Aminofurazans as potent inhibitors of AKT kinase

Article abstract of DOI:10.1016/j.bmcl.2009.01.002

AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

Full text of DOI:10.1016/j.bmcl.2009.01.002

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1508–1511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aminofurazans as potent inhibitors of AKT kinase
a
a
a
a
Meagan B. Rouse a, , Mark A. Seefeld , Jack D. Leber , Kenneth C. McNulty , Lihui Sun ,
William H. Miller a, ShuYun Zhang b, Elisabeth A. Minthorn c, Nestor O. Concha d,
Anthony E. Choudhry e, Michael D. Schaber e, Dirk A. Heerding a
*
a Oncology Chemistry, GlaxoSmithKline, Collegeville, PA 19426, USA
b Oncology Biology, GlaxoSmithKline, Collegeville, PA 19426, USA
c Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Collegeville, PA 19426, USA
d Computational and Structural Chemistry, GlaxoSmithKline, Collegeville, PA 19426, USA
e Enzymology and Mechanistic Pharmacology, GlaxoSmithKline, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have pre-
viously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical tri-
als in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this
scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of
GSK690693.
Received 11 November 2008
Revised 26 December 2008
Accepted 6 January 2009
Available online 9 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
AKT
PKB
Kinase inhibitor
Serine/threonine kinase
Activation of AKT is mediated by tyrosine kinase receptors and
phosphoinositide 3-kinase (PI3K) which recruit AKT to the plasma
membrane.1 Subsequent phosphorylation of the two regulatory
sites (ser473/thr308) generates the active enzyme, which serves
to phosphorylate various downstream proteins.2 As such, AKT
plays a vital role in cell growth, differentiation, and division and
is activated in many human tumors; including prostate, breast,
and ovarian.3,4 Therefore, inhibition of the AKT signaling pathway
offers an attractive strategy for oncology therapy.
We recently described the lead optimization effort around the
aminofurazan series of inhibitors which led to the identification
of GSK690693, a compound that has been evaluated as an intrave-
nous (iv) agent in clinical trials (Fig. 1).5 This effort focused primar-
ily on optimizing the back pocket (C-4) and amino ether side chain
(C-7) substitution. In the follow-up effort, we were interested in
exploring distinct areas of the scaffold to obtain compounds with
improved pharmacokinetic or pharmacodynamic properties to that
of GSK690693. The present communication describes our efforts
focused on the C-6 side chain series of analogs.
underwent selective amination to afford ethyl aminopyridine 5.
Exposure to SnCl2 reduced the nitro group while incorporating a
chlorine adjacent to the pyridine nitrogen in 6.7 The imidazole ring
in intermediate 8 was established through an EDC-mediated cou-
pling with cyanoacetic acid followed by cyclodehydration under
acid conditions. Installation of the aminofurazan in compound 9
was realized through a two step procedure involving oxime forma-
tion followed by cyclodehydration.5,8,9
We reasoned that a 6-position side chain could interact with the
same Glu 236 residue that was identified in the corresponding 7-
position series (see Fig. 2b).5 Alternatively, this side chain may
associate with distinct neighboring residues, such as Asp293 or
Asn280. To ascertain the preferred binding mode, we investigated
side chains containing both carbon and oxygen linkages. Bromo-
pyridine 9 served as the common intermediate for this series of
OH
NH2
4
N
The synthetic route to the C-6 position analogs is illustrated in
Scheme 1. Nucleophilic substitution onto nitropyridine 2 regiose-
lectively afforded nitrophenol 3.6 Treatment with excess phospho-
N
N
O
6
N
N
O7
rus oxybromide generated the dibromo pyridine
4 which
GSK690693
N
H
* Corresponding author. Tel.: +1 610 917 7941.
E-mail address: meagan.b.rouse@gsk.com (M.B. Rouse).
Figure 1. Aminofurazan AKT inhibitor evaluated in clinical trials.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.002

M. B. Rouse et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1508–1511
1509
O2N
O2N
O2N
a
b
N
N
N
MeO
MeO
OH
Br
Br
2
3
4
Cl
O2N
H2N
N
N
c
d
e
EtHN
Br
EtHN
Br
5
6
N
Cl
Cl
NH2
O
Cl
N
N
N
N
N
O
HN
EtHN
f
g,h
N
N
N
NC
N
Br
Br
Br
7
8
9
t
Scheme 1. Reagents and conditions: (a) KOtBu, BuOOH, NH3, THF, 70%; (b) POBr3,
MeCN, 66%; (c) EtNH2, MeOH, THF, 95%; (d) SnCl2, HCl, 60%; (e) cyanoacetic acid,
EDC, NMM, DMF, quant.; (f) AcOH, 100 °C; (g) NaNO2, AcOH, 85%; (h) NH2OH, Et3N,
H2O, 51%.
analogs (Scheme 2). Regioselective vinylation followed by reduc-
tive ozonolysis and Mitsunobu displacement generated the amino-
methyl side chain present in intermediate 10.10 Intermediate 11,
containing terminal amine substitution, arose from vinylation,
ozonolysis, and reductive amination. The extended side chains
contained in intermediate 12 were prepared via hydroboration
using an appropriate vinyl species followed by in situ Suzuki cou-
pling.11 The ether linked side chains leading to intermediate 13
were constructed using an optimized two step procedure. First,
the aryl boronate derived from lithium-halide exchange in the
presence of trimethyl borate was oxidized to give the correspond-
ing pyridone. Subsequent Mitsunobu displacement with a variety
of amino alcohols afforded intermediate 13.10
Scheme 3 depicts the remaining transformations to the final
analogs 14–33. The alkyne moiety was introduced via a standard
Sonogashira coupling.12 While additional back pocket substitution
was explored, the gem-dimethyl alkynol generated the most ideal
activity/selectivity profile.13 Removal of the protecting group from
the pendant amine was affected using hydrazine in the case of
phthalimide protection or either HCl or TFA in the case of the
Boc carbamate.
Table 1 highlights the enzyme and cellular activities for the C-6
substituted aminofurazan analogs. The mechanism based cellular
assay evaluated the ability of these compounds to inhibit the phos-
phorylation of GSK3b, a downstream target of AKT (BT474 cell
Figure 2. (a) Hinge and back pocket binding modes of 32 (yellow) and GSK690693
(green) in AKT2(148–481).18 (b) Comparison of the side chain binding modes of 32
(yellow) and GSK690693 (green) in AKT2(148–481).18
line). Inhibition of proliferation was measured in both tumor cell
lines (BT474 and LNCaP) and non-tumor cells (HFF).14 For the car-
bon linked series (entries 14–19), the aminomethyl side chain in
compound 14 provided the highest level of cellular potency when
compared to the longer chain lengths (15 and 16). Additionally,
Cl
Cl
Cl
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
N
O
N
O
N
O
R
g,h
a,b,c
O
Br
NPhth
13
9
10
f
a,d,e
Cl
Cl
NH2
N
NH2
N
N
N
N
N
N
N
N
O
N
O
n = 1,2
NPhth
N
R1
R2
12
11
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, triethenylboroxin, K2CO3, dioxane–H2O, 75 °C, quant.; (b) Ozone then NaBH4, À78 to 0 °C, 50%; (c) phthalimide, PPh3,
DEAD, THF, 25 °C, 75%; (d) Ozone, À78 °C; (e) R1R2NH, Na(OAc)3BH, MeOH 0 °C, 25–40%—two steps; (f) 9-BBN/vinylphthalimide premix then Pd(OAc)2, dppf, K2CO3, DMF,
75 °C, 20–35%; (g) i—B(OMe)3, À105 °C then nBuLi; ii—H2O2, 3 M NaOH, 50%; (h) ROH, PPh3, DEAD, 25 °C, 30–75%.

1510
M. B. Rouse et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1508–1511
1.2
1
OH
Cl
NH2
N
NH2
N
a,b
0.8
0.6
0.4
0.2
0
N
N
N
N
N
N
N
O
N
O
R
R
(10 - 13)
(14 - 33)
Scheme 3. Reagents and conditions: (a) alkyne, CuI, Pd(PPh3)2Cl2, DMF, Et3N, 80 °C,
50–75%; (b) NH2Me, MeOH, 45–60% for phthalimide; HCl or TFA, MeOH, 25 °C, 50–
70% for Boc.
Vehicle
6.26 mg/kg
12.5 mg/kg
25 mg/kg
Figure 3. Effect of 30 on GSK3b phosphorylation in BT474 xenografts (ip dosing).
acyclic tertiary amine (18) was superior to primary (14), secondary
(17), and cyclic (19) amines. Similarly, for the ether linked series
(entries 20–33), the aminoethyl chain length (20) was more potent
than the longer aminopropyl and aminobutyl chain lengths (21 and
22). In contrast to the carbon linked series, substitution on the pen-
dant amine was not well tolerated (23).
Further exploration revealed that alkyl and aromatic substitu-
tion adjacent to the amine offered significant enhancements in
enzyme and cellular potency with a clear enantiomeric preference
(24–29).16 Interestingly, shifting the position of substitution to
flank the ether oxygen provided aminoethyl (30) and aminopropyl
(32) compounds with comparable enzyme and cellular profiles to
that of GSK690693.
To determine the binding mode for this series, an X-ray co-crystal
structure of compound 32 in the kinase domain of AKT2 (148–481)
was solved.17 Figure 2 depicts an overlay of this compound with that
of GSK690693. As expected, the binding interactions along the hinge
(Ala232) and alkynol (Glu200 and Phe294) residues remained consis-
tent to those previously reported (Fig. 2a).5 However, the side chain
amine showed no apparent binding interactions to the Glu236 resi-
Table 1
SAR for C-6 side chain modifications; compounds 14–33c,d
OH
NH2
N
N
N
N
R
O
N
*
Entry
R
Kinase (IC50 nM, [Ki ])15
Cellular activity (IC50 nM)
Chirality
AKT1
AKT2
AKT3
GSK3ba
BT474b
LNCaPb
HFFb
GSK690693
—
—
—
—
—
—
—
2 [1]
13 [4]
63
25
40
16
9
138
1020
4000
13,200
303
69
806
1090
3520
439
21
296
2020
4720
201
>23,000
24,000
24,000
23,000
5900
14
15
16
17
18
–CH2NH2
–(CH2)2NH2
–(CH2)3NH2
–CH2NHCH3
–CH2N(CH3)2
6
4
3
3
2
70
NT
30
NT
NT
25
375
357
114
30,000
*
19
—
6
200
22
2700
1250
313
30,000
N
20
21
22
23
–O(CH2)2NH2
–O(CH2)3NH2
–O(CH2)4NH2
–O(CH2)2NHCH3
—
—
—
—
5 [6]
16 [115]
63
40 [39]
200 [646]
316
43
NT
NT
NT
1140
30,000
30,000
2600
1080
7120
20,000
3190
497
3180
6160
896
5000
30,000
30,000
17,000
12
79
CH3
NH2
*
O
24
25
R
S
8
13
50
63
NT
NT
400
1210
860
4860
220
560
17,000
28,000
*
*
Ph
NH2
O
O
26
27
R
S
3
5
20
20
17
49
1110
1040
2100
3440
124
176
5000
20,000
Ph
28
29
R
S
0.6 [0.2]
3
10 [2]
20
3
NT
80
310
742
1500
20
101
8000
7000
NH2
Ph
30
31
R
S
1 [0.4]
3 [3]
25 [5]
25 [17]
1
NT
40
190
35
124
27
68
519
20,000
*
*
NH2
O
O
Ph
32
S
2 [0.2]
4 [1.9]
16 [1]
NT
65
41
1
5
11,000
29,000
NH2
33
R
50 [105]
1570
224
85
a
Inhibition of GSK3b phosphorylation (BT474 cells).
Inhibition of proliferation.
Values are the mean of greater than or equal to two experiments.
NT, not tested.
b
c
d

M. B. Rouse et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1508–1511
1511
due (Fig. 2b). This amine was instead associated within a unique
References and notes
region of the active site, in the vicinity of Asp293 and Asn280. Addi-
tionally, the phenyl ring on the side chain displaced the aromatic ring
of the Phe163 residue and engaged in hydrophobic interactions along
the glycine rich loop. This may explain the observed enantiomeric
preference where the aryl group of the antipode would not be prop-
erly oriented to participate in the stacking interaction.
The pharmacokinetics of a representative collection of com-
pounds from this series were examined (18, 28, 30, and 32). These
compounds displayed PK profiles suitable for iv dosing, similar to
that of GSK690693 (data not shown). Unfortunately, there was
no evidence of exposure that would allow for oral administration.
A representative compound (30) was further profiled in a mouse
pharmacodynamic study to evaluate the in vivo potency toward
inhibition of GSK3b phosphorylation in BT474 xenografts (Fig. 3).
This compound showed statistically significant dose dependent
inhibition, comparable to the response observed for GSK690693
(data not shown).5
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In summary, lead optimization around the C-6 position of the
aminofurazan template provided analogs with similar enzyme
and cellular activity profiles to GSK690693. Additionally, a repre-
sentative compound displayed an acceptable dose dependent PD
response in BT474 tumor xenografts. This series also exhibited a
unique binding mode around the amine side chain within the
ATP binding pocket. However, there were no improvements in
the pharmacokinetic profile which would allow for oral adminis-
tration. Development of a series with suitable oral properties is
underway and will be reported in due course.
Acknowledgments
13. Similar trends in kinase selectivity were observed to those previously reported
on this scaffold (see Ref. 5).
14. For a detailed description of the assay methods see Ref. 5.
15. For compounds which approached the limit of detection in our standard IC50
The authors thank Dr. Melissa Dumble for helpful discussions
around in vivo biology and Dr. Kristin Koretke for assistance with
molecular modeling.
*
assay, Ki values were determined. For a detailed protocol describing this assay
see Ref. 5.
16. The chiral amino ether side chains contained in compounds 24–33 were
commercially available, obtained through reduction of the corresponding
amino acid or acquired from chiral resolution of the racemate.
17. Crystallographic data for compound 32 has been deposited with the RSBC
Protein Data Bank, PDB code 3E8D.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.002.
18. Graphics generated using Pymol (http://www.pymol.org).