Synthesis of GSK3β mimetic inhibitors of Akt featuring a novel extended dipeptide surrogate

Article abstract of DOI:10.1016/j.bmcl.2011.09.079

Akt is a cardinal nodal point in PI3K signaling pathway and confers resistance to apoptosis through inactivation of regulatory substrates such as GSK3β. Efforts to inhibit the kinase activity of Akt have largely focused on targeting the ATP-binding domain

Full text of DOI:10.1016/j.bmcl.2011.09.079

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7166–7169
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of GSK3b mimetic inhibitors of Akt featuring a novel extended
dipeptide surrogate
Sujeewa Ranatunga a,b, Juan R. Del Valle a,
⇑
a Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
b Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Akt is a cardinal nodal point in PI3K signaling pathway and confers resistance to apoptosis through inac-
tivation of regulatory substrates such as GSK3b. Efforts to inhibit the kinase activity of Akt have largely
focused on targeting the ATP-binding domain of Akt. Here, we present the design and synthesis of con-
formationally constrained GSK3b mimics featuring a novel extended dipeptide surrogate core. This effort
resulted in the identification of a novel substrate mimetic Akt inhibitor (11) with low micromolar activity
Received 4 August 2011
Revised 16 September 2011
Accepted 19 September 2011
Available online 28 September 2011
in vitro (Akt1 IC50 = 3.1 lM).
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Akt
GSK3
Peptidomimetics
Protein–protein interactions
b Strand
Akt (PKB) is a central protein in the phosphoinositide 3-kinase
(PI3K) signaling pathway and a key regulator of cell growth, cell-
cycle progression, transcription, and metabolism.1–3 Akt is known
to phosphorylate over 20 substrates, many of which are involved
in the induction of apoptosis and in the arrest of cell prolifera-
interaction. Like most kinases, a great deal of effort has also been
devoted to inhibition of the ATP-binding pocket of Akt as a direct
means to abrogate its activity.16–21 Given the highly conserved nat-
ure of the PH and ATP-binding domains across a wide range of ki-
nases, the development of selective Akt inhibitors remains a
challenge.
tion.4,5 Intracellular proteins such as GSK3
ab, Bad, and FoxO are
inactivated upon phosphorylation by Akt and thus prevented from
carrying out their normal biological functions. Although mutations
in the genes encoding Akt are less common, hyperactivation of Akt
frequently occurs due to upstream amplification of PI3K or loss of
PTEN, its most important negative regulator.6–11 Enhanced Akt
activity is the hallmark of several aggressive malignancies
including hormone-refractory prostate cancer, breast cancer, and
pancreatic adenocarcinoma.8,12–15 As a result, Akt has emerged as
an attractive target for the development of novel anticancer thera-
peutics.6,7,16–21
Three homologous isoforms of Akt (Akt1, Akt2, and Akt3) shar-
ing >85% sequence homology have been identified in humans.22–24
Each of these isoforms harbors an N-terminal pleckstrin homology
(PH) domain that acts as a binding site for phosphatidylinositol
3,4,5-triphosphate (PIP3). Upon binding PIP3, Akt undergoes a con-
formational change and localizes to the plasma membrane where it
is activated by the kinases PDK1 and mTORC2. Since constitutive
recruitment of Akt results in its hyperactivation, a number of exist-
ing Akt inhibitors are designed to antagonize the PH domain–PIP3
In contrast to the above strategies, several groups have been
interested in developing Akt inhibitors that specifically target the
substrate binding groove adjacent to the ATP-binding site.25–33
While achieving ligand complementarity in the relevant protein–
protein interaction (PPI) region is expected to be more topochem-
ically demanding, such inhibitors may also exhibit better kinase
selectivity relative to PH and ATP-binding domain antagonists.24
Early work based on GSK3b revealed the minimal peptide sequence
required for Akt1 activity, with several GSK3b-based polypeptides
exhibiting IC50 values in the low to sub-micromolar range
(ꢀ10À0.1
l
M).30,31 A subsequent X-ray crystal structure of a ter-
nary Akt1 complex revealed that the GSK3b peptide adopts a
highly extended conformation when bound to the PPI domain
(Fig. 1).34 Recent efforts to reduce peptide character while main-
taining this extended conformation have led to the identification
of peptidomimetic Akt1 inhibitors with activities comparable to
or better than the parent peptides.25–33 Potent bisubstrate inhibi-
tors that covalently link an ATP-site binder to a GSK3b-based
polypeptide have also been reported.35
Recently, our group reported the synthesis of
a suite of
constrained bicyclic scaffolds designed to mimic the conforma-
⇑
Corresponding author. Tel.: +1 813 745 6142; fax: +1 813 745 6817.
E-mail address: juan.delvalle@moffitt.org (J.R. Del Valle).
tional and electronic properties of highly extended dipeptides.36
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.079

S. Ranatunga, J. R. Del Valle / Bioorg. Med. Chem. Lett. 21 (2011) 7166–7169
7167
KHMDS HPMA,
trisyl-N3, THF,
then AcOH
N3
'
δ α
α
CO2tBu
CO2tBu
N
Bn
1
N
84%
EtO2C
EtO2C
Bn
2
> 20:1 dr
1) PPh3, H2O/THF,
then Boc2O, NEt3 BocHN
2)LiBH4, Et2O
1) H2, Pd(OH)2/C,
MeOH
H
BocHN
CO2tBu
CO2tBu
N
4
2) CDI, NEt3, THF
N
86%
92%
Bn
O
O
OH
2 steps
2 steps
3
H
BocHN
1) 2M aq. NaOH, MeOH
2) HBTU, HOBt, NEt3,
H-Phe-OMe, MeCN
H
N
N
O
CO2Me
O
O
60%
2 steps
5
Figure 1. X-ray crystal structure of a GSK3b peptide (green) bound to Akt1 in the
presence of an ATP-binding site inhibitor (yellow). PDB code 1O6K.
Scheme 1. Synthesis of scaffold 4.36
target scaffold proceeded as shown to give 4 in 66% overall yield
from 1 (five steps).36 Although diffraction quality crystals of 4
could not be obtained, converstion to phenylalanyl derivative 5
afforded a crystalline solid suitable for conformational analysis
by X-ray. Interestingly, this compound exists as a head-to-tail di-
mer in the solid state and features two intermolecular H-bonds
at either end of the bicyclic scaffold. The dipeptide surrogate motif
in compound 5 also exhibits a highly extended conformation with
an N–C(O) dipeptide distance very close that found in a typical
b-strand dipeptide (5.9 vs 5.8–6.0 Å).
A family of five N-terminally modified peptidomimetics were
synthesized as shown in Scheme 2. Although selective acidolysis
of scaffold 4 met with difficulties, we found that the t-butyl ester
could be efficiently hydrolyzed under basic conditions. Condensa-
tion with H-Val-Phe-NHBn and treatment with HCl/dioxane affor-
ded intermediate 6 in high yield. Compounds 7 and 8 were
obtained by coupling to the corresponding amino acids, followed
by Boc acidolysis, amine guanidinylation with Goodman’s reagent,
Molecular modeling and X-ray crystallography indicated that some
of our scaffolds do in fact approximate the conformation of dipep-
tides found in b-sheets. In this Letter, we report the design, synthe-
sis, and biological evaluation of peptidomimetic Akt inhibitors
featuring an extended dipeptide surrogate in place of the central
Thr–Thr motif in the Akt-binding domain of GSK3b.
A series of susbtrate mimetic Akt inhibitors was designed based
on the parent GSK3b peptide sequence (GRPRTTSFAE) shown in
Figure 2.34 First, we elected to replace the conformationally ex-
tended Thr–Thr dipeptide with one of our bicyclic scaffolds in an
effort to enforce a sawtooth peptide backbone arrangement. Intro-
duction of the Val-Phe-NHBn motif at the C-terminus was
prompted by SAR previously carried out by Hamilton and cowork-
ers on a series of GSK3b mimics.25,26 We also chose to probe mod-
ifications at the N-terminus while retaining the biologically
important guanidino group. Finally, we settled on incorporation
of a bicyclic carbamate scaffold as our extended dipeptide surro-
gate due to its conformational properties, ease of synthesis, and
the presence of an oxygen atom to more closely mimic the native
threonine side chain.
1) 2Maq. NaOH, MeOH
Ph
Synthesis of the requisite extended dipeptide surrogate com-
menced with homoproline derivative 1, which is readily available
from pyroglutamic acid (Scheme 1).36,37 Regio- and diastereo-
selective electrophilic azidation under optimized conditions affor-
ded azidoester 2 in high yield. The stereochemical outcome of the
reaction was confirmed by X-ray diffraction. Elaboration into the
O
2) HBTU, HOBt, NEt3,
H-Val-Phe-NHBn
H
H
BocHN
H2N
N
CO2tBu
O
. HCl
N
H
N
4
N
3) 4NHCl/dioxane
O
NHBn
82%
O
O
O
6
O
3 steps
steps
(see below)
H
NH2
NH
N
NH
O
O
H
N
O
H
N
O
H2N
N
N
O
O
N
HN
O
H
N
H
H2N
HN
NH
H2N
HN
NH
HN
O
N
H
O
O
NHBn
O
O
NHBn
7
8
CO2H
CO2H
4 steps: a, b, c, b
37% overall
4 steps: d, e, c, e
41% overall
HO
O
O
O
O
O
H
N
H
N
H
N
H
H
H2N
N
N
NH2
NH
N
NH2
N
H
N
H
N
H
OH
N
H
N
H
O
O
O
O
NH
O
O
H
N
OH
O
O
H
N
O
N
H
N
O
H2N
NH
H2N
NH
HN
N
HN
replace with extended
dipeptide surrogate
O
N
H
HN
N
O
O
O
O
H
NHBn
O
9
O
O
NHBn
HN
O
HN
O
CO2H
CO2H
10
3 steps: f, h, e
22% overall
3 steps: f, g, e
H
N
O
O
25% overall
NH2
NH
H
N
H
N
H
H2N
N
N
N
H
N
H
N
N
N
O
H
H
O
O
H
N
O
O
O
O
O
O
N
H
N
OH
O
HN
N
O
truncation
H
truncation
O
O
NHBn
H2N
R
NH
NH
11
2 steps: i, e
33% overall
O
H
H
H
N
N
N
Scheme 2. Reagents and conditions: (a) HBTU, HOBt, NEt3, Boc-Gly-OH, DMF; (b)
4 N HCl/dioxane; (c) Goodman’s reagent, NEt3, DCM; (d) HBTU, HOBt, NEt3, Boc-
NH(CH2)4CO2H, DMF; (e) TFA/DCM; (f) HBTU, HOBt, NEt3, Fmoc Arg(Boc)2-OH,
DMF; (g) BzCl, NEt3, DCM; (h) BnCOCl, NEt3, DCM; (i) HBTU, HOBt, NEt3, Cbz-
Arg(Boc)2-OH, DMF.
N
N
H
O
O
O
O
O
Figure 2. Design of GSK3b mimics.

7168
S. Ranatunga, J. R. Del Valle / Bioorg. Med. Chem. Lett. 21 (2011) 7166–7169
and final deprotection. Compounds 9 and 10 were prepared via
condensation with Fmoc-Arg(Boc)2-OH, acylation, and Boc depro-
tection. Synthesis of 11 employed Cbz-Arg(Boc)2-OH in place of
the Fmoc derivative. All compounds were purified by RP-HPLC
prior to biological evaluation. Curiously, we noted that each of
the five compounds was an inseparable mixture of diastereomers,
indicating racemization of one of the chiral centers. We later iden-
tified the Phe residue to be the site of stereochemical erosion. This
event occurs during synthesis of H-Val-Phe-NHBn as confirmed by
careful HPLC analysis. Similar epimerization has been observed
previously in a related series of compounds.25
an attractive lead compound for drug design. We are currently
evaluating the whole cell activity and protease stability of 11 to en-
able further development. Structure–activity relationship studies
are also underway and will be reported in due course.
Acknowledgments
We thank Dr. Eileen Duesler (University of New Mexico) for car-
rying out small molecule X-ray diffraction. This work was sup-
ported by the Miles for Moffitt Foundation and the Moffitt
Research Institute.
We next evaluated the ability of compounds 7–11 to inhibit
Akt1 in vitro. Compounds were tested in a single dose duplicate
Supplementary data
model at a concentration of 50
control. Inhibition was measured as
(GRPRTSSFAEG) phosphorylation by His-tagged Akt1 in the pres-
ence of 33P-labeled 10
M ATP. While compound 7 exhibited
almost no activity, compounds 8–11 inhibited Akt1 significantly
at 50 M concentration.
l
M using staurosporine as a
a
function of crosstide
Supplementary data (crystallographic data (excluding structure
factors) for compounds 2 and 5 have been deposited with the Cam-
bridge Crystallographic Data Centre as supplementary CCDC publi-
cations numbers 813013 and 813016. Experimental procedures
and spectral data for all new compounds, and copies of NMR spec-
tra for inhibitors 7–11) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.079.
l
l
Dose-response curves were then generated for the more active
compounds (8–11). As shown in Figure 3, each of the GSK3b mim-
ics inhibited the activity of Akt1 in dose-dependent fashion with
IC50 values in the low micromolar range. Based on the structures
of 7–11, the absence of an N-terminal hydrophobic group has a
negative effect on activity, as does a shortening of the guanidine
tether (see Table 1, 7 vs 8). The most potent inhibitor in the series,
Cbz-Arg-[5,6 carbmate scaffold]-Val-Phe-NHBn (11), exhibited an
References and notes
1. Staal, S. P. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5034.
2. Staal, S. P.; Hartley, J. W.; Rowe, W. P. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 3065.
3. Bellacosa, A.; Testa, J. R.; Staal, S. P.; Tsichlis, P. N. Science 1991, 254, 274.
4. Du, K.; Tsichlis, P. N. Oncogene 2005, 24, 7401.
5. Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.; Nicosia, S. V. Oncogene 2005,
24, 7482.
IC50 of 3.1 lM. The enhanced potency of 11 relative to 9 and 10
may also be due to the increased length of the tether bearing the
aromatic moiety group (Cbz vs benzyl or phenacyl). Compound
11 compares favorably with previously reported inhibitors of Akt
in terms of in vitro activity, despite only harboring one canonical
peptide bond (between Val and Phe).
In summary, we have synthesized a set of GSK3b mimics featur-
ing a novel extended dipeptide surrogate. Compounds 7–11 were
designed to demonstrate the ability of our constrained scaffold to
act as a useful conformational probe. Evaluation of these com-
pounds for activity against Akt1 led to the discovery of 11 as a
low micromolar inhibitor. Given the potential advantages of a sub-
strate mimetic strategy for targeting Akt, compound 11 represents
6. Bellacosa, A.; de Feo, D.; Godwin, A. K.; Bell, D. W.; Cheng, J. Q.; Altomare, D. A.;
Wan, M.; Dubeau, L.; Scambia, G.; Masciullo, V.; Ferrandina, G.; Benedetti
Panici, P.; Mancuso, S.; Neri, G.; Testa, J. R. Int. J. Cancer 1995, 64, 280.
7. Cheng, J. Q.; Godwin, A. K.; Bellacosa, A.; Taguchi, T.; Franke, T. F.; Hamilton, T.
C.; Tsichlis, P. N.; Testa, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9267.
8. Altomare, D. A.; Testa, J. R. Oncogene 2005, 24, 7455.
9. Li, J.; Yen, C.; Liaw, D.; Podsypanina, K.; Bose, S.; Wang, S. I.; Puc, J.; Miliaresis,
C.; Rodgers, L.; McCombie, R.; Bigner, S. H.; Giovanella, B. C.; Ittmann, M.;
Tycko, B.; Hibshoosh, H.; Wigler, M. H.; Parsons, R. Science 1997, 275, 1943.
10. Steck, P. A.; Pershouse, M. A.; Jasser, S. A.; Yung, W. K.; Lin, H.; Ligon, A. H.;
Langford, L. A.; Baumgard, M. L.; Hattier, T.; Davis, T.; Frye, C.; Hu, R.;
Swedlund, B.; Teng, D. H.; Tavtigian, S. V. Nat. Genet. 1997, 15, 356.
11. Parsons, R. Semin. Cell. Dev. Biol. 2004, 15, 171.
12. Hsu, J. H.; Shi, Y.; Hu, L.; Fisher, M.; Franke, T. F.; Lichtenstein, A. Oncogene
2002, 21, 1391.
13. Zinda, M. J.; Johnson, M. A.; Paul, J. D.; Horn, C.; Konicek, B. W.; Lu, Z. H.;
Sandusky, G.; Thomas, J. E.; Neubauer, B. L.; Lai, M. T.; Graff, J. R. Clin. Cancer
Res. 2001, 7, 2475.
14. Page, C.; Lin, H. J.; Jin, Y.; Castle, V. P.; Nunez, G.; Huang, M.; Lin, J. Anticancer
Res. 2000, 20, 407.
15. Crowell, J. A.; Steele, V. E.; Fay, J. R. Mol. Cancer Ther. 2007, 6, 2139.
16. Barnett, S. F.; Bilodeau, M. T.; Lindsley, C. W. Curr. Top. Med. Chem. 2005, 5, 109.
17. Heerding, D. A.; Safonov, I. G.; Verma, S. K. Annu. Rep. Med. Chem. 2007, 42, 365.
18. Li, Q. Expert Opin. Ther. Pat. 2007, 17, 1077.
19. Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-
Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E.
Bioorg. Med. Chem. Lett. 2005, 15, 761.
120
8
9
IC50 = 40.2 (28.4 −56.9) μM
IC50 = 16.3 (11.8 −22.4) μM
100
80
60
40
20
0
10 IC50 = 15.0 (11.5 −19.5) μM
11 IC50 3.1 (1.9 −4.8) μM
=
20. Collins, I. Anti-Cancer Agents Med. Chemi. 2009, 9, 32.
21. Garcia-Echeverria, C.; Sellers, W. R. Oncogene 2008, 27, 5511.
22. Fayard, E.; Tintignac, L. A.; Baudry, A.; Hemmings, B. A. J. Cell Sci. 2005, 118,
5675.
-8
-7
-6
-5
-4
-3
Log [Compound] (M)
23. Breitenlechner, C.; Gassel, M.; Engh, R.; Bossemeyer, D. Oncol. Res. 2004, 14, 267.
24. Kumar, C. C.; Madison, V. Oncogene 2005, 24, 7493.
25. Kayser, K. J.; Glenn, M. P.; Sebti, S. M.; Cheng, J. Q.; Hamilton, A. D. Bioorg. Med.
Chem. Lett. 2007, 17, 2068.
Figure 3. Akt1 dose–response curves for 8–11 (95% confidence interval).
26. Kayser-Bricker, K. J.; Glenn, M. P.; Lee, S. H.; Sebti, S. M.; Cheng, J. Q.; Hamilton,
A. D. Bioorg. Med. Chem. 2009, 17, 1764.
27. Litman, P.; Ohne, O.; Ben-Yaakov, S.; Shemesh-Darvish, L.; Yechezkel, T.;
Salitra, Y.; Rubnov, S.; Cohen, I.; Senderowitz, H.; Kidron, D.; Livnah, O.;
Levitzki, A.; Livnah, N. Biochemistry 2007, 46, 4716.
Table 1
Effect of 7–11 on Akt1 activity at 50
l
M inhibitor concentration
% Akt1 activity at 50 lM
Compound
28. Niv, M. Y.; Rubin, H.; Cohen, J.; Tsirulnikov, L.; Licht, T.; Peretzman-Shemer, A.;
Cna’an, E.; Tartakovsky, A.; Stein, I.; Albeck, S.; Weinstein, I.; Goldenberg-
Furmanov, M.; Tobi, D.; Cohen, E.; Laster, M.; Ben-Sasson, S. A.; Reuveni, H. J.
Biol. Chem. 2004, 279, 1242.
29. Enkvist, E.; Lavogina, D.; Raidaru, G.; Vaasa, A.; Viil, I.; Lust, M.; Viht, K.; Uri, A. J.
Med. Chem. 2006, 49, 7150.
Run 1
Run 2
7
8
9
10
11
89.4
23.9
14.6
21.6
2.2
92.1
21.1
16.0
16.8
2.4
30. Alessi, D. R.; Caudwell, F. B.; Andjelkovic, M.; Hemmings, B. A.; Cohen, P. FEBS
Lett. 1996, 399, 333.

S. Ranatunga, J. R. Del Valle / Bioorg. Med. Chem. Lett. 21 (2011) 7166–7169
7169
31. Obata, T.; Yaffe, M. B.; Leparc, G. G.; Piro, E. T.; Maegawa, H.; Kashiwagi, A.;
Kikkawa, R.; Cantley, L. C. J. Biol Chem. 2000, 275, 36108.
34. Yang, J.; Cron, P.; Good, V. M.; Thompson, V.; Hemmings, B. A.; Barford, D. Nat.
Struct. Mol. Biol. 2002, 9, 940.
32. Freeman, N. S.; Tal-Gan, Y.; Klein, S.; Levitzki, A.; Gilon, C. J. Org. Chem. 2011, 76,
3078.
33. Tal-Gan, Y.; Hurevich, M.; Klein, S.; Ben-Shimon, A.; Rosenthal, D.; Hazan, C.;
Shalev, D. E.; Niv, M. Y.; Levitzki, A.; Gilon, C. J. Med. Chem. 2011, 5154.
35. Lee, J. H.; Kumar, S.; Lawrence, D. S. Chem. Bio. Chem. 2008, 9, 507.
36. Ranatunga, S.; Liyanage, W.; Del Valle, J. R. J. Org. Chem. 2010, 75, 5113.
37. Manzoni, L.; Arosio, D.; Belvisi, L.; Bracci, A.; Colombo, M.; Invernizzi, D.;
Scolastico, C. J. Org. Chem. 2005, 70, 4124.