Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Article abstract of DOI:10.1016/j.bmcl.2005.02.010

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition o

Full text of DOI:10.1016/j.bmcl.2005.02.010

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2409–2413
Design and synthesis of potent pyridazine inhibitors
of p38 MAP kinase
Nuria Tamayo,a,* Lillian Liao,a Martin Goldberg,a David Powers,a Yan-Yan Tudor,a
Violeta Yu,a Lu Min Wong,b Bradley Henkle,b Scot Middleton,b Rashid Syed,a
Timothy Harvey,a Graham Jang,c Randall Hungatea and Celia Domingueza,*
aChemistry Research and Discovery, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
bInflammation, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
cPharmacokinetics and Drug Metabolism, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
Received 11 January 2005; revised 2 February 2005; accepted 3 February 2005
Available online 22 March 2005
Abstract—Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have
activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibi-
tion of LPS-induced TNF-a production in mice and exhibited good efficacy in the rat collagen induced arthritis model.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Chemistry
Inhibitors of p38 mitogen activated protein (MAP) ki-
nase inhibit the production of proinflammatory cyto-
kines, for example, tumor necrosis factor-a (TNF-a)
and interleukin-1b (IL-1b), whose accumulation initiates
a cascade of events leading to inflammation and tissue
destruction in diseases such as rheumatoid arthritis
(RA),1 Crohnꢀs disease,2 inflammatory bowel syndrome,
and psoriasis. The inhibition of TNF-a and IL-1b pre-
sents a useful therapeutic strategy to suppress the
inflammation and prevent joint damage caused by RA,
as shown by the newer biologic therapies for RA (eta-
nercept, infliximab, adalimumab, and anakinra) that
target these cytokines. p38 MAP kinase is a member
of a family of serine–threonine kinases that are activated
by dual phosphorylation of a TGY motif.3 This phos-
phorylation is carried out by dual specificity kinases
(MKK3 and MKK64) in response to extracellular sti-
muli such as osmotic shock, endotoxins (lipopolysacha-
ride, LPS), UV light or cytokines.5
3,4,6-Trisubstituted pyridazines were prepared by reac-
tion of hydrazine with either a 1,4-diketone or a 1,4-
diketoester.6,7 Aromatization of the cyclized adducts
followed by functional group modifications provided
the target analogues.
The preparation of 6-substituted carbon analogues is
shown in Scheme 1. Ketones 1 and 2 were prepared by
reaction of the anion derived from either 2-fluoro-4-meth-
ylpyridine (3) or 4-methyl-2-(methylthio)pyrimidine8 (4)
with the Weinreb amide of 3-trifluoromethylbenzoic
acid (5). Alkylation of the enolates of 1 and 2 with
4-(2-chloroacetyl)-piperidine-1-carboxylic acid benzyl
ester9 led to the 1,4-diketones 6 and 7, respectively.
The cyclization to form the 3,4-dihydropyridazines was
carried out by heating the 1,4-diketones with hydrazine
in t-butanol under reflux followed by evaporation of
the solvent and heating at 180 ꢁC under high vacuum.
Aromatization by oxidation using DDQ in toluene
provided the pyridazines 8 and 9. The pyridazine 8
was converted to the analogue 10 by nucleophilic
displacement of the fluoride in neat (S)-a-methylbenzyl-
amine followed by hydrogenolysis of the protecting
group. Sulfide 9 was oxidized to the sulfone 11 by
heating under reflux with 30% hydrogen peroxide and
sodium tungstate in methanol.10 Nucleophilic displace-
ment of the sulfone with different amines led to 12, 13,
Therefore, pharmacological inhibition of p38 kinase is a
potential therapy for inflammatory conditions due to
excessive cytokine production.
Keywords: p38 Kinase inhibitors; Pyridazines.
*
Corresponding authors. Tel.: +1 805 447 8814; fax: +1 805 480
1337; e-mail: ntamayo@amgen.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.010

2410
N. Tamayo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2409–2413
CF3
CF3
CF3
O
O
N
O
O
ii
i
iii
N
N
O
X
+
N
Y
CF3
N
X
N
N
X
N
X
N
Cbz
Cbz
Y
Y
Y
CF3
3 X = C, Y = F
4 X = N, Y = SMe
5
1 X = C, Y = F
6 X = C, Y = F
8 X = C, Y = F
2 X = N, Y = SMe
7 X = N, Y = SMe
9 X = N, Y = SMe
N
N
vi
N
NH
CF3
CF3
CF3
HN
N
N
N
10
N
N
N
viii, v
vii
N
N
NH
N
N
N
N
N
N
Cbz
Cbz
NR1
NHR1
SO2Me
15 R1 =MeCOPh
12 R1 =Me
11
16 R1 =MeCOpF-Ph
13 R1 =(S)-PhCH(CH3)
14 R1= pF-PhCH(CH3)
v
CF3
CF3
N
N
N
N
ix
NR2
N
N
NH
N
N
NHR1
NHR1
19 R1 =(S)-PhCH(CH3), R2 = (R)-CH3(OH)CO 17 R1 =(S)-PhCH(CH3)
18 R1 =pF-PhCH(CH3)
Scheme 1. Synthesis of piperidine-pyridazines. Reagents and conditions: (i) LDA, THF, À78 ꢁC, 60–64%; (ii) 60% NaH, DMSO, rt, 0.5 h then 4-(2-
chloroacetyl)-piperidine-1-carboxylic acid benzyl ester, rt, 18 h, 26–28%; (iii) (a) N2H4, t-BuOH, reflux, 0.5 h, then neat heated at 180 ꢁC under high
vacuum; (b) DDQ, toluene, rt, 18 h, 32–75%; (iv) (S)-a-methylbenzylamine, 125 ꢁC, 3 h, 70%; (v) Pd/C, H2, rt, 18 h, 31–87%; (vi) 30% H2O2,
Na2WO4Æ2H2O, MeOH, reflux, 18 h, 90%; (vii) R1NH2 neat 125 ꢁC, 57–70%; (viii) ArCOCl, Et3N, DCM, rt, 88–95%; (ix) EDC, HOBt, (R)-lactic
acid, 93%.
and 14. Acylation of 12 with benzoyl chloride or p-fluoro-
benzoyl chloride followed by deprotection of the
Cbz group afforded the analogues 15 and 16. Hydro-
genolysis of 13 and 14 gave pyridazines 17 and 18.
Compound 17 was further acylated with lactic acid to
give 19.
3. Biological results and discussion
Many small molecular inhibitors of p38 MAPK present
a similar structural motif containing a vicinal pyridin-4-
yl/phenyl group attached to a central heterocyclic core.11
Crystallographic studies have shown that these types of
p38 inhibitors bind to the ATP binding pocket of the
enzyme.12 The pyridyl nitrogen forms a key hydrogen
bond interaction with the main chain NHof Met109
and the aromatic group occupies the lipophilic pocket,
which includes residues Ala51, Lys53, Leu75, Ile84,
Leu86, Leu104, and Thr106. However, many of these
inhibitors, although very potent in vitro and in vivo,
have shown significant hepatotoxicity in part due to
their inhibition of cytochrome p450ꢀs.11 Recently, sev-
eral groups have disclosed new analogues that incorpo-
rated an a-substituted aminopyridine or amino-
pyrimidine moiety in place of an unsubstituted pyridine
or pyrimidine with decreased p450 inhibition.13
Our approach was to design and prepare compounds
that incorporated some of these features using a
Synthesis of the 6-amino substituted analogues is illus-
trated in Scheme 2. Ketones 24 and 25 were synthesized
by condensing the anion derived from 2-methylpyrimi-
dine (20) or 4-picoline (21) with the Weinreb amides
22 and 23, respectively. Alkylation of the enolatates
derived from the ketones 24 and 25 with ethyl bromo-
acetate led to the ketoesters 26 and 27, respectively.
Cyclization with hydrazine in t-butanol, followed by
oxidation with bromine in acetic acid afforded hydroxy-
pyridazines 28 and 29. Treatment of 28 and 29 with
phosphorous trichloride gave the chloropyridazines 30
and 31, respectively. The target analogues 32–41 were
then prepared by treatment of the corresponding chloro-
pyridazine with primary or secondary amines.

N. Tamayo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2409–2413
2411
R1
O
R1
X
O
O
O
ii
i
X
O
+
R1
N
OEt
N
N
X
N
22 R1 = mCF3Ph
23 R1 = Napht
24 X = N, R1 = mCF3Ph
25 X = C, R1 = Napht
26 X = N, R1 = mCF3Ph
27 X = C, R1 = Napht
20 X = N
21 X = C
iii, iv
R1
N
R1
X
N
R1
X
N
N
N
N
v
vi
R2
Cl
OH
N
X
N
N
X= N, R1 = mCF3Ph, R2 = s ee Table 2
30 X = N, R1 = mCF3Ph
31 X = C, R1 = Napht
28 X = N, R1 = mCF3Ph
29 X = C, R1 = Napht
X= C, R1 = Napht, R2 = s ee Table 2
Scheme 2. Synthesis of aminopyridazines. Reagents and conditions: (i) LDA, THF, À78 ꢁC, 69–72%; (ii) 60% NaH, DMSO, 0 ꢁC, 45 min then ethyl
bromoacetate, rt, 18 h, 20%; (iii) N2H4, t-BuOH, reflux, 0.5 h, then neat 180 ꢁC under high vacuum, quant; (iv) Br2, AcOH, 95 ꢁC, 2 h, 51–63%; (v)
POCl3, Et3BzNCl, iPr2EtN, 100 ꢁC, 2 h, 58–76%; (vi) R2NH, K2CO3, NMP, 100 ꢁC, 18 h, 10–35%.
pyridazine as a central scaffold.6 In the first series of
analogues (10 and 17) we confirmed what was previously
reported for the imidazole series.14 Both compounds are
very potent in the binding and cellular assays15,16 (see
Table 1). However, their pharmacokinetic properties
are distinct. Using human liver microsomes we deter-
mined that the pyrimidine analogue 17 is more metaboli-
cally stable than 10 (125 vs 750 lL/min/mg), this could
be in part due to the reduced basicity of the pyrimidine
nitrogen compared to the pyridine, decreasing its ten-
dency to oxidation. We also tried to improve potency
by introducing an a-hydroxyamide on the piperidine
ring (19), this analogue although very potent is rapidly
cleared. Using molecular modeling based on the co-
crystal structures of closely related analogues (unpublished
data), we determined the mode of binding of this type of
trisubstituted pyridazines. As expected, the aryl substi-
tuent (mCF3-phenyl and napthyl) of the pyridazine core
fills the previously mentioned hydrophobic pocket
(Ala51, Lys53, Leu75, Ile84, Leu86, Leu104, and
Thr106) and the pyridyl/pyrimydyl nitrogen (p-position)
forms a key hydrogen bond interaction with the NHof
Met109. The phenyl group on the pyridine/pyrimidine
substituent occupies a second hydrophobic pocket
around the linker residues (Met109, Gly110, Ala111,
and Asp112) and residue Leu167. To utilize these impor-
tant hydrophobic interactions we designed a second ser-
ies of compounds having a lipophilic amine linked to the
6-position on the central core (Table 2). With the assist-
ance of co-crystal structures17 and molecular modeling
we postulated that these two types of compounds utilize
very similar protein residue interactions (see Fig. 1).
Although we were able to get a new key interaction be-
tween the amino group on the side chain and the back-
bone of Ser154 (see Fig. 2)18 the new series is in general
less potent than the original carbon analogues (cf. 17
and 32). The pyridine analogues are 10-fold more potent
than the pyrimidine (cf. 32 and 34). Introduction of
substituent at the aromatic ring on 34 did not affect
potency (35). Secondary or tertiary amines were well
tolerated at the 6-position (34 and 39) however, when
the nitrogen at the 6-position is tertiary, the compounds
are less selective against JNK3. Alkylation of the side
chain nitrogen only reduces the potency slightly (34 vs
Table 1. SAR of piperidyl/pyridazines
CF3
N
N
N
X
NR2
NR1
Compd
X
R1
R2
p38 (Ki, nM)
JNK3 (Ki, nM)
THP–TNF (IC50, nM)
10
15
16
17
18
19
C
(S)-PhCH(CH3)
MeCOPh
MeCO-pFPh
H2.1 0.3
H709 15
2970 31
>10,000
>10,000
21.5 0.6
1286 153
1245 54
5.3 0.1
N
N
N
N
N
H1772 82
H1.6 0.2
H10.2 0.5
COCOHMe
(S)-PhCH(CH3)
pF-PhCH(CH3)
(S)-PhCH(CH3)
1584 147
>10,000
93.4 10.4
556 67
6.0 0.9
15.4 1.1

2412
N. Tamayo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2409–2413
Table 2. SAR of aminopyridazines
R1
X
N
N
R2
N
Compd
X
N
R1
R2
p38 (Ki, nM)
JNK3 (Ki, nM)
THP–TNF (IC50, nM)
40.2 3.8
NH
NH
32
mCF3-Ph
56.2 2.8
>10,000
NH2
33
34
35
36
N
C
C
C
mCF3-Ph
Napht
179.3 4.2
7.0 0.5
>10,000
251.2 7.4
50.1 6.3
92.2 (n = 1)
110 17
NH2
NH
735 22
1056 (n = 1)
598 55
NH2
NH
Napht
1.8 (n = 1)
20.4 0.3
NH2
F
NH
Napht
NH
N
NH
37
38
C
C
Napht
Napht
9.2 0.2
81.5 7.9
211 31
5.5 0.3
661 93
NH
NH
65.9 5.2
OH
O
N
39
40
C
C
Napht
Napht
7.3 0.2
160 15
650 29
67.6 3.0
63.6 6.4
HN
N
29.7 1.9
HN
N
N
41
C
Napht
31.8 0.6
72.5 0.7
552.3 51.8
O
O H
Figure 2. X-ray crystal structure of 39 cocrystallized with p38 MAP
kinase.
36 and 37); however, we observed a significant dimini-
shing in cell potency (THP-1) and selectivity when the
Figure 1. Model of 17 and 32 bound in the ATP-binding site of p38
MAP kinase.

N. Tamayo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2409–2413
2413
Table 3. Inhibition of LPS induced TNF-a and IL-1b in human whole
blood
5. Raingeaud, J.; Gupta, S.; Rogers, J. S.; Dickens, M.; Han,
J.; Ulevitch, R. J.; David, R. J. J. Biol. Chem. 1995, 270,
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6. McIntyre, C. J.; Ponticello, G. S.; Liverton, N. J.;
OꢀKeefe, S. J.; OꢀNeill, E. A.; Pang, M.; Schwartz, C.
D.; Claremon, D. A. Bioorg. Med. Chem. Lett. 2002, 12,
689.
7. Tisler, M.; Stanovnik, B. In Comprehensive Heterocyclic
Chemistry; Katritzky, A. R., Rees, C. W., Eds.; Pergamon:
Oxford, 1984; Vol. 3, p 45.
8. Strekowski, L.; Wydra, R. L.; Janda, L.; Harden, D. B.
J. Org. Chem. 1991, 56, 5610.
Compd
WB TNF-a
(IC50, nM)
WB IL-1b
(IC50, nM)
10
17
32
34
35
39
40
5850 8111
1563 738
1827 1667
165 34
453 513
610 243
2420 2369
454 149
974 25
948 28
240 57
547 331
415 132
550 379
9. De Lazlo, S. E.; Mantlo, N. B.; Ponticello, G. S.; Selnick,
H. G.; Liverton, N. J. WO9705877A1; Chem. Abstr. 1997,
126, 225212.
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Grabowski, E. J. J. Org. Chem. 1993, 58, 1672.
11. For a review see: Boehm, J. C.; Adams, J. L. Expert Opin.
Ther. Pat. 2000, 10, 25.
side chain nitrogen is acylated (38 and 41). We observed
a good correlation between the binding and cell potency
(THP-1) for most of these analogues.
To select compounds for further in vivo evaluation, we
measured the activity of the most potent analogues in
inhibiting the LPS-induced release of IL-1b and TNF-a
from human whole blood (Table 3). This shift of
potency in this assay relative to the p38 kinase inhibition
assay provides information on the cell permeability and
protein binding.19
12. Lee, J. C.; Kassis, S.; Kumar, S.; Badger, A.; Adams, J. L.
Pharmacol. Ther. 1999, 82, 389.
13. 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.
14. (a) McLay, I. M.; Halley, F.; Souness, J. E.; McKenna, J.;
Benning, V.; Birrell, M.; Burton, B.; Belvisi, M.; Collis, A.;
Constan, A.; Foster, M.; Hele, D.; Jayyosi, Z.; Kelley, M.;
Maslen, C.; Miller, G.; Ouldelhkim, M.-C.; Page, K.;
Phipps, S.; Pollock, K.; Porter, B.; Ratcliffe, A. J.;
Redford, E. J.; Webber, S.; Slater, B.; Thybaud, V.;
Wilsher, N. Bioorg. Med. Chem. 2001, 9, 537; (b) Liverton,
N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon, D. A.;
Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick,
H. G.; Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga, S.
L.; Agarwal, L.; Dancheck, K.; Forsyth, D. S.; Fletcher,
D. S.; Frantz, B.; Hanlon, W. A.; Harper, C. F.; Hofsess,
S. J.; Kostura, M.; Lin, J.; Luell, S.; OꢀNeil, E. A.;
Orevillo, C. J.; Pang, M.; Parsons, J.; Rolando, A.; Sahly,
Y.; Visco, D. M.; OꢀKeefe, S. J. J. Med. Chem. 1999, 42,
2180.
Based on its potency and pharmacokinetic profile,20 the
pyridazine 39 was evaluated on two models of rheuma-
toid arthritis. In the murine LPS challenge model,21 39
has an ED50 of 1.28 mg/kg when administered orally
1 h prior to LPS challenge.
Compound 39 was also effective in the rat collagen
induced arthritis (CIA) model,22 when administered at
0.3, 1, and 3 mg/kg once a day beginning on day 11
through day 13. Pyridazine 39 inhibited paw swelling
in this model dose dependently with an ED50 of 2.7
mg/kg.
15. Park, Y. W.; Cummings, R. T.; Wu, L.; Zheng, S.;
Cameron, P. M.; Woods, A.; Zaller, D. M.; Marcy, A. I.;
Hermes, J. D. Anal. Biochem. 1999, 269, 94.
16. Whole cell potency in LPS treated THP-1 cells was
determined by measuring secreted TNF-a using an electro-
chemiluminescence immunoassay.
In summary, two series of trisubstituted pyridazines
have been identified as p38 inhibitors. Using molecular
modeling and XR crystallography we were able to deter-
mine that the two different classes occupied a similar
space in the protein binding pocket. Pyridazine 39 from
this series showed oral efficacy in the mouse LPS and rat
CIA in vivo models. Additional SAR studies directed to-
ward increasing potency in the cell-based assays would
be required before any compound from this class could
be advanced further.
17. Unpublished data.
18. Refined crystallographic coordinates for the structure of
p38a complexed with 39 have been deposited with the
Protein Data Bank (http://www.rscb.org) with entry code
1YQJ.
19. There seemed to be no clear explanation for the discrep-
ancy between inhibition of p38a and whole blood potency
in these series. These compounds have very similar
physicochemical characteristics.
References and notes
20. Pharmacokinetic properties of compound 39 in rat (bio-
availability: 30%; t1/2: 2.2 h; Cmax: 0.13 lM @ 10 mg/kg
po). Pyridinylpyridazine 39 was devoid of human p450
inhibition. [Isoenzyme: IC50 (lM); CYP2D6 > 30;
CYP3A4 > 30].
21. Vehicle or compound was administered po to BALB/c
mice (n = 5) 1 h before giving LPS (10 mg/mice, iv). Blood
was collected 90 min later and serum TNF-a levels were
determined using a Biosource ELISA kit. % Inhibition of
TNF-a production was calculated using the formula
[(vehicle control À compound)/(vehicle control)] · 100.
22. Trentham, D. E.; Townes, A. S.; Kang, A. H. J. Exp.
Med. 1977, 146, 857.
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