3-Hydroxypyrimidine-2,4-diones as an Inhibitor Scaffold of HIV Integrase

Article abstract of DOI:10.1021/jm1014378

Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold.

Full text of DOI:10.1021/jm1014378

ARTICLE
pubs.acs.org/jmc
3-Hydroxypyrimidine-2,4-diones as an Inhibitor Scaffold of HIV
Integrase
Jing Tang,† Kasthuraiah Maddali,‡ Mathieu Metifiot,‡ Yuk Y. Sham,† Robert Vince,† Yves Pommier,‡ and
Zhengqiang Wang*,†
†Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
S Supporting Information
b
ABSTRACT: Integrase (IN) represents a clinically validated target
for the development of antivirals against human immunodeficiency
virus (HIV). Inhibitors with a novel structure core are essential for
combating resistance associated with known IN inhibitors (INIs). We
have previously disclosed a novel dual inhibitor scaffold of HIV IN
and reverse transcriptase (RT). Here we report the complete
structure-activity relationship (SAR), molecular modeling, and
resistance profile of this inhibitor type on IN inhibition. These studies
support an antiviral mechanism of dual inhibition against both IN and
RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor
scaffold.
’ INTRODUCTION
HIV infection and the establishment of proviral latency
depend critically on the insertion of viral DNA into host
genome.1-3 This integration process comprises two distinct
steps: 30 processing (30P), in which a sequence-specific endonu-
cleolytic cleavage of the viral DNA generates a CA-OH at the 30
end, and strand transfer (ST), where the processed viral DNA is
inserted into the host genome through a transesterification
reaction using the 30 CA-OH as the nucleophile.4 The virally
encoded enzyme IN catalyzes both reactions and thus represents
an important target for HIV chemotherapeutic intervention. IN
as an antiviral target is rendered particular attractive by the lack of
Figure 1. Structures of best known INIs (1-3) and a novel INI (4).
host cellular counterpart. On the other hand, IN uses the same
active site (DD35E motif) for both steps where either a viral
DNA (in 30P) or a cellular DNA (in ST) serves as the
endogenous substrate. Therefore, inhibitors of IN could also
benefit from a potentially high genetic barrier to resistance
development. In the past two decades, IN-targeted antiviral
research has led to the identification of various inhibitor types,
of which the most prominent scaffolds all feature a diketoacid
(DKA) functionality or its heterocyclic bioisosteres.5-9 These
chemotypes can accommodate highly selective ST inhibition and
have played an essential role in unraveling the pharmacophore
requirements for IN binding.10-15 Extensive research on these
chemotypes culminated with a number of investigational drugs
(Figure 1), including raltegravir (1),16,17 the first and only INI
presently approved for clinical use, elvitegravir (2),18 a promising
candidate in phase III development, and S/GSK-1349572 (3),19,20 a
second-generation IN inhibitor in phase IIb trials.21 In the
meantime, HIV integration also involves a cellular cofactor
LEDGF/p75.22-24 Targeting the protein-protein interaction
between IN and LEDGF/p75 represents an emerging approach
to HIV chemotherapeutic intervention.25 Collectively, inhibitors
targeting IN can substantially enhance the current highly active
antiretroviral therapy (HAART), the standard chemotherapy for
HIV/AIDS.26 However, mutations conferring resistance to these
inhibitors have been generated in cell culture and emerged in
clinical studies,27-29 suggesting that INIs will be confronted with
thesameresistanceissuethathasplaguedHIV/AIDSchemotherapy.
Continued search for structurally novel INIs to combat resis-
tance is therefore urgently needed. Previously, we have reported
that N-3 hydroxylation of pyrimidine-2,4-diones yielded inhibi-
tors dually active against HIV reverse transcriptase (RT) and IN
Received: November 9, 2010
Published: March 07, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
(4, Figure 1).30 Because this scaffold represents a novel chemo-
type for INIs, further SAR efforts on IN inhibition are warranted.
HIV IN is a 32 kDa protein encoded by viral pol gene consist-
ing of three functional domains:31 the N-terminal domain (NTD),
with a conserved “HH-CC”zinc-binding motif, the catalytic core
domain (CCD), containing the key D64-D116-E152 catalytic
triad, and the C-terminal domain (CTD), important for DNA
binding. Crystal structures of each single domain as well as double
domains have been resolved,32-34 albeit without the binding of
viral DNA substrate. Detailed understanding on HIV IN catalysis
requires structural information on a DNA-bound full-length IN
complex, or an intasome, which currently remains elusive due to
major technical barriers35 in obtaining stable HIV intasome.
Recently disclosed crystal structures of a homologous prototype
foamy virus (PFV) intasome36,37 have revealed the requisite
catalytic molecular network comprising IN, viral, and/or host
Scheme 1. Synthesis of Aromatic and Chelating Domainsa
a Reagents and conditions: (a) CH3C(OTMS)dNTMS (BSA), sub-
stituted benzyl alcohol, (HCHO)n, TMSCl, TBAI (cat.), CH2Cl2, rt,
70-90%; (b) Cs2CO3, THF, MeI, 40 °C, 53-63%; (c) NaH, m-CPBA,
THF, rt, 53-85%; (d) NaH, MSH, THF, rt, 54%.
Figure 2. SAR design of lead compound 4. Major structural domains
are highlighted.
Scheme 2. Synthesis of Linker Domaina
a Reagents and conditions: (a) LiAlH4, THF, rt, 67-72%; (b) CBr4, PPh3, THF, rt, 73-80%; (c) CH3C(OTMS)dNTMS (BSA), CH2Cl2; 19-21,
(HCHO)n, TMSCl, TBAI (cat.), rt, 83-89%; (d) NaH, m-CPBA, THF, rt, 62-72%; (e) BSA, benzyl bromide, chlorobenzene, microwave, 160 °C,
50%; (f) BSA, chloromethyl ethyl ether, TBAI (cat.), CH2Cl2, rt, 89%; (g) K2CO3, PhCOCH2Br, MeCN, reflux, 84%; (h) TFA:H2O (9:1), reflux, 76%;
(i) Cs2CO3, 4-F-Ph(CH2)nBr, DMF, 80 °C, 31-63%; (j) Mg, AcOH, MeOH, rt, 48-57%.
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Scheme 3. Synthesis of Hydrophobic Domaina
a Reagents and conditions: (a) NaH, m-CPBA, THF, rt 32-72%; (b) (i) CDI/toluene, reflux, then NH2OBn, reflux, (ii) NaOH/EtOH, reflux, 53%;
(c) benzyl bromide or 4-fluorobenzyl bromide, Cs2CO3, DMF, 80 °C, 58-67%; (d) HBr (48% in H2O), reflux, 77-78%.
DNA substrates and the inhibitor, allowing a largely improved
understanding on the mechanism of catalysis and inhibition of
retroviral IN. Homologousmodels38,39 constructed basedon these
PFV crystal structures have provided valuable details into HIV IN
mechanism of action and formed the basis for structure-based INI
design. Significantly these models corroborate the minimal phar-
macophore embedded in major INIs: a chelating triad capable of
binding two Mg2þ ions and a hydrophobic benzyl moiety. Our
novel IN inhibitors are constructed around a pyrimidine-2,4-dione
ring, a pharmacophore core that has found wide applications in
medicinal chemistry.40-43 Central to our design is to introduce an
OH group on the N-3 position which, along with the two flanking
carbonyl groups, would yield a chelating triad of C(O)N(OH)C-
(O) that mimics a DKA functionality (Figure 2, chelating
domain). On the other hand, the hydrophobic benzyl moiety
can be easily installed via an N-1 alkylation, and the mono- or di-
halogenation of this terminal benzyl group will allow us to explore
the halogen effect IN inhibition (Figure 2, aromatic domain).
Furthermore, to ensure that this benzyl group can fit in the
hydrophobic cavity, its spatial placement over the chelating triad
has to be optimized. This can be achieved through a linker of
different length (Figure 2, linker domain). Finally, the overall
hydrophobicity of the inhibitor could be modified by altering the
C5 and C6 substituents (Figure 2, hydrophobic domain).
’ RESULTS AND DISCUSSION
Chemistry. The chemical synthesis of aromatic and chelating
domains starts with a known 5,6-disubstituted pyrimidine-2,
4-dione 544,45 (Scheme 1). Subsequent selective N-1 alkylation
of 5 via a bis-silylated pyrimidine intermediate with an in situ
generated chloromethyl ether allows the incorporation of the
aromatic domain with an ether linkage (6-10, Scheme 1). As
previously reported,30 the key N-3 hydroxylation was achieved
via a base-mediated meta-chloroperbenzoic acid (m-CPBA) oxida-
tion. To assess the significance of this hydroxyl group on IN
inhibition, an N-3 amino analogue (15) as well as analogues with
an N-3 methyl or methoxy group were also synthesized (10 and
14, Scheme 1).
The synthesis of the linker domain is described in Scheme 2.
Analogues with an ether linkage were easily prepared through a
selective N-1 alkylation with a chloromethyl ether (Scheme 2B).
The requisite alcohols (19-21) were synthesized from com-
mercial carboxylic acids via a direct reduction with LAH. The
construction of all-carbon tethers in the linker domain, however,
proved much more challenging. The BSA-assisted selective N-1
alkylation of pyrimidine-2,4-diones generally involves a chloro-
methyl ether as the alkylating agent. The same reaction with benzyl
bromide was effected only under forcing conditions (microwave,
high temperature, and long reaction time; see Scheme 2C).
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Table 1. Effects of Aromatic and Chelating Domains on IN
Table 3. Effects of Hydrophobic Domain on IN Inhibition
Inhibition
IN IC50 (μM)a
compd R1
linker
R2
R3
30P
STb
IN IC50 (μM)a
11c
51
52
53
54
55
56
64
65
66
67
68
69
70
75
76
H
H
F
CH2OCH2 iPr
CH2OCH2 iPr
CH2OCH2 iPr
CH2OCH2 Et
CH2OCH2 Me
benzyl
>111 21 ( 2
4-F-benzyl >111 7.3 ( 0.8
4-F-benzyl >111 5.5 ( 0.7
compd
R1
R2
30P
STb
11c
12
13
4c
H
OH
OH
OH
OH
H
>111
>111
>111
>111
>111
>111
>111
>111
21 ( 2
4.8 ( 0.7
2.3 ( 0.6
3.5 ( 0.6
>111
H
H
H
F
benzyl
H
>111 8.2 ( 1.0
>111 75 ( 7
>111 36 ( 6
>111 11 ( 0.8
2,4-F
3-Cl-2-F
4-F
CH2OCH2 benzyl
CH2OCH2 benzyl
Et
Et
9
4-F
H
F
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
iPr
iPr
iPr
Et
4-F-benzyl >111 32 ( 6
4-F-benzyl >111 73 ( 12
10
14
15c
4-F
Me
>111
4-F
OMe
NH2
>111
H
H
F
benzyl
benzyl
benzyl
H
>111 28 ( 2
>111 >111
>111 24 ( 3
>111 >111
>111 >111
>111
H
>111
a Concentration inhibiting enzymatic function by 50%. b Mean value (
Et
standard deviation from triplicate experiments. c Reference 30.
H
F
Me
Me
H
Table 2. Effects of Linker Domain on IN Inhibition
H
benzo (fused) >111
F
benzo (fused) >111
>111
a Concentration inhibiting enzymatic function by 50%. b Mean value (
standard deviation from triplicate experiments. c Reference 30.
IN IC50 (μM)a
compd
linker
30P
STb
4c
CH2OCH2
(CH2)2OCH2
(CH2)3OCH2
(CH2)4OCH2
CH2
>111
>111
>111
>111
>111
>111
>111
>111
3.5 ( 0.6
1.6 ( 0.2
3.2 ( 0.5
4.5 ( 0.4
22 ( 0.5
46 ( 6
28
29
30
32
42
43
44
(CH2)2
(CH2)3
0.44 ( 0.1
0.56 ( 0.04
(CH2)4
a Concentration inhibiting enzymatic function by 50%. b Mean value (
standard deviation from triplicate experiments. c Reference 30.
Figure 3. Dose response curves of compounds 4, 9-10 and 12-14.
The synthesis of hydrophobic domain with variable C-5 and
C-6 substituents (Scheme 3A) was achieved through a similar
N-3 hydroxylation of N-1 alkylated intermediates (45-50 and
57-63). Compounds with a slightly different scaffold, the 5,
6-benzopyrimidine-2,4-diones (75-76), were also prepared to
probe the effect of a fused benzene ring on IN inhibition. These
analogues were synthesized via a route depicted in Scheme 3C.
SAR on IN Inhibition. All synthetic analogues were first tested
against recombinant HIV-1 IN using a gel assay. Both 30P and ST
functions were evaluated. As clearly shown in Tables 1-3, our
scaffold tends to selectively inhibit ST as none of the analogues
demonstrated inhibitory activity against 30P.
Generic alkyl halides fail to undergo alkylation with the bis-
silylated pyrimidine intermediate, and base-mediated direct
alkylation normally leads to a mixture of region isomers. As it
turned out, the introduction of all-carbon chain into N-1 requires
multiple protection and deprotection steps (Scheme 2D). In this
event, the N-1 position was first protected with an easily introduced
ethoxymethyl group, which allowed the protection of N-3 position
with an acetophenone group. The N-1 ether linkage was then cleaved
under acidic condition to yield the N-3 protected intermediate
35. Base-mediated direct alkylation led to the incorporation of
the carbon tethers followed by a Mg mediated N-3 deprotection
to yield key N-1 alkylated intermediates 39-41. All N-1 alkylated
pyrimidine-2,4-diones were then oxidized with m-CPBA to
produce desired N-3 hydroxylated compounds (Scheme 2).
Aromatic and Chelating Domains. We have shown previously30
that removing the phenyl ring at the end of the N-1 pendant
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Figure 4. Docking of raltegravir (1, A) and inhibitor 43 (B) in HIV-1 IN CCD in complex with Mg2þ and DNA. The 3-N hydroxyl group
simultaneously chelates to both Mg2þ ions while allowing the placement of the benzyl group into the protein-DNA interfacial hydrophobic pocket.
resulted in a significant loss of inhibition against IN and that
without the OH group the N(3)H analogue is completely
inactive against IN. These findings corroborate the pharmaco-
phore hypothesis that HIV-1 IN inhibition requires minimally a
chelating domain and an aromatic domain. In this study, addi-
tional analogues were synthesized to further study these two
domains. As shown in Table 1 and Figure 3, halogen substitution
of the N-1 benzyl seems to significantly benefit IN binding. A
fluorination at the para position (inhibitor 4) leads to a 6-fold
improvement in IN inhibition over the unsubstituted compound
11. Further improvement in activity was only marginal with a
3-chloro-2-fluoro substituted analogue 13, whereas a 2,4-difluoro
compound 12 showed slightly reduced inhibition when com-
pared to 4 (Table 1). On the other hand, replacing the N-3 OH
group with H, Me, OMe, or even NH2 yielded compounds
completely devoid of inhibitory activity against IN (9-10 and
14-15, Table 1), confirming the critical requirement of the N-3
OH for chelating Mg2þ. The lack of potency with the amino
analogue 15 also reflects an inferior chelating ability of NH2 for
Mg2þ when compared to OH.30
The Linker Domain. Having established the chelating triad and
the N-1 benzyl group as the two major structural determinants of
our novel INI scaffold, our SAR efforts were then centered
around the linker domain as it dictates the angle and distance
between the chelator and benzyl group. Toward this end, we have
designed linkers of different nature and length. Compounds 4
and 28-30 all have an ether linkage with incremental methylene
groups (Table 2). Interestingly, these analogues all exhibited
inhibitory activity in ST assay at low micromolar range, and
demonstrated a discernible binding dependence on the length of
the linker. As shown in Table 2, the 4-atom linker (CH2)2OCH2
(inhibitor 28) conferred the highest inhibition within this series
and a reduced inhibition was achieved with compounds having a
shorter (4) or longer linker (29-30). A similar albeit more sensitive
trend was observed with compounds having an all carbon N-1
linker. In this case, short linkages, such as CH2 (32) and (CH2)2
(42) appeared to pose severe binding difficulties while the 3- and
4-atom linkers (CH2)3 (43) and (CH2)4 (44) conferred a
drastically improved inhibition against ST (Table 2). These
findings strongly imply that the optimal IN inhibition entails a
linker of 3-4 atoms. The nature of the linker (ether linkage vs all-
carbon linkage) was also found to substantially impact IN
inhibition as inhibitors 43 and 44 demonstrated significantly
better inhibition than compound 28. Detailed docking studies on
the inhibitor binding may provide a molecular basis for this
observation.
The Hydrophobic Domain. Synthetic analogues in these
domains vary mainly in the size of the C-5 alkyl group (iPr, Et,
and Me) and fluorine substitution on the C-6 benzyl. These
changes impact most likely the steric hindrance and the overall
hydrophobicity of these inhibitors. Interestingly, a C-5 ethyl
group was found to confer significantly better inhibition than
isopropyl group (53 vs 11, Table 3), indicating that less steric
hindrance at C-5 could benefit IN binding. However, a reversed
effect was observed in compounds with an N-1 linker of CH2,
wherein a C-5 ethyl substitution (67) caused a complete loss of
inhibition when compared to a C-5 isopropyl substitution (66).
On the other hand, swapping C5 and C6 substituents of inhibitor
53 seems to harm IN binding (55 vs 53) as the IC50 value
increased by 4-fold. Notably, a para-fluoro substitution at the C-6
benzyl clearly favors IN inhibition (53 vs 11, 56 vs 55). With a
more dramatic change, a C-5 and C-6 fused benzene ring was
found to render compounds 75-76 completely inactive against
IN inhibition (Table 3). These findings indicate that the hydro-
phobic domain affects IN binding in a less comprehensible way
and changes in this domain impact the activity most likely in
concert with the linker domain.
Modeling. The common mode of inhibitory binding was
explored through molecular docking using a homologous HIV
IN model38 constructed based on the crystal structure of proto-
type foamy virus (PFV) IN.36 Significantly, the new inhibitor 43
fits perfectly into the IN binding site through two major binding
domains also featured in raltegravir (1): the chelation of two
Mg2þ ions and the placement of the 4-F-benzyl group into the
protein-DNA interfacial hydrophobic pocket (Figure 4). The
high degree of similarity shown between the binding of 1 and 43
confirms that our new molecular scaffold can effectively engage
with HIV IN. Interestingly, while residue Y143 contributes
siginificantly to binding of 1 through a π-stacking interaction
(Figure 4A), the same interaction was not observed with our
inhibitor 43 (Figure 4B), suggesting that our inhibitor could be
less susceptible to resistance associated with Y143 mutation. It is
also noteworthy that the linker domain is situated right in the
hydrophobic cleft between residue P145 and viral DNA nucleo-
bases. Therefore, an ether linkage may provide a less favorable
hydrophobic interaction than an all-carbon linker, thus the observed
reduced inhibition with the ether linkage (Table 2, 43 vs 28).
Further validation was achieved by correlating the SAR of
docked inhibitors 4, 11, 28, 29, 30, 32, 42, 43, 44, and 66 as
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Table 5. Fold Resistance of Inhibitors 4 and 13 against IN
Mutants
fold resistancea,b
IN mutants
4c
13
1
G140S/Q148H
Y143R
10/13
7/4
14/17
7/5
148/139
24/28
10/8
N155H
5/6
5/7
a Fold resistance is defined by IC50(mutant)/IC50(WT). b Values from two
independent experiments. c Reference 30.
Table 6. Fold Resistance of Inhibitors 4 and 13 against
Raltegravir-Resistant HIV-1 Strains
Figure 5. Strong correlation of docked 3-hydroxypyrimidine-2,4,-
diones analogues supports a common mode of binding for HIV IN STI
design.
fold resistancea
viruses
major mutations
4b
13c
1
Table 4. Anti-HIV-1 Activity of IN Inhibitors
8070-2
1556-1
4736-2
G140S/Q148H
Y143C
7.3
5.9
3.1
>2.0
>1.7
>5.1
>855
>855
single concentration (10 μM)
dose response
N155H
149
a Fold resistance is defined by EC50(mutant)/EC50(WT)
.
b Reference 30.
CPE
reduction (%)a
cell
EC50 CC50
(μM)b (μM)c
c Toxicity observed (TC50 = 2.0 μM).
compd
viability (%)
TId
4e
100
100
100
100
27
100
100
100
100
83
0.024
0.0080
2.1
>20
>20
>100
>100
ND
ND
ND
ND
ND
ND
ND
>20
>833
>2500
>47
11e
12
13
28
29
30
43
44
49
50
51
IN inhibition in biochemical assays, all-carbon linkers lack the
proper physicochemical properties for the compounds to reach
their target in cell culture. By contrast, with the exception of
compounds having a para-fluorine at the C-6 benzyl group
(49-50, Table 4), INIs with an ether linkage of CH2OCH2 (4,
11-13, and 51) showed antiviral activity at the low micro-
molar to low nanomolar range without appreciable cytotoxi-
city (Table 4). Longer ether linkages that produced excellent
anti-IN activity for compounds 28-30 did not generate
similar activity in the antiviral assay, further demonstrating
that additional CH2s in the linker domain hinder antiviral
activity.
Resistance. To establish the resistance profile of our inhibi-
tors, we first tested two representative inhibitors 4 and 13 against
HIV IN mutants. In this study, major mutants associated with
raltegravir resistance,23,24 namely a G140S/Q148H double mu-
tant and two single mutants Y143R and N155H were employed.
The results are summarized in Table 5. Notably, the G140S/
Q148H double mutant which causes high resistance to raltegravir
(1) yielded a much lower fold resistance to 4 and 13, whereas the
two single mutants confer comparable resistance to 4, 13 and 1,
suggesting a potentially common binding mode for these
inhibitors.
On the basis of these biochemical data, inhibitors 4 and 13
were further evaluated in cell culture against raltegravir-resistant
HIV strains. Unlike the initial antiviral assay wherein HIV-1 IIIB
was used in CEM-SS cells, the resistance studies were carried out
with NL4-3 based viral strains in MT-4 cells. Selected strains
contain major mutations corresponding to the IN mutants
(Table 6 vs Table 5). As shown in Table 6, all three viral strains
show a high level resistance to raltegravir (1). Interestingly,
although likely within experimental variations, the small fold-
resistances observed with inhibitor 4 (Table 6) correlate closely
with resistance observed with IN mutants in biochemical assays
(Table 5). The testing of inhibitor 13 was complicated by its
0.95
NDf
ND
ND
ND
ND
ND
ND
1.1
>105
30
92
12
88
12
97
6.0
48
94
100
92
10
100
100
>18
a Reduction of viral cytopathic effect, indicating inhibitory activity of a
compound. b Concentration inhibiting virus replication by 50%. c Con-
centration resulting in 50% cell death. d Therapeutic index, defined by
CC50/EC50. e Reference 30. f Not determined.
shown in Figure 5. The outstanding correlation observed be-
tween these two sets of IC50 data (expt vs calcd, correlation
coefficiency = 0.82) supports our docking model with a common
mode of binding as a valid platform for HIV IN inhibitor design.
Antiviral Assay. To evaluate the antiviral activity of newly
synthesized INIs, all compounds with an IC50 of 10 μM or lower
from IN ST assay were tested against HIV-1IIIB in CEM-SS cells
using an assay based on viral cytopathic effect (CPE). In this
assay, the reduction of CPE was used to indicate the antiviral
activity of a compound. All selected compounds were first screened
under a single concentration (10 μM). Inhibitors with excellent
CPE reduction and cell viability from this screening assay were
further tested in a dose response fashion, from which the EC50
and CC50 were determined. Table 4 summarizes the antiviral
testing results.
Remarkably, compounds 43 and 44, the two most potent INIs
from IN ST assay with submicromolar IC50s, have shown only
marginal activity against HIV-1 at 10 μM (Table 4). This finding
strongly suggests that despite their ability to confer exceptional
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toxicity observed in MT-4 cells. Notably the same compound did
not show toxicity in CEM-SS cells (TC50 > 100 μM, Table 4). On
the other hand, antiviral assay with two HIV-1NL4-3 strains
containing major mutations associated with NNRTI resistance,
K103N or Y181C, revealed a fold-resistance of 16 and 48,
respectively, to inhibitor 4.46 These results suggest that the
overall antiviral activity of 4 could reflect a concerted yet not
necessarily balanced inhibition to both RT and IN.
1H NMR (600 MHz, CDCl3) δ 7.28 (m, 6H), 7.20 (m, 2H), 7.00 (d, J =
7.8 Hz, 2H), 5.14 (s, 2H), 4.59 (s, 2H), 4.10 (s, 2H), 2.79 (septet, J = 6.6
Hz, 1H), 1.21 (d, J = 6.6 Hz, 6H). MS (ESIþ) m/z: 365.17 (M þ 1).
6-Benzyl-1-((2,4-difluorobenzyloxy)methyl)-5-isopropylpyrimidine-
2,4(1H,3H)-dione (7). This compound was prepared as a white solid
following the procedure described for the preparation of 6; yield 88%.
1H NMR (600 MHz, CDCl3) δ 9.32 (s, 1H), 7.34 (m, 3H), 7.26 (t, J =
7.8 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 6.86 (t, J = 9.0 Hz, 1H), 6.81 (d, J =
9.0 Hz, 1H), 5.20 (s, 2H), 4.64 (s, 2H), 4.16 (s, 2H), 2.87 (m, 1H), 1.28
(d, J = 6.6 Hz, 6H). MS (ESI-) m/z: 399.42 (M - 1).
’ CONCLUSION
6-Benzyl-1-((3-chloro-2-fluorobenzyloxy)methyl)-5-isopropylpyri-
midine-2,4(1H,3H)-dione (8). This compound was prepared as a white
solid following the procedure described for the preparation of 6; yield
90%. 1H NMR (600 MHz, CDCl3) δ 8.96 (b, 1H), 7.36 (m, 3H), 7.33 (t,
J = 7.8 Hz, 2H), 7.10 (m, 3H), 5.21 (s, 2H), 4.69 (s, 2H), 4.16 (s, 2H), 2.87
(m, 1H), 1.28 (d, J = 6.6 Hz, 6H). MS (ESI-) m/z: 415.87 (M - 1).
6-Benzyl-1-((4-fluorobenzyloxy)methyl)-5-isopropylpyrimidine-2,
4(1H,3H)-dione (9). This compound was prepared as a white solid
following the procedure described for the preparation of 6; yield 89%.
1H NMR (600 MHz, CDCl3) δ 8.97 (b, 1H), 7.34 (t, J = 7.2 Hz, 2H),
7.30 (m, 3H), 7.07 (d, J = 7.8 Hz, 2H), 7.03 (m, 2H), 5.20 (s, 2H), 4.61
(s, 2H), 4.16 (s, 2H), 2.88 (septet, J = 7.2 Hz, 1H), 1.28 (d, J = 7.2 Hz,
6H). MS (ESIþ) m/z: 383.18 (M þ 1).
Through complete SAR and docking studies we have demon-
strated that the N-3 OH group and the 4-F-benzyl moiety on
the N-1 side chain are the key structural determinants for
IN inhibition, and that the linker domain sits in a hydrophobic
cleft between DNA nucleobases and P145. Therefore, both the
nature and the length of the linker greatly impact IN binding,
with the optimal IN inhibition entailing a 3-4 atom all-carbon
linkage. However, studies in cell culture revealed that inhibi-
tors with an all-carbon linker generally lack antiviral activity,
presumably due to unfavored physicochemical properties.
Resistance studies with raltegravir-resistant IN mutants as
well as HIV-1NL4-3 viral strains demonstrated low level resis-
tances toward inhibitors 13 and 4, likely due to their anti-RT
activity. In the end, compounds (4, 11, and 13) featuring a
CH2OCH2 linker demonstrate low micromolar IN inhibition
and nanomolar anti-HIV activity, and are suitable for further
development.
6-Benzyl-1-((4-fluorophenethoxy)methyl)-5-isopropylpyrimidine-2,
4(1H,3H)-dione (25). This compound was prepared as a white solid
following the procedure described for the preparation of 6; yield 89%.
1H NMR (600 MHz, CDCl3) δ 8.89 (b, 1H), 7.27 (t, J = 7.2 Hz, 2H),
7.20 (m, 1H), 7.08(m, 2H), 6.99 (d, J = 7.8 Hz, 2H), 6.91 (t, J = 8.4 Hz,
2H), 5.04 (s, 2H), 3.94 (s, 2H), 3.71 (t, J = 6.6 Hz, 2H), 2.74 (m, 3H),
1.20 (d, J = 6.6 Hz, 6H). MS (ESI-) m/z: 395.45 (M - 1).
6-Benzyl-1-((3-(4-fluorophenyl)propoxy)methyl)-5-isopropylpyri-
midine-2,4(1H,3H)-dione (26). This compound was prepared as a white
solid following the procedure described for the preparation of 6; yield
83%. 1H NMR (600 MHz, CDCl3) δ 8.97 (b, 1H), 7.29 (t, J = 7.8 Hz,
2H), 7.21 (t, J = 7.8 Hz, 1H), 7.05 (m, 4H), 6.89 (t, J = 9.0 Hz, 2H), 5.05
(s, 2H), 4.11 (s, 2H), 3.50 (t, J = 6.0 Hz, 2H), 2.87 (septet, J = 7.2 Hz,
1H), 2.56 (t, J = 7.8 Hz, 2H), 1.78 (m, 2H), 1.23 (d, J = 6.6 Hz, 6H). MS
(ESI-) m/z: 409 (M - 1).
6-Benzyl-1-((4-(4-fluorophenyl)butoxy)methyl)-5-isopropylpyrimi-
dine-2,4(1H,3H)-dione (27). This compound was prepared as a white
solid following the procedure described for the preparation of 6; yield
84%. 1H NMR (600 MHz, CDCl3) δ 10.01 (b, 1H), 7.34 (t, J = 7.8 Hz,
2H), 7.26 (t, J = 7.2 Hz, 1H), 7.10 (m, 4H), 6.94 (d, J = 9.0 Hz, 2H), 5.23
(s, 2H), 4.16 (s, 2H), 3.58 (t, J = 6.6 Hz, 2H), 2.86 (septet, J = 7.2 Hz,
1H), 2.58 (t, J = 7.2 Hz, 2H), 1.63 (m, 2H), 1.56 (m, 2H), 1.27 (d, J =
7.2 Hz, 6H). MS (ESI-) m/z: 423.51 (M - 1).
’ EXPERIMENTAL SECTION
Chemistry. General Procedures. All commercial chemicals were
used as supplied unless otherwise indicated. Dry solvents (THF, Et2O,
CH2Cl2, and DMF) were dispensed under argon from an anhydrous
solvent system with two packed columns of neutral alumina or molecular
sieves. Flash chromatography was performed on a Teledyne Combiflash
RF-200 with RediSep columns (silica) and indicated mobile phase. All
reactions were performed under inert atmosphere of ultrapure argon
with oven-dried glassware. 1H and 13C NMR spectra were recorded on a
Varian 600 MHz spectrometer. Mass data were acquired on an Agilent
TOF II TOS/MS spectrometer capable of ESI and APCI ion sources.
Analysis of sample purity was performed on a Varian Prepstar SD-1
HPLC system with a Varian Microsorb-MW 100-5 C18 column (250
mm ꢀ 4.6 mm): solvent A = H2O, solvent B = MeCN; flow rate =
1.0 mL/min; method: linear 30-95% (B) over 25 min. All tested
compounds have a purity g96%.
6-Benzyl-1-(ethoxymethyl)-5-isopropylpyrimidine-2,4(1H,3H)-dione
6-Benzyl-1-(benzyloxymethyl)-5-isopropylpyrimidine-2,4(1H,3H)-
dione (6).44 To a suspension of paraformaldehyde (144 mg, 4.80 mmol)
in TMSCl (2.0 mL) was added benzyl alcohol (518 mg, 4.80 mmol) at
room temperature. The reaction mixture was stirred until a clear solution
was formed. The solution was concentrated under reduced pressure to
give benzyl chloromethyl ether as oil, which was taken to the next step
without further purification. To a suspension of pyrimidine 5 (976 mg
4.00 mmol) in 16 mL of anhydrous DCM was added BSA (2.16 mL, 8.80
mmol) at room temperature. The resulting mixture was stirred until a
clear solution was achieved. The freshly prepared benzyl chloromethy
ether was added to this solution followed by the addition of a catalytic
amount of TBAI. The reaction mixture was kept overnight and then
quenched by adding a saturated aqueous solution of NaHCO3. The
aqueous phase was extracted with CH2Cl2 (20 mL ꢀ 3). The combined
organic extracts were dried over Na2SO4 and concentrated under
reduced pressure. The resultant residue was subjected to combiflash
(Hex/EtOAc, 2:1) to afford compound 6 (1.25 g, 86%) as a white solid.
(33). This compound was prepared as a white solid following the procedure
described for the preparation of 6; yield 89%. H NMR (600 MHz,
CDCl3) δ 8.68 (b, 1H), 7.29 (t, J = 7.8 Hz, 2 H), 7.22 (m, 2H), 7.06 (d,
J = 7.8 Hz, 1H), 5.05 (s, 2H), 4.11 (s, 2H), 3.56 (q, J = 7.2 Hz, 2H), 2.81
(septet, J = 7.2 Hz, 1H), 1.22 (d, J = 6.6 Hz, 6H), 1.11 (t, J = 7.2 Hz, 3H);
MS (ESI-) m/z: 301.37 (M - 1).
1
1-(Benzyloxymethyl)-5-methylpyrimidine-2,4(1H,3H)-dione (48).
This compound was prepared as a white solid following the procedure
1
described for the preparation of 6; yield 82%. H NMR (600 MHz,
CDCl3) δ 9.21 (b, 1H), 7.34 (m, 5H), 7.10 (s, 1H), 5.21 (s, 2H), 4.66 (s,
2H), 4.62 (s, 2H), 1.91 (s, 3H). MS (ESI-) m/z: 245.10 (M - 1).
5-Benzyl-1-(benzyloxymethyl)-6-ethylpyrimidine-2,4(1H,3H)-dione
(49). This compound was prepared as a white solid following the procedure
1
described for the preparation of 6; yield 78%. H NMR (600 MHz,
CDCl3) δ 9.64 (s, 1H), 7.31 (m, 4H), 7.24 (m, 3H), 6.99 (m, 3H), 5.43
(s, 2H), 4.67 (s, 2H), 4.08 (s, 2H), 2.73 (q, J = 6.6 Hz, 2H), 1.06 (t, J =
6.6 Hz, 3H). MS (ESI-) m/z: 349.62 (M - 1).
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5-Benzyl-6-ethyl-1-((4-fluorobenzyloxy)methyl)pyrimidine-2,4(1H,3H)-
dione (50). This compound was prepared as a white solid following the
procedure described for the preparation of 6; yield 80%. 1H NMR (600
MHz, CDCl3) δ 9.51 (s, 1H), 7.29 (t, J = 7.2 Hz, 4H), 7.20 (d, J = 6.6 Hz,
2H), 7.00 (t, J = 6.6 Hz, 2H), 5.41 (s, 2H), 4.62 (s, 2H), 4.08 (s, 2H),
2.72 (q, J = 6.6 Hz, 2H), 1.07 (t, J = 6.6 Hz, 3H). MS (ESI-) m/z:
367.15 (M - 1).
(dd, J = 21.2 Hz), 72.8, 70.9, 33.5, 28.2, 20.4. HRMS (ESI-) calcd for
C22H23FN2O4 [M - H]- 399.1715, found 399.1705 (E = 2.4 ppm).
6-Benzyl-1-(benzyloxymethyl)-3-hydroxy-5-isopropylpyrimidine-2,
4(1H,3H)-dione (11). This compound was prepared as a white solid
following the procedure described for the preparation of 4; yield 53%.
1H NMR (600 MHz, CDCl3) δ 7.32-7.30 (m, 7H), 7.02 (J = 7.2 Hz,
2H), 5.30 (s, 2H), 4.67 (s, 2H), 4.19 (s, 2H), 2.90 (septet, J = 7.2 Hz,
1H), 1.28 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 162.2,
151.9, 148.4, 137.3, 135.3, 129.1, 128.5, 128.0, 127.8, 127.3, 127.2, 119.8,
73.0, 71.8, 33.5, 28.2, 20.4. HRMS (ESI-) calcd for C22H24N2O4 [M -
H]- 381.1809, found 381.1803 (E = 1.5 ppm).
6-Benzyl-1-((2,4-difluorobenzyloxy)methyl)-3-hydroxy-5-isopropyl-
pyrimidine-2,4(1H,3H)-dione (12). This compound was prepared as a
white solid following the procedure described for the preparation of 4;
yield 73%. 1H NMR (600 MHz, CDCl3) δ 7.18 (m, 1H). 7.20 (m, 2H),
7.14 (t, J = 7.2 Hz, 1H), 6.97 (d, J = 7.8 Hz, 2H), 6.73 (td, J = 1.8, 9.6 Hz,
1H), 6.67 (td, J = 1.8, 9.6 Hz, 1H), 5.13 (s, 2H), 4.55 (s, 2H), 4.04 (s,
2H), 2.75 (m, 1H), 1.13 (d, J = 6.6 Hz, 6H). 13C NMR (150, CDCl3) δ
163.8 (d, J = 11.7 Hz), 162.1(dd, J = 7.8, 19.5 Hz), 160.4 (d, J = 11.7 Hz),
148.3, 135.2, 131.5 (d, J = 7.8 Hz), 129.2, 127.2, 120.4 (d, J = 15.6 Hz),
120.0, 111.2 (dd, J = 3.9, 21.2 Hz), 104.1 (t, J = 25.2 Hz), 72.7, 64.9, 33.4,
28.3, 20.3. HRMS (ESI-) calcd for C22H22F2N2O4 [M - H]-
415.1475, found 415.1473 (E = 0.45 ppm).
6-Benzyl-1-((3-chloro-2-fluorobenzyloxy)methyl)-3-hydroxy-5-iso-
propylpyrimidine-2,4(1H,3H)-dione (13). This compound was prepared
as a white solid following the procedure described for the preparation of
4; yield 85%. 1H NMR (600 MHz, CD3OD) δ 7.32 (t, J = 6.6 Hz, 1H),
7.23 (m, 3H), 7.18 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.00 (d, J =
7.8 Hz, 2H), 5.29 (s, 2H), 4.67 (s, 2H), 4.11 (s, 2H), 2.87 (m, 1H), 1.22
(d, J = 7.2 Hz, 6H). 13C NMR (150, CDCl3) 163.7 (d, J = 11.7 Hz),
162.1 (t, J = 11.7 Hz), 160.4 (t, J = 11.7 Hz), 159.7, 144.2, 135.5, 131.8
(d, J = 11.7 Hz), 129.1, 127.3(t, J = 16.2 Hz), 120.5 (d, J = 11.1 Hz),
118.2, 116.6, 111.1 (dd, J = 3.5, 21.3 Hz), 103.9 (t, J = 25.1 Hz), 72.9, 65.5,
33.4, 28.3, 20.4. HRMS (ESI-) calcd for C22H22ClFN2O4 [M - H]-
431.1179, found 431.1181 (E = -0.83 ppm).
6-Benzyl-1-((4-fluorophenethoxy)methyl)-3-hydroxy-5-isopropyl-
pyrimidine-2,4(1H,3H)-dione (28). This compound was prepared as a
white solid following the procedure described for the preparation of 4;
yield 66%. 1H NMR (600 MHz, CDCl3) δ 7.34 (t, J = 7.2 Hz, 2H), 7.29
(d, J = 7.2 Hz, 1H), 7.14 (m, 2H), 7.03 (d, J = 7.8 Hz, 2H), 6.97 (t, J = 8.4
Hz, 2H), 5.19 (s, 2H), 4.04 (s, 2H), 3.82 (t, J = 6.6 Hz, 2H), 2.88 (septet,
J = 7.2 Hz, 1H), 2.81 (t, J = 6.6 Hz, 2H), 1.28 (d, J = 6.6 Hz, 6H). 13C
NMR (150 MHz, CDCl3) δ 162.5 (d, J = 16.2 Hz), 160.7, 155.5, 152.1,
148.4, 135.4, 134.2 (d, J = 2.9 Hz), 130.3 (d, J = 7.8 Hz), 129.2, 127.2,
119.8, 115.2 (d, J = 21.2 Hz), 72.9, 70.0, 35.1, 33.3, 28.2, 20.4. HRMS
(ESIþ) calcd for C23H25FN2O4 [M þ H]þ 413.1871, found 413.1868
(E = 0.76 ppm).
6-Benzyl-1-((4-fluorobenzyloxy)methyl)-5-isopropyl-3-methylpyr-
imidine-2,4(1H,3H)-dione (10). A mixture of compound 9 (60 mg, 0.16
mmol), methyl iodide (27 mg, 0.19 mmol), and Cs2CO3 (61 mg, 0.19
mmol) in 2 mL of THF was stirred under 40 °C for 3 h and then allowed
to cool to room temperature. The reaction mixture was quenched by
adding 5 mL of H2O and extracted with EtOAc (10 mL ꢀ 3). The
combined organic phases were dried over Na2SO4 and then concen-
trated under reduced pressure. The resultant residue was purified with
Combiflash to give compound 10 (40 mg, 63%) as clean oil. 1H NMR
(600 MHz, CDCl3) δ 7.25 (t, J = 7.8 Hz, 2H), 7.21 (m, 3H), 6.99 (d, J =
7.2 Hz, 2H), 6.95 (t, J = 8.4 Hz, 2H), 5.16 (s, 2H), 4.53 (s, 2H), 4.09 (s,
2H), 3.28 (s, 3H), 2.87 (septet, J = 7.2 Hz, 1H), 1.22 (d, J = 7.2 Hz, 6H).
13C NMR (150 MHz, CDCl3) δ 163.2, 161.8, 161.5, 152.6, 146.0, 135.4,
133.2 (d, J = 3.3 Hz), 129.6 (d, J = 7.8 Hz), 129.2, 127.3 (d, J = 7.4 Hz),
118.9, 115.4 (d, J = 21.2 Hz), 73.7, 71.0, 33.4, 28.2, 27.9, 20.4. MS (ESI-)
m/z: 395.45 (M - 1).
6-Benzyl-1-((4-fluorobenzyloxy)methyl)-5-isopropyl-3-methoxypy
rimidine-2,4(1H,3H)-dione (14). This compound was prepared follow-
1
ing the procedure described for the preparation of 10; yield 53%. H
NMR (600 MHz, CDCl3) δ 7.27 (t, J = 8.4 Hz, 2H), 7.24 (m, 3H), 6.99
(d, J = 7.2 Hz, 2H), 6.96 (t, J = 8.4 Hz, 2H), 5.14 (s, 2H), 4.55 (s, 2H),
4.08 (s, 2H), 3.94 (s, 3H), 2.85 (septet, J = 7.2 Hz, 1H), 1.22 (d, J = 7.2 Hz,
6H). MS (ESI-) m/z: 411.45 (M - 1).
3-Amino-6-benzyl-1-(benzyloxymethyl)-5-isopropylpyrimidine-2,
4(1H,3H)-dione (15).30 To a solution of 6 (60 mg, 0.16 mmol) in l.0 mL
of THF was added NaH (31 mg, 0.82 mmol) at 0 °C. This reaction
mixture was allowed to warm to room temperature over 1 h and then
cooled to 0 °C followed by the addition of O-(mesitylsulfonyl)hydroxyl-
amine47 (MSH, 66 mg, 0.32 mmol). After stirring for 0.5 h, this reaction
mixture was stirred at rt overnight. The reaction was quenched by adding
10 mL of H2O; the aqueous phase was then extracted with ethyl acetate
(10 mL ꢀ 3). The combined organic extracts were dried over Na2SO4
and concentrated under reduced pressure. The resultant residue was
subjected to Combiflash (Hex/EtOAc, 1:1) to give compound 15 (22 mg,
1
54% based on consumed start material) as a white solid. H NMR
(600 MHz, CDCl3) δ 7.33-7.26 (m, 8H), 7.04-7.03 (d, J = 7.2 Hz,
2H), 5.24 (s, 2H), 4.66 (s, 2H), 4.19 (s, 2H), 2.90 (septet, J = 7.2 Hz, 1H),
1.29 (d, J = 6.6 Hz, 6H). HRMS (ESI-) calcd for C22H25N3O3 [M - H]-
380.1969, found 380.1976 (E = -1.9 ppm).
6-Benzyl-1-((4-fluorobenzyloxy)methyl)-3-hydroxy-5-isopropylpyri
midine-2,4(1H,3H)-dione (4).47 To a solution of 9 (100 mg, 0.26 mmol)
in 6 mL of THF was added NaH (31 mg, 1.30 mmol) at 0 °C. This
reaction mixture was allowed to warm to room temperature over 1 h and
then cooled to 0 °C followed by the addition of m-CPBA (135 mg, 0.79
mmol). This reaction mixture was allowed to warm to room temperature
and stirred overnight. After the reaction was quenched by adding 10 mL
of H2O, the aqueous phase was acidified with 1N HCl to pH = 7 and
then extracted with ethyl acetate (10 mL ꢀ 3). The combined organic
extracts were dried over Na2SO4 and concentrated under reduced pressure.
The resultant residue was subjected to Combiflash (Hex/EtOAc, 1:1) to
give compound 4 (38 mg, 72% based on consumed start material) as
6-Benzyl-1-((3-(4-fluorophenyl)propoxy)methyl)-3-hydroxy-5-iso-
propylpyrimidine-2,4(1H,3H)-dione (29). This compound was prepared
as a white solid following the procedure described for the preparation of
4; yield 71%. 1H NMR (600 MHz, CDCl3) δ 7.33 (m, 2H), 7.26 (m,
1H), 7.07 (m, 2H), 7.01 (m, 2H), 6.95 (m, 2H), 5.19 (s, 2H), 4.18 (s, 2H),
3.58 (m, 2H), 2.89 (septet, J = 7.2 Hz, 1H), 2.60 (m, 2H), 1.82 (m, 2H),
1.29 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 162.9, 162.1,
160.5, 152.4, 148.5, 137.2 (d, J = 3.3 Hz), 135.4, 129.7 (d, J = 7.8 Hz),
129.2, 127.3 (d, J = 7.8 Hz), 119.9, 115.2 (d, J = 20.6 Hz), 73.0, 68.4,
33.5, 31.3, 31.1, 28.2, 20.4. HRMS (ESIþ) calcd for C24H27FN2O4
[M þ H]þ 427.2028, found 427.2026 (E = 0.38 ppm).
6-Benzyl-1-((4-(4-fluorophenyl)butoxy)methyl)-3-hydroxy-5-iso-
propylpyrimidine-2,4(1H,3H)-dione (30). This compound was prepared
as a white solid following the procedure described for the preparation of
4; yield 64%. 1H NMR (600 MHz, CDCl3) δ 7.34 (t, J = 7.2 Hz, 2H),
7.28 (d, J = 7.2 Hz, 1H), 7.11 (dd, J = 1.8, 7.8 Hz, 2H), 7.07 (d, J = 7.8 Hz,
2H), 6.96 (t, J = 8.4 Hz, 2H), 5.21 (s, 2H), 4.19 (s, 2H), 3.60 (t, J = 6.0
1
a white solid. H NMR (600 MHz, CDCl3) δ 7.33-7.26 (m, 5H),
7.04-6.99 (m, 4H), 5.03 (s, 2H), 4.64 (s, 2H), 4.19 (s, 2H), 2.90
(septet, J = 7.2 Hz, 1H), 1.29 (d, J = 6.6 Hz, 6H). 13C NMR (150 MHz,
CDCl3) δ 163.3, 162.4, 161.6, 152.1, 148.3, 135.2, 133.1(d, J = 3.3 Hz),
129.7 (d, J = 7.8 Hz), 129.2, 127.2 (d, J = 6.8 Hz), 119.9, 115.4
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ARTICLE
Hz, 2H), 2.91 (septet, J = 7.2 Hz, 1H), 2.58 (t, J = 7.2 Hz, 2H), 1.63 (m,
2H), 1.58 (m, 2H), 1.29 (d, J = 6.6 Hz, 6H). 13C NMR (150, CDCl3) δ
162.7, 162.0, 160.4, 152.2, 148.5, 137.8 (d, J = 3.3 Hz), 135.4, 129.7 (d,
J = 7.8 Hz), 129.2, 127.3 (d, J = 7.8 Hz), 119.8, 115.0 (d, J = 20.7 Hz),
73.1, 69.3, 34.7, 33.4, 29.2, 28.9, 28.1, 20.3. HRMS (ESIþ) calcd for
C25H29FN2O4 [M þ H]þ 441.2184, found 441.2164 (E = 4.6 ppm).
6-Benzyl-1-(4-fluorobenzyl)-3-hydroxy-5-isopropylpyrimidine-2,
4(1H,3H)-dione (32). This compound was prepared as a white solid
following the procedure described for the preparation of 4; yield 72%.
1H NMR (600 MHz, CDCl3) δ 7.30 (t, J = 7.2 Hz, 2H), 7.05-7.03 (m,
3H), 6.98 (d, J = 7.2 Hz, 2H), 6.94 (m, 2H), 4.88 (s, 2H), 3.83 (s, 2H),
2.81 (septet, J = 7.2 Hz, 1H), 1.22 (d, J = 6.6 Hz, 6H). HRMS (ESIþ)
calcd for C21H21FN2O3 [M þ H]þ 369.1609, found 369.1608 (E =
0.26 ppm).
6-Benzyl-1-(4-fluorophenethyl)-3-hydroxy-5-isopropylpyrimidine-2,
4(1H,3H)-dione (42). This compound was prepared as a white solid
following the procedure described for the preparation of 4; yield 62%.
1H NMR (600 MHz, CDCl3) δ 7.26-7.24 (m, 2H), 7.21 (m, 1H), 6.97
(m, 2H), 6.89-6.88 (m, 2H), 6.85-6.84 (m, 2H), 3.79 (s, 2H), 3.73 (t,
J = 7.8 Hz, 2H), 2.82 (septet, J = 7.2 Hz, 1H), 2.68 (t, J = 7.8 Hz, 2H),
1.29 (d, J = 6.6 Hz, 6H). HRMS (ESIþ) calcd for C23H23FN2O3 [M þ
H]þ 383.1765, found 383.1768 (E = 0.38 ppm).
1.93 (s, 3H). HRMS (ESI-) calcd for C13H14N2O4 [M - H]-
263.1026, found 263.1023 (E = 1.3 ppm).
5-Benzyl-1-(benzyloxymethyl)-6-ethyl-3-hydroxypyrimidine-2,4(1H,3H)-
dione (55). This compound was prepared as a white solid following the
1
procedure described for the preparation of 4; yield 53%. H NMR
(600 MHz, CDCl3) δ 7.22 (m, 4H), 7.19 (m, 3H), 7.12 (m, 3H), 5.42 (s,
2H), 4.61 (s, 2H), 3.76 (s, 2H), 2.68 (q, J = 7.2 Hz, 2H), 0.99 (t, J = 7.2 Hz,
3H). HRMS (ESI-) calcd for C21H22N2O4 [M - H]- 365.1507, found
365.1484 (E = 6.2 ppm).
5-Benzyl-6-ethyl-1-((4-fluorobenzyloxy)methyl)-3-hydroxypyrimi-
dine-2,4(1H,3H)-dione (56). This compound was prepared as a white
solid following the procedure described for the preparation of 4; yield
59%. 1H NMR (600 MHz, CDCl3) δ 7.13 (m, 3H), 7.07 (m, 4H), 6.83
(t, J = 6.6 Hz, 2H), 5.28 (s, 2H), 4.48 (s, 2H), 3.67 (s, 2H), 2.72 (q, J =
6.6 Hz, 2H), 1.07 (t, J = 6.6 Hz, 3H). HRMS (ESI-) calcd for
C21H21FN2O4 [M - H]- 383.1485, found 383.1394 (E = 4.8 ppm).
1-Benzyl-6-(4-fluorobenzyl)-3-hydroxy-5-isopropylpyrimidine-2,
4(1H,3H)-dione (64). This compound was prepared as a white solid
following the procedure described for the preparation of 4; yield 70%.
1H NMR (600 MHz, CDCl3) δ 7.33 (t, J = 7.2 Hz, 2H), 7.30 (m, 1H),
7.11 (d, J = 7.2 Hz, 2H), 7.06-7.04 (m, 2H), 7.02-7.00 (m, 2H), 4.98
(s, 2H), 3.86 (s, 2H), 2.84 (septet, J = 7.2 Hz, 1H), 1.29 (d, J = 6.6 Hz,
6H). HRMS (ESIþ) calcd for C21H21FN2O3 [M þ H]þ 369.1609,
found 369.1606 (E = -1.9 ppm).
6-Benzyl-1-(3-(4-fluorophenyl)propyl)-3-hydroxy-5-isopropylpyri-
midine-2,4(1H,3H)-dione (43). This compound was prepared as a white
solid following the procedure described for the preparation of 4; yield
72%. 1H NMR (600 MHz, CDCl3) δ 7.21 (m, 3H), 6.94 (m, 2H), 6.87
(t, J = 8.4 Hz, 2H), 6.75 (d, J = 7.8 Hz, 2H), 3.71 (s, 2H), 3.58 (t, J = 7.2
Hz, 2H), 2.78 (septet, J = 7.2 Hz, 1H), 2.46 (t, J = 7.2 Hz, 2H), 1.79 (m,
2H), 1.29 (d, J = 6.6 Hz, 6H). HRMS (ESIþ) calcd for C23H25FN2O3
[M þ H]þ 397.1922, found 397.1924 (E = -0.51 ppm).
1,6-Bis(4-fluorobenzyl)-3-hydroxy-5-isopropylpyrimidine-2,4(1H,3H)-
dione (65). This compound was prepared as a white solid following the
procedure described for the preparation of 4; yield 70%. 1H NMR (600
MHz, CDCl3) δ 7.10-7.08 (m, 2H), 7.06-7.04 (m, 2H), 7.02-6.99
(m, 4H), 4.94 (s, 2H), 3.89 (s, 2H), 2.84 (septet, J = 7.2 Hz, 1H), 1.28 (d,
J = 6.6 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 163.1 (d, J = 31.2 Hz),
162.2, 161.4 (d, J = 31.2 Hz), 151.8, 148.3, 132.2 (d, J = 3.3 Hz), 130.4 (d,
J = 2.9 Hz), 128.6 (d, J = 7.8 Hz), 127.6 (d, J = 8.4 Hz), 119.7, 116.4 (d, J =
21.8 Hz), 116.1 (d, J = 21.8 Hz), 46.6, 33.6, 28.2, 20.4. HRMS (ESIþ) calcd
for C21H20F2N2O3 [M þ H]þ 387.1515, found 387.1502 (E = 3.3 ppm).
1,6-Bibenzyl-3-hydroxy-5-isopropylpyrimidine-2,4(1H,3H)-dione
(66). This compound was prepared as a white solid following the procedure
6-Benzyl-1-(4-(4-fluorophenyl)butyl)-3-hydroxy-5-isopropylpyrimi-
dine-2,4(1H,3H)-dione (44). This compound was prepared as a white
solid following the procedure described for the preparation of 4; yield
65%. 1H NMR (600 MHz, CDCl3) δ 7.34 (t, J = 7.2 Hz, 2H), 7.28 (d, J =
7.8 Hz, 1H), 7.05 (m, 4H), 6.94 (t, J = 8.4 Hz, 2H), 3.92 (s, 2H), 3.73 (m,
2H), 2.87 (septet, J = 7.2 Hz, 1H), 2.51 (t, J = 7.2 Hz, 2H), 1.56 (m, 2H),
1.51 (m, 2H), 1.27 (d, J = 7.2 Hz, 6H). HRMS (ESIþ) calcd for
C24H27FN2O3 [M þ H]þ 411.2078, found 411.2081 (E = -0.62 ppm).
1-(Benzyloxymethyl)-6-(4-fluorobenzyl)-3-hydroxy-5-isopropylpyri-
midine-2,4(1H,3H)-dione (51). This compound was prepared as a white
solid following the procedure described for the preparation of 4; yield
74%. 1H NMR (600 MHz, CDCl3) δ 7.32-7.27 (m, 5H), 7.00-6.98
(m, 4H), 5.28 (s, 2H), 4.67 (s, 2H), 4.14 (s, 2H), 2.81 (septet, J = 7.2 Hz,
1H), 1.27 (d, J = 6.6 Hz, 6H). HRMS (ESI-) calcd for C22H23FN2O4
[M - H]- 399.1715, found 399.1706 (E = 2.1 ppm).
1
described for the preparation of 4; yield 53%. H NMR (600 MHz,
CDCl3) δ 7.39-7.34 (m, 4H), 7.32-7.30 (m, 2H), 7.12 (d, J = 7.2 Hz,
2H), 7.06 (d, J = 7.8 Hz, 2H), 5.00 (s, 2H), 3.91 (s, 2H), 2.90 (septet, J =
7.2 Hz, 1H), 1.29 (d, J = 7.2 Hz, 6H). HRMS (ESIþ) calcd for
C21H22N2O3 [M þ H]þ 351.1703, found 351.1689 (E = 4.1 ppm).
1,6-Bibenzyl-5-ethyl-3-hydroxypyrimidine-2,4(1H,3H)-dione (67).
This compound was prepared as a white solid following the procedure
1
described for the preparation of 4; yield 43%. H NMR (600 MHz,
CDCl3) δ 7.31-7.27 (m, 3H), 7.26-7.23 (m, 3H), 7.04 (d, J = 6.6 Hz,
2H), 7.00 (d, J = 6.6 Hz, 2H), 4.91 (s, 2H), 3.80 (s, 2H), 2.47 (q, J =
7.2 Hz, 2H), 1.00 (t, J = 7.2 Hz, 3H). HRMS (ESIþ) calcd for C20H20-
N2O3 [M þ H]þ 337.1547, found 337.1534 (E = 3.8 ppm).
6-(4-Fluorobenzyl)-1-((4-fluorobenzyloxy)methyl)-3-hydroxy-5-iso-
propylpyrimidine-2,4(1H,3H)-dione (52). This compound was prepared
as a white solid following the procedure described for the preparation of
4; yield 72%. 1H NMR (600 MHz, CDCl3) δ 7.28-7.27 (m, 6H), 7.00
(d, J = 7.2 Hz, 2H), 5.24 (s, 2H), 4.61 (s, 2H), 4.12 (s, 2H), 2.83 (septet,
J = 7.2 Hz, 1H), 1.25 (d, J = 6.6 Hz, 6H). HRMS (ESI-) calcd for
C22H22F2N2O4 [M - H]- 417.1620, found 417.1620 (E = 0.10 ppm).
6-Benzyl-1-(benzyloxymethyl)-5-ethyl-3-hydroxypyrimidine-2,
4(1H,3H)-dione (53). This compound was prepared as a white solid
following the procedure described for the preparation of 4; yield 70%.
1H NMR (600 MHz, CDCl3) δ 7.33-7.27 (m, 8H), 7.03 (d, J = 7.8 Hz,
2H), 5.26 (s, 2H), 4.65 (s, 2H), 4.15 (s, 2H), 2.51 (q, J = 7.8 Hz, 2H),
1.06 (t, J = 7.2 Hz, 3H). HRMS (ESI-) calcd for C21H22N2O4 [M - H]-
367.1652, found 367.1641 (E = 3.1 ppm).
6-Benzyl-5-ethyl-1-(4-fluorobenzyl)-3-hydroxypyrimidine-2,4(1H,3H)-
dione (68). This compound was prepared as a white solid following
the procedure described for the preparation of 4; yield 48%. 1H NMR
(600 MHz, CDCl3) δ 7.38-7.35 (m, 2H), 7.32 (m, 1H), 7.15-7.13 (m,
2H), 7.07 (d, J = 7.8 Hz, 2H), 7.04 (m, 2H), 4.94 (s, 2H), 3.88 (s, 2H),
2.52 (q, J = 7.2 Hz, 2H), 1.07 (t, J = 7.2 Hz, 3H). HRMS (ESIþ) calcd for
C20H19FN2O3 [M þ H]þ 355.1452, found 355.1442 (E = 3.0 ppm).
1-Benzyl-3-hydroxy-5-methylpyrimidine-2,4(1H,3H)-dione (69).
This compound was prepared as a white solid following the procedure
described for the preparation of 4; yield 32%. 1H NMR (600 MHz, CDCl3)
δ 7.35-7.31 (m, 3H), 7.28 (d, J = 7.2 Hz, 2H), 6.94 (s, 1H), 4.94 (s,
2H), 1.90 (s, 3H). HRMS (ESIþ) calcd for C12H12N2O3 [M þ H]þ
233.0921, found 233.0926 (E = -2.3 ppm).
1-(Benzyloxymethyl)-3-hydroxy-5-methylpyrimidine-2,4(1H,3H)-dione
(54). This compound was prepared as a white solid following the procedure
1-(4-Fluorobenzyl)-3-hydroxy-5-methylpyrimidine-2,4(1H,3H)-dione
1
described for the preparation of 4; yield 37%. H NMR (600 MHz,
(70). This compound was prepared as a white solid following the
1
CDCl3) δ 7.32-7.28 (m, 5H), 7.06 (s, 1H), 5.25 (s, 2H), 4.60 (s, 2H),
procedure described for the preparation of 4; yield: 41%. H NMR
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Journal of Medicinal Chemistry
ARTICLE
(600 MHz, CDCl3) δ 7.30-7.27 (m, 2H), 7.08-7.05 (m, 2H), 6.96 (s,
1H), 4.85 (s, 2H), 1.89 (s, 3H). HRMS (ESI-) calcd for C12H11FN2O3
[M - H]- 251.0826, found 251.0822 (E = 1.8 ppm).
’ ACKNOWLEDGMENT
This research was supported by the Center for Drug Design at the
University of Minnesota, the Center for Cancer Research, Intramural
Program of the National Cancer Institute, and an NIH grant from
IATAP (Intramural AIDS Targeted Antiviral Program). We thank
Roger Ptak at Southern Research Institute for the antiviral assay and
the Minnesota Supercomputing Institute for computing resources.
Biology. In Vitro Integrase Catalytic Assays. Expression and pur-
ification of the recombinant IN in Escherichia coli were performed as
previously reported28,48 with addition of 10% glycerol to all buffers.
Preparation of oligonucleotide substrates has been described.49 Inte-
grase reactions were performed in 10 μL with 400 nM of recombinant
IN, 20 nM of 50-end [32P]-labeled oligonucleotide substrate, and inhibitors
at various concentrations. Solutions of 10% DMSO without inhibitors were
used as controls. Reactions were incubated at 37 °C (60 min) in buffer
containing 50 mM MOPS, pH 7.2, 7.5 mM MgCl2, and 14.3 mM
2-mercaptoethanol. Reactions were stopped by addition of 10 μL of loading
dye (10 mM EDTA, 98% deionized formamide, 0.025% xylene cyanol, and
0.025% bromophenol blue). Reactions were then subjected to electrophoresis
in 20% polyacrylamide-7 M urea gels. Gels were dried, and reaction products
were visualized and quantitated with a Typhoon 8600 (GE Healthcare, Little
Chalfont, Buckinghamshire, UK). Densitometric analyses were performed
using ImageQuant from Molecular Dynamics Inc. The concentrations at
which enzyme activity was reduced by 50% (IC50) were determined using
“Prism” software (GraphPad Software, San Diego, CA) for nonlinear
regression to fit dose-response data to logistic curve models.
’ ABBREVIATIONS USED
IN, integrase; HIV, human immunodeficiency virus; INI, inte-
grase inhibitor; SAR, structure-activity relationship; 30P, 30 pro-
cessing; ST, strand transfer; DKA, diketoacid; HAART, highly
active antiretroviral therapy; AIDS, acquired immunodeficiency
syndrome; RT, reverse transcriptase; NTD, N-terminal domain;
CCD, catalytic core domain; CTD, C-terminal domain; PFV,
prototype foamy virus; m-CPBA, meta-chloroperbenzoic acid;
CPE, cytopathic effect
’ REFERENCES
(1) Bagasra, O. A unified concept of HIV latency. Expert Opin. Biol.
Ther. 2006, 6, 1135–1149.
HIV-1 Assay. The HIV cytoprotection assay used CEM-SS cells and
the IIIB strain of HIV-1. Briefly virus and cells were mixed in the presence of
test compound and incubated for 6 days. The virus was pretitered such
that control wells exhibit 70-95% loss of cell viability due to virus
replication. Therefore, antiviral effect or cytoprotection was observed
when compounds prevent virus replication. Each assay plate contained
cell control wells (cells only), virus control wells (cells plus virus), com-
pound toxicity control wells (cells plus compound only), and compound
colorimetric control wells (compound only) as well as experimental
wells (compound plus cells plus virus). Cytoprotection and compound
cytotoxicity were assessed by MTS (CellTiter 96 Reagent, Promega,
Madison WI) and the EC50 (concentration inhibiting virus replication
by 50%), CC50 (concentration resulting in 50% cell death) and a
calculated TI (therapeutic index CC50/EC50) were provided. Each assay
included the HIV RT inhibitor AZT as a positive control.
Modeling and Docking. All modeling studies were carried out using the
Schrodinger Modeling suite50 based on our previous homology model38 of
HIV-1 IN CCD-DNA complex using Glide with standard precision
protocol.51 The Mg2þ ions and the interfacial hydrophobic pocket between
the HIV-1 IN and DNA were defined as required constraints. The van der
Waals radii of nonpolar atoms for each of the ligands were scaled by a factor
of 0.8 to account for structure variability to specific ligand binding. Valida-
tion of the docking protocol was carried out by redocking both raltegravir
and elvitegravir into the ligand binding site with structure comparison to the
earlier reported mode of binding in PFV IN complex.36 To further validate
our structural model for HIV IN inhibitor design, correlation of the
experimental and predicted IC50s for a common mode of binding was
performed for the most active compounds (4, 11, 28, 29, 30, 32, 42) based
on the linear response method52 implemented within Liason.50
(2) Chun, T.-W.; Stuyver, L.; Mizell, S. B.; Ehler, L. A.; Mican,
J. A. M.; Baseler, M.; Lloyd, A. L.; Nowak, M. A.; Fauci, A. S. Presence of
an inducible HIV-1 latent reservoir during highly active antiretroviral
therapy. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13193–13197.
(3) Finzi, D.; Hermankova, M.; Pierson, T.; Carruth, L. M.; Buck, C.;
Chaisson, R. E.; Quinn, T. C.; Chadwick, K.; Margolick, J.; Brookmeyer,
R.; Gallant, J.; Markowitz, M.; Ho, D. D.; Richman, D. D.; Siliciano, R. F.
Identification of a reservoir for HIV-1 in patients on highly active
antiretroviral therapy. Science 1997, 278, 1295–1300.
(4) Engelman, A.; Mizuuchi, K.; Craigie, R. HIV-1 DNA integration:
mechanism of viral DNA cleavage and DNA strand transfer. Cell 1991,
67, 1211–1221.
(5) Pommier, Y.; Johnson, A. A.; Marchand, C. Integrase inhibitors
to treat HIV/AIDS. Nature Rev. Drug Discovery 2005, 4, 236–248.
(6) Cotelle, P. Patented HIV-1 integrase inhibitors (1998-2005).
Recent Pat. Anti-Infect. Drug Discovery 2006, 1, 1–15.
(7) Henao-Mejia, J.; Goez, Y.; Patino, P.; Rugeles, M. T. Diketo acids
derivatives as integrase inhibitors: the war against the acquired immunode-
ficiency syndrome. Recent Pat. Anti-Infect. Drug Discovery 2006, 1, 255–265.
(8) Dubey, S.; Satyanarayana, Y. D.; Lavania, H. Development of
integrase inhibitors for treatment of AIDS: an overview. Eur. J. Med.
Chem. 2007, 42, 1159–1168.
(9) Marchand, C.; Maddali, K.; Metifiot, M.; Pommier, Y. HIV-1 IN inhi-
bitors: 2010 update and perspectives. Curr.Top.Med.Chem.2009,9,1016–1037.
(10) Dayam, R.; Sanchez, T.; Neamati, N. Diketo Acid Pharmaco-
phore. 2. Discovery of Structurally Diverse Inhibitors of HIV-1 Inte-
grase. J. Med. Chem. 2005, 48, 8009–8015.
(11) Dayam, R.; Sanchez, T.; Clement, O.; Shoemaker, R.; Sei, S.;
Neamati, N. β-Diketo acid pharmacophore hypothesis. 1. Discovery of a
novel class of HIV-1 integrase inhibitors. J. Med. Chem. 2005, 48, 111–120.
(12) Deng, J.; Sanchez, T.; Neamati, N.; Briggs, J. M. Dynamic
Pharmacophore Model Optimization: Identification of Novel HIV-1
Integrase Inhibitors. J. Med. Chem. 2006, 49, 1684–1692.
(13) Barreca, M. L.; Ferro, S.; Rao, A.; De Luca, L.; Zappala, M.;
Monforte, A.-M.; Debyser, Z.; Witvrouw, M.; Chimirri, A. Pharmaco-
phore-Based Design of HIV-1 Integrase Strand-Transfer Inhibitors.
J. Med. Chem. 2005, 48, 7084–7088.
’ ASSOCIATED CONTENT
S
Supporting Information. Additional experimental de-
b
tails and NMR, MS, and HPLC data for 31, 34-41, 57-63,
and 72-76. This material is available free of charge via the
Internet at http://pubs.acs.org.
(14) Mustata, G. I.; Brigo, A.; Briggs, J. M. HIV-1 integrase pharma-
cophore model derived from diverse classes of inhibitors. Bioorg. Med.
Chem. Lett. 2004, 14, 1447–1454.
’ AUTHOR INFORMATION
(15) Carlson, H. A.; Masukawa, K. M.; Rubins, K.; Bushman, F. D.;
Jorgensen, W. L.; Lins, R. D.; Briggs, J. M.; McCammon, J. A. Developing a
Dynamic Pharmacophore Model for HIV-1 Integrase. J. Med. Chem. 2000,
43, 2100–2114.
Corresponding Author
*Phone: þ1 (612) 626-7025. Fax: þ1 (612) 625-8154. E-mail:
wangx472@umn.edu.
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Journal of Medicinal Chemistry
ARTICLE
(16) Cocohoba, J.; Dong, B. J. Raltegravir: the first HIV integrase
inhibitor. Clin. Ther. 2008, 30, 1747–1765.
(37) Hare, S.; Vosb, A. M.; Claytonb, R. F.; Thuringb, J. W.;
Cummingsb, M. D.; Cherepanov, P. Molecular mechanisms of retroviral
integrase inhibition and the evolution of viral resistance. Proc. Natl. Acad.
Sci. U.S.A. 2010, 107, 20057–20062.
(17) Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi,
M.; Ferrara, M.; Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D. J.;
Jones, P.; Kinzel, O.; Laufer, R.; Monteagudo, E.; Muraglia, E.; Nizi, E.;
Orvieto, F.; Pace, P.; Pescatore, G.; Scarpelli, R.; Stillmock, K.; Witmer,
M. V.; Rowley, M. Discovery of Raltegravir, a Potent, Selective Orally
Bioavailable HIV-Integrase Inhibitor for the Treatment of HIV-AIDS
Infection. J. Med. Chem. 2008, 51, 5843–5855.
(38) Tang, J.; Maddali, K.; Pommier, Y.; Sham, Y. Y.; Wang, Z. Scaffold
rearrangement of dihydroxypyrimidine inhibitors of HIV integrase: docking
model revisited. Bioorg. Med. Chem. Lett. 2010, 20, 3275–3279.
(39) Krishnan, L.; Li, X.; Naraharisetty, H. L.; Hare, S.; Cherepanov, P.;
Engelman, A. Structure-based modeling of the functional HIV-1 intasome
and its inhibition. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15910–15915.
(40) Tanaka, H.; Hayakawa, H.; Haraguchi, K.; Miyasaka, T.; Walker,
R. T.; De Clercq, E.; Baba, M.; Stammers, D. K.; Stuart, D. I. HEPT: from
an investigation of lithiation of nucleosides towards a rational design of
non-nucleoside reverse transcriptase inhibitors of HIV-1. Adv. Antiviral
Drug Des. 1999, 3, 93–144.
(18) Shimura, K.; Kodama, E. N. Elvitegravir: a new HIV integrase
inhibitor. Antiviral Chem. Chemother. 2009, 20, 79–85.
(19) Vandekerckhove, L. GSK-1349572, a novel integrase inhibitor for
the treatment of HIV infection. Curr. Opin. Invest. Drugs 2010, 11, 203–212.
(20) Min, S.; Song, I.; Borland, J.; Chen, S.; Lou, Y.; Fujiwara, T.;
Piscitelli, S. C. Pharmacokinetics and safety of S/GSK1349572, a next-
generation HIV integrase inhibitor, in healthy volunteers. Antimicrob.
Agents Chemother. 2009, 54, 254–258.
(41) Pontikis, R.; Benhida, R.; Aubertin, A.-M.; Grierson, D. S.;
Monneret, C. Synthesis and Anti-HIV Activity of Novel N-1 Side Chain-
Modified Analogs of 1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-
thymine (HEPT). J. Med. Chem. 1997, 40, 1845–1854.
(21) Prada, N.; Markowitz, M. Novel integrase inhibitors for HIV.
Expert Opin Invest. Drugs 2010, 19, 1087–1098.
(42) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Inouye,
N.; Baba, M.; Shigeta, S.; Walker, R. T.; Clercq, E. D.; Miyasaka, T.
Synthesis and Antiviral Activity of 6-Benzyl Analogs of 1-[(2-Hydro-
xyethoxy)methyl]-5-(phenylthio)thymine (HEPT) as Potent and Se-
lective Anti-HIV-1 Agents. J. Med. Chem. 1995, 38, 2860–2865.
(43) Tanaka, H.; Baba, M.; Hayakawa, H.; Sakamaki, T.; Miyasaka,
T.; Ubasawa, M.; Takashima, H.; Sekiya, K.; Nitta, I.; Shigeta, S.; Walker,
R. T.; Balzarini, J.; Clercq, E. D. A new class of HIV-1 specific
6-substituted acyclouridine derivatives: synthesis and anti-HIV-1 activity
of 5- or 6-substituted analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenyl-
thio)thymine (HEPT). J. Med. Chem. 1991, 34, 349–357.
(44) Wang, Z.; Bennett, E. M.; Wilson, D. J.; Salomon, C.; Vince, R.
Rationally Designed Dual Inhibitors of HIV Reverse Transcriptase and
Integrase. J. Med. Chem. 2007, 50, 3416–3419.
(45) Danel, K.; Larsen, E.; Pedersen, E. B.; Vestergaard, B. F.;
Nielsen, C. Synthesis and Potent Anti-HIV-1 Activity of Novel 6-Ben-
zyluracil Analogs of 1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-
thymine. J. Med. Chem. 1996, 39, 2427–2431.
(46) These assays were done in MAGI R5 cells with NNRTI TNK-
651 as a control. The observed fold-resistances for the control were 22
and 72 for K103N and Y181C mutants.
(47) Bernardes, G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B. G.
Facile Conversion of Cysteine and Alkyl Cysteines to Dehydroalanine
on Protein Surfaces: Versatile and Switchable Access to Functionalized
Proteins. J. Am. Chem. Soc. 2008, 130, 5052–5053.
(48) Leh, H.; Brodin, P.; Bischerour, J.; Deprez, E.; Tauc, P.; Brochon,
J. C.; LeCam, E.; Coulaud, D.; Auclair, C.; Mouscadet, J. F. Determinants
of Mg2þ-dependent activities of recombinant human immunodeficiency
virus type 1 integrase. Biochemistry 2000, 39, 9285–9294.
(49) Semenova, E. A.; Johnson, A. A.; Marchand, C.; Davis, D. A.;
Yarchoan, R.; Pommier, Y. Preferential inhibition of the magnesium-
dependent strand transfer reaction of HIV-1 integrase by R-hydroxy-
tropolones. Mol. Pharmacol. 2006, 69, 1454–1460.
(22) Cherepanov, P.; Ambrosio, A. L. B.; Rahman, S.; Ellenberger,
T.; Engelman, A. Structural basis for the recognition between HIV-1
integrase and transcriptional coactivator p75. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 17308–17313.
(23) Hombrouck, A.; De Rijck, J.; Hendrix, J.; Vandekerckhove, L.;
Voet, A.; De Maeyer, M.; Witvrouw, M.; Engelborghs, Y.; Christ, F.; Gijsbers,
R.; Debyser, Z. Virus evolution reveals an exclusive role for LEDGF/p75 in
chromosomal tethering of HIV. PLoS Pathog. 2007, 3, 418–430.
(24) Engelman, A. Host cell factors and HIV-1 integration. Future
HIV Ther. 2007, 1, 415–426.
(25) Christ, F.; Voet, A.; Marchand, A.; Nicolet, S.; Desimmie, B. A.;
Marchand, D.; Bardiot, D.; Van der Veken, N. J.; Van Remoortel, B.;
Strelkov, S. V.; De Maeyer, M.; Chaltin, P.; Debyser, Z. Rational design
of small-molecule inhibitors of the LEDGF/p75-integrase interaction
and HIV replication. Nature Chem. Biol. 2010, 6, 442–448.
(26) Yeni, P. UpdateonHAARTinHIV. J. Hepatol. 2006, 44, S100–S103.
(27) Metifiot, M.; Marchand, C.; Maddali, K.; Pommier, Y. Resis-
tance to integrase inhibitors. Viruses 2010, 2, 1347–1366.
(28) Metifiot, M.; Maddali, K.; Naumova, A.; Zhang, X.; Marchand,
C.; Pommier, Y. Biochemical and Pharmacological Analyses of HIV-1
Integrase Flexible Loop Mutants Resistant to Raltegravir. Biochemistry
2010, 49, 3715–3722.
(29) da Silva, D.; Van Wesenbeeck, L.; Breilh, D.; Reigadas, S.; Anies,
G.; Van Baelen, K.; Morlat, P.; Neau, D.; Dupon, M.; Wittkop, L.; Fleury,
H.; Masquelier, B. HIV-1 resistance patterns to integrase inhibitors in
antiretroviral-experienced patients with virological failure on raltegravir-
containing regimens. J. Antimicrob. Chemother. 2010, 65, 1262–1269.
(30) Tang, J.; Maddali, K.; Dreis, C. D.; Sham, Y. Y.; Vince, R.;
Pommier, Y.; Wang, Z. N-3 Hydroxylation of Pyrimidine-2,4-diones
Yields Dual Inhibitors of HIV Reverse Transcriptase and Integrase. ACS
Med. Chem. Lett. 2011, 2, 63–67.
(31) Esposito, D.; Craigie, R. HIV integrase structure and function.
Adv. Virus Res. 1999, 52, 319–333.
(50) Maestro version 9.1, G version; Macromodel version 9.8;
Liason version 5.6, version 9.1; Schrodinger, LLC: New York.
(51) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;
Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry,
J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach for
rapid, accurate docking and scoring. 1. Method and assessment of
docking accuracy. J. Med. Chem. 2004, 47, 1139–1149.
(52) Hansson, T.; Aqvist, J. Estimation of binding free energies for
HIV proteinase inhibitors by molecular dynamics simulations. Protein
Eng. 1995, 8, 1137–1144.
(32) Goldgur, Y.; Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Craigie,
R.; Davies, D. R. Three new structures of the core domain of HIV-1
integrase: an active site that binds magnesium. Proc. Natl. Acad. Sci. U.S.
A. 1998, 95, 9150–9154.
(33) Dyda, F.; Hickman, A. B.; Jenkins, T. M.; Engelman, A.; Craigie, R.;
Davies, D. R. Crystal structure of the catalytic domain of HIV-1 integrase:
similarity to other polynucleotidyl transferases. Science 1994, 266, 1981–1986.
(34) O’Brien, C. HIV Integrase Structure Catalyzes Drug Search.
Science 1994, 266, 1946.
(35) Alian, A.; Griner, S. L.; Chiang, V.; Tsiang, M.; Jones, G.; Birkus,
G.; Geleziunas, R.; Leavitt, A. D.; Stroud, R. M. Catalytically-active
complex of HIV-1 integrase with a viral DNA substrate binds anti-
integrase drugs. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8192–8197.
(36) Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P.
Retroviral intasome assembly and inhibition of DNA strand transfer.
Nature 2010, 464, 232–236.
’ NOTE ADDED AFTER ASAP PUBLICATION
An author name was inadvertently omitted in the version of this
paper published ASAP on March 7, 2011. The corrected version
was reposted on March 14, 2011.
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