3-deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth

Article abstract of DOI:10.1016/S0960-894X(00)00640-5

3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of PI3-K and Akt. These data are presented along with IC₅₀ values for the inhibition of the growth of three cancer cell lines.

Full text of DOI:10.1016/S0960-894X(00)00640-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 173±176
3-Deoxy-3-substituted-D-myo-inositol Imidazolyl Ether Lipid
Phosphates and Carbonate as Inhibitors of the
Phosphatidylinositol 3-Kinase Pathway and Cancer Cell Growth
Youhong Hu,a Emmanuelle J. Meuillet,b Margareta Berggren,b Garth Powisb
and Alan P. Kozikowskia,*
aDrug Discovery Program, Department of Neurology, Georgetown University Medical Center,
3900 Reservoir Road NW, Washington, DC 20007, USA
bArizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724, USA
Received 11 September 2000; accepted 31 October 2000
AbstractÐ3-Modi®ed d-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibi-
tors of PI3-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.
# 2001 Elsevier Science Ltd. All rights reserved.
Phosphatidylinositol is known to undergo phosphoryla-
tion at multiple sites to generate diverse phosphoinosi-
tides that in turn regulate several signal transduction
pathways.1 In one of these pathways, PI(4,5)P2 can be
hydrolyzed by PI-PLC to give the water-soluble inositol
trisphosphate I(1,4,5)P3 together with the lipophilic
component diacylglycerol (DAG). DAG is an endogen-
ous activator of protein kinase C (PKC), while I(1,4,5)P3
interacts with a receptor on the endoplasmic reticulum2
to release intracellular Ca2+. In a second pathway, PI 3-
kinase (PI3-K) phosphorylates the 3-position of phos-
phatidylinositols to give a class of 3-phosphorylated PIPs
that have the unique ability to bind to the pleckstrin
homology (PH) domains of a number of signaling proteins
such as Akt. Akt becomes fully activated upon phos-
phorylation by phosphatidylinositol dependent kinases
(PDKs), and it regulates cell survival and proliferation
by phosphorylating a number of downstream targets
such as Bad, an inhibitor of apoptosis.3 Three mammalian
isoforms of Akt have been identi®ed, Akt1, Akt2, and
Akt3. Akt1 has been found to be overexpressed in gastric
adenocarcinomas while Akt2 is overexpressed in breast,
ovarian, and pancreatic cancer.4 Another important
counterpart to PI3-K is the tumor suppressor PTEN.
Mutations in the PTEN tumor suppressor gene appear
to be a common occurrence in a number of human
cancers.5 Thus, chemical modulation of the PI3-K
dependent phospholipid signaling cascade may o€er a
basis for the selective control of cancer cell growth while
minimizing e€ects on normal cells.
In pursuit of this particular strategy for anticancer drug
development, we have reported on the chemistry and
biology of a number of phosphatidylinositol analogues
embodying modi®cations to the 3-position of the inosi-
tol ring, as well as to the lipid portion and the phos-
phate group. Speci®cally, subsequent to our ®nding that
1d-3-deoxyphosphatidyl-myo-inositol (1) was able to
block the growth of HT-29 human colon carcinoma
cells (IC50=35.0 mM),6 the DAG portion of this mole-
cule was replaced by a more stable ether lipid moiety;
certain ether lipids are in fact known to possess anti-
tumor activity of their own. This strategy delivered the
PI analogue 2, which was found to be a reasonably
active inhibitor of both PI3-K (IC50=14.8Æ5.6 mM) and
Akt (IC50=1.5Æ0.3 mM), and to block the growth of
HT-29 colon cancer cells (IC50=2.1 mM).7 Additionally,
we have recently reported that the PI analogues 3 and 6
bearing an axially oriented 3-hydroxymethyl group are
also reasonably good inhibitors of Akt, with compound
6 being more selective than DPIEL (2) for inhibiting
Akt over PI3-K.8 In general, in studies of some of these
PI analogues in vivo, we have been plagued by problems
relating to the poor solubility of these lipids, and by
*Corresponding author. Tel.: +1-202-687-0686; fax: +1-202-687-5065;
e-mail: kozikowa@giccs.georgetown.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00640-5

174
Y. Hu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 173±176
Figure 1. Structures of 3-deoxy-3-substituted PI analogues.
their tendency to form aggregates. Consequently, we have
investigated the synthesis and activity of PI analogues in
which R1 of the ether lipid portion has been changed
from methoxy to the more basic imidazolyl group (Fig.
1, note that the calculated log P for compound 2 is 4.44,
while for 4 it is 3.22). The examination of the e€ect of
chemical modi®cation of the lipid portion was of further
interest to explore as it may play a role in Akt inhibition
as a consequence of alterations in cellular distribution
into cytosol and plasma membrane. Interestingly, in this
connection we note that the structurally related molecule
CPR 1006 belongs to the class of lipid ether drugs and
acts as a PKC inhibitor. It blocks the proliferation of HT
29 human colon cancer cells with an IC50 of 2 mM.9 In
this communication, the preparation of the two phos-
phate containing PI analogues 4 and 5 and the related
carbonate 7 are described, together with a biological
study of their inhibition of Akt, PI3-K, and cancer cell
growth.
The carbonate 7 was prepared using a protocol similar to
that employed in the synthesis of 6.8 Thus, 8a was re¯uxed
in toluene with 1,10-carbonyldiimidazole to furnish a car-
bamate. This carbamate was used directly without further
puri®cation in the coupling reaction with the imidazolyl
ether lipid. The two components were re¯uxed in toluene
in the presence of DBU to give compound 10. Finally,
hydrogenolysis of 10 delivered the desired carbonate 712 in
good purity (Scheme 2).
The three new PI imidazolyl ether lipid phosphates and
carbonate were tested for their ability to inhibit p100/
p85 PI3-K, Akt, and the cell growth of three cell lines
using the methods described previously.8 Data are pre-
sented in Table 1 along with comparison data obtained for
PI analogues 1±3 and 6. It is interesting to note that imi-
dazolyl ether lipid compounds 4 and 5 containing a phos-
phate linker both exhibit reasonably good potency for the
inhibition of PI3-K, while the carbonate 7 appears less
potent. On the other hand, the carbonate 7 is more potent
in inhibiting Akt than the phosphates 4 and 5, although
DPIEL 2 still retains the best inhibitory potency for
Akt. The imidazolyl ether lipid analogue 7 exhibits an
IC50 of 15 mM for inhibition of Akt, and it is about 3-
fold less potent than the carbonate 6 that bears a C-2
methoxy group in the glycerol portion. Both carbonates
6 and 7 are relatively poor inhibitors of PI3-K.
The imidazolyl bearing ether lipid component was pre-
pared from (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-metha-
nol by a known method,10 while the inositol components
8a and 8b were obtained as reported previously.7,8 Com-
ponents 8a and 8b were transformed into the corre-
sponding protected phosphatidylinositols 9a and 9b using
a standard phosphitylation protocol followed by adjust-
ment of the oxidation state of the phosphorus atom.7,8
Finally, hydrogenolysis of the benzyl groups of the inter-
mediates 9a and 9b with 20% Pd(OH)2/C in tert-butanol
gave the desired phosphates 4 and 5 (Scheme 1).11
In terms of the inhibition of cell growth, of the three cell
lines tested, the imidazole bearing compounds 4 and 5
exhibit the best potency for inhibition of the MCF-7
Scheme 1. Synthesis of phosphates 4 and 5.

Y. Hu et al. / Bioorg. Med. Chem. Lett. 11 (2001) 173±176
175
Scheme 2. Synthesis of carbonate 7.
Table 1. IC50 values for inhibition of Akt, PI3-K, and growth of cancer cell lines by compounds 1±7
Compounds
Akt
(IC50, mM)
PI3-K
(IC50, mM)
NIH3T3
(IC50, mM)
HT-29
(IC50, mM)
MCF-7
(IC50, mM)
1
2
3
4
5
6
7
27.4Æ4.4
1.5Æ0.3
7.8Æ0.8
20.5Æ1.8
37.5Æ12
5.0Æ1.9
15.0Æ2.5
38.4Æ4.1
14.8Æ5.6
31.0Æ7.0
7.9Æ3.4
14.0
4.5
2.0
8.2
6.5
2.0
11.5
35.0
2.1
4.5
6.5
15.0
10.0
15.0
10.0
7.2
5.0
2.0
6.0
1.25
20.0
14.5Æ3.2
83.0Æ21.0
49.5Æ4.6
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(breast) cancer cell line; IC50 values of 2.0 and 6.0 mM
were measured, respectively. However, the ether lipid
carbonate 6 is still slightly more potent than either of
these two analogues.
In conclusion, the present work details the synthesis of
novel 3-substituted-d-myo-inositol imidazolyl contain-
ing ether lipid phosphates and a carbonate. The ability
of these compounds to inhibit both Akt and PI3-K were
evaluated, together with the e€ects of these PI ana-
logues to block cell growth in NIH3T3, HT-29 and
MCF-7 cells. Compound 4 was particularly e€ective in
blocking the growth of the MCF-7 cell line.
Acknowledgements
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This work was supported by the National Institutes of
Health (Grant No. CA 61015) and the Department of
Defense (DAMD 17-93-V-3018).
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14.48; 31P (121 MHz, CDCl3/CD3OD 1:1, 85% H3PO4) d 0.58.
Compound 5: [a]D 23.7ꢀ (c 0.81, CHCl3/MeOH 1:1); 1H
NMR (CDCl3/CD3OD 1:1, TMS) d 9.02 (s, 1H), 7.72 (s, 1H),
7.48 (s, 1H), 4.34 (t, 2H, J=5.7 Hz), 4.15 (m, 1H), 3.87 (m,
4H), 3.69 (t, 1H, J=9.3 Hz), 3.46 (m, 2H), 3.33 (m, 1H), 3.18
(t, 1H, J=9.3 Hz), 2.09 (dt, 1H, J=13.5, 3.9 Hz), 1.54 (m, 3H),
1.27 (m, 30H), 0.89 (t, 3H, J=6.0 Hz); 13C NMR (CDCl3/
CD3OD 1:1) d 136.10, 122.38, 120.03, 80.38 (d, J=5.5 Hz),
78.95, 72.55 (d, J=5.0 Hz), 72.39, 69.55, 68.88, 68.35, 64.83 (d,
J=4.6 Hz), 61.87 (d, J=6.6 Hz), 35.91, 32.61, 30.35, 30.03,
26.70, 23.32, 14.32; 31P (121 MHz, CDCl3/CD3OD 1:1, 85%
9. Goekjian, P. G.; Jirousek, M. R. Curr. Med. Chem. 1999, 6,
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.
H3PO4) d 0.68. Anal. calcd for C30H57N2O9P 1.4H2O: C,
55.78; H, 9.33. Found: C, 55.77; H, 9.35.
11. Compound 4: [a]D 8.6ꢀ (c 0.52, CHCl3/MeOH 1:1); H
1
12. Compound 7: [a]D 23.8ꢀ (c 0.49, CHCl3/MeOH 1:1); H
1
NMR (300 MHz, CDCl3/CD3OD 1:1, TMS) d 9.00 (s, 1H),
7.69 (s, 1H), 7.45 (s, 1H), 4.84 (m, 1H), 4.34 (t, 2H, J=5.7 Hz),
4.20 (m, 1H), 4.07 (m, 1H), 3.97 (dd, 1H, J=9.3, 6.0 Hz), 3.87
(m, 3H), 3.73 (t, 1H, J=8.7 Hz), 3.57 (dd, 1H, J=11.1,
8.4 Hz), 3.45 (m, 3H), 2.32 (m, 1H), 1.54 (m, 2H), 1.35±1.24
(m, 30H), 0.89 (t, 3H, J=6.3 Hz); 13C NMR (75 MHz, CDCl3/
CD3OD 1:1) d 136.08, 122.43, 120.08, 78.26 (d, J=6.0 Hz),
75.29, 73.17 (d, J=5.0 Hz), 72.55, 70.79, 70.05, 69.65, 64.95 (d,
J=5.0 Hz). 61.94 (d, J=7.0 Hz), 59.25, 47.73, 32.69, 31.12,
30.43, 30.40, 30.36, 30.17, 30.13, 30.11, 26.77, 23.40, 19.30,
NMR (CDCl3/CD3OD 1:1, TMS) d 9.08 (s, 1H), 7.79 (s, 1H),
7.60 (s, 1H), 4.97 (m, 1H), 4.74 (dd, 1H, J=9.9, 3.0 Hz), 4.61
(m, 1H), 4.23 (m, 1H), 3.89 (m, 3H), 3.75 (t, 1H, J=9.3 Hz),
3.48 (m, 4H), 3.29 (m, 2H), 2.28 (m, 1H), 1.67 (m, 1H), 1.53
(m, 2H), 1.26 (m, 29H), 0.88 (t, 3H, J=6.3 Hz); 13C NMR
(CDCl3/CD3OD 1:1, TMS) d 155.77, 136.95, 122.84, 121.24,
80.67, 75.91, 72.84, 72.69, 71.16, 69.74, 68.99, 67.30, 61.04,
59.55, 33.21, 30.92, 30.66, 30.62, 27.32, 23.87, 14.59.