Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives

Article abstract of DOI:10.1021/jm980565u

Newly synthesized benzamide derivatives were evaluated for their inhibitory activity against histone deacetylase. The structure of these derivatives was unrelated to the known inhibitors, and IC₅₀ values of the active compounds were in the range of 2-50 μM. Structure-activity relationship on the benzanilide moiety showed that the 2'-substituent, an amino or hydroxy group, was indispensable for inhibitory activity. Although the electronic influence of the substituent in the anilide moiety showed only a small effect on inhibitory activity, the steric factor in the anilide moiety, especially at positions 3'and 4', played an important role in interaction with the enzyme. Among these benzamide derivatives, MS-275 (1), which showed significant antitumor activity in vivo, has been selected for further investigation.

Full text of DOI:10.1021/jm980565u

J . Med. Chem. 1999, 42, 3001-3003
3001
Brief Articles
Syn th esis a n d Histon e Dea cetyla se In h ibitor y Activity of New Ben za m id e
Der iva tives
Tsuneji Suzuki,* Tomoyuki Ando, Katsutoshi Tsuchiya, and Nobuyuki Fukazawa
Pharmaceuticals Section, Life Sciences Laboratory, Performance Materials R&D Center, Mitsui Chemicals Inc., 1144, Togo,
Mobara-shi, Chiba 297-0017, J apan
Akiko Saito, Yukiyasu Mariko, Takashi Yamashita, and Osamu Nakanishi
Institute of Biological Science, Mitsui Pharmaceuticals Inc., 1900-1, Togo, Mobara-shi, Chiba 297-0017, J apan
Received October 5, 1998
Newly synthesized benzamide derivatives were evaluated for their inhibitory activity against
histone deacetylase. The structure of these derivatives was unrelated to the known inhibitors,
and IC50 values of the active compounds were in the range of 2-50 µM. Structure-activity
relationship on the benzanilide moiety showed that the 2′-substituent, an amino or hydroxy
group, was indispensable for inhibitory activity. Although the electronic influence of the
substituent in the anilide moiety showed only a small effect on inhibitory activity, the steric
factor in the anilide moiety, especially at positions 3′and 4′, played an important role in
interaction with the enzyme. Among these benzamide derivatives, MS-275 (1), which showed
significant antitumor activity in vivo, has been selected for further investigation.
Ch a r t 1. Structure of Known Histone Deacetylase
Inhibitors
In tr od u ction
Acetylation of histone molecules is catalyzed by two
enzymes: histone acetyltransferase and histone deacety-
lase, which are considered to play an important role in
cell cycle control by acting as a transcriptional coacti-
vator or a transcriptional corepressor.1 Inhibitors of
histone deacetylase such as sodium butyrate,2 trichos-
tatin A,3 and trapoxin4 (Chart 1) have been reported to
induce differentiation of several cancer cell lines and
suppress cell proliferation. Some of these inhibitors or
their related compounds are considered to be effective
agents for the treatment of diseases ascribed to unusual
cell proliferation such as malignant tumors. However
their in vivo antitumor efficiency seems to be limited
due to their instability, low retention, or nonspecific
toxicity.
Ch a r t 2. Structure of MS-275
Recently we reported that a novel benzamide deriva-
tive, MS-275 (Chart 2), which had a structure unrelated
to the known histone deacetylase inhibitors, inhibited
histone deacetylase in vitro and in vivo and showed
remarkable antitumor activity against several human
tumors in vivo.5
acyl chloride hydrochloride 3 by treatment of oxalyl
chloride quantitatively.
Compounds 1 and 4a -e were synthesized by method
A: 3 was reacted with imidazole to form the acylimi-
dazole intermediate, which was not isolated and was
condensed with appropriately substituted aniline in the
presence of trifluoroacetic acid to give 1 and 4a -e in a
50-60% yield. This condensation reaction proceeded
very slowly in the absence of acid.
In this paper we describe the synthesis and structural
requirement for histone deacetylase inhibition of newly
synthesized benzamide derivatives.
Ch em istr y
The new benzamide derivatives 1, 4a -e, and 6a -h
(Table 1) were prepared as outlined in Scheme 1. Con-
densation of 3-(hydroxymethyl)pyridine and 4-(amino-
methyl)benzoic acid using 1,1′-carbonyldiimidazole gave
carboxylic acid 2 in 90% yield, which was converted into
Compounds 6a -h were synthesized by method B: 3
was condensed with appropriately substituted 2-nitro-
aniline in pyridine to give 5 in a 40-50% yield, whose
reduction with SnCl2 gave 6 in a 50-60% yield.
10.1021/jm980565u CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/30/1999

3002 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Brief Articles
Sch em e 1. General Synthetic Procedure for the Preparation of Benzamide Derivativesa
a
Reagents: (a) CDI, DBU, Et3N, THF; (b) oxalyl chloride, toluene; (c) imidazole, THF; (d) substituted aniline, TFA, THF; (e) substituted
2-nitroaniline, pyridine; (f) SnCl2 dihydrate, NH4OAc, MeOH.
Ta ble 1. Inhibition of Histone Deacetylase with 1, 4a -e, and
6a -h a
hydrogen-bonding site or other electrostatic interaction
site and be indispensable to the specific interaction with
the enzyme.
The effect of the substituent at positions 3′, 4′, and 5′
was examined. 3′-Methyl group of 6a caused loss of
activity. Introduction of the 4′-substituent, both the
electron-donating group (6c) and the electron-withdraw-
ing group (6d ), weakened activity similarly. On the
other hand, 5′-substituted derivatives 6e-h showed
almost equal activity to 1. These results suggest that
steric hindrance may play an important role near the
specific binding site (at positions 3′ and 4′). There was
little steric limitation for the 5′-substituent, which may
not interfere in specific binding with the enzyme. On
the other hand, the electronic nature of the 4′- and 5′-
substituents did not change inhibitory activity remark-
ably.
b
compd
R1
R2
R3
R4
IC50 (µM)
1
NH2
H
H
H
NH2
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4.8
>100
>100
>100
>100
2.2
>100
>100
44
40
2.8
4.6
7.7
6.0
140
0.0046
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
6f
6g
6h
H
H
NH2
H
NHAc
OH
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
CH3
OCH3
Cl
Con clu sion
H
H
H
H
H
H
H
H
CH3
OCH3
Cl
In this paper, we reported the synthesis and struc-
tural requirement for histone deacetylase inhibition of
newly synthesized benzamide derivatives. These are
structurally unrelated to the known histone deacetylase
inhibitors, and some showed a 30-fold stronger inhibi-
tory activity than sodium butyrate. Structure-activity
relationship on the benzanilide moiety showed that the
2′-substituent, which might act as a hydrogen-bonding
or other electrostatic interaction site, was indispensable
for inhibitory activity. Although the electronic influence
of the substituent seemed to play a small role, it
appeared that the steric factor in the anilide moiety,
especially at positions 3′ and 4′, had an important role
for interaction with the enzyme. Among these benza-
mide derivatives, MS-275 (1), which showed significant
antitumor activity in vivo,5 has been selected for further
investigation.
F
sodium butyrate
trichostatin A
a
Expressed as the average of at least three determinations.
Concentration required to inhibit by 50%.
b
Resu lts a n d Discu ssion
The synthesized benzamide derivatives 1, 4a -e, and
6a -h were evaluated for their abilities to inhibit
partially purified histone deacetylase. The results are
given as IC50 values and are shown in Table 1. Com-
pound 1 (MS-275) showed a 30-fold stronger inhibitory
activity (IC50: 4.8 µM) than sodium butyrate (IC50: 140
µM), and 1000-fold weaker than trichostatin A (IC50:
0.0046 µM). Removal of the 2′-amino group of benz-
anilide resulted in loss of inhibitory activity (4a ). Among
three regioisomers (1, 4b,c), only 2′-substituted deriva-
tive 1 showed inhibitory activity. These results indicate
that the 2′-amino group of 1 may be involved in the
specific interaction with the enzyme. 2′-Hydroxy deriva-
tive 4e showed almost equal activity to 1, suggesting
that the 2′-substituent of benzanilide might act as a
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Bu¨chi 535
melting point apparatus and were not corrected. Proton
magnetic resonance spectra (1H NMR) were obtained on a
J EOL EX-270 (270 MHz) spectrometer using deuterated
dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 3003
are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS). Infrared spectra (IR) were recorded
on a J ASCO IR 7300 spectrometer. High-resolution mass
spectra (FAB-HRMS) were obtained by M. C. Research Center
Inc. (Yokohama, J apan) on a J EOL SX-102A spectrometer.
(40 mL) were refluxed for 30 min. The mixture was evaporated
to reduce the volume (to 5 mL) and extracted with ethyl acetate
(150 mL). The organic layer was washed with saturated
sodium bicarbonate (100 mL), water (100 mL, three times),
and saline (100 mL), then dried over magnesium sulfate, and
evaporated. Recrystallization from ethanol gave the title
compound (0.31 g, 57% yield): mp 167-168 °C; 1H NMR
(DMSO-d6) δ 2.17 (s, 3H), 4.27 (d, 2H, J ) 6 Hz), 4.68 (s, 2H),
5.10 (s, 2H), 6.68 (d, 1H, J ) 8 Hz), 6.79 (d, 1H, J ) 8 Hz),
7.00 (s, 1H), 7.3-7.5 (m, 3H), 7.79 (d, 1H, J ) 8 Hz), 7.9-8.0
(m, 3H), 8.53 (d, 1H, J ) 3 Hz), 8.59 (s, 1H), 9.59 (s, 1H); IR
(KBr) 3289, 1742, 1637, 1530, 1492, 1257, 1134, 1049, 817,
711 cm-1; FAB-HRMS m/z calcd 391.1770, found 391.1759 (M
+ H)+. Anal. (C22H22N4O3) H, N; C: calcd, 67.68; found, 67.15.
P h ar m acology. In Vitr o In h ibition of Histon e Deacety-
la se. Histone deacetylase fraction was prepared as described
by Yoshida et al.3 Human leukemia K562 (2.5 × 108) cells were
disrupted in buffer-A (15 mM potassium phosphate buffer (pH
7.5) containing 5% glycerol and 0.2 mM EDTA) (15 mL). The
nuclei were collected by centrifugation (35000g, 10 min) and
resuspended with buffer-A (15 mL) containing 1 M (NH4)2SO4.
After sonication, the supernatant was collected by centrifuga-
tion, and ammonium sulfate was added to make the final
concentration 3.5 M. After stirring for 1 h at 0 °C, the
precipitate was collected by centrifugation, dissolved with
buffer-A (4 mL), and dialyzed against buffer-A (2000 mL). The
dialysate was loaded onto a Mono Q HR 5/5 column (Pharma-
cia) equilibrated with buffer-A and eluted with a linear
gradient of 0-1 M NaCl in buffer-A (30 mL). A single peak of
histone deacetylase activity was eluted around 0.4 M NaCl,
and the fraction was stored at -80 °C until use.
Elemental analyses were determined on
CHN2400 elemental analyzer.
a Perkin-Elmer
4-[N -(P yr id in -3-ylm e t h oxyca r b on yl)a m in om e t h yl]-
ben zoic Acid (2). To a suspension of 1,1′-carbonyldiimidazole
(25.6 g, 158 mmol) in THF (120 mL) was added 3-py-
ridinemethanol (17.3 g, 158 mmol) in THF (50 mL) at 10 °C,
and the mixture stirred for 1 h at room temperature. The
resulting solution was added to a suspension of 4-(aminom-
ethyl)benzoic acid (22.6 g, 158 mmol), DBU (24.3 g, 158 mmol),
and triethylamine (22.2 mL, 158 mmol) in THF (250 mL). After
stirring for 5 h at room temperature, the mixture was
evaporated to remove THF and then dissolved in water (300
mL). The solution was acidified with hydrochloric acid (pH 5)
to precipitate a white solid which was collected by filtration,
washed with water (300 mL) and methanol (50 mL), respec-
tively, and dried to give 2 (41.1 g, 91% yield): mp 207-208
°C; 1H NMR (DMSO-d6) δ 4.28 (d, 2H, J ) 5.9 Hz), 5.10 (s,
2H), 7.3-7.5 (m, 3H), 7.7-8.1 (m, 4H), 8.5-8.7 (m, 2H); IR
(KBr) 3043, 1718, 1568, 1434, 1266, 1108, 1037, 984, 756 cm-1
.
4-[N -(P yr id in -3-ylm e t h oxyca r b on yl)a m in om e t h yl]-
ben zoyl Ch lor id e Hyd r och lor id e (3). To a suspension of 2
(40 g, 140 mmol) in toluene (2000 mL) were added DMF (0.8
mL) and oxalyl chloride (24 mL). After the mixture stirred for
4 h, a white solid was collected by filtration, washed with
toluene (500 mL) and diisopropyl ether (500 mL), respectively,
and dried to give the title compound (47.7 g, quantitatively),
which was very hygroscopic and used with no further purifica-
tion.
Inhibition of histone deacetylase was estimated as described
by Yoshida et al.3 with slight modifications. 3H-Labeled histone
was prepared by the method of Yoshida et al.:3 K562 cells (108
cells) were incubated in growth medium (25 mL) containing
0.5 mCi/mL [3H]sodium acetate (152.8 GBq/mmol; NEN) and
5 mM sodium butyrate at 37 °C.
Meth od A: N-(2-Am in op h en yl)-4-[N-(p yr id in -3-ylm eth -
oxyca r bon yl)a m in om eth yl]ben za m id e (1, MS-275). To a
solution of imidazole (0.63 g, 9.2 mmol) in THF (20 mL) was
added 3 (1 g, 2.9 mmol), and the mixture stirred for 1 h at
room temperature. After imidazole hydrochloride was removed
by filtration, 1,2-phenylenediamine (2.52 g, 23.2 mmol) and
trifluoroacetic acid (0.2 mL, 2.6 mmol) were added to the
filtrate and stirred for 15 h. The reaction mixture was
evaporated to remove THF and partitioned between ethyl
acetate (500 mL) and water (400 mL). The organic layer was
washed with water and dried and then purified by silica gel
column chromatography (ethyl acetate) to give 1 (0.62 g, 56%
Histone deacetylase inhibitory activity of test compound was
measured as follows: the mixture (total volume 50 µL)
3
containing the above histone deacetylase fraction (2 µL), H-
labeled histone (100 µg/mL), and test compound (5 µL) was
incubated for 10 min at 37 °C. [3H]Acetic acid, which was
liberated from 3H-labeled histone, was extracted with ethyl
acetate, and radioactivity was measured by a liquid scintilla-
tion counter.
1
yield): mp 159-160 °C; H NMR (DMSO-d6) δ 4.28 (d, 2H, J
Su p p or tin g In for m a tion Ava ila ble: Melting point, 1H
NMR spectra, and IR spectra for compounds 4a -e and 6a -
d ,f-h and HRMS and full 1H NMR spectra for compounds 4d
and 6a ,b,e,g. This material is available free of charge via the
Internet at http://pubs.acs.org.
) 5.9 Hz), 4.86 (s, 2H), 5.10 (s, 2H), 6.60 (t, 1H, J ) 7.3 Hz),
6.78 (d, 1H, J ) 7 Hz), 6.97 (t, 1H, J ) 7 Hz), 7.17 (d, 1H, J )
8 Hz), 7.3-7.5(m, 3H), 7.78 (d, 1H, J ) 8 Hz), 7.93 (d, 2H, J
) 8 Hz), 8.53 (d, 1H, J ) 3.7 Hz), 8.59 (s, 1H), 9.61 (s, 1H); IR
(KBr) 3295, 1648, 1541, 1508, 1457, 1309, 1183, 742 cm-1
Anal. (C21H20N4O3) C, H, N.
.
Refer en ces
Meth od B: N-(2-Am in o-5-m eth ylp h en yl)-4-[N-(p yr id in -
3-ylm eth oxyca r bon yl)a m in om eth yl]ben za m id e (6e). (i)
N-(5-Met h yl-2-n it r op h en yl)-4-[N-(p yr id in -3-ylm et h oxy-
ca r bon yl)a m in om eth yl]ben za m id e (5e). To a solution of
5-methyl-2-nitroaniline (1 g, 6.5 mmol) in pyridine (15 mL)
was added 3 (2 g, 5.9 mmol). After stirring for 1 h, the mixture
was evaporated and partitioned between ethyl acetate and
water. The organic layer was washed with water, dried over
anhydrous sodium sulfate, and then evaporated. The residue
was recrystallized from ethyl acetate to give 5e (1.3 g, 54%
yield): mp 138 °C; 1H NMR (DMSO-d6) δ 2.43 (s, 3H), 4.30 (d,
2H, J ) 5.9 Hz), 5.10 (s, 2H), 7.20-7.24 (m, 1H), 7.39-7.45
(m, 3H), 7.74-7.81 (m, 2H), 7.90-8.00 (m, 4H), 8.53-8.60 (m,
2H), 10.69 (s, 1H); IR (KBr) 3293, 1685, 1603, 1586, 1542, 1497,
(1) Wade, P. A.; Pruss, D.; Wolfe, A. Histone Acetylation: Chromatin
in Action. Trends Biochem. Sci. 1997, 22, 128-132.
(2) Kruh, J . Effects of Sodium Butyrate, a New Pharmacological
Agent, On Cells in Culture. Mol. Cell. Biochem. 1982, 42, 65-
82.
(3) Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. Potent and Specific
Inhibition of Mammalian Histone Deacetylase both In Vivo and
In Vitro by Trichostatin A. J . Biol. Chem. 1990, 265, 17174-
17179.
(4) Kijima, M.; Yoshida, M.; Suguta, K.; Horinouchi, S.; Beppu, T.
Trapoxin, an Antitumor Cyclic Tetrapeptide, Is an Irreversible
Inhibitor of Mammalian Histone Deacetylase. J . Biol. Chem.
1993, 268, 22429-22435.
(5) Saito, A.; Yamashita, T.; Mariko, Y.; Tsuchiya, K.; Ando, T.;
Suzuki, T.; Tsuruo, T.; Nakanishi, O. A Novel Synthetic Inhibitor
of Histone Deacetylase, MS-27-275, with Marked In Vivo
Antitumor Activity against Human Tumors. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 4592-4597. After submission of ref 5, MS-
27-275, the company code of compound 1, had been changed to
MS-275.
1334, 1261, 745 cm-1
.
(ii) N-(2-Am in o-5-m et h ylp h en yl)-4-[N-(p yr id in -3-yl-
m eth oxyca r bon yl)a m in om eth yl]ben za m id e (6e). Com-
pound 5e (0.6 g, 1.38 mmol), SnCl2 dihydrate (1.84 g, 8.15
mmol), and ammonium acetate (1.1 g, 14.3 mmol) in methanol
J M980565U