Novel use of a guanosine prodrug approach to convert 2′,3′-didehydro-2′,3′-dideoxyguanosine into a viable antiviral agent

Article abstract of DOI:10.1128/AAC.46.3.887-891.2002

Transient kinetic studies with human immunodeficiency virus (HIV) type 1 reverse transcriptase suggest that nucleotide analogs containing the 2′,3′-didehydro-2′,3′-dideoxy ribose ring structure present in D4T (stavudine) triphosphate are among the most effective alternative substrates. For unclear reasons, however, the corresponding purine nucleoside, 2′,3′-didehydro-2′,3′-dideoxyguanosine (D4G), was found to be inactive in cell culture. We have found that the previously reported lack of activity of D4G is primarily due to solution instability, and in this report we describe a novel use of a guanosine prodrug approach to stabilize the nucleoside. D4G was modified at the 6 position of the purine ring to contain a cyclopropylamino group yielding the prodrug, cyclo-D4G. An evaluation of cyclo-D4G revealed that the prodrug possessed anti-HIV activity. In addition, cyclo-D4G had increased stability, lipophilicity, and solubility, as well as decreased toxicity relative to D4G, suggesting that further study is warranted.

Full text of DOI:10.1128/AAC.46.3.887-891.2002

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2002, p. 887–891
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.3.887–891.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 3
Novel Use of a Guanosine Prodrug Approach To Convert
2Ј,3Ј-Didehydro-2Ј,3Ј-Dideoxyguanosine into a Viable Antiviral Agent
Adrian S. Ray,1 Zhenjun Yang,2 Chung K. Chu,2 and Karen S. Anderson1*
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066,1 and Department of
Pharmacology and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, Georgia 30602-23522
Received 16 July 2001/Returned for modification 3 October 2001/Accepted 3 December 2001
Transient kinetic studies with human immunodeficiency virus (HIV) type 1 reverse transcriptase suggest
that nucleotide analogs containing the 2؅,3؅-didehydro-2؅,3؅-dideoxy ribose ring structure present in D4T
(stavudine) triphosphate are among the most effective alternative substrates. For unclear reasons, however, the
corresponding purine nucleoside, 2؅,3؅-didehydro-2؅,3؅-dideoxyguanosine (D4G), was found to be inactive in
cell culture. We have found that the previously reported lack of activity of D4G is primarily due to solution
instability, and in this report we describe a novel use of a guanosine prodrug approach to stabilize the
nucleoside. D4G was modified at the 6 position of the purine ring to contain a cyclopropylamino group yielding
the prodrug, cyclo-D4G. An evaluation of cyclo-D4G revealed that the prodrug possessed anti-HIV activity. In
addition, cyclo-D4G had increased stability, lipophilicity, and solubility, as well as decreased toxicity relative
to D4G, suggesting that further study is warranted.
Human immunodeficiency virus type 1 (HIV-1), the caus-
ative agent of AIDS, requires reverse transcriptase (RT) to
copy its single-stranded RNA genome into a double-stranded
DNA copy for integration into the host cell genome. To date,
some of the most successful drugs at treating HIV are nucle-
oside reverse transcriptase inhibitors (NRTIs), which lack a
3Ј-hydroxyl group and serve to chain terminate viral replication
after activation by cellular kinases. However, treatment with
NRTIs is limited by their toxicity to the host (often because of
their interaction with mitochondrial polymerase ␥ [9, 20]) and
the ability of the virus to mutate and gain resistance (3, 14). In
order to avoid the appearance of resistant virus, highly active
antiretroviral therapy has been used, which includes multidrug
combinations (11). However, mutants resistant to combina-
tions of presently available compounds have still arisen (19). In
order to further our ability to treat HIV, new compounds are
needed with different metabolism, resistance, and toxicity pro-
files to supplement agents presently available.
Recently, a guanosine prodrug approach has proven success-
ful in improving the pharmacokinetics of already potently ac-
tive guanosine NRTIs. The guanine ring of both dioxolane
guanosine (DXG) (12) and carbovir (CBV) (26) were func-
tionalized at the 6 position with an amine to make diaminopu-
rine dioxolane (DAPD; compound 1) (1) and abacavir
(1592U89 or Ziagen; compound 2) (5), respectively (Fig. 1).
Abacavir and DAPD are metabolized to guanosine analogs by
deamination and phosphorylated to make the active triphos-
phates (6, 7). The presence of the amino group improved the
lipophilicity, solubility, and oral bioavailability of these
guanosine analogs (1, 5). The presence of the cyclopropyl-
substituted secondary amine of abacavir also improved its ab-
sorption into the central nervous system (5).
Transient kinetic studies have been used to provide insight
into structural modifications of the ribose ring, which are well
tolerated by HIV-1 RT (7, 10, 21, 25). In a study on the
incorporation of D4T triphosphate (D4TTP) by HIV-1 RT
into physiologically relevant primer/templates, it was found
that D4TTP is incorporated as efficiently as the natural sub-
strate dTTP (25). The inability of RT to distinguish between
D4TTP and the natural substrate may be responsible for D4T’s
limited selection of resistance mutations in vitro (13) and in
vivo (8). Similar studies with the active metabolites of the two
presently clinically relevant guanosine prodrug analogs have
shown that DXGTP and CBVTP are incorporated approxi-
mately an order of magnitude less efficiently than dGTP (7,
21). This decreased efficiency of incorporation may suggest
slight differences in the way that these analogs interact with RT
versus the natural deoxynucleoside triphosphate that could be
taken advantage of during the selection of resistant mutants
(25). Taken together, these studies suggest that a guanosine
analog with the same 2Ј,3Ј-didehydro-2Ј,3Ј-dideoxy ribose ring
structure as D4TTP (2Ј,3Ј-didehydro-2Ј,3Ј-dideoxyguanosine
[D4G] TP) would be a better inhibitor than CBV triphosphate
and DXG triphosphate at the RT active site, both as a better
substrate and possibly with respect to the development of re-
sistance.
The kinetic prediction that D4G triphosphate would be a
superior inhibitor of RT has not been tested, although at a
cellular level, the nucleoside D4G has previously been re-
ported to show no anti-HIV activity (2). This report, however,
did not elucidate the reason for the lack of antiviral activity for
D4G, and clearly many factors, such as solubility, stability, and
metabolism may play a role. An ancillary question is if D4G is,
in fact, inactive in cell culture, could it be activated by the use
of a guanosine prodrug approach? In order to address these
questions and gain a better understanding of the guanosine
prodrug approach, D4G and a 6-substituted prodrug (cyclo-
D4G; compound 3) (Fig. 1) were synthesized and evaluated in
* Corresponding author. Mailing address: Department of Pharma-
cology, Yale School of Medicine, 333 Cedar St., P.O. Box 208066, New
Haven, CT 06520-8066. Phone: (203) 785-4526. Fax: (203) 785-7670.
E-mail: karen.anderson@yale.edu.
887

888
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
1H, 5Ј-OH), 3.84 (dd, J ϭ 2.8, 12.8 Hz, H-5Јa), 4.04 (d, J ϭ 12.8
Hz, H-5Јb), 5.06 (d, J ϭ 1.6 Hz, H-4Ј), 5.21 (brs, 2H, NH2), 5.93
(d, J ϭ 6.0 Hz, 2H, H-2Ј, and H-N6), 6.39 (dd, J ϭ 1.4, 6.0 Hz,
1H, H-3Ј), 6.70 (d, J ϭ 1.6 Hz, H-1Ј), and 7.49 (s, 1H, H-8);
HRMS (FAB) (M ϩ 1)ϩ m/z calculated for C13H17N6O2:
289.1439,
found:
289.1422;
anal.
calculated
for
C13H16N6O2·0.3H2O: C, 53.06; H, 5.71; N, 28.56; found: C,
53.27; H, 5.65; and N, 28.47.
Solubility and lipophilicity of D4G and cyclo-D4G. Cyclo-
D4G was far more soluble in phosphate-buffered saline (PBS)
than was D4G. Concentrations of Ͼ60 mM cyclo-D4G could
be achieved in stock solutions, while D4G was soluble only up
to 5 mM. Lipophilicity of these compounds was based upon
their retention times during reverse-phase high-performance
liquid chromatography using a BDS Hypersil C-18 reverse-
phase column (Keystone Scientific) and isocratic gradient (80%
200 mM triethyl ammonium bicarbonate and 20% methanol;
flow rate, 1 ml/min). It was found that cyclo-D4G had much
higher affinity for the C-18 column than D4G (retention times:
deoxyguanosine [dG], 3 min, D4G, 5 min; and cyclo-D4G, 16
min). These features may serve to increase the bioavailability
and overall pharmacokinetic profile of cyclo-D4G over D4G;
however, animal experimentation will be required to confirm
this suggestion.
Anti-HIV activity and toxicity in MT-2 cells. D4G and cyclo-
D4G were compared to other nucleoside analogs in their ac-
tivity against HIV-1 (human T-cell leukemia virus IIIB; R. C.
Gallo) and cytotoxicity in MT-2 cells (human T-cell leukemia
virus-infected human T lymphoblastoid) by previously re-
ported methods (15). The amount of NRTI required to inhibit
50% of HIV infection (50% effective concentration) ranged
from 0.27 ␮M dideoxycytosine (ddC) to 18 ␮M dideoxyinosine
(ddI; data summarized in Table 1). Consistent with previous
results, D4G showed no activity when dissolved in distilled
water (dH2O) (2); however, when dissolved in phosphate-buff-
ered saline (PBS), it was found to have anti-HIV activity.
Surprisingly, D4G’s lack of antiviral activity when dissolved in
FIG. 1. Structures of guanosine prodrug nucleoside analogs:
DAPD (compound 1), abacavir (compound 2), and cyclo-D4G (com-
pound 3).
a number of in vitro assays to assess stability, toxicity, and
anti-HIV activity.
Synthesis of Cyclo-D4G. Cyclo-D4G 3 was synthesized from
guanosine in seven steps (Fig. 2). 2-Amino-6-chloro-9-␤-D-
ribofuranosyl-purine 5 was prepared from guanosine 4 in three
steps (18). Compound 5 was treated with ␣-acetoxyisobutyryl
bromide to give a mixture of 2Ј-acetoxy-3Ј-bromo and 3Ј-ace-
toxy-2Ј-bromo derivative 6, which was treated with activated
zinc to obtain compound 7. Deprotection of compound 7 with
K2CO3 in MeOH/H2O gave the free nucleoside 8 (22), which
was treated with cyclopropylamine in ethanol to afford target
compound 3 [2-amino-6-cyclopropylamino-9-(2Ј,3Ј-dideoxy-␤-
D-glycero-pent-2-enofuranosyl) purine, cyclo-D4G] as a foam
in 83% yield (4): [␣]24 Ϫ16.4° (c 0.64, MeOH); UV (MeOH):
D
284 (ε 1.3 ϫ 104), 260 (ε 8.6 ϫ 103), 224 (ε 1.8 ϫ 104); 1H
nuclear magnetic resonance (NMR) (400 MHz, CDCl3) ␦ 0.61
(m, 2H, CH2), 0.82 (m, 2H, CH2), 2.95 (brs, 1H, CH), 3.30 (brs,
FIG. 2. Synthesis of cyclo-D4G (compound 3). (i) (CH3)2C(OAc)COBr/CH3CN. (ii) Zn/DMF. (iii) K2CO3/H2O/MeOH. (iv) Cyclopro-
pylamine/C2H5OH.

VOL. 46, 2002
TABLE 1. Anti-HIV activity and toxicity in MT-2 cellsa
NOTES
TABLE 2. Stability of nucleoside analogs
889
Results for MT-2
Stability (t1/2 in min)c at pH of:
Nucleoside
Nucleoside
EC50 (␮M)
IC50 (␮M)
nb
2.0
7.4
11
ddC
0.27 Ϯ 0.03
18 Ϯ 4
4 Ϯ 1.4
ϾϾ100
ϾϾ100
Ͼ100
52 Ϯ 10
Ͼ100
16 Ϯ 2
47 Ϯ 14
Ͼ100
3
3
ddI
30
Ͻ2.0
40
Stablea
6,000b
Stable
Stable
Stable
Stable
ddI
D4G
DAPD
CBV
Abacavir
D4T
D4G (dH2O)
D4G (PBS)
Cyclo-D4G
10 Ϯ 4
3
Cyclo-D4G
1.1 Ϯ 0.1
2.5 Ϯ 0.5
2.8 Ϯ 0.7
ND
4.8 Ϯ 1.6
8.6 Ϯ 1.3
3
a Stable ϭ no breakdown observed after 24 h.
b Value estimated by extrapolating from 10% breakdown after 24 h.
c t1/2, half-life.
4
8
4
10
12
7.4). The reason for cyclo-D4G’s increased stability over D4G
is more likely due to the 6-cyclopropylamino group of cyclo-
D4G being less electron withdrawing than the 6-carbonyl
group of D4G, which makes cyclo-D4G more stable than D4G
under acidic conditions. Results showed that degraded D4G is
more toxic to MT-2 cells than the parent intact nucleoside.
Guanine is not toxic enough on its own to explain the toxicity
of degraded D4G (50% infective concentrations [IC50s] of 90
and 15 ␮M, respectively), suggesting that the depurinated al-
dose sugar moiety may be contributing considerable toxicity. A
closer inspection of the structure of the sugar by-product re-
veals that the acyclic open form is a highly active, ␣, ␤-unsat-
urated, Michael acceptor (Fig. 3), which could react with nu-
cleophilic groups on proteins and other cellular components
causing toxicity. It is also possible that the cyclic closed sugar
ring interferes with nucleoside biosynthesis pathways.
a Values represent mean Ϯ standard deviation.
b n ϭ number of independent trials done in triplicate.
c ND ϭ no detectable anti-HIV activity.
d EC50 ϭ 50% effective concentration.
dH2O was accompanied by a twofold increase in toxicity. Sim-
ilar to results found for other nucleoside/prodrug combinations
(CBV and abacavir and published results for DXG and DAPD
[7]), D4G showed twofold-higher activity (when dissolved in
PBS) than did cyclo-D4G. These differences in parent versus
prodrug activities may be related to a number of factors, in-
cluding the abilities of the compounds to be metabolized to
their respective active triphosphate forms. Interestingly, cyclo-
D4G was found to be more than twofold less toxic when com-
pared to D4G. This was in contrast to results obtained with
abacavir and CBV, where it was found that the prodrug was
more toxic in cell culture.
Cytotoxicity in various cell types. Similar to experiments
done in MT-2 cells, toxicity studies were done in Molt-4 (hu-
man peripheral blood, acute lymphoblastic leukemia, ATCC
CRL 1582), CEM (human T lymphoblastoid, ATCC CCL
119), Hep-G2 (human hepatocellular carcinoma, ATCC HB
8065), Vero (monkey kidney, ATCC CRL 1586), 293 (human
transformed primary embryonic kidney, ATCC CRL 1573),
KB (human oral carcinoma, ATCC CCL 17), and HeLa (hu-
man cervical carcinoma, ATCC CCL 2) cells. 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT;
Sigma; catalog no. M-2128) was used to access cell viability,
and then results were verified by manual cell counting. In all
cases the two results were similar, except for cells treated with
abacavir where the MTT dye underestimated the toxicity by
twofold (see discussion below). Once again D4G (dissolved in
PBS) was found to be more toxic than cyclo-D4G in all cell
lines tested. In general, D4G was one of the more toxic com-
pounds tested (in most cases second only to ddC), while cyclo-
D4G showed intermediate-to-low toxicity. Furthermore, it is
interesting that results in T-cell-, liver-, and kidney-derived
cells would suggest that cyclo-D4G would be well tolerated by
Stability at pH 2, 7.4, and 11. The solvent-dependent activity
and toxicity of D4G suggest that it is unstable under certain
conditions and most likely breaks down by an acid-catalyzed
depurination. To further study the pH dependence on stability,
compounds were dissolved in solutions buffered to pH 2, 7.4,
and 11 (using PBS or triethyl ammonium bicarbonate) and
their breakdown was observed over time. Samples at pH 2 were
analyzed over 300 min using thin-layer chromatography using
CH2Cl2/MeOH as a mobile phase. Samples at pH 7.4 and 11
were analyzed over longer time courses using thin-layer chro-
matography, and results for D4G and cyclo-D4G were verified
using reverse-phase high-performance liquid chromatography
(as described above). Results showed that all compounds were
labile at pH 2 but that D4G was greater than an order of
magnitude less stable than ddI and cyclo-D4G (Table 2). All
compounds were stable under basic conditions. Further study
of the breakdown using NMR showed that D4G depurinated
in D2O (pH 6.3), forming three distinct sugar species and
guanine (Fig. 3). This would explain D4G’s lack of activity in
lab water (pH 5 to 6) and activity in buffered solution (PBS, pH
1
FIG. 3. Acid hydrolysis of D4G determined by NMR. H NMR of decomposed D4G (D2O): ␦ ϭ 7.81 (H-8 of guanine), 7.39 (0.03H, H-1 of
aldehyde), 6.32 (0.03H, H-2 of aldehyde), 6.28 (0.03H, H-3 of aldehyde), 6.08 (m, 1H, H-1 of ␣ and ␤), 6.00 (d, J ϭ 3.2 Hz, 0.6H, H-3 of ␤), 5.94
(s, 0.3H, H-3 of ␣), 5.84 (m, 1H, H-2 of ␣ and ␤), 4.93 (s, 0.6H, H-4, of ␤), 4.71 (s, 0.3H, H-4 of ␣), 3.77 to 3.58 (m, 2H, H-5).

890
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. IC50s (in micromolars) for nucleoside analogs in various cell typesa
Cell type
Vero
Nucleoside
Molt-4
CEM
Hep-G2
293
KB
HeLa
ddC
0.6 Ϯ 0.1
NTb
3 Ϯ 1
Ͼ500
25 Ϯ 2
Ͼ500
500 Ϯ 33
180 Ϯ 12
390 Ϯ 26
280 Ϯ 19
Ͼ500
50 Ϯ 10
NT
Ͼ100
NT
100 Ϯ 11
Ͼ500
Ͼ100
ddI
500 Ϯ 33
500 Ϯ 33
500 Ϯ 33
480 Ϯ 31
200 Ϯ 13
500 Ϯ 33
DAPD
Abacavir
D4T
D4G (PBS)
Cyclo-D4G
NT
Ͼ500
NT
NT
Ͼ500
18 Ϯ 3
55 Ϯ 8
35 Ϯ 10
75 Ϯ 11
200 Ϯ 50
190 Ϯ 30
70 Ϯ 11
200 Ϯ 31
480 Ϯ 100
Ͼ500
200 Ϯ 40
350 Ϯ 70
500 Ϯ 75
500 Ϯ 75
180 Ϯ 30
400 Ϯ 60
200 Ϯ 21
450 Ϯ 47
Ͼ500
Ͼ500
a Data obtained from one experiment using MTT dye to quantitate viable cells done in triplicate and verified by an independent experiment using manual cell
counting. Values represent mean Ϯ standard deviation.
b NT ϭ not tested.
these often nucleoside-sensitive cells and tissues (results sum-
marized in Table 3).
Conclusions. In this study it was found that the addition of
the 6-cyclopropylamine substitution to D4G imparted a high
degree of acid stability that the parent compound lacks. This
increase in stability led to cyclo-D4G having stability similar to
that of ddI, an NRTI presently used in the clinic. The en-
hanced solution stability for cyclo-D4G over D4G under acidic
conditions provides an explanation for previous studies sug-
gesting that D4G lacks antiviral activity. The use of a prodrug
approach to improve solution stability in an acidic environment
as in cyclo-D4G has not been previously noted for other
guanosine prodrugs and may be of general utility in the devel-
opment of other guanosine analogs. Stability also appears to be
of importance in the toxicity of cyclo-D4G and D4G. In this
study it was found that degraded D4G was more toxic than the
intact nucleoside. It was also found that the more stable cyclo-
D4G was less toxic than its more labile parent compound. Our
preliminary evidence suggests that the underlying reason for
D4G’s toxicity may be related to the formation of a 2Ј,3Ј-
didehydro-2Ј,3Ј-dideoxy ribose ring by-product of an acid-cat-
alyzed depurination. Due to the presence of this same ribose
moiety in other clinically relevant compounds, its possible tox-
icity should be taken into account in further drug development.
Indeed studies on fluorinated cytidine analogs, which are also
prone to a similar breakdown mechanism, have shown unex-
pectedly high toxicity (17, 23) that may be related to observa-
tions made in this study.
Effect of nucleoside analogs on mitochondrial DNA content.
To specifically address the issue of mitochondrial toxicity of
D4G and cyclo-D4G relative to other FDA-approved NRTIs
(ddC, D4T, and abacavir), CEM cells were grown in the pres-
ence of drug for 8 days and the amount of mitochondrial DNA
was quantitated using previously reported methods (16). Data
for D4G’s effects on mitochondrial DNA content could not be
obtained because of D4G’s high level of general toxicity in
these cells. However, at concentrations up to 50 ␮M, no de-
crease in mitochondrial DNA was detected, suggesting that
D4G’s toxicity is not due to mitochondrial damage. The com-
pounds showed the following order in their ability to decrease
the amount of mitochondrial DNA: ddC ӷ D4T Ͼ cyclo-D4G
ӷ abacavir (data summarized in Fig. 4). Abacavir showed a
slight, but significant, increase in the amount of mitochondrial
DNA at high concentrations. Interestingly, measuring cell vi-
ability by the use of MTT dye, which is metabolized by a
mitochondrial enzyme (24), was found to underestimate the
level of abacavir toxicity (results described above). One might
speculate, based upon these results, that abacavir increases the
number of mitochondria per cell, although the physiological
significance of this observation is unclear. Cyclo-D4G showed
significantly more mitochondrial DNA at 200 ␮M than did
D4T (51% versus 21% of control, respectively).
Although D4G was found to be active under buffered con-
ditions, its acid instability, high toxicity, and low solubility
make it a poor drug candidate. In a novel use of the guanosine
prodrug approach, the prodrug of D4G, cyclo-D4G, was found
to have increased stability and lower toxicity. Cyclo-D4G also
displayed other advantages relative to D4G that have been
previously noted for other guanosine prodrugs, including in-
creased water solubility and lipophilicity with only a slight
decrease in anti-HIV activity. The results shown here warrant
the further study of cyclo-D4G (and other 6-substituted D4G
derivatives) as potential clinically relevant compounds. One
interesting possibility is that the unique combination of a 2Ј,3Ј-
didehydro-2Ј, 3Ј-dideoxy ribose ring with a functionalized gua-
nine base could prove useful in combination therapy.
K.S.A. is supported by NIH grant GM49551. A.S.R. is supported by
NIH, National Research Service Award 5 T32 GM07223 from the
National Institute of General Medical Sciences. C.K.C. is supported by
NIH grant AI25899.
FIG. 4. Bar graph showing the amount of mitochondrial DNA after
incubation with different concentrations of various drugs. Each bar
represents the mean Ϯ standard deviation for two independent exper-
iments done in triplicate.

VOL. 46, 2002
NOTES
891
We sincerely appreciate help from members of Yung Chi Cheng’s
laboratory at Yale University: Ginger E. Dutschman for providing
HIV-1 viral stocks and for training in biohazard level 3 laboratory
practices and Elizabeth Gullen for sharing expertise in the mitochon-
drial DNA inhibition assay and providing various cell lines for toxicity
studies. We thank William B. Parker at Southern Research Institute
for the generous gift of CBV.
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