Beyond the MEK-pocket: Can current MEK kinase inhibitors be utilized to synthesize novel type III NCKIs? Does the MEK-pocket exist in kinases other than MEK?

Article abstract of DOI:10.1016/j.bmcl.2008.10.108

An approach and preliminary results for utilizing legacy MEK inhibitors as templates for a reiterative structural based design and synthesis of novel, type III NCKIs (non-classical kinase inhibitors) is described. Evidence is provided that the MEK-pocket

Full text of DOI:10.1016/j.bmcl.2008.10.108

Bioorganic & Medicinal Chemistry Letters 19 (2009) 226–229
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Beyond the MEK-pocket: Can current MEK kinase inhibitors be utilized
to synthesize novel type III NCKIs? Does the MEK-pocket exist in kinases
other than MEK?
a
a
a
a
a
Haile Tecle a, , Jianxing Shao , Yanhong Li , Michael Kothe , Steven Kazmirski , Julie Penzotti ,
Yuan-Hua Ding a, Jeffrey Ohren b, Deb Moshinsky a, Rocco Coli a, Nidhi Jhawar a, Emilia Bora a,
Suzanne Jacques-O’Hagan a, Joe Wu a
*
a Pfizer Global Research and Development, Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA
b Pfizer Global Research and Development, MS 8118W-330 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:
Received 9 July 2008
Revised 23 October 2008
Accepted 27 October 2008
Available online 31 October 2008
An approach and preliminary results for utilizing legacy MEK inhibitors as templates for a reiterative
structural based design and synthesis of novel, type III NCKIs (non-classical kinase inhibitors) is
described. Evidence is provided that the MEK-pocket or pockets closely related to it may exist in kinases
other than MEK.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Type III NCKIs
MEK
CI-1040, PD-0325901 and related analogs (Table 1) are potent
and exquisitely selective MEK kinase inhibitors. CI-1040 and PD-
0325901 have advanced to the clinic.1–5 The inhibitors are non-
ATP competitive and bind to a unique pocket (designated hence
forth as the M-pocket) adjacent to, but distinct from, the ATP bind-
ing pocket.
binders is very crowded.8 There are 13 approved drugs targeting
kinases and more than 150 in clinical development.9 Some
improvement in selectivity is achieved in DFG-out-pocket inhibi-
tors, for example, Gleevec and Sorafenib.9–12 The number of inhib-
itors that bind to the DFG-out conformation of kinases is rapidly
increasing and the DFG-out pocket approach is no more novel. It
is thus an opportune time to explore, and exploit the potential util-
ity of the M-pocket to design novel inhibitors for kinases other
than MEK. Greater selectivity could herald use of kinase inhibitors
in disease areas other than oncology, for example, Alzheimer dis-
ease, inflammation, etc.
A detailed description of the M-pocket has been reported for an
X-ray co-crystal structure of a ternary complex of PD 0318088, Mg-
ATP and MEK6,7 (PDB ID 1S9J); co-crystal structure of PD 0316684
(PDB ID 3DY7) and PD-0335676 with MEK are shown in Figure 1.
To our knowledge the MEK X-ray structures are the only of their
kind where substrate (ATP) and inhibitor (e.g., PD 0316684) coexist
side by side. This finding raises the question: can MEK inhibitors,
such as PD 0316684, be employed as anchors to which appropriate
linkers/side chains may be attached at the 5 or 6 positions for
interactions beyond the MEK-pocket with amino acid residues un-
ique to a particular kinase or amino acid residues conserved among
kinases other than MEK. Such side chains/interactions in addition
to structural changes in the anchor would then be the basis for a
generalized strategy for designing and preparing non-ATP compet-
itive, type III, kinase inhibitors (Scheme 1).
To test the proposed strategy, we prepared 5-substituted analogs
10a–10e including PF-04622664 (10a),13 via previously described
procedures13,14 as outlined in Schemes 1 and 2. We were able to
get crystals of a ternary complex of PF-04622664, Mg-ATP and
Table 1
Structures of potent legacy Pfizer MEK inhibitors.
Structure
X
Y
R1
1
CI-1040
Cl
F
F
H
H
cPrCH2ONH–
HOCH2CH(OH)CH2ONH–
R
O
PD 0325901
PD 318088
PD 0316684
PD 0188563 CH3
PD 169842 CH3
X
The ATP-pocket is highly conserved among kinases; conse-
quently, the design of selective kinase inhibitors that utilize the
ATP-pocket is very challenging, besides the IP space for ATP-pocket
H
N
Br HOCH2CH(OH)CH2ONH–
F
H
H
H
H
H
HOCH2CH2ONH–
HONH–
HO–
HONH–
HO–
Y
F
I
PD 0335676 Cl
PD 0184264 Cl
* Corresponding author. Tel.: +1 617 551 3026.
E-mail address: haile.tecle@pfizer.com (H. Tecle).
F
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.108

H. Tecle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 226–229
227
Figure 1. Binding modes of PD 0316684 (A) and PD 0335676 (B) in the MEK1 kinase domain. Compound and ATP are shown as yellow sticks. MEK structural features
highlighted in (A) are: glycine-rich loop (blue), alpha C helix (green), activation loop in inactive conformation (red) and catalytic loop (orange). Selected MEK residues are
shown as cyan sticks.
H
N
H
N
R
O
HO
O
HO
O
O
F
O
F
H
N
H
N
A
B
A
B
F
I
F
I
Linker
Linker
Hook
Hook
Anchor
F
F
Furthr modifications on rings A and B
to optimize, potency, selectivity,efficacy,
PK/PD and sefety profiles
Structural biology directed
Optimization of SAR
Scheme 1. Overall chemistry strategy.
HO
O
O
O
O
O
F
F
F
H
N
H
N
H
N
F
F
a, b, c, d
i, j
e, f, g
h
6a, 6b
HO
TBDPSO
Br
H2N
F
I
F
I
F
I
O
F
1
F
5
F
8
F
9
Scheme 2. Synthesis of key intermediates 8 and 9. Reagents and conditions: (a) LiBH4, THSCl, THF (92%); (b) TBDPSCl, CH2Cl2 (84%); (c) s-BuLi, CO2 (86%); (d) 2-fluoro-4-
iodobenzenamine, n-BuLi, HMDS, THF (67%); (e) (Boc)2O, TEA, CH2Cl2 (87%); (f) TBAF/THF (88%); (g) PBr3, BaCO3 (used without purification); (h) NH3/MeOH (55%); (i) 1-
(bromomethyl)-4-(methylsulfonyl)benzene for 6a or RO2H, HATU for 6b; (j) Me3SiOK (15–30%).
H
HO
O
N
O
O
F
H
N
O
O
S
N
Phosphate
N
F
I
H
F
H3C
PF-04679310
Figure 2. Crystal structure of the ternary complex of MEK with ATP and PF-04622664. Compound and ATP are shown as yellow sticks. The proximity of the linker to R234 is
shown as a dashed line with the distance indicated. A schematic of the interactions between compound and protein is shown to the right.

228
H. Tecle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 226–229
0
MEK (PDB ID 3DV3). The crystals diffracted at a resolution of 2.4 ÅA
(Fig. 2). PF-04622664 occupies the same M-pocket as that occupied
by the legacy MEK inhibitors PD 0316684, PD-0318088 and PD-
189 is not in position to interact with the side chain in this structure.
However, previous, in-house generated crystal structures have
shown the guanidine moiety of ARG 189 in alternate conformations
that would interact with appropriately functionalized side chain
such as the sulfonyl group of PF-04679310 (Fig. 3).
Alternatively the side chain of PF-04622664 may be directed to
form a strong interaction with Asp 190. Such interactions could
provide the rational for the design and preparation of inhibitors
against multiple kinases. Selectivity among kinases would arise
from intra-kinase structural differences and or structural modifica-
tions to the anchor and side chain of the inhibitor. The 5-substi-
tuted hydroxamates, 10a–10e, were tested for inhibitory potency
against a proprietary panel of 54 kinases (the KSS); the compounds
show modest propensity to inhibit kinase other than MEK
(Table 2).
0335676. The side chain is on a trajectory to interact with Arg 234
0
and Asp 190 and the terminal OH group of the side chain is 4.8 ÅA
away from Arg 234. An appropriately placed anionic side chain ter-
minus could make a strong salt bridge with the guanidino moiety
of Arg 234. Arg 234 is conserved in 11 kinases: ANKRD3, PIK3R4,
NEK6, NEK7, MKK3, MKK4, MKK5, MKK6, MKK7a, MKK1 and
MKK2. A strong interaction between the side chain and Arg 234
could, thus, potentially lead to inhibitors selective for all or some
of the aforementioned 11 kinases. It is worth noting that Arg 234 is
at the surface and fairly solvent exposed. A nearby Arg, Arg 189, is
conserved in a large number of kinases and may be a better target
for interaction with the side chain. The guanidino moiety of Arg
Figure 3. Model of PF-04679310 in the binding site of the MEK/PD 0316684 complex, highlighting proposed interactions between the sulfonyl of the ligand and the side
chains of R189 and R234 (dashes). Side chains for selected residues are shown as cyan and gray sticks for the PD 0316684 structure. R189 from the PF-04622664 structure is
shown as yellow sticks to highlight the different orientations of this residue in the two structures. A phosphate ion that is observed in some of the MEK structures we
determined and that occupies a space close to the sulfonyl moiety of PF-04679310 is overlaid. The model was obtained by overlaying the phenyl ring of PF-04679310 onto the
phenyl ring of PD 0316884 from the co-crystal structure, followed by protein–ligand minimization using a proprietary version of the AMBER* force field, keeping the protein
rigid and applying GB/SA solvent model with a non-bond cutoff of 14 Å.
Table 2
Binding affinity of select 5-substituted MEK inhibitors against the KSS panel of kinases (results shown for 5 out 54 kinases): % inhibition at 10 lM.
H
HO
N
O
O
F
H
N
R2
F
I
F
Compound
PD 0316684
10A
R2
H
SGK
0
GSK3
0
PAK4
10
EphA2
p38
5
TRKA
14
MK2
AURA
PKC
9
NEK
2
9
9
5
11
2
O
O
0
25
22
13
5
27
0
13
HO
HO
CH2
N
10b
10c
0
0
5
0
0
9
7
1
0
0
2
1
5
0
1
0
0
CH
O
16
31
10
N
MeO
HO
N
N
CH
H
H
N
O
CH
10d
10e
39
60
22
36
33
28
37
22
4
1
0
18
28
29
21
25
27
25
20
O
O
O
N
19
O
CH
HO

H. Tecle et al. / Bioorg. Med. Chem. Lett. 19 (2009) 226–229
229
Table 3
Binding affinity of 5-substituted carboxylic acid intermediates against the KSS panel of kinases (results shown for 15 out 54 kinases): IC50
(lM).
HO
O
F
H
N
R2
F
I
F
Compound R2
SRC
>30
ABL
>30
LCK
FGTR1 ECK AurA VeGFR-2 MAP3K9 BTK CLK1 TRAK-A PKCbII MST2 P38
O
S
6a
>30
>30
>30
>30
1
18
5
>30 >30
2
>30
>30
>30
H
N
O
CH
2
O
H
N
H2N
6b
6c
6d
4
>30
>30
2
>30
3
2
3
4
>30
3
>30
4
>30 >30
13
20
5
2
>30
>30
6
>30
>30
>30
5
CH2
O
NH2
O
N
0.1
0.3
0.5
7
15
3
O
N
H
CH
HO
Cl
O
O
S
O
N
3
4
7
>30
>30
>30
7
>30
10
N
H
CH
H
H
HO
O
HO
N
O
HO
O
HO
N
O
F
O
F
F
O
F
H
N
H
N
H
N
H
F
F
N
f, g
e
h
a, b, c,d
O
O
O
F
I
F
I
F
I
F
I
F
11
F
F
F
15
O
F
17
i
i
k
10b - 10d
6c, 6d
10a
Scheme 3. Synthesis of key intermediates 16 and 17. Reagents and conditions: (a) n-BuLi/I2 (51%); (b) LiTMP/CO2 (83%); (c) Pd2(dba)3, tributyl-(vinyl)-stannane (65%); (d) 2-
fluoro-4-iodobenzenamine, LDA, THF ꢀ78 °C–rt (35%); (e) OsO4/NaIO4 (76%); (f) EDCl/Hunig’s base/HOBt/tBu(Ph)2SiOCH2CH2ONH2 (55%); (g) TBAF (88%); (h) HOCH2CH2OH,
TsOH (77%); (i) H2NR (15–55%); (k) NaBH4 (43%).
2. LoRusso, P. M.; Adjei, A. A.; Varterasian, M.; Gadgeel, S.; Reid, J.; Mitchell, D. Y.;
Considering that legacy carboxylic acid analogs, PD-0169842
Hanson, L.; DeLuca, P.; Bruzek, L.; Piens, J.; Asbury, P.; Van Becelaere, K.;
Herrera, R.; Sebolt-Leopold, J.; Meyer, M. B. J. Clin. Oncol. 2005, 23, 5281.
3. Allen, L. F.; Sebolt-Leopold, J.; Meyer, M. B. Semin. Oncol. 2003, 30, 105.
and PD-0184264, are also potent inhibitors of MEK (e.g., PD
0169842, IC50 = 11 nM and PD-0184264 = IC50 = 5.73 nM)8,9 the 5-
4. Rinehart, J.; Adjei, A. A.; LoRusso, P. M.; Waterhouse, D.; Hecht, J. R.; Natale, R.
substituted carboxylic acid analogs, 6a–6d, were also submitted
to the KSS (Table 3). Interestingly a robust affinity for several ki-
B.; Hamid, Oday; Varterasian, Mary; Asbury, Peggy; Kaldjian, Eric P.; Stephen,
Gulyas; Mitchell, D. Y.; Herrera, R.; Sebolt-Leopold, J. S.; Meyer, M. B. J. Clin.
Oncol. 2004, 22, 4456.
nases, with IC50s ranging from 100 nM to 7
(Scheme 3).
lM were observed
5. Wang, D.; Boerner, S. A.; Winkler, J. D.; LoRusso, P. M. Biochim. Biophys. Acta:
Mol. Cell Res. 2007, 1773, 1248. and references therein.
6. Ohren, J. F.; Chen, H.; Pavlovsky, A.; Whitehead, C.; Zhang, E.; Kuffa, P.; Yan, C.;
McConnell, Patrick; Spessard, Cindy; Banotai, Craig; Mueller, W. Thomas;
Delaney, Amy; Omer, C.; Sebolt-Leopold, J.; Dudley, D. T.; Leung, I. K.; Flamme,
C.; Warmus, J.; Kaufman, M.; Barrett, S.; Tecle, H.; Hasemann, C. A. Nat. Struct.
Mol. Biol. 2004, 11, 1192.
7. Smith, C. K.; Windsor, W. T. Biochemistry 2007, 46, 1358.
8. A cursory examination of the US Patent Collection db shows a list of 3691
patents on kinase inhibitors.
Taken together, the data collected and outlined in this manu-
script is suggestive that the M-pocket or pockets closely related
to it may be present in kinases other than MEK. The results are also
suggestive that our legacy MEK inhibitors could serve as prime
templates for a reiterative structural based design and synthesis
of novel, type III NCKIs (non-classical kinase inhibitors).
9. Alton, G. R.; Lunney, E. A. Expert Opin. Drug Discov. 2008, 3, 595.
10. Okram, B.; Nagle, A.; Adrian, F. J.; Lee, C.; Ren, P.; Wang, X.; Sim, T.; Xie, Y.;
Wang, X.; Xia, G.; Spraggon, G.; Warmuth, M.; Liu, Y.; Gray, N. S. Chem. Biol.
(Camb.) 2006, 13, 779.
11. Thaimattam, R.; Banerjee, R.; Miglani, R.; Iqbal, J. Curr. Pharm. Des. 2007, 13,
2751. and references therein.
Acknowledgment
The authors wish to thank Dr. Theresa Johnson for modeling the
binding mode of PF-04679310 within the MEK-pocket.
12. Kiselyov, A.; Balakin, K. V.; Tkachenko, S. E.; Savchuk, N. P. Mini-Rev. Med. Chem.
2006, 6, 711. and references therein.
13. Isshiki, Y.; Kohchi, Y.; Mizuguchi, E.; Iikura, H.; Matsubara, Y.; Tsujii, S.;
Shimma, N.; Miwa, M.; Aida, S.; Kohchi, M.; Murata, T.; Aso, K. WO 2006011466
A1.
References and notes
1. Sebolt-Leopold, J. S. Curr. Pharm. Des. 2004, 10, 1907. and references therein.
14. Biwersi, C.; Tecle, H.; Warmus, J. S. WO 2001068619 A1.