T CH3). 31P NMR (121 MHz, acetone-d6): δ 21.6, 20.7. HRMS: [M
+ H]+ calcd for C22H31N3O8P, 496.1843; found, 496.1850.
Yield 26% (6 mg). H NMR (600 MHz, CD3OD): δ 7.84 (d, J = 7.4
Hz, 1H, H-6), 6.09 (d, J = 6.6 Hz, 1H, H-1′), 5.90 (d, J = 7.4 Hz, 1H,
H-5), 4.96−5.02 [m, 2H, CH(CH3)2], 4.41 (d, J = 10.3 Hz, 1H, H-
4a′), 4.25−4.28 (m, 1H, H-3′), 3.84−3.95 (m, 3H, H-4b′ and Ala-
CH), 3.61−3.75 (m, 2H, PCH2), 2.48−2.55 (m, 1H, H-2a′), 2.19 (d, J
= 15.0 Hz, 1H, H-2b′), 1.33−1.36 (m, 6H, Ala-CH3). 1.23−1.26 [m,
12H, CH(CH3)2]. 13C NMR (150 MHz, CD3OD): δ 175.3, 175.2
(Ala-CO), 167.7 (C-4), 158.4 (C-2), 143.3 (C-6), 95.6 (C-5), 88.1 (C-
1′), 82.3 (3JP,C = 13.4 Hz, C-3′), 75.0 (C-4′), 70.2 [CH(CH3)2], 66.8
(1JP,C = 137.0 Hz, PCH2), 50.2, 49.9 (Ala-CH), 39.8 (C-2′), 22.0
[CH(CH3)2], 21.4, 21.2 (Ala-CH3). 31P NMR (121 MHz, CD3OD): δ
23.1. HRMS: [M + H]+ calcd for C21H37N5O8P, 518.2374; found,
518.2385.
1′α-(Thymin-1-yl)-2′-deoxy-3′-{[N-(diisoamyl-L-aspartate)]-
(phenoxy)methylphosphonoamidate}-L-threose (33b). Ob-
tained from PMDTT (27 mg, 0.066 mmol, Et3N salt), L-aspartic
acid isoamyl ester hydrochloride (41 mg, 0.133 mmol), and PhOH (31
mg, 0.331 mmol) as a white solid. Yield 60% (25 mg). 1H NMR (500
MHz, acetone-d6): δ 10.00 (s, 1H, NH), 7.68−7.69 (m, 1H, H-6),
7.17−7.35 (m, 5H, Ph), 6.21−6.23 (m, 1H, H-1′), 4.73−4.86 (m, 1H,
NH), 4.34−4.46 (m, 3H, H-3′, H-4a′ and Asp-CH), 4.02−4.17 [m,
6H, PCH2 and OCH2CH2CH(CH3)2], 3.81−3.87 (m, 1H, H-4b′),
2.78−2.82 (m, 2H,Asp-CH2), 2.60−2.65 (m, 1H, H-2a′), 2.12−2.20
(m, 1H, H-2b′), 1.80−1.81 (m, 3H, T CH3), 1.62−1.69 [m, 2H,
OCH2CH2CH(CH3)2], 1.44−1.51 [m, 4H, OCH2CH2CH(CH3)2],
0.88−0.90 [m, 12H, OCH2CH2CH(CH3)2]. 13C NMR (125 MHz,
acetone-d6): δ 172.5, 172.4, 171.1, 171.0 (Asp-CO), 164.4 (C-4),
151.5 (C-2), 137.5, 137.4 (C-6), 130.33, 130.31, 125.44, 125.42,
1′α-(Guanin-9-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-alaninate)]-
(phenoxy)methylphosphonoamidate}-L-threose (35a). Ob-
tained from PMDTG (26 mg, 0.060 mmol, Et3N salt), L-alanine
isopropyl ester hydrochloride (20 mg, 0.12 mmol), and PhOH (28 mg,
1
0.30 mmol) as a white solid. Yield 13% (4 mg). H NMR (300 MHz,
121.80, 121.77, 121.67, 121.64 (Ph), 110.74, 110.67 (C-5), 85.3, 85.2
DMSO-d6): δ 7.90, 7.88 (s, 1H, H-8), 7.15−7.40 (m, 5H, Ph), 6.57
(brs, 2H, NH2), 6.07−6.10 (m, 1H, H-1′), 5.62−5.79 (m, 1H, NH),
4.79−4.88 [m, 1H, CH(CH3)2], 4.36−4.45 (m, 1H, H-3′), 4.16−4.22
(m, 1H, H-4a′), 3.83−4.00 (m, 4H, H-4b′, PCH2 and Ala-CH), 2.63−
2.73 (m, 1H, H-2a′), 2.30−2.42 (m, 1H, H-2b′), 1.12−1.19 [m, 9H,
CH(CH3)2 and Ala-CH3]. 13C NMR (75 MHz, DMSO-d6): 173.1
1
(C-1′), 81.7, 81.6 (C-3′), 73.9, 73.5 (C-4′), 65.7, 64.8 (PCH2, JP,C
=
155.5, 157.0 Hz), 64.51, 64.47, 63.8 [OCH2CH2CH(CH3)2], 51.4
(Asp-CH), 39.7, 39.5 (Asp-CH2), 38.5 (C-2′), 37.97, 37.94, 37.90,
37.88 [OCH2CH2CH(CH3)2], 25.6, 25.5 [OCH2CH2CH(CH3)2],
22.7 [OCH2CH2CH(CH3)2], 12.6 (T CH3). 31P NMR (121 MHz,
P
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(Ala-CO), 157.2 (C-6), 154.1 (C-2), 151.1 (C-4), 150.5 (Ph), 135.6
(C-8), 129.69, 129.66, 124.5, 120.9, 120.8, 120.75, 120.70 (Ph), 116.4
(C-5), 82.2, 82.1 (C-1′), 80.6, 80.4 (C-3′), 72.9, 72.5 (C-4′), 68.10,
68.05 [CH(CH3)2], 65.1 (1JP,C = 154.8 Hz, PCH2), 49.2 (Ala-CH),
37.8, 37.6 (C-2′), 21.5 [CH(CH3)2], 20.4, 20.1 (Ala-CH3). 31P NMR
(121 MHz, DMSO-d6): δ 22.6, 21.9. HRMS: [M + H]+ calcd for
C22H30N6O7P, 521.1908; found, 521.1914.
Metabolic Stability Assay in Human Liver S9 Fraction. The
reaction mixture was prepared in a total volume of 1 mL containing 5
mM of MgCl2, 50 mM of K2HPO4 (pH 7.4), and 100 μM prodrug.
The reaction was initiated by adding 4 mg/mL human liver S9 fraction
(obtained from Gibco) to the reaction mixture and incubated at 37 °C.
At the desired times (0, 10, 20, 30, and 60 min), 100 μL aliquots were
taken and the reaction was stopped by mixing the reaction mixture
sample with 300 μL of acetonitrile. The samples were centrifuged at
13000 rpm for 30 min at 4 °C. Then 10 μL of the supernatant was
analyzed by HPLC on a Shimadzu HPLC system equipped with a
Symmetry C18 column (4.6 mm × 150 mm, 5 μm) and UV detection
at 260 nm. The mobile phase consisted of solvent A (5% acetonitrile in
water, 10 mM ammonium acetate) and solvent B (95% acetonitrile in
water, 10 mM ammonium acetate). Elution was performed using a
linear gradient of solvent B from 0% to 100% for 20 min. The flow rate
was set at 1.0 mL/min. The amount of parent compound was
determined on the basis of the peak area for each time point, and the
percentage remaining was calculated on the basis of the initial amount
measured at 0 h. The half-life of each compound was calculated by
using the GraphPad Prism software.
Metabolic Stability Assay in Human Liver Microsomes. The
stability assay was contracted and carried out by Eurofins (Eurofins ref
0607). Pooled liver microsomes were preincubated with NADPH-
regenerating system (1 mM NADP, 5 mM G6P, and 1 U/mL
G6PDHase) in phosphate buffer (pH 7.4) containing 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 concentration 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 transferred 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 min 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.
HIV Antiviral Assay. Evaluation of the antiviral activity of the
compounds against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4
cells was performed using the MTT assay as previously described.19,20
Stock solutions (10× final concentration) of test compounds were
added in 25 μL volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock- and HIV-infected
cells at the beginning of each experiment. Serial 5-fold dilutions of test
compounds were made directly in flat-bottomed 96-well microtiter
trays using a Biomek 3000 robot (Beckman instruments, Fullerton,
CA). Untreated HIV- and mock-infected cell samples were included as
controls. HIV-1(IIIB) stock (50 μL) at 100−300 CCID50 (50% cell
culture infectious doses) or culture medium was added to either the
infected or mock-infected wells of the microtiter tray. Mock-infected
cells were used to evaluate the effects of test compound on uninfected
cells in order to assess the cytotoxicity of the test compounds.
Exponentially growing MT-4 cells were centrifuged for 5 min at 220 g,
and the supernatant was discarded. The MT-4 cells were resuspended
at 6 × 105 cells/mL, and 50 μL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of mock-and
HIV-infected cells was examined spectrophotometrically using the
MTT assay. The MTT assay is based on the reduction of yellow-
colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics) by mitochondrial dehydrogenase activity in
metabolically active cells to a blue−purple formazan that can be
measured spectrophotometrically. The absorbances were read in an
eight-channel computer-controlled photometer (Infinite M1000,
Tecan) at two wavelengths (540 and 690 nm). All data were
calculated using the median absorbance value of three wells. The 50%
cytotoxic concentration (CC50) was defined as the concentration of
the test compound that reduced the absorbance (OD540) of the mock-
infected control sample by 50%. The concentration achieving 50%
protection against the cytopathic effect of the virus in infected cells was
defined as the 50% effective concentration (EC50).
1′α-(Guanin-9-yl)-2′-deoxy-3′-O-[N,N′-bis(n-propyl-L-
phenylalaninate)]methylphosphonobisamidate]-L-threose
(35c). Obtained from PMDTG (20 mg, 0.045 mmol, Et3N salt) and L-
phenylalanine-O-n-Pr ester hydrochloride (65 mg, 0.27 mmol) as a
1
white solid. Yield 24% (8 mg). H NMR (600 MHz, DMSO-d6): δ
10.56 (brs, 1H, NH), 7.86 (s, 1H, H-8), 7.12−7.27 (m, 10H, Ph), 6.45
(brs, 2H, NH2), 6.04 (dd, J = 8.1, 2.5 Hz, 1H, H-1′), 4.54 (t, J = 11.3
Hz, 1H, NH), 4.20 (t, J = 10.8 Hz, 1H, NH), 4.13−4.16 (m, 1H, H-
3′), 4.07 (d, J = 10.2 Hz, 1H, H-4a′), 3.92−4.04 (m, 4H,
CH2CH2CH3), 3.85−3.91 (m, 2H, PCH2), 3.79 (dd, J = 10.1, 4.3
Hz, 1H, H-4b′), 3.15−3.16 (m, 2H, Phe-CH), 2.76−2.93 (m, 4H, Phe-
CH2), 2.57−2.62 (m, 1H, H-2a′), 2.20 (d, J = 14.6 Hz, H-2b′), 1.45−
1.53 (m, 4H, CH2CH2CH3), 0.75−0.82 (m, 6H, CH2CH2CH3). 13C
NMR (150 MHz, DMSO-d6): 173.0, 172.9 (Phe-CO), 156.9 (C-6),
153.7 (C-2), 150.9 (C-4), 137.3, 137.2 (Ph), 135.8 (C-8), 129.48,
129.47, 128.20, 128.17, 126.55, 126.49 (Ph), 116.4 (C-5), 82.2 (C-1′),
79.8 (3JP,C = 11.2 Hz, C-3′), 72.5 (C-4′), 65.94, 65.87 (CH2CH2CH3),
65.7 (1JP,C = 135.7 Hz, PCH2), 54.2, 53.0 (Phe-CH), 40.1 (Phe-CH2),
37.6 (C-2′), 21.5, 21.4 (CH2CH2CH3), 10.3, 10.2 (CH2CH2CH3). 31
P
NMR (121 MHz, DMSO-d6): δ 20.3. HRMS: [M + H]+ calcd for
C34H45N7O8P, 710.3062; found, 710.3063.
1′α-(Guanin-9-yl)-2′-deoxy-3′-O-[N,N′-bis(n-propyl-L-
alaninate)]methylphosphonobisamidate]-L-threose (35d). Ob-
tained from PMDTG (17 mg, 0.039 mmol, Et3N salt) and L-alanine
isopropyl ester hydrochloride (40 mg, 0.23 mmol) as a white solid.
Yield 23% (5 mg). 1H NMR (300 MHz, DMSO-d6): δ 7.86 (s, 1H, H-
8), 6.59 (brs, 2H, NH2), 6.05 (dd, J = 8.1, 2.5 Hz, 1H, H-1′), 4.84−
4.92 [m, 2H, CH(CH3)2], 4.42−4.59 (m, 2H, NH), 4.35 (brs, 1H, H-
3′), 4.18 (d, J = 9.8 Hz, H-4a′), 3.75−3.90 (m, 3H, H-4b′ and Ala-
CH), 3.56−3.72 (m, 2H, PCH2), 2.59−2.69 (m, 1H, H-2a′), 2.30 (d, J
= 14.7 Hz, H-2b′), 1.27 (t, J = 7.5 Hz, 6H, Ala-CH3), 1.17−1.19 [m,
12H, CH(CH3)2]. 13C NMR (150 MHz, CD3OD): 175.4, 175.2 (Ala-
CO), 159.5 (C-6), 155.3 (C-2), 152.7 (C-4), 138.6 (C-8), 117.3 (C-
5), 85.3 (C-1′), 82.2 (3JP,C = 13.2 Hz, C-3′), 74.7 (C-4′), 70.1
[CH(CH3)2], 66.8 (1JP,C = 137.5 Hz, PCH2), 50.3, 50.0 (Ala-CH),
39.4 (C-2′), 22.0 [CH(CH3)2], 21.21, 21.18 (Ala-CH3). 31P NMR
(121 MHz, DMSO-d6): δ 20.3. HRMS: [M + H]+ calcd for
C22H37N7O8P, 558.2436; found, 558.2448.
31P NMR Stability Experiments in Acidic and Basic pH. Buffer
pH 1: The stability assay toward hydrolysis by aqueous buffer at pH 1
was conducted using in situ 31P NMR (202 MHz). The experiment
was carried out by dissolving prodrug 32b (2.0 mg) in 0.2 mL of
acetone-d6 and then adding buffer (0.3 mL), pH 1 (prepared from
equal parts of 0.2 M HCl and 0.2 M KCl). Next, the sample was
subjected to 31P NMR experiments at 37 °C and the spectra were
recorded every 30 min over 14 h. Buffer pH 8: The stability assay
toward hydrolysis by aqueous buffer at pH 8 was conducted using in
situ 31P NMR (202 MHz). The experiment was carried out by
dissolving prodrug 32b (2 mg) in 0.2 mL of acetone-d6 and then
adding buffer (0.3 mL), pH 8 (prepared from solution of 0.1 M
Na2HPO4 and adjusted to the appropriate pH using 0.1 M HCl). Next,
the sample was subjected to 31P NMR experiments at 37 °C and the
spectra were recorded every 30 min over 14 h.
Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was
carried out by dissolving prodrug 32b (1.0 mg) in 0.10 mL of acetone-
d6 followed by addition of 0.20 mL of Trizma buffer (pH 7.6). After
recording the control 31P NMR (121 MHz) at 25 °C, 0.10 mL of
carboxypeptidase Y (obtained from Sigma-Aldrich, 0.1 mg dissolved in
0.15 mL Trizma) was added to the sample, which was then subjected
to 31P NMR experiments. The spectra were recorded every 1 h over a
period of 16 h at 25 °C. The 31P NMR spectra was processed and
analyzed by the Bruker Topspin 2.1 program.
Q
DOI: 10.1021/acs.jmedchem.6b01260
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HBV Antiviral Assay. Primary Assay. The primary anti-HBV assay
was performed as previously described23,24 with modifications to use
real-time qPCR (TaqMan) to measure extracellular HBV DNA copy
number associated with virions released from HepG2 2.2.15 cells. The
HepG2 2.2.15 cell line is a stable human hepatoblastoma cell line that
contains two copies of the HBV wild-type strain ayw1 genome and
constitutively produces high levels of HBV.22 Briefly, HepG2 2.2.15
cells were plated in 96-well microtiter plates at 1.5 × 104 cells/well in
Dulbecco’s Modified Eagle’s Medium supplemented with 2% FBS, 380
μg/mL G418, 2.0 mM L glutamine, 100 units/mL penicillin, 100 μg/
mL streptomycin, and 0.1 mM nonessential amino acids. Only the
interior wells are utilized to reduce “edge effects” observed during cell
culture; the exterior wells are filled with complete medium to help
minimize sample evaporation. After 16−24 h, the confluent monolayer
of HepG2 2.2.15 cells was washed and the medium was replaced with
complete medium containing various concentrations of a test
compound in triplicate. Lamivudine (3TC) was used as the positive
control, while media alone was added to cells as a negative control
(virus control, VC). Three days later, the culture medium is replaced
with fresh medium containing the appropriately diluted test
compounds. Six days following the initial administration of the test
compound, the cell culture supernatant was collected, treated with
Pronase, and then used in a real-time quantitative TaqMan qPCR
assay. The PCR-amplified HBV DNA was detected in real-time by
monitoring increases in fluorescent signal that result from the
exonucleolytic degradation of a quenched fluorescent probe molecule
that hybridized to the amplified HBV DNA. For each PCR
amplification, a standard curve was simultaneously generated using
dilutions of purified HBV DNA. Antiviral activity was calculated from
the reduction in HBV DNA levels (EC50 and EC90 values determined).
A tetrazolium dye (MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter96 Re-
agent, Promega) uptake assay was then employed to measure cell
viability, which was used to calculate toxicity (CC50).
Rozenski (KU Leuven) for providing HRMS data. Mass
spectrometry was made possible by the support of the Hercules
Foundation of the Flemish Government (grant 20100225-7).
We thank Kristien Erven and Kris Uyttersprot for dedicated
technical assistance. We thank Luc Baudemprez for technical
assistance. Testing against HBV was conducted by Southern
Research Institute using federal funds from the Division of
Microbiology and Infectious Diseases (DMID), NIAID/NIH
under contract HHSN272201100013I entitled “In Vitro
Assessment for Antimicrobial Activity”.
DEDICATION
■
In memory of A. Holy on the occasion of his 80th birthday
ABBREVIATIONS USED
■
PMEA, phosphonomethoxyethyladenine; PMPA, phosphonyl-
methoxypropyladenine; PMT, phosphonomethoxythreosyl;
PMDT, phosphonomethoxydeoxythreosyl; HIV, human im-
munodeficiency virus; HBV, hepatitis B virus; TBSCl, tert-
butylchlorodimethylsilane; BSA, N,O-bis(trimethylsilyl)-
acetamide; TMSOTf, trimethylsilyl trifluoromethanesulfonate;
TCDI, 1,1′-thiocarbonyldiimidazole; DMAP, 4-dimethylamino-
pyridine; AIBN, azobis(isobutyronitrile); DMP, Dess−Martin
periodinane; DAST, diethylaminosulfur trifluoride; THF,
tetrahydrofuran; DMF, N,N-dimethylformamide; TEAB, trie-
thylammonium bicarbonate; TLC, thin layer chromatography;
rt, room temperature
REFERENCES
■
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Secondary Assay. The secondary anti-HBV assay was performed in
a manner similar to the primary assay described above in. However, at
the end of the assay, the cells were processed to isolate total
intracellular DNA using the Qiagen DNeasy Blood and Tissue Kit
following the manufacturer’s protocol. The real-time TaqMan qPCR
assay was then performed using the isolated DNA to measure
reductions in intracellular HBV DNA copy number.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01260.
■
Med. Res. Rev. 2005, 25, 1−20.
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31P NMR spectra of prodrug 32b in pH 1 and pH 8
buffer; 31P NMR spectra of prodrug 32b over time after
incubation with pH 8 buffer; RP-18-HPLC chromato-
gram of prodrug 32b after incubation with pH 8 buffer;
RP-18-HPLC chromatogram of prodrug 32b after
carboxypeptidase Y digestion; NMR spectra for com-
pounds 2−35d (PDF)
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Molecular formula strings (CSV)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +32 16 337387. Fax: +32 16 337340. E-mail: Piet.
Herdewijn@kuleuven.be.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Chao Liu acknowledges the China Scholarship Council (CSC)
for funding (grant 201306220065). We thank Prof. Jef
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