Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b00440

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-Associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing at the C5 position that led to drastically improved RNase H inhibition and significant antiviral activity. Structure-Activity relationship (SAR) concerning primarily the length and flexibility of the two wings revealed important structural features that dictate the potency and selectivity of RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry data also revealed that the RNase H biochemical inhibition largely correlated the antiviral activity.

Full text of DOI:10.1021/acs.jmedchem.7b00440

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Article
Double-winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective
Inhibition against HIV-1 RNase H with Significant Antiviral Activity
Sanjeev Kumar V Vernekar, Jing Tang, Bulan Wu, Andrew D. Huber, Mary C
Casey, Nataliya S. Myshakina, Daniel J. Wilson, Jayakanth Kankanala, Karen
A. Kirby, Michael A Parniak, Stefan G. Sarafianos, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 May 2017
Downloaded from http://pubs.acs.org on May 19, 2017
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Double-winged 3-Hydroxypyrimidine-2,4-diones: Potent and
Selective Inhibition against HIV-1 RNase H with Significant
Antiviral Activity
a
a
# a
b
c
Sanjeev Kumar V. Vernekar , Jing Tang , Bulan Wu , Andrew D. Huber , Mary C. Casey ,
d
a
a
c
Nataliya Myshakina , Daniel Wilson , Jayakanth Kankanala , Karen A. Kirby , Michael A.
Parniak e Stefan G. Sarafianos c, f and Zhengqiang Wang* a
,
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
b
Department of Veterinary Pathobiology, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
c
Department of Molecular Microbiology and Immunology, Christopher S. Bond Life Sciences Center, University of Missouri School of
Medicine, Columbia, MO 65211, USA
d
Department of Natural Science, Chatham University, 1 Woodland Road, Pittsburgh, PA, 15232, USA
e
Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
f
Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA
Abstract
Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H
(
RNase H) remains the only virally encoded enzymatic function yet to be exploited as an
antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted
with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione
(
HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell
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culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing
at the C5 position that led to drastically improved RNase H inhibition and significant antiviral
activity. Structure-activity-relationship (SAR) concerning primarily the length and flexibility of
the two wings revealed important structural features that dictate the potency and selectivity of
RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry
data also revealed a correlation between RNase H biochemical inhibition and the antiviral
activity.
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Introduction
Current management of HIV infection relies primarily on highly active antiretroviral therapy
1
(
HAART) , a combination therapy typically consisting of three antivirals with at least two
distinct mechanisms of action. HAART has proved largely effective with numerous FDA-
approved drugs, particularly those targeting the three virally encoded enzymes: RT, integrase
2
(
IN) and protease (PR). However, since current antiviral therapy does not cure HIV infection,3-4
the required long duration of HAART is expected to eventually lead to the selection of resistant
viral strains and treatment failure. Novel antivirals with unique resistance profiles, particularly
those against viral functions not yet targeted by current HAART, are crucial to combating drug-
resistant viruses. One such novel target is RT-associated RNase H activity.5-6 RT encodes two
distinct domains and enzymatic functions (Figure 1a): a polymerase (pol) domain which carries
out both RNA-dependent and DNA-dependent viral DNA polymerization; and an RNase H
domain which degrades the RNA strand from the RNA/DNA heteroduplex intermediate and
processes primers for the synthesis of both the minus strand and plus strand viral DNA.
Numerous nucleoside RT inhibitors (NRTIs)6-8 and non-nucleoside RT inhibitors (NNRTIs)6, 8-9
targeting the pol domain have been approved by FDA. However, inhibitors of RT-associated
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RNase H have yet to enter the development pipeline, as bona fide RNase H inhibitors remain
elusive. It is noteworthy that while many compounds were reported to inhibit RNase H in
biochemical assays, none conferred antiviral activity via RNase H inhibition. Nevertheless,
attenuated RNase H activities through active site mutation correlated well with reduced levels of
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HIV replication in cell culture, indicating that the functions of RNase H are required for HIV
replication and that small molecules effectively inhibiting RNase H functions in a similar manner
should confer antiviral activities.
(A)
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Figure 1. Targeting HIV RT. (A) Structure of RT (created with PyMOL based on PDB code
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PQU ). The active site of pol is shown in pink and that of RNase H in cyan. The RNA (red) /
DNA (blue) heteroduplex engages with both active sites. Pol is targeted by all current NRTIs and
NNRTIs while bona fide inhibitors of RNase H remain unknown. CN = connection subdomain.
(B) Major chemotypes reported as HIV RNase H active site inhibitors. All chemotypes contain a
chelating triad (magenta); scaffolds 5–7 also feature an aryl or biaryl moiety (cyan) connected
through a methylene or amino linker.
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Current design of RNase H inhibitors exploits mainly the active site, which closely resembles
that of HIV IN,12 and the dependence of catalysis on two divalent metal ions. Accordingly,
reported RNase H inhibitors typically entail a pharmacophore core similar to integrase strand
transfer inhibitors (INSTIs) featuring a chelating triad (Figure 1b).13 Chemotypes known for
active site RNase H inhibition include 2-hydroxyisoquinolinedione (HID, 1),14-16 β-thujaplicinol
(
2),17 dihydroxycoumarin (3),18 diketoacid (4),19 pyrimidinol carboxylic acid (5),20
hydroxynaphthyridine (6)21 and pyridopyrimidone 7.22 Such a strategy has proved viable in
achieving potent RNase H inhibition in biochemical assays. A remaining challenge, however, is
that the observed biochemical inhibition has not been translated into significant antiviral
phenotype in cell culture. While other factors may contribute to the hurdle in achieving antiviral
activity with RNase H-targeting small molecules, the tight binding of much larger DNA/RNA
substrates to RNase H active site may pose a biochemical barrier too steep for the competing
small molecules to overcome. We have long been interested in developing potent and selective
RNase H inhibitors.23-28 Particularly interesting is our reported HPD29-30 subtype 8 designed to
specifically inhibit RNase H. Potent and selective RNase H inhibition was indeed achieved with
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many analogues; however, none showed significant antiviral activity. Interestingly, when 8 is
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docked into the RNase H active site, two distinct binding modes are observed (Figure 2A) with
or without nucleic acid substrate. Without the substrate, 8 binds in a way that the chelating core
(the body) directly interacts with two divalent metal ions while the C-6 biarylamino moiety (the
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wing) makes a key contact with H539 (Figure 2A, left). However, in the presence of the
competing nucleic acid substrate, the wing of 8 is forced to flip, resulting in a possible loss of the
key interaction with H539 (Figure 2A, right). This suggests that 8 may not be able to effectively
compete against the substrate to confer antiviral activity. The two docking poses depicted in
Figure 2A allowed us to envision a double-winged subtype (9) that could make optimized
interactions with H539 with or without substrate. This was confirmed via docking of 9 in the
presence of the substrate (Figure 2B), wherein one of the two wings does interact with both the
H539 and the substrate. However, the synthesis of subtype 9 turned out to be challenging.
Redesign of the C-5 wing by replacing the amino linkage with a synthetically more accessible
2
3
carboxamide linkage generated subtype 10 (Figure 2B). These unsymmetrically double-winged
HPD analogues demonstrated highly potent and selective inhibition against RNase H and
inhibited HIV-1 in cell culture. Herein we report the chemical synthesis, biochemical evaluation
against RNase H, pol and INST, and antiviral activities against HIV-1 of the new HPD subtype
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H539
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S553
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Low synthetic accessibility
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Figure 2. Design of double-winged HPD subtype 10. (A) Docking of single-winged subtype 8
into RNase H active site with (right) or without (left) substrate. With the substrate binding to the
active site, the wing of 8 is forced to flip and the key interaction with H539 is lost. (B)
Introducing a second wing (in blue) at the C-5 position of HPD allows interactions with H539
and nucleic acid substrate (left, docking of 9). Unsymmetrically double-winged subtype 10 is
designed due to synthetic accessibility.
Results and Discussion
Chemistry. Analogues of subtype 10 were synthesized based on our previously reported
procedures (Schemes 1–2).23 The commercially available hydroxyurea 11 was first protected
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with a benzyl group, and the resulting 1-(benzyloxy)urea 12 was subjected to condensation with
diethyl malonate under microwave irradiation to yield cyclic compound 13. Treatment of 13 with
POCl
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preparation of 14 allowed the sequential assembling of the two wings: first the C-6 wing was
introduced by reacting the chloride with amines in the presence of N,N-dimethylaniline under
microwave irradiation to give C-6 amino products 15 in moderate to good yields; then the C-5
wing was assembled via a carboxamidation with commercially available or in situ generated
isocyanates to furnish double-winged compounds 16 in good yields. Alternatively, the C-5
carboxamidation can be achieved via a two-step procedure (Scheme 2) to avoid use of the
unpleasant nitrobenzene as the solvent. In this case, the C-5 site of 15 was first carboxylated with
phenyl chloroformate to give phenylesters 17 which were subsequently aminated with amines
under conventional heating to deliver 16 (Scheme 2). Finally, the synthesis of the desired
compounds 10 was completed through debenzylation via TFA-mediated hydrolysis or catalytic
hydrogenation.
a
Scheme 1 Synthesis of subtype 10
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a
Reagents and conditions: a) KOH, BnBr, MeOH, reflux, 6 h, 91%; b) CH
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, NaOEt,
, N,N-
), PhNO
, 40~50 Psi, MeOH, 4–6 h, 76–92%; g)
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NCl, 50 ºC, 6 h, 88%; d) R -NH
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dimethylaniline, microwave, 170 ºC, 20–25 min, 49-81%; e) R NCO (or R CON
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microwave, 210 ºC, 20–40 min, 52–96%; f) Pd/C, H
TFA, microwave, 120 ºC, 35–55 min, 19–84%.
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a
Scheme 2 Alternative synthesis of chemotype 10
aReagents and conditions: a) ClCO
Ph, pyridine, 60 ºC, 2h, 61–63%; b) amines, dioxane, 120 ºC,
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–12 h, 58–67%; c) Pd/C, H
5–55 min, 19–84%.
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, 40~50 Psi, MeOH, 4–6 h, 76–92%; d) TFA, microwave, 120 ºC,
The design of chemotype 10 analogues concerned mainly the variation of the two wings,
specifically their length (short aryl vs. long biaryl) and the flexibility (flexible when n=1 vs. non-
flexible when n=0). The effects of substituents at the end of each wing were also considered. A
total of 30 analogues were designed and synthesized.
Biology. All analogues of chemotype 10 were first evaluated biochemically for inhibition against
HIV RT-associated RNase H. Due to the tight binding by the substrate to RNase H active site,
achieving ultra-potent RNase H inhibition would be essential for inhibitors to effectively
compete against the substrate. Importantly, RNA cleavage by RNase H is required at multiple
3
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stages of reverse transcription and could involve at least three distinct modes : the random
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internal cleavages, the DNA 3′ end directed and polymerase dependent cleavages and the RNA
5
′ end directed cleavages. Our biochemical assay used the HTS-1 DNA / RNA substrate
specifically designed to probe random internal cleavages, the dominant mode of RNA cutting. In
addition, selective inhibition of the RNase H function of RT is often accompanied by the
inhibition of RT pol. The functions of RT entail a delicate spatial and temporal relationship
between the RNase H and pol domains, and implications of inhibiting one domain on the
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functions of the other are complicated. Moreover, active site inhibitors of RNase H also tend to
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inhibit INST due to similar active site fold and mechanism of catalysis. To gauge the selectivity
in RNase H inhibition by our double-winged compounds, assays measuring the activity of RT
pol and INST were included in our biochemical studies. Finally, selected analogues were also
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tested for cytotoxicity and antiviral potency in a MAGI assay. Previously reported HIV RNase
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H inhibitor trihydroxybenzoyl naphthyl hydrazone (THBNH) was used as the control in all RT
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assays and two FDA-approved INSTIs, raltegravir (RAL) and dolutegravir (DTG) , were used
as controls in both the INST assay and the MAGI antiviral assay. All biological results are
summarized in Tables 1-2. Since the design of chemotype 10 was based on the single-winged
HPD scaffold, compound 8 is listed for comparison.
Biochemical Inhibition. The first set of compounds (10a-c) features a long and nonflexible C5
wing (the left wing) while maintaining the same C-6 wing (the right wing) as in reference
compound 8. Biochemical testing showed that the addition of the new C-5 wing decreased the
RNase H inhibitory potency by 4-5 fold while slightly increasing the potency against RT pol
(10a-c vs 8, Table 1), two undesired trends. The same C-6 wing is preserved with the second set
of compounds (10d-j); however, replacing the long and nonflexible C-5 wing with a short and
flexible wing resulted in drastically improved potencies against RNase H (40–110 nM) along
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with significantly decreased RT pol inhibition (10d, 10f, 10i and 10j, Table 1). These
observations support a desired potent and selective RT RNase H inhibitory profile and indicate
that a short C-5 wing should be preferred over a long one. The next set of compounds (10k-n,
Table 1) features a long C-6 wing with a flexible methylene group. Significantly, all four
analogues exhibited strong biochemical potency against RNase H without inhibiting RT pol at
concentrations up to 10 M. The benefit of the added flexibility in the C-6 wing is evident via
direct comparison of two pairs of analogues: 10k (RNase H IC50 = 0.14 M; pol IC50 > 10 M)
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vs. 10a (RNase H IC50 = 1.0 M; pol IC50 = 1.8 M) and 10n (RNase H IC50 = 0.054 M; pol
IC50 > 10 M) vs. 10d (RNase H IC50 = 0.11 M; pol IC50 >10 M). From this set of
compounds, two additional structure-activity-relationship (SAR) trends concerning the C-5 wing
were revealed: 1) adding a flexible methylene group to the C-5 wing did not produce discernable
inhibitory benefit (10l vs. 10k; 10n vs. 10m); 2) a short wing (10m-n) produced better RNase H
inhibition than a long wing (10k-l), which corroborates the observation from the first two sets of
compounds. Accordingly, our further SAR efforts focused on analogues with a short C-6 wing
(10o-t). When directly compared with corresponding analogues containing a long C-6 wing (10p
vs. 10l; 10q vs. 10m; 10r vs. 10n), the short wing appeared to confer about two-fold higher
potencies. Clearly, both C-5 and C-6 positions prefer a short wing for optimized RNase H
inhibition and the combination of two short wings produced exceptionally potent RNase H
inhibition (10q-t, IC50 = 0.016–0.056 M) and significantly less inhibition against RT pol (IC50
0 M).
Table 1. Biochemical studies of chemotype 10 against RT HIV RNase H and Pol, and INST.
≥
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RT RNase H
IC50 (M) IC50 (M)
RT pol
INST IC50a
R1
R2
Compound
a
a
(M)
NT b
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2.0
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--
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1.0 ± 0.3
1.8 ± 0.5
1
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>100
0.97 ± 0.3
1.6 ± 0.5
>100
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.2 ± 0.3
1.4 ± 0.2
>10
>100
23 ± 9
>100
10c
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.11 ± 0.06
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0
0
.047 ± 0.03
.049 ± 0.02
>10
1
0e
>10
>10
>100
>100
>100
>100
1
0f
0
.040 ± 0.01
1
0g
0.059 ± 0.03
>10
1
0h
0
0
.077 ± 0.03
.050 ± 0.04
8.6 ± 5.7
1
0i
9.5 ± 0.5
>100
1
0j
1
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5
5
6
0
.14 ± 0.09
>10
>10
>10
>10
>10
>100
>100
1
0k
0.11 ± 0.06
1
0l
0
1
2
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8
9
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8
9
0
1
2
3
4
5
6
7
8
9
0
0
.047 ± 0.02
.054 ± 0.02
>100
1
0m
0
1.6 ± 0.7
>100
1
0n
0.12 ± 0.09
0.038 ±
1
0o
>10
>100
>100
1
0p
0
.003
.027 ±
0.002
.032 ±
.006
0
>10
1
0q
0
0.14 ±
0.03
>10
10r
0
0
.23 ±
.04
0
.056 ± 0.02
0.016 ±
>10
>10
1
0s
0t
0
1.5 ± 0.2
1
0
.003
.019 ±
0.005
0
~10
34 ± 8
>100
10u
0.054 ± 0.04
0.019 ±
~10
1
0v
~10
71 ± 33
1
0w
0
.003
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0
.079 ± 0.05
>10
>10
29 ± 11
>100
10x
0.041 ± 0.02
1
0y
10
11
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50
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60
0
.057 ± 0.04
>10
21 ± 6
1
0z
0
.030 ±
.005
0.0040 ±
.002
.038 ± 0.02
>
10
10
2.6 ± 0.5
1
0aa
0
>
3.1 ± 0.7
9.5 ± 1.9
1
0bb
0
0
8.1 ± 1.4
1
0cc
0
.018 ±
7
.7 ± 0.6
3.2 ± 0.5
10dd
0.003
0.2
-
-
-
--
--
0.5
NT
NT
THBNH
RAL
-
>10
0.65 ± 0.1
0
.068 ±
.01
--
--
>10
NT
DTG
0
a
Concentration of a compound inhibiting the enzymatic function by 50%, expressed as the mean
+ standard deviation from at least two independent experiments.
b
NT = not tested.
As for INST, compounds with at least one long wing (10a-p) did not show significant inhibition
at concentrations up to 100 M, with the exception of 10d and 10n. However, when both C-5
and C-6 positions were occupied by short wings, those with a flexible C-5 wing (10r and 10s)
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5
6
inhibited INST at submicromolar concentrations. To minimize the undesired INST inhibition,
further SAR studies involved only analogues with a short and nonflexible C-5 wing (10u-dd) to
probe the effect of phenyl ring substitution. These analogues consist of two subsets: one with a
short and flexible C-6 wing (10u-z) and one with a short and nonflexible C-6 wing (10aa-dd).
These studies demonstrated again that the combination of two short wings consistently conferred
low nanomolar biochemical inhibition against RT RNase H. Particularly notable are analogues
with a para-methyl (10u and 10aa) or para-fluoro (10w and 10bb) substituent which exhibited
unprecedentedly potent RNase H inhibition (IC50 = 0.004–0.030 M). The IC50 of 4 nM with
compound 10bb represents by far the most potent RNase H biochemical inhibition known. In
addition, very little inhibition against RT pol was observed with any of these analogues at
concentrations up to 10 M, with the slight exception of 10cc and 10dd (pol IC50 = 8.1 and 7.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
M, respectively). Interestingly, the flexibility of the C-6 wing also dramatically impacted the
INST inhibition, as compounds with a flexible C-6 wing (10q, 10u-z) did not inhibit INST
significantly (IC50 = 21–>100 M) whereas those with a nonflexible wing (10t, 10aa-dd)
inhibited in the low micromolar range (IC50 = 1.5–9.5 M). Taken together, our biochemical
SAR studies confirmed that introducing a second wing at C-5 substantially improves the RNase
H inhibitory profile and revealed that the combination of a nonflexible short C-5 wing and a
flexible short C-6 wing provides optimized potency and selectivity toward RT RNase H
inhibition.
Antiviral Activity. Selected chemotype 10 analogues inhibiting RNase H with extremely high
potency and selectivity were further evaluated for their ability to inhibit HIV-1 in an antiviral
MAGI assay in parallel with a cytotoxicity assay. This assay measures HIV infection in indicator
32
cells (P4R5) through the expression of a Tat-dependent reporter (β-galactosidase). A total of
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twenty three chemotype 10 analogues (10f, 10i-dd) were tested and the results are summarized
in Table 2. The most important observation from our antiviral assay was that fifteen out of the
twenty three tested analogues inhibited HIV-1 replication in low micromolar range (EC50 = 0.6–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1.4 μM, Table 2). The exceptions include compounds 10k-n, which did not inhibit HIV-1 at
concentrations up to 20 M, strongly suggesting that having a long flexible C-6 wing would
produce undesired antiviral profile. By contrast, a long nonflexible C-6 wing appeared viable for
antiviral activity as shown with compounds 10f, 10i and 10j, and a short C-6 wing was
associated with potent antiviral activity, being flexible (10o, q-r, 10u-v) or nonflexible (10s-t,
1
0aa-dd). On the other hand, the nature of the C-5 wing did not influence the antiviral activity
significantly as shown with compounds 10o, 10p and 10q, though most active compounds from
the antiviral assay feature short wings at both C-5 and C-6 positions.
Table 2. Antiviral activity of selected chemotype 10 analogues against HIV-1 in the MAGI
assay.
Antiviral Profile
Biochemical Inhibition c
EC50 a
RNase H IC50
Compound
b
SI d
CC50 (M)
INST IC50 (M)
(M)
(M)
>
25
>100
>100
>100
>100
ND
0.25
--
--
8
6
.3 ± 0.5
.5 ± 0.003
.7 ± 0.2
20
>20
0.049 ± 0.02
0.077 ± 0.028
0.050 ± 0.036
0.14 ± 0.09
>100
>100
>100
>100
>100
>2.0 x 103
1
0f
0i
0j
0k
0l
>1.3 x 103
1
1
>2.0 x 103
3
1
>
>0.71 x 103
>0.91 x 103
1
>100
0.11 ± 0.06
1
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>
20
20
ND
ND
0.047 ± 0.02
0.054 ± 0.02
0.12 ± 0.09
>100
1.6 ± 0.7
>100
>2.1 x 103
30
10m
10n
10o
>
1
5 ± 1.6
20
.9 ± 0.2
.4 ± 0.04
54 ± 9
>100
>0.83 x 103
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
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9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
>2.6 x 103
>
0.038 ± 0.003
0.027 ± 0.002
0.032 ± 0.006
0.056 ± 0.02
0.016 ± 0.003
0.019 ± 0.005
0.054 ± 0.037
0.019 ± 0.003
0.079 ± 0.050
0.041 ± 0.023
0.057 ± 0.039
0.030 ± 0.005
0.0040 ± 0.002
0.038 ± 0.016
0.018 ± 0.003
>10
>100
1
1
0p
0q
>3.7 x 103
6
52 ± 3
>100
>100
1
0.14 ± 0.03
0.23 ± 0.04
1.5 ± 0.2
34 ± 8
4.4
4.1
1
0r
0s
0.60 ± 0.9
6.6 ± 0.9
7.2 ± 1.1
>100
1
>100
94
1
0t
53 ± 3
>100
1.8 x 103
>1.9 x 103
1
0u
8
.5 ± 0.5
>100
10v
>20
>20
>20
>20
>100
71 ± 33
29 ± 11
>100
3.7 x 103
10w
0.37 x 103
>100
1
1
0x
0y
>2.4 x 103
0.37 x 103
>100
>100
21 ± 6
1
0z
0aa
0bb
0cc
4
.5 ± 0.1
>100
2.6 ± 0.5
3.1 ± 0.7
9.5 ± 1.9
3.2 ± 0.5
0.65 ± 0.14
87
1
6.1 ± 0.4
3.5 ± 0.2
58.4 ± 0.8
>100
0.78 x 103
0.25 x 103
0.18 x 103
<0.065
1
1
1
1.4 ± 0.6
.03
76.8 ± 3.4
NT
10dd
RAL
0
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0
.02
NT
>10
0.068 ± 0.01
<0.0068
DTG
a
Concentration of a compound inhibiting HIV-1 replication by 50%, expressed as the mean +
standard deviation from at least two independent experiments.
b
10
11
12
13
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16
17
18
19
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60
Concentration of a compound causing 50% cytotoxicity, expressed as the mean + standard
deviation from at least two independent experiments.
c
Significant RT pol inhibition was not observed against any of these compounds (pol IC50s >7.7
M).
d
Biochemical selective index, defined as INST IC50 / RNase H IC50
.
To demonstrate the potential correlation between the observed antiviral activity and the
biochemical targets, the IC50s for inhibition against RNase H and INST, and the biochemical
selective index, are also listed in Table 2. Inhibition against RT pol is generally lacking at
concentrations up to 7.7 M, and thus not listed. Interestingly, although the analogue with
nanomolar antiviral activity (10s) inhibited both RNase H and INST potently, the biochemical
inhibition still favored RNase H over INST by a large margin (~4 fold). In fact the vast majority
of the analogues active in our antiviral assay showed either no INST inhibition at 100 M (10f,
10i-j, 10o-q, 10v) or highly selective (>87 fold selectivity) RNase H inhibition over INST
inhibition (10u, 10aa-dd). Compound 10n represents a rare exception where potent inhibition
against RNase H (IC50 = 0.054 M) and INST (IC50 = 1.6 M) did not confer significant
antiviral activity (EC50 > 20 M). Importantly, neither of the two INSTIs, RAL and DTG,
inhibited RNase H in our biochemical assay at concentrations up to 10 M, suggesting that our
RNase H biochemical assay is selective. All these observations are in line with RNase H
inhibition as the potential antiviral mechanism of action. Notably, compared with the ultra-potent
RNase H inhibition (low nanomolar), the level of HIV-1 inhibition was moderate (low
micromolar to submicromolar). This may reflect the underlying challenge for small molecule
inhibitors to compete against the tight binding substrate. Nevertheless, considering that the
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6
single-winged chemotype 8 did not inhibit HIV-1 at 25 M, the consistent antiviral activity
observed with the new double-winged chemotype 10 represents a major improvement and
suggests that carefully designed active site inhibitors of RNase H could cut into the substrate
dominance and confer antiviral activity. As far as cytotoxicity is concerned, with the exception
of the mild cytotoxicity associated with 10o (CC50 = 54 M), 10q (CC50 = 52 M), 10u (CC50
0
1
2
3
4
5
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7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=53 M), 10bb (CC50 = 58 M) and 10dd (CC50 = 77 M), no appreciable cytotoxicity was
observed with chemotype 10 analogues at concentrations up to 100 M.
Conclusions. Based on a previously reported HPD subtype 8 that potently and selectively
inhibited RNase H without inhibiting HIV-1 in cell culture, we have redesigned a new double-
winged HPD chemotype 10 featuring an additional wing at the C-5 position. The biochemical
assays showed that drastically improved RNase H inhibition was achieved when both wings
were short and that the low nanomolar RNase H inhibition was largely selective over RT pol and
INST inhibition. Most importantly, antiviral activity was observed against HIV-1 with these
double winged analogues at submicromolar to low micromolar concentrations. The antiviral
potency appeared to be correlated with the ultra-potent RNase H biochemical inhibition. Further
biophysical and mechanistic studies are currently underway and will be reported in due course.
Nevertheless, the medicinal chemistry reported herein suggests that ultra-potent RNase H
inhibitors could confer significant antiviral activity.
Experimental
Chemistry
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General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents were either purchased (toluene and MeOH) or 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
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
columns (silica) and indicated mobile phase. All moisture sensitive reactions were performed
under an inert atmosphere of ultra-pure 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 Phenomenex Gemini, 5
micron C18 column (250mm x 4.6 mm). HPLC conditions: solvent A = H
TFA, solvent B = MeCN; flow rate = 1.0 mL/min; compounds were eluted with a gradient of
0% MeCN/H O to 100% MeCN for 30 min. Purity was determined by total absorbance at 254
2
O containing 0.1%
2
2
nm. All tested compounds have a purity ≥ 95%.
General procedure 1 for debenzylation via hydrogenation (10a-10dd). To a solution of
compound 16 (100 mg) in MeOH (7.0 mL) was added Pd/C (20 mg, 10%) and the reaction
mixture was degassed and purged with H
2
for three times. The reaction vessel was shaken under
H
2
(40-50 psi) atmosphere at room temperature for appropriate time. The reaction was monitored
by both TLC and MS. The reaction mixture was filtered through a short pad of celite, washed
with MeOH and the solvent was removed in vacco. The solid obtained was further purified by
trituration with MeOH, ethyl acetate and DCM furnished pure compounds 10a-10dd as solid.
General procedure 2 for TFA-mediated debenzylation (10a-10dd). To a microwave reaction
vessel were added compound 16 (0.26 mmol) and TFA (6~10 mL). The reaction vessel was
irradiated at 120 °C for the appropriate time. The reaction was monitored by TLC and MS. The
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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4
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6
reaction mixture was transferred to a round-bottom flask to remove the solvent under reduced
pressure. The crude product was purified by flash chromatography on C18 reverse phase column
(H
2
O-MeOH) or trituration with MeOH, ethyl acetate and DCM yielded pure compounds 10a-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
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8
9
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7
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9
0
1
0dd as solid.
N-([1,1'-Biphenyl]-4-yl)-6-([1,1'-biphenyl]-4-ylamino)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10a). Yield 48%. H NMR (600 MHz, DMSO-d
6
) δ
1
2.52 (s, 1H), 12.17 (s, 1H), 10.45 (s, 1H), 7.76-7.66 (m, 10H), 7.65-7.60 (m, 6H) 7.50-7.35 (m,
1
3
2H); C NMR (150 MHz, DMSO-d
6
) δ 166.2, 162.1, 139.5, 139.3, 137.7, 135.0, 129.0, 128.8,
1
27.6, 127.5, 127.1, 127.0, 126.5, 126.2, 125.4, 120.2, 81.3; HRMS-ESI(-) m/z calcd for
-
C
29
H
21
N
4
O 489.1563 [M-H] , found 489.1563.
4
6
1
d
7
1
-([1,1'-Biphenyl]-4-ylamino)-N-(4'-fluoro-[1,1'-biphenyl]-4-yl)-3-hydroxy-2,4-dioxo-
1
,2,3,4-tetrahydropyrimidine-5-carboxamide (10b). Yield 43%. H NMR (600 MHz, DMSO-
6
) δ 12.51 (s, 1H), 12.06 (s, 1H), 10.46 (s, 1H), 7.76-7.62 (m, 11H), 7.47-7.44 (m, 4H), 7.37-
1
3
.36 (m, 1H), 7.26-7.25 (s, 1H); C NMR (150 MHz, DMSO-d
6
) δ 166.1, 162.4, 160.8, 154.4,
46.9, 139.3, 138.3, 137.6, 136.0, 134.9, 134.1, 129.0, 128.9, 128.2, 128.1, 127.7, 127.5, 127.1,
126.5, 126.2, 125.6, 120.3, 115.7, 115.5, 81.2; HRMS-ESI(-) m/z calcd for C29
H20FN
4
O
4
-
5
07.1469 [M-H] , found 507.1492.
6
-([1,1'-Biphenyl]-4-ylamino)-3-hydroxy-N-(4'-methoxy-[1,1'-biphenyl]-4-yl)-2,4-dioxo-
1
1,2,3,4-tetrahydropyrimidine-5-carboxamide (10c). Yield 54%. H NMR (600 MHz, DMSO-
d
6
1
5
6
) δ 12.52 (s, 1H), 12.00 (s, 1H), 10.42 (s, 1H), 7.74-7.58 (m, 11H), 7.47-7.37 (m, 4H), 6.99-
1
3
.98 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 166.0, 162.0, 158.6, 154.3, 146.8, 139.3, 138.4,
6
36.9, 134.9, 134.9, 132.0, 129.0, 127.7, 127.5, 127.3, 126.6, 126.5, 125.7, 120.3, 114.3, 81.2,
-
5.1; HRMS-ESI(-) m/z calcd for C30
H
23
N
4
O
5
519.1668 [M-H] , found 519.1688.
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N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(biphenylamino)pyrimidine-5-
1
carboxamide (10d). Yield 77%. H NMR (600 MHz, DMSO-d
6
) δ 12.76 (s, 1H), 11.33 (s, 1H),
1
3
1
0.34 (s, 1H), 10.04 (dd, J = 6.0, 4.8 1H), 7.74-7.20 (m, 14H), 4.50 (d, J = 5.4, 2H); C NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
150 MHz, DMSO-d ) δ 167.7, 161.6, 154.0, 147.0, 139.3, 138.1, 135.2, 129.0, 128.4, 127.7,
6
127.5, 127.2, 126.9, 126.5, 125.4, 80.9, 41.9; HRMS-ESI(-) m/z calcd for C24
H
20
N
4
4
O 427.1412
-
[
M-H] , found 427.1414.
N-(4-Fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-
1
(biphenylamino)pyrimidine-5-carboxamide (10e). Yield 19%. H NMR (600 MHz, DMSO-
d ) δ 12.73 (s, 1H), 11.32 (s, 1H), 10.33 (s, 1H), 10.04 (t, J = 6.0, 1H), 7.74-7.15 (m, 13H), 4.48
6
13
(d, J = 6.0, 2H); C NMR (150 MHz, DMSO-d
6
) δ 167.8, 161.6, 161.2 (d, JCF = 240.8 Hz),
154.0, 147.0, 139.3, 138.1, 135.6 (d, JCF = 3.2 Hz), 135.2, 129.2 (d, JCF = 7.7 Hz), 129.0, 127.7,
1
27.5, 126.5, 125.4, 115.1 (d, JCF = 20.6 Hz), 80.9, 41.1; HRMS-ESI(-) m/z calcd for
-
C
24
H19FN
4
O
4
445.1318 [M-H] , found 445.1324.
N-(3-Fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4
1
(biphenylamino)pyrimidine-5-carboxamide (10f). Yield 21%. H NMR (600 MHz, DMSO-d
6
)
δ 12.67 (s, 1H), 10.32 (s, 1H), 10.06 (t, J = 6.0, 1H), 7.72-7.07 (m, 13H), 4.50 (d, J = 6.0, 2H);
13C NMR (150 MHz, DMSO-d
6
) δ 167.8, 162.2 (d, JCF = 241.8 Hz), 161.6, 154.0, 147.0, 142.6
(d, JCF = 6.6 Hz), 139.3, 138.1, 135.2, 130.3 (d, JCF = 7.7 Hz), 129.0, 127.7, 127.5, 126.9, 126.5,
125.4, 123.2 (d, JCF = 3.6 Hz), 113.8 (d, JCF = 21.8 Hz), 113.6 (d, JCF = 20.7 Hz), 80.9, 41.4;
-
HRMS-ESI(-) m/z calcd for C24
H19FN
4
O
4
445.1318 [M-H] , found 445.1315.
N-(2-Fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-
1
(biphenylamino)pyrimidine-5-carboxamide (10g). Yield 48%. H NMR (600 MHz, DMSO-
d ) δ 12.67 (s, 1H), 11.35 (br s, 1H), 10.35 (s, 1H), 10.05 (dd, J = 6.0, 5.4, 1H), 7.74-7.17 (m,
6
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5
5
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5
5
5
5
5
5
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13
1
2
1
3H), 4.54 (d, J = 5.4, 2H); C NMR (150 MHz, DMSO-d ) δ 167.7, 161.6, 160.2 (d, JCF =
6
42.9 Hz), 154.0, 146.97, 139.3, 138.1, 135.1, 129.5 (d, JCF = 4.3 Hz), 129.0 (d, JCF = 10.8 Hz),
29.0, 127.7, 127.5, 126.5, 126.1 (d, JCF = 11.1 Hz), 125.4, 124.4 (d, JCF = 3.3 Hz), 115.2 (d, JCF
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
20.6 Hz), 80.9, 36.0; HRMS-ESI(-) m/z calcd for C24
H19FN
4
4
O 445.1318 [M-H]-, found
445.1324.
N-(3-Chloro-2-fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-
1
(
biphenylamino)pyrimidine-5-carboxamide (10h). Yield 25%. H NMR (600 MHz, DMSO-
d
1
2
6
) δ 12.60 (s, 1H), 11.37 (br s, 1H), 10.35 (s, 1H), 10.10 (dd, J = 6.0, 5.4 Hz, 1H), 7.73-7.20 (m,
13
2H), 4.57 (d, J = 6.0 Hz, 2H); C NMR (150 MHz, DMSO-d
6
) δ 167.8, 161.6, 155.2 (d, JCF
=
=
46 Hz), 154.1, 147.0, 139.3, 138.1, 135.1, 129.2, 129.0, 128.4 (d, JCF = 10.4 Hz), 128.3 (d, JCF
3.4 Hz), 127.7, 127.5, 126.5, 125.4, 125.3 (d, JCF = 4.5 Hz), 119.5 (d, JCF = 17.3 Hz), 80.9, 36.1
-
(
d, JCF = 3.5 Hz); HRMS-ESI(-) m/z calcd for
C
24
H18ClFN
4
O
4
479.0928 [M-H] , found
4
79.0931.
N-(2,4-Difluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(biphenylamino)
1
pyrimidine-5-carboxamide (10i). Yield 55%. H NMR (600 MHz, DMSO-d
6
) δ 12.64 (s, 1H),
1
3
11.35 (br s, 1H), 10.34 (s,1H), 10.04 (s, 1H), 7.73-7.07 (m, 12H), 4.50 (d, J = 5.4 Hz, 2H); C
NMR (150 MHz, DMSO-d ) δ 168.2, 162.7, 162.0, 159.8, 156.8, 154.5, 147.4, 139.8, 138.6,
35.5, 131.3, 129.4, 128.1, 127.9, 126.9, 125.9, 123.0, 122.9, 111.9, 111.8, 104.4, 104.0, 81.3,
6
1
-
36.0; HRMS-ESI(-) m/z calcd for C24
H
18
F
2
N
4
O 463.1223 [M-H] , found 463.1230.
4
N-(2,5-Difluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(biphenylamino)
1
pyrimidine-5-carboxamide (10j). Yield 84%. H NMR (600 MHz, DMSO-d
6
) δ 12.59 (s, 1H),
1
7
1.35 (br s, 1H), 10.35 (s,1H), 10.07 (t, J = 6.0 Hz, 1H), 7.73-7.68 (m, 4H), 7.48-7.37 (m, 5H),
13
.26-7.15 (m, 3H), 4.53 (d, J = 5.4 Hz, 2H); C NMR (150 MHz, DMSO-d ) δ 168.3, 162.0,
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59.3, 157.7, 154.5, 147.4, 139.8, 138.6, 135.5, 129.4, 128.1, 127.9, 126.9, 125.9, 117.2, 116.2,
-
15.6, 115.5, 81.3, 36.3; HRMS-ESI(-) m/z calcd for C24
H
18
F
2
N
4
O
4
463.1223 [M-H] , found
63.1227.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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52
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55
56
57
58
59
60
N-([1,1'-Biphenyl]-4-yl)-6-(([1,1'-biphenyl]-4-ylmethyl)amino)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10k). Yield 92%. H NMR (DMSO-d
6
, 600 MHz) δ
1
7
2.00 (s, 1H), 11.28 (s, 1H), 10.72 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H),
.63-7.59 (m, 6H), 7.46-7.40 (m, 6H), 7.37-7.34 (m, 1H), 7.30 (t, J = 6.6 Hz, 1H), 4.74 (d, J =
1
3
5.4 Hz, 2H); C NMR (DMSO-d
6
, 150 MHz) δ 166.7, 162.3, 155.8, 147.7, 140.2, 140.1, 140.0,
1
38.3, 136.5, 129.4, 129.3, 128.5, 127.9, 127.5, 127.5, 127.4, 127.1, 126.6, 120.6, 80.2, 44.9;
-
HRMS-ESI(-) m/z calcd for C30
H
23
N
4
O 503.1719 [M-H] , found 503.1735.
4
N-([1,1'-Biphenyl]-4-yl)-6-(([1,1'-biphenyl]-4-ylmethyl)amino)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10l). Yield 76%. H NMR (DMSO-d
6
, 600 MHz) δ
1
1.42 (s, 1H), 10.23 (s, 1H), 9.99 (s, 1H), 7.67-7.58 (m, 8H), 7.45-7.41 (m, 4H), 7.40 (d, J = 7.8
13
Hz, 1H), 7.35-7.32 (m, 4H), 4.67 (d, J = 5.4 Hz, 2H), 4.45 (d, J = 6.0 Hz, 2H); C NMR
(
DMSO-d , 150 MHz) δ 168.3, 161.9, 155.6, 147.9, 140.3, 140.1, 139.9, 139.3, 139.2, 136.7,
6
129.4, 129.3, 128.4, 128.2, 128.2, 127.9, 127.7, 127.5, 127.2, 127.1, 127.0, 79.8, 44.7, 41.8;
-
HRMS-ESI(-) m/z calcd for C31
H
25
N
4
O
4
517.1876 [M-H] , found 517.1889.
6
-(([1,1'-Biphenyl]-4-ylmethyl)amino)-3-hydroxy-2,4-dioxo-N-phenyl-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10m). Yield 79%. H NMR (DMSO-d
1.93 (s, 1H), 11.18 (s, 1H), 10.32 (s, 1H), 7.68-7.64 (m, 4H), 7.52 (d, J = 8.4 Hz, 2H), 7.46-7.41
m, 4H), 7.36-7.34 (m, 1H), 7.27 (t, J = 7.8 Hz, 2H), 7.01 (t, J = 7.2 Hz, 1H), 4.72 (d, J = 5.4 Hz,
6
, 600 MHz) δ
1
(
1
3
2
H); C NMR (DMSO-d , 150 MHz) δ 169.1, 161.8, 144.1, 129.4, 129.3, 128.4, 127.9, 127.5,
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427.1406 [M-H]-
1
,
27.1, 120.2, 117.3, 110.0, 80.2, 31.1; HRMS-ESI(-) m/z calcd for C24
H
19
N
4
O
4
found 427.1433.
6
-(([1,1'-Biphenyl]-4-ylmethyl)amino)-N-benzyl-3-hydroxy-2,4-dioxo-1,2,3,4-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
tetrahydropyrimidine-5-carboxamide (10n). Yield 81%. H NMR (DMSO-d
6
, 600 MHz) δ
11.45 (s, 1H), 11.38 (s, 1H), 10.23 (s, 1H), 9.94 (s, 1H), 7.67-7.64 (m, 4H), 7.44 (t, J = 7.8 Hz,
2
1
H), 7.39 (d, J = 7.8 Hz, 2H), 7.36-7.29 (m, 3H), 7.26 (d, J = 7.8 Hz, 2H), 7.21 (t, J = 7.8 Hz,
1
3
H), 4.67 (d, J = 6.0 Hz, 2H), 4.41 (d, J = 5.4 Hz, 2H); C NMR (DMSO-d , 150 MHz) δ 168.2,
6
161.8, 155.4, 147.7, 140.0, 139.9, 139.8, 136.6, 129.4, 128.8, 128.4, 127.9, 127.5, 127.5, 127.4,
-
1
4
27.2, 127.0, 79.7, 44.8, 42.1; HRMS-ESI(-) m/z calcd for C25
H
21
N
4
O
4
441.1563 [M-H] , found
41.1591.
N-([1,1'-Biphenyl]-4-yl)-6-(benzylamino)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10o). Yield 86%. H NMR (DMSO-d
6
, 600 MHz) δ
1
3
1
2.00 (s, 1H), 11.22 (s, 1H), 10.38 (s, 1H), 7.63-7.59 (m, 7H), 7.43-7.38 (m, 4H), 7.34-7.30 (m,
13
H), 4.68 (d, J = 5.4 Hz, 2H); C NMR (DMSO-d
6
, 150 MHz) δ 166.7, 162.3, 155.8, 152.8,
47.7, 140.3, 140.0, 139.6, 138.3, 137.4, 135.3, 134.0, 129.3, 129.2, 128.1, 127.8, 127.5, 127.5,
127.4, 127.2, 126.6, 126.5, 120.6, 119.0, 80.2, 45.2; HRMS-ESI(-) m/z calcd for C24
H
19
N
4
O
4
-
4
27.1406 [M-H] , found 427.1434.
N-([1,1'-Biphenyl]-4-ylmethyl)-6-(benzylamino)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10p). Yield 78%. H NMR (DMSO-d
6
, 600 MHz) δ
1
7
1.41 (s, 1H), 11.32 (s, 1H), 10.23 (s, 1H), 9.96 (s, 1H), 7.61-7.57 (m, 4H), 7.43-7.40 (m, 2H),
1
3
.36-7.28 (m, 8H), 4.61 (d, J = 4.8 Hz, 2H), 4.43 (d, J = 4.8 Hz, 2H); C NMR (DMSO-d , 150
6
MHz) δ 168.2, 161.8, 155.3, 147.7, 140.3, 139.2, 137.4, 129.3, 129.2, 128.2, 128.1, 128.0, 127.8,
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441.1563 [M-H]-,
1
27.7, 127.1, 127.0, 79.7, 45.1, 41.8; HRMS-ESI(-) m/z calcd for C25
H
21
N
4
O
4
found 441.1588.
6
-(Benzylamino)-3-hydroxy-2,4-dioxo-N-phenyl-1,2,3,4-tetrahydropyrimidine-5-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
carboxamide (10q). Yield 84%. H NMR (DMSO-d
6
, 600 MHz) δ 12.03 (s, 1H), 11.88 (s, 1H),
11.33-11.31 (m, 1H), 10.39 (s, 1H), 7.51-7.48 (m, 2H), 7.39-7.37 (m, 2H), 7.34-7.25 (m, 5H),
13
7
1
.03-7.00 (m, 1H), 4.67 (d, J = 6.0 Hz, 2H); C NMR (DMSO-d
6
, 150 MHz) δ 167.0, 166.7,
65.2, 162.3, 157.9, 155.7, 149.1, 147.6, 138.9, 138.8, 137.3, 129.3, 128.1, 127.8, 123.7, 123.5,
-
120.2, 80.2, 45.3; HRMS-ESI(-) m/z calcd for C18
H
15
N
4
O
4
351.1093 [M-H] , found 351.1126.
N-Benzyl-6-(benzylamino)-3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10r). Yield 83%. H NMR (DMSO-d
6
, 600 MHz) δ 11.42 (s, 1H), 11.33 (s, 1H),
10.22 (s, 1H), 9.92 (s, 1H), 7.38-7.36 (m, 2H), 7.30-7.29 (m, 5H), 7.25-7.21 (m, 3H), 4.61 (d, J =
13
6
1
4
.0 Hz, 2H), 4.40 (d, J = 6.0 Hz, 2H); C NMR (DMSO-d
6
, 150 MHz) δ 181.5, 168.2, 161.8,
55.4, 147.7, 139.9, 137.4, 129.2, 128.8, 128.0, 127.8, 127.6, 127.5, 127.2, 96.2, 79.7, 45.1,
-
2.1; HRMS-ESI(-) m/z calcd for C19
H
17
N
4
O
4
365.1250 [M-H] , found 365.1292.
N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(phenylamino)pyrimidine-5-
1
carboxamide (10s). Yield 50%. H NMR (600 MHz, DMSO-d
6
) δ 12.72 (s, 1H), 11.26 (s, 1H),
13
1
0.32 (s, 1H), 10.03 (t, J = 2.4, 1H), 7.43-7.26 (m, 10H), 4.49 (s, 2H); C NMR (150 MHz,
DMSO-d
6
) δ 167.7, 161.6, 154.0, 147.0, 139.3, 135.8, 129.5, 128.4, 127.2, 126.9, 126.5, 124.9,
-
81.7, 41.9; HRMS-ESI(-) m/z calcd for C18
H
16
N
4
O
4
351.1099 [M-H] , found 351.1103.
3
-Hydroxy-2,4-dioxo-N-phenyl-6-(phenylamino)-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10t). Yield 87%. H NMR (DMSO-d
6
, 600 MHz) δ 12.33 (s, 1H), 12.27 (s, 1H),
9
.96 (s, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 6.0 Hz, 2H), 7.32-7.27 (m, 3H), 7.13-6.96 (m,
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
3
1
6
H); C NMR (DMSO-d
6
, 150 MHz) δ 167.3, 165.0, 163.4, 139.4, 129.4, 129.3, 123.2, 120.1,
-
10.0; HRMS-ESI(-) m/z calcd for C17
H
13
N
4
O
4
337.0937 [M-H] , found 337.0961.
-(Benzylamino)-3-hydroxy-2,4-dioxo-N-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
carboxamide (10u). Yield 79%. H NMR (DMSO-d
6
, 600 MHz) δ 11.89 (s, 1H), 11.01 (s, 1H),
10.08 (s, 1H), 7.38-7.34 (m, 4H), 7.31-7.28 (m, 3H), 7.06 (d, J = 7.8 Hz, 2H), 4.63 (d, J = 5.4
13
Hz, 2H), 2.21 (s, 3H); C NMR (DMSO-d
6
, 150 MHz) δ 166.7, 163.4, 162.4, 138.0, 136.6,
1
32.2, 129.6, 129.2, 129.1, 127.8, 120.1, 110.0, 80.3, 44.9, 20.83; HRMS-ESI(-) m/z calcd for
-
C
19
H
17
N
4
4
O 365.1250 [M-H] , found 365.1273.
6
-(Benzylamino)-N-(4-ethylphenyl)-3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10v). Yield 83%. H NMR (DMSO-d
6
, 600 MHz) δ 11.80 (s, 1H), 11.41 (s, 1H),
11.23 (s, 1H), 10.34 (s, 1H), 7.40-7.36 (m, 4H), 7.32-7.29 (m, 3H), 7.10-7.09 (m, 2H), 4.64 (d, J
13
=
5.4 Hz, 2H), 2.50 (q, J = 1.4 Hz, J = 7.5 Hz, 2H), 1.11 (t, J = 7.8 Hz, 3H); C NMR (DMSO-
d
1
3
6
, 150 MHz) δ 166.5, 162.2, 155.7, 147.6, 139.0, 137.4, 136.5, 129.2, 128.5, 128.0, 127.8,
-
20.3, 80.0, 45.2, 27.9, 16.1; HRMS-ESI(-) m/z calcd for C20
H
19
N
4
O
4
379.1406 [M-H] , found
79.1430.
6-(Benzylamino)-N-(4-fluorophenyl)-3-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10w). Yield 78%. H NMR (DMSO-d
6
, 600 MHz) δ 11.95 (s, 1H), 11.42 (s, 1H),
1
0.96 (s, 1H), 10.15 (s, 1H), 7.52-7.50 (m, 2H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 3H), 7.08 (t, J
1
3
= 9.0 Hz, 1H), 4.63 (d, J = 6.0 Hz, 2H); C NMR (DMSO-d
6
, 150 MHz) δ 166.8, 162.4, 159.1,
1
57.5, 137.9, 135.4, 129.1, 127.9, 127.8, 121.9, 121.9, 115.8, 115.7, 80.2, 45.0; HRMS-ESI(-)
-
m/z calcd for C18
H14FN
4
O 369.0999 [M-H] , found 369.1115.
4
6
-(Benzylamino)-N-(2,4-difluorophenyl)-3-hydroxy-2,4-dioxo-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10x). Yield 86%. H NMR (DMSO-d , 600 MHz) δ
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2.03 (s, 1H), 11.49 (s, 1H), 11.04 (s, 1H), 10.40 (s, 1H), 8.20-8.16 (m, 1H), 7.38-7.31 (m, 6H),
1
3
.99 (t, J = 8.4 Hz, 1H), 4.66 (d, J = 5.4 Hz, 2H); C NMR (DMSO-d , 150 MHz) δ 166.7,
6
62.4, 159.7, 155.8, 153.5, 137.3, 129.2, 128.1, 127.8, 123.0, 110.0, 80.1, 45.2; HRMS-ESI(-)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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27
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-
m/z calcd for C18
H
13
F
2
N
4
O
4
387.0905 [M-H] , found 387.0926.
6-(Benzylamino)-3-hydroxy-2,4-dioxo-N-(4-(trifluoromethyl)phenyl)-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10y). Yield 81%. H NMR (DMSO-d
6
, 600 MHz) δ
1
2.31 (s, 1H), 10.83 (s, 1H), 10.21 (s, 1H), 7.72 (d, J = 7.8 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H),
13
7.37-7.35 (m, 2H), 7.32-7.28 (m, 3H), 4.64 (d, J = 4.8 Hz, 2H); C NMR (DMSO-d , 150 MHz)
6
δ 167.2, 162.6, 157.3, 142.7, 137.9, 129.1, 127.9, 127.8, 126.5, 125.8, 119.8, 80.4, 45.3, 20.8;
-
HRMS-ESI(-) m/z calcd for C19
H
14
F
3
N
4
O 419.0967 [M-H] , found 419.1002.
6
-(Benzylamino)-3-hydroxy-N-(naphthalen-1-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10z). Yield 78%. H NMR (DMSO-d
.37 (d, J = 7.2 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.15 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.54 (t,
J = 7.8 Hz, 1H), 7.51-7.48 (m, 2H), 7.37 (t, J = 8.4 Hz, 1H), 7.31-7.30 (m, 3H), 7.22-7.20 (m,
6
, 600 MHz) δ 13.19 (s, 1H), 10.02 (s, 1H),
8
13
1
H), 4.56 (d, J = 4.8 Hz, 2H); C NMR (DMSO-d , 150 MHz) δ 168.1, 165.1, 164.0, 162.6,
6
154.1, 140.8, 135.7, 134.1, 128.9, 128.7, 127.1, 127.0, 126.4, 126.2, 126.1, 125.5, 122.1, 121.5,
-
1
16.3, 82.2, 43.7; HRMS-ESI(-) m/z calcd for C22
H
17
N
4
O
4
401.1250 [M-H] , found 401.1277.
3
-Hydroxy-2,4-dioxo-6-(phenylamino)-N-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10aa). Yield 83%. H NMR (DMSO-d
6
, 600 MHz) δ 12.36 (s, 1H), 12.17 (s, 1H),
9
.94 (s, 1H), 7.51 (d, J = 7.2 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.09 (d,
1
3
J = 7.8 Hz, 3H), 2.23 (s, 3H); C NMR (DMSO-d
6
, 150 MHz) δ 167.1, 163.0, 136.8, 132.1,
1
3
29.7, 129.5, 129.4, 123.1, 120.1, 118.6, 82.0, 20.8; HRMS-ESI(-) m/z calcd for C18
H
15
N
4
O
4
-
51.1093 [M-H] , found 351.1125.
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N-(4-Fluorophenyl)-3-hydroxy-2,4-dioxo-6-(phenylamino)-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10bb). Yield 89%. H NMR (DMSO-d
6
, 600 MHz) δ 12.29 (s, 1H), 12.22 (s,
1
H), 9.92 (s, 1H), 7.58-7.56 (m, 2H), 7.50 (d, J = 6.6 Hz, 2H), 7.32 (t, J = 7.8 Hz, 2H), 7.12 (t, J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
=
7.8 Hz, 3H); C NMR (DMSO-d , 150 MHz) δ 167.1, 163.0, 159.0, 157.4, 155.9, 135.6,
6
129.4, 123.3, 121.9, 115.9, 115.7, 110.0, 93.8, 80.8; HRMS-ESI(-) m/z calcd for C17
H12FN
4
O
4
-
3
55.0843 [M-H] , found 355.0880.
3
-Hydroxy-2,4-dioxo-6-(phenylamino)-N-(4-(trifluoromethyl)phenyl)-1,2,3,4-
1
tetrahydropyrimidine-5-carboxamide (10cc). Yield 84%. H NMR (DMSO-d
6
, 600 MHz) δ
1
7
2.46 (s, 1H), 12.21 (s, 1H), 10.18 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H),
1
3
.46 (d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.2 Hz, 2H), 7.19-7.17 (m, 1H); C NMR (DMSO-d , 150
6
MHz) δ 167.3, 162.9, 142.7, 129.7, 126.6, 125.7, 124.2, 123.4, 120.0, 110.0, 81.8; HRMS-ESI(-)
-
m/z calcd for C18
H
12
F
3
4
N O
4
405.0811 [M-H] , found 405.0847.
3
-Hydroxy-N-(naphthalen-1-yl)-2,4-dioxo-6-(phenylamino)-1,2,3,4-tetrahydropyrimidine-5-
1
carboxamide (10dd). Yield 80%. H NMR (DMSO-d
6
, 600 MHz) δ 12.60 (s, 1H), 12.50 (s,
1
H), 10.54 (s, 1H), 8.27 (d, J = 7.2 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H),
7.69 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.55 (t, J = 7.2 Hz, 1H), 7.48 (t, J = 7.2 Hz,
13
1
H), 7.44-7.41 (m, 2H), 7.37-7.35 (m, 2H), 7.29 (t, J = 7.2 Hz, 1H); C NMR (DMSO-d
6
, 150
MHz) δ 166.9, 163.0, 155.0, 147.3, 136.0, 134.1, 133.7, 130.0, 129.1, 127.2, 126.9, 126.5, 126.3,
126.0, 125.6, 124.3, 121.0, 118.2, 110.0, 81.9; HRMS-ESI(-) m/z calcd for C21
H
15
N
4
4
O 387.1093
-
[
M-H] , found 387.1117.
Biology
Reagents
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Biologicals. Recombinant HIV-1 reverse transcriptase (RT) was expressed and purified as
previously described.36 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 -galactosidase reporter gene under the
control of an HIV LTR promoter.
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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).
RNase H assay
RNase H activity was measured essentially as previously described.31, 37 Full-length HIV RT was
incubated with the RNA/DNA duplex substrate HTS-1 (RNA 5’-gaucugagccugggagcu -3’-
fluorescein annealed to DNA 3’-CTAGACTCGGACCCTCGA -5’-Dabcyl), a high sensitivity
duplex that assesses non-specific internal cleavage of the RNA strand, in the presence of various
concentrations of compound. Results were analyzed using “Prism” software (GraphPad
Software, San Diego, CA) for nonlinear regression to fit dose-response data to logistic curve
models.
RT polymerase assay
RT pol assays were carried out in 96-well plates by measuring the extension of an 18 nucleotide
DNA primer (5'-GTCACTGTTCGAGCACA-3') annealed to a 100 nucleotide DNA template (5'-
ATGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTA
GTCAGTGTGGAATATCTCATAGCTTGGTGCTCGAACAGTGAC-3'). Reactions containing
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