Identification of isothiazole-4-carboxamidines derivatives as a novel class of allosteric MEK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.048

The development of potent, orally bioavailable, and selective series of 5-amino-3-hydroxy-N(1-hydroxypropane-2-yl)isothiazole-4-carboxamidine inhibitors of MEK1 and MEK-2 kinase is described. Optimization of the carboxamidine and the phenoxyaniline group

Full text of DOI:10.1016/j.bmcl.2006.08.048

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5561–5566
Identification of isothiazole-4-carboxamidines derivatives as a novel
class of allosteric MEK1 inhibitors
Hassan El Abdellaoui,* Chamakura V. N. S. Varaprasad, Dinesh Barawkar,
Subrata Chakravarty, Andreas Maderna, Robert Tam, Huanming Chen,
Matt Allan, Jim Z. Wu, Todd Appleby, Shunqi Yan, Weijian Zhang,
Stanley Lang, Nanhua Yao, Robert Hamatake and Zhi Hong
Drug Discovery, Valeant Pharmaceutical Research & Development, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA
Received 2 June 2006; revised 4 August 2006; accepted 7 August 2006
Available online 24 August 2006
Abstract—The development of potent, orally bioavailable, and selective series of 5-amino-3-hydroxy-N(1-hydroxypropane-2-yl)iso-
thiazole-4-carboxamidine inhibitors of MEK1 and MEK-2 kinase is described. Optimization of the carboxamidine and the phenox-
yaniline group led to the identification of 55 which gave good potency as in vitro MEK1 inhibitors, and good oral exposure in rat.
Ó 2006 Elsevier Ltd. All rights reserved.
There are three mitogen-activated protein (MAP) kinase
pathways that control a variety of cellular regulation.
The c-jun kinase pathway (JNK) is important in the reg-
ulation of many transcription factors particularly in
response to cellular stress. The p-38 pathway plays a
critical role in the activation of inflammatory responses.
The third and, by far, the most important and well-un-
derstood signal transduction pathway is the Ras/Raf/
MEK/ERK pathway that controls fundamental cellular
processes such as proliferation, differentiation, survival,
and apoptosis.1,2 The MAPK pathway represents a cas-
cade of phosphorylation events including three pivotal
kinases: Raf, MEK (MAP kinase/ERK kinase), and
ERK (extracellular signal-regulated kinase). The Ras–
Raf–MEK–ERK pathway is activated by a range of
growth factor receptors. When Raf is activated, it phos-
phorylates MEK at two serine residues. The activated
MEK, in turn, phosphorylates the threonine and tyro-
sine residues of MAPK3 or ERK. ERK regulates down-
stream signaling complexes of transcription factors that
affect gene expression.4 Constitutive activation of the
Ras–Raf–MEK–ERK pathway has been demonstrated
in several cancer types, including pancreatic, colon,
lung, and melanoma. Inhibition of this pathway via
MEK1/2 is an attractive strategy for therapeutic inter-
vention in cancer because it has the potential to block
inappropriate signal transduction regardless of the up-
stream position of the oncogenic aberration. Further-
more, ERK1/2 are the only known substrates for
MEK1/2.5–13 These kinases present new opportunities
for the development of novel anti-cancer drugs designed
to be target-specific and probably less toxic than con-
ventional chemotherapeutic agents. A number of drugs
inhibiting Ras, Raf or MEK are currently under clinical
investigation.14,15 Several highly specific MEK1/2 inhib-
itors such as PD98059, U0126, and CI-1040 have been
identified in recent years. These compounds inhibit the
activation of MEK1/2 non-competitively with respect
to ATP. The crystal structure of human MEK-1/2 com-
plexed with an analog of PD184352 demonstrated that
these biaryl amine-based compounds are bound to an
allosteric site adjacent to ATP binding pocket.14,16,17
Previously, we reported the initial results of SAR study
of the cyanoisothiazole scaffold as a MEK-1/2 inhibi-
tor.18 We disclosed the discovery of the potent and selec-
tive MEK-1 and MEK-2 inhibitor (1), which exhibits an
IC50 value of 38 nM and EC50 value of 375 nM. The
optimization of 1 led to a series of potent MEK-1/2 ki-
nase inhibitors, and culminated in the idenfication of the
compound 5-(4-(2,5-dichlorophenoxy)phenylamino)-3-
Keywords: MEK inhibitor; MEK-1; MEK-2; Isothiazole carboxami-
dine; Allosteric inhibitors.
*
Corresponding author. Tel.: +1 949 231 2216; fax: +1 714 641 7233
(H.E.A.); e-mail: HAbdellaoui@Cox.net
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.048

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H. El Abdellaoui et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5561–5566
hydroxy-N-(2- hydroxypropyl)isothiazole-4-carboximi-
damide (55), that showed excellent oral exposure in
the rat. Several derivatives were synthesized in order
to optimize their drug-like properties, that is, their bio-
logical, physicochemical, and pharmacokinetics profile
(PK). This paper describes the structure-guided design
and structure–activity relationships (SAR) of this series.
6, which were cyclized in the presence of bromine to give
the corresponding isothiazoles 7. The carboxamidines 8
were prepared by refluxing 7 in the presence of excess of
amines.18
Optimization of 8 led to a number of potent, orally
active MEK-1/2 kinase inhibitors. The SAR was focused
on changes at three positions: substitutions at either ring
of the phenoxyaniline (R1 and R2) and substitutions at
the carboxamidine (R3).
H
O
NH
H
O
NH
S
N
N
Cl
H
With regard to SAR of 8 we first examined the optimi-
zation of the linker connecting rings B and C in the para
position of phenylamino isothiazoe carboxamidine ser-
ies. We explored a variety of the linkers as we have high-
lighted in Table 1. Except for the gem-dimethyl-linked
15, which showed a reduction in both MEK enzymatic
activity and cell proliferation, activity was retained for
a variety of linkers.19 Interestingly, this series showed
poor to modest rat PK properties, characterized by high
clearance and low oral bioavailability (see Table 1).
H
H
N
Cl
H
N
Cl
HO
HO
S
N
O
Cl
N
1
55
A series of compounds were generated to investigate the
SAR of the 5-aminoaryl-3-hydroxy-N-alkylisothiazole-
4-carboximidamide scaffold. Their inhibitory activities
against both MEK1 and MEK2 were evaluated and
IC50 values were determined using an enzymatic assay
as described elsewhere.18,19 In most cases, the IC50 val-
ues for MEK1 and MEK2 are similar. For simplicity,
we only report the IC50 values against MEK1 here. A
straightforward five-step synthetic approach is detailed
in Scheme 1. Displacement of the fluorine atom in 4-flu-
oronitrobenzenes 3 by a variety of phenols 2 provided a
series of 4-phenoxynitrobenzene intermediates that were
reduced by Fe in the presence of ammonium chloride to
afford anilines 4. Isothiazoles 7 were prepared from the
corresponding isothiocyanates 5, which were either com-
mercially available or freshly prepared from the corre-
The selectivity profiles of MEK-1 inhibitors 9 and 10
were examined. They were assessed against a panel of
85 protein kinases.19] The data confirmed the generally
good selectivity profile of 9 for MEK-1. The excellent
activity of the compound 10 against CHK-2 was some-
what a surprise (see Table 2). This excellent selectivity
may be a result of non-competitive binding nature of
isothiazole cabroxamidine scaffold.19
To circumvent the low oral bioavailability of the iso-
thiazole cabroxamidine series, HPLC/MS/MS analysis
of 9 incubated with rat liver microsomes indicated more
extensive oxidation of the ring C over ring B. Indeed,
any metabolism in the molecule was suppressed by hal-
ogen substitutions in the rings B and C, and improved
the activities as well as rat PK properties (see Table 3).
sponding anilines 4. The isothiocyanates
5 were
reacted with cyanoacetamide in the presence of pow-
dered KOH in DMF to provide the corresponding 2-
cyano-3-(4-phenoxyphenylamino)-3-thioxopropanamides
R2
R2
NH2
OH
NO2
a
R1
R1
+
O
F
4
2
b
3
CN
H
R2
N
NH2
R2
c
NCS
R1
R1
S
O
O
O
6
5
R2
R2
O
O
N
N
S
S
R1
e
R1
d
OH
OH
N
N
H
H
CN
NH
R3
HN
8
7
Scheme 1. General synthesis of 3-hydroxy-5-(4-phenylamino)isothiazole-4-carboxyamidines. Reagents and conditions: (a) K2CO3, DMF, reflux,
16 h; Fe, MeOH, NH4Cl, reflux, 16 h; (b) CSCl2, aq K2CO3, CHCl3, rt, 2 h; (c) cyanoacetamide, KOH, DMF, rt, 16 h; (d) Br2, EtOAc, rt, 1–2 h; (e)
R3-NH2, EtOH, reflux, 16 h.

H. El Abdellaoui et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5561–5566
5563
Table 1. The linker between the rings B and C: MEK-1 activity and oral-IV AUC of 3-hydroxy-N-(2-hydroxy-1-methyl-ethyl)-5-phenylamino-
isothiazole-4-carboxamidine
HO
NH
HN
HO
H
N
A
B
C
S
N
X
Compound
X
IC50 (nM)
EC50 (nM)
IV AUC (ng h/ml)
Oral AUC (ng h/ml)
9
10
11
12
13
14
15
O
33
240
58
42
45
8321
859
1135
1236
220
NH
CH2
C(O)
S
130
100
650
278
1500
7254
2897
6905
8540
9250
120
45
60
265
SO2
C(CH3)2
150
7900
4251
2350
Table 2. Selectivity profile of 9 and 10 (IC50, nM)
Compound MEK1 Aurora Axl CHK2 Flt3 P70S6
Table 3. MEK-1 activity and oral AUC of 3-hydroxy-N-(2-
hydroxy-1-methyl-ethyl)-5-(4-phenoxy-phenylamino)-isothiazole-4-
carboxamidine
9
10
26
266
260
265
455 142
389 13
795
448
2620
1000
HO
NH
HN
HO
H
1
8
N
9
2
7
A
B
C
S
3
6
R2
N
R1
O
Several analogs of 9 containing halogen substituents on
the ring C were investigated (see Table 3). Initial evalu-
ation of mono-substitution in the para position of the
ring C with chlorine, bromine, methylene, trifluorometh-
yl or any group larger than the methyl group revealed no
improvement in potency, but demonstrated large gains
in oral PK. In contrast, any combination of di-halogen
substitutions in the ring C retains potency, and improves
rat PK properties. However, the substitution by different
halogens in ring B did not contribute to either the poten-
cy or rat PK properties. When substitution of both
phenyl rings B and C was simultaneously attempted,
the PK profile improved considerably, but there was
no improvement in potency. In this series, the com-
pounds 16, 17, 18, 20, 27, 38, and 40 exhibited good oral
AUC exposure in rat and retained most of the activities.
The best compound in this series is the compound 25
that showed an IC50 value of 30 nM, EC50 value of
130 nM, and good oral AUC exposure in rat of
47,400 ng h/ml. Importantly, enzymatic profiling of the
compound 25 shows that it binds also non-competitively
with respect to ATP.19
4
5
Compound Ring-C R2 Ring-B R1 IC50 EC50 Oral AUC
(nM) (nM) (ng h/ml)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
6-CF3
H
H
H
H
H
H
44
58
140 28,654
123 13,003
95 20,395
350 21,867
30 15,391
130 900
6-CF3–5-F
6-CF3–5-Cl
5-Cl–8-CF3
6-F–8-CF3
6-NMe2
120
540
140
160
67
5-Cl–6-CF3 2-Cl
5-Cl–6-CF3 2-F
70 5711
88 4500
52
60
5-F–8-CF3
5-Cl–8-Cl
5-Cl–8-F
5-Cl–8-Cl
5-Cl–8-F
5-Cl–8-F
5-Cl–6-Cl
5-F–6-F
H
330 31,270
130 47,400
260 900
H
30
80
H
2-F
2-Me
2-Cl
H
26
220 22,226
350 nd
>10 nd
280
30
75
20
100 5260
42 9427
H
5-Cl–6-Cl
5-F–6-Cl
6-Cl–8-Cl
6-F–8-F
2-Cl
H
27
>10 4275
200 12,567
1450 11,700
128 5135
250 6256
37 5687
145
20
H
H
207
85
5-Cl–7F
5-Cl–7F
H
Additionally, we turned our attention to optimization of
the alkyl substituent at the carboxamidine moiety. Using
2Cl
H
107
64
5-Cl–7-Cl
5-F–7-F
5Cl–6F–8F
140 26,221
150 16,500
78 20,330
332 14,925
250 12,696
(2,5-dichlorophenoxyamino)-3-hydroxyisothiazole
as
H
207
37
the starting scaffold, we evaluated the influence of car-
boxamidine substituents containing a water-soluble
moiety. Most of the compounds in this series displayed
double digit nanomolar IC50 values in the MEK-1 enzy-
matic assay and double digit nanomolar EC50 values
against EGF-stimulated pERK MDA-MB-231 cancer
cells.19 However, there was strong variation in the oral
PK values with only compounds 51, 52, and 55 exhibit-
ing good oral AUC exposure in rat and retaining most
of the activities (see Table 4).
H
5Cl–6F–8F 2F
5-Cl–7-F 1-Me
27
57
The disclosure of the first MEK X-ray crystallographic
structure with (S)-5-bromo-N-(2,3-dihydroxypropoxy)-
3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide
(60)14 (PDB code: 1S9J) revealed an allosteric binding

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H. El Abdellaoui et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5561–5566
Table 4. MEK-1 activity and oral AUC of 5-[4-(2,5-dichloro-phenoxy)-phenylamino]-3-hydroxy-isothiazole-4-carboxamidine
*
NH
HN
H
N
Cl
A
N
B
HO
C
S
O
Cl
Compound
X
IC50 (nM)
32
EC50 (2 M)
47
Oral AUC (ng h/ml)
2876
43
O
N
N
*
*
O
44
45
40
35
617
O
147
284
6862
*
*
N
N
N
46
47
48
34
137
298
113
118
605
1979
262
N
N
*
*
N
2422
N
NH
NH
49
50
58
44
43
49
nd
87
N
N
*
*
O
OH
51
52
46
46
47
54
14,557
12,468
*
*
OH
OH
OH
OH
53
54
35
34
120
22
5105
2020
*
*
OH
OH
OH
55
56
57
58
59
28
56
75
444
564
694
65
23,908
nd
*
*
O
OH
OH
744
40
22,370
14,568
425
OH
*
OH
O
*
*
34

H. El Abdellaoui et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5561–5566
5565
pocket adjacent to the ATP-binding site, providing a
possible platform for the design of ATP non-competi-
tive, selective inhibitors.
OH
OH
O
HN
O
F
H
N
B
A
Br
F
I
F
60
On the assumption that the isothiazoles bind in the same
pocket as hydroxamate 60, an initial working hypothesis
for the equivalency of the hydroxamate and the isothiaz-
ole series was envisaged. Thus, ring A of 60 was consid-
ered the isosteric equivalent of the isothiazole ring and
ring B of 60 that of the attached aminoaryl moiety. In
the crystallographic structure, the B ring of 60 is found
ensconced in a largely hydrophobic environment, with a
face-to-edge interaction with Phe209 of the DFG motif.
Hydrogen bonds form a critical feature of the interac-
tion of 60 with the protein. One of the F atoms in the
A ring forms a hydrogen bond with the backbone NH
of Ser212, the hydroxamate oxygens form a hydrogen-
bonding network with Lys97 and a structural water,
while the diol oxygens interact with Lys97 and one of
the terminal phosphate oxygens.
Figure 1. Overlay of the docked model of 61 (gold carbons) with 60
(gray carbons).
substitution meta to the iodo group enhanced activity
10-fold. Encouragingly, very similar SAR patterns were
observed for the corresponding isothiazole series (see
Table 5). The iodo analog 61 showed submicromolar
activity, while over 10-fold increase in activity was ob-
served when the R1 position was occupied by either a
methyl 62 or a chloro 63 group. Moving the chlorine
in 61 to the R2 position 64 resulted in a 5-fold abroga-
tion of activity.
When the iodo group of 61 is replaced with an aryloxy
moiety, the docking model fails to provide an equivalent
binding pose. It could be speculated that either the aryl-
oxy group is accommodated through induced fit, or that
these compounds are binding at an alternate binding
site. The poor EC50 values for 61–64 were difficult to ex-
plain, but may have been caused by differences in the
cellular uptake of this series of the compounds com-
pared to the aryloxy analogs.
The docked structure of 61, derived using GOLD v. 3.0
software, shows the isothiazole ring to be somewhat off-
set compared to ring A of 60 (Fig. 1). In this orientation,
the nitrogen atom in the isothiazole captures the impor-
tant hydrogen-bonding interaction with Ser212. This
mode of overlay disposes the amidinoalkyl sidechain
in a completely different trajectory compared to the
alkylhydroxamate sidechain, obviating any need to ex-
plain the overlap of non-isoelectronic groups, that is,
the amidine and the hydroxamate groups. However,
no evidence of significant interactions for the amidino
group was discovered in this orientation that would
compensate for the hydrogen-bond network associated
with the hydroxamate sidechain of 60. This overlay sug-
gests that 61 may share the same allosteric binding pock-
et as 60 with an overlap of certain pharmacophoric
features only.
In order to obtain more concrete evidence, X-ray crys-
tallographic experiments were attempted on several iso-
thiazoles. A combination of instability and insolubility
of the isothiazoles in the crystallization buffer conditions
prevented further work in this area. These problems
may also help explain the poor cellular activity obtained
for these compounds. Nevertheless, this computer
Table 5. MEK-1 activity of 3-hydroxy-5-(4-iodophenylamino)-N-iso-
propylisothiazole-4-carboximidamide
Availability of SAR data for a series of 2-anilinobenzo-
ates,14 a precursor series to the hydroxamates represent-
ed by 60, provided an opportunity to test the credibility
of our working hypothesis. In the 2-anilinobenzoate ser-
ies, activity was observed after introducing an iodine
atom in the para position to the nitrogen in ring B. In
a slightly modified series of 2-(4-substituted anilino)-5-
nitrobenzoates, the iodo substitution was associated
with a 15-fold better enzymatic activity than the bromo
substitution. Phenyl, alkyl, alkoxy and thioalkyl substi-
tutions at that position rendered the compounds inac-
NH
R1
HN
H
N
R2
I
HO
S
N
61
Compound
R1
R2
IC50 (nM)
EC50 (nM)
61
62
63
64
H
H
H
H
Cl
510
62
8321
1480
Me
Cl
H
42
200
>10,000
6190
tive. In addition, placing
a
chloro or methyl

5566
H. El Abdellaoui et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5561–5566
10. Zhang, N.; Wu, B.; Eudy, N.; Wang, Y.; Ye, F.; Powell,
D.; Wissner, A.; Feldberg, L. R. Bioorg. Med. Chem. Lett.
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ole compounds could bind to the same allosteric pocket
that a PD184352 analog binds and act as an allosteric
inhibitor for MEK1 and MEK2. The ideas obtained
from this study did, however, provide an excellent plat-
form for the design of alternate, non-labile scaffolds to
be discussed in future publications.
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2004041811, 2004.
In conclusion, we have identified a series of 3-hydro-
xy-4-carboxyalkylamidino-5-arylamino isothiazole car-
boxamidines as a novel class of allosteric MEK-1/2
inhibitors. The present systematic SAR studies of 5-phe-
nylamino-4-cyano-3-hydroxy-isothiazole led to im-
provement in both enzymatic and cellular assays, and
showed promising oral bioavailability.
14. (a) Ohren, J. F.; Chen, H.; Pavlosky, A.; Whitehead,
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The authors thank colleagues from the Drug Develop-
ment Department & PK Group of Dr. Chin-Chung
Lin, the Toxicology Group of Dr. Domenico Vitarella,
the Analytical Group of Dr. Jingfan Huang, and the
HTS Group of Dr. Paul Diaz.
Supplementary data
Experimental details on biological data. Supplementary
data associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2006.08.048.
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19. For IC50 assay, EC50 assay, kinase profiling, and kinetics,
please see Supplementary data.