Iminosugar analogues of phosphatidyl inositol as potential inhibitors of protein kinase B (Akt)

Article abstract of DOI:10.1002/ejoc.201100452

A small virtual library of iminosugar derivatives was evaluated by docking experiments carried out by sampling a protein region corresponding to the phosphoinositide binding site of the PH domain of Akt. Four compounds were selected and efficiently synthesised from a common precursor. All compounds were subjected to preliminary biological evaluation on purified enzyme, and - among them - compound 9 exhibited the best inhibitory activity. A small virtual library of iminosugar-based Akt inhibitors have been designed and evaluated by using docking calculations. Selected compounds have been conveniently synthesised, and preliminary biological evaluation identified compound 9 as a possible lead compound for further development of iminosugar-based Akt inhibitors. Copyright

Full text of DOI:10.1002/ejoc.201100452

FULL PAPER
DOI: 10.1002/ejoc.201100452
Iminosugar Analogues of Phosphatidyl Inositol as Potential Inhibitors of
Protein Kinase B (Akt)
Alexandre Orsato,[a] Emanuela Barbagallo,[a] Barbara Costa,[a] Sandro Olivieri,[a]
Luca De Gioia,[a] Francesco Nicotra,[a] and Barbara La Ferla*[a]
Keywords: Iminosugars / Enzymes / Inhibitors / PH domain / Docking / Glycomimetics / Drug design / Enzyme models
A small virtual library of iminosugar derivatives was evalu-
and efficiently synthesised from a common precursor. All
ated by docking experiments carried out by sampling a pro- compounds were subjected to preliminary biological evalu-
tein region corresponding to the phosphoinositide binding
site of the PH domain of Akt. Four compounds were selected
ation on purified enzyme, and – among them – compound 9
exhibited the best inhibitory activity.
Introduction
diphosphate [PI(3,4)P2] (1) with a specific protein domain,
the so-called pleckstrin homology domain (PH). Re-
cently,[2,5] phosphatidylinositol ether lipid analogues with
3–30 μm IC50 inhibitory activities have been synthesized,
and some of them presented good selectivity towards Akt.
Among them, the carbonate derivative 2[6] (Figure 1)
(5.0Ϯ1.9 μm), although less potent than the corresponding
phosphate, is much more selective, and therefore is used as a
lead compound. According to modeling studies,[6] the axial
hydroxymethyl group in position 3 of the inositol ring of
compound 2 has a strong hydrogen-bonding interaction
Protein Kinase B (Akt) is a protooncogenic serine/threo-
nine kinase involved in the PI3K/Akt transduction pathway.
Its activation promotes cellular proliferation, growth and
survival. Its hyperactivation and overexpression is a com-
mon event in many human cancers,[1,2] and this activation
results in tumor cell survival, resistance to apoptosis, and
promotion of tumor formation.[3] Selective inhibitors of
such enzymes represent, therefore, interesting lead com-
pounds for the development of new anticancer drugs. Akt
activation[4] occurs by the binding of phosphatidylinositol
Figure 1. PH domain substrate (1), inhibitor (2) and members of the iminosugar-based virtual library (3–13).
with the Arg25 residue of the Akt PH domain that seems
[a] Department Biotechnology and Bioscience, University of
Milano-Bicocca,
to be important for the selectivity. The corresponding com-
pound with an equatorial hydroxymethyl group showed a
sixfold reduction in Akt inhibition activity.
Piazza della Scienza 2, 20126 Milano, Italy
Fax: +39-02-64483425
E-mail: Barbara.laferla@unimib.it
5012
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Iminosugars as Potential Inhibitors of Protein Kinase B (Akt)
Previous X-ray crystallographic investigations showed the box boundary. In order to avoid possible artifacts due
that inositol-1,3,4,5-tetrakis(phosphate) (IP4) in the phos- to boundary effects, we carried out also a second set of
phoinositide binding site of the PH domain of Akt is posi- docking experiments, in which the docking box was shifted
tioned with the 2-hydroxy group oriented inside the binding and centered on the protein region where molecules 3, 5,
cleft, and that this is also the orientation of [PI(3,4)P2] and 6 and 7 were docked (hereafter referred to as BOX2; see
[PI(3,4,5)P3] in the binding pocket.[2]
Experimental Section).
Moreover, a network of hydrogen bonds in the binding
pocket involves the three phosphate groups linked to the
equatorial 1-, 3- and 4-hydroxy groups of the inositol ring,
as well as the axial hydroxy group in position 2.[7] These
molecular features of the phosphoinositide binding site of
the PH domain of Akt have been already exploited in pre-
vious studies to design and test Akt inhibitors.[3,6,7] Our
analysis of the X-ray structure of the PH domain of Akt
reveals also that, close to the polar pocket discussed above,
a hydrophobic cleft including residues Ile74, Val83, Ile84
is present, suggesting that this hydrophobic cleft might be
potentially exploited to design novel Akt inhibitors.
Results and Discussion
In light of previous considerations, and with the aim of
generating a new class of inhibitors targeting the phospho-
inositide binding site of the Akt PH domain, we have ini-
tially built and tested, using docking calculations, a small
virtual library of iminosugar-based phosphatidyl inositol
analogues. The library (Figure 1) was designed to include
molecules 3–8, 10, which feature the axial hydroxymethyl
group at C3 of inhibitor 2, which seems crucial for the selec-
Figure 2. Docking experiments sampling a protein region corre-
sponding to the phosphoinositide binding site (BOX1); inositol-
1,3,4,5-tetrakis(phosphate) (green); ligands 3, 5, 6, 7 (blue); ligands
4, 8, 10 (white); ligands 9, 11, 12, 13 (red).
Results from docking calculations carried out on BOX1
and BOX2 are collected in Table 1.
Table 1. Docking energies [kcal/mol] computed for compounds 3–
tivity towards Akt. Moreover, we also designed compounds 13 sampling the protein regions labeled as BOX1 (left) and BOX2
9, 11–13 with an axial carboxy group, which should better
(right).
interact with Arg25, which in previous studies was shown
to be one of the key protein residues involved in the interac-
tion with phosphatidylinositol phosphate ligands. In order
to possibly exploit the hydrophobic characteristics of the
cleft close to the natural ligand binding site (including resi-
dues Ile74, Val83, Ile84, see above), the C2 carbon atom of
inhibitor 2 was substituted with a ring nitrogen atom in
order to allow the chemoselective introduction of hydro-
phobic substituents in this position. Thus, derivatives 4–13,
bearing a substituted ring nitrogen atom with different alkyl
or carbamate groups, were considered. Finally, we planned
to substitute the labile phosphate of the natural substrate
by the more stable carboxymethyl group. Since the phos-
phatidyl moiety is not involved in the interaction with the
PH domain, but is required in vivo to anchor the substrate
Ligand
BOX1
MBE[b]
BOX2
MBE[b]
LBE[a]
LBE[a]
3
4
–4.39
–3.68
–3.25
–2.86
–3.19
–2.84
–6.25
–4.31
–4.40
–6.94
–7.36
–3.86
–2.43
–3.25
–2.30
–2.16
–2.39
–5.56
–3.24
–3.78
–5.67
–6.67
–5.45
–4.81
–6.16
–4.87
–4.88
–5.30
–6.25
–5.52
–5.64
–7.57
–7.05
–4.57
–3.75
–5.00
–3.97
–3.83
–4.46
–6.08
–4.13
–4.75
–6.24
–6.21
5
6
7
8
9
10
11
12
13
[a] Lowest binding energy. [b] Mean binding energy; MBE is com-
puted by using poses within 2 Å of the LBE pose.
According to the computational results, compounds 5, 9,
to the cell membrane, we substituted the lipophilic moiety 12 and 13 were predicted to have the highest affinity for the
with a simple ethyl ester so to avoid water solubility prob- protein, followed by compounds 3, 8, 10 and 11.
lems in the in vitro biological test. In light of the above
considerations, we carried out initial docking calculations eled as BOX1 (Figure 2), molecules 9, 11, 12 and 13 were
on the series of compound members of the library. found to bind in the same pocket that hosts the IP4 ligand
In docking experiments sampling the protein region lab-
In the first series of docking experiments carried out (referred to as pocket 1 hereafter). Compounds 4, 8 and 10
sampling a protein region corresponding to the phospho- were predicted to bind in a pocket partially overlapping the
inositide binding site (hereafter referred to as BOX1), it binding site of the natural ligand (referred to as pocket 2),
turned out that not all compounds were predicted to bind whereas, as discussed above, molecules 3, 5, 6, and 7 were
to the same protein region (see Figure 2). In particular, mo- predicted to bind in a novel pocket not superimposed on
lecules 3, 5, 6 and 7 (blue in Figure 1) were found close to the phosphoinositide binding site (pocket 3). When docking
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B. La Ferla, et al.
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calculations were carried out sampling the protein portion precursor (Schemes 1 and 2), which could be differentially
labeled as BOX2, all ligands were found to bind to the same functionalized at the ring nitrogen atom and selectively oxi-
pocket, which essentially corresponded to pocket 3 of the dized at the primary OH group. Commercially available tet-
BOX1 series of calculations (see Figure 3).
rabenzylglucopyranose was reduced to the diol 14, which
was protected at the primary hydroxy group. In order to
obtain the correct stereochemistry at C6 of the target mole-
cules, inversion of configuration was achieved through a
Mitzunobu reaction with concomitant introduction of the
azido function, precursor of the ring nitrogen atom (com-
pound 16). Removal of the TBDPS group and oxidation of
the free OH group afforded aldehyde 18. The introduction
of the carboxymethyl moiety was done by exploiting a nu-
cleophilic attack of AcOEt-derived enolate on aldehyde 18.
This reaction afforded azido alcohol 19 as an inseparable
diastereoisomeric mixture in an approximate (R)/(S) ratio
Figure 3. Docking experiments sampling a protein region corre-
sponding to BOX2; ligands 3, 4, 5, 6, 7, 8, 9 (blue); inositol-1,3,4,5-
tetrakis(phosphate) (green).
Notably, pocket 3 is sufficiently close to the natural li-
gand binding site to suggest that the interaction between
the tested ligands and the protein might indirectly affect
binding of the natural ligands. All molecules except mole-
cule 13 show better docking scores for the BOX2 binding
site. However, molecules 9, 12 and 13 show very similar
affinity for both BOX1 and BOX2 sites.
Scheme 1. Reagents and conditions: (i) NaBH4, CH2Cl2/EtOH
(1:1), quantitative; (ii) imidazole, TBDPSiCl, CH2Cl2, 97%;
(iii) PPh3, DIAD, DPPA, THF, 75%; (iv) TBAF (1 m), THF, 90%;
(v) Dess–Martin periodinane, CH2Cl2, 79%; (vi) DIPA, tBuLi
(1 m), 0 °C, AcOEt, THF, –78 °C, 95%; (vii) Lindlar catalyst/C, H2,
AcOEt, 86%; (viii) PPh3, DIAD, THF, 0 °C to room temp., 70%.
In the light of the docking results, four molecules (3, 4, 5,
9), which are predicted to bind to the three different binding
pockets, have been selected and synthesized. A preliminary
in vitro biological inhibition assay has also been carried
out.
Compounds 5 and 9 were selected from the group with
the highest affinity for the protein and also as representa-
tives of the group of compounds that bind pockets 3 and
1, respectively. Compound 3 was also selected to evaluate
pocket 3, with the second highest affinity in the group of
compounds binding to this pocket. Compound 4 was se-
lected to evaluate the biological activity of the group of
compounds that bound to pocket 2 in the computational
experiments. The reason for this choice is the lowest binding
affinity of 4 that resulted from the experiment in BOX 2,
which corresponds to the binding to pocket 3. Therefore,
compound 4 has the lowest probability of binding to
pocket 3, which raises the probability of binding to
pocket 2. Even though the highest affinities were obtained
by compounds 12 and 13, the difficulty of purchasing com-
mercially available starting materials for their synthesis
hampered their preparation. The synthesis was designed in
Scheme 2. Reagents and conditions: (i) Pd(OH)2/C, CH3COOH,
H2, MeOH, 67% for 3, quantitative for 4 and 5; (ii) CbzCl,
NaHCO3, MeOH, 25%; (iii) TEMPO, KBr, NaOCl 5%, H2O,
84%; (iv) propionaldehyde, CH3COOH, Na2SO4, Na(AcO)3BH,
1,2-dichloroethane, 52%; (v) cyclohexanecarbaldehyde, CH3-
order to obtain all the target compounds from a common COOH, Na2SO4, Na(AcO)3BH, 1,2-dichloroethane, 79%.
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Iminosugars as Potential Inhibitors of Protein Kinase B (Akt)
of 2:1, determined by NMR spectroscopy on the final cy- their activity may be a consequence of the modification of
clized products. Reduction of the azido function and cycli- the phosphoinositide binding pocket following their bind-
zation through an intramolecular Mitzunobu reaction led ing.
to the desired iminosugar scaffold 21.
Table 2. Inhibition of Akt activity.
The first member of the library was obtained by depro-
tection of 21 through a catalytic hydrogenation [Pd(OH)2/C,
H2]. Target compound 3 was efficiently separated by flash
chromatography from the minor isomer. Compound 9 was
obtained directly from pure compound 3. First the ring ni-
trogen atom was functionalized as a carboxybenzylcarb-
amate, and then the primary hydroxy group was selectively
oxidized to the corresponding carboxylic acid, affording
compound 9 in 21% overall yield. The synthesis of deriva-
tives 4 and 5 was carried out by reductive amination on
precursor 21 with propionaldehyde and cyclohexanecarbal-
dehyde, respectively, followed by catalytic hydrogenation.
This time the diastereoisomeric mixtures were efficiently
separated before the deprotection step. The stereochemistry
at C2 was determined on the deprotected compounds. As
an illustrative example, we report the NMR values of the
coupling constants of compound 3 (major isomer) (Fig-
ure 4). The values (J3,2 = 8.7, J4,3 = 8.7, Hz) were indicative
of a trans-diaxial disposition of the protons, which indi-
cated a 4C1 conformation; moreover, the diaxial disposition
of C(2)–H/C(3)–H allowed us to determine the absolute (S)
configuration of the C2 center.
Ligand
Inhibition [%]
3
4
14.87Ϯ0.133
25.67Ϯ0.882
20.00Ϯ0.577
31.70Ϯ1.670
75.46Ϯ1.059
5
9
2[a]
[a] Known inhibitor.[6]
Conclusions
In our search for Akt-selective inhibitors targeting the
PH domain, we developed a unique iminosugar scaffold
from which a variety of inhibitors have been synthesised by
chemoselective derivatisation of the ring nitrogen atom and
oxidation of the primary hydroxy group. Further members
of the library can be chemoselectively generated by modifi-
cation of the ester group and derivatization of the primary
hydroxy group. The introduction of a lipophilic appendage
at the ring nitrogen atom allowed us to decipher the role of
the hydrophobic cleft close to the natural ligand binding
site revealed by our analysis of the X-ray structure of the
PH domain of Akt. Preliminary biological evaluation
showed inhibitory activities that justify the choice of com-
pound 9 as a lead compound for further development of
iminosugar-based Akt inhibitors.
Figure 4. NMR values of the coupling constants of compound 3.
Experimental Section
Preliminary biological evaluations were performed
through a safe, rapid and reliable method for screening of
inhibitors of Akt: a solid-phase enzyme-linked immuno-
Docking Calculations: The X-ray structure of the human Akt com-
plex was obtained from the Protein Data Bank (code 1H10). Three-
dimensional structures of ligands were generated by molecular me-
sorbent assay (ELISA) that utilizes a synthetic peptide as a chanics optimization using the MMFF94 force field.[8] Docking
calculations were carried out by using AutoDock 4.2.[9] The tor-
substrate for Akt and a polyclonal antibody that recognizes
sional degrees of freedom of the ligands were explicitly considered,
the phosphorylated form of the substrate. All the tested
whereas the protein structure was kept frozen to X-ray atomic coor-
compounds (3, 4, 5 and 9) exhibited a significant inhibitory
dinates. Two boxes were used: BOX1, whose center is fixed on coor-
effect on the activity of the purified enzyme when assayed at
dinates X = 16.677, Y = 25.705, Z = 13.116 (centered in proximity
700 μm (Table 2). Compound 9 showed the best inhibitory
of the natural ligand binding site); BOX2, was centered on coordi-
nates X = 27.627, Y = 19.412, Z = 19.899 (centered in a contiguous
activity, in accordance to the calculated binding energy ob-
tained by docking experiments carried out in BOX1, which
contains the phosphoinositide binding site. This activity
may be ascribed to the presence of the carboxy group, prob-
site of the natural ligand binding site), and its dimension was set
to 20.25ϫ21.75ϫ21 Å3. Atomic charges were calculated within
AutoDock 4.2 by using the Gasteiger-Marsili algorithm.[10] The se-
ably involved in favourable interactions with basic residues arch for best poses was carried out with a Lamarckian genetic algo-
rithm by using the following parameters: run, 100; population size,
300; maximum number of evaluations, 11000000. Two-dimensional
representations of the best protein–ligand adducts were obtained
by using MOE Ligand Interactions Structure-Based Design
Tool.[11] In all in silico experiments, we used the PDB code 1H10
structure that contains the Akt PH domain fragment alone. The
site identified with BOX2 sampling might be not accessible to li-
gands in vivo, since in the whole protein structure it could not be
excluded that the binding site located by docking experiments is
buried. However, we identified another protein with a PH domain
present in the binding pocket. The hydrophobic group pres-
ent on the ring nitrogen atom may also play a role in the
interaction. For instance, compound 3, which lacks this
group, showed the lowest activity. Compounds 4 and 5 both
present a hydrophobic group; in this case the slightly higher
activity observed for compound 4 may be due to the more
flexible propyl group, which may better fit the hydrophobic
cleft. Moreover, from docking calculations, compounds 3
and 5 are predicted to bind the protein region named
pocket 3, close to the phosphoinositide binding site; thus, showing a high sequence similarity to the Akt PH domain (data
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B. La Ferla, et al.
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not shown), in which the region corresponding to BOX2 is access-
ible. While our analysis was underway, a new X-ray structure of
human Akt1 complexed with an allosteric inhibitor was uploaded
in the Protein Data Bank (code 3O96). The structure of the newly
reported PH domain is essentially superimposable with the struc-
ture used in our calculations.
5-Azido-2,3,4,6-tetra-O-benzyl-1-O-tert-butyldiphenylsilyl-5-
deoxy-L-iditol (16): Compound 15 (5.28 g, 6.76 mmol) was dis-
solved in dry THF (20 mL) under argon. Triphenylphosphane
(5.32 g, 20.30 mmol) was added. The reaction mixture was cooled
to 0 °C, and DIAD (3.93 mL, 20.30 mmol) was added dropwise,
resulting in
a yellow precipitate. Diphenylphosphoryl azide
(4.68 mL, 21.64 mmol) was added. After 2 h, the solvent was evap-
orated in vacuo. Purification by flash chromatography (petroleum
ether/ethyl acetate, 9:1) afforded pure compound 16 (4.10 g, 75.2%
yield). [α]2D5 = –1.7 (c = 1.2, CHCl3). 1H NMR (400 MHz): δ = 1.07
[s, 9 H, C(CH3)3], 3.23–3.26 (m, 1 H, 5-H), 3.34 (dd, J = 9.5,
4.8 Hz, 1 H, 6a-H), 3.54–3.58 (m, 2 H, 2-H, 6b-H), 3.82 (dd, J =
7.8, 3.0 Hz, 1 H, 4-H), 3.86–3.91 (m, 2 H, 1a,b-H), 4.04 (dd, J =
7.8, 2.8 Hz, 1 H, 3-H), 4.31 (d, J = 11.8 Hz, 1 H, CHPh), 4.38 (d,
J = 11.9 Hz, 1 H, CHPh), 4.39 (d, J = 11.9 Hz, 1 H, CHPh), 4.54
(d, J = 11.5 Hz, 1 H, CHPh), 4.62 (d, J = 11.8 Hz, 1 H, CHPh),
4.66 (d, J = 11.2 Hz, 1 H, CHPh), 4.76 (d, J = 11.2 Hz, 1 H,
CHPh), 4.78 (d, J = 11.5 Hz, 1 H, CHPh), 7.20–7.80 (m, 30 H, Ar-
H) ppm. 13C NMR (100 MHz): δ = 19.6 [C(CH3)3], 27.3 [C-
(CH3)3], 61.6 [C(5)], 62.6, 70.2 [C(1)], 72.7, 73.5, 75.2, 75.6 (4
CH2Ph), 78.1 [C(2)], 78.6 [C(4)], 79.3 [C(3)], 127.8–130.1 (CHAr),
133.40 (CqAr), 135.8–135.9 (CHAr), 138.0, 138.1, 138.3, 138.4 (4
CqAr) ppm. HRMS: calcd. for C50H56N3O5Si [M + H]+ 806.3984;
found 806.3798.
Synthesis
General Remarks: All solvents were dried with molecular sieves for
at least 24 h prior to use. Thin layer chromatography (TLC) was
performed on silica gel 60 F254 plates (Merck) with UV light detec-
tion when possible, or by charring with a solution of concd. H2SO4/
EtOH/H2O (10:45:45) or a solution of (NH4)6Mo7O24 (21 g),
Ce(SO4)2 (1 g), concd. H2SO4 (31 mL) in water (500 mL). Flash
column chromatography was performed on silica gel 230–400 mesh
(Merck). H and 13C NMR spectra were recorded at 25 °C with a
1
Varian Mercury 400 MHz instrument by using CDCl3 as the sol-
vent unless otherwise stated. Chemical shift assignments, reported
in ppm, were referenced to the corresponding solvent peaks. HR
mass spectra were recorded with a QSTAR elite LC/MS/MS system
with a nanospray ion source. Optical rotations were measured at
room temperature by using an Atago Polax-2 L polarimeter and
are reported in units of 10–1 degcm2 g–1.
2,3,4,6-Tetra-O-benzyl-D-glucitol
(14):
NaBH4
(420 mg,
5-Azido-2,3,4,6-tetra-O-benzyl-5-deoxy-L-iditol (17): Compound 16
11.09 mmol) was added to a stirred solution of 2,3,4,6-tetra-O-
benzyl-d-glucopyranose (2.00 g, 3.70 mmol) in CH2Cl2/EtOH (1:1)
(20 mL). After 48 h, the solvent was evaporated in vacuo, and the
solid was treated with Na2CO3 (10 mL of satd. solution), with stir-
ring, for 10 min. The reaction mixture was diluted with CH2Cl2,
washed with distilled water, dried with Na2SO4 and filtered. The
solvent was evaporated in vacuo to afford compound 14 (2.01 g,
quantitative) as a colorless syrup. [α]2D0 = +3.1 (c = 1.2, CHCl3). 1H
NMR (400 MHz): δ = 3.60 (dd, J = 11.9, 4.8 Hz, 1 H, 1a-H), 3.64–
3.72 (m, 2 H, 6a,b-H), 3.77 (dd, J = 11.9, 4.3 Hz, 1 H, 1b-H), 3.80–
3.86 (m, 2 H, 2-H, 4-H), 3.94 (dd, J = 6.2, 3.6 Hz, 1 H, 3-H), 4.06–
4.10 (m, 1 H, 5-H), 4.54 (d, J = 11.9 Hz, 1 H, CHPh), 4.57–4.59
(m, 2 H, CHPh), 4.63 (d, J = 11.4 Hz, 1 H, CHPh), 4.66 (d, J =
11.5 Hz, 1 H, CHPh), 4.69 (d, J = 11.2 Hz, 1 H, CHPh), 4.70 (d,
J = 11.5 Hz, 1 H, CHPh), 4.75 (d, J = 11.2 Hz, 1 H, CHPh), 7.20–
7.40 (m, 20 H, Ar-H) ppm. 13C NMR (100 MHz): δ = 62.2 [C(1)],
71.1 [C(5)], 71.5 [C(6)], 73.4, 73.6, 73.8, 74.9 (4 CH2Ph), 77.7, 79.4,
79.8 [C(2), C(3), C(4)], 128.0–128.7 (CHAr), 138.0, 138.1, 138.2,
138.4 (CqAr) ppm. HRMS: calcd. for C34H39O6 [M + H]+
543.2741; found 543.2957.
(4.10 g, 5.09 mmol) was dissolved in dry THF (15 mL) under ar-
gon. A solution of TBAF (1 m, 15.26 mL, 15.26 mmol) was added
slowly. After 3 h, the solvent was evaporated in vacuo. Purification
by flash chromatography (petroleum ether/ethyl acetate, 8:2) af-
forded pure compound 17 (2.61 g, 90.4% yield). [α]2D5 = +11.0 (c =
1
1.1, CHCl3). H NMR (400 MHz): δ = 3.40 (dd, J = 9.4, 4.8 Hz,
1 H, 6a-H), 3.47–3.55 (m, 3 H, 2-H, 5-H, 6b-H), 3.56–3.61 (m, 1
H, 1a-H), 3.67–3.73 (m, 1 H, 1b-H), 3.77–3.83 (m, 2 H, 3-H, 4-H),
4.32 (d, J = 11.8 Hz, 1 H, CHPh), 4.36 (d, J = 11.8 Hz, 1 H,
CHPh), 4.49 (d, J = 11.5 Hz, 2 H, 2 CHPh), 4.54 (d, J = 11.5 Hz,
1 H, CHPh), 4.56 (d, J = 11.2 Hz, 1 H, CHPh), 4.62 (d, J =
11.2 Hz, 1 H, CHPh), 4.69 (d, J = 11.5 Hz, 1 H, CHPh), 7.15–7.30
(m, 20 H, Ar-H) ppm. 13C NMR (100 MHz): δ = 61.4 [C(5)], 61.7
[C(1)], 69.8 [C(6)], 72.7, 73.6, 75.0, 75.1 (4 CH2Ph), 78.2, 78.3, 79.2
[C(2), C(3), C(4)], 127.9–128.7 (CHAr), 137.8, 137.9, 137.9, 138.0
(4 CqAr) ppm. HRMS: calcd. for C34H38N3O5 [M + H]+ 568.2806;
found 568.2789.
5-Azido-2,3,4,6-tetra-O-benzyl-5-deoxy-L-idose (18): Compound 17
(2.61 g, 4.60 mmol) was dissolved in CH2Cl2 (20 mL). Dess–Martin
periodinane (2.93 g, 6.90 mmol) was added slowly. After 40 min, a
1:1 solution of satd. NaHCO3/10% Na2S2O3 (100 mL) was added.
After stirring for 30 min, the reaction mixture was diluted with
CH2Cl2 and then washed with water. The organic layer was dried
with Na2SO4, filtered, and the solvent was evaporated in vacuo.
Purification by flash chromatography (petroleum ether/ethyl acet-
ate, 8:2) afforded pure aldehyde 18 (1.98 g, 79.1% yield), which was
immediately used in the next reaction without further characteriza-
tion, due to its low chemical stability.
2,3,4,6-Tetra-O-benzyl-1-O-tert-butyldiphenylsilyl-D-glucitol (15):
Compound 14 (2.00 g, 3.68 mmol) was dissolved in dry CH2Cl2
(12 mL) under argon. Imidazole (550 mg, 8.10 mmol) and
TBDPSiCl (1.04 mL, 4.05 mmol) were added. After 1 h, the reac-
tion mixture was quenched with methanol (0.5 mL), diluted with
H2O, and extracted with CH2Cl2. The organic layer was dried with
Na2SO4, filtered, and the solvent was evaporated in vacuo. Purifi-
cation by flash chromatography (petroleum ether/ethyl acetate, 8:2)
afforded pure compound 15 (2.79 g, 97% yield). [α]2D5 = +12.8 (c =
1.0, CHCl3). 1H NMR (400 MHz): δ = 1.08 [s, 9 H, C(CH3)3], 2.97
Ethyl (3R/S,4S,5S,6R,7R)-7-Azido-4,5,6,8-tetrakis(benzyloxy)-3-hy-
(d, J = 4.9 Hz, 1 H, OH), 3.62–3.64 (m, 2 H, 6a,b-H), 3.76–4.03 droxyoctanoate (19): Diisopropylamine (4.94 mL, 34.99 mmol) was
(m, 6 H, 1a,b-H, 2-H, 3-H, 4-H, 5-H), 4.48–4.59 (m, 5 H, 5 CHPh), dissolved in dry THF (10 mL) under argon. At 0 °C, a solution
4.64–4.70 (m, 3 H, 3 CHPh), 7.20–7.80 (m, 30 H, Ar-H) ppm. 13C of tert-butyllithium (1 m, 21.87 mL, 34.99 mmol) was added. After
NMR (100 MHz): δ = 19.7 [C(CH3)3], 27.3 [C(CH3)3], 63.5, 71.6
[C(1), C(6)], 71.4 [C(5)], 73.4, 73.6, 73.7, 74.6 (4 CH2Ph), 77.8, 78.3,
79.9 [C(2), C(3), C(4)], 127.8–129.9 (CHAr), 133.5, 133.6 (CqAr),
135.8 (CHAr), 138.3, 138.3, 138.4, 138.6 (4 CqAr) ppm. HRMS:
calcd. for C50H57O6Si [M + H]+ 781.3919; found 781.3900.
stirring for 0.5 h, the temperature was reduced to –78 °C, and dry
ethyl acetate (3.42 mL, 34.99 mmol) was added. After stirring for
0.5 h, a solution of compound 18 (1.98 g, 3.50 mmol) in dry THF
(5 mL) was added. After 1 h, the reaction mixture was treated with
a satd. solution of NH4Cl (30 mL), taken to room temperature, and
5016
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Eur. J. Org. Chem. 2011, 5012–5019

Iminosugars as Potential Inhibitors of Protein Kinase B (Akt)
neutralized with 5% HCl. The reaction mixture was dissolved in
CH2Cl2 and washed with water. The organic layer was dried with
Na2SO4, filtered, and the solvent was evaporated in vacuo. Purifi-
cation by flash chromatography (petroleum ether/ethyl acetate, 8:2)
afforded compound 19 as a mixture of stereoisomers [2.18 g, 95.1%
yield, (3R)/(3S) = 2:1]. Major Isomer: 1H NMR (400 MHz): δ =
1.23 (m, 3 H, OCH2CH3), 2.49–2.56 (m, 1 H, 2a-H), 2.72 (dd, J =
16.0, 3.6 Hz, 1 H, 2b-H), 3.50–3.68 (m, 4 H, 4-H, 5-H, 6-H, 7-H),
3.96 (dd, J = 7.0, 3.0 Hz, 1 H, 8a-H), 4.00 (dd, J = 7.0, 3.6 Hz, 1
H, 8b-H), 4.09–4.18 (m, 2 H, CH2CO2Et), 4.26–4.32 (m, 1 H, 3-
H), 4.40–4.49 (m, 2 H, CH2Ph), 4.59–4.72 (m, 5 H, CHPh), 4.80
(d, J = 11.6 Hz, 1 H, CHPh), 7.23–7.40 (m, 20 H, Ar-H) ppm.
13C NMR (100 MHz): δ = 14.4 (OCH2CH3), 38.7, 39.1 [C(2)], 60.8
(OCH2CH3), 61.3, 61.4 [C(7)], 67.5, 69.0 [C(3)], 69.6, 69.9, 73.2,
73.3, 73.4, 74.1, 74.5, 74.7, 74.8, 75.0 [CH2Ph, C(8)], 77.8, 78.1,
78.1, 78.9, 79.0, 79.7 [C(4), C(5), C(6)], 127.9–128.7 (CHAr), 137.6,
137.7, 137.8, 137.9, 138.0, 138.1, 138.2 (CqAr), 172.2, 172.6 [C(1)]
ppm. HRMS: calcd. for C38H44N3O7 [M + H]+ 654.3174; found
654.3748.
172.2 (C=O) ppm. HRMS: m/z = calcd. for C38H44NO6 [M + H]+
610.3163; found 610.4024.
Ethyl [(2S,3S,4R,5R,6S)-3,4,5-Trihydroxy-6-(hydroxymethyl)piper-
idin-2-yl]acetate (3): The diastereoisomeric mixture 21 (26.4 mg,
0.043 mmol) was dissolved in methanol (2 mL). Catalytic amounts
of Pd(OH)2 and acetic acid (0.1 mL) were added, and the reaction
mixture was stirred under H2 overnight. The catalyst was filtered
through a Celite pad (eluent methanol), and the solvent was evapo-
rated in vacuo. Purification by flash chromatography (ethyl acetate/
methanol, 8:2) afforded pure compound 3 as the major isomer
(7.2 mg, 66.7% yield). [α]2D5 = –30.1 (c = 0.7, MeOH). 1H NMR
(400 MHz, CD3OD): δ = 1.26 (t, J = 7.1 Hz, 3 H, OCH2CH3), 2.36
(dd, J = 16.3, 8.8 Hz, 1 H, CHCO2Et), 2.91 (dd, J = 16.3, 3.3 Hz,
1 H, CHCO2Et), 3.10 (t, J = 8.7 Hz, 1 H, 3-H), 3.17 (ddd, J = 9.0,
8.9, 3.4 Hz, 1 H, 2-H), 3.20–3.27 (m, 1 H, 6-H), 3.42 (t, J = 8.7 Hz,
1 H, 4-H), 3.70 (dd, J = 9.2, 5.4 Hz, 1 H, 5-H), 3–75–3.80 (m, 2
H, 7a,b-H), 4.12–4.19 (m, 2 H, OCH2CH3) ppm. 13C NMR
(100 MHz, CD3OD): δ = 14.5 (OCH2CH3), 38.2 (CH2CO2Et),
52.1, 59.0, 73.3, 76.0, 76.3 [C(2), C(3), C(4), C(5), C(6)], 58.1, 61.8,
(OCH2CH3, CH2OH), 174.0 (C=O) ppm. HRMS: calcd. for
C10H20NO6 [M + H]+ 250.1285; found 250.1246.
Ethyl (3R/S,4S,5S,6R,7R)-7-Amino-4,5,6,8-tetrakis(benzyloxy)-3-
hydroxyoctanoate (20): Compound 19 (240 mg, 0.36 mmol) was dis-
solved in ethyl acetate (3 mL). A catalytic amount of Lindlar cata-
lyst was added, and the reaction mixture was stirred under H2 over-
night. The catalyst was filtered through a Celite pad (eluent ethyl
acetate), and the solvent was evaporated in vacuo. Purification by
flash chromatography (petroleum ether/ethyl acetate, 3:7) afforded
compound 20 as a mixture of stereoisomers [190 mg, 85.6% yield,
(3R)/(3S) = 2:1). Major Isomer: 1H NMR (400 MHz): δ = 1.20–
1.30 (m, 3 H, OCH2CH3), 2.49–2.60 (m, 1 H, 2a-H), 2.75 (dd, J =
16.0, 3.7 Hz, 1 H, 2b-H), 3.12 (dt, J = 6.4, 2.4 Hz, 1 H, 7-H), 3.30–
3.45 (m, 2 H, 8a,b-H), 3.61 (dd, J = 6.6, 3.6 Hz, 1 H, 4-H), 3.89
(dd, J = 7.6, 2.5 Hz, 1 H, 6-H), 4.04–4.15 (m, 3 H, OCH2CH3, 5-
H), 4.28–4.36 (m, 1 H, 3-H), 4.03 (d, J = 11.9 Hz, 1 H, CHPh),
4.44 (d, J = 11.9 Hz, 1 H, CHPh), 4.51 (d, J = 11.2 Hz, 1 H,
CHPh), 4.58 (d, J = 11.3 Hz, 1 H, CHPh), 4.64–4.84 (m, 4 H,
CHPh), 7.20–7.40 (m, 20 H, Ar-H) ppm. 13C NMR (100 MHz): δ
= 14.1 (OCH2CH3), 38.5, 38.7 [C(2)], 51.4, 51.5 [C(7)], 60.4
(OCH2CH3), 67.7, 68.8 [C(3)], 72.9, 73.4, 74.1, 74.3, 74.4, 74.5, 74.6
[CHPh, C(8)], 78.6, 78.9, 79.1, 79.4, 79.8, 80.1 [C(4), C(5), C(6)],
127.7, 128.6 (CHAr), 138.1, 138.2, 138.3, 138.5, 138.6 (CqAr),
172.2 [C(1)] ppm. HRMS: calcd. for C38H46NO7 [M + H]+
628.3269; found 628.3371.
Ethyl {(2S,3S,4R,5R,6S)-3,4,5-Tris(benzyloxy)-6-[(benzyloxy)meth-
yl]-1-propylpiperidin-2-yl}acetate (23): The mixture of isomers 21
(115.4 mg, 0.19 mmol) was dissolved in dry 1,2-dichloroethane
(5 mL), then propionaldehyde (41 μL, 0.57 mmol) and acetic acid
(0.108 mL, 1.89 mmol) were added. The reaction mixture was dried
with Na2SO4 and stirred for 0.5 h. Na(AcO)3BH (160.4 mg,
0.76 mmol) was added, and the mixture was stirred overnight. The
reaction mixture was neutralized with a satd. solution of NaHCO3,
dissolved in CH2Cl2, and washed with water. The organic layer was
dried with Na2SO4, filtered, and the solvent was evaporated in
vacuo. Purification by flash chromatography (petroleum ether/ethyl
acetate, 9:1) afforded pure compound 23 (64.0 mg, 51.9% yield) as
the major isomer. [α]2D5 = –38.6 (c = 1.4, CHCl3). 1H NMR
(400 MHz): δ = 0.83 [t, J = 7.3 Hz, 3 H, N(CH2)2CH3], 1.17 (t, J
= 7.1 Hz, 3 H, OCH2CH3), 1.37–1.49 (m, 2 H, NCH2CH2CH3),
2.40–2.53 (m, 2 H, CHCO2Et, NCHCH2CH3), 2.62 (ddd, J = 12.6,
8.6, 6.2 Hz, 1 H, NCHCH2CH3), 2.73 (dd, J = 15.1, 3.9 Hz, 1 H,
CHCO2Et), 3.32–3.44 (m, 3 H, 2-H, 3-H, 6-H), 3.66–3.74 (m, 1 H,
CHOBn), 3.74–3.81 (m, 2 H, CHOBn, 4-H), 3.84 (dd, J = 10.1,
3.4 Hz, 1 H, 5-H), 3.97–4.10 (m, 2 H, OCH2CH3), 4.50–4.78 (m, 6
H, 6 CHPh), 4.89–4.98 (m, 2 H, 2 CHPh), 7.20–7–43 (m, 20 H,
Ar-H) ppm. 13C NMR (100 MHz): δ = 11.90 [N(CH2)2CH3], 14.42
(OCH2CH3), 22.52 (NCH2CH2CH3), 36.15 (CH2CO2Et), 49.99
(NCH2CH2CH3), 56.75, 57.93 [C(2), C(6)], 60.76, 67.84
(OCH2CH3, CH2OBn), 72.98, 73.65, 75.00, 75.50 (4 CH2Ph),
79.08, 80.94, 84.37 [C(3), C(4), C(5)], 127.6–129.6 (CHAr),138.45,
138.45, 139.05, 139.05 (CqAr), 172.25 (C=O) ppm. HRMS: calcd.
for C41H50NO6 [M + H]+ 652.3416; found 652.3633.
Ethyl
{(2S/R,3S,4R,5R,6S)-3,4,5-Tris(benzyloxy)-6-[(benzyloxy)-
methyl]piperidin-2-yl}acetate (21): Compound 20 (170 mg,
0.27 mmol) was dissolved in dry THF (1 mL) under argon. The
reaction mixture was chilled to 0 °C, and triphenylphosphane
(140 mg, 0.54 mmol) was added. DIAD (0.10 mL, 0.54 mmol) was
added dropwise, and the reaction mixture was stirred overnight.
The solvent was removed in vacuo, and purification by flash
chromatography (petroleum ether/ethyl acetate, 7:3) afforded com-
pound 21 as a mixture of stereoisomers [0.12 g, 70.4% yield, (2S)/
Ethyl
[(2S,3S,4R,5R,6S)-3,4,5-Trihydroxy-6-(hydroxymethyl)-1-
(2R) = 2:1]. Major Isomer: 1H NMR (400 MHz): δ = 1.21–1.24 (m, propylpiperidin-2-yl]acetate (4): Compound 23 (64.0 mg,
3 H, OCH2CH3), 2.32 (dd, J = 15.5, 8.1 Hz, 1 H, CHCO2Et), 2.82 0.098 mmol) was dissolved in ethyl acetate (2 mL) and methanol
(dd, J = 15.5, 4.3 Hz, 1 H, CHCO2Et), 3.20–3.28 (m, 2 H, 2-H, 3- (4 mL). Catalytic amounts of Pd(OH)2 and acetic acid (0.1 mL)
H), 3.49–3.58 (m, 1 H, CHOBn), 3.62–3.68 (m, 2 H, 4-H, CHOBn),
3.77 (bt, J = 9.9 Hz, 1 H, 5-H), 3.79 (dd, J = 9.4, 5.9 Hz, 1 H, 6-
H), 4.01–4.15 (m, 2 H, OCH2CH3), 4.52–4.69 (m, 5 H, CHPh),
4.74 (d, J = 10.8 Hz, 1 H, CHPh), 4.91 (d, J = 11.4 Hz, 1 H,
CHPh), 4.94 (d, J = 11.1 Hz, 1 H, CHPh), 7.20–7.40 (m, 20 H, Ar-
H) ppm. 13C NMR (100 MHz): δ = 14.4 (OCH2CH3), 29.9
were added, and the reaction mixture was stirred under H2 over-
night. The catalyst was filtered through a Celite pad (eluent meth-
anol), and the solvent was evaporated in vacuo to afford pure com-
pound 4 (29.2 mg, quant.). [α]2D5 = +2.1 (c = 0.3, MeOH). 1H NMR
(400 MHz, CD3OD): δ = 0.84 [t, J = 7.4 Hz, 3 H, N(CH2)2CH3],
1.25 (t, J = 7.1 Hz, 3 H, OCH2CH3), 1.30–1.47 (m, 2 H,
(CH2CO2Et), 50.7, 54.3 [C(2), C(6)], 60.8, 65.6 (OCH2CH3, NCH2CH2CH3), 2.42–2.54 (m, 2 H, CHCO2Et, NCHCH2CH3),
CH2OBn), 73.0, 73.8, 75.5, 75.7 (CH2Ph), 80.4, 82.2, 83.5 [C(3),
C(4), C(5)], 127.7–128.8 (CHAr), 138.3, 138.4, 138.4, 138.8 (CqAr),
2.66–2.77 (m, 1 H, NCHCH2CH3), 2.86 (dd, J = 16.4, 4.2 Hz, 1
H, CHCO2Et), 3.10–3.21 (m, 2 H, 2-H, 6-H), 3.24 (t, J = 9.6 Hz,
Eur. J. Org. Chem. 2011, 5012–5019
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5017

B. La Ferla, et al.
FULL PAPER
1 H, 3-H), 3.39 (t, J = 9.0 Hz, 1 H, 4-H), 3.73 (d, J = 7.4 Hz, 2 H,
CH2OH), 3.82 (dd, J = 9.5, 6.2 Hz, 1 H, 5-H), 4.13 (q, J = 7.2 Hz,
0.219 mmol) were added. After stirring overnight, the solvent was
evaporated in vacuo. Purification by flash chromatography (ethyl
2 H, OCH2CH3) ppm. 13C NMR (100 MHz, CD3OD): δ = 11.62, acetate) afforded pure compound 22 (10.5 mg, 25.1% yield). [α]2D5
14.50 [N(CH2)2CH3, OCH2CH3], 24.06 (NCH2CH2CH3), 34.98 = +83.3 (c = 0.1, MeOH). 1H NMR (400 MHz, CD3OD): δ = 1.19
(CH2CO2Et), 49.67, 57.58, 61.75 (NCH2CH2CH3, OCH2CH3, (t, J = 7.1 Hz, 3 H, OCH2CH3), 2.88 (dd, J = 16.8, 5.9 Hz, 1 H,
CH2OH), 55.76, 62.15, 70.44, 72.66, 77.57 [C(2), C(3), C(4), C(5), CHCO2Et), 3.12 (dd, J = 16.4, 8.5 Hz, 1 H, CHCO2Et), 3.39 (dd,
C(6)], 174.31 (C=O) ppm. HRMS: calcd. for C13H26NO6
J = 7.8, 6.7 Hz, 1 H, 3-H), 3.56 (dd, J = 8.2, 6.7 Hz, 1 H, 4-H),
3.67 (dd, J = 8.3, 6.1 Hz, 1 H, 5-H), 3.86–4.00 (m, 3 H, 2-H,
CH2OH), 4.01–4.11 (m, 2 H, OCH2CH3), 4.35–4.46 (m, 1 H, 6-H),
5.03–5.13 (m, 2 H, CH2Ph), 7.26–7.45 (m, 5 H, Ar-H) ppm. 13C
[M + H]+ 292.1755; found 292.1720.
Ethyl {(2S,3S,4R,5R,6S)-3,4,5-Tris(benzyloxy)-6-[(benzyloxy)meth-
yl]-1-(cyclohexylmethylene)piperidin-2-yl}acetate (24): Compound
21 (86.5 mg, 0.142 mmol) was dissolved in dry dichloroethane
(4 mL), then cyclohexanecarbaldehyde (0.051 mL, 0.426 mmol)
and acetic acid (0.081 mL, 1.42 mmol) were added. The reaction
mixture was dried with Na2SO4 and stirred for 0.5 h. Na(OAc)3BH
(120.2 mg, 0.567 mmol) was added, and the mixture was stirred
overnight. The reaction mixture was neutralized with a satd. solu-
tion of NaHCO3, dissolved in CH2Cl2, and washed with water. The
organic layer was dried with Na2SO4, filtered, and the solvent was
evaporated in vacuo. Purification by flash chromatography (petro-
leum ether/ethyl acetate, 9:1) afforded pure compound 24 (72.3 mg,
79.4% yield). [α]2D5 = –18.3 (c = 1.0, CHCl3). 1H NMR (400 MHz):
δ = 0.63–0.82 [m, 2 H, 2 CH(Cy)], 1.15 (t, J = 7.1 Hz, 3 H,
OCH2CH3), 1.06–1.40 [m, 3 H, 3 CH(Cy)], 1.58–1.80 [m, 6 H, 6
CH(Cy)], 2.21–2.32 (m, 1 H, NCHCy), 2.35–2.51 (m, 2 H,
CHCO2Et, NCHCy), 2.69 (dd, J = 15.5, 4.7 Hz, 1 H, CHCO2Et),
3.22–3.30 (m, 1 H, 6-H), 3.31–3.38 (m, 1 H, 3-H), 3.38–3.49 (m, 1
H, 2-H), 3.67–3.75 (m, 1 H, 4-H), 3.75–3.82 (m, 2 H, 5-H, 7a-H),
3.85 (dd, J = 10.1, 3.4 Hz, 1 H, 7b-H), 3.91–4.00 (m, 1 H,
OCHCH3), 4.00–4.09 (m, 1 H, OCHCH3), 4.54–4.63 (m, 3 H, 3
CHPh), 4.64–4.71 (m, 2 H, 2 CHPh), 4.73 (d, J = 10.8 Hz, 1 H,
CHPh), 4.93 (d, J = 10.9 Hz, 2 H, 2 CHPh), 7.24–7.37 (m, 20 H,
Ar-H) ppm. 13C NMR (100 MHz): δ = 14.27 (OCH2CH3), 26.19,
26.26, 27.00, 31.29, 31.39 [5 CH2(Cy)], 37.00 [CH(Cy)], 36.03
(CH2CO2Et), 54.51, 60.47, 67.35 (OCH2CH3, NCH2Cy, CH2OBn),
56.26, 58.69 [C(2), C(6)], 78.41, 80.85, 84.38 [C(3), C(4), C(5)],
72.84, 73.35, 74.89, 75.45 (4 OCH2Ph), 127.4–128.5 (CHAr), 138.5,
138.6, 138.9, 138.9 (4 CqAr), 172.2 (C=O) ppm. HRMS: calcd. for
C45H56NO6 [M + H]+ 706.4102; found 706.4029.
NMR (100 MHz, CD3OD):
δ = 14.43 (OCH2CH3), 36.15
(CH2CO2Et), 54.88, 59.46, 72.48, 74.17, 76.81 [C(2), C(3), C(4),
C(5), C(6)], 58.94, 61.76, 68.31 (CH2OH, OCH2CH3, OCH2Ph),
128.96–129.53 (CHAr), 137.86 (CqAr), 157.88 (CO2Bn), 174.09
(CO2Et) ppm. HRMS: calcd. for C18H26NO8 [M + H]+ 384.1653;
found 384.1774.
Ethyl
[(2S,3S,4R,5R,6S)-1-(Benzyloxycarbonyl)-6-carboxy-3,4,5-
trihydroxypiperidin-2-yl]acetate (9): To a solution of compound 22
(10.5 mg, 0.027 mmol) in water (1.5 mL), TEMPO (0.27 mg,
0.00175 mmol) and KBr (9.8 mg, 0.0822 mmol) were added. The
reaction mixture was cooled to 0 °C, and 5% NaOCl (0.54 mL,
0.363 mmol) was added. After stirring for 4 h, methanol (1 mL)
was added, the reaction mixture was acidified with HCl (5%), and
the solvent was removed in vacuo. The white solid was suspended
in ethanol, the suspension was filtered, and the filtrate was concen-
trated in vacuo. Purification by flash chromatography (ethyl acet-
ate/methanol, 8:2) afforded pure compound 9 (9.7 mg, 84.4%
yield). [α]2D5 = +83.3 (c = 0.1, MeOH). 1H NMR (400 MHz,
CD3OD): δ = 1.20 (t, J = 7.1 Hz, 3 H, OCH2CH3), 2.66–2.87 (m,
2 H, CH2CO2Et), 3.52–3.65 (m, 2 H, 3-H, 4-H), 3.75–3.87 (m, 1
H, 5-H), 4.06 (dd, J = 13.6, 6.6 Hz, 2 H, OCH2CH3), 4.19–4.39
(m, 1 H, 2-H), 4.56 (d, J = 5.8 Hz, 1 H, 6-H), 5.10 (m, 2 H,
OCH2Ph), 7.23–7.41 (m, 5 H, HAr) ppm. 13C NMR (100 MHz,
CD3OD): δ = 14.45 (OCH2CH3), 38.33 (CH2CO2Et), 56.73, 61.13,
72.50, 75.49, 78.26 [C(2), C(3), C(4), C(5), C(6)], 61.69, 68.50
(OCH2CH3, OCH2Ph), 128.8–129.5 (CHAr), 135.8 (CqAr), 149.8,
157.3, 173.8 (CO2 H, CO2Bn, CO2Et) ppm. HRMS: calcd. for
C18H23NO9Na [M + Na]+ 420.1265; found 420.1181.
Ethyl [(2S,3S,4R,5R,6S)-1-(Cyclohexylmethylene)-3,4,5-trihydroxy-
Akt Inhibition Assay: The Akt kinase activity assay was performed
by employing a commercial ELISA kit designed specifically to
screen inhibitors or activators of Akt/PKB, purchased from Assay
Designs (Ann Arbor, MI 48108, USA; Cat. No. EKS-400A).
Briefly, the substrate, which is readily phosphorylated by purified
Akt, is precoated at 2.5 μg per well on the wells of the microtiter
plate. The samples to be assayed are added. Specifically, 30 μL of
each of the following was added to appropriate wells: blank (kinase
assay dilution buffer), negative control [inhibitor diluent alone (dis-
tilled water)], samples (inhibitors at 700 μm), positive control at
50 μm. This was followed by the addition of ATP to initiate the
reaction, which was terminated 90 min later. An antibody, which
binds specifically to the phosphorylated substrate, was added to
the wells and incubated for 60 min. After washing, this antibody
was subsequently bound by a peroxidase-conjugated secondary an-
tibody, which was added to the wells for 30 min. The assay was
developed with tetramethylbenzidine (TMB) substrate, and a col-
our developed in proportion to the Akt phosphotransferase ac-
tivity. The colour development was stopped with acid stop solution,
and the intensity of the colour was measured in a microplate reader
at 450 nm. As positive control of the test, a known Akt inhibitor
[1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octa-
decyl-sn-glycerocarbonate, compound 2 (Figure 1), purchased from
Calbiochem (San Diego, CA, USA; Cat. No. 124005)] was used.
6-(hydroxymethyl)piperidin-2-yl]acetate
(5):
Compound
24
(72.3 mg, 0.113 mmol) was submitted to catalytic hydrogenation
according to the same procedure used for the synthesis of com-
pound 3, affording pure compound 5 (40.0 mg, 100% yield). [α]2D5
= –38.0 (c = 4.1, MeOH). 1H NMR (400 MHz, CD3OD): δ = 0.66–
0.79 [m, 1 H, CH(Cy)], 0.80–0.92 [m, 1 H, CH(Cy)], 1.11–1.24 [m,
2 H, 2 CH(Cy)], 1.26 (t, J = 7.1 Hz, 3 H, OCH2CH3), 1.28–1.43
[m, 2 H, 2 CH(Cy)], 1.62–1.78 [m, 6 H, 6 CH(Cy)], 2.36 (dd, J =
13.5, 6.1 Hz, 1 H, NCHCy), 2.49 (dd, J = 17.1, 10.5 Hz, 1 H,
CHCO2Et), 2.57 (dd, J = 13.5, 8.1 Hz, 1 H, NCHCy), 2.87 (dd, J
= 17.1, 3.7 Hz, 1 H, CHCO2Et), 3.14–3.23 (m, 2 H, 2-H, 6-H), 3.27
(dd, J = 10.5, 8.5 Hz, 1 H, 3-H), 3.41 (t, J = 9.0 Hz, 1 H, 4-H),
3.74–3.80 (m, 2 H, CH2OH), 3.85 (dd, J = 9.5, 6.1 Hz, 1 H, 5-H),
4.14 (q, J = 7.1 Hz, 2 H, OCH2CH3) ppm. 13C NMR (100 MHz,
CD3OD): δ = 17.31 (OCH2CH3), 29.84, 30.04, 30.58, 34.81, 35.24,
37.09 [5 CH2(Cy), CH2CO2Et], 41.51 [CH(Cy)], 57.69, 60.62, 64.59
(CH2OH, NCH2Cy, OCH2CH3), 58.26, 65.78, 72.96, 74.88, 80.31
[C(2), C(3), C(4), C(5), C(6)], 177.12 (C=O) ppm. HRMS: calcd.
for C17H32NO6 [M + H]+ 346.2224; found 346.2165.
Ethyl [(2S,3S,4R,5R,6S)-1-(Benzyloxycarbonyl)-3,4,5-trihydroxy-6-
(hydroxymethyl)piperidin-2-yl]acetate (22): To a solution of com-
pound 3 (27.3 mg, 0.109 mmol) in methanol (2 mL), benzyl chloro-
formate (0.047 mL, 0.329 mmol) and NaHCO3 (18.4 mg,
5018
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Acknowledgments
CINMPIS Consortium has supported the work with a fellowship
to E. B.; Alßan Programme has supported the work with a fellow-
ship to A. O. E07D400786BR.
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Received: March 31, 2011
Published Online: July 27, 2011
Eur. J. Org. Chem. 2011, 5012–5019
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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