2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics

Article abstract of DOI:10.1016/j.bmcl.2010.01.046

A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis. Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates, PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.

Full text of DOI:10.1016/j.bmcl.2010.01.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1652–1656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics
a
a
a
Qingping Zeng a, Matthew P. Bourbeau a, , G. Erich Wohlhieter , Guomin Yao , Holger Monenschein ,
James T. Rider a, Matthew R. Lee a, Shiwen Zhang b, Julie Lofgren b, Daniel Freeman b, Chun Li c,
Elizabeth Tominey c, Xin Huang d, Douglas Hoffman e, Harvey Yamane f, Andrew S. Tasker a,
Celia Dominguez a, Vellarkad N. Viswanadhan a, Randall Hungate a, Xiaoling Zhang b
*
a Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Chemistry Research and Discovery, Amgen, Inc., 1 Kendall Square, Cambridge, MA 02139, USA
e Small Molecule Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
f Protein Science, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along
with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity
observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis.
Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates,
PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-
dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 December 2009
Revised 8 January 2010
Accepted 11 January 2010
Available online 20 January 2010
The serine/threonine kinase AKT (also named protein kinase B,
or PKB) is a key component in the signaling cascades mediated
by growth factor receptors and one of the most frequently acti-
vated protein kinases in a number of human cancers.1–3 Constitu-
tively activated AKT provides a driving factor for tumor growth
and survival, and is associated with the resistance of many cancers
to current therapies. There are three AKT isoforms in mammalian
kinase selectivity.6 CDK2 is a serine/threonine kinase that regulates
cell cycle transition and cell proliferation.7 To understand desired
selectivity against these kinases, we followed PKA and CDK2 activity
for all subsequent compounds synthesized. This Letter details the
SAR explorations that resulted in the discovery of compounds with
improved enzymatic potency relative to 1. Select compounds also
showed activity in cellular and pharmacodynamic studies.
cells (AKT1/PKB
a
, AKT2/PKBb, and AKT3/PKB
c
), which have shown
The general synthetic approach used for indazole containing
compounds in this Letter is shown in Scheme 1. In order to prepare
3-substituted indazoles, aldehyde 2 was treated with the appropri-
ate alkyl or aryl Grignard reagent (RMgBr) and the resulting alcohol
was oxidized to ketones 3. Reaction of ketones 3 with hydrazine
afforded the corresponding indazoles 4. In the case of unsubstituted
indazoles (R = H), steps a and b were omitted and 2 was treated with
hydrazine directly (step c). Lithiation, followed by quenching with
both distinct and overlapping functions in normal physiology. All
three isoforms possess the transforming ability important for tu-
mor formation, although arising data has suggested AKT1 is the
major isoform contributing to the oncogenic activity.2 Various
groups have pursued AKT1 inhibitors as a promising anti-cancer
therapy with the potential for broad application in areas including
ovarian, breast, and prostate cancers.4,5
We have undertaken a research program with the goal of devel-
oping an AKT1 inhibitor for the treatment of human cancers. Our
work in this area has focused on a chemical series of 5-aminothi-
adiazoles, exemplified by rationally designed lead compound 1
(Fig. 1). In addition to AKT, compound 1 was found to inhibit protein
kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). We followed
PKA and CDK2 selectivity as we developed the 2-aminothiadiazole
series. PKA is highly homologous to AKT in the kinase domain and
is a prototype for the ACG kinase family. It was expected that im-
proved selectivity for PKA might lead to more broadly improved
* Corresponding author.
E-mail address: bourbeau@amgen.com (M.P. Bourbeau).
Figure 1. Original lead compound in thiadiazole series. Data is reported as the
mean SD where n P 3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.046

Q. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1652–1656
1653
enyl)propanoic acid, and the resulting amide was reduced with
LiAlH4. This resulted in partial over-reduction of the isoquinoline,
so the product was treated with palladium on carbon in cyclohex-
ene to oxidize the over reduced product. Treatment with TFA then
afforded isoquinoline 30. 6-Bromo-3-methylisoquinoline was pre-
pared via published methods8 and coupled with vinyltributyltin to
afford olefin 10. The olefin was subjected to ozonolysis followed by
oxidation of the resulting aldehyde to the corresponding carboxylic
acid 11. Following steps a–e, the acid was then converted to dia-
mine 31.
Utilizing the amino acid coupling described in Scheme 1, we
were able to rapidly examine changes to the diamino portion of
the molecule (Table 1).9,10 To establish the preferred stereochemis-
try of the chiral amine, the R-enantiomer of 1 (R-1) was synthe-
sized and found to be less potent in the AKT1 enzymatic assay
than the S enantiomer. We next examined whether introducing
substitutions on the phenyl ring might improve potency. Commer-
cially available S-phenylalanine analogs were used as inputs. Sub-
stitution at the 2 position of the ring with F, Cl, or CF3 led to
compounds with similar or reduced potency compared to 1 (12–
14), and with no selectivity over PKA. In contrast, substitution at
the 3 and 4 positions with F, Cl, or CF3 was found to generally en-
hance potency and, in the case of 17–19, resulted in compounds
that inhibited AKT1 activity with IC50 values in the single digit
nanomolar range. This improvement in activity could be rational-
ized by X-ray crystal structure analysis of several key compounds
(see below). We also synthesized two examples with disubstitu-
tion on the phenyl ring (20 and 21). These compounds also showed
increased enzymatic potency relative to 1, but they were not as po-
tent as the best monosubstituted examples (i.e., 19). No significant
selectivity for PKA or CDK2 was observed in any compound.
Having shown potency could be improved by changes to the
amino phenyl portion of 1, we next examined changes to the 3-po-
sition of the indazole ring (Table 2). Replacement of the methyl
group with larger groups (22–24) resulted in a loss of potency rel-
ative to 1. However, if the methyl group was replaced with a
hydrogen, potency was maintained in most cases (25, 27–29). In
addition to retaining potency, several of these compounds began
to show modest selectivity over PKA. Compound 28, in particular,
showed 20-fold selectivity over PKA. Compounds 28 and 29 also
showed modest selectivity for CDK2 (4–8-fold).
Scheme 1. Synthesis of 1, 12–29. Reagents and conditions: (a) RMgBr, THF, 0 °C,
89–100%; (b) PDC, molecular sieves, CH2Cl2, 25–84%; (c) hydrazine, 117 °C, 65–94%;
(d) t-BuLi, THF, then CO2, 31–94%; (e) PPA, thiosemicarbazide, 90 °C, 53–92%; (f) N-
Boc amino acids, EDCI, HOBt, DMF; (g) LiAlH4, THF, 0 °C to rt; (h) TFA, CH2Cl2, 5–10%
for three steps.
CO2, led to carboxylic acids 5. Conversion to 5-aminothiadiazoles 6
was followed by amide coupling with a commercially available N-
Boc protected amino acid. The resulting amides were reduced using
LiAlH4. TFA deprotection of the primary amine afforded the desired
products 1, 12–29.
A similar approach used to synthesize isoquinoline containing
compounds 30 and 31 is outlined in Scheme 2. Commercially avail-
able carboxylic acid 7 was converted to thiadiazole 8 by treatment
with thiosemicarbazide in polyphosphoric acid. The thiadiazole
was coupled with N-Boc (S)-2-amino-3-(4-(trifluoromethyl)ph-
Table 1
SAR of changes to the phenyl ring of compound 1
Compds
R
AKT1 IC50 (nM) PKA IC50 (nM) CDK2 IC50 (nM)
1
H
H
2-F
2-Cl
2-CF3
3-F
3-Cl
3-CF3
4-Cl
4-CF3
76 12
>2200a
198 10
54 12
NTb
215 150
106 39
247 40
230 190
72 14
NTb
R-1
12
13
14
15
16
17
18
19
20
21
104a
47
491 35
38
7
57 14
198 96
NTb
6
19 0.2
6.1 0.1
15
6.5
11
13
4
2
2
4
17
2
35 22
72 47
8.9
2
Scheme 2. Synthesis of 30 and 31. Reagents and conditions: (a) PPA, thiosemicar-
bazide, 90 °C, 59–76%; (b) N-Boc (S)-2-amino-3-(4-(trifluoromethyl) phenyl)prop-
anoic acid, EDCI, HOBt, DIEA, DMF; (c) LiAlH4, THF, 0 °C; (d) Pd/carbon, 10%,
cyclohexene; (e) TFA, DCM, 2–11% for four steps; (f) vinyltributyltin, Pd(PPh3)4,
dioxane, 92%; (g) O3, DMS/NaHCO3, 7:1 MeOH/DCM, 88%; (h) NaClO2, NaH2PO4ÁH2O,
2-methyl-2-butene, t-BuOH/H2O, 53%.
5.5 0.1
17
70
19
6
7
6
2,4-Dichloro 18
3,4-Difluoro 14
4
3
43 28
27 21
Data reported as the mean SD where n P 3 except (a) tested once; (b) NT = not
tested.

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Q. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1652–1656
Table 2
The effect of changes in the substitution at the 3 position of the indazole
Compound
R1
R2
AKT1 IC50 (nM)
PKA IC50 (nM)
CDK2 IC50 (nM)
1
Me
Et
Cyclopropyl
Phenyl
H
H
H
H
H
H
H
4-CF3
3-CF3
3-CF3
4-F
4-Cl
4-CF3
2,4-Dichloro
76 19
370a
181 24
413 20
54 12
NTb
72 14
NTb
135 115
22
23
24
25
26
27
28
29
237 60
368 86
117 33
373 93
157 56
108 15
158 88
79
7
25
1
NTb
368
24
8
4
74 12
41 26
6.0
40
1
7
24
8
323a
Data reported as the mean SD where n P 3 except (a) tested once; (b) NT = not tested.
Figure 2. (a) X-ray crystal structures of 18 and 27 with PKA triple mutant designed to mimic the active site of AKT. (b) Model of 18 and 27 docked into the PKA active site.
We were able to obtain X-ray co-crystal structures of several
compounds with a triple-mutant construct of PKA’s kinase domain
that mimics the ATP binding pocket of AKT (Fig. 2, residue number-
ing is based on PKA).11–14 These compounds were observed to bind
in a ‘U’ shaped manner with the indazole, forming two hydrogen
bonds with the amino acid strand that links the N-terminal and
C-terminal domains (often referred to as the linker strand or hinge
region), consisting of a hydrogen bond acceptor to the backbone
NH of Ala123 and a hydrogen bond donor to the backbone carbonyl
O of Glu121. The thiadiazole acted as a hydrogen bond acceptor for
the catalytic Lys72, which is essential for ATP hydrolysis. The pri-
mary amine formed a salt bridge with Asp184 (not displayed in
Fig. 2) and also a hydrogen bond with the amide sidechain of
Asn171. The phenyl ring of the diamine sidechain folded back to-
wards the indazole, forming an edge-to-face contact with the thia-
diazole. Substitutions at C3 and C4 of the diamino sidechain phenyl
ring (15–21) appeared to further stabilize the U-shape through
intramolecular Van der Waals contacts with the indazole and im-
proved contacts with residues Met173, Phe327, Leu49, and
Val57. The phenyl ring also formed intermolecular hydrophobic
contacts with Met173 and Glu127 (not shown). A structurally sim-
ilar series has been reported to bind in a slightly different, ‘Z’
shaped conformation in the PKA triple mutant.14
Careful analysis of crystal structures of 18 and 27 with the PKA
triple mutant, as well as modeling of the two compounds docked15
into the wild type PKA active site, indicated a subtle difference in

Q. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1652–1656
1655
Table 3
Several of the more potent compounds were then profiled in
cell-based assays and rat pharmacokinetics (PK) studies (Table 4).
Two cellular assays were utilized; the first assay measured phos-
phorylation of PRAS40 (Thr246) in U87MG cells via an enzyme-
linked immunosorbent assay (ELISA).16,17 The second assay mea-
sured the translocation of FKHRL1 in a cell immunoassay.18,19 Both
PRAS40 and FKHRL1 have been shown to be direct substrates of
AKT. All of the compounds showed activities in the two cellular as-
says, although cellular potency was much lower than enzymatic
potency (30–200-fold lower). This shift in potency was not directly
correlated with protein binding, permeability, or solubility (data
not shown). Additionally, compound 30 was found to inhibit
cellular proliferation in U87MG cells (EC50 = 380 150 nM), which
correlates with the inhibition of PRAS40 and FKHRL1
phosphorylation.20
The effect of changes in the substitution of the phenyl ring of the indazole
Compound
R
AKT1 IC50
(nM)
PKA IC50
(nM)
CDK2 IC50
(nM)
30
31
3
0.1
8
17
53 23
280 40
274 15
607 15
The compounds were tested in a rat iv PK study.21 Compounds
19 and 28 showed high clearance and high volumes of distribution
(>13 L/kg), with half lives of 4.1 and 2.5 h, respectively. Compound
30, on the other hand, showed a moderate clearance (1.5 L/h/kg,
approximately half of rat hepatic blood flow), low volume of distri-
bution, and reasonable oral bioavailability (20%).
We advanced 30 to a mouse liver pharmacodynamic (PD) assay.
This assay measures phospho-FKHRL1 levels in mouse livers stim-
ulated with exogenously administered hepatocyte growth factor
(HGF).22 Inhibition of phospho-FKHRL1 was measured at 1 h after
treatment in the 30 and 100 mg/kg dose groups (Fig. 3a), and the
effect was dose dependent. There was no reduction in phospho-
AKT23 levels (which is stimulated by HGF), suggesting that the
inhibition of phospho-FKHRL1 was a result of the inhibition of
Data reported as SD where n P 3.
the interaction with a key amino acid (the ‘floor residue’, Met173
in the PKA triple mutant and Leu173 in PKA) that offered a ratio-
nale for the kinase selectivity difference observed between 18
and 27. In the triple-mutant crystal structure, the indazole of 18
makes two key hydrophobic contacts with Met173 (Fig. 2a). In
the case of 27, the crystallographic structures demonstrated that
Met173 was able to slightly reorient its sidechain to accommodate
the space vacated by the removal of the methyl group at the 3-po-
sition of the indazole, thereby maintaining hydrophobic contacts
with the indazole of 27. When these compounds were docked into
wild type PKA, the indazole of compound 18 was also predicted to
make two hydrophobic contacts with one of the delta carbons of
Leu173 (Fig. 2b). However, according to the model of 27, Leu173
was only able to maintain one of the hydrophobic interactions with
the indazole, which likely results from the gamma branching of the
leucine and one less rotatable bond compared to the methionine in
AKT. This net loss of one key hydrophobic interaction in PKA likely
accounted for much of the 10–20-fold difference in potency ob-
served for compounds lacking the indazole methyl group, such as
27. Another cause of the loss of PKA activity in the 3-H indazoles
may be that the added space allowed for a single water molecule
to be trapped and desolvated from bulk solvent thereby leading
to an entropic penalty. Differences in the Leu173/Met173 interac-
tions have been previously implicated in AKT/PKA selectivity ob-
served in other chemical series, although the structurally based
rational for the observed selectivity was different.11,14
In addition to compounds featuring an indazole head group, we
also explored the replacement of the indazole with an isoquinoline,
synthesized as outlined in Scheme 2. Compound 30 showed AKT1
potency similar to indazoles such as 19, but no selectivity over PKA
(Table 3). This lack of selectivity over PKA could be explained by
the isoquinoline occupying a similar volume in the active site as
the 3-methyl indazole, also maintaining two hydrophobic contacts
with the floor residues in both AKT1 and PKA. Methylation at the 3
position of the isoquinoline led to a nearly 100 loss in potency (31),
possibly due to disruption of the U-shaped interaction by the
methyl group. Interestingly, 30 showed improved selectivity over
CDK2 (ꢀ20-fold).
Figure 3. (a) Dose–response PD study with 30 measuring inhibition of phosphor-
FKHRL1. (b) Time course PD study of 30 measuring phosphor-FKHRL1 at 30 mg/kg.
Table 4
Enzymatic, cellular activity, and PK data for 19, 28, and 30
Compound AKT enzymatic IC50 (nM) pPRAS40 cellular IC50 (nM) pFKHRL1 cellular IC50 CL (L/h/kg) iv T1/2 (h) iv VSS (L/kg) iv Cmax (ng/mL) po %F po
19
28
30
5.5 0.1
6.0
3.2 0.07
357 180
444 197
247 107
333 39
154a
695 125
2.8
4.9
1.5
4.1
2.5
3.8
13.7
13.3
3.8
1
430
20
Enzyme and cell data reported as the mean SD where n P 3 except (a) tested once.

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Q. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1652–1656
11. Mutations: PKA Val123 was mutated to AKT Ala, and PKA Leu173 was mutated
to AKT Met. Additionally, PKA Lys181 was mutated to AKT Gln, but this residue
AKT itself and not inhibition of an upstream kinase. FKHRL1 has
also been shown to be phosphorylated at T32 by the SGK kinases.24
Compound 30 was tested in an SKG1 enzymatic assay,25 and was
found to be ꢀ700-fold less potent on SGK1 than AKT1 (SGK1 IC50
faces away from the ATP binding pocket. For
a description of a similar
crystallography strategy see: Caldwell, J. J.; Davies, T. G.; Donald, A.; McHardy,
T.; Rowlands, M. G.; Aherne, G. W.; Hunter, L. K.; Taylor, K.; Ruddle, R.;
Raynaud, F. I.; Verdonk, M.; Workman, P.; Garrett, M. D.; Collins, I. J. Med. Chem.
2008, 51, 2147.
2.2 0.6 lM). This further suggested that the inhibition of phos-
pho-FKHRL1 was driven by AKT inhibition. The PD effect was then
examined in a time course study.26 At 30 mg/kg, compound 30
showed effective inhibition of phospho-FKHRL1 at 1, 3, and 6 h
post-dose ( Fig. 3b). Again, there was no reduction in phospho-
AKT levels in this study.
In conclusion, a series of AKT1 inhibitors is described. SAR stud-
ies resulted in compounds with enzymatic inhibition of AKT1 at
single digit nanomolar IC50 values. In some cases, modest selectiv-
ity over PKA was achieved. The origin of this selectivity can be
rationalized by X-ray crystallographic analysis. Key compounds
showed inhibition of AKT cellular activity as measured by the inhi-
bition of phosphorylation of AKT substrates PRAS 40 and FKHRL1.
Compound 30 had favorable rat PK and in mouse liver PD studies,
showed effective inhibition of phospho-FHKRL1 at 30 mg/kg for up
to 6 h post-dose.
12. The coordinates for the co-crystal structures of 18 and 27 have been deposited
with the RCDB and PBD. 18: RCSB ID code RCSB057006, PDB ID code 3L9M. 27:
RCSB ID code RCSB057007, PDB ID code 3L9N.
13. Gabel, M.; Breitenlechner, C. B.; Rüger, P.; Jucknischke, U.; Schneider, T.; Huber,
R.; Bossemeyer, D.; Engh, R. A. J. Mol. Biol. 2003, 329, 1021.
14. Davies, T. D.; Verdonk, M. L.; Graham, B.; Saalau-Bethell, S.; Hamlett, C. C. F.;
McHardy, T.; Collins, I.; Garrett, M. D.; Workman, P.; Woodhead, S. J.; Jhoti, H.;
Barford, D. J. Mol. Biol. 2007, 367, 882.
15. Lee, M. R.; Sun, Y. J. Chem. Theo. Comp. 2007, 3, 1106.
16. U87MG cells in 5% FBS media were incubated with inhibitors in threefold serial
dilutions for 1 h at 37 °C. The cells were lysed and PRAS40 phosphorylation
was quantified by ELISA assay. Phospho-PRAS40 was normalized to total
PRAS40.
17. Kovacina, K. S.; Park, G. Y.; Bae, S. S.; Guzzetta, A. W.; Schaefer, E.; Birnbaum, M.
J.; Roth, R. A. J. Biol. Chem. 2003, 278, 10189.
18. FKHRL1 nuclear translocation was measured in MDA-MB-468 cells stably
expressing a fluorescence-tagged FKHRL1 fusion protein (FKHRL-GFP). Cells
were treated in threefold serial dilutions of test compounds and nuclear
translocation was evaluated by fluorescence microscopy.
19. Brunet, A.; Bonni, A.; Zigmond, M. J.; Lin, M. Z.; Juo, P.; Hu, L. S.; Anderson, M. J.;
Arden, K. C.; Blenis, J.; Greenberg, M. E. Cell 1999, 96, 857.
20. U-87 glioblastoma cells were seeded on 96-well cell culture plate at 6000 cells/
well, and treated with compounds at indicated concentrations for 3 days. Cell
viability was measured by alamarBlueÒ cell staining (Invitrogen, DAL1100).
The assay was run in triplicate.
21. Compounds were dosed in n = 3 male Sprague/Dawley rats. iv dosing: 1 mg/kg
in 100% DMSO for 19 and 28. 2 mg/kg in 100% DMSO for 30. po dosing: 10 mg/
kg 25% PEG 400/5% water for 30.
Acknowledgements
The authors are indebted to John Allen, Christopher Fotsch, and
Isabelle Dussault for their critical review of this Letter.
References and notes
22. Balb/c female mice (n = 3) were dosed with compounds 30 (ip, 25% PEG400,
75% H2O). 1 h post-dose, AKT signaling was stimulated in mouse liver by
1. Luo, J.; Manning, B. D.; Cantley, L. C. Cancer Cell 2003, 4, 20.
2. Dummler, B.; Hemmings, B. A. Biochem. Soc. Trans. 2007, 35, 231.
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hepatocyte growth factor (HGF) via tail vein injection (5 lg). Five minutes post
HGF stimulation, the mice were sacrificed and livers were harvested for protein
lysate preparation and quantitation by Western blot analysis of phospho-
FKHRL1 (Thr32), normalized to total FKHRL1 levels.
4. Li, Q. Expert Opin. Ther. Pat. 2007, 17, 1077.
23. The phospho-AKT specific antibody detects all three isoforms.
24. Brunet, A.; Park, J.; Tran, H.; Hu, L. S.; Hemmings, B. A.; Greenberg, M. E. Mol.
Cell. Biol. 2001, 21, 952.
25. Zhang, X. L.; Zhang, S. W.; Yamane, H.; Wahl, R.; Ali, A.; Lofgren, J. A.; Kendall, R.
L. J. Biol. Chem. 2006, 281, 13949.
5. Heerding, D. A.; Safonov, I. G.; Verma, S. K. Annu. Rep. Med. Chem. 2008, 42, 365.
6. Johnson, D. A.; Akamine, P.; Radzio-Andzelm, E.; Madhusudan; Taylor, S. S.
Chem. Rev. 2001, 101, 2243.
7. Besson, A.; Dowdy, S. F.; Roberts, J. M. Dev. Cell. 2008, 14, 159.
8. Miller, R. B.; Frincke, J. M. J. Org. Chem. 1980, 45, 5312.
26. Balb/c female mice (n = 3) were dosed with compounds 30 (ip, 25% PEG400,
75% H2O). 1, 3, 6, or 16 h post-dose, AKT signaling was stimulated in mouse
9. AKT1, PKA, SGK1, and CDK2 enzymatic activity was measured using
a
previously described method.25 The ATP concentrations were 10
l
M for all
liver by hepatocyte growth factor (HGF) via tail vein injection (5 lg). Five
kinase reactions. Cyclin 2E was used as the cyclin partner for the CDK2 assay.
10. All compounds were characterized by NMR and MS, and were >95% pure by
HPLC.
minutes post HGF stimulation, the mice were sacrificed and livers were
harvested for protein lysate preparation and quantitation by Western blot
analysis of phospho-FKHRL1 (Thr32), normalized to total FKHRL1 levels