N-3 hydroxylation of pyrimidine-2,4-diones yields dual inhibitors of HIV reverse transcriptase and integrase

Article abstract of DOI:10.1021/ml1002162

A new molecular scaffold featuring an N-hydroxyimide functionality and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of human immunodeficiency virus (HIV) was rationally designed based on 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) non-nucleoside RT inhibitors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine ring of HEPT compound to yield a chelating triad which, along with the existing benzyl group, appeared to satisfy major structural requirements for IN binding. In the mean time, this chemical modification did not severely compromise the compound's ability to inhibit RT. A preliminary structure-activity relationship (SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl group on N-1 side chain is more important for IN binding than the one on C-6.

Full text of DOI:10.1021/ml1002162

pubs.acs.org/acsmedchemlett
N-3 Hydroxylation of Pyrimidine-2,4-diones Yields Dual
Inhibitors of HIV Reverse Transcriptase and Integrase
Jing Tang,† Kasthuraiah Maddali,‡ Christine D. Dreis,† Yuk Y. Sham,† Robert Vince,†
Yves Pommier,‡ and Zhengqiang Wang*,†
†Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street SE, Minneapolis, Minnesota
55455, United States, and ‡Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, United States
ABSTRACT A new molecular scaffold featuring an N-hydroxyimide functionality
and capable of inhibiting both reverse transcriptase (RT) and integrase (IN) of
human immunodeficiency virus (HIV) was rationally designed based on 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) non-nucleoside RT inhibi-
tors (NNRTIs). The design involves a minimal 3-N hydroxylation of the pyrimidine
ring of HEPT compound to yield a chelating triad which, along with the existing
benzyl group, appeared to satisfy major structural requirements for IN binding. In
the mean time, this chemical modification did not severely compromise the
compound's ability to inhibit RT. A preliminary structure-activity relationship
(SAR) study reveals that this N-3 OH is essential for IN inhibition and that the benzyl
group on N-1 side chain is more important for IN binding than the one on C-6.
KEYWORDS HIV, integrase, reverse transcriptase, dual inhibitor, rational design
EPT (1)1 and its analogues constitute an important
class of HIV NNRTIs,2 with the best ones being MKC-
H
442 (emivirine, 2)3-5 and TNK-651 (3)6 (Figure 1).
The clinical development of these compounds was halted as
they were found to stimulate liver enzyme CYP 3A4 and cause
prohibitively low bioavailability of protease inhibitors used in
combination therapy.7 Drug-drug interactions of this type pose
a major barrier to successful highly active antiretroviral therapy
(HAART)against HIV/AIDS.8,9 Simplifying regimens by reducing
the number of drugs used in HAART could alleviate this issue.
Toward this end, we have long been interested in identifying
molecular scaffolds with dually inhibitory activities, particularly
against HIV RTand IN.10-12
Figure 1. Representative structures of HEPT type NNRTIs.
Notably the NNRTI binding pocket is largely hydrophobic and
few H-bonds are accommodated. For the HEPT type of NNRTIs,
the H-bond between the N(3)H of the inhibitor (e.g., inhibitor 3,
Figure 4) and the backbone carbonyl group of K101 represents
the major hydrophilic interaction.18 This H-bond contributes to
the NNRTI binding affinity and, more importantly, serves as an
anchor for the inhibitor to adopt optimized conformation so
that hydrophobic interactions can be maximized throughout
the binding pocket. Additional water mediated H-bonds may be
present, albeit of much less significance.3 Therefore, the N(3)H
group is generally considered too important to be modified. In
our design, we hypothesize that hydroxylation of the N-3 of
compound 3 would not significantly compromise its binding
affinity to RT. This hypothesis is based on two observations: (1)
the newly incorporated OH would retain H-bonding ability; and
(2) the NNRTI binding pocket has four consecutive lysine
residues (K101-K104) that could potentially H-bond to the
The design of inhibitors that can accommodate binding to two
distinct biological targets remains an intriguing yet challenging
scientific endeavor.13-15 Our previous work has demonstrated
the viability of constructing RT/IN dual inhibitors from NNRTIs
using a design-in strategy. Significantly, IN binding requires mini-
mally a two metal ion chelating functionality and a hydrophobic
benzyl group (Figure 2). Certain NNRTI scaffolds, including
HEPT16 and BHAP17 (e.g., delavirdine, 9), have structural moi-
eties that are sitting on the P236 loop of the NNRTI binding
pocket and are not directly involved in RT binding. These moi-
eties provide valuable handles (highlighted, Figure 3) for incor-
porating a relatively hydrophilic chelating functionality (boxed)
to satisfy binding to the IN (Figure 3).10,12 Most other NNRTIs are
deeply buried in the highly hydrophobic binding pocket, thus are
less amenable to hydrophilic modifications. In these cases,
designing in structural components for IN binding requires care-
ful considerations of all binding interactions of NNRTIs.
Received Date: September 17, 2010
Accepted Date: October 19, 2010
Published on Web Date: October 22, 2010
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newly introduced OH. Since this minimal modification only
results in an insertion of an oxygen atom to NH, we expect that
the key H-bond can be formed between the OH and one of
these lysine residues, and the overall conformation of the
inhibitor should be largely preserved. On the other hand, such
a simple hydroxylation yields a C(2)O-N(3)OH-C(4)O chelat-
ing triad (11, Figure 4) capable of chelating two magnesium
ions, which along with an existing hydrophobic benzyl group at
N-1 or C-6 will essentially provide the minimal pharmacophore
for IN binding (Figure 4). Therefore, through a simple N-3
hydroxylation we could convert these HEPT type NNRTIs into
RT/IN dual inhibitors.
This design is verified in silico by molecular modeling. All
€
modeling was carried out using the Schrodinger modeling
suite package.19 Docking of all compounds was carried out
using Glide v2.5 at Standard Precision.20 For docking into
HIV-1 IN, both Mg2þ ions and the interfacial hydrophobic
pocket between the HIV-1 IN and DNA were defined as
required constraints. Docking of 3 and 11 reveals their mode
of binding in the NNRTI binding pocket almost identically
(Figure 5A). Interestingly the OH of 11 and NH of 3 are both
situated in close proximity to the K101 backbone (1.7 Å and
1.6 Å respectively). Accordingly, the H-bonding allows both
inhibitors to adopt a nearly identical conformation in the
binding pocket, suggesting that N-3 hydroxylation should
not significantly compromise RT binding. Docking of 11 into
our recent homology model21 of HIV-1 IN catalytic core
domain (CCD) in complex with Mg2þ and DNA indicated
the newly designed inhibitor 11 also showed reasonable IN
binding (Figure 5B). The 3-N hydroxylation of the pyrimidine
ring yields a chelating triad for Mg2þ binding while allowing
the placement of the benzyl group into the protein-DNA
interfacial hydrophobic pocket, a crucial pharmacophore
requirement for potent HIV-1 ST inhibition.22,23
Figure 2. Structures of important IN inhibitors: the chelating
functionality (red) and hydrophobic benzyl group (blue) represent
the minimal pharmacophore.
The designed inhibitors are synthetically accessed through a
route outlined in Scheme 1. The synthesis of advanced inter-
mediates 2, 3 and 29-33 is well-established in the literature24
whereas the key transformation, the N-3 hydroxylation, is rarely
known. This was achieved through deprotonation of HEPT
intermediates and the subsequent treatment with an oxidiz-
ing agent. After screening of a wide array of oxidants it was
found that m-CPBA can effect this N-3 hydroxylation efficiently
(Scheme 1). Adaptation of this method also allowed us to suc-
cessfully prepare an N-3 amino analogue (16), wherein mesityl
sulfonyl-O-hydroxyl amine (MSH)25 wasemployedastheaminat-
ing agent.
Testing of these compounds against recombinant HIV IN
provided the first validation of our design. In this assay both
Figure 3. Previously designed RT/IN dual inhibitors based on
NNRTIs.
Figure 4. The design of novel RT/IN dual inhibitor 11 from NNRTI 3. The H-bond crucial to RT binding is maintained (red) and the conformation of
the molecule remains unchanged. The N-3 hydroxylation yields a chelating triad (blue), a critical structural component for IN binding.
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Figure 5. (A) Mode of binding of 3 (gray) and 11 (green) in the HIV NNRTI binding pocket. Both compounds hydrogen bonded to the
backbone carbonyl oxygen atom of K101. (B) Mode of binding of 11 (green) 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. The electrostatic potential surface of the binding pocket is shown to highlight the hydrophobic regions
(white) in both enzymes.
Scheme 1a
a Reagents and conditions: (a) Zn, I2 (cat.), THF, 89-90%; (b) thiourea, KOtBu, i-PrOH, 40-71%; (c) ClCH2CO2H, AcOH, H2O, 38-78%; (d)
CH3C(OTMS)dNTMS (BSA), chloromethyl ether, TBAI (cat.), CH2Cl2, rt, 51-89%; (e) NaH, m-CPBA, THF, rt, 50-73%; (f) NaH, MSH, THF, rt, 54%.
the 30 processing (30P) and the strand transfer (ST)activities,
the two primary functions of IN, were assessed. As shown in
Table 1, all N-3 hydroxylated compounds (11-15, 17, 18)
selectively inhibit STover 30Pat low micromolar concentrations,
whereas the unmodified compound (3) did not show any inhi-
bitory activity against HIV IN. This observation strongly suggests
that the N-3 hydroxyl group, partially forming the chelating triad
(Figure 4, 11), is crucial to IN binding. Interestingly, the N-3
amino compound (16) also turned out inactive in IN assay. It
is not surprising that the N-3 hydroxylation yields a better
chelating triad than the N-3 amination as O tends to have a
higher affinity for Mg2þ than N. In addition, compounds 17 and
18 demonstrate significantly reduced (ca. 10-fold) inhibition
against STwhen compared with compounds 11-15, indicating
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Table 1. Inhibitory Activities of Compounds 11-18 against HIV IN
IC50a (μM)
Table 2. Anti-RTand Anti-HIVActivities of Inhibitors 11-18
HIV-1
compd
30P
ST
compd
RT IC50a (μM)
EC50b (μM)
CC50c (μM)
TId
11
12
13
14
15
16
17
18
3
>333
>333
>333
>333
>333
>333
>333
>333
>333
21
11
12
13
14
15
16
17
18
3
6.2
>100
0.17
>100
9.4
0.0080
>10
0.024
>10
1.1
>20
>2500
7.3
3.5
>20
>20
>20
>20
>830
>18
5.5
8.2
>333
85
1.5
>10
4.3
13
>4.7
105
>100
0.040
>10
0.013
>333
>1500
a Concentration inhibiting enzyme activity by 50%.
a Concentration inhibiting enzyme activity by 50%. b Concentration
inhibiting virus replication by 50%. c Concentration resulting in 50% cell
death. d Therapeutic index, defined by CC50/EC50
.
Finally these compounds were tested in cell-based antiviral
assay against HIV-1. With the exception of compounds with a
terminal fluorine atom on the C-6 benzyl group (12, 14 and 18),
and the one with N-3 amino group (16), these inhibitors gene-
rally show excellent antiviral potency and safety (Table 2). The
best compounds in this series, compounds 11 and 13, demon-
strate low nanomolar anti-HIV activity that is comparable to or
even better than that of reference compound 3.
In summary, through rational design we have generated a
novel series of anti-HIV compounds that are dually active against
IN and RTat low and sub-micromolar concentrations. Our design
involves a minimal structural modification on a well-known
NNRTI inhibitor scaffold (HEPT) to introduce inhibitory activity
against IN. Major structural determinants for IN binding as well
as a dramatic fluorine effect for RT binding were identified
through preliminary SAR efforts. In the end, two compounds (11
and 13) were found to have low nanomolar inhibitory activities
against HIV-1 in cell culture. Further studies of this newly
designed chemotype as a novel IN inhibitor scaffold are cur-
rently underway and will be reported in due course.
Figure 6. Comparison of newly designed inhibitor 13 with ralte-
gravir 7 in recombinant HIV IN with G140S and Q148H mutants.
that the benzyl group at the N-1 side chain is far more important
for IN binding than the one at the C-6 position of the pyrimidine-
2,4-dione ring (Figure 4, compound 11).
To further characterize the mechanism of action of our
newly designed compounds, a representative compound 13
was tested head-to-head with raltegravir (7, Figure 2) against
a recombinant HIV IN containing major resistant mutations
G140S and Q148H (Figure 6).26,27 This assay revealed that,
although inhibiting IN at a higher concentration when compared
with 7, compound13 does show a similar resistance profile to 7,
further proving that our new inhibitors are targeting IN ST.
Having established these new inhibitors as valid HIV IN
inhibitors, we then studied their ability to inhibit HIV RT.
Through a quick biochemical assay using a commercial RT
assay kit it was found that N-3 hydroxylated compounds
(e.g., 13) and aminated compound (16) all show inhibitory
activity at low or sub-micromolar range (Table 2). Apparently
an N-3 OH or NH2 allows these compound to virtually maintain
the key H-bonding ability as expected, though the unmodified
N(3)H group of compound 3 appears to form better H-bonding
as compound 3 inhibits RTat a considerably lower concentra-
tion. Meanwhile, a dramatic fluorine effect was also observed
with compounds 12, 14 and 18, where a fluorine substitution at
the para position of the C-6 benzyl completely disables RT
binding. By contrast, the para position of the other benzyl
group in N-1 side chain appears to be highly tolerant toward
fluorine substitution (compound 13).
SUPPORTING INFORMATION AVAILABLE Synthesis of
11-18, experimental details, compound characterization (1H, 13C
NMR, HRMS), HPLC analysis for final products and assay methods.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *E-mail: wangx472@umn.edu. Phone:
þ1 (612) 626-7025. Fax: þ1 (612) 625-8154.
Funding Sources: This research was supported by the Center for
Drug Design at the University of Minnesota, the Center for Cancer
Research, National Cancer Institute and an IATAP grant to Y.P., NIH.
ACKNOWLEDGMENT We thank Roger Ptak at Southern Re-
search Institute for the cell-based antiviral assay and the Minnesota
Supercomputing Institute for providing all the necessary computa-
tional resources.
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ABBREVIATIONS RT, reverse transcriptase; IN, integrase;
HIV, human immunodeficiency virus; HEPT, 1-[(2-hydro-
xyethoxy)methyl]-6-(phenylthio)thymine; NNRTI, non-
nucleoside RT inhibitor; SAR, structure-activity relationship;
HAART, highly active antiretroviral therapy; CCD, catalytic
core domain; MSH, mesityl sulfonyl-O-hydroxyl amine; TBAI,
tetrabutylammonium iodide; 30-P, 30 processing; ST, strand
transfer.
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