Identification of 5,6-substituted 4-aminothieno[2,3-d]pyrimidines as LIMK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.07.050

4-Aminobenzothieno[3,2-d]pyrimidines were previously identified in a high throughput screening campaign as LIMK1 inhibitors. Scaffold reversal led to the identification of a series of simple 5,6-substituted 4-aminothieno[2,3-d] pyrimidines with low micromolar inhibition of LIMK1.

Full text of DOI:10.1016/j.bmcl.2011.07.050

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5992–5994
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 5,6-substituted 4-aminothieno[2,3-d]pyrimidines
as LIMK1 inhibitors
⇑
Brad E. Sleebs, George Nikolakopoulos, Ian P. Street, Hendrik Falk, Jonathan B. Baell
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
Cancer Therapeutics-CRC P/L, 4 Research Ave., La Trobe R&D Park, Bundoora, Victoria 3086, Australia
Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Aminobenzothieno[3,2-d]pyrimidines were previously identified in a high throughput screening cam-
paign as LIMK1 inhibitors. Scaffold reversal led to the identification of a series of simple 5,6-substituted
4-aminothieno[2,3-d]pyrimidines with low micromolar inhibition of LIMK1.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 May 2011
Revised 11 July 2011
Accepted 13 July 2011
Available online 23 July 2011
Keywords:
Kinase inhibitors
LIMK1 inhibitors
LIM-1 kinase
The LIMK family of proteins is comprised of two family mem-
bers LIMK1 and LIMK2. These kinases are recognised by two com-
mon zinc finger LIM motifs, a PDZ and a catalytic domain. The
cysteine rich LIM domains are highly conserved and the catalytic
domains share 70% homology.1
LIMK proteins are mediators of actin polymerisation and micro-
tubule disassembly. LIMK 1 and 2 are actin binding kinases that
phosphorylate cofilin and control the balance of polymerization
of globular actin into filamentous actin. Deregulation of this path-
way is governed by protein phosphatase Slingshot homolog 1
(SSH1) that de-phosphorylates cofilin. The finite balance of LIMK
and SSH1 within microenvironments in the cell determines the
reorganisation of the filamentous actin cytoskeleton, and regula-
tion of cell morphology and motility.2
Actin cytoskeleton dynamics has been associated with highly
metastatic and invasive tumor cells. Studies have found that LIMK1
is highly expressed in a number of malignant tumor cell lines, and
has been shown to play a critical role in metastasis. These studies
have led to the belief that disruption of the dynamic equilibrium of
phospho and non-phospho cofilin will result in reduced metastasis
and invasiveness. Hence the development of a small molecule
inhibitor of LIMK1, will not only provide insights into the precise
role of LIMK1, but may provide a novel approach to cancer therapy.
There is good evidence to suggest the LIMK1 is a viable thera-
peutic cancer target. However, only a few small molecule inhibi-
tors of LIMK 1 and 2 have been described in literature. Bristol
Myers Squibb (BMS) disclosed two classes of inhibitor, pyrazolo
series and a 5-thiazolopyrimidine series.3,4 Some of the pyrazolo
series of inhibitors were shown to be potently cytotoxic but it
was determined that this was due to off-target (anti-microtubule)
activity and that LIMK inhibition did not result in inhibition of cel-
lular proliferation.3 As BMS were not interested in an anti-meta-
static program, development of this series was halted. In an
independent study, Scott et al.5 utilised the BMS pyrazolo inhibitor
in a number of invasive functional assays. In this study it was ob-
served that although motility was not affected by inhibition of
LIMK in cancer associated fibroblasts, the inhibitor did disrupt
invadopodia formation, impairing the ability of path generation
by leading cells in collective invasion.
Lexicon pharmaceuticals are the only other organization to dis-
close LIMK1 and 2 inhibitors; however, these compounds are de-
scribed for treatment of ocular hypertension and glaucoma.6–8
Results from this study are ongoing.
More recently we described aminobenzothieno[3,2-d]pyrimi-
dines as potent inhibitors of LIMK1.9,10 Our primary focus in this
campaign was to develop a LIMK1 inhibitor as a potential meta-
static therapeutic. The series originated from a high throughput
screen of a diverse 40000 compound library, whereby a 3-amino-
thieno[2,3b]pyridine-2-carboxamide 1 scaffold was identified and
transformed into a 4-aminopyridine 3 with concomitant increase
in potency (Fig. 1 and Table 1). Through other iterations it was
found that the pyridyl nitrogen atom could be replaced with car-
bon (2 and 4) and further that the thiophene sulfur atom could
be replace with a nitrogen atom (5). Further substitutions around
the aminobenzothieno[3,2-d]pyrimidine core revealed that the
⇑
Corresponding author.
E-mail address: jbaell@wehi.edu.au (J.B. Baell).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.050

B. E. Sleebs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5992–5994
5993
Figure 1. Previous progression summary of LIMK1 inhibitors.
Table 1
LIMK1 inhibition of compounds 1–7
Scheme 1. Synthesis of compounds 12–15. Reagents and conditions: (a) ethyl 2-
cyanoacetate, Et2NH, S8, EtOH, 25 °C; (b) formamide, formamidine acetate, 160 °C;
(c) POCl3, 90 °C; (d) aq NH4OH, dioxane, 100 °C, lW.
a
Compound
IC50
,
lM
Compound
IC50
4
, lM
1
2
3
4
37
33
9
5
6
7
0.30
0.26
Table 2
LIMK1 inhibition
a,b
of early compounds 12–15
7
#
Structure
IC50
#
Structure
IC50
4.0
a
KinaseGlo assay format9,10
12
4.4
13
7–phenyl compounds 6 and 7 gave a marked increase in potency
and could accommodate solubilizing groups to improve physico-
chemical properties.9
14
5.3
15
2.5
Herein we report our efforts on scaffold hopping to give 4-ami-
nothieno[2,3-d] pyrimidines 12–15 (Fig. 2). Investigation of the ret-
ro synthetic route of such a core revealed the synthon was a 2-
aminothiophene. 2-Aminothiophenes are classically constructed
utilizing the multi-component Gewald reaction. The Gewald reac-
tion consists of a base mediated reaction of typically using 2-cya-
noacetate, sulfur and a ketone (Scheme 1). Given that there are a
vast number of ketones readily available, this would allow a simple
point of variation to the initial scaffold to probe SAR.
aKinaseGlo and TR-FRET assay formats9,10
.
bIC50 values are
lM.
Table 3
LIMK1 inhibition
a,b
of 2-substituted compounds 16–24
#
Structure
IC50
#
Structure
IC50
To evaluate the proposal a 5,6-six membered carbocycle 4-ami-
nothieno[2,3-d] pyrimidine 13 was synthesized. The Gewald reac-
tion utilising ethyl 2-cyanoacetate allowed high yielding access to
the intermediate 2-aminothiophene ester 9. The aminoacetate
moiety was then converted to the hydroxypyrimidine 10 using
formamidine acetate at high temperature. Conversion of the
hydroxypyrimidine 10 to the 4-chloropyrimidine 11 was per-
formed using phosphorous oxychloride, and this was subsequently
converted to the 4-aminopyrimdines 12–15 using ammonia in 1,
4-dioxane at 100 °C (Scheme 1).
16
>100
17
>100
>100
>100
18
20
>100
>100
19
21
Compound 13 was evaluated in an 11 point titration to assess
its inhibition of LIMK1 and was shown to possess an IC50 of
4 lM. This activity was comparable to the activity of the amin-
22
24
>100
>100
23
>100
obenzothieno[3,2-d]pyrimidine 4. To further evaluate the thie-
no[2,3-d] pyrimidine scaffold an acyclic, five and seven
membered 5,6-carbocycle were also synthesised and evaluated
for LIMK1 inhibition. The five membered analogue 14 demon-
strated a slight decrease in activity, the acyclic derivative 12 was
similar in activity, while the seven membered analogue 15 dis-
played a slight increase in potency compared with 13 (Table 2).
We then elaborated SAR at the 2 and 4 positions. 2-Substituted
compounds 16–24 were purchased commercially (Table 3), while
the 4-substituted compounds 25–32 (Table 4) were synthesized
utilising the 4-chloro aminothieno[2,3-d] pyrimidine intermediate
with the appropriate amine, analogous to that represented by
ammonium hydroxide in Scheme 1. The results are shown inTables
aKinaseGlo and TR-FRET assay formats.9,10
bIC50 values are
M.
l
3 and 4, and demonstrate the substitution at both the 2 and 4 posi-
tions have a detrimental effect on activity. This led us to believe
that the binding mode of the thieno[2,3-d] pyrimidine compounds
may be similar to the original aminobenzothieno[3,2-d] pyrimidine
series as substituents in equivalent positions also abrogated
activity.9,10
We next focused our attention to the making alterations to the 5
and 6 positions. In order to make the synthesis amenable to pro-
ducing analogues more efficiently, we endeavored to shorten the
initial synthetic route used from the four steps used in Scheme 1
to two steps (Scheme 2). This was made possible by simply using
malonitrile in place of ethyl 2-cyanoacetate. This modification gave
the 2-amino-3-nitrile thiophene 34, which was then transformed
Figure 2. LIMK1 inhibitor scaffold hopping.

5994
B. E. Sleebs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5992–5994
Table 4
LIMK1 inhibition
Table 5
LIMK1 inhibition
a,b
a,b
of 4-substituted compounds 25–32
of 5,6-substituted compounds 35–48
#
Structure
IC50
#
Structure
IC50
#
Structure
IC50
#
Structure
IC50
18
35
1.8
36
25
>100
26
>100
37
39
2.1
3.2
38
40
2.7
11
27
>100
28
>100
29
31
>100
>100
30
32
>100
>100
41
43
45
47
49
51
2.3
42
44c
46
48
50
52
9.1
0.66
3.5
4.3
17
8.6
aKinaseGlo and TR-FRET assay formats.9,10
bIC50 values are
M.
l
2.8
12.7
>20
>20
Scheme 2. Synthesis of compounds 35–48. Reagents and conditions: (a) malonit-
rile, Et2NH, S8, EtOH, 70 °C; (b) formamidine acetate, formamide, 160 °C.
directly into the 4-aminopyrimidine (Scheme 2). However it was
noted that the yields of Gewald reaction using malonitrile, com-
pared with using ethyl 2-cyanoacetate (Scheme 1), were dramati-
cally reduced and more byproducts observed. Using this
synthetic approach analogues 35–52 were produced (Table 5).
Compounds 44 and 45 are mixture of regioisomers as a result of
the lack of regio control from the Gewald reaction. The isomers
were unable to be separated, so were submitted for LIMK1 evalua-
tion as a mixture. Compounds 49–52 were produced from com-
pound 47 either via hydrolysis or aminolysis.
Of the analogues presented in Table 5, it appeared that a num-
ber changes to the 5,6-substitution pattern were tolerated. How-
ever, it appeared any heteroatom substituted into the 5,6-
carbocycle system was slightly detrimental to activity and polar
extensions such as those in 47, 49–52 led to either a slight (52)
or complete (51) loss of activity. Hydrophobicity was clearly pref-
erable for activity and several simple hydrophobic compounds (35,
37–39, 41, 45, 46 and 48) returned single digit micromolar activity.
Indeed, the regioisomeric mixture of compound 44 was the most
potent and returned an IC50 of 660 nM. The regioisomer responsi-
ble for biological activity remains to be determined.
9.5
KinaseGlo and TR-FRET assay formats.9,10
IC50 values are
A mixture of regioisomers.
a
b
c
lM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.050.
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In summary, we have identified 5,6-substituted 4-aminothie-
no[2,3-d] pyrimidines as a promising lead series, which possesses
moderate LIMK inhibition. In particular, compound 44, with a
molecular weight of only 253, exhibits submicromolar LIMK1
inhibitory activity with an IC50 of 0.66 lM and has potential for sig-
nificant optimization of drug-like properties.
Acknowledgments
The authors acknowledge the financial support of the Cancer
Therapeutics CRC, established and supported under the Australian
Government’s Cooperative Research Centres Program; NHMRC IRI-
ISS grant number 361646 and Victorian State Government OIS
grant.