Hugo Kubinyi, www.kubinyi.

de

Thrombin Inhibitor
Design

Hugo Kubinyi
Germany

E-Mail kubinyi@t-online.de
HomePage www.kubinyi.de

Hugo Kubinyi, www.kubinyi.de

Cyclotheonamide - The Merck Design Story

OH
Thrombin inhibitor
from a marine O
NH
sponge NH
O
O HN O
OHC-NH
N H
N O
O

NH

Ketoamide HN NH2

(reacts with the

catalytic serine)

Guanidine
(1TMB) (interaction with Asp189)
 Hugo Kubinyi, www.kubinyi.de

D-Phe-Pro-Arg-CO-
in a macrocyclic
ring, as model for
cyclotheonamide

HN O
O HN O
N H
N O
O

NH

HN NH2
(1AY6)

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors:

First lead derived from a natural product

HN NH2
NH2
NH
{ O
H3C O
O O N
N H O O
} N
N N CH3
H H N N
O H H
O

Cyclotheonamide Ki (thrombin) = 2.8 nM

(partial structure) Ki (trypsin) = 7.8 nM
 Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors: Model Compounds

for Optimization of the P1 residue
D,L-trans
NH2 NH2

O H3C O
H2 N N O
O H O
N N CH3
NH2
N N N
H H H
O O
L 370 518
Ki (thrombin) = 5 300 nM Ki (thrombin) = 0.09 nM
Ki (trypsin) = 855 000 nM Ki (trypsin) = 1 150 nM

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors:

Elimination of the keto-amide group
NH2 NH2
(L-trans) (trans)

HO
N R3 N
R1 N N R N
O O H O R2 O O H

L 372 051 L 371 912

R1 = H, R2 = H, R3 = Me R = NHMe
Ki (thrombin) = 4 nM Ki (thrombin) = 5 nM

L 372 228 L 372 011

R1= NHMe, R2, R3 = -(CH2)3- R=H
Ki (thrombin) = 0.04 nM Ki (thrombin) = 330 nM
Hugo Kubinyi, www.kubinyi.de

L-371,912 (1TOM)

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors:

Optimization of the P3 residue

NH2 NH2

H3C O O
N O H2N O
H N N
N N
H H
L 371 912 L 372 102
Ki (thrombin) = 5 nM Ki (thrombin) = 0.1 nM
Ki (trypsin) = 11 000 nM Ki (trypsin) = 94 nM
Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors:

Further optimization of the P3 residue
O
NH2
O
NH2
N
O
HN O
O
O SO2 N
N N
H
N
H
Ki = 4.7 nM (diastereomer 1) Ki = 0.4 nM
0.28 nM (diastereomer 2)

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors:

Further optimization of the P3 residue
NH2 NH2

OH
O O
HN O O
SO2 N N
N N
H H
L 372 236
Ki (thrombin) = 0.0025 nM L 372 460
Ki (trypsin) = 4 nM Ki (thrombin) = 1.5 nM

„Use“ of an additional pocket Combinatorial library

Hugo Kubinyi, www.kubinyi.de

L-372 236 (green)

L-372 460 (cyan)

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors: Weakly basic

P1 residues
Cl

binding mode
Cl
H of L 372 585:
N N
HN
SO2 O O
N
X
X = CH, R = NH2 NH2 H2 O
Ki = 9 nM R
3.0 Å 2.7 Å
-
L 372 585 O O
X = N, R = NH2
Ki = 3 nM Asp 189
Hugo Kubinyi, www.kubinyi.de

Superposition
of L-371,912
(1TOM, yellow)
with the
dichloro-Phe
analogue
L-372,585
(color-coded)

D.-M. Feng et al.,

J. Med. Chem. 40,
3726-3738 (1997)

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors: Nonbasic

Cl P1 residues

Cl X = CH, R = H
H Ki = 110 nM
N N
HN
SO2 O O

R
L 373 588
H R = CH3
N N
Ki = 38 nM
H2N O O R
R = Cl
R Ki = 3 nM
Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors: Rigidization

to Achiral Molecules
L 372 774
CH3 Ki (thrombin) = 47 nM
O Ki (trypsin) = 2 200 nM
N
N N
H O H
NH2

CH3 L 373 890

O Ki (thrombin) = 0.5 nM
O S N Ki (trypsin) = 570 nM
N N
O H H
O N NH2

NH

Hugo Kubinyi, www.kubinyi.de

Merck Thrombin Inhibitors: Further

Optimization of the Molecule
L 374 087, R = Me
R Ki (thrombin) = 0.5 nM
O Ki (trypsin) = 3 200 nM
O2S N
N N
H H L 375 052, R = Propyl
O
H3C N NH2 Ki (thrombin) = 0.85 nM
Ki (trypsin) = 1 400 nM
stability and enhancement
oral bioavailability of activity

CH3 L 375 378

Ki (thrombin) = 0.8 nM
N O Ki (trypsin) = 1 800 nM
N
N N
H H
O
H3C N NH2
selectivity
Hugo Kubinyi, www.kubinyi.de

Binding Mode of L-374,087

Hugo Kubinyi, www.kubinyi.de

Merck Clinical Candidate/s ?

The phenoxyacetic acid amide
O NHEt (log P = 2.04) had the best overall
profile of all development candi-
N O dates, in vitro (Ki thrombin =
H2N
O 0.74 nM; Ki trypsin = 23 mM) and
O N in vivo (rat thrombosis model;
H
10%, 40% and 63% bioavailability
in rats, dogs and monkeys).
Cl

Me O
O
O2S N NH-tBu
N N N
H H 217th ACS Meeting
O
N Anaheim, CA, 1999
L-376,062
Hugo Kubinyi, www.kubinyi.de

Hoffmann-la Roche Thrombin Inhibitors

R=H
H O
N Ki (thrombin) = 480 nM
S N Ki (trypsin) = 75 000 nM
H
O O R
N
R = benzyl
HN NH2
Ki (thrombin) = 47 nM
Ki (trypsin) = 42 000 nM
O
O O N COOH
S
N Napsagatran (i.v., short
H biological half-life time)
O N
H
Ki (thrombin) = 0.27 nM
N
Ki (trypsin) = 1 900 nM
HN NH2

Hugo Kubinyi, www.kubinyi.de

Binding Mode of the

Hoffmann-La Roche
Thrombin Inhibitor

no significant
variation of
biological activity
after chemical
variation of the
phenylalanine

NAPAP (green)

Roche (red)
Hugo Kubinyi, www.kubinyi.de

Melagatran (Astra)
was one of the first
H thrombin inhibitors
HO N N
N with some oral
O H O O bioavailability

Ki (thrombin) = 2 nM

HN NH2
Ximelagatran (H 376/95) is a double prodrug of
melagatran:
ester group (cleaved by esterases)
amidoxime (reduced by NADH-cytochrome b5
reductase + CYP 2A6)

Hugo Kubinyi, www.kubinyi.de

Boronic Acid Thrombin Inhibitors (DuPont)

H
CH 3CO N N B(OH) 2
N
H
O O R
R = -(CH 2) 3-NH-C(=NH)NH 2 K i = 0.04 nM
(DuP 714)
R = -(CH 2) 4-NH 2 K i = 0.24 nM
R = -(CH 2) 4-C(=NH)NH 2 K i = 0.29 nM
R = -(CH 2) 5-NH 2 K i = 8.1 nM
R = -(CH 2) 3-NH 2 K i = 79 nM
Hugo Kubinyi, www.kubinyi.de

Ac-D-Phe-Pro-boroArg-OH (1LHC)
N-Acetyl-D-Phe-Pro-NH B(OH)2
Phe 227
W
3.1 2.8 O
3.0
O HN W
2.7 3.0
H2N NH2
Gly 219 +
2.5 2.7
O - O Ki = 0.04 nM
Asp 189

Hugo Kubinyi, www.kubinyi.de

Ac-D-Phe-Pro-boroLys-OH (1LHD)

N-Acetyl-D-Phe-Pro-NH B(OH)2
Phe 227

Gly 219 O
O 3.2 W 3.8
+
NH3 2.8
2.6 W
2.9
O
O - O
2.6
Ki = 0.24 nM
Ala 190
Asp 189
Hugo Kubinyi, www.kubinyi.de

Ac-D-Phe-Pro-boroButylamidinoglycine-OH
(1LHE)
N-Acetyl-D-Phe-Pro-NH B(OH)2
Phe 227

2.8 O

O 2.6 W
3.0

Gly 219 H 2 N + NH 2
2.6 2.6

O - O K i = 0.29 nM

Asp 189

Hugo Kubinyi, www.kubinyi.de

Ac-D-Phe-Pro-boroHomolys-OH (1LHF)
N-Acetyl-D-Phe-Pro-NH B(OH)2
Phe 227

Gly 219 2.9 O

O 4.1 3.8 W
+
H3N
3.6
2.9 3.0
O
O
- O Ki = 8.1 nM
Ala 190 Asp 189
Hugo Kubinyi, www.kubinyi.de

Ac-D-Phe-Pro-boroOrnithine-OH (1LHG)
N-Acetyl-D-Phe-Pro-NH B(OH)2
Gly 219

3.3 3.0 O
O +
H3N W
Phe 227
2.9 2.7
O 3.1 W

Ala 190 3.0

O - O Ki = 79 nM
Asp 189

Hugo Kubinyi, www.kubinyi.de

Binding Mode of a
CN-Phe Boronic Ser214 His57
O H N
Acid
(1AUJ) H
H O
O
H O H N N B
N O Ser195
H
H O O H
HN
NC
O H H
Gly216 H
N H O Gly193
O
H H O NH2 Ki = 0.48 nM
Gly219 N
Gln192
Hugo Kubinyi, www.kubinyi.de

Binding
Mode of
a CN-Phe
Boronic
Acid

(1AUJ)

Hugo Kubinyi, www.kubinyi.de

Aldehydes as Reversible
Covalent Thrombin Inhibitors

O H
S N N CHO
O N
H O O R
NH

CVS 1578: R = N NH2

H
Ki = 1.0 nM (1BA8)
NH

CVS 1694 and 1695: R = N NH2

Ki = 4.4 nM and 0.32 nM
(1BB0 and 1CA8) (R and S)
Hugo Kubinyi, www.kubinyi.de

Benzothiazolyl Inhibitor (1DOJ)

Trp215 Ser214 Trp60D
O HN
His57
N
S N HO O
H H N
Me N N O- oxygen
N H N anion Asp102
O
H O O hole
Ser195

O H + Ki = 0.19 nM (1DOJ)
N H2N NH2
M. J. Costanzo et al.,
Gly216 O O- J. Med. Chem. 39,
Asp189 3039-3043 (1996)

Hugo Kubinyi, www.kubinyi.de

ETH Thrombin Inhibitors

O R3 O H R2 R3
R2
+
R1 N + N R4 R1 N N R4
R5 -
O R6 O H R6 R5
O
O H CH3 O O H
CH3
N N CH3 N N
O H Ki = 90 nM O H
tryp/thro = 7.8

HN Ki = 18 000 nM NH2
NH2 tryp/thro = 0.14 HN
Hugo Kubinyi, www.kubinyi.de

Binding
Mode of the
Tricyclic
Succinimide
Inhibitor to
Thrombin

Ki = 90 nM

Hugo Kubinyi, www.kubinyi.de

ETH Thrombin Inhibitor

(U. Obst and F. Diederich,
1997)
O
O
H

N N

O H

Racemate, Ki = 13 nM
tryp/thro = 760 NH2
HN
(+)-Enantiomer, Ki = 7 nM
tryp/thro = 740

(-)-Enantiomer, Ki = 5 600 nM
tryp/thro = 21
Hugo Kubinyi, www.kubinyi.de

Amidrazones as Selective Thrombin Inhibitors

O N
H O
N H N
S N N
O O H S O
O O

N
R
H2N N R=H NH2
R=H
Ki (thrombin) = 6.5 nM R Ki (thrombin) = 52 nM
Ki (trypsin) = 325 nM Ki (trypsin) = 8 650 nM

R = NH2 R = NH2
Ki (thrombin) = 4 320 nM Ki (thrombin) = 1.5 nM
Ki (trypsin) = 81 500 nM Ki (trypsin) = 3 730 nM

Hugo Kubinyi, www.kubinyi.de

Amidrazones as Selective Thrombin Inhibitors

H3C
H N
N
S O
O O
R=H
Ki (thrombin) = 23 nM N
R
Ki (trypsin) = 700 nM NH2
R = NH2 (LB 30 057)
Ki (thrombin) = 0.38 nM
Ki (trypsin) = 3 290 nM
Hugo Kubinyi, www.kubinyi.de

Thrombin Inhibitors With a Benzylamine Group

as P1 Substituent, Derived from LB 30 057
MeO
O
R= H
R N
S N
Ki = 6.6 nM
O O Me

R=

Ki = 3.3 nM NH2

K. Lee et al., Bioorg. Med. Chem. Lett. 8, 2563-2568 (1998)

Hugo Kubinyi, www.kubinyi.de

Boehringer Mannheim Thrombin Inhibitors

O O BM 51.1011
H
N N (1UVS)
S N Ki = 4 µM
H
O O

N BM 14.1248
R1 (1UVT)
N
R1 = H, R2 = CH3
N O
S N Ki = 23 nM
H
O O
R1 = CH3, R2 = H
Ki = 70 nM
R2
R. A. Engh et al., Structure 4, 1353-1362 (1996)
Hugo Kubinyi, www.kubinyi.de

Other Thrombin Inhibitors: 3D Pharmaceuticals

N Ki = 4.1 µM
H
N N (1QBV)
H 2N N
H
O O
R. Bone et al., J. Med. Chem. 41, 2068-2075 (1998)

Cl

O O N NH Ki = 320 nM
S
O O NH2

CH3
T. Lu et al., Bioorg. Med. Chem. Lett. 8, 1595-1600 (1998)

Hugo Kubinyi, www.kubinyi.de

Other Thrombin Inhibitors: 3D Pharmaceuticals

Cl
N
O O
S N
Ki = 11 nM
O O Me
NH
Cl CH3
N NH2
O O
S Ki = 4.6 nM
O O
Cl
CH3 NH Ki = 13 nM
O O
S N NH2
O O
T. Lu et al., Bioorg. Med. Chem.
CH3 Lett. 8, 1595-1600 (1998)
Hugo Kubinyi, www.kubinyi.de

Eli Lilly Thrombin Inhibitors

X R = H, X = O
N (1D3T)
O
R = H, X = H,H
(1D3Q)
R S R = OH, X = H,H
N
O Ki = 10 nM
D. J. Sall et al., J. Med. Chem. 40, 3489-3493 (1997)

NH
LY178550
H2N
N (1D4P)
N
H O
N. Y. Chirgadze et al., Protein Sci. 6, 1412-1417 (1997)

Hugo Kubinyi, www.kubinyi.de

Binding
mode of
a Lilly
inhibitor
 Hugo Kubinyi, www.kubinyi.de

Assembly of Ligands in the Binding Site

N
H2N N
H N no Zn2+ plus Zn2+
NH
Ki (trypsin) > 1 000 / 136 µ M
Ki (tryptase) = 358 / 0.3 µM
Ki (thrombin) > 1 000 / 10.5 µM
N
H2N N
H N
NH no Zn2+ plus Zn2+
Ki (trypsin) = 31.2 / 22.5 µ M
B. A. Katz et al., Ki (tryptase) = 8.8 / 54.5 µM
Nature 391,
608-612 (1998)
Ki (thrombin) = 31 / 0.04 µM

Hugo Kubinyi, www.kubinyi.de

Assembly of Ligands in the Binding Site

H H2N NH
N O
His-57
H 2N N NH
NH N HN N NH
N
O Zn2+
no Zn2+ N O
Ki (Trypsin) = 87.5 µM HN H
Ki (Tryptase) = 5.7 µM
Ki (Thrombin) > 1 000 µM
Ser-195

plus Zn2+
Ki (Trypsin) = 0.005 µM H 2N NH
Asp-189
Ki (Tryptase) = 0.05 µM
Ki (Thrombin) = 0.10 µM (thrombin complexes:
1C1U, 1C1V, 1C1W)
Hugo Kubinyi, www.kubinyi.de

Trypsin + Keto-BABIM Trypsin + BABIM vs. Keto-BABIM

Hugo Kubinyi, www.kubinyi.de

Molecular Evolution of Thrombin Inhibitors

in a Combinatorial Library (n = 15,360)
prepared from
80 aldehydes,
12 amines, and
O 16 isonitriles
N
H
NH O
K. Illgen et al.,
Chem. Biol. 7,
433-441 (2000)

H2 N NH Ki (thrombin) = 2 nM
Hugo Kubinyi, www.kubinyi.de

Combinatorial
O NH Docking
N
H NH2 Ki (thrombin) = 95 nM
Ki (trypsin) = 520 nM
H.-J. Böhm et al., J. Comp.-Aided Mol. Design 12, 1-6 (1998)

O
NH
N
N XU 817
NH2

Ki (thrombin) = 18 nM
C. Dominguez et al., Bioorg. Med. Ki (trypsin) >15 µM
Chem. Lett. 9, 925-930 (1999)

Hugo Kubinyi, www.kubinyi.de

Bloodsucking Animals:
Mosquitos and Bugs
Hugo Kubinyi, www.kubinyi.de

Vampire Bat

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Leech, Hirudo medicinalis

Hugo Kubinyi, www.kubinyi.de

Sequence of
Hirudin and
3D Structure
of the Hirudin-
Thrombin
Complex (1HRT)

Hugo Kubinyi, www.kubinyi.de

1HRT
Hugo Kubinyi, www.kubinyi.de

1HRT

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1HRT
 Hugo Kubinyi, www.kubinyi.de

Structural Optimization of a Hirudin Fragment

in vitro in vivo

Recombinant Hirudin 100 % 100 %

Hirulog-1, D-Phe-Pro-Arg-(Gly)4-Hirudin53-64 20 % 60 %

Hirudin56-65: Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-OH 0.05 % n.d.

-OOC-(CH ) -CO-Tyr-Glu-Pro-Ile-Hyp-Glu-Glu-Smp-Cha-Gln-OH 28 % 290 %

2 2

Hyp = OH Smp = SO-3 Cha =

N
N N
O H O H O

Hugo Kubinyi, www.kubinyi.de

Bivalent Thrombin Inhibitors based on N-Me-Arg

T. Steinmetzer et al., Biol.
Chem. 381, 603-610 (2000)

N
H2N Me
O N
O TGGGGGDYEPIPEEA-Cha-dGlu
H O
H2N N Ki = 37 pM
NH
active site inhibitor spacer exo-site binding
domain