Synthesis of pelorol and analogues: activators of the inositol 5-phosphatase SHIP

Article abstract of DOI:10.1021/ol047316m

(Chemical Equation Presented) A screening program designed to find new antiinflammatory agents has identified the sponge meroterpenoid pelorol (1) as an in vitro activator of the inositol-5-phosphatase SHIP. Pelorol (1) and several functional group analog

Full text of DOI:10.1021/ol047316m

ORGANIC
LETTERS
2005
Vol. 7, No. 6
1073-1076
Synthesis of Pelorol and Analogues:
Activators of the Inositol 5-Phosphatase
SHIP
Lu Yang,† David E. Williams,† Alice Mui,‡ Christopher Ong,‡ Gerald Krystal,§
|
Rob van Soest, and Raymond J. Andersen*,†
Departments of Chemistry and Earth and Ocean Sciences, UniVersity of British
Columbia, VancouVer, B.C., Canada V6T 1Z1, The Jack Bell Research Centre,
VancouVer, B.C., Canada V6H 3Z6, Terry Fox Laboratory, B.C. Cancer Control
Agency, VancouVer, B.C., Canada V5Z 1L3, and Zoologisch Museum,
UniVersity of Amsterdam, Amsterdam, The Netherlands
randersn@interchange.ubc.ca
Received December 29, 2004
ABSTRACT
A screening program designed to find new antiinflammatory agents has identified the sponge meroterpenoid pelorol (1) as an in vitro activator
of the inositol-5-phosphatase SHIP. Pelorol (1) and several functional group analogues have been synthesized from sclareolide (4).
The phosphatidylinositol-3-kinase (PI3K) signaling pathway
plays an important role in the regulation of many cellular
functions, including survival, adhesion, movement, prolifera-
tion, differentiation, and end cell activation.1 A key second
messenger in this pathway is the membrane-associated
phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3), which
is present in low levels in unstimulated cells but is rapidly
synthesized from PI-4,5-P2 by PI3K in response to a diverse
set of extracellular stimuli (Figure 1). To ensure that
activation of the PI3K pathway is appropriately restrained,
the tumor suppressor PTEN hydrolyzes PI-3,4,5-P3 back to
PI-4,5-P2 and the Src homology 2-containing inositol 5-phos-
phatases SHIP, sSHIP, and SHIP2 hydrolyze it to PI-3,4-P2.
Approximately 50% of human cancers contain biallelic
inactivating mutations of the ubiquitously expressed PTEN,
which illustrates the importance of these phosphatases in
preventing uncontrolled cell growth. Similar to PTEN, SHIP2
is expressed in a wide variety of cell types, whereas sSHIP
is restricted to stem cells and SHIP is found only in
hematopoietic (blood) cells.
† University of British Columbia.
‡ The Jack Bell Research Centre.
§ Terry Fox Laboratory.
| University of Amsterdam.
(1) (a) Kalesnikoff, J.; Sly, L. M.; Hughes, M. R.; Buchse, T.; Rauh, M.
J.; Cao, L.-P.; Lam, V.; Mui, A.; Huber, M.; Krystal, G. ReV. Physiol.
Biochem. Pharmacol. 2003, 149, 87-103. (b) Rauh, M. J.; Kalesnikoff,
M.; Hughes, M.; Sly, L.; Lam, V.; Krystal, G. Biochem. Soc. Trans. 2003,
31, 286-291. (c) Rauh, M. J.; Krystal, G. Clin. InVest. Med. 2002, 25,
68-69. (d) Krystal, G. Semin. Immunol. 2002, 12, 397-403.
Figure 1. Enzymatic synthesis and degradation of PI-3,4,5-P3.
10.1021/ol047316m CCC: $30.25
© 2005 American Chemical Society
Published on Web 02/16/2005

Krystal and co-workers have generated mice containing a
homozygous deletion of SHIP (SHIP-/- mice).2 These
animals are viable and fertile but typically do not survive
beyond 14 weeks because of a myeloproliferative disorder.
Experiments with SHIP-/- mice and with SHIP-/- bone-
marrow-derived mast cells (BMMCs) and macrophages
(BMmφs) obtained from these mice, have demonstrated that
SHIP is a negative regulator of immunoglobulin E (IgE) or
Steel Factor induced mast cell activation,3 a negative
regulator of lipopolysaccharide (LPS) induced macro-
phage activation, and a negative regulator of osteoclast
formation and resorptive function. As a result of this last
property, SHIP-/- mice suffer from severe osteoporosis.4
There is also evidence that SHIP acts as a tumor suppressor
in both acute myelogenous leukemia (AML)5 and in chronic
myelogenous leukemia (CML).6
Current attempts to develop drugs based on intervention
in signaling pathways are overwhelmingly biased toward
finding selective kinase inhibitors. There has been some
recent interest in examining the therapeutic potential of
phosphatase inhibitors,7 but there has been virtually no effort
to explore the usefulness of small-molecule phosphatase
activators. The important role of SHIP as a negative regulator
of mast cell and macrophage activation, osteoclast formation,
and resorptive function, as well as in AML and CML,
combined with its occurrence only in hematopoietic cells,
makes it an attractive drug target. We hypothesized that
selective activators of SHIP would be useful experimental
tools and potential drug candidates to provide proof of
principle validation for a new approach to the treatment of
inflammation, osteoporosis, and leukemia.
undescribed when first isolated in our laboratory as a SHIP
activator, but while further biological studies were in progress
it was isolated by Konig’s group, also from D. elegans,13
and by Schmitz’s group from Petrosaspongia metachromia.10
Spectroscopic data obtained for pelorol in the current work
was in complete agreement with the data reported by Konig
and Schmitz.
The limited quantity (∼10 mg) of pelorol (1) available
from the source sponge D. elegans was inadequate to support
detailed in vitro and in vivo evaluation of its ability to
activate SHIP. To satisfy the need for additional material,
confirm the absolute configuration of the natural product,
and generate analogues for SAR, the total synthesis of pelorol
(1) and analogues where the methyl ester at C-20 was
replaced by methyl and ethyl residues was undertaken.
On the basis of sound biogenetic arguments, Schmitz
predicted that the absolute configuration of pelorol (1) was
5S,8R,9R,10S as drawn. Therefore, the starting material
selected for the synthesis of pelorol and analogues was the
commercially available terpenoid (+)-sclareolide (4), which
has the same absolute configurations at C-5, C-9, and C-10
as those predicted for pelorol (1). The synthetic plan
anticipated that the key reaction would involve a biomimetic
carbocation-initiated cyclization of an intermediate I to
generate the C-8/C-21 bond (Scheme 1). Steric bulk associ-
Scheme 1. Retrosyntheic Analysis of Pelorol (1)
Crude extracts of marine invertebrates were screened for
in vitro activation of the SHIP-catalyzed conversion of
inositol-1,3,4,5-tetrakisphosphate (IP4) to inositol-1,3,4-tris-
phosphate (IP3).8 A MeOH extract of the sponge Dactylo-
spongia elegans (Thiele, 1899), collected in Papua New
Guinea,9 showed promising activity in the assay. Bioassay-
guided fractionation of the extract identified pelorol (1) as
the sole SHIP-activating component. Three related mero-
ated with the C-14 methyl was expected to cause preferential
approach of the phenyl ring from the bottom face of C-8 to
form the required trans B/C ring fusion. Synthetic routes to
both C-8 epimers (5 and 15) of II starting from sclareolide
have been reported, and the plan was to examine both as
terpenoids, illimaquinone,10 mamanuthaquinone,11 and dac-
tyloquinone A,12 were also isolated from the D. elegans
extract but were not active in the assay. Pelorol (1) was
(5) Luo, J.-M.; Yoshida, H.; Komura, S.; Ohishi, N.; Pan, L.; Shigeno,
K.; Hanamura, I.; Miura, K.; Iida, S.; Ueda, R.; Naoe, T.; Akao, Y.; Ohno,
R.; Ohnishi, K. Leukemia 2003, 17, 1-8.
(6) Sattler, M.; Salgia, R.; Shrikhande, G.; Verma, S.; Choi, J.-L.;
Rohrschneider, L. R.; Griffin, J. D. Oncogene 1997, 15, 2379-2384.
(7) McCluskey, A.; Sim, A. T. R.; Sakoff, J. A. J. Med. Chem. 2002,
45, 1151-1175.
(8) The SHIP assay was performed in 96-well microtitre plates with 10
ng of recombinant SHIP enzyme per well. SHIP enzyme was incubated
with extract or DMSO for 15 min at 23 °C before addition of 200 mM
inositol-1,3,4,5-tetrakisphosphate. The reaction was allowed to proceed for
20 min at 37°C and the amount of inorganic phosphate released was then
assessed by the addition of Malachite Green reagent followed by an
absorbance measurement at 650 nm.
(2) Helgason, C. D.; Damen, J. E.; Rosten, P.; Grewal, R.; Sorensen, P.;
Chappel, S. M.; Borowski, A.; Jirik, F.; Krystal, G.; Humphries, R. K. Genes
DeV. 1998, 12, 1610-1620.
(3) Huber, M.; Helgason, C. D.; Damen, J. E.; Liu, L.; Humphries, R.
K.; Krystal, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11330-11335.
(4) Takeshita, S.; Namba, N.; Zhao, J. J.; Jiang, Y.; Genant, H. K.; Silva,
M. J.; Brodt, M. D.; Helgason, C. D.; Kalesnikoff, J.; Rauh, M. J.;
Humphries, R. K.; Krystal, G.; Teitelbaum, S. L.; Ross, F. P. Nat. Med.
2002, 8, 943-949.
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Org. Lett., Vol. 7, No. 6, 2005

intermediates in the preparation of direct precursors to the
carbocation I and ultimately cyclized products.
(R ) Et vs R ) OMe) making the elimination reaction
leading to 12 and the Wagner Meerwein rearrangements
leading to 13 competitive with direct trapping of the C-8
carbocation by the arene to give the desired product 11. After
optimization of the reaction conditions, it was found that
the SnCl4-catalyzed cyclization gave consistently high yields
(∼76%) for the conversion of 9 to 11.
(+)-Sclareolide (4) was converted to the aldehyde 5
following the literature procedure (Scheme 2).14 Overman
Scheme 2. Initial Attempts at Biomimetic Cyclizations
Although it was possible to obtain 11 via the aldehyde 5,
the preparation of 5 from sclareolide was cumbersome
because it required multiple chromatographic separations and
preparation of an oxidizing agent that was not commercially
available.14 Therefore, we turned our attention to the aldehyde
15, having an equatorial OH group at C-8 (Scheme 3).16 (+)-
Scheme 3. Synthesis of the Cyclized Intermediate 11
showed in his synthesis of adociasulfate that a highly
nucleophilic arene was required to trap a carbocation such
as I in preference to proton elimination to give an uncyclized
olefin.15 Therefore, trimethoxyphenyllithium 6 (2 equiv) was
added to aldehyde 5, followed by hydrogenolysis of the
resulting epimeric benzyl alcohols, to give the tertiary alcohol
8. Treatment of 8 with SnCl4 gave the cyclization product
10 having the desired stereochemistry in good yield. Unfor-
tunately, all attempts to transform 1,2,4-trialkoxy benzene
10 or various alkyl ether analogues into pelorol failed.
Sclareolide (4) was converted to the diol 14 in excellent yield
(90%) using a one-pot three-step sequence modification of
the literature procedure.16 Swern oxidation cleanly oxidized
the diol 14 to the aldehyde 15.17 Reaction of 15 with
phenyllithium 7 gave the epimeric benzyl alcohols 16 in good
yield. Hydrogenolysis of the mixture 16 cleanly removed
the benzylic alcohols to give 17. Cyclization of 17 using
SnCl4 as a catalyst gave the desired tetracyclic intermediate
11 in high yield, without any trace of the elimination product
12. Dimethyl ether 11 was converted to the catechol 21, and
the entire sequence was repeated with phenyllithium 7a to
give the methyl analogue 22 to provide pelorol analogues
for SAR.
In an attempt to overcome this problem, the coupling
reaction was repeated with dimethoxyethylphenyllithium 7
to give the tertiary alcohol 9. Initial reactions of 9 with SnCl4
gave variable yields of the desired product 11 and the
undesired elimination product 12, while treatment with the
protic acid PPA gave only the unanticipated cyclization
product 13. We envisaged that the formation of 12 and 13
resulted from reduction of the nucleophilicity of the arene
(9) A voucher sample has been deposited at the Zoological Museum,
University of Amsterdam (ZMA POR. 15986).
(10) Kwak, J. H.; Schmitz, F. J.; Kelly, M. J. Nat. Prod. 2000, 63, 1153-
1156.
(11) Swersey, J. C.; Barrows, L. R.; Ireland, C. M. Tetrahedron Lett.
1991, 32, 6687-6690.
(12) Mitome, H.; Nagasawa, T.; Miyaoka, H.; Yamada, Y.; van Soest,
R. W. M. J. Nat. Prod. 2001, 64, 1506-1508.
(13) Goclik, E.; Konig, G. M.; Wright, A. D.; Kaminsky, R. J. Nat. Prod.
2000, 63, 1150-1152.
(14) Quideau, S.; Lebon, M.; Lamidey, A.-M. Org. Lett. 2002, 4, 3975-
3978.
Reaction of 11 with PCC selectively oxidized the C-22
methylene to give methyl ketone 23 in reasonable yield
(Scheme 4). Treatment of 23 with I2 in aqueous NaOH, in
(16) Kuchkova, K. I.; Chumakov, Y. M.; Simonov, Y. A.; Bocelli, G.;
Panasenko, A. A.; Vlad, P. F. Synthesis 1997, 9, 1045-1048.
(17) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651-1660.
(15) Bogenstaetter, M.; Limberg, A.; Overman, L. E.; Tomasi, A. L. J.
Am. Chem. Soc. 1999, 121, 12206-12207.
Org. Lett., Vol. 7, No. 6, 2005
1075

Pelorol (1), dimethylpelorol (28), the analogues 21 and
22, the corresponding methyl ethers 11 and 20, the tri-
methoxy pelorol analogue 10, and the uncyclized precursor
19 were tested for in vitro activation of SHIP and the ability
to suppress degranulation and tumor necrosis factor R
(TNFR) production in murine mast cells stimulated with
IgE.18 Synthetic pelorol (1), the ethyl analogue 21, and the
methyl analogue 22 showed significant activity in all three
assays, but the methyl ethers 10, 11, 19, 20, and 28 were
inactive. The relative effectiveness of the active compounds
in the SHIP activation assay was 22 > 21 ≈ 1, showing
that replacement of the methyl ester at C-20 in pelorol with
a methyl gives enhanced activity. Lack of activity in the
dimethyl ethers 20, 11, and 28 demonstrates that at least one
phenol is required for activity.
Scheme 4. Synthesis of Pelorol (1)
Compound 22 was chosen for further evaluation because
it showed biological activity greater than that of pelorol and
its synthesis was shorter. Side by side evaluation of 22’s
ability to suppress degranulation and TNFR release in
SHIP-/- and SHIP+/+ mast cells showed that it was only
active in the SHIP+/+ cells, indicating that it selectively
targets SHIP. Compound 22 also showed positive effects
comparable to the reference standard dexamethasone in a
standard mouse ear edema assay for topical antiinflammatory
activity and in a mouse model of septic shock.18
In summary, the sponge meroterpenoid pelorol (1) has
been identified as an activator of the inositol-5-phosphatase
SHIP. An efficient synthesis of pelorol (1) and several
analogues has confirmed the structure and absolute config-
uration of the natural product and provided preliminary SAR
for the SHIP-activating pharmacophore. The C-20 methyl
analogue 22 has shown promising in vivo activity in two
mouse models of inflammation, supporting the initial hy-
pothesis that selective small-molecule SHIP activators should
represent a new class of antiinflammatory agents.
an attempt to effect a haloform reaction to give benzoic acid
27, unexpectedly resulted in the near quantitative formation
of the R-ketoacid 26. This anomalous result might be caused
by the steric bulk of the C-8 carbon ortho to the C-22 ketone
preventing the formation of a triiodinated methyl. If a
diiodinated methyl ketone 24 is attacked by hydroxide to
give a tetrahedral intermediate, the diiodomethyl may not
be a good enough leaving group to depart in the normal
fashion to give a carboxylic acid. Instead, an intramolecular
SN2 displacement of iodide can form an epoxide, which after
fragmentation as shown in Scheme 4 can lead to the R-keto
aldehyde 25. Oxidation of 25 via iodination of the aldehyde
hydrate can generate the final R-ketoacid 26. Simply chang-
ing the halogen to Br2 led to a clean transformation of methyl
ketone 23 to the desired benzoic acid 27.
Acknowledgment. Financial support was provided by
NCIC (R.J.A.), NSERC (R.J.A.), and CIHR (R.J.A., A.M.,
C.O.). The authors thank M. Le Blanc for assistance with
collecting D. elegans.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The synthesis of pelorol (1) was completed by esterifica-
tion of 27 with MeI followed by selective cleavage of the
phenyl methyl ethers with BI3 at -78 °C. Synthetic pelorol
(1) was identical by NMR and MS comparison with the
natural product. The [R]D of the synthetic material was -64°
compared with values of -69° reported by Konig and -71°
reported by Schmitz, confirming that the absolute configu-
ration is 5S,8R,9R,10S as predicted by Schmitz.10
OL047316M
(18) Pure compounds were tested at 5 µg/mL. In the SHIP assay, 22
showed >6-fold activation, whereas 1 and 21 showed ca. 2-fold activation.
See Supporting Information for experimental details of the in vivo assays.
A complete description of the biological activity of pelorol and the synthetic
analogues will be published elsewhere.
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