Fused thiophene derivatives as MEK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.10.105

A number of novel fused thiophene derivatives have been prepared and identified as potent inhibitors of MEK. The SAR data of selected examples and the in vivo profiling of compound 13h demonstrates the functional activity of this class of compounds in HT-

Full text of DOI:10.1016/j.bmcl.2011.10.105

Bioorganic & Medicinal Chemistry Letters 22 (2012) 472–475
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fused thiophene derivatives as MEK inhibitors
Victoria E. Laing a, , Daniel C. Brookings a, Rachel J. Carbery a, Jose Gascon Simorte a, Martin C. Hutchings a,
⇑
Barry J. Langham a, Martin A. Lowe a, Rodger A. Allen b, Joanne R. Fetterman c, James Turner a,
Christoph Meier b, Jeff Kennedy b, Mark Merriman b
a Department of Medicinal Chemistry, UCB Pharma, 216 Bath Road, Slough, Berkshire SL1 3WE, UK
b Department of Biology, UCB Pharma, 216 Bath Road, Slough, Berkshire SL1 3WE, UK
c Department of Pharmacology, UCB Pharma, 216 Bath Road, Slough, Berkshire SL1 3WE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of novel fused thiophene derivatives have been prepared and identified as potent inhibitors of
MEK. The SAR data of selected examples and the in vivo profiling of compound 13h demonstrates the
functional activity of this class of compounds in HT-29 PK/PD models.
Received 7 September 2011
Revised 26 October 2011
Accepted 28 October 2011
Available online 6 November 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
MEK
Non-ATP competitive MEK inhibitor
Allosteric MEK inhibitor
The Ras/Raf/MEK/ERK mitogen-activated protein (MAP) kinase
signaling pathway is responsible for the coordination and regula-
tion of cell growth and differentiation in response to extracellular
stimulation.1,2 Raf-activated MAPK/ERK kinase (MEK 1,2 a dual
specificity tyrosine/threonine protein kinase) activates the MAP
kinase known as extracellular signal-regulated kinase (ERK)
through phosphorylation of key tyrosine and threonine residues.3
The Raf/MEK/ERK cascade plays a central role in the regulation of
cell proliferation and survival and this pathway is deregulated in
a high number of human cancers. Inhibition of this signalling cas-
cade is, therefore, an attractive target for small molecule drug
development.4 In particular non-ATP competitive MEK inhibitors
have been reported to exhibit exquisite selectivity, and there are
several compounds advancing into clinical development.5 We
report in this letter our efforts to identify novel, ATP non-compet-
itive, inhibitors of MEK as potential oncology therapeutics.
Pfizer have reported the clinical development of the non-ATP
competitive MEK inhibitor PD0325901, Figure 1.6 Phase I studies
demonstrated significant target engagement with a decrease in
ERK phosphorylation observed in >60% of tumour biopsies col-
lected post treatment. However extended phase I evaluation
showed unacceptable ocular toxicity and a pilot phase II clinical
trial in previously treated patients with non-small cell lung cancer
demonstrated limited efficacy and the trial was consequently
discontinued. During the discovery of PD0325901 Pfizer disclosed
the X-ray co-crystal structure of MEK with the closely related
analogue PD318088.7
The structure provides valuable insight into the mechanism of
non-ATP competitive inhibition of MEK, in particular PD318088
binds in a novel binding pocket separate from, but adjacent to the
Mg-ATP site with the key feature being an electrostatic interaction
between the aryl iodide of PD318088 and the carbonyl of Val127.
Our attempts to identify novel MEK inhibitors began from a de-
sire to utilise proprietary chemical scaffolds developed for other ki-
nase targets.8,9 We initially hypothesised that a thiazole such as 1
Figure 2, could act as a non-ATP competitive MEK inhibitor.
We reasoned that the aryl iodide would form the key electrostatic
interaction with Val127 whilst the carbonyl function would form a
hydrogen bond with the backbone NH of Ser212 analogous to the
4-fluoro in PD318088.11,12 Consequently we were delighted to
discover that upon synthesis of 1 and testing in an in vitro Raf/
MEK/ERK cascade assay10 compound
1 displayed promising
inhibitory activity (IC50 946 nM). To improve both the potency and
physiochemical properties of 1 we targeted the 3-carboxythiophene
OH
H
N
H
N
O
O
O
F
H
N
HO
O
F
H
N
OH
Br
OH
F
I
F
I
F
F
PD 0325901
PD 318088
⇑
Corresponding author.
E-mail address: victoria.jackson@ucb.com (V.E. Laing).
Figure 1. Structures of PD0325901 which entered clinical development and
PD0318088 which was reported in co-crystallization with MEK1, Mg2+ and ATP.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.105

V. E. Laing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 472–475
473
F
inhibition of ERK phosphorylation and the proliferation of HT-29
colon cancer cell lines.
H
N
N
S
Incorporation of the 3-thiophene ester 4 did not improve po-
tency compared to thiazole 1. However the simple primary amide,
6a demonstrated much improved activity in the cascade assay
(13 nM) which translated into potent inhibition of pERK in HT-29
cells (92 nM). However, poor aqueous solubility precluded further
profiling of 6a. Secondary and tertiary amides 6b and 6c improved
solubility but did not increase inhibition in the cascade assay. Polar
polyhydroxylated amide side chains were synthesized and 6d
showed good initial activity in the cascade assay and against pERK
(28 and 43 nM), but this activity was not maintained in the HT-29
proliferation assay. Hydroxamate esters had previously been re-
ported by Pfizer16 to be beneficial in improving cell potency and
solubility and therefore we endeavoured to synthesize hydroxamic
esters in our thiophene series. Pleasingly the dihydroxypropyl
hydroxamate 6f revealed a 20-fold improvement in activity com-
pared to the analogous amide, 6e. However the most potent com-
pounds in the key proliferation assay were found to be
carboxamides containing a basic side chain exemplified by 6g
which demonstrated good activity in the HT-29 proliferation assay
with an IC50 of 113 nM.
I
N
H
O
1
Figure 2. Compound 1, 2-(2-Fluoro-4-iodo-phenylamino)-7,7-dimethyl-5,6,7,
8-tetrahydro-thiazolo[5,4-c]azepin-4-one.
O
O
EtO
EtO
Cl
H
N
NH2
O
O
a
b
S
S
I
N
H
N
H
N
H
O
O
2
3
4
c
R1
O
O
HO
Cl
Cl
H
N
H
N
d
S
S
I
I
N
H
N
H
O
O
Encouraged by the promising initial SAR studies we sought to
improve the synthetic route. In particular we wished to improve
the overall yield and to identify routes which avoided the low
yielding and capricious nucleophilic substitution reaction to intro-
duce the aniline substituent.
6a-g
5
Scheme 1. Reagents and conditions: (a)S8, EtOH, morpholine, ethylcyanoacetate,
45 °C, 51%; (b) Cs2CO3, DMF, 2-Chloro-1-fluoro-4-iodobenzene, 65 °C, 25%; (c) LiOH,
THF, H2O, reflux, 79%; (d) amine R1, EDC, NMM, HOBt, DMF, rt, 40–60%.
Our improved synthetic route is described in Scheme 2. Key to
the synthesis is the reaction between the easily obtained bromo-
dione 7 and thiocarbamoyl intermediate 10. Pleasingly the union
between intermediates 7 and 10 proceeds smoothly in the pres-
ence of caesium carbonate to furnish directly ester 11 in moderate
yield. Hydrolysis of the ester to the acid and subsequent amide for-
mation occur in an identical manner to the previous synthesis.
Alternatively the acid could be reacted with tetrafluorophenol re-
sin and amides synthesized from the resultant resin-bound acti-
vated ester (step g). Importantly the new synthetic route allowed
access to the 2-fluoro substituted anilines which were unobtain-
able by the previous methodology.
Activity data is presented in Table 2. The primary amide 13a
was sixfold more potent than the corresponding chloro analogue
6a in the HT-29 proliferation assay, however aqueous solubility
was still poor. The 3-hydroxypropyl carboxamide 13b was synthe-
sized to bind to the ATP region and demonstrated that moderate
affinity was achieved in the cascade assay. However there was a
20-fold drop in potency when examined in the HT-29 proliferation
assay (IC50 6210 nM). This could have been due to the poor phys-
ico-chemical properties of the compound which resulted in a lack
of cellular permeability. The corresponding ethylhydroxamate 13c
was prepared which improved the permeability of the compound
in the proliferation assay 18-fold compared to the corresponding
amide. It was expected that the hydroxypropyl hydroxamate 13d
analogues 6 (Scheme 1). We hypothesised that substitution at the
thiophene 3 position would allow access to the ATP binding region
offering potential for increased potency and the introduction of po-
lar functionality to improve the physiochemical properties of the
molecules. Therefore, initial efforts were made to synthesize a series
of thiophene-3-carboxamides as described in Scheme 1 with se-
lected SAR data in Table 1.
Gewald13–15 reaction of 6,6-dimethyl-azepane-2,4-dione,
2
gave thiophene intermediate 3 in an acceptable yield (51%). Intro-
duction of the aniline substituent relied on a poor yielding nucleo-
philic displacement of 2-chloro-1-fluoro-4-iodobenzene with 3
(25% yield). Hydrolysis of the ester 4 provided the acid 5 in 79%
yield. The resultant acid could then be simply reacted with a vari-
ety of substituted amines to provide the carboxamides 6a–6g in
yields ranging from 40–60%.
For SAR determination, all compounds were evaluated in a cou-
pled Raf-MEK-ERK IMAP cascade assay.10 Constituently active Raf
phosphorylates and activates MEK which in turn phosphorylates
ERK which phosphorylates a fluorescent labelled Erk-tide sub-
strate, measurement of which provides the basis of the assay. In
this way the activity of the compounds against either active and/
or inactive MEK can be deduced. Compounds showing potency in
the primary assay were further evaluated by measuring both the
Table 1
Exploratory SAR for compounds 1,4 and 6a–g10
Compound
R1
Cascade IC50
HT-29 pERK IC50
HT-29 proliferation IC50
1
4
n/a
O CH2 CH3
NH2
NH CH3
946
2617
13
226
456
28
200
9
18
1539
NT
92
224
45
43
717
76
13
8869
NT
596
1633
1043
710
12110
896
6a
6b
6c
6d
6e
6f
6g
Piperazine
R(-)-NHCH2CHOH(CH2OH)
R(ꢀ)-NH(CH2)2CHOH(CH2OH)
R(ꢀ)-NHOCH2CHOH(CH2OH)
NHCH2C(CH3)2NH2
113
Values are means of two experiments. IC50 is quoted in nM. NT, not tested.

474
V. E. Laing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 472–475
O
O
O
a
Br
N
H
O
O
N
H
EtO
F
H
N
d
7
O
2
S
I
EtO
N
H
O
S
F
9
F
8
S
c
F
b
N
H2N
11
HN
I
I
I
e
10
R1
O
O
HO
F
F
f or g
H
H
N
N
S
S
I
I
N
H
N
H
O
O
12
13a-f
Scheme 2. Reagents and conditions: (a)NBS, NaHSO4, THF, 25 °C, 81%; (b)
Thiophosgene, CHCl3, H2O, 2-fluoro-4-iodoaniline, rt, 88%; (c) Ethylacetoacetate,
NaOEt, EtOH, rt, 87%; (d) Cs2CO3, DMF, 80 °C, 43%; (e) LiOH, THF, H2O, reflux, 79%;
(f) amine R1, EDC, NMM, HOBt, DMF, rt, 40–60%, (g) (i) tetrafluorophenol resin, DMF,
DIC, DMAP, rt, (ii) amine R1, THF, DMF, rt.
Figure 3. Crystal structure of compound 13e bound to MEK-F11 with AMPPNP-Mg2
(PDB code: 3SLS).
Vehicle
Table 2
1500
1000
500
0
200
150
100
50
Exploratory SAR for compounds 11,13a–h10
Compound 13h 50mg/kg
Compounds R1
Cascade
IC50
HT-29
pERK
IC50, nM
HT-29
proliferation
IC50, nM
11
12
13a
13b
13c
13d
O CH2 CH3
OH
NH2
305
14
9
309
7
NT
978
NT
140
18
NT
NT
158
6210
340
1578
NH(CH2)2 CH2OH
NHOCH2CH2OH
R-(ꢀ)-
6
51
0
1
6
NHOCH2CHOH(CH2OH)
(R)-Piperidin-3-
ylamine
18
Hours
13e
13f
22
42
8
5
91
Figure 4. Time dependent reduction of pERK in HT-29 tumour bearing nude mice
after treatment with compound 13h. HT-29 tumour bearing mice were treated with
vehicle (0 h) or 50 mg/kg compound p.o. pERK levels were measured 1, 6, 18 h post
treatment. Drug plasma concentration was measured by LC–MS/MS.
(R)-1-Pyrrolidin-2-yl-
methylamine
(R)-Pyrrolidin-3-
ylamine
22
9
120
54
13g
13h
NHCH2C(CH3)2NH2
14
6
79
Values are means of two experiments. IC50 is quoted in nM. NT, not tested.
1500
1000
500
0
Vehicle
200
150
100
50
Compound 13h 50mg/kg
would further improve the potency relative to the simple carbox-
amide. The hydroxamate 13d was potent in the cascade assay, with
an IC50 of 6 nM, however, the activity greatly diminished in the HT-
29 proliferation assay (IC50 1578 nM). The polar surface area of 13d
was 148 Å, resulting in poor cellular penetration. Incorporation of
basic amides such as piperidine-3-ylamine 13e or 2-methyl-pro-
pane-1,2-diamine 13h led to good activity in the cascade assay
which was maintained through into improved potencies in the
HT-29 proliferation assay, 13h (IC50 79 nM).
A crystal structure of compound 13e bound to MEK was ob-
tained in the presence of AMPPNP-Mg2+ (Fig. 3).17 The compound
binds to MEK through a similar mode to the non-ATP competitive
inhibitor PD318088. The structure reveals an electrostatic interac-
tion between the 40-iodide and backbone carbonyl of Val127 which
is critical to the activity of the MEK inhibitors. Phe209 also forms
an edge to face interaction with the 2-fluoro-4-iodoanilino ring.
The carbonyl of the azepinone hydrogen bonds to the backbone
NH of Val211, and the piperidin-3-ylamine terminal nitrogen inter-
acts with the terminal phosphate of AMP-PNP. Gratifyingly the
crystal structure confirmed our design hypothesis with the aryl
iodide forming the key electrostatic interaction, the carbonyl group
hydrogen bonding to the backbone and the 3-thiophenyl group
interacting with AMP.
**
**
0
1
6
18
Hours
Figure 5. Time dependent reduction of pERK levels in PMA-stimulated whole blood
in HT-29 tumour bearing nude mice after treatment with compound 13h. HT-29
tumour bearing mice were treated with vehicle (0 h) or 50 mg/kg compound p.o.
pERK levels were measured in whole blood 1, 6, 18 h post treatment. Drug plasma
concentration was measured by LC–MS/MS. ⁄⁄p<0.01% versus vehicle control, one-
way ANOVA followed by Bonferroni post test.
Compound 13h was selected for in vivo pharmacokinetic profil-
ing based on its superior potency, good aqueous solubility (210
lM
at pH 7.4) and low in vitro intrinsic clearance 2.6
l
L/min/106 cells
in rat hepatocytes. The in vivo pharmacokinetics of compound
13h were assessed in female nude mice following a single p.o.

V. E. Laing et al. / Bioorg. Med. Chem. Lett. 22 (2012) 472–475
475
administration at 10 mg/kg. Compound 13h had a Cmax of 323 ng/
References and notes
mL and an AUClast of 1239 ng h/mL and based on these data was
progressed to PK/PD studies in nude mice bearing HT-29 tumours.
As seen in Figure 4, treatment with compound 13h, 50 mg/kg
p.o., resulted in inhibition of pERK in the HT-29 tumour. Within
1 h of dosing, pERK was reduced by 25% compared to control and
this reduction increased at 6 h to 40% with pERK levels returning
to control levels at 18 h. Analysis of plasma concentration show
there was more compound present at the 6 h time point than at
1 h, and the compound was cleared by 18 h consistent with a slow
absorption profile. Analysis of pERK inhibition in PMA-stimulated
whole blood (Fig. 5), showed the complete knock down of pErk
at 1 h which was maintained at 6 h, returning to a 35% reduction
of pErk by 18 h. The greater inhibition of pERK in whole blood com-
pared to tumour may be due to poor tumour penetration or the ac-
tion of an efflux mechanism and has yet to be fully elucidated.
In conclusion a novel series of MEK inhibitors have been devel-
oped, and significant enzyme and cellular potency has been
achieved across the series. Compounds with acceptable in vivo
pharmacokinetic properties were identified and examined in a pri-
mary pharmacology model, demonstrating inhibition of pERK in
both tumour and whole blood. Further optimisation and profiling
of these compounds is ongoing.
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Acknowledgments
We thank Caroline Hyden, Reena Halai and Tovah Shaw who
performed the pErk and HT-29 proliferation assays and the DMPK
team for bioanalysis support and in vivo PK.