Synthesis and biological activity of quinolinone and dihydroquinolinone p38 MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.10.097

Synthesis and biological activities of some quinolinone and dihydroquinolinone p38 MAP kinase inhibitors are reported. Modifications to the dihydroquinolinone pharmacophore revealed that dihydroquinolinone may be replaced with a quinolinone pharmacophore

Full text of DOI:10.1016/j.bmcl.2006.10.097

Bioorganic & Medicinal Chemistry Letters 18 (2008) 2222–2226
Synthesis and biological activity of quinolinone and
dihydroquinolinone p38 MAP kinase inhibitors
Meng-Hsin Chen,a,* Patricia Fitzgerald,a Suresh B. Singh,a Edward A. O’Neill,b
Cheryl D. Schwartz,b Chris M. Thompson,b Stephen J. O’Keefe,b
Dennis M. Zallerb and James B. Dohertya
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
Received 5 September 2006; revised 30 October 2006; accepted 31 October 2006
Available online 6 November 2006
Abstract—Synthesis and biological activities of some quinolinone and dihydroquinolinone p38 MAP kinase inhibitors are reported.
Modifications to the dihydroquinolinone pharmacophore revealed that dihydroquinolinone may be replaced with a quinolinone phar-
macophore and lead to enhanced p38 inhibitory activity. From a study of C-7 substitutions by amino acid side chains, a very potent
series of compounds in the p38 enzyme assays was identified. Translation of the in vitro activity into reasonable whole blood activity
can be improved in this series of compounds by judicious modification of the physical properties at appropriate regions of the lead.
Ó 2006 Elsevier Ltd. All rights reserved.
The enzyme p38 is an intracellular mitogen-activated
protein (MAP) kinase which regulates the release and
the actions of pro-inflammatory mediators such as
TNF-a and IL-b1. Once activated, p38 initiates a signal
cascade leading to the synthesis and amplification of
these mediators. Clinical studies have shown that inhibi-
tion of these inflammatory mediators individually is
beneficial for treating rheumatoid arthritis. Therefore,
inhibition of p38 enzyme, conceptually a concerted inhi-
bition of these mediators, may have considerable thera-
peutic advantage.
Cl
Cl
N
N
F
H
O
O
N
S
N
N
N
S
F
F
VX 745
SB 203580
Figure 1. Two p38a inhibitors SB203580 and VX-745.
The discovery of series of triaryl-imidazoles as p38
inhibitors, exemplified by SB2035802,3 (Fig. 1), was sem-
inal. Subsequent investigation by Vertex scientists led to
a structurally diverse set of p38a inhibitors, exemplified
by VX-745 (Fig. 1). This compound exhibited high selec-
tivity for p38a over a variety of other closely related ki-
nases4. The Vertex work encouraged us to design several
new series of compounds related to VX-745. In this
communication, we wish to report the synthesis and
evaluation of substitution with various functional
Cl
Cl
Gly-110
N
Met-109
N
Gly-110
N
Met-109
N
Cl
Ar
Cl
Ar
1
H
H
H
H
1
N
O
N
O
7
7
3
N
3
5
H
5
Leu-108
Leu-108
O
O
His-107
His-107
1
2
Figure 2. Relative binding orientation of dihydroquinazolinone 1 and
dihydroquinolinone 2 to p38a.
groups at C-3, C-5, and C-7 in a series of quinolinone
and dihydroquinolinone compounds that are closely
related to VX-745 structure.
Keywords: p38; MAP Kinase inhibitors.
*
Corresponding author. Tel.: +1 732 594 3304; fax: +1 732 594
9556; e-mail: meng_hsin_chen@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.097

M.-H. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2222–2226
2223
Cl
Recently, Merck scientists have published on several
scaffold variants of VX-745 and demonstrated that these
inhibitors were highly selective toward p38 against close-
ly related kinases, had good potency (p38a, TNFa, and
in human whole blood), and were readily orally bio-
available5. The key ligand–enzyme interactions of the
dihydroquinazolinone series of inhibitors with the en-
zyme were clearly discerned in X-ray crystallographic
studies6. Three hydrogen bonds (H bonds) were
observed in the dihydroquinazolinone (1) series with
the p38a enzyme (Fig. 2). Two of the H bonds result
from the carbonyl oxygen of 1 interacting with the N–
H bonds of Met-109 and Gly-110 of the enzyme back-
bone; a third H bond is formed between the cyclic urea
N–H bond of 1 and His-107 carbonyl oxygen. In con-
trast, in the dihydroquinolinone (2)-series this third H
bond with His-107 is absent (see Fig. 2). Our working
hypothesis was that this missing third H bond may ex-
plain why, in general, the activity of the dihydroquinaz-
olinone series (1) was consistently 3- to 5-fold more
potent than that of the dihydroquinolinone series (2)
(data not shown). In this report, compounds were de-
signed in order to create a third H bond between the
dihydroquinolinone compounds (2) and His-107 in the
enzyme backbone through introduction of an H bond
donor attached to C-3. The syntheses of these designed
p38a inhibitors are summarized in Scheme 1.
Br
R
Br
R
MeO
HO2C
a, b, c
d, e
Cl
Br
NH
R
O
Br
3
Br
MeO
a: R=H
b: R=OMe
4
Br
h
5
Cl
Cl
Br
Cl
O
Cl
g
f
O
N
R
N
R
MeO
MeO
Cl
7
6
Cl
Cl
Cl
Cl
Cl
Cl
i, j
R
O
N
O
N
R
O
N
R
+
HO
N3
Cl
Cl
Cl
8
10
9
a: R=H
b: R=OMe
c: R=OH
i, k
Cl
Cl
O
N
l
9a
H2N
Bromination of 2,6-dibromotoluene 3 with NBS gave
the expected benzylic bromide in quantitative yield
and reaction of this derived benzyl bromide with methyl
methoxyacetate anion followed by saponification affor-
ded the carboxylic acid 4. The cyclization precursor 5
was obtained from conversion of the carboxylic acid 4
into the corresponding acid chloride and then reaction
of the acid chloride with 2,6-dichloroaniline. The resul-
tant amide 5 was converted to dihydroquinolinone 6 in
good yield using Ullmann reaction conditions7. A Suzu-
ki reaction8 between dihydroquinolinone 6 and 2-chlor-
ophenyl boronic acid gave the desired biaryl coupling
product 7. The latter was then subjected to treatment
with boron tribromide. This led to the anticipated
demethylation product 8, and the alcohol was converted
to the azide 9 via the mesylate. The quinolinone 10 was
obtained from a 1,2-elimination reaction on the mesy-
late intermediate just mentioned; this transformation
was effected very smoothly by DBU. The azide 9a on
exposure to Ra-Ni led to the formation of the amino
analog 11. The methoxy quinolinone 13 was prepared
by a bromination and elimination reaction sequence
on compound 7b. Demethylation with boron tribromide
followed by careful chromatographic separation gave
the monohydroxyquinolinone 14 and dihydroxyquinoli-
none 15.
Cl
11
Cl
O
Cl
N
OH
m
10b
Cl
12
Cl
Cl
O
N
OMe
m
a
7b
MeO
Cl
13
Cl
Cl
Cl
Cl
O
N
OH
Cl
+
O
N
OMe
Cl
HO
HO
15
14
Scheme 1. Reagents and conditions: (a) NBS, cat. (BzO)2, CCl4, quant.;
(b) methyl methoxyacetate, LiN(TMS)2; (c) NaOH 66% for the two steps;
(d) oxalyl chloride, cat. DMF, benzene; (e) 2,6-dichloroaniline, TEA,
À78 °C to rt, 78% for the two steps; (f) CuI, K2CO3, DMF, 150 °C,
[R = H (73%), R = OMe (84%)]; (g) 2-chlorophenylboronic acid,
PdCl2(PPh3)2, 2 N Na2CO3, [R = H (71%), R = OMe (78%)]; (h) BBr3,
À78 °C, CH2Cl2 [(R = H (75%), R = OMe (65%), R = OH (15%)]; (i)
MsCl, TEA, 0 °C; (j) NaN3, DMSO; (k) DBU, DMSO, 80 °C, (93%) over
the two steps; (l) Ra-Ni/H2 R = H 55%; (m) BBr3, (93%, 14 65%, 15 28%).
The designed inhibitors were tested in the p38 enzyme
assay and whole blood assay5a. The effects on the inhibi-
tion of p38 MAP kinase from the various substituents
attached on C-3 and on C-7 of the quinolinone and
dihydroquinolinone scaffold are shown in Table 1 be-
low. The five hydroxylated analogs (8a–c, 14, and 15)
were all more potent than the three methoxy-ether ana-
logs (7a, 7b, and 13), suggesting that the hydroxyl group

2224
M.-H. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2222–2226
Table 1. Substituent effects on p38a inhibition at C-3 and C-7 on
dihydroquinolinone (A) and quinolinone (B) analogs
Cl
Cl
Ar
Cl
Cl
Br
a, b, c
d
O
N
O
N
6
Cl
Cl
Cl
Cl
O
N
R
O
N
R
16
17, Ar=Ph
X
X
18, Ar=2-pyrido
19, Ar=3-pyrido
20, Ar=4-pyrido
Cl
Cl
Scheme 2. Reagents and conditions: (a) BBr3, À78 °C, 90%; (b) MsCl,
TEA, 0 °C; (c) DBU, DMSO, 80 °C, 93% over the two steps; (d) Ar–
B(OH)2, PdCl2 (PPh3)2, 2 N Na2CO3, 80 °C, 25–50%.
template (A)
template (B)
Compound Template
X
R
p38a IC50 (nM)
7a
8a
9
A
A
A
B
A
A
A
B
A
B
B
B
B
CH–OMe
CH–OH
CH–N3
CH
H
H
H
H
H
Inactive
27.5
ticularly potent in the p38a assay containing whole
blood. For example, one of the most active against
p38a in the enzyme assay was compound (10b)
(IC50 = 1.5 nM), but it was only moderately active
(75% inhibition at 10 lM) in the whole blood assay.
The observed lower potency in the whole blood assay
may be accounted for by the high lipophilicity9 and
therefore leading to high plasma binding.
15% at 100
6
Inactive
10a
11
7b
8b
10b
8c
CH–NH2
CH–OMe OMe 63% at 1250
CH–OH
CH
OMe
OMe 1.5
9
CH–OH
CH
OH
OH
4
0.7
12
13
14
15
C–OMe
C–OH
C–OH
OMe 29% at 100
OMe 28
To address the loss of potency in the whole blood assay,
another series of compounds was crafted in order to try
to reduce lipophilicity and increase water solubility
through modification of the substituents attached at
C-5 and at C-7. These designed compounds were pre-
pared in the following way. The bromide 16 was pre-
pared from compound 6 as shown in Scheme 2 and
this bromide 16 then served as a convenient intermediate
for palladium(0)-mediated Stille or Suzuki cross-cou-
pling arylation reactions at position C-5 of the
quinolone.
OH
5
which can serve as a hydrogen-bond donor to His-107
interacts with p38a much more effectively than the
methoxy group which lacks this hydrogen bonding
capacity. Alternatively, it could be that the size of the
binding pocket around C-3 is such that the hydroxyl
group is well tolerated but not the methoxy group,
resulting in weaker interactions overall for the latter.
Intriguingly, from inspection of the binding data in Ta-
ble 1, it appears that an amino group at C-3 (compound
11) is not tolerated. We surmise that although the amine
is a small and capable of acting as an H bond donor
group, in the binding pocket the amine moiety may be
binding to the enzyme in its protonated form. The pres-
ence of a cationic charge located near position C-3 of the
quinolone may be detrimental for potent p38a binding.
Table 2. Substituent effects on p38a inhibition at C-5 on quinolinone
analogs
Compound
Ar
P38a IC50 (nM)
17
18
19
20
Phenyl
13.4
350
2-Pyrido
3-Pyrido
4-Pyrido
460
58% at 1000
Interestingly, the simple quinolinone analogs 10a, 10b,
and 12 (hydrogen atoms attached at C-7) were the most
potent p38a inhibitors in the set of compounds investi-
gated in Table 1. A possible explanation for this increase
in potency of the quinolinone analogs is that this in-
crease is due to the increased electron density on the car-
bonyl oxygen in 10a, 10b, and 12 and so stronger H
bonds with Met-109 and Gly-110 N–H on the p38a en-
zyme are formed compared with the dihydroquinolinone
analogs. Similar increases in potency were also seen
when electron-donating groups such as a methoxy or a
hydroxyl group were attached at position C-7 of the
quinolinone (10b and 12). These functional groups at
C7 also can increase the electron density of the carbonyl
oxygen and therefore result in more potent p38a inhib-
itors when compared with those compounds that were
unsubstituted at C-7 (10a). Even though the quinolinon-
es 10a, 10b, and 12 had high potency in the p38a enzyme
inhibition assay, none of the three compounds were par-
Cl
Cl
O
O
N
c, d
a, b
NH2 TFA
12
Cl
Cl
Cl
O
N
O
Amino acid
N
H
22-30
Cl
Scheme 3. Reagents and conditions: (a) K2CO3, N-Boc-2-chloroeth-
ylamine, DMF, 80 °C; (b) TFA; (c) N-Boc amino acids, EDC, HOBt,
TEA; (d) HCl, EtOAc.

M.-H. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2222–2226
2225
Table 3. The effects on binding from varying the amino acid in compounds 22–30 in the p38a inhibition and whole blood assay
Compound
Amino acid
P38a IC50 (nM)
WB IC50 (nM)
22
23
24
25
26
27
28
29
30
L-Pro
0.75
0.9
87
73
D-Pro
1-Amino-1-cyclopropane-carboxylic acid
1.5
0.3
1010
80
Gly
Sar
0.5
200
28
a-Methyl-Ala
L-Ala
0.12
0.16
0.2
32
47
D-Ala
b-Ala
0.57
130
Comparison between the 2-chlorophenyl analog (10a)
with the simple phenyl analog 17, the former was mar-
ginally more potent, but when the C-5 phenyl substitu-
tion was replaced with pyridine, all three pyridyl-
isomers (18, 19, and 20) were at least 10-fold less active
than the original phenyl analogs (17) in the p38a enzyme
assays (Table 2). This replacement by a pyridine for
phenyl ring resulted in weaker hydrophobic interactions
than those encountered by the phenyl group in this seg-
ment of the molecule. This approach did not lead to a
practical solution to the problem and so another tack
was taken.
no group and this may lead to a higher plasma binding
for analog 24 than the compound 27. On the other hand,
the glycine analog 25 and b-alanine analog 30 had sim-
ilar potency in both the p38a enzyme and whole blood
assays. It does appear that the physical properties of
p38a inhibitors seem to play an important role in trans-
lating in vitro enzyme inhibition into functional whole
blood activity. Modification of different physical proper-
ties of p38a inhibitors on the various related pharmaco-
phores is still under active investigation.
Conclusion. A series of quinolinone and dihydroquinoli-
none analogs related to VX-745 structure were pre-
pared. The five hydroxylated analogs 8a–c, 14, and 15,
all capable of hydrogen bonding to the p38a enzyme,
were potent but the methoxy analogs 7a, 7b, and 13 were
less potent and the amino analog 11 was essentially inac-
tive. The most potent compounds in the in vitro assay,
the simple quinolinones 10a, 10b, and 12, lost activity
considerably in the whole blood assays. Introduction
of the pyridyl substituents (18, 19, and 20) for phenyl
at C-5 in the quinolinone series attempting to lower lipo-
philicity was fruitless. However, incorporation of amino
acids on a 2-aminoethoxy sidechain at C-7 (22–30) did
lead to active compounds in both the p38a in vitro
and also in the whole blood assay. Translation of the
in vitro activity into reasonable whole blood activity
can be improved in this series of compounds by judi-
cious modification of the physical properties at appro-
priate regions of the lead.
A series of papers from Merck have outlined that
attachment of a piperidinyl moiety at the C-7 position
of naphthyridinone-, quinolinone-, dihydroquinazoli-
none-, and dihyropyridopyrimidone-derived p38a inhib-
itors is optimal5. By studying the crystal structures
obtained for the four series of inhibitors with p38a,
the piperidinyl group was located in the kinase enzyme
in a phosphate-binding area, under a glycine-rich loop
that participates with an extensive number of water mol-
ecules to form a hydrogen binding network with the en-
zyme6. The intriguing conjecture that appropriate
substituents at the C-7 position could change the physi-
cal properties of these p38a inhibitors has been raised.
Indeed, this seems reasonable if one considers the pub-
lished data on the interactions between several series
of p38a inhibitors with the enzyme6. These data suggest
that piperidinyl at the C-7 position binds well to the en-
zyme at least partly by virtue of their participation with
surrounding water molecules in a H-bonding network
with the enzyme. We followed this line of investigation
by preparing a series of amino acid tethered to an
aminoethoxy moiety at C-7, conceptually reminiscent
of piperidinyl, as shown in Scheme 3. Compound 12
was converted to 22–30 through alkylation, deprotec-
tion, standard peptide coupling reactions10, and removal
of the BOC-protecting group. Biological evaluation of
this series of compounds on p38a is shown in Table 3.
References and notes
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A.; Griswold, D. E.; Gallagher, T. F. Bioorg. Med.
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Gratifyingly this amino acid series of derived inhibitors
was indeed very potent in the enzyme assays regardless
of the substituents, configuration of the stereocenters
or basicity of the amino acid attached.11,12 The amino
acid derivative 24 however was considerably shifted in
its potency in the whole blood assay with respect to
compound 27. Possibly the poor whole blood activity
of 24 may be attributed to the physical properties of this
compound, for compound 24 possesses a less basic ami-

2226
M.-H. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2222–2226
4. (a) Bemis, G.W.; Salituro, F.G.; Duffy, J.P.; harrington,
E.M. U.S. Patent, 6,147,080, 2000; (b) Salituro, F.; Bemis,
G.; Cochran, J. WO 99/64400.
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Hop, C. E. C.; Zaller, D. M.; Schmatz, D. M.; Doherty, J.
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7. Lindley, J. Tetrahedron 1984, 40, 1433.
8. Suzuki, A.; Brown, H. C.. Organic Syntheses via Boranes
V. 3 Suzuki Coupling; Aldrich Chemical Company, 2003.
9. The calculated log D (pH 7.4) values for compounds 10a,
10b, and 12 are 6.34, 6.29, and 6.05, respectively, accord-
ing to the ACD program in the ISIS database.
10. Bodanszky, M.; Bodanszky, A. The Practice of Peptide
Synthesis; Springer-Verlag: Berlin and Heidelberg, 1984.
11. The calculated log D (pH 7.4) values for compounds 22
and 23 is 2.74; 24 is 4.04; 25 is 3.79; 26 is 3.98; 27 is 4.03; 28
and 29 is 3.78; and 30 is 2.94 according to the ACD
program in the ISIS database.
12. The amino group present in compounds 22–30 is proton-
ated at pH 7.4, with the exception of compound 24,
according to calculations using the ISIS ACD program.
The estimated pKa value for compounds 22 and 23 is 9.43;
while the estimated pKa of 24 is 6.71; 25 is 7.72; 26 is 6.71;
27 is 8.30; 28 and 29 is 8.18; and 30 is 8.91.