6-Benzoyl-3-hydroxypyrimidine-2,4-diones as dual inhibitors of HIV reverse transcriptase and integrase

Article abstract of DOI:10.1016/j.bmcl.2011.02.069

N-3-Hydroxylation of pyrimidine-2,4-diones was recently found to yield inhibitors of both HIV-1 reverse transcriptase (RT) and integrase (IN). An extended series of analogues featuring a benzoyl group at the C-6 position of the pyrimidine ring was synthesized. Through biochemical studies it was found that these new analogues are dually active against both RT and IN in low micromolar range. Antiviral assays confirmed that these new inhibitors are active against HIV-1 in cell culture at nanomolar to low micromolar range, further validating 3-hydroxypyrimidine-2,4-diones as a viable scaffold for antiviral development.

Full text of DOI:10.1016/j.bmcl.2011.02.069

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2400–2402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
6-Benzoyl-3-hydroxypyrimidine-2,4-diones as dual inhibitors of HIV
reverse transcriptase and integrase
Jing Tang a, Kasthuraiah Maddali b, Christine D. Dreis a, Yuk Y. Sham a, Robert Vince a, Yves Pommier b,
Zhengqiang Wang a,
⇑
a Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St SE, Minneapolis, MN 55455, United States
b Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
N-3-Hydroxylation of pyrimidine-2,4-diones was recently found to yield inhibitors of both HIV-1 reverse
transcriptase (RT) and integrase (IN). An extended series of analogues featuring a benzoyl group at the
C-6 position of the pyrimidine ring was synthesized. Through biochemical studies it was found that these
new analogues are dually active against both RT and IN in low micromolar range. Antiviral assays con-
firmed that these new inhibitors are active against HIV-1 in cell culture at nanomolar to low micromolar
range, further validating 3-hydroxypyrimidine-2,4-diones as a viable scaffold for antiviral development.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 27 January 2011
Revised 14 February 2011
Accepted 16 February 2011
Available online 18 February 2011
Keywords:
HIV
6-Benzoyl-3-hydroxypyrimidine-2,4-diones
Dual inhibitors
Reverse transcriptase
Integrase
Human immunodeficiency virus (HIV) infects an estimated 33
million people globally and poses a huge healthcare challenge with
high mortality and morbidity rates.1 As effective HIV vaccines re-
main elusive despite tremendous efforts,2,3 chemotherapy contin-
ues to provide the main leverage in battling HIV/AIDS. Current
standard therapy, the highly active antiretroviral therapy
(HAART),4,5 involves the use of multiple antivirals with orthogonal
mechanisms of action to create a large genetic barrier to resistance.
Its success in clinically managing HIV/AIDS notwithstanding,
HAART requires nearly perfect adherence6,7 which can be difficult
to achieve with the complex dosing of multi-target therapy. Subop-
timal compliance generally leads to treatment failure and develop-
ment of multi-drug resistance.6,7 Drug–drug interactions are
among the most prominent complications of HAART.8 Simplifying
HAART regimens to improve adherence has been a subject of tre-
mendous research efforts and interests.9,10 Conceptually the sim-
plest form of multi-target therapy would be a single structure
compound inhibiting multiple viral targets. We have previously re-
ported various examples11–15 of single structure dual inhibitors of
HIV-1 RT and IN. Of particular interest was our recently disclosed
RT/IN dual inhibitor scaffold14 based on pyrimidine-2,4-dione
non-nucleoside RT inhibitors (NNRTIs). In this case, N-3 hydroxyl-
ation of known 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio) thy-
mine (HEPT) type NNRTIs yielded new compounds active against
IN while retaining inhibition against RT (Fig. 1, chemotype I).14
Interestingly, similar to HEPT, pyrimidine-2,4-diones featuring a
C-6 benzoyl group (chemotype IIa) also represent an important
class of NNRTIs.16–22 It is easily conceived that introducing an
N-3 hydroxy group to these NNRTIs could generate a new molecu-
lar scaffold for inhibitors of RT (RTI) and IN (INI) (Fig. 1, chemotype
IIb). Here we report the synthesis and biological evaluation of this
extended series of 6-benzyol-3-hydroxypyrimidine-2,4-diones.
O
O
R
O
alk
R
O
alk
O
N
O
N
O
3
3
C-6 modification
1
N
1
N
6
6
Abbreviations: HIV, human immunodeficiency virus; RT, reverse transcriptase;
IN, integrase; HAART, highly active antiretroviral therapy; IN, integrase; NNRTI,
non-nucleoside RT inhibitor; HEPT, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)
thymine; 30-P, 30 processing; ST, strand transfer; CCD, catalytic core domain; CPE,
cytopathic effect.
Chemotype II
Chemotype I
IIa
: R = H; RTI
Ia: R = H; RTI
IIb: R = OH; RTI / INI (?)
Ib
: R = OH; RTI / INI
⇑
Corresponding author. Tel.: +1 612 626 7025; fax: +1 612 625 8154.
E-mail address: wangx472@umn.edu (Z. Wang).
Figure 1. Design of the new series of N-3-hydroxypyrimidine-2,4-diones featuring
a C-6 benzoyl group.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.069

J. Tang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2400–2402
2401
Table 1
O
Cl
Biochemical assay results of compounds 17–24 against HIV-1 RT and IN
COOEt
COOEt
Et
O
a
b
Et
Cl
c
HN
Et
N
b
Compd
RT
Ma
IN IC50
30-P
(l
M)
N
H
O
b
Ma
10
l
IC50 (lM)
STc
Cl
N
100
l
1
2
3
17
18
19
20
21
22
23
24
84
78
85
92
46
74
61
94
61
41
68
79
4
60
37
61
14
23
7.6
1.0
––
10
43
0.5
>111
>111
>111
>111
>111
>111
>111
>111
37
25
21
41
4.1 0.2
7.0 0.8
>111
6
3
3
4
OMe
R1
O
R1
OMe
N
Et
Et
O
Et
Cl
N
HN
d
e
N
MeO
N
R1
O
N
H
R1
MeO
O
1
: R1 = H
8: R1 = Me
>111
5
7
: R = H
6: R1 = Me
R1
4
a
b
c
% Inhibition under single concentration.
Concentration inhibiting enzyme activity by 50%.
Mean value standard deviation from three independent measurements.
O
N
O
R1
Et
O
HO
O
Et
HN
N
O
O
R1
N
O
R1
assay. The results clearly demonstrate that these compounds selec-
tively inhibit ST at low micromolar range (Table 1). Most intrigu-
ingly, IN ST inhibition seems to have a high dependence on the
benzene ring in N-1 side chain as the two analogues with an ali-
phatic N-1 side chain (23 and 24) showed no inhibition. Another
major observation was that the extra carbon on the N-1 linker ap-
pears to considerably benefit IN binding as compounds 21 and 22
exhibit significantly higher potency against IN. It was also found
that the fluorine atom on the N-1 benzene tends to slightly favor
IN inhibition (18 vs 17, 19 vs 20). These findings conform to the
common pharmacophore of IN ST inhibitors in which a fluoroben-
zyl group with a specific spatial arrangement to a chelator is a re-
quired structural determinant.15,24–28
To confirm the binding of our compounds to both RT and IN, a
representative inhibitor (20) was docked into both the NNRTI bind-
ing pocket (A)14 and the IN catalytic core domain (CCD) (B)29 using
Glide v2.5 at Standard Precision.30 Significantly, the N-3 OH of 20 is
hydrogen bonded to the K101 backbone in the NNRTI binding
pocket, allowing the inhibitor to adopt a conformation where the
C-6 benzene ring interacts favorably with the Y188, and the N-1
side chain sits in an open channel (Fig. 2,A). On the other hand,
the same inhibitor was found to fit nicely into the IN CCD in com-
plex with Mg and viral DNA (Fig. 2,B). The deprotonation of the N-3
OH group of the pyrimidine ring allows the chelation of two Mg2+
ions and the placement of the N-1 benzyl group into the protein–
DNA interfacial hydrophobic pocket. These docking studies corrob-
orate the observed dual inhibitory activities and suggest that this
chemotype has the ability to engage with both RT and IN.
O
n
n
R2
R2
1 = R2 = H, n = 1
R1 = R2 = H, n = 1
: R1 = H, R2 = F, n = 1
18
19: R1 = Me, R2 = F, n = 1
20: R1 = Me, R2 = H, n = 1
21: R1 = H, R2 = F, n = 2
22: R1 = Me, R2 = F, n = 2
9
17
:
:
R
: R1 = H, R2 = F, n = 1
10
11: R1 = Me, R2 = F, n = 1
12: R1 = Me, R2 = H, n = 1
13: R1 = H, R2 = F, n = 2
14: R1 = Me, R2 = F, n = 2
f
g
O
R1
O
R1
Et
HO
O
Et
HN
N
O
O
N
R1
N
R1
O
O
O
: R1 = H
16: R1 = Me
: R1 = H
15
23
24: R1 = Me
Scheme 1. Synthesis of compounds 17–24. aReagents and conditions: (a) urea,
NaOMe, MeOH, reflux, 75%; (b) POCl3, dimethylaniline, 89%; (c) MeONa, MeOH,
81%; (d) (i) substituted phenyl acetonitrile, NaH, DMF, rt; (ii) NaH, DMF, air, 84–
87%; (e) HCl, MeOH, reflux, 85–93%; (f) CH3C(OTMS)@NTMS (BSA), benzyl alcohol
substrate, (HCHO)n, TMSCl, TBAI (cat.), CH2Cl2, rt; or (for 15, 16) BSA, chloromethyl
ethyl ether, TBAI (cat.), CH2Cl2, rt, 74–94%; (g) NaH, m-CPBA, THF, rt, 38–68%.
The new chemotype was synthesized via a route described in
Scheme 1. The synthesis involves key intermediates 7 and 8 which
were prepared starting from readily available malonate 1 following
reported procedures.16–18 The N-1 ether linkage was then intro-
duced through a regio-selective alkylation with a chloromethyl
ether to give compounds 9–16. N-3-hydroxylation was achieved
via a base-mediated m-CPBA oxidation14 to produce final com-
pounds 17–24.
Finally, the antiviral activity of these new compounds was eval-
uated in CEM-SS cells with the IIIB strain of HIV-1 using an assay
based on viral cytopathic effect (CPE). The reduction of CPE was
used to indicate the inhibition of viral replication. Compounds
were first tested at a single concentration (10 lM) for antiviral
activity and cell viability. The active ones were then tested in dose
Final compounds 17–24 were biochemically evaluated against
recombinant HIV-1 RT and IN. For the RT assay, all compounds
were first screened at two different concentrations (100 and
10 lM) and the promising ones were then assayed in dose re-
response fashion.
sponse fashion. It was observed from these assays that except for
21 these newly synthesized analogues generally inhibit RT at low
micromolar concentrations (Table 1). Notably, the 3,5-dimethyl
group on the benzene ring of the C-6 substituent appears to signif-
icantly benefit RT binding (19 vs 18, 20 vs 17, 24 vs 23). The same
effect was observed with the HEPT NNRTI GCA-18623 where the di-
methyl group on the C-6 benzene provides additional van der
Waals interactions with the roof of the binding pocket. Meanwhile,
the fluorine atom on the benzene ring of the N-1 side chain was
found to hamper RT inhibition (18 vs 17, 19 vs 20), and the extra
carbon in the N-1 linker also conferred a substantial decrease in
RT inhibition (21 vs 18, 22 vs 19).
As shown in Table 2, except for compound 22, all new com-
pounds yielded 100% inhibition against HIV-1 at 10 lM without
appreciable cytotoxicity (Table 2). Further testing in dose–
response fashion found that these compounds inhibit HIV-1 at
low micromolar to low nanomolar range. The parallel cytotoxicity
assay showed that all final compounds are generally safe with
favorable therapeutic indices. Interestingly, compound 24 which
inhibited RT with the highest potency in the series while lacking
anti-IN activity, and compound 21 which showed the best anti-
IN activity with only marginal inhibition against RT, both demon-
strated excellent antiviral activity. The overall anti-HIV-1 activity
of the rest of the compounds likely reflects the dual inhibitory
activities against both RT and IN.
The ability of these new analogues to inhibit HIV-1 IN was
assessed using a biochemical gel assay. Both the 30 processing
(30-P) and the strand transfer (ST) functions were evaluated in this
In summary, based on recently disclosed N-3-hydroxypyrimi-
dine-2,4-diones as a new chemotype for dual inhibitors of HIV-1

2402
J. Tang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2400–2402
Figure 2. Inhibitory modes of binding for compound 20 within (A) the NNRTI binding pocket and (B) the active site of IN CCD in complex with Mg2+ and DNA. The
electrostatic potential surface of the binding pocket is shown to highlight the hydrophobic regions (white) in both enzymes.
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Table 2
Antiviral assay results of compounds 17–24 against HIV-1
Compd
10
l
M
Dose response
% CPEa
reduction
% Cell
viability
EC50
M)
CC50
TId
b
c
(
l
(lM)
17
18
19
20
21
22
23
24
100
100
100
100
100
56
98
100
100
100
100
81
1.4
2.0
>100
>100
28
33
>100
––
>71
>50
78
540
290
––
0.36
0.061
0.34
––
100
100
100
100
2.3
94
77
41
2600
0.030
a
b
c
Viral cytopathic effect.
Concentration inhibiting viral replication by 50%.
Concentration causing 50% cell death.
d
Therapeutic index, defined by CC50/EC50
.
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RT and IN, we have synthesized an extended series of analogues
featuring a benzyol group at the C-6 position of the pyrimidine.
Through biochemical studies and cell culture antiviral assays it
was found that these new analogues inhibit RT, IN and HIV-1 at
nanomolar to low micromolar range. Molecular modeling also sug-
gests that these compounds can fit nicely into both the NNRTI
binding pocket and IN CCD. These results provide further support
on 3-hydroxypyrimidine-2,4-diones as a valid scaffold for antiviral
development.
Acknowledgments
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This research was supported by the Center for Drug Design at
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viral assays and the Minnesota Supercomputing Institute for mod-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.069.
References and notes
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