Synthesis, antiviral activity, and stability of nucleoside analogs containing tricyclic bases

Article abstract of DOI:10.1016/j.ejmech.2009.04.003

A series of 3,9-dihydro-9-oxo-5H-imidazo[1,2-A]purine nucleosides (tricylic nucleosides) were synthesized from 9-[4-α-(hydroxymethyl)cyclopent-2-ene-1-α-yl]guanine (CBV) 5, (-)-β-D-(2R,4R)-1,3-dioxolane-guanosine (DXG) 6, 3′-azido-3′-deoxy-guanosine (AZG)

Full text of DOI:10.1016/j.ejmech.2009.04.003

European Journal of Medicinal Chemistry 44 (2009) 3845–3851
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiviral activity, and stability of nucleoside analogs containing
tricyclic bases
Franck Amblard a, Emilie Fromentin a, Mervi Detorio a, Alexander Obikhod a, Kimberly L. Rapp a,
,
Tamara R. McBrayer b, Tony Whitaker b, Steven J. Coats b, Raymond F. Schinazi a
*
a Center for AIDS Research, Veterans Affairs Medical Center, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine,
1670 Clairmont Road, Atlanta, GA 30033, 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:
A series of 3,9-dihydro-9-oxo-5H-imidazo[1,2-A]purine nucleosides (tricylic nucleosides) were synthe-
Received 16 February 2009
Received in revised form
30 March 2009
Accepted 2 April 2009
Available online 8 April 2009
sized from 9-[4-
a
-(hydroxymethyl)cyclopent-2-ene-1-
a
-yl]guanine (CBV) 5, (ꢀ)-
b-D-(2R,4R)-1,3-dioxo-
lane-guanosine (DXG) 6, 30-azido-30-deoxy-guanosine (AZG) 7, and 20-C-methylguanosine 8. Their in vitro
activity against HIV and HCV was evaluated and correlated to their ability to degrade to their purine
counterpart.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Nucleosides
Tricyclic
Stability
1. Introduction
cirrhosis, and hepatocellular carcinoma. Currently the only
approved therapy for chronic HCV infection is pegylated interferon-
The alkylation–condensation of guanosine nucleosides 1 with
phenylacyl bromide is known to produce the corresponding 3,9-
dihydro-9-oxo-5H-imidazo[1,2-A]purine nucleoside 2 (or simply
called tricyclic nucleoside). This reaction has been extensively
studied using various substrates such as 20-deoxyguanosine 1a [1]
and guanosine 1b, the cyclopropyl-guanosine 1c [2], 9-(2-phos-
phonyl–methoxyethyl)guanine (PMEG) 1d [3] and also the FDA
approvedganciclovir(GCV)1eandacyclovir(ACV) 1f [4–13](Scheme
1). The corresponding highly conjugated compounds 2 show some
fluorescent properties and, surprisingly in some cases, exhibited
potent and selective antiviral activity. Over the years, various studies
have established the antiviral influence of the substitution on the
tricylic moiety. The data obtained from more than 70 ACV and GCV
analogs, indicated that compounds 3 and 4 were as active as their
parent congeners against herpes simplex virus (HSV) [14] (Fig. 1).
Because of these unexpected results for such highly modified
nucleoside analogs, we investigated other guanosine derivatives
that target viruses of major public health concern such as HIV and
hepatitis C virus (HCV). HCV has infected an estimated 170 million
individuals worldwide and is a major cause of chronic liver disease,
a in combination with ribavirin, a modality often poorly tolerated
and effective in only half of the genotype 1 population [15,16]. On
the other hand an estimated 33 million people worldwide live with
HIV. In 2007, it is estimated HIV killed an estimated 2.1 million
people, including 330,000 children. Although treatments for HIV
can slow the course of the disease, there is currently no vaccine or
cure. For both HIV and HCV there is significant need for new and
more effective therapies. Based on the literature [17,18] and our
own antiviral expertise, we investigated the influence of this
tricyclic modification on 9-[4- -(hydroxymethyl)-cyclopent-2-ene-
a
1- b-D-(2R,4R)-1,3-dioxolane-guanosine
a
-yl]guanine (CBV) 5, (ꢀ)-
(DXG) 6, 30-azido-30-deoxyguanosine (AZG) 7, and 20-C-methyl-
guanosine 8 all well known for their anti-HIV [19] or anti-HCV
activities [20–23] (Fig. 2).
2. Results and discussion
2.1. Chemistry
CBV 5 [24], DXG 6, AZG 7 [25], and 20-C-methylguanosine 8 [26],
were synthesized according to literature procedures (Fig. 2). The
corresponding tricyclic derivatives were prepared by reacting
nucleoside analogs 5–8 with an appropriate bromoketone in the
presence of sodium hydride following the previously reported
* Corresponding author. Tel.: þ1 404 728 7711; fax: þ1 404 417 1535.
E-mail address: rschina@emory.edu (R.F. Schinazi).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.003

3846
F. Amblard et al. / European Journal of Medicinal Chemistry 44 (2009) 3845–3851
O
N
O
O
N
N
N
N
N
N
N
NH
N
OMe
R
a)
N
H
N
N
H
N
Sugar
1
NH2
HO
O
Sugar
3
2
O
O
P
HO
HO
HO
N
HO
N
O
OMe
O
N
N
N
HO
HO
H
O
c
d
HO
X
OH
4
a X=H
Sugar =
b X= OH
Fig. 1. Structure of tricyclic derivatives of ACV and GCV.
HO
HO
Scheme 1. (a) NaH, BrCH2(CO)R, DMF, rt.
O
HO
O
e
f
method for this alkylation–condensation reaction (Scheme 2). All
the synthesized tricyclic nucleoside analogs, 9–40, have been
characterized by 1H NMR while their purity and nominal mass were
checked by LC/MS before biological evaluation.
The similarities of antiviral profiles between parent nucleosides
and corresponding tricyclic nucleoside, also observed with ACV and
GCV derivatives, lead us to question the stability of the electron rich
tricycles in cells or in solution. Indeed, several groups have sug-
gested that this kind of tricycle might serve as a prodrug of the
parent nucleoside [5] however; no data has ever been generated to
support this hypothesis.
Since nucleosides are prodrugs that require conversion to the
corresponding triphosphate anabolite via cellular kinases, the
cellular level of phosphorylation for compound 38, which showed
significant activity against HCV, was evaluated. However, after
2.2. Antiviral evaluation and discussion
From our SAR effort (Table 1), several observations can be made.
First, the antiviral selectivity seemed to be related to the structure
of the sugar moiety. Indeed, the tricyclic compounds 36–39 with
a 20-C-methylribo sugar showed selective anti-HCV activity, like its
parent nucleoside 8. On the other hand, compounds 9–34 synthe-
sized from HIV active compound CBV 5, DXG 6 and AZG 7 showed
selective anti-HIV activity. The second important point is that
antiviral activities correlate with the electron richness of the
annulated aryl group of the tricyclic derivative. Thus, compounds
bearing an electron donor group such as 4-MeO-Ph (36), 4-NEt2-Ph
(37), 4-NMe2-Ph (38), 2-thiophenyl (39) showed EC50 values
incubation of compound 38 at 50 mM in Huh-7 cells for 4 h no
tricyclic-monophosphate (MP), -diphosphate (DP), -triphosphate
(TP) or even simple compound 38 were detected by LC–MS/MS. On
the other hand, significant levels of the parents compound 8 and its
MP, DP and TP were detected intra and extracellular, indicating that
the observed activity does not come from the triphosphate of 38, but
from conversion to 8-TP. To confirm these results, the stability of
various tricyclic compounds in solution was studied using LC–MS/
MS for detection of potential products. Compound 33, one of the
most potent compound against HIV, was unstable in water with
a half-life of 25 min. However, the same compound 33 was more
stable in MeOH with only 1% conversion to 7 after 72 h. Compounds
32, 36 and 39 which displayed lower antiviral activity also reverted
to parent nucleoside at slower rates (respectively 60% of 7, 30% of 8
and 60% of 8 after 3 weeks). These differences in the rate of
decomposition could certainly explain the variations of activity in
against HCV below 10 mM. In the same manner, tricyclic compounds
obtained from CBV, DXG, and AZG with a 2-thiophenyl (14, 27),
4-NEt2-Ph (12, 25, 33), 4-NMe2-Ph (13, 26, 34), 4-MeO-Ph (9, 18, 32)
group possessed anti-HIV activity similar to their corresponding
parent nucleoside analogs. However, other substitutions such as
ethyl (16, 35), 4-CN-Ph (30) or 4-Cl-Ph (22) showed less or no
activity in all cases. The influence of the electronic properties of the
substitution observed in our case seems to be in accordance with
the literature since electron withdrawing substituents such as
4-NO2-Ph, naphtalenyl, 3-MeO-Ph or 4-CF3-Ph are not active in the
ACV/GCV series [14]. Finally, it is noteworthy that the most potent
compounds in each series demonstrated a level of potency similar
to the parent nucleoside.
O
N
O
N
O
N
O
N
N
N
N
NH
NH2
NH
NH2
N
N
N
NH
NH2
a)
R2
HO
HO
O
N
R1
N
H
N
R1
N
O
9-40
5-8
DXG
6
CBV
5
HO
HO
HO
O
O
O
N
N
O
NH
NH2
NH
NH2
a
b
R1
=
N
O
HO
N
N
HO
N
O
HO
O
O
HO OH
2’-C-methylguanosine
8
N3
AZG
7
HO OH
N3
c
d
Fig. 2. Structure of CBV, DXG, AZG and 20-C-methylguanosine.
Scheme 2. (a) NaH, BrCH2(CO)R, DMF, rt, 10–50%.

F. Amblard et al. / European Journal of Medicinal Chemistry 44 (2009) 3845–3851
3847
Table 1
In vitro anti-HCV activity, anti-HIV activity and cellular toxicity of nucleoside analogs 9–40. nd: not determined.
Compound
R1
R2
Anti-HIV activity in human PBM cells (
EC50 EC90
0.043 0.29
mM)
Anti-HCV activity Huh-7 cells (
m
M)
Cytotoxicity (CC50 in mM)
EC50
EC90
PBM
7.0
>100
>100
>100
>100
CEM
VERO
CBV, 5
9
10
11
12
13
14
15
DXG, 6
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
AZG, 7
31
32
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a
a
a
a
a
a
a
4-MeO-Ph
4-Me-Ph
4-Br-Ph
4-NEt2-Ph
4-NMe2-Ph
2-thiophenyl
3-thiophenyl
0.62
5.0
12.3
4.9
30.1
43.2
0.13
0.11
0.64
2.6
0.51
31.5
0.65
0.47
7.1
24.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15.5
2.4
>100
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
Et
Ph
7.1
3.45
20.7
8.5
4.9
43.5
23.9
89.6
26.8
23.9
4-MeO-Ph
3-MeO-Ph
2-MeO-Ph
4-Me-Ph
4-Cl-Ph
>100
43.5
14.7
>100
>100
51.8
1.25
1.5
16
4-F-Ph
2,4-F-Ph
4-NEt2-Ph
4-NMe2-Ph
2-thiophenyl
3-thiophenyl
4-N3-Ph
4-CN-Ph
0.25
0.61
3.0
80.8
63.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.91
2.6
6.6
11.4
69.3
0.18
8.4
>100
1.98
94.1
7.0
c
c
c
c
Ph
69.6
56.7
>100
21.8
4-MeO-Ph
4-NEt2-Ph
4-NMe2-Ph
1.3
13.5
14.1
74.6
33
34
0.04
0.14
>100
0.34
0.54
>100
72.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2’-C-MeG, 8
35
36
37
38
2.3
>100
d
d
d
d
d
d
Et
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>10
ꢂ10
ꢃ10
>10
>10
nd
9.5
25.6
>10
>100
>100
>100
>100
>100
>100
4-MeO-Ph
4-NEt2-Ph
4-NMe2-Ph
2-Thiophenyl
3-Thiophenyl
4.4
6.9
ꢂ10
39
40
44.3
>100
each series. A more stable tricyclic compound will more slowly
convert to its active parent and conversely, a less stable tricyclic
compound will generate more of its active nucleoside parent.
With these surprising stability results, we decided to turn our
attention on to the tricyclic ACV analog 6-(4-MeOPh)-TACV 3,
which, according to the literature, shows a similar potency and
selectivity against HSV-1 and HSV-2 as ACV [14]. Compound 3 was
synthesized following the published procedure and evaluated for
activity against HSV-1. First of all, 3 showed some activity against
4. Experimental procedure
4.1. Synthesis
Anhydrous solvents were purchased from Aldrich Chemical
Company, Inc. Reagents were purchased from commercial sources.
Unless noted otherwise, the materials used in the examples were
obtained from readily available commercial suppliers or synthe-
sized by standard methods known to one skilled in the art of
chemical synthesis. 1H NMR spectra were taken on a Varian Unity
Plus 400 spectrometer at room temperature and reported in parts
per million downfield from internal tetramethylsilane. Signal
multiplicities are represented by s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quadruplet), br (broad), bs
(broad singlet), m (multiplet). All J values are in hertz. Mass
spectral analyses were performed on a Micromass TOF instrument
(Hewlett-Packard HPLC driven electrospray MS instrument).
Analytical HPLC analyses were performed on a Hewlett-Packard
HPLC with a Phenomenex Gemini-NX column (2 mm ꢁ 50 mm,
HSV-1 (EC50 ¼ 12.2
m
M, EC90 ¼ 23.7
m
M) even though 3 appears less
potent than ACV in our assay (EC50 ¼ 0.075
m
M, EC90 ¼ 0.7 M) and
m
less potent than what has been reported. Using the method
previously described, the stability of compound 3 in water was
studied and we found that it slowly converted to ACV (4% after 4
days and 35% after 3 weeks).
3. Conclusions
Despite the abundance of literature concerning this type of
tricyclic nucleosides, we are the first to report the instability of
some 3,9-dihydro-9-oxo-5H-imidazo[1,2-A]purine nucleosides in
aqueous media. These results indicate that the activity of the
electron rich tricyclic nucleosides was derived from their ability to
convert to the active parent nucleoside, thus acting as prodrugs.
More studies will be needed in order to determine if the differences
in solubility and polarity of these electron rich tricyclic nucleosides
translate into any advantage for the in vivo delivery of therapeuti-
cally important nucleoside analogs.
3 mm, C18, 110 Å). Mobile phase flow was 0.7 ml/min with a 3.5 min
gradient from 96% aqueous media (0.05% formic acid) to 96%
CH3CN (0.05% formic acid) with a 5.5 min total acquisition time and
190–360 nm PDA detection.
4.1.1. General procedure for the synthesis of compounds 9–40
To a solution of 5–8 (1 equiv) in anhydrous DMF (20 ml/1 mmol
of 5–8) was added sodium hydride as 60% suspension in oil
(1.1 equiv). After being stirred for 2 h at room temperature, the

3848
F. Amblard et al. / European Journal of Medicinal Chemistry 44 (2009) 3845–3851
resulting solution was treated with bromoketone (1.3 equiv) and
stirred overnight. After methanolysis, the volatiles were evaporated
and the residual solid was chromatographed on silica gel column
using CH2Cl2–MeOH (9:1 or 8:2). If necessary, the crude material,
after chromatography, was then washed with a minimum volume
of MeOH to afford the desired compounds 9–40.
7.95–7.96 (m, 1H), 8.08 (s, 1H), 13.00 (br s, NH). LCMS Calcd for
17H15N5O2S 353.4; observed (M þ 1) 354.5.
C
4.1.1.8. 3-( -1,3-Dioxolanyl)-3,9-dihydro-6-ethyl-9-oxo-5H-imidazo-
b-D
[1,2-a]purine (16). 1H NMR (DMSO-d6)
d
1.2 (t, 3H, J ¼ 7.6 Hz, CH3),
2.55–2.61 (m, 2H, CH2), 3.41–3.29 (m, 2H, CH2), 4.16–4.21 (m, 1H),
4.45–4.48 (m, 1H), 5.01–5.02 (m, 1H), 5.12–5.15 (m, 1H), 6.26 (d, 1H,
J ¼ 5.2 Hz), 7.33 (s, 1H), 8.00 (s, 1H), 8.27 (s, 1H), 13.01 (br s, NH).
LCMS Calcd for C17H15N5O2S 305.3; observed (M þ 1) 306.4.
4.1.1.1. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(4-methoxyphenyl)-9-oxo-5H-imidazo-[1,2-a]purine (9). 1H NMR
(DMSO-d6)
d 1.58–1.64 (m, 1H), 2.58–2.66 (m, 1H), 2.83–2.87
(m, 1H), 3.39–3.43 (m, 2H), 3.76 (s, 3H, CH3), 4.73 (br s, 1H), 5.43–
5.47 (m, 1H), 5.91–5.93 (m, 1H), 6.12–6.14 (m, 1H), 6.98 (d, 2H,
J ¼ 8.8 Hz), 7.71 (s, 1H), 7.80 (d, 2H, J ¼ 8.8 Hz), 7.95 (s, 1H), 12.91 (br
4.1.1.9. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-phenyl-9-oxo-5H-imi-
dazo-[1,2-a]purine (17). 1H NMR (DMSO-d6)
d
3.57–3.60 (m, 2H,
CH2), 4.19–4.24 (m, 1H), 4.50–4.53 (m, 1H), 5.04 (t, 1H, J ¼ 2.8 Hz),
5.14 (t, 1H, J ¼ 6.0 Hz), 6.29 (d, 1H, J ¼ 4.0 Hz), 7.34–7.38 (m, 1H),
7.43–7.47 (m, 2H), 7.88 (d, 2H, J ¼ 7.2 Hz), 8.06 (s, 1H), 8.20 (s, 1H),
13.05 (br s, NH). LCMS Calcd for C17H15N5O5 353.3; observed
(M þ 1) 354.4.
s, NH); 13C NMR (DMSO-d6)
d 34.8, 48.5, 55.9, 59.6, 64.7, 102.5, 115.1,
116.4,121.1,127.2,129.6, 130.1,137.3, 139.3, 146.9, 150.3, 152.0,160.3;
LC–MS Calcd for C20H18N5O3 377.4; observed (M þ 1) 378.3.
4.1.1.2. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(4-methylphenyl)-9-oxo-5H-imidazo-[1,2-a]purine (10). 1H NMR
4.1.1.10. 3-(
(4-methoxyphenyl)-9-oxo-5H-imidazo-[1,2-a]purine (18). 1H NMR
(DMSO-d6) 3.56–3.60 (m, 2H, CH2), 3.77 (s, 3H, CH3), 4.19–4.24 (m,
1H), 4.49–4.53 (m, 1H), 5.02–5.05 (m, 1H), 5.12–5.15 (m, 1H), 6.29
(d, 1H, J ¼ 4.8 Hz), 7.01 (d, 2H, J ¼ 8.8 Hz), 7.81 (d, 2H, J ¼ 8.8 Hz),
8.05 (s, 1H), 8.06 (s, 1H), 12.98 (br s, NH); 13C NMR (DMSO-d6)
b-D-1,3-Dioxolanyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(DMSO-d6)
d 1.58–1.65 (m, 1H), 2.30 (s, 3H, CH3), 2.58–2.66 (m,
1H), 2.83–2.87 (m, 1H), 3.39–3.43 (m, 2H), 4.72 (br s, 1H), 5.43–
5.47 (m, 1H), 5.91–5.94 (m, 1H), 6.12–6.15 (m, 1H), 7.24 (d, 2H,
J ¼ 7.6 Hz), 7.74–7.78 (m, 3H), 8.09 (s, 1H), 12.90 (br s, NH). LC–MS
Calcd for C20H19N5O3 361.4; observed (M þ 1) 362.5.
d
d
56.0, 61.8, 71.2, 79.9, 102.6, 106.2, 115.1, 115.9, 120.9, 127.2, 129.8,
4.1.1.3. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
137.0, 146.3, 150.3, 151.9, 160.3; LCMS Calcd for C18H17N5O5 383.4;
observed (M þ 1) 384.5.
(4-bromophenyl)-9-oxo-5H-imidazo-[1,2-a]purine (11). 1H NMR
(DMSO-d6)
d 1.58–1.65 (m, 1H), 2.59–2.67 (m, 1H), 2.83–2.87
(m, 1H), 3.39–3.43 (m, 2H), 4.69–4.73 (m, 1H), 5.43–5.47 (m, 1H),
5.92–5.94 (m, 1H), 6.13–6.15 (m, 1H), 7.65 (d, 2H, J ¼ 8.8 Hz), 7.79–
7.85 (m, 3H), 8.26 (s, 1H), 13.01 (br s, NH). LC–MS Calcd for
C19H16BrN5O2 425.3/427.4; observed (M þ 1) 426.1/428.2.
4.1.1.11. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(3-methoxyphenyl)-9-
oxo-5H-imidazo-[1,2-a]purine (19). 1H NMR (DMSO-d6)
d
3.56–3.60
(m, 2H, CH2), 3.80 (s, 3H, CH3), 4.19–4.24 (m, 1H), 4.49–4.53 (m, 1H),
5.03–5.05 (m, 1H), 5.12–5.15 (m, 1H), 6.29 (d, 1H, J ¼ 4.8 Hz), 6.92
(d, 1H, J ¼ 7.2 Hz), 7.32–7.37 (m, 1H), 7.45–7.48 (m, 2H), 8.05 (s, 1H),
8.27 (s, 1H), 13.05 (br s, NH). LCMS Calcd for C18H17N5O5 383.4;
observed (M þ 1) 384.4.
4.1.1.4. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(4-diethylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine
NMR (DMSO-d6)
(12). 1H
d
1.03–1.07 (m, 6H, 2 ꢁ CH3), 1.58–1.65 (m, 1H),
2.57–2.66 (m, 1H), 2.83–2.87 (m, 1H), 3.31–3.36 (m, 4H, 2 ꢁ CH2),
3.39–3.43 (m, 2H, CH2), 4.72 (br s, 1H), 5.43–5.47 (m, 1H), 5.91–5.94
(m, 1H), 6.12–6.15 (m, 1H), 6.67 (d, 2H, J ¼ 8.0 Hz), 7.63–7.68 (m,
3H), 7.85 (s, 1H), 12.82 (br s, NH). LCMS Calcd for C23H26N6O2 418.5;
observed (M þ 1) 419.4.
4.1.1.12. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(2-methoxyphenyl)-9-
oxo-5H-imidazo-[1,2-a]purine (20). 1H NMR (DMSO-d6)
d
3.56–3.60
(m, 2H, CH2), 3.95 (s, 3H, CH3), 4.19–4.23 (m, 1H), 4.49–4.51 (m, 1H),
5.03–5.05 (m, 1H), 5.10–5.13 (m, 1H), 6.29 (d, 1H, J ¼ 3.6 Hz), 7.03–
7.07 (m, 1H), 7.15–7.17 (m, 1H), 7.34–7.39 (m, 1H), 7.82–7.84 (m, 1H),
7.92 (s, 1H), 8.04 (s, 1H), 13.05 (br s, NH). LCMS Calcd for C18H17N5O5
383.4; observed (M þ 1) 384.4.
4.1.1.5. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(4-dimethylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine (13). 1H
4.1.1.13. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(4-methylphenyl)-
2.30
NMR (DMSO-d6)
d 1.58–1.65 (m, 1H), 2.58–2.66 (m, 1H), 2.83–2.87
9-oxo-5H-imidazo-[1,2-a]purine (21). 1H NMR (DMSO-d6)
d
(m, 1H), 2.92 (s, 6H, 2 ꢁ CH3), 3.39–3.43 (m, 2H), 4.71 (t, 1H,
J ¼ 5.6 Hz), 5.43–5.47 (m, 1H), 5.91–5.94 (m, 1H), 6.12–6.15 (m, 1H),
6.74 (d, 2H, J ¼ 8.8 Hz), 7.67 (d, 2H, J ¼ 8.8 Hz), 7.77 (s, 1H), 7.88
(s, 1H), 12.8 (br s, NH). LCMS Calcd for C21H22N6O2 390.4; observed
(M þ 1) 391.4.
(s, 3H, CH3), 3.56–3.60 (m, 2H, CH2), 4.19–4.23 (m, 1H), 4.47–4.50
(m, 1H), 5.03–5.04 (m, 1H), 5.14–5.17 (m, 1H), 6.29 (d, 1H,
J ¼ 4.4 Hz), 7.24 (d, 2H, J ¼ 8.4 Hz), 7.75 (d, 2H, J ¼ 8.4 Hz), 8.00
(s, 1H), 8.07 (s, 1H), 13.02 (br s, NH). LCMS Calcd for C18H17N5O4
367.4; observed (M þ 1) 368.5.
4.1.1.6. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(2-thienyl)-9-oxo-5H-imidazo-[1,2-a]purine (14). 1H NMR (DMSO-
4.1.1.14. 3-(
b
-D-1,3-Dioxolanyl)-3,9-dihydro-6-(4-chlorophenyl)-9-
3.55–3.60
d6)
d
1.59–1.65 (m, 1H), 2.58–2.66 (m, 1H), 2.83–2.87 (m, 1H),
oxo-5H-imidazo-[1,2-a]purine (22). 1H NMR (DMSO-d6)
d
3.39–3.43 (m, 2H, CH2), 4.71 (br s, 1H), 5.43–5.47 (m, 1H), 5.91–5.94
(m, 1H), 6.12–6.14 (m, 1H), 7.12–7.15 (m, 1H), 7.57–7.58 (m, 1H), 7.62
(d, 1H, J ¼ 5.2 Hz), 7.79 (s, 1H), 7.88 (s, 1H), 13.19 (br s, NH). LCMS
Calcd for C17H15N5O2S 353.4; observed (M þ 1) 354.5.
(m, 2H, CH2), 4.19–4.22 (m, 1H), 4.47–4.49 (m, 1H), 5.03–5.04
(m, 1H), 5.14–5.19 (m, 1H), 6.29 (d, 1H, J ¼ 4.4 Hz), 7.48 (d, 2H,
J ¼ 8.4 Hz), 7.89 (d, 2H, J ¼ 8.4 Hz), 7.98 (s, 1H), 8.17 (s, 1H), 13.04
(br s, NH). LCMS Calcd for C17H14ClN5O4 387.8; observed (M þ 1)
388.1/390.2.
4.1.1.7. 3-[4-(Hydroxymethyl)-2-cyclopenten-1-yl]-3,9-dihydro-6-
(3-thienyl)-9-oxo-5H-imidazo-[1,2-a]purine (15). 1H NMR (DMSO-
4.1.1.15. 3-(
b
-D-1,3-Dioxolanyl)-3,9-dihydro-6-(4-fluorophenyl)-9-
3.57–3.60
d6)
d
1.59–1.65 (m, 1H), 2.58–2.64 (m, 1H), 2.83–2.87 (m, 1H),
oxo-5H-imidazo-[1,2-a]purine (23). 1H NMR (DMSO-d6)
d
3.39–3.43 (m, 2H, CH2), 4.70–4.73 (m, 1H), 5.44–5.47 (m, 1H), 5.92–
5.95 (m, 1H), 6.12–6.15 (m, 1H), 7.67–7.68 (m, 2H), 7.79 (s, 1H),
(m, 2H, CH2), 4.19–4.23 (m, 1H), 4.51 (d, 1H, J ¼ 9.2 Hz), 5.03–5.05
(m, 1H), 5.12 (t, 1H, J ¼ 6.0 Hz), 6.29 (d, 1H, J ¼ 4.4 Hz), 7.29–7.33

F. Amblard et al. / European Journal of Medicinal Chemistry 44 (2009) 3845–3851
3849
(m, 2H), 7.91–7.95 (m, 2H), 8.05 (s, 1H), 8.21 (s, 1H), 13.09 (br s, NH).
LCMS Calcd for C17H14N5O4 371.3; observed (M þ 1) 372.2.
4.1.1.24. 3-(3-Azido-2,3-dideoxy-
6-(4-methoxyphenyl)-9-oxo-5H-imidazo-[1,2-a]purine (32). 1H NMR
(DMSO-d6) 2.46–2.52 (m, 1H), 2.83–2.90 (m, 1H), 3.53–3.61 (m, 2H,
b-D-erythro-pentofuranosyl)-3,9-dihydro-
d
4.1.1.16. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(2,4-difluorophenyl)-9-
CH2), 3.77 (s, 3H, OCH3), 3.88–3.90 (m, 1H), 4.54–4.59 (m, 1H), 5.11–5.17
(m, 1H), 6.21 (t, 1H, J ¼ 6.4 Hz), 7.01 (d, 2H, J ¼ 8.6 Hz), 7.81 (d, 2H,
J¼ 8.6 Hz), 8.06 (s, 1H), 8.15 (s, 1H), 12.93 (br s, NH). 13C NMR (DMSO-d6)
oxo-5H-imidazo-[1,2-a]purine (24). 1H NMR (DMSO-d6)
3.56–3.59
d
(m, 2H, CH2), 4.19–4.23 (m, 1H), 4.50 (d, 1H, J ¼ 9.2 Hz), 5.03–5.05
(m, 1H), 5.14 (br s, 1H), 6.29–6.31 (m, 1H), 7.24–7.29 (m, 1H), 7.43–
7.49 (m, 1H), 7.81 (d, 1H, J ¼ 3.2 Hz), 7.92–7.98 (m, 1H), 8.03 (s, 1H),
13.01 (br s, NH). LCMS Calcd for C17H13F2N5O4 389.3; observed
(M þ 1) 390.3.
d
36.9, 55.9, 61.4, 61.8, 83.2, 85.2, 102.6, 115.1, 116.4, 121.0, 127.2, 129.8,
137.5, 146.8, 150.2, 151.9, 160.3; LCMS Calcd for C19H18N8O4 422.4;
observed (M þ 1) 423.5.
4.1.1.25. 3-(3-Azido-2,3-dideoxy-
dihydro-6-(4-diethylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine
(33). 1H NMR (CDCl3)
b-D-erythro-pentofuranosyl)-3,9-
4.1.1.17. 3-(
b
-D-1,3-Dioxolanyl)-3,9-dihydro-6-(4-diethylaminophenyl)-
9-oxo-5H-imidazo-[1,2-a]purine (25). 1H NMR (DMSO-d6)
d
1.07
d
1.06–1.25 (m, 6H, 2 ꢁ CH3), 2.30–2.35 (m,
(t, 6H, J ¼ 6.8 Hz, 2 ꢁ CH3), 3.27–3.37 (m, 4H, 2 ꢁ CH2), 3.57–3.60
(m, 2H, CH2), 4.18–4.23 (m, 1H), 4.49–4.52 (m, 1H), 5.03–5.04 (m,
1H), 5.11 (t, 1H, J ¼ 6.0 Hz), 6.28 (d, 1H, J ¼ 4.8 Hz), 6.68 (d, 2H,
J ¼ 8.8 Hz), 7.64 (d, 2H, J ¼ 8.8 Hz), 7.86 (s, 1H), 8.03 (s, 1H), 12.81
(br s, NH). LCMS Calcd for C21H24N6O4 424.5; observed (M þ 1)
425.6.
1H), 3.10–3.15 (m, 1H), 3.22–3.29 (m, 2H, CH2), 3.77 (d, 1H,
J ¼ 12.4 Hz), 4.06 (d, 1H, J ¼ 12.4 Hz), 4.19 (s, 1H), 4.44–4.46 (m, 1H),
6.10–6.14 (m, 1H), 6.58 (d, 2H, J ¼ 8.6 Hz), 7.36 (d, 2H, J ¼ 8.6 Hz),
7.55 (s,1H), 7.65 (s,1H). LCMS Calcd for C22H25N9O3 463.5; observed
(M þ 1) 464.6.
4.1.1.26. 3-(3-Azido-2,3-dideoxy-
hydro-6-(4-dimethylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine
(34). 1H NMR (DMSO-d6)
3.61 (m, 2H, CH2), 3.87–3.90 (m, 1H), 4.52–4.59 (m, 1H), 5.11–5.17
(m, 1H), 6.18–6.23 (m, 1H), 6.75 (d, 2H, J ¼ 8.6 Hz), 7.68 (d, 2H,
J ¼ 8.6 Hz), 7.91 (s, 1H), 8.13 (s, 1H), 12.85 (br s, NH). LCMS Calcd for
C20H21N9O3 435.5; observed (M þ 1) 436.5.
b-D-erythro-pentofuranosyl)-3,9-di-
4.1.1.18. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(4-dimethylaminophenyl)-
9-oxo-5H-imidazo-[1,2-a]purine (26). 1H NMR (DMSO-d6)
d
2.92
d
2.81–3.02 (m, 8H, 2 ꢁ CH3, CH2), 3.53–
(s, 6H, 2 ꢁ CH3), 3.57–3.60 (m, 2H, CH2), 4.19–4.23 (m, 1H), 4.49–
4.52 (m, 1H), 5.03–5.04 (m, 1H), 5.12 (t, 1H, J ¼ 6.0 Hz), 6.28 (d, 1H,
J ¼ 4.8 Hz), 6.74 (d, 2H, J ¼ 8.8 Hz), 7.68 (d, 2H, J ¼ 8.8 Hz), 7.90
(s, 1H), 8.02 (s, 1H), 12.84 (br s, NH). LCMS Calcd for C19H20N6O4
396.4; observed (M þ 1) 397.3.
4.1.1.27. 3-(3-azido-2,3-dideoxy-
dihydro-6-ethyl-9-oxo-5H-imidazo-[1,2-a]purine (35). 1H NMR
(DMSO-d6)
b-D-erythro-pentofuranosyl)-3,9-
4.1.1.19. 3-(
b
-D-1,3-Dioxolanyl)-3,9-dihydro-6-(2-thienyl)-9-oxo-5H-
imidazo-[1,2-a]purine (27). 1H NMR (DMSO-d6)
d
3.56–3.60 (m, 2H,
d
0.79 (s, 3H, CH3), 1.19 (t, 3H, J ¼ 7.6 Hz, CH3), 2.56–2.60
CH2), 4.19–4.23 (m, 1H), 4.49–4.51 (m, 1H), 5.02–5.04 (m, 1H), 5.12
(t, 1H, J ¼ 6.4 Hz), 6.28 (d, 1H, J ¼ 4.4 Hz), 7.13–7.15 (m, 1H), 7.58
(d, 1H, J ¼ 3.2 Hz), 7.62–7.64 (m, 1H), 7.91 (s, 1H), 8.04 (s, 1H), 13.17
(br s, NH). LCMS Calcd for C15H13N5O4S 359.4; observed (M þ 1)
360.5.
(m, 2H, CH2), 3.62–3.67 (m, 1H), 3.77–3.86 (m, 2H), 3.95–4.00 (m,
1H), 5.10 (s, OH), 5.15–5.17 (m, OH), 5.25 (d, J ¼ 6.8 Hz, OH), 5.82
(s,1H, H-10), 7.31 (s,1H), 8.21 (s,1H),12.91 (br s, NH). LCMS Calcd for
C15H19N5O5 349.3; observed (M þ 1) 350.2.
4.1.1.28. 3-(
phenyl)-9-oxo-5H-imidazo-[1,2-a]purine (36). 1H NMR (DMSO-d6)
0.80 (s, 3H, CH3), 3.62–3.70 (m, 1H), 3.77–3.88 (m, 4H), 3.96–4.00
b-D-2-C-Methyl-ribofuranosyl)-3,9-dihydro-6-(4-methoxy-
4.1.1.20. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(3-thienyl)-9-oxo-5H-
imidazo-[1,2-a]purine (28). 1H NMR (DMSO-d6)
d
3.57–3.60 (m, 2H,
d
CH2), 4.19–4.23 (m, 1H), 4.49–4.52 (m, 1H), 5.02–5.04 (m, 1H), 5.12–
5.15 (m, 1H), 6.29 (d, 1H, J ¼ 5.6 Hz), 7.67–7.68 (m, 2H), 7.97 (s, 1H),
8.04 (s, 1H), 8.10 (s, 1H), 13.06 (br s, NH). LCMS Calcd for
C15H18N5O4S 359.4; observed (M þ 1) 360.3.
(m, 1H), 4.06–4.10 (m, 1H), 5.10 (s, OH), 5.17–5.19 (m, OH), 5.29 (d,
J ¼ 6.4 Hz, OH), 5.86 (s, 1H, H-10), 7.00 (d, 2H, J ¼ 8.8 Hz), 7.81 (d, 2H,
J ¼ 8.8 Hz), 8.05 (s, 1H), 8.25 (s, 1H), 13.05 (br s, NH); 13C NMR
(DMSO-d6) d 20.7, 55.9, 60.0, 72.2, 79.3, 83.0, 91.2, 102.5, 115.2, 116.1,
121.0, 127.2, 129.8, 137.2, 146.9, 150.2, 151.9, 160.3; LCMS Calcd for
4.1.1.21. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(4-azido-phenyl)-9-oxo-
C20H21N5O5 427.4; observed (M þ 1) 428.3.
5H-imidazo-[1,2-a]purine (29). 1H NMR (DMSO-d6)
d
3.56–3.60 (m,
2H, CH2), 4.19–4.23 (m, 1H), 4.51 (d, 1H, J ¼ 9.2 Hz), 5.00–5.03 (m,
1H), 5.12 (t, 1H, J ¼ 6.0 Hz), 6.28 (d, 1H, J ¼ 4.8 Hz), 7.19 (d, 2H,
J ¼ 8.4 Hz), 7.91 (d, 2H, J ¼ 8.4 Hz), 8.04 (s, 1H), 8.20 (s, 1H), 13.07
(br s, NH). LCMS Calcd for C17H14N8O4 394.3; observed (M þ 1)
395.4.
4.1.1.29. 3-(
ylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine (37). 1H NMR
(DMSO-d6)
(m, 4H, 2 ꢁ CH2), 3.63–3.68 (m, 1H), 3.78–3.87 (m, 2H), 3.95–4.00
(m, 1H), 5.07 (s, OH), 5.17–5.19 (m, OH), 5.28 (d, J ¼ 6.8 Hz, OH), 5.86
(s, 1H, H-10), 6.67 (d, 2H, J ¼ 8.8 Hz), 7.63 (d, 2H, J ¼ 8.8 Hz), 7.84
(s, 1H), 8.22 (s, 1H), 12.85 (br s, NH). LCMS Calcd for C23H28N6O5
468.5; observed (M þ 1) 469.6.
b-D-2-C-Methyl-ribofuranosyl)-3,9-dihydro-6-(4-dieth-
d
0.79 (s, 3H, CH3), 1.00–1.13 (m, 6H, 2 ꢁ CH3), 3.33–3.40
4.1.1.22. 3-(
b
-
D
-1,3-Dioxolanyl)-3,9-dihydro-6-(4-cyano-phenyl)-9-oxo-
5H-imidazo-[1,2-a]purine (29). 1H NMR (DMSO-d6)
d
3.57–3.60 (m,
2H, CH2), 4.19–4.24 (m, 1H), 4.50–4.53 (m, 1H), 5.03–5.04 (m, 1H),
5.11–5.15 (m, 1H), 6.29 (d, 1H, J ¼ 4.4 Hz), 7.91–7.93 (m, 2H), 8.05–
8.08 (m, 3H), 8.47 (s, 1H), 13.25 (br s, NH). LCMS Calcd for
C18H14N6O4 378.3; observed (M þ 1) 379.2.
4.1.1.30. 3-(
thylaminophenyl)-9-oxo-5H-imidazo-[1,2-a]purine (38). 1H NMR
(DMSO-d6)
b-D-2-C-Methyl-ribofuranosyl)-3,9-dihydro-6-(4-dime-
d
0.79 (s, 3H, CH3), 2.92 (s, 6H, 2 ꢁ CH3), 3.63–3.68 (m,
1H), 3.79–3.97 (m, 2H), 3.96–4.01 (m, 1H), 5.08 (s, OH), 5.17–5.19
(m, OH), 5.28 (d, J ¼ 6.8 Hz, OH), 5.86 (s, 1H, H-10), 6.74 (d, 2H,
J ¼ 8.8 Hz), 7.67 (d, 2H, J ¼ 8.8 Hz), 7.90 (s, 1H), 8.25 (s, 1H), 12.89 (br
s, NH). LCMS Calcd for C21H24N6O5 440.4; observed (M þ 1) 441.5.
4.1.1.23. 3-(3-Azido-2,3-dideoxy-
dihydro-6-phenyl-9-oxo-5H-imidazo-[1,2-a]purine (31). 1H NMR
(DMSO-d6) 2.46–2.52 (m, 1H), 2.82–2.89 (m, 1H), 3.53–3.61 (m,
b-D-erythro-pentofuranosyl)-3,9-
d
2H, CH2), 3.87–3.90 (m, 1H), 4.52–4.59 (m, 1H), 5.12–5.16 (m, 1H),
6.21 (t, 1H, J ¼ 6.0 Hz), 7.35–7.39 (m, 1H), 7.43–7.47 (m, 2H), 7.87–
7.89 (m, 2H), 8.16 (s, 1H), 8.20 (s, 1H), 13.02 (br s, NH). LCMS Calcd
for C18H16N8O3 392.4; observed (M þ 1) 393.3.
4.1.1.31. 3-(
9-oxo-5H-imidazo-[1,2-a]purine (39). 1H NMR (DMSO-d6)
3H, CH3), 3.64–3.68 (m, 1H), 3.79–3.87 (m, 2H), 3.96–4.00 (m, 1H),
b-D-2-C-Methyl-ribofuranosyl)-3,9-dihydro-6-(2-thienyl)-
d
0.80 (s,

3850
F. Amblard et al. / European Journal of Medicinal Chemistry 44 (2009) 3845–3851
5.09 (s, OH), 5.17–5.19 (m, OH), 5.29 (d, J ¼ 6.8 Hz, OH), 5.85 (s, 1H,
H-10), 7.12–7.15 (m, 1H), 7.56 (d, 1H, J ¼ 2.4 Hz), 7.62 (d, 1H,
J ¼ 5.2 Hz), 7.89 (s, 1H), 8.25 (s, 1H), 13.23 (br s, NH). LCMS Calcd for
C17H17N5O5S 403.4; observed (M þ 1) 404.5.
was conditioned with 750
Nucleoside mono-, di- and triphosphates were removed by
sequential washes with 750 l of 100 mM KCl, 120 mM KCl and
400 mM KCl, respectively. The cartridge was rinsed with 150 l of
the corresponding buffer between each. The fractions were
collected for de-phosphorylation.
ml of ddH2O and 300 ml 20 mM KCl.
m
m
4.1.1.32. 3-(
b
-
D
-2-C-Methyl-ribofuranosyl)-3,9-dihydro-6-(3-thienyl)-
9-oxo-5H-imidazo-[1,2-a]purine (40). 1H NMR (DMSO-d6)
d
0.79 (s,
The internal standard (3TC, 10 ml of a solution at 1 mM) was
3H, CH3), 3.64–3.68 (m, 1H), 3.78–3.85 (m, 2H), 3.97–4.02 (m, 1H),
5.09 (s, OH), 5.17–5.19 (m, OH), 5.28 (d, J ¼ 6.8 Hz, OH), 5.85 (s, 1H,
H-10), 7.67–7.68 (m, 2H), 7.95–7.96 (m, 1H), 7.95 (s, 1H), 8.26 (s, 1H),
13.01 (br s, NH). LCMS Calcd for C17H17N5O5S 403.4; observed
(M þ 1) 404.4.
added to the fractions at this point. The pH was lowered to 4.5 by
the addition of 400 mM ammonium acetate buffer, and phosphate
groups were removed by addition of 2 unit of type XA sweet potato
acid phosphatase per ml eluent and incubation at 37 ꢄC for 1 h. To
remove salt from the resulting fractions, the samples were loaded
onto Waters OASIS HLB cartridges pre-conditioned with 2 ml
MeOH and 2 ml ddH2O, and washed with 3 ml of ddH2O. Nucleo-
side analogs of interest were eluted with 1 ml of methanol and
dried under a stream of air. The residue was reconstituted with
4.2. LC/MS/MS method for stability studies
The HPLC system was a Dionex Packing Ultimate 3000 modular
LC system consisting of a quaternary pump, vacuum degasser,
thermostated autosampler, and thermostated column compart-
ment (Dionex, CA). A TSQ Quantum Ultra triple quadrupole mass
spectrometer (Thermo Electron Corp.) was used for detection.
Thermo Xcalibur software version 1.3 was used to control the HPLC,
the mass spectrometer and to perform data analysis. The separation
100
with 100
ultrafiltration (COSTAR filters 0.2
m
l. The other half of the samples was directly reconstituted
l of water containing 100 nM 3TC which were filtered by
m). Fractions from both SPE and
m
m
direct reconstitution were injected onto the LC–MS/MS system for
intracellular nucleoside detection. The method described previ-
ously (Section 4.2) was used.
was performed on a Hypersil GOLD-C18 column (50 ꢁ 1 mm, 3
mm
particle size) (Thermo Scientific, Waltham, MA, USA). The mobile
phase A consisted of 10 mM ammonium formate buffer, pH 3
adjusted using formic acid. An alternative mobile phase A consisted
of water only. The mobile phase B consisted of methanol. The initial
4.2.2. Conditions for stability studies
A known concentration of the analogs was diluted in two
separate fractions, water and methanol, directly from a stock
solution synthesized the same day and injected onto the LC/MS/MS
using the method previously described in Section 4.2. Injections
were performed every 15 min for the first two hours, then every
hour, every day and finally every week. Half of the sample was kept
at room temperature and the other half was kept in the autosam-
pler at 4 ꢄC. Standard calibration of the nucleosides 7, 8 and ACV
ranging from 10 to 1000 nM were used in order to calculate the
percentage of G analog formation.
conditions were 95% A and 5% B at 50 ml/min. From 1 min to 2 min B
was increased from 5% to 95% and decreased to 5% after 4 min. The
total run time was 15 min (including time for column regenera-
tion). The column temperature was kept constant at 30 ꢄC. Analytes
were protonated by electrospray ionization (ESI) in positive mode.
Selected Reaction Monitoring (SRM) mode was used for the
acquisition. The intensity of selected product ion in the MS/MS
spectrum of each compound was optimized using direct infusion of
the analytes in the corresponding mobile phase (at concentration
4.3. Antiviral activity evaluation
100
mM) separately into the instrument using a syringe pump at
5
m
l/min. The sheath, ion sweep and auxiliary gas (nitrogen) were
The antiviral activity of compounds 9–40 was evaluated against
HIV [27] in activated primary human PBM cells and in Huh-7 cells
with a HCV subgenomic RNA replicon system [28]. Cytotoxicity was
evaluated in the HCV replicon and normal PBM cells, along with
CEM and Vero cells [29]. Compound 3 was evaluated for activity
against HSV-1 (strain F) by plaque reduction assay in Vero cells
using methodologies described previously [30].
set at 50, 0.5 and 1 arbitrary units (au) respectively. The collision
gas (argon) pressure was set at 1.5 mTorr. The spray voltage was
4000 V. The capillary was heated at 300 ꢄC. 0.1 s scan time was used
for all transitions. The collision-induced dissociation (CID) was used
at ꢀ6 V. Tube lens was 107 V and collision energy was 20 V. The
following m/z transitions were used for 38 m/z 441 / 295, 32 m/z
423 / 282, 33 m/z 464 / 323, 7: m/z 293 / 152, 36 m/z 428 /
m/z 282, 39 m/z 404 / 258, 8: m/z 298 / 152, 3 356 / 282 and
ACV 226 / 152. 3TC was used as internal standard, with the
following SRM: m/z 230 / 112.
Acknowledgments
This work was supported in part by NIH grant 5R01-AI-071846,
5P30-AI-50409 (CFAR), 5R37-AI-041980 and by the Department of
Veterans Affairs. We would like also to thank Dr. Ethel Garnier for
helpful discussions and critical reading of the manuscript.
Dr. Schinazi is the founder and a shareholder of RFS Pharma, LLC.
Emory University received no funding from RFS Pharma, LLC to
perform this work and vice versa.
4.2.1. Detection of nucleoside MP, DP and TP intracellularly
4.2.1.1. Cell culture and drug extraction. The 20-C-methyl analogs
were incubated at 50 m
M in Huh-7 cells at 37 ꢄC for 4 h. The cells
were washed twice with 1 ꢁ ice cold PBS and lysed using 60%
methanol. After centrifugation to remove cellular debris, the
supernatant was evaporated to dryness under a stream of air. Dried
extracts were maintained at ꢀ80 ꢄC until analysis.
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