Fluorescent compounds for imaging of blood vessels and blood flow, and an in vivo screen for pro- and anti-angiogenic agents

Abstract

The present invention discloses fluorescent compounds and a method for their use for selective imaging of blood vessels and blood flow. By applying these fluorescent compounds and the imaging process to a zebrafish model, the present invention further provides methods and procedures for the discovery, selection, and characterization of pro- and anti-angiogenic agents.

Claims

1. A method of visualizing and imaging blood vessels or blood flow in a test subject wherein the test subject is a selected part or the entirety of a dead or living animal, including a human, comprising; a) administering fluorescent compounds to the test subject to achieve inclusion of the fluorescent compounds in the test subject's blood vessels and/or blood serum wherein the fluorescent compounds are selected from; 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, 4-(3-(7-(3-Bromopropoxy)-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, (11-((4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoyl)oxy)undecyl)triphenylphosphonium, Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate, 5-Chloropentyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4, 5-dihydro-1H-pyrazol-1-yl)benzoate, (3R,4R, 5S,6R)-6-(Acetoxymethyl)-3-(4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate, 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoic acid, (3-((3-(1-(4-Carboxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)oxy)propyl)triphenylphosphonium, Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoate, 2,5-Dioxopyrrolidin-1-yl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate, 4-(3-(6-Methoxy-2-oxo-2H-chrornen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-N-((3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)benzamide, 3-(1-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-methoxy-2H-chromen-2-one, (3-((4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoyl)oxy)propyl)triphenylphosphonium, (Z)-5((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzamido)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide, Diethyl 4,4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazole-1,5-diyl)dibenzoate, 3-(2-(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)hydrazinyl)propyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate hydrochloride, 3-Bromopropyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4, 5-dihydro-1H-pyrazol-5-yl)benzoate, (Z)-3-((2-(5-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)ethyl)amino)propyl 4-(3-(6-methoxy-2-oxo-2H-chrornen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate hydrochloride, 4-(5-(4-(Ethoxycarbonyl)phenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4, 5-dihydro-1H-pyrazol-1-yl)benzoic acid, 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-N-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)benzohydrazide, Ethyl 4-(5-(3,4-dimethoxyphenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzoate, and Ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylate; and pharmaceutically acceptable salts, solvates, enantiomers, non-covalent complexes, and mixtures thereof, or pharmaceutical formulations containing the compound; b) irradiating an observation area of choice in the animal with a light source to excite the included fluorescent compounds; c) revealing of blood vessels, or motion of blood flow in the animal when the fluorescent compounds emit fluorescence, and; d) observing and recording fluorescent images of the blood vessels or blood flow with a microscope or a camera.

2. A method of claim 1 wherein the test subject is a zebrafish.

3. A method to identify, select, characterize, or collectively screen for substances that inhibit or promote blood vessel formation or angiogenesis in living, wild type or genetically altered zebrafish, comprising; a) exposing a test zebrafish to a selected substance or mixture of substances under a pre-determined set of conditions appropriate for the interaction between the substance(s) and a zebrafish, followed by; b) subjecting the zebrafish to the method in claim 2, and; c) examining the resulting images of blood vessels or blood flow to determine the effect on zebrafish blood vessel formation, or angiogenesis, caused by the selected substance(s).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Fluorescent compounds of this invention are vital and can be systematically cleared by the test animal after administration. After staining with ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate (10 M) for 1 hour, continuous culture of tested zebrafish in RO water allows the dye to clear away. Pictures show incremental fading of green fluorescence over time within the first 6 hours.

(2) FIG. 2: Confirmation of the anti-angiogenic effect of Sunitinib using the method of this invention. This figure contains microscopic images of zebrafish, treated with Sunitinib followed by staining with 4-(5-(4-(ethoxycarbonyl)phenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (20 M), showing increasing deficiency of vascular development upon treatment with higher Sunitinib dose. (a) control; (b) 1 M Sunitinib; (c) 2.5 M Sunitinib; (d) 5 M Sunitinib. Whole fish images were made by combining three segmental images and the scale bar is 100 m. In each experiment, (a) to (c), four pictures are shown, from top to bottom respectively: bright field whole fish, green fluorescence whole fish, and 2 times magnified images (scale bar is 50 m) of the inset at two different focus points.

(3) FIG. 3: Fluorescent compounds of this invention display specific binding affinity to certain elements of the vascular system. This figure shows a series of Native PAGE results of BSA, cow blood serum, and goldfish (Carassius auratus) blood serum. (a) Before staining with ethyl 1,3-diphenyl-1H-pyrazole-4-carboxylate, visualized with UV (left panel) and fluorescence (right panel, under microscope, approximate position of focus shown by horizontal lines, scale bar is 200 m), showing no UV or fluorescence. (b) After staining with ethyl 1,3-diphenyl-1H-pyrazole-4-carboxylate (25 M), visualized with UV (middle panel), fluorescence (right panel, under microscope, approximate position of focus shown by horizontal lines, scale bar is 200 m), and Coomassie brilliant blue (left panel) in that order, showing UV as well as matching blue fluorescence characteristic of ethyl 1,3-diphenyl-1H-pyrazole-4-carboxylate in both the cow and the fish blood bands.

(4) FIG. 4: Acute toxicity of diethyl 4,4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazole-1,5-diyl)dibenzoate on zebrafish over a five-day regimen. Calculated LC.sub.50 is 272 M using the Benchmark Dose Software (BMDS) developed by the US Environmental Protection Agency. Regular working concentration of this compound for staining zebrafish blood vessels is 10-25 M.

DETAILED DESCRIPTION OF THE INVENTION

(5) This invention provides a method of visualizing and imaging blood vessels and blood flow in a test subject. The said test subject is either a selected part or the entirety of a dead or living animal, including a human. Detailed steps of implementing this method include: (a) administration of fluorescent compounds described herein and below to the test subject, (b) irradiating the observation area of choice in the test subject with an appropriate light source to excite the administered fluorescent compounds, (c) reveal of blood vessels, or motion of blood flow when appropriate, in the test subject when the fluorescent compounds emit fluorescence, and, (d) observation and recording of the fluorescent images of the blood vessels and blood flow with appropriate devices such as a microscope and a camera.

(6) Fluorescent compounds suitable for the method taught above include compounds of Formula I:

(7) ##STR00019## Wherein;
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from hydrogen, alkyl, substituted saturated or unsaturated alkyl, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; an unsubstituted or substituted aryl, and an unsubstituted or substituted, aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
Q-Z is either CC, CC, CN, CN, NC, or NC;
and pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures thereof, including both R and S enantiomeric forms and racemic mixtures thereof, as well as a pharmaceutical Formulation containing the compound.

(8) Compounds of Formula I include compounds of Formula IV:

(9) ##STR00020## Wherein;
Ar.sup.1 and Ar.sup.2 are independently selected from aryl or heteroaryl optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
R.sup.10 and R.sup.11 are independently selected from hydrogen, halogen, nitro, cyano, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(10) Specific examples of compounds of Formula IV include: 1) 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, 2) 4-(3-(7-(3-Bromopropoxy)-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, 3) (11-((4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoyl)oxy)undecyl)triphenylphosphonium, 4) Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate 5) 5-Chloropentyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate, 6) (3R,4R,5S,6R)-6-(Acetoxymethyl)-3-(4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzamido)tetrahydro-2H-pyran-2,4,5-triyltriacetate, 7) 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoic acid, 8) (3-((3-(1-(4-Carboxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)oxy)propyl)triphenylphosphonium, 9) Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoate, 10) 2,5-Dioxopyrrolidin-1-yl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate, 11) 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-N-((3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)benzamide, 12) 3-(1-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-6-methoxy-2H-chromen-2-one, 13) (3-((4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoyl)oxy)propyl)triphenylphosphonium, 14) (Z)-5-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzamido)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide, 15) Diethyl 4,4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazole-1,5-diyl)dibenzoate, 16) 3-(2-(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)hydrazinyl)propyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate hydrochloride, 17) 3-Bromopropyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzoate, 18) (Z)-3-((2-(5-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)ethyl)amino)propyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate hydrochloride, 19) 4-(5-(4-(Ethoxycarbonyl)phenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, 20) 4-(3-(6-Methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-N-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)benzohydrazide, and 21) Ethyl 4-(5-(3,4-dimethoxyphenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzoate, and 22) Ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylate.

(11) Compounds of Formula I include compounds of Formula VII:

(12) ##STR00021## Wherein;
Ar.sup.1 and Ar.sup.3 are independently selected from aryl or heteroaryl optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
R.sup.10 and R.sup.11 are independently selected from hydrogen, halogen, nitro, cyano, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(13) Specific examples of compounds of Formula VII include: 23) Ethyl 4-(5-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate, and 24) Ethyl 4-(5-(7-(3-bromopropoxy)-2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate.

(14) Compounds of Formula I include compounds of Formula IX:

(15) ##STR00022## Wherein;
R.sup.1 and R.sup.2 are independently selected from hydrogen, alkyl, substituted saturated or unsaturated alkyl; an unsubstituted or substituted aryl, and an unsubstituted or substituted, aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
Q is independently chosen from nitrogen or oxygen;
R.sup.12 and R.sup.13 are independently selected from hydrogen, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(16) Specific examples of compounds of Formula IX include: 25)N-Benzyl-1,3-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxamide, 26) Ethyl 1-(4-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-4-carboxylate, and 27) 4-(4-(Ethoxycarbonyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid.

(17) Compounds of Formula I include compounds of Formula X:

(18) ##STR00023## Wherein;
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from hydrogen, alkyl, substituted saturated or unsaturated alkyl, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; an unsubstituted or substituted aryl, and an unsubstituted or substituted, aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(19) Compounds of Formula X include compounds of Formula XIII:

(20) ##STR00024## Wherein;
Ar.sup.1 and Ar.sup.2 are independently selected from aryl or heteroaryl optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
R.sup.10 and R.sup.11 are independently selected from hydrogen, halogen, nitro, cyano, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(21) Specific examples of compounds of Formula XIII include: 28) Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-5-yl)benzoate, 29) 3-(5-(3,4-Dimethoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)-6-methoxy-2H-chromen-2-one, and 30) 4-(5-([1,1-Biphenyl]-4-yl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)benzoic acid.

(22) Compounds of Formula X include compounds of Formula XV:

(23) ##STR00025## Wherein;
Ar.sup.1 and Ar.sup.3 are independently selected from aryl or heteroaryl optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
R.sup.10 and R.sup.11 are independently selected from hydrogen, halogen, nitro, cyano, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(24) Specific examples of compounds of Formula XV include: 31) Ethyl 4-(5-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-phenyl-1H-pyrazol-1-yl)benzoate.

(25) Compounds of Formula X include compounds of Formula XVII:

(26) ##STR00026## Wherein;
R.sup.1 and R.sup.2 are independently selected from hydrogen, alkyl, substituted saturated or unsaturated alkyl; an unsubstituted or substituted aryl, and an unsubstituted or substituted, aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
Q is independently chosen from nitrogen or oxygen;
R.sup.12 and R.sup.13 are independently selected from hydrogen, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(27) Specific examples of compounds of Formula XVII include: 32) Ethyl 1,3-diphenyl-1H-pyrazole-4-carboxylate, 33) 5-((1,3-Diphenyl-1H-pyrazole-4-carboxamido)methyl)-1-ethyl-2-((1E,3E)-3-(1-ethyl-3,3-dimethylindolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-3H-indol-1-ium, 34) Ethyl 1-(4-fluorophenyl)-3-phenyl-1H-pyrazole-4-carboxylate, and 35)N-Benzyl-1-(4-fluorophenyl)-3-phenyl-1H-pyrazole-4-carboxamide.

(28) Compounds of Formula X include compounds of Formula XVIII:

(29) ##STR00027## Wherein;
R.sup.1, R.sup.2, and R.sup.4 are independently selected from hydrogen, alkyl, substituted saturated or unsaturated alkyl; an unsubstituted or substituted aryl, and an unsubstituted or substituted, aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, halogen, cyano, and nitro; Wherein R.sup.5 and R.sup.6 are independently chosen from hydrogen, an optionally substituted saturated or unsaturated alkyl; and an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted 8-12 bicyclic membered ring, an unsubstituted or substituted aryl, heterocycloalkyl, or heteroaryl group having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.6, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro;
Q is independently chosen from nitrogen or oxygen;
R.sup.12 and R.sup.13 are independently selected from hydrogen, alkyl, optionally substituted saturated or unsaturated alkyl, a substituted or unsubstituted aryl; S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, and C(O)NR.sup.5R.sup.6; and an aromatic or non-aromatic, 3-8 membered monocyclic ring, or 8-12 bicyclic membered ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted with 1-6 substituents independently selected from the group consisting of OH, R.sup.5, OR.sup.5, OC(O)R.sup.5, NR.sup.5R.sup.6, S(O).sub.1-2R.sup.5, SO.sub.2NR.sup.5R.sup.6, NR.sup.5SO.sub.2R.sup.6, C(O)R.sup.5, C(O)OR.sup.5, C(O)NR.sup.5R.sup.6, NR.sup.5C(O)R.sup.6, NR.sup.5C(O)OR.sup.6, R.sup.5P(R.sup.6).sub.3, halogen, cyano, and nitro.

(30) Specific examples of compounds of Formula XVIII include: 36) 4-Chloro-6-(4-(4-methoxyphenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol, 37) 3-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)-1H-pyrazole-5-carboxylic acid, 38) 3-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-5-carboxamide, 39) 3-(5-Chloro-2,4-dihydroxyphenyl)-N-(3-((3-(3-fluorophenyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)-4-(4-methoxyphenyl)-1H-pyrazole-5-carboxamide, 40) 5-((3-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)-1H-pyrazole-5-carboxamido)methyl)-1-ethyl-2-((1E,3E)-3-(1-ethyl-3,3-dimethylindolin-2-ylidene)prop-1-en-1-yl)-3,3-dimethyl-3H-indol-1-ium, 41) Ethyl 1-(4-(benzylcarbamoyl)phenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-4-carboxylate, and 42) Sodium 1-(4-(benzylcarbamoyl)phenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-4-carboxylate.

(31) The said method of visualizing and imaging blood vessels and blood flow of this invention includes a method wherein the test subject is zebrafish (Danio rerio).

(32) This invention further provides a method to identify, select, characterize, or collectively screen for substances that inhibit or promote blood vessel formation or angiogenesis in living, wild type or genetically altered zebrafish. Detailed procedure of implementing this method of screening starts with exposing test zebrafish to selected substance or mixture of substances under a pre-determined set of conditions appropriate for the interaction between the substance(s) and the fish. The fish is then subjected to the said method of visualizing and imaging blood vessels and blood flow of this invention. The resulting images of blood vessels and blood flow are examined to determine the extent of effect on zebrafish blood vessel formation, or angiogenesis, caused by the selected substance(s).

Definitions

(33) Unless noted otherwise, the chemical, biological, pharmacological, and other technical terms used herein are consistent with the uses of these terms in contemporary technical journals, patents, textbooks, and other references devoted to the appropriate art. For example, definitions and explanations of organic chemistry terms may be found in standard text such as the latest edition of March's Advanced Organic Chemistry, John Wiley & Sons, Inc, New York. (e.g. 5th Ed., 2001). In the interest of clarity and the convenience of the reader, the definitions of some terms frequently used herein are listed below.

(34) Alkyl refers to a branched or straight chain hydrocarbon group containing from one to 16 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne group. The term saturated alkyl is intended to include groups having exclusively single carbon-carbon bonds. The term unsaturated alkyl is specifically intended to include groups having any degree or level of unsaturation, i.e., groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds.

(35) The terms halo and halogen as used herein to identify substituent moieties, represent fluorine, chlorine, bromine or iodine, preferably chlorine or fluorine.

(36) The term alkoxy as used alone or in combination herein refers to a straight or branched chain alkyl group covalently bonded to the parent molecule through an O-linkage containing from one to 22 carbon atoms.

(37) The term alkoxyalkyl refers to an alkyl group substituted with an alkoxy group.

(38) The term haloalkyl is a substituted alkyl, preferably a substituted lower alkyl, substituted with one or more halogen atoms, and preferably is a C.sub.1 to C.sub.4 alkyl substituted with one to three halogen atoms.

(39) The term alkanoyl as used alone or in combination herein refers to an acyl radical derived from an alkanecarboxylic acid.

(40) The term aminocarbonyl means an amino-substituted carbonyl (carbamoyl or carboxamide) wherein the amino group can be a primary, secondary (mono-substituted amino) or tertiary amino (di-substituted amino) group.

(41) The term cycloalkyl refers to stable, saturated or partially unsaturated monocyclic, bridged monocyclic, bicyclic, and spiro rings of 3 to 15 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclohexyl, bicyclooctyl, bicyclononyl, spirononyl and spirodecyl. The term optionally substituted as it refers to cycloalkyl herein indicates that the cycloalkyl group may be substituted at one or more substitutable ring positions by one or more groups independently selected from alkyl, hydroxy, alkoxy, nitro, monoalkylamino, dialkylamino, cyano, halo, haloalkyl, alkanoyl, am inocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy and aryl and optionally substituted aryl. A cycloalkyl group can have one or more carbon-carbon double or triple bonds in the ring so long as the ring is not rendered aromatic by their presence. The term saturated cycloalkyl is intended to include cyclic rings having exclusively single carbon-carbon bonds. The term unsaturated cycloalkyl is specifically intended to include cyclic rings having any degree or level of unsaturation, i.e., groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds.

(42) Bicyclic includes spirocyclic, ortho-fused and bridged bicyclic systems. Spirocyclic refers to a pair of rings having a single atom in common. Ortho-fused refers to a pair of rings having two adjacent atoms in common. Bridged bicyclic refers to a pair of rings having at least three adjacent atoms in common.

(43) The term heterocycloalkyl as used herein refers to a stable, saturated, or partially unsaturated, monocyclic, bridged monocyclic, bicyclic, and spiro ring system containing carbon atoms and other atoms selected from nitrogen, sulfur and/or oxygen. Preferably, a heterocycloalkyl is a 5 or 6-membered monocyclic ring or an 8-12 membered bicyclic ring which consists of carbon atoms and contains one, two, or three heteroatoms selected from nitrogen, oxygen and/or sulfur. The term optionally substituted as it refers to heterocycloalkyl herein indicates that the heterocycloalkyl group may be substituted at one or more substitutable ring positions by one or more groups independently selected from alkyl, alkoxy, nitro, monoalkylamino, dialkylamino, cyano, halo, haloalkyl, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy and aryl, said aryl being optionally substituted by halo, alkyl, alkoxy, nitro, cyano, haloalkyl, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido, alkoxyalkyl, alkoxycarbonyl, and alkylcarbonyloxy groups. The heterocycloalkyl group may be attached to the parent structure through a carbon atom or through any heteroatom of the heterocycloalkyl that results in a stable structure. A heterocycloalkyl group can have one or more carbon-carbon double bonds or carbon-carbon triple bonds, or carbon-heteroatoms double bonds in the ring as long as the ring is not rendered aromatic by their presence. The term saturated heterocycloalkyl is intended to include heterocyclic rings having exclusively single bonds in the ring. The term unsaturated heterocycloalkyl is specifically intended to include heterocyclic rings having any degree or level of unsaturation, i.e., groups having one or more double bonds, groups having one or more triple bonds and groups having mixtures of single, double and triple bonds.

(44) The term heteroaryl as used herein refers to a stable, aromatic monocyclic or bicyclic ring system containing carbon atoms and other atoms selected from nitrogen, sulfur and/or oxygen. Preferably, a heteroaryl is a 5 or 6-membered monocyclic ring or an 8-12 membered bicyclic ring which consists of carbon atoms and contains one, two, or three heteroatoms selected from nitrogen, oxygen and/or sulfur. The term optionally substituted as it refers to heteroaryl herein indicates that the heteroaryl group may be substituted at one or more substitutable ring positions by one or more groups independently selected from alkyl, alkoxy, nitro, monoalkylamino, dialkylamino, cyano, halo, haloalkyl, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy and aryl, said aryl being optionally substituted by halo, alkyl and alkoxy groups. Examples of such heteroaryl groups are isoxazolyl, imidazolyl, thiazolyl, isothiazolyl, pyridyl, furyl, pyrimidinyl, pyrazolyl, pyridazinyl, furazanyl and thienyl. The heteroaryl group may be attached to the parent structure through a carbon atom or through any heteroatom of the heteroaryl that results in a stable structure.

(45) The term aryl when used alone or in combination refers to an unsubstituted or optionally substituted monocyclic, bicyclic or tricyclic aromatic hydrocarbon ring systems. Preferred are optionally substituted phenyl or naphthyl groups. The aryl group may optionally be substituted at one or more substitutable ring positions by one or more groups independently selected from alkyl, alkoxy, nitro, monoalkylamino, dialkylamino, cyano, halo, haloalkyl, alkanoyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkyl amido, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy and aryl, said aryl being optionally substituted by halo, alkyl and alkoxy groups. Preferably, the aryl group is phenyl optionally substituted with up to five and usually with one or two groups.

(46) Unless otherwise defined, the term optionally substituted as used herein, refers to the substitution of a ring system at one or more positions with one or more groups selected from: C.sub.1-24 alkyl, C.sub.1-24 alkoxy, an optionally substituted phenyl, cyano, halo, C.sub.1-24 alkoxycarbonyl, C.sub.1-24 alkyl carbonyloxy, mono- & bis-(C.sub.1-24 alkyl)-carboxamide, C.sub.1-24 alkyl amido, nitro, and mono- & bis-(C.sub.1-24 alkyl)-amino.

(47) Chelate refers to the chemical entity formed by the coordination of a compound to a metal ion at two (or more) points.

(48) Disease refers to any disease, disorder, condition, symptom, or indication that is not a normal body function.

(49) Optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which the event does not.

(50) Pharmaceutically acceptable refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

(51) Pharmaceutically acceptable excipient, carrier or adjuvant refers to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one chemical entity of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

(52) Pharmaceutically acceptable vehicle refers to a diluent, adjuvant, excipient or carrier.

(53) Pharmaceutical formulation, or synonymously medicament, means a composition containing one or more pharmaceutically active compounds, e.g. one or more chemical entities of the present disclosure, and one or more pharmaceutically acceptable vehicles.

(54) Prodrug refers to a derivative of a therapeutically effective compound that requires a transformation within the body to produce the therapeutically effective compound.

(55) Protecting group refers to a compound that when introduced into a molecule by chemical modification of a functional group reduces or prevents that reactivity. A list of protecting groups can be found in Green et. al., Protective Groups in Organic Chemistry, (Wiley and Sons, 4th ed. 2006).

(56) The term salt refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include but are not limited to: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, and the like. It is possible that a salt that is not pharmaceutically acceptable may be used as a chemical intermediate, but those situations will be note when and where they occur in these teachings.

(57) Solvate refers to the compound formed by the interaction of a solvent and a compound. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.

(58) Stereoisomer refers to an isomer that differs in the arrangement of the constituent atoms in space. Stereoisomers that are mirror images of each other and optically active are termed enantiomers, and stereoisomers that are not mirror images of one another are termed diastereoisomers. A mixture of equal amounts of the two stereoisomers of an optically active substance, such as two enantiomers where such a mixture does not rotate plane-polarized light refers to as racemic mixture.

(59) Subject includes mammals, such as humans. The terms patient, human, and subject are used interchangeably and synonymously herein.

(60) Substituted refers to a molecule in which one or more hydrogen atoms are replaced with one or more non-hydrogen atoms, functional groups or moieties.

(61) General Synthetic Procedures:

(62) Chemical entities of the present disclosure can be prepared by methods well known in the art from readily available starting materials using the following general methods and procedures. The skilled artisan will appreciate that where typical or preferred process conditions, such as, reaction temperatures, times, mole ratios of reactants, solvents, pressures, are given, other process conditions can also be used unless otherwise stated. Reaction conditions may vary with the reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

(63) Furthermore, chemical entities of the present disclosure can contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers, and enriched mixtures thereof, are included within the scope of the present disclosure, unless otherwise indicated. Pure stereoisomers, and enriched mixtures thereof, can be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

(64) Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable reaction conditions for protection and deprotection of certain functional groups are well known in the art. For example, the protecting groups which are described in T. W. Greene and P. G. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

(65) Chemical entities of the present disclosure can be isolated and purified by techniques well known in the art, such as extraction, evaporation, distillation, trituration, chromatography, and recrystallization. General synthetic schemes to prepare the compounds of the present disclosure are presented in the reaction schemes provided herein.

(66) A compound of Formula III can in general be prepared using the teaching in Scheme 1 (R.sup.4=C(O)OEt). See, for reviews on the synthesis of coumarin, Hepworth, J. et. al., in Comprehesive Heterocyclic Chemistry, Pergamon Press, 2nd edition, 1996; Vekariya, R. and Patel, H.; Synth. Comm., 2014, 44(19), 2756-2788. A General method for the synthesis of substituted pyrazolines can be found in Sharma, S. et. al.; Chem. Sci. Trans., 2014, 3(3), 861-875, and references cited therein.

(67) Preparation of coumarin ketoester 3 can be achieved by treating an appropriately substituted aldehyde/ketone 1 with diethyl 3-oxopentanedioate 2 under Knoevenagel condensation reaction conditions. Treatment of intermediate 3 with aldehyde 4, followed by cyclization with hydrazine 6 can afford pyrazoline-coumarin compounds 7. Certain starting materials 1, 4 and 6 are commercially available or can be prepared by methods of the art.

(68) ##STR00028##

(69) A compound of Formula IV can be prepared as illustrated in Scheme 2. Preparation of substituted coumarin intermediates 10 can be achieved by treating an appropriately substituted coumarin-ketone 8 with a substituted aldehyde 9 under aldol condensation reaction conditions. Treatment of intermediate 10 with a substituted hydrazine 11 under cyclization reaction conditions can afford pyrazoline-coumarin compounds 12. Certain starting materials 8, 9 and 11 are commercially available or can be prepared by methods of the art.

(70) ##STR00029##

(71) A compound of Formula VII can be prepared as illustrated in Scheme 3. Treatment of a substituted coumarin-aldehyde 13 with a substituted ketone 14 can provide the corresponding unsaturated coumarin-ketone compound 15. Preparation of pyrazoline substituted compounds 17 can be achieved by treating 15 with substituted hydrazines 16 under cyclization reaction conditions. Certain starting materials 13, 14 and 16 are commercially available or can be prepared by methods known to those skilled in the art.

(72) ##STR00030##

(73) A compound of Formula IX can be prepared as illustrated in Scheme 4. Treatment of a substituted keto-ester 18 with formaldehyde can provide the corresponding unsaturated keto-ester compound 19. Preparation of pyrazoline substituted compounds 21 can be achieved by treating 19 with substituted hydrazines 20 under cyclization reaction conditions to provide the corresponding substituted pyrazoline derivatives 21. Amidation or transesterification of compound 21 can provide the final pyrazoline derivatives 23. Certain starting materials 18, 20 and 22 are commercially available or can be prepared by methods known to those skilled in the art.

(74) ##STR00031##

(75) A compound of Formula XIII can be prepared as illustrated in Scheme 5. Pyrazoles can in general be synthesized by the oxidation of pyrazolines. See, for examples, Bapat, J. et. al.; Aust. J. Chem., 1972, 25, 1321-3; Nakamichi, N. et. al.; Org. Lett., 2002, 4 (22), 3955-3957, and references cited therein.

(76) Treatment of a substituted coumarin-ketone 8 with an aldehyde 9 under aldol condensation reaction conditions can provide the corresponding unsaturated coumarin derivative 10. Preparation of pyrazoline substituted compounds 12 can be achieved by treating a coumarin-ketone 10 with a substituted hydrazine compound 11 under cyclization reaction conditions to provide the corresponding intermediate 12. Oxidation of 12 can provide the substituted pyrazole compounds 24. Certain starting materials 8, 9 and 11 are commercially available or can be prepared by methods known to those skilled in the art.

(77) ##STR00032##

(78) A compound of Formula XV can be prepared as illustrated in Scheme 6. Treatment of a substituted coumarin-aldehyde 13 with a substituted ketone 14 can provide the corresponding unsaturated coumarin-ketone compound 15. Preparation of pyrazoline substituted intermediates 17 can be achieved by treating an appropriately substituted compound 15 with substituted hydrazines 16 under cyclization reaction conditions to provide the corresponding final pyrazoline derivative 17. Oxidation of 17 can provide the pyrazole compounds 25. Certain starting materials 13, 14 and 16 are commercially available or can be prepared by methods known to those skilled in the art.

(79) ##STR00033##

(80) A compound of Formula XVII can be prepared as illustrated in Scheme 7. A general methodology can be found in Crosscurt, A. et. al.; J. Agric. Food Chem., 1979, 27(2), 406-409, and references therein. Treatment of a substituted keto-ester 18 with formaldehyde can provide the corresponding unsaturated keto-ester compound 19. Preparation of pyrazoline substituted compounds 21 can be achieved by treating 19 with substituted hydrazines 20 under cyclization reaction conditions to provide the corresponding substituted pyrazoline derivatives 21. Amidation or transesterification of compound 21 can provide the pyrazoline derivatives 23. Oxidation of 23 can provide pyrazole derivatives 26. Certain starting materials 18, 20 and 22 are commercially available or can be prepared by methods known to those skilled in the art.

(81) ##STR00034##

(82) A compound of Formula XVIII can be prepared as illustrated in Scheme 8. Treatment of a substituted phenolic compound 27 with a substituted acetic acid 28 can provide the corresponding ketone derivative 29, which after cyclization with a derivative of oxalic acid the corresponding cyclized material 30 can be prepared. Preparation of the pyrazole substituted compounds 31 can be achieved by treating 30 with hydrazine under cyclization reaction conditions to provide the corresponding substituted pyrazole derivatives 31. Amidation or transesterification of compounds 31 can provide the pyrazole derivatives 33. Certain starting materials 27, 28 and 32 are commercially available or can be prepared by methods known to those skilled in the art.

(83) ##STR00035##

EXAMPLES

Example 1 (Formula IV)

Synthesis of 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid

(84) ##STR00036##

3-Acetyl-6-methoxy-2H-chromen-2-one

(85) To a stirred solution of ethyl acetoacetate (750 mg, 5 mmol) in ethyl ether (10 mL) at 0 C. was added piperidine (250 mg, 3 mmol). 2-Hydroxy-5-methoxybenzaldehyde (750 mg, 5 mmol) was then added dropwise. The mixture was stirred for 20 min. at 0 C., followed by overnight at room temperature. The resultant yellow suspension was filtered. The solid was washed with ether and dried to give 3-acetyl-6-methoxy-2H-chromen-2-one as a yellow powder (950 mg, 82%).

3-Cinnamoyl-7-methoxy-2H-chromen-2-one

(86) To a 2-neck RB flask equipped with a condenser under nitrogen were added 3-acetyl-7-methoxy-2H-chromen-2-one (200 mg, 0.9 mmol), benzaldehyde (120 mg, 1.2 mmol), piperidine (20 mg, 0.18 mmol) and chloroform (3 mL). The mixture was stirred under reflux for 48 hrs, cooled to rt and cold methanol (2 mL) was added with stirring. The solid was collected, washed with ether and dried under vacuum to give yellow powder. Yield: 180 mg, 61%.

4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid

(87) To a 2-neck RB flask equipped with a condenser under nitrogen were added 3-cinnamoyl-7-methoxy-2H-chromen-2-one (100 mg, 0.32 mmol), 4-hydrazinylbenzoic acid (60 mg, 0.39 mmol), acetic acid (0.25 mL, 4.16 mmol) and ethanol (20 mL). The mixture was stirred under reflux overnight and then cooled to rt. Water (10 mL) was added over 10 minutes with stirring. The solid was collected, washed with cold ethanol and ether and dried under vacuum to give orange solid. Yield: 52 mg, 36%.

Example 2 (Formula IV)

Synthesis of ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1,5-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylate

(88) ##STR00037##

(89) Ethyl 2-(7-hydroxy-2-oxo-2H-chromene-3-carbonyl)-3-phenylacrylate (80 mg, 0.22 mmol, see, for preparation, Vitorio, F. et. al. New J. Chem., 2015, 39, 2323-2332), phenylhydrazine (25 g, 0.23 mmol), acetic acid (0.1 mL) and ethanol (1 mL) were placed in a 5 mL pressured flask. The reaction mixture was heated in an oil bath at 100 C. for 10 hours. The solvent was removed under reduced pressure and the residue was purified by column chromatography (EtOAc/hexane=2:1). Yield: 36%.

Example 3 (Formula VII)

Synthesis of ethyl 4-(5-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate

(90) ##STR00038##

7-Hydroxy-3-methyl-2H-chromen-2-one

(91) To a 10 mL RB flask was added 2,4-dihydroxybenzaldehyde (1 g), sodium propionate (1.5 g), piperidine (0.1 mL) and propionic anhydride (2.5 mL). The mixture was heated to 170 C., poured into ice water, and 1N HCl was added to bring the pH to ca. 3. The solid was collected by filtration, mixed with 1 mL of sulfuric acid, and poured into ice water. The solid was collected, washed with H.sub.2O and ether, and dried under vacuum. Yield: 85%.

3-Methyl-2-oxo-2H-chromen-7-ylacetate

(92) To a 2-neck RB flask were added 7-hydroxy-3-methyl-2H-chromen-2-one (759 mg, 0.34 mmol), acetic acid (2 mL), pyridine (0.07 mL) and dichloromethane (14 mL). The mixture was stirred at rt overnight and then evaporated under reduced pressure. The residue was purified by chromatography (eluted with EtOAc/hexane=1:1). Yield: 72%.

7-Hydroxy-2-oxo-2H-chromene-3-carbaldehyde

(93) To a 2-neck RB flask equipped with a condenser under nitrogen were added NBS (2.78 g), AIBN (102 mg), and benzene (21 mL). The mixture was stirred at 85 C. for 10 minutes and 3-methyl-2-oxo-2H-chromen-7-yl acetate (1.37 g) was added and stirring was continued overnight. Benzene was evaporated, NaOAc (4.62 g) and HOAc (20 mL) were added, and the mixture was stirred under reflux for 4 hrs, then partitioned in EtOAc and water. The organic layer was evaporated, the residue was stirred with 4N HCl for 30 minutes. The solid was collected, washed with ether and dried under vacuum. Yield: 75%.

(E)-7-hydroxy-3-(3-oxo-3-phenylprop-1-en-1-yl)-2H-chromen-2-one

(94) To a RB flask were added acetophenone (63 mg, 0.52 mmol), 7-hydroxy-2-oxo-2H-chromene-3-carbaldehyde (100 mg, 0.52 mmol), and acetic acid (1 mL). The mixture was stirred to solution, 1 drop of conc. H.sub.2SO.sub.4 was added, and stirring was continued at 60 C. overnight. The mixture was cooled to rt and ethanol (2 mL) was added with continuous stirring. Five minutes later the solid was collected by filtration, washed with cold ethanol and ether, and dried under vacuum to give yellow solid. Yield: 60 mg, 32%.

Ethyl 4-(5-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzoate

(95) (E)-7-hydroxy-3-(3-oxo-3-phenylprop-1-en-1-yl)-2H-chromen-2-one (100 mg, 0.34 mmol), ethyl 4-hydrazinylbenzoate (74 mg, 0.41 mmol), acetic acid (164 mg, 2.72 mmol) and ethanol (3 mL) was stirred under reflux for 2 days. The resultant solid was collected by filtration and purified by column chromatography (silica gel, eluted with EtOAc/hexanes 1:2) to give yellow powder. Yield: 66 mg, 38%.

Example 4 (Formula IX)

Synthesis of ethyl 1,3-diphenyl-1H-pyrazole-4-carboxylate and N-benzyl-1,3-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxamide

(96) ##STR00039##

Ethyl 2-benzoylacrylate

(97) Ethyl benzoylacetate (10.0 g, 52.0 mmol), diisopropylammonium trifluoroacetate (11.2 g, 52.0 mmol), TFA (0.4 mL, 5.2 mmol), and THF (150 mL) were placed in a 500 mL RB flask. Paraformaldehyde (6.2 g, 208.1 mmol as monomer) and THF (150 mL) were then added, and the reaction mixture was heated to reflux with stirring overnight. The resulting precipitate was removed by filtration and washed with ethyl acetate. The filtrate was combined with the wash and evaporated under reduced pressure. Column chromatography (CH.sub.2Cl.sub.2/hexane=1:2) of the crude product over silica gel gave a yellow liquid. Yield: 66%.

Ethyl 1,3-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylate

(98) Ethyl 2-benzoylacrylate (6.3 g, 30.8 mmol), phenylhydrazine (3.3 g, 30.8 mmol), acetic acid (14.1 mL) and ethanol (61.7 mL) were placed in a 250 mL round-bottom flask. The reaction mixture was heated to reflux for 3 hours. After the reaction, the solvent was removed under reduced pressure. The resulting residue was extracted with dichloromethane, washed with brine, and dry with MgSO.sub.4. Column chromatography (CH.sub.2Cl.sub.2:hexane=1:1) of the crude product over silica gel gave a yellow liquid. Yield: 70%.

1,3-Diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylic acid

(99) Ethyl 1,3-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylate (120 mg, 0.41 mmol) was dissolved in THF (0.7 mL) and NaOH (30 mg, 0.42 mmol), followed by H.sub.2O (0.7 mL) was added. The mixture was stirred until reaction is complete. THF was evaporated. The residue was adjusted to pH2 with diluted HCl and extracted with EtOAc. The organic layers were dried (MgSO.sub.4) and evaporated. The residue was triturated in CH.sub.2Cl.sub.2. Yield: 82%.

N-benzyl-1,3-diphenyl-4,5-dihydro-1H-pyrazole-4-carboxamide

(100) 1,3-Diphenyl-4,5-dihydro-1H-pyrazole-4-carboxylic acid (150 mg, 0.56 mmol), EDC (160 mg, 0.84 mmol) and HOBt (110 mg, 0.84 mmol) were dissolved in DMF (1.9 mL). Benzylamine (300 mg, 0.56 mmol) and triethylamine (0.16 mL, 1.13 mmol) were added. The mixture was allowed to react for 24 hr and then poured into water. The precipitate was collected, washed with water and recrystallized from CH.sub.2Cl.sub.2. Yield: 39%.

Example 5 (Formula XIII)

Synthesis of ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-5-yl)benzoate

(101) ##STR00040##

(102) Ethyl 4-(3-(6-methoxy-2-oxo-2H-chromen-3-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl) benzoate (100 mg, 0.21 mmol, made by a similar process as Example 1) was dissolved in acetic acid (1 mL) and 10% PdC (20 mg) was added. The mixture was heated to 80 C. and stirred for 6.5 hrs. The mixture was filtered through celite and the celite pad was washed with methanol. The filtrate was evaporated and triturated with 2 mL of methanol over 2 hr. The solid was collected by filtration, washed with cold methanol and ether, and dried under vacuum to give yellow powder. Yield: 50 mg, 50%.

Example 6 (Formula XVIII)

Synthesis of 3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-5-carboxamide

(103) ##STR00041##

6-Chloro-7-hydroxy-3-(4-methoxyphenyl)-4-oxo-N-phenyl-4H-chromene-2-carboxamide

(104) 6-Chloro-7-hydroxy-3-(4-methoxyphenyl)-4-oxo-4H-chromene-2-carboxylic acid (0.20 g, 0.58 mmol, for synthesis see Dymock, B., J. Med. Chem. 2005, 48, 4212-4215), EDC (0.17 g, 0.87 mmol), CH.sub.2Cl.sub.2 (2 mL), and aniline (0.13 g, 1.44 mmol) were placed in a 5.0 mL RB flask. The reaction mixture was stirred overnight. The precipitate was collected by filtration, washed with cold water and ether and dried under vacuum. Yield: 100 mg, 41%.

3-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-5-carboxamide

(105) 6-Chloro-7-hydroxy-3-(4-methoxyphenyl)-4-oxo-N-phenyl-4H-chromene-2-carboxamide (0.10 g, 0.24 mmol), hydrazine hydrate (0.1 mL), and ethanol (2.3 mL) were placed in a 5.0 mL RB flask. The mixture was heated to reflux with stirring overnight. The solvent was removed under reduced pressure. The residue was washed with cold water and ether and dried under vacuum. Yield: 90 mg, 90%.

Example 7

Anti-Angiogenic Activity of Sunitinib

(106) Zebrafish embryos were collected and washed with RO water prior to 2 hours post fertilization (hpf) and then incubated in RO water at 28 C. until 24 hpf. Embryos were placed into 4 single wells of a 24-well plate with five embryos per well. Each well was treated respectively with 1 mL of either 0.05% DMSO as control or Sunitinib in one of three concentrations: 5 M, 2.5 M, and 1 M in water. In one variation of this experiment, phenylthiourea (PTU) was co-administered to a final concentration of 200 M to inhibit embryonic pigmentation. The plate was incubated for additional 48 hours, the liquid in each well was removed and the embryos were washed with 1 mL of water. 4-(5-(4-(Ethoxycarbonyl)phenyl)-3-(6-methoxy-2-oxo-2H-chromen-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid (1 mL at 20 M) was added to each well and the plate was incubated at 28 C. for one hour. The liquid in each well was removed and the embryos were washed with 1 mL of water. The embryos were picked up one by one using a pipette, each placed on a glass slide, and temporarily anesthetized with 10 L of 7.5% tricaine (MS-222). The embryo was embedded in 20 L of 3% methyl cellulose and imaged using a Zeiss M2 microscope and the Axiocam HRC image system.