Inhibitors and substrates of thrombin

Details Inhibitors and substrates of thrombin Inhibitors and substrates of thrombin

1998-12-03

2002-05-14

Celsa, Bennett (Department: 1627)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S017400, C530S330000, C530S331000

Reexamination Certificate

active

06387881

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to thrombin inhibitors and substrates.
2. Description of the Related Art
Thrombin, the last enzyme in the coagulation system, cleaves soluble fibrinogen to fibrin, which is then crosslinked and forms an insoluble gel forming the matrix for a thrombus. When a vessel is damaged, the above process is necessary to stop bleeding. Under normal circumstances there is no measurable amount of thrombin present in plasma. Increase of the thrombin concentration can result in formation of clots, which can lead to thromboembolic disease, one of the most common serious medical problems of our time.
Thrombin contributes to haemostatic control by means of several biological reactions. In addition to its primary function, the conversion of fibrinogen to fibrin, thrombin activates Factor XIII, which is responsible for the crosslinking of fibrin. Thrombin also acts by means of a positive feedback mechanism involving the activation of Factors V and VIII, which both are necessary for its own formation from prothrombin. Thrombin has another essential role: its binding to platelets initiates platelet release and aggregation which is responsible for primary haemostasis.
Fibrinolysis is the process which causes an enzymatic dissolution of fibrinogen and fibrin clots. Plasma contains a protein, plasminogen, which under the influence of various activators is converted to plasmin, a proteolytic enzyme, the activity of which resembles that of fibrin. Plasmin breaks down fibrin to fibrin degradation products.
Under normal conditions, the fibrinolysis system is in balance with the coagulation system. Small thrombi formed in the blood stream can be dissolved enzymatically and the circulation through the vessels can be restored by the activation of the fibrinolytic system in the body. If the fibrinolytic activity is too high, it may cause or prolong bleeding and if it is too low compared to the activity of the coagulation system, there is a risk of thrombosis.
The reactions of thrombin are further controlled by natural inhibitors in plasma. The most important of these are antithrombin III and heparin. These two compounds have been isolated and are therapeutically and prophylactically used in conditions where there is an imbalance in the haemostatic mechanisms with risk for prothrombin activation.
Mainly two types of therapeutic agents are used for the prevention of thrombosis. The heparins act by accelerating the inhibition of thrombin by antithrombin III. Coumarin derivatives, the oral anticoagulants, e.g. Warfarin, prevent the generation of thrombin by blocking the post-translational vitamin K-dependent &ggr;-carboxylation in the synthesis of prothrombin. Neither Heparin nor Warfarin are ideal. Heparin must be given parenterally and as it functions as a cofactor to antithrombin III it has no effect without this inhibitor. The effect of Warfarin develops very slowly and individual doses must be adjusted by frequent tests. None of these anticoagulants is specific for thrombin, they also inhibit other serine proteases and both of them may cause bleeding if the doses are not correctly balanced.
Thus, direct acting, specific thrombin inhibitors, having oral activity would be useful alternatives to the above anticoagulants. Much research in this area has resulted in the synthesis of different kinds of inhibitors of thrombin.
By imitating amino acid sequences of fibrinogen, the important natural substrate of thrombin, several good short peptide substrates for thrombin have been synthesized. The very first developed sequence with affinity for the active site of thrombin was Phe—Val—Arg [1] which mimics the fibrinogensequence preceding the bond split by thrombin. This sequence has later been improved to give D—Phe—Pro—Arg and D—Phe—Pip—Arg which have been used in chromogenic substrates, e.g. D—Phe—Pro—Arg—pNA and D—Phe—Pip—Arg—pNA [1] and in inhibitors of thrombin, e.g. the peptide aldehyde D—Phe—Pro—Arg—H [2], the irreversible inhibitor D—Phe—Pro—Arg—CH
2
Cl [3], inhibitors with a ketomethylene bond e.g. D—Phe—Pro—Arg—k—Gly—piperidide [4] and in the recently synthesized peptide boronic acid inhibitors e.g. Z—D—Phe—Pro—boroArg [5] and the nitrile: Boc—D—Phe—Pro—ArgCN [6].
Thus, D—Phe—Pro—Arg has been considered the best sequence for about 15 years, and it has been shown to have very good affinity for the active site of thrombin, in substrates (Km around 10
−6
M) as well as in inhibitors (Ki 10
−7
M to 10
−9
M).
SUMMARY OF THE INVENTION
We have now found that by exchanging Phe, in the D—Phe—Pro—Arg sequence, for some unnatural, aromatic amino acids, with a specified structure, and by using these new sequences to construct novel substrates and inhibitors we obtained significantly improved substrate and inhibitor properties. The new substrates show better kinetic constants (Km and kcat) and the inhibitors better inhibition constant (Ki).
Reduction of blood pressure is a side effect observed in many of the previous thrombin inhibitors containing Arg or Arg analogues like Gpa and Apa [7]. This side effect which in some compounds can be disturbingly serious is believed to depend on the positively charged guanidino or amidino group of the side chain of Arg or its analogues. Surprisingly, this side effect of inhibitors in the present application is markedly reduced even when the inhibitors have an Arg or Arg analogue.
We have also surprisingly found that by changing the side chain to a non-basic alkyl or alkylaryl group of a certain size, the affinity for thrombin is still very good although the affinity for other serine proteases is greatly reduced, i.e. these inhibitors/substrates are more specific for thrombin than corresponding compounds containing Arg. With this non-basic side chain the blood pressure lowering side effect is greatly reduced.
The present invention provides thrombin inhibitors and substrates derived from D—Phe—Pro—Arg or its analogues wherein Phe is substituted by
and Arg may be substituted by
Suitably, the inhibitors/substrates are of formula I, in which
X=H, CH
3
or an N-protecting group, e.g. Ac, Bz, Cbz, Boc;
Y=[CH
2
]
n
—Q,
where Q=H, amino, amidino, imidazole, guanidino or isothioureido and n=1-5, preferably 3-5, or C
3
-C
9
alkyl and C
5
-C
10
aryl or alkylaryl optionally substituted by up to three groups selected from hydroxy and C
1
-C
4
alkoxy;
R
1
=H, OH, CH
2
Cl, CH
2
—CH
2
—CO—pip, CF
2
—CF
2
—CO—pip,
CH
2
—CH
2
—CO—Pro—NHEt, CF
2
—CF
2
—CO—Pro—NHEt or a chromophoric group e.g. pNA, MCA,
R
2
and R
3
may be the same or different and are selected from the group consisting of OH, OR
6
and NR
6
R
7
, or R
2
and R
3
taken together represent the residue of a diol; where R
6
and R
7
, which may be the same or different, are C
1
-C
10
alkyl, phenyl or C
6
-C
10
arylalkyl,
R
4
and R
5
may be the same or different and are selected from R
2
, R
3
, Gly—pip, Ala—pip or Gly—Pro—NHEt;
Ar
1
and Ar
2
may be the same or different and are selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl, indolyl and saturated groups corresponding to these, optionally substituted by up to three groups selected from C
1
-C
3
alkyl and C
1
-C
3
alkoxy,
L
1
and L
2
may be the same or different and are selected from the group consisting of CH
2
, CH
2
—CH
2
, O—CH
2
, S—CH
2
,
Ar—L taken together may mean H, diphenyl-methyl, fluorenyl or saturated groups corresponding to these, but one of the Ar—L cannot be H when the other Ar—L means H or benzyl;
or its C
1
-C
3
alkyl substituted derivatives, where R
8
═CH
2
, CH
2
—CH
2
, S—CH
2
, S—C(CH
3
)
2
or CH
2
—CH
2
—CH
2
.
Preferably the Phe substitute is Dpa, Nal or Dba and preferable Arg substitute includes Irg, Gpa, Apa and non-basic amino acids such as Pgl, Mbg, Chg.
Examples of compounds which may be preferably used in the invention include:
Ac—D—&bgr;Nal—Pro—boroArg pinanediol ester
Z—D—Dpa—Pr

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