WO2008131586A1

WO2008131586A1 - Uses of 5-methyl-1-(substituted phenyl)-2(1h)-pyridones as anti-inflammatory and tnf-alpha-blocking agents

Info

Publication number: WO2008131586A1
Application number: PCT/CN2007/001423
Authority: WO
Inventors: Yuenian Shi
Original assignee: Dieretech Investment Limited
Priority date: 2007-04-27
Filing date: 2007-04-27
Publication date: 2008-11-06
Also published as: CN101472588A

Abstract

The present invention discloses anti-TNF-a activities of compounds in the 5-methyl-1-(substituted phenyl)-2(1H)-pyridone family. An representative example of 5-methyl-(1-substituted phenyl)-2(1H)-pyridones is 1-(3'-fluorophenyl)-5-methyl-2(1H)-pyridone, AKF-PD. Accordingly, there is provided a method of using one or more 5-methyl-1-(substituted phenyl)-2(1H)-pyridones to treat TNF-a mediated diseases such as rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel disease (Crohn's).

Description

USES OF 5-METHYL-l-(SUBSTITUTED PHENYL) -2 (IH) -PYRIDONES AS ANTI-INFLAMMATORY AND TNF-alpha-BLOCKING AGENTS

FIELD OF THE INVENTION

[0001] This invention is related to the uses of compositions comprising 5-methyl-1- (substituted phenyl) -2 (IH) -pyridones .

BACKGROUND OF THE INVENTION

[0002] Inflammation is a common pathological event and inflammatory diseases cover many medical conditions . Inflammatory process is a complex processes. Many mediators have been identified to have roles in the inflammatory process. Certain inflammatory diseases apparently have a central mediator: such as cytokine, tumor necrosis factor-alpha (TNF-α) , and therefore these inflammatory diseases are also called TNF-α mediated diseases: such as rheumatoid arthritis (RA) .

[0003] TNF-α is a 17 kDa, multi-functional peptide produced by a wide variety of cells during host responses to tissue or organ injury including microbial infections and neoplastic diseases. Activated macrophages are a major cellular source for TNF-α, although other cell types, e.g., T cells, mast cells, neutrophils, endothelial cells, and astrocytes, can be stimulated to secrete TNF-α. Enhanced TNF-α synthesis is associated with the development of numerals inflammatory diseases, such as rheumatoid arthritis (RA) , as well as numerals other inflammatory diseases, such as, psoriasis, psoriatic arthritis, asthma, sepsis, and inflammatory bowel disease (Crohn' s) .

[0004] Rheumatoid arthritis (RA) , a systemic disease, is characterized by a chronic inflammatory reaction in the synovium of joints. The severe inflammatory reaction can cause the degeneration of cartilage and erosion of juxta-articular bone. Plenty data indicates that TNF-α is a central in the pathogenesis of RA and a good therapeutic target for pharmacological intervention. Last decade, three high profile protein TNF-α blockers: etanercept (Enbrel) , inflixmab

(Remicade) , adalimumab (Humira) , have been approved by various regulatory agencies. These injectable protein drugs have been remarkably successful, however their limitation have been recognized gradually (Anti-TNF-α therapies: the next generation,

Palladino MA. et al, Nat Rev. Drug Del, 2(9) 736-746, 2003).

Therefore, there are many efforts trying to develop an alternative anti-TNF-α therapy: orally active, small molecule based therapy.

[0005] Asthma is characterized by the presence of an inflammatory cell infiltrate in the bronchial mucosa consisting of activated mast cells and T cells. Several cytokines are considered to play a pivotal role in this response, particularly interleukin (IL) -4, IL-5, IL- 6, TNF-alpha. TNF-α is recognized as an important mediator in many cytokine-dependent inflammatory events and affects immune response and airway inflammation. Recent studies suggest that allergen exposure is the major primary cause of asthma, and that the global increases in asthma prevalence are due to increases in exposure to aeroallergens . It is known that TNF-α is released in allergic responses from both mast cells and macrophages . Concentrations of TNF-α in bronchoalveilar lavage fluid from asthma patients are much higher than that of normal healthy person.

[0006] Sepsis, also known as systemic inflammatory response syndrome (SIRS) , is a serious medical condition caused by the body's response to an infection. Sepsis can lead to widespread inflammation and blood clotting. Inflammation may result in redness, heat, swelling, pain, and organ dysfunction or failure. Blood clotting during sepsis causes reduced blood flow to limbs and vital organs, and can lead to organ failure or gangrene (damage to tissues) . Experimental animal sepsis shock models demonstrated that administration of TNF-α blocking agents can significantly reduced sepsis-associated death. TNF-α is one the major pre-inflammatory cytokines that triggers sepsis.

[0007] A number of compounds that exhibit anti-inflammatory and anti-fibrotic activities have been described. U.S. patents 3,839,346, 4,052,509, 4,042,699 published a total of 29 compounds with the following pyridone-like formula (structural formula I) :

wherein A is an aromatic group. These compounds have good anti-inflammatory and analgestic activities and can reduce serum levels of uric acid and glucose. One compound in particular, 5-methyl-1-phenyl-2 (IH) -pyridone, has the best activity and low toxicity.

[0008] U.S. patent 5,310,562 reported anti-fibrotic activity for 5-methyl-1-phenyl-2 (IH) -pyridone (PIRFENIDONE, PFD). U.S. patents 5,518,729 and 5,716,632 extended the anti-fibrotic activity to 44 compounds of N-substituted 2 (IH) -pyridone

(structural formula I) or N-substituted 3 (IH) -pyridone .

[0009] US patent 5,962,478 reported anti-TNF-α activity for pirfenidone and additional 44 closely related compounds.

[00010] A Chinese patent ZL02114190.8 described the identification and synthesis of many new 5-methyl-1-

(substituted phenyl) -2 (IH) -pyridones compounds having the following general structural formula (structural formula II) :

II wherein if n = 1, the substituent R can be F, Br, I. If n = 2, R can be F, Cl, Br, I, straight-chain alkyl group, straight- chain alkoxy group, or straight-chain halogenated alkyl group. The relative position of R groups can be ortho, meta or para.

[00011] U.S. patents 5,716,632 and 5,962,478 listed 7 structural formulas of 5-methyl-l- (substituted phenyl) -2 (IH) - pyridone originally described by Gadekar in U.S. patent 4,042,699. For these "substituted phenyl" compounds, Gadekar established a structure-activity relationship that teaches non- substituted phenyl as the compound with the best biological activities. In a US pending patent, we demonstrated that the substituted phenyl compounds having structural formula II, actually, have more desirable anti-fibrotic activity. Thus, it is of interest to determine whether substituted phenyl compounds having structural formula II would have any desirable anti-TNF-α activity.

SUMMARY OF THE INVENTION

[00012] The present invention describes anti-TNF-α effects of compounds in the 5-methyl-l- (substituted phenyl) -2 (IH) -pyridone family. A serious representative substituted phenyl pyridones were synthesized according to the process described in Chinese patent ZL02114190.8 , the disclosure of which is incorporated herein by reference. These pyridones, for example, are 5- methyl-1- (3' -fluorophenyl) -2 (IH) -pyridone (AKF-PD), l-(3'- bromophenyl) -5-methyl-2 (IH) -pyridone (AKF-BR) , 1- (3 ' - chlorophenyl) -5-methyl- 2 (IH) -pyridone (AKF-CL), l-(3'- methylphenyl) -5 -methyl-2 (IH) -pyridone (AKF-CH₃), and l-(3'- methoxyphenyl) -5 -methyl-2 (IH) -pyridone (AKF-OCH₃). The compounds were tested on mouse macrophages. AKF-PD was further investigated in animal model of rheumatoid arthritis (RA) . The results presented herein demonstrate that 5-methyl-l- (substituted phenyl) -2 (IH) -pyridones can be potentially used for treatment of TNF-α-mediated inflammatory diseases such as rheumatoid arthritis with much less toxic effect. [00013] In one embodiment, the present invention provides a use of one or more compound having the general structural formula (structural formula II) , for treating TNF-α mediated diseases, such as rheumatoid arthritis,

wherein when n=l, the substituent R can be F, Br, I, a nitro group, an alkyl group, an alkoxy group, or a halogenated alkyl group; when n=2, the substituent R can be F, Cl, Br, I, an alkyl group, an alkoxy group, or a halogenated alkyl group.

[00014] The present invention also provides a pharmaceutical composition comprising a 5-methyl-1- (substituted phenyl) -2 (IH) - pyridone having the structural formula II, wherein the compound is present in an amount effective for treating TNF-α mediated diseases such as rheumatoid arthritis.

[00015] The present invention also provides a method of treating TNF-α mediated diseases, e.g., rheumatoid arthritis, comprising the administration of a compound having the structural formula II, wherein n=l and the substituent R is F, i.e AKF-PD.

[0016] The present invention also provides uses of a composition comprising a 5-methyl-l- (substituted phenyl) -2 (IH) - pyridone having the structural formula II as disclosed herein for treating TNF-α mediated diseases, such as rheumatoid arthritis .

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Aforementioned and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which: FIG. 1: Inhibition of TNF-α mRNA synthesis in mouse macrophage by AKF-PD and PFD.

FIG. 2: Inhibition of TNF-α protein synthesis and secretion in macrophage by AKF-PD. FIG. 3: Inhibition of TNF-α protein synthesis and secretion in macrophage by five compounds of 5-methyl-1- (substituted phenyl) -2 (IH) -pyridone .

FIG. 4 : Reduction of serum level of TNF-α in rat arthritis model by AKF-PD. FIG. 5: Reduction of paw tissue TNF-α levels in rat arthritis model by AKF-PD.

DETAILED DESCRIPTION OF THE INVENTION

[0018] In the present invention, the term "anti-TNF-α mediated diseases" means treating diseases, which have TNF-α as a crucial pathological factor, such as rheumatoid arthritis. The term "anti-inflammatory" means managing all immune responses, especially local immune response, excluding TNF-α mediated diseases.

[0019] It was shown previously that 5 -methyl-1-phenyl-2 (IH) - pyridone (pirfenidone) is the compound with desirable anti-TNF- α and anti-inflammatory activities, which is consistent with Gadekar's structure-activity relationship: non-substituted phenyl is better than substituted phenyl. Thus, it is unexpected to find 5-methyl-1- (substituted phenyl) -2 (IH) - pyridones (structural formula II) , especially AKF-PD exhibit more potent anti-TNF-α and anti-inflammatory activities and much less toxicity than 5 -methyl-1- (non-substituted phenyl) - 2 (IH) -pyridone (pirfenidone) as shown herein.

[0020] Representative examples of 5 -methyl-1- (substituted phenyl) -2 (IH) -pyridones were synthesized according to the process described in Chinese patent ZL02114190.8, the disclosure of which is incorporated herein by reference. These pyridones are 1- (3 ' -fluorophenyl) -5-methyl-2 (IH) -pyridone (AKF- PD) , 1- (3' -bromophenyl) -5 -methyl-2 (IH) -pyridone (AKF-BR) , 1-

(3' -chlorophenyl) -5-methyl-2 (IH) -pyridone (AKF-CL) , 1- (3' - methylphenyl) -5 -methyl-2 (IH) -pyridone (AKF-CH3) , and 1- (3' - methoxyphenyl) -5-methyl-2 (IH) -pyridone (AKF-OCH3) . It was determined that anti-TNF-α activities of these 5 -methyl-1-

(substituted phenyl) -2 (IH) -pyridone are enhanced compared to

PIRFENIDONE, a 5-methyl-1- (non-substituted phenyl) -2 (IH) - pyridone. Among the above compounds, the most potent one is 1-

(3' -fluorophenyl) -5 -methyl-2 (IH) -pyridone (AKF-PD) . Moreover, the toxicity of 5 -methyl-1- (substituted phenyl) -2 (IH) -pyridone may be drastically decreased. As presented herein, the LD₅₀ for 1- (3 ' -fluorophenyl) -5-methyl-2 (IH) -pyridone is only 30% percent as that of PIRFENIDONE. It is important to note that TNF-α mediated diseases, such as rheumatoid arthritis and dermatitis, such as eczema, are either chronic or recurrent diseases. It is expected that any treatment is going to be a lengthy process, which warrants a long-term pharmacological intervention. Therefore, just like any long-term drug use, it is very desirable for clinical applications to have anti-TNF-α or anti- inflammatory agent drugs with low toxicity.

[0021] Other examples of 5-methyl-1- (substituted phenyl) - 2 (IH) -pyridones (structural formula II) include, but are not limited to, the following compounds:

[0022] When n=l and R=Br, the compounds can be l-(2'- bromopheny1) -5 -methyl-2 (IH) -pyridone, 1- (3' -bromophenyl) -5- methyl-2 (IH) -pyridone, or 1- (4 ' -bromophenyl) -5-methyl-2 (IH) - pyridone .

[0023] When n=l and R=F, the compounds can be l-(2'- fluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' -fluorophenyl) -5- methyl-2 (IH) -pyridone, or 1- (4' -fluorophenyl) -5-methyl-2 (IH) - pyridone .

[0024] When n=l and R=I, the compounds can be l-(2'- iodophenyl) -5 -methyl-2 (IH) -pyridone, 1- (3' -iodophenyl) -5- methyl-2 (IH) -pyridone, or 1- (4' -iodophenyl) -5-methyl-2 (IH) - pyridone .

[0025] When n=2 and R=F, Br, or Cl, the compounds can be 1- (2' ,3' -dibromophenyl) -5-methyl-2 (IH) -pyridone, l-(2',4'- dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,5' - dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,6'- dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (3 ' , 4 ' - dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,5' - dibromophenyl) -5-methyl-2 (IH) -pyridone, l-(2',3'- dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 4' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,5' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (3 ' , 4 ' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, l-(3',5'- dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,3' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2 ' , 4 ' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 5' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' - difluorophenyl) -5-methyl-2 (IH) -pyridone, l-(3',4'- difluorophenyl) -5-methyl-2 (IH) -pyridone, or l-(3',5'- difluorophenyl) -5-methyl-2 (IH) -pyridone.

[0026] When n= 1 or 2 and R= trifluoromethyl, the compounds can be 5-methyl-l- (2 ' -trifluoromethylphenyl) -2 (IH) -pyridone, 5- methyl-1- (4' -trifluoromethylphenyl) -2 (IH) -pyridone, 1- (2' , 3' - bis-trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,4' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,5' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,4' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, or 1- (3' , 5' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone.

[0027] When n= 1 or 2 and R= methyl, the compounds can be 5- methyl-1- (2' -methylphenyl) -2 (IH) -pyridone, 5-methyl-l- (3 '- methylphenyl) -2 (IH) -pyridone, 1- (2' ,3' -dimethylphenyl) -5- methyl-2 (IH) -pyridone, 1- (2' ,4' -dimethylphenyl) -5-methyl-2 (IH) - pyridone, 1- (2' , 5' -dimethylphenyl) -5 -methyl-2 (IH) -pyridone, 1- (2' ,6' -dimethylphenyl) -5 -methyl-2 (IH) -pyridone, 1- (3' ,4' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, or l-(3',5'- dimethylphenyl) -5 -methyl-2 (IH) -pyridone.

[0028] When n= 1 or 2 and R= methoxyl, the compounds can be

1- (2' -methoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (3' - methoxyphenyl) -5 -methyl -2 (IH) -pyridone, 1- (2' ,3' - dimethoxyphenyl) -5 -methyl-2 ( IH) -pyridone , 1- ( 2 ' , 4 ' - dimethoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 5' - dimethoxyphenyl) -5 -methyl-2 (IH) -pyridone, 1- (2' , 6' - dimethoxyphenyl) -5 -methyl-2 (IH) -pyridone , 1- ( 3 ' , 4 ' - dimethoxyphenyl) -5 -methyl-2 (IH) -pyridone, or l-(3',5'- dimethoxyphenyl) -5 -methyl-2 (IH) -pyridone.

[0029] In one embodiment, the present invention provides a method of treating TNF-α mediated diseases, comprising the step of administrating a composition comprising one or more compound having the general structural formula II as shown above, wherein when n=l, the substituent R can be F, Br, I, a nitro group, an alkyl group, an alkoxy group, or a halogenated alkyl group,- wherein when n=2, the substituent R can be F, Cl, Br, I, an alkyl group, an alkoxy group, or a halogenated alkyl group,- and wherein the relative position of R is ortho, meta or para.. Preferably, when n=l, R is F, Br, or I; when n=2, R is F, Cl, Br, I, alkyl group, alkoxy group, or halogenated alkyl group, for example, R can be a straight-chain alkyl group, a branch- chain alkyl group, a straight-chain alkoxy group, or a straight-chain halogenated alkyl group. In general, the straight-chain alkyl group or branched-chain alkyl group has 1 to about 6 carbons, more preferably with 1-4 carbons.

[0030] According to the method of the present invention, the composition comprising one or more of the compounds disclosed herein can be administered at a daily dosage of about 25 mg to about 6,000 mg. The composition of the present invention can be administered by standard procedure. For example, the composition can be administered through oral, injectable or topical routes. Similarly, the composition of the present invention can be prepared into any appropriate pharmaceutical formulations according to standard techniques. For example, the composition can be formulated as solution, tablet, capsules, suppository, inhaler, suspension, gel, cream, or ointment.

[0031] In another embodiment, there is provided a method of treating TNF-α mediated diseases, such as rheumatoid arthritis, comprising administering to a subject a composition comprising a 5-methyl-1- (substituted phenyl) -2 (IH) -pyridone having the structural formula II, wherein n=l and the substituent R is F. For example, the substituent is a Fluoro at position 3. The composition can be formulated and administered according to standard procedures .

[0032] The present invention also provides a composition comprising a 5-methyl-1- (substituted phenyl) -2 (IH) -pyridone in an amount effective for treating TNF- ( mediated diseases, such as rheumatoid arthritis. The 5-methyl-1- (substituted phenyl) - 2 (IH) -pyridone has the general structural formula II as shown above, wherein when n=l, the substituent R can be F, Br, I, a nitro group, an alkyl group, an alkoxy group, or a halogenated alkyl group; wherein when n=2, the substituent R can be F, Cl, Br, I, an alkyl group, an alkoxy group, or a halogenated alkyl group; and wherein the relative position of R is ortho, meta or para. Preferably, when n=l, R is F, Br, or I; when n=2, R is F, Cl, Br, I, alkyl group, alkoxy group, or halogenated alkyl group. For example, R can be a straight-chain alkyl group, a branch-chain alkyl group, a straight-chain alkoxy group, or a straight-chain halogenated alkyl group. In general, the straight-chain alkyl group or branched-chain alkyl group has 1 to about 6 carbons, more preferably with 1-4 carbons. In one embodiment, the amount effective for treating TNF-α mediated diseases comprises a daily dosage of about 25 mg to about 6,000 mg. Preferably, the amount effective for treating TNF-α mediated diseases comprises a daily dosage of about 50 mg to about 2,000 mg, more preferably about 100 mg to about 1,000 mg. [0033] The present invention being generally described, will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1.

Inhibition of TNF-α mRNA synthesis in mouse macrophage by AKF- PD and PFD

[0034] Experimental Design: 264.7 RAW cells, mouse macrophages, are pre-treated with AKF-PD or pirfenidone (PFD) for 4 hours at the following doses: 0, 10 μg/ml, 100 μg/ml, and 500 μg/ml. 264.7 RAW cells are then stimulated by treatment with lipopolysaccharide (LPS, endotoxin) at 5 ng/ml for 15-20 hours . The cells are then harvested and mRNA is isolated from cells. Expression of TNF-α mRNA is assayed by a quantitative real time PCR analysis using the TaqMan PCR core reagent kit (Applied Biosysterns) .

[0035] Results: As shown by Fig.l, both AKF-PD and PFD inhibit LPS-stimulated TNF-α mRNA synthesis in mouse macrophage. Approximately 50% inhibition was reached at the dose of 500 μg/ml. In Fig.l, the mouse TNF-α gene expression in the control non-treated cells was taken as 1 and regarded as control. All the other values were expressed as fold increase of the control. The mouse beta actin gene was used as endogenous control. In Fig.l, each column with error bar represents the mean mRNA level ± SD of duplicate samples. Statistical comparisons for all three AKF doses relative to control indicate p < 0.01; PFD at the doses of 100 and 500 μg/ml relative to control indicate P < 0.01. Similar to PFD, AKF-PD can effectively block LPS induced TNF-α production by macrophages. EXAMPLE 2 .

Inhibition of TNF-α protein synthesis and secretion in macrophage by AKF-PD

[0036] Experimental Procedure: The effect of AKF-PD on TNF-α protein synthesis and secretion was analyzed in LPS-stimulated murine macrophage-like cell line RAW264.7. Cells were pre- treated with AKF-PD for 4 hours at the following doses: 0, 30 μg/ml, 100 μg/ml, and 300 μg/ml . 264.7 RAW cells were then stimulated by the treatment with lipopolysaccharide (LPS, endotoxin) at 100 ng/ml for 8 hours. Cell lysate was prepared from the harvested cells. Media from the cell culture were collected, concentrated 10-fold. Both cell lysate and media were subjected to ELISA assay (OptEIA™) specific for mouse TNF- α according to the manufacturer's instructions (BD Biosciences) .

[0037] Results: As shown by Fig .2 , treatment of the cells with AKF-PD at the dose of 100 μg/ml resulted in a 64% inhibition of secreted levels of TNF-α. At the dose of 300 μg/ml, AKF-PD achieved a robust 89% inhibition of secreted levels of TNF-α (Fig.2A). AKF-PD also suppressed the cellular levels of TNF-α but with less inhibitory effect, achieving approximately 50% inhibition at dose of 300 μg/ml (Fig.2B). Therefore, the exposure of RAW264.7 cells to AKF-PD exerted a significant and dose-dependent suppression of levels of both cell-associated and secreted TNF-α. In Fig.2, each column with error bar represents the average of triplicate TNF-α assay results and the standard deviation (SD) . Statistical comparisons for AKF-PD treated cells relative to the controls indicate P < 0.001 for secreted levels at all three doses; and P < 0.01 for cellular levels at doses of 100 and 300 μg/ml, respectively. EXAMPLE 3 .

Inhibition of TNP-α protein synthesis and secretion in macrophage by a serious 5 methyl-1- (substituted phenyl) -2 (IH) - pyridone compounds

[0038] Experimental Procedure: The effect of five compounds of 5 -methyl-1- (substituted phenyl) -2 (IH) -pyridone : 5-methyl-1- (3' -fluorophenyl) -2 (IH) -pyridone (AKF-PD) , 1- (3' -bromophenyl) - 5-methyl-2 (IH) -pyridone (AKF-BR), 1- (3 ' -chlorophenyl) -5-methyl- 2 (IH) -pyridone (AKF-CL), 1- (3 ' -methylphenyl) -5-methyl- 2 (IH) - pyridone (AKF-CH3) , and 1- (3 ' -methoxyphenyl) -5-methyl- 2(1H)- pyridone (AKF-OCH3) on TNF-α protein synthesis and secretion was analyzed in LPS-stimulated murine macrophage-like cell line RAW264.7. Cells were pre-treated with AKF compounds for 4 hours at the dose of 30 μg/ml . 264.7 RAW cells were then stimulated by the treatment with lipopolysaccharide (LPS, endotoxin) at 100 ng/ml for 8 hours. Media from the cell cultures were collected, concentrated 10-fold, and subjected to ELISA assay (OptEIA™) specific for mouse TNF-α according to the manufacturer's instructions (BD Biosciences) .

[0039] Results: As shown by Fig. 3, all 5-methyl-l-

(substituted phenyl) -2 (IH) -pyridone compounds inhibited LPS- stimulated secreted levels of TNF-α. At the dose of 30 μg/ml,

AKF-PD and AKF-OCH3 achieved a biggest suppressive effect with

56% and 63% inhibition of TNF-α production, respectively.

Compounds AKF-CL, AKF-BR, and AKF-CH3 also suppressed, but with less inhibitory effect, on secreted levels of TNF-α. In Fig. 3, each column with error bar represents the average of triplicate

TNF-α assay results and the standard deviation (SD) .

Statistical comparisons for AKFs treated cells relative to the controls indicate P < 0.001 for all treatments. EXAMPLE 4 .

Reduction of serum level of TNF-α in rat arthritis model by AKF- PD

[0040] Experimental procedure: the effect of AKF-PD on suppression of in vivo TNF-α was evaluated in adjuvant-induced arthritis (AIA) in rat, which is a widely used animal model of human rheumatoid arthritis (RA) . Ten-week old male Lewis rat were used. Complete Freund' s-type adjuvant (CFA) was prepared by suspending heat-killed Mycobacterium butyricum (Difco Laboratories) in incomplete Freund' s adjuvant at 7.5 mg/ml . CFA-induced arthritis was stimulated by injection of 0.1 ml of the CFA emulsion intradermalIy at the base of the tail, as described (Arthritis Rhem 39:1677, 1996). There were total of three groups of rats: normal control (healthy) group, adjuvant/normal saline treated group, and adjuvant/AKF-PD treated group. Each group had 6 rats. To both adjuvant treated groups, 500 mg/kg AKF-PD or normal saline were directly administrated by gavage to rats daily from day 2 to day 14 when animals were sacrificed. Sera were collected and subjected to TNF-α ELISA analysis (ChemiKine) .

[0041] Result: As indicated by Fig 4, animals injected with adjuvant only had significantly higher serum levels of TNF-α than did normal controls (47.9 ng/ml versus 8.1 ng/ml) . Induced arthritis animals treated with AKF-PD had significantly suppressed serum TNF-α levels (12.9 ng/mL) , which are comparable to the level in normal controls (8.1 ng/mL) . In Fig 4, the columns with error bar show the mean and SD of six rats.

EXAMPLE 5.

Reduction of paw tissue TNF-α levels in rat arthritis model by AKF-PD

[0042] Experimental procedure: the procedure for CFA-induced arthritis and AKF-PD treatment is exactly same as the EXAMPLE 4.

Animals were sacrificed at dav 14 following adjuvant and AKF-PD treatment. The paw tissues were harvested and stored at -80°C. After paw tissues thawed, skin was removed, minced, weighed, and extracted at 4⁰C with 5M urea (100 mg wet weight tissue/5 ml buffer) . The extraction was processed by ammonium sulfate 5 precipitation. The partially purified extracts were then frozen immediately until further analysis . Protein concentration in the paw extracts was measured by Bradford Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) .

.0 [0043] Results: As shown by Fig.5, similar to what observed in sera (Fig. 4) , AKF-PD treatment significantly suppressed paw levels of TNF-α from 8.1 ng/ml in the arthritis rats to 3.4 ng/ml in AKF-PD treated rats. In Fig. 5, each column with error bar shows the mean and SD of six rats.

L5

EXAMPLE 6.

AKF-PD prevents development of adjuvant arthritis

[0044] We have used adjuvant rat arthritis model to evaluate 20 the effect of systemic administration of AKF-PD on the development of arthritis .

[0045] Experimental procedure: Ten-week old male Lewis rats were treated with complete Freund' s-type adjuvant (CFA) as 5 described in EXAMPLE 4. The rats were treated daily with AKF-PD, 500 mg/kg or normal saline by gavage from day 2 to day 14 when animals were sacrificed. Rats treated with CFA alone usually develop arthritis (swollen paw) within 2-week following the inoculation with adjuvant. 0

[0046] Summarized in Table 1, adjuvant injection resulted in an average of 5 g decrease in body weight while untreated normal animals gained an average of 41 g during the two-week period of the study. Administration of AKF-PD to adjuvant treated rats 5 prevented the loss of body weight and caused an average weight gain of 37 g. All of the control animals treated with adjuvant alone (8/8) developed arthritis 10-14 days following the inoculation of the adjuvant. None of the animals treated with adjuvant plus AKF-PD developed arthritis (0/8) .

Table 1. Arthritis development frequency and paw diameters

EXAMPLE 7.

Comparison of Acute Toxicity of 5-methyl-1- (3' -fluorophenyl) - 2 (IH) -pyridone (AKF-PD) and PIRFENIDONE

[0047] Experimental procedure: Male and female Kun Ming (KM) mice weighing between 18g-22g were acquired from the animal facility of Hsiang-Ya Medical College, the Central South University. Fifty Kun Ming mice were randomly assigned into 5 groups with 5 male and 5 female mice for each group. The animals went through fasting with a normal water supply before starting drug treatment (either AKF-PD or PIRFENIDONE) . The drug was administrated orally by gavage . The volume of liquid drug was 20ml/kg body weight. The range of dosage for AKF-PD or PFD administrated was from 1071 mg/kg to 6000 mg/kg. The dosage difference between two adjacent doses was 1:0.65 (a dose vs the next lower dose) . All animals were maintained under a regular condition. Acute toxic reaction and death within 14 days of post drug treatment were recorded. Autopsy was performed on all dead animals and visual examination was performed on all organs .

[0048] LD₅₀ was calculated according to Bliss method. Acute toxicity LD₅₀ for 1- (3 ' -fluorophenyl) -5-methyl-2 (IH) -pyridone was 2979.89 mg/kg with a 95% confidence limit of 2402.70-- 3695.73 mg/kg (Table 2). LD₅₀ for PIRFENIDONE was 955.4 mg/kg with a 95% confidence limit of 550.9--1656.7 mg/kg (Table 3). The results of Table 3 are very close to those reported in the literature: 997.7 mg/kg (U.S. patent 5,310,562) and 1112 mg/kg (Pharmaceutical Care and Research 5:4823 (2005)). These results indicate that the toxicity for AKF-PD is only one third of that of PIRFENIDONE (2978mg/kg vs 955mg/kg) .

TABLE 2. LD₅₀ of 5-methyl-1- (3' -fluorophenyl) -2 (IH) -pyridone

TABLE 3. LD₅₀ of PIRFENIDONE

Claims

1. Use of compounds, 5-methyl-1- (substituted phenyl) -2 (IH) pyridones having a formula II

for the preparation of a pharmaceutical composition for treatment of TNF-α mediated diseases.

2. The use of claim 1, wherein when n=l, the substituent R is selected from the group consisting of F, Br, I, a nitro group, an alkyl group, an alkoxy group, and a halogenated alkyl group,- when n=2, the substituent R is selected from the group consisting of F, Cl, Br, I, an alkyl group, an alkoxy group, and a halogenated alkyl group, and wherein the relative position of R is ortho, meta or para.

3. The use of claim 1 or 2 , wherein n=l or 2, R is selected from the group consisting of straight-chain alkyl group, branch-chain alkyl group, straight-chain alkoxy group, and straight-chain halogenated alkyl group.

4. The use of claim 3, wherein the straight-chain alkyl group or branched-chain alkyl group has 1 to about 6 carbons .

5. The use of claims 1, 2, 3 or 4, wherein the composition include one or more compounds selected from the group consisting of 1- (2 ' -bromophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' -bromophenyl) -5-methyl-2 (IH) -pyridone, I- (4' - bromophenyl) -5-methyl-2 (] w) -pyridone, 1- (2 ' -fluorophenyl) - 5-methyl-2 (IH) -pyridone, 1- (3' -fluorophenyl) -5-methyl- 2 (IH) -pyridone, 1- (4 ' -fluorophenyl) -5-methyl-2 (IH) - pyridone, 1- (2' -iodophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' - iodophenyl) -5 -methyl-2 (IH) -pyridone, 1- (4' -iodophenyl) -5- methyl-2 (IH) -pyridone, 1- (2' , 3' -dibromophenyl) -5-methyl- ^■ 2 (IH) -pyridone, 1- (2' ,4' -dibromophenyl) -5 -methyl-2 (IH) - pyridone, 1- (2' , 5' -dibromophenyl) -5 -methyl-2 (IH) -pyridone, 1- (2' ,6' -dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,4' - dibromophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,5' - dibromophenyl) -5-methyl-2 (IH) -pyridone, l-(2',3'- dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2 ' , 4 ' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,5' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,4' - dichlorophenyl) -5-methyl-2 (IH) -pyridone, l-(3',5'- dichlorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2 ' , 3 ' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,4' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 5' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' - difluorophenyl) -5-methyl-2 (IH) -pyridone, l-(3',4'- difluorophenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,5' - difluorophenyl) -5-methyl-2 (IH) -pyridone, 5-methyl-1- (2' - trifluoromethylphenyl) -2 (IH) -pyridone, 5-methyl-1- (4' - trifluoromethylphenyl) -2 (IH) -pyridone, 1- (2' ,3' -bis- trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, l-(2',4'- bis-trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 5' -bis-trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 6' -bis-trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (3 ' , 4 ' -bis-trifluoromethylphenyl) -5-methyl-2 (IH) -pyridone, or 1- (3' ,5' -bis-trifluoromethylphenyl) -5-methyl-2 (IH) - pyridone, 5-methyl-l- (2' -methylphenyl) -2 (IH) -pyridone, 1- (3' -methylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,3' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,4' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,5' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, l-(2',6'- dimethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,4' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (3' ,5' - dimethylphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' - methoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (3' - methoxyphenyl) -5 -methyl-2 (IH) -pyridone , 1- (2 ' , 3 ' - dimethoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,4' - dimethoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' , 5' - dimethoxyphenyl) -5-methyl-2 (IH) -pyridone, 1- (2' ,6' - dimethoxyphenyl) -5 -methyl-2 (IH) -pyridone, 1- (3' ,4' - dimethoxyphenyl) -5-methyl-2 (IH) -pyridone, l-(3',5'- dimethoxyphenyl) -5-methyl-2 (IH) -pyridone.

6. The use of claim 1, wherein the composition is administered at a daily dosage of about 25 mg to about 6,000 mg.

7. The use of claim 1 , wherein the TNF-α mediated diseases are selected from the group consisting of rheumatoid arthritis, psoriatic arthritis, asthma, sepsis, psoriasis, and inflammatory bowel disease (Crohn's) .

8. The use of claim 1, wherein the pharmaceutical composition is in one or more form(s) selected from the group consisting of oral administration, injection, ophthalmic application, and / or topical application.

9. The use of claim 1, wherein the amount of the compounds having the formula II contained in the pharmaceutical composition effective for treating TNF-α-mediated diseases is at a daily dosage of about 25 mg to about 6,000 mg.

10. The use of claim 1, wherein the amount of the compounds having the formula II contained in the pharmaceutical composition effective for treating TNF-α-mediated diseases is at a daily dosage of about 50 mg to about 2000 mg. 11 The use of claim 1, wherein the amount of the compounds having the formula II contained in the pharmaceutical composition effective for treating TNF-α-mediated diseases is at a daily dosage of about 100 mg to about 1000 mg.

12 A use of a low toxic compound, 5-methyl-1- (substituted phenyl) -2 (IH) -pyridone having a formula II,

wherein n=l and the substituent R is F, for the preparation of a pharmaceutical composition for treatment of TNF-α mediated diseases.

13 The use of claim 12, wherein the substituent R is a Fluoro at position 3, i.e 1- (3' -fluorophenyl) -5-methyl-2 (IH) - pyridone .

14 The use of claim 12 or 13, wherein the pharmaceutical composition comprises the effective amount of the compound having the formula II at a daily dosage of about 25 mg to about 6, 000 mg.

15 The use of claim 12 or 13, wherein the pharmaceutical composition comprises the effective amount of the compounds having the formula II at a daily dosage of about 50 mg to about 2000 mg.

16 The use of claim 12 or 13, wherein the pharmaceutical composition comprises the amount effective of the compounds with formula II at a daily dosage of about 100 mg to about 1000 mg. The use of claim 12 or 13, wherein the TNF-α mediated diseases is selected from the group consisting of rheumatoid arthritis (RA), psoriatic arthritis, and inflammatory bowel disease (Crohn's) .

The use of claim 12 or 13, wherein the pharmaceutical composition is in one or more form(s) the group consisting of oral administration, injection, ophthalmic application, and / or topical application.