Discovery of N-[2-hydroxy-6-(4-methoxybenzamido)phenyl]-4- (4-methyl-1,4-diazepan-1-yl)benzamide (darexaban, YM150) as a potent and orally available factor Xa inhibitor

Article abstract of DOI:10.1021/jm200868m

Inhibitors of factor Xa (FXa), a crucial serine protease in the coagulation cascade, have attracted a great deal of attention as a target for developing antithrombotic agents. We previously reported findings from our optimization study of a high-throughpu

Full text of DOI:10.1021/jm200868m

Article
pubs.acs.org/jmc
Discovery of N-[2-Hydroxy-6-(4-methoxybenzamido)phenyl]-4-
(4-methyl-1,4-diazepan-1-yl)benzamide (Darexaban, YM150) as a
Potent and Orally Available Factor Xa Inhibitor
Fukushi Hirayama,*,† Hiroyuki Koshio,† Tsukasa Ishihara,† Shunichiro Hachiya,† Keizo Sugasawa,†
Yuji Koga,† Norio Seki,† Ryouta Shiraki,† Takeshi Shigenaga,† Yoshiyuki Iwatsuki,† Yumiko Moritani,†
Kenichi Mori,† Takeshi Kadokura,‡ Tomihisa Kawasaki,‡ Yuzo Matsumoto,§ Shuichi Sakamoto,∥
and Shin-ichi Tsukamoto†
†Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
‡Development, Astellas Pharma Inc., 17-1, Hasune 3-chome, Itabashi-ku, Tokyo 174-8612, Japan
§Drug Discovery Research, Astellas Pharma Inc., 2-3-11, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8411, Japan
∥Technology Supply Chain & Manufacturing, Astellas Pharma Europe B.V., Elisabethhof 19, 2350 AC Leiderdorp, The Netherlands
ABSTRACT: Inhibitors of factor Xa (FXa), a crucial serine protease in the
coagulation cascade, have attracted a great deal of attention as a target for
developing antithrombotic agents. We previously reported findings from
our optimization study of a high-throughput screening (HTS) derived lead
compound 1a that resulted in the discovery of potent amidine-containing
FXa inhibitors represented by compound 2. We also conducted an
alternative optimization study of 1a without incorporating a strong basic amidine group, which generally has an adverse effect on
the pharmacokinetic profile after oral administration. Replacement of 4-methoxybenzene with a 1,4-benzodiazepine structure and
introduction of a hydroxy group at the central benzene led to the discovery of the potent and orally effective factor Xa inhibitor
14i (darexaban, YM150). Subsequent extensive study revealed a unique aspect to the pharmacokinetic profile of this compound,
wherein the hydroxy moiety of 14i is rapidly transformed into its glucuronide conjugate 16 (YM-222714) as an active metabolite
after oral administration and it plays a major role in expression of potent anticoagulant activity in plasma. The distinctive, potent
activity of inhibitor 14i after oral dosing was explained by this unique pharmacokinetic profile and its favorable membrane
permeability. Compound 14i is currently undergoing clinical development for prevention and treatment of thromboembolic
diseases.
trypsin-like serine protease that plays a central role in the blood
coagulation cascade. Located at the convergence point of the
intrinsic and extrinsic coagulation cascades, FXa is responsible
for the proteolysis of prothrombin to catalytically active
thrombin. Thrombin has several thrombotic functions,
including the proteolytic activation of fibrinogen to fibrin,
activation of platelets, and feedback activation of several
coagulation factors.3 As such, inhibition of thrombin generation
by a FXa inhibitor is recognized as an attractive target for
anticoagulant development. Many small-molecule FXa inhib-
itors have been reported to date,4 and the excellent
anticoagulant effects of FXa inhibitors without significant
hemorrhagic adverse reactions have been demonstrated in
animal models5 as well as recent clinical trial data obtained
regarding advanced oral FXa inhibitors such as rivaroxaban,6
apixaban,7 betrixaban,8 and edoxaban.9 Of these, rivaroxaban
was the first to be approved in U.S., EU, Canada, and several
other countries for the prevention of venous thromboembo-
lism.
INTRODUCTION
■
Intravascular clot formation followed by blood flow blockage in
the vital organs causes thromboembolic disorders such as
myocardial infarction, unstable angina, deep vein thrombosis,
pulmonary embolism, and ischemic stroke. One of the most
effective methods for treating and preventing these life
threatening conditions has been the use of anticoagulants
including unfractionated and low-molecular-weight heparins
and warfarin. While warfarin has historically been the sole orally
effective anticoagulant used worldwide, its many clinical
limitations, such as slow onset and offset of action, narrow
therapeutic window, and interaction with many foods and drugs
resulting in a need for coagulation monitoring, have long
prompted physicians to limit its use.1 Several of these
drawbacks are caused by its mechanism of inhibiting the
biosynthesis of vitamin K dependent coagulation factors,
highlighting the clear unmet need for anticoagulants with
alternative mechanisms that are orally active, clinically safe, and
require less monitoring.2
Numerous efforts to identify such a novel anticoagulant have
been made over the past several decades, and factor Xa (FXa)
has attracted substantial attention as a target enzyme. FXa is a
Received: July 3, 2011
Published: October 13, 2011
© 2011 American Chemical Society
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Figure 1. Optimization from HTS hit compound 1a.
a
Scheme 1. Synthesis of 1,2-Phenylenediamide and Anthranilic Diamide Derivatives
a
Reagents and conditions: (a) 4-(4-methyl-1,4-diazepan-1-yl)benzoic acid, WSC, HOBt, DMF, room temperature; (b) 4-methoxybenzoyl chloride,
pyridine, room temperature; (c) 4-(4-methyl-1,4-diazepan-1-yl)aniline or 4-methoxyaniline, toluene, reflux; (d) 4-methoxybenzoyl chloride or 4-(4-
methyl-1,4-diazepan-1-yl)benzoyl chloride, pyridine, room temperature.
In our search for orally bioavailable FXa inhibitors, we
conducted high-throughput screening (HTS) of the library of
compounds in the collection of Yamanouchi Pharmaceutical
Co., Ltd. (now Astellas Pharma Inc.) and identified compound
1a as a novel lead compound10 with a unique 1,2-phenyl-
enediamide scaffold despite its limited activity (Figure 1). We
previously reported on the structural optimization of
compound 1a by way of the introduction of an amidine
group leading to the potent and selective series of novel
amidine FXa inhibitors represented by compound 2 (YM-
203552).11,12 Learning from setbacks experienced in clinical
development of amidine-containing FXa inhibitors,4 due to the
low oral absorption caused by high polarity, we attempted to
identify another type of FXa inhibitor from the same lead
compound 1a. Here, we describe the results of this
optimization study without incorporation of an amidine moiety,
thereby leading to discovery of the novel FXa inhibitor 14i
(darexaban, YM150).12 We also report on its unique property
of exerting activity after oral administration mainly via its active
metabolite 16 (YM-222714),13 the glucuronide conjugate of
compound 14i.
derivatives 8a and 8b were prepared by treatment of
commercially available benzoxazine derivative 6 and the
corresponding anilines under reflux in toluene followed by
acylation with benzoyl chlorides.
The synthesis of substituted 1,2-phenylenediamide deriva-
tives 14a−q is shown in Scheme 2. Condensation of 2-
nitroaniline derivatives (9a−h) with 4-methoxybenzoyl chlo-
ride or 4-(4-methyl-1,4-diazepane-1-yl)benzoyl chloride11 gave
the intermediate nitro derivatives 10a−h. Reaction of 9e with
4-(4-methyl-1,4-diazepane-1-yl)benzoyl chloride generated a
phenyl ester intermediate by acylation of the hydroxy group in
9e. The desired amide intermediate 10e was obtained by acyl
transformation under basic conditions (NEt3, CH3CN). Under
the same conditions, the hydroxyl group in 9f was also acylated
by treatment of 2.2 equiv of benzoyl chloride and afforded
diacyl derivative 10f. Catalytic hydrogenation of the nitro group
of the intermediates 10a−h gave the corresponding aniline
derivatives, followed by condensation with 4-(4-methyl-1,4-
diazepane-1-yl)benzoyl chloride11 or 4-methoxybenzoyl chlor-
ide to give the 1,2-phenylenediamide derivatives 14a, 14b, 14d,
and 14g−n. The compounds containing carboxyl (14h) or
hydroxyl groups (14k and 14m) were prepared by conversion
from the protected precursors 14g, 14j, and 14l, respectively.
Compound 14o was prepared from 14i via benzylammonium
intermediate to avoid reaction of 14o with bromoacetate at the
terminal amine. The ethyl ester derivative 14o was then
transformed to the corresponding acid (14p) and alcohol
CHEMISTRY
■
1,2-Phenylenediamide derivative 5 and anthranilic diamide
derivatives 8a and 8b were synthesized as shown in Scheme 1.
Compound 5 was obtained from condensation between 4-
methoxybenzoyl chloride and aniline 4.11 Anthranilic diamide
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a
Scheme 2. Synthesis of Substituted 1,2-Phenylenediamide Derivatives
a
Reagents and conditions: (a) (1) 4-methoxybenzoyl chloride or 4-(4-methyl-1,4-diazepan-1-yl)benzoyl chloride, pyridine, room temperature; (2)
for 10e, NEt3, CH3CN, 70 °C; (b) 4-methoxybenzoyl chloride, NEt3, 1,2-dichloroethane, room temperature; (c) 4-methoxybenzoyl chloride,
pyridine, dichloromethane, −78 to 0 °C; (d) (1) H2, Pd−C, MeOH, or for 14l, ammonium chloride, iron powder, EtOH, H2O, reflux; (2) 4-
methoxybenzoyl chloride or 4-(4-methyl-1,4-diazepan-1-yl)benzoyl chloride, pyridine, room temperature; (e) 4-methoxybenzoyl chloride, pyridine,
room temperature; (f) aq NaOH, MeOH, room temperature; (g) H2, Pd−C, AcOH; (h) (1) benzyl bromide, MeOH, CHCl3 room temperature;
(2) bromoacetate, K2CO3, DMF, 100 °C; (3) H2, Pd−C, AcOH; (i) NaBH4, MeOH, THF, 60 °C.
(14q) by alkaline hydrolysis and reduction with NaBH4−
MeOH, respectively. Some compounds (14c and 14f) were
prepared via intermediate aniline compounds (13a and 13b)
synthesized from protected phenilendiamine derivatives 1121 or
by selective amide formation of 12. The 3-hydroxy-1,2-
phenylenediamide derivatives 15a−i in Scheme 3 were also
Scheme 4. Chemical Conversion of Compound 14i into
Glucuronide Conjugate 16
a
a
Scheme 3. Synthesis of 3-Hydroxy-1,2-phenylenediamide
Derivatives
Reagents and conditions: (a) (1) methyl 2,3,4-tri-O-acetyl-α-D-
a
glucopyranosyluronate bromide, DBU, MeOH, CHCl3, room temper-
ature; (2) Na2CO3, MeOH, H2O, room temperature
hydrolysis of the methyl ester of the product by Na2CO3 in
MeOH and H2O. Compound 16 was then purified using
chromatography on ODS as a sodium salt, and subsequent
precipitation from weakly acidic water yielded the compound in
free form.
RESULT AND DISCUSSION
■
a
Reagents and conditions: (a) (1) H2, Pd−C, MeOH; (2) acid
Optimization of Lead Compound 1a. Compounds in
this report were evaluated based on IC50 values for inhibition of
human FXa enzymatic activity. Selected compounds were
further evaluated based on CT2 values for the prolongation of
prothrombin time (PT) using mouse plasma as an indicator of
in vitro anticoagulant activity. CT2 values were defined as the
concentration required to double clotting time. To validate oral
efficacy, ex vivo anticoagulant activities after oral administration
in mice (100 mg/kg) were also determined based on PT-
prolonging effects and expressed as ratios of the PT of the
compound-treated mice plasma with those in the vehicle
treated group.
chloride, pyridine, room temperature; (b) H2, Pd−C, AcOH.
prepared from the common intermediate 10e in the same
manner.
Synthesis of 16, the glucuronide conjugate of 14i, was
accomplished as follows (Scheme 4): Glycosylation of 14i was
accomplished using methyl 2,3,4-tri-O-acetyl-α-D-glucopyrano-
syluronate bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in a mixed solvent of MeOH and CHCl3 at room
temperature with simultaneous deacetylation of the sugar
moiety. Although significant amounts of 14i remained in this
glycosylation step, despite using excess equivalents of reagents,
14i was easily removed by extraction with organic solvent after
Table 1 shows the results of an initial structure−activity
relationship (SAR) study for lead compound 1a. We previously
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Table 1. Initial SAR for HTS Hit Compound 1a
a
Inhibitory activity against human purified FXa. IC50 values are represented by the average of three or more separate determinations with an average
b
standard error of the mean of <15%. CT2 values are defined as the concentration required to double clotting time and represent the average of three
or more separate determinations with the average standard error of the mean being <10%. Prothrombin time using mouse plasma. The relative
c
d
e
prothrombin time compared with that measured using normal mice plasma at 0.5 and 2.0 h after oral administration (100 mg/kg, n = 3). See ref 11.
f
ND: not determined.
reported on the 1,4-benzodiazepane structure as an excellent
template for docking in the S4 pocket of active site of FXa.11,14
We therefore adapted this template to non-amidine compound
1b, which was derived from lead compound 1a by removing the
methoxy moiety on the central benzene,11 and the resulting
compound 5 bearing a 1,4-benzodiazepane instead of a
methoxy group showed a 40-fold increase in FXa inhibitory
activity (IC50 = 4224 nM for 1b vs IC50 = 103 nM for 5).
We then explored compounds containing the reversed amide
linkage in compound 5. Transformation of the amide linkage
attached to the 4-(1,4-diazepane)benzene resulted in a 5-fold
decrease in potency (IC50 = 539 nM for 8a), whereas
transformation of the amide linkage attached to the 4-
Table 2. Effects of Substituents on Central Phenyl Moiety of
Compound 5
PT/control
d
PT
CT
a
2 b
IC50 (nM),
factor Xa
(μM),
c
compd
X
PT
0.5 h
2.0 h
5
H
103
62.6
73.4
118
213
2.8
3.7
7.5
7.1
13
1.38
1.50
1.35
1.25
1.39
1.32
1.38
1.34
methoxybenzene showed only slightly reduced activity (IC50
=
14a
14b
14c
14d
14f
14h
14i
3-OMe
4-OMe
5-OMe
6-OMe
140 nM for 8b). 1,2-Phenylenediamide 5 and anthranilic
diamide 8b, both showing preferable FXa inhibitory activity,
were further evaluated in vitro and for ex vivo anticoagulant
activity. Compound 5 showed 2-fold more potent in vitro
anticoagulant activity than compound 8b (2.8 μM for 5 vs 5.9 μM
for 8b) and exhibited approximately 1.4-fold PT-prolonging
effects at both 0.5 and 2.0 h after oral administration, whereas
8b lacked any oral efficacy ex vivo. On the basis of these results,
we selected 1,2-phenylenediamide 5 for further structural
optimization.
We first attempted to introduce substituents to the central
phenyl moiety of compound 5 (Table 2). To explore the
tolerability of the substituents, a methoxy moiety was
introduced at each position. Results showed that the
compounds bearing the methoxy moiety at the 3- and 4-
position (14a and 14b) had slightly improved FXa inhibitory
activity (IC50 of 62.6 and 73.4 nM, respectively) compared with
compound 5, whereas compounds modified at the 5- and 6-
position (14c and 14d) exhibited diminishing FXa inhibitory
activity (IC50 of 118 and 213 nM, respectively). Introduction of
the methoxy moiety exerted no positive effect on in vitro and ex
vivo anticoagulant activity (14d was not tested). Taken
together, these observations indicated that the 3- and 4-
position of the central benzene were likely to be preferable to
others when introducing substituents.
e
e
ND
ND
f
3-COOH
4-COOH
3-OH
99.2
9.0
1.7
4.1
1.4
4.7
4.3
2.8
3.0
1.05
1.00
3.49
1.04
1.02
1.43
0.99
1.36
1.08
1.00
2.09
1.00
1.01
1.22
0.98
1.31
31.1
54.6
50.3
157
155
123
14k
14m
14n
14p
14q
4-OH
5-OH
3-F
3-OCH2COOH
3-OCH2CH2OH
g
106
a
Inhibitory activity against human purified FXa. IC50 values are
represented by the average of three or more separate determinations
b
with an average standard error of the mean of <15%. CT2 values are
defined as the concentration required to double clotting time and
represent the average of three or more separate determinations with
c
the average standard error of the mean being <10%. Prothrombin
d
time using mouse plasma. The relative prothrombin time compared
with that measured using normal mice plasma at 0.5 and 2.0 h after
e
f
oral administration (100 mg/kg, n = 3). ND: not determined. SEM =
±23.4. SEM = ±18.2.
g
Given our previous findings that the carboxyl group in
certain FXa inhibitors provides favorable effects on activity after
oral dosing,11,14,15 we attempted to introduce a carboxyl group
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Table 3. Effects of the Distal Phenyl Moiety
a
Inhibitory activity against human purified FXa. IC50 values are represented by the average of three or more separate determinations with an average
b
standard error of the mean of <15%. CT2 values are defined as the concentration required to double clotting time and represent the average of three
or more separate determinations with the average standard error of the mean being <10%. Prothrombin time using mouse plasma. The relative
prothrombin time compared with that measured using normal mice plasma at 0.5 and 2.0 h after oral administration (100 mg/kg, n = 3). ND: not
c
d
e
determined.
to the central benzene ring of compound 5. While introduction
at the 3-position (14f) had no effect on FXa inhibitory activity
(IC50 = 99.2 nM), in vitro anticoagulant activity was decreased
(CT2 = 9.0 μM). In contrast, introduction at the 4-position
(14h) increased the FXa inhibitory activity 3-fold (IC50 = 31.1
nM) and slightly improved the in vitro anticoagulant activity
(CT2 = 1.7 μM). However, both compounds lost their PT
prolongation effect after oral dosing.
We then attempted to introduce a phenolic hydroxy group to
the benzene ring as a similar acidic substituent. Addition at the
3-position (14i) led to modest improvement in the FXa
inhibitory activity but decreased anticoagulant activity in vitro
(IC50 = 54.6 nM, CT2 = 4.1 μM). However, despite these
marginal effects, compound 14i was found to have a
surprisingly potent PT prolongation effect after oral admini-
stration in mice (3.49-fold at 0.5 h and 2.09-fold at 2.0 h). In
contrast, 4-hydroxy regioisomers 14k lost all activity after oral
administration in mice, despite showing the more potent PT
prolongation effects in vitro. Then 5-hydroxy regioisomer 14m
was explored for confirmation, and it gave the same ex vivo
results as compound 14k. While other compounds substituted
at the 3-position with introduction of several functional
moieties (14n, 14p and 14q) retained in vitro activity, they
did not produce a similar remarkable effect ex vivo as that
achieved with 14i. These results indicate that the introduction
of a phenolic hydroxyl moiety at the 3-position on the central
benzene improves ex vivo activity after oral dosing over other
substituents, an extremely potent effect that is presumed to
reflect its preferable pharmacokinetic profile given that the in
vitro potency for 14i was not remarkably high compared with
other analogues evaluated in this study.
Table 3 shows the results of structural modification of the
distal benzene for the S1 ligand of 14i. Given the findings from
an initial combinatorial modification study in which 4-
substituted benzenes showed potent factor Xa inhibitory
activity compared to the 2- and 3-substituted regioisomer,16
optimization was focused on the 4-position. Replacement of the
methoxy with a methylthio (15a) or hydroxy (15c) group
reduced the potency of factor Xa inhibitory activity. While
halogen substituents such as chloro (15e) and bromo (15f)
displayed in vitro activities (IC50 of 64.2 and 59.8 nM and CT2
of 5.3 and 6.7 μM, respectively) similar to that of the methoxy
analogue 14i, the fluoro substituent (15d) had reduced activity
(IC50 = 349 nM). However, despite demonstration of
acceptable activity, both the chloro and bromo analogues
showed diminished activity after oral administration and
expressed no activity comparable to that of 14i.
We then transformed benzene into thiophene, a well-known
bioisostere for benzene,17 and introduced methoxy (15g),
chloro (15h), or bromo (15i) substituents. These thiophene
derivatives also showed similar in vitro activities (IC50 of 86.1,
41.3, 41.4 nM and CT2 of 3.8, 5.8, 7.1 μM, respectively) to
those noted with the benzene analogue, and just as was
observed with the benzene analogues, the methoxy derivative
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15g exerted enhanced ex vivo anticoagulant activity after oral
dosing (2.17-fold at 0.5 h and 1.90-fold at 2.0 h). Taken
together, these data suggested that both a methoxy moiety at
the S1 aromatic ligand and a hydroxy moiety on the central
benzene in this series are essential for expression of excellent
activity after oral administration. In this way, we successfully
identified compound 14i as the most potent and orally effective
FXa inhibitor in this series.
Table 5. In Vitro Activities of Selected Compound 14i and
Its Active Metabolite 16
b
CT2 (μM),
a
c
Ki (μM)
PT
plasma
kallikrein
FXa
thrombin trypsin
mouse human
14i
16
0.031
0.020
>100
>100
>100
>100
11
17
4.1
2.5
1.2
0.95
Profiles of Selected Compound 14i. Given that the
observed in vitro and ex vivo activity relationships implied
favorable pharmacokinetic properties for compound 14i, we
next evaluated plasma concentration after oral dosing to rats.
The surprisingly low concentrations of 14i in the collected
plasma samples at all time points measured suggested the
potential generation of active metabolites. After intensive
examination, we identified glucuronide conjugate 16 as a
major metabolite of 14i in much higher concentration than its
parent compound (approximately 50- and 30-fold higher in
a
Inhibitory constant for human enzymes. Ki values are the average of
four separate determinations with the average standard error of the
mean being <10%. CT2 values are defined as the concentration
required to double clotting time and represent the average of three or
more separate determinations with the average standard error of the
mean being <10%. Prothrombin time using mouse and human
plasma.
b
c
to related compounds with similar in vitro activity, we evaluated
several biological and physiological parameters of the
representative compounds (Table 6). Interestingly, values in
Cmax and AUC, respectively, Table 4, Figure 2). To confirm the
Table 4. Pharmacokinetic Parameters of 14i and 16 in Rats
after Oral Administration of 100 mg/kg 14i
a
Table 6. Comparisons of 14i and Related Compounds in
MLM Stability, Membrane Permeability, and Lipophilicity
Tmax (h)
Cmax (ng/mL)
AUC0−24h (ng·h/mL)
b
PAMPA at pH
MLM stability, CLint
a
c
compd
6.5, Pe (10−6 cm/s)
(mL min−1 kg−1
)
CLogP
14i
16
2.0
0.5
174.8
1291.8
9439.5
38539.2
14i
16
30.0
<0.2
42.0
47.5
<0.3
1907
90.6
2.30
−1.03
1.95
a
Monomaeate salt of 14i was used.
5
2047
2205
1705
14a
14k
2.84
1.41
a
b
pION membrane lipid was used at donor buffer, pH 6.5 (n = 2). In
vitro metabolism with mouse liver microsomes in the presence of the
c
NADPH-generating system (n = 2). CLogP values were calculated
using the software ACD/LogP, version 9.0.
the parallel artificial membrane permeability assay (PAMPA)20
differed starkly between compounds 14i and 14k. Despite both
containing hydroxy moieties, the 3-hydroxy derivative 14i
demonstrated favorable membrane permeability (Pe = 30.0 ×
10−6 cm/s at pH 6.5), while the 4-hydroxy derivative 14k
showed a low value (Pe < 0.3 × 10−6 cm/s at pH 6.5). The
considerable discrepancy between 14i and 14k in activity after
oral administration may be attributed to the differing PAMPA
values. In terms of metabolic stability to mouse liver
microsomes (MLMs), only glucuronide conjugate 16 showed
adequate stability (CLint = 90.6 mL min−1 kg−1), with the other
compounds liable to be metabolized (CLint > 1700 mL min−1
kg−1). Taken together, these findings suggest that the
remarkable oral activity of compound 14i may be due to its
rapid conversion to the metabolically stable glucuronide
conjugate 16 after oral absorption, because of its favorable
permeability, subsequently exerting steady anticoagulant
activity. Further, as many previous reports have mentioned,15,21
the markedly low lipophilicity of compound 16 (CLogP =
−1.03) may contribute to its excellent anticoagulant activity
after oral dosing due to factors including low plasma protein
binding. Additionally, glucuronidation is generally recognized as
a detoxication process, and subsequently generated glucuronide
conjugates are biologically nontoxic;22 therefore, some measure
of safety may be achieved with 14i given that it exerts its in vivo
activity as the glucuronide conjugate 16.
Figure 2. Plasma concentration−time profiles of 14i and 16 in rats
following oral administration of 100 mg/kg 14i (mean ± SD, n = 3).
Monomaleate salt of 14i was used in this study.
activity of metabolite 16, we evaluated the chemically
synthesized compound 16 and found that glucuronide
conjugate 16 surprisingly maintained slightly more potent in
vitro activity relative to parent compound 14i (IC50 = 28.6 nM
for 16 vs IC50 = 54.6 nM for 14i; CT2 = 2.5 μM for 16 vs CT2 =
4.1 μM for 14i). In addition, the high selectivities of 14i and 16
were confirmed against other related serine proteases, such as
trypsin, thrombin, and kallikrein, and potent anticoagulant
activities of 14i and 16 were also confirmed using human
plasma. Detailed in vitro activities are summarized in Table 5.18
The above observations clarified the unique behavior of 14i
after oral administration, wherein the compound is immediately
converted to its glucuronide conjugate 16 after administration
and anticoagulant activity in the blood is mainly exerted by this
active metabolite. This pharmacokinetic profile was confirmed
in a clinical trial that also identified the glucuronide conjugate
16 as the main active metabolite in human subjects.19
To further understand the SAR, in which 14i shows
distinctive, potent activity after oral administration compared
Molecular Modeling Study of Compounds 14i and 16
in Factor Xa. Figures 3 and 4 show inhibitors 14i and 16
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Figure 3. Docking study of 14i (A, top view; B, side view) and 16 (C, top view; D, side view) in the active site of FXa depicting the surface of FXa.
Surface color codes are as follows: magenta, hydrogen bond potential; green, hydrophobic; blue, mildly polar. The hydroxy group of 14i at the
Figure 4. Docking study of 14i (A) and 16 (B) in active site of FXa. Essential amino acids are depicted with hydrogen bonds shown as dotted lines.
docked in the active site of FXa. The proposed binding
model indicated that the 4-methoxyphenyl moiety deeply
occupied the S1 pocket, while the 1,4-diazepane moiety fits in
the S4 pocket formed by the residues Phe174, Trp215, and
Tyr99. The model further suggested that the NH group of the
amide bond linked to the methoxyphenyl moiety was within an
H-bond distance to the backbone carbonyl group of Gly216.
Likewise, the carbonyl group of the amide bond connected to
the 4-(1,4-diazepane)benzene was located within an H-bond
distance to the backbone NH group of Gly218. These binding
elements shape inhibitors 14i and 16 into an L conformation,
with the hydroxy group at the central benzene of 14i pointing
away from the protein surface and facing out toward the wide
space of the solvent. As a result, glucuronidation of 14i at the
hydroxy moiety forms without colliding with any residual
portions of FXa, and the glucuronic acid moiety in 16 is
exposed to the solvent, thereby stabilizing the moiety by
hydration. In addition, the docking mode indicated that the
carboxyl group of the glucuronic acid moiety forms hydrogen-
bonding and ionic interaction with basic amino acid residues
Arg143 and Lys147. These docking conformational features
explain FXa inhibitory activity expression of the glucuronide
conjugate 16.
CONCLUSION
■
Optimization of the lead compound 1a by incorporation of 1,4-
benzodiazepane led to compound 5, with 60-fold enhancement
in FXa inhibitory activity. We further optimized its ex vivo
activity by modifying the central and distal benzenes, leading to
the discovery of 14i, which demonstrated excellent anticoagu-
lant activity after oral administration. Subsequent extensive
studies on 14i revealed a unique pharmacokinetic feature in the
rapid generation of glucuronide conjugate 16 as an active
metabolite after oral administration of 14i, with the activity in
the blood predominantly attributed to 16. The favorable
membrane permeability of 14i and high metabolic stability of
active metabolite 16 may contribute to potent activity
expression after oral administration of 14i. The maleate salt
of 14i is currently under clinical development for treatment and
prevention of thromboembolic diseases.
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Article
mixture was stirred at room temperature for 4 h. The reaction mixture
was concentrated under reduced pressure, and the residue was
chromatographed on silica gel, eluting with MeOH/CHCl3 (1/8). The
solution containing purified compound was acidified with 4 N HCl/
EtOAc and concentrated under reduced pressure. The solid was
suspended with EtOH and filtered to give 8b (626 mg, 32%) as a
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were purchased from
commercial sources and used without further purification. H NMR
1
spectra were measured using a JEOL JMN-LA-300 or JEOL JMN-EX-
400 spectrometer, and chemical shifts were expressed in δ (ppm) units
using tetramethylsilane as an internal standard (in 1H NMR
description, s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, and br = broad peak). Mass spectra were recorded with a
Hitachi M-80 or JEOL JMS-DX300 spectrometer. Elemental analysis
was performed on a Yanaco MT-5 microanalyzer (C, H, N) and
Yokogawa IC-7000S ion chromatographic analyzer (S, halogens).
Melting points were measured with a Yanaco MP-500P melting point
apparatus without correction. Purification with ODS column
chromatography was performed on YMC gel (ODS-A 120-230/70).
For elemental analysis, all tested compounds were within ±0.4% of the
theoretical value unless otherwise noted. Purity values for all tested
compounds were found to be above 95% from the high-performance
liquid chromatography (HPLC) analyses (column, TSKgel ODS-
80TM; UV detection, 254 nm; eluent, CH3CN/0.01 M aq KH2PO4 or
CH3CN/0.01 M aq HClO4, with appropriate isocratic conditions
selected for each compound; flow rate, 1 mL/min).
1
colorless solid. H NMR (400 MHz, DMSO-d6) δ: 2.11−2.23 (1H,
m), 2.29−2.40 (1H, m), 2.78 (3H, s), 3.03−3.24 (2H, m), 3.38−3.49
(2H, m), 3.50−3.59 (2H, m), 3.76 (3H, s), 3.79−3.82 (2H, m), 3.89−
3.98 (2H, m), 6.91 (2H, d, J = 8.8 Hz), 6.97 (2H, d, J = 8.8 Hz), 7.22
(1H, t, J = 8.3 Hz), 7.58 (1H, t, J = 8.3 Hz), 7.62 (2H, d, J = 8.8 Hz),
7.79 (2H, d, J = 8.8 Hz), 7.95 (1H, d, J = 8.3 Hz), 8.61 (1H, d, J = 8.3
Hz), 10.47 (1H, s), 10.84 (1H, s), 11.84 (1H, s). FAB MS m/z 459
(M + H)+. Anal. (C27H30N4O3·HCl·0.8H2O) C, H, N, Cl.
4-Methoxy-N-(3-methoxy-2-nitrophenyl)benzamide (10a). To
a stirred solution of 3-methoxy-2-nitroaniline 9a (900 mg, 5.35 mmol)
in pyridine (30 mL) at room temperature was added 4-methoxy-
benzoyl chloride (1.11 g, 6.51 mmol). After the mixture was stirred at
room temperature for 18 h, the solvents were removed under reduced
pressure. The residue was dissolved in EtOAc, and the solution was
washed with 1% aqueous NaHCO3 and 0.1 M aqueous HCl, dried
over Na2SO4, and then concentrated under reduced pressure. The
resulting residue was chromatographed on silica gel, eluting with
CHCl3/MeOH (100:1) to give 10a (0.70 g, 43%) as a colorless solid.
1H NMR (DMSO-d6) δ: 3.84 (3H, s), 3.90 (3H, s), 7.02−7.13 (3H,
m), 7.20 (1H, d, J = 8.4 Hz), 7.57 (1H, t, J = 8.8 Hz), 7.89 (2H, d, J =
9.2 Hz), 10.26 (1H, s). FAB MS m/z 303 (M + H)+.
4-Methoxy-N-(2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}phenyl)benzamide Hydrochloride (5). A solution of N-
(2-aminophenyl)-4-(4-methyl-1,4-diazepan-1-yl)benzamide (4)11 (810
mg, 2.50 mmol) and 4-methoxybenzoyl chloride (470 mg, 2.80 mmol)
in pyridine (20 mL) was stirred at room temperature for 26 h. The
reaction mixture was concentrated under reduced pressure, and the
residue was chromatographed on silica gel, eluting with MeOH/
CHCl3 (1/10). The solution containing purified compound was
acidified with 4 N HCl/EtOAc and concentrated under reduced
pressure. The solid was dissolved in H2O and lyophilized to give 5
4-Methoxy-N-(4-methoxy-2-nitrophenyl)benzamide (10b). Com-
pound 10b was prepared from 4-methoxy-2-nitroaniline 9b and 4-
methoxybenzoyl chloride according to the procedure for the pre-
1
paration of 10a in 92% yield. H NMR (300 MHz, CDCl3) δ: 3.88
1
(3H, s), 3.89 (3H, s), 7.02 (2H, d, J = 8.8 Hz), 7.30 (1H, dd, J = 2.9,
9.3 Hz), 7.73 (1H, d, J = 2.9 Hz), 7.95 (2H, d, J = 8.8 Hz), 8.89 (1H,
d, J = 9.3 Hz), 11.05 (1H, s). FAB MS m/z 303 (M + H)+.
4-Methoxy-N-(2-methoxy-6-nitrophenyl)benzamide (10c). Com-
pound 10c was prepared from 9c and 4-methoxybenzoyl chloride
according to the procedure for the preparation of 10a in 81% yield. 1H
NMR (300 MHz, CDCl3) δ: 3.88 (3H, s), 3.95 (3H, s), 6.98 (2H, d,
J = 8.8 Hz), 7.18 (1H, d, J = 8.2 Hz), 7.28 (1H, t, J = 8.2 Hz), 7.58
(1H, d, J = 8.2 Hz), 7.91 (2H, d, J = 8.8 Hz), 8.38 (1H, s). FAB MS
m/z 303 (M + H)+.
Ethyl 4-[(4-Methoxybenzoyl)amino]-3-nitrobenzoate (10d). Com-
pound 10d was prepared from 9d and 4-methoxybenzoyl chloride
according to the procedure for the preparation of 10a in 8% yield. 1H
NMR (300 MHz, DMSO-d6) δ: 1.35 (3H, t, J = 7.1 Hz), 3.86 (3H, s),
4.37 (2H, q, J = 7.1 Hz), 7.13 (2H, d, J = 8.8 Hz), 7.96 (2H, d, J = 8.8
Hz), 8.07 (1H, d, J = 8.5 Hz), 8.28 (1H, d, J = 8.5 Hz), 8.47 (1H, s),
10.92 (1H, s). FAB MS m/z 345 (M + H)+.
N-(2-Hydroxy-6-nitrophenyl)-4-(4-methyl-1,4-diazepan-1-
yl)benzamide (10e). To a stirred solution of 4-(4-methyl-1,4-
diazepan-1-yl)benzoic acid11 hydrochloride (16.30 g, 60.2 mmol) in
EtOAc (160 mL) was added thionyl chloride (14.30 g, 120.2 mmol)
and DMF (0.88 g 12.0 mmol). After being stirred at 40 °C for 3 h, the
reaction mixture was concentrated under reduced pressure. To the
solution of the residue in acetonitrile (130 mL) at 5 °C was added a
solution of 2-amino-3-nitorophenol 9e (8.35 g, 54.2 mmol) and
pyridine (9.73 mL, 120.4 mmol) in acetonitrile (60 mL), and the
mixture was stirred at 5 °C for 21 h. The resulting precipitate was
filtered and dried under reduced pressure to give 2-amino-3-
nitrophenyl 4-(4-methyl-1,4-diazepan-1-yl)benzoate hydrochloride
(21.4 g, 87.4%). 1H NMR (400 MHz, DMSO-d6) δ: 2.15−2.22
(1H, m), 2.34−2.45 (1H, m), 2.79 (3H, d, J = 5.0 Hz), 3.05−3.22
(2H, m), 3.40−3.61 (4H, m), 3.79−3.88 (1H, m), 3.95−4.03 (1H, m),
6.69−6.75 (1H, m), 6.93 (2H, d, J = 9.0 Hz), 7.05 (2H, br), 8.00 (2H,
d, J = 9.0 Hz), 11.12 (1H, br)
(1.18 g, 86%) as a colorless solid. H NMR (400 MHz, DMSO-d6)
δ:2.13−2.21 (1H, m), 2.24−2.34 (1H, m), 2.78 (3H, d, J = 3.9 Hz),
3.04−3.21 (2H, m), 3.40−3.54 (4H, m), 3.68−3.75 (1H, m), 3.84
(3H, s), 3.86−3.96 (1H, m), 6.85 (2H, d, J = 8.8 Hz), 7.06 (2H, d, J =
8.8 Hz), 7.24−7.27 (2H, m), 7.57−7.66 (2H, m), 7.87 (2H, d, J = 8.8
Hz), 7.96 (2H, d, J = 8.8 Hz), 9.89 (1H, s), 10.14 (1H, s), 10.57 (1H,
br). FAB MS m/z 459 (M + H)+. Anal. (C27H30N4O3·1.3HCl·2.2H2O)
C, H, N, Cl.
2-Amino-N-[4-(4-methyl-1,4-diazepan-1-yl)phenyl]-
benzamide (7a). A solution of 4-(4-methyl-1,4-diazepan-1-yl)-
aniline23 (1.00 g, 4.90 mmol) and 2H-3,1-benzoxazine-2,4(1H)-
dione (6, 0.80 g, 4.90 mmol) in toluene (10 mL) was refluxed for
5 h. The reaction mixture was filtered, and the filtrate was concentrated
under reduced pressure. The residue was chromatographed on silica
gel, eluting with 25% aqueous ammonia/MeOH/CHCl3 (0.1/1/10) to
give 12 (816 mg, 52%) as a yellow solid. 1H NMR (300 MHz, CDCl3)
δ: 1.96−2.06 (2H, m), 2.38 (3H, s), 2.54−2.59 (2H, m), 2.69−2.73
(2H, m), 3.49 (2H, t, J = 6.2 Hz), 3.55−3.60 (2H, m), 5.49 (2H, s),
6.66−6.73 (4H, m), 7.20−7.24 (1H, m), 7.34−7.38 (2H, m), 7.43−
7.47 (1H, m), 7.57 (1H, s). FAB MS m/z 325 (M + H)+
2-[(4-Methoxybenzoyl)amino]-N-[4-(4-methyl-1,4-diazepan-
1-yl)phenyl]benzamide Hydrochloride (8a). Compound 8a was
prepared from 7a and 4-methoxybenzoyl chloride according to the
procedure for the preparation of 5 in 81% yield. 1H NMR (400 MHz,
DMSO-d6) δ: 2.11−2.22 (1H, m), 2.26−2.34 (1H, m), 2.79 (3H, d,
J = 4.9 Hz), 3.05−3.23 (2H, m), 3.37−3.51 (4H, m), 3.65−3.75 (1H,
m), 3.77−3.83 (1H, m), 3.84 (3H, s), 6.80 (2H, d, J = 8.8 Hz), 7.11
(2H, d, J = 8.8 Hz), 7.22−7.28 (1H, m), 7.54 (2H, d, J = 8.8 Hz),
7.56−7.62 (1H, m), 7.88 (2H, d, J = 8.8 Hz), 7.95 (1H, d, J = 7.9 Hz),
8.56 (1H, d, J = 7.9 Hz), 10.38 (1H, s), 10.88 (1H, s), 11.99 (1H, s).
FAB MS m/z 459 (M + H)+. Anal. (C27H30N4O3·1.8HCl·2.2H2O) C,
H, N, Cl.
4′-Methoxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}benzanilide Hydrochloride (8b). A solution of 4-(4-
methyl-1,4-diazepan-1-yl)benzoic acid11 (1.10 g, 4.06 mmol) in
thionyl chloride (10 mL) was stirred at 60 °C for 2 h. The reaction
mixture was concentrated under reduced pressure. To the stirred
solution of 7b24 (930 mg, 3.84 mmol) in pyridine (10 mL,) was added
a solution of the residue in 1,2-dichloroethane (10 mL), and the
To a stirred solution of the resulting ester (2.0 g, 4.92 mmol) in
acetonitrile (20 mL) was added triethylamine (1.37 mL, 9.84 mmol).
After the mixture was stirred at 70 °C for 6 h, a solution of sodium
hydroxide (197 mg, 4.92 mmol) in water (2 mL) and water (20 mL)
was added to the solution. The acetonitrile was removed by heating,
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followed by addition of more water (10 mL), and the mixture was
stirred at room temperature for 14 h. The resulting precipitate was
filtered to give 10e (1.57 g, 82.6%) as a yellow solid. H NMR (400
Ethyl 2-Amino-3-[(4-methoxybenzoyl)amino)benzoate
(13b). To a stirred solution of ethyl 2,3-diaminobenzoate 12 (6.07
g, 33.7 mmol) in pyridine (17 mL) and dichloromethane (17 mL) was
added a solution of 4-methoxybenzoyl chloride (6.04 g, 35.4 mmol) in
dichloromethane (35 mL) at −70 °C. The reaction mixture was
allowed to stir at 0 °C for 2 h and concentrated under reduced
pressure. The residue was partitioned between CHCl3 and 5% aqueous
NaHCO3. The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The resulting residue was
chromatographed on silica gel, eluting with MeOH/CHCl3 (1/100) to
give 13b (7.35 g, 88%) as a brown solid. 1H NMR (300 MHz, CDCl3)
δ: 1.39 (3H, t, J = 7.1 Hz), 3.88 (3H, s), 4.35 (2H, q, J = 7.1 Hz), 5,92
(2H, s), 6.72 (1H, t, J = 7.9 Hz), 6.97 (2H, d, J = 9.0 Hz), 7.42−7.56
(2H, m), 7.81−7.91 (3H, m).
1
MHz, DMSO-d6) δ: 1.86−1.95 (2H, m), 2.29 (3H, s), 2.45−2.52 (2H,
m), 2.65 (2H, t, J = 4.4 Hz), 3.51 (2H, t, J = 6.0 Hz), 3.60 (2H, t, J =
4.4 Hz), 6.76 (2H, d, J = 9.2 Hz), 7.21−7.28 (2H, m), 7.35 (1H, dd,
J = 6.8 Hz, 2.4 Hz), 7.84 (2H, d, J = 9.2 Hz), 9.53 (1H, br). FAB MS
m/z 371 (M + H)+.
4-[(4-Methoxybenzoyl)amino]-3-nitrophenyl 4-Methoxy-
benzoate (10f). Compound 10f was prepared from 9f and 2.2
equiv of 4-methoxybenzoyl chloride according to the procedure for the
preparation of 10a in 62% yield. 1H NMR (400 MHz, CDCl3) δ: 3.91
(3H, s), 3.92 (3H, s), 7.01 (2H, d, J = 8.8 Hz), 7.04 (2H, d, J = 8.8
Hz), 7.59 (1H, dd, J = 2.5, 9.3 Hz), 7.98 (2H, d, J = 8.8 Hz), 8.16 (2H,
d, J = 8.8 Hz), 8.18 (1H, d, J = 2.9 Hz), 9.10, (1H, d, J = 9.3 Hz),
11.27 (1H, s). FAB MS m/z 423 (M + H)+.
N-[4-(Benzyloxy)-2-nitrophenyl]-4-(4-methyl-1,4-diazepan-
1-yl)benzamide (10g). A solution of 4-(4-methyl-1,4-diazepan-1-
yl)benzoic acid11 hydrochloride (3.25 g, 12.0 mmol) in thionyl
chloride (17 mL, 233 mmol) was stirred at 60 °C for 0.5 h and
concentrated under reduced pressure. To the residue was added a
solution of 4-benzyloxy-2-nitroaniline 9g (2.47 g, 10.1 mmol) in
pyridine (100 mL). After the mixture was stirred at room temperature
for 14 h, the resulted precipitate was collected by filtration and washed
with pyridine. The solid was dissolved in CHCl3, washed with
saturated aqueous NaHCO3 solution, dried over Na2SO3, and
concentrated under reduced pressure to give 10g (2.23 g, 48%) as a
red-brown solid. 1H NMR (300 MHz, CDCl3) δ: 1.99−2.09 (2H, m),
2.39 (3H, s), 2.53−2.60 (2H, m), 2.70−2.75 (2H, m), 3.57 (2H, t, J =
6.3 Hz), 3.62−3.68 (2H, m), 5.12 (2H, s), 6.74 (2H, d, J = 9.0 Hz),
7.29−7.47 (6H, m), 7.82 (1H, d, J = 2.9 Hz), 7.86 (2H, d, J = 9.0 Hz),
8.93 (1H, d, J = 9.5 Hz), 11.03 (1H, s). FAB MS m/z 461 (M + H)+
N-(2-Fluoro-6-nitrophenyl)-4-(4-methyl-1,4-diazepan-1-yl)-
benzamide (10h). To a stirred solution of 4-(4-methyl-1,4-diazepan-
1-yl)benzoic acid11 hydrochloride (4.62 g, 17.1 mmol) was added
thionyl chloride (17 mL). After being stirred at 60 °C for 70 min, the
reaction mixture was concentrated under reduced pressure. To the
residue was added a solution of 2-fluoro-6-nitroaniline 9h (890 mg,
5.70 mmol) and N,N-dimethylpyridin-4-amine (700 mg, 5.70 mmol)
in pyridine (60 mL), and the mixture was stirred at 80 °C for 4 days.
The reaction mixture was concentrated under reduced pressure, and
the resulting residue was dissolved with CHCl3 followed by washing
with 5% aqueous NaHCO3 and concentration under reduced pressure.
The residue was chromatographed on silica gel, eluting with MeOH/
CHCl3 (1/99 to 10/90) to give 10h (400 mg, 6.3%) as a yellow solid.
1H NMR (300 MHz, CDCl3) δ: 1.98−2.09 (2H, m), 2.38 (3H, s),
4-Methoxy-N-(3-methoxy-2-{[4-(4-methyl-1,4-diazepan-1-
yl)benzoyl]amino}phenyl)benzamide Hydrochloride (14a). To
a solution of 10a (500 mg, 1.65 mmol) in MeOH (35 mL) at room
temperature was added 10% Pd−C powder (50 mg), and the mixture
was treated with hydrogen at 1 atm for 16 h. The reaction mixture was
filtered through a pad of Celite, and the filtrate was concentrated
under reduced pressure to give crude N-(2-amino-3-methoxyphenyl)-
4-methoxybenzamide (420 mg) which was used without further
purification. To a stirred solution of 4-(4-methyl-1,4-diazepan-1-
yl)benzoic acid11 hydrochloride (570 mg, 2.11 mmol) in thionyl
chloride was added DMF (0.1 mL). After the solution was stirred at
50 °C for 0.5 h, the solvents were removed under reduced pressure. A
mixture of the resulting residue and crude N-(2-amino-3-methoxy-
phenyl)-4-methoxybenzamide (420 mg) prepared above in pyridine
(15 mL) was stirred for 19 h at room temperature, and the reaction
mixture was concentrated under reduced pressure. The residue was
dissolved with CHCl3, washed with 5% aqueous NaHCO3, dried over
Na2SO4, and concentrated under reduced pressure. The resulting
residue was chromatographed on silica gel, eluting with 25% aqueous
ammonia/MeOH/CHCl3 (1/10/100), and lyophilized after being
dissolved with dilute aqueous HCl to give 14a (170 mg, 17%) as a
1
colorless solid. H NMR (400 MHz, DMSO-d6) δ: 2.11−2.40 (2H,
m), 2.79 (3H, d, J = 4.9 Hz), 3.04−3.22 (2H, m), 3.38−3.97 (12H,
m), 6.85(2H, d, J = 8.8 Hz), 6.95 (1H, d, J = 8.3 Hz), 7.02 (2H, d, J =
8.7 Hz), 7.29 (1H, t, J = 8.3 Hz), 7.42 (1H, d, J = 8.3 Hz), 7.84 (2H, d,
J = 8.8 Hz), 7.92 (2H, d, J = 8.8 Hz), 9.48 (1H, s), 9.73 (1H, s), 10.58 (1H,
s). FAB MS m/z 489 (M + H)+. Anal. (C28H32N4O4·1.8HCl·3.8H2O) C,
H, N, Cl.
4-Methoxy-N-(4-methoxy-2-{[4-(4-methyl-1,4-diazepan-1-
yl)benzoyl]amino}phenyl)benzamide Hydrochloride (14b). Com-
pound 14b was prepared from 10b according to the procedure for the
1
preparation of 14a in 42% yield as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.09−2.22 (1H, m), 2.28−2.42 (1H, m), 2.77
(3H, d, J = 4.4 Hz), 3.01−3.21 (2H, m), 3.37−3.54 (4H, m), 3.70−
3.97 (8H, m), 6.80−6.88 (3H, m), 7.05 (2H, d, J = 8.8 Hz), 7.33 (1H,
d, J = 2.9 Hz), 7.43 (1H, d, J = 8.8 Hz), 7.86 (2H, d, J = 8.8 Hz), 7.98
(2H, d, J = 8.8 Hz), 9.87 (1H, s), 10.07 (1H, s), 10.93 (1H, s). FAB
MS m/z 489 (M + H)+. Anal. (C28H32N4O4·1.3HCl·1.0H2O) C, N,
Cl. For H: calcd, 6.42%; found, 7.17%.
2.53−2.59 (2H, m), 2.68−2.75 (2H, m), 3.50−3.68 (4H, m), 6.72
(2H, d, J = 9.2 Hz), 7.29 (1H, dt, J = 5.0, 8.4 Hz), 7.46 (1H, ddd, J =
1.4, 8.4, 9.7 Hz), 7.80−7.92 (3H, m), 9.00 (1H, s). FAB MS m/z 373
(M + H)+
4-Methoxy-N-(2-amino-5-methoxy)benzamide (13a). To a
stirred solution of N-(2-amino-4-methoxyphenyl)-2,2,2-trifluoroace-
toamide 1125 (1.21 g, 5.18 mmol) in 1,2-dichloroethane (52 mL) at
0 °C was added triethylamine (1.57 g, 15.5 mmol) and 4-
methoxybenzoyl chloride (1.33 g, 7.80 mmol). After the mixture was
stirred at room temperature for 13 h, ethanol (3 mL) and brine were
added. The organic layer was separated and concentrated under
reduced pressure. To a solution of the residue in methanol (52 mL)
and H2O (13 mL) at room temperature was added K2CO3, and the
mixture was stirred at 60 °C for 5 h. The reaction mixture was cooled
to room temperature and concentrated under reduced pressure. H2O
was added to the residue, and the solution was extracted with CHCl3.
The organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The resulting residue was chromatographed on silica
gel, eluting with MeOH/CHCl3 (0/100 to 5/100) to give 13a (0.89 g,
4-Methoxy-N-(5-methoxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (14c). Com-
pound 14c was prepared from 13a according to the procedure for the
1
preparation of 14a in 50% yield as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.10−2.22 (1H, m), 2.26−2.42 (1H, m), 2.77
(3H, d, J = 4.9 Hz), 3.02−3.20 (2H, m), 3.36−3.55 (4H, m), 3.70−
3.85 (7H, m), 3.87−3.97 (1H, m), 6.81−6.87 (3H, m), 7.05 (2H, d,
J = 8.8 Hz), 7.28 (1H, d, J = 2.9 Hz), 7.46 (1H, d, J = 8.8 Hz), 7.88 (2H,
d, J = 8.8 Hz), 7.95 (2H, d, J = 8.8 Hz), 9.83 (1H, s), 10.11 (1H, s),
10.86 (1H, s). FAB MS m/z 489 (M + H)+. Anal. (C28H32-
N4O4·1.2HCl·1.3H2O) C, H, N, Cl.
4-Methoxy-N-(6-methoxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (14d). Com-
pound 14d was prepared from 10c according to the procedure for the
1
63%) as a brown solid. H NMR (300 MHz, CDCl3) δ: 3.40 (2H, s),
1
3.76 (3H, s), 3.87 (3H, s), 6.63 (1H, dd, J = 2.9, 8.6 Hz), 6.81 (1H, d,
J = 8.6 Hz), 6.96 (2H, d, J = 8.8 Hz), 7.32 (1H, d, J = 2.9 Hz), 7.87
(2H, d, J = 8.8 Hz), 8.18 (1H, s). FAB MS m/z 273 (M + H)+.
preparation of 14a in 48% yield as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.08−2.22 (1H, m), 2.27−2.41 (1H, m), 2.75
(3H, d, J = 4.9 Hz), 3.00−3.20 (2H, m), 3.34−3.52 (4H, m),
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3.68−4.00 (8H, m), 6.82 (2H, d, J = 8.8 Hz), 6.93 (1H, d, J = 8.3 Hz),
7.06 (2H, d, J = 8.8 Hz), 7.29 (1H, t, J = 8.3 Hz), 7.46 (1H, d, J = 8.3
Hz), 7.77 (2H, d, J = 8.8 Hz), 8.02 (2H, d, J = 8.8 Hz), 9.51 (1H, s),
9.72 (1H, s), 10.93 (1H, s). FAB MS m/z 489 (M + H)+. Anal.
(C28H32N4O4·1.2HCl·1.3H2O) C, H, N, Cl.
reduced pressure. To the resulting residue was added 5% aqueous
NaHCO3, and the appropriate portion was extracted with CHCl3. The
organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was chromatographed on silica
gel, eluting with 25% aqueous ammonia/MeOH/CHCl3 (1/10/100)
and crystallized from EtOH to give the free form of 14i (1.74 g, 62%).
The HCl salt of 14i (5.50 g, 90%) was prepared as a colorless solid by
precipitation of the free form of 14i (5.68 g) from aqueous 0.5 N HCl
Ethyl 3-[(4-Methoxybenzoyl)amino]-2-{[4-(4-methyl-1,4-dia-
zepan-1-yl)benzoyl]amino}benzoate Hydrochloride (14e).
Compound 14e was prepared from 13b according to the procedure
1
1
for the preparation of 14a in 20% yield as a colorless solid. H NMR
(100 mL). H NMR (400 MHz, DMSO-d6) δ: 2.08−2.31 (2H, m),
(400 MHz, DMSO-d6) δ: 1.14 (3H, t, J = 7.3 Hz), 2.08−2.34 (2H, m),
2.80 (3H, d, J = 4.4 Hz), 3.04−3.22 (2H, m), 3.38−3.58 (4H, m),
3.66−3.77 (1H, m), 3.83 (3H, s), 3.86−3.98 (1H, m), 4.16 (2H, q, J =
7.3 Hz), 6.86 (2H, d, J = 8.8 Hz), 7.06 (2H, d, J = 8.8 Hz), 7.41 (1H, t,
J = 7.8 Hz), 7.68 (1H, dd, J = 1.5 Hz, 7.8 Hz), 7.82−7.90 (3H, m),
7.94 (2H, d, J = 8.7 Hz), 10.00 (1H, s), 10.11 (1H, s), 10.30 (1H, s).
FAB MS m/z 531 (M + H)+.
2.80 (3H, s), 3.03−3.24 (2H, m), 3.36−3.60 (4H, m), 3.62−4.02 (5H,
m), 6.81 (1H, d, J = 8.3 Hz), 6,85 (2H, d, J = 8.3 Hz), 7.04 (2H, d, J =
8.3 Hz), 7.14 (1H, d, J = 8.3 Hz)), 7.24 (1H, d, J = 8.3), 7.86 (2H, d, -
J = 8.3 Hz), 7.92 (2H, d, J = 8.3 Hz), 9.52 (1H, s), 9.66 (1H, s), 9.80
(1H, s), 10.20 (1H, s). FAB MS m/z 475 (M + H)+. Anal.
(C27H30N4O4·1.0HCl) C, H, N, Cl.
4-[(4-Methoxybenzoyl)amino]-3-{[4-(4-methyl-1,4-diazepan-
1-yl)benzoyl]amino}phenyl 4-Methoxybenzoate (14j). Com-
pound 14j was prepared from 10f according to the procedure for
3-[(4-Methoxybenzoyl)amino]-2-{[4-(4-methyl-1,4-diazepan-
1-yl)benzoyl]amino}benzoic Acid Hydrochloride (14f). To a
stirred suspension of 14e (500 mg, 0.88 mmol) in MeOH (9.4 mL)
was added 1 M aqueous NaOH (1.9 mL). After being stirred at room
temperature for 4 h, the reaction mixture was acidified with 1 M
aqueous HCl and concentrated under reduced pressure. The resulting
residue was directly chromatographed on ODS gel, eluting with
CH3CN/0.01 M aqueous HCl (0/100 to 50/50) gradually and
1
the preparation of 14a in 64% yield as a colorless solid. H NMR(300
MHz, DMSO-d6) δ: 1.95−2.06 (2H, m), 2.37 (3H, s), 2.51−2.58 (2H,
m), 2.66−2.72 (2H, m), 3.50 (2H, t, J = 6.3 Hz), 3.55−3.61 (2H, m),
3.81 (3H, s), 3.89 (3H, s), 6.67 (2H, d, J = 9.0 Hz), 6.76 (1H, dd, J =
2.6, 8.8 Hz), 6.91 (2H, d, J = 9.0 Hz), 6.95 (2H, d, J = 8.8 Hz), 7.40
(1H, d, J = 2.4 Hz), 7.43 (1H, d, J = 8.8 Hz), 7.88 (2H, d, J = 7.9 Hz),
7.91 (2H, d, J = 7.9 Hz), 8.04 (2H, d, J = 9.0 Hz), 9.20 (1H, s), 9.53
(1H, s). FAB MS m/z 609 (M + H)+.
1
lyophilized to give 14f (346 mg, 73%) as a colorless solid. H NMR
(400 MHz, DMSO-d6) δ: 2.00−2.42 (2H, m), 2.77 (3H, d, J = 4.4
Hz), 2.97−3.25 (2H, m), 3.30−4.20 (9H, m), 6.87 (2H, d, J = 8.8 Hz),
7.05 (2H, d, J = 8.8 Hz), 7.40 (1H, t, J = 7.8 Hz), 7.75 (1H, d, J = 7.3
Hz), 7.88−7.94 (5H, m), 10.10 (1H, s), 10.33 (1H, s), 10.91 (1H, s).
FAB MS m/z 503 (M + H)+. Anal. (C28H30N4O5·1.2HCl·1.5H2O) C,
H, N, Cl.
Ethyl 4-[(4-Methoxybenzoyl)amino]-3-{[4-(4-methyl-1,4-dia-
zepan-1-yl)benzoyl]amino}benzoate (14g). Compound 14g was
prepared from 10d according to the procedure for the preparation of
14a in 82% yield as a colorless solid. 1H NMR (300 MHz, DMSO-d6)
δ: 1.34 (3H, t, J = 6.9 Hz), 2.15−2.32 (2H, m), 2.76 (3H, s), 3.15−
3.36 (6H, m), 3.82−3.90 (5H, m), 4.34 (2H, q, J = 6.9 Hz), 6.86 (2H,
d, J = 8.8 Hz), 7.07 (2H, d, J = 8.8 Hz), 7.83−7.86 (2H, m), 7.93 (2H,
d, J = 8.8 Hz), 7.99 (2H, d, J = 8.8 Hz), 8.23 (1H, s), 10.11 (1H, s),
10.32 (1H, s). FAB MS m/z 531 (M + H)+.
N-(4-Hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}phenyl)-4-methoxybenzamide Hydrochloride (14k). Com-
pound 14k was prepared from 14j according to the procedure for the
1
preparation of compound 14f in 46% yield as a colorless solid. H
NMR (400 MHz, DMSO-d6) δ: 1.06 (3H, t, J = 7.4 Hz), 2.05−2.48
(2H, m), 2.79 (3H, s), 3.00−3.60 (8H, m), 3.64−4.00 (5H,m), 4.33
(1H, s), 6.63 (1H, dd, J = 2.4, 8.8 Hz), 6.84 (2H, d, J = 8.8 Hz), 7.05
(2H, d, J = 8.8 Hz), 7.20 (1H, d, J = 2.4 Hz), 7.27 (1H, d, J = 8.8 Hz),
7.81 (2H, d, J = 8.8 Hz), 7.94 (2H, d, J = 8.8 Hz), 9.50 (1H, s), 9.68
(1H, s), 9.91 (1H, s), 10.37 (1H, s). FAB MS m/z 475 (M + H)+.
Anal. (C28H30N4O5·1.0HCl·1.0C2H6O·1.0H2O) C, N, Cl. For H:
calcd, 6.84%; found, 6.24%.
N-[5-(Benzyloxy)-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl]-4-methoxybenzamide (14l). A stirred
suspension of 10g (1.15 g, 2.50 mmol), ammonium chloride (0.14 g
2.62 mmol), and iron powder (0.70 g, 12.5 mmol) in EtOH (30 mL)
and H2O (15 mL) was refluxed for 1 h. The reaction mixture was
filtered through a pad of Celite, and the filtrate was concentrated
under reduced pressure. The suspension of the residue in saturated
aqueous NaHCO3 was extracted with CHCl3, and the organic layer
was washed with brine, dried over Na2SO4, and concentrated under
reduced pressure to give crude N-[2-amino-4-(benzyloxy)phenyl]-4-
(4-methyl-1,4-diazepan-1-yl)benzamide (1.15 g) which was used
without further purification. Compound 14l was prepared from
above aniline derivative according to the same procedure for the
4-[(4-Methoxybenzoyl)amino]-3-{[4-(4-methyl-1,4-diazepan-
1-yl)benzoyl]amino}benzoic Acid Hydrochloride (14h). Com-
pound 14h was prepared from 14g according to the procedure for the
1
preparation of 14f in 35% yield as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.10−2.22 (1H, m), 2.29−2.43 (1H, m), 2.77
(3H, d, J = 4.9 Hz), 3.02−3.23 (2H, m), 3.37−3.57 (4H, m), 3.71−
3.98 (5H, m), 6.86 (2H, d, J = 8.8 Hz), 7.06 (2H, d, J = 8.8 Hz), 7.82
(2H, s), 7.94 (2H, d, J = 8.8 Hz), 8.00 (2H, d, J = 8.8 Hz), 8.21 (1H,
s), 10.12 (1H, s), 10.34 (1H,s), 10.94 (1H,s). FAB MS m/z 503 (M +
H)+. Anal. (C28H30N4O5·1.9HCl·2.3H2O) C, H, N, Cl.
N-[2-Hydroxy-6-(4-methoxybenzamido)phenyl]-4-(4-meth-
yl-1,4-diazepan-1-yl)benzamide Hydrochloride (14i). To a
solution of 10e (2.14 g, 5.77 mmol) in MeOH (43 mL) at room
temperature was added 10% Pd−C powder (54.2% water wet, 0.47 g),
and the mixture at 30 °C was treated with hydrogen at ambient
pressure until the absorption of hydrogen had stopped. The reaction
mixture was filtered through a pad of Celite, and the filtrate was
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel, eluting with MeOH/CHCl3 (1/20 to 1/10) to
give N-(2-amino-6-hydroxyphenyl)-4-(4-methyl-1,4-diazepan-1-yl)-
benzamide (1.61 g, 82%) as a pale brown solid. 1H NMR (400
MHz, DMSO-d6) δ: 1.85−1.94 (2H, m), 2.26 (3H, s), 2.43 (2H, t, J =
5.6 Hz), 2.61 (2H, t, J = 4.8 Hz), 3.51 (2H, t, J = 6.0 Hz), 3.58 (2H, t,
J = 4.8 Hz), 4.68 (2H, s), 6.16 (1H, dd, J = 7.6 Hz, 1.2 Hz), 6.24 (1H,
dd, J = 8.0 Hz, 1.2 Hz), 6.70−6.81 (3H, m), 7.86 (2H, d, J = 8.8 Hz),
8.93 (1H, br), 8.94 (1H, s).
1
preparation of 14i in 81% yield as pale brown solid. H NMR (300
MHz, CDCl3) δ: 1.91−2.06 (2H, m), 2.38 (3H, s), 2.52−2.58 (2H,
m), 2.67−2.73 (2H, m), 3.49 (2H, t, J = 6.2 Hz), 3.55−3 61 (2H, m),
3.82 (3H, s), 4.68 (2H, s), 6.54 (1H, dd, J = 2.8, 8.7 Hz), 6.69 (2H, d, -
J = 9.0 Hz), 6.95 (2H, d, J = 8.8 Hz), 7.19−7.36 (7H, m), 7.88 (2H, d,
J = 9.0 Hz), 8.00 (2H, d, J = 8.8 Hz), 8.76 (1H, s), 9.63 (1H, s).
N-(5-Hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}phenyl)-4-methoxybenzamide Hydrochloride (14m). To
a suspension of 14l (0.98 g, 1.70 mmol) in AcOH (30 mL) at room
temperature was added 10% Pd−C powder (300 mg), and the mixture
was treated with hydrogen at ambient pressure for 24 h. The reaction
mixture was filtered through a pad of Celite, and the filtrate was
concentrated under reduced pressure. The residue was suspended in
saturated aqueous NaHCO3, extracted with CHCl3, washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was crystallized from EtOH and the aqueous solution of the
resulting crystal was lyophilized after being acidified with 1 N HCl.
A solution of N-(2-amino-6-hydroxyphenyl)-4-(4-methyl-1,4-diaze-
pan-1-yl)benzamide (2.03 g, 5.96 mmol) and 4-methoxybenzoyl
chloride (1.12 g, 6.00 mmol) in pyridine (60 mL) was stirred at room
temperature for 3 days. The reaction mixture was concentrated under
1
14m (558 mg, 59%) was obtained as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.11−2.35 (2H, m), 2.80 (3H, d, J = 5.4 Hz),
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3.02−3.21 (2H, m), 3.38−3.58 (4H, m), 3.67−3.96 (5H, m), 6.66
(1H, dd, J = 2.4, 8.8 Hz), 6.84 (2H, d, J = 8.8 Hz), 7.04 (2H, d, J = 8.8
Hz), 7.15 (1H, d, J = 2.4 Hz), 7.30 (1H, d, J = 8.8 Hz), 7.85 (2H, d, -
J = 8.8 Hz), 7.90 (2H, d, J = 8.8 Hz), 9.52 (1H, s), 9.71 (1H, s), 9.95
(1H, s), 10.36 (1H, s). FAB MS m/z 475 (M + H)+. Anal.
(C27H30N4O4·1.0HCl·2.0 H2O) C, H, N, Cl
under reduced pressure. The residue was chromatographed on silica
gel, eluting with 25% aqueous ammonia/MeOH/CHCl3 (0/0/100 to
1/10/100). The purified product (150 mg) was dissolved with a
mixtutre of EtOH (3 mL) and 1 M aqueous HCl (0.4 mL) and
concentrated under reduced pressure. The residue was crystallized
from H2O to give 14q (107 mg, 27%) as a colorless solid. Mp 174−
176 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.12−2.22 (1H, m),
2.26−2.39 (1H, m), 2.79 (3H, d, J = 3.9 Hz), 3.05−3.21 (2H, m),
3.39−3.55 (4H, m), 3.66−3.79 (3H, m), 3.81 (3H, s), 3.90−3.97 (1H,
m), 4.11 (2H, t, J = 4.9 Hz), 4.86 (1H, s), 6.86 (2H, d, J = 8.8 Hz),
6.97 (1H, d, J = 7.4 Hz), 7.04 (2H, d, J = 8.8 Hz), 7.27(1H, t, J = 8.3
Hz), 7.42 (1H, d, J = 8.3 Hz), 7.86 (2H, d, J = 8.7 Hz), 7.92 (2H, d,
J = 8.8 Hz), 9.55 (1H, s), 9.89 (1H, s), 10.67 (1H, s). FAB MS m/z
519 (M + H)+. Anal. (C29H34N4O5·1.0HCl·1.2H2O) C, H, N, Cl.
N-(2-Hydroxy-6-{[4-(methylsulfanyl)benzoyl]amino}phenyl)-
4-(4-methyl-1,4-diazepan-1-yl)benzamide Hydrochloride
(15a). Compound 15a was prepared from 10e and 4-
(methylsulfanyl)benzoyl chloride according to the procedure for
preparation of 14i in 46% yield as a colorless solid. Mp 264−268 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.09−2.41 (2H, m), 2.52 (3H, s),
N-(3-Fluoro-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}phenyl)-4-methoxybenzamide Hydrochloride (14n). Com-
pound 14n was prepared from 10h according to the procedure for
1
the preparation of 14i in 53% yield as a colorless solid. H NMR
(400 MHz, DMSO-d6) δ: 2.16−2.36 (2H, m), 2.79 (3H, d, J = 5.9
Hz), 3.05−3.21 (2H, m), 3.30−3.57 (4H, m), 3.66−3.78 (1H, m),
3.82 (3H, s), 3.88−3.99 (1H, s), 6.85 (2H, d, J = 9.3 Hz), 7.03 (2H, d,
J = 8.8 Hz), 7.13−7.18 (1H, m), 7.31−7.37 (1H, m), 7.55−7.59 (1H,
m), 7.89 (2H, d, J = 8.8 Hz), 7.94 (2H, d, J = 8.7 Hz), 9.72 (1H, s),
10.03 (1H, s), 10.42 (1H, s). FAB MS m/z 477 (M + H)+. Anal.
(C27H29N4O3F·1.0HCl·1.4H2O) C, H, N, Cl, F
Ethyl (3-[(4-Methoxybenzoyl)amino]-2-{[4-(4-methyl-1,4-di-
azepan-1-yl)benzoyl]amino}phenoxy)acetate Hydrochloride
(14o). To a stirred solution of the free form of 14i (500 mg, 1.05
mmol) in MeOH (11 mL) and CHCl3 (11 mL) at room temperature
was added benzyl bromide (428 mg, 2.50 mmol). The mixture was
stirred for 22 h. The resulting precipitate was filtered and washed with
the MeOH/CHCl3 (50/50) to give crude 1-benzyl-4-[4-({2-hydroxy-
6-[(4-methoxybenzoyl)amino]phenyl}carbamoyl)phenyl]-1-methyl-
1,4-diazepan-1-ium bromide (770 mg) as a solid, which was used
without further purification. To the stirred solution of the solid in
DMF at room temperature, ethyl bromoacetate (210 mg, 1.30 mmol)
and potassium carbonate (174 mg, 1.30 mmol) were added, and the
mixture was stirred at 100 °C for 1 h. The reaction mixture was cooled
to room temperature and filtered. The filtrate was concentrated under
reduced pressure to give crude 1-benzyl-4-[4-({2-(2-ethoxy-2-
oxoethoxy)-6-[(4-methoxybenzoyl)amino]phenyl}carbamoyl)phenyl]-
1-methyl-1,4-diazepan-1-ium as a solid, which was used without further
purification. To the stirred solution of the solid in acetic acid (16 mL)
at room temperature was added 10% Pd−C powder (100 mg), and the
mixture was treated with hydrogen under 2.9 atm at room temperature
for 3 h. The reaction mixture was filtered through a pad of Celite, and
the filtrate was concentrated under reduced pressure. The resulting
residue was chromatographed on silica gel, eluting with 25% aqueous
ammonia/MeOH/CHCl3 (0/0/100 to 1/10/100) and chromato-
graphed on ODS, again eluting with 0.001 M aqueous HCl/EtOH (0/
100 to 30/70). The purified solid was lyophilized after being dissolved
with dilute aqueous HCl to give 14o (350 mg, 59%) as a colorless
2.78 (3H, s), 3.00−3.23 (2H, m), 3.37−3.57 (4H, m), 3.67−3.82 (1H,
m), 3.87−4.00 (1H, m), 6.78−6.90 (3H, m), 7.14 (1H, t, J = 8.3 Hz),
7.24 (1H, d, J = 8.3 Hz), 7.35 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 8.3 Hz),
7.93 (2H, d, J = 8.8 Hz), 9.57 (1H, s), 9.68 (1H, s), 9.91 (1H, s), 10.72
(1H, s). FAB MS m/z 491 (M + H)+. Anal. (C27H30N4O3S·1.0HCl·0.5
H2O) C, H, N, S, Cl.
4-(Benzyloxy)-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-
yl)benzoyl]amino}phenyl)benzamide (15b). Compound 15b
was prepared from 10e and 4-(benzyloxy)benzoyl chloride according
to the procedure for preparation of 14i in 62% yield as a colorless
1
solid. H NMR (300 MHz, CDCl3) δ: 1.96−2.08 (2H, m), 2.38 (3H,
s), 2.53−2.59 (2H, m), 2.68−2.75 (2H, m), 3.50−3.66 (4H, m), 5.15
(2H, s), 6.73−6.75 (3H, m), 6.82−6.99 (2H, m), 7.08 (2H, d, J = 9.0
Hz), 7.30−7.50 (5H, m), 7.82 (2H, d, J = 9.0 Hz), 7.95 (2H, d, J = 8.8
Hz), 8.61 (1H, s), 9.83 (1H, s). FAB MS m/z 551 (M + H)+.
4-Hydroxy-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (15c). Com-
pound 15c was prepared from 15b according to the procedure for
preparation of 14m and crystallized from EtOH−H2O in 58% yield as
a colorless solid. Mp 195−197 °C. 1H NMR (400 MHz, DMSO-d6) δ:
1.06 (3H, t, J = 6.8 Hz), 2.08−2.40 (2H, m), 3.78 (3H, s), 3.04−3.57
(8H, m), 3.67−3.82 (1H, m), 3.87−3.99 (1H, m), 4.36 (1H, s), 6.77−
6.90 (5H, m), 7.13 (1H, t, J = 7.9 Hz), 7.23 (1H, d, J = 7.9 Hz), 7.76
(2H, d, J = 8.8 Hz), 7.93 (2H, d, J = 8.8 Hz), 9.59 (1H, s), 9.65 (1H,
s), 9.72 (1H, s), 10,21 (1H, s), 10,76 (1H, s). FAB MS m/z 461 (M +
H)+. Anal. (C26H28N4O4·1.0HCl·1.0C2H6O·0.3H2O) C, H, N, Cl.
4-Fluoro-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (15d). Com-
pound 15d was prepared from 10e and 4-fluorobenzoylchloride
according to the procedure for preparation of 14i in 57% yield as a
colorless solid. This compound was obtained by crystallization from
1
amorphous solid. H NMR (400 MHz, DMSO-d6) δ: 1.21 (3H, t,
J =7.3 Hz), 2.01−2.22 (1H, m), 2.26−2.42 (1H, m), 2.78 (3H, d,
J = 4.9 Hz), 3.02−3.22 (2H, m), 3.35−4.02 (9H, m), 4.18 (2H, q,
J = 7.3 Hz), 4.83 (2H, s), 6.86 (2H, d, J = 9.3 Hz), 6.92 (1H, d, J = 7.3
Hz), 7.04 (2H, d, J = 8.8 Hz), 7.27 (1H, t, J = 8.3 Hz), 7.49 (1H, d,
J = 7.8 Hz), 7.86 (2H, d, J = 8.8 Hz), 7.93 (2H, d, J = 8.8 Hz), 9.57
(1H, s), 9.86 (1H, s). FAB MS m/z 561 (M + H)+
(3-[(4-Methoxybenzoyl)amino]-2-{[4-(4-methyl-1,4-diaze-
pan-1-yl)benzoyl]amino}phenoxy)acetic Acid Hydrochloride
(14p). Compound 14p was prepared from 14o according to the
procedure for preparation of 14f in 76% yield as a colorless solid. This
compound was obtained by crystallization from H2O. Mp 186−188,
1
EtOH−H2O. Mp 196−198 °C. H NMR (400 MHz, DMSO-d6) δ:
1.06 (3H, t, J = 6.8 Hz), 2.07−2.40 (2H, m), 2.78 (3H, s), 3.00−3.22
(2H, m), 3.35−3.60 (6H, m), 3.67−3.81 (1H, m), 3.85−4.00 (1H, m),
4.35 (1H, s), 6.82−6.85 (3H, m), 7.13−7.17 (1H, m), 7.22 (1H, d, J =
7.8 Hz), 7.32−7.37 (2H, m), 7.93 (2H, d, J = 8.8 Hz), 7.95−7.99 (2H,
m), 9.56 (1H, s), 9.68 (1H, s), 10.01 (1H, s), 10.65 (1H, s). FAB MS
m/z 463 (M + H)+. Anal. (C26H27N4O3F·1.0HCl·1.0C2H6O) C, H, N,
Cl, F
1
2.16−2.30 °C. H NMR (400 MHz, DMSO-d6) δ: 2.16−2.30 (2H,
m), 2.78 (3H, s), 3.15−3.58 (6H, m), 3.78−3.88 (5H, m), 4.75 (2H,
s), 6.86 (2H, d, J = 9.3 Hz), 6.94 (1H, d, J = 7.3 Hz), 7.04 (2H, d, J =
8.8 Hz), 7.28 (1H, t, J = 7.8 Hz), 7.50 (1H, d, J = 7.9 Hz), 7.85 (2H, d,
J = 8.8 Hz), 7.95 (2H, d, J = 8.8 Hz), 9.85−9.98 (2H, m). FAB MS m/
z 533 (M + H)+. Anal. (C29H32N4O6·0.9HCl·0.5H2O) C, H, N, Cl.
N-[3-(2-Hydroxyethoxy)-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl]-4-methoxybenzamide Hydrochloride
(14q). To a stirred solution of 14o (370 mg, 0.71 mmol) in THF
(14 mL) at room temperature was added NaBH4 (216 mg, 5.70
mmol). The reaction mixture was heated to 60 °C, and MeOH (1.86
g, 58.0 mmol) in THF (14 mL) was added dropwise and stirred for 10
min. After the reaction was quenched with H2O (2 mL), the mixture
was concentrated under reduced pressure. The residue was dissolved
with CHCl3, washed with brine, dried over Na2SO4, and concentrated
4-Chloro-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (15e). Com-
pound 15e was prepared from 10e and 4-chlorobenzoyl chloride
according to the procedure for preparation of 14i in 51% yield as a
colorless solid. This compound was crystallized from EtOH−H2O. Mp
1
257−258 °C. H NMR (400 MHz, DMSO-d6) δ: 1.06 (3H, t, J = 6.8
Hz), 2.10−2.37 (2H, m), 2.79 (3H, s), 3.02−3.22 (2H, m), 3.35−3.58
(6H, m), 3.66−3.80 (1H, m), 3.85−3.99 (1H, m), 4.34 (1H, s), 6.82−
6.86 (3H, m), 7.13−7.17 (1H, m), 7.22 (1H, d, J = 8.3 Hz), 7.58 (2H,
d, J = 8.3 Hz), 7.89−7.93 (4H, m), 9.53 (1H, s), 9.68 (1H, s), 10.03
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(1H, s), 10.45 (1H, s). FAB MS m/z 479 (M + H)+. Anal.
(C26H27N4O3Cl·1.0HCl·0.3H2O·1.0C2H6O) C, H, N, Cl.
was concentrated under reduced pressure to remove CH3CN and then
acidified with AcOH (pH 4−5). The resulting precipitate was filtered
and washed with H2O to give 16 (3.18 g, 38%) as a colorless solid. 1H
NMR (400 MHz, DMSO-d6) δ: 1.75−2.00 (2H, m), 2.45 (3H, s),
2.65−3.70 (12H, m), 3.75 (1H, d, J = 9.3 Hz), 3.84 (3H, s), 4.95 (1H,
d, J = 7.3 Hz), 5.21 (1H, s), 5.43 (1H, s), 6.75 (2H, d, J = 8.8 Hz),
7.02−7.11 (3H, m), 7.33 (1H, t, J = 8.3 Hz), 7.62 (1H, d, J = 8.3 Hz),
7.86 (2H, d, J = 8.3 Hz), 7.91 (2H, d, J = 8.3 Hz), 9.66 (1H, s), 9.99
(1H, s). FAB MS m/z 651 (M + H)+. Anal. (C33H38N4O10·2.5H2O)
C, H, N,
In Vitro Assay for Inhibition of Factor Xa. The hydrolysis rates
of synthetic substrates were assayed by continuously measuring
absorbance at 405 nm at 37 °C with a microplate reader (model 3550,
Bio-Rad, U.S.). Reaction mixtures (125 μL) were prepared in 96-well
plates containing chromogenic substrates (S-2222) and an inhibitor in
either 0.05 M Tris-HCl, pH 8.4, or 0.15 M NaCl. Reactions were
initiated with 25 μL of enzyme solution. The concentration of
inhibitor required to inhibit enzyme activity by 50% (IC50) was
calculated from dose−response curves in which the logit trans-
formation of residual activity was plotted against the logarithm of
inhibitor concentration.
Enzyme Selectivity of 14 and 16i. Reaction mixtures were
prepared in 96-well plates containing the chromogenic substrate and
test compound. The reaction was initiated by the addition of enzyme,
and the color was continuously monitored at 405 nm using a
microplate reader SpectraMax 340PC (Molecular Devices, CA, U.S.)
at 37 °C. Each enzyme was used at final concentration as follows: 4.2
mU mL−1 FXa, 0.20 U mL−1 thrombin, 1.0 U mL−1 trypsin, and 1.7
mU mL−1 plasma kallikrein. The enzymatic activities were assessed by
the amidolysis of the following chromogenic substrates for the
corresponding protease: S-2222 for FXa and trypsin, S-2238 for
thrombin, and S-2302 for plasma kallikrein. The rate of substrate
hydrolysis (mOD min−1) was measured at 37 °C. The mode of
inhibition was estimated from a Lineweaver−Burk plot. The Ki was
determined from a Dixon plot by plotting the reciprocal of the initial
reaction velocities at different substrate concentrations against
different inhibitor concentrations.
Prothrombin Time Assays in Vitro. After collection of citrated
blood samples, platelet-poor plasma was prepared by centrifugation at
3000 rpm for 10 min and stored at −40 °C until use. Plasma clotting
times were measured using a KC10A coagulometer (Amelung Co.,
Lehbrinsweg, Germany) at 37 °C. Prothrombin time (PT) was
measured using Orthobrain thromboplastin (OrthoDiagnostic Systems
Co., Tokyo, Japan), and values for each test sample were compared
with coagulation times of a distilled water control. The concentration
required to double the clotting time (CT2) was estimated from each
individual concentration−response curve. Each measurement was
performed three times and represented as the mean value.
4-Bromo-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)benzamide Hydrochloride (15f). Com-
pound 15f was prepared from 10e and 4-bromobenzoyl chloride
according to the procedure for preparation of 14i in 54% yield as a
colorless solid. This compound was crystallized from EtOH−H2O. Mp
263−268 °C . 1H NMR (400 MHz, DMSO-d6) δ: 2.10−2.36 (2H, m),
2.79 (3H, s), 3.02−3.23 (2H, m), 3.37−3.58 (4H, m), 3.67−3.80 (1H,
m), 3.86−3.98 (1H, m), 6.82−6.86 (3H, m), 7.13−7.17 (1H, m), 7.22
(1H, d, J = 7.8 Hz), 7.72 (2H, d, J = 8.3 Hz), 7.83 (2H, d, J = 8.3 Hz),
7.92 (2H, d, J = 8.8 Hz), 9.53 (1H, s), 9.68 (1H, s), 10.03 (1H, s),
10.49 (1H, s). FAB MS m/z 523, 525 (M + H)+. Anal.
(C26H27N4O3Br·1.0HCl·0.3H2O) C, H, N, Br, Cl.
N-(3-Hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]-
amino}phenyl)-5-methoxythiophene-2-carboxamide Hydro-
chloride (15g). Compound 15g was prepared from 10e and 5-
methoxythiophene-2-carbonyl chloride according to the procedure for
preparation of 14i in 39% yield as a colorless solid. This compound
1
was crystallized from EtOH−H2O. Mp 245−247 °C . H NMR (400
MHz, DMSO-d6) δ: 2.10−2.40 (2H, m), 2.80 (3H, d, J = 2.4 Hz),
3.03−3.25 (2H, m), 3.38−3.59 (4H, m), 3.68−3.82 (1H, m), 3.85−
4.01 (4H, m), 6.40 (1H, d, J = 3.9 Hz), 6.80 (1H, dd, J = 1.5, 7.8 Hz),
6.86 (2H, d, J = 8.8 Hz), 7.10−7.18 (2H, m), 7.53 (1H, d, J = 3.9 Hz),
7.94 (2H, d, J = 8.8 Hz), 9.57 (1H, s), 9.63 (1H, s), 9.83 (1H, s),
10.61 (1H, s). FAB MS m/z 481 (M
+
H)+. Anal.
(C25H28N4O4S·1.0HCl·1.0H2O·0.5C2H6O) C, H, N, S, Cl.
5-Chloro-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)thiophene-2-carboxamide Hydrochlor-
ide (15h). Compound 15h was prepared from 10e and 5-
chlorothiophene-2-carbonyl chloride according to the procedure for
preparation of 14i in 37% yield as a colorless solid. This compound
1
was crystallized from EtOAc. Mp 247−248 °C. H NMR (400 MHz,
DMSO-d6) δ: 2.11−2.38 (2H, m), 2.80 (3H, d, J = 3.9 Hz), 3.04−3.23
(2H, m), 3.42−3.60 (4H, m), 3.67−3.80 (1H, m), 3.87−3.98 (1H, m),
6.79−6.88 (3H, m), 7.10−7.18 (2H, m), 7.24 (1H, d, J = 3.9 Hz), 7.72
(1H, d, J = 3.9 Hz), 7.95 (2H, d, J = 8.8 Hz), 9.58 (1H, s), 9.67 (1H,
s), 10.17 (1H, s), 10.49 (1H, s). FAB MS m/z 485 (M + H)+. Anal.
(C24H25N4O3SCl·1.0HCl·0.5H2O) C, H, N, S, Cl.
5-Bromo-N-(3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)-
benzoyl]amino}phenyl)thiophene-2-carboxamide Hydrochlor-
ide (15i). Compound 15i was prepared from 10e and 5-
bromothiophene-2-carbonyl chloride according to the procedure for
1
preparation of 14i in 40% yield as a colorless solid. H NMR (400
MHz, DMSO-d6) δ: 2.11−2.38 (2H, m), 2.80 (3H, d, J = 4.3 Hz),
3.05−3.23 (2H, m), 3.38−3.58 (4H, m), 3.68−3.80 (1H, m), 3.88−
3.98 (1H, m), 6.81−6.86 (3H, m), 7.11−7.17 (2H, m), 7.33 (1H, d,
J = 3.9 Hz), 7.66 (1H, d, J = 4.4 Hz), 7.94 (2H, d, J = 8.8 Hz), 9.55
(1H, s), 9.65 (1H, s), 10.13 (1H, s), 10.48 (1H, s). FAB MS m/z 529,
531 (M + H)+. Anal. (C24H25BrN4O3S·1.3HCl) C, H, N, S, Cl. For Br:
calcd, 13.85%; found, 12.94%.
Ex Vivo Studies. The test drug was dissolved or suspended in
0.5% methyl cellulose and orally administered to male ICR mice (mass
range: 30−37 g) at a dose of 100 mg/kg using a gastric tube. Citrated
blood was collected from the inferior vena cava 0.5 and 2.0 h after oral
administration, and platelet-poor plasma was prepared by centrifuga-
tion for measurement of PT. All data were expressed as relative-fold
values, compared with the baseline value of vehicle-treated mice.
Pharmacokinetic Study. Monomaleate salt of 14i suspended in
0.5% methylcellulose was orally administered to male F344/DuCrlCrlj
rats (aged 6 weeks, Charles River Japan) at a dose of 100 mg/kg under
nonfasted conditions. Three animals were assigned to each sampling
point. Blood samples were collected at 0.5, 1, 2, 4, 8, and 24 h after
administration and centrifuged to obtain the plasma fraction. The
plasma samples were then deproteinized with acetonitrile containing
internal standards for each analyte. After centrifugation, the super-
natant was evaporated to dryness, and the residue was reconstituted in
mobile phase and injected into an LC−MS/MS apparatus to
determine the plasma concentrations of 14i and 16. Dose and plasma
concentrations were expressed as free form.
3-[(4-Methoxybenzoyl)amino]-2-{[4-(4-methyl-1,4-diazepan-
1-yl)benzoyl]amino}phenyl β-D-Glucopyranosiduronic Acid
(16). To a stirred solution of the free form of 14i (5.69 g, 12.0
mmol) in MeOH (50 mL) and CHCl3 (60 mL) at room temperature
were added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 7.18 mL, 48.0
mmol) and methyl 2,3,4-tri-O-acetyl-α-D-glucopyranosyluronate bro-
mide (14.3 g, 36.0 mmol). After the mixture was stirred at room
temperature for 2 h, DBU (7.18 mL, 48.0 mmol) and methyl 2,3,4-tri-
O-acetyl-α-D-glucopyranosyluronate bromide (14.3 g, 36.0 mmol)
were added again, and the mixture was stirred for 3 h. H2O (200 mL)
was added to the reaction mixture and extracted with CHCl3. The
organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. To the solution of the resulting residue in
MeOH (100 mL) at room temperature was added a solution of
Na2CO3 (12.7 g) in H2O (200 mL), and the mixture was stirred for
1 h. Then the mixture was washed with EtOAc (200 mL) 3 times to
remove unreacted starting material 14i, and the aqueous layer was
concentrated under reduced pressure. The residue was directly
chromatographed on ODS gel, eluting with CH3CN/H2O (0/100 to
30/70) gradually, and the solutions containing purified target material
PAMPA. The PAMPA Evolution instrument from pION Inc. was
used in this study. In PAMPA, a “sandwich” is formed from a 96-well
microtiter plate (pION Inc., part no. 110163) and a 96-well filter plate
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(Millipore, IPVH) such that each composite well is divided into two
chambers: donor at the bottom and acceptor at the top, separated by a
125 μm thick microfilter disk (0.45 μm pores) and coated with a 20%
(w/v) dodecane solution of a lecithin mixture (pION Inc., part no.
110669). Drug samples were introduced as 10 mM DMSO stock
solutions in a 96-well polypropylene microtiter plate. The robotic
liquid handling system draws a 5 μL aliquot of the DMSO stock
solution and mixes it into an aqueous buffer solution including 10%
(v/v) of DMSO so that the final typical sample concentration is 50
μM. The drug solutions were filtered using a 96-well filter plate
(Corning, PVDF) and added to the donor compartments. The donor
solutions were adjusted in pH 6.5 (NaOH-treated universal buffer,
pION Inc., part no. 110151), while the acceptor solution had the same
pH 7.4 (pION Inc., part no. 110139). The plate sandwich was formed
and allowed to incubate at 25 °C for 2 h in a humidity-saturated
atmosphere. On completion of the prescribed incubation time, the
sandwich plates were separated and both the donor and acceptor
compartments were assayed for the amount of material present by
comparison with the UV spectrum (270−400 nm) obtained from
reference standards. Mass balance was used to determine the amount
of material remaining in the membrane barrier, and permeability (Pe)
was calculated using PAMPA Evolution software (pION Inc.).
Mouse Liver Microsomal Stability. For estimation of stability
against mouse hepatic CYPs, compounds (0.2 μM) were incubated
with male CD1 mouse liver microsomes (0.2 mg/mL) protein in the
presence of NADPH (1 mM) and EDTA (0.1 mM) in phosphate
buffered saline (100 mM) at 37 °C. Incubations were conducted for 0
and 30 min. Control incubations were conducted by omitting NADPH
from the incubation reaction. The percentage compound remaining
was determined after analysis by LCMS.
Docking of 14i to Factor Xa. For docking of 14i, the coordinates
of factor Xa with a ligand similar to 14i was used (PDB code
1MQ5).26 The docking was done using GOLD with default
parameters. The docking mode with the highest score was employed.
The methoxy group of 14i in the S1 pocket was manually adjusted to
be consistent with the reported X-ray structures of other compounds
with a methoxy group in the S1 pocket (PDB codes 2BQ7,27 2P16,28
2XCO,29 3CS730) . The coordinates of the residues with alternative
charged states or flipped conformation were reassigned using
Protonate3D implemented in MOE. The coordinates were further
minimized using MOE under conditions where the OPLS-AA force
field parameters and generalized Born implicit solvation model were
employed.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-29-863-6712. Fax: +81-29-852-5387. E-mail:
fukushi.hirayama@astellas.com.
ACKNOWLEDGMENTS
■
We thank Dr. Tadashi Hashimoto for pharmacokinetic analysis
and Dr. Katsuhiro Yamano for the useful discussion on MLM
stability and PAMPA. We also express our thanks to Dr. David
Barrett for proofreading the manuscript.
ABBREVIATIONS USED
■
FXa, factor Xa; HTS, high-throughput screening; PT,
prothrombin time; SAR, structure−activity relationship;
PAMPA, parallel artificial membrane permeability assay;
MLM, mouse liver microsome
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