Current Medicinal Chemistry, 2002, 9, 2005-2032 2005

Fostriecin: Chemistry and Biology

D.S. Lewy†, C.-M. Gauss, D.R. Soenen and D.L. Boger*

Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, California 92037, USA

Abstract: A review of the current status of the chemistry and biology of fostriecin (CI-920) is provided.
Fostriecin is a structurally unique, naturally-occurring phosphate monoester that exhibits potent and
efficacious antitumor activity. Initially it was suggested that its activity could be attributed to a direct, albeit
weak, inhibition of the enzyme topoisomerase II. However, recent studies have shown that fostriecin inhibits
the mitotic entry checkpoint through the much more potent and selective inhibition of protein phosphatase 2A
(PP2A) and protein phosphatase 4 (PP4). In fact, it is the most selective small molecule inhibitor of a protein
phosphatase disclosed to date. The contribution, if any, that topoisomerase II versus PP2A/PP4 inhibition
makes to fostriecin's antitumor activity has not yet been fully defined. Initial phase I clinical trials with
fostriecin never reached dose-limiting toxicity or therapeutic dose levels and were halted due to its storage
instability and unpredictable chemical purity. Hence, the total synthesis of fostriecin has been pursued in order
to confirm its structure and stereochemistry, to provide access to quantities of the pure natural product, and to
access key partial structures or simplified/stable analogs. Several additional natural products have been
isolated which contain similar structural features (phospholine, phoslactomycins, phosphazomycin,
leustroducsins, sultriecin, and cytostatin), and some exhibit comparable biological properties.

INTRODUCTION novel, clinically unexplored mechanisms worthy of pursuit

for introduction of a new class of antitumor agents. Herein,
Fostriecin (1, CI-920), Fig. (1), is a structurally novel we review the present status of the chemistry and biology of
phosphate ester produced by Streptomyces pulveraceus that fostriecin.
is active in vitro against leukemia (L1210, IC50 = 0.46
µM), lung, breast, and ovarian cancer, and which exhibits 3 15 17
OH
2 4 14
efficacious in vivo antitumor activity. It has been H2O3PO OH
16 18
investigated in a Phase I clinical trial at NCI that was halted 5
8 10 13
even before dose-limiting toxicities or therapeutic plasma O O
6
7 9 11
12
levels were reached when concerns regarding drug purity and Me OH
storage stability proved problematic with the naturally- 1, Fostriecin
occurring material. Although fostriecin inhibits DNA
topoisomerase II (IC50 = 40 µM) through a novel, non- R1 R2
DNA-strand cleavage mechanism, the activity is weak and it H2 O3PO OH
does not induce G2 arrest like other topoisomerase II
inhibitors. Thus, it is unlikely this initially suggested target O O
is responsible for the antitumor activity of 1. Instead, Me OH
fostriecin has been shown to inhibit the mitotic entry
checkpoint through more potent and selective inhibition of 2, R1 = R2 = H
protein phosphatases 1 (PP1), 2A (PP2A), and 4 (PP4) 3, R1 = R2 = OH
(IC50 = 45 µM, 1.5 nM, and 3 nM, respectively). Notable
in this regard is the fact that fostriecin is the most selective Fig. (1).
protein phosphatase inhibitor known to date, exhibiting a
10 4 -fold selectivity for PP2A/PP4 versus PP1. Also
contributing to its selective antitumor properties, fostriecin ISOLATION AND STRUCTURE ELUCIDATION
was shown to be transported into tumor cells via the reduced
folate carrier system. Inhibition of the mitotic entry Fostriecin (1), along with two structurally related
checkpoint and selective protein phosphatase inhibition are compounds PD 113,270 (2) and PD 113,271 (3), were
isolated from the fermentation broth of an unidentified
actinomycete found in a Brazilian soil sample [1,2]. The
*Address correspondence to this author at the Department of Chemistry organism was characterized as Streptomyces pulveraceus
and The Skaggs Institute for Chemical Biology, The Scripps Research subspecies fostreus ATCC 31906. It was found that
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;
FAX: 1-858-784-7550; E-mail: boger@scripps.edu fostriecin and its congeners were unstable above pH 8 and
†This article is dedicated in memory of David and all that he meant to us. very labile in dilute acid. Therefore, they could only be

0929-8673/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd.

2006 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
obtained in salt form, and isolation steps had to be
performed within the pH range 5.5–7.5. The fermentation
broth was filtered at pH 6.5 and passed through an ion-
exchange column. Dissolution of the filtrate residue in
acetone and purification on a Diaion HP-20 resin column
(water eluent) yielded two fractions which were combined
and further purified. The resulting mixture was
chromatographed on a C18-reverse phase column utilizing 5–
7% CH3CN–0.05 M phosphate buffer (pH 6.8). The eluate
was filtered and desalted, and the resulting solution was
lyophilized at pH 6.5 to afford 40 g of fostriecin as a nearly
white solid. The purity of this product was about 90%, and
a sample of more than 96% purity was prepared by C8- or
C18-reverse phase chromatography using 2 or 10% CH3CN
in 0.1 M phosphate buffer (pH 6.8).
Preliminary characterization of fostriecin and its
congeners 2 and 3 revealed that all three compounds were
monosodium salts of phosphate monoesters containing a
conjugated triene and an α,β-unsaturated δ-lactone [1,3].
Elemental analysis showed that 1 was composed of 6.4%
phosphorus, and dephosphorylation by alkaline phosphatase
demonstrated that it was a monosubstituted phosphate ester.
The IR spectra of the three isolates were nearly identical,
displaying a prominent peak at 1710 cm–1 , which was
attributed to the presence of an α,β-unsaturated δ-lactone.
FABMS analysis of fostriecin revealed a molecular weight
of 452 for the monosodium salt which, together with
elemental analysis and 1 3 C and 1 H NMR spectral
information, led to the assignment of a molecular formula of
C19H26O9PNa.
The 13 C NMR spectrum of 1 displayed characteristic
couplings indicative of two- and three-bond couplings of
three assignable carbon atoms to phosphorus. Inspection of
the 1H NMR spectrum established the Z,Z,E configuration
of the triene system based on first-order analysis of coupling
constants. Consistent with model trienes [4], the four triene
protons with a vicinal single bond and cis coupling (J = 10–
R3
OR1 OR2
O O
Me OH
4, R 1 = PO3 H2 , R2 = Ac, R3 = OAc
5, R 1 = R 2 = H, R3 = OH
OH
OH H2 O3 PO OH
NaO2 C Me OH
6 of fostriecin by Boger, et al. [5], this designation of the 5R
OH
OH
O O OHC
O 8
7
Fig. (2).
Boger et al.
11 Hz) appeared as apparent triplets, whereas the one
observable proton with a vicinal single bond and trans
couplings (J vic = 11 Hz, J trans = 15 Hz) appeared as a
doublet of doublets. 1H NMR decoupling data indicated that
the conjugated triene chain terminated with an allylic
hydroxymethyl group and that an isolated trans carbon–
carbon double bond connected the 5,6-dihydropyran-2-one
ring to the quaternary carbon C8. In addition, a 1 H NMR
signal at δ 4.17 corresponded to a proton adjacent to a
phosphate. Simple chemical transformations further
confirmed the connectivity of fostriecin. Acetylation of the
natural product provided 4, Fig. (2). As mentioned above,
dephosphorylation of 1 with alkaline phosphatase afforded 5.
Hydrolysis of the 5,6-dihydropyran-2-one ring yielded the
carboxylic acid 6 and treatment of 5 with sodium periodate
provided the ketone 7 and aldehyde 8 as cleavage products
[3].
The relative and absolute stereochemistry of 1 was
determined later by Boger, et al. [5]. The relative
stereochemistry between the quaternary C8 carbon and C9,
which contains the phosphate ester, was elucidated upon
formation of the five-membered cyclic phosphodiester 9,
Fig. (3), and observation of NOE’s (2D 1 H– 1 H ROESY
NMR, D2O, 400 MHz, δ) between H6,7 (5.93–6.00) and H9
(4.34), as well as between the C8-CH3 (1.40) and H1 0
(1.75–1.90). This established the cis relationships of the
C6–C7 vinyl side chain with H9 and the C8-CH3 with the
C9 side chain on the five-membered cyclic phosphodiester
and a syn-1,2-diol relationship between C8 and C9.
Derivatization of fostriecin to provide the C9/C11 acetonide
10 led to the elucidation of the relative stereochemistry of
the 1,3-diol. The acetonide methyl groups displayed similar
13 C NMR chemical shifts of δ 24.49 and 24.89 (CDCl ,
3
100 MHz) within the range characteristic of an anti-1,3-diol
acetonide (23.96–25.22) and different from the distinct
chemical shifts observed with syn-1,3-diol acetonides
(18.67–19.98 and 29.74–30.16) [6,7]. In addition,
diagnostic NOE cross-peaks in the 2D 1H–1H NOESY NMR
spectrum of 10 were seen between one acetonide methyl
group (1.35) and H9 (3.72) and between the other acetonide
methyl group (1.39) and H11 (4.73). This data further
supported the assignment of an anti-1,3-diol acetonide
adopting a twist boat conformation. Consistent with this
assignment, H9 (J(H 10 ) = 6.3, 9.6 Hz) and H11 (J(H 10 ) =
6.3, 9.5 Hz) exhibited 1H NMR coupling constants expected
of this twist boat conformation and the observed NOEs.
In order to establish the absolute stereochemistry, (5R)-7
was synthesized through an unambiguous method by
Hokanson, et al. [3]. Comparison (TLC, [α]25D, IR, NMR)
with the analogous degradation product from fostriecin led
to the assignment of the C5 R configuration. During the
final assignment of the relative and absolute stereochemistry
stereocenter was confirmed by an alternative enantioselective
synthesis of (5R)-7. Additionally, the assignment of the
absolute configuration at C11 was established by correlation
(NMR, IR, MS, Chiralcel OD-H HPLC) of the protected
triol 11, derived from degradation of the natural product,
with the (R)-(+)-tribenzoate 11 prepared from (R)-(+)-1,2,4-
butanetriol. The absolute stereochemical determinations at
C5 and C11, combined with the relative configuration
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2007

13C NMR
OH δ = 24.49 and 24.89
OH
nOe's
nOe's
H H OH
a b O O H
1 H
O O O
Me O O O
P ONa Me OH
C8/C9 syn diol c d C9/C11 anti diol
O 9 10
PMBO2C
OBz OBz 11 7
OBz O O COCH3 OH
HO
C11 R configuration C5 R configuration

(a) 4-BrC 6H4COCl, pyr, 70%. (b) alkaline phosphatase, 100%; Me 2C(OMe) 2, TsOH, 40%. (c) alkaline phosphatase; TBDPSCl; NaIO4; NaBH4 ; BzCl;
O3; NaBH4 ; BzCl. (d) alkaline phosphatase, 100%; NaIO4.

Fig. (3). Assignment of relative and absolute stereochemistry for fostriecin.

assignments, provided the complete stereochemical
assignments of fostriecin as 5R,8R,9R,11R.
SYNTHETIC STUDIES
In an initial attempt to establish the stereochemistry of
the natural product, Just and O'Connor prepared a
diastereomer of dephosphorylated fostriecin containing the
5R,8R,9S,11R configuration [8]. The α,β-unsaturated δ-
lactone 13, containing the previously established 5R
stereocenter, was prepared in three steps from the known
methyl ester 12, Fig. (4a). The known thioacetal 14,
obtained in four steps from 1,2-O -isopropylidene-D-
glucofuranose and protected as the cyclopentanone ketal, was
tranformed to the cis bromoalkene 1 5 by thioacetal
deprotection and a Wittig reaction (61% for two steps) Fig.
(4b). This provided the C9 and C11 stereocenters from
naturally-occurring D-glucofuranose. Carbonate
saponification (100%), oxidative diol cleavage, and
subsequent oxidation with PCC/CH3OH (60%) yielded the
methyl ester 16. In order to complete the installation of the
Z,Z,E-triene, 16 was coupled to the TBS derivative of
commercially available (2E)-penten-4-yn-1-ol utilizing a
modified Sonogashira reaction (88%), and the resulting
alkyne 17 was reduced by catalytic hydrogenation utilizing
Brown's catalyst (NaBH4, Ni(OAc)2). Conversion of 18 to
the β-ketophosphonate 19 and Wadsworth–Horner–Emmons
coupling of 13 and 19 proceeded in 50% yield, affording the
C6–C7 trans olefin. Subsequent treatment of the α,β-
unsaturated ketone with trimethylaluminum provided the
tertiary alcohol 20 (65%) containing an 8R stereocenter
derived from a chelation-controlled nucleophilic addition.
Removal of the protecting groups provided 21, and its 13C
NMR spectrum (D2O, 400 MHz) was found to be different
from that of the fostriecin degradation product 5.
In our own efforts, a total synthesis of fostriecin was
viewed as an opportunity to not only confirm our
stereochemical assignments, but also to address the issues of
chemical purity and storage stability currently limiting
clinical trials of the natural product. In addition, it could
provide access to more stable structural analogs not
accessible by degradation or functionalization of fostriecin
itself and was designed to be sufficiently general as to be
adaptable to the structurally-related leustroducsins,
phoslactomycins/phospholine, sultriecin, and cytostatin, for
which no synthetic efforts have yet been described. The
strategy for our total synthesis of fostriecin is highlighted in
Fig. (5) [9]. The key elements include the late stage
deprotection and sequential introduction of the C-terminus
lactone and C9 phosphate, a Wadsworth–Horner–Emmons
installation of the C6–C7 trans double bond, and stepwise
assembly of the sensitive Z,Z,E-triene. The absolute
stereochemistry of the C9 center was set by utilizing an
optically active starting material, the C5 and C11

OH
SEt
p-TsOH
MeO2C SEt SEt
< 35% O O
OH OH
SEt
12
MsCl,
95%
Et3N

Br 2, Et2O/H2O
SEt
O O CHO O O

13 SEt
2008 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

(Fig. 4). contd.....

OR1 OR1 O O Br
Ph3P=CHBr
O 61%, 2 steps O
R2
O O
O O 15
R 1 = H, R2 = CH(SEt) 2 (14)
R1/R 1 = C 5H8, R2 = CH(SEt) 2 NaOH 100%
R1 /R1 = C5H8, R 2 = CHO

OTBS

OTBS O O Br

(Ph3P) 4Pd, CuI

R
O O 88%
R = CH(OH)CH2OH
M eO2C NaIO4
MeOH, PCC R = CHO
17 DMF, 60% R = CO2Me (16)

NaBH4,
OTBS
Ni(OAc) 2 O O
R

O
(MeO)2POCH2Li R = OM e (18)
R = CH2PO(OMe) 2 (19)

50% 13, NaH

OTBDPS
O O

O O
O
65% AlMe3

OTBDPS
O O

O O
Me OH 20

OH
OH OH

O O
Me OH 21
Fig. (4).

stereocenters were installed by Sharpless AD reactions, and
the labile C8 center was installed enlisting a Felkin–Anh
versus chelation-controlled nucleophilic addition.
The 5R stereogenic center found in the lactone was
installed by a Sharpless asymmetric dihydroxylation (AD)
reaction [10] on p-methoxybenzyl 5-hexenoate (22, 79%,
88–92% ee), Fig. (5). Acid-catalyzed lactone formation
(75%) via 23, conversion to the α-phenylselenyl lactone
(50%), and subsequent oxidation to the corresponding
selenoxide provided the unsaturated lactone 24 following in
situ elimination (85%) [5]. Reduction of the lactone with
Dibal-H and formation of the isopropyl acetal yielded 25 in
a 90% overall yield. Removal of the primary alcohol
protecting group (91%) and subsequent oxidation with
TPAP–NMO provided the key aldehyde 26 (90%).
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2009

6 W adsw orth-Horner-E mmons 4 Wit tig Synthesi s of C1-C6

S harpless A D 17 S harpless A D TFA , 75%
OR
OR OR 14 PM BO 2C PM BO 2C
18 79%, 90% ee
HO CH 2OR
8 10 13
RO O 5 Stille coupling R =H
22 TB DP SCl, 89%
- O 3 Still -G enna ri R = T BD PS (23)
7 Me
R
Fel kin Addition 2 Sha rple ss AD H 2O2
1 from D -Glu
OT BD PS 85% O T BD PS
OR O O O O
OR OR
24
RO R=H
P PhSe Br, Dibal-H ;
RO O CH O 90%
RO L D A, 50% R = S ePh PPT S, i-PrO H
O O
C 1-C6 C7-C18

T PA P-N M O
OR OR
90% OR
O O C HO O O
CHO Bu 3S n OR
R = T BDPS (25)
O OR 26 Bu 4N F, 91%
OH R=H
C 1-C6 C8-C12 C 16-C18
OH O TBS
D ibal -H Sha rple ss AD T BSO T f, E t3N
RO PM BO PMBO PM BO
O M sCl, E t3N (D H QD ) 2AQ N
O OH 90%
O OH 88%
O O
73% >83%, > 10:1
PM BO C(=N H)CCl3
28 30
R = H (27) 29
95% Br
R = P MB

OR OT BS CO 2M e T ES O OT BS R TE SO O T BS Br
90% 94% , 2 ste ps
P M BO P MB O RO
Diba l-H CBr 4, PPh 3
B u 3SnH , (Ph 3P )4P d 84%
D DQ , 95% R = PM B
E t3S iOT f De ss-M a rti n
R = H (31) R = CH 2O H (32) R= H
95% 100% Ac 2O , 93%
R = TE S R = CH O (33) R = Ac (34)

OT BD PS O T BDP S
T E SO O T BS T ES O OT BS 82% >98:2 TE S O O TBS Br
Et O A cO
P (E tO )2PO CH2 Li R Bu 3Sn O T BDP S
Et O De ss-M a rt in 90% , 2 steps
O O 40 R = CH2O A c (37) 36 35
Diba l-H , 98%
R = CH2O H (38)
26, t-BuO K, tolue ne, 91% De ss-Ma rtin, 91%
R = CHO (39)
O T BD PS OT BD PS
T E SO O TBS TE SO O TB S
M e Li-Ce Cl3
O O 41 O O 42
96%
O Me OH
3:1
93% P PT S-E tOH
O T BD PS OT BD PS
OR O TB S A g 2CO 3, 81% OH O T BS

O O RO O
1
M e OT BS Me OR
R = E t, R 1 = H (43)
PCl3 ; PM BO H; R = H (48) T BSO T f, 70% 1
H2O2, 91% R = P O(OP MB) 2 (49) R = E t, R = T BS (46)
a q H Cl, 50% R = H , R 1 = T BS (47)
H F;
43
H F-Pyr

OH OR
H 2O 3PO OH OR OR

O O O O
M e OH Me OH
R = H (44)
1, fostrie cin A c2O , 70%
R = Ac (45)
Fig. (5). First total synthesis of fostriecin.
2010 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
The C7–C18 subunit of 1 was prepared by first
assembling C8–C12 with introduction of the C9 and C11
stereocenters, followed by a stepwise introduction of the
sensitive triene, and finally conversion to the β -
ketophosphonate 39 for linkage to C1–C6. The stepwise
order of the triene introduction permitted the assessment of
the relative stability of the C12–C13 Z-alkene, the C12–C15
Z,Z-diene, and the C12–C17 Z,Z,E-triene, and offered the
potential of postponing the introduction of any of its
sensitive segments to a late stage of the synthesis. The
absolute stereochemistry of the C9 center was set by
utilizing the optically active hydroxylactone 27, available
from D-glutamic acid in two steps [11,12]. The absolute
stereochemistry at C11 was set utilizing a Sharpless AD
reaction on the advanced intermediate 28 (>83% yield,
>10:1 ee), with clean delivery of the C3 alcohol cis to the
C5 hydroxymethyl substituent. Selective protection of the
C3 secondary alcohol over the anomeric alcohol was
achieved by direct, low temperature (–78 °C) treatment of 29
with TBSOTf (1.0 equiv, CH2Cl2) in the presence of Et3N
(3.0 equiv), providing lactol 30 in 90% yield. Subsequent
condensation with the Still–Gennari phosphonate [13]
((CF 3 C H 2 O) 2 POCH 2 C O 2 Me, KHMDS, 18-crown-6)
afforded 31 (88%) which contains the first cis olefin of the
sensitive Z,Z,E-triene. TES protection of the secondary
alcohol (95%), followed by stepwise reduction–oxidation,
provided aldehyde 33. The cis olefin at C14–C15 was
introduced utilizing a two-step protocol which enlisted a
Corey–Fuchs reaction [14] (94% from 32), an intervening
protecting group exchange, followed by a selective reduction
of 34 with Bu3 SnH–Pd(PPh 3 )4 [15–17] to provide the Z-
bromoalkene 35 (84%). In the initial efforts to elaborate the
cis bromide to the requisite cis,cis,trans-triene, typical Stille
coupling conditions [18,19] provided low yields for the
homologation of 3 5 with vinylstannane 36 [20,21].
However, the Stille coupling conducted in i-Pr 2 N E t
provided the key triene intermediate 37 in excellent yield
(82%) with near perfect stereochemical integrity (>98:2).
Deprotection of the C8 alcohol and oxidation to the
aldehyde, followed by addition of α -lithio diethyl
methylphosphonate to the aldehyde 39 and oxidation of the
secondary alcohol with Dess–Martin periodinane [22],
afforded the key β-ketophosphonate 40 (90% from 39).
This set the stage for a Wadsworth–Horner–Emmons
reaction to unite the C1–C6 and C7–C18 subunits, which
was carried out smoothly to furnish the desired trans α,β-
unsaturated ketone 41 in 91% yield. Direct addition of MeLi
to 41 via Felkin–Anh attack was anticipated to provide 42.
However, treatment of 4 1 with MeLi in THF gave
predominantly the undesired 1,4-adduct [23] (90%, >10:1).
Alternative conditions including MeLi/18-crown-6 [24],
MeLi/LiCl, or MeTi(OiPr)3 [25], either did not substantially
improve the selectivity or led to no reaction. Ultimately, it
was found that when ketone 41 was introduced as a toluene
solution into a MeLi/CeCl3 [26] slurry in THF, the 1,2-
adduct 42 was formed (>20:1) in 96% yield as a mixture of
two diastereomers (3:1, 1H NMR). Correlation of the major
isomer with natural product derivatives established its
stereochemistry as 8R derived from a Felkin–Anh addition.
Exchange of isopropyl acetal to an ethyl acetal, with
concomitant deprotection of the C9 secondary alcohol to
Boger et al.
allow for the introduction of the phosphate ester, yielded 43
(93%). However, prior to completion of the synthesis,
correlation of synthetic and naturally-derived 44 and 45 was
carried out to ensure that the intermediates bore the correct
relative and absolute configurations. Reprotection of the
secondary alcohol 43 as its OTBS ether (TBSOTf, Et3 N,
CH 2 Cl 2 , –78 °C, 4 h, 77%), followed by aqueous acid
hydrolysis of the acetal (0.5 N aq HCl–acetone 1:4, 50%),
provided the lactol which was cleanly oxidized to the lactone
(Ag2CO 3–Celite, 80%). Deprotection (HF–pyr, THF–pyr,
25 °C, 4 d, 32%) furnished a 3:1 mixture of C8
diastereomers of 44, of which the major C8 diastereomer
correlated with 5 derived from the natural product [5]. In
addition, the 9,11,18-triacetate 45 was prepared from the
naturally-derived 5 by acetylation (Ac2 O, 70%) and
correlated with the major isomer of a sample of 45 prepared
from synthetic 43 (i. Bu4NF; ii. Ac2O; iii. 0.5 N aq. HCl–
acetone 1:4; iv. Ag2CO3–Celite).
In the course of handling 42 and its related C9 OTBS
derivative, a base-catalyzed migration of the C9 silyl group
to the adjacent C8 tertiary alcohol was observed. We took
advantage of this observation and developed conditions
whereby treatment of 43 with TBSOTf (2.0 equiv, 5 equiv
of 2,6-lutidine, –20 °C, 45 min) provided predominantly
46. Hydrolysis to the lactol 47, and oxidation to the lactone,
cleanly provided 48, at which point the separation of the C8
diastereomers was straightforward. Thus, only four
intermediates in the total synthesis (42, 43, 46, and 47) were
prepared and characterized as the 3:1 mixture of C8
diastereomers. Global silyl ether deprotection of 48 was
accomplished by treatment with HF–pyr in THF–pyr (25
°C, 4 d) with a solid NaHCO3 workup, providing the tetraol
4 4 which also proved to be spectroscopically and
chromatographically indistinguishable from naturally-derived
5. Conversion of 48 to the bis-PMB protected C9 phosphate
occurred upon treatment with PCl3 (5 equiv, pyridine, 25
°C), followed by PMBOH (15 equiv) and subsequent
phosphite oxidation (50 equiv H2O2–H2O), to provide 49 in
a superb conversion (91%). Subsequent global deprotection
(HF–pyr) provided fostriecin identical in all aspects with
authentic material.
BIOLOGICAL PROFILE OF FOSTRIECIN
Mode of Action
Inhibition of Topoisomerase II
The biological activity and preclinical studies of
fostriecin have been reviewed [27]. Fostriecin was initially
reported to inhibit macromolecular synthesis in the L1210
cell line [28]. Experimental data suggested that the mode of
action was through the inactivation of an essential
component of the cell cycle responsible for mediating DNA
and RNA synthesis. Subsequently, it was proposed that
fostriecin's antitumor activity and macromolecular synthesis
inhibition could be attributed to the direct inhibition of
topoisomerase II (topo II) [29]. The role that topo II plays in
the cell cycle, as well as its mechanism of action, have been
reviewed [30].
Fostriecin: Chemistry and Biology
Topo II functions to unwind double-stranded DNA
during two distinct phases of the cell cycle. In the S phase,
topo II relaxes supercoiled DNA generated for transcription
and DNA synthesis, but this function is thought to be
nonessential and can be replaced by topoisomerase I. In the
M phase, topo II is required for the unwinding of sister
chromatids and subsequent chromatid segregation [31]. The
enzyme functions by passing an intact DNA helix through a
transient double-stranded break which it generates by
cleaving a phosphodiester bond in each strand of a second
DNA helix [30].
Classical inhibitors of topo II (topo II poisons),
including the epipodophyllotoxins (etoposide and
teniposide) [32–34] and 4'-(9-acridinylamino)methanesulfon-
m-anisidide (m-AMSA) [32,33,35], induce irreversible DNA
strand cleavage during replication in the S phase of the cell
cycle [36–38]. These agents stabilize the covalent interaction
between topo II and double-stranded DNA, serving to trap
the enzyme–DNA complex. It is the trap of this complex
and subsequent DNA cleavage, rather than the inhibition of
the catalytic activity of topo II, that is important, and a cell's
sensitivity to such drugs can be expected to be directly
proportional to the level of topo II activity. The classical
inhibitor’s activity as an anticancer drug is the result of
induced G2 arrest and/or apoptosis in replicating cells. Other
topo II inhibitors do not stabilize topo II–DNA complexes.
Rather, they prevent the binding of the enzyme to the DNA,
or block additional steps in the topo II catalytic cycle. A
cell's sensitivity to such catalytic inhibitors, therefore,
should be inversely proportional to the level of the enzyme
activity [37,39,40]. The mechanism of such inhibitors,
which include the bis(2,6-dioxopiperazines) [41,42],
merbarone [43], aclarubicin [43], amsacrine [44,45], suramin
[46], quinobenoxazine [47], chloroquine [48], and
novobiocin [49] have not been as well elucidated as those of
the classical inhibitors. It has been proposed that fostriecin's
properties could be attributed to a direct inhibition of the
topo II enzyme prior to or without an interaction with DNA.
However, recent studies suggest that the drug's site of action
may be upstream of topo II within the cell cycle control
pathways.
In 1984, a series of studies that attempted to elucidate
fostriecin’s mode of action were reported by groups within
Warner–Lambert/Parke–Davis. Fry, et al. showed that
L1210 leukemia cells exposed to 10 µ M fostriecin
demonstrated a decreased rate of DNA, RNA, and protein
synthesis. The reduction in nucleic acid synthesis was
shown not to be the result of nucleic acid precursor
depletion, and 50 µM of fostriecin had no effect on DNA or
RNA polymerase activity. In addition, adding DNA to drug-
treated cells restored DNA polymerase activity, indicating
that the limiting factor was the quantity of functional DNA
[50,51]. Gedik, et al. also found a strong and delayed
inhibition of DNA synthesis by fostriecin in human HeLa
cells [27,52]. It was also shown that transport of fostriecin
into L1210 leukemia cells occurs through the reduced folate
carrier system which contributes to its properties [50].
The inhibition of topo II catalytic activity by fostriecin
was first observed in a supercoiled DNA decatenation assay
using partially purified topo II from Ehrlich ascites
Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2011
carcinoma. The minimum inhibitory concentration (MIC)
was determined to be 50 µM with a modest K i,app of 110
µM. Unlike the classical topo II inhibitors, fostriecin did
not induce DNA strand breaks [29]. Several subsequent
reports have described studies which have tried to establish
fostriecin’s similarities to known topo II catalytic inhibitors.
Withoff, et al. reported a significant correlation between
decreased topo IIα mRNA levels and increased fostriecin
sensitivity in human small-cell lung carcinoma cells [53],
supporting fostriecin's classification as a topo II catalytic
inhibitor. In addition, an etoposide-resistant GLC4/ADR cell
line (etoposide IC 50 = 10.2 µ M), characterized by a
decreased topo II level, showed a modest 3-fold increase in
sensitivity to fostriecin (IC50 = 4.1 µM) relative to the wild-
type GLC4 cell line (etoposide IC50 = 0.16 µM, fostriecin
IC 50 = 11.2 µM) [54]. However, a related study reported
only a 1.3-fold increased sensitivity to fostriecin in a
teniposide-resistant CCRF-CEM cell line also characterized
by a decreased topo II level [55].
The effects of various catalytic inhibitors on the cell
cycle distribution have been evaluated and could potentially
be used to determine whether fostriecin’s profile merits its
classification as an inhibitor of topo II. Merbarone,
RP60475F, SN 22995, and ICRF-187 induced G2 arrest
without accumulation of cells in another stage, while the
bis(2,6-dioxopiperazine) derivative ICRF-193 caused CHO
cells to pass through the S phase and G2 checkpoint and
enter mitosis, resulting in polyploidy and tangled chromatin
[55,40,56]. Clearly there is some variability in the point of
the cell cycle at which these drugs act. However, both
observations are consistent with inhibited topo II activity
[40,57]. Flow cytometry studies with fostriecin have
produced results that, in some cases, vary significantly from
those of the topo II catalytic inhibitors. Initial studies with
L1210 cells showed an increase in the G2/M fraction and a
constant S fraction at 1.5 µM (growth inhibitory but not
highly toxic) and 5 µM, but a 15 µM concentration of drug
produced a lower proportion of cells in the G2/M phase and
a greater number in the S phase [29,51]. Hotz, et al. reported
that a concentration of 1 µM fostriecin resulted in significant
accumulation in the S phase for both promyelotic (HL-60)
and lymphocytic (MOLT-4) leukemia cells. At
concentrations between 5 and 500 µM, the drug triggered
endonucleotic DNA degradation (apoptosis) in HL-60 cells,
resulting in cell death with little change in phase proportions
versus control, whereas no apoptosis was observed in
MOLT-4 cells at any concentration [58].
The drug also displays additional characteristics that
differ from those expected for a topo II catalytic inhibitor. In
a study similar to that of the etoposide-resistant GLC4/ADR
cell line (vida supra), no cross-resistance or increased
sensitivity to fostriecin was observed in a K562 cell line
with a 26-fold resistance to etoposide, whereas a 3.4-fold
sensitivity was exhibited in the presence of the topo II
catalytic inhibitor ICRF-187. Alone, this lack of cell
sensitivity to fostriecin casts doubt on its classification as a
catalytic inhibitor. However, the catalytic inhibitor
merbarone also showed results (4-fold resistance) that would
not be predicted [59]. Nonetheless, additional studies also
cast doubt on topo II as fostriecin’s primary target. It has
been reported that the catalytic inhibitors merbarone,
2012 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

aclarubicin, and the bis(2,6-dioxopiperazine) derivatives can up to 800 µM fostriecin in CHO cell extracts [61], and,
inhibit etoposide- and m-AMSA-mediated DNA–topo II unlike fostriecin’s effects on L1210 cells (vida supra),
complex formation in vitro [42,43,60]. These findings were merbarone and SN 22995 caused no inhibition of DNA
mirrored by Chen, et al. for teniposide-mediated DNA–topo synthesis in CCRF-CEM cells [55].
II complex formation with merbarone (IC50 = 66 µM),
aclarubicin (IC50 = 2.5 µM), RP60475F (IC50 = 7.2 µM), Inhibition of Ser/Thr Protein Phosphatases
and SN 22995 (IC 5 0 = 19.8 µ M) (two amsacrine
derivatives), whereas no inhibition was observed with More recent studies suggest a different mechanism for
fostriecin (up to 200 µM) under identical conditions. In fostriecin’s antitumor effects, implicating the Ser/Thr protein
addition, Frosina, et al. reported that no inhibitory effect on phosphatase family as the key cellular target [62]. This
topo II’s decatenating activity was detected in the presence of family is a highly homologous (35–65%) class of

Acidic group interacting with dinuclear metal center mimics Inhibitor IC 50 /PP1 IC 50 /PP2A selectivity: PP1/PP2A
substrate phosphate
Fostriec in 45 µM 1.5 nM 10 4-10 5
Elect rophile like microcystin-LA H-bond to Arg221
dehydroalanine?
Microcystin-L A 0.1 nM 0.1-1 nM 1-0.1
OH
H2 O3 PO OH
Tautomyc in 0.2 nM 1 nM 0.2
O O
Me OH
T hr substrate M e Hydrophobic tail tha t binds in (–)-Calyculin 0.5-2 nM 0.1-1 nM ca. 1
mimic substrate binding site

Oka daic acid 20 nM 0.2 nM 100

Proximal OH that mimics substrateThr -OH or displaces PP2A
active site bound water nucleophile? selectivity?
Ca ntharidin 470 µ M 40 µ M 10

CO2 H Me O Forms covalent Michael adduct with thiol of Cys273 of PP1 (x-ray)
N
HN NH
O
OM e O
O

NH HN
H H
N N
O
M icrocystin-LA O
O
CO2 H

O OH O

O OH O OH H
O O
O
O H O
OH O O Me
O
Me O N T automycin
H
(Me )2 N OH N

(–)-Calyculin

N O
H2 O3 PO
C
OH
O

OH OH O Me

O OH
H
O O
HO
O O O
O HO
O OH O O
O H
OH
Common M otifs: O kadaic acid
O Me 1. acidic group: carboxylate (masked) or phosphate
Me 2. methyl substituent
Cantharidin 3. hydrophobic segment

Fig. (6). Structural features of fostriecin fitting the pharmacophore model.

Fostriecin: Chemistry and Biology
bimetalloenzymes of which several subtypes have been
identified. These include PP1, PP2A, PP2B (calcineurin),
PP2C, PP4, PP5, PP6, and PP7. PP1 and PP2A have been
the most studied [63–69]. Because of the high degree of
homology that exists in the family, differentiating which
enzyme is responsible for regulation of a particular
biochemical pathway has proved challenging. The use of
natural product inhibitors that target individual members of
the family has become an indispensible tool in determining
specific function [70], Fig. (6). As discussed in detail
below, fostriecin is a potent and selective inhibitor of two of
the phosphatases, PP2A and PP4, both of which are
implicated in regulating the progression of the cell cycle
[65,71,72]. The fact that fostriecin and other known PP2A-
selective inhibitors, including okadaic acid and calyculin,
induce premature entry into mitosis is further evidence
against the topo II inhibition model [73–76], as topo II is
not required for entry into mitosis [77]. The PP2A substrate
p34cdc2 kinase, together with cyclin B, however, comprises
the maturation promoting factor (MPF), which is the key
enzyme involved in initiating the M phase of the cell cycle
[78–80].
Binding Affinity of Fostriecin for Ser/Thr Protein
Phosphatases
A number of studies have established that fostriecin is a
potent inhibitor of the Ser/Thr protein phosphatases PP1,
PP2A, and PP4 (Table 1). These studies also demonstrated
that the molecule is on the order of 100–10,000 times more
selective for PP2A and its close homolog PP4, than PP1,
making it the most selective inhibitor known for any
member of this class of phosphatases. To date, the only
other PP2A-selective inhibitors known are okadaic acid
(~100:1), cantharidin (~10:1) [81], and calyculin (~5:1).
Table 1. Fostriecin Inhibition of Ser/Thr
Phosphatases
Substrate IC50 PP1 IC50 PP2A
1a phosphorylase a 45 nM 1.5 nM
2b phosphorylase a 131 µM 3.2 nM
3c phsophorylase 4 µM 40 nM
4d phosphohistone 58 µM 5.6 nM
aref 86. bref 127. cref 62. dref 82.
Okadaic acid inhibits both PP2A and PP4 phosphatases
with the same IC50 (0.2 nM) [70], but microcystin-LR is
PP4-selective (8 pM vs 0.1 nM for PP2A). This selectivity
is interesting considering that PP2A and PP4 are ~65%
homologous. Unlike fostriecin, okadaic acid and cantharidin
do not inhibit topoisomerase. Fostriecin does not inhibit
PP2B (calcineurin) or protein tyrosine phosphatases, as is
typical of the other PP1/PP2A-specific natural product
inhibitors. Notably, fostriecin and cantharidin are established
antitumor agents while okadaic acid is categorized as a
tumor promoter. However, the tumor promoting activity of
okadaic acid was determined in a mouse skin model at high
concentrations (123 µM). Experiments in CHO cells at
Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2013
lower concentrations (1–50 nM), similar to those at which
fostriecin was tested, indicated that okadaic acid arrests cell
growth, and no changes in cell morphology were detected
(72 h at 2 nM okadaic acid) [82]. Furthermore, 8 nM
okadaic acid prevented transformed NIH 3T3 cells from
forming colonies, indicating reversion to a normal
phenotype [83].
Implication of PP2A/PP4 in G2/M Checkpoint Control
Regardless of the origin of the selectivity demonstrated
by fostriecin for PP2A and PP4, links have been established
between its inhibition of these enzymes and interference with
normal regulation of mitosis [82,84–86]. Although PP2A
and PP4 share 65% sequence homology, their functions are
independent [65,72,86]. There are two proposals on the
possible role of fostriecin-mediated PP2A inhibition in
regulating the tumor cell's entry into mitosis. One suggests
that fostriecin induces premature mitosis, whereas the second
suggests that it prevents mitosis from beginning. Less is
known about the function of PP4, although it has been
shown to be involved in centrosome-mediated mitotic
spindle formation, and this will be described below in the
discussion of the PP2A inhibition mechanisms.
Premature Mitosis Hypothesis
A basic outline of the cell cycle is represented in Fig.
(7). Cells proceed through a resting G1 phase (first gap),
followed by the S phase during which the synthesis of DNA
and histones occur. These first two stages are followed by
the "quality control" phase, G2 (second gap), in which
transcriptional errors are corrected [87]. Entry into the M
(mitotic) phase, during which cell division occurs, is
regulated by the maturation promoting factor (MPF)
complex which consists of the Ser/Thr kinase p34cdc2 and
Protein cyclin B [79,88]. When activated, the complex is thought to
stimulate normal chromosome condensation [84]. Many
antitumor agents interfere with the normal cell cycle control
IC50 PP4 mechanism or mitotic entry checkpoint that occurs at the
interface between the G2 and M phases. Usually the agents
3 nM do so by initiating G2 arrest, a normal control mechanism
- for preventing a cell from undergoing division with
imperfect DNA [87].
-
chromosome condensation
-
M
MPF MPF
inactive active
Mitotic Entry
Checkpoint
G2 G1
DNA repair
S
DNA and histone
synthesis
Fig. (7). Schematic of the cell cycle.
An early study of fostriecin's effects on the cell cycle
showed that at a 375 µM concentration it induced baby
hamster ovary (BHK) cells to enter mitosis prematurely
2014 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
rather than suffer G2 arrest, as evidenced by chromosome
condensation, separation of spindle poles, and formation of
asters [62]. Premature mitosis with damaged, incomplete, or
incorrect DNA sequences ultimately results in cell death
[89,90]. Okadaic acid also instigated BHK21 cells to
proceed through the mitotic entry checkpoint prematurely at
0.5 µM concentration [75]. Subsequently, several studies
were undertaken to define how this process occurs with
fostriecin and its implications.
Based on substantial precedent, one question deserving
investigation was the nature of fostriecin's effect on the
regulation of p34cdc2 kinase, histone H1, and histone H3, all
of which are implicated in stimulating chromosome
condensation [84]. Roberge and coworkers discovered that
FT210 cells in G 2 arrest underwent chromosome
condensation without active p34cdc2 or histone H1 when
treated with fostriecin (≥25 µM) or okadaic acid (1 µM).
However, an increase in phosphorylated histones 2A and 3
was observed. In the presence of the kinase inhibitor
staurosporine, fostriecin was not able to induce chromosome
condensation, implicating activation of H2A and H3 in
stimulating this process. Pulse-chase experiments with 32P
also showed that, in mitotic cells treated with fostriecin,
there was an increase in the amount of radioactive label
incorporated into H2A and H3 compared with controls. This
suggested that either H2A and H3 phosphatases were
inhibited or H2A and H3 kinases, as yet unidentified, were
activated. An additional interesting finding in this study was
that p34cdc2 kinase activity was decreased in FT210 cells in
the presence of fostriecin. By contrast, BHK cells showed an
increase in p34cdc2 kinase activity when treated with
fostriecin (375 µM) [62]. Okadaic acid also stimulated an
increase in p34cdc2 kinase activity in BHK21 cells [75].
A second investigation led to the discovery that the
filamentous protein vimentin, which is commonly found in
developmental and tumor cell lines, was
hyperphosphorylated in cells administered with 50–200 µM
fostriecin [85]. The same effect has been observed with
okadaic acid in rat brain tumor cells and with calyculin A in
NIH3T3 fibroblasts [91–93]. During mitosis,
hyperphosphorylation of vimentin destabilizes the normal
cytoplasmic filament network by causing the formation of
aggregates. Although vimentin is phosphorylated by p34cdc2
kinase in mitosis [94–96], the results of this study indicate
that hyperphosphorylation of vimentin occurs even in cells
where p34cdc2 activity is suppressed. Ultimately, one of the
isoforms of protein kinase C (PKC) was implicated as the
enzyme responsible for vimentin hyperphosphorylation.
Protein kinase C is known to be deactivated by PP2A and is
implicated in affecting cell morphology. Indeed, the
overactivation of PKC effected by the phorbol esters and
related compounds results in tumor formation [97,98]. It has
been suggested that the tumor promoting activity of okadaic
acid is also the result of PKC activation through the
inhibition of PP2A [85,99].
G2 Arrest Hypothesis
The G2 arrest hypothesis is based on the observation that
fostriecin exerts its antitumor effects at low concentrations
Boger et al.
(0.5–15 nM) in CHO cells [27,82,100,101]. The antitumor
activity occurs at a level that completely inhibits PP2A but
not PP1. If some PP1 is available, the cells will not proceed
into mitosis because they are damaged, resulting in G2
arrest. The mechanism by which fostriecin-induced G2 arrest
occurs is likely through disruption of the normal changes
that take place in the cytoskeleton during mitosis. The two
major abnormalities noted by Honkanen and coworkers were
aberrant mitotic spindle formation and atypical centrosome
replication [82]. Normally, the microtubules which form the
mitotic spindle radiate from phosphorylated centrosomes.
Fostriecin-, okadaic acid-, and cantharidin-treated cells
displayed numerous spindle poles extending from multiple
centrosomes. All three of these agents are PP2A-selective,
and mutant Drosophila which do not express PP2A also
exhibited multiple centrosomes and associated spindles
[102].
It is unclear whether exclusive inhibition of PP2A or
PP4 results in fostriecin’s activity, or if inhibition of both
enzymes is necessary for optimal activity. In Drosophila and
mammalian cells, centrosomes are stained by PP4-sensitive,
but not PP2-sensitive, antibodies, and PP4 is also thought
to be necessary for the initiation of microtubule growth from
centrosomes [65,72]. Therefore, PP4 may also play a role in
fostriecin’s antitumor effects. In conclusion, considerable
evidence supports the inhibition of protein phosphatases as
fostriecin’s mode of action.
Is There a Pathway Connecting PP2A and Topo II?
Some of the known substrates of PP2A are enzymes
which have been shown to regulate topo II. It is possible
that by inhibiting PP2A, fostriecin may indirectly affect the
activation of topo II, Fig. (8). PP2A is a negative regulator
of MPF both directly, via dephosphorylation of a threonine
residue of active MPF, and indirectly, through deactivation
Inactive MPF
Cyclin B
TP
TP
YP
p34cdc2
wee1
cdc25
Inactive MPF
Active MPF
Cyclin B Cyclin B
TP PP2A T
T T
Y Y
p34cdc2
MO15
p34cdc2
Activates
Activates
Cas ein Kinas e 2 Topoisomerase II
Fig. (8). Activation of MPF and a biochemical pathway linking
topoisomerase II and MPF.
Fostriecin: Chemistry and Biology
of the phosphatase cdc25 and dephosphorylation of the
inhibitory kinase wee1, which must be dephosphorylated in
order to be active. PP2A's main function is to phosphorylate
tyrosine and threonine residues on MPF, keeping MPF in a
hyperphosphorylated, inactive state [103–107]. There may be
a link between activated MPF and topoisomerase II through
MPF-mediated activation of the intermediary enzyme casein
kinase 2 (CK2), which is the major kinase known to
phosphorylate topo II [108,109]. The other major enzyme
that phosphorylates topo II is protein kinase C, which is
also known to regulate MPF [110–113].
Fostriecin May Fit the Okadaic Acid Pharmacophore
Model for PP1/PP2A Inhibition
Several groups have proposed a common pharmacophore
based on extensive SAR of the natural product inhibitors of
PP1 and PP2A, which include okadaic acid, the calyculins,
the microcystins and nodularins, cantharidin, and
tautomycin, Fig. (6). Further support for this model comes
from two X-ray crystal structures: the first, of the phosphate
mimic tungstate coordinated with the metal ions in the
catalytic core of PP1, and the second, of the toxin
microcystin-LR complexed in the PP1 active site [114,115].
Conserved features of this pharmacophore are (1) an acidic
moiety – either a phosphate or a carboxylate – proposed to
interact with the phosphatase metal ions (as does tungstate
in the X-ray), (2) a methyl group proximal to the acidic
moiety, proposed to mimic the methyl group in
phosphothreonine, and (3) an extended hydrophobic segment
(except for cantharidin which is a µM inhibitor), thought to
mimic the hydrophobic amino acid residues adjacent to
phosphothreonine in endogenous proteins which bind
PP1/PP2A [116–120]. Notably, the elements of the
pharmacophore are also present in fostriecin and are
highlighted in Fig. (6).
Unfortunately, systematic studies establishing whether
the pharmacophore features are required for binding of
fostriecin with PP1 or PP2A have not yet been conducted.
However, some indirect information is available which
supports this tentative pharmacophore correlation. For
example, dephosphorylated fostriecin shows considerably
diminished cytotoxic and antitumor activity. A naturally-
occurring congener of fostriecin, PD 113,270 (2), which
lacks only the C18 primary alcohol, is equipotent in
cytotoxic assays, nearly as efficacious in antitumor assays in
vivo, and is transported into cells by the reduced folate
carrier at least as effectively, and perhaps better, than 1. The
studies also indicate there may be additional recognition or
selectivity elements not yet implicated as part of the
pharmacophore which are more easily identified within the
structure of fostriecin than other natural product inhibitors.
The PP1/PP2A-mediated phosphate cleavage is catalyzed in
a single step by a dinuclear metal site activating a bridging
water molecule for hydroxide nucleophilic attack on the
bound substrate, and active site residues involved in the
substrate activation (PP1, His125, and Asp95) have been
clearly identified. The comparison of the inhibitors that
display significant and potent PP2A versus PP1 selectivity
(fostriecin, okadaic acid) with the remainder which do not
reveals that these two uniquely bear a hydroxy group
immediately adjacent to the anionic group on the same
Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2015
carbon bearing the important methyl substituent. The role of
this hydroxy group is presently not well understood, but
could serve as a mimic of the substrate Thr-OH or, more
provocatively, serve to displace the enzyme's metal bound
active site water nucleophile.
Additional studies indicate some analogy between an
important recognition element not directly implicated as part
of the pharmacophore and a similar structural feature present
in the other natural product inhibitors. Acetylation of the
C11 alcohol of fostriecin, located α to the hydrophobic
triene, destroys activity. Similarly, a calyculin analog
displayed a dramatic increase in IC50’s with PP1 and PP2A
when its C11 and C13 alcohols, adjacent to its hydrophobic
tetraene, were protected as the acetonide [117]; and
phosphatase binding was abolished in tautomycin analogs in
which the C22 hydroxyl, also located near its purportedly
hydrophobic spiroketal segment, was removed by
deoxygenation or β-elimination [121]. Based on studies
illustrating that this residue is important in recognition,
molecular modeling has indicated that the calyculin and
tautomycin alcohols may form key hydrogen bonds with
Arg221 of PP1 [115,120,122,123].
Specificity for PP2A/PP4– Does it Reside in the β 12–β 13
Loop?
The remarkable selectivity for PP2A and PP4
demonstrated by fostriecin is noteworthy. The elements of
the pharmacophore common to the natural product inhibitors
are only thought to affect binding affinity and not
selectivity. One hypothesis that has emerged is that a
segment of the phosphatase catalytic pocket, known as the
β12–β13 loop, is responsible for the selectivity imparted by
the inhibitors. The catalytic pockets of PP1 and PP2A show
>90% sequence homology except in this region, and assays
of the inhibitors with β12–β13 deletion mutants of PP1
indicate that the loop is critical for binding [124]. In
addition, most of the inhibitors are believed to form specific
contacts with this area based on SAR data [70].
Interestingly, the only study regarding fostriecin which has
attempted to address this issue suggests that the β12–β13
loop is not implicated in fostriecin’s selectivity.
Shenolikar and Honkanen constructed a chimera of
human PP1α (CRHM2) in which the entire amino acid
sequence of the β12–β13 loop was replaced with that of
PP2A (bovine) [124]. Fostriecin, okadaic acid, tautomycin,
microcystin-LR, calyculin A, and endogenous protein
inhibitors of PP1 (I–1, I–2, NIPP1), which are substrates of
PP2A, were assayed against phosphorylase a with
recombinant human PP1α , and CRHM2. The results
showed that all of the natural product inhibitors bound
CRHM2 and PP1α with comparable affinity suggesting that
the β 1 2 – β 13 loop is not involved in the selective
recognition. In contrast, the endogenous proteins inhibited
PP1 at nM concentrations while CRHM2 was effectively not
inhibited, which was similar to reported assays with PP2A.
Therefore, the loop was implicated in preferential PP1
recognition of the endogenous inhibitors. This disparity is
especially interesting when considered with data reported for
another PP1 β12–β13 loop chimera. Previously, Zhang and
coworkers substituted a short segment of the loop in PP1
2016 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

(GEFD, residues 274–277) with the corresponding residues Fostriecin was reported to inhibit the decatenation of
(YRCG) of PP2A. Okadaic acid showed a lower IC50 than kinetoplast DNA by partially purified topoisomerase II in an
for PP1 with this mutant suggesting that the origin of its in vitro assay with an MIC of 50 µM (Table 2). Several
PP2A selectivity could arise from interactions with this natural or semisynthetic analogs of the natural product were
segment. In addition, the IC50's of microcystin and calyculin examined in the same assay. Addition of a hydroxyl group
were also affected, showing an increase with the mutant at the C4 position on the lactone ring increased potency 4-
enzyme [125]. Shenolikar and Honkanen concede that there fold. A 2-fold increase in potency was observed upon
may be a conformational difference in this region between removing the phosphate ester or opening the lactone ring to
the chimera and PP2A arising from incorporating the whole the free carboxylic acid. Acetylation of the primary and
loop sequence. Perhaps a more conclusive study would be to secondary hydroxyl groups decreased the inhibition to an
test the inhibitors against a PP2A chimera constructed with IC50 of 200 µM (4-fold) [29]. Notably, these trends do not
the loop of PP1 and determine whether fostriecin’s affinity follow those observed in functional cytotoxic assays.
is considerably diminished.
Table 2. Topoisomerase II Inhibition
Another finding by Honkanen and coworkers indicated
that fostriecin did not competitively inhibit PP1 in cells R1 R4
treated with both fostriecin and microcystin-LR. They OR2 OR 3
concluded that fostriecin must be binding a different site
than microcystin [126]. However, since fostriecin only binds O O
PP1 with µM affinity, whereas microcystin-LR has low nM Me OH
affinity and binds with a covalent linkage to the enzyme, it
may not be surprising that competitive inhibition is not Topoisomerase II
observed. Fostriecin was tested for PP1 and PP2A Compound R1 R2 R3 R4
µ M))
Inhibition (MIC (µ
inhibition against the known enzyme substrate
phosphorylase a, and it is difficult to imagine that its
phosphate group is not coordinating to the active site Fostriecin H PO3H 2 H OH 50
phosphate-binding metal ions. Site-directed mutagenesis PD 113,270 H PO3H 2 H H
experiments with key active site residues as have been
performed with the other inhibitors should confirm whether PD 113,271 OH PO3H 2 H OH 12.5
fostriecin binds in the active site.
PD 114,027a H PO3H 2 H OH
Undoubtedly, it will prove useful to probe the origin of PD 114,631 H H H OH 25.0
fostriecin’s selectivity with the preparation of structural
analogs. Fostriecin is structurally simple compared to the PD 116,243 H H H OH
other natural product inhibitors and these issues of binding
PD 116,245a OH PO3H 2 H OH 25.0
affinity and selectivity can be most easily and systematically
examined with its analogs. Finally, fostriecin's most PD 116,250 H PO3H 2 Ac OAc 200
significant feature that does not correspond to the
pharmacophore model is the unsaturated lactone. It is PD 116,251 OAc PO3H 2 Ac OAc
tempting to speculate that this group is necessary for the aLactone opened to carboxylic acid
observed selectivity, and to suggest a possible interaction
with the β12–β13 loop. For example, the enone may act as a
Michael acceptor analogous to the dehydroalanine moiety in Several lines of evidence indicate that fostriecin
the microcystins, or the latent acid may form ionic penetrates cells via the reduced folate carrier system. The
associations with active site residues. cultivation of a fostriecin-resistant L1210 cell line
(L1210/CI-920 r) and its cross-resistance to methotrexate,
which was shown to be derived from an impaired transport,
Biological Properties led to the initial discovery that 1 is transported into cells by
the reduced folate carrier system. It was demonstrated that
Fostriecin exhibits potent cytotoxic activity against the drug is a potent inhibitor of methothrexate influx
mouse leukemia (L1210, IC50 = 0.46 µΜ) and is about one exhibiting a mixture of competitive and noncompetitive
order of magnitude less active against human colon inhibition. When 50 µM of fostriecin was administered to
adenocarcinoma cells (HCT-8) [27]. Initial evidence of the in L1210 cells over 3-min intervals, the methothrexate influx
vivo anticancer activity of 1 was obtained against P388 rate fell to 37% of the control cells (Table 3). Interestingly,
leukemia-bearing mice (% T/C = 246). The drug was also this inhibition by fostriecin is transiently irreversible, but
active against B16 melanoma, but its most efficacious in reversed upon addition of thiols (dithiothreitol). The authors
vivo activity was observed with L1210-bearing mice, where suggest this may represent reversible covalent attachment of
an i.p. dose of 6.25 mg/kg/day on days 1–9 was curative fostriecin to the reduced folate carrier by a Cys thiol Michael
[51,100]. Fostriecin was devoid of antibacterial activity addition to the α,β-unsaturated δ-lactone. Removal of the
when tested against 11 organisms at 500 µg/mL [2], but it primary hydroxyl group showed little effect on the activity,
did exhibit significant antimycotic effects versus certain while addition of a hydroxyl group at the C4 position on the
yeasts with MIC values ranging from 30 to 300 µg/mL lactone ring decreased the inhibition of methothrexate entry
[127]. over 50% versus fostriecin. Opening the lactone ring lowered
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2017

Table 3. Fostriecin SAR Studies of Reduced-Folate Carrier, Cytotoxicity, and in vivo Antitumor Activity

R1 R4
OR2 OR 3

O O
Me OH

Methotrexate influx µ M))

Cytotoxicity (IC50 (µ Antitumor Activity (% T/C)a
Rate (% of control)
Compound R1 R2 R3 R4 L1210 HCT-8 L1210 P388

Fostriecin H PO3H 2 H OH 36.6 ± 4.4 0.46 5.1 207 246

PD 113,270 H PO3H 2 H H 31.5 ± 13.4 0.50` 3.4 180 178

PD 113,271 OH PO3H 2 H OH 57.2 ± 6.7 1.8 9.0 121 175

PD 114,027b H PO3H 2 H OH 71.2 ± 7.8 >2.1 120

PD 114,631 H H H OH 89.2 ±5.9 5.5 (6.7)c 9.9

PD 116,243 H H H OH

PD 116,245b OH PO3H 2 H OH 8.1

PD 116,250 H PO3H 2 Ac OAc 84.7 ± 1.4 >100

PD 116,251 OAc PO3H 2 Ac OAc

a(% T/C) represents increase in life-span of treated mice versus control. Sixty-day survivors not included.
bLactone opened to carboxylic acid.
cRepresents data from two separate experiments.

the activity 2-fold. Either C8 dephosphorylation or
acetylation of the C11 and C18 alcohols abolished most of
the inhibitory activity [50,51].
The cytotoxicity of fostriecin and several analogs were
investigated in both L1210 and HCT-8 cell lines (Table 3).
The parent compound proved to be an order of magnitude
more potent in the L1210 assay, but the structure–activity
trends were similar in both cell lines. Removal of the
primary hydroxyl group at C18 had little effect on the drug
potency and addition of a hydroxyl group at the C4 position
on the lactone ring decreased the cytotoxicity approximately
3- to 4-fold. Opening the lactone ring virtually abolished
activity. Removal of the phosphate led to an order of
magnitude decrease in activity in the L1210 cell line, but
only a 2-fold decrease for the HCT-8 cells. Concomitant
hydroxylation of the C4 position and lactone hydrolysis
resulted in greater than a 16-fold loss of activity. As noted
in previous studies, acetylation of the C11 and C18 alcohols
abolished the activity [29,51,100].
Tunac, et al. reported that fostriecin and its natural
product analogs PD 113,270 (2) and PD 113,271 (3) showed
excellent antitumor activity in murine tumor models (Table
3). The L1210 tumor-bearing mice treated with fostriecin
(i.p., 6.25 mg/kg/injection, daily × 9) exhibited increased
life spans of 207% relative to control animals (% T/C).
Removal of the primary hydroxyl group lowered activity
slightly, whereas adding a secondary alcohol at the C4
position lowered the activity more significantly versus the
parent compound. Against P388, a similar but slightly
different trend was observed, where both the removal of the
primary alcohol and the addition of a hydroxy group at the
C4 position had the same effect of lowering efficacy [2]. The
results of a more extensive optimization of the in vivo
activity are summarized in Table 4, which lists the efficacy
of fostriecin and the different analogs (i.p. administration,
L1210). The parent compound showed strong activity and
was "curative" on a daily dosing regimen of 5 or 9 days
duration. Removal of the primary hydroxyl group increased
potency 3-fold, but the efficacy was unchanged or lower
compared to fostriecin. Hydroxylation of the C4 position
reduced the efficacy whereas dephosphorylation or hydrolysis
of the lactone to the carboxylic acid virtually abolished
activity. Acetylation of the primary and C11 secondary
alcohols of fostriecin or PD 113,271 also abolished activity
[51,100].
The antimycotic properties of fostriecin and four analogs
have also been examined (Table 5). The drug was effective
against multiple yeast strains, with MIC values in the range
of 30–300 µg/µL. PD 113,270, which is devoid of the
primary alcohol, shows similar activity. Hydroxylation at
the C4 position of the lactone increased the antimycotic
activity 10-fold. Removal of the phosphate group from
fostriecin or PD 113,271 resulted in no activity [127].
CLINICAL STUDIES
A phase I clinical trial and pharmacokinetic study of
fostriecin was conducted in which the drug was administered
intravenously over 60 min on days 1–5 at 4-week intervals.
The doses used ranged from 2 to 20 mg/m2 /day. The
2018 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

Table 4. Antitumor Activity of Fostriecin and Related Analogs

R1 R4
OR2 OR 3

O O
Me OH

Compound R1 R2 R3 R4 Dose (mg/kg/inj.) Schedule % T/Ca 60-day survivors

Fostriecin H PO3H 2 H OH 40 Days 3-7 267 0/6

25 Days 3-7 250 1/6

12.5 Days 3-7 289 1/6

12.5 Days 3-11 307 0/6

6.25 Days 1-9 Curative 6/6

PD 113,270 H PO3H 2 H H 8.12 Days 3-7 250 0/6

6.25 Days 3-7 271 0/6

6.00 Days 1-9 226 0/6

1.5 Days 1-9 180 0/6

PD 113,271 OH PO3H 2 H OH 25 Days 3-7 180 0/6

10 Days 3-7 140 0/6

17.5 Days 3-11 190 0/6

6.0 Days 1-9 128 0/6

6.0 Days 1-9 123 0/6

3.12 Days 1-9 128 0/6

PD 114,027b H PO3H 2 H OH 25c Days 3-7 100 0/6

PD 114,631 H H H OH 50c Days 3-7 109 0/6

PD 116,250 H PO3H 2 Ac OAc 50c Days 3-7 101 0/6

PD 116,251 OAc PO3H 2 Ac OAc 50c Days 3-7 102 0/6

a(% T/C) represents increase in life-span of treated mice versus control. Sixty-day survivors not included.
bLactone opened to carboxylic acid.
cHighest dose tested.

predominant toxicities included elevated liver transaminases inherent drug instability and unpredictable purity in the
and serum creatinine, but these effects showed only a limited clinical supply of the natural product.
increase with increasing doses, often recovered during drug
administration, and were fully reversible. Other frequent side Table 5. Antimycotic Activity of Fostriecin and Related
effects were grade 1–2 nausea/vomiting, fever, and mild Analogs
fatigue. Mean fostriecin plasma half-life was 0.36 h (initial;
95% Cl, 0–0.76 h) and 1.51 h (terminal; 95% Cl, 0.41–2.61 Compound Antimycotic Activity (MIC, µ g/mL)
h). A metabolite was detected in plasma and urine, and the
authors proposed that this may be dephosphorylated
Fostriecin 30-300
fostriecin. Antitumor activity was not observed; however,
the plasma concentrations reached in the patients were below PD 113,270 30-300
even those needed to cause in vitro growth inhibition, and PD 113,271 3-30
the dose-limiting toxicity was not reached with 20
mg/m2/day [128,129]. Although fostriecin was formulated PD 114,631 no activity
as the sodium ascorbate salt to increase its stability to PD 116,243 no activity
oxidation [101], clinical trials were suspended due to
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2019

3 15 17
2 4 14 16
18
H2O3PO OH
5 8 10
13 21 19
O O 7 9 11 20
6 12
OH R

H2N
O

46, R = H 53, R = O

O O

47, R = O 54, R = O

O
O

48, R = 55, R = O
O
O
O
56, R = O
49, R = O
O
O
57, R = O
50, R = O
O
O
58, R = O
51, R = O

O 59, R = OH

52, R = O

Fig. (9).

RELATED NATURAL PRODUCTS
Phospholine
Phospholine (46), Fig. (9), an amphoteric phosphate
monoester containing an α,β-unsaturated δ-lactone and a
conjugated diene system, was isolated from the culture broth
of Streptomyces hygroscopius subsp. luteolus subsp. nov.
by Ozasa, et al. It was obtained by gel filtration, butanol
extraction, and reverse phase chromatography. The molecular
formula and molecular weight were determined to be
C25H40NO8P (MW 513) from high resolution FABMS data
and elemental analysis. A positive test for ninhydrin and
ammonium molybdate-perchloric acid indicated the presence
of an amino and a phosphate group, respectively. The IR
absorbance at 1720 cm–1 suggested an α,β-unsaturated δ-
lactone and at 1100 cm–1 indicated a phosphate moiety. The
characteristic UV absorbance at 234 nm was indicative of the
α,β-unsaturated δ-lactone and/or a conjugated diene, and
enzymatic hydrolysis with alkaline phosphatase implied the
presence of a phosphate monoester. Phospholine's
connectivity was determined by 1H NMR, 13C NMR, 1H–
1 H COSY, 1 3 C – 1 H COSY, and HMBC spectra.
Phospholine was tested for in vitro cytotoxicity against
L1210, P388, and EL-4 tumor cell lines and was found to
be 100-fold less potent (IC50 = 2–3.4 µg/mL) than that of
mitomycin C (IC50 = 0.02–0.07 µg/mL) [130,131].
Phoslactomycins A–F
Phoslactomycins A (47), B (46), and C–F (48–51), Fig.
(9), phosphate monoesters containing an α,β-unsaturated δ-
lactone and a conjugated diene system, were isolated from
the culture broth of Streptomyces nigrescens by Fushimi, et
al. They were obtained by butanol extraction, gel filtration,
and reverse phase chromatography. The molecular formula
and molecular weight for each compound were determined
from high resolution FABMS, 1H, 13C, and 31P NMR data
(phoslactomycins A, D, and E), from high resolution
FABMS, 1H, 13C NMR data (phoslactomycin B), or from
FABMS and 1H NMR (phoslactomycins D and F) (Table
6). The functional groups present in the phoslactomycins A–
F were also initially elucidated utilizing a combination of
color reactions (ninhydrin: primary amino group,
ammonium molybdate–perchloric acid: phosphate group).
2020 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
Since the UV and 1H NMR spectra of the phoslactomycins
resembled each other so closely, they were assumed to be
structural analogs. The major component of the isolated
material was phoslactomycin E, therefore its structure was
elucidated first.
Table 6. Molecular Formulas and Molecular Weights of
Phoslactomycins A–F
Phoslactomycin Molecular Formula Molecular Weight
A (47) C 29H 46O 10NP 599
B (46) C 25H 40O 8NP 513
C (48) C 30H 48O 10NP 613
D (49) C 31H 50O 10NP 627
E (50) C 32H 50O 10NP 639
F (51) C 32H 52O 10NP 641
The IR spectrum of phoslactomycin E (50) showed major
absorptions at 1724, 1076, and 1250 cm–1 denoting the
presence of an α,β-unsaturated δ-lactone, P–O, and P=O,
respectively. The UV spectra (absorption at 233 nm)
suggested the presence of an α,β-unsaturated δ-lactone. Its
connectivity was determined based on interpretation of 1H
NMR, 13 C NMR, 1 H– 1 H COSY, 13 C– 1 H COSY, DEPT,
decoupling difference (DDS), and 3 1 P spectra. The
configuration of the conjugated diene system was established
as Z,Z on the basis of the coupling constants (J 12,13 = 8.6
Hz, J 14,15 = 9.5 Hz) and the C6–C7 stereochemistry was
determined to be E (J6,7 = 15.1 Hz). The chemical shift of
C5-H (400 MHz, δ 5.10) indicated the formation of a
lactone ring including C1 to C5 which, the authors pointed
out, is almost identical to the chemical shift value of the
corresponding proton in fostriecin. The 1H and 13C NMR
spectra suggested the linkage of a cyclohexane ring to a
carbonyl carbon and its presence was confirmed by detecting
cyclohexane carboxylic acid in the alkaline hydrolysate of
phoslactomycin E by GC–MS. Analysis of the 13C and 2D
COSY NMR data (phoslactomycin A, C, D, F) or the 1 H
and 13 C NMR data (phoslactomycin B) revealed that
phoslactomycin E and all five congeners contained the same
carbon skeleton from C1 to C25. The only structural
difference between the compounds was the acyl moiety
bound to C18. By interpretation of the 2D COSY NMR
spectra, the different acyl subunits for phoslactomycins A–F
were assigned. Phoslactomycin B was shown to be identical
in structure to phospholine [131].
The phoslactomycins A–F showed strong activity against
various fungi, especially phytopathogenic fungi such as
Botrytis cinerea and Alternaria kikuchiana. The antibacterial
activity, however, was weak. Comparison of the
antimicrobial activity between the six phoslactomycin
compounds showed almost an identical profile. Since the
difference in the chemical structures was ascribed to a
substituent bound to a cyclohexane ring, the substituent is
considered not to be important for antimicrobial activity
[132,133].
Boger et al.
Phosphazomycin C
Phosphazomycin C, an equimolar complex of two
phosphate monoesters, each containing an α,β-unsaturated δ-
lactone and a conjugated diene system, was isolated from the
culture broth of Streptomyces sp. HK-803 by Tomiya, et al.
It was obtained by gel filtration, butanol extraction, and
reverse phase chromatography. The molecular formula of
each component was determined to be C30H48NO10P (MW
613) from FABMS data and elemental analysis. It gave
positive reactions to ninhydrin, Lemieux, and anisaldehyde–
sulfuric acid reagents. The connectivity of the two
components of phosphazomycin C (phosphazomycin C1 (48)
and phosphazomycin C2 (52)), Fig. (9), were determined
based on interpretation of 1 H NMR, 13 C NMR, 1 H– 1 H
COSY, 13 C– 1 H COSY, 1 H– 31 P COSY and long-range
proton decoupling (LSPD) spectra. Phosphazomycin C1 was
shown to be identical in structure to phoslactomycin C
[133]. Accordingly, the phosphazomycin C complex showed
potent antifungal activity [134].
Leustroducsins A–C
Leustroducsins A–C (53–55), Fig. (9), phosphate
monoesters containing an α,β-unsaturated δ-lactone and a
conjugated diene system, were isolated from the culture
broth of Streptomyces platensis SANK 60191 by Kohama,
et al. They were obtained by ethyl acetate extraction and
preparative reverse phase chromatography. The molecular
formula and molecular weight for leustroducsins A, B, and
C was determined to be C3 2 H 5 2 N O 1 0 P (MW 641),
C 34H 56NO 10P (MW 669), and C34H 56NO 10P (MW 669),
respectively, from high resolution liquid secondary ion mass
spectrometry (LSI–MS). The functional groups present in
the leustroducsins A–C reacted positively on TLC to iodine,
sulfuric acid, ninhydrin, and ammonium molybdate–
perchloric acid, indicating the presence of amino and
phosphorus groups. The initial UV, IR, 1H and 13C NMR
spectra of the leustroducsins resembled those of the
phoslactomycins so closely that compounds 53–55 were
assumed to be structural analogs. The structural assignment
of the acyl chains, the only novel portion of the molecules
throughout the phospholine, phoslactomycin,
phosphazomycin C, and leustroducsin series, was made
based on 1H and 13C NMR, MS/MS, and LSI–MS/MS. In
addition to the three novel leustroducsins isolated from the
bacterial broth, the previously reported phoslactomycins F
(51) [132,133], I-h (56), I-i (57), and I-j (58) [135] were also
identified [136,137].
A single congener of the leustroducsins was obtained
from treating a mixture of leustroducsins A, B, and C and
phoslactomycin F with porcine liver esterase (leustroducsin
H, 59), Fig. (9). The absolute configuration of the common
core structure 59 was elucidated by Shibata, et al. utilizing
the Mosher ester method. Both protected (+)-(R)- and (–)-(S)-
MTPA esters of the 11-hydroxy group (60) were prepared,
Fig. (10). The proton signals of each derivative were
assigned by COSY, and the systematic arrangement of
positive and negative ∆δ's revealed the R configuration at
C11. An analogous assignment of the configuration at C18
utilizing the (+)-(R)- and (–)-(S)-MTPA esters of the 18-
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2021

OMTPA
determined [138]. The stereochemical assignments also
O H correlated with the absolute configuration determined for
O fostriecin at C5, C8, C9, and C11 [5].
OHC
N 11

OH The leustroducsins A–C were isolated while screening for

O compounds which induce the production of colony-
stimulating factors (CSF) in KM-102 cells [136]. It was
Br O demonstrated that the growth of CSF-dependent cell lines
60
were significantly stimulated by media of KM-102 treated
O with leustroducsin A, B, or C, whereas the activity of the
18 OM TPA
phoslactomycins F, I-h, I-i, and I-j was slightly lower. The
O H OBz E D 50 of these compounds correlated positively with
16
O hydrophobicity, hence leustroducsins B and C were the most
OHC
N potent inducers. The cytotoxicity of leustroducsin B against
H various cell lines was approximately two-fold more potent
than the CSF-inducing ability, suggesting that these
compounds may have limited potential as therapeutic agents.
O Like the phoslactomycins, the leustroducsins showed no
61 antibacterial activity but did exhibit significant antifungal
O activity.
MTPAO OBz
H
HO
OHC 9 Sultriecin
N 8
H OBz Sultriecin (64), Fig. (11), a sulfate monoester containing
an α,β-unsaturated δ-lactone and a conjugated triene system,
O was isolated from the culture broth of Streptomyces
roseeiscleroticus by Ohkuma, et al. as the sodium salt. It
O 62 was obtained by butanol extraction and reverse phase
chromatography. The molecular formula and molecular
H weight was determined to be C23H33O8SNa (MW 492) from
N O FABHRMS and FABMS data. Both the sultriecin
OHC
monosodium salt, as well as its desulfated derivative,
showed positive responses to iodine and sulfuric acid, while
demonstrating negative results for ninhydrin and anthrone
MTPAO 5 tests. The UV absorbance maxima (260, 269, 279 nm)
indicated the presence of a triene while the IR spectrum
MeOOC
63
(1720 and 1260 cm–1) was indicative of an α,β-unsaturated
δ-lactone and a sulfate, respectively. The structure clearly
Fig. (10).
resembled that of fostriecin, but differed in the MS, NMR,
hydroxy group (61) established the S configuration. The and in the absence of a phosphate group as shown by
relative stereochemistry on the cyclohexane ring, and hence negative Hanes reaction. The connectivity of 64 was
the absolute configuration of C16 (R), was determined by determined based on interpretation of 1H NMR, 13C NMR,
1 H– 1 H COSY, and 13 C– 1 H COSY spectra. Sultriecin
examination of the coupling constants of 61. The remaining
relative stereochemistry of the (+)-MTPA of leustroducsin H demonstrated moderate and broad spectrum in vitro
derivative 61 was determined from the NOESY spectrum. It antifungal activity. In addition, 64 and the desulfated
was deduced that both the C7 and H9 of the acetonide ring derivate 65 exhibited weak in vitro cytotoxicity against
and the H4 and H5 of the lactone were in a cis relationship. various cancer cell lines. However, both analogs inhibited
RNA and protein, but not DNA, synthesis in cultured
In order to assign the absolute configuration of the C9
OH
stereocenter, the protected (+)-(R)- and (–)-(S)-MTPA esters OR OH
of the 9-hydroxy group (62) were prepared which led to the
assignment of the R configuration. According to the relative O O
stereochemistry mentioned above, the absolute configuration
Me 64, R = SO3 H
of C8 was assigned as R. Finally, the absolute configuration
of C5 was assigned as S based on proton assignments of 65, R = H
both the (+)-(R)- and (–)-(S)-MTPA esters of the 5-hydroxy Me
group from the lactone derivative 6 3 . The absolute H2O3P OH
configuration of leustroducsin H was hence determined to be
4S,5S,8R,9R,11R,16R,18S. Since all the leustroducsins and O O
phoslactomycin F possess the same core structure, the Me Me
absolute configurations of the four isolates could be 66
Fig. (11).
2022 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
L1210 murine leukemia cells. The natural product 6 4
demonstrated in vivo antitumor activity comparable to
mitomycin C in mice innoculated with P388 and B16
melanoma, but 65 was devoid of activity against P388
leukemia. The antitumor activity, as well as the
macromolecular biosynthesis inhibition, is strikingly similar
to the activity profile of fostriecin [139].
Cytostatin
Cytostatin (66), Fig. (11), is a phosphate monoester
containing an α,β-unsaturated δ-lactone and a conjugated
triene system. The compound was discovered in the cultured
broth of Streptomyces sp. MJ654-NF4 by Amemiya, et al.
as the sodium salt. It was obtained by ethyl acetate
extraction, size-exclusion chromatography, and reverse phase
chromatography. The molecular formula and molecular
weight was determined to be C21H33O7PNa (MW 427) from
high resolution FABMS. Cytostatin showed positive
responses to ferric chloride and anisaldehyde-H2SO4 reagents
on TLC. In addition, a positive color reaction to ammonium
molybdate–perchloric acid indicated the presence of a
phosphate ester. The UV absorbance maxima (260, 267, 277
nm) indicated the presence of a triene while the IR spectrum
(1715 cm–1) was indicative of an α,β-unsaturated ester or
lactone. The connectivity of 66 was determined based on
interpretation of 1 H NMR, 13 C NMR, 31 P NMR, DEPT,
1 H– 1 H COSY, HOHAHA, LSPD, and HMBC spectra.
Cytostatin was intially identified as a novel inhibitor of B16
melanoma cell adhesion to laminin and collagen type IV in
the extracellular matrix. An in vitro cytotoxicity assay
showed that cytostatin is 5- to 10-fold less toxic to the B16
cell line than to other tumor cells [140,141]. Yamazaki, et
al. demonstrated that the natural product increased the rate of
DNA fragmentation in FS3 fibrosarcoma cell, thereby
causing apoptosis [142]. Another study reported that
cytostatin was cytostatic to both B16 and EL-4 cell lines
with an IC50 of 1–2 µg/µL and cytotoxic at a concentration
of 10 µg/µL [143]. In in vivo studies conducted by Ishizuka
and coworkers, cytostatin inhibited lung metastasis of B16-
F10 and B16-BL6 melanoma cells by 76.5 and 72.9%,
respectively, at an i.p. dose of 1.25 mg/kg in BDF1 mice
[140,144]. It has recently been demonstrated that cytostatin
inhibits cell adhesion through the selective noncompetitive
inhibition of serine/threonine phosphatase PP2A (IC50 =
0.09 µ g/mL) [145] which, considering its structural
similarities to fostriecin, is not unexpected.
CONCLUSIONS
Fostriecin is a naturally-occurring potent and efficacious
antitumor agent that most likely derives its activity through
the selective inhibition of protein phosphatases 2A and/or 4
(PP2A and/or PP4), resulting in inhibition of the mitotic
entry checkpoint. Contributing to its activity is an active
transport into tumor cells by the reduced folate carrier.
Fostriecin also inhibits topoisomerase II, albeit much more
weakly, by a mechanism which is not yet clear, and whether
this also contributes to its antitumor properties remains to
be established. Inhibition of the mitotic entry checkpoint via
Boger et al.
selective protein phosphatase inhibition is a novel, clinically
unexplored mechanism worthy of pursuit for introduction of
a new class of antitumor agents. Unfortunately, phase I
clinical trials on fostriecin failed to reach therapeutic dose
levels when the trials were suspended due to concerns over
its inherent storage stability and an unpredictable purity in
the clinical supplies of the natural product. These issues,
along with the opportunity to probe the mechanism of
action, may now be addressed with the development of a
total synthesis of the natural product that is amenable to the
preparation of key substructure analogs that may be used to
define its pharmacophore.
ADDENDUM
Several important papers have appeared while this review
was at the publishers. Chavez and Jacobsen also recently
completed a total synthesis of fostriecin [146]. Their
approach integrates highly effective asymmetric catalytic
reactions to generate key chiral building blocks, and efficient
coupling reactions to enable their convergent assembly. The
synthetic plan, shown in Fig. (12), involves the assembly of
four fragments of similar complexity. Epoxyketone 68 plays
a central role in the synthetic strategy serving not only as the
source of the C9 stereocenter but also as a lynchpin for
joining the left-hand vinyl lactol 67 and the right hand
triene-diol fragment. Key features of the synthesis include
the use of the hydrolytic kinetic resolution (HKR) to provide
enantiomerically pure epoxyketone 68, an asymmetric
catalytic hetero Diels–Alder reaction to afford
enantiomerically pure lactol 67, a highly diastereoselective
1,2-addition used to introduce the C8 stereocenter, and a
catalytic asymmetric transfer hydrogenation was used to set
the C11 stereocenter. A late stage Stille coupling followed
by phosphorylation of the C9 alcohol provided the Z,Z,E-
triene system and the C9 phosphate, respectively.
Highly enantioenriched epoxyketone 68 was prepared by
epoxidation of methyl vinyl ketone and subsequent
hydrolytic kinetic resolution [147] of this epoxide using
(salen)CoIIIOAc 73 under an oxygen atmosphere (>99% ee,
80%). Chiral building block 67 was accessed through a Cr-
catalyzed asymmetric hetero Diels–Alder reaction recently
developed in the Jacobsen group [148]. 1-Benzyloxy-1,3-
butadiene underwent reaction with triisopropylpropar-
galdehyde in the presence of the tridentate Cr Schiff base
catalyst 74 (3 mol%) to provide the cycloadduct 75 in good
yield, diastereoselectivity and enantioselectivity.
Desilylation and acidic workup gave the more stable trans-
acetal, which was recrystallized to provide the highly
enantioenriched alkyne 76 (99% ee).
The acetal 76 was converted to the more robust isopropyl
derivative 7 7 , and was subjected to a one-pot
hydrozirconation-Zn transmetallation sequence developed by
Wipf [149] followed by coupling with epoxyketone 68, and
resulted in the isolation of the desired 1,2-addition product
78 in >30:1 diastereoselectivity favoring the desired
chelation control product in modest yield (45%). The epoxy
alcohol was protected as the triethylsilyl ether 79 (TESCl,
Im., DMF, 99%) and was subsequently coupled with
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2023

Me Chiral Epoxide
Asym. H DA 17
14 18 OH
H H NaH O3 PO OH
N N 10 13
Adama ntyl 8 Stille Coupling
Co O O
t
Bu O t N O OH Asym. Hydrogenation
O Bu 1,2-Add' n
Cr Chelation Control 1
Epoxide Ring Opening
t
Bu t
Bu Cl
O
73 74 OR

Bu3 Sn 70
RO O M
67 O
(S,S)-73 (2 mol%) O OH
O S S
HOAc (4 mol%) + OH
OR O
O 68 H
O 2 Balloon O O
O 69
5-25 o C, 48 h
(R)-(+)-68 H TMS
40% yield 71 72
TIPS
>99% ee

TIPS 1) TBAF, T HF Cp 2Z r(H)Cl;

(R ,S)-74 (3 mol%) 2) TsOH M e2Z n;
H
+
36 h, RT BnO O 3) recryst., 65%, RO O 68
O 45%, >30:1 dr
75 TIPS 99%ee
OBn H
72
90% yield R = Bn (76)
71 90%
89% ee, >95:5 dr R = i Pr (77)

n-BuLi, 69, HO S S
T HF OH S S
O PPTS
i 89% A cetone/H2 O X O
PrO O i
PrO O
Me OR Me OT ES Me O TES T MS
TM S
80
E t3SiCl R = H (78)
MnO2 X = H, OH (81)
99% R = T ES (79)
X = O (82)
65%, 2 ste ps
Ts
Ph
N
Ru
Ph N
H
85
PM BO OR
PhI(O2 CCF3 )2 RO O A gNO3 , NIS

93%, >95:5 dr O O 95%

70% O O Me OT ES
Me OT ES TM S
T MS
T BSOT f R = H (86)
PM BO (C=NH)CCl3 R = H (83)
Trityl-BF4 , 83% 80% R = TBS (87)
R = PMB (84)

RO OT BS HO OTBS
"H N=NH" 70, PdCl2
O O O O
Me OT ES I Me OTES
85% 85%
90 I
D DQ R = PMB (88)
100% R = OH (89)

OH
OR NaHO 3PO OH
R' O OTBS HF;
HF, Pyr, 45% O O
O O
M e OH
Me OTE S

PCl3 ; PM BOH; R' = H (91) 1, fostriecin

t
BuOOH, 65% R' =PO (OPMB)2 (92)
Fig. (12). Jacobsen total synthesis of fostriecin.
2024 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

(+)-Ipc2 BOM e
4 M etathesis MgBr
1 Asym. Allylation 17 OR
OH –100 ˚C; OsO4, NMO;
H2 O3 PO OH 14 18 OH C TMS
H2 O2 , NaOH NaIO4, SiO2
8 10 13
O O 5 Suzuki Coupling 93
Me OH 2 Sharpless AD T BDPSCl, Im. R = H (94)
3 Asym. Allylation 48%, 2 steps R = TBDPS (95)
1
O
PPh 3
RO OR X OT BDPS EtO OT BDPS
OHC + Me EtO2 C AD-mix β;
BR2 OR
O
Me OR 56%, 2 steps , PPTS
C5–C13 C14–C18 96 97
Me OMe
73%, 2 steps, 3:1 dr
OR
O O O O
RO2 C Me Me
OTBDPS OTBDPS
E tO2C
OR NBS, AgNO3 ; EtO2 C Br LiAlH4
98 HN NH 99
C7–C13 C14–C18 58%, 3 ste ps

O
O (+)-Ipc2BOMe
O O O O MgBr O O Cl
Me PPh3 Me
OTBDPS H OT BDPS Me OT BDPS i
–100 ˚C; Pr2 NEt
R Br Br Br
8 8% H2 O2, NaOH 71%, 2 steps
OHC 10 2 1 03
98% de
Swern R = CH2 OH (100) OH
86% R = CHO (101)

T BDPSO Br
O O OH OTBDPS
Me OTBDPS Montmorillo nit e K-10
Br O O O O O
Grubb's ca t.
Me 65%, 2 steps Me OH
10 4 O Br
O 10 5 106 Pd(PPh 3) 4
Ag2 O
O O
74%
BH
O
TBD PSCI, Im O B
OH OT BDPS O OTBDPS
[Rh(cod)Cl] 2
10 7 108 109
PCy3 , Et3 N
63%, 2 ste ps
O
TMSEO P
T MSEO OP
OP O OP
OH OP PCl3 , pyr;
T MSCH2 CH2 OH; HF•pyr
O O 1, fostriecin
O O H2 O2 CH3 CN/H2O
Me OTBS
Me OR 83% 52%
P = TBDPS
TBSOT f R = H (110)
2,6-lutidine 112
86% R = TBS (111)

Fig. (13). Falck total synthesis of fostriecin.

fragment 69 through epoxide ring opening (69, n-BuLi, butyldimethylsilyl ether 87 (TBSOTf, CH2Cl 2, 80%) and
THF, 89%). Acetal hydrolysis (PPTS, acetone/H2 O) was converted to iodoacetylene 88 (AgNO3, NIS, 95%). The C9
followed by selective lactol oxidation (MnO2, CH2Cl2) to alcohol was deprotected at this stage under oxidative
afford lactone 82 (65%, 2 steps). Dethioketalization using conditions (DDQ, quantitative yield) and the iodoacetylene
Stork’s protocol [150] (bis(trifluoroacetoxy)iodosobenzene, reduced to the vinyl iodide 90 using a very selective diimide
70%) was followed by protection of the ß-hydroxy ketone as reduction [152] (o-nitrobenzenesulfonylhydrazide, THF/i-
the p-methoxybenzyl ether 84 (PMBO(C=NH)CCl3, trityl PrOH, 85%).
BF 4 , 83%). The C11 stereocenter was installed using
Noyori’s asymmetric catalytic transfer hydrogenation catalyst Stille coupling of the vinyl iodide with stannane
85 [151] and provided the propargyl alcohol 86 in excellent fragment 70 [153] installed the Z,Z,E-triene system with
yield and diastereoselectivity (92% yield, >95:5 dr). The complete retention of olefin stereochemistry in the presence
resulting propargyl alcohol was protected as the tert- of the free C9 alcohol under ligand free conditions
Fostriecin: Chemistry and Biology
(PdCl2(CH3CN)2, DMF, 85%). Phosphorylation of 91 was
completed by treatment of the free alcohol with PCl3 in
pyridine followed by addition of PMBOH and subsequent
oxidation using t-BuOOH [154] to provide 92 (65%).
Global deprotection (HF, CH3 CN; then Pyr) provided
fostriecin (45%) identical in all aspects to authentic material.
Soon after the reported synthesis by Chavez and
Jacobsen, Reddy and Falck also disclosed a total synthesis
of fostriecin [155]. The strategy, shown in Fig. (13), is
based on late stage introduction of the sensitive unsaturated
lactone, phosphate and triene functional groups. The
unsaturated lactone was formed by ring-closing metathesis
and the Z,Z,E-triene was established by Suzuki coupling of
the corresponding Z-vinyl bromide and a Z,E-dienylboronic
acid derivative. The four stereocenters found in fostriecin
were constructed by two asymmetric allylations (C5, C11)
and a Sharpless asymmetric dihydroxylation of a C8–C9
trisubstituted double bond.
The synthesis commenced with a low temperature
asymmetric allylation [156] of 93 [157] with (+)-B -
methoxydiisopinocampheylborane and allylmagnesium
bromide to provide alcohol 94 [158] in 98% ee. TBDPS
protection of the alcohol (48%, 2 steps) and dihydroxylation
followed by oxidative cleavage of the resultant diol (OsO4,
NMO; NaIO4, SiO2) afforded aldehyde 96. Homologation to
the α,β-unsaturated ester (E)-97 was achieved by Wittig
reaction with (carboethoxyethylidene)triphenylphosphorane
(56%, 2 steps). Next, the C8 and C9 stereocenters were
established by Sharpless asymmetric dihydroxylation [159]
(AD-mix β) with 3:1 diastereoselectivity. Diol protection as
the corresponding 1,2-isopropylidene allowed
chromatographic separation of the diastereomers (73%, 2
steps). Bromination of alkyne 98 [160] followed by diimide
cis-hydrogenation [161] provided vinyl bromide 99.
Reduction of the ester in 99 (LiAlH 4 , 58%, 3 steps)
followed by Swern oxidation afforded aldehyde 101 in good
yield (86%). Subsequent Wittig homologation
(OHCCHPPh3, 88%) established the C6–C7 double bond of
fostriecin.
Construction of the unsaturated lactone was initiated by
an asymmetric allylation of α,β-unsaturated aldehyde 102
((+)-Ipc2BOMe, allylmagnesium bromide; H2O2, 98% de).
Acylation of the newly-formed alcohol (acryloyl chloride, i-
Pr2NEt, 71%, 2 steps) provided the ring-closing metathesis
substrate 104 in good yield and treatment with Grubb’s
catalyst [162] ([1,3-bis-(2,4,6-trimethylphenyl)-4,5-
dihydroimidazol-2-ylidene] [benyzlidene] ruthenium (IV)
dichloride) formed the α,β-unsaturated lactone of fostriecin.
Subsequent acetonide deprotection of 105 (Montmorillonite
K-10 [163], 65%, 2 steps) provided 106, set for introduction
of the triene system.
The C14–C18 subunit was prepared from 107 [164] via
silyl protection (TBDPSCl, Im.) and [Rh(cod)Cl]2-mediated
trans-addition [165] of pinacolborane to the terminal alkyne
of 108 (63%, 2 steps). Suzuki–Miyaura coupling [166] of
subunits 106 and 109 (Pd(PPh3 ) 4 , Ag2 O) afforded the
fostriecin carbon backbone with complete olefin
stereoretention in 74% yield. Selective protection of the C8
alcohol (TBSOTf, 2,6-lutidine, 86%) by the method first
Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2025
detailed by Boger [9] was found necessary to prevent cyclic
phosphate formation during the phosphorylation procedure
[9]. As such, 111 was subjected to Evans’ three-stage
phosphorylation protocol [154] (PCl 3 , pyr;
TMSCH2CH2OH; H2O2) to provide protected fostriecin 112
in 83% yield. Global desilylation was achieved by treatment
with excess HF·pyr at room temperature followed by
NaHCO3 quench to afford natural (+)-fostriecin (52%).
The most recent total synthesis of fostriecin was
completed by Imanishi and coworkers [167]. The key bond
construction steps, shown in Fig. (14), include a Horner–
Wadsworth– Emmons reaction of fragment C1–C6 with C7–
C13 to create the C6–C7 double bond, acid-catalyzed
cyclization of the C5 alcohol to form the δ-lactone and Stille
coupling to form the Z,Z,E-triene. The use of (R)-malic acid
as starting material established the absolute stereochemistry
of C11 and a Sharpless asymmetric dihydroxylation
established the remaining two chiral centers of fragment C7–
C13. The stereochemistry at C5 was set by asymmetric
reduction of the corresponding ketone with (R)-BinalH.
Alcohol 113 was synthesized from (R)-malic acid
according to literature procedure [168]. Subsequent Swern
oxidation and Wittig reaction provided unsaturated ester 114
(86%). Reduction to the allylic alcohol and benzyl
protection yielded the tri-substituted olefin 115 for Sharpless
asymmetric dihydroxylation. The C8 and C9 chiral centers
were formed with high diastereoselectivity (95:5 anti:syn,
92%) with the (DHQD)2PHAL ligand, thereby establishing
three of the four chiral centers of fostriecin. Acetonide
formation followed by selective removal of the terminal
acetonide with zinc nitrate [169] afforded 117 (81%, 2
steps). Full orthogonal protection of fragment C7–C13 was
completed by selective 4-methoxybenzyl protection of the
primary alcohol (Bu2SnO, PMBCl, Bu4NI; 84%) and TBS
protection of the remaining secondary alcohol (TBSOTf,
2,6-lutidine, 100%).
Fragment C7–C13 was coupled with fragment C1–C6
before performing the Stille coupling to avoid complications
with the acid-labile conjugated triene in the course of the
synthesis. As such, the benzoyl group of 119 was removed
by basic hydrolysis (3 M KOH–MeOH–THF (1:2:2), 87%)
and the resultant alcohol 120 was oxidized to the aldehyde
(TPAP, NMO). Subsequent Horner–Wadsworth–Emmons
coupling [170] (DBU, LiCl) provided vinyl ketone 121 with
complete stereocontrol (79%, 2 steps). The last stereocenter
of fostriecin was introduced by (R)-BinalH reduction [171]
of the C5 ketone, affording 1 2 2 with high
diastereoselectivity (>20:1, 85%). Acid treatment of 122
effected a facile lactonization (92%), and a two-step
procedure through selenide 124 (NaHMDS, TMSCl,
PhSeBr, 94%; H 2 O 2 , NaHCO 3 , 94%) introduced the
unsaturation in the δ-lactone.
Oxidative removal of the PMB protecting group (96%)
and a buffered Dess-Martin oxidation of the resulting
primary alcohol set the stage for introduction of the Z,Z,E-
triene system. Wittig reaction (Ph3 PCH 2 I 2 , NaHMDS,
HMPA) of 126 afforded iodo-olefin 127 in modest yield
(61%, 2 steps) and selectivity (4:1 Z:E). It was determined
that acetonide removal was essential before introduction of
2026 Current Medicinal Chemistry, 2002, Vol. 9, No. 22 Boger et al.

4 Asym. Hyd rogenation 1 from R-M alic ac i

d 17 OH
OH O
H2 O3 PO OH 14 HO2 C BH3•Me2 S, 87%
O Swern;
18 CO2 H
8 10 13 HO
O O 5 Stille Coupling Me2 C(OMe) 2, 95%
PPh3
Me OH 2 Sharpless AD 113
3 Horner– CO2 Me
1 86%
Wadsworth–
E mmons
(DHQD)2 PHAL
OH OH I OR K2OsO2 (OH) 4
+ O K3Fe(CN) 6 OBz OH O Me2 C(OMe)2
O O O O pTsOH;
Bu3 Sn R
Me OH Zn(NO3 )2 •6H2 O
C1–C13 C14–C18 K2CO3 Me OH 81%, 2 steps
Me
MeSO2N H2 116
t
OH OH OH BuOH–H2 O

+ OHC HO2 C 92%, 95:5 anti:syn

EtO2 C CHO CO2 H
O
Me OH LiAlH4 ; R = CO2 Me (114)
(MeO)2 OP R-malic acid BzCl, Et3 N
R = CH2 OBz (115)
C1–C6 C7–C13 84%, 2 steps

Bu2 SnO ; TBSOTf

O O PMBCl, Bu4 NI O O 2,6-lutidine O O TPAP, NM O;
Me OH Me Me
OH O TBS
BzO 100 % DBU, L iCl
84% BzO RO
OH O O
117 118 OPM B OPMB
(Me O)2 P
3 M KOH R = Bz (119) (CH2 )3 CO2 Et
87% R = H (120) 79%, 2 steps

OTBS
O O O O OPMB
Me OTBS R-BinalH Me pTsOH 94%
OTBS
85%, >20:1 92% O O O NaHMD S
E tO 2C 3 EtO2 C Me O
OPMB 3 OPMB TM SCl
O 121 122 123
OH PhSeBr

OTBS OTBS OTBS

PhSe OPMB OPMB O
H2O2 Ph 3P CHI
NaHCO3 DDQ, 96% HM PA
O O O O O O O O O
Me O 94% Me O Dess–Martin Me O 61%, 2 steps
124 125 126 4:1 Z:E

T BSO I
TBSOTf,
OH OH I 2,6-lutidin e; OR 1 OR 2 I
1M H C l–M eOH TE SOTf PdCl2 (Me CN) 2
O O O O O O O
89% separation OTBDPS
Me O Me OH Me O TES
127 128 56% Z-i some r
SnBu3 80%
1M HCl R1 = TES, R2 = TBS (129)
70% R1 = H, R 2 = T BS (130)
O
PMBO P
O TBDPS OT BDPS
OH OTBS PCl3 , pyr PMBO O OTBS
PMBOH; HF–CH3 CN;
1, fostriecin
O O t O O
BuOOH, 56% Pyr, 38%
Me OTES Me O TES
131 132

Fig. (14). Imanishi total synthesis of fostriecin.

the acid-labile triene system. As such, treatment of 127 with separation of the Z-isomer (56%). Selective deprotection of
1 M HCl–MeOH provided simultaneous removal of the the C9 TES group (1 M HCl–THF–MeCN (1:3:6), –10ºC)
acetonide and TBS protecting groups. Phosphorylation of affords 130, intersecting the late stage intermediate first
the C9 alcohol required selective protection of the tertiary disclosed in the Jacobsen synthesis.
and allylic secondary alcohols. All alcohols were protected
by sequential treatment with TBSOTf (2,6-lutidine, –78 ºC) Several groups have also recently published synthetic
and TESOTf (–78 to 25 ºC) in one pot, followed by work toward fostriecin. Esumi and coworkers [172]
Fostriecin: Chemistry and Biology Current Medicinal Chemistry, 2002, Vol. 9, No. 22 2027

completed the synthesis of 8,9-acetonide dephospho- Waldmann have published the synthesis of the
fostriecin and Cossy and coworkers have detailed the (4S,5S,6S,9S,10S,11S)-isomer of cytostatin [175]. The
synthesis of a protected C1-C12 subunit of fostriecin [173]. synthesis was designed to have maximal flexibility since the
Kobayashi et al. [174] have also disclosed a study on the relative and absolute configuration of cytostatin is unknown.
dihydroxylation of a series of dienes as synthetic Installation of the chiral centers with asymmetric Evans
intermediates for fostriecin. alkylation (C6), asymmetric Evans aldol addition [176] (C4,
While this review was at the publishers, Bialy and C5, C9, and C10), and enantioselective reduction of an

O O OH M OMCl, i Pr 2N Et
OH
2 Evans' Aldol Swern; 97%
Still–Gennari 3 Evans' Aldol
O N
5 4 134, Bu2 BOTf, LiBH3OH, 78%
H2 O3 PO OH 17 Me i Me Me
1 Me Pr2 NEt; H2 O2
6 Ph Me
O O 9 11 6 Stille Coupling 133 68%, 2 steps 135
H
Asym. Me Me
Alkylation 1 4 Asym. Hydrogenation
OR OMOM 9-BBN; T BSO O MOM OH
(4S,5S,9S,10S,11S)-isomer of 66
H2 O2, NaOH Swern;

83% 134, Bu2 BOTf,

Me Me Me Me i
Pr 2NE t; H2 O2
Me
OH I 138
H2O3 PO TBSCl R = H (136)
Me
+ Im., 97%
O O Bu 3Sn C14–C18 R = T BS (137)
H
Me Me
OR OMOM OH O O Me3 Al
C1–C13
(H2NM eOMe)+Cl-
N O
O O 84%
O OR OR OR O Me Me Me
Me Ph
+ O N
TBSCl R = H (139) H Ph
Me Me Me
Ph Me Et3N, DMAP
R = TBS (140) Ph
C3–C13 1 34 86%, 3 steps
N O
B
(R )-145
n Me
TBSO OMOM OR O BuLi T BSO OMOM OMOM O TBSO OMOM OMOM O
SiMe3 borax BH3 •Me2 S
NMe 96%
95%, 2 steps
Me Me Me OM e Me Me Me TM S Me Me Me

MOM Cl R = H (141) 143 144

O
i
Pr2 NEt R = MOM (142)
92% (CF3CH2 O)2 P CO2M e
TBSO OMOM OMOM OH HO OMO M OM OM OTBDPS O OMOM OM OM OTBDPS 18-c-6,
TBDPSCl, Im.; Dess–Ma rtin KHMDS

HF•pyr, THF 93% 92%

Me Me Me RT, 40 min Me Me Me Me Me Me
146 88%, 2 steps 147 148
OFm
FmO
MO MO MOMO OT BDPS Me P O
Me i
CBr4 OH OTBDPS (FmO) 2PN Pr2 O OTBDPS
HF•pyr
tetrazole;
O O I 2, pyr, H2O O O 82%
Me Me Me OH , 82 ˚C H 95% H
Me O O Me Me Me Me
149 83% 150 151

OFm OFm Me
FmO FmO
Me P O Me P O Bu3 Sn 154
NIS, AgNO3
O OH O OH I
100% PdCl2 (CH3CN)2
O O HN NH O O
H H 62%
Me Me 63% Me Me
152 153

OFm ONa
FmO HO
P O Me P O
Me Et3N , CH3CN (1:5)
O OH Me O OH Me
+
Na Dowex resin
O O O O
H 85% H
Me Me Me Me
155 (4S,5S,9S,10S,11S)-isomer of 66

Fig. (15). Waldmann synthesis of a cytotastin isomer.

2028 Current Medicinal Chemistry, 2002, Vol. 9, No. 22
acetylenic ketone [177] (C11) allows access to every possible
isomer. The C2–C3 (Z)-double bond was formed by a Still-
Gennari coupling and the C13–C14 bond was established by
Stille coupling.
Chiral alcohol 133 was prepared by Evans asymmetric
alkylation as previously described [178] (134, NaHMDS,
allyl iodide; LiAlH4) and subjected to Swern oxidation. The
corresponding aldehyde was used in an asymmetric aldol
condensation with the dibutylboron enolate of
acyloxazolidinone 134 [179] to provide 135 in good yield
(68%, 2 steps) and with high diastereoselectivity. Trace
amounts of the undesired diastereomers were readily
separated by recrystallization. Protection of the C5 alcohol
as the MOM ether and reductive removal of the chiral
auxiliary with in situ generated LiBH3 OH [180] (LiBH4 ,
H2O) afforded alcohol 136 in 78% yield. TBS protection of
the primary alcohol (TBSCl, Im., 97%), hydroboration of
the terminal double bond (9-BBN; H2O2, NaOH; 83%) and
Swern oxidation provided the aldehyde for the second
asymmetric syn aldol condensation. As such, the C9 and
C10 stereocenters were installed with high
diastereoselectivity under the same conditions used for the
C5 and C6 chiral centers (134, Bu2BOTf, i-Pr2NEt; H2O2).
The primary TBS group was cleaved under the aldol reaction
conditions but was readily resilylated (TBSCl, Et3 N ,
DMAP; 86%, 3 steps). Conversion of acyloxazolidinone
1 4 0 to fully protected Weinreb amide 1 4 2 (Me3 A l ,
(H 2 N M e O M e ) + Cl – , 84%; MOMCl, i - Pr 2 NEt, 92%)
provided the starting material for preparation of the C11
acetylenic ketone. The desired ketone was prepared by
treatment of Weinreb amide 142 with lithium
trimethylsilylacetylide followed by borax-mediated TMS
deprotection (95%, 2 steps). After screening several reagents
for the asymmetric reduction of the C11 ketone, the best
conversion was obtained with two equivalents of (R)-145
[Parker, Corey 177]. The last stereocenter was thus
established as a single diastereomer in 96% yield.
The next stage of the synthesis required formation of the
unsaturated lactone. The propargylic alcohol was protected as
the TBDPS ether, a blocking group which would survive the
Still-Gennari olefination and acid-mediated cyclization
conditions as well as allow selective deprotection of the
primary TBS ether. Thus, treatment of the TBDPS
propargylic ether with HF·pyr (THF, 25 ºC, 40 min; 88%, 2
steps) selectively cleaved the primary TBS ether and Dess–
Martin oxidation [22] provided aldehyde 148 in 93% yield.
Condensation with the Still–Gennari phosphonate [13]
((CF3CH2O)2P(O)CH2CO2Me, 18-c-6, KHMDS) provided
Z-olefin 149 in 92% yield. Treatment of 149 with CBr4 (2-
propanol, 82 ºC) caused deprotection of the MOM ethers and
simultaneous cyclization to unsaturated lactone 150, likely
due to in situ formation of HBr [181].
Next, the C9 phosphate was installed by phosphitylation
((FmO)2PNiPr2, tetrazole) and subsequent oxidation (I2, pyr,
H 2 O; 95%). Selective deprotection of the propargylic
TBDPS ether with HF·pyr proceeded smoothly to provide
alkyne 152 in 82% yield. Reduction of the triple bond to the
(Z)-vinyl iodide proved problematic, as a variety of reagents
caused reduction of the α,β-unsaturated lactone and ring
Boger et al.
opening. After much experimentation, it was found that
conversion to the alkynyl iodide with AgNO3 followed by
reaction with in situ generated diimide [161] ((KO2CN) 2,
HOAc, 2-propanol/dioxane) afforded vinyl iodide (Z)-153
(63%). The sensitive Z,Z,E-triene was then formed by Stille
coupling with Z,E-dienyl stannane 154 without additional
phosphine [182] ([PdCl2 (CH 3 CN) 2 ], DMF/THF, 62%).
Treatment of the phosphate triester 155 with excess
triethylamine induced β-elimination of the fluorenylmethyl
phosphate ester [183] and the (4S ,5S ,9S ,10S ,11S ) -
diastereomer of cytostatin was obtained in 85% yield. It was
not reported whether this isomer correlates with the natural
product. However, this isomer of 66 was established to be
an effective inhibitor of PP2A (IC50 = 33 nM) comparable
or more potent than the natural product itself (IC50 = 200
nM).
Since this review was originally submitted, a number of
studies continue to strengthen the expectation that selective
inhibition of PP2A among the phosphatases is the target to
address for the treatment of cancer. PP2A is the protein
phosphatase that fostriecin is uniquely selective against.
PP2A has one of the most highly expressed catalytic
subunits and it combines to form a large repertoire of
heteromultimeric holoenzymes composed of the catalytic
subunit (PP2Ac), a structural A subunit, and at least three
distinct B subunits encoded by at least 13 genes and
multiple splice variants. Recent studies make it clear that a
major function of these regulatory subunits is to target the
PP2A holoenzyme to distinct intracellular locations,
signaling complexes, and substrates, including those
involved in regulation of the cell cycle [184, 185]. The
breadth of PP2A function reported since this review was
submitted extends its role from regulating the initiation of
DNA replication to regulating cell death by apoptosis and it
is this latter role that may be most closely linked to a
potential treatment of cancer. Several recent studies implicate
PP2A as the phosphatase that associates with several
proteins involved in apoptosis including Bcl-2, caspase-3,
and the adenovirus E4orf4 protein [186]. In particular, PP2A
is emerging as a target with powerful antiapoptotic activity
that is mediated by a regulating subunit (R5/B56). The
indirect evidence suggests a role for PP2A in promoting cell
survival through inhibition of apoptosis [187, 188].
ACKNOWLEDGEMENTS
We gratefully acknowledge the financial support of the
National Institute of Health (CA93456), the Skaggs Institute
for Chemical Biology, the award of a NIH postdoctoral
fellowship (C.-M.G.), and a Bristol-Myers Squibb
fellowship (D.R.S.). D.R.S. is a Skaggs Fellow.
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