N-(2-Amino-phenyl)-4-(heteroarylmethyl)-benzamides as new histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.10.050

A variety of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benzamides were designed and synthesized. These compounds were shown to inhibit recombinant human HDAC1 with IC₅₀ values in the sub-micromolar range. In human cancer cells growing in culture these compounds induced hyperacetylation of histones, induced the expression of the tumor suppressor protein p21^WAF1/Cip1, and inhibited cellular proliferation. Certain compounds of this class also showed in vivo activity in various human tumor xenograft models in mice.

Full text of DOI:10.1016/j.bmcl.2007.10.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6729–6733
N-(2-Amino-phenyl)-4-(heteroarylmethyl)-benzamides
as new histone deacetylase inhibitors
Arkadii Vaisburg,a,* Isabelle Paquin,a Naomy Bernstein,a Sylvie Frechette,a
Frederic Gaudette,a Silvana Leit,a Oscar Moradei,a Stephane Raeppel,a Nancy Zhou,a
Giliane Bouchain,a Soon Hyung Woo,c Zhiyun Jin,a Jeff Gillespie,a James Wang,a
Marielle Fournel,b Pu Theresa Yan,b Marie-Claude Trachy-Bourget,b
Marie-France Robert,b Aihua Lu,b Jimmy Yuk,b Jubrail Rahil,b A. Robert MacLeod,e
Jeffrey M. Besterman,b Zuomei Lib and Daniel Delormed
aMethylGene Inc., Department of Chemistry, 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
bMethylGene Inc., Department of Biology, 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
cAuspex Pharmaceuticals, 1261 Liberty Way, Vista, CA 92081-8356, USA
dNeurochem, 275 Armand-Frappier Blvd., Laval, Que., Canada H7V 4A7
eTakeda San Diego, 10410 Science Center Drive, San Diego, CA 92121, USA
Received 9 September 2007; revised 15 October 2007; accepted 15 October 2007
Available online 18 October 2007
Dedicated to the memory of Naomy Bernstein, our colleague and friend.
Abstract—A variety of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benzamides were designed and synthesized. These compounds were
shown to inhibit recombinant human HDAC1 with IC50 values in the sub-micromolar range. In human cancer cells growing in culture
these compounds induced hyperacetylation of histones, induced the expression of the tumor suppressor protein p21WAF1/Cip1
and inhibited cellular proliferation. Certain compounds of this class also showed in vivo activity in various human tumor xenograft
,
models in mice.
Ó 2007 Elsevier Ltd. All rights reserved.
Histone acetylation/deacetylation is essential for chro-
matin remodeling and regulation of gene transcription
in eukaryotic cells. Histone deacetylases (HDACs) and
histone acetyltransferases (HATs) are enzymes that cat-
alyze the deacetylation (associated with transcriptional
silencing) and acetylation (associated with transcrip-
tional activation), respectively, of lysine residues located
in the NH2 terminal tails of core histones.1 Perturba-
tions of this balance in the form of histone deacetylation
are often observed in human tumors.2 Thus, inhibition
of HDACs has emerged as a novel therapeutic strategy
against cancer.3 Small molecules of different classes such
as MGCD0103 (1) (MethylGene Inc.),4,5 CRA-024781
(2) (Celera Genomics),6 PXD-101 (3) (Curagen/Topo-
Target),7 LBH-589 (4) (Novartis AG),8 and MS-275
(5) (Syndax Pharmaceuticals/Schering AG)9 are potent
HDAC inhibitors, demonstrating in vivo antitumor effi-
cacy and are currently undergoing clinical trials (Fig. 1).
Me
N
Me
O
N
O
N
N
H
NH2
H
N
NHOH
NHOH
H
N
N
O
O
O
O
MGCD0103(1)
CRA-024781 (2)
O
H
N
O
S
H
N
NHOH
O
N
PXD-101 (3)
LBH-589 (4)
Me
H
O
Keywords: Histone deacetylase inhibitors; HDAC; Benzamides; Cancer
cell proliferation.
O
H
O
N
H
NH2
N
H
N
NHOH
*
Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337
0550; e-mail: vaisburga@methylgene.com
N
O
O
MS-275 (5)
SAHA
Figure 1.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.050

6730
A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6729–6733
O
Suberoylanilide hydroxamic acid Zolinza(TM) (vorino-
stat, SAHA, Merck)10 has recently been approved for
the treatment of advanced cutaneous T-cell lymphoma
(CTCL).
CHO
CHO
(a), (b)
N
NH2
H
N
O
11
12
In our research program directed toward design, synthe-
sis, and biological evaluation of novel HDAC inhibitors
with adequate ‘drug-like’ properties, we identified sev-
eral distinct classes of such molecules: arylsulfona-
mide-based hydroxamates exemplified by compound
(6),11 arylsulfonamide-based amino-anilides such as
(7),12 long-chain SAHA-like x-substituted hydroxamic
acids like (8),13 and 2-amino-phenylamides of x-substi-
tuted alkanoic acids such as (9).14 All these classes of
compounds while possessing good in vitro HDAC-
inhibitory activity showed, however, marginal in vivo
efficacy, which was attributed to their relatively short
half-life and poor bioavailability (data not shown). Ef-
forts to overcome these shortcomings led us to the novel
class of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benz-
amides (10) (Fig. 2), which not only retained high
HDAC in vitro potency, but showed significant
improvement in antitumor in vivo behavior.
O
O
(c)
O
N
H
OH
N
O
NH2
H
O
13
14
(d), (e) or (f)
O
N
O
N
N
X
NH2
(g), (h)
H
N
X
N
OH
O
O
16: X = CH; 17: X = N
15a: X = CH; 15b: X = N; 15c: X = CCH3
(b)
O
N
NH2
H
N
N
Me
O
18
Scheme 1. Reagents and conditions: (a) p-aminomethylbenzoic acid,
AcOH, 5 min, reflux, 49%; (b) 1,2-phenylenediamine, BOP, Et3N,
DMF, 31–49%; (c) p-aminomethylbenzoic acid, Et3N, H2O, 3 h, 40 °C
100%; (d) HCOOH, reflux, 6 h (X = CH), 96%; (e) NaNO2, HCl, 0 °C
(X = N), 96%; (f) Ac2O, reflux, 1 h, then AcOH, reflux, 48 h
(X = CCH3), 43%; (g) (2-amino-phenyl)-carbamic acid tert-butyl ester,
BOP, Et3N, DMF (58%, X = CH; 62%, X = N); (h) TFA/CH2Cl2
(74%, X = CH; 19%, X = N).
The first series of target compounds representing the
class of N-(2-amino-phenyl)-4-(heteroarylmethyl)-benz-
amides (10) was the set of compounds bearing a car-
bonyl group on the left-hand side heterocyclic moiety
such as compounds 12, 16–18. Their synthesis is pre-
sented in Scheme 1, while the detailed procedures are de-
scribed in Delorme et al.15 It is worth mentioning that
all these HDAC inhibitors as well as the ones described
below bear the N-(2-amino-phenyl)-benzamide fragment
which has been shown to be the pharmacophore.9b Re-
moval of the amino group is detrimental to the
HDAC-inhibitory activity. The amino group, however,
can be replaced by an OH-group without the loss of po-
tency and only a few other subtle substitutions in that
fragment are allowed.9b
pounds 20, 22, and 24–29 (Scheme 2). We also explored
a replacement of the fused benzene ring in compounds
16, 24, and 26 for a thiophene thus preparing the corre-
sponding thienopyrimidines 31, 34, and 36 (Scheme 3).
To further investigate possible replacements within this
class of molecules, we substituted the endocyclic nitro-
gen atom as an attachment point between heterocycles
and the benzamide fragments, for a carbon atom, which
resulted in the design and synthesis of the ‘carbon ana-
logues’ 38, 41–43 (Scheme 4). 4-(4-Oxo-chroman-3-
ylidenemethyl)-benzoic acid methyl ester (40) turned
out to be the key intermediate in the synthesis of target
molecules 41–43. Thus, the RhCl3 mediated isomeriza-
tion of its double bond16 followed by hydrolysis and a
coupling with 1,2-phenylenediamine afforded compound
41. Reduction of the double bond using phenylsulfonyl
hydrazine was the key procedure in the synthesis of
the target 42, while hydrogenation of 40 in the presence
of palladium on charcoal ultimately allowed for the for-
mation of the compound 43 devoid of the carbonyl
group.
The second series of the synthesized target compounds
was the series of molecules bearing two carbonyl groups
on the left-hand side heterocyclic moiety such as com-
O
NHOH
O
O
O
S
N
N
H
O
O
H
S
NH2
MeO
N
H
6
8
OMe
MeO
7
OMe
O
NHOH
O
NH2
H
O
HO
N
N
O
The data in Table 1 demonstrate the ability of these
compounds to inhibit recombinant HDAC1 with an
IC50 range of 0.1–1.0 lM (measured using BocLys(ace-
tyl)AMC as substrate).17 Also, these compounds dem-
onstrated good in vitro antiproliferative potency
(measured with MTT reagent) in the human colon can-
cer cell line (HCT116), with desirable selectivity (6- to
>50-fold) over normal human mammary epithelial cells
N
O
N
O
9
O
Y
N
X
NH2
H
N
10
O
Figure 2.

A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6729–6733
6731
O
O
O
O
O
(c), (d), (b)
(a), (b)
N
NH2
N
NH2
H
N
H
N
O
NH
O
O
O
O
O
O
O
19
20
21
22
O
O
O
(e), (f)
(b)
N
NH2
N
H
NH2
N
H
N
OH
OH
N
H
O
N
H
O
O
O
O
14
24
23
(g), (h), (b)
25:
26:
28:
R = Me;
R = Et;
R =
O
O
Me
N
N
N
NH2
27: R =
H
N
Me
O
N
R
29:
R =
O
25 - 29
OMe
Scheme 2. Reagents and conditions: (a) p-aminomethylbenzoic acid, AcOH, reflux, 68%; (b) 1,2-phenylenediamine, BOP, Et3N, DMF, 10–27%; (c)
methyl 4-(bromomethyl)benzoate, Cs2CO3, DMF, 40%; (d) AcOH, HCl, reflux, 91%; (e) ClCO2Et, Pyr, 0 °C; (f) NaOH, MeOH, H2O, reflux, 90%
(over two steps); (g) MeI, K2CO3, DMF, 85%; (h) NaOH, THF, H2O, 96%. Compounds 26–29 were obtained similarly to the compound 25 using in
the step (g) ethyl iodide, (2-chloroethyl)-dimethylamine, 4-(2-chloroethyl)-morpholine or 1-bromomethyl-4-methoxy-benzene, respectively, instead of
methyl iodide.
O
N
O
S
S
(a), (b), (c)
N
NH2
NH
H
N
N
O
30
31
O
O
O
S
(d), (e), (f)
S
N
NH2
N
(c)
H
N
S
OMe
OH
N
H
O
N
H
O
NH2
32
O
O
33
34
(g), (b)
O
O
S
S
N
N
NH2
(c)
H
N
OH
N
O
N
O
O
Et
O
Et
35
36
Scheme 3. Reagents and conditions: (a) methyl 4-(aminomethyl)benzoate, K2CO3, DMF, 100%; (b) LiOH, THF, H2O, 80–87%; (c) 1,2-
phenylenediamine, BOP, Et3N, DMF, 31–75%; (d) triphosgene, Et3N, DCM, À78 °C to rt; (e) methyl 4-(aminomethyl)benzoate, Et3N, DCM, 91%
(two steps); (f) NaOH, MeOH, reflux, then H2O, reflux, 95%; (g) EtI, K2CO3, DMF, 94%.
O
O
O
NH2
(a), (b), (c)
H
N
NH2
H
N
O
O
O
38
40
37
39
41
(e), (b), (c)
O
O
O
O
X
(d)
NH2
(f) or (g), (b), (c)
H
N
OMe
O
O
O
42: X = CO; 43: X = CH2
Scheme 4. Reagents and conditions: (a) methyl-4-bromomethylbenzoate, LDA 2 M, THF, 17%; (b) LiOH, THF, H2O, 61–98%; (c) 1,2-
phenylenediamine, BOP, Et3N, DMF, 10–69%; (d) methyl-4-formylbenzoate, AcOH, H2SO4, 82%; (e) RhCl3ÆH2O, EtOH, 16 h, 70 °C, 59%; (f)
PhSO2NHNH2, DMF, 100 °C, 42% (X = CO); (g) Pd/C, H2, MeOH, DMA, 59% (X = CH2).
(HMEC). Consistent with inhibiting cellular HDACs,
these inhibitors induced histone hyperacetylation in
T24 human cancer cells (EC50 ꢀ 1 lM), in a dose-depen-
dent fashion. Also consistent with their mechanism
of action, these compounds induced the expression
of the cyclin-dependent kinase inhibitor p21WAF1/Cip1

6732
A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6729–6733
Table 1. In vitro profile of histone deacetylase inhibitors
Compound HDAC1 MTT MTT H4-Ac p21
HCT116 HMEC EC50
6-membered one (compounds 22 and 24). Alkylation
of the nitrogen in 24 generally did not affect
the in vitro parameters (compounds 25–29); their
enzymatic and cellular potencies remained micro- or
sub-micromolar with comparable numbers for H4-acet-
ylation or p21WAF1/Cip1 induction. Thienopyrimidine
based HDAC inhibitors 31, 34, and 36 showed similar
overall in vitro profiles when compared to their benzo
annelated analogues (compounds 16, 24, and 26) with
the most attractive being 34. The carbon analogues 38,
41–43 did not look superior relative to the best
compounds with a C–N linker (16, 25–26, or 34): the
IC50 values in cytotoxicity assay against human normal
mammary epithelial cells (HMEC) were lower, indicat-
ing the potential for toxicity by these molecules. In
addition to that, compound 43 (lacking the carbonyl
group on the left-hand side) exhibited only micromolar
potencies in all the assays (Table 1).
IC50
induction
EC50
(lM)
IC50
(lM)
IC50
(lM)
(lM)
(lM)
12
16
17
18
20
22
24
25
26
27
28
29
31
34
36
38
41
42
43
0.1
0.1
0.2
0.08
0.2
0.1
0.06
0.1
0.2
0.3
0.4
0.1
0.2
0.06
0.3
0.1
0.03
0.09
1
0.5
0.4
0.6
0.6
5
37
28
1
2
1
1
2
3
2
2
1
0.6
1
5
3
1
1
3
2
1
1
3
<1
1
17
>50
>50
21
1
5
0.3
0.8
0.7
0.4
1
1
18
1
>42
25
1
1
>25
>25
>25
>50
29
1
1
3
0.8
0.7
0.4
0.8
0.6
0.3
0.9
2
1
1
1
>50
8
1
<1
1
Selected compounds underwent pharmacokinetic evalu-
ation in female Sprague–Dawley rats. The compounds
were dosed at 2.5 mg/kg (iv) or 5.0 mg/kg (po) with
the dosing vehicles being acidified saline or acidified sal-
ine/PEG400. Compounds 27, 31, 34, and 36 turned out
to be reasonable with respect to their half-lives and oral
availability (Table 2).
9
6
23
2
3
HDAC1: Inhibition of recombinant HDAC1.
MTT: Cytotoxicity/proliferation of human cancer HCT116 cells and
human normal mammary epithelial (HMEC) cells.
H4-Ac: Relative effective concentration of compounds in induction of
histone H4 acetylation in T24 human cancer cells, relative to MS-275
at 1 lM. Human T24 cells were treated with compounds at 0, 1, 5, and
25 lM for 16 h. Cells were harvested and histones were acid-extracted.
Histones were analyzed by SDS–PAGE and immunoblotted with
antibodies specific for either H4 histones or acetylated H4 histones.
Compounds 24, 31, and 34 were further evaluated
in vivo in several human tumor xenograft models
[A549 (non-small cell lung), DU145 (prostate), and
SW48, HCT116 (colon)] in nude mice (6 animals per
group). Compounds were administered once daily for
14 days by either intraperitoneal (ip) injection or orally
(po). The compounds demonstrated significant antitu-
mor activity in all models tested (% tumor growth inhi-
bition vs vehicle treated control animals) by either
dosing route at well tolerated doses (Table 3).
p21WAF1/Cip1
: Relative effective concentration of compounds in
induction of p21WAF1/Cip1 in T24 human cancer cells, relative to MS-
275 at 1 lM. Human cancer T24 cells were treated with compounds at
concentrations at 0, 1, 5, and 25 lM or DMSO alone for 16 h before
whole cell lysates were harvested and analyzed by Western blot using
p21WAF1/Cip1 antibody. Tubulin level was analyzed to reveal protein
loading.
We designed and synthesized a novel class of HDAC
inhibitors, the N-(2-amino-phenyl)-4-(heteroarylmeth-
yl)-benzamides. These compounds exhibit in vitro anti-
proliferative activities in numerous human cancer cells,
but not normal cells, induce p21WAF1/Cip1 expression
(EC50 ꢀ 2 lM). In addition, they caused apoptosis and
G2/M cell cycle arrest in HCT116 human colon cancer
cell line (data not shown).
The first compound from the mono-carbonyl series,
compound 12, already exhibited an in vitro profile sim-
ilar to the one of MS-275, which was used as a positive
control in our experiments. Changing the ring from 5- to
6-membered (proceeding from compound 12 to com-
pounds 16 and 17) or adding a methyl group to the car-
bon between the pyrimidine nitrogens (compound 18)
did not affect the enzymatic or cellular activities.
Table 2. Pharmacokinetic profile of selected compounds
Compound
T12 iv (h)
F%
12
18
27
31
34
36
0.6
0.9
2.1
1.4
1.3
1.2
34
16
38
47
47
64
The first compound from the di-carbonyl series, com-
pound 20, although only 2-fold less potent against the
HD1 enzyme, showed, however, weaker cytotoxic activ-
ity and was inferior in terms of induction of histone
acetylation and p21WAF1/Cip1 when compared with its
mono-carbonyl counterpart, compound 12. The cyto-
toxicity of the HDAC inhibitors and the ability to
induce acetylation of H4 histones have been improved
when the 5-membered ring (in 20) was replaced by the
Table 3. In vivo antitumor activity of 24, 31, and 34
Compound
Dose (mg/kg)
% tumor growth inhibition
24
24
31
34
34
20 (ip)
40 (ip)
80 (po)
40 (ip)
80 (po)
51% (HCT116)
59% (HCT116)
42% (A549), 45% (DU145)
61% (HCT116)
55% (A549), 67% (DU145)

A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6729–6733
6733
8. (a) Maiso, P. et al. Cancer Res. 2006, 66, 5781; (b) Giles,
F. J. et al. Blood 2004, 104, 499A.
and hyperacetylation of core histones, and cause cell
cycle arrest and apoptosis of human cancer cells.
9. (a) Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.;
Tsuchiya, K.; Ando, T.; Suzuki, T.; Tsuruo, T.; Nakani-
shi, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4592; (b)
Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukasawa, N.; Saito,
A.; Mariko, Y.; Yamashita, T.; Nakanishi, O. J. Med.
Chem. 1999, 42, 3001; (c) U.S. Patent 6174905, 2001; (d)
Ryan, Q. C.; Headlee, D.; Acharya, M.; Sparreboom, A.;
Trepel, J. B.; Ye, J.; Figg, W. D.; Hwang, K.; Chung, E. J.;
Murgo, A.; Melillo, G.; Elsayed, Y.; Monga, M.; Kalnit-
skiy, M.; Zwiebel, J.; Sausville, E. A. J. Clin. Oncol. 2005,
23, 3912.
The most promising HDAC inhibitor of this series was
found to be compound 34. It has an excellent in vitro
profile and shows significant antitumor activity in vivo
in several human cancer xenograft models. Compound
34 is metabolically stable, does not show any major
binding to receptors, channels or enzymes that can pre-
dict potential toxicity liabilities (data not shown), and
possesses acceptable physiochemical and pharmacoki-
netic characteristics.
10. (a) Kim, Y. B.; Lee, K. H.; Sugita, K.; Yoshida, M.;
Horinouchi, S. Oncogene 1999, 18, 2461; Review: (b)
Meinke, P. T.; Liberator, P. Curr. Med. Chem. 2000, 8,
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Kelly, W. K.; Richon, V. M.; O’Connor, O.; Curley, T.;
MacGregor-Curtelli, B.; Tong, W.; Klang, M.; Schwartz,
L.; Richardson, S.; Rosa, E.; Drobnjak, M.; Cordon-
Cordo, C.; Chiao, J. H.; Rifkind, R.; Marks, P. A.; Scher,
H. J. Clin. Oncol. 2005, 23, 3923.
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´
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Gaudette, F.; Paquin, I.; Bouchain, G.; Bernstein, N.;
Raeppel, F.; Saavedra, O.; Woo, S. H.; Vaisburg, A.;
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5. Vaisburg, A. Discovery and Development of MGCD0103—
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in Patent Application WO 2003/024448, 2003.
16. Cachia, P.; Darby, N.; Eck, Ch. R.; Money, Th. J. Chem.
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17. The full length human HDAC1 was cloned into a
baculoviral expression vector to generate a C-terminal
FLAG-tagged enzyme. Recombinant HDAC1-FLAG was
expressed in Sf9 cells (Spodoptera frugiperda) and purified
using an affinity resin (antiFLAG M2 agarose) followed
by size exclusion chromatography. The enzymatic reaction
was followed by using substrate BocLys(acetyl)AMC.
The de-acetylated product was captured fluorometrically
upon trypsin hydrolysis of the AMC moiety, which is
accompanied by fluorescence change (kex = 360 nm and
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kem = 470 nm).