Design, synthesis, docking, and biological evaluation of novel diazide-containing isoxazole- and pyrazole-based histone deacetylase probes

Article abstract of DOI:10.1021/jm2001025

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC₅₀ of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.

Full text of DOI:10.1021/jm2001025

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, Docking, and Biological Evaluation of Novel
Diazide-Containing Isoxazole- and Pyrazole-Based Histone
Deacetylase Probes
Raghupathi Neelarapu,||,† Denise L. Holzle,||,‡ Subash Velaparthi,†,1 He Bai,† Michael Brunsteiner,†,1
Sylvie Y. Blond,*,‡,§ and Pavel A. Petukhov*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612, United States
‡Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland, Chicago, Illinois 60612, United States
§UMRS-940, Institut Universitaire d’Hꢀematologie (IUH) Saint Louis, Institut de Gꢀenꢀetique Molꢀeculaire, 27 rue Juliette Dodu,
75010 Paris, France
S Supporting Information
b
ABSTRACT: The design, synthesis, docking, and biological
evaluation of novel potent HDAC3 and HDAC8 isoxazole- and
pyrazole-based diazide probes suitable for binding ensemble
profiling with photoaffinity labeling (BEProFL) experiments in
cells is described. Both the isoxazole- and pyrazole-based probes
exhibit low nanomolar inhibitory activity against HDAC3 and
HDAC8, respectively. The pyrazole-based probe 3f appears to
be one of the most active HDAC8 inhibitors reported in the
literature with an IC50 of 17 nM. Our docking studies suggest
that unlike the isoxazole-based ligands the pyrazole-based
ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the
antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that
they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or
enhance cellular cytotoxicity, or affect cell permeability.
’ INTRODUCTION
the parent HDAC inhibitors. For example, the diazide probe 1
(Figure 1), which was designed by adding a diazide portion to
SAHA through a linker and used in our recent BEProFL
approach, showed a 17-fold decrease in HDAC8 inhibitory
activity compared to SAHA.5
Further extension of the BEProFL approach to cell-based
studies would also require probes that are not highly cytotoxic
and, therefore, evaluation of antiproliferative properties of the
probes in the cell-based studies needs to accompany the evalua-
tion of the HDAC activity of the probes against recombinant
proteins in vitro. Design of isoform-selective inhibitors within an
HDAC class is particularly challenging because of the similarity
between isoforms within the class. In this paper, we decided to
focus on diazide probes for the two class I isoforms HDAC8 and
HDAC3, which inhibitors may be of particular value for the
treatment of neuroblastoma and leukemia,6,7 and gastric, pros-
tate, and colorectal cancer,8 respectively.
Histone deacetylases (HDACs)1 are well established enzy-
matic targets for multiple therapeutic applications.2 It is widely
accepted that therapeutic application of HDAC inhibitors
depends on their HDAC class and isoform selectivity profile,3,4
making isoform selective inhibitors an important issue in the
design and development of novel HDAC-based therapeutics.
One of the key challenges in HDAC inhibitor design is the
limited amount of information regarding the binding modes
(poses) available to the highly solvent exposed surface binding
groups (SBG) of HDAC inhibitors. To address this problem, we
recently developed a binding ensemble profiling with photo-
affinity labeling (BEProFL) approach that utilizes a diazide probe
to map experimentally the multiple binding poses of the SBGs of
HDAC ligands.5 The method requires availability of the active
and preferably isoform-selective HDAC probes containing an
aromatic azide group, which can covalently cross-link to the
protein of interest upon UV-irradiation, and an aliphatic azide
group, which can react with a biotin or fluorescent tag for further
purification or visualization. The design of the diazide-containing
HDAC probes appears to be nontrivial because the extra bulk of
the diazide group may affect the HDAC activity and selectivity of
As a starting point for the design of HDAC3 diazide probes,
we decided to use recently reported isoxazole-based inhibitors
Received: November 19, 2010
Published: May 06, 2011
r
2011 American Chemical Society
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Figure 1. Structure of BEProFL probe 1 and general structure of novel isoxazole-based compounds 2 and pyrazole-based compounds 3.
the probes next to the protein surface. In an opposite orientation
of the five-membered heterocycles, the large portions of the SBG
would not make any pronounced interactions with the protein
surface. An amide group was chosen as connecting unit between
the heterocyclic and linker portions of the ligands. On one hand,
it provides the necessary versatility of the synthesis and, on the
other hand, the amide NH is known to form hydrogen bond with
the side chain of Asp101 at the gorge region of the binding
site.10,11 Excellent potential of the design described above is
supported by a series of highly HDAC3 active isoxazole-based
HDAC inhibitors.9 Until recently,12 the activity of the published
HDAC8 inhibitors was mostly in the high nanomolar to low
micromolar range, leaving us no other options than to try a novel
4-aminopyrazole scaffold in an attempt to find high affinity
probes for HDAC8.
Synthesis. A practical and scalable synthetic route for diazide
moiety 8 was furnished following the procedure described earlier
for 8 by Hosoya et al.13 Several modifications were done to the
original procedure to improve the yields, regioselectivity, and
ease of handling. The synthesis of 8 is outlined in Scheme 1.
Dimethyl 5-aminoisophthalate (4) was treated with excess
LiAlH4 in THF to give the corresponding amino-diol 5. We
replaced the conventional diazotization conditions, NaNO2/
AcOHꢀH2O/NaN3, with t-BuONO and TMS-N3 in CH3CN14
for amine to azide transformation. In the former method, the
polar nature of the resulting azido-diol 6 made the aqueous
workup tedious, resulting in loss of the product 6 in the water
fractions, particularly during large scale preparations. In our
optimized conditions, the azido-diol 6 was obtained with a
90% yield by a direct chromatography purification of the reaction
mixture after evaporation of the organic solvent. We also
optimized the next step: the selective conversion of the azido-
diol 6 to diazido-alcohol 7. The reported method for this
conversion involves a two-step procedure: a selective bromina-
tion using TPP/CBr4 in DMF followed by a nucleophilic azide
displacement reaction. However, we noted that, in our hands, the
bromination step reproducibly yielded only 50% or less of the
required monobromide along with a 10% of a dibromide and
some starting material still unreacted after 3 h at 0 °C. Prolonged
reaction conditions and/or elevated temperatures led to the
formation of the undesired dibromide as the major product.
Besides, it was difficult to recover the starting material from the
byproduct of the reaction, TPPO, as both appeared with the
same Rf on TLC. The newly adopted one-step conversion of
alcohol to azide using bis(2,4-dichlorophenyl) chlorophosphate,
DMAP, and NaN3 in DMF15 gave the diazido-alcohol 7 in 75%
yield along with 10% of a triazide (based on conversion) and with
a 10% starting material recovery. The optimized conditions also
Figure 2. Design of isoxazole- and pyrazole-based diazide probes.
that are particularly active against HDAC3,9 whereas for the
design of HDAC8 active probes we explored a novel 4-amino-
pyrazole-based scaffold. Herein, we report the design, synthesis,
docking, and biological evaluation of active HDAC3 and
HDAC8 diazide probes suitable for photolabeling experiments
in cells.
’ RESULTS AND DISCUSSION
Probe Design. Our preliminary docking studies showed that
HDAC inhibitors with linear scaffolds can be accommodated by
both HDAC3 and HDAC8 binding sites. The next step was to
ensure that the highly lipophilic diazide portions of the probes
necessary for the BEProFL are efficiently hidden in the ridges
surrounding the binding site. The overall design of the probes
is shown in Figure 2. The zinc binding group (ZBG) was chosen
as in SAHA and many other similar hydroxamic acid-based ligands,
as it is known for its excellent Zinc(II) complexing properties.
The flexible aliphatic chain in the linker allows the ligands to find
the most appropriate place for their SBG. The next portion of the
ligand, a five-membered heterocycle, was designed so that it can
provide the necessary kink and facilitate the placement of the
SBG containing the diazide next to the protein surface. The latter
is necessary for both the binding and the efficient cross-linking of
the diazide portion upon UV irradiation in BEProFL. Two
heterocycles drew our attention based on the criteria described
above and ease of synthesis, an isoxazole and a 4-aminopyrazole
ring. The two substituents form an angle of ca. 145°, allowing
the substituents in the isoxazole and the pyrazole rings to be
positioned complementary to the pseudospherical shape of the
protein. We envisioned that the heteroatoms in the ring systems,
as being more hydrophilic, may be oriented toward the solvent,
whereas the more hydrophobic portions of the heterocycles
would be hidden in one of the grooves on the protein surface,
thus facilitating the desired placement of the diazide portion of
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Scheme 1. Synthesis of Diazide Moietiesa
a Reagents and conditions: (a) LiAlH4, THF, 0 °Cꢀrt, 8 h; (b) t-BuONO, TMS-N3, MeCN, 0 °Cꢀrt, 2 h; (c) Bis(2,4-dichlorophenyl) chlorophosphate,
DMAP, NaN3, DMF, rt, 4 h; (d) Ts-Cl, Et3N, 0 °C, 2 h; (e) LiBH4, THF, 50 °C, 48 h; (f) 2N NaOH, MeOHꢀTHF (1:1), 1 h.
Scheme 2. Synthesis of 2aꢀ2ba
a Reagents and conditions: (a) ethyl chlorooximidoacetate, Na2CO3, THFꢀH2O (1:1), 0 °Cꢀrt, 18 h; (b) 2N NaOH, MeOHꢀTHF (1:1), 0 °Cꢀrt,
1 h; (c) EDC, HOBt, methyl 7-aminoheptanoate hydrochloride, DIPEA, CH2Cl2, rt, 4 h; (d) TFA, CH2Cl2, rt, 4 h; (e) NaNO2, AcOHꢀH2O, 0 °C,
10 min, then NaN3, 0 °Cꢀrt, 1 h; (f) 12, DCC, HOBt, DMAP, CH2Cl2, 0 °Cꢀrt, 6 h; (g) NH2OH, KOH, MeOH, 0 °Cꢀrt, 3 h.
allowed us to avoid possible formation of an amine byproduct as
TPP is known to reduce the azides to amines via phosphazene
formation. Treatment of 7 with equivalents of tosyl chloride in
the presence of triethylamine in CH2Cl2 at 0 °C afforded the
required diazido-tosylate 8 in 85% yield in 2 h. It is worth
mentioning that an increase in temperature of the reaction from
0 °C to rt resulted in an unwanted byproduct.
The synthesis of the diazido-acid 12 was initiated from a
common precursor 4 following a synthetic route similar to that
used to prepare diazido-tosylate 8 (Scheme 1). After consider-
able experimentation using different metal hydride reducing
agents such as LiAlH4, NaBH4, and LiBH4, the required regio-
selective reduction of 4 to amino-ester 9 was achieved using
LiBH4 in THF at 50 °C for 48 h. Azido-ester 10 and diazido-ester
11 were prepared in high yields employing the same reaction
conditions described for the synthesis of azido-diol 6 and
diazido-alcohol 7, respectively. The subsequent basic hydrolysis
of ester group of 11 produced the desired diazido-acid 12.
Having an efficient access to both the diazide moieties, we
moved on to design and synthesis of the isoxazole-based HDAC
inhibitors (Scheme 2). The ester 16 was prepared according to
the procedure described earlier.9 1,3-Dipolar cycloaddition of
ethyl chlorooximidoacetate to N-Boc alkyne 13 in the presence
of Na2CO3 in THFꢀH2O afforded the 5-arylisoxazole-3-car-
boxylic acid ethylester (14) in 85% yield. This reaction was
preferred over triethylamine-mediated cycloaddition reaction
because the latter was reported earlier to give a low 40% yield
for the same ethylester 14.9 Hydrolysis of 14 followed by an
amide coupling reaction of the resulting acid 15 with methyl
7-aminoheptanoate gave the key intermediate ester 16. Com-
pound 16 was treated with TFA in CH2Cl2, resulting in forma-
tion of an aromatic amine 17 that was transformed to azide 18
using NaNO2/NaN3 in AcOHꢀH2O. The amide coupling
reaction between amine 17 and acid 12 under the standard
EDC/HOBt/DIPEA conditions was slow and gave only low
yields (up to 15%) of 19 after prolonged reaction time.
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Scheme 3. Synthesis of 3aꢀ3fa
a Reagents and conditions: (a) H2, Pd/C, MeOH, overnight; (b) monometyl suberate, EDC, HOBt, DIPEA, CH2Cl2, 0 °Cꢀrt, 6 h; (c) 8, K2CO3,
acetone, reflux, 6 h; (d) NH2OH, KOH, 0 °Cꢀrt, 3 h; (e) BnBr or 4-nitrobenzyl bromide, NaH, DMF, 0 °Cꢀrt, 4 h; (f) SnCl2 2H2O, MeOH, reflux,
3
2 h; (g) NaNO2, AcOHꢀH2O (9:1), 0 °C, 10 min, NaN3, 0 °Cꢀrt, 1 h; (h) (Boc)2O, Et3N, CH2Cl2, rt, 4 h; (i) 12, EDC, HOBt, DIPEA, CH2Cl2,
0 °Cꢀrt, 6 h.
An alternative coupling reaction using DCC/HOBt/DMAP in
CH2Cl2 resulted in 19 with a much better yield of 70%. Further
treatment of the esters 18 and 19 with freshly prepared NH2OH
in the presence of KOH in MeOH gave the desired hydroxamates
2a and 2b, respectively.
The strategy for the synthesis of pyrazole inhibitors is pre-
sented in Scheme 3. It involves an amide coupling reaction
between 4-aminopyrazole (21), derived from the corresponding
commercially available 4-nitropyrazole (20) by hydrogenolysis,
and monomethyl suberate to give the ester 22.16 Alkylation of 22
with 8 in presence of K2CO3 in refluxing acetone afforded ester
23. Compounds 24 and 25 were obtained by alkylating 22 with
benzyl bromide or 4-nitrobenzyl bromide in the presence of NaH
in DMF, respectively. Reduction of the nitro group of 25 using
Boc-L-Lys(Ac)-AMC and commercial available recombinant
human HDAC3/NCoR2. The HDAC3/8 activity profiles are
summarized in Tables 1 and 2. All newly synthesized HDAC
inhibitors/probes tested had IC50s ranging from 17 to 707 nM.
As expected, the isoxazole-based probes 2a and 2b showed
HDAC3 selectivity over HDAC8, with 2b being the most potent
HDAC3 inhibitor with HDAC3 and HDAC8 IC50s of 45 and
651 nM, respectively, and an IC50 HDAC8/HDAC3 ratio above
14. Isoxazole-based probe 2a inhibited HDAC3 and HDAC8
with IC50s of 73 and 707 nM, respectively. Both probes 2a and 2b
maintained the HDAC3/8 activity and selectivity comparable to
that of SAHA.
Among the pyrazole-based inhibitors, the simplest benzyl-
substituted compound 3a inhibited HDAC3 and HDAC8 with
IC50s of 44 and 76 nM, respectively. Introduction of a nitro group
at the 4-position of the benzyl group of 3a resulted in compound
3b that showed slightly lower activity for both isoforms, 59 nM
and 82 nM against HDAC3 and HDAC8, respectively, whereas
the corresponding azido compound 3c exhibited a 2.0- and
2.7-fold better potency, 22 and 28 nM for HDAC3 and HDAC8,
respectively. Overall, compounds 3aꢀ3c exhibited an inhibitory
activity against HDAC3 comparable to that of SAHA but a better
double digit nanomolar activity against HDAC8. Introduction of
a bulky Boc-protected amino group in 3d decreased the HDAC
activity by about 10-fold, with an IC50 of 191 nM for HDAC3 and
147 nM for HDAC8 isoform. Replacement of the Boc group with
a lipophilic aromatic diazide via a rigid amide bond in 3e further
decreased the activity for both HDAC3 and HDAC8 to 432
and 487 nM, respectively. Comparison of the activity data of
3bꢀ3c with 3dꢀ3e clearly shows that the presence of the bulky
SnCl2 2H2O in refluxing methanol resulted in aniline 26, a key
3
intermediate for compounds 27ꢀ29. Diazotization of amino
group of aniline 26 followed by an azide displacement reaction
with NaN3 gave the corresponding azido compound 27. Treat-
ment of 26 with Boc anhydride and Et3N in CH2Cl2 furnished
the carbamate 28. Ester 29 was obtained by an amide coupling
reaction between 26 and diazido-acid 12. Treatment of the
methyl esters 23ꢀ25 and 27ꢀ29 with KOH/NH2OH in MeOH
gave the corresponding hydroxamates 3f and 3aꢀ3e, respectively.
Activity Assays. The isoxazoles 2aꢀ2b and pyrazoles 3aꢀ3f
were tested for inhibition of HDAC3 and HDAC8 isoforms, and
the procedure is identical to that published previously.5 The
inhibition of HDAC8 was measured using the fluorescent
acetylated HDAC substrate Fluor de Lys and commercially
available recombinant human HDAC8, whereas the inhibition
of HDAC3 was measured using the fluorescent HDAC substrate
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Table 1. HDAC3 and HDAC8 Isoform Inhibitory Activity (IC50, nM) of Isoxazole-Based Compounds 2a and 2b
a Ratio of IC50 (nM)
Table 2. HDAC3 and HDAC8 Isoform Inhibitory Activity (IC50, nM) of Pyrazole-Based Compounds 3aꢀ3f
a Ratio of IC50 (nM)
substituent in the para position of the terminal phenyl ring of 3a
leads to the lower activities for both HDAC3 and HDAC8
isoforms. This observation led us to the modification of 3- and
5-positions of the phenyl group of 3a and replacement of the
phenyl group with a 3-azido-5-azidomethyl phenyl group, result-
ing in 3f. To our surprise, compound 3f was 8-fold more active
toward HDAC8 than for HDAC3, with IC50s equal to 17 and 128
nM, respectively.
Docking. To gain insights into the possible reasons for activity
and selectivity of the two diazide probes 2b and 3f that would also
facilitate the further BEProFL experiments, we docked these
compounds to HDAC8 crystal structures available in the Protein
Data Bank. Although we also docked these ligands to homology
models of HDAC3, we decided not to discuss these results here
as they may not represent the true binding poses. Indeed, the lack
of any structural information on the HDAC3-NCoR2 complex,
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Figure 3. (A) 2b docked to HDAC8 PDB:1T64 (B) 3f docked to HDAC8 PDB:1VKG. Description of the grooves (G1ꢀG3) and ridges (R1ꢀR3)
formed by the protein surface of HDAC8 is the same as in ref 5.
Figure 4. MOE analysis of the interactions between HDAC8 receptors and ligands 2b (A) and 3f (B): (green) hydrophobic, (light-purple) polar,
(blue ring) basic, (red ring) acidic, (purple ring) solvent exposed.
which formation is required for histone deacetylase activity of
HDAC3, the likely malleability of the protein surface of HDAC3
(based on malleability of the surface of HDAC8), and the relative
imprecision of homology models for interpretation of already
theoretical docking poses, make these results highly hypothetical
and very unreliable. The HDAC8 docking poses and MOE
analysis of 2b and 3f are shown in Figures 3 and 4, respectively.
The best scoring pose of both compounds are found with
receptor structures based on a HDAC8 protein structure with
an open second binding pocket. The best docking score for probe
2b was obtained for PDB:1T64, and in case of 3f, PDB:1VKG.
The interactions between probe 2b and the binding site are
consistent with those expected by design, i.e., ZBG interacts with
the zinc atom, the amide NH between the linker and SBG forms
hydrogen bond with the side chain COOꢀ of Asp101, the
heterocycle provides the important kink, the heteroatoms in
the isoxazole point toward the solvent, whereas its opposite side
points toward the protein, the amide group connecting the
terminal phenyl ring forms hydrogen bond with Lys145, and
the phenyl ring and the diazide portion slide along the surface.
The SBG of 2b is linear and rigid and therefore has limited
options in adjusting itself to the landscape of the protein surface
and is mostly solvent exposed. Probe 3f, on the other hand, may
have its SBG buried in a second binding pocket, leading to
extensive nonpolar interactions between the ligand and the
protein and a πꢀcation charge transfer interactions between
the terminal phenyl ring of 3f and Arg37. In comparison to probe
2b, probe 3f is much less solvent exposed (Figure 4). The
interpretation of these two poses agrees with the measured
activities of the two compounds, as 3f, which is more active
against HDAC8 than 2b, appears to find more favorable inter-
actions with HDAC8, while the opposite is true for 2b. The
binding of the SBG of the compounds in the pyrazole series to
the secondary pocket is consistent with the SAR. Ligands with
the relatively small substituents in the terminal phenyl ring
3aꢀ3c and 3f show activity below 82 nM for HDAC8, with
probe 3f peaking at 17 nM. As the size and rigidity of the
substituent increases in ligands 3d and 3e, their activity drops to
147 and 487 nM, respectively. Overall, availability of the second
site for the binding of ligands is supported by X-ray crystal-
lographic data, the upside-down pose of the probe 1 detected
with the BEProFL approach,5 binding of the linkerless HDAC
ligands,12 and the recent modeling studies by Wiest et al.17
HDACs and MMPs Activity Profiling. We also conducted a
preliminary HDAC profiling of 2b and 3f for the remaining class I
HDACs 1 and 2, class II HDACs 4ꢀ7and9ꢀ10, and class IV HDAC
11 isoforms and a representative set of matrix metalloproteases
(MMPs) 1, 3, 9, 14 (Table 3 and the Supporting Information).
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Table 3. A Preliminary Screening for Inhibitory Activity (IC50, nM) of 2b and 3f against HDAC 1, 2, and 4ꢀ7, and 9ꢀ11 Isoformsa
and a Representative Set of Metalloproteases (MMPs) 1, 3, 9, and 14
IC50 (nM)
2b
IC50 ratios
target
control inhibitorb
3f
2b/TSA 2b/SAHA
3f/TSA 3f/SAHA
HDAC1c
8.22 ( 1.45 (TSA),
22.37 ( 4.7 (SAHA)
68 ( 1.9 (TSA),
82.2 ( 1.22
99.1 ( 12.5
10.0
12.1
3.67
4.43
HDAC2c
780 ( 93
1000 ( 180
11.5
14.7
100.0 ( 0.25 (SAHA)
7.80
NDe1.67
10.0
NDe4.74
HDAC3d
HDAC4f
HDAC5f
HDAC6f
HDAC7f
HDAC8d
HDAC9f
HDAC10f
HDAC11f
MMP1f
122
8.08
2.31
139
432
379
892
274
4510
3.54
3.11
932
115
110
47.0
2230
20.4
119
16.0
NDe1.48
32.5
NDe0.0386
20.1
11.3
13.9
0.20
7.62
0.05
0.41
37.8
704
140
476
NAg
NAg
NAg
NAg
1.88
35.0
776
68.7
12.4
116
8.35
34.2
108000
12100
22900
9330
540000
1590
458000
22800
ND
MMP3f
MMP9f
MMP14f
ND
ND
ND
a See Tables 1ꢀ2 for inhibitory activity of 2b and 3f against HDAC3 and HDAC8, respectively. b First number: TSA was used as a control inhibitor in
HDAC 1, 2, 4ꢀ7, and 9ꢀ11 assays. Galardin (GM6001) was used as a control inhibitor in MMPs 1, 3, 9, and 14 assays. Second number: SAHA was used
as an additional control inhibitor for class I isoforms. c IC50 values expressed as mean ( standard deviation of at least two independent experiments.
d IC50 data are given in Tables 1 and 2. e ND: IC50 for TSA were not determined for HDAC3 and HDAC8. f IC50 is a single measurement estimate
provided by Reaction Biology (see Supporting Information). g NA: The remaining activity of the MMPs was above 70% at the highest tested
concentration (10 mM).
Table 4. Antiproliferative Activity (EC50) of 2b, 3a, 3c and 3f towards HepG2, Hela, and SH-SY5Y Carcinoma Cell Lines
HepG2a
48 h
Helaa
48 h
SH-SY5Ya
48 h
compd
24 h
72 h
24 h
72 h
24 h
72 h
SAHA
2b
>50
>50
>50
>50
>50
8.4 ( 1.8
11.5 ( 2.5
12.6 ( 1.6
12.7 ( 1.3
24.3 ( 1.5
3.8 ( 1.1
4.9 ( 1.4
5.5 ( 1.4
5.9 ( 1.3
13.4 ( 1.4
43.0 ( 2.3
26.5 ( 1.1
46.6 ( 1.2
38.3 ( 1.1
30.6 ( 3.3
14.2 ( 1.4
10.7 ( 1.2
10.6 ( 2.3
9.9 ( 1.4
4.7 ( 1.2
4.7 ( 1.1
5.5 ( 1.3
4.6 ( 1.3
15.0 ( 1.1
>50
>50
>50
>50
>50
25.6 ( 1.5
17.4 ( 8.3
23.0 ( 1.1
23.2 ( 2.2
23.1 ( 1.3
20.8 ( 3.1
32.2 ( 1.1
22.7 ( 1.1
23.6 ( 1.4
35.9 ( 1.1
3a
3c
3f
23.7 ( 1.1
a EC50 in μM concentration
The IC50s of 2b and 3f were compared to those obtained for TSA
for HDAC isoforms 1, 2, 4ꢀ7, and 9ꢀ11, SAHA for class I HDACs,
and galardin (GM6001) for MMPs. Neither of the two compounds
exhibits a pronounced level of activity against the MMPs. When
compared to activity of SAHA and TSA against class I, II, and IV
HDACs, probe 3f demonstrated superior selectivity for HDAC8
(Table 3). Specifically, IC50s of 3f for all HDAC isoforms tested was
between 3.11- and 119-fold higher than those of SAHA or TSA for
the same HDAC isoforms except for HDAC8, for which the ratio of
IC50s was 0.0386. A somewhat similar but less pronounced trend was
observed for 2b. When compared to SAHA or TSA, 2b was relatively
less potent against all HDAC isoforms, with only 1.67-, 1.48-, and
1.88-fold difference for HDAC3, HDAC8, and HDAC9, respectively.
Within class I HDACs, 2b and 3f were clearly more selective for
HDAC3 and HDAC8, respectively, compared to SAHA.
secondary objective was to demonstrate that the probes are
compatible with cell-based experiments and have no effect on the
integrity or the fitness of the cell. Specifically, it was important to
show that the probes are cell membrane permeable, not overly
cytotoxic, and that treatment of cells with them leads to
physiological outcomes expected for HDAC inhibitors. Only
compounds that meet those criteria can be used in future
experiments as photoaffinity probes to assess the binding poses
favored in the intracellular environment.
We explored the antiproliferative activity of the HDAC3 active
diazide isoxazole probe 2b and the potent pyrazole inhibitor/probes
3a, 3c, and 3f in three carcinoma cell lines of human origin. Our
results show that the hepatocarcinoma HepG2 cells, cervical
carcinoma Hela cells, and neuroblastoma SH-SY5Y cells are all
sensitive to micromolar concentrations of the isoxazole and pyrazole
HDAC inhibitors (Table 4). The antiproliferative action of the most
HDAC8 active pyrazole inhibitor 3f (EC50ꢀ48 h = 24ꢀ36 μM) is in
Cell-Based Studies. In the addition to our primary goal,
design of potent HDAC3 and HDAC8 BEProFL probes, our
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the same range compared to that obtained for the most HDAC3
active isoxazole inhibitor 2b (EC50ꢀ48 h = 11ꢀ32 μM) and SAHA
(EC50ꢀ48 h = 8ꢀ26 μM). Also, the compounds 3a and 3c, which
inhibit HDAC3 and HDAC8 with comparable potencies
(HDAC8/HDAC3 = 1.3ꢀ1.7, Tables 2ꢀ3), exert very comparable
acetylation being sustained throughout 24 h post-treatment at
low micromolar concentrations (Figure 5). These results suggest
that 2b efficiently targets nuclear HDACs.
In an effort to delineate pathways responsible for the cellular
death observed in the presence of our HDAC inhibitors, we
looked at the activation/cleavage of caspase 3. Inhibitor 2b and
the control compound SAHA induced cleavage of caspase 3 by
14 h with maximum activation at 24 h (Figure 6). Taken
together, our results indicate that in HepG2 cells, the inhibitor
2b successfully inhibits nuclear HDACs within 6 h of treatment
(Figure 5), induces caspase 3 cleavage within 14 h of treatment
(Figure 6), and cell death within 24ꢀ48 h (Table 4) as efficiently
as SAHA does. This is consistent with another study where the
apoptotic effect of 10 μM SAHA appears after a 12ꢀ16 h lag-
phase in HepG2 cells and reached a maximum at about 36 h.18
SAHA has been shown to induce programmed cell death in
HepG2 cells through the acetylation of p5318 and the decrease of
BCL-219 and we sought to investigate whether 2b was exerting its
pro-apoptotic effect via the same mechanism. HepG2 cells were
treated with SAHA or 2b and the acetylation of p53 was
monitored (Figure 6). In our conditions, the level of acetylated
p53 is already significant in nontreated cells and neither SAHA
nor 2b induced additional time and dose-dependent hyperace-
tylation of p53 in total cell extracts. Similar results were obtained
independently on human lung carcinoma cells.20 The data
reported by Carlisi et al.18 actually show a modest 2-fold increase
in p53-acetylation, and very faint bands for acetylated-Lysine320
antiproliferative activity toward the three cell lines (EC50ꢀ48 h
=
10ꢀ23 μM), indicating that difference in HDAC3/HDAC8 iso-
form activity observed in vitro has little impact on the value of EC50s.
Comparison of the antiproliferative activity of azide 3c and diazides
3f and 2b with that of the nonazide inhibitor 3a suggests that the
presence of an azide or a diazide group by itself does not enhance cell
death and the antiproliferative activity observed is likely due to the
inhibition of cellular HDACs of class I.
The inhibitory effect of SAHA and 2b on cellular HDACs was
assessed by monitoring the acetylation status of histone H4 in the
HepG2 cell line using Western blot analysis. Both SAHA and 2b
caused a marked increase in acetylation of histone H4 by 6 h, with
Figure 5. Western blot detection of the acetylation of histone H4
following treatment with the diazide probe 2b in HepG2 cells.
Figure 6. The isoxazole HDAC inhibitor/probe 2b induces apoptosis in HepG2 cells independent of p53 acetylation: (A) Western blotting to detect
the activation of caspase 3 and the acetylation of p53 in the treated HepG2 cells; (B) histograms representing the acetylation of p53 protein in the cell
extracts.
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Figure 7. Differential neuroprotective and cytotoxic effects of HDAC inhibitors in unstressed (dark-gray) or treated with compound 30 differentiated
SH-SY5Y cells (light-gray). **p < 0.01, ***p < 0.001 when compared to vehicle DMSO control (0 μM HDAC inhibitor).
We sought to ascertain the in situ effects of these inhibitors on
nuclear HDACs by monitoring the acetylation status of histone
H4 in retinoic acid-differentiated SH-SY5Y cells. Our results show
that treatment with 1 μM of probes 2a, 2b, and 3f induce
hyperacetylation of histone H4 in differentiated SH-SY5Y cells
(Figure 8). Furthermore, this effect is also observed in cells
stressed with compound 30.26ꢀ28 These results indicate that the
three probes 2a, 2b, and 3f efficiently target nuclear HDACs in
normal and stress conditions in differentiated human neuro-
blastoma cells.
Figure 8. Small-molecule HDAC inhibitors 2a, 2b, and 3f induce
accumulation of acetylated histone H4 in a differentiated SH-SY5Y
cellular model.
p53 and acetylated-Lys373/382 p53 immunoprecipitated from
nuclear extracts with the p53 DO1 antibody. Another study
’ CONCLUSIONS
performed in neuroblastoma cells also shows a modest acetyla-
tion of p53 upon treatment with 3 mM valproic acid, a pan
HDAC inhibitor.21 We do not exclude that SAHA or 2b acts
directly on the acetylation level of p53 and its function in the
nucleus but did not pursue further investigation, as this effect is
very modest in our conditions.
Two types of scaffolds were explored for design of novel
HDAC3 and HDAC8 BEProFL diazide probes. A total of eight
inhibitors/probes were synthesized, tested for HDAC3 and
HDAC8 activity, and docked to HDAC8. Probes 2b and 3f were
profiled against class I, II, and IV HDAC isoforms and MMPs 1,
3, 9, and 14 and compared to a set of control inhibitors. Four
novel active HDAC3 and HDAC8 probes/inhibitors 2b, 3a, 3c,
and 3f were tested for their antiproliferative and neuroprotective
activity and compared to that of SAHA. Both the isoxazole and
pyrazole scaffolds were found to be excellent starting points for
the design of potent HDAC3 and HDAC8 BEProFL probes,
respectively. The synthetic route to the diazide intermediates 8
and 12 was optimized for gram scale, making the final probes/
inhibitors readily accessible in few synthetic steps in a convergent
manner. A facile route for the synthesis of the pyrazole-based
inhibitors developed in this study can be extended to a wide
range of other 4-aminopyrazole-based inhibitors.
Despite the presence of relatively bulky and lipophilic diazide
moieties necessary for BEProFL, both the isoxazole- and the
pyrazole-based probes exhibit excellent low double-digit nano-
molar inhibitory activity against HDAC3 and HDAC8. Having
an IC50 of 17 nM against HDAC8, the meta-diazide pyrazole-
based probe 3f not only shows 8-fold selectivity for HDAC8 vs
HDAC3 but is also one of the most active HDAC8 inhibitors
reported to date. The rest of the novel pyrazole-based HDAC
inhibitors also displayed excellent low nanomolar activity against
HDAC3 and HDAC8, although they were also lacking isoform
The HDAC inhibitor SAHA is FDA approved for treating
T-cell lymphoma,22 yet, in cell and mouse research studies,
can be protective to stressed neuronal cells.23,24 We, thus, next
investigated whether the diazide isoxazole 2b exerted a neuro-
protective effect in a retinoic acid differentiated SH-SY5Y human
neuroblastoma cell model treated with 30 (LY294002), a widely
used inhibitor of the phosphatidylinositol 3-kinase (PI3K)/AKT
survival pathway.25 The HDAC inhibitors display differential
cytotoxic and neuroprotective effects when the cells are stressed
with 100 μM of 30 (Figure 7). The two isoxazoles 2a and 2b and
the pyrazole 3f protect cells from stress induced by compound
30 at low micromolar concentrations, but only the isoxazole 2a is
not overly cytotoxic to retinoic acid-differentiated neuroblasto-
ma cells at the highest concentrations tested (Figure 7). The
pyrazole 3f more than doubles the amount of cell survival at 0.5
and 1 μM concentrations yet shows subtle toxicity at 20 μM. The
compound 3a, which does not contain an azide, and the
compound 3c, which contains a monoazide substituent, are both
non-neuroprotective, yet both are not toxic to the cells indicating
that the single azide has no cytotoxic properties in differentiated
neuroblastoma cells.
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selectivity. Both probes 2b and 3f showed no pronounced
activity against MMPs and were generally not HDAC class
selective based on the comparison of IC50 values. Probe 3f was
more selective for HDAC8 compared to TSA and SAHA. Within
class I HDACs, 2b and 3f were respectively HDAC3 and
HDAC8 isoform-selective compared to SAHA. Our docking
studies suggest that unlike the isoxazole-based ligands the
pyrazole-based ligands may occupy the second binding site of
HDAC8. This theoretical observation is consistent with our
previous BEProFL studies as well as published X-ray data and
molecular modeling studies of the HDAC8 linkerless ligands.
Comparison of antiproliferative and neuroprotective proper-
ties and investigation of the pathways typically triggered by
HDAC inhibitors showed that the presence of an azide or a
diazide group neither interfere with the neuroprotection nor
enhance the cellular cytotoxicity exerted through inhibition of
intracellular HDACs. Taken together, cell-based studies show
that the isoxazole- and pyrazole-based inhibitors and azide and
diazide probes are able to freely enter the cell nucleus and trigger
mechanisms known to respond to HDAC inhibition such as
accumulation of acetylated H4 within 6 h, activation of caspase 3
within 14 h, and cell death of otherwise highly proliferative
human cancer cell lines within 24ꢀ48 h of treatment at low
micromolar concentrations. Thus, the toxicity of these HDAC
inhibitors/probes can be managed by allowing their use at
submicromolar concentrations and 0ꢀ6 h treatment in BEProFL
experiments to identify the binding poses of the inhibitors in
living cells. Unlike SAHA, probe 2a, 2b, and 3f did not exert
cytotoxic effects in neuronal-like cells and even protected these
cells from chemically induced apoptosis, making them interest-
ing candidates for further thorough mechanistic studies in
normal and stress conditions. Our future plans include BEProFL
experiments with recombinant HDAC3/NCoR2 and HDAC8
proteins to improve our understanding of the binding poses of
the probes and generate more data for further refinement of the
docking approaches for HDACs and solvent exposed binding
sites in general, extension of the BEProFL approach to experi-
ments in cells, completion of the HDAC isoform profiling, and
extensive investigation of SAR of the promising pyrazole-based
scaffold identified in this study.
systems were used as eluent for chromatography unless it is mentioned
otherwise.
(3-Amino-5-hydroxymethylphenyl)methanol (5). To a solution of
dimethyl 5-aminoisophthalate 4 (10 g, 47.8 mmol) in dry THF
(200 mL) at 0 °C was added LiAlH4 (4.9 g, 143 mmol) portionwise.
The mixture was stirred for 8 h at rt, cooled to 0 °C, and quenched by
slow addition of aqueous KOH solution (20%, 20 mL) followed by ethyl
acetate (200 mL). The mixture was filtered through a pad of Celite and
rinsed with ethyl acetate. The filtrate was dried over Na2SO4, filtered,
and concentrated under reduced pressure. The resulting crude residue
was purified by flash chromatography (100:0 f 90:10, EtOAc:MeOH)
1
to give 5 as a pale-yellow solid (6.6 g, 90%). H NMR (400 MHz,
methanol-d4): δ 6.70 (s, 1H), 6.65 (s, 2H), 4.50 (s, 4H). 13C NMR (100
MHz, methanol-d4): δ 147.0, 141.9 (2C), 114.8, 112.4 (2C), 63.6 (2C).
(3-Azido-5-hydroxymethylphenyl)methanol (6). To an ice cooled
solution of amino-diol 5 (6.5 g, 42.4 mmol) in dry acetonitrile was added
t-BuONO (5.3 g, 60 mmol) followed by TMS-N3 (6.2 mL, 50 mmol)
dropwise. The resulting mixture was stirred for 2 h at rt and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (30:70 f 0:100, hexanes:EtOAc) to give azido-diol
6 as orangeꢀyellow solid (6.9 g, 91%). 1H NMR (400 MHz, methanol-d4):
δ 7.13 (s, 1H), 6.97 (s, 2H), 4.60 (s, 4H). 13C NMR (100 MHz,
methanol-d4): δ 143.4 (2C), 139.8, 120.9, 115.1 (2C), 62.7 (2C).
(3-Azido-5-azidomethylphenyl)methanol (7). This reaction was
carried out on 1ꢀ2 g scale repeatedly as per the requirement of the
diazido-alcohol 7. To a stirred solution of azido-diol 6 (2 g, 11.2 mmol)
in anhydrous DMF (20 mL) were added at rt NaN3 (2.9 g, 45 mmol)
and DMAP (1.7 g, 13.4 mmol) followed by bis(2,4-dichlorophenyl)-
chlorophosphate (5 g, 12.3 mmol) and the stirring was continued for 4 h.
Brine (40 mL) was added to the reaction mixture and extracted with
ethyl acetate (3 ꢁ 50 mL). The organic fractions were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
residue was purified by flash chromatography (70:30 f 50:50, hexanes:
EtOAc) to afford the desired diazido-alcohol 7 (1.5 g, 75%) as yellow oil
along with triazide (0.22 g, 10%), based on 88% conversion and 12% of
starting material recovery. 1H NMR (400 MHz, CDCl3): δ 7.08 (s, 1H),
7.02 (s, 1H), 6.89 (s, 1H), 4.70 (s, 2H), 4.35 (s, 2H), 2.15 (bs, 1H). 13C
NMR (100 MHz, CDCl3): δ 143.2, 140.6, 137.3, 122.3, 117.3, 116.6,
64.0, 53.9.
Toluene-4-sulfonic Acid 3-Azido-5-azidomethylbenzyl Ester (8). A
solution of 7 (1.5 g, 7.4 mmol) and triethylamine (2.6 mL, 18.4 mmol)
in CH2Cl2 was treated with TsCl (1.6 g, 18.4 mmol) at 0 °C. The
reaction was monitored by TLC while maintaining the temperature
at 0 °C (otherwise undesired products were obtained). The reaction
was quenched with water (20 mL) and extracted with CH2Cl2
(2 ꢁ 20 mL). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residual oil was immediately purified by flash chromatography
(90:10 f 70:30, hexanes:EtOAc) to give the diazido-tosylate 8 as pale-
yellow oil (2.25 g, 85%). 1H NMR (400 MHz, CDCl3): δ 7.80 (d, J =
8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 6.98 (s, 1H), 6.92 (s, 1H), 6.83 (s,
1H), 5.05 (s, 2H), 4.32 (s, 2H), 2.46 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 145.1, 141.1, 138.1, 136.0, 133.0, 129.9 (2C), 127.9 (2C),
124.0, 118.9, 118.4, 70.6, 53.9, 21.6.
3-Amino-5-hydroxymethylbenzoic Acid Methyl Ester (9). To a solution
of dimethyl 5-aminoisophthalate 4 (5 g, 24.0 mmol) in dry THF at
0 °C was added portionwise LiBH4 (1.1 g, 50.0 mmol). The reaction
mixture was heated at 50 °C for 48 h and quenched by adding MeOH at
0 °C. The solvent was evaporated, and the crude solid was purified by
flash chromatography (30:70 f 0:100, hexanes:EtOAc) to give amino-
ester 9 as pale-yellow solid (3.05 g, 71%). 1H NMR (400 MHz,
methanol-d4): δ 7.33 (s, 1H), 7.26 (s, 1H), 6.94 (s, 1H), 4.55
(s, 2H), 3.87 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 167.7, 147.9,
142.7, 130.6, 117.8, 117.0, 114.4, 63.5, 51.2.
’ EXPERIMENTAL SECTION
Chemistry Materials and Methods. All the reagents and
solvents were obtained from commercial sources and used without
further purification. 1H NMR and 13C NMR spectra were recorded on
Bruker spectrometers at 300/400 and 75/100 MHz, respectively.
Chemical shifts were reported on δ scale in ppm with solvent indicated
as the internal reference. Coupling constants were reported in Hz and
the standard abbreviations indicating multiplicity were used as follows:
s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, and
m = multiplet. Mass spectrometry experiment was carried out on Agilent
1100-MSD instrument. The purity of all the final compounds was
confirmed to be g95% as it was analyzed by Shimadzu LCMS-2020
with UV detector at 214 and 254 nm using Kinetex C18 column
(100 mm ꢁ 3.0 mm, 2.6 μ) eluting with mixtures of waterꢀacetonitrile.
TLC was performed with Merck 60F254 silica gel plates. Chromatogra-
phy purification was performed on Biotage-Isolera Four instrument
using prefilled KP-Sil (normal phase) and KP-C18-HS (reverse phase)
SNAP cartridges with UV detection at 254 and 280 nm. Hexanesꢀethyl
acetate (normal phase) and H2OꢀMeOH (reverse phase) solvent
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3-Azido-5-hydroxymethylbenzoic Acid Methyl Ester (10). To an ice
cooled solution of amino-ester 9 (3 g, 16.6 mmol) in dry acetonitrile
(40 mL) was added dropwise t-BuONO (2.4 g, 23.2 mmol) followed by
TMS-N3 (2.3 g, 20.0 mmol). The resulting mixture was stirred at rt for
1 h, concentrated, and the crude product was purified by flash chro-
matography (70:30 f 50:50, hexanes:EtOAc) to give azido-ester 10 as
pale-yellow solid (3.1 g, 91%). 1H NMR (400 MHz, CDCl3): δ 7.72 (s,
1H), 7.54 (s, 1H), 7.18 (s, 1H), 4.69 (s, 2H), 3.91 (s,3H), 2.87 (bs, 1H).
13C NMR (100 MHz, CDCl3): δ 166.2, 143.4, 140.4, 131.8, 124.0,
118.8, 64.0, 52.4.
3-Azido-5-azidomethylbenzoic Acid Methyl Ester (11). To a stirring
solution of azido-alcohol 10 (3 g, 14.5 mmol) in anhydrous DMF
(15 mL) were added at rt NaN3 (4.7 g, 72.4 mmol) and DMAP
(2.6 g, 21.3 mmol) followed by bis(2,4-dichlorophenyl)chloropho-
sphate (7.07 g, 17.4 mmol) and the stirring was continued for 4 h.
Brine (40 mL) was added to the reaction mixture and extracted with
ethyl acetate (3 ꢁ 50 mL). The organic fractions were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
residue was purified by flash chromatography (90:10 f 70:30, hexanes:
EtOAc) to afford diazido-ester 11 as pale-yellow solid (3.1 g, 92%). 1H
NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 7.65 (s, 1H), 7.14 (s, 1H),
4.40 (s, 2H), 3.93 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 165.7, 141.2,
137.9, 132.4, 125.3, 122.6, 119.6, 53.8, 52.5.
3-Azido-5-azidomethylbenzoic Acid (12). An ice cooled solution of
11 (3 g, 12.9 mmol) in THFꢀMeOH (20 mL, 1:1, v/v) was treated with
2N NaOH (10 mL, 20.0 mmol) solution for 2 h. The reaction mixture
was diluted with ethyl acetate (30 mL) and treated with 2N HCl solution
(20 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude residue was purified
by flash chromatography (60:40 f 20:80, hexanes:EtOAc) to give
diazido-acid 12 as white solid (2.5 g, 89%). 1H NMR (400 MHz,
CDCl3): δ 7.83 (s, 1H), 7.73 (s, 1H), 7.22 (s, 1H), 4.45 (s, 2H). 13C
NMR (100 MHz, CDCl3): δ 170.8, 141.5, 138.2, 131.5, 125.8, 123.5,
120.2, 53.8.
Method A. General amide coupling reaction using EDC/HOBt/
DIPEA/CH2Cl2: To a stirred solution of an acid (1 equiv) in anhydrous
CH2Cl2 was added EDC (1.1 equiv) followed by HOBt (1.05 equiv) at
0 °C. After 1 h, a solution of amine/amine hydrochloride (1 equiv) and
DIEPA (3 equiv) in CH2Cl2 was added to the reaction mixture dropwise
at 0 °C. The mixture was allowed to stir at rt for 4ꢀ6 h (monitored by
TLC), treated with 2N HCl solution, and extracted with CH2Cl2. The
organic layer was washed with saturated aqueous NaHCO3 solution
followed by brine. The organic extracts were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
further purified by flash chromatography.
to stir at rt for 1 h. The reaction was diluted with ethyl acetate (20 mL)
and acidified with 2N HCl (20 mL). The organic layer was separated,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
The crude acid (15) obtained as white solid was directly used for
coupling reaction with methyl 7-aminoheptanoate hydrochloride fol-
lowing the method A to afford 16 as white solid (3 g, 72%). 1H NMR
(400 MHz, CDCl3): δ 7.72 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H),
6.90 (bs, 1H), 6.88 (s, 1H), 6.82 (bs, 1H), 3.67 (s, 3H), 3.46 (q, J = 6.8,
13.6 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.65 (m, 4H), 1.54 (s, 9H), 1.38
(m, 4H). 13C NMR (100 MHz, CDCl3): δ 174.1, 171.3, 159.2, 159.0,
152.3, 140.7, 126.8 (2C), 121.3, 118.4 (2C), 98.1, 81.1, 51.5, 39.4, 33.9,
29.2, 28.7, 28.3 (3C), 26.5, 24.7.
7-{[5-(4-Azidophenyl)isoxazole-3-carbonyl]amino}heptanoic Acid
Methyl Ester (18). A solution of 16 (2 g, 4.5 mmol) in CH2Cl2 (20 mL)
was treated with trifloroacetic acid (1 mL, 13.5 mmol) for 4 h at rt. The
solvent was evaporated, and the residue was azeotroped with toluene
(3 ꢁ 10 mL) under reduced pressure. The crude aniline 17 obtained as
pale yellow solid was directly used in the next step without any further
purification.
To a solution of 17 (0.5 g, 1.5 mmol) in AcOHꢀH2O (9:1, v/v,
8 mL) at 0 °C was added NaNO2 (0.12 g, 1.74 mmol) and the mixture
was stirred for 10 min. To this orangeꢀred colored solution was then
added portionwise NaN3 (0.14 g, 2.17 mmol) at the same temperature,
and stirring was continued for additional 1 h. The reaction mixture was
neutralized by adding saturated aqueous NaHCO3 solution and ex-
tracted with ethyl acetate (3 ꢁ 25 mL). The combined organic extracts
were washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (90:10 f 70:30, hexanes:EtOAc) to afford 18 (0.38
g, 71%). 1H NMR (400 MHz, CDCl3): δ 7.60 (d, J = 8.4 Hz, 2H), 7.10
(d, J = 8.4 Hz, 2H), 6.93 (bs, 1H), 6.91 (s, 1H), 3.65 (s, 3H), 3.45 (q, J =
6.6, 13.3 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.70ꢀ1.47 (m, 4H),
1.40ꢀ1.30 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 174.9, 170.9,
159.3, 158.8, 127.47, 123.45, 119.69, 98.96, 51.48, 39.44, 33.92, 29.26,
28.72, 26.52, 24.76. MS (ESI) m/z 372.2 (M þ H)þ.
7-({5-[4-(3-Azido-5-azidomethylbenzoylamino)phenyl]isoxazole-
3-carbonyl}amino)heptanoic Acid Methyl Ester (19). To a mixture
of aniline 17 (500 mg, 1.45 mmol) and diazido-acid 12 (475 mg,
2.17 mmol) in anhydrous CH2Cl2 (10 mL) was added HOBt (315 mg,
2.32 mmol). After the solution was cooled to 0 °C in an ice bath,
DCC (480 mg, 2.32 mmol) was added followed by DMAP (90 mg,
0.07 mmol). The reaction solution was stirred at rt for 6 h. The mixture
was concentrated under reduced pressure, and the crude residue
obtained was purified by flash chromatography (95:05 f 80:20,
1
hexanes:EtOAc) to afford 19 as white solid (550 mg, 70%). H NMR
5-(4-tert-Butoxycarbonylaminophenyl)isoxazole-3-carboxylic Acid
Ethyl Ester (14). To a solution of alkyne 13 (3 g, 13.8 mmol) in
THFꢀH2O (1:1, 60 mL) was added Na2CO3 (18 g, 170.0 mmol) in one
portion at 0 °C. A solution of 2-chloro-2-hydroxyiminoacetic acid ethyl
ester (5.3 g, 35.0 mmol) in THF (40 mL) was added slowly over 18 h
using a syringe pump. The reaction mixture was diluted with ethyl
acetate (50 mL) and washed with water followed by brine. The organic
fractions were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude residue was purified by flash chromatog-
raphy (90:10 f 70:30, hexanes:EtOAc) to give the isoxazole 14 as pale-
(400 MHz, CDCl3): δ 9.81 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.70 (d, J =
8.1 Hz, 2H) 7.65 (s, 1H), 7.63 (s, 1H), 7.59 (s, 1H), 7.09 (m, 2H), 6.85
(s, 1H), 4.19 (s, 2H), 3.60 (s, 3H), 3.37 (m, 2H), 1.58ꢀ1.54 (m, 4H),
1.33 (br s, 4H). 13C NMR (100 MHz, CDCl3): δ 170.32, 169.87, 160.20,
158.79, 141.46, 140.20, 138.15, 137.71, 128.63, 1256.41, 122.83, 120.02,
118.64, 100.50, 53.29, 33.64, 29.84, 28.49, 26.45, 25.33. MS (ESI) m/z
546.1 (M þ H)þ.
7-(1H-Pyrazol-4-ylcarbamoyl)heptanoic Acid Methyl Ester (22). An
amide coupling reaction between aminopyrazole 21 (1 g, 12.0 mmol)
and monomethylsuberate (2.27 g, 12.0 mmol) following method A
1
1
yellow solid (3.8 g, 83%). H NMR (400 MHz, CDCl3): δ 7.70 (d,
resulted in 22 as pale-brown solid (2.1 g, 69%). H NMR (400 MHz,
J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.04 (s, 1H), 6.81 (s, 1H), 4.44
(q, J = 8.0, 16.0 Hz, 2H), 1.54 (s, 9H), 1.45 (t, J = 8.0, Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 171.5, 160.1, 156.8, 152.5, 141.0, 126.8 (2C),
121.0, 118.4 (2C), 98.9, 81.0, 62.1, 28.2 (3C), 14.1.
7-{[5-(4-tert-Butoxycarbonylaminophenyl)isoxazole-3-carbonyl]-
amino}heptanoic Acid Methyl Ester (16). An ice cooled solution of 14
(3.0 g, 9.4 mmol) in THFꢀMeOH (20 mL, 1:1) was treated with 2N
NaOH solution (7 mL, 14 mmol), and the reaction mixture was allowed
DMSO-d6): δ 12.50 (s, 1H), 9.82 (s, 1H), 7.95 (s, 1H), 7.81 (s, 1H),
3.54 (s, 3H), 2.28 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.55ꢀ1.50
(m, 4H), 1.29ꢀ1.24 (m, 4H). 13C NMR (100 MHz, DMSO-d6):
δ 173.3, 168.8, 130.5, 121.7, 118.82, 51.6, 35.9, 33.8, 28.8, 25.5, 24.2.
MS (ESI) m/z 254.2 (M þ H)þ.
7-[1-(3-Azido-5-azidomethylbenzyl)-1H-pyrazol-4-ylcarbamoyl]-
heptanoic Acid Methyl Ester (23). A mixture of 22 (200 mg,
0.79 mmol), 8 (300 mg, 0.84 mmol), and powdered K2CO3 (220 mg,
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1.60 mmol) in acetone (15 mL) was refluxed for 6 h. The mixture was
cooled to rt and concentrated, leaving a residue which was dissolved in
ethylacetate and washed with water followed by brine. The organic
fractions were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by flash chromatog-
raphy (80:20 f 50:50, hexanes:EtOAc) to furnish 23 as pale-yellow
solid (260 mg, 75%). 1H NMR (400 MHz, CDCl3): δ 7.99 (s, 1H), 7.43
(s, 1H), 7.42 (s, 1H), 7.27(s, 1H), 6.91 (s, 1H), 6.81 (s, 1H), 5.30
(s, 2H), 4.31 (s, 2H), 2.36ꢀ2.30 (m, 4H), 1.72ꢀ1.64 (m, 4H), 1.34 (m,
4H). 13C NMR (100 MHz, CDCl3): δ 174.4, 170.5, 141.2, 139.1, 138.1,
130.6, 123.4, 121.9, 121.4, 118.0, 117.9, 55.5, 53.9, 51.5, 36.3, 33.9, 28.7,
28.6, 25.4, 24.6. MS (ESI) m/z 440.2 (M þ H)þ.
organic fractions were washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude residue was
purified by flash chromatography (70:30 f 50:50, hexanes:EtOAc) to
afford 27 (220 mg, 68%). 1H NMR (400 MHz, CDCl3): δ 8.18 (s, 1H),
8.08 (s, 1H), 7.42 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz,
2H), 5.17 (s, 2H), 3.65 (s, 3H), 2.29ꢀ2.19(m, 4H), 1.68ꢀ1.60 (m, 4H),
1.23 (br s, 4H). 13C NMR (100 MHz, CDCl3): δ 174.3, 170.6, 139.0,
133.0, 130.3, 129.3, 121.6, 121.3, 119.4, 55.7, 53.5, 36.4, 33.9, 28.7, 28.7,
24.7. MS (ESI) m/z 385.2 (M þ H)þ.
7-[1-(4-Azidobenzyl)-1H-pyrazol-4-ylcarbamoyl]heptanoic Acid Methyl
Ester (28). To a stirred solution of 26 (200 mg, 0.56 mmol) and
triethylamine (120 μL, 0.84 mmol) in CH2Cl2 (10 mL) was added Boc-
anhydride (150 mg, 0.69 mmol) at rt. After 4 h, the mixture was
concentrated and the crude residue was purified by flash chromatogra-
phy (80:20 f 50:50, hexanes:EtOAc) to furnish 28 (210 mg, 82%). 1H
NMR (400 MHz, CDCl3): δ 8.23 (s, 1H), 7.80 (s, 1H), 7.59 (s, 1H),
7.36 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 6.94 (s, 1H), 5.17 (s,
2H), 3.81 (s, 3H), 2.29ꢀ2.21 (m, 4H), 1.64ꢀ1.56 (m, 4H), 1.47 9 (s,
9H), 1.23 (br s, 4H). 13C NMR (100 MHz, CDCl3): δ 174.4, 70.6,
152.6, 138.4, 130.4, 130.4, 129.3, 128.0, 121.6, 121.1, 118.9, 55.5, 51.5,
36.4, 33.9, 29.7, 28.8, 28.3, 27.9, 25.4, 24.7. MS (ESI) m/z 459.3
(M þ H)þ.
7-(1-Benzyl-1H-pyrazol-4-ylcarbamoyl)heptanoic Acid Methyl Es-
ter (24). To a solution of 22 (200 mg, 0.79 mmol) in anhydrous DMF
(5 mL) at 0 °C was added portionwise NaH (22 mg, 0.95 mmol). After
10 min, benzylbromide (150 mg, 0.88 mmol) was added to the reaction
mixture at the same temperature. After 4 h stirring at rt, the reaction was
quenched with ice water and extracted with ethyl acetate (3 ꢁ 10 mL).
Organic layer was further washed with water followed by brine. The
combined organic fractions were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude residue was purified
by flash chromatography (75:25 f 50:50, hexanes:EtOAc) to afford 24
(190 mg, 70%). 1H NMR (400 MHz, CDCl3): δ 7.95 (s, 1H), 7.43 (s,
1H), 7.36ꢀ7.30 (m, 3H), 7.27ꢀ7.23 (m, 3H), 5.25 (s, 2H), 3.67 (s, 3H),
2.36ꢀ2.30 (m, 4H), 1.72ꢀ1.68 (m, 4H), 1.83 (m, 4H). MS (ESI) m/z
344.2 (M þ H)þ.
7-{1-[4-(3-Azido-5-azidomethylbenzoylamino)benzyl]-1H-pyrazol-
4-ylcarbamoyl}heptanoic Acid Methyl Ester (29). Compound 29
(190 mg, 61%) was obtained by a coupling reaction between acid 12
(122 mg, 0.56 mmol) and amine 26 (200 mg, 0.56 mmol) following
method A. 1H NMR (400 MHz, CDCl3): δ 8.84 (s, 1H), 8.24 (s, 1H),
8.00 (s, 1H), 7.79 (s, 1H), 7.58 (s, 1H), 7.50 (d, J = 8.1 Hz, 2H), 7.40 (s,
1H), 7.34 (s, 1H), 7.06 (d, J = 8.2 Hz, 2H), 5.30 (s, 2H), 4.35 (s, 2H),
2.30ꢀ2.23 (m, 4H), 1.66ꢀ1.54 (m, 4H), 1.27 (br s, 4H). 13C NMR
(CDCl3, 75 MHz): δ 174.4, 170.7, 164.7, 141.4, 138.1, 137.7, 137.0,
132.6, 130.6, 128.4, 123.2, 121.6, 121.4, 121.1, 121.1, 117.9, 55.8, 53.9,
36.6, 36.4, 33.9, 31.5, 28.8, 28.7, 25.4, 24.6, 65.3, 162.3, 160.7, 125.6. MS
(ESI) m/z 560.2 (M þ H)þ.
7-[1-(4-Nitrobenzyl)-1H-pyrazol-4-ylcarbamoyl]heptanoic Acid Methyl
Ester (25). To a solution of 22 (1.6 g, 6.32 mmol) in anhydrous DMF
(15 mL) at 0 °C was added portionwise NaH (175 mg, 7.6 mmol). After
10 min, 4-nitrobenzylbromide (1.5 g, 7.0 mmol) was added to the
reaction mixture at the same temperature. After 4 h stirring at rt, the
reaction was quenched with ice water and extracted with ethyl acetate
(3 ꢁ 30 mL). Organic layer was further washed with water followed by
brine. The combined organic fractions were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude residue was
purified by flash chromatography (70:30 f 40:60, hexanes:EtOAc) to
give 25 as pale yellow solid (1.62 g, 66%). 1H NMR (400 MHz, DMSO-
d6): δ 8.17 (d, J = 8.3 Hz, 2H),8.05 (s, 1H), 7.43 (s, 1H), 7.34 (s, 1H),
7.32 (s, 1H), 5.34 (s, 2H), 3.66 (s, 3H), 2.33ꢀ3.29 (m, 4H), 1.71ꢀ1.64
(m, 4H), 1.23 (br s, 4H). 13C NMR (100 MHz, CDCl3): δ 174.3, 170.4,
147.6, 143.7, 130.8, 128.2 (2C), 124.0 (2C), 122.0, 121.7, 55.3, 51.5,
36.4, 33.9, 28.7, 28.6, 25.3, 24.6. MS (ESI) m/z 389.2 (Mþ þ H).
7-[1-(4-Aminobenzyl)-1H-pyrazol-4-ylcarbamoyl]heptanoic Acid Methyl
General Method for the Preparation of Hydroxamate. All
the following hydroxamates were prepared from their corresponding
methyl ester precursors on 100ꢀ200 mg scale with ca. 60ꢀ70% yield.
To a solution of hydroxylamine hydrochloride (200 mmol) in MeOH
at 0 °C was added portionwise powdered KOH (205 mmol). The
solution was stirred for 1 h at rt after the addition was complete. The
precipitate was filtered off, and the filtrate containing NH2OH in MeOH
was added dropwise to an ice cooled solution of methyl ester (1 mmol)
in MeOH (5 mL). An additional amount of powdered KOH (10 mmol)
was added to the reaction mixture, and the white solution was stirred for
3 h at rt. MeOH was concentrated at rt under reduced pressure. The
residual solid was treated with saturated aqueous NH4Cl solution
(20 mL) and extracted with ethyl acetate (3 ꢁ 25 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
on Biotage using reverse phase C-18 column (90:10 f 10:90, H2O:
MeOH). The purity of all hydroxamates was found to be g95% as it was
analyzed by reversed-phase LCMS.
Ester (26). A mixture of 25 (1.6 g, 4.1 mmol) and SnCl2 2H2O (2.8 g,
3
12.4 mmol) in methanol (20 mL) was heated at 70 °C for 2 h. The pH of
the reaction mixture was adjusted to 7ꢀ8 by addition of saturated
aqueous NaHCO3 solution. The resulting mixture was extracted
with CHCl3 (3 ꢁ 20 mL). The organic phase was washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
crude residue was purified by flash chromatography (100:00 f 90:10,
CH2Cl2:MeOH) to give aniline 26 as orangeꢀyellow solid (1.4 g, 95%).
1H NMR (400 MHz, methanol-d4): δ 7.83 (s, 1H), 7.48 (s, 1H), 7.02
(d, J = 8.0 Hz, 2H), 6.68 (d, J = 8.0 Hz, 2H), 5.10 (s, 2H), 3.65 (s, 3H),
2.30 (m, 4H), 1.64 (m, 4H), 1.36 (s, 4H). 13C NMR (100 MHz,
methanol-d4): δ 174.6, 171.7, 147.4, 129.9, 129.3, 128.7, 125.5, 121.7,
120.7, 115.1, 115.0, 55.4, 50.7, 35.7, 33.4, 28.5, 28.4, 25.3, 24.4.
5-(4-Azidophenyl)isoxazole-3-carboxylic Acid (6-Hydroxycarbamoylhex-
yl)amide (2a). 1H NMR (400 MHz, DMSO-d6): δ 10.39 (s, 1H), 8.81
(br s, 1H), 8.66 (s, 1H), 7.92 (m, 2H), 7.56 (s, 1H), 7.36 (m, 2H), 3.24
(q, J = 6.5, 13.0 Hz, 2H), 1.94 (t, J = 7.2 Hz, 2H), 1.52ꢀ1.45 (m, 4H),
1.17 (br s, 4H). 13C NMR (100 MHz, DMSO-d6): δ 170.7, 169.9, 160.2,
158.9, 144.3, 143.3, 131.3, 129.8, 128.0, 126.8, 123.5, 120.5, 100.0, 32.7,
29.2, 28.6, 26.6, 25.5. MS (ESI) m/z 395.1 (M þ Na)þ.
7-[1-(4-Azidobenzyl)-1H-pyrazol-4-ylcarbamoyl]heptanoic Acid Methyl
Ester (27). To a solution of 26 (300 mg, 0.84 mmol) in AcOHꢀH2O
(9:1, v/v, 5 mL) at 0 °C was added NaNO2 (70 mg, 1.01 mmol) in one
portion. The resulting orangeꢀred colored mixture was stirred at the
same temperature for 10 min. To this was then added portionwise NaN3
(85 mg, 1.3 mmol), and stirring was continued for additional 1 h. The
reaction mixture was neutralized by adding saturated aqueous NaHCO3
solution and extracted with ethyl acetate (3 ꢁ 20 mL). The combined
5-[4-(3-Azido-5-azidomethylbenzoylamino)phenyl]isoxazole-3-
carboxylic Acid (6-Hydroxycarbamoylhexyl)amide (2b). 1H NMR
(400 MHz, DMSO-d6): δ 10.62 (s, 1H), 10.19 (s, 1H), 8.77 (m, 1H),
8.64 (s, 1H), 7.92 (m, 4H), 7.73 (s, 1H), 7.59 (s, 1H), 7.39 (s, 1H), 7.25
(s, 1H), 4.59 (s, 2H), 3.25 (q, J = 6.9, 13.3 Hz, 2H), 2.94 (t, J = 7.3 Hz,
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2H), 1.92 (t, J = 7.1 Hz, 2H), 1.52ꢀ1.45 (m, 4H), 1.19 (br s, 4H).
13C NMR (100 MHz, DMSO-d6): δ 170.4, 169.3, 160.0, 158.9, 141.6,
140.7, 138.3, 137.1, 129.6, 126.8, 122.4, 120.9, 118.4, 99.5, 53.2, 32.7,
29.2, 28.7, 26.6, 25.5. MS (ESI) m/z 547.2 (M þ H)þ.
of inhibitor for 5 min. The assay mixture was treated with 25 μL of
10 μM HDAC substrate KI-178 (BIOMOL) for 25 min at rt. The
reaction was quenched with 50 μL of 50 μg/mL trypsin and 4 μM
trichostatin A in KI-143 (BIOMOL) for 30 min. The plate was read on a
POLARstar OPTIMA (BMG labtech) microplate reader at excitation
wavelength 360 nm and emission wavelength 460 nm.
10 ng HDAC3/NCoR2 (BPS Bioscience) was diluted with KI-311
(BIOMOL) and preincubated with 30 μL of inhibitor for 5 min then
10 μL, 125 μM HDAC fluorescent substrate Boc-L-Lys (Ac)-AMC
(Chem-Impex) was added, and the mixture incubated for 30 min at
room temperature. The reaction was quenched with 50 μL of 1 mg/mL
trypsin and 4 μM trichostatin A in KI-143 (BIOMOL) for 30 min. The
plate was read on an OptimaPolar Starmicroplate reader (BMG labtech)
at excitation wavelength 360 nm and emission wavelength 460 nm.
The IC50 values were determined using the GraphPad Prism 5
software (GraphPad Software Inc., La Jolla, CA).
Octanedioic Acid (1-Benzyl-1H-pyrazol-4-yl)amide Hydroxyamide
(3a). 1H NMR (400 MHz, DMSO-d6): δ 10.35 (s, 1H), 10.24 (s, 1H),
9.92 (s, 1H), 7.97 (s. 1H), 7.41 (s, 1H), 7.35ꢀ7.28 (m, 3H), 7.23ꢀ7.20
(m, 2H), 5.26 (s, 2H), 2.23ꢀ2.17 (m, 2H), 1.95ꢀ1.91(m, 2H),
1.55ꢀ1.47(m, 4H), 1.24 (m, 4H). 13C NMR (100 MHz, methanol-
d4): δ 171.8, 171.5, 136.3, 130.4, 128.6 (2C), 127.9, 127.4 (2C), 121.9,
121.4, 55.9, 35.9, 32.5, 28.4, 28.3, 25.3, 25.1. MS (ESI) m/z 345.2
(M þ H)þ.
Octanedioic Acid Hydroxyamide [1-(4-Nitrobenzyl)-1H-pyrazol-4-
yl]amide (3b). 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.93
(s, 1H), 8.99 (s, 1H), 8.20 (d, J = 8.6 Hz, 2H), 8.06 (s, 1H), 7.45 (s, 1H),
7.40 (d, J = 8.6 Hz, 2H), 5.44 (s, 2H), 2.21 (t, J = 7.3 Hz, 2H), 1.92 (t,
J = 7.3 Hz, 2H), 1.53ꢀ1.45 (m, 4H), 1.26 (br s, 4H) ppm. 13C NMR
(100 MHz, DMSO-d6): δ 170.7, 170.4, 146.7, 144.1, 130.4, 130.3, 127.5,
122.9, 121.5, 120.9, 53.7, 34.5, 31.7, 27.8, 27.7, 24.6, 24.5. MS (ESI) m/z
390.2 (M þ H)þ.
The description of assays against HDAC 1, 2, 4ꢀ7, 9ꢀ11 and MMPs
1, 3, 9, 14 is given in the Supporting Information.
Cell Culture. HepG2 (ATCC) hepatocarcinoma cells and Hela
(ATCC) cervical adenocarcinoma cells were grown in D7 medium
containing DMEM supplemented with 7% FBS, 2 mM L-glutamine,
100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM sodium
pyruvate. SH-SY5Y (ATCC) neuroblastoma cells were grown in SH-
SY5Y medium containing equal amounts of MEM/EBSS and Ham’s F12
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin,
100 μg/mL streptomycin, 1% NEAA, and 0.1% sodium bicarbonate.
Cells were grown in 10 cm2 dishes to a density of 80% confluence and
were differentiated in the SH-SY5Y media (1% FBS 2 mM L-glutamine,
100 U/mL penicillin, 100 μg/mL streptomycin, 1% NEAA, 0.1%
sodium bicarbonate) supplemented with 10 μM retinoic acid (RA,
Sigma) for 7ꢀ8 days. The differentiating medium was replaced every
2ꢀ3 days. Cells were maintained at 37 °C in a humidified atmosphere
supplemented with 5% CO2.
Antiproliferation Assays. Hepatocarcinoma HepG2 cells were seeded
at 1.2 ꢁ 104 cells/well into 96 well plates in D7 medium. Hela cells were
seeded at 0.2 ꢁ 104 cells/well into 96-well plates in D7 medium.
Neuroblastoma SH-SY5Y cells were seeded at 0.9 ꢁ 104 cells/well into
96-well plates in 10% FBS SH-SY5Y medium. After 24 h, the medium
(D7 prepared with phenol red free DMEM for HepG2 and Hela cells,
10% SH-SY5Y medium containing equal amounts of phenol red
free DMEM and Ham’s F12 supplemented with 10% FBS, 2 mM
L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1% NEAA,
and 0.1% sodium bicarbonate for the neuroblastoma cells) was changed
to a medium containing either vehicle (DMSO) or 0.5ꢀ50 μM final
concentrations of HDAC inhibitors. After 24, 48, or 72 h incubation, cell
viability was measured using the XTT cell proliferation assay (Roche).
Data from at least three independent experiments were analyzed using
the GraphPad Prism 5.0 software and EC50s deduced from the best fits
of the data using the nonlinear regression with variable slope equations.
Western Blot Analysis in HepG2 Cells. Hepatocarcinoma HepG2 cells
were seeded at 2.7 ꢁ 105 cells/well into 6-well plates in D7 media. After
24 h, the media was changed to fresh D7 media and the cells were
incubated at 37 °C. After 1 h incubation, cells were treated with vehicle
(DMSO) or 1, 5, or 10 μM 2b or SAHA. Cells were then incubated in the
dark for 6 h, after which the cells were harvested and the cell pellets
frozen and stored at ꢀ80 °C. Cells were lysed in ice cold 1% CHAPSO
lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%
CHAPSO, complete EDTA-free protease inhibitor cocktail from
Roche) for 45 min at 4 °C. Clarified lysates were quantified by the
method of Bradford31, and 40 μg of total protein per lysate was loaded
on a Tris-glycine 12.5% acrylamide gel and submitted to SDS PAGE
and electrophoretically transferred onto PVDF membrane (Millipore)
using a Trans-blot SD semidry electrophoretic transfer cell (Biorad).
Membranes were blocked in Tris buffered saline solution containing
Octanedioic Acid [1-(4-Azidobenzyl)-1H-pyrazol-4-yl]amide Hy-
1
droxyamide (3c). H NMR (400 MHz, DMSO-d6): δ 10.31 (s, 1H),
9.87 (s, 1H), 8.64 (s, 1H), 7.95 (s, 1H), 7.39 (s, 1H), 7.26 (d, J = 8.1 Hz,
2H), 7.10 (d, J = 8.1 Hz, 2H), 5.24 (s, 2H), 2.19 (t, J = 7.4 Hz, 2H),
1.92 (t, J = 7.2 Hz, 2H), 1.52ꢀ1.40 (m, 4H), 1.23 (br s, 4H). 13C NMR
(100 MHz, DMSO-d6): δ 170.7, 170.4, 138.9, 133.1, 129.8, 128.5 (2C),
121.4, 120.4, 118.4 (2C), 54.1, 35.0, 31.7, 27.8, 27.7, 24.6, 24.5. MS
(ESI) m/z 386.2 (M þ H)þ.
{4-[4-(7-Hydroxycarbamoylheptanoylamino)pyrazol-1-ylmethyl]-
phenyl}Carbamic Acid tert-Butyl Ester (3d). 1H NMR (400 MHz,
DMSO-d6): δ 10.31 (s, 1H), 10.06 (s, 1H), 9.86 (s, 1H), 9.29 (s, 1H),
8.63 (s, 1H), 7.88 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz,
2H), 5.14 (s, 2H), 2.19 (t, J = 7.2 Hz, 2H), 1.92 (t, J = 7.1 Hz, 2H),
1.50ꢀ1.46 (m, 4H), 1.26 (br s, 4H). 13C NMR (100 MHz, DMSO-d6):
δ 169.9, 169.6, 153.2, 139.5, 131.4, 128.7, 122.5, 120.5, 118.6, 79.5, 55.0,
35.9, 32.7, 28.8, 25.6, 25.5. MS (ESI) m/z 482.2 (M þ Na)þ.
Octanedioic Acid {1-[4-(3-Azido-5-azidomethylbenzoylamino)benzyl]-
1H-pyrazol-4-yl}amide Hydroxyamide (3e). 1H NMR (400 MHz,
DMSO-d6): δ 10.38 (s, 1H), 10.34 (s, 1H), 9.89 (s, 1H), 7.93 (s,
1H), 7.74 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.63 (s, 1H), 7.40 (s, 1H),
7.34 (s, 1H), 7.23 (d, J = 8.2 Hz, 2H), 5.22 (s, 2H), 4.58 (s, 2H), 2.21
(t, J = 7.2 Hz, 2H), 1.92 (t, J = 7.1 Hz, 2H), 1.55ꢀ1.45 (m, 4H), 1.23
(br s, 4H). 13C NMR (100 MHz, DMSO-d6): δ 171.0, 169.5, 164.6,
140.4, 138.6, 133.4, 128.6, 124.4, 122.4, 120.8, 118.3, 53.2, 49.1, 32.7,
28.8, 25.6, 25.5. MS (ESI) m/z 560.2 (M þ H)þ.
Octanedioic Acid [1-(3-Azido-5-azidomethylbenzyl)-1H-pyrazol-4-
yl]amide Hydroxyamide (3f). 1H NMR (400 MHz, DMSO-d6): δ 10.31
(bs, 1H), 9.89 (bs, 1H), 8.60 (s, 1H), 8.06 (s, 1H), 7.42 (s, 1H), 7.06 (s,
1H), 7.02 (s, 1H), 6.94 (s, 1H), 5.47 (s, 2H), 4.46 (s, 2H), 2.20 (t, J =
8.0 Hz, 2H), 1.93 (t, J = 8.0 Hz, 2H), 1.53ꢀ1.44 (m, 4H), 1.26 (m, 4H)
ppm. 13C NMR (100 MHz, DMSO-d6): δ 170.1, 169.6, 147.3, 146.0,
132.0, 128.9, 124.2, 122.7, 121.4, 54.4, 49.1, 35.9, 32.7, 28.9, 28.8, 25.6,
25.5 ppm. MS (ESI) m/z 441.2 (M þ H)þ.
Modeling Methods. Using Surflex-dock29 with the -pgeomx flag
for more exhaustive docking, and otherwise default parameters, we
docked all ligands to multiple crystal structures of HDAC8, choosing the
best scoring as the final binding pose. For scoring we used a variation of a
fragment based scoring approach that only considers interactions of the
SBG and the linker, but not the ZBG. More details about the
methodology are given elsewhere.30
Biological Materials and Methods. HDAC 3 and 8 Activity
Assay. HDAC inhibition assay was performed in 96-well half-area
microplate (Corning). 0.375U HDAC8 (BIOMOL) was diluted with
HDAC8 assay buffer KI-311 (BIOMOL) and preincubated with 10 μL
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0.5% Tween 20 and 5% nonfat milk and then probed for total p53
(monoclonal antibody, Millipore), acetylated Lys373-p53 (Upstate),
acetylated Lys8-Histone H4 (Upstate), cleaved caspase 3 Asp175 (Cell
Signaling Technology), or GAPDH (Chemicon). Membranes were
washed and subsequently incubated with the appropriate peroxidase
conjugated secondary antibody (anti-Rabbit IgG or anti-Mouse IgG,
GE). The detection was done using the SuperSignal West Pico
chemiluminescence substrate (Pierce). Blots were analyzed on a G:
Box gel documentation system and the images captured using the Gene
Snap software (Syngene, Frederick, MD). Band intensities were quanti-
fied using the Image J freeware from the NIH Web site (http://rsbweb.
nih.gov/ij/).
Neuroprotection Assays. Differentiated SH-SY5Y cells were seeded
at 2 ꢁ 104 cells/well into 96-well plates in 1% FBS media containing
5 μM retinoic acid. After 24 h, the medium was changed to 1% phenol
red free SH-SY5Y media containing phenol red free DMEM and Ham’s
F12 supplemented with 1% FBS, 2 mM L-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin, 1% NEAA, and 0.1% sodium
bicarbonate with 5 μM retinoic acid containing either vehicle
(DMSO) or 0.5ꢀ50 μM final concentrations of HDAC inhibitors in
the absence (Figure 8, dark gray) or presence (Figure 8, light gray) of
100 μM of 30. After 24 h incubation, cell viability was measured using
the XTT cell proliferation kit II (Roche Applied Science). Data are
presented as the average percent survival of four replicates þ standard
deviation, relative to vehicle (100%). Statistical analysis was performed
using one-way ANOVA with Dunnett’s secondary analysis.
Biocomputing, Visualization, and Informatics at the University of
California, San Francisco (supported by NIH P41 RR-01081),
Open Eye Scientific Software, Santa Fe, NM, and Surflex package
from Prof. Ajay N. Jain, San Mateo, CA (supported by NIH R01
GM070481). We thank Aditya Sudheer Vaidya and Hazem
Abdelkarim for conducting HDAC1 and HDAC2 inhibitory
assays for SAHA, TSA, 2b, and 3f.
’ ABBREVIATIONS USED
HDAC, histone deacetylase; BEProFL, (B)inding (E)nsemble
(Pro)filing with (F)photoaffinity (L)abeling; SBG, surface
binding group; ZBG, zinc binding group; SAHA, suberoylanilide
hydroxamic acid
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’ ASSOCIATED CONTENT
S
Supporting Information. Activity assay protocols for
b
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’ AUTHOR INFORMATION
Corresponding Author
*For P.A.P.: phone, 312-996-4174; fax, 312-996-7107; E-mail,
pap4@uic.edu. For S.Y.B.: phone, þ33 (0)1 42 49 48 78; fax,
þ33 (0)1 42 49 48 38; E-mail, sylvie.blond@inserm.fr.
Present Addresses
1Present address for S.V.: Translational Chemistry & Medicine,
Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road (SP30ꢀ3156), La Jolla, CA.
92037. For M.B.: Research Center Pharmaceutical Engineering,
Inffeldgasse 21a/II, 8010 Graz, Austria.
Author Contributions
These authors contributed equally.
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’ ACKNOWLEDGMENT
This study was funded by the National Cancer Institute/NIH
Grant R01CA131970 (P.A.P. and S.Y.B.) and, in part by the
INSERM, the University Denis Diderot/Paris 7 and a Chaire of
Excellence from the Agence Nationale pour la Recherche, France
(S.Y.B.). Molecular modeling studies were in part perfor-
med with the UCSF Chimera package from the Resource for
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Journal of Medicinal Chemistry
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