Synthesis and antiviral evaluation of 2-amino-6-carbamoylpurine dioxolane nucleoside derivatives and their phosphoramidates prodrugs

Article abstract of DOI:10.1016/j.bmc.2014.10.003

The synthesis of 9-(β-d-1,3-dioxolan-4-yl)2,6-diaminopurine nucleoside phosphoramidate prodrugs as well as various 2-amino-6-carbamoylpurine dioxolane derivatives and their phosphoramidates prodrugs is reported. Their ability to block HIV and HBV replicat

Full text of DOI:10.1016/j.bmc.2014.10.003

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral evaluation of 2-amino-6-carbamoylpurine
dioxolane nucleoside derivatives and their phosphoramidates
prodrugs
Jong Hyun Cho a, Lavanya Bondana a, Mervi A. Detorio a, Cathy Montero a, Leda C. Bassit a,
Franck Amblard a, Steven J. Coats b, Raymond F. Schinazi a,
⇑
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, and Veterans Affairs Medical Center,
1670 Haygood Drive, NE, Atlanta, GA 30322, USA
b RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 9-(b-D-1,3-dioxolan-4-yl)2,6-diaminopurine nucleoside phosphoramidate prodrugs as
Received 5 August 2014
Revised 24 September 2014
Accepted 1 October 2014
Available online xxxx
well as various 2-amino-6-carbamoylpurine dioxolane derivatives and their phosphoramidates prodrugs
is reported. Their ability to block HIV and HBV replication along with their cytotoxicity toward HepG2,
human lymphocyte, CEM and Vero cells was also assessed.
Published by Elsevier Ltd.
Keywords:
b-D-1,3-Dioxolan-4-yl nucleosides
DAPD
Antiviral agent
Anti-HIV
Anti-HBV
1. Introduction
purine nucleoside phosphoramidate prodrug 4 displayed submi-
cromolar activities against HBV and HIV while its corresponding
Nucleoside reverse transcriptase inhibitors (NTRI) are the back-
bone in fixed dose combinations now called highly active antiretro-
viral therapy (HAART) for human immunodeficiency virus type 1
(HIV-1).1 Despite the effectiveness of these drugs, resistance can
result from their long-term use and latent toxicity remains
an issue.2 Therefore, studies on novel nucleoside analogs with
improved efficacy, resistance profile and safety are continuously
needed to improve clinical outcome. Among all the modified
nucleoside 3 was devoid of activity (Fig. 1).5 (B) Conversion of
hydrophilic functional groups (such as amino or hydroxy) into cor-
responding lipophilic groups (such as amides or esters) in order to
improve cell penetration. Thus,
L-1,3-dixolane-cytidine (L-OddC)
bearing
a
fatty acid group at its N4-position demonstrated
significantly improved antitumor activity (170-fold) in vitro when
compared to the parent nucleoside.6 Fatty acyl derivatives of
(ꢀ)-20,30-dideoxy-30-thiacytidine (3TC) and (ꢀ)-20,30-dideoxy-5-
fluoro-30-thiacytidine [(ꢀ)-FTC] were 36- and 24-fold, respectively,
more potent against HIV when compared to 3TC and (ꢀ)-FTC.7
Herein, we combine both approaches to potentially improve the
antiviral potency of DAPD. Thus, we report the synthesis and anti-
viral evaluation of DAPD phosphoramidate prodrugs as well as
their 2-amino-6-carbamoylpurine derivatives as a potential strat-
egy to increase intracellular delivery of DXG-TP.
nucleosides prepared over the years, b-
sides appeared to be very promising family. Therefore,
9-(b- -1,3-dioxolan-4-yl)2,6-diaminopurine (DAPD) 1, a prodrug
of 9-(b- -1,3-dioxolan-4-yl)guanine (DXG, 2), was evaluated for
D-1,3-dioxolan-4-yl nucleo-
a
D
D
the treatment of HIV-1 infected persons in phase 2 clinical studies.
However, the study was abandoned due to slow enrollment and
high dosing regimens (500 mg twice a day). Over the years of nucle-
oside analog development, two main approaches have been utilized
to improve the potency of a compound and potentially decrease the
administered dose: (A) formation of a monophosphate prodrug
to bypass the rate limiting first phosphorylation step.3,4 For
2. Results and discussion
We first envisaged to prepare the desired 2-amino-6-car-
bamoylpurine dioxolane nucleoside phosphoramidate prodrugs
10a–i from DAPD in a 2 steps sequence by (1) formation of the
phosphoramidate prodrug 5; (2) introduction of the carbamoyl
instance, (ꢀ)-b-
D-(2R,4R)-1,3-dioxolane-2-amino-6-aminopropyl
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmc.2014.10.003
0968-0896/Published by Elsevier Ltd.
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

2
J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
R1
HN
N
N
N
N
N
N
O
O
N
R2
HO
N
H
P O
N
O
O
O
OPh
O
O
R1 = NH2 , R2 = NH2 DAPD, 1
4
R1 = OH, R2 = NH2 DXG, 2
EC50 = 0.088 µM (HIV)
EC50 = 0.8 µM (HBV)
R1 = NH2 , R2 =
, 3
HN
Figure 1.
moiety. However, reaction of DAPD 1 with phosphoramidate chlo-
ride 68 in the presence of t-BuMgCl afforded DAPD prodrug 5 in
only 45% yield (Scheme 1). To further complicate this approach,
acylation reactions with 5 would either proceed to only about
50% or when forced gave a substantial amount of the N2,N6-diacy-
lated product. These low yields combined with the difficulty of
purifying 5 using repeated tedious silica gel column chromatogra-
phy lead us to design an alternative sequence which also allowed
us to prepare and evaluate the 2-amino-6-carbamoylpurine diox-
olanes 9a–i (Scheme 2).
chemoselective N6-acylation was investigated using oleoyl chloride
as the model system for optimization (Table 1). Interestingly, the
reaction of 7 with oleoyl chloride (1.1 equiv) in the presence of
pyridine (entry 2), DMAP (entry 4), Et3N (entry 6), imidazole (entry
7) or their combination (entries 3 and 5) lead to the formation of
50-O-TBS-2-amino-6-oleoylpurine dioxolane 8i in poor to moderate
yields along with N2,N6-diacylated DAPD in ca. 5–25% yields. On
the other hand, treatment of 7 with oleic acid chloride (1.1 equiv)
in the presence of N-methylimidazole (entry 1),9 provided the
desired compound 8i in 80% yield with less than 5% of the
N2,N6-diacylated byproduct. Using these optimized conditions,
compound 7 was reacted with a variety of acyl chlorides to
give the corresponding 50-O-TBS-2-amino-6-carbamoylpurine
Thus, DAPD 1 was reacted with t-butyldimethylsilyl chloride
(TBSCl) in the presence of imidazole in pyridine to give 50-O-TBS-
2,6-diaminopurine dioxolane 7 in 89% yield. With 7 in hand, its
NH2
NH2
N
N
O
O
N
N
O
P O
N
HO
N
NH2
N
HN
N
NH2
a
O
O
OPh
O
O
1
5
Scheme 1. Reagents and reaction conditions: (a) (EtOAlaNH)P(@O)(OPh)Cl 6, t-BuMgCl, THF, ꢀ78 °C then rt, 8 h.
NH2
N
NH2
N
N
N
N
N
TBSO
HO
N
NH2
N
NH2
O
O
a
b
O
O
89%
78-86%
7
1
NHR
NHR
N
N
N
N
N
N
HO
TBSO
N
NH2
N
NH2
c
O
O
O
93-97%
O
9a i
-
8a-i
a
: R = Acetyl
NHR
N
b: R = Butanoyl
c: R = Hexanoyl
O
O
N
O
P O
d
: R = Octanoyl
e: R = Decanoyl
f: R = Lauroyl
N
O
HN
d
68-82%
N
NH2
OPh
g
: R = Myristoyl
O
h: R = Palmitoyl
i: R = Oleoyl
10a-i
Scheme 2. Reagents and reaction conditions: (a) TBSCl, imidazole, pyridine, 0 °C then rt, 12 h; (b) RCl, NMI, CH2Cl2, 0 °C then rt, 12 h; (c) Et3N–3HF, THF, rt, 12 h; (d)
(NHAlaOEt)P(@O)(OPh)Cl 6, NMI, ꢀ78 °C then rt, 8–12 h.
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
Condition for optimization of the N6-acylation of 7
O
NH2
N
CH3(CH2)7CH=CH(CH2)7
N
NH
N
N
N
N
TBSO
TBSO
N
NH2
N
NH2
O
O
oleoyl chloride
O
O
7
8i
Entry
Conditions
Yield (%)
1
2
3
4
5
6
7
NMI, CH2Cl2, 12 h
Pyridine, 12 h
DMAP (0.1 equiv), pyridine, 12 h
DMAP (2.0 equiv), CH2Cl2, 12 h
DMAP (0.1 equiv), Et3N (2.0 equiv), CH2Cl2, 12 h
Et3N (2.0 equiv), CH2Cl2, 12 h
Imidazole (2.0 equiv), CH2Cl2, 12 h
80
Tracea
5a
43a
18a
21a
8b
Reactions provided N2,N6-diacylated compounds in 20–30% yields and ca. 5–15% of starting material (7) was recovered.
Most of 7 was recovered.
a
b
dioxolane derivatives 8a–i in 78–86% isolated yields. The final phe-
Generating phosphoramidate prodrug of DAPD 5 barely increased
DAPD’s potency against HIV (EC50 of 0.45 vs 0.25 M) but, more
interestingly, boosted its potency against HBV by a factor of 40
(EC50 of 0.3 vs 12
M). C1 to C11 N6-carbamoyl derivative 9a–f, 9i
and their corresponding prodrugs 10a–f, 10i displayed either equal
or less potency than DAPD against HIV and HBV. On the other hand,
phosphoramidate prodrugs 10g and 10h, with EC50’s of 0.041 and
nyloxy phosphoramidate dioxolane derivatives (10a–i) were
obtained after removal of the 50-silyl group with Et3N-3HF and sub-
sequent coupling with phosphoramidate chloride reagent 6.
Both anti-HIV and anti-HBV potency of DAPD monophosphate
prodrug 5, as well as 2-amino-6-carbamoylpurine dioxolanes
(9a–i) and their phosphoramidate dioxolane prodrugs (10a–i)
were evaluated in primary human lymphocytes and HepG2 cells,
respectively. In addition, cytotoxicity was determined in human
peripheral blood mononuclear (PBM) cells, human lymphoblastoid
CEM, African Green monkey Vero cells and HepG2 cells.10 The
antiviral activity and cytotoxicity are summarized in Table 2.
l
l
0.083 lM, respectively, were 3–11 times more potent than DAPD
1 or DAPD prodrug 5 against HIV while their anti-HBV potency
was in the same range as DAPD prodrug 5. Unfortunately, these
compounds also displayed cytotoxicity in various cell lines that
ranged from a CC50 of 7.5 to 71.0 lM.
Table 2
In vitro antiviral activity and cytotoxicity of compounds 9a–i and phosphoramidate prodrugs 5, 10a–i
O
R2
N
NH
N
O
P
OPh
O
R1O
N
NH2
N
N
H
O
PD =
O
O
Compd
R1
R2
Anti-HIV-1 activity (
l
M)
Anti-HBV activity (
lM)
Cytotoxicity, CC50 (lM)
EC50
EC90
EC50
EC90
PBM
CEM
Vero
HepG2
AZT
1
5
9a
10a
9b
10b
9c
10c
9d
10d
9e
10e
9f
10f
9g
10g
9h
10h
9i
NA
H
PD
H
PD
H
PD
H
PD
H
PD
H
PD
H
PD
H
NA
NA
NA
CH3
0.0037
0.45
0.25
0.56
19.0
9.2
1.2
34.0
2.2
0.047
7.6
1.5
>10
12.0
0.30
>10
9.3
>10
3.3
>10
1.3
>10
0.55
>10
0.43
>10
0.34
>10
0.39
>10
0.33
>10
0.1
>10
>100
2.8
>100
>100
67.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
67.0
60.0
14.0
>100
9.9
14.0
>100
>100
>100
>100
>100
>100
>100
89
>100
16.0
65.0
13.0
13
12.0
4.6
7.5
>100
12
13
56.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
87.0
>100
13.0
53.0
45.0
7.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.5
12.0
93.0
38.0
15.0
>100
33.0
56.0
6.6
65.0
9.4
18.0
2.9
27.0
0.89
6.0
0.50
13.0
2.1
>10
>10
>10
>10
>10
>10
>10
P10
>10
P10
>10
P10
>10
8.8
CH3
CH3(CH2)2
CH3(CH2)2
CH3(CH2)4
CH3(CH2)4
CH3(CH2)6
CH3(CH2)6
CH3(CH2)8
CH3(CH2)8
CH3(CH2)10
CH3(CH2)10
CH3(CH2)12
CH3(CH2)12
CH3(CH2)14
CH3(CH2)14
CH3(CH2)7CH@CH(CH2)7
CH3(CH2)7CH@CH(CH2)7
21
0.42
26.0
1.5
3.6
0.98
6.5
0.041
1.2
0.083
3.5
>100
9.5
>100
71.0
56.0
57.0
>100
ND
PD
H
PD
H
13.0
90
>100
19
>10
7.9
>10
5.8
>100
7.9
76
11
ND
10i
PD
0.77
10
10
13
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

4
J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
In conclusion, this work confirms the potential of the phosphor-
10.0 Hz, 1H), 4.19 (dd, J = 1.6, 5.6 Hz, 1H), 3.79 (d, J = 3.2 Hz, 2H),
0.84 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) d 159.8, 155.7, 151.5, 134.9, 112.6, 104.7, 78.6, 70.5, 63.1,
25.8, 18.2, ꢀ5.37, ꢀ5.39; MS-ESI+ m/z 367 (M+H+); HRMS-ESI+
calcd for C15H31N6O3Si (M+H+) 367.1907, found 367.1908.
amidate prodrug approach to reveal or enhance the antiviral activ-
ity of nucleosides. In our hands, DAPD phosphoramidate prodrug 5
was 40 times more potent than DAPD against HBV. Combination of
this approach with the introduction of long fatty ester chains
known to mask polar groups allowed us to even further increase
their potency against HIV. However, this augmentation of potency
came, in this case, with an increase of cellular toxicity.
3.1.3. (Z)-N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)
oxy)methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)octadec-8-
enamide (8i)
To a solution of compound 7 (2.70 g, 7.37 mmol) in 50 mL of
anhydrous CH2Cl2 was added N-methylimidazole (2.0 equiv,
1.21 g, 14.7 mmol) and oleoyl chloride (1.1 equiv, 3.05 g,
8.11 mmol) at 0 °C under N2 atmosphere. After stirring for 12 h
at rt, the reaction mixture was treated with 10 mL of MeOH, stirred
for 30 min at rt and then concentrated under reduced pressure. The
residue was purified on silica gel column chromatography
(hexane/EtOAc = 4:1 to 1:4 v/v) to give compound 8i (3.72 g,
5.90 mmol) in 80% yield. 1H NMR (400 MHz, CDCl3) d 8.66 (br,
1H), 8.07 (s, 1H), 6.34 (dd, J = 1.6, 5.2 Hz, 1H), 5.36–5.32 (m, 2H),
5.16 (br, 2H), 5.12 (t, J = 3.2 Hz, 1H), 4.42 (dd, J = 1.2, 9.6 Hz, 1H),
4.22 (dd, J = 5.2, 9.6 Hz, 1H), 3.95–3.88 (m, 2H), 2.79 (t, J = 7.6 Hz,
2H), 2.20–1.98 (m, 4H), 1.78–1.70 (m, 2H), 1.40–1.24 (m, 20H),
0.91–0.85 (m, 12H), 0.08 (s, 6H); 13C NMR (100 MHz, CDCl3) d
173.3, 159.7, 153.1, 149.9, 138.0, 130.1, 129.9, 129.95, 116.0,
106.0, 79.4, 71.7, 63.5, 38.0, 32.1, 29.93, 29.90, 29.7, 29.54, 29.48,
29.3, 27.39, 27.36, 26.1, 25.0, 22.8, 18.7, 14.3, ꢀ5.17, ꢀ5.19;
MS-ESI+ m/z 631(M+H+); HRMS-ESI+ calcd for C33H58N6O4NaSi
(M+Na+) 653.4188, found 653.4181.
3. Experimentals
3.1. General
Nuclear magnetic resonance (NMR) spectra (1H, 13C and 31P)
were recorded on a Varian Unity Plus 400 MHz and a Bruker
Ascend™ 400 MHz Fourier transform spectrometer at rt, with tet-
ramethylsilane (TMS) as an internal standard. Chemical shifts (d)
are reported in parts per million (ppm), and signals are quoted as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br
(broad), dd (doublet of doublets), or ddd (doublet of doublets of
doublets). The phosphoramidates are an approximate 50:50 mix-
ture of diastereomers (RP/SP) and the 13C NMR data is reported as
observed, that is, some carbon signals overlap. High-resolution
mass spectra (HRMS) were recorded on a Micromass Autospec
high-resolution mass spectrometer with electrospray ionization
(ESI). Thin-layer chromatography (TLC) was performed on
0.25 mm silica gel. Purifications were carried out on silica gel col-
umn chromatography (60 Å, 63–200 lm, or 40–75 lm).
3.1.4. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)acetamide (8a)
Compound 8a was prepared using the same procedure as for
compound 8i; yield: 78%; 1H NMR (400 MHz, CDCl3) d 8.55 (s,
1H), 8.07 (s, 1H), 6.34 (dd, J = 1.6, 5.2 Hz, 1H), 5.12 (t, J = 3.2 Hz,
1H), 5.03 (br, 2H), 4.42 (dd, J = 1.2, 10.0 Hz, 1H), 4.23 (dd, J = 5.2,
10.0 Hz, 1H), 3.94–3.86 (m, 2H), 2.56 (s, 3H), 0.90 (s, 9H), 0.09 (s,
6H); 13C NMR (100 MHz, CDCl3) d 170.7, 159.6, 153.0, 149.8,
138.2, 115.9, 106.0, 79.5, 71.8, 63.5, 39.3, 26.1, 25.9, 18.8,
-5.13, -5.16; MS-ESI+ m/z 409 (M+H+); HRMS-ESI+ calcd for
3.1.1. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-hydroxy-9H-purin-
9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)
propanoate (5)
To a solution of DAPD (0.10 g, 0.40 mmol) in THF (10 mL) was
added t-butylmagnesium chloride (1.19 mL, 1.0 M in THF,
1.19 mmol) at ꢀ78 °C under argon atmosphere. After stirred for
30 min, the reaction mixture was additionally stirred at rt for
30 min. To the solution was added phosphoramide chloride 6
(0.24 g, 0.80 mmol) at ꢀ78 °C under argon atmosphere. The reac-
tion temperature was warmed to rt over 1 h and then stirred for
6 h. The mixture was quenched with saturated ammonium chlo-
ride (1.0 mL) at 0 °C, and then stirred for 10 min. The resulting
solution was adsorbed on silica gel and purified by silica gel col-
umn chromatography (CH2Cl2:MeOH = 20:1 to 10:1 v/v) to give
compound 5 in 45% yield (0.09 g, 0.18 mmol). 1H NMR (400 MHz,
CD3OD): d 7.99–7.98 (s, 1H), 7.37–7.27 (m, 2H), 7.23–7.13 (m,
3H), 6.37–6.33 (m, 1H), 5.36–5.32 (m, 1H), 4.63–4.60 (m, 1H),
4.38–4.30 (m, 3H), 4.14–4.06 (m, 2H), 3.76–3.71 (m, 1H), 1.35–
1.19 (m, 6H); 13C NMR (100 MHz, CD3OD) d 173.6, 160.6, 156.2,
151.3, 150.6, 135.8, 129.2, 124.6, 119.9, 112.6, 103.3, 79.6, 70.6,
64.6, 60.9, 50.1, 18.9, 13.0; 31P NMR (162 MHz, CD3OD) d 3.91,
3.64; MS-ESI+ m/z 508 (M+H+); HRMS-ESI+ calcd for C20H27N7O7P
(M+H+) 508.1708, found 508.1704.
C
17H29N6O4Si (M+H+) 409.2008, found 409.2014.
3.1.5. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)butyramide (8b)
Compound 8b was preparedusing the same procedureas for com-
pound 8i; yield: 84%; 1H NMR (400 MHz, CD3OD) d 8.29 (s, 1H), 6.45
(d, J = 4.0 Hz, 1H), 5.07 (t, J = 2.4 Hz, 1H), 4.42 (d, J = 10.0 Hz, 1H), 4.20
(dd, J = 5.2, 10.0 Hz, 1H), 3.92–3.84 (m, 2H), 2.41 (t, J = 5.2 Hz, 2H),
1.71–1.65 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H), 0.87 (s, 9H), 0.03 (s, 3H),
0.02 (s, 3H); 13C NMR (100 MHz, CD3OD) d 175.7, 157.7, 154.6,
151.4, 140.2, 116.8, 107.0, 81.5, 73.0, 64.1, 40.2, 26.6, 20.1, 19.6,
14.2, ꢀ5.1, ꢀ5.2; MS-ESI+ m/z 437 (M+H+); HRMS-ESI+ calcd for
C
19H33N6O4Si (M+H+) 437.2327, found 437.2327.
3.1.6. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)hexanamide (8c)
Compound 8c was prepared using the same procedure as for
compound 8i; yield: 85%; 1H NMR (400 MHz, CDCl3) d 8.85 (s,
1H), 8.09 (s, 1H), 6.35 (dd, J = 1.2, 5.2 Hz, 1H), 5.20 (br, 2H), 5.12
(t, J = 3.2 Hz, 1H), 4.41 (dd, J = 1.2, 9.6 Hz, 1H), 4.22 (dd, J = 5.2,
9.6 Hz, 1H), 3.94–3.87 (m, 2H), 2.77 (t, J = 7.6 Hz, 2H), 1.78–1.70
(m, 2H), 1.40–1.32 (m, 4H), 0.95–0.88 (m, 12H), 0.09 (s, 6H); 13C
NMR (100 MHz, CDCl3) d 173.2, 159.8, 152.9, 150.0, 138.0, 115.8,
106.0, 79.5, 71.8, 63.4, 37.9, 31.6, 26.1, 24.8, 22.7, 18.8, 14.2,
ꢀ5.1, ꢀ5.2; MS-ESI+ m/z 465 (M+H+); HRMS-ESI+ calcd for
3.1.2. 9-((2R,4R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-1,3-
dioxolan-4-yl)-9H-purine-2,6-diamine (7)
To a solution of DAPD (2.0 g, 7.93 mmol) in 25 mL of anhydrous
pyridine was added imidazole (0.65 g, 9.52 mmol) and TBDMSCl
(1.20 g, 7.93 mmol) at 0 °C under N2 atmosphere. After stirring
for 12 h at rt, the reaction mixture was treated with 10 mL of
MeOH, stirred for 30 min at rt and then concentrated under
reduced pressure. The residue was purified on silica gel column
chromatography (CH2Cl2 to CH2Cl2/MeOH = 10:1 v/v) to give com-
pound 7 (2.58 g, 7.06 mmol) in 89% yield. 1H NMR (400 MHz,
DMSO-d6) d 7.85 (s, 1H), 6.98 (br, 2H), 6.20 (dd, J = 1.6, 5.6 Hz,
1H), 6.05 (br, 2H), 5.04 (t, J = 3.6 Hz, 1H), 4.45 (dd, J = 1.6,
C
21H37N6O4Si (M+H+) 465.2461, found 465.2460.
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
3.1.7. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
3.1.12. (Z)-N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)octadec-8-enamide (9i)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)octanamide (8d)
Compound 8d was prepared using the same procedure as for
compound 8i; yield: 82%; 1H NMR (400 MHz, CDCl3) d 8.98 (s,
1H), 8.09 (s, 1H), 6.35 (dd, J = 1.2, 4.8 Hz, 1H), 5.33 (br, 2H), 5.12
(t, J = 3.2 Hz, 1H), 4.42 (dd, J = 1.2, 10.0 Hz, 1H), 4.22 (dd, J = 4.8,
10.0 Hz, 1H), 3.92–3.86 (m, 2H), 2.77 (t, J = 7.6 Hz, 2H), 1.76–1.69
(m, 2H), 1.42–1.28 (m, 8H), 0.92–0.84 (m, 12H), 0.08 (s, 6H); 13C
NMR (100 MHz, CDCl3) d 173.3, 159.9, 153.0, 150.0, 138.0, 115.9,
105.9, 79.4, 71.7, 63.5, 37.9, 31.8, 29.3, 29.2, 26.0, 25.1, 22.7,
18.7, 14.2, ꢀ5.21, ꢀ5.24; MS-ESI+ m/z 493(M+H+); HRMS-ESI+ calcd
for C23H41N6O4Si (M+H+) 493.2946, found 493.2953.
To a solution of compound 8i (2.20 g, 3.49 mmol) in 50 mL of
anhydrous THF was added Et3N-3HF (5.0 equiv, 0.36 g, 4.60 mmol)
at 0 °C under N2 atmosphere. After stirring for 12 h at rt, the reac-
tion mixture was concentrated under reduced pressure. The resi-
due was purified on silica gel column chromatography (CH2Cl2 to
CH2Cl2/MeOH = 20:1 v/v) to give compound 9i (1.71 g, 3.32 mmol)
in 95% yield. 1H NMR (400 MHz, DMSO-d6) d 10.10 (s, 1H), 8.08 (s,
1H), 6.43 (br, 2H), 6.27 (dd, J = 1.2, 5.6 Hz, 1H), 5.38–5.29 (m, 2H),
5.14 (t, J = 6.4 Hz, 1H), 5.04 (t, J = 3.2 Hz, 1H), 4.49 (dd, J = 1.2,
9.6 Hz, 1H), 4.20 (dd, J = 5.6, 10.0 Hz, 1H), 3.59 (dd, J = 3.2, 6.0 Hz,
1H), 2.53–2.48 (m, 2H), 2.04–1.92 (m, 4H), 1.60–1.50 (m, 2H),
1.36–1.18 (m, 22H), 0.86–0.81 (m, 3H); 13C NMR (100 MHz,
DMSO-d6) d 171.6, 159.9, 153.7, 150.0, 137.7, 129.6, 116.8, 105.5,
78.7, 70.4, 61.1, 36.1, 31.3, 29.13, 29.09, 28.83, 28.73, 28.69,
28.62, 28.58, 26.62, 26.57, 24.81, 22.09, 13.95; MS-ESI+ m/z 517
(M+H+); HRMS-ESI+ calcd for C27H45N6O4 (M+H+) 517.3501, found
517.3497.
3.1.8. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)decanamide (8e)
Compound 8e was prepared using the same procedure as for
compound 8i; yield: 85%; 1H NMR (400 MHz, CDCl3) d 8.93 (s,
1H), 8.06 (s, 1H), 6.32 (dd, J = 1.2, 5.2 Hz, 1H), 5.32 (br, 2H), 5.09
(t, J = 3.2 Hz, 1H), 4.40 (dd, J = 1.2, 9.6 Hz, 1H), 4.18 (dd, J = 5.2,
9.6 Hz, 1H), 3.90–3.82 (m, 2H), 2.75 (t, J = 7.6 Hz, 2H), 1.74–1.66
(m, 2H), 1.36–1.16 (m, 12H), 0.88–0.82 (m, 12H), 0.05 (s, 6H);
13C NMR (100 MHz, CDCl3) d 173.4, 159.8, 153.1, 150.0, 138.0,
116.0, 105.9, 79.3, 71.7, 63.5, 37.9, 32.0, 29.6, 29.4, 26.0, 25.0,
22.8, 18.7, 14.3, ꢀ5.22, ꢀ5.25; MS-ESI+ m/z 521(M+H+); HRMS-
ESI+ calcd for C25H45N6O4Si (M+H+) 521.3267, found 521.3266.
3.1.13. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)acetamide (9a)
Compound 9a was prepared using the same procedure as for
compound 9i; yield: 93%; 1H NMR (400 MHz, DMSO-d6) d 9.87 (s,
1H), 8.16 (s, 1H), 7.27 (br, 2H), 6.30 (d, J = 3.6 Hz, 1H), 5.14 (t,
J = 5.4 Hz, 1H), 5.05 (t, J = 3.2 Hz, 1H), 4.53 (dd, J = 1.2, 9.6 Hz,
1H), 4.22 (dd, J = 4.8, 9.6 Hz, 1H), 3.60 (m, 2H), 2.20 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) d 173.7, 156.1, 153.1, 150.0, 137.8,
115.5, 105.5, 79.0, 70.5, 61.1, 24.7; MS-ESI+ m/z 295 (M+H+);
HRMS-ESI+ calcd for C11H15N6O4 (M+H+) 295.1148, found
295.1149.
3.1.9. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)dodecanamide (8f)
Compound 8f was prepared using the same procedure as for
compound 8i; yield: 74%; 1H NMR (400 MHz, CDCl3) d 9.09 (s,
1H), 8.08 (s, 1H), 6.32 (d, J = 4.4 Hz, 1H), 5.34 (br, 2H), 5.10 (t,
J = 3.2 Hz, 1H), 4.39 (d, J = 10.0 Hz, 1H), 4.20 (dd, J = 5.6, 10.0 Hz,
1H), 3.92–3.84 (m, 2H), 2.70 (t, J = 7.6 Hz, 2H), 1.74–1.66 (m, 2H),
1.38–1.18 (m, 16H), 0.90–0.82 (m, 12H), 0.06 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 173.1, 159.9, 152.9, 150.0, 138.0, 116.0,
105.9, 79.4, 71.8, 63.4, 37.9, 32.1, 29.8, 29.7, 29.6, , 29.5, 29.4,
26.0, 25.1, 22.8, 18.7, 14.3, ꢀ5.22, ꢀ5.25; MS-ESI+ m/z
549(M+H+); HRMS-ESI+ calcd for C27H49N6O4Si (M+H+) 549.3583,
found 549.3579.
3.1.14. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)butyramide (9b)
Compound 9b was prepared using the same procedure as for
compound 9i; yield: 94%; 1H NMR (400 MHz, DMSO-d6) d 10.12
(s, 1H), 8.09 (s, 1H), 6.45 (br, 2H), 6.27 (d, J = 4.4 Hz, 1H), 5.14 (t,
J = 6.0 Hz, 1H), 5.04 (t, J = 3.2 Hz, 1H), 4.51 (d, J = 9.6 Hz, 1H), 4.20
(dd, J = 5.2, 9.6 Hz, 1H), 3.59 (dd, J = 3.2, 5.2 Hz, 2H), 1.62–1.56 (m,
2H), 0.91 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d
171.6, 156.0, 153.7, 150.0, 137.7, 116.9, 105.5, 78.7, 70.4, 38.0,
18.3, 13.6; MS-ESI+ m/z 323 (M+H+); HRMS-ESI+ calcd for
C13H19N6O4 (M+H+) 323.1463, found 323.1462.
3.1.10. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)tetradecanamide (8g)
Compound 8g was prepared using the same procedure as for
compound 8i; yield: 82%; 1H NMR (400 MHz, CDCl3) d 8.66 (br,
1H), 8.08 (s, 1H), 6.34 (dd, J = 1.2, 4.8 Hz, 1H), 5.14–5.11 (m, 3H),
4.42 (dd, J = 1.2, 9.6 Hz, 1H), 4.22 (dd, J = 5.2, 10.0 Hz, 1H), 3.93–
3.86 (m, 2H), 2.79 (t, J = 7.6 Hz, 2H), 1.75–1.69 (m, 2H), 1.40–1.20
(m, 20H), 0.92–0.85 (m, 12H), 0.09 (s, 6H); 13C NMR (100 MHz,
CDCl3) d 173.3, 159.7, 153.0, 150.0, 138.0, 116.0, 106.0, 79.4,
71.8, 63.5, 38.0, 32.1, 29.84, 29.71, 29.65, 29.55, 29.51, 26.1, 25.1,
22.9, 18.8, 14.3, ꢀ5.15, ꢀ5.18; MS-ESI+ m/z 577 (M+H+); HRMS-
ESI+ calcd for C29H53N6O4Si (M+H+) 577.3889, found 577.3892.
3.1.15. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)hexanamide (9c)
Compound 9c was prepared using the same procedure as for
compound 9i; yield: 96%; 1H NMR (400 MHz, CD3OD) d 8.21 (s,
1H), 6.34 (dd, J = 1.2, 4.8 Hz, 1H), 5.16 (t, J = 2.8 Hz, 1H), 4.52 (dd,
J = 1.2, 9.6 Hz, 1H), 4.25 (dd, J = 4.8, 10.0 Hz, 1H), 3.82–3.74 (m,
2H), 2.56 (t, J = 7.6 Hz, 2H), 1.75–1.67 (m, 2H), 1.40–1.30 (m, 4H),
0.92 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 174.6,
161.8, 154.6, 150.9, 140.0, 116.5, 107.1, 81.3, 72.5, 62.4, 38.3,
32.6, 26.1, 23.6, 14.1; MS-ESI+ m/z 351 (M+H+); HRMS-ESI+ calcd
for C15H23N6O4 (M+H+) 351.1781, found 351.1775.
3.1.11. N-(2-Amino-9-((2R,4R)-2-(((tert-butyldimethylsilyl)oxy)
methyl)-1,3-dioxolan-4-yl)-9H-purin-6-yl)palmitamide (8h)
Compound 8h was prepared using the same procedure as for
compound 8i; yield: 86%; 1H NMR (400 MHz, CDCl3) d 10.30 (br,
1H), 8.11 (s, 1H), 8.07 (s, 1H), 7.20–6.80 (br, 1H), 6.35 (d,
J = 4.8 Hz, 1H), 5.13 (t, J = 3.2 Hz, 1H), 4.46 (dd, J = 0.8, 9.6 Hz,
1H), 4.23 (dd, J = 5.2, 10.0 Hz, 1H), 3.94–3.86 (m, 2H), 2.97 (br,
2H), 1.77–1.70 (m, 2H), 1.44–1.20 (m, 24H), 0.92–0.84 (m, 12H),
0.09 (s, 6H); 13C NMR (100 MHz, CDCl3) d 176.8, 157.0, 153.6,
149.9, 138.0, 116.3, 106.0, 79.7, 71.9, 63.5, 37.4, 32.1, 29.9, 29.8,
29.78, 29.5, 26.1, 25.3, 22.8, 18.7, 14.3, ꢀ5.18, ꢀ5.20; MS-ESI+ m/
3.1.16. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)octanamide (9d)
Compound 9d was prepared using the same procedure as for
compound 9i; yield: 95%; 1H NMR (400 MHz, CD3OD) d 8.22 (s,
1H), 6.36 (dd, J = 1.2, 5.2 Hz, 1H), 5.11 (t, J = 2.4 Hz, 1H), 4.51 (dd,
J = 1.2, 10.0 Hz, 1H), 4.25 (dd, J = 5.6, 10.0 Hz, 1H), 3.82–3.74 (m,
2H), 2.57 (t, J = 7.2 Hz, 2H), 1.75–1.66 (m, 2H), 1.44–1.26 (m, 8H),
0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 174.7,
161.9, 154.7, 151.1, 140.1, 116.6, 107.2, 81.3, 72.5, 62.4, 38.4,
33.0, 30.42, 30.35, 26.4, 23.8, 14.6; MS-ESI+ m/z 379 (M+H+);
z
605 (M+H+); HRMS-ESI+ calcd for C31H57N6O4Si (M+H+)
605.4210, found 605.4205.
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

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J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
HRMS-ESI+ calcd for C17H27N6O4 (M+H+) 379.2091, found
379.2088.
175.1, 157.8, 154.7, 152.2, 151.7, 141.5, 139.9, 130.8, 126.3,
121.5, 121.3, 117.0, 104.9, 81.5, 72.4, 66.0, 62.4, 51.6, 40.2, 30.9,
20.5, 14.6; 31P NMR (162 MHz, CD3OD) d 5.23, 4.96; MS-ESI+ m/z
578 (M+H+); HRMS-ESI+ calcd for C24H33N7O8P (M+H+) 578.2130,
found 578.2123.
3.1.17. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)decanamide (9e)
Compound 9e was prepared using the same procedure as for
compound 9i; yield: 97%; 1H NMR (400 MHz, CD3OD) d 8.20 (s,
1H), 6.34 (dd, J = 1.2, 5.2 Hz, 1H), 5.11 (t, J = 2.4 Hz, 1H), 4.51 (dd,
J = 1.2, 10.0 Hz, 1H), 4.25 (dd, J = 5.6, 10.0 Hz, 1H), 3.82–3.74 (m,
2H), 2.56 (t, J = 7.6 Hz, 2H), 1.74–1.68 (m, 2H), 1.42–1.24 (m, 12H),
0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 174.6, 161.9,
154.6, 151.0, 140.0, 116.5, 107.2, 81.3, 72.5, 62.4, 38.4, 33.2, 30.75,
30.68, 30.56, 30.45, 26.4, 23.9, 14.6; MS-ESI+ m/z 407 (M+H+);
HRMS-ESI+ calcd for C19H31N6O4 (M+H+) 407.2398, found 407.2401.
3.1.22. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-hexanamido-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10c)
Compound 10c was prepared using the same procedure as for
compound 10i; yield: 75%; 1H NMR (400 MHz, CD3OD) d 8.10 (s,
0.5H), 8.08 (s, 0.5H), 7.32–7.07 (m, 5H), 6.39–6.36 (m, 1H), 5.32–
5.30 (m, 1H), 4.66–4.64 (m, 1H), 4.36–4.27 (m, 3H), 4.14–4.00
(m, 2H), 3.91–3.66 (m, 1H), 2.58–2.53 (m, 2H), 1.76–1.66 (m,
2H), 1.40–1.26 (m, 6H), 1.23–1.14 (m, 4H), 0.94–0.90 (m, 3H);
13C NMR (100 MHz, CD3OD) d 175.0, 174.5, 161.9, 154.8, 152.1,
151.0, 139.6, 130.8, 126.2, 121.5, 121.2, 116.5, 104.8, 81.1, 72.0,
66.0, 62.4, 51.5, 38.4, 32.7, 26.1, 23.7, 20.6, 20.5, 14.6, 14.5; 31P
NMR (162 MHz, CD3OD) d 5.07, 4.82; MS-ESI+ m/z 606 (M+H+);
HRMS-ESI+ calcd for C26H37N7O8P (M+H+) 606.2447, found
606.2436.
3.1.18. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)dodecanamide (9f)
Compound 9f was prepared using the same procedure as for
compound 9i; yield: 93%; 1H NMR (400 MHz, CD3OD) d 8.37 (s,
1H), 6.52 (d, J = 4.0 Hz, 1H), 5.12 (t, J = 2.4 Hz, 1H), 4.49 (dd,
J = 1.2, 10.0 Hz, 1H), 4.26 (dd, J = 5.2, 10.0 Hz, 1H), 3.84–3.76 (m,
2H), 2.48 (t, J = 7.2 Hz, 2H), 1.76–1.68 (m, 2H), 1.46–1.22 (m,
16H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 174.5,
157.7, 154.6, 152.5, 140.2, 116.9, 107.2, 81.6, 72.9, 62.3, 38.3,
33.2, 30.9, 30.8, 30.7, 30.6, 30.5, 26.8, 23.9, 14.6; MS-ESI+ m/z 435
(M+H+); HRMS-ESI+ calcd for C21H34N6O4Na (M+Na+) 457.2541,
found 457.2538.
3.1.23. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-octanamido-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10d)
Compound 10d was prepared using the same procedure as for
compound 10i; yield: 72%; 1H NMR (400 MHz, CD3OD) d 8.10 (s,
0.5H), 8.09 (s, 0.5H), 7.32–7.07 (m, 5H), 6.40–6.37 (m, 1H), 5.34–
5.31 (m, 1H), 4.66–4.64 (s, 1H), 4.34–4.28 (m, 3H), 4.14–4.00 (m,
2H), 3.91–3.66 (m, 1H), 2.58–2.52 (m, 2H), 1.75–1.67 (m, 2H),
1.44–1.26 (m, 9H), 1.23–1.15 (m, 5H), 0.91–0.88 (m, 3H); 13C
NMR (100 MHz, CD3OD) d 175.0, 174.5, 161.9, 154.8, 152.1,
151.0, 139.6, 130.8, 126.2, 121.5, 121.2, 116.5, 104.8, 81.1, 72.0,
66.0, 62.4, 51.5, 38.4, 32.7, 26.1, 23.7, 20.6, 20.5, 14.6; 31P NMR
(162 MHz, CD3OD) d 5.10, 4.85; MS-ESI+ m/z 634 (M+H+); HRMS-
ESI+ calcd for C28H41N7O8P (M+H+) 634.2758, found 634.2749.
3.1.19. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
dioxolan-4-yl)-9H-purin-6-yl)tetradecanamide (9g)
Compound 9g was prepared using the same procedure as for
compound 9i; yield: 93%; 1H NMR (400 MHz, CDCl3) d 8.98 (br,
1H), 8.01 (s, 1H), 6.28 (d, J = 4.8 Hz, 1H), 5.45 (br, 2H), 5.21 (s,
1H), 4.49 (d, J = 9.6 Hz, 1H), 4.25 (dd, J = 5.2, 10.0 Hz, 1H), 3.97–
3.88 (m, 2H), 2.68 (t, J = 7.2 Hz, 2H), 1.74–1.66 (m, 2H), 1.40–1.20
(m, 22H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
173.2, 159.8, 152.6, 149.7, 138.5, 115.4, 106.0, 79.8, 71.3, 61.6,
37.9, 33.9, 32.1, 29.88, 29.77, 29.72, 29.55, 25.1, 22.9, 14.3; MS-
ESI+ m/z 463 (M+H+); HRMS-ESI+ calcd for C23H39N6O4 (M+H+)
463.3021, found 463.3027.
3.1.24. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-decanamido-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10e)
Compound 10e was prepared using the same procedure as for
compound 10i; yield: 76%; 1H NMR (400 MHz, CD3OD) d 8.09 (s,
0.5H), 8.08 (s, 0.5H), 7.31–7.07 (m, 5H), 6.39–6.35 (m, 1H), 5.33–
5.30 (m, 1H), 4.66–4.63 (m, 1H), 4.35–4.27 (m, 3H), 4.14–4.00 (m,
2H), 3.92–3.65 (m, 1H), 2.58–2.53 (m, 2H), 1.76–1.66 (m, 2H),
1.42–1.26 (m, 13H), 1.23–1.15 (m, 5H), 0.91–0.88 (m, 3H); 13C
NMR (100 MHz, CD3OD) d 175.0, 174.5, 161.9, 154.7, 152.1, 151.0,
139.6, 130.8, 126.2, 121.5, 121.2, 116.4, 104.8, 81.1, 72.0, 66.0,
62.4, 51.5, 38.5, 33.2, 30.7, 30.5, 26.4, 23.9, 20.6, 20.5, 14.6; 31P
NMR (162 MHz, CD3OD) d 5.04, 4.79; MS-ESI+ m/z 662 (M+H+);
HRMS-ESI+ calcd forC30H45N7O8P (M+H+) 662.3067, found 662.3062.
3.1.20. (2S)-Ethyl 2-(((((2R,4R)-4-(6-acetamido-2-amino-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10a)
Compound 10a was prepared using the same procedure as for
compound 10i; yield: 68%; 1H NMR (400 MHz, CD3OD) d 8.20 (s,
0.5H), 8.19 (s, 0.5H), 7.33–7.24 (m, 2H), 7.20–7.17 (m, 1H), 7.15–
7.12 (m, 2H), 6.54–6.50 (m, 1H), 5.35–5.30 (m, 1H), 4.65–4.61
(m, 1H), 4.38–4.29 (m, 3H), 4.13–4.03 (m, 2H), 3.92–3.68 (m,
1H), 2.26 (br, 3H), 1.32–1.30 (m, 2H), 1.22–1.16 (m, 6H); 13C
NMR (100 MHz, CD3OD) d 186.0, 175.1, 157.8, 154.7, 152.1,
151.4, 141.5, 139.9, 130.9, 126.3, 121.5, 121.4, 117.0, 105.5,
104.9, 81.5, 72.3, 66.0, 62.5, 51.6, 24.8, 20.5, 14.6; 31P NMR
(162 MHz, CD3OD) d 5.19, 4.91; MS-ESI+ m/z 550 (M+H+); HRMS-
ESI+ calcd for C22H29N7O8P (M+H+) 550.1823, found 550.1810.
3.1.25. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-dodecanamido-
9H-purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)
phosphoryl)amino)propanoate (10f)
Compound 10f was prepared using the same procedure as for
compound 10i; yield: 70%; 1H NMR (400 MHz, CDCl3) d 9.84 (br,
1H), 8.05 (s, 0.5H), 8.04 (s, 0.5H), 7.30–7.08 (m, 5H), 6.89 (br,
2H), 6.38 (m, 1H), 5.34–5.31 (m, 1H), 4.58–4.60 (s, 1H), 4.43–
4.26 (m, 3H), 4.19–3.90 (m, 3H), 2.88 (br, 2H), 1.78–1.70 (m, 2H),
1.43–1.18 (m, 23H), 0.89–0.86 (m, 3H); 13C NMR (100 MHz, CDCl3)
d 173.7, 156.8, 153.7, 150.7, 150.2, 137.9, 129.8, 125.2, 120.3,
120.2, 116.3, 103.8, 80.0, 71.3, 65.2, 61.8, 50.4, 47.9, 37.5, 32.1,
29.9, 29.8, 29.6, 25.3, 22.9, 21.1, 21.0, 14.3; 31P NMR (162 MHz,
CDCl3) d 3.40, 3.16; MS-ESI+ m/z 690 (M+H+); HRMS-ESI+ calcd
for C32H49N7O8P (M+H+) 690.3379, found 690.3375.
3.1.21. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-butyramido-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10b)
Compound 10b was synthesized using the same procedure as
10i; yield: 79%; 1H NMR (400 MHz, CD3OD) d 8.21 (s, 0.5H), 8.19
(s, 0.5H), 7.34–7.21 (m, 2H), 7.20–7.18 (m, 1H), 7.16–7.10 (m,
2H), 6.56–6.53 (m, 1H), 5.35–5.30 (m, 1H), 4.64–4.60 (m, 1H),
4.39–4.30 (m, 3H), 4.14–4.03 (m, 2H), 3.92–3.69 (m, 1H), 2.47 (t,
J = 7.2 Hz, 2H), 1.78 (m, 2H), 1.32–1.31 (m, 2H), 1.23–1.19 (m,
3H), 1.04–0.99 (m, 3H); 13C NMR (100 MHz, CD3OD) d 175.7,
Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003

J.H. Cho et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
3.1.26. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-tetradecanamido-
9H-purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10g)
0.89–0.86 (m, 3H); 13C NMR (100 MHz, CDCl3) d 173.7, 173.2,
159.8, 153.1, 150.7, 150.0, 138.0, 130.2, 129.8, 125.2, 120.3,
120.2, 116.1, 103.8, 103.7, 80.0, 70.8, 65.2, 64.9, 61.8, 50.4, 38.1,
32.1, 29.9, 29.7, 29.6, 29.5, 29.4, 27.4, 25.0, 22.9, 21.3, 21.2, 14.3;
31P NMR (162 MHz, CDCl3) d 3.41, 3.15; MS-ESI+ m/z 772 (M+H+);
HRMS-ESI+ calcd for C38H59N7O8P (M+H+) 772.4183, found
772.4157.
Compound 10g was prepared using the same procedure as for
compound 10i; yield: 72%; 1H NMR (400 MHz, CD3OD) d 8.11 (s,
0.5H), 8.09 (s, 0.5H), 7.32–7.07 (m, 5H), 6.41–6.37 (m, 1H), 5.34–
5.31 (m, 1H), 4.66–4.63 (m, 1H), 4.36–4.28 (m, 3H), 4.14–4.00
(m, 2H), 3.91–3.66 (m, 1H), 2.58–2.53 (m, 2H), 1.78–1.68 (m,
2H), 1.43–1.24 (m, 21H), 1.23–1.15 (m, 5H), 0.91–0.88 (m, 3H);
13C NMR (100 MHz, CD3OD) d 175.1, 174.6, 162.0, 154.8, 152.1,
151.0, 139.7, 130.8, 126.2, 121.5, 121.3, 116.4, 104.9, 81.1, 72.0,
66.0, 62.4, 51.6, 38.5, 33.2, 30.95, 30.92, 30.89, 30.8, 30.6, 30.4,
26.4, 23.9, 20.6, 20.5, 14.6; 31P NMR (162 MHz, CD3OD) d 5.14,
4.88; MS-ESI+ m/z 718 (M+H+); HRMS-ESI+ calcd for C34H53N7O8P
(M+H+) 718.3703, found 718.3690.
4. Biological testing
Compounds were evaluated against HIV-1 and HBV as previ-
ously reported.9 Cytotoxicity was also determined in various cell
systems as described previously.9
Acknowledgments
3.1.27. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-palmitamido-9H-
purin-9-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)
amino)propanoate (10h)
We thank Tyana Singletary for excellent technical assistance.
This work was supported in part by NIH grant 5P30-AI-50409
(CFAR), and by the Department of Veterans Affairs. Dr. Schinazi is
the founder and a major shareholder of RFS Pharma, LLC. Emory
received no funding from RFS Pharma, LLC to perform this work
and vice versa.
Compound 10h was prepared using the same procedure as for
compound 10i; yield: 78%; 1H NMR (400 MHz, CDCl3) d 9.97 (br,
1H), 8.08 (s, 0.5H), 8.07 (s, 0.5H), 7.30–7.08 (m, 5H), 6.92 (br,
2H), 6.40–6.38 (m, 1H), 5.34–5.30 (m, 1H), 4.58–4.56 (s, 1H),
4.42–4.24 (m, 3H), 4.22–4.07 (m, 2H), 4.03–3.90 (m, 1H), 2.88
(br, 2H), 1.77–1.70 (m, 2H), 1.44–1.17 (m, 31H), 0.89–0.86 (m,
3H); 13C NMR (100 MHz, CDCl3) d 173.7, 156.8, 153.7, 150.7,
150.1, 137.9, 129.8, 125.1, 120.3, 120.2, 116.3, 103.7, 80.0, 71.3,
65.2, 61.7, 50.4, 39.3, 37.5, 32.1, 29.9, 29.84, 29.81, 29.76, 29.5,
25.3, 22.9, 21.1, 21.0, 14.3; 31P NMR (162 MHz, CDCl3) d 3.42,
3.19; MS-ESI+ m/z 746 (M+H+); HRMS-ESI+ calcd for C36H57N7O8P
(M+H+) 746.4012, found 746.4001.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.003.
References and notes
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Clercq, E. J. Clin. Virol. 2001, 22, 73; (d) Mehellou, Y.; De Clercq, E. J. Med. Chem.
2010, 53, 521.
3.1.28. N-(2-Amino-9-((2R,4R)-2-(hydroxymethyl)-1,3-
2. Related reviews (a) Miller, V. Antivir. Ther. 2001, 6, 1; (b) Wainberg, M. A.;
White, A. J. Antivir. Ther. 2001, 6, 11; (c) Shafer, R. W. Clin. Microbiol. Rev. 2002,
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Antiviral Ther. 2003, 8, 489; (f) Wainberg, M. A.; Turner, D. J. Acquir. Immune
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Holte, S.; Routy, J. P.; Daar, E. S.; Markowitz, M.; Collier, A. C. N. Engl. J. Med.
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53, 53; (j) Marcelin, A.-G.; Delaugerre, C.; Wirden, M.; Viegas, P.; Simon, A.;
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dioxolan-4-yl)-9H-purin-6-yl)palmitamide (9h)
Compound 9h was prepared using the same procedure as for
compound 9i; yield: 96%; 1H NMR (400 MHz, DMSO-d6) d 9.84 (s,
1H), 8.16 (s, 1H), 7.26 (br, 1H), 6.29 (d, J = 4.4 Hz, 1H), 5.15 (t,
J = 6.4 Hz, 1H), 5.05 (t, J = 3.2 Hz, 1H), 4.52 (d, J = 9.6 Hz, 1H), 4.21
(dd, J = 5.6, 9.6 Hz, 1H), 3.61–3.59 (m, 2H), 2.47 (t, J = 7.2 Hz, 2H),
1.53 (t, J = 5.8 Hz, 2H), 1.30–1.17 (m, 28H), 0.84 (t, J = 6.8 Hz, 3H);
13C NMR (100 MHz, DMSO-d6) d 186.5, 156.1, 153.0, 150.0, 137.7,
115.5, 105.5, 78.9, 70.5, 61.1, 47.3, 36.2, 33.9, 31.3, 29.1, 29.0,
28.9, 28.7, 24.9, 22.1, 14.0; MS-ESI+ m/z 491 (M+H+); HRMS-ESI+
calcd for C25H43N6O4 (M+H+) 491.3340, found 491.3340.
3.1.29. (2S)-Ethyl 2-(((((2R,4R)-4-(2-amino-6-((Z)-octadec-8-
enamido)-9H-purin-9-yl)-1,3-dioxolan-2-yl)methoxy)
(phenoxy)phosphoryl)amino)propanoate (10i)
To a solution of compound 9i (0.052 g, 0.100 mmol) in 10 mL of
anhydrous THF was added phosphoramidate chloride 6 (2.0 equiv,
0.058 g, 0.20 mmol) and N-methylimidazole (4.0 equiv, 0.033 g,
0.40 mmol) at ꢀ78 °C under argon atmosphere. After stirring for
12 h at rt, the solution was concentrated under reduced pressure.
The residue was dissolved in EtOAc (20 mL) and washed with
aqueous 0.5 M HCl solution (5 mL ꢁ 2) and brine (10 mL). The solu-
tion was dried over Na2SO4 for 3 h at rt and filtered. The filtrate
was concentrated under reduced pressure and purified on silica
gel column chromatography (CH2Cl2/MeOH = 40:1 to 20:1 v/v) to
give compound 10i (0.062 g, 0.081 mmol) in 81% yield. 1H NMR
(400 MHz, CDCl3) d 8.37 (s, 0.5H), 8.34 (s, 0.5H), 7.93 (s, 1H),
7.33–7.10 (m, 5H), 6.35–6.33 (m, 1H), 5.39–5.30 (m, 3H), 5.07
(br, 2H), 4.60–4.55 (m, 1H), 4.47–4.25 (m, 3H), 4.18–4.10 (m,
2H), 4.03–3.91 (m, 1H), 3.84–3.65 (m, 1H), 2.84–2.77 (m, 2H),
2.02–1.98 (m, 4H), 1.78–1.71 (m, 2H), 1.44–1.20 (m, 27H),
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Please cite this article in press as: Cho, J. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.003