Design, Synthesis, and Biological Evaluations of Hydroxypyridonecarboxylic Acids as Inhibitors of HIV Reverse Transcriptase Associated RNase H

Article abstract of DOI:10.1021/acs.jmedchem.6b00465

Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of chelating two divalent metal ions in the RNase H active site. The hydroxypyridonecarboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis, and biological evaluations of a novel variant of the hydroxypyridonecarboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety. Biochemical studies show that most analogues consistently inhibited HIV RT-associated RNase H in the low micromolar range in the absence of significant inhibition of RT polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC₅₀ = 10 μ). Docking and crystallographic studies corroborate favorable binding to the active site of HIV RNase H, providing a basis for the design of more potent analogues.

Full text of DOI:10.1021/acs.jmedchem.6b00465

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Article
Design, Synthesis and Biological Evaluations of Hydroxypyridone Carboxylic
Acids as Inhibitors of HIV Reverse Transcriptase-Associated RNase H
jayakanth kankanala, Karen A. Kirby, Feng Liu, Lena G. Miller, Eva Nagy, Daniel
J. Wilson, Michael A Parniak, Stefan G. Sarafianos, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00465 • Publication Date (Web): 20 Apr 2016
Downloaded from http://pubs.acs.org on April 21, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Design, Synthesis and Biological Evaluations of
Hydroxypyridone Carboxylic Acids as Inhibitors of HIV
Reverse Transcriptase-Associated RNase H
a
b
a
c
c
Jayakanth Kankanala , Karen A. Kirby , Feng Liu , Lena Miller , Eva Nagy , Daniel
a
c
b
a
J. Wilson , Michael A. Parniak , Stefan G. Sarafianos and Zhengqiang Wang*
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
b
c
Department of Molecular Microbiology and Immunology and Department of Biochemistry, University of Missouri School
of Medicine, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, USA
Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
Abstract
Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H
(
RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails
chemotypes capable of chelating two divalent metal ions in the RNase H active site. The
hydroxypyridone carboxylic acid scaffold has been implicated in inhibiting homologous
HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the
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design, synthesis and biological evaluations of a novel variant of the hydroxypyridone
carboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety.
Biochemical studies show that most analogues consistently inhibited HIV RT-associated
RNase H in the low micromolar range in the absence of significant inhibition of RT
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polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC50
0 M). Docking and crystallographic studies corroborate favorable binding to the active
site of HIV RNase H, providing a basis for the design of more potent analogues.
=
1
1
. Introduction
Current HIV antivirals target three of the four virally encoded enzymes (RT polymerase,
1
IN and protease), as well as viral entry proteins and cellular co-receptors. These drugs
2
form the basis for current combination drug therapy of HIV infection (HAART) by virtue
of their distinct mechanisms of action. However, since current HIV therapeutics involve
only a few of possible targets, the efficacy of HAART is plagued by the inevitable
emergence of HIV mutants resistant to current drugs. Thus, the development of antivirals
targeting underexplored and unvalidated processes required for HIV replication may be
key to combating current drug-resistant viral strains. One such target is the RT associated
3
4
RNase H activity. RT encodes two distinct enzymatic functions : the polymerase activity
that carries out both RNA and DNA dependent DNA polymerization; and the RNase H
function that selectively degrades RNA from the RNA/DNA heteroduplex intermediate.
5
The well-known drug families of nucleoside RT inhibitors (NRTIs) and non-nucleoside
6
RT inhibitors (NNRTIs) all target the DNA polymerase function of RT; the RNase H
function remains a clinically unvalidated target as none of its inhibitors has advanced to
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development pipeline. Nevertheless, the crucial role of RNase H in HIV replication has
been well recognized as research showed that active site mutations associated with reduced
8
RNase H biochemical activity conferred attenuated HIV replication in cell culture. This
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suggests that potent and selective RNase H inhibition should lead to effective antiviral
activity.
The main medicinal chemistry challenge of targeting HIV RNase H is the the lack of
appropriate chemical scaffolds supporting specific RNase H inhibition and potent antiviral
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activity. Reported RNase H inhibitors generally target the active site by using a chelating
triad (Figure 1a; in magenta) for competitive binding to divalent metals, e.g.
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hydroxyisoquinolinedione (HID, 1), -thujaplicinol (2), and dihydroxycoumarin (3).
More potent and selective RNase H inhibition was achieved with structurally more
elaborate chemotypes, such as diketoacid 4,13 pyrimidinol carboxylic acid 5,14
1
5
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hydroxynaphthyridine 6 and pyridopyrimidone 7, all featuring a hydrophobic aromatic
moiety seemingly important for antiviral activity. Compounds 6–7 are among the very few
RNase H inhibitors with reported antiviral activity. Exploiting a chelating core for
competitive metal binding is a general medicinal chemistry approach for inhibiting a wide
17
array of enzymes homologous to HIV RNase H. For example, the hydroxypyridone
carboxylate core (blue, Figure 1b) for current study has been implicated in influenza
1
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endonuclease inhibitor 8 as well as the FDA-approved HIV IN strand transfer inhibitor
1
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(
INSTI) dolutegravir (DTG) 9 ( Figure 1b). Based on the pharmacophore model of RNase
H inhibitors 4—7, chemotype 10 was designed for selectively inhibiting RNase H. We
report herein the chemical synthesis, biochemical and antiviral evaluations, molecular
modeling, and co-crystal structure of 10.
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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) hydroxypyridone carboxylate (blue) is implicated in influenza
endonuclease inhibitor 8 and HIV INSTI 9. Active site RNase H inhibitor chemotype 10
based on the same chelting core (blue) is designed. An N-1 aryl or biaryl methyl group is
included to mimick scaffolds 5–7.
2
. Results and Discussion
Chemistry. The pyridone carboxylate chelating core was constructed using a modification
1
8
of a previously reported approach. The synthesis began with a benzyl etherification of
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the commercially available ethyl chloroacetoacetate (12). The resulting ether intermediate
(13) was subjected to a one-carbon homologation to yield enamine 14. The subsequent
Claisen condensation followed by an immediate cyclization produced the pyrone
intermediate 15. The conversion of the pyrone ring into pyridone (16) was effected by
heating with an aromatic amine (R = aryl), which upon saponification afforded pyridone
carboxylic acid 17. At this stage, biaryl N-1 substituents were constructed via a Suzuki
coupling reaction with a para-bromopenyl substrate (17, R = 4-bromobenzyl). The final
step of the synthesis involved a debenzylation to yield the designed analogues (10a-z).
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a
Scheme 1 . Synthesis of hydroxylpyridinone carboxylic acids 10a–z.
a
Reagents and conditions: a) NaH, THF, r.t, 16 h, 95% ; b) DMF DMA, toluene, r.t, 30%;
o
c) Ethyl formate, KOtBu, THF, r.t, 75%; d) R-NH
2
, EtOH, 100 C, 30-40 min, MW, 40-
o
8
5%; e) 2N NaOH, EtOH, 90 C, 4-6 h, 65-95%; f) ArB(OH)
2
, K
2
CO
3
, Pd(PPh
3
4
) ,
o
o
DME/H O (4:1), 150 C, 30-40 min, MW, 60-80%; g) TFA, 80 C, 20 min, MW, 40-87%.
2
Additional carboxylate derivatives were prepared to provide preliminary structure-activity-
relationship (SAR) data (Scheme 2). Direct debenzylation of pyridone carboxylate 16d
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provided an ester analogue (18), and a carboxamide analogue (20) was prepared from 16d
via amination and debenzylation. A structurally more elaborate N-benzyl substituted
carboxamide (22) was synthesized via a three step sequence involving saponification,
amide coupling and debenzylation.
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a
Scheme 2 . Synthesis of hydroxylpyridinone carboxylate analogues.
a
o
Reagents and conditions: a) 7N NH / MeOH, r.t, 48 h, 70%; b) TFA, 80 C, 20 min, MW,
3
o
65—78% c) 2N NaOH, EtOH, 90 C, 4 h, 87% d) benzyl amine, HATU, DIPEA, DMF,
r.t, 12 h, 72%.
Biology: All compounds were evaluated biochemically for inhibition in HIV RT RNase H
and polymerase assays. Selected compounds were also tested in an HIV IN strand transfer
assay. Antiviral screening was conducted using a HIV-1 single replication cycle assay.
RNase H inhibition: Inhibition of RT-associated RNase H inhibitory activity was
evaluated in a biochemical assay20 using three different oligonucleotide duplexes as
substrates to probe three distinct modes of RNase H cleavages: HTS-1 for internal
cleavages which likely represent the primary mode of RNase H cleavage; HTS-2 for the
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DNA 3’ end-directed cleavages allowing specific RNA cuts 17—18 nucleotides
downstream from the polymerase active site; and HTS-3 for the RNA 5’ end-directed
cleavages of recessed RNA templates. Assay results are summarized in Table 1. Most of
the hydroxypyridone carboxylate analogues inhibited the internal cleavage (HTS-1) by
RNase H in the low- to sub-micromolar range (IC50 = 0.65–18 M), with some exceptions
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(
10a–c, 10n–p). Secondly, analogues bearing a two-ring (10r–t) or a biaryl (10r–z)
substituent at N-1 position demonstrated significantly more potent inhibition (IC50 = 0.65–
.7 M) than those with a single-ring substituent (10b–p, IC50 = 12–>25 M), with 10q
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being the sole exception. N-1 unsubstituted (10a) and phenyl substituted (10b) analogues
were both inactive. These observations strongly corroborate the pharmacophore model that
an aryl or biaryl ring separated from the chelating core with a one-atom linker is required
for RNase H inhibition and that the biaryl confers better inhibition than the aryl (Fig. 1a,
5
–7). Third the two submicromolar inhibitors, 10x and 10y, both have H-bond acceptors
and / or donors at the terminus of the biaryl substituent, suggesting interaction of the biaryl
with the nucleic acid substrate.
The broadest range of inhibition across all compounds was noted with the HTS-1 substrate,
which is not surprising since this substrate was designed to enable highly sensitive
21
detection of inhibitors. No inhibition was observed using the more rigorous substrates
HTS-2 or HTS-3 for analogues with a single ring N-1 substituent (10b–p). For analogues
with a two-ring N-1 substituent, the same substrate bias was observed with compounds
1
0r–u and 10z, whereas others (10v–y) showed nearly equal inhibition with all three
substrates.
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In addition, the biochemical assessment of RNase H inhibition was also carried out with a
catalytically active RNase H domain fragment (Table 1, RNase H Fragment). Nearly all
analogues inhibited the RNase H fragment in low micromolar range. The data with this
fragment confirmed that biaryl substitution confers better inhibition than aryl.
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Our SAR analysis was expanded by exploring the importance of the carboxylate functional
group by synthesizing analogues of 10d: the ester 18, the carboxamide 20 and the 2,4-
diflurobenzyl carboxamide 22. The biochemical assay results showed that esterification
abrogated inhibitory activity (compound 18), whereas the inhibitory activity was preserved
with carboxamide modification (20 and 22). Importantly, the 2,4-difluorobenzyl
carboxamide 22 provided significantly better inhibition of RT polymerase and RNase H
activities compared to the parent carboxylic acid 10d. This same modification may also
provide integrase inhibition, leading to multi-target efficacy in a single compound.
Table 1. Biochemical assay results of N substituted pyridone-carboxylic acid analogues
(10) against HIV RT
a
RNH IC50 (M)
RNH
Fragment
RT Pol IC50 a
e
Compd
R
HTS-1b
HTS-2c HTS-3d
(M)
IC50 (M)
10a
10b
H
>25
>25
>25
>25
>25
>25
22 ± 2.3
>25
>25
>25
1
0c
>25
>25
>25
11 ± 1.5
>25
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0d
0e
17
>25
>25
7.8 ± 0.6
5.3 ± 0.5
>25
1
16 ± 4.5
>25
>25
>25
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1
0f
17 ± 5.2
>25
>25
9.8 ± 0.7
>25
1
0g
13 ± 2.1
17
>25
>25
>25
>25
>25
>25
6.8 ± 0.9
7.8 ± 0.8
4.7 ± 0.5
>25
>25
>25
1
0h
1
0i
14
1
0j
12
>25
>25
4.2 ± 0.4
>25
10k
18 ± 2.8
>25
>25
>25
>25
>25
9.8 ± 0.5
>25
>25
>25
1
0l
1
0m
18 ± 4.5
>25
>25
>25
>25
11 ± 1.9
>25
>25
1
0n
>25
>25
>25
1
0o
>25
>25
>25
>25
>25
25 ± 2.0
10p
0q
>25
14 ± 1.1
4.8 ± 0.2
>25
>25
1
0
5 ±
.5
20 ±
2.2
1
2.5 ± 0.2
4
.3 ±
5.4 ±
1.1
1
0r
2.7 ± 0.6
7.0 ± 0.7
1.9 ± 0.1
3.7 ± 0.6
8.5 ± 1.5
19 ± 5.5
0.4
13 ±
1
0s
>25
2
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.1 ±
.5
4.9 ±
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10t
1.5 ± 0.2
8.2 ± 0.1
5.1 ± 0.3
1.8 ± 0.1
>25
25 ±
.5
1
0u
7.7 ± 2.4
2.5 ± 0.4
>25
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1
2.8 ±
0.6
2.9 ±
0.3
1
0v
1.8 ± 0.3
4.4 ± 0.5
2.2 ± 0.1
3.2 ± 0.8
4
0.6
.4 ±
3.9 ±
0.4
1
0w
3.2 ± 0.3
0.90 ±
1.2 ± 0.90 ±
0.1
1
1
0x
0y
1.0 ± 0.01
1.1 ± 0.1
0
.05
0.2
0
0
.65 ±
.05
1.5 ±
0.2
1.0 ±
0.2
1.3 ± 0.05
2.6 ± 0.6
1.2± 0.1
>25
1
0.6
0 ±
15 ±
6.2
1
0z
3.6 ± 0.2
18
--
--
--
>25
>25
>25
>25
>25
>25
>25
2
2
0
2
12 ± 1.8
7.5 ± 0.4
>25
6.8 ± 0.7
3.2 ± 0.2
4.5 ±
4.9 ±
1.0
2.4 ± 0.1
0
.8
a
IC50: concentration of a compound producing 50% inhibition, expressed as mean ±
standard deviation from at least three independent experiments.
b
Substrate that measures internal cleavage.
c
Substrate that measures DNA 3’ end directed cleavage.
d
Substrate that measures RNA 5’ end directed cleavage.
e
Reconstituted HIV RNase H domain.
Polymerase inhibition: All compounds were evaluated for inhibition of RT RNA-
dependent DNA polymerase activity. A sharp SAR trend was noted in which the N-1
unsubstituted analogue 10a and all one-ring substituted analogues (10b–q) showed no
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inhibitory activity (at concentrations up to 25 M) whereas most two-ring substituted
analogues (10r–t and 10v–y) inhibited RT pol with potencies nearly equal to RNase H
inhibition. Although the mechanism of this polymerase inhibition remains unclear, we
speculate that these inhibitors may block the polymerase active site metal-binding triad to
prevent facile catalysis of nucleotide incorporation.
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Order-of-addition RNase H inhibition assays: To determine whether lead analogues retain
inhibitory activity when added after the RNA/DNA substrate we performed order-of-
addition assays to examine the effect of various pre-incubation conditions on RT-
associated RNase H inhibition. We tested six of the most potent inhibitors of RT-associated
RNase H using β-thujaplicinol for comparison under conditions similar to those run for the
in vitro RNase H inhibition assays in which the RNA/DNA substrate is in excess of the RT
(Table 1). Generally, the inhibitors are most potent against RNase H activity when they are
added to RT before the RNA/DNA substrate (Table 2). When the RT/substrate complex is
formed before addition of compound, the inhibitor potency is decreased in all cases,
2
2
including the previously published β-thujaplicinol. Under these conditions, analogue 10x
is the most potent of the inhibitors against cleavage of the pre-formed RT/substrate
complex (>50% inhibition), suggesting it can compete with the substrate for binding to RT.
When these assays were carried out using RT in excess of the RNA/DNA substrate, which
more closely resembles actual conditions in the virion, generally the analogues more
efficiently maintain effective inhibition against the pre-formed RT/substrate complex.
Under conditions of excess RT compared to RNA/DNA substrate, 10y is the most potent
inhibitor of the RT/substrate complex, as it blocks 80% of the RT-asociated RNase H
activity (Table 3).
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4
4
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4
4
4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Order-of-addition RNase H inhibition assay results
%
Inhibition
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1. Pre-Incubation
(10 min at 37 °C)
. Reaction Intiation
RT + Inhibitor RT + MgCl
2
+
RT + Inhibitor + RT + HTS-1
HTS-1
Inhibitor
2
HTS-1 +
MgCl
HTS-1
MgCl
2
Inhibitor +
MgCl
2
2
(10 min at 37 °C)
1
0qa
69% ± 5%b
58% ± 0%
75% ± 5%
68% ± 9%
95% ± 1%
97% ± 1%
100% ± 1%
52% ± 17%
51% ± 7%
84% ± 1%
36% ± 12%
68% ± 10%
79% ± 9%
99% ± 1%
27% ± 4%
35% ± 3%
38% ± 2%
37% ± 7%
66% ± 8%
68% ± 6%
79% ± 8%
21% ± 13%
28% ± 5%
18% ± 13%
21% ± 19%
50% ± 7%
14% ± 3%
12% ± 4%
10r
0t
0v
10x
0y
β-thujaplicinol
1
1
1
aFinal reaction conditions: 100 nM HIV RT, 250 nM HTS-1, 6 mM MgCl
inhibitor.
, 30 µM
2
bValues are reported as mean ± standard deviation from two independent experiments.
Table 3. Order-of-addition RNase H inhibition assay results in the presence of excess RT
%
Inhibition
1. Pre-Incubation
10 min at 37 °C)
. Reaction Intiation
RT + Inhibitor
RT + HTS-1
(
2
HTS-1 +
Inhibitor +
MgCl
2
MgCl
2
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(2 min at 37 °C)
0qa
70% ± 9%b
1
58% ± 3%
61% ± 8%
15% ± 5%
59% ± 6%
74% ± 4%
80% ± 1%
98% ± 4%
1
0r
82% ± 1%
42% ± 1%
78% ± 2%
91% ± 0%
92% ± 1%
100% ± 0%
10
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23
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25
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27
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1
0t
0v
0x
0y
1
1
1
β-thujaplicinol
aFinal reaction conditions: 300 nM HIV RT, 50 nM HTS-1, 0.5 mM EDTA, 6 mM MgCl
0 µM inhibitor.
,
2
3
bValues are reported as mean ± standard deviation from two independent experiments.
Table 4. Comparison of biochemical inhibition against RT RNase H, RT pol and INST
a
Compd
RT RNase H IC50 (M)
RT Pol IC50 (M)
IN IC50 (M)
10a
>25
>25
>25
>25
>100
1
0b
0c
10d
0e
0f
37
1
>25
17
>25
>25
>25
>25
35
35
1
16 ± 4.5
17 ± 5.2
34
1
>100
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1
0g
0h
0i
10j
0k
0l
0m
0n
13 ± 2.1
17
>25
>25
>100
67
1
1
14
>25
36
0
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8
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0
12
>25
25
1
18 ± 2.8
>25
>25
44.5 ± 18.1
>100
>100
>100
>100
>100
>100
23
1
>25
1
18 ± 4.5
>25
>25
1
>25
1
0o
>25
>25
1
0p
>25
>25
10q
2.5 ± 0.2
2.7 ± 0.6
7.0 ± 0.7
1.5 ± 0.2
7.7 ± 2.4
2.5 ± 0.4
3.2 ± 0.3
>25
1
0r
0s
8.5 ± 1.5
19 ± 5.5
1.8 ± 0.1
>25
1
2.26 ± 0.87
14
10t
0u
0v
0w
10x
0y
0z
18
1
17
1
2.2 ± 0.1
3.2 ± 0.8
22
1
7.79 ± 1.93
16.0 ± 6.5
>100
35.0 ± 10.0
>100
32
0.90 ± 0.05
0.65 ± 0.05
3.6 ± 0.2
>25
1.1 ± 0.1
1.2± 0.1
>25
1
1
>25
2
2
0
2
12 ± 1.8
7.5 ± 0.4
>25
2.4 ± 0.1
>100
a
Data with HTS-1 as substrate
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HIV integrase strand transfer inhibition: HIV IN and RNase H share similar active site
configurations, thus inhibitors of these enzymatic functions entail overlapping
pharmacophore features. Selective inhibition of RNase H over INST is therefore
particularly important in RNase H inhibitor discovery. To assess the selectivity profile of
the new pyridone chemotype, all analogues were tested for INST activity (Table 4).
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While most of the compounds showed some INST inhibitory activity, this inhibitory
activity was much weaker (14 M - > 100 M) than that provided against RT RNase H,
suggesting that the hydroxypyridone carboxylate core represents an interesting chemotype
for future development of specific inhibitors of HIV RNase H.
Antiviral assay: All compounds were screened in a previously reported single replication
cycle antiviral assay 23 to gauge their ability in inhibiting HIV replication in cell culture.
Although most compounds did not inhibit HIV-1 replication in this assay, analogue 10r
inhibited HIV-1 replication in a dose-dependent manner (Figure 2, EC50 = 10 M) without
cytotoxicity at concentrations up to 25 M (CC50 > 25 M). This is an important and
exciting observation as very few RNase H inhibitors to date have been reported to possess
antiviral activity in cell-based assays.
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0
Figure 2. Dose response antiviral effect of 10r against HIV-1. Experiments were done in
triplicate with each data point. The antiviral EC50 is calculated to be 10 M.
Molecular modeling: Docking analysis was performed with Glide XP(v.6.4)24-25 using two
metal sites as a constraint. The predicted binding mode of compound 10r in the active site
of RNase H suggests an interaction between the 4-oxo-1,4-dihydropyridine carboxylic acid
core (chelating triad) and the two active site metal cations (coordinated to essential active
site residues D443, E478, D498 and D549). The hydroxyl group at 5-position of 10r can
form H-bonding interactions with H539 which would stabilize inhibitor positioning near
the active site metal-chelating residues D443, E478, D498 and D549, all of which are
crucial for RT-RNase H activity. The flexible methylene linker at N1 position of the
pyridone core induces a conformational flexibility in the inhibitor that allows Π-stacking
interaction between the naphthalene ring of the ligand and RNase H residues H539 and
A538 and L560. This interaction may limit the conformational flexibility of the loop with
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H539 and thus impact on efficiency of RNase H catalytic activity. Notably, a similar
binding mode was observed for RT complexes with dsDNA or RNA-DNA hybrids as
26
27
reported by Harrison et.al and Sarafianos et.al. The predicted binding mode of 10r is
shown in Figure 3a within the active site of RNase H. Compound 10y was predicted to
bind in an identical fashion to that of 10r. Compounds 10r and 10y were observed to
overlay with the pyrimidinol carboxylic acid core (5) and mimic the interactions within the
RNaseH to a greater extent as depicted in Figure 3b and 3c.
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Figure 3. Molecular modeling of analogues 10r and 10y at the RNase H active site and
comparison to crystal structure of the HIV RNase H domain in complex with pyrimidinol
carboxylic acid. a) Predicted binding mode of 10r within active site of RNase H (PDB
code: 3HYF). Active site residues are highlighted in green sticks with metal ions as
spheres. Some chelating and H-bond interactions are depicted as black dotted lines. b)
Overlay of 10r (yellow) on the crystal structure of RNase H with pyrimidinol carboxylic
acid (magenta). c) Overlay of 10y (yellow) on the crystal structure of RNase H with
pyrimidinol carboxylic acid (magenta).
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0
Crystal structure of HIV RT in complex with 10y: In order to further understand the
molecular details of HIV RT-associated RNase H inhibition by hydroxypyridone
carboxylate analogues, we solved the crystal structure of HIV RT in complex with the most
potent RNase H inhibitor from the in vitro assays, 10y, at 2.9 Å resolution. The asymmetric
unit comprises two RT molecules, and therefore two distinct RNase H active sites.
Analogue 10y is only observed at one RT active site in the asymmetric unit, most likely
due to partial occlusion of one RNase H active site by the fingers subdomain of the second
RT molecule. Unlike other RT/RNase H active site-directed inhibitor complex structures,
28-29
the RT/10y complex was crystallized without a non-nucleoside RT inhibitor (NNRTI),
leaving the positions of the two p66 Thumb domains in a closed conformation, similar to
other reported unliganded RT structures.30-34
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Figure 4. X-ray crystal structure of HIV RT in complex with analogue 10y. Cross-eyed
stereo view of 10y (cyan) bound at the RNase H active site of HIV RT. The RNase H
domain of RT is shown in orange, the p51 in light gray. Conserved active site residues are
2+
shown as sticks, and Mg ions are shown as magenta spheres. Chelating and H-bond
interactions are indicated by dashed lines. Cartoon was prepared by PyMOL35 and
crystallographic coordinates have been submitted to the Protein Data Bank (PDB ID:
5
J1E).
2
+
In the occupied RNase H active site, two Mg ions are bound by conserved active site
2
+
residues D443, E478, D498, and D549. Analogue 10y chelates both Mg ions through the
carboxylate, carbonyl, and hydroxyl groups of the pyrimidone (Figure 4), in a manner
29
similar to that of a previously reported RT/pyrimidinol carboxylic acid inhibitor. 10y
interacts directly with RT through interactions between the hydroxyl group of the pyridone
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and H539, and the sulfonamide group of the N-1 substituted biaryl moiety with K540
(Figure 4). These additional interactions with the RT enzyme likely provide increased
stability to the RT/10y complex and may potentially explain the potent RNase H inhibition
observed for 10y in the in vitro assays (Table 1).
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Compared to other analgoues, 10y was found to be the most potent inhibitor of RT-
associated RNase H inihibiton. Structural insights suggest that the length provided by the
N-1 substituted biaryl moiety could be important for RNase H inhibition, since many of
the shorter phenyl-substituted analogues (10b-q) were less potent. It also appears that
charge may also contribute to potent inhibition, as other biaryl-substituted compounds
without charged groups (such as 10w) were less effective inhibitors of RNase H activity.
Furthermore, para substitution of the biaryl moiety relative to the pyridone ring seems to
position the biaryl group in a favorable position to have potential interactions with RT,
which may not be achievable with ortho-substituted analogues such as 10u and may
explain this analogue’s decreased potency.
Conclusions
New hydroxypyridone carboxylate analogues featuring an N-1 benzyl or biarylmethyl
moiety were designed and synthesized as potential inhibitors of HIV RT associated RNase
H. In vitro biochemical assays showed that analogues with a two-ring substituent at N-1
are significantly more potent than those with a one-ring substituent against all three modes
of RNase H cuts as well as the RT polymerase function. While some analogues also
inhibited strand transfer activity of HIV IN, this inhibition was substantially less than that
for RT RNase H inhibition, suggesting that the pyridone chemotype may represent an
2
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interesting scaffold for development of RNase H-specific inhibitors. Importantly,
compound 10r exhibited significant inhibitory activity in a cell-based antiviral assay with
an EC50 of 10 M. Molecular docking of 10r and the crystal structure of RT/10y
corroborate for hydroxypyridone carboxylate analogues a mechanism of active site binding
for RNase H inhibition. The mechanism of the observed polymerase inhibition remains
unclear. These results indicate that the hydroxypyridone carboxylate chemotype previously
implicated in the inhibition of INST and influenza endonuclease can be valuable in the
discovery of HIV antivirals targeting the RT-associated RNase H.
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Experimental
Chemistry: General Procedures. All commercial chemicals were used as supplied unless
otherwise indicated. Flash chromatography was performed on a Teledyne Combiflash RF-
2
00 with RediSep columns (silica) and indicated mobile phase. All moisture sensitive
reactions were performed under an inert atmosphere of ultra-pure argon with oven-dried
1
13
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. 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
compounds were eluted with a gradient of 5% MeCN/H
O from 5-30 min followed by 100% MeCN from 35-40 min. Purity was determined by
total absorbance at 254 nm. All tested compounds have a purity ≥ 98%.
2
O, solvent B = MeCN; flow rate = 1.0 mL/min;
2
O for 0-5 min and to 95% MeCN/
H
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Ethyl-4-(benzyloxy)-3-oxobutanoate (13). To a suspension of 60% NaH (66.83 mmol,
2
.2 eq) in THF (20 mL) at 0 °C was added benzyl alcohol (30.37 mmol, 1.05 eq) and the
resulting mixture was stirred at r.t for 2h. To this mixture was then added ethyl-4-
chloroacetoacetate (30.37 mmol, 1.0 eq) dropwise over 30 min and the resulting clear
yellow solution was stirred at r.t overnight before it was cooled to 5 °C, acidified to pH =
0
1
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0
4
using 5% HCl and extracted with the EtOAc (2 x 25 mL). The combined organics were
washed with brine, dried over Na SO and concentrated in vacuo to yield the desired
2
4
product (13) as orange oil. The crude material was used for next step without further
1
purification. Yield: 95% (6.8 g). H NMR (600 MHz, CDCl
3
) δ 7.40-7.28 (m, 5H), 4.59 (s,
2
H), 4.18 (q, J = 7.1 Hz, 2H), 4.14 (s, 2H), 3.54 (s, 2H), 1.25 (t, J = 7.1 Hz, 3H).
4
-Benzyloxy-2-(1-dimethylaminomethylidene)-3-oxo-butyric acid ethyl ester (14). To
a solution of 13 (28.36 mmol, 1.0 eq) in toluene (30 mL) was added DMF DMA (42.54
mmol, 1.5 eq) and the resulting mixture was stirred at r.t for 12 h. The reaction mixture
was quenched by adding water and was acidified (pH = 4) using 5% HCl. The aqueous
solution was extracted using EtOAc (2 x 20 mL). The combined organics were washed
with NaHCO
3
, and brine, dried over Na
2
SO and concentrated in vacuo. The crude mixture
4
was purified using CombiFlash with eluent starting from hexane/EtOAc (4:1) to
hexane/EtOAc (1:1) to yield the desired product (14) as yellow oil. Yield: 30% (2.45 g);
1H NMR (600 MHz, DMSO-d6) δ 7.67 (s, 1H), 7.38-7.30 (m, 4H), 7.28 (t, J = 6.8 Hz, 1H),
4
.45 (s, 2H), 4.30 (s, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.24 (brs, 3H), 2.73 (brs, 3H), 1.17 (t,
J = 7.1 Hz, 3H).
General method for the synthesis of pyrone: Ethyl-5-(benzyloxy)-4-oxo-4H-pyran-3-
carboxylate (15). To a suspension of potassium tert-butoxide (1M in THF; 15.79 mmol,
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2
.0 eq) in THF (14 mL) was added ethyl formate (33.8 mmol, 4.3 eq) and the mixture was
stirred at r.t for 30 min before compound 13 (7.89 mmol, 1.0 eq) in THF (14 mL) was
added dropwise over 10 min. The resulting mixture was stirred at r.t for 8 h and quenched
by adding 2N HCl (12 mL). After extraction with the EtOAc (2 x 20 mL), the combined
10
11
12
13
14
15
16
17
18
19
20
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22
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organics were washed with NaHCO
3
(2 x 10 mL), water (30 mL), brine (30 mL), dried
over Na SO and concentrated in vacuo to leave brownish yellow oil. This crude product
2
4
was triturated by adding hexanes to yield the desired product (15) as brown solid. Yield:
1
75% (1.62 g); H NMR (600 MHz, DMSO-d6) δ 8.72 (d, J = 0.8 Hz, 1H), 8.30 (d, J = 0.8
Hz, 1H), 7.49 -7.33 (m, 5H), 4.95 (s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1, 3H).
General method for the synthesis of 4-oxo-dihydropyridine esters (16a-16s). To a
solution of 15 (1.0 eq) in EtOH (5 mL) was added amine (2.0 eq) and the resulting mixture
was heated at 100 °C for 40 min under microwave conditions. After the solvent was
removed in vacuo the residue was purified using CombiFlash to produce the crude product
as colorless oil which was triturated with hexanes/EtOAc to yield the titled compound as
colorless solid (16a-16s).
Ethyl-1-benzhydryl-5-(benzyloxy)-4-oxo-1,4-dihydropyridine-3-carboxylate (16a).
1H NMR (600 MHz, DMSO-d6) δ 8.18 (d, J = 1.8 Hz, 1H), 7.48 – 7.40 (m, 6H), 7.36 –
7
.31 (m, 3H), 7.29-7.26 (m, 3H), 7.11 (d, J = 7.0 Hz, 4H), 6.92 (s, 1H), 4.93 (s, 2H), 4.14
(q, J = 7.1 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H).
General procedure for saponification: (17a-17s). To a solution of 16 (1.0 eq) in EtOH
(5 mL) was added 2N NaOH (10 mL). The resulting mixture was heated at 90 °C for 6 h
and was then cooled to r.t and concentrated in vacuo. The resulting solution was acidified
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(
pH = 3) using 2N HCl. The precipitate was collected via filtration, washed with water,
and then dried to obtain the titled compound as colorless solid (17).
1
1
-Benzhydryl-5-(benzyloxy)-4-oxo-1, 4-dihydropyridine-3-carboxylic acid (17a). H
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
NMR (600 MHz, CD OD) δ 8.41 (s, 1H), 7.48 (s,1H), 7.42-7.39 (m, 6H), 7.27-7.26 (m,
3
5H), 7.06 (d, J = 3.9 Hz, 4H), 6.92 (s, 1H), 5.05 (s, 2H).
General procedure for Suzuki coupling (17w-17z). The mixture of 17l (1.0 eq), boronic
acid (1.6 eq), Pd (PPh
3
)
4
(0.065 eq), K
2
CO
3
(3.6 eq) in DME/H O (4:1) was irradiated at
2
150 °C for 30 min under microwave conditions. The black residue formed was filtered
through celite and the solvent was concentrated in vacuo. The resulting aqueous solution
was acidified (pH = 3) using 2N HCl. A white precipitate was obtained via filtration,
washed with water and dried under vacuum to furnish the desired compound as colorless
solid (17w-17z).
1
-((4'-(Aminomethyl)-[1,1'-biphenyl]-4-yl)methyl)-5-(benzyloxy)-4-oxo-1,4-
1
dihydropyridine-3-carboxylic acid (17w). H NMR (600 MHz, DMSO-d6): δ 8.79 (s,
1
H), 8.18 (s, 1H), 7.67 (d, J = 7.8 Hz, 2H), 7.75 – 7.60 (m, 4H), 7.52 – 7.35 (m, 4H), 7.35
–
7.17 (m, 5H), 5.41 (s, 2H), 5.11 (s, 2H), 4.08 (s, 2H).
5
-(Benzyloxy)-1-(4-fluorobenzyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (19). To
a solution of 16d (90 mg) in methanol was added NH in MeOH (7N, 4 mL) and the
3
resulting solution was stirred at r.t for 48 h. A white precipitate was collected via filtration,
washed with methanol, and then dried under high vacuum to yield the carboxamide as
1
colorless solid (19). Yield: 70%. H NMR (600 MHz, DMSO-d6): δ 9.47 (s, 1H), 8.52 (s,
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H), 7.81 (s, 1H), 7.44 (s, 1H), 7.35 (m, 7H), 7.24 – 7.15 (m, 2H), 5.22 (s, 2H), 5.00 (s,
H).
General procedure for amide coupling: 5-(benzyloxy)-N-(2, 4-difluorobenzyl)-1-(4-
10
11
12
13
14
15
16
17
18
19
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fluorobenzyl)-4-oxo-1, 4-dihydropyridine-3-carboxamide (21). To a mixture of 17d
(1.0 eq) in DMF (5 mL) was added HATU (1.2 eq), DIPEA (1.2 eq), amine (1.2 eq) and
the resulting reaction mixture was stirred at r.t for 12 h. The reaction was quenched with
water and extracted with the EtOAc (2 x20 mL). The combined organics were washed with
1
N HCl (2 x 10 mL), NaHCO
3
(2 x 10 mL), water (30 mL), brine (30 mL) and dried over
Na SO . The solvent was removed in vacuo to produce the crude product as yellow solid
2
4
which was purified using CombiFlash with 0-100% EtOAc as an eluent to furnish the titled
1
compound as colorless solid (21). Yield: 72%. H NMR (600 MHz, DMSO-d6) δ 10.61 (t,
J = 5.8 Hz, 1H), 8.57 (d, J = 1.7 Hz, 1H), 7.87 (d, J = 1.7 Hz, 1H), 7.37 (m, 7H, ), 7.22 (m,
4
H), 7.05 (dd, J = 8.4, 6.4 Hz, 1H), 5.26 (s, 2H), 5.02 (s, 2H), 4.51 (d, J = 5.8 Hz, 2H).
General procedure for O-debenzylation: (10a-10z, 18, 20, 22). A mixture of 17 (1.0 eq)
in TFA (2 mL) was irradiated at 80 °C for 20 min under microwave conditions. The solvent
was removed in vacuo to produce the crude product as pale yellow solid which was
triturated in methanol and ether to produce the titled compound as colorless solid (10a-10z,
1
5
8, 20, 22).
-Hydroxy-4-oxo-1, 4-dihydropyridine-3-carboxylic acid (10a). H NMR (600 MHz,
1
13
DMSO-d6): δ 12.74 (s, 1H), 9.84 (s, 1H), 8.31 (s, 1H), 7.69 (s, 1H). C NMR (150 MHz,
DMSO-d6) δ 171.8, 166.6, 148.1, 137.0, 122.4, 113.3. HRMS-ESI (−) m/z calculated for
-
C
6
H
4
NO
4
154.0146 [M-H] , found: 154.0148.
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5
5
5
6
1
5
-Hydroxy-4-oxo-1-phenyl-1,4-dihydropyridine-3-carboxylic acid (10b). H NMR
(
600 MHz, DMSO-d6): 10.22 (s, 1H), 8.46 (d, J = 2.3 Hz, 1H), 8.05 (d, J = 2.3 Hz, 1H),
1
3
7
1
.74-7.68 (m, 2H), 7.63-7.52 (m, 3H). C NMR (150 MHz, DMSO-d6) δ 171.8, 166.0,
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
48.8, 142.9, 139.6, 130.3, 129.6, 125.8, 123.9, 113.8. HRMS-ESI (−) m/z calculated for
-
C
12
H
8
NO
4
230.0459 [M-H] , found: 230.0468.
1
1
-Benzyl-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10c). H NMR (600
MHz, DMSO-d6): δ 10.06 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.51
1
3
–
7.31 (m, 5H), 5.35 (s, 2H). C NMR (150 MHz, DMSO-d6) δ 170.8, 165.0, 148.3, 139.9,
135.4, 128.8, 128.4, 127.9, 125.5, 113.0, 59.8. HRMS-ESI (−) m/z calculated for
-
C
13
H10NO
4
244.0615 [M-H] , found: 244.0627.
1
1
-(4-Fluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10d). H
NMR (600 MHz, DMSO-d6): 10.06 (s, 1H), 8.68 (d, J = 2.0 Hz, 1H), 7.84 (d, J = 2.0 Hz,
1
3
1H), 7.51 (dd, J = 8.5, 5.5 Hz, 2H), 7.45 (t, J = 8.8 Hz, 2H), 5.33 (s, 2H). C NMR (150
MHz, DMSO-d6) δ 171.3, 166.3, 148.9, 140.6, 132.2, 130.7, 116.5, 115.8, 115.6, 113.5,
-
5
9.5. HRMS-ESI (−) m/z calculated for C13
H
9
FNO
4
262.0521 [M-H] , found: 262.0531.
1
-(1-(4-Fluorophenyl) ethyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid
1
(
10e). H NMR (600 MHz, DMSO-d6): δ 10.05 (s, 1H), 8.64 (d, J = 2.2 Hz, 1H), 7.83 (d,
J = 2.2 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.35 – 7.08 (m, 2H), 5.78 (q, J = 7.1 Hz, 1H), 1.86
1
3
(
d, J = 7.1 Hz, 3H). C NMR (150 MHz, DMSO-d6) δ 171.1, 166.1, 163.0, 148.9, 138.9,
135.3, 129.5, 123.6, 116.1, 113.3, 64.9, 19.3. HRMS-ESI (−) m/z calculated for
-
C
14
H11FNO
4
276.0678 [M-H] , found: 276.0682.
1
-(4-Fluoro-3-methylbenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid
1
(
10f). H NMR (600 MHz, DMSO-d6): δ 10.06 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H), 7.82 (d,
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J = 2.1 Hz, 1H), 7.39 (dd, J = 7.2, 1.6 Hz, 1H), 7.34 (m, 1H), 7.21 (m, 1H), 5.29 (s, 2H).
13C NMR (150 MHz, DMSO-d6) δ 171.1, 166.2, 161.8, 160.2, 148.7, 140.2, 131.9, 131.6,
1
2
28.2, 127.9, 125.2, 113.3, 59.5, 14.2. HRMS-ESI (−) m/z calculated for C14
H11FNO
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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-
76.0678 [M-H] , found: 276.0687.
1
-(2,4-Difluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10g).
1H NMR (600 MHz, DMSO-d6): δ 10.11 (s, 1H), 8.60 (d, J = 1.7 Hz, 1H), 7.76 (d, J = 2.0
Hz, 1H), 7.57 (dd, J = 15.2, 8.6 Hz, 1H), 7.41 – 7.31 (m, 1H), 7.17 (td, J = 8.5, 2.2 Hz,
1
3
1
H), 5.42 (s, 2H). C NMR (150 MHz, DMSO-d6) δ 171.2, 166.1, 161.5, 160.1, 148.7,
140.6, 140.4, 132.3, 125.6, 119.2, 113.3, 112.4, 104.6, 54.2. HRMS-ESI (−) m/z calculated
-
for C13
H
8
F
2
NO
4
280.0427 [M-H] , found: 280.0425.
1
1
-(4-Chlorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10h). H
NMR (600 MHz, DMSO-d6): 10.08 (s, 1H), 8.69 (d, J = 1.8 Hz, 1H), 7.83 (d, J = 1.8 Hz,
1
3
1H), 7.48 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 5.34 (s, 2H). C NMR (150 MHz,
DMSO-d6) δ 171.5, 166.5, 149.1, 140.6, 135.1, 133.8, 130.5, 129.4, 126.0, 113.7, 59.6.
-
HRMS-ESI (−) m/z calculated for C13
H
9
ClNO
4
278.0226 [M-H] , found: 278.0222.
1
-(3,4-Dichlorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10i).
1H NMR (600 MHz, dmso): 10.08 (s, 1H), 8.72 (s, 1H), 7.87 (s, 1H), 7.81 (d, J = 8.5 Hz,
1
3
2
H), 7.68 (s, 1H), 7.43 (d, J = 8.5 Hz, 2H), 5.33 (s, 2H). C NMR (150 MHz, DMSO-d6)
δ 171.6, 166.5, 149.1, 140.9, 140.7, 137.0, 131.9, 131.6, 131.0, 128.9, 126.0, 113.8, 59.1.
-
HRMS-ESI (−) m/z calculated for C13
H
8
Cl
2
NO
4
311.9836 [M-H] , found: 311.9843.
1
-(2,4-Dichlorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10j).
1H NMR (600 MHz, DMSO-d6): 10.12 (s, 1H), 8.58 (d, J = 2.1 Hz, 1H), 7.74 (d, J = 2.2
1
3
Hz, 1H), 7.50 (dd, J = 8.3, 2.2 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 5.47 (s, 2H). C NMR
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(
150 MHz, DMSO-d6) δ 171.7, 166.4, 149.0, 141.2, 134.7, 134.1, 132.7, 131.8, 129.8,
1
28.6, 126.2, 113.7, 57.8. HRMS-ESI (−) m/z calculated for C13
H
8
Cl
2
NO 311.9836 [M-
4
-
H] , found: 311.9844.
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
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6
7
8
9
0
1-(3-Chloro-4-fluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid
1
(10k). H NMR (600 MHz, DMSO-d6): δ 10.07 (s, 1H), 8.72 (s, 1H), 7.87 (s, 1H), 7.80 (d,
1
3
J = 6.5 Hz, 1H), 7.49-7.45 (m, 2H), 5.31 (s, 2H). C NMR (150 MHz, DMSO-d6) δ 170.9,
1
65.9, 158.0, 148.5, 140.1, 133.3, 130.7, 129.2, 125.3, 119.8, 117.4, 113.2, 58.5. HRMS-
-
ESI (−) m/z calculated for C13
H
8
ClFNO
4
296.0131 [M-H] , found: 296.0134.
1
1
-(4-Bromobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10l). H
NMR (600 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.68 (d, J = 1.5 Hz, 1H), 7.83 (d, J = 1.5 Hz,
1
3
1
H), 7.61 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 5.33 (s, 2H). C NMR (150 MHz,
DMSO-d6) δ 170.9, 165.9, 148.5, 140.9, 131.8, 130.2, 125.5, 121.9, 113.1, 59.1. HRMS-
-
ESI (−) m/z calculated for C13
H
9
BrNO
4
321.9720 [M-H] , found: 321.9727.
5
-Hydroxy-4-oxo-1-(3-(trifluoromethyl)benzyl)-1,4-dihydropyridine-3-carboxylic
1
acid (10m). H NMR (600 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.75 (d, J = 2.1 Hz, 1H),
7
1
1
.90-7.89 (m, 2H), 7.75 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz,
1
3
H), 5.43 (s, 2H). C NMR (150 MHz, DMSO-d6) δ 171.0, 165.9, 148.5, 140.2, 136.8,
32.2, 130.0, 129.5, 125.4, 125.0, 124.9, 113.2, 59.1. HRMS-ESI (−) m/z calculated for
-
C
14
H
9
F
3
NO
4
312.0489 [M-H] , found: 312.0484.
5
-Hydroxy-4-oxo-1-(pyridin-4-ylmethyl)-1,4-dihydropyridine-3-carboxylic
acid
1
(10n). H NMR (600 MHz, CD
3
OD): 8.74 (d, J = 4.9 Hz, 2H), 8.60 (d, J = 2.0 Hz, 1H),
1
3
7
.69 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 4.9 Hz, 2H), 5.57 (s, 2H). C NMR (150 MHz,
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DMSO-d6) δ HRMS-ESI (−) m/z calculated for C12
H
9
N
2
4
O 245.0568 [M-H] , found:
2
45.0570.
5
-Hydroxy-1-(4-(methylsulfonyl)benzyl)-4-oxo-1,4-dihydropyridine-3-carboxylic
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
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43
44
45
46
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59
60
1
acid (10o). H NMR (600 MHz, DMSO-d6): δ 10.11 (s, 1H), 8.73 (d, J = 2.1 Hz, 1H), 7.96
(
d, J = 8.3 Hz, 2H), 7.86 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 5.48 (s, 2H), 3.21
1
3
(
s, 3H). C NMR (150 MHz, DMSO-d6) δ 171.6, 166.5, 141.8, 141.3, 140.9, 129.2, 128.1,
1
26.1, 113.8, 87.2, 59.9, 44.4. HRMS-ESI (−) m/z calculated for C14
H
12NO S 322.0391
6
-
[
M-H] , found: 322.0382.
1
-(4-Carboxybenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10p).
1H NMR (600 MHz, DMSO-d6): δ 13.04 (s, 1H), 10.09 (s, 1H), 8.69 (d, J = 2.0 Hz, 1H),
7
.96 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 2.1 Hz, 1H), 7.48 (d, J = 8.2 Hz, 2H), 5.44 (s, 2H).
13C NMR (150 MHz, DMSO-d6) δ 171.2, 167.0, 166.2, 148.8, 140.5, 131.0, 130.1, 128.4,
288.0514 [M-H]-
128.0, 125.9, 113.4, 59.7. HRMS-ESI (−) m/z calculated for C14
H10NO
6
,
found: 288.0515.
5
-Hydroxy-1-(4-methoxybenzyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid (10q).
1H NMR (600 MHz, DMSO-d6): δ 10.04 (s, 1H), 8.64 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 2.1
1
3
Hz, 1H), 7.40 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 5.26 (s, 2H), 3.75 (s, 3H). C
NMR (150 MHz, DMSO-d6) δ 171.0, 166.3, 159.7, 148.6, 139.7, 130.0, 127.6, 125.7,
1
14.5, 113.3, 59.8, 55.4. HRMS-ESI (−) m/z calculated for C14
H
12NO
274.0721 [M-H]-,
5
found: 274.0722.
5
-Hydroxy-1-(naphthalen-1-ylmethyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid
1
(
10r). H NMR (600 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.64 (d, J = 1.8 Hz, 1H), 8.07 (d,
J = 8.3 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H),
2
9
ACS Paragon Plus Environment