Synthesis and anti-HIV activity of GS-9148 (2′-Fd4AP), a novel nucleoside phosphonate HIV reverse transcriptase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.11.125

GS-9148 (2′-Fd4AP, 4) has been identified as a nucleoside phosphonate reverse transcriptase (RT) inhibitor with activity against wild-type HIV (EC₅₀ = 12 μM). Unlike many clinical RT inhibitors, relevant reverse transcriptase mutants (M184V, K6

Full text of DOI:10.1016/j.bmcl.2007.11.125

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1120–1123
Synthesis and anti-HIV activity of GS-9148 (20-Fd4AP), a
novel nucleoside phosphonate HIV reverse transcriptase inhibitor
Constantine G. Boojamra,* Richard L. Mackman, David Y. Markevitch, Vidya Prasad,
Adrian S. Ray, Janet Douglas, Deborah Grant, Choung U. Kim and Tomas Cihlar
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
Received 13 November 2007; accepted 30 November 2007
Available online 5 December 2007
Abstract—GS-9148 (20-Fd4AP, 4) has been identified as a nucleoside phosphonate reverse transcriptase (RT) inhibitor with activity
against wild-type HIV (EC50 = 12 lM). Unlike many clinical RT inhibitors, relevant reverse transcriptase mutants (M184V, K65R,
6-TAMs) maintain a susceptibility to 20-Fd4AP that is similar to wild-type virus. The 20-fluorine group was rationally designed into
the molecule to improve the selectivity profile and in preliminary studies using HepG2 cells, compound 4 showed no measurable
effect on mitochondrial DNA content indicating a low potential for mitochondrial toxicity.
Ó 2007 Elsevier Ltd. All rights reserved.
Over the past decade, great strides have been made in
the treatment of HIV infection through use of drug com-
binations known as highly active anti-retroviral therapy
(HAART). HAART regimens usually include three or
more drugs with inhibitory activity against either HIV
reverse transcriptase (RT) or HIV protease.1 Currently,
most HAART regimens include a backbone of two
nucleos(t)ide reverse-transcriptase inhibitors (N(t)RTIs)
with a third agent added from the non-nucleoside re-
verse-transcriptase inhibitor (NNRTI) or protease
inhibitor classes (PI).2
NH2
N
N
NH2
N
N
N
N
N
N
O
O
H2O3P
H2O3P
H2O3P
O
1, PMPA (tenofovir)
2, d4AP
NH2
N
N
NH2
N
N
N
N
O
O
N
O
PO3H2
O
N
F
3, L-d4AP
4, 2'-Fd4AP
Tenofovir-DF (VireadÒ) is an orally available prodrug
of PMPA (1, Fig. 1) and is currently the only clinically
approved NtRTI for treatment of HIV infection. While
regimens containing Viread have demonstrated excellent
efficacy and safety profiles,3 a subset of treatment-expe-
rienced patients harbor tenofovir-resistant virus either
through presence of the K65R RT mutation or multiple
thymidine-analog mutations (TAMs) in RT (4.3% and
8–31%, respectively).4 The cyclic nucleoside phospho-
nate, d4AP (2, Fig. 1), identified earlier,5 demonstrated
a superior resistance profile compared to tenofovir and
other NRTIs (Table 1).6
Figure 1. Tenofovir (1); cyclic analog, d4AP (2); and targets L-d4AP
(2) and 20-Fd4AP (3).
The N(t)RTIs are metabolized to their corresponding
triphosphates (in the case of NRTIs) and diph-
osphophosphonates (in the case of NtRTIs). These
phosphorylated species inhibit RT through incorpora-
tion into the growing DNA chain and subsequent termi-
nation. Unfortunately, many of the same triphosphates
exhibit mitochondrial toxicity mediated through inhibi-
tion of DNA polymerase-c (pol-c).7 As a result, patients
treated with N(t)RTIs may experience changes in body
fat distribution, hyperlipidemia, peripheral neuropathy,
and lactic acidosis. Unfortunately, d4AP 2, despite its
excellent resistance profile, was shown to have mito-
chondrial toxicity commensurate with its anti-HIV
activity, limiting any further development of this
Keywords: HIV; Phosphonate; Antiviral; Resistance.
*
Corresponding author. Tel.: +650 522 5277; fax: +650 522 5899;
e-mail: DBoojamra@gilead.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.125

C. G. Boojamra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1120–1123
1121
Table 1. Anti-HIV activity, enzymatic potency, cytotoxicity, and resistance profile of nucleoside phosphonates 1–4
Compound
WT HIVa EC50/lM WT RTb IC50/lM MT-2c CC50/lM K65Rd Fold res. 6TAMsd Fold res. M184Vd Fold res.
PMPA (1)
d4AP (2)
L-d4AP (3)
3.6 (1.5)
2.1 (1.0)
5.9 (0.3)
0.38 (0.20)
0.60 (0.16)
—
>500
>1000
>1000
>1000
—
4.3 (1.5)
2.9 (1.1)
3.6 (2.0)
1.1 (0.2)
2.2 (0.4)
1.2 (0.7)
8.8 (3.7)
2.9 (1.0)
13.7 (2.3)
4.3 (0.4)
3.0 (0.4)
>50
0.7 (0.2)
0.9 (0.6)
7.1
20-Fd4AP (4) 12.3 (3.4)
1.9 (0.8)
0.4 (0.2)
0.05 (0.03)
0.8 (0.3)
1.2 (0.7)
0.7 (0.2)
20-Fd4Ae
AZTf
2.2 (1.3)
0.16 (0.12)
>200
a Antiviral activity in MT-2 cells infected with HIV-1 IIIb. Values are results of at least 2 experiments, standard deviation is given in parentheses.
b Data were obtained for the corresponding diphosphophosphonate or triphosphate of compound shown.
c Cytotoxicity in uninfected MT-2 cells.
d Resistance determined in MT-2 cell lines.
e See Ref. 17.
f AZT = 30-azido-20,30-dideoxythymidine.
nucleoside phosphonate as an anti-HIV drug.8 There-
fore, we sought to design analogs of 2 that preserve
the favorable resistance profile, while abrogating the
mitochondrial toxicity.
(catalytic oxidation15 or KMnO45a) did not proceed
smoothly. Thus, a sequential protection/deprotection se-
quence was required. Compound 9 was 50-OH protected
as a bis-monomethoxytrityl derivative 10, followed by
30-OH silylation to give 11, and then the trityl groups
were removed under acidic conditions. Optimal yields
were obtained when the deprotection mixture was di-
luted with a high-boiling alcohol, such as 1-butanol, be-
fore concentration in vacuo; this ensured complete
scavenging of the monomethoxytrityl cation. The result-
ing monomethoxytrityl-butyl ether could then be re-
moved by trituration with Et2O. Alcohol 12 was then
oxidized by treatment with TEMPO/BAIB16 to give car-
boxylic acid 13. We attempted to remove the silyl ether
of 13 under mineral acid conditions in order to simplify
isolation of this polar, water-soluble product. Unfortu-
nately, the material was refractory to deprotection with
acid and required treatment with excess TBAF in THF.
Fortunately, tetrabutylammonium carboxylate salt of
this product precipitated from the deprotection mixture
allowing for isolation of TBAF-free product. A salt ex-
change was then performed to remove the tetrabutylam-
monium salts by coevaporating three times from 1 N
HCl in THF followed by trituration with THF. Com-
pound 14 (HCl salt) was obtained in 35% yield from 9
over the six steps.
Two strategies have been reported in the literature with
regard to mitigating the toxicity of nucleosides. First, L-
nucleosides demonstrate improved toxicological profiles
over their D-nucleoside counterparts.9 Second, 20-b-F
nucleosides appear to be weaker inhibitors of DNA
polymerase-c than their non-fluorinated analogs,10 and
thus are expected to have reduced mitochondrial toxic-
ity.11 Consequently, our targets became the L isomer
of d4AP (3), and the 20-fluorine substituted derivative,
20-Fd4AP (4). Since F is a relatively conservative substi-
tution for H, we reasoned that 4 was likely to preserve
the desirable resistance profile of 2. Herein we disclose
the chemistry developed for synthesis of 3 and 4, and
our initial biological characterization.
The synthesis of analog 3 (L-d4AP, Scheme 1) proceeded
analogously to 2, reported earlier.5 Since 20-deoxy-L-
adenosine (6) was not available in substantial amounts,
it was synthesized according to literature procedure12
from 20-deoxy-L-ribose (5). The identical synthetic se-
quence to that reported for 2 was then carried out, with
7 being the pivotal intermediate for subsequent intro-
duction of the phosphonate functionality. L-d4AP (3)
was obtained in 3% overall yield over 12 steps. C-18
gravity purification was necessary to obtain salt-free
material after phosphonic ester deprotection.
It was serendipitous that 14 was transformed to its HCl
salt for characterization since attempts to prepare the
glycal 15 from the tetrabutylammonium carboxylate salt
of 14 by decarboxylative dehydration led to formation
of 9-(furan-2-yl)-9H-purin-6-amine, presumably via
elimination of HF. However, when care was taken to en-
sure complete conversion of 14 to the HCl salt, clean
conversion to the desired glycal ensued (determined by
LC–MS). Glycal 15 was activated with phenylselenyl
chloride and underwent subsequent AgClO4-mediated
glycosylation with diethyl hydroxymethylphospho-
Our strategy toward 4 exploited a similar glycal interme-
diate to 7, except with the fluorine atom incorporated
(compound 15, Scheme 2). Starting from 8,13 compound
9 was obtained employing the method of Schaerer
et al.14 Selective oxidation of the 50-carbon of 9 in the
presence of the unprotected 30-OH via known methods
NH2
NH2
NH2
N
O
N
N
N
N
N
N
HO
OH
N
N
O
N
O
O
O
PO3H2
ref. 12
N
ref. 5a
ref. 5b
OH
OH
N
OH
5
6
7
3
Scheme 1. Synthesis of L-d4AP. Glycal 7 was a pivotal intermediate in synthesis of 3.

1122
C. G. Boojamra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1120–1123
NHMMTr
N
N
iii
NH2
N
N
NHMMTr
N
N
N
N
N
N
N
O
O
O
O
OBz
N
ii
MMTrO
TBSO
ref. 14
i
MMTrO
HO
10
HO
BzO
BzO
F
F
HO
F
F
13
11
8
9
NH2
N
N
NH2
N
N
NH2·HCl
N
N
N
N
N
N
N
N
N
O
O
N
N
N
·HCl
vii
iv
v, vi
O
O
O
viii, ix
x, xi
N
O
HO
TBSO
13
HO
HO
TBSO
N
F
HO
F
F
F
14
15
12
NH2
N
N
NHPiv
NHPiv
N
N
N
N
N
N
N
N
O
P
·NH3
O
P
O
O
HO
HO
O
EtO
EtO
O
N
O
O
xii
EtO
EtO
N
O
xiii, xiv
P
F
PhSe
F
F
4 (monoammonium salt)
16
17
Scheme 2. Reagents and conditions: (i) MMTrCl, pyridine, 93%; (ii) TBSCl, imidazole, DMF, 84%; (iii) HCOOH/MeOH/CH2Cl2 (2:1:1) 2.5 h then
excess n-butanol, 74%; (iv) TEMPO, BAIB, CH2Cl2, H2O, 80%; (v) TBAF, THF; (vi) 1 N HCl in THF, 76% from 13; (vii) DMF-dineopentylacetal,
DMF, 130 °C, 3–4 min; (viii) PhSeCl, THF, ꢀ78 °C to rt; (ix) HOCH2PO3Et2 then AgClO4 followed by filtration and evaporation; (x) NH3/MeOH,
then evaporation; (xi) PivCl, pyridine, 13% from 14; (xii) O3, CCl4, ꢀ15 °C then purge excess O3 and 50 °C for 3 h, 78%; (xiii) NaOMe, MeOH, 100%;
(xiv) TMSBr, DMF, 55 °C then excess 2,6-lutidine, MeOH and evaporation, then conc’d NH4OH, 72% from 17.
nate,5b in a sequence that proceeded stereoselectively to
yield the 40-b-phosphonomethoxy-30-a-phenylselenide
isomer.
used to cleave the pivaloyl group and dealkylate the
phosphonic acid. The free phosphonic acid was con-
verted to its monoammonium salt with excess aqueous
NH3 and the final product isolated by gravity C-18 chro-
matography eluting with neat H2O. In order to deter-
Under the conditions of glycal formation, the nucleo-
base was transformed into its formamidine derivative.
This functional group was cleaved in situ with NH3 in
MeOH immediately after the phosphonate was intro-
duced. The resulting product was protected by treat-
ment with PivCl in pyridine to provide 16 in 13%
overall yield from 14. It was noteworthy that the trans-
formation of 14 to 15 was unusually sensitive to reaction
time and internal temperature. As a consequence, the
reaction was performed by submersing a thin-walled,
round-bottom flask into a preheated oil bath (130 °C)
for 3–4 min and then rapidly transferring it into a
cooling bath. Successful transformations could be
performed on up to 100-mg scale, whereas larger scale
reactions led to the elimination of HF, likely due to
the inability to precisely control the internal reaction
temperature.
mine
the
inhibition
of
isolated
RT,
the
diphosphophosphonate of this compound was synthe-
sized according to literature procedure.20
The compounds were tested for their whole-cell anti-
HIV activity in MT-2 cells infected with wild-type, 6-
TAMs or K65R RT mutants (Table 1). The panel also
included the M184V RT mutant, which is found in
45% of the HAART treatment-experienced population.4
While L-d4AP, which to the best of our knowledge
represents the first reported antiviral L-nucleoside
phosphonate, retained good wild-type anti-HIV activity
relative to PMPA and d4AP, it was considerably less
active against the M184V mutant. Given the prevalence
of this mutation clinically, our interest shifted to
20-Fd4AP (4).
Formation of the d4 functionality from the phenyl sele-
nide 16 was also non-trivial. Standard methods such as
H2O2 did not efficiently oxidize the selenium and less
well-known methods (e.g., Ti(O–i-Pr)4 and t-BuO2H)18
led to complex mixtures. The oxidized phenylselenide
While compound 4 was 6-fold less potent against wild-
type virus than d4AP itself and 3-fold less potent than
PMPA (Table 1, column 2), it retained excellent activity
against several RT mutants. The marginal loss in WT
anti-viral potency (EC50) compared to phosphonates 1
and 2 compares nicely with the 3- to 5-fold reduced inhi-
bition of RT by the active metabolites (Table 1, column
3). Thus, for this series of adenine phosphonate analogs,
it would appear that the permeability and phosphoryla-
tion properties are similar.
19
of 16, which ultimately formed with O3 in CCl4 at
ꢀ15 °C, was stable and could be isolated at room tem-
perature. This oxidized product was forced to eliminate
to the desired d4 product by heating the CCl4 solution to
50 °C, after completely purging residual O3 from the
reaction flask. Only the desired d4 derivative was iso-
lated with no evidence for formation of the 30–40 regio-
isomer. Finally, standard deprotection conditions were
Compound 4 experienced no loss of potency against
the K65R RT mutant compared with wild-type virus,

C. G. Boojamra et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1120–1123
1123
guidelines available at: <http://aidsinfo.nih.gov>. Also see
Ref. 1.
3. Gallant, J. E.; DeJesus, E.; Arribas, J. R.; Pozniak, A. L.;
Gazzard, B.; Campo, R. E.; Lu, B.; McColl, D.; Chuck,
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Med. 2006, 354, 251.
whereas d4AP (2) was 3-fold, and PMPA (1) 4-fold
less active. The loss of activity toward the 6TAMs
mutant was comparable between the fluorinated 4
and non-fluorinated 1 analogs (ꢁ3- to 4-fold), but
slightly improved over PMPA (8.8-fold). No loss of
potency was observed for the natural D analogs to-
ward the M184V RT mutant. Overall the resistance
profile of the fluorinated analog 4 is improved over
both PMPA and d4AP.
4. (a) McColl, D. #393, 7th International Congress on HIV
Drug Therapy, Glasgow, 2004; (b) Johnson, V. A.; Brun-
´
Vezinet, F.; Clotet, B.; Conway, B.; Kuritzkes, D. R.;
Pillay, D.; Schapiro, J. M.; Telenti, A.; Richman, D. Top.
HIV Med. 2005, 13, 125.
For comparison, 20-Fd4A, the nucleoside analog of
phosphonate 4, was tested and found to be approxi-
mately 6-fold more potent against wild-type HIV. Much
of this difference in potency is once again due to the
approximate 5-fold improvement in potency toward
RT inhibition (IC50 = 1.9 lM vs 0.4 lM for the active
metabolites of 4 and 20-Fd4A, respectively).6 In earlier
work from this group, the d4 nucleoside phosphonate
series of analogs, when compared to their d4 nucleoside
counterparts, consistently demonstrate a small loss in
RT inhibition of <10-fold. In this respect, d4 nucleoside
phosphonates are quite good ‘bioisosteres’ of the d4
nucleoside monophosphates. This is also reflected in
the similar resistance profiles between 20-Fd4A and 4
noted in Table 1.
5. (a) Zemlicka, J.; Gasser, R.; Freisler, J. V.; Horwitz, J. P.
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Initial toxicological studies have yielded promising re-
sults. Compound 4 showed no cytotoxicity to MT-2 cells
up to 1 mM. Perhaps most importantly, compound 4
demonstrated no depletion of mitochondrial DNA from
HepG2 cells when tested at up to 300 lM for 14 days.8
Complete biological and toxicological profiling of this
compound will be published concomitant with this
work.
Compound 4, 20-Fd4AP, is a new nucleoside phospho-
nate derivative with a good resistance profile (no resis-
tance by M184V and K65R, minimal resistance from
6-TAMs) and promising toxicological profile compared
to its non-fluorinated analog d4AP (2) and PMPA (1). A
derivative of
investigation.
4
is currently undergoing clinical
Acknowledgments
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The authors gratefully acknowledge the assistance of
Dr. Will Watkins and Dr. Steve Krawczyk for useful
chemistry discussions in the development of the method-
ology to make compound 4. C.G.B. would also like to
thank Jackson Soos for his comments on this
manuscript.
`
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