Discovery of N-(2-Aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino) methyl]benzamide(MGCD0103), an orally active histone deacetylase inhibitor

Article abstract of DOI:10.1021/jm800251w

The design, synthesis, and biological evaluation of N-(2-aminophenyl)-4- [(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide 8 (MGCD0103) is described. Compound 8 is an isotype-selective small molecule histone deacetylase (HDAC) inhibitor that selectively inhibits HDACs 1-3 and 11 at submicromolar concentrations in vitro. 8 blocks cancer cell proliferation and induces histone acetylation, p21^ciP/waf1 protein expression, cell-cycle arrest, and apoptosis. 8 is orally bioavailable, has significant antitumor activity in vivo, has entered clinical trials, and shows promise as an anticancer drug.

Full text of DOI:10.1021/jm800251w

4072
J. Med. Chem. 2008, 51, 4072–4075
Discovery of N-(2-Aminophenyl)-
4-[(4-pyridin-3-ylpyrimidin-
2-ylamino)methyl]benzamide (MGCD0103),
an Orally Active Histone Deacetylase
Inhibitor
Nancy Zhou, Oscar Moradei, Stephane Raeppel,
Silvana Leit, Sylvie Frechette, Frederic Gaudette,
Isabelle Paquin, Naomy Bernstein, Giliane Bouchain,
Arkadii Vaisburg,* Zhiyun Jin, Jeff Gillespie, James Wang,
Marielle Fournel, Pu T. Yan, Marie-Claude Trachy-Bourget,
Ann Kalita, Aihua Lu, Jubrail Rahil, A. Robert MacLeod,§
Zuomei Li, Jeffrey M. Besterman, and Daniel Delorme†
Figure 1. Small molecule HDAC inhibitors in clinical development.
MethylGene Inc., 7220 Frederick-Banting, Montre´al, Que´bec H4S
2A1, Canada
ReceiVed March 7, 2008
Abstract: The design, synthesis, and biological evaluation of N-(2-
aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benza-
mide 8 (MGCD0103) is described. Compound 8 is an isotype-selective
small molecule histone deacetylase (HDAC) inhibitor that selectively
inhibits HDACs 1-3 and 11 at submicromolar concentrations in vitro.
8 blocks cancer cell proliferation and induces histone acetylation,
p21cip/waf1 protein expression, cell-cycle arrest, and apoptosis. 8 is orally
bioavailable, has significant antitumor activity in vivo, has entered
clinical trials, and shows promise as an anticancer drug.
Figure 2. Pharmacophore model of aminophenyl benzamides 7.
Histone acetylation/deacetylation is essential for chromatin
remodeling and epigenetic regulation of gene transcription in
eukaryotic cells. Histone acetyltransferases (HATs) are enzymes
that catalyze histone acetylation, which is associated with
transcriptional activation.1 Histone deacetylases (HDACs) are
enzymes that catalyze the deacetylation of lysine residues located
in the NH2 terminal tails of core histones, which is associated
with transcriptional silencing.1 There are 18 known human
histone deacetylases, grouped into four classes based on the
structure of their accessory domains. Class I (HDACs 1-3 and
8), II (HDACs 4-7, 9, and 10), and IV (HDAC 11) enzymes
are Zn2+-dependent enzymes and are called HDACs, while class
III enzymes (also known as sirtuins) are defined by their
dependency on NAD+. Perturbations of histone deacetylation
have been observed in human tumors, and inhibition of HDACs
has emerged as a novel and validated therapeutic strategy against
cancer.1 2
Small molecule inhibitors of HDACs belonging to different
classes such as SAHA (1) (vorinostat, Merck & Co.),3 recently
approved for the treatment of advanced cutaneous T-cell
lymphoma (CTCL), CRA-024781 (2) (Pharmacyclics),4 PXD-
101 (3) (CuraGen & TopoTarget),5 LBH-589 (4) (Novartis
AG),6 R-306465 (5) (Janssen Pharmaceuticals),7 and MS-275
(6) (Syndax Pharmaceuticals/Schering AG)8 are in various stages
of development and have demonstrated in vivo antitumor
efficacy (Figure 1).
We have identified several distinct classes of novel HDAC
inhibitors with development potential: arylsulfonamide-based
hydroxamates,9 long-chain ω-substituted hydroxamic acids,10
arylsulfonamide-based aminoanilides,11 2-aminophenylamides
of ω-substituted alkanoic acids,12 chalcone type13 or cinnamic
acid14 aminobenzamides. All of these classes of compounds
inhibited HDAC enzyme activity in vitro and in many cases
demonstrated significant antitumor efficacy in vivo. Further
studies revealed the novel class of N-(2-aminophenyl)-4-
(arylaminomethyl)benzamides (7) (Figure 2), which not only
retained potent enzymatic and cellular inhibition of HDAC
activity in vitro but also displayed significant improvements in
pharmacokinetic properties and antitumor efficacy in vivo in
human tumor xenograft models.14,15
Structure-activity relationships (SAR) of aminoanilides 7
were thoroughly investigated14–17 and are summarized in Figure
2. Extensive optimization of the left-hand-side aromatic (het-
eroaromatic) ring of 7, as well as the types and positions of
substituents attached to that ring, resulted in the discovery of a
potent HDAC inhibitor N-(2-aminophenyl)-4-[(4-pyridin-3-
ylpyrimidin-2-ylamino)methyl]benzamide 8 (MGCD0103), which
became the MethylGene clinical candidate. The aminophenyl-
benzamide 8 was obtained via a short reaction sequence18
starting from commercially available 3-acetylpyridine (9),
4-aminomethylbenzoic acid methyl ester hydrochloride salt (10),
and 1,2-phenylenediamine (11) (Scheme 1).
* To whom correspondence should be addressed. Phone: (514) 337-3333,
ext 233. Fax: (514) 337-0550. E-mail: vaisburga@methylgene.com.
The reaction of 9 with N,N-dimethylformamide dimethyl
acetal provided 3-dimethylamino-1-pyridin-3-ylpropenone (12)
as the first synthetic intermediate. Treatment of the amino ester
10 with pyrazole-1-carboxamidine hydrochloride in a basic
§
Present address: Takeda San Diego, 10410 Science Center Drive, San
Diego, CA 92121.
†
Present address: Neurochem, 275 Armand-Frappier Blvd, Laval, Que´bec
H7V 4A7, Canada.
10.1021/jm800251w CCC: $40.75
2008 American Chemical Society
Published on Web 06/21/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4073
Table 1. Inhibitory Activity of Compound 8 against HDAC Enzymesa
Scheme 1
enzyme
mean IC50 ( SEM (µM)
HDAC1
HDAC2
HDAC3
HDAC11
0.15(0.02
0.29(0.08
1.66(0.69
0.59(0.23
a IC50 values of 8 against HDACs 4-8 are greater than 10 µM.
Table 2. In Vitro Activity Profile of Compound 8
assay
cell line potency (n ) 4)
inhibition of cell proliferation (IC50, µM)
HCT116
A549
Du145
HMEC
T24
0.29(0.02
0.9 (0.04
0.67(0.07
20
(
2
H3Ac induction (EC50, µM)a
1.38(0.38
0.45(0.09
<1
p21cip/waf1 protein expression (EC50, µM)b HCT116
cell cycle arrest (G2/M) (EC50, µM)
apoptosis induction (at 1 µM)
HCT116
HCT116
c
+
a H3Ac ) histone H3 acetylation. Effective concentration of 8 for
induction of histone H3 acetylation (relative to 6 at 1 µM). b Effective
c
concentration of 8 for induction of p21cip/waf1 (relative to 6 at 1 µM).
) apoptosis positive.
+
xenograft models. Compound 8 demonstrated significant anti-
tumor activity in multiple models in a dose-dependent manner
(Table 4). There were no significant effects on body weight at
efficacious doses.
medium produced 4-guanidinomethylbenzoic acid methyl ester
(13). This second intermediate reacted with the enaminoketone
12 to form methyl 4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)m-
ethyl)benzoate (14), which was hydrolyzed to provide 4-((4-
(pyridin-3-yl)pyrimidin-2-ylamino)methyl)benzoic acid (15).
Acid 15 underwent an amide coupling reaction with 1,2-
phenylenediamine (11), forming 8.
The aminophenyl benzamide 8 is an isotype selective HDAC
inhibitor targeting HDACs 1-3 and 11 isoforms and is inactive
against HDACs 4-8 (Table 1).
Compound 8 has been evaluated in four single agent phase
I trials. In 23 patients with leukemia or myelodysplastic
syndrome (MDS), oral doses of 8 below 80 mg/m2 given three
times weekly were well-tolerated, and a complete marrow
response was observed in three patients.19 In patients with
advanced solid tumors (n ) 28), 8 was well-tolerated at doses
of 12.5, 20, 27, 36, and 45 mg/m2 given three times weekly for
2 of 3 weeks. Disease stabilization was noted in five patients.
Dose-dependent pharmacodynamic activity and a dose-inde-
pendent half-life were observed in this study.20 Another phase
I study in patients with leukemia or MDS (n ) 24) demonstrated
that 8 given twice weekly at oral doses (40, 53, 66, and 83
mg/m2) was well-tolerated, and four patients experienced stable
disease. Significant inhibition of total HDAC activity in
peripheral blood mononuclear cells was observed in the majority
of these patients.
The most prominent side effects of 8 are fatigue and
gastrointestinal. Fatigue is usually low grade but is occasionally
dose limiting. When necessary, fatigue is usually successfully
managed by dose modification. Gastrointestinal side effects are
also usually low grade and may include anorexia, nausea,
vomiting, or diarrhea and can also often be managed successfully
by supportive care or dose modification. Myelosuppression is
generally rare but does occur in some patients with hematologic
malignancies who have undergone significant prior chemotherapy.
In conclusion, we designed, synthesized, and evaluated the
biological activity of novel HDAC inhibitors, which resulted
in the discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyri-
midin-2-ylamino)methyl]benzamide (8). Out of 11 known non-
NADH+-dependent HDAC isotypes, 8 selectively inhibits
HDACs 1-3 and 11 at submicromolar concentrations in vitro.
It also potently blocks the proliferation of cancer cells and does
not affect the proliferation of the normal cells. At submicromolar
concentrations, aminophenylbenzamide 8 induces hyperacety-
lation of histones, cell-cycle arrest, expression of p21cip/waf1
protein, and apoptosis. Compound 8 has favorable pharmaco-
kinetic parameters across a number of species (mouse, rat, and
dog) and demonstrates antitumor efficacy in human tumor
xenograft models in mice. Compound 8 has entered clinical trials
The biological activity of this molecule was explored by
examining its effects on cancer cell proliferation, histone
acetylation, the protein expression of p21cip/waf1, and apoptosis
(Table 2). Compound 8 differentiated between cancer and
normal cells as exemplified by inhibition of cell proliferation
in HCT116 human colon cancer cells, A549 human lung cancer
cells, DU145 human prostate cancer cells, but not in HMEC
human normal mammary epithelial cells. The aminophenyl
benzamide 8 potently induced acetylation of H3 histones,
p21cip/waf1 protein expression, cell-cycle arrest at G2/M stage,
and apoptosis.
Pharmacokinetic studies of orally administered 8 (or its
dihydrobromide salt) were carried out in female CD-1 mice,
female Sprague-Dawley rats, and male beagle dogs. The
pharmacokinetic parameters are shown in Table 3. The oral
bioavailability was 12% in mice and 47% in rats. Pharmaco-
kinetic parameters in mice and rats were linear with the dose
administered and independent of vehicles used. In dogs,
pharmacokinetic parameters were nonlinear with the dose and
dependent on the vehicles used. A wide range of bioavailability
(1-92%) was observed in dogs. The compound was quickly
absorbed, with a short Tmax across all species tested. The terminal
phase elimination half-life for 8 administered iv was 0.6 h in
mice, 0.7 h in rats, and 1.3 h in dogs. The clearance in the
mice (4.3 (L/h)/kg) was high, but the clearance in rats (1.7 (L/
h)/kg) and dogs (2.0 (L/h)/kg) was reasonable. Steady state
volume of distribution was low in mice (0.34 L/kg) and higher
in rats (0.91 L/kg) and dogs (0.80 L/kg).
The in vivo antitumor activity of orally administered 8 (or
its dihydrobromide salt) was evaluated in several human tumor

4074 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Letters
Table 3. Pharmacokinetic Parameters of Compound 8 in Mouse, Rat, and Doga
dose iv
(mg/kg)
dose po
(mg/kg)
Vss
(L/kg)
Tmax
po (h)
,
Cmax
po (µM/(mg/kg))
,
AUC,
po (µM·h/(mg/kg))
parameter
T1/2 iv (hr)
CL (L/(kg ·h))
F (%)
mouse
rat
dog
2.5-15
2.5-5
10
2.5-15
2.5-50
7-30
0.6
0.7
1.3
4.3
1.7
2.0
0.35
0.91
0.80
0.25
0.59
0.29-2.0
0.086
0.30
0.0029-1.0
0.071
0.63
0.011-1.2
12
47
1-92
a The dosing vehicles in mice were 0.05-0.1 N HCl in saline (both iv and po); in rats were 0.02-0.1 N HCl in saline (iv) and 0.02-0.1 N HCl in saline
with 0.5% hydroxypropylmethylcellulose and 0.01% Tween-80, or 0.5% carboxymethylcellulose buffered with 50 mM acetate buffer at pH 4.0 (po); and in
dogs were 0.2-0.3 N HCl in saline with PEG400 (40% v/v) (iv), 0.1-0.2 N HCl in saline, 0.1-0.2 N HCl in saline with PEG400 (40% v/v), or 0.5%
hydroxyethylcellulose buffered at pH 4.0 with 50 mM acetate (po).
Table 4. In Vivo Antitumor Activity of 8 against Human Tumor
Xenografts in Nude Mice (Oral Dosing)
For ELISA analysis, the levels of histone acetylation were
evaluated by ELISA: black plates (Nunc 437111) were coated
with antihistone antibodies (Chemicon, Millipore, Billerica MA,
product H11-4) and blocked with 1% BSA + 0.1% TritonX-
100 in PBS. To detect H3Ac, an amount of 2 µg of purified
histones was incubated in the coated plate with rabbit antiacetyl-
H3 (Upstate, Millipore, Billerica MA, product 06-599, 1:1000).
The signals were normalized with total H3: 0.5 µg of purified
histones was mixed in separate wells with rabbit anti-H3
(Abcam, Cambridge MA, product ab1791, 1:5000). Binding to
H3Ac or H3 was revealed with HRP-coupled goat antirabbit
(Sigma, Oakville Ontario, Canada, product A-0545) using
Amplex-Red (Molecular Probes, Invitrogen, Burlington Ontario,
Canada, product A12222) as a substrate.
tumor model
SW-48
tumor type
8 ((mg/kg)/day)
TGIb (%)
colon carcinoma
60a
90a
120a
60
90
40
64
70
93
30
64
56
73
4
A431
vulval carcinoma
NSCLC
A549
60
DU145
prostate carcinoma
40
70
80
47
82
a Dihydrobromide salt. b Tumor growth inhibition.
and shows promise as an anticancer drug in combinations and
as a single agent in hematological and solid malignances.
For cell culture experiments, human mammary epithelial cells
(HMEC) were obtained from BioWhittaker (Walkersville,
Maryland). All other cell lines were from American Type
Culture Collection (Manassas, Virginia). Human normal and
cancer cells were cultured following the vendor’s instructions.
The HDAC enzyme in vitro assay was based on a homoge-
neous fluorescence release assay.21 Purified recombinant HDAC
enzymes were incubated with compounds diluted in various
concentrations for 10 min in assay buffer (25 mM HEPES, pH
8.0, 137 mM NaCl, 1 mM MgCl2, 2.7 mM KCl) at room
temperature. The fluorescent substrate Boc-Lys(ε-Ac)-AMC
(Bachem, Torrance CA, I-1875) was added to the mixture for
further incubation at 37 °C. The concentration of the substrate
and the incubation time varied for different isotypes of HDAC
enzymes. A 20 min trypsin incubation at room temperature
allowed the release of the fluorophore from the deacetylated
substrate. The fluorescent signal was detected by fluorometer
(Molecular Devices, Sunnyvale, CA, GeminiXS): 360 nm
excitation, 470 nm emission, 435 nm cutoff. The IC50 values
of the compounds were determined by analyzing dose-response
inhibition curves from 6-21 independent experiments.
For histone extraction, cultured cells or frozen pieces of tumor
(after crunching) were lysed in lysis buffer [10 mM Tris-HCl,
pH 8.0, 1.5 mM MgCl2, 5 mM MgCl2, 0.5% NP-40; protease
inhibitors pepstatin (1 µ g/mL), leupeptin (2 µ g/mL), aprotinin
(2 µ g/mL), TLCK(5 µ g/mL), PMSF (5 µ g/mL), DTT (5 µ
g/mL), and 5 mM Na Butyrate] on ice for 10 min. Samples
were centrifuged at 350g for 15 min, washed in the same lysis
buffer once more, and resuspended in cold water. Non-histone
proteins were precipitated with 3.3% H2SO4 for 1 h. To extract
histones, samples were centrifuged at maximum speed for 5
min and the supernatant was added to 10 volumes of acetone
and incubated overnight at -20 °C. Histones were then
precipitated by centrifugation at maximum speed for 5 min, the
supernatant was removed, and the pellet was resuspended in
distilled water. Protein concentrations of histones were deter-
mined using the BioRad protein assay reagent, and equal
quantities from each sample were analyzed by enzyme-linked
immunosorbant assay (ELISA).
For flow cytometric cell cycle analysis, cells were treated
with inhibitors for 16 h, harvested, and fixed with 70% ethanol
at -20 °C. Nucleic acids from fixed cells were treated with
RNaseIII and stained with propidium iodide (50 µ g/mL).21
DNA content was measured by using a fluorescence-activated
cell cytometer (FACScan, Becton-Dickinson, Franklin Lakes,
NJ) and analyzed using CellQuest Pro software. The MTT
cytotoxic assay was performed as described.21 Compounds at
various concentrations were added to cells in 96-well plates.
Cells were incubated for 72 h at 37 °C in 5% CO2. MTT (Sigma)
was added at a final concentration of 0.5 mg/mL and incubated
with the cells for 4 h before an equal volume of solubilization
buffer (50% N,N-dimethylformamide, 20% SDS, pH 4.7) was
added onto cultured cells. After overnight incubation, solubilized
dye was quantified by colorimetric reading at 570 nm using a
reference at 630 nm. OD values were converted to cell numbers
according to a standard growth curve of the relevant cell line.
The concentration that reduced cell numbers to 50% of those
of DMSO-treated cells was determined as MTT IC50. All
experiments were performed at least three times independently.
Apoptosis of cancer cell lines was analyzed by the “Cell
Death Detection ELISA Plus” kit (Roche).21 Cells were treated
with antisense oligonucleotides for 4 h at 37 °C, after which
cells were washed with PBS and returned to serum-containing
culture medium. Transfection was repeated every 24 h. After
48 h after the initial transfection with antisense oligonucleotides,
cells were harvested and ELISA was performed following
manufacturer’s protocol. Results were read in SPECTRAMAX
190 (Molecular Devices) at 405 nm. Reference was set at 490
nm.
For in vivo antitumor efficacy and drug pharmacokinetic
studies,21 approximately 2 million human cancer cells were
injected subcutaneously (sc) in the flank of female CD-1 nude
mice (aged 8-10 weeks, Charles River Laboratories, Wilm-
ington, MA) and were allowed to form solid tumors. Tumor
fragments were serially passaged a minimum of three times
before use in the experiments described. Tumor fragments
(about 30 mg) were implanted sc through a small surgical
incision on the flank of the mice while under general
anesthesia. HDAC inhibitors were dissolved in vehicles and

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4075
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dosed as solution (PBS acidified with 0.1 N HCl for oral
dosing). Vehicles and inhibitors were administered daily, and
tumor volumes and body weight were monitored three times
weekly for g2 weeks. Each experimental group contained
six to eight animals. At the end of each experiment, blood
was collected and white blood cell counts were performed.
Student’s t-test was used for statistical analysis. For phar-
macokinetic study, blood was collected from animals at
various time points and plasma samples were analyzed in-
house using an Agilent 1100 HPLC system coupled with an
MDS Sciex API2000 triple quadrupole mass spectrometer.
Acknowledgment. The authors are grateful to Dr. Robert
De´ziel for his critical review of the manuscript and Dr. Lori
Martell for her assistance in preparing the manuscript and
stimulating discussions.
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