Design and synthesis of 3-substituted benzamide derivatives as Bcr-Abl kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.11.042

A series of 3-substituted benzamide derivatives structurally related to STI-571 (imatinib mesylate), a Bcr-Abl tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML), was prepared and evaluated for antiproliferative activity against the Bc

Full text of DOI:10.1016/j.bmcl.2005.11.042

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1421–1425
Design and synthesis of 3-substituted benzamide derivatives
as Bcr-Abl kinase inhibitors
Tetsuo Asaki,* Yukiteru Sugiyama, Taisuke Hamamoto, Masaya Higashioka,
Masato Umehara, Haruna Naito and Tomoko Niwa
Discovery Research Laboratories, Nippon Shinyaku Co., Ltd, 14 Nishinosho-Monguchi-Cho, Kisshoin,
Minami-ku, Kyoto 601-8550, Japan
Received 12 August 2005; revised 8 November 2005; accepted 10 November 2005
Available online 5 December 2005
Abstract—A series of 3-substituted benzamide derivatives structurally related to STI-571 (imatinib mesylate), a Bcr-Abl tyrosine
kinase inhibitor used to treat chronic myeloid leukemia (CML), was prepared and evaluated for antiproliferative activity against
the Bcr-Abl-positive leukemia cell line K562. About ten 3-halogenated and 3-trifluoromethylated benzamide derivatives were iden-
tified as highly potent Bcr-Abl kinase inhibitors. One of these, NS-187 (9b), is a promising new candidate Bcr-Abl inhibitor for the
therapy of STI-571-resistant chronic myeloid leukemia.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Chronic myeloid leukemia (CML) is caused by uncon-
trolled signals from a constitutively activated Bcr-Abl
protein tyrosine kinase.1 STI-571 (imatinib mesylate,
Gleevecꢁ or Glivecꢁ; Fig. 1), a specific inhibitor of
Bcr-Abl kinase, produces clinical remission in CML pa-
tients with minimal toxicity.2,3 This selective inhibition
of Bcr-Abl kinase by STI-571 has been a successful ther-
apeutic strategy for CML because of the high efficacy
and mild side effects of this compound.4,5 Crystallo-
graphic studies have revealed that STI-571 binds to
the kinase domain of c-Abl only when the domain
adopts the inactive ÔclosedÕ conformation, which is re-
quired for optimal inhibition of the kinase activity.6,7
However, despite the good hematological and cytoge-
netic responses obtained, primary refractory disease
and secondary resistance still occur with STI-571, par-
ticularly in patients with advanced-phase disease.8
Although several mechanisms have been proposed to ac-
count for this resistance, including increased expression
of Bcr-Abl protein, amplification of the Bcr-Abl gene,
and overexpression of the multidrug resistance P-glyco-
protein,9–12 point mutations in the Bcr-Abl gene itself
account for most cases of resistance. To date, more than
30 different clinically relevant point mutations, such as
N
H
N
N
N
N
N
NH
STI-571
O
N
H
N
N
N
CF3
N
N
Me2N
NH
NS-187 (9b)
O
Figure 1. Chemical structures of STI-571 and NS-187 (9b).
the one at position 255 within the kinase domain, have
been identified.13–16
Nagar et al.7 have already reported the crystal structure
of the kinase domain of c-Abl with STI-571 bound.
They showed that STI-571 forms six hydrogen bonds
with the protein, and that the majority of contacts are
mediated by van der Waals interactions, which lock
the c-Abl kinase into its inactive conformation. When
we closely examined the published crystal structure, we
found a hydrophobic pocket formed by amino acid res-
idues Ile-293, Leu-298, Leu-354, and Val-379 around the
Keywords: Tyrosine kinase; Inhibitor; Bcr-Abl; Structure–activity;
Imatinib.
*
Corresponding author. Tel.: +81 75 321 9168; fax: +81 75 321
9039; e-mail: t.asaki@po.nippon-shinyaku.co.jp
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.042

1422
T. Asaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1421–1425
phenyl ring adjacent to the piperazinylmethyl group of
STI-571. As part of an effort to identify compounds with
improved antiproliferative activity against Bcr-Abl-
positive leukemia cell lines, we first focused on this
hydrophobic pocket, and we introduced various hydro-
phobic substituents at the above-mentioned phenyl ring
in STI-571. We found that 3-halogenated and 3-trifluo-
romethylated benzamides displayed significantly in-
creased activity compared to unsubstituted STI-571. In
this report, we describe structure–activity studies of
3-substituted benzamide derivatives, including a potent
Bcr-Abl kinase inhibitor, NS-187 (9b; Fig. 1).
described.17 Displacement reaction of 8 with various
cyclic amines afforded the desired final products 9a–f.
The antiproliferative activities of the 3-substituted ben-
zamides synthesized were evaluated in vitro against
Bcr-Abl-positive and Bcr-Abl-negative leukemia cells
(Table 1). Bcr-Abl-positive K562 cells and Bcr-Abl-neg-
ative U937 cells were plated at 1 · 103 cells/well and
5 · 103 cells/well, respectively, in 96-well plates and incu-
bated with serial dilutions of the compounds for 3 days.
Antiproliferative activity was determined by the assay
with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoli-
um bromide (MTT),19,20 and the IC50 values were esti-
mated by fitting the data to a logistic curve.
All 3-substituted benzamides reported herein were
synthesized as described below. The synthesis of pyri-
dine-tailed derivatives 5a–e is illustrated in Scheme 1.
Esterification of the commercially available carboxylic
acids 1a–e, subsequent a-bromination with N-bromo-
succinimide, and coupling with 1-methylpiperazine
afforded compounds 2a–e. Acid chlorides 3a–e were
obtained by hydrolysis of the ethyl esters 2a–e followed
by reaction with thionyl chloride. The benzoylation of
the aniline 417 with 3a–e in pyridine solvent afforded
the corresponding amides 5a–e.
All compounds tested showed more-potent antiprolifer-
ative activity against K562 cells than did STI-571,
whereas they had only weak antiproliferative activity
against U937 cells, with IC50 values of micromolar order
and generally about the same as that of STI-571. These
data indicate that our newly synthesized compounds
inhibited Bcr-Abl more specifically than did STI-571.
The introduction of a fluoro substituent at the 3-posi-
tion of the phenyl ring (5a; IC50 = 63 nM against K562
cells) yielded an approximately 3-fold improvement in
antiproliferative activity compared to STI-571
(IC50 = 182 nM). To our surprise, the 3-chloro, 3-bro-
mo, and 3-iodobenzamide analogues (5b–d) had even
higher potency. The 3-bromophenyl analogue 5c, in par-
ticular, exhibited an IC50 value as low as 7 nM, 26-fold
lower than that of STI-571. The most potent derivative
Pyrimidine-tailed derivatives 9a–f were synthesized as
shown in Scheme 2. a-Bromination of 4-methyl-3-trifluo-
romethylbenzoic acid 1e by NaBrO3/NaHSO3 reagent18
followed by reaction with thionyl chloride yielded acid
chloride 6. Compound 6 was converted to the benzyl
bromide 8 through condensation with the aniline 7, which
was prepared from 5-acetylpyrimidine as previously
N
R1
R1
R1
H
N
N
R1
d, e
a, b, c
f
N
·2HCl
COCl
N
N
N
N
N
CO2H
1a-e
CO2Et
N
NH
2a-e
3a-e
5a-e
O
: R1 = F
: R1 = Cl
: R1 = Br
: R1 = I
1-3a, 5a
1-3b, 5b
1-3c, 5c
1-3d, 5d
1-3e, 5e
N
H
N
N
N
: R1 = CF3
NH2
4
Scheme 1. Reagents and conditions: (a) H2SO4, EtOH, reflux; (b) NBS, cat. (PhCO)2O2, CCl4, reflux; (c) 1-methylpiperazine, K2CO3, THF, rt;
(d) 1 N NaOH, reflux, then aq HCl; (e) SOCl2, reflux; (f) 4, pyridine, rt.
N
N
CF3
H
CF3
H
N
N
N
N
N
N
CF3
CF3
a, b
c
Br
d
N
N
R2
Br
CO2H
COCl
NH
N
NH
1e
6
8
9a-f
O
O
:
9a
R
2 = 4-methylpiperazin-1-yl
9b : R2 = (3S)-3-(dimethylamino)pyrrolidin-1-yl
H
R2 = (3R)-3-(dimethylamino)pyrrolidin-1-yl
:
:
N
N
N
9c
R2 = (3R)-3-(dimethylaminomethyl)pyrrolidin-1-yl
9d
9e
9f
N
R
R
2 = (3S)-3-(dimethylaminomethyl)pyrrolidin-1-yl
2 = 3-(dimethylamino)azetidin-1-yl
:
:
NH2
7
Scheme 2. Reagents and conditions: (a) NaBrO3, NaHSO3, EtOAc; (b) (COCl)2, cat. DMF, CH2Cl2, rt; (c) 7, K2CO3, dioxane, rt; (d) cyclic amines,
K2CO3, DMF, rt.

T. Asaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1421–1425
1423
Table 1. Antiproliferative activity of 3-substituted benzamide deriva-
tives
pIC50 ¼ 1:280ðꢀ0:732Þp ꢁ 2:059ðꢀ0:542Þ
n = 6, s = 0.263, r = 0.925, F = 23.6
ð1Þ
N
H
In this and the following equations, n is the number of
compounds, s is the standard error, r is the correlation
coefficient, F is the ratio of the variances of the calculat-
ed and observed values, and the figures in parentheses
are the 95% confidence intervals. According to Eq. 1,
hydrophobic 3-substituents increase the activity, indicat-
ing the existence of hydrophobic interactions between
the 3-substituent and the hydrophobic amino acids, such
as Ile-293, Leu-298, Leu-354, and Val-379. The activity
of the compounds was also highly correlated with
VerloopÕs steric parameter, B1,21 which represents the
minimum width of the substituent:
N
N
X
R1
3
2
N
R2
4
NH
1
O
a
IC50 (nM)
Compound
X
R1
R2
K562 cells U937 cells
N
STI-571
5a
CH
CH
H
F
182
63
10
7
14,000
8000
N
N
N
N
N
N
N
Me
Me
Me
Me
Me
Me
Me
N
N
N
N
N
N
pIC50 ¼ 1:337ðꢀ0:592ÞB1 ꢁ 3:549ðꢀ1:039Þ
n = 6, s = 0.210, r = 0.953, F = 39.3
ð2Þ
5b
CH Cl
CH Br
9000
According to Eq. 2, the inhibitory activity of the com-
pounds increases with the minimum width of the 3-sub-
stituent. Since the 3-substituent is located adjacent to
the methylpiperazine group, its steric effect appears to
restrict the rotation of the methylpiperazine group and
thereby increase the activity. Presumably, these two fac-
tors work cooperatively to enhance the activity of the
compound.
5c
5000
5d
CH
I
10
5
6000
5e
CH CF3
4000
To compensate for the increased hydrophobicity caused
by the introduction of the trifluoromethyl group in 5e,
the distal pyridine ring was selected for further modifica-
tion. According to the crystal structure of the c-Abl ki-
nase domain with bound STI-571, Tyr-253 is located
very close to the distal pyridine ring, and the resulting
interaction stabilizes the inactive form of the kinase;
so that a structural modification that increases the bulk
in this region would be expected to be unfavorable. The
pyridine ring was therefore replaced by the more hydro-
philic pyrimidine ring. Pyrimidine-tailed derivative 9a
displayed activity (IC50 = 4 nM) similar to that of the
original pyridine-tailed derivative 5e, which suggests
that pyrimidinyl substitution is compatible with high
activity.
9a
N
N
N
N
N
N
CF3
CF3
CF3
CF3
CF3
CF3
4
5000
N
N
Me2N
9b
11
4
10,000
9000
9cb
9db
9eb
9fb
Me2N
Me2N
N
11
9
20,000
>100,000
>100,000
Me2N
N
N
The crystal structure of the kinase domain of c-Abl in
complex with STI-571 reported by Nagar et al.7 reveals
that the piperazine moiety in STI-571 interacts with the
carbonyl oxygen atoms of Ile-360 and His-361 through
hydrogen bonding. In the light of the importance of this
interaction, we focused on replacement of the piperazine
moiety in 9a with other cyclic amines in an effort to iden-
tify even-more-potent compounds. The optically pure 3-
(dimethylamino)pyrrolidine derivatives 9b and 9c, and
3-(dimethylaminomethyl)pyrrolidine derivatives 9d and
9e, and the 3-(dimethylamino)azetidine derivative 9f,
which may function as piperazine isosteres,22 were syn-
thesized and evaluated for antiproliferative activity.
The pyrrolidine derivative 9c had excellent potency
(IC50 = 4 nM), comparable to that of 9a. Compound
9b, the enantiomer of 9c, as well as other pyrrolidine
derivatives 9d and 9e, also had superior antiproliferative
activity profiles (IC50 = 11, 11, and 9 nM, respectively),
17
Me2N
a IC50 values represent the concentration which inhibits cell prolifera-
tion by 50%.
b The biological activity of the monohydrochloride salts was evaluated.
in this series was the 3-trifluoromethylbenzamide 5e,
which, with an IC50 value of 5 nM, was 36-fold more po-
tent than STI-571 in the cellular activity profile.
To elucidate the structural factors responsible for
enhancing the inhibitory activity of STI-571, we studied
the correlation of the antiproliferative activity of STI-
571 and 5a–e with various physicochemical parameters
of the 3-substituents with Excel2000 (Microsoft). We
found a high correlation of activity with the hydropho-
bic substituent parameter p.21

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T. Asaki et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1421–1425
tyrosine kinase inhibitors. We found 3-halogenated
and 3-trifluoromethylated benzamide derivatives to be
much more potent than unsubstituted STI-571. We
identified the clinical candidate 9b (NS-187) as a highly
potent Bcr-Abl kinase inhibitor. Compound 9b also
showed a potent inhibitory effect against E255K
Bcr-Abl. Furthermore, 9b was a potent inhibitor of
Lyn kinase, whose overexpression is also associated with
STI-571 resistance.24,25 These detailed pharmacological
properties of 9b have already been published.20 We
expect that 9b will advance to clinical trials.
hydrophobic pocket
Ile293
NS- 187
Leu298
Val379
Leu354
His361
Ile360
Tyr253
Acknowledgments
close contact
We thank Mr. Arihiro Oyamada, Dr. Hironori Otsu,
Mr. Kohei Kagayama, Dr. Hiroki Hayase, Mr. Jiro Shi-
kaura, and Mr. Takara Ino for performing the synthe-
ses, Dr. Akira Matsuura, Mr. Shinichi Tada, and Dr.
Jun Segawa for practical guidance, and Dr. Gerald E.
Smyth for helpful suggestions during the preparation
of the manuscript.
Figure 2. Docking model of Abl in complex with 9b (NS-187).
Hydrophobic amino acids are shown in green, and hydrogen-bonding
interactions are shown as blue broken lines. The amino acid close to
the pyrimidine ring of 9b is shown in white. The figure was prepared
with PyMOL version 0.97 (DeLano Scientific).
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