Diphenylmethylene hydroxamic acids as selective class IIa histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.010

We have identified a series of diphenylmethylene hydroxamic acids as novel and selective HDAC class IIa inhibitors. The original hit, N-hydroxy-2,2-diphenylacetamide (6), has sub-micromolar class IIa HDAC inhibitory activity, while the rigidified oxygen a

Full text of DOI:10.1016/j.bmcl.2009.08.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5684–5688
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diphenylmethylene hydroxamic acids as selective class IIa histone
deacetylase inhibitors
a
b
c
Pierre Tessier a, David V. Smil a, Amal Wahhab a, , Silvana Leit , Jubrail Rahil , Zuomei Li ,
*
Robert Déziel a, Jeffrey M. Besterman c
a MethylGene Inc., Department of Medicinal Chemistry, 7220 Frederick-Banting, Montréal, Québec, Canada H4S 2A1
b MethylGene Inc., Department of Lead Discovery, 7220 Frederick-Banting, Montréal, Québec, Canada H4S 2A1
c MethylGene Inc., Department of Molecular Biology, 7220 Frederick-Banting, Montréal, Québec, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
We have identified a series of diphenylmethylene hydroxamic acids as novel and selective HDAC class IIa
inhibitors. The original hit, N-hydroxy-2,2-diphenylacetamide (6), has sub-micromolar class IIa HDAC
inhibitory activity, while the rigidified oxygen analogue, N-hydroxy-9H-xanthene-9-carboxamide (13),
Received 4 July 2009
Revised 3 August 2009
Accepted 4 August 2009
Available online 7 August 2009
is slightly more selective for HDAC7 with an IC50 of 0.05
lM. Substitution of 6 allows for the modulation
of selectivity and potency amongst the class IIa HDAC isotypes.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HDAC inhibitors
HDAC class IIa selective inhibitors
HDAC4 inhibitors
HDAC5 inhibitors
HDAC7 inhibitors
The dynamic and reversible acetylation of lysine residues on the
N-terminal tails of histones is tightly regulated and controlled by
histone acetyltransferases (HATs) and deacetylses (HDACs).1 Based
on their specificity and catalytic mechanisms, eukaryotic HDACs
are divided into two main groups; group I, the zinc-dependent
amidohydrolases, and group II, NAD+-dependent enzymes (also
designated as HDAC class III, or sirtuins 1–7). Group I consists of
eleven subtypes, HDAC1-11, sharing close catalytic site homology.
Group I is further subdivided into class I (HDAC1, 2, 3 and 8), class
IIa (HDAC4, 5, 7, and 9), class IIb (HDAC 6, and 10), and class IV
(HDAC11), the last of which shares properties with both class I
and class II enzymes.2
HDAC class I enzymes are ubiquitously expressed, and are pre-
dominantly nuclear enzymes. They are reported to regulate differ-
entiation, proliferation, cell cycle, protein turnover, and apoptosis.3
Inhibitors of HDAC class I enzymes have demonstrated benefits in
cancer therapy either as approved drugs (SAHA, or ZolinzaÒ/Vori-
nostat),4a or as agents in different stages of clinical development,
such as MGCD0103,4b and LBH589 amongst others.4c–e
with other proteins.2c,5c HDAC6, a member of the class IIb enzymes,
deacetylates
a
-tubulin,5d however the cellular substrates of the
class IIa HDACs are not well characterized.5b In addition, the func-
tion of HDAC class IIa is not as well defined, but they may play an
essential role as transcriptional regulators of various developmen-
tal and differentiation processes.2c
A few reports have appeared in the literature describing selec-
tive HDAC6 inhibitors6 such as 1,6a–c 2,6d 6e and 46f (Fig. 1). How-
3
ever, it was not until the report of Jones and co-workers7a,b and the
identification of the unnatural trifluoroacetamide lysine substrate,
Boc-L-Lys-MCA, that a reliable classification of HDAC class II inhib-
itors was feasible. Consequently, the trifluoromethyl ketone class
of compounds, such as 5 (Fig. 1) have emerged as selective class
II HDAC inhibitors.7c–e
In order to understand the function of the different HDAC en-
zymes, there is still a dire need for class and isoform selective HDAC
inhibitors. Despite the reports of selective HDAC class II and HDAC
class IIb selective inhibitors, to the best of our knowledge, there is
no report of HDAC class IIa selective inhibitors. Our initial strategy
for the discovery of HDAC class IIa selective inhibitors involved the
screening of a variety of small hydroxamic acids with diverse topol-
ogy utilizing acetyl-Boc-lysine for detection of class I inhibitory
HDAC class II enzymes are expressed in a limited number of cell
types, and either shuttle between the nucleus and cytoplasm (class
IIa), or are mainly cytoplasmic (class IIb).5a,b The N-terminus of
class II HDACs is involved in gene regulation through its interaction
activity, and Boc-Lys-(e-trifluormethylacetyl)-AMC for the detec-
tion of class IIa inhibitory activity. From this exercise, lead com-
pound 6 (Fig. 2) was determined to have class IIa activity in the
high nanomolar range. Although reported to have weak activity for
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: wahhaba@methylgene.com (A. Wahhab).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.010

P. Tessier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5684–5688
5685
O
S
NHOH
O
O
HN
OH
O
HN
N
H
N
3
O
O
S
O
S
O
H
HO
N
N
NH2
N
1
N
H
S
O2
4
CF3
O
O
N
N
N
H
N
NHOH
BocHN
Ph
O
S
N
O
O
O
5
2
Figure 1. Examples of HDAC6 selective inhibitors.
OH
O
HOHN
O
NH
HO
N
HN
LAQ
6
O
N
N
H
N
OH
N
H
N
NH2
H
H
O
N
N
O
MGCD0103
SAHA (Zolinza(R), Vorinostat)
Figure 2. Examples of HDAC inhibitors with different selectivity profile.
HDAC8,8 this compound was previously unknown as an HDAC class
IIa inhibitor. Using both the acetyl-Boc-Lysine and Boc-Lys-( -trif-
ester and subsequently transformed to the hydroxamic acid, com-
pound 11, using established protocols employing potassium
hydroxide and hydroxylamine.9
Rigidification of the diphenyl system was investigated as we
considered analogs of 6 with the phenyl rings linked through the
ortho position by a carbon (12), oxygen (13), or nitrogen atom
(14), Table 3. The carbon and oxygen acids were commercially
available, while the nitrogen analog required synthesis from acri-
dine-9-carboxylic acid as detailed in Scheme 1.
e
luormethylacetyl)-AMC substrates, an HDAC Class I and IIa activity
profile was generated comparing lead 6 to several known HDAC
inhibitors with different selectivity profiles (Fig. 2, and Table 1).
The unique aspect of 6 appears to be the bifurcated diphenyl
moiety, and it was decided that initial analogs should focus on
three main areas of interest; namely, the modification of the site
of bifurcation, rigidification of the diphenyl system and substitu-
tion on the phenyl rings.
Various commercial acids were available with a degree of vari-
ation around the branch point of compound 6. These acids were
either converted directly to the hydroxamic acid by coupling with
hydroxylamine using BOP reagent in pyridine, compounds 7, 8, 9,
and 10 (see Table 2), or were first converted to the methyl or ethyl
Table 2
SAR around the branch point of lead 6 on HDAC class IIa isoforms
(n)
NHOH
R1 R2
O
Table 1
Compd
n
R1
R2
HDAC class IIa isoform IC50
(l
M)a,b
Selectivity of lead 6 on HDAC isoformsa
4
5
7
MG
HDAC isoforms IC50
(lM)
6
0
0
0
1
0
0
Ph
H
H
H
H
Me
H
0.75
>10
>10
>10
4.5
0.14
>10
>10
>10
1.2
0.39
>10
>10
>10
4.8
1b
2b
4c
5c
7c
7c
8
Me
CH2Ph
Ph
Ph
Cy
6d
LAQ
MGCD0103
SAHA
>10
0.008
0.2
6.6
0.75
0.50
>10
>10
0.14
0.19
>10
0.39
2.24
>10
>10
9d
10
11
0.01
0.18
0.20
>10
>10
>10
0.14
1.30
a
a
Values are average of at least two determinations. Standard deviations were
Values are average of at least two determinations. Standard deviations were
within 30% of the IC50 values.
within 30% of the IC50 values.
b
b
Compounds showed no inhibition against HDAC1 and 2 when screened at
The substrate used is acetyl-Boc-Lysine.
c
20 lM inhibitor concentration.
The substrate used is Boc-Lys-(
e
-trifluormethylacetyl)-AMC.
Compound 6 was not active against HDAC6 (IC50 >10 M); HDAC8 IC50 = 66
as reported in Ref. 8.
c
d
Ref. 15.
Ref. 16.
l
lM,
d

5686
P. Tessier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5684–5688
Table 3
HO
O
OH
SAR of rigid analogs of lead 6 on HDAC class IIa isoformsa
H2SO4
O
HCOOH
OH
N
F
F
F
F
20
21
H
X
BOP,
NH2OH-HCl
THF/MeOH
HOHN
O
Compd
X
HDAC class IIa isoforms IC50 (lM)
F
F
4
5
7
19
6
12
13
14
H
CH2
O
0.75
2.32
0.25
1.91
0.14
1.46
0.11
0.47
0.39
0.26
0.05
0.35
Scheme 2. Synthesis of p-F-substituted analogue 19.
NMe
O
SI
O
O
aValues are average of at least two determinations. Standard deviations were within
30% of the IC50 values.
F
F
BF3-OEt2
Et2O
F
F
NaH,THF
55oC
23
24
O
OMe
O
OH
O
F
OH
O
H
CF3SO3Me
DCM
MeI, K2CO3
DMF
Jones'
F
F
F
F
Reagent
BOP, NH2OH-HCl
TFH/MeOH
N
N
N
acetone,
0°C
16
15
26
25
OMe
OMe
O
O
O
NHOH
NH2OH, KOH
THF,MeOH
Zn, NH4Cl
F
EtOH,80°C
+
22
Scheme 3. Synthesis of m-F-substituted analogue 22.
N
Me
Me
-
18
17
CF3SO3
O
NHOH
For p-methylsulfonamide substituted analogue 27, the proce-
dure began with nitration of methyl 2,2-diphenylacetate 28,
employing a solution of fuming nitric acid11 to exclusively give
the para substitution product 29. The nitro groups of 29 were then
reduced with palladium on carbon under hydrogen to produce ani-
line 30, which was reacted with methyl sulfonyl chloride in pyri-
dine to yield 31. Finally, ester 31 was converted to 27 with
potassium hydroxide and hydroxyl amine (Scheme 4). Compounds
32 and 33 were accessible from the corresponding commercial
acids which were converted to their respective esters and were
then taken to the desired hydroxamic acids (Table 4).
All compounds prepared for this study were profiled against
HDAC class IIa, class IIb, and class I enzymes (data not shown).12
SAR around the branch point of 6 confirmed our initial belief that
it was this moiety that gave the molecule its unique HDAC class
IIa activity/selectivity profile (Table 2). Replacement of one of the
phenyl rings with a methyl (7) or ethyl (not shown) group reduced
the activity against HDAC class IIa enzymes. In addition, displace-
N
Me
14
Scheme 1. Synthesis of the rigid nitrogen analogue 14.
Initial methylation of acid 15 was effected through the use of
methyl iodide to give ester 16. Quartenization of the nitrogen did
not occur as expected, and it was necessary to use methyl triflate
to afford 17, which upon reduction of the central ring with zinc
and ammonium chloride gave the dihydroacridine 18. Finally, ester
18 was transformed with hydroxylamine and potassium hydroxide
to hydroxamic acid 14 (Scheme 1).
Next, the effect of substitution on the phenyl rings of 6 on the
HDAC activity and selectivity was investigated. In order to avoid
the introduction of a chiral center, these initial substitutions were
limited to those that would afford the molecule a plane of symme-
try. Somewhat surprisingly, considering the apparent simplicity of
the scaffold of 6, the literature demonstrated a relative paucity of
procedures on how to build the bifurcated system effectively. As
a result, it was necessary to employ a disparate set of conditions
to construct the starting materials that were not commercial.
For the synthesis of the p-fluoroanalogue 19, the difluro-benz-
hydrol 20 was commercial and could be converted to the corre-
sponding acid 21 employing a mixture of concentrated sulfuric
acid and formic acid.10 Acid 21 was then transformed to hydroxa-
mic acid 19 via BOP coupling with hydroxylamine HCl (Scheme 2).
The meta-fluoro substituted isomer 22 required a somewhat
more involved procedure. Initially, the m-difluro benzophenone
23 was treated with trimethylsulfoxonium iodide while heating
to afford epoxide 24. Epoxide 24 was quickly converted to the cor-
responding aldehyde 25 via a 5 min treatment with BF3ÁOEt2,
which was in turn transformed to acid 26 through the use of Jone’s
reagent. Hydroxamic acid 22 was obtained utilizing the usual BOP
and hydroxylamine hydrochloride coupling (Scheme 3).
O
OMe
O
OMe
H2/Pd/C
MeOH
fuming HNO3
O2N
NO2
OMe
28
29
O
OMe
O
MeSO2Cl
O2
S
O2
Pyridine
O
S
H2N
NH2
N
H
N
30
31
H
NHOH
KOH/NH2OH
THF/MeOH
O2
S
O2
S
N
H
N
27
H
Scheme 4. Synthesis of p-substituted sulfonamide 27.

P. Tessier et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5684–5688
5687
Schuetz et al.13 studying the crystal structure of cdHDAC7 en-
zyme, reported that the catalytic domain of class IIa HDACs have
a unique active site topology and an enlarged active site pocket.
Our SAR data is in agreement with the crystal structure report
and point to the presence of a hydrophobic pocket in the active site
of class IIa HDACs that is absent in class I and class IIb enzymes.
This pocket accommodates binding of compounds with aromatic
groups (compare 6 and 11) suggesting that pi-stacking may play
a role in conferring the activity and selectivity of these class IIa
inhibitors.
Table 4
SAR of substituted analogs of lead 6 on HDAC class IIa isoformsa
n(0,1)
R2
R1
O
OH
N
H
R2
R1
n(0,1)
We also tested our inhibitors for their ability to inhibit HDAC
activity within intact cells. The inhibitory activity of these com-
pounds was measured in 293T cells using acetyl-Boc-Lysine sub-
Compd
R1
R2
n
HDAC class IIa isoforms IC50 (lM)
4
5
7
strate for HDAC class I and Boc-Lys-(
e-trifluormethylacetyl)-AMC
6
19
22
27
32
33
H
F
H
H
H
F
H
CH2
H
0
0
0
0
1
0
0.75
1.10
0.7
>10
0.21
0.96
0.14
0.28
0.09
>10
0.11
0.21
0.39
1.76
0.44
>10
0.11
1.4
substrate for HDAC class IIa (Table 5).14
For all examples, inhibition of HDAC class I cellular activity is
non-existent as would be expected. Moreover, cellular class IIa
inhibitory activity tracks well versus inhibition of individual re-
combinant class IIa enzymes, suggesting that these inhibitors are
cell permeable. Compound 32, which had the best recombinant
class IIa enzymatic inhibitory activity, also showed a fivefold
improvement in the class IIa intracellular inhibitory activity versus
NHSO2Me
O
Cl
a
Values are average of at least two determinations. Standard deviations were
within 30% of the IC50 values.
ment of the branched site with a methylene unit (8 and 9), or
replacement of the phenyl ring with a cyclohexyl group (10) com-
pletely destroyed activity. However, addition of a methyl group to
the branch point (11) appears to be tolerated, albeit with an
approximate one log decrease in activity.
Rigidification of 6, while tolerated somewhat, was generally
detrimental or not beneficial to activity. This effect was not uni-
form across the class IIa isoforms and did lead to a modification
of the selectivity profile. Linking of the phenyl rings with either a
carbon (12) or nitrogen (14) had the effect of reducing HDAC 4
and 5 inhibitory activities by several fold, while HDAC7 activity
was almost unchanged. Alternatively, linking with oxygen (13) left
HDAC 4 and 5 inhibitory activities basically unchanged while giv-
ing a modest sixfold increase to potency against HDAC7 (Table 3).
While compound 13 was slightly superior to our initial lead (6), the
scaffold of 6 is more versatile to build the SAR and was used to gen-
erate more analogs.
Substitution on the phenyl rings appears to be fairly well-toler-
ated and can be used to modulate HDAC class IIa selectivity
(Table 4). When fluoro groups are added at the para position (19),
HDAC 4 and 5 inhibitory activities were slightly decreased while
HDAC 7 inhibitory activity was reduced by fivefold. The same trend
was observed when the bulkier chloro groups were used instead of
fluoro (33); but when this group was changed to the more polar
methyl sulfonamide, compound 27, all activity was lost. As the fluo-
rines are shifted to the meta position (22), the activity was improved
over the para, but when compared to the parent compound only a
very modest enhancement for HDAC 5 inhibitory potency was ob-
served. Some improvement in the HDAC class IIa inhibitory activity
was observed, more noticeably against HDAC 4 and 7, when the phe-
nyl groups were replaced with dihydrobenzofuran moieties (32).
the lead 6, with IC50 values of 4.6 and 0.9 lM, respectively. Further
optimization of this class of inhibitors is ongoing.
In conclusion, we have identified diphenylmethylene hydroxa-
mic acids as a new class of selective class IIa HDAC inhibitors.
The original hit, N-hydroxy-2,2-diphenylacetamide (6) has class
IIa HDAC activity in the sub-micromolar range. While the rigidified
oxygen analogue, N-hydroxy-9H-xanthene-9-carboxamide (13), is
slightly more selective for HDAC7 with an IC50 of 0.05 lM. Substi-
tution of 6 is tolerated and allows for the modulation of selectivity
amongst the HDAC class IIa isotypes. These inhibitors demon-
strated cellular HDAC class II inhibitory activity in accordance with
their enzymatic potencies.
Acknowledgments
The authors are grateful for the important support, contribution
and assistance of Mr. Sylvain Lefebvre (protein expression and
purification), Mr. Aihua Lu, Ms. Anne-Marie Lemieux, and Ms. An-
drea J. Petschner (assay development and substrate studies), Mr.
Martin Allan (synthesis of substrates), Mr. Yves A. Chantigny (syn-
thesis of compound 12), Dr. Celia Dominguez and the CHDI Foun-
dation, Inc.
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Compd
293T Class I IC50
l
Mb
293T Class IIa IC50 l
Mc
6
10
13
19
32
27
>50
>50
>50
>50
>50
>50
4.6
16
3.0
3.5
0.9
>50
a
Values are average of at least two determinations. Standard deviations were
within 30% of the IC50 values.
b
Acetyl-Boc-Lysine substrate.
Boc-Lys-(e-trifluormethylacetyl)-AMC.
c

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HDAC5 (620-1122), HDAC6, and HDAC7 (438-915) are N-terminal His-tagged.
The enzymeswere incubated withthe compounds inassaybuffer (25 mMHepes,
pH 8.0, 137 mM NaCl, 1 mM MgCl2 and 2.7 mM KCl) for 10 min at ambient
temperature in black 96-well plates. The substrate was added into enzyme-
compound mixture and incubated at 37 °C. Reaction was quenched by adding
trypsin and TSA to a final concentration of 1 mg/mL and 1
lM, respectively.
Fluorescence was measured using fluorimeter (SPECTRAMAX GeminiXS,
a
Molecular Devices). The 50% inhibitory concentrations (IC50) for inhibitors
were determined by analyzing dose–response inhibition curves with GraFit; (c)
At the time we were investigating different substrates for determining the
activity of HDAC class II enzymes, Jones et al. (Bioorg. Med. Chem. Lett. 2008, 18,
1814) published their obervations that purified mammalian HDAC class IIa
enzymes could not be isolated and are contaminated with other deacetylases.
Our results and those of Jones et al. demonstrated that the BOC-
L-Lys(e-
trifluoroacetyl)-MCA is selective class IIa substrate and allowed the
a
development of assays to measure HDAC class IIa activity even in the presence
of class I enzymes for naked enzymes and even in intact cells; (d) Our studies
7. (a) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.; Neddermann, P.;
Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.; Carfí, A.; Koch, U.; De Francesco, R.;
Steinküler, C.; Gallinari, P. Proc. Natl, Acad. Sci. U.S.A. 2007, 104, 17335; (b)
Nielsen, T. K.; Hildmann, C.; Riester, D.; Wegener, D.; Schwienhorst, A.; Ficner,
R. Acta Cryst. 2007, F63, 270; (c) Jones, P.; Altamura, S.; De Francesco, R.;
Gallinari, P.; Lahm, A.; Neddermann, P.; Rowley, M.; Serafini, S.; Steinküler, C.
Bioorg. Med. Chem. Lett. 2008, 18, 1814; (d) Jones, P.; Bottomley, M. J.; Carfí, A.;
Cecchetti, O.; Ferrigno, F.; Lo Surdo, P.; Ontoria, J. M.; Rowley, M.; Scarpelli, R.;
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8. KrennHrubec, K.; Marshall, B. L.; Hedglin, M.; Verdin, E.; Ulrich, S. M. Bioorg.
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9. Fleming, I.; Iqbal, J.; Krebs, E.-P. Tetrahedron 1983, 39, 841.
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11. Ling, C.; Minato, M.; Lahti, P. M.; van Willigen, H. J. Am. Chem. Soc. 1992, 114,
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12. (a) For experimental details see: Tessier, P.; Leit, S.; Smil, D.; Deziel, R.; Ajamian,
A.; Chantigny, Y.A.; Dominguez, C. International Patent application WO 07/
122115, 2008; (b) The enzymatic assay followed the fluorescent signal obtained
from the HDAC catalyzed deacetylation of coumarin-labeled lysine. The
showed that human HDAC4,
trifluormethylacetyl)-MCA substrate at a much higher turn-over rate than
HDAC8 did. A plot of the enzyme reactivity (Kcat/Km
MÀ1 minÀ1) versus HDAC1,
2, 3, 4, 5, 7, and 8 enzymes utilizing both acetyl-Boc-Lysine and Boc-Lys-(
trifluormethylacetyl)-MCA substrates clearly demonstrated the extraordinary
5
and
7
processed the Boc-Lys-(e-
,
l
e-
specificity of the Boc-Lys-(e-trifluormethylacetyl)-MCA substrate for HDAC4, 5,
and 7 enzymes (class IIa).
13. Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.; Loppnau, P.;
Mazitschek, R.; Kwiatkowski, N. P.; Lewis, T. A.; Maglathin, R. L.; McLean, T. H.;
Bochkarev, A.; Plotnikov, A. N.; Vedadi, M.; Arrowsmith, C. H. J. Biol. Chem.
2008, 283, 11355.
14. (a) For details of the HDAC class I whole cell assay see: Li, Z.; Besterman, J.M.;
and Bonfils, C. International Patent application WO 2007/135471 A1, 2007; (b)
For details of the HDAC class II whole cell assay see Jubrail Rahil, Aihua Lu
Patent Application US 2008/0199897, 2008; (c) The whole cell assay was done
in cultured Human Embryonic Kidney cells (293T), which were treated with
inhibitors for 16 h and then incubated with the substrate Boc-Ac-Lys-AMC, for
measuring class I HDAC activity or Boc-Lys-(
measuring HDAC class IIa activity. After 90 min at 37 °C, the reaction was
quenched with trypsin and TSA to a final concentration of 1 mg/mL and 1 M,
e-trifluormethylacetyl)-AMC for
l
respectively. The cells were lysed with 1% NP-40. Fluorescence was read at Ex
360 nm, Em 470 nm, using GeminiXS fluorimeter.
substrate used for HDAC1, 2, 3, 6, and 8 was Boc-Lys(
e
-acetyl)-AMC (Bachem
15. Campbell, K. J. Chem. Soc. 1946, 25.
Biosciences Inc.) and Boc-Lys-( -trifluormethylacetyl)-AMC (synthesized
in-house) for HDAC4, 5, and 7. Recombinant enzymes expressed in baculovirus
were used. HDAC1, and 2 were C-terminal FLAG-tagged and HDAC4 (612-1034),
e
16. Tanaka, K.; Matsuo, K.; Nakanishi, A.; Hatano, T.; Izeki, H. Chem. Pharm. Bull.
Engl. 1983, 31, 2810.