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1. WO2020191502 - USE OF EMOXYPINE AND DERIVATIVES THEREOF FOR TREATING KIDNEY DISORDERS AND INFLAMMATORY BOWEL DISEASE

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

USE OF EMOXYPINE AN D DERIVATIVES THEREOF FOR TREATING KIDNEY DISORDERS AND INFLAMMATORY BOWEL DISEASE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefits of priority to United States Provisional Patent Application No. 62/824,674, filed March 27, 2019, titled USE OF EMOXYPINE AN D DERIVATIVES THEREOF FOR TREATING KIDNEY DISORDERS AN D IN FLAMMATORY BOWEL DISEASE, the contents of which are hereby expressly incorporated into the present application by reference in their entirety.

Field of Invention

The present invention relates to the use of emoxypine and derivatives of emoxypine for treating kidney disorders and/or inflammatory bowel disease.

Background

[0001] Chronic kidney disease (CKD) is a form of kidney disease

characterized by damage to the kidneys that worsens over time, often as result of renal fibrosis. This results in gradual loss of kidney functions, so that excess fluid and waste from the blood remain in the body and may cause other health

problems.

[0002] Early on, an individual with CKD is generally asymptomatic. As the disease progresses, however, symptoms can include muscle cramps, nausea and vomiting, loss of appetite, swelling in your feet and ankles, and tiredness.

Symptoms of acute kidney failure include abdominal and back pain, diarrhea and vomiting, and fever.

[0003] There is currently no cure for renal fibrosis or CKD. Treatment of CKD generally focus on controlling the risk factors, the goal of therapy is simply to slow down or halt the progression of CKD.

[0004] The murine model of UUO surgical intervention is a well-characterized experimental model of renal injury that ultimately leads to tubulointerstitial fibrosis - a common characteristic of many chronic nephropathies - depending on the duration of obstruction. In this model, the progression of renal fibrosis is highly predictable and reproducible, leading to significant fibrosis and nephron loss in a relatively short period of 7 to 14 days (Eddy et al., Investigating mechanisms of chronic kidney disease in mouse models, Pediatr Nephrol. 2012 August;

27(8) : 1233-47; Grande M T and Lopez-Novoa J M, Fibroblast activation and myofibroblast generation in obstructive nephropathy, Nat Rev Nephrol., 2009 June; 5(6) : 319-28).

[0005] Inflammatory Bowel Disease (IBD) is characterized by an inflamed colon and/or small intestine. Most commonly, IBD is Crohn's Disease, ulcerative colitis (UC), or indeterminate colitis, when it is unclear if the inflammation is due to Crohn's disease or ulcerative colitis.

[0006] Ulcerative Colitis effects the colon only, and is marked by showing no healthy areas in the colon.

[0007] Crohn's disease is a type of inflammatory bowel disease (IBD) that primarily involves the small and large intestine, but may also affect any other part of the gastrointestinal tract. In its mild forms, Crohn's disease causes scattered, shallow ulcers in the inner surface of the bowel. In more serious cases, deeper and larger ulcers can develop, causing scarring and stiffness and possibly narrowing of the bowel, sometimes leading to obstruction. Deep ulcers can puncture holes in the bowel wall, leading to infection in the abdominal cavity (peritonitis) and in adjacent organs.

[0008] There is currently no cure for IBD, and there is no single treatment that works for every individual. 5-aminosalicylic acid, also known as 5-ASA, is currently a global standard for treatment of IBD. Used in combination with other drugs, the goal of such treatment is to reduce the inflammation that triggers the individual's signs and symptoms, and to improve long-term prognosis by limiting complications.

[0009] The murine models of intestinal inflammation are well-characterized experimental models of intestinal immune dysregulation that ultimately lead to colitis - a common characteristic of inflammatory bowel diseases. In these models, the progression of colonic inflammation is highly predictable and reproducible, leading to significant infiltration of the lamina propria with inflammatory cells and tissue damage of the colonic mucosa (Kiesler et al., Experimental models of inflammatory bowel diseases, Cell Mol Gastroenterol Hepatol, 2015; 1 : 154-70). In one such model, 2,4,6-trinitrobenzene sulfonic acid (TNBS) is used to induce colitis in mice. Ethanol and TNBS are co-administered intra-rectally to rats, as the ethanol is used as a means to effectively disrupt intestinal barrier and enable the interaction of TNBS with colon tissue proteins. (Antoniou et al., The TNBS-induced colitis animal model: An overview, Ann Med Surg (Lond). 2016 Nov; 11 : 9-15.) TNBS-induced colitis in mice constitutes an animal model of ulcerative colitis (UC) with high degree of similarity to the histopathological characteristics and distribution of inflammation described in human ulcerative colitis.

[0010] The present invention provides novel uses of emoxypine and its derivatives, typically studied as potential therapies for other pathologies, for the treatment and/or alleviation of renal fibrosis or CKD and IBD.

Summary of Invention

[0011] In one embodiment, the present invention provides methods and uses of Emoxypine in the prophylaxis or treatment of renal fibrosis or kidney disease in a subject.

[0012] In another embodiment, the present invention provides methods and uses of a derivative of Emoxypine in the prophylaxis or treatment of renal fibrosis or kidney disease in a subject.

[0013] In a further aspect, the kidney disease is chronic kidney disease.

[0014] In a further embodiment, the present invention provides methods and uses of Emoxypine in the prophylaxis or treatment of colitis or inflammatory bowel disease in a subject.

[0015] In another embodiment, the present invention provides methods and uses of a derivative of Emoxypine in the prophylaxis or treatment of colitis or inflammatory bowel disease in a subject.

[0016] In a further aspect, the inflammatory bowel disease is Crohn's disease.

[0017] In a yet further aspect, the inflammatory bowel disease is ulcerative colitis.

Brief Description of the Figures

[0018] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0019] FIG. 1 shows a comparison of the change in mean body weight in grams for study groups of C57BL/6 mice in a CKD study consisting of a sham surgery control group "Sham + Vehicle (Control)", a surgery vehicle control group "Vehicle (Control)", an Emoxypine treatment group, and a positive control treatment group, Telmisartan.

[0020] FIG. 2 shows an evaluation of renal function consisting of the plasma urea nitrogen (BUN) in mg/dl_ for the study groups of C57BL76 mice in the CKD study consisting of the sham surgery control group "Sham + Vehicle (Control)", the surgery vehicle control group "Vehicle (Control)", the Emoxypine treatment group, and the positive control treatment group.

[0021] FIG. 3 shows an evaluation of renal function consisting of the

Creatinine in mg/dL for the study groups of C57BL/6 mice in the CKD study consisting of the sham surgery control group "Sham + Vehicle (Control)", the surgery vehicle control group "Vehicle (Control)", Emoxypine treatment group, and the positive control treatment group.

[0022] FIG. 4 shows a comparison of the mean weight difference between the left (ligated UUO) and right (non-ligated) kidneys in grams for the study groups of C7BL/6 mice in the CKD study consisting of the sham surgery control group "Sham + Vehicle (Control)", the surgery vehicle control group "Vehicle (Control)", the Emoxypine treatment group, and the positive control treatment group, Telmisartan.

[0023] FIG. 5 shows an evaluation of renal fibrosis and interstitial damage with the level of fibrosis indicated by Histology Scores of 0 (normal), 1 (light), 2 (moderate) or 3 (severe) in three randomly selected fields of renal cross-sections stained with Sirius Red at x20 magnification for the study groups of C57BL76 mice in the CKD study consisting of the sham surgery control group, the surgery vehicle control group, the Emoxypine treatment group, and the positive control treatment group.

[0024] FIG. 6 shows the reduction in renal fibrosis for the study groups of C57BL/6 mice in the CKD study consisting of the sham surgery control group, the surgery vehicle control group, the Emoxypine treatment group, and the positive control treatment group.

[0025] Figure 7 is a line graph comparing the mean percentage change in body weights from baseline (± the standard error of the mean, or SEM) for experimental treatment groups of mice in an IBD study, comparing test compound Emoxypine with a "Naive" control group, a "TNBS-Vehicle" control group, and a "5-ASA" positive control group.

[0026] Figure 8 is a line graph comparing the mean Disease Activity Index (DAI) data, which includes daily measurement of body weight and evaluation of stool consistency, from baseline (± the standard error of the mean, or SEM) for experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0027] Figure 9 is a line graph comparing the mean fecal consistency from baseline (± the standard error of the mean, or SEM) for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0028] Figure 10 is a line graph comparing the mean occult positivity from baseline (± the standard error of the mean, or SEM) for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0029] Figure 11 is a column graph showing an indication of disease severity with a comparison of the mean colon length in centimeters for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0030] Figure 12 is a column graph showing an indication of disease severity with a comparison of the mean colon weight in grams for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0031] Figure 13 is a column graph showing an indication of disease severity with a comparison of the mean colon weight/length ratio in milligrams/centimeter for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0032] Figure 14 is a line graph showing an indication of disease progression with a comparison of the daily survival rate in percentage for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

[0033] Figure 15 is a column graph showing a Histopathology severity score, which evaluats of colitis, bowel wall inflammation, leukocyte infiltration, high vascular density, bowel wall thickening, disruption of normal crypt architecture and epithelial ulceration, with a comparison of the Histopathology Scores for the experimental treatment groups of mice in the IBD study, comparing test compound Emoxypine with the "Naive" control group, the "TNBS-Vehicle" control group, and the "5-ASA" positive control group.

Detailed Descrmtion

[0034] Without being limited to any particular theory, the inventors believe that compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof act as an actoprotector, has antihypoxic effects, and downregulates HIF1 alpha.

[0035] Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of it's A subunit.

Oxygen deprivation initiates a wide range of responses to restore oxygen homeostasis in the affected tissues. These adaptive responses are aimed to increase oxygen supply and compensate for the loss of energy. The transcription factor HIF-1 helps to restore oxygen homeostasis at a cellular, local, and systemic level. HIF-1 functions by regulating many of the genes involved in angiogenesis, erythropoiesis, glycolysis, iron metabolism, and cell survival. In addition to its role in oxygen homeostasis, HIF-1 has been implicated as a critical factor in the pathogenesis of tumor vascularization, and ischemia. Under normoxic conditions, HIF-la is degraded. Under hypoxic conditions, HIF-la protein is stabilized and initiates a multistep pathway of activation (Stroka et al., The FASEB Journal, Vol 15, Nov 2001, 2445-2453).

[0036] Throughout the following description, specific details are set forth in order to provide a more thorough understanding to persons skilled in the art.

However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0037] Emoxypine, 2-Ethyl-6-methyl-3-hydroxypyridine, is known in the art as an antioxidant. The chemical structure of Emoxypine is as follows:

[0038] A derivative of Emoxypine is shown below as Formula (I), with a chemical structure as follows:


[0039] In certain applications, each R3 to R5 is independently a hydrogen, an optionally substituted Ci to C4 alkyl, or optionally substituted phenyl; and R6 is a hydrogen, an optionally substituted Ci to C4 alkyl, or is -NR^2, wherein

R1 is a hydrogen, a Ci to C4 alkyl, or a phenyl,

R2 is hydroxy(Ci to Cie)alkyl, a (5-hydroxy-trimethylpyridin-2-yloxy)- (C2 to C4)alkyl, a C3 to C7 cycloalkyl, a benzyl, a phenyl, a (Ci to C4 alkyl)phenyl, a nitrophenyl, a halophenyl, a benzo[d][l,3]-dioxol-5-yl, or a pyridine-2-yl; or

R1 and R2 are linked to each other to form a 5-membered ring or a 6- membered ring, thereby forming a heterocyclic compound, and are any one selected from pyrrolidine-l-yl, 4-methylpiperazine-l-yl, morpholino, and 1H- pyrrol-l-yl.

[0040] In one embodiment the compound of formula (I) is 2-ethyl-6-methyl-3-hydroxypyridine, wherein R3 and R4 are H, R5 is -CH2-CH3, R6 is -CH3.

[0041] Definitions: (partially from 2018/0258043 and 2015/0291596 and 9822097)

[0042] "Acyl" means a carbonyl containing substituent represented by the formula— C(O)— R in which R is H, alkyl, a carbocycle, a heterocycle, carbocycle- substituted alkyl or heterocycle-substituted alkyl wherein the alkyl, alkoxy, carbocycle and heterocycle are as defined herein. Acyl groups include alkanoyl (e.g. acetyl), aroyl (e.g. benzoyl), and heteroaroyl.

[0043] "Alkyl" means a branched or unbranched, saturated or unsaturated (i.e. alkenyl, alkynyl) aliphatic hydrocarbon group, having up to 12 carbon atoms unless otherwise specified. When used as part of another term, for example

"alkylamino", the alkyl portion is preferably a saturated hydrocarbon chain, however also includes unsaturated hydrocarbon carbon chains such as

"alkenylamino" and "alkynylamino. "Alkylphosphinate" means a— P(0)R-alkyl group wherein R is H, alkyl, carbocycle-alkyl or heterocycle-alkyl. Examples of preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, n-heptyl, 3-heptyl, 2-methylhexyl, and the like. The terms "lower alkyl" "Ci-C4alkyl" and "alkyl of 1 to 4 carbon atoms" are synonymous and used interchangeably to mean methyl, ethyl, 1-propyl, isopropyl, cyclopropyl, 1-butyl, sec-butyl or t-butyl. Unless specified, substituted, alkyl groups may contain one (preferably), two, three or four substituents which may be the same or different. Examples of the above substituted alkyl groups include, but are not limited to; cyanomethyl, nitromethyl, hydroxymethyl, trityloxymethyl,

propionyloxymethyl, aminomethyl, carboxymethyl, carboxyethyl, carboxypropyl, alkyloxycarbonylmethyl, allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl, ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl, bromomethyl, iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl), 2-amino(iso-propyl), 2-carbamoyloxyethyl and the like. The alkyl group may also be substituted with a carbocycle group. Examples include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl groups, as well as the corresponding-ethyl, -propyl, -butyl, -pentyl, -hexyl groups, etc. Preferred substituted alkyls are substituted methyls e.g. a methyl group substituted by the same substituents as the "substituted Cn-C alkyl" group. Examples of the

substituted methyl group include groups such as hydroxymethyl, protected

hydroxymethyl (e.g. tetrahydropyranyloxymethyl), acetoxymethyl,

carbamoyloxymethyl, trifluoromethyl, chloromethyl, carboxymethyl, bromomethyl and iodomethyl.

[0044] As used herein, the term "alkoxy" or "alkyloxy" refers to an -O-alkyl group. For example, the term "Ci-e alkoxy" or "Ci-e alkyloxy" refers to an— O— (Ci-6 alkyl) group; and the term "Ci-4 alkoxy" or "Ci-4 alkyloxy" refers to an— 0— (Ci-4 alkyl) group; For another example, the term "C1-2 alkoxy" or "C1-2 alkyloxy" refers to an— 0— (Ci-2 alkyl) group. Examples of alkoxy include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like. The alkoxy or alkyloxy group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable

substituents.

[0045] As used here, the term "haloalkoxy" refers to an— O-haloalkyl group. For example, the term "Ci-6 haloalkoxy" refers to an— 0— (Ci-6 haloalkyl) group. For another example, the term "Ci-4 haloalkoxy" refers to an— 0— (Ci-4 haloalkyl) group; and the term "C1-2 haloalkoxy" refers to an— 0— (C1-2 haloalkyl) group. For yet another example, the term "Ci haloalkoxy" refers to a methoxy group having one, two, or three halogen substituents. An example of haloalkoxy is— OCFs or— OCHF2.

[0046] As used herein, the term "cycloalkoxy" or "cycloalkyloxy" refers to an — O-cycloalkyl group. For example the term "C3-7 cycloalkoxy" or "C3-7 cycloalkyloxy" refers to an— 0— (C3-7 cycloalkyl) group. For another example the term "C3-6 cycloalkoxy" or "C3-6 cycloalkyloxy" refers to an— 0— (C3-6 cycloalkyl) group.

Examples of cycloalkoxy include C3-6 cycloalkoxy (e.g., cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexanoxy, and the like). The cycloalkoxy or cycloalkyloxy group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.

[0047] As used here, the term "Ce-io aryloxy" refers to an— 0— (Ce-io aryl) group. An example of a Ce-io aryloxy group is -O-phenyl [i.e., phenoxy]. The Ce- lo aryloxy y group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.

[0048] "Amidine" or "amidino" means the group— C(NH)— NRR wherein each R is independently H, OH, alkyl, alkoxy, a carbocycle, a heterocycle, a carbocycle-substituted alkyl or a heterocycle-substituted alkyl; or both R groups together form a heterocycle. A preferred amidine is the group— C(NH)— NH2.

[0049] "Amino" denotes primary (i.e.— NH2), secondary (i.e.— NRH) and tertiary (i.e.—NRR) amines wherein R is independently alkyl, a carbocycle (e.g. aryl), a heterocycle (e.g. heteroaryl), carbocycle-substituted alkyl (e.g. benzyl) or a heterocycle-substituted alkyl or alternatively two R groups together with the nitrogen atom from which they depend form a heterocycle. Particular secondary and tertiary amines are alkylamine, dialkylamine, arylamine, diarylamine, aralkylamine and diaralkylamine. Particular secondary and tertiary amines are methylamine, ethylamine, propylamine, isopropylamine, phenylamine, benzylamine dimethylamine, diethylamine, dipropylamine and diisopropylamine.

[0050] "Amino-protecting group" as used herein refers to a derivative of the groups commonly employed to block or protect an amino group while reactions are carried out on other functional groups on the compound. Examples of such protecting groups include carbamates, amides, alkyl and aryl groups, imines, as well as many N-heteroatom derivatives which can be removed to regenerate the desired amine group. Preferred amino protecting groups are Boc, Fmoc and Cbz. Further examples of these groups are found in T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", 2nd ed., John Wiley & Sons, Inc., New York, N.Y., 1991, chapter 7; E. Haslam, "Protective Groups in Organic Chemistry",

J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981. The term "protected amino" refers to an amino group substituted with one of the above amino-protecting groups.

[0051] "Aryl" when used alone or as part of another term means a carbocyclic aromatic group whether or not fused having the number of carbon atoms

designated or if no number is designated, up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean, J. A., ed) 13th ed. Table 7-2 [1985]). In a particular embodiment aryl may be phenyl. Substituted phenyl or substituted aryl denotes a phenyl group or aryl group substituted with one, two, three, four or five, such as 1-2, 1-3 or 1-4 substituents chosen, unless otherwise specified, from halogen (F, Cl, Br, I), hydroxy, protected hydroxy, cyano, nitro, alkyl (for example Ci-Ce alkyl), alkoxy (for example Ci-Ce alkoxy), benzyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, aminomethyl, protected aminomethyl, trifluoromethyl,

alkylsulfonylamino, arylsulfonylamino, heterocyclylsulfonylamino, heterocyclyl, aryl, or other groups specified. One or more methyne (CH) and/or methylene (CF ) groups in these substituents may in turn be substituted with a similar group as those denoted above. Examples of the term "substituted phenyl" includes but is not limited to a mono- or di(halo)phenyl group such as 2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-fluorophenyl and the like; a mono- or di(hydroxy)phenyl group such as 4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 3- or 4-nitrophenyl; a cyanophenyl group, for example, 4-cyanophenyl; a mono- or di(lower alkyl)phenyl group such as 4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl, 4-(iso-propyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the like; a mono or

di(alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl, 3-methoxy-4-benzyloxyphenyl, 3-methoxy-4-(l-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl, 4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 3- or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected

carboxy)phenyl group such 4-carboxyphenyl; a mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2-(aminomethyl)phenyl or 2,4-(protected

aminomethyl)phenyl; or a mono- or di(N-(methylsulfonylamino))phenyl such as 3-(N-methylsulfonylamino))phenyl. Also, the term "substituted phenyl" represents disubstituted phenyl groups where the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted phenyl groups where the substituents are different, for example 3-methoxy-4-benzyloxy-6-methyl sulfonylamino, 3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstituted phenyl groups where the substituents are different such as 3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Substituted phenyl groups include 2-chlorophenyl, 2-aminophenyl, 2-bromophenyl, 3-methoxyphenyl, 3-ethoxy-phenyl, 4-benzyloxyphenyl, 4-methoxyphenyl, 3-ethoxy-4-benzyloxyphenyl, 3,4-diethoxyphenyl, 3-methoxy-4-benzyloxyphenyl, 3-methoxy-4-(l-chloromethyl)benzyloxy-phenyl, 3-methoxy-4-(l-chloromethyl)benzyloxy-6-methyl sulfonyl aminophenyl groups. Fused aryl rings may also be substituted with any (for example 1, 2 or 3) of the substituents specified herein in the same manner as substituted alkyl groups.

[0052] "Aralkyl" as used herein refers to an alkyl group aubstituted with an aryl group.

[0053] "Carbamoyl" means an aminocarbonyl containing substituent represented by the formula— C(0)N(R)2 in which R is H, hydroxyl, alkoxy, alkyl, a carbocycle, a heterocycle, carbocycle-substituted alkyl or alkoxy, or heterocycle-substituted alkyl or alkoxy wherein the alkyl, alkoxy, carbocycle and heterocycle are as herein defined. Carbamoyl groups include alkylaminoecarbonyl (e.g.

ethylaminocarbonyl, Et-NH— CO— ), arylaminocarbonyl (e.g. phenylaminocarbonyl), aralkylaminocarbonyl (e.g. benzoylaminocarbonyl) a heterocycleaminocarbonyl (e.g. piperizinylaminocarbonyl), and in particular a heteroarylaminocarbonyl (e.g. pyridylaminocarbonyl).

[0054] "Carbocyclyl", "carbocyclic", "carbocycle" and "carbocyclo" alone and when used as a moiety in a complex group such as a carbocycloalkyl group, refers to a mono-, bi-, or tricyclic aliphatic ring having 3 to 14 carbon atoms and preferably 3 to 7 carbon atoms which may be saturated or unsaturated, aromatic or non-aromatic. Preferred saturated carbocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups and more preferred are cyclopropyl and cyclohexyl and most preferred is cyclohexyl. Preferred unsaturated carbocycles are aromatic e.g. aryl groups as previously defined, the most preferred being phenyl. The terms "substituted carbocyclyl", "substituted carbocycle" and

"substituted carbocyclo" unless otherwise specified mean these groups substituted by the same substituents as the "substituted alkyl" group.

[0055] "Carboxy- protecting group" as used herein refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of such carboxylic acid protecting groups include 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzhydryl, 2, 2', 4,4'-tetramethoxybenzhydryl, alkyl such as t-butyl or t-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, beta-(trimethylsilyl)ethyl, beta-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, l-(trimethylsilylmethyl)prop-l-en-3-yl, and like moieties. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the condition of subsequent reaction(s) on other positions of the molecule and can be removed at the appropriate point without disrupting the remainder of the molecule. In particular, it is important not to subject a carboxy- protected molecule to strong nucleophilic bases, such as lithium hydroxide or NaOH, or reductive conditions employing highly activated metal hydrides such as LiAIhU. (Such harsh removal conditions are also to be

avoided when removing amino-protecting groups and hydroxy-protecting groups, discussed below.) Preferred carboxylic acid protecting groups are the alkyl (e.g. methyl, ethyl, t-butyl), allyl, benzyl and p-nitrobenzyl groups. Similar carboxy-protecting groups used in the cephalosporin, penicillin and peptide arts can also be used to protect a carboxy group substituents. Further examples of these groups are found in T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", 2nd ed., John Wiley & Sons, Inc., New York, N.Y., 1991, chapter 5; E. Haslam, "Protective Groups in Organic Chemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981, Chapter 5. The term

"protected carboxy" refers to a carboxy group substituted with one of the above carboxy- protecting groups.

[0056] As used herein, the term "cycloalkyl" refers to saturated or

unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings (e.g., monocyclics such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclics including spiro, fused, or bridged systems (such as bicyclo[l. l. l]pentanyl, bicyclo[2.2.1]heptanyl,

bicyclo[3.2.1]octanyl or bicyclo[5.2.0]nonanyl, decahydronaphthalenyl, etc.). The cycloalkyl group has 3 to 15 carbon atoms. In some embodiments the cycloalkyl may optionally contain one, two or more non-cumulative non-aromatic double or triple bonds and/or one to three oxo groups. In some embodiments, the bicycloalkyl group has 6 to 14 carbon atoms. For example, the term "C3-14 cycloalkyl" refers to saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 14 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[l. l. l]pentanyl, or cyclodecanyl); and the term "C3-7 cycloalkyl" refers to saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 7 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,

bicyclo[l. l. l]pentan-l-yl, or bicyclo[l. l. l]pentan-2-yl). For another example, the term "C3-6 cycloalkyl" refers to saturated or unsaturated, non-aromatic, monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 6 ring-forming carbon atoms. For yet another example, the term "C3-4 cycloalkyl" refers to cyclopropyl or cyclobutyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings (including aryl and heteroaryl) fused to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclopentene, cyclohexane, and the like (e.g., 2,3-dihydro-lH-indene-l-yl, or lH-inden-2(3H)-one-l-yl). The cycloalkyl group optionally can be substituted by 1 or more (e.g., 1 to 5) suitable substituents.

[0057] As used herein, the term "fluoroalkyl" refers to an alkyl group having one or more fluorine substituents (up to perfluoroalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by fluorine). For example, the term "Ci-2 fluoroalkyl" refers to a C1-2 alkyl group having one or more fluorine substituents (up to perfluoroalkyl, i.e., every hydrogen atom of the C1-2 alkyl group has been replaced by fluorine). For another example, the term "Ci fluoroalkyl" refers to a Ci alkyl group (i.e., methyl) having 1, 2, or 3 fluorine substituents). Examples of fluoroalkyl groups include CF3, C2F5, CH2CF3, CH F2, CH2F, and the like.

[0058] As used here, the term "fluoroalkoxy" refers to an— O-fluoroalkyl group. For example, the term "C1-2 fluoroalkoxy" refers to an—O—C1-2 fluoroalkyl group. For another example, the term "Ci fluoroalkoxy" refers to a methoxy group having one, two, or three fluorine substituents. An example of Ci fluoroalkoxy is— OCF3 or -OCHF2.

[0059] "Guanidine" means the group— NH— C(NH)— NHR wherein R is H, alkyl, a carbocycle, a heterocycle, a carbocycle-substituted alkyl, or a heterocycle-substituted alkyl. A particular guanidine group is— NH— C(NH)— NH2.

[0060] As used herein, the term "halo" or "halogen" group is defined to include fluorine, chlorine, bromine or iodine.

[0061] As used herein, the term "haloalkyl" refers to an alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by a halogen atom). For example, the term "Ci-6 haloalkyl" refers to a Ci-e alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by a halogen atom). For another example, the term "Ci-4 haloalkyl" refers to a Ci-4 alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by a halogen atom); the term "C1-3 haloalkyl" refers to a C1-3 alkyl group having one or more halogen substituents (up to perhaloalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by a halogen atom); and the term "C1-2 haloalkyl" refers to a Ci-2 alkyl group (i.e. methyl or ethyl) having one or more halogen substituents (up to perhaloalkyl, i.e., every hydrogen atom of the alkyl group has been replaced by a halogen atom). For yet another example, the term "Ci haloalkyl" refers to a methyl group having one, two, or three halogen substituents. Examples of haloalkyl groups include CF3, C2F5, CH F2, CH2F, CH2CF3, CH2CI and the like.

[0062] As used herein, the term "halocycloalkyl" refers to a cycloalkyl group having one or more halogen substituents (up to perhalocycloalkyl, i.e., every hydrogen atom of the cycloalkyl group has been replaced by a halogen atom). For example, the term "C3-4 halocycloalkyl" refers to a cyclopropyl or cyclobutyl group having one or more halogen substituents. An example of halocycloalkyl is 2-fluorocyclopropan-l-yl.

[0063] "Heterocyclic group", "heterocyclic", "heterocycle", "heterocyclyl", or "heterocyclo" alone and when used as a moiety in a complex group such as a heterocycloalkyl group, are used interchangeably and refer to any mono-, bi-, or tricyclic, saturated or unsaturated, aromatic (heteroaryl) or non-aromatic ring having the number of atoms designated, generally from 5 to about 14 ring atoms, where the ring atoms are carbon and at least one heteroatom (nitrogen, sulfur or oxygen) and preferably 1 to 4 heteroatoms. "Heterocyclosulfonyl" means a— SO2-heterocycle group; "heterocyclosulfinyl" means a— SO-heterocycle group. Typically, a 5-membered ring has 0 to 2 double bonds and 6- or 7-membered ring has 0 to 3 double bonds and the nitrogen or sulfur heteroatoms may optionally be oxidized (e.g. SO, SO2), and any nitrogen heteroatom may optionally be quaternized.

Preferred non-aromatic heterocycles include morpholinyl (morpholino), pyrrolidinyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2H-pyranyl,

tetrahydropyranyl, thiiranyl, thietanyl, tetrahydrothietanyl, aziridinyl, azetidinyl, 1-methyl-2-pyrrolyl, piperazinyl and piperidinyl. A "heterocycloalkyl" group is a heterocycle group as defined above covalently bonded to an alkyl group as defined above. Preferred 5-membered heterocycles containing a sulfur or oxygen atom and one to three nitrogen atoms include thiazolyl, in particular thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, in particular l,3,4-thiadiazol-5-yl and l,2,4-thiadiazol-5-yl, oxazolyl, preferably oxazol-2-yl, and oxadiazolyl, such as l,3,4-oxadiazol-5-yl, and l,2,4-oxadiazol-5-yl. Preferred 5-membered ring heterocycles containing 2 to 4 nitrogen atoms include imidazolyl, preferably imidazol-2-yl; triazolyl, preferably

1.3.4-triazol-5-yl; l,2,3-triazol-5-yl, l,2,4-triazol-5-yl, and tetrazolyl, preferably lH-tetrazol-5-yl. Preferred benzo-fused 5-membered heterocycles are benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Preferred 6-membered heterocycles contain one to three nitrogen atoms and optionally a sulfur or oxygen atom, for example pyridyl, such as pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl, preferably pyrimid-2-yl and pyrimid-4-yl; triazinyl, preferably l,3,4-triazin-2-yl and

1.3.5-triazin-4-yl; pyridazinyl, in particular pyridazin-3-yl, and pyrazinyl. The pyridine N-oxides and pyridazine N-oxides and the pyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl and the l,3,4-triazin-2-yl groups, are a preferred group.

Substituents for optionally substituted heterocycles, and further examples of the 5-and 6-membered ring systems discussed above can be found in W. Druckheimer et al., U.S. Pat. No. 4,278,793.

[0064] "Heteroaryl" alone and when used as a moiety in a complex group such as a heteroaralkyl group, refers to any mono-, bi-, or tricyclic aromatic ring system having the number of atoms designated where at least one ring is a 5-, 6-or 7-membered ring containing from one to four heteroatoms selected from the

group nitrogen, oxygen, and sulfur, and preferably at least one heteroatom is nitrogen ( Lang's Handbook of Chemistry, supra). Included in the definition are any bicyclic groups where any of the above heteroaryl rings are fused to a benzene ring. Heteroaryls in which nitrogen or oxygen is the heteroatom are preferred. The following ring systems are examples of the heteroaryl (whether substituted or unsubstituted) groups denoted by the term "heteroaryl": thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, tetrazolo[l,5-b]pyridazinyl and purinyl, as well as benzo-fused derivatives, for example benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoimidazolyl and indolyl. A particularly preferred group of "heteroaryl" include; l,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-l,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-l,3-thiazol-2-yl sodium salt, l,2,4-thiadiazol-5-yl, 3-methyl-l,2,4-thiadiazol-5-yl, l,3,4-triazol-5-yl, 2-methyl-l,3,4-triazol-5-yl, 2-hydroxy-l,3,4-triazol-5-yl, 2-carboxy-4-methyl-l,3,4-triazol-5-yl sodium salt, 2-carboxy-4-methyl-l,3,4-triazol-5-yl, l,3-oxazol-2-yl, l,3,4-oxadiazol-5-yl, 2-methyl-l,3,4-oxadiazol-5-yl, 2-(hydroxymethyl)-l,3,4-oxadiazol-5-yl, l,2,4-oxadiazol-5-yl, l,3,4-thiadiazol-5-yl, 2-thiol- 1,3, 4-thiadiazol-5-yl, 2-(methylthio)-l,3,4-thiadiazol-5-yl, 2-am ino- 1,3,4-thiadiazol-5-yl, lH-tetrazol-5-yl, l-methyl-lH-tetrazol-5-yl, 1-(1-(dimethylamino)eth-2-yl)-lH-tetrazol-5-yl, l-(carboxymethyl)-lH-tetrazol-5-yl, 1-(carboxymethyl)-lH-tetrazol-5-yl sodium salt, l-(methylsulfonic acid)-lH-tetrazol- 5-yl, l-(methylsulfonic acid)-lH-tetrazol-5-yl sodium salt, 2-methyl-lH-tetrazol-5-yl, l,2,3-triazol-5-yl, l-methyl-l,2,3-triazol-5-yl, 2-methyl-l,2,3-triazol-5-yl, 4-methyl-l,2,3-triazol-5-yl, pyrid-2-yl N-oxide, 6-methoxy-2-(n-oxide)-pyridaz-3-yl,

6-hydroxypyridaz-3-yl, l-methylpyrid-2-yl, l-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl, l,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl, l,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl, 2,5-dihydro-5-oxo-6-hydroxy-astriazin-3-yl, 2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt, 2,5-dihydro-5-oxo-6- hydroxy-2-methyl-astriazin-3-yl sodium salt, 2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl, 2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl, 2,5-dihydro-5-oxo-as-triazin-3-yl, 2,5-dihydro-5-oxo-2-methyl-as-triazin-3-yl, 2,5-dihydro-5-oxo-2,6-dimethyl-as-tnazin-3-yl, tetrazolo[l,5-b]pyridazin-6-yl and 8-aminotetrazolo[l,5-b]-pyridazin-6-yl. An alternative group of "heteroaryl" includes;

4-(carboxymethyl)-5-methyl-l,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-l,3-thiazol-2-yl sodium salt, l,3,4-triazol-5-yl, 2-methyl-l,3,4-triazol-5-yl, lH-tetrazol- 5-yl, l-methyl-lH-tetrazol-5-yl, l-(l-(dimethylamino)eth-2-yl)-lH-tetrazol-5-yl, 1-(carboxymethyl)-lH-tetrazol-5-yl, l-(carboxymethyl)-lH-tetrazol-5-yl sodium salt,

1-(methylsulfonic acid)-lH-tetrazol-5-yl, l-(methylsulfonic acid)-lH-tetrazol-5-yl sodium salt, l,2,3-triazol-5-yl, l,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl, l,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl, 2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt, 2,5-dihydro-5-oxo-6-hydroxy- 2-methyl-as-triazin-3-yl, tetrazolo[l,5-b]pyridazin-6-yl, and 8-aminotetrazolo[l,5-b]pyridazin-6-yl.

[0065] "Heteroaralkyl" as used herein refers to an alkyl group substituted with a heteroaryl group.

[0066] As used herein, the term "hydroxylalkyl" or "hydroxy a Iky I" refers to an alkyl group having one or more (e.g., 1, 2, or 3) OH substituents. The term "Ci-6 hydroxylalkyl" or "Ci-6 hydroxyalkyl" refers to a Ci-6 alkyl group having one or more (e.g., 1, 2, or 3) OH substituents. The term "Ci-4 hydroxylalkyl" or "Ci-4 hydroxyalkyl" refers to a Ci-4alkyl group having one or more (e.g., 1, 2, or 3) OH substituents; the term "Ci-3hydroxylalkyl" or "C1-3 hydroxyalkyl" refers to a C1-3 alkyl group having one or more (e.g., 1, 2, or 3) OH substituents; and the term "Ci-2 hydroxylalkyl" or "Ci-2hydroxyalkyl" refers to a C1-2 alkyl group having one or more (e.g., 1, 2, or 3) OH substituents. An example of hydroxylalkyl is— CH2OH or— CH2CH2OH.

[0067] "Hydroxy-protecting group" as used herein refers to a derivative of the hydroxy group commonly employed to block or protect the hydroxy group while

reactions are carried out on other functional groups on the compound. Examples of such protecting groups include tetrahydropyranyloxy, benzoyl, acetoxy,

carbamoyloxy, benzyl, and silylethers (e.g. TBS, TBDPS) groups. Further examples of these groups are found in T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", 2nd ed., John Wiley & Sons, Inc., New York, N.Y., 1991, chapters 2-3; E. Haslam, "Protective Groups in Organic Chemistry", J. G. W.

McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981. The term "protected hydroxy" refers to a hydroxy group substituted with one of the above hydroxy- protecting groups.

[0068] As used herein, the term "oxo" refers to =0. When an oxo is

substituted on a carbon atom, they together form a carbonyl moiety [— C(=0)— ].

[0069] "Pharmaceutically acceptable salts" include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like, and organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid and the like.

[0070] "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.

Particularly preferred are the ammonium, potassium, sodium, calcium and

magnesium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, TEA, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,

dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.

[0071] "Phosphinate" means— P(0)R— OR wherein each R is independently H, alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl. Particular phosphinate groups are alkylphosphinate (i.e.— P(0)R— O-alkyl), for example— P(0)Me-OEt.

[0072] "Sulfamoyl" means— SO2— N(R)2 wherein each R is independently H, alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl. Particular sulfamoyl groups are alkylsulfamoyl, for example methylsulfamoyl (— SO2— NHMe); arylsulfamoyl, for example phenylsulfamoyl; aralkylsulfamoyl, for example benzylsulfamoyl.

[0073] "Sulfinyl" means a— SO— R group wherein R is alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl. Particular sulfinyl groups are alkylsulfinyl (i.e.— SO-alkyl), for example methylsulfinyl; arylsu Ifinyl (i.e.— SO-aryl) for example phenylsulfinyl; aralkylsulfinyl, for example benzylsulfinyl.

[0074] "Sulfonamide" means— NR— SO2— R wherein each R is independently

H, alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl), a carbocycle or a heterocycle. Particular sulfonamide groups are alkylsulfonamide (e.g.— NH— S02-alkyl), for example methylsulfonamide; arylsulfonamdie (i.e.— NH— SCh-aryl) for example phenylsulfonamide; aralkylsulfonamide, for example

benzylsulfonamide.

[0075] "Sulfonyl" means a— SO2— R group wherein R is alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl. Particular sulfonyl groups are alkylsulfonyl (i.e.— SCh-alkyl), for example methylsulfonyl; arylsulfonyl, for example phenylsulfonyl; aralkylsulfonyl, for example benzylsulfonyl.

[0076] The phrase "and salts and solvates thereof" as used herein means that compounds of the inventions may exist in one or a mixture of salts and solvate forms. For example a compound of the invention may be substantially pure in one particular salt or solvate form or else may be mixtures of two or more salt or solvate forms.

[0077] Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, compounds of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0078] If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the

diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[0079] It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, fragmentation, decomposition, cyclization, elimination, or other reaction.

[0080] The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative

substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible

substituents of organic compounds.

[0081] As used herein, the term "optionally substituted" means that

substitution is optional and therefore includes both unsubstituted and substituted atoms and moieties. A "substituted" atom or moiety indicates that any hydrogen on the designated atom or moiety can be replaced with a selection from the indicated substituent group (up to that every hydrogen atom on the designated atom or moiety is replaced with a selection from the indicated substituent group), provided that the normal valency of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group (i.e., CH3) is optionally substituted, then up to 3 hydrogen atoms on the carbon atom can be replaced with substituent groups.

[0082] The invention also relates to prodrugs of the compounds of Formula I. Thus certain derivatives of compounds of Formula I which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of Formula I having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as "prodrugs". Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and

Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. E. B. Roche, American Pharmaceutical Association).

[0083] Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of Formula I with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985), or in Prodrugs: Challenges and Reward, 2007 edition, edited by Valentino Stella, Ronald Borchardt, Michael Hageman, Reza Oliyai, Hans Maag, Jefferson Tilley, pages 134-175 (Springer, 2007).

[0084] Moreover, certain compounds of Formula I may themselves act as prodrugs of other compounds of Formula I.

[0085] Also included within the scope of the invention are metabolites of compounds of Formula I, that is, compounds formed in vivo upon administration of the drug.

[0086] The compounds of Formula I (including salts thereof) include all stereoisomers and tautomers. Stereoisomers of Formula I include cis and trans isomers, optical isomers such as R and S enantiomers, diastereomers, geometric isomers, rotational isomers, atropisomers, and conformational isomers of the compounds of Formula I, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs).

Also included are acid addition or base addition salts wherein the counterion is optically active, for example, D-lactate or L-lysine, or racemic, for example, DL-tartrate or DL-arginine.

[0087] Similar compounds can be found, for example, in US patent no.

9889139.

[0088] The pharmaceutically acceptable salt may be an acid-added salt formed from either an organic acid selected from the group consisting of an oxalic acid, a maleic acid, a fumaric acid, a malic acid, a tartaric acid, a citric acid, a benzoic acid, methanesulfonic acid and camphorsulfonic acid, or an inorganic acid selected from the group consisting of a hydrochloric acid, a sulfuric acid, a phosphoric acid, and a hydrobromic acid.

[0089] In some embodiments, the pharmaceutical composition according to the present invention may further include a carrier, an excipient, and a diluent which are conventionally used in preparing a pharmaceutical composition.

[0090] Examples of the carrier, the excipient, and the diluent are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water,

methylhydroxybenzoate, propylhydroxylbenzoate, talc, magnesium stearate, and mineral oil.

[0091] The pharmaceutical composition according to the present invention may be prepared in various formulations according to various conventional methods. For example, the pharmaceutical composition may be prepared in an oral formulation, an external-use formulation, a suppository formulation, or a sterile injection formulation. The oral formulation may be powder, granule, tablet, capsule, suspension, emulsion, syrup, or aerosols.

[0092] During preparation, a diluent or an excipient, such as fillers, thickeners, binders, wetting agents, disintegrants, or surfactants, may be used. For use as a solid preparation for oral administration, tablets, pills, powders, granules, capsules and so on are included, and these solid preparations may be prepared by mixing with at least one of the above-mentioned compounds, for example, starch, calcium carbonate, sucrose, or lactose, or gelatin.

[0093] In addition to the simple excipient, lubricants, such as magnesium stearate and talc, may also be used herein. A liquid preparation for oral

administration may be a suspension, a liquid for internal use, an emulsion, or a syrup, and includes various excipients, such as, for example, a wetting agent, a sweetening agent, an aromatic and a preservative, in addition to simple diluents such as water and liquid paraffin widely used in the art. A preparation for non-oral administration includes a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. A vegetable oil such as propylene glycol, polyethylene glycol, or olive oil, or an injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspension. A base of the suppository that may be used herein may include witepsol, macrogol, Tween 61, cacao butter, laurin butter or glycerogelatin.

For Treatment of Kidnev Disorders

[0094] The inventor has found that Emoxypine and its derivatives thereof are useful in inhibiting renal fibrosis and may be useful in the prophylaxis and/or treatment of kidney disease. In some embodiments, it is found that in the murine model of UUO surgical intervention, the level of fibrosis is inhibited or alleviated with use of Emoxypine. Based on the experimental results described herein, given their similar chemical structures, it can be soundly predicted that the derivatives of Emoxypine, as described above, may also be useful in some embodiments in the prophylaxis and/or treatment of kidney disease and chronic kidney disease.

[0095] The example and data below show the effects of inhibiting or alleviating renal fibrosis using Emoxypine. In the CKD study, a therapeutically effective amount of Emoxypine was administered. The Emoxypine was formulated with a pharmaceutically acceptable vehicle for the purpose of delivery and

absorption.

[0096] The current gold standard for treating kidney disease is administering the pharmacologic compound Telmisartan, which was used as a positive control in the experimental example described herein.

[0097] Telmisartan, 2-(4-{ [4- Methyl-6-( 1-methyl- 1H- l,3-benzodiazol-2-yl)-2-propyl-lH-l,3-benzodiazol-l-yl]methyl}phenyl)benzoic acid, is an angiotensin receptor blocker known in the art for treating hypertension. The chemical structure of Telmisartan is as follows:


[0098] In one aspect, the present invention provides a use and method of treatment or prophylaxis of renal fibrosis or kidney disease in a subject with

Emoxypine, or a derivative thereof as discussed above (including a

pharmaceutically acceptable variation thereof). The kidney disease may be chronic kidney disease.

[0099] In an embodiment, the amount of Emoxypine or derivative thereof used is between 20 and 2000 mg per kg, more preferably between 120 to 200 mg per kg of the subject. In a preferred embodiment, the amount of Emoxypine or derivative thereof used is between 140 to 180 mg per kg of the subject. In a further preferred embodiment, the amount of Emoxypine or derivative thereof used is between 150 to 170 mg per kg of the subject. In a yet further preferred embodiment, the amount of Emoxypine or derivative thereof used is between 155 to 165 mg per kg of the subject. In a still further preferred embodiment, the amount of Emoxypine or derivative thereof used is about 160 mg per kg of the subject.

[00100] The Emoxypine, or derivative/pharmaceutically acceptable variation thereof, may be administered to the subject orally, intravenously or in a manner known in the art. The Emoxypine, or derivative/pharmaceutically acceptable variation thereof, may also be administered with one or more pharmaceutically acceptable excipients.

[00101] Use in Combination

[00102] In another aspect, the present invention provides a use and method of treatment or prophylaxis of chronic kidney disease in a subject with one or more of Suplatast Tosylate, Iguratimod, Repirinast, Lobenzarit, Actarit, Ifenprodil, Bemithyl, Bromantane, Udenafil, Istradefyllne, and Cenicriviroc, in combination with

Emoxypine or a derivative thereof. In another aspect, the present invention provides a use and method of treatment or prophylaxis of chronic kidney disease in a subject with one or more of Suplatast Tosylate, Iguratimod, Repirinast,

Lobenzarit, Actarit, Ifenprodil, Bemithyl, Bromantane, Udenafil, Istradefyllne, and Cenicriviroc in combination with Emoxypine or a derivative thereof and one or more of a cholesterol lowering drug, an antihypertension drug, or erythropoietin.

[00103] Examples of cholesterol lowering drugs for use in combinations include Atorvastatin (Lipitor), Fluvastatin (Lescol), Lovastatin, Pitavastatin (Livalo),

Pravastatin (Pravachol), Rosuvastatin calcium (Crestor), Simvastatin (Zocor) and niacin, Alirocumab (Praluent), Evolocumab (Repatha), Alirocumab (Praluent), and Evolocumab (Repatha). Examples of antihypertension drugs for use in

combinations include antihypertensives, calcium channel blockers, ACE inhibitors angiotensin II receptor blockers, diuretics, and beta blockers. Examples of known angiotensin II receptor antagonists include both angiotensin I receptor subtype antagonists and angiotensin II receptor subtype antagonists. Suitable antiotensin II receptor antagonists include losartan and valsartan. Suitable calcium channel blockers include, for example, verapamil, diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine, and bepridil. Diuretics include, for example, furosemide, diuril, amiloride, and hydrodiuril. Losartan, candesartan, telmisartan, valsartan, olmesartan, irbesartan, and the like can be used as a hypotensive agent.

[00104] The term "effective amount" used herein refers to the amount of an active ingredient sufficient to confer a desired prophylactic or therapeutic effect in a treated subject. In one aspect, an effective amount for inhibiting or alleviating hepatic steatosis, lobular inflammation, hepatocellular ballooning or NASH-derived HCC improves or reduces one or more symptoms, conditions or progression thereof. In some embodiments, the symptoms, conditions or progression are determined and evaluated using methods known in the art that measure various disease progress-related indexes, for example by analyzing liver sections via immunohistochemical staining. In some embodiments, the effective amount is determined by persons skilled in the art evaluating, for example, the administration route and frequency, body weight and species of the subject receiving the pharmacologic compound. In some embodiments, an effective amount of the pharmacologic compound is formulated with a pharmaceutically acceptable vehicle and administered to the subject.

[00105] The term "pharmaceutically acceptable" used herein means that the vehicle is known in the art as compatible with the pharmacologic compound while also being safe to the subject receiving the treatment. In some embodiments, the pharmaceutically acceptable vehicle is determined by persons skilled in the art evaluating, for example, the solubility of the pharmacologic compound in said vehicle.

[00106] Exemplary embodiments of the present invention are further described with reference to the following example, which is intended to be illustrative and not limiting in nature.

[00107] Example 1 - Chronic Kidney Disorder Study

[00108] Materials and Methods

[00109] Male, 9-12 week old C57BL/6 mice of 23-25 grams body weight were used. All mice were acclimated for a minimum of 5 days prior to the start of the study and housed individually in microisolators throughout the study in a 12: 12 light-dark cycle on a standard maintenance mouse chow diet (Harlan Teklad 2018) with food and water given ad libitum. Prior to surgery, all mice were weighed for baseline body weight.

[00110] The UUO surgical intervention was performed according to methods known in the art (Le Meur Y et al., Macrophage accumulation at a site of renal inflammation is dependent on the M-CSF/c-fms pathway, J Leukocyte Biol., 72: 530-537; 2002).

[00111] All mice were first anesthetized by intraperitoneal injection with a rodent cocktail (ketamine lOmg/mL and Xyaline lmg/mL) in normal saline (10pL/g body weight). Pedal reflex and movement of the vibrissae were used to determine the state of unconsciousness. A state of unconsciousness was confirmed after a period of about 5 minutes in all animals. All mice were then shaved on the left side of the abdomen. The shaved area was first swabbed with iodine and then swabbed with alcohol. A vertical incision was made through the skin with a #22 scalpel and the skin was then retracted. A further incision of about 2.5cm was then made through the peritoneum avoiding any major blood vessels. The peritoneum was then retracted and the left kidney was exposed.

[00112] The left kidney was then brought to the surface by hooking the ureter directly beneath the kidney with sterile forceps and gently manipulating the kidney upward. The ureter was ligated at two points directly below the kidney with 5-0 surgical silk with excess suture cut away and discarded post-ligation. The kidney was then gently placed back to its correct anatomical position and the abdomen was lavaged with lmL sterile saline to replenish fluid loss. The peritoneum and then skin were sutured with 5-0 Mersilene, and the incision site was gently wiped with iodine. All mice were then placed individually in clean cages that were set on top of a thermal blanket until recovery at about 30-60 minutes later. In some examples, sham surgery was performed as a control by following all steps of the UUO surgical intervention procedure except ligation.

[00113] Following surgery, the mice were divided into individual study groups of 8 mice each and, following post-surgical recovery, administered a once-daily oral treatment for 14 days. The mice in two study groups (hereafter known as

"treatment groups") had all received the UUO surgical ligation and were treated individually with a distinct pharmacologic compound as set out in Table 1. The mice in two study groups were treated individually with a pharmaceutically acceptable vehicle with no active ingredient. The pharmaceutically acceptable vehicle in all groups was 0.5% carboxymethyl cellulose (CMC). All mice were sacrificed with C02 on post-surgical day 15.

[00114] Table 1


[00115] The dose selected for the animal studies was determined by taking the maximum known human daily dose, dividing by the average weight of an adult

(~60-70 kg) to get a human mg/kg dose. Then that number was multiplied by 12 to convert to a mouse dose based on conventional dosing tables. See Nair and Jacob, J Basic Clin Pharm March 2016-May 2016, 7(2) : 27-31.

[00116] The following measurements and assessments were taken for each mouse.

[00117] Body weight: The body weights were measured on days 1, 2, 5, 8, 10 and 14 using a laboratory balance.

[00118] Serum collection : A blood sample was then collected from all mice and plasma analyzed for urea nitrogen and creatinine. Plasma was stored at -80 °C for possible future analysis.

[00119] Kidney weight: The UUO was then examined in situ to ensure that the surgical ligation ties remained patent. Both the ligated (UUO) and non-ligated kidneys were removed for analysis. Weights of both kidneys were measured using a laboratory balance.

[00120] Histopathology: Formalin-fixed kidney cross-section samples were subjected histopathological scoring with Sirius Red staining, and imaged at a magnification of x20, using standard techniques. All three sections were stained and evaluated. The analysis was performed by a board-certified veterinarian pathologist. The presence of interstitial damage and severity score was assessed according to the following criteria : 0= normal, 1= light, 2= moderate, 3= severe.

[00121] Values are arithmetic means. Comparison between the study group and positive control group was performed using a two-tailed, heteroscedastic (two-sample unequal variance) Student's T-Test. A p-value of <0.05 was considered statistically significant.

[00122] Results

[00123] Body Weight Evaluation: Results of the evaluation of mean body weight change are shown in FIG. 1 and Table 2. Body weights were measured on post-surgical days 1, 2, 5, 8, 10 and 14 as previously described. The mean body weight change of each study group was calculated using the body weight average of all mice in each study group.

[00124] Table 2. Mean Body Weight Change Following Surgery (g)

[00125] All study groups showed an initial decrease in mean body weight change followed by an increase in mean body weight change until sacrifice.

[00126] The mean body weight change on post-surgical day 14 for the

Emoxypine treatment group showed a more positive value than the positive control Telmisartan treatment group. This indicated a greater increase in body weight in the Emoxypine treatment group versus post-surgical day 1.

[00127] Renal Function Evaluation : Renal function and disease progression was evaluated by BUN (blood urea nitrogen) as previously described. The BUN of each study group consisted of the BUN average of all mice in each study group. Results are shown in FIGS. 2 and 3. and Table 3

[00128] Table 3. Results of Renal Function Evaluation.


[00129] The BUN for both treatment groups, Telmisartan and Emoxypine, decreased in comparison to the surgery vehicle control group, though not in comparison to the sham vehicle group.

[00130] Kidney Weight Evaluation: Kidney weight indication, which indicates functional and pathological changes in the kidney, was evaluated by measuring the weight of both the ligated (UUO) and non-ligated kidneys as previously described. The ligated and non-ligated kidney weights of each study group consisted of the respective kidney weight averages of all mice in each study group. Results are shown in Table 4 and FIG. 4.

[00131] Table 4. Kidney Weights for Study Groups.

[00132] The ligated kidney weights for all treatment groups and the surgery vehicle control group were increased in comparison to the sham surgery control group. The ligated kidney weights for the Emoxypine treatment group was higher in comparison to the ligated kidney weight of the positive control treatment group and slightly lower in comparison to the ligated kidney weight of the surgery vehicle control group.

[00133] The increase in weight of the ligated kidney in comparison to the non-ligated kidney was greater in the Emoxypine treatment group as compared to the positive control treatment group. In particular, the positive control Telmisartan (5 mg/kg) showed the greatest numerical reduction in the disparity between the two kidneys.

[00134] Renal Fibrosis Evaluation: Renal fibrosis and interstitial damage was evaluated by histochemical staining of renal cross-sections with Sirius Red as previously described. The Histology Score of each study group consisted of the histology score average of all mice in each study group. Results are shown in Table 5 and FIGS. 5 and 6.

[00135] Table 5. Sirius Red Stain Scoring.

[00136] The p-value of the Exmoypine treatment group was statistically significant and the Histology Scores for treatment group Emoxypine was the same as the positive control treatment group.

[00137] Conclusions

[00138] Oral administration of Emoxypine (160 mg/kg) showed significant reduction in fibrosis as compared to vehicle.

[00139] Oral administration of Telmisartan at 5 mg/kg also showed reduction in BUN levels and fibrosis, though the former was not statistically significant.

Telmisartan appeared to reduce the disparity in kidney size in the UUO-model, but the significance of this effect was not determined.

Example 2 - Treatment of Inflammatory Bowel Disease

[00140] The inventor has found that Emoxypine and derivatives thereof may be useful in the prophylaxis and/or treatment of inflammatory bowel disease. In some embodiments, it is found that in the murine model of TNBS-induced colitis, the level of colonic inflammation is inhibited or alleviated with use of Emoxypine. Based on the experimental results described herein, given their similar chemical structures, it can be soundly predicted that derivatives of Emoxypine, as described above, may also be useful in some embodiments in the prophylaxis and/or treatment of colitis or inflammatory bowel disease.

[00141] 5-ASA (5-Aminosalicylic Acid), 5-Amino-2-hydroxybenzoic acid, is an aminosalicylate anti-inflammatory drug known in the art for treating inflammatory bowel disease such as ulcerative colitis and for maintaining remission in Crohn's disease. The chemical structure of 5-Aminosalicylic Acid is as follows:


[00142] The example and data below show the effects of inhibiting or

alleviating colitis by administering a therapeutically effective amount of Emoxypine. [00143] In one aspect, the present invention provides a use and method of treatment or prophylaxis of colitis or inflammatory bowel disease (IBD) in a subject with Emoxypine or a derivative thereof as discussed above. The IBD may be

Crohn's disease or ulcerative colitis, among others.

[00144] In a preferred embodiment, the amount of Emoxypine or derivative thereof used is between 0.1 to 30 mg per kg of the subject.

[00145] In a further preferred embodiment, the amount of Emoxypine or derivative thereof used is between 5 to 20 mg per kg of the subject.

[00146] In a still further preferred embodiment, the amount of Emoxypine or derivative thereof used is about 13 mg per kg of the subject.

[00147] The Emoxypine, or the derivative/pharmaceutically acceptable salt thereof, may be administered to the subject orally, intravenously or in a manner known in the art. The Emoxypine, or the derivative/pharmaceutically acceptable salt thereof, may also be administered with one or more pharmaceutically

acceptable excipients.

[00148] Use in Combination

[00149] In another aspect, the present invention provides a use and method of treatment or prophylaxis of colitis or inflammatory bowel disease in a subject with Emoxypine or a derivative thereof in combination with one or more of: anti inflammatory drugs, immune system suppressors, antibiotics, anti-diarrheals, pain relievers, iron supplements, vitamin B-12 shots, and calcium and vitamin D supplements.

[00150] The anti-inflammatory drugs are often used in the first step in the treatment of inflammatory bowel disease. They include corticosteroids and/or oral 5-aminosalicylates.

[00151] Oral 5-aminosalicylates include sulfasalazine (for example Azulfidine), which contains sulfa, and mesalamine (for example Asacol HD, Delzicol or others). While oral 5-aminosalicylates have been widely used in the past to treat Crohn's disease, it is now generally considered of limited benefit.

[00152] Corticosteroids, such as prednisone and budesonide (for example Entocort EC), may help reduce inflammation in the body, though it has not been shown to work for everyone with Crohn's disease. It is prescribed, typically, only if the subject hasn't responded to other treatments. Corticosteroids may be used for short-term (three to four months) symptom improvement and to induce remission. Corticosteroids may also be used in combination with an immune system

suppressor.

[00153] Immune system suppressor also reduce inflammation in a subject, but they target the subject's immune system, which produces the substances that cause inflammation. For some, a combination of these drugs works better than one drug alone. Such immunosuppressant drugs used with Emoxypine may comprise one or more of azathioprine, mercaptopurine, infliximab, adalimumab, certolizumab pegol and vedolizumab.

[00154] Azathioprine (for example Azasan or Imuran) and mercaptopurine (for example Purinethol or Purixan) are the most widely used immunosuppressants for treatment of inflammatory bowel disease. As they tend to lower resistance to infection and inflammation of the liver, taking them requires a subject to be followed up closely with a doctor and to have his/her blood checked regularly to look for such side effects. Their use may also cause nausea and vomiting.

[00155] Infliximab (for example Remicade), adalimumab (for example Humira) and certolizumab pegol (for example Cimzia) are called TNF inhibitors or biologies. They work by neutralizing an immune system protein known as tumor necrosis factor (TNF).

[00156] Vedolizumab was recently approved for use in treatment of Crohn's disease. Vedolizumab works like natalizumab, but appears not to carry a risk of brain disease.

[00157] Antibiotics have been used in the past to reduce the amount of drainage and sometimes heal fistulas and abscesses in subjects with Crohn's disease. Some researchers also think antibiotics may help reduce harmful intestinal bacteria that may play a role in activating the intestinal immune system, leading to inflammation. Frequently prescribed antibiotics include ciprofloxacin (for example Cipro) and metronidazole (for example Flagyl).

[00158] In addition to controlling inflammation, Emoxypine or a derivative thereof may also be used with other medications described below to help relieve other signs and symptoms.

[00159] Anti-diarrheals includes fiber supplements, such as psyllium powder (for example Metamucil) or methylcellulose (for example Citrucel), may be used to help relieve mild to moderate diarrhea by adding bulk to stool. For more severe diarrhea, loperamide (for example Imodium A-D) may be used.

[00160] Pain relievers, for mild pain, such as acetaminophen (for example Tylenol or others) may be used. However, other common pain relievers, such as ibuprofen (for example Advil, Motrin IB and others) and naproxen sodium (for example Aleve) should not be used as these drugs tend to make a subject's symptoms worse, and can make the disease worse as well.

[00161] If the subject has chronic intestinal bleeding, the subject may develop iron deficiency anemia and may need to take iron supplements.

[00162] Crohn's disease can cause vitamin B-12 deficiency. As such, Vitamin B-12 shots may be used with Emoxypine or a derivative thereof to help prevent

anemia, promote normal growth and development, since Vitamin B-12 s is essential for proper nerve function.

[00163] Calcium and vitamin D supplements may also be used with

Emoxypine. Crohn's disease and the steroids used to treat it can increase a subject's risk of osteoporosis. Calcium supplement with added vitamin D may help alleviate this osteoporosis.

[00164] Embodiments of the present invention are further described with reference to the following examples which is intended to be illustrative and not limiting in nature.

[00165] Inflammatory Bowel Disease Study

[00166] Materials and Methods

[00167] The test compound Emoxypine was obtained from Toronto Research Chemicals, Toronto, ON, Canada M3J2K8. The Positive control 5-aminosalicylic acid (5-ASA) was obtained from Sigma Aldrich, USA. The vehicle used was 0.5% CMC.

[00168] The dose selected for the animal studies was determined by taking the maximum known human daily dose, dividing by the average weight if an adult (~60-70 kg) to get a human mg/kg dose. Then that number was multiplied by 12 to convert to a mouse dose based on conventional dosing tables. See Nair and Jacob, J Basic Clin Pharm March 2016-May 2016, 7(2) : 27-31.

[00169] Thus, working backwards to arrive at the human dose:

Emoxypine = 160 mg/kg divide 12 = 13.3 mg/kg.

[00170] Healthy young female SJL mice were used for the study. At the commencement of the study, the mice were between 8-9 weeks of age, weighing 20-22g. All the mice were obtained from The Jackson Laboratory, Bar Harbor, Maine 04609 USA.

[00171] The mice were maintained in a controlled environment with a temperature of 70-72° F, humidity 30-70%, with a photo cycle of 12 hours of light and 12 hours of dark. They were provided with TEKLAD 2018-Global 18% diet and Arrowhead drinking water ad libitium.

[00172] After seven days of acclimatization, mice were grouped according to their body weight. Three groups of fifteen mice each were challenged intra-rectally with 100 pi of 2.0% TNBS in 50 % EtOH and another group of ten mice were challenged intra-rectally with 100 pi of 50% EtOH and serve as No-TNBS control. The experimental groups were as follows:

[00173] Table 6: Experimental Design


[00174] The mice were challenged intra-rectally with 100 mI of 2% TNBS in 50% ethanol under light anesthesia with ketamine/xylazine. The test compounds were administrated an hour prior to intra-rectal administration of TNBS as per scheduled daily dosing.

[00175] Emoxypine were prepared in 0.5% CMC and administrated orally once-a-day from day 1 to 7. 5-ASA was also prepared in 0.5% CMC and administrated orally once a day beginning on Day 1 to Day 7. Vehicle and no-TNBS control groups received 0.5% CMC orally from Day 1 to Day 7.

[00176] The following measurements and assessments were taken for each mouse.

[00177] Body Weight: The body weights were measured daily for 1-7 days using a laboratory balance.

[00178] Disease Activity Index: The clinical assessment of the mice was performed beginning Day 2 (a day after the intra-rectal administration of TNBS). The clinical assessment includes body weight, stool consistency and the presence of blood in the stools and scored according to Table 7.

[00179] Table 7: Disease Activity Index


[00180] Serum Collection : on Day 8, blood samples were collected from all the surviving mice and were processed for serum and stored at -80°C.

[00181] Colon Length and Weight: the mice were euthanized using CO2 asphyxiation and colon from the colocecal junction to the anus was removed, washed and cleaned of all fecal matter using PBS. The colon length and weight were measured and then preserved in 10% NBF for histopathology.

[00182] Histopathology: Formalin fixed colon samples were submitted to affiliated histopathology laboratory for histopathological analysis subjected to hematoxylin and eosin (H & E) staining using standard techniques. Each colon was trimmed from both ends and mid. All three sections were stained and evaluated.

[00183] A board certified veterinarian pathologist assessed the presence of colitis and severity score according to the following criteria :

0= no sign of inflammation

1= very low level of inflammation

2= low level of leukocytic infiltration, low level of inflammation

3= high level of leukocytic infiltration, high vascular density, inflammation and thickening of colon wall

4= transmural infiltration, loss of goblet cells, high vascular density, crypt abscesses, thickening of colon wall and ulceration.

[00184] The data is presented as the mean ± standard error (SEM) obtained from Microsoft Excel or GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA). The data was analyzed using two-way ANOVA using Bonferroni post-test. Differences between groups were considered significant at p<0.05.

[00185] Results

[00186] Body Weight

[00187] The percentage changes in body weights are presented in Figure 7 and Table 8. The decrease in body weight gains were observed from Day 3 till Day 5 and then started recovering. Significant differences were observed with the groups treated with Emoxypine (160 mg/kg po) and 5-ASA (100 mg/kg po). They showed significant improvement beginning on Day 4 as compared to the TNBS-vehicle group.

[00188] Table 8: Percent Change in Body Weight


[00189] Disease Activity Index

[00190] The Disease Activity Index (DAI) data are presented in Figure 8 and Table 9. The DAI included daily measurement of body weight and evaluation of stool consistency. No significant differences were observed between the Emoxypine treatment group and TNBS-vehicle group though the response was better with Emoxypine (160 mg/kg) than the 5-ASA (100 mg/kg) treated group.

[00191] Table 9: Disease Activity Index


[00192] Fecal consistency and occult positivity

[00193] This data is presented in Figures 9-10, and Tables 10-11. No significant differences were observed in fecal consistency between the Emoxypine treatment group and TNBS-vehicle group, whereas significant improvements were observed in occult positivity with the groups treated with Emoxypine (160 mg/kg) and 5-ASA (100 mg/kg) on Day 7 as compared to TNBS-vehicle.

[00194] Table 10: Fecal Consistency


[00195] Table 11 : Occult Positivity


[00196] Colon Length, weight and weight/length ratio

[00197] This data is presented in Figures 11-13, and Tables 12-14. Significant differences were observed in colon length between treatments groups and vehicle treated group, whereas in colon weight Emoxypine (160 mg/kg) showed significant improvement as compared to TNBS-vehicle. The ratio between colon weight and length were significant in the Emoxypine treatment group as compared to TNBS- vehicle.

[00198] Table 12: Colon Length, cm



[00199] Table 13: Colon Weight, g

[00200] Table 14: Colon Weight/Length ratio, mg/cm


[00201] Percent Survival

[00202] The percent survival data is presented in Figure 14 and Table 15. The percent survival was higher with the treatment group treated with Emoxypine (160 mg/kg) than 5-ASA (100 mg/kg).

[00203] Table 15: Percent Survivability


[00204] Histopathology

[00205] Rating data are presented in Figure 15 and Table 16. The data demonstrate that Emoxypine, like 5-ASA, was statistically significantly reducing cell morphological changes associated with disease. The 3 main categories include (i) quality and dimension of inflammatory cell infiltrates, (ii) epithelial changes and (iii) overall mucosal architecture.

[00206] Table 16: Histopathological severity score


[00207] Conclusions

[00208] In conclusion, oral administration of Emoxypine at 160 mg/kg showed improvement in colitis as well as in the loss of body weight, DAI, colon length, weight and weight/length ratio as compared to TNBS-vehicle.

[00209] Oral administration of 5-ASA at 100 mg/kg also showed improvement in the colitis, loss of body weight and disease activity index as compared to TNBS- vehicle.

[00210] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.