Novel thrombin inhibitors incorporating non-basic partially saturated heterobicyclic P1-arginine mimetics

Article abstract of DOI:10.1016/S0960-894X(03)00030-1

The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetrahydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetrahydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P₁-arginine side-chain mimetics is described. The binding mode of the most potent candidate in the series co-crystallized with human α-thrombin, which exhibited an in vitro K_i of 140 nM and more that 478-fold selectivity against trypsin, is discussed.

Full text of DOI:10.1016/S0960-894X(03)00030-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 789–794
Novel Thrombin Inhibitors Incorporating Non-basic Partially
Saturated Heterobicyclic P1-Arginine Mimetics
Lucija Peterlin-Masic,a Gregor Mlinsek,b Tomaz Solmajer,b,c Alenka Trampus-Bakija,d
Mojca Stegnard and Danijel Kikelja,*
aFaculty of Pharmacy, University of Ljubljana, Asˇkerc˘eva 7, 1000 Ljubljana, Slovenia
bNational Institute of Chemistry, Hajdrihova 19, 1115 Ljubljana, Slovenia
cLek Pharmaceutical d.d., Drug Discovery, Verovsˇkova 57, 1526 Ljubljana, Slovenia
dUniversity Medical Centre, Department of Angiology, Riharjeva 24, 1000 Ljubljana, Slovenia
Received 10 October 2002; revised 5 December 2002; accepted 8 January 2003
Abstract—The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetra-
hydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetra-
hydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P1-arginine side-chain mimetics is described. The binding
mode of the most potent candidate in the series co-crystallized with human a-thrombin, which exhibited an in vitro Ki of 140 nM
and more that 478-fold selectivity against trypsin, is discussed.
# 2003 Elsevier Science Ltd. All rights reserved.
Thrombin is a multifunctional serine protease with
trypsin-like specificity, and plays a central role in
thrombosis and hemostasis by regulating the blood
coagulation cascade and platelet activation processes.
Serving as the terminal enzyme of the cascade, throm-
bin cleaves fibrinogen to fibrin, which ultimately com-
bines with platelets and other components to form a
blood clot. The limited efficacy and the side effects of
established antithrombotics, including warfarin,
heparin and acetylsalicylic acid, have provided the
impetus for developing alternative anticoagulants.1
Inhibition of thrombin is a prime target for therapeutic
intervention of thrombosis. During the last decade, the
search for orally bioavailable, potent and selective low
molecular weight thrombin inhibitors has become one
of the most intensively studied areas in drug
discovery.2
pocket and consequently most thrombin inhibitors
contain a P1 guanidine or amidine moiety.1,2,4 It was
often observed that thrombin inhibitors with strongly
basic P1 moieties have low selectivity and poor oral
bioavailability, resulting in metabolic instability and
poor absorbtion after peroral application.1,2,4 In order
to overcome the high basicity of the P1 guanidine, ami-
dine and aliphatic amine moieties, various amino het-
erocycles have been investigated as P1 arginine
mimetics. Among them, some bicyclic aromatic (ami-
no)heterocycles, for example 1-isoquinolinamine,5 3-
benzisoxazolamine,6 indole,7 benzimidazole,7 imi-
dazo[1,2-a]pyridine,8 indazole7,9 and pyrrolo[3,2-b]pyri-
dine10 were recently employed as P1 moieties in
tripeptidomimetic thrombin inhibitors.
In a previous paper we reported thrombin inhibitors
featuring P1 4,5,6,7-tetrahydrobenzothiazol-2-amine
moiety.11a In this article, we report the design, synthesis,
in vitro biological activities and a binding mode of novel
selective thrombin inhibitors incorporating different
weakly basic, partially saturated P1-heterobicyclic argi-
nine side-chain mimetics.11b
The enzyme S1 site of thrombin is a deep pocket with
Asp 189 at its bottom, capable of forming ionic and
hydrogen-bond interactions with positively charged
residues such as arginine (I) and lysine.3 Thus, throm-
bin prefers an arginine as P1 moiety in its S1 specify
Our approach to designing thrombin inhibitors based
on the d-Phe-Pro-Arg motif was to explore the S1
pocket of thrombin with weakly basic, partially satu-
*Corresponding author. Tel.: +386-1-476-9561; fax: +386-1-476-
9500; e-mail: danijel.kikelj@ffa.uni-lj.si
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00030-1

790
L. Peterlin-Masˇic˘ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 789–794
rated heterobicyclic arginine mimetics, which would
increase the selectivity of inhibitors for thrombin
against trypsin. l-Proline (a preferred ligand for inter-
action at the YPPW loop) was used as the P2 moiety
and diverse bicyclic arginine side chain mimetics as P1
moieties (Fig. 1). Ideally, our partially saturated het-
erobicyclic P1-arginine mimetics would exhibit a range
of pKa values, participate in hydrogen bonding inter-
actions with Asp 189, and benefit from hydrophobic
interactions at the S1 binding pocket. As it has been
shown previously, potent inhibitors with neutral P1
residues would require the use of lipophilic residues at
P3, in order to add more hydrophobic binding energy to
compensate losses at P1.12
Chemistry
The preparation of arginine mimetics 1, 3, 6 and 7 is
outlined in Scheme 1. Condensation of N-(4-oxocyclo-
hexyl)acetamide (8)16 with dimethyl-formamide dime-
thyl acetal (DMFDMA) provided enamino ketone 916,17
which, with guanidine hydrochloride, hydrazine hydrate
and N-methyl-hydrazine, afforded acetylated hetero-
cycles 10, 11 and 12. Final basic hydrolysis of 10 gave
5,6,7,8-tetrahydro-2,6-quinazolinediamine (3)16 whereas
acid hydrolysis of 11 and 12 afforded 5-amino-4,5,6,7-
tetrahydroindazole (1)17 and 5-amino-2-methyl-4,5,6,7-
tetrahydroindazole (6) as hydrochloride salts. Base-cat-
alyzed reaction of enamino ketone 9 with glycine and
acetic anhydride gave diamide 13 which, after basic
hydrolysis, afforded 5-amino-4,5,6,7-tetrahydro-iso-
indole (7).17
Arginine mimetics prepared as a part of this study are
listed in Figure 1. Their calculated pKa values13 range
from weakly basic 2-aminotetrahydroquinazoline 4
(pKa=4.7) and tetrahydroindazole 2 (pKa=4.0) to the
non-basic tetrahydroquinazoline 5 (pKa=2.4) and H-
bond donor tetrahydroisoindole 7 (pKa=2.9). A con-
venient synthetic approach to these novel partially
saturated, heterobicyclic arginine side-chain mimetics
1–7, containing a five- or six-membered N-heterocyclic
ring optionally substituted by amino or methyl group,
has been reported by us previously.14ꢀ16 We expected
that a bulky and lipophilic cyclohexane ring of III
would introduce conformational rigidity into the P1-
moiety of the inhibitor and confer selectivity for
thrombin against trypsin, since the selectivity pocket
S1 of thrombin is slightly larger and more lipophilic
than that of trypsin, in which it is narrowed by
Ser 190.
Scheme 2 outlines the synthesis of arginine mimetics 2, 4
and 5 with an aminomethyl group bound to the cyclo-
hexane ring.14,15 Condensation of N-[(4-oxo-cyclohex-
yl)methyl]acetamide (14)14 with DMFDMA and
reaction of the resulting enamino ketone 15 with guani-
dine hydrochloride, formamidine hydrochloride and
hydrazine hydrate afforded protected heterocycles 16,
17 and 18, which, on hydrolysis, gave 6-(amino-methyl)-
5,6,7,8-tetrahydro-2-quinazolinamine (4), 6-(amino-
methyl)-5,6,7,8-tetrahydroquinazolinamine (5) and
4,5,6,7-tetrahydroindazol-5-ylmethan-amine (2).14
Scheme 1. Reagents and conditions: (a) DMFDMA, Et3N, toluene,
reflux, 7 h; (b) guanidine hydrochloride/NaOEt, abs EtOH, reflux, 3 h;
(c) aq NaOH, MeOH, reflux 16 h; (d) hydrazine hydrate, EtOH, rt,
16 h; (e) 6 M HCl, reflux, 6 h; (f) methylhydrazine, EtOH, rt, 16 h; (g)
glycine, KOH, abs EtOH, reflux, 2 h; then Ac2O, reflux, 1 h.
Figure 1. Evolution of thrombin inhibitors incorporating weakly
basic, partially saturated heterobicyclic arginine side-chain mimetics,
with their calculated pKa values.

L. Peterlin-Masˇic˘ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 789–794
791
Km.18c The selectivity for thrombin against trypsin was
compared on the basis of the ratios Ki(trypsin)/Ki(thrombin)
.
The inhibitors which showed the most promising results
in amidolytic test were tested in standard clotting assays
including the thrombin time (TT), activated partial
thromboplastin time (APTT) and prothrombin time
(PT) determinations, which were used as a qualitative in
vitro indicator of potential antithrombotic activity.
Results and Discussion
Moderate to good levels of thrombin inhibition, with
Ki’s between 140 and 690 nM were observed in vitro, for
the best inhibitors. All new targets, which were tested
against FXa, were selective against this enzyme. Com-
pounds 23–38 also demonstrated good levels of selec-
tivity against trypsin.
Regarding structure–activity relationship of inhibitors
with different amino-linked P1 heterobicyclic residues
and fixed P2 and P3 moieties, in vitro potency decreased
as a function of the P1 group in the following order:
tetrahydroindazole (35, Ki=5.4 mM, pKa=3.7)>N-
methyltetrahydroindazole (37, Ki=12.8 mM, pKa
=3.2)>tetrahydrisoindole (36, Ki=27.3 mM, pKa=2.9)
>2-aminotetrahydroquinazoline (38, Ki >35.1 mM,
pKa=4.4). A methylene linker between the cyclohexane
ring and the amino group, which allows substantial
rotational freedom of the P1 part, was beneficial for
inhibitory activity against thrombin. Thus, compound
23 exhibited a 38-fold greater inhibitory potency than
the inhibitor 35. It is evident that, among tested P1
arginine side-chain mimetics the 4,5,6,7-tetra-
hydroindazole moiety is preferred for binding in the S1
selectivity pocket of thrombin.
Scheme 2. Reagents and conditions: (a) DMFDMA, Et3N, toluene,
reflux, 7 h; (b) guanidine hydrochloride/NaOEt, abs EtOH, reflux, 3 h;
(c) aq NaOH, MeOH, reflux 16 h; (d) formamidine hydrochloride/
NaOEt, abs EtOH, reflux, 4 h; (e) hydrazine hydrate, EtOH, rt, 16 h;
(f) 6 M HCl, reflux, 6 h.
The final coupling and elaboration reactions between
19–22 and the various arginine mimetics 1–7 are out-
lined in Scheme 3. Coupling reactions were performed
in DMF at room temperature using N-ethyl-N0-(3-
dimethylaminopropyl)-carbodiimide (EDC), 1-hydroxy-
benzotriazole (HOBt) as amide bond forming reagents
and N-methylmorpholine as base. Optional final depro-
tection of N-tert-butyloxycarbonyl (Boc) group was
effected by hydrogen chloride in glacial acetic acid
solution.
In the 4,5,6,7-tetrahydroindazole series, the most potent
compound 23, possessing 4,5,6,7-tetrahydroindazol-5-
ylmethanamine to fill the selectivity pocket, proline in
the central part and a benzylsulfonyl group attached to
d-cyclohexylalanine as the P3 part of the inhibitor, was
found to be the most potent, with Ki value of 140 nM
and more than 478-fold selectivity for thrombin over
trypsin. It doubled the TT, APTT and PT at con-
centrations of 11.0, 42.3 and 78.3 mM, respectively. The
replacement of the benzylsulfonyl group in compound
23 by an N-carboxymethyl group in 24 brought a 2-fold
decrease in inhibitory potency (Ki=280 nM), a decrease
in selectivity for thrombin over trypsin and higher
activity in all plasma-based clotting assays. The com-
pound 24 doubled the TT, APTT and PT at concentra-
tions of 4.9, 28.0 and 55.3 mM, respectively. Inhibitor
25, featuring Boc-d-3,3-(Ph)2-Ala at P3, displayed a Ki
for thrombin of 530 nM and the corresponding free
amino analogue 26 inhibited thrombin with a Ki of
360 nM. Analogue 27, with Boc-d-3,4-Cl2-Phe in the P3
position, had an inhibition constant of 3.3 mM and the
Ki of the corresponding free amino analogue 28 was 5-
fold lower (Ki=0.69 mM).
The in vitro biological activity of the inhibitors 23–38 is
summarized in Table 1. The ability of new thrombin
inhibitors to inhibit the enzymatic action of thrombin,
trypsin and factor Xa was measured with the amidolytic
enzyme assay using S-2238 as a chromogenic sub-
strate.18a Values of Ki were calculated according to
Cheng and Prusoff,18b based on IC50 values, or from a
relation between reaction velocity equations in the
absence and presence of inhibitor using the relevant
Scheme 3. Coupling and elaboration to targets 23–38. Reagents and
conditions: (a) 1–7, HOBt, N-methylmorpholine, EDC, DMF; rt, 12–
15 h; (b) optional clevage of Boc protecting group: for 26, 28, 31 and
33: HClg, HOAc, rt, 0.5 h.
In the 2-aminoquinazoline series, the most potent ana-
logue 30, which featured Boc-d-3,3-(Ph)2-Ala at P3,

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L. Peterlin-Masˇic˘ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 789–794
Table 1. Inhibitory potencies of compounds 23–38
Compd
R4
R3
R2
R1
Ki (mM)
Selectivity
thrombin/trypsin
APTT
PT
TT
Thrombin
Trypsin
FXa
(mM)
23
24
25
26
27
28
SO2CH2Ph
CH2COOH
H
H
Ph
Ph
H
Chx
Chx
Ph
Ph
0.14
0.28
0.53
0.36
3.33
0.69
>68.3
19.9
>75.4
84.8
85.2
82.1
>75.4
>75.4
>478
71
ND81
602
>21
>99
42.3
28
105
43
NDNDND
NDNDND
78.3
55.3
29.2
59.8
11
4.9
a
Boc
H
Boc
H
216.6
>68.3
>68.3
7.7
3,4-Cl2–Ph
3,4-Cl2–Ph
H
29
30
31
32
33
SO2CH2Ph
H
Ph
Ph
H
Chx
Ph
Ph
3,4-Cl2–Ph
3,4-Cl2–Ph
>35.1
4.5
12.9
>35.1
>35.1
>68.3
>75.4
a
NDNNDDND
NDNNDDND
32
NDNNDDND
NDNNDDND
a
Boc
H
Boc
H
409
>68.3
>68.3
57.6
>75.4
>75.4
NDNDND
H
34
35
36
37
38
SO2CH2Ph
SO2CH2Ph
SO2CH2Ph
SO2CH2Ph
SO2CH2Ph
H
H
H
H
H
Chx
Chx
Chx
Chx
Chx
289
5.38
342
111
1.2
NDNDND
NDNDND
>68.3
415.8
730.3
>68.3
>75.4
>13
27.3
ND15 ND ND ND
ND57 ND ND ND
12.8
>35.1
>75.4
NDNNDDND
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; Chx, cyclohexyl.
aDue to precipitation measurements, were not possible.
displayed a Ki for thrombin of 4.5 mM and the corre-
sponding free amino analogue 31 inhibited thrombin
with a Ki of 12.9 mM. Analogues 29, 32 and 33 demon-
strated no inhibitory activity against thrombin (Ki
>35.1 mM) and trypsin (Ki >68.3 mM). Similarly, com-
pound 34 with tetrahydroquinazoline at P1 was also
devoid of inhibitory activity against thrombin
(Ki=289 mM).
The inhibitor 23 forms seven hydrogen bonds to the
surrounding residues and two bonds to water molecules.
As illustrated in Figure 3, both 4,5,6,7-tetra-
hydroindazole nitrogens (N1 and N2) form hydrogen
bonds with Od1 Asp189 while contacts to Od2 Asp189
are not present. The bond length between N2 and Od1
Asp189 is 2.72 A and between N1 and Od1 Asp189 is
2.67 A. At the S1 specify pocket other interactions of the
P1 residue include hydrogen bonds between tetra-
hydroindazole N1 and Ala190 (3.09 A) and hydrogen
bond between N2 of tetrahydroindazole and a water
molecule 34 (3.40 A).
Crystals of thrombin with the inhibitor 23 were pre-
pared using 23 as a diastereomeric mixture with respect
to the stereogenic center at C5 of the 4,5,6,7-tetra-
hydroindazole ring (Fig. 2)19. Based on the electron
density we concluded that both R and S configurations
can bind into the S1 pocket. Although our QM/MM
calculations indicate that R configuration binds energe-
tically more favorably, the electron density is slightly
more compatible with the S configuration.
The oxygen atom of the cyclohexylalanine carbonyl
group forms a weak hydrogen bond to Gly216 (3.20A) of
the thrombin b sheet. The aminomethyl group on the
tetrahydroindazole moiety participates in
a weak
hydrogen bond interaction to the Og Ser 215 (3.51 A) of

L. Peterlin-Masˇic˘ et al. / Bioorg. Med. Chem. Lett. 13 (2003) 789–794
793
interactions with Asp189. However, thrombin inhibitors
with non-basic P1 moieties should posses better pharma-
cokynetic profiles than inhibitors with highly basic P1
moities.21 The results of our studies towards orally bioa-
vailable thrombin inhibitors based on 23 are in progress.
In conclusion, we have designed and evaluated a novel
class of non-covalent thrombin inhibitors incorporating
novel, weakly basic, partially saturated heterobicyclic
P1-arginine side-chain mimetics, while maintaining the
intrinsically potent P2-proline and P3-lipophilic phar-
macophores in the S2 and S3 pockets. Potent and selec-
tive thrombin inhibitors were identified, with Ki for
thrombin of 140 nM and a more that 478-fold selectivity
against trypsin for the most potent candidate 23, which
serves, supported by the results of the crystal structure
of 23 complexed in the thrombin active site, as an
attractive lead for further SAR development.
Figure 2. Connolly surface map of the X-ray structure of the a-
thrombin-inhibitor 23 complex at 2.4 A resolution. The inhibitor is
shown as sticks. Colors green, blue, red and yellow identify carbon,
nitrogen, oxygen and sulphur, respectively.
Acknowledgements
The authors thank Dr. T. B. Tschopp and Dr. B. Steiner
(Pharma Division, Preclinical Research, F. Hoffmann
La Roche Ltd, Basel, Switzerland) for biological testing
of some compounds, Dr. W. Bode (Max Planck Institut
fur Biochemie, Martinsried, Germany) for X-ray struc-
tures and Dr. Roger Pain (Jozef Stefan Institute, Ljubl-
jana, Slovenia) for critical reading of the manuscript.
Financial support of this work by the Ministry of Edu-
cation, Science and Sport of the Republic of Slovenia
(Grant No P0787-502 and L3-2034) and by Lek Phar-
maceuticals d.d., Ljubljana is gratefully acknowledged.
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