Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.094

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitor

Full text of DOI:10.1016/j.bmcl.2009.02.094

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2244–2248
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT
kinase inhibitors
a
a
a
a
Mark A. Seefeld a, , Meagan B. Rouse , Kenneth C. McNulty , Lihui Sun , Jizhou Wang ,
Dennis S. Yamashita a, Juan I. Luengo a, ShuYun Zhang b, Elisabeth A. Minthorn c,
Nestor O. Concha d, 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, Analytical and Structural Sciences, 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:
A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead opti-
mization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in
AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds
showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target
GSK3b.
Received 28 January 2009
Revised 23 February 2009
Accepted 24 February 2009
Available online 27 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
AKT
Kinase
AKT, also known as protein kinase B (PKB), is a serine/threonine
kinase from the AGC family of kinases that exists in three homolo-
gous human isoforms (AKT1, AKT2, AKT3). AKT plays a critical role
in signal transduction and apoptotic pathways effecting cell sur-
vival and proliferation.1 Over-expression of AKT has an anti-apop-
totic effect in many cell types.2 In addition, PTEN, a critical negative
regulator of AKT, is mutated or lost in many cancers including
breast, ovarian, prostate carcinomas and glioblastomas.3 Therefore,
inhibition of the AKT signalling pathway offers a viable strategy for
the treatment of cancers.4
b,c
O
a
N
H
N
H
N
O
N
1
2
Br
O
B
d
N
N
N
SO2Ph
N
SO2Ph
3
4
In a previous communication,5 we described the discovery and
development of an aminofurazan series of AKT inhibitors. Although
potent AKT inhibition was achieved, this series suffered from poor
oral exposure in preclinical species. Therefore, alternative chemo-
types were examined to try to find AKT inhibitors suitable for oral
delivery. Compounds based on pyrrolopyridinyl thiophene carbox-
amides (chemotype 7, Scheme 1) were identified as low nanomolar
ATP-competitive inhibitors of AKT1 through a screening of the GSK
kinase inhibitor collection. Pan-AKT activity is likely given the
sequence similarity of AKT2 (90%) and AKT3 (88%) to AKT1 in the
kinase domain.6,7
O
S
Br
e
O
OH
S
Br
NHBoc
N
R
H
5
R
6a (R = H)
6b (R = Br)
(R = H or Br)
HN
O
f,g,h
N
7 (R = H)
8 (R = Br)
S
N
NH2
H
R
Synthesis of pyrrolopyridinyl thiophene amides began with the
construction of boronate 4.8 7-Azaindole (1) was oxidized to the
Scheme 1. Reagents and conditions: (a) MCPBA, CHCl3 59%; (b) (CH3SO2)2O,
(CH3)4NBr, DMF 40%; (c) PhSO2Cl, Et3N, DCM 95%; (d) Pd(dppf)2Cl2, bis(pinacola-
to)diboron, KOAc, DMF, 81%; (e) PyBrop, Et3N, 1,1-dimethylethyl(2-amino-2-
phenylethyl) carbamate, DCM, rt, 80%; (f) Pd(PPh3)4, K2CO3, 4, dioxane/H2O, 70 °C,
65–79%; (g) NaOH, MeOH/THF/H2O, rt; (h) HCl, dioxane, rt, quant.
* Corresponding author. Tel.: +1 610 917 7936; fax: +1 610 917 4206.
E-mail address: mark.a.seefeld@gsk.com (M.A. Seefeld).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.094

M. A. Seefeld et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2244–2248
2245
Table 1
Effect of pyrrolopyridine and thiophene regiochemistry on AKT1 enzyme and
mechanistic activities for compounds 7 and 9–11
a
b
Compound Structure
AKT1 IC50
(nM)
pGSK3b IC50
(nM)
HN
O
N
7
9
6
190
240
S
N
NH2
H
H
N
O
S
16
N
N
NH2
H
S
O
HN
10
11
40
25
1000
1100
N
H
N
NH2
Figure 1. X-ray structure of 7 co-crystallized in AKT2 (146–481).
S
O
HN
N
HN
a-d
NH2
HN
+
S
Br
N
CHO
S
H2N
NHBoc
N
H
NH2
a
Values are measured against AKT1 at or below Km for ATP; n P 2.
Inhibition of phosphorylation of GSK3b in BT474 cells.
b
13
12
14
N-oxide 2, which was treated with methanesulfonic anhydride and
tetramethyl ammonium bromide to afford 4-bromo-pyrrolopyridine.
Reaction with phenylsulfonyl chloride provided the 4-bromo
pyrrolopyridine 3. Formation of the boronic ester9 using bis
(pinacolato)diboron gave 4. The coupling partner 6 was prepared
by the PyBrop-mediated reaction of commercially available Boc-1-
amino-2-phenyl ethylamine with bromothiophene carboxylate 5.
Suzuki coupling of 4 and 6, followed by sequential deprotection
under basic and then acidic conditions, yielded amide 7.
Compound 8 was made by coupling boronate 4 with the dibromothi-
ophene intermediate 6b. Regiochemical assignment of 8 was
confirmed by NMR studies. Compounds 9–11 were similarly
prepared using bromothiophene 2-carboxylic acids and 1-(phenyl-
sulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-
rolo[2,3-b]pyridine10 as appropriate.
Initially, we were interested to determine if 7 represented the
optimal orientation of the pyrrolopyridine, therefore we conducted
a regioisomeric assessment of the core thiophene and its activity
relationship with the pyrrolopyridine moiety (Table 1). The 2,5-
disubstituted thiophene 7 gave slightly improved AKT enzyme
inhibition relative to regioisomers 9–11. Compounds 7 and 9 dem-
onstrated a more pronounced effect on the inhibition of phosphor-
ylation levels of downstream target GSK3b.11 The attachment of
the thiophene to the pyrrolopyridine at either the 3-position or
the 4-position had little effect on overall activity with a minor pref-
erence for the 4-position regiochemistry as measured in the cell-
based mechanistic assay (pGSK3b). The modest effect of these
regioisomers on AKT activity suggests that the thiophene acts pri-
marily as a lipophilic link between the pyrrolopyridine hingebind-
er and the amide functionality.
HN
e,b-d
H
N
S
N
NH2
O
R
HO
O
19 (R = H)
20 (R = Br)
NHBoc
Br
18
16
HO
NHBoc
S
S
Br
CO2H
NHBoc
f
R
R
g,b-d
S
5
15
(R = H or Br)
HN
H
N
N
NH2
R
17 (R = H)
Scheme 2. Reagents and conditions: (a) NaBH4, EtOH/DCM, rt, 75% (b) Pd(PPh3)4,
K2CO3, 4, dioxane/H2O, 70 °C, 65–79%; (c) NaOH, MeOH/THF/H2O, rt; 85–95% (d)
HCl, dioxane, rt, quant (e) PyBrop, Et3N, DCM, rt, 80%; (f) DPPA, t-BuOH, dioxane,
70 °C, 60%; (g) DIAD, 16, PPh3, THF, rt, 75%.
was constructed by combining aldehyde 12 and amine 13 under
reductive amination conditions and further elaborating as
described in Scheme 2. Amino thiophene 15 was obtained by sub-
jecting 5 to Curtius reaction conditions.14 The Boc-protected amine
15 underwent a Mitsunobu reaction with alcohol 16 to ultimately
provide amine 17. Alternatively, thiophene 15 was deprotected
and coupled to carboxylic acid 18 using PyBrop. A subsequent
Suzuki reaction and deprotection steps yielded reverse amides 19
and/or 20. Substituents at position-3 (R, Table 2) were appended
using a Suzuki reaction with a Boc-protected version of bromide
8 and the appropriate boronate.
We next turned our attention to understanding the interactions
of the linking amide of 7 with AKT. An X-ray co-crystal structure of
0
7 (AKT2 IC50 = 50 nM) was determined to 2.3 ÅA resolution in AKT2
(Fig. 1).12 Based on 0this structure, it evident that the amide car-
bonyl of 7 was 4.4 ÅA away from the terminal nitrogen of Lys181.
We proposed that the amide carbonyl interacts with this lysine
residue of AKT2, possibly via a molecule water. Although we were
unable to detect this interaction in our co-crystal structure, others
have observed it in similar systems.13
The amide functionality of 7 clearly imparts a beneficial effect
as the levels of AKT1 activity and mechanistic activity (inhibition
of GSK3b phosphorylation) were significantly reduced in its
absence (14 and 17, Table 2). The reverse amide compound 19
We then investigated various replacements of the amide linker
in 7 with methylamine and reverse amide linkers. Compound 14

2246
M. A. Seefeld et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2244–2248
Table 2
Table 3
Thiophene substituent and amide regiochemistry effects on AKT1 and GSK3b for
compounds 7, 8, 14, 17and 19–25
Effect of amines 8 and 26–35 on AKT activity
HN
HN
O
N
S
2
5
N
S
X
N R
Z
H
NH2
Br
3
R
Z
a
b
Compound
R
AKT1 IC50 (nM)
pGSK3b IC50 (nM)
a
b
Compound
R
X
AKT1 IC50 (nM)
pGSK3b IC50 (nM)
NH2
26
158
>10,000
>10,000
500
*
7
H
H
H
H
Br
Br
CH3
c-Pr
Ph
C@O
CH2
NH
NH
NH
C@O
C@O
C@O
C@O
NH
NH
CH2
C@O
C@O
NH
NH
NH
NH
6
190
14
17
19
20
8
21
22
23
100
250
3
25
8
8
6
50
>10,000
>10,000
560
4700
500
120
800
10,000
*
27
8
40
8
Ph
Ph
NH2
*
*
*
*
NH2
28
29
30
31
32
33
34
3
780
NHCH3
N
Ph
Ph
Ph
24
C@O
NH
NH
63
63
6000
2500
20
8
>10,000
1500
4800
80
N(CH3)2
H
N N
25
C@O
NH2
a
*
Values are measured against AKT1 at or below Km for ATP; n P 2.
Inhibition of phosphorylation of GSK3b in BT474 cells.
20
1
NH2
NH2
b
Ph
Ph
produced comparable levels of enzyme inhibition, but with
reduced inhibition of AKT mediated GSK3b phosphorylation.
The bromine substituent on reverse amide 20 was less tolerated.
The potential for the thiophene ring to rotate 180° from the orienta-
tion shown in Figure 1 cannot be discounted and may contribute to
the difference in activity of 20 relative to 8. Small lipophilic function-
ality (Br, Me and c-Pr; 8, 21, 22, respectively) was tolerated at posi-
tion R on the thiophene of amide 7. Larger aromatic substituents
(23–25) reduced AKT1 activity. This was in keeping with the com-
pact lipophilic environment of the ATP binding pocket.
*
*
*
*
Ph
Ph
Ph
NHCH3
NHEt
2
260
4
1100
2100
NH(i-Pr)
35
10
a
Values are measured against AKT1 at or below Km for ATP; n P 2.
Inhibition of phosphorylation of GSK3b in BT474 cells.
b
We then systematically investigated the nature of the amide
substituent (Table 3). The bromo group on the thiophene ring
was left intact as a potential source for further structural elabora-
tion as we evaluated amide group modifications. Removing (26) or
shifting the position of the phenyl substituent (27) reduced AKT
activity and was deleterious to inhibition of GSK3b phosphoryla-
tion. Lengthening the alkyl amine (30, 31) also proved to be of
no benefit. Secondary amines (28, 33) were tolerated, however
more sterically demanding amines (34, 35) caused a reduction in
activity, presumably by interfering with the ability of the pendant
amine to bind to Asp293. Tertiary amine 29 showed no detectable
activity in the pGSK3b assay. Primary amine 32 exhibited the best
combination of enzyme and mechanism-based activity, and was
selected for further investigation (Table 4).
The required diamines were prepared from commercially avail-
able amino acids (Scheme 3). Reduction of the carboxylic acid of
36 with BH3ꢀTHF followed by a Mitsunobu reaction between the
resultant alcohol and phthalimide provided a differentially pro-
tected diamine which when treated with HCl in dioxane gave 37.
Cyclohexyl (40) and pyridyl (41) analogs (Table 4) were similarly
prepared.
The (R)-enantiomer 38 was found to be several hundred times
less active in the AKT kinase assays than the corresponding (S)-
enantiomer 39. The most potent combination of enzyme and cellu-
lar activity was provided by small lipophilic functionality. Electron
withdrawing groups on the phenyl ring (43-48), in general, showed
an improvement in the inhibition of proliferation in cell lines that
have been previously demonstrated to be sensitive to AKT (BT474,
LNCaP)15,16 relative to cell lines that are nontumor derived (HFF).
However, considerable inhibition of HFF cells nonetheless remains
and may indicate activities unrelated to AKT inhibition. Subn-
anomolar AKT1 inhibition was required to demonstrate good anti-
proliferative activity from compounds of this series. For
compounds that approached the tight binding limit of the IC50
assay, we used an alternative assay format (Kꢁi )17 which allowed
for measurement of subnanomolar potencies. This assay was run
in parallel with our standard high throughput AKT enzyme assay.11
In order to characterize the pharmacokinetic performance of
representative 5-pyrrolopyridinyl-thiophene amides, compounds
8
and 32 were evaluated in iv/po crossover studies in rat
(Table 5). These two compounds differ structurally by only a meth-
ylene group, yet this minor change appears to be responsible for a
several fold increase in AKT activity. The PK profile for compound 8
shows moderate clearance and oral exposure with a consequence
of modest oral bioavailability. The benzyl compound (32) shows
generally poorer PK. The oral bioavailability of benzyl compound
32, although similar to phenyl compound 8 in average value, ran-
ged from 6% to 70%. This variable oral bioavailability is character-
istic of all the benzyl analogs tested in Table 4. While the PK profile
of the benzyl compounds were less than optimal they were suffi-
cient to conduct PD studies. Compound 44 exhibited potent overall
in vitro activity and was therefore chosen to be tested for a phar-
macodynamic (PD) response in BT474 tumor bearing xenograft
mice. The effect of compound 44 on the inhibition of GSK3b phos-
phorylation was measured at three doses (6.25, 12.5 and 25 mg/kg)
following a single ip injection. Tumors were harvested 4 h post
dose. Figure 2 illustrates the dose-dependent reduction in pGSK3b
levels produced by compound 44 in mice relative to vehicle.

M. A. Seefeld et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2244–2248
2247
Table 4
AKT activity and cell potency derived from benzyl group manipulations
HN
O
R
N
S
N
NH2
*
H
Br
Compound
Chirality
R
Kinase Activitya (nM)
Cellular Activityc IC50 (nM)
AKT1 IC50 [Kꢁi ]
AKT2 IC50 [Kꢁi ]
AKT3 IC50
pGSK3bb
BT474
LNCaP
HFF
8
( )
( )
R
S
S
S
S
S
S
S
Phenyl
Benzyl
Benzyl
Benzyl
Cyclohexyl-CH2-
4-Pyridyl-CH2-
4-Methoxybenzyl
2-Fluorobenzyl
3-Flourobenzyl
4-Fluorobenzyl
2-Trifluoromethylbenzyl
3-Trifluoromethylbenzyl
4-Trifluoromethylbenzyl
8 [20]
1 [0.04]
32 [20]
1 [0.06]
1 [0.79]
[2]
125 [125]
13 [8]
500 [800]
10 [3]
25 [6]
[40]
63 [40]
8
9 [0.63]
6 [2]
20
3
40
2
3
NT
2
2
2
2
2
500
80
4000
200
750
45
2700
21
440
750
720
39
18
66
24
78
1200
410
1900
90
32
38
39
40
41
42
43
44
45
46
47
48
6000
100
1900
1900
1000
220
90
180
50
170
2000
4500
230
2600
2300
1900
120
240
500
NT
3600
890
260
260
490
730
530
1300
3 [5]
1
1 [0.03]
1 [0.06]
3 [0.05]
1 [0.10]
2 [2]
S
S
S
25 [4]
10 [4]
79 [16]
60
60
1200
1
3
440
NT = not tested.
a
Values are an average of at least 2 measurements.
Inhibition of phosphorylation of GSK3b in BT474 cells.
Inhibition of cell proliferation.
b
c
O
Table 6
a,b,c
Activity of 44 on various kinases
O
N
H
H2N
CO2H
36
NPhth
Kinase family
AGC
Kinasesa
44, IC50 (nM)
AKT1b
AKT2b
AKT3
0.03
8
2
37
Scheme 3. Reagents and conditions: (a) BH3ꢀTHF, 0 °C, 89%; (b) DIAD, phthalimide,
PKC
a
40
PPh3, THF, rt, 95%; (c) HCl, dioxane, rt, quant.
ROCK1
2
CMGC
CAMK
TK
GSK3b
20,000
25,000
2500
794
10,000
25,000
P38
a
0.3
0.25
0.2
MAPKAPK2
PIM-1
EGFR
SYK
a
All kinase IC50s were assayed at or below Km for ATP.
Values determined as Kꢁi .
b
0.15
0.1
The selectivity profile of compound 44 was evaluated against a
0.05
0
panel of >50 different kinases, of which representative examples
are shown (Table 6). The results depict a pattern of moderate to poor
selectivity against the AGC family of kinases where a high homology
with the AKT active site exists. A much better selectivity profile was
achieved against protein kinases from other kinase families.
In summary, we have demonstrated that pyrrolopyridinyl
thiophene amides are potent pan-AKT inhibitors. Compounds from
Vehicle
25
12.5
6.25
(mg/kg)
Figure 2. Effect of 44 on GSK3b phosphorylation levels in BT474 xenograft mice
after 4 h. Vehicle: 5% dextrose, pH 4.0.
Table 5
iv/po crossover studies for compounds 8 and 32 in conscious male rats (n = 3)
HN
HN
O
O
N
S
N
S
N
N
NH2
H
NH2
H
Br
Br
8
32
*
Compound
iv/poa,b (mg/kg)
Cmax (ng/mL)
Cl (mL/min/kg)
16.7
T1/2 (h)
AUC(0-t) (ng h/mL)
%F
8
iv (2)
po (10)
iv (2)
1800
550
712
248
3.6
4.1
3.8
ND
3248
3397
893
32
38
32
49
po (10)
664
a
iv: fed, solution in 1% DMSO, 20% encapsin in saline. po: fasted, solution in 1% DMSO, 20% PEG400 in water; pH 3.0–3.5.
Blood samples were collected over 24 h.
b

2248
M. A. Seefeld et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2244–2248
Choudhry, A. E.; Copeland, R. A.; Lai, Z.; Schaber, M. D.; Tummino, P. J.; Strum, S.
L.; Wood, E. R.; Duckett, D. R.; Eberwein, D.; Knick, V. B.; Lansing, T. J.;
McConnell, R. T.; Zhang, S-Y.; Minthorn, E. A.; Concha, N. O.; Warren, G. L.;
Kumar, R. J. Med. Chem. 2008, 51, 5663.
this series showed cellular activity that correlated well with inhi-
bition of phosphorylation of the AKT downstream target GSK3b.
Dose-dependent inhibition of GSK3b phosphorylation was demon-
strated in an in vivo pharmacodynamic model for a representative
compound. The oral bioavailability of compounds across this series
in preclinical species demonstrated a significant improvement
from our earlier series of AKT inhibitors.5 The co-crystal structure
of 7 will be used to guide further structural refinements with a goal
of improving kinase selectivity and PK parameters.
6. Kumar, C. C.; Madison, V. Oncogen 2005, 24, 7493.
7. Allosteric inhibitors have recently been described that show AKT isoform
selectivity. See reference: . Bioorg. Med. Chem. Lett. 2009, 19, 834.
8. Seefeld, M. A.; Hamajima, T.; Jung, D. K.; Nakamura, H.; Reid, P. R.; Reno, M. J.;
Rouse, M. B.; Heerding, D. A.; Tang, J.; Wang, J.; PCT Patent App. WO
2007076423 A2, 2007.
9. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
10. Huang, S.; Li, R.; Connolly, P. J.; Emanuel, S.; Middleton, S. A. Bioorg. Med. Chem.
Lett. 2006, 16, 4818.
11. For details of in vitro kinase inhibition assays and assays used to measure
inhibition of GSK3b phosphorylation see: Rhodes, N.; Heerding, D. A.; Duckett,
D. R.; Eberwein, D. J.; Knick, V. B.; Lansing, T. J.; McConnell, R. T.; Gilmer, T. M.;
Zhang, S. Y.; Robell, K.; Kahana, J. A.; Geske, R. S.; Kleymenova, E. V.; Choudhry,
A. E.; Lai, Z. V.; Leber, J. D.; Minthorn, E. A.; Strum, S. L.; Wood, E. R.; Huang, P.
S.; Copeland, R. A.; Kumar, R. Cancer Res. 2008, 68, 2366.
12. Crystallographic data for compound 7 has been deposited in the PDB with
deposition code 3E87. The level of resolution does not permit the assignment
of specific stereochemistry.
Acknowledgments
The authors would like to thank Melissa Dumble, Kim Robell,
Dana Levy, Michael Schaber, Anthony Choudhry, Zhihong Lai,
Rakesh Kumar and Jason Kahana for helpfull discussions. We
would also like to thank Kristin Koretke for molecular modelling
assistance.
13. Lin, X.; Murray, J. M.; Rico, A. C.; Wang, M. X.; Chu, D. T.; Zhou, Y.; Del Rosario,
M.; Kaufman, S.; Ma, S.; Fang, E.; Crawford, K.; Jefferson, A. B. Bioorg. Med.
Chem. Lett. 2006, 16, 4163.
Supplementary data
14. Thornton, T. J.; Jarman, M. Synthesis 1990, 4, 295.
15. Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer
Ther. 2001, 1, 85.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.094.
16. Nakatani, K.; Thompson, D. A.; Barthel, A.; Sakaue, H.; Liu, W.; Weigel, R. J.;
Roth, R. A. J. Biol. Chem. 1999, 274, 21528.
References and notes
17. Kꢁi values were determined against human or mouse AKT1 and AKT2 as
follows: Either AKT1 (30
concentration 1 nM) in buffer
concentrations. AKT and inhibitor were incubated for 60 min at room
temperature. The reaction was initiated by adding 8 L of substrate mix in
buffer A containing GSK3 peptide (final concentration 10 M), ATP (final
concentration 50 M), and
33P-ATP (final concentration 0.03 mCi/mL). After
120 min, the reaction was stopped with 40 L of 1% H3PO4. The stopped
reaction was transferred to phosphocellulose filter plates and washed 5 times
l
L, final concentration 0.33 nM) or AKT2 (30
lL, final
1. For reviews on AKT see: (a) Bellacosa, A.; Kumar, C.; Di Cristofano, A.; Testa, J. R.
Adv. Cancer Res. 2005, 94, 29; (b) Li, Q.; Zhu, G.-D. Curr. Top. Med. Chem. 2002, 2,
939.
2. Jetzt, A.; Howe, J. A.; Horn, M. T.; Maxwell, E.; Yin, Z.; Johnson, D.; Kumar, C. C.
Cancer Res. 2003, 63, 6697.
3. (a) Sansal, I.; Sellers, W. R. J. Clin. Oncol. 2004, 22, 2954; (b) Hsu, J. H.; Shi, Y.; Hu,
L.; Fisher, M.; Franke, T. F.; Lichtenstein, A. Oncogene 2002, 21, 1391.
4. (a) Lindsley, C. W.; Barnett, S. F.; Yaroschak, M.; Bilodeau, M. T.; Layton, M. E.
Curr. Top. Med. Chem. 2007, 7, 1349; (b) Heerding, D. A.; Safonov, I. G.; Verma, S.
K. Ann. Rep. Med. Chem. 2007, 42, 365.
A
were added to inhibitor at various
2 lL
l
a
l
l
c-
l
with 100 lL of 0.5% H3PO4. Microscint20 cocktail was added to the dried filter
plates and read using a Perkin Elmer Microbeta liquid scintillation counter. Kiꢁ
values were calculated using the following equation:
5. Heerding, D. A.; Rhodes, N.; Leber, J. D.; Clark, T. J.; Keenan, R. M.; LaFrance, L.
V.; Li, M.; Safonov, I. G.; Takata, D. T.; Venslavsky, J. W.; Yamashita, D. S.;
IC50 ¼ ½Eꢂ=2 þ Kꢁi ꢀ ð1 þ ½Sꢂ=KmÞ: