Theoretical and experimental design of atypical kinase inhibitors: Application to p38 MAP kinase

Article abstract of DOI:10.1021/jm050346q

Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant to the anticipated binding interaction for the fragments 1-methyl-

Full text of DOI:10.1021/jm050346q

5
728
J. Med. Chem. 2005, 48, 5728-5737
Theoretical and Experimental Design of Atypical Kinase Inhibitors:
Application to p38 MAP Kinase
Kim F. McClure,* Yuriy A. Abramov,* Ellen R. Laird, John T. Barberia, Weiling Cai, Thomas J. Carty,
Santo R. Cortina, Dennis E. Danley, Alan J. Dipesa, Kathleen M. Donahue, Mark A. Dombroski,
Nancy C. Elliott, Christopher A. Gabel, Seungil Han, Thomas R. Hynes, Peter K. LeMotte,
Mahmoud N. Mansour, Eric S. Marr, Michael A. Letavic, Jayvardhan Pandit, David B. Ripin,
Francis J. Sweeney, Douglas Tan, and Yong Tao
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, Connecticut 06340
Received April 13, 2005
Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and
their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant
to the anticipated binding interaction for the fragments 1-methyl-1H-benzotriazole 5, 3-methyl-
benzo[d]isoxazole 3, and 3-methyl-[1,2,4]triazolo[4,3-a]pyridine 4, pyridine 1, and 1,3-dimethyl-
1
,3-dihydro-benzoimidazol-2-one 2 are reported. The design considerations and synthesis of
p38 inhibitors based on these H-bond acceptor fragments is detailed. Comparative evaluation
of the pyridine-, benzimidazolone-, benzotriazole-, and triazolopyridine-based inhibitors shows
the triazoles 20 and 25 to be significantly more potent experimentally than the benzimidazolone
after which they were modeled. An X-ray crystal structure of 25 bound to the active site shows
that the triazole group serves as the H-bond acceptor but unexpectedly as a dual acceptor,
inducing movement of the crossover connection of p38R. The computed descriptors for the
hydrophobic and π-π interaction capacities were the most useful in ranking potency.
Introduction
tures is a hydrogen bond between the inhibitor and the
flexible strand referred to as the crossover connection.11
This connection is also described as the hinge region
and connects the N-terminal and C-terminal lobes of the
kinase.
In the development of potential drug candidates, lead
compounds are subject to a variety of modifications with
the goal of improving the druglike qualities of the
molecule. In many instances, the changes include
substitution of one group for another or bioisosteric
Previously, we described a series of benzimidazolone
p38 inhibitors,12 wherein we proposed that the imid-
azolone carbonyl participated in this important H-bond
with Met109NH of the crossover connection. The re-
mainder of the molecule is believed to occupy the ATP
binding site of p38R in a manner analogous to the
1
-3
changes.
When one designs bioisosteres it is impor-
tant to consider the steric and electronic properties of
the group being replaced. Knowledge of the presence or
absence of hydrogen bonding (H-bonding) interactions
is also important in contemplating molecular replace-
ments. Intermolecular hydrogen bonding plays a crucial
role in a ligand-protein binding; it outlines the specific-
ity of the interaction and counterbalances desolvation
penalties of both the ligand and the binding site. Herein
we describe a computational evaluation of several
heterocyclic systems intended to replace a typically
conserved H-bonding pattern of a ligand-kinase binding
interaction. While our work is specific to the p38R
kinase enzyme, the approach is applicable to other
protein-ligand interactions in general.
9
prototypical pyridyl-imidazole inhibitors (see Figure 1;
left). To model such benzimidazolone inhibitors into the
published active site seen with pyridyl-imidazole inhibi-
tors, small movements of the protein were required to
accommodate the span of the benzimidazolone group
between the core imidazole ring and the H-bond donor
Met109NH (see Figure 1; right). Although we could not
structurally confirm this mode of binding, it is supported
by the structure-activity relationship (SAR), which,
despite the greater spatial requirements of the benz-
imidazolone, parallels that of the pyridyl-imidazoles.12
Since the only published H-bond acceptor alternatives
to pyridine, at that time, were very close-in permuta-
tions such as pyrimidines, the benzimidazolone nucleus
represented a unique example of a significantly larger
group serving in the same role.
As part of our effort to improve our benzimidazolone
p38 inhibitors we set out to find outright replacements
for the benzimidazolone group. Herein we report a
computational evaluation of these alternatives and the
experimental follow-up of selected analogues compared
with their pyridine and benzimidazolone counterparts.
One of the concerns with the hypothetical binding
mode for the benzimidazolones (shown in Figure 1) had
The stress-activated protein kinase p38R is part of a
complex cytokine-signaling pathway. Small-molecule
inhibitors of p38R have demonstrated antiinflammatory
4
-6
properties in vivo
and have great potential in the
treatment of conditions such as rheumatoid arthritis,
psoriasis, COPD, and inflammatory bowel disease. The
X-ray crystal structure of the enzyme has been solved
with various inhibitors bound in the ATP binding
7
-10
pocket.
One of the key interactions in these struc-
*
To whom correspondence should be addressed. K.F.M.-Phone:
(
860) 441-8223. Fax: (860) 715-4610. E-mail: kim.f.mcclure@pfizer.com.
Y.A.A.-Phone (860) 715-6005. Fax: (860) 441-4734. E-mail:
yuriy.a.abramov@pfizer.com.
1
0.1021/jm050346q CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/05/2005

Design of Atypical Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5729
Figure 1. Cartoon representation (left) of p38R-SB-203580 complex 1IAN and model (right) of a benzimidazolone in the active
site of p38R. See Supporting Information for details on the model.
contributions. Because of the long-range nature of
electrostatic interactions, these were considered beyond
simply their contribution to H-bonding.
Calculations were performed to generate a set of
quantum chemical and classical descriptors relevant to
each of these interactions as discussed below. Potential
Figure 2. Structure of H-bond acceptor fragments.
steric issues resulting from the larger relative size of
template 2 were ignored, since they could not be treated
by these calculations.
to do with the added size of the benzimidazolone H-bond
acceptor group relative to pyridine. In thinking of
alternative H-bond acceptors that could be used, an
emphasis was placed on molecules that were intermedi-
ate in size relative to pyridine and benzimidazolone.13
Another concern was with the dual acceptor potential
of the imidazolone carbonyl oxygen and whether this
potential is realized in the H-bond contact with the
crossover connection Met109NH and neighboring resi-
dues. The available structural information in the lit-
erature at the time only exemplified single H-bond
acceptor or acceptor donor patterns. A loss in potency
should accompany compounds with unsatisfied H-bond
acceptor sites.
Electrostatics. The electrostatic complementarity is
determined by a favorable matching of the electrostatic
features on buried surfaces of the binding molecules,
which are responsible for the H-bonding and salt-bridge
interactions. It plays a crucial role in ligand recognition
of the kinase ATP-binding site. At the same time, the
water medium is by far the best medium for the
favorable electrostatic interaction both with a ligand
and with a protein. As a result, a strong ligand-protein
electrostatic interaction would contribute to the binding
free energy only by minimization of the desolvation
penalties.16 For this reason, the electrostatic properties
of the proposed templates 3-5 were evaluated relative
to those of the known active ligands 1 and 2, as well as
to water. A separation of the ligand-protein electro-
static interactions into close- and long-range contribu-
tions was adopted for the analysis. For close contacts,
the H-bonding capacities of the ligand were expected
to best match those of the water molecules. The elec-
trostatic long-range behavior of the ligand should be
evaluated relative to the corresponding properties of the
water molecules displaced by the ligand in the active
site.
With this in mind, our thinking centered on benzo-
fused hetereocyclic systems smaller than benzimid-
azolone that would still project two potential H-bond
1
4
acceptors. Three ideas fitting our criteria, along with
pyridine 1 and dimethylbenzimidazolone 2, are shown
in Figure 2. Because single heteroatom benzofused
systems would either have hydrogens (e.g., 2H-isoindole)
or have orthogonally disposed lone pairs (e.g., isobenzo-
furan, benzo[c]thiophene) relative to the benzimid-
azolone carbonyl oxygen, all our proposed replacements
separated the H-bond acceptors onto two adjacent
atoms. While other possibilities could also have been
explored, the unusual dual acceptor feature being
proposed warranted a cautious approach given the lack
of precedent.
The molecular electrostatic potential (MEP) associ-
ated with acceptor or donor atoms is a simple measure
of the H-bonding capacity of the molecules.1
7-20
As
shown in Figure 3 the negative MEP surface is concen-
trated at the terminal heteroatom(s) of each ring
system. Interestingly, the triazoles and benzisoxazoles
show an uneven distribution of electron density on one
of the two adjacent heteroatoms, which is reflected in
the values of the corresponding atomic charges fitted
to MEP (ESP charges, Table 1). Among all acceptors
considered in this study, the oxygen of the isolated water
molecule has the highest effective charge, which is
comparable in magnitude with the total charge of the
nitrogen acceptors of the triazolopyridine.
Computational Approach
The templates 3-5 were theoretically compared with
the known reference templates pyridine and the benz-
imidazolone 2. The best template should display optimal
nonbonding interactions with the kinase, which are
crucial for the ATP-site recognition and binding: H-
bonding, hydrophobic, and π-π interaction.15,16 With the
exception of the hydrophobic one, these interactions can
be approximated in terms of electrostatic and van der
Waals (short-range repulsion and dispersion) enthalpic

5
730 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
McClure et al.
Figure 3. Molecular electrostatic potentials of the H-bond fragments (Figure 2) on the 0.002 au isodensity surface. The potentials
are calculated at the B3LYP/6-311G(d,p) level of theory and shown with identical chromatic scales (red is more negative, greater
electron density; blue is more positive, less electron density).
Table 1. Calculated Molecular and Atomic Properties
compound
pyridine
benzoimidazolone
benzoisoxazole
ESP charges
µ, D
µ/V, D/Å3
∆Gsolv, kcal/mol
logP
ASA-H (Å2)
η (eV)
1
2
3
N, -0.62
O, -0.53
O, -0.17
N, -0.37
2.20
2.13
3.17
0.026
0.013
0.024
-5.2
-6.7
-5.6
0.73
1.46
2.09
108.2
311.7
239.6
5.78
5.06
5.57
a
4
5
benzotriazole
triazolopyridine
water
N3 , -0.38
4.09
5.54
2.07
0.031
0.043
0.107
-8.3
-11.9
-7.3
1.06
0.94
206.5
187.4
5.50
5.23
a
N2 , -0.21
a
N3 , -0.44
a
N2 , -0.32
6
O, -0.74
a
Numbered to be consistent with compounds 20 and 25.
The long-range behavior of a ligand MEP contributes
to the interaction with remote areas of the kinase. It
can be mathematically presented by expansion over the
electrostatic moments (the multipole expansion).21,22 In
the case of the neutral molecules, the molecular dipole
moment, µ, describes the major contribution to the long-
range electrostatic interactions. The calculated dipole
moments for the templates are listed in Table 1. All
dipole moments are approximately parallel to each other
on the external electric field F imposed by the unper-
turbed kinase. This leads to the formation of induced
molecular dipole moment, ∆µ, which contributes to the
attractive electrostatic interaction with the kinase. The
above considerations for the molecular dipole moment
µ are also applicable to the ∆µ. This moment can be
approximated as ∆µ ) /2RF, where R is a molecular
polarizability. Since R is linearly correlated with the
molecular volume,23 the R/V values are quite similar for
all the ligands under consideration and are equal to
∼0.10 at the B3LYP/6-311G(d,p) level of theory. Since
the field F is also considered to be fixed for a given
protein conformation, ∆µ/V should be a uniform prop-
erty for all the ligands.
Thus, with respect to the overall electrostatic proper-
ties in water, the benzotriazole and triazolopyridine
display superior characteristics, which are crucial for
the recognition component of binding. On the other
hand, the calculated solvation energies are also the
highest for these ligands (Table 1), which correspond
to the higher penalties to be paid upon ligand desolva-
tion in the active site.
1
22
(not shown) and describe directionality of the binding
to the ATP site; this is an important factor in the ligand/
kinase molecular recognition. The contribution of these
dipole moments to the overall binding free energies
should be evaluated relative to the sum of the dipole
moments of the water molecules displaced by the
templates 1-5 in the cavity. This allows for the intro-
duction of an approximate correction for the actual
binding site desolvation penalty. This correction de-
pends on the molecular van der Waals volume, V, which
varies for the fragments considered in the current study.
In the simplest case, the dipole moments of the dis-
placed waters are uniformly aligned along the µ of the
template. Despite the obvious simplification, this allows
for the harmonization of all the dipole values to one
scale by calculating µ/V descriptors (Table 1). For the
fragments under consideration, this property is the
Hydrophobic Interaction. Molecular recognition
due to the hydrophobic interactions is determined by
shape matching and lipophilic (hydrophobic) comple-
mentarity. In contrast to the electrostatics, the hydro-
phobic interaction with the binding site introduces the
3
highest for the triazolopyridine (0.043 D/Å ) and the
3
16
benzotriazole (0.031 D/Å ), demonstrating that the
major contribution to the binding free energy. This
triazoles have the most favorable long-range electro-
interaction at room temperature is believed to be
entropy-driven due to reorganization of water molecules
static properties for binding. At the same time, the µ/V
3
value of 0.107 D/Å for the water molecule reflects the
at the interface with the hydrophobic (nonpolar) sur-
faces.2
4,25
The hydrophobic interaction capacity can be
superior capacity of the aqueous medium for attractive
electrostatic interactions.
evaluated by the water-accessible hydrophobic surface
25
The electrostatic descriptors considered thus far
reflect the properties of the free unperturbed molecules.
However, polarization of the molecular charge density
is an additional phenomenon, which needs to be con-
sidered with respect to the ligand/protein electrostatic
interaction. It takes place upon binding as a response
area of the molecule, ASA-H, and the octanol-water
partitioning coefficient, logPow.26 The corresponding
properties are shown in Table 1. Based on both descrip-
tors, the highest hydrophobicity was found for the
benzimidazolone and benzoisoxazole and the lowest one
for pyridine. The triazole templates display comparable

Design of Atypical Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5731
Scheme 1. Synthesis of Pyridine Derivativea
hydrophobic parameters, the benzotriazole being some-
what more hydrophobic.
π-π Interactions. This is an attractive medium-
range interaction between aromatic (π-electron) molec-
ular fragments, which takes its origin from dispersion
2
7
and electrostatic forces. These same fragments also
usually contribute to the hydrophobic interaction energy
with the binding site. The molecular recognition due to
π-π interactions is determined by the orientation
priorities of the interacting fragments, which are dic-
tated by the intermolecular electrostatic forces. The
strength of the π-π attraction is predominantly deter-
a
Reagents and conditions: (a) K2CO3, acetonitrile, 22 °C, 2 h,
then 70 °C, 18 h, 60%.
Scheme 2. Synthesis of Benzimidazolone Derivativea
27
mined by dispersion interactions and thus by a degree
of aromaticity of the ligand. The latter property can be
evaluated by absolute chemical hardness, η, as ap-
proximated by using Koopmans’ theorem: η ) (LUMO
2
8
-
HOMO)/2. Here HOMO and LUMO are, respec-
tively, energies of the highest occupied and lowest
unoccupied molecular orbitals. The highest aromaticity
(
η) was found for the pyridine and benzoisoxazole mole-
cules, followed by the benzotriazole and triazolopyridine.
Once the computational evaluation of the various
fragments was completed, the challenge was to use this
information to identify those potentially superior to the
benzimidazolone 2. Although it is possible to rank the
fragments according to their hydrophobic and π-π
interaction capacities in the order of benzoisoxazole >
benzotriazole > triazolopyridine and in the opposite
order for their electrostatic properties, it is their com-
bined effect on the free energy of binding that is
meaningful. The hydrophobic and π-π interaction
properties should predominate in determining the bind-
ing potency. As compared to the benzimidazolone 2,
the three proposed fragments should be reasonable
substitutes since they all have superior π-π interaction
capacities balanced by reduced capacities for hydropho-
bic interactions. Ultimately, the ability of these frag-
ments to replace the benzimidazolone group would
require experimental follow-up.
a
Reagents and conditions: (a) oxalyl chloride, cat. N,N-di-
methylformamide, CH2Cl2, 22 °C, 16 h, then Et3N, MeOH, 22 °C,
6 h, 96%; (b) isopropylamine, CH2Cl2, 22 °C, 2 h, 91%; (c) H2,
Pd/C, 22 °C, 1 h, 100%; (d) phosgene, PhMe/CH2Cl2, 22 °C, 16 h,
6%; (e) NaH, dimethyl sulfoxide, 22 °C, 1 h, then MeI, 22 °C, 48
1
5
h, 63%; (f) LiBH4, THF, MeOH 80 °C, 18 h, 67%; (g) TEMPO,
N-chlorosuccinimide, tetrabutylammonium chloride, pH ) 8.6
buffer, CH2Cl2, 22 °C, 16 h, 68%; (h) 8, K2CO3, acetonitrile, 22 °C,
2
h, then 50 °C, 36 h, 11%.
Scheme 3. Synthesis of Benzotriazole Derivativea
1
6
Synthesis of Kinase Inhibitors
In contemplating the experimental evaluation of the
proposed atypical kinase templates, we planned to
incorporate these fragments into inhibitors within a
constant framework and determine their biological
activity in a p38R enzyme inhibition assay. The benzo-
isoxazole fragment was eliminated from synthetic con-
sideration because any eventual inhibitor incorporating
this group would be more lipophilic (thus potentially
more metabolically unstable) than the compounds it was
designed to replace. Since in vitro microsomal stability
was frequently problematic for the benzimidazolone
series, we had little interest in compounds potentially
less stable. The constant framework chosen for the
remaining compounds was 4-(4-fluoro-phenyl)-oxazole.
While it is known that using oxazole as the central
ring is not optimal for potency verses p38R, it does
eliminate the H-bond donor capacity and tautomer
ambiguity associated with using an imidazole core. The
final consideration was with the connectivity of the
proposed H-bond acceptor with the central oxazole ring
and the nature of any substituent. Previous work in our
labs on benzimidazolone analogues indicated that a
highly favorable substitution pattern is isopropyl, meth-
a
Reagents and conditions: (a) acetone, AcOH, THF/N,N-
dimethylformamide, 2 h, 22 °C, then NaBH(OAc)3, 2.5 h, 36%; (b)
6 N HCl, aqueous NaNO , 0 °C, 1 h, 79%; (c) oxalyl chloride,
CH2Cl2, cat. N,N-dimethylformamide, 22 °C, 4 h, then (i-Pr)2NEt,
NHMeOMeHCl, 22 °C, 16 h, 77%; (d) DIBAL-H, PhMe, -78 to 0
C, 1.5 h, 67%; (e) 8, K2CO3, CH3CN, 22 °C, 4 h, then 70 °C, 14 h,
2%.
2
°
6
yl with the connection to the central ring at C5 (see 15
in Scheme 2 for numbering). However, based on the
localization of the electron density on N3 of the benzo-
triazole system, modeling relative to the benzimid-
azolone suggested that the better point of attachment
for the triazoles would be at C6 rather than C5 (see 20
in Scheme 3 for numbering). For consistency, isopropyl
was chosen as the substituent for the benzotriazole
fragment. The triazolopyridine was matched to the
benzotriazole, and the pyridine analogue was used as a
reference agent to archetypal pyridyl-imidazole p38
inhibitors.
1
2

5
732 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
McClure et al.
Scheme 4. Synthesis of Triazolopyridine Derivativea
Table 2. p38R Enzyme Inhibition Results
p38R
IC50 (nM)
n/SD
(nM)
∆∆G/N
compound
(kcal/mol)a
9
1
2
1460
30
5/345
9/3.5
10/0.1
18/0.5
0.44
0.40
0.51
0.47
5
0
0.9
5.0
CP-808844 25
a
∆∆G ) -RT ln(IC50); N is the number of non-hydrogen atoms.
a
Reagents and conditions: (a) hydrazine hydrate, PEG/2-
butanol/water, 98 ° C, 29 h, 87%; (b) isobutyryl chloride, reflux, 3
h, then NaOH freebase, 74%; (c) i-PrMgCl, THF, -6 to 6 °C, 0.5
h, then N,N-dimethylformamide, reflux, 3 h, 78%; (d) 8, K2CO3,
CH3CN, 22 °C, 16 h, then 70 °C, 24 h, 98%.
The syntheses of the four compounds used in the
comparison are shown in Schemes 1-4. As shown in
Scheme 1, the known pyridine analogue was prepared
2
9-31
in a single step
from commercially available 4-
pyridylcarboxaldehyde.
Figure 4. X-ray structure of 25 bound to p38R.
As shown in Scheme 2, the synthesis of the benz-
imidazolone 15 starts from commercially available
-fluoro-3-nitrobenzoic acid by esterification, reaction
with isopropylamine, and hydrogenation to give 11 in
7% overall yield. Imidazolone ring formation with
23 to 24 occurs. The structure of each isomer was
confirmed by X-ray crystallographic determination.
Reaction of the aldehyde 23 as before gave 25 in 98%
yield.
4
8
phosgene and alkylation with methyl iodide gave the
benzimidazolone intermediate 12 in 30% yield. Reduc-
tion with lithium borohydride and N-chlorosuccinimide/
TEMPO oxidation gave the aldehyde 14 in 45% yield.
Finally, reaction of 14 with 8 gave 15 in poor yield as
result of the need to balance the thermal requirement
for [3 + 2] reaction and the decomposition of the
isonitrile under the conditions.
The synthesis of the benzotriazole starts with the
selective reductive amination of 3,4-diaminobenzoic acid
with acetone, exploiting the difference in reactivity of
the amines due the presence or absence of conjugation
with the carboxylic acid (see Scheme 3). The regio-
chemistry of reaction was confirmed by X-ray crystal-
lographic analysis of 17. Reaction of 17 with sodium
nitrite and hydrochloric acid cleanly gave the benzo-
triazole acid 18. Conversion of the acid 18 to the
Weinereb amide followed by DIBAL-H reduction gave
the aldehyde 19 in 51% yield. The target compound was
again prepared by reaction of the aldehyde with 8; this
time the better yield is presumed to arise from the
ability of the [3 + 2] intermediate adduct to form
without heating. Subsequent elimination of the sulfinic
acid requires heating.
Results and Discussion
With the synthesis complete, the compounds were
32
evaluated in a p38R isolated enzyme assay (see Table
12
29-31
2). Although the pyridyl analogue 9 is known,
the
p38R activity has not been reported, and 9 was included
as a reference agent to put the activity of the other three
compounds into perspective. The most appropriate
comparisons can be made between the triazole ana-
logues 20 and 25 and with the benzimidazolone 15 after
which they were conceived. The superior potency of the
triazoles relative to the benzimidazolone 15 eliminated
our initial concerns about their dual atom H-bond
acceptor capacity, but confirmation of the binding mode
was not obtained until the X-ray structure determina-
tion of a complex between p38R and 25 was achieved.36
As shown in Figure 4, this revealed that both triazole
nitrogens serve as H-bond acceptors as a result of
movement of the crossover connection residues, which
allows Gly110NH, in addition to Met109NH, to partici-
pate as an H-bond donor. Similar movement has been
seen recently with other p38 inhibitors;1
0,37
however, to
our knowledge this represents the first example of a
kinase inhibitor where two distinct atoms act as H-bond
acceptors with the crossover connection region. In light
of these results, it is also likely that the carbonyl group
in the benzimidazolone 15 acts as a dual H-bond
acceptor as is seen with other carbonyl-containing p38
inhibitors.10
Finally, the triazolopyridine 25 (CP-808844) was
assembled as shown in Scheme 4. Reaction of hydrazine
and 2,5-dibromopyridine in aqueous 2-butanol-PEG
mixture followed by acylation and cyclodehydration in
neat isobutyryl chloride gave the bromo triazolopyridine
2
2. The transformation of the bromide to the aldehyde
In terms of making comparisons between 9, 15, 20,
and 25 beyond strictly potency as a way of prioritizing
follow-up work, it is possible to crudely evaluate their
went smoothly to the desired aldehyde 23 by Grignard
3
3
exchange followed by reaction with N,N-dimethyl-
formamide provided that the workup conditions were
neither too basic nor acidic. Under aqueous workup
conditions that strayed significantly from pH 7, com-
binding efficiencies without regard for potential differ-
ences in binding mode or steric interactions.3
9,40
One
simple ligand evaluation method can be performed using
3
4,35
40
plete Dimroth-like rearrangement
of the aldehyde
the maximum ligand affinity scale. For this, the IC50

Design of Atypical Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5733
values are converted to the free energy of binding as
hydrophobic and π-π interaction capacities were the
most useful in ranking the potency.
∆∆G ) -RT ln(IC50). All ligands, except 9, fit the ∆∆G
dependence for the number of non-hydrogen atoms, N,
determined for a wide range of the tight-binding ligands.
Comparison of the ligands was performed in terms of
the free energy of binding per heavy atom, ∆∆G/N
Computational Details
The geometries of the five fragments were fully optimized
in the gas phase at the B3LYP/6-31G(d) level of theory. The
molecular dipole moments, isotropic polarizabilities, and atomic
charges were evaluated by single-point calculations at the
B3LYP/6-311G(d,p) level. The ESP atomic charges were
40
(Table 2). The higher this property the more efficiently
the non-hydrogen atoms drive the affinity. In agreement
with the IC50 data, the highest ∆∆G/N values were
found for the triazole-based ligands. These data also
suggest that the smallest ligand, 9, is more efficient
than the largest one, 15. This observation seems to
indicate that 9 is favorable for further optimization, but
given its absolute potency, it is not clear that this
compound would be a better choice than the triazoles.
4
1
calculated according to the CHELPG algorithm. The polar-
4
2,43
ized continuum model (PCM)
was adopted at the HF/6-
31+G(d) level of theory for the geometry optimizations in
water, followed by single-point calculation of the solvation free
energy, ∆Gsolv, and absolute chemical hardness, η, descriptors.
The majority of the quantum chemical calculations were
4
4
performed by the Gaussian98/03 program suites. The mo-
lecular electrostatic potentials were calculated at the B3LYP/
In terms of actually trying to understand the potency
outcome and which calculated properties were the
most predictive, the situation is more complex. While
the MEP alone correctly predicted the trend in potency
for a series of compounds reported by Johnson &
6
-311G(d,p) level of theory and plotted using Spartan’02
45
software.
The octanol-water partition coefficients, logP, were pre-
46
dicted using ACD/labs 6.00 software package. The van der
Waals molecular volume, V, and water-accessible hydrophobic
19,20
surface area, ASA-H, descriptors were evaluated by the MOE
Johnson,
the triazole examples herein highlight the
47
software. Only atoms with absolute values for the ESP atomic
need to also evaluate the capacity for hydrophobic and
-
charges of less than 0.2 e were considered in calculating the
π-π interactions. The superior electrostatic properties
ASA-H. The molecular geometries optimized in solution were
adopted for these calculations.
(higher H-bonding capacity and higher µ/V, Figure 3,
Table 1) notwithstanding, the triazolopyridine 25 is less
potent than the benzotriazole 20. This observation is
consistent with the fact that H-bond and electrostatic
interactions generally do not contribute much to the
overall free energy of binding as a result of the trade
offs with desolvation. On the other hand, the potency
of the two triazoles can be correctly ranked by their
capacity for hydrophobic and π-π interactions (Table
Experimental Section
Melting points were determined using a B u¨ chi B-545
melting point apparatus and are uncorrected. H NMR spectra
1
were obtained using a Varian Unity 400 or 500 MHz. Chemical
shifts are reported in parts per million (δ) relative to residual
chloroform (7.24 ppm), dimethyl sulfoxide (2.49 ppm), or
methanol (3.30 ppm) as an internal reference. Coupling
constants (J) are reported in hertz (Hz). The peak shapes are
denoted as follows: s, singlet; d, doublet; t, triplet; m, mul-
tiplet; br, broad. APcI mass spectra were recorded by direct
flow analysis for positive and negative atmospheric pressure
chemical ionization (APcI) scan modes using a Waters APcI/
MS model ZMD mass spectrometer equipped with Gilson 215
liquid handling system. GCMS mass spectral data were
obtained using the electron impact ionization technique of the
parent molecular mass using a Hewlett-Packard 6890 series
GC system equipped with a Hewlett-Packard 5973 mass
selective detector. LCMS molecular weight identification was
recorded by positive and negative electrospray ionization (ESI)
scan modes using a Waters/Micromass ESI/MS model ZMD/
LCZ mass spectrometer equipped with Gilson 215 liquid
handling system and Hewlett-Packard 1100 diode array detec-
tor. Small-molecule X-ray structures were collected on a
Bruker APEX or a Siemens P4 diffractometer. Normal-phase
silica gel flash chromatography was performed using Biotage
Flash40 or 25 systems. Reverse-phase purification was con-
ducted with a Waters 600 system with a 486 tunable detector.
Elemental analyses were performed at Quantitative Technolo-
gies Inc., Whitehouse, NJ. The term “concentrated” refers to
the removal of solvent under reduced pressure using a rotory
evaporator.
1
6
1
). These types of interactions are especially important
given the conformational changes of the activation loop
upon ligand binding with the Tyr35 side-chain “closing“
down on the binding site (Figure 4). The resulting “T-
9
shaped” orientation of the tyrosine phenol and the
pyridine ring of 25 is not far from the ideal for π-π
interactions: the distance between ring centers is 5.9
Å in comparison with 5.0 Å for the corresponding
2
7
benzene dimer at equilibrium. Even at this distance,
there is an important attractive contribution for the π-π
interaction energy between benzene molecules esti-
2
7
mated at about 1.5 kcal/mol. With respect to the
potency of the triazoles relative to the benzimidazolone
15, the superior electrostatic properties of the triazole
may contribute, but more likely there are steric con-
straints that prevent 15 from reaching its full potential.
The latter interpretation is consistent with the observa-
tion that the benzimidazolone 15 displays the lowest
∆∆G/N value among all the ligands under consideration
(Table 2).
4
-[4-(4-Fluorophenyl)-5-oxazolyl]-pyridine (9).29,31 To a
Conclusion
stirred solution of 4-pyridinecarboxaldehyde (0.53 g 5.0 mmol)
in 10 mL of acetonitrile was added R-(p-toluensulfonyl)-4-
fluorobenzylisonitrile (1.45 g, 5.0 mmol) followed by potassium
carbonate (0.9 g, 6.5 mmol). The mixture was stirred at 22 °C
for 2 h and at 70 °C for 16 h. The mixture was cooled to 22 °C,
diluted with water, and extracted with methylene chloride. The
organic extracts were dried over sodium sulfate and filtered.
The filtrate was concentrated, and the crude solid was purified
by flash chromatography, eluting with ethyl acetate and
hexanes (1:1) to give 0.73 g (60%) of 4-[4-(4-fluorophenyl)-5-
In this work, we set out to use a set of computationally
derived descriptors to evaluate a series of heterocyclic
fragments for their possible use in atypical kinase
inhibitors. In the end, we were able to identify signifi-
cantly more potent compounds that at the same time
have superior calculated physicochemical properties
than the benzimidazolone group that they were de-
signed to replace. While a complex interplay exists
between the calculated parameters used, descriptors for
29,31
oxazolyl]-pyridine
as a white solid. LCMS (m/z): 241 (M
1
+ H). H NMR (400 MHz, CDCL ): δ 8.60 (dd, J ) 4.6, 1.7
3

5
734 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
McClure et al.
Hz, 2 H), 8.00 (s, 1 H), 7.60 (m, 2 H), 7.46 (dd, J ) 4.6, 1.7 Hz,
H), 7.12 (m, 2 H).
-Fluoro-3-nitrobenzoic Acid Methyl Ester (10). To a
stirred solution of 4-fluoro-3-nitrobenzoic acid (50.0 g, 0.27 mol)
in 500 mL of methylene chloride was added 1 mL of N,N-
dimethylformamide, followed by oxalyl chloride (26 mL, 0.30
mol), and the mixture was stirred at 22 °C for 16 h. The yellow
solution was concentrated to a yellow semisolid and used in
the subsequent step.
carboxylic acid methyl ester as a white solid. LCMS (m/z): 249
1
2
(M + H). H NMR (400 MHz, CDCl
3
): δ 7.81 (d, J ) 8.3 Hz, 1
H), 7.64 (s, 1 H), 7.12 (d, J ) 8.3 Hz, 1 H), 4.69-4.76 (m, 1 H),
.91 (s, 3 H), 3.43 (s, 3 H), 1.52 (d, J ) 7.0 Hz, 6 H). Anal.
Calcd for C13 : C, 62.89; H, 6.50; N, 11.28. Found: C,
2.82; H, 6.47; N, 11.34.
5-Hydroxymethyl-1-isopropyl-3-methyl-1,3-dihydro-
4
3
16 2 3
H N O
6
benzoimidazol-2-one (13). To a stirred solution of 1-iso-
propyl-3-methyl-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carbox-
ylic acid methyl ester (10.5 g, 42.3 mmol) in 140 mL of tetra-
hydrofuran was added lithium borohydride (1.84 g, 84.6 mmol)
at 22 °C. The mixture was heated to 80 °C, and methanol (6.8
mL, 16.9 mmol) was added dropwise with gas evolution. The
resulting mixture was heated at 80 °C for 18 h before being
cooled to 22 °C. The hazy mixture was diluted with saturated
sodium bicarbonate and extracted with methylene chloride
(4×). The combined organic extracts were washed with water
and brine, dried over sodium sulfate, and filtered. The filtrate
was concentrated to give 6.27 g (67%) of 5-hydroxymethyl-1-
To a stirred solution of the crude acid chloride in 200 mL of
methylene chloride was added dropwise a mixture of 74 mL
of triethylamine and 86 mL of methanol. The mixture was
stirred for 16 h at 22 °C and diluted with aqueous sodium
bicarbonate, and the layers were separated. The organic phase
was washed with saturated aqueous bicarbonate (3×), water
(
1×), and brine (1×) and dried (Na
2 4
SO ) and filtered. The
filtrate was concentrated to a yellow oil, which solidified to
give 52.4 g (96%) of 4-fluoro-3-nitrobenzoic acid methyl ester
1
as a yellow solid. H NMR (400 MHz, CDCl
3
): δ 8.72 (dd, J )
8
. 0, 2.1 Hz, 1 H), 8.30 (d, J ) 8. 7, 4.2, 2.1 Hz, 1 H), 7.37 (dd,
J ) 10.3, 8.7 Hz, 1 H), 3.96 (s, 3 H). Anal. Calcd for C
FNO : C, 48.25; H, 3.04; N, 7.03. Found: C, 48.27; H, 2.83;
N, 6.89.
-Amino-4-isopropylaminobenzoic Acid Methyl Ester
11). To a stirred, cold (0 °C) solution of 4-fluoro-3-nitrobenzoic
isopropyl-3-methyl-1,3-dihydro-benzoimidazol-2-one as a white
1
H
6
-
solid. LCMS (m/z): 221 (M + H). H NMR (400 MHz, CDCl
3
):
8
δ 7.01-7.10 (m, 3 H), 4.68-4.75 (m, 3 H), 3.33 (s, 3 H), 1.50
(d, J ) 7.0 Hz, 6 H). Anal. Calcd for C12 : C, 65.43; H,
.32; N, 12.72. Found: C, 65.29; H, 7.43; N, 12.55.
1-Isopropyl-3-methyl-2-oxo-2,3-dihydro-1H-benzo-
imidazole-5-carbaldehyde (14). To stirred solution
4
16 2 2
H N O
7
3
(
acid methyl ester (5.0 g, 25.1 mmol) in 80 mL of methylene
chloride was added isopropylamine (4.2 mL, 49.7 mmol). The
cold bath was removed after 3 h, and the mixture was stirred
at 22 °C for 2 h. The mixture was diluted with water and
extracted with methylene chloride (3×). The combined organic
a
of 5-hydroxymethyl-1-isopropyl-3-methyl-1,3-dihydro-benzo-
imidazol-2-one (8.1 g, 36.8 mmol) in 200 mL of methylene
chloride was added in the following order: TEMPO (1.7 g, 11.0
mmol), tetrabutylammonium chloride (3.1 g, 11.0 mmol),
N-chlorosuccinimide (9.3 g, 69.9 mmol), and 400 mL of pH 8.6
2 4
extracts were washed with water and brine, dried (Na SO ),
32
and filtered. The filtrate was concentrated to give 5.5 g (91%)
of 4-isopropylamino-3-nitrobenzoic acid methyl ester as a
yellow solid.
3 2 3
buffer (NaHCO -K CO ). The resulting mixture was stirred
at 22 °C for 18 h. The layers were separated, and the aqueous
layer was extracted with methylene chloride (3×). The com-
bined organic extracts were washed with aqueous sodium
A mixture of 4-isopropylamino-3-nitrobenzoic acid methyl
ester (4.5 g, 18.9 mmol), 60 mL of ethanol, and palladium on
carbon (0.25 g) was shaken under a 40 psi atmosphere of
hydrogen gas for 1 h. The mixture was filtered through nylon
and concentrated to give 3.9 g (100%) of 3-amino-4-isopropyl-
bicarbonate (1×), water (1×), and brine (1×), dried (Na
2 4
SO ),
and filtered, and the filtrate was concentrated to a dark yellow
solid. The material was purified by flash chromatography,
eluting with 2:1 hexanes/ethyl acetate to give 5.5 g (68%) of
1-isopropyl-3-methyl-2-oxo-2,3-dihydro-1H-benzoimidazole-5-
aminobenzoic acid methyl ester as an off-white solid. LCMS
1
carbaldehyde as a white solid. LCMS (m/z): 219 (M + H).
1
H
(
m/z): 209 (M + H). H NMR (400 MHz, CDCl
3
): δ 7.56 (d, J
)
8.3 Hz, 1 H), 7.39 (s, 1 H), 6.58 (d, J ) 8.3 Hz, 1 H), 3.83 (s,
H), 3.67-3.70 (m, 1 H), 1.24 (d, J ) 6.6 Hz, 6 H). Anal. Calcd
: C, 63.44; H, 7.74; N, 13.45. Found: C, 63.19;
NMR (400 MHz, CDCl ): δ 9.92 (s, 1 H), 7.58 (dd, J ) 8.3, 1.7
3
3
Hz, 1 H) 7.51 (s, 1 H), 7.21-7.25 (m, 1 H), 4.71-4.78 (m, 1 H),
3.44 (s, 3 H), 1.54 (d, J ) 7.0 Hz, 6 H). Anal. Calcd for
for C11
16 2 2
H N O
H, 7.47; N, 13.09.
-Isopropyl-3-methyl-2-oxo-2,3-dihydro-1H-benzo-
12 14 2 2
C H N O : C, 66.04; H, 6.47; N, 12.84. Found: C, 65.99; H,
6.23; N, 12.80.
1
imidazole-5-carboxylic Acid Methyl Ester (12). To a
stirred solution of 3-amino-4-isopropylaminobenzoic acid meth-
yl ester (3.7 g, 17.8 mmol) in 50 mL of methylene chloride was
added phosgene (9.4 mL, 20% in toluene) dropwise. The
mixture was stirred at 22 °C for 16 h; the reaction was
quenched with saturated aqueous bicarbonate, and the mix-
ture was extracted with methylene chloride (3×). The com-
bined organic extracts were washed with water and brine,
5-[4-(4-Fluoro-phenyl)-oxazol-5-yl]-1-isopropyl-3-meth-
yl-1,3-dihydrobenzoimidazol-2-one (15). To a stirred sus-
pension of 1-isopropyl-3-methyl-2-oxo-2,3-dihydro-1H-benzo-
imidazole-5-carbaldehyde (0.20 g, 0.92 mmol) in 3 mL of
acetonitrile was added R-(p-toluensulfonyl)-4-fluorobenzyl-
isonitrile (0.53 g, 1.8 mmol) and powdered potassium carbonate
(0.25 g, 1.8 mmol). The mixture was heated at 50 °C for 18 h
before adding another equivalent each of R-(p-toluensulfonyl)-
4-fluorobenzylisonitrile (0.26 g, 0.92 mmol) and potassium
carbonate (0.13 g, 0.92 mmol). The mixture was heated at 50
°C for an additional 18 h. The mixture was cooled to 22 °C,
diluted with water, and extracted with ethyl acetate (3×). The
combined organic extracts were washed with water and brine,
dried (Na
give a tan solid. This material was suspended in a mixture of
mL of hexanes, 1 mL of ethyl acetate, and 0.5 mL of
2 4
SO ), and filtered. The filtrate was concentrated to
8
methylene chloride and stirred for 16 h at 22 °C. The
suspension was filtered, and the solids were collected and dried
to give 2.35 g (56%) of 1-isopropyl-2-oxo-2,3-dihydro-1H-
benzoimidazole-5-carboxylic acid methyl ester as a white solid.
To a stirred solution of 1-isopropyl-2-oxo-2,3-dihydro-1H-
benzoimidazole-5-carboxylic acid methyl ester (0.54 g, 2.3
mmol) in 5 mL of dimethyl sulfoxide was added sodium hydride
2 4
dried (Na SO ), and filtered, and the filtrate was concentrated
to a dark yellow solid. This material was purified by flash
chromatography, eluting with 3:2 hexanes/ethyl acetate to give
0.2 g of an off-white foam. This material was further purified
by reverse-phase HPLC (MS C18 Xterra 5 µm, 30 mm × 100
mm; 42 mL/min; 214 nM detection), eluting from 6:4 water/
acetonitrile to 2:8 water/acetonitrile (with 0.1% TFA) over 10
min. The desired material had a retention time of 6.3 min.
This material was taken-up in ethyl acetate and washed with
aqueous sodium bicarbonate (1×), water, and brine, dried
(60%, 0.11 g, 2.8 mmol). The resulting mixture was stirred for
1 h at 22 °C before methyl iodide (0.3 mL, 4.6 mmol) was
added. The mixture was stirred for 48 h at 22 °C; the reaction
was quenched with water, and the mixture was extracted with
methylene chloride (3×). The combined organic layers were
washed with water and brine, dried (Na
2
SO
4
), and filtered,
2 4
(Na SO ), and filtered, and the filtrate was concentrated to
and the filtrate was concentrated to give 0.65 g of a yellow
solid. This material was purified by flash chromatography,
eluting with 3:2 hexanes/ethyl acetate to give 0.36 g (63%) of
give 100 mg of a white foam. Final purification was then
achieved by flash chromatography, eluting with 55:45 hexanes/
ethyl acetate to give 36 mg (11%) of 5-[4-(4-fluoro-phenyl)-
oxazol-5-yl]-1-isopropyl-3-methyl-1,3-dihydrobenzoimidazol-2-
1-isopropyl-3-methyl-2-oxo-2,3-dihydro-1H-benzoimidazole-5-

Design of Atypical Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5735
1
1
one as a white solid. LCMS (m/z): 352 (M + H). H NMR (500
z): 190 (M + H). H NMR (400 MHz, CDCl
3
): δ 10.17 (s, 1 H),
MHz, CDCl
3
): δ 7.98 (s, 1 H), 7.67-7.70 (m, 2 H), 7.28-7.31
m, 1 H), 7.19 (d, J ) 1.6 Hz, 1 H), 7.08-7.14 (m, 3 H), 4.73-
.79 (m, 1 H), 3.38 (s, 3 H), 1.56 (d, J ) 6.7 Hz, 6 H). Anal.
: C, 68.36; H, 5.16; N, 11.96. Found C,
8.17; H, 4.93; N, 11.56.
-Amino-3-isopropylaminobenzoic Acid (17). To a stirred
8.18 (d, J ) 8.7 Hz, 1H), 8.12 (d, J ) 1.2 Hz, 1 H), 7.88 (dd, J
) 8.7, 1.2 Hz, 1 H), 5.16 (m, 1 H), 1.77 (d, J ) 6.6 Hz, 6 H).
Anal. Calcd for C10
Found: C, 63.11; H, 5.65; N, 22.22.
-[4-(4-Fluoro-phenyl)-oxazol-5-yl]-1-isopropyl-1H-
(
4
11 3
H N O: C, 63.48; H, 5.86; N, 22.21.
Calcd for C20
H
18FN
3
O
2
6
6
4
benzotriazole (20). To a stirred solution of 3-isopropyl-3H-
benzotriazole-5-carbaldehyde (0.19 g, 1.0 mmol) and R-(p-
toluensulfonyl)-4-fluorobenzylisonitrile (0.32 g, 1.1 mmol) in
3.3 mL of acetonitrile was added powdered potassium carbon-
ate at 22 °C. The resulting suspension was stirred for 4 h at
22 °C and for 14 h at 70 °C. The mixture was cooled to 22 °C,
diluted with water, and extracted with ethyl acetate (3×). The
combined organic extracts were washed with brine and dried
over sodium sulfate. Filtration and concentration of the filtrate
gave a yellow oil, which crystallized upon standing. The crude
solid was purified by flash chromatography, eluting with ethyl
acetate and hexanes (65:35), to give 300 mg of an off-white
solid. Recrystallization of this material from hot ethyl acetate
and hexanes gave 0.20 g (62%) of 6-[4-(4-fluoro-phenyl)-oxazol-
solution of 3,4-diaminobenzoic acid (25.0 g, 0.16 mol) in 300
mL of tetrahydrofuran and 45 mL of N,N-dimethylformamide
was added 12 mL (0.16 mol) of acetone and 9.4 mL of acetic
acid. The mixture was stirred at 22 °C for 2 h before sodium
triacetoxyborohydride (41.8 g, 0.20 mol) was added portion-
wise. The resulting mixture was stirred for 2.5 h before being
filtered through Celite and rinsed with tetrahydrofuran. The
filtrate was concentrated to a dark oil, which was mixed with
water to give a tan precipitate. The solids were collected by
vacuum filtration, suspended in a mixture of diisopropyl ether,
methanol, and ethyl acetate, and stirred at 22 °C for 16 h.
The solids were collected by vacuum filtration, and rinsed with
ethyl acetate and water to give after drying 11.7 g (36%) of
-amino-3-isopropylaminobenzoic acid as an off-white solid
discolors over time), which was crystallized for X-ray deter-
mination by slow evaporation from ethyl acetate and hexanes;
mp ) 151.8-152.1 °C. H NMR (500 MHz, CDCl
J ) 7.7 Hz, 1 H), 7.44 (s, 1 H) 6.72 (d, J ) 7.7 Hz, 1 H), 3.65-
.70 (m, 1 H), 1.26 (d, J ) 6.2 Hz, 6 H). Anal. Calcd for
O: C, 61.84; H, 7.27; N, 14.42. Found C, 61.76; H,
.30; N, 14.34.
-Isopropyl-3H-benzotriazole-5-carboxylic acid (18).
4
(
5-yl]-1-isopropyl-1H-benzotriazole as a white solid. APCI MS
1
(m/z): 323 (M + H). H NMR (500 MHz, CDCl
3
): δ 8.05 (d, J
) 8.8 Hz, 1 H), 8.03 (s, 1 H) 7.81 (s, 1 H), 7.66 (m, 2 H), 7.57
(d, J ) 8.8 Hz, 1 H), 7.12 (m, 2 H), 5.06 (m, 1 H), 1.72 (d, J )
1
3
): δ 7.55 (d,
4
6.7 Hz, 6 H). Anal. Calcd for C18H15FN O: C, 67.07; H, 4.69;
3
N, 17.38. Found: C, 66.68; H, 4.50; N, 17.37.
C
10
H
14
N
2
6-Bromo-3-isopropyl[1,2,4]triazolo[4,3-a]pyridine (22).
A mixture of 2,5-dibromopyridine (35.5 g, 150 mmol) and
hydrazine hydrate (55 wt %, 85.5 mL, 1.5 mol) in poly(ethylene
7
3
To a stirred, cold (0 °C) suspension of 4-amino-3-isopropyl-
aminobenzoic acid (11.7 g, 60.2 mmol) in 125 mL of 6 N
hydrochloric acid was added dropwise a solution of sodium
nitrite (6.2 g, 90.4 mmol) in 50 mL of water. The mixture was
stirred for 1 h at 0 °C and filtered, and the solids were collected
and rinsed with water. The solids were dried to give 9.8 g (79%)
n
glycol) (average M ca. 300, 150 mL), 2-butanol (30 mL), and
water (150 mL) was refluxed for 24 h. The solution was cooled
to room temperature. Water (450 mL) was added. The result-
ing slurry was stirred in an ice/water bath for 1 h and filtered.
The cake was washed with cold water (2 × 100 mL) and dried
in a vacuum oven (35 °C) for 18 h. 5-Bromopyridin-2-yl-
hydrazine (24.0 g, yield 85%) was obtained as a white needle-
of 3-isopropyl-3H-benzotriazole-5-carboxylic acid as a tan solid.
LCMS (m/z): 206 (M + H). H NMR (500 MHz, CDCl
1
+
1
3
): δ 8.47
type crystalline solid. GCMS (m/z): 187 (M ). H NMR (400
(
1
s, 1 H), 8.19 (d, J ) 8. 3 Hz, 1 H), 8.14 (m, 1 H), 5.2 (m, 1 H),
.81 (d, J ) 6.7 Hz, 6 H). Anal. Calcd for C10 : C, 58.53;
H, 5.40; N, 20.48. Found: C, 58.20; H, 5.31; N, 20.40.
3
MHz, CDCl ): δ 8.14 (d, J ) 2.0 Hz, 1 H), 7.55 (dd, J ) 8.7,
11
H N
3
O
2
2.0 Hz, 1 H), 6.66 (d, J ) 7.7 Hz, 1 H), 5.89 (brs, 1 H), 3.65
(brs, 2 H).
3
-Isopropyl-3H-benzotriazole-5-carbaldehyde (19). To
A mixture of isobutyryl chloride (218 mL, 2.08 mol) and
5-bromopyridin-2-yl-hydrazine (43.4 g, 231 mmol) was stirred
under reflux for 3 h. The reaction slurry was cooled to room
temperature, and hexane (220 mL) was added. The slurry was
stirred at room temperature for 15 min and then filtered. The
cake was washed with hexane (3 × 70 mL) and dried in a
vacuum oven (30 °C) for 24 h. 6-Bromo-3-isopropyl[1,2,4]-
triazolo[4,3-a]pyridine hydrochloride (58.9 g, yield 92%) was
obtained as an off-white powder. Aqueous NaOH (1 N, 86 mL)
was added to a mixture of 6-bromo-3-isopropyl[1,2,4]triazolo-
[4,3-a]pyridine hydrochloride (23.4 g, 8.46 mmol) in water (130
mL) and dichloromethane (130 mL). The biphasic mixture was
stirred at room temperature for 15 min. The bottom layer was
separated, and the top aqueous layer was extracted with
dichloromethane (60 mL). The combined organic layers were
a stirred mixture of 3-isopropyl-3H-benzotriazole-5-carboxylic
acid (1.42 g, 6.9 mmol) in 140 mL of methylene chloride and
0
(
.015 mL of N,N-dimethylformamide was added oxalyl chloride
0.78 mL, 9.0 mmol) at 22 °C. The mixture became a solution
after 1 h and was stirred an additional 4 h at 22 °C. N,N-
Diisopropylethylamine (4.20 mL, 24.2 mmol) and N,O-di-
methylhydroxylamine hydrochloride (0.88 mg, 9.00 mmol)
were added, and the mixture was stirred for 16 h at 22 °C.
The mixture was poured into a separatory funnel and washed
with aqueous sodium hydrogen phosphate, saturated aqueous
bicarbonate, 0.1 N HCl, and brine. The organic layer was dried
over sodium sulfate and filtered. The filtrate was concentrated
to give 1.5 g of a yellow oil. This oil was purified by flash
chromatography eluting with ethyl acetate and hexanes 2:1
to give 1.32 g (77%) of 3-isopropyl-3H-benzotriazole-5-carbox-
ylic acid methoxy-methyl-amide as a light yellow solid. LCMS
4
washed with 1:1 brine-water (60 mL), dried (MgSO ), and
concentrated. The residual oil was recrystallized from ethyl
acetate (50 mL) and hexane (200 mL). 6-Bromo-3-isopropyl-
1
(
m/z): 249 (M + H). H NMR (500 MHz, dimethyl sulfoxide-
): δ 8.16 (s, 1 H), 8.09 (d, J ) 8.3 Hz, 1 H), 7.55 (d, J ) 8.3
Hz, 1 H), 5.30 (m, 1 H), 3.56 (s, 3 H), 3.33 (s, 3 H), 1.64 (d, J
6.7 Hz, 6 H). To a stirred, cold (-78 °C) solution of 3-iso-
[
1,2,4]triazolo[4,3-a]pyridine (16.5 g, yield 81%) was obtained
d
6
1
as an off-white powder. H NMR (400 MHz, CDCl
3
): δ 8.06
(
3
s, 1 H), 7.64 (d, J ) 9.5 Hz, 1 H), 7.24 (d, J ) 9.5 Hz, 1 H),
)
.33 (m, J ) 7.0 Hz, 1 H), 1.52 (d, J ) 7.0 Hz, 6 H). Anal.
: C, 45.02; H, 4.20; N, 17.50. Found: C,
5.05; H, 4.06; N, 17.34.
3-Isopropyl-[1,2,4]triazolo[4,3-a]-6-pyridinecarbox-
propyl-3H-benzotriazole-5-carboxylic acid methoxy-methyl-
amide (4.26 g, 17.2 mmol) in 20 mL of toluene was added
diisobutylaluminum hydride (17.2 mL; 1 M in toluene, 17.2
mmol) dropwise under a nitrogen atmosphere. After 1.5 h, the
reaction was warmed to 0 °C with an ice bath; 10 mL of 1 N
HCl was slowly added; the mixture was diluted with water
and extracted with ethyl acetate (3×). The combined organic
extracts were washed with water and brine, dried over sodium
sulfate, and filtered. The filtrate was concentrated to give a
yellow oil, which was purified by flash chromatography, eluting
with ethyl acetate and hexanes 1:2, to a yellow oil, which
crystallized on standing to give 2.2 g (67%) of 3-isopropyl-3H-
benzotriazole-5-carbaldehyde as light yellow solid. LCMS (m/
Calcd for C
4
9
H
10BrN
3
aldehyde (23). A solution of 6-bromo-3-isopropyl[1,2,4]triazolo-
[4,3-a]pyridine (5.00 g, 20.8 mmol) in THF (low water, 50 mL)
was cooled to -8 °C using an acetone/dry ice bath. A solution
of isopropylmagnesium chloride in THF (2.0 M, 12.5 mL, 25
mmol) was added via a syringe over a period of 5 min. The
resulting brownish slurry was stirred between -6 and 6 °C
for 30 min. N,N-Dimethylformamide (anhydrous, 3.9 mL, 50
mmol) was added via a syringe over a period of 1 min. The
cooling bath was replaced with a heating bath. The slurry was

5
736 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
McClure et al.
heated to reflux and stirred under reflux for 3 h. The reaction
mixture was cooled to 10 °C, and dichloromethane (100 mL)
was added. The slurry was slowly poured into a stirred and
cooled (15 °C) 10 wt % aqueous citric acid solution (120 mL).
The biphasic mixture was stirred for 150 min. The organic
layer was separated, and the aqueous layer was extracted with
dichloromethane (60 mL). The combined organic extracts were
(3) Thornber, C. W. Isosterism and Molecular Modification in Drug
Design. Chem. Soc. Rev. 1979, 8, 563-580.
(
4) Badger, A. M.; Griswold, D. E.; Kapadia, R.; Blake, S.; Swift, B.
A.; Hoffman, S. J.; Stroup, G. B.; Webb, E.; Rieman, D. J.;
Gowen, M.; Boehm, J. C.; Adams, J. L.; Lee, J. C. Disease-
modifying activity of SB 242235, a selective inhibitor of p38
mitogen-activated protein kinase, in rat adjuvant-induced ar-
thritis. Arthritis Rheum. 2000, 43, 175-183.
washed with 1:1 v/v brine-water (30 mL), dried (MgSO
4
), and
(5) Jackson, J. R.; Bolognese, B.; Hillegass, L.; Kassis, S.; Adams,
J.; Griswold, D. E.; Winkler, J. D. Pharmacological effects of SB
concentrated. The brownish residual solid was recrystallized
from ethyl acetate (15 mL) and hexane (15 mL). 3-Isopropyl-
2
20025, a selective inhibitor of P38 mitogen-activated protein
kinase, in angiogenesis and chronic inflammatory disease mod-
els. J. Pharmacol. Exp. Ther. 1998, 284, 687-692.
[
1,2,4]triazolo[4,3-a]-6-pyridinecarboxaldehyde (3.07 g, yield
8%) was obtained as a beige powder and crystallized for X-ray
determination by slow evaporation from ethyl acetate and
7
(
6) Wadsworth, S. A.; Cavender, D. E.; Beers, S. A.; Lalan, P.;
Schafer, P. H.; Malloy, E. A.; Wu, W.; Fahmy, B.; Olini, G. C.;
Davis, J. E.; Pellegrino-Gensey, J. L.; Wachter, M. P.; Siekierka,
J. J. RWJ 67657, a Potent, Orally Active Inhibitor of p38
Mitogen-Activated Protein Kinase. J. Pharmacol. Exp. Ther.
hexanes; mp ) 180.7-180.8 °C. R
0
1
f
(silica gel; ethyl acetate) )
.18. GCMS (m/z): 189 (M ). H NMR (500 MHz, CDCl ): δ
0.02 (s, 1 H), 8.52 (s, 1 H), 7.86 (d, J ) 9.8 Hz, 1 H), 7.74 (d,
+
1
3
1999, 291, 680-687.
J ) 9.8 Hz, 1 H), 3.50 (m, J ) 6.7 Hz, 1 H), 1.60 (d, J ) 6.7
Hz, 6 H). Anal. Calcd for C10 O: C, 63.48; H, 5.86; N,
2.21. Found: C, 63.33; H, 5.76; N, 22.18.
-Isopropyl-[1,2,4]triazolo[1,5-a]pyridine-6-carbalde-
(
7) Tong, L.; Pav, S.; White, D. M.; Rogers, S.; Crane, K. M.; Cywin,
C. L.; Brown, M. L.; Pargellis, C. A highly specific inhibitor of
human p38 MAP kinase binds in the ATP pocket. Nat. Struct.
Biol. 1997, 4, 311-316.
11 3
H N
2
2
hyde (24). 2-Isopropyl-[1,2,4]triazolo[1,5-a]pyridine-6-carb-
aldehyde (24) was synthesized following the same reaction
conditions as those for 23, except that instead of being
quenched with 10 wt % aqueous citric acid, 1 N HCl or water
was used. Stirring the aqueous mixture for 2 h leads to
complete rearrangement. The product was crystallized for
X-ray determination by slow evaporation from ethyl acetate
(8) Wilson, K. P.; McCaffrey, P. G.; Hsiao, K.; Pazhanisamy, S.;
Galullo, V.; Bemis, G. W.; Fitzgibbon, M. J.; Caron, P. R.;
Murcko, M. A.; Su, M. S. The structural basis for the specificity
of pyridinylimidazole inhibitors of p38 MAP kinase. Chem. Biol.
1997, 4, 423-431.
(
9) Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.; Cobb,
M. H.; Young, P. R.; Abdel-Meguid, S.; Adams, J. L.; Goldsmith,
E. J. Structural basis of inhibitor selectivity in MAP kinases.
Structure 1998, 6, 1117-1128.
and hexanes; mp ) 99.7-99.9 °C. R
f
(silica gel; ethyl acetate)
): δ 10.02 (s, 1 H), 9.02 (s,
H), 7.98 (d, J ) 9.3 Hz, 1 H), 7.76 (d, J ) 9.3 Hz, 1 H), 3.33
1
(10) Stelmach, J. E.; Liu, L.; Patel, S. B.; Pivnichny, J. V.; Scapin,
G.; Singh, S.; Hop, C. E.; Wang, Z.; Strauss, J. R.; Cameron, P.
M.; Nichols, E. A.; Keefe, S. J.; Neill, E. A.; Schmatz, D. M.;
Schwartz, C. D.; Thompson, C. M.; Zaller, D. M.; Doherty, J. B.
Design and synthesis of potent, orally bioavailable dihydro-
quinazolinone inhibitors of p38 MAP kinase. Bioorg. Med. Chem.
Lett. 2003, 13, 277-280.
)
0.56. H NMR (500 MHz, CDCl
3
1
(
m, J ) 6.7 Hz, 1 H), 1.48 (d, J ) 6.7 Hz, 6 H).
6-[4-(4-Fluoro-phenyl)-oxazol-5-yl]-3-isopropyl-[1,2,4]-
triazolo[4,3-a]pyridine (25). To a stirred solution of 3-iso-
propyl-[1,2,4]triazolo[4,3-a]-6-pyridinecarboxaldehyde (0.30 g,
1
.58 mmol) in 5 mL of acetonitrile was added R-(p-toluen-
sulfonyl)-4-fluorobenzylisonitrile (0.55 g, 1.90 mmol) followed
by powdered potassium carbonate (0.33 g, 2.37 mmol) at 22
(11) Wang, Z.; Harkins, P. C.; Ulevitch, R. J.; Han, J.; Cobb, M. H.;
Goldsmith, E. J. The structure of mitogen-activated protein
kinase p38 at 2.1-Å resolution. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 2327-2332.
°
°
C. The mixture was stirred for 16 h at 22 °C and 24 h at 70
C. The mixture was cooled to 22 °C, diluted with water, and
(
12) Dombroski, M. A.; Letavic, M. A.; McClure, K. F.; Barberia, J.
T.; Carty, T. J.; Cortina, S. R.; Csiki, C.; Dipesa, A. J.; Elliott,
N. C.; Gabel, C. A.; Jordan, C. K.; Labasi, J. M.; Martin, W. H.;
Peese, K. M.; Stock, I. A.; Svensson, L.; Sweeney, F. J.; Yu, C.
H. Benzimidazolone p38 inhibitors. Bioorg. Med. Chem. Lett.
extracted with chloroform (3×). The combined organic extracts
were washed with water and brine, dried (sodium sulfate), and
filtered. Concentration of the filtrate gave 0.59 g of a yellow
solid, which was purified by flash chromatography, eluting
with chloroform/methanol (97:3), to give 0.5 g (98%) of 6-[4-
2004, 14, 919-923.
(
13) de Dios, A.; Shih, C.; de Uralde, B. L.; S a´ nchez, C.; del Prado,
M.; Cabrejas, L. M. M.; Pleite, S.; Blanco-Urgoiti, J.; Mar ´ı a Jos e´
Lorite; Nevill, C. R., Jr.; Bonjouklian, R.; York, J.; Vieth, M.;
Wang, Y.; Magnus, N.; Campbell, R. M.; Anderson, B. D.;
McCann, D. J.; Giera, D. D.; Lee, P. A.; Schultz, R. M.; Li, L. C.;
Johnson, L. M.; Wolos, J. A. Design of Potent and Selective
(4-fluoro-phenyl)-oxazol-5-yl]-3-isopropyl-[1,2,4]triazolo[4,3-a]-
1
pyridine as a white solid: mp 213 °C. H NMR (500 MHz,
CDCl
3
): δ 8.21 (s, 1 H), 8.04 (s, 1 H), 7.85 (d, J ) 9.8 Hz, 1 H),
.64 (m, 2 H), 7.42 (d, J ) 9.8 Hz, 1 H), 7.16 (m, 2 H), 3.34 (m,
O:
7
1
2
-Aminobenzimidazole Based p38R MAP Kinase Inhibitors
with Excellent in Vivo Efficacy. J. Med. Chem. 2005, 48, 2270-
273.
H), 1.53 (d, J ) 7.3 Hz, 6 H). Anal. Calcd for C18H15FN
4
C, 67.07; H, 4.69; N, 17.38; F, 5.89. Found: C, 67.07; H, 4.62;
N, 17.48; F, 6.02.
2
(
14) Nobeli, I.; Price, S. L.; Lommerse, J. P. M.; Taylor, R. Hydrogen
bonding properties of oxygen and nitrogen acceptors in aromatic
heterocycles. J. Comput. Chem. 1997, 18, 2060-2074.
15) Mao, L.; Wang, Y.; Liu, Y.; Hu, X. Molecular Determinants for
ATP-binding in Proteins: A Data Mining and Quantum Chemi-
cal Analysis. J. Mol. Biol. 2004, 336, 787-807.
(16) Davis, A. M.; Teague, S. J. Hydrogen Bonding, Hydrophobic
Interactions, and Failure of the Rigid Receptor Hypothesis.
Angew. Chem., Int. Ed. 1999, 38, 736-749.
Acknowledgment. The authors would like to thank
Dr. Jon Bordner and Ivan Samardjiev for the small-
molecule X-ray structure determinations on compounds
(
17, 23, and 24. We also thank Chris Williams from
Chemical Computing Group Inc. for his support. We
are grateful to Drs. Frank M. DiCapua and Alan
Mathiowetz for the review of the manuscript and helpful
discussions.
(17) Murray, J. S.; Ranganathan, S.; Politzer, P. Correlations between
the Solvent Hydrogen Bond Acceptor Parameter â and the
Calculated Molecular Electrostatic Potential. J. Org. Chem.
1991, 56, 3734-3737.
(
18) Kollman, P.; McKelvey, J.; Johansson, A.; Rothenberg, S.
Theoretical Studies of Hydrogen-Bonded Dimers. Complexes
Supporting Information Available: Details on construc-
tion of the p38 homology model and X-ray crystallographic data
for compounds 17, 23, and 24. This material is available free
of charge via the Internet at http://pubs.acs.org.
Involving HF, H
CH NH, H CS, H
O . J. Am. Chem. Soc. 1975, 97, 955-965.
2
O, NH
2
3
, HCl, H
2
S, PH
3
, NCN, HNC, HCP,
2 4 6 6
-
2
2
CO, CH
4
, CF H, C
3
2
H
2
, C H , C H , F , and
+
H
3
(
19) Henry, J. R.; Rupert, K. C.; Dodd, J. H.; Turchi, I. J.; Wadsworth,
S. A.; Cavender, D. E.; Schafer, P. H.; Siekierka, J. J. Potent
inhibitors of the MAP kinase p38. Bioorg. Med. Chem. Lett. 1998,
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