WO2009055002A1 - Combined inhibitors of cyclooxygenase and semicarbazide-sensitive amine oxidase (ssao) (vascular adhesion protein, vap-1) - Google Patents

Abstract

Compounds are disclosed which inhibit both semicarbazide-sensitive amine oxidase (SSAO) activity and cyclooxygenase (COX) activity. The compounds combine residues or pharmacophores of SSAO inhibitors with residues or pharmacophores of COX inhibitors in order to create the dual inhibitors. Methods of treatment of diseases, such as inflammation, are also disclosed.

Description

COMBINED INHIBITORS OF CYCLOOXYGENASE AND SEMICARBAZIDE-SENSITIVE AMINE OXIDASE (SSAO) (VASCULAR

ADHESION PROTEIN, VAP l)

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the priority benefit of United States

Provisional Patent Application No. 60/982,356 filed October 24, 2007. The entire contents of that application are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT [0002] Not applicable.

TECHNICAL FIELD

[0003] This application relates to compounds which inhibit both cyclooxygenase (COX) enzymes and semicarbazide-sensitive amine oxidase (SSAO, also known as vascular adhesion protein- 1 or VAP-I), for treatment of inflammation, inflammatory diseases and autoimmune disorders.

BACKGROUND

[0004] Cyclooxygenase (COX) is a general term for a group of enzymes known to be involved in inflammatory responses. Cyclooxygenase exists in at least two isoforms, COX-I and COX-2. (A third isoform, COX-3 or COX-Ib, arises from a splice variant and subsequent frameshift mutation in COX-I, although this isoform appears to have activity different from COX-I and COX-2.) Inhibitors of COX enzymes have anti-inflammatory, anti-pyretic, and analgesic effects. Such inhibitors include non-steroidal anti-inflammatory drugs (NSAIDs), a class that contains well- known drugs such as aspirin, ibuprofen, and celecoxib (Celebrex®, a trademark of Pfizer Corp., New York, NY).

[0005] Many other enzymes are also involved in inflammatory responses; one such enzyme is designated semicarbazide-sensitive amine oxidase (SSAO). Cloning and sequencing of the SSAO gene showed that it was identical to the sequence of human vascular adhesion protein- 1 (VAP-I), a type 2, 180 kD homodimeric endothelial cell adhesion molecule. Both the membrane-bound VAP-I protein and the soluble SSAO enzyme have amine oxidase enzymatic activity. Membrane-bound VAP-I can function both as an amine oxidase and a cell adhesion molecule, while SSAO, as its name implies, functions as an amine oxidase. Information regarding SSAO and some molecules which have been proposed as SSAO inhibitors for treating various diseases can be found in US 6,624,202, US 2003/0125360, WO 2003/06003, WO 98/53049, US 5,512,442, US 5,580,780, US 6,066,321, WO 93/25582, WO 2005/014530, US 2005/0096360, WO 2005/082343, US 2006/0025438, PCT/US2007/008187, and United States Patent Application No. 11/731 ,819. [0006] The current invention describes a novel approach to generate nonsteroidal anti-inflammatory drugs with greater efficacy than current NSAIDs by combining critical chemical elements of cyclooxygenase (COX) inhibitors with chemical scaffolds that provide semicarbazide sensitive amine oxidase (SSAO) inhibition into a single molecular entity, i.e., a dual-acting enzyme inhibitor. The combined entity uses COX inhibition to target soluble pathways of inflammation with SSAO inhibition to target cellular pathways of inflammation. Thus, dual-acting COX/SSAO enzyme inhibitors provide the anti-inflammatory, analgesic and antipyretic activities associated with COX inhibition as well as the anti-inflammatory activities of SSAO, affording the potential for a synergistic effect in inhibiting inflammation.

SUMMARY OF THE INVENTION

[0007] SSAO inhibitors can block inflammation and autoimmune processes, as well as other pathological conditions associated with an increased level of the circulating amine substrates and/or products of SSAO. Cyclooxygenase inhibitors, such as COX-I and COX-2 inhibitors, can also block inflammatory processes. [0008] In one embodiment, the invention embraces combined SSAO/COX inhibitors. The combined SSAO/COX inhibitors can be formed from the residue of an SSAO inhibitor covalently linked to a residue of a COX inhibitor. The combined SSAO/COX inhibitors can be formed from a pharmacophore of an SSAO inhibitor covalently linked to the residue of a COX inhibitor. The combined SSAO/COX inhibitors can be formed from a pharmacophore of a COX inhibitor covalently linked to the residue of an SSAO inhibitor. The combined S SAO/COX inhibitors can be formed from a pharmacophore of an SSAO inhibitor covalently linked to a pharmacophore of a COX inhibitor. The combined SSAO/COX inhibitors can be formed from a pharmacophore of an SSAO inhibitor combined with a pharmacophore of a COX inhibitor, where the two pharmacophores share a similar or identical functional group which form a group of overlapping atoms, and where the similar or identical functional group is present in the combined inhibitors. [0009] The invention also embraces any salt of any of the foregoing embodiments. The invention also embraces any solvate of the foregoing embodiments, such as hydrates. The invention also embraces any enantiomer or diastereomer of the foregoing embodiments.

[0010] In another embodiment, the combined SSAO/COX inhibitor is selected from compounds of formula I:

or of formula II:

where R, is selected from -COOH, -CH₂COOH, and -CH(CH₃)COOH; and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

[0011] In another embodiment, the combined SSAO/COX inhibitor is selected from compounds of the formulas

where R₂ is selected from

and

where the asterisk (*) indicates the point of attachment of the R2 group to the remainder of the molecule, and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

[0012] In another embodiment, the residue of the SSAO inhibitor used for the combined inhibitor is derived from

and

[0013] In another embodiment, the SSAO pharmacophore used for the combined inhibitor is selected from

[0014] In another embodiment, the residue of the COX inhibitor used for the combined inhibitor is derived from carprofen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, loxoprofen, naproxen, naproxen sodium, oxaprozin, vedaprofen, acetylsalicylic acid, salicylic acid, choline salicylate, diflunisal, bendazac, diclofenac, etodolac, ibuprofen, ketorolac, nabumetone, sulindac, tolmetin, meclofenamic acid, meclofenamate sodium, and tolfenamic acid, phenylacetic acids, flunixin, indomethacin, nabumetone, ketorolac, and etodolac.

[0015] In another embodiment, the COX pharmacophore used for the combined inhibitor is selected from

and

[0016] In another embodiment, the COX pharmacophore used for the combined inhibitor is selected from -COOH, -CH₂COOH, and -CH(CH₃)COOH. [0017] In another embodiment, the combined SSAO/COX inhibitor is selected from the group consisting of:

and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

[0018] In another embodiment, a direct covalent linkage can link a residue of an SSAO inhibitor with a residue of a COX inhibitor. In another embodiment, a direct covalent linkage can link a pharmacophore of an SSAO inhibitor to the residue of a COX inhibitor. In another embodiment, a direct covalent linkage can link a pharmacophore of a COX inhibitor to the residue of an SSAO inhibitor. In another embodiment, a spacer can covalently link a residue of an SSAO inhibitor with a residue of a COX inhibitor. In another embodiment, a spacer can covalently link a pharmacophore of an SSAO inhibitor to the residue of a COX inhibitor. In another embodiment, a spacer can covalently link a pharmacophore of a COX inhibitor to the residue of an SSAO inhibitor. In another embodiment, the spacer is a Ci-Ci₂ substituted or unsubstituted alkylene or heteroalkylene chain, where the chain can be branched or linear. In another embodiment, the spacer is selected from methylene (-CH₂-), ethylene (-CH₂CH₂-), branched or linear propylene(-CH₂CH₂CH₂- or -CH(CH₃)-CH₂-), branched or linear butylene, branched or linear pentylene, branched or linear hexylene, branched or linear heptylene, branched or linear octylene, branched or linear nonylene, branched or linear decylene, branched or linear undecylene, branched or linear dodecylene, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, and hexaethylene glycol.

[0019] In another embodiment, the invention embraces treatment of inflammation, inflammatory responses, inflammatory diseases, or immune disorders by administering a therapeutically effective amount of a combined SSAO/COX inhibitor. In another embodiment, the invention embraces treatment of multiple sclerosis (including chronic multiple sclerosis); synovitis; systemic inflammatory sepsis; inflammatory bowel diseases; Crohn's disease; ulcerative colitis; Alzheimer's disease; atherosclerosis; rheumatoid arthritis; juvenile rheumatoid arthritis; pulmonary inflammatory conditions; asthma; skin inflammatory conditions and diseases; contact dermatitis; liver inflammatory and autoimmune conditions; autoimmune hepatitis; primary biliary cirrhosis; sclerosing cholangitis; autoimmune cholangitis; alcoholic liver disease; Type I diabetes and/or complications thereof; Type II diabetes and/or complications thereof; atherosclerosis; ischemic diseases such as stroke and/or complications thereof; myocardial infarction; acute pain; chronic pain; signs and symptoms of osteoarthritis; chronic obstructive pulmonary disorder (COPD); and acute respiratory distress syndrome (ARDS); by administering a therapeutically effective amount of a combined SSAO/COX inhibitor. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is multiple sclerosis. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is chronic multiple sclerosis. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is the inflammatory complications resulting from stroke. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is signs and symptoms of osteoarthritis.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to combined inhibitors of semicarbazide- sensitive amine oxidase and cyclooxygenase (COX). The SSAO inhibitors can be inhibitors of the SSAO activity of soluble SSAO, the SSAO activity of membrane- bound VAP-I, binding to membrane-bound VAP-I, or any two of those activities, or all three of those activities. The COX inhibitors can be inhibitors of COX-I, COX-2, COX-3 (COX-Ib), or any two of those enzymes, or all three of those enzymes. The combined inhibitors can have any one or more of the functions of the separate inhibitors. While the current invention contemplates using the molecules disclosed herein as both SSAO and COX inhibitors, novel molecules disclosed herein that display only SSAO-inhibitory activity can be used as SSAO inhibitors, while novel molecules disclosed herein that display only COX-inhibitory activity can be used as COX inhibitors.

[0021] A "residue of an SSAO inhibitor" is defined as the remaining portion of an SSAO inhibitor molecule after an atom or a functional group has been removed or modified to open a valence for attachment of said residue of an SSAO inhibitor to another atom, functional group, or molecular fragment. The molecular fragment can be the residue of an COX inhibitor.

[0022] A "residue of a COX inhibitor" or "residue of a cyclooxygenase inhibitor" is defined as the remaining portion of a COX inhibitor molecule after an atom or a functional group has been removed or modified to open a valence for attachment of said residue of a COX inhibitor to another atom, functional group, or molecular fragment. The molecular fragment can be the residue of an SSAO inhibitor.

[0023] A "pharmacophore" is defined as an ensemble of steric and/or electronic features that is necessary to ensure interaction(s) with a specific biological target molecule or structure, and that are responsible for the biological activity or biological effect resulting from the interaction(s). A pharmacophore can be provided by a portion of a molecule, where that portion is essential for that molecule's biological activity.

[0024] A "combined SSAO/COX inhibitor" is defined as either the combination of a residue of an SSAO inhibitor with a residue of a COX inhibitor, or as the addition of a pharmacophore of an SSAO inhibitor to the residue of a COX inhibitor, or as the addition of a pharmacophore of a COX inhibitor to the residue of an SSAO inhibitor, or as the addition of a pharmacophore of an SSAO inhibitor to a pharmacophore of a COX inhibitor. The components are combined via a covalent linkage. When combining pharmacophores, the components of one pharmacophore can overlap with a similar or identical unit of the other pharmacophore, so that the same functional group forms a part of two different pharmacophores, as described in more detail below. When combining a residue of an SSAO inhibitor with a residue of a COX inhibitor, or a pharmacophore of an SSAO inhibitor to the residue of a COX inhibitor, or a pharmacophore of a COX inhibitor to the residue of an SSAO inhibitor, the two components can be directly linked covalently, or can be covalently linked by means of a spacer, such as a Ci-Ci₂ substituted or unsubstituted alkylene or heteroalkylene chain, where the chain can be branched or linear. Examples of alkyl chains include, but are not limited to, a methylene (-CH₂-), ethylene (-CH₂CH₂-), propylene (-CH₂CH₂CH₂- or -CH(CH₃)-CH₂-), butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, or dodecylene spacer (where the alkylene chain can be branched or linear). Examples of heteroalkylene chains include, but are not limited to, ethylene glycol (-0-CH₂CH₂-O -), diethylene glycol (-0-CH₂CH₂-O-CH₂CH₂-O-), and tri-, terra-, penta-, and hexa-ethylene glycol linkers. [0025] The invention includes all salts of the compounds described herein, as well as methods of using such salts of the compounds. The invention also includes all non-salt forms of any salt of a compound described herein, as well as other salts of any salt of a compound named herein. In one embodiment, the salts of the compounds comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which retain the biological activity of the free compounds and which can be administered as drugs or pharmaceuticals to humans and/or animals. The desired salt of a basic compound may be prepared by methods known to those of skill in the art by treating the compound with an acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, sulfonic acids, and salicylic acid. Salts of basic compounds with amino acids, such as aspartate salts and glutamate salts, can also be prepared. The desired salt of an acidic compound can be prepared by methods known to those of skill in the art by treating the compound with a base. Examples of inorganic salts of acid compounds include, but are not limited to, alkali metal and alkaline earth salts, such as sodium salts, potassium salts, magnesium salts, and calcium salts; ammonium salts; and aluminum salts. Examples of organic salts of acid compounds include, but are not limited to, procaine, dibenzylamine, N- ethylpiperidine, N,N'-dibenzylethylenediamine, and triethylamine salts. Salts of acidic compounds with amino acids, such as lysine salts, can also be prepared. [0026] The invention includes all stereoisomers of the compounds described herein, including enantiomers and diastereomers. The invention includes all enantiomers of any chiral compound disclosed, in either substantially pure levorotatory or dextrorotatory form, or in a racemic mixture, or in any ratio of enantiomers. The invention includes any diastereomers of the compounds described herein in diastereomerically substantially pure form and in the form of mixtures in all ratios. For compounds disclosed as an E isomer, the invention also includes the Z isomer; for compounds disclosed as the Z isomer, the invention also includes the E isomer. The invention also includes all solvates of the compounds described herein, including all hydrates of the compounds described herein. The invention also includes all polymorphs, including crystalline and non-crystalline forms of the compounds described herein. The invention also includes all salts of the compounds described herein, particularly pharmaceutically-acceptable salts. Metabolites and prodrugs of the compounds described herein are also embraced by the invention. In all uses of the compounds disclosed herein, the invention also includes use of any or all of the stereochemical, enantiomeric, diastereomeric, E or Z forms, solvates, hydrates, polymorphic, crystalline, non-crystalline, salt, pharmaceutically acceptable salt, metabolite and prodrug variations of the compounds as described. Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers of the compound depicted. Compounds can be isolated with varying degrees of enantiomeric or diastereomeric purity; for example, a preparation may comprise at least about 95% of the desired isomer; at least about 98% of the desired isomer; at least about 99% of the desired isomer; or at least about 99.5% of the desired isomer. [0027] For certain of the compounds described herein, such as the propionic acid derivative COX inhibitors, specific stereochemical forms may be desired. For example, the S-isomer of naproxen is used as a COX inhibitor, as the R-isomer shows toxicity. Chiral separation techniques (such as chiral HPLC) can be used to separate enantiomers. Standard tests well-known in the art can be used to screen compounds for toxicity; enantiomerically pure compounds that display favorable toxicity characteristics can be selected, while enantiomerically pure compounds with unacceptable toxicity can be discarded.

SSAO inhibitors

[0028] Various semicarbazide-sensitive amine oxidase inhibitors can be used in the combined inhibitors of the invention, in order to provide the residue or pharmacophore of an SSAO inhibitor. Exemplary SSAO inhibitors are disclosed in published International Patent Application No. WO 2005/014530 and United States

Patent Application Publication No. US 2005/0096360; published International Patent

Application No. WO 2005/082343 and United States Patent Application Publication

No. US 2006/0025438; and International Patent Application

No. PCT/US2007/008187 and United States Patent Application No. 11/731,819.

COX inhibitors

[0029] Various COX inhibitors can be employed in the invention to provide the residue or pharmacophore of a COX inhibitor, including, but not limited to:

[0030] propionic acid derivatives, exemplified by carprofen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, loxoprofen, naproxen, naproxen sodium, ketorolac (which can also be classified as an acetic acid derivative), oxaprozin, and vedaprofen;

[0031] salicylic acid derivatives, exemplified by acetylsalicylic acid (aspirin), choline salicylate, and diflunisal;

[0032] acetic acid derivatives exemplified by bendazac, diclofenac, etodolac, ibuprofen, ketorolac (which can also be classified as a propionic acid derivative), nabumetone, sulindac, and tolmetin; [0033] anthranilic acids, exemplified by meclofenamic acid, meclofenamate sodium, and tolfenamic acid;

[0034] phenylacetic acids;

[0035] aminonicotinic acids, exemplified by fiunixin;

[0036] naphthyl-containing compounds, such as nabumetone; and

[0037] indole analogs, exemplified by indomethacin, and etodolac.

[0038] The propionic acid derivatives and acetic acid derivatives represent a preferred class of compounds, as these compounds are relatively permissive with regard to the range of benzylic substitutions that are allowed. The propionic acid derivatives are also preferred from a cardiovascular safety perspective, as recent analyses have demonstrated the benign cardiovascular safety profile of the combined

COX-I and COX-2 inhibitors relative to certain selective COX inhibitors.

Exemplary combinations of SSAO inhibitors and COX inhibitors [0039] "Permissive" sites on an SSAO inhibitor can be used to introduce residues or pharmacophores of a COX inhibitor, and "permissive" sites on a COX inhibitor can be used to introduce residues or pharmacophores of an SSAO inhibitor, thus combining the SSAO inhibitor molecule and the COX inhibitor molecule into a single molecule. Alternatively, overlapping functional groups in a COX pharmacophore and an SSAO pharmacophore can serve as a common portion of a combined inhibitor. Examples of the combination of the SSAO inhibitors LJP 1586 and LJP 1414 with COX inhibitors naproxen and ibuprofen are shown in Scheme 1, Scheme 2, Scheme 3, and Scheme 4 below.

Combined inhibitor Scheme 1

[0040] In the example in Scheme 1, the SSAO inhibitor pharmacophore is the portion

while the COX inhibitor pharmacophore is the portion

The benzene ring bearing the 2-aminomethyl-3-fluoroallyl moiety in the combined inhibitor is both a portion of the pharmacophore of the SSAO inhibitor and of the pharmacophore of the COX inhibitor; the two pharmacophores thus overlap at that ring. Accordingly, the overlapping portion can be used in the combined inhibitor as a common scaffold for both the remainder of the SSAO inhibitor pharmacophore and the remainder of the COX inhibitor pharmacophore.

LJP 1586 Ibuprofen

Combined inhibitor

Scheme 2

[0041] The COX pharmacophore in Scheme 2 is

and the aromatic ring of the COX pharmacophore overlaps with the pharmacophore of the SSAO inhibitor,

in the combined inhibitor.

Scheme 3

[0042] In Scheme 3, the SSAO inhibitor pharmacophore

combines with the COX inhibitor pharmacophore

where the phenyl portion of the pharmacophore of the SSAO inhibitor overlaps with one of the aromatic rings of the naphthyl portion of the COX pharmacophore.

Combined inhibitor Scheme 4

[0043] In scheme 4, the SSAO inhibitor pharmacophore

is combined with the COX inhibitor pharmacophore

(with overlap at the phenyl ring) to form the combined pharmacophore. [0044] As is illustrated in Schemes 1-4, an overlapping functional group, such as a phenyl ring, of one moiety (an SSAO inhibitor or pharmacophore; or a COX inhibitor or pharmacophore) can be "swapped in," along with its pharmacophoric substituents, into the other moiety (a COX inhibitor or pharmacophore; or an SSAO inhibitor or pharmacophore), where the corresponding functional group is "swapped out" (along with its non-pharmacophoric substituents) to create a combined inhibitor. For example, in Scheme 4, the 2-amino-N-benzylacetamide ring system is swapped into the ibuprofen molecule, replacing the isobutylbenzene ring system of the ibuprofen. Similar "swaps" can be performed between the SSAO inhibitors or pharmacophores and the COX inhibitors or pharmacophores disclosed herein, to create combined SSAO/COX inhibitors.

General Synthetic Methods

[0045] The compounds of the invention can be prepared by various synthetic routes; examples of these syntheses are detailed below.

[0046] For compounds that combine the SSAO inhibitor LJP- 1363:

with a COX inhibitor having a benzoic acid function (such as the salicylates, and meclofenamic acid and tolfenamic acid), the chemistry as outlined in Scheme 5 can be used.

Scheme 5 [0047] For compounds that combine the SSAO inhibitor LJP- 1363 with a

COX inhibitor having a phenylacetic acid function (such as diclofenac), the chemistry as outlined in Scheme 5 can be used, but with the substitution of 4-cyanophenylacetic acid for 4-cyanobenzoic acid; see Scheme 5 A.

Scheme 5A

[0048] For compounds that combine the SSAO inhibitor LJP- 1363 with a

COX inhibitor having a 2-phenylpropionic acid function (such as carprofen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, loxoprofen, naproxen, naproxen sodium, and vedaprofen, which includes 2-naphthylpropionic acid-containing compounds as well), the chemistry as outlined in Scheme 6 can be used.

Suzuki coupling

Scheme 6

[0049] For compounds that combine the SSAO inhibitor LJP- 1586 with a

COX inhibitor having a benzoic acid function (such as the salicylates, and meclofenamic acid and tolfenamic acid), the chemistry as outlined in Scheme 7 can be used. NHBoc NC'

Wittig reaction "NHB_OC HCI/H₂O

NH₃ HCI

HOOC

Scheme 7

[0050] For compounds that combine the SSAO inhibitor LJP-1586 with a

COX inhibitor having a phenylacetic acid function (such as diclofenac), the chemistry as outlined in Scheme 8 can be used to obtain the starting material 4-cyanomethylphenylacetic acid; the chemistry of Scheme 7 can then be used, with the substitution of 4-cyanomethylphenylacetic acid for 2-(4-cyanophenyl)acetic acid.

Scheme 8

[0051] For compounds that combine the SSAO inhibitor LJP-1586 with a

COX inhibitor having a 2-phenylpropionic acid function (such as carprofen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, loxoprofen, naproxen, naproxen sodium, and vedaprofen, which includes 2-naphthylpropionic acid-containing compounds as well), the chemistry as outlined in Scheme 9 can be used.

Suzuki coupling

Me₃SiCHN₂ 1 HBr/DCM DCM 2 t-BuOAc/HCICU

Scheme 9

Assays for COX inhibitory activity

[0052] Assays for the activity of cyclooxygenase-1 (COX-I, also known as

Prostaglandin endoperoxide H synthase- 1 or PGHS-I) and cyclooxygenase-2 (COX- 2, also known as Prostaglandin endoperoxide H synthase-2 or PGHS-2) are well- known in the art; see, e.g., Glaser, K. et al., Eur. J. Pharmacol. 1995 JuI 25;281(l):107-l 1. Commercial assays are available; see, for example, URL World- wide-web.cerep.fr/cerep/users/pages/catalog/catalog_detail.asp?test=726 for COX- 1 assays, and URL World-wide-web cerep.fr/cerep/users/pages/catalog/catalog_detail.asp?test=727 for COX-2 assays. COX-I and COX-2 assays can be used to test the cyclooxygenase inhibitory activity of the compounds of the invention.

Assays for SSAO activity and MAO-A, MAO-B activity

[0053] Assays to determine SSAO inhibitory activity are also known in the art; see, e.g., Lizcano JM. et al. (1998) Biochem J. 331 :69. This procedure for assaying in vitro inhibition of SSAO activity is also described below in the Examples. The assay of Holt, A. et al. (1997) Anal. Biochem. 244: 384 (see the Examples below) can be used to determine monoamine oxidase inhibitory activity, in order to determine the selectivity of SSAO inhibitory activity over MAO-A or MAO-B inhibitory activity.

Treatment of Diseases

[0054] The compounds discussed herein are useful for treating inflammation and inflammatory conditions, and for treating immune and autoimmune disorders. The compounds are also useful for treating one or more of a variety of diseases caused by or characterized by inflammation or immune disorders. Thus the compounds can be used to treat diseases caused by inflammation, and can also be used to treat diseases which cause inflammation. The compounds are used to treat mammals, preferably humans. "Treating" a disease with the compounds discussed herein is defined as administering one or more of the compounds discussed herein, with or without additional therapeutic agents, in order to prevent, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease. "Therapeutic use" of the compounds discussed herein is defined as using one or more of the compounds discussed herein to treat a disease, as defined above. A "therapeutically effective amount" of a compound is an amount of the compound, which, when administered to a subject, is sufficient to prevent, reduce, or eliminate either the disease or one or more symptoms of the disease, or to retard the progression of the disease or of one or more symptoms of the disease, or to reduce the severity of the disease or of one or more symptoms of the disease. A "therapeutically effective amount" can be given in one or more administrations.

[0055] The subjects which can be treated with the compounds and methods of the invention include vertebrates, preferably mammals, more preferably humans. [0056] Diseases which can be treated with the compound and methods of the invention include inflammation, inflammatory responses, inflammatory diseases and immune disorders. It should be noted that inflammatory diseases can be caused by immune disorders, and that immune disorders are often accompanied by inflammation, and therefore both inflammation and immune disorders may be treated simultaneously by the compounds and methods of the invention. Diseases which can be treated with the compounds and methods of the invention include, but are not limited to, multiple sclerosis (including chronic multiple sclerosis); synovitis; systemic inflammatory sepsis; inflammatory bowel diseases; Crohn's disease; ulcerative colitis; Alzheimer's disease; atherosclerosis; rheumatoid arthritis; juvenile rheumatoid arthritis; pulmonary inflammatory conditions; asthma; skin inflammatory conditions and diseases; contact dermatitis; liver inflammatory and autoimmune conditions; autoimmune hepatitis; primary biliary cirrhosis; sclerosing cholangitis; autoimmune cholangitis; alcoholic liver disease; Type I diabetes and/or complications thereof; Type II diabetes and/or complications thereof; atherosclerosis; ischemic diseases such as stroke and/or complications thereof; myocardial infarction; acute pain; chronic pain; signs and symptoms of osteoarthritis; chronic obstructive pulmonary disorder (COPD); and acute respiratory distress syndrome (ARDS). In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is multiple sclerosis. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is chronic multiple sclerosis. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is the inflammatory complications resulting from stroke. In another embodiment, the inflammatory disease or immune disorder to be treated by the present invention is signs and symptoms of osteoarthritis. [0057] The Examples below provide procedures for determining the activity of compounds of the invention in treating and/or preventing various diseases.

Modes of administration

[0058] The compounds described for use in the present invention can be administered to a mammalian, preferably human, subject via any route known in the art, including, but not limited to, those disclosed herein. Methods of administration include but are not limited to, intravenous, oral, intraarterial, intramuscular, topical, via inhalation (e.g. as mists or sprays), via nasal mucosa, subcutaneous, transdermal, intraperitoneal, gastrointestinal, and directly to a specific or affected organ. Oral administration is a preferred route of administration. The compounds described for use herein can be administered in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, emulsions, dispersions, food premixes, and in other suitable forms. The compounds can also be administered in liposome formulations. The compounds can also be administered as prodrugs, where the prodrug undergoes transformation in the treated subject to a form which is therapeutically effective. Additional methods of administration are known in the art. [0059] The compounds of the present invention may be administered in an effective amount within the dosage range of about 0.1 μg/kg to about 300 mg/kg, or within about 1.0 μg/kg to about 40 mg/kg body weight, or within about 1.0 μg/kg to about 20 mg/kg body weight, preferably between about 1.0 μg/kg to about 10 mg/kg body weight. Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided dosage of two, three or four times daily.

[0060] The pharmaceutical dosage form which contains the compounds described herein is conveniently admixed with a pharmaceutically acceptable carrier, such as a non-toxic pharmaceutical organic carrier or a non-toxic pharmaceutical inorganic carrier. Typical pharmaceutically-acceptable carriers include, for example, mannitol, urea, dextrans, lactose, potato and maize starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid, and other conventionally employed acceptable carriers. The pharmaceutical dosage form can also contain non-toxic auxiliary substances such as emulsifying, preserving, or wetting agents, and the like. A suitable carrier is one which does not cause an intolerable side effect, but which allows the compound(s) to retain its pharmacological activity in the body. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott, Williams & Wilkins (2000). Solid forms, such as tablets, capsules and powders, can be fabricated using conventional tableting and capsule-filling machinery, which is well known in the art. Solid dosage forms, including tablets and capsules for oral administration in unit dose presentation form, can contain any number of additional non-active ingredients known to the art, including such conventional additives as excipients; desiccants; colorants; binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulfate. The tablets can be coated according to methods well known in standard pharmaceutical practice. Liquid forms for ingestion can be formulated using known liquid carriers, including aqueous and non-aqueous carriers such as sterile water, sterile saline, suspensions, oil-in-water and/or water-in-oil emulsions, and the like. Liquid formulations can also contain any number of additional non-active ingredients, including colorants, fragrance, flavorings, viscosity modifiers, preservatives, stabilizers, and the like. For parenteral administration, the compounds for use in the invention can be administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent or sterile liquid carrier such as water, saline, or oil, with or without additional surfactants or adjuvants. An illustrative list of carrier oils would include animal and vegetable oils (e.g., peanut oil, soy bean oil), petroleum-derived oils (e.g., mineral oil), and synthetic oils. In general, for injectable unit doses, sterile liquids such as water, saline, aqueous dextrose and related sugar solutions, and ethanol and glycol solutions such as propylene glycol or polyethylene glycol are preferred liquid carriers. [0061] The pharmaceutical unit dosage chosen is preferably fabricated and administered to provide a concentration of drug in the blood, tissues, organs, or other targeted region of the body which is therapeutically effective for use in treatment of one or more of the diseases described herein. The optimal effective concentration of the compounds of the invention can be determined empirically and will depend on the type and severity of the disease, route of administration, disease progression and health, mass and body area of the patient. Such determinations are within the skill of one in the art. The compounds for use in the invention can be administered as the sole active ingredient, or can be administered in combination with another active ingredient.

Kits

[0062] The invention also provides articles of manufacture and kits containing materials useful for treating diseases such as inflammatory diseases, autoimmune diseases, multiple sclerosis (including chronic multiple sclerosis); synovitis; systemic inflammatory sepsis; inflammatory bowel diseases; Crohn's disease; ulcerative colitis; Alzheimer's disease; atherosclerosis; rheumatoid arthritis; juvenile rheumatoid arthritis; pulmonary inflammatory conditions; asthma; skin inflammatory conditions and diseases; contact dermatitis; liver inflammatory and autoimmune conditions; autoimmune hepatitis; primary biliary cirrhosis; sclerosing cholangitis; autoimmune cholangitis; alcoholic liver disease; Type I diabetes and/or complications thereof; Type II diabetes and/or complications thereof; atherosclerosis; ischemic diseases such as stroke and/or complications thereof; myocardial infarction; acute pain; chronic pain; signs and symptoms of osteoarthritis; chronic obstructive pulmonary disorder (COPD); and acute respiratory distress syndrome (ARDS); or for inhibiting SSAO enzyme activity (whether the enzyme activity is due either to soluble SSAO enzyme or membrane-bound VAP-I protein, or due to both) and/or inhibiting binding to VAP-I protein and/or inhibiting COX activity (of COX-I, COX-2, or COX-3/COX- Ib isoforms, or any two isoforms, or all isoforms). The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition having an active agent which is effective for treating diseases or for inhibiting SSAO or VAP-I enzyme activity or binding to VAP-I protein or inhibiting COX activity. The active agent in the composition is one or more of the combined SSAO/COX inhibitor (or SSAO inhibitor or COX inhibitor) compounds disclosed herein. The label on the container indicates that the composition is used for treating diseases such as inflammatory or autoimmune diseases, or for inhibiting SSAO or VAP-I enzyme activity or binding to VAP-I protein or inhibiting COX activity, and may also indicate directions for either in vivo or in vitro use, such as those described above.

[0063] The invention also provides kits comprising any one or more of the combined SSAO/COX inhibitor (or SSAO inhibitor or COX inhibitor) compounds disclosed herein. In some embodiments, the kit of the invention comprises the container described above. In other embodiments, the kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein (such as methods for treating autoimmune or inflammatory diseases, and methods for inhibiting SSAO or VAP-I enzyme activity or binding to VAP-I protein or inhibiting COX activity). [0064] In other aspects, the kits may be used for any of the methods described herein, including, for example, to treat an individual with autoimmune or inflammatory disease, such as multiple sclerosis or ischemic disease (such as stroke) and the sequelae thereof.

[0065] The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety.

[0066] The invention will be further understood by the following nonlimiting examples. It should be noted that, while the compounds are typically described as salts, the disclosure expressly includes the non-salt forms of the compounds, as well as any other salt of the compound. EXAMPLES

Example 1

In vitro inhibition of SSAO activity

[0067] SSAO activity is measured as described (Lizcano JM. et al. (1998)

Biochem J. 331.69). Briefly, rat lung or human umbilical cord (UC) homogenates are prepared by chopping the freshly removed tissue into small pieces and washing them thoroughly in PBS. The tissue is then homogenized 1 :10 (w/v) in 10 mM potassium phosphate buffer (pH 7.8) and centrifuged at lOOOg at 4⁰C for 10 minutes; the supernatants are kept frozen until ready to use. Lung or UC homogenate is preincubated with clorgyline and pargyline at 1 μM to inhibit MAO-A and MAO-B activity, respectively, and SSAO inhibitors (in particular, the combined SSAO/COX inhibitors of the invention) are generally present at InM - lOμM. The reaction is initiated by addition of 20 μM ¹⁴C- benzylamine as substrate. The reaction is carried out at 37°C in a final volume of 300 μl of 50 mM potassium phosphate buffer (pH 7.2) and stopped with 100 μl of 2 M citric acid. Radioactively labeled products are extracted into toluene/ethyl acetate (1 :1, v/v) containing 0.6% (w/v) 2,5- diphenyloxazole (PPO) before liquid scintillation counting.

Example 2 Comparison of inhibition of the SSAO activity ofSSAO/VAP-1 versus MAO-A and

MAO-B activities.

[0068] The specificity of SSAO inhibitors, in particular the combined

SSAO/COX inhibitors of the invention, is tested by determining their ability to inhibit MAO-A and MAO-B activities in vitro. Recombinant human MAO-A and human MAO-B enzymes are obtained from BD Biosciences (MA, USA). MAO activity is measured using the colorimetric method essentially as described (Holt, A. et al. (1997) Anal. Biochem. 244: 384). A pre-determined amount of inhibitor diluted in 0.2 M potassium phosphate buffer, pH 7.6, is added to each well, if required. The amount of inhibitor varies in each assay but is generally at a final concentration of between 1 nM and 1 mM. Controls lack inhibitor. The following agents are then added to a final reaction volume of 200 μl in 0.2 M potassium phosphate buffer, pH 7.6: 0.04 mg/ml of MAO-A or 0.07 mg/ml MAO-B enzyme, 15 μl of 10 raM tyramine substrate (for MAO-A), or 15 μl 100 mM benzylamine substrate (for MAO- B), and 50 μl of freshly made chromogenic solution. The chromogenic solution contains 750 nM vanillic acid (Sigma#V-2250), 40OnM 4-aminoantipyrine (Sigma # A-4328) and 12 U/ml horseradish peroxidase (Sigma # P-8250) in order to cause a change of 0.5 OD A490 nm per hour. This is within the linear response range for the assay. The plates are incubated for 60 min at 37°C. The increase in absorbance, reflecting MAO activity, is measured at 490 nm using microplate spectrophotometer (Power Wave 40, Bio-Tek Inst.). Inhibition is determined as percent inhibition compared to control after correcting for background absorbance and IC_5O values are calculated using GraphPad Prism software. Clorgyline and pargyline (inhibitors of MAO-A and MAO-B, respectively) at 1 μM, are added to some wells as positive controls for MAO inhibition. The ability of compounds of the invention to inhibit SSAO activity versus MAO activity is determined.

Example 3

Inhibition of collagen-induced arthritis in mice

[0069] Collagen-induced arthritis (CIA) in mice is widely used as an experimental model for rheumatoid arthritis (RA) in humans. CIA is mediated by autoantibodies to a particular region of type II collagen and complement. The murine CIA model which can be used for this study is called antibody-mediated CIA, and can be induced by i.v. injection of a combination of different anti-type II collagen monoclonal antibodies (Terato K., et al. (1995). Autoimmunity. 22:137). Several compounds have been used to successfully block inflammation in this model, including anti-αl βl and anti-α2β2 integrins monoclonal antibodies (de Fougerolles A.R. (2000) J. Clin. Invest. 105: 721).

[0070] In this example, arthrogen-collagen-induced arthritis antibody kits are purchased from Chemicon International (Temecula, CA) and arthritis is induced using the manufacturer's protocol. Mice are injected i.v. with a cocktail of 4 anti-collagen Type II monoclonal antibodies (0.5 mg each) on day 0, followed by i.p. injection of 25 μg lipopolysaccharide (LPS) on day 2. Mice develop swollen wrists, ankles, and digits 3-4 days after LPS injection, with disease incidence of 90% by day 7. Severity of arthritis in each limb is scored for 3-4 weeks as follows: 0 = normal; 1 = mild redness, slight swelling of ankle or wrist; 2 = moderate redness and swelling of ankle or wrist; 3 = severe redness and swelling of some digits, ankle and paw; 4 = maximally inflamed limb. Animals are divided in 3 groups of 10 animals: vehicle, methotrexate (MTX)-treated, and compound-treated. Animals in the vehicle group are injected i.p. with phosphate buffer saline (PBS), once daily for 10 days (starting on day 0). MTX (3 mg/kg) is administered i.p. starting on day 0 and continuing every other day (Mon., Weds., Fri.) for the duration of the experiment. Administration of compounds is initiated at day 0 and continued until day 10.

Example 4 Inhibition of experimental autoimmune encephalomyelitis in mice by SSAO and

SSAO/COX inhibitors

[0071] SSAO/VAP-1 is expressed on the endothelium of inflamed tissues/organs including brain and spinal cord. Its ability to support lymphocyte transendothelial migration may be an important systemic function of SSAO/VAP-1 in inflammatory diseases such as multiple sclerosis and Alzheimer's disease. An analysis of the use of SSAO inhibitors, and in particular the SSAO/COX inhibitors disclosed herein, to treat inflammatory disease of the central nervous system (CNS) is performed through the use of an experimental autoimmune encephalomyelitis model (EAE) in C57BL/6 mice. EAE in rodents is a well-characterized and reproducible animal model of multiple sclerosis in human (Benson J. M. et al. (2000) J. Clin. Invest. 106:1031). Multiple sclerosis is a chronic immune-mediated disease of the CNS characterized by patchy perivenular inflammatory infiltrates in areas of demyelination and axonal loss. As an animal model, EAE can be induced in mice by immunization with encephalitogenic myelin antigens in the presence of adjuvant. The pathogenesis of EAE comprises presentation of myelin antigens to T cells, migration of activated T cells to the CNS, and development of inflammation and/or demyelination upon recognition of the same antigens.

[0072] Twenty female C57BL/6 mice are immunized subcutaneously (s.c). with myelin oligodendrocyte glycoprotein 35-55 (MOG peptide 35-55) in Complete Freund Adjuvant (CFA) on day 0, followed by i.p. injections of 500 ng pertussis toxin (one pertussis toxin injection on day 0, a second pertussis toxin injection on day 2). Groups of 10 mice receive a compound of the invention, once daily i.p. for 30 consecutive days), or vehicle control (once/day for 30 consecutive days) all starting from one day after the immunization and all administered i.p. Then animals are monitored for body weight, signs of paralysis and death according to a 0-5 scale of scoring system as follows: 1 = limp tail or waddling gait with tail tonicity; 2 = waddling gait with limp tail (ataxia); 2.5 = ataxia with partial limb paralysis; 3 = full paralysis of one limb; 3.5 = full paralysis of one limb with partial paralysis of second limb; 4 = full paralysis of two limbs; 4.5 = moribund; 5 = death.

Example 5

Inhibition of carrageenan-induced rat paw edema

[0073] Carrageenan-induced paw edema is used extensively in the evaluation of anti-inflammatory effects of various therapeutic agents and is a useful experimental system for assessing the efficacy of compounds to alleviate acute inflammation (Whiteley PE and Dalrymple SA, 1998. Models of inflammation: carrageenan- induced paw edema in the rat, in Current Protocols in Pharmacology. Enna SJ, Williams M, Ferkany JW, Kenaki T, Porsolt RE and Sullivan JP, eds., pp 5.4.1-5.4.3, John Wiley & Sons, New York). The full development of the edema is neutrophil- dependent (Salvemini D. et al. (1996) Br. J. Pharmacol. 118: 829). [0074] Female Sprague Dawley rats are used in groups of 8 - 12 and compounds of the invention are administered orally at 50mg/kg 60 minutes prior to carrageenan exposure. The control group is administered orally an equal volume of vehicle (PBS). Edema in the paws is induced as previously described by injecting 50 μl of a 0.5% solution of carrageenan (Type IV Lambda, Sigma) in saline with a 27-G needle s.c. in the right foot pad. (See Whiteley P.E. and Dalrymple S.A. (1998), Models of inflammation: carrageenan-induced paw edema in the rat, in Current Protocols in Pharmacology, Enna SJ, Williams M, Ferkany JW, Kenaki T, Porsolt RE and Sullivan JP, eds., pp 5.4.1-5.4.3, John Wiley & Sons, New York) The size of the tested foot of each animal is measured volumetrically with a plethysmometer before induction of edema, and at 60, 120, 180, 240 and 360 min after carrageenan induction. Example 6

Inhibition of oxazolone-induced colitis

[0075] Oxazolone-induced colitis is a TH2 -mediated process that closely resembles ulcerative colitis and is responsive to anti-IL4 therapy ((Strober W. et al (2002) Annu. Rev. Immunol. 20: 495, Boirivant M. et al. (1998) J. Ex. Med 188: 1929). Oxazolone colitis is induced as described (Fuss I.J. et al. (2002) J. Immunol. 168: 900). Briefly, mice are pre-sensitized by epicutaneous application of 1% oxazolone (4-ethoxymethylene-2-phenyl-2oxazolin-5-one, Sigma) in 100% EtOH (200 μl) on day 0, followed by intrarectal administration of 0.75% oxazolone in 50% EtOH (100 μl) to anesthetized SJL/J male mice on day 5 through a 3.5 F catheter inserted 4 cm proximal to the anal verge. Mice are divided in two treatment groups and injected i.p. twice a day with either PBS (control) or a compound of the invention. Injections are initiated at day 0 and are continued through day 12. Disease progression is evaluated by monitoring body weight and survival.

Example 7

Acute Toxicity Studies

[0076] Oral (p.o.) and intravenous (i.v.) LD₅₀ values for the compounds of the invention are determined in mice. Six-week old C57B1/6 female mice are divided in groups of five and administered a single i.v., p.o. or i.p. injection of compound dissolved in PBS (10-100 mg/kg in 100 μl i.v.; 30-1000 mg/kg p.o.; 30-500 mg/kg in 200 μl i.p.). Control groups are administered the same volume of PBS p.o., i.v., or i.p. Appearance and overt behavior are noted daily, and body weight is measured before compound administration (Day 1) and on Days, 3, 5 and 7. After seven days, animals are euthanized and their liver, spleen and kidneys are weighed.

Example 8

Inhibition of concanavalin A-induced liver injury

[0077] Prevention of inflammation by administration of compounds of the invention is assessed in the concanavalin A (Con A) murine model of liver injury. Con A activates T lymphocytes and causes T cell-mediated hepatic injury in mice. Tumor necrosis factor alpha is a critical mediator in this experimental model. T-cell- mediated liver injury involves the migration of immune cells, notably CD4+ T lymphocytes, into liver tissue. Balb/c mice are inoculated with 10 mg/kg concanavalin A administered i.v. in 200 μl pyrogen-free saline as described (Willuweit A. et al. (2001) J Immunol. 167:3944). Previous to Con A administration, animals are separated into treatment groups and injected i.p with either PBS, or different concentrations of compound of the invention (e.g., 20 mg/kg). Liver damage is evaluated by determining serum levels of liver enzymes such as transaminase and alkaline phosphatase, hepatic histopathology, and levels of different inflammatory cytokines in plasma and liver tissue.

[0078] This procedure is used to screen for compounds which inhibit the development of liver damage as compared to control animals.

Example 9

Effect of compounds of the invention in a mouse model of Alzheimer 's disease [0079] Alzheimer's disease (AD) is characterized clinically by a dementia of insidious onset and pathologically by the presence of numerous neuritic plaques and neurofibrillary tangles. The plaques are composed mainly of β-amyloid (AB) peptide fragments, derived from processing of the amyloid precursor protein (APP). Tangles consist of paired helical filaments composed of the microtubule-associated protein, tau. Transgenic mice carrying a pathogenic mutation in APP show marked elevation of AJJ-protein level and AB deposition in the cerebral cortex and hippocampus from approximately 1 year of age (Hsiao K. et al. (1996) Science 274:99). Mutant PS-I transgenic mice do not show abnormal pathological changes, but do show subtly elevated levels ofthe A1342/43 peptide (Duff K, et al. (1996) Nature 383:710). Transgenic mice derived from a cross between these mice (PS/APP) show markedly accelerated accumulation of AB into visible deposits compared with APP singly transgenic mice (Holcomb L. et al. (1998) Nat Med 4:97). Further, a recent study indicates that in these mice, inflammatory responses may be involved in the AB depositions (Matsuoka Y. et al. (2001) Am J Pathol. 158(4): 1345). [0080] The PS/APP mouse, therefore, has considerable utility in the study of the amyloid phenotype of AD and is used in studies to assess efficacy ofthe compounds of the invention to treat Alzheimer's patients. Mice are injected with vehicle (e.g., PBS) or a compound of the invention (at, e.g., 10-20 mg/kg), and are evaluated by analysis of memory deficits, histological characteristics of sample tissues, and other indicators of disease progression.

Alternate Alzheimer 's model: Assessing efficacy in amyloid-B-induced autoimmune encephalitis

[0081] The abnormal processing and extracellular deposition of amyloid-B

(Aβ) peptide, is a defining characteristic of Alzheimer's disease (AD). Recent evidence suggests that vaccination of transgenic mouse models of AD with Aβ causes a marked reduction in brain amyloid burden (e.g. Schenk D et al. (1999) Nature 400:173). Moreover, a recently published report suggests that vaccination with Aβ can, in certain circumstances, determine an aberrant autoimmune reaction to Aβ within the CNS, resulting in a perivenular inflammatory encephalomyelitis (Furlan R et al (2003) Brain 126:285).

[0082] Evaluation of the efficacy of compounds of the invention is carried out in the Aβ-induced autoimmune encephalomyelitis model. Thirty female C57BL/6 mice are immunized subcutaneously (s.c). with 100 μg of Aβ 1-42 peptide in Complete Freund Adjuvant (CFA) on day 0, followed by i.p. injections of pertussis toxin (one pertussis toxin injection on day 0, a second pertussis toxin injection on day T). Groups of 10 mice receive either a compound of the invention (10 mg/kg/dose, twice daily for 18 consecutive days), methotrexate (2.5 mg/kg/day, three times a week, till day 18) or vehicle control (twice/day for 18 consecutive days), all starting from one day after the immunization and all administered i.p. Then animals are monitored for body weight, signs of paralysis and death according to a 0-5 scale of scoring system as follows: 1 = limp tail or waddling gait with tail tonicity; 2 = waddling gait with limp tail (ataxia); 2.5 = ataxia with partial limb paralysis; 3 = full paralysis of one limb; 3.5 = full paralysis of one limb with partial paralysis of second limb; 4 = full paralysis of two limbs; 4.5 = moribund; 5 = death. Example 10

Effect of compounds of the invention in murine models of Type I diabetes mellitus [0083] It is widely accepted that proinflammatory cytokines play an important role in the development of type 1 diabetes. Thus, compounds of the invention can be used to treat patients suffering from this disease. A mouse with diabetes induced by multiple low doses of streptozotocin (STZ) can be used as an animal model for type 1 diabetes. STZ is used to induce diabetes in C57BL/6J mice. Briefly, STZ (40 mg/kg) or citrate buffer (vehicle) is given i.p. once daily for 5 consecutive days as described (Carlsson P.O. et al. (2000) Endocrinology. 141(8):2752). Compound administration (i.p. 10 mg/kg, twice a day) is started 5 days before STZ injections and continues for 2 weeks. Another widely used model is the NOD mouse model of autoimmune type 1 diabetes (Wong F.S. and Janeway CA. Jr. (1999) Curr Opin Immunol. 11(6):643. Female NOD mice are treated with daily injections of a compound of the invention (20 mg/kg/day) from week 10 through week 25. The effect of the compounds of the invention in preventing the development of insulitis and diabetes in NOD-scid/scid females after adoptive transfer of splenocytes from diabetic NOD females is also assessed. For both the STZ and NOD models, the incidence of diabetes is monitored in several ways, including monitoring of blood glucose levels. Insulin secretion is assessed in pancreatic islets isolated from experimental mice. Cytokine production is measured in mouse sera. Islet apoptosis is assessed quantitatively. [0084] This procedure is used to screen for compounds which inhibit development of diabetes as compared to control animals.

Example 1 1

Effect of compounds of the invention in models of airway inflammation. [0085] Anti-inflammatory compounds such as SSAO inhibitors and

SSAO/COX inhibitors, can have beneficial effects in airway inflammatory conditions such as asthma and chronic obstructive pulmonary disease. The rodent model here described has been extensively used in efficacy studies. Other murine models of acute lung inflammation can also be used to test the compounds of the invention. [0086] For the evaluation of the effects of SSAO^'inhibitors and SSAO/COX inhibitors in preventing airway inflammation, three groups of sensitized rats are studied. Animals are challenged with aerosolized OVA (ovalbumin) after intraperitoneal administration of the vehicle saline, a compound of the invention, or a positive control (e.g. prednisone) twice daily for a period of seven days. At the end of the week animals are anesthetized for measurements of allergen-induced airway responses as described (Martin J. G. et al. (2002) J Immunol. 169(7):3963). Animals are intubated endotracheally with polyethylene tubing and placed on a heating pad to maintain a rectal temperature of 36°C. Airflow is measured by placing the tip of the endotracheal tube inside a Plexiglas box (~250 ml). A pneumotachograph coupled to a differential transducer is connected to the other end of the box to measure airflow. Animals are challenged for 5 min with an aerosol of OVA (5% w/v). A disposable nebulizer will be used with an output of 0.15 ml/min. Airflow is measured every 5 min for 30 min after challenge and subsequently at 15 -min intervals for a total period of 8 h. Animals are then sacrificed for bronchoalveolar lavage (BAL). BAL is performed 8 h after challenge with five instillations of 5 ml of saline. The total cell count and cell viability is estimated using a hemacytometer and trypan blue stain. Slides are prepared using a Cytospin and the differential cell count is assessed with May-Grunwald-Giemsa staining, and eosinophil counts by immunocytochemistry.

Alternate model of airway inflammation: assessing the effect of compounds of the invention

[0087] LPS-induced pulmonary inflammation in rats is a widely used model of airway inflammation (e.g. Billah M et al J. Pharmacol. Exp. Ther. (2002) 302:127). Animals fasted overnight are orally dosed with either a compound of the invention (30 mg/kg), or vehicle 2 h before the LPS challenge. Using a Penn-Centry microspray needle, 0.1 ml of a 100-μg/ml LPS solution in saline is injected into the trachea of anesthetized male Sprague-Dawley rats (250-300 g). Animals not challenged with the LPS solution receive 0.1 ml of saline. Afterward, all animals are returned to their cages and allowed food and water ad libitum. At appropriate time points after intratracheal challenge with LPS, animals are surgically prepared with a tracheal cannula. Surgery is performed under anesthesia. The airways are flushed with 2 x 2 ml of 0.9% saline and the two washings pooled. [0088] Lavage fluid is centrifuged (350g, 4°C, 7 min), the supernatant is aspirated, erythrocytes are lysed, and the white cell pellet is washed three times in phosphate-buffered saline containing 10% heat-inactivated fetal calf serum and 10 μg/ml DNase I. After the washes, the pellet is resuspended again in the same buffer. Total cell counts are performed using a hemacytometer. Differential cell counts are conducted on Cytospin-prepared slides stained with Fisher's Leukostat stain. At least 200 cells are assessed per slide using standard morphological criteria to define mononuclear, neutrophilic, and eosinophilic cells.

Example 12

Efficacy in model of systemic inflammation

[0089] Evaluation of the efficacy of compounds of the invention is carried out in a model of endotoxemia (Pawlinski R et al. (2003) Blood 103:1342). Sixteen female C57B1/6 mice (eight to ten weeks old) are divided in two treatment groups: group A animals are administered 500 μl of PBS orally; group B animals are administered 100 mg/kg of a compound of the invention in 500 μl of PBS orally. Thirty minutes after oral administration of compound, inflammation is induced in all animals by administering i.p. 5 mg/kg of LPS (Ol 11 :B4, Sigma) in PBS. Blood samples (~ 50 μl) are collected from the retro-orbital sinus at 0 (before oral administration of compound), 1, 2, 4, and 8 hrs after LPS injection. Each sample is immediately diluted ¹A in PBS. Half of the diluted sample is used to prepare blood smear and the other 50 μl is centrifuged and serum is collected. Sera samples are used to determine ILl, IL6 and TNFa levels by ELISA. Animal survival rates are recorded for the next 3 days.

Example 13

Inhibition of cutaneous inflammation in the SCID mouse model of psoriasis [0090] Recent establishment of the SCID-human skin chimeras with transplanted psoriasis plaques has opened new vistas to study the molecular complexities involved in psoriasis. This model also offers a unique opportunity to investigate various key biological events such as cell proliferation, homing in of T cells in target tissues, inflammation and cytokine/chemokine cascades involved in an inflammatory reaction. The SCID mouse model has been used to evaluate the efficacy of several compounds for psoriasis and other inflammatory diseases (Boehncke W.H. et al. (1999) Arch Dermatol Res. 291(2-3): 104).

[0091] Transplantations are to be done as described previously (Boehncke,

W.H. et al. (1994) Arch. Dermatol. Res. 286:325). Human full-thickness xenografts are transplanted onto the backs of 6- to 8-week-old CB 17 SCID mice (Charles River). For the surgical procedure, mice are anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 5 mg/kg xylazine. Spindle-shaped pieces of full-thickness skin measuring 1 cm in diameter are grafted onto corresponding excisional full-thickness defects of the shaved central dorsum of the mice and fixed by 6-0 atraumatic monofilament sutures. After applying a sterile petroleum jelly-impregnated gauze, the grafts are protected from injury by suturing a skin pouch over the transplanted area using the adjacent lateral skin. The sutures and over-tied pouches are left in place until they resolve spontaneously after 2-3 weeks. Grafts are allowed 2 weeks for acceptance and healing. Thereafter, daily intraperitoneal injections are performed between days 15 and 42 after transplantation. Mice are injected with either vehicle (PBS), dexamethasone (0.2 mg/kg body weight), or a compound of the invention (at, e.g., 20 mg/kg body weight) in a final volume of 200 μl. Mice are sacrificed at day 42, and after excision with surrounding mouse skin the grafts are formalin-embedded. Subsequently, routine hematoxylin-and-eosin staining is performed, and the grafts are analyzed with regard to their pathological changes both qualitatively (epidermal differentiation, inflammatory infiltrate) and quantitatively (epidermal thickness).

Example 14

Oral bioavailability studies in rodents

[0092] Oral bioavailability studies in mice and rats are to be performed using the following procedure. Briefly, C57B1/6 female mice and Sprague Dawley female rats are administered 50 mg/kg of different compounds of the invention by oral gavage. Animals are bled at different time intervals after compound administration and the levels of inhibitor in plasma are determined using the inhibition assay described herein. Example 15

Dose-response effect from in vivo administration ofSSAO/VAP-1 inhibitors [0093] In vivo inhibition of SSAO is assessed in rat aorta and lungs, two of the tissues where SSAO activity is highest. Six week old female Sprague Dawley rats are to be administered 0, 0.1, 1, 10 and 50 mg/kg of a compound of the invention in 2.5 ml/kg PBS by oral gavage. Four hours after compound administration the animals are euthanized and their aortas and lungs are removed and frozen in liquid nitrogen. Tissues are homogenized in 0.1 M potassium phosphate pH 7.8 buffer (30 ml/g for aorta and 20 ml/g for lung) and centrifuged at 1000 x g for 15 min. Supernatants are collected and used in the radioactive assay following the protocol described by Lizcano J.M. et al. (1998) Biochem. J. 331 :69. Enzymatic reactions are initiated by incubating a 200 μl aliquot of the tissue homogenate with 20 μl of 0.4 mM ¹⁴C- labeled benzylamine substrate (6 mCi/mmol specific activity, Pharmacia) for 30 min at RT. The assay is stopped by addition of 100 μl of 2 M citric acid, the assay volume is extracted with 5 ml toluene:ethyl acetate (1 :1) containing 0.6% (w/v) 2,5- diphenyloxdazole (PPO), and an aliquot of the organic layer is counted by liquid scintillation. Because SSAO and MAO-B are both active towards benzylamine, control samples are run concomitantly so that MAO-B and SSAO activities can be identified. SSAO is inhibited with 0, 10, 50 and 500 μM of semicarbazide for MAO-B determinations, and MAO-B is inhibited with 0, 5, and 100 μM of pargyline for SSAO determinations. The inhibitors are added to the tissue supernatant prior to addition of benzylamine.

Example 16

Blocking of in vitro adhesion by SSAO/VAP-1 inhibitors. [0094] These studies are carried out in order to determine whether

SSAO/VAP-1 transfected into endothelial cells will retain the adhesion function and whether it plays any role in the adhesion of freshly isolated human PBMCs to these cells. Moreover, the studies are also designed to determine whether blocking of SSAO/VAP-1 will have an impact on the level of adhesion between these two cell types. Adhesion assays are performed using cells labeled with the fluorescent dye Calcein-AM (Molecular Probes, OR, USA) as per the manufacturer's instructions. Briefly, rat lymph node high endothelial cells (HEC; isolation and culture is described in Ager, A. (1987) J. Cell Sci. 87: 133) are plated overnight in 96-well plates (2,000 cells/well). PBMCs (peripheral blood mononuclear cells) (1x10⁷) are labeled with 1 ml of 10 μM Calcein-AM for 1 hr at 37°C, washed three times with RPMI, and added to the 96 well plates containing monolayers of HEC cells mock-transfected or transfected with full-length human SSAO/VAP-1 (60,000 PBMCs are plated per well containing 2,000 HEC cells). Adhesion is carried out for 3 hr at 37°C. Non-adherent cells are removed by washing three times with RPMI and fluorescence is measured in a fluorescence plate reader at an excitation wavelength of 485 nm and emission wavelength of 530 nm. Several controls are to be included, such as HEC cells and PBMCs (labeled and unlabeled) alone.

[0095] The next experiments are designed in order to investigate whether blocking the enzymatic catalytic site will have any effect on the adhesion function of SSAO/VAP-1, and whether or not SSAO/COX inhibitors according to the invention will mediate an adhesion-inhibiting effect. Published results suggest that blocking SSAO enzymatic activity with semicarbazide inhibited lymphocyte rolling under laminar sheer on cardiac endothelial monolayers (Salmi et al. Immunity (2001) 14:265). These studies can thus be repeated using the adhesion assay as described above to evaluate the inhibitors of the invention. Adhesion blockers can include an anti-human VAP-I monoclonal antibody (Serotec, Oxford, UK), neuramidase (a sialidase, because SSAO/VAP-1 is a sialoglycoprotein; Sigma), and several function- blocking antibodies to rat adhesion molecules (CD31- PECAM, CD54-ICAM-1, CD92P-P Selectin). Controls can include the SSAO inhibitor semicarbazide (Sigma), MAO-A and MAO-B inhibitors (clorgyline and pargyline, respectively; Sigma), and mouse IgGl and IgG2 isotype controls (BD, USA). Antibodies (10 μg/ml) and neuramidase (5 mU) are incubated with the HECs for 30 min at 37°C; excess antibody is washed away prior to the addition of the labeled PBMCs. Small-molecule inhibitors are pre-incubated the same way at ICiQ₀ concentrations, but the amounts present in the supernatant are not washed away to preserve the ICioo concentration during the adhesion step. Example 17

Inhibition of lipopolysaccharide (LPS)-induced endotoxemia [0096] In sepsis exposure of endothelial cells of all organs to elevated levels of LPS and inflammatory cytokines leads to upregulation of adhesion molecules and chemokines, which results in an increase in the tethering, rolling and transmigration of leukocytes (Pawlinski R. et al. (2004) Blood 103:1342). LPS-induced endotoxemia is a well-characterized model of systemic inflammation and thus can be used to investigate the putative role of SSAO inhibition in these inflammatory mechanisms. Sepsis is to be induced in C57B1/6J female mice by i.p. administration of 5 mg/kg of LPS. Sixty minutes prior to LPS injections, 200 μl of vehicle (PBS) or 50 mg/kg of (2-phenylallyl)hydrazine are administered orally to the animals. Dexamethasone is administered i.p, at a concentration of 3mg/kg 1 hr prior to disease induction. Blood is drawn from the retroorbital plexus of anesthetized animals and sera is collected and frozen until time of cytokine measurements. IL-I β, TNF-α, and IL-6 concentrations are determined by ELISA using commercial kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

[0097] The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety. In particular, WO 2005/014530, US 2005/0096360, WO 2005/082343, US 2006/0025438, PCT/US2007/008187, and United States Patent Application No. 11/731,819 are hereby incorporated herein by reference in their entirety, especially with respect to the SSAO inhibitors described therein and the examples and experimental procedures described therein. [0098] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A combined S SAO/COX inhibitor selected from the group consisting of a residue of an SSAO inhibitor covalently linked to a residue of a COX inhibitor, a pharmacophore of an SSAO inhibitor covalently linked to the residue of a COX inhibitor, a pharmacophore of a COX inhibitor covalently linked to the residue of an SSAO inhibitor, or a pharmacophore of an SSAO inhibitor covalently linked to a pharmacophore of a COX inhibitor; and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

2. The combined inhibitor of claim 1, wherein the combined S SAO/COX inhibitor is a pharmacophore of a COX inhibitor covalently linked to the residue of an SSAO inhibitor.

3. The combined inhibitor of claim 1, wherein the residue of the SSAO inhibitor is derived from

4. The combined inhibitor of claim 1, wherein the SSAO pharmacophore is selected from:

5. The combined inhibitor of claim 1, wherein the residue of the COX inhibitor is derived from: carprofen, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, loxoprofen, naproxen, naproxen sodium, oxaprozin, vedaprofen, acetylsalicylic acid, salicylic acid, choline salicylate, diflunisal, bendazac, diclofenac, etodolac, ibuprofen, ketorolac, nabumetone, sulindac, tolmetin, meclofenamic acid, meclofenamate sodium, and tolfenamic acid, phenylacetic acids, flunixin, indomethacin, nabumetone, ketorolac, and etodolac.

6. The combined inhibitor of claim 1, wherein the COX pharmacophore is selected from:

7. A compound of the formulas

or

where R, is selected from -COOH, -CH₂COOH, and -CH(CH₃)COOH; and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

. A compound of the formulas

where R₂ is selected from

and

where the asterisk (*) indicates the point of attachment of the R2 group to the remainder of the molecule, and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.

9. A combined SSAO/COX inhibitor of claim 1, selected from the group consisting of:

and any salt, isolated stereoisomer, mixture of stereoisomers in any ratio, or racemic mixture thereof.