Phosphatidylinositol 3-phosphate mimics based on a sulfoquinovose scaffold: Synthesis and evaluation as protein kinase B inhibitors

Article abstract of DOI:10.1002/ejoc.201402664

New sulfoquinovose analogues of phosphatidylinositol 3-phosphate have been synthesised based on a sulfoquinovose scaffold as potential protein kinase B (PKB) inhibitors. The synthetic strategy involved the introduction into glucose of a thioacetate group at the 6-position and of an azide group at the anomeric position as precursors of the sulfonate and phosphoramidate moieties present in the final compounds. The synthesised compounds were tested in vitro on isolated PKB by means of ELISA assays and for their anti-proliferative activity against the human ovarian carcinoma cell line IGROV-1. Sulfoquinovose derivatives 2b and 2c showed inhibitory activity in the low micromolar range.

Full text of DOI:10.1002/ejoc.201402664

Job/Unit: O42664
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FULL PAPER
DOI: 10.1002/ejoc.201402664
Phosphatidylinositol 3-Phosphate Mimics Based on a Sulfoquinovose Scaffold:
Synthesis and Evaluation as Protein Kinase B Inhibitors
Luca Gabrielli,[a][‡][Ilaria Calloni, Giulia Donvito, Barbara Costa, Noemi Arrighetti,
[a]
[a]
[a]
[b]
b]
[c]
[c]
[a]
Paola Perego, Diego Colombo, Fiamma Ronchetti, Francesco Nicotra, and
Laura Cipolla*[
a]
Dedicated to C.I.N.M.P.I.S. on the occasion of its 20th anniversary
Keywords: Medicinal chemistry / Antitumor agents / Carbohydrates / Inhibitors / Enzymes / Phospholipids
New sulfoquinovose analogues of phosphatidylinositol 3-
phosphate have been synthesised based on a sulfoquinovose
scaffold as potential protein kinase B (PKB) inhibitors. The
synthetic strategy involved the introduction into glucose of a
thioacetate group at the 6-position and of an azide group at
the anomeric position as precursors of the sulfonate and
phosphoramidate moieties present in the final compounds.
The synthesised compounds were tested in vitro on isolated
PKB by means of ELISA assays and for their anti-proliferative
activity against the human ovarian carcinoma cell line IG-
ROV-1. Sulfoquinovose derivatives 2b and 2c showed inhibi-
tory activity in the low micromolar range.
Introduction
phosphate and phosphatidylinositol 4-phosphate), which in
turn cause PKB translocation to cell membranes followed
by binding of the pleckstrin homology (PH) domain of
PKB, a required step for PKB activation. The PKB PH
domain affinity decreases from phosphatidylinositol 3,4,5-
tris-phosphate to phosphatidylinositol 3,4-bis-phosphate
and phosphatidylinositol 3-phosphate. However, previous
work by Kozikowski and co-workers showed that phos-
phate groups may be absent in targeted PH domain phos-
Protein kinases are a vast class of enzymes that catalyse
the specific phosphorylation of protein substrates, transfer-
ring the γ-phosphate group from ATP (adenosine triphos-
phate) to the hydroxy groups of the serine, threonine (Ser/
Thr kinases) or tyrosine (Tyr kinases) side-chains. Some im-
portant protein kinases, including the PI3K and PKB (also
referred to as Akt) protein kinases, have been involved in
abnormal cell signalling in tumour cells, in which they have
been shown to be improperly activated. Among these pro-
teins, PKB is a key element of the PI3K-Akt-mTOR axis.
Several studies have highlighted PKB as a crucial player in
cell survival and may also be involved in the drug resistance
of tumour cells. PKB activation involves phosphatidylino-
sitol 3-kinase (PI3K),[ which phosphorylates the 3-posi-
tion of phosphatidylinositols (phosphatidylinositol 4,5-bis-
[4]
phatidylinositol mimics. Given these premises, the search
[5–7]
for kinase inhibitors
has attracted growing interest, fo-
[
1]
[8]
cused mainly on ATP-binding-site inhibitors. Because the
ATP binding site is highly conserved in kinases, the design
of selective inhibitors is particularly challenging. An alter-
[
2]
native approach has been directed towards the development
3]
[9,10]
of substrate-competitive inhibitors.
A third, highly
promising strategy for PKB inhibition is to target the PH
domain, and a few such inhibitors have been synthesised.[
In fact, because the PH domain is less recurrent than the
ATP binding site, the targeting of the PH domain of PKB
may result in more selective inhibitors than the ATP pocket
10]
[
a] Dip. di Biotecnologie e Bioscienze, Università degli Studi di
Milano-Bicocca,
P.za Scienza 2, 20126 Milano, Italy
E-mail: laura.cipolla@unimib.it
[
10]
http://www.unimib.it/go/102/Home/English
b] Molecular Pharmacology Unit, Fondazione IRCCS Istituto
Nazionale dei Tumori,
ligands. Moreover, clinical trials of a few PKB inhibitors
showed side-effects associated with the alteration of the in-
[
[
[
[
11]
sulin/glucose metabolism. Thus, new compounds featur-
ing better pharmacological profiles are still needed. Refer-
ring to our recent studies on glucose-based PKB inhibi-
via Amadeo 42/via Venezian 1, 20133 Milan, Italy
c] Dipartimento di Biotecnologie Mediche e Medicina
Traslazionale, Università degli Studi di Milano,
Via Saldini 50, 20133 Milano, Italy
[
12]
‡] Present address: Dip. di Scienze Chimiche, Università degli
Studi di Padova,
tors,
which highlight the relevance of an acidic group
mimicking the 3-phosphate of phosphatidylinositol 3-phos-
phate (PI3P, 1, Figure 1), we propose here new sulfonate
analogues of phosphatidylinositol 3-phosphate (1) as poten-
Via Marzolo 1, 35131 Padova, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402664.
Eur. J. Org. Chem. 0000, 0–0
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L. Cipolla et al.
FULL PAPER
tial PKB inhibitors with the 6-deoxy-6-sulfoglucose (sulfo-
quinovose) skeleton, which mimics PI3P (Figure 1, com-
pounds 2a–c). The sulfonate group at the 6-position is in-
tended as the 3-phosphate mimic, whereas the anomeric
phosphoramidate possessing three different chain lengths
substitutes the diacyl glycerol (DAG) moiety of PI3P (1).
Figure 1. Structures of phosphatidylinositol 3-phosphate and its
analogues as potential PKB inhibitors.
Results and Discussion
Chemical Synthesis
Scheme 1. Synthetic routes towards the target inhibitors 2a–c.
The introduction of the phosphoramidate group requires
as precursor a β-configured anomeric azide, whereas the mation for details), however, the sulfoquinovose 5 could not
sulfonate group at C-6 may be introduced by oxidation of be efficiently converted into azide 6 (see the Supporting In-
the corresponding 6-deoxy-6-mercapto derivative, in turn formation for details) due to the inefficient hydrolysis of
obtained via its sulfur-protected thioacetate. Hence, the tar- the methyl glucoside. Finally, route c (Scheme 1) allowed an
get compounds may be synthesised following three different efficient synthesis of the target sulfoquinovoses 2a–c. In this
strategies (Scheme 1): route a) introduction of the phos- strategy, precursor groups of the phosphoramidates and of
phoramidate group followed by the introduction of a thiol the sulfonate (azide and thioacetate, respectively) had to be
moiety for further oxidation to the corresponding sulfonate, in place before further manipulation (key intermediate 8,
route b) introduction of the sulfonate in the first steps of Scheme 1, route c).
the synthesis followed by the introduction of the anomeric
The detailed synthetic steps are presented in Scheme 2.
azide for further manipulation to the desired phosphor- Glucose was converted into tosylate 11 following literature
[
13]
amidate, and route c) introduction of both precursors (the procedures. Briefly, d-glucose was first treated with tosyl
anomeric azide and 6-thioacetate) in the first steps followed chloride (TsCl) in pyridine (Py) to protect selectively the
by group modifications to give the final functionalities. primary hydroxy group, affording compound 10. Treatment
Given the efficacy of the introduction of the phosphoram- with acetic anhydride and a catalytic amount of 4-(dimeth-
[
12]
idate moiety into glucose reported in the literature,
the ylamino)pyridine (DMAP) afforded fully protected deriva-
synthetic strategy initially envisaged for the preparation of tive 11. Subsequently, the thioacetate moiety was intro-
sulfoquinovose derivatives 2a–c involved the introduction of duced as the sulfonate precursor by treating compound 11
the anomeric phosphoramidate groups in the early stages with potassium thioacetate in dry DMF to afford com-
of the synthesis (Scheme 1, route a, intermediate 3) and the pound 7 (35% overall yield form d-glucose). The azido
introduction of the sulfonate moiety later in the synthesis group was introduced as the phosphoramidate precursor by
(see also the Supporting Information).
treatment of thioacetate 7 with hydrobromic acid in acetic
The synthesis proceeded efficiently until the deprotection acid (HBr/AcOH) in dry dichloromethane. The glycosyl
of phosphoramidate 3a. To introduce the thioacetate moi- bromide 12 was treated without purification with sodium
ety at C-6, activation towards nucleophilic substitution via azide and tetrabutylammonium hydrogen sulfate (TBAHS)
the corresponding 6-tosylate was attempted, however, the in a biphasic mixture of aq. satd. NaHCO and dichloro-
3
tosylate could be obtained only in very low yields (see the methane to afford the key precursor 8 (53% yield over two
Supporting Information for details) and the synthetic route steps, α/β 5:95). Alkyl phosphoramidates 9a–c were ob-
was abandoned. Therefore the synthetic strategy was re- tained by Staudinger reaction of azide 8 with the corre-
versed following route b: first, the sulfonate group was in- sponding trialkyl phosphite. The products were obtained
serted at the 6-position (Scheme 1, route b, intermediate 5) with retention of the configuration at the anomeric posi-
1
from methyl α-d-6-tosylglucoside (see the Supporting Infor- tion, as confirmed by H NMR spectroscopy. Subsequent
2
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Phosphatidylinositol 3-Phosphate Mimics
introduction of the sulfonate moiety[14,15] was achieved by
selective Oxone™ oxidation in AcOH to afford peracetyl-
ated sulfoquinovoses 13a–c. Final cleavage of the acetate
groups under Zèmplen conditions (cat. Na, dry MeOH)
gave the desired compounds 2a–c.
Figure 2. In vitro PKB1 Kinase Assay (ELISA) of compounds 2a–
c at the indicated concentrations. The mean inhibition is also re-
ported for each compound. The control includes DMSO at a con-
centration of 0.1%.
Table 1. Concentration of test compounds producing 50% inhibi-
tion (IC50) of PKB1 activity in the ELISA assay.
Compound
IC50 [μm]
2a
2b
2c
Ͼ100
33.4Ϯ20.18
6.4Ϯ0.97
In contrast, sulfoquinovoses 2b and 2c both showed anti-
proliferative activity, the IC50 values being around 40 μm.
Table 2. Sensitivity of ovarian carcinoma IGROV-1 cells to sulfo-
quinovoses 2a–c.[
a]
Compound
IC50 [μm]
Scheme 2. Synthetic steps to title sulfoquinovoses 2a–c.
2
2
2
a
b
c
Ͼ100
39.7Ϯ22.12
48.7Ϯ28.07
0.31Ϯ0.1
Biological Evaluation
Cisplatin
[
a] Cell sensitivity was evaluated by growth inhibition assays based
Sulfoquinovose derivatives 2a–c were tested for in vitro
inhibitory activity against PKB1 (isoform 1 of protein kin-
on cell counting. Cells were seeded and 24 h later they were exposed
to the compounds for 72 h. At the end of treatment, cells were
ase B) by using an in vitro Kinase Assay Kit (AKT/PKB counted by using a cell counter.
kinase Assay/Inhibitor Screening Kit, CycLex). Figure 2
Therefore it can be concluded that compounds 2b and 2c
are active in ELISA and cell sensitivity assays.
shows the effect of compounds 2a–c tested at three different
concentrations (1, 10 and 100 μm). The results show that
the PKB1 activity was unaffected by compound 2a, whereas
both compounds 2b and 2c elicited an inhibitory effect that
was concentration-dependent. The IC50 values were deter-
mined (Table 1) and show that the inhibitory effect seemed
Conclusions
In this paper we have presented our synthetic efforts to
to be related to the lipophilic chain length because com- achieve an efficient synthesis of sulfonate analogues of
pound 2c is the most potent inhibitor of PKB1 activity phosphatidylinositols based on a sulfoquinovose scaffold.
(IC50 = 6.4 vs. 33.4 μm for 2b).
Their preliminary biological activities were assessed against
The synthesised compounds 2a–c were also tested in vi- isolated PKB (ELISA assay) and for their ability to inhibit
tro for their anti-proliferative activity against the human the proliferation of human ovarian carcinoma cells. Com-
ovarian carcinoma cell line IGROV-1. The conventional pounds 2b and 2c showed inhibitory properties, however,
anti-tumour agent cisplatin was used as the reference com- their anti-proliferative activity towards ovarian carcinoma
pound because it is very potent in inhibiting proliferation cells was lower than that of the conventional cytotoxic
in such a cell line. The cell sensitivity assays (Table 2) indi- agent cisplatin, which is used in the first-line treatment of
cated that compound 2a was inactive as no proliferation ovarian carcinoma. Further experiments are necessary to
inhibition was observed up to 100 μm drug concentration. verify the PH domain binding.
Eur. J. Org. Chem. 0000, 0–0
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L. Cipolla et al.
FULL PAPER
2 3
CH
), 3.68 (ddd, J4,5 = 9.5, J6ЈЈ,5 = 6.4, 3J6Ј,5 = 2.8 Hz, 1
3
3
OCH
H, 5-H), 3.20 (dd, J6Ј,6ЈЈ = 14.3, J6Ј,5 = 2.8 Hz, 1 H, 6Ј-H), 3.05
Experimental Section
2
3
General Methods: Reactions were carried out by using commer-
cially available starting materials and solvents without further puri-
fication. All solvents were dried with molecular sieves for at least
2
6Ј,6ЈЈ = 14.3, 3J6ЈЈ,5 = 6.4 Hz, 1 H, 6ЈЈ-H), 2.31 (s, 3 H,
J
(
dd,
CH
H, CH
NMR (CDCl
66.00 (s, CH
C-5), 68.62 (d, C-3), 67.63 (d, JC,P = 10.1 Hz, C-2), 66.52 (d, C-
), 58.81, 58.81 (2 t, 2 OCH CH ), 26.44 (t, C-6), 26.06 (q,
CH COS), 16.78, 16.68, 16.64 (3q, 3 CH CO), 12.23, 12.16 (2 q, 2
OCH ) ppm. P NMR (162 MHz, CDCl ): δ = 5.18 ppm. MS:
m/z = 522.4 [M + Na] .
3
COS), 2.07 (s, 3 H, CH
3
CO), 2.03 (s, 3 H, CH
CH
COS), 166.58 (s, CH
3
CO), 1.99 (s, 3
13
3
CO), 1.31 (q, J = 7.2 Hz, 6 H, 2 OCH
2
3
) ppm.
C
2
4 h prior to use. When dry conditions were required, the reaction
was performed under Ar. TLC was performed on silica gel 60 F254
plates (Merck) charring with a solution containing conc. H SO
EtOH/H O (5:45:45) or with an oxidant mixture composed of
NH )Mo 24 (21 g), Ce(SO (1 g) and conc. H SO (31 mL) in
water (500 mL). Flash column chromatography was performed on
3
): δ = 190.51 (s, CH
3
3
CO),
1
3 3
CO), 165.92 (s, CH CO), 78.59 (d, C-1), 70.17 (d,
/
3
2
4
2
4
2
3
(
4
7
O
4
)
2
2
4
3
3
31
2
CH
3
3
1
13
31
silica gel (230–400 mesh, Merck). Routine H, C and P NMR
+
1
13
spectra were recorded at 400 MHz ( H), 100.57 MHz ( C) and
62 MHz, respectively, with a Varian Mercury spectrometer. Chem-
Peracetylated Sulfoquinovose 13a: Potassium peroxymonosulfate
Oxone; 1.5 g, 2.4 mmol, 3 equiv.) and sodium acetate trihydrate
1.6 g, 12 mmol, 15 equiv.) were added to a stirred solution of com-
1
(
(
ical shifts are reported in parts per million downfield from TMS
as internal standard; J values are given in Hz. For all compounds,
1
13
pound 9a (396 mg, 0.79 mmol,1 equiv.) in glacial AcOH (8 mL).
After 4 h, the acetic acid was co-evaporated with toluene under
reduced pressure and the residue purified by flash column
chromatography (EtOAc/EtOH, 10:0Ǟ6:4) to afford compound
the assignments of the H and C NMR spectra were based on
D proton–proton and carbon–proton shift-correlation spectra,
2
respectively. Carbon signals of the C10 chains of the phosphoramid-
ate moiety have been omitted from the 13C NMR spectral descrip-
1
3
1
=
3a (251 mg, 62% yield). H NMR (CD
3
OD): δ = 5.26 (app. t, J3,2
tions. Mass spectra were recorded with a System Applied Biosys-
tems MDS SCIEX spectrometer (Q TRAP, LC/MS/MS, turbo ion
spray or Q STAR elite nanospray). Full ESI-MS were recorded
with a Thermo LTQ spectrometer with a direct inlet; relative per-
centages are shown in brackets.
Glucosyl Bromide 12: Compound 7[16] (2.5 g, 6.0 mmol, 1 equiv.)
was dissolved in dry dichloromethane (61 mL) and cooled to 0 °C.
A 33% HBr solution in AcOH (8.5 mL, 48 mmol, 8 equiv.) was
slowly added and the mixture was stirred at room temperature
overnight. Then the reaction was diluted with icy water and washed
3
3
3
J3,4 = 9.3 Hz, 1 H, 3-H), 4.95 (app. t, J3,2 = J1,2 = 9.3 Hz, 1
3
3
H, 2-H), 4.91 (app. t, J3,4 = J4,5 = 9.3 Hz, 1 H, 4-H), 4.64 (app.
3
3
t, J1,2 = JH,P = 9.3 Hz, 1 H, 1-H), 4.19–3.99 (m, 4 H, 2 CH
.06–2.97 (m, 2 H, 6-H), 2.04 (s, 3 H, CH CO), 2.03 (s, 3 H,
CH CO), 1.98 (s, 3 H, CH CO), 1.30 (t, J = 7.0 Hz, 6 H, 2
OCH ) ppm. C NMR (CD OD): δ = 170.36 (s, CH CO),
70.18 (s, CH CO), 169.99 (s, CH CO), 82.18 (d, C-1), 73.32 (d,
C-3), 71.80 (d, C-5), 71.69 (d, C-2), 71.07 (d, C-4), 62.92, 62.92 (2
t, 2 OCH CH ), 52.13 (t, C-6), 22.75 (q, CH CO), 19.45 (q,
CH CO), 19.29 (q, CH CO), 15.14, 15.14 (2 q, 2 OCH CH ) ppm.
P NMR (162 MHz, CD OD): δ = 6.7 ppm. MS: m/z = 504.1 [M –
H]–.
2 3
CH ),
3
3
3
3
1
3
2
CH
3
3
3
1
3
3
2
3
3
3
3
2
3
with satd. Aq. NaHCO
3
until neutrality. The organic layers were
SO and the solvent evaporated under
3
1
3
dried with anhydrous Na
reduced pressure. The crude was used directly in the next reaction
without further purification.
2
4
Sulfoquinovose 2a: A catalytic amount of Na was added to a solu-
tion of compound 13a (70 mg, 0.14 mmol, 1 equiv.) in dry MeOH
Azide 8: TBAHS (2.05 g, 6.04 mmol, 1 equiv.) and NaN
3
(1.18 mg,
(
2 mL). After 2 h, Amberlite IR 120 H+ was added to neutrality.
18.1 mmol, 3 equiv.) were added to a stirred solution of crude 12
The resin was then filtered off and the solvent evaporated to dry-
ness. The crude was purified by flash column chromatography (iso-
3
in dichloromethane (25 mL) and aq. satd. NaHCO (25 mL). The
reaction was stirred vigorously overnight at room temperature. The
organic phase was collected, dried with anhydrous Na SO , filtered
and the solvent evaporated. The crude product was purified by
flash column chromatography (petroleum ether/AcOEt, 9:1Ǟ6:4)
4
propanol/aq. 33% NH OH, 6:4) to afford compound 2a (44 mg,
2
4
1
3
3
8
3% yield). H NMR (D
2
O): δ = 4.13 (app. t, J1,2 = JH,P = 9.1 Hz,
3
1
H, 1-H), 4.02–3.91 (m, 4 H, 2 OCH CH ), 3.60 (app. t, J4,5 =
2 3
3
3
3
1
3,4 4,5
J6ЈЈ,5 = 9.1 Hz, 1 H, 5-H), 3.31 (app. t, J = J = 9.1 Hz, 1 H,
to give compound 8 (1.27 g, 53% yield over two steps). H NMR
2
3
3
4-H), 3.20 (d, J
= 14.4 Hz, 1 H, 6Ј-H), 3.12–3.04 (m, 2 H, 2-
6ЈЈ,5
= 9.1 Hz, 1 H, 6ЈЈ-H), 1.15–
2 3 6
1.08 (m, 6 H, 2 OCH CH ) ppm. C NMR ([D ]DMSO): δ = 81.41
(d, C-1), 73.74 (d, C-4), 71.20 (d, C-5), 70.99 (d, C-3), 69.92 (d, C-
2), 61.94 (t, OCH CH ), 61.76 (t, OCH CH ), 50.12 (t, C-6), 13.07
(q, OCH CH ), 13.01 (q, OCH CH ) ppm. P NMR (162 MHz,
(
CDCl
3
): δ = 5.12 (app. t, J3,2 = J3,4 = 9.5 Hz, 1 H, 3-H), 4.93
6Ј,6ЈЈ
2
3
3
3
3
3
H, 3-H), 2.88 (dd, J
= 14.4, J
(app. t, J3,4 = J4,5 = 9.5 Hz, 1 H, 4-H), 4.85 (app. t, J3,2 = J1,2
6Ј,6ЈЈ
1
3
3
=
9.5 Hz, 1 H, 2-H), 4.55 (d, J1,2 = 9.5 Hz, 1 H, 1-H), 3.66–3.71
2
(
m, 1 H, 5-H), 3.17–3.21 (m, 1 H, 6Ј-H), 3.06 (dd, J6Ј,6ЈЈ = 14.5,
3
J
5,6ЈЈ = 6.3 Hz, 1 H, 6ЈЈ-H), 2.28 (s, 3 H, CH
CH CO), 1.92 (s, 3 H, CH
CO) ppm. 13C NMR (CDCl
94.45 (s, CH COS), 169.98 (s, CH CO), 169.54 (s, CH
69.11 (s, CH CO), 87.49 (d, C-1), 74.95 (d, C-5), 72.38 (d, C-3),
0.55 (d, C-2), 69.83 (d, C-4), 30.33 (t, C-6), 29.75 (q, CH COS),
0.47, 20.47, 20.47 (3 q, 3 CH CO) ppm. MS: m/z = 412.3 [M +
3
COS), 2.00 (s, 6 H,
): δ =
CO),
2
3
2
3
3
1
2
1
1
7
2
3
3
3
2
3
2
3
–
D
2
O): δ = 8.57 ppm. MS: m/z = 378.2 [M – H] .
3
3
3
3
Peracetylated Dibutyl Phosphoramidate 9b: Tributyl phosphite
450 μL, 1.8 mmol, 2 equiv.) was added dropwise to a solution of
compound 8 (350 mg, 0.923 mmol, 1 equiv.) in dry dichlorometh-
ane (9 mL) at 0 °C. The reaction mixture was stirred overnight and
then concentrated to dryness. The crude was purified by flash col-
3
(
3
+
Na] .
Peracetylated Diethyl Phosphoramidate 9a: Triethyl phosphite
290 μL, 1.66 mmol, 2 equiv.) was added dropwise to a solution of
compound 8 (320 mg, 0.83 mmol, 1 equiv.) in dry dichloromethane
9 mL) at 0 °C. The reaction mixture was stirred overnight and then
(
umn chromatography (petroleum ether/EtOAc, 6:4) to afford com-
1
pound 9b (370 mg, 74% yield). H NMR (CDCl
3
): δ = 5.08 (app.
3
3
3
3
(
t,
J
3,2
=
J
3,4 4,5
3,4 = 9.5 Hz, 1 H, 3-H), 4.80 (app. t, J = J =
3
3
concentrated to dryness. The product was purified by flash column
chromatography (petroleum ether/EtOAc, 2:8) to afford compound
9.5 Hz, 1 H, 4-H), 4.73 (app. t, J3,2 = J1,2 = 9.5 Hz, 1 H, 2-H),
4.43–4.35 (m, 1 H, 1-H), 3.91–3.78 (m, 4 H, 2 OCH CH CH CH ),
3.60–3.54 (m, 1 H, 5-H), 3.09 (br. d, J6Ј,6ЈЈ = 14.1 Hz, 1 H, 6Ј-H),
2
2
2
3
1
3
2
9
=
a (396 mg, 95% yield). H NMR (CDCl
3
): δ = 5.20 (app. t, J3,2
3
3
3
2
3
J3,4 = 9.5 Hz, 1 H, 3-H), 4.91 (app. t, J3,4 = J4,5 = 9.5 Hz, 1 2.94 (dd, J6Ј,6ЈЈ = 14.1, J6ЈЈ,5 = 6.4 Hz, 1 H, 6ЈЈ-H), 2.20 (s, 3 H,
3
3
H, 4-H), 4.82 (app. t, J3,2 = J1,2 = 9.5 Hz, 1 H, 2-H), 4.51 (dd,
CH
3
COS), 1.95 (s, 3 H, CH
3
CO), 1.91 (s, 3 H, CH
3
CO), 1.87 (s, 3
), 1.33–1.22
3
1,2 = 9.5, 3
J
J
H,P = 11.1 Hz, 1 H, 1-H), 4.14–3.95 (m, 4 H, 2
H, CH
3
CO), 1.58–1.47 (m, 4 H, 2 OCH
2
CH CH CH
2
2
3
4
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/KAP1
Date: 26-08-14 16:39:58
Pages: 7
Phosphatidylinositol 3-Phosphate Mimics
4,5
J J J =
3,2 = 3 3,4 = 9.5 Hz, 1 H, 3-H), 4.90 (app. t, 3J3,4 = 3
(
m,
4
H,
CH CH
COS), 170.12 (s, CH
CO), 82.43 (d, C-1), 74.01 (d, C-5), 72.69 (d, C-3), 71.50 (d,
2
OCH
CH ) ppm. C NMR (CDCl
CO), 169.81 (s, CH
CH
2
CH
2
CH
3
), 0.84–0.78 (m,
): δ = 194.27 (s,
CO), 169.69 (s,
6
H,
2
t, 3
9.5 Hz, 1 H, 4-H), 4.80 (app. t, J3,2 = J1,2 = 9.5 Hz, 1 H, 2-H),
4.54–4.44 (m, 1 H, 1-H), 4.11–3.81 [m, 4 H, 2 OCH (CH
CH(CH ], 3.71–3.63 (m, 1 H, 5-H), 3.49 (br. s, 1 H, NH),
COS), 2.05 (s, 3 H,
CO), 1.96 (s, 3 H, CH CO),
(CH CH(CH ], 1.52–1.39 [m, 2
], 1.37–0.68 [m, 32 H,
2
1
3
3
3
OCH
CH
2
2
2
3
3
3
3
3
2
2
)
CH
3
6
3 2
)
3
JC,P = 10.6 Hz, C-2), 70.35 (d, C-4), 66.28, 66.22 (2 t, 2 3.17–3.06 (m, 2 H, 6-H), 2.29 (s, 3 H, CH
3
OCH
OCH
2
CH
2
CH
CH
2
CH
CH
3
), 32.19 (t, OCH
), 30.22 (q, CH COS), 20.55 (q, CH
CO), 18.63 (t, OCH CH
CH ), 13.54, 13.54 (2 q,
) ppm. P NMR (162 MHz, CDCl ): δ =
2
CH
2
CH
2
CH
3
), 32.12 (t,
CO), 20.50 1.74–1.53 [m, 4 H, 2 OCH
CH CH ), H, OCH CH (CH CH(CH
OCH CH (CH CH(CH
(s, CH COS), 170.49 (s, CH
CH CO), 82.58 (d, C-1), 74.04 (d, C-5), 72.56 (d, C-3), 71.47 (d,
C-2), 70.29 (d, C-4), 66.67 [t, OCH (CH CH(CH ], 30.42 (t, C-
), 30.08 (q, CH COS), 27.80, 25.16 [2 t, 2 OCH CH (CH
CH(CH
162 MHz, CDCl
CH
3
CO), 2.01 (s, 3 H, CH
3
3
2
CH
2
2
3
3
3
2
CH
2
2
)
5
3 2
)
(q, CH
3
CO), 20.44 (q, CH
8.59 (t, OCH CH CH
CH CH CH
.31 ppm. MS: m/z = 578.6 [M + Na] .
3
2
2
2
3
2
2
2
2
)
5
3
)
2
1
2
3
1
2
2
2
3
2
2
2
2
)
5
3
)
2
] ppm. C NMR (CDCl
3
): δ = 194.39
31
OCH
5
2
2
2
3
3
3
3 3
CO), 169.91 (s, CH CO), 169.71 (s,
+
3
2
2
)
6
3 2
)
Peracetylated Sulfoquinovose 13b: Potassium peroxymonosulfate
Oxone; 904 mg, 1.45 mmol, 3 equiv.) and sodium acetate tri-
6
3
2
2
2
)
(
31
5
3 2 3
) ], 20.97, 20.70, 20.63 (3 q, 3 CH CO) ppm. P NMR
hydrate (983 mg, 7.23 mmol, 15 equiv.) were added to a stirred
solution of compound 9b (268 mg, 0.48 mmol, 1 equiv.) in glacial
acetic acid (8 mL). After 4 h, the acetic acid was co-evaporated
with toluene under reduced pressure and the residue was purified
by flash column chromatography (EtOAc/EtOH, 10:0Ǟ8:2) to af-
ford compound 13b (230 mg, 86% yield). H NMR (CD
): δ = 5.30 ppm. MS: m/z = 746.8 [M + Na]+.
3
(
Peracetylated Sulfoquinovose 13c: Potassium peroxymonosulfate
(Oxone; 1.8 g, 2.90 mmol, 3 equiv.) and sodium acetate trihydrate
(1.96 g, 14.4 mmol, 15 equiv.) were added to a stirred solution of
compound 9c (690 mg, 0.95 mmol, 1 equiv.) in glacial acetic acid
(10 mL). After 4 h, the solvent was evaporated under reduced pres-
sure and the residue purified by flash column chromatography (pe-
1
3
OD): δ =
3
3
3
5
.28 (app. t, J3,2 = J3,4 = 9.4 Hz, 1 H, 3-H), 4.94 (app. t, J3,2 =
3
3
3
J1,2 = 9.4 Hz, 1 H, 2-H), 4.87 (app. t, J3,4 = J4,5 = 9.4 Hz, 1 H,
3
3
4
5
-H), 4.69 (app. t, J1,2 = JH,P = 9.4 Hz, 1 H, 1-H), 4.17–4.09 (m, troleum ether/EtOAc, 1:9, and EtOAc/EtOH, 8:2) to give com-
1
H, 5-H, 2 OCH
CO), 2.02 (s, 3 H, CH
.61 (m, 4 H, 2 OCH CH CH
0.97–0.92
) ppm. 13C NMR (CD
CO), 169.83 (s, CH
2
CH
2
CH
2
CH
3
), 3.01 (br. s, 2 H, 6-H), 2.05 (s, 3 pound 13c (615 mg, 88% yield). H NMR (CD
3
OD): δ = 5.27 (app.
3
3
3
3
H, CH
1
OCH
OCH
CH
7
(
3
3
3
CO), 1.97 (s, 3 H, CH
CH ), 1.47–1.37 (m, 4 H, 2
(m, H,
OD): δ = 170.25 (s, CH(CH
CO), 81.92 (d, C-1), (s, 3 H, CH
3.40 (d, C-3), 71.86 (d, JC,P = 11.8 Hz, C-2), 71.43 (d, C-5), 71.00 OCH CH (CH
d, C-4), 66.99, 66.99 (2 t, 2 OCH CH CH CH ), 52.31 (t, C-6), 170.16 (s, CH CO), 170.02 (s, CH
CH ), 19.45 (q, CH CO), 19.37 (d, C-1), 73.36 (d, C-3), 71.82 (d, JC,P = 10.1 Hz, C-2), 71.54 (d,
CO), 18.43, 18.43 (2 t,
3
CO), 1.69–
t,
J
3,2
=
J
3,4 = 9.1 Hz, 1 H, 3-H), 4.94 (app. t,
9.1 Hz, 1 H, 2-H), 4.91–4.84 (m, 1 H, 4-H), 4.67 (app. t, J1,2
J
3,2
=
J
1,2
=
=
3
2
2
2
3
3
2
CH
CH
2
CH
2
CH
3
),
6
2
J
H,P = 9.1 Hz, 1 H, 1-H), 4.21–3.99 [m, 4 H, 2 OCH
], 3.06–2.95 (m, 2 H, 6-H), 2.04 (s, 3 H, CH CO), 2.03
CO), 1.97 (s, 3 H, CH CO), 1.80–0.77 [m, 38 H, 2
CH(CH OD):
] ppm. 13C NMR (CD
CO), 169.78 (s, CH CO), 81.97
2 2 6
(CH ) -
2
2
CH
2
CH
3
3
3
)
2
3
3
CO), 170.09 (s, CH
3
3
3
3
3
2
2
2
)
5
3
)
2
3
δ =
2
2
2
3
3
3
3
3
1.87, 31.87 (2 t, 2 OCH
2
CH
2
CH
2
3
3
(q, CH
3
CO), 19.21 (q, CH
3
2
C-5), 71.03 (d, C-4), 67.70, 67.43 [2 t, 2 OCH
2
(CH
(CH
2
)
)
6
CH(CH
CH(CH
3
)
)
2
],
],
OCH
OCH
6
2
CH
CH
2
CH
2
CH
3
), 12.72 (q, OCH
2
CH
2
CH
2
CH
3
), 12.66 (q, 52.47 (t, C-6), 28.46, 28.09 [2 t, 2 OCH CH
2
2
2
5
3
2
CH
2
CH
3
) ppm. 31P NMR (162 MHz, CD
3
OD): δ = 19.50, 19.34, 19.16 (3 q, 3 CH
3
CO) ppm. P NMR (162 MHz,
31
2
2
–
): δ = 4.75 ppm. MS: m/z = 728.4 [M – H]–.
.66 ppm. MS: m/z = 560.2 [M – H] .
CDCl
3
Sulfoquinovose 2b: A catalytic amount of Na was added to a solu-
tion of 13b (113 mg, 0.201 mmol, 1 equiv.) in dry methanol (3 mL).
After 2 h, Amberlite IR 120 H was added to neutrality. The resin
Sulfoquinovose 2c: A catalytic amount of Na was added to a solu-
tion of compound 13c (195 mg, 0.267 mmol, 1 equiv.) in dry meth-
anol (3 mL). After 4 h, Amberlite IR 120 H+ was added to neu-
trality. The resin was then filtered off and the solvent evaporated to
+
was removed by filtration and the solvent evaporated to dryness.
The crude was purified by flash column chromatography (EtOAc/
EtOH, 3:7) to afford compound 2b (50 mg, 57% yield). H NMR (EtOAc/EtOH, 5:5) to afford compound 2c (82 mg, 51% yield). H
dryness. The crude was purified by flash column chromatography
1
1
3
3
3
3
(
CD
3
OD): δ = 4.31 (app. t, J1,2 = JH,P = 8.5 Hz, 1 H, 1-H), 4.18– NMR (CD
CH CH CH ), 3.82–3.76 (m, 1 H, 5-H), 3.42 H), 4.26–4.02 [m, 4 H, 2 OCH
2,3 = 8.5 Hz, 1 H, 3-H), 3.35 (br. d,
3
OD): δ = 4.35 (app. t, J1,2 = JH,P = 8.5 Hz, 1 H, 1-
CH (CH CH(CH ], 3.82 (app.
t, J6Ј,5 = J4,5 = 8.5 Hz, 1 H, 5-H), 3.46 (app. t, J3,2 = J3,4
4
(
.06 (m, 4 H, 2 OCH
2
2
2
3
2
2
2
)
5
3 2
)
app. t, 3
J
3,4
=
3
J
2
J
6Ј,6ЈЈ
=
3
3
3
3
=
3
3
2
1
3
1
2.9 Hz, 1 H, 6Ј-H), 3.20 (app. t, J3,2 = J1,2 = 8.5 Hz, 1 H, 2-H),
.13 (app. t, J3,4 = J4,5 = 8.5 Hz, 1 H, 4-H), 3.01 (dd, J6Ј,6ЈЈ
8.5 Hz, 1 H, 3-H), 3.38 (d, J6Ј,6ЈЈ = 14.1 Hz, 1 H, 6ЈЈ-H), 3.21 (app.
3
3
2
3
3,2 3,4 4,5
J = J J = J =
1,2 = 8.5 Hz, 1 H, 2-H), 3.15 (app. t, 3
3
3
=
t,
3
2.9,
J
6ЈЈ,5 = 8.4 Hz, 1 H, 6ЈЈ-H), 1.70–1.61 (m, 4 H, 2 8.5 Hz, 1 H, 4-H), 3.04–2.94 (m, 1 H, 6Ј-H), 1.77–0.73 [m, 38 H, 2
1
3
OCH
2
CH
2
CH
2
CH
6
3
), 1.49–1.37 (m, 4 H, 2 OCH
2
CH
2
CH
C
2
CH
NMR
3
), OCH
2 2 6 3 2 3
(CH ) CH(CH ) ] ppm. C NMR (CD OD): δ = 83.89 (d,
13
0.97–0.92 (m,
H, OCH CH CH CH ) ppm.
2
2
2
2
3
C-1), 77.09 (d, C-3), 73.74 (d, C-2), 73.24 (d, C-5), 73.04 (d, C-4),
OD): δ = 83.95 (d, C-1), 77.16 (d, C-3), 73.76 (d, 3JC,P
=
66.99, 65.20 [2 t, OCH
(CH
)
6
CH(CH
)
2
], 53.06 (t, C-6) ppm.
31
P
(CD
3
2
2
3
1
0.1 Hz, C-2), 73.41 (d, C-5), 73.04 (d, C-4), 66.91, 66.76 (2 t, 2
NMR (162 MHz, CD
H] .
3
OD): δ = 7.00 ppm. MS: m/z = 602.6 [M –
–
OCH
OCH
1
D
2
CH
2
CH
CH
2
CH
CH
3
), 52.99 (t, C-6), 32.03, 31.95 (2 t,
2
2
CH
2
2
3
2 2 2 3
), 18.44, 18.42 (2 t, OCH CH CH CH ), 12.69,
) ppm. 3 P NMR (162 MHz,
1
PKB ELISA Activity Assay: The inhibitory activities of compounds
2a–c were tested by employing the CycLex AKT/PKB Kinase As-
say/Inhibitor Screening Kit (CycLex, Eppendorf, Milano, Italy).
Plates were pre-coated with “AKTide-2T”, which can be efficiently
phosphorylated by PKB1. The detector antibody specifically de-
tects the phosphorylated “AKTide-2T”. In particular, to perform
the test, the samples were dissolved in distilled water (2a) or DMSO
(2b, 2c), and diluted in kinase buffer to a final concentration of
100, 10, or 1 μm. The final concentration of DMSO in the well was
0.1%. A control experiment contained DMSO and showed that its
2.64 (2 q, 2 OCH
O): δ = 8.65 ppm. MS: m/z = 434.2 [M – H] .
2
CH
2
CH
2
CH
3
–
2
Peracetylated Diisodecyl Phosphoramidate 9c: Triisodecyl phosphite
1.3 mL, 2.28 mmol, 2 equiv.) was added dropwise to a solution of
compound 8 (444 mg, 1.14 mmol, 1 equiv.) in dry dichloromethane
12 mL), at 0 °C. The reaction mixture was stirred overnight and
(
(
then concentrated to dryness. The crude was purified by flash col-
umn chromatography (petroleum ether/EtOAc, 5:5) to afford com-
pound 9c (690 mg, 84% yield). H NMR (CDCl
1
3
): δ = 5.18 (app.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5

Job/Unit: O42664
/KAP1
Date: 26-08-14 16:39:58
Pages: 7
L. Cipolla et al.
FULL PAPER
effect in PKB1 inhibition was close to zero. Compounds were
added together with the constitutive active form of human PKB1
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(
25 milliunits/well) and allowed to phosphorylate the bound sub-
2+
strate following the addition of Mg and ATP. The amount of
phosphorylated substrate was measured by binding it with the hor-
seradish peroxidase conjugate of an anti-phospho-AKTide-2T
monoclonal antibody, which then catalysed the conversion of the
chromogenic substrate tetramethylbenzidine from the colourless re-
duced form to the yellow oxidised product after the addition of the
quenching reagent. The absorbance of the resulting solution was
determined spectrophotometrically at λ = 450 nm and was related
to the PKB1 activity in the tested solution. Staurosporine (Sigma–
Aldrich, Milano, Italy) was employed as “inhibitor control”, as
indicated in the assay protocol.
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were seeded in duplicate into 6-well plates and exposed to the drug
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pound 2a in water, compounds 2b and 2c in DMSO, cisplatin in
saline. The compounds were then added to the culture medium.
The DMSO concentration in the medium never exceeded 0.5% and
did not affect cell growth. After 72 h of drug incubation, the cells
were harvested for counting with a cell counter. IC50 is defined as
the drug concentration producing a 50% decrease in cell growth.
Three independent experiments were performed.
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Acknowledgments
This work was supported by Fondazione Cariplo (grant number
2011-0490).
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Received: May 30, 2014
6
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O42664
/KAP1
Date: 26-08-14 16:39:58
Pages: 7
Phosphatidylinositol 3-Phosphate Mimics
Kinase Inhibitors
L. Gabrielli, I. Calloni, G. Donvito,
B. Costa, N. Arrighetti, P. Perego,
D. Colombo, F. Ronchetti, F. Nicotra,
L. Cipolla* ........................................ 1–7
Phosphatidylinositol 3-Phosphate Mimics
Based on a Sulfoquinovose Scaffold: Syn-
thesis and Evaluation as Protein Kinase B
Inhibitors
Novel protein kinase B inhibitors have
been designed as phosphatidylinositol
3-phosphate mimics and synthesised by us-
ing a 6-deoxy-6-sulfoglucose scaffold.
Keywords: Medicinal chemistry / Anti-
tumor agents / Carbohydrates / Inhibitors /
Enzymes / Phospholipids
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7