Total Synthesis and Reassessment of the Phosphatase-Inhibitory Activity of the Antitumor Agent TMC-69-6H

Article abstract of DOI:10.1002/anie.200352268

The unexpected selectivity proflie of I (17R)-and (17S)-TMC-69-6H (see formula) suggests that N-hydroxypyridone derivatives constitute a promising new lead structure in the search for selective phosphatase inhibitors. An unprecedented Pd-catalyzed C-C bond formation to a pyranone derivative was a key step in the synthesis of enantiopure (17R)-and (17S)-TMC-69-6H.

Full text of DOI:10.1002/anie.200352268

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Chemie
Phosphatase Inhibitors
tases. Although pyridone 1 is inherently labile (it degrades
within 2 weeks at 08C), hydrogenation of its triene moiety
results in significantly improved stability and even further
enhanced potency.[4] Importantly, TMC-69-6H (2) thus
formed also exhibits a remarkable efficacy against P388
murine leukemia and B16 melanoma in nude mice.[4] Outlined
belowis the first total synthesis of the 17 R and 17S isomers of
2 in enantiomerically pure form. The absolute stereochemis-
try at this remote stereogenic center in the natural product 1
and its derivative 2 has not yet been established.[4]
Total Synthesis and Reassessment of the
Phosphatase-Inhibitory Activity of the
Antitumor Agent TMC-69-6H**
Alois Fürstner,* Fabian Feyen, Heino Prinz, and
Herbert Waldmann
The family of the Cdc25 dual specific protein phosphatases is
critically involved in cell-cycle control.[1] Their physiological
substrates are cyclin-dependent kinases, which trigger key
transitions in the process of eukaryotic cellular division.
Therefore the homologous Cdc25 enzymes exert crucial
regulatory functions at the crossroads between cellular
proliferation, cell cycle arrest, and apoptosis. Their oncogenic
properties together with the fact that Cdc25A and -B are
overexpressed in many human tumors render these isoen-
zymes molecular targets of utmost interest in the quest for
anticancer drugs.[1–3]
The readily available pyranone 4[5] served as a key
building block en route to 2 (Scheme 2). Its enone moiety
allowed the stereoselective installation of the methyl group by
Despite this widely recognized fact, only a rather limited
number of small molecules are presently known that might
qualify as lead structures in the search for selective inhibitors
of Cdc25 phosphatases.[3] One such compound is TMC-69 (1),
a 2-pyridone derivative isolated from the culture broth of the
fungus Chrysosporium sp. TC 1068 (Scheme 1).[4] According
to the literature, this compound is distinguished by excellent
activity (IC50 values in the lowmicromolar range) and by a
surprising selectivity for Cdc25A and -B over other phospha-
Scheme 2. Synthesis of (17R)-2: [{(allyl)PdCl}2] (0.5%), 10 (1.5%),
Et3N, DMF, 65%, 96% ee; b) TBSCl, Et3N, 18 h, 78%; c) Me2CuLi,
THF, À708C; d) LiHMDS, (R)-15, 69% (over both steps); e) H2, Pd/C,
EtOH; f) TBAF, THF, 69% (over two steps); g) HN(SiMe3)2, TMSCl
cat., reflux; h) 1) [(pyridine)MoO5(hmpa)], CH2Cl2; 2) saturated aque-
ous EDTA-Na, EtOAc, 61% (over three steps). DMF=N,N-dimethyl-
formamide, TBS=tert-butyldimethylsilyl, HMDS=hexamethyldisil-
azide, TBAF=tetrabutylammonium fluoride, TMS=trimethylsilyl,
HMPA=hexamethylphosphortriamide, EDTA=ethylenediaminetetra-
acetate.
Scheme 1. TMC-69 and TMC-69-6H. Retrosynthetic analysis for 2.
1,4-addition, followed by attachment of the lateral chain by
olefination of the remaining ketone. More importantly,
however, the allylic leaving group in 4 opened several options
for the introduction of the heteroaromatic ring. Rather than
taking recourse to known oxo-carbenium cation chemistry,[6]
[*] Prof. A. Fürstner, Dipl.-Chem. F. Feyen
Max-Planck-Institut für Kohlenforschung
45470 Mülheim an der Ruhr (Germany)
Fax: (+49)208-306-2994
E-mail: fuerstner@mpi-muelheim.mpg.de
À
we decided to rely on palladium-catalyzed C C bond
Priv.-Doz. H. Prinz, Prof. H. Waldmann
Max-Planck-Institut für Molekulare Physiologie
44227 Dortmund (Germany)
formations because they allowcontrol of the stereochemical
course of the envisaged allylation process more accurately
(Scheme 2).
[**] We thank Dr. J. Kohno, Tanabe Seiyaku Co. Ltd, Japan, for providing
an authentic sample of TMC-69-6H for comparison. Financial
support by the DFG (Leibniz award) and the Fonds der Chemischen
Industrie is acknowledged with gratitude.
Surprisingly, though, palladium-catalyzed transformations
of 6-hydroxy-6H-pyran-3-one derivatives are scarce and seem
to be restricted to reactions with O nucleophiles.[7] To the best
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of our knowledge, no Pd-catalyzed C-arylation of such a
steps (97% ee). Subsequent hydrogenation of 8 over Pd/C
followed by cleavage of the TBS groups furnished a separable
mixture of the desired product 9 and its C10 epimer in 69%
yield (over two steps). The axial orientation of the alkyl chain
on the tetrahydropyran ring in 9 was evident from an analysis
of the pertinent coupling constants and NOESY data
(Scheme 4).[15]
substrate with a phenol derivative has been described
previously. Gratifyingly, however, (Æ )-4 reacts smoothly
and regioselectively at the enol site of 4-hydroxypyridone
3[8] in the presence of [Pd(PPh3)4] catalyst and Et3N in DMF
to give the tricyclic product (Æ )-5 in 89% yield. This novel
À
palladium-catalyzed C C bond formation is accompanied by
a spontaneous 1,4-addition of the 4-OH group to the enone
entity of the emerging product. Not only are we unaware of
any precedence for this transformation, but this result must
also be seen in the context of previous reports, which show
that 2-pyridones tend to react with allylic substrates at their N
atom in the presence of palladium catalysts.[9]
Attempts to perform this reaction enantioselectively with
rac-4 as the substrate and the chiral diphosphane 10[10] as
Scheme 4. Schematic representation of compound 9 with characteris-
tic NOESY data. Pertinent coupling constants: 3JH7,H8 =10.4 Hz,
3
3JH10,H11ax =2.5 Hz, JH10,H11eq 4 1 Hz (TMC-69-6H numbering).
For the final N-oxidation to the desired hydroxamic acid
derivative,[16] compound 9 was heated at reflux with hexa-
methyl disilazane and the resulting bis(silyl ether) was treated
with the peroxo complex [(pyridine)MoO5(hmpa)].[17] Aque-
ous work-up with EDTA to sequester all metal cations then
completed the first total synthesis of (17R)-TMC-69-6H
((17R)-2).
As mentioned above, no secured information concerning
the absolute stereochemistry of the remote stereogenic center
on the lateral chain of compound 2 derived from natural
sources was available. Therefore the same sequence of
reactions was repeated with ketone (À )-5 and the antipodal
sulfone (S)-15 to give (17S)-2 in similar overall yield. The
ligand to Pd were unrewarding (~ 30% ee). However, the use
of 10 in combination with enantiomerically enriched (S)-4
(81% ee), which is easily prepared on a multigram scale by a
lipase-catalyzed dynamic resolution,[11] served our purpose
very well, and delivered the tricyclic ketone (À )-5 in good
yield and with excellent enantiomeric purity (96% ee)
(Scheme 2).
The cyclic ether moiety of 5 acts as a temporary protecting
group for the enone which can be released by a retro-Michael
reaction upon treatment with oxophilic reagents in the
presence of a base.[12] Optimal results were obtained with
TBSCl in combination with Et3N. Exposure of the resulting
disilyl ether derivative 6 to Me2CuLi afforded the somewhat
labile trans-disubstituted ketone 7, which was subjected to a
Julia-Kocienski olefination reaction[13] with the lithiated
sulfone (R)-15 (prepared from (S)-citronellene 11 as shown
in Scheme 3).[14] HPLC analyses of alkene 8 (E/Z 1:1) on
chiral columns and comparison with the racemic series
confirmed that no racemization had occurred during these
diastereomeric compounds (17R)-2 and (17S)-2 thus
obtained, however, are virtually indistinguishable by NMR
spectroscopy and match the literature data reported for
TMC-69-6H very well.[4] Even a direct comparison of their
spectra recorded at 600 MHz with those of an authentic
sample did not allowthe rigorous assignment of the absolute
stereochemistry of TMC-69-6H at that site.[18]
Since this excellent match leaves no doubt about the
integrity of the synthetic materials, we were surprised to find
that their phosphatase inhibitory activity deviates significantly
from the reported profile both in terms of potency and
selectivity. In contrast to what has been reported, the
compounds prepared by total synthesis as well as an authentic
sample of 2 all turned out to be only rather weak inhibitors of
Cdc25A (IC50 > 30 mm) in our assay. Instead, they exhibit
promising activities against the tyrosine protein phosphatase
PTB1B, the dual specific phosphatase VHR, and the serine/
Scheme 3. Reagents and conditions: a) O3, CH2Cl2, À788C; then
Me2S, 98%; b) (EtO)2P(O)CH2COOMe, NaH, THF, À788C, 87%;
c) DIBAL-H, Et2O, 91%; d) H2, Pd/C, 88%; e) 1-phenyl-1H-tetrazole-5-
thiol, DEAD, PPh3, THF, 68%; f) (NH4)6Mo7O24·4H2O (0.1 equiv),
H2O2 (10 equiv), EtOH, 93%. DIBAL-H=diisobutylaluminum hydride,
DEAD=diethyl azodicarboxylate.
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Table 1: IC50 values [mm] of authentic and synthetic TMC-69-6H and its
Keywords: heterocycles · natural products · palladium ·
phosphatases · structure–activity relationships
.
immediate precursors against different phosphatases.[a]
Compound
Cdc25A
PTP1B
VHR
PP1
authentic 2
(17R)-2
(17S)-2
550
3.2Æ1.6
4Æ2.0
7.0Æ3.5
6Æ3
6Æ3
45Æ23
32Æ16
550
550
8.5Æ4.5
8Æ4
3.5Æ1.7
11Æ5.5
23Æ11
5.5Æ3
9Æ5
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(17S)-9
30Æ5
(17S)-8b[b]
11Æ5.5
32Æ16
[a] The enzymatic activity was determined by hydrolysis of para-nitro-
phenyl phosphate in standard buffers for PTP1B,[22a] VHR,[22b] and PP1.[22b]
[b] The isomer with an E configuration between the CH2O group of the
tetrahydropyran and C11 was tested.
threonine phosphatase PP1 (Table 1). PTP1B is a key
negative regulator of insulin-receptor activity, and PTP1B-
inhibitors are expected to enhance insulin sensitivity and act
as effective therapeutics for the treatment of Type II diabetes,
insulin resistance, and obesity. The vaccinia VH1-related
phosphatase VHR is a physiological regulator of extracellular
regulated kinases of the MAP (mitogen-activated protein)
kinase family and influences signaling through the MAP
kinase pathway. PP1 is a major eukaryotic phosphatase that
regulates diverse cellular processes such as signal trans-
duction, cell-cycle progression, protein synthesis, muscle
contraction, carbohydrate metabolism, and transcription.
These phosphatases have been subject to intense research
activities aimed at the development of inhibitors for bio-
logical studies and drug development.[19–22] Thus, PP1 inhib-
itors are expected to be potent anticancer drugs and have
been used clinically for the treatment of cancers.[21] In
particular, PTB1B is currently a major target of medicinal
chemistry research in the pharmaceutical industry.[19]
Notably, TMC-69-6H incorporates a structural framework
not present in any of the PTP1B and PP1 inhibitors developed
so far. Thus, it provides a unique and unprecedented lead
structure for the development of a newseries of selective
phosphatase inhibitors. Its proven activity in cellular assays
(see above) renders such research activities particularly
promising. Our preliminary results also seem to indicate
that the presence of the N-OH group in the heterocycle,
though not strictly required for phosphatase inhibition,
enhances the potency of such compounds (compare, for
instance, the IC50 values determined for (17S)-2 with the data
for the corresponding amide (17S)-9).
Although we are unable to reconcile these findings with
the literature reports concerning the physiological activity of
TMC-69-6H,[4] the excellent reproducibility of our results, the
internal control against authentic 2, the consistency within the
individual series, and the established validity of our Cdc25
assay[3b] leave no room for interpretation. These data also
make clear that N-hydroxy-2-pyridone derivatives constitute
a promising newclass of selective phosphatase inhibitors and
certainly deserve further studies to establish pertinent
structure–activity relationships and assess their potency in
more detail.[23,24] Synthesis-driven studies along these lines are
underway and will be reported in due course.
[14] For the regioselective ozonolysis of citronellene and related
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[15] The corresponding coupling constants of the minor product
formed in the hydrogenation step are in full accord with an
equatorial orientation of its alkyl side chain: 3JH7,H8 = 10.0 Hz,
3JH10,H11ax = 11.3 Hz, 3JH10,H11eq = 2.6 Hz.
Received: July 1, 2003 [Z52268]
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[18] Unfortunately, the optical rotations did also not allowus to make
a final decision. The optical rotation reported for TMC-69-6H is
[a]25 = + 143.4 (c = 0.48, MeOH);[4] the optical rotation of the
authentic sample of 2 made available to us, however, was lower
than this reported value. When corrected for the residual water
in the sample, we record an [a]20 value of ꢀ + 99 (c = 0.18,
MeOH). The data of our analytically pure synthetic compounds
are as follows: (17R)-2: [a]20 = + 102.3 (c = 0.60, MeOH); (17S)-
2: [a]20 = + 88.6 (c = 0.50, MeOH).
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