2-Alkylamino- and alkoxy-substituted 2-amino-1,3,4-oxadiazoles-O-Alkyl benzohydroxamate esters replacements retain the desired inhibition and selectivity against MEK (MAP ERK kinase)

Article abstract of DOI:10.1016/j.bmcl.2008.10.015

This paper reports a second generation MEK inhibitor. The previously reported potent and efficacious MEK inhibitor, PD-184352 (CI-1040), contains an integral hydroxamate moiety. This compound suffered from less than ideal solubility and metabolic stabilit

Full text of DOI:10.1016/j.bmcl.2008.10.015

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6171–6174
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Alkylamino- and alkoxy-substituted 2-amino-1,3,4-oxadiazoles—O-Alkyl
benzohydroxamate esters replacements retain the desired inhibition
and selectivity against MEK (MAP ERK kinase)
a
a
a
a
c
Joseph S. Warmus a, , Cathlin Flamme , Lu Yan Zhang , Stephen Barrett , Alexander Bridges , Huifen Chen ,
Richard Gowan b, Michael Kaufman a, Judy Sebolt-Leopold b, Wilbur Leopold b, Ronald Merriman b,
Jeffrey Ohren c, Alexander Pavlovsky c, Sally Przybranowski b, Haile Tecle a, Heather Valik b,
Christopher Whitehead c, Erli Zhang c
*
a Department of Chemistry, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
b Department of Oncology Pharmacology, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
c Discovery Technologies Structural and Analytical Sciences, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper reports a second generation MEK inhibitor. The previously reported potent and efficacious
MEK inhibitor, PD-184352 (CI-1040), contains an integral hydroxamate moiety. This compound suffered
from less than ideal solubility and metabolic stability. An oxadiazole moiety behaves as a bioisostere for
the hydroxamate group, leading to a more metabolically stable and efficacious MEK inhibitor.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 23 June 2008
Revised 30 September 2008
Accepted 2 October 2008
Available online 7 October 2008
Keywords:
Oxadiazole
Hydroxamic ester
MEK
MAP ERK kinase
Bioisostere
CI-1040
The mitogen-activated protein kinase pathway is thought to be
essential in cellular growth and differentiation. The family of mito-
gen-activated protein (MAP) kinases can be activated in response
to a wide variety of extracellular stimuli.1,2 In particular, MEK
(MAP kinase kinase) is an attractive target.3 This kinase is activated
by a cascade of phosphorylation events downstream from ras. Its
only known substrates are the MAP kinases, ERK-1 and ERK-2. These
kinases phosphorylate a number of transcription factors that control
cell growth (e.g., ELK-1). Because of its substrate selectivity in the ras
pathway, MEK is an ideal target for intervention of proliferative
An orally active, potent inhibitor of MEK (CI-1040) was reported by
this group.4 This compound inhibits MEK in a colon 26 cell based
assay with an IC50 of 53 nM. CI-1040was shown to not be competitive
with ATPand is exquisitelyselectivefor MEK (IC50 > 10 lM against27
other kinases).4a However, this compound suffers from poor solubil-
ity and also shows a metabolic liability (human liver microsomes). It
was believed that these problems were due in a large part to the key
hydroxamic ester moiety. In order to circumvent this liability, a
survey of five-membered heterocycles was undertaken. After investi-
gating a number of heterocyclic systems, a 5-amino-1,3,4-oxadiazole
was found to have moderate activity against MEK (Table 1).
The synthesis of this oxadiazole11 is shown in Scheme 1. The an-
ion of 4-iodo-2-methyl aniline was prepared using LiHMDS. This
anion was added to the Li salt of 2,3,4-trifluorobenzoic acid in
the presence of excess LiHMDS to afford the anthranilic acid 4. This
substitution occurs exclusively at the 2-position of the benzoic acid
due to coordination of the aniline anion with the carboxylate. For-
mation of the hydrazide followed by condensation with cyanogen
bromide affords the amino-oxadiazole 1. Attempts to reductively
aminate the oxadiazole 1 proved to be difficult. A number of alde-
hydes and conditions were tried, but no generally applicable set of
conditions were found.
diseases.3
H
N
O
O
Cl
C26 cellular IC50 = 53 nm
solubility = <1 μg/mL
HLM = 8-15 min
H
N
F
I
F
CI-1040
* Corresponding author at present address: Pfizer Global Research and Develop-
ment, CVMED Chemistry, Eastern Point Road, Groton, CT 06340, USA. Tel.: +1 860
686 9354; fax: +1 860 686 0001.
E-mail address: Joseph.Warmus@Pfizer.com (J.S. Warmus).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.015

6172
J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6171–6174
Table 1
Initial 1,3,4-oxadiazole data
Table 2
Effect of 20 F on cellular activity
R
NH2
NH
N
N
N
N
O
O
W
H
N
H
N
2'
X
F
Y
F
I
4'
F
F
a
a
Compound
W
X
Y
C26 IC50 (M)
Compound
R
C26 IC50 (M)
1
6
7
8
CH3
H
H
Cl
H
I
I
I
Br
0.210
0.040
0.018
0.140
1
2
3
H
0.21
1.50
10
F
F
F
CH2CH2OH
CONHCH2CH2CH3
a
Western blot analysis was used to measure MEK inhibitory activity in cultured
C26 colon carcinoma cells.
a
Western blot analysis was used to measure MEK inhibitory activity in cultured
C26 colon carcinoma cells.
Two derivatives were made, compounds 2 and 3, in rather low
yields after extended reaction times. Neither of these substitutions
was found to be advantageous. During this investigation, it was
noted that a fluorine in the 20-position of the anthranilic acid
greatly increased potency in the hydroxamate series.4b,4c We inves-
tigated this substitution and found that a similar effect occurred in
the oxadiazole series (Table 2). Simple replacement of the 20
methyl with a fluorine, compound 6, provided a 5-fold increase
in activity, and provided a compound of similar potency to CI-
1040. Addition of a chlorine 7 increased potency slightly. The
importance of the 40-iodo is exemplified by 8, a simple bromo sub-
stitution gives only moderate activity.
In order to circumvent the poor reactivity of the 5-amino-1,3,4-
oxadiazole, we required a new synthesis. The hydrazide 9 was trea-
ted with CDI to provide the intermediate oxazolidinone 10.5 This
was then treated with various amines to give the semicarbazides
11,6 which could be cyclized with either triphenylphosphine or
polymer bound triphenylphosphine and carbon tetrachloride7 to
provide the desired 5-amino-oxadiazoles 12. In the case of 12b–d
Compound 12a was further evaluated in an ex vivo pharmaco-
dynamic assay. This assay analyzed p-ERK suppression in tumors
following an oral dose of inhibitor. Mice bearing subcutaneously
implanted colon 26 tumors were treated with one dose of the
inhibitor and after each time point the tumors were excised and
Western blots were used to measure p-ERK levels versus vehicle
treated controls. As can be seen in Figure 1, 12a showed superior
inhibition of p-ERK levels relative to CI-1040. Most noticeably, at
10 h, 12a completely suppresses phosphorylation of ERK by MEK
at 100 mg/kg dose, and even at 12.5 mg/kg dose, outperforms CI-
1040 (100 mg/kg). A high level of inhibition was retained at the
highest dose even at 24 h post-dose.
We next investigated the effect of 12a in vivo. Colon C26 tumors
were grown to 200 mg over 8 days, then drug (or vehicle) was
administered PO from days 9 to 15 (see Table 4). Compared to
CI-1040 (BID, 100 mg/kg), 12a not only provided 3/3 complete re-
Table 3
(Table 3), the
x-amino BOC was removed using HCl in methanol.
SAR for a series a substituted oxadiazoles
In this case, no opening of the oxadiazole was observed (Scheme 2).
The results of the SAR of the substituted amino oxadiazoles is
shown in Table 3. The goal of this series of compounds was to
maintain or increase potency with a concomitant increase in solu-
bility and metabolic stability. As can be seen, this series proved to
be metabolically stable in a human liver microsome assay. Affect
on solubility for the most part was disappointing, with the major-
ity of analogs having a solubility on par with CI-1040. Compound
12a proved to be the most soluble, and retained an activity 2-fold
better than CI-1040.
R
NH
n
N
O
N
F
H
N
F
I
F
Solubilitya
g/mL)
HLMb t1/2
(min)
a
Compound
R
n
C26 IC50
(M)
(l
6
H
0
2
2
3
4
2
2
3
4
5
2
3
3
3
0
0
0
0.040
0.027
0.015
0.076
0.045
0.140
0.015
0.028
0.012
0.120
0.032
0.080
0.153
0.311
0.067
0.091
0.770
<3
6.7
<3
5.4
ND
ND
<3
<3
<3
<3
40
40
40
40
ND
ND
40
40
40
40
40
35
40
40
40
40
40
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
NHCH3
NH2
NH2
NH2
NH(CH3)2
OH
OH
OH
OH
Morpholine
OMe
R
O
CO2H
F
H
N
c
a
H2N
+
F
F
F
I
I
F
4 R = OH
5 R = NHNH2
HN
b
4.7
NH2
R
<3
<3
<3
<3
3.5
8.8
N
N
N
NH(CH2CH3)2
Morpholine
O
N O
d or e
H
N
H
N
(rac)CH2CHOHCH2CHOH
(S)CH2CHOHCH2CHOH
CH(CH2CHOH)2
F
I
F
I
F
F
1
a
Solubility determined by measuring the concentration of the compound in a
saturated solution over time. In this procedure, increasing aliquots of a 10 mM
solution of a compound in DMSO are added to a series of 15 vials containing 1.8 mL
of 50 mM sodium phosphate buffer at pH 6.5. The solutions are prepared over the
range of 1–60 mg/mL and are allowed to stand overnight at ambient temperature.
The samples are vortexed, then HPLC analysis is performed.
2 R =(CH2)2OH
3 R =CONHn-Pr
Scheme 1. Reagents and conditions: (a) LiHMDS, THF, ꢀ78 °C to rt, 90%; (b)
hydrazine, PyBop, CH2Cl2, THF, rt, 94%; (c) CNBr, NaHCO3, water, dioxane, rt, 86%;
(d) hydroxyacetaldehyde, NaB(OAc)3H, DCE, rt, 64 h, 35%; (e) propyl isocyanate,
toluene, 130 °C, 52%.
b
Human liver microsomal stability, maximum t1/2 is 40 min for this assay.

J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6171–6174
6173
sponses, but also showed a net weight gain for the animals in the
study. The Net log10 Kill is also improved relative to CI-1040.
We have obtained a co-crystal structure8 of the ternary com-
plex of a related inhibitor PD-318088 (13), MgATP, and MEK1
(62–393) (Fig. 2a). Selectivity for these inhibitors can be
explained via binding of CI-1040 to a novel inhibitor binding site
located in an interior hydrophobic pocket adjacent to but distinct
from the MgATP binding site. This binding region has low
sequence homology to other known protein kinases.
One key interaction with the enzyme is a dipolar interaction of
the 4-fluoro atom of PD-318088 (13) and the backbone amide of
Ser 212.9 The hydroxyl groups of the sidechain are also positioned
to allow hydrogen bonding directly to ATP and to Lys 97. The struc-
ture suggests that the PD-318088-like inhibitors act by preventing
the autophosphorylation of MEK and the phosphorylation of ERK
substrate by trapping MEK in a ‘‘closed” but catalytically inactive
conformation.
O
H
N
HN
N
O
O
H2N
F
H
N
a
F
H
b
N
F
I
F
I
F
F
9
10
O
HN
O
R
HN
R
N
N
NH
c
F
HN
O
H
F
H
N
N
F
I
F
I
F
F
11
12
Scheme 2. Reagents and conditions: (a) CDI, DMF, 93%; (b) RNH2, ethanol, 100 °C,
67–99%; (c) PPh3, Et3N, CCl4, CH2Cl2, 45 °C, 31–98%.
12.5 mg/kg
100
25 mg/kg
90
50 mg/kg
80
100 mg/kg
70
60
50
40
30
20
10
0
12a @12a @12a @12a @ CI-
6 h 10 h 24 h 36 h 1040
@ 10
h
Time Post Single Dose
Figure 1. Ex vivo assay of 12a compared to CI-1040 in colon 26 tumors.
Table 4
In vivo test results
Drug
Dosea
Schedule
% Weight
changeb
CRc
PRd
T–Ce
Net
log10 Killf
Vehicle
Vehicle
12a
12a
12a
0
0
Days 9–15
BID;Days 9–15
Days 9–15
Days 9–15
Days 9–15
BID;Days 9–15
BID;Days 9–15
BID;Days 9–15
BID;Days 9–15
ꢀ3.6 (14)
ꢀ4.0 (14)
ꢀ4.5 (14)
ꢀ0.1 (16)
+
0/3
0/3
3/3
2/3
0/3
3/3
3/3
0/3
0/3
0/3
0/3
—
1/3
1/3
—
—
0/3
0/3
—
—
ꢀ0.6
ꢀ0.6
0.3
0.3
0.0
0.4
0.2
0.1
0.1
300
200
100
150
100
50
9.1
8.7
6.0
10.2
7.9
7.4
12a
12a
ꢀ4.0 (14)
+
+
12a
CI-1040g
100
ꢀ4.8 (18)
a
Dose is in mg/kg, oral gavage. Vehicle for 0331257-2B was 5% EtOH + 5% Tween
80 in saline.
b
Maximum treatment related weight loss, expressed as a percent of mean group
weight at initial treatment. Value in parentheses indicates the day the maximum
weight loss was recorded. A net weight gain is represented by a ‘‘+”.
c
A complete response represents a tumor that decreased in mass to less than
62 mg during the study.
d
A partial response represents a tumor that decreased in mass by at least 50%
compared to the tumor mass at initial treatment.
e
T–C is the difference, in days, for the median treated and control tumors to
reach 750 mg.
f
Net log10 tumor cell kill represents the change in tumor burden during therapy.
A negative value indicates a net increase in tumor mass during therapy, while a
positive value indicates a net tumor burden reduction during therapy. Values near
zero indicate tumor stasis during therapy.
g
CI-1040 data from separate test, included for comparison. Median control
tumor size at treatment was 245 mg. Vehicle for CI-1040 was 0.5% hydroxymeth-
ylcellulose + 0.2% Tween 80 in water.
Figure 2. X-ray co-crystal structures of PD-0318088 and 12b.

6174
J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6171–6174
American Chemical Society: Washington, DC, 2008; MEDI-147.; (c) Leopold, J.
A crystal structure of 12b10 shows that the interaction of the en-
S. Abstracts of Papers, 229th ACS National Meeting, San Diego, CA, United States,
March 13–17, 2005; American Chemical Society: Washington, DC, 2005; MEDI-
310.; (d) Spicer, J. A.; Rewcastle, G. W.; Kaufman, M. D.; Black, S. L.; Plummer,
M. S.; Denny, W. A.; Quin, J., III; Shahripour, A. B.; Barrett, S. D.; Whitehead, C.
E.; Milbank, J. B. J.; Ohren, J. F.; Gowan, R. C.; Omer, C.; Camp, H. S.; Esmaeil, N.;
Moore, K.; Sebolt-Leopold, J. S.; Pryzbranowski, S.; Merriman, R. L.; Ortwine, D.
F.; Warmus, J. S.; Flamme, C. M.; Pavlovsky, A. G.; Tecle, H. J. Med. Chem. 2007,
50, 5090.
zyme with the fluorine is preserved. Importantly, an internal
hydrogen bond from the aniline NH to the nitrogen of the oxadiaz-
ole positions the second nitrogen to efficiently bond to Lys 97,
mimicking the hydroxamate binding mode (Fig. 2b).
In conclusion, we have shown that an oxadiazole moiety is a
useful replacement for a hydroxamic ester in a series of MEK inhib-
itors. While solubility was not greatly improved, the compounds
were metabolically stabile. In addition, these inhibitors proved
superior to CI-1040 in both ex vivo pharmacodynamic assays and
in vivo experiments in C26 tumors.
5. (a) Romine, J. L.; Martin, S. W.; Meanwell, N. A.; Gribkoff, V. K.; Boissard, C. G.;
Dworetzky, S. I.; Natale, J.; Moon, S.; Ortiz, A.; Yeleswaram, S.; Pajor, L.; Gao, Q.;
Starrett, J. E., Jr. J. Med. Chem. 2007, 50, 528; (b) Almasirad, A.; Tabatabai, S. A.;
Faizi, M.; Kebriaeezadeh, A.; Mehrabi, N.; Dalvandi, A.; Shafiee, A. Bioorg. Med.
Chem. Lett. 2004, 14, 6057.
6. (a) Saegusa, Y.; Harada, S.; Nakamura, S. J. Heterocycl. Chem. 1988, 25, 1337; (b)
Levins, C. G.; Wan, Z.-K. Org. Lett. 2008, 10, 1755.
7. (a) Yang, Y.-H.; Shi, M. Toxicol. Lett. 2005, 46, 6285; (b) Dhar, T. G. M.; Liu, C.;
Pitts, W. J.; Guo, J.; Watterson, S. H.; Gu, H.; Fleener, C. A.; Rouleau, K.; Sherbina,
N. Z.; Barrish, J. C.; Hollenbaugh, D.; Iwanowicz, E. I. Bioorg. Med. Chem. Lett.
2002, 12, 3125.
References and notes
1. Lewis, T. S.; Shapiro, P. S.; Ahn, N. G. Adv. Cancer Res. 1998, 74, 49.
2. Cobb, M. H.; Goldsmith, E. J. J. Biol. Chem. 1995, 270, 14843.
8. Ohren, J. F.; Pavlovsky, A.; Chen, H.; Whitehead, C.; Kuffa, P.; Yan, C.;
McConnell, P.; Zhang, E.; Spessard, C.; Banotai, C.; Delaney, A.; Omer, C.;
Sebolt-Leopold, J.; Dudley, D.; Flamme, C.; Warmus, J. S.; Kaufman, M.; Barrett,
S. D.; Tecle, H.; Hasemann, C. Nat. Struct. Mol. Biol. 2004, 11, 1193. PDB ID Code
1S9J.
9. (a) Razgulin, A. V.; Mecozzi, S. J. Med. Chem. 2006, 49, 7902; (b) Olsen, J. A.;
Banner, D. W.; Seiler, P.; Wagner, B.; Tschopp, T.; Obst-Sander, U.; Kansy, M.;
Müller, K.; Diederich, F. ChemBioChem 2004, 5, 666; (c) Dunitz, J. D.; Taylor, R.
Eur. Chem. J. 1997, 3, 89.
3. (a) Herra, R.; Sebolt-Leopold, J. S. Trends Mol. Med. 2002, 8, S27; (b) Kohno, M.;
Pouyssegur, K. J. Ann. Med. 2006, 38, 200; (c) Thompson, N.; Lyons, J. Curr. Opin.
Pharmacol. 2005, 5, 350; (d) Wallace, E. M.; Lyssikatos, J.; Blake, J. F.; Seo, J.;
Yang, H. W.; Yeh, T. C.; Perrier, M.; Jarski, H.; Marsh, V.; Poch, G.; Livingston, M.
G.; Otten, J.; Hingorani, G.; Woessner, R.; Lee, P.; Winkler, J.; Koch, K. J. Med.
Chem. 2006, 49, 441; (e) Wallace, E. M.; Lyssikatos, J. P.; Yeh, T.; Winkler, J. D.;
Koch, K. Curr. Top. Med. Chem. 2005, 5, 215.
4. (a) Sebolt-Leopold, J. S.; Dudley, D. T.; Herrera, R.; Van Becelaere, 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; (b) Tecle, H. Abstracts of Papers, 235th
ACS National Meeting, New Orleans, LA, United States, April 6–10, 2008;
10. PDB ID Code 3EQB.
11. All compounds were fully characterized by1H NMR and MS.