Pyridazine based inhibitors of p38 MAPK

Article abstract of DOI:10.1016/S0960-894X(01)00834-4

Trisubstituted pyridazines were synthesized and evaluated as in vitro inhibitors of p38 MAPK. The most active isomers were those possessing an aryl group α and a heteroaryl group β relative to the nitrogen atom in the 2-position of the central pyridazine.

Full text of DOI:10.1016/S0960-894X(01)00834-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 689–692
Pyridazine Based Inhibitors of p38 MAPK
Charles J. McIntyre,a,* Gerald S. Ponticello,a Nigel J. Liverton,a Stephen J. O’Keefe,b
Edward A. O’Neill,b Margaret Pang,b Cheryl D. Schwartzb and David A. Claremona
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Inflammation Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 1 October 2001; accepted 7 December 2001
Abstract—Trisubstituted pyridazines were synthesized and evaluated as in vitro inhibitors ofp38 MAPK. The most active isomers
were those possessing an aryl group a and a heteroaryl group b relative to the nitrogen atom in the 2-position ofthe central pyr-
idazine. Additionally, substitution in the 6-position ofthe central pyridazine with a variety ofdialkylamino substituents afforded a
set ofinhibitors having good (p38 IC 50 1–20 nM) in vitro activity. # 2002 Elsevier Science Ltd. All rights reserved.
The discovery ofnew agents of r the treatment ofcyto-
kine mediated inflammatory diseases such as rheuma-
toid arthritis (RA) or Crohn’s disease has been the
driving force behind our search for novel, selective,
small molecule inhibitors ofp38 mitogen-activated
protein kinase (p38 MAPK).1 By inhibiting p38 MAPK,
the intracellular signaling cascade leading to excessive
production ofthe pro-inflammatory cytokine, tumor
necrosis factor a (TNF-a), is suppressed.2 Reduction of
TNF-a levels by treatment with either anti-TNF-a anti-
bodies (Remicade1) or a soluble TNF-a receptor pro-
tein (Enbrel1) has proved to be disease modifying in RA
therapy.3
the a-position and the attached nitrogen containing
aromatic heterocycle located b to the unsubstituted
nitrogen in the imidazole ring.1,5 Furthermore, X-ray
crystallography has demonstrated the necessity ofa
hydrogen bond acceptor on the central ring in order to
achieve high affinity for the ATP-binding pocket of
p38 MAPK.6
Unlike the five-membered p-electron excessive hetero-
cycles, there are limited reports ofp38 MAPK inhibitors
that possess a p-electron deficient central ring.7 In an
effort to better delineate the nature ofsmall molecule
inhibitors ofp38 MAPK, pyridazine based inhibitors
analogous to the tetrasubstituted imidazole class were
prepared (Schemes 1–3). In each case an amine con-
taining substituent was installed distal to the aryl and
heteroaryl functionalities. Like the tetrasubstitued imi-
dazoles, the nitrogen atoms ofpyridazine based inhibi-
tors possess the ability to function as a hydrogen bond
acceptor in the ATP-binding pocket ofp38 MAPK.
Many small molecule inhibitors ofp38 MAPK have
been reported that contain a central trisubstituted p-
electron excessive aromatic heterocycle such as pyra-
zole, pyrrole, furan, or imidazole.4 Additionally, potent
inhibitors ofp38 MAPK were developed that were
based on the tetrasubstituted imidazole heterocycle, 1
(Fig. 1).1
A common feature of tetrasubstituted imidazole inhibi-
tors (1, p38 IC50 0.11 nM; 2, p38 IC50 12,000 nM) is the
presence ofboth pendant aryl and heteroaryl uf nction-
alities which are positioned distal to a 4-substituted
piperidine. An important element ofimidazole contain-
ing inhibitors is that the isomer (1) having the greatest
intrinsic activity is that which has an aromatic ring in
*Corresponding author. Fax: +1-215-652-3971; e-mail: charles_
mcintyre@merck.com
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00834-4

690
C. J. McIntyre et al. / Bioorg. Med. Chem. Lett. 12 (2002) 689–692
The preparation ofthe required 3,4,6-trisubstituted
pyridazines was accomplished using a classical synthetic
approach, which relied on the reaction ofhydrazine with
either a 1,4-diketone or a 1,4-keto acid.8 Subsequent
aromatization ofthe heterocycle ofllowed by pendant
group modifications gave the compounds desired for
testing. Schemes 1–3 summarize three strategies that
were used for the preparation of 3,4,6-trisubstituted
pyridazine analogues.9
solution of 6, 30% hydrogen peroxide, sodium tung-
state, methanol and ethyl acetate.11 Nucleophilic dis-
placement1 with neat (S)-(À)-a-methylbenzylamine gave
8, which was deprotected using 30% hydrogen bromide
in acetic acid to furnish, after conversion to the dihy-
drochloride salt, the desired pyridazine, 9.
Scheme 2 shows the preparation ofpyridazine 16,7c
which is, due to the transposition ofthe aryl and het-
eroaryl substituents, isomeric with pyridazine 9. Inter-
mediate 12 was prepared by reacting the Grignard
reagent12 derived from 3-trifluoromethylbenzyl chloride
(10) with Weinreb amide 11. The remaining steps in the
synthesis of 16 were carried out in a manner similar to
that described for the preparation of 9 (Scheme 1).
The synthesis of 97c is illustrated in Scheme 1. Alkyl-
ation ofthe enolate of 31 with the Cbz protected 4-(2-
chloroacetyl)piperidine (47c) gave the 1,4-diketone, 5.
Cyclization with hydrazine in ethanol gave the 3,4-
dihydropyridazine, which was aromatized with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)10 in tol-
uene to give 6. Selective oxidation ofthe sulfide ( 6) to
the sulfone (7) was accomplished by heating at reflux a
Scheme 3 illustrates a complementary synthetic pathway
which provided analogues with an amine directly
Scheme 1. Reagents and conditions: (i) 95% NaH, DMSO, rt, 1 h then 4, rt, 18 h, 71%; (ii) N2H4, EtOH, 24 h, rt; (iii) DDQ, toluene, rt 24 h, 82%;
(iv) 30% H2O2, Na2WO4 2H2O, MeOH, EtOAc, reflux, 24 h, 72%; (v) (S)-(À)-a-methylbenzylamine, 125 ꢀC, 3 h, 86%; (vi) 30% HBr–HOAc, rt, 1
.
h, 67%; (vii) HCl–Et2O (1 N), 0.5 h rt, 41%.
Scheme 2. Reagents and conditions: (i) Mg, Et2O, I2 (cat), reflux, 0.5 h, then 11, reflux, 4 h, 15%; (ii) 95% NaH, DMSO, rt, 0.25 h then 4, rt, 18 h,
.
21%; (iii) N2H4, EtOH, 5 h, rt; (iv) DDQ, toluene, rt 0.5 h, 81%; (v) 30% H2O2, Na2WO4 2H2O, MeOH, EtOAc, reflux, 18 h, 49%; (vi) (S)-(À)-a-
methylbenzylamine, 125 ꢀC, 3 h, 94%; (vii) 30% HBr–HOAc, rt, 1 h, 30%; (viii) HCl–Et2O (1 N), 0.5 h rt, 22%.

C. J. McIntyre et al. / Bioorg. Med. Chem. Lett. 12 (2002) 689–692
691
Scheme 3. Reagents and conditions: (i) 60% NaH, DMSO, then methyl bromoacetate, rt, 2 h, 86%; (ii) dioxane, 6 N hydrochloric acid, rt, 24 h,
.
78%; (iii) N2H4, EtOH, reflux, 4 h, 41%; (iv) 30% H2O2, Na2WO4 2H2O, MeOH, EtOAc, reflux, 24 h, 92%; (v) (S)-(À)-a-methylbenzylamine,
reflux, 24 h, 91%; (vi) DDQ, MeCN, rt, 24 h, 92%; (vii) POCl3, 125ꢀC, 24 h, 83%; (viii) amine, 90 ꢀC, 24 h.
attached to the pyridazine heterocycle in the 6-position.
Addition ofmethyl bromoacetate to the previously
employed enolate ofketone 3 gave the alkylated pro-
duct, 17. Hydrolysis ofthe ester and cyclization with
hydrazine in ethanol provided pyridazinone 19. Inter-
mediate 22 was subsequently synthesized from 19 using
an approach similar to those described in Schemes 1 and
2. Further elaboration to the key chloropyridazine, 23,
was effected using phosphorus oxychloride.13 Treatment
of 23 with primary or secondary amines14 gave a series
ofpyridazines possessing a nitrogen-linked substituent
in the 6-position.
tetrasubsituted imidazole series ofinhibitors. These
substituents were found to impart both activity and
selectivity for p38 MAPK over other protein kinases
such as c-Raf.1 While retaining these two key elements
ofthe tetrasubstituted imidazoles and using the pyrid-
azine scaffold, we sought a rapid and flexible approach
that would allow for the preparation of final products
having a variety ofnitrogen-linked substituents in the 6-
position (see Scheme 3). Table 2 shows a set ofsecond-
ary amines that were thus installed on the pyridazine
framework. This strategy allowed for the quick assess-
ment ofthe requisite structural and electronic attributes
ofthe pendant group in the 6-position ofthe central
pyridazine.
The trend observed for the tetrasubstituted imidazole
series1 ofp38 MAPK inhibitors, regarding the arrange-
ment ofsubstituents around the central heterocycle, was
found to be true for the analogous 3,4,6-trisubstituted
pyridazines 9 and 16. In each case a piperidine sub-
stituent was positioned distal to the pendant aryl and
heteroaryl groups. Similarly, the pyridazine with the
aryl ring a and the heteroaryl ring b to the pyridazine
nitrogen in the 2-position showed greater in vitro inhi-
bitory activity for p38 MAPK (9, p38 IC50 0.15 nM)
than the isomeric compound where the aryl and hetero-
aryl rings were interchanged (16, p38 IC50 4.81%@1000
nM).15 Table 1 summarizes p38 MAPK inhibition data
for the two isomeric tetrasubstituted imidazoles and
their trisubstituted pyridazine counterparts.
The introduction ofnitrogen-linked amine substituents
in the 6-position ofthe central pyridazine afforded a set
ofcompounds which, like the carbon-linked piperidine,
9, were potent in vitro inhibitors ofp38 MAPK. The
maintenance ofinhibitory activity in going rfom a car-
bon-linked to a nitrogen-linked functionality suggests a
Table 2. Amine derivatives of 24
Compd
Amine
p38 IC50 (nM)
2.5
24a
24b
24c
24d
24e
24f
24g
1.4
20.1
7.4
The 3-trifluoromethylphenyl and the 2-substituted pyr-
imidine substituents were previously optimized in the
Table 1. Summary ofp38 MAPK inhibition data
2.6
Compd
p38 IC50 (nM)
0.6
1
2
9
16
0.11
12,000
0.15
1.9
4.81%@1000

692
C. J. McIntyre et al. / Bioorg. Med. Chem. Lett. 12 (2002) 689–692
Table 3. Kinase selectivity for compound 24e
3. (a) Physicians Desk Reference, 54th ed.; Medical Econom-
ics: Montvale, 2000; pp 927 and 1413. (b) Seymour, H. E.;
Worsley, A.; Smith, J. M.; Thomas, S. H. L. Br. J. Clin.
Pharmacol. 2001, 51, 201. (c) Keystone, E. C. Rheu. Dis. Clin.
of North Am. 2001, 27, 427.
Kinase p38
c-RafJNK2
a1 JNK2a2
LCK
IC50
2.6 nM 29%@5000 nM 820 nM 1400 nM 22%@15,000 nM
4. For review see: Boehm, J. C.; Adams, J. L. Exp. Opin.
Ther. Pat. 2000, 10, 25.
remarkable tolerance in the 6-position for a range of
substituents, each possessing distinctive structural and
electronic features. One of the first compounds prepared
in this series, the morpholine derivative 24e, was tested
to evaluate its selectivity for p38 MAPK over several
other protein kinases. These results are illustrated in
Table 3.16
5. Owens, R. J.; Lumb, S. In Novel Cytokine Inhibitors; Higgs,
G. A., Henderson, B., Eds.; Birkhauser: Basel, 2000; p 201.
6. (a) Lisnock, J. M.; Tebben, A.; Frantz, B.; O’Neill, E. A.;
Croft, G.; O’Keefe, S. J.; Li, B.; Hacker, C.; de Laszlo, S.;
Smith, A.; Libby, B.; Liverton, N.; Hermes, J.; LoGrasso, P.
Biochemistry 1998, 37, 16573. (b) Lee, J. C.; Kassis, S.;
Kumar, S.; Badger, A.; Adams, J. L. Pharmacol. Ther. 1999,
82, 389.
7. (a) Mantlo, N. B.; Hwang, C. K.; Spohr, U. D. WO9932448;
Chem. Abstr. 1999, 131, 58754. (b) Adams, J. L.; Boehm, J. C.;
Hall, R. WO0025791; Chem. Abstr. 2000, 132, 334471. (c)
Claremon, D. A.; Ponticello, G. S. WO0031065; Chem. Abstr.
2000, 133, 4673. (d) Adams, J. L.; Boehm, J. C. WO0040243;
Chem. Abstr. 2000, 133, 89539.
8. Tisler, M.; Stanovnik, B. In Comprehensive Heterocyclic
Chemistry; Katritzky, A. R., Rees, C. W., Eds.; Pergamon:
Oxford, 1984; Vol. 3, p 45.
9. All final products gave satisfactory 1H NMR, MS, and
In addition to the kinase selectivity experiments, 24e
was assessed in two cell-based assays ofTNF- a release.
A substantial decrease in activity (human monocyte17
IC50 292 nM, human whole blood1 IC50 1200 nM) ver-
sus the value for p38 MAPK inhibition (IC50 2.6 nM)
was observed which suggests this pyridazine derivative
was not effectively penetrating cells.
In summary, although 24e showed favorable in vitro
potency and selectivity versus p38 MAPK, additional
SAR studies directed towards increasing activity in the
cell-based assays would be required before a trisubstituted
pyridazine could advance as a small molecule drug can-
didate for intervention in TNF-a mediated disorders.
CHN analysis. Representative data follows for 9 and 24e. For
1
.
.
9 2HCl 0.95H2O: H NMR (300 MHz, CD3OD) d 7.86 (d, br,
1H), 7.86–7.70 (m, 3H), 7.64–7.52 (m, 3H), 7.36–7.22 (m, 5H),
4.80 (s, br, 1H), 3.66–3.56 (m, 2H), 3.48–3.38 (m, 1H), 3.32–
3.18 (m, 2H), 2.38–2.25 (m, 4H), 1.46 (d, br, 3H); MS m/z
+
.
505.25 (MH ). Anal. calcd for C28H27F3N6 2HCl 0.95H2O:
.
C, 56.56; H, 5.24; N, 14.13. Found: C, 56.61; H, 5.27; N,
1
.
.
14.07. For 24e HCl 1.55H2O: H NMR (500 MHz, CD3OD) d
8.40 (s, br, 1H), 8.06 (s, br, 1H), 7.80 (d, J=7 Hz, 1H), 7.76 (s,
1H), 7.64–7.57 (m, 2H), 7.30–7.24 (m, 2H), 7.23–7.16 (m, 3H),
6.95 (s, br, 1H), 4.50 (s, br, 1H), 3.90 (m, 4H), 3.83 (m, 4H),
1.35 (s, br, 3H); MS m/z 507.27 (MH+). Anal. calcd for
Acknowledgements
The authors wish to thank Dr. John A. McCauley
(Merck Research Laboratories) for helpful editorial
review ofthis manuscript.
.
.
C27H25F3N6O HCl 1.55H2O: C, 56.80; H, 5.14; N, 14.72.
Found: C, 56.82; H, 4.76; N, 14.54.
10. Buckle, D. R. In Encyclopedia of Reagents for Organic
Synthesis; Paquette, L. A., Ed.; John Wiley and Sons: Chi-
chester, 1995; Vol. 3, p 1699.
References and Notes
11. Blacklock, T. J.; Sohar, P.; Butcher, J. W.; Lamanec, T.;
Grabowski, E. J. J. J. Org. Chem. 1993, 58, 1672.
12. Schon, U.; Kehrbach, W.; Hachmeister, B.; Buschmann,
G.; Kuhl, U. G. U.S. 4,755,520; Chem. Abstr. 1987, 106,
156761.
13. Aldous, D. L.; Castle, R. N. In Pyridazines; Castle, R. N.,
Ed.; John Wiley and Sons: New York, 1973; p 221.
14. Nagagome, T. In Pyridazines; Castle, R. N., Ed.; John
Wiley and Sons: New York, 1973; p 468.
15. The inhibition ofp38 kinase was determined according to
a published method18 using an enzyme concentration of0.5 nM.
16. All enzyme inhibition assays were carried out using meth-
ods described in ref1 and the references cited therein.
17. Chin, J.; Kostura, M. J. J. Immunol. 1993, 151, 5574.
18. LoGrasso, P. V.; Frantz, B.; Rolando, A. M.; O’Keefe,
S. J.; Hermes, J. D.; O’Neill, E. A. Biochemistry 1997, 36,
10422.
1. Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon,
D. A.; Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Sel-
nick, H. G.; Smith, G. R.; Tebben, A.; Vacca, J. P.; Varga,
S. L.; Agarwal, L.; Dancheck, K.; Forsyth, A. J.; Fletcher,
D. S.; Frantz, B.; Hanlon, W. A.; Harper, C. F.; Hofsess, S. J.;
Kostura, M.; Lin, J.; Luell, S.; O’Neill, E. A.; Orevillo, C. J.;
Pang, M.; Parsons, J.; Rolando, A.; Sahly, Y.; Visco, D. M.;
O’Keefe, S. J. J. Med. Chem. 1999, 42, 2180.
2. (a) Lee, J. C.; Laydon, J. T.; Mcdonnell, P. C.; Gallagher,
T. F.; Kimar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.;
Heys, J. R.; Landvatter, S. W.; Strickler, J. E.; McLaughlin,
M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.;
Adams, J. L.; Young, P. R. Nature 1994, 372, 739. (b) Lee, J.
C.; Young, P. R. J. Leuk. Bio. 1996, 59, 152. (c) Wang, H.;
Tracey, K. J. In Inflammation: Basic Principles and Clinical
Correlates, 3rd ed.; Gallin, J. I., Snyderman, R., Eds.; Lippin-
cott, Williams & Wilkins: Philadelphia, 1999; p 471.