3-Hydroxypyrimidine-2,4-diones as Selective Active Site Inhibitors of HIV Reverse Transcriptase-Associated RNase H: Design, Synthesis, and Biochemical Evaluations

Article abstract of DOI:10.1021/acs.jmedchem.5b01879

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains an unvalidated antiviral target. A major challenge of specifically targeting HIV RNase H arises from the general lack of selectivity over RT polymerase (pol) and integrase (IN) strand transfer (ST) inhibitions. We report herein the synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to achieve selective RNase H inhibition. Biochemical studies showed the two subtypes with an N-1 methyl group (9 and 10) inhibited RNase H in low micromolar range without siginificantly inhibiting RT polymerase, whereas the N-1 unsubstituted subtype 11 inhibited RNase H in submicromolar range and RT polymerase in low micromolar range. Subtype 11 also exhibited substantially reduced inhibition in the HIV-1 INST assay and no significant cytotoxicity in the cell viability assay, suggesting that it may be amenable to further structure-activity relationship (SAR) for identifying RNase H inhibitors with antiviral activity.

Full text of DOI:10.1021/acs.jmedchem.5b01879

Article
pubs.acs.org/jmc
3
-Hydroxypyrimidine-2,4-diones as Selective Active Site Inhibitors of
HIV Reverse Transcriptase-Associated RNase H: Design, Synthesis,
and Biochemical Evaluations
†
†
‡
‡
§
†
†
Jing Tang, Feng Liu, Eva Nagy, Lena Miller, Karen A. Kirby, Daniel J. Wilson, Bulan Wu,
Stefan G. Sarafianos, Michael A. Parniak, and Zhengqiang Wang*
§
‡
,†
†
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 55455, United States
Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219,
‡
United States
§
Department of Molecular Microbiology and Immunology and Department of Biochemistry, University of Missouri School of
Medicine, Christopher S. Bond Life Sciences Center, Columbia, Missouri 65211, United States
*
S Supporting Information
ABSTRACT: Human immunodeficiency virus (HIV) reverse
transcriptase (RT) associated ribonuclease H (RNase H)
remains an unvalidated antiviral target. A major challenge of
specifically targeting HIV RNase H arises from the general lack
of selectivity over RT polymerase (pol) and integrase (IN)
strand transfer (ST) inhibitions. We report herein the
synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to
achieve selective RNase H inhibition. Biochemical studies showed the two subtypes with an N-1 methyl group (9 and 10)
inhibited RNase H in low micromolar range without siginificantly inhibiting RT polymerase, whereas the N-1 unsubstituted
subtype 11 inhibited RNase H in submicromolar range and RT polymerase in low micromolar range. Subtype 11 also exhibited
substantially reduced inhibition in the HIV-1 INST assay and no significant cytotoxicity in the cell viability assay, suggesting that
it may be amenable to further structure−activity relationship (SAR) for identifying RNase H inhibitors with antiviral activity.
INTRODUCTION
However, the critical role of RNase H in HIV replication has
long been recognized and confirmed through recent experi-
ments showing that active site mutations associated with
attenuated RNase H biochemical activity conferred reduced
HIV replication in cell culture. A similar antiviral phenotype
can be expected if RNase H is selectively and potently inhibited
■
HIV encodes three enzymes crucial for viral replicaton: RT, IN,
1
and protease (PR). Antivirals targeting these enzymes have
successfully transformed HIV from an inevitably fatal disease
into a clinically manageable chronic infection. One particularly
successful aspect of HIV chemotherapy is the approval of
multiple classes of antivirals, which allows effective combina-
tion therapy termed as highly active antiretroviral therapy
HAART). However, due to their long duration HAART
8
2
3
by small molecules.
RNase H belongs to the retroviral integrase super family
9
(
RISF) with an active site fold and catalytic mechanism highly
(
homologous to integrase. Accordingly, efforts in RNase H
inhibition have mostly focused on targeting the active site with
a pharmacophore core similar to INSTIs. The pharmacophore
critically features a chelating triad (magenta) designed to bind
two divalent metals (Figure 1a). Reported RNase H inhibitor
regimens can be plagued by the emergence of resistant HIV
mutants. Mechanistically novel antivirals against underexplored
and unvalidated viral targets will add strategically to HAART
repertoire enabling continued efficacy, especially against drug-
resistant viruses that may emerge with current HAART
regimens. One such novel target is RT associated RNase H
1
0
11
types include 2-hydroxyisoquinolinedione (HID, 1), β-
1
2
13
4
,5
thujaplicinol (2), dihydroxycoumarin (3), diketoacid
(DKA) 4, pyrimidinol carboxylic acid 5, hydroxynaphthyr-
16 17
activity. RT is a well-established drug target with many FDA-
14
15
6
approved nucleoside RT inhibitors (NRTIs) and non-
nucleoside RT inhibitors (NNRTIs) constituting the corner-
7
idine 6, and pyridopyrimidone 7. Importantly, structurally
more elaborate inhibitor types 4−7 also feature a hydrophobic
aromatic moiety (cyan) conferring more potent and selective
RNase H inhibition. Unfortunatley, the biochemical inhibition
observed with these inhibitors typically does not translate into
antiviral activity in cell culture, possibly reflecting a steep
stone of HAART. However, these drugs all target the
polymerase domain which carries out both RNA-dependent
and DNA-dependent viral DNA polymerization. Significantly,
RT also encodes an RNase H domain which selectively
degrades the RNA strand from the RNA/DNA heteroduplex
intermediate during reverse transcription. In stark contrast to
the success of polymerase targeting antivirals, no inhibitors of
RT-associated RNase H have entered clinical development.
Received: December 4, 2015
Published: March 1, 2016
©
2016 American Chemical Society
2648
DOI: 10.1021/acs.jmedchem.5b01879
J. Med. Chem. 2016, 59, 2648−2659

Journal of Medicinal Chemistry
Article
Figure 1. Design of active site RNase H inhibitors. (a) Major chemotypes reported as HIV RNase H active site inhibitors. All chemotypes contain a
chelating triad (magenta); scaffolds 4−7 also feature an aryl or biaryl moiety (cyan) connected through a methylene or amino linker; (b) newly
designed active site RNase H inhibitor chemotypes 9−11 featuring a chelating triad and a biaryl group to satisfy the pharmacophore requirements for
selective RNase H inhibition.
2
3
biochemical barrier of competing against much larger DNA/
NNRTIs. The key 3-OH group was typically introduced in
1
7
18
20−22
RNA substrates. Recent studies by Corona et al. on a series
of DKAs demonstrated that specific interactions with highly
conserved amino acid residues in the RNase H domain
contributed critically to selective RNase H inhibition and
effective targeting of HIV reverse transcription in cells, hence
offering important insights for the rational design of RNase H
inhibitors. However, the most potent antiviral activity was
observed with inhibitors 6−7 presumably via tight binding to
the very last step via a base-mediated N-hydroxylation.
Structure−activity relationship (SAR) relying on this synthetic
route suffers from two major drawbacks: the very early
introduction of structural diversity at the C6 aromatic domain
and sometimes the low efficiency of the N-hydroxylation
reaction. The new design substitutes a heteroatom linker (NH
or O) for the methylene group, which implicates a much more
divergent synthesis where the C6 aromatic domain can be easily
diversified via reacting a library of biaryl amine or alcohol
nucleophiles with 6-chloropyrimidine-2,4-dione intermediates
(15 and 16, Scheme 1). In addition, this new synthetic route
also exploits the symmetrical nature of malonate for the ring
construction (13 to 14) to allow the early introduction of a
protected N−OH without the complication of regioselectivity
issues. This eliminates the need for the late N-hydroxylation
reaction. Overall, the concise synthesis began with the readily
available hydroxyurea (12), which was selectively o-protected.
The resulting benzyl protected hydroxyurea (13) was then
cyclized with malonate to afford 14, which was chlorinated at
C6 position to produce key intermediate 15. For final
compounds with a N-1 methyl group, the methylation was
conducted at this stage with methyl iodide to yield another 6-
chloro intermediate 16. The synthesis of 6-chloropyrimidine-
1
7
RNase H. Intriguingly, these inhibitors encompass a highly
privileged biaryl moiety connected to the chelating core via a
one atom linker, a distinct pharmacophore feature that could be
key in providing tight RNase H binding. This pharmacophore
hypothesis was corroborated by our redesigned HID subtype
with a biaryl moiety that conferred potent RNase H inhibition
19
and significant antiviral activity.
We have previously developed a 3 hydroxypyrimidine-2,4-
diones (HPD) chemotype (8, Figure 1b) demonstrating
exceptional antiviral activity against HIV-1 likely by dually
20−22
inhibiting RT polymerase (pol) and INST.
Based on the
aforementioned pharmacophore model for RNase H inhibition,
we have redesigned the HPD chemotype with the goal of
achieving selective RNase H inhibition (Figure 1b). Key to the
redesign is the introduction of a biaryl group at C6 position
through different linkers (subtypes 9−11). In addition, the new
design also involves two structural simplifications: removal of
the C5 isopropyl group crucial for the allosteric binding to RT
pol; and substitution of the N-1 position with either a small
methyl group (9 and 10) or H (11). These simplifications aim
at minimizing inhibitor binding to the RT pol. We report
herein the chemical synthesis and biochemical evaluations of
these three subtypes.
2
,4-dione intermediates then allowed us to conduct two
different versions of amination/etherification reactions. It
turned out that for the N-1 unsubstituted chloride 15, direct
amination was effected thermally via a microwave reaction with
biaryl amines (23) under the influence of N,N-dimethylaniline.
However, the same reaction with biaryl alcohols (24) failed to
deliver desired products. On the other hand, amination of the
N-1 methylated chloride 16 required a completely different set
of conditions. In this event, the reaction was effected with
LDA/HMPA at low temperature. Significantly, under the same
conditions the biaryl alcohols also reacted to afford desired
intermediates 18. The final step of the synthesis aimed at a
clean benzyl deprotection and was achieved via catalytic
hydrogenation (g) or TFA treatment (h). The requisite
RESULTS AND DISCUSSION
■
Chemistry. Previously reported HPD chemotypes all
featured a methylene linker at C6 position and were
synthesized based largely on the well-documented HEPT
2
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DOI: 10.1021/acs.jmedchem.5b01879
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Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of HPD Analogues 9−11
markedly different from those of the N-1 methyl subtypes (9−
1
0): (1) In general 11 appears to be significantly more potent,
with all analogues inhibiting the RNase H in namomolar range
IC = 0.15−0.51 μM, HTS-1), even slightly more potent that
(
50
compound 7 used as a control (Table 3), whereas IC s for
5
0
subtypes 9−10 are mostly in the low micromolar range; (2)
while a substantial bias toward the inhibition of the internal
cleavage (HTS-1 as substrate) was observed for most analogues
of subtypes 9−10 (Tables 1−2), subtype 11 tends to inhibit all
three modes of RNase H cleavages nearly equally (Table 3). In
addition, comparison between the two N-1 methyl substituted
subtypes 9 and 10 revealed that the amino linkage at C6
(
subtype 9, Table 1) conferred slightly better potency and
considerably less substrate bias than the ether linkage (subtype
0, Table 2). Notably a number of subtype 10 analogues
1
(
Table 2, 10b−f, 10i, 10l, 10o−r, 10w−y) did not inhibit
RNase H with HTS-2 and/or HTS-3 as substrates. As for the
biaryl hydrophobic moiety, although many analogues within
each subtypes were synthesized, the impact of the substitution
on the terminal phenyl ring (Me, CF , OMe, OH, CN, F, Cl,
3
a
Reagents and conditions: (a) KOH, BnCl, MeOH, reflux, 6 h, 91%;
b) CH (COOEt) , NaOEt, MW, 150 °C, 20 min, 58%; (c) POCl ,
etc.) turned out to be marginal. Interestingly, replacing the
terminal aryl group with a saturated ring (compound 9z)
completely abrogated biochemical inhibition, suggesting that
the biaryl moiety may be required for activity. Nevertheless, the
RNase H inhibition was further evaluated biochemically against
a catalytically active RNase H domain with HTS-1 as substrate.
The potency observed from this assay generally agreed with
that of full-length RT with HTS-1 substrate, confirming the
observed RNase H inhibition.
(
2
2
3
BnEt NCl, 50 °C, 6 h, 88%; (d) Cs CO , MeI, DMF, seal tube 80 °C,
2
3
2
3
h, 68%; (e) (for 17−18) Ar′-ArNH / Ar′-ArOH, LDA, HMPA,
2
THF, − 78 °C to rt, overnight; (f) (for 19) Ar′-ArNH , N,N-
2
dimethylaniline, MW, 170 °C, 30−40 min; (g) Pd/C, H , 50 Psi,
2
MeOH, 3−4 h; (h) TFA, MW, 120 °C, 30 min; (i) Pd(PPh ) 1.5%
3
4
(
mol), DMF, 2 M Na CO , MW, 160 °C, 30 min.
2 3
libraries of biaryl amines (23) and alcohols (24) were curated
through commercial sources or quick chemical synthesis via a
standard Suzuki coupling reaction.
Biology. All final compounds of subtypes 9−11 were
evaluated biochemically for inhibition against RT-associated
RNase H and pol as well as INST. To assess the multiple
distinct modes of action of RNA cleavage by RNase H at
multiple stages of reverse transcription, our primary bio-
chemical assays employed three different oligonucleotide
substrates: HTS-1, which measures the random internal
cleavages likely representing the majority of RNase H events;
HTS-2, that gauges the DNA 3′ end directed (polymerase
dependent) cleavages; and HTS-3, which evaluates the RNA 5′
end directed (polymerase independent) cleavages for degrading
the recessed RNA template. These assays were performed with
recombinant p66/p51 RT heterodimer, and the observed
inhibitory activity could be influenced by the spatial and
temporal interactions between RNase H and pol domains. To
confirm RNase H inhibition, a secondary assay was conducted
with substrate HTS-1 using a catalytically active isolated RNase
H domain fragment. The compounds were also evaluated for
inhibition of RT pol activity. An INST assay was also
conducted to determine the inhibitory selectivity of our new
compounds for RNase H over INST. Finally, although antiviral
activity against HIV-1 was not achieved with these analogues,
cytoxicity was evaluated, and the CC50 values could add
significantly to the assessment of the suitability of compounds
for further medicinal chemistry efforts.
In parallel to the RNase H assays, a classic RT pol assay was
also conducted for all analogues with poly(rA) as template,
3
oligo(dT) as primer, and [ H]dTTP as nucleotide substrate.
For subtype 9 (Table 1), 16 out of 26 analogues were
completely inactive in this assay, while the other 10 compounds
inhibited RT pol but with much reduced potency compared to
RNase H inhibition (HTS-1). An even more dramatic
observation was made with subtype 10 (Table 2) of which all
analogues were inactive at the highest testing concentration (25
μM) with the lone exception of 10x which moderately inhibited
RT pol (IC50 = 14 μM). The general lack of RT pol inhibition
with these two subtypes suggests that selective inhibition of
RT-associated RNase H could be achieved. In contrast,
analogues with the N-1 unsubstituted chemotype 11 all
inhibited RT pol at submicromolar to low micromolar
concentrations (Table 3). Nevertheless, other than 11k even
these compounds demonstrated considerable selectivity inhibit-
ing RNase H over pol, particularly analogues 11a−c, 11i−j, 11l
and 11o which inhibited pol 8−50-fold less potently than
RNase H. This observation further confirms that our
redesigned HPD chemotypes provide structural approaches
toward selective RNase H inhibition. Finally, cell viability assays
showed that most analogues of the two N-1 methylated
subtypes 9−10 were significantly cytotoxic at low micromolar
1
9
concentrations (CC = 3.0−10 μM), whereas cytotoxicity was
50
not observed for any analogue of the N-1 unsubstituted subtype
11 at concentrations up to 25 μM, suggesting that 11 may be a
superior chemotype for further antiviral development compared
to chemotypes 9−10.
Detailed RNase H assay results are summarized in Tables
−3. Overall, newly synthesized HPD analogues of subtypes
−11 all potently inhibited RT-associated RNase H activity
1
9
To further characterize the inhibitory selectivity toward
RNase H, we also evaluated our new compounds in a
biochemical assay that measures HIV INST activity. This is a
critical measurement due to the high degree of homology
between RNase H and IN in active site fold and catalytic
with IC50 values ranging from nanomolar to single digit
micromolar concentrations. This observation provides a strong
validation to our inhibitor design. As for as SAR is concerned,
the inhibition profile of the N-1 unsubstituted subtype (11) is
2
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Table 1. Biochemical Inhibitory Activity of Compounds 9a−z Against HIV RT RNase H and Polymerase
a
IC : concentration of a compound producing 50% inhibition, expressed as mean of at least three independent experiments (standard deviation was
50
b
c
<
10% and is not indicated for ease of viewing). Substrate that measures internal cleavage. Substrate that measures DNA 3′ end directed cleavage.
d
e
f
Substrate that measures RNA 5′ end directed cleavage. Reconstituted HIV RNase H domain. CC : concentration of a compound causing 50%
50
g
h
cytotoxicity. No inhibition at concentrations up to 25 μM. Not determined.
mechanism. The INST assay results and the comparison
between INST and RNase H inhibition are summarized in
Table 4. Remarkably, six out of nine tested compounds of the
two N-1 methyl subtypes 9 and 10 exhibited potent inhibition
in single digit micromolar range (IC = 1.0−7.1 μM), with the
The activity and selectivity profiles of subtype 11 are
manifested with a few selected analogues as shown in Table 5.
Overall, these compounds demonstrated exceptional potency in
inhibiting RNase H (IC = 0.15−0.40 μM). Although they also
5
0
inhibited RT pol, the micromolar activity (IC = 5.7−7.3 μM)
5
0
50
other three (9t, 9w, and 10g) showing slightly lower potency
IC50 = 12−30 μM). By contrast, compounds of the N-1
unsubstituted chemotype 11 were either weakly active (11b,
1c, and 11k, IC50 = 35−93 μM) or completely inactive at
reflects a sizable selectivity (14−49 fold) strongly favoring
RNase H inhibition. Given the tendency of RNase H inhibitors
to influence RT pol activity, this level of selectivity can be hard
to achieve. In addition, none of these four analogues inhibited
INST in a biochemical assay, amounting to a dramatic
selectivity for RNase H inhibition over INST inhibition
(selectivity index = 230 − >670). Furthermore, these analogues
did not show appreciable cytotoxicity at concentrations up to
25 μM in cell viability assay. Although significant antiviral
activity was not observed in our cell-based assays, the
exceptional potency and selectivity as revealed from the
(
1
concentrations up to 100 μM (11i, 11j, and 11o). The two
exceptions are 11l and 11m which inhibited INST quite
strongly at low micromolar concentrations (IC50 = 1.2−1.3
μM). These results suggest that the N-1 methyl substitution
generally benefits binding to INST active site and that the N-1
unsubstituted subtype 11 represents the preferred subtype of
HPD for selective RNase H inhibition.
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Table 2. . Biochemical Inhibitory Activity of Compounds 10a−y Against HIV RT RNase H and Polymerase
a
IC : concentration of a compound producing 50% inhibition, expressed as mean of at least three independent experiments (standard deviation was
50
c
d
<
10% and is not indicated for ease of viewing). Substrate that measures DNA 3′ end directed cleavage. Substrate that measures RNA 5′ end
e
f
g
directed cleavage. Reconstituted HIV RNase H domain. CC : concentration of a compound causing 50% cytotoxicity. No inhibition at
concentrations up to 25 μM. Not determined.
50
h
19,25
detailed biochemical profiling suggest that HPD subtype 11
may present an important path toward the generation of
specific inhibitors of HIV RT-associated RNase H.
pharmacophore of active site RNase H inhibitors,
corroborating the active site inhibition mechanism.
hence
Mode of Binding. A molecular model of HIV-1 RT in
complex with representative inhibitor 11i was constructed. It
suggests possible interactions between this chemotype and
conserved RNase H active site residues (Figure 2). Key to this
binding mode is the interaction between the HPD core (the 2-
CO-3-OH-4-CO chelating triad) and the two metal
CONCLUSIONS
■
New HPD chemotypes featuring a C-6 biary moiety were
designed and synthesized as inhibitors of HIV RT associated
RNase H domain. Detailed biochemical profiling revealed that
all three newly designed subtypes can support potent RNase H
inhibition and that the N-1 unsubstituted subtype 11 is strongly
favored over the N-1 methyl subtypes 9−10 due to the
nanomolar potency toward RNase H inhibition, the exceptional
selectivity over RT pol and INST inhibitions, and the lack of
cytotoxicity at concentrations up to 25 μM. Although antiviral
activity was not achieved, these highly favorable biochemical
charateristics lay a solid foundation for potentially developing
subtype 11 as RNase H targeting anitivirals through further
SAR.
2
+
cofactors (Mn ) which are coordinated to the active site
acidic residues D443, E478, D498, and D549. The molecular
model also suggests a potential interaction between the 1-NH
of the HPD core and the imidazole of the highly conserved
H539. In addition, the biaryl moiety is positioned to possibly
interact with K540 as well as other protein residues or nucleic
acid substrate near the small hydrophobic region of the RNase
H. This predicted binding mode conforms to the general
2
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Table 3. Biochemical Inhibitory Activity of Compounds
Table 4. Biochemical Inhibitory Activity of Selected
Compounds Against HIV IN
1
1a−t Against HIV RT RNase H and Polymerase
a
b
c
compd
RNase H IC50 (μM)
INST IC50 (μM)
SI
9
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
b
0.85
0.75
0.60
1.2
2.2 ± 0.66
4.4 ± 0.5
4.6 ± 2.1
30 ± 6.2
7.1 ± 2.6
25 ± 8.8
12 ± 3.2
7.1 ± 2.1
1.0 ± 0.28
93 ± 34
44
2.6
1.1
g
k
7.7
t
25
v
1.1
6.5
w
1.6
16
0g
0h
0p
1b
1c
1i
1j
1k
1l
1m
1o
2.2
5.5
0.90
2.3
7.9
0.43
233
116
>667
>500
184
3.9
0.40
0.38
0.15
0.20
0.19
0.31
0.51
0.20
−
>100
>100
35
1.2
1.3
2.6
>100
>500
−
d
L-731988
0.40 ± 0.05
a
b
Data with HTS-1 as substrate. Expressed as mean ± standard
deviation from at least three independent experiments. Selectivity
index defined as IC50 IN/IC50 RNase H. Stereotypical DKA
compound, reported as a selective INSTI.
c
d
2
4
Table 5. Complete Activity and Selectivity Profile of Selected
Subtype 11 Compounds
a
RNase H IC50
RT Pol IC (μM) INST IC5 0b (μM)
CC50
(μM)
50
(SI)
b
compd
(μM)
(SI)
1
1
1
1
1b
1i
0.40
0.15
0.20
0.20
5.7 (14)
7.3 (49)
6.3 (32)
93 (230)
>100 (>670)
>100 (>500)
>100 (>500)
>25
>25
>25
>25
1j
1o
6.6 (33)
a
b
Data with HTS-1 as substrate. Selectivity index, defined as IC RT
5
0
pol or IN/IC50 RNase H.
a
IC : concentration of a compound producing 50% inhibition,
50
expressed as mean of at least three independent experiments (standard
deviation was <10% and is not indicated for ease of viewing).
b
c
Substrate that measures internal cleavage. Substrate that measures
d
DNA 3′ end directed cleavage. Substrate that measures RNA 5′ end
directed cleavage. Reconstituted HIV RNase H domain. CC :
concentration of a compound causing 50% cytotoxicity. Not
determined.
e
f
50
g
CHEMISTRY
Figure 2. Molecular model of HIV-1 RT in complex with 11i shows
possible interactions between this compound and indicated residues.
RNase H active site residues are shown as orange sticks, 11i as green
sticks, Mn2 ions as purple spheres, and p51 is shown as gray cartoon.
■
General Procedures. All commercial chemicals were used as
supplied unless otherwise indicated. Dry solvents were either
purchased (toluene and dioxane) or dispensed under argon from an
anhydrous solvent system with two packed columns of neutral alumina
or molecular sieves (THF and DMF). Flash chromatography was
performed on a Teledyne Combiflash RF-200 with either RediSep
columns (silica) or reverse-phase HP C18 Aq chromatography
columns and indicated mobile phase. All moisture-sensitive reactions
+
were performed under an inert atmosphere of ultrapure argon with
1
13
oven-dried glassware. H and C 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.
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Analysis of sample purity was performed on a Varian Prepstar SD-1
HPLC system with a Phenomenex Gemini, 5 μm C18 column (250
mmol), and N,N-dimethylaniline (3.0 mmol, 3.0 equiv). The mixture
was irradiated at 170 °C for 25 min until the completion of the
reaction as monitored by both TLC and LC-MS. The reaction mixture
was concentrated in vacuo. The residue was purified with Teledyne
ISCO reverse-phase HP C18 Aq chromatography columns or via
recrystallization (MeOH) to provide the desired compound 19 (54−
85%) as offwhite solid.
mm × 4.6 mm). HPLC conditions: solvent A: H O with 0.1% TFA;
2
solvent B: MeCN; flow rate 1.0 mL/min; compounds were eluted with
a gradient of 20% MeCN/H O with 0.1% TFA to 100% MeCN for 20
2
min. All tested compounds have a purity ≥96%.
3
-(Benzyloxy)-6-hydroxypyrimidine-2,4(1H,3H)-dione (14). To a
20 mL microwave reaction vessel were added 1-(benzyloxy)urea 13
3-(Benzyloxy)pyrimidine-6-([1,1′-biphenyl]-4-ylamino)-2,4-
1
(
1H,3H)-dione (19a). H NMR: (DMSO-d ) δ 7.69 (m, 4H), 7.54 (m,
(
1.0 g, 6.0 mmol), freshly prepared sodium ethoxide (6.0 mmol, 1.0
6
2
1
H), 7.47 (m, 3H), 7.41 (m, 3H), 7.35 (m, 4H), 4.99 (s, 2H), 4.89 (s,
equiv) in 5.5 mL of anhydrous ethanol, and diethyl malonate (6.0
mmol, 1.0 equiv). The mixture was stirred at rt for 5 min, irradiated at
H).
General Procedure for the Synthesis of 3-Hydroxy-1-
150 °C for 20 min, and then cooled to room tempeture. After the
methyl-6-aminopyrimidine-2,4(1H,3H)-dione (9). To a suspen-
sion of 17 (50 mg) in 5 mL MeOH in hydrogenation Parr Shaker was
added carefully 10 mg of Pd/C (20%). The reaction mixture was
solvent was removed under reduced pressure, the residue was
dissolved in cold water, and the solution was acidified to pH = 4−5
with 2 N HCl. The precipitate was filtered off, washed with Et O and
2
degassed using vacuum and refilling with H (40−50 psi) for three
2
small amount of cold MeOH, and dried in vacuo to give compound 14
1
times before it was shaken under 40−50 psi H atmosphere at room
2
as light-yellow solid in 58% yield. H NMR (600 MHz, DMSO-d ) δ
6
temperature for 4 h. The reaction mixture was filtered through a pad of
Celite, and the solvent was removed. The residue was purified via
trituration with MeOH or Teledyne ISCO reverse-phase HP C18 Aq
chromatography columns to yield the desired compound as white solid
1
1.50 (s, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.51 (m, 3H), 4.03 (t, J = 7.2
13
Hz, 1H), 4.98 (s, 2H), 3.74 (s, 2H); C NMR (150 MHz, DMSO-d6)
δ 165.8, 162.4, 148.7, 134.4, 129.3, 128.8, 128.4, 77.5, 41.0.
3
-(Benzyloxy)-6-chloropyrimidine-2,4(1H,3H)-dione (15). To a
(
82−90% yield).
solution of 14 (2.0 g, 8.5 mmol) in POCl (20 mL) was added
3
6
-([1,1′-Biphenyl]-4-ylamino)-3-hydroxy-1-methylpyrimidine-2,4-
BnEt NCl (17 mmol, 2.0 equiv), and the mixture was stirred at 50 °C
3
1
(1H,3H)-dione (9a). H NMR (600 MHz, DMSO-d ) δ 10.15 (s, 1H),
6
for 6 h. After cooled down to room temperature, the reaction mixture
was poured onto ice. The precipitate was filtered off, washed
8
.51 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.46 (t,
J = 7.2 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 4.71
squentially by water, Et O, cold MeOH, and dried in vacuo to give
−
2
(
3
s, 1H), 3.42 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]
1
18 15
3
3
compound 15 as light-yellow solid in 85% yield. H NMR (600 MHz,
08.1040, found 308.1045.
-([1,1′-Biphenyl]-3-ylamino)-3-hydroxy-1-methylpyrimidine-2,4-
DMSO-d ) δ 7.52 (d, J = 7.8 Hz, 2H), 7.40 (m, 3H), 5.97 (s, 1H),
6
6
13
5
.00 (s, 2H); C NMR (150 MHz, DMSO-d ) δ 158.45, 148.1, 142.9,
1
6
(1H,3H)-dione (9b). H NMR (600 MHz, DMSO-d ) δ 10.15 (s, 1H),
6
1
34.2, 129.5, 128.9, 128.4, 99.9, 77.3.
3
8
.53 (s, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.8 Hz, 3H), 7.48 (t,
-(Benzyloxy)-6-chloro-1-methylpyrimidine-2,4(1H,3H)-dione
16). To a solution of 15 (0.50 g, 2.0 mmol) in 5 mL DMF was added
Cs CO (1.3 g, 4.0 mmmol) and MeI (0.57 g, 4.0 mmol), and the
J = 7.8 Hz, 2H), 7.39 (m, 1H), 7.24 (s, 1H), 4.70 (s, 1H), 3.84(s, 3H);
HRMS (ESI-) calcd for C H N O [M − H]- 308.1040, found
(
17
15
3
3
2
3
3
08.1039.
mixture was stirred at 80 °C for 3 h when 15 was consumed
completely as indicated by TLC. The mixture was cooled to room
temperature, and the reaction was quenched by adding 10 mL of
3
-Hydroxy-1-methyl-6-((4′-methyl-[1,1′-biphenyl]-4-yl)amino)-
1
pyrimidine-2,4(1H,3H)-dione (9c). H NMR (600 MHz, DMSO-d ) δ
6
1
0.14 (s, 1H), 8.50 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.8
Hz, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.73 (d, J
8.4 Hz, 2H), 4.68 (s 1H), 3.42 (s, 3H), 3.40 (s, 3H); HRMS (ESI-)
H O.The aqueous was extracted with EtOAc (15 mL × 3), and the
2
combined organics were washed with brine (10 mL × 3) and dried
=
over anhydrous Na SO . Solvent was removed in vacuo, and the
−
2
4
calcd for C H N O [M − H] 322.1197, found 322.1200.
18
17
3
3
residue was purified by flash chromatography on silica gel to give the
3
-Hydroxy-1-methyl-6-((4′-methyl-[1,1′-biphenyl]-3-yl)amino)-
1
1
desired compound 16 (0.32 g, 60%) as offwhite solid. HNMR (600
pyrimidine-2,4(1H,3H)-dione (9d). H NMR (600 MHz, DMSO-d ) δ
6
MHz, CD OD) δ 7.56 (d, J = 7.8 Hz, 2H), 7.38 (m, 3H), 6.03 (s, 1H),
10.15 (s, 1H), 8.52 (s, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4
Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 4.68 (s,
1H), 3.43 (s, 3H), 2.33 (s, 3H); HRMS (ESI-) calcd for C H N O
3
3
5.00 (s, 2H).
General Procedure for the Synthesis of 3-(Benzyloxy)-1-
1
8
17
3
methyl-6-aminopyrimidine-2,4(1H,3H)-dione (17). To a solution
of a biarylamine (0.20 mmol) in dry THF (3 mL) at −78 °C was
added LDA (0.48 mmol). The mixture was stirred at −78 °C for 30
min followed by the addition of HMPA (1.0 mL) and compound 15
[
M − H]- 322.1197, found 322.1185.
3
-Hydroxy-1-methyl-6-((4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-
1
amino)pyrimidine-2,4(1H,3H)-dione (9e). H NMR (600 MHz,
DMSO-d ) δ 10.18 (s, 1H), 8.56 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H),
6
(
0.40 mmol). The resulting mixture was stirred at −78 °C for 30 min,
7.80 (t, J = 7.8 Hz, 4H), 7.35 (d, J = 8.4 Hz, 2H), 4.77 (s, 1H), 3.42 (s,
−
then room temperature overnight before it was quenched by adding 10
mL of water. The aqueous was extracted with EtOAc (10 mL × 3),
and the combined organics were washed with brine and dried over
Na SO . After the solvent was removed in vacuo, the residue was
3H); HRMS (ESI-) calcd for C H F N O [M − H] 376.0987,
18
14
3
3
3
found 376.0990.
3-Hydroxy-6-((4′-hydroxy-[1,1′-biphenyl]-3-yl)amino)-1-methyl-
1
pyrimidine-2,4(1H,3H)-dione (9f). H NMR (600 MHz, DMSO-d ) δ
6
2
4
purified by flash column (Hexanes: EtOAc = 1:1) to give compound
7 (55−83%).
-(Benzyloxy)- 6-([1,1′-biphenyl]-4-ylamino)-1-methylpyrimi-
10.14 (s, 1H), 9.58 (s, 1H), 8.49 (s, 1H), 7.47 (d, J = 6.0 Hz, 2H), 7.43
(m, 2H), 7.14 (s, 1H), 6.84 (d, J = 6.0 Hz, 2H), 4.66 (s, 1H), 3.43 (s,
1
3
3H); HRMS (ESI-) calcd for C17
324.0993.
H N O [M − H]- 324.0990, found
15 3 4
1
dine-2,4(1H,3H)-dione (17a). H NMR: (DMSO-d ) δ 8.66 (s, 1H),
6
7
6
.74 (m, 2H), 7.69 (m, 2H), 7.53 (m, 2H), 7.48 (m, 2H), 7.40 (m,
H), 4.97 (s, 2H), 4.70 (s, 1H), 3.46 (s, 3H), 3.30 (s, 3H).
General Procedure for the Synthesis of 3-(Benzyloxy)-1-
3-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-4-yl)amino)-1-methyl-
1
pyrimidine-2,4(1H,3H)-dione (9g). H NMR (600 MHz, DMSO-d ) δ
6
10.63 (s, 1H), 8.48 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.61 (t, J = 9.0
Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 4.66 (s,
methyl-6-oxopyrimidine-2,4(1H,3H)-dione (18). This chemotype
was synthesized following the same procedure as described for the
synthesis of 17.
1H), 3.77 (s, 3H), 3.42 (s, 3H); HRMS (ESI-) calcd for C18
H
N
O
17
3
4
−
[M − H] 338.1146, found 338.1150.
6
-([1,1′-Biphenyl]-4-yloxy)-3-(benzyloxy)-1-methylpyrimidine-
3-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-4-yl)amino)-1-methyl-
1
1
2
2
2
,4(1H,3H)-dione (18a). H NMR: (CDCl ) δ 7.67 (m, 2H), 7.61 (m,
pyrimidine-2,4(1H,3H)-dione (9h). H NMR (600 MHz, DMSO-d ) δ
6
3
H), 7.58 (m, 2H), 7.49 (m, 2H), 7.41 (m, 4H), 7.20 (m, 2H), 5.14 (s,
H), 4.87 (s, 1H), 3.58 (s, 3H).
10.15 (s, 1H), 8.52 (s, 1H), 7.71 (d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.2
Hz, 1H), 7.30 (t, J = 8.4 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.18 (s,
1H), 6.92 (dd, J = 3.0, 8.4 Hz, 1H), 4.70 (s, 1H), 3.80 (s, 3H), 3.42 (s,
3H); HRMS (ESI-) calcd for C H N O [M − H]- 338.1146, found
General Procedure for the Synthesis of 3-(Benzyloxy)-6-
aminopyrimidine-2,4(1H,3H)-dione (19). To a microwave reaction
vessel were added compound 15 (1.0 mmol), biaryl amine (1.5
18
17
3
4
338.1157.
2
654
DOI: 10.1021/acs.jmedchem.5b01879
J. Med. Chem. 2016, 59, 2648−2659

Journal of Medicinal Chemistry
Article
3
-Hydroxy-6-((2′-methoxy-[1,1′-biphenyl]-4-yl)amino)-1-methyl-
4′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
1
1
pyrimidine-2,4(1H,3H)-dione (9i). H NMR (600 MHz, DMSO-d ) δ
1
7
4-yl)amino)-[1,1′-biphenyl]-3-carbonitrile (9t). H NMR (600 MHz,
6
0.17 (s, 1H), 8.51 (s, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.59 (m, 1H),
.33 (m, 1H), 7.28 (d, J = 7.8 Hz, 2H), 7.13 (d, J = 8.4 Hz, 1H), 7.05
DMSO-d ) δ 10.20 (s, 1H), 8.59 (s, 1H), 8.14 (s, 1H), 7.02 (d, J = 8.4
6
Hz, 1H), 7.79 (d, J = 7.8 Hz, 3H), 7.65 (t, J = 7.8 Hz, 1H), 7.34 (d, J =
(
(
t, J = 7.8 Hz, 1H), 4.72 (s, 1H), 3.78 (s, 3H), 3.45 (s, 3H); HRMS
ESI-) calcd for C H N O [M − H]- 338.1146, found 338.165.
7.8 Hz, 2H), 4.78 (s 1H), 3.43 (s, 3H), 3.42 (s, 3H); HRMS (ESI-)
−
calcd for C H N O [M − H] 333.0993, found 333.1003
1
8
17
3
4
18 14
4
3
3
-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-3-yl)amino)-1-methyl-
3′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
1
1
pyrimidine-2,4(1H,3H)-dione (9j). H NMR (600 MHz, DMSO-d ) δ
4-yl)amino)-[1,1′-biphenyl]-4-carbonitrile (9u). H NMR (600 MHz,
6
1
0.20 (s, 1H), 8.54 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4
Hz, 2H), 7.47 (d, J = 6.0 Hz, 3H), 7.47 (d, J = 6.0 Hz, 1H), 7.35 (d, J
8.4 Hz, 2H), 4.69 (s, 1H), 3.78 (s, 3H); HRMS (ESI-) calcd for
DMSO-d ) δ 10.17 (s, 1H), 8.57 (s, 1H), 7.93 (t, J = 8.4 Hz, 2H), 7.86
6
(d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.8 Hz, 1H),
7.31 (d, J = 7.2 Hz, 1H), 4.70 (s, 1H), 4.01 (t, J = 7.2 Hz, 1H), 3.42 (s,
3H); HRMS (ESI-) calcd for C H N O [M − H]- 333.0993, found
=
−
C H N O [M − H] 338.1146, found 338.1149.
18
17
3
4
18 14
4
3
3
-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-3-yl)amino)-1-methyl-
333.1004.
1
pyrimidine-2,4(1H,3H)-dione (9k). H NMR (600 MHz, DMSO-d ) δ
3′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
6
1
1
7
1
3
3
0.15 (s, 1H), 8.52 (s, 1H), 7.51 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H),
.38 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 7.8 Hz,
H), 7.15 (s,1H), 6.94 (dd, J = 1.8, 7.8 Hz, 2H), 4.67 (s, 1H), 3.80 (s,
H), 3.43 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]-
4-yl)amino)-[1,1′-biphenyl]-3-carbonitrile (9v). H NMR (600 MHz,
DMSO-d ) δ 10.18 (s, 1H), 8.59 (s, 1H), 8.12 (s, 1H), 8.00 (d, J = 7.8
6
Hz, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.58 (m,
2H), 7.53 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 7.2 Hz, 1H), 4.66 (s, 1H),
3.43 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]−
18
17
3
4
38.1146, found 338.1156.
-Hydroxy-6-((2′-methoxy-[1,1′-biphenyl]-3-yl)amino)-1-methyl-
1
8
14
4
3
3
333.0993, found 333.1009.
1
pyrimidine-2,4(1H,3H)-dione (9l). H NMR (600 MHz, DMSO-d ) δ
1
6
6-((4-(3,5-Dimethylisoxazol-4-yl)phenyl)amino)-3-hydroxy-1-
1
0.14 (s, 1H), 8.50 (s, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.37 (s, 1H), 7.35
methylpyrimidine-2,4(1H,3H)-dione (9w). H NMR (600 MHz,
(
t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H),
CD OD) δ 7.42 (d, J = 6.0 Hz, 2H), 7.39 (d, J = 6.0 Hz, 2H), 4.93
3
7
1
.17 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 9.0 Hz, 1H), 7.03 (t, J = 7.8 Hz,
H), 4.82 (s 1H), 3.77 (s, 3H), 3.42 (s, 3H); HRMS (ESI-) calcd for
(s, 1H), 3.57 (s, 3H), 2.42 (s, 3H), 2.27 (s, 3H); HRMS (ESI-) calcd
for C H N O [M − H]- 327.1099, found 327.1115.
16
16
4
4
C H N O [M − H]- 338.1146, found 338.1149.
6-((2-(3,5-Dimethylisoxazol-4-yl)phenyl)amino)-3-hydroxy-1-
18
17
3
4
1
6
-((2′,4′-Dimethoxy-[1,1′-biphenyl]-4-yl)amino)-3-hydroxy-1-
methylpyrimidine-2,4(1H,3H)-dione (9x). H NMR (600 MHz,
1
methylpyrimidine-2,4(1H,3H)-dione (9m). H NMR (600 MHz,
DMSO-d ) δ 10.13 (s, 1H), 8.46 (s, 1H), 7.47 (d, J = 9.0 Hz, 2H),
7
9
HRMS (ESI-) calcd for C H N O [M − H]- 368.1252, found
3
CD OD) δ 7.47 (t, J = 6.0 Hz, 1H), 7.51 (t, J = 6.0 Hz, 1H), 7.12
3
6
(d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 5.39 (s, 1H), 3.33 (s,
.23 (dt, J = 2.4, 8.4, 3H), 6.64 (d, J = 2.4 Hz, 1H), 6.60 (dd, J = 2.4,
.0 Hz, 1H), 4.65 (s 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.42 (s, 3H);
3H), 2.18 (s, 3H), 2.04 (s, 3H) ; HRMS (ESI-) calcd for H N O
1
6
16
4
4
[M − H]- 327.1099, found 327.1088.
19
19
3
5
3-Hydroxy-1-methyl-6-((3-(pyrimidin-5-yl)phenyl)amino)-
1
68.1272.
pyrimidine-2,4(1H,3H)-dione (9y). H NMR (600 MHz, DMSO-d ) δ
6
6
-((3′,5′-Dimethoxy-[1,1′-biphenyl]-4-yl)amino)-3-hydroxy-1-
10.63 (s, 1H), 10.06 (s, 1H), 9.18 (s, 1H), 9.10 (s, 2H), 8.69 (s, 1H),
7.68 (m, 1H), 7.64 (m, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8
Hz, 1H), 4.72 (s, 1H), 3.44 (s, 3H); HRMS (ESI-) calcd for
1
methylpyrimidine-2,4(1H,3H)-dione (9n). H NMR (600 MHz,
DMSO-d ) δ 10.17 (s, 1H), 8.57 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H),
6
−
7
(
1
.29 (d, J = 9.0 Hz, 2H), 6.78 (m, 2H), 6.47 (s, 1H), 5.00 (s, 1H), 3.78
C H N O [M − H] 310.1018, found 310.1020.
15
13
5
3
s, 6H)); 13C NMR (150 MHz, DMSO-d ) δ 161.3, 159.7, 152.6,
6
3-Hydroxy-1-methyl-6-((3-(tetrahydrofuran-3-yl)phenyl)amino)-
1
50.3, 142.0, 138.7, 137.4, 128.2, 125.1, 105.0, 99.9, 77.7, 55.7, 30.7;
pyrimidine-2,4(1H,3H)-dione (9z). H NMR (600 MHz, DMSO-d ) δ
6
HRMS (ESI-) calcd for C H N O [M − H]- 368.1252, found
10.63 (s, 1H), 8.42 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.13 (m, 2H),
7.08 (d, J = 8.4 Hz, 2H), 4.47 (d, J = 1.2 Hz, 1H), 4.01 (t, J = 7.2 Hz,
1H), 3.92 (m, 1H), 3.78 (q, J = 7.2 Hz, 1H), 3.54 (t, J = 7.2 Hz, 1H),
3.39 (m, 1H), 3.31 (s, 3H); HRMS (ESI-) calcd for C H N O [M
19
19
3
5
368.1261.
6
-((2′,6′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxy-1-
1
methylpyrimidine-2,4(1H,3H)-dione (9o). H NMR (600 MHz,
1
5
17
3
4
−
DMSO-d ) δ 10.11 (s, 1H), 8.45 (s, 1H), 7.39 (t, J = 7.8 Hz, 1H),
− H] 302.1219, found 302.1217.
6
7.30 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.08 (m, 2H), 6.73
General Procedure for the Synthesis of 3-Hydroxy-1-
methyl-6-oxopyrimidine-2,4(1H,3H)-dione (10). This chemotype
was synthesized following the same procedure as described for the
synthesis of 9.
(
(
d, J = 8.4 Hz, 2H), 4.79 (s, 1H), 3.67 (s, 6H), 3.40 (s, 3H); HRMS
−
ESI-) calcd for C H N O [M − H] 368.1252, found 368.1255.
19
19
3
5
6
-((3′,4′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxy-1-
1
methylpyrimidine-2,4(1H,3H)-dione (9p). H NMR (600 MHz,
6-([1,1′-Biphenyl]-4-yloxy)-3-hydroxy-1-methylpyrimidine-2,4-
1
DMSO-d ) δ 10.14 (s, 1H), 8.51 (s, 1H), 7.51 (t, J = 7.8 Hz, 2H),
(1H,3H)-dione (10a). H NMR (600 MHz, DMSO-d ) δ 10.42 (s,
6
6
7.67 (t, J = 7.2 Hz, 1H), 7.19 (m, 3H), 7.03 (t, J = 8.4 Hz, 1H), 4.64
1H), 7.81 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 7.8 Hz, 2H), 7.49 (t, J = 7.8
Hz, 2H), 7.41 (t, J = 7.8 Hz, 3H), 4.53 (s, 1H), 3.32 (s, 3H); HRMS
(
s, 1H), 3.82 (s, 3H), 3.77 (s, 3H); HRMS (ESI-) calcd for
−
C H N O [M − H]- 368.1252, found 368.1261.
(ESI-) calcd for C H N O [M − H] 309.0954, found 309.0960.
19
19
3
5
17 14
2
4
6
-((2′,4′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxy-1-
6-([1,1′-Biphenyl]-3-yloxy)-3-hydroxy-1-methylpyrimidine-2,4-
1
1
methylpyrimidine-2,4(1H,3H)-dione (9q). H NMR (600 MHz,
(1H,3H)-dione (10b). H NMR (600 MHz, CDCl ) δ 8.37 (s, 1H),
3
CD OD) δ 7.33 (m, 2H), 7.26 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.4
7.59 (m, 4H), 7.47 (t, J = 7.8 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 7.33
(m, 1H), 7.10 (dd, J = 7.8, 1.8 Hz, 1H), 4.96 (s, 1H), 3.62 (s, 3H),
3.42 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]-
3
Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 6.51 (m, 2H), 4.96 (s, 1H), 4.72 (s,
3
H), 3.74 (s, 3H), 3.72 (s, 3H); HRMS (ESI-) calcd for C H N O
19 19 3 5
17 14
2
4
[
M − H]- 368.1252, found 368.1255.
309.0954, found 309.0885.
6
-((3′,5′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxy-1-
6-([1,1′-Biphenyl]-2-yloxy)-3-hydroxy-1-methylpyrimidine-2,4-
1
1
methylpyrimidine-2,4(1H,3H)-dione (9r). H NMR (600 MHz,
(1H,3H)-dione (10c). H NMR (600 MHz, CDCl ) δ 7.50 (dd, J = 2.4,
3
DMSO-d ) δ 10.14 (s, 1H), 8.52 (s, 1H), 7.51 (m, 2H), 7.50 (t, J =
6.0 Hz, 1H), 7.67 (m, 2H), 7.41 (m, 2H), 7.37 (m, 3H), 7.20 (m, 1H),
6
7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 6.75 (s, 2H), 6.50 (s, 1H), 4.66
4.78 (s, 1H), 3.42 (s, 3H); HRMS (ESI-) calcd for C H N O [M −
17
14
2
4
(
s, 1H), 3.78 (s, 6H), 3.43 (s, 3H); HRMS (ESI-) calcd for
H]- 309.0954, found 309.0958.
C H N O [M − H]- 368.1252, found 368.1263.
3-Hydroxy-1-methyl-6-((4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-
19
19
3
5
1
4
′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
oxy)pyrimidine-2,4(1H,3H)-dione (10d). H NMR (600 MHz,
1
4
-yl)amino)-[1,1′-biphenyl]-4-carbonitrile (9s). H NMR (600 MHz,
CDCl ) δ 8.41 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.63 (t, J = 9.0
3
DMSO-d ) δ 10.12 (s, 1H), 8.67 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H),
Hz, 4H), 7.19 (t, J = 7.8 Hz, 2H), 4.88 (s, 1H), 3.56 (s, 3H); HRMS
(ESI-) calcd for C H F N O [M − H]- 377.0755, found 377.0747.
6
7
2
)
.97 (d, J = 7.8 Hz, 2H), 7.89 (d, J = 7.8 Hz, 2H), 7.44 (t, J = 8.4 Hz,
H), 4.88 (s, 1H), 4.01 (t, J = 7.2 Hz, 1H), 3.51 (s, 3H); HRMS (ESI-
calcd for C H N O [M − H]- 333.0993, found 333.0998.
18
13
3
2
4
3-Hydroxy-1-methyl-6-((4′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)-
1
oxy)pyrimidine-2,4(1H,3H)-dione (10e). H NMR (600 MHz,
18
14
4
3
2
655
DOI: 10.1021/acs.jmedchem.5b01879
J. Med. Chem. 2016, 59, 2648−2659

Journal of Medicinal Chemistry
Article
CD OD) δ 7.77 (m, 2H), 7.73 (m, 2H), 7.67 (m, 1H), 7.62 (t, J = 7.8
Hz, 1H), 7.52 (m, 1H), 7.26 (m, 1H), 4.68 (s, 1H), 3.50 (s, 3H);
3-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-2-yl)oxy)-1-methylpyr-
3
1
imidine-2,4(1H,3H)-dione (10q). H NMR (600 MHz, CDCl
) δ 8.32
3
HRMS (ESI-) calcd for C H F N O [M − H]- 377.0755, found
(s, 1H), 7.49 (dd, J = 1.8, 7.8 Hz, 1H), 7.51 (dt, J = 2.4, 7.8 Hz, 2H),
7.31 (m, 1H), 7.18 (dd, J = 1.8, 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H),
6.89 (m, 2H), 4.76 (s, 1H), 3.79 (s, 3H), 3.43 (s, 3H); HRMS (ESI-)
calcd for C H N O [M − H]- 339.0986, found 339.0992.
18
13
3
2
4
377.0763.
3
-Hydroxy-1-methyl-6-((4′-(trifluoromethyl)-[1,1′-biphenyl]-2-yl)-
1
oxy)pyrimidine-2,4(1H,3H)-dione (10f). H NMR (600 MHz,
1
8
16
2
5
CD OD) δ 7.75 (d, J = 7.8 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.60
3-(Benzyloxy)-6-((2′-methoxy-[1,1′-biphenyl]-2-yl)oxy)-1-methyl-
3
1
(
m, 2H), 7.55 (m, 1H), 7.40 (d, J = 7.8 Hz, 1H), 4.57 (s, 1H), 3.42 (s,
pyrimidine-2,4(1H,3H)-dione (10r). H NMR (600 MHz, DMSO-d )
6
3
H); HRMS (ESI-) calcd for C H F N O [M − H]- 377.0755,
δ 10.34 (s, 1H), 7.51 (dt, J = 1.8, 7.8 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H),
7.40 (m, 1H), 7.37 (m, 1H), 7.34 (dt, J = 1.8, 7.8 Hz, 1H), 7.14 (dd, J
= 1.8, 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.96 (t, J = 1.8, 7.8 Hz,
1H), 4.43 (s, 1H), 3.64 (s, 3H), 3.10 (s, 3H); HRMS (ESI-) calcd for
C H N O [M − H]- 339.0986, found 339.0990.
18
13
3
2
4
found 377.0761
3
-Hydroxy-6-((4′-hydroxy-[1,1′-biphenyl]-4-yl)oxy)-1-methylpyri-
1
midine-2,4(1H,3H)-dione (10g). H NMR (600 MHz, CD OD) δ
7
4
3
.59 (m, 2H), 7.39 (m, 2H), 7.18 (m, 2H), 6.79 (d, J = 8.4 Hz, 2H),
1
8
16
2
5
.78 (s, 1H), 3.45 (s, 3H); HRMS (ESI-) calcd for C H N O [M −
17
14
2
5
4′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
1
H]- 325.0830, found 325.0835.
4-yl)oxy)-[1,1′-biphenyl]-4-carbonitrile (10s). H NMR (600 MHz,
3
-Hydroxy-6-((3′-hydroxy-[1,1′-biphenyl]-3-yl)oxy)-1-methylpyri-
CDCl ) δ 7.83 (m, 6H), 7.38 (d, J = 8.4 Hz, 2H), 4.76 (s, 1H), 3.56 (s,
3
1
midine-2,4(1H,3H)-dione (10h). H NMR (600 MHz, CD OD) δ
7
J = 7.8 Hz, 1H), 7.19 (dd, J = 2.4, 9.0 Hz, 1H), 7.07 (d, J = 8.4 Hz,
3
3H); HRMS (ESI-) calcd for C H N O [M − H]- 324.0990, found
17
15
3
4
.59 (d, J = 8.4 Hz, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.45 (s, 1H), 7.26 (t,
3
24.0993.
4
′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
1
3
3
H), 7.03 (s, 1H), 6.80 (dd, J = 2.4, 7.8 Hz, 1H), 4.72 (s, 1H), 3.45 (s,
1
4
-yl)oxy)-[1,1′-biphenyl]-3-carbonitrile (10t). H NMR (600 MHz,
H); HRMS (ESI-) calcd for C H N O [M − H]- 325.0830, found
25.0839.
-Hydroxy-6-((2′-hydroxy-[1,1′-biphenyl]-2-yl)oxy)-1-methylpyri-
17
14
2
4
CD OD) δ 8.01 (s, 1H), 7.96 (d, J = 6.6 Hz, 1H), 7.80 (d, J = 7.8 Hz,
3
2
7
H), 7.73 (s, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H),
.37 (d, J = 8.4 Hz, 2H), 4.74 (s, 1H), 3.55 (s, 3H), HRMS (ESI-)
3
1
midine-2,4(1H,3H)-dione (10i). H NMR (600 MHz, DMSO-d ) δ
6
calcd for C H N O [M − H]- 334.0833, found 334.0840.
18
13
3
4
1
1
3
1.08 (s, 1H), 10.39 (s, 1H), 7.57 (m, 2H), 7.46 (m, 6H), 4.45 (s,
3
′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
H), 3.18 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]-
1
17
14
2
4
4
-yl)oxy)-[1,1′-biphenyl]-4-carbonitrile (10u). H NMR (600 MHz,
25.0830, found 325.0833.
CD OD) δ 7.86 (d, J = 6.0 Hz, 2H), 7.83 (d, J = 6.0 Hz, 2H), 7.75 (d,
3
3
-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-4-yl)oxy)-1-methylpyr-
J = 12.0 Hz, 1H), 7.67 (m, 2H), 7.35 (d, J = 6.0 Hz, 1H), 4.74 (s, 1H),
1
imidine-2,4(1H,3H)-dione (10j). H NMR (600 MHz, CDCl ) δ 7.63
3
3
3
.58 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]-
1
8
13
3
4
(
d, J = 9.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.12 (d, J = 7.8 Hz, 2H),
.05 (d, J = 9.0 Hz, 2H), 5.00 (s, 1H), 3.87 (s, 3H), 3.62 (s, 3H);
HRMS (ESI-) calcd for C H N O [M − H]- 339.0986, found
34.0833, found 334.0833.
′-((1-Hydroxy-3-methyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-
7
3
18
16
2
5
1
4
-yl)oxy)-[1,1′-biphenyl]-3-carbonitrile (10v). H NMR (600 MHz,
3
39.0890
CDCl ) δ 7.8 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.64 (d, J =
3
3
-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-4-yl)oxy)-1-methylpyr-
1
7.8 Hz, 1H), 7.55 (m, 3H), 7.27 (s, 1H), 7.13 (m, 1H), 4.86 (s, 1H),
imidine-2,4(1H,3H)-dione (10k). H NMR (600 MHz, CD OD) δ
3
3
3
.56 (s, 3H): HRMS (ESI-) calcd for C H N O [M − H]-
1
8
13
3
4
7.72 (d, J = 8.4 Hz, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 9.0 Hz,
2H), 7.17 (d, J = 6.6 Hz, 1H), 7.12 (s, 1H), 6.93 (d, J = 8.4 Hz, 1H),
4.78 (s, 1H), 3.84 (s, 3H), 3.56 (s, 3H); HRMS (ESI-) calcd for
34.0833, found 334.0840.
-((4′-Chloro-[1,1′-biphenyl]-4-yl)oxy)-3-hydroxy-1-methylpyri-
6
1
midine-2,4(1H,3H)-dione (10w). H NMR (600 MHz, DMSO-d )
6
C H N O [M − H]- 339.0986, found 339.0989.
18
16
2
5
1
0.47 (s, 1H), δ 7.80 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 7.2 Hz, 2H), 7.47
3
-Hydroxy-6-((2′-methoxy-[1,1′-biphenyl]-4-yl)oxy)-1-methylpyr-
1
(t, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 4.51 (s, 1H), 3.41 (s,
imidine-2,4(1H,3H)-dione (10l). H NMR (600 MHz, CD OD) δ
3
−
3
H); HRMS (ESI-) calcd for C H ClN O [M − H] 343.0491,
7
.63 (d, J = 8.4 Hz, 2H), 7.33 (t, J = 8.4 Hz, 1H), 7.39 (dd, J = 1.2, 7.2
Hz, 1H), 7.25 (d, J = 7.8 Hz, 2H), 7.08 (d, J = 7.8 Hz, 1H), 7.03 (t, J =
.2 Hz, 1H), 4.78 (s, 1H), 3.80 (s, 3H), 3.57 (s, 3H); HRMS (ESI-)
calcd for C H N O [M − H]- 339.0986, found 339.0991.
17 13
2
4
found 343.0483.
6
-((4′-Chloro-[1,1′-biphenyl]-2-yl)oxy)-3-hydroxy-1-methylpyri-
7
1
midine-2,4(1H,3H)-dione (10x). H NMR (600 MHz, DMSO-d ) δ
6
18
16
2
5
1
0.36 (s, 1H), 7.57 (m, 3H), 7.45 (m, 4H), 7.40 (m, 1H), 7.33 (s,
1H), 4.34 (s, 1H), 3.29 (s, 3H); HRMS (ESI-) calcd for
C H ClN O [M − H]- 343.0491, found 343.0485.
3
-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-3-yl)oxy)-1-methylpyr-
1
imidine-2,4(1H,3H)-dione (10m). H NMR (600 MHz, CDCl ) δ
7
Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H), 4.97 (s, 1H), 3.87 (s, 3H), 3.63 (s,
3
.55 (d, J = 7.8 Hz, 1H), 7.51 (m, 3H), 7.29 (s, 1H), 7.05 (d, J = 7.2
17 13
2
4
3
-Hydroxy-1-methyl-6-(4-(pyridin-4-yl)phenoxy)pyrimidine-2,4-
1
(1H,3H)-dione (10y). H NMR (600 MHz, CD OD) δ 8.61 (d, J = 6.0
3
H); HRMS (ESI-) calcd for C H N O [M − H]- 339.09867,
3
18
16
2
5
Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 7.75 (d, J = 6.6 Hz, 2H), 7.43 (d, J
found 339.1004.
=
C
9.0 Hz, 2H), 5.47 (s, 1H), 3.56 (s, 3H); HRMS (ESI-) calcd for
[M − H]- 310.0833, found 310.0828.
General Procedure for the Synthesis of 3-Hydroxy-6-
3
-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-3-yl)oxy)-1-methylpyr-
1
imidine-2,4(1H,3H)-dione (10n). H NMR (600 MHz, CDCl ) δ 7.59
H N O
16 13 3 4
3
(
d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H),
aminopyrimidine-2,4(1H,3H)-dione (11). This chemotype was
synthesized following the same procedure as described for the
synthesis of 9.
7
7
.33 (s, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.10 (m, 2H), 6.96 (dd, J = 2.4,
.8 Hz, 2H), 4.96 (s, 1H), 3.87 (s, 3H), 3.63 (s, 3H); HRMS (ESI-)
calcd for C H N O [M − H]- 339.0986, found 339.0989.
18
16
2
5
3
-Hydroxy-6-((2′-methoxy-[1,1′-biphenyl]-3-yl)oxy)-1-methylpyr-
3-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-3-yl)amino)-
1
1
imidine-2,4(1H,3H)-dione (10o). H NMR (600 MHz, CDCl ) δ 8.37
pyrimidine-2,4(1H,3H)-dione (11a). H NMR (600 MHz, DMSO-d
6
)
3
(
s, 1H), 7.49 (m, 2H), 7.37 (dt, J = 1.2, 8.4 Hz, 1H), 7.33 (m, 1H),
δ 10.56 (s, 1H), 10.07 (s, 1H), 8.36 (s, 1H), 7.66 (d, J = 9.0 Hz, 2H),
7.62 (d, J = 6.6 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 7.2 Hz,
7
5
.30 (dd, J = 1.2, 7.2 Hz, 1H), 7.06 (m, 2H), 6.99 (d, J = 7.8 Hz, 1H),
.04 (s, 1H), 3.82 (s, 3H), 3.61 (s, 3H);); 13C NMR (150 MHz,
1H), 7.26 (d, J = 8.4 Hz, 2H), 4.91 (s 1H); HRMS (ESI-) calcd for
−
CDCl ) δ 160.8, 158.2, 156.3, 150.9, 146.2, 141.3, 130.5, 130.0, 129.6,
C
16
H
13
N
3
O
3
[M − H] 294.0957, found 294.0961.
3
1
28.4, 122.2, 120.9, 119.0, 111.2, 79.3, 55.5, 29.5; HRMS (ESI-) calcd
3-Hydroxy-6-((4′-methyl-[1,1′-biphenyl]-4-yl)amino)pyrimidine-
1
for C H N O [M − H]- 339.0986, found 339.0991.
2,4(1H,3H)-dione (11b). H NMR (600 MHz, CD OD) 7.63 (d, J =
3
18
16
2
5
3
-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-2-yl)oxy)-1-methylpyr-
7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.24
(d, J = 7.8 Hz, 2H), 2.38 (s, 3H); HRMS (ESI-) calcd for C H N O
3
1
imidine-2,4(1H,3H)-dione (10p). H NMR (600 MHz, CDCl ) δ 7.47
3
17 15
3
(
(
(
3
dd, J = 3.6, 6.0 Hz, 1H), 7.42 (m, 2H), 7.30 (d, J = 9.0 Hz, 2H), 7.17
dd, J = 3.6, 6.0 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 4.78 (s, 1H), 3.81
s, 3H), 3.42 (s, 3H); HRMS (ESI-) calcd for C H N O [M − H]-
[M − H]- 308.1035, found 308.1035.
3-Hydroxy-6-((4′-hydroxy-[1,1′-biphenyl]-3-yl)amino)-1-methyl-
1
pyrimidine-2,4(1H,3H)-dione (11c). H NMR (600 MHz, CD OD) δ
18
16
2
5
3
39.0986, found 339.0981.
7.50 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.43 (s, 1H), 7.27
2
656
DOI: 10.1021/acs.jmedchem.5b01879
J. Med. Chem. 2016, 59, 2648−2659

Journal of Medicinal Chemistry
Article
(
(
d, J = 7.8 Hz, 2H), 7.17 (m, 1H), 5.09 (s, 1H), 2.39 (s, 3H); HRMS
ESI-) calcd for C H N O [M − H]- 308.1035, found 308.1042.
(d, J = 7.8 Hz, 2H), 4.96 (s, 1H); HRMS (ESI-) calcd for C H N O
3
1
7
12
4
[M − H]- 319.0837, found 319.0831.
1
7
15
3
3
6
-((4′-Ethyl-[1,1′-biphenyl]-4-yl)amino)-3-hydroxypyrimidine-2,4-
6-((4′-Fluoro-[1,1′-biphenyl]-3-yl)amino)-3-hydroxypyrimidine-
1
1
(1H,3H)-dione (11d). H NMR (600 MHz, DMSO-d ) δ δ 10.05 (s,
2,4(1H,3H)-dione (11o). HNMR (600 MHz, DMSO-d ) δ 10.78 (s,
6
6
1
4
H), 8.46 (s, 1H), 7.66 (d, J = 9.0 Hz, 2H), 7.57 (m, 2H), 7.30 (m,
H), 4.89 (s, 1H), 2.66 (q, J = 7.8 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H);
1H), 10.07 (s, 1H), 8.34 (s, 1H), 7.68 (m, 2H), 7.45 (m, 1H), 7.40
(m, 1H), 7.29 (t, J = 8.4 Hz, 2H), 7.19 (d, J = 7.8 Hz, 1H), 4.84 (s
1H); HRMS (ESI-) calcd for C16
found 312.0870.
H12FN O
[M − H]− 312.0863,
3 3
HRMS (ESI-) calcd for C H N O [M − H]- 322.1197, found
3
18
17
3
3
22.1200.
6
-((4′-Chloro-[1,1′-biphenyl]-4-yl)amino)-3-hydroxypyrimidine-
3
-Hydroxy-6-((4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)amino)-
1
1
2
1
1
1
,4(1H,3H)-dione (11p). H NMR (600 MHz, DMSO-d ) δ 10.67 (s,
pyrimidine-2,4(1H,3H)-dione (11e). H NMR (600 MHz, DMSO-d )
δ 10.04 (s, 1H); 8.64 (s, 1H), 7.89 (m,, 2H), 7.79 m, 2H), 7.75 (m,
2
6
6
H), 10.03 (s, 1H), 8.42 (s, 1H), 7.70−7.28 (m, 8H), 4.91 (s, 1H);
3C NMR (150 MHz, DMSO-d ) δ 160.8, 149.5, 149.1, 138.2, 138.0,
H), 7.32 (m, 2H), 4.94 (s, 1H); HRMS (ESI-) calcd for
6
34.5, 132.1, 128.9, 128.1, 127.6, 122.4, 76.4; HRMS-ESI(−) m/z
C H F N O [M − H]- 362.0758, found362.0761.
17
12
3
3
3
calcd for C H ClN O [M − H]-328.0494, found 328.0502.
3
-Hydroxy-6-((4′-hydroxy-[1,1′-biphenyl]-4-yl)amino)pyrimidine-
16 12
3
3
1
6
-((4′-Chloro-[1,1′-biphenyl]-3-yl)amino)-3-hydroxypyrimidine-
2
1
4
1
,4(1H,3H)-dione (11f). H NMR (600 MHz, DMSO-d ) δ 10.61 (s,
6
1
2
,4(1H,3H)-dione(11q). H NMR (600 MHz, DMSO-d ) δ 10.73 (s,
H), 10.01 (s, 1H), 9.53 (s, 1H), 8.30 (s, 1H), 7.59−6.83 (m, 8H),
6
.84 (s, 1H); 13C NMR (150 MHz, DMSO-d ) δ 160.8, 157.0, 149.6,
1H), 10.03 (s, 1H), 8.37 (s, 1H), 7.69−7.22 (m, 8H), 4.87 (s, 1H);
6
13
C NMR (150 MHz, DMSO-d ) δ 160.8, 149.7, 148.9, 140.1, 138.8,
48.9, 136.5, 136.4, 130.2, 127.5, 126.8, 122.9, 115.7, 75.8; HRMS-
6
1
38.3, 132.6, 130.1, 128.9, 128.5,122.8, 121.8, 120.6, 76.2; HRMS-
ESI(−) m/z calcd for C H N O 310.0833 [M − H]-, found
3
16
13
3
4
ESI(−) m/z calcd for C H ClN O [M − H]- 328.0494, found
3
10.0833.
16 12
3
3
28.0500.
3
-Hydroxy-6-((3′-hydroxy-[1,1′-biphenyl]-4-yl)amino)pyrimidine-
1
2
1
4
1
,4(1H,3H)-dione (11g). H NMR (600 MHz, DMSO-d ) δ 10.72 (s,
6
H), 10.06 (s, 1H), 9.52 (s, 1H), 8.53 (s, 1H), 7.61−6.75 (m, 8H),
BIOLOGY
■
(
.92 (s, 1H); 13C NMR (150 MHz, DMSO-d ) δ 160.9, 157.8, 149.8,
6
Reagents. Biologicals. Recombinant HIV-1 reverse transcriptase
26
49.3, 140.8, 137.6, 136.2, 129.9, 127.5, 122.5, 117.2, 114.3, 113.2,
RT) was expressed and purified as previously described. The
7
6.1; HRMS-ESI(−) m/z calcd for C H N O 310.0833 [M − H]-,
16
13
3
4
catalytically active RNase H domain fragment of HIV-1 RT was
expressed from plasmid pCSR231 (a generous gift from Dr. Daria
Hazuda, Merck, West Point, PA) and purified as previously
found 310.0838.
3
-Hydroxy-6-((2′-hydroxy-[1,1′-biphenyl]-4-yl)amino)pyrimidine-
1
2
7
2
1
4
1
,4(1H,3H)-dione (11h). H NMR (600 MHz, DMSO-d ) δ 10.58 (s,
6
described. P4R5 HIV infection indicator cells were obtained from
the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH
(p4R5.MAGI from Dr. Nathaniel Landau). These cells express CD4,
CXCR4, and CCR5 as well as a β-galactosidase reporter gene under
the control of an HIV LTR promoter.
H), 10.00 (s, 1H), 9.52 (s, 1H), 8.29 (s, 1H), 7.55−6.84 (m, 8H),
.86 (s, 1H); 13C NMR (150 MHz, DMSO-d ): δ 160.0, 154.3, 149.7,
6
48.9, 136.4, 134.8, 130.1, 130.0, 128.4, 126.9, 122.0, 119.4, 116.0,
7
5.8; HRMS-ESI(−) m/z calcd for C H N O 310.0833 [M − H]-,
16
13
3
4
found 310.0838.
Chemicals. DNA and RNA oligonucleotides for the preparation of
RNA/DNA duplexes for assay of RNase H activity were purchased
from Trilink (San Diego, CA).
3
-Hydroxy-6-((3′-methoxy-[1,1′-biphenyl]-4-yl)amino)-
1
pyrimidine-2,4(1H,3H)-dione (11i). H NMR (600 MHz, DMSO-d )
δ 10.61 (s, 1H), 10.07 (s, 1H), 8.37 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H),
7
1
HRMS (ESI-) calcd for C H N O [M − H] 324.1063, found
3
6
RNase H Assay. RNase H activity was measured essentially as
2
8
.35 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz,
previously described. Three different RNA/DNA duplex substrates
were used, each assessing a different mode of RNase H cleavage. HTS-
1 (RNA 5′-gaucugagccugggagcu-3′-fluorescein annealed to DNA 3′-
CTAGACTCGGACCCTCGA-5′-Dabcyl) is a high sensitivity duplex
that assesses nonspecific internal cleavage. HTS-2 (RNA 5′-
cugguuagaccagaucugagccugggagcu-3′-fluorescein annealed to DNA
3′-GGTCTAGACTCGGACCCTCGA-5′-Dabcyl) provides a duplex
with a recessed DNA 3′-terminus and measures 3′-DNA directed or
polymerase directed RNase H cleavage. HTS-3 (RNA 5′-accagaucu-
gagccugggagcu-3-fluorescein annealed to DNA 3′-GAC-
CAATCTGGTCTAGACTCGGACCCTCGA-5′-Dabcyl) measures
5′-RNA-directed RNase H cleavage.
H), 7.15(s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.83 (s 1H), 3.80 (s, 3H);
−
17
15
3
4
24.1070.
3
-Hydroxy-6-((4′-methoxy-[1,1′-biphenyl]-3-yl)amino)-
1
pyrimidine-2,4(1H,3H)-dione (11j). H NMR (600 MHz, DMSO-d )
δ 10.62 (s, 1H), 10.17 (s, 1H), 8.36 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H),
7
6
.41 (d, J = 7.8 Hz, 2H), 7.37 (m, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.00
(
d, J = 8.4 Hz, 2H), 4.83 (s 1H),;3.78 (s, 3H), HRMS (ESI-) calcd for
−
C H N O [M − H] 324.1063, found 324.1071.
17
15
3
4
6
-((3′,5′-Dimethoxy-[1,1′-biphenyl]-4-yl)amino)-3-hydroxypyri-
1
midine-2,4(1H,3H)-dione (11k). H NMR (600 MHz, DMSO-d ) δ
1
(
4
6
0.47 (s, 1H), 9.84 (s, 1H), 8.21 (s, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.09
d, J = 8.4 Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H), 6.30 (t, J = 8.4 Hz, 1H),
RT Polymerase Assay. HIV RT polymerase activity was
determined in the presence and the absence of inhibitor using 10
3
.70 (s, 1H), 3.61 (s, 6H); HRMS (ESI-) calcd for C H N O [M −
μM [ H]-TTP and 40 nM poly(rA)-oligo(dT)16 (both obtained from
18
17
3
5
H]- 354.1095, found 354.1089.
PerkinElmer, Waltham, MA) in 50 mM Tris-HCl, pH 7.4 (37 °C)
6
-((2′,4′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxypyri-
containing 60 mM KCl and 5 mM MgCl . Reactions were initiated by
2
1
midine-2,4(1H,3H)-dione (11l). H NMR (600 MHz, DMSO-d ) δ
1
7
the addition of 10 nM WT or mutant RT and carried out for 20 min at
37 °C. Reactions were quenched by 200 μL ice cold 10% TCA
containing 20 mM sodium pyrophosphate and filtered using a 1.2 μm
glass fiber filter 96-well plates (Millipore, Billerica, MA) followed by
sequentially wash with 10% TCA and ethanol. The extent of
radionucleotide incorporation was determined by liquid scintillation
spectrometry.
6
0.63 (s, 1H), 10.07 (s, 1H), 8.41 (s, 1H), 7.45 (t, J = 7.8 Hz, 1H),
.39 (s, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.16
(
d, J = 7.8 Hz, 1H), 6.73 (s, 1H), 6.69 (d, J = 7.8 Hz, 1H), 5.04 (s,
1
3
H), 3.86 (s, 6H); HRMS (ESI-) calcd for C H N O [M − H]-
18
17
3
5
54.1095, found 354.1101.
-((2′,6′-Dimethoxy-[1,1′-biphenyl]-3-yl)amino)-3-hydroxypyri-
6
1
midine-2,4(1H,3H)-dione (11m). H NMR (600 MHz, DMSO-d ) δ
1
(
7
HIV IN Assay. HIV integrase was expressed and purified as
6
2
9
0.54 (s, 1H), 9.98 (s, 1H), 8.25 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.36
t, J = 7.8 Hz, 1H), 7.08 (m, 2H), 7.03 (d, J = 7.8 Hz, 1H), 6.74 (d, J =
.8 Hz, 2H), 4.91 (s, 1H), 3.72 (s, 6H); HRMS (ESI-) calcd for
previously reported. Inhibition assays were performed using a
29
modified protocol of our reported method. Briefly, 2.1 μL of
compound suspended in DMSO was placed in duplicate into a Black
96-well nonbinding plate (corning 3991). Compounds were plated in
duplicate to a final concentration of 0.13−100 μM. To each well of the
plate, 186.9 μL of reaction mixture without DNA substrate was added
C H N O [M − H]- 354.1095, found 354.1089.
18
17
3
5
4
′-((1-Hydroxy-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-
1
amino)-[1,1′-biphenyl]-4-carbonitrile (11n). H NMR (600 MHz,
DMSO-d ) δ 10.72 (s, 1H), 10.05 (s, 1H), 8.55 (s, 1H), 7.90 (d, J =
(10 mM HEPES pH 7.5, 10% glycerol w/v, 10 mM MnCl , 1 mM
6
2
7.8 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.31
DTT, 1 μM integrase). The enzyme was incubated with inhibitor for
2
657
DOI: 10.1021/acs.jmedchem.5b01879
J. Med. Chem. 2016, 59, 2648−2659

Journal of Medicinal Chemistry
Article
1
0 min at 25 °C after which the reaction was initiated by the addition
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ASSOCIATED CONTENT
■
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Supporting Information
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AUTHOR INFORMATION
Corresponding Author
E-mail: wangx472@umn.edu. Phone: +1 (612) 626-7025.
■
*
Notes
The authors declare no competing financial interest.
5
2, 5781−5784.
ACKNOWLEDGMENTS
(16) Williams, P. D.; Staas, D. D.; Venkatraman, S.; Loughran, H. M.;
■
Ruzek, R. D.; Booth, T. M.; Lyle, T. A.; Wai, J. S.; Vacca, J. P.;
Feuston, B. P.; Ecto, L. T.; Flynn, J. A.; DiStefano, D. J.; Hazuda, D. J.;
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and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-
This research was supported by the National Institutes of
Health (AI100890 to S.G.S., M.A.P. and Z.W.) and partially by
the Center for Drug Design, University of Minnesota.
ABBREVIATIONS USED
1
,8-naphthyridin-2(1H)-one scaffold. Bioorg. Med. Chem. Lett. 2010,
■
2
(
0, 6754−6757.
HIV, human immunodeficiency virus; RT, reverse transcriptase;
RNase H, ribonuclease H; pol, polymerase; IN, integrase; ST,
strand transfer; HPD, 3-hydroxypyrimidine-2,4-dione; SAR,
structure−activity relationship; PR, protease; HAART, highly
active antiretroviral therapy; NRTIs, nucleoside RT inhibitors;
NNRTIs, nonnucleoside RT inhibitors; DKA, diketoacid; HID,
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Identification of Highly Conserved Residues Involved in Inhibition
2
-hydroxyisoquinolinedione; RISF, retroviral integrase super-
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