p38 MAP kinase inhibitors. Part 6: 2-Arylpyridazin-3-ones as templates for inhibitor design

Article abstract of DOI:10.1016/j.bmcl.2006.08.074

p38 inhibitors based on 3,4-dihydropyrido[4,3-d]pyrimidazin-2-one template were synthesized and their SAR explored. Benchmark compounds 30, 35, and 36 were found to be potent against the enzyme. Crystal structure of p38 in complex with 30 indicated a key

Full text of DOI:10.1016/j.bmcl.2006.08.074

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5809–5813
p38 MAP kinase inhibitors. Part 6: 2-Arylpyridazin-3-ones as
templates for inhibitor design
Swaminathan R. Natarajan,a,* Stephen T. Heller,a Kiyean Nam,a Suresh B. Singh,a
Giovanna Scapin,a Sangita Patel,a James E. Thompson,b
Catherine E. Fitzgeraldb and Stephen J. O’Keefeb
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 15 July 2006; revised 15 August 2006; accepted 15 August 2006
Available online 30 August 2006
Abstract—p38 inhibitors based on 3,4-dihydropyrido[4,3-d]pyrimidazin-2-one template were synthesized and their SAR explored.
Benchmark compounds 30, 35, and 36 were found to be potent against the enzyme. Crystal structure of p38 in complex with 30
indicated a key p-stacking interaction with the pendant tyrosine residue-35 in the glycine-rich loop.
Ó 2006 Elsevier Ltd. All rights reserved.
It has been shown that inhibition of p38 MAP kinase is
key to blocking the pro-inflammatory signal transduc-
tion cascade initiated upon exposure of leukocytes to a
number of extracellular stress stimuli.1–4 Although a
number of structurally different inhibitors have been
reported5 to inhibit p38 with varying degrees of selectiv-
ity, none has reached commercial status.6 This commu-
nication describes the utilization of 2-aryl pyridazinone
as a suitable template for design of novel, selective p38
inhibitors.
Thr-106 ‘gate-keeper’ residue lining the characteristic
hydrophobic pocket attainable only by a and b isoforms
of p38 MAP kinase.
Cl
Cl
N
Cl
Cl
N
O
F
O
N
R
S
F
X
HN
N
S
F
F
1 : X = N (VX-745)
2 : X = NH
pyrimido-pyridazine core
3 : R = H
VX-745 (1)7 and its congeners8 (Fig. 1) are potent and
highly selective inhibitors of p38 MAP kinase. They
have been shown to occupy the ATP binding site of
p38 MAP kinase in a unique fashion.9 The carbonyl
group of these inhibitors induces a re-orientation of
the amide bond between Met-109 and Gly-110 in the
hinge region in order to form a dual hydrogen bond
interaction. In almost all closely related kinases the res-
idue adjacent to Met-109 bears a larger side chain which
prevents facile amide bond re-orientation. A second
hydrogen bond, thus, cannot be formed leading to poor
binding affinity of these compounds for other kinases.
The origin of the reported more than 1000-fold selectiv-
ity exhibited by these inhibitors is attributed to this sui
generis ‘peptide flip’ in combination with a small
4 : R =
N
N
N
pyrido-pyrimidine core
R
Cl
Cl
N
Cl
Cl
N
O
N
O
N
7
7
HN
X
X
Cl
F
F
5 : R = H; X = CH
6 : R = Me; X = CH
8
9
: X = CH
: X = N
Keywords: p38 Inhibitors; MAP Kinase.
*
7 : R = H; X = N
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.2006.08.074

5810
S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5809–5813
Inhibitor design began with an analysis of X-ray crystal-
lographic data and site-directed mutagenesis studies.10
Thus, two essential structural elements are required of
a putative p38 inhibitor. First, a carbonyl group should
be utilized as the anchor point. The kinase restructuring
induced upon binding with the carbonyl group is critical
for selectivity. Second, an aryl substituent suitably teth-
ered to the main scaffold that enables its entry into the
characteristic hydrophobic pocket-1, the entrance to
which is lined by gate-keeper residue Thr-106. In addi-
tion to the above, occupation of a second partial hydro-
phobic pocket is expected to enhance potency and
provide a handle for the manipulation of physical
properties.
2-pyridazinone based inhibitors.11 It is expected that 2-
pyridinones and 4-pyridinones can also be used
similarly.11
The choice of a 2-chlorophenyl or 2,6-dichlorophenyl
group as the substituent that is expected to occupy
hydrophobic pocket 2 was based on prior SAR history.8
The synthesis of pyridazine-based scaffold is shown in
Scheme 1. Thus, condensation of maleic anhydride with
2-chlorophenyl hydrazine gave hydroxy pyridazinone
derivative 10 which was treated with phosphorus oxy-
chloride at reflux to furnish the chloro derivative 11.
Displacement of the chloride was expected to provide
an avenue to explore the requirement of a suitable tether
carrying the hydrophobic substituent. Other analogs
were synthesized in a similar fashion. Several approach-
es to gain access of the hydrophobic pocket-1 were ex-
plored. Biaryls such as 14a and 14b did not inhibit the
kinases (no activity at 20 lM). One-atom tethers such
as aryl amines, aryl sulfides and aryl ethers gave prom-
ising results (Table 1). Aryl amines 15, 17, and 18 regis-
ter IC50s in the half-micromolar range. Small alkyl
groups, chlorine, and fluorines were tolerated, while
larger substituents such as methoxy and phenyl groups
Selection criteria for an appropriate scaffold were driven
by the net dipole moment contributed by the designed
template. The hinge region peptide backbone in p38
MAP kinase is organized in a staggered arrangement
of the amide bonds as expected in the natural state. A
re-orientation of the amide bond between Met-109 and
Gly-110 is required in order to accommodate the car-
bonyl anchor of any designed inhibitor (Fig. 2). It is pro-
posed that the re-organization of amide dipoles within
the peptide backbone becomes facile with templates
characterized by a high dipole moment. Credence for
this proposal can be gleaned from a simple comparison
of p38 inhibitory activities of structurally isomeric com-
pounds 2 and 3 (Fig. 1). Both are expected to make sim-
ilar contacts in the p38 active site thereby implying
equipotency. However, 2 inhibits p38 (IC50 of 0.8 nM)
with 30-fold greater binding affinity than 3 (IC50 of
30 nM). The net dipole moments for the scaffolds pyrim-
ido-pyridazinone and pyrido-pyrimidone in 2 and 3,
respectively, are calculated to be 9.2 and 3.0 Debye
units, respectively.11 It can be speculated that the sub-
stantially higher dipole in 2 enables a much more facile
re-orientation of amide dipoles in the hinge region and
contributes to the stability of the re-organized peptide
backbone resulting in higher binding affinity. The
considerations outlined above, in part, led to design of
O
a
Cl
+
O
Cl
O
N
N
b
HN
NH2
O
OH
10
11
12
c
Cl
Cl
O
N
O
N
N
N
X
Cl
13
14a : X = F
14b : X = Cl
H N
ASP108
O
e
d
N H
MET109
O
Cl
Cl
O
N
N
O
N
H N
N
GLY 110
O
X
NH
F
F
Cl
H N
15
F
Y
ASP108
O
20 : X = S; Y =H
24 : X = O; Y= F
HN
N H
MET109
O
N
Scheme 1. General synthesis of pyridazinone scaffolds. Reagents and
conditions: (a) EtOH, reflux 12 h, 70%; (b) POCl3, 120 °C, 10 h, 82%;
(c) 1.2 equiv of boronic acid, 2.5 equiv of Cs2CO3, 0.1 equiv of
Pd(0)(PPh3)4, DMF/H2O (4:1, v/v), 100 °C, 2 h, 80%; (d) 2.5 equiv of
2-F–Ph–SH or 2,4-di-F–PhOH, 2.5 equiv of Cs2CO3, DMF, 150 °C,
0.5 h, 65–79%; (e) 1.5 equiv of 2-Cl–Ph–NH2, 0.1 equiv of Pd2(dba)3,
0.2 equiv of BINAP, 1.1 equiv of NaO-t-Bu, dioxane, 90 °C, 10 h, 65%.
H
O
Cl
N
Cl
GLY 110
Figure 2. Representation of inhibitor induced re-orientation of hinge
region of p38-MAP kinase.

S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5809–5813
Table 1. Accessing the hydrophobic pocket-1: explorations into
optimal tether lengths and substituent requirements
5811
Cl
Cl
X2
O
N
O
N
a
N
O
N
X1
O
N
N
N
NH
N
Cl
Cl
O
Y
Z1
15
30
Z2
b
Compound X1 X2
Y
Z1
Z2 p38-a p38-b
(nM) (nM)
15
16
17
18
19
20
21
22
23
24
25
26
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
NH
NH
NH
NH
NH
S
Cl
Ph
Et
Me
OMe
F
H
H
H
H
H
H
F
790
NA
560
760
2400
140
310
990
NA
NA
450
920
450
850
NA
NA
NA
NA
NA
Cl
Cl
O
N
O
N
N
N
c
N
NH
N
NH2
Cl
Cl
O
R
Me Cl
Me Cl
S
F
S
H
Cl 2000
28
Cl
Cl
Cl
H
H
H
O
Cl
F
F
NA
NA
3500
NA
Scheme 2. Elaboration of aryl amine 15. Reagents and conditions: (a)
1.5 equiv of NaH, 1.2 equiv of 3-Cl–propyl phthalimide, 0 °C, 3 h,
65%; (b) 5 equiv of NH2NH2, 75 °C, 2 h, 70%; (c) 1.2 equiv of R–
COOH, 1.2 equiv of HOBt, 2.0 equiv of EDC, 2.0 equiv of TEA, rt,
10 h.
O
F
NH–CH2 Me
CF3
H
H
Me Cl O–CH2
NA, no inh. at 20 lM. All numbers reported are mean of two inde-
pendent titrations.
were clearly detrimental. Aryl sulfide 20 inhibited both
isoforms of p38 (a and b) with an IC50 140 and
450 nM, respectively. Aryl sulfides with a chlorine sub-
stitution in the para position (22) is sixfold less potent
than corresponding fluorine analog. It was surprising
that none of the aryl ethers (23 and 24) displayed any
p38 inhibitory activity even though comparable aryl
amines were potent. It is thought that the inherent lack
of rotational bias and physical inability to access the
hydrophobic pocket might contribute to its lack of
activity. Two-atom tethers (25 and 26) were sub-opti-
mal. Based on modeling aryl amine 15 and aryl sulfide
20 within the active site of p38, it was observed that
the glycine-rich loop does not interact with the inhibitor.
It was also conceived that elaboration under this loop
will pre-orient the aryl substituent for facile entry into
the hydrophobic pocket-1. Making contact with Asp-
˚
168 situated 6–9 A from the one-atom tether affords
another opportunity to enhance potency by means of
a salt bridge (Scheme 2).
Aryl amine 15 provided a convenient starting point for
exploring these ideas. The phthalimide derivative 30
with a 3-carbon linker was found to be surprisingly po-
tent against both p38-a and p38-b. Corresponding 2C
linker was not optimal. Unravelling the phthalimide
yielded 28 with a concomitant drop-off in potency.
The phthalimide moeity is thought to be involved in a
p–p stacking interaction with tyrosine residue-35 on
the G-rich loop. Figure 3 represents an X-ray crystal
structure section of the complex between unactivated
p38 and 30.12 The G-rich loop is highlighted in green
color. The face-to-face interaction made by the pendant
Tyr-35 in the G-rich loop with the phthalimide moiety
Figure 3. X-ray Crystal structure of p38-a in complex with 30.
of 30 is obvious. The lack of potency for the correspond-
ing 2C linker can be explained due to its inability to ac-
cess a favorable stacking interaction that the 3C analog
30 is able to achieve. It can also be speculated that mere
occupation of the space under the G-rich loop is suffi-
cient to pre-orient the 2-chlorophenyl substituent for
facile access into the hydrophobic pocket thus leading
to higher potencies.

5812
S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5809–5813
Table 2. Potency augmentation on designed pyridazinone templated
p38 inhibitors: SAR explorations to optimize interactions under the
G-rich loop
Thus, analysis of the binding modes of various p38
inhibitors has enabled the design of novel, potent, and
selective p38 inhibitors based on a 2-aryl pyridazinone
scaffold. Investigations to optimize pharmacokinetic
profiles and functional activities are currently ongoing
and are expected to yield compounds of clinical
importance.
Cl
O
N
N
R
N
Cl
Z1
References and notes
Compound
R
p38-a (nM)
p38-b (nM)
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27a
168
135
OH
28
29
197
490
211
600
NH2
NH
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MeO
OMe
OMe
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O
N
N
30
5
9
15
20
O
O
O
31a
O
NH2
32
33
34
35
36
20
35
11
8
41
29
31
29
2
N
H
O
O
H
N
N
H
H
N
N
H
7. Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E.
M. US Patent 2000, 6,147,080.
O
O
N
H
NH
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Stelmach, J. E.; O’Neill, E. A.; Schwartz, C. D.; Thomp-
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N
H
20
CF3
a Z1 = H for all compounds except for 27 and 31 where Z1 = F.
Due to the comparable potency of 28 with a 2C hydroxy
analog 27, it was assumed that the amine is not posi-
tioned to make a salt bridge with the proximal aspartic
acid residue-168. In order to explore the facility of estab-
lishing this salt bridge amide analogs 32–35 were synthe-
sized. Most of these compounds inhibited both isoforms
of p38 with potency in the low nanomolar range. It is
believed that the high potency displayed by these com-
pounds primarily arises from their ability to occupy
the space under the G-rich loop rather than through a
salt bridge as the original intent for which they were de-
signed for. The equipotency of designed analog 36,
which cannot form a salt bridge with proximal Asp-
168, lends credence to the suggestion made above. It
was also encouraging to note that analogs in Table 2
did not show any significant activity (no inh. at
10 lM) when screened against an extensive panel of
known kinases particularly p38-c, p38-d, JNKs, and
ERKs.
9. (a) Wilson, K. P.; McCaffrey, P. G.; Hsiao, K.; Pazhan-
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S. R. Natarajan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5809–5813
5813
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tion_g03.htm. The calculated dipole moments of
a
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2005, 10, 1017.
pyridazinone, 2-pyridinone, and 4-pyridinone was 3.9,
4.7, and 7.1 Debye units, respectively.
11. Partial charges and dipole moments were derived from the
geometry optimization of each scaffold with the Hartree-
Fock method using 6-31G** basis set. For further infor-
12. Coordinates for the X-ray crystal structure have been
deposited. Accession codes are rscb038982 and PDB ID is
2I0H.