Synthesis and biological activity of 2H-quinolizin-2-one based p38α MAP kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.03.069

The development and synthesis of potent p38α MAP kinase inhibitors containing a 2H-quinolizin-2-one platform is described. Evolution of the 2H-quinolizin-2-one series from an early lead to solving off target activity and pharmacokinetic issues is also discussed.

Full text of DOI:10.1016/j.bmcl.2010.03.069

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2765–2769
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of 2H-quinolizin-2-one based p38
a
MAP
kinase inhibitors
a
a
b
Robert M. Tynebor a, , Meng-Hsin Chen , Swaminathan R. Natarajan , Edward A. O’Neill ,
*
James E. Thompson b, Catherine E. Fitzgerald b, Stephen J. O’Keefe b, James B. Doherty a
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
b Department of Inflammation Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The development and synthesis of potent p38
platform is described. Evolution of the 2H-quinolizin-2-one series from an early lead to solving off target
activity and pharmacokinetic issues is also discussed.
a
MAP kinase inhibitors containing a 2H-quinolizin-2-one
Received 8 February 2010
Revised 16 March 2010
Accepted 17 March 2010
Available online 21 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
p38a inhibitors
MAP kinase
Over the last decade the pharmaceutical industry invested sig-
nificant resources in developing a therapy to regulate tumor necro-
we have investigated a new structural class of potent and selective
p38 inhibitors utilizing 6-(2,4-difluorophenyl)-1-phenyl-2H-
a
sis factor (TNF-
a
)
for the treatment of such indications as
quinolizin-2-one (Fig. 1) scaffold as a pharmacophore, which is
the subject of this communication.
rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel
disease.1 Current TNF-
a
treatments include monoclonal antibodies
Small molecule p38a inhibitors made a structural transition
Infliximab (RemicadeÒ), Adalimumab (HumiraÒ), and the fusion
from the tetrasubstituted imidazole series (2)6 to pyrimido pyrid-
azinone derived compounds with the discovery of VX-745 (3)
(Fig. 2).7 Investigations into the binding mode of 3 revealed several
unique features including a novel induced fit by causing a rotation
or ‘flip’ of the peptide bond between Met-109 and Gly-110. As
shown in Figure 2, the tetrasubstituted imidazoles bind to the
protein Etanercept (EnbrelÒ).2 Although current therapies success-
fully reduce TNF-a levels, long term patient compliance is compro-
mised by safety, cost, and/or efficacy.1,3 Currently no small
molecule therapy has successfully reached the market.2
It has been demonstrated that inhibiting p38
a mitogen-acti-
vated protein (MAP) kinase delays the onset of joint disease in
p38
a
active site by utilizing a pyrimidine nitrogen to hydrogen
active site by using
animal models of arthritis by arresting the over production of
bond with Met-109 while 3 binds to the p38
a
pro-inflammatory cytokines such as TNF-a 4
.
In peripheral mono-
a carbonyl group to hydrogen bond with Met-109. The second
available lone pair on the carbonyl oxygen induces a unique ‘flip’
of the peptide between Met-109 and Gly-110. The newly reorga-
nized enzyme conformation is then stabilized by the uniquely po-
nuclear blood cells, the p38 MAP kinase pathway is activated by
a variety of external stress stimuli, such as heat shock, osmotic
stress, lipopolysaccharide (LPS) and other cytokines.5 Cell surface
receptors recognize these stress stimuli and initiate a signal trans-
duction cascade that proceeds through p38 MAP kinase. The up-
stream activators of p38 MAP kinase are MKK3 and MKK6 while
the downstream substrates include MAPKAP kinase-2 and heat
shock protein (HSP)-27. The end result is the production of pro-
inflammatory cytokines, edema, and joint destruction. Inhibiting
lar scaffold of 3.8,9 MAP kinases outside the p38
a,b,c isoform
family fail to adopt the ‘flipped’ conformation due to larger side
chains present at the amino acid residue 110 that make proper en-
zyme rotation energetically unfavorable. As a result, 3 possessed
unprecedented selectivity over a broad range of kinases as well
as other closely related members of the MAP kinases family.9
It was also hypothesized that the piperidine of 2 interacts with
Asp-168 via a water bridged hydrogen bonding interaction. This
hypothesis helped drive binding and functional potency in the
quinazolinone (4) series by utilizing a piperazine to interact with
Asp-168. Although functional activity and physical properties
were enhanced by the basic amine, such inhibitors were plagued
of the p38
TNF- production and afford an opportunity to slow the progres-
sion of TNF- mediated inflammatory diseases. In our laboratory
a pathway is therefore expected to down regulate
a
a
* Corresponding author. Tel.: +1 215 652 3636; fax: +1 215 652 3971.
E-mail address: Robert_tynebor@merck.com (R.M. Tynebor).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.069

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R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2765–2769
Figure 1. 6-(2,4-Difluorophenyl)-1-phenyl-2H-quinolizin-2-one.
Figure 2. p38a binding schematic for tetrasubstituted imidazole 2, 3, and quinazolinone 4.
with potent ion channel activity. Evolution of small molecule p38
inhibitors focused on replacing the piperazine group of 4.
a
Removing the basic amine from compounds 4 and 5 success-
fully reduced MK-499 activity of quinolizin-2-one 6 to less than
Previous research revealed the entire piperazine substituent
could be replaced with a simple amino group without a significant
loss in functional potency.10 However this required that the core
template had sufficient polarity to achieve good cell potency.
Replacing the urea derived core of 4 with a naphthyridinone and
the piperazine with an amino group lowered log D for compound
5 while maintaining good activity in the LPS stimulated THP-1 cells
20
l
M, but unfortunately other liabilities such as pregnane X
M) and a long half life in dog
receptor (PXR) activation (EC50 3.3
l
(20 h) and monkey (>100 h) precluded further development of 6.
Previous research indicated chemically diverse substituted A-rings
were well tolerated.15 Therefore, we sought to introduce chemi-
cally diversity to the A-ring position in an attempt to reduce PXR
activation and improve pharmacokinetic properties.
and human whole blood (hWB) TNF-
a
release functional assays.
The first series of A-ring modified quinolizin-2-one derivatives
focused on the 2,4,6 substitution pattern. Synthesis of such deriv-
atives began with preparation of benzyl bromide 8 via a reaction of
the mesylate derived from 7 with LiBr (Scheme 1). Treatment of
bromide 8 with activated zinc generated the zinc bromide deriva-
tive in situ which was coupled with 2,6 dibromopyridine to yield
diaryl methylene 9. Suzuki coupling between pyridyl bromide 9
Encouraged by the success of 5 we hypothesized that the isomeric
quinolizin-2-one 6 derivative would sufficiently increase the core
polarity and dipole moment to excise the amine of 5. In the event,
quinolizin-2-one 6 increased scaffold polarity as indicated by the
lower log D value in Table 1, producing biological activity compa-
rable to compounds 4 and 5.
Table 1
Evolution of p38a inhibitors
p38a6,11 IC50 (nM)
THP-l/TNF-a12 IC50 (nM)
hWB/TNF-a6 IC50 (nM)
Log D
Ca2+ IC50
(l
M)
iKrb Ki (
lM)
a
4
5
6
2.6
0.6
7.1
4.0
1.0
5.6
76
34.3
75.5
4.2
2.7
2.5
0.94
4.6
>30
0.87
5.1
>20
a
Inhibition of diltiazem binding.13
Inhibition of MK-499 binding to hERG in HEK293 cells.14
b

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2765–2769
2767
Scheme 1. Synthesis of 2,4,6 substituted A-ring 2H-quinolizin-2-one derivatives. Reagents and conditions: (a) MsCI, TEA, CH2CI2, 0 °C warm to rt; (b) LiBr, DMF, 90 °C 45 min,
75% two steps; (c) Zn, THF, 0 °C warm to rt, 1 h; (d) Pd(PPh3)4 90 °C, 1 h, 68% two steps; (e) 2,4 difluoro phenyl boronic acid, Pd(PPh3)4, toluene/EtOH/2 M Na2CO3 (10:1:1),
89%; (f) Zn(CN)2, tris(dibenzylideneacetone)dipalladium, 1,10-bis(diphenylphosphino)ferrocene, DMF/H2O (100:1), 120 °C, 1 h, 96%; (g) LiHMDS, ꢀ78 °C, 1 h, THF, 84%; (h)
TBAF, THF, 0 °C, 89%; (i) NMP, 90 °C, 38%; (j) 2 N KOH, dioxane, 90 °C, 24 h, 83%; (k) EDC, hobt, NH4OH, NMP, 75%; (l) oxalyl chloride, MeOH, 0 °C, 90%.
benzylic anion of 10 and was quenched with TMS propenyl ethyl
ester to yield the protected diarylbutynone 11. Removal of the
TMS protecting group and thermal cyclization generated the 2H-
quinolizin-2-one platform 12.16
Table 2
Biological activity of 2,4,6 substituted A-ring 2H-quinolizin-2-one derivatives
p38
(nM)
a
IC50
THP-l/TNF-a12 IC50
(nM)
hWB/TNF-a6 IC50
(nM)
PXR EC50
M)
(l
Nitrile 12 not only served as a valuable synthetic intermediate
for future manipulations but also eliminated the PXR activation
associated with compound 6 (Table 2). Although the nitrile pos-
sessed only weak functional activity, this discovery encouraged
the exploration of other heteroatom containing functional groups
at this position. Hydrolysis of nitrile 12 to carboxylic acid 13 and
EDC coupling with ammonium hydroxide yielded amide 14. Amide
12 1.5
14 20
15 1.5
6490
180
6490
N/A
980
N/A
>30
>30
>30
and 2,4 difluoro phenyl boronic acid was used to introduce the
C-ring. The nitrile of 10 was installed via a Pd2(dba)3, dppf, and zinc
cyanide coupling with the A-ring bromide. LiHMDS generated the
14 maintained moderate p38a enzyme potency and improved
Scheme 2. Synthesis of 2,4,6 substituted A-ring 2H-quinolizin-2-one bioisostere derivatives. Reagents and conditions: (a) oxalyl chloride, DMF, DCM, 0 °C, quant.; (b) N2H4,
CH2CI2, rt, quant.; (c) trimethylorthoacetate, 120 °C, MeOH, 1.5 h; (d) triethylorthoformate, 110 °C, MeOH, 2 h, three step yield 6%; (e) cyanogen bromide, MeOH, 90 °C, 2 h,
three step yield 9%; (f) EDC, hobt, N0-hydroxyethanimidamide, NMP, 90 °C, 6 h, 16%.

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R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2765–2769
Table 3
2,5 Substituted analogs 22 and 23 were synthesized by bromi-
nating 4-fluoro-3-methylbenzonitrile with N-bromosuccinimide
and benzoyl peroxide to yield 21 (Scheme 3). Using a similar syn-
thetic protocol as Schemes 1 and 2, bromide 21 was synthetically
modified to yield derivatives 22–23.
Biological activity of 2,4,6 substituted A-ring 2H-quinolizin-2-one bioisostere
derivatives
p38
(nM)
a
IC50
THP-l/TNF-a12 IC50
(nM)
hWB/TNF-a6 IC50
(nM)
PXR EC50
M)
(l
17 14
950
265
270
430
2660
775
1580
690
>30
>30
>30
>30
2,5 Substituted analogs possessed PXR activity and enzyme po-
tency comparable to the 2,4,6 substituted series, while functional
activity dramatically improved (Table 4). For example, THP-1 and
WB activity of oxadiazoles 22 and 23 increased 10–30-fold and
15–60-fold, respectively when compared to 2,4,6 substituted ana-
logs 17 and 18. As with other 2H-quinolizin-2-one derivatives,
compounds 22 and 23 possessed Ca and iKr ion channel activity
18 5.3
19 6.8
20 8.8
whole blood potency to sub micromolar levels. Methyl ester 15
possessed 1.5 nM p38 activity but failed to improve functional
a
greater then 20 lM and 30 lM, respectively.
activity, therefore it was believed that metabolic stability of com-
pounds 12, 14, and 15 were responsible for the large shift in func-
tional activity. As a result, we explored suitable bioisostere
replacements, such as oxadiazoles, for compounds 12, 14, and 15.
Oxadiazole bioisosteres 17–20 were synthesized from carbox-
ylic acid intermediate 13 (Scheme 2). Treatment of acid 13 with
oxalyl chloride and catalytic amounts of DMF yielded the acid chlo-
ride derivative, which reacted with hydrazine hydrate to provide
benzhydrazide 16. The crude benzhydrazide intermediate was
refluxed in trimethyl orthoacetate to yield oxadiazole 17 in low
yield. Similarly, oxadiazoles 18 and 19 were synthesized from
intermediate 16 using triethylorthoformate and cyanogen
bromide, respectively. EDC/hobt amide coupling between 13 and
N0-hydroxyethanimidamide and cyclization yielded oxadiazole 20
in a one pot reaction.
Pharmacokinetic properties of compounds 22 and 23 were eval-
uated in several species (Table 5). Compound 22 possessed good
oral bioavailability, low clearance, and high AUC in all species
tested. However, the half life of compound 22 in the dog IV study
was still comparable to lead compound 6. Compound 23 also pos-
sessed good oral bioavailability, low clearance, and high AUC, and a
shorter half life in the dog pharmacokinetic studies. Therefore,
compound 23 possessed the most desirable profile with potent
functional activity, no ion channel or PXR off target activity, excel-
lent kinase selectivity,17 and a PK profile suitable for oral dosing in
several species.
Efficacy was measured using the LPS induced arthritic rodent
model to gauge the ability of 23 to decrease high levels of TNF-
a
Table 5
Oxadiazole bioisosteres 17–20 retained an excellent PXR profile
Pharmacokinetic profiles of 22 and 23
(EC50 >30 lM) and improved WB and THP-1 potency relative to
compounds 14 and 15, but possessed functional activity several or-
ders of magnitude weaker than lead compound 6 (Table 3). A-rings
possessing a 4 substituted oxadiazole demonstrated that PXR
Species
Cl (ml/min/kg)
AUC (
l
M h)
T1/2 (h)
F (%)
22
23
Rata
3.1
0.49
3.6
25.6
49.3
5.1
1.8
30.4
5.9
100
45
47
Dogb
Monkeyb
activity can be successfully eliminated while retaining p38
a en-
Rata
2.4
0.88
0.90
21.7
58.6
23.9
1.4
12.0
9.5
68
66
55
zyme potency, however other substitution patterns were explored
to also increase functional activity. Previous manuscripts indicated
an ortho substituted A-ring was critical for maintaining good en-
zyme potency.15 Shifting the oxadiazole to the 5-position could ex-
ploit this group’s PXR reducing properties and take advantage of
the potency enhancing effects associated with a 2 fluoro group.
Dogb
Monkeyb
a
Iv 1 mg/kg, po 2 mg/kg, PEG-200/water 70:30 (v/v).
Iv 0.25 mg/kg, ethanol/PEG-200/water (10:40:50) (v/v/v) po 2 mg/kg, 0.5%
methylcellulose + 0.02% SDS.
b
Scheme 3. Synthesis of 2,5 substituted A-ring 2H-quinolizin-2-one derivatives. Reagents: (a) NBS, benzoyl peroxide, CCl4, reflux, 75%.
Table 4
Biological activity of 2,5 substituted A-ring 2H-quinolizin-2-one derivatives
THP-l/TNF-a12 IC50 (nM)
hWB/TNF-a 6 IC50 (nM)
PXR EC50 (nM)
Ca2+ IC50
(l
M)
iKrb Ki (
lM)
a
p38a
IC50 (nM)
22
23
14.7
13.0
23.8
25.4
62.2
45.4
22.8
26.8
>30
>30
>20
>20
a
Inhibition of diltiazem binding.13
Inhibition of MK-499 binding to hERG in HEK293 cells.14
b

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2765–2769
2769
Figure 3. Reduction of TNF-a levels in rat LPS induced arthritic model at 4 and 24 h.
7. (a) Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E. M. U.S. Patent
6147,080, 2000.; (b) Salituro, F.; Bemis, G.; Cochran, J. WO 99/64400.
8. (a) Natarajan, R. S.; Doherty, J. B. Curr. Top. Med. Chem. 2005, 5, 987; (b)
Herberich, B.; Cao, G.-Q.; Chakrabarti, P. P.; Falsey, J. R.; 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,
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5809.
(Fig. 3). At the 4 h time point both 1 mg/kg and 3 mg/kg displayed
>90% reduction of TNF- levels, while 0.03 mg/kg still reduced
THF- levels by an average of 50%. The 24 h time point showed sig-
nificant reduction in TNF- levels at 30 and 10 mg/kg and moder-
ate reduction at 1 and 3 mg/kg. Although the rat t1/2 was 1.4 h, the
excellent physical properties and potent functional activity of com-
pound 23 allowed for excellent coverage in the 24 h LPS model.
In conclusion, we were able to successfully eliminate off target
activity associated with piperazine 4 by removing the basic amine.
Adjusting the core polarity to maintain functional potency led to
a
a
a
11. A SPA-bead based assay was carried out using mouse p38. Compounds were
serially diluted into a 96 well plate containing a MOPS based p38 assay buffer.
The assay was initiated by addition of cold ATP, 33P ATP (gamma) and biotin
the discovery of the novel 2H-quinolizin-2-one class of p38a inhib-
itors. Initially this series was plagued with high PXR activity and
long half life, but SAR optimization of 6 solved such issues while
maintaining excellent potency. Moreover, 23 proved efficacious
in the LPS induced arthritic rat model.
labeled GST-ATF2 substrate (4 lM). After incubation at 30 °C for 3 h, the
reaction was stopped by addition of a PBS based quench buffer with 2ꢁ moles
of SPA beads over the amount of substrate used. The extent of phosphorylation
of GST-ATF2 was measured using a topcount reader and subtracted from
background. IC50s are means of two experiments.
12. Anti human TNF-
a was coated on immulon four plates. THP-1 cells
References and notes
(density = 2.5 ꢁ 106/mL) were suspended into 96-well plates containing
a
PBS based medium. Compound was added as solution in DMSO followed by
addition of LPS. The reaction was incubated for 4 h at 37 °C under CO2. TNF-
release was measured in the supernatants by ELISA. Reported IC50s are means
from three measurements.
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