Inhibition of eEF2-K by thieno[2,3-b]pyridine analogues

Article abstract of DOI:10.1016/j.bmcl.2010.02.005

Several series of thieno[2-3-b]pyridine analogues were synthesized and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF2-K). Modifications around several regions of the lead molecules were made, with a ring fusion adjacent to the nitrogen on the thienopyridine core being critical for activity. The most active compound 34 shows an IC₅₀ of 170 nM against eEF2-K in vitro.

Full text of DOI:10.1016/j.bmcl.2010.02.005

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2283–2286
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of eEF2-K by thieno[2,3-b]pyridine analogues
a
a
b
b
b
Jeffrey W. Lockman a, , Matthew D. Reeder , Kazuyuki Suzuki , Kirill Ostanin , Ryan Hoff , Leena Bhoite ,
*
Harry Austin b, Vijay Baichwal b, J. Adam Willardsen a
a Department of Medicinal Chemistry, Myriad Pharmaceuticals, 305 Chipeta Way, Salt Lake City, UT 84108, United States
b Department of Discovery Biology, Myriad Pharmaceuticals, 305 Chipeta Way, Salt Lake City, UT 84108, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Several series of thieno[2-3-b]pyridine analogues were synthesized and screened for inhibitory activity
against eukaryotic elongation factor-2 kinase (eEF2-K). Modifications around several regions of the lead
molecules were made, with a ring fusion adjacent to the nitrogen on the thienopyridine core being critical
for activity. The most active compound 34 shows an IC50 of 170 nM against eEF2-K in vitro.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 4 January 2010
Revised 28 January 2010
Accepted 1 February 2010
Available online 6 February 2010
Keywords:
Kinase
Thieno[2,3-b]pyridine
eEF2-K
Eukaryotic elongation factor-2 kinase (eEF2-K; EC 2.7.11.20),
also called Ca2+/calmodulin-dependent protein kinase III, is a
member of the alpha kinase family of atypical kinases.1,2 Phosphor-
ylation of elongation factor eEF-2 at T56 and T58, catalyzed by
eEF2-K, leads to decreased translocation of tRNA and mRNA within
the ribosome and transient inhibition of protein synthesis.3,4 The
activity of eEF2-K increases significantly during mitosis, correlat-
ing well with decreased protein synthesis during cell division.5
eEF2-K activity has been shown to be upregulated in numerous
malignant cell lines with a high level of activity seen in MCF7 cells
derived from breast carcinoma.6 Increased activity was also dem-
onstrated in breast carcinoma tissue with little or no activity ob-
served in normal breast tissue. Growth factors including IGF-1
and EGF stimulate the activity of eEF2-K, and cell proliferation in-
duced by these growth factors can be mitigated by eEF2-K
inhibition.
The natural product rottlerin has shown a growth inhibitory ef-
fect and cytotoxicity in glioma cell lines at a concentration coinci-
dent with its inhibition of eEF2-K.7 Rottlerin is known to inhibit
other kinases, notably PKCd, but its cytotoxic timecourse is consis-
tent with its antiproliferative effects being due to inhibition of
eEF2-K and not PKCd. Inhibition of eEF2-K has also been correlated
with antiproliferative effects using a series of 2-methylimidazo-
lium derivatives.8 NH125, the most active of these compounds,
showed IC50 = 60 nM in vitro against eEF2-K and cytotoxicity
against a panel of cancer cell lines with IC50 values of 700 nM to
el, where at 1 mg/kg the mean survival was increased 29% over
vehicle. A series of 1,3-selenazine derivatives have also been syn-
thesized and screened against eEF2-K.9 The most potent of these
compounds (TS-2) showed IC50 = 360 nM and was selective for
eEF2-K over a panel of similar kinases.
A high-throughput screen for eEF2-K inhibition was undertaken
using a fluorometric, coupled-enzyme assay that monitored ADP
release. An alternate assay based on polarization of a fluorescently
labeled peptide substrate was used to validate primary screening
hits.10 These assays led to the identification of one structural class
of inhibitors: thieno[2,3-b]pyridine-containing heterocycles exem-
plified by 1 and 2 (Scheme 1). Similar compounds have been
shown to possess antianaphylatic properties,11 neurotropic activ-
ity,12 and activity against I B kinase-b.13 Compounds 1 and 2
j
showed shifts in their IC50 against eEF2-K as the ATP concentration
in the in vitro assay was increased from 2- to 50-times K M, sug-
gesting that both were ATP-competitive inhibitors of eEF2-K.
The thieno[2,3-b]pyridine cores were divided into two sub-clas-
ses depending on the position of the fused ring around the pyridine
ring: compounds with the fused ring at the 3- and 4-positions rel-
ative to the pyridyl nitrogen such as 1 and those such as 2 with the
N
NH2
D
B
N
B
N
D
NH2
O
C
A
C
3.6 lM. This compound was also assayed in a P388 xenograft mod-
NH2
S
S
N
N
A
O
1
2
* Corresponding author. Tel.: +1 801 214 7885.
E-mail address: jeffrey.lockman@myriadpharma.com (J.W. Lockman).
Scheme 1. Lead molecules. 1, IC50 = 0.22 lM; 2, IC50 = 2.5 lM.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.005

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J. W. Lockman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2283–2286
Table 1
fused ring at the 2- and 3-positions. Both sub-classes were further
divided into four zones (A, B, C, and D) and an SAR was developed
around each zone.
Analogues inspired by 1 were synthesized using variations on a
variety of known routes.14–16 Compounds related to 2 were gener-
ated using a one-pot aldol-heterocyclization approach (Scheme
2).17 Treatment of a ketone with KOH in methanol was followed
In vitro activities of compounds 2–15: zone B aliphatic and phenyl substitution
R
NH2
NH2
S
O
N
by slow addition of an aldehyde, leading to an a–b-unsaturated ke-
Compound
R
IC50 (lM)
tone in situ that was cyclized with 2-cyanothioacetamide in the
presence of NaOMe to generate 2-mercaptopyridine intermediate
3. To generate D-region amino amides 4, 3 was treated with 2-bro-
moacetamide and NaOMe.18 Reaction of 3 with chloroacetonitrile
followed by condensation with formamide yielded D-region 4-
aminopyrimidines 5.19
2
6
7
8
9
10
11
12
13
14
15
H
Me
i-Bu
Cy-Pr
2.5
1.1
0.86
2
rac-CH(CH3)CH2CH2C@CMe2
3,4-Cl2-Ph
4-Cl-Ph
10
0.99
1.1
>20
0.86
1.3
1.2
The fluorometric assay used in the HTS screen10 was utilized for
subsequent assay of designed compounds. None of the variants of
compound 1 exhibited potent in vitro inhibition of eEF2-K. Even
small changes were not tolerated. Morpholine to thiomorpholine
or N-methyl piperazine in zone A, cyclohexyl to cycloheptyl or
cyclopentyl in zone B, conversion of zone C to an indole or furan,
and all secondary or tertiary amides or N-alkylated pyrimidines
Ph
4-COOH-Ph
2-F, 5-OMe-Ph
3-Me-Ph
in zone D led to compounds with greater than 10 lM activity.
Table 2
In vitro activities of compounds 16–31: further B-region modification
Modifications in the B-region of 2 produced many potent com-
pounds. Increasing the size of the B-region group with an aliphatic
chain appeared to increase potency, but the effect tapered off with
chain size and branching (Table 1). Very large groups were not tol-
erated, especially when highly flexible (e.g., 9). Substituted phenyl
rings gave similar results to midsized alkyl substitution. The parent
phenyl substitution (e.g., 12) abrogated activity but all other
Compound
R
IC50 (lM)
R
NH2
NH2
O
S
N
groups tested gave moderate activity, approximately 1
out regard to electronics.
lM, with-
16
17
18
19
20
21
22
23
24
Furan-2-yl
0.64
0.32
0.46
0.94
0.99
1.1
1.4
>20
>20
E-CH@CH-furan-2-yl
3-Me-thiophen-2-yl
Pyrid-3-yl
1-Me-pyrrol-2-yl
Furan-3-yl
Thiazol-2-yl
3-Me-pyrid-2-yl
CH2CH2Ph
B-region heterocycles were then synthesized and screened (Ta-
ble 2). Most of the compounds tested showed activities within a
narrow threefold range, but a subtle dependence on B-region sub-
stituent size was again noted. Five-membered heterocycles with
substitution directed away from the thienopyridine core exhibited
the best activity. This preference was not electronic in nature, as
furans containing electron-poor groups such as chloro (25) and tri-
fluoromethyl (26) showed similar activities to those with similarly
sized electron-rich ethyl (28) and hydroxymethyl (29) substitution.
The effect also appears to have a conformational component, as
phenethyl derivative 24 is inactive while alkenylfuran 17 is among
the most potent compounds tested. Further work is needed to ex-
plain why methyl substitution is favored in a phenyl system (15:
R
O
NH2
NH2
S
O
N
25
26
27
28
29
30
31
5-Cl
5-CF3
Benzofuran-2-yl
5-Et
CH2OH
0.28
0.30
0.33
0.66
0.80
1.1
IC50 = 1.2 lM, 12: IC50 >20 lM) while the reverse is seen with pyr-
idyl substitution (19: IC50 = 0.94 lM, 23: IC50 >20 lM).
4,5-Me2
4-Me,5-COOMe
>20
R3
R3
NH2
R1
R2
R1
R2
CN
S
O
R2
c
a, b
NH2
O
R1
In vitro eEF2-K activity of the analogues of 2 showed a clear
dependence on the size of the A-region (Table 3). The effect was
similar to that observed in the B-region but more dramatic: mod-
erate bulk was optimal while larger or smaller groups led to a
drop-off in activity. When the B-region was occupied by a furan-
2-yl group, the potency of the compounds related to the size of
the A-region ring: 9 > 8 > 7 ꢀ 6 ꢁ 10. Further, compounds with
non-cyclic A-regions lost all potency. Compound 34 was the most
potent compound tested in vitro.
As the lead compounds included both amino amides and pyrim-
idines in zone D, both sets of compounds were made with several
analogues of 2. The potency of the compounds varied considerably
when the amino amide was replaced with the aminopyrimidine,
but the effect was not unidirectional (Table 4). A significant
S
N
3
N
4
d, e
N
R3
N
R1
R2
N
S
NH2
5
Scheme 2. Reagents and conditions: (a) R3CHO, KOH, MeOH; (b) 2-cyanothioac-
etamide; (c) 2-bromoacetamide, NaOMe, MeOH; (d) chloroacetonitrile, NaOMe,
MeOH; (e) formamide.

J. W. Lockman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2283–2286
2285
Table 3
Table 5
In vitro activities of compounds 32–38: zone A modifications
Cytotoxicity of selected compoundsa
Compound
IC50
(lM)
Cytotoxicity EC50
HCT-116
(lM)
O
A549
NH2
NH2
1
2
17
25
34
43
0.22
2.5
0.32
0.28
0.17
4.7
29
38
19
100
17
>100
6.7
38
15
64
n/d
17
n
S
O
N
R
Compound
n
R
IC50
(
lM)
a
n/d = not done.
32
33
16
34
35
1
2
3
4
5
rac-Me
>20
1.1
0.640
0.170
>20
H
H
H
H
pounds with similar in vitro IC50 values. Compound 2 exhibited
considerably more cytotoxicity than would be predicted by its
in vitro activity, suggesting off-target effects in cells. Selectivity
is an important element of kinase-based drug design, and we
planned on screening compounds with IC50 <100 nM against a pa-
nel of phenotypically relevant kinases. No compounds tested, how-
ever, met this threshold and consequently no such analysis was
performed.
O
NH2
NH2
O
S
R
N
In conclusion, a series of thieno[2,3-b]pyridine-containing com-
pounds was synthesized and screened against eEF2-K. Chemistry
was developed that allowed for modification of the core structure
in all regions of the lead molecule. A D-region amino amide or 4-
aminopyrimidine was critical to in vitro potency. Both the A- and
B-regions showed a preference for substituents of a certain size,
with fused cyclooctyl and cyclononyl rings preferred in the A-re-
gion and five-membered heterocycles substituted away from the
central thienopyridyl core in the B-region. These compounds were
found to be ATP competitive and the best show sub-micromolar
in vitro inhibition of eEF2-K.
R
36
37
38
2,4-(MeO)2Ph
CH2-[2,4-(MeO)2Ph]
CH2C6H11
>10
>10
>10
improvement in activity was seen upon cyclization with the
change of 11 to 42, but a greater decrease in potency was seen
when 27 was changed to yield 41. Additionally, for 21/39, 16/40,
and 33/43 the effect of modifying zone D was negligible.
Selected compounds were tested in cellular assays to determine
both eEF2-K inhibition and cytotoxicity. In an ELISA assay that
monitored the levels of phospho-EF2, the lead compound 1 exhib-
ited an IC50 that ranged from 2 to 10
logues tested, only compound 34 showed any cellular kinase
activity when tested at 100 M.
Selected compounds were screened for 72 h cytotoxicity in both
HCT-116 and A549 cell lines (Table 5). Compounds showed cyto-
toxicity at 50–400-fold greater concentrations than their in vitro
inhibition of eEF2-K. This is the expected cell shift for these com-
pounds, as they are ATP competitive and the in vitro assays were
Selected compounds were screened for cytotoxicity in both
HVT-116 and A549 cell lines. Cytotoxicities were similar in the
two cell lines, and showed correlation with in vitro potency. The
best cytotoxicity in HCT-116 cells was observed with compounds
lM. Of the synthesized ana-
34 (EC50 = 17 lM) and 17 (EC50 = 19 lM). These compounds adhere
to each of Lipinski’s rule-of-five,20 and consequently are the first
true drug-like inhibitors of eEF2-K described.
l
Acknowledgments
We thank the Myriad Pharmaceuticals analytical chemistry
group for their work on the purification and characterization of de-
scribed compounds, and Drs. Kraig Yager, Robert Carlson, and Mark
Anderson for their support of this work.
run at 2.5
lar ATP levels are 1–10 mM. The best cytotoxicity in HCT-116 cells
was observed with compounds 34 (EC50 = 17 M) and 17
(EC50 = 19 M).
lM ATP, approximately twice substrate KM, while cellu-
l
l
The observed cytotoxicity generally tracked with in vitro eEF2-
K inhibition, although 25 was 4–5-fold less potent than other com-
References and notes
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Table 4
In vitro activities of compounds 39–43: zone D modifications
R
R
NH2
N
N
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a
b
NH2
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N
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n
n
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10. Full-length GST fusion eEF2-K produced in the E. coli system was used for
in vitro inhibition assays including HTS. Its calmodulin-stimulated kinase
activity towards myelin basic protein (MBP) was monitored using a previously
developed fluorometric method (Ostanin, K.; Hunsaker, T. US7338775). In
order to eliminate false positives, all compounds were also subjected to a
Compound
b
n
R
IC50 (lM)
a
a
b
2
39
2
2
2
2
1
H
2.5
3.7
16
27
11
33
40
41
42
43
2-Furanyl
0.64
0.33
1.1
0.35
>20
0.11
4.7
2-Benzofuranyl
4-Cl-phenyl
2-Furanyl
1.1

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J. W. Lockman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2283–2286
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(1 equiv), and KOH (2 equiv) are stirred in MeOH at rt for 3 h. 2-Cyanothioac-
etamide (1 equiv) and NaOMe (3 equiv) are added and the solution heated at
reflux overnight. Chloroacetonitrile (5 equiv) and NaOMe (1 equiv) are added
and the reaction mixture heated at reflux for 3 h. The mixture is cooled, diluted
with DCM, run through a silica plug, and concentrated. Formamide (6 mL/
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