Synthesis and evaluation of 3′-azido-2′,3′-dideoxypurine nucleosides as inhibitors of human immunodeficiency virus

Article abstract of DOI:10.1016/j.bmcl.2009.11.031

Based on the promising drug resistance profile and potent anti-HIV activity of β-d-3′-azido-2′,3′-dideoxyguanosine, a series of purine modified nucleosides were synthesized by a chemical transglycosylation reaction and evaluated for their antiviral activity, cytotoxicity, and intracellular metabolism. Among the synthesized compounds, several show potent and selective anti-HIV activity in primary lymphocytes.

Full text of DOI:10.1016/j.bmcl.2009.11.031

Bioorganic & Medicinal Chemistry Letters 20 (2010) 60–64
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 30-azido-20,30-dideoxypurine nucleosides
as inhibitors of human immunodeficiency virus
Hong-wang Zhang a, Steven J. Coats b, Lavanya Bondada a, Franck Amblard a, Mervi Detorio a, Ghazia Asif a,
Emilie Fromentin a, Sarah Solomon a, Aleksandr Obikhod a, Tony Whitaker b, Nicolas Sluis-Cremer c,
John W. Mellors c, 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,
Atlanta, GA 30033, USA
b RFS Pharma LLC, 1860 Montreal Road, Tucker, GA 30084, USA
c Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
a r t i c l e i n f o
a b s t r a c t
Based on the promising drug resistance profile and potent anti-HIV activity of b-D
-30-azido-20,30-dide-
Article history:
Received 16 October 2009
Revised 9 November 2009
Accepted 10 November 2009
Available online 13 November 2009
oxyguanosine, a series of purine modified nucleosides were synthesized by a chemical transglycosylation
reaction and evaluated for their antiviral activity, cytotoxicity, and intracellular metabolism. Among the
synthesized compounds, several show potent and selective anti-HIV activity in primary lymphocytes.
Published by Elsevier Ltd.
Keywords:
HIV-1
Nucleoside
Antiviral
Transglycosylation
HIV-1 has spread worldwide and causes excessive morbidity
and mortality in the absence of treatment. Nucleoside reverse
transcriptase inhibitors (NRTIs) are the oldest and most successful
class of agents used to treat HIV-1 infection. NRTIs continue to be
the cornerstone of combination antiretroviral therapy (ART)1 that
suppresses viremia and improves longevity and quality of life for
persons infected with HIV-1. After long-term treatment, however,
drug toxicity and the emergence of drug-resistant viral variants
can limit the effectiveness of ART.2 Thus, the discovery of novel
anti-HIV agents with low toxicity and favorable resistance profiles
is necessary for continued success and further advances in ART.
Structural variations in the pyrimidine and purine moiety of NRTI
may impart different activity, resistance and toxicity profiles.3
30-Azido-30-deoxythymidine (AZT, Zidovudine) is a widely uti-
lized anti-HIV drug despite its bone marrow toxicity which occurs
in about 5% of the patients (Fig. 1)4 We recently showed that the
corresponding purine derivative 30-azido-20,30-dideoxyguanosine
(AZG) with the same 30-azido sugar5 was a potent and selective
inhibitor of HIV-1 with a superior resistance profile compared to
AZT6 This discovery led to the evaluation of the antiviral activity,
resistance profile, intracellular metabolism and toxicity of a series
of 30-azido-20,30-dideoxypurine nucleosides. It should be noted that
a previous study reported on the anti-HIV-1 activity of a limited
number of purine 30-azido-nucleosides in MT-4 cells.7 Our work
differs from this study in that we synthesized a larger number of
base modified analogs and correlated antiviral activity in human
lymphocytes to cellular pharmacology and toxicity. In this regard,
previous discovery efforts in this area have mostly neglected the
importance of pharmacology and toxicity in NRTI development.
A previous study of 30-azido-purine nucleosides found the
chemical transglycosylation reaction to be a poor synthetic strat-
egy resulting in a
/b mixture of nucleosides in less than 25% yield.8
Herein, we report the development of
a transglycosylation
approach to efficiently prepare various 30-azido-20,30-dideoxypu-
rine nucleoside analogs. Further we evaluated the anti-HIV activity
O
N
NH2
N
O
N
H3C
N
N
NH
NH
NH2
HO
HO
HO
O
N
O
O
O
O
N3
N3
N3
AZG
AZT
AZC
* Corresponding author. Tel.: +1 404 728 7711.
E-mail address: rschina@emory.edu (R.F. Schinazi).
Figure 1. Some important 30-azido nucleosides.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.11.031

H.-w. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 60–64
61
and cellular pharmacology in human peripheral blood mononu-
clear (PBM) cells and cytotoxicity in PBM, CEM (a human-T-cell-
derived cell line), and Vero (kidney epithelial cells from the African
green monkey) cells.
A later investigation of the chemical transglycosylation with
AZT11b concluded that the resulting mixture of
and b anomers
were obtained in less than 25% yield and the desired b anomer
a
was difficult to obtain in high purity.
Initially, the 30-azido purine analogs were prepared by a multi-
step traditional sugar modification method.9 Briefly, AZG was pre-
pared from guanosine 1, by protection of the 2-amino group as iso-
butyl amide 2, followed by 50-hydroxy protection to give benzoyl
ester 3 (Scheme 1). Inversion of the 30-hydroxyl group was found
to be problematic due to benzoyl group migration10 to the 30-hy-
droxy group and formation of N3-30 cyclic adduct, but compound
4 could be obtained after a difficult chromatographic separation.
Activation as the mesylate and displacement by azide anion gave
protected 30-azido nucleoside, 5, which was deprotected with so-
dium methoxide to give AZG.9,11
Our initial efforts, following literature precedent,13 involved
presilylation of purine bases and AZT with bistrimethylsilyl acet-
amide (BSA), followed by TMSOTf-promoted transglycosylation
that resulted in very low yields. 50-Hydroxy protection with ben-
zoyl chloride,14 silylation of purine bases such as 9 with bistri-
methylsilyl acetamide, and transglycosylation in acetonitrile in
the presence of TMSOTf under Vorbruggen type conditions at
70 °C for 12–24 h resulted in a much improved 30% yield of 1:1.1
mixture of b and
a anomers as an inseparable mixture. After depro-
tection of the 50-position, the anomers were readily separated by
column chromatography on silica gel. However, the inability to
differentiate the electrophilicity of the 50-benzoyl removal versus
6-chloro displacement limited the usefulness of this approach.
A switch to TBS protection of the 50-position of AZT gave 8 that
greatly broadened the scope of the nucleophile tolerated for
6-chloro displacement (Scheme 3). Silylation with BSA and
After much optimization to simplify purifications and improve
yields, AZG was obtained in a 9% yield over six steps from guano-
sine, 1. In order to prepare analogs which vary the substituent at
the 6-position of the purine ring, we initially prepared the
6-chloro-30-azido purine by treatment with phosphorous
oxychloride, but found the reaction to be unreliable providing
yields ranging from 8% to 32%.10
Conversion of the 6-oxo group of 5 to the tosylate 6 was accom-
plished in 99% yield and subsequent displacement by various
nucleophiles occurred without incident (Scheme 2). Several 6-
substituted 2-amino 30-azido-20,30-dideoxy purines, 7, were syn-
thesized in good yield by this approach from intermediate 6. An
alternate 10-step nucleoside modification methodology to AZG
was also explored, but was found to suffer from most of the same
shortcomings described above.12 Thus, side reactions, difficult
purifications, low overall yield, and the lengthiness of the synthesis
of AZG led us to pursue a more efficient and concise methodology.
The transglycosylation reaction is a two-step, one-pot transfor-
mation that involves the glycosyl transfer from a nucleoside to a
heterocyclic base in an intermolecular fashion. It is most often uti-
lized to transfer the sugar moiety of a more readily available
pyrimidine nucleoside to the corresponding purine base. The
chemical transglycosylation with AZT has been reported with
adenosine13a and availability of AZT made this approach attractive.
transglycosylation with 2-amino-6-chloropurine
9 gave the
anomeric mixture of 10 and 11 that was inseparable by silica gel
chromatography although obtained in slightly better 40% yield.
Deprotection of the 50-TBS group of nucleosides 10 and 11
provided a more easily separated b anomer 10, which was readily
converted to nucleosides such as 2,6-diamino purine 12.15 Direct
transglycosylation from TBS protected AZT with silylated purine
bases was found to be problematic as trace AZT (with an
EC50 = 5–50 nM vs HIV-1LAI in PBM cells) often led to false hits.
However, this two-step approach involving two column purifica-
tions was useful for larger scale re-synthesis of interesting
30-azido-purine nucleosides.
We sought a readily available 30-azido nucleoside with signifi-
cantly reduced anti-HIV activity as a glycosyl transfer agent. As
outlined in Scheme 4, 50-TBS protected AZU (AzddU) 13, was found
to meet these requirements in that it was available in three steps
based on a known procedure16 and it had a weaker potency against
HIV in cell culture than AZT (AZU; EC50 = 0.2
PBM cells).
lM; HIV-1LAI; human
O
O
N
N
NH
NH
O
O
a
b
HO
N
HO
N
N
NH2
N
N
O
O
H
OH
OH
1
2
O
N
O
N
N
N
NH
O
NH
BzO
BzO
BzO
N
OH
d
c
N
N
H
N
H
O
O
OH
3
4
O
N
O
N
N
N
N
NH
O
NH
NH2
e
HO
N
N
H
O
O
N3
N3
5
AZG
Scheme 1. Reagents and conditions: (a) TMSCl, (i-BuCO)2O, pyridine, rt, 4 h, 75%; (b) Bz2O, DMF, Et3N, rt 2 h, 74%; (c) (i) Tf2O, DCM, pyridine, 0 °C, (ii) H2O, (iii) MeOH,
NaHCO3; 32%; (d) (i) MsCl, DCM, DMAP, 0 °C, 40 min, (ii) NaN3, DMF, 120 °C, 2 h, 65%, two steps; (e) NaOCH3/MeOH, DCM 45 °C, 4 h, 82%.

62
H.-w. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 60–64
R1
N
TosO
N
N
N
N
O
N
b
a
BzO
5
N
HO
N
N
H
NH2
O
O
N3
N3
6
7
Scheme 2. Reagents and conditions: (a) TosCl or TPPSCl, Et3N, DMAP, DCM, rt, 18 h 99%; 80%; (b) (i) R1H, (ii) NaOMe/MeOH, DCM 45 °C, 18 h, 55–73%.
O
Cl
H3C
N
N
NH
Cl
N
TBSO
N
N
a
N
O
HO
N
N
NH2
O
+
O
N
NH2
H
N3
N3
8
9
10 β anomer +
11 α anomer
NH2
N
N
N
b
HO
N
NH2
O
N3
12
Scheme 3. Reagents and condition: (a) (i) BSA, CH3CN, 78 °C, 30 min, then TMSOTf, 75 °C, 18 h, 43%, (ii) TBAF, THF, rt, 4 h; (b) (i) anomer separation on silica gel, 43%, (ii) NH3/
CH3OH, 110 °C, 18 h, 71%.
O
N
O
N
NH
NH
b
HO
a
O
TBSO
O
O
O
OH
N3
dU
13
R1
N
R1
N
N
N
N
N
N
R2
HO
R2
TBSO
c
N
O
O
14 β anomer +
15 α anomer
N3
N3
16
Scheme 4. Reagents and condition: (a) (i) TBSCl, imidazole, DMF, rt, overnight, 80%, (ii) DIAD, TPP, 80 °C, 30 min, 75%, (iii) LiN3, DMF, 100 °C, 24 h, 70%; (b) purine base, BSA,
TMSOTf, CH3CN, 70 °C, 18 h, 34–61%; (c) (i) TBAF/acetic acid (v/v: 1:0.2), THF, rt, 4–6 h, (ii) anomer separation on silica gel, (iii) nucleophilic reagents in the case of 2- and/or
6-chloro purines in one to three steps in 20–95% yield.
Transglycosylation of the dU derived 13 proceeded smoothly to
give a 1:1.1 ratio of the mixture of b and anomers 14 and 15,
again, as an inseparable mixture (Scheme 4). During the course
of reaction, monitoring by LC/MS revealed not only the and b
cytotoxicity was determined in PBM, CEM, and Vero cells.18 The
results are summarized in Table 1 along with the synthetic scheme
utilized to prepare each analog. A significant number of the synthe-
sized compounds showed good anti-HIV activity without enhance-
ment of cytotoxicity compared to the natural purine bases analogs
30-azido-20,30-dideoxyadenosine (AZA) and AZG.
a
a
anomers, but also other isomers of the same molecular weight.
We observed this with both AZT and AZU under transglycosylation
reactions. These isomers were tentatively assigned as N-7 and/or
possibly N-3 attachment of the sugar unit to the purine base. Over
the course of extended heating these minor isomers decomposed
or were converted to the desired N-9 isomers. The use of 2-ami-
no-6-chloropurine17 (43%) or 2,6-dichloropurine (41%) as the gly-
cosyl acceptor in the transglycosylation allows for subsequent
displacement of the chlorine atom(s) by appropriate nucleophile(s)
and preparation of diversity at the 2 and 6-position.
Compounds 10, 12, and 17–19 were previously studied in MT4
cells against HIV-1IIIB and were reported to have low lM anti-HIV
activity with little or no toxicity toward the MT4 cells. We ob-
served a significant increase in activity in PBM cells and some mild
to moderate toxicity was also noted in PBM, CEM, and Vero cells.
We made seven additional analogs by variation of the substituent
at the 6-position while holding the 2-NH2 group constant. The 6-
NH-allyl, 20, and 6-NCH3-allyl, 21, displayed good activity with
Anti-HIV activity of the synthesized 30-azido-purine nucleoside
analogs was evaluated against HIV-1LAI in human PBM cells and
an EC50 of 0.5 and 0.4
lM, however, the NCH3-allyl, 21 showed in-
creased toxicity in both CEM and Vero cells. The related 6-NH-

H.-w. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 60–64
63
Table 1
Anti-HIV activity and cytotoxicity of selective synthesized compounds in cellular assay for compounds 10–40
Compds
R1
R2
Anti-HIV activity (
l
M)18
Cytotoxicity; IC50
CEM
(l
M)
Synthesis
Scheme
Based on 16
EC50
EC90
PBM
Vero
AZT
AZU
AZG
AZA
10
12
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
—
—
OH
NH2
Cl
NH2
—
—
NH2
H
0.005
0.16
0.18
0.4
0.02
0.6
2.0
2.7
0.8
0.2
1.4
1.1
8.8
4.3
1.5
8.5
>10
>10
>10
16.2
1.3
>100
>100
100
14.3
69.3
>100
86.2
>100
>100
>100
>100
>100
>100
27.0
>100
>100
>100
>100
>100
19.9
>100
2.1
>100
>100
>100
>100
>100
30.1
>100
13.3
>100
>100
>100
50.6
26
NA
NA
1
4 via 27
3; 4
3 via 10
2; 4
>100
>100
>100
>100
>100
>100
>100
>100
32.0
>100
>100
>100
>100
>100
67.9
>100
27.1
>100
>100
>100
>100
>100
34.4
>100
21.8
>100
>100
>100
74.3
>100
54.0
32.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
23.3
51.1
4.2
>100
35.8
>100
35.3
>100
30.1
9.4
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
H
Cl
Cl
Cl
Cl
Cl
Cl
F
0.1
0.05
0.57
0.21
1.5
0.5
0.4
OCH3
N(CH3)2
NHc-pr
NHallyl
N(Me)allyl
NHpropargyl
NH(CH2)5OH
NHC(CH3)2CH2OH
NHCH(CH3)CH2OH
NH(CH2)2OH
Cl
2
2; 4 via 10
2; 4 via 10
2
4 via 10
2; 4 via 10
2
2
2
4
2
4
3
3
3
3
1.7
86.5
52.5
59.9
2.1
0.4
0.3
NHallyl
Cl
OCH3
1.5
9.3
26.3
>100
>100
>100
51.5
>100
94.7
>100
10.0
39.6
>100
>100
55.3
50.3
>100
15.0
>100
45.8
>100
2.6
NH2
NHCH3
NHc-pr
N(CH3)2
NH2
OCH3
Cl
NH2
OCH3
NHc-pr
3
4
4
4
F
F
7.2
OCH3
OCH3
OCH3
12.4
13.2
59.7
35.0
>100
>100
3 via 31
3 via 30
3 via 33
propargyl analog, 22 and four 6-alkyl hydroxy analogs 23–26 were
significantly less active and displayed no toxicity.
Such broad activity across this series of compounds led us to
investigate whether these 6-substituted analogs could be con-
verted to 30-azido guanosine (AZG) in the PBM cells used for the
anti-HIV assay. To test this hypothesis, PBM cells were exposed
2,6-bis-methoxy analog 39, displaying an anti-HIV EC50 of
13.2 M and no cytotoxicity up to 100 M.
l
l
Many of the synthesized 30-azido purine nucleosides showed
potent anti-HIV activity with low or no toxicity. In addition, a num-
ber of the compounds proved to be acting as prodrugs for AZG in
PBM cells. Any potential benefit of these various prodrugs to a
future clinical candidate will be the subject of future studies. In
this regard, the 2,6-diamino compound 12 deserves attention. This
compound is between 4 times (EC50) and 10 times (EC90) more
potent than AZG (see Table 1), but it is substantially converted to
AZG by adenosine deaminase. Based on these promising results,
further exploration of purine modifications on 30-azido nucleosides
and their effects on anti-HIV activity, cellular pharmacology, and
cytotoxicity are underway.
to the nucleoside analog at 50 lM for 4 h at 37 °C in a 5% CO2 atmo-
sphere. After washing the cells and extraction of metabolites the
supernatant was dried and analyzed by LC/MS/MS. For each sample
attempts to detect the nucleoside analog triphosphate (TP) along
with the parent AZG-TP were made. 6-Cl (10), 6-NH2 (12), 6-OMe
(17), 6-N(CH3)2 (18), 6-NHcyclopropyl (19), and 6-NH-allyl (20),
were all found to be converted to AZG-TP under these conditions
with such efficiency that we were unable to detect the nucleoside
analog in the TP form except in the case of 12, where we detected
low levels of 12-TP along with significant AZG-TP.
Acknowledgments
To determine if these 6-substituted compounds are converted
to AZG by the enzyme adenosine deaminase, nucleoside analogs
were subjected to 0.2 units per mL of commercially available puri-
fied enzyme for 120 min and measured spectrophotometrically at
265 or 285 nm.19 Nucleosides 10, 12, 17, 19, and 20 were found
to be substantially or completely converted to AZG by adenosine
deaminase. Compound 18, the 6-N,N-dimethyl analog was found
to be resistant to adenosine deaminase under the conditions
tested.
This study was supported by grants from the National Institutes
of Health (R01-AI-071846 to J. W. M and 5P30-AI-50409 and 5R37-
AI-041980 to R. F. S.) and by the Department of Veterans Affairs (R.
F. S.). R. F. S. is a founder and major shareholder of RFS Pharma, LLC.
J. W. M. is a consultant to RFS Pharma and owns share options in
RFS Pharma. All other authors have no competing interests.
References and notes
We next investigated analogs of AZA with variation of substitu-
ents at the 2 and 6-position of the purine base. The 6-Cl (27) and 6-
NH-allyl (28) had a similar anti-HIV profile to AZA; however, 27
displayed more cytotoxicity. Nine AZA analogs, 29–37, were pre-
pared with a halogen at the 2-position, but did not have any signif-
icant anti-HIV activity. Two of the 2-fluoro analogs, 35 and 37 and
the 2,6-dichloro analog 29 showed an increase in cytotoxicity. The
2-methoxy analogs 38–40 showed the best overall anti-HIV activ-
ity of the 2-substituents tested to date in the AZA series with the
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amino-6-chloropurine (250 mg, 1.47 mmol) and 50-TBSAZU, 13 (200 mg,
0.54 mmol) in acetonitrile (6 mL) in oven dried glassware was added BSA (2 mL,
8.17 mmol). The resulting heterogeneous mixture was heated at 85 °C under
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nitrogen pressure for 18 h. The reaction mixture was cooled to rt, poured into
saturated NaHCO3 (50 mL), extracted with ethyl acetate (10 mL ꢀ 2), and the
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Appl. EP 505181 A1 19920923, 1992, 14pp.; (d) Rideout, J. L.; Freeman, G. A.;
Short, S. A.; Almond, M. R.; Collins, J. L. Eur. Pat. Appl. EP 421739, A1 19910410,
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(s, 6H). a
-Isomer: 1H NMR (CDCl3, 400 MHz): d 8.05 (s, 1H), 6.24 (dd, J = 2.8 Hz,
J = 7.2 Hz, 1H), 5.10 (s, 2H), 4.30 (m, 2H), 3.76 (dd, J = 3.2 Hz, J = 11.2 Hz, 1H), 3.71
(dd, J = 3.6 Hz, J = 11.6 Hz, 1H), 2.82 (m, 1H), 2.61 (dt, J = 2.8 Hz, J = 14.4 Hz, 1H),
0.90 (s, 9H), 0.08 (s, 6H).
12. Timoshchuk, V. A.; Hogrefe, R. I.; Vaghefi, M. Nucleosides Nucleotides Nucleic
Acids 2004, 23, 171.
13. (a) Imazawa, M.; Eckstein, F. J. Org. Chem. 1978, 43, 3044; (b) An enzymatic
transglycosylation approach from AZT is shown in Ref. 7.
14. (a) Mag, M.; Engels, J. W. Tetrahedron 1994, 50, 10225; (b) Wigerinck, P.; Van
Aerschot, A.; Janssen, G.; Claes, P.; Balzarini, J.; De Clercq, E.; Herdewijn, P. A. M.
J. Med. Chem. 1990, 33, 868.
18. (a) Schinazi, R. F.; Sommadossi, J. P.; Saalmann, V.; Cannon, D. L.; Xie, M.-W.;
Hart, G. C.; Smith, G. A.; Hahn, E. F. Antimicrob. Agents Chemother. 1990, 34,
1061; (b) Stuyver, L. J.; Lostia, S.; Adams, M.; Mathew, J.; Pai, B. S.; Grier, J.;
Tharnish, P.; Choi, Y.; Chong, Y.; Choo, H.; Chu, C. K.; Otto, M. J.; Schinazi, R. F.
Antimicrob. Agents Chemother. 2002, 46, 3854.
19. Reaction conditions were 50 mM potassium phosphate, pH 7.4, with 50 lM
nucleoside analog in 0.5 mL at 25 °C. Reaction time was 7 min with 0.002 units
15. Herdewijn, P.; Van Aerschot, A. Tetrahedron Lett. 1989, 30, 855.
16. (a) Colla, L.; Herdewijn, P.; De Clercq, E.; Balzarini, J.; Vanderhaeghe, H. Eur. J.
Med. Chem. 1985, 20, 295; (b) Moharram, S.; Zhou, A.; Wiebe, L. I.; Knaus, E. E. J.
Med. Chem. 2004, 47, 1840.
of enzyme and 120 min with 0.2 units of enzyme (the unit definition of
adenosine deaminase is one unit will deaminate 1.0 lmol of adenosine to
inosine per min at pH 7.5 at 25 °C). 20-Deoxyadenosine was the positive control
which was 59% deaminated to 20-deoxyinosine under the given conditions in
7 min with 0.002 units of enzyme. 20-Deoxyguanosine was the negative
control.
17. 9-((2R/S,4S,5S)-4-Azido-5-((tert-butyldimethylsilyloxy)-methyl)tetrahydrofuran-
2-yl)-6-chloro-9H-purin-2-amine, 14/15 (R1 = Cl; R2 = NH2): To a solution of 2-