Novel thrombin inhibitors incorporating weakly basic heterobicyclic P1-arginine mimetics: optimization via modification of P1 and P3 moieties.

Article abstract of DOI:10.1016/j.bmcl.2004.03.085

Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P(1)-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors 17-29 and enantiomerically pure thrombin inhibitors 30-33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-ylamine.

Full text of DOI:10.1016/j.bmcl.2004.03.085

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3251–3256
Novel thrombin inhibitors incorporating weakly basic
heterobicyclic P1-arginine mimetics: optimization via
modification of P1 and P3 moieties
a
Andreja Kranjc, Lucija Peterlin-Masic, Janez Ilas, Andrej Prezelj,
b
ꢀ ꢀ a
ꢀ a
ꢀ
Mojca Stegnarc and Danijel Kikelja,*
aFaculty of Pharmacy, University of Ljubljana, Aꢀskerꢀceva 7, 1000 Ljubljana, Slovenia
bLek Pharmaceuticals d.d., Drug Discovery, Verovꢀskova 57, 1526 Ljubljana, Slovenia
cUniversity Medical Centre, Department of Angiology, Zaloꢀska 7, 1525 Ljubljana, Slovenia
Received 27 January 2004; revised 16 March 2004; accepted 29 March 2004
Abstract—Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating
weakly basic heterobicyclic P1-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors 17–29
and enantiomerically pure thrombin inhibitors 30–33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-
tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-yl-
amine.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The development of an orally active thrombin inhibitor
as an anticoagulant is one of the major focuses of the
current pharmaceutical research.1 Thrombin is the last
enzyme in a cascade of trypsine-like plasma serine pro-
teases that are involved in blood coagulation and plays a
pivotal role in both fibrin generation penultimate to clot
formation and platelet activation.2 Although the cur-
rently available anticoagulant agents, including heparin,
warfarin, and acetylsalicylic acid, have provided
remarkable achievements in the treatment of thrombo-
embolic diseases, these drugs have considerable limita-
tions associated with a risk of bleeding and the
inconvenience posed by the need for routine coagulation
monitoring and/or parenteral administration. A key
strategy to overcome these limitations has been directed
toward the discovery of small-molecule inhibitors of the
coagulation cascade enzymes, which possess the phar-
macokinetic properties required for oral administration
once or twice daily.3 Due to its central role in throm-
bosis and hemostasis, thrombin is a prominent target in
the development of new anticoagulants with desired
properties.
The typical feature of the variety of small-molecule
direct thrombin inhibitors is the presence of a highly
basic guanidine or amidine moiety. However, strongly
basic P1 groups will tend to hinder absorption across the
gut wall and may be associated with high plasma
clearance and unwanted side effects.4 In the effort to
produce orally bioavailable thrombin inhibitors we have
focused on design and synthesis of compounds that
incorporate weakly basic S1 binding moieties.5 Toward
this goal we previously reported on series of proline-
based thrombin inhibitors6 and 3-amino-2-pyridinone
acetamide thrombin inhibitors7 incorporating weakly
basic partially saturated heterobicyclic P1-arginine
mimetics. In the last series inhibitors with 4,5,6,7-tetra-
hydro-1,3-benzothiazole-2,6-diamine and 6-amino-
methyl-4,5,6,7-tetrahydroindazole P1 moieties exhibited
the best binding affinities for thrombin and excellent
selectivity against trypsin (Fig. 1). Based on the X-ray
structures of complexes of thrombin inhibitors 1 and 2
with human thrombin,7 our effort was directed toward
optimization of the binding affinity of lead structures 1
and 2.
In this article, as a part of optimization strategy of leads
1 and 2, we disclose the design, synthesis and biological
profiles of novel pyridinone acetamide thrombin inhib-
itors 17–33 incorporating weakly basic P1-heterobicyclic
Keywords: Thrombin; Thrombin inhibitors; Arginine mimetics.
* Corresponding author. Tel.: +386-1-4769561; fax: +386-1-4258031;
e-mail: danijel.kikelj@ffa.uni-lj.si
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.085

3252
A. Kranjc et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3251–3256
NH2
NHCOCH3
NH2*HC l
NH2*HBr
Thrombin Ki =120 nM
Trypsin Ki =260,000 nM
1:R=
c
a
b
*2HBr
S
S
56%
N
100%
Br
O
O
O
NH2
N
O
O
O
S
N
R
NH
N
N
H2N
H
H
7
8
9
10
O
Thrombin Ki =170 nM
Trypsin Ki => 500,000 nM
Scheme 1. Reagents and conditions: (a) 6 M HCl, reflux, 6 h; (b) Br2,
47% HBr, 0 ꢁC, 15 min, then rt, 30 min; (c) thiosemicarbazide, 47%
HBr, rt, 30 min, then reflux, 1.5 h.
2:R =
N
N
H
Figure 1. Structure of lead compounds 1 and 2.
O
O
O
a
O
R
b
N
N
S
H2N
O
O
N
H
O
O
O
12
11
arginine side-chain mimetics and different lipophilic P3
residues. The arginine side-chain mimetics of low bas-
icity used in this study are 4,5,6,7-tetrahydro-1,3-benzo-
thiazol-2-amine derivatives 3 and 4, 4,5,6,7-tetrahydro-
indazole 5 and 2-imino-4,5,6,7-tetrahydro-1,3-benzo-
thiazol-3(2H)-ylamine (6) (Fig. 2). Their calculated pKa
values8 range from 3.17 [2-imino-4,5,6,7-tetrahydro-1,3-
benzothiazol-3(2H)-yl-amine (6)] to 6.5 [4,5,6,7-tetra-
hydro-1,3-benzothiazol-2-amine (4)].
O
O
c
O
Arginine
mimetic
O
O
N
N
S
S
N
N
N
H
OH
R
R
H
H
O
O
14
13
Scheme 2. Reagents and conditions: (a) RSO2Cl,9 CH2Cl2, Et3 N, 0 ꢁC
to rt, 2h; (b) HCl g, EtOAc, 0 ꢁC, 20 min; (c) arginine mimetics 3–6,
EDC, HOBt, NMM, DMF, rt, 12h.
As the crystal structures of thrombin inhibitors 1 and 2
complexed with human thrombin revealed a preferential
binding of R enantiomers of inhibitors 1 and 2 to the
active site of thrombin,7 we prepared also the pure
enantiomers 30–33 of the inhibitors 1 and 20 and mea-
sured their in vitro inhibitory potency (Table 2). In the
synthesis of the pure enantiomers, optical resolution of
( )2,6-diamino-4,5,6,7-tetrahydrobenzothiazole (3) with
A convenient synthetic approach to conformationally
restricted arginine side chain mimetics 3–5 has been
reported by us previously.5a–e;6 The synthetic route to
3-amino-2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-
6(2H)-ylamine (6) based on a general synthesis of
2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3-ylamines5f
is summarized in Scheme 1. Acidic hydrolysis of N-(4-
oxocyclohexyl)acetamide (7)5b gave 4-aminocyclohexa-
none (8), which provided 3-bromo derivative 9 after
bromination with bromine. In the next step a classical
Hantzsch thiazole synthesis applying thiosemicarbazide
as an S–C–N synthon afforded compound 10. Scheme 2
outlines the synthetic route to P3–P2 pyridinone syn-
thons 13 and the ultimate coupling reactions between
P3–P2 synthons 13 and the P1-arginine mimetics, which
afforded final inhibitors 14. The coupling reactions were
performed using EDC, HOBt, and N-methylmorpholine
in dry DMF at room temperature.
L
- and D-tartaric acid was employed affording (S)-2,6-
diamino-4,5,6,7-tetrahydrobenzothiazole
(15)
and
(R)-2,6-diamino-4,5,6,7-tetrahydrobenzothiazole (16).10
The ability of new thrombin inhibitors to inhibit the
enzymatic action of thrombin, trypsin, and factor Xa
was measured with the amidolytic enzyme assays using
chromogenic substrates.11a Values of Ki were calculated
according to Cheng and Prusoff,11b based on IC50 values
or from a relation between reaction velocity equations in
the absence and presence of inhibitor, using the relevant
H3CCONH
H2N
pKa = 6.1
N
3
H3C
N
S
[ ]n
S
n = 2, 3
NH2
NH2
n = 0,1
H3CCONH
O
O
O
S
N
pKa = 6.5
[ ]n
H2N
N
N
N
H
R
H
S
O
N
Het
NH2
4
H3CCONH
H3CCONH
S
NH2
R1
R2
N
pKa = 4.0
N
H2N
H
H2N
N
R1 = 4-F, R2 = H
N
NH2
N
H
5
R1 = 4-CH3, R2 = H
R1 =3-CF3, R2 = H
R1 = 4-F, R2 = 2-Cl
NH2
pKa = 3.17
N
S
S
NH
6
NH
Figure 2. Evolution of 3-amino-2-pyridinone acetamide thrombin inhibitors incorporating weakly basic, partially saturated heterobicyclic
P1-arginine side chain mimetics with their calculated pKa values8 and different P3 lipophilic residues.

A. Kranjc et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3251–3256
Table 1. Inhibitory potencies of lead compounds 1, 2, and thrombin inhibitors 17–29
3253
O
O
O
R1
S
N
R2
N
N
H
H
O
Compound R1
R2
Ki (lM)
Selectivity
thrombin/
trypsin
2 · APTT
PT
(lM)
TT
Thrombin Trypsin FXa
S
1
2
CH2
CH2
0.12
0.17
260
>500
ND
92
2166
36.5
20.5
41.4
30.6
6.6
NH2
N
N
NH
103
>2941
5.2
S
17
18
NH2
H3CCH2CH2
H3CCH2CH2
3.8
ND
ND
28
ND
ND
ND
ND
N
S
N
S
2.30
64.3
200
ND
ND
NH2
19
20
H3CCH2CH2CH2
1.34
50.2179
38
ND
ND
ND
NH2
NH2
N
S
CH2
CH2
0.100
>800
>300
>8000
28.7
57.7
5.1
F
N
N
NH
21
22
0.149
0.100
258
246
314
80
1725
2460
15.8
31.3
34
56
3.9
F
S
12
CH2
H3C
NH2
N
N
NH
23
24
0.102
0.157
>300
1923
228
69
>2900
1097
18.8
94
26.8
3.4
CH2
H3C
F
S
CH2
ND
93.8
NH2
N
Cl
Cl
N
CH2
F
NH
25
26
0.157
0.652
>300
>200
425
75
81
>1910
>300
50
120
435
10.8
CH2
S
428
125
NH2
N
F3C
F3C
N
CH2
NH
27
28
0.562
0.042
252
757
65
447
293
ND
36.6
S
NH
NH
CH2
2.71 >200
2.4
ND
N
NH2
S
29
CH2
0.022
8.06
131
201
22.6
ND
ND
F
N
NH2
APTT: concentration of inhibitor required to double the activated partial thromboplastin time in human plasma.
PT: concentration of inhibitor required to double the prothrombin time in human plasma.
TT: concentration of inhibitor required to double the thrombin time in human plasma.
ND: not determined.

3254
A. Kranjc et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3251–3256
Table 2. Inhibitory potencies of compounds 30–33
O
O
O
S
N
R2
R1
N
H
O
Compound
R1
R2
Ki (lM)
Selectivity
thrombin/
trypsin
2 · APTT
(lM)
Thrombin
0.071
Trypsin
FXa
H
N
S
30
31
CH2
CH2
263
294
225.2
3704
253
9.52
ND
NH2
N
H
N
S
1.16
370.6
NH2
N
H
N
S
32
33
CH2
CH2
0.077
1.44
>700
>700
>500
>9091
>123
20.1
ND
F
F
NH2
N
H
N
S
176.4
NH2
N
Km.11c The selectivity for thrombin over trypsin was
compared on the basis of the ratios Ki(trypsin)/Ki
(thrombin). In addition, the inhibitors were tested in
standard clotting assays including the thrombin time
(TT), activated partial thromboplastin time (2 · APTT)
and prothrombin time (PT) determinations, which were
used as qualitative in vitro indicators of potential anti-
thrombotic activity.
improve the binding affinity and retain selectivity we
synthesized a series of compounds with different benzyl-
sulfonamido P3 parts substituted on phenyl ring bearing
2-amino-4,5,6,7-tetrahydrothiazole arginine mimetic 3
or 4,5,6,7-tetrahydroindazole arginine mimetic 5 in P1
part of the inhibitors. From Table 1, it is clearly seen
that p-fluoro- and p-methylbenzylsulfonamido deriva-
tives 20–23 showed the best Ki values and excellent
selectivity. It is also evident that both arginine mimetics
used in the study contribute equally to the binding
affinity in respect of Ki values for thrombin, although
when comparing 2 · APTT values of inhibitors 20–23 it
can be concluded that tetrahydroindazole inhibitors 21
and 23 show a slightly superior anticoagulant activity
in vitro. Introduction of o-chloro-p-fluorobenzyl moiety
in inhibitors 24 and 25 led to a slight decrease in binding
affinity. Additionally, the 2 · APTT values increased
significantly comparing to values for 20–23, which is
probably due to increased lipophilicity. Incorporation of
trifluoromethylbenzyl R2 substituent in both series
afforded compounds 26 and 27 with a 5-fold loss in
thrombin inhibition compared to the lead compounds 1
and 2.
The in vitro inhibitory potencies of inhibitors 17–33
along with those of the lead compounds 1 and 2 are
summarized in Tables 1 and 2. Starting from the lead
structure 1 we synthesized analogues 17–19 with n-pro-
pyl and n-butyl group in P3 part of the molecules to
examine the contribution of different lipophilic residues
in P3 part to binding affinity. The resulting compounds
turned out to be of one order of magnitude less active
than 1, clearly demonstrating the importance of the
interactions of the benzyl group in 1 with the lipophilic
distal pocket of thrombin. This finding was not a sur-
prise because the X-ray crystal structure of compounds
1 and 2 complexed with human thrombin revealed a
good fit of the benzyl group in the distal pocket of the
enzyme. A methylene linker between the cyclohexane
ring and the amino group in R1 substituent of com-
pound 17 was not beneficial for the inhibitory potency
as compound 18 with amino group bound directly to the
cyclohexane ring had a slightly better binding affinity
than compound 17. On the contrary, in the tetrahydro-
indazole type of inhibitors lacking the terminal amino
group the methylene linker between the heterocycle and
amino group, which allows substantial rotation freedom
of the P1 part is favorable for inhibitory activity against
thrombin.7
In an effort to obtain further improvement of Ki values
modifications were performed also in P1 part of the
inhibitors. In compounds 28 and 29 3-amino-2-imino-
4,5,6,7-tetrahydro-1,3-benzothiazol-6(2H)-ylamine (6)
was introduced as a new arginine mimetic. This modi-
fication resulted in an improved affinity for thrombin
with Ki values for inhibitors 28 and 29 of 42and 22nM,
respectively. Although 29 displayed the best Ki value,
in vitro clot inhibition (2 · APTT) was better with the
compound 28, which is likely due to the differences in
physicochemical properties of 28 and 29 (e.g., increased
lipophilicity in compound 29 and consequently a higher
binding to plasma proteins3b). The new arginine mimetic
In a further step the attention was turned to substituted
benzyl moieties in P3 part of the inhibitors. In order to

A. Kranjc et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3251–3256
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is weakly basic (calculated pKa is 3.17), which is crucial
in an effort to produce orally bioavailable thrombin
inhibitors. Unfortunately, compounds 28 and 29 showed
also better affinity for trypsin than compounds 17–27.
However, as their affinity for thrombin is much higher
than for trypsin they still demonstrate a moderate level
of selectivity.
Based on the crystal structure of 1 complexed to human
thrombin, evidencing a preferential binding of R enan-
tiomer to the active site of thrombin, we prepared the
single enantiomers 30–33 of the existing racemates 1 and
20. The Ki values confirmed that the R enantiomer binds
with approximately 20-fold higher affinity to the
thrombin active site than the S enantiomer. The selec-
tivity of the R enantiomers 30 and 32 was also increased
in comparison to racemates 1 and 20. Thus, compounds
30 and 32 are potent and selective thrombin inhibitors
with Ki values of 71 nM and 77 nM, respectively. The
2 · APTT values for compound 30 is significantly lower
than for compound 33, which again could be explained
on the basis of a higher lipophilicity of the inhibitor 33.
In conclusion, optimization of lead compounds 1 and 2
led to novel noncovalent thrombin inhibitors 20, 23, and
29 in racemic form and inhibitors 30–33 as single
enantiomers, which all incorporate weakly basic hete-
robicyclic P1-arginine side-chain mimetics. This study
confirmed a preferential binding of R enantiomer of
inhibitor 1 in the thrombin active site, which was earlier
proposed on the basis of a crystal structure.
Acknowledgements
4. Sanderson, P. E. J.; Stanton, M. G.; Dorsey, B. D.; Lyle,
T. A.; McDonough, C.; Sanders, W. M.; Savage, K. L.;
Naylor-Olsen, A. M.; Krueger, J. A.; Lewis, S. D.; Lucas,
B. J.; Lynch, J. J.; Yan, Y. W. Bioorg. Med. Chem. Lett.
2003, 13, 795.
Financial support of this work by the Ministry of
Education, Science and Sport of the Republic of
Slovenia and by Lek Pharmaceuticals d.d., Ljubljana is
gratefully acknowledged.
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