Benzimidazole and imidazole inhibitors of histone deacetylases: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmcl.2010.03.092

A series of N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamides (5a-5ab) and N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamides (12a-s) were designed, synthesized, and found to be nanomolar inhibitors of human histone deacetylases. Multiple compounds bearing an N1-piperidine demonstrate EC₅₀s of 20-100 nM in human A549, HL60, and PC3 cells, in vitro and in vivo hyperacetylation of histones H3 and H4, and induction of p21^waf. Compound 5x displays efficacy in human tumor xenograft models.

Full text of DOI:10.1016/j.bmcl.2010.03.092

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3138–3141
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzimidazole and imidazole inhibitors of histone deacetylases: Synthesis
and biological activity
Jerome C. Bressi, Ron de Jong, Yiqin Wu, Andy J. Jennings, Jason W. Brown, Shawn O’Connell, Leslie W. Tari,
*
Robert J. Skene, Phong Vu, Marc Navre, Xiaodong Cao, Anthony R. Gangloff
Takeda San Diego, 10410 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamides (5a–5ab) and
N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamides (12a–s) were designed,
synthesized, and found to be nanomolar inhibitors of human histone deacetylases. Multiple compounds
bearing an N1-piperidine demonstrate EC50s of 20–100 nM in human A549, HL60, and PC3 cells, in vitro
and in vivo hyperacetylation of histones H3 and H4, and induction of p21waf. Compound 5x displays effi-
cacy in human tumor xenograft models.
Received 29 January 2010
Revised 25 March 2010
Accepted 26 March 2010
Available online 30 March 2010
Keywords:
Histone deacetylase inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
HDAC
Oncology
The reversible acetylation of
e
-N-acetyl groups of lysine resi-
identify novel HDAC inhibitors focused on N1-substituted benzimi-
dazoles and imidazoles as binding moiety chemotypes.
dues, in the N-terminal tails of core histones, plays a critical role
in the regulation of gene expression by altering the accessibility
of transcriptional factors to DNA via conformational changes in
the structure of the nucleosome.1–3 This restructuring of chromatin
is regulated by the balance of histone acetyl transferase (HAT) and
histone deacetylase (HDAC) activity.4,5 Perturbations of this bal-
ance have been linked to cancer, and inhibition of HDACs results
in the expression of genes that produce growth arrest, terminal dif-
ferentiation, and/or apoptosis in a variety of cancer cells.6 Inhibi-
tion of HDACs has been indicated as a unique mechanism for
cancer chemotherapy, and a number of small-molecule inhibitors
have advanced into clinical trials.7–9
The synthesis of N-hydroxy-3-[3-(1-substituted-1H-benzoimida-
zol-2-yl)-phenyl]-acrylamides12 (5a–ab, Scheme 1) begins with
nucleophilic aromatic substitution of 1-fluoro-2-nitro-benzene (1)
with the appropriate primary amine followed by reduction of the
nitro group under standard conditions to provide N-substituted-
benzene-1,2-diamines (2). Condensation of the aniline (2) with
NH2
NO2
a, b
NH
R1
F
The structure of the HDAC active site is comprised of a narrow,
hydrophobic tunnel originating at the protein–solvent interface
andleading 8 Å intoa cavitythatcontainsthecatalyticZn2+. Atypical
HDAC inhibitor consists of a bidentate chelators tethered to a bind-
ing moiety that interacts with the mouth of the tunnel at the pro-
tein–solvent interface.10 Utilizing crystal structures of HDAC-2 and
HDAC-8,11 as well as capitalizing on published SAR, we designed
and synthesized a variety of compounds building off of an N-hydrox-
ycinnamamide. Structure-guided designs suggested that substi-
tuted five-membered rings projecting from the 3-position of N-
hydroxycinnamamide would best facilitate productive interaction
between a binding moiety and the protein surface. Our efforts to
1
2
O
O
O
N
c
H
OH
N
R1
OH
3
4a-4ab
N
O
d, e
N
NHOH
R
5a-5ab
Scheme 1. Reagents and conditions: (a) R1NH2, Et3N, DMF, 60 °C, 3–48 h; (b)
powdered Zn, AcOH, EtOH, reflux; (c) 2, AcOH, EtOH, reflux, 24–48 h; (d) NH2OTHP,
EDCI, HOBT, DIEA, DMF, rt, 18 h; (e) CSA, MeOH, rt, 1 h.
* Corresponding author. Tel.: +1 858 731 3531; fax: +1 858 550 0526.
E-mail addresses: agangloff@takedasd.com, anthony.gangloff@takedasd.com
(A.R. Gangloff).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.092

J. C. Bressi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3138–3141
3139
3-(3-formyl-phenyl)-acrylic acid13 (3) and subsequent insitu cycliza-
tion and oxidation provides 3-[3-(1-substituted-1H-benzoimidazol-
2-yl)-phenyl]-acrylic acids (4a–ab). Treatment of 4a–ab with
O-(tetrahydropyran-2-yl)-hydroxylamine in the presence of EDCI
and HOBt followed by treatment with camphorsulfonic acid yields
the desired N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-
2-yl)-phenyl]-acrylamides (5a–ab).
The synthesis of N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imi-
dazol-2-yl)-phenyl]-acrylamides14 (12a–s, Scheme 2) begins with
acylation of the appropriate amino acid ester with 3-bromo-benzoyl
chloridefollowedbysaponificationtoprovidethedesired carboxylic
acids (9). R3 is installed by treatment of 9 with the appropriate anhy-
dride to provide the Dakin–West intermediate (10). Imine formation
with the R4 bearing primary amine and 10 followed by in situ cycli-
zation via microwave irradiation provides the desired 2-(3-bromo-
phenyl)-1,4,5-trisubstituted-1H-imidazole (11a–s). Standard Heck
coupling conditions of 11a–s with N-(tetrahydro-pyran-2-yloxy)-
acrylamide (7) and CSA mediated tetrahydropyran (THP) cleavage
yields the desired N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-
imidazol-2-yl)-phenyl]-acrylamides (12a–s). Substituting the appro
priate reagents, N-hydroxy-3-[3-(2,3,5-trisubstituted-3H-imidazol-
4-yl)-phenyl]-acrylamides14 (17a–f) may be synthesized utilizing
this same methodology as depicted in Scheme 3.
N-Methyl and N-ethyl piperidine derivatives, 5w–5ab, gave similar
results. The cell potency that the piperdine moiety conveyed to the
benzimidazole was also realized with N-hydroxy-3-[3-(1,4,5-tri-
substituted-1H-imidazol-2-yl)-phenyl]-acrylamides (Table 2).
N-Hydroxy-3-[3-(2,3,5-trisubstituted-3H-imidazol-4-yl)-phenyl]-
acr>ylamides experienced diminished binding activity compared
with their structural isomers (Table 3). PK and toxicity screens
showed benzimidazoles 5 as the most promising for further devel-
opment compared to imidazoles 12 (data not shown).
To verify the mode of action of the benzimidazole and imidazole
analogues, compounds showing potent activity in the HL60 cell
viability assay were evaluated for their ability to induce hyperacet-
ylation of histones H3 and H4 in HL60 leukemia cells. At cell effec-
tive concentrations, inhibitor treatment led to induction of histone
H3 and H4 acetylation. With increasing dilution of inhibitor drop-
ping below the effective concentration, histone acetylation re-
turned to background levels similar to DMSO-treated control. In
general, the EC50s in the cellular histone acetylation in HL60 cells
correlated well with HL60 cell viability EC50s. This is exemplified
by compounds 5x (H3 EC50 = 0.10
lM, H4 EC50 = 0.11
lM) and
5aa (H3 EC50 = 0.13 M, H4 EC50 = 0.10
l
lM) in Figure 1.
A hallmark of HDAC inhibition is the induction of p21waf, which
is suspected to mediate the antiproliferative effects observed with
intracellular HDAC inhibition. Compounds 5x and 5aa were evalu-
ated for their ability to activate the p21waf promoter using an engi-
neered cell line containing a stably integrated luciferase reporter
gene under control of the human p21 promoter. Compounds 5x
All compounds were screened against purified recombinant hu-
man HDAC-2, -6, and -8 enzymes and IC50s were determined using
known concentrations of enzyme with tBOC(Ac)-Lys-AMC as a sub-
strate15 (Tables 1–3). Compounds were also evaluated in human
A549 lung, HL60 leukemia, and PC3 prostate cancer cell lines (Ta-
bles 1 and 2). EC50 determinations were calculated from remaining
NADH levels after a 72 h compound incubation and a MTS colori-
metric readout.
and 5aa induced p21waf activity at EC50
s of 01.0 lM and
0.078 lM, respectively. This is the effective concentration of com-
pound that results in 50% of the maximum p21waf induction com-
pared with standard HDAC inhibitor trichostatin-A.
Chemistry efforts started with a small focused library of benzim-
idazoles, 5a–5t (Table 1). With few exceptions, the enzyme activity
for distinct isozymes was 10–100 nM and selectivity across iso-
zymes was less than eightfold. The SAR could not be rationalized gi-
ven that the binding moiety interacts predominantly with the
protein–solvent interface. Cell viability for 5a–5t was relatively flat
with the exception of 5t, which demonstrated low-micromolar
EC50s across all cell lines. Synthetic resolution of the enantiomers,
5u and 5v, realized a 2–6-fold potency increase for the R-isomer.
Compound 5x was further evaluated in a xenograft pharmaco-
dynamic assay. PANC-1 tumor bearing mice received a single intra-
peritoneal dose of 50 mg/kg of 5x. Relative to vehicle control,
treatment with 5x resulted in a substantial increase in the acetyla-
tion of histones H3 and H4 in the tumor tissue (Fig. 2). Histone
acetylation started to subside after 8 h and returned to background
levels around 24 h. Similar results were obtained with 5aa (data
not shown).
Compound 5x was also evaluated in a human HCT116 colon
xenograft mouse model and exhibited evidence of tumor growth
inhibition at the highest dose of 40 mg/kg (Table 4). Compound
5x demonstrated a maximum T/C of 45% with mild, temporary
weight losses observed in the cohorts of animals. In the PC3
prostate model using similar conditions, compound 5x exhibited
similar tumor growth inhibition (data not shown).
O
6
O
a
Cl
NHOTHP
7
R2
O
O
b, c
Br
Br
Cl
HOOC
N
H
O
O
H
N
H2N
R6
a, b
8
d
9
O
OH
d
R5
O
R5
14
Br
R2
R2
O
13
c
N
R3
Br
e
R6
R3
N
H
Br
O
N
H
N
N
R4
R6
O
R5
Br
N
R7
O
R5
10
11a-s
R2
15
R6
16a-f
N
O
f, g
R3
N
O
R5
e, f
N
R4
NHOH
N
R7
NHOH
12a-s
17a-f
Scheme 2. Reagents and conditions: (a) NH2OTHP, Et3N, DMF, 0 °C, 18 h; (b)
NH2R2CH2COOCH3, Et3N, DCM, rt, 2 h; (c) LiOH, MeOH, rt, 2 h; (d) [R3C(O)]2O,
DMAP, Et3N, 60 °C, 30 min then HOAc, 60 °C 30 min; (e) R4NH2, HOAc with
microwave irradiation at 200 °C for 2 h; (f) 7, Et3N, Pd(OAc)2, P(o-Tol)3, DMF, 110 °C,
1 h; (g) CSA, MeOH, rt, 1 h.
Scheme 3. Reagents and conditions: (a) R6C(O)Cl, Et3N, DCM, rt, 2 h; (b) LiOH,
MeOH, rt, 2 h; (c) [3-BrPhC(O)]2O, DMAP, Et3N, 60 °C, 30 min then HOAc, 60 °C,
30 min; (d) R7NH2, HOAc with microwave irradiation at 200 °C for 2 h; (e) 7, Et3N,
Pd(OAc)2, P(o-Tol)3, DMF, 110 °C, 1 h; (f) CSA, MeOH, rt, 1 h.

3140
J. C. Bressi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3138–3141
Table 1
HDAC enzyme inhibition and cell viability data for N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamide analogues
Compd
R1
HDAC-2
IC50 (nM)
HDAC-6
IC50 (nM)
HDAC-8
IC50 (nM)
A549
EC50
HL60
EC50
PC3
EC50 (lM)
a
a
a
b
b
b
(l
M)
(l
M)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
Me
i-Pr
Cyclohexyl
–CH2CH2N(Me)2
–CH2CH2N(Et)2
–CH2CH2N(i-Pr)2
–CH2CH2-N-morpholine
–CH2-4-piperdine
–CH2CH2-N-piperdine
–CH2CH2-N-pyrrolidine
Ph
4-Cl-Ph
4-OMe-Ph
–CH2CH2Ph
–CH2CH2CH2Ph
Bn
100
120
100
79.0
100
25.0
79.0
79.0
100
160
200
40.0
63.0
50.0
100
63.0
31.0
20.0
63.0
40.0
63.0
40.0
79.0
25.0
79.0
100
63.0
100
40.0
50.0
160
63.0
200
400
310
100
63.0
50.0
50.0
63.0
25.0
25.0
40.0
20.0
16.0
160
120
120
160
200
160
120
120
160
25
12
3.1
2.0
1.0
0.5
1.0
0.8
5.0
1.2
>50
0.3
0.5
1.6
0.8
0.5
0.3
1.2
5.0
3.1
3.1
0.3
0.1
0.3
0.08
0.04
0.16
0.06
0.03
0.08
10
7.9
2.5
1.6
2.5
1.2
3.1
5.0
>50
0.6
0.6
4.0
2.5
1.2
0.4
4.0
1.2
10
79.0
25.0
63.0
31.0
40.0
50.0
160
5.0
3.1
7.9
4.0
5.0
25
>50
1.2
3.1
16.
6.3
4.0
2.5
12
10.0
16.0
63.0
50.0
40.0
7.90
12.0
50.0
25.0
63.0
20.0
12.0
12.0
25.0
10.0
16.0
20.0
10.0
31.0
6.3
16
31
(R)-
(S)-
a
-Me-Bn
5s
5t
a-Me-Bn
16
–( )-3-piperdine
–(R)-3-piperdine
–(S)-3-piperdine
–( )-N-Me-3-piperdine
–(R)-N-Me-3-piperdine
–(S)-N-Me-3-piperdine
–( )-N-Et-3-piperdine
–(R)-N-Et-3-piperdine
–(S)-N-Et-3-piperdine
2.5
1.0
2.5
0.5
0.3
1.2
0.5
0.2
0.6
0.5
0.1
0.6
0.1
0.05
0.3
0.1
0.05
0.2
5u
5v
5w
5x
5y
5z
5aa
5ab
50.0
79.0
79.0
79.0
a
IC50 values are the mean of at least three experiments, and statistical error limits have been calculated and amount to 10% or less.
EC50 values are the mean of at least two experiments, and statistical error limits have been calculated and amount to 25% or less.
b
Table 2
HDAC enzyme inhibition and cell viability data for N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamide analogues.
Compd
R2
R3
R4
HDAC-2
IC50 (nM)
HDAC-6
IC50 (nM)
HDAC-8
IC50 (nM)
A549
EC50
HL60
EC50
PC3
EC50 (lM)
a
a
a
b
b
b
(lM)
(
lM)
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
Ph
Ph
Ph
Ph
Me
Me
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Me
Me
Me
Me
Bn
–CH2CH2-N-morpholine
–CH2CH2Ph
10.0
5.00
5.00
6.30
5.00
31.0
7.90
10.0
7.90
6.30
12.0
7.90
6.30
10.0
10.0
5.00
31.0
7.90
12.0
63.0
63.0
63.0
100
79.0
120
79.0
79.0
79.0
100
79.0
63.0
63.0
100
79.0
50.0
160
63.0
120
79.0
20.0
50.0
31.0
31.0
79.0
25.0
100
50.0
40.0
50.0
16.0
79.0
50.0
63.0
20.0
200
6.3
0.8
0.6
0.3
0.5
4.0
1.0
0.4
0.3
0.4
4.0
0.8
0.2
0.1
2.5
0.3
>50
0.4
0.3
0.3
0.2
0.04
0.03
0.04
0.3
1.0
0.3
0.1
0.05
0.1
1.0
0.5
0.1
0.08
0.2
1.0
0.5
0.1
0.04
0.6
0.2
5.0
0.2
0.08
–(R)-N-Me-3-piperdine
–(R)-N-Et-3-piperdine
–(R)-N-i-Pr-3-piperdine
–CH2CH2-N-morpholine
–CH2CH2Ph
–(R)-N-Me-3-piperdine
–(R)-N-Et-3-piperdine
–(R)-N-i-Pr-3-piperdine
–CH2CH2-N-morpholine
–CH2CH2Ph
–(R)-N-Me-3-piperdine
–(R)-N-Et-3-piperdine
–CH2CH2-N-morpholine
–CH2CH2Ph
–CH2CH2-N-morpholine
–CH2CH2Ph
Ph
Me
Me
Me
Me
Me
Bn
Bn
Bn
Bn
Me
Me
Me
Me
Me
0.3
0.03
0.04
0.03
0.3
0.2
0.02
0.03
0.2
0.05
1.6
Bn
Me
Me
Me
31.0
100
0.08
0.04
–(R)-N-Et-3-piperdine
a
IC50 values are the mean of at least three experiments, and statistical error limits have been calculated and amount to 10% or less.
EC50 values are the mean of at least two experiments, and statistical error limits have been calculated and amount to 25% or less.
b
Table 3
HDAC enzyme inhibition data for N-hydroxy-3-[3-(2,3,5-trisubstituted-3H-imidazol-4-yl)-phenyl]-acrylamide analogues
Compd
R5
R6
R7
HDAC-2
IC50 (nM)
HDAC-6
IC50 (nM)
HDAC-8
IC50 (nM)
a
a
a
17a
17b
17c
17d
17e
17f
Me
Me
Ph
Ph
Bn
Bn
Me
Me
Me
Me
Me
Me
–CH2CH2Ph
–(R)-N-Et-3-piperdine
–CH2CH2Ph
–(R)-N-Et-3-piperdine
–CH2CH2Ph
–(R)-N-Et-3-piperdine
79
500
79
630
40
120
400
79
630
63
100
500
50
400
63
120
500
100
a
IC50 values are the mean of at least three experiments, and statistical error limits have been calculated and amount to 10% or less.

J. C. Bressi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3138–3141
3141
Table 4
In vivo antitumor activity of compound 5x against human HCT116 colon carcinoma
xenografta
Compd
Doseb (mg/kg)
%T/Cc (%)
D
% Body weightd (day 6)
5x
5x
5x
15
25
40
66
47
45
+1.7
À2.5
À3.6
a
Each treatment group contained 10 mice.
Compounds were administered intraperitoneally, qdx5 per week, in total 15
b
doses.
c
%T/C values were calculated when vehicle-treated mice reached 1 g median
size, which was 10 days after the last dose.
d
Figure 1. Induction of histone H3 and H4 acetylation by compounds 5x and 5aa in
HL60 leukemia cells. Cells were treated for 4 h with 2.5-fold serial dilutions of
compound. Control DMSO-treated cells are indicated with hyphen. Histone
acetylation in whole cell lysates was analyzed by Western blotting. PCNA
antibodies were used as a control for protein loading. Dose–response signals were
quantified in order to calculate the inhibitor concentration giving the half-maximal
response.
Maximum body weight loss at the nadir.
graft mouse model. Overall, the compounds described add to the
body of knowledge of HDAC inhibitors with novel substituents at
the protein–solvent interface which confer not only enzymatic
activity but also cellular and in vivo activity.
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Figure 2. Increased acetylation of histones in PANC-1 xenograft in nude mice after
administration of 50 mg/kg 5x. At indicated time points, tumors were harvested
and whole tissue lysates were prepared and analyzed by Western blotting. Histone
acetylation was detected by antibodies specific for acetylated Histone H3 or H4,
respectively. Actin antibodies were used as a control for protein loading.
11. 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.;
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12. Synthetic details and characterization data can be found in: Bressi, J. C.; Brown,
J. W.; Cao, S. X.; Gangloff, A. R.; Jennings, A. J.; Stafford, J. A.; Vu, P. H.; Xiao, X.-Y.
U.S. Patent 7169,801, 2007.
In conclusion, a series of benzimidazole and imidazole cinnam-
oyl hydroxamates were shown to inhibit recombinant human
HDACs in the low nanomolar range. Incorporation of an N1-piper-
dine into both benzimidazole and imidazole scaffolds conveyed
cellular potency and resulted in analogues demonstrating killing
of human cancer cells in the sub-micromolar range. Compounds
5x and 5aa induced acetylation of histones H3 and H4 in vitro
and in vivo as well as induced p21waf activity. Compound 5x also
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