Design and synthesis of orally available MEK inhibitors with potent in vivo antitumor efficacy

Article abstract of DOI:10.1016/j.bmcl.2012.02.026

The structure-based design, synthesis, and biological evaluation of two novel series of potent and selective MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic dihydroindolone core provided compounds wit

Full text of DOI:10.1016/j.bmcl.2012.02.026

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2411–2414
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of orally available MEK inhibitors with potent in vivo
antitumor efficacy
⇑
Mark E. Adams , Michael B. Wallace, Toufike Kanouni, Nicholas Scorah, Shawn M. O’Connell,
Hiroshi Miyake, Lihong Shi, Petro Halkowycz, Lilly Zhang, Qing Dong
Takeda California, 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:
The structure-based design, synthesis, and biological evaluation of two novel series of potent and selec-
tive MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic
dihydroindolone core provided compounds with high potency and good drug-like pharmaceutical prop-
erties. Further scaffold modification afforded a series of dihydroindolizinone inhibitors, including an
orally available advanced preclinical MEK inhibitor with potent in vivo antitumor efficacy.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 December 2011
Revised 7 February 2012
Accepted 10 February 2012
Available online 20 February 2012
Keywords:
MEK inhibitor
Kinase
Oncology
Dihydroindolone
Dihydroindolizinone
The RAS/RAF/MEK/ERK cascade plays a central role in the sig-
naling required for cellular transformation and proliferation.1
Downstream in the signaling pathway, the MEK kinases catalyze
the phosphorylation of the MAPK substrates ERK1 and ERK2, and
thus the inhibition of MEK kinases have the potential to control cell
growth. MEK inhibitors could conceivably be utilized as therapeu-
tic agents in hyperproliferative disorders.2 In a previous Letter, we
discussed the structure-based design and synthesis of pyrrole de-
rived MEK inhibitors.3 In continuation of this research, we elabo-
rated on the pyrrole moiety to develop bicyclic compounds
which are orally bioavailable and efficacious in tumor xenograft
models.
the pyrrole. We reasoned that rotationally locking the carbonyl
moiety at the 5-position of pyrrole 1 by the formation of a bicyclic
core (Fig. 1, dashed line) would increase potency by making favor-
able hydrophobic interactions with Ile216 while limiting molecu-
lar conformations. Previous work determined that substitution at
the 4-position of the pyrrole was tolerated3 without any unfavor-
able conformational perturbation of the neighboring hydroxamate
group.
Val127
The key binding features of a representative compound (1) from
the initial pyrrole series3 in the MEK allosteric site are illustrated in
Figure 1.4 Hydrogen bonding interactions are observed between
the hydroxyamate oxygens and Lys97. The exocyclic acetate car-
bonyl forms a hydrogen bond with the backbone NH’s of both
Val211 and Ser212. The iodoaniline moiety5 binds in a lipophilic
pocket, where the iodo group makes an electrostatic interaction
with the backbone carbonyl of Val127.
H
N
Lys97
O
F
I
+
NH3
O
HN
O
Ser212
N
H
N
HO
HN
HN
O
O
It was our goal to improve the in vitro potency and microsomal
stability of compound 1 (IC50 = 18 nM, Colo205 EC50 = 12 nM, RLM
t1/2 = 14 min) by exploring modification at the 4- and 5-positions of
1
O
Ile216
Val211
Ring Fusion
⇑
Corresponding author. Tel.: +1 858 731 3643; fax: +1 858 550 0526.
E-mail address: mark.adams@takedasd.com (M.E. Adams).
Current address: Shanghai Hengrui Pharmaceuticals Co, Ltd, 279 Wenjing Road,
Shanghai 200245, China.
Figure 1. Key interactions of pyrrole compound 1 in the MEK allosteric site.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.026

2412
M. E. Adams et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2411–2414
O
F
O
R1
O
F
Br
F
HN
H
( )n
H2N
O
N
O
O
O
a
NH
b
n = 1,2
O
NH
. HCl
+
R1
O
NH
.HCl
R1
O
n
3a R = I
4a-b
2
3b R = Br
5a R1 = I, n = 2
5b R1 = Br, n = 2
5c R1 = I, n = 1
1 = Br, n = 1
5d
R
F
F
F
F
R1
R1
R1
O
O
O
HN
HN
n
O
HN
HN
d
c
O
e
O
f
O
HO
R
N
H
R
N
N
N
H
N
N
O
O
n
O
O
n
n
7a-d
8-16
17-19
6a-d
Scheme 1. Synthesis of compounds 8–19. Reagents and conditions: (a) EtOAc, reflux, (60–96%); (b) 1-NaHCO3 wash; 2-THF, reflux, (17–24%); (c) Me2SO4, Cs2CO3, DMF, (65–
90%); (d) 1 N NaOH, THF, MeOH, (57–80%); (e) R-ONH2, EDCI/HOBt, Et3N, DMF, (16–50%); (f) 1-TMS-acetylene, PdCl2(dppf), Et3N, CuI, THF; 2-TBAF, THF, (19–80%).
Table 1
Selected data for bicyclic N-methyl pyrrole analogs
F
F
I
R1
O
O
HN
HN
O
R
O
N
H
HO
N
H
N
N
8-19
1
O
( )n
O
R1
n
MEK1 IC50 (nM)
Colo205 EC50 (nM)
A375 EC50 (nM)
HLM/RLM t1/2, (min)
10
11
11
Compd
R
1
8
9
—
—
I
I
I
I
I
I
Br
Br
Br
CCH
CCH
CCH
—
2
2
2
1
1
1
2
2
2
2
2
1
18
14
17
20
8.9
11
54
18
76
52
32
41
36
12
1.0
2.0
2.5
2.0
5.0
9.0
34
53
58
10
14
1.0
2.5
3.0
4.0
7.0
13
31
76
72
15
72
34
36/14
3.7/5.5
22/36
36/26
10/14
64/54
86/54
25/20
76/70
94/85
34/38
>200/>200
107/150
–(CH2)2OH
(S)-CH2CHOHCH2OH
(R)-CH2CHOHCH2OH
–(CH2)2OH
(S)-CH2CHOHCH2OH
(R)-CH2CHOHCH2OH
–(CH2)2OH
(S)-CH2CHOHCH2OH
(R)-CH2CHOHCH2OH
–(CH2)2OH
(R)-CH2CHOHCH2OH
–(CH2)2OH
10
11
12
13
14
15
16
17
18
19
62
17
The bicyclic pyrroles 8–19 were synthesized according to the
general route shown in Scheme 1.6 The phenol displacement of
ethyl 3-imino-3-phenoxypropanoate hydrochloride 2 with a halo-
substituted aniline (3a–b) in refluxing ethyl acetate gave the
imidate HCl salts 4a–b.7 The free base of intermediates 4a–b under-
went a cyclization with either 3-bromocyclohexane-1,2-dione8 or
3-bromocyclopentane-1,2-dione9 in refluxing THF to furnish the
bicyclic core compounds 5a–d. The resulting bicyclic pyrroles were
then subjected to a selective N-methylation with dimethyl sulfate
to give 6a–d, followed by the saponification with aqueous sodium
hydroxide to give the carboxylic acids 7a–d. The subsequent
hydroxamides 8–16 were made via the EDC/HOBt mediated cou-
pling of 7a–d with selected amines in the presence of triethylamine.
Compounds 17–19 were prepared from their respective iodides via
a Sonogashira reaction with trimethylsilyl-acetylene, followed by
TMS-deprotection with tetrabutylammonium fluoride.
(Colo205 EC50 = 1.0 nM and 2.0 nM, respectively); however, micro-
somal stability was reduced (HLM t1/2 < 10 min). To improve the
microsomal stability of 8 and 11 we designed less lipophilic ana-
logs. The diol compounds (9, 10, 12, and 13) showed improved
microsomal stability, albeit with no improvement in potency. As
expected, compounds from the less lipophilic 5,5 series displayed
higher microsomal stability than analogous 5,6 compounds. We
also examined replacements for the iodine on the phenyl ring,
due to its high molecular weight, lipophilic nature, and potential
for metabolic instability. However, both the bromo and acetylene
analogs 14–19 showed no potency advantages in enzymatic assays,
and a 5- to 15-fold loss of activity in the cell assays. The in vitro po-
tency of compound 17, however, is comparable to the initial pyr-
role 1, and has the advantage of being devoid of the iodine moiety.
After successful demonstration of the advantages of the addi-
tional ring fusion in the pyrrole series, a similar strategy was
employed in the context of known pyridone-based inhibitors12 to
develop a series of dihydroindolizinone compounds 27–34 (Table
2). The additional fused semi-saturated ring would occupy similar
The in vitro properties of compounds 8–19 are presented in
Table 1. Both the 5,6 and 5,5-bicyclic analogs of the pyrrole com-
pound
1 (compounds 8 and 11) improved cellular potency

M. E. Adams et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2411–2414
2413
Table 2
Selected data for dihydroindolizinone analogs
F
R1
O
HN
O
R
R2
N
H
N
O
n
R1
R2
n
MEK1 IC50 (nM)
Colo205 EC50 (nM)
A375 EC50 (nM)
HLM/RLM t1/2, (min)
10
11
11
Compd
R
27
28
29
30
31
32
33
34
–(CH2)2OH
–(CH2)2OH
(S)-CH2CHOHCH2OH
(R)-CH2CHOHCH2OH
–(CH2)2OH
–(CH2)2OH
–(CH2)2OH
–(CH2)2OH
I
I
I
I
F
F
F
F
1
2
1
1
1
1
1
1
25
57
94
39
69
29
38
109
1.0
12
44
27
12
5.0
1.0
3.0
3.0
30
139
69
33
13
>200/124
111/41
>200/>200
>200/>200
128/>200
134/62
CCH
F
I
I
I
H
Me
Cl
2.0
5.0
99/40
47/54
MeO
N
O
OH
N
O
Cl
N
O
O
O
O
O
O
b
EtO
EtO
a
d
c
EtO
OEt
EtO
OEt
O
O
21
20
22
23
F
I
F
I
F
I
F
H2N
I
O
Cl
N
O
O
HN
N
O
HN
N
f
t-BuO
NH2
g
O
HN
HO
e
O
O
HO
N
H
HO
t-BuO
N
H
O
O
O
N
O
33
26
24
25
Scheme 2. Synthesis of compound 34. Reagents and conditions: (a) 1-Et3N, MgCl2, 2-NaH, MeI, Benzene, (80%); (b) Et3N, rt, (22%); (c) POCl3, DMAP, reflux, (63%); (d) 1 N
NaOH, THF, MeOH, (95%); (e) LiHMDS, THF, 0 °C, (22%); (f) EDCI, HOBt, Et3N, DMF, (84%); (g) TFA, (92%).
space as the fused ring of the bicyclic pyrrole series, and it could
potentially make favorable hydrophobic interactions with Ile216
and Met219.
Table 3
Rat PK parameters for select compounds17
Compd CL (mL/min/
kg)
Vdss (mL/
kg)
AUC/dose (ng h/mL)(mg/
kg)
F (%)
The dihydroindolizinones 27–34 were made according to the
general route for the synthesis of compound 33 shown in Scheme
2.13,14 Diethyl 3-oxopentandioate 20 formed a bidentate complex
with magnesium chloride in the presence of triethylamine, which
allowed a mono-methylation with sodium hydride and iodometh-
ane to furnish 21.15 The subsequent cyclization was accomplished
by stirring 21 in triethylamine at room temperature for 5 days to
give the bicyclic intermediate 22 in modest yields. Chlorination
with POCl3 gave 23, which was then hydrolyzed to the acid 24 with
1 N NaOH. Next, displacement of the chlorine on 24 with 2-fluoro-
4-iodoaniline added the hydrophobic pocket substituent to pro-
duce 25. The protected sidechain O-(2-(tert-butoxy)ethyl)-hydrox-
ylamine16 was coupled with 25 using typical EDCI/HOBt conditions
to give the intermediate 26, which was deprotected with neat TFA
to give the final compound 33.
The in vitro data of compounds 27–34 are presented in Table 2.
Compound 27 showed excellent cellular potency (Colo205
EC50 = 1.0 nM) and microsomal stability, but expanding the five-
membered fused ring to six-membered ring (28) or changing the
alcohol tail to a diol (29, 30) decreased enzymatic and cellular
activity. At R2, the analogs with F and Me (27, 33) were more po-
tent in cellular assays than the compounds with H or Cl (32, 34).
The pharmacokinetic properties in rats were determined for se-
lected compounds as shown in Table 3. The 5,6-bicyclic pyrrole
1
10
17
19
27
33
52
23.7
5.9
2896
1158
596
144
88
1670
512
262
204
46
12
61
51
21
29
15.6
12.9
21.4
1186
745
971
compound 10 showed moderate clearance with 12% oral bioavail-
ability. This is consistent with poor membrane permeability which
we attributed to the increased hydrogen bond donor count due to
the diol group. In support of this, the bicyclic pyrrole compounds
17 and 19 with one less hydrogen bond donor showed reduced
clearance and concomitant increased oral exposure of 61% and
51%, respectively. The dihydroindolizinone compounds 27 and 33
exhibited moderate exposure, with % F of 21% and 29%, respec-
tively. None of these bicyclic compounds were found to inhibit a
broader panel of kinases or CYP-450s at inhibitor concentrations
up to 10 l
M.18
The pharmacodynamic properties of compounds 17 and 33 in
HT-29 tumors are shown in Figure 2. Compound 17 showed nearly
complete inhibition of phosphorylation of the downstream sub-
strates ERK-1/2 at 30 mpk at 4 h. In comparison, compound 33

2414
M. E. Adams et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2411–2414
2005, 272, 3491; (e) Pratilas, C. A.; Taylor, B. S.; Ye, Q.; Viale, A.; Sander, C.; Solit,
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120.00
100.00
80.00
60.00
40.00
20.00
0.00
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2. (a) Herrera, R.; Sebolt-Leopold, J. S. Trends Mol. Med. 2002, 8, S27; (b) Sebolt-
Leopold, J. S.; Herrera, R. Nat. Rev. Cancer 2004, 4, 937; (c) Montagut, C.;
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4. Protein Data Bank code is 3MBL for the X-ray co-crystal structure of compound
1 in the MEK allosteric site.
5. (a) Barrett, S. D.; Bridges, A. J.; Flamme, C. M.; Kaufam, M.; Doherty, A. M.;
Kennedy, R. M.; Marston, D.; Howard, W. A.; Smith, Y.; Warmus, J. S.; Tecle, H.;
Dudley, D. T.; Saltiel, A. R.; Fergus, J. H.; Delaney, A. M.; Lepage, S.; Leopold, W.
R.; Przybranowski, S. A.; Sebolt-Leopold, J.; Van Becelaere, K. Bioorg. Med. Chem.
Lett. 2008, 18, 6501; (b) Warmus, J. S.; Flamme, C.; Zhang, L. Y.; Barrrett, S.;
Bridges, A.; Kaufman, M.; Tecle, H.; Gowan, R.; Sebolt-Leopold, J.; Leopold, W.;
Merriman, R.; Przybranowski, S.; Valik, H.; Chen, J.; Ohren, J.; Pavlovsky, A.;
Whithead, C.; Ahang, E. Bioorg. Med. Chem. Lett. 2008, 18, 6171.
-
10 mpk 30 mpk 1.0 mpk 3.0 mpk 10 mpk
17 17 33 33 33
Vehicle
Figure 2. Pharmacodynamic data of compounds 17 and 33 in human colon
adenocarcinoma HT-29 tumors at 4 h.
6. Full experimental procedures for compounds 8–19 are contained within the
following patent application: Adams, M. E.; Dong, Q.; Kaldor, S. W.; Stafford, J.
A.; Wallace, M. B. PCT Int. Patent Appl. WO 08/148034, 2008.
7. Danswan, G.; Kennewell, P. D.; Tully, W. R. J. Heterocycl. Chem. 1989, 26, 293.
8. Harmata, M.; Bohnert, G.; Kurti, L.; Barnes, C. Tetrahedron Lett. 2002, 43, 2347.
9. Sha, C.-K.; Ho, W.-Y. J. Chin. Chem. Soc. 1999, 46, 469.
Rx: QD (12-25) PO
1000
10. 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
100
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
Control
showed almost identical IC50 when assayed at 400
lM or 10 lM ATP, only IC50
7/10 CR
results assayed at 400 M ATP were listed. Based on KmATP for MEK1 at 20 lM
l
33 (10 mg/kg)
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
10
15
20
25
11. 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 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 versus 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.
12. (a) Spicer, J. A.; Rewcastle, G. W.; Kaufman, M. D.; Black, S. L.; Plummer, M. S.;
Denny, W. A.; Quin, J.; 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; (b) 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.
Day Post Tumor Implant
Figure 3. HL-60 (NRAS) xenograft response at MTD for compound 33.
gave a p-ERK-1/2 inhibition dose response with nearly complete
4-h biomarker suppression at 3.0 mpk that correlated with com-
pound plasma concentration. An HL-60 xenograft study was
conducted with compound 33 at the maximum tolerable dose
(Fig. 3). Daily oral administration of 10 mpk of 33 induced signifi-
cant tumor regression in the promyelocytic leukemia xenograft
model.
In summary, we designed and synthesized dihydroindolone and
dihydroindolizinone analogs as potent and selective MEK inhibi-
tors. Optimization of the series led to compounds that are orally
bioavailable and efficacious in tumor xenograft models. Further
evaluation of lead compounds as pre-clinical candidates is ongoing.
13. (a) Full experimental procedures for compounds 27–34 are contained within
the following patent application: Adams, M.E.; Dong, Q.; Kaldor, S.W.; Kanouni,
T.; Scorah, N.; Wallace, M.B. PCT Int. Patent Appl. WO 09/064675, 2009.; (b)
Adams, M.E.; Dong, Q.; Kaldor, S.W.; Kanouni, T.; Scorah, N.; Wallace, M. B U.S.
Patent Appl. 09/0124595, 2009.
14. (a) Chikkanna, D.; McCarthy, C.; Moebitz, H.; Pandit, C.; Sistla, R.; Subramanya,
H. U.S. Patent Appl. 09/0275606, 2009.; (b) Waykole, L.M.; Karpinski, P.H. PCT
Int. Patent Appl. WO 11/067348, 2011.
Acknowledgments
The authors thank the following scientists for their valuable
assistance: Melinda Manuel, Victoria A. Feher, Jeffrey A. Stafford,
Stephen W. Kaldor, Patrick Vincent and Keith Wilson.
15. Caliskan, E.; Cameron, D. W.; Griffiths, P. G. Aust. J. Chem. 1999, 52, 1013.
16. Cusack, K.; Salmeron-Garcia, J.-A.; Gordon, T. D.; Barberis, C. E.; Allen, H. J.;
Bischoff, A. K.; Ericsson, A. M.; Friedman, M. M.; George, D. M.; Roth, G. P.;
Talanian, R. V.; Thomas, C.; Wallace, G. A.; Wishart, N.; Yu, Z. PCT Int. Patent
Appl. WO 05/110410, 2005.
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17. Compounds were administered intravenously and orally at 1 and 5 mg/kg,
respectively.
18. 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.