Leptostatin: A synthetic hybrid of the cytotoxic polyketides callystatin A and leptomycin B

Article abstract of DOI:10.1016/j.bmcl.2005.09.084

Four stereoisomeric hybrids of the polyketide natural products callystatin A and leptomycin B have been prepared by parallel synthetic routes involving chiral allenylstannane methodology. Like their natural counterparts, these hybrids exhibit nanomolar le

Full text of DOI:10.1016/j.bmcl.2005.09.084

Bioorganic & Medicinal Chemistry Letters 16 (2006) 320–323
Leptostatin: A synthetic hybrid of the cytotoxic polyketides
callystatin A and leptomycin B
James A. Marshall,a,* Ann M. Mikowski,a Matthew P. Bourbeau,a
Gregory M. Schaafa and Frederick Valerioteb
aDepartment of Chemistry, University of Virginia, Charlottesville, VA 22904, USA
bJosephine Ford Cancer Center, Detroit, MI 48202, USA
Received 10 September 2005; revised 29 September 2005; accepted 29 September 2005
Available online 3 November 2005
Abstract—Four stereoisomeric hybrids of the polyketide natural products callystatin A and leptomycin B have been prepared by
parallel synthetic routes involving chiral allenylstannane methodology. Like their natural counterparts, these hybrids exhibit nano-
molar levels of cytotoxicity toward HCT-116 human colon cancer cells.
Ó 2005 Elsevier Ltd. All rights reserved.
Natural products of the polyketide family have attracted
a great deal of attention in recent years because of their
high levels of activity against various human tumor cell
lines.1 A number of the more potent members with cyto-
toxicities in the nanomolar to picomolar range feature a
branched chain of 17–19 carbons attached to the a0 po-
sition of an a-pyranone terminus. The relatively simple
structures of these compounds, their scarce availability,
and high levels of biological activity render them attrac-
tive targets for total synthesis. Within the past several
years syntheses of leptomycin B,2 callystatin A,3 lepto-
furanin D,4 kazusamycin A,5 and ratjadone6 have been
reported (see Fig. 1). Typically the stereochemical issues
surrounding such efforts have been well resolved by
existing aldol and allylmetal methodology.7 As a possi-
ble candidate for development as an antitumor drug,
callystatin A presents a particularly attractive profile
in consideration of its more modest structural complex-
ity and its reported IC50 value of 5 pM against KB tu-
mor cells. Not surprisingly then, no fewer than eight
total syntheses have been reported to date along with
numerous segment assemblages.3
bioactivity, is the a-pyranone terminus. Several studies
support the notion that the a,b-unsaturated lactone is
the pharmacophore for callystatin A, leptomycin B,
and ratjadone. Kobayashi and co-workers performed
structure–activity relationship studies on callystatin A
and found that analogs lacking the pyranone double
bond showed a dramatic decrease in activity against
KB cells.8 In addition, both a nitro Michael adduct9
and a saturated lactone10 derivative of leptomycin
showed greatly diminished activity against HeLa cells.
Furthermore, ratjadone analogs lacking the lactone car-
bonyl exhibited no biological activity against three dif-
ferent cell lines.11 The idea of the lactone as the
pharmacophore also applies to compounds that have
an a,b-unsaturated lactone but are not members of the
leptomycin family. For example, a recent study by Bo-
ger and co-workers on fostriecin, a novel polyketide a-
pyranone with significant antitumor activity, showed a
200-fold decrease in activity upon reduction of the con-
jugated double bond.12 Additional studies conducted on
both leptomycin and ratjadone showed that the mecha-
nism of action is a Michael addition to the a,b-unsatu-
rated lactone by a cysteine residue in the protein
CRM1, which is involved in the export of proteins from
the nucleus to the cytoplasm.1,13
A significant reoccurring feature among these natural
products, and one that is essential to high levels of
We recently completed total syntheses of callystatin A
and leptofuranin D by parallel nonaldol routes in which
an a-pyranone progenitor was attached to the intact
branched polyketide chain.4,14 The successful application
of the methodology employed in these syntheses
Keywords: Chiral allenylstannane; Cytotoxic compounds; Diastereo-
selective additions; Polyketide; Colon cancer; Lactone.
*
Corresponding author. Tel.: +1 434 924 7997; fax: +1 434 924
7993; e-mail: jam5x@virginia.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.084

J. A. Marshall et al. / Bioorg. Med. Chem. Lett. 16 (2006) 320–323
321
O
O
O
O
Me
Me
Me Me Me Me
Me
Me
Me
Me Me Me
Me
CO H
Me
2
O
OH
O
OH
Callystatin A
Leptomycin B
O
O
O
O
Me
Me
Me
Me Me Me
Me
Me
OH
H
H
Me
O
Me
Me
O
O
Me
Leptofuranin B, R = CH OH
2
Leptofuranin D, R = CHO
Ratjadone
R
Me
OH
Figure 1. Cytotoxic polyketide natural products with an a-pyranone terminus.
OPiv
prompted our speculation on the possibility of inter-
changing the pyranone segment of leptomycin and its
congeners with the acyclic polyketide chain of callystatin
A to produce a synthetic nonnatural hybrid Ôleptostatin.Õ
The concept was particularly appealing since by simple
variations on our allenylstannane methodology15 we
could produce all possible stereoisomers (Ôleptostatin
1–4Õ in Fig. 2) of the pyranone segment and thereby probe
a previously unexplored correlation of lactone stereo-
chemistry and biological activity.
OPiv
H
O
OPiv
•
+
2
OPMB
LA
H
Me
OH
OH
OPMB
Me
Me
Bu Sn
3
(M)-1
OPMB
(S,R)-3
(S,S)-3
LA = MgBr (89%); (S,R)-3: (S,S)-3 = 75:25
LA = SnCl (80%); (S,R)-3: (S,S)-3 = 25:75
2
4
OPiv
OPiv
H
O
OPiv
+
We prepared the C6–C22 segment of the aforemen-
tioned leptostatin analogs as previously described.14
For the pyranone segment we employed variations on
the methodology used in our synthesis of leptofuranin
D.4 Accordingly, MgBr2-promoted addition of the alle-
nylstannane (M)-1 to the p-methoxybenzyloxy aldehyde
2 afforded a roughly 3:1 mixture of the syn and anti-ad-
ducts (S,R)-3 and (S,S)-3. The use of SnCl4 in this reac-
tion led to the same two adducts as a 1:3 mixture.15 By
using the allenylstannane (P)-1 in the addition reaction
we obtained the enantiomeric syn and anti-adducts
(R,S)-3 and (R,R)-3. At this stage, the syn and anti-ad-
ducts were not separable (see Scheme 1).
2
OPMB
LA
Me
H
OH
OH
OPMB
Me
Me
Bu Sn
3
(P)-1
OPMB
(R,S)-3
(R,R)-3
LA = MgBr (84%); (R,S)-3: (R,R)-3 = 75:25
LA = SnCl (90%); (R,S)-3: (R,R)-3 = 25:75
2
4
Scheme 1. Synthesis of four stereoisomeric pyranone precursors.
from ethers 6 afforded the unsaturated alcohols 7 in
high overall yield for all diastereomers. Swern oxida-
tion of these alcohols produced the aldehydes 8 repre-
senting the C1–C6 pyranone segment of the targeted
leptostatins. Wittig condensation with the phosphoni-
um reagent 9 was smoothly effected with KO–t-Bu as
the base to yield the (E,Z)-dienes 10 contaminated by
trace amounts (4–10%) of inseparable (E,E)-isomers.
These impurities were gradually removed through chro-
matographic purification of succeeding intermediates.
Cleavage of the terminal TES grouping of 10 and
iodinolysis of the resulting alcohol 11 gave iodides
12, which were directly converted to the borates 13
in situ (see Scheme 2).
Removal of the pivalic ester group of 3 with DIBAL-H
led to the isomeric diols 4, which were readily separat-
ed by flash chromatography. Lindlar hydrogenation16
of the derived TBS ethers 5 followed by PMB removal
O
O
O
1
O
4
O
O
O
O
Me
Me
Me
Me
5
Attachment of the acyclic polyketide segment 14 of
callystatin A to the pyranone precursor segments 13
was efficiently effected through Suzuki coupling with
Pd(dppf)Cl2 as the precatalyst. The closing steps of the
syntheses entailed oxidation of the alcohols 15 with
the Dess–Martin periodinane reagent and deprotection
of the silyl ethers 16 with a HF–NEt3 complex to form
the diols 17. The final conversion of diols 17 to
Leptostatin-4
Leptostatin-1
Leptostatin-2 Leptostatin-3
R
6
Me
Me Me Me Me
OH OTBS
22
Me
Me
Figure 2. Nonnatural hybrids of callystatin A and leptomycin.

322
J. A. Marshall et al. / Bioorg. Med. Chem. Lett. 16 (2006) 320–323
OPiv
OR
OTBS
DMSO, (COCl)
1. DIBAL-H
2
1. H /Lindlar cat.
2
OTBS
OR
2. TBSOTf,
2,6-lutidine
Et N, -78ÞC
3
2.DDQ, CH Cl Me
OH
OR
2
2
Me
Me
OPMB
OPMB
6 R = PMB (93-99%)
7 R = H (82-85%)
4 R = H
5 R = TBS (85-87%)
3
+
-
OTBS
OTBS
OTBS
Ph P
Br
3
OTBS
Me
OTBS
Me
OTES
Me
I
2,
PPh Im
3,
OTBS
Me
9
Me
1. KO-t-Bu,toluene
2. PPTS, MeOH
Me
Me
H
O
Me
I
Me
OR
8 (93-96%)
12 (87-94%)
10 R = TES (72-84%)
11 R = H (71-75%)
OTBS
OTBS
B-MeO-9-BBN,
t-BuLi, Et O, -78 ÞC
Me
2
Me
-
Me
B
+
Li
OMe
13
Scheme 2. Synthesis of the four stereoisomeric C1–C11 fragments of leptostatins 1–4.
leptostatins 1–4 was effected by means of FetizonÕs re-
agent.17 This method proved to be faster and produced
yields of lactones higher than those of the previously
employed MnO2 methodology (see Scheme 3).14
The IC50 values of the four leptostatin isomers and cal-
lystatin A against human HCT-116 colon cancer cells
are summarized in Table 1. Leptostatins-1 and 3 are
syn isomers and leptostatins-2 and 4 are anti. It will be
noted that the introduction of a methyl substituent at
C4 significantly diminishes cytotoxicity relative to cal-
lystatin A. Interestingly, the C4/C5 configuration of
the isomer with the highest activity (leptostatin-3) is
enantiomeric to that of leptomycin and its congeners.
Kobayashi and Kalesse found that altering the C5
stereocenter in callystatin and ratjadone, respectively,
decreased the activity against KB8 and HEPG2 tumor
cells.11 It is important to note that the lactone in these
studies lacks the methyl group found in both leptomycin
and leptostatin; the added methyl group may cause
OTBS
OTBS
Me
Me Me Me Me
Pd(dppf)Cl
Me
2
+
Me
I
K PO , DMF
3
4
OH OTBS
-
Me
B
14
+
Li
OMe
13
OTBS
OTBS
Me
1. Dess-Martin periodinane
Me
2. (HF) NEt
3
3
Me Me Me Me
Me
Me
Table 1. Cytotoxicity toward HCT-116 cells
OH
OTBS
15 (86-98%)
OR
1
O
O
4
O
O
Me
Me
5
OR
Me
Ag CO /Celite
2
3
Leptostatin-1
Leptostatin-2
Me
Me Me Me Me
O
O
C H , reflux (75%)
6
6
Me
Me
O
O
Me
Me
O
OR
16 R = TBS (71-86%)
17 R = H (72-80%)
Leptostatin-4
Leptostatin-3
O
Compound
IC50 (nM)a
O
Me
Leptostatin-1
Leptostatin-2
Leptostatin-3
Leptostatin-4
Callystatin A
3.0
2.0
Me
Me Me Me Me
Me
Me
0.2
30
O
OH
18
0.03
aAverage of two determinations.
Scheme 3. Completion of the leptostatin 1–4 syntheses.

J. A. Marshall et al. / Bioorg. Med. Chem. Lett. 16 (2006) 320–323
323
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.09.084.
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