Novel Series of Dihydropyridinone P2X7 Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.5b00365

Identification of singleton P2X7 inhibitor 1 from HTS gave a pharmacophore that eventually turned into potential clinical candidates 17 and 19. During development, a number of issues were successfully addressed, such as metabolic stability, plasma stability, GSH adduct formation, and aniline mutagenicity. Thus, careful modification of the molecule, such as conversion of the 1,4-dihydropyridinone to the 1,2-dihydropyridinone system, proper substitution at C-5″, and in some cases addition of fluorine atoms to the aniline ring allowed for the identification of a novel class of potent P2X7 inhibitors suitable for evaluating the role of P2X7 in inflammatory, immune, neurologic, or musculoskeletal disorders.

Full text of DOI:10.1021/acs.jmedchem.5b00365

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Article
A Novel Series of Dihydropyridinone P2X7 Receptor Antagonists
Francisco Lopez-Tapia, Keith Walker, Christine Brotherton-Pleiss, Joanie Caroon, Dov Nitzan, Lee
Lowrie, Shelley Gleason, Shu-Hai Zhao, Jacob Berger, Debra A. Cockayne, Deborah Phippard,
Rebecca Suttmann, William L. Fitch, David L. Bourdet, Pankaj Rege, Xiaojun Huang, Scott
Broadbent, Charles Dvorak, Jiang Zhu, Paul Wagner, Fernando Padilla, Brad Loe, and Alam Jahangir
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00365 • Publication Date (Web): 13 Oct 2015
Downloaded from http://pubs.acs.org on October 29, 2015
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A Novel Series of Dihydropyridinone P2X7
Receptor Antagonists
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Francisco Lopez-Tapia*, Keith A. M. Walker, Christine Brotherton-Pleiss, Joanie Caroon, Dov
Nitzan, Lee Lowrie, Shelley Gleason, Shu-Hai Zhao, Jacob Berger, Debra Cockayne, Deborah
Phippard, Rebecca Suttmann, William L. Fitch, David Bourdet, Pankaj Rege, Xiaojun Huang,
Scott Broadbent, Charles Dvorak, Jiang Zhu, Paul Wagner, Fernando Padilla, Brad Loe, Alam
Jahangir
HoffmannꢀLa Roche Inc, 340 Kingsland St, Nutley, NJ 07110, USA
ABSTRACT: Identification of singleton P2X7 inhibitor 1 from HTS gave a pharmacophore that
eventually turned into potential clinical candidates 17 and 19. During development, a number of
issues were successfully addressed, such as metabolic stability, plasma stability, GSH adduct
formation and aniline mutagenicity. Thus, careful modification of the molecule, such as
conversion of the 1,4ꢀdihydropyridinone to the 1,2ꢀdihydropyridinone system, proper
substitution at Cꢀ5”, and in some cases addition of fluorine atoms to the aniline ring allowed for
the identification of a novel class of potent P2X7 inhibitors suitable for evaluating the role of
P2X7 in inflammatory, immune, neurologic or musculoskeletal disorders.
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INTRODUCTION
The P2X7 receptor (P2X7R) is an ATP ligandꢀgated ion channel expressed in a wide variety of
cells involved in inflammatory and immune responses, and which mediates a major cellular
pathway for the processing and release of the proꢀinflammatory cytokines ILꢀ1β and ILꢀ18.1
Studies of human tissue or mouse models suggest that P2X7 may play important roles in a
number of inflammatory, immune, neurologic or musculoskeletal disorders, such as multiple
sclerosis, amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, cancer,
ischemia, neuropathic and inflammatory pain, rheumatoid arthritis, glomerulonephritis,
pulmonary fibrosis, and graftꢀversusꢀhost disease.2 Based on these findings several P2X7R
antagonists have been advanced into clinical trials for rheumatoid arthritis, however, these efforts
failed to demonstrate efficacy in early proof of concept clinical studies.3 Nonetheless, because
the P2X7R is implicated in a number of other disorders (vide supra), new potent and selective
P2X7R antagonists remain of high value.2 During the course of the investigations, several
P2X7R antagonists have been reported, some of them containing the adamantyl group or similar
lipophilic groups.4 In this report, we present our efforts on the identification and characterization
of potent and selective P2X7 antagonists based on novel chemical templates with no large
lipophilic groups, and with good physicochemical properties and a good safety profile. These
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novel P2X7 antagonists may be useful for further biological exploration into the role of P2X7 in
inflammation, neurologic, musculoskeletal or immuneꢀmediated disorders.2
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RESULTS AND DISCUSSION
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The Roche screening library was assayed in 1321N1 cells expressing human P2X7R
stimulated with BzATP using a FLIPR Ca2+ flux assay. One of the hits from this screen was the
singleton 1,4ꢀdihydropyridinone 1 (Table 1) with an IC50 of 104 nM. However, after initial
optimization, it was found these aryl acetamides had low metabolic stability, e.g., 1 and 2 HLM
CLint were 168 and 153 µL/min/mg protein, respectively. Structure modification to the
corresponding ureas considerably improved metabolic stability while maintaining potency, e.g.,
3 and 4 had HLM CLint of 22 and 8.2 µL/min/mg protein, with IC50 values of 39 and 12 nM,
respectively. When testing the separated enantiomers, potency was found to reside only in the
(S)ꢀenantiomer (Table 2). The XꢀRay crystal structure of 9 is shown,5 in which the fluorophenyl
ring is pseudoaxial, and the dihedral angle between the urea carbonyl and the N1ꢀC6 bond is 11o.
Table 1. Potency and Metabolic Stability of 1,4-Dihydropyridinones 1-
4a
b,c
c
c
Entry1
X
Y
R IC50
HLM CLint
RLM CLint
O
X
Y
N
(nM)
104
92
(µL/min/mg protein)
(µL/min/mg protein)
O
O
O
I
CH2
H
H
H
F
168
153
22
548
490
78
1
2
3
4
R
Br CH2
Br NH
Me NH
39
12
8.2
25
aCompounds 1-3 are racemic, and 4 is the (S)ꢀenantiomer (vide infra);
bFLIPR Ca2+ flux assay using 1321N1 cells expressing human P2X7R
c
stimulated with BzATP. Mean value of n ≥ 2 in presence of a positive
control.
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Table 2. Potency of (R) and (S)-Enantiomers for Compounds 5-9, and X-Ray Crystal Structure
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of Compound 95
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Entry
Configuration
IC50 (nM)a
38
S
R
S
R
S
5
6
7
8
9
3800
33
>10000
40
aMean value of n ≥ 2 in presence of a positive control.
Unfortunately, during DMPK profiling of compounds from this series, it became apparent that
the urea compounds were both positive in the glutathione (GSH) trapping assay (e.g., 10, Table
3) and unstable in human plasma (e.g., 13). In the GSH trapping assay, the resulting adduct
contained the glutathione moiety as part of ring A (Table 3) in both human and rat liver
microsomes. For the characterization of the GSH adduct, the assay for compound 10 showed a
major chromatographic peak (Figure 1) due to the GSH adduct. In the figure, unreacted 10 elutes
at 16.58 min. The major GSH adduct (GSH-1) has retention time of 10.74 minutes and shows
[M+H]+ ion at m/z 740, corresponding formally to a loss of a CH2 (ꢀ 14 Da) plus 2 hydrogens (ꢀ2
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Da) and addition of a GSH (+307 Da). The peak at 14.8 minutes is due to a minor desꢀmethyl
metabolite. A second, minor GSH adduct elutes at 10.2 minutes. The other peaks are
background. Figure 2 shows the MS/MS spectrum of GSH-1. A characteristic from compounds
of this urea series is that they all show a fragmentation to give an isocyanate (leftꢀside of the
molecule) and an arylꢀpyridinone (rightꢀside of the molecule). For GSH-1, the corresponding
isocyanate [M+H]+ ion is at m/z 549, a loss of 191 Da (i.e., loss of the fluorophenylꢀpyridinone).
The other ion species are due to classic6 GSH adduct fragmentation with loss of glycine (75 Da)
from the intact precursor ion to give m/z 665 or from the intermediate m/z 549 to give m/z 474, or
loss of pyroglutamate (129 Da) from the intact precursor ion to give m/z 611, or from the
intermediate m/z 549 to give m/z 420. Our hypothesis for the formation of GSH-1 is shown in
Figure 3. The first step is cytochrome P450 oxidative demethylation. This common reaction is
supported by observation of minor amounts of the desꢀmethyl metabolite in the assay mixture.
This intermediate is setup for further oxidative dehydrogenation to give the reactive quinoneꢀ
imine, which by addition of GSH gives the corresponding adduct with the observed molecular
weight. The regiochemistry is not proven by this data, other isomeric adducts are possible. For
all compounds tested, the observed results on the GSH adduct show that in no case was there any
evidence for addition of GSH to unmetabolicallyꢀactivated molecules, e.g., compound 14 (Figure
4), in which no GSH addition to the 1,4ꢀpyridinone ring was observed.
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Hypothesizing that replacement of the methoxy group para to the anilide nitrogen could
reduce the glutathione adduct formation, halide compounds 11 and 12 were prepared and tested
in the GSH assay (Table 3).7 However, fluoride 11 gave no improvement in the extent of GSH
adduct formation, whereas chloride 12 showed only 50% reduction in its relative formation.8
More elaborate compound 13 with an improved cLogP of 2.76 with respect to 12 (cLogP 4.1)
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had further reduced formation of the GSH adduct in both human and rat liver microsomes (4 and
5% relative UV peak area in HLM and RLM, respectively). However, while changes in the
aniline portion of the molecule showed potential for circumventing the GSH liability, the plasma
instability, which could be attributed to a facile enzymatic hydrolysis of the urea group, needed
to be addressed by modification of the urea linkage.9 To this end compound 15 (Table 3), in
which the 1,4ꢀdihydropyridinone was modified to a 1,2ꢀdihydropyridinone system, was prepared
and assessed in the plasma stability assay. Gratifyingly, compound 15 was not only completely
stable in both human and rat plasma, but importantly potency was maintained and the GSH
adduct formation was now below detectable levels (Table 3). Since the responsible group for
trapping the GSH was still present in compound 15 (vide supra), the undetectable GSH adduct
formation could be due to a further reduction of cLogP, from 2.76 for 13 to 1.97 for 15, which
presumably reduces binding to metabolizing enzymes.10
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Table 3. Relative Glutathione Adduct Formation and Plasma Stability
b
Entry
X
R1
R2
IC50 % Relative GSH Plasma stabilityb
UV peak areaa
(nM) HLM
RLM
33
Br MeO
Me
Me
Me
9
28
27
15
Not Determined
Not Determined
Not Determined
10
11
12
Br
Br
F
32
37
37
Cl
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Me Cl
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ꢀ
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4
5
50% degradation in human plasma at
4h
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15
ꢀ
ꢀ
23
BQL
BQL Stable in both human and rat plasma
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a% Relative GSH adduct UV peak area with respect to the total area of the GSH adduct, other
metabolites and parent compound, bMean value of n ≥ 2 in presence of a positive control.
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16.58
420000
400000
380000
P
360000
340000
320000
300000
280000
260000
240000
220000
200000
180000
160000
140000
120000
10.74
GSH-1
GSH-1
GSH-2
7.04
6
8
10
12
Time (min)
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16
18
Figure 1. LC/UV chromatogram of the GSH trapping assay on compound 10.
Figure 2. Collision induced decomposition MS/MS spectrum of GSH-1.
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O
O
Br
Br
O
H
H
N
N
N
P450
N
O
O
HO
O
O
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F
F
10
P450
O
O
Br
Br
H
N
N
N
O
GS
HO
N
GSH
O
O
O
O
F
F
GSH-1
Figure 3. Hypothesis for formation of GSH-1.
Figure 4. Compound 14 showed no GSH adduct formation.
This interesting new scaffold was profiled further. A summary of SAR for the new 1,2ꢀ
dihydropyridinone series, from which 15 was identified, is presented in Figure 5, and
representative cases are exemplified in Table 4.11 Thus, the dihydropyridinone ring nitrogen can
be substituted with relatively small alkyl groups, such as methyl or hydroxyethyl (e.g., 17, Table
4); replacement of Cꢀ5 with NH is tolerated (e.g., 20); the phenyl group at Cꢀ4 only allows small
substituents with 3’ꢀfluoro (e.g., 16) or 4’ꢀfluoro substitution (e.g., 15) being optimal. Only
halides or small alkyl or alkoxy groups are tolerated at positions 2”, 3” and 4” (e.g., 16ꢀ18, 2112),
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whereas groups of different size at position 5” are allowed (e.g., 15, 17ꢀ20), and even fusion of
the 4”ꢀ5” bond to form a bicyclic system (e.g., 22) is acceptable. The finding that a variety of
groups are acceptable at Cꢀ5” allowed the modulation of physicochemical properties of
compounds to achieve desired parameters, for example for attaining acceptable rat SDPK or
overcoming the mutagenic liability (vide infra).
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These analogs proved to be active in a human whole blood (HWB) ILꢀ1β assay (Table 4).
However, consistent with other published P2X7 antagonists,13 the 1,2ꢀdihydropyridinone
compounds showed no correlation of potency between rat and human. For example, the IC50 of
15 at the human P2X7 receptor was 23 nM (FLIPR Ca2+ flux assay), whereas the IC50 at the rat
P2X7 was greater than 10,000 nM. This behavior precluded development of a robust in vivo
pharmacology package in rodent models, for example in models of inflammatory/rheumatoid
arthritis. However, in a cynomologous nonꢀhuman primate (NHP) ex vivo PK/PD assay (Figure
6), sustained inhibition of the proꢀinflammatory cytokine ILꢀ1β was observed to correlate with
total plasma concentration of 15, providing evidence for P2X7 receptor target engagement in
vivo and ability to mediate inhibition of ILꢀ1β.
Figure 5. SAR for 1,2ꢀdihydropyridinone series. Shown are replacements or substitutions that
maintain potency FLIPR IC50 within 3 times, unless otherwise indicated.
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Table 4. Inhibition of P2X7 Activity by the 1,2-Dihydropyridinone Series
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Entry
X
Y
R1
R2
R3
R4
F
FLIPR IC50 HWB IC50
(nM)a
(nM)a
Me CH2
Br CH2
H
H
H
H
Cl
OCH2CH2OH
OMe
Cꢀ4’
Cꢀ3’
23
60
15
16
OMe
9
107
Me CH2 Me
H
H
F
Cꢀ4’
Cꢀ4’
8
13
27
17
18
F
CH2 Me
H
26
Me CH2 Me
H
H
H
H
Cꢀ4’
Cꢀ4’
30
22
17
33
19
20
Me NH Me
Me CH2
H
ꢀ
Me
ꢀ
H
ꢀ
OMe
ꢀ
Cꢀ4’
ꢀ
9
ꢀ
21
22
ꢀ
ꢀ
23
47
aMean value of n ≥ 2 in presence of a positive control.
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Figure 6. Representative NHP ex vivo PK/PD assay performed with compound 15 at 10 mg/kg.14
Each point indicates mean value of n ≥ 2. Ex vivo NHP WB ILꢀ1β IC50 300 nM.
Table 5 presents further profiling of compound 15. As noted above, 15 is highly potent in the
FLIPR Ca2+ flux assay as well as in the HWB ILꢀ1β assay (IC50 60 nM). Rat single dose
pharmacokinetics showed excellent absorption and systemic exposure (F 74% and AUC/dose
1550 ng*h/ml/mg/kg); and, as predicted from the glutathione assay (vide supra), the HLM and
RLM covalent binding (CVB) assays were negative. In addition, compound 15 showed excellent
selectivity against other P2X receptors (Table 5), and no hits over 33% inhibition at 10 uM were
found from a panel of 78 receptors as performed by the commercial receptor scan at CEREP (see
Supporting Information for detailed report).
Table 5. Compound 15 Rat SDPK (iv 0.5, po 2 mg/kg), CVB and P2X Selectivitya
HWB 1Lꢀ1β
IC50(nM)
F
po AUC/dose po Cmax
IV CL
Vdss
t1/2
(h)
CVBb
P2Xc
Selectivity
FLIPR IC50
(ng*h/mL/mg (ng/mL)
/kg)
(mL/min (L/kg)
/kg)
RLM/
HLM
60
74%
1550
330
8.4
2.2
4.0
Negative
> 10 µM
15
an ≥ 2 per time point, bCVB results with absence and presence of NADPH: RLM 2.6 and 38; and HLM 2.8 and
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18 pmoleꢀEqs/mg protein, respectively. Internal Guideline: Flag above 100 pmoleꢀEqs/mg protein, cP2X selectivity
against the P2X1, P2X2, P2X3, P2X2/3, P2X4, P2X5 receptors
.
6
7
An enantioselective synthesis of 15 is presented in Scheme 1. Thus, pyrandione 2315 was
8
9
subjected to an enantioselective methanolysis using organocatalyst 24 to obtain chiral monoacid
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o
25 in 97% ee.16 Regioselective formylation of 25 with LDA and methyl formate at ꢀ45 C
furnished 26, which was cyclized smoothly with ammonium acetate in the presence of acetic
acid to obtain 1,2ꢀdihydropyridinone 27 with good overall yield from pyrandione 23 (48% yield
for 3 steps) and preserving the enantiomeric purity from 25. Hydrolysis of 27 by the action of
lithium hydroxide in water/methanol, followed by treatment with thionyl chloride afforded acid
chloride 28. The aniline portion was prepared starting with 4ꢀmethylꢀ3ꢀnitroꢀphenol 29, which
was alkylated and reduced to aniline 30 with 86% overall yield. Aniline 30 was then selectively
chlorinated at Cꢀ4 with Nꢀchlorosuccinimide to obtain 31 in 70% yield. Treatment of aniline 31
with acid chloride 28, followed by methanolysis of the acetyl protecting group gave desired 15
with 89% yield for 2 steps.
Scheme 1. Enantioselective Synthesis of Compound 15a
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aReagents and conditions: a) MeOH (10 eq), 2 mol% 24, 2ꢀMeꢀTHF, rt, then treatment with
toluene/hexane, 97% ee; b) LDA, HCO2Me, THF, ꢀ45 oC; c) NH4OAc, HOAc, 80 oC, 48% 3 steps,
97% ee; d) LiOH, H2O, MeOH, 86%, 96% ee; e) SOCl2, DMF, DCM; f) Br(CH2)2OAc, K2CO3,
DMA; g) H2, 5% Pd/C, EtOH, 5 psi, 86% 2 steps ; h) NCS, CH3CN, 70%; i) 28, 2,6ꢀlutidine, Meꢀ
THF; j) cat. NaOMe, MeOH, 89% 2 steps.
Although compound 15 was observed to be stable in both human plasma (4 h) and blood (3
h); and in vitro metabolism (HLM and HH) and in vivo metabolic studies [NHP plasma and urine
(10 mg/kg)] did not show any signs of hydrolysis of the amide bond, a one month stability
assessment showed slow degradation of 15 in the solid phase to produce free aniline 32. This
was also observed in formulations at different pH systems. Because of this finding, aniline 32
was assessed in the Ames mutagenicity assay,17 which was positive. Even though parent
compound 15 was Ames negative, we considered that it would not be possible to control the
level of aniline present in the final drug product to the required level. As a consequence further
development of compound 15 was discontinued.
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NH2
6
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8
Cl
O
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OH
32
Our focus shifted to molecules that contained anilines devoid of mutagenic liability. It is
known that mutagenic activity can be diminished or even eliminated by addition of fluorine
atoms in the aniline ring,18 as often predicted by the in silico tools DEREK and MCASE.19 Thus,
compounds 33 and 34 (Figure 7), which had excellent HWB potency but contained Ames
positive anilines, were modified to fluoroꢀanalogs 17 and 18. Gratifyingly, potency was
maintained, and not only had the corresponding anilines been predicted to be negative in both
DEREK and MCASE, but also were shown to be negative in the Ames mutagenicity assay
(Figure 7).
During our efforts to identify additional nonꢀmutagenic anilines, we found that some, although
predicted to be positive in both DEREK and MCASE, were negative in the Ames assay.
Examples of compounds containing such anilines are presented in Figure 8.
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Figure 7. Addition of fluorine retains potency and gives Ames negative anilines.
N
O
N
O
H
N
O
H
N
N
F
O
H
N
H
N
H
N
NH
O
O
O
O
N
O
O
O
N
N
O
N
O
N
N
F
N
F
N
F
O
OH
HO
36
35
19
20
HWB IC50 17 nM
HWB IC50 33 nM
HWB IC50 21 nM
HWB IC50 17 nM
Figure 8. Compounds containing anilines predicted to be positive in DEREK and MCASE, but
negative in the Ames mutagenicity assay.
The knowledge gained from compounds with Ames negative anilines allowed the selection of
17 and 19 as potential clinical candidates. Both compounds had excellent potency in the HWB
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assay, good solubility, good overall rat PK profile, and good safety as measured by CYP
inhibition, GSH adduct formation, and the aniline Ames mutagenicity assay (Table 6).
7
8
9
Table 6. Potency, Solubility, CYP Inhibition, GSH Adduct formation, Aniline Ames
Mutagenicity Results, and Rat Pharmacokinetic Parameters for Compounds 17 and 19a
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60
cLogP/ FLIPR HWB Solubc
CYP
IC50 (µg/mL) Inhd
GSH
Aniline
Ames
Rat PK
IV 0.5 mg/kg
PO 2 mg/kg
PSAb
IC50
(nM)
(nM)
(µM)
2.5/82
8
13
322
>32
Tracee
Negative F 64%, CLf 17, t1/2
1.0 h, Vssg 1.0,
17
19
AUCh 1121
0.9/131
33
17
1025
>50
Negative Negative F 20%, CLf 23, t1/2
4.0 h, Vssg 1.2,
AUCh 279
aExperimental values are an average of n ≥ 2, bcLogP and PSA (polar surface area) values
were obtained from ChemBioDraw Ultra 13.0, cSolubility was obtained using LYSA
(lyophilization solubility assay),20 dA fluorescenceꢀbased CYP inhibition assay was utilized to
generate IC50 values for inhibition of CYP3A4, CYP2D6, and CYP2C9,21 e0.2% HLM/RLM
relative UV peak area, fmL/min/kg, gL/kg, hPO ng*h/mL
Schemes 2 and 3 present the preparation of 17 and 19, respectively. Thus, chiral acid 26 (vide
supra) was subjected to cyclization in the presence of methylamine to furnish Nꢀmethylꢀ
dihydropyridinone 37 in 100% ee after crystallization from MTBE.16b Ester hydrolysis, followed
by treatment with thionyl chloride gave acid chloride 39. For the aniline, 1ꢀbromoꢀ2ꢀfluoroꢀ4ꢀ
methylꢀ5ꢀnitroꢀbenzene 40 was converted to the corresponding boronate ester, which was
reduced to aniline 41 in 58% yield. Suzuki coupling with chloropyridazinone 42 furnished 43 in
93% yield. Final coupling of aniline 43 with acid chloride 39 furnished desired amide 17. For the
preparation of 19 (Scheme 3), 4ꢀmethylꢀ3ꢀnitroꢀaniline 44 was transformed to its diazonium salt,
which by treatment with bisꢀethoxycarbonylꢀmalonamide 45 afforded triazinedione ester 46.
Hydrolysis with aqueous sodium hydroxide followed by decarboxylation with mercaptoacetic
acid at 180 oC gave 47 in 58% overall yield for 4 steps. Alkylation with (R)ꢀglycidylmethyl ester,
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followed by reduction of the nitro group furnished aniline 48 with 54% yield for 2 steps. Final
coupling of 48 with acid 38 in the presence of HATU/DIPEA afforded desired amide 19.
Scheme 2. Enantioselective Synthesis of Compound 17a
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o
aReagents and conditions: a) MeNH2/water (40% weight, 3.0 eq), AcOH, 65 C, then recrystallization
from MTBE, 44% overall, 100% ee; b) LiOH, H2O, MeOH, 86%; c) SOCl2 (2 eq), DCM, DMF (1 drop);
d) bis(pinacolato)diboron (1.2 eq), KOAc (3.0 eq), Pd(dppf)Cl2 (0.5 mol %) dioxane, 100 oC, 91%; e) H2,
o
Pd/C, THF, 58%; f) 42 (1.0 eq), Cs2CO3 (3 eq), Pd(Ph3P)4 (10 mol %), DMF/H2O, microwave, 120 C,
93%; g) 39 (1.25 eq), pyridine (1.25 eq), DCM, 43%
Scheme 3. Preparation of Compound 19a
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aReagents and conditions: a) NaNO2 (1.05 eq), HCl (conc, 8 eq), CH2(CONHCO2Et)2 o(45, 1.0
o
eq), H2O, ꢀ5 C; b) aq KOH, THF/EtOH; c) NaOH, EtOH/H2O; d) HSCH2CO2H, 180 C, 58%
overall 3 steps; e) (R)ꢀ(ꢀ)ꢀglycidylmethyl ether (3.0 eq), silica gel (1.5 g/ 1.0 g 47, 200ꢀ400 mesh),
o
DMF, 80 C, 64%; f) Fe (5 eq), NH4Cl (5 eq), EtOH/H2O, 85 oC, 84%; g) 38 (1.0 eq), HATU (1.0
eq), DIPEA (2.0 eq), DMF, 80 oC, 39%.
CONCLUSION
In summary, identification of a singleton P2X7 inhibitor 1 from HTS revealed a
pharmacophore that was eventually turned into potential clinical candidates 17 and 19. During
development, a number of different properties were optimized and issues circumvented, such as
metabolic stability, plasma stability, GSH adduct formation and aniline mutagenicity. Thus,
iterative modifications of the molecule, such as conversion of the 1,4ꢀdihydropyridinone to the
1,2ꢀdihydropyridinone, proper substitution at Cꢀ5”, and addition of fluorine atoms to the aniline
ring allowed the identification of potent and selective P2X7 antagonists that may be useful for
efforts aimed at evaluating the biology of P2X7 and its role in inflammatory, immune,
neurologic or musculoskeletal disorders.2
EXPERIMENTAL SECTION
General Methods. All reagents and solvents were purchased from commercial sources and
1
used without further purification. H NMR spectra were measured using a Bruker NMR Avance
400 MHz or Bruker NMR Avance 300 MHz spectrometer, and chemical shifts were expressed in
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δ (ppm) units using tetramethylsilane as an internal standard. Mass spectra were recorded on an
Agilent AG1 Single Quad G6140A spectrometer.
7
8
9
Purity values for all tested compounds were found to be above 95% from the HPLC analyses.
In general, the nomenclature used in this section is based on IUPAC as noted above, or,
alternatively, based on ChemDraw.
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The preparation of compound 1 represents a typical procedure used for the synthesis of
4-phenyl-2,3-dihydro-1H-pyridine-4-one
amides.
1-[2-(2-Iodo-4,5-dimethoxy-phenyl)-
solution of (2ꢀiodoꢀ4,5ꢀ
acetyl]-2-phenyl-2,3-dihydro-1H-pyridin-4-one (1). To
a
dimethoxyphenyl)ꢀacetic acid (2.03 g, 6.3 mmol) in DCM (20 mL) was added oxalyl chloride
(0.6 mL, 6.93 mmol) under argon. One drop of DMF was added and the resulting mixture was
stirred at rt for 3.5 h. The mixture was evaporated to dryness. Dichloroethane was added and
the resulting solution was evaporated to dryness. This was repeated and the resulting residue
was dried under vacuum to afford 2.2 g of (2ꢀiodoꢀ4,5ꢀdimethoxyphenyl)ꢀacetyl chloride.
To a solution of 4ꢀmethoxypyridine (305.7 mg, 2.8 mmol) in THF (6 mL) was added at ꢀ25 to ꢀ
30° C phenyl magnesium bromide (3M in ether, 1.03 mL, 3.08 mmol) under an argon
atmosphere. After 5 minutes, (2ꢀiodoꢀ4,5ꢀdimethoxyphenyl)ꢀacetyl chloride (953.9 mg, 2.8
mmol) was added slowly keeping the temperture between – 25 to ꢀ30 °C. The reaction mixture
was allowed to stir at the same temperature for 2.5 h. An aqueous solution of HCl (2N) was
added to the reaction mixture at ꢀ 30°C and the resulting mixture was allowed to warm to rt and
stirred at rt for 10 minutes. The mixture was extracted with EtOAc (2x). The combined organic
extract was washed with saturated sodium chloride solution, dried over Na2SO4 and concentrated
in vacuo. Purification by flash chromatography (6:4 hexane/EtOAc) provided 1 (869 mg, 65%).
LCMS (M + H)+ = 478. 1H NMR (300 MHz, DMSOꢀd6) δ ppm 2.73 (dt, J=16.44, 1.67 Hz, 1
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H) 3.17 (dd, J=16.48, 7.06 Hz, 1 H) 3.71 (s, 3 H) 3.75 (s, 3 H) 3.88 ꢀ 4.00 (m, 1 H) 4.22 (d,
J=16.95 Hz, 1 H) 5.32 (dd, J=8.48, 1.51 Hz, 1 H) 5.96 (d, J=6.97 Hz, 1 H) 6.90 (s, 1 H) 7.17 ꢀ
7.41 (m, 6 H) 8.21 (dd, J=8.29, 1.32 Hz, 1 H).
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The preparation of compound 4 represents a typical procedure used for the synthesis of
4-phenyl-2,3-dihydro-1H-pyridine-4-one ureas. (2S)-N-(4,5-dimethoxy-2-methyl-phenyl)-2-
(4-fluorophenyl)-4-oxo-2,3-dihydropyridine-1-carboxamide (4). Step 1. [(1R,5R)-5-methyl-
2-(1-methyl-1-phenyl-ethyl)cyclohexyl]
(2S)-2-(4-fluorophenyl)-4-oxo-5-triisopropylsilyl-
2,3-dihydropyridine-1-carboxylate. Following Comins’ procedure,22 to a solution of 4ꢀ
methoxyꢀ3ꢀtriisopropylsilanylꢀpyridine (7.11 g, 26.8 mmol) in toluene (193 mL) at −25 °C. was
added
a
solution
of
[(1R,5R)ꢀ5ꢀmethylꢀ2ꢀ(1ꢀmethylꢀ1ꢀphenylꢀethyl)cyclohexyl]
carbonochloridate (7.9 g, 26.8 mmol) in toluene (160 mL). After 15 minutes the mixture was
cooled to −78 °C. and 1.0M 4ꢀfluorophenylmagnesium bromide in THF (30.6 mL, 30.6 mmol)
was added slowly. The mixture was stirred at −78 °C. for 1 hour. 2N HCl was added and the
mixture was allowed to warm to room temperature and was stirred for additional 15 minutes. The
mixture was extracted with ethyl acetate, washed with water and brine, dried over MgSO4,
filtered and concentrated to dryness. Purification of the residue by flash chromatography
(hexane/ethyl acetate 9:1) gave [(1R,5R)ꢀ5ꢀmethylꢀ2ꢀ(1ꢀmethylꢀ1ꢀphenylꢀethyl)cyclohexyl] (2S)ꢀ
2ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ5ꢀtriisopropylsilylꢀ2,3ꢀdihydropyridineꢀ1ꢀcarboxylate (13.14 g, 81%)
1
as a white solid. H NMR (300 MHz, DMSOꢀd6) δ ppm 0.54 (br. s., 1 H) 0.75 (d, J=6.40 Hz, 3
H) 0.77 ꢀ 0.83 (m, 1 H) 0.98 (dd, J=7.35, 1.70 Hz, 18 H) 1.16 (s, 3 H) 1.21ꢀ1.45 (m, 5H) 1.29 (s,
3 H) 1.47 ꢀ 1.65 (m, 2 H) 1.75 (d, J=12.81 Hz, 1 H) 1.97 ꢀ 2.12 (m, 1 H) 2.34 (d, J=15.07 Hz, 1
H) 2.86 (dd, J=15.82, 7.54 Hz, 1 H) 4.38 (br. s., 1 H) 4.73 (td, J=10.64, 4.33 Hz, 1 H) 6.88 ꢀ 7.11
(m, 4 H) 7.14 ꢀ 7.24 (m, 1 H) 7.24 ꢀ 7.36 (m, 4 H) 7.84 (s, 1 H).
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Step 2 (S)-2-(4-Fluoro-phenyl)-2,3-dihydro-1H-pyridin-4-one. To a solution of [(1R,5R)ꢀ5ꢀ
5
6
methylꢀ2ꢀ(1ꢀmethylꢀ1ꢀphenylꢀethyl)cyclohexyl]
(2S)ꢀ2ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ5ꢀ
7
8
9
triisopropylsilylꢀ2,3ꢀdihydropyridineꢀ1ꢀcarboxylate (13.14 g, 21.9 mmol) in methanol (160 mL)
was added 25% sodium methoxide in methanol (49.6 mL, 218.6 mmol). The mixture was heated
at reflux for 16 h. After cooling to room temperature, oxalic acid (78 g, 867 mmol) was added
and the mixture was stirred for 2 h. The solvent was evaporated to dryness. The crude residue
was partitioned between ethyl acetate and water. The organic layer was washed with water, then
brine, dried over MgSO4, filtered and concentrated to dryness. Purification by flash
chromatography (EtOAc/ hexane gradient, 0 – 95%) gave (S)ꢀ2ꢀ(4ꢀfluorophenyl)ꢀ2,3ꢀdihydroꢀ
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1
1Hꢀpyridinꢀ4ꢀone (2.34 g, 56%, ee 98.9%)23 as a yellow solid. LCMS (M + H)+ = 192. H NMR
(300 MHz, CDCl3) δ ppm 2.45 ꢀ 2.58 (m, 1 H) 2.63 ꢀ 2.80 (m, 1 H) 4.75 (dd, J=14.51, 5.09 Hz, 1
H) 5.08 (br. s., 1 H) 5.13 (d, J=7.54 Hz, 1 H) 7.02 ꢀ 7.15 (m, 2 H) 7.20 ꢀ 7.31 (m, 1 H) 7.32 ꢀ
7.44 (m, 2 H).
Step
3.
(2S)-N-(4,5-dimethoxy-2-methyl-phenyl)-2-(4-fluorophenyl)-4-oxo-2,3-
dihydropyridine-1-carboxamide (4). To a solution of 4,5ꢀdimethoxyꢀ2ꢀmethylꢀaniline ( 84 mg,
0.5 mmol) in 10 mL of toluene was added pyridine (0.6 mL) and DMAP (61 mg) followed by
phosgene ( 0.34 mL of 20% in toluene). The mixture was stirred at room temperature for 2 h.
The solution was filtered and the filtrate concentrated in vacuo. The residue was diluted with
DCM and concentrated in vacuo again to provide 1ꢀisocyanatoꢀ4,5ꢀdimethoxyꢀ2ꢀmethylꢀbenzene
(96 mg, 99%) as a white crystalline solid.
(S)ꢀ2ꢀ(4ꢀFluoroꢀphenyl)ꢀ2,3ꢀdihydroꢀ1Hꢀpyridinꢀ4ꢀone (80 mg, 0.42 mmol) was dissolved in 5
mL of dry THF and cooled to – 40° C. A solution of nꢀBuLi (0.22 mL of 2.5 M, 0.55 mmol) was
added and the mixture was allowed to stir for 30 min. 1ꢀisocyanatoꢀ4,5ꢀdimethoxyꢀ2ꢀmethylꢀ
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benzene (96 mg, 0.50 mmol) was added and the mixture was stirred for 1 h and then allowed to
warm to room temperature and stir at room temperature for 2 h. The reaction was quenched with
saturated NH4Cl (aq.) and water. The mixture was extracted with EtOAc. The organic extracts
were dried over Na2SO4 and concentrate in vacuo. The residue was purified by flash
chromatography (silica, 1:1 hexane: EtOAc) to afford (2S)ꢀNꢀ(4,5ꢀdimethoxyꢀ2ꢀmethylꢀphenyl)ꢀ
2ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ2,3ꢀdihydropyridineꢀ1ꢀcarboxamide 4 (95 mg, 54%) as a yellow foam.
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1
LCMS (M + H)+ = 385. H NMR (500 MHz, DMSOꢀd6) δ ppm 1.93 (s, 3 H) 2.70 (d, J=16.48
Hz, 1 H) 3.22 (dd, J=16.48, 7.32 Hz, 1 H) 3.69 (s, 3 H) 3.72 (s, 3 H) 5.11 ꢀ 5.32 (m, 1 H) 5.85 (d,
J=6.71 Hz, 1 H) 6.75 (s, 1 H) 6.79 (s, 1 H) 7.12 ꢀ 7.33 (m, 4 H) 8.24 (dd, J=8.54, 1.22 Hz, 1 H)
9.08 (s, 1 H).
2-(3-Amino-4-methyl-phenoxy)ethyl acetate (30). A 12ꢀL 3ꢀneck round bottom flask flushed
with nitrogen was charged with 4ꢀmethylꢀ3ꢀnitroꢀphenol (350 g, 2.29 mol) followed by an
addition funnel over a period of 1 h. K2CO3 (325 mesh, 441.5 g, 3.20 mol) and DMA (700 mL).
To the mixture was added 2ꢀbromoethyl acetate (438.9 g, 2.63 mol) via After the addition was
complete, the reaction mixture was stirred at 65 °C for 24 h, and then cooled to 15 °C. To the
reaction mixture was then added slowly 2.7 L of MTBE and 3.8 L of water. The mixture was
stirred for 10 min, and the organic layer was separated and washed with 4 X 2.1 L of water. The
organic layer was then distilled at atmospheric pressure to a final volume of ca. 1 L. To this
residue was added 800 mL of EtOH and the atmospheric distillation was continued to remove
additional 750 mL of the distillate with the final internal temperature of 82 °C. This solution
was used as is in the next step.
1
Note: A small aliquot of the ethanolic solution was concentrated to record a H NMR of the
intermediate 2ꢀ(4ꢀmethylꢀ3ꢀnitroꢀphenoxy)ethyl acetate in CDCl3 (300 MHz): 7.54 (1H, d, J= 3
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Hz), 7.23 (1H, s), 7.09 (1H, d, J1= 3 Hz, J2= 2.0 Hz), 4.44 (2H, d, J= 6 Hz), 4.21 (2H, d, J=
6Hz), 2.53 (3H, s), 2.11 (3H, s).
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9
A 25ꢀL reactor, flushed with 3 cycles of vacuum and nitrogen, was charged with 55 g of 20%
Pd(OH)2 on carbon and then flushed again with 3 cycles of vacuum and nitrogen. To this vessel
was charged ethanolic solution of intermediate 2ꢀ(4ꢀmethylꢀ3ꢀnitroꢀphenoxy)ethyl acetate from
above. The mixture so obtained was then stirred under hydrogen atmosphere (3–4 psi) at 45–50
°C for 8 h. Upon complete conversion, the mixture was cooled to 20 °C, purged with nitrogen
and filtered through a pad of 200 g of SolkaꢀFloc® and the filtercake was washed with 1.3 L of
ethanol. The combined filtrate and washes were concentrated at 40 °C under vacuum to remove
ca. 1.5 L of ethanol to obtain a slurry, which was cooled to 0 °C and then aged for 1 h. The
solids were filtered on a sintered glass funnel and washed with 1 L of iceꢀcold ethyl acetate. The
wet cake was dried in a vacuum oven at 40 °C for 18 h to obtain 409.9 g (85% yield) of 2ꢀ(3ꢀ
aminoꢀ4ꢀmethylꢀphenoxy)ethyl acetate, 30, as a beige colored solid (Mp: 98–100 °C).
2-(5-Amino-2-chloro-4-methyl-phenoxy)ethyl acetate (31). To an iceꢀcold (0–7 °C) slurry of
30 (500 g, 2.39 mol) in 2.5 L of acetonitrile was added in three equal portions Nꢀ
chlorosuccinimide (NCS, 367 g, 2.75 mol). After ca. 2–3 h, a solution of Na2SO3 (173 g, 1.38
mol) in water (1.0 L) was added such that the reaction temperature was maintained below 10 °C.
The solution so obtained was then added slowly to 12 L of iceꢀcold water over ca. 2 h such that
the reaction temperature was maintained below 10 °C. The resulting slurry was stirred at 4–10
°C for 1 h and then filtered on a sintered glass funnel. The wet cake was washed with 2x 2.5 L
of cold water followed by 2x 1.0 L of hexanes, and then dried in a vacuum oven at ca. 45 °C for
18 h to obtain 530 g (91% crude yield) of 2ꢀ(5ꢀaminoꢀ2ꢀchloroꢀ4ꢀmethylꢀphenoxy)ethyl acetate,
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31. The material was recrystallized from 8 volumes of 1:2 ethyl acetate/ hexanes with ca. 80%
recovery.
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1H NMR (300 MHz, CDCl3): 7.01 (1H, s), 6.30 (1H, s), 4.43 (2H, t, J= 6 Hz), 4.16 (2H, t, J= 6
Hz), 3.60 (2H, broad s), 2.10 (3H, s), 2.08 (3H, s).
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(4S)-N-[4-chloro-5-(2-hydroxyethoxy)-2-methyl-phenyl]-4-(4-fluorophenyl)-2-oxo-3,4-
dihydro-1H-pyridine-5-carboxamide (15). To a suspension of (4S)ꢀ4ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoꢀ
3,4ꢀdihydroꢀ1Hꢀpyridineꢀ5ꢀcarboxylic acid,16 (10 g, 42.5 mmol) in 50 mL of dichloromethane at
ambient temperature was added DMF (30ꢁL, 387 ꢁmol) followed by slow addition of SOCl2
(6.21 mL, 85.1 mmol). The resulting mixture was stirred at ambient temperature for at 2 h,
during which time the gas evolution had ceased and the reaction mixture had turned
homogenous. The reaction mixture was concentrated under reduced pressure at 50 °C to a
minimum volume. The concentrated mixture was further evaporated with 2 x 54 mL of toluene
to obtain the (4S)ꢀ4ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoꢀ3,4ꢀdihydroꢀ1Hꢀpyridineꢀ5ꢀcarbonyl chloride, 28, as
a thick orange oil, which was dissolved in 54 mL of MeTHF.
In separate flask, 2ꢀ(5ꢀaminoꢀ2ꢀchloroꢀ4ꢀmethylꢀphenoxy)ethyl acetate, 31 (10.8 g, 40.4 mmol)
was dissolved in 54 mL of MeTHF, and to this solution was added 2,6ꢀdimethylpyridine (5.2
mL, 44.7 mmol). To this solution, cooled to 0–4 °C with an ice/water bath, was added via an
addition funnel the MeTHF solution 28 from above. After the addition was complete (ca. 15
min), the addition funnel was rinsed with 2 mL of MeTHF and the rinse was added to the
reaction mixture, which was then stirred for ca. 2 h to obtain a slurry. The reaction mixture was
allowed to warm to ambient temperature and to the reaction flask was slowly added 15 mL of 1:2
mixture of brine:1N HCl to obtain a biphasic mixture. The organic layer was separated, and then
washed twice with 15 mL 1:1, brine:water followed by 15 mL of 1.05 M sodium carbonate and
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finally with 15 mL of brine. The organic layer was concentrated under vacuum at 40 °C, and
residue so obtained was azeotroped with MeOH such that a solution of acetylated 15 in ca. 45
mL of MeOH was obtained. To this methanol solution was added via a syringe pump 25 weight
% methanolic solution of sodium methoxide (4.45 mL, 19.46 mmol). After ca. 3 h at ambient
temperature, 145 mL of water was added at a rate of 76 mL/h such that internal temperature was
< 35 °C. After the water addition was complete the slurry so obtained was stirred at ambient
temperature for at 2 h, and then filtered on a sintered glass funnel. The wet cake was washed
with 35–40 mL of 15% (v/v) methanol/water, then dried in vacuum oven at 60 °C for 18 h
providing 14.93 g (89–91% yield) of (4S)ꢀNꢀ[4ꢀchloroꢀ5ꢀ(2ꢀhydroxyethoxy)ꢀ2ꢀmethylꢀphenyl]ꢀ
4ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoꢀ3,4ꢀdihydroꢀ1Hꢀpyridineꢀ5ꢀcarboxamide, 15. MS (M+H) = 419. 1H
NMR (300 MHz, DMSOꢀd6): 9.88 (1H, broad s), 9.08 (1H, broad s), 7.29–7.22 (3H, m), 7.17–
7.10 (3H, m), 4.83 (1H, bs), 4.27 (1H, d, J= 6.6 Hz), 4.06–3.95 (2H, m), 3.72ꢀ3.60 (2H, m), 3.00
(1H, dd, J1= 6 Hz, J2= 6 Hz), 2.42 (1H, d, J= 3 Hz), 1.94 (3H, s).
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13C NMR (75 MHz, DMSOꢀd6): 169.03, 164.12, 160.96 (d, JCꢀF= 242 Hz), 151.62, 138.14,
138.10, 136.07, 133.03, 130.57, 128.51, 128.41, 125.80, 117.46, 115.35, 115.07, 112.35, 111.55,
70.64, 59.28, 35.08, 16.46.
The preparation of compound 16 represents a general procedure used for the synthesis of
(4S)-2-oxo-4-aryl-3,4-dihydro-1H-pyridine-5-carboxamides. Step 1. Preparation of 4-(3-
fluorophenyl)-2-oxo-3,4-dihydro-1H-pyridine-5-carboxylic acid. To a solution of 6ꢀchloroꢀ
nicotinic acid (3.2 g, 20 mmol) in 16 mL of THF was added (3ꢀfluorophenyl)magnesium
bromide ( 16 mL of 1M in THF) at 0 °C over 30 min. The resulting reaction mixture was stirred
at room temperature for 48 h, before cooling to ꢀ10 °C. To the cooled mixture was added 30 mL
of acetic acid. The reaction mixture was warmed to room temperature and 100 mL of water was
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added. The mixture was stirred at room temperature for 2h and extracted with DCM (3 X 300
mL). The combined organic layer was concentrated, washed with hexane:DCM( 9:1, 100 mL)
and decanted. The solids were dried under vacuum to afford 1.8 g of 4ꢀ(3ꢀfluorophenyl)ꢀ2ꢀoxoꢀ
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3,4ꢀdihydroꢀ1Hꢀpyridineꢀ5ꢀcarboxylic acid. LCMS (M+H)+ = 236, (MꢀH)+ = 234;. H NMR
(300 MHz, Methanolꢀd4) δ ppm 2.64 (dd, J=16.77, 1.70 Hz, 1 H) 3.03 (dd, J=16.58, 8.67 Hz, 1
H) 4.17 (d, J=7.16 Hz, 1 H) 6.85 ꢀ 6.97 (m, 2 H) 7.04 (d, J=7.91 Hz, 1 H) 7.21 ꢀ 7.35 (m, 1 H)
7.54 (s, 1 H).
Step 2. Preparation of (4S)-N-(2-bromo-4,5-dimethoxy-phenyl)-4-(3-fluorophenyl)-2-oxo-
3,4-dihydro-1H-pyridine-5-carboxamide (16). A solution of 4ꢀ(3ꢀfluorophenyl)ꢀ2ꢀoxoꢀ3,4ꢀ
dihydroꢀ1Hꢀpyridineꢀ5ꢀcarboxylic acid (150 mg, 0.64 mmol) in DCM ( 10 mL) was treated
sequentially with 3 drops DMF and 2 equiv. oxalyl chloride. The resulting reaction mixture was
stirred for 30 min. before concentration under vacuum. The residue was reꢀdissolved in DCM
and to the resulting solution was added 2ꢀbromoꢀ4,5ꢀdimethoxyꢀaniline (150 mg, 0.65 mmol)
and 2 equiv. TEA. The reaction mixture was stirred at room temperature for 24 h.
Chromatography (30 – 100% EtOAc/ hexane gradient) provided crude product, which was
further purified by chiral HPLC to provide 80 mg of a frontꢀrunning, more active isomer,
assigned as the desired product, (4S)ꢀNꢀ(2ꢀbromoꢀ4,5ꢀdimethoxyphenyl)ꢀ4ꢀ(3ꢀfluorophenyl)ꢀ2ꢀ
oxoꢀ3,4ꢀ dihydroꢀ1Hꢀpyridineꢀ5ꢀcarboxamide, 16. LCMS (M + H)+ = 449, 451. H NMR (300
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MHz, CDCl3) δ ppm 2.75 (d, J=15.64 Hz, 1 H) 3.13 (dd, J=16.58, 8.48 Hz, 1 H) 3.81 (s, 3 H)
3.87 (s, 3 H) 4.18 (d, J=6.97 Hz, 1 H) 6.89 (s, 1 H) 6.94 ꢀ 7.06 (m, 2 H) 7.12 (d, J=7.91 Hz, 1 H)
7.29 ꢀ 7.39 (m, 1 H) 7.42 (s, 1 H) 7.57 (d, J=5.27 Hz, 1 H) 7.99 (s, 1 H) 8.44 (d, J=5.09 Hz, 1 H).
(4S)-4-(4-Fluorophenyl)-1-methyl-2-oxo-3,4-dihydropyridine-5-carboxylic acid (38). To a
solution of methyl (4S)ꢀ4ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ2ꢀoxoꢀ3,4ꢀdihydropyridineꢀ5ꢀcarboxylate
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3716b (7.18 g, 27 mmol) in 65 mL of methanol at room temperature under argon was added
lithium hydroxide hydrate (1.5 g, 35 mmol) followed by water (11 mL). The resulting reaction
mixture was warmed at 60 °C for 18 h, then cooled to room temperature and concentrated under
vacuum. Water and ether were added. The aqueous phase was washed with ether and the
ethereal layer discarded. The aqueous layer was then acidified to pH 2 with 6 N HCl. The
aqueous phase was extracted with ether (2X). The combined ethereal extracts were washed with
brine, dried over MgSO4, filtered and evaporated in vacuo to provide the title compound, 38
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(6.27 g, 93%) as a white foam. LCMS (M + H)+ = 250. H NMR (400 MHz, CDCl3) δ 7.56 (s,
1H), 7.08ꢀ7.22 (m, 2H), 6.89ꢀ7.05 (m, 2H), 4.13 (d, J=8.59 Hz, 1H), 3.23 (s, 3H), 3.00 (dd,
J=8.34, 16.42 Hz, 1H), 2.79 (dd, J=1.52, 16.67 Hz, 1H).
4-Fluoro-2-methyl-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine
(41).
Step 1. A mixture of 4ꢀbromoꢀ5ꢀfluoroꢀ2ꢀnitrotoluene, 40 (4.9 grams, 0.02moles, 1equiv.),
bis(pinacolato) diboron (6.38 grams, 0.0251 moles, 1.2 equiv.), potassium acetate (6.17 grams,
0.062 moles, 3.0 equiv.) and [1,1’ꢀbis(diphenyphosphino)ferrocene]ꢀdichoropalladium(II) (1:1)
complex with dichoromethane (0.85 g, 0.0001 moles) in anhydrous dioxane (25 ml) was heated
in sealed reaction vessel under argon for 1 hour at 100 °C. To the reaction mixture was added 50
ml water and the resultant mixture was filtered through pad of celite, washed and extracted with
ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate,
filtered and concentrated under vacuum. Chromatography on silica gel, eluting with 20% ethyl
acetate in hexanes afforded 5.36 grams of 2ꢀ(2ꢀfluoroꢀ4ꢀmethylꢀ5ꢀnitrophenyl)ꢀ4,4,5,5ꢀ
°
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tetramethylꢀ1,3,2ꢀdioxaborolane (0.0191 moles, 91%). mp 95ꢀ96 C. H NMR (400 MHz,
CDCl3) δ ppm 1.37 (s, 12 H) 2.63 (s, 3 H) 7.01 (d, J=9.60 Hz, 1 H) 8.43 (d, J=5.56 Hz, 1 H).
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Step 2. To a solution of 2ꢀ(2ꢀfluoroꢀ4ꢀmethylꢀ5ꢀnitrophenyl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolane (2.95 grams, 0.0105 moles) in 50 mL THF was added 100 mg of 10% palladium
on carbon under nitrogen. The reaction mixture was evacuated under vacuum, then filled with
hydrogen gas via balloon, and stirred under the hydrogen atmosphere at room temperature for 4
hours. After the reaction was complete, the reaction mixture was filtered through celite, and
purified by chromatography on silica, eluting with 20% ethyl acetate in hexanes to give 1.54 g 4ꢀ
fluoroꢀ2ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀphenyl amine, 41 (0.006 moles,
58%). mp 91ꢀ93 °C. 1H NMR (300 MHz, CDCl3) δ ppm 1.36 (s, 12 H) 2.17 (s, 3 H) 3.53 (br. s.,
2 H) 6.78 (d, J=9.82 Hz, 1 H) 7.00 (d, J=4.91 Hz, 1 H).
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4-Chloro-2-methyl-2H-pyridazin-3-one (42). Step 1. A solution of 4,5ꢀdichloroꢀ2ꢀmethylꢀ
2Hꢀpyridazinꢀ3ꢀone, (5 g, 28 mM) in 20 ml ethanol was added dropwise to anhydrous hydrazine
(3.5 ml, 112 mM) that was stirring in 20 ml ethanol at 3 °C. The reaction mixture was stirred at 0
°C an additional 3 hours, then filtered and the precipitate washed with ether to provide 0.68 g
slightly impure product. The filtrate was cooled in the freezer overnight, filtered and washed
with ether to provide an additional 2.37 g of product. The collected solids were combined to
provide 3.05 g of impure 4ꢀchloroꢀ5ꢀhydrazinoꢀ2ꢀmethylꢀ2Hꢀpyridazinꢀ3ꢀone, which was used as
is for the next step. LCMS (M + H)+ = 175.
Step 2. A solution of 4ꢀchloroꢀ5ꢀhydrazinoꢀ2ꢀmethylꢀ2Hꢀpyridazinꢀ3ꢀone ( 2.5 g, 14.3 mM )
and CuSO4.5H2O ( 20 g ) in 100 ml H2O was heated at 90 °C for 45 minutes. The reaction
mixture was allowed to cool and extracted into DCM. The combined organic extracts were
washed with water, then brine and dried over sodium sulfate and concentrated in vacuo.
Chromatography on silica gel using a 0% ꢀ 20% EtOAc/Hexane gradient afforded 0.74 g (35%)
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of 4ꢀchloroꢀ2ꢀmethylꢀ2Hꢀpyridazinꢀ3ꢀone, 42. LCMS (M + H)+ = 145. 1H NMR (300 MHz,
CDCl3) δ ppm 3.86 (s, 3 H) 7.38 (d, J=4.52 Hz, 1 H) 7.67 (d, J=4.14 Hz, 1 H).
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4-(5-Amino-2-fluoro-4-methylphenyl)-2-methyl-2H-pyridazin-3-one (43). A microwave
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vial was charged with
a
mixture of 4ꢀfluoroꢀ2ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ
[1,3,2]dioxaborolanꢀ2ꢀyl)ꢀphenylamine, 41 (0.104 g, 0.415 mmol), 4ꢀchloroꢀ2ꢀmethylꢀ2Hꢀ
pyridazinꢀ3ꢀone, 42, (0.06 g, 0.415 mmol), cesium carbonate (0.405 g, 1.245 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.048 g, 0.041 mmol) in a 1:1 solution of DMFꢀwater
(3 mL). The vial was sonicated while argon was bubbled into the solution for 10 min. Then the
vial was heated under microwaves at 120 °C for 20 min. After cooling to room temperature, the
mixture was extracted with ethyl acetate (3x15 mL), combined organics were washed with brine
(3x5 mL), dried over magnesium sulfate, filtered and concentrated. The residue was purified by
flash chromatography eluting with a 0 to 100% ethyl acetate /hexanes gradient to yield the title
compound, 43 (0.109 g, 93%). LCMS: (M+H)+ = 234; 1H NMR (400 MHz, CDCl3) δ ppm 2.20
(s, 3 H) 3.87 (s, 3 H) 6.89 (dd, J=11.12, 0.51 Hz, 1 H) 7.10 (d, J=6.57 Hz, 1 H) 7.34 (dd, J=4.29,
1.77 Hz, 1 H) 7.80 (d, J=4.04 Hz, 1 H).
(S)-4-(4-Fluorophenyl)-1-methyl-6-oxo-1,4,5,6-tetrahydropyridine-3-carboxylic acid [4-
fluoro-2-methyl-5-(2-methyl-3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-amide (17). To a
solution of (S)ꢀ4ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ6ꢀoxoꢀ1,4,5,6ꢀtetrahydropyridineꢀ3ꢀcarboxylic acid,
38 (0.12 g, 0.48 mmol) in dichloromethane (2 mL), containing a catalytic amount of DMF (1
drop), was added dropwise thionyl chloride (0.07 mL, 0.962 mmol) and the mixture stirred at
room temperature for 2 h. Then the solvents were evaporated under vacuum, residue suspended
in toluene (10 mL) and concentrated again (twice) and residue dried in high vacuum for 30 min
to afford acid chloride intermediate 39, which was used as is. The residue was dissolved in
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