Identification of triazolopyridazinones as potent p38α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.067

Structure-activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38α mitogen activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency was observed for 2,6-disubstituted phenyl m

Full text of DOI:10.1016/j.bmcl.2011.11.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1226–1229
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of triazolopyridazinones as potent p38a inhibitors
Brad Herberich a, , Claire Jackson a, Ryan P. Wurz a, Liping H. Pettus a, Lisa Sherman b, Qiurong Liu b,
Bradley Henkle b, Christiaan J.M. Saris b, Lu Min Wong b, Samer Chmait c, Matthew R. Lee c,
Christopher Mohr c, Faye Hsieh d, Andrew S. Tasker a
⇑
a Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Inflammation, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38a mitogen
Received 14 October 2011
Revised 14 November 2011
Accepted 18 November 2011
Available online 23 November 2011
activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency
was observed for 2,6-disubstituted phenyl moieties and N-ethyl triazolopyridazinone cores due to key
contacts with Leu108, Ala157 and Val38. Further investigation led to the identification of three com-
pounds, 3g, 3j and 3m that are highly potent inhibitors of LPS-induced MAPKAP kinase 2 (MK2) phos-
phorylation in 50% human whole blood (hWB), and possess desirable in vivo pharmacokinetic and
kinase selectivity profiles.
Keywords:
p38
Kinase
Ó 2011 Elsevier Ltd. All rights reserved.
Inflammation
The production of TNF
through the activation of p38
a
and other cytokines is triggered
mitogen-activated protein (MAP)
long sought an orally available small molecule that decreases cyto-
kine production by the inhibition of p38
.3 Earlier efforts from our
a
a
kinase by multiple external stimuli including stress and a variety
of cytokines.1 Excessive production of cytokines has been impli-
cated in many chronic inflammatory diseases, such as rheumatoid
arthritis and psoriasis. Although there are a number of marketed
p38 program at Amgen have focused on pyrazolopyridinones (1,
Fig. 1) and triazolopyridines 2.4 In this communication, we outline
our development of a novel class of triazolopyridazinone-derived
p38a inhibitors (3, Fig. 1) designed to combine the excellent
anti-tumor necrosis factor
a
(TNF
a
) biological therapies, including
potency and pharmacokinetic profiles of compounds 1 and 2.
Previous SAR investigations of 1 and 2 demonstrated a cyclo-
propyl ring to be the optimum amide substitutent;4 we therefore
etanercept, adalimumab and infliximab, that provide relief from
the symptoms of several inflammatory diseases,2 researchers have
R2
H
H
N
H
N
N
N
N
N
N
N
O
N
O
N
O
N
N
O
N
O
F
F
R1
Ar
F
F
1
2
3
p38α IC50 = 2.3 nM
Rat CL = 220 mL/h/kg
p38α IC50 = 2.5 nM
Rat CL = 370 mL/h/kg
Figure 1. Pyrazolopyridinone-, triazolopyridine- and triazolopyridazinone-derived p38a inhibitors.
⇑
Corresponding author.
E-mail address: brad.herberich@amgen.com (B. Herberich).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.067

B. Herberich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1226–1229
1227
H
Cl
N
Cl
Cl
N
a
b
c
d
H2N
N
N
N
N
ArCHO
N
Cl
N
H
4
O
N
O
N
O
R1
5
R1
6
Br
O
B
Br
Br
O
N
N
R2
e
f
g
N
N
N
O
+
N
N
N
N
O
N
O
N
R1
R1
R1
Ar
Ar
Ar
O
N
H
7
8
9
R2
10 R2= H
11 R2= F
H
N
N
N
N
O
N
O
R1
Ar
3a-n
Scheme 1. General synthetic scheme towards triazolopyridazinone benzamides 3a–n. Reagents and conditions: (a) NaOH, H2O, 80 °C, 85%; (b) MeI or EtI, K2CO3, DMF,
65–98%; (c) hydrazine hydrate, 70 °C, 30–48%; (d) (i) EtOH, reflux, (ii) chloramine T hydrate, THF, 65 °C, 57–82%; (e) Br2, AcOH, 80 °C, 47–95%; (f) NEt3, THF, 60–95%; (g)
PdCl2(PPh3)2, 2 M Na2CO3 (aq), dioxane, 120 °C, 26–73%.
Table 1
focused our present efforts on an investigation of triazolopyridaz-
SAR of modifications of Ara
inone core, tolyl ring, and aryl ring substituents (R1, R2 and Ar,
Fig. 1).
H
N
Scheme
1 outlines the synthesis of triazolopyridazinones
N
N
(3a–n) starting from commercially available 3,6-dichloropyrid-
azine. Hydrolysis under basic aqueous conditions gave pyridazi-
none 4. N-alkylation of the resultant pyridazinone with methyl
or ethyl iodide and subsequent SNAr displacement of the chloride
with hydrazine hydrate afforded 6. The triazolopyridazinone core
7 was prepared by condensation of 6 with a substituted benzalde-
hyde to form the corresponding hydrazone followed by oxidative
cyclization in the presence of chloramine T.5 Dibromination of 7
followed by mono-debromination under basic conditions gave
N
O
N
F
O
3
Ar
Compound
Ar
IC50 (nM)
hWB TNFa/IL-8
p38
a
F
3a
3b
3c
1.3 0.2
1.4 0.4
2.0 0.6
21.4 0.9
Cl
the desired a-bromide 9. The synthesis of 3a–n was subsequently
completed via Suzuki coupling with boronic esters 10 or 11.6,7
The in vitro potency of 3a–n was evaluated by measuring the
inhibition of activating transcription factor 2 (ATF2) phosphoryla-
F
8.5 1.5
Me
Cl
F
11.3 6.0
tion by p38
a as well as the inhibition of TNFa-induced IL-8 pro-
duction in 50% human whole blood (hWB).4a For advanced
compounds, on-mechanism activity was verified by measuring
their ability to modulate LPS-driven MAPKAP kinase 2 (MK2) phos-
phorylation in 50% human whole blood, an event that is currently
understood to be mediated solely by p38 MAP kinase.8 The
3d
3e
6.3 2.0
51.7 5.8
112 40.4
F
F
influence of the triazolopyridazinone 3-substitutent on p38
activity was first investigated with compounds 3a–e (Table 1).
The 2,6-disubstituted analogs (3a–c) exhibited greater potency in
a
16.9 5.3
F
the p38a enzyme and TNFa/IL8 human whole blood assays relative
to the corresponding 2,4- or 2,5-disubstitued analogs (3d–e). This
increase in activity for the 2,6-substituted analogs may be a result
a
The IC50 data are mean values derived from at least three independent dose–
response curves standard deviation.
of a better van der Waals contacts with Leu108 and Ala157 of p38
(see Fig. 2).
a
Further SAR studies concerning substitution of the tolyl ring and
at N5 of the triazolopyridazinone core are outlined in Table 2.
Introduction of a fluorine on the tolyl ring of 3a (2,6-di-F-Ph) and
3b (2-Cl-6-F-Ph) to give 3f and 3g resulted in improved cell-shifts
and an increase of potency in the human whole blood assay. In
general, increasing the steric bulk at N5 from methyl to ethyl
increased the activity of the compounds (3i–j vs 3a,b) in both
potency with the ethyl substitution at N5 was also observed in the
fluorinated tolyl ring analogs (3l–n vs 3f–h). The compounds in Ta-
ble 2 were evaluated in the LPS/pMK2 assay. When the R1 and R2
substituents were kept constant, the 2-Cl-6-F-phenyl-substituted
analogs (3g, 3j and 3m) exhibited greater activity than the corre-
sponding 2,6-di-F-phenyl (3f, 3i and 3l) or 2-Me-6-F-phenyl (3h,
3k, 3n) substituted analogs.
A crystal structure of the unphosphorylated p38/3j complex re-
veals hydrogen bond interactions between the carbonyl of the
cyclopropyl amide and Asp168, and between NH of the cyclopropyl
amide and Glu71.9 Similar to pyrazolopyridinone-derived inhibi-
the enzyme and human whole blood TNFa/IL-8 assays, a result
which can be understood by the ability of the methyl terminus of
the ethyl group to engage the side chain of Val38; while the methyl
group is unable to form similar contacts (see Fig. 2). The increase in

1228
B. Herberich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1226–1229
Figure 2. X-ray crystal structure of 3j with unphosphorylated p38a.
tors and in contrast to phthalazine-derived inhibitors,4a,10 the
N-ethylpyridinone core induces a reorganization of the P-loop.
The carbonyl of the triazolopyridazinone ring participates in a
water mediated hydrogen bond to the backbone NH of Ala34 and
to the ammonium terminus of Lys53. The methyl group of the tolyl
ring sits between the side chains of Thr106 and Val38, while the
ethyl group of the N-ethylpyridazinone makes contact with
Val38. The cyclopropyl group resides next to Phe169. The 2-
chloro-6-fluoro phenyl ring is perpendicular to the triazolopyridaz-
inone core and the fluorine group interacts with the side chain of
Leu108, while the chloride substituent engages Ala157. An alanine
residue on the floor of the ATP binding site is present in only seven
kinases within the kinome.11 Hence, by effectively exploiting both
features of Ala157 and Thr106 (a relatively small gatekeeper) com-
pound 3j is highly selective against a broad panel of kinases (see
below).
Table 2
SAR modifications of Ar, R1, and R2
R2
H
N
N
N
5
N
O
N
O
R1
Ar
Compound Ar
R1
R2
IC50 (nM)
hWB TNF
/IL-8a pMK2b
p38aa
a
F
F
3f
Me
Me
Me
Et
F
4.1 3.0 7.2 1.8
18.5
9.8
To evaluate its kinase selectivity, compound 3j was tested
Cl
against 442 kinases at a concentration of 10 lM using the Ambit
F
3g
F
1.1 0.6 2.6 0.001
2.8 0.4 12.3 2.6
1.0 0.1 4.8 2.2
1.0 0.3 3.2 0.9
1.6 0.4 14.1 11.7
1.9 0.3 3.9 1.5
Biosciences Kinomescan.12 This experiment showed that 3j had
moderate binding (POC <50%) to only one kinase, RET (V804M)13
Me
(14% of control), outside the p38a, b, and c isoforms (0, 0, 14% of
F
3h
3i
F
18.0
7.8
control, respectively).
The in vivo pharmacokinetic profiles of 3g, 3j and 3m are shown
in Table 3. Fluorination of the tolyl ring had little impact on the
pharmacokinetic properties of the analogs (3m vs 3j). Exchanging
a methyl for an ethyl group on the triazolopyridazinone core in-
creased the exposure and bioavailability (3g vs 3m).
Compounds 3g, 3j and 3m were subjected to CYP3A4 and
CYP2D6 inhibition assays as inhibition of these enzymes had been
observed by related N-ethyl pyrazolopyridinone analogs.4a The 3-
fluorotolyl analogs 3m and 3g showed low CYP3A4 inhibition with
F
F
H
H
H
F
Cl
Me
F
F
3j
Et
4.9
F
3k
3l
Et
15.8
8.0
an estimated IC50 of 11.8 and 25.8
sessed an estimated IC50 of >27 M. All three compounds demon-
strated an estimated IC50 of >27 M against CYP2D6. Compounds
3g, 3j and 3m were tested for hERG binding through a 3H-dofetilide
lM, respectively while 3j pos-
l
l
F
Et

B. Herberich et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1226–1229
1229
5. (a) Thiel, O. R.; Achmatowicz, M. M.; Reichelt, A.; Larsen, R. D. Angew. Chem., Int.
Ed. 2010, 49, 8395; (b) Reichelt, A.; Falsey, J. R.; Rzasa, R. M.; Thiel, O. R.;
Achmatowicz, M. M.; Larsen, R. D.; Zhang, D. Org. Lett. 2010, 12, 792.
6. For syntheses of boronic esters 10 and 11 see: Aston, N. M.; Bamborough, P.;
Buckton, J. B.; Edwards, C. D.; Holmes, D. S.; Jones, K. L.; Patel, V. K.; Smee, P. A.;
Somers, D. O.; Vitulli, G.; Walker, A. L. J. Med. Chem. 2009, 52, 6257.
7. Procedures for the preparation of 3j: A suspension of 3,6-dichloropyridazine
(25.50 g, 171.2 mmol) in 100 mL of water was treated with NaOH (15.06 g,
376.6 mmol) and heated at 80 °C for 2 h. The resulting red solution was
allowed to cool to rt and was then acidified to pH 1 with concentrated HCl
(aq). The off-white solid was washed with water and Et2O and then dried
under vacuum overnight to afford 6-chloropyridazin-3(2H)-one (4, 19.13 g,
Table 2 (continued)
Compound Ar
R1
R2
IC50 (nM)
hWB TNF
/IL-8a pMK2b
p38aa
a
Cl
F
3m
Et
Et
F
F
1.2 0.2 1.6 0.4
6.9
Me
F
3n
1.0 0.1 3.4 2.1
13.6
85% yield).
A mixture of 4 (2.55 g, 19.54 mmol) and EtI (1.88 mL,
23.44 mmol) in 10 mL of DMF at rt was treated with K2CO3 (8.10 g,
58.61 mmol). The reaction mixture stirred 48 h at rt and then H2O was
added and the mixture was extracted with EtOAc. The combined organic
layers were washed with water, dried over Na2SO4, filtered and concentrated
to give 6-chloro-2-ethylpyridazin-3(2H)-one (5, 2.70 g, 87% yield). A solution
of 5 (2.70 g, 17.03 mmol) in hydrazine hydrate (4.14 mL, 85.13 mmol) was
heated at 70 °C. After 2 h, the reaction mixture was loaded directly on to a
silica gel column and eluted with 0–10% MeOH in CH2Cl2 to afford 2-ethyl-
a
The IC50 data are mean values derived from at least three independent dose–
response curves standard deviation.
b
The pMK2 IC50 data are an average of two values.
Table 3
Pharmacokinetic profiles of selected compounds in male Sprague–Dawley ratsa
6-hydrazinylpyridazin-3(2H)-one (6, 1.26 g, 48% yield) as
a light-yellow
solid. mixture of (163 mg, 1.06 mmol) and 2-chloro-6-
A
6
Cmpd
IV (2.0 mg/kg in DMSO)
PO (2.0 mg/kg)b
fluorobenzaldehyde (168 mg, 1.06 mmol) in 8 mL of EtOH was heated to
reflux. After 1 h, the reaction mixture was concentrated, the solid was
resuspended in THF (8 mL) and chloramine T-hydrate (265 mg, 1.16 mmol)
was added. The mixture was heated at 65 °C for 4 h. The reaction mixture
was allowed to cool to rt and H2O was added. The mixture was extracted
with EtOAc. The combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated. The crude material was purified by silica gel
chromatography (0–50% EtOAc in hexanes) to afford 3-(2-chloro-6-
fluorophenyl)-5-ethyl-[1,2,4]triazolo[4,3-b]pyridazin-6(5H)-one (7, 222 mg,
CL (mL/h/kg) Vdss (mL/kg) t1/2 (h) AUC (0–1) (ngꢂh/mL) F (%)
3g
3j
3m
526
787
686
2423
2545
2704
3.40
2.76
2.76
3190
1423
1370
83
55
42
a
Values are for an average of three rats.
Vehicle: 1% Pluronic F68, 1% HPMC, 15% hydroxypropyl b-cyclodextrin, 83%
b
water.
72% yield).
A mixture of 7 (222 mg, 0.76 mmol) and Br2 (0.19 mL,
3.79 mmol) in 3 mL of acetic acid was heated at 80 °C for 2 h. The
reaction was cooled to rt, water was added and the mixture was extracted
with EtOAc. The combined organic layers were washed with H2O and
saturated NaHCO3 (aq) and then dried over anhydrous Na2SO4, filtered and
concentrated to afford 7,8-dibromo-3-(2-chloro-6-fluorophenyl)-5-ethyl-7,8-
dihydro-[1,2,4]triazolo[4,3-b]pyridazin-6(5H)-one (8, 285 mg, 83% yield) as a
yellow oil. A solution of 8 (285 mg, 0.63 mmol) in 3 mL of THF at rt was
treated with NEt3 (0.26 mL, 1.89 mmol). After 1 h, H2O was added and the
mixture was extracted with EtOAc. The combined organic layers were dried
over anhydrous Na2SO4, filtered and concentrated to give 7-bromo-3-(2-
chloro-6-fluorophenyl)-5-ethyl-[1,2,4]triazolo[4,3-b]pyridazin-6(5H)-one (9,
displacement assay. All three compounds had IC50 values of
>30
lM.
In conclusion, a new class of triazolopyridazinone p38
a
inhibi-
tors, based on earlier pyrazolopyridinone and triazolopyridine
scaffolds, was designed and synthesized. SAR investigations of
the aryl group at the 3-position of the triazolopyridazinone led to
the identification of the 2-chloro-6-fluoro-phenyl substituent,
which makes contacts with Leu108 and Ala157, and results in ana-
194 mg, 83% yield) as
a yellow oil. In a microwave tube was placed 9
(194 mg, 0.52 mmol), 10 (190 mg, 0.63 mmol), PdCl2(PPh3)2 (18 mg,
0.026 mmol) and 2 M Na2CO3 (aq, 1.3 mL, 2.6 mmol) and 2.5 mL of
logs possessing superior p38a inhibitory activity in whole cells.
dioxane. The mixture was heated in
a microwave at 120 °C for 25 min.
Further optimization of related compounds resulted in the prepa-
ration of 3g, 3j, and 3m, which displayed single digit nanomolar
potency in the huWB LPS/pMK2 assay, favorable pharmacokinetic
profiles, high selectivity against other kinases, and low CYP and
hERG inhibition.
After cooling to rt, H2O was added and the mixture was extracted with
EtOAc. The combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated. The crude material was purified by HPLC
(Phenomenex 150 ꢀ 30 mm Luna column) eluting with 5–100% CH3CN in
water with 0.1% TFA at 35 mL/min over 15 min to afford 3-(3-(2-chloro-6-
fluorophenyl)-5-ethyl-6-oxo-5,6-dihydro-[1,2,4]triazolo[4,3-b]pyridazin-7-yl)-
N-cyclopropyl-4-methylbenzamide (3j, 69 mg, 28% yield) as an off-white
solid. 1H NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.75 (dd, J = 7.92, 1.86 Hz,
1H), 7.67 (d, J = 1.76 Hz, 1H), 7.61 (td, J = 8.31, 5.87 Hz, 1H), 7.46 (d,
J = 8.02 Hz, 1H), 7.37 (d, J = 8.02 Hz, 1H), 7.25–7.30 (m, 1H), 6.34 (br s, 1H),
Supplementary data
4.29 (dq, J = 14.79, 7.13 Hz, 1H), 3.91–4.02 (m,
1 H), 2.90 (tq, J = 7.02,
Supplementary data (X-ray crystallography) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.11.067.
3.47 Hz, 1H), 2.33 (s, 3H), 1.04 (t, J = 7.04 Hz, 3 H), 0.83–0.90 (m, 2H), 0.58–
0.65 (m, 2H). 19F NMR (400 MHz, CDCl3) d ꢁ105.90. MS (ESI, pos. ion) m/z:
466.0 (M+1).
8. Haar, E. Structure 2003, 11, 611.
9. The X-ray coordinates have been deposited in the RCSB Protein Data Bank
database, RCSB ID code: rcsb068453 and PDB ID code: 3U8W). See
Supplementary data for crystallographic protocol and refinement statistics.
10. Herberich, B.; Cao, G.-Q.; Chakrabarti, P.; Falsey, J.; Pettus, L.; Rzasa, R. M.;
Reed, A. B.; Reichelt, A.; Sham, K.; Thaman, M.; Wurz, R. P.; Xu, S.; Zhang, D.;
Hsieh, F.; Lee, M. R.; Syed, R.; Li, V.; Grosfeld, D.; Plant, M. H.; Henkle, B.;
Sherman, L.; Middleton, S.; Wong, L. M.; Tasker, A. S. J. Med. Chem. 2008, 51,
6271.
11. Only 7 out of the 518 kinases have alanine as the floor reside in ATP binding
pocket. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
Science 2002, 298, 1912.
12. Ambit Biosciences: http://www.kinomescan.com/
13. The V804M mutation of the RET kinase has been identified in patients with
medullary thyroid cancer.
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