Azadideoxyadenosine: Synthesis, enzymology, and anti-HIV activity

Article abstract of DOI:10.1016/j.bmcl.2006.04.085

Synthesis of an azanucleoside, a new analogue of dideoxyadenosine, is described. This compound is only slowly deaminated by mammalian adenosine deaminase and it is a substrate for adenosine kinase. It exhibits in vitro anti-HIV activity.

Full text of DOI:10.1016/j.bmcl.2006.04.085

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4099–4101
Azadideoxyadenosine: Synthesis, enzymology,
and anti-HIV activity
Abdumalik A. Nishonov, Xiaohui Ma and Vasu Nair*
The Center for Drug Discovery and Department of Pharmaceutical and Biomedical Sciences,
The University of Georgia, Athens, GA 30602, USA
Received 12 April 2006; revised 26 April 2006; accepted 27 April 2006
Available online 12 May 2006
Abstract—Synthesis of an azanucleoside, a new analogue of dideoxyadenosine, is described. This compound is only slowly
deaminated by mammalian adenosine deaminase and it is a substrate for adenosine kinase. It exhibits in vitro anti-HIV activity.
Ó 2006 Elsevier Ltd. All rights reserved.
NH2
There is continuing interest in the synthesis of new
nucleoside analogues with potential antiviral activity.
Synthesis and anti-HIV activities of new dideoxynucle-
osides, of both the D- and L-related families, in which
the furanose oxygen is transposed to a new endocyclic
position within the cyclopentane ring have been the
subject of a number of studies.1,2 The family of nucle-
oside analogues referred to as carbocyclic nucleosides
has been the subject of intense investigation, both in
synthesis and antiviral studies.3–7 For example,
the enantiomerically pure carbocyclic analogue of
20-deoxyadenosine (Fig. 1) has been synthesized and
showed activity against DNA viruses.8,9
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
HO
HO
N
HO
O
N
OH
OH
OH
Figure 1. Structures of three related adenine nucleosides (L to R)
20-deoxyadenosine,
oxyadenosine.
carba-20-deoxyadenosine,
and
EtOOC
HO
30-azadide
OH
OH
HOOC
OH
HOOC
Conformational studies on the pyrrolidine and
4-hydroxyproline rings have indicated substantial sim-
ilarity to the cyclopentyl and tetrahydrofuran ring
structures.10 While a few pyrrolidine nucleosides have
been synthesized,11,12 antiviral or other biological
studies of these compounds are largely lacking. Also,
some interesting azanucleosides, such as the title com-
pound (Fig. 1), have not been synthesized or exam-
ined for antiviral activity. This communication
describes the synthesis, enzymology and in vitro
anti-HIV studies of 30-aza-20,40-dideoxyadenosine, a
new carbocyclic nucleoside analogue.
b
a
N
H
N
N
H .HCl
3
H .HCl
1
2
OTs
OH
EtOOC
EtOOC
OAc
c
e
d
N
N
N
Ts
Ts
4
Ts
6
5
TBDPSO
OTs
TBDPSO
OH
g
N
f
N
Ts
Ts
8
The starting compound for the synthesis (Scheme 1) was
trans-4-hydroxy-L-proline (1). Epimerization of trans-4-
7
Scheme 1. Reagents and conditions: (a) Ac2O, AcOH, reflux, 5.5 h,
2 N HCl, reflux, 3 h, 65%; (b) absolute EtOH, HCl (gas), reflux, 5 h,
67%; (c) Et3N, Py, TsCl, ꢀ5 °C, 1 h, refrigerated 12 h, 5 h at rt, 68%;
(d) (Et)4N+ꢀOAc, toluene, reflux 12 h, 75%; (e) LiBH4, THF, 0 °C
30 min, rt 12 h, 76%; (f) t-TBDPSCl, imidazole, DMAP, dry CH2Cl2,
at 0 °C then 18 h at rt, 47%; (g) p-TsCl, CH2Cl2, DMAP, 48 h, rt, 99%.
Keywords: Azanucleoside; Synthesis; Adenosine deaminase; Kinase
phosphorylation; Anti-HIV activity.
*
Corresponding author. Tel.: +1 706 542 6293; fax: +1 706 583
8283; e-mail: vnair@rx.uga.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.085

4100
A. A. Nishonov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4099–4101
NH2
NH2
N
N
N
N
TBDPSO
OTs
N
N
HO
N
N
TBDPSO
N
b
a
N
H
N
Ts
8
Ts
9
10
c
NH2
N
NH2
N
N
N
N
N
N
N
HO
HO
d
N
N
OH
CH2CH2CN
12
11
Scheme 2. Reagents and conditions: (a) adenine, K2CO3, 18-crown-6,
DMF, 80 °C, 12 h, 59%; (b) HBr, HOAc, phenol, 90 °C, 16 h, 88%; (c)
acrylonitrile, 0 °C, 1 h, to rt, 4 h, 58%; (d) MeOH–CHCl3 (1:1),
m-CPBA, ꢀ78 °C, K2CO3, 3 h, rt, 16 h, 90%.
Figure 2. Lineweaver–Burk plot of the deamination of compound 12.
Table 1. Kinetic parameters for substrate activity for 12 compared to
adenosine toward bovine adenosine deaminase
Substrate
ka
(nm) (lM)
Km
Vmax
Vmax/Km
hydroxy-L-proline under the conditions described13 gave
the cis- or allo-isomer 2. The product crystallized from
aqueous HCl as its hydrochloride salt, which was free
from the trans-compound. The hydrochloride salt 2
was esterified with ethanol to 3, and the amine and
hydroxyl functionalities were then blocked with tosyl
groups to give 4.
lmole minꢀ1 relative (%)
mg proteinꢀ1
Adenosine 265
Compound 12 265
32 2.7
131 15.4
246 9.1
12 0.8
100
1.2
a Deamination carried out in phosphate buffer (pH 7) and monitored at
265 nm, the wavelength of maximum difference between substrate
and product.
Displacement (SN2) of the tosyloxy group of 4 with ace-
tate ions gave the inverted acetate 5, which was converted
to 6 with lithium borohydride. Selective protection of the
primary hydroxyl group with TBDPS (to give 7), followed
by tosylation of the secondary hydroxyl functionality,
gave key intermediate 8. For the main coupling step to
the aza nucleoside (Scheme 2), intermediate 8 was treated
with adenine in the presence of 18-crown-6 and K2CO3 in
DMF,14,15 which produced nucleoside 9. The structure of
9 was confirmed by NMR data and NOE experiments.
Reductive cleavage of the N-tosyl protecting group of 9
was carried out with HBr in acetic acid in the presence
of phenol. Under these reaction conditions, the silyl pro-
tecting group was also removed to produce the azanucle-
oside 10. Cyanoethylation of 10 with acrylonitrile gave
the cyanoethyl derivative 11, which was oxidized with
77% m-chloroperbenzoic acid in MeOH/CHCl3 (1:1) to
give the desired hydroxylamine product 12. Purification
of the crude product by crystallization from ethanol gave
the pure target compound. Its structure was confirmed by
1H and 13C NMR, UV, and HRMS data.16 Compound 12
was optically active and dextrorotatory.
Compound 12 was tested in a PBMC-based, microtiter
anti-HIV assay against the clinical isolate, HIV-1TEKI
(NSI phenotype) and HIV-1NL4-3 (SI phenotype). All
antiviral determinations were performed in triplicate with
serial 1/2 log10 dilution of the test materials (six to nine
concentrations total).17 The overall performance of both
assays was validated by the MOI-sensitive positive con-
trol compound, AZT, exhibiting the expected level of
antiviral activity [IC50 = 1.98 nM (HIV-1TEKI) and
1.75 nM (HIV-1NL4-3), CC50 >1000 nM, PBMC].
Compound 12 was found to have anti-HIV activity
[IC50 = 3.52 lM (HIV-1TEKI) and 3.11 lM (HIV-1NL4-3)].
The CC50 value in PBMC was 46.6 lM.
In summary, an azadideoxyadenosine analogue of 20-
deoxyadenosine was synthesized, and its structure and
stereochemistry were confirmed by optical rotation,
1
UV, H and 13C NMR spectroscopy, and HRMS data.
This compound exhibits resistance toward deamination
by ADA. It possesses in vitro anti-HIV activity with
IC50s in the low micromolar range. As the compound
is phosphorylated by AK, it seems likely that the anti-
HIV activity could be attributed to the inhibition of
HIV reverse transcriptase by its 50-triphosphate.
One key question pertaining to compound 12 was its
stability toward deamination by mammalian adenosine
deaminase (ADA). Kinetic parameters were obtained
for 12 and compared to adenosine as the positive stan-
dard. Compound 12 was a poor substrate for ADA
(Fig. 2 and Table 1). It is not an inhibitor of this enzyme.
Phosphorylation studies using mammalian adenosine
kinase (AK) showed that 12 was a substrate for AK and
was slowly converted to its monophosphate, the
formation of which could be analyzed by HPLC analysis.
Acknowledgments
This project was supported by the National Institutes of
Health (NIAID). The contents of this paper are solely
the responsibility of the authors and do not necessarily
represent the official views of the NIH. One of us

A. A. Nishonov et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4099–4101
4101
16. cis-4-(6-Amino-9H-purin-9-yl)-D
-prolinol (12). Acrylo-
(V.N.) also thanks the Terry Endowment and the
Georgia Research Alliance for their support.
nitrile (0.25 ml, 1.58 mmol) was added dropwise over
15 min to a cooled (ice-salt bath) solution of 10 (0.37 g,
1.58 mmol) in H2O (10 ml). Stirring was continued for
1 h and for an additional 4 h with the bath removed.
The reaction mixture was evaporated to dryness and the
residue crystallized from methanol to give N-(cyano-
References and notes
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ethyl)-cis-4-(6-amino-9H-purin-9-yl)-D-prolinol
(11)
(0.26 g, 0.91 mmol, 58%). 1H NMR (500 MHz,
CD3OD + CDCl3): d 1.99 (m, 1H), 2.70 (m, 4H), 2.83
(m, 1H), 2.91 (m, 1H), 3.43 (d, 1H), 3.67 (m, 2H), 5.17
(m, 1H), 8.28 (s, 1H), 8.59 (s, 1H). A solution of 11
(0.076 g, 0.27 mmol) in methanol and chloroform (1:1,
10 ml) was cooled to ꢀ78 °C, and m-chloroperbenzoic
acid (0.068 g, 0.4 mmol) and K2CO3 (0.55 g, 0.4 mmol)
were added. After stirring for 3 h at ꢀ78 °C, the
reaction mixture was warmed to room temperature
and stirred for an additional 16 h. The resulting
suspension was filtered through a MgSO4 pad and
concentrated. The residue was purified by column
chromatography on silica gel (CHCl3–MeOH, 10:1)
and then crystallized from ethanol to give pure cis-4-
(6-amino-9H-purin-9-yl)prolinol (12) as a white solid
(0.06 g, 0.24 mmol, 90%): mp 201–202 °C. 1H NMR
(500 MHz, CD3OD): d 2.00 (m, 1H), 2.76 (m, 1H), 3.05
(br s, 1H), 3.44, 3.78, 3.83–3.86 (m, 3H), 5.26 (br s, 1H),
8.23 (s, 1H), 8.50 (s, 1H); 13C NMR (125 MHz,
CD3OD) d: 32.5, 48.2, 61.0, 63.4, 68.8, 118.4, 140.2,
149.0, 152.2, 155.9, HRMS-FAB calcd for C10H15N6O2
24
(M+H)+: 251.1256, found: 251.1252. ½aꢁD +30.5 (c 2 in
MeOH); UV kmax 261 nm (e 15,700).
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