Phenyl substituted 3-hydroxypyridin-2(1H)-ones: Inhibitors of influenza A endonuclease

Article abstract of DOI:10.1016/j.bmc.2013.08.053

Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is recognized as an attractive target for the development of new agents for the treatment of influenza infection. Our earlier study employing small molecule fragment screening using a high-resolution crystal form of pandemic 2009 H1N1 influenza A endonuclease domain (PA_N) resulted in the identification of 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating ligand at the active site of the enzyme. In the present study, several phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity as measured by a high-throughput fluorescence assay. Two of the more potent compounds in this series, 16 and 18, had IC₅₀ values of 11 and 23 nM in the enzymatic assay, respectively. Crystal structures revealed that these compounds had distinct binding modes that chelate the two active site metal ions (M1 and M2) using only two chelating groups. The SAR and the binding mode of these 3-hydroxypyridin-2-ones provide a basis for developing a new class of anti-influenza drugs.

Full text of DOI:10.1016/j.bmc.2013.08.053

Accepted Manuscript
Phenyl substituted 3-hydroxypyridin(1H)-2-ones: Inhibitors of influenza A en‐
donuclease
Ajit K. Parhi, Amy Xiang, Joseph D. Bauman, Disha Patel, R.S.K. Vijayan,
Kalyan Das, Eddy Arnold, Edmond J. LaVoie
PII:
S0968-0896(13)00756-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.053
BMC 11069
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 June 2013
15 August 2013
23 August 2013
Please cite this article as: Parhi, A.K., Xiang, A., Bauman, J.D., Patel, D., Vijayan, R.S.K., Das, K., Arnold, E.,
LaVoie, E.J., Phenyl substituted 3-hydroxypyridin(1H)-2-ones: Inhibitors of influenza A endonuclease, Bioorganic
& Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.053
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Phenyl substituted 3-hydroxypyridin(1H)-2-ones: Inhibitors of influenza A
endonuclease
Ajit K. Parhi, a,b Amy Xiang,b Joseph D. Bauman,c Disha Patel,a,c R. S. K. Vijayan,c Kalyan Das,c
Eddy Arnold,c and Edmond J. LaVoiea*
a
Department of Medicinal Chemistry, Ernest Mario School of Pharmacy,
Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020, U.S.A.
bTaxis Pharmaceuticals Inc., North Brunswick, New Jersey, U.S.A.
cCenter for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology,
Rutgers University, Piscataway, New Jersey, 08854-5627, U.S.A.
Keywords: Antiviral; influenza A, pyridinones, 3-hydroxypyridin-2-ones, endonuclease
Abstract: Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is
recognized as an attractive target for the development of new agents for the treatment of influenza
infection. Our earlier study employing small molecule fragment screening using a high-resolution
crystal form of pandemic 2009 H1N1 influenza A endonuclease domain (PAN) resulted in the
identification of 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating ligand at the active site of
the enzyme. In the present study, several phenyl substituted 3-hydroxypyridin-2(1H)-one compounds
were synthesized and evaluated for their ability to inhibit the endonuclease activity as measured by a
high-throughput fluorescence assay. Two of the more potent compounds in this series, 16 and 18, had
IC50 values of 11 and 23 nM in the enzymatic assay, respectively. Crystal structures revealed that these
compounds had a superimposable binding mode that chelates the two active site metal ions (M1 and
M2) using only two chelating groups. The SAR and the binding mode of these 3-hydroxypyridin-2-ones
provide a basis for developing a new class of anti-influenza drugs.
*Corresponding author. Tel.: +1 (848) 445 2674; fax +1 (732) 445 6312
Email address: elavoie@pharmacy.rutgers.edu (E.J. LaVoie)
1

Antivirals play a major role as a prophylactic measure and for treatment of seasonal and pandemic
influenza infections. There are two current classes of antivirals available for the treatment of influenza.
These include, adamantanes, which target the M2 ion-channel protein, as well as zanamivir and
oseltamivir, which target neuraminidase (NA). However, adamantanes are not currently recommended
in treating influenza due to widespread drug resistance. Oseltamivir (Tamiflu) is the most widely used
oral anti-influenza drug; however, oseltamivir-resistant seasonal influenza A strains are in circulation.1
When accompanied by compensatory mutations, these mutant viruses exhibit fitness comparable to
wild-type influenza A and remain resistant to oseltamivir.2 With the emergence of oseltamivir resistant
strains of influenza A, H1N1,3 H5N14 and H7N95, there is a pressing need for new antiviral therapies.
The heterotrimeric influenza RNA-dependent RNA polymerase (RdRP) is an attractive target for anti-
influenza therapy. RdRP, which is composed of polymerase acidic (PA), polymerase basic (PB1) and
polymerase basic 2 (PB2) polypeptide chains, is responsible for the replication and transcription of viral
genes into viral mRNA. This requires hijacking of 5’-capped RNA fragment from host pre-mRNAs in a
process called “cap-snatching” in which the 5’-cap is bound at the PB2 subunit and the mRNA is
cleaved 10-13 nucleotides downstream by the endonuclease activity of the PA subunit (6 for review).
The cleaved end of the capped RNA fragment is then used to prime the transcription of the viral mRNA
by RdRP.7 Cap-snatching is an attractive target for drug therapy since it is essential for replication of
influenza A, B, and C viruses, and the host cell has no analogous activity.
Inhibition of the endonuclease activity, which resides at the N-terminal domain of the PA subunit, PAN
(residues 1-197), by small molecules has been the focus of several studies. Structural characterization of
PAN and complexes with various inhibitors has provided opportunities for structure-based design of
effective endonuclease inhibitors as potential influenza drugs.8-11 Inhibitors of PAN include 2,4-
dioxobutanoic acid derivatives,12,13 flutimide and its derivatives,14,15 5-hydroxy-1,6-dihydropyrimidine-
4-carboxylic acid derivatives,16 and tetramic acid derivatives.17 Crystal structures of many of these
inhibitors have revealed a two metal binding mode using three chelating groups.
We have recently conducted a fragment screening campaign using a new crystal form of pandemic
H1N1 PAN to identify novel chemical scaffolds inhibiting the endonuclease activity (Sagong et al.,
2013; Bauman et al., submitted).18,19 Here, we report the development of a 3-hydroxypyridin-2(1H)-one
series of compounds with strong inhibitory activity in the enzymatic assay. Structural biology in
combination with efficient chemistry allowed for rapid exploration of the hydroxypridinone scaffold to
identify key ligand-protein interactions necessary for higher potency.
1. Chemistry
4-Phenyl- and 4-(p-fluorophenyl)-3-hydroxypyridin(1H)-2-one, 1 and 2, were prepared from 4-bromo-
2,3-dimethoxypyridine as outlined in Scheme 1. Commercially available, 2,3-dimethoxypyridine was
converted as previously described to 4-bromo-2,3-dimethoxypyridine,20 which was then treated with
either phenylboronic acid or p-fluorophenylboronic acid under Suzuki-coupling conditions to provide
2

either 4-phenyl- or 4(p-fluorophenyl)-2,3-dimethoxypyridine in good yield. Treatment of these
dimethoxypyridine derivatives with excess BBr3 provided 1 and 2.
Scheme 1. Preparation of 4-phenyl-3-hydroxypyridin(1H)-2-one and 4-(p-fluorophenyl)-3-hydroxypyridin(1H)-2-one.
Reagents and conditions: (a) n-BuLi, THF, Br2, - 78 ºC, then rt, (b) p-Z-Phenylboronic acid Pd(PPh3)4, K2CO3, Dioxane:H2O
(3:1), 100 ºC; (c) BBr3, CH2Cl2, rt.
The requisite halogenated intermediates that were used for the preparation of 5- and 6-phenyl substituted
3-hydroxypyridin(1H)-2-ones were synthesized as outlined in Scheme 2. Intermediate A was prepared
as previously described.21 Alternatively, 5-bromo-2,3-dimethoxypyridine, Intermediate B could be
Scheme 2. Methods for the preparation of 5- and 6-bromo 3-hydroxypyridin(1H)-2-ones. Reagents and conditions: (a) m-
CPBA, CH2Cl2, rt; (b) POCl3, CH2Cl2, rt; (c) EtONa, EtOH, 80 ºC; (d) I2, H2O, K2CO3, rt; (e) CH3I, K2CO3, THF, 0-20 ºC;
(f) Br2 (0.5 equiv.), AcOH, NaOAc, -32-0 ºC.
formed together with 6-bromo-2,3-dimethoxypyridine (intermediate D) by bromination of 2,3-
dimethoxypyridine and these two monobrominated products were separated by chromatography.22 We
also prepared 6-iodo-2,3-dimethoxypyridine, intermediate C, by an initial iodination of 2-bromo-3-
hydroxypyridine, followed by formation of its O-methyl ether and subsequent selective displacement of
its 2-bromo substituent with sodium ethoxide as described in the literature.23
3

Using either intermediate A or B, Suzuki-coupling with phenylboric acid, (p-fluorophenyl)boronic acid,
or (p-cyanophenylboronic acid) as outlined in Scheme 3 provided good yields of their respective phenyl
dialkoxypyridines. Treatment of these phenyl dialkoxypyridines with excess BBr3 provided the 3-
hydroxypyridin-2-ones, 3-5. In the presence of NaN3 in acetic acid/dimethylformamide, 5 was
converted to the 3-hydroxy-5-[4-(tetrazoly-5-yl)phenyl]pyridin(1H)-2-one, 6.
Scheme 3. Preparation of substituted 5-phenyl-3-hydroxypyridin(1H)-2-ones. Reagents and conditions: (a) p-Z-
Phenylboronic acid Pd(PPh3)4, K2CO3, Dioxane:H2O (3:1), 100 ºC; (b) BBr3, CH2Cl2, rt; (c) NaN3, AcOH, DMF, sealed tube,
120 ºC.
The synthesis of 6-phenyl substituted 3-hydroxypyridin(1H)-2-ones was similarly accomplished as
outlined in Scheme 4. Using either intermediate C or D, Suzuki-coupling was performed to provide the
desired 6-phenyl 2,3-dialkoxypyridine derivatives. Treatment of these 2,3-dialkoxypyridine derivatives
with excess BBr3 provided 7-9. In the presence of NaN3 in acetic acid/dimethylformamide, 9 was
converted to the 3-hydroxy-5-[4-(tetrazoly-5-yl)phenyl]pyridin(1H)-2-one, 10.
Scheme 4. Preparation of substituted 6-phenyl-3-hydroxypyridin(1H)-2-ones. Reagents and conditions: (a) p-Z-
Phenylboronic acid Pd(PPh3)4, K2CO3, Dioxane:H2O (3:1), 100 ºC; (b) BBr3, CH2Cl2, rt; (c) NaN3, AcOH, DMF, sealed tube,
120 ºC.
The synthesis of 5,6-diphenylpyridin(1H)-2-ones wherein the phenyl substituents had identical
substituents could be prepared from 5,6-dibromo-2,3-dimethoxypyridine as outlined in Scheme 5.
Suzuki coupling using 5,6-dibromo-2,3-dimethoxypyridine, which was prepared as previously
described23, gave the diphenyldimethoxypyridine intermediates, which in the presence of excess BBr3
provided 11-13. Treatment of 13 with NaN3 in acetic acid and dimethylformamide in a sealed tube at
120 ºC provided 5,6-bis-[(tetrazoly-5-yl)phenyl]-3-hydroxypyridin(1H)-2-one, 14.
4

The synthesis of 5-(p-fluorophenyl)-6-(p-cyanophenyl)-2,3-dimethoxypyridine was accomplished as
illustrated in Scheme 5. Bromination of 5-(p-fluorophenyl)-2,3-dimethoxypyridine provided the 6-
bromo derivative, which upon Suzuki-coupling with p-cyanophenylboronic acid gave 5-(p-
fluorophenyl)-6-(p-cyanophenyl)-2,3-dimethoxypyridine. Treatment of this dimethoxypyridine with
BBr3 gave 15, which was converted in the presence of azide to 16. In a similar manner as illustrated in
Scheme 5, 6-(p-fluorophenyl)-5-(p-cyanophenyl)-2,3-dimethoxypyridine was prepared and converted to
17. Treatment of 17 with NaN3 in acetic acid and dimethylformamide gave 5-[(4-tetrazol-5-yl)phenyl]-
6-(p-fluorophenyl-3-hydroxypyridin(1H)-2-one, 18.
Scheme 5. Methods for the preparation of substituted 5,6-diphenyl-3-hydroxypyridin(1H)-2-ones. Reagents and conditions:
(a) Br2 (2 equiv.), AcOH, NaOAc, 0 ºC; (b) p-Z-Phenylboronic acid for 9-11 and p-fluorophenylboronic acid for 13 and 15,
Pd(PPh3)4, K2CO3, Dioxane:H2O (3:1), 100 ºC; (c) BBr3, CH2Cl2, rt; (d) NaN3, AcOH, DMF, sealed tube, 120 ºC
2. Pharmacology and Structure-Activity Relationships
Each phenyl substituted or diphenyl substituted 3-hydroxypyridin-2-one was evaluated for its capacity to
inhibit influenza A endonuclease. Using a high-throughput 96-well plate based assay as previously
employed in the evaluation of a series of 3-hydroxyquinolin-2-ones,18 the relative potential to block
endonuclease cleavage by PAN was determined. A TaqMan-like oligonucleotide containing a 6-
carboxy-fluorescein (FAM) fluorophore at the 5’-end followed by 19 nucleotides and a minor groove
binding non-fluorescent quencher (MGBNFQ, Applied Biosystems) at the 3’-end was employed in these
5

studies. When excited, MGBNFQ quenches the fluorescence of FAM via fluorescence resonance energy
transfer. Upon cleavage of the oligonucleotide, the quencher is no longer coupled to the fluorophore,
and therefore, FAM fluoresces. This assay was used to determine the inhibitory IC50 values for these 3-
hydroxypyridin-2-ones.
The results of these assays are summarized in Table 1. These data indicate that the presence of either a
4-fluorophenyl or 4-(1H-tetrazol-5-yl)phenyl at the 5-position of 3-hydroxypyridin-2(1H)-one, as in 4
Table 1. Inhibition assay of influenza A endonuclease by phenyl substituted 3-hydroxypyridin(1H)-2-
ones. IC50 is the mean of triplicate measurements ± standard error of the mean (SEM).
Compd
1
IC50 (µM)
35 1.76
>200
2.1 0.107
X
Y
Z
Phenyl
H
H
Phenyl
H
H
H
H
H
H
Phenyl
4-Fluorophenyl
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
0.730 0.047
3.83 0.288
0.368 0.041
0.87 0.040
0.430 0.010
0.802 0.044
0.085 0.008
0.047 0.005
0.041 0.003
0.103 0.009
0.285 0.027
0.136 0.013
0.011 0.0005
0.054 0.003
0.023 0.001
4-Fluorophenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
H
4
5
6
7
H
H
H
Phenyl
4-Fluorophenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
Phenyl
4-Fluorophenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
4-Fluorophenyl
4-Fluorophenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
8
9
10
11
12
13
14
15
16
17
18
4-Fluorophenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
4-Cyanophenyl
4-(Tetrazol-5-yl)phenyl
4-Fluorophenyl
4-Fluorophenyl
and 6, is associated significant enzyme inhibition (IC50 < 1.0 µM). There was little difference in relative
enzyme inhibition among 6-phenyl-3-hydroxypyridin(1H)-2-ones wherein the phenyl moiety was
unsusbstituted, 7, or had a 4-cyano substituent, 9. However, there was a slight increase in activity
observed with the p-fluorophenyl derivative, 8, and a significant increase in potency with a p- (tetrazol-
5-yl) moiety, 10. The substitution of either a 4- phenyl or 4-fluorophenyl substituent at the 4-position
of 3-hydroxypyridin-2(1H)-one, 1 and 2, is clearly associated with a dramatic loss in potency as an
enzyme inhibitor in comparison to the isomers where these substituents are at either the 5- or 6-position.
All of the 5,6-diphenyl substituted 3-hydroxypyridin(1H)-2-ones exhibited significant activity as
inhibitors of influenza A endonuclease. The 5,6-bisphenyl and 5,6-bis(4-fluorophenyl) derivatives 11
6

and 12 are much more potent than the monophenyl substituted derivatives 3 and 7 or their analogous 4-
fluorophenyl derivatives 4 and 8. 5,6-bis(4-Tetrazol-5-yl)phenyl-3-hydroxypyridin(1H)-2-one, 14, was
the least active of these 5,6-diphenyl substituted derivatives with an IC50 of 0.285 µM. A single 4-
(tetrazol-5-yl)phenyl together at either the 5- or 6-position combined with a 4-fluorophenyl moiety at the
adjacent 5- or 6-position, was associated with the greatest inhibitory activity with IC50 values of 11 and
23 nM for 16 and 18, respectively. These studies indicate that significant enhancement in enzyme
inhibition can be achieved by the placement of phenyl groups at both the 5- and 6-position. In addition,
the selection of substituents on these phenyl moieties can have a profound impact on potency.
Cytotoxicity studies were performed using Madin-Darby Canine Epithelial (MDCK) Cells and human
embryonic kidney (A293) cells. There was no correlation between the potency of inhibition of influenza
endonuclease activity and relative cytotoxic activity. In MDCK cells, 13 and 17 had IC50 values of 40
µM, 15 and 16 had IC50 values of 50 µM. In this same cell line, 3 and 7 had IC50 values of 80 and 60
µM, the remaining twelve compounds have IC50 values >100 µM. In the human A293 cell line,
compounds 3, 7, 9-11, 13 and 15-17 have IC50 values that are ≥28 µM, while 6 has an IC50 value of 80
µM and 1, 2, 4, 5, 8, 12, 14 and 18 have IC50 values >100 µM. While at an early stage of development,
these data do not indicate any major concern that would interfere in future studies directed toward the
establishment of ex-vivo antiviral data for this series of compounds.
Crystal structures of PAN in complex with compounds of this series were routinely determined as part of
a structure-based drug design effort.18 In brief, the pyridinone ring chelates to the active site metals, M1
and M2, in a mode that allows for π-stacking with His41. The pyridinone nitrogen is within hydrogen-
bonding distance (2.9 Å for both) to a coordinating water of M2 and to the catalytic residue Glu80.
During the course of fragment screening a third metal was discovered to exist in the active site (M3),
which can make cation-π interactions with a phenyl ring substituted at the 6-position of the pyridinone.
Crystal structures of the two most potent compounds, 16 and 18, bound to the endonuclease active site
are described here. Soaking of 18 into apo crystals of PAN revealed the compound bound to the active
site metal in a flipped orientation compared to what was detected previously (Figure 1-A). The
pyridinone ring nitrogen makes an electrostatic interaction with Tyr130 (4.1 Å). The p-fluorophenyl
forms a cation-π interaction with M3 and makes hydrophobic interactions with the side chains of Ala20,
Tyr24, and Ile38. The electron density for Tyr24 is weak, indicating that the residue is flexible and
further optimization at the 6-position may strengthen this interaction and thereby will improve the
potency. The 4-(tetrazol-5-yl)phenyl forms hydrophobic interactions with the side chains of Lys34,
Ala37, and Ile38. The tetrazolyl group displaces an ethylene glycol molecule, previously detected in
other crystal structures of PAN, to form bidentate hydrogen bonds to Arg84. When the 4-(tetrazol-5-
yl)phenyl was substituted at the 5-position, compound 16 (Figure 1-B) retains the standard orientation
seen in previous structures of compounds in this series.18 The greater potency of 16 in comparison to 18
can be attributed to the optimal position of the pyridinone nitrogen and its favorable interactions with
metal chelating atoms.
7

Figure 1. Stereoview images of crystal structures of 16 and 18 bound to PAN. Metal-coordinating bonds are depicted as
black dashed lines, hydrophobic and cation-π interactions are grey, and hydrogen or electrostatic bonds as blue dashed lines.
Residues with significant structural changes upon binding of a ligand are depicted with the apo structure shown in orange.
Electron density calculated from an omit map is contoured at 4.0σ. (A) Crystal structure of 18 bound to PAN. Ethylene
glycol, as detected in other PAN crystal structures, is shown in light green. (B) Crystal structure of 16 bound to PAN.
3. Conclusion
Several 5- and 6-phenyl substituted 3-hydroxypyridin(1H)-2-ones have significant activity as inhibitors
of influenza A endonuclease. The presence of phenyl substituents at both the 5- and 6-positions of 3-
hydroxypyridin(1H)-2-one resulted in a further enhancement in their potency as inhibitors. Among the
8

diphenyl 3-hydroxypyridin(1H)-2-ones evaluated, the two more potent analogs, 16 and 18, had a tetrazo-
5-yl moiety at the para-position of either the 5- or 6-phenyl substituent, respectively. The binding mode
of each of these compounds as determined by X-ray crystallography differed dramatically and was
influenced by the interaction of this tetrazole within the active site of the enzyme. This observation is of
major importance in understanding the structure-activity of similarly substituted 3-hydroxypyridin(1H)-
2-ones. It also provides critical insights that could prove useful in efforts to further the development of
3-hydroxypyridin(1H)-2-ones as potent inhibitors of influenza A endonuclease.
4. Chemistry Experimental
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 (300 MHz) and
13C (75 MHz) NMR spectra were done in CDCl3, Methanol-d4, and DMSO-d6 and recorded on a Varian
Unity Inova (300 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. HRMS experiments were conducted by Washington University
Resource for Biomedical and Bioorganic Mass Spectrometry Department of Chemistry.
4.1.1 4-Phenyl-3-hydroxypyridin-2(1H)-one (1)
To a solution of (80 mg, 0.37 mmol) in CH2Cl2 (3.0 ml) under nitrogen, boron tribromide (1.0 ml of a
1.0 M solution in CH2Cl2) was added. After addition was completed, the reaction mixture was stirred
for 16 hours at room temperature. Dichloromethane was removed from the reaction mixture followed
by addition of HCl (3N). The resulting solid was filtered. The solid was redissolved in CH2Cl2 and
washed with NaHCO3 and brine. The organic phase was then dried and evaporated under reduced
pressure to afford the crude product which was purified by ISCO flash chromatography using 10%
MeOH in DCM to furnish the pure product (25 mg) as white solid, yield: 36%. mp 186-188 °C; 1H
NMR (300 MHz, DMSO-d6) δ: 7.62 (d, J = 7.5 Hz, 2H), 7.41 (m, 2H), 7.33 (m, 1H), 6.92 (d, J = 6.3 Hz,
1H), 6.25 (d, J = 6.3 Hz, 1H). 13C NMR (75 MHz, DMSO d6) δ: 172.5, 159.4, 143.9, 136.7, 129.3,
128.9, 128.4, 123.9, 107.6. HRMS Calcd for C11H9NO2 (M + H)+ 188.0706. Found 188.0706.
4.1.2 2,3-Dimethoxy-4-phenylpyridine
A mixture of 4-bromo-2,3-dimethoxypyridine (100 mg, 0.46 mmol) , phenylboronic acid (84 mg, 0.69
mmol), Pd(PPh3)4 (58 mg, 0.05 mmol) and K2CO3 (125 mg, 0.92 mmol) in 1,4- dioxane (3.0 ml) and
H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 hours.
The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
9

resulting residue was purified by ISCO flash chromatography using 10% EtOAc in hexane to give 80
1
mg (81%) of the desired product. H NMR (300 MHz, CDCl3) δ: 7.92 (d, J = 5.1 Hz, 1H), 7.58 (d, J =
7.2 Hz, 2H), 7.41 (m, 3H), 6.88 (d, J = 5.1Hz, 1H), 4.05 (s, 3H), 3.63 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ: 176.1, 158.4, 142.3, 140.7, 135.9, 128.9, 128.4, 128.2, 118.3, 60.3, 53.7.
4.1.3 4-Bromo-2,3-dimethoxypyridine
To a solution of 2,3-dimethoxypyridine (1.59 g, 10.77 mmol) in THF (30 ml) at -75 oC was added n-
BuLi (10.6 ml, 1.6 M solution in hexane) dropwise. The reaction mixture was stirred at 0 oC for 1 h and
cooled again to -70 °C before the addition of Br2 (0.6 ml, 10.77 mmol). The reaction mixture was
stirred at -70 oC for 2 h and then was allowed to warm to room temperature. The reaction was then
quenched with water, and was diluted with ethyl acetate. The layers were separated and the organic
layer was washed with sodium thiosulphate and water. The organic layer was dried with Na2SO4,
concentrated under reduced pressure to afford a brown oil. Purification in ISCO using 10% EtOAc in
hexane furnished the product (1.2 g, 51% yield. 1H NMR (300 MHz, CDCl3) δ: 7.66 (d, J = 5.4 Hz, 1H),
7.01 (d, J = 5.4 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 172.3, 158.4, 141.1,
126,1, 121.9, 60.4, 53.9.
4.2.1 4-(p-Fluorophenyl)-3-hydroxypyridin-2(1H)-one (2)
To a solution of 2,3-dimethoxy-4-(p-fluorophenyl)pyridine (190 mg, 0.81 mmol) in CH2Cl2 (5.0 ml)
under nitrogen, boron tribromide (1.5 ml of 1.0 M solution in CH2Cl2) was added. After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered.
The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine. The organic phase was then
dried and evaporated under reduced pressure to afford the crude product which was purified in ISCO
1
using 10% MeOH in DCM to furnish the pure product (100 mg) as white solid, yield: 60%. H NMR
(300 MHz, DMSO-d6) δ: 11.83 (bs, 1H), 7.71 (m, 1H), 7.50 (m, 1H), 7.30-7.19 (m, 2H), 6.96 (d, J = 4.8
Hz, 1H), 6.30 (m, 1H). HRMS Calcd for C11H8FNO2 (M + H)+ 206.0612. Found 206.0609.
4.2.2 2,3-Dimethoxy-4-(4-fluorophenyl)pyridine
A mixture of 4-bromo-2,3-dimethoxypyridine (100 mg, 0.46 mmol), 4-fluorophenylboronic acid (84 mg,
0.6 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol) and K2CO3 (125 mg, 0.92 mmol) in 1,4- dioxane (3.0 ml)
and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16
hours. The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and
EtOAc (3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was
concentrated. The resulting residue was purified by ISCO flash chromatography using 20% EtOAc in
1
hexane to give 95.5 mg (89%) of the desired product. H NMR (300 MHz, CDCl3) δ: 7.89 (d, J = 5.1
Hz, 1H), 7.57-7.53 (m, 2H), 7.13-7.07 (m, 2H), 6.83 (d, J = 5.4 Hz, 1H), 4.02 (s, 3H), 3.61 (s, 3H). 13C
NMR (75 MHz, CDCl3) δ: 176.2, 164.4, 161.1, 158.5, 141.2, 140.8, 130.8, 130.7, 118.1, 115.5, 115.2,
60.3, 53.7.
10

4.3.1 5-Phenyl-3-hydroxypyridin-2(1H)-one (3)
To a solution of 2,3-dimethoxy-5-phenylpyridine (100 mg, 0.465 mmol) in CH2Cl2 (3.0 ml) under
nitrogen, boron tribromide was added (1.0 ml of 1.0 M solution in CH2Cl2). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed and the reaction mixture was diluted with ethyl acetate. The solution was washed with
NaHCO3 and brine. The organic phase was then dried and evaporated under reduced pressure to afford
the crude product which was purified in ISCO using 10% MeOH in DCM to furnish the pure product
(21 mg) as white solid, yield: 24%. mp 175-177 oC; 1H NMR (300 MHz, DMSO-d6) δ: 9.33 (bs, 1H),
7.62-7.38 (m, 5H), 7.30 (s, 1H), 7.20 (s, 1H). 13C NMR (75 MHz, DMSO d6) δ: 172.5, 158.3, 147.9,
137.3, 129.6, 126.0, 121.9, 115.6, 107.7. HRMS Calcd for C11H9NO2 (M + H)+ 188.0706. Found
188.0708.
4.3.2. 2,3-Dimethoxy-5-phenylpyridine
A mixture of 5-bromo-2,3-dimethoxypyridine (100 mg, 0.46 mmol) , phenylboronic acid (84 mg, 0.69
mmol), Pd(PPh3)4 (58 mg, 0.05 mmol) and K2CO3 (125 mg, 0.92 mmol) in 1,4- dioxane (3.0 ml) and
H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 0C and stirred for 16 h. The
reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc (3x),
and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using DCM to give 100 mg (99.9%)
1
desired product. H NMR (300 MHz, CDCl3) δ: 7.97 (s, 1H), 7.53-7.36 (m, 5H), 7,24 (s, 1H), 4.06 (s,
3H), 3.92 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 176.1, 153.8, 144.0, 138.0, 134.9, 130.8, 129.6, 128.9,
127.4, 126.8, 116.4, 56.2, 54.2.
4.3.3. 5-Bromo-2,3-dimethoxypyridine
To a 25 ml flask containing (63.8 mg, 1.2 mmol) of sodium ethoxide was added 3 ml dry ethanol. The
mixture was stirred for 30 minutes. 2-Chloro-3-methoxy-5-bromopyridine (230 mg, 1.04 mmol) was
added to the mixture and heated to 60 oC for 16 h. The reaction was allowed to cool and the solvent was
removed. Ethyl acetate (50 ml) was added to the residue. The ethyl acetate solution was washed with
water and then brine. The organic solvent was dried and concentrated under reduced pressure, and
purified by ISCO flash chromatography using 10% ethyl acetate in hexane to give 220 mg (97% yield)
1
of the desired product. H NMR (300 MHz, CDCl3) δ: 7.71 (s, 1H), 7.09 (s, 1H), 4.37 (qt, 2H), 3.82 (s,
3H), 1.38 (t, 3H).
4.3.4. 2-Chloro-3-methoxy-5-bromopyridine
Phosphoryl chloride (POCl3) (4.8 ml, 52.9 mmol) was added to a solution of 5-bromo-3-
methoxypyridine oxide (540 mg, 2.6 mmol) in 15 ml DCM. The reaction mixture was stirred at room
temperature overnight. The reaction mixture was washed with saturated NaHCO3and brine. The
organic layer was dried, concentrated under reduced pressure and purified using ISCO flash
1
chromatography using 50% ethyl acetate in hexane to provide 369 mg product (62% yield). H NMR
(300 MHz, CDCl3) δ: 8.05 (s, 1H), 7.34 (s, 1H), 3.93 (s, 3H).
11

4.3.5. 5-Bromo-3-methoxypyridine oxide
A solution of 3-(methoxy)-5-bromopyridine (2.0 g, 10.6 mmol.) and meta-chloroperbenzoic acid
(mCPBA) (2.5 g, 14.8 mmol).) in dichloromethane (50 ml) was stirred at room temperature for 3 h. The
reaction mixture was washed with 15 ml 2N KOH followed by brine. The organic layers were dried
over anhydrous MgSO4 and concentrated under reduced pressure to give product as white solid (1.06 g,
51%). mp 201 oC, 1H NMR (300 MHz, CDCl3) δ: 8.01 (s, 1H), 7.91 (s, 1H), 7.04 (s, 1H), 3.85 (s, 3H).
4.4.1. 5-(4-Fluorophenyl-3-hydroxypyridin-2(1H)-one (4)
To a solution of 2,3-dimethoxy-5(4-fluorophenyl)pyridine (60 mg, 0.257 mmol) in CH2Cl2 (3.0 ml)
under nitrogen, boron tribromide (1.0 ml, 1.0 M solution in CH2Cl2) was added. After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed and the reaction mixture was diluted with ethyl acetate. The solution was washed with
NaHCO3, water, and brine. The organic phase was then dried and evaporated under reduced pressure to
afford the crude product which was purified in ISCO using 10 % MeOH in DCM to furnish the pure
product (28 mg) as off-white solid, yield: 53%. mp 136-138 oC; 1H NMR (300 MHz, DMSO-d6) δ: 9.33
(bs, 1H), 7.66 (m, 2H), 7.30 (m, 3H), 7.17 (s, 1H). 13C NMR (75 MHz, DMSO-d6) δ: 172.5, 158.3,
148.0, 133.9, 128.1, 128.0, 121.8, 116.4, 116.1, 115.5, 107.6. HRMS Calcd for C11H8FNO2 (M + H)+
206.0612. Found 206.0614.
4.4.2. 2,3-Dimethoxy-5-(4-fluorophenyl)pyridine
A mixture of 5-bromo- 2,3-dimethoxypyridine (651 mg, 3.0 mmol) ,4-fluorophenylboronic acid (630
mg, 4.5 mmol), Pd(PPh3)4 (404 mg, 0.35 mmol) and K2CO3 (828 mg, 6.0 mmol) in 1,4- dioxane (6.0
ml) and H2O (2 .0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 24
h. The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 30-70% EtOAc in hexane to give
1
665 mg (95% yield) desired product. H NMR (300 MHz, CDCl3) δ: 7.90 (s, 1H), 7.51-7.45 (m, 2H),
7.19-7.11 (m, 3H), 4.06 (s, 3H), 3.94 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 176.3, 164.2, 160.9, 144.2,
134.9, 129.9, 128.6, 128.5, 116.4, 116.1, 115.8, 55.8, 53.8.
4.5.1. 5-(p-Cyanophenyl-3-hydroxypyridin-2(1H)-one (5)
To a solution of 2,3-dimethoxy-5(4-cyanophenyl)pyridine (100 mg, 0.417 mmol) in toluene (3.0 ml)
under nitrogen, boron tribromide (1.2 ml of 1.0 M solution in CH2Cl2) was added. After addition was
completed, the reaction mixture was heated to 100 oC in a sealed tube for 2 h and then at room
temperature overnight. The solvent was removed from the reaction mixture and the resulting solid was
1
purified in ISCO using 10 % MeOH in DCM to furnish the pure product (86 mg) yield: 60%. H NMR
(300 MHz, Acetone d6) δ: 7.67 (s, 4H), 7.36 (s, 1H), 7.10 (s, 1H). HRMS Calcd for C12H8N2O2 (M +
H)+ 212.0580. Found 212.0578.
12

4.5.2. 2,3-Dimethoxy-5-(4-cyanophenyl)pyridine
A mixture of 5-bromo-2,3-dimethoxypyridine (570 mg, 2.63 mmol) ,4-cyanophenylboronic acid (578
mg, 3.94 mmol), Pd(PPh3)4 (311 mg, 0.27 mmol) and K2CO3 (717 mg, 5.2 mmol) in 1,4- dioxane (6.0
ml) and H2O (1.5 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 h.
The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 20% EtOAc in hexane to give 495
1
mg (78% yield) desired product. H NMR (300 MHz, CDCl3) δ: 7.74 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H),
7.41 (d, J = 8.4 Hz, 2H), 7.0 (s, 1H), 3.84 (s, 3H), 3.73 (s, 3H). 13C NMR (75 MHz, CDCl3) δ: 176.2,
154.7, 144.3, 142.6, 135.4, 132.7, 128.7, 127.3, 118.8, 115.7, 110.9, 55.8, 54.0.
4.6.1. 5-[4-(1H-tetrazol-5-yl)phenyl]-3-hydroxypyridin-2(1H)-one (6)
To a sealed tube equipped with a small stirring bar was added 4-(5,6-dihydroxypyridin-3-yl)benzonitrile
(86 mg, 0.40 mmol), DMF (3.0 ml), NaN3 (104 mg, 1.6 mmol) followed by AcOH (1.0 ml). The
resulting mixture was heated to 120 oC overnight. After the completion of the reaction, the solvent was
removed under vacuum. Addition of 1N HCl and stirring produced a solid which was filtered to give
1
the pure product (35 mg, 34% yield). H NMR (300 MHz, DMSO-d6) δ: 7.96 (m, 3H), 7.53 (d, J = 9.0
Hz, 2H), 7.22 (s, 1H), 7.16 (s, 1H). HRMS Calcd for C12H9N5O2 (M + H)+ 256.0929. Found 256.0827.
4.7.1. 6-Phenyl-3-hydroxypyridin-2(1H)-one (7)
To a solution of 2-ethoxy-3-methoxy-6-phenylpyridine (150 mg, 0.65 mmol) in CH2Cl2 (2.0 ml) under
nitrogen, was added boron tribromide (1.0 ml of a 1.0 M solution in CH2Cl2). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered.
The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine. The organic phase was then
dried and evaporated under reduced pressure to afford the crude product which was purified in ISCO
using 10% MeOH in DCM to furnish the pure product (41 mg) as white solid, yield: 34%. mp 167-169
oC; 1H NMR (300 MHz, DMSO-d6) δ: 11.8 (bs, 1H), 9.19 (s, 1H), 7.66 (d, J = 7.8 Hz, 2H), 7.26-7.37
(m, 3H), 6.80 (d, J = 7.5 Hz, 1H), 6.45 (d, J = 7.5 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ: 172.5,
159.5, 147.1, 136.2, 134.4, 129.4, 129.0, 126.7, 116.7. HRMS Calcd for C11H9NO2 (M + H)+ 188.0706.
Found 188.0703.
4.7.2. 2-Ethoxy-3-methoxy-6-phenylpyridine
A mixture of 6-iodo-2-ethoxy-3-methoxypyridine (200 mg, 0.717 mmol), phenylboronic acid (131 mg,
1.07 mmol), Pd(PPh3)4 (127 mg, 0.11 mmol) and K2CO3 (195 mg, 1.43 mmol) in 1,4- dioxane (3.0 ml)
and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 h.
The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 15% EtOAc in hexane to give 150
13

1
mg (91%) of the desired product. H NMR (300 MHz, CDCl3) δ: 7.97 (d, J = 5.4 Hz, 2H), 7.44-7.39
(m, 2H), 7.34-7.26 (m, 2H), 7.06 (d, J = 8.1 Hz, 1H), 4.60 (qt, 2H), 3.88 (s, 3H), 1.50 (t, 3H). 13C NMR
(75 MHz, CDCl3) δ: 176.2, 153.2, 145.1, 143.2, 138.9, 128.5, 127.8, 126.0, 117.9, 112.5, 61.8, 55.1,
14.2.
4.7.3. 6-Iodo-2-ethoxy-3-methoxypyridine
To a solution of 6-iodo-2-bromo-3-methoxypyridine (1.5 g, 4.8 mmol) in EtOH (100 ml), NaOEt (487.6
mg) in EtOH (20 ml) was added for 30 min. This reaction was heated to 100 °C for 2 h. After cooling
to room temperature, ethanol was removed under reduced pressure and diluted with 50 ml water. The
resulting residue was extracted with CH2Cl2 (3x), washed with brine, dried over Na2SO4 and evaporated
under reduced pressure to give a crude oil, which was purified by ISCO flash chromatography using
20% EtOAc in hexanes, to provide recovered starting material (400 mg) and 6-iodo-2-ethoxyl-3-
1
methoxylpyridine: 600 mg (yield: 45%). H NMR (300 MHz, CDCl3) δ: 7.22 (d, J = 7.8 Hz, 1H), 7.78
(d, J = 7.8 Hz, 1H), 4.45 (qt, J = 7.5 Hz, 2H), 3.86 (s, 3H), 1.45 (t, J = 4.5 Hz, 3H).
4.7.4. 6-iodo-2-bromo-3-methoxypyridine
To a solution of 6-iodo-2-bromopyridin 3-ol (1.7 g, 5.9 mmol) in THF (50 ml), K2CO3 (1.22 g, 8.85
mmol) was added, and the mixture was stirred for 10 min at 0 °C in an ice bath. To this mixture CH3I
(1.0 g, 7.0 mmol) was then added slowly. The reaction mixture was stirred for 16 h at room
temperature. The reaction mixture was poured into ice water (100 ml), then extracted with EtOAc (3x),
1
dried in Na2SO4 and evaporated under reduced pressure to afford a white solid 1.5 g (yield: 80.96%). H
NMR (300 MHz, CDCl3) δ: 7.50 (d, J = 8.1 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H).
4.7.5. 6-Iodo-2-bromopyridin 3-ol
To a solution of 2-bromopyridin 3-ol (2.4 g, 13.79 mmol) in H2O (34 ml) was added potassium
carbonate (2.85 g, 20.7 mmol) followed by iodine ( 3.8 g, 15.2 mmol) and this mixture was stirred at
room temperature overnight. This mixture was cooled to 0 °C, then slowly quenched by 2N HCl to pH
6. The resulting precipitate was collected by filtration, washed with water and dried to give 2-bromo-6-
iodopyridine-3-ol (3 g, 73% yield). LC/MS: 300 (M +H).
4.8.1. 6-(4-Fluorophenyl-3-hydroxypyridin-2(1H)-one (8)
To a solution of 2-ethoxy-3-methoxy-6-(4-fluorophenyl)pyridine (57 mg, 0.245 mmol) in CH2Cl2 (3.0
ml) under nitrogen, was added boron tribromide (1.0 ml of a 1.0 M solution in CH2Cl2). After addition
was completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered.
The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine. The organic phase was then
dried and evaporated under reduced pressure to afford the crude product which was purified in ISCO
using 5% MeOH in DCM to furnish the pure product (35 mg) as white solid, yield: 70%. mp 159-165
°C; 1H NMR (300 MHz, DMSO-d6) δ: 7.57-7.53 (m, 2H), 7.12 (t, J = 8.7 Hz, 2H), 6.84 (d, J = 7.5 Hz,
1H), 6.27 (d, J = 6.9 Hz, 1H). HRMS Calcd for C11H8FNO2 (M + H)+ 206.0612. Found 206.0608. .
4.8.2. 2-Ethoxy-3-methoxy-6-fluorophenylpyridine
14

A mixture of 6-iodo-2-ethoxy-3-methoxypyridine (530 mg, 2.44 mmol), 4-fluorophenylboronic acid
(512 mg, 3.67 mmol), Pd(PPh3)4 (416 mg, 0.36 mmol) and K2CO3 (673 mg, 4.88 mmol) in 1,4-
dioxane (6.0 ml) and H2O (2.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and
stirred for 16 h. The reaction mixture was cooled to room temperature and partitioned between NaHCO3
and EtOAc (3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was
concentrated. The resulting residue was purified by ISCO flash chromatography using 20% EtOAc in
1
hexane to give 551 mg (97%) of the desired product. H NMR (300 MHz, CDCl3) δ: 7.95 (m, 2H),
7.27-7.05 (m, 4H), 4.59 (qt, 2H), 3.91 (s, 3H), 1.47 (t, 3H).
4.9.1. 6-(4-Cyanophenyl-3-hydroxypyridin-2(1H)-one (9)
To a solution of 2-ethoxy-3-methoxy-6-(4-cyanophenyl)pyridine (150 mg, 0.59 mmol) in CH2Cl2 (5.0
ml) under nitrogen, was added boron tribromide (2.0 ml, 1.0 M solution in CH2Cl2). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered.
The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine. The organic phase was then
dried and evaporated under reduced pressure to afford the crude product which was purified in ISCO
using 10 % MeOH in DCM to furnish the pure product (90 mg) as tan solid, yield: 72%. mp 161-163
°C; 1H NMR (300 MHz, DMSO-d6) δ: 7.88 (s, 4H), 6.85 (d, J = 7.5 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H).
13C NMR (75 MHz, DMSO-d6) δ: 172.5, 159.1, 148.0, 138.8, 134.6, 133.3, 127.3, 119.4, 116.9, 111.1,
107.6. HRMS Calcd for C1289N2O2 (M + H)+ 213.0659. Found 213.0651. .
4.9.2. 2-Ethoxy-3-methoxy-6-(4-cyanophenyl)pyridine
A mixture of 6-iodo-2-ethoxy-3-methoxypyridine (150 mg, 0.894 mmol) , 4-cyanophenylboronic acid
(197 mg, 1.34mmol), Pd(PPh3)4 (155 mg, 0.136 mmol) and K2CO3 (276 mg, 2.0 mmol) in 1,4-dioxane
(3.0 ml) and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for
16 h. The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and
EtOAc (3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was
concentrated. The resulting residue was purified by ISCO flash chromatography using 20% EtOAc in
1
hexane to give 185 mg (82% yield) desired product. H NMR (300 MHz, CDCl3) δ:8.07 (d, J = 8.4 Hz,
2H), 7.70 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 4.59 (qt, 2H), 3.93 (s,
3H), 1.51 (t, 3H). 13C NMR (75 MHz, CDCl3) δ: 172.2, 153.5, 144.5, 143.0, 142.5, 132.4, 126.3, 119.1,
117.5, 113.7, 110.9, 62.1, 55.9, 14.6.
4.10.1. 6-[4-(1H-tetrazol-5-yl)phenyl]-3-hydroxypyridin-2(1H)-one (10)
To a sealed tube equipped with a small stirring bar was added 6-(4-cyanophenyl-3-hydroxypyridin-
2(1H)-one (50 mg, 0.235 mmol), DMF (2.0 ml), NaN3 (61 mg, 0.94 mmol) followed by AcOH (1.0 ml).
The resulting mixture was heated to 120 °C overnight. After the completion of the reaction, the solvent
was removed under vacuum. Addition of 1N HCl and stirring produced a solid which was filtered to
1
give the pure product (51 mg, 85% yield). H NMR (300 MHz, DMSO-d6) δ: 8.07 (d, 2H), 7.83 (d, J =
8.1 Hz, 2H), 7.76 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.59 (d, J = 7.5 Hz, 1H). 13C NMR (75
15

MHz, DMSO-d6) δ: 172.5, 159.4, 135.5, 127.6, 127.2, 114.3, 107.6. HRMS Calcd for C12H9N5O2 (M +
H)+ 256.0829. Found 256.0820.
4.11.1. 5,6-Diphenyl-3-hydroxypyridin-2(1H)-one (11)
To a solution of 2,3-dimethoxy-5,6-diphenylpyridine (130 mg, 0.45 mmol) in CH2Cl2 (6.0 ml) under
nitrogen, was added boron tribromide (2.0 ml of a 1.0 M solution in CH2Cl2). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered.
The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine. The organic phase was then
dried and evaporated under reduced pressure to afford a solid (60 mg), yield: 55%. .1H NMR (300 MHz,
DMSO-d6) δ: 11.89, 9.31, 7.58 (m, 6H), 7.23 (t, 1H), 7.15 (m, 1H), 7.07 (t, 1H), 6.7 (d, J = 7.2 Hz, 1H).
13C NMR (75 MHz, DMSO-d6) δ: 172.5, 158.7, 146.9, 139.2, 134.7, 130.6, 130.1, 128.8, 128.7, 127.1,
119.3, 107.7. HRMS Calcd for C17H13NO2 (M + H)+ 264.1019. Found 264.1020. mp 249-255 °C.
4.11.2. 2,3-Dimethoxy-5,6-diphenylpyridine
A mixture of 5,6-dibromo-2,3-dimethoxypyridine (150 mg, 0. 5 mmol, phenylboronic acid (242 mg, 2.0
mmol), Pd(PPh3)4 (231 mg, 0.2 mmol) and K2CO3 (276 mg, 2.0 mmol) in 1,4- dioxane (5.0 ml) and
H2O (1.5 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 h. The
reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc (3x),
and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 10% EtOAc in hexane to give 130
1
mg of the desired product, yield: 89%. H NMR (300 MHz, CDCl3) δ: 7.4 (m, 2H), 7.29 (m, 3H), 7.24-
7.21 (m, 5H), 7.12 (s, 1H), 4.15 (s, 3H), 3.96 (s, 3H). LC/MS: 292.23 (M + H).
4.11.3. 5,6-Dibromo-2,3-dimethoxypyridine
To a solution of commercially available 2,3-dimethoxypyridine (2.0 g, 14.7 mmol) and NaOAc (5.4 g,
44 mmol) in AcOH (25 ml ) at 0 °C was added a solution of bromine (2.0 ml, 36.6 mmol) in AcOH (0.5
ml). The cooling bath was removed and the reaction was then stirred at room temperature for 16 h. The
mixture was poured into crushed ice followed by neutralization with 25% aqueous NaOH solution; the
aqueous phase was extracted with CH2Cl2 (3x). The combined organic phases were dried over Na2SO4
and concentrated. Purification by ISCO flash chromatography using a gradient of 10-15% EtOAc in
hexanes provided the desired product 3.86 g, tan solid (89%): 1H NMR (300 MHz, CDCl3) δ: 7.18 (s,
1H), 3.98 (s, 3H), 3.84 (s, 3H).
4.12.1. 5,6-bis(4-Fluorophenyl)-3-hydroxypyridin-2(1H)-one (12)
To a solution of 2,3-dimethoxy-5,6-bis(4-fluorophenyl)pyridine (100 mg, 0.3 mmol) in CH2Cl2 (3.0 ml)
under nitrogen, was added boron tribromide (1.0 M solution in CH2Cl2) (1.5 ml). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered,
which was redissolved in CH2Cl2 and washed with NaHCO3 and brine, dried using anhydrous sodium
sulfate and evaporated under reduced pressure to afford a solid (80 mg), yield: 87%. mp 152-157 °C; 1H
16

NMR (300 MHz, DMSO-d6) δ: 7.66 (m, 2H), 7.6 (m, 2H), 7.46 (m, 2H), 7.20 (d, J = 6.0 Hz, 2H), 7.08
(m, 1H), 6.7 (d, 1H), 6.28 (d, 1H). HRMS Calcd for C17H11F2NO2 (M + H)+ 300.0831. Found 300.0830.
4.12.2. 2,3-bis(4-Fluorophenyl)-5,6-dimethoxypyridine
The mixture of 5,6-dibromo-2,3-dimethoxypyridine (260 mg, 0.87 mmol), 4-fluorophenylboronic acid
(367 mg, 3.0 mmol), Pd(PPh3)4 (303 mg, 0.26 mmol) and K2CO3 (414 mg, 3.0 mmol) in 1,4- dioxane
(4.0 ml) and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for
16 h. The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and
EtOAc (3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was
concentrated. The resulting residue was purified by ISCO flash chromatography using 10% EtOAc in
1
hexane to give 210 mg of the desired product, yield: 73%. H NMR (300 MHz, CDCl3) δ: 7.35 (m, 2H),
7.20 (m, 2H), 7.20 (m, 2H), 7.17 (m, 2H), 7.04-6.98 (m, 4H), 4.18 (s, 3H), 3.01 (s, 3H). LC/MS:
328.205(M + H).
4.13.1. 5,6-bis(4-Cyanophenyl)-3-hydroxypyridin-2(1H)-one (13)
To a solution of 2,3-dimethoxy-5,6-bis(4-cyanophenyl)pyridine (50 mg, 0.146 mmol) in CH2Cl2 (3.0 ml)
under nitrogen, was added boron tribromide (1.0 M solution in CH2Cl2) (1.5 ml). After addition was
completed, the reaction mixture was stirred for 16 h at room temperature. Dichloromethane was
removed from the reaction mixture followed by addition of HCl (3N). The resulting solid was filtered,
which was redissolved in CH2Cl2 and washed with NaHCO3 and brine, dried using anhydrous sodium
sulfate and evaporated under reduced pressure to afford a solid (15 mg), yield: 33%. mp 159-162 °C; 1H
NMR (300 MHz, MeOD-d4) δ: 7.62 (m 4H), 7.3 (m, 4H), 7.03 (s, 1H). HRMS Calcd for C19H11N3O2
(M + H)+ 314.0924. Found 314.0920.
4.13.2. 2,3-bis(4-Cyanophenyl)-5,6-dimethoxypyridine
The mixture of 5,6-dibromo- 2,3-dimethoxypyridine (100 mg, 0.338 mmol), 4-cyanophenylboronic acid
(102 mg, 0.7 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol) and K2CO3 (136 mg, 1.0 mmol) in 1,4-dioxane (3.0
ml) and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 h.
The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 30% EtOAc in hexane to give 56
1
mg of the desired product, yield: 49%. H NMR (300 MHz, CDCl3) δ: 7.56 (d, J = 8.1 Hz, 2H), 7.47 (d,
J = 9.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 7.0 (s, 1H), 4.05 (s, 3H), 3.91 (s, 3H).
13C NMR (75 MHz, CDCl3) δ:172.3, 153.7, 144.3, 144.0, 143.6, 142.0, 132.4, 131.7, 130.5, 130.4,
128.1, 119.6, 118.7, 118.5, 111.2, 111.1, 58.2, 56.2.
4.14.1. 5,6-bis[4-(1H-tetrazol-5-yl)phenyl]-3-hydroxypyridin-2(1H)-one (14)
To a sealed tube equipped with a small stirring bar was added 5,6-bis(4-cyanophenyl)-3-
hydroxypyridin-2(1H)-one (100 mg, 0.319 mmol), DMF (2.0 ml), NaN3 (61 mg, 0.94 mmol) followed
by AcOH (1.0 ml). The resulting mixture was heated to 120 °C overnight. After completion of the
reaction, the solvent was removed under vacuum. Addition of 1N HCl and stirring produced a solid
which was filtered to give the pure product (45 mg, 35% yield). mp 180-182 °C;1H NMR (300 MHz,
17

DMSO-d6) δ: 7.96 (bs, 2H), 7.85-7.77 (m, 4H), 7.18 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.84
(s, 1H). HRMS Calcd for C19H13N9O2 (M + H)+ 400.1265. Found 400.1262. .
4.15.1. 5-(4-Cyanophenyl)-6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (15)
To a solution of 5-(4-cyanophenyl)-6-(4-fluorophenyl)-2,3-dimethoxypyridine (150 mg, 0.45 mmol) in
CH2Cl2 (4.0 ml) under nitrogen, was added boron tribromide (2.0 ml of a 1.0 M solution in CH2Cl2).
After addition was completed, the reaction mixture was stirred for 20 h at room temperature.
Dichloromethane was removed from the reaction mixture followed by addition of HCl (3N). The
resulting solid was filtered. The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine.
The organic phase was then dried and evaporated under reduced pressure to afford the crude product
which was purified in ISCO using 5% MeOH in DCM to furnish the pure product (85 mg) as white
solid, yield: 62%. mp 255-257 °C; 1H NMR (300 MHz, DMSO-d6) δ: 9.39 (bs, 1H), 7.60 (d, J = 8.1 Hz,
2H), 7.14-7.03 (m, 6H), 6.77 (s, 1H). HRMS Calcd for C18H11FN2O2 (M + H)+ 307.0877. Found
307.0870.
4.15.2. 5-(4-Cyanophenyl)-6-(4-fluorophenyl)-2,3-dimethoxypyridine
A mixture of 6-bromo-5-(4-cyanophenyl)-2,3-dimethoxypyridine (150 mg, 0.47 mmol) , 4-
fluorophenylboronic acid (99 mg, 0.70 mmol), Pd(PPh3)4 (70 mg, 0.06 mmol) and K2CO3 (129 mg,
0.94 mmol) in 1,4-dioxane (3.0 ml) and H2O (1.0 ml) was degassed for 30 min. This mixture was
heated to 100 0C and stirred for 16 h. The reaction mixture was cooled to room temperature and
partitioned between NaHCO3 and EtOAc (3x), and washed with NaCl (1x). The organic phase was
dried over Na2SO4 and was concentrated. The resulting residue was purified by ISCO flash
1
chromatography using 25% EtOAc in hexane to give 150 mg (95% yield) desired product. H NMR
(300 MHz, CDCl3) δ: 7.51 (m, 2H), 7.35-7.33 (m, 2H), 7.27-7.22 (m, 2H), 6.99 (s, 1H), 6.89 (t, J = 8.7
Hz, 2H), 4.08 (s, 3H), 3.92 (s, 3H).
4.15.3. 6-Bromo-5-(4-cyanophenyl)-2,3-dimethoxypyridine
To a solution of 2,3-dimethoxy-5-(4-cyanophenyl)pyridine (350 mg, 1.46 mmol) in acetic acid (8.0 ml)
was added sodium acetate (402 mg, 2.9 mmol). The reaction mixture was cooled to 0 °C and Br2 (238
mg) in acetic acid (1.0 ml) was added dropwise. The reaction mixture was stirred at room temperature
for 16 h. To this mixture 25% NaOH was added at 0 °C until pH 6 and then extracted with
dichloromethane three times. Organic layer wad dried, concentrated under reduced pressure and
purified by ISCO using 100% DCM to afford pure product 310 mg (66 % yield) as an oil. 1H NMR (300
MHz, CDCl3) δ: 7.70 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 6.99 (s, 1H). 13C NMR (75 MHz,
CDCl3) δ: 172.5, 153.4, 143.9, 132.3, 130.7, 120.7, 118.8, 111.9, 56.5, 54.4.
4.16.1 5-[4-(1H-tetrazol-5-yl)phenyl]-6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (16)
To a sealed tube equipped with a small stirring bar was added 5-(4-cyanophenyl)-6-(4-fluorophenyl)-3-
hydroxypyridin-2(1H)-one (45 mg, 0.141 mmol), DMF (2.0 ml), NaN3 (38 mg, 0.56 mmol) followed by
18

AcOH (0.1 ml). The resulting mixture was heated to 120oC for 4 h. After the completion of the
reaction, the solvent was removed under reduced pressure. Addition of 3N HCl and stirring produced a
solid which was filtered to give the pure product (15 mg, 31% yield). mp 185-188 °C; 1H NMR (300
MHz, DMSO-d6) δ: 9.4 (bs, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.8 Hz, 4H), 7.10 (m, 2H), 6.86
(s, 1H). HRMS Calcd for C18H12FN5O2 (M + H)+ 350.1048. Found 350.1057.
4.17.1. 5-(4-Fluorophenyl)-6-(4-cyanophenyl)-3-hydroxypyridin-2(1H)-one (17)
To a solution of 5-(4-fluorophenyl)-6-(4-cyanophenyl)-2,3-dimethoxypyridine (240 mg, 0.72 mmol) in
CH2Cl2 (3.0 ml) under nitrogen, was added boron tribromide (2.0 ml of a 1.0 M solution in CH2Cl2).
After addition was completed, the reaction mixture was stirred for 16 h at room temperature.
Dichloromethane was removed from the reaction mixture followed by addition of HCl (3N). The
resulting solid was filtered. The solid was redissolved in CH2Cl2 and washed with NaHCO3 and brine.
The organic phase was then dried and evaporated under reduced pressure to afford the crude product
which was purified in ISCO using 5% MeOH in DCM to furnish the pure product (183 mg) as off-white
solid, yield: 83%. mp 255-258 °C; 1H NMR (300 MHz, DMSO-d6) δ: 1H NMR (300 MHz, DMSO-d6)
δ: 7.75 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 7.2 Hz, 4H), 6.82 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) δ: 172.5, 147.0, 132.7, 132.5, 132.2, 132.1, 131.6, 119.2, 116.1, 115.7, 107.6.
HRMS Calcd for C18H11FN2O2 (M + H)+ 307.0877. Found 307.0844.
4.17.2. 5-(4-Fluorophenyl)-6-(4-cyanophenyl)-2,3-dimethoxypyridine
A mixture of 5-bromo-6-(p-cyanophenyl)-2,3-dimethoxypyridine (360 mg, 1.13 mmol) , 4-
fluorophenylboronic acid (237 mg, 1.69 mmol), Pd(PPh3)4 (173 mg, 0.15 mmol) and K2CO3 (307 mg,
2.26 mmol) in 1,4- dioxane (3 ml) and H2O (1.5 ml) was degassed for 30 min. This mixture was heated
to 100 °C and stirred for 16 h. The reaction mixture was cooled to room temperature and partitioned
between NaHCO3 and EtOAc (3x), and washed with NaCl (1x). The organic phase was dried over
Na2SO4 and was concentrated. The resulting residue was purified by ISCO flash chromatography using
1
25% EtOAc in hexane to give 340 mg (90% yield) of the desired product. H NMR (300 MHz, CDCl3)
δ: 7.49-7.40 (m, 4H), 7.13-7.09 (m, 2H), 7.02-6.95 (m, 3H), 4.07 (s, 3H), 3.92 (s, 3H). 13C NMR (75
MHz, CDCl3) δ: 163.8, 160.5, 153.1, 144.17, 143.76, 141.56, 135.47, 135.42, 132.86, 131.54, 131.31,
131.20, 130.50, 129.05, 127.94, 120.16, 118.96, 115.96, 115.57, 110.58, 56.01, 53.88.
4.17.3. 6-(4-Cyanophenyl)-5-bromo-2,3-dimethoxypyridine
To a mixture of 2,3-dimethoxy-6-(4-cyanophenyl)pyridine (210 mg, 0.875 mmol) in acetic acid (3.0 ml)
was added sodium acetate (217 mg, 1.6 mmol). The reaction mixture was cooled to 0 °C and Br2 (0.045
ml) in acetic acid (1.0 ml) was added dropwise. The reaction mixture was stirred at room temperature
for 12 h. To this mixture 25% NaOH was added at 0 °C until pH 6 and then extracted with DCM three
times. Organic layer wad dried, concentrated under reduced pressure and purified by ISCO using 20%
1
EtOAC in hexane to afford pure product 360 mg (91% yield). H NMR (300 MHz, CDCl3) δ: 7.51 (d, J
= 8.7 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 4.07 (s, 3H), 3.89 (s, 3H).
4.17.4 2,3-Dimethoxy-6-(4-cyanophenyl)pyridine
19

A mixture of 6-bromo-2,3-dimethoxypyridine (434 mg, 2.0 mmol) , 4-cyanophenylboronic acid (440
mg, 3.0 mmol), Pd(PPh3)4 (346 mg, 0.3 mmol) and K2CO3 (552 mg, 4.0 mmol) in 1,4-dioxane (3 ml)
and H2O (1.0 ml) was degassed for 30 min. This mixture was heated to 100 °C and stirred for 16 h.
The reaction mixture was cooled to room temperature and partitioned between NaHCO3 and EtOAc
(3x), and washed with NaCl (1x). The organic phase was dried over Na2SO4 and was concentrated. The
resulting residue was purified by ISCO flash chromatography using 25% EtOAc in hexane to give 200
1
mg desired product. H NMR (300 MHz, CDCl3) δ: 8.04 (d, J = 8.7 Hz, 2H), 7.6 (d, J = 8.4 Hz, 2H),
7.36 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 4.09 (s, 3H), 3.90 (s, 3H).
4.17.5 6-Bromo-2,3-dimethoxypyridine
To a solution of 2,3-dimethoxypyridine (5.0 g, 36 mmol) in AcOH (10.0 ml), NaOAC (9.78 g, 71.94
mmol) was added and the mixture was cooled to -32 °C. After 30 minutes, Br2 (1.0 ml) in AcOH (5 ml)
was added dropwise for 30 minutes. After the addition the reaction mixture was stirred for 2 h at -20 °C.
To this mixture 25% NaOH was added at 0 °C until pH 6 and then extracted with DCM three times.
Organic layer wad dried, concentrated under reduced pressure and purified by ISCO using 10% EtOAC
in hexane to afford a mixture of 5- and 6-bromo intermediates along with starting material which can be
separated to afford the 6-bromo-2,3-dimethoxypyridine (1.4 g). mp 172-175 °C; 1H NMR (300 MHz,
CDCl3) δ: 6.95 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.96 (s, 3H), 3.83 (s, 3H).
4.18.1 5-(4-Fluorophenyl)-6-[4-(1H-tetrazol-5-yl)phenyl]- -3-hydroxypyridin-2(1H)-one (18)
To a sealed tube equipped with a small stirring bar was added 5-(4-fluorophenyl)-6-(4-cyanophenyl)-3-
hydroxypyridin-2(1H)-one (50 mg, 0.16 mmol), DMF (2.0 ml), NaN3 (42 mg, 0.64 mmol) followed by
AcOH (1.0 ml). The resulting mixture was heated to 120 °C overnight. After completion of the
reaction, the solvent was removed under vacuum. Addition of 1N HCl and stirring produced a solid
1
which was filtered to give the pure product (30 mg, 54% yield). H NMR (300 MHz, DMSO-d6) δ: 7.83
(d, J = 8.1 Hz, 2H), 7.14 (d, J = 8.1 Hz, 2H), 7.09-7.00 (m, 4H), 6.81 (s, 1H). HRMS Calcd for
C18H12FN5O2 (M + H)+ 350.1048. Found 350.1058.
5.
Expression, Purification and Crystallization
Pandemic H1N1 endonuclease (residues 1-204) was expressed in BL21 (RIL) cells (Stratagene). The
BL21 cells 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. The dual hexahis tag was then removed by HRV14 3C protease cleavage. S2C 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 a 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% PEG8k, 200 mM ammonium sulfate, 1 mM manganese chloride,
20

10 mM magnesium acetate, 10 mM taurine, and 50 mM sodium fluoride. Trays are stored at 20 degrees
Celsius and crystals form within a few hours and grow to maximum size in one to two weeks.
6.
Endonuclease Assay
The Influenza A PAN domain has been shown to cleave ssRNA as well as ssDNA. To demonstrate the
inhibition of endonuclease cleavage by PAN, a high throughput assay was developed (U.S. Patent
Application Serial Number 13/554,709). A TaqMan-like oligonucleotide was used containing a 6-
carboxy-fluorescein (FAM) fluorophore at the 5’-end followed by 19 nucleotides and a minor groove
binding non-fluorescent quencher (MGBNFQ, Applied Biosystems) at the 3’-end. When excited by
light at a wavelength of 488 nm, MGBNFQ quenches the fluorescence of FAM via fluorescence
resonance energy transfer. If the endonuclease cleaves the oligonucleotide, the quencher is no longer
coupled to the fluorophore, and therefore, FAM fluoresces. This assay can be performed in a high-
throughput (e.g. 96 well plate) format. The assay can be used to evaluate the inhibitory characteristics
of compounds that are found to bind PAN and to screen libraries of drug-like compounds. The assay uses
the probe 6FAM-TGGCAATATCAGCTCCACA-MGBNFQ.
The assay can be performed in a 40 µl reaction volume with 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM
MgSO4, 0.05 mM MnSO4, 1 mM DTT, 0.75 mM CHAPS, 50 nM probe, and 25 nM endonuclease. The
reaction mixture is set up as a master mix with the buffer, probe, and protein on ice. The inhibitor is
then added to a maximum DMSO concentration of 2.5% (v/v) and serial dilutions are made on ice.
Varioskan Fluorometer (Thermo Scientific), set to an excitation of 488 nm and emission of 518 nm, is
used to measure the fluorescence of the samples at 37 °C. Fluorescence is measured at various time
points (5, 120, and 240 minutes) during the 37 °C incubation. Activity/inhibition is calculated based on
the change in fluorescence over time using Prism Graphpad non-linear regression analysis.
7.
Compound Soaking, Data Collection and Processing
Crystal structures of compounds 16 and 18 were determined in complex with influenza A 2009 H1N1
influenza A endonuclease enzyme as previously described.18 The soaks of 16 and 18 were performed by
taking crystals and 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, and 10% (v/v) ethylene glycol. 80-100 mM L-arginine was included to improve
solubility of the compounds. Crystals were then soaked with the ligand for 2-17 hours at 20 °C before
placing 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 HKL2000.24 The structure was solved and refined using the software
PHENIX.25
8.
Cytotoxicity Assays. Cytotoxicity was determined using the MTT-microtiter plate tetrazolinium
cytotoxicity assay (MTA). The human embryonic kidney 293 (HEK293) cell line was provided by Dr.
Zue-Hung Hsu (formerly at Columbia University, presently at Beijing National academy). The Madin-
Darby Canine Kidney (MDCK) epithelial cells were obtained from Prof. Patrick Sinko (Rutgers
21

University). The cytotoxicity assay was performed using 96-well microtiter plates. Cells were grown in
suspension at 37 °C in 5% CO2 and maintained by regular passage in DMEM media. For determination
of IC50, cells were exposed continuously for four days to varying concentrations of drug in triplicate
wells, each seeded with 3,000 cells. Each assay was performed with a control that did not contain any
drug. The MTT assays were performed at the end of the fourth day.
Acknowledgements
This study was supported by a research agreement between Prodaptics Pharmaceuticals, Inc. and
Rutgers, The State University of New Jersey (E.A. and E.J.L.). We are grateful to Angela A. Liu in the
Department of Pharmacology at Rutgers Robert Wood Johnson School of Medicine for performing the
cytotoxicity assays. The Bruker Avance III 400 MHz NMR spectrometer used in 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.
Supplementary data
Representative kinetic curves associated with the inhibition of endonuclease actitivy for six compounds
is provided in Figure 1S. X-ray data and refinement statistics for the analysis performed with 18. The
parameters associated with the analysis of the X-ray data for 16 are reported elsewhere.19
References
1.
2.
3.
Moscona, A. N. Engl. J. Med. 2005, 353, 2633.
Bloom, J. D.; Gong, L. I.; Baltimore D. Science 2010, 328, 1272.
Memoli, M. J.; Davis, A. S.; Proudfoot, K.; Chertow, D. S.; Hrabal, R. J.; Bristol, T.;
Taubenberger, J. K. J. Infect. Dis. 2010, 203, 348.
4.
5.
Govorkova, E. A.; Baranovich, T.; Seiler, P.; Armstrong, J.; Burnham, A.; Yi, G.; Peiris, M.;
Webby, R. J.; Webster, R. G. Antiviral Res 2013, d, 297.
Hu, Y.; Lu, S.; Song, Z.; Wang, W.; Hao, P.; Li, J.; Zhang, X.; Yen, H.; Shi, B.; Li, T.; Guan,
W.; Xu, L.; Liu, Y.; Wang, S.; Zhang, X.; Tian, D.; Zhu, Z.; He, J.; Huang, K.; Chen, H.; Zheng,
L.; Li, X.; Ping, J.; Kang, B.; Xi, X.; Zha, L.; Li, Y.; Zhang, Z.; Peiris, M.; Yuan, Z. Lancet
2013, Epub May 29, 2013. DOI:10.1016/S0140-6736(08)61345-8
6.
7.
8.
Das, K.; Aramini, J. M.; Ma, L-C.; Krug, R. M.; Arnold, E. Nat .Struc. Mol. Biol. 2010, 17, 530.
Plotch, S. J.; Bouloy M.; Ulmanen, I.; Krug R.M. Cell 1981, 23, 847.
Yuan, P; Bartlam, M.; Lou, Z.; Chen, S.; Zhou, J.; He, X.; Lv, Z.; Ge, R.; Li, X.; Deng, T.;
Fodor, E.; Rao, Z.; Liu, Y. Nature 2009, 458, 909.
9.
Dias, A.; Bouvier, D.; Crépin, T.; McCarthy, A. A.; Hart, D. J.; Baudin, F.; Cusack, S.; Ruigrok,
R.W.H. Nature 2009, 458, 914.
22

10.
11.
12.
DuBois, R. M.; Slavish, P. J.; Baughman, B. M.; Yun, M-K.; Bao, J.; Webby, R. J.; Webb, T. R.;
White, S.W. PLoS Pathog. 2012, 8, e1002830.
Kowalinski, E.; Zubieta, C.; Wolkerstorfer, A.; Szolar, O. H. J.; Ruigrok, R. W. H.; Cusack, S.
PLoS Pathog. 2012, 8, e10002831.
Tomassini, J.; Selnick, H.; Davies, M.E.; Armstrong, M.E.; Bladwin, J.; Bourgeois, M.;
Hastings, J.; Hazuda, D.; Lewis, J.; McClements, W.; Ponticello, G.; Radzilowski, E.; Smith, G.;
Tebben, A.; Wolfe, A. Antimicrob. Agents Chemother. 1994, 38, 2827.
Hastings, J.C.; Selnick, H.; Wolanski, B.; Tomassini, J.E. Antimicrob. Agents Chemother. 1996,
40, 1304.
Tomassini, J.; Davies, M.E.; Hastings, J.; Lingham, R.; Mojena, M.; Raghoobar, S.L.; Singh,
S.B.; Tkacz, J.S.; Goetz, M.A. Antimicrob. Agents Chemother. 1996, 40, 1189.
Singh, S.B. Tetrhedron Lett. 1995, 36, 2009.
13.
14.
15.
16.
Baughman, B. M. B.; Slavish, P.J.; DuBois, R.; Boyd, V.A.; White, S.W.; Webb, T.R. ACS
Chem Biol 2012, 7, 526.
17.
18.
19.
20.
21.
22.
23.
Parkes, K.E.B.; Ermert, P.; Fässler, J.; Ives, J.; Martin, J.A.; Merrett, J.H.; Obrecht, D.;
Williams, G.; Klumpp, K. J. Med. Chem. 2003, 46, 1153.
Sagong, H.Y.; Parhi, A.; Bauman, J.D.; Patel, D.; Vijayan, R.S.K., Das, K.; Arnold, E.; LaVoie,
E.J. ACS Med. Chem. Lett., 2013, 4, 547.
Bauman J. D.; Patel, D.; Baker, S.F.; Vijayna, R.S.K.; Xiang, A., Parhi; A.K.; Martinez-Sobrido,
L.; LaVoie, E.J.; Das, K.; Arnold, E. ACS Chem Biol. 2013, 8, revised and submitted.
Bell, M. P.; O'Dowd, C. R.; Rountree, J. S. S.; Trevitt, G. P.; Harrison, T.; McFarland, M. M.,
PCT Int. Appl. (2011), WO 2011055115 A1 20110512
Aronov, A.; Come, J.; Cottrell, K.; Grey, R.; LeTiran, A.; Marone, V.; Martinez-Botella, G.;
Messersmith, D.; Huck, E.P.PCT Int. Appl. (2010), WO 2010135014 A1 20101125.
Wolkenberg, S.; Barrow, J. C.; Harrison, Scott T.; Trotter, B. W.; Nanda, K. K.; Manley, P. J.;
Zhao, Z. PCT Int. Appl. (2011), WO 2011109261 A1 20110909.
Bergman, J.M.; Coleman, P.J.; Fraley, Mark E.; Mercer, S.P.; Reger, T.S.; Roecker, A.J.; Steen,
J.T. PCT Int. Appl. (2009), WO 2009020642 A1 20090212.
24.
25.
Otwinowski, Z.; Minor, W. Methods in Enzymology 1997, 276, 307.
Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.;
Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner,
R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta
Crystallogr. D Biol. Crystallogr. 2010, 66, 213.
23

Phenyl substituted 3-hydroxypyridin(1H)-2-ones: Potential inhibitors of influenza A
endonuclease.
Ajit K. Parhi, Amy Xiang, Joseph D. Bauman, Disha Patel, R. S. K. Vijayan, Kalyan Das,
Eddy Arnold and Edmond J. LaVoie
X = phenyl or p-fluoropheny, Y = Z = HX = Z = H; Y = phenyl, p-fluorophenyl, p-cyanophenyl or p-(tetrazol-5-yl)phenylX
= Y = H; Z = phenyl, p-fluorophenyl, p-cyanophenyl or p-(tetrazol-5-yl)phenylX = H; Y = Z = phenyl, p-fluorophenyl, p-
cyanophenyl or p-(tetrazol-5-yl)phenylX = H; Y = p-cyanophenyl or p-(tetrazol-5-yl)phenyl; Z = p-fluorophenylX = H; Y =
p-fluorophenyl; Z = p-cyanophenyl or p-(tetrazol-5-yl)phenyl
24