Design of potent and selective 2-aminobenzimidazole-based p38α MAP kinase inhibitors with excellent in vivo efficacy

Article abstract of DOI:10.1021/jm048978k

We report the design and discovery of a 2-amino-benzimidazole-based series of potent and highly selective p38α inhibitors. The lead compound 1 had low-nanomolar activity in both ATP competitive enzyme binding and inhibition of TNFα release in macrophages.

Full text of DOI:10.1021/jm048978k

2270
J. Med. Chem. 2005, 48, 2270-2273
Design of Potent and Selective
2-Aminobenzimidazole-Based p38r MAP
Kinase Inhibitors with Excellent in Vivo
Efficacy
Alfonso de Dios,*,† Chuan Shih,‡
Beatriz Lo´pez de Uralde,† Concepcio´n Sa´nchez,†
Miriam del Prado,† Luisa M. Mart´ın Cabrejas,†
Sehila Pleite,† Jaime Blanco-Urgoiti,†
Mar´ıa Jose´ Lorite,† C. Richard Nevill Jr.,‡
Rosanne Bonjouklian,‡ Jeremy York,‡ Michal Vieth,‡
Yong Wang,‡ Nicholas Magnus,‡
Robert M. Campbell,‡ Bryan D. Anderson,‡
Denis J. McCann,‡ Deborah D. Giera,‡ Paul A. Lee,‡
Richard M. Schultz,‡ Li C. Li,‡ Lea M. Johnson,‡ and
Jeffrey A. Wolos‡
Eli Lilly and Co., Lilly S.A., Avenida de la Industria, 30,
28108 Alcobendas, Madrid, Spain, and Lilly Research
Laboratories, Eli Lilly and Co., Lilly Corporate Center,
Indianapolis, Indiana 46285
Received December 17, 2004
Abstract: We report the design and discovery of a 2-amino-
benzimidazole-based series of potent and highly selective p38R
inhibitors. The lead compound 1 had low-nanomolar activity
in both ATP competitive enzyme binding and inhibition of
TNFR release in macrophages. Compound 18 showed excellent
pharmacokinetics properties and oral activity in the rat
collagen induced arthritis model compared with other p38
reference compounds. A SAR strategy to address CyP3A4
liability is also described.
Figure 1. Hybrid design, known p38 inhibitors, and X-ray
structure of 1 (green) bound to the ATP-binding site of p38R
and superposed onto X-ray structure of SB203580 3 (yellow).
Over the past decade, the pursuit of p38R MAP kinase
inhibitors has received an extraordinary level of atten-
tion in the pharmaceutical industry1 and in the medici-
nal chemistry community.2 A unique combination of
well-established pharmacology, clinical efficacy,3-6 and
the opportunity to utilize structure-based drug design7
has made this a highly attractive target for therapeutic
intervention.8
Figure 1), reduces IL-1â and TNFR levels in vitro and
in vivo.15 Several other lead molecules (e.g., SB242235
4,5,16 RWJ67657 5,6 and L-790070 6,11,17 etc.) have been
advanced to preclinical or clinical studies. In the past 3
years many disclosures present scaffolds with differ-
ent structural features;18 however, only Boehringer’s
BIRB79619 seems to offer a distinct profile in terms of
binding kinetics (slow binding using an allosteric do-
main). In this communication we show that a novel
design combined with a strategy to address some well-
known CyP450 liabilities20 provides great advantage in
terms of in vivo efficacy.
Kinase screening efforts at Lilly led to the identi-
fication of modestly potent hits with C-6 substituted
2-aminobenzimidazoles as the generic core structure (2,
R ) R1SO2 in some cases, Figure 1). Docking studies
indicated the potential for the C-2 amino group and N-3
imine nitrogen in the benzimidazole core to interact with
the kinase hinge region in the ATP binding cleft. On
the basis of the known X-ray structures for triaryl-
imidazoles such as 3 and the docking hypothesis of 2,
we envisioned that a hybrid molecule 1 (Figure 1) would
have potent p38R activity. Because of the larger size of
benzimidazole compared to the 4-pyridyl, it was ex-
pected that binding of 1 would require a rearrangement
in the flexible p38R hinge (from His107 to Ala111) or a
more probable rearrangement of the Lys53 side chain.
We were pleased to discover that the first molecule
designed and synthesized in this series, 1, turned out
There is overwhelming evidence indicating that p38R
plays a dominant role in the pathogenesis of acute and
chronic inflammatory responses.1,2 Activation of p38R
occurs in monocytes and macrophages under different
stress-related stimuli. Subsequent phosphorylation of
downstream effectors and transcriptional factors leads
to the biosynthesis of potentially deleterious proinflam-
matory cytokines such as TNFR and IL-1â.9 The clinical
proof of concept in rheumatoid arthritis achieved with
VX-7453 and BIRB-7964 validates the MAP kinase
pathway as a useful mechanism for intervention in
inflammatory disease.
The seminal work by SmithKline Beecham10 (initially
followed by Merck11 and RWJ12) with the aid of X-ray
crystallography and mutagenesis studies13 has revealed
the structural basis for much of the observed SAR
around the well-known pyridinylimidazole p38 phar-
macophore.14 The prototypical inhibitor, SB203580 (3,
* To whom correspondence should be addressed. Phone: +34-91-
6633412. Fax: +34-916633411. E-mail: a.de_dios@lilly.com.
† Eli Lilly and Co., Spain.
‡ Eli Lilly and Co., Indiana.
10.1021/jm048978k CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/19/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2271
Scheme 1a
Metal-halogen exchange and addition to Weinreb
amides 9a,b24 led to the silyloxy ketone intermediates
11a that underwent copper(II)-catalyzed oxidative cy-
clization with the appropriate aldehyde to yield final
products in just four steps. Alternatively, a more ef-
ficient route could be used. Palladium-catalyzed cou-
pling of the corresponding iodide 8 with arylalkynes
(10a-c) followed by oxidation using KMnO4 led to
diketones 11b. In this case, no oxidating copper reagent
was required to afford the final desired C-2 substituted
imidazoles 1 and 12-23 (Table 1). For the synthesis of
N-1 substituted imidazoles (Scheme 2), the key inter-
mediate was found to be the C-6 carboxaldehyde 8e that
was prepared from 8a via lithiation and DMF addition.
Imine formation and van Leusen cyclization25 using
TosMIC reagent 2426 afforded the desired derivatives.
Inhibition of p38R was determined for compounds in
Table 1 using recombinant human p38R in a standard
filter binding protocol using ATP[γ-33P] and EGFR 21-
mer peptide as substrate. Almost all of the compounds
showed potent p38R inhibition with IC50 values ranging
from 1 to 30 nM. Only N-1 piperidyl substituted imid-
azole 27 showed decreased activity, indicating that our
SAR did not parallel that of SB242235 (4)16 and only
smaller substitutions were well tolerated (25).
a Reagents and conditions (yields for 1): (a) R1SO2Cl, NaOH,
t
CH3CN/H2O, 90%; (b) PhLi, BuLi, MeI, THF, -78 °C; (c) NIS,
i
AcOH, 84%; (d) PrMgCl, THF, -78 °C, then 9a,b, 0 °C, 50%; (e)
10a-c, Pd(PPh3)2(AcO)2, CuI, DMSO, Et3N, room temp, 86%; (f)
KMnO4, MgSO4/NaHCO3, acetone, 35 °C, 83%; (g) R3CHO (or
paraformaldehyde for R3 ) H), Cu(OAc)2, NH4OAc, AcOH, 90 °C,
62% from 11a or R3CHO, NH4OAc, nBuOH, 55 °C, 78% from 11b.
(h) Some compounds were isolated as mesylate salts: MsOH,
MeOH, 98% for 14 (see Supporting Information).
Scheme 2a
For lead compound 1, the observed IC50 was equiva-
lent to the Ki, which showed promise for good cell
activity within this series. Deletion of C-2 imidazole
substitution and introduction of small alkyl groups at
that position and/or fluorine atoms in the phenyl ring
also led to potent inhibitors (12-18), which is consistent
with previous observations.14
Introduction of bulkier sulfonyl derivatives (18, 22)
or a sulfamoyl group (23) was not detrimental for
binding relative to 1, indicating that larger or polar
groups could be accommodated in the small lipophilic
pocket close to Gly110. In general, this series showed
equally potent activity against p38â2, some cross-
reactivity against JNKs (see Table 2), and high selectiv-
ity against the other kinases screened in the panel.27
Functional inhibition of TNFR in murine peritoneal
macrophages was determined using LPS stimulation in
the presence of test compounds (Table 1). Compounds
1, 12-19, and 21-23 were potent inhibitors of TNFR
release in the cell-based assay (IC50 ) 2-60 nM). N-1
substituted imidazoles 25 and 27 showed decreased cell
activity that for 27 correlates with decreased enzyme
activity. The C-2 piperidyl derivative 20 had a dramatic
loss in cell-based potency presumably because of poor
cell permeability. N-Isobutyl substitution at the pi-
peridyl nitrogen, 21, restored the cell activity.28
a Reagents and conditions: (yields for 27): (a) PhLi, tBuLi,
DMF, THF, from -78 °C to 0 °C, 76%; (b) R4NH2, DMF, room
t
temp, 95%; (c) 24, BuNH2, MeOH, reflux, 25%; (d) concentrated
HCl, reflux, then aqueous NaOH to pH 7, 43%.
to be a potent inhibitor of p38R (Ki ) 5 nM, ATP-
competitive) and p38â. The binary complex of 1 bound
to rat p38R was obtained (Figure 1) and confirmed the
predicted binding,21 showing a clear shift in the hinge.
The N-3 imine nitrogen acted as the key hydrogen bond
i
acceptor for Met109 amide N-H, and the Pr sulfonyl
group was exposed to the small lipophilic pocket in
proximity to Gly110. Interestingly, it was not clear that
a hydrogen bond existed between imidazole N-3 and
terminal NH2 in Lys53 because the shorter distance
with Lys53Nꢀ was with one of the fluorine atoms (2.83
Å). Broader kinase profiling with 1 showed that it had
remarkable selectivity with the exception of JNK2 (vide
infra). As hypothesized, 1 shifted the flexible hinge to
accommodate the benzimidazole moiety but did not
produce the Gly110 carbonyl flip described for other
inhibitors.17 This peptide flip seems to serve as a source
of p38 selectivity vs JNKs because of the restriction in
the Ramachandran space for the analogue Asp residue
in JNKs.17 The fact that p38 accommodated 1 without
the Gly110 flip may very well explain the JNK2 activity.
The synthesis of our inhibitors is outlined in Schemes
1 and 2. Starting from commercially available 2-amino-
benzimidazole, sulfonylation at N-1 followed by regio-
selective iodination with NIS afforded 6-iodobenz-
imidazoles 8a-d.22 The tert-butyl sulfone was synthe-
To assess potential drug-drug interaction for this
series as early as possible,20 in vitro CyP3A4 inhibition
was determined. Increasing the steric hindrance around
the C-2 imidazole carbon greatly reduced the CyP3A4
inhibition (compare compounds 12-16 in Table 1). C2-
unsubstituted analogues (12 and 13) were found to be
potent inhibitors, while ethyl and isopropyl analogues
t
and especially the Bu derivative 16 led to reduced
activity at the 3A4 isoform.29
Plasma exposure was measured after oral adminis-
tration in the rat. Besides the low exposure observed
for C-2 unsubstituted 12 and 13, it is interesting to
compare the modest exposure and bioavailability for 1
i
sized by lithiation of the Pr sulfone derivative of 7
followed by alkylation with MeI to afford 8b after the
iodination step.23

2272 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Letters
Table 1. Biochemical, in Vivo, and Rat Pharmacokinetics Data for Aminobenzimidazole-Based P38R Inhibitors (See Scheme 1 for
Structure)
cell TNFR inh
mice TNFR inh
(iv LPS), oralc
rat oral AUC0-inf
(10 mg/kg),c
µM h
a
p38R IC50
,
IC50,a nM, mouse CyP3A4 inh
compd
R1
R2
R3
nM (n)
macrophages (n)
IC50,b µM
TMED50,d mg/kg
1
iPr
H
H
2,6-F2-C6H4
4.9 ( 0.4 (17)
4.2 ( 0.5 (8)
4.3 ( 1.8 (5)
4.7 ( 1.7 (3)
4.5 ( 1.7 (4)
15.9 ( 2.5 (7)
4.4 ( 0.6 (5)
4.8 ( 1.7 (3)
15.3 ( 5.0 (3)
2.7 ( 0.4 (3)
10.3 ( 0.4 (202)
31.8 ( 7.8 (4)
65.7 ( 3.1 (4)
47.2 ( 12.8 (4)
28.6 ( 5.6 (4)
31.1 ( 5.6 (4)
6.2 ( 1.8 (4)
1.6 ( 0.2 (4)
3.4 ( 1.2 (4)
2885 (2)
27.9 ( 7.6 (4)
6.0 (2)
8.8 ( 3.3 (3)
720 ( 154 (4)
4457 ( 2083 (4)
6.3
5.2
5.4
12
iPr
H
1.6
19.8
9.6
3.7
13e
14f
15f
16e
17f
18f
19f
20
iPr
4-F
4-F
4-F
4-F
H
0.8
1.4
iPr
Et
iPr
tBu
4.4
6.5
NTg
12.8
13.5
14.6
28.3
17.0
NTg
NTg
0.9
iPr
6.1
4.9
iPr
31.8
NTg
NTg
NTg
NTg
5.3
3.9
iPr
2-F, 4-F tBu
2.2
tBu
iPr
2-F, 4-F tBu
5.3
H
H
H
H
H
2,6-Cl2-C6H4
4-piperidyl
2.5
iPr
NTg
7.3
21
iPr
1-iBu-4-piperidyl 5.5 ( 0.9 (3)
22e
23e
25
C5H9
NMe2
2,6-F2-C6H4
2,6-F2-C6H4
19.9 ( 5.2 (3)
22.8 ( 10.9 (3)
20.7 ( 5.5 (3)
125 ( 23.2 (4)
13.0
NTg
NTg
NTg
5.1
12.5
NTg
NTg
9.8
see Scheme 2
see Scheme 2
NTg
NTg
27
a Mean values ( SEM of at least three independent determinations. b n ) 1. c Vehicle: 1% NaCMC/0.25% Tween-80 in water. d Threshold
minimal effective doses (mg/kg). e Bismesylate salt. f Monomesylate salt. g Not tested.
Table 2. Selectivity Profile of 1
Table 3. Rat in Vivo Data for Selected p38 Inhibitorsa
TNFR inh,b CIA, paw swelling CIA, histology,
kinase assay
kinase assay
IC50 (nM)
kinasea
IC50 (nM)b
kinase
compd
rat joint, %
TMED50, mg/kgc
TMED50, mg/kgc
p38â2
11.9 ( 0.1
>20000
rCAMKII
PKAR
>20000
>20000
>20000
>20000
>20000
>20000
17
81
1.5
1.5
15
5.0
>30
8.1
4.2
30
7.0
>30
p38γ
18
82
JNK1R1
JNK2R2
MAPKAPK2
TGFâ RI
4422 ( 1653
141.7 ( 38.2
>20000
E/CDK2
PKCâII
PI3K
BIRB796
SB242235
RWJ67657
55
NDd
<50
>20000
VEGFR2
a Vehicle: see Table 1. b 3 mg/kg oral dose. c Oral bid dosing
a Human kinases unless otherwise indicated. b Mean values (
SEM of at least three determinations.
for 14 days, therapeutic, p < 0.05 for all cases. d Not determined.
cartilage destruction) compares favorably with that of
other advanced molecules in clinical development. Com-
pound 18 showed remarkable efficacy in this model with
a TMED50 of 1.5 mg/kg for inflammation and 4.2 mg/kg
for histology (10- and 7-fold better than BIRB796,
respectively). Similar half-life (6-8 h) and increased
exposure in 18 with respect to 17 may explain the 2-fold
increase in histology efficacy within the aminobenz-
imidazoles.
In summary, we have developed a potent series of
2-aminobenzimidazole based p38R MAP kinase inhibi-
tors. The binding mode based on X-ray data and
preliminary SAR trends have been outlined, as well as
the molecular basis for in vitro activity, reduced CyP3A4
liability, enhanced rat oral exposure, and improved in
vivo efficacy compared with other advanced triaryl
imidazoles.
(F ) 39%) with the excellent data for the 2,6-dichloro
analogue 19 (3-fold higher AUC, F ) 55%). Additionally,
tBu substitution (16-18) led to readily absorbed and
highly bioavailable compounds.
Analysis of plasma from a rat bioavailability study
of 12 resulted in the identification of major oxidative
metabolites derived from imidazole ring opening (data
not shown). This exposure data suggest that both tert-
butyl and 2,6-dichlorophenyl substitutions might act as
protecting groups of the C-2 imidazole carbon. This
position seems to be prone to extensive oxidation and
ring opening,29 leading to reduced oral exposure.
To evaluate how the observed cell activity could
translate to in vivo acute TNFR inhibition, compounds
were orally dosed to Balb/c mice followed by iv LPS
administration for 2 h (Table 1). From these dose-
response studies, a TMED50 dose30 (threshold minimum
50% effective dose) was determined. TMED50 values
ranged from 2 to 20 mg/kg. Unsubstituted imidazoles
12 and 13 were some of the less potent derivatives,
which correlates with more extensive oxidative metabo-
lism and lower plasma exposure (vide supra).
On the basis of a combination of in vitro, in vivo
activity, and rat oral exposure, 17 and 18 were consid-
ered for further evaluation. Both compounds showed
potent inhibition of acute LPS-induced TNFR production
in rat joints as measured in the rat synovial lavage fluid
(p < 0.05). In a 14-day rat collagen induced arthritis
model (CIA, therapeutic dosing), both compounds showed
excellent dose-dependent inhibition after oral adminis-
tration (Table 3).31 Our data show that activity obtained
for these aminobenzimidazoles in inflammation (rat
ankle size) and histopathology scores (bone erosion and
Acknowledgment. The authors thank Harold Brooks
and Scott Watkins for Ki determination, Alfonso Espada
for HPLC data, and James Wikel, Andrea Zaliani, Luis
Casarrubios, and Chafiq Hamdouchi for useful sugges-
tions.
Supporting Information Available: Crystallographic
information for 1, experimental procedures for compound
preparation, and biological assay protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
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3-(4-pyridyl)-1H-pyrrolo[2,3-b]pyridine (RWJ 68354): a potent
and selective p38 kinase inhibitor. J. Med. Chem. 1998, 41,
4196-4198.
(26) Sisko, J.; Mellinger, M.; Sheldrake, P. W.; Baine, N. H. An
efficient method for the synthesis of substituted Tosmic precur-
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(27) Additionally, 1 showed no significant activity against other 30
different kinases (data not shown) when tested at 1 µM in
duplicate.
(28) Measured log D values (pH 7) for both derivatives were found
to be 0.09 and 1.74, indicating that a cell permeability problem
for the polar piperidyl was overcome by increasing lipophilicity
with the N-isobutyl residue.
(29) This steric effect may prevent the coordination of the heme iron
to imidazole N1 or N3, and therefore, it might be a tentative
explanation of this effect. See: Dalvie, D. K.; Kalgutkar, A. S.;
Khojasteh-Bakht, S. C.; Obach, R. S.; O’Donnell, J. P. Invited
review: Biotransformation reactions of five-membered aromatic
heterocyclic rings. Chem. Res. Toxicol. 2002, 15, 269-299.
(30) TMED50 is defined as the lowest dose where statistically
significant efficacy was achieved, and there was at least 50%
inhibition compared to vehicle control.
(13) Gum, R.; McLaughlin, M. M.; Kumar, S.; Wang, Z. L.; Bower,
M. J.; Lee, J. C.; Adams, J. L.; Livi, G. P.; Goldsmith, E. J.;
Young, P. R. Acquisition of sensitivity of stress-activated protein
kinases to the p38 inhibitor, SB 203580, by alteration of one or
more amino acids within the ATP binding pocket. J. Biol. Chem.
1998, 273, 15605-15610.
(14) For a review, see: Jackson, P. F.; Bullington, J. L. Pyridinyl
imidazole based p38 MAP kinase inhibitors. Curr. Top. Med.
Chem. 2002, 2, 1021-1035. For a recent disclosure, see: Revesz,
L.; Blum, E.; Di Padova, F. E.; Buhl, T.; Feifel, R.; Gram, H.;
(31) TMED50 was calculated from the AUC (paw swelling) or com-
posite histology score using JMP5.1.1.
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