Continued exploration of the triazolopyridine scaffold as a platform for p38 MAP kinase inhibition

Article abstract of DOI:10.1016/j.bmcl.2009.11.114

The structure based drug design, synthesis and structure-activity relationship of a series of C6 sulfur linked triazolopyridine based p38 inhibitors are described. The metabolic deficiencies of this series were overcome through changes in the C6 linker fr

Full text of DOI:10.1016/j.bmcl.2009.11.114

Bioorganic & Medicinal Chemistry Letters 20 (2010) 469–473
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Continued exploration of the triazolopyridine scaffold as a platform
for p38 MAP kinase inhibition
a,
Kevin D. Jerome a, , Paul V. Rucker , Li Xing a, Huey S. Shieh a, John E. Baldus a, Shaun R. Selness a,
*
Michael A. Letavic b,à, John F. Braganza b,§, Kim F. McClure b
a Pfizer Global Research and Development, St. Louis Laboratories, 700 Chesterfield Pkwy. W., Chesterfield, MO 63017, USA
b Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure based drug design, synthesis and structure–activity relationship of a series of C6 sulfur
linked triazolopyridine based p38 inhibitors are described. The metabolic deficiencies of this series were
overcome through changes in the C6 linker from sulfur to methylene, which was predicted by molecular
modeling to be bioisosteric. X-ray of the ethylene linked compound 61 confirmed the predicted binding
orientation of the scaffold in the p38 enzyme.
Received 4 September 2009
Revised 20 November 2009
Accepted 23 November 2009
Available online 26 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
p38
Kinase
Triazolopyridines
Inhibition of the p38
a
mitogen-activated protein (MAP) kinase
Our strategy has been similar to those employed by other orga-
nizations in the search for novel p38 MAP kinase inhibitors: (1)
determine a privileged scaffold that provides potent compounds
and permits the construction of an SAR grid, (2) build in drug-able
properties that will drive both in vitro potency and in vivo expo-
sure, (3) identify key functionality that imparts kinase selectivity.
In earlier reports from our organization, we disclosed such a
as a viable drug target has garnered a great deal of attention from
multiple drug companies and academic institutions worldwide.1
MAP protein kinase p38 is a dual specificity kinase that has four
known isoforms (a, b,
c
, and d), with their expression varying
amongst cell types of the immune system; and p38
a
being the
chief isoform implicated in inflammatory disease.2 To date there
are now over half a dozen US approved kinase inhibitors, but none
yet approved that specifically target p38 as a mediator of inflam-
matory cytokines. Preclinical studies strongly support that selec-
scaffold targeting p38a, manifested in the oxazole substituted
triazolopyridine chemotype 1.5 In addition, many other p38 inhib-
itors have been reported in the literature, including VX-745.6 Here-
in we report our current progress with the triazolopyridine series,
specifically around the replacement of the oxazole functionality
with a series of isosteric linear chains.
tive inhibition of p38
factor (TNF- ), interleukin-1b (IL-1b) and other cytokine produc-
tion. Because TNF and IL-1b have been implicated as key cyto-
a can effectively modulate tumor necrosis
a
a
kines in inflammatory diseases, p38 has been pursued as a kinase
target for such diseases as rheumatoid arthritis (RA), Crohn’s dis-
ease, inflammatory bowel syndrome (IBS), and psoriasis.3 One cur-
rent treatment option for these inflammatory diseases is with
Structure-based design was used to combine key binding
elements of the triazolopyridine compound CP-808844 1 and VX-
745 2 to design compound 3, utilizing a C6 sulfur atom linker as
an oxazole bioisosteric replacement (Fig. 1).
biological agents known for shutting down TNF-
a function with
great success.4 Hence it is anticipated that a small molecule p38
a
F
F
F
S
MAP kinase inhibitor should provide therapeutic value for the
treatment of such inflammatory conditions.
N
F
S
N
O
N
F
Cl
SBDD
N
* Corresponding author. Tel.: +1 636 247 8655; fax: +1 636 247 0966.
E-mail address: kevin.d.jerome@pfizer.com (K.D. Jerome).
N
N
N
N
N
O
N
Cl
Present address: Genomics Institute of the Novartis Research Foundation (GNF),
10675 John Jay Hopkins Dr., San Diego, CA 92121, USA.
CP-808844
VX-745
à
3
Present address: Johnson & Johnson PRD-La Jolla, 3210 Merryfield Row, San
1
2
Diego, CA 9212, USA.
§
Present address: Pfizer Global Research and Development, La Jolla Laboratories,
Figure 1. Structure based drug design of C6 sulfur linked triazolopyridines from CP-
808844 (1) and VX-745 (2).
10770 Science Center Drive, San Diego, CA 92121, USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.114

470
K. D. Jerome et al. / Bioorg. Med. Chem. Lett. 20 (2010) 469–473
F
The synthesis of the initial bromo triazolopyridine scaffold was
F
F
S
neutral alumina
SH
F
achieved through two different routes. In both routes, the initial
step required the conversion of the di-bromo pyridine, 4, to the
hydrazine, 5, which afforded solely the regioisomer shown
(Scheme 1). In Method A, a one pot synthesis of the bromo triazol-
opyridine core was achieved by acylation of 5 to give the in situ
formation of the corresponding hydrazide, followed by dehydra-
tion to the triazolopyridine.7
Method B shows an alternate two step synthesis of the bromo
triazolopyridine core from the hydrazine, 5. Acylation of 5 formed
the hydrazide, which was isolated, followed by dehydration to the
triazolopyridine. Although Method B contains an additional syn-
thetic step, it was found to be more general than the one pot route
in Method A. The bromo triazolopyridine cores synthesized, 6–15,
as well as the method used to synthesize them, are shown in Table
1.
2 eq
S
F
DMSO, 40ºC
90% yield
F
16
Scheme 2. Synthesis of aryl disulfides from thiols.
R2
O
R3
R2
O
Cl
R2
WCl6
,
NaI
S
S
2 eq
S
R3
R4
R4
MeCN
R4
R3
Scheme 3. Synthesis of aryl disulfides from sulfonyl chlorides.
Table 2
Aryl disulfides from sulfonyl chlorides
The sulfur linked triazolopyridines were synthesized from the
bromo triazolopyridine cores, 6–15, and various aryl disulfide com-
pounds. These aryl disulfides were synthesized by two methods.
The initial method (Scheme 2) involved the dimerization of 2,4-
difluorophenyl thiol using neutral alumina8 to form the disulfide
16.
Compounds
R2
R3
R4
Yield (%)
17
18
19
CF3
CF3
Cl
Br
F
Cl
H
H
Cl
82
79
77
The preparation of additional aryl disulfides was accomplished
through a method that permitted the use of the more commercially
abundant sulfonyl chlorides, using WCl6 and NaI to provide their
corresponding disulfides (Scheme 3, Table 2).9
Aryl disulfides 16–19 were then allowed to react with the cor-
responding Grignards derived from 6 to 15 (Scheme 4, Table 3).
The HCl salt of the triazolopyridine could be formed by treatment
R3
R2
S
Br
1)
2)
i-PrMgCl, THF, 0ºC to rt;
R3
HCl
R4
R2
R1
N
R2
R1
N
N
N
S
N
S
N
R4
R4
R3
3) HCl in dioxane
O
Cl
R1
Scheme 4. Synthesis of sulfur linked triazolopyridines.
Br
Br
(or carboxylic
acid, oxalyl
chloride)
Br
Br
hydrazine hydrate
1-propanol
reflux
N
N
N
with 4 N HCl in dioxane following work-up. If the starting bromo
triazolopyridine core was an HCl salt, 2 equiv of Grignard reagent
were necessary.
84% yield
i-Pr2NEt,
HN
HN
NH2 dioxane/toluene
0 ºC to r.t.
NH
O
R1
4
5
Triazolopyridine cores, 9 and 11–14, were further elaborated,
using known chemistry, to obtain additional compounds shown
in Table 3. Although the sulfur linked triazolopyridines afforded
O
R1
HO
POCl3
100ºC
(one pot)
EDCI, CH2Cl2
some single digit nM inhibitors of p38
a, the series as a whole suf-
fered from a poor metabolic and pharmacokinetic profile. How-
ever, 3 did show p38
a inhibition of 79 nM, had 89% human liver
and 46% rat liver microsomal stability10 and was the only com-
pound in the series to show significant in vivo activity (61% inhibi-
tion of rat LPS@ 5 MPK, 4 h).11 Therefore, 3 was considered as
validation that a linear linkage could potentially substitute for
Br
Br
Br
HCl
R1
SOCl2, dioxane
100ºC
N
N
R1
N
HN
O
N
N
NH
N
N
Method A
the oxazole in a potent triazolopyridine p38a inhibitor, warranting
further study to improve metabolic stability and pK properties, and
R1
Method B
ultimately the in vivo activity of the series.
Scheme 1. Alternate syntheses of the bromo triazolopyridine Scaffold.
It was determined through metabolic identification experi-
ments that the most prevalent metabolic soft spot was oxidation
of the sulfur linkage to the sulfoxide and/or the sulfone. It was
hypothesized that if bulky groups were installed to C5 and C7 of
the triazolopyridine core of 3, the sulfur atom would be sterically
shielded and thereby metabolism would be blocked. Based on
modeling studies, the C5 carbon of the triazolopyridine would be
well tolerated for substitution. In contrast, substitution of the C7
position would interfere with the binding conformation of the
Table 1
Bromo triazolopyridine cores
Compounds
R1
Method
6
7
8
iso-Propyl
t-Butyl
A
A
B
A
A
A
B
A
B
B
1-Methylcyclopropyl
2-Methylpent-4-en-2-yl
2,6-DiF-Ph
2-Me-4-Br-Ph
3-COOMe-Ph
2-Me-5-COOMe-Ph
4-COOMe-Ph
4-Piperidine
9
2,4-difluorophenyl group towards the lipophilic pocket of p38a.
10
11
12
13
14
15
Triazolopyridine 3 was halogenated12 (Fig. 2) to obtain the mod-
eled analogs. Consistent with the predictions by molecular model-
ing, compound 48 showed good p38a activity (Table 4), and any
substitution at R6 (C7 of the triazolopyridine) resulted in loss of
activity.

K. D. Jerome et al. / Bioorg. Med. Chem. Lett. 20 (2010) 469–473
471
Table 3
Sulfur linked triazolopyridines
Compd
Core
Disulfide
R1
R2
R3
R4
p38a IC50 (nM)
3
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
6
7
8
9
9
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
17
18
19
i-Pr
t-Bu
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
CF3
CF3
Cl
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Br
F
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
79.3
4.8
24.9
16.3
1740
7.2
1-Me-cyclopropyl
2-Me-pent-4-en-2-yl
1,1-DiMe-3,4-butanediol
2,4-Diflurophenyl
2-Me-4-Br-phenyl
2-Me-4-vinyl-phenyl
2-Me-4-(1,2-diOHÁEt)-Ph
2-Me-4-(CH2NH-(CH2)2OH-Ph
3-COOMe-phenyl
3-COOH-phenyl
3-CONH2-phenyl
2-Me-5-COOMe-phenyl
2-Me-5-COOH-phenyl
2-Me-5-CONH2-phenyl
2-Me-5-CONHMe-Ph
2-Me-5-CONHCH2-CONH2-Ph
2-Me-5-CONH(CH2)2-OH-phenyl
4-COOMe-Ph
4-COOH-Ph
4-CONH2-Ph
4-CONHMe-Ph
4-Piperidine
N-COCH2OAc-4-piperidine
N-COCH2OH-4-piperidine
i-Pr
i-Pr
i-Pr
10
11
11
11
11
12
12
12
13
13
13
13
13
13
14
14
14
14
15
15
15
6
141
106
28.3
33.3
181
7420
241
7.8
2920
60.8
39.9
46.5
20.5
322
1170
201
293
3770
367
212
>10,000
>10,000
8240
6
6
Table 4
Halogenated analogs of triazolopyridine 3
Human and rat liver microsomal data showed that the haloge-
nation hypothesis failed to improve upon the metabolic stability
of the compounds. At this point, we sought to replace the sulfur
linkage altogether with an alternative connection that might afford
better in vivo stability. Given the large size of the sulfur atom and
the flexibility of the sulfur–carbon bond, the distance afforded by
the sulfur linker is suggested to be between that of a methylene
and ethylene unit. Upon evaluating a number of alternative linkers
by molecular modeling, based on their ability to maintain the rel-
ative binding conformations of the hinge binding core and the lipo-
philic aryl group, several triazolopyridine analogs were
Compounds
R5
R6
P38
a
IC50 (nM)
HLM
RLM
(% remaining)
(% remaining)
3
48
49
50
51
H
Cl
H
Cl
Br
H
H
Cl
Cl
Cl
79.3
12.3
889
3380
6080
89
65
71
39
24
46
23
37
20
9
F
F
X
synthesized and tested for p38
a potency and in liver microsomal
stability assays (Fig. 3, Table 5).
In modeling studies, the methylene and ethylene linkers ap-
peared to be a suitable replacement for sulfur. Upon evaluation,
N
these replacements gave potent p38
a
inhibition, with 57 and 58
N
N
showing similar p38 potency as 3. Compound 57 in particular
a
Figure 3. Alternate triazolopyridine bioisosteric linkers.
also showed a very favorable metabolic stability profile and there-
fore stood out as the most promising sulfur replacement linker.
On the other hand, from modeling studies the ketone moiety
appeared too rigid, imposing a restricted conformation of the 2,4-
difluorophenyl that disrupted its interaction with the aryl pocket.
This hypothesis was supported, as 52 showed a significant drop
Table 5
Alternate triazolopyridine bioisosteric linkers
Compounds
X
P38
a
IC50 (nM) HLM
RLM
(% remaining) (% remaining)
in p38
a potency. The amide function appeared to have to adopt
3
52
53
54
55
56
57
58
S
79.3
89
46
a cis conformation in order to preserve the binding interactions,
and such energy penalty is reflected in the loss of activity of 55.
The loss of activity of the sulfonyl linker 54 cannot be explained
by steric effects, since little conformational change was suggested
C@O
S@O
SO2
(C@O)NH
(C@O)CH2 7370
1050
9680
>10,000
>10,000
74
88
100
ND
ND
ND
96
100
ND
ND
ND
95
CH2
CH2CH2
26.1
85.0
53
54
F
F
S
R6
R5
from sulfur to sulfonyl and the protein sub-environment is open to
accommodate the sulfonyl oxygen atoms. The effect could be elec-
trostatic. One possibility is that the strong electron withdrawing
sulfonyl group attenuates the hydrogen bond accepting ability of
N
N
N
Figure 2. Halogenation of triazolopyridine 3.

472
K. D. Jerome et al. / Bioorg. Med. Chem. Lett. 20 (2010) 469–473
the nitrogen atoms of the triazole. Such hydrogen bond recognition
with the kinase hinge is critical for inhibitor potency, and is sensi-
tive to the electron density distribution of the heterocyclic core.13
Additionally, the sulfonyl oxygen atoms, each carrying a negative
point charge and would interact unfavorably with the vicinal
Asp168 on the p38
a
activation loop (OÁ Á ÁO distance is ꢀ4.2 Å per
modeling). Both of the rationales would predict the potency trend
observed for 3, 53 and 54, with the sulfoxide exhibiting an inter-
mediate electrostatic property between the sulfur and the sulfonyl
linkers. It was noted, however, that the metabolic stability of 53
was much improved over 3.
Encouraged by our results from 57, we continued to explore our
options for the construction of carbon based bridges. The synthetic
preparation of 61 is delineated in Scheme 5.14 Conversion of bro-
mide 59 to intermediate 60 was accomplished by conversion to
the Grignard reagent using isopropyl magnesium chloride and sub-
sequent trapping with DMF to provide the aldehyde. This aldehyde
was then transformed into the ethylene bridge using a Wittig
homologation and hydrogenation. The use of 2-chloro-4,6-dime-
thoxy-1,3,5-triazine as an activating agent to furnish the desired
amide 61 provided slightly better overall yields than the conven-
tional HATU coupling in this instance, owing to the insolubility
of the acid precursor. The intermediate carboxylic acid was sus-
pended in THF and treated with 4-methyl-morpholine followed
by treatment with 2-chloro-4,6-dimethoxy-1,3,5-triazine. The
resulting suspension was allowed to stir at room temperature fol-
lowed by work-up with ammonium hydroxide and subsequent
normal phase silica purification.15
Figure 4. Crystal structure of compound 61 bound to p38a.
The crystal structure confirmed the binding models generated
by molecular docking for the triazolopyridine compounds. As ex-
pected, 61 is bound at the ATP binding site of p38a kinase, which
is located at the junction between the N- and C-terminal domains.
The following protein residues define the binding site of 61: the
glycine-rich loop (31–38), the crossover peptide (107–111), C-ter-
Typified in Figure 4, we were able to utilize compound 61, a po-
tent p38
a inhibitor of 40nM, by obtaining a co-crystal with the
minal end of a a-helix (61–78), a b-strand from the N-terminal do-
p38
a
enzyme.16 The binary complex has been refined to 2.0 Å res-
main (49–53) and a b-strand from the C-terminal domain (162–
171) and a flexible loop (84–86). The triazolopyridine ring anchors
to the hinge region, and due to the small gate residue T106, the 2,4-
difluorophenyl ring is able to insert into the hydrophobic pocket
formed by residues Val 38, Ala 51, His 107, Leu 108, Met 109 and
Leu 167. The benzamide moiety is exposed to the solvent region.
Consistent with inhibitor structures of other chemical classes, the
peptide bond connecting Met109 and Gly110 exhibits a flipped
conformation from its apo state for the formation of the bidentate
hydrogen bond.13 The 1,2-nitrogen atoms of the triazolopyridine
core serve as hydrogen bond acceptors that receive protons from
the backbone NH of Met109 and Gly110.
olution with Rwork = 23.6% (Rfree = 27.5%) with good stereochemis-
try (rms deviation from ideality of 0.011 Å in bond lengths and
1.35° in bond angles). This co-crystallization permitted us to verify
our binding model hypothesis and further demonstrate that carbon
bridges, in this case ethylene, are sufficient substitutes for the sul-
fur atom linkage.
F
1) i-PrMgCl, THF, 0ºC; DMF
0ºC to r.t.
Br
2) NaHMDS, THF, -20ºC
F
N
In summary, the evolution of C6 linkers of triazolopyridine
F
F
N
N
OMe
p38
linked compounds such as 3 were first modeled, then found to be
potent p38 inhibitors, but they lacked sufficient metabolic stabil-
ity. Shown to be bioisosteric through modeling, methylene linked
inhibitors such as 57 were found to be not only potent in p38
a inhibitors was described. Using structure based design, sulfur
OMe
O
PPh3Br
O
N
3) Pd/C 10%, Hydrogen
(57% yield, 3 steps)
N
N
59
a
60
a
,
4) NaOH, water-THF, 50ºC
but also metabolically stable. An X-ray of the ethylene linked com-
pound 61 confirmed the binding models for the triazolopyridine
series.
5) NMM, 2-chloro-4,6-
dimethoxy-1,3,5-triazine,
THF
6) NH4OH
(55% yield, 3 steps)
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9. Firouzabadi, H.; Karimi, B. Synthesis 1999, 500.
10. Metabolic stability was assessed in vitro by incubating 2
lM test compound
14. (a) Full disclosure of chemistry methods is made in the following patents:
Rucker, P. V.; Jerome, K. D.; Selness, S. R.; Baldus, J. E.; Xing, L. PCT Int. Appl.
2006, 141 p, WO 2006018735.; (b) Rucker, P. V.; Jerome, K. D.; Selness, S. R.;
Baldus, J. E.; Xing, L. PCT Int. Appl. 2006, 161 p, WO 2006018727.
15. Kaminski, Z. J. Synthesis 1987, 10, 917.
with human or rat liver microsomes, NADPH and buffer at 37 °C for 45 min and
measuring percent compound remaining by a precipitation procedure followed
by LC–MS analysis.
16. The atomic coordinates for the X-ray structure of p38
a in complex with 61
have been deposited in the RCSB: rcsb id code—RCSB055916; PDB code—3KF7.