Potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide for treating inflammatory bowel disease

Abstract

The present invention provides the compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide potassium salt, which has the formula (I): [Formula 1] The compound is believed to have the structure of TIFF2023524518000020.tif34128. The compound is provided in solid (e.g., crystalline or amorphous) or aqueous solution form. Pharmaceutical compositions containing the compound are also provided. The compound finds use as a pharmaceutical, particularly for the treatment or prevention of inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to the novel compound potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide. This compound has found particular use in the treatment and / or prevention of inflammatory bowel disease.

Background Art

[0002] Inflammatory Bowel Disease (IBD) is a group of heterogeneous diseases and disorders characterized by chronic inflammation of the gastrointestinal wall. Symptoms caused by chronic inflammation include abdominal pain, diarrhea, general malaise, and loss of appetite and poor absorption of nutrients from food, which often results in weight loss. IBD has two main forms, namely ulcerative colitis (UC), which typically begins in the distal colon and rectum and can spread continuously to affect the entire colon (pancolitis), and Crohn's disease (CD), which most commonly affects the ileum and ascending colon. Indeterminate Colitis (IC) can also be considered a form of IBD. IBD is classified as IC when the disease state cannot be distinguished between CD and UC.

[0003] Currently available IBD treatments mainly aim at reducing symptoms and maintaining remission. In many cases, long-term maintenance therapy is required to prolong remission. Primary treatment often involves the use of aminosalicylates and / or corticosteroids. Secondary treatments include immunosuppressants, Tumor Necrosis Factor (TNF) inhibitors, and integrin inhibitors. Secondary treatments can be used as monotherapy or in combination with one or more primary or secondary treatments. In many cases, surgical intervention is required.

[0004] In addition, immunomodulatory drugs have been shown to be promising for the treatment of IBD. N-alkyl 1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxanilide (hereinafter referred to as N-alkylquinoline-3-carboxanilide) is a particularly promising class of compounds that have been shown to have immunomodulatory properties. The immunomodulatory properties and therapeutic potential of N-alkylquinoline-3-carboxanilide were first reported in the 1980s (see, for example, Patent Document 1). One member of this class is lacosamide, which has been reported to be beneficial in the treatment of Crohn's disease (see, for example, Non-Patent Document 1 and Patent Document 2).

[0005] Despite initial promise, the clinical success of N-alkylquinoline-3-carboxanilide has been limited by insufficient efficacy, toxicity, and instability. For example, Non-Patent Document 2 reported that N-alkylquinoline-3-carboxanilide is chemically reactive towards nucleophiles and renders them unstable in their neutral form. N-alkylquinoline-3-carboxamides such as lacosamide have also been shown to be readily metabolized by cytochrome P450 (CYP) enzymes into various active metabolites that potentially have different potencies, toxicities, and physicochemical properties (see, for example, Non-Patent Document 3). In particular, lacosamide did not receive marketing authorization in Europe for the treatment of relapsing-remitting multiple sclerosis due to concerns about human safety and insufficient efficacy (EMA 2014 Public Assessment Report for Medicinal Products - EMA / 451905 / 2014).

[0006] Unfortunately, for many patients, available IBD treatments have no effect in reducing symptoms and slowing the progression of the disease. In addition, many of the available treatments cause serious adverse effects such as an increased risk of infection, liver inflammation, nausea and illness, weight gain, and rarely progressive multifocal leukoencephalopathy. Therefore, there is a significant clinical need for more effective therapeutic and prophylactic treatments for IBD. In particular, there is a need for an effective treatment that is easy to manage side effects while providing clinical benefit to patients suffering from IBD.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0009] The present invention provides the potassium salt of the compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. This compound has the following structure:

[0010]

Chemical Formula

[0011] and is considered to have.

[0012] The inventors have found that the compounds according to the present invention are effective in the treatment and / or prevention of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Furthermore, the inventors have surprisingly found that the compounds according to the present invention have particularly beneficial physical properties, namely beneficial properties that give significantly more advantages than the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

[0013] For example, in solid form, the free acid 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide exists as crystals in the form of long needles, and these needles visibly aggregate. On the other hand, the inventors have found that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a powdery crystal having a tabular habit. This material is much easier to handle than the long needles of the free acid compound.

[0014] Further physicochemical advantages of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide are described below in this specification.

[0015] Due to the effectiveness of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and its very advantageous physicochemical properties, its use in effective therapeutic treatments for reducing the symptoms of inflammatory bowel diseases, particularly Crohn's disease and ulcerative colitis, and prolonging disease remission becomes possible.

[0016] The present invention further provides a compound of formula (I) for use as a medicament.

[0017] The present invention further provides a compound of formula (I) for use as a medicament in the treatment and / or prevention of inflammatory bowel diseases.

[0018] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) and at least one pharmaceutically acceptable excipient. The composition may optionally comprise one or more additional therapeutic agents.

[0019] The present invention further provides a method of treating and / or preventing inflammatory bowel disease, comprising administering a pharmaceutically effective amount of a compound of formula (I) to a subject suffering from or at risk of developing inflammatory bowel disease.

[0020] The use of a compound of formula (I) in the manufacture of a medicament for the treatment and / or prevention of inflammatory bowel disease is also provided herein. The present invention further provides a kit comprising a compound of formula (I) together with one or more pharmaceutically acceptable excipients and optionally one or more additional therapeutic agents. The kit of the present invention finds use in the treatment and / or prevention of inflammatory bowel disease.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

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Figure 9

Figure 10

Mode for Carrying Out the Invention

[0022] The inventors have found that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is effective in the treatment and / or prevention of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Furthermore, the inventors have surprisingly found that the compounds according to the invention have beneficial physical properties, namely beneficial properties that give significantly more advantages than the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

[0023] Advantageous crystalline form In the solid form, the free acid 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide forms crystals which are long needles and appear to be aggregated. On the other hand, the inventors have found that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a powdery crystal having a tabular habit. This material is much easier to handle than the long needles of the free acid compound.

[0024] The potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide has been found to generally have a particle size distribution (PSD) of less than 10 microns, which is significantly smaller than the needle length of 200 - 300 microns of the 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid. The PSD of the potassium salt is also advantageously smaller than other salt forms. For example, the PSD of both the sodium salt and the lithium salt has been found to be 10 - 20 microns.

[0025] Accordingly, the present invention further provides a compound of formula (I) having a D50 size in the range of 0.5 - 7 μm and a D90 size in the range of 5 - 10 μm. Preferably, the compound has a D50 size in the range of 2 - 5 μm and a D90 size in the range of 7 - 9 μm.

[0026] The inventors further established that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide has better thermal stability than the free acid of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. The free acid melts at 178 °C and decomposes at 260 °C, while the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide melts at 347 °C and only decomposes at that temperature. The increased thermal stability provides flexibility useful in the manufacturing process and also allows for an extended shelf life of the compound.

[0027] In differential scanning calorimetry (DSC) analysis, there was no significant endotherm prior to melting starting at approximately 343 °C. The maximum endothermic heat flow was observed at 361.3 °C.

[0028] Accordingly, the present invention provides a compound in crystalline form of formula (I), wherein the crystalline form is characterized by a differential scanning calorimetry trace recorded at a heating rate of 10 °C / min showing a maximum value of the endothermic heat flow having a peak at 361.3 ± 2 °C.

[0029] For example, the crystalline form can be characterized by a differential scanning calorimetry trace that substantially coincides with that shown in Figure 1.

[0030] A further advantageous property of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is that it exists in a non-hydrated form and is not hygroscopic.

[0031] The preferred solid form of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a crystalline salt having a powder X-ray diffractogram with peaks characteristic of 2θ = 6.9 ± 0.2°, 15.6 ± 0.2°, 24.9 ± 0.2° and 28.4 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 20.9° ± 0.2°, 24.2 ± 0.2°, 25.2 ± 0.2°, 25.6 ± 0.2° and 27.3 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 13.9 ± 0.2°, 17.8 ± 0.2°, 23.6 ± 0.2°, 26.8 ± 0.2°, 29.2 ± 0.2° and 34.0 ± 0.2°.

[0032] An exemplary XRPD trace of the crystalline salt is shown in Figure 2. Accordingly, the present invention provides a compound which is a compound of formula (I) in crystalline form, wherein the crystalline form is characterized by a powder X-ray diffraction pattern whose peak positions substantially coincide with the peak positions of the pattern shown in Figure 2.

[0033] Beneficial formation of the crystalline form The inventors have surprisingly found that potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide can be provided in crystalline form from a wide range of solvents. When the compound was crystallized from any of ethanol, acetone, ethyl acetate, acetonitrile, THF, toluene, methanol / water 50:50 and isopropanol / water 90:10, the same type of crystals were formed. The fact that crystals can be consistently formed from a wide variety of solvents imparts useful flexibility to the manufacturing process. Other salts that have been studied do not exhibit such flexibility in different solvents. For the parent free acid compound, it has been found that there are at least two polymorphic forms and the form produced depends on the crystallization solvent.

[0034] Accordingly, the present invention provides a method for preparing potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide by crystallization from a solvent or a solvent mixture, wherein the solvent is selected from any one or more of ethanol, acetone, ethyl acetate, acetonitrile, THF, toluene, methanol / water 50:50, isopropanol / water 90:10 and isopropanol. Preferred solvents include ethanol, acetone, ethyl acetate, acetonitrile, isopropanol / water 90:10 and isopropanol.

[0035] 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide can be prepared using methods known to those skilled in the art of organic chemistry. Specific methods for preparing 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and its potassium salt according to the present invention are described in the Examples section of this specification.

[0036] Solubility in Aqueous Solvents An important characteristic for any drug compound is its solubility in water. This solubility determines how easily the compound can be administered in solution. Solubility also determines how well the compound can be absorbed into the patient's body and distributed around the body. Thus, solubility has a significant impact on the bioavailability of the drug.

[0037] 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid has been found to have relatively low solubility. For example, in PBS (pH 7.4), 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid is so poorly soluble that it cannot be quantified. On the other hand, the inventors have found that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide dissolves up to 0.91 μg / ml. Similarly, in simulated colonic fluid (SCF), the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is more than twice as soluble (7.99 μg / ml compared to 3.44 μg / ml).

[0038] The inventors have further demonstrated in an in vivo study that the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is absorbed through the intestine into the plasma in rats.

[0039] This beneficial increase in solubility confers advantages to the dissolved form of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

[0040] A further advantageous property of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is that it is stable in aqueous solution. This is also the case when it is a slurry in water (above its solubility limit).

[0041] Accordingly, the present invention provides an aqueous solution containing potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide in water. The aqueous solution may be buffered, for example. For example, the buffered solution according to the present invention may be a solution in phosphate-buffered saline (PBS). The solution according to the present invention may be an aqueous pharmaceutical composition.

[0042] The compound of formula (I) can also be prepared in a solid amorphous form. Such forms are also known as amorphous.

[0043] Clinical efficacy: The compounds of the present invention exhibit surprising efficacy in the treatment or prevention of the symptoms and onset of inflammatory bowel disease. In particular, the inventors have found that the compounds according to formula (I) exhibit surprisingly beneficial properties in the treatment or prevention of IBD such as Crohn's disease and ulcerative colitis.

[0044] As will be discussed in more detail below, the inventors have found that potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is effective in inhibiting inflammation / edema associated with inflammatory bowel disease, as evaluated in a CD4+ adoptive transfer-induced inflammatory bowel disease in mice. Furthermore, mice orally treated with potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide showed a significant increase in CYP1A1 mRNA expression compared to untreated animals, indicating that the compound is effective in activating the aryl hydrocarbon receptor (AhR) in the colon.

[0045] The inventors have further found that 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide reduced weight loss and had a protective effect against the onset of clinical symptoms of ulcerative colitis in an in vivo mouse model of ulcerative colitis. The inventors have also found that 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is effective in reducing colonic shortening, which indicates a reduction in edema associated with ulcerative colitis. The surprising effectiveness of DELAQ enables its use in the effective treatment and prevention of IBDs such as Crohn's disease and ulcerative colitis.

[0046] Accordingly, the present invention provides a compound of formula (I) for use as a medicament.

[0047] The present invention further provides a compound of formula (I) for use as a medicament in the treatment and / or prevention of inflammatory bowel disease.

[0048] The present invention further provides a method of treating and / or preventing inflammatory bowel disease, comprising administering a pharmaceutically effective amount of a compound of formula (I) to a subject suffering from or at risk of developing inflammatory bowel disease.

[0049] Use of a compound of formula (I) in the manufacture of a medicament for the treatment and / or prevention of inflammatory bowel disease is also provided herein.

[0050] N-Desalkylquinoline-3-carboxanilide has previously been reported to be an active metabolite of N-alkylquinoline-3-carboxanilides such as laclademod and taslademod. In isolated form, N-desalkylquinoline-3-carboxanilide has been reported to be unsuitable for in vivo administration due to insufficient stability and low water solubility (see, for example, Tuvesson et al., 2005, Drug Metab. Dispos, 33:866-872, 2005, WO 2012 / 050500, and Mariout et al., 2017, Tox. Appl. Pharm., 326, 54-65).

[0051] The inventors have found that 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide, particularly the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide, is surprisingly effective for the treatment and prevention of IBD, particularly CD and UC, and has good stability and water solubility.

[0052] Pharmaceutical composition While the compounds of formula (I) can be administered alone, they are preferably present in a composition, particularly a pharmaceutical composition. The pharmaceutical compositions of the invention comprise a compound of formula (I) and one or more pharmaceutically acceptable excipients.

[0053] The pharmaceutical compositions of the present invention suitable for oral administration can be presented as separate units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient, as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions. The compounds of formula (I) may also be presented as a bolus, a lozenge or a paste. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical treatises, for example, Remington’s Pharmaceutical Sciences by E.W. Martin. Also, see Wang, Y.J. and Hanson, M.A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

[0054] Pharmaceutical compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including skin, intraoral, sublingual and intraocular) administration, although the most suitable route may depend, for example, on the type of IBD under treatment.

[0055] Pharmaceutical compositions for rectal administration can be presented as suppositories with carriers such as cocoa butter, synthetic glyceride esters, or polyethylene glycol. Such carriers are typically solid at room temperature but liquefy and / or dissolve in the rectal cavity to release the drug.

[0056] For example, in the human body, under suitable conditions, certain compounds are known that can be converted by dealkylation or hydrolysis to 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. Known compounds that can be converted in this way are 5-chloro-N-ethyl-4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide (known as lacipimod):

[0057]

Chem.

[0058] is.

[0059] Preferably, the composition of the present invention contains ralimod in an amount of less than 10 mole percent (mol%) of the total number of moles of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and ralimod present in the composition. More preferably, the composition containing the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide contains ralimod in an amount of less than 5 mol%. For example, it is less than 4, 3, 2, or 1 mol% (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mol%) of ralimod. Even more preferably, the composition of the present invention is substantially free of ralimod.

[0060] Preferably, the composition of the present invention containing the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide contains ralimod in an amount of less than 10% by weight of the total weight of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and ralimod present in the composition. More preferably, ralimod is present in the composition of the present invention in an amount of less than 5% by weight. For example, it is less than 4, 3, 2, or 1% by weight (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1% by weight) of ralimod.

[0061] Even more preferably, the composition of the present invention is substantially free of ralimod.

[0062] The composition of the present invention may comprise one or more additional therapeutic agents. Examples of additional therapeutic agents that may be present in the composition of the present invention include aminosalicylates (e.g., mesalazine, olsalazine, sulfasalazine, balsalazide), corticosteroids (e.g., prednisolone, prednisone, methylprednisolone, budesonide, hydrocortisone and beclomethasone dipropionate), immunosuppressants (e.g., azathioprine, mercaptopurine, methotrexate, cyclosporine and tacrolimus), anti-TNF agents (e.g., infliximab, adalimumab and golimumab), antibiotics (e.g., ciprofloxacin and metronidazole), anti-integrin agents (e.g., vedolizumab and natalizumab), interleukin inhibitors (e.g., ustekinumab), Janus kinase inhibitors (e.g., tofacitinib, filgotinib, upadacitinib, and TYK2 inhibitors such as BMS-986165), but are not limited thereto.

[0063] Inflammatory bowel disease The compounds of formula (I) and the pharmaceutical compositions of the present invention find use in the treatment of IBD, such as CD and UC.

[0064] Accordingly, a compound of formula (I) according to the present invention, or a composition of the present invention, may be administered to a subject having IBD such as CD or UC. The subject may be a human subject, such as a human patient.

[0065] The subject may have IBD that can be classified as refractory, relapsing or refractory-relapsing. For example, the subject may have refractory, relapsing or refractory-relapsing CD or UC. Additionally or alternatively, the subject may have IBD that is partially or completely resistant to established IBD treatments such as aminosalicylates and corticosteroids. For example, the IBD may be CD or UC that is partially or completely resistant to aminosalicylate and / or corticosteroid treatment or prophylaxis. Additionally or alternatively, the subject may have experienced, or be at risk of experiencing, an adverse reaction to established IBD treatments such as aminosalicylates and corticosteroids.

[0066] The compounds of formula (I) according to the present invention, and the compositions of the present invention, can be administered to a subject known or suspected to be at risk of developing IBD. For example, a subject having a known or suspected genetic predisposition to the development of IBD such as CD or UC. For example, the compounds of formula (I), or the compositions of the present invention, can be administered to a subject in need of prolonging remission of IBD and / or delaying the progression of IBD.

[0067] The compounds of formula (I) and the compositions of the present invention have been found to be useful in a method for treating or preventing IBD, which method comprises administering a compound of formula (I) or a composition of the present invention to a subject having IBD such as CD or UC. In certain embodiments, the method for treating or preventing IBD comprises administering a compound of formula (I) or a composition of the present invention to a subject known or suspected to be at risk of developing IBD.

[0068] In certain embodiments, the treatment or prevention method comprises delivering a compound of formula (I) or a pharmaceutical composition of the present invention to the small intestine and / or large intestine of the subject. For example, delivering a compound of formula (I) or a pharmaceutical composition of the present invention to one or more of the duodenum, jejunum, and ileum, and / or one or more of the cecum, ascending colon, transverse colon, descending colon, and / or sigmoid colon. The treatment or prevention method may also comprise administering a compound of formula (I) or a composition of the present invention to the subject orally or rectally.

[0069] The compounds of formula (I) also find use in the manufacture of a medicament for the treatment or prevention of IBD. For example, the compounds of formula (I) can be used in the manufacture of a medicament for the treatment or prevention of CD or UC.

[0070] Delivery to the small intestine and / or large intestine The composition according to the present invention can be adapted for the selective release of the compound of formula (I) in the small intestine or large intestine after rectal or oral administration. For example, in certain embodiments, the compound of formula (I) or the pharmaceutical composition of the present invention is administered locally to the small intestine and / or large intestine. This can be achieved by the use of specific coatings and / or formulations.

[0071] The composition of the present invention can have an enteric coating. Enteric coatings are known that protect the active ingredient in the composition from attack and degradation in the stomach and allow release in the intestine. The optimal coating for any particular formulation depends on the exact purpose of use, and the coating can be adjusted to release the active ingredient within a particular region of the intestine or at a particular time after ingestion.

[0072] The composition of the present invention can be adapted to release the compound of formula (I) in the small intestine, for example, in one or more of the duodenum, jejunum, and ileum. Additionally or alternatively, the composition of the present invention can be adapted to release the compound of formula (I) in the large intestine, for example, in one or more of the cecum, ascending colon, transverse colon, descending colon, and / or sigmoid colon.

[0073] The composition of the present invention can preferably be in solid or semi-solid form and include an enteric coating adapted to release the compound of formula (I) in the small intestine and / or large intestine. Such formulations can contain one or more intermediate layers between the active ingredient and the outer enteric coating. In certain embodiments, the composition of the present invention can release a portion of its contents in one or more specific regions of the small intestine and a further portion of its contents in one or more specific regions of the large intestine.

[0074] Dosing schedule The amount of the compound of formula (I) required to achieve a therapeutic effect will vary depending on the particular route of administration and the characteristics of the subject under treatment, such as species, age, weight, gender, medical condition, specific IBD and its severity, as well as other relevant medical and physical factors. A conventional skilled physician can readily determine and administer an effective amount of the compound of formula (I) necessary for the treatment or prevention of IBD.

[0075] The compound of formula (I) can be administered daily (including several times a day), every two or three days, weekly, every two, three or four weeks, or as a high single dose depending on the subject being treated and the IBD.

[0076] Preferably, the compound of formula (I) (excluding the mass of any counterions or solvents) can be administered in an amount of about 1 to 1000 mg per administration. For example, 1, 5, 10, 15, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mg.

[0077] In certain embodiments, the compound of formula (I) is administered as a composition. Preferably, the composition is a pharmaceutical composition of the present invention.

[0078] The compound of formula (I) can be used as the sole active ingredient in the present invention, but it is also possible to use it in combination with one or more additional therapeutic agents, and the use of such a combination provides an embodiment of the present invention. Such additional therapeutic agents can be agents useful for the treatment or prevention of IBD, or other pharmaceutically active materials. Such agents are known in the art. Examples of additional therapeutic agents for use in the present invention include those described herein.

[0079] One or more additional therapeutic agents may be used simultaneously with, sequentially, or separately from the administration of the dosage of the compound of formula (I). The individual components of such combinations can be administered separately at different times during the course of treatment, or can be administered simultaneously in a divided or single combined form. One skilled in the art can readily determine and administer the effective amount of one or more therapeutic agents necessary to have the desired therapeutic effect.

[0080] The compound of formula (I) can be administered as an oral or rectal dosage, and thus the dosage of the compound of formula (I) can be in a form suitable for delivery of the compound of formula (I) to the small intestine and / or large intestine.

[0081] Preferred unit dosage compositions for use according to the present invention contain an effective dosage of the compound of formula (I) or an appropriate fraction thereof. Release of the compound of formula (I) from a particular composition can also be sustained, for example, if the composition contains suitable controlled release excipients.

[0082] Kit The present invention provides a kit comprising a compound of formula (I), one or more pharmaceutically acceptable excipients, and optionally one or more additional therapeutic agents useful for the treatment or prevention of IBD. Examples of such additional therapeutic agents include those described herein as being suitable for use in the present invention, and are optionally present in the pharmaceutical compositions of the present invention as additional therapeutic agents.

[0083] The kits of the present invention find use in the treatment and prevention of IBD, particularly CD and UC.

[0084] To avoid doubt, the compound of formula (I) present in the kits according to the present invention is in a form and amount suitable for use according to the present invention. Suitable pharmaceutical compositions and formulations are described herein. One skilled in the art can readily determine the amount of the compound of formula (I) suitable for inclusion in the kits of the present invention and suitable for use according to the present invention.

[0085] Further aspects of the present invention The inventors also provide the sodium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. This compound has the following structure:

[0086]

Chemical formula

[0087] and is considered to have.

[0088] The inventors have found that the compounds of formula (II) are effective in the treatment and / or prevention of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Furthermore, the inventors have surprisingly found that the compounds of formula (II) have beneficial physical properties, namely beneficial properties that give significantly more advantages than the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

[0089] For example, in solid form, the free acid 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide exists as crystals in the form of long needles, and these needles visibly aggregate. On the other hand, the inventors have found that the sodium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a powdery crystal with a tabular habit. This material is much easier to handle than the long needles of the free acid compound.

[0090] Further physicochemical advantages of the sodium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide are described below in this specification.

[0091] Due to the effectiveness of sodium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and its advantageous physicochemical properties, its use in an effective therapeutic treatment for reducing the symptoms of inflammatory bowel diseases, particularly Crohn's disease and ulcerative colitis, and prolonging disease remission becomes possible.

[0092] The present invention further provides a compound of formula (II) for use as a medicament.

[0093] The present invention further provides a compound of formula (II) for use as a medicament in the treatment and / or prevention of inflammatory bowel diseases.

[0094] The present invention also provides a pharmaceutical composition comprising a compound of formula (II) and at least one pharmaceutically acceptable excipient. The composition may optionally contain one or more additional therapeutic agents.

[0095] The present invention further provides a method for treating and / or preventing inflammatory bowel diseases, comprising administering a pharmaceutically effective amount of a compound of formula (II) to a subject suffering from or at risk of developing inflammatory bowel diseases.

[0096] The use of a compound of formula (II) in the manufacture of a medicament for the treatment and / or prevention of inflammatory bowel diseases is also provided herein. The present invention further provides a kit comprising a compound of formula (II) together with one or more pharmaceutically acceptable excipients and optionally one or more further therapeutic agents. The kit of the present invention finds use in the treatment and / or prevention of inflammatory bowel diseases.

[0097] The inventors have established that sodium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide has better thermal stability than the free acid form of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide. The free acid melts at 178 °C and decomposes at 260 °C, while sodium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide melts at 356 °C and only decomposes at approximately 350 °C. The increased thermal stability provides flexibility useful in the manufacturing process and also allows for an extended shelf life of the compound.

[0098] A preferred solid form of sodium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide is a crystalline salt having a powder X-ray diffractogram with peaks characteristic of 2θ = 7.2 ± 0.2°, 24.5 ± 0.2°, 26.4 ± 0.2° and 26.5 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 9.3 ± 0.2°, 15.9 ± 0.2°, 23.1 ± 0.2° and 25.2 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 12.2 ± 0.2°, 18.9 ± 0.2°, 22.2 ± 0.2° and 29.7 ± 0.2°.

[0099] The inventors also provide a lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide. This compound has the following structure:

[0100]

Chemical formula

[0101] and is believed to have.

[0102] The inventors have found that the compounds of formula (III) are effective in the treatment and / or prevention of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Furthermore, the inventors have surprisingly found that the compounds of formula (III) have beneficial physical properties, namely beneficial properties that provide significant advantages over the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

[0103] For example, in solid form, the free acid 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide exists as crystals in the form of long needles, and these needles visibly aggregate. On the other hand, the inventors have found that the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a powdery crystal with a tabular habit. This material is much easier to handle than the long needles of the free acid compound.

[0104] Further physicochemical advantages of the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide are described hereinbelow.

[0105] Due to the effectiveness of the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide and its advantageous physicochemical properties, its use in effective therapeutic treatments for reducing the symptoms of inflammatory bowel diseases, particularly Crohn's disease and ulcerative colitis, and prolonging disease remission becomes possible.

[0106] The present invention further provides a compound of formula (III) for use as a medicament.

[0107] The present invention further provides a compound of formula (III) for use as a medicament in the treatment and / or prevention of inflammatory bowel diseases.

[0108] The present invention also provides a pharmaceutical composition comprising a compound of formula (III) and at least one pharmaceutically acceptable excipient. The composition may optionally comprise one or more additional therapeutic agents.

[0109] The present invention further provides a method of treating and / or preventing an inflammatory bowel disease, comprising administering a pharmaceutically effective amount of a compound of formula (III) to a subject suffering from or at risk of developing an inflammatory bowel disease.

[0110] The use of a compound of formula (III) in the manufacture of a medicament for the treatment and / or prevention of an inflammatory bowel disease is also provided herein. The present invention further provides a kit comprising a compound of formula (III) together with one or more pharmaceutically acceptable excipients and optionally one or more additional therapeutic agents. The kit of the present invention finds use in the treatment and / or prevention of an inflammatory bowel disease.

[0111] The inventors have established that the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide has better thermal stability than the free acid of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. The free acid melts at 178 °C and decomposes at 260 °C, while the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide melts at 362 °C and only decomposes at approximately 360 °C. The increased thermal stability provides flexibility useful in the manufacturing process and also allows for an extended shelf life of the compound.

[0112] The preferred solid form of the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is a crystalline salt having a powder X-ray diffractogram with peaks characteristic of 2θ = 6.7 ± 0.2°, 21.2 ± 0.2°, 24.3 ± 0.2° and 25.9 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 10.4 ± 0.2°, 15.8 ± 0.2°, 27.0 ± 0.2° and 28.6 ± 0.2°. The powder X-ray diffractogram may have additional characteristic peaks at 2θ = 19.8 ± 0.2°, 20.0 ± 0.2° and 30.6 ± 0.2°.

[0113] Equivalents The present invention is broadly and generically described herein. Those skilled in the art will appreciate that all parameters, dimensions, materials and configurations described herein are exemplary, and that actual parameters, dimensions, materials and / or configurations will depend on the particular one or more uses for which the teachings of the invention are employed. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it is to be understood that the invention may be practiced otherwise than as specifically described and claimed within the scope of the appended claims and their equivalents. The present invention is directed to each and every individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits and / or methods, if not mutually inconsistent, is included within the scope of the present invention. Further, each of the narrower species and subgeneric groups within the general disclosure also forms part of the present invention. This includes the general description of the invention under any conditions or negative limitations that exclude any subject from the genus, whether or not the excluded subject is specifically recited herein.

[0114] Incorporation by reference The contents of the papers, patents and patent applications, as well as other documents and electronically available information referred to or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The applicant reserves the right to physically incorporate any and all materials and information from such papers, patents, patent applications, or other physical and electronic documents into this application.

[0115] The following examples illustrate the present invention.

Example

[0116] Example 1: Synthesis of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (Example Compound 1) Step a): Synthesis of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid

[0117]

Chemical formula

[0118] A mixture of methyl 5-chloro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxylate (25 g, 0.0934 mol) and aniline (17.4 g, 0.0333 mol, 2 eq) in toluene (600 mL) was stirred at 100 °C for 17 h. Total conversion to the product was revealed by HPLC. The reactants were removed from the heat and the product was precipitated. The reaction mixture was left at room temperature for 2 days. The soft solid cake was suspended in n-heptane (500 mL), stirred for 5 min, and then the solid was filtered off. The solid was washed with a 1:1 mixture of toluene and n-heptane (1000 mL) to give crude 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide. The product was recrystallized from toluene, washed with heptane, and then further purified by column chromatography (100% petroleum ether -> 100% DCM). Final recrystallization from AcCN gave the desired product (21.5 g, 70% yield).

[0119] LC / MS: M+H = 329.10. 1 1H NMR (400 MHz, DMSO-d6) δ: 12.75 (1H, s), 7.73 (1H, m), 7.65 (3H, m), 7.44 (3H, m), 7.22 (1H, m), 3.70 (s, 3H).

[0120] Step b) Preparation of the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid (300 mg) was suspended in ethanol (6.0 mL) and 5 M aqueous potassium hydroxide (0.198 mL, ca. 1.1 eq) was added. The resulting suspension was shaken well by hand and then stirred and temperature cycled from 40 °C to ambient temperature for 48 h.

[0121] The product was isolated by filtration, washed with ethanol (2 x 1 mL), and dried to constant weight at 45 °C under vacuum. White crystals were obtained. The yield was 251 mg.

[0122] Example 2: Crystallization of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide (Example Compound 1) from various solvents 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide free acid (20 mg) was suspended in a solvent (400 μL), and 5 M aqueous potassium hydroxide solution (about 1.1 equivalents) was added. The mixture was shaken well by hand and then temperature-cycled at ambient temperature to 40 °C for at least 18 to 24 hours. The solid product was isolated by filtration.

[0123] The solvents investigated were as follows.

[0124] [Table 1]

[0125] In each case, a crystalline salt was formed. All solids were analyzed by X-ray Powder Diffraction analysis (XRPD). When the salt was isolated from each of the solvents investigated, the same polymorphic form was obtained.

[0126] Example 3: Characterization of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide (Example Compound 1) a) Solution NMR A sample of Example Compound 1 was dissolved in DMSO-d6 and analyzed by 1 1H NMR (400 MHz). Peaks were observed at δ: 13.09 (1H, s), 7.65 (2H, m), 7.35 (1H, m), 7.24 (3H, m), 7.05 (1H, m), 6.81 (1H, m), 3.49 (s, 3H).

[0127] Salt formation was confirmed by the change in the splitting pattern compared to the free API.

[0128] b) Elemental analysis Elemental analysis was performed as follows. For the determination of CHN, a "Vario Micro Cube" (Elementar) was used. The chlorine content was measured using a Metrohm Model 883 Plus ion chromatograph. The potassium content was determined using an ICP-OES Model Spectro Arcos (Spectro). Analyses were performed on replicate samples of Example Compound 1.

[0129] The results of the elemental analysis were as follows.

[0130] [Table 2]

[0131] The predicted values shown in the table are the expected values for the monopotassium salt without water of hydration. As can be seen in the table, the potassium level was found to be 10.6% w / w on average from two experiments. These results were consistent with the monopotassium salt without water of hydration. (Theoretical value: 10.7%).

[0132] c) Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) For the TGA analysis, a sample of approximately 5 - 10 mg of Example Compound 1 was accurately weighed into a pierced Tzero aluminum pan and sealed with a pinhole lid using a crimper. Next, the sample was loaded into the chamber of the TGA thermal analyzer at ambient temperature. A TA Instruments TGA5500 device was used. Then, the sample was heated from 20 °C to 400 °C at a rate of 20 °C / min. The purge gas used was nitrogen at a flow rate of 50 cm 3 / min.

[0133] For DSC analysis, approximately 2 mg of the sample was accurately weighed into a perforated Tzero aluminum pan and sealed using a universal crimper. Next, the sample was loaded into the chamber of the DSC analyzer at ambient temperature. The DSC2500 apparatus from TA Instruments was used. Subsequently, the sample was heated from 30 °C to 400 °C at a rate of 20 °C / min. The purge gas used was nitrogen with a flow rate of 50 cm 3 / min.

[0134] The DSC trace of Example Compound 1 is shown in Figure 1. The numerical findings are as follows. The findings for the free acid compound are shown in the third column for comparison.

[0135]

Table 3

[0136] The STA data did not show a sharp weight loss corresponding to an observable endotherm. A slight weight loss, just below 1% at approximately 300 °C, may correspond to some moisture trapped within the structure. However, this indicated that the sample was neither hydrated nor solvated. The onset of decomposition corresponded to a melting that started at approximately 347 °C. The TGA data showed a slight progressive weight loss of 0.6% from the onset corresponding to surface moisture. There was no significant endotherm prior to melting starting at approximately 343 °C from the DSC data. The maximum endothermic heat flow was observed at 361.3 °C.

[0137] Overall, these data demonstrate that Example Compound 1 was not hydrated and had good stability up to a temperature of 347 °C.

[0138] d) X-ray powder diffraction analysis (XRPD) A sample of about 5 - 10 mg of Example Compound 1 was gently compressed on an XRPD zero background single obliquely cut silica sample holder. The sample was then loaded into a Philips X-Pert PRO diffractometer and analyzed using the following experimental conditions.

[0139]

Table 4

[0140] For certain replicate experiments, slower scan rates were also used over the range of 4 - 40° two-theta as detailed below.

[0141]

Table 5

[0142] The sample was confirmed to be crystalline by XRPD. The XRPD trace is shown in Figure 2.

[0143] The peaks in the XRPD trace were as follows.

[0144]

Table 6

[0145] A sample of Example Compound 1 was stored in a 20 mg / 400 μL aqueous slurry at ambient temperature for 48 hours, recovered, dried by evaporation, and re-examined by XRPD. There was no change in the X-ray diffractogram, indicating that the salt does not tend to disproportionate.

[0146] e) Optical microscopy The crystals of Example Compound 1 were observed under an optical microscope and compared with the crystals of the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (described in Example 1a above).

[0147] A photograph of the crystals of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide is shown in Figure 3a. It can be seen that the crystals are large needles with a length of 200 - 300 microns. The bar in the photograph indicates a length of 100 μm.

[0148] A photograph of the crystals of Example Compound 1 is shown in Figure 3b. The crystals appear to be plate-like with a particle size distribution of less than 10 microns. The bar in the photograph indicates a length of 20 μm.

[0149] The free acid compound appeared to be aggregated and this could be seen with the naked eye. On the other hand, the potassium salt was powdery with a plate-like habit. This material was easier to handle than the long needles that make up the free acid compound.

[0150] f) Solubility and stability in aqueous solution The solubility and stability at room temperature of Example Compound 1 and the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (described in Example 1a above) were measured in PBS (pH 7.4) and simulated colonic fluid (SCF) at ambient temperature. The SCF was the product FaSSCof obtained from Biorelevant.com prepared according to the manufacturer's instructions. FaSSCof contained 0.15 mM sodium cholate, 0.3 mM phospholipid, 0.1 mM oleate, 120 mM sodium hydroxide, 45 mM TRIS and 76 mM maleate.

[0151] Saturated solutions were prepared in these solvents and the concentration / impurity profiles were determined by HPLC-UV using a calibration curve in the range of 1 - 150 μg / ml at 300 nm or 320 nm. Visual evaluations were also performed after each step over the test period.

[0152]

Table 7

[0153] As can be seen from the table, Example Compound 1 has better solubility than the corresponding free acid in both of these solvents.

[0154] The samples were also analyzed for the presence of impurities. After 48 and 72 hours, all impurities were below the lower limit of quantification.

[0155] g) Hygroscopicity Dynamic Vapour Sorption (DVS) analysis was performed on samples of Example Compound 1.

[0156] Approximately 20 mg of the sample was placed in a wire mesh vapour sorption balance pan and loaded onto an "IgaSorp" vapour sorption balance (Hiden Analytical Instruments). The sample was then dried by maintaining an environment of 0% humidity until no further weight change was recorded.

[0157] The sample was then subjected to a step profile of 0 - 90% RH in 10% RH increments. The desorption isotherm was from 90% RH to 0% RH in 10% RH steps. Next, the weight change during the sorption / desorption cycle was monitored to measure the hygroscopicity of the sample. The analysis was performed using the following parameters.

[0158] Analysis mode: F1 Waiting time: 99% Sample temperature: 25 °C Minimum timeout: 30 minutes Maximum timeout: 180 minutes Gas flow rate: 250 ml / min The results showed that the sample was non - hygroscopic and had a slight reversible weight gain of less than 0.2% when analyzed up to 80% RH.

[0159] Example 3: Biological Activity Example 3a): Activity of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide in the inhibition of inflammation / edema associated with inflammatory bowel disease The efficacy of potassium 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenylquinoline-3-carboxamide in the inhibition of inflammation / edema associated with inflammatory bowel disease was evaluated in a CD4+ adoptive transfer-induced inflammatory bowel disease in mice.

[0160] Details of the mice were as follows.

[0161] Species / strain or variety: Fox Chase C.B-17 SCID and Balb / C Supplier: Charles River Age / weight (Wt) at arrival: CB-17 SCID - 6 - 7 weeks old Balb / C - 11 - 12 weeks old Sex: Female Acclimation: At least 7 days of acclimatization after arrival Housing: 5 animals / cage On test day - 1, SCID mice were weighed and evenly distributed into treatment groups based on body weight.

[0162] On test day 0, Balb / C mice were terminated and spleens were harvested for cell isolation (using the SCID IBD cell isolation protocol). After cells were sorted, each animal in the treatment groups received at least 4×10 + CD45RB 高 cells (200 μl / mouse injection) by IP injection of CD4 5 CD45RB + 高 cells. Subsequently, a naive group of mice was followed through an experiment without cell injection. The naive group consisted of 5 animals.

[0163] ​On the 21st day of the test, treatment with 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide potassium salt (1 mg / kg, daily from the 21st day to the 49th day) was initiated. The compound was formulated as a 0.1 mg / mL suspension containing sodium carboxymethylcellulose (1%, w / v). Mice were administered either 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide potassium salt (Example Compound 1) or vehicle. Each of these groups contained 10 animals.

[0164] On the 49th day of the test, the animals were anesthetized with isoflurane, blood was collected, and then they were sacrificed by cervical dislocation. The entire colon was removed, measured, and weighed. The colon was analyzed for its interferon-γ and IL-22 levels. The results are shown in Figure 5. The inflammation in the colon was also scored by visual and histopathological evaluation. The results are shown in Figure 6.

[0165] Each animal was weighed at 3- or 4-day intervals, and the average body weights of the three groups of mice are shown in Figure 4. As can be seen in the figure, mice treated with Example Compound 1 had less weight loss than mice treated with vehicle alone. In Figure 5, it can be seen that mice treated with Example Compound 1 had lower levels of inflammatory markers than mice treated with vehicle alone. Similarly, in Figure 6, it can be seen that mice treated with Example Compound 1 had fewer signs of inflammation than mice treated with vehicle alone. * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01). The results in Figures 5 and 6 indicate that Example Compound 1 has a local anti-inflammatory effect in the colon of animals.

[0166] Example 3b): Activation of the aryl hydrocarbon receptor (AhR) in the colon of mice after administration of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide potassium salt The possibility that potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (Example Compound 1) activates the aryl hydrocarbon receptor (AhR) in the colon after oral administration was evaluated in wild-type (WT) mice.

[0167] Details of the mice were as follows.

[0168] Number of animals: Ordered 51 (50 for the test + 1 additional) Species / strain or breed: C57Bl / 6 Age / weight (Wt) at arrival: 6 - 7 weeks old Sex: Female Housing: 5 animals / cage On the 0th day of the test, the animals were weighed and evenly distributed into treatment groups based on their body weight.

[0169] Also, on the 0th day of the test, the treatment was started (the outline of the treatment schedule is shown in the following table). On the 14th day of the test, the animals were euthanized by cervical dislocation followed by asphyxiation by CO2 inhalation. The entire colon was removed, collected, and prepared for qPCR analysis of CYP1A1 (normalized to GAPDH & ACTB).

[0170]

Table 8

[0171] Example Compound 1 was formulated as a suspension of 0.1 mg / ml, 0.01 mg / ml, or 0.001 mg / ml containing sodium carboxymethyl cellulose (1%, w / v).

[0172] CYP1A1 qPCR results

[0173]

Table 9

[0174] As shown in the table, WT mice treated with the Example Compound 1 salt at 1.0 and 0.1 mg / kg had a significant increase in CYP1A1 mRNA expression compared to untreated animals, indicating activation of AhR. Furthermore, the results show that the increase in liver CYP1A1 expression was less than the increase in CYP1A1 in the colon, indicating that Example Compound 1 has a local AhR activation effect.

[0175] Example 3c): In Vivo Pharmacokinetics of Potassium Salt of 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide An in vivo pharmacokinetic study was performed in rats to determine whether the potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (Example Compound 1) could be systemically absorbed and detected after oral administration.

[0176] Four male Sprague Dawley rats, dosed at approximately 225 g - 250 g, were administered 1 mg / kg of Example Compound 2 formulated as a 0.1 mg / mL suspension containing sodium carboxymethylcellulose (1%, w / v). Samples were taken at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after dosing.

[0177] Approximately 250 μL of blood was sampled into K3EDTA vials at each blood sampling, and approximately 100 μL of plasma was also prepared.

[0178] Plasma samples were prepared by mixing 50 μL of plasma with 250 μL of internal standard solution (20 ng / ml of phenacetin in ACN containing 1% formic acid in ACN), mixing, and centrifuging (20 minutes, 4000 rpm).

[0179] Plasma samples were transferred to Waters Ostro96 well plates and drawn through the plates by applying a positive pressure of 6 - 8 psi for 10 minutes. 100 μL of the supernatant was further diluted with 50 μL of UP water and the samples were subjected to analysis.

[0180] Standards and QC samples were prepared in blank rat colon homogenates and blank rat plasma. The standards were spiked with analyte at concentrations of 0.1 - 10,000 ng / ml, and the QC samples were spiked with analyte at concentrations of 3, 30, 300, and 3000 ng / ml and otherwise treated as samples.

[0181] Levels of Example Compound 1 in plasma at various time points are shown in Figure 7. It can be seen that the compound is detected in plasma and rapidly removed.

[0182] Example 3d): Activity of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide in a murine model of ulcerative colitis Ulcerative colitis was modeled in C57Bl / 6 mice by administering dextran sulfate sodium (DSS) in drinking water (1.5% w / v) for 5 days. Mice were monitored daily for weight loss and clinical signs of disease starting at the initiation of DSS administration for 10 days.

[0183] Animals in different treatment groups were administered vehicle, 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid (1 mg / kg as an aqueous suspension of 0.1 mg / mL containing CMC-Na (carboxymethylcellulose sodium, 2% w / v)), or anti-TNFα antibody (anti-mouse TNFα antibody clone XT3.11). Mice in one control group were not administered DSS, 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid, or vehicle (referred to herein as "naïve animals"). Vehicle and 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid were administered daily starting on either day -7 or day 9. The anti-TNFα antibody was administered at 500 μg / treatment on days 0, 2, 4, and 6. After the end of day 10, the colon was removed and measured. The length and weight of the colon were also evaluated.

[0184] Compared with naive animals, animals given DSS water showed clinical signs of disease including weight loss and diarrhea, as well as total pathological signs of disease at the end of the experiment. Furthermore, the length of the colon was significantly decreased in diseased animals compared to naive animals, while the weight increased. The length was short and the weight increased. The length ratio indicates edema associated with ulcerative colitis.

[0185] As shown in FIGS. 8-10, daily oral treatment with 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid (referred to as "Example" in FIGS. 8-10) resulted in a significant decrease in the clinical score of colitis on day 9 of the test, a reduction in weight loss, and a significant increase in colon length at the end of the experiment compared to vehicle-treated controls, indicating an effective effect of the treatment.

[0186] Example 4: Sodium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (Example Compound 2) 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid (300 mg) was suspended in ethanol (6.0 mL), and 5M aqueous sodium hydroxide solution (0.198 mL, about 1.1 equivalents) was added. The resulting suspension was shaken well by hand and then stirred, and the temperature was cycled from 40 °C to ambient temperature for 48 hours.

[0187] The product was isolated by filtration, washed with ethanol (2 × 1 mL), and dried under vacuum at 45 °C to a constant weight. White crystals were obtained. The yield was 240 mg.

[0188] When the sodium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide was analyzed by thermogravimetric analysis (TGA) using the method described in Example 3c) above, the numerical findings were as follows. The findings for the free acid compound are shown in the third column for comparison.

[0189] [Table 10]

[0190] A sample of about 5 - 10 mg of Example Compound 2 was gently compressed on an XRPD zero - background single - slant cut silica sample holder. The sample was then loaded into a Philips X - Pert PRO diffractometer and analyzed using the experimental conditions described in Example 3d) above. The sample was confirmed to be crystalline by XRPD. The peaks in the XRPD trace were as follows.

[0191]

Table 11

[0192] A sample of Example Compound 2 was stored in a 20 mg / 400 μL aqueous slurry at ambient temperature for 48 hours, recovered, dried by evaporation, and re - examined by XRPD. There were slight changes in the X - ray diffractogram. This indicates that the salt showed a tendency to disproportionate, albeit slowly.

[0193] The crystals of Example Compound 2 were observed under an optical microscope and compared with the crystals of the free acid compound 5 - chloro - 4 - hydroxy - 1 - methyl - 2 - oxo - N - phenyl - quinoline - 3 - carboxamide (described in Example 1a) above).

[0194] The sodium salt of 5 - chloro - 4 - hydroxy - 1 - methyl - 2 - oxo - N - phenyl - quinoline - 3 - carboxamide was found to be powdery with a tabular habit and a particle size distribution of less than 20 microns. This material is easier to handle than the long needles that make up the free acid compound.

[0195] The hygroscopicity of Example Compound 2 was analyzed using the method described in Example 3g) above. The results showed that the sample was non - hygroscopic and had a slight reversible weight gain of 1% when analyzed up to 80% RH.

[0196] Example 5: Lithium Salt of 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (Example Compound 3) 5-Chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide free acid (300 mg) was suspended in acetone (6.0 mL), and a solution of lithium hydroxide (24 mg) in water (0.2 ml) was added. The resulting suspension was shaken well by hand and then stirred, and temperature-cycled from 40 °C to ambient temperature for 48 hours.

[0197] The product was isolated by filtration, washed with ethanol (2 × 1 mL), and dried under vacuum at 45 °C to a constant weight. White crystals were obtained. The yield was 225 mg.

[0198] When the lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide was analyzed by thermogravimetric analysis (TGA) using the method described in Example 3c) above, the numerical findings were as follows. The findings for the free acid compound are shown in the third column for comparison.

[0199] [Table 12]

[0200] A sample of about 5 - 10 mg of Example Compound 3 was gently compressed on an XRPD zero-background single-bevel cut silica sample holder. The sample was then loaded into a Philips X-Pert PRO diffractometer and analyzed using the experimental conditions described in Example 3d) above. The sample was confirmed to be crystalline by XRPD. The peaks in the XRPD trace were as follows.

[0201] [Table 13]

[0202] A sample of Example Compound 3 was stored in a 20 mg / 400 uL aqueous slurry at ambient temperature for 48 hours, recovered, dried by evaporation, and re-examined by XRPD. There were slight changes in the X-ray diffraction pattern, indicating that the salt showed a tendency to disproportionate, albeit slowly.

[0203] The crystals of Example Compound 3 were observed under an optical microscope and compared with the crystals of the free acid compound 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide (described in Example 1a above).

[0204] The lithium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide was found to be a powder with a tabular habit and a particle size distribution of less than 20 microns. This material is easier to handle than the long needles that make up the free acid compound.

[0205] The hygroscopicity of Example Compound 3 was analyzed using the method described in Example 3g) above. The results showed that the sample was slightly hygroscopic, with a slight reversible weight gain of less than 0.25% when analyzed up to 80% RH.

Claims

1. A compound of potassium salt of 5-chloro-4-hydroxy-1-methyl-2-oxo-N-phenyl-quinoline-3-carboxamide.

2. The compound according to claim 1, which is a solid.

3. The compound according to claim 2, which is a crystal.

4. The compound according to claim 3, having a powder X-ray diffractogram with characteristic peaks at 2θ = 6.9 ± 0.2°, 15.6 ± 0.2°, 24.9 ± 0.2° and 28.4 ± 0.2°.

5. The compound according to claim 4, wherein the powder X-ray diffractogram has additional characteristic peaks at 2θ = 20.9° ± 0.2°, 24.2 ± 0.2°, 25.2 ± 0.2°, 25.6 ± 0.2° and 27.3 ± 0.2°.

6. The compound according to claim 2, which is amorphous.

7. An aqueous solution containing the compound according to claim 1.

8. The compound according to any one of claims 1 to 6, for use as a medicament.

9. The aqueous solution according to claim 7, for use as a medicament.

10. The compound according to any one of claims 1 to 6, for use as a medicament for the treatment or prevention of inflammatory bowel disease.

11. The aqueous solution according to claim 7, for use as a medicament for the treatment or prevention of inflammatory bowel disease.

12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 or the aqueous solution according to claim 7 and at least one pharmaceutically acceptable excipient.

13. The pharmaceutical composition according to claim 12, further comprising one or more additional therapeutic agents.

14. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 for the treatment or prevention of inflammatory bowel disease.

15. A pharmaceutical composition comprising the aqueous solution according to claim 7 for the treatment or prevention of inflammatory bowel disease.

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