p38 MAP kinase inhibitors. Part 5: Discovery of an orally bio-available and highly efficacious compound based on the 7-amino-naphthyridone scaffold

Article abstract of DOI:10.1016/j.bmcl.2006.06.084

A new sub-class of p38 inhibitors represented by 7-amino-naphthyridone have been discovered. Benchmark compound 16 potently inhibited p38 in vitro, was functionally active, and displayed excellent pharmacokinetic profiles in two animal species. Compound 1

Full text of DOI:10.1016/j.bmcl.2006.06.084

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5468–5471
p38 MAP kinase inhibitors. Part 5: Discovery of an orally
bio-available and highly efficacious compound based
on the 7-amino-naphthyridone scaffold
Swaminathan R. Natarajan,a,* Luping Liu,a Mark Levorse,a James E. Thompson,b
Edward A. O’Neill,b Stephen J. O’Keefe,b Kalpit A. Vora,b Raymond Cvetovich,c
John Y. Chung,c Ester Carballo-Janed and Denise M. Viscoe
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Inflammation Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Process Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of AnimalPharmacolgy, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
eDepartment of LAR, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 13 May 2006; revised 7 June 2006; accepted 8 June 2006
Abstract—A new sub-class of p38 inhibitors represented by 7-amino-naphthyridone have been discovered. Benchmark compound
16 potently inhibited p38 in vitro, was functionally active, and displayed excellent pharmacokinetic profiles in two animal species.
Compound 16 reduced inflammation in animal disease models at EC50 doses as low as 0.2 mpk.
Ó 2006 Elsevier Ltd. All rights reserved.
In preceding communications1 the development of a
novel series of p38 MAP kinase inhibitors originating
Cl
Cl
N
Cl
Cl
N
from quinazolinones were described. This class of p38
MAP kinase inhibitors was originally inspired by semi-
nal work done at Vertex Pharmaceuticals which yielded
them a clinical candidate VX-745 (1).2 The subject of
this communication is an SAR analysis on the naph-
thyridone platform and the subsequent discovery of a
new sub-class, the 7-amino-naphthyridones, as potent,
orally bio-available, and efficacious p38 inhibitors (see
Fig. 1).
O
F
O
N
R
S
F
7
X
N
HN
S
F
F
1 : X = N (VX-745)
2 : X = NH
3 : R = H
4 : R =
N
N
N
pyrimido-pyridazinone
core
pyrido[3,2-d]pyrimidone core
The pyrido[3,2-d]pyrimidone core (3 and 4) is an isomeric
scaffold derived from VX-745 (1). Significant potency
could only be attained via the installation of a C7-substi-
tuent which predisposes the pendant sulfide moiety in
order to enable a facile entry into the hydrophobic pock-
et past Thr-106 in the p38 ATP binding site. Based on
predicted binding mode of these compounds in the
Cl
Cl
NH
Cl
Cl
NH
O
N
O
N
7
7
HN
X
X
4
F
F
5 : X = CH
6 : X = N
7 : X = CH
8 : X = N
Keywords: 7-Amino-naphthyridones; p38 MAP kinase inhibitors;
Peptide flip; Rat LPS challenge.
quinazolinone core and
pyrido[4,3-d]pyrimidone core
quinolinone core and
1,6-naphthyridone core
*
Corresponding author. Tel.: +1 732 594 0939. e-mail:
ravi_natarajan@merck.com
Figure 1. Novel p38 inhibitors: VX-745 and homologs.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.084

S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5468–5471
5469
active site this design was further refined by direct
attachment of the aryl substituent onto the scaffold.
The resulting quinazolinones (5) and pyrido[4,3-d]pyr-
imidones (6) represented a promising new series of p38
inhibitors. The viability of this class of inhibitors criti-
cally depended on a C7-substituted piperidine append-
age for functional activity. On account of metabolic
lability represented by the pendant piperidine append-
age and the C4-benzyllic moiety, these compounds gen-
erally suffered from high clearances and poor overall
pharmacokinetic profiles in all animal species. The latter
liability was addressed by a scaffold re-design resulting
in the quinolinones (7) and 1,6-naphthyridones (8).3
The liability arising from the pendant piperidine
appendage, which is critical for functional activity, still
remained. Key compounds in this class are typified by
9 and 10, where the introduction of a bulky iso-propyl
group on the piperidine slowed down metabolism and
enabled efficacy. Although 9 and 10 embody many desir-
able features of a clinical compound, there still remained
much room for improvement over the above compounds
and a solution to restore functional activity to these p38
inhibitors based on the naphthyridone platform without
making use of a piperidine appendage was highly sought
after (see Fig. 2).
sponding 7-bromo compound.4 Table 1 depicts activities
of three C7-unsubstituted naphthyridinone analogs.
Although compound 11 displayed potent in vitro activ-
ity, functional activity was modest in human whole
blood. It was encouraging to note that compounds 12
and 13 showed similar potent functional activity. It
can be reasoned that substitution with fluorines contrib-
uted to lower calculated logDs and enabled greater
aqueous solvation or increased fraction of protein un-
bound drug which translated into observed functional
potencies. Compound 11 inhibited TNF-a release in
LPS-stimulated human whole blood with an IC50 of
100 nM. In rats 11 was 57% orally bioavailable with
an AUC (PO/1 mpk) of 1.5 lM, Cmax of 1 lM, and
t1/2 of 2 h. With the above rat pharmacokinetic profile
it was anticipated that 11 could effectively inhibit
TNF-a production in a rat LPS challenge model at
3 mpk oral QD dose. Thus, rats were treated with a
3 mpk oral dose of 11 and were injected with LPS
1.5 h prior to euthanasia. At the end of the experiment
plasma samples were pooled and the levels of circulating
TNF-a were determined at three different time points. A
61% inhibition of TNF-a observed at the 6 h time point
gave rise to optimism. However, inhibition at the 16 and
24 h time points was only 37% and 27%, respectively.
The lack of sustained inhibition was disappointing.
SAR investigations began with C7-unsubstituted naph-
thyridones (Table 1). Synthesis of these derivatives was
achieved readily by Pd-mediated reduction of corre-
Treatment in a liver microsomal assay revealed that the
unsubstituted naphthyridine analogs above (Table 1)
were turned over rapidly. In the case of compound 12
it was hypothesized that formation of corresponding
N-oxide derivative 14 could be a facile first step in the
metabolic process. Accordingly an authentic sample of
proposed N-oxide 14 was synthesized by oxidation of
11 with MCPBA as shown in Scheme 1.
N
N
Cl
Cl
Cl
Cl
O
N
O
N
N
Cl
Cl
Compound 14 displayed remarkable rat pharmacokinet-
ic profile. Thus, a 1 mpk po dose of 14 in SD rats
showed 98% bioavailability with an AUC of 49 lM,
Cmax of 3.5 lM, and half-life of 9.9 h. Since N-oxides
can be reduced in vivo, the above oral dose experiment
was also monitored for the formation of 11 and no
evidence for the reductive process could be observed.
N-oxide 14 inhibited p38 with an IC50 of 12 nM and
TNF-a release from LPS stimulated monocytes with
an IC50 of 200 nM. Given the good oral bioavailability
and potency, it was not surprising that a single 3 mpk
F
9
F
10
Figure 2. Key quinolinone and 1,6-naphthyridone p38 inhibitors.
Table 1. p38 inhibitory properties for 1,6-naphthyridones
X1
O
X2
N
N
Cl
Cl
Cl
Cl
O
N
O
N
Z1
a
N
N+
O-
F
F
Z2
Z2
F
F
Compound
X1
X2
Z1
p38
(nM)
THP-1
(nM)
WB
(nM)
11
14
11
12
13
Cl
Cl
F
Cl
H
F
F
F
F
F
F
F
1.5
9.3
9.7
6.6
40.5
7.0
101
86.1
145
Scheme 1. Synthesis of pyrimido[4,5-d]pyrimidone-based p38 inhibi-
tors. Reagents and conditions: (a) 1.5 equiv of MCPBA, MeCN, rt,
5 h, 85%.

5470
S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5468–5471
oral dose of 14 could effectively lower TNF-a produc-
tion by 80% for 24 h in a rat LPS challenge PD assay.
From the observed profile of 11 and 14 above it became
obvious that metabolism at the naphthyridinone
N-atom was a key factor contributing to poor therapeu-
tic efficacy of the C7-unsubstituted naphthyridinone
compounds. Alternative means to modulate the charac-
ter of naphthyridinone scaffold in order to improve
metabolic profiles was therefore sought.
F
F
F
F
O
N
O
N
Br
F
NH2
F
a,b
N
N
F
F
18
16
Three targetted analogs, pyridone 15, amino-pyridine
16, and aminomethyl-pyridine 17, were synthesized
(Table 2) in order to ameliorate purported in vivo
metabolism at the naphthyridone N-atom. Treatment
of bromide 18 with potassium hydroxide at 110 °C yield-
ed a meager 10% of desired pyridone 15. Although 15
potently inhibited TNF-a release in human whole blood
(IC50 = 121 nM), only a 70% reduction in circulating
TNF-a levels was observed at the 3 h time point with
a 10 mpk oral dose in a rat LPS challenge assay. The hy-
droxy pyridine moiety could have contributed to poor
absorption or enabled conjugation and rapid clearance.
Scheme 2. Synthesis of amino-pyridine 16. Reagents and conditions:
(a) 3.0 equiv of PMB-NH2, MeCN, 150 °C, 5 h, 85%; (b) TFA, reflux,
10 h, 79%.
low clearance of 1.6 mL/min/kg, Cmax of 3.5 lM and
at 0.9 L/kg, a small volume of distribution. Conjecture
has it that a small volume of distribution could be
thought of as a safety feature. The compound would
be expected to localize in the vasculature and it is spec-
ulated that p38-mediated processes in the rest of the
body not pertaining to the arrest of inflammation could
be spared. In male rhesus macaques the oral bioavail-
ability was 22% with a half-life of 11.6 h, a low clearance
of 1.3 mL/min/kg, and similar low volume of distribu-
tion of 0.9 L/kg.
Amino-pyridine derivative 16 and aminomethyl-pyri-
dine 17 potently inhibited TNF-a release in human
monocytes. Speculations over metabolic demethylation
of 17 to 16 re-directed our attention to the simple ami-
no-pyridine derivative for further study. The synthesis
of compound 16 is shown in Scheme 2. Bromo deriva-
tive 18 was synthesized in an analogous manner as de-
scribed previously. Direct displacement of bromide in
18 with ammonia did not yield 16 in appreciable yield.
Access to 16 was achieved indirectly by first displace-
ment of the bromide with PMB amine by simply heating
at 150 °C. The PMB group was removed by treatment
with TFA to give desired analog 16. It was gratifying
to note that amino-pyridine derivative 16 was stable in
microsomal assays and displayed excellent pharmacoki-
netic profiles in rats and monkeys.
For in vivo evaluation 16 was initially screened in a
mouse LPS challenge assay.4 12- to 16-week-old female
Balb/c mice were used in these studies. The compound
was formulated in 0.5% methylcellulose and adminis-
tered orally 22.5 h prior to LPS challenge. The animals
were sacrificed 1.5 h after challenge and the resulting
plasma samples collected were analyzed by ELISA to
measure amount of circulating TNF-a. It was found
that 16 was very effective at suppressing the production
of TNF-a (>90% inhibition) for up to 24 h at doses as
low as 0.3 mpk. A similar LPS challenge experiment
was carried out in rats using dexamethasone as a posi-
tive control. One panel of rats was given a 3 mpk oral
dose of 16, while a second panel of rats was given a
1 mpk dose of dexamethasone. Rats in both panels were
injected with LPS at three time points. At the 6 h, 16 h,
and 24 h time points a 3 mpk dose of 16 inhibited TNF-
a production by 97%, 95%, and 92%, respectively, while
a 1 mpk dose of dexamethasone inhibited TNF-a pro-
duction, 92%, 30%, and 28%, respectively. In another
rat LPS challenge study doses of 0.1, 0.3, 1, and
3 mpk of 16 inhibited TNF-a production, 53%, 75%,
79%, and 92%, respectively, at the 24 h time point
(Fig. 3). It was thus inferred that 16 was equally effica-
cious, if not better than dexamethasone at arresting
LPS induced inflammatory processes in rats.
In male SD rat 16 showed a 68% oral bioavailability
when dosed as a 0.5% methylcellulose suspension.
AUC at 1 mpk was 12 lM with a half life of 6.5 h,
Table 2. p38 inhibitory properties for 1,6-naphthyridones
R
F
O
N
N
In order to test the efficacy in an animal disease model,
16 was dosed in rats in a therapeutic manner after dis-
ease progression was induced by means of suitable adju-
vant. Seven-week-old female lewis rats were used. On
day 0 rats were weighed, paw volumes measured, and
radiographed. Adjuvant was injected into the left hind
paw. Onset of disease was allowed to occur for 12 days
when the animals were re-weighed, the paw volumes re-
measured, and radiographed.
F
Compound
–R
OH
p38 (nM)
1.6
THP-1 (nM)
WB (nM)
121
15
16
4.5
1.0
0.69
34
NH2
N
H
17
0.51
7.3
73

S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5468–5471
5471
35000
30000
25000
20000
15000
10000
5000
0
peutic efficacy in the AIA disease model was determined
to be 0.22 mpk (Fig. 4). It is also worthy to note that 16
did not display affinities for ion channels and this bodes
well from a cardiovascular safety point of view for this
class of inhibitors.
In summary, the goal of achieving functional activities
on the naphthyridinone platform without resorting to
use of a piperidine appendage has been demonstrated.
Compound 16 is a highly effective compound for block-
ing p38-mediated inflammatory processes. Follow-up
studies and identification of appropriate drug candidates
in this area are underway.
References and notes
1. (a) Natarajan, S. R.; Wisnoski, D. D.; Singh, S. B.;
Stelmach, J. E.; O’Neill, E. A.; Schwartz, C. D.; Thompson,
C. M.; Fitzgerald, C. E.; O’Keefe, S. J.; Kumar, S.; Hop, C.
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Strauss, J. R.; Cameron, P. M.; Nichols, E. A.; O’Keefe, S.
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Thompson, C. M.; Zaller, D. M.; Doherty, J. B. Bioorg.
Med. Chem. Lett. 2003, 13, 277; (c) Natarajan, S. R.;
Wisnoski, D. D.; Thompson, J. E.; O’Neill, E. A.; O’Keefe,
S. J.; Zaller, D. M.; Doherty, J. B. Bioorg. Med. Chem. Lett.
2, in press.
Vehicle 24 hrs
16: 3 mpk
16: 1 mpk
16: 0.3 mpk 16: 0.01 mpk
Treatments
Figure 3. Dose titration of 16 in a rat LPS challenge assay.
90
80
70
60
50
40
Right Paw
30
EC50
0.2250
2. Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E.
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0.01
0.1
1
10
Dosage (mpk)
Figure 4. Dose titration of 16 for inhibition of paw swelling in AIA
animal disease model.
The animals were orally dosed at 0.1, 0.3, 1, and 3 mpk
once daily for the next ten days. On day 21 animals were
re-weighed, paw volumes measured, and radiographed.
A dose dependent inhibition of swelling bserved in the
secondary paw at the four doses was 38%, 54%, 65%,
and 80% respectively. The ED50 for the observed thera-