A novel diketo phosphonic acid that exhibits specific, strand-transfer inhibition of HIV integrase and anti-HIV activity

Article abstract of DOI:10.1016/j.bmcl.2006.12.009

We have synthesized novel phosphonic acid analogues of β-diketo acids. Interestingly, the phosphonic acid isostere, 2, of our anti-HIV compound, 1, was an inhibitor of only the strand transfer step, in stark contrast to 1. Compound 2 had lower anti-HIV ac

Full text of DOI:10.1016/j.bmcl.2006.12.009

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1266–1269
A novel diketo phosphonic acid that exhibits specific,
strand-transfer inhibition of HIV integrase and anti-HIV activity
Guochen Chi,a Vasu Nair,a,* Elena Semenovab and Yves Pommierb
aThe Center for Drug Discovery and the Department of Pharmaceutical and Biomedical Sciences, University of Georgia,
Athens, GA 30602, USA
bLaboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, MD 20892, USA
Received 5 October 2006; revised 1 December 2006; accepted 4 December 2006
Available online 15 December 2006
Abstract—We have synthesized novel phosphonic acid analogues of b-diketo acids. Interestingly, the phosphonic acid isostere, 2, of
our anti-HIV compound, 1, was an inhibitor of only the strand transfer step, in stark contrast to 1. Compound 2 had lower anti-HIV
activity than 1, but was more active and less toxic than the phosphonic acid analogue of L-708906. These isosteric compounds rep-
resent the first examples of b-diketo phosphonic acids of structural, synthetic, and antiviral interest.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The pol gene of the human immunodeficiency virus
(HIV) encodes viral enzymes that are required for HIV
replication.1–3 Drug design and discovery work directed
at inhibitors of two of these enzymes, HIV reverse trans-
criptase (RT) and HIV protease (PR), have created clin-
ically useful compounds for the treatment of acquired
immunodeficiency syndrome (AIDS).4–8 However, re-
search efforts on drug discovery pertaining to the third
enzyme of the pol gene, HIV integrase, have not pro-
duced a single approved drug whose mechanism of ac-
tion is inhibition of HIV integrase.9–11 As HIV
integrase has no human counterpart and is important
in HIV replication, it remains a key viral target for the
design and discovery of new anti-HIV agents.
which occurs in the nucleus, is identified as strand trans-
fer. Following staggered nicking of chromosomal DNA
and joining of each 30-end of the recessed viral DNA to
the 50-ends of the host DNA, there is repair, which com-
pletes the integration process. The strand transfer step,
occurring in the nucleus, is partitioned from the 30-pro-
cessing step in the cytoplasm.
While many structurally diverse compounds have been
reported to be inhibitors of HIV integrase,8–10,17–28 only
a few compounds of one group, the b-diketo acids, and
their related compounds, represent the most convincing,
biologically validated inhibitors23,24 of this viral enzyme.
Nair and coworkers have designed a conceptually new
b-diketo acid with a nucleobase scaffold, compound 1,
which is a powerful inhibitor of both 30-processing and
strand transfer steps of HIV-1 integrase and which
exhibits remarkably potent in vitro anti-HIV activity
(Fig. 1).29,30
HIV-1 integrase is encoded at the 30-end of the HIV pol
gene and is a relatively small (32 kDa) viral protein. Its
catalysis results in the incorporation of HIV DNA into
host chromosomal DNA.12–16 This process begins in the
cytoplasm where the viral DNA, produced by reverse
transcription, is assembled on HIV integrase. Following
this assembly, there is specific endonucleolytic cleavage
of two nucleotides from each 30-end of double-stranded
viral DNA. This step, referred to as 30-processing, pro-
duces tailored and recessed viral DNA. The next step,
Phosphonic acids have been viewed commonly as mim-
ics of carboxylic acids, particularly with reference to bio-
logical activity. For example, amino phosphonic acids,
isosteres of amino acids, reveal diverse biological prop-
erties.31 With this concept in mind, we designed the
b-diketo phosphonic acids, 2 and 3, the phosphorus-
based isosteres of compounds 1 and L-708906. In this
report, we describe the methodology for the syntheses
of 2 and 3, and discuss their integrase inhibition and
antiviral data.
Keywords: Synthesis; Phosphonates; HIV integrase inhibitors; Mech-
anism of inhibition; 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 ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.009

G. Chi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1266–1269
1267
phonyl group and the poor solubility of 13 in acetone.35
The problem was circumvented by conversion of 13 to
its protonated form by ion-exchange chromatography
with Dowex 50 · 8 in methanol to afford 14. When com-
pound 14 was heated under reflux with 5 equiv of NaI in
acetone for 24 h and the resulting crystalline precipitate
was collected and washed with acetone, the target phos-
phonate 2 (monosodium salt) was produced in a highly
purified form (51% yield). Compound 3 was synthesized
by a similar procedure to compound 2. The structures of
Ph
Ph
O
O
OH
O
OH
O
N
OH
O
OH
N
O
O
O
Ph
1
Ph
O
L708906
O
Ph
Ph
OH
O
OH
O
N
OH
OH
OH
OH
1
2 and 3 were confirmed by H, 13C, and 31P NMR, and
P
N
P
HRMS data.34
O
O
O
O
Ph
Integrase inhibition studies were conducted with recom-
binant wild-type HIV-1 integrase and a 21-mer oligonu-
cleotide substrate following a previously described
procedure.36,37 Target compound 2 was not an inhibitor
Ph
2
3
Figure 1. Structures of two biologically active diketo acids (top) and
their phosphonic acid isosteres (bottom).
of the 30-processing step of HIV integrase (IC50
>
333 lM) but inhibited the strand transfer step (IC50
20.2 7.2 lM).38 The integrase inhibition activity of 2 is
in sharp contrast to that of compound 1, which is a potent
inhibitor of both the 30-processing and strand transfer
steps of integrase (IC50 3.7 1.0 lM and 0.2 0.1 lM,
respectively,29,39 and 14.4 1.6 lM and 4.5 2.5 lM,
respectively38).
Although the b-diketo acid 1 was synthesized, using as
the key step, the condensation of acetyl uracil 4 with
dimethyl oxalate under basic conditions,29 the related
reaction of 4 with trimethyl phosphonoformate was
unsuccessful (Scheme 1). The reason for this may be
the instability of the C(O)–P(O) bond of trimethyl phos-
phonoformate under basic conditions.32 Therefore, an
alternative synthetic route was devised (Scheme 2).
Compound 2 was tested in a PBMC-based, microtiter
anti-HIV assay against the clinical isolates, HIV-1TEKI
(NSI phenotype) and HIV-1NL4-3 (SI phenotype).29
Antiviral determinations were performed in triplicate
with serial 1/2log10 dilution of the test materials (six
to nine concentrations total). The overall performance
of both assays was validated by the MOI-sensitive posi-
tive control compound, AZT. As summarized in Table
1, in vitro anti-HIV studies against HIV-1 isolates in
PBMC showed that compound 2 (highest test concen-
tration = 200 lM) was active with IC50 values in the
low lM range and with antiviral efficacy data [therapeu-
tic indices, TI = CC50/IC50 of >50 (HIV-1TEKI) and >62
(HIV-1NL4-3)]. Cell viability data showed only mild cel-
lular cytotoxicity at the highest test concentrations;
however, a CC50 (>200 lM) was not reached. In con-
trast, compound 3 was significantly less active and more
toxic [TI = 5.8 (HIV-1TEKI) and 4.0 (HIV-1NL4-3)]. The
control compound, AZT (highest test concentra-
tion = 1 lM), gave therapeutic indices of >504 (HIV-
1TEKI) and >573 (HIV-1NL4-3).
Thus, 5-formyl uracil 7 was benzylated to give 8 (89%).
Compound 8 was condensed with ethyl diazoacetate in
the presence of tin (II) chloride33 to afford 9, which
was transformed into its protected form 10 (28% yield
from 8). Reduction of 10 to the corresponding aldehyde
with diisobutylaluminum hydride at ꢀ78 ꢁC, followed
by a Pudovik reaction with dimethyl phosphite and tri-
ethylamine, proceeded to form the phosphonate 11 (50%
for two steps). The ketal group of 11 was deprotected to
produce the key intermediate 12, which was purified by
HPLC (65% yield) and fully characterized by multinu-
clear NMR and HRMS data.34 Dess–Martin oxidation
of 12 produced diketone 6, which exists largely in the
enolic form. Compound 6 was deprotected by stirring
with NaI in acetone for 3 days.35 The resulting precipi-
tate was collected and washed with acetone to afford the
sodium methyl phosphonate 13 (67% yield). However,
compound 13 could not be further deprotected with
NaI, even under acetone reflux conditions, possibly be-
cause of the presence of the negative charge on the phos-
In summary, we have developed a methodology for the
synthesis of a new b-diketo phosphonic acid, 2, an iso-
stere of our highly active, anti-HIV compound, 1. In
complete contrast to 1, compound 2 specifically inhibits
only the strand transfer step of WT HIV-1 integrase.
Anti-HIV data show that the exchange of the carboxyl-
ate functionality with a phosphonate results in a reduc-
tion in in vitro anti-HIV activity. The anti-HIV data in
PBMC may be a reflection of the difference in integrase
inhibitory activity of 1 compared to 2 and the result of
decreased cellular permeability of phosphonate 2 com-
pared to the less polar carboxylate 1. Interestingly, com-
pound 2 is more anti-HIV active and less toxic than the
phosphonic acid analogue of L-708906 (compound 3).
Finally, these isosteric compounds represent the first
O
O
OH
O
O
O
O
O
BnN
O
CH3
BnN
O
CH3
MeOC COMe
t-BuONa
O
N
N
Bn
Bn
5
4
O
OH
O
O
O
O
O
OMe
OMe
MeOC P(OMe)2
BnN
O
CH3
BnN
O
P
O
N
N
Bn
Bn
t-BuONa
6
4
Scheme 1. The difference in the reactivity of 4 with dimethyl oxalate
compared to trimethyl phosphonoformate.

1268
G. Chi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1266–1269
O
O
O
O
O
O
O
BnN
O
H
BnN
O
OEt
HN
H
a
b
N
N
O
N
H
Bn
Bn
9
8
7
O
OH
O
O
N
O
O
O
O
OMe
OMe
f
BnN
O
OEt
BnN
P
c
d, e
O
N
O
Bn
Bn
11
10
O
OH
O
O
OH
O
N
OMe
OMe
OMe
g
BnN
O
P
BnN
O
P
h
OMe
O
O
N
Bn
Bn
12
6
O
OH
O
OH
O
O
OMe
ONa
OMe
OH
j
i
BnN
O
P
BnN
P
2
O
O
N
O
N
Bn
Bn
13
14
Scheme 2. Synthesis of integrase inhibitor 2. Reagents and conditions: (a) BnBr/DMF/K2CO3; (b) N2CHCO2Et/SnCl2/CH2Cl2; (c) ethylene glycol/
CH(OEt)3/PTS; (d) dibal-H/toluene/ꢀ78 ꢁC; (e) HP(O)(OMe)2/Et3N/MeOH; (f) PTS/acetone/H2O; (g) Dess–Martin periodinane/CH2Cl2; (h) NaI/
acetone; (i) Dowex50Wx8-100/MeOH; (j) NaI/acetone/reflux.
Table 1. Antiviral data for compounds of Figure 1 in PBMC
Cancer Institute, Center for Cancer Research. The con-
tents of this paper are solely the responsibility of the
authors and do not necessarily represent the official
views of the NIH. We thank the SRI for the anti-HIV
screening data.
Compound
HIV-1 isolate
cell line
IC50
CC50
TI
1a
HIV-1TEKI
(PBMC)
50 nM
>200 lM
>200 lM
88.3 lM
>200 lM
>200 lM
46.9 lM
46.9 lM
>4000
>10,000
16
HIV-1NL4-3
(PBMC)
<20 nM
5.5 lM
4.0 lM
3.2 lM
8.1 lM
11.8 lM
L708906b
2
HIV-1 (IIIB)
(MT-4)
References and notes
HIV-1TEKI
(PBMC)
>50
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Acknowledgments
This project was supported by Grant No. RO1 AI 43181
from the National Institutes of Health (NIAID) and by
the Intramural Research Program of the NIH, National

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ra-hydropyrimidin-5-yl)-1-hydroxy-3-oxopropylphosph-
onate (12). 1H NMR (CDCl3): 8.29 (s, 1H), 7.30–7.50
(m, 10H), 5.18 (s, 2H), 5.05 (d, 1H, J = 15), 5.02 (d, 1H,
J = 15), 4.61 (m, 1H), 3.87 (d, 3H, J = 11.0), 3.86 (d, 3H,
J = 10.5), 3.57–3.64 (m, 1H), 3.42–3.48 (m, 1H).
13CNMR (CDCl3): 194.9 (d, J = 17.6), 160.8, 150.9,
149.3, (136.2, 134.3, 129.5, 129.2, 129.1, 128.7, 128.5,
128.1, phenyl ring), 111.7 (d, J = 2.31), 64.3 (d,
J = 169.4), 53.8, (53.72, 53.68, 53.66, 53.61, P-OCH3),
45.1, 43.9 (d, J = 3.82). 31PNMR (CDCl3): 26.5. FAB-
HRMS: calcd for C23 H26 N2O7P (M+H)+ 473.1478; Found
473.1469. Data for 3-(1,3-dibenzyl-2,4-dioxo-1,2,3,4-tetrahy-
dro-pyrimidin-5-yl)-1-hydroxy-3-oxoprop-1-enylphosphonic
acid (2) as its monosodium salt: mp 224 ꢁC (dec). 1H
NMR (CD3OD): 8.58 (s, 1H), 7.22–7.39 (m, 11H), 5.12 (s,
2H), 5.10 (s, 2H). 13CNMR (CD3OD): 188.1 (d, J = 14.5),
185.9 (d, J = 190.4), 161.5, 152.5, 150.6, (138.3, 137.1,
130.2, 129.6, 129.5, 129.3, 128.7, phenyl ring), 111.2 (d,
J = 4.90), 104.5 (d, J = 24.4), 54.4, 45.7. 31PNMR
(CD3OD): 0.82. FAB-HRMS: calcd for C21H19N2NaO7P
(M+H)+ 465.0828; Found 465.0840. Data for 3-(3,5-
bis(benzyloxy)phenyl)-1-hydroxy-3-oxoprop-1-enyl- phos-
phonic acid (3) as its monosodium salt: mp 201 ꢁC (dec).
1H NMR (CD3OD): 7.29–7.46 (m, 10H), 7.25 (d, 2H,
J = 2.5), 6.89 (d, 1H, J = 7.0), 6.85 (t, 1H, J = 2.5), 5.12 (s,
4H). 13CNMR (CD3OD): 193.3 (d, J = 13.4), 185.0 (d,
J = 191.3), [161.8, 140.0 (d, J = 4.27), 138.4, 129.7, 129.2,
128.9, 108.3, 107.8, phenyl ring], 101.6 (weak), 71.5.
31PNMR (CD3OD): 0.91. FAB-HRMS: calcd for
C23H21NaO7P (M+H)+ 463.0923; Found 463.0927.
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38. Magnesium chloride-based assay.
39. Manganese chloride-based assay.