Synthesis and biological evaluation of cytostatin analogues.

Article abstract of DOI:10.1039/b305308n

Analogues of the serine/threonine phosphatase 2A inhibitor cytostatin were synthesized and evaluated as inhibitors of various phosphatases in order to establish a basic structure-activity relationship (SAR) of the natural product.

Full text of DOI:10.1039/b305308n

Synthesis and biological evaluation of cytostatin analogues†
ab
Laurent Bialy* and Herbert Waldmannab
a
Max-Planck-Institut für molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Str. 11, 44227
Dortmund, Germany. E-mail: herbert.waldmann@mpi-dortmund.mpg.de; Fax: 49 231 133 2499; Tel: 49
2
31 133 2400
b
Universität Dortmund, Fachbereich 3, Organische Chemie, 44221 Dortmund, Germany
Received (in Cambridge, UK) 12th May 2003, Accepted 3rd June 2003
First published as an Advance Article on the web 20th June 2003
Analogues of the serine/threonine phosphatase 2A inhibitor
cytostatin were synthesized and evaluated as inhibitors of
various phosphatases in order to establish a basic structure–
activity relationship (SAR) of the natural product.
truncated analogue was synthesized starting from alcohol 3. The
alcohol was protected as the methoxymethyl (MOM) ether and
after cleavage of the tert-butyldiphenylsilyl (TBDPS) group,
oxidation to the corresponding aldehyde was carried out.
8
Subsequent Still–Gennari olefination yielded (Z)-configured
Protein phosphorylation and dephosphorylation reactions are
employed by living organisms for the regulation of innumerable
cellular processes, and aberrant protein phosphorylation con-
tributes to the development of many human diseases inducing
unsaturated ester 5 (Scheme 2). 5 was converted to the lactone
with hot aqueous HCl. Treatment with phosphoramidite
9
(FmO)
2
PN(iPr)2 (Fm = 9-fluorenylmethyl) and subsequent
oxidation with meta-chloroperbenzoic acid (m-CPBA) gave the
desired phosphotriester which was deprotected with an excess
1
cancer and diabetes. Phosphorylation states are governed by
protein kinases (PKs), which catalyze protein phosphorylation,
and protein phosphatases (PPs), which are responsible for
dephosphorylation. Naturally occurring PP inhibitors have
widely been used to antagonize PP action in biology experi-
2
ments. In the light of this experience and the fact that potent
and selective inhibitors of this enzyme class still are in short
supply the development of small-molecule inhibitors of PPs is
3
emerging as a very rapidly growing area of investigation.
4a
Cytostatin 1, isolated from a Streptomyces strain, inhibits
the serine/threonine phosphatase 2A at submicromolar concen-
tration. It belongs to a family of natural products related to
fostriecin,5 which all inhibit PP2A with an unprecedented
selectivity compared to other natural products like okadaic acid,
4b
6
calyculin or the microcystins. Given the importance of PP
inhibitors it is of major interest to medicinal chemistry and
clinical biology to gain insight into the structural parameters
that underlie the biological activity of these natural products.
7
Based on the total synthesis of cytostatin (Scheme 1), we
now describe the synthesis of structural analogues of the
molecule and their evaluation as phosphatase inhibitors.
Dephosphorylated cytostatin is not active as a phosphatase
inhibitor.4b To establish whether the lactone and phosphate
moieties alone would suffice to confer activity, a corresponding
Scheme 2 Synthesis of cytostatin analogues: (i) 10 eq. MOMCl, 13 eq.
(
iPr)
room temp., 15 h, 90% over 2 steps; (iii) 1.5 eq. Dess–Martin periodinane,
1.5 eq. NaHCO , CH Cl , room temp., 90 min; (iv) 2 eq. (CF CH O)
P(O)CH C(O)Me, 4 eq. 18-crown-6, 1.5 eq. KHMDS, THF, 278 °C, 35
min, aldehyde, 278 °C, 3 h, 80% over 2 steps; (v) 1 M HCl, H O, THF, 15
h, 60 °C, 88%; (vi) 4 eq. (iPr )NP(OFm) , 3 eq. tetrazole, CH Cl , 270 min,
0 eq. m-CPBA, 278 °C, 0 °C, 90 min; 88%; (vii) NEt /CH CN 1/4.8 (v/v),
room temp., 18 h, 75%; (viii) 6.1 eq. (b-CE) PN(iPr) , 4.45 eq. tetrazole,
CH CN, 0 °C to room temp., 105 min; 6.1 eq. I , THF/pyridine/H O 7/2/1,
room temp., 5 min, quantitative; (ix) 70% HF.pyridine/THF, 17/83, room
temp., 24 h, 84%; (x) NEt /CH CN 1/4.6 (v/v), room temp., 15 h, 85% (7),
8% (9), 49% (10), quantitative (12, 14), 92% (16);: (xi) 3 eq.
iPr) NP(OFm) , 2.7 eq. tetrazole, CH CN/CH Cl 5/4 (v/v), room temp.,
30 min, 3 eq. I , THF/pyridine/H O 7/2/1 (v/v), 5 min, 95%; (xii)
2 2 2
NEt, CH Cl , 0 °C, 1 h, room temp., 13 h; (ii) 1.2 eq. TBAF, THF,
1
3
2
2
3
2
2
-
2
2
2
2
2
2
1
3
3
2
2
3
2
2
3
3
9
(
3
2
2
3
2
2
2
2
HF.pyridine/THF 1/4.75 (v/v), room temp., 24 h, then 1/2.4, room temp., 8
h, 82%; (xiii) Ac O, pyridine, cat. DMAP, 1 h, 60%; (xiv) 1.5 eq. N-
iodosuccinimide (NIS), 0.15 eq. AgNO , DMF, 90 min, quantitative; (xv)
Scheme 1 Synthesis of (4S,5S,6S,9S,10S,11S)-cytostatin (1).
2
3
+
2
2 +
†
Electronic supplementary information (ESI) available: experimental
1.73 eq. K ( OOCNNNCOO ) K, 3.47 eq. HOAc, 2-propanol/dioxane
details. See http://www.rsc.org/suppdata/cc/b3/b305308n/
11/1 (v/v), 870 min, 63%.
1
872
CHEM. COMMUN., 2003, 1872–1873
This journal is © The Royal Society of Chemistry 2003

View Article Online
of triethylamine in dry acetonitrile to yield the phosphate 6 as
monotriethylammonium salt. The advanced intermediate 4 was
used to generate more complex analogues. The mono-protected
phosphate 7 could readily be synthesized by making use of the
less base-labile b-cyanoethyl protecting group. After phosphit-
2 2 2 2
ylation with the phosphoramidite (NCCH CH O) PN(iPr) ,
oxidation with I /pyridine/water, cleavage of the TBDPS group
2
with HF–pyridine and treatment with an excess of triethylamine
in dry acetonitrile, the phosphodiester 7 was obtained in high
yield. Also the 9-fluorenylmethyl protected phosphotriester 8
was cleanly deprotected to alkyne 9 under basic conditions. In
order to assess the importance of the C-11–OH group for
biological activity acetylated compound 10 was synthesized in
Fig. 1 SAR of (4S,5S,6S,9S,10S,11S)-cytostatin.
1
1. The triene moiety is not essential for PP2A inhibition. The
presence of a (Z)-configured double bond with a hydrophobic
group attached (here an iodine) seems to be sufficient for high
PP2A-inhibitory activity.
2
steps in satisfactory yield from alcohol 8. The iodoalkyne 11
and iodoalkene 13, both intermediates in the synthesis of the
natural product, were deprotected under standard conditions to
the phosphates 12 and 14. The saturated lactone 15 was obtained
as a by-product during the diimide reduction of iodoalkyne 11
to iodoalkene 13. The corresponding deprotected phosphate 16
was obtained by treatment with triethylamine and was con-
sidered useful to evaluate the relevance of the double-bond for
biological activity.
Notably protein phosphatase 1 (PP1) was not inhibited by any
of the cytostatin analogues in a p-NPP based assay identical to
the PP2A assay. This demonstrates that variation of the
structure of the natural product can be carried out without
compromising the high selectivity described for the natural
products of the fostriecin class. Furthermore VHR, PTP1B and
CD45 were also not inhibited by any of the analogues. Clinical
phase I trials of fostriecin have been stopped due to impurities
in the natural product samples.15 Most probably the triene
moiety in fostriecin and cytostatin is responsible for the
instability of the natural products upon storage. Thus, the
finding that it may be replaced by simpler structural elements
without loss of biological activity is of particular importance for
the design of more stable but still active analogues.16
This research was supported by the Fonds der Chemischen
Industrie. L. B. is grateful to the Fonds der Chemischen
Industrie for a scholarship.
The cytostatin analogues were evaluated by means of in vitro
inhibition assays of the serine–threonine phosphatases of type
2
A (PP2A) and 1 (PP1). These are of particular interest because
cytostatin has been described as a highly selective PP2A, but
not PP1, inhibitor.4d In addition, the inhibition of protein
tyrosine phosphatases PTP1B (which is considered as a
potential target for diabetes therapy ) and CD45 (a positive
regulator of T-cell activation and therefore a potential target for
treatment of autoimmune diseases and suppression of graft
rejection ), and the dual-specifity phosphatase VHR (which
dephosphorylates ERK, a member of the ras signal transduction
pathway ) was examined. The enzymatic activity was deter-
10
11
12
Notes and references
mined by hydrolysis of para-nitrophenyl phosphate in standard
‡ The assay was performed using the commercially available BIOMOL
GREEN™ CD45 Tyrosine Phosphatase Assay Kit.
13a
13a
13b
13b
buffers for PP2A
1
,
PP1,
VHR,
PTP1B,
and of a
1
2
T. Hunter, Cell, 2000, 100, 113.
(a) K. Hinterding, D. Alonzo-Díaz and H. Waldmann, Angew. Chem.
Int. Ed., 1998, 37, 668; (b) J. E. Sheppeck II, C.-M. Gauss and A. R.
Chamberlin, Bioorg. Med. Chem., 1997, 5, 1739.
See for instance: J. S. Lazo, D. C. Aslan, E. C. Southwick, K. A. Cooley,
A. P. Ducruet, B. Joo, A. Vogt and P. Wipf, J. Med. Chem., 2001, 44,
3
4
042.
acetylated derivative 10 is totally inactive (entry 5), while the
saturated lactone 16 and the phosphodiester 7 show very weak
activities with IC50 values in the high micromolecular range
4 (a) M. Amemiya, M. Ueno, M. Osono, T. Masuda, N. Kinoshita, C.
Nishida, M. Hamada, M. Ishizuka and T. Takeuchi, J. Antibiot., 1994,
47, 536; (b) M. Kawada, M. Amemiya, M. Ishizuka and T. Takeuchi,
Biochim. Biophys. Acta, 1999, 1452, 209.
(
entries 3, 8). From these results, a basic structure–activity
5
review: D. S. Lewy, C.-M. Gauss, D. R. Soenen and D. L. Boger, Curr.
Med. Chem., 2002, 9, 2005and references cited therein.
J. E. Sheppeck, II, C.-M. Gauss and A. R. Chamberlin, Bioorg. Med.
Chem., 1997, 5, 1739.
relationship can be derived (Fig. 1). The unsaturated lactone is
necessary for the biological activity of the natural product. It is
prudent to speculate that covalent modification of the enzyme
may take place, for example by nucleophilic attack of a cysteine
residue in a Michael-type reaction.14 Furthermore, the phos-
phate group must be fully deprotected, suggesting that a very
tight interaction of the phosphate with the enzyme is required
for inhibition. The C-11-hydroxy group seems to be essential
for activity, as suggested by the lack of inhibition by the
truncated compound 6 (entry 2) and the acetylated compound
6
7
8
L. Bialy and H. Waldmann, Angew. Chem. Int. Ed., 2002, 41, 1748.
W. C. Still and C. Gennari, Tetrahedron Lett., 1983, 41, 4405.
9 Y. Watanabe, T. Nakamura and H. Mitsumoto, Tetrahedron Lett., 1997,
38, 7407.
10 A. Cheng, N. Dubé, F. Gu and M. L. Tremblay, Eur. J. Biochem., 2002,
2
69, 1050.
1
1 A. I. Lazarovitz, S. Poppema, Z. Zhang, M. Khandaker, C. E. Le Feuvre,
S. K. Singhal, B. M. Garcia, N. Ogasa, A. M. Jevnikar and M. H. White,
Nature, 1996, 380, 717.
Table 1 IC50 values (in µM) for inhibition of different phosphatases with
cytostatin analogues
12 T. Ishibashi, D. P. Bottaro, A. Chan, T. Miki and S. A. Aaronson, Proc.
Natl. Acad. Sci. USA, 1992, 89, 12170.
1
3 (a) A. Takai and G. Mieskies, Biochem. J., 1991, 275, 233; X. Huang,
M. R. Swingle and R. E. Honkanen, in Methods in Enzymology, Vol.
Entry Compound PP2A
PP1
VHR
PTP1B CD45
3
15, (Ed.: K. Palczewski), Academic Press, San Diego, p. 589; (b) T.
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
> 25a
> 100
1
2
3
4
5
6
7
8
a
1
6
7
0.033 ± 0.003
> 100
42 ± 7
> 25
> 25
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 25
> 100
> 100
> 100
> 100
> 100
> 100
> 100
Hamaguchi, A. Masuda, T. Morino and H. Osada, Chem. Biol., 1997, 4,
279.
a
a
a
a
a
a
a
a
a
> 100
> 100
> 100
> 100
b
—
14 The possible role of a cysteine residue in the PP2A inhibition of
fostriecin has been discussed: D. R. H. Evans and J. A. Simon, FEBS
Letters, 2001, 498, 110.
15 R. S. de Jong, N. H. Mulder, D. R. A. Uges, D. Th. Sleijfer, F. J. P.
Höppener, H. J. M. Groen, P. H. B. Willemse, W. T. A. van der Graaf
and E. G. E. de Vries, Brit. J. Canc., 1999, 79, 882.
9
0.37 ± 0.05
> 100a
a
b
10
12
14
16
> 100
—
0.079 ± 0.009 > 100
0.039 ± 0.004 > 100
ca. 100
> 100a
a
> 100
b
> 100
—
Highest concentration investigated. b Not investigated.
16 In this respect we note that cytostatin derived inhibitors 9, 12 and 14 are
significantly more stable upon storage that the natural product.
CHEM. COMMUN., 2003, 1872–1873
1873