Design and synthesis of 4-[(s-triazin-2-ylamino)methyl]-N-(2-aminophenyl)-benzamides and their analogues as a novel class of histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.009

Inhibition of histone deacetylases (HDAC) is emerging as a new strategy in human cancer therapy. The synthesis and biological evaluation of a variety of 4-(heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides is presented herein. From the different series

Full text of DOI:10.1016/j.bmcl.2007.12.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1067–1071
Design and synthesis of 4-[(s-triazin-2-ylamino)methyl]-N-
(2-aminophenyl)-benzamides and their analogues as a novel
class of histone deacetylase inhibitors
a
a,
a
a
a
*
´
´ ´
Isabelle Paquin, Stephane Raeppel, Silvana Leit, Frederic Gaudette, Nancy Zhou,
Oscar Moradei,a Oscar Saavedra,a Naomy Bernstein,a Franck Raeppel,a
Giliane Bouchain, Sylvie Frechette, Soon H. Woo, Arkadii Vaisburg,a
a
a
a,ꢀ
´
Marielle Fournel,b Ann Kalita,b Marie-France Robert,b Aihua Lu,b
Marie-Claude Trachy-Bourget,b Pu Theresa Yan,b Jianhong Liu,b Jubrail Rahil,b
A. Robert MacLeod,b,ꢁ Jeffrey M. Besterman,b Zuomei Lib and Daniel Delormea,§
aMethylGene Inc., Department of Medicinal Chemistry, 7220 Frederick-Banting, Montre´al, QC., Canada H4S 2A1
bMethylGene Inc., Department of Biology, 7220 Frederick-Banting, Montre´al, QC., Canada H4S 2A1
Received 16 October 2007; revised 5 December 2007; accepted 6 December 2007
Available online 21 December 2007
Abstract—Inhibition of histone deacetylases (HDAC) is emerging as a new strategy in human cancer therapy. The synthesis and
biological evaluation of a variety of 4-(heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides is presented herein. From the dif-
ferent series bearing a six-membered heteroaromatic ring studied, the s-triazine series showed the best HDAC1 enzyme and
in vitro anti-proliferative activities with IC50 values below micromolar range. Some of these compounds can also significantly reduce
tumor growth in human tumor xenograft models in mice.
Ó 2007 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) are prominent targets
for cancer therapy. Evidence has shown that HDAC
inhibitors arrest cell growth and lead to differentiation
and apoptosis in tumor cells.1 HDACs are zinc enzymes
that catalyze the removal of the acetyl group from the
e-amino groups in histone lysine residues. These are
located at the N-terminal tails of histones and their
hydrolysis into positively charged amine groups leads
to changes in the chromatin structure, blocking the
access of transcription factors to DNA during gene
expression. Recently, ZolinzaTM from Merck (also
known as SAHA or Vorinostat)2 was the first HDAC
inhibitor approved by the FDA for the treatment of ad-
vanced cutaneous T-cell lymphoma (CTCL). Methyl-
Gene’s isotype specific HDAC inhibitor MGCD0103
(Fig. 1) is currently undergoing clinical evaluation in so-
lid tumors and hematological malignancies.3
Therefore, in our effort to identify new therapeutic
agents for the treatment of cancer, the design and
synthesis of selective HDAC inhibitors4 has become
one of our major goals. Thus, we have recently disclosed
a new series of N-(2-aminophenyl)cinnamide- and
benzamide-type derivatives 1a–c which inhibit HDAC
enzymes and exhibit in vitro anti-proliferative activities
in human tumor cell lines (Fig. 1).5 As a continuation
of these efforts, we report herein the efficient and
straightforward synthesis of new compounds of type 2,
bearing a six-membered heteroaromatic ring in the
amino-benzylic position.
Keywords: Histone deacetylases (HDACs); HDAC inhibitor; Anti-
proliferative activity; s-Triazine; Benzamides.
*
Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337
0550; e-mail: raeppels@methylgene.com
ꢀ
Present address: Auspex Pharmaceuticals, 1261 Liberty Way, Vista,
CA 92081-8356, USA.
ꢁ
Present address: Takeda San Diego, 10410 Science Center Drive, San
Diego, CA 92121, USA.
§
We also report the evaluation of these compounds as
HDAC inhibitors and potential anti-tumor agents.
Present address: Neurochem Inc., 275 Armand-Frappier Blvd, Laval,
Que., Canada H7V 4A7.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.009

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I. Paquin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1067–1071
O
N
H
N
OH
N
N
N
H
NH2
H
H
N
O
N
SAHA (ZolinzaTM
)
MGCD0103
O
R
O
X
X
R
N
H
NH2
H
S
n
N
NH2
X
N
H
NH2
H
N
N
H
N
1a, n = 1
1b, n = 0
N
H
O
O
O
O
1c
2, X = N and/or CH
Figure 1.
Table 1. Pyridine series (3–7)
A new pyridine-based series of HDAC inhibitors was
synthesized following the procedures described in
Scheme 1 (compounds 3–6).5a The thiopyridinyl inter-
mediate was synthesized by alkylation of pyridine-2-
thiol with methyl 4-(bromomethyl)-benzoate in the pres-
ence of base. The O-alkylation of 6-methyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile with the same alkylating
agent gave the methyl ester. Fluorine substitution of
2,6-difluoropyridine with methyl 4-aminomethylbenzo-
ate generated the ester intermediate.
X
NH2
H
N
O
Compound
X
HDAC1a
MTT HCT116b
IC50 (lM)
IC50 (lM)
3
0.3
1
N
S
Finally, palladium cross-coupling reaction between
2-chloro-6-methoxypyridine and methyl 4-amino-
methyl-benzoate was used to prepare the aminopyridine
intermediate. The ester intermediates were then hydro-
lyzed and coupled with 1,2-phenylenediamine to afford
the corresponding final compounds (3–6). Compound
7 (Table 1) was obtained similarly to the synthesis of 6.
CN
4
5
6
7
0.3
0.1
0.1
0.1
0.5
1
H3C
F
N
O
N
N
H
A new series of 4-[(pyrimidin-2-ylamino)methyl]-benz-
amides was also synthesized (Table 2, compounds
8–12). Compounds 8–10 were synthesized via a reduc-
tive amination6 as a key step, and an amide coupling
with 1,2-phenylenediamine, starting from the substi-
tuted 2-aminopyrimidines and 4-formylbenzoic acid.
Compound 11 was prepared in three steps (Scheme 2).
0.8
0.7
MeO
MeO
N
N
H
N
N
H
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation of human cancer HCT116 cells.
CN
The first step was the Suzuki coupling of 3,4-dim-
ethoxyphenylboronic acid with 2-amino-4,6-dichloro-
pyrimidine, followed by a reductive amination reaction
with the 4-formylbenzoic acid; the final amide coupling
with 1,2-phenylenediamine afforded the desired material
11. In a similar fashion, Suzuki coupling between 2,4-
dichloropyrimidine and 3,4-dimethoxyphenylboronic
acid, followed by the chlorine displacement with methyl
4-(aminomethyl)-benzoate, then saponification and
amide coupling with 1,2-phenylenediamine yielded the
desired compound 12.
H3C
N
H
O
F
N
F
c, e
b, e
MeO
N
Cl
R3
N
N
SH
R2
R1
R4
X
a, e
d, e
NH2
H
N
3: X = S, R1 = R2= R3 = R4 = H
4: X = O, R1 = Me, R2 = R3 = H, R4 = CN;
5: X = NH, R1 = F, R2 = R3 = R4 = H;
6: X = NH, R1 = MeO, R2= R3 = R4 = H
O
Scheme 1. Reagents and conditions: (a) methyl 4-(bromomethyl)-
benzoate, NaH, DMF, 0 °C–rt; (b) methyl 4-(bromomethyl)-benzoate,
K2CO3, DME, reflux; (c) methyl 4-(aminomethyl)-benzoate hydrochlo-
Pyrimidine 13 (Table 3) was synthesized using the same
procedures as used in the synthesis of 9. Few purine
derivatives were also generated (compounds 14–16,
Table 3). For example, the chlorine substitution of
6-chloro-9H-purin-2-amine or 2,6-dichloro-9H-purine
by methyl 4-(aminomethyl)-benzoate followed by a
˚
ride, K2CO3, MS 4 A, 18-crown-6, DMF, 110 °C; (d) methyl
4-(aminomethyl)-benzoate, Pd(OAc)2, NaO-t-Bu, 2-(dibutylphosphi-
no)-biphenyl, toluene, 110 °C; (e) i—LiOHÆH2O, THF/H2O, rt; ii—1,
2-phenylenediamine, BOP, Et3N, DMF.

I. Paquin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1067–1071
1069
Table 2. Pyrimidine series (8–12)
Table 3. Purine series (14–16)
R2
N
X
NH2
H
N
O
R1
N
N
H
NH2
H
N
Compound
X
HDAC1a
MTT HCT116b
IC50 (lM) IC50 (lM)
O
Compound R1
R2
HDAC1a
MTT
N
N
IC50 (lM) HCT116b
IC50 (lM)
13
0.2
0.2
1
MeO
N
H
NH2
8
9
H
CH3
H
CH3
0.7
0.3
2
1
0.6
N
N
14
10
10
OCH3
OCH3 0.1
N
HN
MeO
H
N
11
12
Cl
H
0.1
0.4
0.1
NH2
N
MeO
MeO
N
15
16
0.3
2
n
-Bu
N
H
0.05
N
N
Cl
MeO
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation of human cancer HCT116 cells.
N
N
0.05
0.1
n-Bu
N
H
N
N
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation of human cancer HCT116 cells.
Cl
N
N
Cl
N
Cl
Cl
N
NH2
R1
R2
N
X
a, c-e
X
a, b, e
N
N
N
a-d
N
N
N
H
NH2
H
N
n-Bu
N
H
NH2
N
Cl
HN
H
N
N
11: R1 =3,4-dimethoxyphenyl, R2= Cl
N
12: R1 =3,4-dimethoxyphenyl , R2= H
15: X = NH2
16: X = Cl
O
O
Scheme 2. Reagents and conditions: (a) 3,4-dimethoxyphenylboronic
acid, Ph3P, Pd(OAc)2, Na2CO3, DME, reflux; (b) 4-formylbenzoic
acid, Bu2SnCl2, PhSiH3, THF; (c) methyl 4-(aminomethyl)-benzoate,
DIPEA, DMF, 120 °C; (d) LiOHÆH2O, THF/H2O; (e) 1,2-phenylene-
diamine, BOP, Et3N, DMF.
Scheme 3. Reagents: (a) methyl 4-(aminomethyl)-benzoate hydrochlo-
ride, NaHCO3, H2O; (b) n-butanol, Ph3P, DEAD; (c) LiOHÆH2O,
THF/H2O; (d) 1,2-phenylenediamine, BOP, DIPEA, DMF.
from cyanuric chloride according to the pathways A
or B as indicated in Table 4 (also in Scheme 4).
Mitsunobu reaction with n-butanol gave the methyl es-
ter intermediates. Then, the hydrolysis and the final
amide coupling generated the desired compounds 15
and 16 (Scheme 3).
All compounds were initially screened for their ability to
inhibit recombinant human HDAC1. We targeted this
isotype following the results of our work and of others
which implicated HDAC1 for both transcriptional
repression and chromatin remodeling.7 The compounds
were screened as described earlier, using BocLys(acetyl)-
AMC as substrate. The in vitro anti-proliferative activ-
ities of the synthesized compounds against HCT116
human colon cancer cell line were evaluated using
3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bro-
mide (MTT). The first series studied, compounds 3–7
(Table 1), showed good activity against HDAC1 and
good anti-proliferative activity, both in the sub-micromo-
lar range. The inhibitory potency (IC50) was not sensitive
to the small structural changes on the pyridine ring, such
as in compounds 5 and 6, the heteroatom linker in the
benzylic position (compounds 3–5), or the pyridine nitro-
gen position (e.g., 6 vs 7). The in vitro anti-proliferative
activity (MTT assay) followed the same trend.
To complete the SAR, the HDAC inhibitors of the s-tri-
azine series were prepared (compounds 17–25, Table 4).
The intermediate 26 was generated via a mono-substitu-
tion reaction of the chlorine atom in cyanuric chloride
with methyl 4-(aminomethyl)-benzoate (Scheme 4). In
pathway A the NH2-group was first introduced using
ammonia gas in a sealed flask and the indan-2-yl amino
group was introduced at higher temperature to generate
the compound 29. In pathway B, the indan-2-yl amino
group was introduced first followed by the cyclopropyl
amino group incorporation, to obtain compound 30.
The ester functionalities of compounds 29 and 30 were
hydrolyzed and then coupled with 1,2-phenylenediamine
using BOP reagent to give final compounds 17 and 23.
Compounds 18–22 and 24–25 were synthesized starting

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I. Paquin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1067–1071
Table 4. s-Triazine series (17–25)
R2
N
N
R1
N
N
NH2
H
N
H
O
Compound
R1
R2
Synthesis
pathway
HDAC1a
MTT HCT116b
H4 Acc (T24)
MTT HMECb
IC50 (lM)
IC50 (lM)
EC50 (lM)
IC50 (lM)
17
18
19
20
21
22
23
24
25
NH2
NH2
NH2
NH2
NH2
NH2
A
A
A
B
A
A
B
B
B
0.2
0.6
1
8
11
NH
0.2
0.7
0.3
0.8
0.5
0.8
1
0.8
1
NH
0.1
21
N
H
N
0.1
1
10
0.04
0.2
1
>50
>40
18
N
5
O
N
0.03
0.08
0.07
1
NH
NH
NH
N
H
2
1
>50
16
O
O
N
0.6
1
N
N
H
Human T24 cells were treated with compounds at 0, 1, 5, 25 lM for 16 h. Cells were harvested and histones were acid-extracted.
Histones were analyzed by SDS–PAGE and immunoblotting with antibodies specific for either H4 histones or acetylated H4 histones.
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation of human cancer HCT116 cells and human normal mammary epithelial (HMEC) cells.
c Relative effective concentration of compounds in induction of histone H4 acetylation in T24 human cancer cells, relative to pyridin-3-ylmethyl 4-(2-
aminophenylcarbamoyl)benzylcarbamate (MS-275),8 which was used as a positive control in these experiments at 1 lM.
Cl
N
NH2
N
Cl
N
N
N
N
a
b
Pathway A
N
N
Cl
N
H
Cl
N
N
H
OMe
OMe
26
Cl
Cl
27
NR3R4
N
O
O
NR3R4
Cl
d
c
e
N
N
N
N
N
f
Pathway B
R1R2N
N
N
H
R1R2N
N
N
NH2
R1R2N
N
N
H
N
H
H
OMe
OMe
O
O
O
29: R1 = indanyl-2-yl, R2 = R3 =R4 = H
17: R1 = indanyl-2-yl, R2 = R3 =R4 =H
23: R1 = indanyl-2-yl, R2 = R3 = H, R4 = cyclopropyl
28: R1 = indanyl-2-yl,R2 = H
30: R1 = indanyl-2-yl, R2 = R3 = H, R4 = cyclopropyl
Scheme 4. Reagents and conditions: (a) methyl 4-(aminomethyl)-benzoate hydrochloride, DIPEA, THF, À78 °C; (b) NH3 gas, 1,4-dioxane, sealed
flask, 70 °C; (c) R1R2NH, THF or 1,4-dioxane, sealed flask, 120–140 °C; (d) R1R2NH, i-Pr2NEt, THF; (e) NH3 or R3R4NH2, THF or 1,4-dioxane,
sealed flask, 120–140 °C; (f) i—LiOHÆH2O, THF/H2O; ii—1,2-phenylenediamine, BOP, Et3N, DMF.

I. Paquin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1067–1071
1071
Similarly, in the pyrimidine series (Table 2), the size and
the electronic effects of substituents have a minimal
effect on HDAC1 inhibitory activity (0.05–0.3 lM),
except for compound 8 which shows slightly weakened
cytotoxicity (2 lM). Replacing the pyrimidine ring with
a purine system (compounds 14–16, Table 3) generates
compounds that are at least as potent as the compound
13 against the HDAC1 enzyme. With respect to the
activity in the cell proliferation assay it looks like the
polar fragments are detrimental and the hydrophobic
substituents are favorable (e.g., 14 vs 15 and 15 vs 16).
having HDAC1 inhibitory potency and anti-prolifera-
tive activity in the sub-micromolar range. These inhibi-
tors selectively induced hyperacetylation of histones in
whole cells. Compounds 18, 22, and 25 showed signifi-
cant anti-tumor activity in vivo in HCT116 human co-
lon cancer xenograft models in mice. These results
represent an important step toward the development
of selective HDAC small molecule inhibitors with favor-
able drug-like characteristics.
References and notes
Finally, the s-triazine series (compounds 17–25, Table 4)
can accommodate a plethora of substituents. The struc-
tural diversification brought about increased potency
against HDAC1 with IC50 less than 0.2 lM and with
concomitant increase in anti-proliferative activity. Fur-
thermore, as the data in Table 4 show, the anti-prolifer-
ative activity of these compounds is selective to the
cancer cell line tested (HCT116) with a toxicity index
that in some cases exceeds a 100-fold when compared
to a normal cell line (HMEC). The optimal R1 substitu-
ent producing the best HDAC1 inhibitory activity was
found to be either indanyl-2-amino- (23) or iso-indoli-
nyl- (21). Likewise, the optimal R2 substituent seems
to be an amino- or alkylamino- group. In general, com-
pounds of the triazine series show better profiles than
their pyridine, pyrimidine, and purine analogues.
1. (a) Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074; (b)
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A representative set of compounds (17, 18, 22, and 25)
were profiled against the different HDAC isoforms. In
class I, the compounds showed potent activity against
HDAC1, 2, and 3 in the sub-micromolar range, and
were very weak or inactive against HDAC 8. The same
compounds exhibited no activity against the class II
HDACs (HDAC4–7). Consistent with the ability of
the s-triazine-based compounds to inhibit cellular
HDAC, they were able to induce histone acetylation in
T24 human bladder cancer cells (Table 4) and induce
p21WAF1/Cip1 (data not shown). A few HDAC small
molecule inhibitors from the s-triazine series were evalu-
ated in vivo in HCT116 (human colon) tumor xenograft
model in mice. Compounds were administered daily by
intraperitoneal (ip) injection. Compound 25 displayed
anti-tumor activity with 49% tumor growth inhibition
relative to vehicle control when dosed at 20 mg/kg per
day for two weeks. Compounds 18 and 22 showed both
around 50% inhibition when dosed at 40 mg/kg. The
three compounds displayed little toxicity as revealed
by body and spleen weight measurements and white
blood cell counts.
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A novel class of selective HDAC class I inhibitors of
general structure 2 was designed and the synthesis of
these new compounds was developed. s-Triazine-based
analogues turned out to be the most active compounds