Design, synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part II: Exploration of 6-6 fused rings as alternative S1 moieties

Article abstract of DOI:10.1016/j.bmc.2009.10.024

A series of cis-1,2-diaminocyclohexane derivatives possessing a 6-6 fused ring for the S1 moiety were synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure-activity relationship (SAR), and physicochemical properties are reported herein, together with the discovery of compound 45c, which has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, including a reduced negative food effect.

Full text of DOI:10.1016/j.bmc.2009.10.024

Bioorganic & Medicinal Chemistry 17 (2009) 8221–8233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and SAR of cis-1,2-diaminocyclohexane derivatives
as potent factor Xa inhibitors. Part II: Exploration of 6–6 fused rings
as alternative S1 moieties
b
b
a
b
Kenji Yoshikawa a, , Shozo Kobayashi , Yumi Nakamoto , Noriyasu Haginoya , Satoshi Komoriya ,
Toshiharu Yoshino a, Tsutomu Nagata b, Akiyoshi Mochizuki b, Kengo Watanabe b, Makoto Suzuki a,
Hideyuki Kanno c, Toshiharu Ohta a
*
a R&D Division, Daiichi Sankyo Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
b R&D Division, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Daiichi Sankyo RD Associe Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cis-1,2-diaminocyclohexane derivatives possessing a 6–6 fused ring for the S1 moiety were
synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure–activity relationship (SAR), and
physicochemical properties are reported herein, together with the discovery of compound 45c, which
has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, includ-
ing a reduced negative food effect.
Received 15 August 2009
Revised 10 October 2009
Accepted 13 October 2009
Available online 17 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anticoagulant
Factor Xa inhibitor
Solubility
Food effect
1. Introduction
own formation. It is anticipated that selective inhibition of fXa will
provide an antithrombotic effect by diminishing the amplified for-
Thromboembolic disorders including acute myocardial infarc-
tion, deep vein thrombosis, pulmonary embolism, and ischemic
stroke are the leading cause of morbidity and mortality in devel-
oped countries. Anticoagulants currently in clinical use for the pre-
vention and treatment of these diseases include parentally
administered unfractionated heparin and low molecular weight
heparins (LMWHs), as well as orally administered warfarin (cou-
marins). Warfarin inhibits the biosynthesis of prothrombin, coagu-
lation factor VII, IX, and X. This mechanism leads to a slow onset of
action. Additionally, careful monitoring of the clotting time to
achieve efficacy and dose titration to minimize excessive bleeding
are necessary.1 Thus, a novel anticoagulant which can be adminis-
tered more conveniently and safely is desired.2,3 Factor Xa (fXa) is a
key serine protease located at the convergence of the intrinsic and
extrinsic pathways.4 In the prothrombinase complex consisting of
fXa, factor Va, and Ca2+, fXa catalyzes the conversion of prothrom-
bin to thrombin. Thrombin is responsible for the formation of fibrin
clots, the activation of platelets, and the feedback activation of
other coagulation factors, resulting in the amplification of its
mation of thrombin without compromising normal hemostasis and
platelet activation because the basal thrombin level is maintained.5
Therefore, fXa is a particularly attractive target and extensive clin-
ical trials are now ongoing for a number of clinical candidates.6,7
In the preceding paper, we reported8 the exploration of 5–6
fused ring systems as alternative S1 moieties for compound A, a
potent fXa inhibitor selected as a clinical candidate (Fig. 1),9 to re-
duce the negative food effect. The study indicated that the food ef-
fect could be reduced by improving solubility. We continued the
Compound A
O
N
S4 moiety
S
anti-fXa activity IC50 2.3 nM
PTCT2 in plasma 0.33 µM
Solubility pH 1.2 124 µg/ml
pH 6.8 14 µg/ml
Bioavailability (monkey) 68%
AUC ratio (fed/fasted) = 0.17
Cl
O
N
H
N
N
HN
N
H
O
S1 moiey
* Corresponding author.
E-mail address: yoshikawa.kenji.t6@daiichisankyo.co.jp (K. Yoshikawa).
Figure 1. Properties of compound A.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.024

8222
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
b
method I
method II
S
O
N
O
N
O
O
N
O
N
O
N
O
N
OLi
c
a
4
O
O
N
S
2a-c,f
N
S
Boc
2d,e,g-k
Boc
N
N
H
N
N
N
HN
Ar
H
H
N
N
NH2
NH2
H
HN
3a-c,f
Ar
5a-k
1
6
Cl
HO2C
Cl
Cl
EtO2C
N
Cl
Cl
N
Cl
HO2C
N
N
ArCO2H =
HO2C
HO2C
N
HO2C
HO2C
N
2a
2b
2c
2d
O
2e
2f
O
H
O
O
N
O
Cl
Cl
HO2C
N
Cl
HO2C
O
Cl
EtO2C
S
Cl
N
HO2C
N
H
N
H
N
2g
H
2i
2h
2j
2k
Scheme 1. Reagents and conditions: (a) ArCO2H (2a–c, f), EDCÁHCl, HOBt, DMF; (b) (i) HCl, EtOH, CH2Cl2; (ii) 4, EDCÁHCl, HOBt, DMF; (c) ArCO2H (2d, e, g–k), EDCÁHCl, HOBt,
DMF. In the case of 2d and 2k, corresponding carboxylic acids were prepared by hydrolysis prior to use.
optimization of our clinical candidate, compound A, aiming to
obtain a more potent derivative possessing better PK profiles. In
this paper, we report the results of the exploration of 6–6 fused
ring systems as alternative S1 moieties, as well as the finding of
a novel S4 moiety, which resulted in the discovery of 45c.
14 with methyl acrylate afforded 15, which was employed in a
cyclization reaction to give 2d.14
Reduction of nitro group of 16 afforded 2-amino-5-chlorobenz-
aldehyde (17), which was then cyclized to give 2e (Scheme 3).
Michael addition of morpholine to alkyne 18 provided 19. Sepa-
rately, diazonium salt 21 was prepared by treating aniline 20 with
sodium nitrite. Compounds 19 and 21 were employed in the cycli-
zation affording cinnoline 22,15 which was hydrolyzed to give 2f.
Michael addition of 23 to 24 and successive Horner–Emmons
type cyclization provided 25,16 which was hydrolyzed to afford
carboxylic acid 2g (Scheme 4).
Michael addition of 26 to 27 afforded arylaminoacrylate, which
was cyclized to quinolone 28 in a manner similar to a Conrad–
Limpach reaction17 (Scheme 5). Quinolone 28 was hydrolyzed to
give 2h. Knoevenagel condensation of 29 with diethyl malonate,
followed by reduction of the nitro group and successive cyclization
provided 2-quinolone 31, which was hydrolyzed to give 2i. Reac-
tion of amide 32 with ethyl chlorooxoacetate (33) gave 34, which
was hydrolyzed to afford 2j. 4-Chloroaniline (35) was treated with
chlorosulfonylisocyanate, and subsequent intramolecular cycliza-
tion gave benzothiadiazin-3(4H)-one 1,1-dioxide 36. Ring opening
of 36 in 50% sulfuric acid, followed by condensation with 33, affor-
2. Chemistry
Compounds 5a–k were synthesized as shown in Scheme 1. In
method I, starting material amine 18 was reacted with carboxylic
acid 2 to give 3. Removal of the Boc group on amide 3, followed
by condensation with lithium 5-methyl-4,5,6,7-tetrahydrothiazol-
o[5,4-c]pyridine-2-carboxylate (4)10 afforded target compound 5.
In method II, condensation of amine 68 with S1 fragment 2 led to
target compound 5. In the case of 2d and 2k, corresponding car-
boxylic acids were prepared by hydrolysis prior to use.
Reissert reaction11 of 7 gave nitrile 8, which was hydrolyzed to
afford 2b (Scheme 2). Phenethylamide 10 was prepared by the
treatment of 9 with formic acid. Bischler–Napieralski reaction12
of 10 directly afforded isoquinoline 11, which was hydrolyzed to
give carboxylic acid 2c. Imine 14 was synthesized by the reaction
of aldehyde 12 and sulfonamide 13. Baylis–Hillman reaction13 of
Cl
b
a
Cl
Cl
N
NC
N
HO2C
N
7
8
2b
Cl
OHC
c
Cl
e
d
NH2
Cl
Cl
NH
N
N
MeO2C
MeO2C
MeO2C
HO2C
9
10
11
2c
Cl
Cl
Cl
Cl
H
Cl
+
Cl
O
S NH2
O
f
g
h
Cl
N
H
MeO2C
N
MeO2C
O
12
13
NH
Ts
14
Ts
2d
15
Scheme 2. Reagents and conditions: (a) (i) m-CPBA, CH2Cl2; (ii) TMSCN, ClCONMe2, CH2Cl2; (b) concd HClaq; (c) HCO2H, EDCÁHCl, HOBt, NMM, CH2Cl2; (d) (i) (COCl)2, CH2Cl2;
(ii) FeCl3; (iii) concd H2SO4, MeOH; (e) concd HCl aq; (f) toluene, MS 4 Å, reflux; (g) methyl acrylate, DABCO, THF; (h) 13, K2CO3, DMF.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8223
OHC
O2N
Cl
OHC
H2N
Cl
O
a
b
Cl
N
HO2C
N
2e
16
17
O
c
N
OEt
OEt
18
O
19
N
Cl
e
N
f
N
Cl
N
EtO2C
H2N
Cl
HO2C
d
N2+
22
Cl
2f
BF4-
20
21
Scheme 3. Reagents and conditions: (a) NaHSO3, Na2CO3, H2O, MeOH; (b) AcONH4, glyoxylic acid, EtOH; (c) morpholine, CH2Cl2; (d) (i) NaNO2, HCl, H2O; (ii) NaBF4; (e)
CH3CN, reflux; (f) NaOH, H2O, THF.
44. The condensation of 3a or 3c with 44 afforded 45a or 45c,
O
O
P
O
respectively.
Cl
HO
a
Hydrolysis of 469 and successive amidation with dimethyl-
amine gave 47, which was then treated with HCl and condensed
with dihydropyrrolothiazole 44, to afford 48 (Scheme 7).
EtO
O
Cl
O
+
O
EtO2C
24
Cl
23
25
O
b
3. Results and discussion
HO2C
Anti-fXa activity (IC50), anticoagulant activity (PTCT2), solubil-
ity, and distribution coefficient (log D) of the synthesized com-
pounds are shown in Table 1. Several potent compounds were
orally administered to rats at the dose of 10 mg/kg and the anti-
fXa activities in plasma were measured at 0.5, 1, 2, and 4 h later
after administration. The maximum anti-fXa activity among them
is shown in Table 1.
2g
Scheme 4. Reagents and conditions: (a) NaH, THF; (b) LiOH, THF, EtOH, H2O.
Naphthalene variant 5a exhibited strong anti-fXa activity and
improved solubility. The introduction of nitrogen at the 1-position
(5b) of the naphthalene moiety caused a drastic decrease in activ-
ity, while introduction at the 3- or 4-position (5c or 5d) sustained
strong activity. In the case of diazanaphthalene derivatives, 3,4-
diazanaphthalene (cinnoline) 5f displayed almost the same
ded 38. Compound 2k was finally obtained by cyclization of 38
with NaOMe.
Substitution of dibromide 4018 with sulfonamide 39 gave dihy-
dropyrrolothiazole 41 (Scheme 6). Removal of the sulfonyl group,
followed by reductive methylation, gave 43, which was treated
with tert-BuLi and then successively with carbon dioxide to give
O
O
O
MeO
O
Cl
a
b
Cl
Cl
+
OMe
H2N
MeO2C
Cl
N
HO2C
N
27
26
H
H
2h
28
O2N
H
H
O2N
Cl
c
d
e
Cl
Cl
O
N
O
N
OHC
EtO2C CO2Et
HO2C
EtO2C
29
31
2i
30
O
O
O
O
f
g
Cl
Cl
OEt
Cl
N
N
H2N
H2N
Cl
+
O
EtO2C
N
H
34
HO2C
Cl
N
H
33
32
2j
O
H2N S
O
O
O
O
O
O
Cl
O
h
O
Cl
i
S
j
S
k
Cl
HN
Cl
S
H2N
N
H2N
EtO
O
N
H2N
N
MeO2C
N
H
35
H
H
O
37
36
38
2k
Scheme 5. Reagents and conditions: (a) (i) MeOH, reflux; (ii) Ph2O, 240 °C; (b) NaOH, dioxane, H2O; (c) diethyl malonate, NaHCO3, Ac2O; (d) Fe, AcOH; (e) NaOH, H2O, THF,
EtOH; (f) (i) Py; (ii) Ac2O, AcOH; (g) LiOH, THF, H2O; (h) (i) ClSO2NCO, EtNO2; (ii) AlCl3; (i) 50% H2SO4aq; (j) 33, Et3N, AcOH; (k) MeONa, MeOH.

8224
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
magnitude of activity as compound A, while 1,3-diazanaphtalene
(quinazoline) 5e was found to be much less potent. Compound 5f
also exhibited significant improvement of solubility in the neutral
pH region. The anti-fXa activity of chromene 5g decreased. 6–6
Fused ring derivatives possessing an NH moiety, which was ex-
pected to make a hydrogen bond between Gly218 of fXa like in
compound A,9 were then examined. Quinolone 5h, 1H-quinazo-
lin-4-one 5j, and benzo[1,2,4]thiadiazine 5k showed very strong
anti-fXa activity. However, these compounds did not exhibit strong
anti-fXa activity in a rat ex vivo assay except for 5j. The results of
the metabolic stability and permeability measurements are shown
in Table 2. Unfortunately, it was revealed that the introduction of
an NH moiety to increase the anti-fXa activity also resulted in a de-
crease in permeability (5h, 5i, and 5j).
Prospective compounds, 5a, 5c, 5f, 5h, and 5j, which showed
strong rat ex vivo activity and/or in vitro activity were adminis-
tered to monkeys to obtain the PK profiles (Table 3). Compounds
5a and 5c exhibited relatively large AUC, compared to more polar
compounds like 5f, 5h and 5j. The negative food effects of 5a, 5c,
and 5f, which showed improved solubility, were smaller than that
of compound A. Since 5a and 5c showed better PK profiles than
the other compounds, further modification was conducted.
Assuming that a smaller S4 moiety is preferable to further in-
crease solubility, a dihydropyrrolo[3,4-d]thiazole (DHPT) struc-
ture was incorporated to the scaffolds of 5a, 5c, and compound
A. The assay results of these compounds are summarized in
Table 4.
The introduction of DHPT improved solubility at pH 6.8 in all
the compounds. Although the in vitro anti-fXa activity was slightly
decreased, the anticoagulant activity of 45c was comparable to
compound A. In addition, 45c showed the highest rat ex vivo activ-
ity among those compounds. Considering its anti-fXa activity, com-
pound 45c exhibited relatively strong anticoagulant activity
compared with 45a and 48. It is probably due to the lower lipophil-
icity of compound 45c. As previously described,8,9 it is assumed
that higher lipophilicity causes higher non-specific protein binding
and larger decrease of anticoagulant activity in plasma. The PTCT2/
IC50 ratio and log D value and protein binding were well correlated
among these compounds (Table 5).
The food effect of 45c in monkeys and dogs were then examined
(Table 6). Compound 45c exhibited a substantially smaller food ef-
fect than compound A in both monkeys and dogs. Moreover, the
AUC of 45c was significantly larger in dogs compared to compound
A.
Figure 2. X-ray crystal structure of 45c in fXa. (A) Surface of fXa S4 site and 45c
(CPK model) are illustrated. The S4 site consisted of Try99, Phe174, and Trp215. (B)
The binding mode utilized in the S1 site is shown. There are water-mediated
hydrogen bonds (blue line), an aromatic CHÁ Á ÁO hydrogen bond (pink line), and Cl-
aromatic contact (green line). Ser195, Gly218, and Tyr228 are depicted in the ball
and stick model.
The X-ray crystal structure of 45c in fXa was obtained at a res-
olution of 1.8 Å. Figure 2A and B show the binding mode of 45c in
intramolecular
hydrogen bond
(N-N 2.74A)
intramolecular
hydrogen bond
(N-N 2.74A)
N
O
Ser195
H2O
water-mediated
hydrogen bond
(N-O 2.51A, O-O 2.76A)
H
N
N
N
H
N
H
S
N
O
O
hydrophobic
interaction
in S4 site
Cl-aromatic interaction
(Cl-centeroid of benzene 3.60A)
O
Cl
S-O
Gly218
interaction
(S-O 3.10A)
Tyr228
HO
CH
O
hydrogen bond
(C-O 3.29A)
Figure 3. Major interactions between 45c and fXa and intramolecular interactions.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8225
O
S
O
2HBr
S
N
a
Br
Br
O
S N
O
b
c
S
N
S
N
NH2
N
O
S
N
d
S
+
N
HN
N
N
O Li
39
40
41
42
43
44
O
O
N
Boc
e
O
Cl
N
X
Cl
S
H
HN
N
X
H
N
HN
O
N
O
3a (X = C)
3c (X = N)
45a (X = C)
45c (X = N)
Scheme 6. Reagents and conditions: (a) NaH, DMF; (b) 47% HBr aq, PhOH; (c) HCHO, Et3N, AcOH, NaBH(OAc)3, CH2Cl2; (d) (i) tert-BuLi, THF; (ii) CO2 gas; (e) (i) HCl, EtOH; (ii)
44, EDCÁHCl, HOBt, DMF.
4. Conclusions
O
OEt
O
O
N
O
a
Exploration of 6–6 fused rings as alternative S1 moieties and the
modification of S4 moiety were conducted. An S1 moiety having
nitrogen at the 3- or 4-position was acceptable, but was unaccept-
able at the 1-position. On the contrary, an NH moiety at the 1-po-
sition contributed to strong anti-fXa activity. However, these
compounds did not exhibit good PK profiles, probably due to insuf-
ficient permeability. In the course of further optimization, we have
identified the novel S4 moiety dihydropyrrolo[3,4-d]thiazole,
which led to the discovery of compound 45c. Compound 45c
possesses strong anticoagulant activity, good physicochemical
properties, excellent PK profiles, and a reduced food effect in both
monkeys and dogs compared with compound A. The X-ray crystal
structure of 45c in fXa revealed its binding mode to the enzyme
and also indicated the existence of intramolecular interactions.
The findings described in this and the previous reports are valuable
for further optimization, which will be reported in due course.
Cl
Boc
Cl
Boc
N
N
H
HN
46
H
HN
47
N
N
H
H
O
N
b
O
Cl
S
N
H
N
HN
N
N
48
H
O
Scheme 7. Reagents and conditions: (a) (i) NaOH, THF, EtOH, H2O; (ii) MeNH2ÁHCl,
EDCÁHCl, HOBt, Et3N, DMF; (b) (i) HCl, EtOH; (ii) 44, EDCÁHCl, HOBt, DMF.
5. Experimental section
5.1. Chemistry
the S4 and S1 sites, respectively. The major interactions are sum-
marized in Figure 3.
(i) Interactions of 45c with fXa (Fig. 2A and B): The DHPT moiety
closely fits into the S4 site, the 3-azanaphthalene moiety fits into
the S1 site, and cyclohexane moiety acts as a linker to connect
these two motifs (Fig. 2A).19 This binding mode is similar to those
observed previously in our laboratory.9,20 On the other hand, sev-
eral specific binding modes were observed for compound 45c with
fXa. There is one water-mediated hydrogen bond between the
nitrogen of 3-azanaphthalene and the hydroxyl group of Ser195
(Fig. 2B).21 As well, the aromatic CH moiety in azanaphthalene
seems to make a CHÁ Á ÁO hydrogen bond22 with the oxygen of
Gly218 (Fig. 2B). It should be noted that the nitrogen of 3-azanaph-
thalene and the adjacent carbonyl oxygen are located in the oppo-
site direction. This conformation is important to fit the chlorine
atom into the cavity of the S1 site. Similar to other inhibi-
tors,21,23–25 the chlorine makes contact with the benzene ring of
Tyr228 which is located at the bottom of the S1 site. (Fig. 2B).
(ii) Intramolecular interactions of 45c (Fig. 3): The distance be-
tween nitrogen of azanaphthalene and adjacent amide nitrogen is
2.74 Å, indicating that there is an intramolecular hydrogen bond.26
The distance between the sulfur of thiazole and the oxygen of the
adjacent carbonyl group is 3.10 Å, which is slightly shorter than the
sum of the van der Waals radius (3.25 Å). This result indicates the
existence of an S–O interaction.20,27,28 We assume that this interac-
tion contributes to constrain the rotation of the S4 moiety.29 As
well, an intramolecular hydrogen bond between thiazole nitrogen
and the adjacent amide hydrogen seems to work cooperatively.30
5.1.1. General
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. Melting points
were determined on a YANACO MP-J3 or a BUCHI B-545 and are
uncorrected. 1H NMR spectra were recorded on a JEOL JNM-EX-
400 spectrometer, and chemical shifts are given in ppm (d) from
tetramethylsilane as the internal standard. FAB mass spectra were
recorded on a JEOL JMS-HX110 spectrometer. ESI mass spectra
were recorded on a SCIEX API-150EX spectrometer. IR spectra were
recorded on a HITACHI 270-30 or HORIBA FT-720 spectrometer.
Column chromatography was performed with Merck Silica Gel 60
(particle size 0.060–0.200 or 0.040–0.063). Thin-layer chromatog-
raphy (TLC) was performed on Merck pre-coated TLC glass sheets
with Silica Gel 60 F254
.
5.1.2. tert-Butyl {(1R,2S,5S)-2-[(6-chloro-2-naphthoyl)amino]-
5-[(dimethylamino)carbonyl]cyclohexyl}carbamate (3a)
To a solution of tert-butyl (1R,2S,5S)-2-amino-5-[(dimethyl-
amino)carbonyl]cyclohexylcarbamate (1)8 (0.17 g, 0.60 mmol) in
DMF (5 mL) were added 6-chloronaphtalene-2-carboxylic acid
(2a) (0.14 g, 0.68 mmol), 1-hydroxybenzotriazole (HOBt) (91 mg,
0.59 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCÁHCl) (0.13 g, 0.68 mmol). After stirring for
22 h, the mixture was concentrated in vacuo. The residue was par-
titioned between AcOEt and water. The organic layer was dried

8226
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
Table 1
Assay results of 6–6 fused ring derivatives
O
N
O
S
N
H
N
HN
N
S1
Compd
S1
fXa IC50 (nM)
PTCT2 (
human plasma
l
M)a in
Solubility (
l
g/mL)
log Db (pH 6.8)
Ex vivo anti-fXa
activityc (%)
pH 1.2
124
pH 6.8
Cl
A
2.3
0.33
14
61
2.8
62
81
N
H
O
4
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3
5a
5.7
0.55
990
2.9
2
1
O
O
O
O
O
O
O
5b
5c
5d
5e
5f
4000
3
NTd
0.28
0.44
>20
0.24
1.5
>1000
>1000
>1000
NT
43
2.6
2.3
2
NT
95
38
NT
64
43
N
N
N
41
4.1
870
3.3
35
110
NT
N
N
NT
1.7
2.7
N
N
>1000
>1000
730
270
O
O
5g
Cl
Cl
5h
5i
0.43
12
0.34
0.56
0.63
0.89
>1000
>1000
970
73
1.8
2.1
1.6
0.8
17
4.2
63
1
N
H
O
O
H
N
23
O
O
Cl
Cl
N
5j
1.4
4.9
>1000
>1000
N
H
O
O
O O
S
N
5k
>1000
N
H
a
b
c
Anticoagulant activities were evaluated with the human plasma clotting time doubling concentration for prothrombin time (PTCT2).
n-Octanol to the Japanese Pharmacopoeia Second Fluid (pH 6.8) distribution coefficient.
The maximum anti-fXa activity in plasma after oral administration.
d
Not tested.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8227
Table 2
Table 5
Metabolic stability and permeability
The PTCT2/IC50 ratio, log D value, and protein binding
Compd
Metabolic stabilitya (nmol/min/mg)
Permeability AT ratiob
Compd
PTCT2/IC50 ratio
log D
Protein binding (%)
A
0.032
0.029
0.022
0.026
0.059
0.007
0.017
0.004
>30
>30
>30
>30
>30
4.3
45a
45c
48
77
46
100
2.7
2.2
2.6
70
48
68
5a
5c
5f
5g
5h
5i
5.2
15.4
5.1.3. N-{(1R,2S,5S)-2-[(6-Chloro-2-naphthoyl)amino]-5-[(dimeth-
ylamino)carbonyl]cyclohexyl}-5-methyl-4,5,6,7-tetrahydrothiazolo-
[5,4-c]pyridine-2-carboxamide hydrochloride (5a)
5j
a
Metabolic stabilities were evaluated by the initial velocity of disappearance
(Vini) in rat liver microsomes.
Permeabilities were measured using Caco-2 monolayers. Relative permeabili-
ties are shown as a ratio compared to atenolol.
To a solution of 3a (0.27 g, 0.57 mmol) in CH2Cl2 (10 mL) was
added saturated HCl ethanolic solution (10 mL) and the mixture
was stirred for 1.5 h. The solvent was evaporated and to the res-
idue was added Et2O. The resultant precipitate was collected by
filtration. This solid was dissolved in DMF (7 mL) and lithium
5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylate
(4)10 (0.11 g, 0.54 mmol), HOBt (70 mg, 0.46 mmol), and EDCÁHCl
(0.10 g, 0.52 mmol) were added. After stirring for 23 h, the mix-
ture was concentrated in vacuo. The residue was partitioned be-
tween AcOEt and water. The organic layer was dried over Na2SO4
and concentrated in vacuo. The residue was chromatographed
(MeOH/CH2Cl2 = 1/9). The obtained compound was dissolved in
MeOH and 1 N HCl ethanolic solution (0.30 mL, 0.30 mmol) was
added. The solvent was evaporated and to the residue was added
AcOEt. The resultant precipitate was collected by filtration to give
the title compound (0.13 g, 0.21 mmol, 37%) as a pale yellow
amorphous solid. 1H NMR (DMSO-d6) d: 1.45–1.60 (1H, m),
1.70–1.90 (3H, m), 1.90–2.10 (2H, m), 2.81 (3H, s), 2.91 (3H, s),
3.00 (3H, s), 3.00–3.40 (5H, m), 3.25–3.45 (1H, br), 4.10–4.20
(1H, m), 4.40–4.70 (2H, m), 7.59 (1H, dd, J = 8.8, 2.2 Hz), 7.87
(1H, d, J = 8.5 Hz), 7.96 (1H, d, J = 8.5 Hz), 8.02 (1H, d, J = 8.8
Hz), 8.10 (1H, d, J = 2.2 Hz), 8.33 (1H, s), 8.43 (1H, d, J = 8.1 Hz),
8.52 (1H, d, J = 7.3 Hz). MS (FAB) m/z: 554 (M+H)+. Anal. Calcd
for C28H32ClN5O3SÁHClÁ5/4H2O: C, 54.86; H, 5.84; Cl, 11.57; N,
11.42; S, 5.23. Found: C, 54.81; H, 5.90; Cl, 11.28; N, 11.22;
S, 5.19.
b
Table 3
PK profiles in monkeys (1 mg/kg, po) (n = 3)
Compd
AUCfasted (ng h/mL)
AUCfed (ng h/mL)
Ratio fed/fasted
A
985
520
517
182a
0
172
346a
333a
97a
0.17
0.67
0.64
0.53
—
5a
5c
5f
5h
5j
NTb
NT
271
—
a
Cassette dosing: 4 or 5 compounds were administered at the same time. The
dose was 1 mg/kg for each compound.
b
Not tested.
over Na2SO4 and concentrated in vacuo. The residue was chro-
matographed (MeOH/CH2Cl2 = 1/19) to give the title compound
(0.27 g, 0.57 mmol, 95%) as a pale yellow amorphous solid. 1H
NMR (CDCl3) d: 1.30–1.59 (10H, m), 1.71–1.94 (2H, m), 2.17 (1H,
br s), 2.35 (1H, br s), 2.66 (1H, br s), 2.96 (3H, s), 3.09 (3H, s),
4.00–4.20 (1H, m), 4.20–4.30 (1H, m), 4.75–4.95 (1H, m), 7.44
(1H, d, J = 9.0 Hz), 7.70–7.95 (5H, m), 8.31 (1H, s). MS (FAB) m/z:
474 (M+H)+.
Table 4
Assay results of 45a, 45c, and 48
N
O
S4
N
H
N
S1
H
Compd
45a
S4
S1
fXa IC50 (nM)
PTCT2 (
lM) in human plasma
Solubility (
l
g/mL)
log D
2.7
Ex vivo anti-fXa activity (%)
pH 1.2
pH 6.8
O
O
O
Cl
Cl
S
S
S
12
0.92
0.44
>1000
>1000
100
71
90
N
N
N
O
N
N
45c
9.5
120
220
2.2
O
O
N
N
Cl
48
3.4
0.34
270
2.6
68
N
H

8228
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
Table 6
Food effect on AUC in monkeys and dogs
Compd
Monkey (po 1 mg/kg)
Dog (po 10 mg/head)
AUCfed (ng h/mL)
AUCfasted (ng h/mL)
AUCfed (ng h/mL)
Ratio fed/fasted
AUCfasted (ng h/mL)
Ratio fed/fasted
A
45c
988
723
172
322
0.17
0.45
301
1823
40
778
0.13
0.43
5.1.4. 7-Chloro-N-{(1S,2R,4S)-4-[(dimethylamino)carbonyl]-2-
{[(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)car-
bonyl]amino}cyclohexyl}quinoline-3-carboxamide
matographed (CH2Cl2) to give another portion of the title com-
pound (0.800 g, 4.24 mmol, 28%) as pale yellow crystals. 1H NMR
(DMSO-d6) d: 7.94 (1H, dd, J = 9.0, 2.2 Hz), 8.09 (1H, d, J = 8.5 Hz),
8.15 (1H, d, J = 9.0 Hz), 8.29 (1H, d, J = 2.2 Hz), 8.63 (1H, d,
J = 8.5 Hz). MS (FAB) m/z: 189 (M+H)+.
hydrochloride (5d)
(i) Hydrolysis step: Compound 2d (443 mg, 2.00 mmol) was dis-
solved in THF (12 mL) and H2O (1.5 mL), to the solution was added
LiOH (72 mg, 3.0 mmol) and the mixture was stirred for 17 h. The
solvent was evaporated to give lithium 7-chloro-quinoline-3-
carboxylate.
5.1.6. 6-Chloroquinoline-2-carboxylic acid (2b)
Compound 8 (1.73 g, 9.17 mmol) was dissolved in concd HCl
aqueous solution (40 mL), and the solution was refluxed for 19 h.
After the mixture was cooled to room temperature, the resultant
precipitate was collected by filtration and washed with water to
give the title compound (1.81 g, 8.72 mmol, 95%) as a colorless so-
lid. 1H NMR (DMSO-d6) d: 7.87 (1H, dd, J = 9.0, 2.4 Hz), 8.10–8.20
(2H, m), 8.24 (1H, d, J = 2.2 Hz), 8.52 (1H, d, J = 8.5 Hz). MS (FAB)
m/z: 208 (M+H)+.
(ii) Condensation step: The residue was dissolved in DMF
(15 mL) and to the solution were added N-{(1R,2S,5S)-2-amino-
5-[(dimethylamino)carbonyl]cyclohexyl}-5-methyl-4,5,6,7-tetra-
hydrothiazolo[5,4-c]pyridine-2-carboxamide (6)8 (320 mg, 0.876
mmol) in DMF (15 mL), HOBt (118 mg, 0.876 mmol), and EDCÁHCl
(252 mg, 1.31 mmol). After stirring for 2 days, the mixture was
concentrated in vacuo. The residue was partitioned between
CH2Cl2 and water. The organic layer was washed successively
with saturated NaHCO3 aqueous solution and brine, and dried
over Na2SO4. The solvent was evaporated and the residue was
chromatographed (MeOH/CH2Cl2 = 1/19). The obtained solid was
dissolved in EtOH (2 mL) and 1 N HCl ethanolic solution
(0.8 mL) was added. After stirring for 30 min, the solvent was
evaporated and to the residue was added Et2O. The resultant pre-
cipitate was collected by filtration to give the title compound
(131 mg, 0.200 mmol, 36%) as a pale orange solid. 1H NMR
(DMSO-d6) d: 1.45–1.60 (1H, m), 1.65–2.05 (5H, m), 2.81 (3H, s),
2.91, 2.92 (total 3H, each s), 3.02, 3.04 (total 3H, each s), 3.08–
3.38 (2H, m), 3.41–3.52 (1H, m), 3.67–3.74 (1H, m), 4.10–4.13
(2H, m), 4.37–4.47 (1H, m), 4.65–4.74 (2H, m), 7.74 (1H, dd,
J = 8.8, 2.0 Hz), 8.12–8.16 (2H, m), 8.46–8.51 (1H, m), 8.70–8.79
(2H, m), 9.18 (1H, dd, J = 7.8, 2.0 Hz), 11.52, 11.73 (total 1H, each
br s). IR (ATR) cmÀ1: 3396, 3251, 3058, 2931, 2865, 2700–2300,
1635, 1542, 1517, 1450, 1363, 1309, 1253. MS (ESI) m/z: 555
(M+H)+. Anal. Calcd for C27H31ClN6O3SÁ1.8HClÁ2H2O: C, 49.38; H,
5.65; Cl, 15.11; N, 12.80; S, 4.88. Found: C, 49.56; H, 5.71; Cl,
15.08; N, 12.67; S, 5.07.
5.1.7. rac-Methyl 4-chloro-N-formylphenylalaninate (10)
rac-Methyl 4-chlorophenylalaninate hydrochloride (9) (2.00 g,
8.00 mmol) was suspended in CH2Cl2 (20 mL), and to the suspen-
sion were added EDCÁHCl (1.60 g, 8.35 mmol), HOBt (1.23 g,
8.03 mmol), N-methylmorpholine (1.90 mL, 17.3 mmol), and for-
mic acid (0.30 mL, 8.0 mmol), followed by stirring for 15 min. Addi-
tion of formic acid (0.30 mL) and stirring for 15 min were repeated
three times. The mixture was diluted with CH2Cl2. The organic
layer was washed with water and dried over Na2SO4. The solvent
was evaporated and the residue was chromatographed (MeOH/
CH2Cl2 = 1/40) to give the title compound (1.21 g, 5.01 mmol,
63%) as a yellow oil. 1H NMR (CDCl3) d: 3.10 (1H, dd, J = 13.9,
5.6 Hz), 3.18 (1H, dd, J = 13.9, 5.9 Hz), 3.75 (3H, s), 4.95–5.00
(1H, m), 6.07 (1H, br s), 7.05 (2H, d, J = 8.3 Hz), 7.27 (2H, d,
J = 8.3 Hz), 8.18 (1H, s). MS (FAB) m/z: 242 (M+H)+.
5.1.8. Methyl 7-chloroisoquinoline-3-carboxylate (11)
Compound 10 (1.45 g, 6.00 mmol) was dissolved in CH2Cl2
(40 mL), and oxalyl chloride (0.570 mL, 6.65 mmol) was added
dropwise, followed by stirring at room temperature for 30 min.
The mixture was cooled to À10 °C and ferric chloride (1.17 g,
7.21 mmol) was added. The mixture was stirred at room tempera-
ture for 4 days. To the mixture was added 1 N HCl aqueous solu-
tion, and the mixture was diluted with CH2Cl2. The organic layer
was separated and dried over Na2SO4. The solvent was evaporated
and the residue was dissolved in MeOH (38 mL). Concd sulfuric
acid (2 mL) was added to the solution and the mixture was re-
fluxed for 20 h. Saturated NaHCO3 aqueous solution was added
to the mixture and the mixture was extracted with CH2Cl2. The or-
ganic layer was dried over Na2SO4. The solvent was evaporated and
the residue was chromatographed (EtOAc) to give the title com-
pound (0.250 g, 1.13 mmol, 19%) as a colorless solid. 1H NMR
(CDCl3) d: 4.07 (3H, s), 7.74 (1H, dd, J = 8.8, 2.0 Hz), 7.94 (1H, d,
J = 8.8 Hz), 8.06 (1H, d, J = 2.0 Hz), 8.59 (1H, s), 9.28 (1H, s). Anal.
Calcd for C11H8ClNO2: C, 59.61; H, 3.64; Cl, 16.00; N, 6.32. Found;
C, 59.40; H, 3.61; Cl, 16.22; N, 6.23.
5.1.5. 6-Chloroquinoline-2-carbonitrile (8)
6-Chloroquinoline (7) (2.50 g, 15.3 mmol) was dissolved in
CH2Cl2 (25 mL), and m-chloroperbenzoic acid (m-CPBA) (70%,
3.71 g, 15.0 mmol) was added to the solution under ice cooling, fol-
lowed by stirring at room temperature for 1 h. The mixture was di-
luted with CH2Cl2, washed successively with sodium thiosulfate
aqueous solution and NaOH aqueous solution, and dried over
Na2SO4. The solvent was evaporated, and the residue was dissolved
in CH2Cl2 (40 mL). To the solution were added trimethylsilyl cya-
nide (2.00 mL, 15.9 mmol) and N,N-dimethylcarbamoyl chloride
(1.50 mL, 16.3 mmol). The mixture was refluxed for 9 h. To the
mixture were added additional trimethylsilyl cyanide (1.00 mL,
7.95 mmol) and N,N-dimethylcarbamoyl chloride (0.800 mL,
8.69 mmol), and the mixture was refluxed for an additional 16 h.
The mixture was diluted with CH2Cl2, and 10% K2CO3 aqueous solu-
tion (40 mL) was added, followed by stirring for 30 min. The organ-
ic layer was separated and dried over Na2SO4. The solvent was
evaporated, and the residue was triturated with CH2Cl2 and col-
lected by filtration to give the title compound (1.77 g, 9.38 mmol,
61%) as colorless crystals. The filtrate was concentrated, and chro-
5.1.9. 7-Chloroisoquinoline-3-carboxylic acid hydrochloride
(2c)
Compound 11 (0.230 g, 1.04 mmol) was dissolved in concd HCl
aqueous solution (10 mL) and the solution was refluxed for 18 h.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8229
After the mixture was cooled to room temperature, the resultant
precipitate was collected by filtration and washed with water to
give the title compound (0.21 g, 0.86 mmol, 83%) as a colorless
solid. 1H NMR (DMSO-d6) d: 7.90–8.00 (1H, m), 8.29 (1H, d,
J = 8.5 Hz), 8.44 (1H, s), 8.72 (1H, s), 9.45 (1H, d, J = 6.6 Hz). MS
(FAB) m/z: 208 (M+H)+.
(4.0 mL) and the mixture was stirred for 15.5 h. After cooling,
water was added and the resultant precipitate was collected by fil-
tration. The precipitate was washed with EtOH to give the title
compound (74 mg, 0.35 mmol, 8.7%) as a yellow amorphous solid.
1H NMR (DMSO-d6) d: 8.00–8.10 (2H, m), 8.32 (1H, s), 9.59 (1H, d,
J = 2.4 Hz). MS (EI) m/z: 208 (M+H)+.
5.1.10. N-[(E)-2,4-Dichlorobenzylidene]-4-methylbenzene-
sulfonamide (14)
5.1.14. Ethyl (E)-3-(morpholin-4-yl)-2-acrylate (19)
Morpholine (1.70 mL, 19.5 mmol) was added dropwise to a
solution of ethyl propiolate (18) (2.00 mL, 19.6 mmol) in CH2Cl2
under ice cooling. After stirring at room temperature for 1 h, the
mixture was concentrated in vacuo and the residue was chromato-
graphed (MeOH/CH2Cl2 = 1/20) to give the title compound (3.72 g)
as a yellow oil. 1H NMR (CDCl3) d: 1.26 (3H, t, J = 7.1 Hz), 3.21 (4H,
t, J = 5.1 Hz), 3.71 (4H, t, J = 5.1 Hz), 4.14 (2H, q, J = 7.1 Hz), 4.70
(1H, d, J = 13.4 Hz), 7.36 (1H, d, J = 13.4 Hz). MS (FAB) m/z: 186
(M+H)+.
To
a solution of 2,4-dichlorobenzaldehyde (12) (3.50 g,
20.0 mmol) in toluene (80 mL) were added 4-methylbenzenesulf-
onamide (13) (3.42 g, 20.0 mmol) and molecular sieves (4 Å, pow-
der, 300 mg) and the mixture was refluxed for 2 h. After the solvent
was evaporated, Et2O was added to the residue. The resultant pre-
cipitate was collected by filtration and washed with Et2O to give
the title compound (2.56 g, 7.80 mmol, 39%) as a colorless solid.
The filtrate was evaporated and the same procedure gave another
portion of the title compound (3.60 g, 11.0 mmol, 55%). 1H NMR
(CDCl3) d: 2.45 (3H, s), 7.32 (1H, dd, J = 8.6, 2.0 Hz), 7.36 (2H, d,
J = 8.6 Hz), 7.49 (1H, d, J = 2.0 Hz), 7.89 (2H, d, J = 8.6 Hz), 8.09
(1H, d, J = 8.6 Hz), 9.42 (1H, s).
5.1.15. Ethyl 7-chlorocinnoline-3-carboxylate (22)
3-Chloroaniline (20) (2.00 g, 15.7 mmol) was dissolved in a
mixed solvent of water (30 mL) and concd HCl aqueous solution
(3.5 mL), and sodium nitrite (1.30 g, 18.8 mmol) was added under
ice cooling. After stirring for 10 min, concd HCl aqueous solution
(5.30 mL) and sodium tetrafluoroborate (6.90 g, 62.8 mmol) were
added. The mixture was stirred for 30 min under ice cooling. The
resultant precipitate was collected by filtration and washed suc-
cessively with water, MeOH, and Et2O to give 3-chloro-
benzenediazonium tetrafluoroborate (21) (2.63 g, 11.6 mmol,
74%) as a colorless solid. This compound was used for the next
reaction without further purification.
Compound 19 (1.45 g, 7.83 mmol) was dissolved in acetonitrile
(100 mL), and to the solution was added compound 21 (1.73 g,
7.64 mmol). The mixture was stirred at room temperature for 1 h
then refluxed for 7 days. The solvent was evaporated and the resi-
due was chromatographed twice (EtOAc/CH2Cl2 = 1/10, then
EtOAc/hexane = 1/1) to give the title compound (0.250 g,
1.06 mmol, 14%) as a yellow solid. 1H NMR (CDCl3) d: 1.53 (3H, t,
J = 7.1 Hz), 4.62 (2H, q, J = 7.1 Hz), 7.80 (1H, dd, J = 8.8, 2.0 Hz),
7.95 (1H, d, J = 8.8 Hz), 8.64 (1H, s), 8.68 (1H, d, J = 2.0 Hz).
5.1.11. Methyl 2-{(2,4-dichlorophenyl){[(4-methylphenyl)sul-
fonyl]amino}methyl}acrylate (15)
To a solution of 14 (2.55 g, 7.77 mmol) in THF (50 mL) were
added 1,4-diazabicyclo[2.2.2]octane (DABCO) (87.5 mg, 0.780
mmol) and methyl acrylate (2.8 mL, 31 mmol) and the mixture
was refluxed for 16 h. The solvent was evaporated and the residue
was dissolved in CH2Cl2. The organic layer was washed succes-
sively with 1 N HCl aqueous solution, water, and brine and dried
over Na2SO4. The solvent was evaporated and the residue was
chromatographed (EtOAc/hexane = 1/2) to give the title compound
(2.03 g, 4.90 mmol, 63%) as a colorless solid. 1H NMR (CDCl3) d:
2.39 (3H, s), 3.63 (3H, s), 5.66 (1H, d, J = 8.0 Hz), 5.85 (1H, d,
J = 8.0 Hz), 5.87 (1H, s), 6.28 (1H, s), 7.06 (1H, dd, J = 8.2, 1.9 Hz),
7.17 (2H, d, J = 8.2 Hz), 7.23–7.30 (2H, m), 7.61 (2H, d, J = 8.2 Hz).
MS (FAB) m/z: 414 (M+H)+.
5.1.12. Methyl 7-chloroquinoline-3-carboxylate (2d)
To a solution of 15 (1.66 g, 4.00 mmol) in DMF (30 mL) were
added 4-methylbenzenesulfonamide (13) (0.14 g, 0.80 mmol) and
K2CO3 (1.11 g, 8.00 mmol) and the mixture was stirred for 16 h
at 90 °C. The solvent was evaporated and the residue was parti-
tioned between EtOAc and water. The organic layer was washed
with brine and dried over Na2SO4. The solvent was evaporated
and the residue was chromatographed (EtOAc/hexane = 1/4) to
give the title compound (0.630 g, 2.84 mmol, 71%) as a colorless so-
lid. 1H NMR (CDCl3) d: 4.02 (3H, s), 7.58 (1H, dd, J = 8.6, 2.2 Hz),
7.88 (1H, d, J = 8.6 Hz), 8.16 (1H, d, J = 1.9 Hz), 8.83 (1H, d,
J = 1.9 Hz), 9.45 (1H, d, J = 2.2 Hz). MS (FAB) m/z: 222 (M+H)+.
5.1.16. 7-Chlorocinnoline-3-carboxylic acid (2f)
To a solution of compound 22 (0.23 g, 0.97 mmol) in THF (9 mL)
was added 1 N NaOH aqueous solution (3.0 mL, 3.0 mmol) and the
mixture was stirred for 16 h. After 10% citric acid aqueous solution
was added, the solvent was evaporated. The residue was triturated
with water and collected by filtration to give the title compound
(0.16 g, 0.77 mmol, 79%) as a yellow solid. 1H NMR (DMSO-d6) d:
8.02 (1H, dd, J = 8.8, 2.0 Hz), 8.34 (1H, d, J = 8.8 Hz), 8.70 (1H, br
s), 8.90 (1H, s). MS (FAB) m/z: 209 (M+H)+.
5.1.17. Ethyl 7-chloro-2H-chromene-3-carboxylate (25)
4-Chloro-2-hydroxybenzaldehyde (23) (510 mg, 3.26 mmol)
was dissolved in THF (40 mL), and NaH (60% in oil, 157 mg,
3.91 mmol) was added, followed by stirring at room temperature
for 2 h. To the mixture was added a solution of ethyl 2-(diethoxy-
phosphoryl)acrylate (24) (769 mg, 3.26 mmol) in THF (10 mL), and
the mixture was stirred at room temperature for 2 h and then
refluxed overnight. After the mixture was cooled to room temper-
ature, water and Et2O were added. The organic layer was separated,
dried over Na2SO4, and concentrated in vacuo. The residue was
chromatographed (EtOAc/hexane = 1/10) to give the title com-
pound (247 mg, 1.03 mmol, 32%) as a colorless solid. 1H NMR
(DMSO-d6) d: 1.33 (3H, t, J = 7.1 Hz), 4.27 (2H, q, J = 7.1 Hz), 4.99
(2H, d, J = 1.2 Hz), 6.85 (1H, d, J = 1.2 Hz), 6.89 (1H, dd, J = 8.1,
2.0 Hz), 7.04 (1H, d, J = 8.1 Hz), 7.38 (1H, br s). MS (EI)
m/z: 238 (M)+.
5.1.13. 6-Chloroquinazoline-2-carboxylic acid (2e)
NaHSO3 (10.4 g, 59.8 mmol) and Na2CO3 (5.13 g, 48.4 mmol)
were dissolved in water (200 mL) and heated to 60 °C with stirring.
To this solution was added dropwise a solution of 5-chloro-2-nitro-
benzaldehyde (16) (2.04 g, 11.0 mmol) in MeOH (40 mL). The mix-
ture was stirred for 40 min at 60 °C then refluxed for 2.5 h. After
cooling, Et2O was added and the organic layer was separated,
washed with water, and dried over Na2SO4. The solvent was evap-
orated to give 2-amino-5-chlorobenzaldehyde (17) (0.63 g,
4.0 mmol, 37%) as a yellow oil. This compound was used for the
next reaction without further purification.
To a solution of 17 (0.63 g, 4.0 mmol) in EtOH (12 mL) was
added ammonium acetate (328 mg, 4.25 mmol) and the mixture
was warmed to 35–40 °C. To this mixture was added dropwise a
solution of glyoxylic acid monohydrate (0.40 g, 4.4 mmol) in water

8230
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
5.1.18. 7-Chloro-2H-chromene-3-carboxylic acid (2g)
Compound 2g was synthesized from 25 according to the proce-
dure used to prepare 2f. A pale yellow solid. Yield 99%. 1H NMR
(DMSO-d6) d: 4.92 (1H, d, J = 2.0 Hz), 6.95 (1H, d, J = 2.0 Hz), 7.01
(1H, dd, J = 8.1, 2.2 Hz), 7.35 (1H, d, J = 8.1 Hz), 7.44 (1H, br s).
MS (EI) m/z: 210 (M)+.
5.1.24. Ethyl 6-chloro-4-oxo-1,4-dihydroquinazoline-2-
carboxylate (34)
To
a solution of 2-amino-5-chlorobenzamide (32) (2.50 g,
14.7 mmol) in pyridine (15 mL) was added ethyl 2-chloro-2-
oxoacetate (33) (2.00 mL, 17.9 mmol) and the mixture was stir-
red at room temperature for 18 h. The mixture was concentrated
in vacuo and the residue was dissolved in AcOH (50 mL). To the
solution was added Ac2O (5.0 mL) and the mixture was refluxed
for 16 h. The solvent was evaporated and EtOH was added to the
residue. The resultant precipitate was collected by filtration to
give the title compound (2.71 g, 10.7 mmol, 73%) as a pale yellow
solid. 1H NMR (DMSO-d6) d: 1.35 (3H, t, J = 7.1 Hz), 4.38 (2H, q,
J = 7.1 Hz), 7.85 (1H, d, J = 8.6 Hz), 7.91 (1H, dd, J = 8.6, 2.3 Hz),
8.10 (1H, d, J = 2.3 Hz), 12.85 (1H, br s). MS (ESI) m/z: 253
(M+H)+.
5.1.19. Methyl 6-chloro-4-oxo-1,4-dihydroquinoline-2-
carboxylate (28)
To a solution of 4-chloroaniline (26) (12.8 g, 100 mmol) in
MeOH (150 mL) was added dimethyl acetylenedicarboxylate (27)
(13.5 mL, 110 mmol). The mixture was refluxed for 8 h and concen-
trated in vacuo. The residue was dissolved in diphenyl ether
(70 mL) and the solution was stirred at 240 °C for 4 h. After cooling,
hexane and Et2O were added. The resultant precipitate was col-
lected by filtration to give the title compound (11.1 g, 46.7 mmol,
47%) as a yellow powder. 1H NMR (DMSO-d6) d: 3.97 (3H, s), 6.66
(1H, br s), 7.76 (1H, dd, J = 9.0, 2.5 Hz), 7.90–8.05 (2H, m), 12.28
(1H, br s). MS (ESI) m/z: 238 (M+H)+.
5.1.25. 6-Chloro-4-oxo-1,4-dihydroquinazoline-2-carboxylic
acid (2j)
Compound 2j was synthesized from 34 according to the proce-
dure used to prepare 2f. A colorless solid. Yield 85%. 1H NMR
(DMSO-d6) d: 7.50–8.20 (3H, m), 12.44 (1H, br s). MS (ESI) m/z:
265 (M+H+CH3CN)+.
5.1.20. 6-Chloro-4-oxo-1,4-dihydroquinoline-2-carboxylic acid
(2h)
Compound 2h was synthesized from 28 according to the proce-
dure used to prepare 2f. A yellow solid. Yield 47%. 1H NMR (DMSO-
d6) d: 6.90–7.05 (1H, m), 7.90–8.05 (2H, m), 10.10–10.30 (1H, m),
12.13 (1H, br s). MS (ESI) m/z: 224 (M+H)+.
5.1.26. 7-Chloro-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-
dioxide (36)
To a solution of chlorosulfonylisocyanate (15.0 g, 106 mmol) in
nitroethane (120 mL) was added dropwise 4-chloroaniline (35)
(11.3 g, 90.0 mmol) in nitroethane (20 mL) at À78 °C over 10 min.
The mixture was allowed to warm to 0 °C and AlCl3 (15.0 g,
112 mmol) was added to the mixture in one portion. After the mix-
ture became a clear solution, it was allowed to warm to room tem-
perature. The mixture was heated at 115 °C for 25 min and poured
into ice-water. The resultant precipitate was collected by filtration
and washed successively with water and Et2O to give the title com-
pound (14.7 g, 63.2 mmol, 60%) as a pale brown powder. 1H NMR
(DMSO-d6) d: 7.26 (1H, d, J = 8.8 Hz), 7.69 (1H, dd, J = 8.8, 2.0 Hz),
7.82 (1H, d, J = 2.0 Hz), 11.39 (1H, s).
5.1.21. Diethyl (4-chloro-2-nitrobenzylidene)malonate (30)
To a solution of 4-chloro-2-nitrobenzaldehyde (29) (10.0 g,
53.9 mmol) in Ac2O (25 mL) were added diethyl malonate
(10.0 mL, 66.2 mmol) and NaHCO3 (8.34 g, 100 mmol), and the
mixture was stirred at 100 °C overnight. After cooling, 5% Na2CO3
aqueous solution and EtOAc were added to the mixture. The or-
ganic layer was separated, washed with brine, and dried over
Na2SO4. The solvent was evaporated and the residue was recrys-
tallized from EtOH to give the title compound (8.06 g, 24.6 mmol,
46%) as pale brown crystals. 1H NMR (CDCl3) d: 1.05–1.13 (3H,
m), 1.29–1.38 (3H, m), 4.08–4.17 (2H, m), 4.29–4.38 (2H, m),
7.39 (1H, d, J = 8.3 Hz), 7.61 (1H, dd, J = 8.3, 2.1 Hz), 8.09 (1H, s),
8.20 (1H, d, J = 2.1 Hz). MS (FAB) m/z: 328 (M+H)+. Anal. Calcd
for C14H14ClNO6: C, 51.31; H, 4.27; Cl, 10.82; N, 4.27. Found: C,
51.43; H, 4.29; Cl, 10.59; N, 4.36.
5.1.27. 2-Amino-5-chlorobenzenesulfonamide (37)
To compound 36 (7.83 g, 33.7 mmol) was added 50% sulfuric
acid (270 mL, 2.53 mol) and the mixture was heated at 130–
140 °C for 3 h. After cooling, the mixture was poured into ice-water
(600 mL) and 40% NaOH aqueous solution (500 mL) was added. The
pH of the mixture was adjusted to 3 with 1 N HCl aqueous solution.
The water layer was extracted with EtOAc (twice). The combined
organic layer was washed with water, dried over Na2SO4, and con-
centrated in vacuo. The residue was chromatographed (MeOH/
CH2Cl2 = 1/10) to give the title compound (6.31 g, 30.5 mmol,
97%) as a colorless solid. 1H NMR (DMSO-d6) d: 5.99 (2H, br s),
6.82 (1H, d, J = 8.8 Hz), 7.27 (1H, dd, J = 8.8, 2.7 Hz), 7.38 (2H, br
s), 7.50 (1H, d, J = 2.7 Hz). IR (ATR) cmÀ1: 3444, 3392, 3361, 3282,
1626, 1601, 1533, 1477, 1408, 1304, 1254, 1140, 1111, 881, 818.
MS (FAB) m/z: 207 (M+H)+.
5.1.22. Ethyl 7-chloro-2-oxo-1,2-dihydroquinoline-3-
carboxylate (31)
To a solution of 30 (8.00 g, 24.5 mmol) in AcOH (90 mL) was
added iron powder (8.20 g, 147 mmol) and the mixture was stir-
red at 80 °C for 7 h. After cooling, the mixture was filtered
through a Celite pad and the filtrate was concentrated in vacuo.
The resultant precipitate was collected by filtration to give the ti-
tle compound (4.19 g, 16.6 mmol, 68%) as a colorless solid. 1H
NMR (DMSO-d6) d: 1.31 (3H, t, J = 6.8 Hz), 4.27 (2H, q,
J = 6.8 Hz), 7.27 (1H, d, J = 7.8 Hz), 7.34 (1H, s), 7.86 (1H, d,
J = 7.8 Hz), 8.50 (1H, s), 12.10 (1H, s). MS (FAB) m/z: 252
(M+H)+. Anal. Calcd for C12H10ClNO3: C, 57.27; H, 4.01; Cl,
14.09; N, 5.57. Found: C, 57.00; H, 3.97; Cl, 14.09; N, 5.51.
5.1.28. Ethyl 2-[2-(aminosulfonyl)-4-chloroanilino]-2-
oxoacetate (38)
To a solution of 37 (7.83 g, 33.7 mmol) in glacial acetic acid
(3.0 mL) was added Et3N (2.80 mL, 20.1 mmol). To the solution
was carefully added ethyl 2-chloro-2-oxoacetate (33) (1.12 mL,
10.0 mmol). After stirring for 1 h, water (10 mL) and concd HCl
aqueous solution were added. The resultant precipitate was col-
lected by filtration and washed with water (twice). Recrystalliza-
tion of this solid (2.17 g) from EtOH (12 mL) gave the title
compound (710 mg, 2.31 mmol, 23%) as colorless crystals. 1H
NMR (DMSO-d6) d: 1.33 (3H, t, J = 7.1 Hz), 4.33 (2H, q, J = 7.1 Hz),
5.1.23. 7-Chloro-2-oxo-1,2-dihydroquinoline-3-carboxylic acid
(2i)
Compound 2i was synthesized from 31 according to the proce-
dure used to prepare 2f. A colorless solid. Yield 94%. 1H NMR
(DMSO-d6) d: 7.32–7.55 (2H, m), 8.05 (1H, s), 8.93 (1H, s), 13.80
(1H, br s). MS (FAB) m/z: 224 (M+H)+. Anal. Calcd for
C10H5ClNO3Á0.2H2O: C, 53.10; H, 2.41; Cl, 15.67; N, 6.19. Found:
C, 52.99; H, 2.69; Cl, 15.57; N, 6.09.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8231
7.77 (1H, dd, J = 8.8, 2.4 Hz), 7.87 (1H, d, J = 2.4 Hz), 7.87 (2H, br s),
8.30 (1H, d, J = 8.8 Hz), 10.76 (1H, br s). IR (ATR) cmÀ1: 3298, 3236,
1745, 1709, 1583, 1523, 1390, 1346, 1259, 1194, 1159, 760, 710,
602, 499. MS (FAB) m/z: 307 (M+H)+.
to room temperature and the solvent was evaporated to give the
title compound (1.08 g, quant.) as a brown solid. This solid was
used for the next reaction without further purification. 1H NMR
(DMSO-d6) d: 2.52 (3H, s), 3.73 (2H, t, J = 3.2 Hz), 3.87 (2H, t,
J = 3.2 Hz).
5.1.29. Methyl 7-chloro-4H-1,2,4-benzothiadiazine-3-
carboxylate 1,1-dioxide (2k)
5.1.34. N-{(1R,2S,5S)-2-[(6-Chloro-2-naphthoyl)amino]-5-[(di-
methylamino)carbonyl]cyclohexyl}-5-methyl-5,6-dihydro-4H-
pyrrolo[3,4-d]thiazole-2-carboxamide hydrochloride (45a)
Compound 45a was synthesized from 3a and 44 according to
the procedure used to prepare 5a. A pale yellow solid. Yield 18%.
A pale yellow solid. 1H NMR (DMSO-d6) d: 1.48–1.56 (1H, m),
1.71–1.84 (3H, m), 1.95–2.04 (2H, m), 2.81 (3H, s), 3.00 (3H, s),
3.02 (3H, s), 3.06–3.15 (2H, m), 4.13–4.14 (1H, m), 4.52–4.63
(4H, m), 7.60 (1H, d, J = 8.5 Hz), 7.87 (1H, d, J = 8.8 Hz), 7.96 (1H,
d, J = 8.5 Hz), 8.01 (1H, d, J = 8.8 Hz), 8.10 (1H, s), 8.32 (1H, s),
8.45 (1H, d, J = 8.1 Hz), 8.51 (1H, d, J = 7.3 Hz). MS (FAB) m/z: 540
(M+H)+. Anal. Calcd for C27H30ClN5O3SÁ0.9HClÁ2.7H2OÁ0.1Et2O: C,
52.34; H, 5.95; Cl, 10.71; N, 11.14; S, 5.10. Found: C, 52.08; H,
5.55; Cl, 10.72; N, 11.19; S, 5.48.
To a solution of 38 (700 mg, 2.28 mmol) in MeOH (3.0 mL) were
added NaOMe (156 mg, 2.89 mmol) in MeOH (3.0 mL) and phenol-
phthalein (small amount). After stirring for 29 h, NaOMe (160 mg,
2.96 mmol) was added and the mixture was stirred for an addi-
tional 18.5 h. Water (6.0 mL) and 1 N HCl aqueous solution
(5.85 mL) were added to the mixture. The resultant precipitate
was collected by filtration to give the title compound (491 mg,
1.79 mmol, 78%) as a colorless solid. 1H NMR (DMSO-d6) d: 3.94
(3H, s), 7.80 (1H, d, J = 9.0 Hz), 7.83 (1H, dd, J = 9.0, 2.2 Hz), 7.96
(1H, d, J = 2.2 Hz), 12.91 (1H, br s). IR (ATR) cmÀ1: 3248, 1734,
1595, 1520, 1475, 1441, 1336, 1306, 1263, 1167, 1146, 1109,
939, 825, 806, 715, 565. MS (FAB) m/z: 275 (M+H)+.
5.1.30. 5-(Phenylsulfonyl)-5,6-dihydro-4H-pyrrolo[3,4-
d]thiazole (41)
Benzenesulfonamide (39) (638 mg, 4.06 mmol) and 4,5-bis(bro-
momethyl)thiazole (40) (1.10 g, 4.06 mmol) were dissolved in DMF
(10 mL) and cooled to 0 °C. To this solution was added NaH (60% in
oil, 357 mg, 8.93 mmol), and the mixture was stirred for 3 h at
room temperature. To the mixture were added CH2Cl2 and H2O,
and the organic layer was separated, dried over Na2SO4, and con-
centrated in vacuo. The residue was chromatographed (EtOAc/
CH2Cl2 = 1/9) to give the title compound (137 mg, 0.515 mmol,
13%) as a colorless solid. 1H NMR (CDCl3) d: 4.60–4.63 (2H, m),
4.70–4.73 (2H, m), 7.52–7.64 (3H, m), 7.88–7.92 (2H, m), 8.71
(1H, s). MS (FAB) m/z: 267 (M+H)+. Anal. Calcd for C11H10N2O2S2:
C, 49.60; H, 3.78; N, 10.52. Found: C, 49.28; H, 3.79; N, 10.41.
5.1.35. 7-Chloro-N-{(1S,2R,4S)-4-[(dimethylamino)carbonyl]-2-
{[(5-methyl-5,6-dihydro-4H-pyrrolo[3,4-d]thiazol-2-yl)car-
bonyl]amino}cyclohexyl}isoquinoline-3-carboxamide hydro-
chloride (45c)
Compound 45c was synthesized from 3c and 44 according to
the procedure used to prepare 5c. A colorless solid. Yield 40%.
1H NMR (DMSO-d6) d: 1.47–1.56 (1H, m), 1.71–1.75 (3H, m),
1.95–1.99 (1H, m), 2.12–2.15 (1H, m), 2.78 (3H, s), 2.95 (3H, s),
2.98 (1H, br s), 3.05 (3H, s), 4.19–4.22 (1H, m), 4.44–4.52 (3H,
m), 4.74–4.88 (2H, m), 7.87 (1H, dd, J = 8.8, 1.7 Hz), 8.24 (1H, d,
J = 8.8 Hz), 8.36 (1H, d, J = 1.7 Hz), 8.58 (1H, s), 8.90–8.92 (2H,
m), 9.30 (1H, s), 12.65–12.75 (1H, m). MS (FAB) m/z: 541
(M+H)+. Anal. Calcd for C26H29ClN6O3SÁ1.3HClÁ1.3H2O: C, 51.04;
H, 5.42; Cl, 13.33; N, 13.78; S, 5.24. Found: C, 51.29; H, 5.54;
Cl, 13.28; N, 13.40; S, 5.56.
5.1.31. 5,6-Dihydro-4H-pyrrolo[3,4-d]thiazole dihydrobromide
(42)
A mixture of 41 (800 mg, 3.00 mmol), phenol (0.800 mL) and 47%
HBr aqueous solution (5.00 mL) was refluxed for 2 h. After cooling,
EtOAc and H2O were added. The water layer was separated and con-
centrated in vacuo. To the residue was added EtOAc and the resul-
tant precipitate was collected by filtration to give the title
compound (521 mg, 2.52 mmol, 60%) as a colorless solid. 1H NMR
(DMSO-d6) d: 4.42 (2H, br s), 4.56 (2H, br s), 9.14 (1H, s). MS (FAB)
m/z: 127 (M+H)+. Anal. Calcd for C5H6N2SÁ2HBr: C, 20.85; H, 2.80;
Br, 55.49; N, 9.73. Found: C, 20.67; H, 2.78; Br, 55.74; N, 9.39.
5.1.36. tert-Butyl {(1R,2S,5S)-2-{[(5-chloroindol-2-yl)carbonyl]-
amino}-5-[(dimethylamino)carbonyl]cyclohexyl}carbamate (47)
To a solution of ethyl (1S,3R,4S)-3-[(tert-butoxycarbonyl)amino]4-
{[(5-chloroindol-2-yl)carbonyl]amino}cyclohexanecarboxylate (46)9
(4.00 g, 8.62 mmol) in THF (50 mL) and EtOH (50 mL) was added
1 N NaOH aqueous solution (37 mL) at 0 °C. The mixture was al-
lowed to warm to room temperature and stirred for 5 h. The mix-
ture was then neutralized with 1 N HCl aqueous solution (37 mL)
at 0 °C and concentrated in vacuo. The resultant precipitate was
collected by filtration to give (1S,3R,4S)-3-[(tert-butoxycar-
bonyl)amino]-4-{[(5-chloroindol-2-yl)carbonyl]amino}cyclohex-
anecarboxylic acid (7.99 g, 18.3 mmol). 7.89 g (18.1 mmol) of this
solid was dissolved in DMF (200 mL) and to the solution were
added dimethylamine hydrochloride (3.69 g, 45.2 mmol), EDCÁHCl
(6.94 g, 36.2 mmol), HOBt (2.44 g, 18.1 mmol), and Et3N (6.30 mL,
45.2 mmol). After stirring for 14.5 h, the solvent was evaporated
in vacuo. The residue was partitioned between EtOAc and water,
and the water layer was extracted with EtOAc. The combined or-
ganic layer was washed with brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was chromatographed (EtOAc/
hexane = 1/19) to give the title compound (8.18 g, 17.7 mmol,
96%) as a gray amorphous solid. 1H NMR (CDCl3) d: 1.52 (9H, s),
1.62–1.79 (1H, m), 1.80–1.97 (2H, m), 2.05–2.38 (2H, m), 2.58–
2.72 (1H, m), 2.95 (3H, s), 3.07 (3H, s), 3.91–4.10 (1H, m), 4.18–
4.27 (1H, m), 4.68–4.88 (1H, m), 6.80 (1H, s), 7.22 (1H, dd,
J = 8.8, 1.5 Hz), 7.27 (1H, d, J = 1.5 Hz), 7.35 (1H, d, J = 8.8 Hz),
7.59 (1H, s), 7.98 (1H, s), 9.50 (1H, s).
5.1.32. 5-Methyl-5,6-dihydro-4H-pyrrolo[3,4-d]thiazole (43)
To a suspension of 42 (357 mg, 1.24 mmol) in CH2Cl2 (5 mL)
were added Et3N (0.407 mL, 2.92 mmol), AcOH (0.167 mL,
2.92 mmol), formalin (0.188 mL, 2.19 mmol), and NaBH(OAc)3
(464 mg, 2.19 mol). After stirring for 30 min, CH2Cl2 and saturated
NaHCO3 aqueous solution were added. The organic layer was sep-
arated, dried over Na2SO4, and concentrated in vacuo. The residue
was chromatographed (MeOH/CH2Cl2 = 1/19) to give the title com-
pound (150 mg, 1.07 mmol, 86%) as a brown oil. 1H NMR (CDCl3) d:
2.67 (3H, s), 3.95–3.99 (2H, m), 4.01–4.05 (2H, m), 8.69 (1H, s). MS
(ESI) m/z: 141 (M+H)+.
5.1.33. Lithium 5-methyl-5,6-dihydro-4H-pyrrolo[3,4-
d]thiazole-2-carboxylate (44)
To a solution of 43 (771 mg, 5.50 mmol) in THF (10 mL) was
added tert-butyl lithium (1.54 M pentane solution, 3.93 mL,
6.05 mmol) at À78 °C under argon atmosphere. After stirring for
1 h at 0 °C, the mixture was cooled to À78 °C again and CO2 gas
was bubbled into it over 20 min. The mixture was allowed to warm

8232
K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
Table 7
5.1.37. 5-Chloro-N-{(1S,2R,4S)-4-[(dimethylamino)carbonyl]-2-
{[(5-methyl-5,6-dihydro-4H-pyrrolo[3,4-d]thiazol-2-yl)carbonyl]-
amino}cyclohexyl}indole-2-carboxamide hydrochloride (48)
Compound 48 was synthesized from 47 and 44 according to the
procedure used to prepare 5a. A colorless solid. Yield 34%. 1H NMR
(DMSO-d6) d: 1.45–1.55 (1H, m), 1.67–1.83 (3H, m), 1.91–1.99 (2H,
m), 2.79 (3H, s), 2.97 (3H, s), 3.01–3.08 (1H, m), 3.05 (3H, s), 4.07–
4.13 (1H, m), 4.35–4.91 (5H, m), 7.03 (1H, d, J = 1.5 Hz), 7.15 (1H,
dd, J = 8.7, 2.1 Hz), 7.40 (1H, d, J = 8.7 Hz), 7.66 (1H, d, J = 2.1 Hz),
8.36 (1H, d, J = 7.8 Hz), 8.40 (1H, d, J = 8.3 Hz), 11.77 (1H, s),
12.25 (1H, br s). MS (ESI) m/z: 529 (M+H)+. Anal. Calcd for
C25H29ClN6O3SÁHClÁ1.4H2O: C, 50.83; H, 5.60; Cl, 12.00; N, 14.23;
S, 5.43. Found: C, 50.85; H, 5.33; Cl, 12.00; N, 14.37; S, 5.74.
Crystal and diffraction data of human fXa with 45c
Crystal parameters
Space group
a (Å)
b (Å)
c (Å)
Resolution (Å)
Rsym (%)
Completeness (%)
No. of reflections, redundancy
P212121
56.1
72.2
79.2
1.8
4.8 (26.6)a
94.5 (79.5)a
28,855, 2.66
Refinement
No. of protein atoms (occupancy –0)
2239
Average B value for protein and ligand atoms (Å2)
30.9, 39.2
25.0–1.8
19.6
Range of data
R value
Rfree
23.0
5.2. Biological assays
(Not weighted) rmsd from ideality
Bond length (Å)
Bond angle (Å)
0.025
2.290
Assays of the in vitro anti-fXa activity, in vitro anticoagulant
activity and rat ex vivo anti-fXa activity assay were performed as
previously described.8
a
Figures in parentheses represent statics in the last shell of data (highest
resolution).
5.3. Metabolic stability and permeability
Metabolic stability and permeability were measured as previ-
ously described.8
(25% PEG6000/25% glycerol/0.3 M AcONa/2.5 mM CaCl2/0.1 M male-
ate imidazole, pH 5.0/1 mM of compound 45c. After 1 day, the crystal
was picked up and directly exposed to soak solution 2, and the soaking
was continued. The entire soaking process was performed at 20 °C.
5.4. Distribution coefficient and protein binding
Distribution coefficient and protein binding were measured as
previously described.9
5.8. X-ray data collection and processing
The soaked crystal was flash-cooled in liquid nitrogen and cen-
tered in a gaseous nitrogen stream. The X-ray data set was col-
lected at 100 K on an R-Axis IIc imaging plate detector (Rigaku)
using an RU200 rotating anode generator (Rigaku). Data processing
was carried out with Mosflm31 and scala.32
5.5. Pharmacokinetic analysis in cynomolgus monkey
Pharmacokinetic analysis was performed as previously
described.8
5.6. Pharmacokinetic analysis in beagle dogs
5.9. Structure solution and crystallographic refinement
Male beagle dogs (8–11 months old, n = 5) were fasted over-
night. The test compounds were administered orally (10 mg/head,
ca. 1 mg/kg, in water suspension, 4 mL/kg, via a stomach tube) in a
fasted or non-fasted condition. For the non-fasted condition, ca.
270 g of solid food (TC-2, Aixia) was given to each dog 1–2 h before
the oral administration. The chemical composition of TC-2 is as fol-
lows: protein 23%; fat 9%; carbohydrate, 51%. Blood sample collec-
tion and measurement of the compound concentration in plasma
were performed in a similar manner to that was employed in the
pharmacokinetic analysis in cynomolgus monkeys.8
The previously reported Gla-less fXa structure (PDB code:
1HCG33) was used as the initial structure. Phase refinement and
model improvement were carried out with refmac34 and Turbo
Frodo.35 Stereochemistry checks indicated that the refined protein
model was in good agreement with the expectations within each
resolution range. The statistics of the data processing and crystal-
lographic refinement are shown in Table 7. The atomic coordinates
have been deposited with the Protein Data Bank (PDB code: 3IIT).
Acknowledgements
5.7. Preparation of the crystal
We are grateful to anticoagulant group in Biological Research
Laboratories II. We thank Dr. Kiyoshi Nakayama for helpful discus-
sions and editorial assistance in preparing the manuscript.
Purified human Gla-less fXa was purchased from Hematologic
Technologies Inc. Without further purification, the purchased pro-
tein sample was dialyzed against 5 mM maleate imidazole, pH 5.0/
4 mM CaCl2/10 mM benzamidine, and concentrated to 7.5 mg/mL
with microcon-10 (Millipore Co.). Concentrated Gla-less fXa was
mixed with an equal volume of reservoir solution (15% PEG6000/
1 mM CaCl2/0.3 M AcONa/0.1 M maleate imidazole, pH 5.0) and va-
por-equilibrated against the same solution at 20 °C. Under these con-
ditions, the crystal did not form spontaneously, so micro- and
macroseeding methods were needed to obtain crystals of the appro-
priate size. The resultant benzamidine/Gla-less fXa crystal was ex-
posed to a two-step soaking method described below to obtain
complex crystals with compound 45c. The benzamidine/Gla-less
fXa crystal was dialyzed in a microdialysis button against soak solu-
tion 1 (20% PEG6000/15% glycerol/0.3 M AcONa/2.5 mM CaCl2/0.1 M
maleate imidazole, pH 5.0) for 5 h and then against soak solution 2
Supplementary data
Supplementarydata(characterizationof furtherintermediatesas
well as final compounds are supplied) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.2009.
10.024.
References and notes
1. Hirsh, J.; Poller, L. Arch. Int. Med. 1994, 154, 282.
2. Klauss, V.; Spannagl, M. Curr. Drug Targets 2006, 7, 1285.
3. Desai, S. S.; Massad, M. G.; DiDomenico, R. J.; Abdelhady, K.; Hanhan, Z.; Lele,
H.; Snow, N. J.; Geha, A. S. Recent Patents Cardiovasc. Drug Disc. 2006, 1, 307.
4. Casimiro-Garcia, A.; Dudley, D. A.; Heemstra, R. J.; Filipski, K. J.; Bigge, C. F.;
Edmunds, J. J. Expert Opin. Ther. Patents 2006, 16, 119.

K. Yoshikawa et al. / Bioorg. Med. Chem. 17 (2009) 8221–8233
8233
5. Kaiser, B. Drugs Future 1998, 23, 423.
6. Turpie, A. G. G. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 1238.
7. Garcia, D. Thromb. Res. 2009, 123, S50.
8. Yoshikawa, K.; et al. Bioorg. Med. Chem. 2009, in press, doi:10.1016/
j.bmc.2009.10.023.
9. Nagata, T.; Yoshino, T.; Haginoya, N.; Yoshikawa, K.; Nagamochi, M.; Kobayashi,
S.; Komoriya, S.; Yokomizo, A.; Muto, R.; Yamaguchi, M.; Osanai, K.; Suzuki, M.;
Kanno, H. Bioorg. Med. Chem. 2009, 17, 1193.
10. Haginoya, N.; Komoriya, S.; Osanai, K.; Yoshino, T.; Nagata, T.; Nagamochi, M.;
Muto, R.; Yamaguchi, M.; Nagahara, T.; Kanno, H. Heterocycles 2004, 63, 1555.
11. McEwen, W. E.; Cobb, R. L. Chem. Rev. 1955, 55, 511.
12. A modified condition was employed: Larsen, R. D.; Reamer, R. A.; Corley, E. G.;
Davis, P.; Grabowski, E. J. J.; Reider, P. J.; Shinkai, I. J. Org. Chem. 1991, 56, 6034.
13. Basavaiah, D.; Rao, P. D.; Hyma, R. S. Tetrahedron 1996, 52, 8001.
14. Kim, J. N.; Lee, H. J.; Lee, K. Y.; Kim, H. S. Tetrahedron Lett. 2001, 42, 3737.
15. Kanner, C. B.; Pandit, U. K. Tetrahedron 1981, 37, 3513.
16. Kleschick, W. A.; Heathcock, C. H. J. Org. Chem. 1978, 43, 1256.
17. Reitsema, R. H. Chem. Rev. 1948, 43, 43.
18. Hariri, M. A.; Galley, O.; Pautet, F.; Fillion, H. Eur J. Org. Chem. 1998, 4, 593.
19. (Dimethylamino)carbonyl group was not observed clearly in this crystal,
however it is assumed to locate toward exterior of enzyme.
24. Maignan, S.; Guilloteau, J.-P.; Choi-Sledeski, Y. M.; Becker, M. R.; Ewing, W. R.;
Pauls, H. W.; Spada, A. P.; Mikol, V. J. Med. Chem. 2003, 46, 685.
25. Chan, C.; Borthwick, A. D.; Brown, D.; Burns-Kurtis, C. L.; Campbell, M.;
Chaudry, L.; Chung, C.; Convery, M. A.; Hamblin, J. N.; Johnstone, L.; Kelly, H. A.;
Kleanthous, S.; Patikis, A.; Patel, C.; Pateman, A. J.; Senger, S.; Shah, G. P.;
Toomey, J. R.; Watson, N. S.; Weston, H. E.; Whitworth, C.; Young, R. J.; Zhou, P.
J. Med. Chem. 2007, 50, 1546.
26. A similar intramolecular hydrogen bond was exploited in quinoline-derived
oligoamide to stabilize its conformation: Jiang, H.; Léger, J.-M.; Dolain, C.;
Guionneau, P.; Huc, I. Tetrahedron 2003, 59, 8365.
27. Nagao, Y.; Hirata, T.; Goto, S.; Sano, S.; Kakehi, A.; Iizuka, K.; Shiro, M. J. Am.
Chem. Soc. 1998, 120, 3104.
28. Honda, T.; Tajima, H.; Kaneko, Y.; Ban, M.; Inaba, T.; Takeno, Y.; Okamoto, K.;
Aono, H. Bioorg. Med. Chem. Lett. 2008, 18, 2939.
29. The torsion angle of O–C–C–S is 2.5°.
30. The existence of intramolecular hydrogen bonds between amide NH and
thiazole nitrogen were indicated in thiazole containing cyclic peptides:
Bertram, A.; Blake, A. J.; Turiso, F. G.-L.; Hannam, J. S.; Jolliffe, K. A.;
Pattenden, G.; Skae, M. Tetrahedron 2003, 59, 6979.
31. Leslie, A. G. W. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography;
Daresbury Lab Eds.; Daresbury, UK, 1992; No. 26.
20. Haginoya, N.; Kobayashi, S.; Komoriya, S.; Yoshino, T.; Suzuki, M.; Shimada, T.;
Watanabe, K.; Hirokawa, Y.; Furugori, T.; Nagahara, T. J. Med. Chem. 2004, 47, 5167.
21. Adler, M.; Kochanny, M. J.; Ye, B.; Rumennik, G.; Light, D. R.; Biancalana, S.;
Whitlow, M. Biochemistry 2002, 41, 15514.
22. (a) Taylor, R.; Kennard, O. J. Am. Chem. Soc. 1982, 104, 5063; (b) Pierce, A. C.; ter
Haar, E.; Binch, H. M.; Kay, D. P.; Patel, S. R.; Li, P. J. Med. Chem. 2005, 48, 1278.
23. Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J.; Schlemmer, K.-
H.; Reinemer, P.; Perzborn, E. J. Med. Chem. 2005, 48, 5900.
32. Collaborative Computational Project. The CCP4 Suite: Programs for Protein
Crystallography. Acta Crystallogr., Sect. D 1994, 50, 760–763.
33. Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.; Park, C. H.; Bode, W.;
Huber, R.; Blankenship, D. T.; Cardin, A. D.; Kisiel, W. J. Mol. Biol. 1993, 232,
947.
34. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., Sect. D 1997, 53,
240.
35. Jones, T. A. Methods Enzymol. 1985, 115, 157.