JPWO2013018685A1 - Lactam derivatives and their pharmaceutical use - Google Patents

Abstract

The object of the present invention is to provide a highly safe treatment or prevention agent for fibrosis that has no phototoxicity and has reduced hepatotoxicity and gastrointestinal disorders. The present invention provides a lactam derivative represented by the following chemical formula (I).

Description

  The present invention relates to a lactam derivative and its pharmaceutical use.

  Fibrosis is a disease that adversely affects the function of various organs, mainly due to the overproliferation of fibroblasts and the overproduction and accumulation of extracellular matrix components, particularly the lungs, skin, digestive tract, It affects the kidneys and liver.

  It is known that once fibrosis develops, not only the function of the fibrotic organ is reduced, but also the whole body is seriously damaged. For example, in the case of fibrosis that develops on the skin, in severe cases, the growth of scar tissue in the skin is not limited to the appearance abnormality of the affected area, but affects the motor function of the limbs.

  Renal fibrosis leads to renal failure at the end stage, requiring treatment by artificial dialysis or kidney transplantation. Liver fibrosis leads to liver sclerosis and cirrhosis at the end stage, many of which progress to liver cancer. However, in pulmonary fibrosis, respiratory failure occurs, and in the worst case, death occurs.

  Currently, immunosuppressants and anti-inflammatory agents such as corticosteroids are mainly used for the treatment of fibrosis.

  In recent years, as a drug effective for idiopathic pulmonary fibrosis, pirfenidone has obtained the world's first manufacturing and marketing approval. Pirfenidone has been recognized to have an antifibrotic effect in affected organs of various fibrotic disease animal models (Non-Patent Documents 1 to 4).

Oku et al., European Journal of Pharmacology, 2008, 590, p. 400-408 Garcia et al., Journal of Hepatology, 2002, 37, p. 797-805 Shimizu et al., Kidney International, 1998, 54, p. 99-109 Shetlar et al., Journal of Laboratory and Clinical Medicine, 1998, vol. 132, p. 491-496

  However, the mechanism involved in the control of fibrosis is different from the mechanism of action of immunosuppressants and anti-inflammatory agents, so fibrosis itself is not possible with immunosuppressive and anti-inflammatory agents prescribed for the treatment of fibrotic diseases. In reality, it cannot be fundamentally prevented or improved, and it has not always been effective in reducing or preventing fibrosis.

  In addition, pirfenidone, a therapeutic agent for idiopathic pulmonary fibrosis, is known to have phototoxicity, hepatotoxicity, and digestive system disorders as serious side effects. If these side effects are observed, the drug must be discontinued. It was the present situation that we did not get.

  Accordingly, an object of the present invention is to provide a highly safe treatment or prevention agent for fibrosis that has no phototoxicity and has reduced hepatotoxicity and digestive system disorders.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a lactam derivative having an antifibrotic effect, having no phototoxicity, and having reduced hepatotoxicity and digestive system damage. Successfully completed the present invention.

That is, the present invention provides a lactam derivative represented by the following chemical formula (I).

  The present invention also provides a medicament, particularly a therapeutic or prophylactic agent for fibrosis, containing the lactam derivative represented by the above formula (I) as an active ingredient.

  The lactam derivative of the present invention has an antifibrotic effect on in vitro models and animal models of fibrosis, has no phototoxicity, and has reduced hepatotoxicity and digestive system damage. It can be used as a highly safe treatment or prevention agent for fibrosis.

It is a figure which shows the inhibitory effect with respect to the amount of hydroxyproline in the lung tissue of the compound of Example 1 in a mouse bleomycin-induced pulmonary fibrosis model.

The lactam derivative of the present invention is characterized by being represented by the following chemical formula (I).

  Unless otherwise specified, the above lactam derivatives are isomers due to the presence of asymmetric carbon (R-form, S-form, α-form, β-form, enantiomer), optical isomers having optical activity (D-form, L-form, d Body, l-form, (+)-form, (-)-form), a mixture of these in any ratio, and a racemic mixture.

  The above lactam derivative can be synthesized, for example, according to the production method shown in Scheme 1.

The above lactam derivative obtained by the following production method can be isolated and purified by known means. Known isolation and purification methods include, for example, solvent extraction, recrystallization, or chromatography.

  In the above scheme 1, X represents a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[First step]
The N-arylpyridone compound represented by the formula (IV) is represented by the general formula (III) in the presence of a metal catalyst, a ligand, and a base in a suitable solvent with respect to the hydroxypyridine represented by the formula (II). It can be synthesized by a coupling reaction in which the represented aryl halide is allowed to act.

  Examples of the aryl halide include those having a leaving group that allows the coupling reaction to proceed smoothly, such as iodobenzene, bromobenzene, or chlorobenzene, with iodobenzene or bromobenzene being preferred.

  1-20 mol is preferable with respect to 1 mol of hydroxy pyridine shown by a formula (II), and, as for the usage-amount of said aryl halide, 1-10 mol is more preferable.

  Examples of the metal catalyst include a palladium catalyst such as tris (dibenzylideneacetone) dipalladium, palladium acetate or tetrakis (triphenylphosphine) palladium, or a copper catalyst such as cuprous oxide, copper iodide or copper powder. Copper catalysts are preferred.

  The amount of the metal catalyst used is preferably 0.001 to 1 mol, more preferably 0.01 to 0.5 mol, per 1 mol of hydroxypyridine represented by the formula (II).

  The ligand is appropriately selected according to the metal catalyst. Examples of the ligand for the palladium catalyst include triphenylphosphine, 2,2′-bis (diphenylphosphino) -1,1′-binaphthyl, and 4, Examples include phosphine derivatives such as 5-bis (diphenylphosphino) -9,9-dimethylxanthene, and 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene is preferable. Moreover, when using a copper catalyst, it is preferable not to add a ligand.

  0-1 mol is preferable with respect to 1 mol of hydroxypyridines represented by Formula (II), and, as for the usage-amount of a ligand, 0-0.5 mol is more preferable.

  Examples of the base include alkali metal carbonates such as sodium carbonate and potassium carbonate, alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate, alkali metal phosphates such as sodium phosphate and potassium phosphate, and phosphoric acid. Alkali metal hydrogen phosphates such as sodium hydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate or potassium dihydrogen phosphate are listed, and alkali metal carbonates are preferred.

  0.5-10 mol is preferable with respect to 1 mol of hydroxypyridines represented by Formula (II), and, as for the usage-amount of a base, 1-5 mol is more preferable.

  As the reaction solvent, a solvent that does not normally inhibit the reaction is appropriately selected. For example, ether solvents such as tetrahydrofuran (hereinafter referred to as THF), 1,4-dioxane or ethylene glycol dimethyl ether, and aromatic hydrocarbon solvents such as benzene or toluene. An aprotic polar solvent such as N, N-dimethylformamide (hereinafter referred to as DMF) or dimethyl sulfoxide (hereinafter referred to as DMSO) or the absence of a solvent may be mentioned.

  0-200 degreeC is preferable and, as for reaction temperature, 30-150 degreeC is more preferable.

  The reaction time is appropriately selected according to conditions such as the reaction temperature, but satisfactory results are usually obtained in about 1 to 72 hours.

[Second step]
The above lactam derivative can be synthesized by a reduction reaction for reducing the diene of the N-arylpyridone compound represented by the formula (IV) in an appropriate solvent. Examples of the reduction reaction include catalytic hydrogenation in which a metal catalyst is allowed to act in a hydrogen gas atmosphere.

  Examples of the metal catalyst used for catalytic hydrogenation include platinum dioxide, palladium / activated carbon, and palladium hydroxide / activated carbon, with platinum dioxide being preferred.

  The amount of the metal catalyst used is preferably 0.05 to 200% by weight, more preferably 1 to 100% by weight, based on 1 mole of the N-arylpyridone compound represented by the formula (IV).

  The pressure of hydrogen gas is preferably 1 to 10 atm, and more preferably 1 to 5 atm.

  As the reaction solvent, a solvent that does not normally inhibit the reaction is appropriately selected. For example, an alcohol solvent such as methanol, ethanol or 2-propanol, an ester solvent such as ethyl acetate or n-propyl acetate, THF, 1,4-dioxane. Alternatively, an ether solvent such as ethylene glycol dimethyl ether, a halogen solvent such as dichloromethane, chloroform or 1,2-dichloroethane, water, or a mixed solvent thereof can be used, and an alcohol solvent is preferable.

  The concentration of the N-arylpyridone compound represented by the formula (IV) in the reaction solution is preferably 0.001 to 5 mol / L, and more preferably 0.05 to 2 mol / L.

  0-100 degreeC is preferable and, as for reaction temperature, 10-60 degreeC is more preferable.

  The reaction time is appropriately selected according to conditions such as the reaction temperature, but satisfactory results are usually obtained in about 30 minutes to 48 hours.

  The above lactam derivative has an excellent antifibrotic effect, does not have phototoxicity, has reduced liver toxicity and gastrointestinal disorders, and can be used as a highly safe pharmaceutical. It can be preferably used as a therapeutic or prophylactic agent for symptom.

  Fibrosis is a disease in which fibrotic lesions are formed in tissues mainly due to overproliferation of fibroblasts and overproduction and accumulation of extracellular matrix components, and adversely affect the function of various organs, for example, , Liver fibrosis, cirrhosis, liver necrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, myocardial fibrosis, renal fibrosis, vascular fibrosis or skin scars, but the above lactam derivatives are especially pulmonary fibrosis It is preferably used for treatment or prevention.

  Pulmonary fibrosis is caused by various stimuli that damage the alveolar epithelium or basement membrane, resulting in fibrotic lesions in the lungs due to overproliferation of fibroblasts and overproduction and accumulation of extracellular matrix components during the repair process. Diseases that are formed, such as lung fibrosis seen late in pneumonia due to viral infection, pulmonary fibrosis caused by the progression of hypersensitivity pneumonia, idiopathic pulmonary fibrosis or radiation pulmonary fibrosis, The lactam derivative is preferably used particularly for the treatment or prevention of idiopathic pulmonary fibrosis.

  The effectiveness of the above lactam derivative for treating or preventing fibrosis can be evaluated using an in vitro model test. Examples of in vitro model tests to be used include, for example, a test using collagen gel in which fibroblasts derived from various organs such as lung, liver, kidney, and skin are cultivated (Pulmonary Pharmacology & Therapeutics, 2009, Vol. 22, p.487-491, British Journal of Pharmacology, 2010, 159, 304-315, Kidney International, 2006, 70, p.298-305, Archives of Dermatological, Research 20, Year 20. 304, p.217-222). When activated in the process of tissue fibrosis, fibroblasts become hyperproliferative and produce excessive amounts of extracellular matrix components and acquire contractility. When fibroblasts are embedded and cultured in a collagen gel, the collagen gel contracts as the fibroblasts contract due to fibroblast activation (for example, addition of TGF-β1), and the collagen gel area decreases. Therefore, by measuring the collagen gel area, the contractility of fibroblasts, that is, the activation of fibroblasts can be evaluated.

  Moreover, it can be evaluated using a disease state model animal that the above lactam derivative is effective in treating or preventing fibrosis. Examples of pathological model animals used include bleomycin-induced pulmonary fibrosis model with bleomycin (European Journal of Pharmacology, 2008, 590, pp. 400-408), carbon tetrachloride cirrhosis model (Journal of Hepatology, 2002) 37, p. 797-805), renal fibrosis model with ammonium metavanadate (Biochemical Pharmacology, 2002, 64, p. 517-525), chronic renal failure model (Kidney International, 1998, 54, p. 99-109) or keloid model (Journal of Laboratory and Clinical Medicine, 1 1998, No. 132 Vol., P.491-496) and the like. The bleomycin-induced pulmonary fibrosis model is a common model of pulmonary fibrosis, but its pathological condition is considered to be close to idiopathic pulmonary fibrosis.

  The effectiveness of the above lactam derivative for treating or preventing fibrosis is measured by measuring the area of the collagen gel, which is an index of fibroblast contractility, using the above in vitro model test. Can be evaluated. Further, by using the above-mentioned disease state model animal, for example, a decrease in the amount of tissue hydroxyproline, which is a characteristic index of fibrosis, can be evaluated as an index.

  Phototoxicity generally refers to the property that a drug absorbed in the skin, when activated by a photochemical reaction caused by light energy, acquires toxicity and damages tissue.

  The fact that the above lactam derivative does not have phototoxicity can be evaluated by, for example, a phototoxicity test using mice or a phototoxicity test using guinea pigs or rabbits.

  Hepatotoxicity refers to local pain, joint pain, gastrointestinal disorders, general malaise, jaundice, hepatomegaly, ascites, spleen enlargement, edema, hepatic encephalopathy, hepatic sensation due to exposure of the liver to the drug taken. It causes symptoms such as coma or tenderness, and it has been reported that hepatotoxicity observed with pirfenidone is caused by induction of cytochrome P450 (hereinafter, CYP) (EUROPEAN MEDICINES AGENCY, CHMP assessment report, 2010, Procedure No. EMEA). / H / C / 002154).

  The low drug metabolizing enzyme CYP-inducing action of the lactam derivative can be evaluated, for example, in a CYP induction test using human frozen hepatocytes (Nippon Pharmacology, 2009, Vol. 134, p. 330-333).

  Gastrointestinal disorders are those that cause symptoms such as upper abdominal discomfort, abdominal pain, abdominal bloating, loss of appetite, nausea, vomiting or diarrhea depending on the drugs taken.

  The low digestive system damage action of the lactam derivative can be evaluated by, for example, a test on gastric excretion ability using a pigment in rats, a test on small intestine transport ability using carbon powder, or a test on ferret emesis.

  The above lactam derivative improves or treats the pathology of fibrosis when administered to mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys or humans), particularly humans. Or a preventive effect can be exhibited.

  The above lactam derivative can be administered orally or parenterally in a non-added state or as a preparation containing a pharmaceutically acceptable carrier.

  Examples of dosage forms for oral administration of the above-mentioned preparations containing lactam derivatives include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and microcapsules). ), Syrups, emulsions or suspensions. Examples of dosage forms for parenteral administration include injections, infusions, drops, and suppositories. In addition, the above lactam derivative may be a suitable base (eg, butyric acid polymer, glycolic acid polymer, butyric acid-glycolic acid copolymer, butyric acid polymer and glycolic acid polymer mixture or poly In combination with glycerol fatty acid ester), it is possible to obtain a sustained-release preparation, which is effective in treating fibrosis. In addition, in order to improve the bioavailability and stability of the lactam derivative, a drug delivery system including a preparation technique such as microcapsule, micronization or packaging can be used.

  A pharmaceutical dosage form containing the above lactam derivative as an active ingredient can be prepared according to a method generally used in the pharmaceutical field. In this case, it can be prepared by containing excipients, binders, lubricants, disintegrating agents, sweeteners, surfactants, suspending agents, emulsifiers and the like that are generally used in the pharmaceutical field as necessary. .

  Tablets can be prepared, for example, containing excipients, binders, disintegrants or lubricants, and pills and granules can be prepared, for example, with excipients, binders or disintegrants. It can be made to contain. The preparation of powders and capsules can be carried out, for example, by containing excipients, and the preparation of syrups can be carried out, for example, by containing sweeteners, and preparation of emulsions and suspensions. Can be carried out, for example, by containing a surfactant, a suspending agent or an emulsifier.

  Examples of the excipient include lactose, glucose, starch, sucrose, microcrystalline cellulose, licorice powder, mannitol, sodium bicarbonate, calcium phosphate or calcium sulfate. Examples of the binder include starch paste, gum arabic, gelatin, tragacanth, carboxymethylcellulose, sodium alginate, and glycerin. Examples of the disintegrant include starch and calcium carbonate. Examples of the lubricant include magnesium stearate, stearic acid, calcium stearate, and purified talc. Examples of the sweetening agent include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, and simple syrup. Examples of the surfactant include sodium lauryl sulfate, polysorbate 80, sorbitan monofatty acid ester or polyoxyl 40 stearate. Examples of the suspending agent include gum arabic, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, and bentonite. Examples of the emulsifier include gum arabic, tragacanth, gelatin, and polysorbate 80.

  Furthermore, when preparing a preparation containing the above lactam derivative into the above dosage form, colorants, preservatives, fragrances, flavoring agents, stabilizers, thickeners and the like that are commonly used in the pharmaceutical field are added. It may be added.

  The dose of the above lactam derivative varies depending on the target therapeutic effect, administration method, age or body weight, and thus cannot be defined unconditionally. However, the daily oral dose is usually 100 to 10,000 mg as an active ingredient. Is preferable, and 200-8000 mg is more preferable. What is necessary is just to divide and administer this 1 to 5 times a day.

  EXAMPLES Hereinafter, although an Example is shown and this invention is explained in full detail, this invention is not limited to these.

¹H-NMR (400MHz, CDCl₃)δ: 7.51-7.46 (2H, m), 7.43-7.36 (3H, m), 7.27 (1H, dd, J=9.3, 2.7 Hz), 7.12-7.10 (1H, m), 6.61 (1H, d, J=9.3 Hz), 2.11 (3H, s).
MS (ESI)： 186 ([M+H]⁺).Example 1 Synthesis of 5-methyl-1-phenylpiperidin-2-one:
[Step 1] Synthesis of 5-methyl-1-phenylpyridin-2 (1H) -one:
Bromobenzene (28.8 mL) was added to a mixture of 2-hydroxy-5-methylpyridine (10.0 g, 92.0 mmol), cuprous oxide (0.656 g, 4.58 mmol) and potassium carbonate (13.9 g, 101 mmol). 275 mmol) was added and the inside of the reaction vessel was purged with argon, followed by stirring at 120 ° C. for 17 hours. Cuprous oxide (0.197 g, 1.38 mmol), potassium carbonate (4.18 g, 30.2 mmol) and bromobenzene (8.64 mL, 82.5 mmol) were added to the reaction mixture, and the reaction vessel was filled with argon. After the replacement, the mixture was stirred at 120 ° C. for 24 hours. The reaction mixture was filtered through celite, the obtained filtrate was concentrated under reduced pressure, and water was added to the residue. After extraction with dichloromethane, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Dichloromethane, hexane and diethyl ether were added to the resulting residue for recrystallization, and the precipitated solid was collected by filtration. The obtained solid and filtrate were purified by amine silica gel column chromatography (ethyl acetate / n-hexane = 3/7 to 17/3), dried together, and 5-methyl represented by the following chemical formula. 15.2 g (yield 89%) of -1-phenylpyridin-2 (1H) -one was obtained.

¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.51-7.46 (2H, m), 7.43-7.36 (3H, m), 7.27 (1H, dd, J = 9.3, 2.7 Hz), 7.12-7.10 (1H, m), 6.61 (1H, d, J = 9.3 Hz), 2.11 (3H, s).
MS (ESI): 186 ([M + H] ⁺ ).

¹H-NMR (400MHz, CDCl₃)δ: 7.42-7.36 (2H, m), 7.27-7.23 (3H, m), 3.57 (1H, ddd, J=11.9, 4.9, 1.8 Hz), 3.33 (1H, dd, J=11.9, 10.2 Hz), 2.66-2.50 (2H, m), 2.21-2.09 (1H, m), 2.01-1.93 (1H, m), 1.67-1.57 (1H, m), 1.07 (3H, d, J=6.8 Hz).
MS (ESI)： 190 ([M+H]⁺).
IR (KBr, cm^-1)： 3050, 2935, 2899, 2873, 2827, 1646, 1589, 1490, 1458, 1412, 1354, 1277, 1219, 1181, 1149, 1076, 1027, 774, 737.
Mp: 61-62 ℃[Second Step] Synthesis of 5-methyl-1-phenylpiperidin-2-one:
To a solution of 5-methyl-1-phenylpyridin-2 (1H) -one (3.00 g, 16.2 mmol) in ethanol (30 mL) was added platinum dioxide (0.368 g, 1.62 mmol) and at room temperature under a hydrogen atmosphere. Stir for 13 hours. The reaction mixture was filtered through Celite, and the obtained filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 9 / 11-3 / 1) and represented by the following chemical formula: 5-methyl-1-phenylpiperidin-2-one (hereinafter, implementation) 3.06 g (quantitative) of the compound of Example 1) was obtained.

¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.42-7.36 (2H, m), 7.27-7.23 (3H, m), 3.57 (1H, ddd, J = 11.9, 4.9, 1.8 Hz), 3.33 (1H, dd, J = 11.9, 10.2 Hz), 2.66-2.50 (2H, m), 2.21-2.09 (1H, m), 2.01-1.93 (1H, m), 1.67-1.57 (1H, m), 1.07 (3H, d, J = 6.8 Hz).
MS (ESI): 190 ([M + H] ⁺ ).
IR (KBr, cm ^-1 ): 3050, 2935, 2899, 2873, 2827, 1646, 1589, 1490, 1458, 1412, 1354, 1277, 1219, 1181, 1149, 1076, 1027, 774, 737.
Mp: 61-62 ℃

(Example 2) Effect on contraction of collagen gel in which lung fibroblasts are embedded and cultured:
The effect of the compound of Example 1 on the contraction of collagen gel in which HFL-1 cells, human lung fibroblast HFL-1 cells, were cultured. Collagen gel contraction is described in Pulmonary Pharmacology & Therapeutics, 2009, Vol. 22, p. The method described in 487-491 was partially modified and evaluated.

Rat type I collagen (Becton Dickinson) dissolved in 0.02N acetic acid was diluted with penicillin / streptomycin-containing low glucose Dulbecco's Modified Eagle Medium (hereinafter DMEM) and suspended in the same medium. Mixed with cells (Human Science Research Resource Bank). The mixture was neutralized with 1N NaOH and 0.5 mL (collagen concentration: 2.5 mg / mL, cell density: 3 × 10 ⁵ cells / mL) was dispensed into each well of a 24-well flat bottom plate. Incubated for minutes to allow the mixture to gel. DMEM was added to the top of the collagen gel and further incubated at 37 ° C. for 24 hours.

  Thereafter, the collagen gel was transferred to each well of a 12-well flat bottom plate to which 0.96 mL of DMEM was added and floated, and the collagen gel in each well was photographed with a digital camera. Subsequently, 0.12 mL of the compound of Example 1 diluted with DMSO and diluted with DMEM was added to each well (final compound concentration 3000 μmol / L, DMSO final concentration 0.5%), and incubated at 37 ° C. for 1 hour. Thereafter, 0.12 mL of TGF-β1 (R & D Systems) was added to each well (final concentration: 3 ng / mL), further incubated at 37 ° C. for 24 hours, and then the collagen gel was photographed with a digital camera. In addition, TGF-β1 non-added and compound non-added were used as untreated controls, and TGF-β1 added and no compounds added were used as TGF-β1-treated controls.

The area of the photographed collagen gel image was measured using particle analysis version 3 (Sumitomo Metal Technology). From the obtained area values, the area ratio after addition of the compound was determined for each gel when the addition before compound addition was 1, and the inhibition rate (%) against the collagen gel contraction caused by the addition of TGF-β1 was calculated using the following formula. Calculated.
Inhibition rate (%) = (1 − ([area ratio of untreated control] − [area ratio of each gel])
/ ([Area ratio of untreated control] − [area ratio of TGF-β1 treated control])) × 100

  As a result, the shrinkage of the collagen gel caused by the addition of TGF-β1 was statistically significantly suppressed by the addition of 3000 μmol / L of the compound of Example 1 (suppression rate 75.4 ± 28.1% (average value). ± standard deviation); P <0.01, t test).

  From this result, it was clarified that the compound of Example 1 inhibits the contraction of lung fibroblasts, and thus exhibits a fibrosis inhibiting action.

Example 3 Evaluation in a mouse bleomycin-induced pulmonary fibrosis model:
The effect of the compound of Example 1 on the amount of hydroxyproline in lung tissue of a mouse bleomycin-induced pulmonary fibrosis model was examined. As a mouse bleomycin-induced pulmonary fibrosis model, European Journal of Pharmacology, 2008, Vol. 590, p. The method described in 400-408 was used with some modifications.

  Bleomycin (Nippon Kayaku) was administered to ICR mice (male, 13 weeks old, Nippon Charles River) at a dose of 10 mg / kg once daily for 5 days in the tail vein to induce fibrosis. Mice that did not induce fibrosis (normal group) were similarly administered with physiological saline.

  To the mice in which fibrosis was induced, the compound of Example 1 was orally administered three times a day (100 mg / kg / day) at a dose of 33 mg / kg for 10 days from the start of bleomycin administration. The compound of Example 1 was used after being suspended in a 0.5% methylcellulose solution. A 0.5% methylcellulose solution was similarly administered to the normal group and the solvent group.

  The day after the administration of the compound of Example 1, the left lung was collected after exsanguination under isoflurane anesthesia. The amount of hydroxyproline in lung tissue, which is an index of fibrosis, was quantified as follows by partially modifying the Woessner method (Archives of Biochemistry and Biophysics, 1961, Vol. 93, p. 440-447). 1 mL of distilled water was added to the collected left lung (total) and homogenized, and 1 mL of concentrated hydrochloric acid was added thereto and heated at 120 ° C. for 24 hours. Thereafter, 2.4 mL of 5N NaOH was added for neutralization, and this was used as a measurement sample. A sample for measurement was appropriately diluted with distilled water, 1 mL of a 1.4% chloramine T solution was added and reacted at room temperature for 20 minutes, and then 3.15 M perchloric acid and 20% paradimethylaminobenzaldehyde / 2-methoxyethanol. 1 mL of each solution was added and reacted at 60 ° C. for 20 minutes. Then, it immediately cooled in the water bath and measured the light absorbency. The absorbance was measured at a measurement wavelength of 570 nm with a microplate reader (Bio-Rad, Model 680). The amount of hydroxyproline in the lung tissue (left lung) was calculated from a calibration curve (1 to 15 μg) obtained by linear regression analysis.

  The results of quantitative determination of the amount of hydroxyproline in lung tissue are shown in FIG. The values in the figure are average values ± standard error (n = 10). “Normal” in the figure indicates a group (normal group) in which 0.5% methylcellulose solution was orally administered to mice that do not induce fibrosis, and “solvent” represents 0.5% methylcellulose in mice that have induced fibrosis. The group (solvent group) to which the solution was orally administered is shown, and the “compound of Example 1” represents the group in which 100 mg / kg / day of the compound of Example 1 was orally administered to mice in which fibrosis was induced. # Indicates a statistically significant comparison with the normal group (Aspin-Welch t-test) (#: P <0.05), and * indicates a comparison with the solvent group (Aspin-Welch's t-test). t-test) indicates statistical significance (**: P <0.01).

  The amount of hydroxyproline in the lung tissue of the solvent group was statistically significantly higher than that of the normal group, indicating that bleomycin administration caused lung fibrosis. By administering the compound of Example 1 to mice in which fibrosis was induced by bleomycin administration, the amount of hydroxyproline in lung tissue was suppressed to a level equivalent to that in the normal group, and the value was statistically higher than that in the solvent group. Significantly decreased.

  From this result, it was revealed that the compound of Example 1 exhibits an excellent fibrosis inhibiting effect.

Example 4 Phototoxicity assessment of compounds in mice:
The phototoxic effect of the compound of Example 1 in mice was compared with pirfenidone. BALB / c mice (female, 7 weeks old, Charles River, Japan) were orally administered at a dose of 500 and 1000 mg / kg of the compound of Example 1 or 500 mg / kg of pirfenidone. The compound of Example 1 and pirfenidone were used in suspension in a 0.5% methylcellulose solution. The solvent group was similarly administered with a 0.5% methylcellulose solution.

Immediately after administration, 5 out of 10 patients in each group were placed under an ultraviolet irradiation device (Dermalley-200 Type A / NB and Delmale-200 Type A / B; Terumo Clinical Supply Co., Ltd.). 20 J / cm ² : Irradiance of about 1.4 mW / cm ² , irradiation for 4 hours) (ultraviolet irradiation animals), and the remaining 5 cases in each group were not irradiated (non-irradiated animals). After completion of UV irradiation (after 0.5, 24, 48 and 72 hours), the degree of erythema and edema that developed on the skin of the auricle was visually observed. Non-irradiated animals were also observed and compared to evaluate the presence or absence of phototoxicity.

  In the solvent group and the 500 and 1000 mg / kg administration groups of the compound of Example 1, erythema and edema were not observed in both the ultraviolet-irradiated animals and the non-irradiated animals. On the other hand, in the 500 mg / kg administration group of pirfenidone, erythema and edema were not observed in all non-irradiated animals, but erythema was observed 24 hours after irradiation in 4 out of 5 animals irradiated with ultraviolet rays.

  From this result, it was revealed that pirfenidone exhibits phototoxicity, whereas the compound of Example 1 does not exhibit phototoxicity.

(Example 5) Drug metabolism enzyme CYP induction test using frozen human hepatocytes:
A drug metabolizing enzyme CYP induction test using frozen human hepatocytes was carried out, and the inducing action of the compound of Example 1 on the drug metabolizing enzyme CYP was evaluated.

A suspension of human frozen hepatocytes (Xenotech) was prepared using Hepatocyte Isolation Kit (Xenotech). A 48-well plate was seeded with 1.2 × 10 ⁵ cells per well and pre-cultured for 48 hours. After the preculture, the compound of Example 1 and pirfenidone were cultured in CP medium (Celsis) containing 10, 100 and 1000 μmol / L, respectively, for 24 hours. The negative control group was cultured in CP medium (Celsis) containing 0.1 (v / v)% DMSO for 24 hours.

  Cells were washed with KHB Buffer (Celsis) 24 hours after exposure to the compound of Example 1 and pirfenidone. After washing, 0.3 mL of 125 μmol / L Testosterone-containing KHB Buffer was added and reacted at 37 ° C. for 30 minutes. 20 μL of the supernatant after the reaction was collected and diluted with 180 μL of ice-cooled KHB Buffer, which was used as a measurement sample.

20 μL of a sample for measurement was collected, 180 μL of 0.1% formic acid aqueous solution was added, and after stirring, the sample was subjected to LC / MS / MS analysis. Analysis by HPLC was performed under the following conditions. For the HPLC system, Agilent 1100 series (Agilent) was used. CAPCELL PAK MGIII 2.0 mm ID × 50 mm C18 (Shisei) was used for the column. The mobile phase was 0.1% formic acid / acetonitrile. As the MS / MS system, API-4000 (Applied Biosystems) was used. From the chromatogram obtained by LC / MS / MS analysis, the amount of 6β-hydroxytestosterone, which is a metabolite of testosterone, was quantified, and CYP3A4 enzyme activity (pmol / min / 10 ⁶ cells) was calculated.

  The results are shown in Table 1. In addition, it shows that the induction | guidance | derivation effect | action with respect to the drug metabolizing enzyme CYP of a compound is so high that the value of CYP3A4 enzyme activity is large. In addition, the CYP3A4 enzyme activity when treated with a compound when the CYP3A4 enzyme activity of the negative control group not exposed to the compound was set to 1.00 was shown as the induction ratio.

  From this result, it was clarified that the compound of Example 1 has a lower inducing action on the drug metabolizing enzyme CYP than pirfenidone. This suggested that the compound of Example 1 had low liver toxicity.

Example 6 Evaluation of Compound Gastrointestinal Disorders in Rats:
The effects of the compound of Example 1 on the digestive system damage were compared with pirfenidone by a gastric emptying ability test in rats. A single dose of 30 mg / kg of the compound of Example 1 or 30 mg / kg of pirfenidone was orally administered to SD rats (male, 6 weeks old, Charles River, Japan). The compound of Example 1 and pirfenidone were used in suspension in a 0.5% methylcellulose solution. The solvent group was similarly administered with a 0.5% methylcellulose solution.

Rats were fasted for 18 hours or more from the day before administration of the test compound. Sixty minutes after administration of the test compound, a 0.1% Evans blue solution was orally administered in a volume of 0.2 mL (200 μg). 30 minutes after administration of the 0.1% Evans blue solution, the rats were euthanized by cervical dislocation, immediately laparotomized, and the stomach was removed. The extracted stomach was immersed in 4 mL of 6.3 mol / L urea solution and shaken. Further acetone _8mL, 20% ZnSO 4 _0.4mL, was added in the order of 1N NaOH 0.4 mL, repeated shaking. Thereafter, the stomach was removed from the solution, and the volume of the solution was measured using a graduated cylinder. The solution was filtered through filter paper, and the absorbance at a wavelength of 620 nm was measured with a microplate reader (VersaMax, Molecular Devices Corp.). The concentration of Evans blue dye in the solution was determined using a calibration curve prepared from the standard solution for the calibration curve. The amount of Evans blue pigment contained in the solution was calculated and used as the amount of pigment remaining in the stomach. The amount of pigment remaining in the stomach and the stomach excretion ability were calculated from the following formulas.
Gastric residual pigment amount (μg) = concentration (μg / mL) × volume (mL)
Stomach excretion (%) = (1−residual pigment amount (μg) / amount of Evans blue administered (μg)) × 100

  Further, the digestive system damage action of the compound of Example 1 was compared with pirfenidone by a small intestine transportability test in rats. A single dose of 30 mg / kg of the compound of Example 1 or 30 mg / kg of pirfenidone was orally administered to SD rats (male, 6 weeks old, Charles River, Japan). The compound of Example 1 and pirfenidone were used in suspension in a 0.5% methylcellulose solution. The solvent group was similarly administered with a 0.5% methylcellulose solution.

Rats were fasted for 18 hours or more from the day before administration of the test compound. 60 minutes after administration of the test compound, 5% carbon powder suspension (liquid obtained by suspending carbon powder (Wako Pure Chemical Industries) at a ratio of 5% in 10% gum arabic (Wako Pure Chemical Industries)) in a volume of 1 mL. Orally administered. Thirty minutes after administration of the 5% carbon powder suspension, the rats were euthanized by cervical dislocation and immediately laparotomized, and the stomach and intestinal tract were excised while removing the mesentery. The intestinal tract was stretched, and the entire length of the small intestine (from the stomach pylorus to the cecal opening) and the distance from the stomach pylorus to the tip of the carbon powder suspension were measured using a measure. The transfer rate of 5% carbon powder suspension with respect to the entire length of the small intestine was calculated as the small intestine transport ability (%) by the following formula.
Small intestine transportability (%) = distance from stomach pylorus to tip of carbon powder suspension / total length of small intestine x 100

  Table 2 shows the results of the gastric emptying ability test and the small intestine transport ability test. The values in the table represent mean values ± standard error (n = 8). “Solvent” in the table indicates a group (solvent group) orally administered with a 0.5% methylcellulose solution, “Compound of Example 1” indicates a group orally administered with 30 mg / kg of the compound of Example 1, and “ “Pirfenidone” indicates a group to which 30 mg / kg of pirfenidone was orally administered.

  From this result, it was clarified that the compound of Example 1 had less influence on gastric excretion ability and small intestine transport ability than pirfenidone. This suggested that the compound of Example 1 had a low digestive system disorder action.

  The novel lactam derivative of the present invention has an excellent antifibrotic effect, does not have phototoxicity, and has reduced liver toxicity and gastrointestinal disorders. Therefore, it can be used as a highly safe pharmaceutical. In particular, it can be used as a therapeutic or prophylactic agent for fibrosis.

Claims (4)

A lactam derivative represented by the following chemical formula (I):

  A pharmaceutical comprising the lactam derivative according to claim 1 as an active ingredient.   A therapeutic or prophylactic agent for fibrosis comprising the lactam derivative according to claim 1 as an active ingredient.   A therapeutic or prophylactic agent for pulmonary fibrosis comprising the lactam derivative according to claim 1 as an active ingredient.

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