WO2004072013A1 - Hydroxyeicosadienoic acid compound - Google Patents

Hydroxyeicosadienoic acid compound Download PDF

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Publication number
WO2004072013A1
WO2004072013A1 PCT/JP2004/001312 JP2004001312W WO2004072013A1 WO 2004072013 A1 WO2004072013 A1 WO 2004072013A1 JP 2004001312 W JP2004001312 W JP 2004001312W WO 2004072013 A1 WO2004072013 A1 WO 2004072013A1
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Prior art keywords
compound
elastase
hydroxyeicosadienoic
acid
acid compound
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PCT/JP2004/001312
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French (fr)
Japanese (ja)
Inventor
Tohru Tanami
Naoya Ono
Tomomichi Chonan
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Taisho Pharmaceutical Co.,Ltd.
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Publication of WO2004072013A1 publication Critical patent/WO2004072013A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to a hydroxyeicosagen acid conjugate, and more particularly, has an erasase inhibitory effect, is easy to purify, is easy to handle, is physically stable, and is useful as a pharmaceutical. Is a hydroxyeicosadic acid compound which is easy. Conventional technology
  • elastase neutrophil elastase
  • Elastase is an enzyme that mainly degrades proteins such as elastin, collagen, proteodarican, and fibronectin, which constitute the stroma of in vivo connective tissues such as lung, cartilage, vascular wall, skin, and ligament. It has also been shown to act on other proteins and cells.
  • elastase In vivo, elastase is controlled by its endogenous inhibitor proteins, a1-proteazyme, ⁇ 2-macroglobulin, secretory leukocyte protease, and the like. Maintains homeostasis. However, if the balance between elastase and endogenous inhibitors is impaired by a decrease in inhibitory levels of elastase at the site of inflammation, control of elastase action is disrupted and tissue is damaged.
  • elastase Diseases that have been suggested to be involved in the pathogenesis of elastase are: emphysema, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pulmonary fibrosis, chronic interstitial pneumonia, chronic bronchitis, chronic airway infection , Diffuse panbronchiolitis, bronchiectasis, asthma, rheumatism, nephritis, liver failure, chronic rheumatoid arthritis, arthrosclerosis, osteoarthritis, psoriasis, periodontitis, atherosclerosis, organ transplantation Rejection, premature rupture, bullous disease, shock, sepsis, systemic erythematosus, Crohn's disease, disseminated intravascular coagulation, cerebral infarction, heart disease, ischemia-reperfusion injury observed in renal disease, corneal scar Tissue formation and spondylitis are known.
  • elastase inhibitors are useful as therapeutic or preventive agents for these diseases.
  • research has been actively conducted in recent years, and various elastase inhibitors have been reported, but their effects are not necessarily satisfactory.
  • No clinically useful drug has yet been found as an elastase inhibitor composed of a fatty acid derivative.
  • this compound is a viscous oily substance and has a problem with stability over time, so that it is difficult to use it as a medicine. Disclosure of the invention
  • An object of the present invention is to provide a compound which has an elasase inhibitory activity, is easy to purify, is easy to handle, is physically stable, and is easily used as a pharmaceutical.
  • the present inventors have conducted intensive studies on (5Z, 14Z) -1 (16R) -16-hydroxyeicosa-5,14-genic acid esters and salts for the purpose of solving the above problem.
  • 5Z, 14Z)-(16R)-(16-hydroxyeicosa) -5,14-genic acid lysine salt represented by (I) is a specific, colorless powder, They have found that they have good physicochemical properties and are physiologically useful, and have completed the present invention.
  • the present invention provides a compound of the formula (I)
  • the hydroxyeicosadic acid compound of the present invention can be obtained by the method described in W099 / 59964, (5Z, 14Z)-(16R) —16-hydroxyeicosa-5,14-genic acid, It can be easily obtained by reacting (L) -lysine in an inert solvent such as methanol or ethanol.
  • the compounds of the present invention can be administered systemically or topically orally or parenterally, such as intrarectally, subcutaneously, intramuscularly, intravenously, and transdermally.
  • it can be orally administered in the form of tablets, powders, granules, powders, capsules, solutions, emulsions, suspensions, etc., which can be produced by a conventional method.
  • preparations for intravenous administration aqueous or non-aqueous solutions, emulsions, suspensions, solid preparations which are dissolved in an injection solvent immediately before use and the like can be used.
  • the compound of the present invention can also be formulated by forming an inclusion compound with a, j8 or arcyclodextrin or methylated cyclodextrin.
  • the aqueous or non-aqueous solution, emulsion, suspension and the like can be administered by injection or the like.
  • the dosage varies depending on age, body weight, etc., but is 0.0 mg / kg / day / adult for adults, administered once or several times a day.
  • Example 1 the effects of the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited by these descriptions.
  • Example 1 the effects of the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited by these descriptions.
  • Test Example 1 [Test of elastase production by stimulation with fMLP (N-formyl Met-Leu-Phe)] The compound (compound (1)) obtained in Example 1 and Example 18 in the specification of W099 / 59964 The compound (compound (1)) was used as the test drug, and rat neutrophils were dissolved in 1% case In solution was prepared 15 to 18 hours after intraperitoneal administration (120 ml / kg). After decapitation, cells were collected by peritoneal washing. Use cold PBS as the washing solution. Collect the peritoneal lavage fluid, centrifuge, and resuspend in HBSS at a cell concentration of 1 x cell Zml.
  • cytochalasin B final concentration for the purpose of priming.
  • f is the ML P a group without added plus 0.4% ethanol solution 10. after stirring, and incubated an additional 10 minutes. The reaction was stopped on ice, and the culture supernatant was collected by centrifugation.
  • Elastase activity in the culture supernatant is an elastase-specific substrate
  • the activity of inhibiting the release of Elasase was calculated by the following equation.
  • Inhibition rate (%) ⁇ 1- (A-C) / (B-C) ⁇ X100
  • A represents the fluorescence intensity when stimulated with ⁇ MLP (1 / iM).
  • B represents the fluorescence intensity when fMLP (1 and the compound were added.
  • C represents the fluorescence intensity when fMLP was not added.
  • the 50% inhibitory concentration (IC50 value) was calculated from the concentration-inhibition curve.
  • a stability test of the test drug was performed at 6, 2, and 3 months later.
  • the residual ratio of the test drug was measured as follows. Precisely weigh about lmg of each test drug and heat
  • UV absorption spectrophotometer (Detection wavelength: 200 nm)
  • Table 3 shows the residual ratio of compound (I) and compound (II) when stored for 3 months under heating (40 ° C) and heating and humidification (40%, 75% RH).
  • Compound (II) had a very low residual ratio, indicating poor stability.
  • the compound (I) of the present invention was found to have a high residual ratio and to be stable.
  • the compound according to the present invention has a sufficient elastase inhibitory effect, is easy to purify, and is physically stable. Therefore, according to the present invention, emphysema, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, cystic pulmonary fibrosis, chronic interstitial pneumonia, chronic bronchitis, chronic airway infection, diffuse panbronchiolitis, bronchiectasis, Asthma, inflammation, nephritis, liver failure, rheumatoid arthritis, joints Sclerosis, osteoarthritis, psoriasis, periodontitis, atherosclerosis, rejection in organ transplantation, premature rupture of water, bullous disease, shock, sepsis, systemic lupus erythematosus, Crohn's disease, disseminated It has become possible to provide useful medicines for diseases such as intravascular coagulation, cerebral infarction, heart disease, renal disease, ischemia-reperfusion injury, formation of corneal scar

Abstract

The hydroxyeicosadienoic acid compound represented by the formula (I). (I) The compound has sufficient elastase inhibitory activity, is east to purify, and is physically stable. It is usable as a useful medicine.

Description

明細書 ヒドロキシエイコサジェン酸化合物 技術分野  Description Hydroxyeicosadic acid compound Technical field
本発明は、 ヒドロキシエイコサジェン酸ィ匕合物に関し、 更に詳しくは、 エラス夕一 ゼ阻害作用を有し、 精製が容易で取り扱いやすく、 物理的にも安定であり、 医薬品と しての利用が容易なヒドロキシエイコサジェン酸化合物である。 従来技術  The present invention relates to a hydroxyeicosagen acid conjugate, and more particularly, has an erasase inhibitory effect, is easy to purify, is easy to handle, is physically stable, and is useful as a pharmaceutical. Is a hydroxyeicosadic acid compound which is easy. Conventional technology
リンパ球の一種である好中球から出されるプロテア一ゼは主に細菌等の外来微生物 や損傷細胞の分解等の役割を有し生体防御反応において重要な役目を担っている。 セ リンプロテア一ゼの一種である好中球エラスタ一ゼ (以下、 単にエラスタ一ゼと称す る） は、 感染や炎症性疾患時に出現する好中球の顆粒から大量に放出される。 エラス タ一ゼは、 主として肺、 軟骨、 血管壁、 皮膚、 靱帯などの生体内結合組織の間質を構 成するタンパク質エラスチン、 コラーゲン、 プロテオダリカン、 フイブロネクチンな どを分解する酵素である。 また、 その他のタンパク質や細胞にも作用する事が明らか にされてきている。  Proteases released from neutrophils, a type of lymphocyte, mainly play a role in degrading foreign microorganisms such as bacteria and damaged cells, and play an important role in biological defense reactions. Neutrophil elastase (hereinafter simply referred to as elastase), a type of serine protease, is released in large amounts from neutrophil granules that appear during infections and inflammatory diseases. Elastase is an enzyme that mainly degrades proteins such as elastin, collagen, proteodarican, and fibronectin, which constitute the stroma of in vivo connective tissues such as lung, cartilage, vascular wall, skin, and ligament. It has also been shown to act on other proteins and cells.
生体内でエラス夕ーゼは、 内因性インヒビタ一タンパク質である a 1—プロテア一 ゼインヒピ夕一、 α 2—マクログロプリン、 分泌性白血球プロテアーゼィンヒピ夕一 などによってその作用が制御されつつ生体の恒常性を維持している。 しかしながら，炎 症部位でのエラス夕—ゼの過剰放出ゃィンヒビターレベルの低下によりエラス夕一ゼ と内因性インヒビタ一のバランスが損なわれると、 エラスターゼ作用の制御が崩れ組 織が傷害される。  In vivo, elastase is controlled by its endogenous inhibitor proteins, a1-proteazyme, α2-macroglobulin, secretory leukocyte protease, and the like. Maintains homeostasis. However, if the balance between elastase and endogenous inhibitors is impaired by a decrease in inhibitory levels of elastase at the site of inflammation, control of elastase action is disrupted and tissue is damaged.
エラスタ一ゼの病態への関与が示唆されている疾患としては、 肺気腫、 成人呼吸窮 迫症候群、 突発性肺線維症、 囊胞性肺線維症、 慢性間質性肺炎、 慢性気管支炎、 慢性 気道感染、 びまん性汎細気管支炎、 気管支拡張症、 喘息、 塍炎、 腎炎、 肝不全、 慢性 関節リュウマチ、 関節硬化症、 変形性関節炎、 乾せん、 歯周炎、 ァテローム性動脈硬 化症、 臓器移植における拒絶反応、 早期破水、 水疱症、 ショック、 敗血症、 全身性ェ リテマト一デス、 クローン病、 播種性血管内凝固症、 脳梗塞、 心疾患、 腎疾患時に認 められる虚血再灌流障害、 角膜瘢痕組織の形成、 脊椎炎などが知られている。 従って、 エラスターゼ阻害剤はこれらの疾患の治療剤、 あるいは予防剤として有用 である。 かかる期待のもとに近年盛んに研究が行われており、 種々のエラスターゼ阻 害剤が報告されているが、 その作用は必ずしも十分とは言えない。 また、 脂肪酸誘導 体からなるエラスタ一ゼ阻害物質としては未だ臨床上有用な薬剤は見い出されていな い。 Diseases that have been suggested to be involved in the pathogenesis of elastase are: emphysema, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, pulmonary fibrosis, chronic interstitial pneumonia, chronic bronchitis, chronic airway infection , Diffuse panbronchiolitis, bronchiectasis, asthma, rheumatism, nephritis, liver failure, chronic rheumatoid arthritis, arthrosclerosis, osteoarthritis, psoriasis, periodontitis, atherosclerosis, organ transplantation Rejection, premature rupture, bullous disease, shock, sepsis, systemic erythematosus, Crohn's disease, disseminated intravascular coagulation, cerebral infarction, heart disease, ischemia-reperfusion injury observed in renal disease, corneal scar Tissue formation and spondylitis are known. Therefore, elastase inhibitors are useful as therapeutic or preventive agents for these diseases. With such expectations, research has been actively conducted in recent years, and various elastase inhibitors have been reported, but their effects are not necessarily satisfactory. No clinically useful drug has yet been found as an elastase inhibitor composed of a fatty acid derivative.
W099/59964号に開示された下記式  The following formula disclosed in W099 / 59964
で表される （5 Z, 14Z) - (16R) 一 16—ヒドロキシエイコサー 5， 14一ジェン酸
Figure imgf000004_0001
(5 Z, 14Z)-(16R)-16-hydroxy eicosa 5,14
Figure imgf000004_0001
は、 エラス夕ーゼ阻害作用を有し、 上記疾患に対し有用な化合物である。 しかしなが ら、 この化合物は粘性の油性物質であり、 また経時的な安定性に問題があるため、 医 薬品として利用することが困難である。 発明の開示 Is a compound that has an Elasase inhibitory activity and is useful for the above diseases. However, this compound is a viscous oily substance and has a problem with stability over time, so that it is difficult to use it as a medicine. Disclosure of the invention
本発明の目的は、 エラス夕ーゼ阻害作用を有し、 精製が容易で取り扱いやすく、 物 理的にも安定であり、 医薬品としての利用が容易な化合物を提供することにある。 本発明者らは、 前記課題の解決を目的として、 （5 Z, 14Z) 一 (16R) —16—ヒ ドロキシエイコサ— 5, 14ージェン酸のエステル体や塩等について鋭意検討した結果.. 式 ( I) で表される (5 Z, 14Z) 一 (16R) —16—ヒドロキシエイコサ— 5, 14- ジェン酸のリジン塩のみが、 驚くべきことに、 特異的に、 無色粉末であり、 極めて良 好な物理化学的性質を有し、 かつ生理学的にも有用であることを見出し本発明を完成 した。  An object of the present invention is to provide a compound which has an elasase inhibitory activity, is easy to purify, is easy to handle, is physically stable, and is easily used as a pharmaceutical. The present inventors have conducted intensive studies on (5Z, 14Z) -1 (16R) -16-hydroxyeicosa-5,14-genic acid esters and salts for the purpose of solving the above problem. Surprisingly, only the (5Z, 14Z)-(16R)-(16-hydroxyeicosa) -5,14-genic acid lysine salt represented by (I) is a specific, colorless powder, They have found that they have good physicochemical properties and are physiologically useful, and have completed the present invention.
すなわち、 本発明は、 式 （I)
Figure imgf000004_0002
That is, the present invention provides a compound of the formula (I)
Figure imgf000004_0002
で表されるヒドロキシエイコサジェン酸化合物である。 発明を実施するための最良の形態 It is a hydroxy eicosadic acid compound represented by these. BEST MODE FOR CARRYING OUT THE INVENTION
本発明のヒドロキシエイコサジェン酸化合物は、 W099/59964号に記載の方法により 得られる （5 Z, 14Z) - (16R) —16—ヒドロキシエイコサ— 5, 14—ジェン酸に、 メタノール、 エタノールなどの不活性溶媒中、 （L) 一リジンを反応させることによ り容易に得られる。 The hydroxyeicosadic acid compound of the present invention can be obtained by the method described in W099 / 59964, (5Z, 14Z)-(16R) —16-hydroxyeicosa-5,14-genic acid, It can be easily obtained by reacting (L) -lysine in an inert solvent such as methanol or ethanol.
本発明の化合物は、 全身的または局所的に経口または直腸内、 皮下、 筋肉内、 静脈 内経皮等の非経口的に投与される。 例えば、 通常の方法により製造することができる 錠剤、 粉剤、 顆粒剤、 散剤、 カプセル剤、 液剤、 乳剤、 懸濁剤等の形で経口投与する ことができる。静脈内投与の製剤としては、水性または非水性溶液剤、乳剤、懸濁剤、 使用直前に注射溶媒に溶解して使用する固形製剤等を用いることができる。 また、 本 発明の化合物は、 a、 j8 もしくはアーシクロデキストリンまたはメチル化シクロデキ ストリン等と包接ィ匕合物を形成させて製剤ィ匕することもできる。 更に、 その水性また は非水性溶液剤、 乳剤、 懸濁剤等を注射等により投与することができる。 投与量は年 齢、 体重等により異なるが、 成人に対し 0.0卜 100mg/kg/dayであり、 これを 1日 1回 または数回に分けて投与する。 実施例  The compounds of the present invention can be administered systemically or topically orally or parenterally, such as intrarectally, subcutaneously, intramuscularly, intravenously, and transdermally. For example, it can be orally administered in the form of tablets, powders, granules, powders, capsules, solutions, emulsions, suspensions, etc., which can be produced by a conventional method. As preparations for intravenous administration, aqueous or non-aqueous solutions, emulsions, suspensions, solid preparations which are dissolved in an injection solvent immediately before use and the like can be used. The compound of the present invention can also be formulated by forming an inclusion compound with a, j8 or arcyclodextrin or methylated cyclodextrin. Further, the aqueous or non-aqueous solution, emulsion, suspension and the like can be administered by injection or the like. The dosage varies depending on age, body weight, etc., but is 0.0 mg / kg / day / adult for adults, administered once or several times a day. Example
以下、 本発明の効果を実施例および試験例を挙げてより具体的に説明するが、 本発 明はこれらの記載によつてなんら制限されるものではない。 実施例 1  Hereinafter, the effects of the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited by these descriptions. Example 1
(5 Z, 14Z) 一 (16R) 一 16—ヒドロキシエイコサ— 5, 14—ジェン酸 Lーリ ジン塩の製造  (5Z, 14Z) One (16R) One 16-Hydroxyeicosa-5,14-L-lysine acid
W099/59964号の明細書の実施例 18に記載された （5Z， 14Z) - (16 ) —16—ヒ ドロキシエイコサー 5, 14一ジェン酸 (1.012g, 3.12mmol) と L—リジン (455mg, 3.11mmol) のエタノール （120 ml) 溶液を混合し、 室温にて 3時間攪拌後、 減圧乾固 した。 残留物に石油エーテル (120 ml) を加え、 室温にて 5分間攪拌、 濾過した後、 固形物を減圧乾燥して、 無色粉末の標記化合物 (1.32g) を得た。  (5Z, 14Z)-(16) -16-hydroxyeicosa 5,14-monoic acid (1.012 g, 3.12 mmol) and L-lysine (described in Example 18 of the specification of W099 / 59964) A solution of 455 mg (3.11 mmol) in ethanol (120 ml) was mixed, stirred at room temperature for 3 hours, and evaporated to dryness under reduced pressure. Petroleum ether (120 ml) was added to the residue, the mixture was stirred at room temperature for 5 minutes, filtered, and the solid was dried under reduced pressure to give the title compound (1.32 g) as a colorless powder.
Ή -匪 R(CD30D,300MHz) (5 pm:0.91 (t, J=6.8Hz, 3H), 1.24-1.74(m, 22H) , 1.78-1.90 (m, 2H)， 1.98-2.14(m, 6H), 2.18(t, J=7.6Hz, 2H), 2.91 (t, J=7.5Hz, 1H) , 3.52(t, J=6.1Hz, 1H), 4.32-4.42 (m, 1H) , 5.26-5.50 (m, 1H) Ή-Marauder R (CD 3 0D, 300MHz) (5 pm: 0.91 (t, J = 6.8Hz, 3H), 1.24-1.74 (m, 22H), 1.78-1.90 (m, 2H), 1.98-2.14 (m , 6H), 2.18 (t, J = 7.6Hz, 2H), 2.91 (t, J = 7.5Hz, 1H), 3.52 (t, J = 6.1Hz, 1H), 4.32-4.42 (m, 1H), 5.26 -5.50 (m, 1H)
IR(KBr) ： 2928, 2856, 2143, 1584, 1516, 1466, 1407, 1350, 1322, 1202, 1163, 1019, 912， 864, 736, 707, 667, 547, 480, 428, 415 cm"1 IR (KBr): 2928, 2856, 2143, 1584, 1516, 1466, 1407, 1350, 1322, 1202, 1163, 1019, 912, 864, 736, 707, 667, 547, 480, 428, 415 cm " 1
m.p. :176. -181.5°C 比較例 mp: 176.-181.5 ° C Comparative example
実施例 1の L一リジンの代わりにプロ力イン、 コリン、 2—アミノエタノール、 ピ ペラジン、 ベンザチン、 トリエタノールァミン、 トリス （ヒドロキシメチル） ァミノ メタン、 N—メチル一D—ダルカミン、 エチレンジァミン、 シクロへキシルァミン、 3—アミノビリジン、 2—ァミノ一 4—ピコリン、 ベンジルァミン、 L一オル二チン 又は酸ィ匕カルシウムを用い、 実施例 1と同様な方法で、 W099/59964号の明細書の実施 例 18に記載された （5 Z， 14 Z ) 一 (16R) 一 16—ヒドロキシエイコサ— 5， 14—ジ ェン酸の塩を得た。それぞれの塩の状態を表 1に示す。比較例で得られた塩の状態は、 油状物質またはアモルファスであり、 取り扱いが困難であり、 医薬品として利用する ことが困難である。 表 1  In place of L-lysine in Example 1, pro-force, choline, 2-aminoethanol, piperazine, benzathine, triethanolamine, tris (hydroxymethyl) amino methane, N-methyl-1-D-dalkamine, ethylenediamine, cyclo Using hexylamine, 3-aminoviridine, 2-amino-14-picoline, benzylamine, L-orditin or calcium oxydiamine, in the same manner as in Example 1, the example of the specification of W099 / 59964 is used. The salt of (5Z, 14Z) -1 (16R) -1 16-hydroxyeicosa-5,14-genic acid described in 18 was obtained. Table 1 shows the state of each salt. The state of the salt obtained in the comparative example is an oily substance or an amorphous state, is difficult to handle, and is difficult to use as a pharmaceutical. table 1
Figure imgf000006_0001
試験例 1 [fMLP (N- formy卜 Met-Leu-Phe)刺激によるエラス夕一ゼ産生試験] 実施例 1で得た化合物 （化合物（1) ) および W099/59964号の明細書の実施例 18の化 合物 （化合物（Π) ) を被験薬物とし、 ラット好中球は生理食塩水に溶解した 1 %カゼ イン溶液を腹腔内投与（120ml/kg) 15から 18時間後に調製した。 断頭後、 腹腔洗浄に より細胞を回収した。 洗浄液は冷 PBSを使用する。 腹腔洗浄液を回収し、 遠心処理し HBSSを用いて細胞濃度、 1 X 細胞 Zmlに再懸濁する。
Figure imgf000006_0001
Test Example 1 [Test of elastase production by stimulation with fMLP (N-formyl Met-Leu-Phe)] The compound (compound (1)) obtained in Example 1 and Example 18 in the specification of W099 / 59964 The compound (compound (1)) was used as the test drug, and rat neutrophils were dissolved in 1% case In solution was prepared 15 to 18 hours after intraperitoneal administration (120 ml / kg). After decapitation, cells were collected by peritoneal washing. Use cold PBS as the washing solution. Collect the peritoneal lavage fluid, centrifuge, and resuspend in HBSS at a cell concentration of 1 x cell Zml.
細胞をプライミングする目的でサイトカラシン B (最終濃度 を添加した。 96ゥエル培養プレートにゥエル当たり 190 1加え、規定濃度（1 X10— 7Mから 3 Χ1(Γ5 Μ)の被験薬物を添加し 5%C02、 37度で培養した。 10分後、 20 MfMLPを 10 Lを加えた。 f ML Pを加えない群には 0.4%エタノール溶液 10 を加えた。攪拌後、 さらに 10分間培養した。 反応は氷上で停止し、 培養上清は遠心により回収した。 培養上清中のエラス夕ーゼ活性測定 Cells were added to cytochalasin B (final concentration for the purpose of priming. 96 Ueru culture plates 190 1 added per Ueru, determined by adding test drugs prescribed concentration (1 X10- 7 3 Χ1 (Γ 5 Μ from M) 5 % C0 2, and cultured at 37 °. after 10 minutes, a 20 MfMLP was added 10 L. f is the ML P a group without added plus 0.4% ethanol solution 10. after stirring, and incubated an additional 10 minutes. The reaction was stopped on ice, and the culture supernatant was collected by centrifugation.
培養上清中のエラス夕ーゼ活性はエラス夕ーゼ特異的な基質  Elastase activity in the culture supernatant is an elastase-specific substrate
(N-s uc c i ny 1 -L-a 1 any 1 -L-a 1 any 1 -L- r o 1 i ne-v 1 ine-MCA； Peptide Institute, Inc. 大 阪) 0.12mMを用いて 50mMトリス溶液 （pH8.0) 中で測定した。 培養上清 50 1と基質 溶液 50, 1を加え、 37度 30分間インキュベートした。 エラスタ一ゼ活性は励起波長 360nm、 吸収波長 480nmで測定した。 (Ns uc ciny 1 -La 1 any 1 -La 1 any 1 -L-ro 1ine-v 1 ine-MCA; Peptide Institute, Inc. Osaka) 50 mM Tris solution (pH 8.0 ). The culture supernatant 50 1 and the substrate solution 50, 1 were added, and incubated at 37 ° C for 30 minutes. Elastase activity was measured at an excitation wavelength of 360 nm and an absorption wavelength of 480 nm.
エラス夕ーゼ遊離抑制活性 (阻害率) は以下の式によって算出した。 The activity of inhibiting the release of Elasase (inhibition rate) was calculated by the following equation.
阻害率 (%) = {1-(A-C)/(B-C)} X100  Inhibition rate (%) = {1- (A-C) / (B-C)} X100
Aは〖MLP (1 /iM) で刺激した時の蛍光強度を表す。 Bは fMLP (1 と化合物が 添加した時の蛍光強度を表す。 Cは fMLPを添加しない時の蛍光強度を表す。 50%阻害 する濃度 （IC50値） は濃度-阻害曲線から算出した。  A represents the fluorescence intensity when stimulated with ΔMLP (1 / iM). B represents the fluorescence intensity when fMLP (1 and the compound were added. C represents the fluorescence intensity when fMLP was not added. The 50% inhibitory concentration (IC50 value) was calculated from the concentration-inhibition curve.
結果 Result
IC50値を表 2に示した。 化合物（II)と化合物（I)はほぼ同等の IC50値を示した。 よ つて、 本願発明の化合物（I)は、 十分なエラス夕ーゼ活性を示すことが証明された。 表 2  The IC50 values are shown in Table 2. Compound (II) and compound (I) showed almost the same IC50 value. Therefore, it was proved that the compound (I) of the present invention exhibited a sufficient elastase activity. Table 2
被験薬 IC50値 ( M)  Test drug IC50 value (M)
化合物 (II) 12 5  Compound (II) 12 5
化合物 (I) 16 9 試験例 2 [安定性試験]  Compound (I) 16 9 Test Example 2 [Stability test]
試験例 2と同様に、 化合物 (I)および化合物（II)を被験薬物とし、 これらを加温 (40°C) および加温加湿'（40°C、 75 RH) で 3ヶ月保存したときの、 保存開始から 1 W As in Test Example 2, Compound (I) and Compound (II) were used as test drugs, and these were stored for 3 months in warming (40 ° C) and warming and humidifying (40 ° C, 75 RH). , From the start of saving 1 W
6 ヶ月後、 2ヶ月後、 3ヶ月後における被験薬物の安定性試験を実施した。 被験薬物の 残存率測定は下記の要領で行った。 被験薬物各約 lmgを精密に量り取り、 加温条件 A stability test of the test drug was performed at 6, 2, and 3 months later. The residual ratio of the test drug was measured as follows. Precisely weigh about lmg of each test drug and heat
(40°C) では遮光 （アルミ箔） 密栓、 加温 ·加湿条件 （40°C、 75 %RH) では遮光 （アルミ 箔） 開栓のネジロ試験管に入れ保存した。 定量は以下の手順で行った。 所定の保存期 間が終了した試験管に 50%水/ァセトニトリル混液 10mlを加えて溶解後， 液体クロマ トグラフで定量した。 At (40 ° C), light-shielded (aluminum foil) was sealed and heated and humidified (40 ° C, 75% RH). The quantification was performed according to the following procedure. After the specified storage period, 10 ml of a 50% water / acetonitrile mixture was added to the test tubes, dissolved, and quantified by liquid chromatography.
HPLC条件 HPLC conditions
カラム： CAPCELL PAK UG120, 5 m, Φ 4 . 6 X 150 画 (SHISEID0) Column: CAPCELL PAK UG120, 5 m, Φ 4.6 X 150 screen (SHISEID0)
カラム温度： 4(TC  Column temperature: 4 (TC
検出：紫外吸光光度計 （検出波長： 200nm)  Detection: UV absorption spectrophotometer (Detection wavelength: 200 nm)
流速： 1. Oml/min ―  Flow rate: 1. Oml / min ―
注入量： I O I  Injection volume: I O I
移動相： 0. 1 %リン酸水溶液/ァセトニトリル ( 2 ： 3 )  Mobile phase: 0.1% phosphoric acid aqueous solution / acetonitrile (2: 3)
結果 Result
加温 （40°C) および加温加湿 （40で、 75%RH) で 3ヶ月保存したときの化合物（I)及 び化合物 (I I)の残存率を表 3に示した。 化合物（I I)は、 残存率が非常に低く、 安定性 が悪いことが示された。 それに対し、 本願発明の化合物 (I)は、 残存率が高く、 安定で あることが示された。  Table 3 shows the residual ratio of compound (I) and compound (II) when stored for 3 months under heating (40 ° C) and heating and humidification (40%, 75% RH). Compound (II) had a very low residual ratio, indicating poor stability. In contrast, the compound (I) of the present invention was found to have a high residual ratio and to be stable.
？ S 3? S 3
Figure imgf000008_0001
産業上の利用可能性
Figure imgf000008_0001
Industrial applicability
本発明に係る化合物は十分なエラス夕ーゼ阻害作用を有し、 かつ精製が容易で物理 的にも安定である。 従って、 本発明により肺気腫、 成人呼吸窮迫症候群、 突発性肺線 維症、 嚢胞性肺線維症、 慢性間質性肺炎、 慢性気管支炎、 慢性気道感染、 びまん性汎 細気管支炎、 気管支拡張症、 喘息、 塍炎、 腎炎、 肝不全、 慢性関節リュウマチ、 関節 硬化症、 変形性関節炎、 乾せん、 歯周炎、 ァテロ一ム性動脈硬化症、 臓器移植におけ る拒絶反応、 早期破水、 水疱症、 ショック、 敗血症、 全身性エリテマトーデス、 クロ ーン病、 播種性血管内凝固症、 脳梗塞、 心疾患、 腎疾患時に認められる虚血再灌流障 害、 角膜瘢痕組織の形成、 脊椎炎などの疾患に対して有用な医薬を提供することが可 能となった。 The compound according to the present invention has a sufficient elastase inhibitory effect, is easy to purify, and is physically stable. Therefore, according to the present invention, emphysema, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, cystic pulmonary fibrosis, chronic interstitial pneumonia, chronic bronchitis, chronic airway infection, diffuse panbronchiolitis, bronchiectasis, Asthma, inflammation, nephritis, liver failure, rheumatoid arthritis, joints Sclerosis, osteoarthritis, psoriasis, periodontitis, atherosclerosis, rejection in organ transplantation, premature rupture of water, bullous disease, shock, sepsis, systemic lupus erythematosus, Crohn's disease, disseminated It has become possible to provide useful medicines for diseases such as intravascular coagulation, cerebral infarction, heart disease, renal disease, ischemia-reperfusion injury, formation of corneal scar tissue, and spondylitis .

Claims

請求の範囲 The scope of the claims
1. 式 （I)
Figure imgf000010_0001
1. Formula (I)
Figure imgf000010_0001
で表されるヒドロキシエイコサジェン酸ィ匕合物。 A hydroxyeicosadiene acid conjugate represented by the formula:
PCT/JP2004/001312 2003-02-14 2004-02-09 Hydroxyeicosadienoic acid compound WO2004072013A1 (en)

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WO2015071766A1 (en) * 2013-11-15 2015-05-21 Dignity Sciences Limited Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids

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Publication number Priority date Publication date Assignee Title
JP2002515480A (en) * 1998-05-15 2002-05-28 ユニバーシティ オブ バーモント Novel analogs of 16-hydroxyeicosatetraenoic acid

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Publication number Priority date Publication date Assignee Title
JP2002515480A (en) * 1998-05-15 2002-05-28 ユニバーシティ オブ バーモント Novel analogs of 16-hydroxyeicosatetraenoic acid

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Publication number Priority date Publication date Assignee Title
WO2015071766A1 (en) * 2013-11-15 2015-05-21 Dignity Sciences Limited Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids
US20150152034A1 (en) * 2013-11-15 2015-06-04 Dignity Sciences Limited Pharmaceutically Acceptable Salts of Fatty Acids
CN105899485A (en) * 2013-11-15 2016-08-24 尊严科学有限公司 Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids
JP2016538288A (en) * 2013-11-15 2016-12-08 ディグニティ サイエンシス リミテッド Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids
US10017453B2 (en) * 2013-11-15 2018-07-10 Ds Biopharma Limited Pharmaceutically acceptable salts of fatty acids
CN105899485B (en) * 2013-11-15 2018-10-19 尊严科学有限公司 The pharmaceutically acceptable salt of how unsaturated hydroxy fatty acid
CN109232278A (en) * 2013-11-15 2019-01-18 Ds生物制药有限公司 The pharmaceutically acceptable salt of how unsaturated hydroxy fatty acid
EP3546446A1 (en) * 2013-11-15 2019-10-02 DS Biopharma Limited Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids
US10544088B2 (en) 2013-11-15 2020-01-28 Ds Biopharma Limited Pharmaceutically acceptable salts of fatty acids
JP2020055825A (en) * 2013-11-15 2020-04-09 ディーエス バイオファーマ リミテッド Pharmaceutically acceptable salts of polyunsaturated hydroxy fatty acids

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