Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent

Article abstract of DOI:10.1016/j.bmcl.2011.01.062

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Full text of DOI:10.1016/j.bmcl.2011.01.062

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1795–1801
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel allosteric MEK inhibitor CH4987655
as an orally available anticancer agent
Yoshiaki Isshiki a, , Yasunori Kohchi a, Hitoshi Iikura b, Yasuaki Matsubara a, Kohsuke Asoh a,
⇑
Takeshi Murata a, Masami Kohchi a, Eisaku Mizuguchi b, Shinji Tsujii a, Kazuo Hattori a, Takaaki Miura a,
Yasushi Yoshimura a, Satoshi Aida a, Masanori Miwa a, Ryoichi Saitoh b, Naoaki Murao b, Hisafumi Okabe a,
Charles Belunis c, Cheryl Janson c, Christine Lukacs c, Verena Schück c, Nobuo Shimma a
a Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
b Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
c Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, 07110 NJ, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differ-
entiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel
MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional
optimization including metabolic stability, physicochemical properties and safety profiles were effec-
tively performed and led to the identification of a clinical candidate for an orally available potent MEK
inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benz-
amide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo
antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignifi-
cant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK pro-
file and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 October 2010
Revised 8 December 2010
Accepted 17 January 2011
Available online 21 January 2011
Keywords:
Allosteric MEK inhibitor
Orally available anticancer agent
[1,2]Oxazinan-3-one ring structure
The mitogen-activated protein kinase (MAPK) pathway, includ-
ing the Ras/Raf/MEK/ERK signaling cascade, is one of the most
important pathways involved in cell proliferation and differentia-
tion.1 Aberrant activation of the MAPK pathway in tumor cells,
such as K-Ras or B-Raf mutation, is frequently observed2,3 and,
therefore, the components of this pathway have promise as targets
for antitumor drugs.4,5
Although several MEK inhibitors were clinically evaluated, clear
clinical efficacy has not yet been observed. The development of
CI-1040 (Pfizer Inc.), the first MEK inhibitor tested clinically, was
terminated due to its insufficient efficacy and poor PK profile such
as inter-patient variability, which may be a result of its low water
solubility and metabolic instability, especially against hydrolysis of
the hydroxamate moiety.6 PD0325901 (Pfizer Inc.), the second
MEK inhibitor, exhibited improved water solubility and stability
against this metabolism. Oral administration to human of this
drug, however, still afforded a certain amount of COOH metabo-
lites. Furthermore, PD0325901 exhibited several side effects such
as visual disturbance and syncope which implied the drug has an
effect on the CNS.7 These implied a demand for novel MEK inhibi-
tors with superior efficacy and better physicochemical, metabolic
and safety profiles. Thus, we initiated research to create a novel
MEK inhibitor with the required profiles taking CI-1040 as a lead
structure (Fig. 1).
Wabnitz et al. studied in vitro and in vivo metabolism of
CI-1040 revealing that the hydroxamate moiety of CI-1040 is
hydrolyzed by both cytochrome P450 enzymes and non-P450
amidase.8 Although a preliminary attempt to replace the hydroxa-
mate moiety with another functional group resulted in a reduction
of MEK inhibitory activity (data not shown), we thought modifica-
tion of another position might contribute to solving the problem of
the hydrolysis by affecting the recognition by the metabolic
H
R2
N
O
HO
O
R1
R1
O
H
N
H
N
hydrolysis
in human
F
I
F
I
F
F
*
CI-1040
PD0325901 R1 = F, R2
R1 = Cl, R2 =
HO
OH
*
=
⇑
Corresponding author.
E-mail address: isshikiysa@chugai-pharm.co.jp (Y. Isshiki).
Figure 1. Structure of 1st and 2nd clinically tested MEK inhibitors and the major
metabolic pathway.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.062

1796
Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
enzymes. From the point of maintenance of MEK inhibitory activity
and synthetic feasibility, we focused on the modification of 5-posi-
tion. The 5-substituted derivatives were synthesized via common
intermediate 4 which was prepared starting with iodination of
benzoic acid 19 followed by vinylation10 and nucleophilic aromatic
substitution.11 We discovered that the oxime ether derivatives
10
10
10
10
CH4858061
CH4858060
PD0325901
CI-1040
1
0.1
1
COOH metabolite
1
0.1
1
0.1
COOH metabolite
COOH metabolite
COOH metabolite
0.1
0.01
0.01
0.01
0.01
0.001
0.001
0.001
0.001
0
6
12
18
24
0
6
12
Time (h r)
18
24
0
6
12
18
24
0
6
12
18
24
Time (hr)
Time (hr)
Time (h r)
Figure 2. Time course of plasma concentration after single oral administration of CI-1040, PD0325901, CH4858060 and CH4858061 (2 mg/kg) in cynomolgus monkeys
(n = 2). Values for drug concentration in plasma are given as the mean (
g/ml); N: intact CI-1040; 4: COOH metabolite of CI-1040; j: intact PD0325901; h: COOH metabolite
of PD0325901; ꢀ: intact CH4858060 (6); }: COOH metabolite of CH4858060 (6); d: intact CH4858061 (7); s: COOH metabolite of CH4858061 (7).
l
HO
F
c
O
HN
HO
O
HO
O
HO
O
H2N
HO
O
O
F
BF3.K
HO
I
NH2
HO
H
N
F
O
H
N
F
F
F
F
F
F
I
R1
F
e
b
a
I
N
F
R2
O
5
F
F
2
F
3
F
F
4: R1 = vinyl
1
6 (CH4858060): R2= I
d
5: R1 = formyl
f
7 (CH4858061): R2= ethynyl
HO
NH2
HO
O
O
HN
O
HN
O
HO
O
10: R3 =
11: R3 =
O
HO
F
*
O
F
H
N
H
N
O
4
S
g
*
R3
N
j for 10
k for 11
l for 12
R1
F
I
H
N
F
I
12: R3 =
*
F
F
O
8: R1 = vinyl
h
9: R1 = formyl
Scheme 1. Reagents and conditions: (a) I2, MnO2, Ac2O, AcOH, H2SO4, 50 °C, 94%; (b) Pd(dppf)Cl2, MeOH, reflux, 98%; (c) LiHMDS, THF, ꢀ78 °C–rt, 94%; (d) OsO4 (cat.), NaIO4,
NaHCO3, THF–H2O, rt, 93%; (e) (i) WSC, HODhbt, THF, 91%; (f) (i)TMS–acetylene, (PPh3)2PdCl2, CuI, THF, 79%; (ii) TBAF, THF, 74%; (g) WSC, HODhbt, DMF, 91%; (h) OsO4 (cat.),
NaIO4, DMA, THF–H2O, 56%; (j) 1-aminooxy-2-methyl-propan-2-ol, THF–CH2Cl2, 52%; (k) (i) O-(2-methylsulfanyl-ethyl)-hydroxylamine, THF–CH2Cl2, 90%; (ii) oxone, MeOH–
H2O, 63%; (l) (i) O-(2-amino-ethyl)-hydroxylamine, THF–CH2Cl2, 90%; (ii) N-methoxy-diacetamide, DMF, MeOH, 83%.
Table 1
Biological and physicochemical profiles of 5-substituted benzamide MEK inhibitors (part 1)
Core structure
Compound R1
R2
In vitro IC50 (nM)
Solubility mLM hLM
pH 6.5 t1/2 t1/2
(min) (min)
In vivo antitumor efficacy
MEK HT-29 QG56
(CRC) (NSCLC)
TGI(@MTD)
in HT-29 p.o.,
qd ꢁ 14 days,
n = 6 (%)
MTD
(mg/kg)
(l
M)
HO
O
HO
N
N
*
*
6
7
I
100
80
160
160
8.3
237
135
65 82
80 82
100
O
O
HN
O
HO
HO
Ethynyl 235 150
415
15
>200
F
H
N
N
*
O
10
11
I
I
37
67
40
27
128
24
>360 >360 85
100
R1
F
R2
O
S
O
1
>360
90 98
>200
N
*
F
O
H
N
N
*
12
O
I
48
86
199
16
>360 >360 99
>200
O
CRC: colorectal cancer; NSCLC: non small cell lung cancer; mLM: mouse liver microsome; hLM: human liver microsome, TGI: tumor growth inhibition; MTD: maximum
tolerable dose.
Detailed procedures for each assay listed in Tables 1–4 are described in the Supplementary data.

Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
1797
Figure 3. Ternary complex of CH4858061/MEK1/ATP (PDB code: 3OS3). (A) Overview; green: CH4858061; white: MEK1; orange: ATP; yellow dotted lines: intermolecular H-
bondings and electrostatic interactions; (B) close-up of area around the oxime-ether side chain.
HO
HO
HO
O
O
O
HN
O
HN
O
HN
O
HO
F
F
F
OH
H
N
H
N
H
N
HO
HO
6
6
9
O
N
O
a
b
c
HN
F
I
R
F
O
I
F
I
F
F
F
*
15
14
16: R =
O
d
O
*
17: R =
HO
HO
NH2
HO
HO
O
O
O
H
N
O
NH2
HN
O
O
HN
O
HN
NH2
O
F
F
F
O
N
H
O
H
N
H
N
H
N
O
9
9
9
h
k
e
R
F
R
F
R
F
I
I
I
F
F
F
N
O
H
N
*
*
18 : R =
O
N
*
N
O
21 : R =
22 : R =
23 : R =
*
N
f
O
H
H
O
N
19 : R =
l
j
O
O
N
O
O
N
24 (CH4987655) :R =
g
*
N
*
20 : R =
*
O
O
O
Scheme 2. Reagents and conditions: (a) Cu(OTf)2, toluene, 60 °C, 81%; (b) NaBH3CN, TFA, MeOH, rt, 46%; (c) TsOH (cat.), THF; (d) NaBH4, TFA, THF, two steps 80%; (e) CH2Cl2,
THF, 90%; (f) BH3–pyridine, CHCl2COOH, CH2Cl2, 93%; (g) CH3COOH, EDC, HODhbt, Et3N, CH2Cl2, 53%; (h) THF, MeOH, 80%; (j) BH3–pyridine, CHCl2COOH, CH2Cl2, rt, then 1,2-
dichloroethane, 60 °C, 90%; (k) THF, MeOH, 80%; (l) BH3–pyridine, CHCl2COOH, CH2Cl2, rt, then 1,2-dichloroethane, 60 °C, 91%.
CH4858060 (6) and CH4858061 (7) showed significantly higher
metabolic stability of the hydroxamate side chain compared to
CI-1040 and PD0325909112 after oral administration in monkeys
as shown in Figure 2. Considering the fact that PD0325901 possess-
ing hydrophilic 2,3-dihydroxypropyl group in its hydroxamate
part still gives a certain amount of COOH metabolite in monkeys,
it can be concluded that the oxime side chain newly introduced
at 5-position dominantly contributes to the improvement of the
metabolic stability against hydrolysis. This interesting remote-
control nature of the metabolic stability encouraged us to further
modify these oxime ether derivatives (Scheme 1).
Despite the excellent metabolic stability, CH4858060 (6) and
CH4858061 (7), however, still had several drawbacks including
insufficient water solubility, low human liver microsome stability,
and weak enzyme inhibitory activity as well as antitumor activity
both in vitro and in vivo. Chemical modification of a terminal part
of the oxime side chain, such as introduction of alkyl (10), sulfonyl
(11) or amide (12) moiety, did not lead to significant improvement
(Table 1).
To overcome these problems, a 3D-structure of the ternary
complex of 7/ATP/MEK1 using X-ray crystallography was utilized.
Compound 7 is located in almost the same position as other known
allosteric MEK inhibitors and a hydrogen bond network among
ATP, MEK1 and 7 is observed (Fig. 3A). The oxime ether side chain
is located near the activation loop and a certain amount of space
was noticed around it (Fig. 3B). It was thought that this space could
be utilized for further modification of the lead compounds 6 and 7
to obtain higher activity and better physicochemical properties.
In order to fill up the space, we designed several types of deriv-
atives, having different degrees of conformational flexibility and
bulkiness of the C-5 substituents. The Z-oxime ether 14 was suc-
cessfully prepared from the corresponding E-isomer 6 by treating
with Cu(OTf)2 under heating13 and the alkoxylamines 15 and 19
were prepared by reducing the corresponding oxime-ethers.
Reductive etheration of the benzaldehyde 9, typically acetal-
formation followed by reduction, afforded the benzylether 17. The
alkoxylamides 20 and 22 as derivatives possessing a branched or
cyclic type of side chain were prepared by acylation of the

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Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
Table 2
Biological and physicochemical profiles of 5-substituted benzamide MEK inhibitors (part 2)
Core structure
Compound
R
In vitro IC50 (nM)
Solubility
pH 6.5
mLM
t1/2
(min)
hLM
t1/2
(min)
In vivo antitumor efficacy
MEK
HT-29
(CRC)
QG56
TGI (@MTD)
in HT-29 p.o.,
qd ꢁ 14 days,
n = 6 (%)
MTD
(mg/kg)
(lM)
(NSCLC)
HO
O
HO
HO
N
*
*
6
100
15
80
160
6
8.3
237
65
82
95
100
O
HN
O
O
N
F
14
2.4
75
>360
>360
12.5
H
N
*
O
*
17
15
14
19
4.2
29
20
94
728
606
>360
128
>360
>360
105
93
50
HO
HO
R
F
I
H
N
100
F
O
*
O
N
20
22
8.6
1.5
1.3
7.5
3.5
116
216
36
64
113
105
25
O
O
N
*
7.2
>360
>360
12.5
O
Table 3
Biological and physicochemical profiles of 5-substituted benzamide MEK inhibitors (part 3)
Core structure
Compound
R
In vitro IC50 (nM)
Solubility
mLM t1/2 (min)
hLM t1/2 (min)
pH 6.5 (
lM)
MEK
7.2
HT-29 (CRC)
1.3
QG56 (NSCLC)
HO
O
O
N
*
22
3.5
216
>360
>360
HN
O
F
H
N
O
N
N
*
*
*
25
26
27
85
20
560
17
57
3063
56
156
284
57
124
>360
126
185
>360
38
R
F
I
O
O
F
>250
42
O
O
N
O
O
HO
HO
O
HN
O
HN
25: R =
26: R =
N
*
O
O
O
F
F
H
N
H
N
9
15
O
O
N
N
a
*
b
O
O
R
F
I
F
I
O
F
F
27
Scheme 3. Reagents and conditions: (a) preparation of 25: 4-amino-butylic acid methylester hydrochloride, NaBH3CN, MeOH, rt–reflux, 47%; preparation of 26:
(i) ethanolamine, CH3COOH, NaBH3CN, MeOH, 84%; (ii) N,N-disuccinimidyl carbonate, Et3N, THF, CH3CN, 42%; (b) N,N-disuccinimidyl carbonate, Et3N, THF, CH3CN, 12%.
HO
corresponding alkoxylamine 19 or reduction of the oxime-ether 21
followed by simultaneous intra-molecular cyclization (Scheme 2).
The physicochemical and biological profiles of the representa-
tive derivatives are summarized in Table 2. As compared with
E-oxime, the Z-oxime as well as the conformationally more flexible
benzyl ether and alkoxy-amine derivatives showed higher water
solubility, metabolic stability, and antitumor activity. The im-
proved enzyme inhibitory activity of the Z-oxime 14 as compared
with the E-oxime 6 suggested favorable conformation of the C-5
substituent that fits to the binding pocket. Interestingly, the cyclic
O
HN
O
F
H
N
O
N
F
I
O
F
CH4987655
Figure 4. Structure of CH4987655.

Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
1799
Table 4
In vitro profile of CH4987655
Enzyme
inhibition (IC50
Tumor cell
anti-proliferation (IC50)
Physicochemical property
In vitro safety profile
)
MEK
Other 400
kinases
COLO205 HT29
QG56
MIA
C32
Solubility
Liver microsome
stability (half life)
Drug–drug
interaction
Genotoxicity Cardio-
toxicity
(CRC,
(CRC,
(NSCLC,
PaCa-2
(Panc.,
(melanoma, pH 6.5
BrafV600E
)
BrafV600E
)
HrasQ61L
)
BrafV600E
)
KrasG12C
)
5.2 nM <50% inhibition 0.86 nM
at 10
1.7 nM
9.5 nM
3.3 nM
8.4 nM
88
l
M
Mouse
>360 min 116 min
Human
CYP3A4
IC50 >100
Ames MNT
negative
hERG not
significant
lM
lM
Figure 5. X-ray crystal structure of CH4987655/MEK1/AMP-PNP (PDB code: 3ORN). (A) Overview; green: CH4987655; white: MEK1; orange: AMP-PNP; yellow dotted lines:
intermolecular H-bondings and electrostatic interactions; (B) close-up view around the newly introduced 3-oxo-oxazinan ring sub-structure.
derivative, isoxazolidinone 22, exhibited the highest enzyme
inhibitory activity and antitumor activity among these derivatives.
Evaluation of the cyclic system of 22 by modifying in particular
the pattern of the heteroatom arrangement revealed that the O–N–
C(@O)–C sub-structure was important for exhibiting the desired
biological activity. For example, replacement of the ether oxygen
by a methylene carbon dramatically reduced the MEK inhibitory
activity (Table 3, Scheme 3).
Further optimization of 22 led us eventually to identify
CH4987655 (24) (Scheme 2 and Fig. 4) as a clinical candidate hav-
ing a unique 3-oxo-[1,2]oxazinan-2-ylmethyl group at the 5-posi-
tion. The in vitro profiles of CH4987655 are summarized in Table 4.
CH4987655 inhibits MEK with an IC50 of 5 nM, but did not inhibit
H-bonding network observed between CH4987655 and AMP-PNP
in the MEK1 complex structure (Fig. 5A). The slower dissociation
of CH4987655 from MEK1 could explain the observed superior
pharmacodynamics of CH4987655 to PD0325901 in cynomolgus
monkeys showing the longer pharmacodynamic duration with the
lower IC50 value for pERK formation in PBMC (IC50 = 7.03 ng/mL
for CH4987655 vs IC50 = 32.8 ng/mL for PD0325901).15 The details
of the interaction and the role of the newly introduced [1,2]oxaz-
inan-3-one ring structure with MEK1 is now under investigation.
We examined the MEK inhibition status of CH4987655 in both
tumor and brain in a HT-29 human colon cancer xenograft at the
maximum tolerable dose (MTD), and compared it with that of
PD0325901. Both CH4987655 and PD0325901 strongly inhibited
pERK formation in tumor (Fig. 6A), and CH4987655 showed strong
tumor regression at this dose (Fig. 7). Interestingly, a distinct differ-
ence was seen between the two drugs in the target inhibition in the
brain. CH4987655 did not inhibit MEK in mouse brain at MTD, but
PD0325901 did (Fig. 6B). This result can be explained by the very
low distribution of CH4987655 to the brain as compared with
the plasma concentration in a rat experiment (Fig. 6C).
the other 400 kinases at 10 lM. It showed strong in vitro anti-pro-
liferative activity against a broad range of tumor cells without
genotoxicity or hERG and CYP inhibition in vitro. Sufficient water
solubility and in vitro metabolic stability was also observed.
X-ray crystal structure analysis of the ternary complex of
CH4987655/MEK1/AMP-PNP showed H-bonding interaction of
the hydroxamate oxygens with AMP-PNP through Lys97, and elec-
trostatic interactions of the 4-fluorine with the backbone NHs of
Val211 and Ser212, and 40-iodine with backbone carbonyl of
Val127 (Fig. 5A). Notably, the newly introduced [1,2]oxazinan-3-
one ring structure occupies well the aforementioned open-space
(Fig. 5B), surrounded by five amino acid residues, Gly210, His188,
Arg189, Asn221 and Met219. In particular, the terminal aminocar-
bonyl moiety of Asn221 has moved greatly from its original posi-
tion in the structure of CH4858061/MEK1/ATP to form a tighter
space fitting the [1,2]oxazinan-3-one ring structure. Indeed, sur-
face plasmon resonance (SPR) analysis revealed the higher affinity
of CH4987655 for MEK1 compared with that of PD0325901 both in
the absence and the presence of ATP, largely due to a slower disso-
ciation (Table 5). Also, as reported previously for PD0325901,14 the
binding affinity of CH4987655 was significantly increased in the
presence of ATP (Table 5), consistent with the intermolecular
CH4987655 demonstrated antitumor activity in a wide range of
human cancer xenograft models. Strong tumor regression was ob-
served in 70% of the models tested.16 Examples of such strong anti-
tumor efficacy with remarkable tumor regression are shown in
Table 5
Binding kinetics of MEK inhibitors to MEK1 in the presence or absence of ATP
Compound
ꢀATP
+ATP (50
KD (nM)
lM)
KD (nM)
Koff (/s)
Koff (/s)
CH4987655
PD0325901
8.7
31
8.00E-03
1.70E-02
0.24
0.4
6.00E-05
2.00E-04
The binding kinetics was measured by SPR analysis as described in the Supple-
mentary data.

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Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
1200
1000
800
600
400
200
0
2 h
CH PD
4 h
CH PD
7 h
CH PD
24 h
CH PD
(C)
(A)
(B)
Rat, 1 mg/kg, po
Plasma
V
V
V
V
V
V
V
V
Cerebrum
Cerebellum
Spinal cord
pERK
(Thr202/Tyr204)
total ERK
2 h
CH PD
4 h
CH PD
7 h
CH PD
24 h
CH PD
pERK
(Thr202/Tyr204)
2
8
24
48
total ERK
Figure 6. MEK inhibition status in HT-29 human colon cancer xenograft model after oral once-a-day treatment with CH4987655 (CH) at MTD (= 6.25 mg/kg), PD0325901
(PD) at MTD (25 mg/kg) and vehicle (V). (A) MEK inhibition status in tumor tissue; (B) MEK inhibition status in brain in mice; (C) distribution after oral administration of
CH4987655 (1 mg/kg) into brain (cerebrum, cerebellum and spinal cord) as compared with into plasma in rat experiment.
116%
117%
137%
Figure 7. Antitumor effect of CH4987655 in human cancer xenograft models. Human tumor cell lines were subcutaneously transplanted into CAnN.Cg-Foxn1 nu/CrlCrlj mice.
After confirmation of tumor implantation, the mice were randomly allocated into vehicle and drug-treated groups. CH4987655 was orally administered once-a-day at the
maximum tolerated dose (MTD) for 14 days. Each group consisted of 6 mice. Values for tumor volume are given as the mean S.D.; s: vehicle; : CH4987655. Percent tumor
growth inhibition is shown to the right of each treatment group and values >100% indicate tumor regression.
Figure 7. Furthermore, CH4987655 in combination with various
antitumor agents enhanced the antitumor activity.16 Concurrent
Vehicle
CH 1 mg/kg (1/6 x MTD)
Everolimus 20 mg/kg
CH + Everolimus
oral treatment with CH4987655 (1 mg/kg = 1/6 MTD) and everoli-
mus, the mTOR inhibitor, in HCT116 tumor-bearing mice showed
greater antitumor activity compared to single-agent treatments
showing clear tumor regression (Fig. 8).
1000
As expected, oral CH4987655 showed higher metabolic stability
than PD0325901 in cynomolgus monkeys: namely, the generation
of COOH metabolites was much less in CH4987655. Exposures
were dose–proportional and the AUC was about twice as high as
that of PD0325901 (Fig. 9, Table 6).
P < 0.0001
P = 0.0003
This favorable PK profile was also confirmed by a phase 1 clin-
ical trial in healthy volunteers. CH4987655 was rapidly absorbed
after oral administration with a Tmax of about 1 h and the disposi-
tion was biphasic with a terminal t1/2 of about 25 h. Drug expo-
sures were clearly dose–proportional within the tested dose, and
very low inter-subject variability was observed. The effects of the
target inhibition in PBMC were exposure-dependent and were
greater than 80% at 4 mg. Good PK/PD correlation was observed.17
In summary, we discovered that the introduction of a unique
oxime-ether side chain at 5-position of benzamide core structure
dramatically improves the metabolic stability against hydrolysis
of previously reported MEK inhibitors. Starting from such metabol-
ically stable oxime-ethers 6 and 7 and utilizing the 3D-structural
information, we identified a novel selective and potent MEK inhib-
itor, CH4987655 (24), possessing a unique 3-oxo-oxazinane ring
100
14
17
20
23
26
Days after cell inoculation
Figure 8. Antitumor effect of CH4987655 in combination with everolimus in
HCT116(CRC, KrasG13D) human cancer xenograft model. CH4987655 (1 mg/kg (1/6
MTD)) and everolimus (Sequoia Research Products Ltd) (20 mg/kg) were adminis-
tered orally once daily for 11 days. Each group consisted of 5 mice (BALB-nu/nu
(Crlj), male). Values for tumor volume are given as the mean S.D. ꢀ: vehicle;
everolimus (20 mg/kg); CH4987655 (1 mg/kg = 1/6 MTD); CH4987655
(1 mg/kg) + everolimus (20 mg/kg); combo versus CH4987655: P = 0.0003; combo
versus everolimus: P <0.0001. Percent tumor growth inhibition is shown to the
right of each treatment group and values >100% indicate tumor regression.
:
:
:

Y. Isshiki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1795–1801
1801
10
10
1
PD0325901
CH4987655
1
0.1
COOH metabolite
HO
O
COOH metabolite
O
HN
O
F
H
N
0.1
O
N
F
I
0.01
0.001
0.01
0.001
F
CH4987655
0
6
12
Time (h r)
18
24
0
6
12
18
24
Time (h r)
Figure 9. Time course of plasma concentration of CH4987655 and PD0325901 together with their COOH metabolites after oral single administration in cynomolgus monkeys
(n = 4); Values for dug concentration in plasma are given as the mean S.D.; d: intact CH4987655; s: COOH metabolite of CH4987655; j: intact PD0325901; h: COOH
metabolite of PD0325901.
Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.;
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Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
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Quiros, R. M.; Gattuso, P.; Ain, K. B.; Prinz, R. A. Cancer Res. 2003, 63, 4561.
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Table 6
PK parameters of CH4987655 and PD0325901 after oral single administration into
cynomolgus monkeys
Compound
Dose
(mg/kg)
Tmax
(h)
Cmax
g/ml)
AUCinf
gꢂh/ml)
2.2
7.4
t1/2
(h)
MRTinf
(h)
(l
(l
1.5
3
6
1.3
2.5
1.5
0.4
1
2.1
6.6
6
5
8.4
8.8
7.3
CH4987655
PD0325901
10.4
6
1.1
1.7
7.9
5.5
7.3
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M.; Delaney, A. M.; Kaufman, M.; LePage, S.; Leopold, W. R.; Przybranowski, S.
A.; Sebolt-Leopold, J.; Becelaere, K. V.; Doherty, A. M.; Kennedy, R. M.; Marston,
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structure at the 5-position. CH4987655 shows slow dissociation
from MEK, high metabolic stability and remarkable in vivo antitu-
mor efficacy with clear target inhibition in tumor but not in the
brain. Combination therapy with the PI3K/Akt/mTOR pathway
inhibitor significantly enhances the in vivo efficacy, exhibiting
clear tumor regression. Favorable PK/PD profiles were observed
in
a healthy volunteer study. Taken together, we expect
CH4987655 to be clinically effective with manageable toxicity. A
phase 1 clinical study with solid tumor patients is currently in
progress.
Acknowledgments
8. Wabnitz, P. A.; Mitchell, D.; Wabnitz, D. A. M. Pharm. Res. 2004, 21, 1670.
9. (a) Lulinski, P.; Skulski, L. Bull. Chem. Soc. Jpn. 1999, 72, 115; (b) Koyano, H.;
Iikura, H.; Isshiki, Y.; Kohchi, Y. WO 2006109661.
We are grateful to N. Oikawa, H. Koyano, T. Hayase, S. Takeda,
K. Morikami, K. Yoshinari, Y. Itezono, N. Nakayama, H. Shirai,
H. Shindo, T. Mitsui, T. Ueto, R. Takano, N. Inagaki, Y. Inagaki,
Y. Tomii, E. Hashimoto, R. Watanabe, K. Hamada, Y. Tachibana,
N. Ishii, A. Kubota, H. Suzuki, T. Shiga, H. Matsumoto, T. Tsuno,
A. Kawashima and all people who contributed to this project.
10. Molander, G. A.; Rivero, M. R. Org. Lett. 2002, 4, 107.
11. (a) Chen, M. H. G.; Magano, J. WO 2000064856.; (b) Chen, M. H.; Beylin, V. G.;
Iakovleva, E.; Kesten, S. J.; Magano, J.; Vrieze, D. Synth. Commun. 2002, 32, 411.
12. CI-1040 was prepared according to the description (Example 95) of WO99/
01426. PD0325901 was prepared according to the description (Example 39) of
WO02/06213.
13. As the first screening, several kinds of Brönsted and Lewis acid such as HCl,
TsOH, PPTS, BF3OEt2, Mg(OTf)2, Cu(OTf)2 were attempted. Then metal
screening, including Cu, Sc, Y, Hf, Zn, Sn as triflate, followed by solvent
screening was performed to reveal Cu(OTf)2/toluene as the best condition for
the isomerization of the oxime ether 6.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.062.
14. Smith, C. K.; Windsor, W. T. Biochemistry 2007, 46, 1358.
15. Yoshimura, Y.; Ishii, N.; Sakamoto, H.; Miura, T.; Isshiki, Y.; Ishikawa, Y.; Miwa,
M.; Shimma, N.; Okabe, H.; Aoki, Y.; AACR, annual meeting, Abstract no. 2920,
2009.
References and notes
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