The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901

Article abstract of DOI:10.1016/j.bmcl.2008.10.054

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzam ide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-b enzamide PD 0325901.

Full text of DOI:10.1016/j.bmcl.2008.10.054

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6501–6504
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901
Stephen D. Barrett a, Alexander J. Bridges a, David T. Dudley b, Alan R. Saltiel b, James H. Fergus b,
Cathlin M. Flamme a, Amy M. Delaney b, Michael Kaufman a, Sophie LePage b, Wilbur R. Leopold b,
Sally A. Przybranowski b, Judith Sebolt-Leopold b, Keri Van Becelaere b, Annette M. Doherty a,
Robert M. Kennedy a, Dan Marston a, W. Allen Howard Jr. a, Yvonne Smith a,
a
Joseph S. Warmus a,c, , Haile Tecle
*
a Department of Chemistry, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
b Oncology Pharmacology, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
c Pfizer Global Research and Development Eastern Point Road, Groton, CT 06340, USA
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 benzhydroxamate esters derived from their precursor anthranilic acids have been pre-
pared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopro-
pylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity
in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-
1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result,
development of the compound was terminated. Optimization of the diphenylamine core and modifica-
tion of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted
in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-
phenylamino)-benzamide PD 0325901.
Received 30 September 2008
Revised 6 October 2008
Accepted 10 October 2008
Available online 15 October 2008
Keywords:
MEK
CI-1040
PD 0325901
Mitogen-activated protein kinase
MAP/ERK kinase
ERK
Ó 2008 Elsevier Ltd. All rights reserved.
Hydroxamate ester
Benzhydroxamate ester
Oncology
Anthranilic acid
The mitogen-activated protein (MAP) kinase intracellular sig-
naling pathways are involved in the regulation of various cellular
functions.1,2 Levels of phosphorylated MAP kinases are increased
by a multitude of mitogenic stimuli, including growth factors cou-
pled through tyrosine kinase receptors, hormones that bind to G
protein coupled receptors, cytokines, and phorbol esters. This sug-
gests signaling through MAP kinases is essential to multiple cellu-
lar functions.3 One of these pathways, the Raf/MEK/ERK pathway,
plays a major role in the regulation of cellular growth, differentia-
tion, and proliferation, therefore, modulation of the cascade could
be a useful approach for treating proliferative disorders such as
cancer.4 Raf-activated MAP/ERK kinase (MEK) activates the MAP
kinase known as extracellular signal-regulated kinase (ERK)
through a highly specific double phosphorylation.5 The pharmaco-
logical role of MEK has been widely investigated using the weak
in vitro MEK inhibitor PD 0098059, Fig. 1 a compound discovered
in our laboratories.6
In the past, the role of this compound was limited to in vitro
study in that exposure levels required for such a weak inhibitor
to modulate target in vivo were unattainable due to its poor
solubility. In this article we describe the discovery of the new
series of benzhydroxamate esters from which emerged CI-
1040 (compound 24), the first MEK inhibitor to enter clinical
trials.7,8 We furthermore summarize the optimization of the
benzhydroxamate series that culminated in the identification
of the second-generation clinical candidate PD 0325901 (com-
pound 34).9
Scheme 1 describes the general procedure for preparing the
benzhydroxamate esters.13 The appropriate 2-fluorobenzoic acid
(1) and aniline derivatives are treated with excess base in a solvent
(e.g., LiHMDS in THF) to give a substituted N-phenylanthranilic
acid (2). Coupling of the appropriate hydroxylamine to an N-
phenylanthranilic acid core using PyBOP, BOP, or Ph2POCl afforded
the desired products (3).
In cases where a free hydroxyl or diol was protected as an allyl
ether or acetal, respectively, deprotection was accomplished using
pTsOH in MeOH/H2O.
* Corresponding author. 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.2008.10.054

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S. D. Barrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6501–6504
Table 1
Activity of the 2-phenylamino-benzoic acids.
Compound
R1
R2
R3
R4
R5
R6
R7
Kinase cascade IC50 (nM)
5
6
7
8
9
10
11
12
13
14
H
H
H
H
H
H
H
H
F
Cl
H
H
H
H
F
H
H
F
H
H
H
H
H
H
F
H
H
F
H
H
H
H
H
H
H
F
Me
Me
I
H
H
Me
Me
Me
Me
Me
H
H
H
I
H
H
H
H
H
H
I
I
H
H
I
I
I
I
I
554
390
>10,000
>10,000
3050
15
166
518
5.8
6.8
Scheme 1. General procedure for the preparation of the benzhydroxamates.
The goal of the medicinal chemistry program was to identify
lead matter that possesses in vivo activity when administered or-
ally in an animal model. We believed that it would be necessary
to identify novel compounds with activity in the low nanomolar
range for both a kinase cascade assay and the whole cell assay. A
kinase cascade assay was employed as the primary screen for po-
tential MEK inhibitors through the discovery of CI-1040.
H
H
F
F
I
This assay evaluates 32P phosphate incorporation into myelin
basic protein (MBP) by MAPK in the presence of MEK. MAPK is only
weakly active in the basal state, and is activated upon phosphory-
lation by ATP-activated MEK. Order of addition experiments were
required to determine whether inhibition of MBP phosphorylation
in the assay is due to MEK or MAPK inhibition.6 X-ray crystallogra-
phy data previously reported has confirmed the benzhydroxa-
mates bind as ternary MEK-ATP-inhibitor complexes.10
Table 2
Activity of the 4-fluoro-N-substituted-2-(4-iodo-phenylamino)-benzamides.
The secondary screen was a whole cell (K-Balb or murine colon
26) assay that measured levels of phosphorylated ERK (pERK) after
treatment with compound and stimulation of the kinase pathway
with platelet-derived growth factor (PDGF) versus DMSO vehicle-
treated controls. The K-Balb cell line is a K-ras transformed murine
fibroblast line that is fast growing, easy to handle, and gives a
strong response to growth factor stimulation, thus making it quite
suitable for measuring the suppression of pERK levels with
inhibitors.
Compound
R
R3 R1 R5
Kinase cascade K-Balb cell IC50 (nM)
IC50 (nM)
1 h
24 h
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
Me
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
H
H
H
F
F
F
H
H
F
F
F
Me 7.96
Cl 2.8
3.55
1.59
11.8
4.18
4.42
8.18
23.5
54.9
6.48
2.45
26.4
75.8
119
624
645
50
Me 26
Me 20
Me 18
Me 35
Me 27
Cl
Me 7.0
Cl 2.3
Me 7.8
Me 10
Me 23
Me 48
Et
47
nPr
136
232
257
84.6
172
35.4
113
326
661
1780
iPr
–CH2cPr
–CH2cPr
–CH2cPr
–CH2cPr
–CH2cPr
–CH2cBu
–CH2cC5H9
High-throughput screening identified a novel iodoanthranilic
acid (5) as a potent inhibitor of MEK (Table 1). These acids gener-
ally showed weak activity in cells at 10 lM regardless of their ki-
13
nase inhibition potencies. The N-phenylanthranilic acid template
was used to systematically study substituent effects on the diphe-
nylamine core. It was evident that the iodo substituent was favored
at the 40 position, as evidenced by compounds (7–9). This series
also indicated that a substituent, in this case a methyl, was desired
at the 20 position, as evidenced by compounds 6 and 9. Addition-
ally, a halo substituent at the 4 position of ring A, in particular a
fluorine (compound 10), was optimal. X-ray data10 published after
this work had been completed revealed that the fluorine has a
dipolar interaction with the Ser212 amide backbone hydrogen in
the MEK1 structure.11
Later synthetic efforts in our laboratories that are yet unpub-
lished have sought to exploit this key interaction. Another favorable
substitution on the A-ring is fluorine at position R1. Analog pair (10/
13) demonstrate the fluorine’s moderate potency-enhancing effect.
Carboxylic acid isosteres were investigated in order to obtain
cellular potency with this class of inhibitors. Benzhydroxamic acid
(R=H) analogs (15–16) are potent inhibitors in both the kinase and
cellular assays (Table 2).
F
F
–CH2cC6H11 Br
F
1140
A variety of small alkyl and cycloalkylmethyl esters (17–28)
are likewise potent in both assays. With the cycloalkylmethyl es-
ters, potencies are diminished with increasing ring size from
cyclopropyl to cyclohexyl. The ester series distinguishes itself
from the acid series when compounds are incubated with the
K-Balb cells for 24 h before stimulation with growth factor. Loss
in cell potency over time is in general more pronounced with
the benzhydroxamic acids than with the O-substituted ester ana-
logs. We speculate the esters are more resistant to metabolic pro-
cesses within the cells such as hydrolysis and O-glucuronidation
leading to a lower rate of metabolic degradation of parent com-
pound concentrations and thus less receding of cell activity over
time. Analog (24), CI-1040, emerged from the field to demon-
strate in vivo activity with oral administration in a mouse tumor
model.12 This compound advanced into Phase 2 but failed for
inadequate efficacy caused by insufficient systemic exposure.
The clinical trials of CI-1040 thus failed to validate MEK as a via-
ble anticancer drug target, and it was clear that a backup medic-
inal chemistry program should more aggressively address the
physical and pharmacokinetic property issues connected with
the termination of the drug’s development.
Figure 1. Structure of MEK inhibitor PD 0098059.

S. D. Barrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6501–6504
6503
The solubility of (24) is less than 1
l
g/mL in, pH 6.5, phosphate
the cyclopropylmethyl hydroxamate ester analog (29) a 35-fold in-
crease in colon 26 cell MEK inhibition. The same substitution in the
hydroxyethyl hydroxamate ester pair (analogs 30 and 31) provides
a 50-fold improvement in cell potency and for the first time gave a
subnanomolar cellular MEK inhibitor. The two SAR advances also
provided the benefit of improved aqueous solubility (Table 3). Nei-
ther the fluorine at R5 nor the hydroxyethyl side chain provided
significant increases in solubility in and of themselves as seen in
comparisons between the analog pairs (24/29) and (24/30). A com-
parison of analogs (29), (30), and (31) show that the two substitu-
tions together provide a modest improvement in solubility in the
pH 6.5 buffer. Synergy between the R5 fluorine and the dihyroxy-
propyl side chain clearly give the most dramatic increases in aque-
ous solubility as observed for the racemate-enantiomeric trio (33),
(34), and (35).
A comparison between key physical properties and in vitro
metabolism of CI-1040 (24) and PD 0325901 (34) is presented in
Table 4.
The second-generation MEK inhibitor demonstrates signifi-
cantly greater stability over CI-1040 (24) when assayed with either
human liver microsomes or with human hepatocytes. PF-0325901
(34) also shows higher permeability than (24). The in vitro profiles
suggest PD 0325901 (34) should be able to achieve higher systemic
exposures than CI-1040 (24) in vivo.
buffer, making it clear that low in vivo exposure is at least partly
due to solubility limitations. In animals and humans, a high-dose
formulation of CI-1040 was administered up to three times daily
in order to achieve sustained plasma levels required for the target
modulation required to observe expected efficacy. The goal of the
backup program was the optimization of both in vitro potency
and aqueous solubility that would translate to a lower dose admin-
istered once daily to achieve sustained plasma concentrations re-
quired to validate MEK as an anticancer target.
In order to increase solubility in the second-generation MEK
inhibitor series, a number of polar hydroxylamine side chains were
prepared and were coupled with some of the most potent N-
phenylanthranilic acids to produce the series of hydroxylated and
dihydroxylated benzhydroxamate ester MEK inhibitors described
in Table 3. We made two significant SAR observations with this
work. First, a comparison between the colon 26 cell potencies of
CI-1040 and its hydroxyethyl hydroxamate analog (30) reveal a
10-fold improvement due to the hydroxyethyl over the cyclopro-
pylmethyl side chain. The improvement in cell potency of the race-
mic dihydroxypropyl hydroxamate ester analog (32) over CI-1040
is not as dramatic. The second critical finding was the discovery
that substitution of fluorine for chlorine at R5 in CI-1040 gives
Table 3
Both CI-1040 and PD 0325901 were tested for the ability to in-
hibit pERK in subcutaneously implanted murine colon 26 tumors
in mice 24 h after a single oral administration of compound at var-
ious doses. Consistent with the in vitro potency and ADME profiles,
PD 0325901 (34) clearly differentiates itself from CI-1040 (24) ex
vivo.
Activity and solubility of the 3,4-difluoro-2-(2-halo-4-iodo-phenylamino)-N-(2-
hydroxy-ethoxy)-benzamides.
The compound significantly suppresses pERK levels in the tu-
mors at doses lower than those for which effect can be seen for
CI-1040. At 100 mpk, CI-1040 provides only 14% inhibition of
pERK. In contrast, PD 0325901 inhibits tumor pERK levels 84% at
50 mpk. Even at 12.5 mpk, PD 0325901 inhibits pERK 59% one
day after the dose is administered. These data translate to the cor-
responding colon 26 mouse in vivo model (Table 5). All mice show
some response at the MTD (QD, 25 mg/kg, 14 days) of PD 0325901,
whereas to achieve a similar result, mice must be dosed TID with
300 mg of CI-1040 for 14 days. The MTD for CI-1040 cannot be
achieved in this model.
Compound
R
R5
C26 IC50
(nM)
Sol (pH 6.5)
g/mL)
(l
24 (CI-1040)
29
30
31
32
33
–CH2cPr
Cl
F
Cl
F
Cl
F
F
35
1.0
3.5
0.07
19
0.48
0.33
0.82
<1
<1
<1
5
–CH2cPr
–CH2CH2OH
–CH2CH2OH
( )-CH2CHOH(CH2OH)
( )-CH2CHOH(CH2OH)
R-(ꢁ)-CH2CHOH(CH2OH)
S-(+)-CH2CHOH(CH2OH)
—
147
190
255
34 (PD 0325901)
35
F
Both the foundation established by the study of the ERK signal-
ing pathway using the in vitro pharmacological probe PD
00985096 and the identification of Parke–Davis collection screen
hit, the N-phenylanthranilic acid (2), provided the medicinal chem-
istry program a suitable small molecule template to begin pursuing
an orally administered in vivo active MEK inhibitor for the treat-
ment of cancer. Optimization of the substitutions around the
diphenylamine core for in vitro potency and selection of a ben-
zhydroxamate ester that retained robust MEK inhibition activity
in cells over time produced the first orally active MEK inhibitor
Table 4
Comparison of the physical and metabolic properties of CI-1040 and PD 032590.
Compound
HLM
HuHep
LogD Caco-2
C26 IC50 Sol (pH 6.5)
t1/2 (min) t1/2 (min)
(cm/s)
(nM)
(lg/mL)
24 (CI-1040)
34 (PD 0325901) >100
11.5
100
370
>5
4.6
16.2 ꢀ 10ꢁ6 3.5
<1
190
72.7 ꢀ 10ꢁ6 0.33
Table 5
In vivo comparison of antitumor activity by orally dosed CI-1040 and PD 0325901 in advanced stage C26 mouse colon carcinoma.
Compound
% pERK Inhibition in subcutaneous-implanted murine C26 tumors in mice 24 hours post-dose (PO)
Dosea (mg/kg/treatment)
Treatment schedule
Total daily dose (mg/kg)
% Complete responseb
% Partial responseb
T-Cc (days)
24 (CI-1040)
75
300
TID ꢀ 14 days
225
900
10
70
20
30
10.8
12.7
34 (PD 0325901)
1.6
25
QD ꢀ 14 days
1.6
25
20
70
0
30
7.7
15.4
a
A maximum tolerated dose (MTD) is defined as the highest non-lethal dose that does not produce a weight loss greater than 15%. A minimal effective dose (MED) is
defined as the dose that produces at least 20% response rate (CR + PR), or stasis in tumor growth. CI-1040 doses are the MED and the highest dose given. Sufficient exposure
cannot be obtained to establish the MTD for CI-1040. The doses of PD 0325901 are the MED and the MTD. The vehicle for both compounds is 0.5% hydroxypropylmethyl-
cellulose + 0.2% Tween 80 in water.
b
A complete response (CR) is defined as a 100% reduction of initial tumor mass. Partial response (PR) is defined as at least 50% reduction in tumor mass.
Tumor growth delay (T-C) is the difference in days for the control and treated tumors to reach a mass of 750 mg.
c

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S. D. Barrett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6501–6504
in an animal model.12 CI-1040 (24) suffered from low systemic
exposure because of its insolubility and rapid metabolism, and
these liabilities prevented the drug from achieving sufficient effi-
cacy in the clinic. The medicinal chemistry program addressed
the pharmacokinetic issues in the discovery of PD 0325901 (34)
by altering the structure to improve aqueous solubility. In so doing,
new synergies were realized by combining the optimized diphe-
nylamine core substitutions with small mono- and dihydroxylated
hydroxamate ester side chains. This lead to both improved physical
properties, as well as a greatly improved pharmacological profile
and clinical candidate.
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