Structure-based design and synthesis of pyrrole derivatives as MEK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.058

A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design. Optimization of the series led to the identification of potent inhibitors with good pharmaceutical properties.

Full text of DOI:10.1016/j.bmcl.2010.05.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4156–4158
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design and synthesis of pyrrole derivatives as MEK inhibitors
*
Michael B. Wallace , Mark E. Adams, Toufike Kanouni, Clifford D. Mol, Douglas R. Dougan, Victoria A. Feher,
Shawn M. O’Connell, Lihong Shi, Petro Halkowycz, Qing Dong
Takeda San Diego, 10410 Science Center Drive, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design.
Optimization of the series led to the identification of potent inhibitors with good pharmaceutical
properties.
Received 28 March 2010
Revised 12 May 2010
Accepted 14 May 2010
Available online 20 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
MEK
Pyrrole
Oncology
Br
The mitogen activated protein kinase (MAPK) signaling path-
ways are involved in controlling cellular processes including prolif-
eration, differentiation, and apoptosis.1 The RAF-MEK-ERK signal
transduction pathway plays a central role in mediating the trans-
mission of growth-promoting signals from several growth factor
receptors. MEK occupies a strategic downstream position in the
RAF-MEK-ERK pathway, catalyzing the phosphorylation of its only
known substrates, ERK1 and ERK2.2 Overexpression and activation
of MEK/ERK have been associated with several types of human can-
cers.3 The inhibition of MEK has the potential to control cell growth
and provide a treatment for various cancers.4
I
Cl
F
HN
O
HN
O
OH
F
F
F
O
O
N
H
N
H
HO
OH
N
N
1
2
Lys97
Val127
NH
O
+
I
F
NH3
Several MEK inhibitors have been evaluated in the clinic,
including PD0325901 (1) and AZD6244 (2).5 The reported X-ray
co-crystal structure of a close analogue of PD0325901 (3) demon-
strates this class of inhibitors binds in an allosteric pocket adjacent
to the ATP binding site,6 showing key H-bonding interactions of the
hydroxamate oxygens with Lys97, and H-bonding of a fluorine
with the backbone NHs of Val211 and Ser212. The iodo group
makes an important electrostatic interaction with the backbone
carbonyl of Val127. Hydrophobic interactions in the pocket formed
by Ile141, Met143, Val127, and Phe209 are also observed.
HN
O
OH
F
F
O
HO
N
H
HN
Br
H
3
Ser212
O
N
O
Val211
We set out to design novel MEK allosteric site inhibitors that
would incorporate the key interactions with Lys97 and the hydro-
phobic pocket, while making the important Val211 and Ser212
H-bonding interactions through an exocyclic carbonyl on a five-
member ring heterocyclic scaffold. We initially tested this idea with
the 3-aminopyrrole compounds 8a and 8b, which were synthesized
according to the general method depicted in Scheme 1. The Michael
addition of an N-acetylamino acid methyl ester to tert-butyl acry-
late in the presence of potassium tert-butoxide proceeded with a
subsequent Dieckmann cyclization to form the b-keto ester
compound 4.7 Acid catalyzed condensation with 2-fluoro-4-iodo-
aniline was followed by tautomerization to the enamine 5,8 which
was oxidized with DDQ to produce the pyrrole compound 6. Finally,
acidic hydrolysis of the tert-butyl ester gave the carboxylic acid 7,
and hydroxamic ester formation by HATU coupling led to the target
compounds 8a and 8b.
* Corresponding author. Tel.: +1 858 731 3598; fax: +1 858 550 0526.
E-mail address: michael.wallace@takedasd.com (M.B. Wallace).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.058

M. B. Wallace et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4156–4158
4157
O
O
F
R1
H
N
O
O
O
O
a
O
O
O
a
+
R1
N
H
NH
. HCl
O
NH
. HCl
O
N
O
I
O
O
4a R1 = H
4b
9
R
1 = Me
F
F
F
F
I
I
I
O
HN
N
O
O
I
HN
N
HN
O
HN
NH
O
O
O
c
b
R1
R1
O
N
c
b
R1
O
O
O
O
6aR1 = H
5a R1 = H
11a R1= H
11b R1= Cl
11c R1 = F
6bR1 = Me
d
e
10
5b R1 = Me
F
F
F
HN
N
F
I
I
O
I
O
HN
I
O
HN
O
O
HN
e, f
d
R
N
HO
N
H
R1
H
R1
f
g
HO
HO
N
N
N
R1
R1
O
O
O
O
7aR1 = H
7bR1 = Me
8a R1 = H
13-26
12 a-c
8b R1 = Me
Scheme 2. Synthesis of compounds 13–26. Reagents and conditions: (a) 2-fluoro-
4-iodoaniline, EtOAc, reflux; (b) 1-bromo-2,3-butanedione, THF, reflux; (c) Me2SO4,
Cs2CO3, DMF; (d) N-chlorosuccinimide, CH2Cl2; (e) CsF, DMSO, 180 °C; (f) NaOH; (g)
R-NH2, HATU, CH2Cl2.
Scheme 1. Synthesis of compounds 8a and 8b. Reagents and conditions: (a) KOtBu,
THF, rt, 18 h; (b) 2-fluoro-4-iodoaniline, pTsOH, benzene, reflux, 1 h; (c) DDQ,
toluene, reflux, 30 min; (d) 50% TFA/CH2Cl2, 4 h; (e) CH2CHOCH2CH2ONH2, HATU,
CH2Cl2; (f) 25% 1 N HCl/THF, 1 h.
for the diols 15 and 16 were comparable to the alcohol 13, suggest-
ing limited productive interaction of the additional hydroxy group
with the ATP phosphates. However, the diols showed improved
microsomal stability and solubility. Activity of the S-isomer diols
was only marginally better than the R-isomers for this series.
Addition of a halide group (F, Cl) at the pyrrole 4-position had little
effect on activity. The amide 18 was less potent than its corre-
sponding hydroxamic ester 13, but amide R-groups were well tol-
erated when incorporated in a 4- or 5-member ring (24–26).
Compounds from this series did not inhibit other kinases tested
The N-acetyl pyrrole compounds 8a and 8b (Table 1) demon-
strated excellent enzymatic activity,9 but showed only weak,
micromolar activity in our cellular assays.10 This was due to the
inherent chemical instability of the series: pyrrole N-deacetylation
was observed over the course of the assay incubation. To address
this issue, it was necessary to modify the central core while retain-
ing all of the key binding elements. We envisioned this could be
achieved with a modification to a 2-aminopyrrole scaffold (shown
in compounds 13–26).
The synthesis of 2-aminopyrrole compounds 13–26 is outlined
in Scheme 2.11 2-Fluoro-4-iodoaniline was heated with ethyl
3-imino-3-phenoxypropionate hydrochloride to give the imidate
salt 9.12 Reaction with 1-bromo-2,3-butanedione13 led to the forma-
tion of the substituted pyrrole scaffold 10. Pyrrole N-methylation
with dimethylsulfate gave compound 11a. Optionally, the 4-posi-
tion of the pyrrole could be chlorinated with NCS to give 11b, which
could further be converted to the fluoride 11c by microwave heating
in the presence of cesium fluoride. Finally, ester hydrolysis to the
carboxylic acid 12 followed by amide or hydroxamic ester formation
produced the target compounds 13–26.
with inhibitor concentrations up to 10 l
M.14
The pharmacokinetic properties of several compounds were
determined in rats (Table 3). The alcohol 13 showed medium to
Table 2
Selected data for 5-acetyl N-methyl pyrrole analogues
F
I
O
HN
R
N
R1
The in vitro data for compounds 13–26 is presented in Table 2.
Compound 13 showed comparable enzyme activity to its pyrrole
isostere 8b, but also displayed potent cellular activity. The activity
O
R1 MEK1 Colo205 A375
Compd
R
HLM/
RLM t
(min)
9
10
10
IC50
M)
EC50
M)
EC50
M)
½
(
l
(
l
(
l
Table 1
Selected data for N-acetyl pyrrole analogues
12a
13
14
15
16
17
18
19
20
21
22
23
24
25
26
OH
H
H
H
H
H
H
H
0.042 7.10
2.65
0.014 36/14
0.176 43/13
123/7
F
NHO(CH2)2OH
NHO(CH2)3OH
(R)-NHOCH2CHOHCH2OH
(S)-NHOCH2CHOHCH2OH
NHOCH(CH2OH)2
NH(CH2)3OH
NHO(CH2)2OH
(R)-NHOCH2CHOHCH2OH Cl 0.032 0.023
(S)-NHOCH2CHOHCH2OH Cl 0.013 0.018
(R)-NHOCH2CHOHCH2OH
(S)-NHOCH2CHOHCH2OH
3-OH-azetidine
0.018 0.012
0.046 0.104
0.020 0.033
0.011 0.011
0.019 0.020
0.090 0.128
I
O
HN
0.036 >200/29
0.017 150/68
0.033 90/16
0.313 95/10
0.028 7/6
0.046 52/23
0.029 38/30
0.072 107/28
0.048 115/49
0.097 15/13
0.039 55/64
0.127 14/9
R
R1
N
O
Cl 0.018 0.007
R1
H
MEK1 IC50
M)
Colo205
EC50
HLM/RLM t
(min)
9
Compd
R
½
10
(
l
(l
M)
F
F
H
H
H
0.029 0.045
0.019 0.058
0.030 0.032
0.007 0.037
0.013 0.084
7a
7b
8a
8b
OH
OH
0.220
>50
21
3.1
10
29/20
29/21
3/6
Me 0.072
0.011
NHO(CH2)2OH
NHO(CH2)2OH Me 0.020
H
3-NH2-azetidine
(R)-3-OH-pyrrolidine
8/2

4158
M. B. Wallace et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4156–4158
Table 3
supported by the Director, Office of Science, Office of Basic Energy
Sciences, Materials Sciences Division, of the U.S. Department of En-
ergy (DOE) under Contract No. DE-AC02-05CH11231 at Lawrence
Berkeley National Laboratory. We thank the staff at ALS for their
support in the use of the synchrotron beam lines.
Selected rat PK parameters for compounds 13, 15, and 2015
Compd
CL (mL/min/kg)
Vdss (mL/kg)
MRTiv (h)
MRTpo (h)
F (%)
13
15
20
52
48
7.1
2896
2102
361
0.93
0.72
0.85
6.4
7.4
4.1
46
19
40
References and notes
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Sebolt-Leopold, J.; Dudley, D. T.; Leung, I. K.; Flamme, C.; Warmus, J.; Kaufman,
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9. MEK1 Enzyme Assay: Inhibition of compounds relative to MEK1 were
determined using a cascade assay method in 384 well format under the
following reaction conditions: test compounds serial diluted in DMSO were
diluted into assay buffer (50 mM HEPES pH 7.3, 10 mM NaCl, 10 mM MgCl2,
0.01% Brij35, 1 mM DTT) and added into ERK1, fluorescent labeled ERK1
substrate: IPTTPITTYFFFK-5FAM-COOH, and the reaction was initiated with 1
nM MEK1 and 400 lM ATP or 10 lM ATP. Reaction product was determined
quantitatively by fluorescent polarization using progressive IMAP beads from
molecular devices. Inhibition constants (IC50) were calculated using standard
mathematical models. An ERK1 assay was also conducted to rule out that
inhibition was due to ERK1 in the cascade assay. Since all compounds tested
Figure 1. X-ray co-crystal structure of compound 13 in the MEK1 allosteric site.
high plasma clearance and reasonable oral bioavailability (46%).
The R-isomer diol 15 showed lower oral bioavailability; whereas
the 4-chloropyrrole 20 exhibited much reduced plasma clearance
and improved oral bioavailability.
showed almost identical IC50 when assayed at 400
lM or 10 lM ATP, only IC50
results assayed at 400 M ATP were listed. Based on KmATP for MEK1 at 20 lM
l
determined using direct assay method (not shown), no potency shift when
compounds were assayed at 10 Â Km and 0.5 Â Km ATP concentration indicated
compounds were not ATP competitive inhibitors.
10. A375 and Colo205 EC50s were generated using a cellular colorimetric MTS
assay which measures newly produced NADH. Briefly, human cancer cell lines
were seeded between 3000 and 10,000 cells per 96 well and incubated for 16 h
The co-crystal structure of compound 13 in the MEK1 allosteric
site is shown in Figure 1.16 The acetyl group oxygen makes impor-
tant hydrogen bonding interactions with the backbone NHs of
Val211 and Ser212 (2.9 and 3.0 Å, respectively). The acetyl methyl
group packs tightly with the surface of the protein. As with the
anthranilic hydroxamate inhibitors, the hydroxamide oxygens
interact with Lys97. There is little density for the terminal hydroxyl
group, indicating some flexibility in this region. The 2-fluoro-4-
iodoaniline piece sits in the hydrophobic pocket formed by
Ile141, Met143, Val127, and Phe209.
in
a humidified 5% CO2 atmosphere incubator at 37 °C. Cells were then
incubated with an eleven point dilution of test compound in duplicate for 72 h
and subsequently assayed for NADH levels via the CellTiter 96-AQueousÒ kit
(Promega) which utilizes a MTS tetrazolium salt conversion. The resulting
colorimetric reaction was read on a spectrophotometer (Molecular Devices) at
OD 490 nm and EC50 values of compound concentration vs. total NADH levels
were calculated in Activity Base (IDBS). It is important to note the A375 and
Colo205 cell lines both posses the BRAF(V600E) mutation making them reliant
upon MEK signaling for survival. All compounds listed were also tested against
the PC3 cell line whose survival is independent of MEK signaling and served as
a control for MEK inhibitory selectivity. The EC50s generated for all compounds
listed were at a minimum 50-fold higher in the PC3 cell line.
In conclusion, we have designed and characterized a series of
novel pyrrole-based MEK inhibitors. Future reports will describe
modifications of this scaffold for optimization of in vivo properties.
11. Full experimental procedures for compounds 13–26 are contained within the
following patent application: Adams, M. E.; Dong, Q.; Kaldor, S. W.; Kanouni, T.;
Wallace, M. B. PCT Int. Patent Appl. WO 08/055236, 2008.
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13. Matulenko, M. A.; Lee, C.-H.; Jiang, M.; Frey, R. R.; Cowart, M. D.; Bayburt, E. K.;
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A. O.; Yu, H.; Kohlhaas, K. L.; Alexander, K. M.; McGaraughty, S.; Wismer, C. T.;
Mikusa, J.; Marsh, K. C.; Snyder, R. D.; Diehl, M. S.; Kowaluk, E. A.; Jarvis, M. F.;
Bhagwat, S. S. Bioorg. Med. Chem. 2005, 13, 3705.
Acknowledgements
The authors thank Keith Wilson for project leadership. We also
thank the following scientists for their valuable experimental
assistance: Lilly Zhang (DMPK), Melinda Manuel (DMPK), Bi-Ching
Sang (cloning), Ken Bragstad (protein purification), and Gyorgy
Snell (crystallography data collection). The X-ray crystallography
data reported here is based on research conducted at the Advanced
Light Source (ALS) using beam line ALS 5.0.2. The staff at ALS is
14. Kinase Panel: Abl1, AKT3, c-RAF, CamK1
D
, CDK2/cyclinA, cMet, cSRC, EGFR,
GSK3b, IR, JAK3, P38 , PDGFRb, PDK1, PKC
a
a, PLK3, Syk, Tie2.
15. Compounds were administered intravenously and orally at 1 and 5 mg/kg,
respectively.
16. Protein Data Bank code is 3MBL.