1,7-naphthyridine 1-oxides as novel potent and selective inhibitors of p38 mitogen activated protein kinase

Article abstract of DOI:10.1021/jm200975u

The design, synthesis, and ability to inhibit p38 MAP kinase by a novel series of naphthyridine N-oxides will be described. Some of these compounds showed a significant reduction in the LPS-induced TNF production in human whole blood. Structure-activity relationship studies revealed that N-oxide oxygen was essential for activity and was probably a determinant factor for its marked selectivity against other related kinases. After an extensive SAR exercise, several compounds from this series were identified as very potent p38 inhibitors. In vivo efficacy of some derivatives was demonstrated to reduce TNF levels in an acute murine model of inflammation (ED₅₀ = 0.5 mg/kg in LPS-induced TNF production when dosed orally 1.5 h prior to LPS administration). The oral efficacy was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally (ED ₅₀ < 1 mg/kg).

Full text of DOI:10.1021/jm200975u

Article
pubs.acs.org/jmc
1,7-Naphthyridine 1-Oxides as Novel Potent and Selective Inhibitors
of p38 Mitogen Activated Protein Kinase†
Wenceslao Lumeras,‡,# Laura Vidal,‡ Bernat Vidal,‡ Cristina Balague,§ Adelina Orellana,§
́
§
∥
⊥
,‡
́
́
́
Monica Maldonado, Maria Dominguez, Victor Segarra, and Francisco Caturla*
́
‡Department of Medicinal Chemistry, Department of Biology, Department of ADME, and Department of Computational and
§
∥
⊥
Structural Drug Discovery, Almirall Research Center, Almirall S.A., Ctra. Laurea
Barcelona, Spain
̀
Miró 408, E-08980 Sant Feliu de Llobregat,
S
* Supporting Information
ABSTRACT: The design, synthesis, and ability to inhibit p38α MAP kinase by a
novel series of naphthyridine N-oxides will be described. Some of these compounds
showed a significant reduction in the LPS-induced TNFα production in human
whole blood. Structure−activity relationship studies revealed that N-oxide oxygen
was essential for activity and was probably a determinant factor for its marked
selectivity against other related kinases. After an extensive SAR exercise, several
compounds from this series were identified as very potent p38α inhibitors. In vivo
efficacy of some derivatives was demonstrated to reduce TNFα levels in an acute
murine model of inflammation (ED50 = 0.5 mg/kg in LPS-induced TNFα production when dosed orally 1.5 h prior to LPS
administration). The oral efficacy was further demonstrated in a chronic model of adjuvant arthritis in rats with established
disease when administered orally (ED50 < 1 mg/kg).
a simultaneous double interaction with the backbone amide
NH of Met109 and Gly110.14 In this sense, we selected two
starting points for our rational design: Merck inhibitor 1
(Figure 1a)15 and aminopyridine N-oxide 2 (Figure 1b).16
Merck inhibitor 1 is derived from a dihydroquinolinone
core17,18 and is characterized by the presence of two pendent
aromatic rings that are critical for the enzymatic potency. This
p38α MAP kinase inhibitor displayed potent activity (p38α
IC50 = 0.74 nM) and selectivity for p38α over a variety of other
closely related kinases, including ERK1 and JNK1−3 (>10000
fold).15 On the other hand, aminopyridine N-oxide 2, that was
derived from Bayer p38α inhibitor pyridinones,9,20 is also a
potent (p38α IC50 = 21 nM) and selective p38α inhibitor. The
X-ray crystal structure of these two compounds cocrystallized
with unphosphorylated p38α15,16 revealed the formation of two
key hydrogen bond contacts between an oxygen atom of the
ligands (carbonyl for 1 and N-oxide for 2) and the backbone
amide NH of Met109 and NH of Gly110, inducing a
conformational change in the hinge region of the enzyme
(“Gly flip”). Moreover, both molecules show a fine-tuned
occupation of the hydrophobic pocket I placed near the
gatekeeper Thr106. Overlaying X-ray crystal structures of 1 and
2 clearly revealed the similarities of the key hydrogen bond
interactions of both compounds with the hinge region of the
enzyme, strengthening the pseudobicyclic disposition of
aminopyridine N-oxides through the formation of an intra-
molecular hydrogen bond between the carbonyl oxygen and the
amino group and placing the pendent aryl rings of 2 in a
INTRODUCTION
■
The p38α mitogen activated protein (MAP) kinase is an
intracellular serine/threonine (Ser/Thr) kinase that is activated
by a range of environmental stimuli such as TNFα, IL-1β, and
stress.1,2 Activation of p38α occurs through bisphosphorylation
by the dual-specificity Ser/Thr MAP kinases MKK3 and MKK6
on the Thr180-Gly181-Tyr182 motif located on the activation
loop.2,3 In its activated state, p38α phosphorylates a range of
intracellular protein substrates that post-transcriptionally regulate
the biosynthesis of TNFα and IL-1β. The pathophysiological
consequence of excessive production of TNFα and IL-1β is
thought to be significant mediation of the progression of many
inflammatory diseases such as rheumatoid arthritis, psoriasis, and
inflammatory bowel disease.4−7 The inhibition of p38α MAP
kinase can efficiently regulate both the release and the activity of
those proinflamatory cytokines, and it has been seen as an
opportunity to discover novel anti-inflammatory drugs.8
Since the first report in 1994 of the pyridylimidazole-based
p38α inhibitor SB203580,9 a variety of compounds have
advanced into clinical trials for rheumatoid arthritis, reaching
proof-of-concept phase.10 The robust preclinical validation of
the target along with the reported efficacy for VX-74511,12
spurred the conceptualization of novel p38 MAPK inhibitors
with improved pharmacological profiles. As of today, there is
scarce information on the clinical results obtained for the most
advanced compounds in rheumatoid arthritis, although a recent
report on pamapimod has unveiled a transient reduction in
inflammation that may have compromised overall efficacy.13
As part of our strategy to identify novel, potent, and selective
p38α MAP kinase inhibitors, we focused our interest in the
unprecedented level of selectivity shown by ligands that display
Received: July 21, 2011
Published: October 14, 2011
© 2011 American Chemical Society
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Figure 1. (a) Schematic representation of key interactions of the dihydroquinolinone-based Merck’s inhibitor 1 in the active site of p38α, derived
from X-ray complex 1OVE.15 (b) Schematic representation of key interactions of aminopyridine N-oxide inhibitor 2 in the active site of p38α,
derived from X-ray complex 3HRB.16
a
conformation similar to that of 1 within the p38α active site
(Figure 1).
Scheme 1
In light of these two crystal structures, we assume that the
exquisite selectivity of these compounds relays an optimum
interaction with the hydrophobic pocket I, which is particularly
accessible in p38α because of the size of the gatekeeper amino
acid (Thr106), as well as the ability of the ligands to force a Gly
flip, which is an almost exclusive feature of p38α. Other related
kinases contain bulkier residues at this position, which makes
the conformational change virtually impossible,14 giving some
explanation to the high degree of selectivity achieved by
compounds 1 and 2.
With the aim of identifying novel, potent, and selective p38α
inhibitors based on the X-ray studies of these structures in the
catalytic site of the enzyme, a bicyclic core B emerged as a
logical step forward from A in the design of conformationally
more rigid analogues, keeping the unique binding mode
through the N-oxide oxygen atom (Figure 2). Additionally,
a
Reagents and conditions: (a) (R3 = H) AcOH glacial, microwave,
100 °C, 32−85%; (b) (R3 ≠ H) H2SO4 conc, MgSO4 anh, PhMe,
115 °C, 32−73%.
Regarding the piperidine substituted naphthyridines, some of
them were derivatized on the piperidine nitrogen (alkylation/
acylation), as depicted in Scheme 2, to afford compounds 23,
24, and 29.
a
Scheme 2
Figure 2. Derivation of the novel naphthyridines N-oxide core B.
Merck’s quinolinones C confirmed that a bicyclic core could
provide access to good p38α inhibitors, and therefore, we
propose naphthyridine N-oxides B as a suitable integrated
alternative.
a
Reagents and conditions: (a) (R = Me, 23) HCHO, HCOOH,
80 °C; (b)(R = iPr, 24) iPrBr, KI, K2CO3, ACN, 80 °C; (c) (R = Ac, 29)
AcCl, Et3N, CH2Cl2.
For the preparation of R3 substituted naphthyridines 30−36,
having a heteroatom as the attachment point with the
naphthyridine core, we followed the synthetic pathway shown
in Scheme 3. The amino group of the corresponding
aminopyridines 38a−c16 was acylated with pivaloyl chloride.
Subsequent addition of the tert-butyl acetate enolate over the
diaryl ketone group furnished an intermediate that was
submitted to acidic cyclization affording naphthyridinones
39a−c. These intermediates were converted into the
corresponding N-oxides 40a−c via a smooth oxidation with
mCPBA, and the latter compounds were treated with POCl3 to
install a chlorine atom at position 2 of the naphthyridine
CHEMISTRY
■
Naphthyridines 3−20 (R3 = H) were prepared by a variant of
the Friedlander cyclizationa following the synthetic route
̈
depicted in Scheme 1. Starting from the corresponding
aminopyridines,16 the cyclization with acetaldehyde was carried
out using microwave irradiation in glacial AcOH. In the case of
R3 substituted naphthyridines (R3 ≠ H) 21, 22, 25−28, the
optimum conditions for the cyclization of the starting
aminopyridines with the corresponding methylketones consist
of the combination of acid catalysis (concentrated H2SO4) and
a desiccant (MgSO4).
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a
(p38α IC50 = 42 nM; LPS-induced TNFα release in THP-1
cells, IC50 = 14 nM).
Scheme 3
On the other hand, based on the parent aminopyridine
series,16 we were aware of the limited solubility of these
molecules, as can be exemplified by compound 4 (27 μg/mL at
pH 7.4; 41 μg/mL at pH 1). Moreover, as we have learned
from the previous SAR studies with the aminopyridine series,16
the location of the hydrophobic pocket II near the solvent
exposed area allows the introduction of large substituents on
the para position of the R2 aromatic ring. Therefore, we could
use this position to introduce polar groups in order to increase
the aqueous solubility and, potentially, to improve the PK
behavior. However, as can be seen in Table 1, naphthyridines
are much more sensitive than the corresponding amino-
pyridines toward the substitution on this position and none of
the compounds tested gave an appropriate enzymatic potency
or improved physicochemical properties (compounds 12−15).
The postulated binding mode of these ligands (Figure 1b for
putative aminopyridines) within the p38α active site suggests
that not very large substituents would be tolerated in the para
position of the phenyl group in R1 because of its proximity to
the back pocket of the enzyme.22 However, ortho substitution
on this group still allows a potentially interesting SAR
exploration with medium size substituents. A small number of
compounds (16−20) were synthesized, keeping the ortho,or-
tho-disubstitution pattern in R2, and their potency in the
enzymatic assay was measured (Table 2).
Compound 16 (Table 2), bearing an ortho-methoxy group,
gave an enzymatic potency comparable to that of compound 11
(Table 1) but slightly inferior to that of compound 19
(Table 2). This fact showed that although a methoxy group is
allowed, the size of the ortho substituents at R1 was important.
In this sense, taking into account the loss in potency in
compounds 17 and 18 (Table 2), CF3 is clearly too bulky a
substituent while an ortho-chlorine atom on R1 is allowed,
matching the best pocket I occupancy with compound 20
(p38α IC50 = 14 nM, THP-1 cells IC50 = 44 nM, and TNFα in
HWB IC50 = 360 nM).
After this preliminary exploration, compound 20 was judged
as the one bearing the best substitutions on R1 (2-Cl,4-F-Ph)
and R2 (2,6-diCl). Therefore, it was worth testing its selectivity
against a panel of 55 tyrosine and serine/threonine kinases,23
showing an impressive selectivity for p38α/β over all tested
kinases (e.g., c-Raf, JNK-1−3, MK2, IC50 > 10 μM).
At this point, having in mind the structural motifs developed
by Merck (Figure 1a), regarding the introduction of basic
substituents in position 2, we contemplated the opportunity
offered by our naphthyridine core.
In the case of Merck naphthyridones, quinolinones, and
dihydroquinazolinones,17,18 the introduction of a piperidinyl
moiety confers aqueous solubility and increased p38α
inhibition. The increased potency is presumed to arise from a
salt bridge interaction between the protonated piperidine
nitrogen and the carboxylate group of Asp168. Therefore, we
prepared diversely substituted naphthyridines in the R3 position
(Table 3). All piperidine based substitutions in R3 displayed
excellent potencies in the enzymatic (subnanomolar range in
most cases) and cellular assays. The inhibition of TNFα in a
more complex system like human whole blood (hWB) was also
excellent with values in the low nanomolar range (compounds
21, 22, and 28).
a
Reagents and conditions: (a) pivaloyl chloride, DIEA, dioxane, 110 °C;
(b) LDA, AcOtBu, THF, −78 °C; (c) HCl, 100 °C; (d) mCPBA,
CH2Cl2, room temp; (e) POCl3, 110 °C; (f) R3H, base, 110−130 °C.
system. The introduction of different R3 groups was done by
nucleophilic displacement of the chlorine atom, affording the
desired naphthyridines 30−36.
RESULTS AND DISCUSSION
■
In light of the high similarity of the bicyclic core design with the
already studied aminopyridine series,16 we followed the main
part of the trends identified in that series in order to speed up
the naphthyridines SAR exploration. Taking into account that
the putative non-N-oxide aminopyridines retained a consid-
erable inhibitory potency, we considered it interesting to test
the potency of the corresponding non-N-oxide naphthyridines.
In this sense, naphthyridine 3 (Table 1) showed some degree
of p38α inhibition in the micromolar range, similar to that in
the aminopyridine series but far from the potency conferred by
the N-oxide group, as we will see next.
Subsequently, we oxidized the nitrogen on the 7 position.
This oxygen atom would be responsible for the postulated
interactions with the hinge region, affording compound 4 with
a reasonable inhibitory potency (p38α IC50 = 68 nM) in the
enzymatic assay. We considered this initial value as the proof-
of-concept of our bicyclic core design, and preliminary SAR
expansion of this series was undertaken to further explore its
full potential. Following the same exploratory sequence as in
the former aminopyridine series, we started to explore the best
occupation of the hydrophobic pocket II (Figure 1). We already
knew that ortho substitution on this part of the molecule is
mandatory to get an orthogonal conformation of the aromatic
ring with respect to the central core, leading to the best
occupancy of the pocket without clashing with the hinge region.
For this reason, all compounds shown in Table 1 have at
least one ortho substituent. In this sense, compounds 8−11
possessing two ortho substituents were markedly more potent
than the monosubustituted derivatives 4−7. In particular,
compound 10, with an ortho,ortho-dichloro substitution, is the
most potent one in both the enzymatic and cellular assays
The microsomal oxidative metabolism of these molecules
was low, and following Merck’s exploratory route,17,18 we
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Table 1. 4-(2,4-Difluorophenyl)-8-aryl-1,7-naphthyridine 7-Oxides: R2 Group Exploration
a
Values are reported as the geometric mean of at least two independent determinations along with the geometric SD (in parentheses). nd = not
b
determined. n = 1.
decided to test the effect exerted by the substitution of the
piperidine nitrogen over the potency and ADME properties.
The N-alkyl derivatives 23−25 showed potencies similar to
those of their unsubstituted analogues.
Table 2. 8-(2,6-Dichlorophenyl)-4-aryl-1,7-naphthyridine 7-
Oxides: R1 Group Optimization
The introduction of basic aromatic groups (compounds 26
and 27) or removal of the basic character by forming an
acetamide (compound 29) has a profound negative effect on
the enzymatic activity, confirming the importance of the
postulated salt bridge interaction with Asp168.
a
IC50 (nM) obtained in the enzymatic and cellular
Following our SAR exploration over the R3 position, we
decided to study the effect of having a heteroatom as the
attachment point with the naphthyridine core. For those
compounds, shown in Table 4, the potency in all assays was
maintained or even increased, becoming clear that the main
requisite was the presence of a basic nitrogen at the appropriate
position (see drop in potency for compounds 31 and 36).
To confirm the proposed binding mode for our naphthyr-
idine N-oxide scaffold B (Figure 2), compound 21 was
cocrystallized with unphosphorylated p38α.24 The X-ray
structure (Figure 3b) revealed a binding mode compatible to
assays
TNFα cell
(THP-1)
compd
X
R1
2-MeO-Ph 77 (1.19)
p38α
TNFα HWB
16
17
18
19
20
F
nd
292 (1.72)
nd
Cl 3-CF3-Ph
Cl 4-CF3-Ph
Cl 2-Cl-Ph
>10000
>10000
37 (1.03)
nd
nd
nd
137 (1.15)
37 (1.34)
>1000
360 (1.39)
Cl 2-Cl,4-F-Ph 14 (1.26)
a
Values are reported as the geometric mean of at least two
independent determinations along with the geometric SD (in
parentheses). nd = not determined.
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Table 3. 8-(2,6 -Halophenyl)-4-aryl-2-(piperidin-4-yl)-1,7-naphthyridine 7-Oxides: R3 Group Optimization
a
Values are reported as the geometric mean of at least two independent determinations along with the geometric SD (in parentheses).
that for the previously described aminopyridines (Figure 1b),
confirming that ring closure to render a naphthyridine bicyclic
core was a convenient structural modification. Moreover,
the X-ray structure displays a Gly flip, generating a double
hydrogen bond interaction with the backbone NH of Met109
and the NH of Gly110, as in the aminopyridine series. A
comparison of X-ray crystal structures of 2124 (Figure3b) with
that of Merck inhibitor 1 (Figure 3a)15 clearly confirmed the
similarities of the key hydrogen bond interactions of both
compounds with the hinge region of the enzyme. More
interestingly, it was confirmed that the formation of a salt
bridge with Asp168 may be responsible for the extremely high
enzymatic potencies reached. Although it is true that the
distance between piperidine nitrogen and the carboxylic group
of Asp168 exceeded what is usually accepted for this kind of
interaction, the distribution of the positive charge on the
carbons adjacent to the nitrogen should account for this salt
bridge being responsible of the potency boost.
The ADME profiling of some representative derivatives
showed a great difference between compounds with and
without a basic R3. As shown in Table 5, compounds 21 and 30
displayed very high clearance and distribution volumes together
with very low plasma levels, in contrast with the pharmacoki-
netic behavior shown by Merck naphthyridones, quinolinones,
and dihydroquinazolinones,17,18 with basic pendent groups.
However, unsubstituted naphthyridine 4, among the most
potent compounds, showed sustained levels, low clearance
(Cl = 2.7 mL min−1 kg−1), and good bioavailability (F = 38%).
Therefore, because of its balanced potency/ADME profile, 4
was selected for further characterization.
Furthermore, compound 4 presented a low oxidative
metabolic rate in rat (9%) and human (<5%) microsomes
and did not inhibit any of the most relevant P450 isoforms
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Table 4. 4-(2-Chlorophenyl)-8-(2,6-halophenyl)-2-substituted-1,7-naphthyridine 7-Oxides: R3 Group Optimization
a
Values are reported as the geometric mean of at least two independent determinations along with the geometric SD (in parentheses).
Figure 3. (a) X-ray crystal structure of Merck inhibitor 1 (shown in green, taken from the published X-ray structure).15 (b) X-ray overlay of
compounds 2 (shown in cyan)16 and 21 (shown in yellow)24 complexed to p38α active site. The conformational change of Gly110 can be seen in
both proteins, revealing significant hydrogen bond interactions between Met109/Gly110 NHs and N-oxide oxygen in both cases. Comparing X-ray
crystal structures of 2124 with that of Merck inhibitor 1 (Figure 1a)15 clearly confirmed the similarities of the key hydrogen bond interactions of both
compounds with the hinge region of the enzyme besides the formation of a salt bridge with Asp168.
(1A2, 3A4, 2C9, 2C19, and 2D6) at 10 μM. On the other hand,
compound 4 was tested for in vitro cytotoxicity against a
Chinese hamster ovary (CHO) cell line, showing a clean
cytotoxic profile (30% at 200 μM).
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a
Table 5. Pharmacokinetic Parameters of 4, 21, and 30 in Male Wistar Rat after Intravenous (iv) and Oral (po) Administration
b
e
Cmax po
(μM)
tmax po
(h)
AUC0−6 po
t1/2 iv
(h)
F0−6
c
d
compd dose po (mg/kg) dose iv (mg/kg)
(μM h)
Cl iv (mL min−1 kg−1
)
Vss iv (L/kg)
(%)
4
10
10
10
1
1
1
5.3
0.2
3.5
0.3
0.3
29.5
0.48
7.4
2.3
4.6
2.7
102
59
1.7
38
16
21
30
13.7
10.3
0.09
0.05
<10
a
b
c
d
e
Results expressed as the mean ± SD of n = 2. AUC = area under the curve. Cl = clearance. Vss = volume of distribution steady state. F =
bioavailability.
Mercury plus NMR spectrometer at 400 MHz. Samples were dissolved
in deuterated chloroform (CDCl3) or deuterated dimethylsulfoxide
(DMSO-d6), and tetramethylsilane (TMS) was used as reference.
Analytical thin-layer chromatography (TLC) was performed on
Merck silica gel 60 F254. Compounds were visualized by UV light and/
or stained with either potassium permanganate or cerium molybdate
solutions followed by heating. Flash column chromatography was
performed on SDS silica gel 60 (particle size of 40−63 μm).
HPLC analysis was performed on a Waters Alliance 2795
chromatograph equipped with a Waters 2996 diode array detector
and a Waters ZQ mass spectrometer detector. HPLC analysis was
conducted with the following parameters: Chromatography was
performed on a Symmetry C18 column (100 mm × 2.16 mm, 3.5
μm). The mobile phase, at a flow of 0.4 mL/min, was a 20 min binary
gradient of water (containing 0.01 M ammonium formate at pH 3.0)
and a mixture acetonitrile−methanol 50:50 (containing 0.01 M
ammonic formiate 0.01M) (0−95%). The total run time was 26 min.
The retention time (tR) is expressed in min, and UV chromatograms
were processed at 210 nm with blank subtraction. All key compounds
were proven by this method to show ≥95% purity.
In Vivo Pharmacological Evaluation. To evaluate the
compound’s efficacy, 4 was tested in vivo in an acute model
based on rat-LPS induced TNFα and in an adjuvant-induced
arthritis (AIA) assay as a chronic disease model, proving to be
superior to aminopyridine 2 and to standard reference
compounds BIRB-79625,26 and VX-74511,12 (Table 6). In the
Table 6. Summary of in Vivo Efficacy Assays
AIA ED50 (mg/kg) or %
a
a
compd rat LPS-induced TNF ED50 (mg/kg)
inhibition
4
2
0.5 (0.32−0.79)
1.03 (0.5−2.07)
7.9 (2.7−22.8)
<1
4.5 (1.7−11.8)
8.6 (3.8−19.6)
>100
BIRB-796
VX-745
nd
a
Results represent the calculated ED50 along with the lower and upper
95% confidence limits (in parentheses). nd = not determined.
rat-LPS induced TNFα model, compounds were dosed orally
1 h prior to LPS administration and the amount of TNFα in
plasma was measured 1.5 h later (coinciding with the peak of
TNFα production). Compound 4 dose-dependently inhibited
TNFα production with an ED50 of 0.5 mg/kg.
The adjuvant-induced arthritis (AIA) model was selected
as a chronic disease model to evaluate compound efficacy
(Table 6). Upon arthritis induction, compounds were
administered orally once daily for 7 days. Compound 4 dose-
dependently inhibited arthritis progression with ED50 < 1 mg/
kg. These results demonstrate a superior efficacy of 4 versus
other clinically tested reference compounds like BIRB-796 and
VX-745, when dosed in the same manner.
General Procedure for the Synthesis of 1,7-Naphtyridines
3−20 (R3 = H) (Scheme 1). 4-(2,4-Difluorophenyl)-8-(2-
methylphenyl)-1,7-naphthyridine (3). To a solution of 20 mg
(0.062 mmol) of 37a16 in 0.4 mL of glacial AcOH was added 0.3 mL
of acetaldehyde, and the mixture was heated in a microwave system
(“Initiator Sixty” from Biotage) at 100 °C for 45 min. The cooled
mixture was poured into 50 mL of H2O and the pH adjusted to 6−7
using 2 M NaOH. The solution was extracted with EtOAc. The
organic extract was washed with brine, dried (Na2SO4), and
evaporated. Purification by flash chromatography, eluting with
hexane/EtOAc (4:1) gave 12 mg (56%) of 1. LCMS (m/z): 333
1
[M + 1]+. H NMR (CDCl3) δ 2.15 (s, 3H), 7.03−7.17 (m, 2H),
7.33−7.54 (m, 7H), 8.67 (d, J = 5.8 Hz, 1H), 9.04 (d, J = 4.2 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2-methylphenyl)-1,7-naphthyri-
dine 7-Oxide (4). This compound was prepared from 37b16 as
described in the synthesis of 3 but heating at 110 °C for 18 h. Yield:
45%. LCMS (m/z): 349 [M + 1]+. 1H NMR (CDCl3) δ 2.18 (s, 3H),
7.03−7.18 (m, 2H), 7.30−7.54 (m, 7H), 8.33 (d, J = 8.1 Hz, 1H), 8.96
(d, J = 5.7 Hz, 1H).
CONCLUSIONS
■
A novel series of naphthyridine N-oxides were designed,
synthesized, and tested for their ability to inhibit p38α MAP
kinase. The proposed binding mode for this novel series of
inhibitors is consistent with the crystallographic results
obtained from inhibitor 21 within the p38α active site. Some
of these compounds showed a significant reduction in the LPS-
induced TNFα production in THP-1 cells and in hWB.
The appropriate balance between potency and pharmacokinetics
of compound 4 coupled to its excellent behavior in a chronic
model of arthritis highlights the potential of this compound to be
further profiled in various inflammatory conditions.
4-(2,4-Difluorophenyl)-8-(2-methoxyphenyl)-1,7-naphthyri-
dine 7-Oxide (5). This compound was prepared from 37c16 as
described in the synthesis of 3. Yield: 55%. LCMS (m/z): 365
1
[M + 1]+. H NMR (CDCl3) δ 3.80 (s, 3H), 7.02−7.21 (m, 4H),
7.33−7.59 (m, 5H), 8.32 (d, J = 7.8 Hz, 1H), 8.96 (d, J = 4.1 Hz, 1H).
8-(2-Chlorophenyl)-4-(2,4-difluorophenyl)-1,7-naphthyri-
dine 7-Oxide (6). This compound was prepared from 37d16 as
described in the synthesis of 3. Yield: 85%. LCMS (m/z): 369
[M + 1]+. 1H NMR (DMSO-d6) δ 7.30−7.41 (m, 1H), 7.45−7.70 (m,
8H), 8.38 (d, J = 7.9 Hz, 1H), 8.94 (d, J = 4.0 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2-fluorophenyl)-1,7-naphthyri-
dine 7-Oxide (7). This compound was prepared from 37e16 as
described in the synthesis of 3. Yield: 51%. LCMS (m/z): 353
EXPERIMENTAL SECTION
■
Chemistry. Nonaqueous reactions were performed under argon or
nitrogen atmosphere at room temperature unless otherwise noted. All
commercial reagents and anhydrous solvents were purchased from
Aldrich and were used without further purification or distillation unless
otherwise stated.
1
[M + 1]+. H NMR (DMSO-d6) δ 7.30−7.42 (m, 1H), 7.50−7.74
(m, 8H), 8.38 (d, J = 6.0 Hz, 1H), 8.96 (d, J = 4.0 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2,6-dimethylphenyl)-1,7-naph-
thyridine 7-Oxide (8). This compound was prepared from 37f16 as
described in the synthesis of 3. Yield: 53%. LCMS (m/z): 363 [M +
1
Routine H nuclear magnetic resonance spectra were recorded on
1
1]+. H NMR (CDCl3) δ 2.03 (s, 6H), 7.04−7.17 (m, 1H), 7.11
the following instruments: Varian Gemini 300 MHz, Varian Mercury
plus NMR spectrometer operating at a frequency of 200 MHz, Varian
(d, J = 7.9 Hz, 1H), 7.22−7.26 (m, 2H), 7.35 (d, J = 6.0 Hz, 1H),
7905
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(21). An amount of 0.2 mL (3.85 mmol) of H2SO4 98% was added
dropwise to a suspension of 200 mg (0.506 mmol) of 37p,16 400 mg
(3.7 mmol) of anhydrous MgSO4, and 252 mg (1.11 mmol) of tert-
butyl 4-acetylpiperidine-1-carboxylate in 6 mL of toluene, and the
mixture was vigorously stirred in a preheated oil bath at 115 °C. After
60 min, the mixture was cooled and the solvent discarded. The residue
was washed with EtOAc, dissolved in MeOH, and filtered through a
sintered glass to eliminate most of the inorganic salts. Evaporation and
purification by reverse phase chromatography, eluting with H2O/ACN
(0.1% of HCOOH) (from 0% to 50% of ACN), gave 180 mg. Yield:
7.38−7.47 (m, 2H), 7.53 (dd, J = 2.2, 7.9 Hz, 1H), 8.36 (d, J = 7.9 Hz,
1H), 8.96 (d, J = 6.0 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2,6-dimethoxyphenyl)-1,7-naph-
thyridine 7-Oxide (9). This compound was prepared from 37g16 as
described in the synthesis of 3. Yield: 33%. LCMS (m/z): 395 [M +
1]+. 1H NMR (CDCl3) δ 3.74 (s, 6H), 6.76 (d, J = 7.8 Hz, 2H), 7.02−
7.16 (m, 2H), 7.33 (d, J = 6.1 Hz, 1H), 7.35−7.53 (m, 2H), 7.47 (d,
J = 7.9 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.94 (d, J = 6.1 Hz, 1H).
8-(2,6-Dichlorophenyl)-4-(2,4-difluorophenyl)-1,7-naphthyr-
idine 7-Oxide (10). This compound was prepared from 37h16 as
described in the synthesis of 3. Yield: 57%. LCMS (m/z): 403 [M + 1]+.
1H NMR (CDCl3) δ 7.04−7.20 (m, 2H), 7.39−7.61 (m, 6H), 8.33 (d,
1
73%. LCMS (m/z): 486 [M + 1]+. H NMR (CDCl3) δ 1.55−1.94
(m, 4H), 2.64−2.77 (m, 2H), 2.84−3.00 (m, 1H), 3.08−3.20 (m, 2H),
7.02−7.16 (m, 2H), 7.29−7.55 (m, 6H), 8.28 (d, J = 8 Hz, 1H).
4-(2-Chloro-4-fluorophenyl)-8-(2,6-dichlorophenyl)-2-piper-
idin-4-yl-1,7-naphthyridine 7-Oxide (22). This compound was
prepared from 37q16 as described in the synthesis of 3. Yield: 36%.
J = 8.2 Hz, 1H), 8.96 (d, J = 6.4 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2,6-difluorophenyl)-1,7-naphthyr-
idine 7-Oxide (11). This compound was prepared from 37i16 as
described in the synthesis of 3. Yield: 71%. LCMS (m/z): 371
[M + 1]+. 1H NMR (CDCl3) δ 7.06−7.16 (m, 4H), 7.37−7.55
(m, 3H), 7.57 (dd, J = 2.3, 6.1 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.97
(d, J = 6.1 Hz, 1H).
1
LCMS (m/z): 502 [M + 1]+. H NMR (CDCl3) δ 2.04−2.10 (m,
2H), 2.25−2.36 (m, 2H), 3.00−3.10 (m, 2H), 3.19 (m, 1H), 3.28−
3.34 (m, 2H), 7.16−7.26 (m, 2H), 7.35−7.54 (m, 6H), 8.28 (d, J = 7.7
Hz, 1H).
4-[4-(2,4-Difluorophenyl)-7-oxido-1,7-naphthyridin-8-yl]-3-
methylphenol (12). This compound was prepared from 37j16 as
described in the synthesis of 3. Yield: 32%. LCMS (m/z): 365
2-(1-tert-Butylpiperidin-4-yl)-4-(2-chloro-4-fluorophenyl)-8-
(2,6-dichlorophenyl)-1,7-naphthyridine 7-Oxide (25). This
compound was prepared from 37r16 as described in the synthesis of
3. Yield: 32%. LCMS (m/z): 558 [M + 1]+. 1H NMR (CDCl3) δ 1.08
(s, 9H), 1.75−1.81 (m, 2H), 1.93−1.96 (m, 2H), 2.17−2.23 (m, 2H),
2.78 (m, 1H), 3.06−3.10 (m, 2H), 7.21 (m, 1H), 7.29 (s, 1H), 7.36−
7.45 (m, 4H), 7.49−7.53 (m, 2H), 8.25 (d, J = 9.2 Hz, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-2-pyridin-4-yl-
1,7-naphthyridine 7-Oxide (26). This compound was prepared
from 37s16 as described in the synthesis of 3. Yield: 51%. LCMS
(m/z): 496 [M + 1]+. 1H NMR (CDCl3) δ 7.19−7.32 (m, 1H), 7.38−
7.53 (m, 4H), 7.54−7.62 (m, 2H), 7.79 (d, J = 6.0 Hz, 2H), 7.86
(s, 1H), 8.31 (d, J = 7.4 Hz, 1H), 8.71 (d, J = 6.0 Hz, 2H).
4-(2-Chloro-4-fluorophenyl)-8-(2,6-dichlorophenyl)-2-pyri-
din-3-yl-1,7-naphthyridine 7-Oxide (27). This compound was
prepared from 37t16 as described in the synthesis of 3. Yield: 50%.
1
[M + 1]+. H NMR (CDCl3) δ 2.09 (s, 3H), 6.64−6.69 (m, 2H),
7.04−7.16 (m, 3H), 7.38−7.44 (m, 2H), 7.54 (dd, J = 1.7, 8.0 Hz,
1H), 8.37 (d, J = 6.2 Hz, 1H), 9.02 (d, J = 4.1 Hz, 1H).
4-(2,4-DifluorophenyI)-8-[2-methyl-4-(2-morpholin-4-
ylethoxy)phenyl]-l,7-naphthyridine 7-Oxide (13). This com-
pound was prepared from 37k16 as described in the synthesis of
3 but heating at 110 °C for 18 h. Yield: 14%. LCMS (m/z): 478
[M + 1]+.
4-[4-(2,4-Difluorophenyl)-7-oxido-1,7-naphthyridin-8-yl]-N-
(2-methoxyethyl)-3-methyl-benzamide (14). This compound
was prepared from 37l16 as described in the synthesis of 3 but
heating at 110 °C for 18 h Yield: 23%. LCMS (m/z): 450 [M + 1]+.
N-{4-[4-(2,4-Difluorophenyl)-7-oxido-1,7-naphthyridin-8-yl]-
3-methylphenyl}methanesulfonamide (15). This compound was
prepared from 37m16 as described in the synthesis of 3. Yield: 21%.
LCMS (m/z): 442 [M + 1]+. 1H NMR (CDCl3) δ 2.15 (s, 3H), 3.08 (s,
3H), 7.04−7.23 (m, 5H), 7.39−7.45 (m, 2H), 7.57 (dd, J = 2.3, 6.1 Hz,
1H), 8.00 (brs, 1H), 8.37 (d, J = 7.8 Hz, 1H), 8.99 (d, J = 6.1 Hz, 1H).
8-(2,6-Difluoropheny1)-4-(2-methoxypheny1)-1,7-naphthyr-
idine 7-Oxide (16). This compound was prepared from 37n16 as
described in the synthesis of 3. Yield: 45%. LCMS (m/z): 365
1
LCMS (m/z): 496 [M + 1]+. H NMR (CDCl3) δ 7.20−7.29 (m,
2H), 7.32−7.59 (m, 6H), 7.84 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.29
(d, J = 7.4 Hz, 1H), 8.67 (d, J = 4.9 Hz, 1H), 9.19 (s, 1H).
4-(2-Chlorophenyl)-8-(2,6-difluorophenyl)-2-piperidin-4-yl-
1,7-naphthyridine 7-Oxide (28). This compound was prepared
from 37u16 as described in the synthesis of 3. Yield: 50%. LCMS
1
(m/z): 452 [M + 1]+. H NMR (CDCl3) δ 1.62−1.89 (m, 2H), 1.99
1
[M + 1]+. H NMR (CDCl3) δ 3.78 (s, 3H), 7.08−7.18 (m, 4H),
(dd, J = 13.7, 3.5 Hz, 2H), 2.70−2.88 (m, 2H), 2.88−3.10 (m, 1H),
3.12−3.29 (m, 2H), 7.04−7.19 (m, 2H), 7.27 (s, 2H), 7.32−7.65 (m,
6H), 8.26 (d, J = 7.0 Hz, 1H).
7.27−7.31 (m, 1H), 7.37 (d, J = 4 Hz, 1H), 7.47−7.60 (m, 3H), 8.27
(d, J = 6 Hz, 1H), 8.94 (d, J = 4 Hz, 1H).
8-(2,6-Dichlorophenyl)-4-[3-(trifluoromethyl)phenyl]-1,7-
naphthyridine 7-Oxide (17). This compound was prepared from
37o16 as described in the synthesis of 3. Yield: 57%. LCMS (m/z):
Synthesis of 4-(2-Chloro-4-fluorophenyl)-8-(2,6-dichloro-
phenyl)-2-(1-methylpiperidin-4-yl)-1,7-naphthyridine 7-Oxide
(23) (Scheme 2). 0.017 mL (0.47 mmol) of formic acid and 0.034
mL (0.47 mmol) of formaldehyde (37% in water) were added to 34
mg (0.06 mmol) of 22, and the mixture was heated at 80 °C for 4 h
and at room temperature for 16 h. Purification by reverse phase
chromatography, eluting with H2O/ACN (0.1% of HCOOH) (from
0% to 50% of ACN) gave 5.4 mg (17%). LCMS (m/z): 516 [M + 1]+.
1H NMR (CDCl3) δ 1.76−1.87 (m, 4H), 2.00−2.08 (m, 2H), 2.27 (s,
1
435 [M + 1]+. H NMR (CDCl3) δ 7.42 (d, J = 6.3 Hz, 1H), 7.44−
7.57 (m, 3H), 7.73−7.76 (m, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.80−7.88
(m, 2H), 8.34 (d, J = 8.0 Hz, 1H), 8.97 (d, J = 6.3 Hz, 1H).
8-(2,6-Dichlorophenyl)-4-[4-(trifluoromethyl)phenyl]-1,7-
naphthyridine 7-Oxide (18). This compound was prepared from
37m16 as described in the synthesis of 3. Yield: 64%. LCMS (m/z):
1
435 [M + 1]+. H NMR (CDCl3) δ 7.41 (d, J = 5.7 Hz, 1H), 7.46−
3H), 2.76−2.80 (m, 1H), 2.80−2.93 (m, 2H), 7.05−7.11 (m, 2H),
7.38−7.52 (m, 5H), 7.55−7.60 (m, 1H), 8.24 (d, J = 4.2 Hz, 1H).
Synthesis of 4-(2-Chloro-4-fluorophenyl)-8-(2,6-dichloro-
phenyl)-2-(1-isopropylpiperidin-4-yl)-1,7-naphthyridine 7-
Oxide (24) (Scheme 2). 127 mg (1.04 mmol) of 2-bromopropane,
8.63 mg (0.05 mmol) of potassium iodide, and 71 mg (0.52 mmol) of
potassium carbonate were sequentially added to a solution of 131 mg
(0.26 mmol) of 22 in 2.6 mL of acetonitrile, and the mixture was
stirred at 80 °C for 7 h. After cooling, the reaction mixture was filtered
through a sintered glass to eliminate most of the inorganic salts.
Evaporation and purification by reverse phase chromatography, eluting
with H2O/ACN (0.1% of HCOOH) (from 0% to 50% of ACN), gave
89 mg (67%). LCMS (m/z): 544 [M + 1]+. 1H NMR (CDCl3) δ 1.05
(d, J = 5.8 Hz, 6H), 1.73−1.83 (m, 2H), 1.93−1.96 (m, 2H), 2.22−
2.27 (m, 2H), 2.69−2.75 (m 1H), 2.76−2.83 (m, 1H), 2.91−2.94 (m, 2H),
7.57 (m, 3H), 7.68 (d, J = 7.9 Hz, 2H), 7.75 (d, J = 7.9 Hz, 1H), 7.88
(d, J = 8.0 Hz, 2H), 8.34 (d, J = 7.9 Hz, 1H), 8.97 (d, J = 3.6 Hz, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-1,7-naphthyri-
dine 7-Oxide (19). This compound was prepared from 37n16 as
described in the synthesis of 3. Yield: 13%. LCMS (m/z): 401 [M +
1
1]+. H NMR (CDCl3) δ 7.33−7.58 (m, 8H), 7.59−7.68 (m, 1H),
8.29 (d, J = 7.4 Hz, 1H), 8.97 (d, J = 4.4 Hz, 1H).
4-(2-Chloro-4-fluorophenyl)-8-(2,6-dichlorophenyl)-1,7-
naphthyridine 7-Oxide (20). This compound was prepared from
37o16 as described in the synthesis of 3. Yield: 16%. LCMS (m/z):
1
419 [M + 1]+. H NMR (CDCl3) δ 7.27−7.57 (m, 8H), 8.30 (d, J =
5.6 Hz, 1H), 8.98 (d, J = 4.0 Hz, 1H).
General Procedure for the Synthesis of 1,7-Naphtyridines
21, 22, 25−28 (Scheme 1). 8-(2,6-Dichlorophenyl)-4-(2,4-
́
difluorophenyl)-2-(piperidin-4-yl)-l,7-naphthyridine 7-Oxide
7906
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7.17−7.22 (m, 1H), 7.27 (s, 1H), 7.35−7.44 (m, 4H), 7.48−7.51 (m,
2H), 8.25 (d, J = 8.0 Hz, 1H).
was diluted with water (50 mL) and extracted with EtOAc (3 × 40
mL). The organic layer was dried over sodium sulfate and the solvent
removed under reduced pressure to yield the title compound (0.27 g,
Synthesis of 2-(1-Acetylpiperidin-4-yl)-4-(2-chlorophenyl)-
8-(2,6-difluorophenyl)-1,7-naphthyridine 7-Oxide (29)
(Scheme 2). To a solution of 50 mg (0.15 mmol) of 28 in 1.5 mL
of CH2Cl2 were added 0.035 mL (0.25 mmol) of Et3N and 0.014 mL
(0.20 mmol) of acetyl chloride. The mixture was stirred under argon at
room temperature for 48 h. The reaction mixture was quenched by the
addition of 1 mL of H2O and stirred at room temperature for 10 min.
Then it was diluted with EtOAc, washed with 2 N HCl, brine, dried,
and evaporated. Purification by reverse phase chromatography, eluting
with H2O/ACN (0.1% of HCOOH) (from 0% to 70% of ACN), gave
1
85%) as a solid. LCMS (m/z): 387 [M + 1]+. H NMR (300 MHz,
DMSO-d6) δ 6.64 (s, 1H), 7.43−7.12 (m, 5H), 7.80−7.45 (m, 3H),
8.17 (d, J = 6.7 Hz, 1H).
4-(2-Chlorophenyl)-8-(2,6-difluorophenyl)-l,7-naphthyridin-
2(1H)-one 7-Oxide (40b). This compound was prepared from 39b
following the experimental procedure described for the synthesis of
1
40a. Yield: 98%. LCMS (m/z): 385 [M + 1]+. H NMR (300 MHz,
CDCl3) δ 6.56 (bd, J = 1.6 Hz, 1H) 7.71−6.93 (m, 8H), 8.18−7.99
(m, 1H), 8.89 (bs, 1H).
1
50 mg (65%). LCMS (m/z): 494 [M + 1]+. H NMR (CDCl3) δ
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-l,7-naphthyridin-
2(1H)-one 7-Oxide (40c). This compound was prepared from 39c
following the experimental procedure described for the synthesis of
1.66−1.83 (m, 2H), 1.99 (dd, J = 10.9, 5.1 Hz, 2H), 2.10 (s, 3H),
2.69−2.88 (m, 1H), 2.98−3.11 (m, 1H), 3.14−3.26 (m, 1H), 3.85 (d,
J = 14.8 Hz, 1H), 4.44−4.64 (m, 1H), 7.03−7.17 (m, 2H), 7.31−7.66
(m, 7H), 8.27 (d, J = 7.1 Hz, 1H).
1
40a. Yield: 91%. LCMS (m/z): 417 [M + 1]+. H NMR (300 MHz,
CDCl3) δ 6.57 (s, 1H), 7.07 (d, J = 7.1 Hz, 1H), 7.69−7.32 (m, 7H),
8.07 (d, J = 7.0 Hz, 1H), 8.27 (bs, 1H).
General Procedure for the Synthesis of 1,7-Naphtyridines
30−36 (R3 ≠ H) (Scheme 3). 4-(2,4-Difluorophenyl)-8-(2,6-
difluorophenyl)-l,7-naphthyridin-2(1H)-one (39a). (a) To a
solution of 38a16 (1.5 g, 4 mmol) and diisopropylethylamine (1.5
mL, 8.6 mmol) in 10 mL of dioxane under argon was carefully added
pivaloyl chloride (1 mL, 8.1 mmol) in 5 mL of dioxane. After the
addition was completed, the reaction mixture was stirred at 110 °C for
18 h. The mixture was diluted with EtOAc, washed with water,
aqueous 4% sodium bicarbonate, brine, dried over sodium sulfate and
the solvent removed under reduced pressure. The residue obtained
was triturated with pentane to afford a solid that was filtered to yield
1.58 g (88%) of N-[4-(2,4-difluorobenzoyl)-2-(2,6-difluorophenyl)-
pyridin-3-yl]-2,2-dimethylpropanamide. LCMS (m/z): 431 [M + 1]+.
(b) Subsequently, n-BuLi (1.6 M in hexanes, 8.8 mL, 14.1 mmol)
was added dropwise to a solution of diisopropylamine (2 mL, 14.2
mmol) in dry tetrahydrofuran (10 mL) at −78 °C under argon, and
the resulting mixture was stirred at room temperature for 20 min.
Then the reaction mixture was cooled to −78 °C and tert-butyl acetate
(1.9 g, 14.1 mmol) in dry tetrahydrofuran (5 mL) was added.
Afterward, the compound obtained in the previous step (1.6 g, 3.52
mmol) in dry tetrahydrofuran (10 mL) was added and the mixture
stirred overnight at room temperature. Subsequently, the solvent was
removed under reduced pressure and the residue was partitioned
between EtOAc (80 mL) and water (80 mL). The organic solution
was washed with brine, dried over anhydrous sodium sulfate and the
solvent removed under reduced pressure to yield 3-(2,4-difluorophe-
nyI)-3-[2-(2,6-difluorophenyl)-3-(2,2-dimethylpropiony1amino)-
pyridin-4-yl]-3-hydroxypropionic acid tert-butyl ester (1.9 g, 99%) as a
pale yellow solid. LCMS (m/z): 547 [M + 1]+.
2-Chloro-4-(2,4-difluorophenyl)-8-(2,6-difluorophenyl)-l,7-
naphthyridine 7-Oxide (41a). A mixture of the intermediate
compound 40a (0.361 g, 0.9 mmol) and phosphorus oxychloride (5
mL) was heated at 110 °C in a sealed tube for 2 h. The mixture was
cooled, poured into ice−water and the pH adjusted to 10−11 with
concentrated aqueous ammonia. The solution was extracted with
EtOAc (2 × 100 mL), the organic layer was washed with brine, dried
over sodium sulfate, and the solvent removed under reduced pressure.
The residue was crystallized with isopropanol to yield compound 41a
(0.34 g, 78%) as a yellow solid. LCMS (m/z): 405 [M + 1]+. 1H NMR
(300 MHz, DMSO-d6) δ 7.46−7.25 (m, 2H), 7.80−7.53 (m, 5H), 7.81
(s, 1H), 8.49 (d, J = 7.4 Hz, 1H).
2-Chloro-4-(2-chlorophenyl)-8-(2,6-difluorophenyl)-l,7-
naphthyridine 7-Oxide (41b). This compound was prepared from
40b following the experimental procedure described for the synthesis
of 41a. Yield: 55%. LCMS (m/z): 403 [M + 1]+. 1H NMR (300 MHz,
CDCl3) δ 7.11 (m, 2H), 7.41−7.29 (m, 3H), 7.67−7.42 (m, 4H), 8.28
(d, J = 7.4 Hz, 1H).
2-Chloro-4-(2-chlorophenyl)-8-(2,6-dichlorophenyl)-l,7-
naphthyridine 7-Oxide (41c). This compound was prepared from
40c following the experimental procedure described for the synthesis
of 41a. Yield: 68%. LCMS (m/z): 435 [M + 1]+. 1H NMR (300 MHz,
CDCl3) δ 7.10 (m, 2H), 7.25−7.38 (m, 3H), 7.39−7.60 (m, 4H), 8.20
(d, J = 7.4 Hz, 1H).
4-(2,4-DifluorophenyI)-8-(2,6-difluorophenyl)-N-piperidin-4-
yl-l,7-naphthyridin-2-amine 7-Oxide (30). (a) A mixture of
compound 41a (179 mg, 0.40 mmol) and tert-butyl 4-amino-
piperidine-1-carboxylate (188 mg, 0.9 mmol) in ethoxyethanol (2
mL) was heated at 130 °C in a sealed tube for 3 h. The mixture was
cooled, diluted with EtOAc (40 mL), washed with water, brine, and
dried over sodium sulfate, and the solvent was removed under reduced
pressure. The residue was purified by column chromatography on
silica gel, eluting with hexane/EtOAc, 1/4, to yield intermediate 4-[8-
(2,6-difluorophenyl)-4-(2,4-difluorophenyl)-7-oxy-l,7-naphthyridin-2-
ylamino]piperidine-1-carboxylic acid tert-butyl ester (100 mg, 46%) as
a solid. LCMS (m/z): 569 [M + 1]+.
(c) In the next step, a mixture of the previous intermediate (1.9 g,
3.52 mmol) and 5 M aqueous solution of HCl (30 mL) was stirred at
100 °C overnight. The mixture was carefully neutralized with a
saturated solution of potassium carbonate in water, and the
precipitated solid was filtered to yield the title compound (1.2 g,
1
88%) as a solid. LCMS (m/z): 371 [M + 1]+. H NMR (CDCl3) δ
6.78 (s, 1H), 7.11 (d, J = 5.3 Hz, 1H), 7.25−7.62 (m, 6H), 8.48 (d, J =
5.3 Hz, 1H), 8.55 (bs, 1H).
(b) A mixture of the previous intermediate compound (100 mg,
0.17 mmol) and a solution of hydrochloric acid in dioxane (4M, 3 mL)
was stirred for 90 min at room temperature. The solvent was removed
under reduced pressure, and the residue was partitioned between
EtOAc (30 mL) and a saturated solution of sodium bicarbonate (30
mL). The organic solution was washed with brine, dried over
anhydrous sodium sulfate and the solvent removed under reduced
pressure to yield the title compound (60 mg, 74%) as a solid. LCMS
4-(2-Chlorophenyl)-8-(2,6-difluorophenyl)-l,7-naphthyridin-
2(1H)-one (39b). This compound was prepared from 38b14
following the experimental procedure described for the synthesis of
39a. Yield: 60% (over the three steps). LCMS (m/z): 370 [M + 1]+.
1H NMR (300 MHz, CDCl3) δ 6.78 (s, 1H), 7.20−7.04 (m, 3H), 7.36
(m, 1H), 7.52 (m, 4H), 8.48 (d, J = 5.3 Hz, 1H), 8.56 (bs, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-l,7-naphthyridin-
2(1H)-one (39c). This compound was prepared from 38c14 following
the experimental procedure described for the synthesis of 39a. Yield:
1
(m/z): 469 [M + 1]+. H NMR (CD3OD) δ 1.36−1.43 (m, 2H),
1.92−1.99 (m, 2H), 2.1 5 (s, 1H), 2.42−2.53, (m, 2H), 2.99−3.06 (m,
2H), 3.57−3.66 (m, 1H), 6.81 (s, 1H), 7.12−7.23 (m, 4H), 7.47−7.64
(m, 3H), 8.09 (d, J = 6.6 Hz, 1H).
1
50% (over the three steps). LCMS (m/z): 401 [M + 1]+. H NMR
(300 MHz, CDCl3) δ 6.78 (bd, J = 1.6 Hz, 1H), 7.11 (d, J = 5.3 Hz,
1H), 7.65−7.34 (m, 7H), 8.39 (bs, 1H), 8.48 (d, J = 5.3 Hz, 1H).
4-(2,4-Difluorophenyl)-8-(2,6-difluorophenyl)-l,7-naphthyri-
din-2(1H)-one 7-Oxide (40a). A mixture of intermediate 39a (0.3 g,
0.81 mmol), acetic acid (8 mL), and 30% hydrogen peroxide (2 mL)
was heated at 95 °C in a sealed tube for 16 h. The reaction mixture
4-(2-Chlorophenyl)-8-(2,6-difluorophenyl)-2-morpholino-
l,7-naphthyridin-2-amine 7-Oxide (31). 31 was obtained as a
white solid (70 mg, 35% yield) from intermediate compound 41b (177
mg) following the experimental procedure described for the synthesis
of 30a. LCMS (m/z): 454 [M + 1]+. 1H NMR (CDCl3) δ: 3.47−3.63
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(m, 4H), 3.69−3.80 (m, 4H), 6.84 (s, 1H), 7.01−7.11 (m, 2H), 7.14
(d, 1H), 7.31−7.37 (m, 1H), 7.39−7.51 (m, 3H), 7.55−7.63 (m, 1H),
8.06 (d, 1H).
10 μg/mL. Compounds were resuspended in 100% DMSO at 1 mM
and titrated thereof in 10× dilutions in medium. Controls included
unstimulated and stimulated cells treated with the highest concen-
tration of compound vehicle (1% DMSO). Cells were incubated for
5 h at 37 °C in a 5% CO2 atmosphere. Cell supernatant was recovered
by centrifugation and diluted 5-fold prior to testing in a standard
human TNFα ELISA (RnD systems).
Inhibition of TNFα Production Induced by LPS in Human
Whole Blood assay. Healthy volunteer donor blood was collected
by venipuncture in heparinized tubes. An amount of 2 μL of 10-fold
compound concentrations in 100% DMSO was mixed with 200 μL of
LPS-stimulated blood in microtiter plates. Controls included
unstimulated and stimulated blood treated with the highest
concentration of compound vehicle (1% DMSO). Plates were
incubated for 24 h at 37 °C with shaking. Supernatant was recovered
by centrifugation and diluted 1 in 6 prior to testing in a standard
TNFα ELISA (RnD systems).
Data were analyzed by nonlinear regression (Hill equation) to
generate a dose−response curve. The calculated IC50 value
corresponds to the concentration of the test compound causing a
50% decrease in the maximal TNFα production (absolute IC50).
In Vivo Assays. LPS-Induced TNFα in the Rat. One hour prior
to LPS administration, rats were dosed orally with the compounds
suspended in 0.5% methylcellulose/0.1% Tween-80. LPS (5 mg/kg)
was administered intraperitoneally, and 1.5 h later, rats were
anesthesized and retroorbital blood was collected in heparin tubes.
Plasma was separated by centrifugation and diluted one-fifth prior to
assaying in a standard rat TNF-α ELISA (RnD Systems).
Adjuvant-Induced Arthritis (AIA) Model. Arthritis was induced
by intraplantar administration of 100 μL of Mycobacterium tuberculosis
suspension (5 mg/mL in paraffin oil) in the left paw of male Wistar
rats (day 0). On day 11 after inoculation, animals were weighed and
paw volume was measured by plesthimometry. Animals with left paw
volumes ranging between 3.5 and 5 mL and right paw volumes
between 2 and 3 mL were randomized in the required treatment
groups (7 animals/group). Treatment was started and continued for 7
consecutive days. On each day animals were weighed and
appropriately dosed orally with the indicated doses of compounds
suspended in 0.5% methylcellulose/0.1% Tween 80. Paw volumes
were monitored every other day. A healthy control group was
inoculated with paraffin oil on day 0 and monitored thereafter for paw
volumes and weight. At the end of the study, animals were euthanized
with CO2. Inhibition percentage was calculated using last day
contralateral (right) paw volumes.
ADME Methods. In Vitro Studies. Metabolic Stability. Meta-
bolic stability of test compounds was evaluated in rat and human liver
microsomes. Test compounds (5 μM) were incubated in liver
microsomes (1 mg/mL protein) at 37 °C for 30 min in the presence
and absence of NADPH (cofactor). Test compounds from the
incubations were analyzed using UPLC−MS/MS. The metabolic rate
was calculated by comparison of the area of the parent compound in
the incubations in the presence and absence of NADPH.
CYP Inhibition. The potential to inhibit CYP1A2, 2C9, 2C19,
2C6, and 3A4 was evaluated in human microsomes in the presence of
the appropriate substrates (1A2, ethoxyresorufin; 2C9, diclofenac;
2C19, S-mephenytoin;. 2D6, dextromethorphan; 3A4, testosterone).
Human liver microsomes (0.1 mg/mL protein for all isoforms with the
exception of 2C19 (0.5 mg/mL protein)) were incubated at 37 °C for
10 min (1A2, 2C9, and 3A4), 30 min (2D6), or 60 min (2C19) with
various concentrations of test compounds (0, 1, 5, and 25 μM) in the
presence of an NADPH-generated system. To determine the activity,
the amount of metabolite formed for each substrate was quantified
by UPLC−MS/MS at the different concentrations of the tested
compounds.
4-(2-ChlorophenyI)-8-(2,6-dichlorophenyl)-2-piperazin-l-yl-
l,7-naphthyridine 7-Oxide (32). 32 was obtained as a white solid
(52% yield) from intermediate compound 41c following the
experimental procedure described for the synthesis of 30. LCMS
1
(m/z): 485 [M + 1]+. H NMR (CDCl3) δ: 2.83−2.92 (m, 4H),
3.46−3.55 (m, 4H), 6.85 (s, 1H), 7.14 (d, 1H), 7.32−7.42 (m, 2H),
7.42−7.52 (m, 4H), 7.55−7.62 (m, 1H), 8.02 (d, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-N-piperidin-4-yl-
l,7-naphthyridin-2-amine 7-Oxide (33). 33 was obtained as a
white solid (68%) from intermediate compound 41c following the
experimental procedure described for the synthesis of 30. LCMS
(m/z): 499 [M + 1]+. 1H NMR (CDCl3) δ 1.12−1.38 (m, 3H), 1.93−
1.97 (m, 2H), 2.32−2.54 (m, 2H), 2.92−3.09 (m, 2H), 3.44−3.63 (m,
1H), 4.80 (bd, J = 6.6 Hz, 1H), 6.48 (s, 1H), 7.12 (d, J = 7.1 Hz, 1H),
7.31−7.50 (m, 6H), 7.52−7.62 (m, 1H), 8.02 (d, J = 7.1 Hz, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyI)-N-(l,2,2,6,6-pen-
tamethylpiperidin-4-yl)-1,7-naphthyridin-2-amine 7-Oxide
(34). 34 was obtained as a white solid (6%) from intermediate
compound 41c following the experimental procedure described for the
1
synthesis of 30a. LCMS (m/z): 569 [M + 1]+. H NMR (CDCl3) δ
1.10 (s, 6H), 1.14−1.32 (m, 2H), 1.46 (s, 6H), 1.83 (m, 3H), 2.20 (s,
3H), 4.11 (m, 1H), 4.59 (bd, J = 7.9 Hz, 1H), 6.45 (s, 1H), 7.11 (d, J =
7.1 Hz, 1H), 7.27−7.51 (m, 6H), 7.52−7.63 (m, 1H), 8.02 (d, J = 7.1
Hz, 1H).
4-(2-ChlorophenyI)-8-(2,6-dichlorophenyl)-2-(piperidin-4-
yloxy)-l,7-naphthyridine 7-Oxide (35). 35 was obtained as a white
solid (31%) from intermediate compound 41c following the
experimental procedure described for the synthesis of 30a. LCMS
1
(m/z): 500 [M + 1]+. H NMR (CDCl3) δ 1.48−1.67 (m, 2H), 1.96
(m, 2H), 2.52 (m, 2H), 3.06 (m, 2H), 3.70 (s, 1H), 4.63−4.81 (m,
1H), 6.83 (s, 1H), 7.28 (d, J = 7.2 Hz, 1H), 7.45 (m, 6H), 7.59 (m,
1H), 8.17 (d, J = 7.2 Hz, 1H).
4-(2-Chlorophenyl)-8-(2,6-dichlorophenyl)-2-(2-ethoxye-
thoxy)-1,7-naphthyridine 7-Oxide (36). 36 was obtained as a
white solid (82%) from intermediate compound 41c following the
experimental procedure described for the synthesis of 30a. LCMS
1
(m/z): 489 [M + 1]+. H NMR (CDCl3) δ 1.20 (m, 3H), 3.42−3.56
(m, 2H), 3.65 (m, 2H), 4.18−4.35 (m, 2H), 6.92 (s, 1H), 7.22−
7.32 (m, 1H), 7.33−7.54 (m, 6H), 7.54−7.64 (m, 1H), 8.18 (d, J =
7.2 Hz, 1H).
Biological Methods. p38α Kinase Inhibition Assay. Enzy-
matic activity assay was performed in 96-well microtiter plates
(Corning, catalog number 3686) using a total volume of 50 μL of an
assay buffer composed of 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1.75
mM Na3VO4.
Various concentrations of the test compound or vehicle controls
were preincubated for 1 h with 0.055 μg/mL of the human p38α
(SAPKa) enzyme (obtained from University of Dundee, U.K.). The
reaction started by addition of biotinylated ATF2 substrate and ATP in
concentrations around their Km values (final concentration 0.62 and 60
μM, respectively) and took place for 1 h at 25 °C. Addition of the
detection reagents, streptavidin−XL665 and anti-phospho residue
antibody coupled to europium cryptate, caused the juxtaposition of the
cryptate and the XL665 fluorophore, resulting in fluorescence energy
transfer (FRET). The FRET intensity depends on the amount of
bounded cryptate antibody, which is proportional to the extent of
substrate phosphorylation. FRET intensity was measured using Victor
2V spectrofluorometer.
Data were analyzed by nonlinear regression (Hill equation) to generate
a dose−response curve. The calculated IC50 is the concentration of the test
compound that caused a 50% decrease in the maximal FRET intensity.
Inhibition of TNFα Production Induced by LPS in the
Human Monocytic Cell Line THP-1 Assay. For this purpose, 2 ×
105 cells/well were plated in tissue-culture-treated round-bottom 96-
well plates in RPMI (containing 10% FCS, 2mM L-Gln, 10 mM Hepes
buffer, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L NaHCO3, and
β-mercaptoethanol 50 μM), together with compounds at the desired
test concentration and LPS (Sigma, L2630) at a final concentration of
In Vivo Studies. PK Studies in Rats. Test compounds were
administered to Wistar rats (1 mg/kg iv, 10 mg/kg po). Blood samples
were drawn from the retro-orbital plexus at specific time points. After
centrifugation (3000 rpm, 10 min, 4 °C), plasma was separated and
stored at −20 °C until analysis. Samples were prepared by protein
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precipitation followed by UPLC−MS/MS analysis. PK parameters
models of arthritis, bone resorption, endotoxin shock and immune
function. J. Pharmacol. Exp. Ther. 1996, 279, 1453−1461.
(5) Feldmann, M.; Brennan, F. M.; Maini, R. N. Role of cytokines in
rheumatoid arthritis. Annu. Rev. Immunol. 1996, 14, 397−440.
(6) Rutgeerts, P.; D’Haens, G.; Targan, S.; Vasiliauskas, E.; Hanauer,
S. B.; Present, D. H.; Mayer, L.; van Hogezand, R. A.; Braakman, T.;
DeWoody, K. L.; et al. Efficacy and safety of retreatment with anti-
tumour necrosis factor antibody (Infliximab) to maintain remission in
Chron’s disease. Gastroenterology 1999, 117, 761−769.
were calculated using WinNolin.
ASSOCIATED CONTENT
■
S
* Supporting Information
Kinase selectivity panel for compound 20. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
(7) Foster, M. L.; Halley, F.; Souness, J. E. Potential of p38 inhibitors
in the treatment of rheumatoid arthritis. Drug News Perspect. 2000, 13,
488−497.
†Coordinates and structure factors have been deposited in the
RCSB Protein Data Bank (access code 3MW1 for complex of
p38α with 21).
(8) (a) Kumar, S.; Boehm, J.; Lee, J. C. p38 MAP kinases: key
signaling molecules as therapeutic targets for inflammatory diseases.
Nat. Rev. Drug Discovery 2003, 2, 717−726. (b) Mayer, R. J.; Callahan,
J. F. p38 MAP kinase inhibitors: a future therapy for inflammatory
diseases. Drug Discovery Today: Ther. Strategies 2006, 3 (1), 49−54.
(9) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.;
Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.; Heyes, J. R.;
Landvatter, S. W.; Strickler, J. E.; McLaughlin, M. M.; Siemens, I. R.;
Fisher, S. M.; Livi, G. P.; White, J. R.; Adams, J. L.; Young, P. R. A
protein kinase involved in the regulation of inflammatory cytokine
biosynthesis. Nature 1994, 372, 739−746.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 34 93 291 35 83. Fax: 34 93 291 34 20. E-mail: juan.
francisco.caturla@almirall.com.
Present Address
#Eli Lilly & Co. Centro de Investigacio
́n Lilly, S.A. Avda. de la
Industria, 30, 28108 Alcobendas (Madrid), Spain.
(10) (a) Weisman, M.; Furst, D.; Schiff, M.; Kauffman, R.; Merica,
E.; Martin-Munley, S. FRI0018 a double-blind, placebo controlled trial
of VX-745, an oral p38 MAP kinase inhibitor, in patients with
rheumatoid arthritis (RA). Ann. Rheum. Dis. 2002, 61 (Suppl. 1).
(b) Goldstein, D. M.; Kuglstatter, A.; Lou, Y.; Soth, M. J. J. Med. Chem.
2010, 53, 2345−2353.
(11) Ferraciola, G. F. VX-745 Vertex Pharmaceuticals. Curr. Opin.
Anti-Inflammatory Immunomodulatory. Invest. Drugs 2000, 2, 74−77.
(12) Haddad, J. J. VX-745 (Vertex Pharmaceuticals). Curr. Opin.
Invest. Drugs 2001, 2, 1070−1076.
(13) Cohen, S. B.; Cheng, T. T.; Chindalore, V.; Damjanov, N.;
Burgos-Vargas, R.; Delora, P.; Zimany, K.; Travers, H.; Caulfield,
J. P. Evaluation of the efficacy and safety of pamapimod, a p38 MAP
kinase inhibitor, in a double-blind, methotrexate-controlled study of
patients with active rheumatoid arthritis. Arthritis Rheum. 2009, 60 (2),
335−44.
(14) For further discussion on the “flip” binding mode of unrelated
series, see the following: (a) Fitzgerald, C. E.; Patel, S. B.; Becker, J.
W.; Cameron, P. M.; Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin,
G. Nat. Struct. Biol. 2003, 10, 764. (b) Natarajan, S. R.; Heller, S. T.;
Nam, K.; Singh, S. B.; Scapin, G.; Patel, S.; Thompson, J. E.;
Fitzgerald, C. E.; O’Keefe, S. J. Bioorg. Med. Chem. Lett. 2006, 16 (22),
5809.
ACKNOWLEDGMENTS
■
We thank Josep Maria Huerta for spectroscopic data and
Ramo
́
edge Phil Leonard, Marieke Lamers, and Martin Fisher from
BioFocus DPI (formerly at Sareum) for their kind assistance in
the crystallographic studies of compound 21.
ABBREVIATIONS USED
■
MAP, mitogen activated protein; TNFα, tumor necrosis factor
α; IL-1β, interleukin 1β; MKK3, mitogen activated protein
kinase kinase-3; MKK6, mitogen activated protein kinase
kinase-6; RA, rheumatoid arthritis; ERK1, mitogen activated
protein kinase 1; JNK1−3, c-Jun N-terminal kinase 1−3; MK2,
mitogen activated protein kinase activated protein kinase-2;
COPD, chronic obstructive pulmonary disease; TMEDA,
tetramethylethylenediamine; HBTU, 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; S-Phos,
2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; mCPBA, m-
chloroperbenzoic acid; DIEA, Hunig’s base; LDA, lithium
̈
diisopropylamide; LPS, lipopolysaccharide; THP-1, human
acute monocytic leukemia cell line; SAR, structure−activity
relationship; HWB, human whole blood; CHO, Chinese
hamster ovary; hERG, human ether-a-go-go-related gene;
HEK, human embryonic kidney; AIA, adjuvant-induced
arthritis; ATF2, activating transcription factor 2; RPMI, Roswell
Park Memorial Institute medium
(15) Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron, P. M.;
Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G. Structural basis for
p38α MAP kinase quinazolinone and pyrido-pyrimidine inhibitor
specificity. Nat. Struct. Biol. 2003, 10, 764−769.
́
(16) Lumeras, W.; Caturla, F.; Vidal, L.; Esteve, C.; Balague, C.;
Orellana, A.; Dominguez, M.; Roca, R.; Huerta, J. M.; Godessart, N.;
Vidal, B. Design, synthesis and structure-activity relationships of
aminopyridine N-oxides, a novel scaffold for the potent and selective
inhibition of p38 mitogen activated protein kinase. J. Med. Chem. 2009,
52, 5531−5545.
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