Thrombin active site inhibitors: Chemical synthesis, in vitro and in vivo pharmacological profile of a novel and selective agent BMS-189090 and analogues

Article abstract of DOI:10.1016/S0960-894X(01)00664-3

A series of structurally novel small molecule inhibitors of human α-thrombin was prepared to elucidate their structure- activity relationships (SAR), selectivity and activity in vivo. BMS-189090 (5) is identified as a potent, selective, and reversible inh

Full text of DOI:10.1016/S0960-894X(01)00664-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 41–44
Thrombin Active Site Inhibitors: Chemical Synthesis, In Vitro and
In Vivo Pharmacological Profile of a Novel and Selective Agent
BMS-189090 and Analogues
Jagabandhu Das,* S. David Kimball, Joyce A. Reid, Tammy C. Wang, Wan F. Lau,y
Daniel G. M. Roberts, Steven M. Seiler, William A. Schumacher and Martin L. Ogletree
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 16 July 2001; accepted 14 September 2001
Abstract—A series of structurally novel small molecule inhibitors of human a-thrombin was prepared to elucidate their structure–
activity relationships (SAR), selectivity and activity in vivo. BMS-189090 (5) is identified as a potent, selective, and reversible
inhibitor of human a-thrombin that is efficacious in vivo in a mice lethality model, and in inhibiting both arterial and venous
thrombosis in a rat model. # 2001 Elsevier Science Ltd. All rights reserved.
Thrombin is the serine protease that occupies the cen-
tral position in the blood coagulation cascade.1 It pro-
teolytically cleaves A-a and B-b chains of fibrinogen
with release of fibrin monomer which are polymerized
and covalently linked by thrombin activated factor
XIIIa. In addition thrombin amplifies the coagulation
cascade by activating factors V and VIII, and activates
platelets through its surface receptors leading to platelet
shape change and aggregation.2 Accordingly, direct
inhibition of thrombin has attracted considerable atten-
tion as a therapeutic target for the management of
thrombotic diseases including arterial and venous
thrombosis, transient ischemic attack, stroke and
coronary artery bypass surgery.3
thrombin inhibitors have been developed. We have been
able to modify these compounds to obtain highly potent
and selective reversible inhibitors of thrombin active site
that demonstrate good antithrombotic activity in vivo.
The synthesis of this class of compounds follows a gen-
eral synthetic route that is illustrated in Scheme 1 with
the preparation of 5 (BMS-189090).
Treatment of tosylate 17 with 1.5 equiv of sodium
azide followed by removal of BOC-protecting group
and subsequent coupling of the crude amine with Na-
BOC-l-benzyloxyserine afforded azide 2 in 92% yield.
Over the past decades development of direct acting
thrombin inhibitors4 has been an area of intense
research. Our drug discovery effort in this arena has
focused on discovering structurally novel, reversible and
direct acting inhibitors that have no electrophile. By
combining crystallographic data from the structure of
Argatroban with trypsin,5 and the initial SAR of a
novel series of tripeptide inhibitors,6 we designed a new
template for building thrombin active site inhibitors
(Fig. 1). This template is widely amenable to synthetic
modification, and has provided a motif from which new
*Corresponding author. Tel.: +1-609-252-5068; fax: +1-609-252-
6804; e-mail: jagabandhu.das@bms.com
yCurrent address: Pfizer Central Research, Eastern Point Road, Gro-
ton, CT 06140, USA.
Figure 1. Schematic representation of novel inhibitor design from
Argatroban and BMS-183507.
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00664-3

42
J. Das et al. / Bioorg. Med. Chem. Lett. 12 (2002) 41–44
Table 2. SAR from the P1–P2 linker modification
Compd
X
Thrombin Compd
IC50 (mM)10
X
Thrombin
IC50 (mM)10
10
11
12
13
CONHCH2
CH2SCH2
CH2S
2.8
1.4
7.4
0.39
14 CH2NHCOCH2
15 CH2NHCO(CH2)2
16 CH2NHCO(CH2)3 14
0.08
0.05
Scheme 1. Synthesis of 5: (a) NaN3, DMSO, 70 ꢀC; (b) TFA, CH2Cl2,
0 ꢀC; (c) EDAC, HOBt, BOC-l-Ser(OBn)-OH, DMF, NMM; (d)
CH2Cl2, Et3N, naphthalene-2-sulfonyl chloride; (e) 10% Pd–C, H2,
EtOH; (f) EDAC, HOBt, (S)-N-CBZ-nipecotic acid, NMM, DMF; (g)
10% Pd–C, H2 (55 psi), EtOH, AcCl; (h) DMF, i-Pr2NEt, pyrazole-1-
carboxamidine.
CH2NHCO
only 5-fold drop in activity (Table 1). Accordingly
further optimization studies were undertaken with 10.
BOC-deprotection followed by reaction of the crude
amine with naphthalene-2-sulfonyl chloride and sub-
sequent hydrogenation of the chromatographically purified
azide formed amine 3 in 66% yield. Coupling of the
crude amine 3 with (S)-Na-CBZ-nipecotic acid8 formed
amide 4 in 77% yield. Removal of CBZ and benzyloxy
protecting groups under hydrogenolysis conditions,
followed by reaction of the amine with pyrazole-1-carbox-
amidine9 in DMF in presence of Hunig’s base afforded 5
(BMS-189090) in 64% overall yield after purifications
by reverse-phase preparative HPLC.
Table 2 outlines the SAR observed with the modifica-
tion of the side chain connecting the pyrrolidine ring at
P2 and the guanidine moiety that binds to an aspartic
acid residue (Asp) in the specificity pocket of thrombin.
Replacement of the –CONHCH2– linker of 10 with
thioether linkages (11 and 12) has a modest effect in
thrombin inhibitory activity in vitro. In contrast, repla-
cement by a reverse amide linker –CH2NHCO– (13)
resulted in a 7-fold increase in activity. In this reverse
amide series 4-5 methylene linker (14 and 15) appears to
be optimal for thrombin inhibitory activity.
Compound 5 and its analogue were tested for their
ability to inhibit thrombin hydrolysis10 of the chromo-
genic substrate S-2238 (IC50, Tables 1–5).
In order to further optimize the potency we investigated
the effect of introducing a constrained hydrophobic ring
binding to the P1 specificity pocket of thrombin (Table
3). Introduction of a 3-substituted piperidine ring (17,
mixture of diastereomers) resulted in a 12-fold increase
in activity relative to 13. The corresponding 4-sub-
stituted piperidine analogue 19 is 3-fold more potent
than 7. Synthesis of the enantiomerically pure diaster-
eomers of 17 (5 and 18) demonstrates that the thrombin
inhibitory activity resides primarily with the S-nipecot-
amide analogue 5 (BMS-189090). Extending the linker
chain by a methylene unit in both 17 and 19 (data not
shown) significantly attenuated the activity in vitro
while the 4-benzamidine analogue 21 is found to be
equipotent to 5 which is attributed to the increased
hydrophobic interactions of the phenyl ring and strong
hydrogen bond interaction of the amidine moiety with
Asp189 residue in the P1 specificity pocket of thrombin.
The prototypical analogue 6 (BMS-184919, Fig. 1) of
the series has modest potency against thrombin in vitro.
In contrast, the diastereomeric analogue 7 is roughly 3-
fold more potent than 6. The preferred stereochemistry
of the azacycloalkyl ring at P2 (2S,4S) is the mirror
image of the preference anticipated from the solid state
structure of Argatroban. Removal of the hydroxy-
methyl group at P3 in both 6 and 7 resulted in a
substantial drop in potency. More importantly sub-
stitution of the pipecolic acid moiety at P2 of 7 with
synthetically more accessible S-proline (10) resulted in
Table 1. SAR from the P2 azacycloalkyl modification
Further optimization on activity of 5 relied on mod-
ification of P3 substituent, and is reported in Table 4.
Substitution of the P3 a-carbon with either hydrogen or
a methyl group (22 and 23) significantly reduced the
activity relative to the hydroxymethyl substituted ana-
logue 5. Neither the threonine or allo-threonine derived
analogues (24 and 25, respectively) were as potent as 5.
In contrast the methylaspartate derived analogue 26 was
found to be equipotent to 5. Presumably the carbo-
methoxy group in 26 is engaged in a similar kind of
hydrogen bond interaction as the hydroxymethyl
function in 5 with backbone Gly219 of thrombin.12
Compd
R
Stereochemistry
Thrombin
IC50 (mM)10
6
7
8
9
10
CH2OH
CH2OH
H
H
CH2OH
2R,4R
2S,4S
2R,4R
2S,4S
S-Pro
1.7
0.62
27
2.9
2.8

J. Das et al. / Bioorg. Med. Chem. Lett. 12 (2002) 41–44
Table 3. SAR from the P1–P2 linker modification
Table 5. Enzyme selectivity of selected thrombin inhibitors11
43
Compd
Thrombin Trypsina Plasmina
IC50 (mM)10
tPAa
Factor Xaa
13
5
21
0.39
8
8000
4
658
0.4
500
>800
300
500
200
>1700
300
0.018
0.013
0.038
0.018
>18,000 >18,000
200
4900
13,000
700
6900
200
Argatroban
Efegatran
Compd
R
Thrombin Compd
IC50 (mM)10
R
Thrombin
IC50 (mM)10
aSelectivity ratio: IC50/IC50 (thrombin).
iv; 29 mpk po) was found to be similar to that of Efe-
gatran (ID50=0.21 mpk, iv; 23 mpk po) and sig-
nificantly superior to that of Argatroban (ID50=1.6
mpk, iv; >100 mpk po). BMS-189090 competitively
17
0.032
19
0.11
5
0.016
0.50
20
21
0.20
and
reversibly
inhibited
thrombin
in
vitro
(Ki=3.44Æ0.05 nM)14 without showing time dependent
kinetics like Efegatran which is characterized as a slow-
binding tight inhibitor. In gel-filtered platelets BMS-
189090 inhibited thrombin induced platelet aggregation
with a pA2 value of 8.61 (n=2). A modified thrombin
time (TT) was used to determine the direct inhibition of
thrombin activity in a protein-rich plasma environment.
In this assay, BMS-189090 doubled thrombin clotting
time in vitro at 61Æ2.3 nM( n=3).
18
0.013
Selected inhibitors of this class were evaluated for their
specificity over other serine proteases including trypsin,
t-PA, plasmin and Factor Xa (Table 5). A clinically
useful thrombin inhibitor should not modulate the
fibrinolytic processes through inhibition of plasmin and
t-PA.
In pentobarbital-anesthetized rats BMS-189090 inhib-
ited thrombosis in a dose-dependent manner (Fig. 2).
Thrombosis was induced in either the carotid artery by
topical FeCl2 application or in the vena cava by a com-
bination of blood stasis and endothelial disruption
caused by hyptonic saline infusion.15 Near complete
inhibition of venous thrombosis (98%) was obtained at
a dose of 0.5 mg/kg+25 mg/kg/min iv. A lesser degree of
As shown in Table 5, several analogues in this series (5,
13, and 21) exhibited excellent selectivity over other
serine proteases. Of particular interest is 5 (BMS-
189090), which is the most potent and most selective
thrombin inhibitor in the series. The selectivity profile of
BMS-189090 is comparable to that of Argatroban and
significantly superior to that of Efegatran. The crystal
structure of the ternary complexes of human a-throm-
bin with hirugen and BMS-189090 showed that this
inhibitor binds in an antiparallel fashion to thrombin,
like PPACK and Argatroban. These findings were
reported in a previous communication.12
Because of its potency and selectivity, BMS-189090 was
selected for further evaluations in vitro and in vivo. In a
thrombin-induced lethality model in anesthetized mice13
the in vivo potency of BMS-189090 (ID50=0.11 mpk,
Table 4. SAR from the P3 modification
Compd
R
Thrombin Compd
IC50 (mM)10
R
Thrombin
IC50 (mM)10
22
23
5
H
CH3
CH2OH
0.31
0.075
0.018
24
25
26
CH(Me)OH (S)
CH(Me)OH (R)
CH2COOMe
0.65
0.50
0.012
Figure 2. Effect of BMS-189090 on venous and arterial thrombosis in
anesthetized rats.

44
J. Das et al. / Bioorg. Med. Chem. Lett. 12 (2002) 41–44
5. Bode, W.; Huber, R.; Rydel, T. J.; Tulinsky, A. In Throm-
bin Structure and Function; Berliner, L. J., Ed.; Plenum: New
York; p 3. Stubbs, M. T.; Bode, W. Perspect. Drug Discov.
Des. 1994, 1, 431.
6. Tabernero, L.; Chang, C. Y.; Ohringer, S. L.; Lau, W. F.;
Iwanowicz, E. J.; Han, W.-C.; Wang, T. C.; Seiler, S. M.;
Roberts, D. G. M.; Sack, J. S. J. Mol. Biol. 1995, 246, 14.
7. Das, J.; Floyd, D. M.; Kimball, S. D.; Duff, K. J.; Lago,
M. W.; Krapcho, J.; White, R. E.; Obermeier, M. T.; More-
land, S.; McMullen, D.; Normandin, D.; Hedberg, S. A.;
Schaeffer, T. R. J. Med. Chem. 1992, 35, 2610.
8. (S)-Na-CBZ-nipecotic acid was prepared from (À)-ethylni-
pecotate. d-Tartarate salt in two steps: (a) K2CO3, CBZ-Cl,
Et2O–H2O, and (b) MeOH, 1 N aq NaOH. For preparation of
(À)-ethylnipecotate. d-Tartarate see: Akkerman, A. M.;
DeJongh, D. K.; Veldstra, H. Receuil 1951, 70, 899.
9. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. J. Org. Chem.
1992, 57, 2497.
10. In vitro inhibition of thrombin catalytic activity using 10
mMsubstrate s-2238 ( d-Phe-Pip-Arg-pNA) was measured at rt
after 3 min incubation with inhibitor. For a description of the
assay, see: Balasubramanian, N.; St. Laurent, D. R.; Federici,
M. E.; Meanwell, N. A.; Wright, J. J.; Schumacher, W. A.;
Seiler, S. M. J. Med. Chem. 1993, 36, 300.
Figure 3. Effect of BMS-189090 on bleeding time in anesthetized rats.
inhibition of arterial thrombosis (53%) required a
higher dose of 1 mg/kg+50 mg/kg/min iv. The efficacy
of BMS-189090 in arterial thrombosis was comparable
to that obtained with Argatroban.15 BMS-189090 also
increased bleeding time by 180% in mesenteric arteries
that were punctured with a hypodermic needle,15 but the
dose required to significantly increase bleeding time (3
mg/kg+150 mg/kg/min iv) exceeded maximal doses for
inhibition of venous and arterial thrombosis (Fig. 3).
11. Inhibition of bovine pancreatic trypsin was measured in
an assay containing 2 mMCaCl , 50 mMTris pH 8.0 and 30
2
mMcarbobenzyloxy-Val-Gly-Arg-pNA as substrate. The inhi-
bitor was combined with trypsin assay buffer and incubated
for 3 min, after which the substrate was added and absorption
was measured using a microplate reader (Molecular devices
Vmax) at 405 nM. Inhibition of human plasmin was measured
in an assay containing 145 mMNaCl, 5 mMKCl, 1 mg/mL
polyethylene glycol (PEG-8000), 30 mM N-(2-hydro-
xyethyl)piperazine-N0-ethanesulfonic acid, pH 7.4 and 100 mM
S-2251 (d-Val-Leu-Lys-pNA). The inhibitor was incubated
with the enzyme for 3 min, after which the substrate was
added and the rate of hydrolysis was measured as for throm-
bin and trypsin. Inhibition of tissue plasminogen activator (t-
PA) was measured in an assay similar to that of human plas-
min except using 100 mMmethanesulfonyl- d-cyclohexylty-
rosyl-Gly-Arg-pNA as substrate. Inhibition of Factor Xa was
measured in an assay similar to that of human plasmin except
using 100 mMS-2222 as substrate.
12. Malley, M. F.; Tabernero, L.; Chang, C. Y.; Ohringer, S.;
Roberts, D. G. M.; Das, J.; Sack, J. S. Protein Sci. 1996, 5,
221.
13. The model involved iv challenge with human a-thrombin
(10–20 units/mouse) in anesthetized mice treated with the
inhibitor either 10 min (iv) or 60 min (po) prior to thrombin
challenge. ID50 is the concentration of the inhibitor required
for 50% mice survival.
In conclusion, we have described SAR of a structurally
novel series of compounds leading to BMS-189090 as
potent, highly selective, and reversible inhibitor of
thrombin. The data provided show that BMS-189090 is
efficacious in protecting mice from thrombin-induced
lethality, in vivo and in inhibiting arterial and venous
thrombosis in rats.
References and Notes
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Hemostasis and Thrombosis; Colman, R. W., Marder, V. J.,
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1994; pp 3–18.
2. Coughlin, S. R. Nature 2000, 407, 258.
3. Vacca, J. P. Curr. Opin. Chem. Biol. 2000, 4, 394. Stein, B.;
Fuster, V.; Halperin, J. L.; Chesebro, J. H. Circulation 1989,
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Med. Chem. 1995, 3, 999. Kimball, S. D. Handb. Exp. Phar-
macol. 1999, 132, 367. Menear, K. Curr. Med. Chem. 1998, 5,
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14. Ki was determined by measuring enzyme activity at three
different substrate (S) concentrations and six different inhi-
bitor (I) concentrations, curve fitting the data to V=Vmax[S]/
{Km(1+[I]/Ki)+[S]}.
15. The experimental rats models were performed as described
in: Schumacher, W. A.; Heran, C. L.; Steinbacher, T. E. J.
Cardiovasc. Pharmacol. 1996, 28, 19.