Orally active factor Xa inhibitors: 4,5,6,7-tetrahydrothiazolo[5,4-c] pyridine derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.03.036

In our investigation of factor Xa inhibitors, a series of 1-(6-chloronaphthalen-2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiazolo[5,4-c] pyridine-2-carbonyl)piperazines 3a-i were synthesized. In vitro inhibitory activities of the compounds against factor Xa and coagulation are summarized. Among the compounds, 3c and 3d, possessing a carbamoyl or N-methylcarbamoyl moiety, showed potent inhibitory activities when administered orally to rats.

Full text of DOI:10.1016/j.bmcl.2004.03.036

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2935–2939
Orally active factor Xa inhibitors: 4,5,6,7-tetrahydrothiazolo-
[5,4-c]pyridine derivatives
Noriyasu Haginoya,a,* Syozo Kobayashi,a Satoshi Komoriya,a Yumiko Hirokawa,b
Taketoshi Furugorib and Takayasu Nagaharab
aMedicinal Chemistry Research Laboratory, Daiichi Pharmaceutical Co. Ltd, 1-16-13, Kita-Kasai,
Edogawa-ku, Tokyo 134-8630, Japan
bNewProduct Research Laboratory 2, Daiichi Pharmaceutical Co. Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
Received 13February 2004; revised 11 March 2004; accepted 11 March 2004
Abstract—In our investigation of factor Xa inhibitors, a series of 1-(6-chloronaphthalen-2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiaz-
olo[5,4-c]pyridine-2-carbonyl)piperazines 3a–i were synthesized. In vitro inhibitory activities of the compounds against factor Xa
and coagulation are summarized. Among the compounds, 3c and 3d, possessing a carbamoyl or N-methylcarbamoyl moiety, showed
potent inhibitory activities when administered orally to rats.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
tion where the intrinsic and extrinsic pathways in the
coagulation cascade meets one another, fXa inhibitors
theoretically block the coagulation cascade evoked in
either pathway.3 Up to the present, a lot of fXa inhibi-
tors have been synthesized. Such fXa inhibitors can be
classified into two groups, that is, amidine derivatives
exemplified by DX-9065a (1)4 and nonamidine deriva-
tives such as compound 2.5 As already described in
many reports, most of the amidine inhibitors are not
sufficiently absorbed in intestinal tracts.6 Therefore, a
trend in the synthetic study of fXa inhibitors seems to be
shifted to nonamidine derivatives from amidine deriva-
tives (Fig. 1).7
Anti-coagulants have been prescribed for the treatment
of thromboembolism, which is a major cause of such
ischemic diseases as cardiac infarction, cerebral infarc-
tion, deep vein thrombosis and unstable angina. Hepa-
rin and warfarin are standard anti-coagulants. However,
heparin is not suitable for long-term treatment of the
above diseases because of parenteral administration and
side effects such as bleeding tendency.1 Although war-
farin is administered orally and is able to be used for
long-term administration, the drug needs continuous
administration for several days to exhibit anti-coagula-
tion activity and also needs frequent monitoring to
maintain that activity at appropriate levels.2 Therefore,
a novel anti-coagulant should satisfy the following
requirements: a good absorption rate in intestinal tracts,
a fast onset of action and a low risk of bleeding.
In our synthetic study of nonamidine type fXa inhibi-
tors, we surveyed bicyclic heterocycle structures that can
satisfy the three-dimensional requirements in the active
site of fXa as well as does the 4-(piperidino)pyridine
moiety of 2. In this line of investigation, we have found
4,5,6,7-tetrahydrothiazolopyridine derivatives 3 that
show potent fXa inhibitory activity in both in vitro and
ex vivo assays.
In order to develop such an anti-coagulant, we have
studied factor Xa (fXa) inhibitors. FXa is a serine pro-
tease that cleaves prothrombin to produce thrombin, a
pivotal serine protease in the blood coagulation cascade.
In addition, since fXa is situated at the confluent posi-
2. Chemistry
Keywords: Factor Xa inhibitor; 4,5,6,7-Tetrahydrothiazolo[5,4-c]pyr-
idine.
Synthetic routes to compounds 3a–i and 10 listed in
Table 1 were shown in Scheme 1. Reaction of 3-chloro-
1-ethoxycarbonyl-4-piperidone (4)8 with thioformamide
* Corresponding author. Tel.: +81-3569-67435; fax: +81-3569-68723;
e-mail: hagin9kg@daiichipharm.co.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.036

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N. Haginoya et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2935–2939
of 2-chloronaphthalene (7). Compound 8 was treated
CO2H
H2N
with tert-butyl 1-piperazinecarboxylate and the resulting
compound was deprotected to give 1-(6-chloronaph-
thalene-2-sulfonyl)piperazine (9a). On the other hand,
compound 8 was treated with 2-ethoxy-carbonylpiper-
azine to obtain 1-Boc-4-(6-chloronaphthalen-2-yl)sulfon-
ylpiperazine-2-carboxylic acid (9b). Compound 9b was
converted to 4-(6-chloronaphthalen-2-yl)sulfonyl-2-car-
bamoylpiperazines 9c–i by reactions with a series of
amines.
Me
NH
N
NH
O
DX-9065a (1)
O
Cl
N
N
N
O
S
S
O
O
N
2
Piperazine derivative 9a was condensed with carboxylic
acid lithium salt 6a. Following treatment of the product
with hydrochloric acid afforded 1-(6-chloronaphthalen-
2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-
2-yl)carbonylpiperazine (3a), which was converted to 3b
by reductive methylation. Compound 3b was alkylated
further with methyl iodide to give quaternalized com-
pound 10.
R2
O
S
Cl
N
R1
N
N
N
O
3
Figure 1. Structures of DX-9065a (1), 2 and 3.
Condensation of a series of 3-carbamoylpiperazines 9c–i
with carboxylic acid 6b afforded 1-(6-chloronaphthalen-
2-yl)sulfonyl-4-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-
2-yl)carbonyl-3-carbamoylpiperazines 3c–i.
gave 5-ethoxycarbonyl-4,5,6,7-tetrahydro-thiazolo[5,4-c]-
pyridine (5). After removal of the ethoxycarbonyl group
of 5 by alkali hydrolysis, the resulting 4,5,6,7-tetrahy-
drothiazolo[5,4-c]pyridine was protected with di-tert-
butyl dicarbonate then treated with n-BuLi and CO2 to
give lithium 5-Boc-4,5,6,7-tetrahydrothiazolo[5,4-c]-
pyridine-2-carboxylate (6a). Compound 5 was reduced
with LiAlH4 and then treated with n-BuLi and CO2 to
give lithium 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]-
pyridin-2-carboxylate (6b).
3. Results and discussion
In Table 1, in vitro anti-fXa, anti-thrombin and anti-
coagulant activities are summarized. All compounds
showed selective anti-fXa activity comparing with anti-
thrombin activity. Compounds 3a (R1 ¼ H, R2 ¼ H)
showed IC50 value of 60 nM as anti-fXa activity. N-
Methyl derivative 3b showed approximately 3-fold
(6-Chloronaphthalen-2-yl)sulfonylchloride (8) was syn-
thesized via b-sulfonylation and successive chlorination
Table 1. In vitro anti-fXa, anti-thrombin and anti-coagulant activities of the synthesized compounds
R2
O
O
I-
S
Cl
S
Cl
Me
Me
N
N
N+
R1
N
N
N
O
N
N
S
O
S
O O
3a-i
10
Compound
R1
R2
Anti-fXa IC50
(nM)a
Anti-thrombin
IC50 (nM)a
PTCT2 in human
plasma (lM)b
PTCT2 in rat
plasma (lM)b
3a
3b
3c
3d
3e
H
H
H
60
22
2900
560
11.2
6.0
4.4
3.0
16.1
10.0
14.0
6.3
Me
Me
Me
Me
CONH2
CONHMe
CONHCH2CONH2
24
27
1000
1350
23880 .8
3
8.5
O
3f
Me
25
1000
2.1
4.3
CONH
N
O
3g
3h
Me
Me
CONH-iPr
CONMe2
45
130
820
6000
8.8
7.0
8.0
11.4
3i
Me
640
7.8
1300
3200
NTc
0.8
NTc
1.6
CONH
H
10
N,N-Dimethyl
a The measuring methods of anti-fXa and anti-thrombin activity were described in Refs. 9,10.
b Anti-coagulant activities in human and rat plasma were evaluated with the plasma clotting time doubling concentration (PTCT2).11
c NT ¼ not tested because of the weak anti-Xa and anti-thrombin activity.

N. Haginoya et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2935–2939
2937
R1
O
S
N
N
O
N
OLi
Cl
EtO2C
a)
b)
c)
S
N
N
6a: R1 = Boc
CO2Et
5
6b: R1 = Me
4
R2
R2
O
R3
N
Cl
S
Cl
N
R1
N
N
N
O
N
S
S
O
O
O
Cl
Cl
e)
f)
d)
h)
9a: R2 = R3 = H
3a: R1 = H
R 2 = H
ClSO2
6a
7
8
9b: R2 = CO2H
R 3 = Boc
h)
g)
3c-i: R1 = Me
9c-i: R2 =carbamoyls
6b
R 2 = carbamoyls
R3 = H
O
O
I-
S
Cl
S
Cl
N
Me
N
i)
j)
N+
MeN
N
N
3a
N
N
S
S
O
Me
O
O
O
3b
10
ꢀ
Scheme 1. Syntheses of 3a–i and 10. Reagents and conditions: (a) thioformamide, molecular seaves 4 A/EtOH, 51%; (b) (i) 5 N NaOH, then (Boc)2O,
55%, (ii) n-BuLi/ether, then CO2 gas, 85%; (c) (i) LiAlH4/ether, 40%, (ii) n-BuLi/ether, then CO2 gas, quant; (d) (i) c.H2SO4, (ii) SOCl2/DMF, two
steps 17%; (e) (i) tert-butyl 1-piperazinecarboxylate, Et3N/CH2Cl2, (ii) satd HCl/EtOH, two steps quant; (f) (i) 2-ethoxycarbonylpiperazine, Et3N/
CH2Cl2, (ii) (Boc)2O/THF, (iii) aq NaOH/EtOH, three steps 34%; (g) a series of amines, WSCI, HOBt/DMF, (ii) satd HCl/EtOH, two steps 71–99%;
(h) WSCI, HOBt/DMF, 30–61%; (i) HCHO, AcOH, Et3N/CH2Cl2, 71%; (j) CH3I/DMF, 56%.
higher anti-fXa activity and 2-fold higher PTCT2
activity when compared with 3a.
trast, compound 3i with
a
lipophilic N-(cyclo-
hexylmethyl)carbamoyl group showed markedly
decreased activity as compared with carbamoyl deriva-
tive 3c, suggesting that lipophilicity of those substituents
is likely to affect the potency. These results indicate that
the carbamoyl and N-substituted carbamoyl groups
may direct to the outer side of fXa but not to the inner
side of fXa when these inhibitors bind to fXa.
We synthesized a series of carbamoyl derivatives 3c–i
based on the following assumption. Since the piperazine
ring of compound 3b may be a linker connecting the
5-chloronaphthalene and tetrahydrothiazolopyridine
moieties, introduction of a carbamoyl group on the
piperazine ring may not produce a new steric problem in
the inhibitor–fXa interactions. We therefore expected
that the hydrophilic carbamoyl group might associate
with water molecules surrounding the complex of the
inhibitor and fXa. Alternatively, the carbamoyl group
might make hydrogen bonds with fXa. In either case,
such a contribution of the carbamoyl group would sta-
bilize the inhibitor–fXa interactions, leading to an ele-
vation in potency of anti-fXa activity. As a result,
carbamoyl and N-methylcarbamoyl derivatives 3c and
3d exhibited almost equipotent anti-fXa activity in
comparison with 3b. N-(Carbamoylmethyl)- and
N-(morpholinecarbonymethyl)carbamoyl derivatives 3e
and 3f also showed similar potency to that of 3b.
With respect to PTCT2, there is some inconsistency
between potency in human plasma and that in rat
plasma. For instance, compound 3c showed potent
PTCT2 activity in human plasma, while they did not
exhibit paralleled potency in rat plasma. For the reason
why such a discrepancy occurred, we thought possibly
due to nonspecific plasma protein binding.
In Table 2, ex vivo anti-fXa and anti-coagulant activities
were shown, compound 3c displayed 67.4% of anti-fXa
activity at 1 h after oral administration of 30 mg/kg to
rats, and also showed evident prolongation of pro-
thrombin time (PT) (1.13-fold). Compound 3d showed
higher potency in ex vivo anti-fXa activity (89.5% at 1 h)
in the same condition. The activity of both compounds
sustained for 6 h. On the other hand, compounds 3b, 3e
and 3f, which were equipotent to 3c or 3d in in vitro
anti-fXa activity, showed only weak ex vivo anti-fXa
activity in rats (data not shown). Quaternary ammo-
nium salt 10 showed the most potent in vitro anti-fXa
and PTCT2 activities, however the compound 10
exhibited only 20–30% of ex vivo anti-fXa activity.
The discrepancy would be attributable to its poor
availability.
However, N-isopropylcarbamoyl analogue 3g and N,N-
dimethylcarbamoyl 3h were less active. Furthermore,
N-(cyclohexylmethyl)carbamoyl derivative 3i had
markedly decreased activity. Being different from our
expectation, introduction of a series of carbamoyl
groups did not elevate anti-fXa activity. However, it is
interesting that the size of the N-substituents of the
carbamoyl group did not affect the potency: for
instance, carbamoyl derivatives 3c and N-(morpholine-
carbonymethyl)carbamoyl 3f were equipotent. In con-

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N. Haginoya et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2935–2939
Table 2. Ex vivo anti-fXa and anti-coagulant activities for compounds 3c and 3d
Compound
At 30 mg/kg (p.o.) to rats
Anti-fXa activity (%)a
3
Prolongation effect of PT (fold)a
1 h
1 h
h
6 h
3
h
3c
3d
67.4 1.5
89.5 0.8
55.8 1.5
73.7 2.0
24.7 6.0
23.5 2.6
1.13 0.02
1.35 0.01
1.09 0.02
1.14 0.02
1.05 0.00
1.04 0.01
a The measuring methods of ex vivo anti-fXa and anti-coagulant activities were described in Ref. 12. Values expressed as mean S.E. from four rats.
Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.;
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In the in situ intestinal loop of rats, compounds 3c and
3d were shown to be absorbed in 75% and 86%,
respectively.13 The result suggested good oral absorption
rates of 3c and 3d, which reflected on their ex vivo
activity.
4. Conclusion
The 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine
derivatives were synthesized. The class of compounds
showed potent and highly specific inhibitory activity for
factor Xa. Introduction of carbamoyl groups on the
piperazine ring improved in vivo activity markedly
without substantial change in anti-fXa activity. Indeed,
compounds 3c and 3d showed anti-fXa activity and anti-
coagulation activity in ex vivo test after oral adminis-
tration.
8. Wender, P. A.; White, A. W. Tetrahedron 1983, 39, 3767.
9. Anti-fXa activity in vitro. In vitro anti-fXa activity was
measured by using
a chromogenic substrate S-2222
(Chromogenix, Inc.) and human fXa (Cosmo Bio-ERL).
Aqueous DMSO (5% V/V; 10 lL) or inhibitors in aqueous
DMSO (10 lL) and 0.05 U/mL human fXa (10 lL) were
mixed with 0.1 M Tris–0.2 M NaCl–0.2% BSA buffer
(pH 7.4; 40 lL). The reaction was started by the addition
of 0.75 M S-2222 (40 lL). After the mixture was stirred for
10 s at rt, the increase of optical densities (OD/min) were
measured at 405 nm. Anti-fXa activity (inhibition %) was
calculated as follows: Anti-fXa activity ¼ 1 ) [(OD/min) of
sample/(OD/min) of control]. The IC50 value was obtained
by plotting the inhibitor concentration against the anti-
fXa activity.
Acknowledgements
The authors acknowledge Dr. J. Furukawa, Dr.
S. Kunitada and Dr. Y. Watanabe for useful discussion.
References and notes
€
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10. Anti-thrombin activity in vitro. In vitro anti-thrombin
activity was measured by using chromogenic substrate
S-2266 (Chromogenix, Inc.) and human thrombin (Sigma
Chemical, Inc.). The detail method was according to the
one for anti-fXa activity.
2. Verstrate, M.; Wessler, S. In Thrombosis in Cardiovascular
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12. Anti-fXa activity and anti-coagulant activity ex vivo. Male
wister rats were fasted overnight. Synthetic compounds
were dissolved in 0.5% (w/v) methylcellose solution and
administered orally to rats with a stomach tube. For
control rats, 0.5% (w/v) methylcellose solution was
administered orally. Rats were anesthetized with halo-
thane at several time points when blood samples were
collected in the presence of trisodiumcitrate. After blood
samples were centrifuged, the platelet poor plasma sam-
ples were used for measuring their anti-fXa activities or
anti-coagulant activities. Anti-Xa activity: Plasma (5 lL)
was mixed with 0.1 M Tris–0.2 M NaCl–0.2% BSA buffer
(pH 7.4; 40 lL), H2O (5 lL) and 0.1 U/mL human fXa
(10 lL). The reaction was started by the addition of 0.75 M

N. Haginoya et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2935–2939
2939
S-2222 (40 lL). After the mixture was stirred for 10 s at rt,
the increase of optical densities (OD/min) were measured
at 405 nm. Anti-fXa activity (inhibition %) was calculated
as follows: Anti-fXa activity ¼ 1 ) [(OD/min) of sample/
(OD/min) of control]. Anti-coagulant activity: Plasma
(20 lL) was mixed with inhibitors in saline (20 lL) in the
process tube. The coagulation was started by the addition
of SIMPLASTIN (40 lL). Anti-coagulant activity was
evaluated with the prolongation rate of prothrombin time
versus control.
13. Absorption rates in rat intestine were measured according
to the method described in the following: Hori, R.; Okano,
T.; Kato, M.; Maegawa, H.; Inui, K. J. Pharm. Pharma-
col. 1988, 40, 646.