Compositions and therapeutic methods for the treatment of complement-associated diseases

Abstract

The invention comprises methods of modulating the complement cascade in a mammal and for treating and/or preventing diseases and disorders associated with the complement pathway by administering a compound of Formula I or Formula II, such as, for example, 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one or a pharmaceutically acceptable salt thereof.

Claims

1. A compound selected from: 2-{4-[2-(3,3-difluoropyrrolidin-1-yl)ethoxy]-3,5-dimethylphenyl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one; methyl N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}carbamate; and stereoisomers, tautomers, pharmaceutically acceptable salts, and hydrates thereof.

2. The compound of claim 1, wherein the compound is 2-{4-[2-(3,3-difluoropyrrolidin-1-yl)ethoxy]-3,5-dimethylphenyl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one.

3. The compound of claim 1, wherein the compound is methyl N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}carbamate.

4. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising the compound of claim 2 and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition comprising the compound of claim 3 and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 demonstrates that RVX000222 reduces expression of complement component 3, 4 and 5 at the mRNA level in Huh-7 cells treated simultaneously with cytokines that induce complement expression during inflammation. mRNA level was determined by TaqMan real-time PCR and is normalized to the level of cyclophilin mRNA. Data is the mean of triplicate samples.

(2) FIG. 2 demonstrates that RVX000222 reduces expression of complement component 3, 4 and 5 at the mRNA level in HepG2 cells treated simultaneously with cytokines that induce complement expression during inflammation. mRNA level was determined by TaqMan real-time PCR and is normalized to the level of cyclophilin mRNA. Data is the mean of triplicate samples.

(3) FIG. 3 RVX000222 reduces expression of complement component 3, 4 and 5 at the mRNA level in Huh-7 cells pre-treated with cytokines that induce complement expression during inflammation. mRNA level was determined by TaqMan real-time PCR and is normalized to the level of cyclophilin mRNA. Data is the mean of triplicate samples.

(4) FIG. 4 RVX000222 reduces expression of complement component 3, 4 and 5 at the mRNA level in HepG2 cells pre-treated with cytokines that induce complement expression during inflammation. mRNA level was determined by TaqMan real-time PCR and is normalized to the level of cyclophilin mRNA. Data is the mean of triplicate samples.

(5) FIG. 5 demonstrates that 30 uM RVX000222 reduces secretion of C3, C4, and C5 proteins by Huh-7 cells treated simultaneously with interleukin 6 (IL-6). IL-6 induces complement expression during inflammation. Protein levels were quantitated by ELISA. Data is the mean of duplicate samples.

(6) FIG. 6: demonstrated that 30 uM RVX000222 reduces secretion of C3, C4, C5 and C9 proteins by primary human hepatocytes treated simultaneously with interleukin 6 (IL-6). IL-6 induces complement expression during inflammation. Protein levels were quantitated by ELISA. Data is the mean of duplicate samples.

(7) FIG. 7A: RVX000222 reduces complement activity in clinical samples as measured by AH50 assay. FIG. 7B: RVX000222 reduces complement activity in clinical samples as measured by CH50 assay.

DESCRIPTION OF EMBODIMENTS

(8) In certain embodiments, the method for modulating the complement system in a subject in need thereof comprises administering a therapeutically effective amount of at least one compound of Formula I or Formula II as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(9) In certain embodiments, the method for treating complement-associated diseases or disorders in a subject in need thereof comprises administering a therapeutically effective amount of at least one compound of Formula I or Formula II as described herein or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

Definitions

(10) As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. The following abbreviations and terms have the indicated meanings throughout:

(11) The term compound of Formula I refers to compounds having the general structure:

(12) ##STR00003## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, wherein: R.sub.1 and R.sub.3 are each independently selected from alkoxy, alkyl, amino, halogen, and hydrogen; R.sub.2 is selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, halogen, and hydrogen; R.sub.5 and R.sub.7 are each independently selected from alkyl, alkoxy, amino, halogen, and hydrogen; R.sub.6 is selected from amino, amide, alkyl, hydrogen, hydroxyl, piperazinyl, and alkoxy, wherein the alkoxy is optionally substituted with one or more groups chosen from amide, amine, aryl, benzyloxy, carbamate, carboxy, heterocyclyl, hydroxyl, methoxy, and sulfonamide; and W is CH or N.

(13) In some embodiments, W is CH in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, and R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.6, and R.sub.7, are as defined in the foregoing paragraph.

(14) In some embodiments, R.sub.6 in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is selected from alkoxy optionally substituted with one or more groups chosen from amide, amine, aryl, benzyloxy, carbamate, carboxy, heterocyclyl, hydroxyl, methoxy, and sulfonamide, and R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.7, and W are as defined in any of the two foregoing paragraphs.

(15) In some embodiments, R.sub.6 in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is selected from hydrogen, methoxy,

(16) ##STR00004##
wherein n is 1, 2, or 3; R.sub.8 is selected from hydrogen or C.sub.1-C.sub.6 alkyl substituted with one or more groups selected from methyl, phenyl, and pyridinyl; R.sub.9 and R.sub.10 are independently selected from unsubstituted C.sub.1-C.sub.6 alkyl, wherein R.sub.9 and R.sub.10 may be joined together with N to form a 3- to 12-membered ring; and R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.7, and W are as defined above for the compound Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(17) In some embodiments, R.sub.6 in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is selected from 2-(hydroxy)ethoxy, 2-(pyrrolidin-1-yl)ethoxy, 4-isopropylpiperazin-1-yl, and 2-(isopropylamino)ethoxy, and R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.7, and W are as defined above for the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(18) In some embodiments, R.sub.6 in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is 2-(hydroxy)ethoxy, and R.sub.1, R.sub.2, R.sub.3, R.sub.5, R.sub.7, and W are as defined above for the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(19) In some embodiments, R.sub.1 and R.sub.3 in the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, are both methoxy, R.sub.2, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and W are as defined above for the compound of Formula I or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(20) In some embodiments, the compound of Formula I is selected from: 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 2-{3,5-dimethyl-4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-5,7-di methoxy-3,4-dihydroquinazolin-4-one; 2-(3,5-dimethyl-4-{2-[(propan-2-yl)amino]ethoxy}phenyl)-5,7-dimethoxy-3,4-dihydroquinazolin-4-one; 5,7-dimethoxy-2-{4-[4-(propan-2-yl)piperazin-1-yl]phenyl}-3,4-dihydroquinazolin-4-one; 5,7-dimethoxy-2-{3-methoxy-5-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-3,4-dihydroquinazolin-4-one; 2-{3,5-dimethyl-4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-5,7-di methoxy-3H,4H-pyrido[2,3-d]pyrimidin-4-one; 2-{4-[2-(3,3-difluoropyrrolidin-1-yl)ethoxy]-3,5-dimethylphenyl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one; N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}-2-methylpropanamide; 5,7-dimethoxy-2-[4-(piperazin-1-yl)phenyl]-3,4-dihydroquinazolin-4-one; 2-(4-hydroxy-3,5-dimethylphenyl)-5,7-dimethoxy-3,4-dihydroquinazolin-4-one; N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}acetamide; methyl N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}carbamate; 2-[4-(2,3-di hydroxypropoxy)-3,5-dimethylphenyl]-5,7-dimethoxy-3,4-dihydroquinazolin-4-one; N-(2-(4-(5,7-Dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethyl)-4-methylbenzamide; 2-(4-(5,7-Dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethyl-phenoxy)ethyl methylcarbamate; 2-(4-(5,7-Dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethyl-phenoxy)ethyl propylcarbamate; N-(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethyl)methanesulfonamide (RVX002093); 4-chloro-N-(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethyl)benzenesulfonamide; N-(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethyl)-4-methoxybenzenesulfonamide; 2-(4-(2-aminoethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; N.sup.1-(2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)ethyl)-N.sup.2-methylphthalamide; 2-(4-(2-hydroxyethoxy)-3-methylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 2-(4-(benzyloxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 6-bromo-2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)quinazolin-4(3H)-one; 6-bromo-2-(4-hydroxy-3,5-dimethylphenyl)quinazolin-4(3H)-one; 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-6-methoxyquinazolin-4(3H)-one; 5,7-dichloro-2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)quinazolin-4(3H)-one; 5,7-dimethoxy-2-(4-(2-methoxyethoxy)-3,5-dimethylphenyl)quinazolin-4(3H)-one; N-(2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)acetamide; 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxypyrido[2,3-d]pyrimidin-4(3H)-one; 5,7-dimethoxy-2-(4-methoxy-3-(morpholinomethyl)phenyl)quinazolin-4(3H)-one; 2-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 5,7-dimethoxy-2-(4-(morpholinomethyl)phenyl)quinazolin-4(3H)-one; N-(4-(5,7-di methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)phenyl)-2-hydroxyacetamide; 2-(4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)acetic acid; N-(4-(5,7-di methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenyl)-2-hydroxyacetamide; 5,7-dimethoxy-2-(4-((4-methylpiperazin-1-yl)methyl)phenyl)quinazolin-4(3H)-one; 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-6,7-dimethoxyquinazolin-4(3H)-one; 2-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 2-(3-chloro-4-(2-hydroxyethoxy)phenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 2-(4-(6,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy)acetamide; N-(2-(4-hydroxy-3,5-dimethylphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)acetamide; 2-(4-(bis(2-hydroxyethyl)amino)phenyl)-6,7-dimethoxyquinazolin-4(3H)-one; 2-(4-(bis(2-hydroxyethyl)amino)phenyl)-5,7-dimethoxyquinazolin-4(3H)-one; 5,7-dimethoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)quinazolin-4(3H)-one (RVX000255); 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)quinazolin-4(3H)-one; 2-(3,5-dimethyl-4-(2-morpholinoethoxy)phenyl)quinazolin-4(3H)-one; 2-(3,5-dimethyl-4-(2-morpholinoethoxy)phenyl)-5,7-dimethoxyquinazolin-4(3H)-one; and stereoisomers, tautomers, pharmaceutically acceptable salts, and hydrates thereof.

(21) In some embodiments, the compound of Formula I is 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (RVX000222) (also known as RVX-208)

(22) ##STR00005##
or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(23) In certain embodiments, the compound of Formula I is 2-{3,5-dimethyl-4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(24) ##STR00006##
or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(25) In other embodiments, the compound of Formula I is 2-(3,5-dimethyl-4-{2-[(propan-2-yl)amino]ethoxy}phenyl)-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(26) ##STR00007##
or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(27) In yet other embodiments, the compound of Formula I is 5,7-dimethoxy-2-{4-[4-(propan-2-yl)piperazin-1-yl]phenyl}-3,4-dihydroquinazolin-4-one

(28) ##STR00008##
or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(29) In some embodiments, the compound of Formula I is 5,7-dimethoxy-2-{3-methoxy-5-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-3,4-dihydroquinazolin-4-one

(30) ##STR00009## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(31) In some embodiments, the compound of Formula I is 2-{3,5-dimethyl-4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-5,7-dimethoxy-3H,4H-pyrido[2,3-d]pyrimidin-4-one

(32) ##STR00010## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(33) In some embodiments, the compound is 2-{2-[(dimethylamino)methyl]-1H-indol-5-yl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(34) ##STR00011## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(35) In some embodiments, the compound of Formula I is 2-{4-[2-(3,3-difluoropyrrolidin-1-yl)ethoxy]-3,5-dimethylphenyl}-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(36) ##STR00012## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(37) In some embodiments, the compound of Formula I is N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}-2-methylpropanamide

(38) ##STR00013## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(39) In some embodiments, the compound of Formula I is 5,7-dimethoxy-2-[4-(piperazin-1-yl)phenyl]-3,4-dihydroquinazolin-4-one

(40) ##STR00014## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(41) In some embodiments, the compound of Formula I is 2-(4-hydroxy-3,5-dimethylphenyl)-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(42) ##STR00015## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(43) In some embodiments, the compound of Formula I is N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}acetamide

(44) ##STR00016## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(45) In some embodiments, the compound of Formula I is methyl N-{2-[4-(5,7-dimethoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-2,6-dimethylphenoxy]ethyl}carbamate

(46) ##STR00017## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(47) In some embodiments, the compound of Formula I is 2-[4-(2,3-dihydroxypropoxy)-3,5-dimethylphenyl]-5,7-dimethoxy-3,4-dihydroquinazolin-4-one

(48) ##STR00018## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(49) The term compound of Formula II refers to compounds having the general structure:

(50) ##STR00019## or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, wherein: R.sub.A and R.sub.B are independently selected from hydrogen, methyl, (CH.sub.2).sub.nR.sub.F, (CH.sub.2).sub.nOR.sub.F, and CH.sub.2C(O)OR.sub.G; R.sub.C is selected from hydrogen, para-halogen, and OCH.sub.2O or OCH.sub.2CH.sub.2O connected to the ortho and meta positions or connected to the meta and para positions of the phenyl ring; R.sub.D and R.sub.E are independently selected from hydrogen and methyl; R.sub.F is selected from methyl, ethyl, and CH.sub.2CH.sub.2OCH.sub.3; R.sub.G is selected from methyl, Ethyl, n-propyl, isopropyl, n-butyl, and tert-butyl; and n is selected from 1, 2, 3, and 4.

(51) In some embodiments, R.sub.C is para-Cl.

(52) In some embodiments, the compound of Formula II is selected from: 6,6-dimethyl-4-phenyl-9-methyl-6H-thieno[3,2-f]-s-triazolo[4,3-a][1,4]diazepine; 4-(3,4-mehylenedioxyphenyl)-9-methyl-6H-thieno[3,2-f]-s-triazolo[4,3-a][1,4]diazepine; 9-methyl-4-phenyl-6H-thieno[3,2-f]-s-triazolo[4,3-a][1,4]diazepine; (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (JQ1); and stereoisomers, tautomers, pharmaceutically acceptable salts, and hydrates thereof.

(53) In some embodiments, the compound of Formula I or Formula II is in the form of a solvate. In some embodiments, the compound of Formula I or Formula II is in the form of a hydrate. In some embodiments, the compound of Formula I or Formula II is in the form of a chelate. In some embodiments, the compound of Formula I or Formula II is in the form of a pharmaceutically acceptable salt. In some embodiments, the compound of Formula I or Formula II is in crystalline form. In some embodiments, the compound of Formula I or Formula II is a polymorph or a pseudopolymorph. In some embodiments, the compound of Formula I or Formula II is in the form of an unsolvated polymorph, such as, e.g., an anhydrate. In some embodiments, the compound of Formula I or Formula II is in the form of a conformational polymorph. In some embodiments, the compound of Formula I or Formula II is amorphous. In some embodiments, the compound of Formula I or Formula II is in the form of a non-covalent complex. In some embodiments, the compound of Formula I or Formula II is in the form of a solvate of a salt. In some embodiments, the compound of Formula I or Formula II is in the form of a chelate of a salt. In some embodiments, the compound of Formula I or Formula II is in the form of a hemi-hydrate. In some embodiments, the compound of Formula I or Formula II is in the form of a monohydrate.

(54) In some embodiments, a prodrug is administered to a patient to become a compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, e.g., upon metabolic processing of the prodrug. Examples of prodrugs include derivatives of functional groups, such as a carboxylic acid group, in the compounds of Formula I or Formula II. Exemplary prodrugs of a carboxylic acid group include, but are not limited to, carboxylic acid esters such as alkyl esters, hydroxyalkyl esters, arylalkyl esters, and aryloxyalkyl esters.

(55) A solvate is formed by the interaction of a solvent and a compound, and the compounds of Formula I or Formula II may be in the form of a solvate. Similarly, a salt of the compounds of Formula I or Formula II may be in the form of a solvate of salt. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.

(56) A chelate is formed by the coordination of a compound to a metal ion at two (or more) points. The compound of Formula I or Formula II may be in the form of a chelate. Similarly, a salt of a compound of Formula I or Formula II may be in the form of a chelate.

(57) A non-covalent complex may be formed by the interaction of a compound of Formula I or Formula II and another molecule wherein a covalent bond is not formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also called ionic bonding).

(58) A dash (-) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, CONH.sub.2 is attached through the carbon atom.

(59) By optional or optionally is meant 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 is does not. For example, optionally substituted aryl encompasses both aryl and substituted aryl as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable.

(60) The term acyl term as used herein refers to a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl, or heteroaryl. Exemplary acyl groups include, but are not limited to, acetyl, formyl, propionyl, benzoyl, and the like.

(61) The term aldehyde or formyl as used herein refers to CHO.

(62) The term alkenyl as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-22, 2-8, or 2-6 carbon atoms, referred to herein as (C.sub.2-C.sub.22)alkenyl, (C.sub.2-C.sub.8)alkenyl, and (C.sub.2-C.sub.6)alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-butene)-pentenyl.

(63) The term alkoxy as used herein refers to an alkyl group attached to an oxygen (O-alkyl-). Alkoxy groups also include an alkenyl group attached to an oxygen (alkenyloxy) or an alkynyl group attached to an oxygen (alkynyloxy) groups. Exemplary alkoxy groups include, but are not limited to, groups with an alkyl, alkenyl or alkynyl group of 1-22, 1-8, or 1-6 carbon atoms, referred to herein as (C.sub.1-C.sub.22)alkoxy, (C.sub.1-C.sub.8)alkoxy, and (C.sub.1-C.sub.6)alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy and ethoxy.

(64) The term alkyl as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-22, 1-8, or 1-6 carbon atoms, referred to herein as (C.sub.1-C.sub.2)alkyl, (C.sub.1-C.sub.8)alkyl, and (C.sub.1-C.sub.6)alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

(65) The term alkynyl as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-22, 2-8, or 2-6 carbon atoms, referred to herein as (C.sub.2-C.sub.22)alkynyl, (C.sub.2-C.sub.8)alkynyl, and (C.sub.2-C.sub.6)alkynyl, respectively. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.

(66) The term amide as used herein refers to the structure NR.sub.aC(O)(R.sub.b) or C(O)NR.sub.bR.sub.c, wherein R.sub.a, R.sub.b and R.sub.c are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. The amide can be attached to another group through the carbon, the nitrogen, R.sub.b, or R.sub.c. The amide also may be cyclic, for example R.sub.b and R.sub.c, may be joined to form a 3- to 12-membered ring, such as a 3- to 10-membered ring or a 5- or 6-membered ring. The term amide encompasses groups such as sulfonamide, urea, ureido, carbamate, carbamic acid, and cyclic versions thereof. The term amide also encompasses an amide group attached to a carboxy group, e.g., -amide-COOH or salts such as -amide-COONa, an amino group attached to a carboxy group (e.g., -amino-COOH or salts such as -amino-COONa).

(67) The term amine or amino as used herein refers to the structure NR.sub.dR.sub.e or N(R.sub.d)R.sub.e, where R.sub.d and R.sub.e are independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. The amino can be attached to the parent molecular group through the nitrogen. The amino also may be cyclic, for example any two of R.sub.d and R.sub.e may be joined together or with the N to form a 3- to 12-membered ring (e.g., morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salt of any amino group. Exemplary amino groups include alkylamino groups, wherein at least one of R.sub.d or R.sub.e is an alkyl group.

(68) The term aryl as used herein refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system. The aryl group can optionally be fused to one or more rings selected from aryls, cycloalkyls, and heterocyclyls. The aryl groups of this invention can be substituted with groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups also include, but are not limited to a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as (C.sub.6)aryl.

(69) The term arylalkyl as used herein refers to an alkyl group having at least one aryl substituent (e.g., -aryl-alkyl-). Exemplary arylalkyl groups include, but are not limited to, arylalkyls having a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as (C.sub.6)arylalkyl.

(70) The term aryloxy as used herein refers to an aryl group attached to an oxygen atom. Exemplary aryloxy groups include, but are not limited to, aryloxys having a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as (C.sub.6)aryloxy.

(71) The term arylthio as used herein refers to an aryl group attached to an sulfur atom. Exemplary arylthio groups include, but are not limited to, arylthios having a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as (C.sub.6)arylthio.

(72) The term arylsulfonyl as used herein refers to an aryl group attached to a sulfonyl group, e.g., S(O).sub.2-aryl-. Exemplary arylsulfonyl groups include, but are not limited to, arylsulfonyls having a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as (C.sub.6)arylsulfonyl.

(73) The term benzyl as used herein refers to the group CH.sub.2-phenyl.

(74) The term bicyclic aryl as used herein refers to an aryl group fused to another aromatic or non-aromatic carbocylic or heterocyclic ring. Exemplary bicyclic aryl groups include, but are not limited to, naphthyl or partly reduced forms thereof, such as di-, tetra-, or hexahydronaphthyl.

(75) The term bicyclic heteroaryl as used herein refers to a heteroaryl group fused to another aromatic or non-aromatic carbocylic or heterocyclic ring. Exemplary bicyclic heteroaryls include, but are not limited to 5,6- or 6,6-fused systems, wherein one or both rings contain heteroatoms. The term bicyclic heteroaryl also encompasses reduced or partly reduced forms of fused aromatic system wherein one or both rings contain ring heteroatoms. The ring system may contain up to three heteroatoms, independently selected from oxygen, nitrogen, and sulfur. The bicyclic system may be optionally substituted with one or more groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Exemplary bicyclic heteroaryl's include, but are not limited to, quinazolinyl, benzothiophenyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl, phthalazinyl, benzotriazolyl, benzopyridinyl, and benzofuranyl.

(76) The term carbamate as used herein refers to the form R.sub.gOC(O)N(R.sub.h), R.sub.gOC(O)N(R.sub.h)R.sub.i, or OC(O)NR.sub.hR.sub.i, wherein R&, R.sub.h and R.sub.i are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. Exemplary carbamates include, but are not limited to, arylcarbamates or heteroaryl carbamates (e.g., wherein at least one of R.sub.g, R.sub.h and R.sub.i are independently selected from aryl or heteroaryl, such as pyridine, pyridazine, pyrimidine, and pyrazine).

(77) The term carbonyl as used herein refers to C(O).

(78) The term carboxy as used herein refers to COOH or its corresponding carboxylate salts (e.g., COONa). The term carboxy also includes carboxycarbonyl, e.g. a carboxy group attached to a carbonyl group, e.g., C(O)COOH or salts, such as C(O)COONa.

(79) The term cyano as used herein refers to CN.

(80) The term cycloalkoxy as used herein refers to a cycloalkyl group attached to an oxygen.

(81) The term cycloalkyl as used herein refers to a saturated or unsaturated cyclic, bicyclic, or bridged bicyclic hydrocarbon group of 3-12 carbons, or 3-8 carbons, referred to herein as (C.sub.3-C.sub.8)cycloalkyl, derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclohexenes, cyclopentanes, and cyclopentenes. Cycloalkyl groups may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Cycloalkyl groups can be fused to other cycloalkyl saturated or unsaturated, aryl, or heterocyclyl groups.

(82) The term dicarboxylic acid as used herein refers to a group containing at least two carboxylic acid groups such as saturated and unsaturated hydrocarbon dicarboxylic acids and salts thereof. Exemplary dicarboxylic acids include alkyl dicarboxylic acids. Dicarboxylic acids may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Dicarboxylic acids include, but are not limited to succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, maleic acid, phthalic acid, aspartic acid, glutamic acid, malonic acid, fumaric acid, (+)/()-malic acid, (+)/() tartaric acid, isophthalic acid, and terephthalic acid. Dicarboxylic acids further include carboxylic acid derivatives thereof, such as anhydrides, imides, hydrazides (for example, succinic anhydride and succinimide).

(83) The term ester refers to the structure C(O)O, C(O)OR.sub.j-, R.sub.kC(O)OR.sub.j, or R.sub.kC(O)O, where 0 is not bound to hydrogen, and R.sub.j and R.sub.k can independently be selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, cycloalkyl, ether, haloalkyl, heteroaryl, and heterocyclyl. R.sub.k can be a hydrogen, but R.sub.j cannot be hydrogen. The ester may be cyclic, for example the carbon atom and R.sub.j, the oxygen atom and R.sub.k, or R.sub.j and R.sub.k may be joined to form a 3- to 12-membered ring. Exemplary esters include, but are not limited to, alkyl esters wherein at least one of R.sub.j or R.sub.k is alkyl, such as OC(O)-alkyl, C(O)O-alkyl-, and -alkyl-C(O)O-alkyl-. Exemplary esters also include aryl or heteoraryl esters, e.g. wherein at least one of R.sub.j or R.sub.k is a heteroaryl group such as pyridine, pyridazine, pyrmidine and pyrazine, such as a nicotinate ester. Exemplary esters also include reverse esters having the structure R.sub.kC(O)O, where the oxygen is bound to the parent molecule. Exemplary reverse esters include succinate, D-argininate, L-argininate, L-lysinate and D-lysinate. Esters also include carboxylic acid anhydrides and acid halides.

(84) The term ether refers to the structure R.sub.lOR.sub.m, where R.sub.l and R.sub.m can independently be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, and ether. The ether can be attached to the parent molecular group through R.sub.l or R.sub.m. Exemplary ethers include, but are not limited to, alkoxyalkyl and alkoxyaryl groups. Ethers also includes polyethers, e.g., where one or both of R.sub.l and R.sub.m are ethers.

(85) The terms halo or halogen or Hal as used herein refer to F, Cl, Br, or I.

(86) The term haloalkyl as used herein refers to an alkyl group substituted with one or more halogen atoms. Haloalkyls also encompass alkenyl or alkynyl groups substituted with one or more halogen atoms.

(87) The term heteroaryl as used herein refers to a mono-, bi-, or multi-cyclic, aromatic ring system containing one or more heteroatoms, for example 1-3 heteroatoms, such as nitrogen, oxygen, and sulfur. Heteroaryls can be substituted with one or more substituents including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Heteroaryls can also be fused to non-aromatic rings. Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl, pyrimidilyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl, phenyl, isoxazolyl, and oxazolyl. Exemplary heteroaryl groups include, but are not limited to, a monocyclic aromatic ring, wherein the ring comprises 2-5 carbon atoms and 1-3 heteroatoms, referred to herein as (C.sub.2-C.sub.5)heteroaryl.

(88) The terms heterocycle, heterocyclyl, or heterocyclic as used herein refer to a saturated or unsaturated 3-, 4-, 5-, 6- or 7-membered ring containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Heterocycles can be aromatic (heteroaryls) or non-aromatic. Heterocycles can be substituted with one or more substituents including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Heterocycles also include bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from aryls, cycloalkyls, and heterocycles. Exemplary heterocycles include acridinyl, benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, furyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl, isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, pyrrolyl, quinolinyl, quinoxaloyl, tetra hydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, thiomorpholinyl, thiopyranyl, and triazolyl.

(89) The terms hydroxy and hydroxyl as used herein refers to OH.

(90) The term hydroxyalkyl as used herein refers to a hydroxy attached to an alkyl group.

(91) The term hydroxyaryl as used herein refers to a hydroxy attached to an aryl group.

(92) The term ketone as used herein refers to the structure C(O)Rn (such as acetyl, C(O)CH.sub.3 or R.sub.n-C(O)R.sub.o. The ketone can be attached to another group through R.sub.n or R.sub.o. R.sub.n or R.sub.o can be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or R.sub.n or R.sub.o can be joined to form a 3- to 12-membered ring.

(93) The term monoester as used herein refers to an analogue of a dicarboxylic acid wherein one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or salt of a carboxylic acid. Examples of monoesters include, but are not limited to, to monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

(94) The term nitro as used herein refers to NO.sub.2.

(95) The term perfluoroalkoxy as used herein refers to an alkoxy group in which all of the hydrogen atoms have been replaced by fluorine atoms.

(96) The term perfluoroalkyl as used herein refers to an alkyl group in which all of the hydrogen atoms have been replaced by fluorine atoms. Exemplary perfluroalkyl groups include, but are not limited to, C.sub.1-C.sub.5 perfluoroalkyl, such as trifluoromethyl.

(97) The term perfluorocycloalkyl as used herein refers to a cycloalkyl group in which all of the hydrogen atoms have been replaced by fluorine atoms.

(98) The term phenyl as used herein refers to a 6-membered carbocyclic aromatic ring. The phenyl group can also be fused to a cyclohexane or cyclopentane ring. Phenyl can be substituted with one or more substituents including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone.

(99) The term phosphate as used herein refers to the structure OP(O)O.sub.2, R.sub.xOP(O)O.sub.2, OP(O)O.sub.2R.sub.y, or R.sub.xOP(O)O.sub.2R.sub.y, wherein R.sub.x and R.sub.y can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, and hydrogen.

(100) The term sulfide as used herein refers to the structure R.sub.zS, where R.sub.z can be alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl. The sulfide may be cyclic, forming a 3 to 12-membered ring. The term alkylsulfide as used herein refers to an alkyl group attached to a sulfur atom.

(101) The term sulfinyl as used herein refers to the structure S(O)O, R.sub.pS(O)O, R.sub.pS(O)OR.sub.q, or S(O)OR.sub.q, wherein R.sub.p and R.sub.q can be alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, hydroxyl. Exemplary sulfinyl groups include, but are not limited to, alkylsulfinyls wherein at least one of R.sub.p or R.sub.q is alkyl, alkenyl, or alkynyl.

(102) The term sulfonamide as used herein refers to the structure (R.sub.r)NS(O).sub.2R.sub.s or R.sub.t(R.sub.r)NS(O).sub.2R.sub.s, where R.sub.t, R.sub.r, and R.sub.s can be, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclyl. Exemplary sulfonamides include alkylsulfonamides (e.g., where R.sub.s is alkyl), arylsulfonamides (e.g., where R.sub.s is aryl), cycloalkyl sulfonamides (e.g., where R.sub.s is cycloalkyl), and heterocyclyl sulfonamides (e.g., where R.sub.s is heterocyclyl).

(103) The term sulfonate as used herein refers to OSO.sub.3. Sulfonate includes salts such as OSO.sub.3Na, OSO.sub.3K and the acid OSO.sub.3H.

(104) The term sulfonic acid refers to SO.sub.3H and its corresponding salts (e.g., SO.sub.3K and SO.sub.3Na).

(105) The term sulfonyl as used herein refers to the structure R.sub.uSO.sub.2, where R.sub.u can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclyl (e.g., alkylsulfonyl). The term alkylsulfonyl as used herein refers to an alkyl group attached to a sulfonyl group. Alkylsulfonyl groups can optionally contain alkenyl or alkynyl groups.

(106) The term thioketone refers to the structure R.sub.vC(S)R.sub.w. The ketone can be attached to another group through R.sub.v or R.sub.w. R.sub.v or R.sub.w can be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or R.sub.v or R.sub.w can be joined to form a 3- to 12-membered ring.

(107) Alkyl groups can be substituted with or interrupted by or branched with at least one group selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, ketone, heteroaryl, heterocyclyl, hydroxyl, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, thioketone, ureido and N. The substituents may be branched to form a substituted or unsubstituted heterocycle or cycloalkyl.

(108) Alkenyl, alkynyl, alkoxy, amino and amide groups can be substituted with or interrupted by or branched with at least one group selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carbonyl, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, thioketone, ureido and N. The substituents may be branched to form a substituted or unsubstituted heterocycle or cycloalkyl.

(109) As used herein, a suitable substituent refers to a group that does not nullify the synthetic or pharmaceutical utility of the compounds of Formula I or Formula II. Examples of suitable substituents include, but are not limited to: C.sub.1-22, C.sub.1-8, and C.sub.1-6 alkyl, alkenyl or alkynyl; C.sub.1-6 aryl, C.sub.2-5 heteroaryl; C.sub.3-7 cycloalkyl; C.sub.1-22, C.sub.1-8, and C.sub.1-6 alkoxy; C.sub.6 aryloxy; CN; OH; oxo; halo, carboxy; amino, such as NH(C.sub.1-22, C.sub.1-8, or C.sub.1-6 alkyl), N(C.sub.1-22, C.sub.1-8, and C.sub.1-6 alkyl).sub.2, NH((C.sub.6)aryl), or N((C.sub.6)aryl).sub.2; formyl; ketones, such as CO(C.sub.1-22, C.sub.1-8, and C.sub.1-6 alkyl), CO((C.sub.6 aryl) esters, such as CO.sub.2(C.sub.1-22, C.sub.1-8, and C.sub.1-6 alkyl) and CO.sub.2 (C.sub.6 aryl). One of skill in art can readily choose a suitable substituent based on the stability and pharmacological and synthetic activity of the compound of the invention.

(110) The term pharmaceutically acceptable carrier as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

(111) The term pharmaceutically acceptable composition as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

(112) The term pharmaceutically acceptable prodrugs as used herein represents those prodrugs of the compounds of the present invention that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of Formula I or Formula II. A discussion is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, ACS Symposium Series, Vol. 14, and in Roche, E. B., ed. Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

(113) The term pharmaceutically acceptable salt(s) refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, matate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

(114) In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.

(115) The compounds of Formula I and Formula II may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term stereoisomers when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols R or S, depending on the configuration of substituents around the stereogenic carbon atom. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated () in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

(116) Individual stereoisomers of compounds for use in the methods of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

(117) Geometric isomers can also exist in the compounds of Formula I and Formula II. The present invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the Z or E configuration wherein the terms Z and E are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the E and Z isomers.

(118) Substituents around a carbon-carbon double bond alternatively can be referred to as cis or trans, where cis represents substituents on the same side of the double bond and trans represents substituents on opposite sides of the double bond. The arrangements of substituents around a carbocyclic ring are designated as cis or trans. The term cis represents substituents on the same side of the plane of the ring and the term trans represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated cis/trans.

(119) The compounds of Formula I and Formula II disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted. For example, any claim to compound A below is understood to include tautomeric structure B, and vice versa, as well as mixtures thereof.

(120) ##STR00020##

(121) As used herein, complement-associated disease, complement-associated disorder and complement-associated condition refers to diseases, disorders and conditions mediated by aberrant activity of one or more of the components of the complement cascade and its associated systems. Exemplary complement-associated diseases include, but are not limited to, atherosclerosis, membranous glomerulonephritis, asthma, organ transplantation rejection, thrombosis, deep vein thrombosis, disseminated venous thromboembolism, disseminated intravascular coagulation, and chronic obstructive pulmonary disease (COPD). Additional exemplary complement-associated diseases include, but are not limited to, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, amyotrophic lateral sclerosis, macular degeneration, lupus nephritis, myasthenia gravis, neuromyelitis optica, anti-phospholipid syndrome, catastrophic anti-phospholipid syndrome, dense deposit disease (type II membranoproliferative glomerulonephritis), Shiga-like toxin-producing E. coli hemolytic uremic syndrome, and abdominal and thoracic aortic aneurysms. Further exemplary complement-associated diseases include, but are not limited to, familial CD59 deficiency, cold agglutinin disease, familial C3 glomerulopathy, C3 glomerulonephritis, complement factor H related protein 5 nephropathy, IgA nephropathy, and hereditary angioedema (HAE).

(122) Subject refers to an animal, such as a mammal, that has been or will be the object of treatment, observation, or experiment. The methods described herein may be useful for both human therapy and veterinary applications. In one embodiment, the subject is a human.

(123) As used herein, treatment or treating refers to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, treatment or treating refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In yet another embodiment, treatment or treating refers to reducing the progression of a disease or disorder, either physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both. In yet another embodiment, treatment or treating refers to delaying the onset of a disease or disorder. For example, treating a cholesterol disorder may comprise decreasing blood cholesterol levels.

(124) As used herein, prevention or preventing refers to a reduction of the risk of acquiring a given disease or disorder or a symptom of a given disease or disorder.

(125) As used herein, modulate, modulation or modulating refers to a downregulation of expression of components of the complement cascade resulting in reduced activity of the complement pathway.

(126) Pharmaceutical Compositions

(127) In certain embodiments, the compound of Formula I or Formula II (or a tautomer, stereoisomer, pharmaceutically acceptable salt, or hydrate thereof) is formulated for oral administration. Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, tablets, or patches, each containing a predetermined amount of a compound of the present disclosure as powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association at least one compound of the present disclosure as the active compound and a carrier or excipient (which may constitute one or more accessory ingredients). The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and must not be deleterious to the recipient. The carrier may be a solid or a liquid, or both, and may be formulated with at least one compound described herein as the active compound in a unit-dose formulation, for example, a tablet, which may contain from about 0.05% to about 95% by weight of the at least one active compound. Other pharmacologically active substances may also be present including other compounds. The formulations of the present disclosure may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.

(128) For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmacologically administrable compositions can, for example, be prepared by, for example, dissolving or dispersing, at least one active compound of the present disclosure as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution, ointment, or suspension. In general, suitable formulations may be prepared by uniformly and intimately admixing at least one active compound of the present disclosure with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, a tablet may be prepared by compressing or molding a powder or granules of at least one compound of the present disclosure, which may be optionally combined with one or more accessory ingredients.

(129) Compressed tablets may be prepared by compressing, in a suitable machine, at least one compound of the present disclosure in a free-flowing form, such as a powder or granules, which may be optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, where the powdered form of at least one compound of the present disclosure is moistened with an inert liquid diluent.

(130) Formulations suitable for buccal (sub-lingual) administration include lozenges comprising at least one compound of the present disclosure in a flavored base, usually sucrose and acacia or tragacanth, and pastilles comprising the at least one compound in an inert base such as gelatin and glycerin or sucrose and acacia.

(131) The amount of active compound administered may be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician. For example, a dosing schedule may involve the daily or twice-daily administration of the encapsulated compound or compounds at a dosage of about 1-100 mg or 100-300 mg of a compound of Formula I or Formula II (or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof).

(132) In another embodiment, intermittent administration, such as on a monthly or yearly basis, of a dose of the encapsulated compound may be employed. Encapsulation facilitates access to the site of action and allows the administration of the active ingredients simultaneously, in theory producing a synergistic effect. In accordance with standard dosing regimens, physicians will readily determine optimum dosages and will be able to readily modify administration to achieve such dosages.

(133) A therapeutically effective amount of a compound or composition disclosed herein can be measured by the therapeutic effectiveness of the compound. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being used. In one embodiment, the therapeutically effective amount of a disclosed compound is sufficient to establish a maximal plasma concentration. Preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.

(134) Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compositions that exhibit large therapeutic indices are preferable.

(135) Data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. Therapeutically effective dosages achieved in one animal model may be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et al., Cancer Chemother. Reports 50(4):219-244 (1966) and Table 1 for Equivalent Surface Area Dosage Factors).

(136) TABLE-US-00001 TABLE 1 Equivalent Surface Area Dosage Factors To: Mouse Rat Monkey Dog Human From: (20 g) (150 g) (3.5 kg) (8 kg) (60 kg) Mouse 1 1/2 1/4 1/6 1/12 Rat 2 1 1/2 1/4 1/7 Monkey 4 2 1 3/5 1/3 Dog 6 4 3/5 1 1/2 Human 12 7 3 2 1

(137) The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Generally, a therapeutically effective amount may vary with the subject's age, condition, and gender, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

(138) Methods of Treatment

(139) The invention provides methods for modulating the complement system in a patient in need thereof. In some embodiments, the methods comprise treating or preventing complement-associated diseases or disorders by administering to a subject (e.g., a mammal, such as e.g., a human) a therapeutically effective amount of at least one compound of the invention, i.e., a compound of Formula I or Formula II, or a tautomer, stereoisomer, pharmaceutically acceptable salt, or hydrate thereof. In certain embodiments, the methods of the invention comprise administering a pharmaceutically acceptable composition, comprising one or more compounds of Formula I or Formula II and a pharmaceutically acceptable carrier.

(140) The invention further provides a method for treating or preventing a complement-associated disease or disorder involving the modulation of one or more genes selected from, for example, Mannose-Binding Lectin (protein C) 2, complement component 9, complement component 6, complement component 8, alpha polypeptide, complement component 4B, complement component 4A, coagulation factor IX, Coagulation factor VII, complement component 4 binding protein-beta, complement component 5, Protein C, coagulation factor XI, kallikrein B, plasma, tissue factor pathway inhibitor, complement component 8, gamma polypeptide, complement component 1-s subcomponent, complement component 8-beta polypeptide, coagulation factor XII, coagulation factor II, coagulation factor XIII B polypeptide, serpin peptidase inhibitor clade E, complement component 2, alpha-2-macroglobulin, complement factor H, complement factor I, complement factor B, complement component 1 R subcomponent, mannan-binding lectin serine peptidase 1, protein S, coagulation factor V, complement component 5a receptor 1, complement component 4 binding protein alpha, serpin peptidase inhibitor clade C member 1, complement component 3, mannan-binding lectin serine peptidase 2, coagulation factor X, coagulation factor VIII, serpin peptidase inhibitor clade D member 1, serpin peptidase inhibitor clade F member 2, plasminogen, bradykinin receptor B2, bradykinin receptor B1, serpin peptidase inhibitor clade A member 5, coagulation factor III, serpin peptidase inhibitor, clade G (C1 inhibitor) member 1, carboxypeptidase B2 (Plasma), fibrinogen beta chain, kininogen 1, complement component (3b/4b) receptor 1, plasminogen activator tissue, complement component (3d/epstein barr virus) receptor 2, thrombomodulin, CD55 molecule, decay accelerating factor for complement, complement component 1 Q subcomponent A chain, or complement component 7, plasminogen activator urokinase, complement factor D, complement component 1 Q subcomponent C chain, CD46 molecule complement regulatory protein, fibrinogen gamma chain, von willebrand factor, CD59 molecule complement regulatory, plasminogen activator urokinase receptor, serpin peptidase inhibitor clade A member 1, coagulation factor XIII A1 polypeptide, complement component 3a receptor 1, fibrinogen alpha chain, complement component 1 Q subcomponent, B chain, and/or coagulation factor II (thrombin) receptor, by administering a therapeutically effective amount of at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

(141) Another embodiment comprises a method for treating or preventing a complement-associated disease or disorder involving the modulation of one or more genes selected from, Mannose-Binding Lectin (protein C) 2, complement component 3, complement component 5, complement factor D, complement factor H, and/or complement component 9.

(142) In one embodiment, the method comprises administering at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, to a subject, such as a human, as a preventative against complement-associated diseases and disorders, such as, for example, atherosclerosis, membranous glomerulonephritis, asthma, organ transplantation rejection, thrombosis, deep vein thrombosis, disseminated venous thromboembolism, disseminated intravascular coagulation, and chronic obstructive pulmonary disease (COPD).

(143) In another embodiment, the method comprises administering at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, to a subject, such as a human, as a preventative against complement-associated diseases and disorders, such as, for example, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, amyotrophic lateral sclerosis, macular degeneration, lupus nephritis, myasthenia gravis, neuromyelitis optica, anti-phospholipid syndrome, catastrophic anti-phospholipid syndrome, dense deposit disease (type II membranoproliferative glomerulonephritis), Shiga-like toxin-producing E. coli hemolytic uremic syndrome, and abdominal and thoracic aortic aneurysms.

(144) In another embodiment, the method comprises administering at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, to a subject, such as a human, as a preventative against complement-associated diseases and disorders, such as, for example, familial CD59 deficiency, cold agglutinin disease, familial C3 glomerulopathy, C3 glomerulonephritis, complement factor H related protein 5 nephropathy, IgA nephropathy, and hereditary angioedema (HAE).

(145) In one embodiment, at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is administered as a preventative to a subject, such as a human, having a genetic predisposition to complement-associated diseases and disorders, such as, for example, atherosclerosis, membranous glomerulonephritis, asthma, organ transplantation rejection, thrombosis, deep vein thrombosis, disseminated venous thromboembolism, disseminated intravascular coagulation, and chronic obstructive pulmonary disease (COPD).

(146) In another embodiment, at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is administered as a preventative measure to a subject, such as a human, having a genetic predisposition to complement-associated diseases and disorders, such as, for example, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, amyotrophic lateral sclerosis, macular degeneration, lupus nephritis, myasthenia gravis, neuromyelitis optica, anti-phospholipid syndrome, catastrophic anti-phospholipid syndrome, dense deposit disease (type II membranoproliferative glomerulonephritis), Shiga-like toxin-producing E. coli hemolytic uremic syndrome, and abdominal and thoracic aortic aneurysms.

(147) In another embodiment, at least one compound of Formula I or Formula II or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, is administered as a preventative measure to a subject, such as a human, having a genetic predisposition to complement-associated diseases and disorders, such as, for example, familial CD59 deficiency, cold agglutinin disease, familial C3 glomerulopathy, C3 glomerulonephritis, complement factor H related protein 5 nephropathy, IgA nephropathy, and hereditary angioedema (HAE)

(148) In another embodiment, the compounds of Formula I or Formula II may be used for the prevention of one complement-associated disease or disorder while concurrently treating another.

EXAMPLES

Example 1: Gene Expression Changes

(149) In this example, mRNA levels from cultured cells were quantitated. The assay can be used to determine the effect of compound(s) on regulating mRNA levels, including those compounds in the present invention. Complement genes are expressed at high endogenous levels, but their expression can also be stimulated with various cytokines in inflammatory conditions. Experiments in this example target both basal and inflammatory complement gene expression. Compound mediated changes in gene expression and resulting mRNA levels are presented in Tables 2, 3 and 4 as well as FIGS. 1, 2, 3 and 4 below.

(150) Huh-7 and HepG2 cells are liver-derived cell lines and are models for what can occur in the liver. Huh-7 cells (JCRB Cell Bank) were introduced to 96-well plates (2.510.sup.5 per well) in 100 L DMEM containing 10% (v/v) FBS, 100 U/mL penicillin, 100 ug/mL streptomycin and 5 ug/mL plasmocin (all reagents from Gibco, except for the former, which was obtained from Invivogen). After 24 h, Huh-7 cells were treated with compounds in the same media formulation used for plating, and supplemented with 0.1% DMSO for the amount of time indicated in tables 2, 3, and 4. For select experiments, 24 h post-plating, cells were treated with cytokines and the compound of interest simultaneously, for a total treatment time of 48 h. Alternatively, 24 h after plating, cells were pre-treated with cytokines for 24 h before adding the compound of interest for 48 h. HepG2 cells (ATCC) were cultured in 96-well plates (2.510.sup.5 per well) in MEM containing 10% FBS, 1 non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin and 5 ug/mL plasmocin. Serum amount was reduced to 0.5% for treatments with compound or cytokines. Timing of treatment of HepG2 cells with compounds and cytokines was as described for Huh-7 cells. Primary human hepatocytes (CellzDirect/Life Technologies) were plated in collagen coated 96-well plates at 70 000 cells/well, then overlaid with Matrigel as recommended by the supplier. Cells were treated with compounds of interest and/or cytokines for the indicated time points in the recommended media supplemented with 0.1% DMSO and 10% FBS (v/v). Cells were harvested by mRNA Catcher PLUS Kit (Life Technologies) followed by real-time PCR using the RNA UltraSense One-Step qRT-PCR System. The level of the mRNA of interest was measured by TaqMan real-time PCR relative to the endogenous control cyclophilin A in the same sample. Data were acquired using the ViiA-7 Real Time PCR System (Applied Biosystems).

(151) Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result. Tables 2 and 3 list the concentration of compounds at which the level of the indicated mRNA is reduced by 50%, as well as the duration of treatment with compound. Table 4 lists the maximum reduction in the indicated mRNA measured in primary human hepatocytes treated with compounds for up to 72 hours. FIGS. 1, 2, 3 and 4 show effects of compounds on cytokine-induced (i.e. inflammatory) expression of complement genes in Huh-7 cells and HepG2 cells.

(152) In addition to genes shown in Tables 2 and 4, other members of the complement and coagulation cascades are assayed via real-time PCR in cultured cells such as, but not limited to, Huh-7, HepG2 and/or primary human hepatocytes.

(153) TABLE-US-00002 TABLE 2 Suppression of complement gene expression in human hepatoma cells. Data are presented as half maximal inhibitory concentrations (IC50) of compounds in micromolar (uM). RVX000222 JQ1 RVX000297 RVX002109 RVX002135 Complement gene Huh-7 HepG2 Huh-7 HepG2 Huh-7 C3 (48 h) 1.90 0.20 0.20 4.70 34.8 2.20 C4a/C4b (48 h) 6.30 4.50 62.2 4.30 C5 (48 h) >30 >10 >50 37.9 C3 (72 h) 25.0 0.49 MBL2 (48 h) 16.3 3.88 0.16 0.03 C1S (48 h) 18.9 9.56 0.3 0.12 C4a/C4b (72 h) 5.40 10.0 0.07 0.25 C5 (72 h) 21.8 20.0 0.27 0.46

(154) TABLE-US-00003 TABLE 3 Suppression of C3 and C4 expression in Huh-7 cells after a 48 h treatment with listed compounds. Data are presented as half maximal inhibitory concentrations (IC50) of compounds in micromolar (uM). mRNA expression in Huh-7 cells: IC.sub.50 (uM) Compound C3 C4 RVX000206 3.6 9.3 RVX000255 20.4 17.4 RVX000344 11.6 15.6 RVX000641 12.6 9.3 RVX000662 29.2 21.1 RVX000668 9.5 15.6 RVX000843 11.1 >50 uM RVX002093 18.5 17.2 RVX002101 11.3 22.0 RVX002103 13.9 13.8 RVX002113 5.8 7.8 RVX002141 15.4 17.8 JQ1 0.18 0.065

(155) TABLE-US-00004 TABLE 4 Downregulation of expression of complement components in primary human hepatocytes from a single donor. mRNA levels were determined at 6, 24, 48, and 72 hours of compound treatment. Values show the percent maximal reduction in gene expression and the associated treatment period (hours). Over this time course, maximum reduction in complement C3, complement C5, and MBL2 mRNA abundance was observed at 24 hours of treatment, and at 72 hours of treatment for complement C1S and complement C2 mRNA levels. Maximum reduction in complement C4 mRNA was observed at 24 hours of treatment with JQ1, versus 72 hours of RVX000222 treatment. Maximum reduction in complement C9 mRNA was found with 48 hours of treatment with JQ1 and 72 hours with RVX000222. Differences in treatment period required for maximum reduction in mRNA levels may be related to mRNA half-life or sensitivity of a particular gene to BET inhibition. Treatment C3 C4 C5 C9 MBL2 C1S C2 30 uM RVX000222 13% (24 h) 44% (72 h) 38% (24 h) 86% (72 h) 92% (24 h) 27% (72 h) 35% (72 h) 0.3 uM JQ1 54% (24 h) 31% (24 h) 67% (24 h) 72% (48 h) 93% (24 h) 22% (72 h) 29% (72 h)

Example 2: In Vivo Studies Using Mouse Models

(156) In this example, chimeric mice with humanized livers were generated by transplanting human hepatocytes into urokinase-type plasminogen activator.sup.+/+/severe combined immunodeficient transgenic mice. Replacement with human hepatocytes can reach 80-90%. This mouse model can be used to determine the effect of compounds, including those compounds in the present invention, on regulating mRNA levels in human hepatocytes in vivo. Mice were treated with 150 mg/kg b.i.d. with RVX000222 or vehicle by oral gavage for 3 days. Livers were harvested and RNA levels determined by real-time PCR using human specific TaqMan primer probes and cyclophilin A as an endogenous control. Table 5 lists the reduction in the levels of the indicated mRNAs. *p<0.05, **p<0.01 versus vehicle treated animals using 2-tailed student's t-tests.

(157) TABLE-US-00005 TABLE 5 RVX000222 reduces mRNA expression levels of complement components 3 (C3), 4 (C4) and 5 (C5) and mannose-binding lectin 2 (MBL2) in humanized livers of chimeric mice treated with 150 mg/kg b.i.d. for 3 days. Numbers represent average % reduction in expression relative to vehicle treated mice (3 mice per group). Compound C3 C4 C5 C9 MBL2 RVX000222 20% 36%* 17% 45%** 61%** Asterisk indicates p < 0.05; two asterisks indicate p < 0.01.

Example 3: Microarray Analysis in Whole Blood

(158) In this example, RNA from human whole blood treated ex vivo was analyzed by microarray. The method can be used to determine the effect of compounds, including those in the present invention, on RNA levels (Table 6).

(159) After obtaining informed consent, whole blood was collected from three healthy volunteers into BD Vacutainer Sodium Heparin tubes and samples were inverted 10 times. Blood samples (1 mL) were combined with 1 mL of RPMI containing 2 mM glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 20% FBS and the compound of interest or vehicle (0.1% DMSO), followed by a 24 h incubation at 37 C. Treated samples were transferred to a PAXgene RNA tube (PreAnalytix/Qiagen), inverted 5 times and frozen. RNA was isolated with the PAXgene RNA kit according to manufacturer's instructions. Microarray analysis was performed by Asuragen (Austin, Tex.) using the Affymetrix Human U133 Plus 2.4 Array. Shown in Table 6 is the mean of 3 independent samples (p<0.01). Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result. Upregulation of negative regulators or downregulation of positive regulators of the pathway will also result in reduced activity of the pathway and thus will constitute a positive result.

(160) TABLE-US-00006 TABLE 6 20 uM RVX000222 alters complement component 3, CD55 and CD59 mRNA levels in ex-vivo treated human blood. Gene % change in expression Complement component 3 (C3) 59% CD55 58% CD59 88%

Example 4: Measure of Secreted Complement Proteins

(161) In this example, protein secretion from cells grown in culture in the presence of compound of interest was analyzed by enzyme linked immunosorbent assay (ELISA). In some cases, cultured cells were treated with cytokines and the compound of interest to mimic an inflammatory state. The method can be used to determine the effect of compounds, including those in the present invention, on the secretion of specific proteins from cells grown in culture under basal and cytokine stimulated (i.e. inflammatory) conditions (Table 7, FIGS. 5 and 6).

(162) Huh-7 cells (JCRB Cell Bank) were introduced to 24-well plates in 500 L DMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 ug/mL streptomycin and 5 ug/mL plasmocin (all reagents from Gibco, except for the former, which comes from Invivogen) at 200 000 cells/well. After 24 h, cells were treated with the compound of interest and/or cytokines in DMEM with 10% FBS containing 0.1% DMSO for a total treatment time of 72 h. Fresh media containing compounds and/or cytokines was introduced in the final 24 h of the experiment. At harvest, media were collected, debris was removed by brief centrifugation, and ELISA assays for the indicated proteins were performed as per the manufacturer's protocol. To correct for differences in cell numbers, values obtained for complement proteins were normalized to values for transferrin. HepG2 cells (ATCC) were cultured in MEM containing 10% FBS, 1 non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin and 5 ug/mL plasmocin. Serum amount was reduced to 0.5% when compounds were present. Treatment combinations and timing were as described above for Huh-7 cells. Primary human hepatocytes (CellzDirect/Life Technologies) were plated in collagen coated 96-well plates at 70 000 cells/well, then overlaid with Matrigel as recommended by the supplier. Cells were treated with compounds of interest with or without the indicated cytokines for a total of 72 h in the recommended media supplemented with 10% FBS and 0.1% DMSO (v/v). Media were collected for measurements of secreted proteins.

(163) The ELISA kits for detection of complement C3, C4, C5 and C9 were obtained from AssayPro (St. Charles, Mo.), while the ELISA reagents for transferrin detection were from Bethyl Laboratories (Montgomery, Tex.). Data were collected on a Thermo Scientific Multiskan GO apparatus. Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result. Table 7: FIGS. 5 and 6 list the amount of C3, C4, C5 and C9 protein that are detected in the media of cells treated with RVX000222 (compared to DMSO).

(164) Quantitation of additional secreted proteins from cultured cells using the ELISA method is being evaluated. This includes, but is not limited to, complement C6, C8, MBL2 or Factor H.

(165) TABLE-US-00007 TABLE 7 Secretion of complement C3, C4, and C5 in Huh-7 and HepG2 cells. Data are the percent maximum reduction in protein levels with standard deviation derived from three independent experiments. C3 C4 C5 Treatment Huh-7 HepG2 Huh-7 HepG2 Huh-7 HepG2 30 uM RVX000222 87 9% 34 5% 79 3% 65 5% 53 7% 77 4% 0.75 uM JQ1 99 1% 54 5% 81 6% 81 9% 50 13% 82 7%

Example 5: Multi-Analyte Profiling

(166) In this example, plasma samples from human subjects treated with placebo or RVX000222 was analyzed by Multi-Analyte Profiling (MAP) technology. The method can be used to determine the effect of compounds, including those in the present invention, on the levels of various analytes in plasma (Table 8).

(167) Plasma collected from twenty RVX000222 treated subjects and ten placebo treated subjects at baseline and terminal time points (26 weeks) (from the previously completed ASSURE clinical trial; NCT01067820, was sent for MAP analysis. Using microsphere-based immuno-multiplexing, each sample was analyzed and the level of 107 different plasma proteins quantitated. The changes in values for each protein analyte were calculated versus the baseline measure, and statistically significant (p<0.05) and trending (0.01>p>0.05) values reported. Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result. Table 8 summarizes changes in plasma analytes observed with 26 week treatment with RVX000222.

(168) TABLE-US-00008 TABLE 8 Changes in plasma analytes from the ASSURE trial (NCT01067820) measured using multi-analyte profiling (week 26 vs. baseline p < 0.05) Change Percent Change P-value Analyte N Baseline Units from baseline from baseline vs baseline Complement Factor H (CFH) 20 570.3 ug/mL 75.35 11.45 0.01 Complement Factor H - 17 2353.5 ug/mL 291.76 10.75 0.003 Related Protein 1 (CFHR1) Complement C3 (C3) 20 1.1 mg/mL 0.1 9.28 0.002

Example 6: Protein Quantitation Using LC-MRM/MS

(169) In this example, plasma samples from human subjects treated with placebo or RVX000222 were analyzed by 1D LC-MRM/MS technology. The method can be used to determine the effect of compounds, including those in the present invention, on the levels of various analytes found in plasma.

(170) Plasma collected from 74 RVX000222 treated subjects and 17 placebo treated subjects at baseline and terminal time points (26 weeks) (from the previously completed ASSURE clinical trial; NCT01067820) was sent for absolute protein quantification. Using mass spectrometric methods, including multiple reaction monitoring (MRM) mass spectrometry (MRM-MS), each sample is analyzed for the presence and amount of 43 different plasma proteins. The changes in values for each protein analyte are calculated versus the baseline measure, and statistically significant (p<0.05) and trending (0.10>p>0.05) values are reported (Table 9). Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result.

(171) TABLE-US-00009 TABLE 9 Changes in plasma analytes from the ASSURE trial (NCT01067820) measured using LC-MRM/MS (week 26 vs. baseline p < 0.10) Change Percent from Change P-value Baseline baseline from vs Protein Peptide (ng/mL) (ng/mL) baseline baseline Complement component C9 37,592 5,034 13.8 0.0001 Complement component C8 1,828 120 8.6 0.0001 alpha chain Complement C5 34,391 2,228 5.3 0.0001 Complement factor 1 17,911 1,205 5.2 0.002 Complement C4-B 159,041 7,492 4.3 0.001 Complement factor H 206,226 8,710 4.2 0.0001 Complement component C8 5,907 341 3.9 0.004 beta chain Complement C3 769,753 34,105 3.6 0.02 Complement C1r sub- 122,295 571 3.6 0.02 component Complement factor B 131,959 4,259 4.2 0.06 Note: Results are shown for peptide with highest concentration; italics indicate median

Example 7: Complement Activity Assays

(172) In this example, serum samples from human subjects treated with placebo or RVX000222 were analyzed by the total hemolytic complement (CH50) assay and the complement alternative pathway (AH50) assay. The method can be used to determine the effect of compounds, including those in the present invention, on the activity of the classical and alternative complement system in clinical samples.

(173) Serum collected from RVX000222 treated subjects and placebo treated subjects at baseline and terminal time points (26 weeks) (from the previously completed ASSURE clinical trial; NCT01067820), was analyzed in the AH50 and CH50 assays. Using the CH50 screening assay to detect the hemolysis of sheep erythrocytes sensitized by specific antibodies, the hemolytic activity of the complement system in serum samples from treated and untreated subjects was measured. Likewise, using specific conditions to activate only the alternative pathway (AH50), activity of the complement response was measured. The degree of complement activation was measured at baseline and terminally to determine if there were any changes in the function of the complement system following drug treatment (FIG. 7). Reduced function of the complement system constitutes a positive result.

Example 8: Protein Quantitation in Clinical Samples Using SOMAScan

(174) In this example, plasma samples from human subjects treated with placebo or RVX000222 are analyzed by the SOMAscan assay (SomaLogic). The method can be used to determine the effect of compounds, including those in the present invention, on the abundance of proteins, including complement components, in clinical samples.

(175) Plasma collected from 47 RVX000222 treated subjects at baseline and terminal time points (26 weeks) (from the previously completed ASSURE clinical trial; NCT01067820) was sent for analysis. Using the SOMAscan technology, each sample is analyzed for the relative presence and amount of 1,310 different proteins. The changes in values for each protein analyte are calculated versus the baseline measure, and statistically significant (p<0.05) values are reported (Table 10). Downregulation of expression of components of the complement cascade will result in reduced activity of the pathway and thus will constitute a positive result.

(176) TABLE-US-00010 TABLE 10 Changes in serum analytes from the ASSURE trial measured using SOMAscan (week 26 vs. baseline p < 0.05). RVX-208 at 200 mg/day Gene % change vs. p-value vs Protein Name Symbol baseline baseline Complement C3b C3 52.7 0.001 C-reactive protein CRP 43.6 0.0001 C5a anaphylatoxin C5 28.7 0.0002 Complement component C9 C9 18.3 0.0001 Mannose-binding protein C MBL2 14.6 0.0002 Complement component C6 C6 14.3 0.0001 Complement C5b-C6 complex C5 C6 12.0 0.0001 Complement C5 C5 11.7 0.0001 Complement component C8 C8 10.1 0.004 Complement factor B CFB 6.8 0.001 Complement C2 C2 6.7 0.001 Complement factor I CFI 6.4 0.01 Complement C1s subcomponent C1S 6.1 0.02 Complement factor H CFH 5.6 0.0001 Complement decay-accelerating CD55 4.0 0.02 factor Mannan-binding lectin serine MASP1 4.8 0.04 protease 1

(177) All references referred to herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.