β-Diketo acids with purine nucleobase scaffolds: Novel, selective inhibitors of the strand transfer step of HIV integrase

Article abstract of DOI:10.1016/j.bmcl.2005.12.093

The HIV pol gene encodes three viral enzymes that are required for its replication. While drug discovery involving the viral targets, reverse transcriptase and protease, has resulted in useful therapeutic agents, such efforts on HIV integrase have not produced a single FDA-approved drug. In the work focused on the discovery of inhibitors of HIV integrase, we have synthesized new β-diketo acids with purine nucleobase scaffolds that are potent inhibitors of the strand transfer steps of wild-type HIV-1 integrase.

Full text of DOI:10.1016/j.bmcl.2005.12.093

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1920–1923
b-Diketo acids with purine nucleobase scaffolds: Novel,
selective inhibitors of the strand transfer step of HIV integrase
Vasu Nair,a,* Vinod Uchila and Nouri Neamatib
aThe Center for Drug Discovery and the Department of Pharmaceutical and Biomedical Sciences,
University of Georgia, Athens, GA 30602, USA
bDepartment of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Received 4 November 2005; revised 22 December 2005; accepted 22 December 2005
Available online 24 January 2006
Abstract—The HIV pol gene encodes three viral enzymes that are required for its replication. While drug discovery involving the
viral targets, reverse transcriptase and protease, has resulted in useful therapeutic agents, such efforts on HIV integrase have not
produced a single FDA-approved drug. In the work focused on the discovery of inhibitors of HIV integrase, we have synthesized
new b-diketo acids with purine nucleobase scaffolds that are potent inhibitors of the strand transfer steps of wild-type HIV-1
integrase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Enzymes of the pol gene of the human immunodeficien-
cy virus (HIV) are required for HIV replication and
therefore represent significant viral targets for the dis-
covery of anti-HIV therapeutic agents.1–5 Drug discov-
ery directed at inhibitors of two of these enzymes,
HIV reverse transcriptase (RT) and HIV protease
(PR), has produced clinically useful compounds for
the treatment of acquired immunodeficiency syndrome
(AIDS).4–8 However, while HIV RT and HIV PR have
been successfully investigated for the development of
clinically useful therapeutic agents, research efforts on
drug discovery pertaining to the third enzyme of the
pol gene, HIV integrase, have not resulted in a single
FDA-approved drug whose mechanism of action is inhi-
bition of HIV integrase.9–11 Nevertheless, because inte-
grase is essential for HIV replication and has no
human counterpart, it remains a significant target for
the discovery of new anti-HIV agents.
reverse transcription, on HIV integrase. Following this
assembly, endonucleatic cleavage of two nucleotides
from each 30-end of double-stranded viral DNA (30-pro-
cessing) produces tailored viral DNA recessed by two
nucleotides. In the next step, which occurs in the nucleus
and is identified as strand transfer, there is staggered
nicking of chromosomal DNA and joining of each
30-end of the recessed viral DNA to the 50-ends of the
host DNA. The strand transfer step, occurring in the
nucleus, is partitioned from 30-processing and is carried
out after transport of the processed, preintegration com-
plex from the cytoplasm into the nucleus.
While a number of structurally diverse compounds have
been reported to be inhibitors of HIV integrase,8–10,17–26
only a few compounds of one group, the b-diketo acids,
represent the most convincing, biologically validated
inhibitors23,24 of this viral enzyme (Fig. 1).
HIV-1 integrase is a 32 kDa protein encoded at the
30-end of the HIV pol gene and is responsible for the
integration of HIV DNA into host chromosomal
DNA.12–16 Prior to the initiation of integration, there
is assembly of viral DNA, previously produced by
In this report, we disclose the synthesis and HIV-1 inte-
grase data of new b-diketo acids with purine nucleobase
scaffolds (Fig. 2) that are inhibitors of the strand trans-
fer steps of HIV-1 integrase. We have discovered that
the nucleobase scaffold, the substituents, and the specific
spatial relationship of substituents in the scaffold are
critical for integrase inhibitory activity involving either
strand transfer inhibition selectivity (as in this case) or
inhibition of both the 30-processing and strand transfer
steps (as in other cases).
Keywords: HIV integrase inhibitors; Purine diketo acids; Mechanism
of inhibition.
*
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.2005.12.093

V. Nair et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1920–1923
1921
F
followed by hydrolysis. Conversion of 11 to 4 involved
a cross-Claisen condensation31 followed by base-cata-
lyzed ester hydrolysis.32 The overall yield of the target
compound 4 from guanine involving 11 steps was
0.56%. The characterization data for compounds 1–4
are summarized.33
NH
N
CO2H
N
O
N
O
O
O
O
F
S-1360
L-731988
Figure 1. Structures of two b-diketo compounds that are inhibitors of
HIV-1 integrase.
Integrase inhibition studies were conducted with re-
combinant wild-type HIV-1 integrase and a 21-mer
oligonucleotide substrate, following
a
previously
described procedure.34 Percent inhibition was
calculated using the following equation: inhibi-
tion = 100 · [1 ꢀ (D ꢀ C)/(N ꢀ C)], where C, N, and
D are the fractions of 21-mer substrate converted to
19-mer (30-processing product) or strand transfer
products for DNA alone, DNA plus integrase, and in-
tegrase plus drug, respectively. The 50% inhibitory
concentrations (IC50) were graphically determined
from the plot of the log of drug concentration versus
% inhibition. Integrase inhibition data are shown in
Table 1. Compounds 3 and 4 with hydrophobic benzyl
groups, and the b-keto acid functionality at the 8- and
2-positions, respectively, were strong inhibitors of the
strand transfer step of HIV-1 integrase. However,
both compounds showed much lower inhibition of
the 30-processing step of HIV integrase action. In
sharp contrast, b-diketo acids with pyrimidine nucleo-
base scaffolds are potent inhibitors of both the 30-pro-
cessing and strand transfer steps.35 For example, 4-
(1,3-dibenzyl-1,2,3,4-tetra-hydro-2,4-dioxo-pyrimidin-5-
yl)-2-hydroxy- 4-oxo-but-2-enoic acid, a b-diketo acid
with a pyrimidine nucleobase scaffold synthesized in
our laboratory, exhibits strong inhibition of both 30-
processing and strand transfer steps (IC50 3.7 and
0.2 lM, respectively).35 Closely related analogs of this
pyrimidine-based inhibitor also exhibit potent anti-
HIV integrase inhibitory activity for both key steps
of integrase enzymology. The reason for this difference
is not entirely clear. However, our molecular modeling
data reveal that the regiochemical arrangement and
preferred conformation of the b-diketo acids with
pyrimidine nucleobase scaffolds allow for more effec-
tive overlap of these diketo acids with both the 30-pro-
cessing and strand transfer pockets within the catalytic
site.35,36 Figure 3 shows the docking of HIV integrase
inhibitor, (4), with the ‘two magnesium model’ of
HIV-1 integrase core structure. The catalytic triad is
colored in blue-green. The Mg2+ ion present in the
crystal structure is shown in green and is coordinated
by Asp64 and Asp116. The modeled Mg2+ ion (red
sphere) is located in the strand transfer pocket of
the active site and is coordinated by Asp64 and the
side-chain carbonyl of Asn155 (shown in orange)
and also interacts with the b-diketo acid moiety.
O
N
HO
HO
O
HO
HO
O
HO
OH
O
O
N
N
N
O
N
N
N
N
N
N
O
O
O
N
N
N
N
N
N
OH
OH
O
4
2
1
3
Figure 2. Compounds investigated for HIV integrase activity showing
the regiochemical arrangement of functional and hydrophobic groups.
The synthesis of these compounds using both known
and new steps is illustrated with one example in which
the b-diketo acid is at the C-2 position (see Scheme 1).
Guanine (5) served as the starting material and was
selectively benzylated at the 9-position (to compound
7) via the intermediate 6.27 Conversion of 7 to interme-
diate 8 required initial protection of the lactam group,28
a radical deamination-halogenation reaction at the 2-
position,29 and then a two-step hydrolytic dehalogen-
ation at the 6-position. Intermediate 8 was benzylated
at N-1 and converted to the 2-acetyl intermediate 11
by a palladium-catalyzed cross-coupling reaction28,30
O
O
10 % Pd/C / MeOH
HCO2NH4
O
N
N
N
NH
N
Ac2O / NMP
NH
NH2
N
N
NH
NH
Ac
N
Br
NH2
N
H
N
NH4OH
BnBr
80%
76%
7
5
1) POCl3/ CH3CN (45%)
2) ButONO
CH2I2 / CH3CN (41%)
6
3) NaOMe (95%)
4) 1N NaOH (78%)
OEt
SnBu3
O
O
NaH /BnBr
O
N
N
NH
I
N
N
N
N
DMF
N
Pd(PPh3)2Cl2
N
N
N
I
N
(49%)
DMF
O
8
(96%)
9
10
1 N HCl
Acetone
(99%)
O
N
O
N
1N NaOH
Table 1. IC50 data (lM) for the inhibition of wild-type HIV-1
integrase by the compounds of Figure 2
N
N
N
NaOBut
N
O
4
N
O
N
MeOH
56%
OH
CH3OCOCOOCH3
26%
30-Processing (lM)
Strand transfer (lM)
CH3
Inhibitors
O
OCH3
1
2
3
4
1000
100
31.5
30
790
10
11
12
4.1
2.7
Scheme 1. Representative example of the synthesis of an integrase
inhibitor with a purine nucleobase scaffold.

1922
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In summary, new b-diketo acids, assembled on purine
nucleobase scaffolds, have been synthesized and found
to exhibit moderate selectivity of inhibition of the strand
transfer step of HIV-1 integrase. Further work with
other novel purine-based inhibitors of HIV integrase is
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Acknowledgments
This project was supported by Grant No. RO1 AI 43181
from 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. We thank Dr. Arthur Cox of our
research group for the molecular modeling data.
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V. Nair et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1920–1923
1923
128.3, 128.8, 132.7, 132.8, 135.9, 146.8, 151.6, 152.6,
155.2, 161.9, 186.2; HRMS: (M+H)+ calcd for
C16H13N4O4 325.0937, found 325.0930. Data for 4-(9-
benzyl-9H-purin-8-yl)-2-hydroxy-4-oxobut-2-enoic acid
(2): mp 162–163 ꢁC. 1H NMR (DMSO, 500 MHz):
5.91 (s, 2H, CH2), , 7.26–7.34 (m, 6H, Ar-H and
179.5. HRMS (M+H)+ calcd for C23H19N4O5
431.1355, found 431.1374. Data for 4-(1,9-dibenzyl-
6,9-dihydro-6-oxo-1H-purin-2-yl)-2-hydroxy-4-oxobut-
2-enoic acid (4): mp 138–139 ꢁC; 1H NMR (CDCl3/
CD3OD, 500 MHz) d 5.33 (s, 2H, CH2), 5.73 (s, 2H,
CH2), 7.11–7.37 (m, 11H, Ar-H and olefinic CH), 7.92
olefinic CH), 9.15 (s,1H, purine C6-H), 9.48 (s, 1H,
purine C2-H).
(s,1H,
purine
C8-H);
13C
NMR
(CDCl3/
13
C NMR (CDCl3/CD3OD, 125 MHz):
CD3OD,125 MHz) d 45.9, 47.9, 124.5, 127.4, 127.5,
127.5, 127.6, 127.7, 128.0, 128.0, 128.1, 128.2, 128.5,
128.6, 128.8, 129.0, 129.1, 134.5, 136.6, 141.9, 145.4,
156.8, 162.8; HRMS (M+H)+ calcd for C23H19N4O5
431.1355, found 431.1372.
d 47.6, 101.4, 127.9, 127.9, 128.9, 129.3, 129.3, 131.7,
134.5, 147.4, 147.5, 152.2, 154.3, 162.0, 172.8, 185.7;
HRMS: (M+H)+ calcd for C16H13N4O4 325.0937,
found 325.0926. Data for 4-(1,9-benzyl-6,9-dihydro-6-
oxo-1H-purin-8-yl)-2-hydroxy-4-oxobut-2-enoic acid (3):
mp 167 ꢁC, decomposes. 1H NMR (DMSO-d6,
500 MHz): 5.27 (s, 2H, CH2), 5.80 (s, 2H, CH2),
7.25 (s, 1H, olefinic CH), 7.27–7.38 (m, 10H, Ar-H),
8.78 (s,1H, purine C2-H). 13C NMR (DMSO-d6,
125 MHz): d 47.9, 49.3, 101.3, 123.9, 127.4, 127.4,
127.5, 127.5, 128.1, 128.2, 128.2, 128.6, 128.9, 129.1,
129.1, 137.1, 137.2, 150.4, 151.5, 156.4, 163.8, 175.9,
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