CN101652136A

CN101652136A - Unit dose formulations and methods of treating thrombosis with an oral factor XA inhibitor

Info

Publication number: CN101652136A
Application number: CN200780050362A
Authority: CN
Inventors: 乌马·辛哈; 斯坦利·J·霍伦巴克; 基思·阿贝
Original assignee: Millennium Pharmaceuticals Inc
Current assignee: Millennium Pharmaceuticals Inc
Priority date: 2006-12-08
Filing date: 2007-11-15
Publication date: 2010-02-17
Also published as: ZA200904758B

Abstract

Unit doses of factor Xa inhibitor compounds and methods of using these compounds for inhibiting blood coagulation in a human patient are taught herein. The unit dose of the factor Xa inhibitor compounds disclosed herein required to inhibit coagulation in a primate is lower than the unit dose required to obtain similar levels of coagulation inhibition in other animal models, such as rodents. Also taught are in vitro assays useful in predicting in vivo antithrombotic activity in humans.

Description

Use oral factor XA inhibitor to treat thrombotic unit dose formulations and method

The opinion of priority

The application's case is advocated the temporary patent application case the 60/873rd of December in 2006 application on the 8th according to 35 U.S.C. § 119 (e), 60/947 of No. 792 and on July 2nd, 2007 application, 629 rights and interests, these application cases all are all to be incorporated herein by reference.

Technical field

The factor Xa inhibitor that the present invention is directed to the use given dose comes the method for anticoagulant.Thereby the present invention also assesses the calibrating of the anti-thrombosis activity of test compounds at the thrombin generation in the measurement blood.

Background technology

Hemostasis is promptly controlled hemorrhage, and it is undertaken by modus operandi or the physiology characteristic by vasoconstriction and blood coagulation.All relate to platelet and blood coagulation although recover in hemostasis and the thrombotic disease, but some component of coagulation cascade mainly is responsible for amplification and is quickened related process in platelet aggregation and the fibrin deposition, and described platelet aggregation and fibrin deposition are the main incidents in thrombosis and the hemostasis.

Grumeleuse forms to relate to fibrinogen is changed into fibrin, and described fibrin becomes network to recover hemostasis at the damage post polymerization.Similar procedure causes blood vessel blockage in thrombotic disease.Fibrinogen is converted into fibrin by catalyzed by thrombin, and wherein fibrin is the end product of series reaction in the coagulation cascade.Thrombin is playing a crucial role in platelet activation also, thereby facilitate thrombosis under the situation of tremulous pulse and venous blood flow.Therefore, suppose that effectively regulating thrombin can produce thrombotic effective adjusting.Several anticoagulant that use at present can directly or indirectly influence thrombin (that is, unfractionated heparin, low molecular weight heparin, heparin sample chemical compound, pentasaccharides and warfarin (warfarin)).Direct or indirect inhibition to thrombin activity also is the focus (by Erikson (Eriksson) and Kui Lun (Quinlan), materia medica (Drugs) 11:1411-1429,2006 comments) of multiple anticoagulant in the clinical development.

Thrombinogen is the presoma of thrombin, and it changes into organized enzyme through factor Xa (fXa).The local activation that tissue factor/factor Xa of factor VIIa mediation produces is increased by factors IX a/ factor VIIla complex, and makes prothrombinase assemble on activated platelet.Factor Xa is unique enzyme of being responsible for continuing to form thrombin in blood vessel structure as the part of thrombinogen multienzyme complex.Factor Xa is a serine protease, is the activated form of its presoma factor X, and for calcium ion in conjunction with, contain a member of gamma-carboxyl glutamate (GLA), vitamin k-dependent and thrombin.With to comprising fibrinogen and PAR receptor (protease activated receptor, Kao Fulin (Coughlin), thrombosis hemostasis periodical (J.Thrombosis Haemostasis) 3:1800-1814,2005) the thrombin difference that multiple proteins substrate works, as if factor Xa have single physiology substrate, i.e. thrombinogen.Because a part factor Xa may be able to produce thrombin (the Man En people such as (Mann) who surpasses 1000 molecules, thrombosis hemostasis periodical (J.Thrombosis.Haemostasis) 1:1504-1514,2003), so direct inhibitive factor Xa may be effective anticoagulant strategy as the mode that indirect Trombin inhibiting forms.This asserts that being based on pivotal role and the Trombin inhibiting protoenzyme of prothrombinase in thrombin is synthetic will have the fact of remarkable effect to whole platelet aggregation and coagulation path.

Such as the activated protease of factor VIIa, factors IX a or factor Xa to himself having the low proteolytic activity.Yet it significantly strengthens its catalytic efficiency through being assembled into cofactor dependency, membrane-bound complex.This effect is the most remarkable to factor Xa, and wherein said effect strengthens 10 ⁵Doubly people such as (Man En () Mann, hematology (Blood) 76 (1): 1-16,1990).Owing to have the proenzyme (1.4 μ M thrombinogens are than 150nM factor Xa) and the activation kinetics of higher concentration in the blood, therefore realize that the amount of the factor Xa of the required inhibition of anticoagulation is less than the amount of thrombin.In the clinical trial that prevention of deep vein thrombosis forms, find that also factor Xa is compared to the circumstantial evidence of thrombin as the hypothesis of the advantage of therapeutic goal.It is Antithrombin III dependency factor Xa inhibitor that sulphur reaches heparin (Fondaparinux), confirmed in 4 routine orthomorphia tests that it surpasses Enoxaparin (enoxaparin) (low molecular weight heparin of a kind of Trombin inhibiting and factor Xa) (Tours pendant people such as (Turpie), internal medicine archives (Archives Internal Medicine) 162 (16): 1833-1840,2002).Therefore, the chemical compound that has shown selective cytokine inhibitory Xa can or be used in some thrombosis disease therapeutic dispensing, for example referring to WO 94/13693 as diagnostic agent in vitro.

Summary of the invention

The relatively simulation that the relative antithrombotic of thrombinogen enzymatic activity is formed the change degree of efficacy levels has made finds the therapeutic activity of factor Xa inhibitor in the mankind.The present invention is directed to the method for factor Xa inhibitor anticoagulant in human patients of using the blood coagulation amount of suppression.Specifically, but the direct inhibitor of the factor Xa that per os uses with the patient total every day amount (" mg/kg ") of overall restatement between per kilogram of body weight about 0.01 is with about 2.0 milligrams to patient's throwing and the time, can effectively suppress the blood coagulation in the human patients.Expect that this dosage will be all effective to any chemical compound of specificity suppressioning factor Xa, and especially effective to the factor Xa inhibitor of hereinafter described formula I, II, III, IV or V.

The present invention is also at the method for the blood coagulation state of assess patient.Expection also can use the method for assessment blood coagulation state to test multiple different classes of anticoagulant except that factor Xa inhibitor.

In one aspect of the invention, described method comprises to the formula I chemical compound of patient's throwing with the blood coagulation amount of suppression:

A-Q-D-E-G-J-X

I，

Wherein:

A is selected from:

(a) C ₁-C ₆Alkyl;

(b) C ₃-C ₈Cycloalkyl;

(c)-N(R ¹，R ²)、N(R ¹，R ²)-C(＝NR ³)-、N(R ¹，R ²)-C(＝NR ³)-N(R ⁴)-、R ¹-C(＝NR ³)-、R ¹-C(＝NR ³)-N(R ⁴)-；

(d) phenyl that replaces through 0-2 R substituent group independently;

(e) naphthyl that replaces through 0-2 R substituent group independently; With

Have the monocycle or the condensed-bicyclic heterocyclic ring system of 5-10 annular atoms, wherein the 1-4 of a loop systems annular atoms is selected from N, O and S, and wherein loop systems can replace through 0-2 R substituent group;

R is selected from:

H, halogen ,-CN ,-CO ₂R ¹,-C (=O)-N (R ¹, R ²) ,-(CH ₂) _m-CO ₂R ¹,-(CH ₂) _m-C (=O)-N (R ¹, R ²) ,-NO ₂,-SO ₂N (R ¹, R ²) ,-SO ₂R ¹,-(CH ₂) _mNR ¹R ²,-(CH ₂) _m-C (=NR ³)-R ¹,-(CH ₂) _m-C (=NR ³)-N (R ¹, R ²) ,-(CH ₂) _m-N (R ⁴)-C (=NR ³)-N (R ¹, R ²), side joint is in containing heteroatomic 3 to 6 yuan of heterocyclic-(CH that 1-4 is selected from N, O and S ₂) _mNR ¹Group ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CF ₃,-OR ²With contain the heteroatomic 5-6 unit heterocyclic ring system that 1-4 is selected from N, O and S, wherein the hydrogen atom of the 1-4 on the heterocyclic ring system can be independently through be selected from by halogen ,-C ₁-C ₄Alkyl ,-C _1-4Alkyl-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

M is the integer of 0-2;

R ¹, R ², R ³And R ⁴Be independently selected from the group that forms by following:

H ,-OR ⁵,-N (R ⁵,-R ⁶) ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkyl phenyl and-C _0-4The alkyl naphthyl, wherein 1-4 hydrogen atom on the annular atoms of phenyl and naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms; Or R ¹And R ², or R ²And R ³Can form 3-8 unit's cycloalkyl or heterocyclic ring system together, wherein heterocyclic ring system can have 3 to 10 annular atomses, wherein have 1 to 2 and encircle and contain 1-4 hetero atom that is selected from N, O and S in loop systems, wherein the hydrogen atom of the 1-4 on the heterocyclic ring system can be independently through being selected from by halogen, C ₁-C ₄Alkyl ,-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

R ⁵And R ⁶Be independently selected from the group that forms by following:

H ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkyl phenyl and-C _0-4The alkyl naphthyl, wherein 1-4 hydrogen atom on the annular atoms of phenyl and naphthyl moiety can be independently through be selected from halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms; Or R ⁵And R ⁶Can form 3-8 unit's cycloalkyl or heterocyclic ring system together, wherein said heterocyclic ring system can have 3 to 10 annular atomses, wherein in loop systems, have 1 to 2 and encircle and contain the hetero atom that 1-4 is selected from N, O and S, wherein the hydrogen atom of the 1-4 on the heterocyclic ring system can be independently through be selected from by halogen ,-C ₁-C ₄Alkyl ,-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

Q is the member who is selected from by the following group that forms:

Direct bond ,-CH ₂-,-C (=O)-,-O-,-N (R ⁷)-,-N (R ⁷) CH ₂-,-CH ₂N (R ⁷)-,-C (=NR ⁷)-,-C (=O)-N (R ⁷)-,-N (R ⁷)-C (=O)-,-S-,-SO-,-SO ₂-,-SO ₂-N (R ⁷)-and-N (R ⁷)-SO ₂-;

R ⁷Be selected from:

H ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4The alkylidene phenyl and-C _0-4The alkylidene naphthyl, wherein 1-4 hydrogen atom on the annular atoms of phenyl and naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms;

D is direct bond or is selected from member by the following group that forms:

(a) independently through 0-2 R ^1aThe phenyl that substituent group replaces;

(b) independently through 0-2 R ^1aThe naphthyl that substituent group replaces; With

(c) have the monocycle or the condensed-bicyclic heterocyclic ring system of 5-10 annular atoms, wherein the 1-4 of a loop systems annular atoms is selected from N, O and S, and wherein loop systems can be through 0-2 R ^1aSubstituent group replaces;

R ^1aBe selected from:

Halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN ,-NO ₂,-(CH ₂) _nNR ^2aR ^3a,-(CH ₂) _nCO ₂R ^2a,-(CH ₂) _nCONR ^2aR ^3a,-SO ₂NR ^2aR ^3a,-SO ₂R ^2a,-CF ₃,-OR ^2aWith contain the heteroaromatic system of heteroatomic 5-6 unit that 1-4 is selected from N, O and S, 1-4 hydrogen atom in the wherein said heteroaromatic system can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms;

R ^2aAnd R ^3aBe independently selected from the group that forms by following:

H ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkylidene-phenyl and-C _0-4Alkylidene-naphthyl, the hydrogen atom of the 1-4 on the annular atoms of wherein said phenyl and described naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms;

N is the integer of 0-2;

E is direct bond or is selected from member by the following group that forms:

-C _1-2Alkylidene-,-O-,-S-,-SO-,-SO ₂-,-C _0-1Alkylidene-C (=O) ,-C _0-1Alkylidene-C (=O)-N (R ⁸)-C _0-1Alkylidene-,-C _0-1Alkylidene-N (R ⁸)-C (=O)-C _0-1Alkylidene-,-N (R ⁸)-C (=O)-N (R ⁸)-and-C _0-1Alkylidene-N (R ⁸)-;

R ⁸Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4Alkylidene aryl;-C _0-4Alkylidene-heteroaryl;-C _1-4Alkylidene-C (=O)-OH ,-C _1-4Alkylidene-C (=O)-O-C _1-4Alkyl and-C _1-4Alkylidene-C (=O)-N (R ^2b,-R ^3b);

R ^2bAnd R ^3bEach is independently selected from the member by the following group that forms naturally:

H ,-C _1-4Alkyl ,-C _0-4Alkylidene-aryl;-C _0-4Alkylidenyl-heterocyclic base, and R ^2bAnd R ^3bCan form together with the N atom that it connected and to contain the heteroatomic 5-8 unit heterocycle that 1-4 is selected from N, O and S, wherein said heterocycle can be through 0-2 R ^1cGroup replaces;

R ^1cBe the member who is selected from by the following group that forms:

Halogen;-C _1-4Alkyl;-CN;-NO ₂-C (=O)-N (R ^2c,-R ^3c);-C (=O)-OR ^2c-(CH ₂) _q-N (R ^2c,-R ^3c);-SO ₂-N (R ^2c,-R ^3c);-SO ₂R ^2c-CF ₃With-(CH ₂) _q-OR ^2c

R ^2cAnd R ^3cBe the member who is selected from by the following group that forms independently of one another:

H;-C _1-4Alkyl; With-C _1-4Alkylidene-aryl;

Q is the integer of 0-2;

G is the member who is selected from by the following group that forms:

(a) C ₂Thiazolinyl or C _3-8Cycloalkenyl group, wherein thiazolinyl and cycloalkenyl group junction point are thiazolinyl carbon atom and wherein-C ₂Thiazolinyl or-C _3-8Cycloalkenyl group is through 0-4 R ^1dGroup replaces;

(b) phenyl, wherein the ring carbon atom of phenylene is through 0-4 R ^1dGroup replaces;

(c) contain 1-4 the first heterocyclic ring system of heteroatomic 3-8 that is selected from N, O and S, a wherein said heterocyclic 0-2 annular atoms can be through 0-4 R ^1dGroup replaces; With

(d) 8-10 unit annelated heterocycles bicyclic system, it contains 1-4 hetero atom that is selected from N, O and S, and 0-2 annular atoms of wherein said condensed-bicyclic system can be through 0-4 R ^1dGroup replaces;

R ^1dBe the member who is selected from by the following group that forms:

H, halogen; C _1-6Alkyl, aryl ,-CN;-NO ₂-(CH ₂) _0-6-NR ^2dR ^3d-SO ₂NR ^2dR ^3d-SO ₂R ^2d-CF ₃-(CH ₂) _0-6-OR ^2d-O-(CH ₂) _1-6OR ^2d-O-(CH ₂) _1-6-C (=O)-O-R ^2d-O-(CH ₂) _1-6-C (=O)-N (R ^2d, R ^3d);-N (R ^5a)-(CH ₂) _1-6-OR ^2d-N (R ^5a)-(CH ₂) _1-6-N (R ^2d, R ^3d);-C (=O)-N (R ^2d, R ^3d);-N (R ^5a)-(CH ₂) _1-6-C (=O)-N (R ^2d, R ^3d);-N ((CH ₂) _1-6-OR ^2d) ₂-N (R ^5a)-(CH ₂) _1-6-OR ^2d-N (R ^5a)-C (=O)-R ^2d-N (R ^5a)-SO ₂-R ^2d-(CH ₂) _0-6-C (=O)-O-R ^2d-(CH ₂) _0-6-C (=O)-N (R ^2d, R ^3d);-(CH ₂) _0-6-C (=NR ^2d)-N (R ^3d, R ^4d);-(CH ₂) _0-6-N (R ^5a) C (=NR ^2d)-N (R ^3d, R ^4d); Contain 1-4 heteroatomic-(CH that is selected from N, O and S ₂) _0-6-N (R ^3d) C _5-6Unit's heterocycle and contain heteroatomic-(CH that 1-4 is selected from N, O and S ₂) _0-6-5-6 unit heterocycle;

R ^5a, R ^2d, R ^3dAnd R ^4dBe the member who is selected from by the following group that forms independently of one another:

H; C _1-6Alkyl; C _1-6Alkylaryl;-CN;-NO ₂Isocyclic aryl;-CN;-NO ₂Or R ^2dAnd R ^3dN atom together with its separate connection forms 5-7 unit heterocycle; Or R ^3dAnd R ^4dForm together with the N atom that it connected and to contain the heteroatomic 5-8 unit heterocycle that 1-4 is selected from N, O and S;

J is direct bond or is selected from member by the following group that forms:

-N (R ⁹)-C (=O)-;-C (=O)-N (R ⁹)-;-O-;-S-;-SO-;-SO ₂-;-CH ₂-;-N (R ⁹)-; With-N (R ⁹)-SO ₂-;

R ⁹Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4Alkyl-isocyclic aryl; Contain 1-4 heteroatomic-(CH that is selected from N, O and S ₂) _0-4-5-6 unit heterocycle;-(CH ₂) _1-6-C (=O)-O-C _1-4-alkyl; With-(CH ₂) _1-6-C (=O)-N (R ^6a, R ^6b);

R ^6aAnd R ^6bEach is independently selected from the member by the following group that forms naturally:

H and-C _1-6Alkyl;

X is the member who is selected from by the following group that forms:

(a) through 0-3 R ^1eThe phenyl that group replaces;

(b) through 0-3 R ^1eThe naphthyl that group replaces; With

(c) 6 yuan of heteroaromatic systems, it contains 1-3 N atom and has 0-3 through 0-3 R ^1eThe annular atoms that group replaces; With

(d) contain the heteroatomic 8-10 unit that 1-4 is selected from N, O and S and condense the heteroaromatic bicyclic system, and the 0-3 of a described annelated heterocycles bicyclic system annular atoms is through 0-3 R ^1eGroup replaces;

R ^1eBe the member who is independently selected from by the following group that forms:

Halogen; CF ₃-C _1-4Alkyl; Isocyclic aryl;-C _0-2Alkylidene-CN;-O-R ^2e-C _0-2Alkylidene-C (=O)-O-R ^2e-C _0-2Alkylidene-C (=O)-N (R ^2e, R ^3e);-C _0-2Alkylidene-NO ₂-C _0-2Alkylidene-N (R ^2e, R ^3e);-C _0-2Alkylidene-SO ₂-N (R ^2e, R ^3e);-C _0-2Alkylidene-SO ₂-R ^2eThree alkylhalide groups;-O-C _0-2Alkylidene-O-R ^2e-C _0-2Alkylidene-O-R ^2e-O-C _1-4Alkylidene-C (=O)-N (R ^2e, R ^3e);-O-C _1-4Alkylidene-C (=O)-O-R ^2e-C _0-2Alkylidene-N (R ^2e)-C (=O)-R ^3e-C _0-2Alkylidene-N (R ^2e)-SO ₂-R ^3e-CH ₂-N (R ^2e)-C (=O)-R ^3e-CH ₂-N (R ^2e)-SO ₂-R ^3e-(CH ₂) _0-6-NR ^2eR ^3e-C (=O)-N (R ^2e, R ^3e);-N ((CH ₂) _1-6-OR ^2e) ₂-N (R ¹⁰)-(CH ₂) _1-6-OR ^2e-N (R ¹⁰)-C (=O)-R ^2e-N (R ¹⁰)-SO ₂-R ^2e-C (=N (R ¹⁰))-N (R ^2e, R ^3e); With contain heteroatomic-(CH that 1-4 is selected from N, O and S ₂) _0-6-5-6 unit heterocycle;

R ¹⁰, R ^2eAnd R ^3eBe the member who is selected from by the following group that forms independently of one another:

H;-C _1-4Alkyl;-C _0-2Alkylidene-O-R ^1g-C _0-2Alkylidene-N (R ^1g,-R ^2g);-C _1-4Alkylidene-isocyclic aryl;-C _1-4Alkylidenyl-heterocyclic; And R ¹⁰And R ^2e, or R ^2eAnd R ^3eTogether with the N atom that it connected can form contain 1-4 be selected from the heteroatomic of N, O and S can be through 0-2 R ^1gThe 5-8 unit heterocycle that group replaces;

R ^1gAnd R ^2gBe the member who is selected from following group independently:

H; Halogen;-C _1-4Alkyl, isocyclic aryl; Heterocyclic radical;-CN;-C (=O)-N (R ^3g) R ^4g-C (=O)-OR ^3g-NO ₂-(CH ₂) _p-NR ^3gR ^4g-SO ₂NR ^3gR ^4g-SO ₂R ^3g-CF ₃With-(CH ₂) _pOR ^3g

P is the integer of 0-2; And

R ^3gAnd R ^4gBe selected from the group that forms by following independently of one another:

H;-C _1-4Alkyl and-C _0-4Alkylidene-isocyclic aryl;

Or its pharmaceutically acceptable salt.

In one embodiment, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between about 0.01 and about 2.0mg/kg between total daily dose.In another embodiment, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between about 0.1 and about 1.5mg/kg between total daily dose.In another embodiment, described amount between about 0.4 and about 1.2mg/kg between.

In one embodiment, throw once a day and the blood coagulation amount of suppression to the patient.In another embodiment, in one day, throw and the blood coagulation amount of suppression, but administration every day for several times, i.e. every day twice (" BID ") or every day three times (" TID ") to the patient.

In one embodiment, the pharmaceutically acceptable salt of factor Xa inhibitor chemical compound is maleate.In another embodiment, formula I chemical compound is a formula II chemical compound:

Or its pharmaceutically acceptable salt.In certain embodiments, described salt is maleate.In certain embodiments, the blood coagulation amount of suppression be between about 0.01 and about 2.0mg/kg between or between about 0.1 and about 1.5mg/kg between or between about 0.4 and about 1.2mg/kg between total daily dose.

In another embodiment, the factor Xa inhibitor chemical compound is a maleate, and it has the structure of formula III:

The present invention is also at the unit dose formulations that comprises medical composition, described medical composition comprises factor Xa inhibitor chemical compound (as mentioned below) or its pharmaceutically acceptable salt of formula I, II, IV or the V of pharmaceutically acceptable supporting agent and blood coagulation amount of suppression, or the salt of formula III.In certain embodiments, the blood coagulation amount of suppression between about 0.01 and about 2.0mg/kg between.In other embodiments, dosage between about 0.1 and about 1.5mg/kg between or between about 0.4 and about 1.2mg/kg between.

The present invention also forms the in vitro calibrating of characteristic at the in vivo antithrombotic of measuring chemical compound.Described calibrating comprises following steps:

A) test compounds is introduced in vitro in whole blood or the plasma sample forming test sample book, described in vitro whole blood or plasma sample comprise tissue factor (TF) but and with the thrombin substrate of detection mode labelling;

B) but by in the described test sample book of monitoring with the thrombin substrate cracking in time of detection mode labelling, measure the thrombin activity level in the described test sample book;

C) but by measuring thrombin activity level in the check sample with the thrombin substrate cracking in time of detection mode labelling in the monitoring check sample, wherein said check sample comprise contain tissue factor (TF) but and with the whole blood or the blood plasma of the thrombin substrate of detection mode labelling;

D) the thrombin activity level in compare test sample and the check sample, the thrombin activity level in the wherein said test sample book are low to show that described test compounds has in vivo anti-thrombosis activity.

Described blood and blood plasma can be handled or handle without anticoagulation through anticoagulation.

In certain embodiments of the present invention, but be Z-Gly-Gly-Arg-AMC with the thrombin substrate of detection mode labelling.

Description of drawings

Figure 1A shows that the patient's body intravascular coagulation enzyme through the anticoagulation processing for the treatment of with warfarin produces and the mutual relation of international normalized ratio (INR).Measure in the time of 10 minutes from the thrombin generation in the patient's (n=137) who accepts to stablize the warfarin therapy the plasma sample.Provide from 1/5 form of the patient's data of accepting the warfarin therapy with INR result.Error bars is showed standard error.

Figure 1B shows generation of patient's body intravascular coagulation enzyme and the mutual relation of activated partial Thromboplastin time (aPTT) through the anticoagulation processing with the Enoxaparin treatment.Measure 16 with the blood plasma thrombin generation among the patient of Enoxaparin treatment.Illustrate according to manufacturer by COATEST LMW calibrating and to measure anti-fXa unit.By student t test each group is compared.

Fig. 1 C shows that the patient's body intravascular coagulation enzyme through the anticoagulation processing for the treatment of with unfractionated heparin (UFH) produces and the mutual relation of aPTT.Measure the blood plasma thrombin generation among 15 patients.Illustrate according to manufacturer by COATEST LMW calibrating and to measure anti-fXa unit.By student t test each group is compared.

Fig. 2 is illustrated in the baboon model thrombotic dose response inhibitory action in vivo, and the evidence of in vitro examining and determine measurable in vivo anti-thrombosis activity is provided.In the bent former times class of infusion mediator or shellfish (betrixaban) afterwards, through the platelet of indium labelling deposition in time in chamber.Infusion thromboblast at individual animal is counted in the standardization chamber through the hematoblastic quantity of labelling.Data representation meansigma methods ± standard deviation.By ANOVA, then carry out the platelet deposition of Du Naite after tests (Dunnett ' s post-test) analysis in the time of 140 minutes.Dosage 3 (26-38ng/ml) compares remarkable reduction (P＜0.05) with contrast and dosage 4 (54-88ng/ml) is compared also significantly reduction (P value＜0.01) with contrast.

Fig. 3 be illustrated in from the whole blood of healthy human volunteer to the dose response inhibitory action of thrombin generation.Before test beginning, whole blood is handled with the shellfish song former times class (in nM) of normal concentration.Displaying is because of the relative fluorescence unit that cracking produced through labelling thrombin substrate.

Fig. 4 A and Fig. 4 B be illustrated in from the whole blood of healthy human volunteer to the dose response inhibitory action of thrombin generation.Before test beginning, reach heparin (in nM) with the sulphur of normal concentration whole blood is handled.Therapeutic anti Blood clotting level used among the concentration range that the sulphur that is illustrated among Fig. 4 B reaches heparin and orthomorphia and the acute coronary artery syndrome patient is corresponding.Displaying is because of the relative fluorescence unit that cracking produced through labelling thrombin substrate.

Fig. 5 shows that use gives the in vitro data of calibrating that blood plasma carried out of the healthy human volunteer of shellfish class of bent former times from per os.In example 8, this is carried out more detailed description.

Fig. 6 shows shellfish class of bent former times and the Enoxaparin of measuring according to the institute that carried out unidirectional venography in the example 9 between the 10th day and the 14th day, and from using trouble venous thromboembolism (VTE) percentage of patients and 95% confidence interval of carrying out the Enoxaparin historical control (B and C) of the research of two-way venography in orthomorphia (total knee arthroplasty) back between the 10th day and the 14th day.In example 9, this is carried out more detailed description.Corresponding to the research details of Enox B hematology (Blood) 102 (11); Report in 2003.Report in the gloomy people's such as (Lassen) of Lay list of references (thrombosis hemostasis periodical (JThromb Haemostasis) 2007 on JIUYUE 15) corresponding to the research details of Enox C.

Fig. 7 is illustrated in the 2nd day, leave hospital and during phlebography with the shellfish class of bent former times of various dose and Enoxaparin treatment back reduction from the baseline thrombin generation.Displaying is by the mean change (standard error of ± meansigma methods) of the baseline relative fluorescence unit in the blood plasma thrombin generation of following up a case by regular visits to and treating generation.The patient who left hospital at the 2nd day also represents with the meansigma methods of described research day.

Fig. 8 is illustrated in the 2nd day, leave hospital and the shellfish class of bent former times of various dose and the active variation of anti-fXa of Enoxaparin during phlebography.Displaying is by measured average (standard error of ± meansigma methods) the anti-fXa activity (U/mL) of following up a case by regular visits to and treating of Coatest LMW heparin calibrating.But the detectable limit of anti-fXa is 0.05U/ml; But the value that will be lower than detectable limit is made as 0.025.The patient who left hospital at the 2nd day also represents with the meansigma methods of described research day.

Fig. 9 is illustrated in the 2nd day, leaves hospital and the bent former times class's concentration (ng/mL) of blood plasma shellfish of the shellfish class of bent former times of various dose during phlebography.

The specific embodiment

Before describing compositions and method, should be appreciated that to the invention is not restricted to described ad hoc approach, scheme, cell strain, calibrating and reagent that it can change.Should also be clear that term desire used herein describes specific embodiment of the present invention, and it limits category of the present invention illustrated in the claims of enclosing never in any form.

Unless otherwise defined, otherwise all technology used herein have the meaning identical with those skilled in the art in the invention's common sense with scientific terminology.Although can use and those similar or equivalent any method and materials as herein described in enforcement of the present invention and the test, describe method for optimizing, device and material now.All technology and patent disclosure case that this paper quotes all are incorporated herein by reference.This paper should be interpreted as and admit that the present invention does not have precedence over the qualification of the disclosure of existing invention.

Unless otherwise indicated, otherwise the present invention will adopt the conventional techniques of tissue culture, immunology, molecular biology, microbiology, cytobiology and recombinant DNA to implement, and described technology is in affiliated territory.For example compile (2001) molecular cloning experiment guide (Molecular Cloning:A Laboratory Manual), the 3rd edition referring to Sa Brooker (Sambrook) and Russell (Russell); Su Beier people such as (Ausubel) difficult to understand compiles (2007) modern molecular biology rules (Current Protocols in Molecular Biology) series of books; Enzymology method (Methods inEnzymology) series of books (new york academic publishing house company limited (Academic Press, Inc., N.Y.)); McPpherson people (1991) PCR 1 such as (MacPherson): practical approach (A Practical Approach) (the IRL publishing house of Oxford University Press (IRL Press at Oxford University Press)); People such as McPpherson (1995) PCR 2: practical approach (A Practical Approach); Ha Luo (Harlow) and blue grace (Lane) are compiled (1999) antibody technique experiment guide (Antibodies, A Laboratory Manual); Fu Leixieni (Freshney) (2005) Zooblast culture medium plinth technical manual (Culture of Animal Cells:A Manual of Basic Techique), the 5th edition; Gai Te (Gait) compiles (1984) oligonucleotide synthetic (Oligonucleotide Synthesis); United States Patent (USP) the 4th, 683, No. 195; Breathe out Metz (Hames) and John Higgins (Higgins) and compile (1984) nucleic acid hybridization (Nucleic Acid Hybridization); Anderson (Anderson) (1999) nucleic acid hybridization (Nucleic Acid Hybridization); Breathe out Metz (Hames) and John Higgins (Higgins) volume (1984) and transcribe and translate (Transcription and Translation); The immobilization of enzyme and cell (Immobilized Cells and Enzymes) (IRL publishing house (1986)); Pei Baer (Perbal) (1984) molecular cloning practical guide (A Practical Guide to Molecular Cloning); This (Calos) of Miller (Miller) and Carlow compiles the gene transfer vector (Gene Transfer Vectors for Mammalian Cells) (cold spring harbor laboratory (Cold Spring Harbor Laboratory)) of (1987) mammalian cell; Ma Jidisi (Makrides) compiles gene transfer and the expression (Gene Transfer and Expression in Mammalian Cells) in (2003) mammalian cell; Mayer (Mayer) and fertile gram (Walker) are compiled the immuno-chemical method (Immunochemical Methods in Cell and Molecular Biology) (academic press, London (AcademicPress, London)) in (1987) cell and the molecular biology; Hertz En Baige people such as (Herzenberg) compiles the Webster handbook (Weir ' sHandbook of Experimental Immunology) that (1996) experiment immunization is learned; Mice embryonic operation experiments guide (Manipulating the MouseEmbryo:A Laboratory Manual), the 3rd edition (publishing house of cold spring harbor laboratory (Cold Spring HarborLaboratory Press) (2002)).

All numeric representations are to change the approximation of ((+) or (-)) with increment 0.1, and described numeric representation is pH value, temperature, time, concentration and molecular weight for example, comprises scope.Should be appreciated that, although always clearly do not stipulate, but all numeric representations are all titled with term " about ".Should be appreciated that, although always clearly do not stipulate, but reagent as herein described only is the equivalent of the known described reagent of illustration and affiliated field.

1. definition

According to the present invention and as used herein, unless clear and definite regulation in addition, otherwise use following implication to limit following term.

As used in description and claims, unless context offers some clarification in addition, otherwise singulative " " and " described " comprise a plurality of indicants.For example, term " cell " comprises a plurality of cells, comprises its mixture.

As used herein, wish that term " comprises " to mean compositions and method comprises described key element, but do not get rid of other key element." basically by ... form " when being used for definitions section compound and method, will mean other key element that the combination of eliminating for the defined purpose has few basic meaning.Therefore, the compositions of being made up of described key element basically will not get rid of to come the contaminant trace species of self-separation and purification process and pharmaceutically acceptable supporting agent, described supporting agent such as phosphate buffered saline, antiseptic etc. as herein defined." by ... form " will mean and eliminate other composition and be used to throw with the essence method step of the present composition or make compositions or realize the trace key element of the procedure of processing of expected results.By the embodiment of each definition in these transitional term all within category of the present invention.

Term " total daily dose " refer in 24 hour period, threw and medicine or the amount of chemical compound.

Term " test sample book " or " calibrating solution " comprise whole blood or blood plasma (handle or handle), tissue factor (" TF ") without anticoagulation through anticoagulation but, with the thrombin substrate and the test compounds of detection mode labelling.Described solution can also contain many suitable solvents, such as dimethyl sulfoxine (DMSO).In certain embodiments, test sample book or calibrating in the solution blood or blood plasma is unprocessed or through the enzyme titration, comprise the reptile toxenzyme, such as reptilase

In another embodiment, blood or blood plasma are not to be rich in platelet.

Term " tissue factor " or TF are present in subendothelial tissue, platelet and the leukocyte, and split and start from the proenzyme thrombinogen to form thrombin be essential.TF also is called Thromboplastin, factor III or CD 142.Used TF is preferably the human TF of human recombinant in the inventive method, and it is from moral spirit medicine (Dade BehringPharmaceuticals) or the U.S. diagnosis company limited (American Diagnostica, Inc.) purchase.Used TF can also be the combination of endogenous TF or endogenous TF and external source TF in the calibrating.

Term " test compounds " refers to the non-peptide compound of molecular weight less than 1500 dalton (Dalton) as used herein.This term also refers to molecular weight less than 5000 daltonian little heparinoid and saccharide parts.

Term " check sample " refers to test sample book or the calibrating solution that does not have test compounds.

Term " without the whole blood of anticoagulation processing " refers to from the collected blood of patient of not accepting to carry out with antithrombotic agent anticoagulant therapy.This term also refers to from accepting anticoagulant therapy but no longer realizes the patient's of any benefit or effect blood.

Term " through the whole blood of anticoagulation processing " or " through the blood plasma of anticoagulation processing " refer to blood or the blood plasma that obtains from the nearest patient who experiences or experiencing anticoagulant therapy.This term also refer to from human donor obtain and be collected in by venipuncture the anticoagulant that contains external source and add (such as, citrate, EDTA or heparin) culture medium in blood.

The term thrombin substrate of detection mode labelling " but with " refers to the thrombin substrate that detectable is carried out labelling of knowing by any number.Described label includes, but is not limited to fluorescent marker, phosphorescent labels, chemiluminescent labels, electrochemiluminescence label and radioactive marker.In some embodiments of the invention, be the Z-Gly-Gly-Arg-AMC that buys from Ba Heng company (Bachem) through labeled substrate.

Term " anti-thrombosis activity " refers to chemical compound and suppresses thrombosis, and the typical case to coagulation parameters, platelet and platelet function measures the ability that produces acceptable effect simultaneously.With improper thrombosis is that the condition of illness of feature comprises the condition of illness that relates to tremulous pulse and vein blood vessel structure.For the coronary blood tubular construction, unusual thrombosis is the feature of formed plaques may rupture, described plaques may rupture is the main cause of acute myocardial infarction and unstable angina, simultaneously the feature of the obstruction coronary artery thrombosis that is still caused by angioplasty (PTCA) in thrombolytic therapy or the percutaneous coronary.

For the vein blood vessel structure, unusual thrombosis is to experience the feature of situation about being observed among the patient of lower limb or abdomen area capital operation, described patient suffers the thrombosis in the vein blood vessel structure usually, and the blood flow that causes arriving affected limb reduces and is the inducement of pulmonary infarction.The feature of the DIC that unusual thrombosis still occurs in vascular system during septic shock, some viral infection and cancer usually, described DIC consumes rapidly and the general blood coagulation for there being thrombin, cause in whole microvessel structure, forming life-threatening thrombi, thereby produce extensive organ failure's the patient's condition.

The practicality that the evidence of anti-thrombosis activity will make anticoagulant therapy have to prevent stored whole blood from solidifying and other biological sample of preventing to be used to test or store solidifies.

Term " thiazolinyl " refers to trivalent straight or branched unsaturated aliphatic group.Term " alkynyl " refers to and comprises at least two straight or branched aliphatic groups by the carbon of three key connecting.If do not specify carbon number, then thiazolinyl and alkynyl refer to the group with 2-12 carbon atom separately.

Term " alkyl " refers to the saturated aliphatic groups that comprises straight chain, side chain and cyclic group, and described group has the appointment carbon number, or if do not specify carbon number, then has 12 carbon atoms at the most.As used herein, term " cycloalkyl " refers to monocycle, dicyclo or the three cycloaliphatic rings with 3 to 14 carbon atoms and preferred 3 to 7 carbon atoms.Term " alkylidene " refers to divalent alkyl, promptly-and CH ₂-.

As used herein, wish that term " carbocyclic ring structure " and " C3-16 carbocyclic ring monocycle, dicyclo or tricyclic structure " or its similar terms mean separately and only have the stabilizing ring structure of carbon atom as annular atoms, wherein said ring structure is the member who is substituted or is unsubstituted who is selected from by the following group that forms: as the stable monocycle (" aryl ") of the aromatic ring with 6 annular atomses; The stable monocycle non-aromatic ring that in ring, has 3 to 7 annular atomses; The stable twin nuclei that in two rings, has 7 to 12 annular atomses altogether, in the ring structure that it all is aromatic ring that wherein said twin nuclei is selected from by two rings, the ring one is the group that the ring structure of aromatic ring and ring structure that two rings all are non-aromatic rings are formed; And the stable tricyclic structure that in three rings, has 10 to 16 atoms altogether, wherein said tricyclic structure is selected from the group that is made up of following ring structure: in the ring three are that two in the ring structure, ring of aromatic ring are that in the ring structure of aromatic ring and the ring three are the ring structures of non-aromatic ring.Can be saturated, fractional saturation or fully saturated independently when in all cases, described non-aromatic ring is in being present in monocycle, dicyclo or tricyclic structure.The example of described carbocyclic ring structure includes, but is not limited to cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, adamantyl, ring octyl group, [3.3.0] double-octane, [4.3.0] bicyclic nonane, [4.4.0] dicyclo decane (naphthalane), [2.2.2] double-octane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl or tetralyl (tetrahydronaphthalene).In addition, ring structure as herein described can specify side group to be connected via any carbon atom that forms rock-steady structure and one or more.With term that the carbocyclic ring structure is used in combination " be substituted " mean the hydrogen atom that is connected with the ring carbon atom of ring structure as herein described can be through at one or more replacements in the specified substituent group of described structure, as long as described replacement will produce stable compound.

The included term " aryl " of term " carbocyclic ring structure " refers to the aromatic ring that is unsubstituted or is substituted, and it is through being selected from following one, two or three substituent groups replacements: low-carbon alkoxy (O-C _1-6Alkyl), low-carbon alkyl (C _1-6Alkyl), low-carbon alkyl amino (NRR, wherein 1 among the R or 2 are low-carbon alkyls), hydroxyl, halogen, cyano group, hydroxyl, sulfydryl, nitro, thio alkoxy (S-alkyl), carboxylic aldehyde radical (OC (O)-H), carboxyl (C (O) OH), alkoxy carbonyl group (C (O)-O-alkyl) and carbamyl (C (O)-NH ₂); Include, but is not limited to isocyclic aryl, heterocyclic aryl and biaryl etc., its all can choose wantonly and be substituted.Preferred aryl groups comprises phenyl, halogen phenyl, low-carbon alkyl phenyl, naphthyl, xenyl, phenanthryl and naphthacenyl.

The included term " aryl alkyl " of term " isocyclic aryl " refers to and one, two or three aryl with appointment carbon number with the alkyl side joint of specifying carbon number.Suitable aryl alkyl includes, but is not limited to benzyl, picolyl, naphthyl methyl, phenethyl, benzhydryl, trityl etc., and it all can be chosen wantonly and be substituted.

As used herein, term " heterocycle " or " heterocyclic ring system " are intended to refer to be selected from and are substituted or are unsubstituted the member by the following group that forms: ring itself has 5-7 member and has 1 to 4 stable monocycle that is selected from the heteroatom of the group that is made up of N, O and S; The stable twin nuclei that has 7 to 12 atoms in two rings altogether, in wherein said two rings at least one has 1 to 4 hetero atom that is selected from the group that is made up of N, O and S, comprises in the described stable monocyclic heterocycles any and hexane or the condensed twin nuclei of phenyl ring; And the stable tricyclic heterocyclic structure that has 10 to 16 atoms in three rings altogether, wherein at least one in three rings has 1 to 4 hetero atom that is selected from the group that is made up of N, O and S.Existing any nitrogen-atoms and sulphur atom all can be through oxidations in the heterocycle of described heterocycle structure.Unless otherwise indicated, otherwise term " heterocycle " or " heterocyclic ring system " comprise aromatic ring, and can be the non-aromatic rings of saturated, fractional saturation or complete saturated non-aromatic ring.Equally, unless otherwise indicated, otherwise term " heterocyclic ring system " comprises that it is not that all rings all contain at least one heteroatomic structure that all rings all contain at least one heteroatomic ring structure and the ring structure, for example comprise in the term " heterocyclic ring system " that a ring is that in phenyl ring and the ring one has one or more heteroatomic twin nucleis, and in two rings each all has at least one heteroatomic twin nuclei.In addition, ring structure as herein described can specify side group to be connected with one or more via any hetero atom that forms rock-steady structure or carbon atom.In addition, term " is substituted " ring carbon atom or one or more hydrogen atoms on the nitrogen-atoms of meaning each ring in the ring structure as herein described can specify the substituent group displacement through one or more, as long as described displacement can produce stable compound.Nitrogen-atoms in the ring structure can be through quaternized, but described chemical compound is belonged to by clearly specifying or is included in the term " pharmaceutically acceptable salt " at specific compound.When the sum of O atom in the single heterocycle and S atom greater than 1 the time, preferably these atoms are not adjacent one another are.Preferably, O atom in the same ring or S atom are no more than 1 in the given heterocycle structure.

The example of monocycle and bicyclic heterocyclic system (alphabet sequence) is an acridinyl, the azocine base, benzimidazolyl, benzofuranyl, benzothienyl (benzothiofuranyl, benzothiophenyl), benzoxazolyl, benzothiazolyl, the benzotriazole base, the benzo tetrazole radical, the benzisoxa oxazolyl, the benzisothiazole base, the benzimidazoline base, carbazyl, 4 α H-carbazyls, carbolinyl, Chromanyl, chromenyl, the cinnolines base, decahydroquinolyl, 2H, 6H-1,5,2-dithiazine base, dihydrofuran also [2,3-β] tetrahydrofuran base, furyl, furan a word used for translation base, imidazolidinyl, imidazolinyl, imidazole radicals, the 1H-indazolyl, the indoline base, the indolizine base, indyl, the 3H-indyl, isobenzofuran-base, different Chromanyl, iso indazolyl, the isoindoline base, isoindolyl, isoquinolyl (benzimidazolyl), isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, the octahydro isoquinolyl, the oxadiazoles base, 1,2,3-oxadiazoles base, 1,2,4-oxadiazoles base, 1,2,5-oxadiazoles base, 1,3,4-oxadiazoles base, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidine radicals, phenanthridinyl, the phenanthroline base, phenazinyl, phenothiazinyl, phenoxazine thiophene base, the phenoxazine base, phthalazinyl, piperazinyl, piperidyl, pteridyl, purine radicals, pyranose, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, the pyrido oxazolyl, the pyridine-imidazole base, the pyrido thiazolyl, pyridine radicals (pyridinyl, pyridyl), pyrimidine radicals, pyrrolidinyl, pyrrolinyl, the 2H-pyrrole radicals, pyrrole radicals, quinazolyl, quinolyl, the 4H-quinolizinyl, quinoxalinyl, quininuclidinyl, tetrahydrofuran base, tetrahydro isoquinolyl, tetrahydric quinoline group, 6H-1,2,5-thiadiazine base, 1,2, the 3-thiadiazolyl group, 1,2, the 4-thiadiazolyl group, 1,2, the 5-thiadiazolyl group, 1,3, the 4-thiadiazolyl group, thianthrene group, thiazolyl, thienyl, the thieno thiazolyl, the thieno oxazolyl, the Thienoimidazole base, thienyl, triazine radical, 1,2, the 3-triazolyl, 1,2, the 4-triazolyl, 1,2, the 5-triazolyl, 1,3,4-triazolyl and ton base.Preferred heterocycle structure includes, but is not limited to pyridine radicals, furyl, thienyl, pyrrole radicals, pyrazolyl, pyrrolidinyl, imidazole radicals, indyl, benzimidazolyl, 1H-indazolyl, oxazoline group or isatin base (isatinoyl).Also comprise the condensed ring and the spiro-compound that contain (for example) above-mentioned heterocycle structure.

As used herein, term " heteroaromatic system " has the definition identical with monocycle and bicyclic system basically, and at least one ring that is described loop systems is that heteroaromatic or described dicyclo have and condensed aromatic series of aromatic carbon ring structure or non-aromatic heterocycle.

As used herein, term " halogen " or " halogen " refer to Cl, Br, F or I substituent group.Term " alkylhalide group " etc. refers to has at least one aliphatic carbon group through the metathetical hydrogen atom of Cl, Br, F or I atom (mixture that comprises different halogen atoms).Three alkylhalide groups for example comprise trifluoromethyl etc.

Term " methylene " refers to-CH ₂-.

Term " pharmaceutically acceptable salt " comprises the deutero-salt of combination from chemical compound and organic or inorganic acid.Described chemical compound is fit to use with free alkali and salt form.In fact, use salt form to be equivalent to use the alkali form; The bronsted lowry acids and bases bronsted lowry addition salts is all within category of the present invention.

" pharmaceutically acceptable acid-addition salts " refer to the biological effectiveness that keeps free alkali and characteristic and not undesirable in biology or others, with mineral acid and the formed salt of organic acid, all example hydrochloric acids of described mineral acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid etc.; Described organic acid such as acetic acid, propanoic acid, glycolic, acetone acid, ethanedioic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, Loprazolam, ethane sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid etc.

" pharmaceutically acceptable base addition salts " comprises from deutero-those salt of inorganic base, such as sodium salt, potassium salt, lithium salts, ammonium salt, calcium salt, magnesium salt, iron salt, zinc salt, mantoquita, magnesium salt, aluminum salt etc.Especially preferred ammonium salt, potassium salt, sodium salt, calcium salt and magnesium salt.Comprise primary amine from the deutero-salt of pharmaceutically acceptable organic nontoxic alkali, secondary amine and tertiary amine, be substituted amine (comprising the naturally occurring amine that is substituted), cyclammonium and basic ion exchanger resin are (such as 2-aminopropane., trimethylamine, diethylamine, triethylamine, tripropyl amine (TPA), ethanolamine, the 2-DEAE diethylaminoethanol, trometamol, hexanamine, lysine, arginine, histidine, caffeine (caffeine), procaine (procaine), Compocillin (hydrabamine), choline, betanin, ethylenediamine, glycosamine, methylglucosamine, theobromine, purine, piperazine, piperidines, N-ethylpiperidine, polyamino resin etc.) salt.Especially preferred organic nontoxic alkali is 2-aminopropane., diethylamine, ethanolamine, trometamol, hexanamine, choline and caffeine.

" biological characteristics " at this paper purpose means in vivo effector or antigen function or the activity that The compounds of this invention is directly or indirectly carried out, and described characteristic is usually by in vitro calibrating displaying.Effector function comprises that receptor or ligand combination, any enzymatic activity or enzyme are regulated activity, any carrier is in conjunction with activity, any hormonal activity, promotion or suppresses cell and extracellular matrix or the adherent any activity of cell surface molecule, or any structure effect.Antigen function comprise have can with epitope or the antigen site at the antibody response of its generation.

In The compounds of this invention and the carbon atom of four different substituents bonds are asymmetric carbon atoms.Therefore, chemical compound can diastereomer, the form of enantiomer or its mixture exists.As herein described synthesizing can adopt racemic modification, enantiomer or diastereomer as initial substance or intermediate.Can or separate the diastereo-isomerism product of described synthetic generation by known other method in the affiliated field by chromatography or crystallization process.Equally, can be by using constructed or separating the enantiomerism product mixtures by known other technology in the affiliated field.Each asymmetric carbon atom that exists in the The compounds of this invention can be in two kinds of configurations (R or S), and the both is within category of the present invention.

2. the method and the unit dose formulations that suppress blood coagulation

An aspect of of the present present invention provides by throw the method that suppresses the human patients blood coagulation with the factor Xa inhibitor chemical compound of blood coagulation amount of suppression to the patient.This relates to following wonderful discovery on the one hand: in primate anticoagulant required factor Xa inhibitor chemical compound disclosed herein unit dose to suppress the required unit dose of degree low than obtain similar blood coagulation in other animal model (such as, rodent).As example and graphic in further describe, in vivo in the calibrating and prove this species specificity by in rat, rabbit, baboon and human plasma, in vitro extending prothrombin time (PT).Than primate, wherein in baboon under the 1 μ M concentration and the twice that in the mankind, under 0.4 μ M concentration, observes PT change, in rat under the concentration of 8.9 μ M and the twice that in rabbit, under 1.6 μ M concentration, obtains PT increase.

The factor Xa inhibitor chemical compound is applicable to treats the mammiferous disease patient's condition of suffering from the blood coagulation disease, such as treating or preventing unstable angina, intractable angina pectoris, myocardial infarction, transient ischemic attack, thrombotic apoplexy, embolic stroke, disseminated inravascular coagulation, comprise that obstruction or restenosis more coronarius are poured in venous thrombosis or the treatment in treatment septic shock, the pulmonary infarction prevention again.In addition, described chemical compound is applicable to that treatment or prevention relate to the generation of factor Xa/ thrombinogen multienzyme complex and/or those diseases of effect.This comprises that coagulation cascade is subjected to activatory multiple thrombosis and short thrombosis state, and its thromboembolic complication and Peripheral arteries that includes, but is not limited to venous thrombosis, pulmonary infarction, myocardial infarction, apoplexy, operation is stopped up.Can by throw obstruction coronary artery thrombosis that other disease for the treatment of with The compounds of this invention or preventing includes, but is not limited to be caused by angioplasty in thrombolytic therapy or the percutaneous coronary, in the vein blood vessel structure thrombosis, DIC, exist thrombin to consume rapidly and the patient's condition (cause forming life-threatening thrombi in whole microvessel structure, thereby produce extensive organ failure), hemorrhagic apoplexy, the kidney of general blood coagulation are dialysed, blood oxygenate and cardiac catheterization.

Therefore, an aspect of of the present present invention provides the method that suppresses to have the blood coagulation that needs human patients, it comprises to described patient throws factor Xa inhibitor chemical compound or its pharmaceutically acceptable salt with the blood coagulation amount of suppression, wherein the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.01 and 2.0mg/kg between total daily dose.The specific embodiment of factor Xa inhibitor chemical compound is showed in as in formula I, II, IV, V and its salt as illustrated in hereinafter (such as, formula III).Other embodiment provides the method that suppresses to have the blood coagulation that needs human patients, it comprises to described patient throws factor Xa inhibitor chemical compound or its pharmaceutically acceptable salt with the blood coagulation amount of suppression, wherein the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.05 and 2.0mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.1 and 2.0mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.1 or 0.2 and 1.5mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.4 and 1.2mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.43 and 1.15mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.45 and 0.55mg/kg between total daily dose.In another embodiment of the present invention, to the mankind throw and the amount of factor Xa inhibitor chemical compound make serum content be about 1 μ M or lower.

Another aspect of the present invention provides the unit dose formulations that comprises medical composition, described medical composition comprises factor Xa inhibitor chemical compound or its pharmaceutically acceptable salt of pharmaceutically acceptable supporting agent and blood coagulation amount of suppression, and wherein the blood coagulation amount of suppression of factor Xa inhibitor chemical compound is the total daily dose between 0.01 to 2.0mg/kg; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.05 and 2.0mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.1 and 2.0mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.2 and 1.5mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.4 and 1.2mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between 0.43 and 1.15mg/kg between total daily dose; Perhaps, the blood coagulation amount of suppression of factor Xa inhibitor chemical compound be between about 0.45 and about 0.55mg/kg between total daily dose.

Further describe composite at title in the part of " composite ".

3. thrombin generation calibrating

People have determined to be used to monitor the current index (activated partial Thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), activated clotting time (ACT), anti-fXa unit) of blood coagulation state at existing anticoagulant (that is, heparin and warfarin).Existing test is responsive inadequately to the treatment concentration of assessing direct fXa inhibitor.

Because the realistic objective of fXa inhibitor is the former multienzyme complex of film bind thrombin, will be to measure the splendid means that give the anticoagulation degree that realized among the patient of direct fXa inhibitor so measure the calibrating of thrombin generation.Shellfish class of bent former times is a kind of biological available fXa inhibitor of per os that is in clinical development advanced stage (II phase), uses it for this hypothesis of checking.Shellfish class of bent former times is the directed competitive inhibitor (Ki=117pM) of avtive spot of human fXa and represents greater than 86 at the associated protein enzyme, 000 times specificity, described protease can be such as thrombin, factor VIIa, factors IX a, activated protein C, organize plasminogen activation factor, fibrinolysin and trypsin.Shellfish class of bent former times is effective inhibitor (Ki=801pM) of thrombinogen multienzyme complex.

Under known calibrating in the field (such as the Xin Ha people such as (Sinha) who all incorporates this paper by reference at ATVB 2003, calibrating described in the 23:1098-1104) the difference part with calibrating described herein is additional step, such as material processed blood or the employing different measuring method with snake venom or needs.For example, the Xin Ha described calibratings of people such as (Sinha) need be with reptilase

Handle blood plasma and remove fibrinogen, or use and be rich in hematoblastic blood plasma, or to measure thrombin indirectly such as the label of thrombin Antithrombin III complex.

An aspect of of the present present invention provides predictive compound whether to have the in vivo in vitro calibrating of anti-thrombosis activity.Can measure anti-thrombosis activity by using whole blood to measure thrombin activity.In Figure 1A, 1B and 1C, the amount of thrombin is associated with anticoagulant active.This calibrating also is associated and more develops in Fig. 2 and example with anticoagulant active in vivo.

Described calibrating relates under the situation that has and do not exist test compounds measures thrombin activity.In one embodiment, preparation calibrating solution.Calibrating solution comprise the whole blood handled without anticoagulation, tissue factor (TF) but with the thrombin substrate of detection mode labelling (such as, Z-Gly-Gly-Arg AMC).Add the test compounds of being paid close attention to calibrating in the solution, but and measure thrombin substrate cracking in time with the detection mode labelling.Can change component is introduced the order of examining and determine in the solution.

In addition, also under the situation that does not have test compounds, use calibrating solution to contrast calibrating.Indicated as the cracked reduction of thrombin substrate, examining and determine decreases shows that test compounds has in vivo anti-thrombosis activity to thrombin activity with respect to contrasting in the calibrating of use test chemical compound.

In alternate embodiment, calibrating does not comprise the TF that external source is added.But stir calibrating solution so that the TF of endogenous existence activation in the cell in the whole blood.

In other embodiment of calibrating, calibrating solution comprises the whole blood handled through anticoagulation or the blood plasma handled through anticoagulation but not the whole blood handled without anticoagulation.

Therefore, an aspect of of the present present invention provides the mensuration chemical compound whether to have the in vivo in vitro calibrating of anti-thrombosis activity, and it comprises:

A) test compounds is introduced in the in vitro sample of the whole blood of handling without anticoagulation forming test sample book, the in vitro sample of the described whole blood of handling without anticoagulation comprise tissue factor (TF) but and with the thrombin substrate of detection mode labelling;

C) but by measuring thrombin activity level in the check sample with the thrombin substrate cracking in time of detection mode labelling in the monitoring check sample, wherein said check sample comprise contain tissue factor (TF) but and with the whole blood of handling without anticoagulation of the thrombin substrate of detection mode labelling;

D) the thrombin activity level in more described test sample book and the described check sample, the thrombin activity level in the wherein said test sample book are low to show that described test compounds has in vivo anti-thrombosis activity.

In other specific embodiment, with a) the whole blood of handling through anticoagulation replace with the whole blood handled without anticoagulation, the blood plasma of handling through anticoagulation or the blood plasma of handling without anticoagulation.

2. factor Xa inhibitor chemical compound

As deriving from the polypeptide of eating the blood organism and not reporting several factor Xa inhibitors for the chemical compound of big polypeptide type inhibitor.Other factor Xa inhibitor comprises the micromolecule organic compound, such as having the substituent nitrogen-containing heterocycle compound of amidino groups, two functional groups of wherein said chemical compound can with the avtive spot of factor Xa in two combine.For example, WO 98/28269 describes and has terminal amidino groups (C (=NH)-NH2) pyrazole compound; WO 97/21437 describes the benzimidazole compound that replaces through basic group, described basic group via the straight or branched alkylidene ,-C (=O)-or-S (=O) ₂-bridge joint group is connected with naphthyl; WO 99/10316 describes the chemical compound with 4-phenyl-N-alkyl amidine-piperidyl and 4-phenoxy group-N-alkyl amidine-piperidyl, and wherein said group is connected with 3-amidino groups phenyl through carboxylic acid amides alkylidene amino bridge; And EP 798295 describes the chemical compound with 4-phenoxy group-N-alkyl amidine-piperidyl, and wherein said group is connected with the amidino groups naphthyl via sulfonamide that is substituted or is unsubstituted or carboxylic acid amides bridge joint group.Other comprises the inhibitor (United States Patent (USP) the 6th, 376, No. 515 and the 6th, 844, No. 367) with the structure that comprises the phenyl-amidino groups, phenyl and the halobenzene base that connect through amido link through the report factor Xa inhibitor.Other factor Xa inhibitor has used halogen pyridine radicals displacement halobenzene base (referring to United States Patent (USP) the 6th, 376,515B2 number and the 6th, 835, No. 739).In certain embodiments of the invention, factor Xa inhibitor is a formula I chemical compound mentioned above.Formula I chemical compound is disclosed in United States Patent (USP) the 6th, 844, and No. 367 and the 6th, 376, in No. 515, it all is incorporated herein by reference.

In some embodiments of the invention, factor Xa inhibitor is a formula IV chemical compound:

Wherein:

Z ' and Z " are the optional C that replaces through hydroxyl, carboxylic acid group or carboxylic acid ester groups independently of one another ₁-C ₆Alkyl;

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

R ^1d1And R ^1d3Be the member who is selected from by the following group that forms independently of one another:

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃And

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

Demonstration formula IV chemical compound is selected from following:

In another embodiment of the present invention, factor Xa inhibitor is a formula V chemical compound:

Wherein:

A-Q is the member who is selected from by the following group that forms:

Wherein Z ' is the optional C that replaces through hydroxyl, carboxylic acid group or carboxylic acid ester groups _1-6Alkyl;

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

Demonstration formula V chemical compound includes, but is not limited to:

In other embodiments of the invention, factor Xa inhibitor is a formula II chemical compound:

With and pharmaceutically acceptable isomer, salt, hydrate, solvate and prodrug derivant.Formula II chemical compound is at United States Patent (USP) the 6th, 376, and No. 515 and the 6th, 835,206 disclose as an example in No. 739, described patent all is incorporated herein by reference.The adopted name of formula II chemical compound is shellfish class of bent former times and uses described title sometimes in full.

In a specific embodiment, the salt of formula II chemical compound is maleate.By the protonated maleate that forms of one or more nitrogen-atoms that make formula II chemical compound.In one embodiment, make amidino groups nitrogen (=NH) the protonated (=NH of formula II ₂ ⁺) to form salt.

In one embodiment, the maleate of formula II chemical compound is represented by formula III:

Described salt also is called shellfish class's maleate of bent former times in this article.

In another embodiment, the invention provides formula III salt with crystallization polymorphic forms.In a preferred embodiment, the powder X ray diffraction pattern of crystallization polymorphic forms has in the following roughly characteristic peak positions at least 4 and more preferably 8: 4.9,9.7,13.8,14.1,15.2,17.6,18.5,20.8,21.6,22.7,24.1,26.3,26.82 θ angles.In another embodiment, the powder X ray diffraction pattern has following roughly characteristic peak positions: 4.9,9.7,11.8,13.8,14.1,15.2,17.6,18.5,19.9,20.8,21.6,22.7,24.1,25.0,26.3,26.82 θ angles.The present invention expects that roughly characteristic peak will have the deviation of about+0.22 θ angle at the most.Please referring to the PCT/US2006/43635 of application on November 7th, 2006, it all is incorporated herein by reference about more disclosure of the polymorph of the salt of formula II chemical compound and salt.

4. composite

One embodiment of the invention are at unit dose formulations.Unit dose formulations comprises medical composition, and described medical composition comprises factor Xa inhibitor as indicated above and pharmaceutically acceptable supporting agent.Factor Xa inhibitor exists with common effective blood coagulation amount of suppression between about 0.1mg/kg and about 2.0mg/kg.In certain embodiments, dosage to throw with once or twice every day, makes that every day, total amount was the blood coagulation effective dose through allotment.In certain embodiments, unit dose formulations is to be used for the per os transmission.

In the processing of thrombosis disease, The compounds of this invention and/or salt can be used for such as the lozenge that is used for oral administration, capsule, suck agent or elixir; Suppository; In the compositions of sterile solution or suspension or injectable dispensing etc., or be incorporated in the formed article.Medication administration method will be decided because of individual difference and on following factor, such as the mammiferous type of treat, its sex, body weight, diet, parallel Drug therapy, overall clinical condition, the specific compound that is adopted and/or the special-purpose of salt, the described chemical compound of employing and/or salt and the other factors that the field of medicaments technical staff is recognized.

Be applicable to that capsule of the present invention can use habitual and known capsule encapsulation technique preparation, the 5th, 735, No. 105 described technology of United States Patent (USP) of described technology such as Stroud people such as (Stroud).The normally general cylindrical hollow casing of capsule, it has enough diameters and length can be assemblied in the capsule so that contain the medical solutions compositions of suitable dosage active agents.Capsular outside can comprise plasticizer, water, gelatin, modified starch, glue, carrageenin (carrageenan) with and composition thereof.Be understood by those skilled in the art which kind of compositions is suitable for.

Except active agents, be applicable to that lozenge of the present invention also can comprise other composition that filler, binding agent, compression agent, lubricant, disintegrating agent, coloring agent, water, Pulvis Talci and those skilled in the art recognize.Lozenge can be the homogenizing lozenge with monolayer nuclear, or has a plurality of layers to realize preferably discharging overview.In some cases, lozenge of the present invention can be through the enteric coating coating.It will be understood by one of ordinary skill in the art that other excipient also is applicable in the lozenge of the present invention.

Be applicable to and of the present inventionly suck agent and comprise that the activating agent of appropriate amount and any filler, binding agent, disintegrating agent, solvent, solubilizing agent, sweeting agent, coloring agent and those skilled in the art think essential any other composition.Thereby of the present inventionly suck agent and when contact, dissolve and release bioactive agent with the patient oral cavity through design.It will be understood by one of ordinary skill in the art that other transmission method also is applicable to the present invention.

Be used for storing or dispensing by making salt with the purity of wanting and physiology go up the composite that mixing such as acceptable supporting agent, excipient, stabilizing agent prepare The compounds of this invention and/or salt, and described composite can continue to discharge or timing discharges the composite form and provides.Being used for the treatment of the supporting agent accepted of purposes or diluent is known by field of medicaments and is described in (for example) Lei Shi pharmacy complete works (Remington ' s Pharmaceutical Sciences), Mike publishing company (MackPublishing Co.) is in (A.R. Zhen Laluo (Gennaro) compiles .1985).Described material is nontoxic to the receiver under dosage that is adopted and concentration, and comprises the buffer such as phosphate, citrate, acetate and other organic acid compound and/or salt; Such as ascorbic acid, low-molecular-weight (less than about 10 residues) the peptide antioxidant of (such as, poly arginine); Protein such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Such as glycine, glutamic acid, aspartic acid or arginic aminoacid; Monosaccharide, disaccharide and comprise other carbohydrate of cellulose or derivatives thereof, glucose, mannose or dextrin; Chelating agen such as EDTA; Sugar alcohol such as mannitol or Sorbitol; Equilibrium ion such as sodium; And/or such as the nontoxic surfactants of tween (Tween), pluronic (Pluronics) or Polyethylene Glycol.

The dose formulations of desiring the The compounds of this invention of being used for the treatment of property dispensing and/or salt must be aseptic.Aseptic being easy to realized by the non-velum filteration of warp such as 0.2 micron membranes or by other conventional process.Composite will use lyophilized form to store usually or store with the aqueous solution form.The pH value of preparation of the present invention usually will be between 3 and 11, and more preferably 5 to 9 and most preferably be 7 to 8.Should be appreciated that using some aforementioned excipients, supporting agent or stabilizing agent to make forms ring type polypeptide chemical compound and/or salt.Although preferred dosing way is injection; but also expect other medication administration method; such as adopt such as suppository, implant pill or small column, aerosol, per os dose formulations (such as; lozenge, capsule and suck agent) and local composite (such as, ointment, drop and transdermal patches) intravenous (fast injection and/or transfusion), subcutaneous, intramuscular, per rectum, per rectum, per nasal or the intraperitoneal dispensing of multiple dosage form.Hope is incorporated no Mycoderma of the present invention in the formed article such as implant into, described formed article can adopt inert material, such as biodegradable polymer or synthetic silicones, for example silicone rubber (Silastic, silicone rubber) or other commercial polymer.

Chemical compound of the present invention and/or salt also can liposome transmission system form throw with, the little liposome of described transmission system such as monolayer, the big liposome of monolayer and multilamellar liposome.Liposome can be formed by the multiple lipid such as cholesterol, 18-amine. or phosphatidylcholine.

Chemical compound of the present invention and/or salt also can be by using antibody, antibody fragment, somatomedin, hormone or transmitting with other targeting moiety of molecules of salt coupling.But also can make the suitable polymers coupling of chemical compound of the present invention and/or salt and conduct targeted drug supporting agent.Described polymer can comprise polyvinylpyrrolidone, pyran co-polymer, poly-hydroxyl-propyl group-Methacrylamide-phenol, poly-hydroxyethyl-agedoite-phenol or the polyethylene glycol oxide-polylysine that replaces through the palmityl residue.In addition, can make chemical compound of the present invention and/or salt and a class be suitable for realizing the biodegradable polymer coupling of the controlled release of medicine, for example, the crosslinked or amphipathic nature block polymer of the copolymer of polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid, poly-epsilon-caprolactone (polyepsiloncaprolactone), poly butyric, poe, poly-acetal, poly-dihydropyran, polyacrylonitrile and hydrogel.Can make polymer and semipermeable polymers substrate be configured as formed article, such as valve, support, pipe, prosthese etc.

5. the preparation of chemical compound

A. the salt of formula II chemical compound

Formula II chemical compound can be changed into various mineral acids and organic acid salt, include, but is not limited to HCl salt, lactate, maleate, phenoxyacetic acid salt, propionate, succinate, adipate, Ascorbate, camphorate, gluconate, phosphate, tartrate, citrate, mesylate, fumarate, oxyacetate, naphthalene-1,5-disulfonate, gentisate and benzene sulfonate.It will be understood by one of ordinary skill in the art that can use other acid to prepare is applicable to the salt that comprises formula I chemical compound of the present invention.Expect that also salt of the present invention can easily change into other salt of the present invention.

Multiple technologies are applicable to the salt that preparation is mentioned above and are that the those skilled in the art is known.For example, make formula II chemical compound and one or more molar equivalents want acid in solvent or solvent mixture (described salt is insoluble in described solvent or solvent mixture) or in solvent, react such as water, remove solvent by evaporation, distillation or lyophilizing subsequently.Perhaps, can make formula II chemical compound pass ion exchange resin, maybe can use identical conventional method that a kind of salt form of product is changed into another kind of salt form to form the salt of being wanted.

According to program preparation formula II chemical compound hereinafter described.Maleate is because of the maleate of its remarkable crystallinity, heat and hydrolytic stability and high-purity selecting type II chemical compound.

B. shellfish class of bent former times

Can according in some distinct methods any with the gram scale (＜1kg) or kilogram scale (＞1kg) preparation formula II chemical compound or shellfish class of bent former times.Hereinafter illustrate the gram full scale process in the example 2.Another gram full scale process is illustrated in United States Patent (USP) the 6th, 844, and in 367B1 number (referring to example 266), described patent all is incorporated herein by reference.

Perhaps, but the program of illustrating in the formula II chemical compound use-case 2 with the kilogram scale preparation.The formation of the dimethylamidine of formula II relates to the nucleophillic attack of deprotonation amine to cyano group, and wherein said deprotonation amine is formed by secondary amine and lithium alkylide.As used herein, term " alkyl " refers to the alkyl with 1 to 8 carbon atom.It will be understood by one of ordinary skill in the art that and can form deprotonation amine by other method, and can be by the formation of multiple other method preparation formula II amidine function.

The solvent that is applicable to the inventive method as indicated above is an aprotic solvent, such as oxolane (THF), ether, dimethoxymethane, dioxanes, hexane, methyl tributyl ether, heptane and cyclohexane extraction.In addition, the formation of deprotonation amine can be carried out being lower than under 10 ℃ the temperature.Amine is carried out nucleophilic addition also can carry out being lower than under 10 ℃ the temperature to form formula I chemical compound.It will be understood by one of ordinary skill in the art that and to use multiple other solvent, reagent and reaction temperature to implement the inventive method.

In addition, although the inventive method that is used for to restrain scale preparation formula II chemical compound is similar with the program of using with the kilogram scale, the increase of reaction scale is no more than 3400%.In addition, in some steps, the amount that reduces excess reagent makes gain in yield.It will be understood by one of ordinary skill in the art that formula I chemical compound can be by other chemical method preparation of gram scale and kilogram scale.

Example

Unless otherwise indicated, otherwise description in full employed abbreviation have following implication:

A%=gross area percentage ratio

Aq.=aqueous solution

Cm=centimetre

D=doublet

DSC=differential scanning calorimetry

EDTA=ethylenediaminetetraacetic acid

Eq.=equivalent (equivalent)

EtOH=ethanol

G=gram

HPLC=high performance liquid chromatography

Hr=hour

Hz=hertz (Hertz)

IR=infrared ray

J=coupling constant

Kg=kilogram

KV=kilovolt

L=liter

LOD=detectability

M=molar concentration

M=multiplet

MA=milliampere

Me=methyl

MeO=methoxyl group

MeOH=methanol

Mg=milligram

Min.=minute

ML=milliliter

Mm=millimeter

MTBE=methyl tributyl ether

N=equivalent (normal)

NM=nanomolar concentration

NMR=nuclear magnetic resonance, NMR

S=unimodal

TDS=total dissolved solid

TGA=thermogravimetry

THF=oxolane

μ M=micro-molar concentration

Twice of BID=every day

Example 1

The preparation of the crystallization polymorphic salt of formula III

A. restrain the preparation of scale

Loading type I free alkali compound (25g in the 1500mL three neck round-bottomed flasks that are equipped with condenser; 1 equivalent) and under agitation add 9: 1EtOH/ water (500mL).The gained slurry is heated to 70 ℃.With solution form (9 of 100mL: 1EtOH/ water) dropwise add maleic acid (12.77g; And after adding 50mL, solution becomes gets obviously clarification 2 equivalents).After the interpolation of finishing maleic acid solution, temperature remained under 80 ℃ last 5 minutes.Make container slowly be cooled to 45 ℃ and interpolation 400mL MTBE.With solution restir 12 hours.Leach the gained precipitate and drying under vacuum.The maleate (14.2g) of productive rate recovery type I chemical compound with 45%.

B. the preparation of kilogram scale

Formula I chemical compound (24.6kg) is packed in the 760L GLMS reactor (reactor A).Add maleic acid (12.7kg, 2.0 equivalents), ethanol (445kg, 18.1 parts) and high-purity water (140kg, 5.7 parts).Reactant mixture is adjusted to 22 ℃ (19-25 ℃), and, then transfers in the damping 780L Haast alloy reactor (Hastelloy reactor) (reactor B) by polishing filter 22 ℃ of following stir abouts 1 hour.Enter in the reactor B with extra ethanol (about 45kg) flushing reactor A pump and pipeline by polishing filter.Filtrate is concentrated, up to residue about 140L (5.7 parts by volume) under vacuum with the maximum temperature of 45 ℃ of warm ethylene glycol baths (reactor heating chuck).Sampling is used for producing NMR to the reactor B content, and described NMR shows ethanol: the mol ratio of the maleate of formula I chemical compound is 26.The reactor volume that reclaims up to obtaining about 140L (5.7 parts by volume) will be concentrated in high-purity water (49kg, the 2.0 parts) reactor B of packing into and under the vacuum.NMR indication ethanol in the production: the mol ratio of the maleate of formula I chemical compound is 14.Concentrate under high-purity water (49kg, 2.0 parts) and the vacuum of packing into once more and reclaim the reactor volume that obtains about 140L.NMR shows ethanol in the production: the mol ratio of the maleate of formula I chemical compound is 5.The temperature of reactor B content is adjusted to 22 ℃ (19-25 ℃) and the formation of range estimation affirmation slurry.Reactant mixture at 22 ℃ (19-25 ℃) following stir about 2 hours, and then is filled into and is equipped with 30 " on the whizzer of filter cloth.Enter 30 " in the whizzer by polishing filter with two parts of high-purity waters (each about 30kg) flushing reactor B pump and pipeline.Sampling is used for producing HPLC to filter cake, and described HPLC shows that product purity is 99.1A%, and maximum contaminant is 0.26A%, and therefore need not recrystallize.Filter cake (33.1kg) is dry under vacuum with the maximum temperature of 40 ℃ of warm ethylene glycol baths (heat drier chuck).After about 30.5 hours, LOD analysis indication solvent is 0% in the production.Desciccate is discharged (26.4kg) and storage under 2-8 ℃.The productivity ratio expection of end product is higher slightly, is 85% (expection 50-80%).By measuring the purity of measuring maleate that exists with HPLC, and find purity＞99% through hydrolysis amidine content.

¹H?NMR(DMSO-d ₆)：δ3.0(s，3H)，3.2(s，3H)，3.82(s，3H)，7.2(d，1H，J＝9.0Hz)，7.42(s，1H)，7.68(d，1H，J＝8.0Hz)，7.95-8.15(m，2H)，8.12(m)，8.18(m，1H)，8.42(s，1H)，9.0(s，1H)，11.0(s，1H)，11.2(s，1H)；IR(KBr，cm ^-1)：3300，1685，1600，1515，1380，1270，1200，1100，1050，880，800，710。

Example 2

The preparation of formula II chemical compound

A. restrain the preparation of scale

Preparation formula F chemical compound (455g, 1.0 equivalents) in THF (4.67kg, 10.3 parts) slurry and be adjusted to＜10 ℃.Be prepared as follows the dimethylformamide lithium: hexyl lithium (2.3N/ hexane, 2.45L, 5.5 equivalents) is added in the dimethylamine solution (2N/THF, 2.8L, 5.5 equivalents) of maintenance＜10 ℃.Dimethylformamide lithium solution packed into contain in the slurry of formula F chemical compound, keep temperature of reaction kettle＜10 ℃.By HPLC monitoring reaction process in producing, described HPLC confirms amount＜1.0A% of formula F.Preparation NaHCO ₃(490g, 1.1 parts, 5.7 equivalents) and Na ₂CO ₃(490g, 1.1 parts, 4.5 equivalent eq.) buffer solution in deionized water (6.6kg, 14.51 parts), and above-mentioned reactant mixture is transferred in aqueous solution of＜5 ℃ of this maintenances.Product precipitation is separated out and the gained slurry is adjusted to 20 ℃ through 12 hours period.Cross filter solid, and with the gained wet cake with 3.5kg (7.7 parts) deionized water wash.Use coarse sintering glass platform filter to leach solid, and wash with cold (0-5 ℃) dehydrated alcohol (628g, 1.4 parts).Product is dry down at 30-35 ℃.Obtain 458g (productive rate 73%) desciccate.

B. the preparation of kilogram scale

Preparation formula F chemical compound in 780L Haast alloy reactor (reactor A) (31.5kg, 1.0 equivalents) in THF (251kg, 8.0 parts) slurry and be adjusted to 0 ℃ (3 to 3 ℃).2M dimethylamine (161.0kg, 5.0 equivalents) that will be in THF and THF (63kg, 2 parts) pack in the 1900L GLMS reactor (reactor B) and be adjusted to 0 ℃ (3 to 3 ℃) under at utmost stirring.In hexyl lithium (2.3M, 97.2kg, the 4.5 equivalents) reactor B of slowly packing into, keep 10 ℃ maximum temperature simultaneously.With THF (3.2kg) flushing pump and pipeline in reactor B.The reactor B content is adjusted to 0 ℃ (3 to 3 ℃), then transfers in the reactor A, keep reactor A temperature≤10 ℃ simultaneously.With THF (31.4kg, 1.0 parts) flushing reactor B pump and pipeline.The reactor A content is adjusted to 0 ℃ (3 to 3 ℃), and under described temperature, stirs until finish (1-2 hour) by the HPLC confirmatory reaction.Behind the stir about 1 hour, HPLC analyzes indication residue 0A% initial substance (standard in the production: maximum 1A%) in the production.The reactor A content is adjusted to-5 ℃ (8 to-3 ℃).Make water carry out cleaning in the production of reactor B.The aqueous solution (NaHCO in water (236kg, 7.5 parts) with two parts of previous preparations ₃(35.0kg, 1.1 parts) and the Na in water (236kg, 7.5 parts) ₂CO ₃(35.0kg, 1.1 parts)) in the reactor B of packing into and be adjusted to-3 ℃ (0 to 6 ℃).The reactor A content is transferred in the reactor B by heat insulation pipeline, and the temperature that keeps reactor B is-8 ℃ to maximum 5 ℃.With cold [5 ℃ (8 to-3 ℃)] THF (31.4kg, 1.0 parts) flushing reactor A pump and pipeline.The reactor B content was adjusted to 22 ℃ (19-25 ℃) and stir about 3 hours.Range estimation confirms that slurry forms, and the reactor B content is filled into is equipped with 30 " on the whizzer of filter cloth.With drinking water (63kg, 2 parts) flushing reactor B pump and pipeline to being equipped with 30 " on the whizzer of filter cloth.Wet cake (66.5kg) shifted back in the reactor B and last about 1 hour in drinking water (1005kg, 32 parts), carrying out slurry washing under 22 ℃ (19-25 ℃).Product is filled into 30 " on the whizzer (in the production cleaning and be equipped with after the filter cloth) and with drinking water (63kg, 2 parts) flushing reactor B pipeline and pump.Sampling is used for the TDS test to aqueous rinse solution, is found to be 0.46%.Reactor B pump, pipeline and wet cake are further washed with cold [0 ℃ (3 to 3 ℃)] ethanol (44kg, 1.39 parts).Maximum temperature dry wet filter cake under vacuum with 35 ℃ of water-baths (heat drier chuck).LOD is 0% in the production after doing after about 24 hours, and discharges product (24.8kg), productive rate 76.7%.HPLC shows that purity is 98%, and dechlorination impurity is 1.14%.

Example 3

The preparation of formula F chemical compound

Synthesizing of step 1.2-nitro-N-(5-chloro-pyridine-2-yl)-5-methoxyl group-Benzoylamide (C)

5-methoxyl group-2-nitrobenzoic acid (A) (25.0kg, 1.0 equivalents), 2-amino-5 chloropyridine (B) (16.3kg, 1.0 equivalents) and acetonitrile (87.5kg, 3.5 parts) are packed in the 380L GLMS reactor.Adjust reactant mixture to 22 ℃ (19-25 ℃), and add anhydrous pyridine (30.0kg, 3.0 equivalents).With acetonitrile (22.5kg, 0.9 part) flushing pump and pipeline, and reactor content is adjusted to 19-22 ℃ temperature.Phosphorous oxychloride (23.3kg, 1.20 equivalents) is packed in the reactor content by dosing pump, keep the temperature of 25 ℃ (22-28 ℃) simultaneously.With acetonitrile (12.5kg, 0.5 part) flushing dosing pump and pipeline, keeping temperature simultaneously is 25 ℃ (22-28 ℃).Reactant mixture becomes settled solution by slurry usually after adding about 1/3POCl3.Add when finishing, it becomes muddy.After interpolation is finished, reactant mixture was descended stir about 1 hour at 25 ℃ (22-28 ℃), this moment, the reaction of HPLC analysis confirmation was finished.Cooling solution to 15 ℃ (12-18 ℃) and the drinking water of slowly packing into (156.3kg, 6.25 parts) keep reaction temperature simultaneously between 12 and 30 ℃.Then reactant mixture is adjusted to 22 ℃ (19-25 ℃) and stir about stopped until heat release in 5 hours.Range estimation confirms that slurry forms and reactor content is filled on the pressure nutsch filter (nutsche) that is equipped with filter cloth.With two parts of drinking waters (each 62.5kg, 2.5 parts) washing reaction device, pump and pipeline to pressure nutsch filter.The filtrate pH value is 7.Product (41.8kg) is dry under vacuum with the maximum temperature of 50 ℃ of water-baths (heat drier chuck).After about 12 hours, LOD analysis indication solvent is 0.72% in the production.Discharge desciccate (C) (34.4kg), HPLC indication productive rate be 88.2% and purity be 99.1%.

Synthesizing of step 2.2-amino-N-(5-chloro-pyridine-2-yl)-5-methoxyl group-Benzoylamide (D)

The Compound C of in 780L Haast alloy reactor, packing into (33kg, 1.0 equivalents), 5% platinum/carbon (sulfuration, 0.33kg, 0.010 part) and dichloromethane (578kg, 17.5 parts).Begin to stir and reactor content is adjusted to 22 ℃ (19-25 ℃).With reactor with about 30psi pressurized with hydrogen and mild heat reactant mixture to 28 ℃ (25-31 ℃).Under about 30psi, under 28 ℃ (25 to 31 ℃, maximum 31 ℃), carry out the hydrogenation of reactor content, finish until the HPLC Indicator Reaction.16.5 after hour, confirming not exist initial substance (0.472A%) to think that afterwards reaction finishes.Reactor content is filtered by damping Celite pad (with the 0.2-0.5kg kieselguhr of 20-55kg dichloromethane damping) remove platinum catalyst, " preparation in the flash of light filter (sparkler filter) that described Celite pad is 8.With two parts of dichloromethane (each 83kg, 2.5 parts) flushing reactor and bed of diatomaceous earth.Be transferred to filtrate in the 570L GLMS reactor and under atmospheric pressure be concentrated into about 132L (4 parts by volume) therein.The ethanol of packing into (69kg, 2.1 parts) and under atmospheric pressure continue to be concentrated into about 99L (3 parts by volume).NMR indication dichloromethane content is 39% in the production.Reinstall ethanol (69kg, 2.1 parts) and continue to be concentrated into about 99L (3 parts by volume) again.NMR indication dichloromethane content is 5% in the production.Then reactant mixture is adjusted to 3 ℃ (0 to 6 ℃), stir about 1 hour, and with the gained slurries filtration to the chuck pressure nutsch filter that is equipped with filter cloth.With cold [3 ℃ (0-6 ℃)] ethanol (26kg, 0.8 part) flushing reactor, pump and pipeline.Wet cake (36.6kg) is dry down at 40-50 ℃ under vacuum with the maximum temperature of 50 ℃ of water-baths (heat drier chuck).12.5 it is 0.1% that the LOD after hour analyzes the indication solvent.Discharge desciccate (D) (26.4kg), productive rate is 89.5%.HPLC shows that purity is 98.4A%, and dechlorination impurity is 0.083%.

Synthesizing of step 3.N-(5-chloro-pyridine-2-yl)-2-(4-cyano group-benzoyl-amino)-5-methoxyl group-benzamide hydrochloride salt (F)

4-cyano-benzoyl chloride (E) (17.2kg, 1.1 equivalents) and THF (92kg, 3.5 parts) pack in 780L Haast alloy reactor.Reactor content stirring under 22 ℃ (19-25 ℃) is all dissolved until all solids.Gained solution is transferred in the receptor of bottom and with THF (26kg, 1 part) flushing reactor.In Compound D (26.4kg, 1 equivalent), THF (396kg, 15 parts) and pyridine (2.90kg, the 0.4 equivalent) cleaning reactor of packing into.With THF (34kg, 1.3 parts) flushing pump and pipeline.4-cyano-benzoyl chloride/THF solution is packed in the reactor by dosing pump, keep temperature＜30 ℃ and wash with THF (about 10kg).Stirred the gained yellow slurry about 2 hours down at 22 ℃ (19-25 ℃).HPLC display type D compounds content is 0% in the production of carrying out after 2 hours, and Indicator Reaction is finished.With slurries filtration to the pressure nutsch filter that is equipped with filter cloth.With three parts of ethanol (each about 15kg) flushing reactor, pump, pipeline and wet cake.Discharge wet cake (65.4kg) and shift back in the reactor, under 22 ℃ (19-25 ℃), carry out slurry washing and last about 1 hour with ethanol (317kg, 12 parts).With slurries filtration to pressure nutsch filter, and with two parts of ethanol (each about 15kg) and two parts of THF (each about 15kg) flushing reactor, pump, pipeline and wet cake.Wet cake is dry under vacuum with the maximum temperature of 40 ℃ of warm ethylene glycol baths (heat drier chuck).After dry 14.5 hours, LOD is 0.75%.The drying material is ground (sieve mesh 0.125 ") produced the 31.8kg product, with its under vacuum dry 10.5 hours again.Dried LOD is 1.8%, and discharges product (31.5kg), and productive rate is 74.8% (expection 60-90%).HPLC shows that purity is 100%.

Example 4

Measure the bioassay of anti-thrombosis activity

This case description is measured the in vivo in vitro calibrating of anti-thrombosis activity.Used hereinafter described establish the predictive value that baboon model is in vivo checked calibrating.Chemical compound used in the described calibrating is corresponding to formula II compound or its salt.

Materials and methods

For in vitro studying, the blood that is collected into by venipuncture in 3.2% (1: 9 volume ratio) trisodium citrate from minimum 10 donors produces the blood plasma pond.Used specific coagulation zymolyte is Z-GGR-AMC (Bachem).Tissue factor (according to Novi (Innovin), " TF ") is available from moral spirit (Dade Behring).Anti-human TF IgG of rabbit and purified reorganization TF diagnose company limited (American Diagnostica) from the U.S..COATEST LMW heparin/heparin test kit is from Chomogenix.The anticoagulant source of being tested is as follows: Enoxaparin Sodium (An Wante drug company (Aventis Pharmaceuticals)), fondaparinux sodium (GlaxoSmithKline PLC company (Glaxo Smith Kline)), bivalirudin (bivalirudin) (pharmaceuticals (The Medicines Company)), fXa inhibitor C 921-78 (Bei Tesi A (Betz, A.) etc. people's bioid is learned (Biochem.), 1999; 38-14582-14591); Profit cuts down Luo Shaban (rivaroxaban) and profit is cut down husky class (razaxaban).

Under the situation that does not have and exist various anticoagulant and in patient, open thrombin generation in the beginning blood plasma pond by adding according to Novi and calcium with warfarin treatment.By experience the continuous signal measurement thrombin generation that 10 minute period produced by cracking thrombin substrate in FlexStation fluorescence reader (molecular probe company (Molecular Probes)).Fluorescence signal (maximum-minimum relative fluorescence unit) is measured anti-fXa unit with the unit (RFU) of thrombin generation or arbitrary unit (AU) oblatio and by Coatest.By using the anti-human TF IgG of rabbit to carry out immunoblotting, carry out subsequently spectrodensitometry and with purified human TF (the residue 1-263 that in baculovirus, expresses) relatively carry out according to TF concentration in the Novi quantitatively.

For the stripped checking of calibrating, collect blood plasma from 168 patients that accept anticoagulant therapy.Individuality comprises medically stable inpatient or attends the out-patient clinic and accept the individuality (warfarin dosage last about 7 days change) of warfarin.Only when the general measure of predetermined venesection and traditional anticoagulation test (INR, aPTT etc.), just collect blood, and other acupuncture is not carried out in this research.Regather 5mL blood and be placed in the test tube that the sodium citrate anticoagulation is handled.Then with twice of sample centrifugalize collecting the blood plasma part, and then described blood plasma is stored in rapidly under-80 ℃.

The result

Calibrating is to having the anticoagulant sensitivity of different mechanism of action.In healthy donor's blood plasma, add unfractionated heparin (UFH) or low molecular weight heparin (LMWH) produces dose-dependent inhibition in calibrating.Similarly, directly thrombin or fXa inhibitor also cause with the dose proportional inhibition active.Show of the effect of the specific inhibitor of thrombin or factor Xa in the table 1 to the inductive blood plasma thrombin generation of TF.

Table 1

Medicine	??IC ₅₀
Medicine	??IC ₅₀	Sulphur reaches heparin	??160nM
The thrombin inhibitor bivalirudin	??2.4μg/mL	Sulphur reaches heparin	??160nM
The thrombin inhibitor bivalirudin	??2.4μg/mL	FXa inhibitor C 921-78	??17nM
FXa inhibitor profit is cut down Luo Shaban	??80nM	FXa inhibitor C 921-78	??17nM
FXa inhibitor profit is cut down Luo Shaban	??80nM	FXa inhibitor profit is cut down husky class	??125nM

This calibrating also is applicable to the blood coagulation resisting function of measuring short-term and long-term anticoagulation processing patient.APTT through the patient of UFH or LMWH treatment measures relevant with the inhibition to thrombin generation.For the conventional tag thing of thrombin generation (according to the TAT and the F 1.2 of ELISA measurement), this bioassay provides the outstanding dependency of measuring with INR.It the results are shown among Figure 1A, 1B and the 1C.The degree of thrombin generation (AU) when measuring in the plasma sample 10 minutes, described blood sample sample is from the patient of (a) warfarin treatment, and at draw from 137 patients' INR value (Figure 1A), (b) patient of Enoxaparin treatment, at drawing (Figure 1B) from 16 patients' aPTT value and (c) patient of unfractionated heparin (UFH) treatment, at aPTT value drawing (Fig. 1 C) from 15 patients.

Example 5

Shellfish class of bent former times and sulphur reach the comparison of heparin

The evaluation of carrying out the calibrating of whole blood prothrombinase comes comparison shellfish class of bent former times and sulphur to reach heparin, i.e. the indirect inhibitor of fXa.To reach heparin the same with sulphur, and shellfish class of bent former times suppresses platelet-mediated thrombinogen enzymatic activity in the described test macro in the dose dependent mode.

Materials and methods

The amide hydrolysis calibrating (Xin Ha (Sinha), people such as the U Europe medical journals of Chinese (Eur.J.Pharmacol.) 2000 that suppress purified human fXa and associated protein enzyme according to previous report; 114:2313-6).For prothrombinase suppresses active mensuration, human plasma thrombinogen-2 available from blood techniques company (Haematologic Technologies) and thrombin substrate B oc-Asp (OBzl)-Pro-Arg-AMC.HCl available from Ba Heng company (Bachem).Use Dynafit software (Biokin) to drive the data analysis of kinetic parameter.Shellfish class of bent former times shows that the Ki of 0.117 (nM) and the Ki of prothrombinase are 0.801 (nM).

Adopt the calibrating described in the example 4.In arteriovenous shunt, incorporate (the people's clinical research periodical (J.Clin.Invest.) 1993 such as the Chinese gloomy (Hanson) SR of test shellfish class of bent former times in the baboon model of thrombosis device into; 92:2003-12).Binary (two component) thrombosis device uses warp ¹¹¹The platelet of In labelling and warp ¹²⁵The fibrinogen of I labelling is measured the thrombus growth on dacron patch and the expanding chamber.Before used the multiple anticoagulant of this scale-model investigation (common and low molecular weight heparin, thrombin and fXa inhibitor).

The result

The shellfish class of bent former times of 4 dosage (0.05,0.12,0.21 and 0.49mg/kg) is observed dose-dependent inhibition to venous thrombosis.During described time-histories, carry out the stripped measurement of blood plasma thrombin generation, aPTT, PT, activated clotting time (" ACT ") and anti-fXa unit.Opposite with the anti-thrombosis activity that observes (30 to 89% platelet depositions suppress, and 0 to 87% fibrin deposition suppresses), the extension minimum of shellfish class of bent former times treatment back coagulation parameter.Its result shows in following table 2.

Table 2

It is every milliliter 0.31 unit that anti-fXa unit is lower than three quantitation limit and maximum dose levels than low dosage.The template bleeding time of arbitrary animal of handling through shellfish class of bent former times is all undisturbed.The stripped parameter relevant with anti-thrombosis activity is to the proportional inhibition (coefficient R of the dosage of blood plasma thrombin generation ²=0.99); It suppresses (lowest dose level) 13% and suppresses in the scope of (maximum dose level) to 72%.Therefore, this novel prothrombinase bioassay is predicted in vivo anti-thrombosis activity.

Following table 3 provides in the baboon model in vivo thrombotic dose response inhibition.Table 3 also provides the anti-fXa activity in the baboon plasma sample during infusion contrast or the test compounds.

Table 3

The administration group	The plasma concentration scope (ng/ml) of shellfish class of bent former times	Platelet deposition suppresses %	The inhibition % that fibrin forms
The administration group		Platelet deposition suppresses %	The inhibition % that fibrin forms	Contrast	??0	??0.00	??0.00
??1	??7-10	??29.71	??-0.38	Contrast	??0	??0.00	??0.00
??1	??7-10	??29.71	??-0.38	??2	??13-21	??44.20	??37.50
??3	??26-38	??65.22	??71.97	??2	??13-21	??44.20	??37.50
??3	??26-38	??65.22	??71.97	??4	??54-88	??88.77	??87.12

Fig. 2 provides other evidence of in vitro examining and determine measurable in vivo anti-thrombosis activity.Specifically, Fig. 2 shows in the baboon model in vitro thrombotic dose response inhibition.Between infusion shellfish schedule of bent former times, warp ¹¹¹The platelet of In labelling is deposition (n=3 of each dosage) in venous chamber in time.To control animal infusion mediator (n=4).

Fig. 3,4A and 4B show shellfish class of bent former times compared to sulphur reach heparin in human whole blood to open the inhibition of the thrombin generation of beginning by tissue factor.Cracking by the fluorogen substrate comes quantitative thrombin activity.Reach heparin and measure dose response by in from 11 healthy donors' whole blood, adding not commensurability shellfish class of bent former times and sulphur the inhibition of thrombin generation.The relative fluorescence unit (RFU) that shows representative donor measures.

Originally studies show that the feature in vitro and in vivo of shellfish class of bent former times as effective inhibitor of purified human fXa and thrombinogen multienzyme complex.Specifically, to relevant people albuminoid enzyme (such as, thrombin and activation of protein C) have a specificity greater than 86,000 times.Also there is dose-dependent inhibition in the thrombin generation that tissue factor in the human whole blood is opened the beginning and in arteriovenous thrombosis primate model also there is dose-dependent inhibition in ongoing thrombosis, and coagulation parameter or bleeding time do not prolong.Therefore, compared to the canonical measure of blood coagulation, this calibrating has more predictability to the in vivo effect in the animal model.

Example 6

The effect of inhibition prediction fXa inhibitor in the in vivo model of venous thrombosis (DVT) to prothrombinase and insoluble factor Xa

This example is through designing to test following hypothesis: will predict that at the effectiveness of the fXa inhibitor that is incorporated into the fXa in the thrombinogen multienzyme complex in vivo antithrombotic forms effect.Test the ability of thrombinogen multienzyme complex in 8 kinds of fXa inhibitor inhibition human plasmas, described 8 kinds of fXa inhibitor are from having a series of active 4 kinds of structure unique chemical medicine series at fXa.The cracking subsequently of thrombin generation and specific coagulation zymolyte is used as the active measurement of prothrombinase, and (2 * lag) required inhibitor concentration define to suppress active 2 times of prolongations by the time that produces the maximum thrombin generation of realization.Also measure the in vitro rabbit PT time.Measure in vitro rabbit prothrombin time (PT) by in plasma in rabbit, adding Thromboplastin.Inhibition (Huo Lunbaqi people such as (Hollenbach), thrombosis hemostasis (Thromb.Haemost.) 1994 according to previous described assessment rabbit DVT model; 71:357) and related with the plasma concentration of medicine.

Materials and methods

Synthetic compound (people such as (Zhu) Zhu; The current proposition of medical chemistry (Curr.Top.Med.Chem.) 2001; 2:101-119; Bei Tesi people's bioids such as (Betz) is learned (Biochem.), 1999; 36:14582-91).After forming paranitroanilinum, peptide substrates Z-D-Arg-Gly-Arg pNA (Diapharma) suppresses 50% required compound concentration (IC50) (Xin Ha people such as (Sinha), Europe medicine (Eur.J.Pharmacol.) 2000 at the amidohydrolase activity of in Spectramax plate reader (molecular device (Molecular Devices)), measuring under the 405nm the human fXa of purified solubility (blood techniques company (Haematologic Technologies)); 395:51-59).In the hematoblastic pooled human plasma of handling with reptilase of shortage, after peptide thrombin substrate Pefachrome TG (Pan Defa (Pentapharm)) cracking, under 405nm, measure thrombin formation (Xin Ha people such as (Sinha), ATVB that paranitroanilinum assessment prothrombinase causes; 2003; 23:1098-1104).The compound concentration that causes the time increase twice that realizes maximum thrombin generation is recorded as 2 * lag concentration.Increase to measure and form effect by measuring at the thrombotic antithrombotic of rabbit vein by means of inserting thrombosis on the cotton thread that conduit in the left thigh vein places caval vein.Express the chemical compound infusion and reduce (Huo Lunbaqi (Hollenbach) people of etc.ing, thrombosis (Thromb.Haemost.) 1994 that stop blooding with respect to the thrombus weight of mediator tester after 2 hours; 71:357).

By the drug level in the LC-MS/MS measurement plasma in rabbit.(ACL 3000 to use Thromboplastin C+ (Dade) to measure the rabbit prothrombin time on automatic blood coagulation timer, instrument experiment chamber (InstrumentationLaboratory)) (Xin Ha people such as (Sinha), Europe medicine (Eur.J.Pharmacol.), 2000; 395:51-59).

The result

All chemical compounds suppress 50% with solubility fXa under the concentration below the 10nM.Yet the ranking order that suppresses the effectiveness of solubility fXa is different from the required ranking order of the former multienzyme complex of Trombin inhibiting.The results are shown in the table 4.

Table 4

Numbering	Compounds category	??fXa?IC ₅₀??(nM)	Prothrombinase 2 * lag (ì M)	Plasma concentration among the DVT (ì M)	Thrombosis suppresses (%)	Rabbit PT2 * variation (ì M)
Numbering	Compounds category	??fXa?IC ₅₀??(nM)	Prothrombinase 2 * lag (ì M)	Plasma concentration among the DVT (ì M)	Thrombosis suppresses (%)	Rabbit PT2 * variation (ì M)	??1	The ketone group thiazole	??0.5	??0.18	??0.06	??94	??7
??2	Aminobenzamide	??1.3	??0.22	??1.14	??37	??2.7	??1	The ketone group thiazole	??0.5	??0.18	??0.06	??94	??7
??2	Aminobenzamide	??1.3	??0.22	??1.14	??37	??2.7	??3	Aminobenzamide	??0.7	??0.24	??1.65	??47	??1.7
??4	Aminobenzamide	??0.4	??0.25	??1.04	??47	??1.0	??3	Aminobenzamide	??0.7	??0.24	??1.65	??47	??1.7
??4	Aminobenzamide	??0.4	??0.25	??1.04	??47	??1.0	??5	Aminobenzamide	??0.8	??0.34	??3.39	??41	??1.5
??6	The naphthalene pyrazoles	??4.4	??0.92	??5.2	??11	??4.7	??5	Aminobenzamide	??0.8	??0.34	??3.39	??41	??1.5
??6	The naphthalene pyrazoles	??4.4	??0.92	??5.2	??11	??4.7	??7	1, the 2-phenylenediamine	??3.5	??1.35	??4.6	??19	??8.8
??8	Isoxazole	??8.2	??1.66	??9.2	??0	??64	??7	1, the 2-phenylenediamine	??3.5	??1.35	??4.6	??19	??8.8

2 * lag value that prothrombinase suppresses changes needed concentration (R with 2 times that realize rabbit PT ²=0.57) also there is bad dependency between.The variation that fXa suppresses activity or rabbit PT all can not be predicted the activity in venous thrombosis (DVT) model in vivo.On the contrary, can be according to the effectiveness in the chemical compound prothrombinase calibrating in vitro, the effect that chemical compound is in vivo suppressed thrombus growth roughly is divided into 3 levels.Chemical compound 1 has minimum 2 * lag value of 0.18 μ M, and is the most effective in vivo thrombogenesis inhibitor, is 94% inhibition under the plasma concentration of 65nM.2 * lag the value of second group of chemical compound in the prothrombinase calibrating is in the scope of 0.22 to 0.34 μ M, and it is in vivo thrombosis inhibition 37 to 47% under the plasma concentration of 1.04 to 3.39 μ M scopes.The 3rd compounds is for least effective thrombinogen enzyme inhibitor (2 * lag value is greater than 0.92 μ M) and even can not significantly suppress in vivo thrombosis under the plasma concentration of 9.2 μ M.2 * lag the value that obtains in the calibrating of described data show prothrombinase but not to the inhibition of solubility fXa or the prolongation of rabbit PT time to the tool predictability of the fXa inhibition effect in the rabbit DVT model in vivo.

Because the restriction of current anticoagulant, so factor Xa (fXa) meeting point that is arranged in the external and inherent path of blood coagulation makes it become new therapy to research and develop attractive target.About the research of the structure-activity relation (SAR) of potential fXa inhibitor, the calibrating of measuring the inhibition of solution phase fXa proteinase activity is obvious starting point.Yet the ability that chemical compound suppresses the purified fXa of solubility is not the in vivo interactional true reflection of inhibitor and enzyme.This be because fXa in the maximum contribution that occurs during with factor Va combination coagulation process as the part of thrombinogen multienzyme complex, this moment, proteinase activity increased by 300,000 times with respect to solution phase fXa.The activity increase of fXa is extremely difficult in the thrombinogen multienzyme complex suppresses, and therefore any assessment that the anti-fXa of chemical compound is renderd a service should comprise the evaluation that prothrombinase is suppressed.Can in lacking hematoblastic blood plasma (PPP), measure the thrombinogen enzymatic activity, and under the described conditions, consider that also plasma proteins is in conjunction with the effect that chemical compound is renderd a service.Another of the effect of assessment chemical compound antagonism coagulation process more direct mode is to use the calibrating of in vitro condensing, such as prothrombin time (PT) and activated partial Thromboplastin time (aPTT).

Example 7

The evaluation of bioassay in a plurality of cumulative dose studies

Fig. 5 shows the in vitro data of calibrating that the blood plasma from the healthy human donor who gives shellfish class of bent former times with per os carries out.Acceptance 40,80 in continuous separately 10 days per 12 hours of 9 individualities or 120mg medicament capsule.Contrast individual (12) is with placebo or the treatment of non-anticoagulant medicine tester.In opening the in vitro biology calibrating of thrombin generation of beginning, tissue factor estimates from the described individual plasma sample that obtains.Also use the drug level of high performance liquid chromatography and tandem mass spectrometry analysis plasma sample.The result shows the dose response mutual relation between the inhibition of shellfish class's plasma drug level of bent former times and thrombin generation in the biology calibrating.

Example 8

Human treatment's concentration of shellfish class of bent former times

Carry out the thrombin generation calibrating described in example 4, wherein chemical compound being exsomatized is added in the human whole blood.By the PT value and the aPTT value that lack in the various species of compound determination that added in the hematoblastic blood plasma.Go up analyzing samples at ACL 3000plus (Coalter (Coulter)), use Thromboplastin C+ measures PT and use

FS activation PTT reagent is measured aPTT.

Shellfish class of bent former times and sulphur reach TAT and the F1.2 that heparin (fXa inhibitor indirectly) all significantly suppresses in the human whole blood and produce.Reach the treatment level (200nM) of heparin compared to sulphur, two kinds of labels that the more effective Trombin inhibiting of the bent former times class of shellfish (200nM) produces.

Materials and methods

In three animal models, estimate the shellfish class of bent former times of a plurality of dosage.First model measurement places the grumeleuse on the cotton thread of rabbit abdominal part caval vein to increase, and it is compared to the inhibitory action of thrombi shellfish class of bent former times with the effect of the super therapeutic dose of Enoxaparin (LMW heparin).

Second model relatively shellfish class of bent former times under the arterial flow condition at FeCl ₃Keep the ability of unobstructed blood vessel and the ability of Enoxaparin or clopidogrel (clopidogrel) (anti-platelet agents) realization same function in the rat carotid artery thrombosis that causes.

The 3rd scale-model investigation is built in warp on dacron patch in the thigh arteriovenous shunt and the expanding chamber to the baboon body ¹¹¹The inhibitory action of the platelet deposition of In labelling.In all models of being tested, shellfish class of bent former times and Enoxaparin with the intravenous infusion form throw with and the clopidogrel oral administration last 3 days.In all models, measure exsomatize PT and aPTT.The strict standard that has the dose response anti-thrombosis activity of tremulous pulse and venous thrombosis and shellfish class of bent former times generation in described model each in three models.The super treatment level that advantageously compares shellfish class's effect of bent former times and Enoxaparin and clopidogrel.

Increase and measure by measuring the thrombotic effect of rabbit vein by means of inserting thrombosis on the cotton thread that conduit in the left thigh vein places caval vein, as the Huo Lunbaqi people such as (Hollenbach) who all is incorporated herein by reference, thrombosis hemostasis (Thromb.Haemost.) 1994; 71:357-362 is described.Reduction with respect to mediator tester statement infusion chemical compound thrombus weight after 2 hours.Also breathe out people such as (Sinha) referring to suffering, Europe medicine (Eur.J.Pharmacology) 395:51-59 (2000), it all is incorporated herein by reference.

Rat FeCl ₃Model is from KURZ (Kurz), human thrombomodulins such as K.D. research (Thromb.Res.) 1990; 60 (4): 269-280 revises, and it utilizes left neck artery, describedly all is incorporated herein by reference with reference to case.Implement stricture of artery with silk ligature, thereby the blood flow that will slightly damage reduces 50%, uses 50%FeCl at duration of experiment subsequently ₃By pulse Doppler blood flow probe monitor blood flow and at FeCl ₃Used the back record 90 minutes.Intravenous throwing and Enoxaparin and Bei Qu former times class.Four times a day oral administration and clopidogrel.At (the Oregon Health ﹠amp of Univ Oregon Health ﹠ Science; Science University) according to the Chinese gloomy (Hanson), people's clinical research periodical (J.Clin.Invest.) 1993 such as S.R.; The described program of 92:2003-2012 is carried out the baboon model, describedly all is incorporated herein by reference with reference to case.In simple terms, bring out thrombosis by two different thrombosis devices incorporating in external thigh artery-vein (A-V) shunt.Artery model utilizes the thrombosis on the high shear stress environment promotion dacron patch.The vein model utilizes the thrombosis in the low shear stress environment promotion expanding chamber.Respectively with platelet and fibrin with ¹¹¹In and ¹²⁵I fibrinogen labelling is in advance measured platelet deposition and fibrin accumulation.

The result

Astoundingly, different with rodent model, the effect in the primate obtains under much lower dosage, wherein the prolongation minimum of PT.In vitro prolongation by PT in rat, rabbit, baboon and the human plasma and aPTT is the prover species specificity also.Under 8.9,1.6,1 and 0.4 μ M concentration, obtain 2 of PT * variation respectively.Data show the shellfish class's dosage of bent former times that suppresses thrombin generation in the human blood and provide with shellfish class's dosage of bent former times with the similar blood coagulation resisting function of baboon of every kilogram of 0.05-0.49mg dosed administration can be enough to prevent venous thrombosis among the mankind.The relatively simulation of the PT intensity of variation of antagonism thrombosis efficacy levels also makes us predict: the human treatment that can obtain shellfish class of bent former times is active and need not to change simultaneously stripped coagulation parameter.Data are presented in the table 5.

Table 5

Example 9

Shellfish class of bent former times and Enoxaparin prevent the comparison in the random experiment of venous thromboembolism incident behind total knee arthroplasty

This research comprises the patient of 215 experience total knee arthroplastys (TKR), give 15mg or 40mg shellfish bent former times class dosage or every day twice subcutaneous give 30mg Enoxaparin (Enox) dosage with 2: 2: 1 ratio random packet to accept every day twice per os with described patient, last 10 to 14 days.Research concealment 15 and 40mg administration, but do not conceal shellfish class of bent former times and Enox.Venous thromboembolism (" VTE ") (forcing symptom or asymptomatic venous thrombosis being arranged or the symptom pulmonary infarction is arranged on the unidirectional phlebography) for taking place in main effect terminal point at the 10th day to the 14th day.The safety terminal point comprises that the phlebography postoperative takes place serious and clinical significantly not serious hemorrhage all day.By the powerful and hemorrhage terminal point of the central ruling of ignorant independence committee (blinded, independent central adjudication committee) ruling.

Materials and methods

Shellfish class of bent former times in described research with the gelatine capsule form throw with.Described capsule contains shellfish class's maleate of bent former times, dextrose monohydrate and magnesium stearate.

The operation back was carried out first administration and twice (BID) administration every day thereafter of shellfish class of bent former times in 6 to 8 hours.Operation back is subcutaneous throwing and Enoxaparin and threw in per thereafter 12 hours and once (q 12h) after 12 to 24 hours.Unless satisfy the stopping criterion of scheme regulation, otherwise continue treatment 10 to 14 days.After leaving hospital, the patient hauls oneself willingly into and studies medicine.Throw and the final dose of studying medicine in predetermined pressure venography morning (the 10th day to the 14th day) of operation lower limb.

When screening, throwing in the 2nd day with the dosage after 1 to 4 hour in morning of research medicine, leave hospital day and forced phlebography in the 10th day to the 14th day before obtain and be used to assess the blood sample that plasma drug level and drug effect are measured.Also use the shellfish class of bent former times of high performance liquid chromatography and tandem mass spectrometry analysis plasma sample.Based on analysis the 0.100-50.0ng/mL scope is verified described method to 0.2mL blood plasma.The calibration standard curve that uses the weighted least squares regression analysis to produce carries out quantitatively.

Except that activated partial Thromboplastin time (aPTT), prothrombin time (PT) and international normalized ratio (INR), also suppress calibrating and anti-xa activity measurement blood coagulation resisting function by the thrombin generation described in the example 4.The calibrating of anti-Xa unit is to change 96 hole culture plate forms into from Coatest LMW heparin test kit (Diapharma) modification and with it.In this calibrating, duplicate standard verification sample and patient's sample and quantitation limit are 0.05 anti-Xa U/mL.By in patient's blood plasma (0.1mL) of handling through citrate anticoagulation blood, adding the generation that tissue factor (according to Novi, the moral spirit) and calcium open the beginning thrombin.Measure the formation of thrombin through the cracking of 10 minute period by specific coagulation zymolyte (Z-GGR-AMC, Ba Heng company).In FlexStation fluorescence reader (molecular device), measure relative fluorescence unit and use it for quantitative thrombin generation.All patient's plasma samples are reported with the version from each individual baseline relative fluorescence unit with triplicate calibrating and to the inhibition of thrombin generation.

The result

Observe shellfish class of bent former times to the inhibition of thrombin generation and the dosage and the concentration dependent effect of anti-fXa level.175 patients' (81.4%) effect and safety evaluation are as shown in table 6.

Table 6

Main effect and safety results	The bent former times 15mgBID of class of shellfish	The bent former times 40mg of the class BID of shellfish	??Enox?30mg?BID
Main effect and safety results	The bent former times 15mgBID of class of shellfish	The bent former times 40mg of the class BID of shellfish	??Enox?30mg?BID	??VTE％(95％CI)	??20(12-32)	??15(8-27)	??10(3-23)
Severe haemorrhage % (95%CI)	??0(0-4)	??0(0-4)	??2(0-12)	??VTE％(95％CI)	??20(12-32)	??15(8-27)	??10(3-23)
Severe haemorrhage % (95%CI)	??0(0-4)	??0(0-4)	??2(0-12)	Clinical significantly not serious hemorrhage	??0(0-4)	??2(0-8)	??5(1-16)

Fig. 6 shows in this research shellfish class of bent former times that measured with unidirectional phlebography and the VTE ratio and 95% confidence interval of Enoxaparin between the 8th day and the 10th day, and Enoxaparin be in hospital the VTE ratio and 95% confidence interval of contrast, described Enoxaparin is in hospital contrasting data from the research of using the two-way venography of carrying out between the 10th day and the 14th day behind the TKR.Fig. 9 describes (the 2nd day) after the administration for the second time, leave hospital and the blood plasma shellfish class's concentration of bent former times during venography.As expecting,, increase after the administration several times of mean drug concentration pro-and as if when leaving hospital, reach steady statue (median is the 4th day) two shellfishes class's dosage groups of bent former times according to the long half-lift institute of shellfish class of bent former times.Shellfish class of bent former times represents dose dependent and the concentration dependent effect (Fig. 7 and Fig. 8) to Trombin inhibiting generation and anti-Xa level.15mg shellfish class of bent former times is similar with the effect that Enoxaparin observes to the effect that thrombin generation suppresses, and the being seen effect of 40mg shellfish song former times class is more remarkable.On the contrary, antagonism xa activity being seen effect is similar to the effect of 40mg shellfish class of bent former times and Enoxaparin and less than the effect of 15mg shellfish class of bent former times.Other Blood clotting parameter of not appreciable impact (that is, aPTT, PT and INR) is treated by class of bent former times with shellfish.

Shellfish class of bent former times effectively prevents the VTE behind the TKR and is safe and well tolerable.

Should be appreciated that, although the present invention is described in conjunction with the foregoing description, above stated specification and example meant for illustration and do not limit category of the present invention.Others, advantage and modification in the category of the present invention will be apparent to those skilled in the art in the invention.

Claims

1. an inhibition has the method for the blood coagulation that needs human patients, and it comprises to described patient throws factor Xa inhibitor chemical compound with the formula I of blood coagulation amount of suppression:

A-Q-D-E-G-J-X

I，

Wherein:

A is selected from:

(a) C ₁-C ₆Alkyl;

(b) C ₃-C ₈Cycloalkyl;

(d) phenyl that replaces through 0-2 R substituent group independently;

(e) naphthyl that replaces through 0-2 R substituent group independently; With

Have the monocycle or the condensed-bicyclic heterocyclic ring system of 5 to 10 annular atomses, the 1-4 of a wherein said loop systems annular atoms is selected from N, O and S, and wherein said loop systems can replace through 0-2 R substituent group;

R is selected from:

H, halogen ,-CN ,-CO ₂R ¹,-C (=O)-N (R ¹, R ²) ,-(CH ₂) _m-CO ₂R ¹,-(CH ₂) _m-C (=O)-N (R ¹, R ²) ,-NO ₂,-SO ₂N (R ¹, R ²) ,-SO ₂R ¹,-(CH ₂) _mNR ¹R ²,-(CH ₂) _m-C (=NR ³)-R ¹,-(CH ₂) _m-C (=NR ³)-N (R ¹, R ²) ,-(CH ₂) _m-N (R ⁴)-C (=NR ³)-N (R ¹, R ²), side joint is in containing heteroatomic 3 to 6 yuan of heterocyclic-(CH that 1-4 is selected from N, O and S ₂) _mNR ¹-group ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CF ₃,-OR ²With contain the heteroatomic 5-6 unit heterocyclic ring system that 1-4 is selected from N, O and S, the hydrogen atom of the 1-4 on the wherein said heterocyclic ring system can be independently through be selected from by halogen ,-C ₁-C ₄Alkyl ,-C _1-4Alkyl-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

M is the integer of 0-2;

H ,-OR ⁵,-N (R ⁵,-R ⁶) ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkyl phenyl and-C _0-4Alkyl naphthyl, the hydrogen atom of the 1-4 on the annular atoms of wherein said phenyl and described naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms; Or R ¹And R ², or R ²And R ³Can form 3-8 unit's cycloalkyl or heterocyclic ring system together, wherein said heterocyclic ring system can have 3 to 10 annular atomses, wherein have 1 to 2 and encircle and contain 1-4 hetero atom that is selected from N, O and S in described loop systems, the hydrogen atom of the 1-4 on the wherein said heterocyclic ring system can be independently through being selected from by halogen, C ₁-C ₄Alkyl ,-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

R ⁵And R ⁶Be independently selected from the group that forms by following:

H ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkylidene-phenyl and-C _0-4Alkylidene-naphthyl, the hydrogen atom of the 1-4 on the annular atoms of wherein said phenyl and described naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms; Or R ⁵And R ⁶Can form 3-8 unit's cycloalkyl or heterocyclic ring system together, wherein said heterocyclic ring system can have 3 to 10 annular atomses, wherein in described loop systems, have 1 to 2 and encircle and contain the hetero atom that 1-4 is selected from N, O and S, the hydrogen atom of the 1-4 on the wherein said heterocyclic ring system can be independently through be selected from by halogen ,-C ₁-C ₄Alkyl ,-CN ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl and-NO ₂Member's displacement of the group that forms;

Q is the member who is selected from by the following group that forms:

R ⁷Be selected from:

H ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-C _0-4Alkylidene-phenyl and-C _0-4Alkylidene-naphthyl, the hydrogen atom of the 1-4 on the annular atoms of wherein said phenyl and described naphthyl moiety can be independently through be selected from by halogen ,-C _1-4Alkyl ,-C _2-6Thiazolinyl ,-C _2-6Alkynyl ,-C _3-8Cycloalkyl ,-C _0-4Alkylidene-C _3-8Cycloalkyl ,-CN and-NO ₂Member's displacement of the group that forms; D is direct bond or is selected from member by the following group that forms:

(a) independently through 0-2 R ^1aThe phenyl that substituent group replaces;

(c) have the monocycle or the condensed-bicyclic heterocyclic ring system of 5 to 10 annular atomses, the 1-4 of a wherein said loop systems annular atoms is selected from N, O and S, and wherein said loop systems can be through 0-2 R ^1aSubstituent group replaces;

R ^1aBe selected from:

R ^2aAnd R ^3aBe independently selected from the group that forms by following:

N is the integer of 0-2;

E is direct bond or is selected from member by the following group that forms:

-C _1-2-alkylidene-,-O-,-S-,-SO-,-SO ₂-,-C _0-1-alkylidene-C (=O) ,-C _0-1-alkylidene-C (=O)-N (R ⁸)-C _0-1-alkylidene-,-C _0-1-alkylidene-N (R ⁸)-C (=O)-C _0-1-alkylidene-,-N (R ⁸)-C (=O)-N (R ⁸)-and-C _0-1-alkylidene-N (R ⁸)-;

R ⁸Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4Alkylidene-aryl;-C _0-4Alkylidene-heteroaryl;-C _1-4Alkylidene-C (=O)-OH ,-C _1-4Alkylidene-C (=O)-O-C _1-4Alkyl and-C _1-4-alkylidene-C (=O)-N (R ^2b,-R ^3b);

H ,-C _1-4Alkyl ,-C _0-4Alkylidene-aryl;-C _0-4Alkylidenyl-heterocyclic base, and R ^2bAnd R ^3bCan form together with the N atom that it connected and to contain the heteroatomic 5-8 unit heterocycle that 1-4 is selected from N, O and S,

Wherein said heterocycle can be through 0-2 R ^1cGroup replaces;

R ^1cBe the member who is selected from by the following group that forms:

Halogen;-C _1-4Alkyl;-CN;-NO ₂-C (=O)-N (R ^2c,-R ^3c);-C (=O)-OR ^2c-(CH ₂) _q-N (R ^2c,-R ^3c);-SO ₂-N (R ^2c,-R ^3c);-SO ₂R ^2c-CF ₃With-(CH ₂) _q-OR ^2cR ^2cAnd R ^3cBe the member who is selected from by the following group that forms independently of one another:

H;-C _1-4Alkyl; With-C _1-4Alkylidene-aryl;

Q is the integer of 0-2;

G is the member who is selected from by the following group that forms:

(a) C ₂Thiazolinyl or C _3-8Cycloalkenyl group, wherein said thiazolinyl and cycloalkenyl group junction point are thiazolinyl carbon atom and wherein said-C ₂Thiazolinyl or-C _3-8Cycloalkenyl group is through 0-4 R ^1dGroup replaces;

R ^1dBe the member who is selected from by the following group that forms:

H, halogen; C _1-6Alkyl, aryl ,-CN;-NO ₂-(CH ₂) _0-6-NR ^2dR ^3d-SO ₂NR ^2dR ^3d-SO ₂R ^2d-CF ₃-(CH ₂) _0-6-OR ^2d-O-(CH ₂) _1-6OR ^2d-O-(CH ₂) _1-6-C (=O)-O-R ^2d-O-(CH ₂) _1-6-C (=O)-N (R ^2d, R ^3d);-N (R ^5a)-(CH ₂) _1-6-OR ^2d-N (R ^5a)-(CH ₂) _1-6-N (R ^2dR ^3d);-C (=O)-N (R ^2dR ^3d);-N (R ^5a)-(CH ₂) _1-6-C (=O)-N (R ^2d, R ^3d);-N ((CH ₂) _1-6-OR ^2d) ₂-N (R ^5a)-(CH ₂) _1-6-OR ^2d-N (R ^5a)-C (=O)-R ^2d-N (R ^5a)-SO ₂-R ^2d-(CH ₂) _0-6-C (=O)-O-R ^2d-(CH ₂) _0-6-C (=O)-N (R ^2dR ^3d);-(CH ₂) _0-6-C (=NR ^2d)-N (R ^3d, R ^4d);-(CH ₂) _0-6-N (R ^5a) C (=NR ^2d)-N (R ^3d, R ^4d); Contain 1-4 heteroatomic-(CH that is selected from N, O and S ₂) _0-6-N (R ^3d) C _5-6Unit's heterocycle and contain heteroatomic-(CH that 1-4 is selected from N, O and S ₂) _0-6-5-6 unit heterocycle;

H, C _1-6Alkyl, C _1-6-alkylaryl ,-CN;-NO ₂Isocyclic aryl ,-CN;-NO ₂Or R ^2dAnd R ^3dN atom together with its separate connection forms 5-7 unit heterocycle; Or R ^3dAnd R ^4dForm together with the N atom that it connected and to contain the heteroatomic 5-8 unit heterocycle that 1-4 is selected from N, O and S;

J is direct bond or is selected from member by the following group that forms:

R ⁹Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4-alkyl-isocyclic aryl; Contain 1-4 heteroatomic-(CH that is selected from N, O and S ₂) _0-4-5-6 unit heterocycle;-(CH ₂) _1-6-C (=O)-O-C _1-4Alkyl; With-(CH ₂) _1-6-C (=O)-N (R ^6aR ^6b); R ^6aAnd R ^6bEach is independently selected from the member by the following group that forms naturally:

H and-C _1-6Alkyl;

X is the member who is selected from by the following group that forms:

(a) through 0-3 R ^1eThe phenyl that group replaces;

(b) through 0-3 R ^1eThe naphthyl that group replaces; With

Halogen; CF ₃-C _1-4Alkyl; Isocyclic aryl;-C _0-2Alkylidene-CN;-O-R ^2e-C _0-2Alkylidene-C (=O)-O-R ^2e-C _0-2Alkylidene-C (=O)-N (R ^2e, R ^3e);-C _0-2Alkylidene-NO ₂-C _0-2-alkylidene-N (R ^2e, R ^3e);-C _0-2Alkylidene-SO ₂-N (R ^2e, R ^3e);-C _0-2Alkylidene-SO ₂-R ^2eThree alkylhalide groups;-O-C _0-2Alkylidene-O-R ^2e-C _0-2Alkylidene-O-R ^2e-O-C _1-4Alkylidene-C (=O)-N (R ^2e, R ^3e);-O-C _1-4Alkylidene-C (=O)-O-R ^2e-C _0-2Alkylidene-N (R ^2e)-C (=O)-R ^3e-C _0-2Alkylidene-N (R ^2e)-SO ₂-R ^3e-CH ₂-N (R ^2e)-C (=O)-R ^3e-CH ₂-N (R ^2e)-SO ₂-R ^3e-(CH ₂) _0-6-NR ^2eR ^3e-C (=O)-N (R ^2e, R ^3e);-N ((CH ₂) _1-6-OR ^2e) ₂-N (R ¹⁰)-(CH ₂) _1-6-OR ^2e-N (R ¹⁰)-C (=O)-R ^2e-N (R ¹⁰)-SO ₂-R ^2e-C (=N (R ¹⁰))-N (R ^2e, R ^3e); With contain heteroatomic-(CH that 1-4 is selected from N, O and S ₂) _0-6-5-6 unit heterocycle;

H;-C _1-4Alkyl;-C _0-2Alkylidene-O-R ^1g-C _0-2Alkylidene-N (R ^1g,-R ^2g);-C _1-4Alkylidene-isocyclic aryl;-C _1-4Alkylidenyl-heterocyclic; And R ¹⁰And R ^2e, or R ^2eAnd R ^3eTogether with the N atom that it connected can form contain 1-4 be selected from N, O and S hetero atom and can be through 0-2 R ^1gThe 5-8 unit heterocycle that group replaces;

R ^1gAnd R ^2gBe the member who is selected from following group independently:

P is the integer of 0-2; And

H; C _1-4Alkyl and-C _0-4Alkylidene-isocyclic aryl;

Or its pharmaceutically acceptable salt;

The described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.01 and about 2.0mg/kg between total daily dose.

2. method according to claim 1, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.1 and about 1.5mg/kg between total daily dose.

3. method according to claim 1, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.4 and about 1.2mg/kg between total daily dose.

4. method according to claim 1, the pharmaceutically acceptable salt of wherein said factor Xa inhibitor chemical compound is maleate.

5. method according to claim 1 is wherein thrown and described factor Xa inhibitor chemical compound to described patient once a day.

6. method according to claim 1 is wherein thrown and described factor Xa inhibitor chemical compound to described patient twice of every day or every day three times.

7. method according to claim 1, wherein said factor Xa inhibitor chemical compound has formula IV:

Wherein:

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃And

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

Method according to claim 1, wherein said chemical compound is selected from the group that is made up of following:

Or its pharmaceutically acceptable salt.

9. method according to claim 1, wherein said factor Xa inhibitor chemical compound has formula V:

Wherein:

A-Q is the member who is selected from by the following group that forms:

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃And

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

10. chemical compound according to claim 9, wherein said chemical compound is selected from the group that is made up of following:

Or its pharmaceutically acceptable salt.

11. an inhibition has the method for the blood coagulation that needs human patients, it comprises to the factor Xa inhibitor chemical compound of described patient's throwing with the formula II of blood coagulation amount of suppression:

Or its pharmaceutically acceptable salt, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.01 and about 2.0mg/kg between total daily dose.

12. method according to claim 11, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.1 and about 1.5mg/kg between total daily dose.

13. method according to claim 12, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.4 and about 1.2mg/kg between total daily dose.

14. method according to claim 12, the pharmaceutically acceptable salt of wherein said factor Xa inhibitor chemical compound is maleate.

15. a unit dose formulations that comprises medical composition, described medical composition comprise the factor Xa inhibitor chemical compound of the formula I of pharmaceutically acceptable supporting agent and blood coagulation amount of suppression:

A-Q-D-E-G-J-X

I，

Wherein:

A is selected from:

(a) C ₁-C ₆Alkyl;

(b) C ₃-C ₈Cycloalkyl;

(d) phenyl that replaces through 0-2 R substituent group independently;

(e) naphthyl that replaces through 0-2 R substituent group independently; With

Have the monocycle or the condensed-bicyclic heterocyclic ring system of 5-10 annular atoms, the 1-4 of a wherein said loop systems annular atoms is selected from N, O and S, and wherein said loop systems can replace through 0-2 R substituent group;

R is selected from:

M is the integer of 0-2;

R ⁵And R ⁶Be independently selected from the group that forms by following:

Q is the member who is selected from by the following group that forms:

R ⁷Be selected from:

D is direct bond or is selected from member by the following group that forms:

(a) independently through 0-2 R ^1aThe phenyl that substituent group replaces;

(c) have the monocycle or the condensed-bicyclic heterocyclic ring system of 5-10 annular atoms, the 1-4 of a wherein said loop systems annular atoms is selected from N, O and S, and wherein said loop systems can be through 0-2 R ^1aSubstituent group replaces;

R ^1aBe selected from:

R ^2aAnd R ^3aBe independently selected from the group that forms by following:

N is the integer of 0-2:

E is direct bond or is selected from member by the following group that forms:

R ⁸Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4Alkylidene-aryl;-C _0-4Alkylidene-heteroaryl;-C _1-4Alkylidene-C (=O)-OH ,-C _1-4Alkylidene-C (=O)-O-C _1-4Alkyl and-C _1-4Alkylidene-C (=O)-N (R ^2b,-R ^3b);

R ^1cBe the member who is selected from by the following group that forms:

H;-C _1-4Alkyl; With-C _1-4Alkylidene-aryl;

Q is the integer of 0-2;

G is the member who is selected from by the following group that forms:

R ^1dBe the member who is selected from by the following group that forms:

H, C _1-6Alkyl, C _1-6Alkylaryl ,-CN;-NO ₂Isocyclic aryl ,-CN;-NO ₂Or R ^2dAnd R ^3dN atom together with its separate connection forms 5-7 unit heterocycle; Or R ^3dAnd R ^4dForm together with the N atom that it connected and to contain the heteroatomic 5-8 unit heterocycle that 1-4 is selected from N, O and S;

J is direct bond or is selected from member by the following group that forms:

R ⁹Be the member who is selected from by the following group that forms:

H;-C _1-4Alkyl;-C _0-4Alkyl-isocyclic aryl; Contain 1-4 heteroatomic-(CH that is selected from N, O and S ₂) _0-4-5-6 unit heterocycle;-(CH ₂) _1-6-C (=O)-O-C _1-4Alkyl; With-(CH ₂) _1-6-C (=O)-N (R ^6a, R ^6b); R ^6aAnd R ^6bEach is independently selected from the member by the following group that forms naturally:

H and-C _1-6Alkyl;

X is the member who is selected from by the following group that forms:

(a) through 0-3 R ^1eThe phenyl that group replaces;

(b) through 0-3 R ^1eThe naphthyl that group replaces; With

R ^1gAnd R ^2gBe the member who is selected from following group independently:

P is the integer of 0-2; And

H;-C _1-4Alkyl and-C _0-4Alkylidene-isocyclic aryl;

Or its pharmaceutically acceptable salt;

16. unit dose formulations according to claim 15, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.1 and about 1.5mg/kg between total daily dose.

17. unit dose formulations according to claim 15, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.4 and about 1.2mg/kg between total daily dose.

18. unit dose formulations according to claim 15, the pharmaceutically acceptable salt of wherein said factor Xa inhibitor chemical compound is maleate.

19. unit dose formulations according to claim 15, wherein said factor Xa inhibitor chemical compound has formula IV:

Wherein:

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃And

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

20. unit dose formulations according to claim 19, wherein said chemical compound is selected from the group that is made up of following:

Or its pharmaceutically acceptable salt.

21. unit dose formulations according to claim 15, wherein said factor Xa inhibitor chemical compound has formula V:

Wherein:

A-Q is the member who is selected from by the following group that forms:

R ^1aBe selected from the group that forms by following:

H ,-F ,-Cl and Br;

R ^1d2And R ^1d4Each is H naturally;

H ,-Cl ,-F ,-Br ,-OH and-OCH ₃And

R ^1eBe the member who is selected from by the following group that forms:

-F ,-Cl ,-Br ,-OH ,-CH ₃With-OCH ₃,

Or its pharmaceutically acceptable salt.

22. unit dose formulations according to claim 21, wherein said chemical compound is selected from the group that is made up of following:

Or its pharmaceutically acceptable salt.

23. a unit dose formulations that comprises medical composition, described medical composition comprise the factor Xa inhibitor Compound I I of pharmaceutically acceptable supporting agent and blood coagulation amount of suppression:

24. unit dose formulations according to claim 23, the described blood coagulation amount of suppression of wherein said factor Xa inhibitor chemical compound be between about 0.1 and about 1.5mg/kg between total daily dose.

25. method according to claim 10, the pharmaceutically acceptable salt of wherein said factor Xa inhibitor chemical compound is maleate.

26. judge whether chemical compound has the in vivo in vitro calibrating of anti-thrombosis activity, and it comprises for one kind:

B) but measure thrombin activity level in the described test sample book by monitoring described in the described test sample book with the thrombin substrate cracking in time of detection mode labelling;

C) but described in the monitoring check sample, measure thrombin activity level in the described check sample with the thrombin substrate cracking in time of detection mode labelling, wherein said check sample comprise contain tissue factor (TF) but and with the whole blood or the blood plasma of the thrombin substrate of detection mode labelling;

27. calibrating according to claim 26, wherein said test sample book and described check sample comprise whole blood.

28. calibrating according to claim 27, wherein said whole blood is handled through anticoagulation.

29. calibrating according to claim 27, wherein said whole blood is handled without anticoagulation.

30. calibrating according to claim 26, wherein said test sample book and described check sample comprise blood plasma.

31. calibrating according to claim 30, wherein said blood plasma is handled through anticoagulation.

32. calibrating according to claim 30, wherein said blood plasma is handled without anticoagulation.

33. calibrating according to claim 26, but be Z-Gly-Gly-Arg-AMC wherein with the thrombin substrate of detection mode labelling.