Synthesis and biological activity of 3-hydroxy(phosphono)methyl-bearing phosphatidylinositol ether lipid analogues

Article abstract of DOI:10.1016/S0040-4039(02)00354-4

Two 3-hydroxy(phosphono)methyl-bearing phosphatidylinositol ether lipid analogues were synthesized and shown to be inhibitors of Akt and PI3-K. These compounds were also shown to inhibit the growth of HT-29 human colon cancer cells and MCF-7 human breast

Full text of DOI:10.1016/S0040-4039(02)00354-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2835–2838
Synthesis and biological activity of
3-hydroxy(phosphono)methyl-bearing phosphatidylinositol
ether lipid analogues
Haiying Sun,a Gaddam Bapu Reddy,a Clifford George,b Emmanuelle J. Meuillet,c
Margareta Berggren,c Garth Powisc and Alan P. Kozikowskia,*
aDrug Discovery Program, Department of Neurology, Georgetown University Medical Center, 3970 Reservoir Road, NW,
Washington, DC 20007, USA
bNaval Research Laboratory, 4555 Overlook Ave, SW, Washington, DC 20375, USA
cArizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724, USA
Received 14 December 2001; accepted 15 February 2002
Abstract—Two 3-hydroxy(phosphono)methyl-bearing phosphatidylinositol ether lipid analogues were synthesized and shown to
be inhibitors of Akt and PI3-K. These compounds were also shown to inhibit the growth of HT-29 human colon cancer cells and
MCF-7 human breast cancer cells. © 2002 Elsevier Science Ltd. All rights reserved.
Phosphatidylinositol 3-kinase (PI3-K) phosphorylates
the 3-position of phosphatidylinositol (PI), PI(4)P, and
PI(4,5)P2 to give rise to three signaling phospholipids:
PI(3)P, PI(3,4)P2, and PI(3,4,5)P3, respectively.1 These
3-phosphorylated PIs have the unique ability to bind to
specific protein domains, the so-called pleckstrin
homology (PH) domains, of a number of signaling
proteins. One of the most extensively studied of the PH
domain-regulated signaling proteins acting downstream
of PI3-K is the proto-oncogenic serine/threonine kinase
Akt. In particular, while the PH domain of Akt binds
both PI(3,4)P2 and PI(3,4,5)P3 in vitro, only PI(3,4)P2
activates Akt.2 Akt is a proto-oncogene that inhibits
apoptosis by phosphorylating a number of downstream
targets, thus, the inhibition of Akt activation induces
cancer cell apoptosis.3 An important counterpart to
PI3-K is the tumor suppressor PTEN, a protein that is
able to bring about the dephosphorylation of
PI(3,4,5)P3, with specificity being shown for the phos-
phate at the D-3 position of the inositol ring. Muta-
tions in the PTEN tumor suppressor gene appear to be
a common occurrence in a number of human cancers.4
Thus, PI3-K and Akt provide novel targets for drugs to
inhibit the repression of apoptosis in cancer cells and
thereby the opportunity to overcome the effects of the
loss of the tumor suppressor PTEN.
In our previous work,5 we have designed and synthe-
sized some 3-modified PI ether lipid analogues6 (Fig. 1),
and found that all of these compounds exhibit some
activity for the inhibition of Akt and PI3-K. Because of
these favorable results, we deemed it valuable to inves-
tigate the activity of other 3-modified PI ether lipid
analogues with the aim to further improve upon Akt
inhibition and antiproliferative action. In this paper, we
detail the synthesis and biology of analogues that bear
a hydroxy(phosphono)methyl group at position 3.
These compounds thus act as mimics of 3-phosphoryl-
ated PI.
OH
X
OMe
HO
HO
O
O
OC18H37
P
Y
HO
O
OH
1 X = H, Y = H
2 X = CH2OH, Y = H
3 X = H, Y = CH2OH
OH
X
OMe
HO
HO
O
O
OC18H37
Y
HO
O
4 X = CH2OH, Y = H
5 X = H, Y = CH2OH
* Corresponding author. Tel.: 2026870686; fax: 2026875065; e-mail:
kozikowa@georgetown.edu
Figure 1.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00354-4

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H. Sun et al. / Tetrahedron Letters 43 (2002) 2835–2838
Compound 6 was synthesized according to our pub-
lished method from
-quebrachitol.6 After allyl protec-
Biological activity
L
tion of the 3-hydroxymethyl group, the trans-acetonide
was selectively removed to give diol 7. The two
hydroxyl groups of 7 were protected by benzylation,
and the second acetonide was removed to yield diol 8.
After selective p-methoxybenzylation of the 1-OH via a
1,2-O-stannylene intermediate and benzylation of the
2-OH, the allyl group was removed to give alcohol 9.
Dess–Martin oxidation of the hydroxymethyl group in
9 gave an aldehyde. Reaction of this aldehyde with
lithium dibenzyl phosphite (prepared in situ from
dibenzyl phosphite and butyllithium) yielded two phos-
phonates 10 and 11, which can be separated by chro-
matography. Subsequently, the p-methoxybenzyl
groups in 10 and 11 were removed by oxidation with
CAN to give two key intermediates 12 and 13 (Scheme
1).
Data for the two new PI analogues from the PI3-K and
Akt activity studies are presented in Table 1 along with
comparison data obtained previously for compounds 2
and 3.
The two new analogues exhibit comparable activity for
the inhibition of Akt and PI3-K, while 21 is more
selective for Akt than 20. The compounds are more
potent inhibitors of Akt and PI3-K than the hydroxy-
methyl-bearing compounds 2 and 3, which suggests that
the phosphonate moiety may increase their binding
affinity for these two signaling proteins.
The growth inhibition of different cancer cell lines by
compounds 20 and 21 was also tested (Table 2). We
found that these PI analogues have similar cell growth
inhibition effects on HT-29 human colon cancer and
MCF-7 human breast cancer cell lines, but that they are
somewhat less active than compounds 2 and 3. They
also exhibit some activity against the NIH3T3 mouse
embryo derived cell line. The cell growth inhibitory
effects of these PI analogues are probably due to a
combination of their PI3-K and Akt inhibitory activi-
ties along with effects on other cell signaling proteins.6
In order to ascertain the stereochemistry of 12 and 13,
we transformed 6 into phosphonate 14 by Dess–Martin
oxidation and subsequent phosphite addition (Scheme
2). It is interesting that in this case only one isomer was
obtained. Compound 14 proved to be crystalline, and
we established its structure by X-ray analysis (Fig. 2).
Compound 14 was totally deprotected by hydrogena-
tion and subsequent acidification to give 15. By com-
paring the 1H and 13C NMR spectra of 15 with those of
the deprotection products of 12 and 13, we found that
the hydrogenation product of 13 is identical to 15
(Scheme 2).
After solving the problem of stereochemistry, 12 and 13
were phosphorylated by reaction with the ether lipid
phosphoramidite 17 catalyzed by 1H-tetrazole and sub-
sequent oxidation of the phosphite intermediates with
tert-butyl hydroperoxide. Finally the resulting phos-
phates 18 and 19 were completely deprotected by cata-
lytic hydrogenation to give the desired PI analogues 20
and 21 (Scheme 3).7,8
Scheme 2. Reagents and conditions: (a) (i) Dess–Martin perio-
dinane, CH2Cl2, rt, (ii) dibenzyl phosphite, BuLi, THF,
−78°C, 85% for two steps; (b) (i) H2, 20% Pd(OH)2–C,
t-BuOH, 1 atm, rt, (ii) 3N HCl, rt, 82%; (c) H2, 20%
Pd(OH)2–C, t-BuOH, 1 atm, rt, 96%.
HO
O
OH
MeO
HO
ref. 6
a
O
OH
O
O
OH
HO
BnO
6
(L)-Quebrachitol
AllylO
AllylO
HO
O
OH
OH
OBn
OMPM
HO
HO
BnO
BnO
BnO
BnO
b
c
O
BnO 7
BnO
8
BnO
9
O
P
O
P
Y
Y
BnO
BnO
BnO
BnO
X
X
OBn
OMPM
OBn
OH
e
d
BnO
BnO
BnO
BnO
BnO
BnO
10 (X = H, Y = OH) 48%
11 (X = OH, Y = H) 42%
12 (X = H, Y = OH) 88%
13 (X = OH, Y = H) 89%
Scheme 1. Reagents and conditions: (a) (i) NaH, allyl bromide, DMF, 0°C to rt, (ii) AcCl (cat.), CH2Cl2–CH3OH, rt, 72% for two
steps; (b) (i) NaH, BnBr, DMF, 0°C to rt; (ii) AcCl (cat.), CH3OH, rt, 90% for two steps; (c) (i) Bu2SnO, toluene, reflux, then
p-MeOC6H4CH2Cl, CsF, DMF, rt, (ii) NaH, BnBr, DMF, 0°C to rt; (iii) RhCl(PPh3)3 (cat.), DABCO, EtOH, reflux, then 1N
HCl, acetone, reflux, 85% for three steps; (d) (i) Dess–Martin periodinane, CH2Cl2, rt, (ii) dibenzyl phosphite, BuLi, THF, −78°C;
(e) CAN, CH3CN–H2O, 4:1 (v/v), rt.

H. Sun et al. / Tetrahedron Letters 43 (2002) 2835–2838
2837
Figure 2. X-Ray structure of compound 14.
OMe
OMe
Table 2. Effects of compounds 2, 3, 20 and 21 on the
growth inhibition of cell lines in vitro (72 h exposure)
a
HO
OC18H37
(iPr)2N
O
OC18H37
P
16
17
OBn
Compound
IC50 (mM)
O
P
Y
BnO
BnO
X
OBn
HT-29
MCF-7
NIH3T3
O
OMe
b
c
BnO
BnO
O
P O
OBn
OC18H37
2
3
20
21
4.5
7.5
7.8
7.8
5.0
2.0
9.0
8.0
–
–
19.8
19.8
BnO
18 (X = H, Y = OH) 65%
19 (X = OH, Y = H) 51%
O
Y
HO
HO
X
OH
P
O
OMe
HO
HO
O
P O
OC18H37
are structurally related to PI-3-phosphate and act as
reasonably good inhibitors of Akt and PI3-K.
HO
OH
20 (X = H, Y = OH) 95%
21 (X = OH, Y = H) 96%
Acknowledgements
Scheme 3. Reagents and conditions: (a) BnOP(N(i-Pr)2)2,
diisopropylammonium tetrazolide, CH2Cl2, rt, 98%; (b) (i) 12
or 13, 1H-tetrazole, CH2Cl2, rt, (ii) tert-butyl hydroperoxide,
CH2Cl2, rt; (c) H2, Pd(OH)2–C, t-BuOH, 1 atm, rt.
We thank the National Institutes of Health (Grant
CA61015) for support of this research.
Table 1. IC50 values for inhibition of Akt and PI3-K
activity
References
Compound
IC50 (mM)
1. (a) Carpenter, C. L.; Cantley, L. C. Curr. Opin. Cell Biol.
1996, 8, 153–158; (b) Shepherd, P. R.; Reaves, B. J.;
Davidson, H. W. Trends Cell. Biol. 1996, 6, 92–97.
Akt
PI3-K
2
3
20
21
7.890.8
9.191.7
4.591.3
2.5
31.097.0
18.591.7
5.791.2
8.891.7
2. (a) Lemmon, M. A.; Ferguson, K. M.; Schlessinger, J. Cell
1996, 85, 621–624; (b) French, M.; Andjelkovich, M.;
Ingley, E.; Reddy, K. K.; Falck, J. R.; Hemmings, B. A. J.
Biol. Chem. 1997, 272, 8474–8481; (c) Stokoe, D.;
Stephens, L. R.; Copeland, T.; Gaffney, P. R. J.; Resses,
C. B.; Painter, G. F.; Holmes, A. B.; McCormick, F.;
Hawkins, P. T. Science 1997, 277, 567–570; (d) Grishin, N.
V. J. Mol. Biol. 1999, 291, 239–247; (e) Cross, D. A. E.;
Alessi, D. R.; Cohen, P.; Andjelkovich, M.; Hemmings, B.
A. Nature 1995, 378, 785–789; (f) Kauffmann-Zeh, A.;
In conclusion, the present paper details the synthesis
and biological activity of two new 3-hydroxy(phos-
phono)methyl-bearing PI analogues. These compounds

2838
H. Sun et al. / Tetrahedron Letters 43 (2002) 2835–2838
Rodriguez-Viciana, P.; Ulrich, E.; Gilbert, C.; Coffer, P.;
Tetrahedron 1997, 53, 14903–14914; (f) Qiao, L.; Nan, F.;
Kunkel, M.; Gallegos, A.; Powis, G.; Kozikowski, A. P. J.
Med. Chem. 1998, 41, 3303–3306; (g) Qiao, L.; Hu, Y.;
Nan, F.; Powis, G.; Kozikowski, A. P. Org. Lett. 2000, 2,
115–117.
Downward, J.; Evan, G. Nature 1997, 385, 544–548; (g)
Kulik, G.; Klippel, A.; Weber, M. J. Mol. Cell. Biol. 1997,
17, 1595–1606; (h) Paradis, S.; Ailion, M.; Toker, A.;
Thomas, J. H.; Ruvkun, G. Gen. Dev. 1999, 13, 1438–
1452.
6. Hu, Y.; Qiao, L.; Wang, S.; Rong, S.; Meuillet, E. J.;
Berggren, M.; Gallegos, A.; Powis, G.; Kozikowski, A. P.
J. Med. Chem. 2000, 43, 3045–3051.
3. (a) Coffer, P. J.; Jin, J.; Woodgett, J. R. Biochem. J. 1998,
335, 1–13; (b) Staal, S. P. Proc. Natl. Acad. Sci. USA 1987,
84, 5034–5037; (c) Bellacosa, A.; Feo, D. D.; Godwin, A.
K.; Bell, D. W.; Cheng, J. Q.; Altomare, D. A.; Wan, M.;
Dubeau, L.; Scambia, G.; Masciullu, V.; Ferrandina, G.;
Panici, P. B.; Mancuso, S.; Neri, G.; Testa, J. R. Int. J.
Cancer 1995, 64, 280–285; (d) Thompson, F. H.; Nelson,
M. A.; Trent, J. M.; Guan, X. Y.; Liu, Y.; Yang, J. M.;
Emerson, J.; Adair, L.; Wymer, J.; Balfour, C.; Massey,
K.; Weinstein, R.; Alberts, D. S.; Taetle, R. K. K.; Testa,
J. R. Proc. Natl. Acad. Sci. USA 1996, 93, 3636–3641.
4. (a) Cantley, L. C.; Neel, B. G. Proc. Natl. Acad. Sci. USA
1999, 96, 4240–4245; (b) Lee, J.; Yang, H.; Georgescu, M.;
Cristofano, A. D.; Maehama, T.; Shi, Y.; Dixon, J. E.;
Pandolfi, P.; Paveltich, N. P. Cell 1999, 99, 323–334.
5. (a) Kozikowski, A. P.; Ognyanov, V. I.; Fauq, A. H.;
Wilcox, R. A.; Nahorski, S. R. J. Chem. Soc., Chem.
Commun. 1994, 559–560; (b) Kozikowski, A. P.;
Ognyanov, V. I.; Fauq, A. H.; Nahorski, S. R.; Wilcox, R.
A. J. Am. Chem. Soc. 1993, 115, 4429–4434; (c)
Kozikowski, A. P.; Kiddle, J. J.; Frew, T.; Berggren, M.;
Powis, G. J. Med. Chem. 1995, 38, 1053–1056; (d)
Kozikowski, A. P.; Powis, G.; Fauq, A. H.; Tu¨ckmantel,
W.; Gallegos, A. J. Org. Chem. 1994, 59, 963–971; (e)
Kozikowski, A. P.; Qiao, L.; Tu¨ckmantel, W.; Powis, G.
7. Compound 20: [h]D25=−4.6 (c 0.2, CHCl3–CH3OH, 1:1);
1H NMR (300 MHz, CD3OD–CDCl3, 1:1, TMS): l 4.75
(br s, 1H), 4.40 (br d, J=6.9 Hz, 1H), 4.22–4.02 (m, 2H),
4.02–3.88 (m, 2H), 3.83 (t, J=9.3 Hz, 1H), 3.70–3.40 (m,
9H), 1.80 (m, 1H), 1.55 (m, 2H), 1.25 (br s, 30H), 0.88 (t,
J=6.6 Hz, 3H); 13C NMR (75 MHz, CD3OD–CDCl3,
1:1): l 81.07, 79.53, 72.45, 71.80, 70.53, 69.92, 68.64, 67.94,
67.79, 67.76, 58.25, 42.74, 32.45, 30.21, 30.17, 30.05, 30.02,
29.88, 26.54, 23.18, 14.40; 31P NMR (CD3OD–CDCl3, 1:1,
121 MHz, 85% H3PO4): l 23.49, −0.42. Anal. calcd for
C29H60O14P2: C, 50.14; H, 8.71; found: C, 50.14; H, 8.69%.
8. Compound 21: [h]D25=5.1 (c 0.43, CHCl3–CH3OH, 1:1); 1H
NMR (300 MHz, CD3OD–CDCl3, 1:1, TMS): l 4.37 (br s,
1H), 4.35–4.25 (m, 1H), 4.23–3.98 (m, 4H), 3.69 (t, J=9.3
Hz, 1H), 3.53–3.40 (m, 9H), 2.10–1.93 (m, 1H), 1.62–1.50
(m, 2H), 1.26 (br s, 30H), 0.88 (t, J=6.6 Hz, 3H); 13C
NMR (75 MHz, CD3OD–CDCl3, 1:1): l 81.32, 79.72,
77.89, 72.58, 71.72, 71.28, 71.08, 70.10, 68.82, 66.88, 58.33,
44.76, 32.61, 30.36, 30.32, 30.23, 30.18, 30.04, 26.72, 23.34,
14.46; 31P NMR (121 MHz, CD3OD–CDCl3, 1:1, 85%
H3PO4):
l
23.49,
−0.30.
Anal.
calcd
for
C29H60O14P2·1.5H2O: C, 48.28; H, 8.80; found: C, 48.31;
H, 8.84%.