Compounds for the prevention and treatment of bleeding and vascular system instability

Abstract

Thrombolytic therapy and anticoagulant therapy can induce bleeding and vascular instability. In certain aspects, provided herein are methods for treating and / or preventing bleeding and vascular instability comprising administering a compound disclosed herein to a subject in need thereof.

Description

Technical Field

[0001] In certain aspects, provided herein are methods for treating and / or preventing bleeding and vascular instability comprising administering to a subject in need thereof a compound disclosed herein. Background Art

[0002] Thrombolytic and anticoagulant therapies are commonly administered to prevent clot formation and / or to dissolve or break down clots. However, these agents often have side effects of inducing bleeding and / or vascular instability, such as local edema, loss of vascular tone, and disruption of blood-brain barrier function. The compounds disclosed herein can be used to prevent and / or treat bleeding and vascular instability induced by thrombolytic and anticoagulant therapies. Summary of the Invention

[0003] In certain aspects, provided herein are methods for preventing, reducing the risk of, inhibiting, reducing, alleviating, and / or treating bleeding and / or vascular instability in a subject, wherein the bleeding or vascular instability is induced by thrombolytic therapy, anticoagulant therapy, and / or intravascular interventional therapy, wherein the method comprises administering to the subject a therapeutically effective amount of a compound disclosed herein.

[0004] In certain aspects, provided herein are methods of protecting and / or stabilizing the vascular endothelium in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a compound disclosed herein.

[0005] In certain aspects, provided herein are methods of preventing and / or treating blood-brain barrier disruption in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a compound disclosed herein.

[0006] In some embodiments, the compounds disclosed herein for use in any of the disclosed methods are compounds of Formula I, II, III, or IV. In some embodiments, such compounds comprise the structure of Formula IV. In some embodiments, such compounds comprise the structure of Formula IV:

[0007]

[0008] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings illustrate embodiments of the present technology and are non-limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases, various aspects may be shown exaggerated or enlarged to facilitate understanding of particular embodiments.

[0010] Figure 1A-1H shows representative images of triphenyltetrazolium chloride (TTC)-stained coronal brain sections (selected from the median animal in each group) showing tissue infarct (white) and normal tissue (red) in the indicated groups 24 hours after stroke. Figure 1 A and Figure 1 B is a placebo-treated control animal showing Bederson scores of 3 and 5, and significant infarct volume (white). Figure 1 C and ID are animals treated with 1 mg / kg J147 administered intravascularly (iv), showing better Bederson scores of 3 and 3, and non-significant reduction in infarct volume relative to placebo. Figure 1 E and 1F are animals treated with 10 mg / kg ivJ147, showing a great improvement in Bederson scores of 1 and 2, and a great reduction in infarct volume. Figure 1 G and 1H are animals treated with intraperitoneal administration of J147, showing Bederson scores of 2 and 3 and moderate reductions in infarct volume.

[0011] Figure 2 A-2H show images of brains isolated from animals treated with tPA alone, Figure 3 A-3H show images of brains isolated from animals treated with tPA+J147. Unstained coronal sections ( Figure 2 A, 2C, 2E, 2G and Figure 3 Representative images of A, 3C, 3E, and 3G) show intracerebral hemorrhage (red), TTC-stained coronal sections ( Figure 2 B, 2D, 2F, 2H and Figure 3 B, 3D, 3F, and 3H) show tissue infarct (white) and normal tissue (red) in the indicated groups 24 hours after stroke.

[0012] Figure 4A A graph of Bederson scores is shown, which showed a positive trend for tPA+J147-treated animals compared to tPA-only-treated animals (p=0.6254). Figure 4B Shown are scans showing quantitative infarct volume with a positive trend in tPA+J147 treated animals (p=0.061). Figure 4C Graphs showing quantification of bleeding volume, which demonstrated significant improvement in tPA+J147 treated animals (p=0.048).

[0013] Figure 5A TTC-stained coronal sections of brain isolated from rats treated with vehicle or J147 are shown. Red indicates normal tissue and white indicates tissue infarcts in the designated group (white). Rats were subjected to 2 hours of ischemia followed by reperfusion. Infarct volumes were assessed 72 hours after stroke ( Figure 5C) and Bederson score ( Figure 5B (n=7-8 / group). * indicates P<0.05.

[0014] Figure 6A Unstained and TTC-stained coronal sections of brains isolated from rats treated with saline, tPA+vehicle, or tPA+J147 are shown. Red in TTC-stained sections indicates normal tissue, and white indicates tissue infarction in the indicated group. Rats were subjected to 2 hours of ischemia followed by reperfusion. Infarct volume was assessed 72 hours after stroke ( Figure 6C ), Bederson score ( Figure 6B ) and blood volume ( Figure 6D (n=7-8 / group). * indicates P<0.05.

[0015] Figure 7 A shows representative images of double immunofluorescence staining for MMP-9 using endothelial barrier antigen (EBA) in the indicated groups. Quantitative analysis showed that combined treatment (tPA and J147) significantly inhibited MMP9 expression in both single cells and microvessels compared to vehicle or tPA alone. Bar = 100 μm, *P < 0.05. n = 3 animals / group.

[0016] Figure 7 B and 7C show representative images (top) and quantitative analysis (bottom) of brain neutrophil infiltration (A) and microglial expansion (B) in the indicated groups. Bar = 100 μm, *P < 0.05. n = 3 animals / group.

[0017] Figure 8 A and 8B show representative images of double immunofluorescence staining showing fibrin (A, top panel) or platelets (B, top panel) deposited in brain microvessels (labeled by endothelial barrier antigen [EBA] staining). The number of fibrin-positive (A, bottom panel) and platelet-positive (B, bottom panel) vessels was counted as described in the methods.

[0018] Figure 8 C shows representative images of double immunofluorescence staining of PAI-1 (green) and EBA (red) in the indicated groups. The number of PAI-1-positive vessels was counted as described in the Methods. Bar = 100 μm, *P < 0.05. n = 3 animals / group.

[0019] Figure 9 A shows flow cytometric measurement of platelet P-selectin expression in whole blood. Representative gating strategy for platelets in whole blood and histograms of P-selectin (CD62P) expression on platelets in the indicated groups. Data are expressed as mean fluorescence intensity. *P < 0.05 vs. sham group. # < 0.05 vs. saline or tPA group.

[0020] Figure 9 B shows the gating strategy for flow cytometric analysis of total leukocytes (R2, CD45-positive cells included in R1) in whole blood: neutrophils (N; R3) and monocytes (M; R4) are defined by differential expression of CD11b and RP-1 (specific for rat granulocytes); and representative flow cytometric dot plots of platelet-neutrophil (CD42d+CD45+CD11b+PR1+) and -monocyte (CD42d+CD45+CD11b+PR1-) aggregates in the indicated groups. Fluorescein isothiocyanate (FITC)-labeled CD42d (glycoprotein V) mAb was used as a specific platelet marker to identify platelet-neutrophil / monocyte complexes. Quantitative analysis showed that combined treatment significantly attenuated ischemia-induced and delayed tPA-enhanced circulating platelet-neutrophil aggregation, but not platelet-monocyte aggregation. n = 3 rats / group. *P < 0.05 vs sham group; #P < 0.05 vs saline or tPA group. NS; non-significant

[0021] Figure 10A TTC-stained coronal sections of brains isolated from rats treated with vehicle or J147 are shown. Red indicates normal tissue and white indicates tissue infarction (white) in the designated group. Rats were subjected to 2 hours of ischemia followed by administration of J147 or controls, with no reperfusion from TPA administration in all cases.

[0022] Figure 10B Shows Figure 10A Graphical representation of infarct volume (%) assessed 24 hours after stroke for the indicated experiments (n=7-8 / group). DETAILED DESCRIPTION

[0023] The compounds disclosed herein can protect the vascular endothelium (including the blood-brain barrier) from various injuries and can reduce bleeding. In some embodiments, provided herein are compounds for treating and / or preventing bleeding or vascular instability. In certain embodiments, the methods herein include preventing bleeding or vascular instability in a subject, reducing the risk of bleeding or vascular instability, inhibiting, reducing, alleviating or treating bleeding or vascular instability, wherein the bleeding or vascular instability is induced by thrombolytic therapy or anticoagulant therapy.

[0024] Also provided herein are methods for preventing, reducing the risk of, inhibiting, reducing, alleviating or treating tissue damage caused by reperfusion (e.g., reperfusion injury). The mitochondrial permeability transition pore (mPTP) is directly involved in a variety of human diseases. Of particular importance are those caused by the reperfusion injury component of acute ischemia-reperfusion injury (IRI). IRI typically causes organ damage, tissue damage, and organ or tissue dysfunction, and is a leading cause of death and disability worldwide. IRI is typically caused by insufficient blood supply to tissues (ischemia) and subsequent medically mediated or natural recovery (reperfusion). The reperfusion component of IRI (i.e., reperfusion injury) is estimated to cause up to 50% of the damage to tissues and organs affected by ischemia. Reperfusion of ischemic tissues often causes damage as a result of reoxygenation of cells of these tissues and mitochondria in these cells. More specifically, in mitochondria, calcium overload, excessive production of reactive oxygen species (ROS), and changes in pH within the first few minutes after reperfusion trigger a cascade of biochemical changes that lead to prolonged opening of the mPTP in the mitochondrial membrane. This can lead to a catastrophic collapse of mitochondrial function and cell death or apoptosis. Reperfusion injury can affect many organs and tissues, including the heart, kidneys, lungs, liver, skin flaps, ovaries, intestines, stomach, and pancreas. Applicants have determined that the compounds disclosed herein can prevent, alleviate, inhibit, and / or treat reperfusion injury.

[0025] Compound

[0026] In some embodiments, provided herein are compounds for performing the methods disclosed herein. In some embodiments, provided herein are compounds for preventing or treating bleeding and / or vascular instability in a subject. In certain embodiments, the compounds used herein comprise the structure of Formula I:

[0027]

[0028] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof. In some embodiments of Formula I, R 2 is hydrogen (H) or methyl; R 3 is methyl, fluoro-substituted alkyl (e.g., fluoromethyl, difluoromethyl, or trifluoromethyl) or bromo-substituted alkyl (e.g., bromomethyl, dibromomethyl, tribromomethyl); L 3 is a carbonyl group; and R 6 is independently selected at each occurrence from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, hydroxy, methoxy, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy, mercapto, alkylthio, arylthio, carbonyl, carboxyl, aryl, substituted aryl, substituted heterocyclyl, halogen, cyano, cyanoalkyl, amine, methylamine, dimethylamine, nitro, amino, amidino, carbamate, CF3, OCF3, S(O) nR 7 and C(O)R 8 , or two R at adjacent positions 6 combines to form an optionally substituted heteroaryl or heteroalkyl ring fused to the adjacent phenyl moiety; wherein R 7 Selected from H, R 9 、NH2、HNR 9 and NR 9 R 10 ; R 8 Selected from OH, OR 9 、NH2、NHR 9 and NR 9 R 10 ; where R 9 and R 10 is independently at each occurrence optionally substituted alkyl; and n is 1 or 2.

[0029] In certain embodiments of Formula I, R 6 is independently selected at each occurrence from alkyl, substituted alkyl, alkenyl, substituted alkenyl, hydroxy, alkoxy, methoxy, substituted alkoxy, halogen, carbonyl, carboxyl, or C(O)R 8 ; In some such respects, R 6 Each occurrence of L is methyl, methoxy, perfluoromethyl, perfluoromethoxy, hydroxy, Cl, F, or I. In some embodiments of Formula I, L 3 is a carbonyl group, R 3 It's CF 3 , R 2 is H, and R 6 is empty or H at each occurrence. In some embodiments of Formula I, L 3 is a carbonyl group, R 3 It is CF3, R 2 is H, and R 6 In some embodiments of Formula I, L is independently selected from methyl or methoxy. 3 is a carbonyl group, R 3 It is CF3, R 2 is a methyl group, and R 6 is independently selected at each occurrence from methyl or methoxy.

[0030] In some embodiments, the compounds used herein comprise the structure of Formula II:

[0031]

[0032] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein:

[0033] (i)R A2 、RA4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is a methyl group, and R B4 It is a methyl group;

[0034] (ii)R A2 、R A3 、R A5 and R A6 It's H, R A4 is methoxy, R B2 is a methyl group, and R B4 It is a methyl group;

[0035] (iii)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is H, and R B4 It is H;

[0036] (iv)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is a methyl group, and R B4 It is a methyl group;

[0037] (v)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is H, and R B4 It is H;

[0038] (vi)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is H, and R B4 It is a methyl group;

[0039] (vii)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is H, and R B4 It is a methyl group;

[0040] (viii)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is a methyl group, and R B4 It is H;

[0041] (ix)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is a methyl group, and R B4 It is H;

[0042] (x)R A2 、R A3 、R A5 and R A6 It's H, R A4 is COOH, R B2 is a methyl group, and R B4 It is a methyl group;

[0043] (xi)R A2 、R A4 and R A5 It's H, R A3 and R A6 is hydroxyl, R B2 is a methyl group, and R B4 It is a methyl group;

[0044] (xii)R A2 、R A4 and R A6 It's H, R A3 and R A5 is hydroxyl, R B2 is a methyl group, and R B4 It is a methyl group;

[0045] (xiii)R A2 、R A4 and R A5 It's H, R A3 is methoxy, R A6 It's F, R B2 is H, and R B4 It is Cl;

[0046] (xiv)R A3 and R A5 It's H, R A2 and R A6 It's F, R A4 is hydroxyl, R A6 It's F, RB2 is H, and R B4 It is F;

[0047] (xv)R A2 、R A4 and R A6 It's H, R A3 is hydroxyl, R A5 It's F, R B2 is H, and R B4 is F; or

[0048] (xvi)R A2 、R A5 and R A6 It's H, R A3 and R A4 Together they are -O-CH2-O-, R A5 It's F, R B2 is H, and R B4 It's F.

[0049] In some embodiments of the compound of Formula II, R A2 、R A5 and R A6 H, R A3 is methoxy, R B2 and R B4 is methyl, and R A4 is selected from H, NO2, OH, methoxy, phenol, methyl, fluorine (F), N(CH3)2, CHC(CN)2 and O-tert-butyldimethylsilyl (OTBDMS). In some embodiments of the compound of formula II, R A2 、R A4 、R A5 and R A6 H, R A3 is methoxy, R B2 is methyl, and R B4 In some embodiments of the compound of Formula II, R A2 、R A3 、R A5 and R A6 H, R A4 is methoxy, R B2 is methyl, and R B4 In some embodiments of the compound of Formula II, R A2 、R A3 、R A4 、R A5 and R A6 H, R B2 is methyl, and R B4 In some embodiments of the compound of Formula II, R A2、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is H, and R B4 Is H. In some embodiments of the compound of Formula II, R A2 、R A3 、R A4 、R A5 and R A6 H, R B2 is H, and R B4 In some embodiments of the compound of Formula II, R A2 、R A3 、R A4 、R A5 and R A6 H, R B2 is H, and R B4 In some embodiments of the compound of Formula II, R A2 、R A4 、R A5 and R A6 H, R A3 is methoxy, R B2 is H, and R B4 In some embodiments of the compound of Formula II, R A2 、R A4 、R A5 and R A6 H, R A3 is methoxy, R B2 is methyl, and R B4 is H. In some embodiments of the compound of Formula II, R A2 、R A3 、R A4 、R A5 and R A6 H, R B2 is methyl, and R B4 is H. In some embodiments of the compound of Formula II, R A2 、R A3 、R A5 and R A6 H, R A4 is a carboxyl group, R B2 is methyl, and R B4 In some embodiments of the compound of Formula II, R A2 、R A4 、R A5 and R A6 H, R A3 is a carboxyl group, R B2is methyl, and R B4 It is a methyl group.

[0050] In some embodiments, the compounds used herein comprise the structure of Formula III:

[0051]

[0052] or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein R1 is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, dibromomethyl, or tribromomethyl; R2 is methyl, methoxy, hydroxyl, halogen, CF3, OCH3, OCF3, or OCBr3; and R3 and R4 are independently selected from hydrogen, hydroxyl, halogen (e.g., Cl, F, or Br), methyl, methoxy, and amine. In some embodiments of Formula III, R1 is CF3 (trifluoromethyl), R2 is OCH3, and R3 and R4 are methyl. In some embodiments of Formula III, R1 is CF3 (trifluoromethyl), R2 is OCF3, and R3 and R4 are methyl.

[0053] In some embodiments, the compounds used herein comprise the structure of Formula IV below, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof.

[0054]

[0055] The structure of Formula IV is sometimes referred to herein as "J147."

[0056] The following terms have the corresponding definitions listed below.

[0057] "Alkyl" refers to a straight or branched chain alkyl group having from about 1 to about 12 carbon atoms (e.g., methyl, ethyl, propyl, butyl, etc.). "Substituted alkyl" refers to an alkyl group further bearing one or more substituents as described herein (e.g., 1, 2, 3, 4, or even 5). "Optionally substituted alkyl" refers to an alkyl or substituted alkyl group.

[0058] "Cycloalkyl" refers to a ring-containing group containing from about 3 to about 12 carbon atoms. "Substituted cycloalkyl" refers to a cycloalkyl further bearing one or more substituents (e.g., 1, 2, 3, 4, or even 5) selected from alkyl, substituted alkyl, and any of the substituents described herein. "Optionally substituted cycloalkyl" refers to a cycloalkyl or substituted cycloalkyl.

[0059] "Heterocycle," "heterocyclic," and similar terms refer to cyclic (i.e., ring-containing) groups that contain one or more heteroatoms (e.g., N, O, S, etc.) as part of the ring and have from 1 to about 14 carbon atoms. "Substituted heterocycle" and similar terms refer to heterocycles that further bear one or more substituents (e.g., 1, 2, 3, 4, or even 5) as described herein. Exemplary heterocyclic moieties include saturated, unsaturated, and aromatic heteroatom-containing ring systems, such as epoxy, tetrahydrofuran, oxazoline, pyrrole, pyridine, furan, and the like. "Optionally substituted heterocycle" and similar terms refer to heterocycles or substituted heterocycles.

[0060] References to "optionally substituted bicyclic rings" are to bicyclic ring structures known in the art, optionally including substitutions as defined herein.

[0061] "Alkenyl" refers to a straight chain, branched, or cyclic hydrocarbon radical comprising 2 to about 20 carbon atoms, having at least one, 1-3, 1-2, or one carbon-carbon double bond. "Substituted alkenyl" refers to an alkenyl substituted at one or more, e.g., 1, 2, 3, 4, or even 5, positions with substituents as described herein. "Optionally substituted alkenyl" refers to an alkenyl or substituted alkenyl. In some embodiments, the alkenyl is ethenyl or propenyl. In certain embodiments, the substituted alkenyl is substituted ethenyl or substituted propenyl. In some embodiments, the ethenyl or propenyl is substituted with one or more CN moieties. For example, in some embodiments, the substituted ethenyl comprises (CN)2C=CH-.

[0062] "Aryl" refers to an aromatic group having 6 to about 14 carbon atoms. "Substituted aryl" refers to an aryl group further bearing one or more (e.g., 1, 2, 3, 4, or even 5) substituents selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, carbonyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, halogen, trifluoromethyl, pentafluoroethyl, cyano, cyanoalkyl, nitro, amino, amido, amidino, carboxyl, carbamate, SO2X (wherein X is H, R, NH2, NHR, or NR2), SO3Y (wherein Y is H, NH2, NHR, or NR2), or C(O)Z (wherein Z is OH, OR, NH2, NHR, or NR2), and the like. "Optionally substituted aryl" refers to an aryl or substituted aryl group.

[0063] "Aralkyl" refers to an alkyl group substituted with an aryl group. "Substituted aralkyl" refers to an aralkyl group further bearing one or more (e.g., 1, 2, 3, 4, or even 5) substituents selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, and any of the substituents described herein. Thus, aralkyl groups particularly include benzyl, diphenylmethyl, and 1-phenylethyl (-CH(C6H5)(CH3)). "Optionally substituted aralkyl" refers to an aralkyl or substituted aralkyl group.

[0064] "Heteroaryl" refers to an aromatic group containing one or more heteroatoms (e.g., N, O, S, etc.) as part of the aromatic ring, typically having 2 to about 14 carbon atoms, and "substituted heteroaryl" refers to a heteroaryl group further bearing one or more (e.g., 1, 2, 3, 4, or even 5) substituents selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, and any of the substituents listed above.

[0065] "Heteroaralkyl" and "heteroarylalkyl" refer to an alkyl group substituted with one or more heteroaryl groups. "Substituted heteroaralkyl" refers to a heteroaralkyl group further bearing one or more (e.g., 1, 2, 3, 4, or even 5) substituents selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, and any of the substituents described herein. "Optionally substituted heteroaralkyl" refers to a heteroaralkyl or substituted heteroaralkyl group.

[0066] "Halogen" and "halo" refer to fluorine, chlorine, bromine or iodine.

[0067] "Hydroxy" and "hydroxyl group" refer to the functional group -OH.

[0068] "Alkoxy" refers to the group -OR, where R is an alkyl group. "Substituted alkoxy" refers to the group -OR, where R is a substituted alkyl group. "Optionally substituted alkoxy" refers to an alkoxy or substituted alkoxy group.

[0069] "Aryloxy" refers to the group -OR, where R is an aryl group. "Substituted aryloxy" refers to the group -OR, where R is a substituted aryl group. "Optionally substituted aryloxy" refers to an aryloxy or substituted aryloxy group.

[0070] "Mercapto" and "thiol" refer to the functional group -SH.

[0071] "Alkylthio" and "thioalkoxy" refer to the groups -SR, -S(O) n＝1-2 -R, wherein R is alkyl. "Substituted alkylthio" and "substituted thioalkoxy" refer to the groups -SR, -S(O) n＝1-2 -R, wherein R is substituted alkyl. "Optionally substituted alkylthio" and "optionally substituted thioalkoxy" refer to alkylthio or substituted alkylthio.

[0072] "Arylthio" refers to a group -SR where R is an aryl group. "Substituted arylthio" refers to a group -SR where R is a substituted aryl group. "Optionally substituted arylthio" refers to an arylthio or substituted arylthio group.

[0073] "Amino" refers to unsubstituted, monosubstituted, and disubstituted amino groups, including the substituent -NH2, "monoalkylamino" refers to a substituent having the structure -NHR, where R is alkyl or substituted alkyl, and "dialkylamino" refers to a substituent having the structure -NR2, where each R is independently alkyl or substituted alkyl.

[0074] "Amino" refers to the group -C(=NR q )NR r R s , where R q 、R r and R s is independently hydrogen or optionally substituted alkyl.

[0075] Reference to "amido" includes substituents of the structure -C (O) -NR2, wherein each R is independently H, alkyl, substituted alkyl, aryl or substituted aryl, as described above. When each R is H, the substituent is also referred to as "carbamoyl" (i.e., a substituent having the structure -C (O) -NH2). When only one R group is H, the substituent is also referred to as "monoalkylcarbamoyl" (i.e., a substituent having the structure -C (O) -NHR, wherein R is an alkyl or substituted alkyl as described above) or "arylcarbamoyl" (i.e., a substituent having the structure -C (O) -NH (aryl), wherein aryl is as defined above, including substituted aryl). When none of the R groups is H, the substituent is also referred to as "dialkylcarbamoyl" (i.e., a substituent having the structure -C (O) -NR2, wherein each R is independently alkyl or substituted alkyl as described above).

[0076] Reference to a "carbamate" includes substituents of the structure -OC(O)-NR2, wherein each R is independently H, alkyl, substituted alkyl, aryl, or substituted aryl.

[0077] Reference to an "ester group" includes substituents of the structure -OC(O)-OR, wherein each R is independently alkyl, substituted alkyl, aryl, or substituted aryl.

[0078] "Acyl" refers to a group having the structure -C(O)R, where R is hydrogen, alkyl, aryl, etc., as defined herein. "Substituted acyl" refers to an acyl group in which the substituent R is substituted as defined herein. "Optionally substituted acyl" refers to both acyl and substituted acyl groups.

[0079] "Cyanoalkyl" refers to the group -R≡N, where R is optionally substituted alkylenyl.

[0080] As used herein, "substituted" refers to an atom or group of atoms that has been substituted by another atom or group of atoms (i.e., a substituent), and includes all levels of substitution, such as single-, two-, three-, four-, five-, or even six-substitutions, where such substitutions are chemically permitted. Substitutions can occur at any chemically accessible position and on any atom, such as carbon and any heteroatom (e.g., oxygen, nitrogen, or sulfur). For example, substituted moieties include those in which one or more bonds to contained hydrogen or carbon atoms are replaced by bonds to non-hydrogen and / or non-carbon atoms. Substituents may include, but are not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl, alkoxy, aryloxy, and ester groups; sulfur atoms in groups such as thiol, alkyl and aryl sulfide, sulfone, sulfonyl, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and heteroatoms in other groups well known in the art.

[0081] Non-limiting examples of substituents include, but are not limited to, halogen, -OH, -NH2, -NO2, -CN, -C(O)OH, -C(S)OH, -C(O)NH2, -C(S)NH2, -S(O)2NH2, -NHC(O)NH2, -NHC(S)NH2, -NHS(O)2NH2, -C(NH)NH2, -OR, -SR, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -S(O)R, -S(O)2R, -C(O)NHR, -C(S)NHR, -C(O)NRR, -C(S)NRR, -S(O)2NHR, -S(O)2NRR, -C(NR)NHR, -C(NH)NRR, -NH C(O)R, -NHC(S)R, -NRC(O)R, -NRC(S)R, -NHS(O)R, -NRS(O)R, -NHC(O)NHR, -NHC(S)NHR, -NRC(O)NH, -NRC(S)NH, -NRC(O)NHR, -NRC(S)NHR, -NHC(O)NRR, -NHC(S)NRR, -NRC(O)NRR, -NRC(S)NRR, -NHS(O)NHR, -NRS(O)NH, -NRS(O)NHR, -NHS(O)NRR, -NRS(O)NRR, -NHR, -NRR, where R at each occurrence is independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl.Also contemplated are substitutions with optionally substituted hydrocarbyl moieties containing one or more of the following chemical functionalities: -O-, -S-, -NR-, -OC(O)-, -OC(O)-O-, -OC(O)-NR-, -NR-C(O)-, -NR-C(O)-O-, -NR-C(O)-NR-, -SC(O)-, -SC(O)-O-, -SC(O)-NR-, -S(O)-, -S(O)2-, -OS(O)2-, -OS(O) 2-O, -OS(O)2-NR-, -OS(O)-, -OS(O)-O-, -OS(O)-NR-, -O-NR-C(O)-, -O-NR-C(O)-O-, -O-NR-C(O)-NR-, - NR-OC(O)-, -NR-OC(O)-O-, -NR-OC(O)-NR-, -O-NR-C(S)-, -O-NR-C(S)-O-, -O-NR-C(S)-NR-, -NR-OC(S) -, -NR-OC(S)-O-, -NR-OC(S)-NR-, -OC(S)-, -OC(S)-O-, -OC(S)-NR-, -NR-C(S)-, -NR-C(S)-O-, -NR-C( S)-NR-, -SS(O)2-, -SS(O)2-O-, -SS(O)2-NR-, -NR-OS(O)-, -NR-OS(O)-O-, -NR-OS(O)-NR-, -NR-OS(O)2 -, -NR-OS(O)2-NR-, -O-NR-S(O)-, -O-NR-S(O)-O-, -O-NR-S(O)-NR-, -O-NR-S(O)2-O-, -O-NR-S(O)2-NR-, -O-NR-S(O)2-, -OP(O)R2-, -SP(O)R2-, or -NRP(O)R2-, where R at each occurrence is independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0082] In some embodiments, compounds for use herein include isomers of the compounds disclosed herein, including stereoisomers (eg, enantiomers and diastereomers), structural isomers, tautomers, conformational isomers, and geometric isomers.

[0083] Exemplary structural isomers include, for example, but are not limited to, isomers resulting from different attachments of functional groups forming the compounds disclosed herein, such as 1-propyl substituted with 2-propyl, etc. Structural isomers in combination with tautomerism also include bond rearrangements involving double bonds and migration of substituents. For example, tautomerism in combination with 1-3 pleiotropic hydrogen shifts can result in structural isomers.

[0084] Exemplary conformational isomers include, for example, but are not limited to, isomers produced by rotation about a bond where such rotation is hindered to the extent that separable isomers are produced, as is well known in the art.

[0085] Exemplary geometric isomers include double bonds having "E" or "Z" configurations as known in the art.

[0086] The compounds disclosed herein can be readily prepared using suitable synthetic methods. For example, 3-methoxybenzaldehyde can be condensed with 2,4-dimethylphenylhydrazine in methanol using standard hydrazone preparation conditions (e.g., heating in a microwave to accelerate the reaction time). The free NH is then acylated using TFAA (trifluoroacetic anhydride) plus a catalytic amount (0.1%) of DMAP (dimethylaminopyridine), THF (tetrahydrofuran), or DCM (dichloromethane).

[0087] In some embodiments, compound used herein is provided in the form of a pharmaceutically acceptable salt. Compound used herein can be complexed with any suitable inorganic or organic salt using a suitable method. In some embodiments, the salt of the compound used herein is prepared by reacting the compound with a suitable organic or inorganic acid or base. The limiting examples of the organic salts used herein include mesylate, acetate, oxalate, adipate, alginate, aspartate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate (toluenesulfonate), citrate, malate, maleate, fumarate, succinate, tartrate, naphthalenesulfonate, mesylate, 2-naphthalenesulfonate, nicotinate, benzenesulfonate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, glucoheptanoate, glycerophosphate, enanthate, hexanoate, undecanoate, 2-hydroxyethanesulfonate, ethanesulfonate etc. In some embodiments, inorganic salts can be formed from inorganic acids, such as sulfates, bisulfates, hemisulfates, hydrochlorides, chlorates, perchlorates, hydrobromides, hydroiodides, etc. Non-limiting examples of base salts include ammonium salts; alkali metal salts, such as sodium salts, potassium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; salts formed with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, phenylethylamine, etc.; and salts formed with amino acids such as arginine, lysine, etc.

[0088] Thus, in certain embodiments, the methods herein comprise administering to a subject a therapeutically effective amount of a compound of Formula I, II, III, or IV.

[0089] Bleeding and vascular instability

[0090] Provided herein are methods for preventing bleeding and vascular instability, reducing the risk of bleeding and vascular instability, inhibiting, reducing, alleviating and / or treating bleeding and vascular instability. In some embodiments, described herein are methods for preventing, reducing the risk of, inhibiting, reducing, alleviating and / or treating bleeding and vascular instability induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy.

[0091] Bleeding

[0092] In certain embodiments, the methods include preventing bleeding, reducing the risk of bleeding, inhibiting bleeding, reducing bleeding, alleviating bleeding, and / or treating bleeding, which methods, in certain embodiments, comprise administering to a subject having bleeding, suspected of having bleeding, or at risk of having bleeding, a therapeutically effective amount of a compound disclosed herein.

[0093] In certain embodiments, hemorrhage is internal bleeding or external bleeding. In some embodiments, hemorrhage is internal bleeding (such as internal bleeding), for example, wherein the bleeding is invisible and / or is located at and / or contained in the subject's body. The non-limiting examples of internal bleeding include internal bleeding into the chest, abdomen, neck, retroperitoneum, pelvis, uterus, liver, heart, limbs, head, brain, its tissues, etc. In some embodiments, hemorrhage includes myocardial hemorrhage or consists of it. Therefore, in some embodiments, the method includes preventing myocardial hemorrhage, reducing the risk of myocardial hemorrhage, suppressing myocardial hemorrhage, reducing myocardial hemorrhage, alleviating myocardial hemorrhage and / or treating myocardial hemorrhage. In some embodiments, hemorrhage includes cerebral hemorrhage or cerebral hemorrhage or consists of it. Therefore, in some embodiments, the method includes preventing cerebral hemorrhage or cerebral hemorrhage, reducing cerebral hemorrhage or cerebral hemorrhage risk, suppressing, reducing, alleviating and / or treating cerebral hemorrhage or cerebral hemorrhage. In some embodiments, hemorrhage includes intracerebral hemorrhage or intracranial hemorrhage. Therefore, in some embodiments, the method includes preventing intracerebral hemorrhage or intracranial hemorrhage, reducing its risk, suppressing, reducing, alleviating and / or treating intracerebral hemorrhage or intracranial hemorrhage.

[0094] Internal bleeding can be caused by one or more disorders, complications, or conditions, non-limiting examples of which include, for example, ruptured blood vessels due to high blood pressure, aneurysms, varicose veins, ulcers (e.g., peptic ulcers), ectopic pregnancy, surgery, medical procedures, trauma, combinations thereof, etc. In certain embodiments, internal bleeding is caused by or is associated with cancer, a blood disorder, vitamin K deficiency, or a virally induced hemorrhagic fever.

[0095] In some embodiments, bleeding is induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy. In certain embodiments, the bleeding induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy is (directly or indirectly) caused by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy, worsened, aggravated, aggravated and / or amplified bleeding. The compounds disclosed herein can prevent, inhibit, alleviate, reduce the degree of bleeding or delay the onset of bleeding, and the bleeding is (directly or indirectly) caused by thrombolytic therapy, anticoagulant therapy, antiplatelet drugs, anti-inflammatory drugs or intravascular interventional therapy, worsened, aggravated, aggravated and / or amplified. In certain embodiments, the bleeding induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy is observed, diagnosed or suspected during or after the administration of thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy. In certain embodiments, the bleeding induced by antiplatelet drugs or anti-inflammatory drugs is observed, diagnosed or suspected during or after the administration of antiplatelet drugs or anti-inflammatory drugs. Bleeding can be diagnosed by suitable methods, non-limiting examples of which include CT scan, MRI scan or angiography.

[0096] Bleeding can be acute bleeding or chronic bleeding. Bleeding can be mild, moderate or severe. In some embodiments, bleeding is I, II, III or IV class bleeding. In some embodiments, I class bleeding is classified in part by an estimated blood loss of less than ≤ 15% of the total blood volume. In certain embodiments, the methods herein include preventing bleeding, reducing the risk of bleeding, inhibiting bleeding, reducing bleeding, alleviating bleeding and / or treating bleeding, wherein the bleeding is I class bleeding, or wherein the estimated blood loss due to bleeding is equal to or less than 15%, 10%, 5%, 2% or 1% of the total blood volume.

[0097] In certain embodiments, the method for suppressing, reducing, alleviating and / or treating hemorrhage includes reducing bleeding volume. The method herein can reduce bleeding volume by at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or at least 90%. In certain embodiments, the method herein can reduce bleeding volume by 1% to 100%, 1% to 75%, 1% to 50%, 1% to 25%, 1% to 20%, 1% to 10% or 1% to 5%.

[0098] In some embodiments, hemorrhage includes or is composed of hemorrhagic transformation. In some embodiments, hemorrhagic transformation includes hemorrhagic transformation in the brain (for example, cerebral hemorrhagic transformation). Therefore, in some embodiments, prevention of hemorrhage, reducing the risk of bleeding, suppressing, reducing, alleviating and / or treating the method for hemorrhage includes preventing hemorrhagic transformation, reducing the risk of hemorrhagic transformation, suppressing, reducing, alleviating and / or treating the method for hemorrhagic transformation. In some embodiments, method includes preventing the hemorrhagic transformation induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy, reducing its risk, suppressing, reducing, alleviating and / or treating it.

[0099] Vascular instability

[0100] Provided herein are methods for preventing vascular instability, reducing the risk of vascular instability, inhibiting, reducing, alleviating and / or treating vascular instability. In certain embodiments, the methods comprise administering a therapeutically effective amount of a compound disclosed herein to a subject having, suspected of having, or at risk of having vascular instability. In some embodiments, the method for preventing vascular instability comprises protecting the vascular endothelium and / or the blood-brain barrier from damage or injury.

[0101] In some embodiments, vascular instability comprises endothelial dysfunction, non-limiting examples of which include decreased or loss of vascular tone, decreased or loss of hemostasis, edema (e.g., localized swelling), damage and / or dysfunction of the vascular endothelium, damage or disruption of the blood-brain barrier, and the like, and combinations thereof. In some embodiments, vascular instability comprises edema. In some embodiments, the edema is cerebral edema. In some embodiments, the edema comprises swelling of the blood-brain barrier.

[0102] In some embodiments, vascular instability includes reperfusion injury. In some embodiments, reperfusion injury includes damage or impairment of the vascular endothelium caused by reperfusion. In some embodiments, vascular instability includes vascular endothelial injury caused by reperfusion (for example, reperfusion induced by thrombolytic therapy, anticoagulant therapy and / or intravascular interventional therapy; for example, reperfusion injury). In some embodiments, vascular instability includes damage and / or dysfunction of the blood-brain barrier. Damage and / or dysfunction of the blood-brain barrier can also be caused by reperfusion. In some embodiments, the method for treating vascular instability includes stabilizing the vascular endothelium and / or stabilizing the blood-brain barrier during or after reperfusion, and / or during or after giving thrombolytic therapy, anticoagulant therapy, and / or intravascular interventional therapy. Therefore, in some embodiments, the method includes preventing damage and / or dysfunction of the blood-brain barrier, reducing the risk of damage and / or dysfunction of the blood-brain barrier, inhibiting, reducing, alleviating and / or treating damage and / or dysfunction of the blood-brain barrier, wherein the damage and / or dysfunction is induced by thrombolytic therapy, anticoagulant therapy and / or intravascular intervention therapy.

[0103] Thrombolytic therapy, anticoagulation, and endovascular therapy

[0104] In certain embodiments, bleeding or vascular instability is induced, caused (directly or indirectly), worsened, aggravated, exacerbated and / or amplified by the administration (e.g., including self-administration) of one or more of thrombolytic therapy, anticoagulant therapy, antiplatelet drugs, anti-inflammatory drugs or intravascular intervention, which therapy can be administered alone or in combination. Therefore, in some embodiments, the methods herein include preventing bleeding or vascular instability in a subject, reducing its risk, inhibiting, reducing, alleviating or treating bleeding or vascular instability in a subject, wherein the bleeding or vascular instability is induced, caused (directly or indirectly), worsened, aggravated, exacerbated and / or amplified by the administration (e.g., including self-administration) of one or more of thrombolytic therapy, anticoagulant therapy, antiplatelet drugs, anti-inflammatory drugs or intravascular intervention, wherein the method includes administering to the subject a therapeutically effective amount of a compound as described herein.

[0105] In certain embodiments, bleeding or vascular instability is induced, caused (directly or indirectly), exacerbated, aggravated, exacerbated and / or amplified by thrombolytic therapy (e.g., thrombolytic therapy administered to a subject). Therefore, in certain embodiments, the method comprises administering a compound disclosed herein to a subject before, during, or after administering thrombolytic therapy to the subject. Thrombolytic therapy generally comprises administering to a subject a drug or therapy that destroys or dissolves a blood clot. Non-limiting examples of thrombolytic therapy include administering tissue plasminogen activator (TPA), streptokinase, a streptokinase activator, or urokinase to the subject. Thrombolytic drugs may comprise recombinantly expressed proteins. Non-limiting examples of TPA include alteplase (Activase), reteplase (Retavase), or tenecteplase (TNKase, Metalyse). In some embodiments, the streptokinase activator is an anisoylated plasminogen streptokinase activator complex. Non-limiting examples of anisoylated plasminogen streptokinase activator complexes include anistreplase or eminase. In some embodiments, urokinase is a urokinase-type plasminogen activator. An example of a urokinase-type plasminogen activator is saruplase.

[0106] In certain embodiments, bleeding or vascular instability is induced, caused (directly or indirectly), worsened, aggravated, aggravated and / or amplified by anticoagulant therapy (e.g., administering an anticoagulant to a subject). Therefore, in certain embodiments, the method includes administering a compound disclosed herein to a subject before, during, or after administering anticoagulant therapy to the subject. Anticoagulant therapy is typically a drug or therapy administered to a subject to inhibit or prevent the formation of blood clots. Non-limiting examples of anticoagulant therapy include administering vitamin K antagonists, thrombin inhibitors, factor Xa inhibitors, heparin, low molecular weight heparin, derivatives thereof, and the like. Non-limiting examples of vitamin K antagonists include warfarin, acenocoumarol, coumatetralyl, biscoumarol, biscoumarol ethyl ester, phenprocoumon, atromentin, chloroindanedione, diphenylindanedione, phenylindanedione, and thiochlorocoumarin. The limiting examples of thrombin inhibitors include hirudin, bivalirudin, argatroban, dabigatran, efegatran, enogatran, melagatran, ximelagatran, desirudin, lepirudin and antithrombin III. The limiting examples of factor Xa inhibitors include low molecular weight heparin (e.g., bemiparin, cetuximab, dalteparin, enoxaparin, nadroparin, pamaparin, reviparin and tizaparin), apixaban (Eliquis), betrixaban, darixaban, omixaban, fondaparinux, rivaroxaban (Xarelto), edoxaban (Lixiana), imixaban, fondaparinux, edaparinux and heparinoids (e.g., danaparioid, dermatan sulfate and sulodexide). Non-limiting examples of heparin, low molecular weight heparin and heparin derivatives include enoxaparin, dalteparin, tizaparin and danaparin. Other non-limiting examples of anticoagulants include dabigatran (Pradaxa), batroxobin, hementin, clopidogrel, ticlopidine, prasugrel, ticagrelor and aspirin.

[0107] In certain embodiments, bleeding or vascular instability is induced, caused (directly or indirectly), exacerbated, aggravated, exacerbated, and / or amplified by antiplatelet agents, non-limiting examples of which include glycoprotein IIb / IIIa inhibitors (e.g., abciximab, eptifibatide, orbofiban, roxifiban, sibrafiban, and tirofiban), ADP receptor / P2Y 12inhibitors (e.g., thienopyridines (e.g., clopidogrel, purogracillin, and ticlopidine) and nucleotide / nucleoside analogs (e.g., cangrelor, elinogrel, and ticagrelor)), prostaglandin analogs (PGI2) (e.g., beraprost, iloprost, prostacyclin, and treprostinil)), cyclooxygenase (COX) inhibitors (e.g., acetylsalicylic acid / aspirin, aloxiprin, carbaspirin calcium, indobufen, and triflusal), thromboxane inhibitors (e.g., thromboxane synthase inhibitors (e.g., dipyridamole, picostatamide, and terbogrel), receptor antagonists (e.g., terbogrel and trutroban)), cloricromen, ditazol, and vorapaxar.

[0108] In certain embodiments, the bleeding or vascular instability is induced, caused (directly or indirectly), exacerbated, aggravated, exacerbated, and / or amplified by an anti-inflammatory drug, non-limiting examples of which include ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, and indomethacin.

[0109] In certain embodiments, the bleeding or vascular instability is induced, caused (directly or indirectly), exacerbated, aggravated, exacerbated, and / or amplified by an endovascular interventional procedure. Non-limiting examples of endovascular interventional procedures include mechanical thrombectomy.

[0110] Subjects

[0111] The term "subject" refers to a mammal. Any suitable mammal can be treated by the methods or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the subject is a non-human primate or a human. In some embodiments, the subject is a human. The subject can be of any age or at any stage of development (e.g., an adult, a teenager, a child, an infant, or an intrauterine mammal). The subject can be male or female.

[0112] In some embodiments, the subject has bleeding and vascular instability, is suspected of having bleeding and vascular instability or is at risk of having bleeding and vascular instability. In certain embodiments, the subject at risk of bleeding or vascular instability is a subject being administered, has been administered or will be administered a thrombolytic, anticoagulant or intravascular interventional therapy. In certain embodiments, the subject at risk of bleeding or vascular instability is a subject being administered, has been administered or will be administered tissue plasminogen activator (TPA). In certain embodiments, the subject plans to receive or will receive thrombolysis, anticoagulant or intravascular interventional therapy.

[0113] In some embodiments, the subject has or is suspected of having a blood clot (i.e., a thrombus) or thrombosis (e.g., a blood clot blocking a blood vessel). In some embodiments, the subject has or is suspected of having a venous thrombosis or deep vein thrombosis. In some embodiments, the subject has or is suspected of having a blood clot or thrombosis and is administered or plans to be administered thrombolytic therapy (e.g., TPA). In some embodiments, the subject has or is suspected of having a venous thrombosis or deep vein thrombosis and is administered or plans to be administered thrombolytic therapy.

[0114] In some embodiments, the subject has or is suspected of having an ischemic stroke and is administered or is planned to be administered thrombolytic therapy. In some embodiments, the subject has or is suspected of having an ischemic stroke and is administered or is planned to be administered TPA.

[0115] Pharmaceutical composition

[0116] In some embodiments, the composition or pharmaceutical composition comprises a compound disclosed herein. In some embodiments, the composition or pharmaceutical composition comprises a therapeutically effective amount of a compound disclosed herein. In some embodiments, the composition or pharmaceutical composition comprises a compound disclosed herein in an amount ranging from 1 μg to 1000 mg, 1 μg to 100 mg, or 10 μg to 100 μg. In some embodiments, provided herein is a pharmaceutical composition comprising a compound disclosed herein for performing the methods described herein. In some embodiments, the pharmaceutical composition comprises a compound disclosed herein and a pharmaceutically acceptable excipient, diluent, additive, or carrier.

[0117] The pharmaceutical composition can be formulated for a suitable route of administration. In some embodiments, the pharmaceutical composition is formulated for oral, subcutaneous (sc), intradermal, intramuscular, intraperitoneal and / or intravenous (iv) administration. In certain embodiments, the pharmaceutical composition contains formulation materials for changing, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate (e.g., phosphate-buffered saline), or a suitable organic acid); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); diluents; excipients, and / or pharmaceutical adjuvants. In particular, the pharmaceutical composition may comprise any suitable carrier, formulation or ingredient, etc., or a combination thereof, such as "Remington: The Science And Practice of Pharmacy" Mack Publishing Co., Easton, PA, 19th Edition, (1995) (hereinafter referred to as Remington'95), or "Remington: The Science and Practice of Pharmacy", Pharmaceutical Press, Easton, PA, 22nd Edition, (2013) (hereinafter referred to as Remington 2013), the contents of which are incorporated herein by reference in their entirety.

[0118] In certain embodiments, the pharmaceutical compositions comprise suitable excipients, non-limiting examples of which include anti-adherents (e.g., magnesium stearate), binders, fillers, monosaccharides, disaccharides, other carbohydrates (e.g., glucose, mannose, or dextrin), sugar alcohols (e.g., mannitol or sorbitol), coatings (e.g., cellulose, hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose, synthetic polymers, shellac, gelatin, zein, enteric or other polysaccharides), starches (e.g., potato, corn, or wheat starch), silicon dioxide, colorants, disintegrants, flavorings, lubricants, preservatives The invention relates to a pharmaceutical composition comprising ...

[0119] In some embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable additive and / or carrier. Non-limiting examples of suitable additives include suitable pH adjusting agents, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, etc. Non-limiting examples of sulfur-containing reducing agents include those with sulfhydryl groups (e.g., mercaptan), such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salt, sodium thiosulfate, glutathione, and C1-C7 thioalkanoic acid. The limiting examples of antioxidants include isoascorbic acid, butylated toluene, butylated hydroxyanisole, alpha-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate and propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate and sodium metaphosphate. In addition, diluents, additives and excipients may include other common ingredients, for example inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate, and organic salts such as sodium citrate, potassium citrate and sodium acetate.

[0120] Pharmaceutical composition used herein can be stable in the time period of extension (for example, in the magnitude of several months or several years).In some embodiments, pharmaceutical composition comprises one or more suitable preservatives.The limiting examples of preservative include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide etc. and / or its combination.Preservative can include quaternary ammonium compound, for example benzalkonium chloride, benzoxonium chloride (benzoxonium chloride), benzethonium chloride, cetrimide, sepazonium chloride (sepazoniumchloride), cetylpyridinium chloride or domifene bromide. Preservatives may include alkylmercuric salts of thiosalicylic acid, such as thimerosal, phenylmercuric nitrate, phenylmercuric acetate, or phenylmercuric borate. Preservatives may include parabens, such as methylparaben or propylparaben. Preservatives may include alcohols, such as chlorobutanol, benzyl alcohol, or phenylethyl alcohol. Preservatives may include biguanide derivatives, such as chlorhexidine or polyhexamethylene biguanide. Preservatives may include sodium perborate, imidazolidinyl urea, and / or sorbic acid. Preservatives may include stable oxychloro complexes, such as those known and marketed under the trade name Preservatives may include polyethylene glycol-polyamine condensation resins, such as those known and available from Henkel KGaA under the trade name Commercially available. The preservative may include stabilized hydrogen peroxide. The preservative may be benzalkonium chloride. In some embodiments, the pharmaceutical composition is preservative-free.

[0121] In some embodiments, compositions disclosed herein, pharmaceutical compositions or compounds are substantially free of pollutants (e.g., blood cells, platelets, polypeptides, minerals, blood-derived compounds or chemicals, viruses, bacteria, other pathogens, toxins, etc.). In some embodiments, compositions disclosed herein, pharmaceutical compositions or compounds are substantially free of serum and serum pollutants (e.g., serum proteins, serum lipids, serum carbohydrates, serum antigens, etc.). In some embodiments, compositions disclosed herein, pharmaceutical compositions or compounds are substantially free of pathogens (e.g., viruses, parasites or bacteria). In some embodiments, compositions disclosed herein, pharmaceutical compositions or compounds are substantially free of endotoxins. In some embodiments, compositions disclosed herein, pharmaceutical compositions or compounds are sterile. In certain embodiments, compositions disclosed herein or pharmaceutical compositions comprise compounds of Formula I, II, III or IV.

[0122] Pharmaceutical compositions as described herein can be formulated for administration to a subject in any suitable form and / or amount according to the therapy using them. For example, a pharmaceutical composition formulated for parenteral administration (for example, by injection or infusion) can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it can contain preparatons, excipients, additives and / or diluents, such as aqueous or non-aqueous solvents, cosolvents, suspending solutions, preservatives, stabilizers and / or dispersants. In some embodiments, the pharmaceutical composition suitable for parenteral administration can contain one or more excipients. In some embodiments, the pharmaceutical composition is lyophilized into a dry powder form. In some embodiments, the pharmaceutical composition is lyophilized into a dry powder form, which is suitable for reconstructing with a suitable pharmaceutical solvent (for example, water, saline, isotonic buffer (for example, PBS), DMSO, a combination thereof, etc.). In certain embodiments, the reconstructed form of the lyophilized pharmaceutical composition is suitable for parenteral administration (for example, intravenous administration) to mammals.

[0123] In certain embodiments, the pharmaceutical composition is formulated for oral administration and can be formulated as tablets, microtablets, microtablets, micropellets, powders, granules, capsules (e.g., capsules filled with microtablets, micropellets, powders or granules), emulsions, solutions, or the like, or combinations thereof. Pharmaceutical compositions formulated for oral administration may comprise a suitable coating to delay or sustain the release of the active ingredient, non-limiting examples of which include enteric coatings such as fatty acids, waxes, shellac, plastics, methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, sodium alginate, zein, plant fibers, and the like, and combinations thereof.

[0124] In some embodiments, the pharmaceutical compositions described herein can be formulated for topical administration and can include one or more binders and / or lubricants, polyglycols, gelatin, cocoa butter or other suitable waxes or fats. In some embodiments, the pharmaceutical compositions described herein are incorporated into topical formulations comprising a topical carrier that is generally suitable for topical drug administration and comprises any suitable material known to those skilled in the art. In certain embodiments, the topical formulations of the pharmaceutical compositions are formulated for administration of the compound using a topical patch.

[0125] In certain embodiments, one skilled in the art will determine the optimal pharmaceutical composition based on, for example, the intended route of administration, delivery form, and desired dosage (see, for example, Remington '95 or Remington 2013, supra). Pharmaceutical compositions can be prepared by any suitable means, including, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, embedding, or tableting methods (see, for example, the methods described in Remington '95 or Remington 2013).

[0126] Route of administration

[0127] Suitable methods for administering the compositions, pharmaceutical compositions, or compounds disclosed herein to a subject can be used. Suitable formulations and / or routes of administration can be used to administer the compounds disclosed herein or compositions disclosed herein (e.g., see Fingl et al., 1975, "The Pharmacological Basis of Therapeutics," which is incorporated herein by reference in its entirety). Suitable formulations and / or routes of administration can be selected by a medical professional (e.g., a physician) based on, for example, the risk, age, and / or condition of the subject. Non-limiting examples of routes of administration include external or topical (e.g., transdermal or cutaneous (e.g., on the skin or epidermis), in or on the eye, intranasal, transmucosal, in the ear, intraauricular (e.g., behind the eardrum)), enteral (e.g., delivered through the gastrointestinal tract, e.g., orally (e.g., as a tablet, capsule, granules, liquid, emulsion, lozenge, or a combination thereof), sublingually, through a gastric feeding tube, rectally, etc.), by parenteral administration (e.g., parenterally, e.g., intravenously, intraarterially, intramuscularly, intraperitoneally, intradermally, subcutaneously, intracavitary, intracranial, intraarticular, into a joint space, intracardiac (into the heart), intracavernous injection, intralesional (into a skin lesion), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intrauterine, intravaginal, intravesical instillation, intravitreal), etc. or combinations thereof.

[0128] In some embodiments, a compound disclosed herein or a pharmaceutical composition described herein is administered to the lungs, bronchial passages, trachea, esophagus, sinuses, or nasal passages using a suitable method, non-limiting examples of which include intranasal administration, intratracheal instillation, and oral inhalation administration (e.g., by use of an inhaler, such as a single / multi-dose dry powder inhaler, a nebulizer, etc.).

[0129] In some embodiments, compound disclosed herein or pharmaceutical composition disclosed herein is provided to the subject.For example, the composition provided to the subject is sometimes provided to the subject for self-administration or is administered to the subject by another person (for example, a non-medical professional).As another example, the composition can be provided according to the instructions written by a medical practitioner, and the instructions are authorized to provide the composition as described herein or treatment (for example, prescription) to the patient.In another example, compositions can be provided to the subject, wherein the subject is, for example, administered orally, intravenously or by an inhaler self-administered composition.

[0130] Alternatively, a compound or composition disclosed herein may be administered in a local rather than systemic manner, for example, by direct application to the skin, mucosa, or treatment area of interest, including the use of depot or sustained-release formulations.

[0131] In certain embodiments, pharmaceutical compositions comprising a compound disclosed herein are administered alone (e.g., as a single active ingredient (AI) or, for example, as a single active pharmaceutical ingredient (API)). In other embodiments, pharmaceutical compositions comprising a compound disclosed herein are administered in combination with one or more additional AIs / APIs, e.g., as two separate compositions or as a single composition in which the one or more additional AIs / APIs are mixed or formulated in a pharmaceutical composition with the compound disclosed herein.

[0132] Dosage and therapeutically effective amount

[0133] In some embodiments, the amount of the compound disclosed herein (e.g., in a pharmaceutical composition) is a therapeutically effective amount. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound disclosed herein. In some embodiments, a therapeutically effective amount of a compound disclosed herein is administered to a subject. In some embodiments, the therapeutically effective amount of a compound disclosed herein is the amount required to obtain an effective treatment result. In certain embodiments, the therapeutically effective amount of a compound disclosed herein is an amount sufficient to treat or prevent bleeding or vascular instability. The determination of a therapeutically effective amount is fully within the capabilities of those skilled in the art, particularly in accordance with the detailed disclosure provided herein.

[0134] In certain embodiments, a therapeutically effective amount is an amount that is high enough to provide an effective therapeutic effect (e.g., a beneficial therapeutic effect) and low enough to minimize undesirable adverse reactions. Thus, in certain embodiments, the therapeutically effective amount of the compounds disclosed herein may vary with the subject, typically depending on the subject's age, weight, general health, and severity of the condition being treated. Thus, in some embodiments, the therapeutically effective amount is determined empirically. Thus, the therapeutically effective amount of the compound administered to a subject may be determined by one of ordinary skill in the art based on, for example, the effective amount found in animal or clinical studies, physician experience, and recommended dosage ranges or dosing guidelines.

[0135] In certain embodiments, a therapeutically effective amount of a compound disclosed herein is administered at a suitable dose (e.g., at a suitable volume, frequency, and / or concentration, which generally depends on the subject's weight, age, and / or condition) intended to achieve an acceptable therapeutic outcome. In certain embodiments, a therapeutically effective amount of a compound comprises one or more doses selected from the group consisting of at least 0.01 mg / kg (e.g., mg compound / kg subject weight), at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1 mg / kg, at least 10 mg / kg, or at least 100 mg / kg. In certain embodiments, the therapeutically effective amount of the compound is selected from one or more of the following doses: about 0.001 mg / kg (e.g., mg compound / kg subject body weight) to about 5000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.01 mg / kg to 500 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, 100 mg / kg to 1000 mg / kg, g, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 250 mg / kg, 0.1 mg / kg to 150 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 75 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.5 mg / kg to 5 mg / kg, amounts therebetween, and combinations thereof. In some aspects, the therapeutically effective amount of the compound administered to the subject includes about one or more of the following dosages: 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, and amounts and combinations thereof. In some embodiments, the therapeutically effective amount of a compound disclosed herein is about 0.1 mg / kg to about 50 mg / kg, about 1 mg / kg to about 50 mg / kg, or about 1 mg / kg to about 30 mg / kg.

[0136] In certain embodiments, a therapeutically effective amount of TPA is administered in a suitable dose (e.g., in a suitable volume, frequency, and / or concentration, which generally depends on the subject's weight, age, and / or condition) intended to achieve an acceptable therapeutic outcome. In some embodiments, TPA is administered in a dose of 0.1 mg / kg to 1.5 mg / kg. As disclosed herein, when administered with the compounds disclosed herein, TPA can be administered in higher doses because the compounds disclosed herein can inhibit or mitigate the adverse effects of TPA. Thus, in some embodiments, TPA is administered in a dose of 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 30 mg / kg, 0.1 mg / kg to 20 mg / kg, or 0.1 mg / kg to 5 mg / kg. In some embodiments, TPA is administered at a dose of 1.0 mg / kg to 100 mg / kg, 1.0 mg / kg to 50 mg / kg, 1.0 mg / kg to 30 mg / kg, 1.0 mg / kg to 20 mg / kg, or 1.0 mg / kg to 5 mg / kg. In some embodiments, TPA is administered at a dose of 1.5 mg / kg to 100 mg / kg, 1.5 mg / kg to 50 mg / kg, 1.5 mg / kg to 30 mg / kg, 1.5 mg / kg to 20 mg / kg, or 1.5 mg / kg to 5 mg / kg. In some embodiments, TPA is administered at a dose of about 1.6 mg / kg to 100 mg / kg, 1.8 mg / kg to 100 mg / kg, 2.0 mg / kg to 100 mg / kg, 1.6 mg / kg to 50 mg / kg, 1.8 mg / kg to 50 mg / kg, 2.0 mg / kg to 500 mg / kg, 1.6 mg / kg to 30 mg / kg, 1.8 mg / kg to 30 mg / kg, 2.0 mg / kg to 30 mg / kg, 1.6 mg / kg to 20 mg / kg, 1.8 mg / kg to 20 mg / kg, 2.0 mg / kg to 20 mg / kg, 1.6 mg / kg to 5 mg / kg, 1.8 mg / kg to 5 mg / kg, or 2.0 mg / kg to 5 mg / kg. In some embodiments, TPA is administered in combination with a compound disclosed herein. In some embodiments, TPA is administered substantially simultaneously with or prior to the administration of a compound disclosed herein. Substantially simultaneously means that TPA and the compound disclosed herein are administered within a 30 minute period. In some embodiments, TPA is administered after administration of the compound disclosed herein.In certain embodiments, TPA may be administered with a compound disclosed herein at a dose of greater than 20 mg, greater than 30 mg, greater than 40 mg, greater than 50 mg, greater than 90 mg, greater than 100 mg, or greater than 110 mg when administered as a bolus dose, or by infusion over a period of 30 minutes to 10 hours, or 30 minutes to 2 hours.

[0137] In some embodiments, administering a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition comprising a compound disclosed herein comprises administering a suitable dose at a frequency or interval required to obtain an effective therapeutic result. In some embodiments, administering a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition comprises administering a suitable dose in the following manner: every hour, every two hours, every 4 hours, every 6 hours, three times a day, twice a day, once a day, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, a combination thereof, and / or at regular or irregular intervals and / or only at the desired frequency or interval recommended by a medical professional. In some embodiments, a therapeutically effective amount of a compound or pharmaceutical composition is administered continuously, for example, by intravenous administration.

[0138] In some embodiments, the compound of therapeutically effective amount is administered to the subject before, during and / or after the subject receives thrombolytic therapy, anticoagulant therapy or intravascular interventional therapy. In some embodiments, the compound of therapeutically effective amount is administered to the subject at most 3 days before giving thrombolysis, anticoagulant or intravascular interventional therapy, at most 2 days before, at most 1 day before, at most 20 hours before, at most 15 hours before, at most 10 hours before, at most 5 hours before, at most 2 hours before or at most 1 hour before. In some embodiments, 0 to 72 hours, 0 to 48 hours, 0 to 24 hours, 0 to 12 hours, 0 to 6 hours, 0 to 4 hours or 0 to 2 hours before giving thrombolysis, anticoagulant or intravascular interventional therapy. In some embodiments, the compound of therapeutically effective amount is administered during or simultaneously during giving thrombolysis, anticoagulant or intravascular interventional therapy. In some embodiments, the therapeutically effective amount of the compound is administered intermittently or continuously up to 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, one week, 1 month, 3 months, 6 months, 12 months, 18 months, 24 months, or up to 36 months after administration of the thrombolytic, anticoagulant, or intravascular interventional therapy.

[0139] In some embodiments, a therapeutically effective amount of a compound as described herein is administered 1 hour to 1 week after administering thrombolytic therapy, or within a range thereof. In certain embodiments, a therapeutically effective amount of a compound as described herein is administered 1 to 48 hours, 2 to 48 hours, or 4 to 48 hours after administering thrombolytic therapy.

[0140] In some embodiments, thrombolytic therapy includes administering tissue plasminogen activator (TPA). TPA is typically administered within a time period of 30 seconds to 3.5 hours after the subject experiences an ischemic stroke. However, TPA administration can cause adverse, and sometimes fatal, side effects such as bleeding, vascular instability, and blood-brain barrier disruption. As disclosed herein, administration of the compounds described herein can prevent bleeding associated with TPA administration, reduce its risk, inhibit its severity, reduce, alleviate, and / or treat it. Therefore, in some embodiments, a therapeutically effective amount of a compound as described herein is administered simultaneously with TPA, within 0 to 48 hours after TPA administration, or at most 1-3 weeks after TPA administration. In some embodiments, a therapeutically effective amount of a compound as described herein is administered 1 to 24 hours, 2 to 24 hours, 3 to 24 hours, 3.5 to 24 hours, or 4 to 24 hours after TPA administration. In some embodiments, a therapeutically effective amount of a compound as described herein is administered 0 to 8 hours, 0 to 6 hours, or 0 to 4 hours after TPA administration.

[0141] In certain embodiments, administration of the compounds described herein can broaden the window for effective and safe TPA administration after stroke. For example, the benefits of TPA administration are generally considered to outweigh the risk of bleeding only if administered to a subject within 30 seconds to 3 hours after a stroke. In certain embodiments, administering the compounds described herein before, concurrently with, or after TPA administration not only expands the therapeutic window for TPA but also greatly expands the patient population that can be safely treated with TPA.

[0142] Reagent test kit

[0143] In some embodiments, provided herein are kits comprising compounds disclosed herein or pharmaceutical compositions comprising compounds disclosed herein. In some embodiments, the kit comprises one or more doses of pharmaceutical compositions comprising compounds disclosed herein. In some embodiments, the kit comprises one or more packages and / or one or more dispensing devices, which may contain one or more doses of compounds disclosed herein or pharmaceutical compositions thereof as described herein. Non-limiting examples of packaging include metal, glass or plastic containers, syringes or blister packs comprising compounds disclosed herein or compositions described herein. In certain embodiments, the kit comprises a dispensing device such as a syringe or inhaler, which may or may not contain compounds disclosed herein or compositions described herein. The packaging and / or dispenser device may be accompanied by instructions for administration. The packaging or dispenser may also be accompanied by a notice associated with the container (in a form prescribed by a government agency regulating the manufacture, use or sale of the drug), which reflects the form of the drug approved by the agency for human or veterinary administration. For example, such a notice may be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert.

[0144] In some embodiments, the kit or package contains a compound disclosed herein in an amount sufficient to treat a patient for 1 day to 1 year, 1 day to 180 days, 1 day to 120 days, 1 day to 90 days, 1 day to 60 days, 1 day to 30 days, 1-24 hours, 1-12 hours, 1-4 hours, or an amount of time therebetween.

[0145] The test kit optionally includes a product label and / or one or more packaging inserts, which provide the instructions for the description of the component or the in vitro, in vivo or ex vivo use of the component. Exemplary instructions can include instructions for treatment or therapeutic schemes. In certain embodiments, the test kit comprises packaging material, which refers to the physical structure of the test kit components. The packaging material can aseptically keep the components and can be made from the material (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, bottle, tube, etc.) that is generally used for this purpose. The product label or insert include "printed matter", such as paper or cardboard, or individually or fixed to an assembly, test kit or packaging material (e.g., box), or connected to an ampoule, tube or bottle containing the test kit components. The label or insert can additionally include a computer-readable medium, such as an optical disc such as CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, or an electrical storage medium such as RAM and ROM, or a mixture of these media such as magnetic / optical storage media, flash memory media or memory card. The product label or insert may include identification information, dosage, clinical pharmacology of the active ingredient (including mechanism of action, pharmacokinetics (PK) and pharmacodynamics (PD)) of one or more components. The product label or insert may include information identifying the manufacturer, batch number, manufacturer location, date, information about the specified condition, illness, disease or symptom for which the kit components can be used. The product label or insert may include instructions for a clinician or subject to use one or more kit components in a method, treatment regimen or therapeutic regimen. The instructions may include dosage, frequency or duration, and instructions for implementing any method, treatment regimen or therapeutic regimen described herein. The kit may additionally include labels or instructions for implementing any method described herein. The product label or insert may include potential adverse side effects and / or warning information.

[0146] Example

[0147] Example 1 - Suture-induced transient MCAO model

[0148] Male Sprague Dawley rats weighing 280-320 g were used to determine the efficacy and dose response of J147 (Compound IV) to reperfusion using a suture-type transient middle cerebral artery occlusion (MCAO) model. In this model, J147 was administered intravenously (iv) immediately after reperfusion (i.e., approximately 2 hours after the onset of ischemia) at doses of 1 mg / kg, 10 mg / kg, and 30 mg / kg of total body weight. The 10 mg / kg (iv) dose was considered the optimal effective dose in this study.

[0149] Suture-induced temporary MCAO model

[0150] SD rats: 280 to 320 g

[0151] Stroke model: suture occlusion, 2 hours of ischemia, followed by 24 hours of reperfusion

[0152] Bederson score* (tested 24 hours after reperfusion): 0 to 5, from low to high neurobehavioral deficits

[0153] J147: Administer immediately after reperfusion

[0154] J147 stock solution: 6 mg / ml, 30% HS15 / 70% saline

[0155] J147 working solution: stock solution / saline 1:1 (3 mg / ml, 15% HS15 / 85% saline)

[0156] Vehicle: 15% HS15 / 85% saline

[0157] o IV injection (bolus): 3.3 ml / kg body weight.

[0158] o ip injection (bolus): 10 ml / kg body weight

[0159] Rat: Maximum volume (iv., bolus) = 5 ml / kg. (ip., bolus) = 10 ml / kg.

[0160] After a stroke, animals exhibit a variety of neurological deficits. The Bederson scale is a global neurological assessment developed to measure neurological deficits after a stroke. Tests include forelimb flexion, resistance to lateral thrust, and rotational behavior. In this case, behavioral deficits after reperfusion are assessed using the Bederson grading scale of 0-5. This scoring scale is a simple way to show basic neurological deficits. Ischemic animals have significantly more neurological deficits than non-ischemic animals, resulting in higher scores.

[0161] The results are as follows Figure 1Figure 24 is shown in Figure 25. 24 hours after reperfusion, representative images of brain coronal sections AH stained with triphenyltetrazolium chloride (TTC) (selected from the median animal of each group) show tissue infarction (white) in the designated group. Figures A and B are control animals treated with placebo, showing Bederson scores of 3 and 5, and significant infarct volume (white) after reperfusion. Figures C and D are animals treated with 1mg / kg J147 administered intravenously (iv), showing better Bederson scores of 3 and 3, and non-significant reduction relative to the infarct volume of placebo. Figures E and F are animals treated with 10mg / kg J147 administered intravenously, showing a great improvement of Bederson scores of 1 and 2, and a significant reduction in infarct volume after reperfusion. Figures G and H are animals treated with intraperitoneal administration of J147, showing that Bederson scores were 2 and 3, and that infarct volume moderately reduced after reperfusion.

[0162] Example 2 - Embolic MCAO Model

[0163] For the embolic MCAO model (eMCAO), a single 4 μm fibrin-rich clot was placed at the origin of the right MCA via modified PE-50 tubing (0.3 mm outer diameter). Regional cerebral blood flow (rCBF) within the MCA territory (2 mm posterior and 5 mm lateral to the right superior parietal bregma) was monitored using laser Doppler flowmetry (MSP300XP; ADInstruments Inc). Animals were included in the study if their rCBF decreased to 25% or less of baseline.

[0164] Combination therapy: J147 plus tPA, tissue plasminogen activator, the only FDA-approved drug for the treatment of stroke.

[0165] SD rats: 280 to 320 g

[0166] Stroke model: eMCAO, 4μm blood clot

[0167] Bederson scale (tested 24 hours after ischemia): 0 to 5

[0168] J147 (10 mg / kg: 5% DMSO, 70% PEG200, and 25% saline): 4 hours after ischemic onset (intravenous infusion for 1 minute)

[0169] tPA (10 mg / kg (2 mg / ml)): 4 hours after the onset of ischemia, 10% bolus injection over 1 minute, 90% infusion over 30 minutes.

[0170] Two groups: tPA-only and tPA+J147-treated

[0171] The results are shown in Figure 2(animals treated with tPA only) and Figure 3 (Animals treated with tPA+J147.) Representative images of unstained coronal sections (A, C, E, G) showing intracerebral hemorrhage (red) and TTC-stained coronal sections (B, D, F, H) showing tissue infarction (white) are shown in the indicated groups 24 hours after stroke (selected from the median animal in each group). Figure 4A Shown is a graph of Bederson scores showing a positive trend in tPA+J147-treated animals versus tPA-only-treated animals (p=0.6254). Figure 4B Graphical representation showing scan-quantified infarct volume showing a positive trend in tPA+J147-treated animals (p=0.061). Figure 4C Graph showing quantification of bleeding volume, demonstrating significant improvement in tPA+J147 treated animals (p=0.048).

[0172] Example 3 - J147 Dose Response (Extension Study)

[0173] Objective: This study was conducted to determine the dose-dependent therapeutic effect of J147 and to identify the optimal dose.

[0174] J147 stock solution: 6 mg / ml in 30% HS15 / 70% saline

[0175] J147 working solution: 3 mg / ml in 15% HS15 / 85% saline (stock solution / saline 1:1)

[0176] Vehicle: 15% HS15 / 85% saline

[0177] Male SD rats; n = 7-8 / group

[0178] Methods: Male SD rats (280-300 g) were subjected to 2 hours of suture-induced MCAO ischemia followed by reperfusion. J147 compound (1.0, 10, 30 mg / kg) and vehicle ( FIG5 ) were administered 2 hours after the onset of ischemia. Bederson scores were examined before sacrifice 72 hours after the stroke. Infarct volumes were measured in TTC-stained coronal brain sections. Results: J147 treatment significantly reduced infarct volume and neurological deficits after reperfusion in a dose-dependent manner. A dose of 10 mg / kg J147 was selected as the "optimal effective dose" for the treatment of acute ischemic stroke in rats.

[0179] Example 4 - J147 + tPA Extension Study (approximately 80 animals)

[0180] Objective: To determine whether J147 can prevent tPA-associated intracerebral hemorrhage and prolong the therapeutic window of tPA in a clinically relevant rat thromboembolic stroke model.

[0181] • J147 compound: Stock solution: 60 mg / ml DMSO, in 60 ul / vial, stored at -20°C.

[0182] Working solution: 3 mg / ml, in 5% DMSO, 70% PEG200 and 25% saline, prepared just before injection.

[0183] • Warm one vial of stock solution at 37°C and mix with 1140ul of sterile solution (PEG200:saline, 2.8:1).

[0184] Recombinant human tPA (alteplase, 2.2 mg / vial): Reconstitute with 1.1 ml of water (2 mg / ml). 10 mg / kg, 10% bolus injection, 90% over 30 minutes. Via a minipump.

[0185] A. Infarct volume and intracerebral hemorrhage

[0186] Methods: Male Sprague-Dawley rats (280-300 g) underwent thromboembolic middle cerebral artery occlusion (MCAO) and were treated with saline 4 hours after the onset of ischemia, tPA (tissue plasminogen activator, 10 mg / kg, IV) 4 hours after the onset of ischemia, and J147 (10 mg / kg, IV) plus tPA 4 hours after the onset of ischemia. Bederson scores were assessed before sacrifice 72 hours after stroke. Infarct volume, cerebral hemorrhage, and animal mortality were measured.

[0187] Results: 4 hours of tPA treatment alone did not reduce cerebral infarction but worsened hemorrhagic transformation. Combination of J147 with tPA reduced infarct volume and improved cerebral hemorrhage ( Figures 6A-6D The mortality rates in the saline and tPA alone treatment groups were 25% (3 of 12 rats) and 32% (7 of 22 rats), respectively. The mortality rate in the combination treatment group was reduced to 12% (Table 1).

[0188] Conclusions: Combination treatment with J147 plus tPA attenuated tPA-associated bleeding and reduced brain injury after eMCAO. Thus, the combination of J147 and tPA may extend the therapeutic window for tPA administration after an ischemic event, thereby increasing the number of stroke patients who could benefit from tPA use.

[0189] Table 1 Mortality rate

[0190]

[0191] *p<0.05, compared with saline or tPA alone group, log-rank test

[0192] B. Immunohistochemistry

[0193] MMPs, particularly MMP-9, play a key role in stroke-associated blood-brain barrier (BBB) disruption, hemorrhagic transformation, and neuroinflammation following stroke. Double immunohistochemistry was performed to determine the expression of MMP-9 in the brain 24 hours after stroke ( Figure 7 A).

[0194] Methods: At the specified time point, animals were euthanized with CO2 and perfused through the heart with 200mL of ice-cold PBS (0.01M, pH 7.4), followed by perfusion with 4% paraformaldehyde (PFA). The brain was removed and post-fixed overnight at 4°C in a 4% PFA solution. Brain samples were then cryopreserved by immersing them in 10%, 20%, and 30% gradient sucrose solutions at 4°C for 48 hours. After this, the brain was embedded in an optimal cutting temperature (OCT) compound. Coronal sections (15 μm) were cut and fixed on positively charged slides using a cryostat at 0.4-1.4 mm behind the bregma. Twenty coronal sections of a total of 5 sections were used for immunohistochemistry. Primary antibodies: IBa-1 (1:200, Wako), MPO (1:200, Abcam), MMP9 (1:100, R&D), EBA (endothelial barrier antigen, vascular endothelial marker, 1:500, Covance), fibrinogen (1:200, Dako), platelets (1:200, Lifespan), and PAI-1 (1:200, Novus Biologicals) were used. Isotype-matched antibodies were used as negative controls. Immunostaining was performed by conventional methods.

[0195] For quantitative analysis, the number of immunoreactive blood vessels was counted in the ischemic border area. All immunostaining data were analyzed by Image Pro plus software (version 5.1, Media Cybernetics, Inc.) blinded to the experimental group, and the data were expressed as the density of immunoreactive blood vessels relative to the imaging area (mm2). Data were expressed as mean ± standard error of the mean (SEM). GraphPad Prism 8 software package was used for statistical analysis. Unless otherwise stated, multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test. If only two groups were compared, unpaired two-tailed Student's t-test was applied. p < 0.05 was considered statistically significant.

[0196] Results: MMP-9 immunoreactivity was absent in sham-operated rats, whereas it was significantly increased in stroke animals. In the delayed tPA treatment group, it was further increased, present on single cells and brain microvessels (marked by endothelial barrier antigen staining). However, combined treatment with J147 significantly reduced MMP9 expression on single cells and microvessels. We also evaluated neutrophil (MPO) infiltration and microglial (Iba-1) expansion in the brain, both of which are involved in post-ischemic neuroinflammation. Immunostaining showed that no MPO-positive cells were detected in sham-operated rats, but they were significantly induced by stroke and further enhanced by delayed tPA treatment ( Figure 7 B). Combination treatment with J147 significantly reduced neutrophil infiltration into the brain parenchyma after stroke. In addition, the number of Iba-1 positive cells increased significantly after stroke ( Figure 7 C). However, combined treatment with J147 significantly blocked microglial proliferation; in contrast, delayed tPA alone showed no significant effect on microglial expansion in the saline group. In both the saline and tPA groups, the majority of Iba-1-positive cells transformed from highly branched to less branched or amorphous cells, indicating microglial activation. Importantly, this was significantly inhibited by combined treatment with J147.

[0197] Conclusions: J147 treatment attenuates ischemia-induced and delayed tPA-enhanced neuroinflammation.

[0198] After ischemic stroke, intravascular fibrin / fibrinogen deposition and platelet aggregation essentially lead to secondary microvascular thrombosis. Double immunofluorescence staining showed that intravascular fibrin / fibrinogen deposition was rarely detected in sham-operated rats ( Figure 8 A) and platelet accumulation ( Figure 8 B), whereas relatively low levels were detected in saline-treated stroke animals, which may be due to low cerebral perfusion in downstream microvasculature without tPA thrombolysis in this embolic stroke model. However, fibrin / fibrinogen and platelet deposition in downstream microvasculature were significantly increased in stroke rats with delayed tPA, and these increases were significantly attenuated by combined treatment with J147 ( Figure 8 A and 8B).

[0199] Results: Evidence suggests that local upregulation of PAI-1 (plasminogen activator inhibitor-1), a downstream mediator in the NF-κB cascade, in the ischemic brain endothelium contributes to intravascular fibrin / fibrinogen deposition during ischemia / reperfusion-induced acute cerebral ischemic injury. Double immunohistochemistry revealed that combined treatment with J147 significantly reduced ischemia-induced and delayed tPA-enhanced PAI-1 expression in brain endothelial cells observed in the peri-ischemic region of the cerebral cortex ( Figure 8C).

[0200] Conclusions: J147 treatment attenuates delayed tPA-enhanced microvascular thrombosis and thereby prolongs the duration of the therapeutic window when tPA is administered after an ischemic event.

[0201] C. Platelet Activation and Platelet-Leukocyte Interactions

[0202] Elevated platelet activation and platelet-leukocyte interactions contribute significantly to proinflammatory and thrombotic events during ischemia-reperfusion injury after stroke. We used flow cytometry to analyze platelet activation and platelet-leukocyte aggregation in whole blood 24 hours after ischemia onset ( Figure 9 A).

[0203] Methods: 24 hours after stroke, blood was drawn into heparinized capillaries via the retroorbital plexus. Whole blood was diluted 1:10 in flow cytometry staining buffer containing rat Fc-blocking anti-rat CD32 (eBioscience). To assess platelet activation, samples were co-stained with FITC mouse anti-rat CD42d (1:200, RPM.4) for platelets and PE / Cy7 mouse anti-rat CD62P (1:100, RMP-1) for P-selectin expression. To detect platelet-leukocyte aggregate (PLA) formation, samples were co-stained with FITC mouse anti-rat CD42d (1:200, RPM.4), PE mouse anti-rat granulocyte (1:100, RP-1), APC mouse anti-rat CD11b (1:50, WT.5), and PE / Cy7 mouse anti-rat CD45 (1:100, OX-1). Isotype-matched control antibodies are used to distinguish nonspecific background signals from specific antibody signals. TM Samples were analyzed on a C6 flow cytometer.

[0204] Results: Increased platelet activation (as measured by P-selectin expression) and platelet-granulocyte aggregation were observed in saline-treated stroke rats, but IV tPA had no additional effect. However, combined treatment significantly reduced ischemia-induced platelet P-selectin expression and platelet-granulocyte aggregation. Platelet-monocyte aggregates did not change significantly between groups.

[0205] Conclusion: J147 treatment attenuates circulating platelet activation and platelet-neutrophil aggregation in vivo.

[0206] Example 5-J147 alone and embolic MCAO (eMCAO) model

[0207] In this example, eMCAO experiments were performed to determine whether J147 alone (ie, without administration of TPA or other thrombolytic interventions) provides any therapeutic benefit in stroke in the absence of reperfusion induced by TPA.

[0208] SD rats: 280 to 320 g

[0209] Stroke model: eMCAO, 4μm blood clot

[0210] Bederson scale (tested 24 hours after ischemia): 0 to 5

[0211] No tPA treatment.

[0212] J147 was administered 2 hours after stroke onset at 0 mg / ml (vehicle), 1 mg / ml, or 10 mg / ml iv or 30 mg / ml ip. (n=5 / group)

[0213] J147 stock solution: 6 mg / ml (30% HS15 / 70% saline)

[0214] J147 working solution: stock solution / saline 1:1 (3 mg / ml, 15% HS15 / 85% saline)

[0215] Vehicle: 15% HS15 / 85% saline

[0216] In this example, animals were treated with vehicle or J147 in the absence of reperfusion or TPA treatment. Figure 10A and 10B As shown. Representative images of TTC-stained coronal sections showing tissue infarction (white) 24 hours after stroke in the designated groups are shown (selected from the median animal in each group). These experiments show that in the absence of reperfusion (e.g., reperfusion induced by TPA), J147 alone does not provide any significant protection against stroke. In contrast, the previous examples demonstrated the therapeutic effect of administering J147 in combination with TPA.

[0217] Example 6 - Certain non-limiting embodiments

[0218] A1. A method for preventing, reducing the risk of, inhibiting, reducing, alleviating or treating bleeding or vascular instability in a subject, comprising administering to the subject a therapeutically effective amount of a compound having the structure of Formula I:

[0219]

[0220] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof,

[0221] in:

[0222] R 2 selected from H and methyl;

[0223] R 3 is trifluoromethyl or other fluorine-substituted alkyl;

[0224] L 3 is a carbonyl group; and

[0225] R 6 is independently selected at each occurrence from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, hydroxy, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy, mercapto, alkylthio, arylthio, carbonyl, aryl, substituted aryl, substituted heterocyclyl, halogen, cyano, cyanoalkyl, nitro, amino, amidino, carbamate, S(O) n R 7 and C(O)R 8 , or two R6 at adjacent positions combine to form an optionally substituted heteroaryl or heteroalkyl ring fused to the adjacent phenyl moiety;

[0226] R 7 It is H, R 9 、NH2、HNR 9 or NR 9 R 10 ;

[0227] R 8 It's OH, OR 9 、NH2、NHR 9 or NR 9 R 10 ;

[0228] R 9 and R 10 is independently at each occurrence optionally substituted alkyl; and

[0229] n=1 or 2.

[0230] A2. The method of embodiment A1, wherein R 6 is selected at each occurrence from alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, halogen, and C(O)R 8 .

[0231] A3. The method of embodiment A2, wherein R 6 is selected at each occurrence from methyl, methoxy, perfluoromethyl, perfluoromethoxy, hydroxy, Cl, F, and I.

[0232] A4. The method of embodiment A1, wherein the compound has the structure of Formula II:

[0233]

[0234] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein:

[0235] (i)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is a methyl group, and R B4 is methyl; or

[0236] (ii)R A2 、R A3 、R A5 and R A6 It's H, R A4 is methoxy, R B2 is a methyl group, and R B4 is methyl; or

[0237] (iii)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is H, and R B4 is H; or

[0238] (iv)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is a methyl group, and R B4 is methyl; or

[0239] (v)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is H, and R B4 is H; or

[0240] (vi)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is H, and R B4 is methyl; or

[0241] (vii)R A2 、RA4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is H, and R B4 is methyl; or

[0242] (viii)R A2 、R A3 、R A4 、R A5 and R A6 It's H, R B2 is a methyl group, and R B4 is H; or

[0243] (ix)R A2 、R A4 、R A5 and R A6 It's H, R A3 is methoxy, R B2 is a methyl group, and R B4 is H; or

[0244] (x)R A2 、R A3 、R A5 and R A6 It's H, R A4 is COOH, R B2 is a methyl group, and R B4 is methyl; or

[0245] (xi)R A2 、R A4 and R A5 It's H, R A3 and R A6 is hydroxyl, R B2 is a methyl group, and R B4 is methyl; or

[0246] (xii)R A2 、R A4 and R A6 It's H, R A3 and R A5 is hydroxyl, R B2 is a methyl group, and R B4 is methyl; or

[0247] (xiii)R A2 、R A4 and R A5 It's H, R A3 is methoxy, R A6 It's F, R B2 is H, and R B4 is Cl; or

[0248] (xiv)R A3 and R A5 It's H, R A2 and R A6 It's F, R A4 is hydroxyl, R A6 It's F, R B2 is H, and R B4 is F; or

[0249] (xv)R A2 、R A4 and R A6 It's H, R A3 is hydroxyl, R A5 It's F, R B2 is H, and R B4 is F; or

[0250] (xvi)R A2 、R A5 and R A6 It's H, R A3 and R A4 Together they are -O-CH2-O-, R A5 It's F, R B2 is H, and R B4 It's F.

[0251] A5. The method of embodiment A1, wherein the compound has the structure of formula III

[0252]

[0253] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein R1 is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, dibromomethyl or tribromomethyl; R2 is methyl, methoxy, hydroxyl, halogen, CF3, OCH3, OCF3 or OCBr3; and R3 and R4 are independently selected from hydrogen, hydroxyl, halogen (e.g., Cl, F or Br), methyl, methoxy and amine.

[0254] A6.1. The method of embodiment A5, wherein the compound has the structure of Formula IV,

[0255]

[0256] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof.

[0257] A6.2. The method of any one of embodiments A1 to A6.1, wherein the bleeding or vascular instability is induced by thrombolytic therapy, anticoagulant therapy, or endovascular interventional therapy including mechanical thrombectomy.

[0258] A7. The method of any one of embodiments A1 to A6.2, wherein the bleeding comprises internal bleeding.

[0259] A7.1. The method of any one of embodiments A1 to A7, wherein the bleeding comprises Category I bleeding.

[0260] A7.2. The method of any one of embodiments A1 to A7.1, wherein the bleeding comprises an estimated blood loss of equal to or less than 15%, 10%, 5%, 2%, or equal to or less than 1% of total blood volume.

[0261] A8. The method of any one of embodiments A1 to A7.2, wherein the bleeding comprises hemorrhagic transformation.

[0262] A8.1. The method of any one of embodiments A1 to A8, wherein the bleeding comprises a cerebral hemorrhage or intracerebral hemorrhage.

[0263] A8.2. The method of any one of embodiments A1 to A8.1, wherein said inhibiting, reducing, alleviating or treating bleeding comprises reducing the amount of bleeding.

[0264] A9. The method of any one of embodiments A1 to A8.2, wherein the vascular instability comprises endothelial dysfunction.

[0265] A10. The method of any one of embodiments A1 to A9, wherein the vascular instability comprises a decrease or loss of vascular tone or hemostasis.

[0266] A11. The method of any one of embodiments A1 to A10, wherein the vascular instability comprises edema.

[0267] A12. The method of any one of embodiments A1 to A11, wherein the edema comprises cerebral edema or blood-brain barrier swelling.

[0268] A13. The method of any one of embodiments A1 to A12, wherein the vascular instability comprises damage to the vascular endothelium or the blood-brain barrier due to reperfusion.

[0269] A14. The method of any one of embodiments A1 to A13, comprising protecting and / or stabilizing the vascular endothelium in the subject.

[0270] A15. The method of embodiment A14, wherein the vascular endothelium comprises the blood-brain barrier.

[0271] A16. The method of any one of embodiments A1 to A15, comprising preventing or treating a breakdown of the blood-brain barrier in the subject.

[0272] A17. The method of any one of embodiments A1 to A16, wherein the subject is scheduled to receive, is currently receiving, or has received thrombolytic therapy or anticoagulant therapy.

[0273] A18. The method of any one of embodiments A1 to A17, wherein the subject is at risk of bleeding.

[0274] A19. The method of any one of embodiments A6.2 to A18, wherein the thrombolytic therapy comprises administering tissue plasminogen activator (TPA), streptokinase, a streptokinase activator, or urokinase.

[0275] A20. The method of embodiment A19, wherein the streptokinase activator is an anisoylated plasminogen streptokinase activator complex.

[0276] A21. The method of embodiment A20, wherein the anisoylated plasminogen streptokinase activator complex is anistreplase or eminase.

[0277] A22. The method of embodiment A19, wherein the urokinase is urokinase-type plasminogen activator.

[0278] A23. The method of embodiment A22, wherein the urokinase-type plasminogen activator is saruplase.

[0279] A24. The method of embodiment A19, wherein the tissue plasminogen activator is recombinant TPA.

[0280] A25. The method of embodiment A19 or A23, wherein the tissue plasminogen activator is alteplase (Activase), reteplase (Retavase) or tenecteplase (TNKase, Metalyse).

[0281] A26. The method of any one of embodiments A6.2 to A25, wherein the anticoagulation therapy comprises administration of a vitamin K antagonist.

[0282] A27. The method of embodiment 26, wherein the vitamin K antagonist is warfarin.

[0283] A27.1. The method of any one of embodiments A6.2 to A26, wherein the anticoagulation therapy comprises administration of heparin, a derivative thereof, or a low molecular weight heparin.

[0284] A28. The method of embodiment A27.1, wherein the heparin derivative or low molecular weight heparin is selected from enoxaparin, dalteparin, tinzaparin and danaparin.

[0285] A29. The method of any one of embodiments A6.2 to A28, wherein the anticoagulant therapy comprises administering a thrombin inhibitor.

[0286] A30. The method of embodiment A29, wherein the thrombin inhibitor is selected from the group consisting of bivalirudin, argatroban, dabigatran, desirudin, lepirudin, and antithrombin III.

[0287] A31. The method of any one of embodiments A6.2 to A30, wherein the anticoagulation therapy comprises administering a Factor Xa inhibitor.

[0288] A32. The method of embodiment A31, wherein the factor Xa inhibitor is selected from apixaban, fondaparinux, rivaroxaban, edoxaban, and betrixaban.

[0289] A33. The method of any one of embodiments A1 to A32, wherein the method further comprises administering thrombolytic therapy, anticoagulant therapy, or intravascular interventional therapy to the subject.

[0290] A34. The method of any one of embodiments A1 to A33, wherein the subject is a mammal or a human.

[0291] A35. A pharmaceutical composition comprising a compound comprising a structure selected from any one of Formula I, Formula II, Formula III, and Formula IV, for performing the method of any one of Embodiments A1 to A34.

[0292] B1. A method for preventing reperfusion injury, reducing the risk of reperfusion injury, inhibiting, reducing, alleviating or treating reperfusion injury in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound having the structure of Formula III:

[0293]

[0294] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof, wherein R1 is methyl, fluoromethyl, difluoromethyl, trifluoromethyl, bromomethyl, dibromomethyl or tribromomethyl; R2 is methyl, methoxy, hydroxyl, halogen, CF3, OCH3, OCF3 or OCBr3; and R3 and R4 are independently selected from hydrogen, hydroxyl, halogen (e.g., Cl, F or Br), methyl, methoxy and amine.

[0295] B2. The method of embodiment B1, wherein the compound comprises or consists of a structure of formula IV;

[0296]

[0297] or a pharmaceutically acceptable salt, stereoisomer or tautomer thereof.

[0298] B3. The method of embodiment B1 or B2, wherein the reperfusion injury is induced by thrombolytic therapy or anticoagulant therapy.

[0299] B4. The method of embodiment B1 or B2, wherein the reperfusion injury is induced by intravascular interventional therapy.

[0300] B5. The method of embodiment B4, wherein the intravascular intervention comprises mechanical thrombectomy.

[0301] B6. The method of any one of embodiments B1 to B5, wherein the reperfusion injury comprises or consists of ischemia-reperfusion injury.

[0302] B6.1. The method of any one of embodiments B1 to B6, wherein the reperfusion injury comprises or consists of cerebral reperfusion injury, liver reperfusion injury, or myocardial reperfusion injury.

[0303] B7. The method of any one of embodiments B1 to B6.1, comprising protecting and / or stabilizing the vascular endothelium in the subject.

[0304] B8. The method of embodiment B7, wherein the vascular endothelium comprises the blood-brain barrier.

[0305] B9. The method of any one of embodiments B1 to B8, comprising preventing or treating a breakdown of the blood-brain barrier in the subject.

[0306] B10. The method of any one of embodiments B1 to B9, wherein the subject is scheduled to receive, is currently receiving, or has received thrombolytic therapy or anticoagulant therapy.

[0307] B11. The method of any one of embodiments B1 to B10, wherein the subject is at risk of bleeding.

[0308] B12. The method of any one of embodiments B1 to B11, wherein the bleeding comprises internal bleeding.

[0309] B13. The method of any one of embodiments B1 to B12, wherein the bleeding comprises hemorrhagic transformation.

[0310] B14. The method of any one of embodiments B1 to B13, wherein the bleeding comprises bleeding in the brain.

[0311] B15. The method of any one of embodiments B1 to B14, wherein the method comprises reducing bleeding volume.

[0312] B16. The method of any one of embodiments B3 to B15, wherein the thrombolytic therapy comprises administering tissue plasminogen activator (TPA), streptokinase, a streptokinase activator, or urokinase.

[0313] B17. The method of embodiment B16, wherein the streptokinase activator is an anisoylated plasminogen streptokinase activator complex.

[0314] B18. The method of embodiment B17, wherein the anisoylated plasminogen streptokinase activator complex is anistreplase or eminase.

[0315] B19. The method of embodiment B16, wherein the urokinase is urokinase-type plasminogen activator.

[0316] B20. The method of embodiment B19, wherein the urokinase-type plasminogen activator is saruplase.

[0317] B21. The method of embodiment B16, wherein the tissue plasminogen activator is recombinant TPA.

[0318] B22. The method of embodiment B16 or B21, wherein the tissue plasminogen activator is alteplase (Activase), reteplase (Retavase) or tenecteplase (TNKase, Metalyse).

[0319] B23. The method of any one of embodiments B1 to B22, wherein the anticoagulant therapy comprises administering a vitamin K antagonist.

[0320] B24. The method of embodiment B23, wherein the vitamin K antagonist is warfarin.

[0321] B25. The method of any one of embodiments B1 to B24, wherein the anticoagulation therapy comprises the administration of heparin, a derivative thereof, or a low molecular weight heparin.

[0322] B26. The method of embodiment B25, wherein the heparin derivative or low molecular weight heparin is selected from enoxaparin, dalteparin, tinzaparin and danaparin.

[0323] B27. The method of any one of embodiments B1 to B26, wherein the anticoagulant therapy comprises administering a thrombin inhibitor.

[0324] B28. The method of embodiment B27, wherein the thrombin inhibitor is selected from the group consisting of bivalirudin, argatroban, dabigatran, desirudin, lepirudin, and antithrombin III.

[0325] B29. The method of any one of embodiments B1 to B28, wherein the anticoagulation therapy comprises administering a Factor Xa inhibitor.

[0326] B30. The method of embodiment B29, wherein the Factor Xa inhibitor is selected from the group consisting of apixaban, fondaparinux, rivaroxaban, edoxaban, and betrixaban.

[0327] B31. The method of any one of embodiments B1 to B30, wherein the method further comprises administering thrombolytic therapy, anticoagulant therapy, or intravascular interventional therapy to the subject.

[0328] B32. The method of any one of embodiments B1 to B31, wherein the subject is a human.

[0329] B33. A compound comprising the structures of Formula III and Formula IV for use in implementing the method of any one of Embodiments B1 to B32.

[0330] Each patent, patent application, publication, or any other reference or document cited herein is hereby incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control.

[0331] The citation of any patent, patent application, publication or any other document is not an admission that any of the foregoing is relevant prior art nor does it constitute any admission as to the contents or date of such publication or document.

[0332] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0333] All features disclosed herein can be combined in any combination. Each feature disclosed in the specification can be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, disclosed features (e.g., antibodies) are examples of families of equivalent or similar features.

[0334] Unless expressly stated otherwise, the phrase "induced by" encompasses "exacerbated by," "aggravated by," "exacerbated by," and / or "amplified by."

[0335] As used herein, unless the context clearly indicates otherwise, all numerical values or numerical ranges include integers within such ranges and fractions of values or integers within the range. In addition, when a numerical list (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) is described herein, the list includes all intermediate values and fractional values (e.g., 54%, 85.4%) thereof. Thus, for illustration, reference to 80% or higher identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., and 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so on.

[0336] References to integers higher (greater than) or lower than include any number higher or lower than the cited number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., down to the number one (1); and reference to less than 10 includes 9, 8, 7, etc., down to the number one (1).

[0337] As used herein, unless the context clearly indicates otherwise, all values or ranges include fractions of these values and integers within these ranges and fractions of integers within these ranges. Thus, for illustration, reference to a numerical range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so on. Thus, reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so on.

[0338] Reference to a series of ranges includes combining ranges that combine the boundaries of different ranges within the series. Thus, for illustration, reference to a series of ranges, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,000, 500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000, including ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

[0339] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments specifically disclosed in this application and that such modifications and improvements are within the scope and spirit of the present technology.

[0340] This article uses affirmative language to describe numerous embodiments and aspects, and generally discloses the present invention. The present invention also specifically includes embodiments that exclude, in whole or in part, specific themes, such as substances or materials, method steps and conditions, schemes or procedures. For example, in some embodiments or aspects of the methods disclosed herein, some materials and / or method steps are excluded. Therefore, although the present invention is generally not described herein with respect to what is not included in the present invention, aspects of the present invention that are not explicitly excluded are still disclosed herein.

[0341] Some embodiments of the technology described herein can be suitably practiced in the absence of elements not specifically disclosed herein. Thus, in some embodiments, the term "comprising" or "including" can be replaced with "consisting essentially of" or "consisting of" or grammatical variations thereof. A composition "consisting essentially of" means that it contains only the claimed active ingredient (e.g., an active ingredient (AI) or active pharmaceutical ingredient (API); for example, TPA and / or a compound described and claimed herein); the composition may include other ingredients, such as formulation materials, excipients, additives, carriers, preservatives, diluents, solvents, fillers, salts, buffers, coatings, binders, and lubricants; and the composition excludes other APIs that are not claimed.

[0342] The term "one / a kind of" can refer to one or more (for example, "reagent" can mean one or more reagents) of the element it modifies, unless the context clearly describes one / a kind of or more than one / a kind of element in the element. As used herein, the term "about" refers to a value within 10% of the basic parameter (that is, plus or minus 10%), and the term "about" is used to modify each value at the beginning of the value string (that is, "about 1, 2 and 3" refers to about 1, about 2 and about 3). For example, the weight of "about 100 grams" can include the weight between 90 grams and 110 grams. As used herein, the term "substantially" refers to a value modifier, meaning "at least 95%", "at least 96%", "at least 97%", "at least 98%" or "at least 99%" and can include 100%. For example, a composition substantially free of X can include less than 5%, less than 4%, less than 3%, less than 2% or less than 1% X, and / or X can not exist or can not be detected in the composition.

Claims

1. Use of a therapeutically effective amount of a compound having the structure of Formula IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, reducing the risk of, inhibiting, reducing, alleviating or treating bleeding in the brain in a subject suffering from ischemic stroke, wherein the bleeding is induced by thrombolytic therapy comprising administering tissue plasminogen activator (TPA):

2. The method according to claim 1, wherein the bleeding is also induced by intravascular interventional therapy.

3. The use according to claim 2, wherein the intravascular intervention comprises mechanical thrombectomy.

4. Use of a therapeutically effective amount of a compound having the structure of Formula IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, reducing the risk of, inhibiting, reducing, alleviating or treating edema in the brain in a subject suffering from ischemic stroke, wherein the edema is induced by thrombolytic therapy comprising administering tissue plasminogen activator (TPA):

5. Use of a therapeutically effective amount of a compound having a structure of Formula IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for protecting the blood-brain barrier of a subject suffering from ischemic stroke:

6. Use of a therapeutically effective amount of a compound having the structure of Formula IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for protecting vascular endothelium in a subject suffering from ischemic stroke:

7. The method according to any one of claims 5 to 6, wherein the subject is planning to receive, is currently receiving or has received thrombolytic therapy or anticoagulant therapy.

8. The use of claim 7, wherein the subject is at risk of hemorrhage in the brain.

9. The method of claim 8, wherein the bleeding comprises hemorrhagic transformation.

10. The use according to any one of claims 1 to 6, wherein the medicament is for reducing bleeding volume.

11. The use according to any one of claims 1 to 4, wherein the tissue plasminogen activator is recombinant TPA.

12. The use according to any one of claims 1 to 4, wherein the tissue plasminogen activator is alteplase (Activase), reteplase (Retavase) or tenecteplase (TNKase, Metalyse).

13. The method of any one of claims 1 or 4, wherein the subject is also administered anticoagulation therapy or intravascular intervention therapy.

14. The use of any one of claims 1 to 4, wherein the subject is a human.

15. The use of any one of claims 1 to 4, wherein the subject has or is suspected of having a blood clot.

16. The use of claim 1, wherein a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof is administered in combination with a therapeutically effective amount of a second compound capable of treating or preventing reperfusion injury.

17. The use according to claim 16, wherein the second compound is cyclosporin A or a pharmaceutically acceptable salt thereof.

18. The use of any one of claims 16 to 17, wherein the therapeutically effective amount of the second compound administered is lower than the dose required to achieve a comparable therapeutic effect when the second compound is used alone.

19. The use of claim 18, wherein one or more of the side effects associated with administration of the compound or a pharmaceutically acceptable salt thereof together with the second compound are reduced compared to such side effects associated with administration of the second compound alone to achieve a comparable therapeutic effect.

20. Use of a therapeutically effective amount of a compound having the structure of Formula IV or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, reducing the risk of, inhibiting, reducing, alleviating or treating hemorrhage or reperfusion injury in the brain in a subject, wherein the subject suffers from ischemic stroke and is administered tissue plasminogen activator (TPA):

21. The use of claim 20, wherein the compound is administered before, during or after administration of TPA.

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