Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

Article abstract of DOI:10.1016/j.bmcl.2012.06.017

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.

Full text of DOI:10.1016/j.bmcl.2012.06.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5025–5030
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, modeling, biological evaluation and photoaffinity labeling
studies of novel series of photoreactive benzamide probes for histone
deacetylase 2
Aditya Sudheer Vaidya a, , Bhargava Karumudi a, , Emma Mendonca b, Antonett Madriaga a,
Hazem Abdelkarim a, Richard B. van Breemen a, Pavel A. Petukhov a,
⇑
a Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA
b Department of Bioengineering, University of Illinois at Chicago, 851 South Morgan Street, Chicago, IL 60607, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide
probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and
are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit
a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by
the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the
biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are
cell permeable and target the nuclear HDACs.
Received 12 April 2012
Revised 4 June 2012
Accepted 6 June 2012
Available online 18 June 2012
Keywords:
Benzamides
HDAC 1 and 2
Time-dependent inhibition
Photoaffinity labeling
Diazides
Ó 2012 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) are considered viable drug tar-
gets for multiple therapeutic applications including cancer and
neurological diseases.1,2 Recently, Cravatt et al.3 and Gottesfeld
et al.4 described the design and applications of photoaffinity probes
for profiling HDACs in native proteomes and live cells. The scaffold
of the probes included a portion of a pan HDAC inhibitor suberoyl
anilide hydroxamic acid (SAHA), a benzophenone group as a
photoreactive group, and an alkyne handle to attach an azide con-
taining reporter tag via (3+2) cycloaddition. Attempts to use the
same features based on HDAC1 and 2 selective benzamide scaf-
(3+2) cycloaddition with an alkyne moiety of the biotin-containing
reporter group. Based on these features, we have successfully de-
signed and synthesized highly potent and selective probes for
HDAC3 and HDAC8 and demonstrated that they are cell permeable
and exhibit excellent antiproliferative activity against several can-
cer cell lines.8 Our main objective in this study was to design
photoreactive benzamide probes for HDAC2 and evaluate their
activity/selectivity profile for other class I HDAC isoforms.
We hypothesized that a set of potent and selective benzamide-
based probes capable of crosslinking with HDAC2 can be designed
by appropriately decorating benzamides 1 and 2 (Fig. 1) with a
combination of the aryl and alkyl azides. Both 1 and 2 and their
derivatives were reported by Delorme,9 Miller,10 Gangloff,11 and
their colleagues to be active and selective inhibitors of HDACs1
and 2.
folds resulted in probes with HDAC potency above 180
lM in HeLa
cell nuclear lysate.5
We have already established the Binding (E) nsemble (Pro) fil-
ing with (F) photoaffinity (L) abeling approach (BEProFL) where
we have experimentally mapped the multiple binding modes of
diazide based photoreactive probes for HDACs.6 The design of these
probes included decoration of HDAC ligands with a 3-azido-5-azi-
domethylene moiety, a photoaffinity labeling group originally pro-
posed by Suzuki et al.7 for specific labeling of the catalytic portion
of HMG-CoA reductase. The aromatic azido moiety was used as a
photoreactive group and the aliphatic azide was well suited for
Ph
Ph
O
O
N
H
H
N
O
NH2
N
H
NH2
O
⇑
Corresponding author. Tel.: +1 312 996 4174; fax: +1 312 996 7107.
E-mail address: pap4@uic.edu (P.A. Petukhov).
1
2
These authors contributed equally to the work.
Figure 1. Benzamide inhibitors of HDAC 1 and 2.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.017

5026
A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5025–5030
O
R1
O
R4
R2
R3
R1
R2
OH
OH
H
N
O
H2N
R3
O
NHBoc
3, R1 = H, R2 = N3, R3 = H
7, R1 = N3, R2 = H, R3 = H
9, R4 = Ph
1
2
3
4, R1 = N3, R2 = H, R3 = CH2N3
5, R1 = N3, R2 = -O(CH2)2N3 H, R3 = H
6, R1 = H, R2 = NHCO(CH2)2N3, R3 = H
8,
10, R4 = 2-thiophenyl
11, R4 = N3
R = N3, R = H, R = CH2N3
Figure 2. Amine and acid precursors used for synthesis of photoreactive probes.
Substituted benzoic acids 3–8, mono-N-Boc protected pheny-
All the newly synthesized benzamide-based probes had activity
ranging between 70 nM and 55 M and 110 nM and 77 M for
lenediamines 9, 10 and azidoaniline 11 shown in (Fig. 2) were cho-
sen as precursors for the synthesis of the photoreactive probes.
Acid 4, protected phenylendiamines 9, 10, and intermediates 16,
18 and 19 were synthesized as reported previously,7,8,10,12 whereas
benzoic acid 3 was available commercially. The synthesis of pre-
cursors 5, 6, 7, 8, and 11 is shown in Schemes 1 and 2. The synthe-
sis of the probes 1a–g and 2a–b proceeded through an efficient
carbodiimide based coupling reaction between mono-N-Boc pro-
tected phenylendiamines 9–11 and benzoic acids 3–8 followed
by deprotection of the resulting N-Boc products to give the final
probes in 70–80% overall yield13 (Scheme 2).
The inhibitory profile of the probes against class I HDAC iso-
forms was determined using a fluorogenic assay and the results
are given in Table 1. The inhibition of HDAC8 was measured using
the fluorogenic acetylated substrate Fluor de Lys and purified re-
combinant human HDAC8 from Escherichia coli,14 whereas the inhi-
bition of HDAC1–3 was measured using fluorogenic acetylated
l
l
HDAC1 and HDAC2, respectively. All of the probes demonstrated
a robust 2- to 40-fold increase in inhibition of HDAC1 and 2 upon
preincubation with the enzymes for 3 h and 24 h, respectively (Ta-
ble 2). Consistent with the previously reported observation,11
SAHA, a hydroxamate-based inhibitor, did not exhibit time-depen-
dent inhibition. Similar trends were observed with HDAC3 and
HDAC8. In the discussion below we will use only IC50’s obtained
at the maximum preincubation time, unless specified otherwise.
In general, the probes exhibited better activity and selectivity
for HDAC1 and 2 as compared to HDAC3 and HDAC8 (Table 1).
The most HDAC1 and 2 potent probe 1b had an estimated 100-
and 1000-fold selectivity for HDAC1 and 2 as compared to HDAC3
and HDAC8, respectively. In the case of HDAC8, no inhibition was
observed after 5 min, whereas inhibition of HDAC3 varied from
2% for 1g to 21% for 2c at 10 lM concentration of the inhibitors.
After preincubation for 3 h, inhibition of HDAC3 and HDAC8 by
the probes varied from 6.5% for 1g to 56% for 1b and from 4.7%
for 1b to 26% for 1g, respectively. Similarly to the probes, ligand
1 showed pronounced inhibition of HDAC3 and HDAC8, 96% and
24%, respectively, and ligand 2 inhibited 40% of activity of HDAC3
and only 3% of activity of HDAC8. Despite the similarity of probes
substrate Boc-L-Lys(Ac)-AMC and commercially available recombi-
nant human HDAC1–3.15 We also explored the effect of preincuba-
tion with HDAC1, 2, 3, and 8 as it was previously observed that the
potency of the benzamide-based HDAC inhibitors increased with
preincubation with HDAC1–3.11,16 The maximum incubation time
was chosen on the basis of stability of HDAC proteins in the condi-
tions used to determine IC50 values. We found that for HDAC1, 3,
and 8 the maximum incubation time was 3 h, whereas HDAC2 pro-
tein was stable for 24 h. The IC50 values of ligands 1 and 2 deter-
mined in this study vary from those reported previously.10,11 We
attribute this discrepancy to the differences in the assay condi-
tions, the protein sources, substrates, and preincubation times.
The analysis of SAR was facilitated by docking all the probes to
HDAC2 (PDB:3MAX),11 HDAC3 (PDB:4A69),17 and HDAC8
(PDB:1T69)18 using GOLD v.5.1.19,20
2a–c, 2a did not inhibit HDAC8 at 10 lM, whereas both 2b and 2c
inhibited 25% and 22% of activity of HDAC8, respectively.
Gangloff et al.11 suggested that the time-dependent inhibition in
the case of HDAC2 may be explained by the gradual disruption of
the internal hydrogen bond between the aniline hydrogen and car-
bonyl oxygen in the unbound form of the ligand so as to form a
bidentate complex with Zn2+ ion in the bound form. After a preincu-
bation 3 h, increase in inhibition of HDAC1 and HDAC2 by the
probes varied from 1.6-fold for 1d to 17-fold for 1b and from
1.3-fold for 1g to 7.1-fold for 2c, respectively, (Table 2). After
N3
N3
Br
X
Z
O
e
Y
N3
a,b,c,d
H2N
H2N
NHBoc
NHBoc
16
COOH
11
12, X = OH, Y = NH2, Z = COOH
13, X = OH, Y = N3, Z = COOH
14, X = OH, Y = N3, Z = COOMe
O
a
b
c
d
f,g,h
O
OH
Br
OH
N3
N
H
6
15, X = O(CH2)2N3, Y = N3 , Z = COOMe
5,X = O(CH2)2N3, Y = N3 , Z = COOH
O
17
Scheme 1. Reagents and conditions: (a) NaNO2, HCl, NaN3, 5 h, 0 °C–rt, 85%; (b) SOCl2, MeOH, 8 h, 0 °C –rt, 87%; (c) K2CO3, 2-azidoethyl-4-methylbenzene sulphonate,
acetone, 5 h, reflux, 77%; (d) THF/H2O (1:1), KOH, 10 h, 70 °C, 92%; (e) NaN3, Sod. ascorbate, CuI, N,N-dimethylethane-1,2-diamine, EtOH/H2O, reflux, 92%; (f) (i) NaN3, CH3CN,
reflux, 80%; (ii) oxalyl chloride, DCM, 6 h; (g) methyl 4-aminobenzoate, pyridine, DCM, 0 °C–rt, 86%; (h) 2 N NaOH, THF/H2O (8:2), 2 h, rt, 93%.

A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5025–5030
5027
R4
O
O
A
R1
R1
R2
9
10
11
or + b, c
or
OH
N
B
H
NH2
R2
R3
R3
1
2
3
1
2
3
4
3,
4,
1a,
R = H, R = N3, R = H, R = Ph
R = H, R = N3, R = H
1
2
3
1
2
3
4
1b,
1c,
1d,
1e,
1f,
R = N3, R = H, R = CH2N3
R = H, R = N3, R = H,R = 2-thiophenyl
5, R1 = N3, R2 = O(CH2)2N3 H, R3 = H
R = N3, R = H, R = CH2N3, R = 2-thiophenyl
1
2
3
4
1
2
3
1
2
3
4
6,
R = H, R = NHCO(CH2)2N3, R = H
R = N3, R = H, R = CH2N33, R = Ph
1
2
4
R = N3, R = -O(CH2)2N3, R = H, R = Ph
1
2
3
4
R = N3, R = -O(CH2)2N3, R = H, R = 2-thiophenyl
R = H, R = NHCO(CH2)2N3, R = H, R = N3
1
2
3
4
1g,
R4
A
R1
O
R1
O
R1
R2
R3
R2
OH
R2
R3
9
10
N
H
or
+ b, c
H
N
a
H
N
B
O
NH2
O
R3
OH
O
O
1
2
3
4
1
2
3
1
2
3
2a,
R = N3, R = H, R = H, R = Ph
18,
19,
R = N3, R = H, R = H
7,
8,
R = N3, R = H, R = H
2b, R1 = N3, R2 = H, R3 = CH2N3, R4 = Ph
1
2
3
1
2
3
R = N3, R = H, R = CH2N3
R = N3, R = H, R = CH2N3
1
2
3
4
2c,
R = N3, R = H, R = CH2N3 ,R = 2-thiophenyl
Scheme 2. Reagents and conditions: (a) CDI, DBU, TEA, 4-(aminomethyl)benzoic acid, THF, 10 h, 0 °C–rt, 90–92%; (b) EDCI, HOBt, DMF, 12 h, 80 °C, 80–90%; (c) TFA/DCM,
0.5 h, rt, 90–95%.
Table 1
Potency of the probes against class I HDAC isoforms
#
HDAC1 IC50 (nM)
Preincubation time
3 h
140 34
HDAC2 IC50 (nM)
Preincubation time
3 h
1050 81
HDAC3 % inhibition (10
lM)
HDAC8 % inhibition (10 lM)
Preincubation time
3 h
Preincubation time
3 h
5 min
5 min
5130 470
24 h
210 17
110 36
2400 74
10000 490
830 28
750 81
77000 4100
320 32
5 min
5 min
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
1
2100 44
1200 85
6.6
8.7
2.2
NA
4.3
7.6
2.0
7.8
11
21
26
8.8
100
35
56
27
24
16
12
6.5
48
46
47
96
40
100
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
100
NA
4.7
11
14
22
6.6
26
NA
25
22
24
70 5.3
1400 160
13000 320
2700 69
3800 54
55000 1300
780 22
990 53
1210 68
52 4.3
3200 260
18000 910
32000 1700
34000 2300
17000 4400
120000 4900
3800 540
7000 250
7100 220
1200 93
690 57
5200 400
21000 720
6500 120
6600 140
94000 530
1000 70
1100 25
1000 50
350 15
5600 520
21000 100
23000 1400
18000 130
96000 1600
3800 120
2500 280
2800 240
410 16
350 16
300 77
140
8
2
14500 1300
29 1.6
1880 5.2
34 3.2
38000 2000
200 14
14000 1030
ND
740 49
260 4.3
3.0
100
SAHA
NA, no inhibition up to 10 lM concentration; ND, not determined. Data are mean SD of three independent experiments.
preincubation for 24 h, inhibition of HDAC2 further improved to
1.6-fold for 1g to 41-fold for 1e. A comparison of the IC50 ratios
for 3 h versus 5 min and 24 h versus 5 min for HDAC2 (Table 2)
shows that the weakest inhibitors 1c, 1d, and 1g exhibit the least
pronounced change in their IC50 with time. A somewhat similar
but less pronounced trend is observed in the case of HDAC1. In gen-
eral, the trends observed in our case seem to be consistent with the
explanation for the time-dependent inhibition given by Gangloff
et al.11 The difference in the time-dependent inhibition by the
probes that have the same substituent binding to the ‘foot pocket’,
for example, 1a, 1d, 1e, 2a, and 2b, at 3 h versus 5 min and 24 h ver-
sus 5 min suggests that additional factors should be taken into ac-
count. Overall ability of the ligands to adopt the necessary
conformation for induced fit may play a role in addition to the con-
formational flexibility of the benzamide portion of the ligands.
HDAC1 is highly homologous to HDAC2, and, therefore, its
time-dependent inhibition may be explained in a similar fashion.
However, neither HDAC3 nor HDAC8 were reported to have crystal
structures that would contain a binding pocket similar to the ‘foot
pocket’ of HDAC2. The docking of the probes to HDAC2 showed that
their binding poses are essentially the same as that of ligand 1, that
is, the aniline nitrogen and the amide oxygen form a bi-dentate che-
late with Zn2+, whereas the bi-aryl portion occupies the ‘foot pock-
et’ (Figs. 3 and 4).
A comparison of the docking pose of probe 2c in HDAC2,
HDAC3, and HDAC8 shows that, unlike HDAC2 (Fig. 3A), HDAC3
(Fig. 3B) and HDAC8 (Fig. 3C) cannot accommodate 2c such that
it can form a bi-dentate complex with Zn2+ in the catalytic site.
The binding site of HDAC3 in 4A69 is too small for 2c and the probe
is mostly resides outside the binding site. In HDAC8 in 1T69, the
binding site is too short and has a somewhat different shape com-
pared to HDAC2. None of the docking poses of 2c coordinates with

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A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5025–5030
Table 2
Ratios of IC50 for HDACs 1, 2 and 3 with respect to preincubation time and selectivity for HDAC1 versus 2
#
HDAC1 IC50 ratio 3 h/ HDAC2 IC50 ratio 3 h/ HDAC2 IC50 ratio
HDAC3 %inhibition ratio
3 h/5 min
HDAC2/HDAC1 IC50 ratio HDAC2/HDAC1 IC50 ratio
5 min
5 min
24 h/5 min
3 h/3 h
24 h/3 h
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
1
15
17
4.9
4.6
3.5
1.5
5.2
2.6
1.3
3.8
6.4
7.1
3.4
2.7
ND
24
29
7.5
3.2
41
23
1.6
12
20
24
5.3
6.4
12
7.5
9.9
3.7
1.6
2.4
1.7
1.7
1.3
1.1
0.82
6.7
7.5
ND
1.5
1.6
1.7
4.0
1.6
8.5
4.7
1.7
4.9
2.5
2.3
7.9
7.8
0.85
—
0.77
0.31
0.20
1.4
0.41
0.35
0.25
2.7
3.7
1.6
3.3
6.2
4.2
2.2
3.7
4.5
1.0
8.6
52
0.77
2
0.39
7.7
SAHA
ND, not determined.
Figure 3. Probe 2c docked into the active site of (A) HDAC2, (B) HDAC3, and (C) HDAC8.
Zn2+ despite the proximity of the groups necessary for coordina-
tion. After a co-minimization of 2c with the HDAC8, only coordina-
tion between the carbonyl oxygen of 2c and Zn2+ was observed.
Interestingly, although the residues in the foot pocket of HDAC2
and the corresponding residues in HDAC3 (according to sequence
alignment) are the same, the recent X-ray apo-structure of
HDAC317 did not contain a ‘foot pocket’. Schwabe et al.17 noted that
the HDAC3 structure was crystallized in the absence of the ligand
and, therefore, may not be representative of the actual protein–li-
gand complex interactions. In our opinion, the similarity of the
time-dependent inhibition of HDAC2 and HDAC3 and HDAC8 sug-
gests that the latter two isoforms may also adopt the conformation
with a ‘foot pocket’ that can accommodate the benzamide-based li-
gands. The relatively low inhibition of HDAC8 compared to
HDAC1-3 may be rationalized by the difference in the residues at
the entrance to the ‘foot-pocket’ that imposes different steric and
electrostatic requirements on the R4 substituent. In HDAC8, the
opening to the putative ‘foot pocket’ is hindered by the presence
of bulky sidechain of Trp127 as shown in Fig. 3, whereas in HDAC1,
2 and 3 the corresponding residue Leu144 is less bulky and more
flexible and makes the ‘foot pocket’ more accessible to the ligands.
This is also indirectly supported by the SAR—probe 1g is consis-
tently the least active against HDAC1–3 but its inhibition of HDAC8
is comparable to that of 1, 1e, 2b, and 2c.
SAR study to explore how the positioning of the aryl azide and ali-
phatic azide affects the potency and selectivity of the probes. De-
spite the presence of additional azido groups, probes 1a and 1b
were comparable in potency to ligand 1 and probes 2a–c were
more than 2- to 2.5-fold more potent than ligand 2 for HDAC1
and 2. Probes 1c–g were 27- to 1000- and 5- to 550-fold less potent
than ligand 1 for HDAC1 and 2, respectively. In general, probes 1a–
g were found to be less potent than ligand 1 for both HDAC3 and 8,
whereas compounds 2a–c and ligand 2 demonstrated comparable
potency against HDAC3. In HDAC8, the diazide probes 2b and 2c
appear to be more potent than ligand 2 and the monoazide probe
2a was inactive. To gain insights into the plausible explanations for
the difference in potency we compared the docking poses of the
probes with that of ligand 1. We observed that the meta-substitu-
ents R1 and R3 are too close to the residues Phe210, Gly154,
Phe155, Leu276, and Asp104 (Fig. 4).
As a result of this steric interference, the probes are forced to
adopt a conformation where the face-to-face
tween ring B of the probes and Phe155 is disrupted. The loss of
these stacking interactions may explain relatively poor po-
p–p stacking be-
p–p
tency of mono meta-substituted probes 1e and 1f, 830 and
750 nM, respectively, and especially 3,5-disubstituted probes 1c
and 1d, 2.4 and 10 lM, respectively, compared to probes with no
meta-substituents 1a and 1b, 210 and 110 nM, respectively. The
width and shape of the gorge region appears to be important to
gain potency and isoform selectivity as demonstrated by Kozikow-
ski et al.21 in design of tubastatin A, a selective inhibitor of HDAC6.
Cravatt et al.5 attributed the low potency of benzophenone
based benzamide probes to the positioning of the photoreactive
group. Based on their observations, we decided to carry out a small

A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5025–5030
5029
Next we investigated whether the newly designed probes are
capable of crosslinking HDAC2. Photoaffinity labeling studies were
conducted with the probes using commercially available recombi-
nant His-tagged HDAC2. The probes (25
lM) were preincubated
with HDAC2 (1.25 M) for 24 h in photolabeling buffer, exposed
l
to 254 nm UV light for 3 Â 1 min with 1 min resting. A commer-
cially available strained cyclooctyne based biotin tag (BT) was at-
tached to the HDAC2-probe adduct using (3+2) cycloaddition
reaction and the biotinylated HDAC2 was visualized by streptavi-
din-HRP and western blot analysis (Fig. 5). The loading was con-
firmed by using nickel-HRP, which recognized the His-tag of the
recombinant HDAC2 protein.23 To ensure that the biotinylation
was primarily driven through interactions of the probes with the
binding site of HDAC2, we performed competition experiments of
the probes with a known potent HDAC inhibitor Trichostatin A
(125 l
M), which has an IC50 of 68 nM for HDAC2. 8 All of the diaz-
ide probes showed a pronounced decrease in biotinylation in the
presence of five-fold molar excess of the competing ligand. The de-
crease was slightly less pronounced in the case of weakly potent
probe 1g.
Figure 4. Overlay of compounds 1b (green), 1c (magenta), 1g (cyan) and 2c (gold)
in the active site of HDAC2.
We also confirmed that our probes 1a–f and 2a–c are cell per-
meable and capable of inhibiting nuclear HDACs by monitoring
the acetylation status of histone H4 in MDA-MB-231 breast cancer
cell line using previously published procedure.24 H4 is a known nu-
clear target for HDAC1 and HDAC2 in this cell line.25 All of the
probes inhibited deacetylation of histone H4 at 50
tion after a 24 h treatment (Fig. 6A and B).
lM concentra-
In conclusion, two benzamide scaffolds were successfully ex-
plored for design of novel HDAC2 nanomolar potent and selective
photoreactive probes suitable for further BEProFL experiments. A
total of 10 monoazide and diazide containing benzamide probes
were synthesized and tested for their inhibitory activity against
class I HDAC isoforms. All the probes are readily accessible in
few synthetic steps carried out in a convergent manner. The inhi-
bition was measured at two time points for HDAC1, 3, and 8 and
three points for HDAC2. The probes exhibited a 2- to 40-fold in-
crease in inhibition with respect to time for HDAC1 and 2 and
modest increase was observed for HDAC3 and 8. Time-dependent
inhibition of HDAC1, 3, and 8 suggests that these isoforms may also
adopt the ‘foot pocket’ conformation similar to that of HDAC2 to
accommodate the benzamide ligands. The most potent probes ex-
hibit nanomolar activity against HDAC1 and 2. Probe 1b has an IC50
of 70 and 110 nM for HDAC1 and 2, respectively, and shows an
estimated 100- to 1000-fold selectivity for HDAC1 and 2 as com-
pared to HDAC3 and 8. The most active diazide probes 2b and 2c
Figure 5. Characterization of biotinylated HDAC2 and His-tagged HDAC2 using
streptavidin-HRP and nickel-HRP. Western blot analysis of diazide probes 1c, 1d, 1e,
1f, 2b, 2c and 1g (25
lM) photocrosslinked to HDAC 2 (1.25 lM) in the presence or
absence of 125 M of Trichostatin A using streptavidin-HRP and nickel-HRP. Shown
l
is the representative western blot of three independent experiments.
Placement of the aromatic azido group in the ‘foot-pocket’ in 1g led
to poor potency for HDAC1 and 2, 55 and 77 lM, respectively,
slightly less pronounced decrease in inhibition of HDAC3 but not
HDAC8. The docking showed that the R4 azido substituent fits well
in the ‘foot pocket’ and occupies the same space as the R4 phenyl
and 2-thiophenyl substituents in 1a–f. This observation appears
to be consistent with the SAR found by Methot et al.,22 where
non-polar aromatic substituents were found to be preferable com-
pared to polar and/or relatively small substituents R4. The addi-
tional interactions between the carbamate appendage in probes
2a, 2b and 2c and Tyr209 of HDAC2 identified by docking did not
contribute to potency of these ligands, suggesting that this
appendage is likely to remain solvent exposed.
have an IC50 of 0.9 and 1.2 lM and 300 and 350 nM for HDAC1
and 2, respectively, and show approximately 30-fold selectivity
for HDAC1 and 2 as compared to HDAC3 and 8. Docking studies
with HDAC2 indicated that the placement of the azido groups meta
Figure 6. Western blot detection of acetyl H4 in MDA-MB-231 cell lines following a 24 h treatment with probes at 50 lM. (A) Treatment of cells with probes 1a, 1b, 1c, 1d, 1e,
suberoyl anilide hydroxamic acid (SAHA) and parent ligand 1. (B) Treatment of cells with probes 1f, 2a, 2b, 2c, suberoyl anilide hydroxamic acid (SAHA) and parent ligand 1.
Shown is a representative blot of three independent experiments.

5030
A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5025–5030
14. Dowling, D. P.; Gattis, S. G.; Fierke, C. A.; Christianson, D. W. Biochemistry 2010,
49, 5048.
15. HDAC inhibition assay was performed in 96-well opaque half-area microplate
(Corning). Human recombinant HDAC1,2 and 3 (BPS Bioscience) and HDAC8
(purified from E. Coli) were diluted with assay buffer 1 (25 mM Tris–HCl, pH
8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg/mL BSA), so as to have 4,
but not para to the benzamide group in ring B leads to unfavorable
for stacking between ring B and Phe155 orientation of the li-
p–p
gand. Consistent with earlier reports, the presence of the bi-aryl
moiety in the ‘foot-pocket’ was found to be essential for maintain-
ing potency for HDAC1–3. On the other hand, 1g, a probe that lacks
the bi-aryl portion found in 1a–f and 2a–c, was found to be supe-
rior to the bi-aryl-containing probes in inhibiting of HDAC8. As
demonstrated by our photolabeling experiments, all the diazide
probes efficiently photocrosslinked with recombinant HDAC2. Cell
based studies show that the benzamide probes are able to enter the
cell nucleus and trigger accumulation of acetylated H4. Presently,
the probes are being extensively used in mapping the binding site
of HDAC2 via proteomics experiments. Cell based photolabeling
experiments are currently underway to understand how these
probes bind to HDAC complexes in cells.
5
and 1 ng/ll and 8.5 ng/lL stocks of each isoform, respectively. Serial
dilutions of the probes were made in assay buffer 2 (25 mM Tris–HCl, pH
8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2). Ten microlitre of the enzyme
stock was added to 30 lL of the probes and preincubated for different
preincubation times. It was observed the Z-factor for the assays remained
above 0.7 up to 3 h for HDAC1, 3 and 8 and up to 24 h for HDAC2, hence these
preincubation times were chosen for the assays. After preincubation, 10
125 M HDAC fluorescent substrate Boc- -Lys (Ac)-AMC (Chem-Impex) in case
of HDAC1,2 and 3 and 10 L of 25 M BML-KI-178 (Biomol Inc.) in case of
HDAC8 was added, and the mixture incubated for 35 min (HDAC1, 3, 8), 60 min
(HDAC2) at room temperature. The reaction was quenched with 50 L of 1 mg/
mL trypsin and 5 M trichostatin A in assay buffer 1 (25 mM Tris–HCl, pH 8.0,
lL of
l
L
l
l
l
l
137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) and further incubated at room
temperature for 35 min. The plate was read on Synergy 4 hybrid microplate
reader (BioTeck) 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).
Acknowledgments
16. Chou, C. J.; Herman, D.; Gottesfeld, J. M. J. Biol. Chem. 2008, 283, 35402.
17. Watson, P. J.; Fairall, L.; Santos, G. M.; Schwabe, J. W. Nature 2012, 481, 335.
18. Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.; Jennings, A. J.; Luong, C.;
Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang, B. C.; Verner, E.; Wynands, R.; Leahy,
E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M. W.; Swanson, R. V.; McRee,
D. E.; Tari, L. W. Structure 2004, 12, 1325.
19. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727.
20. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins
2003, 52, 609.
This study was funded by the National Cancer Institute/NIH
Grant R01CA131970 and ADDF Grant #20101103. We thank Pro-
fessor Carol Fierke, University of Michigan, MI, for generously pro-
viding us with the plasmid for in-house expression and purification
of HDAC8.
21. Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.; Kozikowski, A. P. J.
Am. Chem. Soc. 2010, 132, 10842.
References and notes
22. Methot, J. L.; Chakravarty, P. K.; Chenard, M.; Close, J.; Cruz, J. C.; Dahlberg, W.
K.; Fleming, J.; Hamblett, C. L.; Hamill, J. E.; Harrington, P.; Harsch, A.;
Heidebrecht, R.; Hughes, B.; Jung, J.; Kenific, C. M.; Kral, A. M.; Meinke, P. T.;
Middleton, R. E.; Ozerova, N.; Sloman, D. L.; Stanton, M. G.; Szewczak, A. A.;
Tyagarajan, S.; Witter, D. J.; Secrist, J. P.; Miller, T. A. Bioorg. Med. Chem. Lett.
2008, 18, 973.
1. Khan, O.; La Thangue, N. B. Immunol. Cell Biol. 2012, 90, 85.
2. Mai, A.; Rotili, D.; Valente, S.; Kazantsev, A. G. Curr. Pharm. Des. 2009, 15, 3940.
3. Salisbury, C. M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1171.
4. Xu, C.; Soragni, E.; Chou, C. J.; Herman, D.; Plasterer, H. L.; Rusche, J. R.;
Gottesfeld, J. M. Chem. Biol. 2009, 16, 980.
5. Salisbury, C. M.; Cravatt, B. F. J. Am. Chem. Soc. 2008, 130, 2184.
6. He, B.; Velaparthi, S.; Pieffet, G.; Pennington, C.; Mahesh, A.; Holzle, D. L.;
Brunsteiner, M.; van Breemen, R.; Blond, S. Y.; Petukhov, P. A. J. Med. Chem.
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Iwata, T.; Maruyama, K.; Endo, M.; Suzuki, M. Org. Biomol. Chem. 2004, 2, 637.
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Petukhov, P. A. J. Med. Chem. 2011, 54, 4350.
9. Paquin, I.; Raeppel, S.; Leit, S.; Gaudette, F.; Zhou, N.; Moradei, O.; Saavedra, O.;
Bernstein, N.; Raeppel, F.; Bouchain, G.; Frechette, S.; Woo, S. H.; Vaisburg, A.;
Fournel, M.; Kalita, A.; Robert, M. F.; Lu, A.; Trachy-Bourget, M. C.; Yan, P. T.;
Liu, J.; Rahil, J.; MacLeod, A. R.; Besterman, J. M.; Li, Z.; Delorme, D. Bioorg. Med.
Chem. Lett. 2008, 10, 1067.
23. Diazide probe (25
was incubated with HDAC2 (1.25
Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1% Triton-X),
m of
l
M) or probe-TSA (25
lM-probe and 125 lM-TSA) mixture
lM) for 24 h in photolabelling buffer (25 mM
exposed to 254 nm UV light 3 Â 1 min with 1 minresting. A solution of 500
l
DBCO-PEG4-biotin conjugate (ClickChemistry tools) inphotolabeling buffer was
added to initiate the (3+2) cycloaddition reaction with HDAC2-probe adduct. The
cycloaddition reaction was carried out for 3 h at room temperature. Western
blotting was done with 1.5
lg of purified protein with 5Â loading buffer
containing 10% SDS, 0.05% bromophenol blue, 50% glycerol, and b-
mercaptoethanol. Protein samples were boiled for 5 min and allowed to cool
before loading on a denaturing 4–15% polyacrylamide gel electrophoresis (SDS–
PAGE). After electrophoresis, protein was transferred to a polyvinylidiene
difluoride membrane (Iblot-Invitrogen). The membrane was incubated for 12 h
10. Witter, D. J.; Harrington, P.; Wilson, K. J.; Chenard, M.; Fleming, J. C.; Haines, B.;
Kral, A. M.; Secrist, J. P.; Miller, T. A. Bioorg. Med. Chem. Lett. 2008, 18, 726.
11. Bressi, J. C.; Jennings, A. J.; Skene, R.; Wu, Y.; Melkus, R.; De Jong, R.; O’Connell,
S.; Grimshaw, C. E.; Navre, M.; Gangloff, A. R. Bioorg. Med. Chem. Lett. 2010, 20,
3142.
with 5% albumin fraction
V
(Sigma–Aldrich) in 1Â Tris based saline
supplemented with 0.1% Tween-20 (TBST). The membrane was washed three
times with TBST and then incubated with Ni-HRP (1:2000) in TBST for 1 h under
room temperature with slight agitation. After three washes in TBST, the
chemilumiscent signal was detected using the enhanced chemiluminescence
(ECL) kit from Pierce (Pierce Biotechnology, Rockford, IL). The membrane was
washed three times with 1 Â phosphate buffer saline supplemented 0.1% Tween-
20 (PBST), and stripped of Ni-HRP by incubating with 5% BSA, 2 M Imidazole in
PBST for 1 h at room temperature. After three washes of PBST, the membrane was
incubated with Streptavidin-HRP (1:5000) in PBST for 1 h. After three washes in
PBST and water, the chemilumiscent signal was detected using the enhanced
chemiluminescence (ECL) kit from Pierce (Pierce Biotechnology, Rockford, IL).
24. MDA-MB-231 cells seeded at 1.0 Â 105 cells/well in 6-plates and grown to 90%
confluence in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1%
penicillin-streptomycin (Mediatech). Cells were treated with either DMSO or
12. Umezawa, N.; Matsumoto, N.; Iwama, S.; Kato, N.; Higuchi, T. Bioorg. Med.
Chem. 2010, 18, 6340.
13. General procedure for synthesis of the probes: To a solution of substituted
benzoic acid 3–8 (1.1 equiv) in anhydrous DMF (5 mL/mmol of amine) was
added EDCI (1.2 equiv) followed by HOBt (1.2 equiv) and stirred at room
temperature for 1 h. Thereafter protected phenylendiamine 9–11 (1 equiv) was
added and the mixture heated at 78 °C overnight. After completion of the
reaction as confirmed by TLC, saturated sodium bicarbonate solution (20 mL/
mmoL of amine) was added and the mixture was extracted with ethylacetate
(30 mL/mmol of amine). The organic layer was washed with water (30 mL/
mmol of amine), dried over anhydrous sodium sulphate and evaporated in
vacuo. The residue was purified using flash chromatography (silica gel, hexane/
ethylacetate gradient) to yield the N-Boc protected probes. The Boc group was
subsequently removed by treating N-Boc protected compound with a mixture
of TFA/DCM (1:1 v/v) at room temperature for 1 h. The solvent was removed in
vacuo and the residue purified using flash chromatography (silica gel, hexane/
ethylacetate gradient) to yield the final probes as solids in 70–80% over all
yield. Spectral data for probe 1b; 1H NMR (400 MHz, DMSO-d6) d (ppm) 9.83
(bs, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.40–7.25 (m, 6H), 7.06–7.04 (m, 1H), 6.86 (d,
J = 8.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) d (ppm). 164.13, 144.42, 143.13,
142.33, 131.33, 130.28(2C), 128.75, 124.49, 124.44, 124.36, 123.96, 123.60,
121.80, 119.36(2C), 117.48. (M+H)+ 336.40. Spectral data for probe 2c; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 9.79 (bs, 1H), 8.00–7.95 (m, 2H), 7.65–7.55 (m,
3H), 7.52–7.35 (m, 4H), 7.33–7.23 (m, 1H), 7.18 (s, 1H), 7.09 (s, 1H), 6.89 (d,
J = 8.4 Hz, 1H), 5.08 (s, 2H), 4.49 (s, 2H) 4.29 (s, 2H). 13C NMR (100 MHz, DMSO-
d6) d (ppm) 165.39, 156.32, 143.35, 140.15, 140.05 140.01, 138.18 138.16,
133.17, 128.89 (2C), 128.74 (2C), 127.95 (2C), 126.81 (2C), 126.19, 125.61,
124.78, 123.91, 118.24, 117.61, 116.95, 64.46, 52.96, 43.68. (M+H)+ 553.60.
benzamide probes at a final concentration of 50 lM and maintained at 37 °C
and 5% CO2 for 24 h. Cells were lysed using 1 Â RIPA buffer (150 mM NaCl, 1%
Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0)
containing protease inhibitor cocktail (Roche) and 1:100 dilution of
phosphatase inhibitor (Sigma) with shearing. Lysates were clarified through
centrifugation at 13,000 rpm at 4 °C. Total protein concentration was
determined via the BCA Protein Assay Kit (Pierce). Lysates were stored at
À20 °C until later use. Sample vials were prepared by aliquoting 25
lg of total
protein and adding 5Â Sample buffer. Sample vials were boiled for 5 min,
cooled to RT, and proteins separated by gel electrophoresis at 80 V. Proteins in
the gel were transferred to PVDF membrane in 4 min using the Invitrogen iBlot
system. Membranes were blocked using 5% Milk in PBST and probed using
anti-GAPDH (1:5000) or anti-acetyl histone H4 (1:1000) overnight at 4 °C.
Membranes were incubated with either anti-mouse (1:5000) or anti-rabbit
(1:5000) in 5% Milk in PBST. Results were visualized using Femto
chemiluminescent substrate (Pierce) in CCD camera.
25. Suzuki, J.; Chen, Y. Y.; Scott, G. K.; Devries, S.; Chin, K.; Benz, C. C.; Waldman, F.
M.; Hwang, E. S. Clin. Cancer Res. 2009, 15, 3163.