Figure 2. Structures of synthetic inhibitors compared with the
known inhibitor, SAHA,3a with the structural motifs required for
HDAC inhibition.2a
Figure 1. Left: Transition state proposed for human HDAC1.3b
Right: Model for binding of phosphonamidate 1.
to Zn.3b This suggested that phosphorus-based TS analogues
could act as potent HDAC inhibitors. To investigate the
analogies between HDAC and Zn protease TSs, phospho-
namidate, phosphonate, and phosphinate SAHA analogues
1-3 (Figure 2) were synthesized and tested as HDAC
inhibitors.
The structural motifs that are significant for inhibitory
activity from structure-activity relationship studies2a and
observed in the X-ray structure3b are (1) a hydrophobic
capping group binding to the outer rim of the tunnel, (2) a
spacer interacting with the hydrophobic walls of the tunnel,
and (3) a functional group serving to chelate the metal cation
in the active site (Figure 2).
The syntheses of phosphonamidate 1, phosphonate 2, and
phosphinate 3 are outlined in Scheme 1. Each synthesis began
with aniline reacting with a six-carbon acid via the acyl
chloride to form anilides 4-6. N-Carbobenzyloxy-6-amino-
hexanoic acid was the starting material for phosphonamidate
1; the monomethyl ester of pimelic acid was used for
phosphonate 2, and 6-bromo-hexanoic acid was used for
phosphinate 3.
The anilide 4 was deprotected by catalytic hydrogenation
to afford the free amine 7, which was coupled with methyl
phosphonic acid methyl ester 9 using EDC to give the
phosphonamidate methyl ester 10. Phosphonamidates are
known to be labile under acidic conditions.6e It is known
that Li+ as a counterion stabilizes phosphonamidates.6b,f To
avoid hydrolysis of phosphonamidates 10 and 1, saponifica-
tion followed by neutralization was monitored by 31P NMR.
The best results were achieved using 15 equiv of 1.5 M LiOH
in MeOH or CH3CN for ester hydrolysis and careful
neutralization to pH 7.1. The lithium salt 1 was stable as a
solid at room temperature under anhydrous conditions. It was
used in the bioassays without further purification.
human HDAC isoforms and, thus, the mechanism of
deacetylation may be conserved as well.1c,3b
Both hydroxamic acid and phosphoramidate motifs are
found in known inhibitors of zinc proteases such as ther-
molysin and carboxypeptidase A. For example, thermolysin
is inhibited by both Leu-NHOH (Ki ) 190 µM) and 2-O3P-
Leu-NH2 (Ki ) 1.3 µM).4 Leu-NHOH binds “backwards”
in the active site, with the side chain in the P1′ site; thus,
the binding contribution due to the leucine side chain is
approximately the same for both inhibitors.5 The anionic form
of the hydroxamic acid (RNOH-) binds to thermolysin,5 and
in both cases, the bidentate anion is coordinated to the zinc.
Therefore, the phosphoramidate is a better TS analogue of
the Zn protease reaction than the hydroxamic acid.
Phosphorus-containing compounds are among the most
potent known inhibitors of metalloproteases.6 Bartlett and
Marlowe demonstrated that phosphonamidates are transition-
state analogue inhibitors of thermolysin.6c The phosphorus
center is tetrahedral with heteroatoms appropriately posi-
tioned to mimic the tetrahedral TS of amide bond hydrolysis
(Figure 1). The phosphorus-heteroatom bonds are longer
than ground-state carbon-heteroatom bonds and thus able
to mimic the longer bonds of the TS.6c
The mechanism of HDAC deacetylation, simply an amide
bond hydrolysis, was proposed to have certain analogies to
zinc protease mechanisms, including a tetrahedral TS bound
(4) (a) Nishino, N.; Powers, J. C. Biochemistry 1978, 17, 2846-2850.
(b) Kam, C.-M.; Nishino, N.; Powers, J. C. Biochemistry 1979, 18, 3032-
3038.
(5) Holmes, M. A.; Matthews, B. W. Biochemistry 1981, 20, 6912-
6920.
(6) (a) Nishino, N.; Powers, J. C. Biochemistry 1979, 18, 4340-4347.
(b) Jacobsen, N. E.; Bartlett, P. A. J. Am. Chem. Soc. 1981, 103, 654-657.
(c) Bartlett, P. A.; Marlowe, C. K. Biochemistry 1983, 22, 4618-4624. (d)
Bartlett, P. A.; Marlowe, C. K. Biochemistry 1987, 26, 8553-8561. (e)
Hanson, J. E.; Kaplan, A. P.; Bartlett, P. A. Biochemistry 1989, 28, 6294-
6305. (f) Morgan, B. P.; Scholtz, J. M.; Ballinger, M. D.; Zipkin, I. D.;
Bartlett, P. A. J. Am. Chem. Soc. 1991, 113, 297-307. (g) Bertenshaw, S.
R.; Rogers, R. S.; Stern, M. K.; Norman, B. H.; Moore, W. M.; Jerome, G.
M.; Branson, L. M.; McDonald, J. F.; McMahon, E. G.; Palomo, M. A. J.
Med. Chem. 1993, 36, 173-176. (h) Yiallouros, I.; Vassiliou, S.; Yiotakis,
A.; Zwilling, R.; Stocker, W.; Dive, V. Biochem. J. 1998, 331, 375-379.
(i) Gall, A. L.; Ruff, M.; Kannan, R.; Cuniasse, P.; Yiotakis, A.; Dive, V.;
Rio, M. C.; Basset, P.; Moras, D. J. Mol. Biol. 2001, 307, 577-586.
The phosphonate precursor, alcohol 8, was obtained by
reduction of the methyl ester 5 with LiAlH4 at -10 °C.
Alcohol 8 was then reacted with methylphosphonic acid 9
using PyBOP as the activating reagent and DIEA as base.7
(7) Campagne, J.-M.; Coste, J.; Jouin, P. J. Org. Chem. 1995, 60, 5214-
5223.
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Org. Lett., Vol. 5, No. 17, 2003