Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as potent p38α MAP kinase inhibitors with excellent in vivo antiinflammatory properties

Article abstract of DOI:10.1016/j.bmcl.2007.10.106

Herein we report investigations into the p38α MAP kinase activity of trisubstituted imidazoles that led to the identification of compounds possessing highly potent in vivo activity. The SAR of a novel series of imidazopyridines is demonstrated as well, resulting in compounds possessing cellular potency and enhanced in vivo activity in the rat collagen-induced arthritis model of chronic inflammation.

Full text of DOI:10.1016/j.bmcl.2007.10.106

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 179–183
Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines
as potent p38a MAP kinase inhibitors with excellent
in vivo antiinflammatory properties
a,
b
b
b
a
Mary Mader,b,* Alfonso de Dios, Chuan Shih, Rosanne Bonjouklian, Tiechao Li,
*
b
a
a
´
´
´
Wesley White, Beatriz Lopez de Uralde, Concepcion Sanchez-Martinez,
a
a
a
´
Miriam del Prado, Carlos Jaramillo, Eugenio de Diego, Luisa M. Martın Cabrejas,
Carmen Dominguez,a Carlos Montero,a Timothy Shepherd,b Robert Dally,b
John E. Toth,b Arindam Chatterjee,b Sehila Pleite,a Jaime Blanco-Urgoiti,a Leticia Perez,a
a
a
a
b
´
´
Mario Barberis, Marıa Jose Lorite, Enrique Jambrina, C. Richard Nevill Jr.,
Paul A. Lee,b Richard C. Schultz,b Jeffrey A. Wolos,b Li C. Li,b
Robert M. Campbellb and Bryan D. Andersonb
aEli Lilly and Co., Centro de Investigacio´n, Lilly S.A., Avenida de la Industria, 30, 28108 Alcobendas, Madrid, Spain
bEli Lilly and Co., Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 28 August 2007; revised 26 October 2007; accepted 29 October 2007
Available online 1 November 2007
Abstract—Herein we report investigations into the p38a MAP kinase activity of trisubstituted imidazoles that led to the identifica-
tion of compounds possessing highly potent in vivo activity. The SAR of a novel series of imidazopyridines is demonstrated as well,
resulting in compounds possessing cellular potency and enhanced in vivo activity in the rat collagen-induced arthritis model of
chronic inflammation.
Ó 2007 Elsevier Ltd. All rights reserved.
Over the last decade, the pursuit of p38a 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.8,9
stress-related stimuli. Subsequent phosphorylation of
downstream effectors and transcriptional factors leads
to the biosynthesis of potentially deleterious pro-inflam-
matory cytokines such as TNFa and IL-1b.10 The clini-
cal proof of concept in rheumatoid arthritis achieved
with VX-7453 and BIRB-7965 validates the MAP kinase
pathway as a useful mechanism for intervention in
inflammatory disease.
There is overwhelming evidence indicating that p38a
plays a dominant role in the pathogenesis of acute and
chronic inflammatory responses.1,2 Activation of p38a
occurs in monocytes and macrophages under different
We have previously reported the design and discovery of
a 2-aminobenzimidazole-based series of potent and
highly selective p38a inhibitors.11 Awareness of poten-
tial CYP activity associated with the imidazole central
core12 led to an SAR around the imidazole C-2, and rec-
ognition that sterically bulky groups such as tert-butyl,
2,6-dichlorophenyl, or 2,6-difluorophenyl resulted in de-
creased inhibition of CyP3A4. The lead compound 1
had low nanomolar activity in both ATP competitive
enzyme binding and inhibition of TNFa release in
macrophages. Expansion of the SAR identified 2
Keywords:
p38
MAP
kinase;
Inflammation;
Imidazole;
Imidazopyridine.
*
Corresponding authors. Tel.: +1 317 277 3400; e-mail:
mader_mary@lilly.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.106

180
M. Mader et al. / Bioorg. Med. Chem. Lett. 18 (2008) 179–183
which showed excellent in vivo activity in the rat
collagen-induced arthritis model (CIA) as compared to
the p38 reference compounds BIRB-796, SB242235,
and RWJ-67657. Herein we report investigations into
the SAR of substitution on C-2, 4, and 5 of the imidaz-
ole to identify additional compounds possessing in vivo
activity. The activity of a series of imidazopyridines is
demonstrated as well, resulting in compounds with po-
tency in the cellular and in vivo assays.
the corresponding alkyl amine in THF. Frequently,
these basic compounds were converted to their mesylate
salts for assay.
Inhibition of p38a was determined using recombinant
human p38a in a standard filter binding protocol using
ATP[c-33P] and EGFR 21-mer peptide as substrate.
Functional inhibition of TNFa in murine peritoneal
macrophages was determined using LPS stimulation in
the presence of test compounds. To assess p38a activity
in cells more directly, RAW 264.7 cells were treated with
test compounds and then stimulated with anisomycin.
The level of p38a activity was detected using a phospho-
MAPKAPK-2 (pMK2) (Thr 334) antibody which re-
acted with a residue specifically phosphorylated by
p38a. The compounds appeared to have greater potency
in the TNFa macrophage assay due to JNK2a2 activ-
ity,11 and therefore we focused on the more direct
read-out of p38 cellular activity, the pMK2 cELISA,
to prioritize compounds for in vivo study.14
The route for preparation of N-sulfonyl analogs of 1
(20, 24–34) has been reported previously.11 The N-alkyl
derivatives in Tables 1–3 were prepared as shown in
Scheme 1. Beginning with nucleophilic aromatic substi-
tution by an alkyl amine on the dihalides 3a13 or 3b, pal-
ladium catalyzed coupling of the halides 4a or 4b with
aryl alkynes (9a–c) was followed by oxidation using
KMnO4, leading to diketones 6a and 6b. Treatment of
the diketones with an aldehyde in the presence of excess
ammonium acetate afforded the trisubstituted imida-
zoles 7a and 7b. Finally, reduction of the nitro moiety
by either sodium dithionite or tin dichloride provided
an amine, which was cyclized by treatment with cyano-
gen bromide to form the C-2 amino benzimidazoles 8a
or imidazopyridines 8b. Alkylamino benzimidazole
products 10, 11, and 14 were prepared via a modified se-
quence (not shown) in which amino benzimidazoles 8a
were converted into chlorides via a Sandmeyer reaction
utilizing tert-butyl nitrite and copper chloride. The chlo-
rides were converted to the alkyl amine in reaction with
In comparison to the initial lead 1, mono- and dialkyla-
tion of the benzimidazole C-20 amino group resulted in
decreased potency in both the enzyme and cellular as-
says for 10 and 11. Removal of the sulfonyl group at
N-10 of the benzimidazole also diminishes activity (13),
but in this instance, C-20 amino alkylation of 14 recovers
2- to 3-fold activity in the enzyme and cell.
Placing an alkyl group at N-10 rather than on the C-20
amine is also well tolerated (15–18), but a phenyl group
is not (19). Interestingly, the C-20 amino moiety is not
essential for enzyme activity (12 vs 13 and 14; 15 and
17 vs 16), and its impact on cellular activity appears
dependent on substitution at N-10. Introduction of
nitrogen into the 6-membered ring of the benzimidazole
Scheme 1. General synthetic scheme for imidazolyl benzimidazoles and imidazopyridines. Reagents and conditions: (a) RNH2 (1.5 equiv), Na2CO3
(1.75 equiv), EtOH, rt, 18 h (84–94%); (b) 9a–c (1.5 equiv), Ph3P, Pd(OAc)2, Et3N, 70–80 °C, 1–3 h (71–85%); (c) KMnO4, MgSO4/NaHCO3,
acetone, 0 °C, 1–2 h (50–82%); (d) R3–CHO (2 equiv), NH4OAc (15 equiv), AcOH, 80 °C (62–97%); (e) Na2S2O4, concd NH4OH, rt, 2 h; (f)
SnCl2Æ2H2O, EtOH, EtOAc, 70 °C, 2 h; (g) CNBr (1.1 equiv), LiOMe (1.5 equiv), 1,2-dichloroethane (43–59% for the two-step reduction/cyclization
process). Some compounds were isolated as mesylate salts: MsOH, MeOH.

M. Mader et al. / Bioorg. Med. Chem. Lett. 18 (2008) 179–183
181
Table 1. Enzymatic and cellular activity for N-10-substituted aminobenzimidazoles (IC50, nM)
Compound
R1
R2
X
p38a
Macrophage TNFa
cELISA pMK2
1
10
i-PrSO2
i-PrSO2
i-PrSO2
H
NH2
NHEt
N(Me)Bn
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
4.9
10.3
527
234
419
1326
493
51.9
47.5
338
3.5
45.7
c
—
156
2489
88.4
131
56.4
16.2
9.9
c
11
—
766
12a
13
c
H
NH2
NHEt
H
—
c
14
H
i-Bu
—
c
15
—
78.3
16a
17b
18a
19
i-Bu
i-Bu
NH2
Me
c
11.2
6.0
—
126
CH2-t-Bu
Ph
NH2
NH2
NH2
NH2
H
c
c
158
3.5
—
—
298
20b
21b
22b
23b
i-PrSO2
i-Bu
i-Bu
3.7
1.3
26.7
<9
N
2.5
4.5
35.0
69.7
N
c
—
CH2-t-Bu
NH2
N
6.4
a Bismesylate salt.
b Monomesylate salt.
c Not tested.
provided a series of imidazopyridines with potent TNFa
macrophage activity (20–23), and was independent of
substitution at N-10 and C-20.
potency is observed when the same position is substi-
tuted by an electronegative chloride (26). An electron
withdrawing group at the ortho site is comparable to
that at para (26 vs 27), hence we focused on the ortho
positions because of earlier findings that demonstrated
ortho substitution decreased CYP activity. Best within
this series is the 2,6-dichloro analog 30, which shows en-
hanced cellular potency relative to the other disubsti-
tuted analogs, 28 and 29. An aromatic group at C-4 of
the imidazole has been shown to occupy the lipophilic
specificity pocket of the ATP binding site,10,15 and addi-
Table 2 demonstrates the impact of variation of substi-
tution at the imidazole C-2 by aromatics or methyl,
while maintaining the amino benzimidazole substitution
constant. No substitution (R1) is required for enzymatic
activity (24), but it does improve cellular potency (2). An
embedded electronegative atom (pyridyl) is well toler-
ated in 25, but a decrease in enzymatic and cellular
Table 2. Enzymatic and cellular activity for 1-(isopropylsulfonyl)benzimidazole-2-amines (IC50, nM)
Compound
R1
R2
p38a
Macrophage TNFa
cELISA pMK2
2c
24
t-Bu
H
4-Pyr
2,4-F2
H
4.4
4.2
6.2
31.8
14.1
71.0
33.1
11.2
1.9
26.2
195
25
26
H
6.1
62.3
376
4-Cl–C6H5
2-CF3–C6H4
2-Cl–6-F–C6H4
2-Cl–6-CF3–C6H4
2,6-Cl2–C6H4
2,6-Cl2–C6H4
Me
H
34.7
26.1
14.5
8.5
27
H
284
28a
29b
30b
31b
32a
33b
34b
H
74.5
398
H
H
15.3
6.7
2.6
5.3
38.9
27.5
4-F
H
c
20.4
7.1
186.4
103.5
46.5
—
225
Me
Me
4-F
2,4-F2
8.8
59.7
a Bismesylate salt.
b Monomesylate salt.
c Not tested.

182
M. Mader et al. / Bioorg. Med. Chem. Lett. 18 (2008) 179–183
Table 3. Enzymatic and cellular activity for imidazopyridine derivatives (IC50, nM)
Compound
R1
R2
R3
p38a
Macrophage TNFa
cELISA pMK2
21b
35b
36a
37b
38b
39b
40a
41b
42b
43b
H
2,6-F2–C6H4
2,6-Cl2–C6H4
t-Bu
H
2.5
3.7
5.1
3.9
3.9
3.2
7.0
7.2
3.6
2.3
1.3
1.7
35.0
64.7
66.4
31.2
41.8
39
H
H
H
H
54.5
<9.0
49.2
<9.0
5.2
H
2,6-Cl2–C6H4
t-Bu
2,4-F2
2,4-F2
H
H
CH3
CH3
CH3
CH3
CH3
2-Cl, 6-F–C6H4
t-Bu
4-F
4-F
4-F
2,4-F2
34.3
c
—
2,6-F2–C6H4
2,6-Cl2–C6H4
t-Bu
<9.0
<9.0
24.1
23.0
20.7
a Bismesylate salt.
b Monomesylate salt.
c Not tested.
tion of halogens to the C-4 aryl improves both enzy-
matic and cellular potency. A stepwise analysis of the
impact of halogenation on the C-4 aromatic was con-
ducted in the series 32–34, with a 2-fold improvement
in enzymatic and macrophage TNFa activity as fluo-
rines are added to the phenyl ring. Fluorination in-
creases activity in the pMK2 cELISA approximately 4-
fold (33 and 34).
To evaluate how the cell activity translated to in vivo
acute TNFa inhibition, compounds were orally dosed
in Balb/c mice followed by iv LPS administration after
2 h. From these dose response studies, a threshold min-
imum 50% effective dose (TMED50) was determined.
The compounds were also evaluated in a 14-day chronic
model of inflammation, the rat CIA,16 and activity was
assessed by effect on paw swelling and by histopathology
scores (bone erosion and cartilage destruction). In the
mouse model, the imidazopyridines 35 and 40 showed
improved potency relative to the benzimidazoles. In
the rat CIA model, both exposure and half-life in rat ap-
peared to influence activity. Compounds 1 and 35 were
significantly less potent than the tert-butyl analogs 2
and 40 in both paw swelling and histology TMED50
(Table 4).
The imidazopyridines 20–23 possessed significant enzy-
matic and cellular potency, and the series was expanded.
Gains in cellular potency were achieved as we focused
on the elements that had been found to be preferred
on the imidazole: at C-2, a di-ortho substituted aromatic
ring or tert-butyl, and at C-4, a fluorinated phenyl (Ta-
ble 3). Varying R1 as isobutyl (21, 35–38) or neopentyl
(39–43) provided compounds that were similarly potent
in the enzyme, as all were single-digit nanomolar inhib-
itors. Greater differences in potency were manifested in
the cellular assays. Aromatic substitution at R2 of the
imidazole was preferred in the isobutyl series for cellular
potency (35 and 37), as the tert-butyl analogs 36 and 38
were 50-fold less potent in the macrophage assay. How-
ever, the neopentyl analogs 40–42 tolerated tert-butyl
and dihalo aromatics equally well in the cellular assays.
An exception is 43, for which the 2,4-difluorophenyl at
C-4 did not provide an increase in cellular potency as
it had in the methyl imidazole series 32–34.
In summary, we have shown that substitution at C-2 of
the imidazole by di-ortho substituted aromatics or tert-
butyl provides analogs with good activity in the enzyme
and cellular assays, and that cellular potency can be in-
creased with addition of fluorines to the imidazole C-4
aromatic ring. Conversion of the benzimidazole
warhead into an imidazopyridine results in increased
in vivo potency in an acute inflammation model,
but activity in the chronic model may be more
dependent on the pharmacokinetic properties of the
compounds.
Table 4. In vivo activity of selected compoundsa
Compound
Mouse TNFa inh.
(po, iv LPS), TMED50 (mg/kg)
CIA, paw swelling
b
TMED50 (mg/kg)
CIA, histology,
b
TMED50 (mg/kg)
Rat oral AUC0ꢀinf
(10 mg/kg) (lM h)
Rat oral
t1/2 (h)
1
5.2
2.2
>30
1.5
15
>30
8.1
5.5
5
8
2
14.6
29.9
87.5
35
40
<2.5
<1.0
>30
1.5
4
10
1.5
a Vehicle: 1%CMC/0/25% Tween 80 in water.
b Oral bid dosing for 14 days, therapeutic, p < 0.05 for all cases.

M. Mader et al. / Bioorg. Med. Chem. Lett. 18 (2008) 179–183
183
M.; Naruo, K.; Kimura, H.; Tanaka, T.; Asahi, S.;
Ohkawa, S. J. Med. Chem. 2005, 48, 5966.
References and notes
9. Zhang, J.; Shen, B.; Lin, A. Trends Pharmacol. Sci. 2007,
28, 286.
1. Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug Discov.
2003, 2, 717.
10. Feldmann, M.; Brennan, F. M.; Maini, R. Ann. Rev.
Immunol. 1996, 14, 397.
2. Chakravarty, S.; Dugar, S. Ann. Rep. Med. Chem. 2002,
37, 177.
11. de Dios, A.; Shih, C.; Lopez de Uralde, B.; Sanchez, C.;
del Prado, M.; Cabrejas, L. M. M.; Pleite, S.; Blanco-
Urgoiti, J.; Lorite, M. J.; 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. J. Med. Chem. 2005, 48, 2270.
12. Adams, J. L.; Boehm, J. C.; Kassis, S.; Gorycki, P. D.;
Webb, E. F.; Hall, R.; Sorenson, M.; Lee, J. C.; Ayrton,
A.; Griswold, D. E.; Gallagher, T. F. Bioorg. Med. Chem.
Lett. 1998, 8, 3111.
13. Prepared by portionwise treatments of 1,3-dibromoben-
zene with cold sulfuric acid (5 mL/g) with ammonium
nitrate (1 equiv) at 0 °C for 10 min as described in Dodic,
N.; Donche, F; Gellibert, F. J. PCT Int. WO 2004/111036
(A1), 2004.
3. Ferracioli, G. F. Curr. Opin. Anti-inflam. Immunomod.
Invest. Drugs 2000, 2, 74.
4. Ding, C. Curr. Opin. Invest. Drugs 2006, 7, 1020.
5. Dominguez, C.; Powers, D. A.; Tamayo, N. Curr. Opin.
Drug Discov. Devel. 2005, 8, 421.
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.
Pharmacol. Exp. Ther. 1999, 291, 680.
7. Wang, Z. L.; Canaragajah, B. J.; Boehm, J. C.;
Kassis, S.; Cobb, M. H.; Young, P. R.; Abdel-
Meguid, S.; Adams, J. L.; Goldsmith, E. J. Structure
1998, 6, 1117.
8. (a) Dhar, T. G. M.; Wrobleski, S. T.; Lin, S.; Furch, J. A.;
Nirschl, D. S.; Fan, Y.; Todderud, G.; Pitt, S.; Doweyko,
A. M.; Sack, J. S.; Mathur, A.; McKinnon, M.; Barrish, J.
C.; Dodd, J. H.; Schieven, G. L.; Leftheris, K. Bioorg.
Med. Chem. Lett. 2007, 17, 5019; (b) Natarajan, S. R.;
Wisnoski, D. D.; Thompson, J. E.; O’Neill, E. A.;
O’Keefe, S. J. Bioorg. Med. Chem. Lett. 2006, 16, 4400;
(c) Miwatashi, S.; Arikawa, Y.; Kotani, E.; Miyamoto,
14. The minimum significant ratio of the enzyme assay is 4,
and that of the cell assays is 2.8.
15. Gill, A.; Cleasby, A.; Jhoti, H. ChemBioChem 2005, 6, 506.
16. Trentham, D. E.; Townes, A. S.; Kang, A. H. J. Exp.
Med. 1977, 146, 857.