Amide analogues of TSA: Synthesis, binding mode analysis and HDAC inhibition

Article abstract of DOI:10.1016/S0960-894X(03)00284-1

The synthesis of new amide type histone deacetylase inhibitors is described, having an (R)-methyl substituent and a diene or saturated structure of the chain linking the hydroxamic acid and dimethylaminobenzoyl groups. The saturated compound shows stronge

Full text of DOI:10.1016/S0960-894X(03)00284-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1861–1864
Amide Analogues of TSA: Synthesis, Binding Mode Analysis
and HDAC Inhibition
K. Van Ommeslaeghe,a G. Elaut,c V. Brecx,a P. Papeleu,c
K. Iterbeke,a P. Geerlings,b D. Tourwea,* and V. Rogiersc
aDepartment of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
bDepartment of General Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
cDepartment of Toxicology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
Received 19 December 2002; revised 31 January 2003; accepted 17 March 2003
Abstract—The synthesis of new amide type histone deacetylase inhibitors is described, having an (R)-methyl substituent and a diene
or saturated structure of the chain linking the hydroxamic acid and dimethylaminobenzoyl groups. The saturated compound shows
stronger HDAC inhibition than the unsaturated analogue. Molecular modeling suggests that the flexibility of the linker chain is
important for an optimal orientation of the dimethylaminobenzoyl group in the enzyme.
# 2003 Elsevier Science Ltd. All rights reserved.
Histone deacetylase (HDAC) inhibitors have recently
attracted considerable interest because of their thera-
peutic potential for the treatment of cell proliferative
diseases.1,2
configuration of the chiral center has been shown to be
important since the enantiomer (S)-TSA is inactive.3,9
We now report our results on the synthesis and HDAC
inhibition of amide analogues having a (R)-methyl
group and a diene unsaturated chain 9 or a saturated
chain 10. The binding mode of these new analogues to
HDLP has been investigated by molecular modeling.
The natural product Trichostatin A (TSA) 1 has been
identified as a potent and specific HDAC inhibitor3 and
its phase 1 biotransformation has been elucidated.4
Structurally simpler TSA analogues have been described
such as SAHA 2,5 the amide analogues 36 and aryl-
ketones 4.7,8 These analogues are generally less potent
than TSA. A major structural difference is the presence
in TSA of an unsaturated chain linking the important
hydroxamate function and the aromatic ring, and the
presence of an (R)-methyl group. The absolute
A synthetic route towards the unsaturated amide is
outlined in Scheme 1. The (R)-stereochemistry of the
chiral center originated from d-alanine.
Reduction of the N,O-dimethylhydroxamate 5 to alde-
hyde 6,10 followed by a Wittig–Horner olefination using
*Corresponding author. Tel.: +32-2-629-3295; fax: +32-2-629-3304; e-mail: datourwe@vub.ac.be
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00284-1

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Scheme 1. Conditions: (a) LiAlH4; (b) (EtO)2POCH2CH¼CHCOOEt, LiOH; (c) TFA; (d) 4-dimethylaminobenzoic acid, TBTU, NEt3; (e) 5 N
HCl, acetone, reflux, overnight; (f) NH2OBn HCl, TBTU, N-methylmorpholine; (g) HFliq, anisole, 0 ꢀC, 1 h; (h) H2, Pd/C.
.
triethyl-4-phosphocrotonate11,12 afforded the unsatu-
rated ester 7 with high (>95%) E stereoselectivity, in
63% yield calculated from 5. The enantiomeric purity
(90% ee) was determined after Boc-deprotection, and
derivatisation with Marfey’s reagent,13 followed by LC–
MS analysis. The peak of the minor isomer was identi-
fied after synthesis of the reference epimeric compound
starting from l-alanine.
obtained after treatment with anhydrous liquid hydrogen
fluoride (76%) and HPLC purification.
HDAC inhibition by analogues 9 and 10 was deter-
mined using [3H]-acetate prelabeled chicken reticulocyte
core histones as enzyme substrate and HDAC from rat
hepatocyte lysate as enzyme source.14,15 The liberated
[3H]-acetate was quantified by scintillation counting and
the results are shown in Table 1.
After removal of the Boc-nitrogen protection, 4-dime-
thylaminobenzoic acid was coupled to the amine. In
contrast to previous reports,14 the use of BOP-Cl as
coupling reagent resulted in low yields (44%). Using
TBTU with a preactivation time of the dimethylamino-
benzoic acid of 2 h, gave a 84% yield of 8. The ester was
hydrolysed in acidic conditions (70%), and the resulting
carboxylic acid was converted to the O-benzyl protected
hydroxamate to using TBTU as coupling reagent
(95%). All attempts to selectivity remove the O-benzyl
protection by hydrogenolysis failed, but resulted in the
saturated analogue 10. The hydroxamic acid 9 was
As a reference compound the amide 3 (n=2), which is
one of the most potent analogues of this type of com-
pounds,16 was used. The inhibition data indicate that
the introduction of unsaturation in the chain resulting
in 9 has a negative effect on the potency of the com-
pound. Saturation of the chain to 10 restores the inhi-
bitory capacity of 10 to a level similar to that of
reference compound 3 (n=2). Compound 10 has an
identical backbone structure as 3 (n=2), but carries an
(R)-methyl substituent as in TSA 1. These results there-
fore indicate a negligible effect of this substituent on the
potency. The decreased potency of (S)-TSA 1, should
therefore be ascribed to an unfavourable interaction of
the methyl group in this enantiomer with the enzyme.
Table 1. HDAC inhibition data
Compd
Structure
HDAC
IC50 (nM)
In order to study the differences in potency of these
compounds, their binding mode in the catalytic site of
the HDLP protein was studied by molecular modeling.
We have developed a model of the active site of HDLP,
based on the published structure of the TSA/HDLP
complex.17 The model consists essentially of all amino
acid residues within a radius of 4.5 A around TSA. This
model allowed the ab initio minimization of TSA 1 in
this active site model at the HF 3-21G(*) level resulting
in an excellent agreement between the calculated bind-
ing conformation and the observed TSA/HDLP com-
plex.18 Since the residues that make up the active site are
conserved in the HDAC family,17 the results of this
model can be extrapolated to other HDAC types,
including rat HDAC.19 The same active site model was
used to model the interactions with the analogues 9 and
10, using the MMFF94 forcefield. The hydroxamic acid
was fixed at the position found in the X-ray structure,
and a systematic conformational search was conducted
a
1 (TSA)
12ꢁ1
3 (n=2)
152ꢁ15
12,187ꢁ1384
359ꢁ92
9
10
aData from ref 20.

K. Van Ommeslaeghe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1861–1864
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Figure 1. Superposition of the low energy conformations of 9 (blue and green) and TSA (red) in the active site model of HDLP; (a) side view,
(b) top view.
Figure 2. Superposition of the low energy conformations of 10 (blue and green) and TSA (red) in the active site model of HDLP; (a) side view,
(b) top view.
over all rotatable bonds. The energy of each generated
conformation was calculated. An in-house routine was
used to find all local minima in the resulting energy
landscape, and the conformations found by this proce-
dure within 20 kcal/mol from the absolute minimum
were further minimized. After minimization, two low
energy conformations within 4 kcal/mol were found for
analogue 9 bound in the active site. Compared to the
bound conformation of TSA (red), the unsaturated
chain has an almost identical orientation in the tube-like
cleft in the enzyme (Fig. 1a). However it appears that
the amide bond region of both conformers of 9 is sig-
nificantly displaced from the corresponding ketone part
in TSA. Consequently, the dimethylaminobenzoyl
group also shifted (Fig. 1b), apparently resulting in less
efficient interactions with the enzyme’s surface. For the
saturated analogue 10, two relevant low energy con-
formers were obtained within 4 kcal/mol. In this case, as
could be expected, considerable more deviations in the
orientation of the saturated chain versus the unsatu-
rated chain in TSA were observed (Fig. 2a). In contrast,
the more flexible saturated chain allows an orientation
of the dimethylaminobenzamide part corresponding
more closely to that in TSA (Fig. 2b). Therefore, in
these structures, it is apparent that the optimal orienta-
tion of the ‘capping’ region of HDAC inhibitors con-
tributes significantly to their potency, and that the
contribution of p–p interactions in the tube-like cleft, if
important, is difficult to evaluate. Similarly, the effect of
the introduction of an (R)-methyl substituent in the
amide analogues is minimal, either due to an imperfect
orientation compared to that in TSA, or due to a lack of
interaction with the enzyme.
In conclusion, the introduction of the (R)-methyl group
and of the diene function, which are present in TSA 1,
into the amide bond analogues does not result in an
increased HDAC inhibitory potency. The optimal
interaction of the aromatic end group with the enzyme
is crucial for this potency, and is influenced by differ-
ences in the flexibility of the spacer chain. Although
these conclusions are derived from a histon deacetylase-
like protein, the similarity in the active site residues in
rat and human HDAC’s allows speculation that they
are also valid for these types and the results are there-
fore valuable for the further design of potent analogues.
Acknowledgements
This work was supported by grants from the Fund for
Scientific Research (FWO-Vlaanderen, Belgium), and
the Research Council (OZR) of the Vrije Universiteit
Brussel, Belgium.

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12. Bonadies, F.; Cardilli, A.; Lattanzi, A.; Orelli, L. R.;
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