Novel aminophenyl benzamide-type histone deacetylase inhibitors with enhanced potency and selectivity

Article abstract of DOI:10.1021/jm701079h

Significant effort is being made to understand the role of HDAC isotypes in human cancer and to develop antitumor agents with better therapeutic windows. A part of this endeavor was the exploration of the 14 ? internal cavity adjacent to the enzyme catalytic site, which led to the design and synthesis of compound 4 with the unusual bis-(aryl)-type pharmacophore. SAR studies around this lead resulted in optimization to potent, selective, nonhydroxamic acid HDAC inhibitors.

Full text of DOI:10.1021/jm701079h

J. Med. Chem. 2007, 50, 5543-5546
5543
Novel Aminophenyl Benzamide-Type Histone
Deacetylase Inhibitors with Enhanced Potency
and Selectivity
Oscar M. Moradei,* Tammy C. Mallais, Sylvie Frechette,
Isabelle Paquin, Pierre E. Tessier, Silvana M. Leit,
Marielle Fournel, Claire Bonfils,
Marie-Claude Trachy-Bourget, Jianhong Liu,
Theresa P. Yan, Ai-Hua Lu, Jubrail Rahil, James Wang,
Sylvain Lefebvre, Zuomei Li, Arkadii F. Vaisburg, and
Jeffrey M. Besterman
MethylGene Inc, 7220 Frederick-Banting,
Figure 1.
Montre´al, QC H4S 2A1, Canada
domain. X-ray structural information on the zinc-dependent
HDACs is available for two bacterial deacetylases, histone
deacetylase-like protein (HDLP)8 and histone deacetylase-like
aminohydrolase (FB188 HDAH),9 as well as human HDAC-7
and 8.10 In both bacteria and human HDACs, X-ray crystal-
lography revealed a tube-like channel which leads to the active
site in which the zinc ion is coordinated to side chains of two
aspartate and one histidine residues. The second coordination
sphere includes tyrosine and two histidine-aspartate dyads,
which coordinate the hydrolytic water molecule (in some class
II enzymes, that tyrosine is replaced by a histidine, and in one
dyad, asparagine replaces aspartate).
Immediately below the active site, there is an internal cavity
(14 Å cavity, Figure 2), originally described as being flexible
and lined primarily with hydrophobic residues. The function of
this cavity is not clear. This internal cavity was described for
HDLP, FB188 HDAH, and hHDAC-8. Obviously, it is of
considerable interest for inhibitor design as this space can
potentially be used for additional binding interactions.11
As typical zinc hydrolases, most of the previously reported
HDAC inhibitors are hydroxamic acids,12 exemplified by 1.13
However, hydroxamate HDAC inhibitors, with the exception
of a few compounds such as tubacin,14 are nonselective
inhibitors targeting both HDAC classes I and II. We identified
nonhydroxamate HDAC inhibitors featuring the 2-aminoben-
zanilide functionality.15 Unlike the hydroxamates, this class of
inhibitors, typified by our clinical compound 2, demonstrated
very interesting isoform selectivity targeting primarily HDAC-1
and 2, with weaker activity against 3 and 11. Compound 2 is
being tested in multiple phase II single-agent clinical trials, and
objective clinical activity has been documented in acute
myelogenous leukemia (AML), myelodysplastic syndrome
(MDS), diffuse large B-cell lymphoma (DLBCL), follicular
lymphoma, and Hodgkin’s Lymphoma (HL). Other compounds
have been identified which belong to the same class, such as 3,
currently in phase II clinical studies.
ReceiVed August 31, 2007
Abstract: Significant effort is being made to understand the role of
HDAC isotypes in human cancer and to develop antitumor agents with
better therapeutic windows. A part of this endeavor was the exploration
of the 14 Å internal cavity adjacent to the enzyme catalytic site, which
led to the design and synthesis of compound 4 with the unusual bis-
(aryl)-type pharmacophore. SAR studies around this lead resulted in
optimization to potent, selective, nonhydroxamic acid HDAC inhibitors.
Reversible histone acetylation is regulated by the opposing
activities of histone acetyltransferases (HATsa) and histone
deacetylases (HDACs). It plays an important role in the dynamic
regulation of the chromatin structure and is essential for
biological processes. The acetylation status of histones is crucial
in modulating gene expression and cell fate,1 and its misregu-
lation is involved in the development of several cancers.2 In
general, histone deacetylation is correlated with transcriptional
repression, whereas histone hyperacetylation facilitates gene
expression.3 Thus, inhibitors of HDAC activity induce histone
hyperacetylation, leading to the transcriptional activation of
suppressed genes, which are associated with cell cycle arrest,
differentiation, or apoptosis in tumor cells. In recent years,
inhibition of HDACs has emerged as a valid strategy to reverse
aberrant epigenetic changes associated with cancer, and several
classes of HDAC inhibitors have been found to have potent
and specific anticancer activities in preclinical and clinical
studies.4 This is exemplified by compound 1 (Figure 1), which
was launched in the U.S. market in October 2006 for the
treatment of cutaneous T-cell lymphoma.
Histone deacetylases constitute an ancient family of enzymes
that can be divided into four groups denoted as classes I-IV.5
Class I enzymes (HDACs 1, 2, 3, and 8) are ubiquitously
expressed, predominantly nuclear, and mainly function as
transcriptional co-repressors. Class II HDACs (4, 5, 6, 7, 9, and
10) are selectively distributed among tissues, suggesting distinct
functions in cellular differentiation and developmental pro-
cesses.6 Sirtuins, or class III, are structurally unrelated NAD-
dependent deacetylase enzymes.7 Finally, HDAC-11, another
recently identified member of the HDAC family, bears low
similarities with HDAC class I and class II and therefore is
classified as a class IV. Classes I, II, and IV are closely related
zinc-dependent enzymes bearing a highly conserved catalytic
We built a homology model of HDAC-1 around the published
coordinates of HDLP.8 Compound 3 was docked into the active
site, with the presumption that the carbonyl-oxygen and the
ortho-NH2 group coordinate to the active site zinc in a seven-
membered ring. Examination of the docked structure revealed
the presence of the cavity, mentioned above in the HDLP crystal
structure, in fortuitous alignment with the para position of the
pharmacophoric NH2 (Figure 2). The structure suggested that
substituents at the para position would fit snugly into this
hydrophobic 14 Å internal cavity.
* To whom correspondence should be addressed. E-mail: moradeio@
methylgene.com. Phone: 514-337-3333, ext. 257.
a Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacety-
lase; HDLP, histone deacetylase-like protein; HDAH, histone deacetylase-
like aminohydrolase; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)-
phosphoniumhexafluorophosphate; AMC, aminomethylcoumarin.
Therefore, the strategy for the further development of isoform-
selective HDAC inhibitors was substitution of the aforemen-
10.1021/jm701079h CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/17/2007

5544 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Letters
Figure 2. Human HDAC-1 homology model with the inhibitor 3 docked in the active site pocket, with its NH2 group placed near the Zn atom
(purple sphere) and bound to His 140 and 141 through hydrogen bonds. The empty cavity below the anilide ring is part of the 14 Å pocket. Key
amino acid residues are depicted in bold style.
Table 1. Inhibition of Recombinant HDAC-1 and HDAC-2
(Representative IC50 Values)
Scheme 1
tioned para position with appropriate moieties. The presence
of Met30 and Cys151 residues lining the walls of the internal
cavity suggested placing of a thienyl substituent in order to
benefit from potential sulfur interactions.16 Thus, lead compound
4 was first synthesized.17 As expected, the para-thienyl substitu-
tion picked up additional beneficial interactions with the enzyme,
which resulted in 27-fold and 5-fold improvement in potency
against recombinant human HDAC-1 and HDAC-2, respec-
tively, compared with its parent analogue 3. In addition to the
gain in potency, we discovered an interesting change in the
specificity profile. This class of novel benzamide inhibitors lost
its activity against HDAC-3 and bore exclusively HDAC-1 and
-2 inhibition. To our knowledge, this is the first HDAC inhibitor
exhibiting such high isoform selectivity among class I enzymes.
These encouraging results led to further attempts to character-
ize the mentioned cavity. Therefore, a series of compounds
bearing this novel 2-amino-5-arylanilide-based benzamide phar-
macophore was synthesized according to Scheme 1 (exemplified
by compound 4).
Bis(aryl) core 6 was prepared by Suzuki coupling of
2-thiophene boronic acid with bromide 5.18 Masked dianiline 6
was then reacted with acid chloride 719 to furnish nitroamide 8.
Reduction of the nitro group rendered 2-amino-5-thienylanilide
4. Compounds 10-17 (Table 1) were synthesized similarly;
however, in some cases, nitroaniline 6 was reacted with the
corresponding acid by BOP20 ((benzotriazol-1-yloxy)tris(di-
methylamino)phosphonium hexafluorophosphate)-promoted con-
densation in the presence of sodium hydride; compounds 16
and 17 were prepared starting from the appropriate 2-aminophe-
nol, while the syntheses of reference compounds 9 and 16 have
been previously described.15
Consistent with the prediction above, adding an aryl group
at the para position to the amine group significantly enhanced
HDAC inhibitory activity against HDAC-1 and HDAC-2, up
to 10-fold. Substituents like phenyl, 2- and 3-thienyl, and 2-furyl
are well tolerated in this position, suggesting that hydrophobic
rather than sulfur interactions in the 14 Å cavity are responsible
for the increase in potency. Interestingly, replacing the NH2
group in 9 and 10 by OH showed the same trend (compounds
16 and 17, respectively). However, a bulkier benzo[b]thienyl
(14) and substitution in the meta position to the NH2 group (15)
were not tolerated.

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5545
Table 2
a Inhibition of recombinant class I HDACs. b Inhibition in whole-cell enzyme, in 293T cells transfected with vector alone. c MTT cytotoxicity/proliferation
of human colon cancer HCT116 cells or human mammary epithelial cells HMEC (representative data).
Figure 3. Dose-dependent inhibition of whole-cell HDAC inhibitory activity by selected thienyl benzamides, their parents (in a or b), and 24 (c)
in human colon HCT116 cells; arrows indicate the maximum % of inhibition of total cellular HDAC activity at 60 µM, which is also shown in (d).
Importantly, the effect of the para substitution on the
biological profile in the smaller HDAC inhibitors (Table 2) was
even more dramatic. For example, 2-thienyl derivatives 19, 21,
and 23 (Table 2) showed increased potency by 20- to 40-fold
relative to the corresponding parent compounds 18 (Pfizer,
CI-994), 20, and 22. Consistent with their increased potency
against HDAC enzymes in vitro, thienyl compounds exhibited
better antiproliferative activity than that of their parent
benzamides against various human cancer cells. Moreover, there
was no concomitant increase in cytotoxicity against normal
human epithelial cells. Also, these compounds inhibited total
HDAC activity in intact cells (293T), with demonstrated
selectivity to class I HDACs. Furthermore, mechanistic studies
are consistent with a competitive mode of inhibition (data not
shown).
Compounds 18-23 were synthesized by BOP-promoted
condensation between the corresponding commercial carboxylic
acids and either 1,2-phenylenediamine or with compound 6,
following the same procedure described in Scheme 1. All
benzamide-type compounds were screened in vitro against
recombinant human HDAC isotypes 1, 2, 3, and 8 (Table 2),
as well as 4, 5, 6, and 7, exhibiting inhibition lower than 50%
when screened at a 20 µM concentration against these class II
HDACs (data not shown). Whole-cell HDAC inhibitory activity
was analyzed in 293T cells. The evaluation of in vitro
antiproliferative activities was performed using 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
against a human cancer cell line (HCT116) and in human
mammary epithelial cells (HMEC).
Isotype selectivity of HDAC inhibitors in intact cells can be
estimated from the amount of inhibited HDAC activity relative
to the total by using the pan-substrate Boc-Lys(acetyl)-AMC.
For example, the hydroxamate pan-inhibitor Novartis’ 24 was
able to inhibit 100% of the total cellular HDAC activity (Figure
3c). On the other hand, benzamide inhibitors, which are known
to be class I specific, inhibit about 90% of the total activity. In
contrast, thienyl derivatives bring about only 75-80% inhibition
of the total cellular HDAC activity (Figure 3a,b,d). This supports
the results obtained from the recombinant enzymatic screening,
which clearly indicate the selectivity of the para-substituted
aminophenyl benzamides as highly specific inhibitors of HDAC-1
and HDAC-2.
In conclusion, the present study demonstrated the rational
design of potent and selective HDAC inhibitors from available

5546 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Letters
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analogues is available. This material is available free of charge via
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JM701079H