2′,3′-didehydro-2′,3′-dideoxynucleosides are degraded to furfuryl alcohol under acidic conditions

Article abstract of DOI:10.1016/j.bmcl.2004.02.031

2^′,3^′-Didehydro-2^′, 3^′-dideoxynucleosides are clinically relevant antiviral agents. These nucleosides could be degraded under acidic conditions. Acidic stability studies showed the D4N had the following increasing stability order: D4GA concerted A-1 mechanism has been proposed for the acidic cleavage of D4-nucleosides. The cleavage products were characterized as furfuryl alcohol and the corresponding nucleobase. Furfuryl alcohol is an agent found in many everyday food products. The biological results demonstrated that furfuryl alcohol had neither anti-HIV activity nor cytotoxicity in vitro, suggesting the acid instability of D4-nucleosides is unlikely to have an impact on the toxicity of these nucleoside analogs in humans.

Full text of DOI:10.1016/j.bmcl.2004.02.031

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2159–2162
0
0
0
0
2
,3 -Didehydro-2 ,3 -dideoxynucleosides are degraded to furfuryl
alcohol under acidic conditions
a,b,
c,à
a,b
Adrian S. Ray, Judy S. Mathew, Karen S. Anderson, Chung K. Chu
c
d
Junxing Shi,
a,b,
*
and Raymond F. Schinazi
a
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30323, USA
Emory University School of Pediatrics and Veterans Affairs Medical Center, Medical Research 151H, 1670 Clairmont Road,
Decatur, GA 30033, USA
b
c
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA
Department of Pharmacology and Biomedical Sciences, College of Pharmacy, The University of Georgia,
Athens, GA 30602-2352, USA
d
Received 30 October 2003; accepted 6 February 2004
0
0
0
0
Abstract—2 ,3 -Didehydro-2 ,3 -dideoxynucleosides are clinically relevant antiviral agents. These nucleosides could be degraded
under acidic conditions. Acidic stability studies showed the D4N had the following increasing stability order: D4G < cyclo-
D4G 6 RVT < D4T with half-lives ranging from less than 2 min to 35 days. A concerted A-1 mechanism has been proposed for the
acidic cleavage of D4-nucleosides. The cleavage products were characterized as furfuryl alcohol and the corresponding nucleobase.
Furfuryl alcohol is an agent found in many everyday food products. The biological results demonstrated that furfuryl alcohol had
neither anti-HIV activity nor cytotoxicity in vitro, suggesting the acid instability of D4-nucleosides is unlikely to have an impact on
the toxicity of these nucleoside analogs in humans.
ꢀ
2004 Elsevier Ltd. All rights reserved.
0
0
0
0
6
2
,3 -Didehydro-2 ,3 -dideoxynucleosides belong to an
cosidic bond cleavage has not drawn much attention.
This is in contrast to the nucleosides bearing natural
sugars, which have been investigated intensively.
important class of antiviral agents, especially for the
treatment of human immunodeficiency virus (HIV)
6–30
In
infection. Among this class of nucleosides,
1
D
-D4T
Stavudine) is an approved anti-HIV agent. Two other
these studies, the cleavage products were assigned as
corresponding nucleobase and sugar without full char-
(
nucleosides, RVT (Reverset,
2
0
D
-D4FC) and
L
-D4FC
Elvucitabine) are in advanced Phase II clinical trials.
acterization. This is true especially for ribo- and 2 -de-
oxyribonucleosides. However, for D4-nucleosides, the
cleavage product formed from the sugar portion is still
3
(
In addition, several analogs of this class, for example,
0
0
0
0
0
0
0
4
31
D
-2 ,3 -didehydro-2 ,3 -dideoxycytidine
-2 ,3 -didehydro-2 ,3 -dideoxyadenosine
(
D
-D4C) and
-D4A) are
ambiguous. Only recently, Ray et al. proposed 2-hy-
0
5
L
(
L
droxy-5-hydroxymethyl-2,5-dihydrofuran (1, Fig. 1) in
two isomeric forms as a major cleavage product and an
a/b unsaturated aldehyde (2, Fig. 1) as a minor product
also potent antiviral agents. All these nucleosides are
metabolized to their triphosphates in vivo, which act as
viral DNA chain terminators, due to the lack of the
0 0 0 0
of
D-2 ,3 -didehydro-2 ,3 -dideoxyguanosine (D-D4G)
0
3 -hydroxyl groups. Although the acidic instability of
D4-nucleosides is well known, the study on their gly-
degradation under neutral aqueous conditions based on
proton NMR. These sugar products could be a possible
reason for degraded D4Gs relatively high cytotoxicity.
Therefore, further investigation of the cleavage of the
D4-nucleosides is warranted. Having an unambiguous
Keywords: HIV; Nucleoside; Furfuryl alcohol; Reverset; Degradation;
Cleavage.
*
Corresponding author. Tel.: +1-404-728-7711; fax: +1-404-728-7726;
e-mail: rschina@emory.edu
Present address: Pharmasset Inc., 1860 Montreal Road, Tucker, GA
HO
HO
O
OH
OH
CHO
3
0084, USA.
1
2
à
Present address: Gilead Sciences, 333 Lakeside Drive, Foster City,
CA 94404, USA.
31
Figure 1. Proposed cleavage products of
D-D4G by Ray et al.
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.031

2160
J. Shi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2159–2162
1
13
structure of the cleavage products of D4-nucleosides
would be helpful in understanding the biological and
pharmacokinetic profiles of these nucleosides, and
should prove useful in designing improved antiviral
agents. We have investigated the acid cleavage of some
D4-nucleosides, and herein report our results on the
susceptibility of these nucleosides to acid catalyzed
degradation, as well as the biological activity of the
cleavage products.
ryl alcohol based on TLC, H, and C NMR data of
this compound as compared to those of an authentic
sample obtained from a commercial source. The other
three D4-nucleosides, D-D4T, D-D4G, and D-cyclo-
D4G, also resulted in furfuryl alcohol as the cleavage
product, which was confirmed by TLC in two develop-
ment systems (CHCl
4:1).
3
/MeOH 95:5, and EtOAc/hexanes
For the stability experiment, an aqueous pH 2 phos-
phate buffer under ambient temperature was utilized for
these nucleosides, and the products were assayed by
reverse phase HPLC. The reaction solutions were also
checked by TLC using the above-mentioned systems at
the last time point (24 h). The TLC results confirmed the
presence of furfuryl alcohol for all the four tested nu-
cleosides under milder acidic conditions.
Two series of experiments were designed for four D4-
nucleosides, namely, RVT,
clo-D4G (Fig. 2). First, the acidic cleavage products
cleavage experiment) were determined, and the second
study focused on the acidic stability of these nucleosides
stability experiment). For the cleavage experiment,
D-D4T, D-D4G, and D-cy-
(
(
harsher conditions were adopted in order to facilitate
the process. When subjected to an acidic aqueous solu-
tion (HOAc) under increased temperature (35–50 ꢁC),
these nucleosides were cleaved to produce two products
as evidenced by thin-layer chromatography (TLC). One
of the products was the corresponding nucleobase, as
confirmed by TLC and HPLC in comparison with an
authentic sample. The other product was scarcely visible
on a TLC plate under UV light, but showed a strong
purple color after spraying with 5% H SO /EtOH and
Cleavage of the D4-nucleosides. RVT (500 mg) was dis-
solved in water (10 mL) and HOAc (0.5 mL) was added.
The solution (pH ꢀ 3–4) was stirred at 35–40 ꢁC for 1 h,
then extracted with CH Cl (3 · 20 mL). The combined
2
2
2 4
extracts were washed with water, dried (Na SO ), fil-
tered, and evaporated to give 125 mg (58%) of product
as colorless oil. As this product was water soluble and
highly volatile, no attempt was made to optimize the
yield. This product was compared with an authentic
sample of furfuryl alcohol purchased from Sigma-Al-
2
4
heating. A cleavage reaction using one of our large
stocked D4-nucleosides, RVT, resulted in the isolation
of a colorless oily product upon extraction of the reac-
tion mixture. This product was determined to be furfu-
1
13
drich Co. (catalog no 18,593-0) using H and C NMR
and TLC (visualized after spraying with 5% H SO in
EtOH and heating), and found to have identical prop-
2
4
erties in all respects. TLC: 0.49 (CHCl /MeOH, 95:5),
3
1
0
.68 (EtOAc/hexanes, 4:1); H NMR (DMSO-d
6
) d 7.58
NH2
O
N
(
d, J ¼ 1:2 Hz, 1H, H-5), 6.38 (dd, J ¼ 1:2 and 3.2 Hz,
F
N
N
N
1H, H-4), 6.27 (d, J ¼ 3:2 Hz, 1H, H-3), 5.19 (br, 1H,
NH
13
D O exchangeable, OH), 4.37 (s, 2H, CH OH);
2
C
2
N
O
O
NH2
HO
HO
NMR (DMSO-d
(d, C-4), 106.9 (d, C-3), 55.6 (t, CH OH).
6
) d 155.4 (s, C-2), 142.1 (d, C-5), 110.3
O
O
2
RVT
D-D4G
The other three D4-nucleosides,
D
D-D4T, D-D4G, and
-cyclo-D4G, were also processed in a similar way
(0.3mL HOAc in 3mL of water, pH ꢀ 3), but on a one-
tenth scale (each with 50 mg) and at a higher tempera-
ture (50 ꢁC) (Scheme 1). Each reaction was monitored by
NH
NH
O
N
N
N
N
TLC using two developing systems (CHCl /MeOH 95:5,
3
N
NH2
HO
HO
O
D-D4T
Figure 2. Structure of D4-nucleosides studied.
O
and EtOAc/Hexanes 4:1) and compared with an
authentic sample of furfuryl alcohol. All the reactions
resulted in the formation of the base and a single
product that has the same R value as furfuryl alcohol by
TLC.
D-cyclo-D4G
f
B
dilute HOAc
HO
O
HO
O
+
Base
3
5—50 °C
B: FC, T, G, c-G
furfuryl alcohol
H+
H+
–
+
O
B H
HO
H
Scheme 1. Acidic cleavage of the D4-nucleosides and the possible mechanism.

J. Shi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2159–2162
2161
Stability of the D4-nucleosides. As D4-nucleosides were
base mechanism, which involves protonation of the
sugar ring oxygen, opening of the sugar ring to form a
Schiff base, water attack on the Schiff base followed by
31
relatively stable under basic and neutral conditions,
only the acidic stability of the nucleosides (RVT,
-D4T, -D4G, and -cyclo-D4G) was investigated. A
7–14
D
D
D
cleavage of the glycosidic bond.
The other, an A-1
small amount of D4-nucleoside (about 1 mg) was dis-
solved in NaH PO buffer (0.05 M, pH 2.0, 200 lL) and
mechanism, involves the protonation of the nucleobase,
followed by the direct cleavage of the glycosidic
2
4
6;15–21
the solution was stirred at ambient temperature for 24 h.
A sample was taken at 1 and 24 h, and checked by
reverse phase HPLC (Varian Prostar 210; Alltech Zor-
bax SB-C18 4.6 · 150 mm (5 lM) column; solvent A:
bond.
Our data fit the A-1 mechanism better and
does not support the Schiff base mechanism, because the
formation of furfuryl alcohol is better explained without
the sugar ring opening. Under our experimental condi-
tions, no sugar ring opening product was detected. The
proposed mechanism is depicted in Scheme 1. Aroma-
tization of the furan ring with a concerted process of
0
.05 M triethyl ammonium bicarbonate; solvent B: 70%
acetonitrile in water; starting from 100% A, by
increasing B to 50% at 15 min, then to 100% B at 20 min,
and maintaining for 25 min). From the HPLC data, the
half-life (t1=2) of the D4N was estimated and is shown in
Table 1.
0
C1 –N1 glycosidic bond cleavage and the release of a
0
proton from C4 is the driving force of the reaction that
only occurs during cleavage of D4-nucleosides.
The biological activity of furfuryl alcohol was evaluated
in comparison with RVT. The anti-HIV-1 activity was
assayed in activated human peripheral blood mono-
nuclear (PBM) cells, and the cytotoxicity was deter-
mined in PBM, CEM, and Vero cells, as described
Although the acidic cleavage products of the D4-
nucleosides proved to be nontoxic, the reason for the
cytotoxicity caused by
still unknown. Since the toxicity was due to the cleavage
of -D4G in neutral aqueous solution, the mechanism
D-D4G in aqueous medium is
D
32
previously.
The mitochondrial toxicity was also
might be different from cleavage under acidic conditions
(mimicking stomach conditions). Thus, the intermedi-
32
examined utilizing the previously described method. In
addition, the three nucleobases, that is, 5-fluorocytosine,
thymine, and guanine, were also tested for cytotoxicity.
It is clear that furfuryl alcohol demonstrated neither
anti-HIV activity nor cytotoxicity in the tested cells
31
ates proposed by Ray et al. could not be excluded.
However, it has been shown that certain purine nucle-
osides can be further hydrolyzed at the imidazole ring of
the nucleobase, giving rise to C8–N9 bond cleavage
21;26
(
Table 2). The nucleobases 5-fluorocytosine and thymine
product even under neutral aqueous conditions.
D
If
this is true, -D4G would produce the cleavage product
were also nontoxic in the three cell lines up to 100 lM,
whereas guanine showed minimal toxicity in CEM and
Vero cells. The mitochondrial toxicity result in liver cells
revealed that furfuryl alcohol has no significant effect on
mitochondrial DNA levels up to 100 lM (data not
shown). Furthermore, the compound did not increase
lactic acid production in these cells.
2,4-diamino-5-formamido-6-hydroxypyrimidine. As this
compound is not commercially available, the prepara-
tion and evaluation of this compound would be neces-
sary to prove this hypothesis, and may provide a clue as
to the cause of
D-D4Gs cytotoxicity.
In conclusion, certain important antiviral D4N are
unstable under acidic conditions. A concerted A-1
mechanism is proposed for the acidic cleavage of D4N.
The cleavage product, furfuryl alcohol, has neither anti-
HIV activity nor cytotoxicity in culture. These results
are consistent with the presence of furfuryl alcohol in
many common food items including popcorn, coffee,
0
For acidic cleavage of ribo- and 2 -deoxyribonucleo-
sides, two mechanisms were postulated. One was a Schiff
Table 1. Estimated half-life of the D4-nucleosides at 20 ꢁC in 0.05 M
2 4
pH 2 NaH PO buffer
Nucleoside
RVT
Half-life (t1=2, min)
33
and peanuts, and its general acceptance as being
45
nontoxic with limited exposure. These studies are most
relevant to degradation, which occurs in the stomach
under acidic conditions and suggests a limited risk of
toxicity associated with D4-nucleoside acid degradation
D
D
D
-D4T
-D4G
-cyclo-D4G
50,000
<2
41
Table 2. Anti-HIV-1 activity and cytotoxicity of furfuryl alcohol and nucleobases relative to Reverset (RVT)
Compound
Anti-HIV-1 activity in PBM cells (lM)
Cytotoxicity (IC50, lM) in
EC50
EC90
PBM
CEM
Vero
RVT
Furfuryl alcohol
0.067
>100
1.9
>100
>100
>100
P100
>100
>100
>100
0.46
>100
>100
>100
>100
64.354.9
30.9
>100
>100
>100
>100
a
a
5-Fluorocytosine
Thymine
ND
ND
ND
ND
a
a
a
a
Guanine
b
ND
0.002
ND
0.02
AZT
Cycloheximide
29.0
0.53
b
a
a
ND
ND
0.08
a
ND: not determined.
Positive controls for anti-HIV activity (AZT) and toxicity (cycloheximide).
b

2162
J. Shi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2159–2162
and the need for enterically coated drugs. Further
studies are needed to characterize and understand the
activities of sugar moieties released during systemic
metabolism of D4-nucleosides, such as those formed
7. Kenner, G. W. In The Chemistry and Biology of Purines,
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3
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from
D-D4T.
9
1
0. Michelson, A. M. The Chemistry of Nucleosides and
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The authors acknowledge Ann Hobbs, Jason P. Grier,
and Phil M. Tharnish for their excellent technical work,
and Drs. Kyoichi A. Watanabe, Jinfa Du, and Lieven
Stuyver for their helpful discussions. R.F.S. is supported
in part by NIH grant 1R37-AI-41980 and the Depart-
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supported by NIH grant GM49551. A.S.R. was sup-
ported by NIH, National Research Service Award 5 T32
GM07223from the National Institute of General
Medical Sciences. R.F.S. and C.K.C. are the founders of
Pharmasset Inc. and did not receive any funding from
the company for these studies. Reverset is currently in
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