Phenyl substituted 4-hydroxypyridazin-3(2 H)-ones and 5-hydroxypyrimidin-4(3 H)-ones: Inhibitors of influenza a endonuclease

Article abstract of DOI:10.1021/jm500958x

Seasonal and pandemic influenza outbreaks remain a major human health problem. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is attractive for the development of new agents for the treatment of influenza infection. Our earlier studies identified a series of 5- and 6-phenyl substituted 3-hydroxypyridin-2(1H)-ones that were effective inhibitors of influenza endonuclease. These agents identified as bimetal chelating ligands binding to the active site of the enzyme. In the present study, several aza analogues of these phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity. In contrast to the 4-aza analogue of 6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one, the 5-aza analogue (5-hydroxy-2-(4-fluorophenyl)pyrimidin-4(3H)-one) did exhibit significant activity as an endonuclease inhibitor. The 6-aza analogue of 5-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (6-(4-fluorophenyl)-4-hydroxypyridazin-3(2H)-one) also retained modest activity as an inhibitor. Several varied 6-phenyl-4-hydroxypyridazin-3(2H)-ones and 2-phenyl-5-hydroxypyrimidin-4(3H)-ones were synthesized and evaluated as endonuclease inhibitors. The SAR observed for these aza analogues are consistent with those previously observed with various phenyl substituted 3-hydroxypyridin-2(1H)-ones.

Full text of DOI:10.1021/jm500958x

Article
pubs.acs.org/jmc
Phenyl Substituted 4‑Hydroxypyridazin-3(2H)‑ones and
5‑Hydroxypyrimidin-4(3H)‑ones: Inhibitors of Influenza A
Endonuclease
Hye Yeon Sagong,† Joseph D. Bauman,‡ Disha Patel,‡ Kalyan Das,‡ Eddy Arnold,‡
and Edmond J. LaVoie*,†
†Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway,
New Jersey 08854-8020, United States
‡Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University,
Piscataway, New Jersey 08854-5627, United States
S
* Supporting Information
ABSTRACT: Seasonal and pandemic influenza outbreaks remain a major human health problem. Inhibition of the endonuclease
activity of influenza RNA-dependent RNA polymerase is attractive for the development of new agents for the treatment of
influenza infection. Our earlier studies identified a series of 5- and 6-phenyl substituted 3-hydroxypyridin-2(1H)-ones that were
effective inhibitors of influenza endonuclease. These agents identified as bimetal chelating ligands binding to the active site of the
enzyme. In the present study, several aza analogues of these phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were
synthesized and evaluated for their ability to inhibit the endonuclease activity. In contrast to the 4-aza analogue of 6-(4-
fluorophenyl)-3-hydroxypyridin-2(1H)-one, the 5-aza analogue (5-hydroxy-2-(4-fluorophenyl)pyrimidin-4(3H)-one) did exhibit
significant activity as an endonuclease inhibitor. The 6-aza analogue of 5-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (6-(4-
fluorophenyl)-4-hydroxypyridazin-3(2H)-one) also retained modest activity as an inhibitor. Several varied 6-phenyl-4-
hydroxypyridazin-3(2H)-ones and 2-phenyl-5-hydroxypyrimidin-4(3H)-ones were synthesized and evaluated as endonuclease
inhibitors. The SAR observed for these aza analogues are consistent with those previously observed with various phenyl
substituted 3-hydroxypyridin-2(1H)-ones.
endonuclease activity, (ii) is involved in viral RNA (vRNA)/
complementary RNA (cRNA) promoter binding, and (iii)
interacts with the PB1 subunit.6 PA has two domains, PAN (a
∼25 kDa N-terminal domain; residues 1−197) and PAC (∼55
kDa C-terminal domain; residues 239−716). Crystal structures
of PAC have been determined in complexes with N-terminal
fragments of PB1.7 The structure of PAN has been solved in
several crystal forms both unliganded and with various
ligands.8−13
Influenza RdRP is responsible for the replication and
transcription of the segmented viral RNA genes. Viral mRNA
transcription involves a cap-snatching mechanism in which the
polymerase binds to the host cellular mRNA via the 5′-cap and
INTRODUCTION
■
Two classes of agents, those targeting the M2 ion-channel and
those targeting neuramindase, are available to treat or prevent
influenza infection. The emergence of resistance to the M2 ion-
channel inhibiting drugs, amantadine and rimantadine, has
limited their clinical utility.1,2 Resistance to neuraminidase
inhibitors, including oseltamivir, has also been observed in
several seasonal influenza A strains.3,4 Drug resistance
mutations can emerge in almost all influenza A subtypes/
strains, including the pandemic 2009 H1N1 virus,5 limiting the
clinical efficacy of the currently approved drugs.
Influenza A contains eight negative-stranded RNA genomic
segments. The three largest genomic RNA segments encode
the viral RNA-dependent RNA polymerase (RdRP) consisting
of the polymerase acidic protein (PA) and polymerase basic
proteins 1 (PB1) and 2 (PB2). The PA subunit: (i) has
Received: July 11, 2014
© XXXX American Chemical Society
A
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cleaves the mRNA 12−13 nucleotides downstream. This
cleaved RNA fragment, which contains the 5′ cap, acts as a
primer for viral mRNA synthesis.14 Cap-snatching is an
important event in the life cycle of all members of the
Orthomyxoviridae family of viruses, including influenza A, B,
and C viruses. Because the host cell has no analogous activity,
inhibitors of cap-snatching may be selective against all influenza
subtypes and strains, including oseltamivir-resistant influenza A
viruses, without interfering with the host cell.
to either 5- or 6-(4-fluorophenyl)-3-hydroxypyrazin-2(1H)-one
(1b, 1c). The 2,3-dichloropyrazine was treated with excess
sodium methoxide to form 2,3-dimethoxypyrazine as previously
described.24 Bromination of 2,3-dimethoxypyrazine with N-
bromosuccimide in DMF provided the 5-bromo derivative,24
which under Suzuki coupling conditions with 4-fluorophenyl-
boronic acid gave 5-(4-fluorophenyl)-2,3-dimethoxypyrazine.
Treatment of this dimethoxypyrazine with a (1:1) mixture of
dioxane and 2 N HCl provided 1.25
Several small molecules have been identified as influenza
endonuclease inhibitors. These include 2,4-dioxobutanoic acid
derivatives,10,11,15,16 5-hydroxy-1,6-dihydropyrimidine-4-carbox-
ylic acid derivatives,11 and flutimide and its deriva-
tives,10,11,15−18 2-hydroxyphenyl amide derivatives,19 as well as
tetramic acid derivatives.20 Recently, in silico screening with in
vitro and ex vivo follow up studies identified hexanetetrones
and trihydroxyphenyls as endonuclease inhibitors with anti-
influenza activity.21 As correctly hypothesized, the endonu-
clease activity of influenza polymerase belongs to the two metal
ion group of phosphate-processing enzymes.20 From an X-ray
crystallographic screening campaign of a fragment library
targeting the influenza A endonuclease enzyme, we identified
the 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelat-
ing ligand at the active site of the enzyme.13 Using this
information, we have reported the structure−activity relation-
ships for two new series of compounds, 3-hydroxypyridin-
2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones, as endonu-
clease inhibitors (Figure 1).22,23
The method used to prepare 6-(4-fluorophenyl)-5-hydrox-
ypyrimidin-4(3H)-one 2 is outlined in Scheme 2. 4,5-
Dimethoxy-6-chloropyrimidine was prepared from 4,6-di-
chloro-5-methoxypyrimidine by treatment with sodium meth-
oxide.26 Suzuki coupling of this intermediate with 4-
fluorophenylboronic acid provided 6-(p-fluorophenyl)-4,5-
dimethoxypyrimidine, which in a 1:1 mixture of dioxane and
2 N HCl produced 5-methoxy-6-(4-fluorophenyl)pyrimidin-
4(3H)-one. Treatment of this 5-methoxypyrimidin-4(3H)-one
with BBr3 in CH2Cl2 provided 2.
The synthetic approach employed for the preparation of
various substituted 2-phenyl 5-hydroxypyrimidin-4(3H)-ones is
outlined in Scheme 3. 2-Chloro-4,5-dimethoxypyrimidine,
prepared from 2,4-dichloro-5-methoxypyrimidine,27 was a
common intermediate for these 2-phenyl 5-hydroxypyrimidin-
4(3H)-ones. Suzuki coupling with the appropriate phenyl-
boronic acid provided the desired 2-phenyl 4,5-dimethoxypyr-
imidine derivative, which was initially hydrolyzed with 1:1
dioxane and 2 N HCl to provide the requisite 2-phenyl 5-
methoxypyrimidin-4(3H)-one. Further treatment of these 5-
methoxypyrimidin-4(3H)-ones with BBr3 in CH2Cl2 provided
the 2-(fluorophenyl) derivatives 3−5 as well as the various 2-
biphenyl derivatives 6−8. In a similar manner, the 2-(4-
cyanophenyl) and 2-(3-cyanophenyl) 5-methoxypyrimidin-
4(3H)-ones were prepared and treated with and BBr3 in
CH2Cl2 to form the 5-hydroxypyrimidin-4(3H)-ones 9 and 10.
Treatment of 9 or 10 with sodium azide in DMF in the
presence of a catalytic amount of acetic acid gave the 5-tetrazoyl
derivatives 11 and 12, respectively.
The preparation of 5-(4-fluorophenyl)-4-hydroxypyridazin-
3(2H)-one is summarized in Scheme 4. Formation of the 2-
methoxymethyl derivative of 4,5-dichloropyridazin-3(2H)-one,
followed by selective replacement of the 4-chloro substituent
with a methoxyl group, provided 2-methoxymethyl-5-chloro-4-
methoxypyridazin-3(2H)-one.28 This MOM-protected chlor-
opyridazin-3(2H)-one was reacted with 4-fluorophenylboronic
acid under Suzuki coupling conditions to provide 2-
methoxymethyl-5-(4-fluorophenyl)-4-methoxypyridazin-
3(2H)-one. Treatment of this 5-(4-fluorophenyl)pyridazin-
3(2H)-one with BBr3 in CH2Cl2 gave 13 in good yield.
The synthesis of various 6-phenyl substituted pyridazin-
3(2H)-ones was accomplished as outlined in Scheme 5.
Commercially available 3,4,6-trichloropyridazine was converted
to 6-chloro-3,4-dimethoxypyridazine,29 which was subsequently
used with either 4-fluorophenylboronic acid or 4-cyanophe-
nylboronic acid under Suzuki coupling conditions to form the
6-phenyl-3,4-dimethoxypyridazine intermediates. Treatment of
these dimethoxypyridazines with a 1:1 mixture of dioxane and 2
N HCl provided the 4-methoxypyridazine-3(2H)-ones, which
were subsequently treated with BBr3 in CH2Cl2 to provide 14
and 15. Treatment of 15 with sodium azide in DMF in the
presence of a catalytic amount of acetic acid provided the 6-(4-
(tetrazol-5-yl)phenyl) derivative, 16 (Table 1).
Figure 1. Structures and of 5-(p-fluorophenyl-3-hydroxypyridin-
2(1H)-one (5-FPhP), 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-
one (6-FPhP), 6-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one (6-
FPhQ), and 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one (7-
FPhQ).
Phenyl substituted derivatives of 3-hydroxypyridin-2(1H)-
ones are among the more potent inhibitors of influenza A
endonuclease. While 4-(4-fluorophenyl)-3-hydroxypyridin-2-
(1H)one did not exhibit significant activity, both 5-(4-
fluorophenyl)-3-hydroxypyridin-2(1H)one and 6-(4-fluoro-
phenyl)-3-hydroxypyridin-2(1H)one had IC50 values <1.0
μM. The present study was undertaken to determine the
relative effects of aza substitution at various positions of phenyl
substituted 3-hydroxypyridin-2(1H)-ones on their potential to
inhibit influenza A endonuclease.
CHEMISTRY
■
The synthesis of 5-(4-fluorophenyl)-1,4-dihydropyrazine-2,3-
dione, 1a, is outlined in Scheme 1. This dione can tautomerize
B
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a
Scheme 1
a
Reagents and conditions: (a) NaOMe (10 mol equiv), MeOH under Ar; (b) NBS (1.05 mol equiv), DMF under Ar; (c) p-fluorophenylboronic
acid, Pd(PPh3)4, Na2CO3, dioxane/H2O (3:1) under Ar; (d) dioxane/2 N HCl (1:1) under Ar.
a
Scheme 2
a
Reagents and conditions: (a) NaOMe (1.1 mol equiv), MeOH under Ar; (b) p-fluorophenylboronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O (3:1)
under Ar; (c) dioxane/2 N HCl (1:1) under Ar; (d) BBr3 in CH2Cl2, 0 °C to rt under Ar.
The structure−activity studies performed with 3-hydroxypyr-
idin-2-ones indicated 6-[(4-tetrazoyl)phenyl]-3-hydroxypyri-
din-2-one had pronounced activity as an endonuclease inhibitor
(IC50 = 0.085 μM).23 On the basis of this observation, 2-
[(tetrazoyl)phenyl] derivatives of 5-hydroxypyrimidin-4(3H)-
one and their 2-(cyanophenyl) precursors were targeted for
synthesis and evaluation as endonuclease inhibitors. The 3-
cyanophenyl derivative 10 is approximately twice as potent
compared to the 4-cyanophenyl isomer 9. However, the 4-
(tetrazoly)phenyl derivative 11 is the most potent of 5-
hydroxypyrimidin-4(3H)-ones evaluated (IC50 = 0.15 μM) with
3-fold greater potency than the 3-(tetrazoly)phenyl derivative
12.
Previously formed crystals of apo pandemic 2009 influenza
endonuclease were soaked in a solution containing 11 as
described previously (Supporting Information, Table 1S).13
The crystal structure revealed that 11 chelates to the two active
site metal ions in a mode similar to the 3-hydroxypyridin-
2(1H)-one containing compounds (Figure 2). The N1 atom of
the pyrimidine ring forms hydrogen bond interactions with a
network of water molecules present near the active site. N3
interacts with Tyr130 through two bridging waters. Similar to
the 5-[4-(tetrazoly)phenyl] containing pyridinone compounds,
the 2-[4-(tetrazoly)phenyl] makes bidentate hydrogen bond
interactions with Arg124.23 The 2-phenyl ring is rotated ∼90°
relative to the 5-phenyl seen previously and is coplanar with the
pyrimidine ring due to the lack of steric restraint from
additional ring substitutions as seen previously.13
RESULTS AND DISCUSSION
■
The tautomeric forms of 5-(4-fluorophenyl)-1,4-dihydropyr-
azine-2,3-dione, 1b and 1c (5- and 6-(4-fluorophenyl)-3-
hydroxypyrazin-2(1H)-one), represent the 4-aza derivatives of
both 5- and 6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one,
respectively. Upon the basis of its NMR spectra, however, the
most dominant tautomeric form of 1 is the 1,4-dihydropyr-
azine-2,3-dione, 1a, with only minor amounts of tautomers 1b
and 1c. This may explain the comparatively weak activity of 1 as
an endonuclease inhibitor. Alternatively, should 1b or 1c form
to an appreciable extent under the enzyme assay conditions, the
presence of a 4-aza substituent adjacent to the 3-hydroxyl group
of either tautomer 1b or 1c may be associated with reduced
intrinsic activity as an inhibitor.
Compound 2 is the 5-aza analogue of 4-(4-fluorophenyl)-3-
hydroxypyridin-2(1H)-one. The lack of activity observed for 2
is consistent with previous structure−activity relationships
observed with various 4-substituted 3-hydroxypyridin-2-ones.
The presence of a phenyl group or other relatively large
substituents at the 4-position of various 3-hydroxypyridin-2-
ones is associated with a dramatic loss in potency as an
inhibitor. It is noteworthy that when the 4-fluorophenyl
substituent is at the 2-postion of 5-hydroxypyrimidin-4(3H)-
ones, which is comparable to the 6-position of 3-hydroxypyr-
idin-2-ones, significant activity in terms of endonuclease
inhibition is observed (IC50 = 0.58 μM).13,23 This 4-
fluorophenyl derivative is more than twice as potent as either
of the 3- or 2-fluorophenyl isomers, 4 and 5. A series of 2-
biphenyl 5-hydroxypyrimidin-4(3H)-ones 6−8 was also
evaluated. Steric factors may be responsible for the decreased
activity observed for the 4- and 2-biphenyl derivatives 6 and 8,
relative to 7.
5-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one 13 is the
6-aza analogue of 4-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-
one. Consistent with the structure−activity relationships
observed with 4-phenyl substituted 3-hydroxy-pyridin-2(1H)-
C
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a
Scheme 3
a
Reagents and conditions: (a) K2CO3 (10 mol equiv), MeOH under Ar; (b) 4-, 3-, and 2-fluorophenylboronic acid (for 3−5) and 4-, 3-, and 2-
biphenylboronic acid (for 6−8), Pd(PPh3)4, Na2CO3, dioxane/H2O (3:1), 4-cyanophenylboronic acid (for 9), 3-cyanophenylboronic acid (for 10),
under Ar; (c) dioxane/2 N HCl (1:1) under Ar; (d) BBr3 in CH2Cl2, 0 °C to rt under Ar; (e) NaN3 (4.0 mol equiv), DMF and a catalytic amount of
AcOH under Ar.
a
Scheme 4
a
Reagents and conditions: (a) MOMCl (1.2 mol equiv), DMAP (0.1 mol equiv), NEt3 (1.4 mol equiv), CH2Cl2, 0 °C to rt under Ar; (b) NaOMe
(1.1 mol equiv), MeOH under Ar; (c) 4-fluorophenylboronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O (3:1) under Ar; (d) BBr3 in CH2Cl2, 0 °C to
rt under Ar.
ones, 13 did not exhibit significant activity as an endonuclease
inhibitor. The various 6-(phenyl)-4-hydroxypyridazin-3(2H)-
ones evaluated in this study are the 6-aza analogues of 5-
(phenyl)-3-hydroxypyridin-2(1H)-ones. These 6-phenylpyrida-
zin-3(2H)-ones 14−16 are more active than the 4-fluorophe-
nylpyrazine derivative 1 but are at least an order of magnitude
less active than analogous 2-phenyl-5-hydroxypyrimidin-4(3H)-
ones. The most active derivative was the 6-(4-tetrazoyl)phenyl-
4-hydroxypyridazin-3(2H)-one 16 (IC50 = 3.0 μM). Interest-
ingly, 5-substituted 3-hydroxypyridin-2(1H)-ones and 6-sub-
stituted 4-hydroxypyridazin-3(2H)ones as well as variously
substituted 3-hydroxyquinolin-2(1H)ones have been previously
established as D-amino acid oxidase (DAAO) inhibitors.30,31
However, none of these similarly structured aza-analogues of 3-
hydroxypyridin-2(1H)-ones that were synthesized in the
present study were evaluated for their relative activity as
DAAO inhibitors.
Data on the relative activity of these aza 3-hydroxypyridin-
2(1H)-one derivatives indicate that 2-phenyl-5-hydroxypyrimi-
din-4(3H)-ones, which are the 5-aza analogues of 6-phenyl-3-
hydroxypyridin-2(1H)-ones, can exhibit comparable activity as
inhibitors of endonuclease. However, 6-phenyl-4-hydroxypyr-
idazin-3(2H)-ones, which are the 6-aza analogues of 5-phenyl-
3-hydroxypyridin-2(1H)-ones, are 2.3−8.2-fold less active.
Although the tautomers of 5-(4-fluorophenyl)-1,4-dihydropyr-
azine-2,3-dione can be viewed as 4-aza-analogues either 5- or 6-
D
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a
Scheme 5
EXPERIMENTAL SECTION
■
Chemistry: General Methods. All reactions, unless otherwise
stated, were done under nitrogen atmosphere. Reaction monitoring
and follow-up were done using aluminum backed Silica G TLC plates
with UV254 (Sorbent Technologies), visualizing with ultraviolet light.
Flash column chromatography was done on a Combi Flash Rf
Teledyne ISCO using hexane, ethyl acetate, dichloromethane, and
methanol. The 1H (400 MHz) and 13C (100 MHz) NMR spectra were
done in CDCl3, methanol-d4, and DMSO-d6 and recorded on a Bruker
Avance III (400 MHz) multinuclear NMR spectrometer. Data is
expressed in parts per million relative to the residual nondeuterated
solvent signals, spin multiplicities are given as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet), dt (doublet of triplets), q
(quartet), m (multiplet), and bs (broad singlet), and coupling
constants (J) are reported in hertz. Melting points were determined
using Mel-temp II apparatus and are uncorrected. Analytical HPLC
was performed on a Shimadzu LC-20AT Prominence liquid
chromatograph using a 150 mm × 4.6 mm Princeton SPHER-100
RP C18 1000A 5 μ column using 0% water for 2 min and a 0−100%
water/methanol gradient over a 5 min period and 5 min at 100%
methanol at a 2.0 mL/min flow rate monitoring UV absorbance at 254
and 296 nm. Using this method of analysis, the purity of all
compounds used in bioassays was determined to be ≥95%. HRMS
experiments were conducted by Washington University Resource for
Biomedical and Bioorganic Mass Spectrometry Department of
Chemistry.
a
Reagents and conditions: (a) NaOMe (2 mol equiv), MeOH under
Ar; (b) 4-fluorophenylboronic acid (for 14), Pd(PPh3)4, Na2CO3,
dioxane/H2O (3:1), 4-cyanophenylboronic acid (for 15) under Ar; (c)
dioxane/2 N HCl (1:1) under Ar; (d) BBr3 in CH2Cl2, 0 °C to rt
under Ar; (e) NaN3 (4.0 mol equiv), DMF, and a catalytic amount of
AcOH under Ar.
Table 1. Inhibition Assay of Influenza A Endonuclease by
Phenyl Substituted 3-Hydroxypyrazin-2(1H)-ones, 5-
Hydroxypyrimidin-4(3H)-ones, and 4-Hydroxypyridazin-
3(2H)-ones
5-(4-Fluorophenyl)pyrazine-2,3(1H,4H)-dione (1). 5-(4-Fluoro-
phenyl)-2,3-dimethoxypyrazine (100 mg, 0.43 mmol) was dissolved
in a mixture of dioxane (5 mL) and 2 N HCl (5 mL). The reaction
mixture was then refluxed for 21 h. After the reaction was completed, it
was cooled to room temperature. Then solvent was removed under
reduced pressure. The resulting white solid was dissolved in satd
NaHCO3. The white suspension was filtered and solid was collected
and dried to give 5-(4-fluorophenyl)pyrazine-2,3(1H,4H)-dione as a
1
white solid (75 mg, 85%); mp 285−287 °C. H NMR (400 MHz,
DMSO-d6) δ 11.55 (s, 1H), 11.46 (s, 1H), 7.57, (dd, J = 9 Hz, J = 5
Hz, 2H), 7.23−7.19 (m, 2H), 6.59 (d, J = 3 Hz, 1H). 13C NMR (100
compd
IC50 (μM)
R1
R2
MHz, DMSO-d6) δ 161.8 (JC,F C,F = 243 Hz), 156.9, 155.7, 128.1,
J
1
59.0
>193
0.58
1.56
1.67
2.24
0.40
1.10
0.52
0.25
0.15
0.48
177
4-FC6H5
127.6 (JC,F = 9 Hz), 121.0, 115.5 (JC,F = 21 Hz), 107.1. 19F NMR (376
MHz, DMSO-d6) δ −114.0. HRMS (ESI) calculated for
C10H7FN2O2Na (M + Na)+ 229.0384, found 229.0382.
2
4-FC6H5
H
3
H
4-FC6H4
4
H
3-FC6H4
5-(4-Fluorophenyl)-2,3-dimethoxypyrazine. 5-Bromo-2,3-dime-
thoxypyrazine (150 mg, 0.69 mmol), (4-fluorophenyl)boronic acid
(144 mg, 1.03 mmol), Pd(PPh3)4 (80 mg, 0.069 mmol), and Na2CO3
(218 mg, 2.06 mmol) were dissolved in a mixture of dioxane (6 mL)
and water (2 mL). The air was evacuated and replaced with N2. Then,
the reaction mixture was refluxed for 5 h. Because the reaction was not
completed, additional (4-fluorophenyl)boronic acid (48 mg, 0.34
mmol) was added. It was then again refluxed for 16 h. It was cooled to
room temperature, and it was diluted with EtOAc and washed with
satd NH4Cl, followed by brine. The organic layer was dried over
Na2SO4 and concentrated under reduced pressure, and the resulting
residue was purified by flash chromatography on silica gel, eluting with
0−10% EtOAc/hexane. This afforded 5-(4-fluorophenyl)-2,3-dime-
5
H
2-FC6H4
6
H
4-[1,1′]-biphenyl
3-[1,1′]-biphenyl
2-[1,1′]-biphenyl
4-CNC6H4
7
H
8
H
9
H
10
H
3-CNC6H4
11
H
4-(CN4H)phenyl
3-(CN4H)phenyl
H
12
H
13
4-FC6H4
14
6.0
H
H
H
4-FC6H4
15
9.3
4-CNC6H4
16
3.0
4-(CN4H)phenyl
1
thoxypyrazine as a white solid (123 mg, 77%); mp 108−110 °C. H
5-FPhP
6-FPhP
0.73
0.43
NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.90 (dd, J = 9 Hz, J = 5 Hz,
2H), 7.15−7.10 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.06 (JC,F
= 247 Hz), 149.43, 149.41, 140.29, 132.66 (JC,F = 3 Hz), 127.77,
127.73, 127.69 (JC,F = 8 Hz), 115. 68 (JC,F = 21 Hz), 54.09, 53.81. 19
F
NMR (376 MHz, CDCl3) δ −113.7. HRMS (ESI) calculated for
C12H12FN2O2 (M + H)+ 235.0877, found 235.0880.
(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one, they were more
than 80 times less potent than either of these 3-hydroxypyridin-
2(1H)-ones. The relative ranking of comparable phenyl
substituted pyridin-2(1H)-ones and their aza derivatives in
terms of endonuclease inhibition is 3-hydroxypyridin-2(1H)-
one ≈ 5-hydroxypyrimidin-4(3H)-ones >4-hydroxypyridazin-
3(2H)-ones ≫1,4-dihydropyrazine-2,3-dione.
5-Bromo-2,3-dimethoxypyrazine. 2,3-Dimethoxypyrazine (565
mg, 4.03 mmol) and NBS (754 mg, 4.23 mmol) were dissolved in
DMF (5 mL). The mixture was stirred for 29 h at room temperature.
After the reaction was completed, DMF was removed by Kugelrohr
distillation. Then, the resulting residue was diluted with DCM, and the
organic layer was washed with satd NaHCO3, followed by brine. The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure to provide 5-bromo-2,3-dimethoxypyrazine as a white solid
E
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Figure 2. Stereoview image of a crystal structure of 11 (yellow) bound to PAN(cyan) superposed with a previously published structure (PDB ID:
4M5U) of 5-(4-(1H-tetrazol-5-yl)phenyl)-6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (green). Metal-coordinating bonds are depicted as black
dashed lines whereas hydrogen or electrostatic bonds are depicted in blue. Electron density calculated from an omit map is contoured at 4.0σ (blue
mesh).
1
(384 mg, 43%); mp 54−56 °C. H NMR (400 MHz, CDCl3) δ 7.86
(s, 1H), 4.03 (s, 3H), 3.88 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
161.2, 150.0, 141.7, 138.1, 56.6, 55.1.
246 Hz), 158.8, 147.4, 143.7, 142.9, 131.7, 131.2 (JC,F = 8 Hz), 115.0
(JC,F = 21 Hz), 58.8. 19F NMR (376 MHz, DMSO-d6) δ −111.8.
HRMS (ESI) calculated for C11H10FN2O2 (M + H)+ 221.0721, found
221.0721.
2,3-Dimethoxypyrazine. 2,3-Dichloropyrazine (1.04 mL, 10 mmol)
was added to MeOH (10 mL). Then the reaction mixture was cooled
to 0 °C. It was treated with NaOMe (5.4 g, 100 mmol) and allowed to
warm to room temperature. The reaction mixture was then stirred for
22 h at room temperature. After the reaction was completed, it was
diluted with DCM. The resulting white suspension was filtered, and
the filtrate was concentrated under reduced pressure. The residue was
diluted with DCM and washed with water, followed by brine. The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure to reveal a colorless liquid. 2,3-Dimethoxypyrazine was
4-(4-Fluorophenyl)-5,6-dimethoxypyrimidine. 4-Chloro-4,5-dime-
thoxypyrimidine (165 mg, 0.95 mmol), (4-fluorophenyl)boronic acid
(99 mg, 1.42 mmol), Pd(PPh3)4 (110 mg, 0.095 mmol), and Na2CO3
(300 mg, 2.84 mmol) were dissolved in a mixture of dioxane (9 mL)
and water (3 mL). The air was evacuated and replaced with N2. Then
the reaction mixture was refluxed for 19 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−10% EtOAc/hexane. This afforded
4-(4-fluorophenyl)-5,6-dimethoxypyrimidine as a white solid (181 mg,
1
crystallized as a white solid (1.13 g, 81%); mp 21−23 °C. H NMR
(400 MHz, CDCl3) δ 7.47 (s, 2H), 3.88 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 150.3, 131.7, 53.4.
1
6-(4-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (2). 6-(4-Fluo-
rophenyl)-5-methoxypyrimidin-4(3H)-one (107 mg, 0.49 mmol) was
dissolved in anhydrous DCM (5 mL). The reaction mixture was
cooled to 0 °C, and the 1 M in DCM BBr3 (5 mL, 5 mmol) was
added. It was then allowed to warm to room temperature and stirred
for 24 h. Then the solvent was removed under reduced pressure. The
resulting residue was diluted with EtOAc, which was washed with satd
NaHCO3, followed by brine. The organic layer was dried over Na2SO4,
followed by concentration under the vacuum. The resulting residue
was purified by flash chromatography on silica gel, eluting with 0−10%
MeOH/DCM to provide 6-(4-fluorophenyl)-5-hydroxypyrimidin-
4(3H)-one as a white solid (50 mg, 50%); mp 285−287 °C. 1H
NMR (400 MHz, DMSO-d6) δ 12.67 (bs, 1H), 9.83 (bs, 1H), 8.20
(dd, J = 9 Hz, J = 6 Hz, 2H), 7.87 (s, 1H), 7.29−7.25 (m, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 161.9 (JC,F = 245 Hz), 158.7, 141.1,
138.5, 136.0, 132.3, 130.7 (JC,F = 8 Hz), 114.8 (JC,F = 21 Hz). 19F
NMR (376 MHz, DMSO-d6) δ −113.0. HRMS (ESI) calculated for
C10H8FN2O2 (M + H)+ 207.0564, found 207.0568.
82%); mp 62−64 °C. H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H),
8.07 (dd, J = 9 Hz, J = 6 Hz, 2H), 7.17−7.11 (m, 2H), 4.07 (s, 3H),
3.73 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.7 (JC,F = 249 Hz),
164.0, 154.3, 152.0,139.4, 131.4 (JC,F = 8 Hz), 131.3, 115.3 (JC,F = 21
Hz), 60.2, 54.2. 19F NMR (376 MHz, CDCl3) δ −111.0. HRMS (ESI)
calculated for C12H12FN2O2 (M + H)+ 235.0877, found 235.0882.
4-Chloro-5,6-dimethoxypyrimidine. 4,6-Dichloro-5-methoxypyri-
midine (300 mg, 1.68 mmol) was added to MeOH (10 mL). Then
the reaction mixture was cooled to 0 °C. It was treated with NaOMe
(99 mg, 1.85 mmol) and allowed to warm to room temperature. The
reaction mixture was then stirred for 19 h at room temperature. After
the reaction was completed, it was put under the vacuum to remove
MeOH. The resulting residue was diluted with EtOAc, which was then
washed with satd NH4Cl, followed by brine. The organic layer was
dried over Na2SO4 and then concentrated. The residue was purified by
flash chromatography on silica gel, eluting with 0−10% EtOAc/hexane
to provide 4-chloro-5,6-dimethoxypyrimidine as a white solid (168 mg,
1
57%); mp 53−55 °C. H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H),
6-(4-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one. 4-(4-Fluoro-
phenyl)-5,6-dimethoxypyrimidine (134 mg 0.57 mmol) was dissolved
in a mixture of 2 N HCl (5 mL) and dioxane (5 mL). The reaction
mixture was then refluxed for 12 h. It was then cooled to room
temperature. The reaction mixture was put under the vacuum to
remove the solvent, which gave white residue. This residue was diluted
with water and filtered. The solid was collected and gave 6-(4-
fluorophenyl)-5-methoxypyrimidin-4(3H)-one as a white solid (109
mg, 87%); mp 204−206 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.76
(s, 1H), 8.07 (s, 1H), 8.04 (dd, J = 9 Hz, J = 6 Hz, 2H), 7.33−7.28 (m,
4.03 (s, 3H), 3.88 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.6,
151.6, 151.3, 138.2, 60.7, 54.8.
2-(4-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (3). 2-(4-Fluo-
rophenyl)-5-methoxypyrimidin-4(3H)-one (58 mg, 0.26 mmol) was
dissolved in anhydrous DCM (5 mL). The reaction mixture was
cooled to 0 °C, and the 1 M in DCM BBr3 (3 mL, 3 mmol) was
added. It was then allowed to warm to room temperature and stirred
for 24 h. Then the solvent was removed under reduced pressure. The
resulting residue was suspended in water. It was filtered, and the solid
was collected and dried under vacuum to provide 2-(4-fluorophenyl)-
5-hydroxypyrimidin-4(3H)-one as a white solid (23 mg, 42%); mp
2H), 3.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.5 (JC,F
=
F
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252−254 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.88 (bs, 1H), 9.64
(bs, 1H), 8.05 (dd, J = 9 Hz, J = 5 Hz, 2H), 7.54 (s, 1H), 7.33−7.29
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.4 (JC,F = 246 Hz),
159.0, 146.9, 143.4, 131.7, 129.4 (JC,F = 9 Hz), 129.1, 115.5 (JC,F = 22
Hz). 19F NMR (376 MHz, DMSO-d6) δ −110.5. HRMS (ESI)
calculated for C10H8FN2O2 (M + H)+ 207.0564, found 207.0566.
2-(4-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one. 2-(4-Fluoro-
phenyl)-4,5-dimethoxypyrimidine (187 mg, 0.799 mmol) was
dissolved in a mixture of 2 N HCl (5 mL) and dioxane (5 mL).
The reaction mixture was then refluxed for 12 h. It was then cooled to
room temperature. The reaction mixture was diluted with EtOAc and
washed with satd NaHCO3, followed by brine. The organic layer was
dried over Na2SO4 and concentrated. The resulting residue was
purified by flash chromatography on silica gel, eluting with 50−100%
EtOAc/hexane to give 2-(4-fluorophenyl)-5-methoxypyrimidin-
4(3H)-one as a white solid (41 mg, 23%); mp 229−231 °C. 1H
NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.68 (s, 1H), 8.08 (dd, J
= 9 Hz, J = 5 Hz, 2H), 7.35−7.30 (m, 2H), 3.79 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 163.6 (JC,F = 247 Hz), 158.1, 148.4, 145.4,
130.4, 129.6 (JC,F = 9 Hz), 129.1, 115.5 (JC,F = 22 Hz), 56.0. 19F NMR
(376 MHz, DMSO-d6) δ −110.2. HRMS (ESI) calculated for
C11H9FN2O2Na (M + Na)+ 243.0540, found 243.0546.
2-(4-Fluorophenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dime-
thoxypyrimidine (500 mg, 2.86 mmol), (4-fluorophenyl)boronic acid
(601 mg, 4.30 mmol), Pd(PPh3)4 (330 mg, 0.29 mmol), and Na2CO3
(910 mg, 8.59 mmol) were dissolved in a mixture of dioxane (12 mL)
and water (4 mL). The air was evacuated and replaced with N2. Then,
the reaction mixture was refluxed for 5 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−10% EtOAc/hexane. This afforded
2-(4-fluorophenyl)-4,5-dimethoxypyrimidine as a white solid (123 mg,
77%); mp 114−116 °C. 1H NMR (400 MHz, CDCl3) δ 8.37 (dd, J =
9 Hz, J = 6 Hz, 2H), 8.12 (s, 1H), 7.16−7.12 (m, 2H), 4.17 (s, 3H),
3.98 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.1 (JC,F = 248 Hz),
159.7, 155.3, 141.0, 137.2, 133.6 (JC,F = 3 Hz), 129.6 (JC,F = 8 Hz),
115.3 (JC,F = 22 Hz), 56.4, 54.0. 19F NMR (376 MHz, CDCl3) δ
−111.9. HRMS (ESI) calculated for C12H12FN2O2 (M + H)+
235.0877, found 235.0878.
MHz, DMSO-d6) δ −112.5. HRMS (ESI) calculated for C10H8FN2O2
(M + H)+ 207.0564, found 207.0565.
2-(3-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one. 2-(3-Fluoro-
phenyl)-4,5-dimethoxypyrimidine (390 mg, 1.67 mmol) was dissolved
in a mixture of 2 N HCl (10 mL) and dioxane (10 mL). The reaction
mixture was then refluxed for 18 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with water, followed by brine. The organic layer was dried over
Na2SO4 and concentrated. The resulting residue was purified by flash
chromatography on silica gel, eluting with 0−100% EtOAc/hexane to
give 2-(3-fluorophenyl)-5-methoxypyrimidin-4(3H)-one as a white
solid (237 mg, 63%); mp 122−124 °C. 1H NMR (400 MHz, DMSO-
d6) δ 12.86 (bs, 1H), 7.91 (d, J = 8 Hz, 1H), 7.87−7.83 (m, 1H), 7.71
(s, 1H), 7.58−7.53 (m, 1H), 7.37 (td, J = 8 Hz, J = 2 Hz, 1H), 3.81 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 162.1 (JC,F = 242 Hz), 157.9,
147.8, 145.5, 134.7, 130.7 (JC,F = 9 Hz), 130.1, 123.2 (JC,F = 9 Hz),
117.5 (JC,F = 22 Hz), 113.8 (JC,F = 24 Hz), 56.1. 19F NMR (376 MHz,
DMSO-d6) δ −112.5. HRMS (ESI) calculated for C11H10FN2O2 (M +
H)+ 221.0721, found 227.0722.
2-(3-Fluorophenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dime-
thoxypyrimidine (300 mg, 1.72 mmol), (3-fluorophenyl)boronic acid
(343 mg, 2.58 mmol), Pd(PPh3)4 (199 mg, 0.17 mmol), and Na2CO3
(546 mg, 5.15 mmol) were dissolved in a mixture of dioxane (12 mL)
and water (4 mL). The air was evacuated and replaced with N2. Then
the reaction mixture was refluxed for 8 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−10% EtOAc/hexane. This afforded
2-(3-fluorophenyl)-4,5-dimethoxypyrimidine as a white solid (393 mg,
1
98%); mp 83−85 °C. H NMR (400 MHz, CDCl3) δ 8.09 (dt, J = 8
Hz, J = 1 Hz, 1H), 8.03 (s, 1H), 8.01−7.98 (m, 1H), 7.38−7.33 (m,
1H), 7.07 (tdd, J = 8 Hz, J = 3 Hz, J = 1 Hz, 1H), 4.09 (s, 3H), 3.90 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 163.1 (JC,F = 242 Hz), 159.5,
154.6 (JC,F =3 Hz), 141.3, 139.8 (JC,F = 8 Hz), 136.9, 129.7 (JC,F = 8
Hz), 123.1 (JC,F = 3 Hz), 116.4 (JC,F = 22 Hz), 114.3 (JC,F = 23 Hz),
56.2, 53.9. 19F NMR (376 MHz, CDCl3) δ −113.5. HRMS (ESI)
calculated for C12H12FN2O2 (M + H)+ 235.0877, found 235.0879.
2-(2-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (5). 2-(2-Fluo-
rophenyl)-5-methoxypyrimidin-4(3H)-one (100 mg, 0.45 mmol) was
dissolved in anhydrous DCM (5 mL). The reaction mixture was
cooled to 0 °C, and then 1 M in DCM BBr3 (4.54 mL, 4.54 mmol)
was added. It was then allowed to warm to room temperature and
stirred for 18 h. Then the solvent was removed under reduced
pressure. The resulting residue was diluted with EtOAc, which was
washed with 2 N HCl, followed by brine. The organic layer was dried
over Na2SO4, followed by concentration under the vacuum. The
resulting residue was purified by flash chromatography on silica gel,
eluting with 0−10% MeOH/DCM to give 2-(2-fluorophenyl)-5-
hydroxypyrimidin-4(3H)-one as a white solid (89 mg, 96%); mp 187−
2-Chloro-4,5-dimethoxypyrimidine. 2,4-Dichloro-5-methoxypyri-
midine (2.37 g, 13.2 mmol) and K2CO3 (1.8 g, 13.2 mmol) were
dissolved in MeOH (50 mL) and stirred for 19 h at room temperature.
The solvent was removed under reduced pressure. The resulting
residue was dissolved in EtOAc and washed with distilled water,
followed by brine. The organic layer was dried over Na2SO4 and
concentrated. The resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−20% EtOAc/hexane to give 2-
chloro-4,5-dimethoxypyrimidine as a white solid (1.70 g, 73%); mp
65−67 °C. 1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 4.03 (s, 3H),
3.88 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 161.2, 150.0, 141.7,
138.2, 56.6, 55.0.
1
189 °C. H NMR (400 MHz, DMSO-d6) δ 12.80 (bs, 1H), 9.73 (bs,
1H), 7.65 (t, J = 7 Hz, 1H), 7.59 (s, 1H), 7.55 (t, J = 7 Hz, 1H), 7.36−
7.30 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 159.3 (JC,F = 248
Hz), 158.2, 144.6, 143.9, 132.2 (JC,F = 8 Hz), 132.1, 130.8, 124.5 (JC,F
= 3 Hz), 121.8 (JC,F = 12 Hz), 116.1 (JC,F = 21 Hz). 19F NMR (376
MHz, DMSO-d6) δ −115.2. HRMS (ESI) calculated for C10H8FN2O2
(M + H)+ 207.0564, found 207.0565.
2-(3-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (4). 2-(3-Fluo-
rophenyl)-5-methoxypyrimidin-4(3H)-one (100 mg, 0.45 mmol) was
dissolved in anhydrous DCM (5 mL). The reaction mixture was
cooled to 0 °C, and then 1 M in DCM BBr3 (4.54 mL, 4.54 mmol)
was added. It was then allowed to warm to room temperature and
stirred for 18 h. Then the solvent was removed under reduced
pressure. The resulting residue was diluted with EtOAc, which was
washed with 2 N HCl, followed by brine. The organic layer was dried
over Na2SO4, followed by concentration under the vacuum. The
resulting residue was purified by flash chromatography on silica gel,
eluting with 0−10% MeOH/DCM to give 2-(3-fluorophenyl)-5-
hydroxypyrimidin-4(3H)-one as a white solid (87 mg, 93%); mp 210−
2-(2-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one. 2-(2-Fluoro-
phenyl)-4,5-dimethoxypyrimidine (400 mg, 1.71 mmol) was dissolved
in a mixture of 2 N HCl (10 mL) and dioxane (10 mL). The reaction
mixture was then refluxed for 18 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with water, followed by brine. The organic layer was dried over
Na2SO4 and concentrated. The resulting residue was purified by flash
chromatography on silica gel, eluting with 0−100% EtOAc/hexane,
followed by 0−10% MeOH/DCM to give 2-(2-fluorophenyl)-5-
methoxypyrimidin-4(3H)-one as a white solid (300 mg, 80%); mp
159−161 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.81 (bs, 1H), 7.70
(s, 1H), 7.66 (t, J = 8 Hz, 1H), 7.60−7.55 (m, 1H), 7.37−7.31 (m,
1
212 °C. H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 8 Hz, 1H),
7.84 (dd, J = 10 Hz, J = 2 Hz, 1H), 7.61 (s, 1H), 7.57−7.52 (m, 1H),
7.36 (td, J = 8 Hz, J = 2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
162.1 (JC,F = 243 Hz), 158.9, 146.5, 143.8, 134.7, 131.5, 130.6 (JC,F = 8
Hz), 123.0, 117.2 (JC,F = 20 Hz), 113.6 (JC,F = 24 Hz). 19F NMR (376
G
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Journal of Medicinal Chemistry
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1H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 159.3 (JC,F
=
7.47 (t, J = 8 Hz, 2H), 7.37 (t, J = 7 Hz, 1H), 4.19 (s, 3H), 3.98 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 159.7, 156.0. 142.5, 141.1, 140.7,
137.3, 136.4, 128.8, 128.1, 127.5, 126.1, 56.4, 54.0. HRMS (ESI)
calculated for C18H17N2O2 (M + H)+ 293.1285, found 293.1286.
2-(3-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (7). 2-(3-Biphenyl)-
5-methoxypyrimidin-4(3H)-one (100 mg, 0.36 mmol) was dissolved
in anhydrous DCM (5 mL). The reaction mixture was cooled to 0 °C,
and the 1 M in DCM BBr3 (3.60 mL, 3.60 mmol) was added. It was
then allowed to warm to room temperature and stirred for 6 h. Then
the solvent was removed under reduced pressure. The resulting
residue was diluted with EtOAc, which was washed with 2 N HCl,
followed by brine. The organic layer was dried over Na2SO4, followed
by concentration under the vacuum. The resulting residue was purified
by flash chromatography on silica gel, eluting with 0−10% MeOH/
DCM to give 2-(3-biphenyl)-5-hydroxypyrimidin-4(3H)-one as a
248 Hz), 157.3, 145.9, 132.4 (JC,F = 9 Hz), 130.8 (JC,F = 8 Hz), 130.2,
124.5 (JC,F = 3 Hz), 121.8, 121.6, 116.1 (JC,F = 22 Hz), 56.0. 19F NMR
(376 MHz, DMSO-d6) δ −115.2. HRMS (ESI) calculated for
C11H10FN2O2 (M + H)+ 221.0721, found 221.0721.
2-(2-Fluorophenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dime-
thoxypyrimidine (300 mg, 1.72 mmol), (2-fluorophenyl)boronic acid
(343 mg, 2.58 mmol), Pd(PPh3)4 (199 mg, 0.17 mmol), and Na2CO3
(546 mg, 5.15 mmol) were dissolved in a mixture of dioxane (12 mL)
and water (4 mL). The air was evacuated and replaced with N2. Then,
the reaction mixture was refluxed for 18 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−30% EtOAc/hexane. This afforded
2-(2-fluorophenyl)-4,5-dimethoxypyrimidine as a white solid (402 mg,
1
white solid (34 mg, 36%); mp 221−223 °C. H NMR (400 MHz,
DMSO-d6) δ 8.30 (s, 1H), 7.99 (d, J = 7 Hz, 1H), 7.88 (d, J = 8 Hz,
1H), 7.81 (d, J = 8 Hz, 2H), 7.66 (s, 1H), 7.63 (t, J = 8 Hz, 1H), 7.52
(t, J = 8 Hz, 2H), 7.43 (t, J = 7 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 158.9, 147.5, 143.7, 140.4, 139.5, 133.1, 132.3, 129.2,
128.9, 128.5, 127.7, 126.8, 126.0, 125.0. HRMS (ESI) calculated for
C16H13N2O2 (M + H)+ 265.0972, found 265.0973.
1
100%); mp 72−74 °C. H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H),
7.97 (td, J = 8 Hz, J = 2 Hz, 1H), 7.34−7.29 (m, 1H), 7.15 (td, J = 8
Hz, J = 1 Hz, 1H), 7.11−7.06 (m, 1H), 4.06 (s, 3H), 3.90 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 161.0 (JC,F = 253 Hz), 159.5, 154.2 (JC,F
=
4 Hz), 140.9, 137.0, 131.4 (JC,F = 2 Hz), 130.9 (JC,F = 8 Hz), 126. 0
(JC,F = 9 Hz), 123.8 (JC,F = 4 Hz), 116.7 (JC,F = 22 Hz), 56.2, 54.1. 19
F
2-(3-Biphenyl)-5-methoxypyrimidin-4(3H)-one. 2-(3-Biphenyl)-
4,5-dimethoxypyrimidine (670 mg, 2.29 mmol) was dissolved in a
mixture of 2 N HCl (15 mL) and dioxane (15 mL). The reaction
mixture was then refluxed for 18 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with water, followed by brine. The organic layer was dried over
Na2SO4 and concentrated. The resulting residue was suspended in hot
chloroform and was filtered. The solid was collected to give 2-(3-
biphenyl)-5-methoxypyrimidin-4(3H)-one as a white solid (493 mg,
77%); mp 209−211 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.97 (br
s, 1H), 8.34 (s, 1H), 8.04 (d, J = 8 Hz, 1H), 7.83−7.80 (m, 3H), 7.73
(s, 1H), 7.59 (t, J = 8 Hz, 1H), 7.51 (t, J = 8 Hz, 2H), 7.41 (t, J = 7 Hz,
1H), 3.82 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 157.9, 148.9,
145.8, 140.4, 139.4, 132.9, 130.5, 129.3, 128.9, 128.8, 127.8, 126.9,
126.2, 125.2, 56.1. HRMS (ESI) calculated for C17H15N2O2 (M + H)+
279.1128, found 279.1129.
NMR (376 MHz, CDCl3) δ −114.6. HRMS (ESI) calculated for
C12H12FN2O2 (M + H)+ 235.0877, found 235.0877.
2-(4-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (6). 2-(4-Biphenyl)-
5-methoxypyrimidin-4(3H)-one (50 mg, 0.18 mmol) was dissolved in
anhydrous DCM (5 mL). The reaction mixture was cooled to 0 °C,
and then 1 M in DCM BBr3 (1.80 mL, 1.80 mmol) was added. It was
then allowed to warm to room temperature and stirred for 18 h. Then
the solvent was removed under reduced pressure. The resulting
residue was diluted with EtOAc, which was washed with 2 N HCl,
followed by brine. The organic layer was dried over Na2SO4, followed
by concentration under the vacuum. The resulting residue was purified
by flash chromatography on silica gel, eluting with 0−10% MeOH/
DCM to give 2-(4-biphenyl)-5-hydroxypyrimidin-4(3H)-one as a
1
white solid (46 mg, 96%); dec 259−261 °C. H NMR (400 MHz,
DMSO-d6) δ 12.85 (bs, 1H), 9.68 (bs, 1H), 8.13 (d, J = 8 Hz, 2H),
7.80 (d, J = 8 Hz, 2H), 7.75 (d, J = 7 Hz, 2H), 7.62 (s, 1H), 7.50 (t, J =
8 Hz, 2H), 7.41 (t, J = 7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
159.1, 147.5, 143.5, 141.9, 139.0, 131.7, 131.3, 129.0, 128.0, 128.5,
126.7, 126.7. HRMS (ESI) calculated for C16H13N2O2 (M + H)+
265.0972, found 265.0973.
2-(3-Biphenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dimethoxy-
pyrimidine (400 mg, 2.29 mmol), 3-biphenylboronic acid (681 mg,
3.44 mmol), Pd(PPh3)4 (264 mg, 0.23 mmol), and Na2CO3 (728 mg,
6.87 mmol) were dissolved in a mixture of dioxane (12 mL) and water
(4 mL). The air was evacuated and replaced with N2. Then the
reaction mixture was refluxed for 8 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−30% EtOAc/hexane. This afforded
2-(3-biphenyl)-4,5-dimethoxypyrimidine as a white solid (670 mg,
2-(4-Biphenyl)-5-methoxypyrimidin-4(3H)-one. 2-(4-Biphenyl)-
4,5-dimethoxypyrimidine (343 mg, 1.73 mmol) was dissolved in a
mixture of 2 N HCl (15 mL) and dioxane (15 mL). The reaction
mixture was then refluxed for 18 h and became a white suspension. It
was then cooled to room temperature. The suspension was filtered and
solid was collected to give 2-(4-biphenyl)-5-methoxypyrimidin-4(3H)-
1
one as white solid (256 mg, 78%); mp 292−294 °C. H NMR (400
1
MHz, DMSO-d6) δ 12.83 (bs, 1H), 8.14 (d, J = 8 Hz, 2H), 7.81 (d, J =
8 Hz, 2H), 7.77−7.72 (m, 3H), 7.50 (t, J = 8 Hz, 2H), 7.42 (t, J = 7
Hz, 1H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.0,
148.8, 145.6, 142.1, 139.0, 131.2, 130.4, 129.0, 128.0, 127.7, 126.74,
126.70, 56.1. HRMS (ESI) calculated for C17H15N2O2 (M + H)+
279.1128, found 279.1128.
100%); mp 72−74 °C. H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H),
8.36 (d, J = 8 Hz, 1H), 8.15 (s, 1H), 7.71 (d, J = 7 Hz, 2H), 7.67 (d, J
= 8 Hz, 1H), 7.54 (t, J = 8 Hz, 2H), 7.49−7.45 (m, 3H), 7.37 (t, J = 7
Hz, 1H), 4.18 (s, 3H), 3.97 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
159.7, 156.1, 141.4, 141.17, 141.15, 137.9, 137.2, 128.9, 128.8, 128.6,
127.4, 127.3, 126.6, 126.4, 56.4, 54.0. HRMS (ESI) calculated for
C18H17N2O2 (M + H)+ 293.1285, found 293.1286.
2-(4-Biphenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dimethoxy-
pyrimidine (400 mg, 2.29 mmol), 4-biphenylboronic acid (681 mg,
3.44 mmol), Pd(PPh3)4 (264 mg, 0.23 mmol), and Na2CO3 (728 mg,
6.87 mmol) were dissolved in a mixture of dioxane (12 mL) and water
(4 mL). The air was evacuated and replaced with N2. Then the
reaction mixture was refluxed for 8 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−30% EtOAc/hexane. This afforded
2-(4-biphenyl)-4,5-dimethoxypyrimidine as a white solid (670 mg,
100%); mp 115−117 °C. 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J =
8 Hz, 2H), 8.15 (s, 1H), 7.70 (d, J = 8 Hz, 2H), 7.67 (J = 7 Hz, 2H),
2-(2-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (8). 2-(2-Biphenyl)-
5-methoxypyrimidin-4(3H)-one (50 mg, 0.18 mmol) was dissolved in
anhydrous DCM (5 mL). The reaction mixture was cooled to 0 °C,
and the 1 M in DCM BBr3 (1.80 mL, 1.80 mmol) was added. It was
then allowed to warm to room temperature and stirred for 18 h. Then
the solvent was removed under reduced pressure. The resulting
residue was diluted with EtOAc, which was washed with 2 N HCl,
followed by brine. The organic layer was dried over Na2SO4, followed
by concentration under the vacuum. The resulting residue was purified
by flash chromatography on silica gel, eluting with 0−10% MeOH/
DCM to give 2-(2-biphenyl)-5-hydroxypyrimidin-4(3H)-one as a
1
white solid (31 mg, 64%); mp 271−273 °C. H NMR (400 MHz,
DMSO-d6) δ 7.70−7.65 (m, 2H), 7.56 (t, J = 8 Hz, 2H), 7.48 (s, 1H),
H
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Journal of Medicinal Chemistry
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7.39 (t, J = 7 Hz, 2H), 7.33 (t, J = 7 Hz, 1H), 7.23 (d, J = 7 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) δ 158.6, 156.9, 148.3, 140.8, 140.5,
139.4, 131.9, 130.4, 130.1, 129.9, 128.6, 128.2, 127.2. HRMS (ESI)
calculated for C16H13N2O2 (M + H)+ 265.0972, found 265.0974.
2-(2-Biphenyl)-5-methoxypyrimidin-4(3H)-one. 2-(2-Biphenyl)-
4,5-dimethoxypyrimidine (309 mg, 1.06 mmol) was dissolved in a
mixture of 2 N HCl (15 mL) and dioxane (15 mL). The reaction
mixture was then refluxed for 18 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with water, followed by brine. The organic layer was dried over
Na2SO4 and concentrated. The resulting residue was purified by flash
chromatography on silica gel, eluting with 0−10% MeOH/DCM to
give 2-(2-biphenyl)-5-methoxypyrimidin-4(3H)-one as colorless oil
Na2CO3(182 mg, 1.72 mmol) were dissolved in a mixture of dioxane
(9 mL) and water (3 mL). The air was evacuated and replaced with
N2. Then the reaction mixture was refluxed for 4 h. After the reaction
was completed, it was cooled to room temperature and it was diluted
with EtOAc and washed with satd NH4Cl, followed by brine. The
organic layer was dried over Na2SO4 and concentrated in vacuo, and
the resulting residue was purified by flash chromatography on silica gel,
eluting with 0−20% EtOAc/hexane. This afforded 4-(4,5-dimethox-
ypyrimidin-2-yl)benzonitrile as a white solid (69 mg, 74%); mp 170−
1
172 °C. H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 9 Hz, 2H), 8.18
(s, 1H), 7.76 (d, J = 9 Hz, 2H), 4.20 (s, 3H), 4.02 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 159.8, 154.0, 141.8, 141.5, 137.0, 132.3, 128.0,
119.0, 113.0, 56.4, 54.2. HRMS (ESI) calculated for C13H12N3O2 (M +
H)+ 242.0924, found 242.0929.
1
(158 mg, 54%). H NMR (400 MHz, DMSO-d6) δ 12.51 (bs, 1H),
3-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (10).
3-(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (50 mg,
0.22 mmol) was dissolved in anhydrous DCM (5 mL). The reaction
mixture was cooled to 0 °C, and then 1 M in DCM BBr3 (2.2 mL, 2.2
mmol) was added. It was then allowed to warm to room temperature
and stirred for 18 h. Then the solvent was removed under reduced
pressure. The resulting residue was suspended in water. It was filtered,
and the solid was collected and dried under vacuum to provide 3-(5-
hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile as a white solid
7.59 (t, J = 8 Hz, 1H), 7.55−7.47 (m, 4H), 7.36 (t, J = 7 Hz, 2H), 7.30
(t, J = 7 Hz, 1H), 7.23 (d, J = 7 Hz, 2H), 3.72 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 157.1, 151.0, 145.4, 140.3, 139.8, 133.0, 131.5,
131.4, 130.03, 130.01, 129.8, 128.6, 128.2, 127.2, 55.8. HRMS (ESI)
calculated for C17H15N2O2 (M + H)+ 279.1128, found 279.1129.
2-(2-Biphenyl)-4,5-dimethoxypyrimidine. 2-Chloro-4,5-dimethoxy-
pyrimidine (500 mg, 2.86 mmol), 2-biphenylboronic acid (851 mg,
4.30 mmol), Pd(PPh3)4 (266 mg, 0.29 mmol), and Na2CO3 (909 mg,
8.58 mmol) were dissolved in a mixture of dioxane (21 mL) and water
(7 mL). The air was evacuated and replaced with N2. Then the
reaction mixture was refluxed for 8 h. After the reaction was
completed, it was cooled to room temperature and it was diluted with
EtOAc and washed with satd NH4Cl, followed by brine. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−30% EtOAc/hexane. This afforded
2-(2-biphenyl)-4,5-dimethoxypyrimidine as colorless oil (377 mg,
45%). 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.84 (dd, J = 7
Hz, J = 1 Hz, 1H), 7.54−7.45 (m, 2H), 7.40 (dd, J = 7 Hz, J = 1 Hz,
1H), 7.30−7.24 (m, 3H), 7.08 (d, J = 7 Hz, 2H), 3.85 (s, 3H), 3.30 (s,
3H). 13C NMR (100 MHz, DMSO-d6) 158.1, 157.2, 142.2, 141.1,
140.0, 137.7, 137.3, 130.4, 130.2, 128.9, 128.6, 127.8, 127.2, 126.2,
56.0, 52.9. HRMS (ESI) calculated for C18H17N2O2 (M + H)+
293.1285, found 293.1290.
1
(27 mg, 58%); mp 294−296 °C. H NMR (400 MHz, DMSO-d6) δ
8.44 (s, 1H), 8.35 (d, J = 8 Hz, 1H), 7.93 (d, J = 8 Hz, 1H), 7.68 (t, J =
8 Hz, 1H), 7.61 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 159.3,
146.4, 143.9, 134.3, 133.4, 131.9, 131.5, 130.4, 129.8, 118.4, 111.7.
HRMS (ESI) calculated for C11H8N3O2 (M + H)+ 214.0611, found
214.0613.
3-(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile. 3-
(4,5-Dimethoxypyrimidin-2-yl)benzonitrile (138 mg, 0.57 mmol)
was dissolved in a mixture of 2 N HCl (5 mL) and dioxane (5
mL). The reaction mixture was then refluxed for 6 h. It was then
cooled to room temperature. The reaction mixture was diluted with
EtOAc and washed with satd NaHCO3, followed by brine. The organic
layer was dried over Na2SO4 and concentrated. The resulting residue
was purified by flash chromatography on silica gel, eluting with 0−10%
MeOH/DCM to give 3-(5-methoxy-6-oxo-1,6-dihydropyrimidin-2-
1
4-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (9). 4-
(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (50 mg,
0.22 mmol) was dissolved in anhydrous DCM (5 mL). The reaction
mixture was cooled to 0 °C, and then 1 M in DCM BBr3 (2.2 mL, 2.2
mmol) was added. It was then allowed to warm to room temperature
and stirred for 18 h. Then the solvent was removed under reduced
pressure. The resulting residue was suspended in water. It was filtered,
and the solid was collected and dried under vacuum to provide 4-(5-
hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile as a white solid
yl)benzonitrile as a white solid (56 mg, 43%); mp 255−257 °C. H
NMR (400 MHz, DMSO-d6) δ 12.93 (bs, 1H), 8.44 (s, 1H), 8.34 (d, J
= 8 Hz, 1H), 7.99 (d, J = 8 Hz, 1H), 7.74−7.70 (m, 2H), 3.82 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 157.9, 147.5, 145.9, 133.9, 133.7,
131.7, 130.7, 130.4, 129.9, 118.3, 111.7, 56.1. HRMS (ESI) calculated
for C12H10N3O2 (M + H)+ 228.0768, found 228.0767.
3-(4,5-Dimethoxypyrimidin-2-yl)benzonitrile. 2-Chloro-4,5-dime-
thoxypyrimidine (100 mg, 0.57 mmol), (3-cyanophenyl)boronic acid
(126 mg, 0.86 mmol), Pd(PPh3)4 (66 mg, 0.06 mmol), and
Na2CO3(182 mg, 1.72 mmol) were dissolved in a mixture of dioxane
(9 mL) and water (3 mL). The air was evacuated and replaced with
N2. Then the reaction mixture was refluxed for 5 h. After the reaction
was completed, it was cooled to room temperature and it was diluted
with EtOAc and washed with satd NH4Cl, followed by brine. The
organic layer was dried over Na2SO4 and concentrated in vacuo, and
the resulting residue was purified by flash chromatography on silica gel,
eluting with 0−20% EtOAc/hexane. This afforded 3-(4,5-dimethox-
ypyrimidin-2-yl)benzonitrile as a white solid (138 mg, 100%); mp
134−136 °C. 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.54 (d, J =
8 Hz, 1H), 8.09 (s, 1H), 7.65 (d, J = 8 Hz, 1H), 7.51 (t, J = 8 Hz, 1H),
4.13 (s, 3H), 3.95 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 159.8,
153.6, 141.7, 138.5, 137.0, 132.8, 131.6, 131.3, 129.2, 118.9, 112.6,
56.4, 54.2. HRMS (ESI) calculated for C13H12N3O2 (M + H)+
242.0924, found 242.0928.
2-(4-(1H-Tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one
(11). 4-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (68
mg, 0.32 mmol) and NaN3 (79 mg, 1.21 mmol) were dissolved in
anhydrous DMF (1 mL). The reaction mixture was treated with 2
drops of acetic acid. It was sealed, and then it was heated at 130 °C for
17 h. The reaction was cooled to room temperature and gave a
brownish suspension. DMF was removed by Kugelrohr distillation.
The resulting residue was suspended in water and filtered to give 2-(4-
1
(15 mg, 32%); mp 324−326 °C. H NMR (400 MHz, DMSO-d6) δ
13.05 (bs, 1H), 9.91 (bs, 1H), 8.18 (d, J = 8 Hz, 2H), 7.95 (d, J = 8
Hz, 2H), 7.64 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 158.8,
132.5, 129.4, 127.6, 127.2, 127.1, 126.7, 118.4, 112.5. HRMS (ESI)
calculated for C11H8N3O2 (M + H)+ 214.0611, found 214.0618.
4-(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile. 4-
(4,5-Dimethoxypyrimidin-2-yl)benzonitrile (53 mg, 0.22 mmol) was
dissolved in a mixture of 2 N HCl (5 mL) and dioxane (5 mL). The
reaction mixture was then refluxed for 5 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with satd NaHCO3, followed by brine. The organic layer was
dried over Na2SO4 and concentrated. The resulting residue was
purified by flash chromatography on silica gel, eluting with 0−5%
MeOH/DCM to give 4-(5-methoxy-6-oxo-1,6-dihydropyrimidin-2-
yl)benzonitrile as a white solid (50 mg, 100%); mp 297−299 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.99 (bs, 1H), 8.19 (d, J = 8 Hz,
2H), 7.96 (d, J = 8 Hz, 2H), 7.75 (s, 1H), 3.81 (s, 3H). 13C NMR (100
MHz, DMSO-d6) 157.8, 147.4, 146.2, 136.5, 132.5, 130.0, 127.8, 118.4,
112.8, 56.1. HRMS (ESI) calculated for C12H10N3O2 (M + H)+
228.0768, found 228.0770.
4-(4,5-Dimethoxypyrimidin-2-yl)benzonitrile. 2-Chloro-4,5-dime-
thoxypyrimidine (100 mg, 0.57 mmol), (4-cyanophenyl)boronic acid
(126 mg, 0.86 mmol), Pd(PPh3)4 (66 mg, 0.06 mmol), and
I
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Journal of Medicinal Chemistry
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(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one as a dark-
brown solid (42 mg, 51%); dec 290−292 °C. H NMR (400 MHz,
DMSO-d6) δ 8.08 (d, J = 9 Hz, 2H), 8.03 (d, J = 8 Hz, 2H), 7.55 (s,
1H). 13C NMR (100 MHz, DMSO-d6) δ 161.0, 160.2, 148.9, 143.3,
133.8, 132.4, 131.6, 126.9, 125.5. HRMS (ESI) calculated for
C11H9N6O2 (M + H)+ 257.0781, found 257.0791.
Na2SO4 and then concentrated. The residue was purified by flash
chromatography on silica gel, eluting with 0−30% EtOAc/hexane to
provide 5-chloro-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-one
1
1
as a white solid (59 mg, 72%); mp 101−103 °C. H NMR (400
MHz, CDCl3) δ 7.85 (s, 1H), 5.45 (s, 2H), 4.08 (s, 3H), 3.44 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 159.1, 155.1, 127.1, 116.9, 81.9, 57.9,
57.8. HRMS (ESI) calculated for C7H10ClN2O3 (M + H)+ 205.0374,
found 205.0384.
2-(3-(1H-Tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one
(12). 3-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (108
mg, 0.50 mmol) and NaN3 (131 mg, 2.02 mmol) were dissolved in
anhydrous DMF (1 mL). The reaction mixture was treated with 2
drops of acetic acid. It was sealed, and then it was heated at 130 °C for
18 h. The reaction was cooled to room temperature and gave a
brownish suspension. It was filtered, and a greenish solid was obtained.
The greenish solid was suspended in 2 N HCl, followed by second
filtration to provide 2-(3-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimi-
4,5-Dichloro-2-(methoxymethyl)pyridazin-3(2H)-one. 4,5-Di-
chloropyridazin-3(2H)-one (200 mg, 1.21 mmol) and 4-dimethylami-
nopyridine (15 mg, 0.12 mmol) were dissolved in anhydrous DCM
(20 mL). Then the reaction mixture was cooled to 0 °C and treated
with NEt3 (0.29 mL, 1.70 mmol), followed by MOMCl (0.110 mL,
1.454 mmol). The reaction mixture was allowed to warm to room
temperature and stirred for 17 h. It was then poured into DCM, and it
was washed with satd NH4Cl, followed by brine. The organic layer was
dried over Na2SO4, which was concentrated. The resulting residue was
purified by flash chromatography on silica gel, eluting with 0−30%
EtOAc/hexane to provide 4,5-dichloro-2-(methoxymethyl)pyridazin-
1
din-4(3H)-one as a beige solid (32 mg, 25%); dec 295−297 °C. H
NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.11 (d, J = 8 Hz, 1H),
7.97 (d, J = 8 Hz, 1H), 7.63 (s, 1H), 7.56 (t, J = 8 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) δ162.3, 159.1, 158.7, 148.0, 143.4, 133.1, 130.4,
128.9, 127.9, 126.5, 125.1. HRMS (ESI) calculated for C11H9N6O2 (M
+ H)+ 257.0781, found 257.0785.
1
3(2H)-one as a white solid (84 mg, 33%); mp 65−67 °C. H NMR
(400 MHz, CDCl3) δ 7.78 (s, 1H), 5.41 (s, 2H), 3.43 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 156.9, 137.0, 136.0, 134.9, 82.4, 58.1.
6-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one (14). 6-(4-Flu-
orophenyl)-4-methoxypyridazin-3(2H)-one (16 mg, 0.074 mmol) was
dissolved in anhydrous DCM (5 mL). The reaction mixture was
cooled to 0 °C, and then 1 M in DCM BBr3 (0.74 mL, 0.74 mmol)
was added. It was then allowed to warm to room temperature and
stirred for 36 h. Then the solvent was removed under reduced
pressure. The resulting residue was suspended in water. It was filtered,
and the solid was collected and dried under vacuum to provide 6-(4-
fluorophenyl)-4-hydroxypyridazin-3(2H)-one as a white solid (5 mg,
35%); mp 281−283 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.10 (bs,
1H), 11.02 (bs, 1H), 7.87 (dd, J = 9 Hz, J = 5 Hz, 2H), 7.30−7.26
(m,2H), 7.19 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 162.6 (JC,F
= 245 Hz), 157.6, 155.4, 145.2, 132.0 (JC,F = 3 Hz), 128.0 (JC,F = 9
Hz), 115.6 (JC,F = 22 Hz), 106.0. 19F NMR (376 MHz, DMSO-d6) δ
−113.0. HRMS (ESI) calculated for C10H8FN2O2 (M + H)+ 207.0564,
found 207.0567.
5-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one (13). 5-(4-Flu-
orophenyl)-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-one (55
mg, 0.21 mmol) was dissolved in anhydrous DCM (10 mL). The
reaction mixture was cooled to 0 °C, and then 1 M in DCM BBr3 (2.1
mL, 2.1 mmol) was added. It was then allowed to warm to room
temperature and stirred for 24 h. Then the solvent was removed under
reduced pressure. This gave 5-(4-fluorophenyl)-4-methoxypyridazin-
3(2H)-one, which was again recharged with 1 M in DCM BBr3 (2.1
mL, 2.1 mmol). The reaction mixture was stirred for 24 h at room
temperature. Then the solvent was again removed under reduced
pressure. The resulting residue was suspended with water and filtered.
The filtered solid was purified by flash chromatography on silica gel,
eluting with 0−10% MeOH/DCM. This gave 5-(4-fluorophenyl)-4-
hydroxypyridazin-3(2H)-one as a white solid (6.2 mg, 14%); mp 274−
1
276 °C. H NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 7.76 (s,
1H), 7.58 (J = 8 Hz, J = 5 Hz, 2H), 7.21−7.17 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 161.2 (JC,F = 243 Hz), 161.9, 154.6, 132.7,
132.4 (JC,F = 8 Hz), 127.2 (JC,F = 4 Hz), 115.8, 114.2 (JC,F = 21 Hz).
19F NMR (376 MHz, DMSO-d6) δ −114.1. HRMS (ESI) calculated
6-(4-Fluorophenyl)-4-methoxypyridazin-3(2H)-one. 6-(4-Fluoro-
phenyl)-3,4-dimethoxypyridazine (122 mg 0.52 mmol) was dissolved
in a mixture of 2 N HCl (5 mL) and dioxane (5 mL). The reaction
mixture was then refluxed for 11 h. It was then cooled to room
temperature. The reaction mixture was diluted with EtOAc and
washed with satd NaHCO3, followed by brine. The organic layer was
dried over Na2SO4 and concentrated. The resulting residue was
purified by flash chromatography on silica gel, eluting with 0−10%
MeOH/DCM to give 6-(4-fluorophenyl)-4-methoxypyridazin-3(2H)-
for C10H8FN2O2 (M + H)+ 207.0564, found 207.0575.
5-(4-Fluorophenyl)-4-methoxy-2-(methoxymethyl)pyridazin-
3(2H)-one. 5-Chloro-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-
one (58 mg, 0.28 mmol), (4-fluorophenyl)boronic acid (140 mg, 0.43
mmol), Pd(PPh3)4 (32 mg, 0.028 mmol), and Na2CO3 (90 mg, 0.85
mmol) were dissolved in a mixture of dioxane (9 mL) and water (3
mL). The air was evacuated and replaced with N2. Then the reaction
mixture was refluxed for 14 h. After the reaction was completed, it was
cooled to room temperature, and it was diluted with EtOAc and
washed with satd NH4Cl, followed by brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo, and the resulting
residue was purified by flash chromatography on silica gel, eluting with
0−30% EtOAc/hexane. This afforded 5-(4-fluorophenyl)-4-methoxy-
2-(methoxymethyl)pyridazin-3(2H)-one as a white solid (56 mg,
74%); mp 123−125 °C. 1H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H),
7.53 (dd, J = 9 Hz, J = 6 Hz, 2H), 7.11−7. 07 (m, 2H), 5.48 (s, 2H),
3.93 (s, 3H), 3.49 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 162.6 (JC,F
= 247 Hz), 161.6, 154.8, 132.3 (JC,F = 8 Hz), 128.5, 125.7, 120.7, 112.9
(JC,F = 22 Hz), 81.6, 57.9, 57.3. 19F NMR (376 MHz, CDCl3) δ
−112.6. HRMS (ESI) calculated for C13H14FN2O3 (M + H)+
265.0983, found 265.0992.
5-Chloro-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-one.
4,5-Dichloro-2-(methoxymethyl)pyridazin-3(2H)-one (84 mg, 0.40
mmol) was added to MeOH (10 mL). Then the reaction mixture
was cooled to 0 °C. It was treated with NaOMe (24 mg, 0.44 mmol)
and allowed to warm to room temperature. The reaction mixture was
then stirred for 18 h at room temperature. After the reaction was
completed, it was put under the vacuum to remove MeOH. The
resulting residue was diluted with EtOAc, which was then washed with
satd NH4Cl, followed by brine. The organic layer was dried over
1
one as a white solid (41 mg, 36%); mp 211−213 °C. H NMR (400
MHz, DMSO-d6) δ 13.05 (br s, 1H), 7.96 (dd, J = 9 Hz, J = 6 Hz,
2H), 7.34−7.29 (m, 2H), 7.28 (s, 1H), 3.92 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 162.7 (JC,F =245 Hz), 156.2, 155.7, 144.3, 132.0
(JC,F = 3 Hz), 128.2 (JC,F = 8 Hz), 115.6 (JC,F = 22 Hz), 104.4, 56.2.
19F NMR (376 MHz, DMSO-d6) δ −112.9. HRMS (ESI) calculated
for C11H10FN2O2 (M + H)+ 221.0721, found 221.0727.
6-(4-Fluorophenyl)-3,4-dimethoxypyridazine. 6-Chloro-3,4-dime-
thoxypyridazine (101 mg, 0.58 mmol), (4-fluorophenyl)boronic acid
(122 mg, 0.87 mmol), Pd(PPh3)4 (67 mg, 0.06 mmol), and
Na2CO3(184 mg, 1.74 mmol) were dissolved in a mixture of dioxane
(15 mL) and water (5 mL). The air was evacuated and replaced with
N2. Then the reaction mixture was refluxed for 3 h. After the reaction
was completed, it was cooled to room temperature and it was diluted
with EtOAc and washed with satd NH4Cl, followed by brine. The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−20% EtOAc/hexane. This afforded
6-(4-fluorophenyl)-3,4-dimethoxypyridazine as a white solid (109 mg,
80%); mp 103−105 °C. 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, J =
9 Hz, J = 5 Hz, 2H), 7.10−7. 06 (m, 2H), 7.00 (s, 1H), 4.14 (s, 3H),
3.93 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.7 (JC,F = 247 Hz),
156.5, 155.7, 148.9, 132.9 (JC,F = 3 Hz), 128.5 (JC,F = 9 Hz), 115.8 (JC,F
J
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Journal of Medicinal Chemistry
Article
= 21 Hz), 104.7, 55.7, 55.2. 19F NMR (376 MHz, CDCl3) δ −112.2.
HRMS (ESI) calculated for C12H12FN2O2 (M + H)+ 235.0877, found
235.0885.
solid, which was treated with 2 N HCl and filtered again. This afforded
6-(4-(1H-tetrazol-5-yl)phenyl)-4-hydroxypyridazin-3(2H)-one as a
white solid (33 mg, 48%); dec 273−275 °C. H NMR (400 MHz,
1
6-Chloro-3,4-dimethoxypyridazine. 3,4,6-Trichloropyridazine (200
mg, 1.09 mmol) was added to MeOH (15 mL). Then the reaction
mixture was cooled to 0 °C. It was treated with NaOMe (117 mg, 2.17
mmol) and allowed to warm to room temperature. The reaction
mixture was then stirred for 10 h at room temperature. After the
reaction was completed, it was put under the vacuum to remove
MeOH. The resulting residue was diluted with EtOAc, which was then
washed with satd NH4Cl, followed by brine. The organic layer was
dried over Na2SO4 and then concentrated. The residue was purified by
flash chromatography on silica gel, eluting with 0−30% EtOAc/hexane
to provide 4-chloro-5,6-dimethoxypyrimidine as a white solid (101 mg,
53%); mp 117−119 °C. 1H NMR (400 MHz, CDCl3) δ 6.71 (s, 1H),
4.08 (s, 3H), 3.88 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 156.9,
151.1, 149.8, 108.4, 56.2, 55.3.
4-(5-Hydroxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile (15).
4-(5-Methoxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile (109 mg,
0.48 mmol) was dissolved in anhydrous DCM (5 mL). The reaction
mixture was cooled to 0 °C, and then 1 M in DCM BBr3 (4.8 mL, 4.8
mmol) was added. It was then allowed to warm to room temperature
and stirred for 20 h. Then the solvent was removed under reduced
pressure. The resulting residue was suspended in water. It was filtered,
and the solid was collected. The solid was dry loaded on silica gel and
was purified by flash chromatography on silica gel, eluting with 0−10%
MeOH/DCM. This afforded 4-(5-hydroxy-6-oxo-1,6-dihydropyrida-
zin-3-yl)benzonitrile as a white solid (75 mg, 73%); mp 305−307 °C.
1H NMR (400 MHz, DMSO-d6) δ 13.29 (s, 1H), 11.15 (bs, 1H), 8.03
(d, J = 9 Hz, 2H), 7.92 (d, J = 9 Hz, 2H), 7.29 (s, 1H). 13C NMR (100
MHz, DMSO-d6) δ 157.7, 154.6, 144.4, 139.8, 132.7, 126.5, 118.6,
111.4, 106.0. HRMS (ESI) calculated for C11H8N3O2 (M + H)+
214.0611, found 214.0615.
DMSO-d6) δ 11.13 (s, 1H), 8.09 (d, J = 8 Hz, 2H), 7.95 (d, J = 7 Hz,
2H), 7.25 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 158.0, 157.7,
154.9, 145.7, 135.8, 128.9, 126.6, 126.2, 105.9. HRMS (ESI) calculated
for C11H9N6O2 (M + H)+ 257.0781, found 257.0791.
Expression, Purification, and Crystallization. Pandemic H1N1
endonuclease (residues 1−204) was expressed and purified as
described previously.13 The BL21 (RIL) cells (Stratagene) were
grown to an OD600 of 0.8 and induced with 0.15 mM IPTG at 17 °C
for 17 h. Cells were harvested by centrifugation and purified on Ni-
NTA (Qiagen) according to manufacturers recommendations, except
the final elution was completed with 500 mM imidazole containing
buffer. The dual hexahis tag was then removed by HRV14 3C protease
cleavage. The endonuclease was further purified by size exclusion
chromatography using HiLoad 26/60 Superdex 75 (GE Healthcare).
The buffer used for size exclusion and the final buffer for storage of the
protein was 100 mM NaCl and 20 mM Tris pH 8.0. The protein was
concentrated to 5 mg/mL using an Ultrafree 10K (Millipore),
aliquoted, and stored at −80 °C.
Crystals are formed by mixing in a 1:1 ratio endonuclease (5 mg/
mL) with crystallization buffer containing 200 mM MES pH 6.7, 27%
PEG8000, 200 mM ammonium sulfate, 1 mM manganese chloride, 10
mM magnesium acetate, 10 mM taurine, and 50 mM sodium fluoride.
Crystals form within a few hours and grow to maximum size in 1−2
weeks at 20 °C.
Compound Soaking, Data Collection, and Processing.
Crystal structures of compounds 11 were determined in complex
with endonuclease as previously described.11 11 was soaked into
preformed crystals by stepwise gradient shifting the surrounding
crystallization solution to 1 mM manganese sulfate, 200 mM HEPES
pH 7.7, 25% (w/v) PEG 8000, 50 mM ammonium sulfate, 5 mM
magnesium acetate, 80 mM L-arginine, 10% DMSO containing 100
mM 11, and 10% (v/v) ethylene glycol. Crystals were then soaked
with the ligand for 2 h at 20 °C before being placed into liquid
nitrogen for storage. X-ray diffraction data collection was performed at
the Cornell High Energy Synchrotron Source (CHESS) F1 beamline.
The diffraction data were indexed, processed, scaled, and merged using
HKL2000.32 The structure was solved and refined using the software
PHENIX.33
Endonuclease Assay. A high throughput endonuclease assay was
performed as described previously.11 The endonuclease assay used a
single-stranded DNA probe with the sequence (6-FAM)-
TGGCAATATCAGCTCCACA(MGBNFQ) (Applied Biosystems).
Reaction buffer contained 50 mM Tris pH 7.5, 50 mM sodium
chloride, 1 mM DTT, 5 mM magnesium chloride, 0.5 mM manganese
sulfate, and 1 mM CHAPS. DNA probe (50 nM), endonuclease (25
nM), and compound were mixed on ice and then placed in a
Varioskan fluorimeter (Thermo Scientific) preincubated at 37 °C to
detect fluorescence with an excitation of 488 nm and emission of 518
nm. Fluorescence is measured at various time points, and activity/
inhibition is calculated with GraphPad Prism 6.0b.
4-(5-Methoxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile. 4-
(5,6-Dimethoxypyridazin-3-yl)benzonitrile (125 mg, 0.52 mmol) was
dissolved in a mixture of 2 N HCl (5 mL) and dioxane (5 mL). The
reaction mixture was then refluxed for 4 h. It was then cooled to room
temperature and then put under reduced pressure. The resulting
residue was suspended in water and filtered. The product 4-(5-
methoxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile was collected as
1
a white solid (82 mg, 70%); mp 271−273 °C. H NMR (400 MHz,
DMSO-d6) δ 13.97 (bs, 1H), 8.02 (d, J = 8 Hz, 2H), 7.94 (d, J = 8 Hz,
2H), 6.62 (s, 1H), 3.96 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
156.3, 155.9, 143.5, 139.7, 132.7, 126.7, 118.6, 111.4, 104.3, 56.3.
HRMS (ESI) calculated for C12H10N3O2 (M + H)+ 228.0768, found
228.0774.
4-(5,6-Dimethoxypyridazin-3-yl)benzonitrile. 6-Chloro-3,4-dime-
thoxypyridazine (298 mg, 1.71 mmol), (4-cyanophenyl)boronic acid
(376 mg, 2.56 mmol), Pd(PPh3)4 (198 mg, 0.17 mmol), and
Na2CO3(543 mg, 5.12 mmol) were dissolved in a mixture of dioxane
(15 mL) and water (5 mL). The air was evacuated and replaced with
N2. Then the reaction mixture was refluxed for 15 h. After the reaction
was completed, it was cooled to room temperature and it was diluted
with EtOAc and washed with satd NH4Cl, followed by brine. The
organic layer was dried over Na2SO4 and concentrated under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy on silica gel, eluting with 0−30% EtOAc/hexane. This afforded
4-(5,6-dimethoxypyridazin-3-yl)benzonitrile as a white solid (130 mg,
31%); mp 187−189 °C. 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 8
Hz, 2H), 7.77 (d, J = 8 Hz, 2H), 7.14 (s, 1H), 4.23 (s, 3H), 4.03 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 157.1, 154.7, 149.1, 140.9, 132.6,
127.3, 118.5, 113.0, 104.9, 55.9, 55.3. HRMS (ESI) calculated for
C13H12N3O2 (M + H)+ 242.0924, found 242.0931.
6-(4-(1H-Tetrazol-5-yl)phenyl)-4-hydroxypyridazin-3(2H)-one
(16). 4-(5-Methoxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile (56
mg, 0.26 mmol) and NaN3 (69 mg, 1.06 mmol) were dissolved in
anhydrous DMF (1 mL). The reaction mixture was treated with 2
drops of acetic acid. It was sealed, and then it was heated at 130 °C for
21 h. The reaction was cooled to room temperature and gave a
brownish suspension. The suspension was filtered and gave a white
ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray data and refinement statistics for the analysis performed
with 11. The atomic coordinates and structure factors are
deposited in the Protein Data Bank (PDB) with ID 4W9S. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1 (848) 445 2674. Fax: +1 (732) 445 6312. Email:
elavoie@pharmacy.rutgers.edu.
K
dx.doi.org/10.1021/jm500958x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
influenza-like virus H17N10 has endonuclease activity. J. Virol. 2013,
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Parhi, A.; Martinez-Sobrido, L.; LaVoie, E. J.; Das, K.; Arnold, E.
Crystallographic fragment screening and structure-based optimization
yields a new class of influenza endonuclease inhibitors. ACS Chem.
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(14) Plotch, S. J.; Bouloy, M.; Ulmanen, I.; Krug, R. M. A unique
Cap(m7CppXm)-dependent influenza virion endonuclease cleaves
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Notes
The authors declare the following competing financial
interest(s): Prof. LaVoie is a co-founder, together with Dr.
Bauman, Dr. Das and Prof. Arnold, of Prodaptics Pharmaceut-
icals, Inc..
ACKNOWLEDGMENTS
■
The Bruker Avance III 400 MHz NMR spectrometer used for
this study was purchased with funds from NCRR Grant no.
1S10RR23698-1A1. Mass spectrometry was provided by the
Washington University Mass Spectrometry Resource with
support from the NIH National Center for Research Resources
grant no. P41RR0954. We thank the laboratories of Ann Stock
and Gaetano Montelione for access to equipment used in this
study. E.A.’s laboratory is grateful for support from NIH grants
R37 AI027690 (MERIT AWARD) and P50 GM103368. X-ray
data collection was conducted at the Cornell High Energy
Synchrotron Source (CHESS). CHESS is supported by the
NSF and NIH/NIGMS via NSF award DMR-0225180, and the
MacCHESS resource is supported by NIH/NCRR award RR-
01646.
(18) Singh, S. B. Total synthesis of flutimide, a novel endonuclease
inhibitor of influenza virus. Tetrhedron Lett. 1995, 36, 2009−2012.
(19) Carcelli, M.; Rogolino, D.; Bacchi, A.; Rispoli, G.; Fisicaro, E.;
Compari, C.; Sechi, C.; Stevaert, A.; Naesens, L. Metal-Chelating 2-
Hydroxyphenyl Amide Pharmacophore for Inhibition of Influenza
Virus Endonuclease. Mol. Pharmaceutics 2013, 11, 304−316,
DOI: 10.1021/mp400482a.
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