CN100430389C

CN100430389C - Inhibition of phsphoinostide 3-dinase beta

Info

Publication number: CN100430389C
Application number: CNB038243628A
Authority: CN
Inventors: S·P·杰克逊; A·D·罗伯逊; V·肯切; P·汤普逊; H·普拉巴哈兰; K·安德逊; B·阿博特; I·贡卡尔夫斯; W·内斯比特; S·舍恩维尔德; D·赛利克
Original assignee: AstraZeneca AB
Current assignee: AstraZeneca AB
Priority date: 2002-08-16
Filing date: 2003-08-18
Publication date: 2008-11-05
Anticipated expiration: 2023-08-18
Also published as: CN101260104A; CN1688575A

Abstract

The present invention relates to a selective depressant of phosphoinositide (PI) 3-kinase Beta, which is applied to the anti-thrombosis therapy, and a method of screening a compound used by a novel anti-thrombosis therapy by detecting the selective inhibitory activity of the PI 3-kinase Beta of the compound.

Description

Inhibition of phosphoinositide 3-kinase beta

Background of invention

I. invention field

The present invention relates generally to the compound that a kind of new antithrombotic forms therapy and is used for new therapy.More particularly, the present invention relates to the selective depressant of phosphoinositide (PI) 3-kinase beta, this selective depressant forms application in the therapy at antithrombotic, and the selective inhibitory activity of the PI 3-kinase beta by detection compound screens and can be used for the method that this new antithrombotic forms the compound of therapy.

II. the description of association area

Thrombocyte is special adhesive cell, and they play an important role in hemostasis.Under normal circumstances, thrombocyte is inadhesion, is not also activated by blood vessel endothelium.Yet breaking of the damage of endothelium or patch makes the mobile blood comprise in collagen, fibronectin and Feng's von willebrand's factor (vWF) in various thrombosis factors.The round-robin thrombocyte bears the acceptor of these thrombosis factors.Zymoplasm, thrombotonin, with vasoconstrictor nerve thromboxane A2 (TxA2). under the situation of blood vessel injury, via glycoprotein (GP) Ib acceptor, at disruptive flat zone position, thrombocyte adhesiveness becomes state of activation (platelet activation) in specific interior subcutaneous adhesion protein Feng's von willebrand's factor (vWF) (thrombocyte adhesiveness) for example, produces some materials and comprises adenosine diphosphate (ADP) (ADP), zymoplasm, thrombotonin and vasoconstrictor nerve thromboxane A2 (TxA2).The adp receptor that is activated is conversely with the GP IIb/IIIa receptor activation on the platelet surface.These acceptors become the position of scleroproein bridge, and thrombocyte is linked together (platelet aggregation) also forms thrombus subsequently.

Therefore, the breaking suddenly or split of the atherosclerotic plaque of Xing Chenging causes the over-drastic platelet adhesion reaction in advance, causes the formation of the platelet thrombus of vascular occlusion.The formation of these thrombus causes myocardial infarction and apoplexy respectively in crown or cerebral circulation, and they represent first cause dead in the industrialization society altogether.Platelet thrombus forms and also to cause multiple other clinical symptom, comprises unstable angina pectoris, sudden death, transient ischemic attack, temporary black-out and four limbs and internal's acute ischemic.Some factors are influential to the increase of the thrombosis possibility of the patch that breaks, the hyperergy that comprises tackiness substrate in (1) patch, (2) existence of tissue factor in the damage, and (3) cause intravascular space to narrow down by congee sample of blood bolt forming process and the direct platelet activation effect of the high-shear that causes.

It is target with the one or more committed steps in the thrombosis process mainly that existing antithrombotic forms therapy.By giving suitable anticoagulant, comprise one or more coumarin derivativess (for example warfarin and temparin) or charged aggregation (for example heparin, r-hirudin or r-hirudin analogue), or, can reduce or eliminate clot in many cases by using antiplatelet drug (for example in acetylsalicylic acid, clopidogrel, ticlopidine, Dipyridamole or several GPIIb/IIIa receptor antagonist a kind of).Yet because side effect is for example hemorrhage, the reason of inaccessible, " white clot " syndrome, stimulation, fertility defective, thrombopenia and hepatic insufficiency again, anticoagulant and platelet suppressant drug are very restricted.In addition, give anticoagulant and platelet suppressant drug for a long time and can significantly increase life-threatening disease or risk of bleeding.

Therefore, for fear of existing antithrombotic aforementioned disadvantages, needing exploitation a kind of is that target and don't the new antithrombotic that hinders normal haemostasis form therapy with the process that the pathology thrombosis is had crucial meaning optionally.

Rheology disorder (high-shear and turbulent flow) plays main effect in promoting the pathology thrombus, therefore a such strategy should be, by being target with the sensation of the mechanicalness in thrombocyte key element, weakens the activation effect of thrombocyte shearing force.Yet, before the present invention,, also be not identified for shearing stress inductive platelet activation rather than signal effect to stopping blooding significant.

Summary of the invention

Therefore, an object of the present invention is to provide and disintegrate the platelet aggregation that takes place and the method for adhesion under high-shear state, described method comprises the kinase inhibitor to the selectivity PI 3-of patient's effective dosage that these needs are arranged.

Another object of the present invention provides a kind of antithrombotic formation method, and described method comprises to the selectivity PI 3-of patient's effective dosage that these needs are arranged kinase beta inhibitor.According to this method, in to by the significant PI3-kinase beta of the platelet activation of shear-induced, can realize thrombotic specificity inhibition is not influenced normal haemostasis by targeting.Therefore the present invention can not involve for example prolongation in bleeding time of side effect that is caused by normal haemostasis destruction.

Therefore, another object of the present invention provides the method for inhibition by the platelet activation of shear-induced, and described method comprises the selectivity PI3-kinase beta inhibitor to patient's effective dosage that these needs are arranged.By PI3-kinase beta inhibitor to patient's effective dosage that these needs are arranged, the for example method of coronary occlusion, apoplexy, acute coronary syndrome, Acute Myocardial Infarction, restenosis, atherosclerosis and unstable angina pectoris of prevention or treatment cardiovascular disorder is provided, and also is one object of the present invention.In this method, the use of PI3-kinase beta inhibitor can be avoided by destroying for example prolongation in bleeding time of side effect that normal haemostasis causes.

The present invention preferably uses the selectivity PI 3-kinase beta inhibitor of determining by following method, described method comprises candidate compound is contacted with isolating PI 3-kinases isotype, detect the inhibition effect of described compound to each isotype, wherein said compound to the described compound of relatively decision of the detection effect of each isotype as selectivity PI 3-kinase beta inhibitor.

Therefore, another object of the present invention provides the screening method of PI 3-kinase beta, described method comprises candidate compound is contacted with isolating PI 3-kinases isotype, detect the inhibition effect of described compound to each isotype, wherein said compound to the described compound of relatively decision of the detection effect of each isotype as selectivity PI 3-kinase beta inhibitor.

In biochemical measurement, with respect to the I PI 3-kinases isotype of other type, the selectivity that selectivity PI 3-kinase beta inhibitor suppresses PI 3-kinase beta is preferably at least about 〉=10 times, more preferably 〉=20 times, and more preferably 〉=30 times.The selectivity I PI 3-kinases of other type like this comprises PI 3-kinases α, γ and δ.

Another object of the present invention relates to antithrombotic method, and described method comprises to the selectivity PI 3-of patient's effective dosage that these needs are arranged kinase beta inhibitor,

Condition is that described inhibitor is not formula (II) compound:

Wherein,

R is H, OH, F, Cl, Br, I, C ₁-C ₆Alkyl, aryl or (CH ₂) _n-aryl;

R ¹Be H, OH, F, Cl, Br, I, C ₁-C ₆Alkyl, C ₃-C ₆Cycloalkyl, CH=CH-aryl, C ≡ C-aryl, (CHR ³) _n-aryl, NR ³-C ₁-C ₆Alkyl, NR ³-cycloalkyl, NR ³-(CHR ³) _n-aryl, (CHR ³) _n-NR ³-alkyl, (CHR ³) _n-NR ³-cycloalkyl, (CHR ³) _n-O-aryl, (CHR ³) _n-O-alkyl, (CHR ³) _n-O-cycloalkyl, O-(CHR ³) _n-aryl, S-(CHR ³) _n-aryl or CO-aryl, wherein n is 0,1 or 2, and described alkyl, cycloalkyl or aryl are optional is replaced by following group: F, Cl, Br, I, CN, CO ₂H, CO ₂R ³, NO ₂, CF ₃, replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted aryl, OCF ₃, OR ³, OSO ₂-aryl, replacement or unsubstituted amine, NHCOR ³, NHSO ₂R ³, CONHR ³Or SO ₂NHR ³And

R ³Be H or replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted aryl

But be selected from except following formula (II) compound:

9-(3-pyridylmethyl) oxygen base-2-morpholinyl-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-140);

7-methyl-9-phenyl amino methyl-2-morpholinyl-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-183);

8-(4-aminomethyl phenyl) 2-)-4 (1H)-quinolones (TGX-113) 4-morpholinyl);

8-(4-fluorophenoxy)-2-(4-morpholinyl)-4 (1H)-quinolones (TGX-121);

2-morpholinyl-8-(phenyl methyl)-4H-1-chromene-4-ketone (TGX-90);

2-(4-morpholinyl)-8-(4-fluoro-2-aminomethyl phenyl) oxygen base-4H-1-chromene-4-ketone (TGX-184);

The 9-[[(2-chloro-phenyl-)-and methyl] amino-7-methyl-2-(4-morpholinyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-167);

The 9-[[(2-p-methoxy-phenyl)-and methyl] amino]-7-methyl-2-(4-morpholinyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-137);

7-methyl-2-(4-morpholinyl)-9-[(phenyl methyl) amino]-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-126);

9-[[(4-fluoro-2-aminomethyl phenyl) amino]-7-methyl-2-(4-morpholinyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-170);

7-methyl-2-(4-morpholinyl)-9-[[(1R)-1-phenylethyl] amino]-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-123);

7-methyl-2-(4-morpholinyl)-9-[(2-pyridylmethyl) amino]-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-161);

The 9-[[(4-chloro-phenyl-) methyl] amino]-7-methyl-2-(4-morpholinyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-108);

2-(4-morpholinyl)-9-(phenyl methyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-040);

7-methyl-9-(N-methyl-N-phenyl) amino methyl-2-(4-morpholinyl)-4H-pyrido [1,2-a] pyrimidin-4-one (TGX-195);

2-(4-morpholinyl)-8-(phenyl methyl) oxygen base-4H-1-chromene-4-ketone (TGX-102);

2-(4-morpholinyl)-8-(phenyl methyl) amino-4H-1-chromene-4-ketone (TGX-204);

2-(4-morpholinyl)-8-phenyl amino-4H-1-chromene-4-ketone (TGX-324);

8-(3-chloro-phenyl-) oxygen base-2-(4-morpholinyl)-4H-1-chromene-4-ketone (TGX-259);

8-(3-aminomethyl phenyl)-2-(4-morpholinyl)-4 (1H)-quinolones (TGX-127);

8-(2-fluorophenyl)-2-(4-morpholinyl)-4 (1H)-quinolones (TGX-143);

(±)-7-methyl-2-morpholine-4-base-9-[1-(3-pyridinylamino) ethyl] pyrido [1,2-a] pyrimidin-4-one (KN-304).

In another object of the present invention, antithrombotic method relates to the selectivity PI 3-kinase beta inhibitor of the formula of using (I):

Wherein

R is H, C ₁-C ₆Branched-chain or straight-chain alkyl or aryl or (CH ₂) _n-aryl;

R ₁Be H, OH, OCH ₃, OCF ₃, F, Cl, CF ₃, C ₁-C ₆Branched-chain or straight-chain alkyl or aryl or (CH ₂) _n-aryl;

R ₂Be H, the C of R or S configuration ₁-C ₆Branched-chain or straight-chain alkyl or aryl or (CH ₂) _n-aryl;

R ₃Be one or more H, F, Cl, Br, I, CN, CO ₂H, CO ₂R, NO ₂, CF ₃, replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted aryl, OCH ₃, OCH ₂F, OCHF ₂, OCF ₃, OR, OSO ₂-aryl, replacement or unsubstituted amine, NHCOR, NHSO ₂R, CONHR or SO ₂NHR;

X is C or N, and Y is N or O.

In another object of the present invention, antithrombotic method relates to the selectivity PI 3-kinase beta inhibitor of the formula of using (III):

Wherein X and Y are respectively C and O, or are respectively C and NH, or all are N;

R is H, OH, OCH ₃, OCF ₃, F, Cl, Br, I, C ₁-C ₆Alkyl, aryl or (CH ₂) _n-aryl;

R ₁, R ₂And R ₃Be H, OH, F, Cl, Br, I, C independently ₁-C ₆Alkyl, C ₃-C ₆Cycloalkyl, CH=CH-aryl, C ≡ C-aryl, (CHR ' ³) _n-aryl, NR ' ³-C ₁-C ₆Alkyl, NR ' ³-cycloalkyl, NR ' ³-(CHR ' ³) _n-aryl, (CHR ' ³) _n-NR ' ³-aryl, (CHR ' ³) _n-NR ' ³-alkyl, (CHR ' ³) _n-NR ' ³-cycloalkyl, (CHR ' ³) _n-O-aryl, (CHR ' ³) _n-O-alkyl, (CHR ' ³) _n-O-cycloalkyl, O-(CHR ' ³) _n-aryl, S-(CHR ' ³) _n-aryl or CO-aryl, wherein n is 0,1 or 2, and described alkyl, cycloalkyl or aryl are optional is replaced by following group: F, Cl, Br, I, CN, CO ₂H, CO ₂R ' ³, NO ₂, CF ₃, replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted aryl, OCF ₃, OR ' ³, OSO ₂-aryl, replacement or unsubstituted amine, NHCOR ' ³, NHSO ₂R ' ³, CONHR ' ³Or SO ₂NHR ' ³And

R ' ³Be H or replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted aryl.

One object of the present invention relates to the have formula new compound of (III):

In another object of the present invention, antithrombotic method relates to the derivative of the 2-morpholino replacement of Medicine-feeding type (I), wherein:

R is H, C ₁-C ₆Branched-chain or straight-chain alkyl or aryl;

R ₁Be H, OH, OCH ₃, OCF ₃, F, Cl, CF ₃, C ₁-C ₆Branched-chain or straight-chain alkyl;

R ₂Be H, the C of R or S configuration ₁-C ₆Branched-chain or straight-chain alkyl or aryl;

R ₃Be one or more H, F, Cl, Br, CN, CO ₂H, CO ₂R, NO ₂, CF ₃, side chain or straight chain C ₁-C ₆Alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted aryl, OCH ₃, OCH ₂F, OCHF ₂, OCF ₃, OR, replacement or unsubstituted amine, NHCOR, NHSO ₂R, CONHR or SO ₂NHR;

X is C or N, and Y is N or O.

In another object of the present invention, antithrombotic method relates to administration and is selected from following PI 3-kinase inhibitor:

(±)-7-methyl-9-{[methyl (phenyl) amino] methyl }-2-morpholine-4-yl pyridines [1,2-a] pyrimidin-4-one (TGX-195) also;

(±)-7-methyl-2-morpholine-4-base-9-(1-phenyl amino ethyl)-pyrido [1,2-a] pyrimidin-4-one (TGX-221);

(±)-7-methyl-2-morpholine-4-base-9-[1-(4-fluorophenyl amino) ethyl] pyrido [1,2-a] pyrimidin-4-one (TGX-224);

(±)-9-[1-(3,4-difluorophenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-237);

(±)-9-[1-(2,5-difluorophenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-238);

(±)-9-[1-(3,5-difluorophenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-239);

(±)-9-[1-(4-fluoro-2-aminomethyl phenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-240);

(±)-9-[1-(4-chloro-phenyl-amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-243);

(±)-9-[1-(3,4-dichlorophenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-244);

(±)-9-[1-(3-fluorophenyl amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-247);

(±)-9-[1-(3-chloro-phenyl-amino) ethyl]-7-methyl-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-248);

(±)-7-methyl-2-morpholine-4-base-9-[1-(2-thiazolyl amino) ethyl] pyrido [1,2-a] pyrimidin-4-one (TGX-261);

(±)-7-methyl-9-[1-(3-aminomethyl phenyl amino) ethyl]-2-morpholine-4-base-pyrido [1,2-a] pyrimidin-4-one (TGX-262);

(±)-7-methyl-2-morpholine-4-base-9-[1-(3-trifluoromethyl amino) ethyl]-pyrido [1,2-a] pyrimidin-4-one (TGX-264); With

(±)-7-methyl-2-morpholine-4-base-9-[1-(2-pyridinylamino) ethyl] pyrido [1,2-a] pyrimidin-4-one (TGX-295);

(±)-2-(1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] and ethyl } amino) phenylformic acid (KN-309);

(±)-2-(1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] and ethyl } amino) methyl benzoate (KN-321);

(±)-2-(1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] and ethyl } amino) benzonitrile (KN-320);

(±)-7-methyl-2-(morpholine-4-yl)-9-(1-{[2-(2H-tetrazolium-5-yl) phenyl] amino } ethyl)-pyrido [1,2-a] pyrimidin-4-one (KN-325);

(±)-2-(4-morpholinyl)-8[1-(phenyl amino) ethyl]-4H-1-chromene-4-ketone (TGX-280).

An object of the present invention is to provide formula (III) compound, wherein R ¹Be selected from CH ₃, C ₂H ₅,

Also relate to a kind of concrete formula (III) compound, wherein R is a methyl, and R ¹Be

Also relate to a kind of concrete formula (III) compound, wherein R is H, and R ¹Be

The present invention also relates to the method for the phosphoinositide 3-kinase among a kind of patient of inhibition, described method comprises to a certain amount of formula (III) compound that can effectively suppress phosphoinositide 3-kinase among the patient of patient's administration.

The present invention also relates to prevent or treat the method for cardiovascular disorder, described method comprises formula (III) compound to patient's effective dosage that these needs are arranged.

The present invention also relates to prevent or treat the method for respiratory disease, described method comprises formula (III) compound to patient's effective dosage that these needs are arranged.

The present invention also relates to prevention or treatment method for cancer, described method comprises formula (III) compound to patient's effective dosage that these needs are arranged.

The present invention also relates to prevent or the method for treatment and leukocyte function obstacle diseases associated, described method comprises formula (III) compound to patient's effective dosage that these needs are arranged.

A purpose of the inventive method relates to the inhibitor below the administration:

A purpose of the inventive method relates to administration 6-methyl-8-[1-(phenyl amino) ethyl]-2-(4-pyridyl)-4H-chromene-4-ketone.

One object of the present invention relates to administration 6-methyl-8-{1-[(2-aminophenyl) amino] ethyl }-2-(4-pyridyl)-4R-chromene-4-ketone.

The present invention also relates to be selected from following new compound:

(±)-7-methyl-2-morpholine-4-base-9-(1-phenyl amino ethyl) pyrido [1,2-a] pyrimidin-4-one;

(±)-2-(1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] and ethyl } amino) phenylformic acid;

(±)-2-(1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] and ethyl } amino) benzonitrile;

(±)-2-({ 1-[7-methyl-2-(morpholine-4-yl)-4-oxo-pyrido [1,2-a] pyrimidine-9-yl] ethyl } amino) methyl benzoate and

(±)-7-methyl-2-(morpholine-4-yl)-9-(1-{[2-(2H-tetrazolium--base) phenyl] amino } ethyl) pyrido [1,2-a] pyrimidin-4-one.

Therefore, provide the method that suppresses PI 3-kinase beta, comprise that wherein said amount can effectively suppress the PI 3-kinase beta among the patient, remains another one purpose of the present invention to a certain amount of a kind of compound with formula (I) of patient's administration.

The accompanying drawing summary

Practical LY294002 of Figure 1A-1E representative certificate or wortmannin suppress PI 3-kinases and eliminate the test-results of thrombocyte to the reaction of acceleration shearing.Application can make us analyze thrombocyte adhesiveness kinetics and the activation on the fixed von Willebrand factor (vWf) (by monitoring cytoplasmic calcium flow) simultaneously based on the moving adhesion mensuration of extracorporeal flow.Briefly, with of the microscopic capillary perfusion of isolating thrombocyte via vWf (100 μ g/ml) bag quilt, and with confocal microscope real-time analysis cytoplasmic calcium flow.

Figure 1A diagram shows, under various blood flow states with the hematoblastic ratio of the irreversible adhesion of the interactional formation of vWf.Figure (i) in, with isolating thrombocyte with the constant shearing rate (600,1800 or 10,000s ^-1) be filled on the vWf matrix, in scheming (ii), allow isolating thrombocyte on vWf matrix, deposit, and make it with 10 000.s subsequently ^-2Shearing rate gradient (↑ Δ γ) quicken.

Figure 1B shows that single thrombocyte is in the lip-deep situation of fixed vWf; The figure shows cytoplasmic calcium flow (Δ [Ca ²⁺] _c) curve and single thrombocyte in time (second) follow displacement (μ m) relation curve.Applying acceleration shearing rate (↑ Δ γ) before, allow isolating thrombocyte on the vWf surface, deposit with 1 second interval.The application point is sheared in arrow (↓) expression: lasting, and the Δ [Ca of the swing that expression thrombocyte experience and fixing adhesion interrelate ²⁺] _cOf short duration, expression thrombocyte experience is followed the Δ [Ca of of short duration fixedly adhesion ²⁺] _cPeak value; Fluctuation, the expression thrombocyte shows minimum Δ [Ca ²⁺] _cWith in the lip-deep rapid traverse of vWf.

Fig. 1 C diagram shows, (after DMSO＜0.25%v/v), 0.5U/ml apyrase, 1mM acetylsalicylic acid or the 25 μ M LY294002 pre-treatment, show the hematoblastic ratio that the vWf of lasting, of short duration or fluctuation status adheres with carrier.

Fig. 1 D shows representative single thrombocyte record, illustrates with GPIb/V/IX to handle the back at the lip-deep GPIb/V/IX dependency of fixed vWf Δ [Ca in conjunction with control agent, ristocetin (1mg/ml) ²⁺] _cBefore analyzing, with the thrombocyte integrin alpha _IIbβ ₃Antagonist, Aggrastat (200nM) handled 10 minutes: fixed is illustrated under 37 ℃ of situations that do not have to shear thrombocyte was placed 10 minutes on the lath surface of vWf bag quilt.In the time of 10 minutes with Δ [Ca ²⁺] _cMonitored 1.5 minutes; Constant γ, expression is with 1800.s ^-1Constant shearing rate under thrombocyte is filled on the vWf matrix; ↑ Δ γ (0-10,000.s ^-1), expression allows thrombocyte fixedly deposit on the vWf surface, makes γ increase to 10 gradually, 000.s with 1 second interval subsequently ^-1The application point is sheared in arrow (↓) expression; + LY294002, after 10 minutes, general ↑ Δ γ puts on the thrombocyte with 20 μ M PI 3-kinase inhibitor LY294002 pre-treatment in expression.

Fig. 1 E shows the GPIb/V/IX hematoblastic ratio of adhering, and performance is with 1 second or 60 seconds high frequency Δ [Ca at interval ²⁺] _cThe reaction of right ↑ Δ γ.

The characteristic of new PI 3-kinase beta isotype selective depressant

Fig. 2 A shows the selectivity of TGX 221, uses isolating PI 3-kinases isotype to carry out, and wherein measures active by producing PI 3-kinases product phosphatidylinositols 3-phosphoric acid (PtdIns (3) P).Top bar graph explanation detects the generation of PtdIns (3) P with tlc and is suppressed by the dose response that the TGX-221 in p110 α and the p110 β isotype produces.The TGX-221 of graphical representation three main thrombocyte PI 3-kinases isotype p110 α, p110 β and p110 γ suppresses dose response curve.

The functional analysis of TGX-221

Fig. 3 A instruction card is shown in awl-plate viscometer by shearing (5000sec ^-1, 2 minutes) activate after, the bar graph that the dose response that TGX-221 produces lipid in the thrombocyte suppresses.After putting on the cone-plate device, draw the thrombocyte sample thereupon and analyze single platelet count with Sysmex KN-21N hematology analyzer.

Fig. 3 B represents explanation in various TGX-221 concentration, with respect to the sample of be untreated (contrast), and the proportional increase of single platelet count.

By with HPLC initially-separate lipid and with clear and definite PtdIns (4,5) P ₂And PtdIns (3,4) P ₂Standard substance is identified the lipid peak value, is determined at lipid products PtdIns (4, the 5) P in the complete thrombocyte ₂And PtdIns (3,4) P ₂Lipid is integrated and is standardized into the TL that applies, and as being untreated the fraction representation of (contrast) sample.

Proof suppresses PI 3-kinases by TGX-221 and eliminates the reaction of thrombocyte to quickening to shear

The bar graph that Fig. 4 A Display Group is analyzed illustrate with 0.5 μ M TGX-221 processing the adhere influence of hematoblastic ratio of vWf, represents to be exposed to 1 second interval to be up to 10,000.s ^-1Shearing rate gradient (↑ Δ γ) after lasting, of short duration or fluctuation status (top described).

Fig. 4 B shows representative signal thrombocyte record, illustrates at the lip-deep GPIb/V/IX dependency of vWf Δ [Ca ²⁺] _cAs mentioned above, constant γ; Thrombocyte is exposed to 1800s ^-1Shearing rate, ↑ Δ γ (10,000.s ^-2); Thrombocyte is exposed to shear and quickens, and ↑ Δ γ (10,000.s ^-2)+TGX-221 (0.5 μ M); General ↑ Δ γ puts on 10 minutes thrombocyte of 0.5 μ m TXG-221 pre-treatment.

Fig. 4 C shows facs analysis, illustrates that the physiology agonist stimulates the back integrin alpha _IIbβ ₃Activation levels (by PAC-1 in conjunction with determining) and P-select proteic surface expression: contrast, thrombocyte by resuspending in Tyrodes damping fluid+1mM CaCl ₂/ MgCl ₂In (no agonist handle); DMSO, agonist stimulate before with thrombocyte 0.25%DMSO (carrier) pre-treatment; TGX-221,0.5mM TGX-221 pre-treatment 10 minutes are used thrombocyte in the agonist stimulation before.Agonist: zymoplasm, lU/ml; ADP, 12.5mM; U46619,1 μ M; Soluble collagen 10 μ g/ml.

Anti-thrombosis activity in the body of TGX-221

Fig. 5 A demonstration, in the Folts model, during per 30 minutes, (a before the administration;-30-0 minute) and administration after (b-d; Difference 1-30,31-60 ﹠amp; 61-90 minute), in the rat (A) and rabbit (B) of Sodital anesthesia, carrier (propylene glycol) or TGX-221 lower the influence of the average quantity of (CFR) to the round-robin cerebral artery blood flow.After administration and administration 90 minutes, to CFR monitoring 30 minutes.N: size of animal.Limit of error is ± 1SEM.

Fig. 5 B demonstration, in electrolytic model, in the rat of Sodital anesthesia, (4 minutes (time-4-0 minute) zero blood flows of 7mA electric current behind electrolytic lesion; Decontroling the artery anchor clamps at 0 minute) carrier (propylene glycol) or TGX-221 be to the influence of carotid artery flow (ml/ minute every 100g body weight).Treat in intravenously bolus injection mode in the 9 minutes time (promptly apply electric current 5 minutes before).Limit of error is the average SEM from replication ANOVA.Fig. 5 B inserts the figure explanation, and in the rat of Sodital anesthesia, treatment is to the influence of carotid artery flow volume (stimulate back 60 minutes during in blood flow area under curve) behind the electrolytic lesion.Limit of error is ± 1SEM.

Fig. 5 C shows, in the rat of the halothane anesthesia for the treatment of with TGX-221, TGX-221+ heparin, acetylsalicylic acid, clopidogrel, clopidogrel+heparin, clopidogrel+heparin+TGX-221, acetylsalicylic acid+heparin and acetylsalicylic acid+heparin+TGX-221 respectively, the comparison in rat tail bleeding time.

Fig. 6 A and 6B show the effect of the TGX-286 of various concentration to the ROS reaction.

Fig. 7 shows the effect that the TGX-286 of various concentration discharges elastoser.

Detailed description of preferred embodiment

Two main thrombocyte adhesiveness acceptor GPIb/V/IX (" GPIb ") and integrin alphas _IIbβ ₃(high-shear and shearing are quickened fast) has unique mechanicalness sensory function with respect to platelet activation under the fluidics state of disturbance.The present inventor finds, the signal transmission by two acceptors is subjected to the adjusting that shearing rate is quickened (↑ Δ γ) fast, comprises the platelet activation by PI 3-kinases dependent signals transmission course.

Therefore, the present inventor has illustrated the decisive signal transfer mechanisms under high-shear state, thereby confirms that PI 3-kinase beta is as the factor that causes platelet activation under the pathology blood flow state.Blocking the existing Antiplatelet therapy method of specific thrombocyte adhesiveness acceptor can not be at pathology and stop blooding normally and discern between the platelet activation.Therefore, this discovery of present inventor, it is the platelet activation that the being selected from property inhibition of PI 3-kinase beta can avoid platelet activation that the pathology increase by shearing rate causes and don't influence to be caused by the physiology agonist, for the antithrombotic treatment method provides a kind of method of new uniqueness, comprise the new compound that is used for this therapy.

Illustrated as Figure 1A, shear environment is regulated the adhesion to vWf of hematoblastic activation and thrombocyte.Compare with the thrombocyte that is exposed to the steady state shearing, be exposed to shearing rate acceleration (↑ Δ γ) and cause more platelet activation to contact with stable adhesion.The more careful inspection of platelet activation being carried out by monitoring cytoplasmic calcium flow, announcement ↑ Δ γ is to GPIb and integrin alpha _IIbβ ₃The transmission of calcium signal has special and praiseworthy influence.Under the situation of integrating plain calcium signal transmission, ↑ Δ γ causes the specific quick startup calcium signal transmission that can continue to be securely bonded to vWf that keeps in thrombocyte.This by shearing the integrin alpha of regulating _IIbβ ₃The generation of calcium signal and platelet agonist (ADP and TXA ₂) irrelevant, but depend on PI 3-kinases (Figure 1B and 1C) fully.↑ Δ γ causes new GPIb calcium signal, and it is before people such as Nesbit, 2002, and J.Biol.Chen., the GPIb calcium transient state of being distinguished among the 277:2965. completely different (Fig. 1 D).Three defined features of ↑ Δ γ GPIb calcium signal comprise (1), and it relies on (Fig. 1 E) for the strictness of the accelerated speed of γ, the frequency of the increase of (2) calcium response (Fig. 1 D) and (3) it to the susceptibility (1D) of PI 3-kinase inhibitor.

Because GPIb and integrin alpha _IIbβ ₃PI 3-kinases is depended in the transmission of calcium signal, so PI 3-kinases causes GPIb and integrin alpha by shearing rate (↑ Δ γ) triggering _IIbβ ₃The elimination of calcium signal.

The uncorrelated PI 3-of structure kinase inhibitor for example LY294002 or wortmannin (seeing below) stops the preliminary research of the ability of shearing the platelet aggregation that causes, for the platelet activation that shearing causes, the imagination of the kinase whose important mechanicalness sensory signal transfer function of PI 3-is proposed.Because these compounds do not show in various PI 3-kinases isotypes, so, do not know that still which special PI 3-kinases isotype or isotype relate to the platelet activation relevant with shearing.

Selectivity of the present invention suppresses the illustrative compound exhibits of PI 3 kinase betas in following:

The derivative that the 2-morpholino of formula (I) replaces is defined in down:

Wherein

X is C or N, and Y is N or O;

R ₃Be one or more H, F, Cl, Br, I, CN, CO ₂H, CO ₂R, NO ₂, CF ₃, replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted aryl, OCH ₃, OCH ₂F, OCHF ₂, OCF ₃, OR, OSO ₂-aryl, replacement or unsubstituted amine, NHCOR, NHSO ₂R, CONHR or SO ₂NHR.

Except formula (I) compound as PI 3-kinase beta activity inhibitor, formula (III) compound that new selectivity suppresses PI 3-kinase beta is defined as follows:

The preferred compound that is used for the inventive method comprises the Pyridopyrimidine derivatives that the 2-morpholino of formula (I) replaces, wherein

R is H, C ₁-C ₆Branched-chain or straight-chain alkyl or aryl;

X is C or N, and Y is N or O.

The example of some concrete formula (I) inhibitor comprises:

(±)-9-[1-(3,5-difluorophenyl amino) ethyl]-7-methyl-2-morpholine-4-yl pyridines [1,2-a] pyrimidin-4-one (TGX-239) also;

(±)-7-methyl-2-morpholine-4-base-9-[1-(2-pyridinylamino) ethyl] pyrido [1,2-a] pyrimidin-4-one (TGX-295).

In the context of this description, term " alkyl " refers to the radical of saturated aliphatic alkyl of straight or branched.Preferably, alkyl has 1-6 carbon atom, for example methyl, ethyl, propyl group, sec.-propyl, butyl, sec-butyl, the tertiary butyl, amyl group, isopentyl, hexyl etc.Alkyl is optional to be selected from following group and to replace by one or more: F, Cl, Br or I; CN; CO ₂R ₃NO ₂CF ₃Replace or unsubstituted C ₁-C ₆Alkyl; Replace or unsubstituted C ₃-C ₆Cycloalkyl; Replace or unsubstituted aryl; OCF ₃, OR ₃, replacement or unsubstituted amine; NHCOR ₃NHSO ₂R ₃CONHR ₃Or SO ₂NHR ₃, R wherein ₃Be H, replacement or do not replace C ₁-C ₆Alkyl, replacement or unsubstituted aryl.

Term " cycloalkyl " refers to non-heterocycle (for example carbocyclic ring) or heterocycle.In this respect, non-heterocyclic example is to replace or unsubstituted cyclopropane, tetramethylene, pentamethylene, hexanaphthene, cyclohexanedione, cyclopentanedione, quinone etc.Suitable heterocycloalkyl comprises and replacing or unsubstituted tetramethyleneimine, piperidines, piperazine, 2-piperidone, azepine hexamethylene-2-ketone and morpholine group.Cycloalkyl is chosen wantonly on one or more positions and replaced by following groups: halogen is F, Cl, Br or I for example; CN; CO ₂R ₃NO ₂CF ₃, replacement or unsubstituted C ₁-C ₆Alkyl; Replace or unsubstituted C ₃-C ₆Cycloalkyl; Replace or unsubstituted aryl; OCF ₃, OR ₃, replacement or unsubstituted amine; NHCOR ₃NHSO ₂R ₃CONHR ₃Or SO ₂NHR ₃, R wherein ₃Be H, replacement or unsubstituted C ₁-C ₆Alkyl, replacement or unsubstituted aryl.

Term " aryl " refers to aromatics or aromatic heterocycle.The example of aryl is a tetramethyleneimine, thiophene, the pyrroles, pyrazoles, imidazoles, 1,2,3-triazoles, 1,2,4-triazole oxazole isoxazole, thiazole, isothiazole, furans, 1,2, the 3-oxadiazole, 1,2, the 4-oxadiazole, 1,2, the 5-oxadiazole, 1,3, the 4-oxadiazole, 1,2,3,4-dislikes triazole, 1,2,3,5-dislikes triazole, 1,2, the 3-thiadiazoles, 1,2, the 4-thiadiazoles, 1,2, the 5-thiadiazoles, 1,3, the 4-thiadiazoles, 1,2,3, the 4-thiatriazole, 1,2,3, the 5-thiatriazole, tetrazolium, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, indenes, naphthalene, indoles, isoindole, indolizine, cumarone, thionaphthene, indazole, benzoglyoxaline, benzothiazole, purine, quinolizine, quinoline, isoquinoline 99.9, cinnolines, 2, the 3-naphthyridine, quinazoline, quinoxaline, naphthyridine, pteridine, fluorenes, carboline, acridine, azophenlyene, and anthracene.Aromatic yl group is chosen wantonly on one or more positions and replaced by following groups: halogen is F, Cl, Br or I for example; CN; CO ₂R ₃NO ₂CF ₃Replace or unsubstituted C ₁-C ₆Alkyl; Replace or unsubstituted C ₃-C ₆Cycloalkyl; Replace or unsubstituted aryl; OCF ₃, OR ₃, replacement or unsubstituted amine; NHCOR ₃NHSO ₂R ₃CONHR ₃Or SO ₂NHR ₃, R wherein ₃Be H, replacement or do not replace C ₁-C ₆Alkyl, replacement or unsubstituted aryl.

Term " selectivity PI 3-kinase beta inhibitor " used in this refers to a kind of compound, its to the inhibition effect of PI 3-kinase beta at least than other isotype 〉=10 of PI 3-kinases family times, preferred 〉=20 times, more preferably 〉=30 times.With traditionally be commonly referred to PI 3-kinase inhibitor for example the compound of LY294002 or wortmannin compare, " selectivity PI 3-kinase beta inhibitor " compound is considered to PI 3-kinase beta is had more selectivity.The compound that selectively suppresses performance of PI 3-kinase beta or active any kind can both be used as the selectivity PI 3-kinase beta inhibitor of the inventive method.

Have been found that the compound that pyridine of the present invention replaces can suppress lipid signal transmission enzyme PI 3-kinases, it regulates the thrombocyte adhesiveness process under the high-shear blood flow state, and thereby show anti-thrombosis activity and below believe other pharmacological characteristics of description.PI 3-kinases produces 3-phosphorylation PI second information, comprises phosphatidylinositols-3-phosphoric acid (PI (3) P), phosphatidylinositols-3,4-bisphosphate (PI (3,4) P ₂), phosphatidylinositols-3,4,5-triphosphoric acid (PI (3,4,5) P ₃).These second information are considered to regulate the cell phenomenon of different range, comprise that glucose is carried, apoptosis stops, cell is grown and the cytoskeleton reorganization.

About the influence of PI 3-kinase beta inhibitor under being correlated with flow state in physiopathology to thrombocyte adhesiveness, the report of not delivering.However, have been found that PI 3-kinases, particularly under the physiology flow state, playing a crucial role aspect the adjusting thrombocyte adhesiveness.Therefore, treat with the thrombocyte of The compounds of this invention and suppress PI 3-kinases, (PI (3) P), (PI (3,4) P ₂) and (PI (3,4,5) P ₃) the formation of phosphorylation lipid, thrombocyte adhesiveness produces significant effect to aspect the vWf model under the flow state reducing.The reduction of this thrombocyte adhesiveness and the expansion of unusual thrombocyte and thrombosis interrelate.Because with shear relevant thrombocyte adhesiveness and activation is significant in artery thrombosis, be an important target so PI 3-kinases is got involved for the treatment of cardiovascular disorder.

These PI 3-kinase inhibitor also have potential treatment application value in other various diseases.For example, at vessel branch is in the vascular smooth muscle cell, Thyberg, 1998, EuropeanJournal of Cell Biology 76 (1): 33-42 and in lung (tracheal smooth muscle cell), people such as Krymskaya, 1999, American Journal of Physiology 277:65-78, PI 3-kinases plays an important role in the hyperplasia that promotes unstriated muscle.The hyper-proliferative of vascular smooth muscle cell plays an important role in the formation of atherosclerotic plaque with in the aggressive blood vessel is disposed the development of back neovascularity intimal hyperplasia.People such as Scwartz, 1984, Progress inCardiovascular Disease 26:355-372; People such as Colwes, 1978, LaboratoryInvestigations 39:141-150.And in asthma and chronic tracheitis, the hyperplasia of tracheal smooth muscle cell causes the development of COPD.Therefore PI 3-kinase inhibitor can be used to pre-preventing restenosis of blood vessel, atherosclerosis and COPD.

PI 3-kinases also plays an important role in regulate tumor cell with in these cell experience apoptosis growths.People such as Sellers, 1999, The Journal of Clinical Investigation104:1655-1661.In addition, PI 3-kinases lipid products PI (3,4, the 5) P that causes by lipid Phosphoric acid esterase PTEN ₃And PI (3,4) P ₂Adjusting out of control, in many human malignant lesions' development, play an important role.People such as Leevers, 1999, Current Opinion in Cell Biology11:219-225.Therefore, PI 3-kinase inhibitor can be used to people's tumor treatment.

PI 3-kinases is at leukocyte function (Fuller etc., 1999, The Journal ofImmunology 162 (11): 6337-6340; Eder etc., 1998, The Journal ofBiological Chemistry 273 (43): 28025-31) and lymphocyte function (Vicente-Manzanares etc., 1999, The Journal of Immunology 163 (7): also play an important role 4001-4012).For example, the white corpuscle that is adhered to the endothelium of inflammation relates to by the signals transmission relevant with PI 3-kinases and activates endogenous white corpuscle integrin.In addition, oxidative burst (Nishioka etc. in the neutrophil, 1998, FEBS Letters 441 (1): 63-66) organize (Kirsch etc. again with cytoskeleton, 1999, Proceedings National Academy ofSciences 96 (11): 6211-6216) as if relate to the kinase whose signal transmission of PI 3-.Therefore, PI 3-kinase inhibitor can be used for reducing the activation of white corpuscle adhesion and inflammation part, therefore can be used for treating acute and/or chronic inflammatory disease.PI 3-kinases also plays an important role at lymphopoiesis with in activating.Fruman etc., 1999, Science 283 (5400): 393-397.The vital role of known lymphocyte in autoimmune disease, PI 3-kinase inhibitor can be used for treating this class disease.

Then the result is compared by for example suppressing active concentration, can be determined relative efficiency as the compound of the inhibitor of enzymic activity according to each compound of predetermined level determinations.Usually, preferred mensuration is to suppress 50% active concentration, i.e. 50% inhibition concentration or " IC in biochemical analysis ₅₀".IC ₅₀Can measure with technology known in the art.

According to the present invention, confirmed that PI 3-kinase beta in the thrombocyte has the crucial platelet activation with shearing causes and forms relevant mechanicalness sensory function with occluding thrombus.PI 3-kinase beta inhibitor TGX-221 is by GPIb and integrin alpha _IIbβ ₃To the analysis of mechanicalness conduction, prove for by two acceptors ↑ calcium current that Δ γ causes goes out, PI 3-kinase beta is absolute prerequisite (Fig. 4 A and Fig. 4 B).Effusive effect is to shear optionally to TGX-221 to calcium, because it does not suppress the GPIb calcium signal transmission (Fig. 4 B) that γ relies on.Dihydro, the hematoblastic priming reaction that other is caused by zymoplasm, collagen and ADP, for example integrin alpha _IIbβ ₃Activation (PAC-1 combination) and α granule secretion (P-selects protein expression) are not subjected to TGX-221 to influence (Fig. 4 C).

Two the main thrombocyte adhesiveness acceptor GPIb of signal transmission control and the integrin alpha of PI 3-kinase beta _IIbβ ₃Dirty, under the rheology disturbed conditions, promote the calcium current of cytosol to go out and platelet activation.Because the mechanicalness sensory function of these acceptors, the inhibition of PI 3-kinase beta can be eliminated occluding thrombus formation and not hinder the desired orthoplastocyte functional response of hemostasis.

For the antithrombotic of studying this compound forms potential, use two distinct thrombotic models; Folts mouse of revising and rabbit model (people such as Folts, 1991, Circulation, IV-3-IV-14) and mouse electrolytic lesion carotid artery model (people such as Bush, 1991, Faseb J., 4:3087).With 2mg/kg invention compound TGX-221 test injection animal for example, (Fig. 5 A and Fig. 5 B) stops the formation of occluding thrombus fully and keeps intestines carotid artery flow volume (Fig. 5 B inserts figure) during back 60 minutes in damage simultaneously in two models.And in the carotid artery of the damage of Folts and electrolysis research, TGX-221 not have to influence (not having display data) to baseline arteriotony, the rhythm of the heart or blood flow.Meaning importantly, as to by tail or ear bleeding time or when the Combined Preparation by for example hemorrhage analysis of carrying out of prolongation that causes of Vitrum AB, acetylsalicylic acid or clopidogrel of other anti-coagulant, TGX-221 does not have disadvantageous effect to the normal haemostasis in these animals.

Explained the crucial mechanicalness sensory function that the platelet activation that causes with shearing and occluding thrombus form the PI 3-kinase beta in the relevant thrombocyte in the present invention of this general introduction.Under the situation of the functional response of not interfering the orthoplastocyte relevant with hemostasis, PI 3-kinase beta inhibitor TGX-221 eliminates the proof that occluding thrombus forms, and the lipid kinase inhibitors of explaining this novelty is as being used for a kind of important new medicament that antithrombotic forms therapy.

Advantageously, in the inventive method of prevention or treatment disease, can be with the significant quantity of a kind of compound form administration with dosage.In preferred embodiments, dosage is preferably with preparation, intramuscular injectable formulations, syrup, suppository, aerosol, sublingual formulation, percutaneous preparation or vaginal suppository in tablet (for example be used for oral, hypogloeeis and orally administering and make tablet), capsule (capsule that for example contains powder, liquid or sustained release formulation), iv formulation, the nose.Preferably, dosage contains the 5-50mg compound of having an appointment, and more preferably contains the 25-300mg compound of having an appointment.

Another aspect of the present invention relates to and contains the The compounds of this invention that pyridine replaces and the pharmaceutical composition of one or more pharmaceutically acceptable carrier and/or solvent.Hereinafter, term " activeconstituents " can be the The compounds of this invention that any pyridine replaces, perhaps their pharmacologically acceptable salt, solvate or functional derivatives.

The administration of this pharmaceutical composition is preferably carried out with any method easily.The administration of dosage can every day, week, the moon or in other suitable timed interval, carries out via oral, intravenously for example, intraperitoneal, intramuscular, subcutaneous, intracutaneous or suppository or by implantation approach (for example with slow release molecule).If active compound is with the tablet form administration, this tablet contains tackiness agent for example tragakanta, W-Gum or gelatin; Disintegrating agent, for example alginic acid; With slipping agent Magnesium Stearate for example.

The pharmaceutical composition of suitable injection comprises aseptic aqueous solution or dispersion liquid and is used for aseptic injectable solution or the sterilized powder of the interim preparation of dispersion liquid, perhaps is suitable for the form of topical application with emulsion or other.Carrier can be solvent or dispersion medium, and it contains for example water, ethanol, polyvalent alcohol (for example glycerine, propylene glycol and liquid macrogol etc.), their suitable mixture and rapeseed oil.For example, by using for example Yelkin TTS of dressing, under the situation of dispersion liquid, pass through to keep desired particle size, and, can keep suitable flowability by using tensio-active agent.Use various antibiotic and anti-mycotic agents for example parabens, chlorobutanol, phenol, Sorbic Acid, Thiomersalate etc., can avoid the pollution that causes by microorganism.Preferably can comprise isotonic agent, for example sugar or sodium-chlor.Postpone sorbent material for example monostearate aluminium and gelatin, the absorption that can prolong injectable composition by in composition, using.

By active compound is mixed the preparation aseptic injectable solution with the amount that requires in suitable volume with above-named various other compositions.Usually, be mixed in the sterile carrier and one or more above-mentioned compositions that contains basic dispersion medium the preparation dispersion liquid by active compound with various sterilizations.Prepare aseptic injectable solution under the situation of sterilized powder, preferred manufacturing procedure is, the active compound powder that vacuum-drying and freeze-drying are produced adds the desired any ancillary component that obtains from previous sterile filtration solution.

Tablet, lozenge, pill, capsule etc. also can contain wedding agent for example resin, gum arabic, W-Gum or gel; Vehicle is for example W-Gum, potato starch, alginic acid etc. of Lin Suanergai, disintegrating agent for example; Slipping agent is Magnesium Stearate for example; With sweeting agent for example sucrose, lactose or asccharin; Perhaps for example spearmint oil, wintergreen oil or cherry flavor of seasonings.When dosage unit form was capsule, except the material of the above-mentioned type, it can contain liquid vehicle.Various other materials can be used as dressing to improve the physical appearance of dosage forms unit.For example can be with tablet, pill or capsule shellac, sugar or both dressings.Syrup or elixir also contain active compound, as the sucrose of sweeting agent, as the Tegosept M of sanitas and propylben, dyestuff and seasonings for example cherry or orange spices.Certainly, any material that is used to prepare any dosage unit form should be pharmacology purified and on usage quantity basic nontoxicity.In addition, active compound can be mixed in delayed release preparation and the formulation.

By with reference to the following embodiment that only states, the present invention is further described as illustrations.Among these embodiment anything should not be used as the restriction of all scopes of the present invention.

Synthetic embodiment

Embodiment 1. synthetic (±)-7-methyl-2-morpholine-4-base-9-(1-phenyl amino ethyl)-pyrido [1,2-a] pyrimidin-4-one (TGX-221:R ₁=CH ₃, R ₂=CH ₃, R ₃=H)

Compound 2: under ice-cold temperature to 2-amino-3-bromo-5-picoline (1) (45g, 0.45mol) add in the solution in methylene dichloride (500mL) the malonyl-dichloro (25mL, 0.25mol).In room temperature mixture was stirred 48 hours.By filtering the faint yellow solid of collecting precipitation, (3 * 100mL) washings, drying under vacuum obtains product 2 (52.5g) then with methylene dichloride.Filtrate decompression is concentrated.The gained resistates is suspended in water and stirred 1 hour.Solution is filtered, then filtrate is used solid NaNCO ₃Neutralization obtains unreacted 2-amino-3-bromo-5-picoline (6g).Need not to be further purified crude compound is used for next synthesis step.

¹H NMR(300MHz，DMSO-d ₆)δ8.72(s，1H)，8.28(s，1H)，5.50(s，1H)，2.33(s，3H).

Compound 3: under ice-cold temperature, to compound 2 (12.75g, 0.05mol) add in the suspension in methylene dichloride (300mL) triethylamine (14mL, 0.1mol), add then methylsulfonyl chloride (5.42mL, 0.07mol).At room temperature reaction mixture was stirred 0.5 hour then.Add morpholine (13mL, 0.15mol) after, reaction mixture was stirred 24 hours under reflux temperature.With the mixture concentrating under reduced pressure, use H then ₂O (300mL) dilution obtains light yellow precipitate.Solid filtering,, be accredited as ketone 3 (6.8g is by HPLC purity assay＞80%) at drying under reduced pressure.Need not to be further purified product 3 is used for next reactions steps.

¹H NMR(300MHz，CDCl ₃)δ8.69(s，1H)，7.84(s，1H)，5.58(s，1H)，3.80(m，4H)，3.70(m，4H)，2.32(s，3H).

Compound 4: in the solution of bromide 3 (35mol) in DMF (70mL), add N, N-diisopropylethylamine (18mL), butyl vinyl ether (13mL) and dichloride 1,1 '-two (diphenylphosphino) ferrocene palladium (II) (1.09g, 1.5mol).Stirred 16 hours at 120 ℃ of following suspension (becoming homogeneous phase after 20 minutes).With reaction mixture cooling and pour in the ice-cold solution of 1M HCl (200mL), stirred then 1 hour.With this solution of dichloromethane extraction, then organic layer is washed with water, at Na ₂SO ₄Last drying ( ^*Avoid using the NaCl solution washing, because solution can become emulsion).After concentrating in a vacuum, black residue with column chromatography purifying (silica gel, 3: 1 ethyl acetate/petroleum ether), is obtained faint yellow solid. ¹HNMR(300MHz，CDCl ₃)δ8.86(s，1H)，7.84(s，1H)，5.63(s，1H)，3.79(m，4H)，3.62(m，4H)，2.77(s，3H)，2.36(s，3H).

TGX 221: to ketone 4 (1mmol) in toluene (10mL) solution add aniline (3mmol) and refluxed 4 hours.Reaction mixture is cooled off gradually, under ice-cold temperature, add sodium borohydride (1mmol) then.At room temperature reaction mixture was further stirred 1 hour.(30mL) dilutes this solution with methylene dichloride, with organic layer water, salt water washing, then at Na ₂SO ₄Last dry.After the vacuum-drying, resistates with column chromatography (silica gel, 3: 1 ethyl acetate/petroleum ether) purifying, is obtained faint yellow solid (productive rate＞60%).

¹H NMR(300MHz，CDCl ₃)δ8.65(s，1H)，7.58(s，1H)，(7.11 br t，2H)，6.68(t，J＝7.5Hz，1H)，6.46(br t，2H)，5.66(s，1H)，5.12(m，1H)，4.24(br s，-NH，1H)，3.80(m，4H)，3.68(m，4H)，2.26(s，3H)，1.57(d，J＝6.7Hz，3H).

Embodiment 2.The chromone derivatives that the preparation pyridine replaces

According to following by Cushman and Nagarathnam, 1990, the improved general method of Tetrahedron Letters31:6497 prepares 8-(replacement)-2-(4-pyridyl)-4H-1-chromene-4-ketone.Briefly, various precursor 2-hydroxy acetophenones (1) are handled with iso methyl nicotinate and derivative,, made the product (3) that pyridine replaces then by the effect of cyclization processed.Then by various linked reactions at R ₁Last importing specified substituent.

Substituting group on methyl phenyl ketone (R) can include but not limited to bromine, hydroxyl, kharophen, methoxymethyl, methyl, ethyl, methoxyl group, trifluoro-methanesulfonyl oxy and ethanoyl substituting group.Substituting group on iso-nicotinate (R ') includes but not limited to chlorine, methyl and amino substituting group.The reagent that is used for condensation reaction comprises that use is at solvent for example tetrahydrofuran (THF) or N, two in the dinethylformamide (trimethyl silyl) Lithamide, sodium hydride, 1,8-diazabicylo [2.2.2] undecane, sodium butylate or sodium methylate.Can use for example 1 among the sulfuric acid in the ethanol, the hydrochloric acid in the methyl alcohol, the DMF of reagent mixture, the trifluoromethanesulfanhydride anhydride in 8-diazabicylo [2.2.2] undecane, the methylene dichloride carries out the cyclization dehydration.

The further reaction of product (3) can be included in the catalytic crosslinking reaction of palladium on the R, and wherein R is that R is the product of aryl, arylamino, alkylamino or ethanoyl to obtain wherein for halogenide or trifluoro-methanesulfonyl oxy.At R is under the situation of ethanoyl functional group, and further reaction can comprise that R is the product of hydroxyethyl or amino-ethyl to obtain wherein for reduction or reduction amination.At R is under the situation of methoxyl group alkyl or hydroxyalkyl functional group, and further reaction can obtain wherein that R is the product of bromine alkyl.At R is under the situation of bromine alkyl, and further reaction can obtain wherein that R is the product of arylamino alkyl or aryloxy alkyl.At R is under the situation of hydroxyl, and further reaction can obtain the product of R=aryloxy wherein or alkoxyl group.Under R was amino situation, further reaction can obtain wherein that R is the product of arylamino or alkylamino.

The following examples further are used for illustrating the present invention.

The preparation of intermediate

Embodiment 2a: 6-methyl-8-ethanoyl-2-(4-pyridyl)-4H-1-chromene-4-ketone

3 '-ethanoyl-2 '-hydroxyl-5 '-methyl acetophenone

(15g, 0.1mol) mixture in methylene dichloride (100ml) is handled with triethylamine (13.9ml), dimethyl aminopyridine (1.22g) and diacetyl oxide (9.5ml), at room temperature stirs then and spends the night with 2-hydroxy-5-methyl benzoylformaldoxime.Then mixture is poured in the water (300ml) also with methylene dichloride (3 * 60ml) extractions.With the saturated NaHCO of mixture that merges ₃Solution washing, dry (Na ₂SO ₄) and remove and desolvate, obtain colorless oil (19.5g).

Under 0 ℃, this product is dissolved in the methylene dichloride (200ml), and handles, at room temperature stirred then 5 days with aluminum chloride (19.5g).Solution is handled with ice (50g) and 2N hydrochloric acid (50ml), at room temperature stirred then 1 hour.Dichloromethane layer is separated, and (2 * 60ml) extract with methylene dichloride with water layer.With the extract that merges with saturated NaCl solution washing, drying (Na ₂SO ₄), and solvent removed, obtain crude product.Product with column chromatography purifying (0-25% ethyl acetate mixture in gasoline), is obtained Huang/green solid (11.4g).

8-ethanoyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone

At-78 ℃ under nitrogen atmosphere; to 3 '-ethanoyl-2 '-hydroxyl-5 '-methyl acetophenone (3.0g; 15.6mmol) add two (dimetylsilyl) Lithamide (solution of 1.0M in THF in the solution in anhydrous THF (100ml); 50ml; 50mmol), under 0 ℃, mixture was stirred 1 hour then.Mixture is cooled to-78 ℃, and the adding iso methyl nicotinate (2.14ml, 15.6mmol).Reaction mixture is heated to room temperature also to be continued to stir to spend the night.Mixture is poured into 1N hydrochloric acid soln (200ml), under vacuum, remove THF.Mixture is filtered then with the neutralization of 1N aqueous sodium hydroxide solution.With filter cake dried overnight under high vacuum.

Use the acetate (40ml) and the vitriol oil (2ml) to handle successively gained solid (3.0g), then 80 ℃ of heating 3 hours.Under cooling,, neutralize with the 1N aqueous sodium hydroxide solution with mixture water (100ml) dilution.With sedimentation and filtration, wash with water and drying under high vacuum.Crude product by the column chromatography purifying, is carried out gradient elution with the mixture of 0-20% methyl alcohol in ethyl acetate, obtain the brown solid.

ES-MS：280.36(M+H)

Also made with similar methods:

By 2, the 3-resacetophenone makes 8-hydroxyl-2-(4-pyridyl)-4H-1-chromene-4-ketone; ES-MS:240.2 (M+H);

Make 8-bromo-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone by 3-bromo-5-methyl-2-hydroxy acetophenone; ES-MS:316.2,318.2 (M+H);

By 2, the 4-resacetophenone makes 7-hydroxyl-2-(4-pyridyl)-4H-1-chromene-4-ketone; ES-MS:240.15 (M+H);

Make 8-acetylaminohydroxyphenylarsonic acid 2-(4-pyridyl)-4H-1-chromene-4-ketone by 3-acetylaminohydroxyphenylarsonic acid 2-hydroxy acetophenone; ES-MS:281.2 (M+H);

Make 8-amino-2-(4-pyridyl)-4H-1-chromene-4-ketone by 3-acetylaminohydroxyphenylarsonic acid 2-hydroxy acetophenone; ES-MS:239.2 (M+H);

8-ethanoyl-6-methyl-2-(2-chloro-6-methyl-4-pyridyl)-4H-1-chromene-4-ketone ES-MS:328.12,330.12 (M+H);

8-ethanoyl-6-methyl-2-(2-amino-4-pyridyl)-4H-1-chromene-4-ketone ES-MS:295.5 (M+H);

6-methyl-8-ethanoyl-2-(3-pyridyl)-4H-1-chromene-4-ketone ES-MS:280.3 (M+H); With

6-methoxyl group-8-methoxymethyl-2-(3-pyridyl)-4H-1-chromene-4-ketone ES-MS:316.2 (M+H).

Embodiment 2b: the Heck coupling of bromo-chromone

8-ethanoyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone (other method)

6-methyl-8-(ethanoyl)-2-(4-pyridyl)-4H-1-chromene-4-ketone

With 8-bromo-2-(4-pyridyl)-6-methyl-4H-1-chromene-4-ketone (0.12g, 0.36mmol), n-butyl vinyl ether (0.047ml, 0.36mmol), triethylamine (0.050ml, 0.36mmol) the mixture N in DMF (5ml) ₂PdCl is used in cleaning then ₂(dppf) (27mg 0.036mmol) handles.With mixture 90 ℃ of heated overnight.Mixture is cooled to room temperature, handles, stir then and spend the night with 1N hydrochloric acid (30ml).With this mixture water (50ml) dilution and freeze-drying.Resistates via C8-HPLC post wash-out, and is separated product, obtained white solid (2.5mg).This product and top described product are just the same.

Embodiment 2c: the reduction of ethanoyl chromone: 8-(1-hydroxyethyl)-2-(4-pyridyl)-6-methyl-4H-1-chromene-4-ketone

(4.6g, 16.5mmol) mixture in methyl alcohol (100ml) spends the night with sodium borohydride (1.22g, 33mmol) processing and reflux with 8-ethanoyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone.Under cooling, add entry (2ml), under the vacuum that solution concentration is extremely approaching dry then.Add entry (100ml), the throw out that forms is filtered, obtain pale solid (4.0g).

Embodiment 2d: the bromination of alcohol: 8-(1-bromotrifluoromethane)-2-(4-pyridyl)-6-methyl-4H-1-chromene-4-ketone

8-(1-hydroxyethyl)-2-(4-the pyridyl)-6-methyl-4H-1-chromene-mixture of 4-ketone (4.0g) in Glacial acetic acid (45ml) handled with 48% hydrobromic acid aqueous solution (34ml) and 80 ℃ of heated overnight, under cooling, mixture is poured in the icy water, with the neutralization of 50% sodium hydroxide, with methylene dichloride (3 * 40ml) extractions.Dry also the removing of the extract that merges desolvated.Resistates via silica gel chromatography, with the mixture wash-out of 0-10% methyl alcohol in ethyl acetate, is obtained pale solid (1.1g).ESI-MS:344.1, (as selection, this product can be by using PBr for 346.1 (M+H) ₃Mixture process alcohol in methylene dichloride obtains).

Embodiment 2e: the methylsulfonylization of alcohol: 8-(1-mesyloxy ethyl)-2-(4-pyridyl)-6-methyl-4H-1-chromene-4-ketone

Then use methylsulfonyl chloride (0.67ml) to handle with triethylamine (1.3ml) earlier in the mixture of 8-(1-hydroxyethyl)-2-(4-pyridyl)-6-methyl-4H-1-chromene-4-ketone (2.4g) in methylene dichloride (100ml) in ice bath, then mixture was stirred 30 minutes down at 0 ℃.(2 * 30ml) wash, dry (Na with 1N hydrochloric acid with solution then ₂SO ₄), remove and desolvate, obtain the oily brown solid, need not it is further purified.

Embodiment 2f:6-methyl-8-brooethyl-2-(4-pyridyl)-4H-chromene-1-ketone: 6-methyl-8-brooethyl-2-(4-pyridyl)-4H-chromene-1-ketone

2 '-hydroxyl-5 '-methyl-3 '-the methoxyl methyl methyl phenyl ketone

(1.0g 6.7mmol) handles with paraformaldehyde (0.18g) and concentrated hydrochloric acid (5ml), then 60 ℃ of heated overnight with 2 '-hydroxyl-5 '-methyl acetophenone.Under cooling, (3 * 30ml) extraction mixtures are with the extract drying (Na that merges with toluene ₂SO ₄) and remove and desolvate, obtain yellow oil.Oily matter handled with methyl alcohol (30ml) and reflux 1 hour.Under cooling, solution evaporation near dry, with resistates chromatogram purification on silicagel column, is used the mixture wash-out of 0-10% ethyl acetate in sherwood oil.The purified product that obtains is a white powder.

8-brooethyl-6-methyl-2-(4-pyridyl)-4H-chromene-1-ketone

(0.76g, (solution of 1.0M in THF, 11.8ml 11.8mmol), stir mixture 1 hour down at 0 ℃ then 3.9mmol) to add two (trimethyl silyl) Lithamide in the solution in THF (30ml) to methyl phenyl ketone under-78 ℃.Mixture is cooled to-78 ℃, add then iso methyl nicotinate (0.53ml, 3.9mmol).Reaction mixture is heated to room temperature also to be continued to stir to spend the night.Mixture is injected 1N hydrochloric acid soln (200ml), remove THF in a vacuum.Mixture with the neutralization of 1N aqueous sodium hydroxide solution, is filtered then.Filter cake dried overnight under vacuum.

With resistates earlier with acetate (6ml) then use Hydrogen bromide (48% solution in water, 6ml) processing, and with mixture 80 ℃ of following heated overnight.Under cooling, mixture is transferred to pH 5 with the 2N sodium hydroxide solution.The gained throw out is filtered, and dry under vacuum.Resistates via silica gel chromatography, is used the mixture wash-out of 0-5% methyl alcohol in ethyl acetate, product is separated obtained pale solid (310mg).LC-MS：332，334(M+H)

Embodiment 2g: 8-trifluoro-methanesulfonyl oxy-2-(4-pyridyl)--4H-1-chromene-4-ketone

In 8-hydroxyl-2-(4-the pyridyl)-4H-1-chromene-mixture of 4-ketone (10mg) in acetonitrile (2ml), add diisopropylethylamine (20uL), add N-phenyltriflimide (20mg) then, at room temperature mixture was stirred 2 hours then.Mixture is adsorbed on silica gel, and the usefulness ethyl acetate via the silicagel column wash-out, obtains being this title compound of brown solid (10mg) as eluent.ESI-MS：372.1(M+H)。

Embodiment 2h: the reduction amination of ketone: 8-1-(phenyl amino) ethyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone (TGX-286)

In 8-ethanoyl-6-methyl-2-(4-the pyridyl)-4H-1-chromene-suspension of 4-ketone (0.28g) in methyl alcohol (30ml), add Glacial acetic acid (2.5ml), aniline (2.5ml) and sodium cyanoborohydride (62mg), then with mixture 70 ℃ of heated overnight.Under cooling, mixture is adsorbed onto on the silica gel and uses the column chromatography purifying, carry out gradient elution with the mixture of 0-10% methyl alcohol in ethyl acetate, obtain brown solid (200mg).

1H NMR(CDCl ₃，300MHz)：δ1.65(d，3H，J＝1.2Hz)，2.39(s，3H)，5.186(m，1H)，6.51(d，2H，J＝7.8Hz)，6.69(t，1H，J＝7.5Hz)，6.94(s，1H)，7.11(t，J＝8.1Hz)，7.65(d，1H，J＝1.8Hz)，7.73(d，2H，J＝6Hz)，7.90(s，1H)，8.80(d，2H，J＝6Hz).

ES-MS：357.3(M+H)，264.3.

Also prepared with similar method:

8-1-(4-fluoro-2-aminomethyl phenyl amino) ethyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone (KN-303); ES-MS:389.3 (M+H)

8-1-(phenyl amino) ethyl-6-methyl-2-(3-pyridyl)-4H-1-chromene-4-ketone (KN-305); ES-MS 357.3 (M+H)

8-1-(6-picoline-2-base is amino) ethyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone (KN-310); ES-MS:372.3 (M+H)

8-1-(3-trifluoromethyl amino) ethyl-6-methyl-2-(4-pyridyl)-4H-1-chromene-4-ketone (KN-322); ES-MS:425.0 (M+H)

8-1-(phenyl amino) ethyl-6-methyl-2-(2-chloro-6-methyl-4-pyridyl)-4H-1-chromene-4-ketone (KN-340); ES-MS:405.44,407.42 (M+H)

Embodiment 2i: the chromone that the bromine alkyl replaces and the reaction of phenol or aniline: 6-methyl-8-phenyl amino methyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-312)

(48mg, 0.15mmol) (48uL 0.52mmol) handles the mixture in acetonitrile (5ml), then this mixture is heated 4 hours at 70 ℃ with aniline with 8-brooethyl-6-methyl-2-(4-pyridyl)-4H-chromene-1-ketone.Under cooling, this mixture is handled with solid carbonic acid potassium (60mg), this mixture is adsorbed onto on the silica gel (1.0g), remove then and desolvate.Resistates is put on the silicagel column, the product mixture wash-out of 0-5% methyl alcohol in ethyl acetate.Product is separated, obtained yellow powder (25mg).

1H NMR(CDCl ₃，300MHz)：δ2.41(s，3H)，4.19(s，1H)，4.71(s，2H)，6.68(d，2H，J＝8.4Hz)，6.77(t，1H，J＝7.2Hz)，6.9(s，1H)，7.21(t，2H，7.5Hz)，7.59(s，1H)，7.67(d，2H，4.8Hz)，7.91(s，1H)，8.74(s，2H).

LC-MS：343.08(M+H)，249.98

Also prepared with similar method:

6-methyl-8-phenoxymethyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-313) ES-MS 344.1

6-methyl-8-(2-pyridyl) amino methyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-315) ES-MS:344.1

6-methyl-8-1-(phenoxy group) ethyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-317) ES-MS 358.1

6-methyl-8-(2-carboxyl) phenyl amino methyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-323) ES-MS 387.04 (M+H)

6-methyl-8-(2-kharophen) phenyl amino methyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-326) ES-MS:413.9 (M+H)

6-methyl-8-[1-(2-carboxyl) phenyl amino] ethyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-334) ES-MS:401.4

Embodiment 2j: 8-(1-(2-aminophenyl amino) ethyl-2-(4-pyridyl)-4H-chromene-1-ketone

With the mixture of 8-(1-mesyloxy) ethyl-6-methyl-2-(4-pyridyl)-4H-chromene-1-ketone (2.4g) in acetonitrile (50ml) with the processing of salt of wormwood (2.4g) and list-Boc phenylenediamine (2.7g), then mixture 70 ℃ of heated overnight.Under cooling, mixture is handled and is adsorbed onto on the silica gel with methylene dichloride.Remove and desolvate, resistates is put on the silicagel column, then with the product mixture wash-out of 0-5% methyl alcohol in ethyl acetate.Product is separated, obtained yellow oil (0.9g).

ES-MS：472.3(M+H)

(mixture of 1-(Boc-2-aminophenyl amino) ethyl-2-(4-pyridyl)-4H-chromene-1-ketone (0.9g) in methylene dichloride (12ml) handled with trifluoroacetic acid (8ml), at room temperature stirs then 1 hour with 8-.Mixture is diluted with methylene dichloride (30ml), and first water (30ml) is used 1N concentrated hydrochloric acid (30ml) extraction then.The aqueous extract that merges is neutralized with sodium hydroxide, and (3 * 50ml) extract with methylene dichloride.Then with organic extract 0.3N aqueous hydrochloric acid (2 * 50ml) extractions again that merge.Extremely dry the aqueous extract freeze-drying that merges then, obtain sorrel solid (0.61g).

ES-MS：372.3

Prepared with similar method:

6-methyl-8-1-(2-trifluoromethyl-benzoglyoxaline-1-yl)-ethyl-2-(4-pyridyl)-4H-chromene-1-ketone (KN-328) ES-MS:450 (M+H)

Embodiment 2k:8-benzyloxy-2-(4-pyridyl)-4H-chromene-1-ketone (KN-335)

Mixture in acetonitrile is handled with bromotoluene with 8-hydroxyl-2-(4-pyridyl)-4H-chromene-1-ketone (52mg) and Anhydrous potassium carbonate (117mg), and reflux is spent the night.Under cooling, mixture is handled with methylene dichloride, be adsorbed onto on the silica gel, and put on the silicon chromatographic column.Product with the mixture wash-out of 0-4% methane in ethyl acetate, is separated desired product, obtained brown solid (17mg).

ES-MS：330.2(M+H)

Prepared with similar method:

7-benzyloxy-2-(4-pyridyl)-4H-chromene-1-ketone (KN-342) ES-MS:330.5 (M+H)

Embodiment 2l:8-benzylamino-2-(4-pyridyl)-4H-chromene-1-ketone (KN-336)

8-amino-2-(4-pyridyl)-4H-chromene-1-ketone (16mg), phenyl aldehyde (40 μ L) and acetate (the 20 μ L) mixture in methyl alcohol (5ml) is handled with sodium cyanoborohydride (5mg), and 70 ℃ of heated overnight.Under cooling solution absorbs to silica gel, via the silica gel chromatography purifying, with the mixture gradient elution of 0-5% methyl alcohol in ethyl acetate.Desired product is separated, obtained yellow solid.ES-MS：329.3(M+H)

Embodiment 2m:8-phenyl amino-2-(4-pyridyl)-4H-chromene-1-ketone (KN-341)

With 8-amino-2-(4-pyridyl)-4H-chromene-1-ketone (20mg), phenyl-boron dihydroxide (30mg, 0.25mmol) and triethylamine (70uL, 0.5mmol) (45mg 0.25mmol) handles, and at room temperature mixture is stirred and spend the night with venus crystals for mixture in methylene dichloride (5ml).Mixture is adsorbed onto on the silica gel, puts on the silicagel column also with the mixture wash-out of 0-5% methyl alcohol in ethyl acetate.Obtain desired compound, be yellow solid (4mg).

ES-MS：315.5(M+H)。

Prepared with similar methods:

8-(3-fluorophenyl amino)-2-(4-pyridyl)-4H-chromene-1-ketone (KN-351) ES-MS:333.3 (M+H)

Embodiment 2n:8-phenyl-6-methyl-2-(4-pyridyl)-4H-chromene-1-ketone (TGX-25S)

With 8-bromo-6-methyl-2-(4-pyridyl)-4H-chromene-1-ketone (0.1g, 0.32mmol), potassiumphosphate (0.2g, 0.95mmol), phenyl-boron dihydroxide (0.042g, 0.35mmol) and PdCl ₂(dppf) (7.8mg, 0.009mmol) mixture heating up of the nitrogen purging in dioxane (6ml) refluxes and spends the night.Filtering mixt under cooling, and filtrate is concentrated into drying.Via C8 HPLC column chromatography purifying, use the mixture of 0-60% acetonitrile in 0.1% dense TFA resistates as eluent.The fraction of purifying is merged, obtain yellow powder (53.4mg).

1H NMR(CDCl ₃，300MHz)：δ2.53(s，3H)，7.03(s，1H)，7.55(m，6H)，7.80(d，J＝5.7Hz，2H)，8.04(s，1H)，8.80(d，J＝5.7Hz，2H).ES-MS：314.3(M+H)

Embodiment 2o: synthetic 6-methyl-8-bromo-3-hydroxyl-2-(4-pyridyl)-4H-chromene-1-ketone

To 3 '-bromo-2 '-hydroxyl-5 '-methyl acetophenone (1.15g) and the mixture of Pyridine-4-Carboxaldehyde (0.54g) in ethanol (10ml) in Dropwise 5 0% sodium hydroxide solution, and at room temperature mixture was stirred 4 hours.Mixture is handled to pH 5.5 with ice-cold Glacial acetic acid, formed throw out, filter and obtain the phenyl styryl ketone intermediate, as yellow solid (0.85g).

Then using 30% (v/v) superoxol (189 μ L) to handle with 2N sodium hydroxide (2.1ml) earlier the solution of phenyl styryl ketone (0.132g) in methyl alcohol (2.3ml) under 0 ℃.Under 4 ℃, this mixture stirring is spent the night.With neutralize this mixture and form throw out of 2N sulfuric acid, its filtration is obtained the brown solid.ES-MS：332.0，334.0(M+H)

Embodiment 3. the Carbostyril derivative that the preparation pyridine replaces

Prepare the quinolone compounds that pyridine of the present invention replaces according to following general method:

Reagent: i. tosic acid, toluene, reflux; Ii.Ph ₂O, heating.

I. ester intermediate (compound 3) is synthetic: with β-oxo-4-pyridine ethyl propionate (compound 1,4.35mmol) [people such as Lesher, 1984, J.Heterocycl.Chem.21 (6): 1849], aniline (compound 2,3.35mmol) and the mixture of tosic acid (0.54mmol) in toluene (30mL) under reflux temperature, heated 18 hours azeotropic removal of water.With solvent removed under reduced pressure, obtain rough yellow oil, with after the purification by flash chromatography of petrol ether/ethyl acetate (1: 1) as eluent, obtain the ester intermediate (compound 3,70-80%).

Ii. quinolone (compound 4) is synthetic: ester intermediate (compound 3) (2.58mmol) was refluxed 20 minutes in diphenyl ether (3mL), be cooled to room temperature and obtain the paste solid with the sherwood oil processing then.Throw out is filtered, with petroleum ether for several times,, as eluent, obtain desired quinolone compounds 4 (60-70%) with ethyl acetate/methanol (9: 1) then via purification by flash chromatography.

8-phenoxy group-2-(4-pyridyl)-4 (1H)-quinolones (KN-319)

By in reflux in toluene with ester intermediate (compound 3, wherein R is OPh) cyclization in position, obtain quinolone (compound 4) (step I):

¹H NMR(400MHz，CDCl ₃)：δ6.64(d，J＝1.9Hz，1H)，7.06(dd，J＝8.0，1.1Hz，1H)，7.15(dd，J＝7.7，0.7Hz，2H)，7.20-7.31(m，2H)，7.44(td，J＝7.7，0.7Hz，2H)，7.57(dd，J＝5.0，1.4Hz，2H)，8.06(dd，J＝8.0，1.1Hz，1H)，8.80(d，J＝5.0Hz，2H)，8.88(br.s，NH)；MS-ES m/e 315(M+H).

8-bromo-2-(4-pyridyl)-4 (1H)-quinolones (KN-343)

Ester intermediate (compound 3, wherein R is Br):

¹H NMR(300MHz，CDCl ₃)δ1.33(t，J＝7.1Hz，3H)，4.25(q，J＝7.1Hz，2H)，5.17(s，1H)，6.31(dd，J＝7.7，1.6Hz，1H)，6.81(td，J＝7.7，1.6Hz，1H)，6.89(td，J＝7.7，1.6Hz，1H)，7.21(d，J＝5.2Hz，2H)，7.54(dd，J＝7.7，1.6Hz，1H)，8.56(d，J＝5.2Hz，2H)，10.16(br.s，NH)；MS-ES m/e 347(M+H).

Quinolone (compound 4):

¹H NMR(400MHz，(CD ₃) ₂SO)δ7.45(t，J＝7.9Hz，1H)，7.49(s，1H)，8.12(d，J＝5.2Hz，2H)，8.14(dd，J＝7.9，1.3Hz，1H)，8.19(dd，J＝7.9，1.3Hz，1H)，8.78(d，J＝5.2Hz，2H)，12.02(br.s，NH)；MS-ES m/e 301(M+H).

KN-343 is an intermediate in quinolone analogs that various other pyridines replace synthetic.

8-(4-fluoro-2-methylphenoxy)-2-(4-pyridyl)-4 (1H)-quinolones (KN-337)

Ester intermediate (compound 3, wherein R is a 4-fluoro-2-methylphenoxy):

¹H NMR(400MHz，CDCl ₃)：δ1.29(t，J＝7.1Hz，3H)，2.26(s，3H)，4.20(q，J＝7.1Hz，2H)，5.07(s，1H)，6.41(dd，J＝7.7，1.9Hz，1H)，6.56(dd，J＝7.7，1.9Hz，1H)，6.69(td，J＝7.7，1.9Hz，1H)，7.76(dd，J＝8.9，5.0Hz，1H)，6.81-6.86(m，2H)，6.97(dd，J＝8.9，3.1Hz，1H)，7.27(dd，J＝4.5，1.5Hz，2H)，8.57(dd，J＝4.5，1.5Hz，2H)，10.25(s，NH)；MS-ES m/e 393(M+H).

Quinolone (compound 4):

¹H NMR(400MHz，CDCl ₃)：δ2.23(s，3H)，6.67(d，J＝2.1Hz，1H)，6.76(dd，J＝8.1，1.0Hz，1H)，6.98(td，J＝8.2，2.8Hz，1H)，7.03-7.07(m，2H)，7.20(t，J＝8.1Hz，1H)，7.62(dd，J＝4.5，1.6Hz，2H)，8.02(dd，J＝8.1，1.0Hz，1H)，8.84(dd，J＝4.5，1.6Hz，2H)，8.86(s，NH)；MS-ES m/e 347(M+H).

8-methoxyl group-2-(4-pyridyl)-4 (1H)-quinolones (KN-344)

By in reflux in toluene with ester intermediate (compound 3, wherein R is OMe) cyclization in position, obtain quinolone (compound 4) (step I):

¹H NMR(400MHz，(CD ₃) ₂SO)δ3.99(s，3H)，6.66(s，1H)，7.25(dd，J＝7.9，1.3Hz，1H)，7.32(t，J＝7.9Hz，1H)，7.70(dd，J＝7.9，1.3Hz，1H)，7.82(dd，J＝4.4，1.7Hz，2H)，8.72(dd，J＝4.4，1.7Hz，2H)；MS-ES m/e253(M+H).

KN-344 is crucial intermediate in the quinolone analogs that various other pyridines replace.

8-phenyl-2-(4-pyridyl)-4 (1H)-quinolones (KN-345)

By in reflux in toluene with ester intermediate (compound, wherein R is a phenyl) cyclization in position, obtain quinolone (compound 4) (step I):

¹H NMR(400MHz，CDCl ₃)δ6.65(d，J＝2.0Hz，1H)，7.38(dd，J＝4.5，1.7Hz，2H)，7.46(td，J＝8.1，1.6Hz，1H)，7.52-7.56(m，3H)，7.58-7.63(m，2H)，8.42(dd，J＝8.1，1.6Hz，2H)，8.75(dd，J＝4.5，1.7Hz，2H)；MS-ES m/e 299(M+H).

8-benzyl-2-(4-pyridyl)-4 (1H)-quinolones (KN-346)

By in reflux in toluene with ester intermediate (compound 3, wherein R is a benzyl) cyclization in position, obtain quinolone (compound 4) (step I):

¹H NMR(400MHz，CDCl ₃)δ4.34(s，2H)，6.52(s，1H)，7.27-7.33(m，3H)，7.37-7.44(m，4H)，7.63(d，J＝6.9Hz，2H)，8.35(d，J＝8.3Hz，1H)，8.67(br.s，2H)；MS-ES m/e 313(M+H).

8-nitro-2-(4-pyridyl)-4 (1H)-quinolones (KN-352)

In synthetic quinolone (compound 4), use the crude ester intermediate (compound 3, wherein R is a nitro) that need not to be further purified:

¹H NMR(400MHz，CDCl ₃)δ6.77(d，J＝2Hz，1H)，7.51(t，J＝7.9Hz，1H)7.65(dd，J＝4.5，1.7Hz，2H)，8.72(dd，J＝7.9，1.6Hz，1H)8.80(ddd，J＝7.9，1.6，0.6Hz，1H)8.89(dd，J＝4.5，1.7Hz，2H)；MS-ESm/e 268(M+H).

8-amino-2-(4-pyridyl)-4 (1H)-quinolones (KN-353)

With 8-nitro-2-(4-pyridyl)-4 (1H)-quinolone (KN-352) hydrogenations, obtain this title compound: MS-ES m/e 238 (M+H) with the mixture of Pd/C in ethanol.

8-naphthyl-2-(4-pyridyl)-4 (1H)-quinolones (KN-350)

Ester intermediate (compound 3, wherein R is a naphthyloxy):

¹H NMR(400MHz，CDCl ₃)：δ1.23(t，J＝7.1Hz，3H)，4.13(q，J＝7.1Hz，2H)，5.01(d，J＝1.6Hz，1H)，6.56(dd，J＝7.8，1.6Hz，1H)，6.77-6.82(m，3H)，6.88(td，J＝7.8，1.6Hz，1H)，7.27(dd，J＝4.5，1.6Hz，2H)，7.37(t，J＝8.1Hz，1H)，7.50-7.54(m，2H)，7.62(d，J＝8.1Hz，1H)，7.85-7.89(m，1H)，8.18-8.21(m，1H)，8.57(dd，J＝4.5，1.6Hz，2H)，10.24(s，NH)；MS-ES m/e 410(M+H).

Quinolone (compound 4):

¹H NMR(400MHz，CDCl ₃)：δ6.69(d，J＝2.1Hz，1H)，6.97(dd，J＝7.6，1.2Hz，1H)，7.16(dd，J＝7.6，1.2Hz，1H)，7.21(t，J＝8.1Hz，1H)，7.48(t，J＝7.9Hz，1H)，7.52-7.55(m，2H)，7.59(td，J＝7.6，1.2Hz，1H)，7.78(d，J＝8.1Hz，1H)，7.96(d，J＝7.9Hz，1H)，8.07(d，J＝7.9Hz，1H)，8.08(d，J＝8.1Hz，1H)，8.79(dd，J＝4.5，1.6Hz，2H)，8.94(br.s，NH)；MS-ES m/e 365(M+H).

Embodiment 4. the Pyridopyrimidinone derivatives that the preparation pyridine replaces

Embodiment 4a

Ordinary test method: the mixture of amine (3.00mmol) and γ-oxo-4-pyridine ethyl propionate (3.00mmol) was heated 20-45 minute down at 180-200 ℃.Subsequently this crude product is carried out column chromatography purifying (SiO ₂, EtOAc), obtain desired pyrimidine compound.Reaction product is between 10-20%.

9-methyl-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one (KN-347)

¹H NMR(CDCl ₃，200MHz)δ2.75(s，3H)；7.03(s，1H)；7.15(t，J＝6.97Hz，1H)，7.69-7.73(bd，J＝6.91Hz，1H)；8.12-8.15(bd，J＝6.05Hz，2H)；8.82-8.84(bd，J＝4.73Hz，2H)；9.01-9.05(bd，J＝7.40Hz，1H).LCMS m/z 238(M ⁺+H).

9-benzyl-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one (KN-349):

¹H NMR(CDCl ₃，300MHz)δ4.50(s，2H)；7.03(s，1H)；7.16(t，J＝7.03Hz，1H)；7.28-7.39(m，5H)；7.56-7.58(m，1H)；8.21(bs，2H)；8.83(bs，2H)；9.02-9.05(m，1H).LCMS m/z 314(M ⁺+H).

9-phenyl-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one (KN-348)

¹H NMR(CDCl ₃，300MHz)δ2.50(d，J＝1.07Hz，3H)；7.00(s，1H)；7.48-7.56(m，4H)；7.71-7.75(m，4H)；7.90(bs，2H)；8.96(m，1H).LCMS m/z 314(M ⁺+H).

9-bromo-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one

¹H NMR(DMSO，300MHz)δ2.42(m，3H)；7.23(s，1H)；8.19(dd，J＝4.48，1.66Hz，2H)；8.40(d，J＝1.95Hz，1H)；8.77(dd，J＝4.56，1.65Hz，2H)；8.83(m，1H).LCMS m/z316(M ⁺).

Embodiment 4b: 9-(2-styroyl) amino-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one (KN-316)

At reflux temperature under nitrogen atmosphere, with br-derivatives 1 (324mg, 1mmol), (R)-(+)-Alpha-Methyl benzylamine 2 (122mg, 1mmol), potassium tert.-butoxide (225mg, 2mmol) and PdCl ₂(dppf) (35mg, 0.05mmol) mixture in THF stirred 20 hours.Dilute the reaction mixture cooling and with ethyl acetate.With organic layer water, salt water washing and at Na ₂SO ₄Last dry.Under vacuum, ethyl acetate layer is concentrated, and with resistates via column chromatography (silica gel, ethyl acetate) purifying, obtain desired product 3.

¹H NMR(300MHz，CDCl ₃)，δ8.78(br s，2H)，8.21(s，1H)，7.92(d，J＝5.9Hz，2H)，7.4-7.26(m，5H)，6.90(s，1H)，6.50(d，J＝5.5Hz，1H，-NH)，6.27(s，1H)，4.60(m，1H)，2.23(s，3H)，1.71(d，J＝6.9Hz，3H).MS(m/z)＝357.13(m+1).

Embodiment 4c: 9-bromo-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one

With iso ethyl nicotinate (5.0ml, 25.91mmol) and 4-ethanoyl morpholine (3.0ml, 25.91mmol) solution in THF (25ml) is with two (trimethyl silyl) Lithamide (7.0g, 41.83mmol) solution-treated in THF (25ml).At room temperature this solution stirring 24 hours.Filter this solution and use ether (3 * 50ml) washings.Filtrate is dissolved in the water (100ml), uses the Glacial acetic acid acidifying, use CH ₂Cl ₂(3 * 30ml) extractions, dry (Na ₂SO ₄), filter and be evaporated to drying.With rough reaction mixture via column chromatography (SiO ₂, ethyl acetate) and purifying, obtain compound 5 into yellow viscous oil shape thing, it is leaving standstill curing down. ¹HNMR (CDCl ₃, 300MHz) the NMR δ 3.47-3.57 of the merging of ketone and enol ether (bm, 8H); 3.95 and 5.77 (s, 1H); 7.44 and 7.61 (dd, J=4.43,1.62Hz, 2H); 8.49 (bd is J=5.93Hz) with 8.64 (dd, J=4.44,1.62Hz) 2H.LCMS m/z 235 (M ⁺+ H).

With 2-amino-3-bromo-5-picoline (1.00g, 4.30mmol), compound 5 (1.20g, 6.40mmol) and the tosic acid monohydrate (203.0mg, 1.07mmol) mixture in toluene (50ml) refluxed 4 days.Remove toluene in a vacuum, with the gained crude reaction mixture via column chromatography (SiO ₂, ethyl acetate) and purifying, obtain compound 5, be yellow mercury oxide (0.66g, 65%).

Embodiment 4d:

90-100 ℃ following in nitrogen atmosphere, with 9-bromo-2-(4-pyridyl)-4H-pyrido [1,2-a] pyrimidin-4-one 1 (232.1mg, 0.73mmol), butyl vinyl ether (0.19ml, 1.08mmol), salt of wormwood (149.2g, 1.49mmol) and Pd (OAc) ₂(4.8mg, 21.6 μ mol) solution stirring in dry DMF (6ml) 2 hours.With this solution of 1M salt acid treatment until mixture be acid (～pH4), then at room temperature should back one-tenth solution stirring 3 hours.With reactant water (10ml) dilution, use NaHCO ₃The neutralization and with water layer CH ₂Cl ₂(3 * 15ml) extractions.The organic extract liquid water that merges (3 * 15ml) washings, dry (Na ₂SO ₄), filter and be concentrated into dried, obtain the brown throw out.This solid is developed with ether, filtered, (3 * 15ml) wash, and dry air obtains desired ketone 6 then, and it is yellow mercury oxide (61.4mg, 30%) with other ether.

¹H NMR(CDCl ₃，300MHz)δ2.50(d，J＝3.29Hz，3H)；2.93(s，3H)；7.03(s，1H)；8.03(d，J＝2.18Hz，1H)；8.16(bd，J＝6.37Hz，2H)；8.83(bs，2H)；9.03(m，1H).LCMS m/z 280(M ⁺+H).

Biological test

Embodiment 1.PI the selectivity of 3-kinase beta

Use the vitro enzyme analysis to measure with respect to other I type PI 3-kinases family member or other associated kinase family enzyme, the TGX-221 selectivity suppresses the active ability of PI 3-kinase beta.

In order to measure TGX-221 to PI 3-kinases α, β or δ isotype or the kinase whose inhibition activity of PI4, with the thrombocyte of washing with 1 * molten born of the same parents' damping fluid (10Mm Tris, pH 7.6,10mM PMSF, 5mM EDTA, 2mM benzamidine, 0.1%Triton X-100) dissolving, then by with 15,000 * g centrifugation 5 minutes with whole cytolysis things clarifications.For each immunoprecipitation, under 4 ℃ with the 1mg lysate with anti--p110 α (1 μ g), anti--p110 β (1 μ g), anti--p110 δ (1 μ g) or anti--PI kinase antibody (2-5 μ g) and 50 μ l 50%A protein spheres slurries overnight incubation.For PI 3-kinase assays, A protein spheres immunity mixture is also used 1x PI 3-kinase assay buffer (20mM Hepes, pH7.2,5mM MgCl with molten born of the same parents' damping fluid washed twice ₂, 0.25mM EDTA) and more than the washed twice, use 40 μ l PI 3-kinases enzyme substrates phosphatidylinositols (PtdIns), 10 μ l ATP mixed solution (0.5 μ l γ then ³²P-ATP+0.5 μ l 10mM ATP), 10 μ l 10x kinase buffer liquid, 15 μ l milliQ H ₂O and 1 μ lTGX-221 (0-10 μ M) at room temperature cultivate the p110 α or the β isotype of 25 μ l immunoprecipitations.For the PI4 kinase assays, A protein spheres immunity mixture is also used molten born of the same parents' damping fluid washed twice, use 1X PI4 kinase assay buffer (20mM Hepes, pH7.5,10mMMgCl then ₂, 0.3%Tron X-100) and washed twice, and add other mensuration composition, as described above.By adding 100 μ l 1M HCl, 200 μ l chloroform/methanol (1: 1) and 500 μ l 2MKCl and passing through with 15, the lipid of 000xg centrifugation extraction in 2 minutes finishes total overall reaction.Analyze the generation of determining PI3 or PI4 kinases lipid products PtdIns (3) P and PtdIns (4) P by TLC.Weeding of grease particle spot and quantize the level that radioactive level produces with accurate mensuration PtdIns (3) P/PtdIns (3) P from the TLC plate then.

Be to measure the ability that TGX-221 suppresses PI 3-kinases γ isotype, with 0.5 μ l p110 γ recombinant protein with 10 μ l 10X kinase buffer liquid (2.5mM EDTA, 200mM HEPES, 50mM MgCl ₂PH7.2), 30 μ l milliQ H ₂O, 40 μ l PI (150 μ l/ml), 10 μ l ATP mixed solution (0.5 μ l γ ³²P-ATP+0.5 μ l 10mM ATP) and 1 μ l TGX-221 (0-10 μ M) incubated at room temperature 20 minutes.Other PI 3-kinases isotype as described above is measured, and finishes reaction and analyzes lipid products.

Active (Panlabs Taiwan) carries out TGX-221 via MDSPharma Services to the kinase whose inhibition of other tyrosine and Ser/Thr.

TGX 221PI shows the IC of 5nM to the 3-kinase beta ₅₀, to the selectivity of＞100 times of two kinds in the thrombocyte other main type i PI 3-kinases isotypes (PI 3-kinases α and PI 3-kinases γ) demonstrations.

E. enzyme	TGX-221 IC ₅₀(μM)
E. enzyme	TGX-221 IC ₅₀(μM)	Lipid kinase p110 α-PI3K p110 β-PI3K p110 γ-PI3K p110 δ-PI3K PI4K SRCA b1 EGF acceptor Fyn HER2 acceptor insulin receptor Ser/Thr kinases casein kinase 2 Cdk2/ cyclin A ERK1 P38 α P70 ⁸⁶The non-selective CaMK of the non-selective PKC of PKA	5 0.005 ＞10 0.1 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10 ＞10

In far-ranging protein kinase, TGX-221 shows＞1000 times selectivity to PI 3-kinase beta.This results are shown in Fig. 2 D.TGX-221 shows the inhibition effect (Fig. 2 D) to PI4 kinases minimum, and optionally suppresses PI 3-kinases lipid generation (Fig. 3 A) in the body, and does not change conventional phosphoinositide, PtdIns, PtdIns (4) P (not display data) and PtdIns (4,5) P ₂(Fig. 3 A).

The inhibition concentration of each test compound is listed in the table below.

Table I: the activity of the anti-PI 3-of the compound of selection kinases isotype

Embodiment 2. shear the platelet aggregation research that causes

Use the awl-panel assembly of customization to measure the platelet aggregation that TGX-221 suppresses PI 3-kinases lipid products and caused by shearing pathology level.

For this analysis, at anti-coagulant [6 volume blood: 1 volume anti-coagulant (90 Trisodium Citrates, 7mM citric acid, pH4.6,140mM glucose and 70mM theophylline)] gather blood under the situation about existing, and with the thrombocyte segregation, wash with improved Baezinger and Majerus method (1974).Briefly, by with 200 χ g with whole blood centrifugation 30 minutes, obtain to be rich in hematoblastic blood plasma (PRP).By centrifugation PRP 10 minutes under 2,000 χ g, make the thrombocyte balling then.Thrombocyte ball resuspending in thrombocyte lavation buffer solution (PWB) [4.3Mm K ₂HPO ₄, 4.3mM Na ₂HPO ₄, 24.3mM NaH ₂PO ₄, pH6.5,113mM NaCl, 5.5mM glucose, 0.5% bovine serum (BSA) and 10mM theophylline] in.With the no phosphoric acid salt Tyrode damping fluid washing platelet twice that contains platelet activation inhibitor theophylline (10mM), inorganic with 0.3mCi/ml down at 37 ℃ then ³²P mark 2 hours.Under the situation that has theophylline (10mM) to exist by with twice of no phosphoric acid salt Tyrode damping fluid washing platelet with incomplete tusion ³²P removes, then the thrombocyte resuspending in the no phosphoric acid salt Tyrode damping fluid that contains 1mM calcium.When increasing TGX-221 (0-500nM) concentration, under 37 ℃, radiolabeled thrombocyte was cultivated 30 minutes.Add vWf (10 μ g/ml) rapidly, make thrombocyte stand 5000s then ^-1Pathology shearing rate 2 minutes.(1997, Cell 89:105-114), analyzes by HPLC, and with thrombocytolysis, extraction also separates according to people such as Stephens.Will with commercially available PtdIns (3,4) P ₂And PtdIns (4,5) P ₂The lipid peak integration that standard substance elutes together and be standardized into the TL that applies, and the fraction representation of sample in contrast.

In order to measure the effect of TGX-221 to the platelet aggregation that causes by the pathology shearing rate, when increasing TGX-221 (0-1 μ M) concentration, the thrombocyte that will be suspended in the washing in the Tyrode damping fluid that contains 1mM calcium (350 * 109) was cultivated 10 minutes down at 37 ℃.Add vWf (10 μ g/ml) immediately, make thrombocyte stand 5000s then ^-1 Pathology shearing rate 5 minutes.Extract the thrombocyte sample out, by using Sysmex ^TMThe KN-21N hematology analyzer is analyzed the single hematoblastic number of incomplete tusion, determines the level of platelet aggregation.All data is normalized to the controlled trial data, and the ratio that is expressed as with respect to check sample increases.TGX-221 suppresses to shear the platelet aggregation that causes effectively in concentration range (ICso=0.05-0.1 μ M), is more or less the same with the lipid (Fig. 3 B) that suppresses the 3-phosphorylation.

Embodiment 3.FACS analyze

Determine to come the effect of TGX-221 with facs analysis to conglutnin activation and platelet activation.To be suspended in the thrombocyte of the washing in the Tyrode damping fluid that contains 1mM CaCl2/1mM MgCl2, select plain or 1 μ g/ml PAC-1 antibody and independent carrier or 0.5 μ M TGX-221, cultivated 10 minutes down at 37 ℃ with anti--P.Thrombocyte was at room temperature stimulated 20 minutes with zymoplasm (1U/ml), ADP (12.5mM), U46619 (1AM) or collagen (10 μ g/ml), at room temperature fix 45 minutes then with 2% Paraformaldehyde 96.With the fixed thrombocyte with Tyrode damping fluid washed twice, the anti-mouse (ab) of puting together with 1 μ g/ml FITC- ₂' antibody cultivated 15 minutes, with Tyrode damping fluid washed twice, resuspending to the final volume of 500 μ l, and is analyzed with FACS in the Tyrode damping fluid.

Embodiment 4. the research of flowing in the body

Use based on the mobile adhesion Analysis and measure TGX-221 the effusive effect of thrombocyte calcium.The thrombocyte (1.5 * 109) that is suspended in the washing among the PWB was cultivated 30 minutes down at 37 ℃ with calconcarboxylic acid pigment, OregonGreen 488 BAPTA-1, AM (1 μ M) and Fura Red, AM (1.25 μ M).Resuspending was used twice of PWB washing platelet before the Tyrode damping fluid that contains 1mM calcium.Before carrying out static state or measuring based on mobile adhesion, with thrombocyte with independent carrier, TGX-221 (0.5 μ M), LY294002 (20 μ M), acetylsalicylic acid (1mM), apyrase (0.5U/ml) or Aggrastat (200nM) cultivation 10 minutes.

Adhesion is measured for static state, places 30 minutes on the surface of the cover glass of vWf dressing at 37 ℃ of following thrombocytes.For flow assay, 600,1800 or 10,000s ^-1Constant shearing rate under thrombocyte is filled on the microscopic capillary of vWf dressing, perhaps thrombocyte is placed on the surface of microscopic capillary of vWf dressing, made it pass through 10 at interval at 1 second then, 000s ^-1Shear gradient.For static and measure based on mobile, respectively between 30 minutes incubation periods with 1 second at interval, or, monitor real-time thrombocyte calcium current and go out with 0.586 at interval up to 175 seconds.

Embodiment 5. the mensuration in improved Folts model

In anesthetized rat and rabbit, with anti-thrombosis activity in the body of the folts model research TGX-221 that improves.Research by University of Melbourne Animal EthicsCommittee according to National Health ﹠amp; The criterion of Medical Research Council ofAustralia is checked and accepted.Use vetanarcol (Nembutal for Sprague-Dawley rat (260-400g); 60mg/kg i.p.; Merial Australia Pty.Ltd., Sydney, NSW Australia) anaesthetizes, and uses too slave (6Rg/kg i.v. of pentobarbitone (15mg/kg i.v.) and sweet smell for New Zealand white rabbit (2-3kg); David Bull Laboratories, Mulgrave, VIC Australia) anaesthetizes.Animal is used additional with O ₂Room air give power ventilation (Ugo Basile ventilator, Comerio, VA, Italy). in process of the test, keep body temperature.By femoral artery is connected to pressure transmitter (Model 1050.1, the AD instrument, Sydney NSW) measures arteriotony, and (1mm i.d. is used for Da Shu ﹠amp around each (contrast and test) artery the blood flow probe to be set; 2.5mm i.d. is used for rabbit; Under meter T206, Transonic Systems Inc., Ithaca, NY, USA); With whole reference records in PowerLab data system (8SP; The AD instrument).The silk suture line is tightened around 1 artery of the probe tip that flows, be used for subsequently narrow.Test preceding 5 minutes intravenously administrable gram match (0.24mg/kg Da Shu ﹠amp; The 1mg/kg rabbit; Enoxaparin Sodium; Aventis Australia Pty.Ltd., Sydney, NSW, Australia).Suture line tightened cause narrowly, make artery blood flow reduce 50%.Then by with tweezers cramping artery 5 times on suture line, with the artery segment endothelium-denuded cell under narrow.The monitoring carotid artery flow reaches 0ml/min until it, and sign forms grumeleuse in stenosis.After 1 minute,, grumeleuse is disappeared and the recovery blood flow with stenosis springing gently.Monitor blood flow reaches 0ml/min and writing time until it again.The reduction (CFRs) that circulates is observed in preceding 30 minutes of administration.After 30 minutes, heavy dose of administration 25ml/kg is as the propylene glycol or the 2mg/kg TGX-221 of carrier, and blood pressure is continued to measure 90 minutes.During this period,, recover after for some time that perhaps disappears, eliminate (by flicking blood vessel) with the physics method when blood flow reaches zero each time, so that recover CFRs if CFR does not disappear.TGX-221 is in 100% rat (n=8) and rabbit (n=9) (Fig. 5 A).

Embodiment 6. the mensuration in the electrolytic model

At left neck artery blood flow probe end, a slice Parafilm embedded be used for ionization below the blood vessel and analyse.Artery is placed on the hook-shaped platinum electrode, then, is fixed to endways on the electrode with inaccessible blood flow, be connected to Grass SD9 stimulate instrument (Model CCU1, the Grass instrument, Quincy, MA USA) passed to the 7mA electric current 4 minutes.Stimulate and finish the back blood flow monitoring 60 minutes.Reduce to zero by blood flow and can determine thrombosis.At (Bush ﹠amp with rat electrolytic lesion carotid artery model; Shebuski, 1990) in, compare with the occlusion rate of the rat (71=10) (Fig. 5 B) 90% of vehicle treatment, damage preceding 5 minutes injection TGX-221 (solution of 2mg/kg in PG) stop occluding thrombus to form (n=6) fully, and kept volume of blood flow (Fig. 5 B inserts figure) in back 60 minutes in damage.In Folts and electrolysis research, TGX-221 is to not influence of the blood flow in original blood pressure, the rhythm of the heart or the unmarred carotid artery.

Embodiment 7. the hemorrhage research of tail in the rat

Replenishing with O ₂Room air in fluothane with rat anesthesia.After administration preceding 15 minutes (15) minute and the

administration

5 and 30 minutes, measure the tail bleeding time.For about with acetylsalicylic acid and the pretreated test of clopidogrel, before irritating the administration of food method for the first time-25 hours are also measured the tail bleeding time.At each time point, will cut on the tail with 5mm the long and dark otch of 1mm, and monitored hemorrhage situation in per 30 seconds, up to stop hemorrhage till (=tail bleeding time).

As shown in Figure 5, to studies show that of rat, TGX-221 does not increase the bleeding time when with＞20 times of minimum treatment concentration administrations.Importantly, when with TGX-221 (20mg/kg injection) during separately or with heparin (100U/kg injection) administration, the rat tail bleeding time uninfluenced (5C).With clopidogrel (10mg/kg is oral)+heparin (100U/kg injection) or acetylsalicylic acid (200mg/kg is oral)+heparin (100U/kg injection) drug combination the time, TGX-221 (2mg/kg injection) does not aggravate to prolong the bleeding time (Fig. 5 C) that is caused by these medicaments.When merging administration with clopidogrel or acetylsalicylic acid, TGX-221 (2mg/kg injection) does not influence the bleeding time yet.

Embodiment 8. PI 3-kinase assays in the body

Determine of the effect of the compound of pyridine replacement with PI 3-kinase assays in the body to PI 3-kinase activity.Use PI 3-kinases to carry out this mensuration as substrate material as enzyme and PI by the human blood platelets immunoprecipitation.As previously described (people such as Susa, 1992, The Journal of BiologicalChemistry 267 (32): 22951-22956.), by measure enzyme in conjunction with [ ³²P] enter PI formation PI ([32P]-3) P, measure PI 3-kinase activity.

The human blood platelets of washing was dissolved in the Triton X-100 dissolving damping fluid (10mM Tutofusin tris, pH 7.4,1%Triton X-100,2mM EDTA, 1mM PMSF) 30 minutes.By with 15,000g centrifugal separating cell solute 10 minutes is removed the undissolvable fraction of TritonX-100.By 500 μ g cytolysis things were mixed 2 hours with the rabbit Chinese People's Anti-Japanese Military and Political College murine antibody and 30 μ l, the 50% a-protein-sepharose pearl of the kinase whose p85/110 form of the anti-PI 3-of 1 μ g, come immunoprecipitation PI 3-kinases under 4 ℃.By with 15; 000g is with 5 seconds of gel beads granulation; with ice-cold Triton X-100 dissolving damping fluid washing 3 times; use PI 3-kinase assay buffer (20mM HEPES then; pH 7.4; 1mM EGTA, 5mMMgCl2) washing is 4 times, with a-protein-sepharose bonded PI 3-kinases segregation.

At N ₂Under will be stored in CHCl ₃The PI drying, with the ultimate density resuspending of 330 μ g/ml in lipid damping fluid (50mM HEPES, pH 7.2,1mM EDTA), and supersound process on ice 6 minutes.By with the PI 3-kinases of immunoprecipitation and 40 μ l PI, 10 μ lATP (1mM) and ³²P-r-ATP (0.5 μ Ci, 1 μ Ci/nmol), 10 μ l 10x kinase buffer liquid are being used H ₂The 100 μ l that O adjusts finally measure in the volume and mix, generation PI ([ ³²P]-3) P.Before adding ATP, TGX was cultivated 5 minutes with PI 3-kinases is pre-.Finish to measure with 100 μ l 1NHC1, and with 200 μ l chloroforms: methyl alcohol (1: 1) and 500 μ l 2M KC1 extract PI ([ ³²P]-3) the P product.By tlc, with containing CHCl ₃: MeOH: HAC: H ₂O (43: 38: 5: 7) (v: v: v: v) solvent system with the PI of chloroform in mutually ([ ³²P]-3) P dissolving, and show with the radio-autograph picture.Then from TLC dish scrape off The PI ([ ³²P]-3) the P spot, use the 1ml methylamine down at 53 ℃: butanols: methyl alcohol (42: 9: 47) (v: v: v) deacetylated 4 hours, measure numerical value with lipid scintillometer (LKB 1209 RackBETA).

Embodiment 9. reorganization is measured based on mobile

Use based on mobile adhesion mensuration and determine the influence of TGX-286 thrombocyte adhesiveness.In red corpuscle reorganization to 50% hematocrit, under 37 ℃ with the thrombocyte of washing with 10,25 or 50nM TGX-286 or contrast damping fluid (0.1%DMSO) pre-treatment 30 minutes.The red blood cell of thrombocyte and reorganization with 1800s ^-1The shearing rate perfusion little slide glass of glass by the vWf-dressing 1 minute.By 1800 ^S-1Wash the cell of removing little adhesion in 10 minutes down, measure the hematoblastic number of adhesion and be expressed as average ± SEM.TGX-286 suppresses the ability of thrombocyte adhesiveness in dose-dependent mode, when with thrombocyte with 10,25 with during the 50nMTGX-286 pre-treatment, show that 51,67 and 86% thrombocyte adhesiveness reduces.

Embodiment 10. be rich in the platelet aggregation that the CD9-antibody of hematoblastic blood plasma causes

In being rich in hematoblastic blood plasma (PRP), determine TGX286 and KN327 effect to the platelet aggregation that causes by antibody CD9.PRP suspension is cultivated with 20-100nM TGX286 or contrast damping fluid (0.1%DMSO), handled with the antibody aliquot then.In the aggregometer of 4 passages, the gathering under stirring was monitored 10 minutes.The gathering level as the light transmission by the measure of the change in the sample cell.IC50 concentration is derived as PRP and assembles the concentration that is subjected to 50% test compound that suppresses.

The whole blood flow assay

Owing to pass through the little and difficult regeneration of thrombus that the thrombocyte of washing forms, determine the restraining effect that TGX-286 forms platelet thrombus with the whole blood flow assay.With 1800s ^-1The shearing rate perfusion little slide glass of glass by the vWf dressing 2 minutes before, with the whole blood of anti-freezing with 50,100 or 200nM TGX-286 or contrast damping fluid (0.1%DMSO) cultivate 30 minutes also attached to jiggle.By at 1800s ^-1Wash down and removed the not thrombocyte of adhesion in 10 minutes, then with the globulolysis of 1% ammonium oxalate adhesion.By measure thrombocyte LDH (U/L) level of whole cell solute with spectrophotometry, directly measure the thrombosis level.The whole blood perfusion was observed on the surface of little slide glass and is rich in hematoblastic thrombus after 2 minutes.Suppress ability that platelet thrombus form in dose-dependent mode in the vWf zone with the TGX-286 pre-treatment.With respect to contrast,, cause thrombosis to reduce by 25,53 and 80% with 50,100 and 200nM TGX-286 pre-treatment whole blood

Embodiment 11. the animal model of ICAO

The people such as Folts that suitably set up, 1982, in the arterial thrombus animal model of Circulation 65:248-255, determine the restraining effect of TGX-286.This model is used to study the interior anticoagulation medicine of body to the influence of response crushing damage succeeded by the setting time of stricture of artery.

The carotid artery of the rat of anesthesia is dissected out, the mobile probe of electromagnetism is put to measure blood flow around artery.Near mobile probe, artery is clamped vessel wall is caused the moderate infringement with the operating forceps of silicone tube with parcel.The plastic barrel of ligature or suitable internal diameter is tightened to produce 70% minimizing of artery diameter around artery.

Thrombocyte forms the occlusive platelet thrombus gradually at the arteries region clustering of narrow damage, regards the minimizing of blood flow aspect as.When thrombosis, elevation of blood pressure is causing that near narrow positions thrombus breaks and embolism takes place.If thrombus does not have the spontaneous generation embolism, narrow zone is shaken gently so that thrombus moves.This causes the unexpected recovery of blood flow.Thrombocyte is assembled once more and is damaged arteries at narrow zone, repeats thrombus-embolism and forms pattern.This violent be that the thrombosis of media takes place succeeded by embolism with the thrombocyte, in blood flow, cause to circulate minimizing (CFR).In case rat produces regular CFR, form compound or vehicle Control via jugular vein administration antithrombotic.

With the dosage of 2.5mg/kg and 4mg/kg via intravenously administrable TGX-286 or KN327, and record blood flow stability.TGX-286 and KN327 with the 14.0mg/kg administration made 80% treatment animal recovery baseline in 10 minutes, represent that this compound is effective aspect the treatment coronary occlusion.

Embodiment 12.TGX-286 with the influence of KN-327 for platelet thrombus formation under flowing

37 ℃ with the whole blood of adding citric acid salt with 50,100 or 200nM TGX-286, KN-327 or the pre-treatment of contrast damping fluid (0.1%DMSO) 10 minutes.With 600s ^-1With the microscopic capillary of hemoperfusion by the bag of the von Willebrand factor-(vWf) quilt 2 minutes.By with 600s ^-1The perfusion damping fluid was removed the not cell of adhesion in 2 minutes, by handle the globulolysis with any adhesion with 1% ammonium oxalate.Analyze serum lactic dehydrogenase (LDH) level (U/L) then by the thrombocytolysis of adding 1%Triton X-100, and with spectrophotometry with adhesion.Compared with the control, with 50,100,200nM TGX-286 pre-treatment whole blood causes thrombotic reduction.

Embodiment 13. the optionally external PI3K enzymatic determination of isotype

Carry out vitro enzyme and measure affinity and the specific primary screen that the selection isotype of medicine is determined in conduct.With the antibody that can discern p100 α (sc-7174) and β (sc-603) isotype that derives from Santa Cruz Biotechnology, α and the β isotype immunoprecipitation from the thrombocyte lysate of PI3K come out.The γ isotype makes as recombinant protein matter in the Kinacia laboratory.With similar method, go out the δ isotype with δ specific antibody (sc-7176) immunoprecipitation from the THP-1 cell.Be with or without under the situation of inhibitor, phosphatidyl-4 is pure and mild with using ³²The standard phosphorylation of p is analyzed, and measures the activity of enzyme in immunoprecipitation.The activity of determining enzyme in the certain limit of inhibitor concentration is determined IC ₅₀Value.

PI3 kinase inhibitor LY294002 (8-phenyl-2-(4-morpholinyl)-4H chromene-4-ketone, Sigma ﹠amp; Num; L9908) IC of the various isotypes of inhibition PI3K ₅₀, (0.5-1.5pM is identical with the value of previous report.

Embodiment 14. neutrophil ROS response

Prepare white cell by human blood

The whole blood of 3ml preservative-free is put into the 50ml Taper Pipe.25 ℃ add down 48ml globulolysis liquid and on hematology nutator reverse the mixing gently 10 minutes, subsequently in the desktop whizzer under 25 ℃ with 350xg centrifugation 10 minutes.Being rich in leukocytic ball resuspending in the physiological saline that replenishes with the phosphate buffered of 10%w/v gel (PBS-gel), and as described above the separation.With the white cell ball once, with 2 * 10 with Hanks balanced salt solution (HBSS) washing ⁶The final cell of/ml is counted resuspending and is existed side by side promptly and to use.

The ROS that measurement derives from neutrophil produces

The generation of reactive oxygen species (ROS) is the active sign of neutrophil.Some chemokines and cytokine promote the generation of ROS by neutrophil.Measure the effect that ROS is produced with the post-stimulatory PI3K inhibitor of chenotactic peptide TGX-286.Via fluorescence activated cell sorting (FACS), by people such as modification Robinson, the method for (pp 9.7.5-9.7.9, Curr.Protocols in Cytometry (1997)), the oxidation of dihydro rhodamine 123 (DHR) in the monitoring cell, the generation that comes measure R OS.The loose load of white cell suspension with 2 μ l 50mM DHR/ml cells (finally being 50mM), and was cultivated 12 minutes down at 37 ℃.The cell of the DHR load of 500 ℃ of aliquots was stimulated 20 minutes down at 37 ℃ with 5 μ l 100uM fmlp effect liquid, and subsequently in quencher on ice.Use and be used for 520 ± 20nm passband that DHR distributes and flow cytometer is set at excitation wavelength is 488nm.Using not the stimulated control sample to differentiate with gate neutrophil population and via the anti-CD14 immunostaining proofreaies and correct.Proofread and correct the fluorescence baseline data with these gate contrast white cells.Analyze the irritation cell sample, and 5000 gate white cells of each sample are monitored green fluorescence (DHR).For the relative value of measure R OS, will from total value, deduct by the value that check sample obtains, and be normalized to the value that obtains by independent fmlp sample (unrestraint agent).

Embodiment 15. neutrophil elastoser discharge

Prepare neutrophil by human blood

The heparinized blood that derives from the healthy volunteer of sample aliquot (24ml) is layered on

With

(Sigma) on the 12ml pad, then at 25 ℃ with 700 * g centrifugation 30 minutes.Collect the just top band that is rich in neutrophil of Histopaque-119 pad and wash with HBSS.Remove remaining red blood corpuscle with 0.2%NaCl by hypotonic lysis.The neutrophil sample existed side by side promptly with the HBSS washed twice use.

The mensuration of neutrophil elastoser exocytosis

The activated neutrophil by discharging certain proteolytic enzyme a series of stimulator being reacted and, and this proteolytic enzyme is to cause tissue and extracellular matrix destructive major cause in inflammatory process.As the indication that proteolytic enzyme discharges, measure TGX-286 to the exocytosis of neutrophilia elastoser.By people such as modification Ciesla, (The Journal of Trauma, 48 (3): method 388-395 (2000)), exocytosis is quantized to elastoser, and is as described below.The release of measuring the neutrophil elastoser by the special elastoser matrix of cracking AAPV-pNA.With isolating neutrophil (6.25 * 10 ⁵Cell) 37 ℃ of pre-down cultivations 5 minutes, stimulated 20 minutes with 0.11M fmlp then.Subsequently with cell suspending liquid with 400 * g centrifugation 5 minutes, extract the gained supernatant liquor then out and keep it.

By adding the supernatant liquor that 100 μ l do not have cell in the single hole on 96 hole microplates, that contain the mixture of the special elastoser chromogenic substrate of 0.33mM AAPV-pNA in 33.3mM hydroxy ethylene piperazine esilate and 0.17mM NaCl, measure the release of elastoser.Emptying aperture also contains elastase inhibitor AAPV-CK (0.17mM).Total reaction volume is 150 μ L, and each is tested with independent AAPV-CK blank to carry out in duplicate.96 hole flat boards were cultivated 60 minutes down at 37 ℃, and measurement is in the absorbancy of 405nm.In order to measure the relative release rate of elastoser, the numerical value that derives from check sample is deducted from total value, and be normalized to the numerical value that obtains by independent fmlp sample (unrestraint agent).

Embodiment 16. cell proliferating determining

Measure the antiproliferative activity of TGX-286 to U937 (monokaryon) clone.Continue 4 days cytotoxicities, and measure the viability of cell with the colorimetric estimation of Metabolic activity by calculating cell number monitoring compound.

Table II below the inhibitor concentration (nM) of test compound in each biological activity listed in.

Table II

Table III

	P110 α isotype	P110 β isotype	Platelet aggregation is to CD9 antibody	Thrombosis suppresses	ROS from neutrophil discharges	Antiproliferative activity is to the U937 cell
	P110 α isotype	P110 β isotype	Platelet aggregation is to CD9 antibody	Thrombosis suppresses	ROS from neutrophil discharges	Antiproliferative activity is to the U937 cell	Method	Embodiment 13	Embodiment 13	Embodiment 10	Embodiment 12	Embodiment 14	Embodiment 16
#	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	Method	Embodiment 13	Embodiment 13	Embodiment 10	Embodiment 12	Embodiment 14	Embodiment 16
#	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	IC50/(nM)	TGX-286	5000	2	100	500	5	5
KN-327			200	250	78	20	TGX-286	5000	2	100	500	5	5

Example of formulations

Embodiment 1. contain the preparation of drug combination and the administration of the compound of pyridine replacement

Some preferred drug substances of the present invention is described below.

The tablet that is used for oral administration:

The component that is used for the tablet of oral administration is listed in following Table IV.Tablet A, B and C make like this: preceding 6 kinds of components of listing in the Table IV are carried out wet granulation with polyvinylpyrrolidone, add Magnesium Stearate then, subsequently compressing tablet.

The tablet that is used for sublingual administration:

The composition as listed Table V below that is used for two kinds of tablets of sublingual administration.Tablet A and B make like this: preceding 6 kinds of components of listing in the Table V are carried out wet granulation with polyvinylpyrrolidone, add Magnesium Stearate then, subsequently compressing tablet.

The tablet that is used for the cheek administration:

The tablet that is used for the cheek administration makes like this: the component that following Table VI is listed is mixed, then with blended component direct compression.

The capsule that powder is filled:

The composition as listed of the capsule that two kinds of powder are filled is below in the Table VII.Capsules A and B make like this: component is mixed, fill two halves formula hard gelatin capsule with the gained mixture.

The capsule of liquid filling:

The composition as listed of the capsule of two kinds of liquid filling is below in the Table VIII.Capsules A makes like this: with Macrogol 4000 BP fusing, active ingredient is dispersed in this melt, fills two halves formula hard gelatin capsule with it.Capsule B makes like this: active ingredient is dispersed in Yelkin TTS and the peanut oil, fills soft elastic gelatin capsule with the gained dispersion liquid.

Extencap:

The capsule that is used for controlled release makes like this: preceding 4 kinds of components that following Table I X is listed are mixed and are extruded, with nodularization of gained extrudate and drying.The exsiccant piller is used as the ethyl cellulose dressing of release-controlled film, the gained piller is filled in the two halves formula hard gelatin capsule.

The intravenous administration preparation:

The intravenous administration preparation that contains the component of listing in the following Table X makes like this: active ingredient is placed citrate buffer, then with hydrochloric acid with the pH regulator of this solution to pH 7.Gained solution is complemented to desired volume, be filled in the aseptic vial of sealing via millipore filter then, sealing once more after the filling.

The intranasal administration preparation:

The intranasal administration preparation that contains the component of listing among the following Table X I makes like this: active ingredient is placed the mixture of hydroxy benzoate, be used in hydrochloric acid in the citrate buffer with the pH regulator of gained solution to pH 7.Gained solution is complemented to desired volume, be filled in the aseptic vial of sealing via millipore filter then, sealing once more after the filling.

The intramuscularly preparation:

The intramuscularly preparation that contains the component of listing among the following Table X II makes like this: with solubilization of active ingredient in Glycofurol.Add phenylcarbinol and dissolving then, adding entry to final volume is 3ml.Then this mixture is filled in the aseptic vial of sealing sealing once more after the filling via millipore filter.

Syrup:

The syrup that contains the component of listing among the Table X III below makes like this: Sodium Benzoate is dissolved in a part of purified water, adds Sorbitol Solution USP then.Add active ingredient and dissolving.Then gained solution is mixed with glycerine, complement to desired volume with purified water.

Suppository:

The suppository that contains the component of listing among the Table X IV below makes like this: 1/5th Witepsol top temperature in 45 ℃ in the pot of steam jacket is melted.Then active ingredient is sieved via 200 μ m screen clothes, use the Silverson mixing tank that is equipped with crop to mix, until obtaining level and smooth dispersion liquid with the matrix of fusing.Keep 45 ℃ temperature, in suspension, add all the other Witepsol H15, it is stirred to guarantee uniform mixing.Then whole suspension is crossed 250 μ m stainless steel meshs, continued to be cooled to 40 ℃ under the stirring.Under 38-40 ℃ temperature, the mixture of 2.0g sample aliquot is filled in the suitable plastic mould.Allow gained suppository be cooled to room temperature.

¹Used active ingredient is a powder, and wherein at least 90% particulate diameter is 63 μ m or lower.

Aerosol:

The aerosol that contains the component of listing among the Table X V below makes like this: active compound is mixed with ethanol, add water for injection.Then this solution is added among a part of Propellant 22, is cooled to-30 ℃, and transfers in the tamping unit.Then aequum is fed in the stainless steel vessel, with all the other propelling agent dilutions.Then valve is assembled on the container.

The hysterophore preparation:

The hysterophore preparation is directly to mix by the component of will be below listing among the Table X VI to make.Hysterophore is by making the compression of gained mixture.

Claims

1. the compound that has formula (III):

Wherein X and Y are N;

R is H or C ₁-C ₄Alkyl;

R ₁Be F, Cl, Br, C ₁-C ₄Alkyl, aryl, NR ' ³-(CHR ' ³) _n-aryl, (CHR ' ³) _n-NR ' ³-aryl, (CHR ' ³) _n-O-aryl or O-(CHR ' ³) _n-aryl, wherein n is 0,1 or 2;

R ₂Be H, F, Cl, Br, C ₁-C ₄Alkyl or CH ₂Ph;

R ₃Be H;

And

R ' ³Be H or C ₁-C ₄Alkyl.

2. the compound of claim 1, wherein R ¹Be selected from CH ₃, C ₂H ₅,

3. the compound of claim 1, wherein R is a methyl, and R ¹Be selected from

4. the compound of claim 1 is used for disintegrating the purposes of the medicine of the platelet aggregation that takes place and adhesion under high-shear state in preparation.

5. the compound of claim 1 is used for the purposes of antithrombotic medicine in preparation.

6. the compound of claim 1 is used for prevention or treatment cardiovascular disorder in preparation by inhibition of phosphoinositide 3-kinase, prevention or treatment respiratory disease, prevention or treatment cancer, the purposes in the medicine of prevention or treatment and leukocyte function obstacle diseases associated.