Biol. Chem. Hoppe-Seyler Vol. 373, pp. 491-496, July 1992

Interactions of Thrombin with Benzamidine-based Inhibitors JÖRG STÜRZEBECHER3, HELMUT ViEWEGb, PETER WiKSTRoMb, DUSAN TURKC AND WOLFRAM BODEC a

b c

Institut für Pharmakologie und Toxikologie, Medizinische Akademie E r f u r t , Nordhäuser Str. 74, D(0)-5010 E r f u r t , FRG Pentapharm AG, Engelgasse 109, CH-4002 Basel, Switzerland Max-Planck-Institut für Biochemie, D ( W ) - 8 0 3 3 Martinsried, FRG

Summary

Trypsin and trypsin-like enzymes cleave C-terminal bonds of the basic amino acids Arg and Lys. Inhibitors of these enzymes have been found not only among Arg and Lys derivatives but also with structurally related benzamidines. Especially cyclic amides of 4-amidinophenylalanine were found to be inhibitors of thrombin. The most potent selective thrombin inhibitor of these type is -(ß-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide. From the X-ray crystal structures of thrombin and trypsin-inhibitor complexes the thrombin complexes formed with inhibitors derived from amidinophenylalanine have been modeled. These models allow valuable predictions to design inhibitors of improved selection and binding properties. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved. Introduction

Inhibitors of proteinases involved in coagulation are potential anticoagulants. Thrombin is the key enzyme of blood coagulation and its inhibition represents an effective interference in blood coagulation. This was demonstrated by means of the naturally occurring thrombin inhibitor hirudin [1]. However, hirudin and its derivatives are polypeptides and their therapeutic use is limited due to their pharmacokinetics, especially with regard to distribution, gastrointestinal absorption and immunogenic properties. Therefore, synthetic, low-molecular weight inhibitors that are absorped orally are of special interest [ 2 ] , Like thrombin several other enzymes in blood are trypsin-like serine proteinases. They are involved in several processes of physiological relevance besides coagulation. Therefore, selectivity for the target enzyme thrombin is a further important requirement for the therapeutic use of an inhibitor. Too low selectivity may lead to undesired side effects and enhance the toxicity.

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492

J. Stürzebecheret al.

Vol. 373 (1992)

Selective, Benzamidine-derived Inhibitors of Thrombin Several competitive reversible inhibitors of trypsin-like enzymes are derived from benzamidine. However, ordinary inhibitors. For the first among amino acids

benzamidine derivatives are no

time selective inhibitors

containing a

benzamidine moiety

specific

of thrombin were at the

found

side chain

[3],

These compounds are isosteric derivatives of arginine in which the basic guanidinoalkyl side chain of arginine is replaced by a benzamidine moiety. The araino

acids amidinophenylglycine,

-aminobutyric acid

araidinophenylalanine,

and amidinophenyl-a-aminovaleric

amidinophenyl-

acid result

from

the

different length of the alkyl side chain. For designing of thrombin inhibitors especially cyclic amides of 4-amidinophenylalanine are key building blocks. Most potent inhibitors were obtained of 4-amidinophenylalanine and atives with Gly

at P2 are

thrombin activity

after variation of the N-terminal

by interposition

of amino

highly potent inhibitors

is reduced

with increasing

acids [ 4 ] .

of thrombin. The

distance due

to the

part Derivanti-

spacer.

Thus, ß-alanine at P2 or Gly-Gly at P2 and ?3 reduce the inhibitory activity. In contrast to peptide substrates and inhibitors, derivatives of 4-amidinophenylalanine with Pro in P 2 or P- possess only poor antithrombin activity. The piperidide containing N-terminal a ß-naphthylsulfonyl-glycyl residue is the most potent synthetic, competitive inhibitor derived from 4-amidinophenylalanine [ 4 ] . The compound, also named NAPAP, inhibits thrombin selectively with a K.^ of 6 nanomolar (Fig. 1). The Revalues for inhibition most of other trypsin-like enzymes are greater than micromolar.

Enzyme Thrombin Factor Xa Factor Xlla Protein Ca Trypsln Tryptase Plasmln sc-tPA Uroklnase PKallikreln GKalllkreln Acrosln Batroxobln

K . , /jmol/l 0,006 7.9 500 4.8 0.69 45 30 430 230 14 93 2.9 1.7

of

H2NX,NH

-S0 2 -NH-CH 2 -CO-NH-C-CO-N / S V y H -

Fig. 1. Chemical formula of NAPAP and its pattern of inhibition of several trypsin-like enzymes. (PKallikrein = plasma kallikrein, GKallikrein = glandular Kallikrein)

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Vol. 373 (1992)

Interactions of Thrombin with Benzamidine-based Inhibitors

493

The high antithrombin activity of NAPAP was demonstrated in blood plasma in experimental animals. Application marked prolongation

of

of this tight-binding inhibitor

clotting time

and

and

causes

antithrombotic effects

[5,

6].

However, the pharmacological requirements of NAPAP and other 4-amidinophenylalanine derivatives are not sufficient for in most benzamidines its

toxicity

intravenous application. Compounds orally or not at all.

vivo use. As is the case

lies between 20 and

A rapid

of this type

with

50 mg/kg body weight

are only slightly

on

absorbed

bolus application is accompanied by a drop

in

blood pressure [6, 7 ] . Therefore, designing of new benzamidine-derived inhibitors with high antithrombin activity, enzyme thrombin and

with comparatively

with high selectivity for the low toxicity is

of great

target

interest.

Furthermore, new compounds should be effective after oral administration with a long biological half-life. Comparison of structure-activity relationships

has shown that the

undesired

effects of benzamidines are due to the basicity of the amidino function However, the

amidino moiety

inhibitors at the

active site of

synthesize compounds with free carbonyl

is necessary

the basic

amidino group.

into the

structure of

[7].

of

to

compounds with

Such compounds

the antithrombin

a

show

However, introduction of the

NAPAP reduces

the

This prompted us

structure, that means

improved pharmacokinetic properties [ 8 ] , oxyl group

primary binding

the enzyme thrombin.

a betain

group besides

for the

carb-

activity

markedly [ 9 ] . Another way to obtain compounds with improved pharmacokinetic properties

was

to modify or to exchange the amidino moiety of NAPAP. Surprisingly, isosteric derivatives containing an

amino, aminomethyl,

guanidino or

guanidinomethyl

group do not possess antithrombin activity. Only the derivative containing substituted amidino moiety, for example an oxamidino group, has affinity

for thrombin [10]. All

a

considerable

further substitutions at the NAPAP

resulted in a drastic loss of antithrombin activity, however, no

molecule

explanation

was apparent for this phenomenon. X-Ray Crystal Structures of Thrombin-Inhibitor Complexes Our receptor-based design of inhibitors is based on the X-ray crystal

struc-

tures of

1.9

thrombin and

thrombin-inhibitor

complexes. At

first, the

A

crystal structure of human α-thrombin in complex with the irreversibly acting inhibitor D-Phe-Pro-ArgCH2Cl

was

trypsin-inhibitor complexes could

established be solved

[11] and [12]. The

the

structures

mode of

of

interaction

with thrombin of benzamidine-derived and several other noncovalently

binding

inhibitors could be explored assuming homologous interactions as with trypsin [12,

13]. These "modeled" complexes

the binding properties of different

allowed some plausible explanations

for

inhibitors and some predictions for

the

synthesis of new inhibitors. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved [14]. Fig. 2 shows the X-ray crystal structure of the thrombin-NAPAP complex. The D-isomer

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494

J. Stürzebecheretal.

Vol. 373 (1992)

\

Fig. 2. Interaction of the NAPAP molecule (thick connections) with the active site of bovine thrombin (thin connections) displayed together with the Connolly surface of thrombin (experimental structure according to [ 1 4 ] ) .

of NAPAP binds into the active site of thrombin in a quite compact form: amidinophenylalanine moiety binds into group

stands

perpendicularly to the

ring is tightly

packed between

its

the specificity pocket; the indole ring of

naphthyl

Trp215; the piperidine

group and

interactions of the C-terminal carbonamide bond

His57. However,

of NAPAP to the active

no site

Serl95 were apparent. Obviously, the inhibitor fits quite perfectly into active site of thrombin leaving almost no space for substituents. the NAPAP molecule could not serve as

The

naphthyl

the

Therefore,

a key building block for designing

of

new potent thrombin inhibitors. To find other key structures allowing introduction of different substituents, X-ray crystal

structures of

the complexes

containing several

derived inhibitors were solved [12, 13, 14]. the piperidide

of

3-amidinophenylalanine. Its

inhibits thrombin with

a

,^-value

NAPAP, in case of the 3-amidino compound it

a compact

conformation

into the

derivative

active

shown in

Fig. 4.

is

(3-TAPAP)

The selectivity

(Fig. 3).The X-ray crystal

of the thrombin complex formed with 3-TAPAP is to

tosyl

in micromolar range.

inhibition is similar to that of NAPAP.

benzamidine-

One of such a key structure

of

structure In contrast

is the L-isomer that binds in

site. The benzaraidine

moiety

is

arranged into the specificity pocket; the tosyl group is almost perpendicular to the indole moiety of Trp215; and the

toluene ring.

the piperidine ring is placed between

However, both

the toluene

ring and

the

His57

piperidine

moiety leave space for additional substituents.

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Vol. 373 (1992)

495

Interactions of Thrombin with Benzamidine-based Inhibitors Enzyme Thrombln Factor Xa Factor XI la Protein Ca Trypsln Tryptase Plasmin sc-tPA Urokinase PKalllkreln GKalllkreln Acrosln Batroxobln

Κ , , μΓποΙ/Ι 0.34 15 280 65 1.2

HN^

61

12 420 12

34

590 0.51 320

^^^

C H O 2 2 N' ^V^ CH 9

^3^·ν^

Fig. 3. Chemical formula of 3-TAPAP and its trypsin-like enzymes.

^/

/T- SOo-NH-C-CO-N

^-y

ή

)

pattern of inhibition of

several

Fig. 4. Active site region of thrombin including Connolly surface of thrombin superimposed with the proposed models of NAPAP (thin line inhibitor) and 3-TAPAP (thick connections) according to [13].

Synthesis of New Derivatives of 3-Amidinophenylalanine We synthesized a number of new compounds starting from 3-amidinophenylalanine as key building block. Substitution of the piperidine ring is well tolerated. Introduction of alkyl but also of charged substituents in 2- and 4-position leads to inhibitors with Revalues in 10~"7 and 1θ"8 molar range. Compounds with large substituents

in 4-position do

not bound to

the thrombin

active

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496

J. Stürzebecheretal.

site while small substituents result molar range.

This result

is

Vol. 373 (1992)

in inhibition constants near the

in agreement

with

the predictions

nano-

from

the

crystal structures. Variation of

the Na-substituent

Furthermore, variation charged groups was

of

enhances

the

carried out.

tolerated, however, compounds

also the

amidino moiety A guanidino

antithrombin

to

other,

or aminomethyl

containing an amino

activity.

especially

less

group is

or a substituted

well

amidino

group are less potent than the isosteric amidino derivative. However,

combi-

nation of different variations results in several highly potent and selective inhibitors of thrombin.

First in vivo

studies indicate

improved

pharmaco-

kinetic properties. In conclusion, derivatives of basic substituted

amino acids containing

benzene ring

thrombin inhibitors.

are

To design

key building

compounds which

benzamidine or a

blocks for f u l f i l the

less

designing

of

pharmacological

requirements their synthesis should be assisted by solving the structures its

of

complexes with thrombin.

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