Pyrrolopyridazine MEK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.10.052

The synthesis and SAR of a series of pyrrolopyridazine MEK inhibitors are reported. Optimal activity was achieved by incorporation of a 4-phenoxyaniline substituent at C4 and an acylated amine at C6.

Full text of DOI:10.1016/j.bmcl.2005.10.052

Bioorganic & Medicinal Chemistry Letters 16 (2006) 628–632
Pyrrolopyridazine MEK inhibitors
Zhong Chen,* Soong-Hoon Kim, Stephanie A. Barbosa, Tram Huynh, David R. Tortolani,
Kenneth J. Leavitt, Donna D. Wei, Veeraswamy Manne, Carolyn S. Ricca,
Johnni Gullo-Brown, Michael A. Poss, Wayne Vaccaro and Mark E. Salvati
Bristol-Myers Squibb, Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 9 September 2005; revised 11 October 2005; accepted 13 October 2005
Available online 3 November 2005
Abstract—The synthesis and SAR of a series of pyrrolopyridazine MEK inhibitors are reported. Optimal activity was achieved by
incorporation of a 4-phenoxyaniline substituent at C4 and an acylated amine at C6.
Ó 2005 Elsevier Ltd. All rights reserved.
Cell growth and differentiation in response to extracellu-
lar stimuli and certain intracellular oncogenes are med-
iated through various intracellular signal transduction
pathways. A key player in this kinase-signaling cascade
from growth factors to the cell nucleus is the mitogen-
activated protein kinase (MAPK) pathway.1,2 This path-
way involves a series of kinases at multiple levels.
Members of the Raf family of kinases are downstream
of Ras. When Raf is activated, it phosphorylates
MEK (MAPK/ERK kinase, or MAP kinase) on two
serine residues. The activated MEK in turn phosphory-
lates the threonine and tyrosine residues of MAPK (or
ERK, extracellular signal-regulated kinase). Activated
MAPK then translocates and accumulates in the nucle-
us, where it effects cell growth and proliferation through
phosphorylation of various substrates.3
Multiple isoforms exist within the MEK family.2,3
Among them, MEK-1 has been extensively studied
and various types of MEK-1 inhibitors have been
reported.6–13 Here we report the synthesis and SAR of
pyrrolopyridazine MEK inhibitors.
The synthesis14 of the pyrrolopyridazine core, as shown
in Scheme 1, started with the formation of the diethyl
O
EtO
OEt
N+
a
b
-C
OEt
O
O
N
H
O
1
O
EtO
Overexpression and overactivation of MEK or ERK
have been found to be associated with various human
cancers.4 It has been demonstrated that inhibition of
MEK prevents activation of ERK and subsequent acti-
vation of ERK substrates in cells, resulting in inhibition
of cell growth stimulation and reversal of the phenotype
of Ras-transformed cells.5 Therefore, inhibition of the
MAPK pathway through inhibition of Raf, MEK, or
ERK presents a unique opportunity to block uncon-
trolled cell growth and, thus, has potential therapeutic
utility in cancer treatment.
CN
c
O
d
OEt
N
N
EtO
N
H
O
NH2
3
2
Cl
N
R
5
4
3
CN
CN
O
e
O
N
6
N
EtO
EtO
N
4
Scheme 1. Reagents and conditions: (a) DBU, acetaldehyde, THF,
55 °C, 17 h, 42%; (b) NaH, DMF, then O-(2,4-dinitrophenyl)hydrox-
ylamine, 10–25 °C, 12 h, 75%; (c) 1,1-diethoxypropionitrile, TsOH,
toluene, reflux, 12 h, then DBU, 80 °C, 1 h, 40%; (d) POCl3, 75 °C, 2 h,
77%; (e) RH, Et3N or K2CO3, THF or DMF, rt to reflux, 1–72 h, 10–
100% depending on R.
Keywords: MEK inhibitor; MEK1; Pyrrolopyridazine.
*
Corresponding author. Tel.: +1 609 252 6336; e-mail:
zhong.chen@bms.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.052

Z. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 628–632
629
pyrrole-2,4-dicarboxylate 1 by condensation of ethyl
isocyanoacetate with acetaldehyde in the presence of
DBU. N-Amination of 1 gave 2, which was condensed
with diethoxypropionitrile and the resulting intermedi-
ate was cyclized under basic condition to give the 3-cya-
nopyrrolopyridazine core 3. Compound 3 was treated
with POCl3 to give the 4-chloro compound 4, which
readily reacted with various nucleophiles to give com-
pounds 5a–l.
the corresponding amides 13a–c. Certain amides were
cyclized to form heterocycles, as illustrated by benzimid-
azole 25. Curtius rearrangement of the carboxylic acid
12 with DPPA, proceeding through the acyl azide, and
then the isocyanate intermediates, afforded either the
carbamate (15a–d) by trapping with an alcohol, or the
urea (14a–e) by trapping with TMS–azide to form the
carbamoyl azide15–17 first, which then reacted readily
with a primary amine to give the urea product. Hydrog-
enolysis of the benzyl carbamate 15e gave the C6 amino
compound 16, which upon acylation yielded the C6 N-
linked amides such as 17a–c. Sulfonylation of 16 gave
the sulfonamide 18, reductive amination gave the benzyl
amine 19, and copper-mediated boronic acid coupling
gave the aniline 20.18 The acid 12 can be decarboxylated
to give 21. The ester 5b can react with methyl Grignard
reagent to give 22, which yielded the C6 hydroxyl
compound 23 upon benzylic hydroperoxide rearrange-
ment.19 Alkylation of 23 with alkyl halides or Mitsunobu
reaction with alcohols afforded the ether compounds such
as 24a and b.
Synthetic modification at C3 is shown in Scheme 2.
Hydrogenation of the C3 cyano group of 12 provided
the corresponding benzyl amine 6, which may be acety-
lated or sulfonylated to give compounds such as 7 or 8.
Modification at C5 is shown in Scheme 3. a-Bromina-
tion of the C5 methyl group of 4 afforded 9, which
underwent substitution reactions to give the alcohol or
ether compounds 10a–d. Subsequent nucleophilic aro-
matic substitution with 4-phenoxyaniline yielded com-
pounds 11a–d.
The C6 position was subjected to the most intense mod-
ifications (Scheme 4). Hydrolysis of the ethyl ester 5b led
to the carboxylic acid 12, which was a good starting
point for further modifications. Amide coupling with
various primary or secondary amines, or anilines, gave
The compounds were tested in an in vitro 96-well plate
assay measuring their inhibitory activity against MEK-
1.20,21 The IC50 values of select compounds are shown
in Tables 1–5.
The SAR of the C3 and C4 positions of the pyrrolopy-
ridazine is generally very tight. Selected modifications to
the C3 cyano group, shown in Scheme 2, led to complete
loss of MEK activity (Table 1). The pyrrolotriazine
compound 2622–24 also showed significantly weaker
MEK activity than the corresponding C–cyano com-
pound 5b, suggesting that the C3 cyano group plays a
critical role for MEK activity.
O
Ar
HN
CN
O
Ar =
N
HO
N
12
Ar
HN
O
a
O
N
H
b
Ar
N
The 4-phenoxyaniline is a key group at the C4 position
for MEK activity. Modifications to this group, such as
one atom extension/contraction or O/N exchange, gen-
erally lead to a dramatic loss of activity (Table 2). Some
exceptions include: (1) a –CH2– in place of the oxygen
between the two phenyl rings (5f); and (2) certain sub-
stituents on the ortho (5k) or meta position of the termi-
nal phenyl ring, which led to a slight improvement of
MEK activity.
HN
HO
N
7
O
NH2
Ar
HN
O
O
N
c
HO
N
S
O
6
N
H
N
HO
N
8
Scheme 2. Reagents and conditions: (a) H2 (1 atm), Pd/C, MeOH,
THF, TFA, rt, 17 h, 100%; (b) Ac2O, Et3N, THF, rt, 10 min, then
NaOH, rt, 2 h, 81%; (c) MeSO2Cl, Et3N, THF, rt, 10 min, 27%.
Although modifications at the C5 methyl are generally
tolerated, no improvement of MEK activity was ob-
served (Table 3).
Br
Cl
N
Cl
N
CN
CN
CN
b
a
O
O
Extensive modifications at C6 yielded diverse SAR (Ta-
ble 4). A wide variety of substituents are tolerated,
although a carbonyl one atom away from the C6 carbon
of the pyrrolopyridazine core is preferred, as is illustrat-
ed by the activity of the ureas (14a,b), carbamates
(15a,b), and N-linked amides (17a–c) being better than
the C-linked amides (13a,b) and ethers (24a,b). Removal
of this amide carbonyl (19) or conversion to a sulfonyl
(18) resulted in a dramatic loss of activity. Both hydro-
phobic groups (17b,c) and hydrophilic ones (14b and
15b) at the other side of the carbonyl can lead to potent
activity. Small groups, such as H (21), OH (23), and
NH2 (16), but not certain bulky ones such as –CMe2OH
N
N
EtO
EtO
4
9
O
OR
OR
HN
Cl
N
CN
c
O
O
N
N
EtO
N
EtO
10a - 10d
11a - 11d
Scheme 3. Reagents and conditions: (a) NBS, benzoyl peroxide, CCl4,
77 °C, 3 h, 99%; (b) ROH, NaHCO3, 25–70 °C, 3 h to 3 days, 80–
100%; (c) 4-phenoxyaniline, Et3N, THF, 70 °C, 1 h, 92%.

630
Z. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 628–632
Ar
Ar
O
HN
HN
R0
CN
CN
O
N
c
N
b
d
Ar =
13c
N
N
R1 = H
R2 =
R1
N
H
N
N
R0 = H, F, OMe.
R2
13a - 13c
Ar
25
HN
Ar
HN
CN
CN
CN
H2N
g
CN
HN
N
N
R
HN
O
N
N
17a - 17c
Ar
O
HN
R
HN
14a - 14e
CN
O
Ar
Ar
HN
HN
15e
R = Bn
N
HO
N
CN
12
Ar
h
e
f
HN
S
O
HN
HN
Ph
N
N
N
15a - 15e
N
CN
a
O
18
O
+H3N
Cl-
O
R
N
Ar
N
HN
Ar
Ar
16
HN
CN
HN
O
CN
i
k
N
HN
EtO
N
N
N
5b
N
Ph
N
19
l
21
Ar
HN
Ar
Ar
Ar
HN
HN
HN
CN
j
CN
CN
CN
m
n
HN
Ph
N
HO
RO
N
N
N
N
N
N
N
HO
20
23
24a, 24b
22
Scheme 4. Reagents and conditions: (a) NaOH, H2O, EtOH, THF, 80 °C, 6 h, 95%; (b) NR1R2, EDC, HOAt, THF, 60 °C, 15 h, 30–99%; (c) CSA,
toluene, 110 °C, 5 h, 33%; (d) (PhO)2PON3, Et3N, 1,4-dioxane, 25 °C, 15 h, then TMSN3, 80 °C, 2 h, then RNH2, 25 °C, 1 h, 50–80%; (e)
(PhO)2PON3, Et3N, 1,4-dioxane, 25 °C, 15 h, then ROH, 75 °C, 4 h, 30–80%; (f) H2 (1 atm), Pd/C, MeOH, rt, 30 min, then HCl (4 M in dioxane, to
form HCl salt), 100%; (g) RCOOH, EDC, HOAt, THF, 60 °C, 15 h, 35–95%; (h) PhSO2Cl, DIEA, 1,2-dichloroethane, rt, 16 h, 27%; (i) PhCHO,
NaBH(OAc)3, 1,2-dichloroethane/AcOH, rt, 16 h, 11%; (j) PhB(OH)2, Cu(OAc)2, Et3N, CH2Cl2, rt, 16 h, 15%; (k) Cu2O, diethyleneglycol, 155 °C,
21 h, 6%; (l) MeMgBr, THF, 0–50 °C, 1 h, 64%; (m) BF3ÆOEt2, H2O2, CH2Cl2, ꢀ5 °C, 1 h, 65%; (n) ROH, Ph3P, DEAD, THF, 0–25 °C, 2 h, 60–90%.
Table 2. MEK inhibitory activity of pyrrolopyridazines with C4
variations
Table 1. MEK inhibitory activity of pyrrolopyridazines with C3
variations
R
OPh
OPh
CN
O
EtO
HN
HN
N
N
R
O
O
N
N
N
R'O
N
EtO
N
Compound
R
MEK IC50 (lM)
26
5a
5b
5c
5d
5e
5f
–NHPh
>25
–NHPh-4-OPh
–NHPh-3-OPh
–NHPh-4-Ph
0.094
2.65
>25
Compound
R
R0
MEK IC50 (lM)
5b
12
6
–CN
–CN
–Et
–H
–H
–H
–H
—
0.094
0.147
>25
–NHPh-4-NHPh
–NHPh-4-CH2Ph
–NHPh-4-COPh
–OPh-4-OPh
2.32
0.068
1.11
5.42
>25
–CH2NH2
–CH2NHAc
5g
5h
5i
7
>25
>25
8
26
–CH2NHSO2Me
—
–NHCH2Ph-4-OPh
–NHPh-4-OCH2Ph
–NHPh-4-OPh-2-OMe
–NHPh-4-OPh-4-Cl
3.47
5j
>25
5k
5l
0.023
0.642
(22), at C6 are tolerated. Certain heterocycles such as
benzimidazole (25) at the C6 position also demonstrated
potent inhibition of MEK.
The 3-cyanopyrrolopyridazines are highly selective
against other kinases such as VEGFR-2.24 For example,
two of the most active MEK inhibitors 14b and 15b both
had less than 5% inhibition against VEGFR-2 at con-
centrations up to 50 lM.
Some of our most active compounds were obtained by
combining the best substituents at C4 and C6. Several
such examples are shown in Table 5.

Z. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 628–632
631
Table 3. MEK Inhibitory activity of pyrrolopyridazines with C5
variations
Table 5. MEK Inhibitory activity of pyrrolopyridazines with optimal
C4 and C6 substituents
O
O
R
R
O
HN
HN
CN
CN
O
EtO
N
HN
N
N
N
N
X
O
Compound
R
MEK IC50 (lM)
Compound
X
R
MEK IC50 (lM)
5b
11a
11b
11c
11d
–H
0.094
0.203
0.191
0.331
0.566
14c
14d
14e
15c
15d
NH
NH
NH
O
–2-OMe
–2-F
0.024
0.033
0.029
0.048
0.076
–OH
–OMe
–OEt
–3-OMe
–2-OMe
–3-OMe
–OCH2CH2OMe
O
Table 4. MEK inhibitory activity of pyrrolopyridazines with C6
variations
Table 6. Cell growth inhibitory activity of select pyrrolopyridazines
O
Compound MEK enzyme MEK2 cell HT-29
IC50 (lM)
Colo205
(lM)
(lM)
(lM)
HN
CN
5b
14a
14b
15a
15b
15c
15d
17b
17c
21
0.094
0.018
0.036
0.023
0.058
0.048
0.076
0.042
0.014
0.241
>20
—
—
—
9.78
7.38
R
N
2.45
7.65
0.50
3.67
0.86
7.64
3.76
3.78
2.35
2.13
1.34
3.90
—
N
2.88
2.06
0.72
2.42
1.86
>10
Compound
R
MEK IC50 (lM)
—
—
12.1
21
23
–H
–OH
0.241
0.181
0.805
0.190
>25
2.50
>10
24a
16
–OMe
–NH2
22
–CMe2OH
–COOEt
5b
0.094
0.147
0.397
0.386
0.018
0.036
0.023
0.058
0.087
0.042
0.014
14.6
12
–COOH
–CONHMe
In conclusion, a series of pyrrolopyridazines have been
prepared as MEK inhibitors. The SAR at the 3–6 posi-
tions have been explored. The most active compounds
have a cyano group at C3 position, 4-phenoxyaniline
with optional ortho or meta substitution at the terminal
phenyl at C4, methyl or alkoxymethyl at C5, and carba-
mate, urea or N-linked amide at C6. These compounds
have potent MEK inhibitory activity and inhibit the
growth of several human tumor cell lines.
13a
13b
14a
14b
15a
15b
17a
17b
17c
18
–CONH(CH2)2-N-morpholine
–NHCONHMe
–NHCONH(CH2)2-N-morpholine
–NHCOOMe
–NHCOO(CH2)2-N-morpholine
–NHCOMe
–NHCOPh
–NHCOPh-3-NHAc
–NHSO2Ph
–NHCH2Ph
19
20
6.13
–NHPh
–O(CH2)2-N-morpholine
–2-Benzimidazolyl
0.361
0.521
0.168
References and notes
24b
25
1. Seeger, R.; Krebs, E. G. FASEB J. 1995, 9, 726.
2. Robinson, M. J.; Cobb, M. H. Curr. Opin. Cell Biol. 1997,
9, 180.
3. Kolch, W. Biochem. J. 2000, 351, 289.
4. Sivaraman, V. S.; Wang, H.; Nuovo, G. J.; Malbon, C. C.
J. Clin. Invest. 1997, 99, 1478.
5. Dudley, D. T.; Pang, L.; Decker, S. J.; Bridges, A. J.;
Saltiel, A. R. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7686.
6. Sebolt-Leopold, J. S.; Dudley, D. T.; Herrera, R.; Van, K.;
Wiland, A.; Gowan, R. C.; Tecle, H.; Barrett, S. D.;
Bridges, A.; Przybranowski, S.; Leopold, W. R.; Saltiel, A.
R. Nat. Med. 1999, 5, 810.
7. Duncia, J. V.; Santella, J. B.; Higley, C. A.; Pitts, W. J.;
Wityak, J.; Frietze, W. E.; Rankin, F. W.; Sun, J.-H.; Earl,
R. A.; Tabaka, A. C.; Teleha, C. A.; Blom, K. F.; Favata,
M. F.; Manos, E. J.; Daulerio, A. J.; Stradley, D. A.;
Horiuchi, K.; Copeland, R. A.; Scherle, P. A.; Trzaskos, J.
Compounds active in the MEK enzyme assay were
tested in cell assays. The MEK2 cells are Rat1 cells
transformed by human MEK-1 cDNA with two acti-
vating mutations (S218E and S222E).25 Compounds
that inhibit the growth of such cells indicate a strong
probability of MEK inhibition. The compounds were
also tested against two human tumor cell lines: HT-29
and Colo205. The cellular inhibitory activities of the
pyrrolopyridazines are generally 10- to 100- fold less
potent than their enzyme activities. It is shown as a
general trend that more active MEK inhibitors are
more potent against tumor cell growth (Table 6).

632
Z. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 628–632
M.; Magolda, R. L.; Trainor, G. L.; Wexler, R. R.;
19. Boger, D. L.; Coleman, R. S. J. Org. Chem. 1986, 51, 5436.
20. Mek kinase assay: An in vitro 96-well plate assay
described in Ref. 21 was adapted with a few modifications.
Each well contained 30 ll assay buffer (Tris–HCl, pH 7.5,
MgCl2, DTT, BSA, ATP, and [g-33P]ATP), 10 ll inhibitor
dilutions or empty DMSO solvent, and 10 ll enzyme
substrate mixture (50–100 ng Mek-EE and 1000–2000 ng
gst-MAPK). The final concentrations in the assay were
20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 0.3 mM DTT,
50 lg BSA, and 10 lM ATP. The plates were incubated at
room temperature for 60 min and reactions were termi-
nated by the addition of 10 ll stop mixture containing
300 mM EDTA and 25 lg BSA. The samples were
subjected to precipitation with TCA containing ATP
(final concentrations: TCA, 3.2% and ATP, 2.3 mM). The
samples were transferred to Packard GF/C 96-well Uni-
filter plates using a Packard Filter Mate 196 Harvester.
Following drying under light, the plates were counted in a
Packard Top count microplate counter.
Hobbs, F. W.; Olson, R. E. Bioorg. Med. Chem. Lett.
1998, 8, 2839.
8. Wityak, J.; Hobbs, F. W.; Gardner, D. S.; Santella, J. B.,
III; Petraitis, J. J.; Sun, J.-H.; Favata, M. F.; Daulerio, A.
J.; Horiuchi, K. Y.; Copeland, R. A.; Scherle, P. A.; Jaffe,
B. D.; Trzaskos, J. M.; Magolda, R. L.; Trainor, G. L.;
Duncia, J. V. Bioorg. Med. Chem. Lett. 2004, 14, 1483.
9. Zhang, N.; Wu, B.; Powell, D.; Wissner, A.; Floyd, M. B.;
Kovacs, E. D.; Toral-Barza, L.; Kohler, C. Bioorg. Med.
Chem. Lett. 2000, 10, 2825.
10. Zhang, N.; Wu, B.; Eudy, N.; Wang, Y.; Ye, F.; Powell,
D.; Wissner, A.; Feldberg, L. R.; Kim, S. C.; Mallon, R.;
Kovacs, E. D.; Toral-Barza, L.; Kohler, C. A. Bioorg.
Med. Chem. Lett. 2001, 11, 1407.
11. Berger, D.; Dutia, M.; Powell, D.; Wu, B.; Wissner, A.;
Boschelli, D. H.; Floyd, M. B.; Zhang, N.; Torres, N.;
Levin, J.; Du, X.; Wojciechowicz, D.; Discafani, C.;
Kohler, C.; Kim, S. C.; Feldberg, L. R.; Collins, K.;
Mallon, R. Bioorg. Med. Chem. Lett. 2003, 13, 3031.
12. Boyle, F. T.; Gibson, K. H.; Poyser, J. P.; Turner, P. PCT
Int. Appl. WO 2000068201, 2000.
13. Hennequin, L.; Francois A. PCT Int. Appl. WO
2004041811, 2004.
14. The synthesis of pyrrolopyridazines has been previously
reported in patent literature: Salvati, M. E.; Barbosa, S.
A.; Chen, Z.; Hunt, J. T. PCT Int. Appl. WO 2003082208,
2003.
15. Lieber, E.; Minnis, R. L.; Rao, C. N. R. Chem. Rev. 1965,
65, 377.
16. Froyen, P. Synth. Commun. 1996, 26, 4549.
17. Tsuge, O.; Urano, S.; Oe, K. J. Org. Chem. 1980, 45, 5130.
18. Chan, D. M. T.; Monaco, K. L.; Wang, R.-P.; Winters,
W. P. Tetrahedron Lett. 1998, 39, 2933.
21. Antonsson, B.; Marshall, C. J.; Montessuit, S.; Arkinstall,
S. Anal. Biochem. 1999, 267, 294.
22. Hunt, J. T.; Bhide, Ra. S.; Borzilleri, R. M.; Qian, L. PCT
Int. Appl. WO 2000071129, 2000.
23. Hunt, J. T.; Mitt, T.; Borzilleri, R.; Gullo-Brown, J.;
Fargnoli, J.; Fink, B.; Han, W.-C.; Mortillo, S.; Vite, G.;
Wautlet, B.; Wong, T.; Yu, C.; Zheng, X.-P.; Bhide, R. J.
Med. Chem. 2004, 47, 4054.
24. Borzilleri, R. M.; Cai, Z.-W.; Ellis, C.; Fargnoli, J.; Fura,
A.; Gerhardt, T. y.; Goyal, B.; Hunt, J. T.; Mortillo, S.;
Qian, L.; Tokarski, J.; Vyas, V.; Wautlet, B.; Zheng, X.;
Bhide, R. S. Bioorg. Med. Chem. Lett. 2005, 15, 1429.
25. Rose, W. C.; Lee, F. Y.; Fairchild, C. R.; Lynch, M.;
Monticello, T.; Kramer, R. A.; Manne, V. Cancer Res.
2001, 61, 7507.