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ArticleSeptember 16, 2014

Phenyl Substituted 4-Hydroxypyridazin-3(2H)-ones and 5-Hydroxypyrimidin-4(3H)-ones: Inhibitors of Influenza A Endonuclease
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2014, 57, 19, 8086–8098

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https://doi.org/10.1021/jm500958x

Published September 16, 2014

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Abstract

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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.

Subjects

Introduction

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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 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, PA_N (a ∼25 kDa N-terminal domain; residues 1–197) and PA_C (∼55 kDa C-terminal domain; residues 239–716). Crystal structures of PA_C have been determined in complexes with N-terminal fragments of PB1. (7) The structure of PA_N 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 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.

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-carboxylic acid derivatives, (11) and flutimide and its derivatives, (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 endonuclease 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 chelating ligand at the active site of the enzyme. (13) Using this information, we have reported the structure–activity relationships for two new series of compounds, 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones, as endonuclease inhibitors (Figure 1). (22, 23)

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-fluorophenyl)-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

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The synthesis of 5-(4-fluorophenyl)-1,4-dihydropyrazine-2,3-dione, 1a, is outlined in Scheme 1. This dione can tautomerize 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-fluorophenylboronic 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)

The method used to prepare 6-(4-fluorophenyl)-5-hydroxypyrimidin-4(3H)-one 2 is outlined in Scheme 2. 4,5-Dimethoxy-6-chloropyrimidine was prepared from 4,6-dichloro-5-methoxypyrimidine by treatment with sodium methoxide. (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 phenylboronic acid provided the desired 2-phenyl 4,5-dimethoxypyrimidine 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 chloropyridazin-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-cyanophenylboronic 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).

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

compd	IC₅₀ (μM)	R₁	R₂
1	59.0	4-FC₆H₅
2	>193	4-FC₆H₅	H
3	0.58	H	4-FC₆H₄
4	1.56	H	3-FC₆H₄
5	1.67	H	2-FC₆H₄
6	2.24	H	4-[1,1′]-biphenyl
7	0.40	H	3-[1,1′]-biphenyl
8	1.10	H	2-[1,1′]-biphenyl
9	0.52	H	4-CNC₆H₄
10	0.25	H	3-CNC₆H₄
11	0.15	H	4-(CN₄H)phenyl
12	0.48	H	3-(CN₄H)phenyl
13	177	4-FC₆H₄	H
14	6.0	H	4-FC₆H₄
15	9.3	H	4-CNC₆H₄
16	3.0	H	4-(CN₄H)phenyl
5-FPhP	0.73
6-FPhP	0.43

Results and Discussion

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The tautomeric forms of 5-(4-fluorophenyl)-1,4-dihydropyrazine-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-dihydropyrazine-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-hydroxypyridin-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.

The structure–activity studies performed with 3-hydroxypyridin-2-ones indicated 6-[(4-tetrazoyl)phenyl]-3-hydroxypyridin-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)

Figure 2. Stereoview image of a crystal structure of 11 (yellow) bound to PA_N(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).

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)-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-phenylpyridazin-3(2H)-ones 14–16 are more active than the 4-fluorophenylpyrazine 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). Interestingly, 5-substituted 3-hydroxypyridin-2(1H)-ones and 6-substituted 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-hydroxypyrimidin-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-hydroxypyridazin-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-dihydropyrazine-2,3-dione can be viewed as 4-aza-analogues either 5- or 6-(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.

Experimental Section

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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.

5-(4-Fluorophenyl)pyrazine-2,3(1H,4H)-dione (1)

5-(4-Fluorophenyl)-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 white solid (75 mg, 85%); mp 285–287 °C. ¹H 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 MHz, DMSO-d₆) δ 161.8 (J_C,FJ_C,F = 243 Hz), 156.9, 155.7, 128.1, 127.6 (J_C,F = 9 Hz), 121.0, 115.5 (J_C,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.

5-(4-Fluorophenyl)-2,3-dimethoxypyrazine

5-Bromo-2,3-dimethoxypyrazine (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-dimethoxypyrazine as a white solid (123 mg, 77%); mp 108–110 °C. ¹H 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 (J_C,F = 247 Hz), 149.43, 149.41, 140.29, 132.66 (J_C,F = 3 Hz), 127.77, 127.73, 127.69 (J_C,F = 8 Hz), 115. 68 (J_C,F = 21 Hz), 54.09, 53.81. ¹⁹F NMR (376 MHz, CDCl₃) δ −113.7. HRMS (ESI) calculated for C₁₂H₁₂FN₂O₂ (M + H)⁺ 235.0877, found 235.0880.

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 (384 mg, 43%); mp 54–56 °C. ¹H 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.

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 crystallized as a white solid (1.13 g, 81%); mp 21–23 °C. ¹H 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.

6-(4-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (2)

6-(4-Fluorophenyl)-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 (J_C,F = 245 Hz), 158.7, 141.1, 138.5, 136.0, 132.3, 130.7 (J_C,F = 8 Hz), 114.8 (J_C,F = 21 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −113.0. HRMS (ESI) calculated for C₁₀H₈FN₂O₂ (M + H)⁺ 207.0564, found 207.0568.

6-(4-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

4-(4-Fluorophenyl)-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, 2H), 3.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.5 (J_C,F = 246 Hz), 158.8, 147.4, 143.7, 142.9, 131.7, 131.2 (J_C,F = 8 Hz), 115.0 (J_C,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.

4-(4-Fluorophenyl)-5,6-dimethoxypyrimidine

4-Chloro-4,5-dimethoxypyrimidine (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 chromatography on silica gel, eluting with 0–10% EtOAc/hexane. This afforded 4-(4-fluorophenyl)-5,6-dimethoxypyrimidine as a white solid (181 mg, 82%); mp 62–64 °C. ¹H 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 (J_C,F = 249 Hz), 164.0, 154.3, 152.0,139.4, 131.4 (J_C,F = 8 Hz), 131.3, 115.3 (J_C,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-methoxypyrimidine (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, 57%); mp 53–55 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 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-Fluorophenyl)-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 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 (J_C,F = 246 Hz), 159.0, 146.9, 143.4, 131.7, 129.4 (J_C,F = 9 Hz), 129.1, 115.5 (J_C,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-Fluorophenyl)-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 (J_C,F = 247 Hz), 158.1, 148.4, 145.4, 130.4, 129.6 (J_C,F = 9 Hz), 129.1, 115.5 (J_C,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-dimethoxypyrimidine (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 chromatography 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 (J_C,F = 248 Hz), 159.7, 155.3, 141.0, 137.2, 133.6 (J_C,F = 3 Hz), 129.6 (J_C,F = 8 Hz), 115.3 (J_C,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.

2-Chloro-4,5-dimethoxypyrimidine

2,4-Dichloro-5-methoxypyrimidine (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 chromatography 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.

2-(3-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (4)

2-(3-Fluorophenyl)-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 210–212 °C. ¹H 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 (J_C,F = 243 Hz), 158.9, 146.5, 143.8, 134.7, 131.5, 130.6 (J_C,F = 8 Hz), 123.0, 117.2 (J_C,F = 20 Hz), 113.6 (J_C,F = 24 Hz). ¹⁹F NMR (376 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-Fluorophenyl)-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 (J_C,F = 242 Hz), 157.9, 147.8, 145.5, 134.7, 130.7 (J_C,F = 9 Hz), 130.1, 123.2 (J_C,F = 9 Hz), 117.5 (J_C,F = 22 Hz), 113.8 (J_C,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-dimethoxypyrimidine (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 chromatography on silica gel, eluting with 0–10% EtOAc/hexane. This afforded 2-(3-fluorophenyl)-4,5-dimethoxypyrimidine as a white solid (393 mg, 98%); mp 83–85 °C. ¹H 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 (J_C,F = 242 Hz), 159.5, 154.6 (J_C,F =3 Hz), 141.3, 139.8 (J_C,F = 8 Hz), 136.9, 129.7 (J_C,F = 8 Hz), 123.1 (J_C,F = 3 Hz), 116.4 (J_C,F = 22 Hz), 114.3 (J_C,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-Fluorophenyl)-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 187–189 °C. ¹H 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 (J_C,F = 248 Hz), 158.2, 144.6, 143.9, 132.2 (J_C,F = 8 Hz), 132.1, 130.8, 124.5 (J_C,F = 3 Hz), 121.8 (J_C,F = 12 Hz), 116.1 (J_C,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-(2-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

2-(2-Fluorophenyl)-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, 1H), 3.80 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 159.3 (J_C,F = 248 Hz), 157.3, 145.9, 132.4 (J_C,F = 9 Hz), 130.8 (J_C,F = 8 Hz), 130.2, 124.5 (J_C,F = 3 Hz), 121.8, 121.6, 116.1 (J_C,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-dimethoxypyrimidine (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 chromatography on silica gel, eluting with 0–30% EtOAc/hexane. This afforded 2-(2-fluorophenyl)-4,5-dimethoxypyrimidine as a white solid (402 mg, 100%); mp 72–74 °C. ¹H 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 (J_C,F = 253 Hz), 159.5, 154.2 (J_C,F = 4 Hz), 140.9, 137.0, 131.4 (J_C,F = 2 Hz), 130.9 (J_C,F = 8 Hz), 126. 0 (J_C,F = 9 Hz), 123.8 (J_C,F = 4 Hz), 116.7 (J_C,F = 22 Hz), 56.2, 54.1. ¹⁹F 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 white solid (46 mg, 96%); dec 259–261 °C. ¹H 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-(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)-one as white solid (256 mg, 78%); mp 292–294 °C. ¹H NMR (400 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.

2-(4-Biphenyl)-4,5-dimethoxypyrimidine

2-Chloro-4,5-dimethoxypyrimidine (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 chromatography 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), 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 white solid (34 mg, 36%); mp 221–223 °C. ¹H 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.

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.

2-(3-Biphenyl)-4,5-dimethoxypyrimidine

2-Chloro-4,5-dimethoxypyrimidine (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 chromatography on silica gel, eluting with 0–30% EtOAc/hexane. This afforded 2-(3-biphenyl)-4,5-dimethoxypyrimidine as a white solid (670 mg, 100%); mp 72–74 °C. ¹H 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-(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 white solid (31 mg, 64%); mp 271–273 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.70–7.65 (m, 2H), 7.56 (t, J = 8 Hz, 2H), 7.48 (s, 1H), 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 (158 mg, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.51 (bs, 1H), 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-dimethoxypyrimidine (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 chromatography 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.

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 (15 mg, 32%); mp 324–326 °C. ¹H 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-dimethoxypyrimidine (100 mg, 0.57 mmol), (4-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 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-dimethoxypyrimidin-2-yl)benzonitrile as a white solid (69 mg, 74%); mp 170–172 °C. ¹H 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.

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 (27 mg, 58%); mp 294–296 °C. ¹H 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-yl)benzonitrile as a white solid (56 mg, 43%); mp 255–257 °C. ¹H 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-dimethoxypyrimidine (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-dimethoxypyrimidin-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-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one as a dark-brown solid (42 mg, 51%); dec 290–292 °C. ¹H 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.

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-hydroxypyrimidin-4(3H)-one as a beige solid (32 mg, 25%); dec 295–297 °C. ¹H 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.

5-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one (13)

5-(4-Fluorophenyl)-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 274–276 °C. ¹H 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 (J_C,F = 243 Hz), 161.9, 154.6, 132.7, 132.4 (J_C,F = 8 Hz), 127.2 (J_C,F = 4 Hz), 115.8, 114.2 (J_C,F = 21 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −114.1. HRMS (ESI) calculated 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 (J_C,F = 247 Hz), 161.6, 154.8, 132.3 (J_C,F = 8 Hz), 128.5, 125.7, 120.7, 112.9 (J_C,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 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 as a white solid (59 mg, 72%); mp 101–103 °C. ¹H 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.

4,5-Dichloro-2-(methoxymethyl)pyridazin-3(2H)-one

4,5-Dichloropyridazin-3(2H)-one (200 mg, 1.21 mmol) and 4-dimethylaminopyridine (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-3(2H)-one as a white solid (84 mg, 33%); mp 65–67 °C. ¹H 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-Fluorophenyl)-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 (J_C,F = 245 Hz), 157.6, 155.4, 145.2, 132.0 (J_C,F = 3 Hz), 128.0 (J_C,F = 9 Hz), 115.6 (J_C,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.

6-(4-Fluorophenyl)-4-methoxypyridazin-3(2H)-one

6-(4-Fluorophenyl)-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)-one as a white solid (41 mg, 36%); mp 211–213 °C. ¹H 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 (J_C,F =245 Hz), 156.2, 155.7, 144.3, 132.0 (J_C,F = 3 Hz), 128.2 (J_C,F = 8 Hz), 115.6 (J_C,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-dimethoxypyridazine (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 chromatography 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 (J_C,F = 247 Hz), 156.5, 155.7, 148.9, 132.9 (J_C,F = 3 Hz), 128.5 (J_C,F = 9 Hz), 115.8 (J_C,F = 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.

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-dihydropyridazin-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.

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 a white solid (82 mg, 70%); mp 271–273 °C. ¹H 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-dimethoxypyridazine (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 chromatography 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 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. ¹H NMR (400 MHz, 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.

Supporting Information

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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.

jm500958x_si_001.pdf (46.06 kb)

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Author Information

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Corresponding Author
- 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; Email: [email protected]
Authors
- Hye Yeon Sagong - Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8020, United States
- Joseph D. Bauman - Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854-5627, United States
- Disha Patel - Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854-5627, United States
- Kalyan Das - Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854-5627, United States
- Eddy Arnold - Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854-5627, United States
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 Pharmaceuticals, Inc..

Acknowledgment

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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.

References

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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2014, 57, 19, 8086–8098

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https://doi.org/10.1021/jm500958x

Published September 16, 2014

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Abstract
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Figure 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).
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Scheme 1
Scheme 1. ^a
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Scheme aReagents 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.
Scheme 2
Scheme 2. ^a
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Scheme aReagents 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.
Scheme 3
Scheme 3. ^a
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Scheme aReagents 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.
Scheme 4
Scheme 4. ^a
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Scheme aReagents 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.
Scheme 5
Scheme 5. ^a
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Scheme aReagents 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.
Figure 2
Figure 2. Stereoview image of a crystal structure of 11 (yellow) bound to PA_N(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).
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References
This article references 33 other publications.
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  Memoli Matthew J; Davis A Sally; Proudfoot Kathleen; Chertow Daniel S; Hrabal Rachel J; Bristol Tyler; Taubenberger Jeffery K
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  BACKGROUND: The 2009 influenza A(H1N1) pandemic called attention to the limited influenza treatment options available, especially in individuals at high risk of severe disease. Neuraminidase inhibitor-resistant seasonal H1N1 viruses have demonstrated the ability to transmit well despite early data indicating that resistance reduces viral fitness. 2009 H1N1 pandemic viruses have sporadically appeared containing resistance to neuraminidase inhibitors and the adamantanes, but the ability of these viruses to replicate, transmit, and cause disease in mammalian hosts has not been fully characterized. METHODS: Two pretreatment wild-type viruses and 2 posttreatment multidrug-resistant viruses containing the neuraminidase H275Y mutation collected from immunocompromised patients infected with pandemic influenza H1N1 were tested for viral fitness, pathogenicity, and transmissibility in ferrets. RESULTS: The pretreatment wild-type viruses and posttreatment resistant viruses containing the H275Y mutation all demonstrated significant pathogenicity and equivalent viral fitness and transmissibility. CONCLUSIONS: The admantane-resistant 2009 pandemic influenza A(H1N1) virus can develop the H275Y change in the neuraminidase gene conferring resistance to both oseltamivir and peramivir without any loss in fitness, transmissibility, or pathogenicity. This suggests that the dissemination of widespread multidrug resistance similar to neuraminidase inhibitor resistance in seasonal H1N1 is a significant threat.
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  Das, K.; Aramini, J. M.; Ma, L.-C.; Krug, R. M.; Arnold, E. Structures of influenza A proteins and insights into antiviral drug targets Nature Struct Mol. Biol. 2010, 17, 530– 538
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  Structures of influenza A proteins and insights into antiviral drug targets
  Das, Kalyan; Aramini, James M.; Ma, Li-Chung; Krug, Robert M.; Arnold, Eddy
  Nature Structural & Molecular Biology (2010), 17 (5), 530-538CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
  A review. The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa, and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the mol. biol. of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance the understanding of the mol. mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the non-structural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.
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  He, X.; Zhou, J.; Bartlam, M.; Zhang, R.; Ma, J.; Lou, Z.; Li, X.; Li, J.; Joachimiak, A.; Zeng, Z.; Ge, R.; Rao, Z.; Liu, Y. Crystal structure of the polymerase PAC–PB1N complex from an avian influenza H5N1 virus Nature 2008, 454, 1123– 1126
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  Yan, 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. Crystal structure of an avian influenza polymerase PA_N reveals an endonuclease active site Nature 2009, 458, 909– 913
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  Dias, A.; Bouvier, D.; Crépin, T.; McCarthy, A. A.; Hart, D. J.; Baudin, F.; Cusack, S.; Ruigrok, R. W. H. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit Nature 2009, 458, 914– 918
  9
  The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit
  Dias, Alexandre; Bouvier, Denis; Crepin, Thibaut; McCarthy, Andrew A.; Hart, Darren J.; Baudin, Florence; Cusack, Stephen; Ruigrok, Rob W. H.
  Nature (London, United Kingdom) (2009), 458 (7240), 914-918CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight sep. segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral mRNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 subunits. Here we describe biochem. and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site is comprised of one histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimization of endonuclease inhibitors as potential new anti-influenza drugs.
10. 10
  DuBois, R. M.; Slavish, P. J.; Baughman, B. M.; Yun, M.-K.; Bao, J.; Webby, R. J.; Webb, T. R.; White, S. W. Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease PLoS Pathog. 2012, 8, e1002830
  10
  Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease
  DuBois, Rebecca M.; Slavish, P. Jake; Baughman, Brandi M.; Yun, Mi-Kyung; Bao, Ju; Webby, Richard J.; Webb, Thomas R.; White, Stephen W.
  PLoS Pathogens (2012), 8 (8), e1002830CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
  Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the mol. mechanism of inhibition by several compds. that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compds. block the enzymic activity of the isolated PA endonuclease domain. Using X-ray crystallog., we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a mol. understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
11. 11
  Kowalinski, E.; Zubieta, C.; Wolkerstorfer, A.; Szolar, O. H. J.; Ruigrok, R. W. H.; Cusack, S. Structural Analysis of Specific Metal Chelating Inhibitor Binding to the Endonuclease Domain of Influenza pH1N1 (2009) Polymerase PLoS Pathogens 2012, 8 (8) e1002831 DOI: 10.1371/journal.ppat.1002831
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  Tefsen, B.; Lu, G.; Zhu, Y.; Haywood, J.; Zhao, L.; Deng, T.; Qi, J.; Gao, G. F. The N-terminal domain of PA from bat-derived influenza-like virus H17N10 has endonuclease activity J. Virol. 2013, 88, 1935– 1941 DOI: 10.1128/JVI.03270-13
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  Bauman, J. D.; Patel, D.; Baker, S.; Vijayan, R. S. K.; Xiang, A.; 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. Biol. 2013, 8, 2501– 2508
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  Crystallographic Fragment Screening and Structure-Based Optimization Yields a New Class of Influenza Endonuclease Inhibitors
  Bauman, Joseph D.; Patel, Disha; Baker, Steven F.; Vijayan, R. S. K.; Xiang, Amy; Parhi, Ajit K.; Martinez-Sobrido, Luis; LaVoie, Edmond J.; Das, Kalyan; Arnold, Eddy
  ACS Chemical Biology (2013), 8 (11), 2501-2508CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
  Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, the authors report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resoln. crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chem. scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, the authors developed a highly potent hydroxypyridinone series of compds. from a fragment hit that defines a new mode of chelation to the active site metal ions. A compd. from the series demonstrating promising enzymic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.
14. 14
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  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. Inhibition of Cap (m⁷GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds Antimicrob. Agents Chemother. 1994, 38, 2827– 2837
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  Tomassini, J.; Selnick, H.; Davies, M. E.; Armstrong, M. E.; Baldwin, J.; Bourgeois, M.; Hastings, J.; Hazuda, D.; Lewis, J.; et al.
  Antimicrobial Agents and Chemotherapy (1994), 38 (12), 2827-37CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
  Synthesis of influenza virus mRNA is primed by capped and methylated (cap 1, m7GpppXm) RNAs which the virus derives by endonucleolytic cleavage from RNA polymerase II transcripts in host cells. The conserved nature of the endonucleolytic processing provides a unique target for the development of antiviral agents for influenza viruses. A series of 4-substituted 2,4-dioxobutanoic acid compds. has been identified as selective inhibitors of this activity in both influenza A and B viruses. These inhibitors exhibited 50% inhibitory concns. in the range of 0.2 to 29.0 μM for cap-dependent influenza virus transcription and had no effect on the activity of other viral and cellular polymerases when tested at 100- to 500-fold higher concns. The compds. did not inhibit the initiation or elongation of influenza virus mRNA synthesis but specifically inhibited the cleavage of capped RNAs by the influenza virus endonuclease and were not inhibitory to the activities of other nucleases. Addnl., the compds. specifically inhibited replication of influenza A and B viruses in cell culture with potencies comparable to the 50% inhibitory concns. obtained for transcription.
16. 16
  Hastings, J. C.; Selnick, H.; Wolanski, B.; Tomassini, J. E. Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors Antimicrob. Agents Chemother. 1996, 40, 1304– 1307
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  Tomassini, J.; Davies, M. E.; Hastings, J.; Lingham, R.; Mojena, M.; Raghoobar, S. L.; Singh, S. B.; Tkacz, J. S.; Goetz, M. A. A novel antiviral agent which inhibits the endonuclease of influenza viruses Antimicrob. Agents Chemother. 1996, 40, 1189– 1193
  17
  A novel antiviral agent which inhibits the endonuclease of influenza viruses
  Tomassini, J. E.; Davies, M. E.; Hastings, J. C.; Lingham, R.; Mojena, M.; Raghoobar, S. L.; Singh, S. B.; Tkacz, J. S.; Goetz, M. A.
  Antimicrobial Agents and Chemotherapy (1996), 40 (5), 1189-1193CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
  A novel anti-influenza virus compd., flutimide, was identified in exts. of a recently identified fungal species, Delitschia confertaspora. The compd., a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously, this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Addnl., the compd. was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compd. further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.
18. 18
  Singh, S. B. Total synthesis of flutimide, a novel endonuclease inhibitor of influenza virus Tetrhedron Lett. 1995, 36, 2009– 2012
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  Total synthesis of flutimide, a novel endonuclease inhibitor of influenza virus
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  Tetrahedron Letters (1995), 36 (12), 2009-12CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
  Flutimide (I) is a completely substituted 1-N-hydroxy-2,6-diketo-Δ3-piperazine isolated from a new species of Delitschia cofertaspora. It selectively inhibits influenza virus A endonuclease activity without affecting any viral transcription activity. Total synthesis of flutimide starting from L-leucine has been described.
19. 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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  Parkes, K. E. B.; Ermert, P.; Fässler, J.; Ives, J.; Martin, J. A.; Merrett, J. H.; Obrecht, D.; Williams, G.; Klumpp, K. Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors J. Med. Chem. 2002, 46, 1153– 1164
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  Chen, E.; Swift, R. V.; Alderson, N.; Feher, V. A.; Feng, G.-S.; Amaro, R. E. Computation-guided discovery of influenza endonuclease inhibitors ACS Med. Chem. Lett. 2014, 5, 61– 64
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  Computation-Guided Discovery of Influenza Endonuclease Inhibitors
  Chen, Eric; Swift, Robert V.; Alderson, Nazilla; Feher, Victoria A.; Feng, Gen-Sheng; Amaro, Rommie E.
  ACS Medicinal Chemistry Letters (2014), 5 (1), 61-64CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
  Influenza is a global human health threat, and there is an immediate need for new antiviral therapies to circumvent the limitations of vaccination and current small mol. therapies. During viral transcription, influenza incorporates the 5'-end of the host cell's mRNA in a process that requires the influenza endonuclease. On the basis of recently published endonuclease crystd. structures, a three-dimensional pharmacophore was developed and used to virtually screen 450,000 compds. for influenza endonuclease inhibitors. Of 264 compds. tested in a FRET-based endonuclease-inhibition assay, 16 inhibitors (IC50 < 50 μM) that span 5 mol. classes novel to this endonuclease were found (6.1% hit rate). To det. cytotoxicity and antiviral activity, subsequent cellular assays were performed. Two compds. suppress viral replication with negligible cell toxicity.
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  Darout, Etzer; Robinson, Ralph P.; McClure, Kim F.; Corbett, Matthew; Li, Bryan; Shavnya, Andrei; Andrews, Melissa P.; Jones, Christopher S.; Li, Qifang; Minich, Martha L.; Mascitti, Vincent; Guimaraes, Cristiano R. W.; Munchhof, Michael J.; Bahnck, Kevin B.; Cai, Cuiman; Price, David A.; Liras, Spiros; Bonin, Paul D.; Cornelius, Peter; Wang, Ruduan; Bagdasarian, Victoria; Sobota, Colleen P.; Hornby, Sam; Masterson, Victoria M.; Joseph, Reena M.; Kalgutkar, Amit S.; Chen, Yue
  Journal of Medicinal Chemistry (2013), 56 (1), 301-319CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  A series of GPR119 agonists based on a 2,6-diazatricyclo[3.3.1.1∼3,7∼]decane ring system is described. Also provided is a detailed account of the development of a multigram scale synthesis of the diazatricyclic ring system, which was achieved using a Hofmann-Loeffler-Freytag reaction as the key step. The basis for the use of this complex framework lies in an attempt to constrain one end of the mol. in the "agonist conformation" as was previously described for 3-oxa-7-aza-bicyclo[3.3.1]nonanes. Optimization of carbamate analogs of the diazatricyclic compds. led to the identification of I as a potent agonist of the GPR119 receptor with low unbound human liver microsomal clearance. The use of an agonist response weighted ligand lipophilic efficiency (LLE) termed AgLLE is discussed along with the issues of applying efficiency measures to agonist programs. Ultimately, soly. limited absorption and poor exposure reduced further interest in these mols.
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  Nagashima, H.; Ukai, K.; Oda, H.; Masaki, Y.; Kaji, K. Studies on syntheses and reactions of methoxypyridazines. II. Methoxylation of 3,4,6-trichloropyridazine Chem. Pharm. Bull. 1987, 35, 350– 356
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  Hondo, T.; Warizaya, M.; Niimi, T.; Namatame, I.; Yamaguchi, T.; Nakanishi, K.; Hamajima, T.; Harada, K.; Sakashita, H.; Matsumoto, Y.; Orita, M.; Takeuchi, M. 4-Hydroxypyridazin-3(2H)-one derivatives as novel d-amino acid oxidase inhibitors J. Med. Chem. 2013, 56, 3582– 3592
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  Duplantier, A. J.; Becker, S. L.; Bohanon, M. J.; Bozilleri, K. A.; Chrunyk, B. A.; Downs, J. T.; Hu, L.-Y.; El-Kattan, A.; James, L. C.; Liou, S.; Lu, J.; Maklad, N.; Mansour, M. N.; Mente, S.; Piotrowski, M. A.; Sakaya, S. M.; Sheehan, S.; Steyn; Strick, S. J.; Williams, V. A.; Zhang, Z. Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent d-amino acid oxidase (DAAO) inhibitors J. Med. Chem. 2009, 52, 3576– 3585
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  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. PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213– 221
  33
  PHENIX: a comprehensive Python-based system for macromolecular structure solution
  Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
  Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
  A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
- PDB: 4M5U
- PDB: 4W9S
Supporting Information
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.
- jm500958x_si_001.pdf (46.06 kb)
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Phenyl Substituted 4-Hydroxypyridazin-3(2H)-ones and 5-Hydroxypyrimidin-4(3H)-ones: Inhibitors of Influenza A EndonucleaseClick to copy article linkArticle link copied!

Journal of Medicinal Chemistry

Abstract

Introduction

Figure 1

Chemistry

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Results and Discussion

Figure 2

Experimental Section

Chemistry: General Methods

5-(4-Fluorophenyl)pyrazine-2,3(1H,4H)-dione (1)

5-(4-Fluorophenyl)-2,3-dimethoxypyrazine

5-Bromo-2,3-dimethoxypyrazine

2,3-Dimethoxypyrazine

6-(4-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (2)

6-(4-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

4-(4-Fluorophenyl)-5,6-dimethoxypyrimidine

4-Chloro-5,6-dimethoxypyrimidine

2-(4-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (3)

2-(4-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

2-(4-Fluorophenyl)-4,5-dimethoxypyrimidine

2-Chloro-4,5-dimethoxypyrimidine

2-(3-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (4)

2-(3-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

2-(3-Fluorophenyl)-4,5-dimethoxypyrimidine

2-(2-Fluorophenyl)-5-hydroxypyrimidin-4(3H)-one (5)

2-(2-Fluorophenyl)-5-methoxypyrimidin-4(3H)-one

2-(2-Fluorophenyl)-4,5-dimethoxypyrimidine

2-(4-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (6)

2-(4-Biphenyl)-5-methoxypyrimidin-4(3H)-one

2-(4-Biphenyl)-4,5-dimethoxypyrimidine

2-(3-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (7)

2-(3-Biphenyl)-5-methoxypyrimidin-4(3H)-one

2-(3-Biphenyl)-4,5-dimethoxypyrimidine

2-(2-Biphenyl)-5-hydroxypyrimidin-4(3H)-one (8)

2-(2-Biphenyl)-5-methoxypyrimidin-4(3H)-one

2-(2-Biphenyl)-4,5-dimethoxypyrimidine

4-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (9)

4-(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile

4-(4,5-Dimethoxypyrimidin-2-yl)benzonitrile

3-(5-Hydroxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile (10)

3-(5-Methoxy-6-oxo-1,6-dihydropyrimidin-2-yl)benzonitrile

3-(4,5-Dimethoxypyrimidin-2-yl)benzonitrile

2-(4-(1H-Tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (11)

2-(3-(1H-Tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (12)

5-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one (13)

5-(4-Fluorophenyl)-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-one

5-Chloro-4-methoxy-2-(methoxymethyl)pyridazin-3(2H)-one

4,5-Dichloro-2-(methoxymethyl)pyridazin-3(2H)-one

6-(4-Fluorophenyl)-4-hydroxypyridazin-3(2H)-one (14)

6-(4-Fluorophenyl)-4-methoxypyridazin-3(2H)-one

6-(4-Fluorophenyl)-3,4-dimethoxypyridazine

6-Chloro-3,4-dimethoxypyridazine

4-(5-Hydroxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile (15)

4-(5-Methoxy-6-oxo-1,6-dihydropyridazin-3-yl)benzonitrile

4-(5,6-Dimethoxypyridazin-3-yl)benzonitrile

6-(4-(1H-Tetrazol-5-yl)phenyl)-4-hydroxypyridazin-3(2H)-one (16)

Expression, Purification, and Crystallization

Compound Soaking, Data Collection, and Processing

Endonuclease Assay

Supporting Information

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Acknowledgment

References

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Abstract

Figure 1

Scheme 1

Scheme 2

Phenyl Substituted 4-Hydroxypyridazin-3(2H)-ones and 5-Hydroxypyrimidin-4(3H)-ones: Inhibitors of Influenza A Endonuclease
Click to copy article linkArticle link copied!