4-Anilino-5-carboxamido-2-pyridone derivatives as noncompetitive inhibitors of mitogen-activated protein kinase kinase

Article abstract of DOI:10.1021/jm0704548

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain,

Full text of DOI:10.1021/jm0704548

5090
J. Med. Chem. 2007, 50, 5090-5102
4-Anilino-5-carboxamido-2-pyridone Derivatives as Noncompetitive Inhibitors of
Mitogen-Activated Protein Kinase Kinase
Julie A. Spicer,*,† Gordon W. Rewcastle,† Michael D. Kaufman,¶ Shannon L. Black,† Mark S. Plummer,¶ William A. Denny,†
John Quin, III,¶ Aurash B. Shahripour,¶ Stephen D. Barrett,¶ Christopher E. Whitehead,¶ Jared B. J. Milbank,¶ Jeffrey F. Ohren,¶
Richard C. Gowan,¶ Charles Omer,¶ Heidi S. Camp,¶ Nadia Esmaeil,¶ Kelley Moore,¶ Judith S. Sebolt-Leopold,¶
Sally Pryzbranowski,¶ Ronald L. Merriman,¶ Daniel F. Ortwine,¶ Joseph S. Warmus,¶ Cathlin M. Flamme,¶
Alexander G. Pavlovsky,¶ and Haile Tecle¶
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The UniVersity of Auckland, PriVate Bag 92019,
Auckland 1142, New Zealand, and Pfizer Global Research and DeVelopment, 2800 Plymouth Rd., Ann Arbor, Michigan 48105
ReceiVed April 17, 2007
A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized
using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect
of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the
4′-iodide were all investigated. This study afforded several compounds which were either equipotent or
more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon
carcinoma (C26) cells, as measured by supression of phosphorylated ERK substrate. Most notably, pyridone
27 was found to be more potent than 1 in Vitro and produced a 100% response rate at a lower dose than 1,
when tested for in ViVo efficacy in animals bearing C26 tumors.
Chart 1. Compound 1 and Analogues
Introduction
The Ras/Raf/MEK/ERK mitogen-activated protein (MAP)
kinase signaling pathway is responsible for the coordination and
regulation of cell growth and differentiation in response to
extracellular stimulation.1-3 Numerous studies have shown that
the MAP kinase pathway plays an integral part in the formation,
progression, and survival of tumors, in addition to participating
in many inflammatory processes. MAP kinase kinase 1 and 2
(MEK1, MEK2) are closely related, dual-specificity, tyrosine/
threonine protein kinases that phosphorylate the downstream
target ERK (a MAP kinase) on specific tyrosine and threonine
residues.4 Therefore, small-molecule inhibitors of MEK have
the potential to be anticancer, antiviral, and antiinflammatory
therapeutics. As a result, this target has been the focus of intense
medicinal chemistry efforts by a number of research groups in
recent years,5-8 with several compounds advancing into clinical
development, including Pfizer compounds CI-1040 (1, Chart
1, also known as PD184352)5 and PD325901 (2a)9 as well as
the Array BioPharma candidate ARRY-142886 (AZD6244).10
availability.16,17 These were addressed by the second-generation
dihydroxyalkyl hydroxamate compound 2a (PD325901), of the
same class, which displayed more potency and improved
bioavailability compared to 1.
Throughout this development process we also sought to
identify additional inhibitor templates that would retain the
significant advantages of 2a but provide an even better
pharmacological profile.
A structural basis for the unique, noncompetitive nature of
MEK inhibition possessed by compounds such as 1 was
provided by the crystal structures of analogues 2b (PD318088)
and 3 (PD334581) bound to MEK1 and MEK2, respectively.18,19
These structures revealed that the inhibitors bind in a novel
allosteric binding pocket separate from, but adjacent to, the
MgATP site. Several key interactions were observed between
the inhibitors and amino acid residues within this binding site.
First, the B ring of the inhibitor was bound within a deep
hydrophobic pocket formed by Met143, Ile141, Leu118, and
Phe209, creating numerous van der Waals interactions with the
amino acid side chains involved, while Phe209 also formed a
critical edge-to-face aromatic interaction with the B ring.
In 1999 the diarylamine 1 was reported to be a highly
selective, potent inhibitor of MEK1 and MEK2. It was found
to utilize a unique noncompetitive mechanism of inhibition and
had significant antitumor activity in ViVo.11-14 On the basis of
its promising preclinical profile, compound 1 was advanced into
clinical trials15 where a number of pharmacological issues were
identified, such as metabolic instability and low overall bio-
* To whom correspondence should be addressed. Phone: +64
9 3737599. Fax: +64 9 3737502. E-mail: j.spicer@auckland.ac.nz.
† The University of Auckland.
¶ Pfizer Global Research and Development.
a Abbreviations: MEK, mitogen-activated protein kinase kinase; ERK,
extracellular signal-regulated kinase; MAP, mitogen-activated protein;
MgATP, magnesium adenosine triphosphate; CoMFA, comparative mo-
lecular field analysis; SAR, structure-activity relationship; pERK, phos-
phorylated extracellular signal-regulated kinase; DMT-MM, 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; PFP-TFA, pentafluoro-
phenyl trifluoroacetate; DABCO, 1,4-diazabicyclo[2.2.2]octane; TFA, tri-
fluoroacetic acid; PyBOP, benzotriazolyloxytris-(pyrrolidino)phosphonium
hexafluorophosphate; BID, twice daily; TID, three times daily.
10.1021/jm0704548 CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/19/2007

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5091
model.20 The docking mode of compounds within the allosteric
inhibitor binding site of MEK1 was used to generate the CoMFA
model which is illustrated in Figure 1.
By providing suggestions for increasing the biological activity
of compounds using the CoMFA contours, the model was useful
in prioritizing the synthesis of novel compounds. The CoMFA
contours within the inhibitor-binding site are shown in Figure
2 and suggest that the bioactivity of novel compounds can be
increased by placing more steric bulk into the green volumes
and less bulk in the yellow volumes, more negative charge in
the red volumes, and less negative charge in the blue. From the
green contours, one can see the importance of having bulky
groups within the iodophenyl binding pocket. In addition, there
is a red region off the 4-fluoro position of the A-ring, signaling
the importance of maintaining the hydrogen bond to the amide
of Ser212.
The general protocol for submitting new compounds for
structure-based drug design involved initially docking the
compounds into the inhibitor-binding site using GOLD then
CoMFA scoring all docking poses of each molecule.20 The
compounds were then prioritized based on consistency of
binding mode, predicted IC50, and ease of synthesis. On the
basis of these observations, we chose to investigate replacement
of the difluorophenyl group of the original class of inhibitors
with a pyridone derivative to determine whether the pyridone
carbonyl group would provide an improved hydrogen bond
interaction with Ser212. We then explored a variety of car-
boxamide side chains as alternatives to the hydroxamate of 1.
In combination with both of the above modifications we also
investigated the effect of substitution on the pyridone nitrogen.
Finally, we looked at structural changes involving replacement
of the 4′-iodo substituent with other groups designed to explore
binding in the hydrophobic pocket while interacting with the
carbonyl oxygen of Val127.
Figure 1. Result of GOLD docking of a set of compounds into the
inhibitor binding site (Purple Connolly Surface) of the ternary complex
of MEK1, MgATP, and 2a (PDB Code: 1S9J). The GOLD docking
model served as alignment rule for Comparative Molecular Field
Analysis (CoMFA).
Second, an important electrostatic interaction between the
backbone carbonyl oxygen of Val127 and the 4′-iodide was
observed at the back of the pocket. Finally, a hydrogen bond
interaction was shown to exist between the 4-fluoro atom of
the inhibitor A ring and the backbone amide of Ser212.
This structural information was combined with Comparative
Molecular Field Analysis (CoMFA) techniques to enable the
structure-guided design of a new class of potent inhibitors of
MEK1 and MEK2, the 4-(phenylamino)-5-carboxamido-2-
pyridones 4, where R1, R2, and Ar were each varied indepen-
dently in order to establish an optimal arrangement of substit-
uents.
For SAR determination, all compounds were first evaluated
in a coupled Raf-MEK-ERK cascade assay, measuring the
inhibition of phosphorylated ERK (pERK) formation as the
terminal endpoint. Specific details are provided in the Experi-
mental Section. Selected compounds were also evaluated in a
cellular assay that measured the inhibition of ERK phosphory-
lation in murine colon carcinoma cells.
Results and Discussion
The key protein-ligand interactions derived from the crystal
structures of MEK1 and MEK2 provided the context for the
structure-guided discovery of new MEK inhibitor templates.
This information was used as a template for docking experiments
to aid in the discovery of novel structures. The docking of
compounds with known potencies was used as an alignment
rule for a Comparative Molecular Field Analysis (CoMFA)
Optimization of the Carboxamide Side Chain. The syn-
thesis of the 4-(phenylamino)-5-carboxamido-2-pyridones 4
Figure 2. CoMFA Contours shown in the MEK1 ternary complex coordinate frame provides insights into the design of novel chemical entities.
The contours suggest that the compounds’ activity can be optimized by placing more negative charge density and steric bulk in the red and green
volumes, respectively, and less charge density and steric bulk in the blue and yellow volumes.

5092 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Spicer et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) AcOH, H2O, reflux, 6 d; (ii) 1 M NaOH,
EtOH, RT, 15 h; (iii) a. PFP-TFA, py, DMA, RT, 2 h, b. amine R1, DIEA,
THF, RT, 2 h.
Notably, the simple amide was nearly as potent in Vitro as 1
and was considerably more potent than 1 in C26 cells. The
latter fact might be attributable to its greater aqueous solubility
and Caco permeability (Table 1). It was expected that the
hydroxyethylhydroxamate would further improve the potency
relative to the simple carboxamide. However, hydroxamate 26
and amide 27 were essentially equipotent. This is in marked
contrast to 37/38 (IC50s 0.0012 µM vs 0.0174 µM) and may
indicate that a tighter H-bond of the pyridone carbonyl to
Ser212 obviates the need to highly optimize the carboxamide
substituent. The hydroxyamides 30 and 31 were slightly less
effective, with 31, the hydroxamate isostere, being 2-3-fold
more potent than the hydroxyethylamide of 30. Table 1 also
lists the structure-guided CoMFA prediction for all of the
molecules in this series. The molecules that were listed as
unable to predict gave inconsistent binding modes and predicted
values.
Substitution on the Pyridone Nitrogen. The NH-pyridone
ester 40 was prepared by heating ethyl 6-chloro-4-(2-fluoro-4-
iodoanilino)nicotinate 11 at reflux in a 3:1 mixture of AcOH/
H2O22 (Scheme 2). Deprotection of ester 40 and coupling of
the resulting acid 41 with amines to give the target amides (42-
44) was carried out under the same conditions (using PFP-
TFA as the coupling agent) as detailed in Scheme 1.
a Reagents and conditions: (i) EtOH, concd HCl (cat.), 90 °C, 15 h; (ii)
a. Me2SO4, CHCl3, 0 °C to reflux, b. TEA, AcOH, EtOH, reflux 2 h; (iii)
1 M NaOH, EtOH, RT, 15 h; (iv) amine R1, DMT-MM, MeOH/THF,
RT, 15 h; (v) a. PFP-TFA, pyridine, DMA, RT, 2 h, b. amine R1, DIEA,
THF, RT, 2 h.
involved the initial selective acid-catalyzed displacement of the
4-chlorine atom of ethyl 4,6-dichloronicotinate 5 with substituted
anilines 6-10 to give intermediate 4-anilino-6-chloropyridine
esters 11-15 (Scheme 1).
The N-methylpyridone esters 16-20 were prepared by
quaternization of the chloropyridine esters 11-15 with dimethyl
sulfate followed by in situ conversion to the pyridones 16-20
with AcOH/TEA.21 Deprotection of ethyl esters 16-20 under
basic conditions gave the acids 21-25 which were coupled with
amines or hydroxylamines, using 4-(4,6-dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium chloride (DMT-MM) (Scheme 1).
Alternatively, the acids were first converted to the corresponding
pentafluorophenyl esters with pentafluorophenyl trifluoroacetate
(PFP-TFA) and then reacted with amines.
Initial efforts focused on the preparation of 2-fluoro-4-iodo-
anilino-substituted pyridones, owing to the previously observed17
potency enhancement vis-a`-vis the 2-chloro-4-iodoanilines.
Thus, ester 16 was prepared according to Scheme 1, further
saponified and derivatized as amides. From our previous work,
it was expected that introduction of a carboxamide would both
increase the potency and improve the pharmacokinetic properties
relative to the acid. Indeed, the simple carboxamide 27 was
highly potent against the Raf-MEK-ERK cascade and was
significantly active in the colon26 cellular assay. Mono- (28)
or dimethylation (29), however, greatly diminished this potency.
Primary amide 42 showed a loss of potency in both isolated
enzyme and cellular assays (Table 2). The hydroxyamides 43
and 44 were also prepared, on the basis that the corresponding
N-methylpyridones 30 and 31 showed appreciable activity
(Table 1). Unfortunately, this was not the case for NH-pyridones,
as 43 and 44 had IC50s of 1.17 and 2.68 µM, respectively,
against the isolated enzyme and both IC50s were >5 µM in the
Table 1. Variation of the Carboxamide Side Chain
cascade
IC50 (µM)
cellular
IC50 (µM)
aq solution
(µg/mL)
Caco
(cm/sec)
COMFA
prediction
compd
R1
1
37
38
39
16
21
26
27
28
29
30
31
cPrCH2ONH
HOCH2CH2ONH
NH2
HOCH2CH2NH
CH3CH2O
0.016
0.0012
0.0174
0.0842
0.669
0.425
0.018
0.023
0.195
0.627
0.206
0.061
0.046
0.002
0.030
0.068
0.063
>5
0.005
0.004
-
<1
7
<1
11
0
43.4
0
21.2
-
0
0.017
0.0009
0.0522
0.0036
0.0465
-
OH
770
610
6
unable to predict
0.0044
unable to predict
0.0624
HOCH2CH2ONH
NH2
CH3NH
(CH3)2N
HOCH2CH2NH
HOCH2CH2CH2NH
0
24.8
-
-
0
31
1200
29
-
0.180
0.095
0.0217
0.0212
0.0014
68
0

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5093
Scheme 4a
Table 2. Substitution on the Pyridone Nitrogen
cascade
IC50 (µM)
cellular
IC50 (µM)
compd
R2
27
42
53
54
55
56
58
60
62
63
64
CH3
H
CH3CH2
CH3CH2CH2
CH3O(CH2)2O(CH2)2
NCCH2CH2CH2
HOCH2CH2
HOCH2CH2CH2
HO2CCH2
0.023
0.100
1.00
2.54
1.13
0.004
0.63
-
0.43
-
4.89
2.54
-
a Reagents and conditions: (i) a. Me2SO4, 120 °C, b. sat. NaHCO3,
CH3CN, RT, 18 h.; (ii) 1 M NaOH, EtOH, RT, 15 h; (iii) Li[Si(CH3)2],
THF, 0 °C, 1 h. (iv) amine R1, PyBOP, CH2Cl2, RT, 15 h.
>5
-
>3
>10
0.091
>10
-
H2CdCHCH2
HOCH2CH(OH)CH2
-
Scheme 5a
-
Scheme 3a
a Reagents and conditions: (i) Alkyne, (Ph3P)2PdCl2, CuI, TEA, THF,
RT, 15 h.; (ii) K2CO3, MeOH/THF, RT, 48 h.; (iii) 5% Pd/C, H2, MeOH,
RT, 15 h.; (iv) KCN, CuI, (Ph3P)4Pd, DMF, 110 °C, 4 h.
a Reagents and conditions: (i) a. NaH, DMF, 0 °C to RT, b. R2X, DMF,
RT; (ii) 1 M NaOH, EtOH, RT, 15 h; (iii) a. PFP-TFA, py, DMA, RT, 2
h, b. ammonia, THF, RT, 2 h; (iv) 1 M HCl, EtOH, RT, 2 h; (v) TFA,
CH2Cl2, RT, 2 h; (vi) OsO4, K3Fe(CN)6, K2CO3, DABCO, tBuOH/H2O,
RT, 15 h.
obtained Via dihydroxylation of the allyl compound 63 with
OsO4 in the presence of K3Fe(CN)6 and 1,4-diazabicyclo[2.2.2]-
octane.23
cellular assay. This drop in activity may be attributed to the
loss of the key hydrogen bond to Ser212 due to partial
tautomerization of the pyridone system, providing further
evidence for the importance of this interaction for inhibition of
the MEK1 and MEK2 protein kinases.
The crystal structures of MEK1 and MEK2 show a significant
area of space adjacent to the 4- and 5-positions of the
diphenylamine A-rings of 2b and 3, which corresponds to the
position occupied by the pyridone series A-ring nitrogen atom.
It was therefore decided to explore the impact of increased
substitution on this nitrogen atom.
Hence, the NH-pyridone ester 40 was alkylated with various
alkyl halides using NaH in DMF to give intermediates 45-52
(Scheme 3). Subsequent hydrolysis and amide coupling to give
final products 53-56 and 63 was performed as described in
Schemes 1 and 2.
In the cases of compounds 57 and 59 the THP and TBDMS
protecting groups were removed using 1 M HCl in EtOH at
room temperature, giving the 4′-hydroxyethyl 58 and 4′-
hydroxypropyl 60 derivatives, respectively. For compound 61,
deprotection of the tert-butyl ester was carried out in TFA/CH2-
Cl2 to give the desired N-acetic acid 62. Compound 64 was
However, even with the presence of a pocket near the
pyridone nitrogen, substitution other than methyl does not appear
to be tolerated (Table 2). Extension of the N-methyl 27 to
N-ethyl 53 or N-propyl 54 resulted in a progressive loss of
activity (IC50s ) 0.023, 1.00, and 2.54 µM, respectively),
although the N-allyl compound 63 was surprisingly potent with
an IC50 of 0.091 µM against the isolated enzyme. Recognizing
that Arg189 lined the potential binding pocket for this position,
the pyridone nitrogen was alkylated with hydroxylated side
chains (Scheme 3) to probe potential hydrogen bonding (direct
or water-bridged) with the Arg residue. However all substitutions
of this type afforded micromolar inhibitors (2.54 to >10 µM),
well below the level of activity possessed by the N-methyl lead
compound 27.
Replacement of the 4′-Iodide. Finally, we investigated the
impact of the (presumably) stronger pyridone H-bond on the
preference for iodine at the 4′-position. The primary amide (R1
) NH2) was set as the best amide side chain and a series of
compounds synthesized with a selection of 4′-substituents
(Scheme 1). An alternative procedure to the desired pyridone
carboxamides 4 involved the quaternization and conversion to
the pyridone being performed first, on ethyl 4,6-dichloronico-

5094 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Table 3. Replacement of the 4′-Iodide
Spicer et al.
the target 4′-saturated alcohol (78). Palladium-catalyzed reaction
of the 4′-iodide 27 with KCN also gave the corresponding 4′-
nitrile (76).
From the results obtained, it was clear that the interaction
which exists between the 4′-iodide and Val127 in both MEK1
and MEK2 crystal structures is critical to the activity of this
series of MEK inhibitors. All compounds were significantly less
potent than the corresponding 4′-iodide 27 (Table 3). For
example, even the closely related bromides (33, 347) had IC50s
against the isolated enzyme in the micromolar range (1.91 µM
for 33 and 3.63 µM for 34) versus the nanomolar range for the
corresponding iodides (0.023 µM and 0.018 µM for 27 and 26,
respectively). These bromides (33, 34) and iodides (27, 26) also
provide an interesting comparison with a series of pyridone-
based inhibitors of MEK prepared and published by workers at
Array BioPharma.7 After completion of this study, we became
aware that both our group and the group at Array Biopharma
had independently24,25 established the utility of the pyridone
template in the preparation of inhibitors of MEK. Array
BioPharma prepared a series of pyridine-2(1H)-ones as MEK
inhibitors, based on a 2′-fluoro-4′-bromo template such as 34,
with variation at either the carboxamide/hydroxamate side chain
or the C5-position of the pyridone ring. From our own work,
however, it appears that the 4′-iodide provides superior potency
compared to the 4′-bromide of the Array BioPharma series. In
the current study, the only sub-micromolar derivatives other than
iodide were the 4′-SMe 72 and 4′-acetylene 74 with IC50s of
0.356 and 0.100 µM, respectively. The acetylene 74 was also a
potent inhibitor in the cellular assay (IC50 ) 0.021 µM).
Although the 4′-hydroxypropyl derivative was designed to
provide an H-bond donor interaction to Val127, it was in fact
inactive, illustrating the overriding preference for nontraditional
donor-acceptor interactions in the context of the highly
hydrophobic binding pocket. The presumed electrostatic interac-
tion of Val127 with the iodine originates from the crystal
structure of 2b,18 and the potent activity of the acetylenic variant
was predicted based on modeling, because of the presence of a
CH-hydrogen bond. Although it was not possible to cocrystallize
a pyridone template with a MEK1 construct, we were able to
obtain a crystal structure of an analogue of 1 with an acetylene
substituent (79), and this is illustrated in Figure 3. The acetylene
cascade
IC50 (µM) IC50 (µM)
cellular
compd
R1
R3
27
32
33
NH2
NH2
NH2
2-F-4-I
0.023
2.97
1.91
3.63
0.004
2-F-4-CH3
2-F-4-Br
-
0.093
0.12
-
-
3.9
0.15
0.021
0.110
-
-
34a HOCH2CH2ONH 2-F-4-Br
35
36
71
72
74
75
76
78
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
2-F-4-CF2CF2CF2CF3 >10
3,4-benzo
3.00
5.72
0.356
0.100
2.62
3,4-dichloro
2-F-4-SCH3
2-F-4-CCH
2-F-4-CH2CH3
2-F-4-CN
>10
2-F-4-CH2CH2CH2OH >10
a Compound 34 has been published previously in ref 7 (as compound
10).
tinate 5, to give the 4-chloropyridone ester 65 (Scheme 4).
Deprotection of the ester 65 was carried out under basic
conditions, followed by reaction of the resultant acid 66 with
substituted anilines (67, 68) in the presence of LiN[Si(CH3)3]2
to give substituted N-methylpyridones 69 and 70 as obtained
in Scheme 1. These acids were then reacted with ammonia to
give the target amides 71 and 72, using benzotriazolyloxytris-
(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) as the
coupling agent. Generally speaking, the overall yield of the
target compounds 4 was superior for Scheme 1 as compared to
Scheme 4.
In the case of amide 27 further elaboration of the iodide was
carried out Via Sonogashira coupling with either TMS-acetylene
or propargyl alcohol to give silyl alkyne or hydroxymethyl
alkyne derivatives (73 and 77, respectively) (Scheme 5).
Deprotection of the trimethylsilyl protecting group of 73 with
K2CO3 in MeOH/THF gave the acetylene (74), followed by
hydrogenation to give the corresponding 4′-ethyl compound (75).
Similarly, hydrogenation of the hydroxymethyl alkyne 77 gave
Figure 3. MEK1 with MgATP and compound 79 bound (PDB Code: 2P55). The acetylene of compound 79 forms a nontraditional hydrogen bond
with the backbone carbonyl oxygen of Val127.

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5095
Table 4. Tumor Levels of pERK after Treatment with Pyridone 27
primary or secondary alkyl amide (27, 30, 31) with little or no
loss of activity. Any substituent other than methyl on the
pyridone nitrogen, however, was detrimental to activity although
the crystal structure indicated a pocket in this area of the MEK1
protein. In the case of 4′-substituents, those compounds (72,
74) which modeling predicted would interact with the protein
in a manner similar to the 4′-iodide of a compound 1-type
template were found to have superior activity.
pyridone 27: % inhibition
of tumor pERK (hours postdose)
dose
6 h
10 h
24 h
200 mpk
100 mpk
50 mpk
95
58
77
91
83
91
0
0
0
Table 5. Antitumor Efficacy of Pyridone 27
Experimental Section
nonspecific
deaths
CR,
%
PR,
%
dosea,b
schedule
T - Ce
c
d
Measurement of ERK Phosphorylation in Vitro. All com-
pounds were first evaluated in a coupled Raf-MEK-ERK cascade
assay, measuring the inhibition of pERK formation as the terminal
endpoint. Each individual IC50 was determined from an 11-point
dose-response curve which was carried out in duplicate. Each IC50
was determined twice using this method.
27
1
50
300
BID, D10-23
TID, D10-23
0/5
0
100
70
-
30
20.9
12.7
a Tumor doubling time was 4.1 days. b Dose is in mg/kg/injection.
c Complete response is defined as a 100% reduction of initial tumor mass.
d Partial response is defined as at least a 50% reduction of initial tumor
mass. e T - C is the difference, in days, for the median treated and control
tumors to reach a fixed evaluation size of 750 mg.
Exponentially growing murine colon 26 carcinoma cells cultured
in the presence of 10% fetal bovine serum were treated with various
concentrations of the test compounds (or vehicle control) for 1 h
at 37 °C. After drug treatment, cells were harvested in a lysis
solution containing 50 mM B-glycerophosphate, 10 mM HEPES,
pH 7.4, 70 mM NaCl, 2 mM EDTA, and 1% SDS. The protein
lysates were diluted 1:15 with supplied assay buffer prior to the
execution of the assay. pERK was measured by ELISA assays
employing phospho-specific antibodies to ERK1 and ERK2 (Assay
Designs Inc, Ann Arbor, MI). Each IC50 was determined from a
7-point dose-response curve which was carried out in duplicate.
Each IC50 was determined four times using this method. The highest
concentration tested for any given compound was 5 µM. The
estimated error is 30% based on historical variability of the control
compound 1.
Measurement of ERK Phosphorylation in Tumors. Ex ViVo
tissue samples were immediately frozen at -80 °C after dissection.
Homogenates were subsequently prepared from frozen tissues by
thawing the samples in lysis buffer (70 mM NaCl, 50 mM glycerol
phosphate, 10 mM HEPES, pH 7.4, 1% Triton X-100, 1 mM Na3-
VO4, 100 µM PMSF, 10 µM leupeptin, and 10 µM pepstatin)
followed by disruption with a Polytron homogenizer. After
centrifugation, supernatants were assayed for protein concentration
and evaluated for pERK levels by standard Western blotting
methodology employing commercially available phospho-specific
antibodies for ERK1 and ERK2.
Measurement of Xenograft Growth Delay. Colon 26 tumor
fragments (approximately 3 mm3 in size) were implanted subcu-
taneously into the right axillae of CD2F1 male mice at 4-6 weeks
old. The tumors were grown to approximately 200 mg over 8 days,
and then the drug (or vehicle) was administered from days 9 to 15.
The vehicle for all compounds was 0.5% hydropropylmethylcel-
lulose and 0.2% Tween 80 in water. All compounds were
administered orally. Tumor size was evaluated periodically by
calliper measurements, generally three times per week. The time
required, in days, for the median-treated and nontreated tumors to
reach a fixed evaluation size of 750 mg was then determined.
Crystallography. The crystal structure of MEK1 in a complex
with MgATP and compound 79 was determined essentially as
previously described.18 The data were collected to a resolution of
2.8 Å on beam-line 17-ID at the Advanced Photon Source in
Argonne, IL.26 The crystallographic data were indexed, scaled and
error reduced using the program HKL200.27 The structure was
determined by Fourier difference methods using the MEK1 structure
1S9J and refined against the crystallographic data using the program
Refmac5 in the CCP4 suite of programs.28,29 The statistics for the
final crystallographic model are 24.0% for the Rwork and 29.5% for
the Rfree value.
forms a nontraditional hydrogen bond between the terminal
carbon of the acetylene and the carbonyl oxygen of Val127 with
a nonbonded distance of 3.07 A.
Activity of the Optimized Pyridone 27 in ViWo. The in ViVo
activity of the best pyridone, amide 27, was evaluated in an ex
ViVo pharmacodynamic assay in colon 26 tumor-bearing mice.
The animals were administered a single dose of drug by oral
gavage over a wide range of doses as summarized in Table 4.
Tumors were excised at the indicated time points and tumor
levels of pERK measured by immunoblotting methodology.
As can be seen in Table 4, treatment with pyridone 27 resulted
in significant inhibition of ERK phosphorylation in tumors
within 6 h of dosing, as reflected by 58% to 95% suppression
over the range of doses tested. Furthermore, this high level of
target suppression was maintained out to 10 h postdosing. By
24 h, control levels of ERK phosphorylation were observed at
all doses of pyridone 27. It is unclear why the degree of target
inhibition did not correlate with dose of drug but may reflect
tumor heterogeneity in this single experiment.
Pyridone 27 was also examined for in ViVo efficacy in animals
bearing C26 tumors treated on a BID dosing schedule. A dose
of 50 mpk administered twice daily proved to be well tolerated
with no lethality or weight loss, whereas the 100 and 200 mpk
BID dosing regimens were not tolerated, as reflected by 40%
and 100% lethality at these two doses, respectively. However,
as summarized in Table 5, pyridone 27 proved to be more
efficacious than 1. Despite being administered at a significantly
lower dose than 1 as well as being administered less frequently,
this compound nonetheless resulted in a complete response rate
of 100% compared to 70% for 1. As is further shown in Table
5, tumor growth delay was also significantly greater for pyridone
27 compared to 1.
Conclusions
Using information derived from both the crystal structures
of MEK1 and MEK218 and molecular modeling, a new series
of inhibitors based on a novel template, the 4-anilino-5-
carboxamido-2-pyridones, was designed and prepared. Replace-
ment of the difluorophenyl group (A-ring) of the diphenylamine
series of inhibitors with a pyridone resulted in analogues (26,
27) which were of similar potency to the corresponding
diphenylamines (37, 38) and more potent than clinical candidate
1. One of these pyridones, 27, was further shown to be more
efficacious than 1 as measured by response rate as well as tumor
growth delay. It was also found possible to replace the
hydroxamate side chain of 1 with a more metabolically stable
Medicinal Chemistry. Analyses were performed by the Micro-
chemical Laboratory, University of Otago, Dunedin, NZ, or by
Quantitative Technologies Inc., Whitehouse, NJ. Melting points
were determined using an Electrothermal Model 9200 or Gallen-
kamp digital melting point apparatus and are as read. NMR spectra
were measured on Bruker AM-400, Bruker Avance 400, or Varian

5096 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Spicer et al.
Unity 400 spectrometers and referenced to Me4Si. Mass spectra
were recorded either on a Varian VG 7070 spectrometer at nominal
5000 resolution or a Finnigan MAT 900Q spectrometer. All final
compound purities were determined to be >95% by HPLC.
Procedure A: Ethyl 6-Chloro-4-(2-fluoro-4-iodoanilino)-
nicotinate (11). Ethyl 4,6-dichloronicotinate 5 (prepared by reaction
of ethyl 4,6-dihydroxynicotinate with POCl3 according to a literature
procedure30) (4.00 g, 18.2 mmol) and 2-fluoro-4-iodoaniline 6 (4.30
g, 18.2 mmol) were dissolved in EtOH (80 mL), to which was added
concd HCl (6 drops). This mixture was heated at 90 °C for 15 h,
and then the solution was cooled, whereupon the desired product
crystallized out of solution as fine needles. The product was isolated
by filtration and washed with 10% Et2O/hexanes to give compound
11 as white needles (3.79 g, 50%): mp (EtOAc/hexanes) 162-
Procedure E: 2,3,4,5,6-Pentafluorophenyl 4-(2-Fluoro-4-
iodoanilino)-1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxy-
late (80). Compound 21 (894 mg, 2.30 mmol) and pyridine (909
mg, 11.5 mmol) were dissolved in DMA (15 mL). To this mixture
was added PFP-TFA (3.22 g, 11.5 mmol), and then the solution
was allowed to stir at RT for 2 h. The DMA solution was diluted
with EtOAc (150 mL) and was washed sequentially with 1 M HCl
(2 × 100 mL), water (100 mL), sat. NaHCO3 (2 × 100 mL), and
brine (100 mL). The EtOAc fraction was then dried (Na2SO4) and
filtered and the solvent removed under reduced pressure to yield a
viscous oil. Purification by flash column chromatography on silica
gel (50% EtOAc/hexanes as eluant) gave compound 80 as a cream
foam (1.22 g, 96%) which was used directly in subsequent steps.
1H NMR [400 MHz, (CD3)2SO] δ 9.03 (s, 1 H), 8.70 (s, 1 H), 7.79
(dd, J ) 10.1, 1.9 Hz, 1 H), 7.62 (br dd, J ) 8.4, 1.0 Hz, 1 H),
7.29 (t, J ) 8.4 Hz, 1 H), 5.36 (d, J ) 1.6 Hz, 1 H), 3.45 (s, 3 H).
LCMS (APCI+) calcd for C19H9F6N2O3 555 (MH+), found 555.
Procedure F: 4-(2-Fluoro-4-iodoanilino)-N-(2-hydroxyethyl)-
1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxamide (30). Com-
pound 80 (600 mg, 1.08 mmol) was dissolved in THF (15 mL) to
which was added DIEA (697 mg, 5.40 mmol), followed by
2-aminoethanol (132 mg, 2.17 mmol). This mixture was stirred at
RT for 2 h, then the solvent was removed under reduced pressure
and the resulting white solid suspended in Et2O. This solid was
collected by filtration and recrystallized from EtOAc/MeOH to give
compound 30 (394 mg, 85%) as a white solid: mp (EtOAc/MeOH)
1
164 °C. H NMR [400 MHz, (CD3)2SO] δ 9.62 (s, 1 H), 8.69 (s,
1 H), 7.82 (dd, J ) 9.9, 1.9 Hz, 1 H), 7.61-7.66 (m, 1 H), 7.33 (t,
J ) 8.5 Hz, 1 H), 6.67 (d, J ) 1.8 Hz), 4.37 (q, J ) 7.1 Hz, 2 H),
1.35 (t, J ) 7.1 Hz, 3 H). LCMS (APCI+) calcd for C14H12ClFIN2O2
421 (MH+), found 421. Anal. (C14H11ClFIN2O2) C, H, N.
Procedure B: Ethyl 4-(2-Fluoro-4-iodoanilino)-1-methyl-6-
oxo-1,6-dihydro-3-pyridinecarboxylate (16). Compound 11 (200
mg, 0.48 mmol) was dissolved in CHCl3 (5 mL) and the solution
cooled (ice/water). Dimethyl sulfate (0.27 mL, 2.86 mmol) was
added and the solution allowed to warm to RT and then heated at
reflux for 20 h. Upon cooling, a mixture of triethylamine (1.41
mL), acetic acid (0.94 mL), and EtOH (0.94 mL) was added and
the reaction heated at reflux for a further 2 h. After cooling, water
(10 mL) was added and the mixture was partitioned between EtOAc
(100 mL) and water (50 mL). The EtOAc layer was washed with
further water (50 mL) and brine (50 mL) and then dried (Na2SO4),
filtered and the solvent removed under reduced pressure. Purifica-
tion by flash column chromatography on silica gel (50% EtOAc/
hexanes as eluant) gave compound 16 as a white solid (143 mg,
1
205-208 °C. H NMR [400 MHz, (CD3)2SO] δ 10.10 (s, 1 H),
8.43 (br s, 1 H), 8.28 (s, 1 H), 7.72 (dd, J ) 10.2, 1.8 Hz, 1 H),
7.56 (br d, J ) 8.5 Hz, 1 H), 7.28 (t, J ) 8.5 Hz, 1 H), 5.58 (s, 1
H), 4.76 (br t, J ) 5.2 Hz, 1 H), 3.50 (q, J ) 5.8 Hz, 2 H), 3.36
(s, 3 H), 3.32-3.26 (m, 2 H). LCMS (APCI+) calcd for C15H16-
FIN3O3 432 (MH+), found 432. Anal. (C15H15FIN3O3) C, H, N.
4-(2-Fluoro-4-iodoanilino)-1-methyl-6-oxo-1,6-dihydro-3-py-
ridinecarboxamide (27). Compound 80 was reacted with concd
NH3 solution in THF according to procedure F. Recrystallization
from EtOAc/MeOH afforded compound 27 as white crystals
1
72%), mp (EtOAc/n-hexane) 169-170 °C. H NMR [400 MHz,
(CD3)2SO] δ 9.31 (s, 1 H), 8.54 (s, 1 H), 7.77 (dd, J ) 10.1, 1.9
Hz, 1 H), 7.60 (br dd, J ) 8.3, 1.0 Hz, 1 H), 7.30 (t, J ) 8.5 Hz,
1 H), 5.46 (s, 1 H), 4.30 (q, J ) 7.1 Hz, 2 H), 3.43 (s, 3 H), 1.32
(t, J ) 7.1 Hz, 3 H). LCMS (APCI+) calcd for C15H15FIN2O3 417
(MH+), found 417. Anal. (C15H14FIN2O3) C, H, N.
1
(84%): mp (EtOAc/MeOH) 283-285 °C. H NMR [400 MHz,
(CD3)2SO] δ 10.40 (s, 1 H), 8.34 (s, 1 H), 7.88 (br s, 1 H), 7.74
(dd, J ) 9.9, 1.9 Hz, 1 H), 7.57 (br d, J ) 7.8 Hz, 1 H), 7.46 (br
s, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H), 5.56 (d, J ) 0.7 Hz, 1 H), 3.36
(s, 3 H). LCMS (APCI+) calcd for C13H12FIN3O2 388 (MH+), found
388. Anal. (C13H11FIN3O2) C, H, N.
Procedure C: 4-(2-Fluoro-4-iodoanilino)-1-methyl-6-oxo-1,6-
dihydro-3-pyridinecarboxylic Acid (21). Compound 16 (140 mg,
0.34 mmol) was suspended in EtOH (10 mL), to which was added
1 M NaOH (10 mL). This mixture was stirred at RT for 15 h and
then diluted with 1 M HCl (50 mL) and the resulting precipitate
extracted into EtOAc (2 × 50 mL). The combined EtOAc fractions
were dried (Na2SO4) and filtered, and the solvent was removed
under reduced pressure to afford compound 21 as a white solid
(132 mg, 100%), mp (acetone/MeOH) 254-257 °C. 1H NMR [400
MHz, (CD3)2SO] δ 13.30 (v br s, 1 H), 9.66 (s, 1 H), 8.52 (s, 1 H),
7.76 (dd, J ) 10.1, 1.9 Hz, 1 H), 7.59 (ddd, J ) 8.4, 1.7, 0.8 Hz,
1 H), 7.31 (t, J ) 8.5 Hz, 1 H), 5.49 (d, J ) 0.7 Hz, 1 H), 3.41 (s,
3 H). LCMS (APCI-) calcd for C13H9FIN2O3 387 (M - H), found
387. Anal. (C13H10FIN2O3) C, H, N.
Procedure D: 4-(2-Fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-
1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxamide (26). To a
mixture of compound 21 (130 mg, 0.34 mmol) and 2-(aminooxy)-
ethanol (52 mg, 0.67 mmol) in MeOH/THF (1:1, 20 mL) was added
DMT-MM (187 mg, 0.67 mmol) and the reaction stirred at RT
for 15 h. All solvent was removed under reduced pressure and the
oily residue partitioned between water (100 mL) and EtOAc (100
mL). The EtOAc fraction was then washed with water (2 × 100
mL) and brine (100 mL), dried (Na2SO4), and filtered and the
solvent removed under reduced pressure. Purification by recrys-
tallization from EtOAc/MeOH gave compound 26 as a white,
crystalline solid (83 mg, 55%): mp (EtOAc/MeOH) 148-151 °C.
1H NMR [400 MHz, (CD3)2SO] δ 11.65 (v br s, 1 H), 9.48 (br s,
1 H), 8.13 (s, 1 H), 7.74 (dd, J ) 10.2, 1.8 Hz, 1 H), 7.58 (br d,
J ) 8.6 Hz, 1 H), 7.28 (t, J ) 8.5 Hz, 1 H), 5.55 (s, 1 H), 4.76 (v
br s, 1 H), 3.90 (t, J ) 4.9 Hz, 2 H), 3.61 (t, J ) 4.9 Hz, 2 H), 3.36
(s, 3 H). LCMS (APCI+) calcd for C15H16FIN3O4 448 (MH+), found
448. Anal. (C15H15FIN3O4) C, H, N.
4-(2-Fluoro-4-iodoanilino)-N,1-dimethyl-6-oxo-1,6-dihydro-3-
pyridinecarboxamide (28). Compound 80 was reacted with
methylamine (40% aq solution) in THF according to procedure F.
Trituration with hexane afforded compound 28 as a white solid
(73%); mp 252-254 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.10
(br s, 1 H), 8.41 (br d, J ) 4.4 Hz, 1 H), 8.22 (s, 1 H), 7.72 (dd,
J ) 10.2, 1.9 Hz, 1 H), 7.57 (dt, J ) 8.4, 0.9 Hz, 1 H), 7.28 (t, J
) 8.4 Hz, 1 H), 5.58 (d, J ) 0.9 Hz, 1 H), 3.36 (s, 3 H), 2.75 (d,
J ) 4.4 Hz, 3 H). HRMS (FAB+) calcd for C14H14FIN3O2 402.0115
(MH+), found 402.0119.
4-(2-Fluoro-4-iodoanilino)-N,N,1-trimethyl-6-oxo-1,6-dihydro-
3-pyridinecarboxamide (29). Compound 80 was reacted with
dimethylamine (40% aq solution) in THF according to procedure
F. Trituration with hexane gave compound 29 as a pale yellow
1
solid (58%); mp 106-111 °C. H NMR [400 MHz, (CD3)2SO] δ
8.21 (s, 1 H), 7.79 (s, 1 H), 7.72 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.56
(dt, J ) 8.4, 1.2 Hz, 1 H), 7.16 (t, J ) 8.4 Hz, 1 H), 5.41 (d, J )
1.2 Hz, 1 H), 3.34 (s, 3 H), 2.98 (s, 6 H). HRMS (FAB+) calcd for
C15H16FIN3O2 416.0271 (MH+), found 416.0270.
4-(2-Fluoro-4-iodoanilino)-N-(3-hydroxypropyl)-1-methyl-6-
oxo-1,6-dihydro-3-pyridinecarboxamide (31). Compound 80 was
reacted with 3-aminopropanol in THF in the presence of DIEA
according to procedure F. Purification by flash chromatography on
silica gel (50% acetone/CH2Cl2) gave compound 31 as a white solid
1
(95%), mp (Et2O) 81-86 °C. H NMR [400 MHz, (CD3)2SO] δ
10.11 (s, 1 H), 8.40 (br s, 1 H), 8.24 (s, 1 H), 7.74 (d, J ) 9.7 Hz,
1 H), 7.58 (d, J ) 8.2 Hz, 1 H), 7.29 (t, J ) 8.5 Hz, 1 H), 5.59 (s,
1 H), 4.48 (t, J ) 5.1 Hz, 1 H), 3.48 (q, J ) 5.7 Hz, 2 H), 3.37 (s,
3 H), 3.30-3.24 (m, 2 H), 1.67 (pentet, J ) 6.7 Hz, 2 H). LCMS

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5097
(APCI+) calcd for C16H18FIN3O3 446 (MH+), found 446. Anal.
(C16H17FIN3O3·0.5H2O) C, H, N.
284 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.39 (s, 1 H), 8.35 (s,
1 H), 7.89 (br s, 1 H), 7.66 (dd, J ) 10.4, 1.7 Hz, 1 H), 7.51-7.44
(br m, 2 H), 7.42 (dd, J ) 8.5, 1.9 Hz, 1 H), 5.55 (s, 1 H), 3.38 (s,
3 H). LCMS (ACPI-) calcd for C13H10BrFN3O2 338, 340 (M -
H), found 338, 340. Anal. (C13H11BrFN3O2) C, H, N.
4-(4-Bromo-2-fluoroanilino)-N-(2-hydroxyethoxy)-1-methyl-
6-oxo-1,6-dihydro-3-pyridinecarboxamide (34). Compound 23
and 2-(aminooxy)ethanol were reacted the presence of DMT-MM
in MeOH according to procedure D. Purification by flash column
chromatography on silica gel (EtOAc followed by 10% MeOH/
CH2Cl2) gave compound 34 (38%); mp (EtOAc/hexane) 124-
128 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.64 (br s, 1 H), 9.50
(br s, 1 H), 8.13 (s, 1 H), 7.69-7.65 (m, 1 H), 7.47-7.41 (m, 2
H), 5.53 (d, J ) 0.7 Hz, 1 H), 4.78 (s, 1 H), 3.91 (t, J ) 5.0 Hz,
2 H), 3.61 (t, J ) 5.0 Hz, 2 H), 3.36 (s, 3 H). LCMS (ACPI-)
calcd for C15H14BrFN3O4 398, 400 (M - H), found 398, 400. Anal.
(C15H15BrFN3O4·0.25 H2O) C, H, N.
2-Fluoro-4-(1,1,2,2,3,3,4,4,4-nonafluorobutyl)aniline (9). A
dispersion of 1,1,1,2,2,3,3,4,4-nonafluoro-4-iodobutane (3.50 g, 10.1
mmol), 2-fluoro-4-iodoaniline 6 (2.00 g, 8.4 mmol), and copper
bronze (1.93 g, 30.4 mmol) in DMSO (10 mL) was stirred at 120 °C
for 15 h. Copper(I) iodide was removed by filtration through celite
which was washed with Et2O (100 mL). Water (100 mL) was then
added to the filtrate, and the mixture was stirred at RT for 5 min.
The organic layer was separated, washed with water (5 × 100 mL)
to remove DMSO, dried (Na2SO4), and concentrated. Flash column
chromatography on silica gel (10% EtOAc/hexane) afforded
compound 9 (1.69 g, 61%). 1H NMR (400 MHz, CDCl3) δ 7.25-
7.14 (m, 2 H), 6.86-6.74 (m, 1 H), 4.07 (br s, 1 H). LCMS (ACPI-)
calcd for C10H4NF10 328 (M - H), found 328.
Ethyl 6-Chloro-4-(2-fluoro-4-methylanilino)nicotinate (12).
2-Fluoro-4-methylaniline 7 and 5 were reacted according to
procedure A. Trituration with 10% Et2O/hexane gave compound
1
12 (44%); mp (EtOH/water) 107-109 °C. H NMR (400 MHz,
CDCl3) δ 9.63 (br s, 1 H), 8.77 (s, 1 H), 7.20 (t, J ) 8.2 Hz, 1 H),
7.05-6.99 (m, 2 H), 6.65 (d, J ) 1.6 Hz, 1 H), 4.41 (q, J ) 7.1
Hz, 2 H), 1.43 (t, J ) 7.1 Hz, 3 H). LRMS (FAB+) calcd for
C15H15N2O2FCl, 309 (MH+), found 309.
Ethyl 4-(2-Fluoro-4-methylanilino)-1-methyl-6-oxo-1,6-dihy-
dro-3-pyridinecarboxylate (17). Compound 12 was reacted ac-
cording to procedure B. Purification by recrystallization (EtOAc/
hexane) afforded compound 17 (84%); mp (EtOAc/hexane) 148-
150 °C. 1H NMR (400 MHz, CDCl3) δ 9.17 (br s, 1 H), 8.20 (s, 1
H), 7.27-7.21 (m, 1 H), 6.99-6.92 (m, 2 H), 5.76 (s, 1 H), 4.35
(q, J ) 7.1 Hz, 2 H), 3.53 (s, 3 H), 2.35 (s, 3 H), 1.39 (t, J ) 7.1
Hz, 3 H). LCMS (ACPI-) calcd for C16H16N2O3 303 (M - H),
found 303. Anal. (C16H17FN2O3) C, H, N.
4-(2-Fluoro-4-methylanilino)-1-methyl-6-oxo-1,6-dihydro-3-
pyridinecarboxylic Acid (22). Hydrolysis of compound 17 was
carried out according to procedure C. Compound 22 was isolated
as a white solid (91%) and used directly in the next step. 1H NMR
[400 MHz, (CD3)2SO] δ 13.20 (v br s, 1 H), 9.47 (br s, 1 H), 8.49
(s, 1 H), 7.34 (t, J ) 8.3 Hz, 1 H), 7.18 (d, J ) 11.7 Hz, 1 H), 7.07
(d, J ) 7.8 Hz, 1 H), 5.33 (d, J ) 1.1 Hz, 1 H), 3.39 (s, 3 H), 2.33
(s, 3 H).
4-(2-Fluoro-4-methylanilino)-1-methyl-6-oxo-1,6-dihydro-3-
pyridinecarboxamide (32). Compound 22 was reacted according
to procedure E. Purification by flash chromatography on silica gel
(60% EtOAc/hexane) gave the intermediate pentafluorophenyl ester
(90%) which was reacted directly with concd NH3 in THF according
to procedure F. Recrystallization from EtOAc/hexane gave com-
pound 32 (84%); mp (EtOAc/hexane) 285-288 °C. 1H NMR [400
MHz, (CD3)2SO] δ 10.14 (s, 1 H), 8.32 (s, 1 H), 7.86 (br s, 1 H),
7.43 (br s, 1 H), 7.31 (t, J ) 8.4 Hz, 1 H), 7.16 (dd, J ) 11.8, 1.1
Hz, 1 H), 7.05 (d, J ) 8.2 Hz, 1 H), 5.38 (d, J ) 1.1 Hz, 1 H),
3.34 (s, 3 H), 2.32 (s, 3 H). LCMS (ACPI-) calcd for C14H13FN3O2
274 (M - H), found 274. Anal. (C14H14FN3O2) C, H, N.
Ethyl 4-(4-Bromo-2-fluoroanilino)-6-chloronicotinate (13).
4-Bromo-2-fluoroaniline 8 and 5 were reacted according to
procedure A. The product was isolated by filtration and washed
with 10% Et2O/hexane, affording compound 13 (58%); mp (EtOH/
Ethyl 6-Chloro-4-[2-fluoro-4-(1,1,2,2,3,3,4,4,4-nonafluorobu-
tyl)anilino]nicotinate (14). Compounds 9 and 5 were reacted
according to procedure A. Purification by flash column chroma-
tography on silica gel (10% EtOAc/hexane as eluant) gave
1
compound 14 (30%) which was used directly in the next step. H
NMR (400 MHz, CDCl3) δ 0.11 (br s, 1 H), 8.86 (s, 1 H), 7.57 (t,
J ) 8.0 Hz, 1 H), 7.46 (d, J ) 9.7 Hz, 2 H), 6.98 (d, J ) 0.7 Hz,
1 H), 4.44 (q, J ) 7.1 Hz, 2 H), 1.44 (t, J ) 7.1 Hz, 3 H).
Ethyl 4-[2-Fluoro-4-(1,1,2,2,3,3,4,4,4-nonafluorobutyl)anilino]-
1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxylate (19). Com-
pound 14 was reacted according to procedure B. Recrystallization
from EtOAc/hexane gave compound 19 (47%). 1H NMR [400 MHz,
(CD3)2SO] δ 9.50 (s, 1 H), 8.35 (s, 1 H), 7.58 (t, J ) 8.3 Hz, 1 H),
7.52 (dd, J ) 10.9, 1.8 Hz, 1 H), 7.34 (d, J ) 8.1 Hz, 1 H), 5.59
(s, 1 H), 4.08 (q, J ) 7.1 Hz, 2 H), 3.22 (s, 3 H), 1.10 (t, J ) 7.1
Hz, 3 H).
1
water) 150-152 °C. H NMR (400 MHz, CDCl3) δ 9.74 (br s, 1
H), 8.81 (s, 1 H), 7.41 (dd, J ) 9.6, 2.2 Hz, 1 H), 7.38-7.35 (m,
1 H), 7.25 (t, J ) 8.3 Hz, 1 H), 6.71 (d, J ) 1.5 Hz, 1 H), 4.42 (q,
J ) 7.1 Hz, 2 H), 1.43 (t, J ) 7.1 Hz, 3 H). LRMS (FAB+) calcd
for C14H12N2O2FBrCl 375 (MH+), found 375.
4-[2-Fluoro-4-(1,1,2,2,3,3,4,4,4-nonafluorobutyl)anilino]-1-
methyl-6-oxo-1,6 -dihydro-3-pyridinecarboxylic Acid (24). Hy-
drolysis of compound 19 was carried out according to procedure
C. Compound 24 was isolated as a white solid (75%) and used
Ethyl 4-(4-Bromo-2-fluoroanilino)-1-methyl-6-oxo-1,6-dihy-
dro-3-pyridinecarboxylate (18). Compound 13 was reacted ac-
cording to procedure B. Recrystallization from EtOAc/hexane gave
1
directly in the next step. H NMR [400 MHz, (CD3)2SO] δ 13.20
1
compound 18 (77%); mp (EtOAc/hexane) 159-162 °C. H NMR
(v br s, 1 H), 10.21 (br s, 1 H), 8.56 (s, 1 H), 7.83 (t, J ) 8.3 Hz,
1 H), 7.74 (d, J ) 10.4 Hz, 1 H), 7.57 (d, J ) 8.2 Hz, 1 H), 5.86
(s, 1 H), 3.44 (s, 3 H).
(400 MHz, CDCl3) δ 9.34 (br s, 1 H), 8.22 (s, 1 H), 7.36-7.25
(m, 3 H), 5.85 (s, 1 H), 4.35 (q, J ) 7.1 Hz, 2 H), 3.54 (s, 3 H),
1.39 (t, J ) 7.1 Hz, 3 H). LCMS (ACPI-) calcd for C15H13BrFN2O3,
367, 369 (M - H), found 367, 369. Anal. (C15H14BrFN2O3) C, H,
N.
4-[2-Fluoro-4-(1,1,2,2,3,3,4,4,4-nonafluorobutyl)anilino]-1-
methyl-6-oxo-1,6 -dihydro-3-pyridinecarboxamide (35). Com-
pound 24 was reacted according to procedure E, then the interme-
diate pentafluorophenyl ester reacted directly with c. NH3 solution
in THF according to procedure F. Purification by recrystallization
(EtOAc/hexane) gave compound 35 (65%); mp (EtOAc/hexane)
4-(4-Bromo-2-fluoroanilino)-1-methyl-6-oxo-1,6-dihydro-3-
pyridinecarboxylic Acid (23). Hydrolysis of compound 18 was
carried out according to procedure C. Compound 23 was isolated
as a white solid (89%) and used directly in the next step. 1H NMR
[400 MHz, (CD3)2SO] δ 13.30 (v br s, 1 H), 9.65 (br s, 1 H), 8.52
(s, 1 H), 7.69 (dd, J ) 10.3, 1.6 Hz, 1 H), 7.51-7.43 (m, 2 H),
5.47 (d, J ) 1.2 Hz, 1 H), 3.41 (s, 3 H).
4-(4-Bromo-2-fluoroanilino)-1-methyl-6-oxo-1,6-dihydro-3-
pyridinecarboxamide (33). Compound 23 was reacted according
to procedure E. Purification by flash column chromatography on
silica gel (60% EtOAc/hexane) gave the intermediate pentafluo-
rophenyl ester (97%) which was reacted directly with concd NH3
in THF according to procedure F. Recrystallization from EtOAc/
hexane gave compound 33 (100%); mp (EtOAc/hexane) 282-
1
112-118 °C. H NMR [400 MHz, (CD3)2SO] δ 10.92 (s, 1 H),
8.40 (s, 1 H), 7.93 (br s, 1 H), 7.79 (t, J ) 8.3 Hz, 1 H), 7.70 (dd,
J ) 11.2, 1.7 Hz, 1 H), 7.53 (d, J ) 7.8 Hz, 2 H), 5.92 (s, 1 H),
3.40 (s, 3 H). HRMS (FAB+) calcd for C17H12F10N3O2 480.0770
(M+), found 480.0762.
Ethyl 6-Chloro-4-(2-naphthylamino)nicotinate (15). Com-
pounds 10 and 5 were reacted according to procedure A. Trituration
with 10% Et2O/hexane yielded compound 15 (68%) which was used
1
directly in the next step. H NMR [400 MHz, (CD3)2SO] δ 9.91
(br s, 1 H), 8.71 (s, 1 H), 8.02 (d, J ) 8.7 Hz, 1 H), 7.97-7.88 (m,

5098 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Spicer et al.
3 H), 7.59-7.48 (m, 3 H), 6.91 (s, 1 H), 4.39 (q, J ) 7.1 Hz, 2 H),
1.37 (t, J ) 7.1 Hz, 3 H).
q, J ) 5.7 Hz, 2 H). LCMS (APCI+) calcd for C14H14FIN3O3 418
(MH+), found 418. Anal. (C14H13FIN3O3‚0.5H2O) C, H, N.
Ethyl 1-Methyl-4-(2-naphthylamino)-6-oxo-1,6-dihydro-3-py-
ridinecarboxylate (20). Compound 20 was prepared from com-
pound 15 according to procedure B. Purification by recrystallization
(EtOAc/hexane) gave compound 20 as a white solid (57%); mp
4-(2-Fluoro-4-iodoanilino)-N-(3-hydroxypropyl)-6-oxo-1,6-di-
hydro-3-pyridinecarboxamide (44). Compound 41 was reacted
according to procedure E, and then the intermediate pentafluo-
rophenyl ester reacted directly with 3-aminoethanol according to
procedure F. Purification by flash chromatography on silica gel
(10% MeOH/CH2Cl2) gave compound 44 as a crystalline white solid
(89%), mp (EtOAc) 253-255 °C. 1H NMR [400 MHz, (CD3)2SO]
δ 11.39 (br s, 1 H), 10.26 (br s, 1 H), 8.47 (br s, 1 H), 7.92 (s, 1
H), 7.74 (dd, J ) 10.2, 1.7 Hz, 1 H), 7.57 (dd, J ) 8.5, 0.8 Hz, 1
H), 7.28 (t, J ) 8.5 Hz, 1 H), 5.49 (s, 1 H), 4.46 (br s, 1 H), 3.48-
3.41 (m, 2 H), 3.24 (q, J ) 6.4 Hz, 2 H), 1.64 (pentet, J ) 6.7 Hz,
2 H). LCMS (APCI+) calcd for C15H16FIN3O3 432 (MH+), found
432. Anal. (C15H15FIN3O3) C, H, N.
1
(EtOAc/hexane) 160-163 °C. H NMR [400 MHz, (CD3)2SO] δ
9.55 (s, 1 H), 8.57 (s, 1 H), 7.97 (d, J ) 8.8 Hz, 1 H), 7.91 (dd, J
) 7.7, 4.7 Hz, 2 H), 7.81 (d, J ) 1.8 Hz, 1 H), 7.55-7.43 (m, 3
H), 5.79 (s, 1 H), 4.32 (q, J ) 7.1 Hz, 2 H), 3.43 (s, 3 H), 1.35 (t,
J ) 7.1 Hz, 3 H). Anal. (C19H18N2O3) C, H, N.
1-Methyl-4-(2-naphthylamino)-6-oxo-1,6-dihydro-3-pyridin-
ecarboxylic Acid (25). Hydrolysis of compound 20 was carried
out according to procedure C. Compound 25 (88%) was isolated
1
as a white solid and used directly in the next step. H NMR [400
MHz, (CD3)2SO] δ 13.27 (v br s, 1 H), 9.95 (br s, 1 H), 8.53 (s, 1
H), 7.96 (d, J ) 8.8 Hz, 1 H), 7.91 (d, J ) 8.8 Hz, 2 H), 7.80 (d,
J ) 1.9 Hz, 1 H), 7.54-7.42 (m, 3 H), 5.81 (s, 1 H), 3.42 (s, 3 H).
1-Methyl-4-(2-naphthylamino)-6-oxo-1,6-dihydro-3-pyridin-
ecarboxamide (36). Compound 25 was reacted according to
procedure E. Purification by flash column chromatography on silica
gel (60% EtOAc/hexane) gave the intermediate pentafluorophenyl
ester (97%), used directly in the next step by reaction with c. NH3
in THF according to procedure F. Recrystallization (MeOH/EtOAc)
gave compound 36 as a white solid (87%); mp (MeOH/EtOAc)
Procedure G: Ethyl 4-(2-Fluoro-4-iodoanilino)-6-oxo-1-[2-
(tetrahydro-2H-pyran-2-yloxy)ethyl]-1,6-dihydro-3-pyridinecar-
boxylate (45). 2-Iodoethanol was protected as the tetrahydropyranyl
ether according to a literature procedure.31 Compound 40 (383 mg,
0.95 mmol) was dissolved/suspended in dry DMF (15 mL), and
the solution cooled (ice/water). NaH (42 mg, 1.05 mmol) was added,
the flask placed under nitrogen, and the resulting mixture allowed
to warm and then stir at RT for 2 h. A solution of the above-
protected iodide (1.22 g, 4.77 mmol) in dry DMF (5 mL) was then
added as a single portion and the entire mixture stirred at RT for
15 h. Water (100 mL) was added and the resulting aqueous
suspension extracted with EtOAc (3 × 50 mL). The combined
EtOAc fractions were then washed with water (2 × 50 mL) and
brine (50 mL), dried (Na2SO4), and filtered. The solvent was
removed under reduced pressure to afford an oil which was purified
by flash column chromatography on silica gel (50% EtOAc/hexanes
as eluant) to give compound 45 as a transparent oil (249 mg, 49%)
1
254-257 °C. H NMR [400 MHz, (CD3)2SO] δ 10.56 (s, 1 H),
8.35 (s, 1 H), 7.95-7.86 (m, 4 H), 7.74 (d, J ) 1.9 Hz, 1 H),
7.53-7.42 (m, 3 H), 7.39 (dd, J ) 8.7, 2.1 Hz, 1 H), 5.86 (s, 1 H),
3.37 (s, 3 H). LCMS (APCI-) calcd for C17H14N3O2 292 (M - H),
found 292. Anal. (C17H15N3O2) C, H, N.
Ethyl 4-(2-Fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-pyridi-
necarboxylate (40). Compound 11 (2.03 g, 4.83 mmol) was
dissolved in acetic acid (75 mL), to which was added water (25
mL). This solution was heated at reflux for 144 h. The mixture
was cooled, and a cream solid crystallized out. This material was
isolated by filtration, washed well with water and hexanes, and
then dried to afford compound 40 as a white solid (1.14 g, 59%),
1
which was used directly in the next step. H NMR [400 MHz,
(CD3)2SO] δ 9.31 (s, 1 H), 8.49 (s, 1 H), 7.78 (dd, J ) 10.2, 1.9
Hz, 1 H), 7.61 (br d, J ) 8.6 Hz, 1 H), 7.31 (t, J ) 7.5 Hz, 1 H),
5.45 (s, 1 H), 4.59 (br s, 1 H), 4.30 (q, J ) 7.0 Hz, 2 H), 4.11 (t,
J ) 5.0 Hz, 2 H), 3.75 (pentet, J ) 5.5 Hz, 1 H), 3.61-3.49 (m,
2 H), 3.40-3.32 (m, 1 H), 1.74-1.33 (m, 6 H), 1.30 (t, J ) 7.1
Hz, 3 H). HRMS (EI+) calcd C21H24FIN2O5 (M+) 530.0714, found
530.0704.
1
mp (acetone/MeOH) 262-264 °C. H NMR [400 MHz, (CD3)2-
SO] δ 11.59 (br s, 1 H), 9.32 (s, 1 H), 8.10 (s, 1 H), 7.77 (dd, J )
10.1, 1.9 Hz, 1 H), 7.61 (ddd, J ) 8.4, 2.0, 0.8 Hz, 1 H), 5.38 (d,
J ) 1.3 Hz, 1 H), 4.27 (q, J ) 7.1 Hz, 2 H), 1.30 (t, J ) 7.1 Hz,
3 H). LCMS (APCI+) calcd for C14H13FIN2O3 403 (MH+), found
403. Anal. (C14H12FIN2O3) C, H, N.
4-(2-Fluoro-4-iodoanilino)-6-oxo-1-[2-(tetrahydro-2H-pyran-
2-yloxy)ethyl]-1,6-dihydro-3-pyridinecarboxamide (57). Hydrol-
ysis of compound 45 was carried out according to procedure C,
giving the intermediate acid (88%) which was reacted directly
according to procedure E. This intermediate pentafluorophenyl ester
was then reacted directly with concd NH3 solution in THF according
to procedure F. Purification by flash chromatography on silica gel
(5% MeOH/CH2Cl2 as eluant) gave compound 57 as an oily cream
4-(2-Fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-pyridinecar-
boxylic Acid (41). Hydrolysis of compound 40 was carried out
according to procedure C, giving compound 41 as a white solid
(99%), used directly in the next step. 1H NMR [400 MHz, (CD3)2-
SO] δ 13.30 (v br s, 1 H), 11.44 (br s, 1 H), 9.75 (br s, 1 H), 8.06
(s, 1 H), 7.76 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.59 (ddd, J ) 8.3, 1.9,
0.9 Hz, 1 H), 7.32 (t, J ) 8.4 Hz, 1 H), 5.40 (d, J ) 1.1 Hz, 1 H).
LCMS (APCI+) calcd for C12H9FIN2O3 375 (MH+), found 375.
4-(2-Fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-pyridinecar-
boxamide (42). Compound 41 was reacted according to procedure
E, and then the intermediate pentafluorophenyl ester reacted directly
with c. NH3 in THF according to procedure F. Recrystallization
from EtOAc/MeOH gave compound 42 as cream needles (87%),
1
solid (76%), used directly in the next step. H NMR [400 MHz,
(CD3)2SO] δ 10.49 (s, 1 H), 8.34 (s, 1 H), 7.93 (br s, 1 H), 7.74
(dd, J ) 10.2, 1.8 Hz, 1 H), 7.57 (dd, J ) 8.7, 1.0 Hz, 1 H), 7.48
(br s, 1 H), 7.30 (t, J ) 8.6 Hz, 1 H), 5.55 (s, 1 H), 4.58-4.52 (m,
1 H), 4.04-3.91 (m, 2 H), 3.82-3.75 (m, 1 H), 3.63-3.35 (m, 3
H), 1.73-1.26 (m, 6 H). HRMS (EI+) calcd C19H21FIN3O4 (M+)
501.0561, found 501.0564.
1
Procedure H: 4-(2-Fluoro-4-iodoanilino)-1-(2-hydroxyethyl)-
6-oxo-1,6-dihydro-3-pyridinecarboxamide (58). Compound 57
(118 mg, 0.24 mmol) was dissolved in EtOH (8 mL), to which
was added 1 M HCl (2 mL). This mixture was stirred at RT for 2
h and then diluted with water (80 mL). The resulting solution was
extracted with EtOAc (3 × 40 mL), and then the combined EtOAc
fractions were washed with water (2 × 50 mL) and brine (50 mL)
and dried (Na2SO4). Filtration and removal of the solvent under
reduced pressure afforded compound 58 as a white solid (95 mg,
97%), mp (EtOAc/MeOH) 212-215 °C. 1H NMR [400 MHz,
(CD3)2SO] δ 10.45 (s, 1 H), 8.27 (s, 1 H), 7.94 (br s, 1 H), 7.74
(dd, J ) 10.1, 1.7 Hz, 1 H), 7.57 (br d, J ) 8.6 Hz, 1 H), 7.48 (br
s, 1 H), 7.29 (t, J ) 8.5 Hz, 1 H), 5.55 (s, 1 H), 4.93 (t, J ) 5.3
Hz, 1 H), 3.85 (t, J ) 5.5 Hz, 2 H), 3.60 (q, J ) 5.4 Hz, 2 H).
Anal. (C14H13FIN3O3) C, H, N.
mp (CH2Cl2/MeOH) 320-325 °C. H NMR [400 MHz, (CD3)2-
SO] δ 11.42 (s, 1 H), 10.53 (s, 1 H), 8.01 (s, 1 H), 7.96 (br s, 1 H),
7.73 (dd, J ) 10.1 Hz, 1 H), 7.57 (br d, J ) 8.4 Hz, 1 H), 7.41 (br
s, 1 H), 7.29 (t, J ) 8.5 Hz, 1 H), 5.47 (s, 1 H). Anal. (C12H9-
FIN3O2) C, H, N.
4-(2-Fluoro-4-iodoanilino)-N-(2-hydroxyethyl)-6-oxo-1,6-di-
hydro-3-pyridinecarboxamide (43). Compound 41 was reacted
according to procedure E, and then the intermediate pentafluo-
rophenyl ester reacted directly with 2-aminoethanol according to
procedure F. Purification by recrystallization from acetone/MeOH
gave compound 43 as a white solid (74%), mp (acetone/MeOH)
254-256 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.38 (br s, 1 H),
10.24 (br s, 1 H), 8.51 (br s, 1 H), 7.96 (s, 1 H), 7.73 (dd, J )
10.1, 1.8 Hz, 1 H), 7.57 (dd, J ) 8.4, 0.9 Hz, 1 H), 7.28 (t, J ) 8.5
Hz, 1 H), 5.48 (s, 1 H), 4.75 (br s, 1 H), 3.48 (br s, 2 H), 3.26 (br

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5099
Ethyl 1-Ethyl-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-
3-pyridinecarboxylate (48). Compound 40 was reacted with NaH
and iodoethane in DMF according to procedure G. Purification by
flash column chromatography on silica gel (50% EtOAc/hexanes
as eluant) gave compound 48 as white needles (61%), mp (EtOAc/
hexanes) 138-142 °C. 1H NMR [400 MHz, (CD3)2SO] δ 9.29 (s,
1 H), 8.52 (s, 1 H), 7.77 (dd, J ) 10.1, 1.9 Hz, 1 H), 7.60 (ddd, J
) 8.4, 2.0, 0.8 Hz, 1 H), 7.31 (t, J ) 8.5 Hz, 1 H), 5.45 (s, 1 H),
4.30 (q, J ) 7.1 Hz, 2 H), 3.93 (q, J ) 7.1 Hz, 2 H), 1.33 (t, J )
7.1 Hz, 3 H), 1.20 (t, J ) 7.1 Hz, 3 H). Anal. (C16H16FIN2O3) C,
H, N.
1-Ethyl-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-pyridi-
necarboxamide (53). Hydrolysis of compound 48 was carried out
according to procedure C, giving the intermediate acid (100%)
which was reacted directly according to procedure E. This
intermediate pentafluorophenyl ester was then reacted directly with
concentrated NH3 solution in THF according to procedure F.
Purification by flash column chromatography on silica gel (5%
MeOH/CH2Cl2 as eluant) gave compound 53 as a white solid (84%),
mp (EtOAc) 260-262 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.38
(s, 1 H), 8.31 (s, 1 H), 7.90 (br s, 1 H), 7.73 (dd, J ) 10.2, 1.9 Hz,
1 H), 7.57 (ddd, J ) 8.4, 1.9, 0.9 Hz, 1 H), 7.46 (br s, 1 H), 7.29
(t, J ) 8.5 Hz, 1 H), 5.55 (d, J ) 0.9 Hz, 1 H), 3.83 (q, J ) 7.1
Hz, 2 H), 1.23 (t, J ) 7.1 Hz, 3 H). Anal. (C14H13FIN3O2) C, H,
N.
MHz, (CD3)2SO] δ 10.43 (s, 1 H), 8.28 (s, 1 H), 7.81 (br s, 1 H),
7.73 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.57 (dd, J ) 8.4, 0.9 Hz, 1 H),
7.48 (br s, 1 H), 7.29 (t, J ) 8.5 Hz, 1 H), 5.55 (s, 1 H), 3.95 (t,
J ) 5.5 Hz, 2 H), 3.63 (t, J ) 5.5 Hz, 2 H), 3.54-3.51 (m, 2 H),
3.43-3.39 (m, 2 H), 3.22 (s, 3 H). Anal. (C17H19FIN3O4) C, H, N.
Ethyl 1-(3-Cyanopropyl)-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-
dihydro-3-pyridinecarboxylate (47). Compound 40 was reacted
with NaH and 4-bromobutyronitrile in DMF according to procedure
G. Purification by flash column chromatography on silica gel (50%
EtOAc/hexanes as eluant) gave compound 47 as a white solid
(67%), mp (EtOAc) 157-159 °C. 1H NMR [400 MHz, (CD3)2SO]
δ 9.30 (s, 1 H), 8.51 (s, 1 H), 7.77 (dd, J ) 10.1, 1.9 Hz, 1 H),
7.61 (br d, J ) 8.4 Hz, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H), 5.46 (s, 1
H), 4.31 (q, J ) 7.1 Hz, 2 H), 3.96 (t, J ) 7.2 Hz, 2 H), 2.53 (t,
J ) 7.2 Hz, 2 H), 1.93 (pentet, J ) 7.2 Hz, 2 H), 1.33 (t, J ) 7.1
Hz, 3 H). Anal. (C18H17FIN3O3) C, H, N.
1-(3-Cyanopropyl)-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-dihy-
dro-3-pyridinecarboxamide (56). Hydrolysis of compound 47 was
carried out according to procedure C, to give the intermediate acid
(100%) which was reacted directly according to procedure E. This
intermediate pentafluorophenyl ester was then reacted directly with
concd NH3 solution in THF according to procedure F. Purification
by flash column chromatography on silica gel (5% MeOH/CH2Cl2
as eluant) gave compound 56 as a white solid (70%), mp (EtOAc)
1
146-150 °C. H NMR [400 MHz, (CD3)2SO] δ 10.36 (s, 1 H),
Ethyl 4-(2-Fluoro-4-iodoanilino)-6-oxo-1-propyl-1,6-dihydro-
3-pyridinecarboxylate (49). Compound 40 was reacted with NaH
and bromopropane in DMF according to procedure G. Purification
by flash column chromatography on silica gel (50% EtOAc/hexanes
as eluant) gave compound 49 as a white solid (54%), mp (EtOAc/
hexanes) 147-150 °C. 1H NMR [400 MHz, (CD3)2SO] δ 9.29 (s,
1 H), 8.50 (s, 1 H), 7.76 (dd, J ) 10.1, 1.9 Hz, 1 H), 7.60 (ddd, J
) 8.4, 2.0, 0.8 Hz, 1 H), 7.31 (t, J ) 8.5 Hz, 1 H), 5.46 (d, J ) 1.3
Hz, 1 H), 4.30 (q, J ) 7.1 Hz, 2 H), 3.85 (t, J ) 7.3 Hz, 2 H), 1.62
(sextet, J ) 7.3 Hz, 2 H), 1.33 (t, J ) 7.1 Hz, 3 H), 0.85 (t, J )
7.4 Hz, 3 H). Anal. (C17H18FIN2O3) C, H, N.
8.28 (s, 1 H), 7.91 (br s, 1 H), 7.73 (dd, J ) 10.2, 1.9 Hz, 1 H),
7.57 (ddd, J ) 8.4, 1.9, 0.9 Hz, 1 H), 7.48 (br s, 1 H), 7.29 (t, J )
8.5 Hz, 1 H), 5.56 (d, J ) 0.9 Hz, 1 H), 3.87 (t, J ) 7.2 Hz, 2 H),
2.55 (t, J ) 7.2 Hz, 2 H), 1.96 (pentet, J ) 7.2 Hz, 2 H). Anal.
(C16H14FIN4O2) H, N. C: calcd, 43.7; found, 44.2.
1-(3-{[tert-Butyl(dimethyl)silyl]oxy}propyl)-4-(2-fluoro-4-io-
doanilino)-6-oxo-1,6-dihydro-3-pyridinecarboxamide (59). 3-Bro-
mopropanol was protected as the tert-butyldimethylsilyl ether
according to a literature procedure.32 Compound 40 was reacted
with NaH and the silyl ether-protected bromide in DMF according
to procedure G, giving a crude product which was purified by
column chromatography on silica gel (5% MeOH/CH2Cl2 as eluant).
Rather than isolating the desired compound 52, hydrolysis to the
intermediate acid occurred in situ. This acid (34%) was therefore
reacted directly according to procedure E. This intermediate
pentafluorophenyl ester was then reacted directly with concd NH3
solution in THF according to procedure F. Purification by chro-
matography on silica gel (50% EtOAc/hexanes as eluant) gave
compound 59 as a white solid (68%), used directly in the next step.
1H NMR [400 MHz, (CD3)2SO] δ 10.36 (s, 1 H), 8.24 (s, 1 H),
7.92 (br s, 1 H), 7.72 (dd, J ) 10.2, 1.8 Hz, 1 H), 7.56 (br d, J )
8.9 Hz, 1 H), 7.46 (br s, 1 H), 7.28 (t, J ) 8.5 Hz, 1 H), 5.55 (s,
1 H), 3.85 (t, J ) 7.1 Hz, 2 H), 3.62 (t, J ) 6.1 Hz, 2 H), 1.85
(pentet, J ) 6.6 Hz, 2 H), 0.86 (s, 9 H), 0.03 (s, 6 H). HRMS
(EI+) calcd C21H29FIN3O3Si (M+) 545.1007, found 545.1019.
4-(2-Fluoro-4-iodoanilino)-1-(3-hydroxypropyl)-6-oxo-1,6-di-
hydro-3-pyridinecarboxamide (60). Deprotection of compound
59 was carried out according to procedure H, giving compound 60
as a white solid which was recrystallized from EtOAc/MeOH
4-(2-Fluoro-4-iodoanilino)-6-oxo-1-propyl-1,6-dihydro-3-py-
ridinecarboxamide (54). Hydrolysis of compound 49 was carried
out according to procedure C, giving the intermediate acid (100%)
which was reacted directly according to procedure E. This
intermediate pentafluorophenyl ester was then reacted directly with
concd NH3 solution in THF according to procedure F. Purification
by flash column chromatography on silica gel (5% MeOH/CH2Cl2
as eluant) gave compound 54 as a white solid (97%), mp (EtOAc)
1
218-220 °C. H NMR [400 MHz, (CD3)2SO] δ 10.43 (s, 1 H),
8.30 (s, 1 H), 7.80 (br s, 1 H), 7.73 (dd, J ) 10.2, 1.9 Hz, 1 H),
7.56 (dd, J ) 8.4, 0.9 Hz, 1 H), 7.46 (br s, 1 H), 7.30 (t, J ) 8.5
Hz, 1 H), 5.55 (d, J ) 0.7 Hz, 1 H), 3.75 (t, J ) 7.3 Hz, 2 H), 1.65
(sextet, J ) 7.4 Hz, 2 H), 0.87 (t, J ) 7.4 Hz, 3 H). Anal. (C15H15-
FIN3O2) C, H, N.
Ethyl 4-(2-Fluoro-4-iodoanilino)-1-[2-(2-methoxyethoxy)-
ethyl]-6-oxo-1,6-dihydro-3-pyridinecarboxylate (50). Compound
40 was reacted with NaH and 1-bromo-2-(2-methoxyethoxy)ethane
in DMF according to procedure G. Purification by flash chroma-
tography on silica gel (1% MeOH/CH2Cl2 as eluant) gave com-
pound 50 as a pale yellow oil (41%). 1H NMR [400 MHz,
(CD3)2SO] δ 9.30 (s, 1 H), 8.43 (s, 1 H), 7.77 (dd, J ) 10.1, 1.9
Hz, 1 H), 7.60 (ddd, J ) 8.3, 1.9, 0.8 Hz, 1 H), 7.31 (t, J ) 8.5
Hz, 1 H), 5.44 (d, J ) 1.3 Hz, 1 H), 4.31 (q, J ) 7.1 Hz, 2 H),
4.06 (t, J ) 5.1 Hz, 2 H), 3.61 (t, J ) 5.2 Hz, 2 H), 3.54-3.50 (m,
2 H), 3.43-3.39 (m, 2 H), 3.22 (s, 3 H), 1.32 (t, J ) 7.1 Hz, 3 H).
HRMS (EI+) calcd C19H22FIN2O5 (M+) 504.0558, found 504.0552.
4-(2-Fluoro-4-iodoanilino)-1-[2-(2-methoxyethoxy)ethyl]-6-
oxo-1,6-dihydro-3-pyridinecarboxamide (55). Hydrolysis of com-
pound 47 was carried out according to procedure C, to give the
intermediate acid (99%) which was reacted directly according to
procedure E. This intermediate pentafluorophenyl ester was then
reacted directly with concd NH3 solution in THF according to
procedure F. Purification by flash column chromatography on silica
gel (50% acetone/CH2Cl2 as eluant) gave compound 55 as a white
1
(86%), mp (EtOAc/MeOH) 220-223 °C. H NMR [400 MHz,
(CD3)2SO] δ 10.40 (s, 1 H), 8.29 (s, 1 H), 7.91 (br s, 1 H), 7.72
(dd, J ) 10.2, 1.8 Hz, 1 H), 7.56 (br d, J ) 8.4 Hz, 1 H), 7.46 (br
s, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H), 5.56 (s, 1 H), 4.58 (t, J ) 5.1
Hz, 1 H), 3.86 (t, J ) 7.2 Hz, 2 H), 3.41 (q, J ) 5.7 Hz, 2 H), 1.79
(pentet, J ) 6.6 Hz, 2 H). Anal. (C15H15FIN3O3) C, H, N.
Ethyl 1-(2-tert-Butoxy-2-oxoethyl)-4-(2-fluoro-4-iodoanilino)-
6-oxo-1,6-dihydro-3-pyridinecarboxylate (46). Compound 40 was
reacted with NaH and tert-butylbromoacetate in DMF according
to procedure G. Purification by flash chromatography on silica gel
(1% MeOH/CH2Cl2 as eluant) gave compound 46 as a white solid
1
(72%), mp (EtOAc/hexanes) 149-151 °C. H NMR [400 MHz,
(CD3)2SO] δ 9.34 (s, 1 H), 8.57 (s, 1 H), 7.78 (dd, J ) 10.0, 1.9
Hz, 1 H), 7.61 (dd, J ) 8.4, 1.0 Hz, 1 H), 7.31 (t, J ) 8.5 Hz, 1
H), 5.43 (d, J ) 1.2 Hz, 1 H), 4.62 (s, 2 H), 4.31 (q, J ) 7.1 Hz,
2 H), 1.42 (s, 9 H), 1.33 (t, J ) 7.1 Hz, 3 H). Anal. (C20H22FIN2O5)
C, H, N.
1
solid (58%), mp (EtOAc/n-pentane) 114-116 °C. H NMR [400

5100 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Spicer et al.
tert-Butyl [5-(Aminocarbonyl)-4-(2-fluoro-4-iodoanilino)-2-
oxo-1(2H)-pyridinyl)]acetate (61). Hydrolysis of compound 46
was carried out according to procedure C but using 1 M K2CO3
instead of 1 M NaOH. The intermediate acid (64%) was reacted
according to procedure E. This intermediate pentafluorophenyl ester
was then reacted directly with concd NH3 solution in THF according
to procedure F. Purification by flash column chromatography on
silica gel (50% acetone/CH2Cl2 as eluant) gave compound 61 as a
1 H), 7.46 (br s, 1 H), 7.29 (t, J ) 8.5 Hz, 1 H), 5.56 (d, J ) 0.9
Hz, 1 H), 4.96 (d, J ) 5.5 Hz, 1 H), 4.70 (t, J ) 4.7 Hz, 1 H), 4.14
(dd, J ) 13.1, 3.5 Hz, 1 H), 3.79-3.70 (m, 1 H), 3.51 (dd, J )
13.1, 8.3 Hz, 1 H), 3.40-3.30 (m, 2 H). Anal. (C15H15FIN3O4) C,
H, N.
4-Chloro-1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxylic Acid
(66). Compound 5 (3.19 g, 14.5 mmol) and dimethyl sulfate (6.0
mL, 63 mmol) were combined in a thick-walled glass tube with a
Teflon cap. The tube was sealed and heated in a 120 °C sand bath.
After 5 h, the reaction was cooled to RT and diluted with acetonitrile
(100 mL) and saturated aq NaHCO3 solution (100 mL). The reaction
mixture was stirred vigorously for 18 h. This mixture was further
diluted with water and was extracted with CH2Cl2 (3 × 100 mL).
The combined extracts were washed with brine (200 mL) and dried
(MgSO4). The solution was filtered and then solvent removed under
reduced pressure to afford ethyl 4-chloro-1-methyl-6-oxo-1,6-
dihydro-3-pyridinecarboxylate 65 (2.65 g, 85% yield) as an oil that
solidified upon standing. This material was then hydrolyzed directly
according to procedure C, followed by crystallization from methanol/
ethyl acetate to give compound 66 (37%) as an off-white solid:
1H NMR [400 MHz, (CD3)2SO] δ 13.02 (br s, 1 H), 8.58 (s, 1 H),
6.58 (s, 1 H), 3.48 (s, 3 H).
Procedure I: Alternative Preparation of 4-(2-fluoro-4-iodoa-
nilino)-1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxylic Acid
(21). Compound 66 (133 mg, 0.709 mmol) and 2-fluoro-4-
iodoaniline 6 (172 mg, 0.726 mmol) were combined in a round-
bottom flask equipped with magnetic stir bar. The flask was
immersed in an ice bath, and lithium bis(trimethylsilyl)amide (1.0
M in THF, 5.0 mL, 5.0 mmol) was added slowly (5 min) with
vigorous stirring under an atmosphere of nitrogen. The reaction
mixture was further stirred for 1 h at 0 °C and 1 h at RT. The
reaction mixture was diluted with 1 M HCl and water and was
extracted with EtOAc (3 × 100 mL). The extracts were dried
(MgSO4) and filtered, and upon concentration compound 21 (114
mg, 41% yield) was isolated directly from the EtOAc by filtration
and drying in Vacuo. Spectral data for this compound was in exact
agreement with that obtained for compound 21 described in
procedure C above.
Procedure J: 4-(3,4-Dichlorophenylamino)-1-methyl-6-oxo-
1,6-dihydro-3-pyridinecarboxamide (71). Compound 66 and 3,4-
dichloroaniline 67 were reacted in the presence of lithium diiso-
propylamide in THF according to procedure I. The resultant
compound 69 was used without further purification in the following
step. Compound 69 (234 mg) was dissolved in CH2Cl2 (5 mL) and
ethanolic ammonia (2 M, 2.0 mL, 4.0 mmol). PyBOP (579 mg,
1.10 mmol) was added in one portion, and the reaction mixture
was stirred overnight at RT. The reaction mixture was quenched
with AcOH (ca. 0.5 mL), diluted with EtOAc (40 mL), and washed
with water (2 × 10 mL) and brine (10 mL). The organics were
dried (MgSO4) and filtered, and the solvent was removed under
reduced pressure. Purification by flash column chromatography on
silica gel (4:1 CH2Cl2/MeOH as eluant) gave compound 71 as a
dark yellow solid (28 mg, 12%); mp (MeOH) > 250 °C. 1H NMR
[400 MHz, (CD3)2SO] δ 10.38 (s, 1 H), 8.30 (s, 1 H), 7.85 (br s,
1 H), 7.57 (d, J ) 8.8 Hz, 1 H), 7.48 (d, J ) 2.4 Hz, 1 H), 7.45 (br
s, 1 H), 7.23 (dd, J ) 8.8, 2.4 Hz, 1 H), 5.70 (s, 1 H), 3.33 (s, 3
H). Anal. (C13H11Cl2N3O2) C, H, N.
4-(2-Fluoro-4-methylsulfanylphenylamino)-1-methyl-6-oxo-
1,6-dihydro-3 -pyridinecarboxylic Acid (70). Compound 66 and
2-fluoro-3-thiomethylaniline 68 were reacted in the presence of
lithium diisopropylamide in THF according to procedure I.
Compound 70 was isolated as a light yellow solid (85%). 1H NMR
[400 MHz, (CD3)2SO] δ 13.22 (v br s, 1 H), 9.43 (s, 1 H), 8.47 (s,
1 H), 7.39 (t, 1 H), 7.26 (dd, 1 H), 7.13 (dd, 1 H), 5.33 (s, 1 H),
3.38 (s, 3 H), 2.48 (s, 3 H). MS (APCI+) calcd for C14H14FN2O3S
309 (MH+), found 309.
1
white solid (93%), used directly in the next step. H NMR [400
MHz, (CD3)2SO] δ 10.40 (s, 1 H), 8.30 (s, 1 H), 7.80 (br s, 1 H),
7.74 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.58 (br d, J ) 8.4 Hz, 1 H), 7.54
(br s, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H), 5.53 (d, J ) 0.8 Hz, 1 H),
4.46 (s, 2 H), 1.43 (s, 9 H). HRMS (FAB+) calcd C18H20FIN3O4
(MH+) 488.0483, found 488.0471.
[5-(Aminocarbonyl)-4-(2-fluoro-4-iodoanilino)-2-oxo-1(2H)-
pyridinyl]acetic Acid (62). Compound 61 was dissolved in a
mixture of CH2Cl2 (10 mL) and trifluoroacetic acid (10 mL) and
stirred at RT for 2 h. All solvent was evaporated under a stream of
nitrogen, and the resulting oil was redissolved in MeOH (10 mL)
to which was added sat. NaHCO3 (10 mL). This mixture was stirred
at RT for 1 h, 1 M HCl (50 mL) added, and the resulting white
precipitate collected by filtration. Purification was carried out by
recrystallization from EtOAc/MeOH to afford compound 62 as a
white solid (38%), mp (EtOAc/MeOH) 296-300 °C. 1H NMR [400
MHz, (CD3)2SO] δ 13.10 (v br s, 1 H), 10.42 (s, 1 H), 8.32 (s, 1
H), 7.86 (br s, 1 H), 7.75 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.58 (dd, J
) 8.4, 0.8 Hz, 1 H), 7.52 (br s, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H),
5.53 (d, J ) 1.0 Hz, 1 H), 4.49 (s, 2 H). Anal. (C14H11FIN3O4‚
0.5H2O) C, H, N.
Ethyl 1-Allyl-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-
pyridinecarboxylate (51). Compound 40 was reacted with NaH
and allyl bromide in DMF according to procedure G. Purification
by column chromatography on silica gel (50% EtOAc/hexanes as
eluant) gave compound 51 as a white solid (78%), mp (EtOAc)
138-141 °C. 1H NMR [400 MHz, (CD3)2SO] δ 9.30 (s, 1 H), 8.45
(s, 1 H), 7.77 (dd, J ) 10.1, 1.9 Hz, 1 H), 7.61 (ddd, J ) 8.4, 2.0,
0.8 Hz, 1 H), 7.31 (t, J ) 8.5 Hz, 1 H), 5.98-5.87 (m, 1 H), 5.47
(d, J ) 1.3 Hz, 1 H), 5.19 (ddd, J ) 10.4, 2.7, 1.3 Hz, 1 H), 5.12
(ddd, J ) 17.2, 3.0, 1.5 Hz, 1 H), 4.54 (br d, J ) 5.5 Hz, 2 H),
4.31 (q, J ) 7.1 Hz, 2 H), 1.32 (t, J ) 7.1 Hz, 3 H). Anal. (C17H16-
FIN2O3) C, H, N.
1-Allyl-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-dihydro-3-pyridi-
necarboxamide (63). Hydrolysis of compound 51 was carried out
according to procedure C, to give the intermediate acid (99%) which
was reacted directly according to procedure E. This intermediate
pentafluorophenyl ester was then reacted directly with concd NH3
solution in THF according to procedure F. Purification by flash
column chromatography on silica gel (5% MeOH/CH2Cl2 as eluant)
gave compound 63 as a white solid (85%), mp (EtOAc) 215-
217 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.43 (s, 1 H), 8.27 (s,
1 H), 7.90 (br s, 1 H), 7.73 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.57 (dd,
J ) 8.3, 0.8 Hz, 1 H), 7.49 (br s, 1 H), 7.30 (t, J ) 8.5 Hz, 1 H),
6.00-5.88 (m, 1 H), 5.55 (s, 1 H), 5.19 (dd, J ) 10.3, 1.3 Hz, 1
H), 5.11 (ddd, J ) 17.2, 2.9, 1.5 Hz, 1 H), 4.41 (d, J ) 5.5 Hz, 2
H). Anal. (C15H13FIN3O2) C, H, N.
1-(2,3-Dihydroxypropyl)-4-(2-fluoro-4-iodoanilino)-6-oxo-1,6-
dihydro-3-pyridinecarboxamide (64). Compound 63 (400 mg,
0.97 mmol) was dissolved in a mixture of tert-butyl alcohol (60
mL) and water (60 mL), and to the resulting solution were added
K3Fe(CN)6 (956 mg, 2.91 mmol), K2CO3 (400 mg, 2.91 mmol),
OsO4 (0.62 mL of a 4% w/w solution in water), and diazabicy-
clooctane (108 mg, 0.97 mmol). The reaction mixture was stirred
at RT for 15 h, poured into 1 M Na2S2O4 (200 mL), and extracted
with EtOAc (3 × 100 mL). The combined EtOAc extracts were
washed with water (100 mL) and brine (100 mL) and dried (Na2-
SO4). The solution was filtered and then solvent removed under
reduced pressure. Purification by flash column chromatography on
silica gel (5% MeOH/CH2Cl2 as eluant) gave compound 64 as a
4-(2-Fluoro-4-methylsulfanylphenylamino)-1-methyl-6-oxo-
1,6-dihydro-3 -pyridinecarboxamide (72). Compound 70 was
reacted according to procedure J. Compound 72 was isolated as a
1
white solid (312 mg, 72%), mp (EtOAc) 210-213 °C. H NMR
1
[400 MHz, (CD3)2SO] δ 10.44 (s, 1 H), 8.26 (s, 1 H), 7.87 (br s,
1 H), 7.73 (dd, J ) 10.2, 1.9 Hz, 1 H), 7.57 (dd, J ) 8.4, 0.9 Hz,
white solid (47%). H NMR [400 MHz, (CD3)2SO] δ 10.16 (s, 1
H), 8.36 (s, 1 H), 7.80 (v br s, 1 H), 7.40 (v br s, 1 H), 7.37 (t, 1

Pyridones as Inhibitors of MAP Kinase Kinase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5101
H), 7.25 (dd, 1 H), 7.10 (dd, 1 H), 5.38 (s, 1 H), 3.33 (s, 3 H), 2.46
(s, 3 H). MS (APCI+) calcd for C14H15FN3O2S 308 (MH+), found
308.
reacted with propargyl alcohol according to procedure K. Purifica-
tion by flash column chromatography on silica gel (5% MeOH/
CH2Cl2 as eluant) gave compound 77 as an off-white solid (89%),
1
used directly in the next step. H NMR [400 MHz, (CD3)2SO] δ
Procedure K: 4-{2-Fluoro-4-[(trimethylsilyl)ethynyl]anilino}-
1-methyl-6-oxo-1,6-dihydro-3-pyridinecarboxamide (73). Com-
pound 27 (200 mg, 0.52 mmol), CuI (2 mg, 0.01 mmol), and
(Ph3P)2PdCl2 (8 mg, 0.01 mmol) were dissolved in THF (10 mL),
and the flask was flushed with nitrogen. A solution of TMS-
acetylene (56 mg, 0.06 mmol) in TEA (2 mL) was added dropwise
over 5 min, and then the reaction was allowed to stir at RT for 15
h. This mixture was diluted with EtOAc (80 mL) which was
subsequently washed with water (3 × 50 mL) and brine (50 mL)
and dried (Na2SO4). Purification by flash column chromatography
on silica gel (10% MeOH/CH2Cl2 as eluant) gave compound 73 as
a pale yellow solid (185 mg, 100%), used directly in the next step.
1H NMR [400 MHz, (CD3)2SO] δ 10.66 (s, 1 H), 8.38 (s, 1 H),
7.92 (br s, 1 H), 7.50 (t, J ) 8.5 Hz, 1 H), 7.48 (br s, 1 H), 7.42
(dd, J ) 11.5, 1.8 Hz, 1 H), 7.30 (dd, J ) 8.3, 1.4 Hz, 1 H), 5.73
(s, 1 H), 3.38 (s, 3 H), 0.23 (s, 9 H). HRMS (EI+) calcd C18H20-
FN3O2Si (M+) 357.1309, found 357.1308.
4-(4-Ethynyl-2-fluoroanilino)-1-methyl-6-oxo-1,6-dihydro-3-
pyridinecarboxamide (74). Compound 73 (185 mg, 0.52 mmol)
was dissolved in a mixture of MeOH (9 mL) and THF (1 mL), to
which was added solid K2CO3 (143 mg, 1.03 mmol). This mixture
was allowed to stir for 15 h at RT and then diluted with EtOAc
(50 mL). The EtOAc solution was washed with water (3 × 50 mL)
and brine (50 mL), dried (Na2SO4) and the solvent removed under
reduced pressure. Purification by flash column chromatography on
silica gel (50% acetone/CH2Cl2 as eluant) gave compound 74 as a
pale yellow-orange solid (108 mg, 73%); mp (CH2Cl2/MeOH) 269-
272 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.60 (s, 1 H), 8.37 (s,
1 H), 7.91 (br s, 1 H), 7.51 (t, J ) 8.4 Hz, 1 H), 7.48 (br s, 1 H),
7.46 (dd, J ) 11.4, 1.7 Hz, 1 H), 7.33 (dd, J ) 8.3, 0.9 Hz, 1 H),
5.71 (s, 1 H), 4.25 (s, 1 H). Anal. (C15H12FN3O2·0.25H2O) C, H,
N.
Procedure L: 4-(4-Ethyl-2-fluoroanilino)-1-methyl-6-oxo-1,6-
dihydro-3-pyridinecarboxamide (75). Compound 74 (77 mg, 0.27
mmol) was dissolved in a mixture of THF (10 mL) and EtOH (10
mL) to which was added 5% Pd/C (10 mg). This mixture was stirred
under an atmosphere of H2 at 60 psi for 18 h at RT, the Pd/C
removed over celite, and the solvent removed under reduced
pressure. Purification by flash column chromatography on silica
gel (50% acetone/CH2Cl2 as eluant) gave compound 75 as a cream
solid (64 mg, 82%), mp (EtOAc) 268-272 °C. 1H NMR [400 MHz,
(CD3)2SO] δ 10.14 (s, 1 H), 8.32 (s, 1 H), 7.84 (br s, 1 H), 7.43
(br s, 1 H), 7.33 (t, J ) 8.4 Hz, 1 H), 7.19 (dd, J ) 11.1, 1.7 Hz,
1 H), 7.08 (dd, J ) 8.2, 1.5 Hz, 1 H), 5.39 (d, J ) 1.1 Hz, 1 H),
3.36 (s, 3 H), 2.62 (q, J ) 7.6 Hz, 2 H), 1.19 (t, J ) 7.6 Hz, 3 H).
Anal. (C15H16FN3O2) C, H, N.
4-(4-Cyano-2-fluoroanilino)-1-methyl-6-oxo-1,6-dihydro-3-py-
ridinecarboxamide (76). Compound 27 (0.20 g, 0.5 mmol) was
added to potassium cyanide (0.23 g, 3.50 mmol), copper(I) iodide
(667 mg, 3.50 mmol), and tetrakis(triphenylphosphine)palladium-
(0) (20 mg) in DMF (30 mL) and heated at 110 °C for 4 h. The
reaction mixture was allowed to cool to RT and filtered through
celite which was washed well with 5% MeOH/EtOAc. The filtrate
and washings were combined and concentrated, washed with water
(100 mL) and brine (100 mL), dried (Na2SO4), and filtered.
Removal of the solvent under reduced pressure followed by
trituration with EtOAc gave an approximately 1:1 mixture of
unreacted compound 27 and the desired product 76. Further
purification by preparative HPLC [90% (H2O/TFA)/(acetonitrile/
TFA)-1% (H2O/TFA)/(acetonitrile/TFA) gradient elution 0.8 mL/
min, pH 2.5-2.6] yielded compound 76 (0.01 g, 7%); mp 261-
266 °C. 1H NMR [400 MHz, (CD3)2SO] δ 11.01 (s, 1 H), 8.39 (s,
1 H), 7.91 (dd and br s, J ) 11.2, 1.7 Hz, 2 H), 7.72 (t, J ) 8.2
Hz, 1 H), 7.66 (dd, J ) 8.6, 1.7 Hz, 1 H), 7.52 (br s, 1 H), 5.97 (s,
1 H), 3.40 (s, 3 H). MS (APCI+) calcd for C14H12N4O2F 287 (MH+),
found 387.
10.57 (s, 1 H), 8.35 (s, 1 H), 7.90 (br s, 1 H), 7.50 (t, J ) 8.5 Hz,
1 H), 7.48 (br s, 1 H), 7.39 (dd, J ) 11.4, 1.8 Hz, 1 H), 7.28 (dd,
J ) 8.3, 1.5 Hz, 1 H), 5.69 (s, 1 H), 5.36 (t, J ) 6.0 Hz, 1 H), 4.30
(d, J ) 5.9 Hz, 2 H), 3.37 (s, 3 H). LCMS (APCI+) calcd for C16H15-
FN3O3 316 (MH+), found 316.
4-[2-Fluoro-4-(3-hydroxypropyl)anilino]-1-methyl-6-oxo-1,6-
dihydro-3-pyridinecarboxamide (78). Compound 77 was hydro-
genated according to procedure L. Purification by flash column
chromatography on silica gel (5% MeOH/CH2Cl2 as eluant) gave
compound 78 as a crystalline cream solid (90%), mp (EtOAc/
MeOH) 214-216 °C. 1H NMR [400 MHz, (CD3)2SO] δ 10.15 (s,
1 H), 8.32 (s, 1 H), 7.85 (br s, 1 H), 7.43 (br s, 1 H), 7.33 (t, J )
8.3 Hz, 1 H), 7.17 (dd, J ) 11.8, 1.7 Hz, 1 H), 7.07 (dd, J ) 8.2,
1.6 Hz, 1 H), 5.40 (s, 1 H), 4.50 (t, J ) 5.1 Hz, 1 H), 3.41 (q, J )
6.0 Hz, 2 H), 3.36 (s, 3 H), 2.63 (t, J ) 7.7 Hz, 2 H), 1.76-1.69
(m, 2 H). LCMS (APCI-) calcd for C16H17FN3O3 318 (MH+), found
318. Anal. (C16H18FN3O3) C, H, N.
Acknowledgment. This work was supported by Pfizer
Global Research and Development (USA) and the Auckland
Division of the Cancer Society of New Zealand.
Supporting Information Available: Elemental analysis, HRMS,
and HPLC data on final compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
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